Queensland University of Technology
School of Natural Resource Sciences
Seed dispersal, germination and fine-scale
genetic structure in the stream lily,
Helmholtzia glaberrima (Philydraceae)
Peter Prentis B.Sc. (Hons).
Submitted in fulfilment of the requirements
for the degree of Doctor of Philosophy
1
July 2005
Abstract
Seed dispersal in aquatic habitats is often considered to be a complex multistage
process, where initial seed shadows are redistributed by water (hydrochory). The roles
of hydrochory in seed dispersal and influencing population genetic structure were
examined in Helmholtzia glaberrima using both ecological and genetic techniques.
Ecological experiments showed that water can redistribute seeds and seedlings over
local scales and that hydrochory can provide the potential for very long distance seed
and seedling dispersal. Patterns of seedling genetic structure were affected by micro-
drainages that direct water flow within populations and influence water-borne seed
dispersal on a local scale. Strong non-equilibrium dynamics and persistent founder
effects were responsible for the patterns of genetic structure observed among
established populations of H. glaberrima. Classical metapopulation models best
described dispersal patterns, while water-borne seed dispersal could potentially explain
patterns of genetic differentiation within a stream system, it could not explain the
distribution of genetic variation among stream systems. The current study found that
although hydrochory influenced seed dispersal and seedling genetic structure within a
population, it had little effect on the spatial pattern of genetic variation among
established populations of H. glaberrima. Moreover, even though prolonged buoyancy
and viability in water provide the potential for long-distance hydrochory, results
presented here do not support the hypothesis that flowing water is an effective long
distance seed dispersal vector for H. glaberrima. Taken together, these results suggest
that the relative importance of gene flow via water-born seed dispersal in H. glaberrima
may be low compared with that of some other riparian species.
2
Keywords: genetic diversity; Helmholtzia; hydrochory; seed dispersal
3
List of manuscripts
1. Prentis, P. J., A. Vesey, N. M. Meyers, and P. B. Mather 2004. Genetic structuring
of the stream lily Helmholtzia glaberrima (Philydraceae) within Toolona Creek,
south-eastern Queensland. Australian Journal of Botany 52: 201—207.
2. Prentis, P. J., N. M. Meyers, and P. B. Mather. In press. The significance of post-
germination buoyancy in Helmholtzia glaberrima and Philydrum lanuginosum
(Philydraceae). Australian Journal of Botany.
3. Prentis, P. J., N. M. Meyers, and P. B. Mather. (In Prep) Seed dispersal and
seedling establishment in the riparian plant Helmholtzia glaberrima. Freshwater
Biology.
4. Prentis, P. J., N. M. Meyers, and P. B. Mather. (In Review) Micro-geographic
landscape features demarcate seedling genetic structure in the stream lily,
Helmholtzia glaberrima (Philydraceae)1. American Journal of Botany.
5. Prentis, P. J., N. M. Meyers, AND P. B. Mather. (In Review) Fine-scale patterns of
genetic diversity and population structure in the stream lily Helmholtzia
glaberrima (Philydraceae) along rainforest streams, south-east Queensland.
Freshwater Biology.
4
Table of Contents
Abstract 1
List of Manuscripts 3
Table of Contents 4
Statement of Original Authorship 7
Acknowledgments 8
Chapter 1: GENERAL INTRODUCTION
Introduction 9
Spatial and temporal dynamics of aquatic habitats 10
Seed dispersal in freshwater habitats 12
Colonisation of isolated habitat patches via long-distance hydrochory 16
Other mechanisms affecting long-distance seed transport 17
Study species and system 22
Account of research progress linking manuscripts 24
References 26 Chapter 2: GENETIC STRUCTURING OF THE STREAM LILY Helmholtzia
glaberrima (PHILYDRACEAE) WITHIN TOOLONA CREEK, SOUTH-EAST
QUEENSLAND
Statement of Joint Authorship 34
Manuscript 1 35
Introduction 37
Materials and Methods 40
Results 43
Discussion 45
References 50
5
Tables and Figures 54
Chapter 3: THE SIGNIFICANCE OF POST-GERMINATION BUOYANCY IN
Helmholtzia glaberrima AND Philydrum lanuginosum (PHILYDRACEAE)
Statement of Joint Authorship 61
Manuscript 2 62
Introduction 64
Materials and Methods 67
Results 72
Discussion 74
References 78
Tables and Figures 81
Chapter 4: SEED DISPERSAL AND SEEDLING ESTABLISHMENT IN THE RIPARIAN
PLANT Helmholtzia glaberrima
Statement of Joint Authorship 84
Manuscript 3 85
Introduction 88
Materials and Methods 91
Results 94
Discussion 96
References 100
Tables and Figures 102
Chapter 5: MICRO-GEOGRAPHIC LANDSCAPE FEATURES DEMARCATE
SEEDLING GENETIC STRUCTURE IN THE STREAM LILY, Helmholtzia glaberrima
(PHILYDRACEAE)
Statement of Joint Authorship 105
6
Manuscript 4 106
Introduction 108
Materials and Methods 110
Results 112
Discussion 114
References 118
Tables and Figures 121
Chapter 6: FINE-SCALE PATTERNS OF GENETIC DIVERSITY AND POPULATION
STRUCTURE IN THE STREAM LILY Helmholtzia glaberrima (PHILYDRACEAE)
ALONG RAINFOREST STREAMS, SOUTH-EAST QUEENSLAND
Statement of Joint Authorship 126
Manuscript 5 127
Introduction 129
Materials and Methods 132
Results 134
Discussion 136
References 140
Tables and Figures 143
Chapter 7: GENERAL DISCUSSION General Discussion 147
References 153
7
Statement of Original Authorship
This work contains no material which has been accepted for the award of any other
degree or diploma in any university or other tertiary institution and, to the best of my
knowledge and belief, contains no material previously published or written by another
person, except where due reference has been made in the text.
Signed
Date
8
Acknowledgments
Firstly, I would like to thank my supervisors for your guidance and encouragement in
helping me complete this project. Many thanks must go both to the ecology and genetics
discussion groups for fruitful discussions on sampling designs and data. My parents
deserve many thanks for their support of my chosen path and providing guidance during
the gloomy PhD blues.
A special thankyou to all the people who volunteered to help me with field and lab work,
particularly Ana Pavasovic, Cameron Schulz, Mark Schutze, Doug Harding, Alex Wilson
and Grant Hamilton. Big thanks must go to Dr Graham Kelly the man whose lectures
and infectious enthusiasm are responsible for my interest in plant biology. Lastly and
mostly importantly, I have to thank my partner Ana for her support, encouragement and
cooking without which I would be a lonely, thin man.
9
CHAPTER 1. General introduction
Introduction
How seeds disperse among isolated habitat patches, establish and
contribute to the genetic pool of new plant populations has long fascinated
botanists (Darwin, 1859). This is because dispersal is one of the primary
processes that influence population dynamics and evolution of plants (Nathan &
Muller-Landau, 2000). In particular the spatial and temporal dynamics of plant
populations will be determined chiefly by the movement of seeds within and
among populations (Husband & Barrett, 1998). At a broader geographic scale,
the range at which seed dispersal is effective will influence the possibility that
extirpated populations are recolonised and the probability that new isolated
habitat patches are colonised (Cain et al., 2000).
Plant species are rarely distributed uniformly in space but often occur as
isolated local populations where favourable conditions for successful
establishment exist (Edwards & Sharitz, 2000; Ellison & Parker, 2002). Dispersal
patterns within and among local populations will determine the extent to which
local populations are interconnected via gene flow (Ouborg et al., 1999). Gene
flow will only occur however, if the seeds dispersing among populations establish
and contribute to future reproduction in the new population (Ouborg et al., 1999).
The level of gene flow among patches will determine the distribution of genetic
variation within and among local populations and whether they function
collectively, or as isolated units (Tero et al., 2003). In situations where dispersal
10
rates among populations are high, gene flow will tend to homogenise gene
frequencies among local populations, so they form a single panmictic unit
(Ouborg et al., 1999). Alternatively, if gene flow is very low or absent, local
populations are likely to diverge and evolve independently due to the diversifying
forces of drift and selection (Tero et al., 2003). A number of factors can influence
the level of seed dispersal and gene flow that occurs among isolated local
populations, including temporal and spatial distribution of favourable habitat
patches, the type of vectors that disperse seeds and the individual life-history
characteristics of a plant species (Pannell and Charlesworth, 1999, 2000). This
review will focus primarily factors acting on aquatic plants restricted to freshwater
habitats.
Spatial and temporal dynamics of aquatic habitats
Many studies have reported that habitat patches are often dispersed
heterogeneously across the landscape (Husband & Barrett, 1998; Levins, 1969;
McCauley, 1989). Spatial discontinuities, including mountains, valleys or rivers
can influence significantly the distribution of many organisms (Lawton, 1993).
Most organisms do not exhibit uniform distributions in space (Lawton, 1993).
Instead organisms frequently occur as isolated local populations confined to
suitable habitat surrounded by a matrix of less favourable habitat (Andrewartha &
Birch, 1954; Dejong, 1995; Dupre & Ehrlen, 2002). This is particularly true for
organisms with narrow habitat requirements such as freshwater aquatic plants,
whose preferred habitat is often distributed like naturally occurring islands in a
11
sea of terrestrial habitat (Barrett, 1985; Husband & Barrett, 1998; Santamaria,
2002).
Freshwater aquatic plant species are restricted to permanent, seasonal or
ephemeral aquatic environments, such as rivers, lakes or ponds (Edwards &
Sharitz, 2000; Ellison & Parker, 2002). Most freshwater aquatic habitats occur as
discrete habitat patches often separated by hundreds or thousands of metres of
unfavourable areas (Barrett et al., 1993). As a result, populations of many
aquatic plant species have spatially disjunct distributions. Freshwater aquatic
habitats in most landscapes are also highly heterogeneous at several spatial and
temporal scales, meaning that the distribution of many aquatic plant species can
also be temporally unstable (Husband & Barrett, 1995, 1998).
Most freshwater aquatic habitats are not static as they are created and
replenished by precipitation or runoff (Brock & Rogers, 1998; Hampe, 2004;
Lopez, 2001; Scarano et al., 2003; Van der Valk, 1981). As rainfall is not usually
evenly distributed in space or time, temporal and spatial fluctuations in water
level occur in freshwater habitats particularly in areas with seasonal climates
(Hampe, 2004; Husband & Barrett, 1998). Consequently, many aquatic habitats
are ephemeral or experience dramatic water level fluctuations among seasons
depending on the frequency and duration of rain or runoff from catchments
(Brock & Rogers, 1998). In periods of heavy rain, freshwater habitats may flood
and connect to other isolated wetland patches; however during drought many
aquatic habitats may disappear. As freshwater aquatic habitats can be
12
ephemeral, aquatic plants inhabiting them may experience alternating wet and
dry conditions (Lopez, 2001).
Alternating wet and dry conditions can influence the persistence of aquatic
plant populations in particular survival during the phase from seed release to
seedling establishment (Husband & Barrett, 1998; Lopez, 2001). Due to the
unpredictable nature of habitats occupied by most aquatic plants, individual
species may have only a short window of opportunity for seed dispersal,
germination and seedling establishment (Hampe, 2004). Many wetland plants,
however, have evolved unusual adaptations to water level changes or are
capable of variable responses (plasticity), promoting survival in heterogeneous
environments (Arber, 1920; Dorken & Barrett, 2004a; Santamaria et al., 2003;
Sculthorpe, 1967; Wells & Pigliucci, 2000). Thus seed dispersal, germination and
seedling establishment in aquatic plants may be less constrained by fluctuations
in water levels and alternating wet and dry conditions than had previously been
thought (Lopez, 2001). Traits that favour increased seed floatation during
flooding, seed survival in water and/or seedling establishment under flooded
conditions should be advantageous to aquatic or riparian plants.
Seed dispersal in freshwater habitats
Patterns of seed dispersal and seedling establishment have important
effects on the dynamics and persistence of plant populations (Barrett et al.,
1993). Dispersal of seeds provides an opportunity for individual plants or
populations to recruit because some seeds are likely to arrive at suitable
13
microhabitats for seedling establishment, thus increasing their potential for
survival (Nathan & Muller-Landau, 2000). Seed dispersal is often a complex
multistage process, with seeds known to be dispersed by a variety of vectors
(primary and secondary; sensu Vander Wall & Longland, 2004) over a number of
different spatial scales (local and long-distance) (Chambers & McMahon, 1994;
Hampe, 2004). Local and long-distance dispersal in complex seed dispersal
systems can often affect different components of the demography and dynamics
of individual plants and populations (Cain et al., 2000).
Seed dispersal in aquatic habitats often occurs as a multistage process,
complicated by each individual plant’s life history characteristics (Hampe, 2004).
Broadly we can classify aquatic plants into four life-history categories; 1) semi-
aquatic/riparian species, 2) emergent aquatic species 3) free-floating aquatic
species and 4) submerged aquatic species (Sculthorpe, 1967). For the purposes
of this review we will concentrate principally on emergent and semi-
aquatic/riparian species. Early life-history traits that affect seed dispersal in
emergent and semi-aquatic species can be quite similar (Boedeltje et al., 2003).
For example many emergent and semi-aquatic wetland species exhibit a limited
capacity for primary seed dispersal due to their reliance on gravity as a vector
(Waser et al., 1982).
The mean dispersal distance for the herbaceous wetland perennial
Sarracenia purpurea, was 12.8 cm with 78% of seeds caught within five
centimetres of focal plants (Ellison & Parker, 2002). Similarly short primary
dispersal distances have also been found in many other wetland herbs (Cain et
14
al., 1998). Reliance solely on short-distance primary seed dispersal in these
species should constrain the potential for movement of seeds among patches,
yet propagules are known to have established in isolated sites following rare
dispersal events across large tracts of unsuitable habitat (Ellisson & Parker,
2002). Understanding dispersal mechanisms that facilitate these rare events is
the first step in understanding colonisation, establishment and dynamics of
spread in emergent or semi-aquatic plant species that otherwise possess limited
dispersal capacity.
Many riparian and emergent aquatic species exhibit prolonged seed
buoyancy (Boedeltje et al., 2003). Consequently seeds in wetland species can be
redistributed by water (Hampe, 2004). Redistribution of seeds in fresh water may
affect the spatial pattern of the seed shadow generated by primary seed
dispersal vectors (Hampe, 2004). Prolonged seed buoyancy may enhance the
potential range of seed dispersal, possibly conferring the capacity to colonise
isolated habitat patches. Redistribution of seeds by fresh water however,
depends strongly on the amount of rainfall or runoff received between seed
release and seedling establishment (Hampe, 2004). For example, if seed release
coincides with dry conditions then there is limited or no opportunity for seed
dispersal by water in ephemeral wetlands. Alternatively if rainfall does occur
during times of seed release then redistribution of seeds by water is highly likely.
Dispersal via primary and secondary dispersal vectors are poorly understood,
particularly systems where initial seed shadows are redistributed by freshwater
(Hampe, 2004).
15
The relative contribution that hydrochory makes to secondary dispersal
and establishment of wetland species remains contentious. Most likely, in many
freshwater herbs water-born seed dispersal contributes little to long-distance
dispersal and if it does, it represents a rare event (Ellison & Parker, 2002). Some
investigators have found evidence that hydrochory can result in effective long-
distance seed dispersal at scales of hundreds to thousands of metres (Kinamoto
et al., 2005; Kudoh & Whigham, 1997; Lonsdale, 1993; Nilsson et al., 1991;
Ridley, 1930). This indicates that in at least some emergent or riparian species
long-distance water-borne seed dispersal is possible. Consequently, rare long-
distance hydrochory may explain colonisation of isolated habitat patches in both
emergent and semi-aquatic species (Tero et al., 2003).
Redistribution of seeds via water however may affect seed germination
and seedling recruitment. Water-borne seed dispersal can affect seed
germination or establishment in at least three ways; first, if seeds dispersed by
water are not flood tolerant then immersion in water for prolonged periods may
reduce seed viability and germination (Edwards et al., 1994; Lopez, 2001;
Middleton, 2000; Scarano et al., 2003). Second, redistribution of seeds by water
will determine if seeds are deposited in suitable microhabitat conditions for
germination and establishment (Boedeltje et al., 2004; Ozinga et al., 2004; Van
der Valk, 1981). Last, if water-born dispersal changes the density of the initial
seed shadow, germination and establishment may be negatively or positively
affected by density dependent processes such as mortality (Debussche &
Isenmann, 1994; McMurray et al., 1997; Nathan & Casagrandi, 2004; Orth et al.,
16
2003; Russo & Augspurger, 2004). Therefore, how seeds of emergent or riparian
species respond to water-borne dispersal and where they end up (deposition
site) will influence potential for colonisation of isolated habitat patches (Nathan &
Muller-Landau, 2000).
Colonisation of isolated habitat patches via long-distance hydrochory
Populations of emergent or riparian plant species often occur in spatially
isolated habitat patches (Edwards & Sharitz, 2000). Many hundreds to
thousands of metres may separate individual wetland habitat patches (Ellison &
Parker, 2002). Thus colonisation of suitable isolated patches will require effective
long-distance dispersal. The capacity of seeds from emergent or riparian species
immersed in water to float for long periods of time (Boedeltje et al., 2003), may
represent an adaptation to long-distance water-borne dispersal. For example,
Fridriksson (1975) found that species with adaptations that promoted buoyancy
were more likely to disperse over distances of greater than 20 km on ocean
currents to colonise the newly emerged volcanic island of Surtsey. In fact, over
78% of the vascular plant taxa that arrived on Surtsey between 1963 and 1972
were known to be dispersed by water (Fridriksson, 1975). This demonstrates that
water-borne dispersal can be an effective method for long-distance dispersal in
plants and the colonisation of isolated habitats. It is unknown however, if
secondary seed dispersal by flowing freshwater can disperse seeds over
relatively long distances allowing colonisation of isolated habitats.
17
Experiments have shown that seeds of many wind dispersed species,
often float and remain viable in runoff, streams and rivers (Ridley, 1930). It is
thought in some of these instances, that seeds can be dispersed much greater
distances via water than by wind (Carlquist, 1967). Furthermore, a recent study
by Hampe (2004) found that secondary dispersal by water changed the
distribution of the bird-mediated seed shadow of the Eurasian tree, Frangula
alnus from a negative exponential towards an extended Poisson distribution. This
indicates that secondary seed dispersal by water can result in regular long-
distance seed dispersal for F. alnus. Secondary water-borne dispersal may
therefore represent an important long-distance dispersal vector, aiding the
colonisation of isolated habitat in some emergent or riparian species. More than
one type of dispersal vector however may result in long-distance secondary seed
dispersal.
Other mechanisms affecting long-distance seed transport
There are several ways that long distance seed dispersal events may
occur including; dispersal by water (Kudoh & Whigham, 1997; Lonsdale, 1993;
Nilsson et al. 1991; Ridley, 1930), by biotic influences (Figuerola & Green, 2002;
Fragosa, 1997; Holbrook et al. 2002; Shilton et al. 1999), wind vectors (Bullock &
Clarke, 2000; Nathan et al. 2002) or rafting on other dead or live organisms
(Nathan & Muller-Landau, 2000). Some researchers assume a link between seed
attributes such as morphology and dispersal (e.g. buoyant seeds imply
hydrochory) (e.g. Hughes et al., 1994). A recent review however, recognised that
18
the morphological attributes a seed may possess does not necessarily determine
which vector will disperse it (Higgins et al. 2003). Moreover, this study suggests
that although dispersal types based on morphological traits provide a useful
framework for describing local dispersal processes, they are often poorly related
to the actual mechanisms responsible for long-distance seed dispersal in many
plant species (Higgins et al. 2003).
Many wind and water dispersed seeds from wetland species are known to
adhere to the fur, feathers or feet of some vertebrate species (Carlquist, 1967;
Darwin, 1859; Figuerola & Green, 2002; Higgins & Richardson, 1999; Ridley,
1930; Vivian-Smith & Siles, 1994). This will confer a potential for long-distance
dispersal by mechanisms other than those determined primarily by morphological
traits (Higgins et al. 2003). For example, it is estimated that approximately one
quarter of the plant species that colonised Easter Island and the Juan Fernandez
Islands were transported in mud attached to the feet of migratory birds (Carlquist,
1967). In addition, the distribution of some wetland plants with buoyant seeds are
strongly correlated with the dispersal patterns and preferred habitat of migratory
waterfowl (Frith et al. 1977) suggesting that waterfowl may act as dispersal
vectors for these species. Further evidence supporting the role that biotic vectors
play in dispersing the seeds of aquatic plants over long distances comes from
population genetic studies (Tero et al., 2003).
Studies of the genetic structure of many aquatic species suggest that
dispersal mechanisms other than water have been important in some long
distance seed dispersal events (Dorken & Barrett, 2004b; Godt & Hamrick, 1998;
19
Tero et al., 2003). A study of the genetic structure of the fennel pondweed
(Potamogeton pectinatus) in Europe found strong evidence that long distance
seed dispersal in this species was not related to hydrochory (Mader et al., 1998).
The study established a correlation between genetic and geographic distance
among ponds not visited by swans (a potential dispersal agent). When ponds
visited by swans were included in the analysis no relationship was evident.
Based on this finding the authors concluded that swan-mediated long-distance
dispersal influenced the pattern of genetic structure observed (Mader et al.,
1998). Therefore, molecular markers in combination with well designed
experiments can be used to examine patterns of dispersal in plant species
(Abbott et al., 2000; He et al., 2004; Mader et al., 1998).
Genetic methods are broadly applicable to investigating both patterns of
local and long-distance seed dispersal in plant species (Cain et al., 2000; He et
al., 2004; Kinamoto et al. 2005; Ouborg et al., 1999; Sork et al., 1999). Studies of
seed dispersal in plant species that have applied genetic markers however, have
usually sampled seedlings or older stages of plant development (Cain et al.,
2000). If genetic studies of dispersal are based on samples collected from
seedlings or later stages of development, they not only provide estimates of seed
dispersal but also plant establishment, thus providing a measure of ‘effective’
seed dispersal (Nathan & Muller-Landau, 2000; but see Godoy & Jordano, 2001).
Effective seed dispersal may be the parameter of greater importance in many
ecological and conservation studies, since seed movement to locations in which
20
establishment fails will not influence the population dynamics and genetic
structure of the recipient population (Nathan, 2006).
Several models have been proposed to explain how dispersal can
influence population structure in plant populations. The types of models proposed
include: island models, fragmented models, stepping stone models, classical
metapopulation models and source/sink models (Gaggiotti and Smouse, 1996;
Kimura 1953; Kimura & Weiss 1964; Levins, 1969; Wright, 1931).
In an island model, all populations form a single panmictic unit with free
gene flow across a genetically uniform population (Wright, 1931). This model
assumes that populations have reached equilibrium, that all are of equal size and
that dispersal rate is uniform among populations irrespective of their relative
proximity in the landscape (Wright, 1951). Where panmixia is operating in an
island model, populations should not show any structure (Wright, 1951). A
fragmented model however, assumes that a formerly continuous population has
been fragmented into remnant populations where no contemporary gene flow
occurs among populations (Slatkin, 1985). Strong genetic structuring is expected
therefore among all populations in a fragmented system (Slatkin, 1985).
In contrast to the island and fragmented models, a stepping-stone model
assumes dispersal is limited by distance and occurs at higher rates among
adjacent local populations (Kimura 1953; Kimura & Weiss 1964). At equilibrium in
a stepping-stone model, genetic differentiation among populations should
increase linearly with increasing geographical distance among populations
21
(Kimura & Weiss 1964). Therefore a signature of isolation by distance is
expected under a stepping-stone model (Slatkin, 1985).
In both source/sink and classical metapopulation models, populations are
not permanent and are subject to extinction/colonization dynamics (Pannell and
Charlesworth, 1999). Source/sink metapopulation models assume that
individuals disperse from more permanent, high-quality source subpopulations
into low quality ephemeral sink populations and that sink populations are likely to
go extinct without ongoing dispersal from source populations (Gaggiotti and
Smouse, 1996). Therefore sink populations are expected to possess lower levels
of genetic diversity relative to source populations. Classical metapopulation
models assume that all patches are ephemeral and may be founded by different
numbers of individuals from single or multiple source patches (Giles and Goudet,
1997). This model also assumes that only limited dispersal occurs among extant
populations. Consequently, levels of genetic variation may decrease or increase
randomly within and among populations under a metapopulation model (Pannell
and Charlesworth, 1999, 2000).
These models provide a number of testable hypotheses about how
different modes of dispersal can influence the distribution of genetic variation
within and among subpopulations. Therefore this genetic information permits
inferences to be made about patterns of dispersal within and among populations.
In conjunction with ecological experiments genetic studies can be used to
elucidate patterns of local and long-distance seed dispersal.
22
Study species and system
The Philydraceae is a small family of chiefly erect herbs that is restricted
to Australia and south east Asia (Clifford, 1983; Hamann, 1998). Family
Philydraceae is closely related to the Pontederiaceae and Haemodoraceae in the
O. Commelinales (Graham et al., 2002). Species within this family are only found
in saturated areas like permanently waterlogged soil, swamps or soaks (Clifford,
1983). The Family Philydraceae comprises a total of six species distributed in 3
genera (Adams 1989). The 3 genera include the montane rainforest genus
Helmholtzia (3 species: H. glaberrima, H. acorifolia and H. novoguineensis) and
2 lowland genera Philydrella (2 species: P. drummondii and P. pygmaea) and
Philydrum (monotypic: P. lanuginosum) (Adams, 1989). The species examined in
this study was H. glaberrima a semi-aquatic riparian plant (Hamann, 1998).
H. glaberrima is a long-lived perennial herb (Philydraceae), (2n =34) that
is known to live for at least ten years and which grows to 2.5 m (Adams 1989).
The species has low levels of protracted flowering throughout the year and
flowers are produced on either single or multiple large pink panicles which are
believed to be pollinated by small insects (Adams 1989). Numerous 4-5 mm long
seeds are produced in capsules throughout the infructescence after pollination
(Adams 1989). Currently, no dispersal vectors are known that disperse the seed
of this plant, but hydrochory has been suggested as a possible dispersal
mechanism (Hamann, 1998).
The stream lily, H. glaberrima (Philydraceae), is a rare but occasionally
locally abundant, riparian species confined to high-elevation dendritic streams in
23
temperate and subtropical rainforest of the McPherson Ranges on the border of
Queensland and New South Wales (Clifford, 1983). Within its range of
approximately 100 km, H. glaberrima is restricted to riparian wetlands and moist
gullies along high elevation creek banks (Hamann, 1998). As H. glaberrima is
restricted to permanently waterlogged areas, it occurs in groups of spatially
isolated patches along stream systems. Consequently, populations at the top of
stream systems are thought to be less isolated from each other than downstream
populations. Although extant populations are spatially isolated from one another
they are interconnected by permanent stream systems (Adams 1989).
In the McPherson Ranges amount of rainfall received decreases with
altitude. Altitudes above 1000 m receive ≤ 3000 mm in annual rainfall, while
areas between 800 – 1000 m receive ≤ 1800 mm annually and areas below
800m receive < 1300 mm of annual rainfall (BOM, 2003). Consequently, the
preferred habitat of H. glaberrima declines with altitude as presumably do the
number and size of H. glaberrima populations.
Rainfall is also highly seasonal in south east Queensland and northern
New South Wales with most rain falling over the summer period between
December and March (BOM, 2003). Many patches occupied by H. glaberrima
are subject to periodic inundation and soil saturation in summer that usually
recedes to moist soil conditions over winter, the same time that many species
disperse their seed in this area (Prentis, personal observation). Therefore H.
glaberrima seeds have a strong possibility of being redistributed by water during
periods of heavy rain.
24
A combination of genetic and ecological approaches were used to explore
the role that hydrochory plays in the dispersal of H. glaberrima seeds over local
and long distances. Ecological approaches were used to examine the potential
for water-borne seed dispersal to transport seeds over local and long distances.
Estimates of genetic diversity among established populations of H. glaberrima
and of seedlings within a population were examined to elucidate the actual role
that water-borne seed dispersal may have on population genetic structure over
local and long-distance scales. These data were used to address the five aims of
this project:
1. To define what model of dispersal can best explain the observed
population structure (manuscript 1)
2. To examine potential for water-borne seed dispersal over local and
long-distance scales (manuscript 3)
3. To investigate the role that post-germination buoyancy plays in
seed dispersal and seedling establishment (Manuscript 2)
4. To test whether microtopographic landscape features that direct
water flow can influence seed dispersal and seedling genetic
structure in local populations (manuscript 4)
5. To quantify the influence that water-borne seed dispersal has on
observed population genetic structure (manuscript 5)
Account of research progress linking manuscripts
25
The first manuscript (chapter 2) examines the model of dispersal which
best explains the observed pattern of population genetic structure in Helmholtzia
along a single stream system. The pattern of genetic structure observed along
this stream system was best explained by classical metapopulation dynamics
and a complex seed dispersal strategy which was hypothesised to be the result
of hydrochory. The subsequent two manuscripts (chapter 3 & 4) investigated the
role of hydrochory to ‘effectively’ disperse seeds and seedlings over local and
long distances. The fourth and fifth manuscripts (chapter 5 & 6) then evaluated if
the hydrochory actually influenced the pattern of genetic structure in Helmholtzia
over a local (seedling genetic structure) and long-distance scale (among stream
systems).
26
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34
Statement of Joint Authorship
Manuscript 1
Prentis, P. J., A. Vesey, N. M. Meyers, and P. B. Mather 2004. Genetic structuring of the
stream lily Helmholtzia glaberrima (Philydraceae) within Toolona Creek, south-eastern
Queensland. Australian Journal of Botany 52: 201—207.
A re-print of the journal article is presented in Appendix 1.
Peter Prentis (Candidate)
• Wrote the manuscript and acted as corresponding author
• Designed and formulated sampling design and experimental protocols
• Undertook all field and laboratory work, analysis and interpretation of data
Amanda Vesey
• Undertook all field work and some lab work
• Provided editing and contributed to the structure of the manuscript
Noel Meyers
• Co-supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
Peter Mather
• Co-supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
35
CHAPTER 2. Genetic structuring of the stream lily Helmholtzia glaberrima
(Philydraceae) within Toolona Creek, south-east Queensland
Peter J. PrentisA,B, Vesey A.A, Meyers N.M.A and Mather P.B.A
A. School of Natural Resource Sciences, Queensland University of Technology, GPO
Box 2434, Brisbane, Qld 4001, Australia.
B. Corresponding author; email: [email protected]
Suggested running title: Population structure in Helmholtzia glaberrima.
36
Abstract. The distribution of genetic variation among five isolated sites of the riparian
species Helmholtzia glaberrima (J.D. Hook) was examined in Toloona Creek (280 13’ S,
153007’E) using dominant AFLP markers. From the 137 fragments assessed, analysis of
molecular variance (AMOVA) showed that most genetic variability occurred within sites
(68%), although high (32%) variation also occurred among sites. Highly significant
pairwise θ estimates among all sampled sites suggests gene flow is restricted in H.
glaberrima. Levels of within site diversity were intermediate and differed significantly
across the sampled sites. Significant levels of linkage disequilibrium were detected at all
sites except TC3. Differences in linkage disequilibrium and genetic diversity among the
sites suggest that sites may be founded by different numbers of colonists. Mantel tests
found no correlation between geographic and genetic distance and significant levels of
linkage disequilibrium were detected at the total site level. This result supports a non-
equilibrium model of population structure. The observed pattern of non-equilibrium
population structure and genetic diversity in H. glaberrima are best explained by a
classical metapopulation model.
37
Introduction
Tero et al. (2003) propose several models to explain how dispersal may influence
population structure in linearly arranged riparian plant subpopulations. These models
include: an island model, a completely fragmented model, a stepping stone model, a
classical metapopulation model and a source/sink model. The island model predicts
populations will exhibit genetic uniformity over all spatial scales, while the fragmented
system model suggests all populations should show significant differentiation due to a
lack of recurrent gene flow. In the stepping stone model, populations in close proximity
should be more genetically similar than populations at greater geographical distance.
While, the classical metapopulation model recognises that patches are ephemeral and
can be founded by differing numbers of individuals from single or multiple source
patches. Consequently, genetic variation may decrease or increase randomly within
patches while high levels of linkage disequilibrium may also be present. The source/sink
model predicts that genetic diversity will be lower in the source populations relative to
the founder patch.
Relatively few studies have examined how dispersal may influence the genetic structure
of riparian angiosperm species. Those studies that address this question report results
that do not always conform to the proposed models. For example, Hibiscus moscheutos
did not conform to the models because the moderate differentiation found in a stream
system was related to the distance from the stream channel and did not conform to the
proposed models (Kudoh and Whigham 1997). Alternatively, Keller (2000) found
extremely low levels of differentiation among sampled populations of Phragmites
australis in the Charles River watershed attributed to gene flow among populations. An
island model of population structure best explains the pattern of genetic structure found
in P. australis. A recent study on Silene tatarica along the Oulankajoki River found high
38
levels of genetic differentiation among sites, which did not conform to an isolation by
distance model (Tero et al. 2003). The authors suggest that the observed population
genetic structure resulted primarily from “classical” metapopulation dynamics and that
populations were not in drift/gene flow equilibrium.
In this paper we studied the genetic structure of a rare but locally abundant, riparian
species H. glaberrima along a single stream to evaluate which models of population
structure best fit this species. H. glaberrima is confined to high-elevation dendritic
streams in the subtropical rainforest of the McPherson Ranges on the border of
Queensland and New South Wales. H. glaberrima is restricted to permanently
waterlogged soil along creek banks, resulting in spatially structured patches that become
increasingly more isolated with distance downstream. Patches of H. glaberrima comprise
dense aggregations of up to 1.6 individuals/m2 (Prentis, Unpublished data). An added
complication is that H. glaberrima is rhizomatous over short distances, so ramets may
contribute to the expansion of patches after colonisation of new sites by propagules
(Harden 1993). Seed is presumably first dispersed via gravity and secondarily by water.
These factors may disperse seeds over very different spatial scales. Gravity dispersal
should increase genetic structure among populations as dispersal by this mechanism is
likely to occur over very small spatial scales. While hydrochoric dispersal may spread
seed over much greater distances, seed movement will be unidirectional resulting in
either increasing or decreasing genetic diversity downstream depending on how patches
are colonised.
The study aimed to test the suitability of various population models (Tero et al. 2003) to
explain the patterns of genetic variation among riparian populations of H. glaberrima
because previous work indicates exceptions to these models (Kudoh and Whigham
39
1997). H. glaberrima was chosen as a model species since populations occur in discrete
patches and are restricted to specific habitat along creek systems. We also sought to
determine whether vegetative growth in H. glaberrima could influence patterns of genetic
structure and which model best suits population structure in this species.
40
Materials and methods
Study species
The stream lily, H. glaberrima (Philydraceae), (2n =34), is a perennial herb, which is
endemic to the understorey of subtropical, warm and cool-temperate rainforest in the
McPherson Ranges with a range of less than 100 km. Within this range the species
exhibits a restricted distribution to high-elevation creek banks. H. glaberrima is a long-
lived (at least 10 years) species that grows to 2.5 m and has low levels of protracted
flowering throughout the year with most individuals flowering between August and
February. Flowers are produced on either single or multiple large, many-branched
panicles which Adams (1989) believes are pollinated by small insects. Numerous 4-5
mm long seeds are produced in capsules throughout the infructescence after pollination.
No dispersal vectors are known to disperse the seed of this species, although Hamann
(1998) suggests hydrochory as a possible dispersal mechanism.
Study site and plant collections
Samples were collected from five populations of H. glaberrima located in the headwaters
of Toolona Creek (Fig. 1) in Lamington National Park (280 13’ S, 153007’E). This single
creek system allowed sampling of both high and low altitude sites, not possible in other
adjacent stream systems. Sampled populations ranged in size from 118 to 651 adult and
juvenile plants (Table 1). In all populations, sampling was based on a concentric circle
design with distances varying from a known reference individual. The 20 m diameter of
this circle was large enough to encompass all individual plants within sites. Samples
were collected from adult plants at intervals of 1, 2, 4, 7, and 10 m in each quadrant of
the circle yielding a total of 21 individuals from each population for genetic analysis.
41
DNA extraction and AFLP analysis
Genomic DNA was extracted from 1.2 g of finely ground plant leaf tissue according to
the protocol of Doyle and Doyle (1988) with slight modifications.
AFLP procedures were performed according to the restriction ligation, PCR reactions
and gel analysis protocols of Adjome-Mardsen et al. (1997) with slight modifications.
Modifications included using the Tru91 (Roche) restriction enzyme (isoschizomer for
Mse1) in place of Taq1 as the dominant cutter. The selective PCR was conducted using
the selective nucleotides combinations: (1) E-AAG/M-AG, (2) E-AAG/M-GA and (3) E-
ATG/M-AG. The AFLP procedure was trialled twice on four samples initially, using the
same primer sets and reproducible loci were detected for this species.
Data analysis
Statistical analyses using variation in AFLP phenotypes were based on the assumptions
that AFLP markers are dominant diploid markers, which conform to Hardy-Weinberg
equilibrium and where no co-migration of fragments occur. A Dice similarity matrix
(Sneath and Sokal 1973) was used to compare the similarity and clonality among
individuals within sites using SPSS (2002). Mean genetic diversity estimates within
populations were calculated three ways: (i) as the percentage of polymorphic loci (P%),
(ii) Shannon’s index of phenotypic diversity (IS) (Lewontin 1972) and (iii) Nei’s (1978)
unbiased expected heterozygosity (HE) using POPGENE Version 1.32 (Yeh et al. 1997). To
lend support to measures of genetic diversity found within sites average Dice similarity
within sites was also calculated and subtracted from one. A Kruskal-Wallace test was
used to examine whether levels of genetic diversity were consistent among sites. To
investigate the influence that patch size and isolation from nearest neighbouring patch
had on diversity indices, a Spearman rank correlation was used. A Monte Carlo
42
simulation process was used to examine linkage disequilibrium between AFLP
phenotypes within and among sampling sites using Lian 3.1(Haubold and Hudson 2000).
The significance of departures from linkage equilibrium in these tests were generated
over 5000 iterations. Linkage disequilibrium was examined to asses if populations had
gone through bottlenecks or were formed by different levels of admixture.
Principal Coordinates Analysis (PCA) was used to visualise how individuals from
sampling sites clustered using Genalex (Peakall and Smouse 2001). A UPGMA
dendogram was constructed using Nei’s unbiased genetic distance (Nei 1978) to
visualise how sampling sites clustered together with TFPGA (Miller 1997). To evaluate
the extent of among population genetic differentiation, data were first analysed using
analysis of molecular variance (AMOVA) in Arlequin 2.000 (Schneider et al. 2000).
Estimates of θ were obtained for each pair of sites using coancestry distance (Reynolds
et al. 1983) in TFPGA. Using the general framework of Hutchinson and Templeton
(1999) we inferred the importance of genetic drift and migration among sampling sites by
constructing scatter plots using pairwise θ comparisons against both in-stream and
straight geographic distance. A Mantel test (Mantel 1967) was then used to evaluate
whether genetic and in-stream or linear distance matrices were significantly correlated in
TFPGA using 1000 perturbations.
43
Results
AFLP polymorphism and patterns
Three primer pairs were used to screen 137 unambiguous AFLP loci. One hundred and
sixteen of these fragments (92%) were polymorphic across the entire survey, with the
primer pairs one, two and three producing 33 (80.5%), 41 (89.1%) and 42 (84%)
fragments, respectively. Five individuals did not amplify yielding a total of 100 individuals
across five sites for the analysis.
Within-site variation
All individuals screened showed unique AFLP banding profiles inferring that individuals
possessed a unique genotype and were not clonal. Table 1 shows the mean average
dice similarity, Nei’s expected heterozygosity, Shannon index of phenotypic diversity and
percentage polymorphic loci at each site. Similarity values among individual plants
ranged between 0.039 and 0.269, while percentage polymorphic loci ranged between
46.8% to 68%. Nei and Shannon indices varied from 0.16 to 0.236 and from 0.239 to
0.352 respectively. A Kruskall-Wallace test showed that Nei and Shannon indices were
significantly different among sampled sites (P<0.05), but differences in diversity levels
among sites were not correlated with either patch size or distance to neighbouring
patches (P>0.2).
Significant levels of linkage disequilibrium were found at all sampling sites except for
TC3, where the result was marginally within the 95% Monte-Carlo confidence interval for
linkage equilibrium. This indicates that sampled sites show different levels of linkage
disequilibrium which is supported by the observed levels of linkage (VD) in Table 2.
Significant linkage disequilibrium was also detected at the total site level (Table 2).
44
Significant linkage disequilibrium suggests populations may have gone through
bottlenecks or have been formed by different levels of admixture.
Among-site distances
PCA analysis showed relationships among populations, with the first two axes
accounting for 28.2% of total variation. This analysis revealed minimal overlap among
sampling sites as individuals within sites tended to cluster together, with sites 4 and 5
clustering independently from sites 1, 2 and 3 (Fig. 2). The UPGMA dendrogram also
illustrated a similar pattern as sites 4 and 5 clustered together and sites 1, 2 and 3
clustered together (Fig. 3). AMOVA results indicate that although the major proportion of
genetic variation was found within populations (68%), a significant proportion was also
distributed among populations (32%). Pairwise θ estimates also confirmed that all sites
were highly different (Table 3). Neither the scatter plots (Fig. 4) or Mantel tests revealed
any significant relationships between genetic and in-stream or linear distance among
populations within the stream system.
45
Discussion
Within-site variation
All Helmholtzia glaberrima individuals sampled possessed unique multilocus genotypes.
This result demonstrates that vegetative growth contributes little to within population
structure or that the effect occurs over a spatial scale of less than one metre. An
alternative explanation is that infrequent mis-scoring of phenotypes led to the false
interpretation that no clones were present. This interpretation is unlikely however, as any
difficult to interpret or ambiguous loci were excluded from the analysis to eliminate this
problem and a minimum of 5 loci were different between any two individuals.
Helmholtzia glaberrima exhibited moderate levels of polymorphic AFLP loci, and
diversity compared to other plants (Gaudeul et al. 2000; Despres et al. 2002, Tero et al.
2003). Two other herbaceous perennial species, Eryngium alpinum and Trollius
europaeus (European globe flower) exhibited similar diversity levels to H. glaberrima, but
unlike these species, no relationship occurred between genetic diversity and population
size or degree of isolation (Gaudeul et al. 2000; Despres et al. 2002). Like H.
glaberrima, other plant species with non-equilibrium dynamics often exhibit a poor
correlation between genetic diversity and population size or isolation (Schmidt and
Jensen 2000; Tero et al. 2003).
Linkage disequilibrium tests showed that four of the five sampled populations of H.
glaberrima were not in equilibrium. High levels of linkage disequilibrium within some, but
not all sites, suggest that patches may result from different dynamics. Theory predicts
that equilibrium dynamics can be disrupted by different levels of admixture and
bottlenecks during patch formation (Tero et al. 2003). Also, patches founded by
individuals arising from a greater number of source patches will exhibit greater levels of
46
genetic diversity relative to other patches (Whitlock and McCauley 1990). Differences in
the level of population admixture or the severity of bottlenecks during patch formation
are two potential causes for observed patterns of linkage disequilibrium and genetic
diversity among sampling sites of H. glaberrima.
Among-site distance
Highly significant genetic differentiation occurred among sampled sites for H. glaberrima
(φst =0.32), in spite of the fact that most genetic variation was present within sites.
Therefore, sampled sites do not form a single panmictic unit. This result demonstrates
that the species does not conform to an island model of population structure.
Similar patterns of variation over small spatial scales have been reported with dominant
markers in other plant species (Hogbin and Peakall 1999; Gaudeul et al. 2000; Despres
et al. 2002). Restricted dispersal among geographically isolated patches has often been
used to explain this pattern of variation. An isolation by distance pattern of population
structuring is likely to develop under low levels of localised gene flow (Hutchison and
Templeton 1999). However, H. glaberrima populations demonstrated strong
differentiation, irrespective of their geographical proximity by linear or in-stream distance.
This suggests that most gene flow among H. glaberrima individuals occurs within
patches. Moreover, it demonstrates that gene flow which occurs among patches is not
related to patch proximity.
Both UPGMA and PCA results indicate that sites TC2 and TC3 and sites TC4 and TC5
respectively cluster more strongly than other sampled sites. The two clusters are also
the most divergent from each other, even though sites TC3 and TC4 are closer to each
other than is site TC2. This relationship indicates that one and two dimensional stepping
47
stone models of dispersal cannot explain H. glaberrima’s population structure.
Furthermore, scatter plots of coancestry distance versus geographic distance (Fig. 4)
show a random scattering of points, indicating that recurrent gene flow has less
influence than drift or non-equilibrium dynamics on the observed pattern of genetic
structure. Two likely explanations may account for this pattern of fine-scale structure: (1)
genetic drift predominates because dispersal is low or absent among sites and/or (2)
different numbers of individuals arising from different numbers of source populations
contribute to founding patches.
Short-distance seed dispersal may result in the strong genetic differentiation among
sampled sites. Seed dispersal among patches may be rare because H. glaberrima seed
lacks specialised structures for long distance dispersal, except potentially by water. In
this situation, genetic drift may cause the random fixation or loss of alleles within
geographically isolated patches if effective population sizes are low. Consequently,
genetic differentiation should increase among patches as private alleles evolve or alleles
are lost within patches. No private alleles were detected, however, among the sampled
sites. In addition the highest frequency of null alleles in any patch was 5%. Thus genetic
drift within sites would seem to be quite weak and the observed population structure
cannot be adequately explained by a fragmented model. Non-equilibrium dynamics
associated with patch formation or dispersal represent more likely causes for the
observed population structuring.
Chance colonisation events of the limited suitable habitat occurring along stream banks
represent a likely explanation for potential differences in population founding numbers in
H. glaberrima, causing populations to be out of equilibrium. Because of the low
probability that seeds with similar genotypes will found a patch, when different H.
48
glaberrima sites are compared highly divergent gene frequencies among sites may
occur. Also the occurrence of linkage disequilibrium at the total population level indicates
that H. glaberrima patches have been colonised by non-equilibrium dynamics. Highly
divergent gene frequencies and high levels of linkage disequilibrium are often associated
with typical non-equilibrium dynamics of both source/sink and classical metapopulations
(Whitlock and McCauley 1990; Boileau et al. 1992). However as genetic diversity did not
decrease downstream as the source/sink model predicted but varied randomly in the
stream system it indicates that classical metapopulation models can best explain the
observed pattern of population structuring in H. glaberrima.
In conclusion, the present study has shown that patches of H. glaberrima display
intermediate levels of genetic diversity even in small patches and that vegetative growth
does not influence patch structure. The random pattern of genetic variation among
patches and high levels of linkage disequilibrium within some sites indicate populations
do not conform to an equilibrium population structure model. Moreover, classical
metapopulation models best explain patterns of genetic variation and differentiation.
Future work should investigate whether H. glaberrima population structure follows
metapopulation or non-equilibrium models in other stream systems. This information will
assist our understanding of the population dynamics in riparian plant species.
49
Acknowledgments
The authors thank Grant Hamilton and Shaun Meredith for assistance with collections
from field sites and Vincent Chand and Natalie Baker for their help with lab work. We are
also grateful to Ana Pavasovic and Dr Grahame Kelly and two anonymous reviewers
who read earlier drafts of this manuscript.
50
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54
Table 1. Population size, isolation of populations from nearest neighbouring
population and within population diversity indices from the sampled H. glaberrima
populations
Populatio
n
Isolation
(m)
Populatio
n size
Nei’s
Index (HE)
Shannon
Index (IS)
Variable
Loci (%)
Average
Similarity
TC1 250 234 0.236 0.352 68 0.174
TC2 68 118 0.177 0.268 54.5 0.087
TC3 40 618 0.159 0.239 46.8 0.105
TC4 32 307 0.195 0.289 54.2 0.131
TC5 21 213 0.178 0.266 49 0.109
55
Table 2. Analysis of linkage disequilibrium within each of the sampled sites and at
the total population level in H. glaberrima
Levels of significance were derived by 5000 iterations using a Monte-carlo simulation
where * < 0.05, ** < 0.025, *** < 0.001
Sites VD VE LMC Significance
TC1 39.3 17.1 21.9 ***
TC2 84 12.5 16.4 ***
TC3 15.6 12.2 15.7
TC4 33.9 14.6 19 ***
TC5 20.6 13.5 17.2 **
Total 60.5 19.4 21.3 **
56
Table 3. Pairwise θ distance values among sampled populations of H. glaberrima
Population TC1 TC2 TC3 TC4 TC5
TC1 0
TC2 0.358 0
TC3 0.312 0.31 0
TC4 0.33 0.381 0.423 0
TC5 0.447 0.539 0.542 0.374 0
57
.
Fig. 1. Map of sampling locations within Toloona Creek
Toolona Gorge
0km 0.5km
N
TC2
TC3
TC1
TC5 TC4 Toolona Creek
Mt Toolona
Mt Wanungara Mt Bithongabal
Lamington National Park
Lamington National Park 1km
58
-40
-30
-20
-10
0
10
20
-20 -10 0 10 20 30
axis 1
axis
2
site 1site 2site 3site 4site 5
Fig. 2. Principal coordinate analysis illustrating genetic differentiation of the H.
glaberrima individuals sampled across the 5 sites. Axis 1 extracted 17.88% of the
variance and axis 2 extracted 10.36% of the variance.
59
Fig. 3. UPGMA dendrogram illustrating sites 1, 2 and 3 clustering with each other and
away from the cluster of sites 4 and 5.
60
Fig. 4. Scatter plots of pairwise θ distance versus in-stream and linear distance
0
0.1
0.2
0.3
0.4
0.5
0.6
0 500 1000 1500 2000 2500 3000
In-stream distance (m)
Coa
nces
try
dist
ance
0
0.1
0.2
0.3
0.4
0.5
0.6
0 500 1000 1500 2000
Linear distance (m)
61
Statement of Joint Authorship
Manuscript 2
Prentis, P. J., N. M. Meyers, and P. B.Mather. In press. The significance of post-
germination buoyancy in Helmholtzia glaberrima and Philydrum lanuginosum
(Philydraceae). Australian Journal of Botany.
Peter Prentis (Candidate)
• Wrote the manuscript and acted as corresponding author
• Designed and formulated sampling design and experimental protocols
• Undertook all field and laboratory work, analysis and interpretation of data
Noel Meyers
• Supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
Peter Mather
• Co-supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
62
CHAPTER 3. The significance of post-germination buoyancy in Helmholtzia
glaberrima and Philydrum lanuginosum (Philydraceae)
Peter J. PrentisA, B, Noel M. MeyersA and Peter B. MatherA
A School of Natural Resource Sciences, Queensland University of Technology, GPO Box
2434, Brisbane, Qld 4001, Australia.
B Corresponding author: E-mail: [email protected]
Running title: Seedling floatation promotes establishment
84
Statement of Joint Authorship
Manuscript 3
Prentis, P. J., N. M. Meyers, and P. B.Mather. (In Prep) Seed dispersal and seedling
establishment in the riparian plant Helmholtzia glaberrima. Freshwater Biology.
Peter Prentis (Candidate)
• Wrote the manuscript and acted as corresponding author
• Designed and formulated sampling design and experimental protocols
• Undertook all field and laboratory work, analysis and interpretation of data
Noel Meyers
• Supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
Peter Mather
• Co-supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
85
CHAPTER 4. Seed dispersal and seedling establishment in the riparian plant
Helmholtzia glaberrima
Peter J. Prentis1, Noel M. Meyers and Peter B. Mather
School of Natural Resource Sciences, Queensland University of Technology, GPO Box 2434,
Brisbane, Qld, 4001 Australia.
1 Corresponding author:
Peter Prentis
School of Natural Resource Sciences,
Queensland University of Technology,
GPO Box 2434,
Brisbane, Qld, 4001 Australia.
Fax: +617 3864 1535
Phone: +617 3864 2186
Running title: Seed dispersal in H. glaberrima
105
Statement of Joint Authorship
Manuscript 4
Prentis, P. J., N. M. Meyers, and P. B. Mather. (In Review) Micro-geographic landscape
features demarcate seedling genetic structure in the stream lily, Helmholtzia glaberrima
(Philydraceae)1. American Journal of Botany.
Peter Prentis (Candidate)
• Wrote the manuscript and acted as corresponding author
• Designed and formulated sampling design and experimental protocols
• Undertook all field and laboratory work, analysis and interpretation of data
Noel Meyers
• Supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
Peter Mather
• Co-supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
106
CHAPTER 5. Micro-geographic landscape features demarcate seedling genetic
structure in the stream lily, Helmholtzia glaberrima (Philydraceae)
Peter J. Prentis,1,2,* and Peter B. Mather1
1School of Natural Resource Sciences, Queensland University of Technology, GPO Box
2434, Brisbane, Qld 4001, Australia.
2School of Earth and Environmental Sciences, University of Adelaide, North Terrace, SA
5005, Australia.
* Corresponding author: Peter Prentis
Email: [email protected]
Phone: +618 8303 5594
Fax: +618 8303 4364
Postal address: School of Earth and Environmental Sciences, University of Adelaide,
North Terrace, SA 5005, Australia
126
Statement of Joint Authorship
Manuscript 5
Prentis, P. J., N. M. Meyers, and P. B.Mather. (In Review) Fine-scale patterns of
genetic diversity and population structure in the stream lily Helmholtzia glaberrima
(Philydraceae) along rainforest streams, south-east Queensland. Freshwater Biology.
Peter Prentis (Candidate)
• Wrote the manuscript and acted as corresponding author
• Designed and formulated sampling design and experimental protocols
• Undertook all field and laboratory work, analysis and interpretation of data
Noel Meyers
• Supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
Peter Mather
• Co-supervised the sampling design and experimental protocols
• Assisted in the interpretation of data
• Provided editing and contributed to the structure of the manuscript
127
CHAPTER 6. Fine-scale patterns of genetic diversity and population structure
in the stream lily Helmholtzia glaberrima (Philydraceae) along rainforest
streams, south-east Queensland
Peter. J. Prentis, Peter. B. Mather, and Noel. M, Meyers
School of Natural Resource Sciences, Queensland University of Technology, GPO
Box 2434, Brisbane, Qld 4001, Australia.
Corresponding author e-mail: [email protected]
Running head: Population structure in a riparian plant
Keywords : AFLP, genetic diversity, landscape, population structure, riparian plant
147
CHAPTER 7. GENERAL DISCUSSION
A number of recent papers have debated the role of genetic and ecological
approaches to understanding seed dispersal in plant species (Cain et al., 2000;
Nathan, 2005; Nathan & Muller-Landau, 2000; Nathan et al., 2003). The authors of
these papers have suggested that genetic and ecological approaches usually
measure different components of seed dispersal. Ecological approaches are
suggested to measure actual or potential seed dispersal, while genetic approaches
usually measure realised seed dispersal or the phase after seedling establishment
(Cain et al., 2000). Most studies however, only use one approach to examine seed
dispersal, thereby overlooking either potential or realised components of seed
dispersal (Nathan, 2005). Information on both potential and realised dispersal is
needed to understand the dynamics of persistence, spread and distribution of plant
populations. For instance seed may often disperse to among sites over very long
distances, but never establish and contribute to realised dispersal among sites.
Ecological data alone in this case would suggest that there is high levels of seed
mediated gene flow among sites, while genetic data alone would suggest there is no
seed dispersal or gene flow occurring among the sites. In combination an ecological
and genetic approach would determine that although a large amount of seeds are
dispersed among the sites, there is no recruitment of these seeds into the breeding
population and therefore no gene flow. Therefore by integrating genetic and
ecological approaches to studying seed dispersal we can determine if seeds are
actually dispersing among populations and if they are then incorporated into the
breeding population at new sites. To this end, the present study investigated seed
dispersal in H. glaberrima using both ecological and genetic approaches.
Ecological approaches indicated primary seed dispersal in H. glaberrima
apparently occurs over short physical distances, which has also been reported in
many other herbaceous species (Cain et al. 1998; Ellison & Parker, 2002). Unlike
148
some herbaceous wetland species, however, the results of the current study support
the contention that water could potentially disperse seeds of riparian or wetland
specialists over both local and long-distances (Ellison & Parker, 2002; Waser et al.
1982). After prolonged periods of immersion in water, nearly all H. glaberrima seeds
tested still floated and over half of these seeds were still viable conferring the
potential for successful long-distance water-borne dispersal. The duration of seed
buoyancy observed here is similar to those observed in other riparian plant species
(Boedeltje et al., 2003; Ridley, 1930).
The capacity of H. glaberrima seeds to refloat after sinking and the prolonged
seedling buoyancy observed may also represent an adaptation to local or long-
distance dispersal via water. Seedlings of some other riparian plants are positively
buoyant (Ridley 1930; Sculthorpe 1967; Van der Valk 1981), which may promote the
probability of seed dispersal or the early anchoring and subsequent establishment of
seedlings (Sculthorpe 1967; Nicol and Ganf 2000). Seedling dispersal would be likely
to occur over a local scale within populations, as long-distance transport in the fast
flowing rocky streams of the McPherson Ranges could damage young leaf tissue.
Nicol and Ganf, (2000) also suggested that seedling dispersal may occur over a local
scale, but only if seedlings dispersed to the permanently moist edges of wetland
habitats.
Seed germination and seedling establishment was strongly reduced by
lowered soil moisture in H. glaberrima, with seedling establishment only occurring
under permanently wet conditions. Hydrological conditions have long been known to
influence seed germination and seedling establishment in aquatic or riparian plant
species (Boedeltje et al., 2003; Keddy and Ellis, 1985; Keddy and Constable, 1986;
Nicol and Ganf 2000). Furthermore, hydrology plays an important role in zonation
and structuring the composition of aquatic vegetation as water levels rise and fall and
soil moisture levels change (Blanch et al., 1999). Therefore, dispersal of seeds in
aquatic or riparian species to areas with unfavourable hydrological conditions is
149
unlikely to result in successful seedling recruitment and establishment.
Consequently, the effectiveness of hydrochory is likely to be limited if seeds or
seedlings of H. glaberrima are not dispersed to permanently wet habitat, as wet/dry
soil conditions inhibit seedling establishment.
Together these ecological data provide evidence that dispersal by water can
move seeds successfully over long distances along streams between isolated
wetland habitats if seeds are dispersed to microhabitat conditions favourable for
seedling establishment. Long-distance seed dispersal along riparian corridors has
been suggested to be an effective method for seed dispersal among streamside
populations in other obligate riparian species (Kudoh & Whigham 1997; Kitamoto et
al. 2005). In fact seeds of some riparian plant species have been dispersed distances
of at least six kilometres in flowing waters along streams (Boedeltje et al., 2003). In
many plant species however, potential long-distance seed dispersal is not realised as
seedling establishment usually does not result. Therefore, genetic data are required
to determine if long-distance seed dispersal has been successful.
Genetic studies showed that classical metapopulation models of dispersal
best explain the pattern of genetic structure observed here in H. glaberrima. Mixed
patterns of local and long-distance dispersal are reported to be common in plant
metapopulations (Jakalaniemi et al., in press). In many cases, most dispersal occurs
on a local scale, but very rare long-distance colonisation or dispersal events do
occur. The patterns of genetic diversity observed in H. glaberrima suggest that nearly
all successful dispersal occurs at a local scale, but that extremely rare long-distance
dispersal events may be responsible for establishment of new patches. Similar
patterns of genetic structure have also been found in the riparian species, Silene
tatarica, which were suggested to arise from mixed patterns of seed dispersal (Tero
et al., 2003). Differences in the amount of local versus long-distance dispersal may
be influenced by the effectiveness of a vector for short or long-distance dispersal or
the presence of more than dispersal vector (Ozinga et al., 2003).
150
On a local scale water-born seed dispersal can strongly influence the pattern
of genetic structure of seedlings within a population, however it had little affect on the
spatial pattern of genetic variation among established populations of H. glaberrima.
Moreover, results presented here do not support the hypothesis that flowing water is
an effective long-distance seed dispersal vector in H. glaberrima. Although ecological
experiments demonstrated seeds of H. glaberrima have many obvious adaptations
for hydrochory including prolonged buoyancy and extended viability in water, water-
borne seed dispersal does not provide effective gene flow among established
populations of H. glaberrima. Recently it has been suggested that dispersal attributes
related to seed characteristics provide a very useful framework for describing local
dispersal processes, but are poorly related to the rarer processes that may move
seeds over long distances (Higgins et al., 2003). The authors refer to the dispersal
vectors responsible for rare long-distance dispersal as non-standard means of
dispersal. It is possible then that long-distance dispersal events responsible for
population founding in H. glaberrima may occur via non standard means.
The pattern of genetic differentiation among stream systems in H. glaberrima,
suggest that rare dispersal events by nonstandard vectors may be responsible for the
colonisation of new patches. The greatest level of genetic variation was found at the
smallest spatial scale, among populations within a single stream system, while only a
small percentage of variation was found among stream systems. If water was
responsible for colonisation of new patches rather than a non-standard vector,
population genetic structure would likely conform to a hierarchical model of gene
flow. As this was not the case, other seed dispersal vectors therefore, must play an
important role in shaping the genetic structure of H. glaberrima in this region and may
prove to be a significant influence on the spatial pattern of genetic variation in this
species as a whole.
More generally, this study has shed new light on the role of hydrochory and
seed dispersal in riparian and wetland habitats. Although the potential for long-
151
distance hydrochory exists in H. glaberrima, it would seem to only occur at a local
scale. This contrasts with previous findings which show that hydrochory is often an
effective long-distance dispersal vector in complex, multistage dispersal processes
(Fridriksson, 1975; Hampe, 2004). These studies however, have only examined
potential seed dispersal and have no genetic data to determine if the plants
dispersing over long-distances have contributed to the gene pool of new populations.
This highlights the importance for incorporating genetic components into studies on
seed dispersal as without this information we gain an insight into potential seed
dispersal. Without a genetic component to the current study, it would be assumed
that hydrochory should maintain gene flow among streamside populations of H.
glaberrima. This is not the case however, as gene flow among established
populations would seem to extremely rare and unrelated to hydrochory.
In H. glaberrima, a non-standard vector would appear to be the most likely
long-distance seed dispersal vector. This highlights the lack of generality that can be
made about seed dispersal in many riparian or wetland systems except to say that
dispersal in these systems is very complex and often involve a number of different
seed dispersal vectors. Currently no mechanistic models of seed dispersal can
explain such complex seed dispersal patterns as those found in many riparian
systems. These models need to be developed in order to understand how seed
dispersal influences spatio-temporal patterns of seedling and population recruitment
in riparian and wetland plants. This information is critically required to gain
knowledge into the persistence of populations and dynamics of population extinction
and recolonisation in riparian and wetland plants. Without an understanding of how
riparian or wetland plants colonise extirpated populations or new patches we cannot
adequately develop management strategies to promote the persistence of these
species in urbanised landscapes. Therefore, H. glaberrima and other riparian species
present a unique opportunity to investigate the processes of population extinction
152
and recolonisation and long-distance seed dispersal by non-standard dispersal
vectors extinction/recolonisation dynamics in plants.
153
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