Hydrochory in willows
Kottler, 1
Sevilleta LTER REU
August 7, 2014
Population genetics of Salix exigua in the Middle Rio Grande in New Mexico, U.S.A.
ABSTRACT
Hydrochory, or the dispersal of seeds and other propagules via water, is one of the
primary dispersal mechanisms of many riparian plant species. It allows gene flow from upstream
to downstream populations, shaping genetic structure.Water-mediated dispersal may increase
genetic diversity in floodplains, but rapidly spreading hydrochorous plants can also decrease
biodiversity by forming monocoltures. Salix exigua, or coyote willow, is a plant native to
western North America found in riparian ecosystems, which can be prolific at colonizing
riverbank areas due to its diverse dispersal modes and ability to reproduce sexually and clonally.
Although important as a stabilizer of stream bank areas, S. exigua can act as an aggressive
colonizer that overcrowds streams which can choke water flow. This population study examines
the genetic structure of S. exigua populations in the riparian ecosystems that surround the Middle
Rio Grande to assess how this potentially invasive native plant migrates across the landscape. I
sampled S. exigua populations at 8 sites on the Sevilleta National Wildlife Refuge along both the
Rio Grande and an artificial drain extension that runs adjacent to it. I then use inter-sequence
simple repeats (ISSR) as molecular markers to detect genetic variation at four loci among and
between willow populations. I predict that hydrochory plays an important role in gene flow for S.
exigua. If that is the case, we should see strong genetic differentiation between populations on
different waterways as well as increased intra-population diversity in downstream populations.
Preliminary results found high genetic differentiation between populations, and also high within
population variance. This project will continue after the REU internship is completed, in order to
complete our data set with the goal of publishing in a scientific journal.
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INTRODUCTION
Hydrochory is the spread of plant propagules via water flow. In riparian ecosystems, it is
the dominant mechanism of dispersal, and as a result, genetic diversity of riparian species is
largely affected by it. In streams, it has been shown that populations situated downstream
accumulate more genotypes relative to upstream populations. Further, it is also shown that
populations situated in confluences contain a greater degree of diversity due to multiple sources.
In riparian systems, hydrochorous plants often fit a linear model of gene flow due to predominant
migration of individuals downstream (Liu et al. 2006, Pollux et al. 2009). Hydrochory allows
plants to spread their propagules over variable distances, depending on the buoyancy of
propagules and water dynamics (Nilsson et al. 2010).
One major issue in riparian ecosystems is invasive species taking over the landscape by
hydrochory and other dispersal mechanisms (Thomas et al. 2005, Kowarik & Saumel 2008).
This can negatively impact plant communities, as it can decrease overall plant biodiversity and
alter hydrological and geological processes. For example, Impatiens glandulifera, or Himalayan
balsam, was established in England and Ireland as an ornamental in the 1800s, but now
dominates the landscape of English and Irish rivers and tributaries (Love et al. 2013). This is also
the case in the Middle Rio Grande, where the majority of riparian land is taken up by invasive
species, such as salt cedar (Tamarix spp.) and Russian olive (Elaeagnus angustifolia)
(Ringold et al. 2008).
Salix exigua, known commonly as coyote willow, is a shrublike member of the
Salicaceae family. It is native to the western part of the United States in wetlands and stream
banks, and reproduces both sexually and clonally. Though genetically distinct from other
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members of the Salicaceae, like most willows it is known to frequently hybridize with closely
related species (Brunsfeld et al. 1991, Chong et al. 1996). An effective colonizer, S. exigua
spreads propagules through hydrochory due to stream flow in natural waterways. It has the
ability to stabilize riverbank areas, but also has the potential to form monocultures, overcrowding
the riverbed. It's dual reproductive strategy and multiple dispersal modes make S. exigua an
interesting case for a population analysis of genetic diversity. Since multiple strategies are being
used to propagate the species, we have the opportunity to understand the relative influences of
different dispersal mechanisms on gene flow and population genetic structure. Learning more
about genetic structure will also be important for management plans as conservation managers
attempt to control invasives. In particular, when land management measures such as salt cedar
removal are taken, monocultures of coyote willows soon establish themselves in the area (Jon
Erz, personal communication, 2014). By understanding the rapid spread of a native species we
can gather clues on how plants become invasive.
For my REU research project, I analyzed genetic diversity and genetic structure of Salix
exigua populations in the Sevilleta National Wildlife Refuge reach of the Middle Rio Grande
(New Mexico, U.S.A.) to infer dispersal patterns. I selected populations along the mainstream of
Rio Grande, and adjacent to an artificial drain extension running parallel to the river about 1km
west. I hypothesize that if hydrochory results in long distance dispersal of willow seeds, then
genetic diversity at downstream sites will be greater and high gene flow will be observed among
populations. I also predict that populations of the artificial and natural streams will be genetically
differentiated. To address this, I will be genotyping individual plants and implement a genetic
structure approach.
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METHODS
Population Sampling
I collected leaf tissue samples from S. exigua samples in eight riparian sites in the La
Joya Waterfowl Management Area and Sevilleta National Wildife Refuge, New Mexico (Figure
1). Four of the sampling units were situated along the west side of the Rio Grande, two on the
west side of the Unit 7 Drain Extension, one on the east side of the drain, and one in a small
trench on the west side of the train tracks adjacent to the Unit 7 Drain Extension. I selected sites
that were 1-2.5 km apart, with the exception of sites S7W/SR1 (0.5km apart) and DR/D7 (0.2km
apart) which I selected due to access limitations and availability of willow populated areas.
Figure 1: S. exigua population sites. Populations STW, S7W, SR1 and SR2 are located
in S. Ladow Waterfowl Management area. A7E, AR, DR and D7 are inside the Sevilleta
National Wildlife Refuge.
For each site, samples were collected along a transect that is adjacent to the given
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waterway. In order to prevent sampling from the same individual, I selected adult trees spaced
approximately four meters apart from each other. I collected a small sample branch from each
specimen to be used for DNA analysis. I also recorded the geographic coordinates of each
individual plant in order to compare genetic distance and geographic distance of samples within
and among sites, and analyze the spatial spread of clonal propagules.
DNA isolation and amplification
Initially I used the Promega DNA extraction protocol on our samples. However, this
protocol yielded high levels of proteins and secondary metabolites that inhibited polymerase
chain reactions (PCR). We determined that cetyl trimethylammonium bromine (CTAB) DNA
extraction protocol (Doyle and Doyle, 1987) was more effective at purifying DNA from crude
samples, so we proceeded with this method. I screened inter-sequence simple repeats (ISSR)
primers based on the University of British Columbia primer list. ISSRs worked well for my study
because they give information about multiple loci simultaneously. Also, as bands were scored as
either present or absent at our focal loci, this method gave us binary data instead of Mendelian
data, which would require more complex statistics for analysis.
To amplify loci from each primer, I used 9.0μl nuclease-free water, 12.0μl Promega
master mix, 1.0μl primer and 2.0μl magnesium chloride for each 1.0μl DNA template. I used an
annealing temperature of 44° C. To characterize the bands, I ran PCR products on a 2% agarose
gel stained with ethidium bromide and used GelAnalyzer to score the resulting bands.
To screen primers, I amplified four S. exigua samples with each test primer, and ran them
on an agarose gel. In my ongoing primer screening process, I am looking for primers that create
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four or more intense, clear bands at which S. exigua is polymorphic. My goal is to determine a
panel of 20 to 40 polymorphic loci to generate a sufficient number of genotypes for detecting
population genetic parameters. This could be accomplished using 2 to10 effective ISSR primers
depending on the number of loci that can be scored in a primer.
Statistical analysis
To evaluate genetic differentiation within and among different populations, I performed
analysis of molecular variance (AMOVA), a statistic similar to ANOVA and Fst, using the
program GeneAlEx 6.5 (Peakall & Smouse, 2012). I counted the number of genotypes for our
four loci, and then determined genotype frequencies for each population and for the entire data
set. In future analysis, I will determine the proportion of clones in each population, calculate
Shannon-Weaver's Index of diversity (I; Shannon & Weaver, 1949), evaluate deviations from
Hardy-Weinberg equilibrium, and determine the heterozygosity (H) and inbreeding coefficient
(F). I will measure the correlation between genetic distance and geographic distance using a
Mantel test.
PRELIMINARY RESULTS
This was the first population analysis conducted using ISSR primers on S. exigua. As
such, the duration of the summer REU project was largely spent developing and refining the
molecular protocol to produce useable PCR products with this species. To date I have tested six
primers and two of them, primers 864 and 840, produce numerous polymorphic bands. In the
remaining time, I was able to genotype a total of 35 individuals from five out of eight
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populations. These individuals were genotyped at four loci using primer 864 (Figure 2).
Figure 2: Polymorphic bands on GelAnalyzer. Column M is a 1Kb DNA ladder, and SR1-1
through 15 and S7-1 through 4 are individuals in two populations. The white dots indicate bands
detected by GelAnalyzer.
There were nine observed genotypes amongst the five populations. The frequencies of
these genotypes are shown in Figure 3.
Figure 3: Genotype frequencies. The legend (R-Z) shows different genotypes, and their
Hydrochory in willows
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proportions in each site are shown as pie charts on the map.
The AMOVA test of genetic variation for this preliminary data found that 72% of genetic
variance occurred within populations while 28% of variance occurred among populations. There
was a significant degree of genetic variance between populations (ΦPT = 0.28, p = 0.001).
DISCUSSION
My preliminary data suggests that my samples populations of Salix exigua are genetically
differentiated from each other. These results lead to similar conclusions as previous research.
Pollux et al.'s (2009) paper on the population genetics of another riparian plant, Sparganium
emersum, also found significant genetic differentiation between populations (Pollux et al. 2009).
The level of intra population genetic variation for S. exigua (72%) was slightly higher than that
in the Pollux study (59.7%). This intra population diversity contradicts previous research
asserting the importance of clonal reproduction in the dispersal strategy of Salix exigua. In their
2005 paper, Douhovnikoff et al. hypothesizes that due to low seedling survival, the predominant
model of S. exigua dispersal was colonization by a few individuals, followed by extensive clonal
reproduction producing 200-325m² clonal stands (Douhovnikoff et al. 2005). To confirm, we
would need to complete our data set with an acceptable sample size. If our completed data set
has similarly high values, it might suggest that propagule pressure from hydrochory and wind
dispersal are more important than previously recognized in the establishment of S. exigua
populations.
Another major finding in similar studies was increased genetic diversity in downstream
populations (Love et al. 2013). While not statistically significant, our data shows more genotypes
Hydrochory in willows
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downstream and a more even distribution of different genotypes. However, no solid conclusions
about the effect of hydrochory on gene flow can be made using this preliminary data. ISSR
analysis is sensitive to low sample sizes, so our preliminary results do not give a comprehensive
understanding of the genetic structure of S. exigua populations in the Sevilleta. In short, we need
to increase the statistical power of our analysis by including all our samples and additional
primers.
We will be genotyping all 120 individuals I sampled, using 2-8 primers. We will compare
ABI capillary gel electrophoresis with our current genotyping protocol to see if there is a
difference. The geographic coordinates of each individual will be incorporated into our analysis,
so we can look at the relationship between genetic and geographic distance. In the future, we also
intend to expand the scale of our study. Currently, we are working on a landscape scale, and can
only interpret the population structures of S. exigua within the Sevilleta reach of the Middle Rio
Grande riparian ecosystem. With our research team that includes my mentor, Brian Alfaro from
U.N.M., and the possible inclusion of Prof. Richard Dodd from U.C. Berkeley (California), we
intend to continue the project to expand the scale of the study. In particular, we will collect from
new sample sites ranging from Colorado to Las Cruces, NM in different watersheds. This will
allow us to examine the genetic differentiation not only between populations, but also between
regions. We will work to tease apart the varying influences of clonal dispersal, wind dispersal,
and hydrochory, and how they play different roles in diverse landscapes among regions.
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WORKS CITED
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