Shahzada Arshid - Univeristy of Kashmir Information Service

STUDIES ON REPRODUCTIVE BIOLOGY
OF MYRIOPHYLLUM SPICATUM L.
IN THE KASHMIR VALLEY
Dissertation submitted to the University of
Kashmir
For award of the Degree of
Master of Philosophy (M. Phil)
IN
BOTANY
By
Shahzada Arshid
M.sc, NET (JRF)
Post Graduate Department of Botany,
Faculty of Biological Science,
University of Kashmir,
Srinagar-190006, J&K India.
2011
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DEPARTMENT OF BOTANY
UNIVERSITY OF KASHMIR
HAZRATBAL, SRINAGAR, KASHMIR - 190006
CERTIFICATE
No ................
Dated...............
Certified that the Dissertation entitled “Studies on
Reproductive Biology of Myriophyllum spicatum L. in the Kashmir
Valley.” submitted to the University of Kashmir, Hazratbal, Srinagar
for award of the degree of Master of Philosophy in Botany, embodies
original research work carried out by Shahzada Arshid under my
supervision. This work has not been submitted in part or in full for this
or any other degree before.
The candidate has worked for the period required under
statutes and has put in the required attendance in the Department.
Prof.(Dr.) Zafar A. Reshi
(Head)
Department of Botany
University of Kashmir,
Srinagar-190006.
..
Dr. Aijaz Ahmad Wani
(Supervisor)
Sr. Asst. Professor
Department of Botany,
University of Kashmir,
Srinagar-190006
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ACKNOWLEDGEMENT
Foremost, my all praise for Almighty Allah, the most
merciful and the most beneficent, for it is indeed through His
blessings alone that the work has been completed.
I feel privileged to express a profound sense of gratitude
and indebtness to Dr. Aijaz Ahmad Wani, (Sr.Assistant
Professor) my esteemed teacher and worthy supervisor for his
sagacious, enthusiastic, intellectual and encouraging
stimulation which provided the guidelines for carrying out
the proposed work. His invaluable scholarly suggestions,
immense interest and the noble guidance throughout the
course of present work provided me specific and sequential
parameters for successfully completing this work. I have been
fortunate enough to be inspired by his dynamic and
innovative nature throughout the period of my study.
I would like to express my deep sense of gratitude to Dr.
Zaffar Ahmad Reshi, Head Deptt. of Botany, for his constant
encouragement, valuable advises and the freedom of work he
provided all throughout my research work.
I am also grateful to my eminent teachers Dr. Irshad A
Nawchoo, Prof. B. A. Wafai, Dr. Inayat-ul-lah Tahir, Dr. A.H.
Wani, Dr. Zahoor Ahmad Kaloo, and Mr. Anzar A. Khuroo
for their kind cooperation and timely help.
I would also like to express a special thanks to my senior
scholars Mr. Aijaz Hassan Ganaie,Fozia Amin and Fayaz
Ahmad for their valuable suggestions and generous help
during the entire course of this study.
Special thanks also goes to my friends Peerzada Arshid
Shabir, Nida Aslam, Shugufta Bashir, Mushtaq Ahmad Bhat,
Tajamul Islam, Zahoor Ahmad Itoo, Chesfeeda, Ishah, Samee
ullaha,Abid Mohiuddin, Hilal Ahmad Bhat, Parveez Amin,
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Mushtaq A Rather, Pervaiz Ahmad Dar, Showkat A, Burhan
Ahad, Waseem and others for their company, support and
encouragement.
I candidly express my profound indebtness from the core
of my heart to my friend Mr.Farooq Ahmad (Research
Scholar,CORD) for his kind and immense help, without his
generous help and support during this study, the dissertation
would have been less illuminating in its final form.
I have no words to express my heartful gratitude to my
beloved and Wonderful parents, (Ab.Hamid Shah and
Dilafroza), who dedicated their entire life for my Success and
are the tower of strength and source of inspiration for me.
They always supported and encouraged me, in the moment of
despair; they came forth to kindle a ray of hope which
rejuvenated me to work with renewed zeal and zest, so I can’t
forget to recollect the kind support which they have provided
throughout my educational career.
To my greatest joy, a special word of thanks goes to my
well wishers especially to my dear and loving Sisters and
Mohd Ashraf shah (Uncle) without whose constant,
affectionate, moral and kind support, and encouragement
that helped me to push through the good and rough times,
from zeroth to infinite, the present work would not have been
here. My special thanks goes to kids Madeeha Ashraf, Sobia
gul, Namat ul ayn, Suhail shafi and Rehana Fayaz.
A sincere thanks also goes to CSIR New Delhi, for
providing financial assistance during the course of study.
(Shahzada Arshid)
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Contents
ACKNOWLEDGEMENT
Page
Chapter 1 INTRODUCTION 1-5
Chapter 2 REVIEW OF LITERATURE 6-32
Chapter 3 MATERIAL AND METHODS 33-43
Chapter 4 OBSERVATIONS 44-110
Chapter 5 DISCUSSION 111-125
Chapter 6 REFERENCES 126-153
No.
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CHAPTER-1
INTRODUCTION
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R
eproductive biology is one of the fundamental fields for
development of conservation protocols for elite and
threatened plant species and on the other hand prevention
protocols for invasive plant species which are increasingly
becoming a threat to our terrestrial and aquatic ecosystems. The knowledge
of reproductive biology is regarded to be of nuclear importance in
developing control methods for aggressive aquatic species (Haynes, 1988).
The studies on various aspects of reproductive biology help us in
understanding the nature, systematics, modes of propagation, adaptation,
hybridization and speciation (Anderson et al., 2002, 2006; Neil and
Anderson, 2005).
Aquatic plants are essential components of healthy ecosystems in freshwater
lakes and ponds, producing oxygen, reducing erosion and regulating nutrient
cycling (Hutchinson, 1975). They provide food for many birds as well as
habitat that supports rich communities of aquatic invertebrates and
vertebrates (Sculthrope, 1967). Invasive aquatic plants, however, are not
native species, and they are often destructive (Vitousek et al., 1996). Non-
native plants are responsible for economic losses and control costs estimated
in one analysis at 137 billion per year in the United States alone (Pimental et
al., 2000). Invasive aquatic plants are noted for their explosive growth
potential (Barrett, 1989) and their ability to grow from a few plants to cover
hundreds of acres in a few years (Groth et al., 1996). Invasive aquatic plants
have caused declines in native plant populations throughout New England
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(Scheldon, 1994).In some water bodies, invasive plants have become so
abundent that they displaced native species (Langeland, 1996). Many
biologists feel invasive species are second only to habitat destruction as the
most serious threat to endangered species globally (Wilcove et al., 1998).
Because of their great growth potential, invasive aquatic plants can block
navigation channels, irrigation ditches and water intake pipes, and can also
reduce aesthetic and recreational value of water bodies, affecting tourism
and real estate values (Catling and Dobson, 1985). In some cases, the plants
have been found to increase breeding habitat for mosquitoes (Eiswerth et al.,
2000). Attempts to eradicate invasive plants once they become established
often have failed (Anonymous, 1993; Growth et al., 1996; Simberloff,
1997), and their management is expensive (Center et al., 1997). Early
identification of invasive plant populations and knowing their reproductive
strategies is critically important to prevent the spread of these plants
(Simberloff, 1997)
The Myriophyllum, commonly known as watermilfoil is a genus of
aquatic mostly fresh water plants of the family Haloragaceae. The genus
name has originated from two Greek words viz: Myrio means many and
phyll means leaves. It is cosmopolitan in distribution (Moody and Les,
2010) and is represented by 68 species (APG II, 2003). Myriophyllum is well
known for its invasive species. The aggressive Myriophyllum spicatum is
native to Europe, Asia and North Africa (Couch and Nelson, 1985) and has
now established on all the continents except Antarctica (Orchard, 1986; Yu
et al., 2002). It is distributed from Spain and UK in the west to China and
Japan in the east and from Finland in the north to Morocco in the south
(Meusel and Jager, 1978) and is introduced and invasive in North America,
South America, India and Australia (Holm et al., 1979).
Myriophyllum spicatum is a herbaceous perennial species. Leaves
occur in whorls around the stem, usually four together at a node (Aiken et
al., 1979) and each leaf is divided into more than 14 pairs of thread like
leaflets. Reproductive characters are the most important characters in the
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identification of the species. In the absence of flowers and fruits, the most
distinctive characters are the reddish tips, more than 14 leaflets on each side
of the central axis of the leaf and the truncated leaf base (Crow and
Hellquist, 2000).
Both sexual as well as asexual modes of reproduction are operative in
Myriophyllum spicatum (Williams, 1975; Grant, 1981). A variety of
propagules and dispersal mechanisms involving seeds, rhizomes, axillary
buds and stem fragments (Sculthrope, 1967; Hutchinson, 1975; Grace, 1993;
Vierssen, 1993) enable the species to grow in varied habitats all over the
temperate areas of the world. The relative importance of sexual vs. clonal
reproduction varies widely among plant species in response to ecological
and/or genetic factors (Eckart, 2002). Very recently there has been much
interest in exploring the role of reproductive modes in geographical
distribution (Peck et al., 1998; Keitt et al., 2001) and the capacity for range
expansion, which in extreme cases manifests as biological invasion (Barret
and Richadson, 1986; Thompson et al., 1995; Pysek, 1997). The most
obvious difference between sexual and clonal reproduction is that sexual
reproduction generates genotypic diversity, whereas strictly asexual clonal
reproduction does not. Limited sexual reproduction can be due to ecological
factors that impair pollination, seed maturation and seedling establishment
(Barrat, 1980).Sexual infertility may also be due to population-level genetic
factors, such as limited mating-type diversity in self-incompatible or
dioecious species (O‘Connel and Eckert, 1999). Understanding the
underlying variations in reproduction may be crucial for predicting the
reproductive potential of the species.
Several working groups (Aiken et al., 1979; Smith and Barko, 1990;
Cook, 1996; Nathan et al., 1993; Alwadie, 2008 ) are working on various
facets of reproductive biology of Myriophyllum. Such studies have revealed
that the inflorescence in Myriophyllum spicatum is a spike and floral
organization of the species is tetramerous (Aiken et al., 1979; Moody and
Les, 2007). The pollination mechanisms operative are: anemophily and
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epihydrophily ( Patten, 1956; Cook, 1996). The seed production is high in
Myriophyllum spicatum and it has been found to play an important role in
the establishment of new genotypes in existing populations (Kimber et al.,
1995; McFerland, 2006), movement of populations into new regions (Arnold
et al., 2000; Figuerola and Green, 2002; Harwell and Orth, 2002) and
reestablishment of populations after episodic declines (Titus and Hoover,
1991; Parveen et al, 1995; Jarvis and Moore, 2008). However studies on
breeding system and pollination in Myriophyllum spicatum are few,
probably due to aquatic habit and small flower size.
Vegetative propagation is widespread in Myriophyllum spicatum and
is ubiquitous among aquatic species regardless of their taxonomic affiliation
(Hutchinson, 1975; Grace, 1993; Les and Philbrick, 1993). Vegetative
reproduction is often assumed to be the dominant mode of reproduction in
water plants (Hutchinson, 1975; Sculthrope, 1967). In Myriophyllum
spicatum different types of vegetative propagules are produced viz;
rhizomes, axillary buds and stem fragments which provide a mechanism for
clonal reproduction with localized to intermediate distance dispersal
(Madsen, 1991). However little work has been done on reproductive
potential, germination and ecology of these propagules.
Notwithstanding the diversity in vegetative and reproductive attributes
and occurrence and abundance of various species of Myriophyllum in
Kashmir valley, the information on reproductive biology and other allied
aspects is meager in the literature. Therefore present study was focused on
distribution pattern, phenotypic variability, floral morphology and
reproductive biology of Myriophyllum spicatum in relation to its habitat
characteristics in order to identify the key habitat specific reproductive
attributes that contribute to the abundance of this species in different aquatic
habitats of the Kashmir valley. Such information is obligatory for effective
management of the species that invade aquatic habitats to damage the native
biodiversity and functioning of these habitats.
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CHAPTER-2
REVIEW OF LITERATURE
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2.1: Distribution
Worldover, the family Haloragaceae comprises of 8 genera and 120
species; and these taxa are extremely diverse in their habit, ranging from
small trees to submerged macrophytes (Moody and Les, 2007). The four
genera (Glischrocaryon, Gonocarpus, Haloragis, Haloragodendron) are
primarily terrestrial, whereas four (Laurembergia, Meizella, Myriophyllum,
Prosperpinaca) are aquatic/semi-aquatic (Table 1).
The Myriophyllum, commonly known as watermilfoil, is among the
species rich (68 spp.) genus of the aquatic ―Core eudicots‖ (APGII, 2003). It
shows a cosmopolitan distribution (except Antarctica), with a centre of
diversity in Australia (42 spp., 34 endemic); North America (14 spp., 7
endemic) and Asia (16 spp., 8 endemic) also harbor a significant continental
diversity and share seven common species as listed in Table 2 (Moody and
Les, 2010). Myriophyllum is well-known for its invasive species. The
aggressive M. spicatum L. (Eurasian watermilfoil) and South American M.
aquaticum (Vell) Verdc. (Parrotfeather) are now established on most
continents and listed as noxious weeds in United States. The North
American endemic M. heterophyllum reportedly is naturalized in Europe
(Wimmer, 1997), Asia (Yu et al., 2002), and also is considered to be
invasive outside its endemic range in the northeast and northwest United
States (Les and Mehrhoff, 1999). Hybridization also has been shown to play
a role in North American invasions with two hybrid lineages recognized viz:
M. spicatum x M. sibiricum and M. heterophyllum x M. laxum (Moody and
Les, 2002).
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Table 1: Distribution, habit and species diversity of Haloragaceae
genera (Moody and Les, 2007).
Genus Distribution Habit No. of
species
1. Glischrocaryon Australia Terrestrial 4
2. Gonocarpus Australia, New Zealand,
S.E. Asia
3. Haloragis Australia, New Zealand.
S. Pacific
Terrestrial 36
Terrestrial *
4. Haloragodendron Australia Terrestrial 5
5. Laurembergia Pantropical Semiaquatic 4
6. Meziella S.W. Australia Aquatic 1
7. Myriophyllum Cosmopolitan Aquatic 60
8. Proserpinaca New World Aquatic 3
*Three species are aquatic.
26
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Table 2. Global distribution of Genus Myriophyllum.
(Meijden, 1969; Meijden and Caspers, 1971; Orchard,1980, 1981, 1986; Aiken, 1981; Yu et al., 2002.)
Continent No. of
species
Australia
Europe
North
America
South
America
Africa
44
6
14
4
Name of species
M. balladoniense, M. caput-medusae, M. decussatum, M. porcatum, M. robustum, M.
salsugineum, M. spicatum, M. triphyllum, M. verrucosum, M. aquaticum, M. trifidum, M.
amphibium, M. pendunculatum, M. tillaeoides, M. dicoccum, M. latifolium, M. muricatum, M.
coronatum, M. costatum, M. filiforme, M. implicatum, M. striatum, M. trachycarpum, M.
alpinum, M. austropygmaeum, M. crispatum, M. drummondii, M. alpinum, M. gracile, M.
integrifolium, M. lapidicola, M. limnophilum, M. lophatum, M. papillosum, M. petraeum, M.
propinquum, M. pygmaeum, M. simulans, M. variifolium, M. votschii, M. artesium, M.
callitrichoides, M. glomeratum, M. muelleri
M. alterniflorum, M. sibiricum, M. spicatum, M. verticillatum, M. aquaticum, M. hterophyllum,
M. alterniflorum, M. quitense, M. sibircum, M. spicatum, M. verticillatum, M. aquaticum, M.
farwellii, M. heterophyllum, M. hippuroides, M. humile, M. laxum, M. pinnatum, M. tenellum,
M. ussuriense.
M. spicatum, M. aquaticum, M. mattogrossense, M. quitense
4 M. spicatum, M. aquaticum, M. mezianum, M. axilliflorum.
Asia 16 M. alterniflorum, M. sibiricum, M. spicatum, M. oguraense, M. verticillatum, M. aquaticum,
M. dicoccum, M. exasperatum, M. tuberculatum, M. heterophyllum, M. bonii, M. siamense, M.
ussuriense,
M. indicum, M. oliganthum, M. tetrandrum.
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From the Indian subcontinent, five species of Myriophyllum have been
reported (Hooker, 1879). In the Kashmir Himalaya, earlier the genus has been
reported to comprise of two species: M. spicatum and M. verticillatum (Kaul
and Zutshi, 1965), while as later on three species have been reported viz. M.
spicatum, M. verticillatum and M. tuberculatum (Kak, 1990).
M. spicatum is native to Europe, Asia and North Africa (Couch and
Nelson, 1985) occurring from Spain and UK in the west to China and Japan is
the east, and from Finland in the North to Morocco in the South (Meusel and
Jager, 1978). It was introduced into North America between 1880‘s and 1940‘s
and now occurs in both Canada and the United States (Reed, 1977; Aiken et
al., 1979; Couch and Nelson, 1985). From the initial point of introduction in
the North America, M. spicatum has spread to 44 states and at least three
Canadian provinces (Creed, 1998) and is now considered a major nuisance
species throughout the Northeast, Northern Midwest and Pacific Northwest of
the United States (Couch and Nelsons, 1985; White et al., 1993). M. spicatum
has spread to 46 states and three Canadian provinces of North America (Jacono
and Richerson, 2003; Kim, 2005). M.spicatum is categorized among the five
most noxious wetland plants (Cronk and Fennessy, 2001) and it is the most
widely managed aquatic weed in the United States (Bartodziej and Ludlow,
1998).
2.2: Taxonomy
The genus Myriophyllum L. belongs to the watermilfoil family,
Haloragaceae, in the order saxifragales (Moody and Les, 2010). Myriophyllum
has been hypothesized as a distinct within Haloragaceae due to a combination
of characters including its aquatic habit (also found in Meionectes, Meziella
and Proserpinaca), tendency towards monoecy (also found in Laurembrgia)
and a fruit that splits at maturity into two or four individual nutlets (not found
elsewhere in the family) (Orchard, 1986). Myriophyllum was found to be
paraphyletic in regard to the monotypic Meziella, in recent phylogenetic
analysis (Moody and Les, 2007). Meziella is similar in habit to Myriophyllum
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ut possesses hermaphrodite flowers and while forming four nutlets, they do
not split at maturity due to a persistent exocarp (Orchard and Keighery, 1993).
The other aquatic Haloragaceae genera (Prosperpinaca and Meionectes) are
distinct, having perfect, two or three merous flowers with a nut and are only
distantly related (Moody and Les, 2007).
A taxonomic confusion exists in the genus Myriophyllum, particularly
with respect to the taxa Myriophyllum spicatum, M. exalbescens and M.
verticillatum (Couch and Nelson, 1983). M. spicatum and M. verticillatum
were first described by Linnaeus in the 1700‘s (Aiken and McNeill, 1980). In
1919, Fernald described a new species for North America, M.exalbescens
(Fernald, 1919). Thereafter Jepson (1925); Hulten (1947); (Patten, 1954, 1956)
and Orchard (1981) found the differences between M. spicatum and M.
exalbescens too significant to warrant separation. Hulten and Patten placed M.
exalbescens within the older taxon as a subspecies, where as Jepson, Nichols
and Orchard preferred the varietal level. Fernald (1919) steadfastly opposed
considering M. spicatum and M. exalbescens as one species. Love (1961); Reed
(1977), Aiken (1979), Aiken and Walz (1979), Aiken et al., (1979) agreed
with Fernald and concluded that the native American species, M. exalbescens
and M. verticillatum, should be separate taxa based on differences in
morphology, physiology and phenology.
All Haloragaceae species are herbs, submersed in quiet waters or rooted
on muddy shores. The similarity of the species has led to much confusion about
species identity and most species in the family cannot be separated using only
individual specimens or without flowers (Johnson et al., 1998). A phylogenetic
study based on nr DNA ITS sequence reveals that M. spicatum is most closely
related to the holarctic species M. sibiricum and M. alterniflorum. There is also
close relationship to M. verticillatum and the M. quitense. All other species
analysed i.e., M. heterophyllum, M. laxum, M. hippuroides, M. tenellum, M.
pinnatum, M. farwellii and M. humile are well separated from M. spicatum
(Moody, 2004).
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M. spicatum was found to hybridize with the M. sibiricum in North
American (Moody & Les, 2002). While M. spicatum and M. sibiricum can be
distinguished by morphological characters related to leaf segments and the
presence (M. sibiricum) or absence (M. spicatum) of turions, hybrids overlap
with both parents in leaf characters and lack turions, thus can only be
distinguished using molecular analysis (Moody and Les, 2010). M. spicatum is
variable in appearance with long stems, and usually 12 to 21 leaflet pairs,
which are not stiff when out of the water, in contrast the very similar M.
sibiricum usually have five to 10 leaf pairs with leaflets that stay rigid when out
of the water, so leaf morphology can be used to separate these two very similar
species successfully (Gerber and Les, 1994).
As per Crow and Hellquist (2000), following taxonomic characters are
used to identify M. spicatum.
1. Leaves pinnately divided, with filiform segments; vegetative stems
elongate.
2. Leaves whorled.
3. Bracts usually twice as long as pistillate flowers.
4. Bracts of upper portion of inflorescence lanceolate, entire to denticulate,
not glaucous.
5. Middle leaves with 12 or more segments on each side of rachis; many of
the uppermost leaves truncate at apex; stem diameter below
inflorescence greater, up to twice the diameter of the lower stem, stem
tips usually reddish; winter buds not formed.
Reproductive characters are the most important characters in the
identification of the species. In the absence of flowers and/or seeds, the most
distinctive characters are the reddish tips, the 12 or more filaments on each side
of the central axis of each leaf and the truncated leaf tips.
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2.3: Phenology
Many factors affect the reproductive success of flowering plants. Among
these factors, the timing, frequency and duration of the flowering period
collectively referred to as phenology is obviously of great importance (Rathcke
and Lacey, 1985). The phenology of a species not only encompasses when,
how often, and how long reproduction takes place but also determines the
degree of reproduction synchrony with other plant species (Rathcke, 1988).
Synchrony among species might be advantageous if the presence of one species
facilitates increase in pollinator visitation and thus fruit/seed set in another
species (Rathcke and Lacey, 1985). Phenology in general and reproductive
phenology in particular is a critical and important trait of a plant because it
determines the growth, developmental pattern and number of potential mates in
a population thus providing a mechanism for reproductive isolation or
speciation over time (Rathcke, 1983; Bronstein et al., 1990). Researchers
always and continuously try to identify environmental factors that correlate
with phenological events such as initiation of flowering, the synchronization of
flowering, the length of the flowering phase and variation in flower abundance
(Opler et al., 1980; Borchert, 1983; Inouye et al., 2002). Environmental factors
that initiate the onset of a particular phenophase include photoperiod,
temperature and precipitation (Rathcke and Lacey, 1985). The same
environmental factors can delimit a particular phenophase including flowering
season in some specific ecoedaphic conditions or environments (Borchert,
1980; Inouye and McGuire, 1991).
Patten (1956) studied the phenological events of Myriophyllum spicatum
in Lake Musconetcong, New Jersey. The inflorescences first appeared on July
11. Two days later these were abundent. Towards the beginning of August, the
plants of this area were in full bloom and August 19, approximated the peak of
the flowering period for the lake as a whole. By the middle of September,
flowering was over and the fruiting spikes were laid just beneath the surface of
water. During October the fruiting was in peak and most of the fruits were
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eginning to drop off from their spikes. Half or more of the year‘s seed crop
was released by the end of November. Many fruits remained attached to the
plants throughout the winter months and quite often were frozen into the ice.
Fruits no longer remained by the late February.
Leikic (1970) found that germination of M. spicatum generally takes
place in March and April in Yugoslavia. The stem elongates during the
vegetative phase, and leaf production commences. Relatively high water
temperatures are necessary for vegetative growth. Branching starts in May and
has a phototropic response which increased with the increase in light intensity.
Flowering started in the end of the May and continued for 2 months with
maximum flowering at the end of June and beginning of July. Lack of sunlight
was found to delay the flowering. Seed production was found to be dependent
upon the seasonal weather conditions; the first mature seeds were recorded at
the end of July, but may last through October. Seed maturation was followed
by senescence of the plant, which ultimately falls to the bottom of the habitat.
Unfavourable conditions for the development of M. spicatum can result in
reduced size, vitality and seed production. During favourable conditions
increase in the number of plants in the population resulted in an increase in
plant height and a decrease in the number of fruits per plant.
Spencer and Leikic (1974) reported flowering in early summer which
continues for several months. Flowering in Myriophyllum dicoccum occurs
from August to September and fruiting from September to October, in M.
ussuriense flowering occur from April to September and fruit formation starts
in the month of July and continues up to November and in M. aquaticum
flowering occurs in May at low altitudes to July at high attitudes and fruit
formation was not observed. Nathan et al., (1993) reported that flowering in M.
spicatum occurs during April and May and fruit formation occurs from May to
November. Aiken et al., (1979) stated that M. spicatum blooms during July to
September and fruit matures late in September.
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2.4: Sexual Reproduction
Reproduction in aquatic angiosperms occurs by both sexual and asexual
means (Wade, 1990). Sexual reproduction (the chief source of hereditary
variation via genetic recombination) in plants is considered to be advantageous
in changing heterogeneous environments, and asexual reproduction (which
perpetuates genetic uniformity) is considered to be more successful in stable or
uniform habitats (Grant, 1981, Williams, 1975). Sexual reproduction is a
primary mode of reproduction for terrestrial ancestors of aquatic plants. A shift
from sexual to asexual (vegetative) reproduction is often associated with the
evolution of aquatic plants, a complete absence of flowering and seed set is
found in only a few aquatic species (Philbrick and Les, 1996). The majority of
aquatic angiosperms retain the ability to flower and seed set (Sculthrope,
1967). Cook (1987) pointed out that of the 12 most notorious aquatic invaders,
only one Trapa natans relies on sexual processes for its reproduction and
success. Out of the remaining 11 species, only Myriophyllum spicatum, Najas
minor and Pistia stratiotes regularly develop seed in their native and adventive
ranges. Salvinia molesta is a sterile hybrid, and the rest have well developed
self-incompatibility mechanisms. The retention of sexual reproduction by
angiosperms in aquatic conditions represents a difficult evolutionary transition
but floral systems of aquatic plants are generally conservative and reflect their
terrestrial heritage (Philbrick and Les, 1996). The aquatic plants betray their
terrestrial ancestors in their sexual phase with the greatest clearity (Sculthrope,
1967).
Sexual reproduction of aquatic plants departs from that of terrestrial
plants. When flowers are adapted to aerial life, aquatic plants have to raise their
inflorescence axis well above the water level, if cross pollination is to be
secured (Arber, 1920). This is not always completely achieved. In Ranunculus
penicillatus var. calcareus, the stem maintaining the flowers above the water
are considerably more fragile than the normal submerged and vegetative ones
and they are easily damaged and lost (Cook, 1966; Dawson, 1980). Arber
Page 49

(1920) reports that Ranunculus fluitans does not hold its peduncles very erect,
and often suffers the inundation of its flowers and thus fails to set seeds while
Ranunculus trichopyllus often opens its flowers under the water surface, but it
seems that it cannot set seeds under these conditions. It was, however,
considered that the failure in setting ripe fruits in this species usually was not
due to inundation, but mainly to genetic reasons. In hybrid populations of
Ranunculus fluitans the abortion of the anthers and abnormal pollen might be
responsible for the seed sterility of the plants (Cook, 1966; Turala-Szybowska,
1977); this feature is also observed in Ranunculus pencillatus (Turala, 1969,
1970; Webster, 1986, 1988). The production of reproductive structures above
the water surface can increase the likelihood of herbivory and mechanical
damage (Spencer and Bows, 1990).
Myriophyllum spicatum reproduces sexually by forming large number of
seeds (Cook, 1987). Sexual reproduction appears unimportant in shaping
population structure of Eurasian water milfoil (M. spicatum) in Minnesota
(Furnier and Mustaphi, 1992); however, significant germination was observed
in Lake George in New York State (Hartleb et al., 1993).
2.4.1: Floral Organization
The sexual stage of M. spicatum starts with the inflorescence which is a
terminal, cylindrical, naked, interrupted spike, indeterminate in development
and often very red (Patten, 1956). All flowers in the spike are whorled, with 4
flowers in each whorl. Male flowers are on upper side of the spike with pinkish
petals, female flowers on lower side without perianth. (Aiken et al., 1979). The
inflorescence is a terminal spike and is born above the water surface; flowers
are small and inconspicuous (Smith and Barko, 1990). The upper flowers on a
spike are staminate and lower pistillate, the transition between appearing abrupt
(Knupp, 1911). The entire spike may sometimes be staminate or pistillate -
dioecious, considered to be a vestigial condition of a completely
hermaphroditic phylogenetic origin of the group (Knupp, 1911 and Hagi,
1926). Hagi (1926) reported that surrounding aqueous medium is the direct
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cause of repression of the male sex in the lower flowers. Each flower is sessile
in the axil of a strong bract, two more delicate bracteoles are also located at the
base of each flower, these three bracts entirely enclose and protect the young
flower and gradually open out as the spike elongates and the flowers mature,
the flowers are verticillate in fours, each whorl being rotated about 45 degrees
from the adjacent ones, so whorls are in two ranks and this rotation produces
the spiral arrangement characteristic of the spikes before elongation (Patten,
1956). The first report of flowering in the field is the appearance of small,
green, compressed spikes at the stem apices, covered at least partially by the
last leaves produced by the vegetative apex (Patten, 1956). This is followed by
elongation of spike axis, elevation of the spike out of the water, pollination and
resubmergence. The spike axis becomes thicker than the normal vegetative
stem and blooming occurs just above the water surface regardless of the depth
(Vaucher, 1841). Aiken (1981) reports that spike axis becomes almost double
in width than the normal vegetative stem.
2.4.2: Pollen morphology, Pollination system and Breeding behaviour
Pollination has been of interest since ancient times. (Cox, 1988).
Theophrastas (1976) deduced that pollen is a desiccant which prevents the
rotting of developing fruits and pointed out the necessity of pollen to fruit
formation. Pollen is a vital link between each generation of flowering plants,
houses the male gametes and is of profound importance in the classification
and taxonomy of angiosperms (Ducker and Knox, 1985).
There is a correlation between pollen morphology and pollination
mechanism (Van Vierssen et al., 1982; Tanaka et al., 2004). A definite
relationship is exhibited between pollen characters and pollination types
especially in entomophily and anemophily. Pollen grains of entomophilous taxa
are characterized by compound apertures i.e. 3-colporate, prolate-spheroidal
shape, generally large, thick walled, sticky and with reticulate tectum, while
pollen grains of anemophilous taxa are with simple apertures i.e. monoporate,
Page 51

spheroidal, small thin walled, dry and with scabrate-areolate tectum (Shuang-
Quan et al., 2001; Parveen et al., 1994).
Underwater pollination systems in angiosperms are derived from aerial
floral systems (Philbrick and Les, 1996). Exine reduction or complete exine
loss appears to be an essential component in the evolution of hydrophily
(Philbrick, 1991). This has been documented in hydrophilous members of
Hydrocharitaceae (Pettitt, 1981). There is a correlation between the variation in
the exine and pollination mechanism (Tanaka et al., 2004). Pollen grains of
hydrophilous taxa are spheroidal non-aperturate coarsely reticulate tectum with
thin exine. The thin elastic exine and reduced or omniapertures (non-aperture)
are considered characters of hydrophilous taxa (Punt, 1986).
Many authors have studies the pollen morphology of some species of
aquatic families such as Lemnaceae (Aiken, 1978), Potamogetonaceae
(Parveen, 1999), Hydrocharitaceae (Takahashi, 1994; Tanaka et al., 2004),
Podostemaceae (Obson et al., 2000), Haloragaceae (Labdolt, 1986; Sorsa,
1988), Najadaceae (Shuang-Quan, 2001), Ceratophyllaceae (Takahashi,1995),
Rubbiaceae (Lacroix and Kemp, 1997; Callitrichaceae (Cooper et al., 2000)
and Nymphaceae (Shiga and Kadono, 2007).
Alwadie (2008) examined pollen morphology of six species of aquatic
angiosperms from Saudi Arabia belonging to five genera distributed in five
families using both light and scanning electron microcopy. These species
include Myriophyllum spicatum, Elodea canadensis, Potamogeton crispus, P.
pectinatus and Ruppia maritima. The pollen grains of M. spicatum are
generally radially symmetrical, isopolar, sub-oblate to oblate, 4-zonocolpate,
colpi short, 4-5 colpate, tectum scabrate-punctate. Exine thick, sexine thicker
than nexine. Parveen (1999) examined the pollen morphology of 16 species of
aquatic angiosperms in 14 families from Karachi using light and scanning
electron microscope including Myriophyllum verticillatum. The Pollen grains
of aquatic families are mostly apolar or isopolar and rarely heteropolar. The
pollen shape is commonly spheroidal, rarely boat shaped (Nymphaea stellate).
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However in the family Juncaceae and in the single species of Typhaceae
(Typha elephantina) pollen grains are united in tetrads. Exine sculpturing is
also extremely varied, ranging from reticulate to regulate, fossulate, scabrate or
olate. Apertures are mostly colpate or porate. However in few taxa non-
aperturate pollen are also found (Potamogeton, Juncus) rarely tricolporate as in
Enhydra fluctuans. Pollen grains of M. verticillatum are radially symmetrical,
isopolar, sub-oblate, 4-5 colpate, colpi short. Exine thick, sexine thicker than
nexine. Tectum scabrate-punctate, scabrae fine.
The flowers of most aquatic angiosperms must be elevated above the
water in order for pollination to occur by entomophily (pollination by insects)
and anemophily (by wind), very rarely pollen transfer occur underwater
(hydrophily) (Riemer, 1984). The transport and capture of pollen in 20% of all
angiosperm families occurs in air and water (Ackerman, 2000). Delpino (1871)
recognized that pollen transfer could take place without animals and divided
abiotic pollination into wind (anemophily) and water pollination (hydrophily).
Whereas anemophily occurs in the air, hydrophily is further divided into
epihydrophily (surface pollination) and hypohydrophily (underwater
pollination) (Knuth, 1906). Additional distinctions can be made for both
epihydrophily and hypohydrophily, especially with respect to whether pollen
and stigmas are wet (Les et al., 1997). The diversity in abiotic pollination
reflects the fact that anemophily and hydrophily have evolved many times in
terrestrial and aquatic plants (Arber, 1920; Sculthrope, 1967; Les et al, 1997;
Niklas, 1997; Raven et al., 1999). Anemophily is much more common than
hydrophily, 98% of the plants that pollinate abiotically are pollinated by wind
(Faegri and Vander Pigl, 1979). Some of the earliest seed plants were
pollinated by wind (Niklas,1997).
Cook (1988) reported that in family Haloragaceae, genus Haloragis,
Myriophyllum, Laurembergia, Prosperpinaca and Vinkia have aquatic species,
all of which are well adapted to wind pollination. All have flowers with
reduced petals and often caducous perianths and dry, powdery pollen liberated
Page 53

from long filamented anthers. There is a trend from bisexual to unisexual
flowers culminating in dioecy. Cook (1996) reported the occurence of
anemophily in different genera of family Haloragaceae including
Myriophyllum. Patten (1956) reported that in Myriophyllum spicatum when the
spikes emerge they exhibit protogynous dichogamy: The stigmas ripening well
in advance of the stamens, thereby favouring cross-pollination, soon after, the
deciduous petals abscise forecasting the ripening of the stamens. The mode of
pollination is the subject of diverse opinions and occurs when the spikes project
out of the water and that the hydrophilous pollination does not occur in M.
spicatum. This is substantiated by the observation that lowermost carpels on
some inflorescences are often submerged, such submerged carpels have always
been observed to abort instead of developing into mature achenes. Many spikes
lean at an angle away from the perpendicular out of the water causing
submergence of some carpels of a whorl while the others remain exposed to the
atmosphere. The submerged carpels abort and the emersed ones develop into
mature seeds at the same node giving an evidence of wind-pollination. It was
also reported by him that self-pollination in M. spicatum is generally ruled out
with the following exception. A single plant usually possesses a number of
flowering stems each of which has lateral branches which also flower. It
happens that the main stems bloom before the laterals so that pollen from the
central spike can ripe at the same time the lateral pistils are receptive. Even
here, though, the chances for cross-pollination would be still greater due to the
greater abundance of pollen freed from other sources. Patten pointed out that
the staminate parts of the spike decline, often abscising completely and the
pistillate portion with the staminate remnant disappears beneath the surface,
along with a decrease in the thickness of the spike axis and develop into the
mature achenes. The spikes slowly disintegrate until they are sufficiently rotted
for the achenes to drop with the aid of wave action.
Arber (1920) states that a certain number of aquatic plants have given up
insect-pollination and taken to anemophily, along with simplification of the
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flower and Myriophyllum is one such genus as evidenced by the structural
adaptation of long anthers swinging on flexible filaments. Hagi (1926) also
favoured wind-pollination. Ludwig (1881) cited wind pollination although he
reported that whole populations of Myriophyllum verticillatum flowers
underwater. Knuth (1906) reported anemophily as well as hydrophily for M.
spicatum. Hagi (1926) reported hydrophily in M. verticillatum. Vaucher (1841)
overlooking the protogynous nature of M. spicatum stated that pollen falls out
of the anthers directly on the stigmas below and hence pollination occurs.
Knupp (1911) is in favour of hydrophily for M. spicatum.
2.4.3: Pollen-Ovule Ratio
The pollen-ovule ratio (P/O) is the ratio of pollen grains produced per
ovule. Cruden (1977) reported that cleistogamous flowers should have the
lowest pollen to ovule ratio‘s and that autogamous flowers will have lower
pollen to ovule ratio‘s than xenogamous flowers i.e., pollen to ovule ratio‘s are
correlated with breeding systems. The more efficient the transfer of pollen, the
lower the pollen to ovule ratios should be. The evolutionary shift from
xenogamy (outcrossing) to autogamy (selfing) has been mediated through
decreased flower size and alterations in floral morphology which reduce the
energetic cost per flower and facilitate self pollination (Ornduff, 1969).
Flowers of self-incompatible and other xenogamous taxa produce more pollen
grains than closely related self-compatible and/or autogamous taxa (Arroyo,
1973; Baker, 1967; Cruden, 1973; Gibbs et al., 1975; Lloyd, 1965; Vries,
1974).
Pollen grain number has to be positively related to ovule number. This
relationship was observed across families (Cruden and Miller-Ward, 1981) and
within families (Small, 1988; Plitman and Levin, 1990; Kirk, 1993; Lopez et
al., 1999). Variation in Pollen grain and/or ovule number may occur within
individuals and within or among populations, or may be more or less constant
(Arnold, 1982; Devlin, 1989). In homoecious species (species with
hermaphroditic flowers), both pollen and ovule number may vary within a plant
Page 55

during the flowering season (Cruden and Lloyed, 1995) and there is no change
in the pollen to ovule ratio (Vasek et al., 1987). Some of the seasonal variation
in pollen grain and ovule number may reflect dichogamy. In populations of
protoandrous plants or those with protoandrous flowers the pollen from the first
flowers to open will not reach a stigma and the plants suffer a loss in
reproductive success through male function. Brunet and Charlesworth (1995)
suggested that this might result in plants investing less in male function in the
first opening flowers and relatively more in female function. Protogyny should
produce the opposite pattern.
Pollen grain number and/or ovule number may vary among populations
of a species. Such variation may be very small and no one has demonstrated a
selective basis for it (Affre et al., 1995; Cruden, 1977; Diaz lifante, 1996;
Ramsey et al., 1994, Vuille, 1988). Such variation may reflect sampling error
or have an environmental basis. In some species there was substantial variation
among populations that reflected difference in breeding system (Affre et al.,
1995; Dahl, 1989; Thomas and Murray; 1981). There may be substantial
difference in pollen to ovule ratio among populations with the same breeding
system that may reflect pollination efficiency (Webb, 1994).
A negative relationship between pollen grain number and size is well
documented (Cruden, 1977; Dulberger and Ornduff, 1980; Cruden and Miller-
ward, 1981; Small, 1988; Mione and Anderson, 1992; Knudsen and Olesen,
1993; Lopez et al., 1999). Many attribute the relationship to a simple trade-off
between number and size (Charnov, 1982).
There is a relationship between pollen number and duration of stigma
receptivity. The number of pollen grains recieved by the stigma is increased by
the length of time it is receptive. Differences in the period of receptivity may
reflect ecological and/or physiological factors or genetic differences among
populations and in many species unpollinated stigmas remain receptive longer
than pollinated stigmas (Devlin and Stephenson, 1984; Richardson and
Stephenson, 1989). The pollen to ovule ratios of plants whose pollen is
Page 56

dispersed in tetrads, polyads and pollinia has lower pollen-ovule ratio than
those dispersed as monads (Cruden, 1977, 1997). The pollen to ovule ratio of
species with secondary pollen presentation are lower than those of species with
primary pollen presentation (Yeo, 1993) and the species that offer only pollen
as a reward produce more pollen per ovule than those that provide nectar as a
reward (Vogel, 1978; Pellmyr, 1985, 1986).
There is a strong correlation between pollen to ovule ratio and breeding
system. Pollen to ovule ratio decreases from obligate xenogamous to
facultative xenogamous to autogamous species. This holds within species
(Dahl, 1989; Affre et al., 1995; and Hannen and Prucher, 1996), generas (Spira,
1980; Vuille, 1987; Feliner, 1991; Sharma et al., 1992) and families (Schlising
et al., 1980; Short, 1981; Webb, 1984; Lawrence, 1985; Preston, 1986; Vasek
et al., 1987; Plitman and Levin, 1990; Feliner, 1991). Cruden (2000) reported
that homoecious species have lower pollen to ovule ratio‘s than those with
other sexual systems and that the pollen to ovule ratio‘s of wind-pollinated
species are higher than those of animal pollinated species with the same
breeding system .Wind pollinated, xenogamous plants produce large numbers
of pollen grains (Pohl, 1937; Proctor and Yeo, 1972), because most flowers
have just a single ovule their pollen to ovule ratio‘s should be high relative to
those of animal pollinated plants.
Philbrick and Anderson (1987) examined the pollen-ovule ratio in genus
Potamogeton. All the ten Potamogeton species studied with aerial flowers are
xenogamous. Ratios in the aerial flowered species extended from a low of
about 3000 grains per ovule in P. robbinsii to a mean high of about 40,000 in
P. zosteriformis. Species with aerial and submerged flowers and those with
regularly submerged flowers possess significantly lower pollen to ovule ratios
than aerial-flowered species.
2.4.4: Fruit and Seed Biology
The fruit of Myriophyllum is called as achene and seed as nutlet (Patten,
1955), where as fruit as nut which breaks at maturity into two or four
Page 57

individual seeds called as nutlets (Orchard, 1986). Fruit is called as Schizocarp
with 4 longitudinal ridges where it splits into 4 one seeded nutlets ( Aiken and
Mc Neilt, 1980 ; Aiken, 1981).
Although seed germination of terrestrial species has been extensively
studied, few studies have examined submerged macrophytes (Coble and Vance,
1987; Hartleb et al., 1993; Lal and Gopal, 1993; Baskin and Baskin, 1988; Ke
and Li, 2006; Hay et al., 2008; Jarris and Moore, 2008). This may be due to the
fact that most submerged macrophytes show dominant clonal reproduction, and
their seeds are rarely observed to grow into mature plants in nature. However,
seeds of submerged macrophytes have been found to play to important roles in
the establishment of new genotypes in existing populations (Kimber et al.,
1995; McFarland, 2006), movement of populations into new regions (Arnold et
al., 2000; Figuerola and Green, 2002; Harwell and Orth, 2002) and
reestablishment of populations after episodic declines (McMillan & Jewelt-
Smith, 1988; Titus and Hoover, 1991; Parveen et al., 1995; Jarvis and Moore,
2008). A recent study on the genetic pattern of Potamogeton malaianus
suggested that bird mediated seed dispersal could greatly influence gene
movements among lakes (Chen et al., 2009).
Germination requirements and dormancy characteristics of species are
often assumed to be adaptations to the particular habitats where the species
occur (Angevine and Chabot, 1979; Meyer et al., 1990). Germination at the
right time and in the right place is important to determine the probability of a
seedling surviving to maturity (Thompson, 1973). Previous studies have
indicated that temperature (Hartleb et al., 1993; Ke and Li, 2006; Hay et al.,
2008; Jarvis and Moore, 2008) and light (Coble and Vance, 1987; Lal and
Gopal, 1993) are important ecological triggers in the seed germination of
submerged species.
Xiao et al., (2010) examined the effect of temperature, water level and
burial depth on seed germination of Myriophyllum spicatum and Potamogeton
malaianus. There was no significant difference in final germination of M.
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spicatum among water level treatments, but P. malaianus germinations at 1cm
and 12 cm water levels where better than at 0 cm water level. Little to no
germination was observed for either species at the temperature of 10 0 C but at
15 0 C germination increased significantly to 66.3-70.6% for M. spicatum and
to 29.4 -48.1% for P. malaianus under all three water level treatments.
Increasing temperature from 15-30 0 C had no significant effect on final
germination of M. spicatum, but enhanced significantly the germination of P.
malaianus. The germination percentage of M. spicatum was 71.3% at 0 cm
burial depth, but decreased to 5.0% and to 2.5% at depths of 1cm and 2cm
respectively, where as germination percentage of P. malaianus were 40%,
23.8%, 12.5%, 7.5% and 1.3% at depths of 0cm, 1cm, 2cm, 3cm, and 5cm
respectively. Analysis of mean time to germination revealed that M. spicatum
is a faster germinator relative to P. malaianus.
Coble and Vance (1987) examined the affect of light quality on the
germination of seeds of M. spicatum. Under favourable conditions, 92.2%
germination rate was observed. The effect of white, red (700 and 725 nm),
green (520 nm), and blue (445nm) light as well as darkness on the germination
was studied. As many as 97% of the seeds germinated under red light (725nm).
Blue light significantly inhibited germination and almost no seeds germinated
in the darkness. The mean percent germination ranged from 77.8% to 97.2%
for those seeds exposed to light wavelength above 500nm.Seeds exposed to
light of 445nm and those left in the dark had lower germination rates of 30.5%
and 5.6% respectively. These studies suggest that seed germination can be
inhibited by an extreme increase or decrease in light intensity. The extremely
high germination rate in the red light and inhibition of germination in the blue
light both indicate the presence of an active Phytochrome system (Hartmann,
1966; Rollin and Maignan, 1966; Rollin, 1970; Malcoste et al., 1972; Mitrakos
and Shropshire, 1972).
Standifer and Madsen (1997) studied the effect of drying period on the
germination of Eurasian watermilfoil seeds. The seeds were subjected to the
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drying treatment for different periods. Maximum percent germination (81.6%)
was observed in seeds of the control treatment while seeds exposed to the 36
week drying period demonstrated the lowest rate of germination (53%). There
is significant downward trend in germination rates as drying period increases.
However a substantial percentage of Eurasian watermilfoil seeds remain viable
following a drying period of up to 36 weeks. The viability of these seeds
following an extensive drying period demonstrates the resilience of these seeds
to desiccation and suggests a potential role for reproduction.
Patten (1955) studied the seed germination of Myriophyllum spicatum by
subjecting the seeds to different conditioning treatments. The seeds of M.
spicatum doesn‘t germinate readily early in the first year unless exposed to
certain conditioning treatments. There is a necessity for a period of after-
ripening which increases the ability of seeds to germinate. Treatments which
were found to enhance after-ripening, and hence germination, in decreasing
order of effectiveness are:-
1. Partial removal of the stony endocarp
2. Scarification
3. Freezing
4. Alternate freezing and drying
5. Drying
6. Exposure to relatively high H-ion and OH-ion concentrations
7. Prolonged exposure to exposure to low temperature
The pericarps are inhibitory to germination. The conditioning treatments
presumably increases germination through some action on pericarps. There is a
minimum temperature below which germination does not occur normally.
There is a prompt germination when water warms in the spring to this
temperature. Cooling during the winter and possible freezing condition the
seeds for such a spring eruption.
Madsen and Boylen (1989) studied the seed germination of M. spicatum
from an oligotrophic and eutrophic lake. The seeds of the eutrophic lake show
Page 60

mean germination of 69% and the seeds of oligotrophic lake showed lower
germination rates and the mean germination of 41% under light conditions. In
oligotrophic lake the seeds not appear sufficient to contribute to the spread of
the population due to the low numbers of seed set and low rate of germination
while as in eutrophic lake there is a potential for seeds to contribute to annual
propagation and expansion of the population due to relatively high seed set
percentages and germination rates.
Hartleb et al., (1993) reported that M. spicatum require high temperature
(above 14 0 C) for germination. Light is not considered as a limiting factor, but
increased sedimentation can greatly suppress germination.
Aiken et al., (1979) examined that flowering and seed production are
common in M. spicatum, but the seeds exhibit prolonged dormancy and their
germination is erratic. Even in areas where the plant is common, no seedlings
have been found (Bates et al., 1985).
Guppy (1897) reported that with proper scarification up to 85% of the
seeds germinate, but under natural conditions seeds may actually have their
germination delayed until at least their second spring. Seedling establishment
appears to be a particularly fragile stage in the life cycle (Patten, 1956). Young
populations of Eurasian watermilfoil produced on an average 112 seeds per
stalk. Despite the high seed production, the germination of seeds is not a
significant factor in reproduction (Washington State Department of Ecology,
2003).
2.5: Asexual Reproduction
Asexual reproduction includes both seed production without fertilization
(agamospermy) and vegetative reproduction (Philbrick and Les, 1996). The
extent of agamospermy among aquatic plants is poorly understood. Vegetative
reproduction is often assumed to be the dominant mode of reproduction in
water plants (Sculthrope, 1967; Hutchinson, 1975). The ability to reproduce
vegetatively is ubiquitous among aquatic species, regardless of their taxonomic
affiliation (Sculthrope, 1967; Hutchinson, 1975; Grace, 1993; Les and
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Philbrick, 1993). Abrahamson (1980) considered that genetically identical
offsprings produced through the process of the vegetative reproduction make
this process similar to the growth than to the reproduction, and ramets
(vegetatively produced progeny) are not always genetically identical to the
parent.
Aquatic plants are more common proportionally in monocots than dicots
(Les and Schneider, 1995). This correlation suggests that clonal growth
contribute to the evolution of aquatic species. Triffney and Niklas (1985)
postulated that the greater proportion of aquatic monocots is associated with
high incidence of rhizomatous growth (i.e., by horizontal underground stems)
in monocots. In contrast, fever dicots are rhizomatous (Grace, 1993).
Most aquatic species show prolific clonal propagation or perennation,
often in association with limited sexual reproduction (Les, 1988; Barrett et al.,
1993). Clonal growth and propagation has been assumed to be more abundant
in aquatic habitats than in terrestrial ones (Sculthrope, 1967; Hutchinson, 1975;
Grace, 1993; Duarte et al., 1994). The wide spread occurrence of clonality
among aquatic plants serves the primary function of increasing genet survival
under conditions that severely inhabit or restrict the success of sexual
reproduction (Sculthrope, 1967; Grace, 1993). These conditions include
decreased pollination success, particularly for submerged species (Duarte et al.,
1994); unstable ecological conditions for seed production, maturation,
germination and seedling success arising from small population size of many
aquatic habitats (Barrett et al., 1993; Les and Philbrick, 1993). Clonal spread
may contribute to the limited success of sexual reproduction by reducing
pollination success in self-incompatible species (Charpentier et al., 2000).
Perennial water plants posses many devices for vegetative reproduction,
including corms, rhizomes, stolens, tubers and turions (Sculthrope, 1967;
Hutchinson, 1975; Grace, 1993; Vierssen, 1993). Annual aquatic plant genera
reproduce vegetatively by extensive lateral growth, fragmentation or the
occasional production of turions (Agami et al., 1986; Sculthrope, 1967). The
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most notable vegetative propagule in aquatic plants is the turion, the well
insulated turions referred to as winter buds; enable the plans to overcome the
stressful winter season (Vierssen, 1993). Turions are compact, club shaped
apices that become detached as the parent shoot system decays and either float
or sink to the bottom and each turion is able to develop into a new plant in the
next spring (Barret-Segretain, 1996). Turions have been reported in many
macrophytic species such as Utricularia vulgaris, Hydrilla verticillata, Lemna
minor, L. trisulca, Spirodela polyrhiza, Hydrocharis morsusranae and many
Potamogeton species (Arber, 1920; Jacobs, 1947; Patten, 1956; Aiken and
Walz, 1979; Sastroutomo, 1981; Klaine and Ward, 1984; Van Wijk and
Trompenaars, 1985; Brux et al., 1987; Dudley, 1987; Spencer and Ksander,
1991; wiegleb et al., 1991).
Fragments play an important role in the vegetative reproduction of
aquatic plants, with each individual node often capable of regeneration (Grier,
1920). The fragments are unspecialized organs of vegetative reproduction. Any
fragment of a shoot of Callitriche containing meristematic tissue is able to
regenerate a new plant (Barret-Segretain, 1996). In Hydrilla verticillata, a few
single node fragments produce new roots and shoots, but every three-node
fragment is able to initiate a new plant (Langeland and Sutton, 1980). Brock et
al., (1989) mentioned that detached shoots of Hottonia palustris can settle
successfully on favourable substrates and give rise to new plants.
Many aquatic plants have specialized vegetative organs of perennation
and these organs are essential for multiplication and the infestation of new
areas. These vegetative organs include rhizomes, runners or stolons and tubers.
The rhizomes of Hippuris vulgaris, Scirpus lacustris, Nuphar lutea, Nymphea
alba may be broken and each fragment bearing buds can develop into a new
plant (Arber, 1920; Heslop-Harrison; 1955; Bartley and Spence, 1989; Smith et
al., 1989). Runners or stolons are stems growing respectively just above the
soil surface, or underground. Each runner or stolon travels away from the
parent plant for some distance, its growing apex eventually becoming erect and
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forms a new plant (Barrat-Segretain, 1996). Both runners and stolons are
generally short lived and their prime function is undoubtedly as reproduction
rather than perennation (Arber, 1920). Tubers are swollen portions of stems,
rhizomes or roots that become independent because the connecting organs
decay during the winter. In the spring adventitious roots and new leaves
develop and each tuber can give rise to a new plant (Barrat-Segretain, 1996).
Stem and rhizome tubers are very frequent. A single plant of Sagittaria
sagittifolia may produce as many as ten tuber bearing stolons (Sculthrope,
1967) and tubers of Potamogeton pectinatus are the most regularly occurring
propagules of this species (Van Wijk, 1989).
Vegetative reproduction is regarded as a more important mechanism of
population expansion than seed dispersal (Kimbel, 1982; Madsen and Boylen,
1989; Madsen et al., 1988). In M. spicatum, asexual propagules provide a
mechanism for short-term clonal reproduction with contiguous to intermediate
distance dispersal (Madsen, 1991). Localized expansion is provided by stolon
growth. Stems which form adventitious roots may establish new plants in the
immediate area of the parent. Stolons located in the upper few centimeters of
the sediment, extend outward from the parent plant and produce new plants in
the immediate area (Nichols and Shaw, 1986).
Fragmentation is another type of vegetative clonal propagation which
provides intermediate to long distance dispersal (Aiken et al., 1979). M.
spicatum exhibit two types of fragmentation: autofragmentation and
allofragmentation. Autofragmentation is the self-induced abscission of shoot
apices, which generally occur after peak biomass has been attained (Madsen et
al., 1988; Madsen, 1997, Kimbel, 1982). Typically, one or more of the
internodes located on the upper 15- 20cm of the apical tip develop roots and
separation occurs shortly thereafter (Aiken et al., 1979). Allofragments are
formed by the mechanical breakage of the parent stem by disturbances in the
water, by boats, swimmers, animals and wave action. After separation from the
parent plant, fragments usually descend to the sediment where they produce
Page 64

oots, anchor and establish new plants, depending on the suitability of in-situ
conditions. Autofragments which have higher total nonstructural carbohydrate
content (TNC), have been shown to grow and over winter better than
allofragments (Kimbel, 1982).
Smith et al., (1967) reported that the most efficient method of
reproduction and spread of M. spicatum is by fragmentation. Either large
mature plant parts break off or float to the new areas or very small portions (5-
15 cm) of the plant tips are abscised, float for a period of time and then lose
their buoyancy and settle to the lake bottom. Roots develop from the abscised
tips which anchor the plant to the soil and under favourable conditions, new
colonies are started. A single 5cm fragment may take root and grow 3.5m or
more in one season. During the second year, multiple stems arise from the
rooted base and may achieve lengths of 7 to 13m and their fragmentation
continuous the spread of the plant. Patten (1956) reported that all new plant
material of M. spicatum in Lake Musconetcong is provided by asexual means
and the most efficient means of the reproduction was by the vegetative
fragments. An excised or an abscised stem will vigorously sprout adventitious
roots, bud profusely and increase in size. Such fragments float at first, but
ultimately sink to a level where the roots are able to grasp the substratum.
During late winter and early spring the plants develop a profusion of small
axillary buds which differ from normal vegetative buds in their size and the
ease with which they detach. Many species of Myriophyllum develop large and
prominent winter buds known as turions (Arber, 1920; Hagi, 1926) or
hibernaculae (Stover, 1951). Morphologically the small axillary buds of M.
spicatum are not turions but physiologically they may be considered. These
buds when detached, germinate into independent young plants. Turions have
been reported in some species of Myriophyllum, such as M. verticillatum and
M. exalbescens (Aiken and Walz, 1979, Arber, 1920). Arber (1920) stated that
under normal conditions it is probably the lowering of water temperature in
winter which induces turion formation. M. spicatum has been stated not to
Page 65

produce turions (Hegi, 1926, Hulten, 1947; Patten, 1956). Sprouting from
rhizomes is another mechanism for revegetation in the spring and effectiveness
of this type of reproduction has been observed in mid April (Patten, 1956).
Madsen et al., (1988) studied the relative importance of various
methods of reproduction for the expansion of Eurasian watermilfoil
populations in Lake George, New York. The study indicated that local spread
was largely dependent on stolons. Stoloniferous expansion was prominent
during mid-summer followed by a decline later in the season. Fragmentation
showed its prominence in September. The relative frequency of successful
expansion by Stolons accounted for 87%, while by fragments accounted for
13% only of the total expansion.
Madsen and Smith (1997) studied the vegetative spread of M. spicatum
in outdoor ponds at Lewisville, Texas. Colonies of M. spicatum expand via
stolon and fragment production. Clonal expansion primarily by stolons
provides Eurasian watermilfoil with a means of localized spread, while
intermediate distance expansion is provided by fragment production. The
predominant mechanism was stolon production. Stolon production accounted
for 74% of the areal expansion while fragments accounted for 26%.
Aiken et al., (1979) reported that in M. spicatum both fragments that
are formed naturally and those formed if the plant in broken by man‘s activities
are viable and have been responsible for the very rapid spread of this weed in
North America. However Nicholos (1991) explained that the problem
associated with fragments generated by harvesting does not survive and grow
as good as naturally produced fragments (Kimbel, 1982). Fragmentation and
winter buds are more important in reproduction. Winter buds are tight leaf
clusters born near the end of the stem; they develop when water temperatures
drop and day length shortens. The buds breakoff and fall to the bottom, where
they over winter. In the spring, the buds grow and elongate to form new plants
(Washington State Department of Ecology, 2003).
Page 66

Asexual reproduction by fragmentation and winter bud formation in
Myriophyllum has also been reported by other workers (Stanley, 1976; Cook,
1987). The fragmentation has been reported either accidental or the result is
abscission. Abscising fragments however, develop roots at the nodes before
separation from the parent plant and float for a period before they sink and
germinate into new plants. The over wintering buds or shoots of milfoil do not
usually consist of compact, abortive leaf tissue generally associated with true
turions.
Page 67

CHAPTER-3
MATERIAL & METHODS
Page 68

3.1: Study Area
The present study has been conducted in the valley of Kashmir which is
situated in the northern area of Indian subcontinent between 33 0 22' and 34 0 50'
N latitudes and 73 0 55' and 73 0 33' E longitudes covering an area of about
16,000 sq.km. The valley is surrounded by girdling chain of Himalayan
mountains, namely the Pir Panjal in the South and the Great Himalayan range
in the South East to North East and West. The climate of the valley is
predominantly temperate and it changes to sub-alpine and alpine in the higher
mountains. The valley of Kashmir also called as paradise on earth has a
network of rivers, glaciated streams, rivers as well as alpine, sub-alpine and
valley lakes which add to its beauty. The alpine lakes are situated above the
tree line and are fed mainly by glaciers. Their basins are rocky and are
completely devoid of macrophytic vegetation and remain covered with ice from
October to May. The sub- alpine lakes are situated in middle of the pine forest.
The main water bodies of the valley include: Anchar lake, Dal Lake, Mansbal
Lake, Wular Lake, Hokarsar and Hygam wetlands.
The River Jhelum is the main river of the Kashmir valley. The major
tributaries of this river system include; Lidder (Anantnag), Rambiara
(Pulwama/ Kulgam), Sind (Ganderbal/ Srinagar) and pohru (Kupwara). In
addition, there are many streams, irrigation channels, ponds and marshes which
support various species of aquatic plants.
3.2: Exploration and Collection of Plant Material
Aquatic habitats in the Kashmir valley were extensively surveyed and
explored for the collection of the plant material of Myriophyllum spicatum. The
sites were selected on the basis of the following criteria.
a. Represent all types of aquatic habitats inhabited by Myriophyllum
spicatum.
b. Abundance of Myriophyllum spicatum.
c. Trophic status and lentic and lotic water bodies.
Page 69

The exact geographical location of the selected sites (Fig. 1) and their
characteristic features are summarized in Table 3. The sites were surveyed
from April 2009 to November 2010. The species was collected from different
habitats.
The specimens of the species from each population were collected
prepared into vouchers using standard herbarium methodology (such as
pressing, drying and preservation etc.) and deposited in the Kashmir University
Herbarium (KASH) at the centre of plant taxonomy (COPT) of the Department
of Botany. The specimens of the species were initially identified with the help
of available literature and described. The description of the species includes
author citation and detailed morphological characterization. The species
identification was got confirmed from Dr. Michael L. Moody of the School of
Plant Biology, University of Western Australia, Crawly, WA 6009 Australia.
Table 3: Salient features of some Kashmir valley Sites selected for Studies
on Myriophyllum spicatum L.
Parameter Dal
Lake
(DL)
Srinagar
Nature Urban
Valley
Lake
Location 3 km of
Srinagar
city
Altitude
(m.a.s.l)
Mansbal
Lake
(ML)
Ganderbal
Rural
Valley
Lake
25 km
NW of
Srinagar
Hygam
Wetlands(HW)
Baramulla
Rural Valley
Wetland
40 km NW of
Srinagar
Shalimar
Stream(SS)
Srinagar
Chanderhama
Irrigation
Canal(CIC)
Baramulla
Stream Irrigation
canal
8 Km NE
of Srinagar
35 Km NW
of Srinagar
1585 1584 1583 1585 1583
Latitude
(North) 34 0 6´ 34 0 15´ 34 0 7´
Longitude
(East)
Area
(Sq.Km)
Maximum
depth (m)
34 0 7´ 34 0 7´
74 0 8´ 74 0 41´ 74 0 14´ 74 0 10´ 74 0 14´
11.5 2.8 - - -
6 12.5 - - -
Source: Surveyor General of India (SGI) Toposheet number =43J/Q.
Page 70

1.
2.
3
Hygam wetland
Chanderhama
irrigation canal
3. Mansbal lake
4.
5.
Dal lake
74 *
Shalimar stream
1
2
4
5
75 *
Fig.1: Map of J&K showing different study sites.
Page 71

3.3: Specie Morphology and Phenotypic Variability
A Random sample of 25 plants of the Myriophyllum spicatum were
drawn from each population (both running and standing waters) and the plants
were analyzed for morphological features and phenotypic variability. The
characters studied include number, shape and dimensions of leaves; petiole
length of leaves; rhizome size and length; stem shape and inter node length;
spike shape and length; peduncle length and number of flowers per spike;
shape and dimensions of flowers, seed morphology and size and number of
seeds per spike and per flower. The variations were stastically analyzed; using
SPSS 10. For microscopic studies Olympus stereo Zoom Trinocular
microscope was used.
3.4: Architectural Analysis
For description of patch characteristics, terminology used by Wolfer
(2004) was followed, according to which ‗plant‘ is defined as a complete unit
of ramets connected by rhizomes originating from a single primary shoot.
‗Ramet‘ is a single module of a clonal plant, consisting of shoots, rhizome and
roots. ‗Spacer length‘ is the rhizome length between two consecutive shoots of
the same plant.
In order to analyse the growth architecture of the species in various populations
(both standing and as well as running) a random sample of 25 plants from each
population was drawn and plants were analysed for spatio-temporal dynamics
and plasticity of clonal architecture. The characters studied include: total length
of rhizome, number of ramets per plant, branching frequency, ramet length,
spacer length and branching frequency of rhizome. The variation if any was
analysed stastically.
3.5: Phenology
Healthy individuals of the species were selected from different
populations, tagged and examined throughout the growing season to study the
life history pattern and mode of reproduction operative in the species in relation
to habitat condition of the study sites. The tagged individuals were monitored
Page 72

to record data on various reproductive phenophases such as initiation of
budding, peduncle growth and anthesis, duration of flowering and seed
formation. The axillary bud formation was examined regularly and their
number was recorded to evaluate the importance of these propagules in the
reproduction and fitness of the species.
3.6: Reproductive Biology
3.6.1: Floral Morphology, Development, Anthesis and Anther
Dehiscence.
A random sample of 25 inflorescences was used for studying the
morphology of flower and inflorescence. The floral structure and
characteristics of floral parts were determined by using magnifying lenses (10x
and 20x) and olympus zoom stereo microscope. Observations on flower and
inflorescence development and blossoming stage were made every day at
regular time intervals. Observations on anthesis and anther dehiscence were
also made at regular intervals, with the help of a 10x magnifying lens.
3.6.2: Stigma Receptivity
Stigma receptivity was checked by fixing stigmas of different ages in
carnoy‘s fixative (3 alcohol: 1 acetic acid) for 3-4 hours. The stigmas were
stained with aniline blue-lactophenol (Hauser and Morison, 1964) and scanned
under light microscope (10x X 20x combination). The stigmas carrying the
germinating pollen grains were considered as receptive. In order to find out
duration of stigma receptivity, the stigmas of varying ages were pollinated
manually by dusting on them the pollen grains obtained in bulk from freshly
dehisced anthers with the help of a sterilized needle. The pollinated stigmas
were fixed in 1: 3 acetic alcohol and subsequently stained in aniline blue-
lactophenol and studied periodically under a microscope. The data obtained
was recorded for determining the duration of stigma receptively. Stigma
receptivity was also determined by visual observation. Stigmas which were
transparent, shining and wet and adhere to 1mm 2 piece of paper were
considered receptive (Teryokin et al., 2002).
Page 73

3.6.3: Pollen Viability and Pollen to Ovule Ratio
Pollen viability was estimated by two methods:
1. Following Stanley and Linskens (1974), mature and undehised anthers
were placed in 1% tetrazolium chloride for one hour and squashed.
2. Dianne and Spicer, (1958) method in which mature and undehisced
anthers were squashed in 1% aniline blue-lactophenol and observed after
15 minutes. The healthy and plump stained pollen were recorded as
viable in both the cases.
Floral buds ready to anthese were collected for estimating pollen-ovule
ratio. Pollen quantity was estimated by squashing one anther (Several times) in
10 drops of distilled water in a cavity block and then shaken with a glass rod.
The following equation was used to calculate the number of pollen per flower.
Where
p x q = r
r x s = t
p = mean pollen count per drop of water
q = number of water drops taken initially in which one anther was squashed.
r = mean number of pollen per anther
s = mean number of anthers per flower
t = total count per flower
Average ovule number per pistil was counted by using dissection
microscope.
Pollen-ovule ratio was calculated following Cruden‘s (1977) method as
follows:-
P/O = pollen count per anther x No. of anthers per flower
Number of ovules per flower
Page 74

3.6.4: Pollen Volume
For calculating pollen volume Zhang‘s (2009) method was followed. The
pollen grains from fifty randomly selected dehisced anthers were put in a drop
of 2% acetocarmine and pollen diameter measurements were taken at 400x
using ocular micrometer. For spherical pollen grains, the volume was
calculated by using the following formula.
V= π D 3
6 where D = Diameter of Pollen grain.
3.6.5: In-vitro Pollen Germination
The in-vitro pollen germination was analyzed in media containing
sucrose, boric acid, calcium nitrate, magnesium sulphate and potassium nitrate
following Brew-Backer and Kwach (1963) with and without modifications
(Table 4) keeping the normality same as in the Brew-Beaker and Kwach
Solution. The pollen grains with their tube length more than their diameter
were considered germinated.
Table4: Combinations of Various Nutrients used for pollen germination of
Myriophyllum spicatum.
S. No Nutrient Combinations
1. 10% sucrose
2. 10% sucrose + boric acid 1 mg/10ml
3. 10% sucrose + calcium nitrate 3 mg/10ml
4. 10% sucrose + potassium nitrate 1 mg/10ml.
5. 10% sucrose + magnesium sulphate 2 mg/10ml
6. 10% sucrose + magnesium sulphate 2 mg/10ml + calcium nitrate
3 mg/10ml
7. 10% sucrose + boric acid 1 mg/10ml + Potassium nitrate 1
mg/10ml
8. 10% sucrose + boric acid 1 mg/10ml + calcium nitrate 3mg/10ml
9. 10% sucrose + boric acid 1 mg/ 10 ml + calcium nitrate 3 mg/ 10
ml + magnesium sulphate 2 mg/ 10 ml + potassium nitrate 1 mg/
10 ml
Final volume in each case is 30 ml.
Page 75

3.7: Pollen Mother Cell Meiosis
For studying pollen mother cell meiosis floral spikes when outside the
leaf sheath were fixed in Carnoy‘s fixative [ethanol: acetic acid (3:1)] between
1000hr-1300hr for 60-90 minutes, transferred to freshly prepared Carnoy‘s
fixative for about 22 hours and then transferred to 70% ethanol for preservation
at 4 0 C. During meiotic analysis a young spike was kept under dissection
microscope and individual floral buds were removed carefully from the spike.
Each floral bud was then taken on a neat and clean slide and a drop of 2%
propionocarmine was put on it for squashing the anthers. Analysis of various
stages of meiosis was done from temporary slides. The photographs were taken
from temporary slides at USIC using Leica DM LS2 microscope.
3.8: Resource Allocation
Individual plants from natural populations were harvested at maturity
(both pre and post pollinated), washed in the tap water and dried using blotting
paper. The ramets were divided into individual parts (shoots, rhizomes and
roots, spikes and seeds). Oven dried at 80 0 for 48 hours (Kawano and Masuda,
1980) and the dry mass (representing the amount of resources allocated) of
each component was calculated using and electronic balance.
3.9: Seed Biology: Seed biology was studied along the following lines:-
3.9.1: Seed Set
For estimating the seed set and reproductive success, plants were selected
at random in different populations, tagged and scored for the number of spikes
per ramet and the number of flowers and seeds per spike following Lubber and
Christenson (1966).
Percentage seed set was calculated as follows:
% age seed set per ramet = Total number of seeds set X 100
Total number of ovules born
Page 76

3.9.2: Seed Viability
Seed viability was analyzed using Tz-test. Twenty seeds of each replicate
were placed in water at room temperature for 24 hours and longitudinally
sectioned. The sections were incubated in dark in 2% aqueous solution of Tz
for 24 hours. Seeds showing strong stained embryo were considered viable.
3.9.3: In - Vitro Seed Germination
For in vitro seed germination studies, the seeds were randomly collected
from natural population and washed with 0.1% mercuric chloride for 5-7
minutes, followed by washing 4-5 times with distilled water and subjected to
different physical and chemical treatments (Table 5). All the experiments were
conducted in temperature, light and humidity controlled incubation cabinets.
The treatment solutions were made using deionized water and analytical grade
chemicals. Each treatment consisted of three replicated of 10 seeds, placed
within closed glass petri-dishes on Whatman No. 1 filter paper moistened with
15 ml of the given treatment solution or distilled water. Seeds planed for dark
treatment were moistened under green light and then quickly wrapped in black
carbon papers to reduce the chances of exposure of light. Emergence of
plumule was used as the indicator of germination and total germination was
recorded at the culmination of the experiment in respect of all the treatments.
Seed germination was also checked by placing 50 healthy and viable seeds in
30 x 10 x 4 inch tray containing 2 kg (wet mass) of sediment and water level
were maintained 1.5 inch above the surface of the sediment. These trays were
placed inside the seed germinator where temperature was maintained at
25±2°c.Seeds were considered to have germinated at the first visible plumule
emergence.
Page 77

Table 5: Physical and chemical treatments to test seed germination in
Myriophyllum spicatum.
S.
Treatment Time period Concentration
No.
(days) (mM)
1. Dry chilling 40 -
2. Wet chilling 40 -
3. Surgical exposure of embryo (epicarp,
mesocarp and endocarp removal)
4. Surgical exposure of embryo + GA3 0.5
5. Surgical exposure of embryo+ GA3 1
6. Surgical exposure of embryo+ GA3 2
7. GA3 1
8. GA3 2
9. Scarification (removal of epicarp and
mesocarp)
10. Scarification + IAA 0.5
11. Scarification + IAA 1
3.10: Reproductive Potential of Shoot Fragments
The reproductive potential of plant fragments was analyzed by placing 6
noded and 3 noded (apical, 5 each) and 6 noded and 3 noded (intermediate, 5
each) shoot fragments in three replicates in trays containing water without
sediment for a period of 15 days. During this period the temperature ranged
between 20 to 25 0 C and regular monitoring during this period was done to
record the number of plantlets formed by the fragments.
3.11: Reproductive Potential of Rhizomes
The reproductive potential of rhizomes was estimated by placing five
rhizome cuttings each 5 cm long (in three replicates) in 25x20x15 cm trays
containing 3kg (dry mass) of sediment and water level was maintained at 2 cm
above the sediment. The trays were kept in the laboratory for three months (
Jan - March 2010) with regular monitoring at room temperature. During this
period the temperature ranged between 8 to 20 0 C. At the culmination of the
experiment the characters studied include total number of plantlets formed per
rhizome cutting.
Page 78

3.12: Axillary Bud Germination
Vegetatively produced axillary buds were used for in vitro germination
studies. The axillary buds were germinated at different temperature regimes,
two varying light exposures and with or without sediment. Each treatment, with
three replicates, included five propagules placed in 1000 ml beakers. Sediment
of 200 g (on dry weight basis) was added to beakers and distilled water was
added to the saturation levels. The beakers were kept inside seed germinator
(Yorko) at different temperature regimes for 10 days. Axillary buds intended
for dark treatment were wrapped in aluminum foil. However the axillary buds
in alternate light and dark treated were not covered with aluminum foil. Total
germination was recorded at the culmination of the experiment in respect of all
the treatments.
3.13: Reproductive Potential of Axillary Buds
The reproductive potential of axillary buds was estimated by placing five
axillary buds (in three replicates) in 25 x 20 x 10 cm trays containing 2 kg
(dry mass) of sediment. The trays were put in the laboratory at room
temperature for one month, with regular monitoring. During this period, the
temperature recorded ranged between 14 to 20 0 C. At the culmination of the
experiment the germinated auxiliary buds were collected and analyzed for
reproductive potential. The characters studied include-total number of plantlets
formed from each axillary bud, number of meristematic branches and newly
formed axillary buds if any.
3.14: Scanning Electron Microscope (SEM) Studies
Double sided conductive tap was fixed to the stub and pollen from
mature undehisced anthers were dusted over it. The dusted material sputter
coated with gold was observed under SEM (S-3000 H). Mature and dry fruits
and seeds were loaded on the stub and coated with gold and observed under
SEM.
3.15: Statistical Analysis
For Statistical analysis software SPSS(10) was used.
Page 79

CHAPTER-4
OBSERVATIONS
Page 80

4.1: Distribution
Myriophyllum spicatum is cosmopolitan, herbaceous perennial
hydrophyte widely distributed in fresh to brackish waters throughout the world
and inhabit a wide variety of habitats Fig. (2)
In the Kashmir valley, the species is distributed in lakes, ponds, rivers and
streams . The species is found in all the major lakes of the valley viz: Dal lake,
Mansbal lake, Anchar lake, Wular lake, Nilnag lake; wetlands viz: Hookersar
wetland and Hygam wetland, river Jhelum, Achabal spring, Shalimar stream,
Chanderhama irrigation canal, Bal kol and almost in all other fresh water
bodies and irrigation chennels. From the above varied habitats of Myriophyllum
spicatum, three standing and two running water sites were selected in the
present study. The characteristic features of these selected sites are summarized
in Table ( 3) and Fig. (1).
4.2: Species Morphology and Taxonomy
Based on observations made in the present study, the overall Morpho-
taxonomical features of Myriophyllum spicatum are summarized as follows ;
Perennial rhizomatous herb, rhizomes terete with prominent nodes and
internodes, at each node of the rhizome adventitious roots arise. Stem 60-117
cm long, 1.5-3 mm in diameter with nodes and internodes, flexible, slender,
greenish or sometimes reddish in colour with internodes ranging from 1.74-3
cm. Leaves in whorls of 4(3-5), 1.74-3.28 × 0.39-0.84, pinnately divided into
24-33 filiform leaflets. Flowers whorled, with 4 flowers in each whorl,
aggregated on 2.73-5.13 cm long aerial spike, with upper male and lower
female flowers. The staminate flowers auxiliary, bracts 3 , shorter than petals, 3
× 1 mm, rhombic to elongate , entire ; petals 4 , pinkish 2 mm long , entire ;
stamens 8. Pistillate flowers without perianth; bracts 3, pectinate; stigmas 4,
plumose, white or light pinkish; ovary tetragonal with 4 deep furrows. Fruit
Page 81

5
4
Source: (Meijden, 1969; Meijden and Caspers, 1971; Orchard, 1975, 1980, 1981, 1986;
Aiken, 1981
1.
Fig. 2: Global distribution of Myriophyllum spicatum.
1. Asia, 2. Europe, 3. Africa, 4. South America, 5. North America, 6. Australia.
3
2
1
6
Page 82

A
3cm=3cm
1cm= 4 cm 1cm= 4 cm
1cm= 4 cm 1cm= 5 cm
Fig. 3: Myriophyllum spicatum (A) Habit (B) Female flower (C) Gynoecium (D) Male
flower ( E) Androecium.
B
C
D E
Page 83

globular 2.2×2.5 mm, dark brown, dehiscing by 4 longitudinal sutures. Seeds
trigonal with two flat sides and an outer convex side, 2.2× 1.5 mm. Fig. (3).
4.3: Phenotypic variability
The species inhabit both standing and running waters and is highly
variable in respect of its quantitative traits. The morphological traits analyzed
in the present study include internodal length, leaf dimensions, number of
leaflets per leaf , mature spike length, peduncle length, number of spikes per
ramet, total number of flowers per ramet, number of male and female flowers
per ramet and number of seeds per ramet. The results are summarized in Table
(6) and Figs. (4-18).
As depicted in the Table (7), a significant amount of variability was observed
for various morphological traits analyzed in different populations. The
differences were more prominent between standing and running water
populations. Myriophyllum spicatum produces longer and narrower leaves in
running water populations and smaller and broader ones in standing water
populations. The species produces longer petioles in running waters as
compared to standing waters. Mature spike length, peduncle length, number of
spikes and flowers per ramet were significantly higher in standing water
populations, on the other hand internodal length was found higher in running
water populations. Seeds were not formed in running water populations
whereas high seed set was recorded in standing water populations.
Dal Lake, Mansbal Lake and Hygam wetland populations were almost at par
with each other with respect to various morphological features analyzed, but
these three standing water populations were significantly different from two
running water populations namely Shalimar stream and Chanderhama irrigation
canal in these features.
Based on Tukeys test, different letters a, b, c and d in the Table (6) and Figures
(4-18) indicate means that are significantly different (Tukeys test≤0.05). The
letter ‗a‘ indicates the smallest mean value whereas letter‗d‘ as the largest mean
value for a particular trait.
Page 84

Feature
Table 6 : Phenotypic variability in morphological traits of Myriophyllum
spicatum from various populations of Kashmir valley.
Internode
length(cm)
Length of
leaves(cm)
Breadth of
leaves(cm)
Length of
apical
leaves(cm)
Breadth of
apical
leaves(cm)
Number of
leaflets per
leaf.
Number of
leaflets per
apical leaves.
Petiole
length(cm)
Mature spike
length(cm)
Peduncle
length(cm)
Number of
spikes per
ramet.
Total number
of flowers per
ramet.
Number of
male flowers
per ramet.
Number of
female
flowers per
ramet.
Number of
seeds per
ramet.
Population
Standing water (Mean±SE) Running water(Mean±SE)
Dal lake Mansbal lake
Hygam
wetland
Shalimar
stream
Chanderhama
irigation canal
b
a
c
d
d
1.89 ± 0.06 1.74 ± 0.03 1.99± 0.07 3.0± 0.12 2.69± 0.15
b
a
b
c
c
2.41 ± 0.26 2.21± 0.11 2.48±0.12 3.28±0.32 3.05±0.29
d
c
b
a
a
0.84±0.08 0.76±0.09 0.64±0.04 0.40±0.03 0.39±0.02
b
a
b
c
c
1.92±0.07 1.74±0.04 2.06±0.08 2.88±0.22 2.83±0.10
c
0.59±0.06
ab
30±3.21
b
29±2.76
a
0.14±0.02
c
5.13±0.50
c
1.41±0.22
b
3.26±0.7
c
50.86±5.46
c
20.8±2.90
bc
30.06±3.58
c
93.5±5.93
c
0.55±0.02
a
28±2.26
a
24±1.51
a
0.11±0.01
c
4.98±0.43
b
1.11±0.10
b
3.33±0.49
c
53.93±5.17
c
21.53±2.34
c
32.40±3.85
c
92±5.22
b
0.49±0.03
bc
32±3.35
b
31±2.47
b
0.20±0.04
b
4.13±0.35
b
1.00±0.21
b
3.2±0.41
b
46.4±4.98
b
18.13±1.64
b
28.26±3.84
b
85±4.50
a
0.39±0.01
c
34±3.17
b
31±2.03
c
0.40±0.05
a
2.86±0.17
a
0.74±0.04
a
1.73±0.35
a
23.5±2.77
a
9.66±1.60
a
13.86±1.93
a
0.39±0.01
c
33±2.08
b
31±2.03
c
0.39±0.05
a
2.73±0.24
a
0.72±0.08
a
1.20±0.41
a
24.5±2.53
a
10.26±0.96
a
14.26±1.73
Different letters a, b, c, d indicate means that are significantly different (Tukeys test ≤ 0.05)
-
-
Page 85

Table 7: ANOVA of phenotypic characters in Myriophyllum spicatum across
different populations.
Character Df F P
Internode length 4 573.84 0.000
Length of leaves 4 61.61 0.000
Breadth of leaves 4 122.58 0.000
Length of apical leaves 4 141.56 0.000
Breadth of apical leaves 4 35.60 0.000
Number of leaflets per leaf 4 10.73 0.000
Number of leaflets per apical leaf 4 28.35 0.000
Petiole length 4 156.65 0.000
Mature spike length 4 156.83 0.000
Peduncle length 4 50.82 0.000
Number of spikes per ramet 4 74.04 0.000
Total number of flowers per ramet 4 176.78 0.000
Number of male flowers per ramet 4 120.32
Number of female flowers per
ramet
4 125.43
Number of seeds per ramet 4 2233.90
0.000
0.000
0.000
Page 86

Internode length (cm)
3.5
3
2.5
2
1.5
1
0.5
0
b
a
c
DL ML HW SS CIC
standing water running water
Population
Fig. 4: Internode length in different standing and running water populations.
Leaf length (cm)
4
3.5
3
2.5
2
1.5
1
0.5
0
b
a
DL ML
standing water
HW
Population
SS CIC
running water
Fig. 5 : Leaf length in different standing and running water populations.
b
d
c
d
c
Page 87

Leaf breadth (cm)
Apical leaf length (cm)
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
3.5
3
2.5
2
1.5
1
0.5
0
d c
DL ML
standing water
HW
Population
SS CIC
running water
Fig. 6: Leaf breadth in different standing and running water populations.
b a
b
c c
DL ML
standing water
HW
Population
SS CIC
running water
Fig.7: Apical leaf length in different standing and running water populations
b
a
a
Page 88

Apical leaf breadth (cm)
Number of leaflets per leaf
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
40
35
30
25
20
15
10
5
0
c
c
b
a a
DL ML
standing water
HW SS CIC
running water
Population
Fig.8: Apical leaf breadth in different standing and running water populations
ab
a
bc
DL ML
standing water
HW SS CIC
running water
Population
Fig.9: Number of leaflets per leaf in different standing and running water populations.
c
c
Page 89

Petiole length (cm)
Number of leaflets per apical leaf
40
35
30
25
20
15
10
5
0
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
b
a
b b b
DL ML HW SS CIC
standing water running water
Population
Fig.10: Number of leaflets per apical leaves in different standing and running water populations.
a
a
b
c c
DL ML HW SS CIC
standing water
Population
running water
Fig.11: Petiole length in different standing and running water populations.
Page 90

Peduncle length (cm)
Mature spike length (cm)
6
5
4
3
2
1
0
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
c
c
b
a a
DL ML
standing water
HW SS CIC
running water
Population
Fig.12: Mature spike length in different standing and running water populations.
c
b b
DL ML HW SS CIC
standing water
running water
Population
Fig.13: Peduncle length in different standing and running water populations.
a
a
Page 91

Number of spikes per ramet
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
b
b b
DL ML HW SS CIC
standing water running water
Population
Fig.14 : Number of spikes per ramet in different standing and running water populations.
Total number of flowers per ramet
70
60
50
40
30
20
10
0
c
c
DL ML HW SS CIC
standing water running water
Population
Fig.15 : Total number of flowers per ramet in different standing and running water
populations.
b
a
a
a
a
Page 92

Number of male flowers per ramet
30
25
20
15
10
5
0
c c
b
DL ML HW SS CIC
standing water running water
Population
Fig.16 : Number of male flowers in different standing and running water populations.
Number of female flowers per ramet
40
35
30
25
20
15
10
5
0
bc
c
b
DL ML HW SS CIC
standing water
running water
Population
Fig.17 : Number of female flowers per ramet in different standing and running water populations.
a
a
a
a
Page 93

Number of seeds per ramet
120
100
80
60
40
20
0
c c
b a a
DL ML HW SS CIC
standing water running water
Population
Fig.18: Number of seeds per ramet in different standing and running water populations.
Page 94

4.4: Growth Architecture
Myriophyllum spicatum shows clonal growth form, consisting of a
branched rhizome and from which arises a network of leafy shoots called
ramets. The whole network of rhizome and ramets arising from it is called as
genet. The number of ramets on each rhizome and the spacer length between
two consecutive ramets are the important features in Myriophyllum spicatum
allowing the plant to some extent to adapt to heterogeneous environments such
as running and standing waters. The various growth architectural features
analyzed in the present study include: total length of rhizome, number of
ramets per plant (genet), number of branches per ramet, spacer length and
average length of ramets Fig. (19).
As the species inhabits both standing and running waters, it showed significant
variability in almost all growth architectural features between standing and
running water populations Table (9).It was observed that total length of
rhizome, branching of ramets, spacer length and average number of ramets per
plant was highest in standing water populations, whereas number of ramets per
plant and number of branches per rhizome was highest in running water
populations. However these features do not show significant differences among
standing water namely Dal lake, Mansbal lake and Hygam wetland populations
or among two running water namely Shalimar stream and Chanderhama
irrigation canal populations. Table (8) Figs. (20-25).
The different letters a, b, c and d in Table (8) and Figs. (20-25) indicate means
that are significantly different (Tukeys test≤ 0.05). The letter ‗a‘ indicate the
smallest mean value whereas letter ‗d‘ indicate the largest mean value of a
particular trait.
Page 95

Table 8 : Growth architecture of Myriophyllum spicatum in different
populations of Kashmir valley
Feature
Total length of
rhizome(cm)
Number
ramets per
plant/genet
Number of
branchs per
ramet
Spacer
length(cm)
Average length
of ramets(cm)
Number of
branchs per
rhizome
Mean±SE
Dal lake
c
18.02±1.26
a
5.86±0.35
d
0.86±0.01
b
3.0±0.14
c
109.46±5.85
a
2.13±0.35
Standing water
Mansbal lake
c
18.59±1.20
a
5.80±0.25
e
0.93±0.02
c
3.20±0.15
c
111.53±5.85
a
2.06±0.25
Population
Hygam
wetland
b
16.01±1.05
a
5.40±0.28
c
0.66±0.01
b
2.96±0.17
b
84.6±3.65
a
2.26±0.45
Shalimar
stream
a
12.26±1.04
b
14.2±0.94
b
0.33±0.01
a
0.83±0.03
a
64.33±3.37
b
3.0±0.37
Running water
Chanderhama
irrigation
canal
a
13.17±1.27
c
15.0±1.06
a
0.26±0.01
a
0.86±0.03
a
66.6±3.68
b
3.13±0.35
Letters a, b , c, d depicted in the table indicate means that are significantly different
(Tukeys test≤ 0.05)
Page 96

Table 9 : ANOVA of Growth architecture of Myriophyllum spicatum across
different populations.
Character
Total length of
rhizome
Number of ramets
per plant/genet
Number of branches
per ramet
Spacer length
Average length of
ramets
Number of branches
per rhizome
df
4
4
4
4
4
4
F
87.66
773.87
7111.83
1764.87
317.62
28.85
P
0.000
0.000
0.000
0.000
0.000
0.000
Page 97

R
A
M
E
T
Spacer length
Genet
Fig .19 : Clonal unit showing different parts.
Branch
Rhizome
Page 98

Total length of rhizome(cm)
25
20
15
10
5
0
c
c
DL ML HW SS CIC
standing water running water
Population
Fig.20: Total length of rhizome in different standing and running water populations.
Number of ramets per plant
18
16
14
12
10
8
6
4
2
0
a a
b
a
DL ML HW SS CIC
standing water running water
Population
Fig.21: Number of ramets per plant in different standing and running water populations.
a
b
c
a
Page 99

Number of branches per ramet
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
d
e
c
DL ML HW SS CIC
standing water running water
Population
Fig.22: Number of branches per ramet in different standing and running water populations
Spacer length(cm)
4
3.5
3
2.5
2
1.5
1
0.5
0
b
c
DL ML HW SS CIC
standing water running water
Population
Fig.23: Spacer length in different standing and running water populations
b
b
a
a
a
Page 100

Average length of ramet(cm)
140
120
100
80
60
40
20
0
c
c
b
DL ML HW SS CIC
standing water running water
Population
Fig. 24: Average length of ramets in different standing and running water populations.
Number of branches of rhizome
4
3.5
3
2.5
2
1.5
1
0.5
0
a
a
a
DL ML HW SS CIC
standing water
running water
Population
Fig.25: Number of branches per rhizome in different standing and running water populations.
a
b
a
b
Page 101

4.5: Phenology
The species is a submerged, perennial herb with pinnately divided
leaves inhabiting both standing and running water habitats. The phenological
behaviour of the species was studied in both standing and running water
populations. The phenology starts with the sprouting of rhizomes and axillary
buds in the first week of March and continues upto first week of April in
standing water populations, whereas sprouting of rhizomes and formation of
nodal plantlets commences in the second week of March and continues upto
second week of April in running water populations. The planlets grow
vegetatively from second week of April upto first week of June in standing
waters, while in running waters the process is completed between second week
of April and second week of July. The mature plantlets enter into the sexual
phase during first week of June and flowering continues upto last week of
September in standing waters. However, in running waters this phase starts
during fourth week of June and lasts upto third week of September. In standing
waters fruiting sets from second week of September and ends in third week of
October. In running waters, however, fruits are not formed. The senescence of
the above sediment parts starts in the second week of October and continues till
first week of December in standing water and in running waters the process
starts during the fourth week of October and continues upto the last week of
December Table (10) and Fig.(26).
Page 102

Table 10 : Phenological behavior of Myriophyllum spicatum in standing and
running water populations in the Kashmir valley.
Phenophase
Sprouting of vegetative
propagules(rhizomes,
axillary buds)
Vegetative growth
Flowering phase
Fruiting phase
Senescence
SW = Standing water
RW = Running water
Habitat
SW
RW
SW
RW
SW
RW
SW
RW
SW
RW
Duration
Week(month)
1 (3) - 1 (4)
2 (3) - 2 (4)
2 (4) - 1 (6)
2 (4 ) - 2 (7)
1(6) - 4 (9)
4(6) - 3(9)
2 (9) - 3(10)
-
2 (10) - 1(12)
4 (10) - 4(12)
Number of days
27
32
62
88
110
81
39
-
54
60
Page 103

Standing water Runing water
Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan
4.6: Reproductive Biology
4.6.1: Sexual Reproduction
4.6.1.1: Floral organization and development
Sprouting
Vegetative growth
Flowering phase
Fruting phase
Senescence
Fig.26: Phenological behaviour of Myriophyllum spicatum in standing and running water populations
The inflorescence of Myriophyllum spicatum is a cylindrical spike, red
in colour, raised on an elongated peduncle. The flowers are arranged
acropetally on the inflorescence axis. The flowers are unisexual; upper flowers
on a spike are staminate whereas lower ones are pistillate. The staminate and
pistillate flowers are tetramerous arising in the axil of a strong bract with two
more delicate bracteoles located at the base of each flower. These three bracts
entirely enclose and protect the young flower. Male flowers are having pinkish
petals, female flowers are without perianth. The morphological organization of
the inflorescence and flowers show slight variation in the number of whorls and
number of flowers in an inflorescence, but number of flowers in a whorl and
Page 104

number of anthers and pistils per flower do not vary. The number of flowers in
each whorl is 4, with 8 anthers per flower in the upper whorls and 4 pistils per
flower in the lower whorls Fig. (27).
The process of flowering can be divided chronologically into following
phases Figs. (28,29).
a) Formation of inflorescence (budding)
b) Peduncle growth
c) Female phase
d) Anthesis- anther dehiscence- male phase
a) Budding: During this stage differentiation of inflorescence axis and
flower bud initiation takes place and it lasts for 7-9 days.
b) Peduncle growth: Accelerated growth of the peduncle takes place during
this phase and it continues upto anthesis/ anther dehiscence. The
peduncle continues to grow for 8-12 days.
c) Female phase: The species is protogynous. This phase lasts for 3-4 days.
During this phase the female flowers open and stigmas look bright,
shining, turgid and wet. The opening of the female flowers occurs
acropetally during this phase, however bracts of the male flowers open
but the opening of the male flowers does not take place and anthers still
does not dehise and remain covered by pinkish petals.
d) Anthesis: Towards the termination of the female phase, the pinkish
petals of the male flowers open and anther dehiscence starts to facilitate
pollination. During this phase the stigmas of the same spike are dry and
shriveled, while as anther dehiscence is at its peak.
4.6.2: Stigma Receptivity
In Myriophyllum spicatum, the spikes after coming out of the leaf sheath
have flowers with androecium enclosed by petals and bract and gynoecium
enclosed by bract only. The stigma begin to come out of the bract on the 2 nd
day after the spikes come out of the leaf sheath and do not have any germinated
Page 105

a b
c
Fig.27 : (a) Inflorescence of Myriophyllum spicatum (b) Tetramerous arrangement of
flowers (c) Male flower (d) Female flower
d
Page 106

Developing spike
enclosed by leaf sheath
Stigmas exerted and
receptive
Anther dehiscence
Floral bud stage Peduncle growth Female phase initiates first
(Protogyny)
Dry, shrivelled
stigmas
Male phase (Anther dehiscence)
Fig.28: Diagrammatic representation of the chronology of spike and floral development in Myriophyllum spicatum.
Page 107

e
a b
c
Fig. 29: Chronology of spike and floral development in Myriophyllum spicatum:
a: Budding stage; b: Peduncle growth; c,d: Female phase; e,f: Male phase.
f
d
Page 108

Table 11: Stigma receptivity of Myriophyllum spicatum at different
developmental stages.
Stages of spike
development after
coming out of the
leaf sheath
0
+1
+2
+3
+4
+5
Pollen load on
stigmatic
surface
0 = on the day spike comes out of the leaf sheath.
Number of
germinated
pollen grains
Percentage
germination±SE
0 0 0
0 0 0
15.46±4.17 0 0
213.73 ±5.45 42.0±4.48 19.50±2.21
309.26±24.50 90.0±4.11 29.31±2.91
171.0±9.34 25.86±5.68 15.18±3.66
+1, +2, +3, +4, +5 = days after spike comes out of leaf sheath.
Beyond 4th day , the stigma shrivels and lose receptivity gradually.
Number of pollen on stigmatic surface
350
300
250
200
150
100
50
0
0Day 1Day 2Day 3day 4Day 5Day
Number of days
Fig.30: Pollen load on stigmatic surface is maximum on 4th day after the spike
come out of the leaf sheath and gradually decreases 4th day onwards.
Page 109

Number of germinated pollen grains
35
30
25
20
15
10
5
0
100
90
80
70
60
50
40
30
20
10
0
0Day 1Day 2Day 3day 4Day 5Day
Number of days
Fig.31: Pollen germination initiates on stigmatic surface on 3rd day after the spike come out
of the leaf sheath and frequency of germination is highest on 4th day and then sharply
declines 4 th day onwards.
Percentage of germinated pollen
grains
0Day 1Day 2Day 3day 4Day 5Day
Number of days
Fig.32 : Percentage of germinated pollen grains is maximum on 4 th day after the spike comes
out of the leaf sheath hence stigma receptivity and steadily decreases 4 th day onwards.
Page 110

a
Fig. 33 : Stigma receptivity a) pollen load on stigma b) germinating pollen grains on
stigmatic surface.
Page 111

pollen during this period. Subsequently, the stigma becomes receptive and the
receptivity lasts for 3-4 days. The number of deposited pollen grains as well as
the percentage germination of the pollen grains was highest on 4 th day after the
spike comes out of the sheathing leaf Table (11) Figs. (30-33).After this the
receptivity of the stigma decreases gradually within one or two days as the
stigma becomes brown and dry and the number of pollen grains deposited on
the stigma and their percentage germination declines.
4.6.3: Pollen morphology and viability
Based on the light and scanning electron microscope studies it was
observed that pollen grains of M. spicatum are small, sub-oblate to oblate,
radially symmetrical, isopolar, 4-zono colpate with scabrate-punctate tectum
Fig (35).The species produces high percentage of healthy, plump and stainable
pollen grains Table (12) and Figs. (34,36), which ranges from 88.56±4.58 to
89.19±4.90. There were no significant difference in the pollen viability
between standing and running water populations. The lentic and lotic waters
have no effect on the viability.
Table 12: Pollen viability of Myriophyllum spicatum in different
populations of the Kashmir valley.
Number of pollen
Population
grains scanned
M±SE
Dal lake 2496±55.46
Mansbal lake 2278±52.59
Hygam wetland 2253±54.53
Shalimar stream 2200±52.88
Chanderhama
irrigation canal
21759±52.8
Number of viable
pollen
M±SE
2247±57.29 88.82±9
2051±52.92 89.06±9.8
2031±54.49 89.19±9.4
1973±52.88 88.66±9.1
1953±52.90 88.56±9.0
Percentage
viability
M±SE
Page 112

4.6.4: Pollen-ovule ratio, pollen diameter and pollen volume
The species produces enormous quantities of pollen grains ranging from
27416.08±1216.48 to 29982.86±2336.10 per flower but only 4 ovules per
flower (1 ovule per carpel) are present. The pollen-ovule ratio worked out
ranges from 6854.02±304.12 to 7495.71±584.02 across different populations.
Pollen volume in the species ranges from 6540.82±387.81µm 3
7076.19±752.81 µm 3 and diameter from 23.2±0.44 to 23.8±0.83 µm Fig. (41).
Pollen-ovule ratio, pollen volume and pollen diameter did not show much
variation across different populations Table(13) and Figs.(37-39).
4.6.5: In-vitro pollen germination
Pollen grains were germinated in media containing sucrose, boric acid,
calcium nitrate, magnesium sulphate and potassium nitrate in different
combinations. Overall In-vitro pollen germination was very low and does not
exceed 15%. The media containing 10% sucrose and calcium nitrate and 10%
sucrose and magnesium sulphate yielded low percentage of germinated pollen
grains,
Percentage viability
120
100
80
60
40
20
0
DL ML HW SS CIC CIc
Population
Fig.34 : Pollen viability of Myriophyllum spicatum in different populations
to
Page 113

a
c
b
Fig.35: Pollen morphology: (a) Light microscopic view (b,c) SEM.
Page 114

a
b
Fig.36: Pollen Viability (a) Tetrazolium chloride test (b) Aniline blue test.
Page 115

Table 13 : Pollen-ovule ratio , Pollen diameter and Pollen volume of
Myriophyllum spicatum in different populations of Kashmir valley.
Feature
Pollenovule
ratio
Pollen
diameter
µm n=
50
Pollen
volume
µm³
n=50
Population
Dal lake Mansbal lake Hygam
wetland
7495.71±548.02
23.8±0.83
7076.19±752.81
Mean±SE
N= number of pollen grain
V= πD 3
6
Pollen - ovule ratio
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
7325±561.36
23.6±0.54
6887.69±474.96
7309±546.84
23.4±0.54
6714.25±474.96
Shalimar
stream
6993.60±455.36
23.2±0.83
6555.21±699.89
DL ML HW SS CIC
Population
Fig.37: Pollen -ovule ratio of Myriophyllum spicatum in different populations.
Chanderhama
irrigation canal
6854.02±304.12
23.2±0.44
6540.82±387.81
Page 116

Pollen diameter(µm)
30
25
20
15
10
5
0
DL ML HW
Population
SS CIC
Fig.38: Pollen diameter of Myriophyllum spicatum in different populations.
Pollen volume(µm)
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
DL ML HW SS CIC
Population
Fig.39: Pollen volume of Myriophyllum spicatum in different populations.
Page 117

Table 14 : In-vitro pollen germination of Myriophyllum spicatum in
different media.
Composition of
medium
S
S+B
S+C
S+P
S+M
S+M+C
S+M+P
S+B+C
S+B+C+M+P
Mean±SE
S = 10% Sucrose (30 ml).
Number of Pollen
grains scanned
200±10
191±24.55
184.66±5.03
187.33±5.68
191.33±19.50
181.33±10.50
209.66±11.06
215.66±18.44
213.00±16.32
Number of pollen
grains
Germinated
10±0.57
13±1.73
5.33±0.57
11.24±0.34
3.76±0.40
7.49±0.49
14.80±0.57
26.30±1.57
33.25±1.63
S+B = 10% Sucrose(15ml) + Boric acid 1mg/10ml of distilled water (15ml).
Percentage
germination
5.16±0.28
6.80±0.39
2.89±0.38
6.33±0.57
1.96±0.10
4.12±0.20
7.06±0.20
12.33±0.57
15.61±0.65
Page 118

S+C = 10% Sucrose (15ml) + Calcium nitrate 3mg/10ml of distilled water(15ml).
S+P = 10% Sucrose(15ml) + Potassium nitrate 1mg/10ml of distilled water(15ml).
S+M = 10% Sucrose(15ml) + Megnesium sulphate 2mg/10ml of distilled water(125ml).
S+M+C =10% Sucrose(10ml) + Megnesium sulphate 2mg/10ml (10ml) + Calcium nitrate
3mg/10ml (10ml).
S+M+P =10% Sucrose (10ml) +Megnesium sulphate 2mg/10ml(10ml) +Potassium nitrate
1mg/10ml (10ml).
S+B+C = 10% Sucr ose(10ml) +Boric acid 1mg/10ml (10ml) + Calcium nitrate 3mg/10ml
(10ml).
S+B+C+M+P = 10% Sucrose(6ml) + Boric acid 1mg/10ml of distilled water (6ml)+ Calcium
nitrate 3mg/10ml of distilled water (6ml) + Megnesium sulphate 2mg/10ml of distilled water
(6ml) + Potassium nitrate 1mg/10ml of distilled water (6ml).
Percentage germination
18
16
14
12
10
8
6
4
2
0
Composition of media
Fig. 40 : In-vitro pollen germination in different media
while media containing 10% sucrose, boric acid , calcium nitrate and 10%
sucrose, boric acid, calcium nitrate, magnesium sulphate and potassium nitrate
yielded highest percentage of germinated pollen Table(14) and Figs.(40,41 )
Page 119

a
b
Fig.41 : (a) Pollen diameter ( b) In-vitro pollen germination.
Page 120

4.6.6: Pollination mechanism
The species produces unisexual flowers on a common spike, the upper
male and lower female flowers. The spikes initially emerge just below the
water surface but finally come out and remain above the water surface. In a
population different spikes of the plant (genet) or different plants are at
different developmental stages viz: budding, female phase, male phase and
fruiting stage and continue to advance from one stage to another. The spikes
bearing receptive stigmas are smaller in length as the male flowers are not yet
fully developed and are around 3-3.5 cm above the water surface, whereas
those having completed the female phase and advanced into the male phase
with dehisced anthers and loaded with pollen are comparatively larger in length
and are around 3.5-6 cm above the water surface. The female and male phases
are distinct in that the male phase follows the female phase and the stigmas lose
their receptivity during male phase and become dry and shriveled. This clearly
depicts cross pollinated nature of the species, where pollen from spikes at male
phase are transferred to the spikes which are yet at female phase bearing
receptive stigmas Figs. (42,44). The first formed spikes in a population fail to
produce seeds due to the non- availability of pollen at the time of stigma
receptivity. As the spikes are well above the water surface, the primary mode
of pollination is anemophily. Entomophily and hydrophily can be ruled out
because it was observed that insects do not visit the spikes and the submerged
carpels abort instead of developing into mature seeds. Another possible mode
of pollination observed in the present study was anemo-ephydrophily where in
pollen grains falling on the water surface sometimes travel through water
currents and reach the receptive stigmas for pollination Fig.(42,44). However,
the major pollination mechanism seems to be anemophily. In running water
populations however, the spikes do not withstand the pressure of flowing water
and remain submerged all along and eventually decay without accomplishment
of effective pollination, hence fail to produce seed.Figs. (43,44).
Page 121

a b
Fig.42: Modes of pollination operative in Myriophyllum spicatum (a) Anemophily (b) Anemo-epihydrophily.
Page 122

Fig.43: Flowers do not open and spikes decay under water.
Page 123

a
b
Fig.44: Position of spikes with respect to water surface and pollination modes
operative in Myriophyllum spicatum (a) standing water (b) running water
Page 124

4.6.7: Fruit and seed morphology
The stereo and scanning electron microscopic analysis revealed that fruit
is a globular dark brown 2.2×2.5 mm schizocarp having smooth surface
architecture with four deep longitudinal sutures and a square shaped scar on
upper side having four elongated points which are the remains of the stigmas.
The lower side (where from fruit is attached to the spike) is circular. Each fruit
contains four individual seeds. The seed is a trigonal nutlet 2.2×1.5 mm with
smooth epicarp and mesocarp and a hard endocarp,having two flat sides and an
outer convex side Fig. (47).
4.6.8: Seed set and viability
The Myriophyllum spicatum produces a copious amount of seeds. The
seed set of the species is presented in Table (15) and Figs.(45,48). It was
observed in the present study that only standing water populations produce
seeds, whereas running water populations are not able to produce seeds because
effective pollination is not possible in running waters. The seed set percentage
in three standing water populations viz: Dal lake, Mansbal lake and Hygam
wetland was recorded as 77.91%, 70.98% and 75.19% respectively, indicating
that good numbers of seeds are produced by the species.
The seed viability of different populations do not very much. The dissected
seeds having healthy embryo and stain deep red in 2% 2, 3, 5 triphenyl
tetrazolium chloride (TTC) Fig. (48) were considered as viable. The percentage
of viable seeds was 90% in Dal lake population, 88% in Mansbal population
and 85% in Hygam wetland population Table (15) and Fig. (46). In running
water populations viz: Shalimar stream and Chanderhama irrigation canal, no
seed set was recorded.
Page 125

Table 15: Seed set and viability of Myriophyllum spicatum in different
populations .
Character
Number of
spikes per
ramet
Number of
flowers per
ramet
Number of
seeds per
ramet
Percentage
seed set
Percentage
seed
viability
Mean±SE
Dal lake
3.26±0.21
30.06±2.21
93.5±5.93
77.91
90.0±5.0
standing water
Mansbal
lake
3.33±0.24
32.40±2.50
92.0±5.22
70.98
88.33±2.88
Population
Hygam
wetland
3.13±0.17
28.26±2.10
85.0±4.50
75.19
85.0±5.00
Shalimar
stream
1.53±0.9
13.86±1.20
0
0
0
running water
Chanderhama
irrigation
canal
1.60±1.00
14.26±1.24
0
0
0
Page 126

Percentage seed set per ramet
90
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
DL ML HW SS CIC
standing water
running water
Population
Fig.45 : Seed set of Myriophyllum spicatum in different standing and running water populations.
Percentage seed viability
DL ML HW
Population
Fig .46: Seed viability of Myriophyllum spicatum
Page 127

a b
c d
Fig. 47: Fruit and seed morphology of Myriophyllum spicatum (a,b) SEM of fruit and
seed (c,d) Stereoscopic view of fruit and seed.
Page 128

a
b
Fig.48 : (a) Spike with fruits attached (b) Seed viability in tetrazolium chloride.
Page 129

4.6.9: Seed germination
Myriophyllum spicatum produce seeds in the month of September-
October. The mature seeds fall in water and then reach to the bottom of the
water body .These seeds get buried in the sediment at the bottom and do not
germinate immediately and remain dormant for long period. However, in order
to work out the dormant nature and germination ability of the seeds, in vitro
studies were carried out. The seeds were subjected to different concentrations
of GA3 and IAA, surgical exposure of the embryo by removal of the seed wall,
removal of epicarp and mesocarp, chilling and incubation under alternating
light and dark and continuous dark conditions in order to break dormancy. The
results are summarized in Table (16) and Fig. (49). The data reveals that seeds
in control and those treated with different concentrations of GA3 and IAA did
not germinate. Moreover, the seeds in which epicarp and mesocarp was
removed did not show any sign of germination. However, good germination
percentage was observed in seeds subjected to both wet and dry chilling. It was
observed that surgical exposure of embryos has a promoting effect on
germination and maximum percentage germination was recorded due to
surgical exposure of embryo plus different concentrations of GA3.
Age of seeds and light regimes also has effect on percentage germination. The
percentage germination of one year old seeds was less as compared to the
current year seeds in all the treatments. The seeds of the current year
germinate more vigorously and more in number as compared to the one year
old seeds provided the seed wall is cut to expose the embryo indicating that
hard endocarp is responsible for their dormancy. Moreover, it was observed
that overall percentage germination was more in alternate light and dark as
compared to continuous dark conditions in all the treatments. This
Page 130

Table 16: Effect of various physical and chemical treatments on germination
of seeds of different ages under alternate light and dark (L) and
continuous dark (D) conditions at 25°c in Myriophyllum spicatum
Treatment
Controll
40 days dry
chilling
40 days wet
chilling
Surgical exposure
of embryo
(epicarp, mesocarp
and endocarp
removal)
Surgical exposure
of embryo +
0.5mM GA3
Surgical exposure
of embryo + 1mM
GA3
Surgical exposure
of embryo + 2 mM
GA3
Age of seed Light regime Percentage
germination±SE
E0 L 0 a
D 0 a
E1 L 0 a
D 0 a
E0 L 31.66±2.88 fg
D 13.33±2.88 bc
E1 L 23.33±2.90 def
D 10.00±0 b
E0 L 33.33±2.90 g
D 16.66±2.88 bcd
E1 L 26.66±2.88 efg
D 13.33±2.88 bc
E0 L 63.00±5.77 hij
D 28.33±2.35 efg
E1 L 55.00±5.0 h
D 16.66±2.88 bcd
E0 L 68.33±5.77 ijk
D 31.66±2.88 fg
E1 L 60.00±5.00 hi
D 20.00±0 cde
E0 L 71.66±7.63 jk
D 33.33±2.90 g
E1 L 66.66±2.88 ij
D 21.66±2.88 cde
E0 L 76.66±5.77 k
D 35.00±2.88 g
E1 L 70.00±5.00 jk
D 23.33±2.88 def
GA3, 1mM E0 L 0 a
D 0 a
E1 L 0 a
D 0 a
GA3, 2mM E0 L 0 a
D 0 a
E1 L 0 a
D 0 a
Page 131

Scarification
(removal of
epicarp and
mesocarp)
Scarification
(removal of
epicarp and
mesocarp) + IAA,
0.5mM
Scarification
(removal of
epicarp and
mesocarp) +IAA,
1mM
E0 L 0 a
D 0 a
E1 L 0 a
D 0 a
E0 L 0 a
D 0 a
E1 L 0 a
D 0 a
E0 L 0 a
D 0 a
E1 L 0 a
D 0 a
df= 47, F=231.60,
P=0.000
E0= Seeds of current year
E1= Seeds collected one year before
GA3= gibberlic acid
IAA= Indole acetic acid
Different letters a, b, c, d, e, f,……… indicate means that are significantly different
(Tukeys test≤0.05)
Percentage germination
90
80
70
60
50
40
30
20
10
0
Treatment
Fig.49 : Percentage germination in various effective treatments.
L, E0
D, E0
D, E1
L, E1
Page 132

a
b
Fig.50: In-vitro seed germination in Myriophyllum spicatum
Page 133

eveals that with the increase in age of the seeds, there is decrease in their
viability and light has promoting effect on seed germination Fig. (50).
Seed germination was also tested by placing fifty healthy seeds in trays
containing sediment and water. After one month of continuous observation, the
seeds failed to germinate establishing the fact that seed germination is not
possible under natural conditions unless the hard endocarp is broken.
4.7: Pollen mother cell meiosis
Myriophyllum spicatum showed 21 perfect bivalents at metaphase.
Anaphasic disjunction was regular with 21 chromosomes at each pole Fig .(51).
The basic chromosome number in the species is 7. Based on x = 7, the species
turned out to be hexaploid with 2n =6x =42. Since 21 bivalents were observed
at metaphase and no multivalent formation was observed, it remains to be seen
whether the species is amphidiploids or not. The actual ploidy level of the
species can only be worked out by root tip mitosis and karyotype analysis
which was not possible in the present study due to small size of chromosomes.
4.8: Resource allocation
The allocation of dry mass to different plant parts in the species across
different populations is summarized in Table (17).
The dry mass allocation to different plant parts differs significantly across
standing and running water populations Table(18) and Fig. (52). The allocation
of dry mass to shoots is highest followed by seeds and spikes and much less is
allocated to undersediment parts in standing water populations. In running
water populations the trend is reverse, the undersediment parts in these
populations allocate higher resources and have higher dry mass as compared to
the standing water populations. The dry mass allocation in these populations is
higher to shoots followed by undersediment parts and spikes. Seeds are not
formed in these populations.
Page 134

a b
c
Fig. 51: (a,b) Metaphase I (2n=42) and (c,d) Anaphase I.
d
Page 135

Table 17 : Resource allocation to different parts in Myriophyllum spictum across different standing and running water
populations in the Kashmir valley.
Dry
weight(mg)↓
Shoots
Undersediment
part
Spikes
Seeds
Dal lake
b
4048±58.15
a
34.33±1.70
b
91.6±5.96
c
436.1±19.84
%age
87.80
0.74
1.98
9.45
Standing water (Mean±SE)
Mansbal
lake
b
4028.2±54.37
a
32.6±1.64
b
86.2±5.41
b
413±18.66
%age
88.33
0.71
1.89
9.05
Hgam
wetland
Population
b
4042.1±54.51
a
33.2±1.09
b
90.9±5.48
bc
419±18.77
%age
88.15
0.72
1.98
Shalimar
stream
a
2295.7±33.17
b
63.8±3.04
a
53±2.58
Running water(Mean±SE)
%age
95.15
2.64
2.58
Chanderhama
irrigation
canal
a
2305±35.74
b
65.4±3.77
a
55.00±2.58
Different letters a, b, c, d in the table indicate means that are significantly different( Tukeys test≤0.05)
9.13
a
0
-
a
0
%age
95.03
2.69
2.26
-
Page 136

Table 18 : ANOVA of Resource allocation in Myriophyllum spicatum across
different populations
Character
Dry weight of shoots
Dry weight of
undersediment parts
Dry weight of spikes
Dry weight of seeds
Percentage
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
b
a b c
b
df
4
4
4
4
b
a b
b
bc
a b
F
3814.27
488.38
177.39
2449.80
DL ML HW SS CIC
Population
a
b a
a
b a
P
0.000
0.000
0.000
0.000
Dry weght of shoots
Dry weight of
undersediment part
Dry weight of spikes
Dry weight of seeds
Fig.52: Resource allocation pattern of Myriophyllum spicatum in different standing and
running water populations.
Page 137

4.9: Vegetative reproduction
Vegetative propagation is the dominant mode of reproduction in
aquatic plants. The speed and ease with which these aquatic weeds spread to
non-indigenous regions confirms the potential of vegetative reproduction in
these plants. The Myriophyllum spicatum also operate various modes of
vegetative reproduction to spread. The different vegetative propagules
produced by this species include rhizomes, axillary buds, plant fragments
and nodal plantlets. As the species inhabits both standing and running
waters, it operates different modes in these two types of habitats. In standing
waters, it reproduces mostly by means of plant fragments, axillary buds and
rhizomes thus helping the species to spread in such type of habitats. In
running waters the most prevalent modes of vegetative reproduction is by
rhizomes, stem fragments and nodal plantlets arising from the older stems.
Axillary bud formation in running waters and nodal plantlet in standing
waters are rare. In lentic waters fragmentation of the stem is the most
important mode of vegetative propagation followed by axillary buds and
rhizomes. In running waters, rhizomes are the most prevalent mode followed
by fragmentation and nodal plantlets.
4.9.1: Reproductive potential of shoot fragments
Fragmentation is the most important mode of vegetative reproduction in
Myriophyllum spicatum. In the present study it was observed that under
natural conditions two types of shoot fragments are formed viz:
autofragments formed by the self induced abscission of shoot apices and
these fragments are generally formed when the plant has attained the peak
biomass, whereas another type viz: allofragments are formed by the
mechanical breakage of the parent stem by disturbances in the water by
boats, swimmers and water currents. To check out the potential of the
fragments to form new plantlets, apical and intermediate fragments with
varying number of nodes were used viz: six noded intermediate, three noded
intermediate, six noded apical and three noded apical. The results of
Page 1

different types of shoot fragments analysed for their reproductive potential
are summarized in Table (19). It was revealed that the six noded
intermediate fragments have the potential to form the maximum number of
plantlets followed by three noded intermediate and six noded apical
fragments. The three noded apical fragments are not able to form any
plantlet. It was also observed that length of six and three noded intermediate
fragments do not increase, hence the number of nodes remains the same,
whereas the length of six and three noded apical fragments increases and
hence the number of nodes also increases, but they show least potential to
form new plantlets, thus showing least reproductive capacity as compared to
intermediate fragments Figs.(53,54). So fragmentation is a type of
vegetative clonal propagation which provides intermediate to long distance
dispersal for the species.
Table 19 : Different types of shoot fragments and their reproductive
potential in Myriophyllum spicatum.
Fragment type
6- noded
intermediate
3- noded
intermediate
6- noded apical
3- noded apical
Mean±SE
()* = initial number of nodes
()** = final number of nodes
Mean initial
length(cm)
13.16 (6) *
6.3 (6)
7.7(6)
2.86(3)
Mean final
length(cm)
13.16(6) **
6.3 (6)
11.58(8.6)
4.16 (4.6)
Total number of
plantlets formed
2.86±0.64
2.3±0.41
0.93±0.11
0
Page 2

Total number of plantlets formed
3.5
3
2.5
2
1.5
1
0.5
0
6 - noded int 3 - noded int 6 - noded api 3 - noded api
4.9.2: Reproductive potential of rhizomes
At the end of the growing season the above sediment parts of
Myriophyllum spicatum undergo senescence but the undersediment rhizome
remain dormant during the winter. In the next growing season the rhizome
gives rise to new leafy shoots which develop into full plants after
undergoing vegetative growth and then enter into the reproductive phase.
The rhizomes have a high potential to form new plantlets, the present study
revealed that each 5 cm long rhizome cutting has the potential to form
6.0±0.62 new plantlets Fig. (54). So rhizomes contribute to the new
recruitments on arrival of the favourable conditions and help in
establishment of new populations. So rhizomes are a means of localized
spread in Myriophyllum spicatum.
4.9.3: Axillary bud germination
Fragment type
Fig.53 : Reproductive potential of different types of shoot fragments
Effect of temperature and sediment on axillary bud germination is
presented in Table (20) and the effect of temperature and light is presented
in Table (21). It was observed that the optimum temperature for axillary bud
germination is 20-25°C, though considerable germination occurs at 15°C;
the germination of axillary buds tends to decrease below 15˚C and is almost
Page 3

absent at 10˚C.The germination beyond 25°C also starts to decline
progressively and
c
a
b
Fig.54 : (a) Reproductive potential of stem fragments to form new plantlets (b)
plantlet with adventitious roots (c) Rhizome giving rise to new plntlets.
Page 4

percentage germination at 30°C is very low. The sediment does not have any
role in axillary bud germination as good percentage germination was
recorded without sediment also. Under alternate light and dark conditions,
axillary buds germinate more rapidly and more in number as compared to
conditions of complete darkness Figs. (55-57).
Table 20 : Effect of temperature and sediment under alternate light and dark
conditions on axillary bud germination.
Temperature 0 c
10
15
20
25
30
Treatment
Sediment/ without
sediment
S
W
S
W
S
W
S
W
S
W
S = with Sediment; W = without Sediment
Percentage germination
±SE
0
0
57±3.86
55.33±3.86
75.53±3.86
68.86±3.88
88.85±3.86
77.76±3.85
37.77±3.85
31.10±3.88
Page 5

Table 21: Effect of temperature and light on percentage axillary bud
germination in Myriophyllum spicatum.
Temperature 0 C
10
15
20
25
30
L=light and dark condition
D=dark condition
Treatment
Light regime
L
D
L
D
L
D
L
D
L
D
Percentage
germination±SE
0
0
42.2±3.81
31.08±3.88
68.88±3.85
51.06±3.88
82.2±3.81
64.4±3.81
31.06±3.86
22.2±3.81
Page 6

%age germination
100
90
80
70
60
50
40
30
20
10
0
15 20 25 30
Temperature˚C
with sediment
without sediment
Fig.55: Effect of temperature and sediment under alternate light and dark
conditions on percentage axillary bud germination.
%age germination
100
90
80
70
60
50
40
30
20
10
0
15 20 25 30
Temperature˚C
alternate light and dark
dark
Fig.56 : Effect of temperature and light on axillary bud germination.
Page 7

c
a
b
Fig.57 : (a) Axillary buds attached to the plant (b) Axillary buds of varying sizes
(c) In-vitro germination of axillary buds.
4.9.4: Reproductive potential of axillary buds
Page 8

The axillary buds of Myriophyllum spicatum are normal vegetative
buds but they differ from the vegetative buds in their size and the ease with
which they detach from the parent plant. Their size is larger than the normal
vegetative buds and detach easily from the parent plant. These buds are
formed during October to November and remain dormant during the chilling
temperature of the Kashmir valley and soon on arrival of the favourable
conditions germinate to give rise to new planlets.
The present study revealed that each axillary bud on germination
produces a single ramet. The newly formed ramet undergoes vegetative
growth and produces 8-12 new axillary buds which in a geometric
progression produce 64-144 new ramets. So Myriophyllum spicatum has a
very high potential to spread through axillary buds in lakes and ponds. Figs.
(58-59).
Axillary bud
germination Vegetative growth
Ramet
Fig.58: Reproductive potential of an axillary bud
bud.
Ramet with
8—12 axillary
buds
Geometric
progressio
n
New Ramets
(64—144)
Page 9

a
b
Fig.59: Potential of axillary buds to form new plantlets (a) axillary buds of
different sizes (b) germinated axillary bud.
Page 10

CHAPTER-5
DISCUSSION
Page 11

5.1: Distribution
The present work was carried out on Myriophyllum spicatum
belonging to the genus Myriophyllum (Haloragaceae) which is an aquatic
genus having cosmopolitan distribution (Moody and Les, 2010). It is native
to Europe, Asia and North Africa (Couch and Nelson, 1985). During the
present study M. spicatum was found in all the major Lakes of the valley viz.
Dal Lake, Mansbal Lake, Anchar Lake, Wular Lake, Nilnag Lake; wetlands
which include Hygam wetland, Hookarsar wetland, river Jhelum, Achabal
spring, Shalimar stream, Chanderhama irrigation canal, Bal kol and almost
in all fresh water bodies and irrigation channels. The observations of Kaul
and Zutshi (1965) and Kak (1990) on the distribution of M. spicatum in
Kashmir valley support our results.
5.2: Species morphology and phenotypic variability
The present study revealed that the species is a perennial rhizomatous
herb with adventitious roots having a long stem distinguished into nodes and
internodes with leaves in whorls of 3-5(4) at nodes, pinnately divided into
25-37 filiform leaflets, with four flowers in each whorl aggregated on aerial
spike with upper male and lower female; staminate flowers axillary with
three bracts, four pinkish petals and eight stamens; pistillate flowers without
perianth, bracts three, pectinate with tetragonal ovary having four deep
furrows and four stigmas; fruit globular schizocarp dark brown dehiscing by
four longitudinal sutures, splitting at maturity into four individual seeds
called nutlets; seed trigonal with two flat and an outer convex side. These
results are in agreement with various studies at global level (Kak, A.M,
1978; Aiken and McNeill, 1980; Aiken, 1981; Haynes, 1988; Orchard, 1986;
Donaldson and Johnson, 1998; Johnson et al., 1998; Crow and Hellquist,
2000 and Moody and Les, 2010).
Aquatic plants, many of which propagate vegetatively tend to possess
lower genetic diversity than terrestrial plants, implying an increased role of
phenotypic plasticity for diversity (Sculthrope, 1967; Barrett et al., 1993).
During the present investigation it was observed that M. spicatum inhabit
Page 12

oth standing as well as running waters. The species in response to different
ecological environments have developed distinct morphological characters
with respect to leaf dimensions, petiole length, mature spike and peduncle
length as well as number of spikes, flowers and seeds per ramet to adapt
itself in different habitats. It was observed that leaf dimensions of the
species do not vary much within different standing/running water
populations but differ significantly between standing and running water
populations. The species produces narrow and longer leaf blades as well as
longer petioles in running waters whereas in standing waters wider and
smaller leaf blades with short petioles are produced. Such variations in leaf
morphology have been reported earlier in Potamogeton by Kaplan (2002,
2008). Puijalon and Bornette (2006) also observed that leaf breadth reduces
significantly in plants exposed to stress of water currents. Mature spike and
peduncle length vary significantly across standing and running water
populations. Mature spike and peduncle length was found smaller in running
water populations as compared to standing water populations. This is
because pressure of the flowing waters impairs the development of spikes as
strong water currents in running waters lead to negative development of
plants due to mechanical stress ( Gants and Caro, 2001; Riis and Biggs,
2003). The number of spikes and flowers per ramet in running water
populations was less as compared to standing water population showing
significant differences because of stress of flowing water, similar results
were obtained by Kautsky (1987) who observed that in Potamogeton
pectinatus floral number per m 2 was highest in sheltered populations and the
number decreased from 945 to 672 per m 2 with increasing exposure to
waves. In standing water populations copious amount of seeds were formed
whereas no seed set was observed in running water populations because the
plants growing in running waters fail to accomplish sexual reproduction and
do not produce seeds (Kaplan, 2002, 2008).
Page 13

5.3: Growth architecture
Majority of the submerged macrophytes form clones, consisting of
complex network of ramets interconnected by rhizome. Clonal morphology
changes in response to environmental factors, both at the level of individual
ramet as well as at clonal level (Puijalon et al., 2008). The present study
revealed that Myriophyllum spicatum showed considerable and significant
variation in clonal growth between standing and running waters, however
various growth architectural features do not vary much among different
standing water populations or running water populations.
In standing water populations branching of the ramets and spacer
length is more as compared to the plants of running water populations. The
ramets in running water populations do not produce many branches because
fast flowing waters impairs the development of branches and there is also a
risk of getting detached from the main axis by pressure of flowing water.
However in running waters, the number of ramets per plant (clone) and the
number of branches per rhizome is more as compared to the plants of
standing waters. Thus in running waters increased number of ramets per
plant, reduced spacer length and increased number of branches per rhizome
could help in the formation of dense canopy and enhance anchorage
efficiency; a strategy of resistance that reduces the effect of pressure of
flowing waters. This dense growth form described as Phalanx growth form
by Lovett-Doust (1987) due to reduced spacer length and increased
branching could allow the plants to occupy favourable patches in a
heterogeneous environment (De kroon et al., 1994 ; Dong and De- kroon,
1994; De-kroon and Hutchings, 1995).The present study is quite in
agreement with the earlier findings for Mentha aquatic, where the species
produces increased number of creeping stems and dense canopy in running
waters to reduce the stress of flowing water (Puijalon et al., 2008). The
benefit of increased number of creeping stems, enhanced anchorage and
formation of dense canopy are responsible for reducing the effect of aero or
Page 14

hydrodynamic forces in many other plant species (Sand-Jenson and Mebus,
1996; Speck, 2003; Lui et al., 2007).
The length/height of ramets in standing water populations mostly
depend upon the depth of water body; while in running waters ramets grow
in the direction of flow. The alignment of creeping stems with the direction
of flow may be induced by different mechanisms. First it could be due to
drag forces exerted by water currents pushing the stems in the flow direction
(Vogel, 1994; Kotshy and Rogers, 2008). Secondly, it could also result due
to higher mortality of the creeping stems growing in the other directions, for
instance due to damages by sandblasting and drifting particles (Cleugh et al.,
1998). Third factor is the different activity of the meristems. The meristems
could grow perfectly in the downstream direction that is partly sheltered
from the water current and submitted to less stressful conditions (Sand-
jenson and Pedersen, 1999), where as the other growth directions could be
inhabited (Puijalone et al., 2008). Total length of rhizome in different
populations could not be measured precisely as it depends upon the nature of
substratum of water body and handling of plant material during collection
(plucking of plant material).
5.4: Phenology
Myriophyllum spicatum overwinters by means of rhizomes and winter
buds. These structures start sprouting during the first week of March and
continue upto second week of April when environmental conditions such as
temperature and light are available, because these factors are important for
the initiation of a particular phenophase (Rathcke and Lacey, 1985). The
vegetative growth commences during April and continues till June; however
in running water populations the vegetative growth phase is longer than in
standing water populations. The flowering commences from June to
September and continues for 1-4 months in standing water populations and
for 1-3 months in running water populations. The longer vegetative phase,
delayed and shorter flowering phase in running waters may be due to the
lower allocation of resources to sexual reproduction and permanent exposure
Page 15

of plants to mechanical stress (Niklas, 1998; Henery and Thomas, 2002; and
Hodges et al., 2004). The fruiting phase completes from September to
October in standing water populations where as in running water populations
fruits are not formed. The senescence starts from second week of October
and continues till December. These phenological events are in agreement
with the work of Patten (1956) and Spencer and Lekic (1974).
The knowledge about life history is very important for effective
management of this species and can be utilized to identify week points in the
plants life cycle and exploit them for long term management (Madsen,
1993). M. spicatum produces flowers, seeds and axillary buds during June-
October. Therefore removal of ramets before June can prove an effective
method for control of this aggressive species. This is supported by the work
of Caffery and Monahan (2006) who reported that in Myriophyllum
verticillatum, removal of turions yielded desirable results in control of this
species as compared to annual treatment with dichlobexil, followed by
mechanical cutting. The present study revealed that knowledge about
various life history traits, such as different types of sexual and vegetative
propagules, their time of formation and germination is very important for
long term management of this aggressive species in the Kashmir valley.
5.5: Sexual Reproduction
Sexual reproduction in Myriophyllum spicatum starts with the
development of an inflorescence which is a terminal spike, red in colour and
raised on an elongated peduncle. The flowers are unisexual, aggregated
acropetally on the inflorescence axis with upper male and lower female
arising in the axil of a strong bract with two more delicate bracteoles located
at the base of each flower. Male flowers are having pinkish petals whereas
female flowers are without perianth. The morphological organization of the
inflorescence and flowers show slight variation but the number of flowers in
each whorl is four. These results are in agreement with works of Cook
(1987); Smith and Barko (1990); Aiken et al., (1979) and Hartleb et al.,
(1993). The process of flowering is chronologically divided into four distinct
Page 16

phases viz: budding, peduncle growth, female phase and male phase and this
is in close agreement with the observations of Patten (1956) and Aiken
(1981).
The stigmas of an inflorescence remain receptive for 3-4 days after
the stigmas come out of the leaf sheath. During this period the anthers are
enclosed by petals and the receptive stigmas are well exerted above the
bracts surrounding the female flowers. The ―female phase‖ is quite distinct
and follows the ―male phase‖ (protogyny). Protogyny is a well known
adaptation to outcrossing in aquatic angiosperms as also demonstrated by
Patten (1956) in Myriophyllum spicatum; Teryokhin et al., (2002) in
different species of Potamogeton and Guo and Cook (1990) in Groenlandia
densa. The long duration of stigma receptivity is attributed to blossoming of
the flowers in female phase acropetally along the axis of the inflorescence.
The spikes having receptive stigmas (female phase) as well as those with
fully opened flowers undergoing anther dehiscence (male phase) are always
situated well above the water surface except few lower female flowers
occasionally. This creates congenial conditions for aerial pollen transfer
from anthers to stigmatic surface. The insect pollinators were never found
visiting Myriophyllum spikes which further strengthen our belief of
anemophily operating in the present species.
The pollen–ovule ratio (p/o) in a flower is a useful tool to study plant
breeding systems (Cruden, 1977). Cruden (1977) observed that higher the
degree of autogamy, the lower the pollen ovule ratio. A number of studies
have more or less confirmed the validity of pollen- ovule ratio as an
indicator of the breeding system (Lopez et al., 1999; Cruden, 2000; Jurgens
et al., 2002). During the present study it was observed that Myriophyllum
spicatum depicts high pollen- ovule ratio which ranges from
6854.02±304.12 to 7495.71±584.02 across different populations, which
shows its anemophillous and outbreeding nature. Our results are well
supported by the findings of Philbrick and Anderson (1987); Cruden (2000)
and Zhang et al., (2009) which state that the species whose pollen-ovule
Page 17

atio ranges from 6,000-8,200 are xenogamous and show anemophily or
epihydrophily. The small amount of variation observed in pollen-ovule ratio
between different populations in the present study may reflect sampling
error or may have environmental basis (Diazlifante, 1996; Affre et al., 1995;
Ramsay et al., 1994; Vuille, 1988 and Cruden, 1977). In the present study,
pollen diameter of the species was found ranging from 23.2±0.44 to
23.8±0.83 µm. Probably due this small size of pollen, the species is able to
produce large amount of pollen grains per flower and hence pollen-ovule
ratio, thus helping in its outbreeding nature. These observations are well
supported by the findings of Zhang et al., (2009); Lopez et al., (1999);
Vonhof and Harder (1995); Knudsen and Olesen (1993); Mione and
Anderson (1992); Small (1988); Cruden and Miller-ward (1981); Ornduff
(1980) and Cruden (1977). These investigators have reported a negative
correlation between pollen grain number and size. Akerman (2000) reported
that small pollen grains are an adaptation to anemophily.
The pollen grains of M. spicatum were found small, spherical,
radially symmetrical, isopolar having four colpi with a rough surface.
Similar results have been reported in earlier studies on M. spicatum by
Alwadie (2008) in Saudi Arabia and Parveen (1999) in Karachi. A strong
correlation exists between pollen morphology and pollination mechanism
(Tanaka et al., 2004; van Vierssen et al., 1982). Pollen grains of
anemophillous taxa are small, spheroidal, and dry with simple apertures and
scabrate tectum (Shuang-Quan et al., 2001; Parveen et al., 1994). Based on
the pollen morphology, the present investigation clearly indicates that M.
spicatum comes under the category of anemophillous taxa. During my field
surveys the mature spikes in M. spicatum were always found above the
surface of water. These spikes were also found at different development
stages. Some spikes were at female phase when stigmas were found
receptive and male flowers completely closed, whereas other spikes were at
male phase showing anther dehiscence and their stigmas have lost the
receptivity. Based on this observation we can draw a conclusion that pollen
Page 18

from male flowers of one spike would be available for pollination to the
receptive stigmas of other spikes thus exhibiting cross pollination. As the
spikes are found well above the water surface, insects do not visit the
flowers and based on pollen morphology and pollen-ovule ratio it is clear
that primary mode of pollination is anemophily. Anemophily in
Myriophyllum has also been reported earlier (Cook, 1988, 1996; Patten,
1956). An interesting alteration to anemophily observed in the present
investigation was anemo-ephydrophily, where in due to the activity of wind
pollen dust falls on the surface of water and due to water currents the pollen
grains are transferred to the lowermost receptive stigmas which are at the
surface of water. However this is not a primary mode of pollination and such
anemo-ephydrophily has also been reported in Potamogeton (Zhang et al.,
2009).
5.6: Fruit and Seed Biology
The fruit and seed of Myriophyllum spicatum has been variously
described by different workers and given different names. Fruit has been
given the name achene and seed as nutlet by Patten (1955), but it is not an
achene because achene is a single seeded indehiscent fruit. Orchard (1986)
called fruit as nut and seed as nutlet but according to the present
investigation it cannot be called as nut because nut is a single seeded
indehiscent fruit. Based on the present stereo and scanning electron
microscopic investigations the fruit is a schizocarp with four deep
longitudinal sutures where from it splits into four individual seeds called
nutlets. Because schizocarpic fruits are intermediate between dehiscent and
indehiscent fruits. The fruit instead of dehiscing rather splits into a number
of segments each containing one or more seeds. Our observations are
strongly supported by the findings of Aiken (1981) .
During the present investigation it was observed that Myriophyllum
spicatum produce a copious amount of seeds and the seed set percentage
ranges from 70.98% to 77.91% across different populations. The seed set
depends upon various factors which include inflorescence size, position of
Page 19

inflorescence with respect to water surface, meiotic behaviour and habitat.
The standing water populations possess large inflorescences which are
always above the water surface and have more than thirty flowers per
inflorescence. The pollen ovule ratio is high and the stigmas remain
receptive for long duration along the whole length of the spike. All these
features including normal meiotic behaviour are factors which favour
effective pollination and in turn high seed set. The anaphasic segregation is
normal and hence the species is able to produce viable pollen and seeds. The
small amount of variation observed in seed set between different standing
water population‘s viz. Dal Lake, Mansbal Lake and Hygam wetland may be
due the eutrophic environment of the Dal Lake as compared to Mansbal
Lake and Hygam wetland. This is supported by the work of Madsen and
Boylin (1989) on Myriophyllum seed ecology from an oligotrophic and
eutrophic Lake. The plants inhibiting running waters do not produce seeds.
Probably the spikes in such populations do not withstand the pressure of
running waters and pollen grains are washed away without causing effective
pollination.
Seed germination in Myriophyllum spicatum was found significantly
influenced by various factors such as physical and chemical treatments, age
of seeds and light conditions. The seeds in which seed coat was not removed
(intact seeds) do not germinate even when treated with different
concentrations of GA3, IAA and also after removal of epicarp and mesocarp.
The study of Patten (1955) that partial removal of stony endocarp of the
Myriophyllum spicatum seeds is effective in causing seed germination is in
close agreement with the present study, however removal of epicarp and
mesocarp causes seed germination is not in agreement with the present
study.
The cold stratification treatment to some extent improved seed
germination. This confirms that dormancy is due to the mechanical barrier of
the seed coat, mainly because of hard stony endocarp of the coat. Low
temperature of the seeds causes rupturing of the seed coat and thus their
Page 20

dormancy is overcome. The results obtained are in conformity with those of
Patten (1955) who also reported that seed germination can be improved by
following cold stratification in M. spicatum. Similar results have been
obtained by Teltscherova and Hejny (1973); Baskin and Baskin (1998) and
Hay et al., (2008) in different species of Potamogeton. Under natural
conditions also, the stratification treatment more closely akin to freezing
winter temperatures of the Kashmir valley may have promotory effect on
seed germination. Cold stratification is an effective way of alleviating
dormancy in many species especially those from temperate regions (Baskin
and Baskin, 1998, 2004) which ensured that germination does not occur
until spring commences when water temperature is more favourable to the
vegetative growth of aquatic plants (Hay et al., 2008).
The seed age has significant effect on its germination. The present
data confirms that M. spicatum seeds do survive desiccation. These results
are in conformity with those of Standifer and Madsen (1997) who reported
that M. spicatum seeds do survive desiccation and viability of seeds is
reduced under storage. The freshly collected seeds as well as those dry
stored upto one year germinate provided the seed coat is cut. Percent
germination however, declines with age of the seed. Similar results were
obtained by Hay et al., (2000, 2008) in case of Potamogeton. These
observations indicate that no after ripening period is needed by the embryo
of M. spicatum and the seeds are an important means of propagation in the
habitats where water dries up for some period during the year. These
observations are in agreement with the work of Kadano (1984) who
observed that Potamogeton distinctus overcomes the long lasting drought in
irrigation reservoirs under warm temperature conditions by the formation of
seeds.
The seed germination varies significantly under continuous dark and
alternate light and dark conditions. During the present study it was observed
that the percentage germination was more in alternate light and dark
conditions as compared to the seeds kept in continuous dark conditions in all
Page 21

the treatments. These observations are in agreement with the results of Coble
and Vance (1987). However, for aquatic plants effects of quality of light are
less clear, although some quantitative effects are known (Spencer et al.,
1971; Sharma and Gopal, 1978).
The seeds do not germinate in trays provided with sediment and
water. The reason for this is the hard seed coat which restricts the water
absorption and low temperature needed by the seeds for their germination
causing prolonged seed dormancy. The ability to germinate under a range of
environmental conditions is one of the reasons why M. spicatum is found in
range of habitats and has wide distribution in the diverse aquatic ecosystems
of the Kashmir valley.
5.7: Pollen mother cell meiosis
Earlier work on pollen mother cell meiosis and chromosome count
reveals that genus Myriophyllum is monobasic having x = 7 (Fedorov, 1969;
Love and Love, 1956; Love and Ritchie, 1966). During the present
investigation it was observed that Myriophyllum spicatum is hexaploid with
2n = 6x =42. The 42 chromosomes pair into 21 bivalents which segregate to
21 chromosomes to each pole without error. The anaphasic disjunction is
regular and normal due to which enormous quantities of viable pollen grains
are produced. The normal meiotic behaviour imposes genetic stability upon
the species and ensures fertility and high pollen output.
5.8: Resource allocation
Every organism allocates its resources for various essential activities,
like maintenance, growth and reproduction (Willson, 1983). The analysis of
resource allocation to different plant parts is useful in predicting allocation
patterns and survival strategies of plants (Madsen, 1991).
The dry mass allocation to different plant parts in Myriophyllum spicatum
varies significantly between standing and running water populations. The
dry mass allocation to shoots and spikes is less, and undersediment parts are
higher in plants inhabiting running waters as compared to plants inhabiting
standing waters. It is presumably because the ramets in running waters do
Page 22

not produce more branches required to avoid the risk of getting detached by
the pressure of running waters. Similarly the development of spikes gets
impaired in such stressful environments. The undersediment parts (rhizome,
roots) spread between the stones and sediment and in turn provide strong
anchorage and protect the plants from completely being washed away. The
rhizomes are mostly the means of propagation in running waters. Permanent
exposure of plants to mechanical stress usually results in reduced size of
leaves, height and consequently biomass allocation (Niklas, 1998; Henery
and Thomas, 2002; Boeger and Poulsan, 2003) and increased allocation to
undersediment parts. Puijalon and Bornette (2006) also observed higher
allocation to below ground organs in running waters. This result in better
resource accumulation in the parts protected from the water currents and is
considered to improve anchorage efficiency (Cook and Emnos, 1996;
Niklas, 1998). A lower allocation to sexual reproduction associated with
delayed flowering is also often observed in running water populations
(Niklas, 1998; Hodges et al., 2004). The plants in standing waters allocate
much of the resources to shoots, seeds and spikes followed by
undersediment parts.
5.9: Vegetative Reproduction
Aquatic plants display a wide range of methods for clonal
propagation which include the formation of rhizomes, runners, stolons,
tubers, fragments and axillary buds. Vegetative reproduction is the dominant
mode of reproduction in water plants (Sculthrope, 1967; Hutchinson, 1975).
During the present study it was observed that Myriophyllum spicatum
operates multiple modes of vegetative propagation by rhizomes, fragments,
axillary buds and nodal planlets.
Rhizomes have a tremendous potential to form new plantlets. The
experimental data obtained during the present investigation reveals that each
5 cm long rhizome cutting has the potential to form 6.0±0.62 new plantlets.
The sprouting of rhizomes is an effective mechanism for revegetation and
effectiveness of this type of reproduction has been observed in March to
Page 23

April of the growing season. So rhizomes are a means of localized spread in
Myriophyllum spicatum. These observations are strongly supported by the
work of Patten (1956); Madsen et al., (1988) and Madsen and Smith (1997)
who obtained almost similar type of results in Myriophyllum, and by Bartly
and Spence (1987) in Scirpus lucustris, Smith et al., (1989) in case of
Nymphea alba. Rhizomes are the most important means of proliferation in
case of running waters. Weigleb and Kadano (1989) opined that highly
disturbed sites are colonized by the species of Potamogeton by means of
rhizomes and stolons. The rhizomatous growth form helps to resist the
damaging forces of waves and tidal currents (Hartog, 1970).
Fragmentation is another important mode of reproduction operative in
the species. During the present study it was observed that Myriophyllum
spicatum produces two types of stem fragments viz: autofragments and
allofragments in natural populations. The autofragments are formed by the
self induced abscission of shoot apices and these fragments are generally
formed when peak biomass has been attained whereas allofragments are
formed by the mechanical breakage of the parent stem. These fragments
provide a means of intermediate to long distance dispersal of the species.
These observations are strongly supported by the work of Aiken et al.,
(1979); Madsen et al., (1988); Madsen (1997) and Kimbel (1982) in
Myriophyllum and by Riis et al., (2009) in case of Potamogeton. The
deweeding produces a large number of allofragments. This is however, a
common practice in different water bodies of the Kashmir valley. These
allofragments are usually more important than seeds for dispersal of the
plant and colonization of open stream beds (Riis, 2008). Moreover, the
reproductive potential of apical and intermediate fragments with varying
number of nodes was also analysed during the present study. It was observed
that among six and three noded apical and intermediate fragments, the six
noded intermediate fragments showed the maximum potential to form the
new plantlets followed by three noded intermediate and six noded apical
fragments. The three noded apical fragments are not able to form any
Page 24

plantlet. Moreover it was also observed that apical fragments showing the
least potential to form new plantlets showed an increase in length and
number of nodes while as intermediate fragments does not showed any
change in length and number of nodes.
These observations reveal that both the number of nodes of the stem
fragment and position of the fragment whether apical or intermediate
determine the reproductive potential of the stem fragments in case of
Myriophyllum spicatum. These observations are in accordance with the work
of Langeland and Sutton (1980) in Hydrilla verticillata in which a single
noded fragment produces roots and shoots but every three noded fragment is
able to initiate a new plant.
Another type of vegetative propagule produced by Myriophyllum
spicatum is axillary bud. The axillary buds of M. spicatum are normal
vegetative buds but they differ from vegetative buds in their size and the
ease with which they detach. Many species of Myriophyllum develop large
and prominent winter buds known as turions. Morphologically the small
axillary buds of M. spicatum are not turions but physiologically they may be
considered. These buds are formed during October-November and remain
dormant during the chilling winter and germinate immediately on arrival of
the spring when the conditions are favourable. The present experimental
studies reveal that the germination of axillary buds is controlled by
temperature and light and the presence of sediment is not obligatory for
germination of axillary buds provided sufficient quantity of water is
available. These observations draw support from Jain et al., (2003) who
observed that turions of Potamogeton crispus are capable of germinating in
any water body irrespective of type of substratum. As demonstrated during
the present study, the optimum temperature for axillary bud germination is
20-25°C. Similar results have been reported in Potamogeton pectinatus by
Madsen and Adams (1988). The germination of these propagules is
inhabited below 10°C and beyond 30°C. Under natural conditions also, the
axillary buds remain dormant during the cold winters of Kashmir valley
Page 25

when the temperature of water bodies is much lower, mostly less than 4°C.
The germination of axillary buds begins from March and proceeds upto
ending April, when the temperature is optimum for their germination. Some
of the ungerminated axillary buds do not germinate after April because
temperature beyond 30°C inhabits germination. The temperature dependence
of turion germination was also observed by Rogers and Breen (1980) in case
of Potamogeton crispus. The experimental studies conducted during the
present study on axillary bud germination revealed that both rate and overall
percentage germination of axillary buds was higher under alternate light and
dark than in continuous dark conditions indicating that axillary bud
germination is also influenced by light. This observation is strongly
supported by the work of Jain et al., (2003) who observed that percentage
germination of turions decreases with increase in the depth of water body
because of presence of low light intensity at deeper depths.
The axillary buds have very high potential to spread Myriophyllum
spicatum locally. Each axillary bud has the potential to form a single
plantlet. The newly formed plantlet (ramet) undergoes vegetative growth and
produce 8-12 new axillary buds which in a geometric progression produce
80-144 new ramets. This could be an important mechanism contributing to
the spread of this species in Lakes and a pond experiencing high predation
pressure from herbivorous fishs as has been reported by Jain et al., (2003)
for different species of Potamogeton. This clearly indicates that a single
axillary bud is capable of establishing a large population in few years. These
axillary buds not only produce plantlets but also spread throughout the space
available and also give rise to new propagules which invade the water bodies
and produce large populations of the species covering the entire water body
as a green mat. The removal of these axillary buds has a great role in
providing long term safety against the spread of this aggressive species. The
axillary bud removal can be carried out during October to November when
these are produced.
Page 26

CHAPTER-6
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Page 27

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