Sex-Role Reversed Pipefish, Syngnathus abaster. - Universidade ...

Reproductive Ecology of the ‘Mildly’
Sex-Role Reversed Pipefish,
Syngnathus abaster.
Carina Santos da Silva
Departamento de Zoologia e Antropologia
2008

Dissertação apresentada à
Faculdade de Ciências da Universidade do Porto
Para a obtenção do grau de Doutor em Biologia
Este trabalho foi apoiado financeiramente pela
Fundação para a Ciência e a Tecnologia (FCT)
através da atribuição de uma bolsa de doutoramento
(SFRH/BD/13171/2003)

“The most beautiful thing we can experience is the mysterious. It is the
source of all true art and all science. He to whom this emotion is a
stranger, who can no longer pause to wonder and stand rapt in awe, is as
good as dead: his eyes are closed.”
Albert Einsten

Para os meus pais,

Agradecimentos
“As palavras tornam-se sempre insuficientes quando com elas queremos
dizer o que nos vai para lá da alma.” Como agora…
Muito para além de um trabalho académico, esta tese contém em
si um objectivo pessoal que nunca teria sido alcançado sem a dedicação,
o esforço e a amizade de um considerável número de pessoas. Agradecer
e realçar o contributo de todas elas, sendo da mais elementar justiça, é no
entanto, também a mais difícil das tarefas. Assim, e porque me falta ‘arte’
para escrever tudo quanto queria, o meu mais profundo agradecimento
será a todos expresso, não somente pelo que aqui deixo, mas pela minha
entrega, empenho, dedicação e amizade nas diversas etapas que a este
percurso se seguirão.
É com grande orgulho que as minhas primeiras palavras vão para
os meus orientadores, que me abriram as ‘portas’ da ciência e me
guiaram e inspiraram ao longo destes anos de trabalho.
Obrigada Professora Natividade. Pela boa disposição, pelo apoio,
pela confiança, e, acima de tudo, por nunca ter deixado este ‘sonho’
desmoronar-se. Foi uma honra ter a Professora como orientadora. Será
muito gratificante continuar a trabalhar consigo, espero nunca a desiludir.
Obrigada Nuno. Por teres apostado (e arriscado!) em mim e por
nunca teres desistido (mesmo quando poderia parecer o mais sensato).
Obrigada pela tua orientação, pela tua dedicação… pela tua paciência.
Aprendi muito contigo, este trabalho também é teu.
Obrigada Professor Almada. Esta tese não teria o mesmo valor
sem a sua experiência e os seus valiosos conselhos. Tenho imensa
admiração pelo seu trabalho.
Quero também agradecer a todos os funcionários da Estação de
Zoologia da Foz, em particular ao sr. Pedro Correia e sr. Sá Pereira, por
i

toda a ajuda prestada durante a realização das experiências, e por terem
tornado possível o que muitas vezes parecia impraticável.
Agradeço ainda a todos os meus colegas da Estação, e muito em
particular ao Rui Sá, pela ajuda, pelo apoio, pelos animados almoços
(sempre com música), pelas gargalhadas e pelas partilhas que fizeram da
passagem pela Foz uma etapa particularmente marcante.
Agradeço ao Professor António Paulo, a simpatia e as ‘dicas’ que
sempre me deu nos momentos de ‘crise’ com as larvas.
Agradeço ao Professor Coimbra a atenção que teve para comigo
ao ceder-me um lugar de gabinete para poder completar a última fase
deste trabalho nas melhores condições.
difícil…
Agradeço aos meus Amigos. Sem eles tudo teria sido muito mais
Romain, le plus grands des mercis! Il y aurait tellement à dire et,
en même temps rien ne serait jamais assez. Cette these te dois tant… je
(!) te dois tant!
Ana, (sra. doutora, Ana Medeiros!) não há palavras que traduzam
a minha amizade e o meu agradecimento. Tu, melhor do que ninguém,
sabes o que este momento representa e a vitória que ele encerra. O meu
muito obrigada pelas infindáveis horas de conversas, risos e lágrimas, por
teres ajudado a manter a minha sanidade mental (dentro de limites
razoáveis!), por teres estado (e continuares) sempre aqui.
Maninha, eis o teu ‘apontamentosinho’! Novamente, faltam-me as
palavras… Como é bom poder fazer-me acompanhar da tua amizade.
Melhor, como é bom, saber que poderei sempre contar com ela! Obrigada
pelas incansáveis revisões dos textos, pelas conversas, pelos disparates
que sempre nos ajudaram a sorrir mesmo quando tudo parecia cinzento.
O teu apoio e a tua ajuda foram determinantes. Espero em breve poder
retribuir de uma forma tão dedicada. Não posso também deixar de
agradecer às ‘tuas’ meninas da UnIGENe, por toda a colaboração!
El-vi-na (!), thank you so much for your precious comments on my
manuscripts! Ainda me lembro do primeiro dia do colégio: “ (…) é difícil,
muito difícil, mas com trabalho é possível” dizias tu. Quem diria onde esse
trabalho nos trouxe! Obrigada pela tua amizade.
ii

Catarina e Ricardo, devo-vos mais do que alguma vez conseguirei
traduzir por palavras. Obrigada por fazerem parte do caminho. Om Shanti!
destes anos.
Teresa, Daniel, obrigada por terem estado presentes ao longo
Armando, a tua t-shirt salvou os ‘meus peixes’! Obrigada pelas
etapas que me ajudaste a superar, pela tua entrega, pela tua força. Muito
do que aqui está foi conseguido em conjunto.
Agradeço às minhas colegas (e amigas), Filipa e Marisa, o
companheirismo (mesma nas saídas de campo mais frustrantes), a
confiança e a motivação com que sempre me brindaram.
Agradeço à Ana Coimbra pelos valiosos conselhos, pelas revisões
dos textos e pela companhia na ‘maratona’ final. A tua ajuda foi muito
importante, um grande obrigada!
Agradeço ao Pedro Romeu o auxílio prestado na última etapa de
elaboração desta tese (quando a internet até já se desligava sozinha e os
textos se pintavam de amarelo!).
Agradeço ao Jorge pel’A capa! A tua ajuda foi preciosa.
Finalmente, agradeço àqueles a quem tudo devo e de quem mais
me orgulho… à minha família.
Avós, obrigado por me terem ensinado o valor das coisas simples.
Obrigada, maninho, pelos abraços nos momentos certos.
Desculpa ter tantas vezes, ‘descarregado’ em ti as frustrações destes
anos.
Pai, mãe… Obrigado por me terem dado o melhor dos exemplos
para traçar o meu caminho. Sem vocês nada disto faria sentido… a vocês
tudo dedico.
iii

Sumário
A família Syngnathidae, com o seu elevado grau de
especialização parental associado a singularidades ecológicas e
comportamentais, constitui um modelo particularmente interessante para o
estudo da selecção sexual. No entanto, a falta de informação disponível
sobre os caracteres mais básicos da sua biologia impossibilitam uma mais
vasta compreensão acerca da direcção da evolução das estratégias
reprodutivas, nomeadamente daquelas que se relacionam com a evolução
dos diferentes padrões de acasalamento e estabelecimento de uma
reversão dos papéis sexuais. Paralelamente, e uma vez que muitas
populações de singnatídeos se encontram actualmente em processo de
acentuado declínio, este tipo de informação torna-se essencial para o
desenvolvimento de planos de protecção e conservação.
Com o presente estudo pretendeu-se, através de distintas
aproximações experimentais, claramente interligadas, caracterizar, de
forma detalhada e extensiva, a ecologia reprodutiva de uma população de
Syngnathus abaster.
Embora as várias espécies de Syngnathidae sejam
frequentemente descritas como extremamente sensíveis e de difícil
reprodução em aquário, após determinação do intervalo de temperatura
óptimo, importante variável física condicionante da reprodução, foram
observados inúmeros episódios de corte e acasalamento. Desta forma, foi
possível monitorizar e descrever os vários estados de desenvolvimento
embrionário e larval em S. abaster, posteriormente comparados com a
descrição do processo análogo em S. acus. Quando integrada com dados
descritos para outras espécies de marinhas, a informação obtida aponta
para uma correlação positiva entre a especialização morfológica das
estruturas incubadoras e o grau de desenvolvimento larvar, aparentando
existir, simultaneamente, uma associação com as diferentes estratégias
de distribuição larvar onde espécies sem marsúpio tendem a produzir
iv

larvas pelágicas enquanto que espécies com marsúpio favorecem o
desenvolvimento de larvas bênticas.
Paralelamente, foi descrito o ritual de corte e acasalamento em S.
abaster. Este segue um padrão similar ao já descrito para espécies da
mesma família. No entanto, são perceptíveis algumas dissemelhanças
nomeadamente no grau de dimorfismo sexual, menos pronunciado, e nos
níveis de actividade dos machos, que cortejam activamente.
Uma vez caracterizada a 'moderada' reversão dos papéis sexuais
desta espécie, foi avaliado o efeito modulador da temperatura da água no
reconhecimento sexual, preferência de parceiros e interacções entre
fêmeas. Para tal, modulando a temperatura da água, foram mimetizados
três períodos temporalmente distintos: imediatamente antes, durante o
inicio e fim da época de reprodução. Os resultados, para além de
evidenciarem a capacidade de reconhecimento e escolha visual de
potenciais parceiros de acasalamento, com ambos os sexos a
apresentarem uma preferência por indivíduos do sexo oposto e de maior
tamanho, mostram como um factor físico pode directamente influenciar,
ainda que de forma distinta, as respostas comportamentais de machos e
fêmeas quando na presença de membros do mesmo sexo. Enquanto os
machos desviam a sua atenção para as fêmeas logo que são expostos a
temperaturas que permitem a ocorrência de reprodução, as fêmeas
exibem um comportamento competitivo, apenas demonstrando um
interesse evidente pelos machos a temperaturas que mimetizam o final do
período de reprodução. Simultaneamente, a temperatura gerou distintas
respostas comportamentais em fêmeas de diferentes tamanhos. Tal facto
dever-se-á, possivelmente, ao facto de as fêmeas maiores, geralmente
dominantes, apresentarem um forte comportamento competitivo a
temperaturas intermédias, potencialmente inibindo a reprodução das mais
pequenas, que apenas investirão neste tipo de interacção a temperaturas
que reflectem o período final da época de reprodução.
Outra variável que se mostrou ser importante na modulação da
expressão de comportamentos reprodutivos, por afectar directamente a
magnitude do investimento de ambos os sexos nos processos de escolha
de parceiros e de competição pelo seu acesso, foi a razão entre sexos
v

(sex-ratio). Um aumento do número de machos resultou no cancelamento
da 'reversão dos papeis sexuais' normalmente observada quando existe
uma igualdade entre o número de indivíduos de cada sexo. Curiosamente,
um excesso do número de fêmeas não originou um aumento global no
nível de competição fêmea-fêmea. Nesta situação, as fêmeas maiores e
preferidas pelos machos, pareceram confiar no seu “sex appeal”
intrínseco, optando por reduzir o nível de competição, e assim evitando
eventuais custos associados. Por sua vez, fêmeas mais pequenas e, por
consequência, menos atraentes, intensificaram as suas interacções
competitivas.
A análise dos padrões de alocação de ovos de fêmeas grandes e
pequenas, quando expostas a diferentes contextos sociais, onde se variou
o número e tamanho de potenciais parceiros, demonstrou que o tamanho
do corpo, para além de afectar as respostas comportamentais das
fêmeas, está também associado a diferentes estratégias de investimento.
Contrariamente às fêmeas pequenas que apresentaram, em todas as
situações testadas, um esforço reprodutivo constante no que respeita ao
número e tamanho dos ovos, as fêmeas maiores aparentaram um controlo
sobre o investimento modelado pela disponibilidade e qualidade dos
potenciais parceiros. No mesmo estudo, foi possível observar que as
fêmeas, grandes e pequenas, não produziram um número de ovos capaz
de totalmente ocupar o marsúpio dos machos, durante o período da
gravidez. A monitorização de uma população selvagem de S. abaster,
durante dois anos, revelou um padrão semelhante. Simultaneamente, as
observações de campo sugeriram a predominância de acasalamentos
entre indivíduos de tamanhos semelhantes, provavelmente resultantes
quer da selecção mútua de parceiros quer do resultado de competição
fêmea-fêmea.
No seu conjunto, os resultados apresentados evidenciam a
existência de um padrão de acasalamento extremamente dinâmico e
complexo, permitindo interessantes inferências potenciadoras de novas
abordagens experimentais que potenciem ainda mais a clarificação de
que variáveis sustentam a estruturação dos padrões reprodutivos
observados e a expressão da reversão dos papéis sexuais.
vi

Abstract
The family Syngnathidae, with its unique specializations for male
parental care, together with the occurrence of sex role reversal in some
species, constitutes an exceptional model for the study of sexual selection
related issues, mate choice and the role of operational sex ratio in the
evolution of sex role reversal. “Syngnathid research” still lacks, however,
access to basic life history data, in order to allow for a full comprehension
of reproductive strategies, namely those related with the evolution of the
different mating patterns and sex-role reversal. Simultaneously, basic life
history information is crucial for the creation of effective protection plans
since many syngnathid species are currently in an overt process of
decline.
The main goal of the present thesis was to characterize, through
distinct but interconnected approaches, the reproductive ecology of a
Southern European population of the black-striped pipefish, Syngnathus
abaster.
Even though syngnathids are sometimes described as difficult
species to maintain and breed in aquaria, it was possible to obtain
numerous breeding episodes once temperature, a key factor in the control
of marine fish reproduction, was correctly adjusted. The embryonic and
larval development of S. abaster was described and then complemented
with observations on the development and early life history behaviour of a
congeneric species, S. acus. When integrated with available information
on other pipefish species, the data obtained suggested a positive
correlation between pipefish pouch specialization and larval development,
associated to distinct early life strategies. Parallelly, the courtship and
mating ritual was described and comparisons with other syngnathids were
established, highlighting differences and similarities in both behavioural
and morphological differences between the sexes.
vii

Once a moderate sex-role reversal was characterized, aquaria
experiments were conducted as to test for the effects of three distinct
water temperatures (mimicking the periods immediately before, during the
early and late periods of the breeding season) on sexual recognition, mate
preference and female-female interactions. Besides highlighting the fact
that both sexes can visually recognize potential mating partners and
exercise mate choice (preferring larger members of the opposite sex),
results showed how a physical factor can directly and differentially
influence male and female behavioural responses when exposed to
conspecifics. While males diverted their attention to females as soon as
exposed to water temperatures allowing for reproduction to occur, females
exhibited a more competitive behaviour, only showing a clear interest on
males at higher temperatures reflecting the end of the breeding period.
Also it was demonstrated that different-sized females (large and small)
adopted distinct temperature-modulated behaviours, possibly because
large dominant females, which showed intense competitive motivations at
intermediate temperatures, constrained the reproduction of smaller ones,
which appeared more willing to compete at temperatures reflecting the late
period of the breeding season.
Sex ratios also proved to be an important variable in the
expression of reproductive behaviours, by directly affecting the extent to
which males and females invested in choosiness and competition. A
surplus of males was even able to ‘un-reverse’ the sex role reversal
observed under a balanced sex ratio. Interestingly, a surplus of females
did not lead to an overall increase in female-female competition since,
again, different-sized females responded differentially according to their
relative mating prospects. While large, preferred females, opted to avoid
the hypothetical costs derived from intense competition, counting on their
‘sex appeal’ for achieving matings, small, less attractive, females
intensified their competitive displays.
The analysis of the egg allocation patterns of large and small
females exposed to distinct mating contexts (controlling for the number
and the size of potential mates) showed that body size, besides affecting
females’ behavioural responses, was also associated to distinct
viii

investment strategies. Contrarily to small females who showed a constant
effort (regarding both the number and the size of the eggs laid) through all
tested situations, large ones invested differentially according to both mate
availability and quality. Interestingly, neither large nor small females
produced enough eggs to fully occupy a male’s marsupium during the
extent of a pregnancy. A two-year monitoring of a wild S. abaster
population, showed a similar pattern, with males rarely being filled up to
capacity. Simultaneously, field observations suggested the occurrence of
size assortative mating probably arising from the combination of female-
female competition and mutual mate choice.
Altogether the presented results highlight a complex mating ‘game’
allowing for several inferences about mating patterns and the expression
of sex role reversal that should motivate further work.
ix

Résumé
La famille Syngnathidae, avec son unique forme de soin parental
et la présence de réversion des rôles sexuels chez certaines espèces,
constitue un modèle exceptionnel pour l’étude de sujets en rapport avec la
sélection sexuelle, le choix de partenaires et le rôle du sex-ratio
opérationnel dans l’évolution de la réversion des rôles des sexes.
La recherche sur les syngnathides manque encore d’information
de base permettant une pleine compréhension des stratégies
reproductrices, notamment celles en rapport avec l’évolution des différents
systèmes d’accouplements et la réversion des rôles sexuels.
Simultanément, information d’histoire de vie est cruciale pour le
développement de mesures de conservation effectives étant donné que
plusieures espèces de syngnathides sont présentement en voie de
disparition.
Le but principal de la présente thèse est une caractérisation
détaillée et approfondie de l’écologie reproductive d’une population
Sud-Européenne de Syngnathus abaster suivant des méthodes distinctes
mais clairement interconnectées.
Bien que les syngnathides soient parfois décris comme des
espèces difficiles à maintenir et à élever en aquarium, il fut possible
d’observer de nombreux épisodes d’élevage des jeunes une fois que la
température identifiée comme le facteur principal déclenchant la
reproduction. Les développements embryonnaire et larvaire de S. abaster
furent ainsi décris et par la suite complétés avec l’étude du développement
larvaire de S. acus. Une fois mises en parallèle, les informations obtenues
mirent en évidence une corrélation positive entre, d’une part, la
spécialisation de la poche d’incubation des mâles (marsupium) et le
développement larvaire et, d’autre part, des stratégies d’histoire de vie
distinctes (espèces avec marsupium à larves benthiques; espèces sans
marsupium à larves pélagiques).
x

De même, le rituel de cour et d’accouplement de S. abaster fut
décrit et comparé avec ceux décrits pour d’autres syngnathides de façon à
mettre en évidence différences et similarités chez les différences
morphologiques et comportementales entre les sexes.
Une fois la réversion des sexes caractérisée, des expériences en
aquarium ont été réalisées de façon à tester les effets de trois
températures distinctes (reproduisant les périodes immédiatement avant,
pendant les premiers et les derniers moments de la saison reproductrice)
dans la reconnaissance sexuelle, les préférences d’accouplements et les
interactions entre femelles. À part avoir mis en évidence le fait que les
sont capables de reconnaître visuellement de potentiels partenaires
sexuels et d’être sélectifs (préférant les plus grands membres du sexe
opposé), les résultats montrèrent comment un facteur physique peut,
directement et de façon différentielle, moduler les réponses
comportementales des deux sexes. Alors que les male montrèrent de
l’intérêt pour le sexe opposé des qu’ils furent soumis à des températures
permettant l’occurrence de reproduction, les femelles firent part d’un
comportement plus compétitif, montrant un net intérêt pour de potentiels
partenaires uniquement à des températures représentant la fin de la
période reproductrice. Par ailleurs, les femelles adoptèrent des
comportements en réponse à la température dépendants de leur taille
possiblement parce-que les grandes femelles, qui s’engagent dans la
compétition en début de saison, contraignent la reproduction des petites
femelles qui semblent ne s’engager dans la compétition que vers la fin de
la période de reproduction.
Aussi les sex-ratios se révélèrent une variable importante dans
l’expression des comportements reproductifs en affectant la mesure dont
les deux sexes investissent en choix et en compétition. Un excès de males
a même inverti la réversion des rôles sexuels observée sous un sex-ratio
équilibré. De façon intéressante, un excès de femelles n’a pas conduit à
une poussée globale de la compétition entre femelles, étant donnée, que
là encore, des femelles de différentes tailles se comportèrent
différemment, en accord avec leurs perspectives d’accouplements à venir.
Alors que les grandes femelles optèrent éviter les hypothétiques couts
xi

advenant d’une compétition intensifiée, contant sur leurs ‘sex-appeal’ pour
se trouver de partenaires, les petites et moins attirantes femelles,
intensifièrent leurs exhibitions compétitives.
L’analyse des patterns d’allocation des œufs chez des grandes et
petites femelles présentées soumises à de différents contextes de
reproduction (contrôlant pour le nombre et la taille de potentiels
partenaires) a révélé que la taille, outre affecter les réponses
comportementales des femelles est aussi associée a de distinctes
stratégies d’investissement. Contrairement aux petites femelles, qui
montrèrent un constant investissement (soit dans le nombre soit dans la
taille des œufs transférés) sous toutes situations testées, les grandes
femelles ont investi différemment, en accord avec le nombre et la taille des
partenaires disponibles. D’une façon intéressant, les femelles (grandes et
petites) ne produisirent pas un nombre d’œufs suffisant pour remplir la
poche marsupiale des males pendant la période de gestation.
Le suivi d’une population de S. abaster dans son milieu naturel a
montré un pattern similaire, avec les males étant rarement remplis jusqu'à
leurs limites. Simultanément, les observations de terrain ont fortement
suggéré l’occurrence d’accouplements taille-assortis, advenant
probablement de la combinaison de compétition chez les femelles et choix
chez les deux sexes. Dans l’ensemble, les résultats ici présentés mettent
en évidence un complexe ‘jeux de reproduction’ permettant plusieures
inférences concernant les patterns d’accouplements et l’expression de la
réversion des rôles sexuels qui devrait motiver de la recherche future.
xii

Contents
1 General Introduction 1
1.1 Theoretical framework 1
1.2 The family Syngnathidae 4
1.3 The black-striped pipefish, Syngnathus abaster 7
1.4 Objectives 9
1.5 List of publications integrating the thesis 11
2 Early Life History and Reproductive Behaviour 13
2.1 Early life history of Syngnathus abaster 13
2.1.1 Abstract 13
2.1.2 Introduction 14
2.1.3 Methods 15
2.1.4 Results 16
2.1.5 Discussion 22
2.1.6 Acknowledgements 24
2.1.7 References 24
xiii

2.2 Development and early life history behaviour of
aquarium-reared Syngnathus acus (Pisces: Syngnathidae)
2.2.1 Abstract 28
2.2.2 Introduction 29
2.2.3 Methods 30
2.2.4 Results 31
2.2.5 Discussion 33
2.2.6 Acknowledgements 35
2.2.7 References 35
2.3 Reproductive behaviour of the black-striped pipefish,
Syngnathus abaster (Pisces; Syngnathidae)
2.3.1 Abstract 38
2.3.2 Introduction 39
2.3.3 Methods 40
2.3.4 Results 41
2.3.5 Discussion 46
2.3.6 Acknowledgements 50
2.3.7 References 51
3 Understanding Sex-Role Reversal (ex situ) 56
3.1 The effect of temperature on mate preferences and
female-female interactions in Syngnathus abaster
3.1.1 Abstract 56
3.1.2 Introduction 57
3.1.3 Methods 58
3.1.4 Results 64
3.1.5 Discussion 70
3.1.6 Acknowledgements 74
3.1.7 References 75
xiv
28
38
56

3.2 Reversing sex-role reversal: compete only when you
must
3.2.1 Abstract 80
3.2.2 Introduction 81
3.2.3 Methods 83
3.2.4 Results 88
3.2.5 Discussion 98
3.2.6 Acknowledgements 102
3.2.7 References 103
3.3 Female reproductive tactics in a sex-role reversed
pipefish: screening for quality and number
xv
80
107
3.3.1 Abstract 107
3.3.2 Introduction 108
3.3.3 Methods 110
3.3.4 Results 114
3.3.5 Discussion 122
3.3.6 Acknowledgements 125
3.3.7 References 126
4 Reproductive Dynamics (in situ) 130
Can the limited marsupium space be a limiting factor for
Syngnathus abaster females? Insights from a population
with size assortative mating
130
4.1 Abstract 130
4.2 Introduction 131
4.3 Methods 133
4.4 Results 135
4.5 Discussion 137
4.6 Acknowledgements 139
4.7 References 140

5 Final Discussion 144
5.1 Towards a new approach on sex-roles 144
5.1.1 Water temperature and the expression of reproductive
behaviours
xvi
145
5.1.2 Sex-ratios and mating competition 148
5.1.3 Mate preferences versus mate choice: the importance
of the social context
149
5.1.4 Assortative-mating and syngnathid evolution 150
5.1.5 Female reproductive investment 151
5.1.6 Multiple mating strategies 153
5.1.7 What causes sex-role reversal? 155
5.1.8 Further research: an endocrine approach to sex-role
reversal
157
5.1.9 A ‘mild’ sex-role reversion 158
5.2 Conservation note 159
6 References 162

List of Figures
1.1 Specimen of Syngnathus abaster. 7
1.2 Geographical distribution of Syngnathus abaster. 8
2.1.1 Ontogenetic events occurring during the embryonic and larval
development of Syngnathus abaster in order of first
appearance. 18
2.1.2 Different developmental stages in Syngnathus abaster. 19
2.1.3 Vertical distribution in the water column displayed by juvenile
Syngnathus abaster during the first 3 weeks after abandoning
the marsupium and juvenile total length during the first 4
weeks. 21
2.2.1 Syngnathus acus newborns, comparison of meristic characters
of Syngnathus acus and Syngnathus abaster newborns,
juvenile growth rate during the first eight weeks and vertical
distribution in the water column displayed by juveniles during
the first five weeks after abandoning the marsupium. 32
xvii

2.3.1 Relationship between male total length and the number of
eggs carried. 42
2.3.2 Female ‘amplifiable ornament’ in isolation and interacting with
another female. 43
2.3.3 Overview of the main behavioural stages of courtship and
mating. 45
2.3.4 Male and female total length relationships in successful and
unsuccessful Syngnathus abaster mating pairs. 50
3.1.1 Experimental set-ups. 61
3.1.2 ‘Relative interest’ of males and females of different size
classes towards consexuals and non-consexuals, at three
different temperatures (15ºC; 18ºC; 24ºC). 66
3.1.3 ‘Relative interest’ towards stimulus fish of different sizes at
three different temperatures (15ºC, 18ºC and 24ºC). 68
3.1.4 Relative interest’ of females of different size classes towards
stimuli females at three different temperatures (15ºC, 18ºC
and 24ºC), ‘relative interest’ aroused by females of different
size classes at three different temperatures (15ºC, 18ºC and
24ºC) and ‘relative interest’ of different size females towards
large and small consexuals. 69
3.2.1 Cluster analyses based on a similarity matrix constructed on
the mean frequencies of four selected behaviours according to
the type of interaction, the sex of the fish and the sex-ratio
treatment. 89
3.2.2 Mean frequencies of four selected behaviours for male and
female inter- and intra-sexual interactions in different sex ratio
treatments. 94
3.2.3 Observed number of male and female successful mating
disruptions in three selected treatments (equal, male and
female biased sex-ratio). 96
xviii

3.2.4 Number of mating pairs observed in three selected treatments
(equal, male and female biased sex-ratio), described
according to the four possible size class combinations of
males and females (large and small). 97
3.2.5 Mean time elapsed until the first spawn in the three selected
treatments (equal, male and female biased sex-ratio). 98
3.3.1 Mean number of mating partners for large and small females in
different ‘mating settings’. 115
3.3.2 Mean number of large and small mating partners for large and
small females within an heterogeneous mating setting. 115
3.3.3 Day of first copulation in different ‘mating settings’. 116
3.3.4 Mean time between copulations (in days) for large and small
females in different ‘mating settings’. 116
3.3.5 Mean number of eggs laid by large and small females in
different ‘mating settings’, mean number of eggs laid in
different ‘mating settings’ and mean number of eggs laid by
large and small females. 118
3.3.6 Mean number of eggs laid by large and small females in
different-size males (large and small). 119
3.3.7 Mean size of the eggs laid by large and small females in
different ‘mating settings’. 119
3.3.8 Mean size of the eggs from first and second batches. 120
4.1 Relationship between male size and male marsupium height;
and male size and male marsupium width. 136
4.2 Relationship between male size and egg size; female size and
egg size; number of eggs carried in large and small males;
number of missing eggs estimated for large and small males. 136
xix

List of tables
2.1.1 ANOVA results on the vertical distribution of Syngnathus
abaster juveniles during the first three weeks after abandoning
the marsupium.
2.2.1 Non-parametric analysis of variance results on the vertical
distribution of Syngnathus acus and Syngnathus abaster
during the first three weeks after abandoning the marsupium.
3.1.1 ANOVA results on the ‘relative interest’ of males and females
of different size classes towards consexuals and non-
consexuals, at three different temperatures (15ºC, 18ºC and
24ºC).
3.1.2 ANOVA results on the ‘relative interest’ of males and females
of different size classes towards large and small potential
mates, at three different temperatures (15ºC, 18ºC and 24ºC).
3.1.3 ANOVA results on the ‘relative interest’ of females of different
size classes towards large and small consexuals, at three
different temperatures (15ºC, 18ºC and 24ºC).
xx
20
34
65
67
67

3.2.1 Recorded mating behaviours and corresponding descriptions 87
3.2.2 MANOVAs results on male and female interactions in the form
of approach, parallel-swimming, flickering and lateral display.
Dependent variables included intra and inter-sexual
interactions.
3.2.3 ANOVA results on the time elapsed until the first mating event,
considering sex-ratio treatment and male size.
3.3.1 ANOVA results on (A) the number of mating partners, (B) the
number of mating partners within the heterogeneous ‘mating
setting’ (T2+2), (C) the elapsed time before the first spawn,
(D) the time elapsed between copulations, (E) the number of
spawned eggs, (F) the number of eggs in the heterogeneous
‘mating setting’ (T2+2) and (G) the size of the eggs.
5.1 Abundance of syngnathids in the Ria de Aveiro estuarine lagoon. 161
xxi
91
97
120

Chapter 1. General Introduction
Chapter 1
General Introduction
1.1 Sexual selection: theoretical framework
In evolutionary terms, the success of individuals depends not only on their
survival, but also on their ability to reproduce and produce successful
offspring. In most animals, mating systems are characterized by
competitive males exhibiting conspicuous morphological or behavioural
characters used to court females and compete over them, and by a
preference in females to mate with those males (Darwin, 1871; Reynolds,
1996). Part of the explanation for this pattern of sex roles lies in the
gamete dimorphism that defines the sexes: males produce numerically
abundant, small, ‘cheap’ sperm whereas females invest in far less, but
large, nutritious, costly oocytes (Parker et al., 1972). Such discrepancy
between the sexes in gametic investment is often maintained or
exaggerated by subsequent female parental care (Clutton-Brock, 1991;
Queller, 1997). As a result, the availability of females limits male
reproductive success and males must compete for access to mates.
1

Chapter 1. General Introduction
Females, in turn, can benefit from being choosy (without loosing mating
opportunities) as they constantly encounter males eager to mate
(Bateman, 1948). According to Darwin (1871) these two behaviours (male
competitiveness and female choosiness) give rise to a process of sexual
selection 1 promoting the evolution of secondary sexual traits in males:
menacing ‘weapons’ or conspicuous ornaments that either improve
intra-sexual competitiveness or increase attractiveness to potential mates.
In some classes of animals, however, a few exceptional cases
occur, in which conventional sex roles are reversed: females compete
more intensely for mates and have “well pronounced sexually secondary
characters such as brighter colours, greater size strength or pugnacity”
(Darwin, 1871).
Understanding the evolution of sex roles and what causes reversals
has developed into one of the most productive and controversial themes of
evolutionary biology. Trivers (1972) decoupled sex roles from anisogamy
and associated them instead to parental investment 2 . According to him, the
higher investing sex would become a limiting resource causing the
members of the lower investing sex to compete for mating opportunities.
The relative investment of the sexes would also affect the criteria of mate
choice: the higher investing sex should be more selective (Trivers, 1972).
According to this theory, it could be expected that in species where
substantial paternal care occurs together with reduced or non-existent
maternal care, females would be under strong sexual selection.
The extent of male care greatly varies among the taxa. Although rare
in mammals, reptiles and invertebrates (Tallamy, 2000), it is a common
1 Sexual selection “depends not on a struggle for existence but on a struggle
between the males for possession of the females; the result is not death to the
unsuccessful competitor, but few or no offspring” (Darwin, 1871).
2 “Any investment by the parent in an individual offspring that increases the
offspring chance of survival (and hence reproductive success) at the cost of the
parent’s ability to invest in other offspring” (Trivers, 1972).
2

Chapter 1. General Introduction
phenomenon in birds (e.g. Moller & Cuervo, 2000), amphibians (e.g.
Summers, 1990) and specially fish 3 , where males are the primary or
exclusive custodians in nearly 70% of the families with parental care
(Gross & Shine, 1981; Gross & Sargent, 1985; Smith & Wootton, 1995).
Although sex role reversal has been increasingly documented in a large
number of birds (e.g. Colwell & Oring, 1988; Oring et al., 1983) and
amphibians (e.g. Verrell & Brown, 1993), such examples are surprisingly
rare in fish (Eens & Pinxten, 2000). In fact, in most fish species with
paternal care, males are still the predominant competitors for mates and
evidence for sex role reversal is scarce (Breder & Rosen, 1966; Thresher,
1984).
Trivers’s theory was thus revised by many workers who suggested
that the operational sex ratio (ratio of sexually active males to sexually
active females at a given time) could predict the occurrence of sex role
reversal much more accurately than relative parental investment does
(Emlen & Oring, 1977). When males have a higher potential mating rate 4
than females the operational sex rate will be male biased and, therefore,
3 The reasons for the prevailing male care in fish are probably twofold. Fish, in
contrast to birds and mammals, have indeterminate growth. Females, may,
therefore have a lot to gain from not caring for young but instead allocate resources
to growth and in turn produce more eggs in the future, as body size correlates
positively with egg production. There should thus be selection pressure on females
to allocate resources to growth instead of care. As sperm is cheaper to produce
than eggs, males are not energetically constrained in gamete production like
females. Furthermore, males often do not reduce their attractiveness to females
when taking the parental burden. Actually, to a female, a male who is already
guarding eggs may be even more attractive than a male without eggs, as her own
eggs experience lower risk of predation in a nest containing other eggs, e.g.
through the dilution effect (see Clutton-Brock & Godfray, 1991; Sargent, 1997).
Thus male care should be a sexually selected trait in fish.
4 The highest possible reproduction rate for an individual given the constraints of
speed of gamete production and parental care and assuming unlimited access to
opposite sex mates (Clutton-Brock, 1991).
3

Chapter 1. General Introduction
males will be the more competitive sex whilst females may afford to be
selective (conventional sex roles). Conversely, when females have a
higher potential reproductive rate than males, the operational sex ratio will
be female biased, therefore, females will be the more active sex in
competition whilst males will be the more active sex in mate choice
(Clutton-Brock & Vincent, 1991; Vincent et al., 1992). In the three-spined
stickleback (Gasterosteus aculeatus) with exclusive paternal care, for
example, males can simultaneously guard ten or more egg clutches for
about two weeks, whereas females can lay out one clutch every 3-5 days.
Consequently the potential reproductive rate of males is higher than that of
females, the operational sex ratio is male-biased and males compete
intensely for mates.
Although relative parental investment may be the prime determinant
of reproductive rates, other behavioral, ecological, physical and
phylogenetic factors may act as constraints (see Vincent et al., 1992).
Fluctuating ecological and physical factors may differentially affect the
reproductive rates of males and females, shifting biases in the operational
sex ratio in space and time (Clutton-Brock, 1991). As such, fish, as
ectothermic organisms, are a particularly sensitive group as their
reproductive rates are expected to be more susceptible to fluctuations in
environmental conditions such as temperature or food. In sand gobies
(Pomatoschistus minutus), for example, temperature influences sexual
differences in the potential reproductive rates causing variation in the
intensity of mating competition (Kvarnemo, 1994).
1.2 The family Syngnathidae
Although relatively rare in fish, a wide variation in the degree of paternal
care together with the occurrence of sex role reversal can be observed
within the family Syngnathidae 5 . Comprising unique morphotypes such as
4

Chapter 1. General Introduction
seahorses, pipefishes and seadragons, this group offers unique
opportunities to explore hypotheses concerning the relationship between
parental investment, sex roles and sexual selection.
Syngnathids are characterized by remarkable adaptations for
paternal care, with females depositing unfertilized 6 eggs into a male’
specialized incubating area that varies in complexity in five steps: i) a
simple unprotected ventral area for egg ‘gluing’, ii) individual membranous
egg compartments, iii) protection of eggs in a pouch with pouch plates, iv)
bilateral pouch folds that contact midline into a closed pouch and v) the
most complex and completely enclosed brooding pouch of seahorses
(Herald, 1959).
Phylogenetic relationships within this family have been studied both
by Herald (1959), based on morphological observations, namely the
position or closure method of the incubating area, and Wilson et al. (2001;
2003), who proposed a new mitochondrial DNA-based phylogeny. Both
approaches agree that the evolutionary radiation of this family was
accompanied by a diversification of structures involved in parental care,
from an ancestral pipefish, that probably presented a rather simple brood
structure.
Regardless of the degree of complexity, all syngnathid brooding
structures were once assumed to provide developing embryos with
protection, osmoregulation and nutrients (Vincent et al., 1992). However, a
recent anatomical study, describing the morphology and ultra-structure of
three distinct incubating structures, refuted this uniform functionality
suggesting that the epithelium has different functions in each type of
enclosure. Specifically, more enclosed pouches were observed to contain
5 The term is derived from Greek, meaning "fused jaw": syn meaning fused or
together, and gnathus meaning jaws.
6 Internal fertilization in males assures paternity and is believed to be one of the
selective pressures behind the evolution of the male brooding structure (Jones &
Avise, 1997; Jones et al., 1998; Jones et al., 1999).
5

Chapter 1. General Introduction
greater anatomical complexity and secretory function (Carcupino et al.,
2002).
Aside from male pregnancy, some syngnathid species show sex-role
reversal, with females competing for access to mates and sometimes
presenting conspicuous secondary sexual characters (e.g. Monteiro et al.,
2002; Silva et al. 2006). Energy investment, brooding-space constraints
and pregnancy length, operating alone or in combination, may limit female
reproductive success thus providing an explanation of such sexual
differences (Berglund & Rosenqvist, 2003). Nevertheless, despite many
attempts to clearly identify the mechanisms behind the evolution of sex-
role reversal in syngnathids, no direct relationship between sex roles and
any of these factors has yet been accurately demonstrated (Wilson et al.,
2003). The multiple occurrence of sex role reversal within different clades,
exhibiting distinct parental adaptations, suggests, instead, a much more
dynamic process underlying the behavioral and morphological
particularities of these fish.
Mating patterns, in turn, seem to be strongly associated with the
direction of sexual selection: sex role reversed syngnathids are
polygamous whereas species with non-reversed patterns of mating
competition are generally monogamous (Vincent et al., 1992; Avise et al.,
2002). Only one exception to this association has been detected so far.
The pipefish Corythoichthys haematopterus is monogamous but
behavioural observations in a wild population suggest sex role reversal
(Matsumoto & Yanagisawa, 2001).
Even though a prominent area, “syngnathid research” still lacks
access to basic life history data that would certainly allow for a better
understanding of the ultimate factors shaping mating patterns and sex role
reversal within this family. Simultaneously, basic life history information is
crucial for the establishment of effective protection plans since syngnathid
populations are currently in an overt process of decline mainly due to
habitat destruction.
6

Chapter 1. General Introduction
1.3 The black-striped pipefish, Syngnathus
abaster
Figure 1.1: Specimen of Syngnathus abaster.
Syngnathus abaster (Risso, 1827) is a small, euryhaline pipefish with a
restricted distribution area that includes the Mediterranean and Black
Seas, northward to southern Bay of Biscay (northern Spain). It can be
found either in coastal areas or in brackish and fresh waters (Cakic et al.,
2002), mainly among sand, mud or eelgrass meadows, between depths of
0.5 to 5 meters, within a temperature range of 8ºC to 24ºC (Dawson, 1986;
Froese & Pauly, 2004). Males can easily be distinguished from females by
the presence of the brood pouch (marsupium) located on the tail, formed
by two skin-folds that contact medially with their free edges.
Studies on S. abaster are scarce and the characteristics of its life
cycle are still superficially known. Campolmi et al. 1996, Tomasini et al.
(1991) and Riccato et al. (2003) presented basic data on this pipefish
reproduction and population structure in three Mediterranean lagoons. All
three studies suggested a short life span, with only one or few reproductive
seasons. Females are batch spawners and males can incubate several
broods during one breeding season. Further references to S. abaster are
restricted to Carcupino et al., (1997) that described the ultra-structural
7

Chapter 1. General Introduction
organization of the male brood pouch epithelium, Cakic et al. (2002), who
presented a biometric analysis of S. abaster populations of the River,
sections within Dawson (1985) and Kuiter (2000) and generalised
comments on syngnathids by various authors with particular incidence in
field guides.
Figure 1.2: Geographical distribution of Syngnathus abaster (as presented
by Dawson, 1986).
The present thesis presents the first comprehensive study on the
reproductive ecology and behaviour of the black-striped pipefish providing
extensive data on distinct but clearly interrelated and complementary
reproductive parameters. The geographical location of the sampled
population (Ria de Aveiro estuarine lagoon 7 ) is of particular interest as it is
7 The Ria de Aveiro estuarine lagoon, situated on the northern coast of Portugal is
the only typical estuarine lagoon in the country. It is influenced by a maritime,
temperate climate with a marked seasonal variation in rainfall and air temperature.
The depth at low tide is only 1 m over most of the lagoon reaching 10 m in the
navigation channels and near the mouth where tidal action mixes fresh with
seawater. Currents are almost insignificant with exception of the mouth, the central
8

Chapter 1. General Introduction
near the northern limit of distribution of this species and thus allows for
future comparisons between populations, perhaps revealing physiological
and behavioral trends along the distribution of this species. Moreover,
when integrated with available information on syngnathid reproduction, the
obtained data on the studied population will certainly allow for a better
understanding of the dynamics underlying the behavioural patterns
observed in this family.
1.4 Objectives
Three main sections were created according to the distinct established
goals of the present thesis:
1. Early Life History and Reproductive Behaviour - Chapter 2
a. Early Life History - Chapters 2.1 and 2.2
The analysis of variation in early life history parameters across
syngnathids is highly important for a better understanding of the
mechanisms and strategies involved in syngnathids evolution and
extraordinary level of diversity. As such, in order to capitalize the
obtained mating events in aquaria, the embryonic and larval
development of S. abaster was monitored and compared with
available data on the early life cycles of other syngnathids, namely
part of the main channels and a few other restricted areas. The Ria is composed of
a wide range of biotopes (e.g. wetlands, salt marshes and mudflats) used as
nursery areas for many valuable species including birds, bivalves, crustaceans and
fish. The abiotic attributes, shallowness, high turbidity, nature of the substratum,
temperature, salinity and oxygen, together with high biotic productivity, offer (or
offered) excellent conditions to temporary or permanent habitat for many fish
species, including syngnathids (for further information on the Ria de Aveiro see, for
example Rebelo, 1992).
9

Chapter 1. General Introduction
the closely related species S. acus. Pregnant males of S. acus were
captured and information on the development and early life history
behaviour of juveniles was gathered in order to assess the
importance and implications of the marsupium of the genus
Syngnathus on juvenile development and exhibited dispersal
strategies (benthic or pelagic). Implications in population dynamics
and connectivity were further discussed.
b. Reproductive behaviour - Chapter 2.3
Objective descriptions of syngnathid reproductive behaviours are
crucial for the understanding of the evolution of sex role reversal in
this family. As such, the courtship and mating ritual of S. abaster
was described to determine whether and to what extent this species
is sex role reversed. Comparisons with other syngnathids were then
established highlighting differences and similarities in both
behavioral and morphological sexual differences.
2. Understanding Sex-Role Reversal (ex situ) - Chapter 3
Several recent studies have shown that conventional sex roles may be
somewhat modified, being less straightforward than predicted in theory. In
sex role reversed species, such modifications are still virtually
undocumented and it is yet not known how individuals with reversed sex
roles respond to variable environmental and social conditions. In this
extent, aquaria experiments were conducted as to test for the effects of
distinct water temperatures (Chapter 3.1) and sex ratios (Chapter 3.2) on
male and female reproductive behaviours. Moreover, since in sex role
reversed species, several factors may constrain female reproduction, egg
allocation patterns were analysed under different contexts of mate
availability and quality (Chapter 3.3).
10

Chapter 1. General Introduction
3. Reproductive Dynamics (in situ) - Chapter 4
Even though a lot of work has been done on sexual selection in
syngnathids, basic data from the field (where the behavioral repertoire of
the fish is modulated by far more variables than in aquaria) is still lacking
for most taxa. This final section presents a continuous monitoring of a wild
population of S. abaster allowing for several inferences about mating
patterns and sexual selection that should motivate additional work from the
hypotheses generated. For example, this species appears to mate
size-assortatively, probably as a consequence of mutual mate choice. This
type of assortative mating likely has important implications for sexual
selection and possibly speciation in syngnathid fishes. In addition, males
were rarely filled to capacity in this species, a surprising observation that
leads to several testable hypotheses.
1.5 List of publications integrating the thesis
I. Silva, K., Monteiro, N. M., Almada, V. C. & Vieira, M. N. 2006. Early life
history of Syngnathus abaster. Journal of Fish Biology, 68, 80-86.
II. Silva, K., Monteiro, N. M., Almada, V. C. & Vieira, M. N. 2006.
Development and early life history behaviour of aquarium reared
Syngnathus acus (Pisces: Syngnathidae). Journal of the Marine Biological
Association of the United Kingdom, 86, 1469-1472.
III. Silva, K., Monteiro, N. M., Vieira, M. N. & Almada, V. C. 2006.
Reproductive behaviour of the black-striped pipefish, Syngnathus abaster
(Pisces; Syngnathidae). Journal of Fish Biology, 69, 1860-1869.
IV. Silva, K., Almada, V. C., Vieira, M. N. & Monteiro, N. M. 2007. The
effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster. Animal Behaviour, 74, 1525-1533.
11

Chapter 1. General Introduction
V. Silva, K., Vieira, M. N., Almada, V. C. & Monteiro, N. M. Reversing
sex-role reversal: compete only when you must. Submitted for publication.
VI. Silva, K., Almada, V. C., Vieira, M. N & Monteiro, N. M. Female
reproductive tactics in a sex-role reversed pipefish: screening for quality
and number. Submitted for publication.
VII. Silva, K., Vieira, M. N., Almada, V. C. & Monteiro, N. M. In Press. Can
the limited marsupium space be a limiting factor for Syngnathus abaster
females? Insights from a population with size assortative mating. Journal
of Animal Ecology.
12

Chapter 2. Early Life History and Reproductive Behaviour
Chapter 2.1
Early life history of Syngnathus
abaster
2.1.1 Abstract
The embryonic and larval development of the pipefish Syngnathus abaster
is described, based on ex situ observations. The full development
sequence lasted 24-32 days (at 18-19ºC), which was shortened to 21 days
at higher temperatures (21-22ºC). Newborn juveniles, with a uniform dark
brown colouration, immediately assumed a benthic spatial distribution.
This vertical distribution pattern remained unchanged at least during the
first 4 weeks, after the release from the marsupium. The apparent absence
of a pelagic life phase might have important repercussions in terms of
population connectivity given increasing fragmentation and degradation of
the eelgrass habitat in the species’ range.
13

2.1. Early life history of Syngnathus abaster
2.1.2 Introduction
The Syngnathidae (pipefishes, pipehorses, seadragons and seahorses)
exhibits one of the most specialized forms of parental care, with females
depositing eggs in a specialized incubating area, located either on the
abdomen (Gastrophori) or tail (Urophori) of the males (Herald, 1959). Even
though male pregnancy is a widespread characteristic in all syngnathids,
the anatomical complexity of the brooding structures varies among
species, from the simplest incubating ventral surface of the Nerophinae,
where eggs are glued without any protective plates or membranes, to the
Hippocampinae sealed brood pouch. This gradient in conspicuous male
parental care structures was used by Herald (1959) in order to propose a
phylogeny of the family, and the main results were later confirmed by
Wilson et al. (2001, 2003), using mitochondrial DNA.
A comparative study on three anatomically distinct brooding
structures highlighted different ultrastructures, suggesting distinct functions
related to different reproductive strategies (Carcupino et al., 2002). The
underlying anatomical variations visible among the various pouch types
may also signal distinct early life histories since the interactions between
the male body and the developing embryos are inversely proportional to
the degree of egg exposure to the external environment. The relative
degree of development exhibited by newborns after release from the
parent may be crucial to immediate survival and larval dispersal.
The main aim of this work was to extend understanding on
syngnathid biology by describing, for the first time, the developmental
sequence, from egg to newborn, of captive black-striped pipefish
Syngnathus abaster Risso, a data deficient species (Baillie et al., 2004).
Simultaneously, the vertical distribution of newborns was analysed, as a
means to evaluate the benthic or pelagic nature of the juveniles, a variable
that has profound implications in the population dynamics of pipefishes
that inhabit increasingly fragmented habitats, such as eelgrass meadows.
14

Chapter 2. Early Life History and Reproductive Behaviour
2.1.3 Methods
S. abaster is a euryhaline species with a restricted distribution area that
includes the Mediterranean and Black Seas, northward to the southern
Bay of Biscay (northern Spain). The fish can be found either in coastal
areas or in brackish and fresh waters (Cakic et al., 2002), mainly among
sand, mud or eelgrass meadows, between depths of 0.5 and 5 m, within a
temperature range of 8–24ºC (Dawson, 1986; Froese & Pauly, 2005).
Males can easily be distinguished from females by the presence of the
brood pouch (marsupium) located on the tail, formed by two skin-folds that
contact medially with their free edges.
Eggs and larvae were obtained (August 2003 to March 2004) from
wild specimens collected in a salt pond reservoir, at the Ria de Aveiro
estuarine lagoon (40º45’ N; 8º40’ W), in Portugal, that successfully
copulated in aquaria (15 male pregnancies were followed). Captive fish
were fed daily with fresh Artemia franciscana nauplii and maintained in 80 l
aquaria, illuminated with natural light, supplemented with 18 W fluorescent
lamps. Due to the ‘gas bubble disease’, common in pipefishes (A.
Berglund, pers. comm.), aeration was performed outside the fish tanks.
Continuously running sea water was physically and biologically filtered and
temperature was maintained within two different ranges, 18-19ºC and
21-22ºC. Substrata consisted mainly of sand and plastic seagrass laid in
order to mimic the original habitat from where the fish were caught. Once
copulation occurred, pregnant males were isolated and eggs were
removed daily from the marsupium (except during the first day, when egg
extraction occurred several times) and immediately preserved in 4%
formalin. Both eggs and larvae were observed and photographed with a
Leica stereomicroscope attached to a digital video camera.
In order to further extend knowledge on syngnathid early life
history, newborn juveniles were maintained in the same conditions as
adults, since they were already capable of eating A. franciscana nauplii.
Juvenile total length (LT) was recorded using a ruler (25 juveniles were
continuously measured during the first 4 weeks). Since this was a simple
15

2.1. Early life history of Syngnathus abaster
and quick handling procedure, no mortality was observed due to fish
manipulation. The vertical distribution of juveniles was also studied by
separately placing individuals inside small 4 l aquaria (40 x 10 x 10 cm),
distinct from those used for pregnant adults and with no water circulation,
divided in three sections: surface (10 cm), middle section (20 cm) and
bottom (10 cm). After an initial ‘resting’ period (c. 45 min), during which
black striped pipefish were allowed to familiarize with the new
surroundings, 10 min observation periods were conducted on the time
juveniles spent in the ‘surface’ and ‘bottom’ sections. In order to assure
data independence, each observation was conducted using different
individuals, from different broods, that were only used once (10 trials
measuring time spent near surface and another 10 trials aimed at
measuring time spent in the bottom section). This experimental design was
repeated during 3 consecutive weeks, always using different individuals.
An orthogonal ANOVA with two factors [time (three levels = 3 weeks) and
vertical position (two levels = surface and bottom)] was conducted. Data
were transformed [ln (x + 0.5)] in order to obtain homogeneity of variances
(Cochran’s test; P > 0.05). All statistical analyses were performed using
Statistica 6.1 (Statsoft).
2.1.4 Results
The bright orange eggs of S. abaster were almost spherical when
spawned, measuring c. 1.6 mm (n= 45; mean ± S.D.= 1.58 ± 0.24 mm;
range: 1.09-2.06 mm), but became quite variable in shape due to different
degrees of compression caused by adjacent eggs. Males carried embryos
for c. 24-32 days (18-19ºC; n= 24, mean ± S.D. number of eggs per males
= 40.67 ± 10.10; range: 18-64) and gave birth, during a 2-3 day period, to
completely independent young measuring c. 18mm (n= 64; mean ± S.D
= 17.67 ± 0.22 mm; range: 14-23 mm). At higher temperatures (21-22ºC)
embryonic and larval development was concluded within 21 days (four
male pregnancies were observed). The main ontogenetic stages are
16

Chapter 2. Early Life History and Reproductive Behaviour
summarized in Figure 2.1.1 and representative pictures of the
developmental process (18-19ºC) are given in Figure 2.1.2.
Cleavage in S. abaster, as in all bony fishes, is meroblastic
(incomplete), leading to a formation of a mass of cells sitting atop of the
yolk. Embryos entered the blastula stage c. 24 h after fertilization with the
blastodisc presenting a regular hemispheric shape. Gastrulation began 72
h after fertilization and was characterized by the flattening of the blastodisc
(epiboly) and the formation of the embryonic shield as a thickening of the
germ ring [Figure 2.1.2 (A)]. By this time, the two edges of the marsupium
folds strongly adhered to each other. The neural tube and the dorsal corda
were apparent in the anterior region of the embryo body from the half
epiboly stage (7 days after fertilization) [Figure 2.1.2 (B)]. During this
period the embryo presented a well developed head, body and tail. The
cephalic region was already discernable and the optic vesicles were
visible. The anlagen of the crystalline lens and the ocular pigmentation
were visible by day 9. Somite formation started c. 8 days after fertilization
and, as development continued, the rudiments of the organs became
visible, with the tail region now free from the yolk as the embryo elongated
and fins became visible. Melanogenesis started on the head region after 8
days of development and, by day 10, some pigmented cells were visible
along the antero-posterior embryonic axis [Figure 2.1.2 (C)]. After 14 days
of development the heart beat and blood vessels were visible and embryos
exhibited some motility, responding to touch. During the subsequent days
fin rays began to differentiate [Figure 2.1.2 (D)] and the embryos started
hatching from the egg envelopes, but remaining within the marsupium
whose edges increased in volume and were now easily separable. As
development proceeded, the yolk was gradually absorbed, with the
elements of the dermal plates visibly developing and the mouth apparatus
becoming elongated, acquiring the typical adult form [Figure 2.1.2 (E), (F)].
Finally, 24-32 days after fertilization, the embryonic development of S.
abaster was completed and fully formed dark brown juveniles, resembling
adult individuals [Figure 2.1.2 (G),(H)], Were released from the marsupium
by sharp bending movements of the male’s body.
17

18
Figure 2.1.1: Ontogenetic events occurring during the embryonic and larval development of Syngnathus
abaster in order of first appearance (a, blastula stage; b, epiboly; c, embryonic shield recognizable; d, cephalic
and caudal dilatation; e, optic vesicles; f, notochord and neural tube differentiation; g, beginning of somite
formation; h, crystalline lens; i, ocular pigmentation; j, tail region free from yolk; k, beginning of melanogenesis; l,
fin differentiation; m, heart beats and visible blood vessels; n, embryo motility; o, development of mouth
apparatus; p, development of fin rays; q, dermal plates; r, brownish colouration; s, hatch from egg envelope; t,
release from marsupium).
2.1. Early life history of Syngnathus abaster

19
Figure 2.1.2: Different developmental stages in Syngnathus abaster: (A) 3 days, (B) 7 days, (C) 13 days, (D) 17
days, (E) 19 days, (F) 21 days, (G) newborn head detail and (H) newborn juveniles. Scale bars: 1 mm.
Chapter 2. Early Life History and Reproductive Behaviour

2.1. Early life history of Syngnathus abaster
A significant difference was found between time spent near the
surface or bottom of the aquaria (Table 2.1.1). Newborn juveniles spent
most of the time near the bottom with only some sporadic movements
towards the surface, followed by a return to the bottom section of the
aquaria. The observed vertical distribution did not significantly change
during the following weeks, but a slight reduction in time spent near the
surface was observed (Figure 2.1.3). Juveniles used for the calculation of
the growth (n = 25) doubled in LT within the first 4 weeks after abandoning
the parental marsupium (mean ± S.D. initial size = 15.60 ± 0.22 mm;
4 weeks old = 3.14 ± 0.28 cm).
No evidence of schooling behaviour was ever observed when
juveniles were maintained together. In fact, corroborating the results of the
vertical distribution experiment, the juveniles, with a cryptic colouration,
were usually observed in close contact with the substratum laid in the
bottom of the aquarium.
Table 2.1.1: ANOVA results on the vertical distribution of Syngnathus
abaster juveniles during the first three weeks after abandoning the
marsupium.
Source DF MS F P
Time 2 5.727 1.45 0.243
Vertical distribution 1 487.569 123.80 < 0.001
Time x V. distribution 2 3.445 0.87 0.423
Residuals 54 3.939
Total 59
20

21
Figure 2.1.3: (A) Vertical distribution in the water column (mean ± S.D. per cent time spent at the
surface and bottom) displayed by juvenile Syngnathus abaster during the first 3 weeks after abandoning
the marsupium and (B) mean ± S.D. juvenile total length during the first 4 weeks.
Chapter 2. Early Life History and Reproductive Behaviour

2.1. Early life history of Syngnathus abaster
2.1.5 Discussion
The observation of the embryonic and larval development of S. abaster
reinforces the notion that the presence of a marsupium contributes to a
higher degree of newborn development, when compared to a
marsupium-lacking pipefish such as Nerophis lumbriciformis (Jenyus)
(Monteiro et al., 2003) or Nerophis ophidion (L.) (Russell, 1976). As also
observed in Syngnathus acusimilis Gunther (Drozdov et al., 1997), S.
abaster larvae hatch from the egg envelope before complete formation of
the mouth apparatus, still exhibiting the final remains of the yolk sack. The
final stages of development occur inside the marsupium, without the
physical constraints of the limited egg space. As a result, marsupium
presenting pipefishes of the genus Syngnathus give birth to fully
developed juveniles while marsupium-lacking pipefishes of the genera
Nerophis and Entelurus produce less developed larvae (Russell, 1976;
Monteiro et al., 2003), whose morphology is still some way from the adult
form. The observed differences in the final degree of development might
have an important role in larvae early life history behaviour. Contrary to N.
lumbriciformis newborn larvae, that immediately showed vertical swim-up
and drift behaviour, using the pectoral fins to rotate along the body axis
(Monteiro et al., 2003), S. abaster juveniles occupy the bottom of the
aquaria shortly after birth, rapidly growing, thus suggesting a benthic
strategy (Figure 2.1.3). During regular monthly samples conducted for >1
year, aimed at collecting fish larvae in the water column of a Portuguese
river estuary (Lima River, northern Portugal), only two juveniles were
captured, even though S. abaster is a common inhabitant of the estuary
(S. Silva, unpubl. data).
Early life history behaviour is of great importance for the species
ecology as it might determine population demography and ‘connectivity’
(Cowen et al., 2000). Since, mainly due to habitat reduction and
environmental degradation (Nagelkerken et al., 2000), the total area
occupied by eelgrass meadows is currently receding in southern Europe
(Duarte, 2002), black striped pipefish populations face new and
22

Chapter 2. Early Life History and Reproductive Behaviour
challenging problems. The observed behaviour of newborn juveniles,
unlike that registered for N. lumbriciformis or N. ophidion, whose larvae
display a clear pelagic life phase (Russell, 1976; Monteiro et al., 2003),
may imply a limited dispersion capability, thus contributing to the ongoing
isolation of geographically distant populations (S. abaster populations
seem to be confined to estuaries and salt pond reservoirs within mainland
Portugal; N.M. Monteiro, pers. obs.). An increasing number of factors (the
destruction and reduction of the habitat, the increasing geographical
distance among populations and the apparent benthic behaviour of the
juveniles) interact and contribute to a decrease in connectivity between
populations.
The role of connectivity patterns via larval dispersal in structuring
marine populations has been a central issue in marine ecology (Palumbi,
1999; Cowen et al., 2000; Armsworth, 2002) and increasing knowledge of
syngnathid early life history would increase the understanding of how
developmental and behavioural processes may affect population
persistence and evolution. Within pipefishes, species without a marsupium
seem to produce smaller and less developed larvae, having a primordial
fin and transparent colouration, that display pelagic early life history
behaviour, e.g. N. lumbriciformis, N. ophidion and Entelurus aequoreus
(L.) (Russell, 1976; Monteiro et al., 2003). Pipefish species with a
marsupium, with the exception of the small juveniles (13-14 mm) produced
by Syngnathus rostellatus Nilsson (Froese & Pauly, 2005), seem to
produce more developed juveniles that immediately take a bottom life on
release from the parent (Syngnathus acus L., Syngnathus typhle L. and S.
acusimilis) (Russell, 1976; Drozdov et al., 1997). In the specific case of the
genus Hippocampus, even though some authors state that seahorses
avoid a planktonic larval phase (Schmid & Senn, 2002), as might be the
case of Hippocampus fuscus Ruppell (Golani & Fine, 2002), several
species are recurrently observed in open sea, (e.g. Hippocampus
mohnikei Bleeker, Hippocampus comes Cantor, Hippocampus
spinosissimus Weber and Hippocampus abdominalis Lesson (Kanou &
Kohno, 2001; Foster & Vincent, 2004).
23

2.1. Early life history of Syngnathus abaster
An interesting observation that, as far as is known, has not yet
been described, deals with filial cannibalism. Even though juveniles were
already able to feed on A. franciscana nauplii (supplied on a daily basis),
the density of newborn was initially observed to dramatically decrease.
The direct observation of adults feeding on juveniles (sometimes their
own) confirmed the hypothesis that filial cannibalism does occur, since
newborn are sufficiently slender to pass through an adult’s mouth opening
(K. Silva, pers. obs.). Even though potentially negligible in the wild, this
phenomenon might be a serious concern in aquarium-reared individuals
whose larvae are smaller than an adult’s mouth. Thus, the type of
substratum used (with plants and crevices) might play an important role in
the rate of juvenile survival during the first development stages.
2.1.6 Acknowledgements
We would like to thank everybody that helped during the laboratorial work,
especially P. Correia. N. Monteiro’s participation was funded by Fundação
para a Ciência e a Tecnologia (FCT-SFRH/BD/2747/2000). K. Silva’s
participation was funded by Fundação para a Ciência e a Tecnologia
(FCT-SFRH/BD/13171/2003). V. Almada’s participation was partially
funded by Programa Plurianual de Apoio às Unidades de Investigação.
2.1.7 References
Armsworth, P. R. (2002). Recruitment limitation, population regulation, and
larval connectivity in reef fish metapopulations. Ecology 83, 1092–1104.
Baillie, J. E. M., Hilton-Taylor, C. & Stuart, S. N. (2004). 2004 IUCN Red
List of Threatened Species. A Global Species Assessment. IUCN, Gland,
Switzerland and Cambridge, United Kingdom.
24

Chapter 2. Early Life History and Reproductive Behaviour
Cakic, P., Lenhardt, M., Mickovic, D., Sekulic, N. & Budakov, L. J. (2002).
Biometric analysis of Syngnathus abaster populations. Journal of Fish
Biology 60, 1562–1569.
Carcupino, M., Baldacci, A., Mazzini, M. & Franzoi, P. (2002). Functional
significance of the male brood pouch in the reproductive strategies of
pipefishes and seahorses, a morphological and ultrastructural comparative
study on three anatomically different pouches. Journal of Fish Biology 61,
1465–1480.
Cowen, R. K., Lwiza, K. M., Sponaugle, S., Paris, C. B. & Olson, D. B.
(2000). Connectivity of marine populations, open or closed? Science 287,
857–859.
Dawson, C. E. (1986). Syngnathidae. In: Fishes of the North-eastern
Atlantic and the Mediterranean (Whitehead, P. J. P., Bauchot, M. L.,
Hureau, J. C., Nielsen, J. & Tortonese, E., eds), pp. 628–639. Paris:
Unesco.
Drozdov, A. L., Kornienko, E. S. & Krasnolutsky, A. V. (1997).
Reproduction and development of the pipefish Syngnathus acusimilis.
Biologiya Morya 23, 304–308.
Duarte, C. M. (2002). The future of seagrass meadows. Environmental
Conservation 29, 192–206.
Foster, S. J. & Vincent, A. C. J. (2004). Life history and ecology of
seahorses, implications for conservation and management. Journal of Fish
Biology 65, 1–61.
Froese, R. & Pauly, D. (2005) FishBase. World Wide Web electronic
publication.
25

2.1. Early life history of Syngnathus abaster
Golani, D. & Fine, M. (2002). On the occurrence of Hippocampus fuscus in
the eastern Mediterranean. Journal of Fish Biology 60, 764–766.
Herald, E. S. (1959). From pipefish to seahorse – a study of phylogenetic
relationships. Proceedings of the Californian Academy of Sciences 29,
465–473.
Kanou, K. & Kohno, H. (2001). Early life history of a seahorse,
Hippocampus mohnikei in Tokyo Bay, Japan. Ichthyological Research 48,
361–369.
Monteiro, N. M., Almada, V. C. & Vieira, M. N. (2003). Early life history of
the pipefish Nerophis lumbriciformis (Pisces, Syngnathidae). Journal of the
Marine Biological Association of the United Kingdom 83, 1179–1182.
Nagelkerken, I., Van Der Velde, G., Gorissen, M. W., Meijer, G. J., Van’t
Hof, T. & Den Hartog, C. (2000). Importance of mangroves, seagrass beds
and the shallow coral reefs as a nursery for important coral reef fishes,
using a visual census technique. Estuarine, Coastal and Shelf Science 51,
31–44.
Palumbi, S. R. (1999). The prodigal fish. Nature 402, 733–735.
Russell, F. S. (1976). The Eggs and Planktonic Stages of British Marine
Fishes. London: Academic Press.
Schmid, M. S. & Senn, D. G. (2002). Seahorses–masters of adaptation.
Vie et Milieu 52, 201–207.
Wilson, A. B., Vincent, A., Ahnesjo, I. & Meyer, A. (2001). Male pregnancy
in seahorses and pipefishes (Family Syngnathidae): Rapid diversification
of paternal brood pouch morphology inferred from a molecular phylogeny.
The Journal of Heredity 92, 159–166.
26

Chapter 2. Early Life History and Reproductive Behaviour
Wilson, A. B., Ahnesjo, I., Vincent, A. C. J. & Meyer, A. (2003). The
dynamics of male brooding, mating patterns, and sex roles in pipefishes
and seahorses (Family Syngnathidae). Evolution 57, 1374–1386.
27

Chapter 2. Early Life History and Reproductive Behaviour
Chapter 2.2
Development and early life history
behaviour of aquarium reared
Syngnathus acus
(Pisces: Syngnathidae)
2.2.1 Abstract
Some notes on development and early life history behaviour of aquarium-
-reared Syngnathus acus are presented and compared with other
syngnathid species, namely S. abaster. Implications in population
dynamics and connectivity are discussed.
28

2.2. Development and early life history behaviour of aquarium reared Syngnathus
acus (Pisces: Syngnathidae)
2.2.2 Introduction
The family Syngnathidae is remarkable for its adaptations for parental
care, with females depositing eggs in a male’s specialized incubating area
where embryos are protected, nourished and osmoregulated (Vincent et
al., 1992). Despite the ubiquitous male brooding of the eggs, the
anatomical organization of the brooding structures varies in complexity,
from a simple ventral surface where eggs are brooded openly without any
protective plates or covering membranes, to a brood pouch (marsupium)
with different degrees of closure culminating in the sealed sack like pouch
of seahorses (Herald, 1959). This gradient in conspicuous male paternal
care structures was used to construct a phylogeny (Herald, 1959) that was
later largely conformed by Wilson et al. (2003) using mitochondrial DNA.
A comparative study of three different brood structures [Nerophis
ophidion, Syngnathus abaster and Hippocampus hippocampus; pouch
types B1, A4i and A5, respectively, (Herald, 1959)] showed that each
structure had a skin with a different ultrastructure, suggesting different
functions that may be related to different reproductive strategies of each
species (Carcupino et al., 2002). Recent work suggested that the
anatomical variations among brooding structures could also signal distinct
early life histories with different degrees of newborn development being
associated with contrasting dispersal potentials (Silva et al., 2006). As a
fact, within pipefish, marsupium lacking species, such as Nerophis
lumbriciformis, seem to produce smaller and less developed planktonic
larvae, with a morphology still distant from the adult (Monteiro et al., 2003),
while species with a marsupium, such as S. abaster, give birth to fully
formed juveniles that immediately assume a benthic distribution on release
from the parent’s pouch (Silva et al., 2006). The ultrastructural changes
related to the increased elaboration of pipefish brooding structures seem,
thus, to be accompanied by an increase in the extent of newborn
development along with a transition from a two-phase life history
29

Chapter 2. Early Life History and Reproductive Behaviour
(planktonic larvae and benthic adults) to an apparently single benthic
strategy (Silva et al., 2006).
Larvae biology and behaviour of most syngnathid species remain,
however, undocumented and variation in early life history parameters
across genera requires thorough analysis in order to correctly assess the
mechanisms and strategies involved in syngnathid evolution. Furthermore,
increasing knowledge on early life history related parameters could provide
powerful insights into the relative vulnerability of different species or
populations to the actual increasing pressures presented through
overexploitation and habitat fragmentation.
In this paper, some notes on the development of aquarium- reared
juveniles of the greater pipefish, Syngnathus acus Linnaeus, a data
deficient species (Baillie et al., 2004), are presented. Simultaneously, the
vertical distribution of newborns was analysed as an indication of the
benthic or pelagic nature of the juveniles, a variable with profound
implications in the population dynamics of pipefish that inhabit increasingly
fragmented habitats, such as eelgrass meadows. Comparisons are also
presented, highlighting similarities and differences between S. acus and
other western European pipefish species, particularly S. abaster
2.2.3 Methods
Syngnathus acus juveniles were obtained from four pregnant males
captured by net in the Ria de Aveiro estuarine lagoon (40º45’ N; 8º40’ W),
in Portugal. Captive males were fed daily with fresh Artemia franciscana
nauplii and maintained in 250-l aquaria, illuminated with natural light,
supplemented with 18 W fluorescent lamps. Due to the ‘gas bubble
disease’ aeration was performed outside the fish tanks. Continuously
running seawater was physically and biologically filtered and temperature
maintained at 18-19ºC. Due to the direct observation of cannibalism,
newborns were separated from males and maintained in 80-l aquaria, in
the same conditions as adults.
30

2.2. Development and early life history behaviour of aquarium reared Syngnathus
acus (Pisces: Syngnathidae)
Total length (LT) of ten juveniles haphazardly collected from the
tank was measured every week up to two months after birth.
The vertical distribution of juveniles was analysed as in Silva et al.
(2006) by separately placing individuals inside small 4-l aquaria
(40x10x10 cm) with no water circulation, divided in three sections: surface
(10 cm), middle section (20 cm) and bottom (10 cm). After an initial
accommodation period (≈45 min), 10 min observation periods were
conducted on the time juveniles spent in the ‘surface’ and ‘bottom’
sections. In order to assure data independence, each observation was
conducted using different individuals that were only used once (ten trials
measuring time spent near the surface and another ten trials aimed at
measuring time spent in the bottom section). This experimental design was
consecutively repeated during five weeks, always using different
individuals. Results were analysed and further compared with data for S.
abaster (Silva et al., 2006) through a three factor non-parametric analysis
of variance [species (2 levels=S. acus and S. abaster) time (3 levels=3
weeks after birth) and vertical position (2 levels=surface and bottom)].
Comparison between S. acus and S. abaster juvenile birth lengths were
assessed through a 2-tailed independent-samples t-test. All statistical
analyses were performed using the Statistical Package for the Social
Sciences 13.0 (SPSS Inc.).
2.2.4 Results
Pregnant males gave birth, during a 2-3 day period, to fully formed
juveniles (Figure 2.2.1.A) measuring approximately 26 mm (LT; N=110,
average=25.94 mm, range:17-30 mm, SD=2.52) already capable of
feeding on Artemia nauplii. Meristic data are presented in Figure 2.2.1.B.
Comparative data for S. abaster is also presented (Froese & Pauly, 2005;
K. Silva, personal observation).
31

Chapter 2. Early Life History and Reproductive Behaviour
Figure 2.2.1: (A) Syngnathus acus newborns. (B) Comparison of meristic
characters of Syngnathus acus and Syngnathus abaster newborns. (C)
Juvenile growth rate during the first eight weeks. Error bars represent
standard deviations. (D) Vertical distribution in the water column displayed
by juveniles during the first five weeks after abandoning the marsupium.
Error bars represent standard deviations.
32

2.2. Development and early life history behaviour of aquarium reared Syngnathus
acus (Pisces: Syngnathidae)
The two congeneric species resemble each other in general morphology,
with, however, some significant differences concerning birth length [S.
acus: N=110, average=25.94 mm, S.D.=2.52; S. abaster: N=64
average=17.67 mm, S.D.=2.22; independent-samples 2-tailed t-test:
N=174, P

Chapter 2. Early Life History and Reproductive Behaviour
Table 2.2.1: Non-parametric analysis of variance results on the vertical
distribution of Syngnathus acus and Syngnathus abaster during the first
three weeks after abandoning the marsupium.
Source of variation
Deegres of
freedom
34
Sum of
squares
Hstatistic
P value
Species 1 143.01 0.134 0.714
Time 2 421.91 0.395 0.821
Vertical distribution 1 99498.70 92.251

2.2. Development and early life history behaviour of aquarium reared Syngnathus
acus (Pisces: Syngnathidae)
way as it has been documented in S. abaster (Silva et al., 2006). Along the
coasts of the Portuguese mainland, these two species seem mainly
confined to estuaries (Monteiro, personal observation). Habitat
fragmentation and/or long distance colonization, as well as restricted
dispersal with isolation by distance have already been pointed out as
important forces able to determine the actual geographical distribution of
certain seahorse species (Lourie et al., 2005).
A final interesting note on S. acus deals with filial cannibalism.
Syngnathus acus newborns were actively preyed upon by adults, including
their own parents. Cannibalism in pipefish has also been reported in S.
abaster (Silva et al., 2006), S. scovelli, S. floridae and S. fuscus (Teixeira
& Musick, 1995), and may be a widespread phenomenon in the genus
Syngnathus.
2.2.6 Acknowledgments
We thank everyone who helped during the fieldwork, especially Alberto
Silva, Armando Jorge, Mario Ramalho and Pedro Correia. Vítor Almada
and Natividade Vieira’s participation were partially funded by Programa
Plurianual de Apoio às Unidades de Investigação. Nuno Monteiro’s
participation was funded by Fundação para a Ciência e a Tecnologia
(FCT-SFRH/BPD/ 14992/2004). Karine Silva’s participation was funded by
Fundação para a Ciência e a Tecnologia (FCT-SFRH/BD/13171/2003).
2.2.7 References
Armsworth, P. R., 2002. Recruitment limitation, population regulation, and
larval connectivity in reef fish metapopulations. Ecology, 83, 1092-1104.
35

Chapter 2. Early Life History and Reproductive Behaviour
Baillie, J. E. M., Hilton-Taylor, C. & Stuart, S. N. (2004). 2004 IUCN Red
List of Threatened Species. A Global Species Assessment. IUCN, Gland,
Switzerland and Cambridge, United Kingdom.
Carcupino, M., Baldacci, A., Mazzini, M. & Franzoi, P., 2002. Functional
significance of the male brood pouch in the reproductive strategies of
pipefishes and seahorses: a morphological and ultrastructural comparative
study on three anatomically different pouches. Journal of Fish Biology, 61,
1465-1480.
Cowen, R.K., Lwiza, K.M., Sponaugle, S., Paris, C.B. & Olson, D.B., 2000.
Connectivity of marine populations: open or closed? Science, NewYork,
287, 857-859.
Dawson, C.E., 1986. Syngnathidae. In: Fishes of the North-eastern
Atlantic and the Mediterranean (ed. P.J.P. Whitehead et al.), pp. 628-639.
Paris: Unesco.
Froese, R. & Pauly, D. (2005). FishBase. World Wide Web electronic
publication.
Herald, E. S., 1959. From pipefish to seahorse - a study of phylogenetic
relationships. Proceedings of the Californian Academy of Sciences 29,
465-473.
Lourie, S.A., Green, D.M. & Vincent, A.C.J., 2005. Dispersal, habitat
differences, and comparative phylogeography of Southeast Asian
seahorses (Syngnathidae: Hippocampus). Molecular Ecology, 14,
1073-1094.
Monteiro, N.M., Almada,V.C. & Vieira, M.N., 2003. Early life history of the
pipefish Nerophis lumbriciformis (Pisces: Syngnathidae). Journal of the
Marine Biological Association of the United Kingdom, 83, 1179-1182.
36

2.2. Development and early life history behaviour of aquarium reared Syngnathus
acus (Pisces: Syngnathidae)
Monteiro, N.M., Almada, V.C. & Vieira, M.N., 2005. Implications of different
brood pouch structures in syngnathid reproduction. Journal of the Marine
Biological Association of the United Kingdom, 85, 1235-1241.
Russell, F. S., 1976. The eggs and planktonic stages of British marine
fishes. London: Academic Press.
Silva, K., Monteiro, N.M., Almada,V.C. & Vieira, M.N., 2006. Early life
history of Syngnathus abaster (Pisces: Syngnathidae). Journal of Fish
Biology, 68, 80-86.
Teixeira, R.L. & Musick, J.A., 1995. Trophic ecology of two congeneric
pipefishes (Syngnathidae) of the lower York River, Virginia. Environmental
Biology of Fishes, 43, 295-309.
Vincent, A., Ahnesjo, I., Berglund, A. & Roseqvist, G., 1992. Pipefishes
and seahorses: are they all sex role reversed? Trends in Ecology and
Evolution, 7, 237-241.
Wilson, A.B., Ahnesjo, I., Vincent, A.C.J. & Meyer, A., 2003. The dynamics
of male brooding, mating patterns, and sex roles in pipefishes and
seahorses (Family Syngnathidae). Evolution, 57, 1374-1386.
37

Chapter 2. Early Life History and Reproductive Behaviour
Chapter 2.3
Reproductive behaviour of the
black-striped pipefish Syngnathus
abaster (Pisces; Syngnathidae)
2.3.1 Abstract
The reproductive behaviour of Syngnathus abaster is described and
compared with those of other syngnathids. The need for standardized
behavioural data is discussed in light of the actual theories of evolution of
mating patterns and sex-role reversal within this family.
2.3.2 Introduction
The Syngnathidae (comprising pipefishes, seahorses and seadragons) is
well known for its unique form of parental care, where females deposit
38

2.3. Reproductive behaviour of the black-striped pipefish Syngnathus abaster
(Pisces; Syngnathidae)
unfertilized eggs into the male’s specialized ventral incubating surface
(Herald, 1959). Together with male pregnancy, some syngnathid species
also exhibit varying degrees of sex-role reversal (Monteiro et al., 2002;
Berglund & Rosenqvist, 2003), with females competing for access to
mates and sometimes presenting conspicuous secondary sexual
characters. These specializations have promoted the use of syngnathids
as models in which to study not only the evolution of parental care (Trivers,
1972; Ridley, 1978), but also to test theoretical assumptions of sexual
selection (Johnstone, 1995).
The phylogenetic relationships among syngnathids have been
studied both by Herald (1959), based on the morphological organization of
the male’s incubating area, and Wilson et al. (2003), using mitochondrial
DNA, producing similar results. Attempts to determine the evolution of
mating patterns or the degree of sex-role reversal, superimposing
information from behavioural and ecological data are, nevertheless, still far
from producing definitive results. Despite the ubiquity of male pregnancy in
syngnathids, mating patterns and sexual dimorphism differ greatly among
species. Thus, comparative studies must be preceded and supported by
objective and reasonably standardized behavioural observations, able to
highlight not only differences between species but also variation among
populations.
In order to further extend knowledge of syngnathid reproduction, a
study area where information on the most basic life-history parameters is
scarce, data were collected on the reproductive biology of the black-striped
pipefish Syngnathus abaster Risso, based on aquarium observations of
individuals captured in the wild. The reproductive behaviour of this species
is poorly understood and information on its reproductive biology is still
insufficient. Tomasini et al. (1991) described factors that influence the
reproductive success of a population of S. abaster in a Mediterranean
lagoon (Mauguio, France), Carcupino et al. (1997) described the
ultrastructural organization of the male brood pouch epithelium and Silva
et al. (2006) presented a description of the embryonic development and
juvenile behaviour. Furthermore, comparisons are presented and
39

Chapter 2. Early Life History and Reproductive Behaviour
discussed, highlighting similarities and differences in the reproductive
biology of the black-striped pipefish when compared to other Western
European pipefish species.
2.3.3 Methods
Syngnathus abaster, a euryhaline species with a restricted distribution that
includes the Mediterranean and Black Sea northward to southern Biscay
(Dawson, 1986), occurs either in coastal areas or in brackish and fresh
waters (Cakic et al., 2002). The black-striped pipefish is a small
brown-green pipefish, with dark or pale spots on the trunk and tail. It can
be found mainly among sand, mud or eelgrass meadows, between depths
of 0.5 and 5 m, within a temperature range of 8 and 24°C. Males of the
Urophori have a brood pouch located ventrally on the tail, which consists
of two skin folds that contact medially with their free edges.
Fish were collected with a hand-net, in a salt pond reservoir, at the
Ria de Aveiro estuarine lagoon (40°45’ N; 8°40’ W), Portugal. One
hundred and seventy-six mature individuals, including males and females,
were captured during a 2-year period (March 2003 to June 2005),
measured for total length (LT) and the number of eggs (visible through the
marsupium folds) was counted in each pregnant male. These individuals
were transported to the laboratory and maintained in several 80 l aquaria,
illuminated with natural light and supplemented with 18 W fluorescent
lamps. The tank substrata consisted mainly of sand and plastic seagrass
laid in order to mimic the original habitat where the fish were caught. Due
to the ‘gas bubble disease’, common in pipefishes, aeration was performed
outside the fish tanks (Monteiro et al., 2002). The continuously running
seawater was physically and biologically filtered and its temperature and
salinity (33‰) maintained constant. Fish were fed daily with fresh Artemia
franciscana nauplii. Adult males and females were initially kept separate in
order to synchronize the ‘disposition to mate’ and simultaneously to allow
the fish to be accustomed to the presence of an observer. An equal
number of females and non-brooding males (not exceeding six individuals)
40

2.3. Reproductive behaviour of the black-striped pipefish Syngnathus abaster
(Pisces; Syngnathidae)
were then placed together to mate. Since no successful mating rituals
were observed in the first courtship trials, with the water temperature
ranging from 14 to 15°C, and considering that tempe rature is one of the
strongest factors in the control of marine fish reproduction (Monteiro et al.,
2001), water temperature was gradually increased to 18-19°C. This is in
agreement with in situ observations (during 2005, adult S. abaster
migrated to salt ponds, in order to reproduce, when temperature reached
values >18°C; K. Silva, pers. obs.). This adjustmen t in water temperature
resulted in the observation of several successful matings. More than 230 h
of ad libitum observations were conducted (Martin & Bateson, 1993), at
random intervals, and the main stages of the courtship rituals were
defined, measured and described. More than 15 h of video-tape recordings
were also used in order to further describe the behavioural patterns of
courtship and mating.
2.3.4 Results
Differences between male and female initiative, flickering movements and
leads were determined using ADERSIML (Almada & Oliveira, 1997). This
computer programme implements a procedure to access the significance
of goodness of fit tests that would usually be addressed using the χ 2
distribution. This procedure was chosen because it allows the analysis of
data where expected frequencies are very low (

Chapter 2. Early Life History and Reproductive Behaviour
marsupium (n=58, r=0.554, P

2.3. Reproductive behaviour of the black-striped pipefish Syngnathus abaster
(Pisces; Syngnathidae)
(Figure 2.3.2). Males presented swollen pouch folds, particularly in the
vicinity of the genital area.
Figure 2.3.2: Female ‘amplifiable ornament’ (A) in isolation and (B)
interacting with another female (increasing contrast in the striped pattern).
The reproductive ritual of S. abaster consisted of three distinct phases,
marked by prominent behavioural changes that can be summarized as
follows. Initial courtship [Figure 2.3.3 (A), (B), (C)]: the first stage of the
courtship ritual was characterized by mutual flickering movements that
affected the entire body rather than just the anterior section of the fish, as
observed in Nerophis lumbriciformis (Jenyns) (Monteiro et al., 2002). It
consisted mainly of rapid and vigorous bends moving along the main axis
of the body. Both females and males approached the opposite sex, without
either of the sexes showing greater initiative (n=17 courtship trials, male:
nine initiatives, female: eight initiatives, P>0.05). Females tended to flicker
first (χ 2 , n=17 courtship trials, male: 3, female: 14, P0.05). After the first flicker, if the opposite sex
flickered in response, both individuals performed rapid side-by-side
vibrations while swimming through the aquarium in a more or less parallel
position (73% of trials). Females tended to lead these short movements
(χ 2 , n=34 leads in 17 courtship trials; male: nine leads, female: 25 leads;
43

Chapter 2. Early Life History and Reproductive Behaviour
P

45
Figure 2.3.3: Overview of the main behavioural stages of courtship and mating: (A) vertical swimming, (B)
crossing, (C) parallel swimming and (D) spawning
2.3. Reproductive behaviour of the black-striped pipefish Syngnathus abaster
(Pisces; Syngnathidae)

Chapter 2. Early Life History and Reproductive Behaviour
this phase (n=17, mean±S.D.=26.0±3.0 s, range:21-31 s), the male slowly
descended to the substratum where it stayed motionless (mean±S.D.
= 95.0±85.4 s), sometimes assuming an S-like position (seven in 17
matings), as also observed in Syngnathus acus L. and Syngnathus typhle
L. (Vincent et al., 1995). After a variable latency period
(mean±S.D.= 15.0±6.4 min), a new courtship ritual might occur. In aquaria,
some pairs (three in eight) were observed to spawn up to three
consecutive times and, interestingly, one female was observed spawning
with three different males within

2.3. Reproductive behaviour of the black-striped pipefish Syngnathus abaster
(Pisces; Syngnathidae)
reduction of courtship displays in the water column was also documented
by Almada & Santos (1995) for blenniids that spawn in habitats subjected
to strong wave action.
Another widespread phenomenon seems to be the occurrence of
disruptions during the first phase of the courtship ritual. During the
aquarium observations, females were observed approaching the courting
pair and beginning to actively flicker or merely following the pair in a
parallel motion, a behaviour that might be viewed as a form of competition
(Matsumoto & Yanagisawa, 2001). Curiously, the intruding female was
usually unable to mate with the courting male. Only in one occasion was
the opposite observed. Nevertheless, these disrupting females were far
more successful in redirecting the other female’s attention, causing the
apparent end of the ongoing courtship display. Similar observations have
been described for Corythoichthys haematopterus (Bleeker) (Matsumoto &
Yanagisawa, 2001), Syngnathus schlegeli Kaup (Watanabe et al., 2000)
and N. lumbriciformis (Monteiro et al., 2002), suggesting that the still
poorly understood female-female interactions, that greatly vary among
syngnathid genera, play an important role in the competition for access to
mates (Berglund & Rosenqvist, 2003). As in N. ophidion and S. typhle
(Berglund & Rosenqvist, 2003), the apparent absence of overtly
aggressive interactions among S. abaster females suggests that female
dominance occurs mainly through sexual signalling, namely a more
contrasted colouration in the trunk (see Figure 2.3.2). This ornamentation
seems to be an amplification of the normal colouration, similar to what has
been described both for S. typhle (Berglund & Rosenqvist, 2003) and N.
lumbriciformis (Monteiro et al., 2002), a phenomenon that might be directly
involved not only in male attraction but also in female-female interactions.
The observed sexual dimorphism in size (females being larger than
males) and behaviour suggest sex-role reversion in the sampled
population of S. abaster (at least under an even sex ratio), a phenomena
already documented for several other syngnathids (Kornienko, 2001;
Monteiro et al., 2002; Berglund & Rosenqvist, 2003) as well as other fish
47

Chapter 2. Early Life History and Reproductive Behaviour
families [Kuwamura, 1985 (Apogonidae); Balshine-Earn & McAndrew,
1995 (Cichlidae); Swenson, 1997 (Gobiidae)].
Recently, several hypotheses have been presented on the evolution
of sex-role reversal in syngnathids, superimposing behavioural information
on phylogenetic data (Wilson et al., 2003). Still far from a definitive result,
genetic evidence suggests a pattern still difficult to analyse within
syngnathids, with sex-role reversal appearing to be largely independent of
pouch complexity (Wilson et al., 2003). These difficulties may well arise
from two distinct factors: 1) observations do not take into account the
natural variation in the degree of sex-role reversal that might exist in
geographically distinct populations within a species. Factors such as the
operational sex ratio (Vincent et al., 1994), mate quality (Berglund &
Rosenqvist, 2003), differences between sexes in the relative ‘time in’
(availability to mate) v. ‘time out’ period (Masonjones & Lewis, 2000), as
well as predation, breeding resources and mate rate encounters are
thought to locally modulate the final expression of the sex-role reversal,
not only in syngnathids (Almada et al., 1995; Forsgren et al., 2004). For
example, S. schlegeli, from Vostok and Amurskii bays, is described as
presenting conventional sex roles by Kornienko (2001), while Watanabe et
al. (2000) present data suggesting that the same species, from Otsuchi
and Funakoshi Bays, is a sexually reversed syngnathid; 2) observations
largely depend on a binary definition of sex-role reversal (reversed v.
conventional). Although S. abaster proved to be sex-role reversed, there is
a considerable difference in the magnitude of some measured variables,
such as the role of the male during courtship ritual, when compared with
other Western European syngnathids, also reported as sex-role reversed
species (Kornienko, 2001; Monteiro et al., 2002; Berglund & Rosenqvist,
2003). S. abaster males have a more active role in courtship, flickering and
sometimes approaching females, when compared to N. lumbriciformis
(Monteiro et al., 2002), a species where males are much more passive.
Moreover, sexual dimorphism is more pronounced in N. lumbriciformis
(Monteiro et al., 2002) and N. ophidion (Berglund & Rosenqvist, 2003),
with differences in colouration among the sexes being observed not only in
the middle region of the body (amplifiable ornament display) but also in the
48

2.3. Reproductive behaviour of the black-striped pipefish Syngnathus abaster
(Pisces; Syngnathidae)
head of the females. Nerophis ophidion females present bright blue colour
markings along the sides of the head while N. lumbriciformis females
exhibit an intricate pattern of bright facial spots (Monteiro et al., 2005a). A
moderate sex-role reversion has also been reported for the tidewater goby,
Eucyclogobius newberryi (Girard), where females display a secondary
sexual trait and compete for mates more intensely than do males.
Nevertheless, males also engage in intrasexual competition for territories
and sometimes also initiate courtship (Swenson, 1997).
More detailed information on syngnathid courtship and mating
patterns, encompassing population heterogeneity and what seems to be
the clinal nature of reproductive behaviours, would certainly allow for a
more conclusive phylogenetic analysis of the evolution of the reproduction
within the Syngnathidae family.
Although genetic data are lacking and observations reported in this
paper are from captive fish, available evidence suggests that this pipefish
is polygynandrous since females were observed mating with different
males, sometimes within a very short time period, and males were also
observed receiving eggs from distinct females. In general, more
polyandrous species show greater degrees of sexual dimorphism, as a
result of an increased intensity in sexual selection (Avise et al., 2002).
Polygynandrous mating systems, however, have been reported for two
sex-role reversed species with intermediate degrees of sexual dimorphism
[S. typhle and Syngnathus floridae (Jordan & Gilbert) Jones & Avise,
2001], as well as for the strongly sexual dimorphic sex-role reversed N.
lumbriciformis (Monteiro et al., 2006), possibly suggesting a more
elaborated pattern than the generally accepted continuum sexual
selection-mating system (Avise et al., 2002).
Clutch size of pregnant S. abaster males (10–64 eggs) are at the
lower end of the range reported for other syngnathids, with Hippocampus
reidi Ginsburg and Hippocampus erectus Perry presenting the largest
reported clutch size (Monteiro et al., 2005b).
The observed significant correlation between male size and egg
number may reflect a LT-dependent reproductive fitness (Figure 2.3.1),
49

Chapter 2. Early Life History and Reproductive Behaviour
with larger males accommodating more offspring, as also reported for S.
typhle (Berglund & Rosenqvist, 2003) and S. schlegeli (Watanabe &
Watanabe, 2002). Accordingly, LT seems also to affect mate choice, as
suggested by the observed significant correlation between male and
female size (n=17 successful mating pairs, r=0.920, P0.05).
Figure 2.3.4 Male and female total length relationships in successful
(black squares: y=0.7774x+2.16) and unsuccessful (white squares)
Syngnathus abaster mating pairs.
2.3.6 Acknowledgments
We would like to thank the anonymous referees for their criticism and
suggestions as well as everyone who helped during the fieldwork,
especially A. Silva, A. Jorge and P. Correia. V. Almada’s participation was
partially funded by Programa Plurianual de Apoio às Unidades de
Investigação. N. Monteiro’s participation was funded by Fundação para a
50

2.3. Reproductive behaviour of the black-striped pipefish Syngnathus abaster
(Pisces; Syngnathidae)
Ciência e a Tecnologia. K. Silva’s participation was funded by Fundação
para a Ciência e a Tecnologia (FCT SFRH/BD/13171/2003).
2.3.7 References
Almada, V. C. & Oliveira, R. F. (1997). Sobre o uso de estatística de
simulação em estudos de comportamento. Análise Psicológica 1, 97–109.
Almada, V. C. & Santos, A. J. (1995). Parental care in the rocky intertidal:
a case study of adaptation and exaptation in Mediterranean and Atlantic
blennies. Reviews in Fish Biology and Fisheries 5, 23–37.
Almada, V. C., Gonçalves, E. J., Oliveira, R. F. & Santos, A. J. (1995).
Courting females: ecological constraints affect sex roles in a natural
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Avise, J. C., Jones, A. G., Walker, D. & DeWoody, J. A. (2002). Genetic
mating systems and reproductive natural histories of fishes: lessons for
ecology and evolution. Annual Review of Genetics 36, 19–45.
Balshine-Earn, S. & McAndrew, B. J. (1995). Sex-role reversal in the
black-chinned tilapia, Sarotherodon melanotheron (Rupel) (Cichlidae).
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Berglund, A. & Rosenqvist, G. (2003). Sex role reversal in pipefish.
Advances in the Study of Behaviour 32, 131–167.
Cakic, P., Lenhardt, M., Mickovic, D., Sekulic, N. & Budakov, L. J. (2002).
Biometric analysis of Syngnathus abaster populations. Journal of Fish
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Carcupino, M., Baldacci, A., Mazzini, M. & Franzoi, P. (1997).
Morphological organization of the male brood pouch epithelium of
Syngnathus abaster Risso (Teleostea, Syngnathidae) before, during, and
after egg incubation. Tissue Cell 29, 21–30.
Dawson, C. E. (1986). Syngnathidae. In: Fishes of the North-eastern
Atlantic and the Mediterranean (Whitehead, P. J. P., Bauchot, M. L.,
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Fiedler, K. (1954). Vergleichende verhaltensstudien an seenadeln,
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relationships. Proceedings of the Californian Academy of Sciences 29,
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Jones, A. G. & Avise, J. C. (2001). Mating systems and sexual selection in
male-pregnant pipefishes and seahorses: insights from
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Johnstone, R. A. (1995). Sexual selection, honest advertisement and the
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Kuwamura, T. (1985). Social and reproductive behavior of three
mouthbrooding cardinalfishes, Apogon doederleini, A. niger and A.
notatus. Environmental Biology of Fishes 13, 17–24.
Kvarnemo, C. & Simmons, L. W. (2004). Testes investment and spawning
mode in pipefishes and seahorses (Syngnathidae). Biological Journal of
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Martin, P. & Bateson, P. (1993). Measuring Behaviour: an Introductory
Guide, 20th edn. Cambridge: Cambridge University Press.
Masonjones, H. D. & Lewis, S. M. (1996). Courtship behavior in the dwarf
seahorse, Hippocampus zosterae. Copeia 3, 634–640.
Masonjones, H. D. & Lewis, S. M. (2000). Differences in potential
reproductive rates of male and female seahorses related to courtship
roles. Animal Behaviour 59, 11–20.
Matsumoto, K. & Yanagisawa, Y. (2001). Monogamy and sex role reversal
in the pipefish Corythoichthys haematopterus. Animal Behaviour 61,
163-170.
Monteiro, N. M., Vieira, N. M. & Almada, V. C. (2001). The breeding
ecology of the pipefish Nerophis lumbriciformis and its relation to latitude
and water temperature. Acta Ethologica 81, 1031–1033.
Monteiro, N. M., Vieira, N. M. & Almada, V. C. (2002). The courtship
behaviour of the pipefish Nerophis lumbriciformis: reflections of and
adaptation to intertidal life. Acta Ethologica 4, 109–111.
Monteiro, N. M., Vieira, N. M. & Almada, V. C. (2005a). Homing behaviour
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Syngnathidae): a true intertidal resident? Estuarine, Coastal and Shelf
Science 63, 93–99.
Monteiro, N. M., Almada, V. C. & Vieira, N. M. (2005b). Implications of
different brood pouch structures in syngnathid reproduction. Journal of the
Marine Biological Association of the United Kingdom 85, 1235–1241.
Monteiro, N. M., Berglund, A., Vieira, N. M. & Almada, V. C. (2006).
Reproductive migrations of the sex role reversed pipefish Nerophis
lumbriciformis (Pisces: Syngnathidae). Journal of Fish Biology 69, 66–74.
Ridley, M. (1978). Parental care. Animal Behaviour 26, 904–932.
Silva, K., Monteiro, N. M., Vieira, M. N. & Almada, V. C. (2006). Early life
history of Syngnathus abaster (Pisces: Syngnathidae). Journal of Fish
Biology 68, 80–86.
Swenson, R. O. (1997). Sex-role reversal in the tidewater goby,
Eucyclogobius newberryi. Environmental Biology of Fishes 50, 27–40.
Tomasini, J. A., Quignard, J. P., Capapé , C. & Bouchereau, J. L. (1991).
Facteurs du succés reproductif de Syngnathus abaster Risso, 1826
(Pisces, Teleostei, Syngnathidae) en milieu lagunaire mediterranéen
(lagune de Mauguio, France). Acta Oecologica 12, 331–355.
Trivers, R. L. (1972). Parental investment and sexual selection. In: Sexual
Selection and the Descent of Man (Campbell, B., ed.), pp. 136–179.
Chicago, IL: Aldine-Atherton.
Vincent, A., Ahnesjo, I. & Berglund, A. (1994). Operational sex rations and
behavioural sex differences in a pipefish population. Behavioral Ecology
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(Pisces; Syngnathidae)
Vincent, A., Berglund, A. & Ahnesjo, I. (1995). Reproductive ecology of five
pipefish species in one eelgrass meadow. Environmental Biology of Fishes
44, 347–361.
Watanabe, S. & Watanabe, Y. (2002). Relationship between male size and
newborn size in the seaweed pipefish, Syngnathus schlegeli.
Environmental Biology of Fishes 65, 319–325.
Watanabe, S., Hara, M. & Watanabe, Y. (2000). Male internal fertilization
and introsperm-like sperm of the seaweed pipefish Syngnathus schlegeli.
Zoological Science 17, 759–767.
Wilson, A. B., Ahnesjo, I., Vincent, A. C. J. & Meyer, A. (2003). The
dynamics of male brooding, mating patterns, and sex roles in pipefishes
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Zar, J. H. (1984). Biostatistical Analysis. Upper Saddle River, NJ:
Prentice-Hall.
55

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Chapter 3.1
The effect of temperature on mate
preferences and female-female
interactions in Syngnathus
abaster
3.1.1 Abstract
Despite much effort to ascertain the consequences of temperature
variation for a wide range of animal performance traits, the effect of
temperature on interactions among organisms is still poorly understood.
The present work tests for a direct influence of water temperature on
sexual recognition, mate preferences and female-female interactions in the
pipefish Syngnathus abaster. Three experiments were conducted by
monitoring time spent in the vicinity of conspecifics at three water
temperatures intended to reflect seawater temperatures before the onset
56

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
of reproduction (15ºC) and during the early (18ºC) and late breeding
season (24ºC). Four major results emerged: (1) S. abaster can visually
discriminate potential mates from fish of the same sex; (2) males and
females responded differently with the former diverting their attention
towards the opposite sex at intermediate (18ºC) and high temperatures
(24ºC), while the latter only showed a significant interest in potential mates
at 24ºC, devoting an equal amount of interest towards both males and
females at 18ºC; (3) at breeding season water temperatures, both sexes
discriminated against smaller partners, preferring larger ones; (4)
different-sized females adopted distinct temperature-modulated
behavioural responses, possibly because large dominant females, which
engage in competition at 18ºC, constrained the reproduction of smaller
ones, which seem to compete only at 24ºC. These results highlight the
importance of temperature as an effective agent in the modulation of S.
abaster reproductive behaviour. Considerations on the ecological
significance of the observed behavioural patterns are also discussed.
3.1.2 Introduction
The decision-making processes involved in mating behaviour can have
profound implications on the dynamics of sexual selection, the mechanism
of evolution and speciation (Andersson 1994). Although traditional
theoretical models have generally ignored the consequences of individual
variation in mate choice, it is known that there are both genetic and
environmental conditions capable of inducing polymorphism in mating
decisions (Jennions & Petrie 1997). In fact, mate choice behaviour is a
contextual phenomenon molded by a variety of factors, such as the
genotype of the choosing individual and/or that of the potential mate or
mates (Puurtinen et al. 2005), time and energy costs of sampling (Milinski
& Bakker 1992), social environment (Galef Jr. & White 2000) and
predation risk (Berglund 1993). Studies in a variety of fish species have
shown that environmental abiotic features, such as oxygen concentrations
57

Chapter 3. Understanding Sex-Role Reversal (ex situ)
(e.g. Pomatoschistus microps: Reynolds & Jones 1999), light (e.g.
Gasterosteus aculeatus: Rick et al. 2006) water turbidity (e.g.
Pomatoschistus minutus: Jarvenpaa & Lindstrom 2004) and temperature,
can be effective agents in the modulation of mating behaviour. In sand
gobies (Pomatoschistus minutus), a trend towards a higher frequency of
female-female interactions was observed in the cold water treatment,
suggesting that females were more liable to compete when the water was
cold. When the water was warm, by contrast, females interacted less and
appeared to concentrate on carefully choosing a partner (Kvarnemo 1996).
Despite much effort to determine the functional consequences of
temperature variation for a wide range of animal performance traits, there
is still little understanding of the influence of thermal variation on
behavioural interactions among organisms (Wilson 2005), a topic of
increasing importance in light of the implications of increasing seawater
temperatures as an expected consequence of global warming (Walther et
al. 2002). The present work tries to determine the influence of water
temperature in the ability of S. abaster to recognize and select mating
partners. Additionally, an alteration in female-female interaction patterns
concurrent with increasing water temperatures was also investigated.
Experiments were conducted using three distinct temperatures, intended
to reflect seawater temperatures before the onset of reproduction and
during the early and late breeding season of a Southern European S.
abaster population. Furthermore, considerations on the ecological
significance of the observed behavioural responses are presented.
3.1.3 Methods
S. abaster is a euryhaline species with a restricted distribution that
includes the Mediterranean, the Black Sea, and the Atlantic coast of South
West Europe up to southern Biscay (Dawson 1986). This black-striped
pipefish occurs either in coastal areas or in brackish and fresh waters
(Cakic et al. 2002), and can be found mainly among sand, mud or eelgrass
meadows, at depths between 0.5 and 5 m, within a temperature range of
58

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
8ºC to 24ºC. Males have a brood pouch located ventrally on the tail
(Urophori) which consists of two skin folds that contact medially with their
free edges. Sex roles seem to be reversed, as females are larger and
apparently more competitive than males, at least under even sex ratio
conditions (Silva et al. 2006).
Fishes were collected with a hand net, in a salt pond reservoir, at
the Ria de Aveiro estuarine lagoon (40º45’N, 8º40’W), in Portugal, and
transported to the laboratory, where they were maintained in 250 litre
aquaria illuminated by natural light supplemented with 18 W fluorescent
lamps. Tank substrata consisted mainly of sand and plastic seagrass laid
in order to mimic the original habitat where the fishes were caught. The
continuously running seawater was physically and biologically filtered and
aeration was performed outside the fish tanks to prevent the ‘gas bubble
disease’, common in pipefishes (Monteiro et al. 2002). Fishes were fed
daily with fresh Artemia franciscana nauplii. Sexually mature males and
females were kept in separate tanks.
Three aquarium experiments were conducted using three distinct
water temperatures intended to mimic the periods immediately before the
onset of reproduction (15ºC), the beginning of the breeding period (18ºC)
and the final stage near the end of the reproductive season (24ºC). These
values were obtained and validated in the field. At 15ºC no pregnant males
were found, with eggs appearing in the marsupium only when
temperatures reached ≈17ºC. Reproduction ceases shortly after
temperature reaches its highest values (≈25ºC). In the stock tanks the
fishes were kept at 18ºC, a temperature at which they breed continuously.
Before the experiments at 15ºC or 24ºC, the fishes were first placed in new
aquaria at 18ºC, after which the temperature was gradually increased or
decreased during a period of 10 to 15 days. After the tanks reached the
desired temperature, the fishes were left to acclimate for a period of one
month before the experiments were conducted. It is important to note that
at 24ºC the fishes continue to breed regularly while at 15ºC reproduction
ceases.
59

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Size cut-offs for ‘large’ and ‘small’ individuals were defined as ½
standard deviation below and above the mean size (total length) for each
sex (♀: mean=9.4 cm, sd=1.38 cm; ♂: mean=8.5 cm, sd=1.26 cm). Large
males and females were longer than 9.1 cm and 10.1 cm respectively.
Small males and females were shorter than 7.9 cm and 8.7 cm
respectively. Since the smallest observed pregnant male was 5.2 cm, it
seems likely that the smallest individual used (6.5 cm) was also mature.
Due to the large number of individuals needed to complete all
three experiments, some of the focal fish were reused in posterior
experiments as stimulus fish. Since stimulus fish were kept inside two
smaller aquaria (ruling out the possibility of chemical communication) and
were also unable to see the focal fish, it can be assumed that this
procedure could not have altered the observed response patterns.
Experiment 1
The first experiment consisted on a tendency evaluation of different-sized
males and females to approach consexual and non-consexual individuals
at three pre-selected water temperatures. Experimental set-up is shown in
Figure 3.1.1.A: a one way mirror between aquarium 1 and the other two
smaller aquaria (2 and 3) allowed the focal fish in aquarium 1 to see the
stimuli fishes without being seen, ruling out the possibility of a response
based on chemical communication. An opaque wall located between the
two smaller aquaria blocked visual contact between the two stimuli
individuals. Two distinct areas were considered in aquarium 1, with Area A
located closer to the one-way mirror (see Figure 3.1.1.A) as opposed to
Area B (‘neutral’ area). The time spent in Area A, directly in front of one of
the two smaller aquaria (aquarium 2 or 3, randomly containing a male or a
female; see Figure 3.1.1) was monitored during 30 min, after an initial
acclimatization period (≈45 min). Tests started only when the observer,
located at 50 cm from aquarium 1, was confident that the focal fish showed
no stress signs and had had the opportunity to see the stimuli fishes,
which were matched for size. Stimuli fishes were randomly allocated to
aquaria 2 and 3 in order to rule out possible effects of other variables on
focal fish behaviour.
60

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
Figure 3.1.1: A. Experimental set-up used in experiments 1 and 2; B.
Experimental set-up used in experiment 3.
61

Chapter 3. Understanding Sex-Role Reversal (ex situ)
The ‘relative interest’ (time spent in Area A in front of aquarium 2 or
3 / 1800 sec) was measured for 20 males and 20 females (10 large and 10
small individuals) for each temperature. Different focal individuals were
used in each trial in order to avoid data dependency. Each set of stimulus
fish (one male and one female) was presented to a pair of focal fish
alternatives (once for a male and once for a female), after which they were
not further re-used. See also the Statistical analysis section.
Experiment 2
Mate preferences for body size were analysed through a protocol similar to
that of experiment 1. Males and females were simultaneously presented
with two potential mates of different sizes (one large and one small), and
‘relative interest’ displayed by focal fishes was recorded for each
temperature. Twenty focal males and 20 focal females (10 large and 10
small individuals) were used. As in experiment 1 the stimuli fishes were
randomly allocated to aquaria 2 and 3. Different focal individuals were
used in each trial in order to avoid data dependency. Each set of stimulus
fish (one large and one small) was presented to a pair of focal fish
alternatives (once for a large and once for a small individual), after which
they were not further re-used. See also the Statistical analysis section.
Experiment 3
From the analysis of data collected in experiments 1 and 2, experiment 3
was planned in order to study the ‘relative interest’ of focal females on
other females with different body sizes (small and large). Experimental set-
up was composed of two aquaria (Figure 3.1.1.B) with one focal female
(Aquarium 1) separated by a one way mirror from a stimulus female
(Aquarium 2). Aquarium 1 was also divided in two areas, one adjacent to
the mirror (area A) and one ‘neutral zone’ (area B). Large and small focal
females (20 large females and 20 small females, for each temperature)
were presented with stimuli females of two size classes (large and small)
and ‘relative interest’ was measured (suggesting female-female
competition). In this experiment, each focal female was exposed only to a
single stimulus female, either a large or a small one. Different focal
62

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
females were used in each trial in order to avoid data dependency. Each
stimulus female was presented to a pair of focal fish alternatives (once for
a large and once for a small individual), after which they were not further
re-used. See also the Statistical analysis section.
Statistical analysis
All statistical analyses were performed using STATISTICA 7.0 (Statsoft).
Data from the three experiments were tested for deviance from normality
and for the homogeneity of variance. Assumptions were met in experiment
2 but no homogeneity of variance was found in experiment 1 and 3
(Levene test; P0.05; experiment 2:
R=0.0140, P>0.05), parametric statistics were used throughout the entire
study.
Experiment 1: orthogonal ANOVA with four factors; temperature
(three levels=15ºC, 18ºC and 24ºC), sex of focal fish (two levels=male and
female), size of focal fish (two levels=large and small) and sex of stimulus
fish (two levels=male and female).
Experiment 2: orthogonal ANOVA with four factors; temperature
(three levels=15ºC, 18ºC and 24ºC), sex of focal fish (two levels=male and
female), size of focal fish (two levels=large and small) and size of stimulus
fish (two levels= large and small).
Experiment 3: orthogonal ANOVA with three factors: temperature
(three levels=15ºC, 18ºC and 24ºC), size of focal female (two levels=large
and small) and size of stimulus female (two levels=large and small).
Post-hoc comparisons were conducted using Newman-Keuls test.
All probabilities are two-tailed and a significance level of 0.05 was used.
63

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Ethical note
All the focal and stimuli fishes were returned to stock tanks after the
experiments and no signs of disturbance or stress could be detected. No
fish died or became sick and no manipulations were used in the study
apart from that needed to transfer the fish from one tank to another with a
small hand net. The study was conducted in agreement with the pertinent
European and Portuguese legislation on animal welfare.
3.1.4 Results
Experiment 1
A significant interaction was found between temperature, sex of the focal
fish and sex of the stimulus fish (See Table 3.1.1). At higher temperatures
(24ºC), both males and females spent more time in front of the opposite
sex (Figure 3.1.2.C,D), while at 15ºC no significant differences were
observed (Figure 3.1.2.A,D). At 18ºC, a distinctive pattern was observed,
with males spending more time in the vicinity of the opposite sex and
females spending an approximately equal amount of time in the vicinity of
both consexuals and non-consexuals (Figure 3.1.2.B,D). In males, ‘relative
interest’ in females increased as soon as water temperature was raised to
18ºC (temperature that mimics the onset of the breeding season) while in
females a clear interest on males was only visible at 24ºC.
Since, at least at higher temperatures, both males and females
seemed to effectively discriminate among sexes (Figure 3.1.2), experiment
2 and 3 were conducted.
Experiment 2
A significant interaction was found between temperature and size of the
stimulus fish (See Table 3.1.2). At lower temperatures (15ºC), no
significant differences were found between the ‘relative interest’ aroused
by either size class. However, as temperatures increased, larger stimuli
fishes obtained more attention from focal individuals. No differences
between temperatures were found for small fishes (see Figure 3.1.3.A,B).
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3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
Furthermore, independently of temperature, males spent more time near
the stimuli fishes than females (see Table 3.1.2).
Table 3.1.1: ANOVA results on the ‘relative interest’ of males and females
of different size classes towards consexuals and non-consexuals, at three
different temperatures (15, 18 and 24ºC) (experiment 1)
Source Df MS F P
Temperature (Temp) 2 0.48 5.33 0.006
Sex of the focal fish (Sex_F) 1 0.16 1.77 0.187
Size of the focal fish (Size_F) 1 0.01 0.06 0.811
Sex of the stimulus fish (Sex_St) 1 0.44 4.84 0.030
Temp * Sex_F 2 0.19 2.12 0.125
Temp * Size_F 2 0.04 0.48 0.621
Sex_F * Size_F 1 0.12 1.39 0.242
Temp * Sex_St 2 0.63 7.00 0.001
Sex_F * Sex_St 1 3.94 43.75 0.000
Size_F* Sex_St 1 0.02 0.20 0.660
Temp * Sex_F * Size_F 2 0.20 2.20 0.116
Temp * Sex_F * Sex_St 2 0.63 6.70 0.001
Temp * Size_F* Sex_St 2 0.01 0.07 0.928
Sex_F * Size_F* Sex_St 1 0.01 0.16 0.692
Temp * Sex_F * Size_F* Sex_St 2 0.16 1.82 0.167
Error 96 0.09
Experiment 3
Three significant interactions were found (see Table 3.1.3):
(1) Temperature and size of the focal females: at 15ºC, no
significant differences were found between different-sized focal females’
responsiveness to stimuli females. On the other hand, at 18ºC, large
females spent significantly more time in Area A than small females. At high
temperatures (24ºC) an inverted pattern was observed, with small females
spending significantly more time in Area A than large females (Figure
3.1.4.A,B).
65

Chapter 3. Understanding Sex-Role Reversal (ex situ)
(2) Temperature and size of the stimuli females: at low
temperatures (15ºC) large females attracted significantly more attention
than small females. No significant differences were found either at 18ºC or
at 24ºC (Figure 3.1.4.C,D).
(3) Size of the focal females and size of the stimulus females:
large females concentrated their attention on females of equivalent size
while small females show no distinct preference (Figure 3.1.4.E,F).
Figure 3.1.2: ‘Relative interest’ of males and females of different size
classes towards consexuals and non-consexuals, at three different
temperatures (A-15ºC; B-18ºC; C-24ºC) and Newman-Keuls test results
(D), with gray squares representing significant differences. The numbers
15, 18, 24 represent the tested temperatures and the two initials (with F for
females and M for males) represent the focal and the stimulus fish,
respectively. Error bars represent standard deviations.
66

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
Table 3.1.2: ANOVA results on the ‘relative interest’ of males and females
of different size classes towards large and small potential mates, at three
different temperatures (15, 18 and 24ºC) (experiment 2).
Source Df MS F P
Temperature (Temp) 2 0.52 5.98 0.004
Sex of the focal fish (Sex_F) 1 0.54 6.16 0.015
Size of the focal fish (Size_F) 1 0.05 0.58 0.450
Size of the stimulus fish (Sex_St) 1 2.91 33.27

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Figure 3.1.3: ‘Relative interest’ towards stimulus fish of different sizes (A)
at three different temperatures (15, 18 and 24ºC) and Newman-Keuls test
results, with gray squares representing significant differences (B). The
numbers 15, 18, 24 represent the tested temperatures and the two initials
(with L for large and S for small) represent the size of the stimulus fish,
respectively. Error bars represent standard deviations.
68

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
Figure 3.1.4
69

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Figure 3.1.4 (previous page): ‘Relative interest’ of females of different
size classes towards stimuli females (A), at three different temperatures
(15, 18 and 24ºC). ‘Relative interest’ aroused by females of different size
classes (C) at three different temperatures (15, 18 and 24ºC). ‘Relative
interest’ of different size females towards large and small consexuals (E).
B, D and F represent Newman-Keuls test results, with gray squares
showing significant differences (for A, C and E, respectively). The numbers
15, 18, 24 represent the tested temperatures and the two initials (with L for
large and S for small) represent the size of the focal and the stimulus
female (for B and D respectively). In F, the two initials represent the size of
the focal and the stimulus female. Error bars represent standard
deviations.
3.1.5 Discussion
Mating discrimination is the ability of individuals of one sex to identify,
assess and selectively mate with conspecific members of the opposite sex
(Andersson 1994; Pfenning 1998). Attempts to quantify mating
preferences and/or intra-sexual interactions often rely on restrained access
experiments where individuals are presented with different stimuli (Wagner
1998). However, objective interpretations of such experiments cannot be
obtained without confirmation of mate discrimination. Furthermore, the
outcome of such experiments may be modified by environmental factors,
such as temperature (e.g. Kvarnemo 1996).
Besides showing that S. abaster can discriminate potential mates
from fish of the same sex using only visual information (observed fish were
kept inside a transparent sealed tank), the results of experiment 1 also
provide evidence on how a physical factor, water temperature, can directly
and differentially modulate the behavioural responses of S. abaster males
and females when exposed to conspecifics (see Figure 3.1.2).
Even though some exceptions can occur (see Kidd et al. 2006),
physical proximity has previously been validated as a reliable indicator of
mate choice (Forsgren, 1992; Kodric-Brown 1993; Berglund 1994;
70

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
Gonçalves & Oliveira 2003; Wong 2004). Curiously, S. abaster males and
females exhibited different response patterns, especially at water
temperatures that mimic the onset of the breeding season. While male
interest in females was expressed at 18ºC, female interest at this
temperature was distributed among both males and females, while at 24ºC
interest towards males was significantly greater than that towards
consexuals. One possible hypothesis to explain theses results would
involve a difference between sexes in the way sexual behaviour and the
underlying motivational mechanisms vary with temperature. It is known
that syngnathid females ready to mate spend a considerable amount of
time interacting with other females (Berglund & Rosenqvist 2003). It is
possible that at 18ºC the mechanisms favouring approaching males and
those involved in female-female interactions compete, so that sexual
motivation is partly masked in females. At 24ºC the motivation to approach
males would become dominant, with females spending less time with
consexuals. In males, which in sex-role reversed syngnathids often have
no need to compete with other males (Berglund & Rosenqvist 2003), the
mechanism could be simpler, basically translated into an increased focus
on females. This difference between male and female response to
temperature makes sense in the environmental conditions where S.
abaster lives. In fact, this species breeds during summer, with
temperatures increasing rapidly to a peak late in the season and dropping
visibly in the beginning of autumn, when reproduction no longer takes
place (K. Silva personal observation). In this context, females that still
have eggs to spawn by the end of the breeding season would likely gain
by minimising time spent in interactions with other females and
concentrating their efforts in courting males. Alternatively, it is possible that
a mating hierarchy is enforced, especially during an initial phase of the
breeding season, thus explaining the later decrease in interest towards
other females.
As in other pipefish species (Berglund & Rosenqvist 2003), body
size proved to be an important trait in S. abaster. According to the results
of experiment 2, at 18ºC and 24ºC (temperatures at which reproduction
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
occurs) both males and females spent more time near larger potential
partners, as opposed to what happened at 15ºC, where no preferences
were observed. Furthermore, as temperature increased, larger individuals
tended to obtain more attention from the focal fish, independently of the
size and sex of the latter. By discriminating against smaller partners,
individuals could benefit in several ways. In S. typhle, larger size means
higher quality in terms of reproductive return for both males and females,
since both sexes accrue direct advantages from mating with larger
individuals (Jones et al. 2000; Berglund et al. 2005).
Results of experiment 2 also showed that males spent significantly
more time in the vicinity of females than females in the vicinity of males
(see Table 3.1.2). Intuitively, in a sex role reversed species, as seems to
be the case of S. abaster (Silva et al. 2006), one would expect females to
be more active than males in approaching the opposite sex. However, it is
important to distinguish between situations in which females have difficulty
finding a male ready to receive eggs and those in which the males have
their brood pouch empty like in experiment 2. In this case males may
express a courting motivation that would be masked in experimental
designs aimed to emphasize female competition. In fact, Silva et al. (2006)
showed that S. abaster males have an active role in mating, approaching
and courting females.
Exhibiting a preference and actually succeeding in pursuing the
mate choice can be two very different things. Individual behaviours may be
governed by factors beyond the individual’s control, such as competition
from others (Berglund et al. 2005). In sex role reversed syngnathids,
where males are generally viewed as a limiting factor and no conspicuous
agonistic interactions occur, females should assess their attractiveness
relative to other females in order to establish a hypothetical status that
reliably translate their intrinsic quality. If this is the case, then the still
poorly understood female-female interactions, that vary considerably
among syngnathid genera, may play an important role in the competition
for access to mates. In N. ophidion and S. typhle, female’s competition for
males is mainly indirect, through dominance hierarchies, with larger
females dominating smaller ones by sexual signalling, namely a more
72

3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
contrasted coloration in the trunk (an amplifier of a previous quality signal
such as body size, which is also correlated with fecundity; Berglund &
Rosenqvist, 2003). Moreover, as predicted in a model of mutual mate
choice as a dynamic game, proposed by Johnstone (1997), which yielded
predictions about mating behaviour under the influence of time constraints,
choice costs and competition for mates, when all individuals are present
from the start of the breeding season, the correlation between the qualities
of individuals pairing at a given time declines throughout the season, so
that mates are more closely matched among individuals who pair early
than among those who pair late. The mean quality of unmated males and
females declines over time, because more attractive individuals tend to
mate sooner. If this is so, then it is possible that in S. abaster the chance
for small females to reproduce appears only near the end of the
reproductive season, when high quality (larger) females have already
mated. A reproductive opportunity for smaller females may also arise from
the fact that at higher temperatures sex roles may be more equal,
decreasing competition between females. For example, in S. typhle, higher
temperatures tend to decrease differences in the potential reproductive
rates of males and females (Ahnesjo, 1995).
Different-sized females may thus adopt distinct reproductive
tactics during the reproductive season, with large dominant females
probably imposing reproduction constraints on small females. An indication
of the disruption of the ‘normal pattern’ (larger females showing more
interest and spending more time near stimulus fish) can be viewed at 24ºC
(end of the breeding season), where smaller females showed a
significantly greater ‘interest’ than larger females. At high latitudes, where
the breeding season tends to be shortened to a few months per year, less
attractive females of a sexual reversed syngnathid species, S. typhle,
reallocate their resources from present to future reproduction (Berglund
1991). Nevertheless, in warmer waters where the breeding season is
considerably longer, such as that of S. abaster in Portugal (K. Silva
unpublished data), inferior quality (smaller) females may be able to avoid
postponing reproduction to the next breeding season since males might
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
still be able to receive eggs near the end of the breeding season (see also
Jones et al. 1999) In N. lumbriciformis, the number of eggs per breeding
male was found to be significantly higher during the last months of the
breeding period when compared to the onset and middle of the
reproductive season. Monteiro et al. (2006) interpreted this result as an
alteration of the egg laying strategy, suggesting that in this cryptic pipefish
there also seems to be a modification of some reproductive parameters
occurring at the end of the breeding season.
There are, however, alternative hypothesis that would explain
the same pattern. Regardless of sex role reversal, in fishes, female
fecundity tends to increase with size (e.g. Berglund et al. 1986; Kraak &
Bakker 1998; Herdman et al. 2004). If the breeding season is also the
main time window when fish can feed and grow, small females may be
forced to allocate a fraction of the reproductive season to growth, limiting
spawning to a shorter period before breeding ceases. It is important to
note, however, that the two hypotheses are not mutually exclusive: it might
well be that competition by larger females and the need to devote part of
the breeding season to growth both favours the alteration of the breeding
interval in smaller individuals.
Besides contributing to a better understanding of potential sources
of variability in mating behaviour, studying the effect of temperature on
syngnathid behaviour and reproduction might be of great importance in a
time when the prospect of global warming is of increasing concern
(Walther et al. 2002).
3.1.6 Acknowledgements
We would like to thank everybody that helped during the laboratorial work,
especially Pedro Correia and Armando Jorge, as well as Jorge Salgueiro
for the review of the manuscript. We also thank Prof Anders Berglund and
one anonymous referee for their suggestions and criticism that greatly
improved the manuscript. Professor Vitor Almada’s participation was
partially funded by Programa Plurianual de Apoio às Unidades de
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3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
Investigação. Nuno Monteiro's participation was funded by Fundação para
a Ciência e a Tecnologia (FCT-SFRH/BPD/14992/2004) and Programa
Plurianual de Apoio às Unidades de Investigação. Karine Silva's
participation was funded by Fundação para a Ciência e a Tecnologia
(FCT-SFRH/BD/13171/2003). This work was partially funded by FCT
(POCI/MAR/60895/2004).
3.1.7 References
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Monteiro, N. M., Vieira, N. M. & Almada, V. C. (2002). The courtship
behaviour of the pipefish Nerophis lumbriciformis: reflections of and
adaptation to intertidal life. Acta ethologica, 4, 109-111.
Monteiro, N. M., Berglund, A., Vieira, M. N. & Almada, V. C. (2006).
Reproductive migrations of the sex role reversed pipefish Nerophis
lumbriciformis (Pisces; Syngnathidae). Journal of Fish Biology, 69, 66-74.
Pfennig, K. S. (1998). The evolution of mate choice and the potential for
conflict between species and mate-quality recognition. Proceedings of the
Royal Society of London B, 265,1743–1748.
Puurtinen, M., Ketola, T. & Kotiaho, J. S. (2005). Genetic compatibility and
sexual selection. Trends in Ecology and Evolution, 20, 157-158.
Reynolds, J. D. & Jones, J. C. (1999). Female preference for preferred
males is reversed under low oxygen conditions in the common goby
(Pomatoschistus microps). Behavioral Ecology, 10, 149-154.
Rick, I. P., Modarressie, R. & Bakker, T. C. (2006). M. UV-wavelengths
affect female mate choice in three-spined sticklebacks. Animal Behaviour,
71, 307-313.
Silva, K., Monteiro, N. M., Vieira, M. N. & Almada, V. C. (2006).
Reproductive behaviour of the black-striped pipefish, Syngnathus abaster
(Pisces; Syngnathidae). Journal of Fish Biology, 69, 1860-1869.
Wagner, W. E. (1998). Measuring female mating preferences. Animal
Behaviour, 55, 1029-1042.
Walther, G. R., Post, E., Convey, P., Menzel, A., Parmesan, C., Beebee,
T. J. C., Fromentin, J. M., Hoegh-Guilberg, O. & Bairlein, F. (2002).
Ecological responses to recent climate change. Nature, 416, 389–395.
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3.1. The effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster
Wilson, R. S. (2005). Temperature influences the coercive mating and
swimming performance of male eastern mosquitofish. Animal Behaviour,
70, 1387-1394.
Wong, B. B. M. (2004). Superior fighters make mediocre fathers in the
Pacific blue-eyed fish. Animal Behaviour, 67, 583–590.
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
Chapter 3.2
Reversing sex-role reversal:
compete only when you must
3.2.1 Abstract
The operational sex-ratio (OSR: ratio of females producing fertilizable eggs
to sexually active males, at a given time) is thought to be a major factor
influencing the intensity of mating competition and sexual selection.
Generally, as females become a limiting resource, the surplus of “ready to
mate” males intensifies male-male competition. Contrastingly, a surplus of
receptive females usually promotes female-female competition, sometimes
resulting in a reversion of sex roles.
The present work tested for a direct influence of tertiary sex-ratios
(as a direct estimation of the OSR) on the reproductive behaviour and
reproductive success of a sex role reversed species, the black-striped
pipefish Syngnathus abaster. Interestingly, males and females exhibited
different response patterns to the three tested sex-ratios (even sex-ratio,
80

3.2. Reversing sex-role reversal: compete only when you must
male biased sex-ratio and female biased sex-ratio). While, a surplus of
males ‘un-reversed’ the sex role reversal observed when both sexes
coexist in similar numbers, an excess of females did not boost
female-female competition. Additionally, different-sized females were
observed to adopt distinct competitive responses. As the proportion of
males decreased, large dominant females, invested less in competitive
interactions than could be expected given the low number of available
mates. Small females on the other hand, were more prone to compete.
Also choosiness related to sex-ratios as the sex in shortage was
more choosy. Nevertheless, results evidenced a size-assortative mating
pattern in all tested sex-ratios, suggesting that only large individual could
afford to be choosy.
3.2.2 Introduction
The operational sex-ratio (OSR: ratio of females producing fertilizable eggs
to sexually active males, at a given time and place) is thought to be of
fundamental importance in predicting the direction of sexual selection
(Emlen & Oring, 1977; Clutton-Brock & Parker, 1992; Kokko & Monaghan,
2001). Theoretical models assume that whenever the OSR deviates from
equality, the sex occurring in “excess” will likely compete more intensely
for access to mates while the sex occurring in “shortage” will be choosier
with respect to potential mates (Emlen & Oring, 1977). Although factors,
such as variation in mate quality and sex differences in mortality patterns
(Kokko & Monaghan, 2001), may considerably complicate this process,
many studies using the overall adult sex-ratio as a direct estimation of the
OSR, have confirmed that, in many taxa, as females become a limiting
resource, the surplus of “ready to mate” males intensifies male-male
competition [e.g. spider mite, Tetranychus urticae, Enders (1993);
Japanese medaka, Oryzias latipes (Grant, 1995); field cricket, Gryllus
pennsylvanicus, (Souroukis & Cade, 1993)]. Conversely, a surplus of
reproductively active females has been proved to promote female-female
competition for access to males, occasionally even resulting in reversed
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
sex roles (sensu Vincent et al., 1992). In the sand goby, Pomatoschistus
minutus, for example, females increased their competitive interactions
under a female biased sex-ratio even though males remained the
predominant competitors (Kvarnemo et al., 1995). In the two-spotted goby,
Gobiusculus flavescens, however, a skew in the sex-ratio towards females
resulted in sex role reversal, with male-male competition and intensive
male courtship behaviour being superseded by female-female competition
and actively courting females (Forsgren et al., 2004).
It could be predicted that sex role reversed species, where
females are the predominant competitors for mates, would exhibit a
mirror-like response pattern to variations in the OSR. A surplus of “ready to
mate” males would promote male-male competition, reversing sex role
reversal, while an excess of receptive females would increase female-
female competition for access to males, thus intensifying sex role reversal.
The family Syngnathidae (comprising pipefishes, seahorses and
seadragons) with its varying degrees of sex-role reversal (Monteiro et al.,
2002; Berglund & Rosenqvist, 2003; Silva et al., 2006) where females
compete for access to mates and sometimes present conspicuous
secondary sexual characters, provides an unique opportunity to test the
accuracy of OSR-based estimates of mating competition and sexual
selection. Such potential remains yet to be fully explored, with very few
studies focusing on the modulation of syngnathid reproductive behaviour
by OSR (e.g. Berglund, 1994; Vincent, 1994; Vincent et al, 1994).
The present work tests for a direct influence of tertiary sex-ratios
(ratio of mature individuals) on the mating behaviour of Syngnathus
abaster (Risso, 1826), a sex role reversed pipefish according to Silva et al.
(2006). Intra and inter-sexual interactions were investigated under an even
(4♀:4♂) and two biased sex-ratios (6♀:2♂; 2♀:6♂). Additionally, mating
competition was directly assessed through the frequency of disruptions
causing the end of ongoing courtship rituals. Four major questions were
addressed: i) Can sex-ratios per se affect courtship and mating
competition in S. abaster? ii) Do males and females exhibit similar
response patterns to the tested sex-ratios? iii) Does body size, a trait that
82

3.2. Reversing sex-role reversal: compete only when you must
is implicated in mate choice in this species (Silva et al., 2007), influence
behavioural strategies? iv) How does choosiness relate to OSR?
3.2.3 Methods
S. abaster is a euryhaline species with a restricted distribution that
includes the Mediterranean, the Black Sea, and the Atlantic coast of
Southwest Europe up to southern Biscay (Dawson 1986). This pipefish
occurs either in coastal areas or in brackish and fresh waters (Cakic et al.,
2002), and can be found mainly among sand, mud or eelgrass meadows,
at depths between 0.5 and 5 m, within a temperature range of 8ºC to 24ºC.
Pipefishes were collected with a hand net, in a salt pond reservoir,
at the Ria de Aveiro estuarine lagoon (40º45’N, 8º40’W), in Portugal. Fish
were transported to the laboratory, where they were maintained in 250 litre
aquaria, illuminated by natural light supplemented with 18 W fluorescent
lamps. Tank substrata consisted mainly of sand and plastic seagrass laid
in order to mimic the original habitat. The continuously running seawater
was physically and biologically filtered and its temperature kept constant at
18-19ºC, a temperature at which S. abaster breeds continuously (Silva et
al., 2007). Fish were fed daily with fresh Artemia franciscana nauplii. In
order to maximize the chance of observing matings in aquaria, mature,
non pregnant, males and females were kept in separate tanks for 3 weeks
prior to the beginning of the experiment.
The experiment involved an even (4♀:4♂) and two biased sex-
ratios (6♀:2♂; 2♀:6♂) where an equal number of large and small
individuals was used. Since only reproductively mature individuals were
used, manipulation of the sex-ratio resulted in a direct change in the
operational sex-ratio.
Size cut-offs for ‘large’ and ‘small’ individuals were defined
according to Silva et al. (2007), as ½ standard deviation below and above
the mean size (total length) for each sex (♀:mean=9.4 cm, sd=1.38 cm;
♂:mean=8.5 cm, sd=1.26 cm, values obtained from prior measurements in
the same population). Large males and females were longer than 9.1 cm
83

Chapter 3. Understanding Sex-Role Reversal (ex situ)
and 10.1 cm, respectively. Small males and females were shorter than
7.9 cm and 8.7 cm respectively.
Individuals were randomly assigned to either one of the three sets
of experimental tanks and were left to acclimatize for one day before the
beginning of the observation period. Ten groups of each sex-ratio
treatment were conducted and, thus, a total of 120 males and 120 females
were needed. Due to the large number of individuals required to complete
all three treatments, and given the inability to capture additional pipefish,
some fish were used twice (35 non-pregnant males and 21 females) but
care was taken in order to allow for a “resting period” of at least one
month. In the case of females, this period allows for the production of new
eggs ready to be spawned (K. Silva, unpublished data). In the case of
males, care was taken so as to re-use only non-pregnant males even
though aquarium kept S. abaster males were observed receiving new egg
clutches only one hour after the release of juveniles. Some of the re-used
males received eggs, which indicates that they were not physiologically
unable to reproduce.
Four easily distinguishable behaviours, occurring during the
courtship and mating rituals of many syngnathid species (Berglund et al.,
1986; Watanabe et al., 2000; Matsumoto & Yanagisawa, 2001; Monteiro et
al., 2002), were selected: Approach, Parallel-swimming, Flickering and
Lateral display (see Table 3.2.1 for additional descriptions). These
behaviours were then assigned to four different interaction categories
[male to male (M→M), female to female (F→F), male to female (M→F),
and female to male (F→M)]. To avoid pseudo-replication, data from each
individual were only used once per category. As an example, in the ten
conducted male biased sex-ratio replicates (with three large and three
small males), a total of fifteen observations were recorded for large males
interacting with males and another fifteen observations for interactions with
females.
Each fish was observed continuously during 10 min periods over 5
consecutive days (50 min overall observation per fish) and the frequency
of each behaviour was recorded. The extension of the observation period
over a few days seemed particularly important in the case of the female
84

3.2. Reversing sex-role reversal: compete only when you must
biased treatment where males could be expected to be more choosy, thus
requiring more time to copulate (see Berglund, 1994). Also, the selected
extent of the observation period would maximize the probability of
observing all the selected behaviours, simultaneously diluting any possible
dependence in their expression (e.g. if a particular behaviour tends to
occur first and/or is more frequent, a short observation period might not
allow for the detection of other less frequent or late occurring behaviours).
Notwithstanding, exaggerated observation periods were avoided in order
to prevent a full occupation of the male’s pouch, a situation that would
modify the number of active males and thus the operational sex-ratio
within a tank.
Given the different numbers of males and females present in the
tested sex-ratios, which imply distinct probabilities of the observed
interactions to occur, the observed frequencies were corrected
accordingly. The observed behaviour frequencies per individual were
divided by the proportion off all individuals of the relevant sex that could be
encountered. For example, 782 male-male interactions in the male biased
treatment were corrected to 1095 [782/(5/7)].
Additionally, the number of successful disruptions of ongoing
courtship rituals [interpreted as a form of intra-sexual competition,
according to Silva et al. (2006)] was estimated for both large and small
males and females. During the observation period, each mating event was
recorded along with the size class of the involved individuals.
Statistical analysis
The mean frequencies of all selected behaviours for all possible interaction
categories were used to create a similarity matrix (fourth root
transformation, similarity measure: Euclidian distance). A Cluster analysis,
using the computer software package PRIMER 5 (Plymouth Routines in
Multivariate Ecological Research), was then conducted (Cluster mode:
Group average) in order to observe a possible relationship between the
tested sex-ratios and the types of interactions (intra and inter-sexual)
85

Chapter 3. Understanding Sex-Role Reversal (ex situ)
performed by each sex. If some of the tested sex-ratios intensified or
depressed the amount of interactions, visible clusters would be formed
helping to understand the underlying dynamics of the observed process. It
could be predicted that female to female interactions would reach the
highest values in the female biased sex-ratios. It could also be predicted
that male to male interactions, in the male biased sex-ratio, would attain
also high values, thus forming a cluster. Furthermore, this approach will
allow for a ‘global ordination’ of the intra- and inter-sexual interactions, for
all tested sex-ratios (e.g. in the female biased sex-ratio, will the amount of
interactions performed by a female be greater toward males or females?
And how do these values relate to male performed interactions in the male
biased sex-ratio?).
Four Multiple Analyses of Variance (MANOVAs), one for each
selected behaviour (approach, parallel swimming, flicker and lateral
display), were conducted to test for possible effects of sex-ratio, sex and
body size on the frequencies of intra and inter-sexual interactions. In order
to obtain homogeneity of variances data were log transformed in the case
of parallel swimming and flickering (intra-sexual interactions) and flickering
(inter-sexual interactions). Lateral display data (inter-sexual interactions)
were square-root transformed. Even though the homogeneity of variances
assumption was not met for approach and lateral display, solely for
intra-sexual interactions, it was decided to continue with the analysis given
the robustness of the F statistic (Lindman, 1974).
Successful disruptions caused by males or females were analysed
with ACTUS (Analysis of Contingency Tables Using Simulation; Estabrook
& Estabrook, 1989). For each sex, a potential association of body size and
sex-ratio treatments was assessed. Simultaneously, using an observed vs.
expected χ 2 analysis (with Bonferroni adjustments), the differences
between disruptions among large and small individuals were also
assessed within each sex-ratio treatment.
A possible association between sex-ratios and mating preferences
was also assessed with ACTUS. The observed number of size-assortative
and non size-assortative couples was scored for each treatment.
Additionally, within the observed non size-assortative mating couples
86

3.2. Reversing sex-role reversal: compete only when you must
formed during the experiment (large males with small females or small
males with large females), a possible association between the sex-ratio
treatment and the size combination between males and females was
assessed.
Finally an orthogonal ANOVA was conducted to determine
whether sex-ratio and male size affected the time elapsed until male’s first
pregnancy [here interpreted as an indirect measurement of male
choosiness, as observed by Berglund (1994)].
Post-hoc comparisons for both ANOVA and MANOVA were
conducted using Newman-Keuls test. All probabilities are two-tailed and a
significance level of 0.05 was used. Apart from otherwise stated, statistical
analyses were performed using Statistica 6.1 (Statsoft).
Table 3.2.1: Recorded behaviours and corresponding descriptions
(according to Silva et al., 2006).
Behaviour Description
Approach Moving towards another fish.
Parallelswimming
Flickering
Lateral display
Two fish swimming through the aquarium in a more or
less parallel position.
Rapid and vigorous bends moving along the main axis of
the body displayed towards another fish.
Temporary colour ornament displayed in a motionless
and parallel position towards another fish.
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
3.2.4 Results
Cluster analysis
The dendrogram resulting from the cluster analysis identified two major
clusters, A and B (Figure 3.2.1). Cluster A, grouped the lowest observed
frequencies, consisting of intra-sexual interactions of males in the equal
and female biased sex-ratios (IME and IMF), together with the female
intra-sexual interactions in the male biased sex-ratio (IFM). Cluster B was
subdivided into group C, including inter-sexual interactions of males in the
female biased sex-ratio (XMF) and inter-sexual interactions of females in
the male biased sex-ratio (XFM), and group D. Within group D, the highest
observed frequencies were gathered in cluster E, comprising intra-sexual
interactions of females in the equal and female-biased sex-ratios (IFE and
IFF) together with intra-sexual interactions of males in the male biased
sex-ratios (IMM). Intermediate values were grouped in cluster F. Even
though the selected behaviours are usually interpreted as part of the
courtship ritual, it seems important to highlight that the highest frequencies
were observed in intra-sexual interactions.
MANOVAs
The MANOVA analyses showed a significant interaction between the
tested sex-ratios, sex and focal fish size (Table 3.2.2) for all considered
behaviours.
Intra-sexual interactions
In the equal sex-ratio treatment, female-female interactions were more
common than male-male interactions (Figure 3.2.2). While females
displayed all behaviour categories towards other females, males were only
seen approaching other males. In female-female interactions, large
females interacted more with other females than smaller ones.
88

3.2. Reversing sex-role reversal: compete only when you must
Figure 3.2.1: Cluster analyses based on a similarity matrix constructed on
the mean frequencies of all selected behaviours (side table; AP=approach,
PS=parallel swimming, FL=flickering, LD=lateral display) according to the
type of interaction (I=intra- and X=inter-sexual interaction) and sex of the
fish (M=male and F=female) and sex-ratio treatment (E=equal,
M=male-biased and F=female-biased). A detailed description of the
signalled sub-clusters can be obtained in the results section.
Contrastingly, no differences were observed between large and small
males.
Biased sex-ratios induced different response patterns on male and
female intra-sexual displays. A male biased sex-ratio resulted in a
pronounced increase in the number of male-male interactions, when
compared to the equal sex-ratio treatment, with males displaying all
selected behaviours. In contrast with the equal sex-ratio treatment, large
and small males significantly differed in activity levels. Large individuals
were more interactive than small ones when considering all behaviours
except lateral display (where no differences were observed). It seems
important to highlight the fact that males exhibited the contrasted stripes
ornament (Figure 3.2.2, Lateral display), typically described only for
89

Chapter 3. Understanding Sex-Role Reversal (ex situ)
females or for males interacting with females (Berglund & Rosenqvist,
2003). Within females, the excess of males resulted in a decrease in
intra-sexual interactions when compared to the equal treatment. No female
was observed swimming parallely or flickering towards other females.
Additionally, small females never displayed the ornament. The behaviours
exhibited by females (approach and ornament display) were significantly
affected by size, with large females being more active than small ones.
Interestingly, an excess of females towards males did not intensify
female-female interactions. Large females displayed less than in the even
treatment (significant differences for approach, parallel swimming and
lateral display). Small females, on the other hand, showed no decrease in
activity, flickering even more often. Still, large females remained more
active than smaller ones (except in approach, where no differences were
observed). Within males, interactions were reduced to approach as in the
equal treatment, and no differences in the level of activity were found
between large and small individuals.
Inter-sexual interactions
The extremely low level of parallel swimming interactions (Figure 3.2.2), in
both sexes and sizes, indicates that this behaviour is primarily expressed
towards the same sex.
In the equal sex-ratio treatment, contrarily to the same-sex
interactions that were almost exclusive to females, both sexes displayed
all selected behaviours towards the opposite sex with the exception of
ornament display in males. Body size affected the level of flickering and
lateral display interactions in females with larger individuals interacting
more than small ones.
The male biased sex-ratio had distinct effects on the way different
sized males interacted with females (Figure 3.2.2). While small individuals
maintained the same level of interactions observed in the equal sex-ratio
treatment, large males increased the frequency of approach and lateral
display interactions. Within females, the more pronounced behavioural
modification when compared to the equal sex-ratio treatment was the
absence of flickering towards males. Additionally, ornament display was
90

3.2. Reversing sex-role reversal: compete only when you must
significantly reduced. Female size only affected the frequency of
approaches, with large females approaching males more often than small
ones.
The female biased sex-ratio had a more subtle effect on
inter-sexual interactions leading only to a decrease in the frequency of
approaches and flickering displayed by large males and the frequency of
approaches by small males. No further differences were detected when
compared to the equal sex-ratio treatment. Within females, size affected
the frequency of approaches and ornament display. Within males, small
males approached females less often than large ones.
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
Table 3.2.2: MANOVAs results on male and female interactions in the
form of approach, parallel-swimming, flickering and lateral display. (A)
Multivariate Tests of Significance and (B) Univariate results are presented.
Dependent variables included intra and inter-sexual interactions. Analyses
were performed independently for each of the selected behaviours.
A Multivariate Tests of Significance
Effect Wilks λ F Effect DF Error DF P
Approach
Sex-ratio 0.60 15482.00 4 214

3.2. Reversing sex-role reversal: compete only when you must
B Univariate results for each dependent variable
Intra-sexual interactions Inter-sexual interactions
Effect DF MS F P MS F P
Approach
Sex-ratio 2 229374 29.047

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Figure 3.2.2
94

3.2. Reversing sex-role reversal: compete only when you must
Figure 3.2.2 (previous page): Mean frequencies of all four selected
behaviours for male and female inter- and intra-sexual interactions in each
sex ratio treatment (E=equal, M=male-biased and F=female-biased).
Newman-Keuls test results are presented with gray squares indicating
significant differences. Error bars represent standard deviations.
Courtship disruptions
The ACTUS analysis on male successful pair disruptions did not
significantly differ from what would be expected if there was no
dependence among the variables male size and sex-ratio treatment
(χ 2 =1.098, DF=5, P=0.471; Figure 3.2.3). Nevertheless, under a
male-biased sex-ratio, large males were more successful in disrupting
courting pairs (χ 2 =27.962, DF=1, P

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Figure 3.2.3: Observed number of male and female successful mating
disruptions in the three selected treatments (equal, male and female
biased sex-ratio).
Mating pairs
Size-assortative mating pairs were always more frequent than pairs
formed by individuals of contrasting size classes (Figure 3.2.4),
independently of sex-ratio treatment (ACTUS; χ 2 = 1.551, DF= 5, P= 0.481;
Equal sex-ratio= 33 to 10 pairs; Female biased sex-ratio= 16 to 4 pairs;
Male biased sex-ratio= 17 to 9 pairs). When considering only the less
common non size-assortative pairs, a significant association was observed
between sex-ratio treatment and the relative size combination of males
and females (ACTUS; χ 2 = 13.081, DF= 5, P < 0.001). In the female biased
sex-ratio, where males are scarce and females more abundant, large
males did not mate with small females. Likewise, in the male biased
sex-ratio, where males are now in excess, large females did not mate with
small males. Interestingly, while small males mated more often with large
females in the female biased sex-ratio, the opposite (small females mating
with large males in the male biased sex-ratio) did not occur more
frequently than expected by chance alone.
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3.2. Reversing sex-role reversal: compete only when you must
Figure 3.2.4: Number of mating pairs observed in the three selected
treatments (equal, male and female biased sex-ratio), described according
to the four possible size class combinations of males and females (L=large
and S=small).
Latency until male’s first pregnancy
Finally, the ANOVA results on the time taken to the first mating,
considering the sex-ratio treatment and male size, revealed a significant
effect of sex-ratio (Table 3.2.3, Figure 3.2.5). No significant interaction with
male size was observed. The time taken until the first spawning event was
significantly higher in the female biased sex-ratio.
Table 3.2.3: ANOVA results on the mean time elapsed until the first
mating event, considering sex-ratio treatment and male size.
Effect DF MS F P
Size 1 0.05 0.04 0.84
Sex ratio 2 11.50 8.65

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Figure 3.2.5: Mean time elapsed until the first spawn in the three selected
treatments (equal, male and female biased sex-ratio). Significant
differences are indicated by an asterisk (*). Error bars represent standard
deviations.
3.2.5 Discussion
There is increasing evidence that mating patterns are, in general, more
plastic than previously thought (e.g. Kvarnemo et al., 1995). The present
study evidenced some of the effects of sex-ratio variations on the
reproductive behaviour of S. abaster males and females. The already
described sex role reversal of this species (Silva et al., 2006) can be easily
observed in the even sex-ratio, where females were clearly more
competitive than males [Figure 3.2.1 and Figure 3.2.2 (Intra-sexual
interactions)], investing intensely in intra-sexual displays probably as a
means to establish a dominance hierarchy based on body size (see also
Silva et al., 2007). In fact, besides engaging more in intra-sexual
interactions, large dominant females were also far more successful than
small ones in disrupting ongoing courtships (Figure 3.2.3). Males, on the
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3.2. Reversing sex-role reversal: compete only when you must
other hand, seemed almost solely interested in females, courting
dynamically [Figure 3.2.1 and Figure 3.2.2 (Inter-sexual interactions)]. As
referred in Silva et al. (2006), this active role in courtship contrasted with
other sex-role reversed pipefishes where males are much more passive
[e.g. Nerophis lumbriciformis (Monteiro et al., 2002)].
Even taking into consideration the sex-role reversal of S. abaster,
at least under an even sex-ratio, it could be expected that a surplus of
males would boost male-male interactions, probably even reversing
sex-role reversal, since females would become the limiting sex. The
presented results seem to corroborate this prediction since the levels of
male intra-sexual interactions significantly rose above female-female
interactions, under a male biased sex-ratio (see Figure 3.2.1).
Interestingly, males were observed displaying the ornament towards other
males, a phenomenon that, at least to the author’s knowledge, has never
been described for syngnathids. Berglund & Rosenqvist (2001) state that
the male’s ornament might have evolved as a correlated response to
selection for ornamentation in females, as males prefer ornamented
females. Nevertheless, male ornament has already been described as a
signalling tool towards females in the sex role reversed pipefish S. typhle
(Berglund et al, 2005). Surprisingly, S. abaster males seem capable of
using the ornament in both contexts, courtship and competition. Since
ornament display was only observed in the male biased sex-ratio
treatment, where males actively compete for female access, it could be
argued that its function is similar to that of female ornament display:
signalling quality towards the opposite sex and potential rivals. As well as
in females (Silva et al., 2007), male size seems also to play a major role in
the outcome of intra-sexual interactions, mainly in the male biased sex-
ratio, where male competition occurs. Larger males interacted more than
small ones (significant differences observed in all considered behaviours
except lateral display) and were also more successful in disrupting mating
pairs. Thus, the male dominance hierarchy seems similar to that of
females, based on size and advertised with the same ornament.
Surprisingly, a shift towards a female biased sex-ratio didn’t
globally intensify female-female competition. Contrarily to what could be
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
expected, large females, usually dominant and prone to intra-sexual
competitive behaviours, interacted even less with other females (with the
exception of flickering where no differences were observed, Figure 3.2.2).
Small females, on the other hand, showed no reduction in the number of
interactions, and flickered more in the female-biased treatment. Since,
when many females were available, males seemed to be “more choosy”
(Figure 3.2.5), preferring larger partners and avoiding mating with smaller
ones (Figure 3.2.4), it could be hypothesised that large (preferred) females
might not need to maintain or increase competitive behaviours, as small
ones do, in order to obtain a mating partner since male choosiness
translates into a higher reproductive confidence. Contrastingly, small
females need to continue taking higher risks in order to obtain a mating
partner (e.g. flickering is a conspicuous behaviour and it’s the only
behaviour that actually significantly rose from the even to the female
biased sex-ratio). The more active behaviour exhibited by small females in
this treatment was also translated into an increased courtship activity.
Game theoretical models predict that the value of winning should
affect how much an individual invests in a contest (Leimar et al., 1991).
Accordingly, Parker (1983) suggested that individuals might vary their
mating tactics according to their relative attractiveness. In S. typhle, small
males, which have a lower competitive ability, courted more and took
greater risks in order to get a partner, than larger, preferred males (Billing
et al. 2007). Also, in the Trinidadian guppy (Poecilia reticulata), attractive
males showed lower levels of courtship as they gained higher reproductive
benefits when compared to less attractive individuals (Reynolds, 1993).
Thus, when exposed to a scarce number of potential mates, S. abaster
large females might opt to decrease competition since temporarily
postponing reproduction and waiting for additional mating opportunities
might not be a high risk strategy given male preference for large partners.
Interestingly, in a parallel experiment, it was observed that large females
laid more eggs when surrounded by a large number of males (K. Silva,
unpublished data). Thus, it seems that the number of available males is an
important variable in the expression of female mating behaviour.
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3.2. Reversing sex-role reversal: compete only when you must
It could be expected that a skewed sex-ratio towards one sex,
besides increasing intra-sexual competition, would also intensify courtship.
Nevertheless, the most drastic alterations were observed in intra-sexual
interactions. This is especially surprising when considering the fact that the
monitored behaviours are usually described as part of the courtship ritual.
Similar findings were obtained in the sand goby, Pomatoschistus minutus,
where behavioural patterns connected to courtship did not change with the
sex-ratio, while competitive interactions were drastically affected
(Kvarnemo, 1996). One possible explanation to this observation might lie
in the fact that intra-sexual interactions may also serve as a cue for mate
choice. Animals obtain information and guide their choice between
potential partners from observing competitive interactions and displays
between them, just as well as from displays directed towards the choosing
individual (Kvarnemo et al., 1995; Berglund & Rosenqvist, 2001). In S.
typhle, for example, males preferred dominant over attractive females,
remembering information from competitive displays and using it, rather
than immediate information from displays (Berglund & Rosenqvist, 2001).
Field observations revealed a size-assortative mating pattern in S.
abaster (Silva et al, 2007), which was also visible in the present aquarium
experiments. Independently of sex-ratio treatment, individuals mated more
often with similar size partners. When only analysing pairs formed by
individuals of contrasting sizes, it became apparent that large individuals
were especially choosier when there was an excess of mating partners.
Small individuals, on the other hand, might not have the opportunity of
being as choosy as large ones do since both sexes have similar
preferences for large partners (Silva et al., 2006). Small females might be
especially limited since large females appear to monopolize male access.
As an example, small males mated significantly more often than could be
expected with large females, in the female biased sex-ratio. In the male
biased sex-ratio, small females did not mate with large males more often
than expected.
Male choosiness was also evidenced in the female biased
sex-ratio where time required until the first egg transfer was significantly
higher. A hypothetical delay in egg transfer could also be expected in the
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
male biased sex-ratio, where females are choosier. Since this delay was
not observed, it seems justifiable to assume that males, more than
females, ultimately control the egg transfer event.
Sex-ratio proved to be an effective variable in the expression of
sex-roles, with a surplus of males being able to ‘un-reverse’ the sex role
reversal observed when both sexes coexist in similar numbers. Indeed,
obtained results showed that S. abaster, males and females, seem able to
actively detect imbalances in the ‘population’ sex-ratio, becoming either
choosier and less competitive towards conspecifics when the opposite sex
is in excess, or more prone to adopt a competitive strategy. Interestingly,
in specific scenarios where hypothetical costs derived from intra-sexual
competition might outweigh the benefits of immediately achieving a
partner, large females seem to count on their ‘sex appeal’, becoming less
competitive towards conspecifics, probably saving resources for future
breeding events.
Altogether, the present results show a highly dynamic system, far
from what could be expected under a binary sex-role definition where
individuals are also usually assumed to be solely competitive or choosy.
The apparently discordant definition on the occurrence of sex-role reversal
in S. schlegeli, reversed according to Watanabe et al. (2000) under a
female biased sex-ratio, and conventional according to Kornienko (2001)
under an even sex-ratio, might just reflect a behavioural plasticity, similar
to that described for S. abaster.
3.2.6 Acknowledgements
We would like to thank everybody that helped during the laboratorial work,
especially Pedro Correia. Professor Vitor Almada’s participation was
partially funded by Programa Plurianual de Apoio às Unidades de
Investigação. Nuno Monteiro's participation was funded by Fundação para
a Ciência e a Tecnologia (FCT-SFRH/BPD/14992/2004) and Programa
Plurianual de Apoio às Unidades de Investigação. Karine Silva's
participation was funded by Fundação para a Ciência e a Tecnologia
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(FCT-SFRH/BD/13171/2003). This work was partially funded by FCT
(POCI/MAR/60895/2004).
3.2.7 References
Berglund, A. (1994). The operational sex-ratio influences choosiness in a
pipefish. Behavioural Ecology 5, 254-258.
Berglund, A. & Rosenqvist G. (2001). Male pipefish prefer dominant over
attractive females. Behavioral Ecology 12, 402-406.
Berglund, A. & Rosenqvist, G. (2003). Sex role reversal in pipefish.
Advances in the Study of Behavior 32, 131-167.
Berglund, A., Rosenqvist, G. & Svensson, I. (1986). Reversed sex roles
and parental energy investment in zygotes of two pipefish (Syngnathidae)
species. Marine Ecology Progress Series 29, 209-215.
Berglund, A., Sandvik, W. M. & Rosenqvist, G. (2005). Sex-role reversal
revisted:choosy females and ornamented, competetive males in a pipefish.
Behavioral Ecology 200, 649-655.
Billing, A. M., Rosenqvist, G. & Berglund, A. (2007). No terminal
investment in pipefish males: only young males exhibit risk-prone courtship
behavior. Behavioral Ecology 18, 535–540.
Cakic, P., Lenhardt, M., Mickovic, D., Sekulic, N. & Budakov, L. J. (2002).
Biometric analysis of Syngnathus abaster populations. Journal of Fish
Biology 60, 1562–1569.
Clutton-Brock, T. H. & Parker, G.A. (1992). Potential reproductive rates
and the operation of sexual selection. Quaterly Review of Biology 67,
437-456.
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Dawson, C. E. (1986). Syngnathidae. In: Fishes of the North-eastern
Atlantic and the Mediterranean (Ed. P. J. P. Whitehead, M. L. Bauchot, J.
C. Hureau, J. Nielsen, & E. Tortonese), pp. 628–639. Paris: Unesco.
Emlen, S.T. & Oring, L.W. (1977). Ecology, sexual selection, and the
evolution of mating systems. Science 197, 215-223.
Endlers, M. M. (1993). The effect of male size and operational sex-ratio on
male mating success in the common spider mite, Tetranychus urticae
Kock (Acari: Tetranychidae). Animal Behaviour 46, 835-846.
Estabrook, C. B. & G. F. Estabrook (1989). ACTUS: a solution to the
problem of small samples in the analysis of two-way contingency tables.
Historical Methods 22: 5–8.
Forsgren, E., Amundsen, T., Borg, A. A. & Bjelvenmark, J. (2004).
Unusually dynamic sex roles in a fish. Nature 429, 551-554.
Grant, J. W. A (1995). Operational sex-ratio, mediated by synchrony of
female arrival, alters the variance of male mating success in Japanese
medaka. Animal behaviour 49, 367-375.
Kokko H. & Monaghan P. (2001). Predicting the direction of sexual
selection. Ecology Letters 4, 159–165.
Kornienko, E. S. (2001). The spawning behaviour of the pipefish
Syngnathus acusimilis. Russian Journal of Marine Biology 27, 54–57
Kvarnemo, C. & Ahnesjo, I. (1996). The dynamics of operational sex-ratios
and competition for mates. Trends in Ecology and Evolution 11, 404–408.
Kvarnemo, C., Forsgren, E. & Magnhagen, C. (1995). Effects of sex-ratio
on intra- and intersexual behaviour in sand gobies. Animal Behaviour 50,
1455–1461.
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Leimar, O., Austad, S. & Enquist, M. (1991). A test of the sequential
assessment game: fighting in the bowl and doily spider Frontinella
pyramitela. Evolution 45, 862–874.
Lindman H. R. (1974). Analysis of Variance in Complex Experimental
Designs. W. H. Freeman & Co, San Francisco, CA.
Matsumoto, K. & Yanagisawa, Y. (2001) Monogamy and sex role reversal
in the pipefish Corythoichthys haematopterus. Animal Behaviour 61,
163-170.
Monteiro, N. M., Vieira, N. M. & Almada, V. C. (2002). The courtship
behaviour of the pipefish Nerophis lumbriciformis: reflections of and
adaptation to intertidal life. Acta ethologica 4, 109-111.
Parker, G. A. (1983). Mate quality and mating decisions. In: Mate Choice
(Ed. P. Bateson ), pp. 141-166. Cambridge University Press, Cambridge.
Reynolds, J. D. (1993). Should attractive individuals court more? Theory
and a test. American Naturalist 141, 914–927.
Silva, K., Monteiro, N. M., Vieira, M. N. & Almada, V. C. (2006).
Reproductive behaviour of the black-striped pipefish, Syngnathus abaster
(Pisces; Syngnathidae). Journal of Fish Biology 69, 1860-1869.
Silva, K., Vieira, M. N., Almada, V. C. & Monteiro, N. M. (2007). The effect
of temperature on mate preferences and female-female interactions in
Syngnathus abaster. Animal Behaviour 74, 1525-1533.
Souroukis K. & Cade W. H. (1993). Reproductive competition and
selection on male traits at varying sex-ratios in the field cricket, Gryllus
pennsylvanicus. Behaviour 126, 45-62.
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Vincent , A. C. J. (1994). Operational sex-ratios in seahorses. Behaviour
128, 153–167.
Vincent, A. C. J., Ahnesjo, I., Berglund, A. & Rosenqvist, G. (1992).
Pipefishes and seahorses: are they all sex role reversed? Trends in
Ecology and Evolution 7, 237–241.
Vincent, A. C. J., Ahnesjo, I. & Berglund, A. (1994). Operational sex-ratios
and behavioural sex differences in a pipefish population. Behavioral
Ecology and Sociobiology 34, 435–442.
Watanabe, S., Hara, M. & Watanabe, Y. (2000). Male internal fertilization
and introsperm-like sperm of the seaweed pipefish (Syngnathus schlegeli).
Zoological Science 17, 759–767.
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Chapter 3.3
Female reproductive tactics in a
sex-role-reversed pipefish:
screening for quality and number
3.3.1 Abstract
The differential allocation hypothesis predicts that individuals should invest
in current reproduction according to both the expected pay-offs from
mating with different-quality mates and their future mating prospects.
Accordingly, many studies over the past decade have shown that females
in many species invest more resources when paired with more attractive
males. Within fish, the family Syngnathidae is a particularly interesting
model group. Large and small pregnant males are likely to vary in their
investment in offspring and thus females, who are often constrained in
their mate choice, should benefit from differentially allocate their eggs in
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3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
large and small partners. The present work tests for the influence of male
quality (large and small body sizes) and number on the egg allocation
pattern of different sized females of the pipefish Syngnathus abaster.
Three major results emerged: i) both large and small females distribute
their eggs among several males, ii) neither large nor small females
produce enough eggs to fully occupy a male’s marsupium during the
extent of a male pregnancy and iii) different sized females exhibit different
allocation strategies, reflecting their distinct mating prospects. Contrarily to
small, less attractive females who show no significant differences both in
the number and the size of the eggs laid in the different experimental
contexts, large, preferred females respond according to both mate quality
and number through a process of serial matings resulting in the deposition
of eggs varying in number and size.
3.3.2 Introduction
Reproductive effort is a central topic in life-history theory (Roff, 1992). In
iteroparous species, a high investment in current reproduction might
reduce the capacity to invest in future reproductive events (Williams, 1966;
Trivers, 1972). According to the differential allocation hypothesis,
individuals facing trade-offs between investment in current and future
reproduction should strategically modulate their reproductive effort
depending on the attractiveness of their mate and the likelihood of finding
a better one in the future (Burley 1986, 1988; Sheldon, 2000). As proxies
for differential allocation, many studies have examined variation in clutch
characteristics (e.g. egg number and size) or parental behaviour, revealing
that females across a vast range of taxa invested more resources when
paired with attractive rather than with non-attractive males [e.g. insects
(Simmons, 1987), amphibians (Reyer et al., 1999), birds (Gorman et al.,
2005) or mammals (Drickamer et al., 2000)]. Surprisingly, given the
diversity and amount of work conducted on parental care in fishes, female
differential allocation has not been reported until very recently. Females of
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
the Banggai cardinalfish (Pterapogon kauderni), for instance, were found
to produce heavier eggs when paired with larger, preferred males (Kolm,
2001). Also, females of the Mediterranean blenniid (Aidablennius sphinx)
were observed spawning more eggs per time unit when paired with large,
preferred males (Locatello & Neat, 2005). A similar pattern has been
described by Skinner & Watt (2007) for the Zebra fish (Danio rerio).
The family Syngnathidae, comprising pipefishes, seahorses and
seadragons, is a particularly interesting group to study reproductive
investment and differential allocation patterns in fish. In this family, females
lay eggs in a specialised incubating structure of the males, who then
undergo a more or less prolonged ‘pregnancy’ providing protection,
aeration, osmoregulation (Carcupino et al., 2002) and nutrients to the
developing brood (Haresign & Shumway, 1981).
Berglund et al. (1986) argued that, in Syngnathus typhle, larger
males carried more eggs and provided more energy per offspring than
smaller individuals. If males do vary in their investment in offspring,
females would benefit from differentially allocating their eggs towards large
or small mating partners. Nevertheless, due to several factors, such as
male choosiness, marsupium limitations or female-female competition,
females might not always be able to of laying eggs in the preferred partner.
In this scenario, only preferred, dominant females would gain from an
increased opportunity to access high quality males. Thus, it seems
plausible that differential egg allocation according to the expected pay-offs
from mating with different-quality males together with the likelihood of
finding a better partner (or any other partner at all) might have evolved, in
females, as an adaptive strategy.
Despite the growing interest in syngnathid reproductive biology,
particularly on sex-role reversed species usually seen as mirror-like
models specially useful to highlight factors affecting the intensity of sexual
selection, there is still no experimental data supporting the hypothesis of
female differential egg allocation. To this extent, the present study
addresses five major questions: i) Do females spawn more eggs than a
male can care for over the extent of a pregnancy? ii) Do females
differentially allocate their reproductive effort (here quantified as the total
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3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
number of spawned eggs and its mean size) according to mate quality? iii)
Does mate availability (one versus several potential partners) influence
female egg allocation? iv) Do females scatter the eggs over multiple
partners when given the opportunity or, instead, do they concentrate their
eggs into one preferred male? v) Do different quality females exhibit
distinct responses to the questions raised above?
3.3.3 Methods
Syngnathus abaster is a euryhaline species that inhabits the
Mediterranean, Black Sea, and the Atlantic coast of Southwest Europe up
to southern Biscay (Dawson 1986). This black-striped pipefish occurs
either in coastal areas or in brackish and fresh waters (Cakic et al. 2002),
and can be found mainly among sand, mud or eelgrass meadows, at
depths between 0.5 and 5 m, within a temperature range of 8ºC to 24ºC.
Males have a brood pouch located ventrally on the tail (Urophori) which
consists of two skin folds that contact medially along their free edges.
Females are larger and more competitive than males under even sex ratio
conditions (Silva et al. 2006a).
Fish were collected with a hand net, in a salt pond storage tank at
the Ria de Aveiro estuarine lagoon (40º45’N, 8º40’W), in Portugal, and
transported to the laboratory where they were maintained in 250 litre
aquaria illuminated by natural light supplemented with 18 W fluorescent
lamps. Tank substrata consisted mainly of sand and plastic seagrass laid
in order to mimic the original habitat where the fish were caught. The
continuously running seawater was physically and biologically filtered and
aeration was performed outside the fish tanks to prevent the ‘gas bubble
disease’, common in pipefishes (Monteiro et al., 2002). Fishes were fed
daily with fresh Artemia franciscana nauplii. Sexually mature males and
females were kept in separate tanks.
The experiment was designed to assess the number of
reproductive partners and the characteristics of the transferred clutches
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
within the time span of a male pregnancy [30 days, according to Silva et al.
(2006b)]. Apart from the estimation of the time required until the first
copulation event, all the other variables were measured during 30 days
after the first mating event. Both large and small females were individually
presented to distinct ‘mating settings’: i) one male, large (T1L) or small
(T1S); ii) four identically sized males, large (T4L) or small (T4S) and iii) four
males, comprising two large and two small individuals (T2+2) in order to
allow females to simultaneously assess males of different sizes. Since the
results were to be interpreted mainly according to male size, and in order
to avoid data dependency, two independent trials were performed in the
heterogeneous group (T2+2L and T2+2S). The number of eggs laid by a
particular female in the two large males could not be assessed and
contrasted to those counted in the two small males that were present in
the same trial. Accordingly, on the T2+2L trial, results were recorded for
large males and on the separate T2+2S trial, values were obtained from
small males. Nevertheless, variables such as the number of pregnant
males within the T2+2 treatment were registered regardless of male size.
Five replicates of each of the 12 experimental units were
conducted [(T1L , T1S , T4L , T4S , T2+2L and T2+2S) x 2 female size
classes]. A total of 240 pipefish were used (30 large and 30 small females;
90 large and 90 small males). No fish was used more than once.
All males presented well-developed brood pouches and carried no
offspring at the beginning of the experiment. Size cut-offs for ‘large’ and
‘small’ individuals were defined according to Silva et al. (2007), as ½
standard deviation below and above the mean size (LT) of each sex
(♀: mean=9.4 cm, sd=1.38 cm; ♂: mean=8.5 cm, sd=1.26 cm). Large
males and females were longer than 9.1 cm and 10.1 cm respectively.
Small males and females were shorter than 7.9 cm and 8.7 cm
respectively.
Males were inspected for eggs three times per day (early morning,
noon and late afternoon). Both time required for the first spawn as well as
time elapsed between mating events was recorded. In each inspection,
newly spawned eggs, corresponding to additional copulation events, were
counted and photographed alongside an external ruler for size reference.
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3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
Good quality photos were imported into UTHSCA© Image Tool and only
perfectly visible eggs were measured in order to calculate the average egg
diameter per clutch. Due to marsupium opacity, good photographs of the
eggs were not possible for all pregnant males.
Statistical analysis
As to answer the first question addressed in this study, an estimation of
the female’s potential reproductive rate (the number of males that each
tested female could fill up to capacity) was calculated for each ‘mating
setting’ (T4L, T4S, T2+2L, T2+2S, T1L and T1S). To this purpose, the area of
the total eggs laid by each female (AE) [total number of eggs x π (egg
diameter/2) 2 ] was divided by the approximately rectangular marsupium
area (AM) of an average large or small male according to the
corresponding ‘mating settings’ (e.g. in the T4L the area of the eggs laid
was divided by the marsupium area of an average large male). A value
smaller than 1 indicates that the total number of eggs deposited during one
month was unable to fully occupy a male’s brood pouch whilst, as an
example, a value of 2 would indicate that the number of eggs was enough
to occupy two males. Marsupium area was estimated according to the
formula AM = marsupium height (HM) x marsupium width (WM) where HM
and WM were inferred from two regressions:
1) HM=83.606 x log (LT) – 134.36, r=0.938, P

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Additionally, in order to verify if in the heterogeneous ‘mating
setting’ (T2+2), females mated more often with large or small males, an
orthogonal ANOVA (A2) was conducted with two factors: female and male
size (two levels=large and small). In this analysis, values from the T2+2L
and T2+2S were used, rather than the total number of pregnant males.
Two orthogonal ANOVAs were also conducted to determine the
possible effects of ‘mate settings’, male and female size on the time
elapsed until the first spawn (A3) as well as between copulations (A4).
ANOVAS were conducted considering two factors: treatment (six
levels=T1L, T1S, T4L, T4S, T2+2L and T2+2S) and female size (two
levels=large and small).
Differential female allocation of the total number of spawned eggs
was tested through an orthogonal ANOVA (A5) with two factors: female
size (two levels = large and small) and ‘mate settings’ (five levels=T1L,
T1S, T4L, T4S and T2+2). Female egg allocation was then analysed within
the T2+2 through an additional orthogonal ANOVA (A6) with two factors:
male and female size (two levels=large and small).
Differential allocation on egg size was also analysed. To this
purpose an orthogonal ANOVA (A7) with two factors, female size (two
levels=large and small) and ‘mate settings’ (six levels=T1L, T1S, T4L, T4S
and T2+2L and T2+2S), was conducted.
In order to evaluate hypothetical differences in size of the eggs laid
in two consecutive spawnings by large females, a t-test for dependent
samples was also performed. Due to the difficulty in measuring all the
eggs in the male marsupium, due to transparency differences, a total value
of 12 consecutive spawnings was used.
All statistical analyses were performed using STATISTICA 7.0
(Statsoft). Data were tested for deviance from normality and for the
homogeneity of variance. Assumptions were not met regarding the size of
the eggs (Cochran’s test; P0.05), parametric statistics were used. Post-hoc
113

3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
comparisons were conducted using Newman-Keuls test in all analyses.
Probabilities were two-tailed and a significance level of 0.05 was used.
3.3.4 Results
The estimated mean value of the potential reproductive rate for both large
and small females was smaller than 1 in every ‘mating setting’, indicating
that the total number of eggs laid during the extent of a male pregnancy
could not fill a male pouch up to full capacity (Large females: T4L=0,67;
T4S=0,90; T2+2L=0,74; T2+2S=0,10; T1L=0,27; T1S=0,63; Small females:
T4L=0,27; T4S=0,38; T2+2L=0,32; T2+2S=0,44; T1L=0,30; T1S=0,54).
When considering female choice on concentrating or dispersing
their eggs (A1), it was found that both large and small females distributed
the eggs among several males, when given the opportunity (Figure 3.3.1).
Additionally, the number of mating partners was neither affected by female
size nor by ‘mating settings’ (Table 3.3.1.A). Within the heterogeneous
mating setting (T2+2), combining two large and two small males, the
number of pregnant males was neither affected by male nor female size
(A2; Figure 3.3.2; Table 3.3.1.B).
When analysing the effects of ‘mate settings’ and female size on
the day of first copulation (A3), only a significant effect of ‘mating settings’
was found (Table 3.3.1.C). Females started depositing eggs since the first
day in all tested treatments, with the exception of the heterogeneous
treatment T2+2S, where it took on average more than 4 days for small
males to receive an egg batch (Figure 3.3.3). It is important to stress that
the increased amount of time taken by females to lay eggs on small males
was probably dependent on the fact that the two large males present in the
T2+2S treatment were the first to receive eggs. Contrastingly, the mean
time elapsed between copulations (A4) was neither affected by male or
female size nor by ‘mating settings’ (Figure 3.3.4; Table 3.3.1.D).
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
Figure 3.3.1: Mean number of mating partners for large and small females
in each ‘mating setting’. Error bars represent standard deviations.
Figure 3.3.2: Mean number of large and small mating partners for large
and small females within the heterogeneous mating setting. Error bars
represent standard deviations.
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3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
Figure 3.3.3: Day of first copulation in each ‘mating setting’ and Newman-
Keuls test results, with gray squares showing significant differences. Error
bars represent standard deviations.
Figure 3.3.4: Mean time between copulations (in days) for large and small
females in each ‘mating setting’. Error bars represent standard deviations.
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
When considering the total number of spawned eggs (A5), a
significant monotonic interaction between female size and ‘mating settings’
was found (Figure 3.3.5; Table 3.3.1.E). As such, the main effects were
also analysed (see Figures 3.3.5.B, and C). Globally, it could be seen that
small females laid a similar number of eggs in all selected treatments even
though a non-significant increase could be observed in the T2+2 (Figure
3.3.5.A). When considering the number of eggs laid by large females, a
trend towards more eggs in treatments with more males could be
observed. This increase in the number of laid eggs is particularly evident in
the T2+2 and T4L, groups where more than one large male was available
(Figure 3.3.5.A). A similar pattern of results can be observed in the ‘mating
settings’ main effects, where the trend towards more eggs in the T2+2 and
T4L is also visible (Figure 3.3.5.B). This egg number increase seems to be
the result of the large females’ ability to lay more eggs, when compared to
the small females (Figure 3.3.5.C), especially in groups where more than
one large male was present.
When interpreting the results within the heterogeneous groups
T2+2 (A6), considering the number of eggs laid in small (T2+2S) and large
males (T2+2L), a significant interaction between female and male size was
found (Figure 3.3.6; Table 3.3.1.F). Small females laid the same number of
eggs independently of male size. Contrastingly, large females deposited
bigger clutches in large males while laying fewer eggs in small mating
partners (Figure 3.3.6).
Finally, a significant interaction between ‘mating settings’ and
female size (A7) was found for the mean size of the eggs (Figure 3.3.7;
Table 3.3.1.G). While small females laid eggs of similar dimensions in all
mating contexts, differences were observed among egg sizes laid by large
females in distinct ‘mating settings’. Even though no egg size differences
were observed between T4L and T4S nor T1L and T1S (neither among
these groups), when simultaneously presented with both large and small
males (T2+2), larger females laid larger eggs in larger partners. In fact, the
size of the eggs laid in the T2+2L treatment was larger than in all other
groups. Contrastingly, small males received smaller eggs in the T2+2S
when compared to T4S and T1S (Figure 3.3.7).
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3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
Figure 3.3.5: A. Mean number of eggs laid by large and small females in
each ‘mating setting’ and Newman-Keuls test results, with gray squares
showing significant differences. The initial L or S represents the size of the
female (large or small, respectively). B. Number of eggs laid in each
‘mating setting’ and Newman-Keuls test results, with gray squares showing
significant differences. C. Number of eggs laid by large and small females.
Error bars represent standard deviations.
Data collected from 13 large females that transferred two egg
batches within 7 days showed that the egg size significantly varied over
spawnings, with the eggs from the first batch being larger than the second
(T-test for dependent samples, 1 st =1.483 mm, 2 nd =1.399 mm, DF=11,
P

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Figure 3.3.6: Mean number of eggs laid by large and small females in
large and small males and Newman-Keuls test results, with gray squares
showing significant differences. The two initials, L (large) and S (small)
represent the size of the female and the male, respectively. Error bars
represent standard deviations.
Figure 3.3.7: Mean size of the eggs laid by large and small females in
each ‘mating setting’. Newman-Keuls test results are presented with gray
squares indicating significant differences. The initial L or S represents the
size of the female (large or small, respectively). Error bars represent
standard deviations.
119

3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
Figure 3.3.8: Mean size of the eggs from first and second batches. Error
bars represent standard deviations.
120

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Table 3.3.1: ANOVA results on (A) the number of mating partners, (B) the
number of mating partners within the heterogeneous ‘mating setting’
(T2+2), (C) the elapsed time before the first spawn, (D) the mean time
elapsed between copulations, (E) the total number of spawned eggs, (F)
the total number of eggs in the heterogeneous ‘mating setting’ (T2+2) and
(G) Mean size of the eggs.
Source DF MS F P
A. Number of mating partners
‘Mating settings’ 2 22.125 25.328 0.0943
Female Size 1 0.0100 0.0114 0.9154
‘Mating settings’ x Female
Size 2 10.125 11.591 0.3259
Error 34 0.8735
B. Number of mating partners within the heterogeneous ‘mating setting’ (T2+2)
Male size 1 0.2000 0.5000 0.4897
Female Size 1

3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
E. Number of spawned eggs
Female size 1 1123.38 85.650 0.0056
‘Mating settings’ 4 747.53 56.994 0.0010
Female size x ‘Mating
settings’ 4 349.23 26.626 0.0463
Error 40 131.16
F. Total number of eggs in the heterogeneous ‘mating setting’ (T2+2)
Female size 1 4.05 0.03283 0.8585
Male size 1 2268.45 183.904

Chapter 3. Understanding Sex-Role Reversal (ex situ)
Together with the decision of dividing their egg clutches through
multiple partners, S. abaster females revealed a particularly interesting
egg allocation pattern, with different-size females showing distinct
investment tactics. Despite the previously described preference for large
males, measured as the time spent by large and small females near a
particular male (Silva et al., 2007), females did not avoid mating with small
individuals. Instead, the preference for large partners, assessed in the
heterogeneous ‘mating setting’ (containing large and small males), was
translated into different mating timings. Females, large and small, opted to
mate sooner with large partners, only then depositing eggs in smaller
males. A non exclusive hypothesis, helping to explain the observed mating
timings, deals with male-male competition in a skewed sex-ratio setting
(4♂:1♀), where large males may have access to females not only due to
female preference but also because of a hypothetically higher hierarchical
status.
Within large females, the preference for larger individuals was
reinforced through differential egg allocation according to male size, with
larger males receiving more and larger eggs. Interestingly, this differential
egg allocation was only visible when large females were allowed to
simultaneously evaluate both large and small males (T2+2). These results
are in accordance with theoretical studies addressing female mate choice
behaviour, which argue that, in the absence of some internal standard,
females may assess male quality through a comparison process (Halliday,
1983; Wiegmann et al., 1999). The differential egg allocation strategy
seems to be restricted to large females since smaller ones showed no
significant differences, both in egg number and size, regardless of the
tested ‘mating settings’.
According to Kolm & Olsson (2003), differential allocation in egg
size according to male quality can be facilitated either through an extended
pair bond or by an ability to rapidly adjust egg size prior to mating. Thus,
decreasing an already defined high egg investment seems highly
improbable. Females of the Banggai cardinalfish, for example, are capable
of rapidly increasing their egg size investment in response to a new, more
attractive, male even after the onset of egg maturation. Contrastingly, they
123

3.3. Female reproductive tactics in a sex-role-reversed pipefish: screening for
quality and number
are unable to adjust egg size in response to a decrease in mate
attractiveness (Kolm & Olsson, 2003).
In S. abaster, when males of different sizes were available, larger
females deposited smaller eggs in smaller partners while transferring
larger eggs into high quality males. These results might suggest that large
females are adjusting egg size according to male quality. Nevertheless,
the observation that egg size diminished over two sequential spawnings
indicates that the observed egg allocation pattern might result mainly from
a process of serial matings: larger males first, smaller males later. This
hypothesis would help justify not only the wide difference in egg size
measured in the T2+2L (large eggs) vs the T2+2S (small eggs) but also
why the other ‘homogeneous’ groups revealed similar values, slightly
smaller than the T2+2L (in these groups, the measured egg size was the
result of the averaging of all laid eggs, large and small).
Interestingly, besides being differentially allocated according to
size, eggs of larger females were also laid in greater number in mating
settings involving more than one available large male (T4L and,
particularly, T2+2). The reduced egg number in ‘mating settings’ involving
only one male could be thought to result from marsupium space
constrains. Nevertheless, the estimations of females’ potential
reproductive rate clearly revealed that, during the extent of a male
pregnancy, females did not spawn enough eggs to fully occupy a male’s
pouch, thus corroborating the in situ observations of Silva et al. (in press).
Furthermore, the fact that the number of eggs laid by large females in T1L
and T1S did not differ from those observed in T4S but were significantly
smaller than those recorded for T2+2 and T4L, strongly suggests that large
females can have a fine-tuned control over their reproductive investment,
being able to ‘deliberately’ save a share of their resources for future, high
quality males.
It is unclear why investing more and larger eggs in larger males is
not a common strategy to both large and small females of S. abaster when
considering the direct benefits they could accrue from mating with high
quality partners (e.g. Berglund et al., 1986). Even considering that smaller
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Chapter 3. Understanding Sex-Role Reversal (ex situ)
females, due to physiological constraints, might not be able to produce as
much nor as large eggs as those laid by large females, a similar pattern of
egg allocation could be expected. The constant reproductive effort through
all ‘mating settings’ strongly suggests that, contrarily to large females,
small females seem to invest heavily in each breeding attempt. Maximizing
investment in current reproduction might well be an adaptive tactic for
smaller females to compensate their lower attractiveness (see Silva et al.,
2007). Contrarily to large preferred females who have increased
opportunities to mate with high quality males, small females might not
have access to a better partner, or any other partner in the future. This
adaptive tactic might be especially relevant in populations located in areas
where the extent of the breeding season raises the opportunity of small
females to reproduce. In areas where the extent of breeding season is
reduced to a few months, small females could opt to allocate resources
into growth rather than reproduction. Large, preferred females, on the
other hand, seem able to simultaneously monitor the number of available
mates and their intrinsic quality, responding accordingly through the
deposition of eggs that vary in number and size. Assuming that the size of
the fish reflects age (Billing et al., 2007) this strongly suggests the
existence of a ‘switch point’ in females’ reproductive tactics through
lifetime.
3.3.6 Acknowledgements
We would like to thank everybody that helped during the laboratorial work,
especially Pedro Correia. Professor Vitor Almada’s participation was
partially funded by Programa Plurianual de Apoio às Unidades de
Investigação. Nuno Monteiro's participation was funded by Fundação para
a Ciência e a Tecnologia (FCT-SFRH/BPD/14992/2004) and Programa
Plurianual de Apoio às Unidades de Investigação. Karine Silva's
participation was funded by Fundação para a Ciência e a Tecnologia
(FCT-SFRH/BD/13171/2003). This work was partially funded by FCT
(POCI/MAR/60895/2004).
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quality and number
3.3.7 References
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and parental energy investment in zygotes of two pipefish (Syngnathidae)
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Berglund, A., Rosenqvist, G. & Svensson, I. (1988). Multiple matings and
paternal brood care in the pipefish Syngnathus typhle. Oikos 51, 184-188.
Billing, A. M., Rosenqvist, G. & Berglund, A. (2007). No terminal
investment in pipefish males: only young males exhibit risk-prone courtship
behavior. Behavioral Ecology 18, 535–540.
Burley, N. (1986). Sexual selection for aesthetic traits in species with
biparental care. American Naturalist 127, 415–445.
Burley, N. (1988). The differential-allocation hypothesis: an experimental
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Cakic, P., Lenhardt, M., Mickovic, D., Sekulic, N. & Budakov, L. J. (2002).
Biometric analysis of Syngnathus abaster populations. Journal of Fish
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Carcupino, M., Baldacci, A., Mazzini, M. & Franzoi, P. (2002). Functional
significance of the male brood pouch in the reproductive strategies of
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study on three anatomically different pouches. Journal of Fish Biology 61,
1465-1480.
Dawson, C. E. 1986. Syngnathidae. In: Fishes of the North-eastern
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Drickamer, L. C., Gowaty, P. A. & Holmes, C. M. (2000). Free female mate
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Fox, C. W. & Rauter, C. M. (2003). Bet-hedging and the evolution of
multiple mating. Evolutionary Ecology Research 5, 273-286.
Gorman, H. E., Orr, K. J., Adam, A. & Nager, R. G. (2005). Effects of
incubation conditions and offspring sex on embryonic development and
survival in the zebra finch (Taeniopygia guttata). Auk 122, 1239–1248.
Halliday, T. R. (1983). The study of mate choice. In: Mate choice, (Bateson
P, ed), pp. 3-32. Cambridge: Cambridge University Press.
Haresign, T. W. & Schumway, S. E. (1981). Permeability of the marsupium
of the pipefish Syngnathus fuscus to [14C]-alpha animo-isobutyric acid.
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Jones, A. G., Rosenqvist, G., Berglund, A. & Avise, C. J. (2000). Mate
quality influences multiple maternity in the sex-role-reversed pipefish
Syngnathus typhle. Oikos 90, 321-326.
Jones, A. G. & Avise, J. C. (2001). Mating systems and sexual selection in
male-pregnant pipefishes and seahorses: insights from microsatellite-
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Kolm, N. (2001). Females produce larger eggs for larger males in a
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Kolm, N. & Olsson, J. (2003). Differential investment in the Banggai
cardinalfish: can females adjust egg size close to egg maturation to match
the attractiveness of a new partner? Journal of Fish Biology 63, 144–151.
Lee, P. L. M. & Hays, G. C. (2004). Polyandry in a marine turtle: females
make the best of a bad job. Proceedings of the National Academy of
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Designs. W. H. Freeman & Co, San Francisco, CA.
Locatello, L. & Neat, F.C. (2005). Reproductive allocation in Aidablennius
sphinx (Teleostei, Blenniidae): females lay more eggs faster when paired
with larger males. Journal of Experimental Zoology 303, 922-926.
Monteiro, N. M., Vieira, N. M. & Almada, V. C. (2002). The courtship
behaviour of the pipefish Nerophis lumbriciformis: reflections of and
adaptation to intertidal life. Acta ethologica 4, 109-111.
Reyer, H.U., Frei, G. & Som, C. (1999). Cryptic female choice: frogs
reduce their clutches when amplexed by undesired male. Proceedings of
the Royal Society of London B 266, 2101–2107.
Roff, D. A. (1992). The Evolution of Life Histories. London: Chapman &
Hall.
Sheldon, B. C. (2000). Differential allocation: tests, mechanisms and
implications. Trends in Ecology & Evolution 15, 397–402.
Silva, K., Monteiro, N. M., Vieira, M. N. & Almada, V. C. (2006a).
Reproductive behaviour of the black-striped pipefish, Syngnathus abaster
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Silva, K., Monteiro, N. M., Almada, V. C. & Vieira, M. N. (2006b). Early life
history of Syngnathus abaster (Pisces: Syngnathidae). Journal of Fish
Biology 68, 80-86.
Silva, K., Vieira, M. N., Almada, V. C. & Monteiro, N. M. (2007). The effect
of temperature on mate preferences and female-female interactions in
Syngnathus abaster. Animal Behaviour 74, 1525-1533.
Silva, K., Vieira, M. N., Almada, V. C. & Monteiro, N. M. (In press). Can the
limited marsupium space be a limiting factor for Syngnathus abaster
females: Insights from a population with size assortative mating. Animal
Ecology.
Simmons, L. W. (1987). Female choice contributes to offspring fitness in
the field cricket Gryllus bimaculatus (De Geer). Behavioral Ecology and
Sociobiology 21, 313–321.
Skinner, A. J, Watt, P. J. (2007). Strategic egg allocation in the zebra fish,
Danio rerio. Behavioural Ecology 18, 905-909.
Trivers R. L. (1972). Parental investment and sexual selection. In:
Campbell B (ed) Sexual selection and the descent of man. Aldine Press,
Chicago, Ill. pp 136–179.
Wiegmann, D. D., Mukhopadhyay, K. & Real, L. A. (1999). Sequential
search and the influence of male quality on female mating decisions.
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Chapter 4. Reproductive Dynamics (in situ)
Can the limited marsupium space
be a limiting factor for Syngnathus
abaster females?
Insights from a population with
size assortative mating.
4.1 Abstract
Some syngnathid species, aside from male pregnancy, show varying
degrees of sex-role reversal, with females competing for access to mates
and sometimes presenting conspicuous secondary sexual characters.
Among other variables, brooding-space constraints are usually considered
as a key element in female reproductive success, strongly contributing to
the observed morphological and behavioural sexual differences.
Nevertheless, a close relationship between sex role reversal and male
130

4.1. Can the limited marsupium space be a limiting factor for Syngnathus abaster
females? Insights from a population with size assortative mating.
brooding space limitation has not yet been accurately demonstrated in field
studies.
The present work, conducted over two consecutive breeding
seasons in a wild population of the sex-role reversed pipefish Syngnathus
abaster, simultaneously analyzed the egg number and occupied space, as
well as the free area in the male’s marsupium. The number of eggs that
would fit in the observed unoccupied space was estimated.
Contrarily to what could be expected given the marked sexual
dimorphism observed in the studied population, where females are larger
and more colourful, male brooding space didn’t appear to limit female
reproduction since neither large nor small individuals presented a fully
occupied pouch. Interestingly, the largest marsupium unoccupied areas
were found in the larger individuals, even though they received more and
larger eggs. Laboratory data also showed that bigger females lay larger
eggs.
Altogether these results suggest the existence of assortative
mating which may result either from: i) the reluctance of larger males
(which tend not to receive small eggs usually laid by small females) to
mate with less quality females, even at the expense of a smaller number of
offspring or, ii) female-female competition which might strongly reduce the
hypothesis of a small female mating with a large male.
The potential impact of temperature on reproduction and
population dynamics is also discussed under the light of the ongoing
climatic changes.
4.2 Introduction
In many sexually reproducing animals one sex clearly competes more
intensely for mates than the other. Differences in the relative intensity of
mating competition are usually attributed to sex differences in potential
reproductive rates that determine which sex limits the reproductive
success of the other (Andersson 1994; Clutton-Brock & Parker 1992;
131

Chapter 4. Reproductive Dynamics (in situ)
Ahnesjo, Kvarnemo & Merilaita 2001). Sex roles are termed ‘conventional’
when males compete among themselves for female access. Contrarily,
when females compete over mates, sex roles are usually described as
reversed (Vincent et al. 1992).
Although uncommon in nature (Balshine-Earn & McAndrew 1995;
Gwynne & Simmons 1990: Colwell & Oring 1988), sex-role reversal can
nevertheless be observed in some species of the Syngnathidae family, a
fish group characterised by a highly specialised male pregnancy whose
costs are believed to exceed those of most vertebrates (Clutton-Brock &
Vincent 1991). Syngnathid females deposit unfertilized eggs into a male’s
specialized ventral incubating surface whose anatomical complexity varies
among species, from a simple incubating ventral surface to a sealed pouch
(Herald 1959). In some pipefish species, females compete for male
access, sometimes presenting conspicuous secondary sexual characters
(Monteiro, Vieira & Almada 2002; Silva et al. 2006). Energy investment,
brooding-space constraints and pregnancy length, operating alone or in
combination, may limit female reproductive success thus providing an
explanation of such sexual differences (Berglund & Rosenqvist 2003).
Despite many attempts to clearly identify the mechanisms behind the
evolution of sex-role reversal in syngnathids, such as whether the male’s
limited brooding space can effectively contribute to sex-role reversal, the
causes are yet to be fully demonstrated, especially in wild populations
where a species behavioural repertoire is modulated by far more variables
than in aquaria.
Laboratory studies showed that brooding-space constraints and
pregnancy length, rather than energy investment, might limit female
reproduction in two Northern-European populations of sex-role reversed
pipefish, Nerophis ophidion and Syngnathus typhle (Berglund, Rosenqvist
& Svensson 1989). In both species, females were found to produce more
eggs than a male can brood during a pregnancy episode (Berglund et al.
1989). The need to contextualize these findings with field observations has
encouraged the present study on a wild population of the sex-role reversed
Syngnathus abaster (Risso, 1827), morphologically similar to S. typhle.
Both the space occupied by eggs and the remaining free area in the
132

4.1. Can the limited marsupium space be a limiting factor for Syngnathus abaster
females? Insights from a population with size assortative mating.
male’s pouch was analysed as a means to test whether the male’s limited
brooding space could indeed constrain female reproductive success.
Furthermore, the importance of male and female size was also considered
in order to investigate potential differences in egg allocation and its
consequences in mating patterns.
4.3 Methods
Syngnathus abaster is a euryhaline species that inhabits the
Mediterranean, Black Sea, and the Atlantic coast of Southwest Europe up
to southern Biscay (Dawson 1986). The black-striped pipefish occurs
either in coastal areas or in brackish and fresh waters (Cakic et al. 2002),
and can be found mainly among sand, mud or eelgrass meadows, at
depths between 0.5 and 5 m, within a temperature range of 8ºC to 24ºC.
Males have a brood pouch located ventrally on the tail (Urophori) which
consists of two skin folds that contact medially with their free edges. Sex
roles seem to be reversed, as females are larger and apparently more
competitive than males, at least under even sex ratio conditions (Silva et
al. 2006).
Bimonthly samples were conducted over two consecutive breeding
seasons in the Ria de Aveiro estuarine lagoon (40º45’N, 8º40’W), in
northern Portugal. Two hundred and ninety five pregnant males were
captured and measured for total length (LT). For each pregnant male, eggs
were counted and macro photographs, including an external ruler for size
reference, were taken in the most transparent area of the marsupium. A
sub- sample of ninety-five individuals (≈32%) was brought to the laboratory
for additional marsupium width and height measurements. All other
captured males were immediately released in the same area where initially
collected. Good quality photographs, available for 121 individuals, were
imported into UTHSCA© Image Tool and perfectly visible eggs were
measured in order to calculate the average egg diameter per male (ED).
The total number of captured fish, including non-pregnant males and
133

Chapter 4. Reproductive Dynamics (in situ)
females was also recorded in each sampling event for sex-ratio
calculation.
The approximately rectangular marsupium area (AM) was
calculated according to the formula: AM=marsupium height (HM) x
marsupium width (WM). HM and WM (measured in the 95 individuals that
were transported to the laboratory specifically for this goal) were inferred
for all males according to the regressions presented in the Results section.
The number of missing eggs, ME [i.e. the number of eggs, with the average
diameter observed for each male, that would fill the unoccupied
marsupium space (UM)], was calculated for each individual according to
the following formula: ME=UM / π (egg diameter/2) 2 , where UM = AM – egg
number x π (egg diameter/2) 2 .
Other approaches for the calculation of the number of eggs that
could fit the available marsupium area could have been selected, namely
those that would consider space between the theoretically spherical eggs
as unusable. Alternatively, the degree of compression sometimes alters
egg sphericity into a cube like form. It could be expected that the most
accurate of these two approaches [UM=AM – egg number x π (egg
diameter/2) 2 or UM=AM – egg number x egg diameter 2 ], when subtracted to
the direct measurement of the actual area occupied by eggs (as
determined using photographs of the marsupium of aquarium kept males,
using UTHSCA© Image Tool), would tend to zero. A t-test of means
against a reference constant (0) showed that the selected approach was
indeed accurate (N=4, DF=3, p=0.64) when compared to the hypothesis
that eggs have a cube-like form or that the space between spherical eggs
cannot be accounted as free space (N=4, DF=3, P=0.03).
Regressions were conducted in order to test for possible
relationships between male and egg size. Additionally, data gathered in
aquarium, using female size and the diameter of the laid eggs, was used
to test for a possible correlation between these two variables.
Taking into consideration that mate size is an important variable in
syngnathid mate choice (Silva et al. in press; Berglund & Rosenqvist
2003), two male groups were considered with the size cut-offs for ‘large’
and ‘small’ pregnant males defined according to Silva et al. (in press), as
134

4.1. Can the limited marsupium space be a limiting factor for Syngnathus abaster
females? Insights from a population with size assortative mating.
½ standard deviation below and above the mean male size (LT)
(mean=8.5 cm, SD=1.26 cm, values obtained from prior measurements in
the same population, N=214 males). Large males were longer than 9.1 cm
whilst small individuals were shorter than 7.9 cm. Accordingly, two one-
way ANOVAS were conducted in order to test for differences between
male size classes (large and small; untransformed data) in the number of
carried eggs and number of ‘missing’ eggs [transformed data: square root
(x+constant)]. The homogeneity of variances assumption was met for all
analyses. All probabilities are two-tailed and a significance level of 0.05
was used.
4.4 Results
The number of captured males and females was not significantly different
during the 29 sampling visits (t-test for dependent samples, ♀=13.24,
♂=15.03, DF=28, P=0.239).
The regression equations used to estimate the marsupium height
and width were: a) Marsupium height: HM=83.606 x log(LT) – 134.36,
r=0.938, P

Chapter 4. Reproductive Dynamics (in situ)
male, was found to be significantly higher in large than in small individuals
[ANOVA: DF (1,88) F=19.79; P

4.1. Can the limited marsupium space be a limiting factor for Syngnathus abaster
females? Insights from a population with size assortative mating.
4.5 Discussion
It is not always clear how to demonstrate that one sex actually limits the
reproductive success of the other. For example, even if a male makes a
substantial investment in time and energy in offspring, either this may not
exceed female investment or time and energy may not be limiting factors
during the breeding period (Berglund et al. 1989).
The present study showed that neither large nor small S. abaster
males presented fully occupied pouches over the breeding season, at least
in the studied population where the number of males and females was
similar. Thus, it seems that the limited marsupium space may not to be a
limiting factor for S. abaster females. The fact that only brooding males
were considered in this work reinforces the obtained results since
otherwise the available areas for females to lay their eggs upon would be
even greater. Also, it could be argued that, even though space is available,
males could refrain from accepting eggs due to physiological or allocation
trade-off reasons. Nevertheless, it is important to stress that several fully
occupied pouches were observed during the two-year sampling period,
showing that males can be filled up to maximum capacity.
Berglund et al. (1989) showed that, in aquaria, S. typhle females,
captured from a population under an even sex ratio, produced far more
eggs than a male could bear during the extent of a pregnancy event. If
these results could be transposed to the field, than a marsupium space
limitation would be expected. These differences between S. abaster and S.
typhle could be interpreted from distinct viewpoints: i) even considering
that S. typhle is a larger species, with males presenting a bigger
marsupium, the far greater number of eggs produced by S. typhle females
(105, as indicated in Monteiro, Almada & Vieira 2005) may still fully occupy
the increased brooding area. Alternatively, ii) the results obtained in
aquaria, during a male pregnancy, and those observed in the wild, during
the full extent of a breeding season, might not be fully comparable. For
instance, in preliminary aquaria observations, a S. abaster female was
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Chapter 4. Reproductive Dynamics (in situ)
able to lay 78 eggs during the extent of a male’s pregnancy. No male
captured in the wild showed more than 62 eggs.
The observed differences in egg number and size among S.
abaster male length classes, with larger individuals receiving more and
larger eggs, seem to be the result of mating with larger females, as
inferred from laboratory data on female size and its laid eggs. Since large
body sizes proved to be a sexually selected trait (Silva et al. in press) it
could be expected that large males would have more mating opportunities
and thus, more occupied pouches than smaller individuals. Interestingly,
the largest unoccupied marsupium areas were found in the larger
individuals group. Apparently discordant, this observation might be the
result of male choosiness since larger males were observed to also prefer
large partners (Silva et al. in press). It should be stressed that the
difference in marsupium occupation observed in large and small males
could also be the result of different trade-offs between egg size and egg
numbers, with a larger empty space in the brood pouch being more
beneficial for large males. Furthermore, it seems that larger males may
also be reluctant in mating with less quality females, even at the expense
of a smaller number of offspring. By mating with large females, which
produce larger eggs, males could gain in offspring quality. Offspring
hatching from larger eggs have been reported to have higher survival,
higher resistance to starvation and increased swimming performance
(Kolm & Ahnesjo 2005). A non-exclusive hypothesis, explaining the
apparent absence of small female eggs in large male’s marsupium, deals
with female-female competition. Large dominant females may strongly
reduce the opportunity of small females to mate with large males, as
suggested in Silva et al. (in press). Small females of S. typhle were also
found to be reproductively constrained by large dominant females
(Berglund & Rosenqvist 2003).
Male mate choice for larger females was not only observed in
sex-role reversed syngnathids [e.g. S. abaster (Silva et al. in press) and S.
typhle (Berglund & Rosenqvist, 2003)] but also in ‘conventional’ sex-role
species outside this family, such as Gasterosteus aculeatus (Kraak &
Bakker 1998), Poecilia reticulata (Herdman, Kelly & Godin 2004) and
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4.1. Can the limited marsupium space be a limiting factor for Syngnathus abaster
females? Insights from a population with size assortative mating.
Pseudomugil signifer (Wong, Jennions & Keogh 2004). Thus, it could be
expected that small males, that also prefer large mates, might be able to
carry eggs laid by large females. Nevertheless, the significant correlation
obtained between male and female size suggests that larger females may
avoid reproducing with small males.
Together, these results suggest the occurrence of assortative
mating, as already proposed in Silva et al. (2006), where S. abaster
courting pairs with more asymmetrical body sizes were less successful in
achieving mating. Size-assortative mating has been observed in many fish
species [e.g. Sarotherodon galilaeus (Ros, Zeilstra & Oliveira 2003),
Gasterosteus aculeatus (Olafsdottir, Ritchie & Snorrason 2006) and
Cichlasoma nigrofasciatum (Beeching & Hopp 1999)]. Assortative mating
is believed to have played a significant role in seahorse speciation process
(Jones et al. 2003). Size assortative mating might also contribute to a
female reproductive limitation since the number of preferred males (that
are also choosy), tend to be smaller, reinforcing sex-role reversal.
It would be interesting to see if a similar pattern of results could be
viewed in other S. abaster populations, especially those inhabiting different
latitudes where the extent of the breeding season markedly differs. It could
be predicted that the limited marsupium space could indeed be a limiting
factor for females, particularly those of small size, in areas where the
extent of the breeding period would not allow for time-scattered
spawnings. Moreover, given the current climatic changes, the local
expression of a species mating system will surely vary with consequences
that cannot yet be foreseen given the small amount of available
information on estuarine and coastal fish population dynamics.
4.6 Acknowledgements
We would like to thank everybody that helped during the laboratorial and
fieldwork, especially Alberto Silva, Armando Jorge and Pedro Correia. We
would also like to thank Anders Berglund and an anonymous referee for
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Chapter 4. Reproductive Dynamics (in situ)
suggestions and criticism. Vitor Almada’s participation was partially funded
by Programa Plurianual de Apoio às Unidades de Investigação. Nuno
Monteiro's participation was funded by Fundação para a Ciência e a
Tecnologia (FCT-SFRH/BPD/14992/2004) and Programa Plurianual de
Apoio às Unidades de Investigação. Karine Silva's participation was
funded by Fundação para a Ciência e a Tecnologia
(FCT-SFRH/BD/13171/2003). This work was partially funded by FCT
(POCI/MAR/60895/2004).
4.7 References
Ahnesjo, I., Kvarnemo, C. & Merilaita S. (2001) Using potential
reproductive rates to predict mating competition among individuals
qualified to mate. Behavioral Ecology, 12, 397-401.
Andersson, M. (1994) Sexual Selection. Princeton, New Jersey: Princeton
University Press.
Balshine-Earn, S. & McAndrew, B. J. (1995) Sex-role reversal in the
black-chinned tilapia, Sarotherodon melanotheron (Ruppel) (Cichlidae).
Behaviour, 132, 861-874.
Beeching, S. C. & Hopp, A. B. (1999) Male mate preference and
size-assortative pairing in the convict cichlid. Journal of Fish Biology, 55,
1001–1008.
Berglund, A., Rosenqvist, G. & Svensson, I. (1989) Reproductive success
of females limited by males in two pipefish species. The American
Naturalist, 133, 506-516.
Berglund, A. & Rosenqvist, G. (2003) Sex role reversal in pipefish.
Advances in the Study of Behaviour, 32, 131-167.
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females? Insights from a population with size assortative mating.
Cakic, P., Lenhardt, M., Mickovic, D., Sekulic, N. & Budakov, L. J. (2002)
Biometric analysis of Syngnathus abaster populations. Journal of Fish
Biology, 60, 1562–1569.
Clutton-Brock, T. H. & Vincent A. (1991) Sexual selection and the potential
reproductive rates of males and females. Nature, 351, 58–60.
Clutton-Brock, T. H. & Parker, G. A. (1992) Potential reproductive rates
and the operation of sexual selection. The Quarterly Review of Biology, 67,
437-456.
Colwell M. A. & Oring, L. W. (1988) Sex ratios and intrasexual competition
for mates in a sex-role reversed shorebird, Wilson’s phalarope
(Phalaropus tricolor). Behavioral Ecology and Sociobiology, 22, 165–173.
Dawson, C. E. (1986) Syngnathidae. Fishes of the North-eastern Atlantic
and the Mediterranean (eds. P. J. P. Whitehead, M. L. Bauchot, J. C.
Hureau, J. Nielsen, & E. Tortonese), pp. 628–639. Paris, Unesco.
Gwynne, T. D. & Simmons, L. W. (1990) Experimental reversal of
courtship roles in an insect. Nature, 346, 172-174.
Herald, E. S. (1959) From pipefish to seahorse - a study of phylogenetic
relationships. Proceedings of the Californian Academy of Sciences, 29,
465–473.
Herdman, E. J. E., Kelly, C. D. & Godin, J. G. J. (2004) Male Mate Choice
in the Guppy (Poecilia reticulata): Do Males Prefer Larger Females as
Mates? Ethology, 110, 97-111.
Jones, A. G., Moore, G. I., Kvarnemo, C., Walker, D. E. & Avise, J. C.
(2003) Sympatric speciation as a consequence of male pregnancy in
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seahorses. Proceedings of the National Academy of Science, 100,
6598-6603.
Kolm, N. & Ahnesjo, I. (2005) Do egg size and parental care coevolve in
fishes? Journal of Fish Biology, 66, 1499-1515.
Kraak, S. B. M. & Bakker, T. C. M. (1998) Mutual mate choice in
sticklebacks: attractive males choose big females, which lay big eggs.
Animal Behaviour, 56, 859-866.
Monteiro, N. M., Vieira, N. M. & Almada, V. C. (2002) The courtship
behaviour of the pipefish Nerophis lumbriciformis: reflections of and
adaptation to intertidal life. Acta ethologica, 4, 109-111.
Monteiro, N.M., Almada, V.C. & Vieira, M.N. (2005) Implications of
different brood pouch structures in syngnathid reproduction. Journal of
Marine Biological Association of the United Kingdom, 85, 1235-1241.
Olafsdottir, G. A., Ritchie, M. G. & Snorrason, S. S. (2006) Positive
assortative mating between recently described sympatric morphs of
Icelandic sticklebacks. Biology Letters, 2, 250–252.
Ros, A. F. H, Zeilstra, I. & Oliveira, R. F. (2003) Mate choice in the Galilee
St. Peter’s fish, Sarotherodon galilaeus, Behaviour, 140, 1173-1188.
Silva, K., Monteiro, N. M., Vieira, M. N. & Almada, V. C. (2006)
Reproductive behaviour of the black-striped pipefish, Syngnathus abaster
(Pisces; Syngnathidae). Journal of Fish Biology, 69, 1860-1869.
Silva, K., Vieira, M. N., Almada, V. C. & Monteiro, N. M. in press. The
effect of temperature on mate preferences and female-female interactions
in Syngnathus abaster. Animal Behaviour.
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4.1. Can the limited marsupium space be a limiting factor for Syngnathus abaster
females? Insights from a population with size assortative mating.
Vincent, A., Ahnesjo, I., Berglund, A. & Rosenqvist, G. (1992) Pipefishes
and seahorses: are they all sex role reversed? Trends in Ecology and
Evolution, 7, 237–241.
Wong, B. B. M., Jennions, M. D. & Keogh, J. S. (2004) Sequential male
mate choice in a fish, the Pacific blue-eye Pseudomugil signifer.
Behavioral Ecology and Sociobiology, 56, 253–256.
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Chapter 5. Final Discussion
Chapter 5
Final Discussion
5.1 Towards a new approach on sex-roles
The observed patterns of Syngnathus abaster reproduction clearly
challenge the oversimplistic binary notion of sex-roles (conventional versus
reversed), highlighting, instead, the flexible nature of the reproductive
behaviours within the family Syngnathidae. Although evidence for intense
female-female competition, and thus sex-role reversal (sensu Vincent et
al., 1992), is repeatedly reported in this thesis (Chapter 2.3, Chapter 3.1
and Chapter 3.2), the behavioural and morphological sexual differences
observed in S. abaster are clearly moderate when compared to other
pipefish also reported as sex-role reversed. For instance, the role of the
male during courtship is much more pronounced than in Nerophis
lumbriciformis where males are more passive (Monteiro et al., 2002). Also
sexual dimorphism is less evident than in other pipefish species where
females are highly modified by sexual selection. The dimorphic banding
coloration of S. fuscus (Roelke & Sogard, 1993), for example, clearly
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Chapter 5. Final Discussion
contrasts with the temporary ornament displayed by both males and
females in S. abaster.
5.1.1 Water temperature and the expression of
reproductive behaviours
Seasonal variations in temperature are likely to affect the intensity of
sexual selection in many ectotherms (Kvarnemo & Ahnesjo, 1996).
Through its impact on physiological processes, which operate within the
bounds of temperature extremes, higher temperatures characteristically
increase the potential reproductive rates of males more than that of
females, thus affecting the operational sex-ratio and, consequently, the
intensity of mating competition (see Chapter 1).
In the sand goby, Pomatoschistus minutus, for example, the time
males spend guarding eggs decreases significantly more with increasing
temperatures than does the time females need to produce a new clutch of
eggs (Kvarnemo, 1994), resulting in a highly male-biased operational
sex-ratio and thus intense male competition in warmer waters (Kvarnemo,
1996). In a similar way, temperature is known to differentially affect the
reproductive rates of males and females in S. typhle (Ahnesjo, 1995). In
this sex-role reversed pipefish, the male brooding time is much longer in
colder than in warmer waters. In females, however, the number of eggs laid
per day (given an unlimited access to mates) is not significantly influenced
by temperature (Ahnesjo, 1995). Even though reduced female-female
competition in warmer waters has already been predicted, the study
presented in Chapter 3.1 is the first to directly explore the potential
influence of temperature on syngnathid reproductive behaviour dynamics.
Through the observation of behavioural interactions at three
distinct water temperatures (mimicking the periods before the onset of the
breeding season, during its beginning and near its end) it was possible to
highlight some of the possible consequences of fluctuating temperatures
on the expression of reproductive behaviours of S. abaster males and
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Chapter 5. Final Discussion
females. It is important to stress out that the referred experiments were
performed within the extent of the breeding season of a Southern
European S. abaster population, which occurs mainly during summer, with
temperatures increasing rapidly to a peak late in the season and dropping
visibly in the beginning of autumn, when reproduction no longer takes
place (K. Silva, unpublished data collected during a three-year monitoring
period of a wild population occurring in the Ria de Aveiro estuarine
lagoon). Data on the reproduction of S. abaster in the field shows a high
correlation with water temperature and no correlation with other tested
variables such as air temperature or salinity. Furthermore, when
temperature is kept above 18ºC, there is continuous reproduction in
aquaria. As such water temperature appears to be a key factor in S.
abaster reproduction and the ultimate cause for the behavioural pattern
obtained in the mentioned study.
Two main results were expected: i) both sexes would show a
particular interest towards possible mates as soon as temperature rises
above the threshold allowing for reproduction to occur, and ii) females,
would compete less intensely at higher temperatures since male
pregnancy is shortened in warmer waters (Chapter 2.1) and thus a less
pronounced sexual difference in potential rates could be predicted.
Temperatures mimicking the beginning of the breeding season
indeed triggered sexual interest in males. In females however, a clear
interest on the opposite sex was only visible at higher temperatures,
reflecting the end of the reproductive period. Such a pattern of results
would make sense in species such as N. lumbriciformis, where males
arrive earlier than females at the intertidal mating grounds (Monteiro et al.,
2006) and thus the interest in the opposite sex might be possibly triggered,
in males and females, at distinct moments of the reproductive period. A
three-year monitoring of a wild population of S. abaster shows however no
evidence for this hypothesis, with males and females arriving
synchronously at the mating pools (K. Silva unpublished data)
A more plausible explanation for the obtained pattern of results
could lie instead in the reversed patterns of competition observed in S.
abaster and what seems to be an intrinsic female disposition to interact
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Chapter 5. Final Discussion
with other females. As mentioned in Chapter 3.1, at temperatures
reflecting the beginning of the breeding period, the mechanisms favouring
male approach and those involved in female-female interactions would
compete, so that sexual motivation would be partly masked in females. In
contrast, as temperature increases, and the reproductive season
approaches its end, females, who still have eggs to spawn would likely
benefit from re-direction their attention. In males, which in sex-role
reversed syngnathids often have little need to compete with other males,
the mechanism appears simpler, basically translated into an increased
focus on females concurrent with warmer waters.
Although this pattern of results suggest an overall reduction in the
intensity of female competition at higher temperatures, the focusing on
female-female interactions shows, however, that only large (and preferred)
females responded according to predictions based on the mentioned
temperature-dependent effects on potential reproductive rates: they
engage intensely in competition at the beginning of the breeding season
(colder waters), and reduce competition as the mate acquisition period
draws to a close (warmer waters). Small females, in opposition, appeared
more liable to compete at temperatures reflecting the late period of the
breeding season. By presenting this behaviour, these less attractive and
less competitive females probably make the most efficient use of an
extending breeding season. Small females probably decrease hypothetical
energetic costs of competition and maximize their body size prior to
reproduction, thus gaining in reproductive success, both in terms of
increased fecundity and increased attractiveness and competitiveness.
Another, non-exclusive, explanation for this higher activity of smaller
females at temperatures mimicking the end of the breeding period could
derive from a low winter survival for these individuals. A young female with
a low chance of surviving would be expected to invest heavily in the
current reproductive season and not to save resources to next year (Billing
et al., 2007).
It could be argued that small females might just mature later. Field
observations on S. abaster reproductive season do not, however,
corroborate this possibility. In fact, it seems that individuals that were born
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Chapter 5. Final Discussion
in the preceding breeding season are able to reproduce at the beginning of
the next reproductive period while young of the year are also able to
reproduce at the end of the reproductive period, if given enough time to
grow. Thus, small mature females seem to always be present during the
reproductive season (K. Silva unpublished data).
5.1.2 Sex-ratios and mating competition
The study presented in Chapter 3.2 clearly illustrates how temporally or
spatially variable sex-ratios can affect the expression of sex-roles in S.
abaster. Such behavioural plasticity has already been reported for species
with conventional sex-roles (e.g. Forsgren et al., 2004) but a reversal in
sex-role reversal remained to be experimentally demonstrated.
Surprisingly, the relationship between sex-ratios and the intensity of
mating competition was not as linear in females as in males where both
large and small individuals assume a prone competitive behaviour when
exposed to a scarce number of potential mates. Indeed, unbalanced
sex-ratios towards a surplus of females differentially modulated the
intensity of competition in different-size females in much the same way as
temperature did: large females were found to decrease competition while
small ones interacted intensely with other females. The balance between
the costs and benefits of competitive behaviour, rather than just the
potential reproductive rates or the relative abundance of the sexes, seem
to ultimately determine the intensity of sexual selection in the primary
competitor sex, constraining the behavioral responses of individuals
according to their own intrinsic quality. Avoiding the hypothetical costs
derived from competitive displays under female-biased mating contexts,
and temporarily postponing reproduction waiting for additional mating
opportunities might only be a high risk strategy for small females with more
uncertain mating prospects. Large females, on the contrary, can possibly
count on their ‘sex appeal’, which boosts the probability to acquire new
matings.
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Chapter 5. Final Discussion
5.1.3 Mate preferences versus mate choice: the
importance of the social context
Preferences for mates with particular phenotypic traits are common in
animals (Andersson, 1994). Exhibiting a preference in ‘two-choice’ tests
where individuals are simultaneously presented with a pair of potential
mates differing in ‘quality’ (as in Chapter 3.1), and actually pursuing the
mate choice when inserted in a social context allowing for individuals to
interact [as in Chapter 3.2, (in-situ) and Chapter 4.1 (ex-situ)], may,
however, be two very different situations. By being choosy individuals
increase the chances of mating with a more desirable partner, but this
strategy also faces costs. Generally, the balance between these costs and
benefits differ between individuals according to their condition, generating
variation in the optimal cost-benefit trade-off (Hoglund & Alatalo, 1995) and
thereby leading to differences in choosiness between different quality
individuals (Jennions & Petrie, 1997; Brooks & Endler, 2001). In species
(or populations) in which there is strong competition for attractive mates
and individuals vary in their ability to access mating partners, the poorest
competitors, which are unlikely to succeed matings without being
disrupted, might minimize the costs of not mating at all by avoiding the
highest-quality partners and instead focus on low-quality ones. In species
with bi-directional mate choice (i.e. both sexes of a species typically
participate in mate choice), things may become even more complex as an
individual’s attractiveness to members of the opposite sex limits might also
limit its ability to acquire mates. As a consequence, individuals may
dynamically adjust their mate selectivity based on their own current mating
quality to avoid searches for unwilling mates. In most cases, competition
and choice combine to make mating strongly assortative with respect to
quality (Johnstone, 1997; Fawcett & Johnstone, 2003).
The hypothesis that individuals vary their selectivity in response to
their perceived mate-getting ability has received little attention, perhaps
because the idea that, in any given species, members of a single sex
choose mates, has dominated the literature on sexual selection for years.
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Chapter 5. Final Discussion
In some cases, the assumption that only one sex actively selects among
mates is reasonable. For example, in the mite species Tetranychus
urticae, males guard duetonymph females and prefer those females that
are closest to eclosion (Everson & Addicott, 1982). The females obviously
have no potential role in choice here, since they are still in their quiescent
form. Few systems however are as simple as the one observed in these
mites. Instead, a number of studies have been leading to the growing
realization that sex-role generally involve both competition and choice in
both sexes, with the overall classification as conventional or sex-role
reversed dependent on the relative strength of these behaviours (e.g.
Amundsen, 2000; Berglund et al., 2005).
In S. abaster, a larger body size generally translates into a higher
quality in terms of both attractiveness and competitiveness in males and
females. True to predictions, the data in this thesis highlights important
differences in the strength of preferences between different-size
individuals who generally end up mating size assortatively, both in aquaria
(Chapter 2.3 and Chapter 3.2) and in the field (Chapter 4). Interestingly,
not even unbalanced sex-ratios, which should, theoretically, relate to
choosiness as the sex in shortage may afford to be selective (see Chapter
1), were able to completely disrupt this pattern. Indeed, contrarily to large
individuals who become particularly choosy when exposed to a larger
number of potential mates, small males and females continued to mate
with similar size partners as the opposite sex, although in surplus, also
exercised mate choice, preferring larger mates.
5.1.4 Assortative-mating and syngnathid evolution
Identifying factors associated with speciation can help to understand why
groups vary in diversity. Recent studies have put forward the hypothesis
that size assortative mating may play a role in the speciation process of
many different fish species, by initiating and maintaining reproductive
isolation [e.g. in the stickleback, Gasterosteus aculeatus (Nagel & Schluter
1998; McKinnon et al. 2004), the sockeye salmon, Oncorhyncus nerka
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Chapter 5. Final Discussion
(Foote & Larkin, 1988), the charr, Salvelinus alpinus (Sigurjonsdottir &
Gunnarsson 1989) and the mosquitofish, Gambusia holbrooki (McPeek,
1992)]. Within syngnathids, Jones et al. (2003) investigated the possibility
that such a situation may have been important in the diversification of the
genus Hippocampus. In seahorses, monogamy forces both males and
females to place a high premium on finding a partner with a similar
reproductive capacity, otherwise eggs or brood pouch space would be
wasted. Reproductive output is positively correlated with size for both
sexes, and selection therefore favours size assortative mating (Vincent &
Sadler, 1995). According to Jones et al., (2003) this mating pattern
coupled with modest disruptive selection on body size can effectively
produce sympatric speciation over quite short time scales.
By demonstrating that social constraints (bi-directional mate
choice and female-female competition) might also lead to size assortative
mating in a non-monogamous syngnathid, the results presented in this
thesis warrant broader attention to the role of size assortative mating in the
speciation process of the whole family Syngnathidae, which is, by far, the
most diverse group in the order Gasterosteiformes, with approximately 230
species described (Dawson, 1985).
5.1.5 Female reproductive investment
Theoretical studies predict that females facing constraints in mate choice
(e.g. due to competition and/or mutual mate choice) should differentially
allocate their reproductive effort according to the attractiveness of their
mates and the likelihood of finding a better one in the future (Burley, 1986,
1988; Sheldon, 2000). Accordingly, the study presented in Chapter 3.3
revealed a complex egg allocation pattern, with different-size females
adopting different investment tactics that reflect their distinct mating
prospects. Large, dominant and preferred females, who have increased
opportunities to mate with high quality partners, seem able to monitor the
number and quality (body size) of available mates, laying more eggs when
in the presence of more than one large male, or ‘deliberately’ saving their
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Chapter 5. Final Discussion
reproductive efforts for future high quality males when presented with
either one (large or small) or four small males. In contrast, the constant
reproductive effort of small females through all mating contexts strongly
suggests that they compensate their lower chances of laying eggs in a
higher quality male, by maximizing the number of eggs laid in each mating
event.
Another important finding presented in Chapter 3.3 deals with the
apparent ability of large females to adjust egg size according to male
quality. When simultaneously presented with both large and small potential
partners, these females laid larger eggs in larger males while depositing
smaller eggs in lower quality individuals. Interestingly, this allocation
appears to be the result of a combination of two processes rather than an
ability to adjust egg size according to male quality: i) serial mating (larger
males first) and ii) reduction in egg size over mating events. Since this
reduction was observed in all mating contexts, it is probably not ‘deliberate’
but rather a result of egg production constraints (e.g. Bagenal 1969;
Kinnison et al., 2001).
Changes in egg size according to the attractiveness of the male
are especially important because they are likely to confound estimates of
paternal effects on offspring fitness (Petrie & Williams, 1993). Larger eggs
containing high absolute amount of egg components might improve
offspring fitness (Kolm & Ahnesjo, 2005), thus generating a correlation
between the phenotype of the male and the performance of the offspring.
A similar association can also arise since female possible gather “good
genes” for their progeny when they mate with high quality males.
It has recently been suggested that mating order effects, such as
those reported for S. abaster, might result in strong directional selection on
male traits (both behavioural and physiological) (e.g. Wedell & Cook, 1998;
Birkhead et al., 1999; Evans & Magurran, 2001). If this is the case then the
male’s active role in courtship observed in S. abaster is likely to be a
selected trait aimed at securing the highest quality portion of a female’s
investment through mating precedence.
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Chapter 5. Final Discussion
5.1.6 Multiple mating strategies
Even though no molecular studies have been conducted and observations
reported in this thesis are from captive fish, the available evidence strongly
suggests that S. abaster mating system is polygynandrous since both
males (Chapter 2.3) and females (Chapter 2.3 and Chapter 3.3) have
multiple mates in the course of a single male pregnancy. Similar
observations have been reported for other Syngnathus species where
males can carry eggs from different females and a female can distribute
ripe eggs among many partners (Berglund et al., 1988; Vincent et al.,
1992, 1995; Jones & Avise 2001). Two types of explanations to this egg
allocation pattern have been postulated in Berglund et al. (1988).
i) It might be a consequence of the time required for the maturation
of the entire egg clutch. In S. abaster, however, some females allocated
their eggs within 24 hours (Chapter 2.3 and Chapter 3.3), within which time
span eggs probably have not time to mature (Wallace & Selman, 1981).
ii) It is an adaptative tactic of the male and/or the female,
increasing own or offspring survival.
According to Berglund et al. (1988) the only acceptable
explanation for a male to strive for multiple matings is to prevent receiving
an entire brood from a female of low genetic quality. Accordingly, Ahnesjo
(1996) referred that partial filling may be a means for males to optimize the
trade-off between the costs of searching for large attractive females and
mating with smaller ones. This however implies that males, rather than
females, control for the number of eggs transferred during copulation. If
this is the case, males may tend to accept fewer eggs from small females
given the possibility that the next mate may be larger. Thus, the male is
assured of some progeny while reserving additional brood space for a
larger female that might appear later (see Jones et al., 2000). Even
considering that the study presented in the Chapter 3.3 of this thesis was
not aimed at answering this question, the current results do not show
significant differences in the number of eggs carried by males S. abaster
when joined with large or small females. Instead, the results suggest that
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Chapter 5. Final Discussion
females of this species (at least large ones) can have a fine-tuned control
over the number of transferred eggs.
If males fall victim of predation, a female faces the risk of loosing
all her offspring if she has allocated all her eggs into a single partner
(Berglund et al., 1988; Tomasini et al., 1991). Also, if minimizing the
variance in the number of produced offspring (“bet-hedging”) is important,
egg allocation among several males would be the predicted strategy
(Berglund et al., 1988). In S. typhle, for example, females seem genetically
predisposed to seek a “bet-hedging” strategy wherein they typically lay
eggs in the pouches of multiple males, particularly when males are
common and the quality of those males is difficult to assess (Jones et al.,
2000). Interestingly, S. abaster females presented with several males did
not have more partners in such contexts (homogenous mating settings
comprising only large or small males, see Chapter 3.3). Instead, large
females adopted a differential allocation strategy according to the quality of
the male.
Finally, spreading the eggs may reduce competition among
siblings in the male’s marsupium in a density dependent fashion (Berlgund
et al., 1988; Ahnesjo, 1996).
Promiscuous mating systems as that here reported for S. abaster
are likely to reduce the penalty for males for having less quality mates,
thus reducing the intensity of sexual selection among females and hence
explaining why Syngnathus species are generally less dimorphic than
marsupium-lacking pipefish (Berglund & Rosenqvist, 2003). In these later,
males receive an entire brood from a single female and thus should be
more pressured to select a more fecund partner, which may result in more
intense sexual selection on females. The large body size and vivid
coloration in females of N. ophidion and N. lumbriciformis, for example, are
probably a good example of this kind of increased sexual selection
pressure.
Variation in sexual dimorphism within a single genus also occurs
and may be far more difficult to explain. Recently, Ripley & Foran (2006)
suggested that differential parental nutrient allocation could explain
differences in sexual traits and behaviours between S. floridae and S.
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Chapter 5. Final Discussion
fuscus. According to these authors, pouch closure is not indicative of the
degree of physiological allocation of nutrients by brooding males to
embryos. Instead, brood pouch physiology varies between related species
with similar brood pouch appearance. In Syngnathus species, where
males contribute to a greater proportion of parental care, Ripley & Foran
(2006) predict a divergence in sexual dimorphism and sex-roles. In
contrast, more balanced investment in progeny between the sexes
predicts comparable reproductive rates for males and females with the
sexes not evolving strongly dimorphic traits and behaviours.
5.1.7 What causes sex-role reversal?
The standard explanation for reversed sex-roles is that males constitute a
bottleneck for female reproduction. Within syngnathids this has been
demonstrated experimentally in a number of species where marsupium
space limitations and pregnancy length proved to constrain female
reproductive success. In other words, females were found to lay more
eggs than a male could accommodate in its marsupium during the extent
of a breeding period [e.g. S. typhle and Nerophis ophidion, Berglund et al.
(1989)]. Interestingly, marsupium space does not seem to physically limit
females in S. abaster, in aquaria (Chapter 3.3) or in the field (Chapter 3.4).
As a fact, neither did females spawn enough eggs to fully occupy a male’s
pouch during the extent of a pregnancy (Chapter 3.3), nor did males
presented, on average, fully occupy pouches over the breeding season
(Chapter 3.4). These observations pose an interesting question: what then
causes the sex-role reversal in S. abaster?
Even though marsupium space, per se, might not be a limiting
factor in (at least in the studied population) other variables might be
limiting females. For instance, males could refrain from accepting eggs
due to physiological reasons. In S. typhle, for example, the folds of the
marsupium merge after 3-4 days subsequent to the first mating event, a
placenta-like tissue is formed and males are no longer receptive (Berglund
et al., 1986). Preliminary observations suggest that also in S. abaster,
155

Chapter 5. Final Discussion
males no longer accept new eggs once a few days have elapsed since
they first reproduce. Caring a developmentally homogenous brood is likely
to be beneficial to males as it will enable them to give birth to all young
simultaneously, thus reducing pregnancy length and hence increasing
potential reproductive rates. The synchronous development of the brood
might also present advantages to the larvae since hypothetical changes in
paternal investment during pregnancy will affect the young in a similar
way. Also, the opening of the skin folds that would allow fully developed
juveniles to leave the marsupium could affect developing larvae.
Allocation trade-off reasons could also make males not accept as
many eggs as they can physically accommodate. If resources provided by
the males are limited, half filled males are likely to have the potential to
provide all offspring with an ample resource supply. An indication that
resources could actually be limiting is the lack of correlation between male
and offspring size in half-filled S. typhle males, whereas in filled males
larger individuals produced larger offspring (Berglund et al., 1988). The
difference in marsupium occupations observed in large and small males in
S. abaster could indeed be the result of different trade-offs between egg
size and egg numbers, with a larger empty space in the brood pouch being
more beneficial for large males carrying larger eggs which probably
demand more resources (Berglund et al., 1986). A field study such as the
one presented cannot, however, give insights on larval survival or size at
birth and, as such, trade-offs mechanisms cannot be accurately assessed
with the presented data.
The fact that reproduction in S. abaster appears strongly
size-assortative could also contribute to a reduction on available mates
helping to explain the moderate sex-role reversal of this species. If larger
males are preferred both by large and small females, then available mating
partners tend to be scarce. Since males are also choosy, a bottleneck for
females can be easily predicted. Other factors such as the number of
produced gametes (and the rate at which they are produced) over the
entire breeding season could also show additional female conditioning.
To fully understand sexual selection in a species, it is essential to
explore the range of spatial or temporal variation in selection regimes. As
156

Chapter 5. Final Discussion
such, it would be particularly interesting to analyse the behavioural and
morphological sexual differences in other S. abaster populations. All else
being equal, differences in the length of the brood pouch as those reported
by Cakic et al. (2002) for S. abaster populations of Ukraine are likely to
affect the extent to which males constrain female reproduction.
A different pattern of results is also likely to occur in latitudes
where the extent of the breeding season markedly differs from that of the
studied population. As mentioned in Chapter 4.1, it could be predicted that
the limited marsupium space could indeed be a limiting factor for females,
particularly those of small size, in areas where the extent of the breeding
period would not allow for time-scattered spawnings.
5.1.8 Further research: an endocrine approach to
sex-role reversal
In most teleosts, 11-ketotestosterone (OT) is the dominant circulating
androgen in breeding males (see Fostier et al., 1987), being high in males
and low in females. In contrast, the estrogen 17β-estradiol (E2) is generally
higher in females than in males. The levels of testosterone (T) and 17α-
hydroxy-20β-dihydroprogesterone (17,20-P), in turn, may be high in both
sexes (Fostier et al., 1983).
In species where the sex-roles are reversed, it could be expected
deviations in this characteristic pattern. To date, however, only one study
has explored this hypothesis. Mayer et al. (1993) measured plasma levels
of androgens 1 by means of radioimmunoassay (RIA) during the breeding
season in three species of pipefish (Nerophis ophidion, Syngnathus typhle,
and Syngnathus acus). Three main results were obtained: i) the levels of
most measured hormones were not consistently different between the
sexes; ii) in the case of the OT pipefish showed the “typical” teleosts
1 T, OT, 11β-hydroxytestosterone (OHT), 11-ketoandrostenedione (OA),
11β-hydroxyandrostenedione (OHA), together with (17,20-P) and E2.
157

Chapter 5. Final Discussion
pattern, in that levels of this androgen were higher in pre-breeding than in
post-breeding males (individuals sampled during the earlier period of the
reproductive cycle, including both courtship and mating phases, and
individuals sampled during the subsequent period of parental care,
respectively) and iii) the observed relatively high levels of E2 in breeding
males indicated a partial reversal of the normal teleosts steroid pattern
between the sexes, although this is somewhat contentious, as the single
highest E2 level was measured in a female.
Overall, the data from this study did not suggest that steroid levels
in pipefishes were markedly different from the “typical” teleosts pattern and
thus that sex-role reversion might not be accompanied by an endocrine
reversal. Further work is however needed in this area. In this extent, the
study of the varying degrees of sex-role reversal occurring in syngnathids
offers unique opportunities to enhance the current understanding of the
neuroendocrine mechanisms mediating behavioural sex differences.
5.1.9 A ‘mild’ sex-role reversion
As data was being collected, it seemed more and more evident that mating
patterns and sex-roles are far more complex than it could be thought
initially. When taken together, the studies presented in this thesis reveal a
striking flexibility in the expression of sex-role reversal, with several
variables (e.g. temperature and sex-ratios) being able to influence the
strength of intra-sexual competition and mate choice in both males and
females of S. abaster. Clearly, in this species, females are the primary
competitor sex but both sexes can end up at different degrees of
choosiness and competition and both are morphologically adapted to
‘advertise’ one’s condition and quality to potential mates and/or rivals, a
pattern fitting more closely into the proposed designation of a “mild”
sex-role reversal than into the binary classification of sex-role reversal.
According to the available evidence, the extent to which individuals
engage in competition and/or choosiness seems not merely a question of
sex, but rather of the relative costs and benefits associated to certain
158

Chapter 5. Final Discussion
behaviours under particular contexts. Females, in particular, seem to be
able to continuously update information regarding their relative “sexiness”
and mating prospects under the surrounding conditions (both physical and
social) of the environment in which they are inserted.
Are S. abaster reproductive patterns unique among syngnathids?
Probably not. A recent study on the pot-bellied seahorse, Hippocampus
abdominalis, indicates that, also in this species, sex-roles are plastic,
being strongly affected by local conditions including sex-ratio and
population density (Wilson & Martin-Smith, 2007). The opportunity to look
for more cases and further explore the potential of syngnathids as
premiers models for the study of sexual selection may however be running
out of time and important research allowing for a fully comprehension of
the complex mating game between males and females may be seriously
compromised.
5.2 Conservation note
Pipefish populations, just as much as seahorses’, are currently in an overt
process of decline as they are directly affected by most of the threatening
processes occurring in the oceans, including overexploitation, incidental
by-catch and habitat degradation or fragmentation (Baillie et al., 2004).
Among other important syngnathid habitats, the vanishing of
marine angiosperm populations, which assume a crucial multifunctional
role in marine ecosystems (see Bell & Harmelin-Vivien, 1983; Almeida,
1994; Nagelkerken et al., 2000), is currently happening at an alarming
rate. Only small, especially distant foci still persist, whose future will be
uncertain, as cumulative human impacts are increasingly felt on estuarine
and coastal areas (Powles et al., 1999). In the Ria de Aveiro estuarine
lagoon, where the fish used for this thesis where caught, large areas of the
lagoon bed were covered by seagrasses (Zostera, Ruppia, Potamogeton)
until 1980. Ever since, seagrass species experienced a marked reduction
such that they are now completely absent from sub-tidal areas and are
found only in restricted inter-tidal zones (Figueiredo et al., 2004).
159

Chapter 5. Final Discussion
The apparent benthic behaviour of newborn S. abaster and S.
acus presented in Chapter 2.1 and 2.2, respectively, unlike that reported
for syngnathid species with pelagic larval phases [e.g. Nerophis
lumbriciformis, Monteiro et al., (2003)], probably implies a limited
dispersion capability, thus contributing to the ongoing isolation of
geographically distant populations. Habitat fragmentation and/or long
distance colonization together with a restrictive dispersal have already
been pointed out as determinant forces on the current geographical
distribution of certain seahorse species (Lourie et al., 2005).
Within mainland Portugal, S. abaster populations seem to be
already confined to estuaries and salt ponds, as in the Ria de Aveiro
(Monteiro, personal observation). In this particular lagoon, the massive
degradation, mainly due do changes in land use patterns, pollution from
industry and fishing ports, paper factories, urban sewerage and cattle
raising, has been leading to a loss of biodiversity. Only 38 of the 55 fish
species reported from the Ria at the in 1992 (Rebelo, 1992) were
observed in more recent samples (Pombo et al., 2002). Within
syngnathids, Syngnathus abaster seems to be a particularly vulnerable
species (see Table 5.1).
Further studies of syngnathids early life strategies are crucial for
understanding the consequences of fragmented habitats on populations.
Basic data on reproduction related-parameters would allow for the
differentiating between anthropogenic effects and natural population
dynamics. As an example, regular population monitoring providing
estimates of abundance, sex-ratio and size structure has documented
significant declines in populations of big-bellied seahorses over different
sites in the Derwent estuary that could be caused by some form of
reproductive limitation related to the death of males in the population
(Martin-Smith & Vincent, 2005).
Another particularly important point in conservation is to be able to
predict what will happen to populations as a result of current or future
environment changes. An advantage of studies aiming at understanding
the behavioural decisions made by individuals at different environmental
conditions, such as those presented in Chapter 3.1 is that it enables to
160

Chapter 5. Final Discussion
prediction of the species behaviour when exposed to novel conditions. To
this extent, the demonstrated importance of water temperature on
syngnathid reproduction means that a climate-induced increase in sea
surface temperature (Walther et al. 2002) may already be affecting
reproduction, a pattern recently documented in two pipefish species,
Entelurus aequaoreus (Kirby et al., 2006) and N. lumbriciformis (Monteiro
et al., 2002). Studying natural variation in reproductive dynamics may thus
provide valuable insights into the expected impacts of long-term climate
warming on reproductive biology of syngnathids and fish in general.
Table 5.1: Abundance of syngnathids in the Ria de Aveiro estuarine
lagoon
2 Rebelo (1992).
Species 1992 2 2002 3
Syngnathus acus Linnaeus, 1758 704 662
Syngnathus typhle Linnaeus, 1758 16 38
Syngnathus abaster Risso, 1826 188 16
Hippocampus hippocampus Linnaeus, 1758 1 0
3 Pombo et al. (2002).
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