the phylogeny and biogeography of the genus zonites - Journal of ...

Journal of Molluscan Studies Advance Access published 20 February 2009
THE PHYLOGENY AND BIOGEOGRAPHY OF THE GENUS ZONITES
MONTFORT, 1810 (GASTROPODA: PULMONATA): PRELIMINARY
EVIDENCE FROM MITOCHONDRIAL DATA
PANAGIOTIS KORNILIOS 1 , NIKOS POULAKAKIS 2,3 , MOYSIS MYLONAS 1,2
AND KATERINA VARDINOYANNIS 2
1 Department of Biology, University of Crete, Vassilika Vouton, PO Box 2208, 71409 Heraklion, Crete, Greece;
2 Natural History Museum of Crete, University of Crete, Knosos Avenue, PO Box 2208, 71409 Herakleio, Crete, Greece; and
3 Department of Ecology and Evolutionary Biology and Yale Institute of Biospheric Studies, Yale University, New Haven, CT 06520-8106, USA
(Received 6 June 2008; accepted 26 November 2008)
ABSTRACT
The genus Zonites, which includes 26 extant species, is distributed in the northeastern Mediterranean
area and exhibits significant diversity and endemism. This is the first phylogenetic study of Zonites,
based on partial mitochondrial DNA sequences of the 16S rRNA gene. A total of 43 specimens,
belonging to 15 species from 39 localities in Greece and Turkey, were included. Analyses revealed
two major clades, corresponding to two distinct geographic regions, west and east of the mid-Aegean
trench. The palaeogeography of the Aegean and the plasticity of the Zonites shells have produced a
great variety of forms that have been identified as different species or subspecies. Our results,
although preliminary, suggest that the taxonomy of Zonites requires revision. We propose an historical
interpretation of the present distribution of Zonites, based on the palaeogeographic history of the
Aegean region.
INTRODUCTION
The family Zonitidae is one of the most species-rich families of
pulmonates. It is represented in Greece by c. 117 species that
belong to 10 genera (Riedel, 1992): Vitrea, Lindbergia, Gyralina,
Aegopinella, Allaegopis, Doraegopis, Balcanodiscus, Oxychilus,
Eopolita and Zonites. The family is diverse in terms of its large
number of species, their significant morphological divergence
and their high endemism. The Greek zonitid fauna is a few
times richer than that of any other area of similar or greater
size and 75% of the worldwide zonitid species are endemic to
Greece (Riedel, 1992).
The genus Zonites Montfort, 1810 includes 28 species, of
which 25 are found in Greece (seven of these also present in
Turkey; 18 exclusively endemic to Greece) (Riedel, 1992;
Riedel & Mylonas, 1995, 1997; Schu¨tt, 2005). Two species
(Z. santoriniensis and Z. siphnicus) are extinct and found only as
subfossils (Riedel, 1985; Riedel & Mylonas, 1997) (Fig. 1).
Zonites is distributed in the northeastern part of the
Mediterranean region. In particular, it is found in central and
southern Peloponnisos (six species), in Evvoia and the adjacent
coast of Attica (two species), in the Aegean islands (15 species,
including the two extinct species) and in western Turkey (10
species, three of which are endemic) (Fig. 1). Zonites is found
only as a fossil in the southwestern Kyklades and in Crete, and
is absent from some areas whose malacofauna has been
thoroughly studied, such as the northern Kyklades, Sterea
Ellada and some islands of the central East Aegean (Riedel,
1992).
Formerly, the genus Zonites was divided into two subgenera:
Zonites, distributed in Greece and western Asia Minor as
already described, and Turcozonites, distributed in southeastern
Turkey and Syria (Riedel, 1987a). These two taxa are now
considered to be distinct genera (Riedel, 1995).
Correspondence: P. Kornilios; e-mail: korniliospan@yahoo.gr
The genus Zonites is believed to be of eastern origin (Riedel,
1987b) and most probably descended from an ancestral form
that was related to Turcozonites, since the supposedly most basal
Zonites species (Z. osmanicus) occurs at the eastern edge of the
range, in Asia Minor (Riedel, 1992).
According to Riedel (1992) the diversification of Zonites
dates from the Neogene, when these snails must have expanded
from Asia Minor to Evvoia and Peloponnisos via the Kyklades
bridge (the islands of the Kyklades complex were at that time
connected above sea level). The numerous species and high
endemism of Zonites may be explained by palaeogeographic
events that formed the present Aegean archipelago during the
Neogene and Pleistocene. The palaeogeographic history of this
region is described in detail by Poulakakis et al. (2005a).
During the early and middle Miocene (23–12 Ma), the
Aegean was part of a continuous landmass known as Agäis. At
12–8 Ma intense tectonic movements probably initiated the
modern configuration of the Aegean, causing the breaking up
of the southern Aegean landmass. At the end of the middle
Miocene (12 Ma), the mid-Aegean trench (east of Crete and
west of Kasos–Karpathos) began to form and was fully
completed during the early late Miocene (c.10–9 Ma). This
phenomenon caused the separation of western from eastern
Aegean islands. Crete, which was connected with the mainland
during the Miocene, became an island 5 Ma, and has not
been connected with the mainland since then. The central
Aegean islands were united in a landmass connected to the
Greek mainland until the Pliocene, although they have had no
direct connection with Peloponnisos since the late Tortonian
(8 Ma). In the late Pleistocene (21,500 year BP), this landmass
(known as the Kyklades bridge) had no connection with the
mainland. The break-up of the Kyklades plateau into separate
islands occurred later, at about 11,500 year BP. Kythira was
partly submerged in the early Pliocene, and did not re-emerge
until the late Pliocene (c.2.5 Ma), while in the Pleistocene it
was probably an island, smaller than today, but not as far
from the coast (Peloponnisos). Karpathos was connected with
Journal of Molluscan Studies (2009) 00: 1–9 doi:10.1093/mollus/eyp003
# The Author 2009. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
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the Turkish mainland via Rodos during the early Pliocene. It
was almost totally submerged during the late Pliocene, while
in the Pleistocene it re-emerged and probably was connected
with Kasos. Rodos remained connected with the Turkish
mainland until the early Pleistocene, when it was finally isolated.
Finally, the isolation of the eastern Aegean islands (e.g.
Samos, Lesvos, Chios) from the Turkish mainland occurred
during the late Pleistocene or Holocene (data from
Creutzburg, 1963; Daams & Van der Weerd, 1980;
Dermitzakis & Papanikolaou, 1981; Meulenkamp, 1985; Boger
& Dermitzakis, 1985; Dermitzakis, 1990; Sondaar, et al., 1998;
Perissoratis & Conispoliatis, 2003).
The systematics of Zonites is based on external (shell) and
internal (genitalia) morphology. Some of the currently recognized
species show significant morphological variation; some
individuals, assigned morphologically to a certain species, also
bear characteristics of other species (e.g. Z. graecus comprises
forms that resemble Z. kobelti or Z. messenicus on Peloponnisos,
while some individuals identified as Z. messenicus bear characters
of Z. graecus and, as Riedel (1992) states, Z. messenicus could be
considered a subspecies of Z. graecus). The nomenclature of
several species has been changed on many occasions in the past
and Riedel (1992) has pointed out the need for further studies
on their biogeography, systematics and nomenclature.
Although numerous studies have used morphological characters
to establish taxonomy and to propose phylogenetic
relationships of terrestrial gastropods in the past, in many cases
their remarkable phenotypic diversity has led to controversial
systematics. Mitochondrial and nuclear genetic markers are
now widely used to study stylommatophoran phylogeny (e.g.
Douris et al., 1995; Parmakelis et al., 2003; Steinke, Albrecht &
Pfenninger, 2004; Parmakelis et al., 2005; Pinceel, Jordaens &
Backeljau, 2005; Ketmaier, Giusti & Caccone, 2006;
Pfenninger, Cordellier & Streit, 2006; Douris et al., 2007). Some
of these studies suggest that shell morphology alone is a poor
P. KORNILIOS ET AL.
Figure 1. Map showing distribution of the genus Zonites, sampling localities (map codes are reported in Appendix) and several important regions
and islands mentioned in text.
2
guide to taxonomy, because shell variation is often continuous
or overlapping, and shells may be phenotypically plastic
(Steinke et al., 2004; Ketmaier et al., 2006; Pfenninger et al.,
2006). Therefore, studies combining traditional taxonomy with
DNA-based results are needed (Pfenninger et al., 2006).
Despite the diversity and endemism of Zonites, little work has
been done on its phylogeny and phylogeography. Nevertheless,
terrestrial molluscs can be particularly informative in studies of
the biogeography of islands, such as the Aegean archipelago
with its complex palaeogeographic history (Mylonas, 1982;
Riedel, 1992; Riedel & Mylonas, 1995).
This preliminary study is the first phylogenetic approach to
the genus Zonites, and includes c. 65% of the extant Zonites
species found in Greece. Since only a relatively small fragment
of a single mitochondrial molecular marker (16S rRNA) was
analysed, this data set does not permit definitive conclusions or
support taxonomic revision. However, it serves our two aims of
testing whether the genetic relationships agree with the current
morphology-based taxonomy, and proposing a hypothetical
biogeographic scenario to explain the present geographic
distribution.
MATERIAL AND METHODS
Sampling of taxa
A fragment of the 16S gene was sequenced from 43 individuals
of Zonites. Two individuals of the genus Turcozonites were used
as outgroup. All sequences were obtained from museum
samples, deposited in the Natural History Museum of Crete,
preserved in 75% alcohol. The sampling localities are shown
in Figure 1 and the specimens are listed in the Appendix. The
identification of species and subspecies was based on shell morphology
and the anatomy of the genitalia (Riedel, 1987a,
1987b, 1992, 1995; Riedel & Mylonas, 1995, 1997).
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DNA extraction and amplification
Total genomic DNA was extracted from foot tissue (tail tip).
After homogenization, the Macherey–Nagel NucleoSpin
Tissue DNA extraction kit was used.
Using the universal primers 16Sar-L and 16Sbr-H (Palumbi
et al., 1991), we amplified a c. 500-bp segment of the 16S
rRNA gene (primary PCR). The amplification of DNA from
samples preserved in 75% alcohol is a difficult procedure, due
to DNA damage. Because none of the specimens produced a
visible PCR product in agarose gel (1%) stained with EtBr, we
applied a nested PCR. With a pair of internal primers, PyrFor
(5 0 -GCGGCAGTACATTGACTGTGC-3 0 ) and PyrRev1
(5 0 -GCCTTAATCCAACATCGAGGT-3 0 ), whose design was
based on published land-snail sequences, we performed a secondary
PCR using the primary PCR product as template. In
this way we amplified a segment of c. 310 bp of the 16S rRNA
gene. PCR conditions for both pairs of primers involved an
initial cycle of denaturation at 948C for 5 min, and 40 subsequent
cycles of 948C for 1 min, 428C for 1 min and 728C for
1 min, in the presence of 1.5 mM MgCl2.
Purification and sequencing
PCR products were purified with the Macherey–Nagel
NucleoSpin Extract II DNA purification kit. Sequencing was
conducted on an ABI PRISM automated sequencer using the
Big-Dye Terminator (v. 3.1) Cycle Sequencing kit and the PCR
primers. Both forward and reverse strands of the PCR product
were sequenced. We performed an NCBI-BLAST search (http://
blast.ncbi.nlm.nih.gov/) to confirm that the obtained sequences
belonged to pulmonate molluscan 16S rRNA.
Alignment and genetic divergence
DNA sequences were aligned using CLUSTAL X (Thompson,
Higgins & Gibson, 1994) and adjusted by eye. All DNA
sequences are deposited in GenBank (accession numbers:
EF568962–EF569006).
Distance-based genetic divergences were estimated in
MEGA v. 2 (Kumar, Tamura & Nei, 2004) using the Kimura
2-parameter model (Kimura, 1981).
Phylogenetic analyses
Three methods were used to conduct the phylogenetic analyses:
Neighbour-Joining (NJ), Maximum Likelihood (ML)
and Bayesian inference (BI).
The selection of the most suitable model of DNA substitution
for the ML and BI analyses was done using Modeltest
3.7 (Posada & Crandall, 1998), under the Akaike Information
Criterion (AIC; Akaike, 1974). The best-fit model was K81 þ
G (Kimura, 1981) (model parameters: ln L ¼ 21855.670, K ¼
93, Rmatrix A/C ¼ 1.00, A/G ¼ 5.37, A/T ¼ 1.97, C/G ¼
1.97, C/T ¼ 5.37, G/T ¼ 1.00, a ¼ 0.41).
NJ analysis was performed with PAUP* (v. 4.0 b10;
Swofford, 2002), with 1000 bootstrap replicates to estimate
statistical support values (Felsenstein, 1985).
ML analyses (Felsenstein, 1981) were conducted using
PAUP* and GARLI v. 0.95 (Zwickl, 2006). In PAUP*, heuristic
ML searches were performed with 10 replicates of random
sequence addition and TBR branch swapping, based on the
successive-approximations strategy of Swofford et al. (1996) and
Sullivan et al. (2005). In GARLI, heuristic ML searches were
performed with 10 5 replicates of random sequence addition
using the GARLI algorithm. Since a ML tree search with such
a complex model would be computationally excessive in
PHYLOGENY OF ZONITES
3
PAUP*, the confidence of the nodes was assessed only in
GARLI based on 1000 bootstrap replicates.
The Bayesian analysis was performed in MrBayes (v. 3.1;
Ronquist & Huelsenbeck, 2003) with two runs and eight
chains for each run, for 10 7 generations and the current tree
was saved every 100 generations. This generated an output of
10 10 4 trees. Stationarity was reached, both in terms of likelihood
scores and parameter estimation, after c. 10 6 generations
and the first 10 4 trees (10% ‘burn-in’ in Bayesian terms) were
discarded as a conservative measure to avoid the possibility of
including random, sub-optimal trees. During tree search, full
parameter estimation was performed, and the posterior probabilities
were calculated as the percentage of samples recovering
any particular clade (Huelsenbeck & Ronquist, 2001),
where probabilities 95% indicate significant support. Two
further independent Bayesian analyses were run so that global
likelihood scores, individual parameter values, topology and
nodal support could be compared to check for local optima.
Testing alternative hypotheses
We tested two explicit hypotheses: (1) the monophyly of
Z. pergranulatus and (2) the monophyly of the species groups to
the east and west of the mid-Aegean trench (Fig. 3). The corresponding
hypothetical trees (a tree constrained to be monophyletic
for Z. pergranulatus and a tree constrained to be
monophyletic for the species east of the mid-Aegean trench)
were generated with PAUP* under the likelihood criterion,
and compared with our optimal topology using the
Shimodaira–Hasegawa (SH) test (Shimodaira & Hasegawa,
1999) as implemented in PAUP* and employing RELL bootstrap
with 1000 replicates.
Calibration of molecular clock and estimation of divergence times
A log-likelihood ratio test (LRT) was used to examine the
clock-like evolution of sequences of the ingroup by calculating
a x 2 statistic (LRT) based on ML values with and without
rate constancy enforced (x 2 ¼ 2 [(2ln LCLOCK) 2 (2ln
LUNCONSTRAINED)], df ¼ number of terminal nodes 2 2)
(Felsenstein, 1981). The divergence times of Zonites lineages
were estimated in the software r8s (v. 1.7.1) (Sanderson, 1997,
2002) with the recommended Powell algorithm using the
Langley–Fitch method (LF), which uses ML to reconstruct
divergence times under the assumption of a molecular clock,
since this hypothesis was not rejected. To estimate substitution
rates, we used a single calibration point based on the assumption
that divergence between Z. festai from Rodos and Z. casius
from Kasos began 3.5 Ma (enforcement of maximum age constraint
on that node), soon after the separation of the Kasos–
Karpathos island group from the island of Rodos, which took
place 3–3.5 Ma (Daams & Van der Weerd, 1980). These
monophyletic lineages are suitable for this calibration, as they
form sister clades with low intra-specific variability. Factors
that could affect clock calibrations include stochastic variation
at low levels of sequence divergence and the possibility of
extinct or unsampled lineages (Emerson, Oromı´ & Hewitt
2000; Emerson, 2002). The separation of the Kasos–Karpathos
island group from the island of Rodos has been successfully
used as a reliable calibration point in the past (Poulakakis
et al., 2005a).
RESULTS
Polymorphisms and genetic divergence
The 16S segment provided 310 bp, containing 150 variable
sites (48.4%), 137 of which (44.2%) were parsimony-
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Table 1. Average genetic divergences (%), based on the Kimura 2-parameter model (Kimura, 1981), within (bold text) and between the
geographical groups.
informative. Excluding the outgroup, there were 129 (41.6%)
variable sites, of which 116 were parsimony-informative
(37.4%). The mean values of A, T, G and C, within the
ingroup, are 38.2%, 34.2%, 16.8% and 10.7%, respectively.
Genetic divergences varied from 0% (individuals of the
same species and of adjacent localities) to 45.7% between individuals
of remote localities (within the ingroup). The mean
value of the genetic divergences was 22.2%. The genetic divergence
values within and between the groups defined according
to their geographic and taxonomic status are shown in
Table 1. Within the groups the divergences vary from 0% to
2.4% (within the Z. pergranulatus pergranulatus group which
includes individuals from four different Aegean islands), while
between the groups, the smallest divergence is that between
the Z. rhodius martensi and the Z. r. elatior groups (1.2%) and
the largest one is that between the Z. smyrnensis group and the
group including all Peloponnesian species (40.4%).
Phylogenetic tree topology
All three phylogenetic analyses produced trees of the same topology
(Fig. 2). A geographic presentation of the phylogenetic
affinities is shown in Figure 3. ML analyses resulted in the
same topology with ln L ¼ 21848.248 and 21849.765 in
GARLI and PAUP*, respectively, while Bayesian inference
resulted in a topology with mean ln L ¼ 21933.843. Both runs
of the Bayesian inference produced identical consensus trees
and the 50% majority-rule consensus tree of all trees remaining
after burn-in is presented in Figure 3. Zonites appears to split
into two major clades: most of the Zonites individuals from the
eastern sampling localities (Turkey and the eastern Aegean
islands) are included in clade A, while clade B includes
mainly the individuals from mainland Greece and the central
Aegean islands. The support values for these main clades (NJ
bootstrap value/ML bootstrap value/BI posterior probability)
are, respectively, 62/55/0.81 for clade A and 91/77/1.00 for
clade B.
Clade B is subdivided into two subclades, B1 and B2, which
comprise specimens from continental Greece (Evvoia,
Peloponnisos, Kythira) along with Lesvos (a case of human
dispersal), and from the Aegean islands, respectively. The
respective support values are 85/76/0.80 and 79/64/0.90.
Finally, two main lineages are present in the islands of subclade
B2: lineage B2.1 (Karpathos, Kasos, Naxos, Amorgos,
P. KORNILIOS ET AL.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1. Chios, Turkey (Z. chl) 2.2
2. Chios, Samos (Z.smy) 20.8 1.9
3. Rodos (Z fes) 24.8 23.3 0
4. Kasos (Z.cas) 24.7 22.9 2.7 0.4
5. Kastelorizo (Z.car) 22.3 24.6 10.2 11.2 0.2
6. Karpathos (Z.r.m) 39.4 37.9 27.6 29.9 27.2 0.7
7. Kasos (Z.r.e) 39.3 38.5 26.5 28.7 25.5 1.2 0
8. Naxos, Amorgos, Kinaros, Levitha (Z.p.p) 36.5 39.2 30.4 30.7 29.0 7.2 7.4 2.4
9. Astypalaia, Astakida (Z.p.c & Z.ast) 32.1 35.6 34.6 35.0 31.4 17.0 15.7 19.7 0.9
10. Anafi (Z.ana) 27.8 33.6 29.8 30.2 28.4 11.3 10.3 13.8 3.8 0
11. Kythira (Z.alg. & Z.par) 35.8 37.3 23.3 25.4 27.2 16.7 16.7 17.7 17.6 16.6 0.3
12. Peloponnisos (Z.gra, Z kob & Z mes) 33.6 40.4 28.9 31.5 31.1 22.5 23.6 21.7 26.9 21.2 10.9 1.8
13. Evvoia (Z.oer) 32.2 38.3 19.7 21.2 23.3 19.8 19.9 19.5 19.1 15.3 11.0 10.8 0
14. Syria (Tur) 41.1 49.3 36.6 35.4 37.5 40.5 38.0 37.7 52.2 43.2 41.6 42.8 37.3 0
Maximum and minimum values between the groups are italicized. Species abbreviations are shown in the Appendix.
4
Kinaros, Levitha) and lineage B2.2 (Anafi, Astipalea,
Astakida) (92/57/0.98 and 99/94/1.00, respectively).
The SH test rejects the hypotheses that Z. pergranulatus is a
monophyletic taxon (P . 0.05), and that the species distributed
east and west of the mid-Aegean trench form monophyletic
groups (P . 0.05).
The existence of a uniform molecular clock implying the
homogeneity of evolutionary rates was tested with the LRT by
calculating a x 2 statistic. This test did not reject the null
hypothesis of a homogeneous clock-like rate for the tree produced
by the Zonites sequences from the Aegean region
(LRT ¼ 2*[1871.03 2 1849.76] ¼ 42.54, df ¼ 43, a ¼ 0.05,
xcritical ¼ 59.30 and P , 0.0001), assuming clock-like evolution
of the involved sequences. According to the calibration reference
point, the diversification of the lineages of Zonites included
in this study occurred at c. 11.7 Ma, during the late Miocene
(Fig. 2).
DISCUSSION
Polymorphism and genetic divergence
The 16S rRNA gene is considered a relatively conserved region
of the mitochondrial DNA. Numerous studies in the past have
shown that land snails exhibit great levels of mitochondrial
diversity, even for this relatively slowly evolving gene (Douris
et al., 1995; Thomaz, Guiller & Clarke, 1996; Parmakelis et al.,
2003, 2005). In our case, the genetic divergences among Zonites
specimens are extremely high (mean 22.2%, maximum
40.4%). However, given the relative small size of the 16S gene
segment analysed, these diversity and divergence estimates
may be biased, particularly since rRNA genes comprise both
conserved and rapidly evolving regions (Smit, Widmann &
Knight, 2007).
Phylogenetic relationships and systematics
The molecular phylogenetic hypothesis agrees to some extent
with the current taxonomy of the genus based on morphological
characters. In most cases, specimens of the same species
group together, even if sampled from different regions or
islands (e.g. Z. pergranulatus pergranulatus from Naxos, Amorgos,
Kinaros and Levitha; Z. algirus from Kythira and Lesvos;
Z. graecus from distant localities of Peloponnisos; Z. chloroticus
from Chios and Turkey; Z. smyrnensis from Samos and Chios).
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Figure 2. Tree summarizing phylogenetic relationships among the 45 individuals studied with the genus Turcozonites from Syria as outgroup. The
tree presented is the one produced from the BI analysis (all methods of analysis, NJ, ML and BI, produced trees of the same topology). Bootstrap
values of NJ/ML analyses are shown, respectively, above branches, and posterior probabilities of BI are below branches. The numbers by some
internal nodes (with relative high statistical support), highlighted with a small circle, indicate age of that node in millions of years.
On the other hand, some forms that have been morphologically
identified as different species group together and exhibit
such small genetic divergence values that the validity of these
species is questionable. As seen in Figures 2 and 3, the lineage
from Peloponnisos (B1 subclade) includes three species that form
a group with low intra-specific divergence (1.8%) compared
with the mean value of the ingroup (22.2%). This result agrees
with the observations that these species (Z. graecus, Z. kobelti,
Z. messenicus) present forms with intermediate morphological
characteristics and that Z. messenicus could be considered a subspecies
of Z. graecus (Riedel, 1992). The genetic divergence
between Z. parnonensis and Z. algirus from Kythira is also small
(0.3%), as are those between Z. pergranulatus cycladicus
(Astypalaia) and Z. astakidae (Astakida) (0.9%) and between
Z. anaphiensis (Anafi) and Z. p. cycladicus/Z. astakidae (2.4%). In
PHYLOGENY OF ZONITES
5
the latter cases, the morphological differentiation is not reflected
at the genetic level, at least as shown in our molecular data.
As seen on subclade B2 (Figs 2 and 3), individuals of
Z. pergranulatus pergranulatus (Naxos, Amorgos, Kinaros,
Levitha) are more closely related to individuals of Z. rhodius
(genetic divergence: 7.3%) than they are to the conspecific
Z. p. cycladicus (Astypalaia) (19.7%). A highly significant result
of the SH test further rejects the hypothesis of monophyly of
Z. pergranulatus in favour of the topology of Figure 2. In other
words, Z. pergranulatus seems to be polyphyletic, suggesting
that two (cryptic) species may be involved.
Although preliminary, our results suggest that revision of the
current taxonomy of the genus Zonites is needed, since there
are many inconsistencies between the molecular and the morphological
groupings.
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Figure 3. A geographical interpretation of the phylogenetic affinities among Zonites species. The mid-Aegean trench is shown as a thick dashed
line. The main clades of the phylogenetic tree are indicated. Long-distance affinities are indicated by narrow dashed lines.
Phylogeography
The tree topology suggests that the genus Zonites follows a phylogeographical
pattern (Figs 2 and 3) that is consistent with
the palaeogeographic history of the Aegean area.
The phylogenetic analysis identifies two major clades,
although it should be noted that one of them (Clade A) exhibits
low statistical support (Fig. 2). These two clades, A and B,
indicate two major lineages within Zonites and correspond to
two separate geographical areas (Fig. 3); they include species
from the east Aegean and from the west, respectively.
Although the low bootstrap values of the eastern Clade A call
for cautious conclusions, we can hypothesize that behind the
existence of these two clades lies a major palaeogeographical
event, the formation of the Mid-Aegean trench at 12–8 Ma.
This isolated the faunas of eastern and western Aegean islands,
and the genetic imprint has been found in phylogeographical
studies of many taxa, e.g. reptiles (Poulakakis et al., 2003,
2005a, b, c), land snails (Parmakelis et al., 2005), Coleoptera
(Fattorini, 2002) and scorpions (Parmakelis et al., 2006).
We suggest that the formation of the mid-Aegean trench was
also the cause of the separation of the putative eastern Clade A
and the western Clade B which, according to our molecular
clock data, began to diverge c. 11.7 Ma, in agreement with the
palaeogeographical data.
The western Clade B is subdivided into subclade B1
(Peloponnisos, Kythira, Evvoia, Lesvos) and subclade B2
(Aegean islands) (Figs 2 and 3). This separation, which seems
to have taken place some 7.9 Ma, reflects the isolation of island
Zonites taxa from those of the Peloponnesian mainland, which
must have occurred in the late Tortonian (8 Ma) (Dermitzakis
& Papanikolaou, 1981).
There are two possible biogeographic scenarios to explain
the existence of Zonites in Evvoia and Peloponnisos (Fig. 1).
The first suggests that the ancestral Zonites form dispersed via
P. KORNILIOS ET AL.
6
the Kyklades land bridge of the central Aegean to the Greek
mainland, and later colonized Evvoia and Peloponnisos. The
second assumes that this genus reached Peloponnisos and
Evvoia by two different pathways, through Crete and the
Kyklades bridge respectively. Our results show that Z. oertzeni
from Evvoia is grouped with the Peloponnesian species. This
implies a more recent common ancestry of the species from
Evvoia and Peloponnisos, possibly supporting the first biogeographic
scenario. Hence, we assume that Zonites inhabited
Sterea Ellada (Fig. 1) in the past and later disappeared from
this region. It should be noted that although southeastern continental
Greece has been thoroughly studied, no Zonites representatives
(living, subfossil or fossil) are known from this area
(field observations of co-authors).
Finally, the grouping of the island of Kythira with
Peloponnisos also reflects its palaeogeographical history, since
it was partly submerged in the Early Pliocene, and did not
re-emerge until the Late Pliocene (about 2.5 Ma;
Meulenkamp, 1985). During the Pleistocene it was probably
an island, smaller than today, but quite close to the coast
(Peloponnisos) (Dermitzakis, 1990). The presence of Z. algirus
from Lesvos (an island near the north coast of Asia Minor) in
the clade of Z. algirus from Kythira, is also expected. Zonites
land snails are not edible, and therefore have no commercial
interest. Nevertheless, Z. algirus, which most likely originated
from southern Peloponnisos, is the one species of the genus that
has been dispersed by humans. It has been introduced into
Lesvos, Turkey, southern France and western Greece (Riedel,
1992; O¨ rstan, 2003).
The other major subclade B2 is also subdivided into
lineages, B2.1 and B2.2 (Figs 2 and 3). This split took place c.
5.7 Ma. Lineage B2.2 includes Z. pergranulatus cycladicus,
Z. astakidae and Z. anaphiensis from Amorgos, Astakida and
Anafi respectively. These three islands are located between the
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central Aegean islands (west of the mid-Aegean trench)
and Kasos-Karpathos (east of the mid-Aegean trench).
Unexpectedly, these taxa are not joined directly with the specimens
from the other Kyklades islands (Naxos, Amorgos,
Levithta and Kinaros) (genetic divergence: 19.7%); instead
the latter taxa are joined first with the specimens of Z. rhodius
from Kasos and Karpathos, forming the lineage B2.1 (genetic
divergence: 7.0%). The results of the Shimodaira-Hasegawa
test confirm this, rejecting the hypothesis that all haplotypes
east of the mid-Aegean trench form a monophyletic group.
This can only be explained by a more recent dispersal event
from Kyklades to Kasos and Karpathos, giving rise to the form
recognized today as Z. rhodius. This recent dispersal, which
probably took place 2.6 Ma, also explains the existence of two
completely different forms of Zonites on Kasos (Z. rhodius and
Z. casius; genetic divergence: 28.7%). Over-seas dispersal of
land snails is most probably the result of transportation by
birds (attached to feathers or in digestive tracts), although
transport by wind (in case of small shells or eggs attached on
leaves) or on natural rafts cannot be excluded (Rees, 1965;
Vagvolgyi, 1975; Gittenberger et al., 2006).
Zonites p. pergranulatus does not seem to have differentiated
into significantly different forms in the central Aegean islands,
since the topology of lineage B2.1 does not reflect the different
islands. This can be explained by the relatively recent isolation
of these islands from each other, because the break-up of the
Kyklades plateau into separate islands occurred at about
11,500 year BP (Perissoratis & Conispoliatis, 2003).
Finally, the eastern clade A, whose existence is not strongly
supported (Fig. 2), shows a subdivision, dating from 9.4 Ma,
into two lineages (Figs 2 and 3): a lineage that comprises
Z. smyrnensis and Z. chloroticus in the north (Chios, Samos,
Turkey) and another that includes Z. festai, Z. casius and
Z. caricus in the south (Kasos, Rodos, Kastelorizo). These two
lineages are separated by a genetic divergence of 23.8%. The
lineage of Z. festai, Z. casius and Z. caricus shows a mean divergence
of only 8.0%, while Z. smyrnensis and Z. chloroticus are
divergent by 20.8%. This high value and the co-existence of
two genetically distant forms of Zonites on Chios (Z. smyrnensis
and Z. chloroticus) can be explained by multiple colonizations
by the genus from Asia Minor.
The genus Zonites has a patchy geographical distribution,
with regions where Zonites snails are completely absent
(Fig. 1). This fact, combined with the genetic connectivity
between regions such as Evvoia and Peloponnisos, as shown in
the phylogenetic tree of this study, suggests that this genus was
probably present in Sterea Ellada in the past (Fig. 1). At
present, this taxon is absent from southeastern continental
Greece, Crete, where it has been reported as a fossil (unpublished)
and certain eastern Aegean islands (absent or reported
only as subfossil). Some of its species are extinct (Z. siphnicus
from Sifnos, Sikinos and Folegandros; Z. santoriniensis from
Santorini) while others are ‘rare’ or ‘in the process of extinction’
(e.g. Z. nikariae from Ikaria; Z. chloroticus from Chios;
Z. rhodius rhodius from Rodos; Z. rhodius martensi from
Karpathos; Z. rhodius elatior from Kasos) (Riedel, 1992; Riedel
& Mylonas, 1997). It has been proposed that the genus Zonites
be protected by including it in the Red Data Book (Riedel &
Mylonas, 1997).
ACKNOWLEDGEMENTS
We are very grateful to all the people that donated samples for
this study or helped in the field: R. Belardinelli, R. Cameron,
A. Parmakelis, K. Triantis and A. Riedel. We would also like
to thank Aristides Kordomenidis for helping with the maps
and James Howlett for linguistic help. Finally, we appreciate
all helpful comments and suggestions of the anonymous
PHYLOGENY OF ZONITES
7
reviewers. This work was funded by the Animal Biodiversity
scholarship from the State Scholarships Foundation of Greece
to P. Kornilios.
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PHYLOGENY OF ZONITES
APPENDIX
The specimens examined in the present study: taxon name, sampling localities and regions, population map codes (Fig. 1), abbreviations and
accession numbers.
Map code Region/Island Locality Species Abbreviations Accession numbers
1 Astypalaia Ag. Panteleimonas Z. pergranulatus cycladicus Z.p.c. EF568962
2 Astypalaia Pontikousa Z. pergranulatus cycladicus Z.p.c. EF568963
3 Astypalaia Panagia Thoma Z. pergranulatus cycladicus Z.p.c. EF568964
4 Astypalaia Vathi Z. pergranulatus cycladicus Z.p.c. EF568965
5 Astypalaia Ag. Ioannis Z. pergranulatus cycladicus Z.p.c. EF568966
6 Astypalaia Z. pergranulatus cycladicus Z.p.c. EF568967
7 Astakida Z. astakidae Z.ast. EF568968
8 Karpathos Volada Z. rhodius martensi Z.r.m. EF568969
9 Karpathos Lastos Z. rhodius martensi Z.r.m. EF568970
10 Karpathos Avlona Z. rhodius martensi Z.r.m. EF568971
11 Naxos Za Cave Z. pergranulatus pergranulatus Z.p.p. EF568972
12 Amorgos Korakas Mts Z. pergranulatus pergranulatus Z.p.p. EF568973
13 Amorgos Profitis Ilias Z. pergranulatus pergranulatus Z.p.p. EF568974
14 Kinaros Z. pergranulatus pergranulatus Z.p.p. EF568975
15 Levitha Kentrika Z. pergranulatus pergranulatus Z.p.p. EF568976
16 Levitha Kentrika Z. pergranulatus pergranulatus Z.p.p. EF568977
17 Rodos Profitis Ilias Z. festai Z.fes. EF568978
18 Rodos Profitis Ilias Z. festai Z.fes. EF568979
19 Kyhtira Mermigaris Z. algirus Z.alg. EF568980
20 Kyhtira Fratsia Z. algirus Z.alg. EF568981
21 Kyhtira Ag. Sofia cave Z. algirus Z.alg. EF568982
22 Kyhtira Mirtidion Monastery Z. parnonensis Z.par. EF568983
23 Kasos Triola Z. casius Z.cas. EF568984
24 Kasos Kremasta Z. casius Z.cas. EF568985
25 Kasos Alonaki Z. casius Z.cas. EF568986
26 Kasos Alonaki Z. casius Z.cas. EF568987
27 Kasos Alonaki Z. rhodius elatior Z.r.e. EF568988
28 Kasos Alonaki Z. rhodius elatior Z.r.e. EF568989
29 Kastelorizo Paleokastro Z .caricus Z.car. EF568990
30 Kastelorizo Ag. Georgios Z. caricus Z.car. EF568991
31 Kastelorizo Navlaka Z. caricus Z.car. EF568992
32 Peloponnisos Githio Z. graecus Z.gra. EF568993
33 Peloponnisos Logastra Z. graecus Z.gra. EF568994
34 Peloponnisos Parori Z. graecus Z.gra. EF568995
35 Peloponnisos Taigetos Mt. Z .kobelti Z.kob EF568996
36 Peloponnisos Ithomi Z. messenicus Z.mes. EF568997
37 Evvoia Frigano Z. oertzeni Z.oer. EF568998
38 Anafi Monastiri Z. anaphiensis Z.ana. EF568999
39 Lesvos Kako Lagathi Z. algirus Z.alg. EF569000
40 Turkey Tus: cesme Z. chloroticus Z.chl. EF569001
41 Chios Z. chloroticus Z.chl. EF569002
42 Chios Z. smyrnensis Z.smy. EF569003
43 Samos Z. smyrnensis Z.smy. EF569004
44 Syria Slun Feh Turcozonites corax Tur. EF569005
45 Syria Slun Feh Turcozonite) corax Tur. EF569006
9
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