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\begin{document}

\title{Hybridization, polyploidy and clonality influence geographic patterns of
diversity and salt tolerance in the model halophyte seashore paspalum
(\emph{Paspalum vaginatum})}



\author[1]{David Goad}%
\author[2]{Ivan Baxter}%
\author[2]{Elizabeth Kellogg}%
\author[3]{Kenneth   Olsen}%
\affil[1]{Washington University in Saint Louis}%
\affil[2]{Donald Danforth Plant Science Center}%
\affil[3]{Washington University}%


\vspace{-1em}



  
  \date{May 04, 2024}


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\selectlanguage{english}
\begin{abstract}
Seashore paspalum (\emph{Paspalum vaginatum} Swartz) is a halophytic
turfgrass and emerging genomic model system for the study of salt
tolerance in cereals and other grasses. Despite recent interest and an
increase in available tools, little is known about the diversity present
in wild populations of \emph{P. vaginatum} and its close relative
\emph{P. distichum}. Variation in ploidy, clonal propagation,
hybridization, and subgenome composition appear to occur in the wild and
may interact to influence geographic patterns of adaptation,
particularly in response to environmental salinity levels. Using 218
accessions representing \textgreater{}170 wild collections from
throughout the coastal southern United States plus existing USDA
germplasm, we employed genotyping-by-sequencing, cpDNA sequencing and
flow cytometry to identify genetic differentiation and ploidy variation.
Within \emph{P. vaginatum}, there are two morphologically distinct
ecotypes: the fine-textured ecotype is diploid and appears to reproduce
in the wild both sexually and by clonal propagation; in contrast, the
coarse-textured ecotype consists largely of clonally-propagating
triploid and diploid genotypes. The coarse-textured ecotype appears to
be derived from hybridization between fine-textured \emph{P. vaginatum}
and an unidentified \emph{Paspalum} species. These clonally propagating
hybrid genotypes are more broadly distributed than clonal fine-textured
genotypes and may represent a transition to a more generalist adaptive
strategy. The triploid genotypes vary in whether they carry one or two
copies of the \emph{P. vaginatum} subgenome, indicating multiple
evolutionary origins. This variation in subgenome composition shows
associations with local ocean salinity levels across the sampled
populations and may play a role in local adaptation.%
\end{abstract}%



\sloppy


\textbf{Introduction}

A hallmark of angiosperm diversification has been the ability of plant
species to adapt to an extremely wide range of environments
characterized by different biotic and abiotic stresses. Among abiotic
stresses, the physiological stress of growing in high salt environments
is particularly challenging for plants. High levels of salt change the
osmotic environment, leading to dehydration; in addition, both Na+ and
Cl- ions are toxic if they accumulate in cells. Nonetheless, a small
number of terrestrial plant species (ca. 2\%) have managed to colonize
and even thrive in saline ecosystems such as beaches, salt marshes, and
estuaries (Glenn, Brown, \& Blumwald, 1999). These plants, collectively
called halophytes, have become important models for the study of salt
tolerance mechanisms and evolution (Cheeseman, 2013).

The term ``salt tolerance'' is often used as though it is a binary
characteristic; a plant is either tolerant or not. However, salt
tolerance in halophytes is likely to be quantitative, with tolerance
tuned to match the local environment (Cheeseman, 2013). Saline
environments are often characterized by broad gradients of environmental
salt concentration, from relatively low concentrations up to (and
occasionally even beyond) the concentration of seawater. Such gradients
are common where fresh and sea water mix, with areas of lower salinity
extending along the coastline and far offshore from the mouths of major
rivers (Fournier, Reager, Dzwonkowski, \& Vazquez-Cuervo, 2019). This
variation offers extensive opportunities for local salinity adaptation
within widely distributed halophytic species; however, whether such
local adaptation occurs and the forces that influence it are largely
unknown. As described below, three common characteristics of halophytic
species that could shape patterns of local salinity adaptation are
clonal propagation, interspecific hybridization and ploidy variation.

Clonal propagation occurs widely in halophytes and has important
implications for geographic patterns of diversity and local salinity
adaptation (Bricker, Calladine, Virnstein, \& Waycott, 2018; Jefferies
\& Rudmik, 1991; R\selectlanguage{ngerman}óis et al., 2015). Depending on the dispersal capacity
of clonal propagules and the frequency at which novel genotypes arise
(e.g., through sexual reproduction or somatic mutation), one of two
reproductive strategies might be expected. If new genotypes arise often
but have limited clonal dispersal, a ``frozen niche'' strategy might be
favored, whereby multiple genetically distinct clonal populations occur,
each restricted to the small geographical area to which it is locally
adapted (Róis et al., 2015; Vrijenhoek, 1979). Alternatively, if the
production of new genotypes is infrequent and/or dispersal potential is
high, a ``general-purpose genotype'' strategy might instead be favored,
whereby a few generalist genotypes are present in multiple environments
across a wide geographic range (Baker, 1965; Bricker, Calladine,
Virnstein, \& Waycott, 2018; Coughlan, Han, Stefanovi\selectlanguage{polish}ć, \selectlanguage{english}\& Dickinson,
2017). The underlying conditions that lead genotypes to follow one
pattern or another remain poorly understood (Vrijenhoek \& Parker,
2009).

In contrast to clonal propagation, interspecific hybridization can
provide access to new ecological niches by introducing novel genetic
variation into a population and/or increasing heterosis (Rieseberg,
Archer, \& Wayne, 1999; Soltis \& Soltis, 2009). Adaptation to saline
environments has been shown to be influenced by natural hybridization in
several wild systems. Specifically, hybrids derived from two halophytic
species may show a relative increase or decrease in tolerance relative
to their parents (Gallego-T\selectlanguage{ngerman}évar, Curado, Grewell, Figueroa, \& Castillo,
2018; Lee, Ayres, Pakenham-Walsh, \& Strong, 2016). It can also produce
a novel halophyte from two non-halophytic parental species (Edelist et
al., 2009; Welch \& Rieseberg, 2002). The relative roles of the genomic
contributions from different parental species for phenotypic and niche
differentiation in hybrids remains an open question (Bar-Zvi, Lupo,
Levy, \& Barkai, 2017). High-throughput genomic techniques can help in
identifying hybrid genotypes, their genomic composition, and the
relative importance of their parental genomes for salinity adaptation.

Polyploidy (i.e. whole-genome duplication) may also play a role in
salinity adaptation. Studies in non-halophytes have suggested that
inducing autopolyploidy in a diploid species (i.e. doubling its genome)
can increase salt tolerance (Chao et al., 2013; Wu, Lin, Jiao, \& Li,
2019). However, in the wild most halophytic polyploids are not
autopolyploids but rather allopolyploids --- having arisen through
interspecific hybridization where each parental species contributes its
entire genome (Ainouche, Baumel, Salmon, \& Yannic, 2004). In such cases
the effect of the genome duplication is confounded with the potential
effects of interspecific hybridization described above (Fort et al.
2016). Systems where repeated natural hybridization events have resulted
in a mix of diploid and polyploid genotypes could potentially be used to
untangle these effects. If such a group also varied with respect to the
relative numbers of genome copies received from each parental species,
whole-genome dosage effects on fitness and other phenotypes could also
be examined (Betto-Colliard, Hofmann, Sermier, Perrin, \& Stöck, 2018;
Harvey, Fjelldal, Solberg, Hansen, \& Glover, 2017; Tan et al. 2016).

The halophytic grass seashore paspalum (\emph{Paspalum
vaginatum}Swartz\emph{)} provides an advantageous system for addressing
how clonality, hybridization and polyploidy interact to shape patterns
of genotypic variation across salinity gradients. First, it has a
worldwide distribution across habitats of varying salinity (Duncan \&
Carrow, 2000). Second, it shows quantitative variation in salt
tolerance, up to and including sea water concentrations (Lee, Duncan, \&
Carrow, 2004; Lee, Carrow, \& Duncan, 2004, 2005; Lee, Carrow, Duncan,
Eiteman, \& Rieger, 2008), although the genetic basis and geographical
distribution of this variation is almost completely unknown. Third, it
spreads vegetatively by stolons and is capable of clonal propagation
under cultivation, suggesting that dispersal of clonal propagules may
affect geographical patterns of genotypic variation in the wild. Fourth,
it has been proposed to undergo hybridization with a closely related
non-salt tolerant species (\emph{P. distichum} L.) (Eudy, Bahri,
Harrison, Raymer, \& Devos, 2017), which could contribute to natural
phenotypic variation in salt tolerance. Finally, although it is believed
to be predominantly diploid (2n = 2x =20) (Duncan and Carrow, 2000;
Eudy, Bahri, Harrison, Raymer, \& Devos, 2017), both diploid and
triploid cytotypes have been reported (Eudy, Bahri, Harrison, Raymer, \&
Devos, 2017), which suggests that ploidy level and subgenome composition
might also contribute to population structure and local salinity
adaptation.

In addition to these life history features, \emph{Paspalum
vaginatum}also provides the benefits of a genome-enabled model system
with experimental tractability and economic value that facilitate
applied studies. It has a manageably-sized \textasciitilde{}600 Mb
diploid genome which has recently been assembled, allowing for genomic
analyses (Eudy, Bahri, Harrison, Raymer, \& Devos, 2017; Qi et al.,
2019). It is closely related to several economically important grass
species in the subfamily Panicoideae, including maize, sorghum and
sugarcane (Morrone et al., 2012), which could facilitate the transfer of
salt tolerance mechanisms to major cereal crops. It is also economically
important in its own right as a turfgrass for salt-affected soils. As
such, it has resources and germplasm available through breeding programs
(Duncan \& Carrow, 2000).

One current limitation of publicly available (USDA) \emph{P.
vaginatum}germplasm is its narrow genetic variability due to genotypic
redundancy among accessions (Eudy, Bahri, Harrison, Raymer \& Devos,
2017) and collection biases towards plants with favorable turfgrass
qualities (Duncan \& Carrow, 2000). Additionally, due to the
decades-long gap between the collection and genotyping of these USDA
samples, it is unclear whether the high degree of genotypic redundancy
in the germplasm collection represents natural clonal propagation in the
wild or post-collection cross-contamination of accessions, which can
easily occur as the aggressive stolons typical of the species invade
neighboring pots (Eudy, Bahri, Harrison, Raymer, \& Devos, 2017).
Genotyping of newly collected material is thus required to quantify the
actual levels of clonal propagation in the wild and geographical
patterns of genotypic diversity.

In this study, we undertook the largest collection of living wild
specimens of \emph{P. vaginatum} and \emph{P. distichum} to date and
analyzed them together with available USDA germplasm to investigate the
nature of genotypic distributions across salinity gradients in the North
American species range. Specifically, we asked the following questions:
1) Do wild \emph{P. vaginatum} populations represent many distinct
genotypes or replicates of a few geographically widespread clones? 2) To
the extent that clonal propagation occurs in the wild, do geographical
and environmental distributions suggest a localized ``frozen niche''
reproductive strategy, a widespread ``general purpose genotype''
strategy, or no discernible pattern? 3) To what extent does
interspecific hybridization and/or ploidy variation in this system
influence patterns of genetic differentiation and diversity in wild
populations? 4) How do clonal structure, ploidy variation and subgenome
composition vary across geographical gradients in salinity? Our findings
provide insight into the roles of genome composition and clonal
propagation in local salinity adaptation in \emph{P. vaginatum} , and
lay the necessary groundwork for future investigations into the genetic
basis of salt tolerance in this genomic model halophytic grass species.

\textbf{Methods}

\textbf{Study system}

\emph{Paspalum vaginatum} is distributed throughout tropical and
subtropical saline ecosystems worldwide (Duncan \& Carrow, 2000). It is
characterized by two common ecotypes that differ in shoot and leaf
morphology, referred to as the `coarse-textured' and `fine-textured'
ecotypes (Fig. 1B; Duncan \& Carrow, 2000). The fine-textured ecotype is
more economically important because its prostrate growth form and
smaller leaves makes it valuable for use as a turfgrass; the
coarse-textured ecotype has also seen limited use as a dune stabilizer.
Within the US, the coarse-textured ecotype is typically found on the
Gulf Coast and Florida, while fine-textured plants are found on the
Atlantic coast from North Carolina to Georgia (Duncan \& Carrow, 2000).
Due to the focus on collecting samples for turf breeding programs, the
USDA \emph{P. vaginatum} germplasm collection is composed almost
entirely of fine-textured accessions (USDA-ARS, 2015).

\emph{Paspalum vaginatum} and its closely related non-halophytic sister
species \emph{P. distichum} together comprise the Disticha clade, which
is distantly related to the other ca. 300 species in the genus (Burson,
1981; Scataglini, Zuloaga, Giussani, Denham, \& Morrone, 2014). The two
species are morphologically very similar and often confused (Bor \&
Guest, 1968; Brummitt, 1983), although they are distinguishable based on
cpDNA and nuclear sequences (Scataglini, Zuloaga, Giussani, Denham, \&
Morrone, 2014). Unlike \emph{P. vaginatum} , \emph{P. distichum} is
believed to be predominantly hexaploid (2n = 60), with tetraploid (2n =
40), pentaploid (2n = 50) and hyperpentaploid (2n = 52, 54, 57, and 58)
cytotypes occurring occasionally in the wild Echarte, Clausen, \& Sala,
1992). A putative diploid \emph{P. distichum} accession has also been
reported (Eudy, Bahri, Harrison, Raymer, \& Devos, 2017)\emph{.}

\textbf{Sampling}

A set of 146 coarse-textured \emph{P. vaginatum} , 18
fine-textured\emph{P. vaginatum} and 10 \emph{P. distichum} plant
samples was collected along the Atlantic and Gulf coasts of the
southeastern United States, from North Carolina to Texas, during June
and July of 2016 and 2017 (Fig. 1A; Table S1). Collection habitats
included beaches, salt marshes, brackish estuaries and freshwater rivers
and lakes. To reduce the chance of repeatedly collecting representatives
of the same clone, localities were separated by at least 1.5 km. One to
three samples were collected in each location. When multiple samples
were collected from a single locality, they were separated by at least
100 m to minimize replicated sampling of a single clone. Samples were
harvested as stolons which were wrapped in a damp paper towel and then
placed in a plastic bag for no longer than one week before planting.
Stolons were propagated in the Donald Danforth Plant Science Center
greenhouses. In addition to the wild-collected material, a set of 40
samples, representing 35\emph{P. vaginatum} (two of which are
coarse-textured) and five \emph{P. distichum} , were obtained from the
USDA National Genetic Resources Program. Additionally, dried tissue for
DNA extraction, representing four \emph{P. vaginatum} accessions and one
\emph{P. distichum}accession, were acquired from Uruguay from Dr. Pablo
Speranza (Universidad de la República de Uruguay).

\textbf{DNA extraction and genotyping-by-sequencing}

Genomic DNA was extracted using the CTAB protocol of Brock, Dönmez,
Beilstein, \& Olsen (2018), which was modified from Webb \& Knapp
(1990). Genotyping-by-sequencing (GBS) libraries were prepared following
Poland \& Rife (2012), using a double digest of \emph{Pst} I
and\emph{Msp} I. Samples were sequenced on three lanes of the Illumina
HiSeq 2500 platform with 1x100 reads at the Roy J. Carver Biotechnology
Center at the University of Illinois Champaign-Urbana.

\textbf{Bioinformatics}

Read processing and variant calling were performed using the Fast-GBS
pipeline with default parameters (Torkamaneh, Laroche, Bastien, Abed, \&
Belzile, 2017), which demultiplexes with sabre v1.000 (Joshi, 2011),
trims with cutadapt v1.14 (Martin, 2011), maps with BWA MEM
v0.7.12-r1039 (Li \& Durbin, 2009) and calls variants with Platypus
v0.8.1 (Rimmer et al., 2014). This produced a large SNP dataset which
was then further filtered with plink v1.90b4.6 (Purcell et al. 2007).
Sites were removed if there were missing calls for more than 10\% of the
samples or had less than 0.01 minor allele frequency. Individuals that
were missing calls at 50\% or more sites were also removed.

To identify clonal replicates among collected plants, all samples were
included in a principal component analysis (PCA) performed with plink
(Purcell et al., 2007); within each visually identified genotype cluster
in the PCA, a pairwise genetic distance matrix was constructed. To
produce a group-specific similarity threshold to identify identical
genotypes, we then created a histogram of distances and heuristically
chose a threshold that separated the first and second peaks (see Fig.
S1). Refined variant datasets were then created with duplicate genotypes
removed; for each set of duplicate genotypes, only the accession with
the highest coverage was retained.

To prevent biases due to multiple collections of the same genotype,
minor allele frequency and missing data filters were repeated in plink
for the dataset consisting of unique genotypes only. An additional PCA
was then performed on this dataset. To assess population structure
within diploid \emph{P. vaginatum} , ADMIXTURE v1.3.0 (Alexander,
Novembre, \& Lange, 2009) analysis was performed on a further reduced
dataset comprising coarse- and fine-textured diploids, with assumed
population numbers ranging from K = 2-6. Cross validation error
estimation was used to determine the optimal K.

\textbf{Geographic and environmental dispersion of clonal genotypes.}

For genotypes with multiple clones collected from the wild, we
calculated pairwise geographic distance between collections within each
clonal genotype based on GPS coordinates of each collection with
the\emph{distHaversine} function in R v3.4.1 (Hijmans, Williams, \&
Vennes, 2019; R Core Team, 2013). To measure pairwise environmental
distance, we calculated Euclidian distance for environmental variables
in three dimensions with the \emph{dist} function in R, as follows:
coordinates for the first and second dimension were obtained as the
first and second principal components in a PCA of the 19 WorldClim
BioClim variables (Fick \& Hijmans, 2017) for each of our collection
locations. Principal components were calculated using the \emph{prcomp}
function in R with scaled unit variance. The values of the first two PCs
explained 50.9\% and 22.7\% of the variance respectively. For the third
coordinate, we downloaded the average sea surface salinity dataset for
the years 2005-2017 from the World Ocean Atlas (Zweng et al., 2019) and
identified the salinity of the ocean water nearest to each collection
site (excluding one sample collected \textgreater{} 40 km from the
coast). All three coordinates where then centered and scaled with
the\emph{scale} function in R before calculating distance.

\textbf{Chloroplast gene sequencing}

To look for differences in maternal subgenome between \emph{P.
distichum} and \emph{P. vaginatum} and to confirm that the
coarse-textured ecotypes are part of the Disticha clade (see Results),
we sequenced the intron of the \emph{rpl16} chloroplast ribosomal genes
of 16 accessions (3 \emph{P.} \emph{distichum} , 13 \emph{P. vaginatum}
) using the F71 and R1661 primers described in Giussani et al. (2009).
The\emph{P. vaginatum} samples included 3 fine-textured and 10
coarse-textured ecotypes. Direct Sanger sequencing of purified PCR
products was performed at the University of Wisconsin Biotechnology
Center using an ABI 3730xl DNA Analyzer. Sequences were aligned against
the \emph{P. vaginatum rpl16} sequence (accession KF853027; Scataglini,
Zuloaga, Giussani, Denham, \& Morrone, 2014) and compared using BioEdit
(Hall, 1999).

\textbf{Ploidy estimation with flow cytometry}

In order to assess genome size variation and ploidy, fresh leaf tissue
was collected from one accession of each unique genotype, except for
fine-textured samples where only two of the 24 unique genotypes were
tested. Tissue was chopped with a razor blade in 1 mL cold LB01
(Dole\selectlanguage{polish}ž\selectlanguage{english}el, Binarov\selectlanguage{ngerman}á, \& Lucretti, 1989) along with maize line B73 tissue,
which was included as an internal standard. Nuclei were stained with
propidium iodide, and samples were run on an Accuri C6 flow cytometer.
Genome sizes were then calculated by taking the ratio of sample DNA to
B73 DNA. These calculations were then divided by the value calculated
for PI 509022 (the \emph{P. vaginatum} reference genome accession, 0.259
relative to B73) to determine relative genome size of each accession
compared to PI 509022.

\textbf{Ploidy estimates with genome-wide heterozygosity}

For GBS-genotyped samples that vary in ploidy, measures of genome-wide
heterozygosity can potentially be used as a complement to flow cytometry
for identifying ploidy variation (Gompert \& Mock, 2017). This approach
is based on the rationale that if all reads are mapped to a diploid
reference genome, any subgenome differences in polyploids will be
recorded as `heterozygous' loci. At increasing ploidy levels, each
additional subgenome will introduce more differences and increase the
number of heterozygous locus calls. This approach can also be used to
identify diploid hybrids, which are identifiable as showing a quantum
increase in heterozygosity with no associated increase in genome size
based on flow cytometry. We employed these strategies to assess ploidy
variation in the \emph{Paspalum} samples, focusing on genotypically
unique accessions with the highest sequence coverage. Genome-wide
heterozygosity was calculated as the individual inbreeding coefficient
(F) with the het function in plink (Purcell et al., 2007).

\textbf{Subgenome comparisons}

Heterozygous genotype calls were also used to infer subgenome identity
using a custom python script
(https://github.com/david-goad/paspalum-hybridization/het\_venn.py). For
this analysis we used the dataset comprising all unique genotypes after
removal of sites with \textless{}0.05 heterozygosity. We calculated the
number of loci that were heterozygous in each of our three morphological
groups (the two \emph{P. vaginatum} ecotypes and \emph{P.
distichum,}excluding three putatively admixed accessions). We then
compared the number of loci with heterozygous calls unique to each group
to those that were shared between groups. Following the logic that
`heterozygous' loci reflect subgenome differences within individuals, we
expected that subgenomes shared between groups would be reflected as
shared `heterozygous' loci. In the case of allopolyploid and diploid
hybrid groups (\emph{P. distichum} and coarse-textured \emph{P.
vaginatum} ; see Results), high numbers of shared loci would indicate
sharing of a subgenome that is not present in the diploid fine-textured
\emph{P. vaginatum} samples.

To investigate the number of subgenome copies received from each parent
in triploid coarse-textured genotypes, we used a custom python script
(https://github.com/david-goad/paspalum-hybridization/triploid\_comp.py)
that counts reads at heterozygous loci in a SAM file (Fig. S2). Based on
the distribution of the relative number of reference vs non-reference
reads at each locus, the number of subgenome copies can be inferred
(Delomas, 2019; Gompert \& Mock, 2017). We chose the highest coverage
individual from each of the three putative triploid genotypes and the
diploid genotype with the greatest number of genotypically-identical
accessions (to serve as a 1:1 control). In cases where a locus was
called as heterozygous in all four of the genotypes, we counted the
number of each read type covering the position in the same file based on
their CIGAR string (the column of the SAM file which identifies the
position of variants in the read). To remove the effect of low-quality
reads (often manifested as singletons) we only compared the two most
common read types. Additionally, sites were excluded if neither of the
two most common reads was identical to the reference genome (to ensure
unambiguous assignment to a subgenome) or if their combined count was
less than 20 (to ensure adequate depth). The number of reference and
alternate reads at each locus was then visualized as a scatter plot for
the raw counts and a histogram for the percent of reads matching the
reference genome. The diploid genome should return a distribution of
loci centered on a 1:1 read count ratio (50\% reference genome reads,
50\% alternate reads). Triploids should return distributions that are
centered on either 1:2 (33.3\% reference genome, 66.7\% alternate) or
2:1 (66.7\% reference genome, 33.3\% alternate) depending on the
subgenome composition (e.g. ABB or AAB respectively, where the reference
genome is A).

\textbf{Correlations with ocean salinity levels}

To test for associations between genotypic variation and local ocean
salinity within \emph{P. vaginatum,} we used the local ocean salinity
measurements from the World Ocean Atlas dataset (Zweng et al., 2019) to
perform two statistical comparisons. First, we used a two-tailed t-test
to test for differences in local ocean salinity between diploids and
triploids. Then we used a fixed-effects linear model to test the effects
of copy number of both the \emph{P. vaginatum} subgenome
(N\textsubscript{Pv}) and the other unidentified subgenome
(N\textsubscript{other}). We compared the models with an interaction
(salinity \textasciitilde{} N\textsubscript{Pv} +
N\textsubscript{other}+ N\textsubscript{Pv}:N\textsubscript{other}) and
without it (salinity \textasciitilde{} N\textsubscript{Pv} +
N\textsubscript{other}). We chose the model without an interaction
because it had a lower AIC (200.27 vs. 201.09) and because the
interaction term was not significant when it was included.

\textbf{Results}

\textbf{Identification of clonal replicates}

The filtered GBS dataset was characterized by 127,282 SNPs in 218
accessions. Principal component analysis (PCA) and application of
genetic similarity threshold criteria to identify clonal replicates (see
Methods; Supporting Information, Fig. S1) revealed a large number of
genotypically identical accessions (Supporting Information, Table S1).
For the coarse-textured \emph{P. vaginatum} ecotype, only 12 unique
genotypes were detected among the 148 accessions, with just three
widely-distributed genotypes accounting for \textgreater{}85\% of the
accessions. This indicates that extensive clonal propagation occurs in
wild populations of this ecotype. Somewhat greater genotypic diversity
was found in the fine-textured ecotype, but extensive clonality was
still present; 24 unique genotypes were identified in 55 accessions.
Four out of eight genotypes in the wild-collected fine-textured
accessions were represented by multiple clonal replicates. For \emph{P.
distichum} , there were four unique genotypes in the collection of 15
accessions, two of which were unique to our wild collections. The final
SNP dataset for all genotypically unique samples across both species,
including wild-collected and USDA samples, totaled 40 genotypes (Table
1). Together these results suggest that clonal propagation occurs
extensively in wild \emph{P. vaginatum} and \emph{P.
distichum}populations.

\textbf{Geographic patterns of clonal propagation}

Having detected extensive clonality among the wild-collected \emph{P.
vaginatum} samples, we next assessed the distribution of geographic and
environmental distances between clones of the same genotype in our wild
collections. Specifically, we investigated the extent to which they
showed patterns consistent with a ``frozen-niche'' strategy (multiple
geographically and environmentally restricted clones) or a
``general-purpose genotype'' strategy (a few broadly distributed
clones). For the fine-textured ecotype, the four genotypes with multiple
clones in the wild tended to have relatively small geographic ranges;
two of the four had genotypes restricted to a single location, while the
two found in multiple locations had a maximum range of 126 km (Table S1;
Fig. 1; Fig. 2A). Consistent with their narrow geographical
distributions, assessments of pairwise environmental distances among
locations (based on local climatic and salinity conditions; see Methods)
indicated a low level of environmental dispersion (Fig. 2B). These
patterns are generally consistent with a ``frozen-niche'' reproductive
strategy for fine-textured \emph{P. vaginatum} .

In contrast to the fine-textured ecotype, genotypes of the
coarse-textured ecotype showed patterns more consistent with a ``general
purpose genotype'' strategy. Not only did a handful of unique genotypes
make up the majority of wild accessions (Table 1), but these genotypes
showed broad distributions with respect to both geography and
environmental variables. With the exception of one genotype that was
restricted to a 1 km area (coarse\_06), the narrowest geographical range
of any coarse-textured clonal genotype was 750 km (coarse\_04), and the
three most common genotypes (coarse\_01, coarse\_02 and coarse\_03) all
had ranges \textgreater{}1400 km (Fig. 1; Fig. 2A). Pairwise
environmental distance measures were similarly higher for the
coarse-textured ecotype compared to fine-textured accessions (Fig. 2B);
this indicates that clonal propagules of these widely dispersed
genotypes are not specializing on particular environments in the
locations where they occur.

\textbf{Genetic differences between genotypes and species}

Following the identification and characterization of clonal variation,
subsequent analyses focused on a reduced dataset composed of one
high-coverage accession per genotype. Removal of the lower coverage
samples in each clonal group allowed more sites to pass the missing data
filter, resulting in an increase to 177,954 variant sites for this
highest-quality dataset. A PCA on the set of 40 unique genotypes
revealed the same groupings as in the total dataset (Fig. 3, Fig. S3).
For both PCAs, most accessions fell into three distinct groups of
tightly clustered accessions, corresponding to \emph{P. distichum} and
the two ecotypes of \emph{P. vaginatum.} For the reduced-sample dataset,
PC 1 explained 31.6\% of the variance and separated \emph{P.
distichum}from \emph{P. vaginatum} ; PC 2 explained 20.2\% of the
variance and primarily separated the coarse- and fine-textured ecotypes
of \emph{P. vaginatum} .

In addition to the three main PCA clusters, two coarse-textured \emph{P.
vaginatum} genotypes were placed as intermediate between the fine- and
coarse-textured accessions along PC2 (Fig. 3, Fig. S3). These two
genotypes corresponded to GRIN accession PI 612771 and two of the
wild-collected accessions which were clones of one genotype (coarse\_05,
Table S1). The intermediate placement of these genotypes suggests
possible admixture between the coarse-textured and fine-textured
ecotypes. To test this hypothesis, we created a reduced SNP dataset
containing only unique diploid \emph{P. vaginatum} genotypes (33
accessions and 117,218 polymorphic sites). ADMIXTURE analysis shows that
the genetically intermediate coarse-textured individuals indeed have
genetic compositions consistent with admixture between coarse- and
fine-textured ecotypes (Fig. S4). This result suggests that there is
occasional gene flow between the two \emph{P. vaginatum} ecotypes.

SNP variation in the \emph{rpl16} intron of the chloroplast genome
confirmed the three major groups identified by the GBS data. In the 1.15
kb aligned sequence, \emph{P. distichum} differed from both ecotypes
of\emph{P. vaginatum} by a total of 6 SNPs and one 10-bp indel, with one
additional SNP segregating within \emph{P. distichum} (Table S2).
Within\emph{P. vaginatum} , accessions of the fine-textured ecotype
carried two unique SNPs that distinguished them from the other two
groups. These results are consistent with the current taxonomy
recognizing \emph{P. vaginatum} and \emph{P. distichum} as two distinct
species (Brummitt, 1983). In addition, the pattern of SNP variation
within \emph{P. vaginatum} indicates that the fine-textured ecotype is
characterized by unique, derived variants.

\textbf{Ploidy variation and hybridization}

Using flow cytometry to estimate genome size on a subset of our samples,
we identified extensive ploidy variation associated with the three
genetically differentiated groups. Within \emph{P. vaginatum} , the
fine-textured ecotype had an average estimated genome size consistent
with diploidy (1.01, when 1.0 is designated as the size of the
diploid\emph{P. vaginatum} reference genome) (Table 1). In contrast, the
relative genome size estimates for coarse-textured samples were
consistent with a mix of diploids (1.04) and triploids (1.56).
For\emph{Paspalum distichum} , samples had higher average relative
genome size estimates overall, consistent with predictions that this
species is predominantly hexaploid and pentaploid (Echarte, Clausen, \&
Sala, 1992). Unlike \emph{P. vaginatum} samples, \emph{P. distichum}
genome size estimates did not form obvious multiples of the \emph{P.
vaginatum}reference genome. Instead, the difference in genome size
between putative pentaploids and hexaploids (2.25 and 2.69,
respectively) suggests a \emph{P. distichum} basal genome size that is
\textasciitilde{}11\% smaller than \emph{P. vaginatum} . When \emph{P.
distichum} relative genome sizes were recalculated assuming a basal
genome size of 0.89 instead of 1.0 as the point of reference, the values
closely match numerical expectations for pentaploid and hexaploid
genomes (2.53 and 3.03, respectively) (Table 1). Together these results
suggest that \emph{P. vaginatum} is composed primarily of diploid and
triploid genotypes, that \emph{P.} \emph{distichum} is largely composed
of pentaploid and hexaploid genotypes, and that the basal genome size of
the latter species is likely \textasciitilde{}11\% smaller than
the\emph{P. vaginatum} reference genome.

By using genome-wide heterozygosity calls from the SNP dataset (F-values
generated by plink) in conjunction with the flow cytometry results
above, we were able to estimate ploidy for all genotypes (see Gompert \&
Mock, 2017). Specifically, individuals containing multiple subgenomes
(e.g. allopolyploids) are expected to show an increase in heterozygosity
with each additional subgenome. The highest genome-wide heterozygosity
values (corresponding to more negative F-values) occurred in \emph{P.
distichum} and were consistent with a combination of allohexaploid and
allopentaploid genotypes (average F = -1.323 and -1.241 respectively)
(Table 1). Conversely, the fine-textured cluster of \emph{P.
vaginatum}(composed entirely of diploids) had the lowest genome-wide
heterozygosity calls (average F = 0.541). For the
coarse-textured\emph{P. vaginatum} group, which flow cytometry data
indicated to be a combination of diploids and triploids, F-values were
consistent with a slight increase in heterozygosity in putative
triploids relative to diploids (average F = -0.520 and -0.279
respectively). Unexpectedly, there was a large difference in F-value
between diploid coarse- and fine-textured accessions (-0.278 and 0.541);
the greater heterozygosity in the coarse-textured genotypes, despite a
lack of difference in genome size, is potentially consistent with
hybridization in the origin of the diploid coarse-textured ecotype.
Collectively these findings support the inference that \emph{P.
vaginatum} not only falls into two genetically distinct subgroups
according to ecotype, but that the coarse-textured ecotype can be
further subdivided by ploidy variation.

\textbf{Subgenome composition of allopolyploids and hybrids}

Because allopolyploid genotypes were identified in both
coarse-textured\emph{P. vaginatum} and in \emph{P. distichum} , we
examined levels of heterozygous SNP sharing between these groups to
assess whether the additional subgenomes in polyploid \emph{P.
vaginatum} might be shared with \emph{P. distichum} . Patterns of
heterozygous locus sharing indicate that this is not the case. Whereas
28,881 heterozygous loci were unique to coarse-textured individuals and
33,585 heterozygous loci were unique to \emph{P. distichum} , only 3713
loci were shared between species (Fig. S5). These patterns are
consistent with PCA results (Fig. 3, Fig. S3) in indicating that
\emph{P. distichum} is no more closely related to coarse-textured
\emph{P. vaginatum} genotypes (regardless of ploidy) than to
fine-textured genotypes. Thus, the triploid coarse-textured \emph{P.
vaginatum} genotypes appear to carry a distinct subgenome from \emph{P.
distichum} from an unknown source.

Heterozygous locus calls further indicated that in addition to ploidy
variation, there were also subgenome composition differences within the
coarse-textured \emph{P. vaginatum} group. By counting the number of
reference versus alternate reads at heterozygous loci, we could estimate
the number of haploid subgenomes within a genotype that are from\emph{P.
vaginatum} or some other source (see Methods). Accession DG076, a
representative diploid coarse-textured genotype (coarse\_03; Table S1),
followed the expected pattern of a 1:1 ratio of reference to alternate
reads (50\% reference; Fig. 4A; Fig. S6A). Among the three triploid
genotypes, one of them (coarse\_01, represented by accession DG117)
showed a 1:2 ratio (33.3\% reference reads; Fig. 4B; Fig. S6B), while
the other two (coarse\_02 and coarse\_04, represented by accessions DG
107 and DG120, respectively) showed 2:1 ratios (66.6\% reference reads;
Fig. 4 C-D; Fig. S6 C-D). This difference among triploid genotypes
indicates that they do not have the same subgenome composition. More
specifically, one genotype appears to carry one copy of the
fine-textured \emph{P. vaginatum} reference genome and two subgenome
copies from an unknown source, whereas the other two genotypes appear to
have the reverse pattern. Our use of the same set of heterozygous loci
in all four of the tested coarse-textured genotypes allows us to infer
that the same two subgenomes are present in all of them, but that the
number of copies of each subgenome differs.

\textbf{Correlations with ocean salinity}

To investigate the potential roles of ploidy and subgenome composition
in adaptation to salt tolerance, we compared the surface salinity of the
ocean nearest to the collection location of each \emph{P.
vaginatum}sample. Salinity levels did not differ significantly between
the locations of diploid and triploid individuals (P = 0.5328, Fig. 5);
thus, ploidy variation alone does not obviously contribute to adaptive
variation in salinity tolerance. In contrast, a linear model that
incorporated both the number of \emph{P. vaginatum} subgenomes (one vs.
two, independent of ploidy) and the number of the unidentified
subgenomes (zero, one, or two) revealed that having two \emph{P.
vaginatum} subgenomes was strongly associated with higher local ocean
salinity (P = 7.6e-11 ), while having an additional copy of the unknown
genome was weakly associated with decreased salinity (P = 0.033, Fig.
5). This finding suggests that the relative proportion of subgenomes
from the halophytic species has a detectable effect on salinity
adaptation. It should be noted, however, that we could not entirely
remove the effect of ploidy from this analysis because after accounting
for the number of \emph{P. vaginatum} subgenomes, a change in the copy
number of the other subgenome necessarily changes ploidy as well (2:0 to
2:1 and 1:1 to 1:2).

\textbf{Discussion}

\emph{Paspalum vaginatum} is a promising new genomic model system for
the study of salt tolerance in cereals and other plant species. Our
population genomic analyses, using new wild samples collected widely
throughout the southern US, and without a bias toward turf-like
(fine-textured) ecotypes, has allowed us to make several key insights
into the genetic diversity, population structure and genomic composition
of this species, with broader implications for mechanisms of salinity
adaption in widespread species. Our findings include the following: 1)
Clonal propagation occurs extensively in both \emph{P.
vaginatum}ecotypes, as well as in the non-halophytic sister species
\emph{P. distichum} (Table 1; Table S1); 2) Clones of the
coarse-textured ecotype are more widely distributed than the
fine-textured ecotype, both geographically and with respect to the
environment (Fig. 1; Fig. 2), suggesting a ``general purpose genotype''
adaptive strategy for the former and a ``frozen niche'' strategy for the
latter; 3) The system is characterized by extensive ploidy variation,
including diploids and allotriploids that vary in their subgenome
composition (Table 1; Fig. S5, S6); and 4) Increasing number of \emph{P.
vaginatum} subgenomes within a genotype are associated with occurrence
in more saline environments (Fig. 5), suggesting a direct effect of the
halophytic species' genome on salinity adaptation. Collectively these
findings suggest that both clonal propagation and
hybridization-associated ploidy variation play key roles in the ability
of this species to grow widely across environments of varying salinity.

\textbf{Differing patterns of clonal propagation}

For species that commonly reproduce clonally, there are two proposed
strategies that can explain geographic distributions of individual
clonal genotypes. The ``frozen niche'' strategy is characterized by
multiple genotypes that are each adapted to a specific geographically
localized environment (Róis et al., 2015; Vrijenhoek 1979). Conversely,
the ``general purpose genotype'' strategy is characterized by a few
genotypes that are widely distributed across a range of environments
(Baker, 1965; Bricker, Calladine, Virnstein, \& Waycott, 2018; Coughlan,
Han, Stefanovi\selectlanguage{polish}ć, \selectlanguage{english}\& Dickinson, 2017). The occurrence of interspecific
hybridization could be an important factor in determining clonal
propagation strategy. Hybrids, which are often obligately asexual, are a
common model system to investigate patterns of clonal distribution;
however, opportunities to investigate differential clonal reproduction
strategies between a hybrid and its parental species are rare as the
parental species are often obligately sexual (Coughlan, Han, Stefanovi\selectlanguage{polish}ć,
\selectlanguage{english}\& Dickinson, 2017). With our collection of clones from both the hybrid
coarse-textured \emph{P. vaginatum} and one of its parent species,
fine-textured \emph{P. vaginatum,} we were able to explicitly test for
and confirm differences in the geographic and environmental ranges of
clonal genotypes.

Hybridization is expected to yield genotypes with increased phenotypic
plasticity, as they can access alleles from both parental species
(Shimizu-Inatsugi et al., 2017). Since high plasticity genotypes should
have increased ability to survive in multiple environments, hybrid
genotypes may thus be more likely to follow the ``general-purpose
genotypes'' strategy than either parental species (Baker, 1965;
Coughlan, Han, Stefanovi\selectlanguage{polish}ć, \selectlanguage{english}\& Dickinson, 2017; Lynch, 1984). Our
findings are consistent with this prediction in that the hybrid
coarse-textured ecotypes follow a ``general-purpose genotype'' pattern
while fine-textured plants follow a ``frozen niche'' pattern. This
indicates that hybridization may have induced a transition in clonal
propagation strategy in \emph{P. vaginatum.} Furthermore, within the
hybrid coarse-textured genotypes, allotriploidy may provide an
additional boost to plasticity relative to diploids as the two most
common and widespread genotypes are both triploids (Fig. 1). This is
consistent with the assumption that allopolyploids have access to even
more potentially adaptive alleles than diploid hybrids.

Differences in the rate of sexual reproduction may also influence clonal
propagation strategy. Recombination during sexual reproduction can
produce novel clonally propagating genotypes. Clones of the most fit
genotype in a given location will then dominate it, resulting in a
``frozen-niche'' pattern (Vrijenhoek \& Parker, 2009). Consistent with
this proposed strategy, fine-textured \emph{P. vaginatum} genotypes are
known to be capable of sexual reproduction in addition to vegetative
clonal propagation (Duncan \& Carrow 2000). Sexual reproduction of
coarse-textured ecotypes has not been tested; however, the low number of
unique genotypes indicate that it is likely rare (although the
presence of admixed individuals suggests that it is possible) (Fig. 3,
Fig. S3, Fig. S4). This lack of post-hybridization recombination could
make it difficult for coarse-textured plants to specialize in a given
location. Additionally, clonal propagation can serve to maintain
plasticity by preventing the loss of heterozygosity in hybrids. Both of
these factors could explain the ``general-purpose genotype'' pattern
seen in coarse-textured clones.

\textbf{Subgenome composition of hybrids and allopolyploids drives
genetic differentiation between groups}

Interspecific hybridization and gene flow can introduce alleles into a
population, which can potentially result in adaptation to novel
environmental conditions. Previous work identified apparent admixture
between salt-tolerant \emph{P. vaginatum} and non-tolerant \emph{P.
distichum} , which could produce genotypes adapted to intermediate
salinity levels (Eudy, Bahri, Harrison, Raymer, \& Devos, 2017). We
found that the two ecotypes of \emph{P. vaginatum} and \emph{P.
distichum} formed distinct genetic groups, with no signatures of gene
flow between \emph{P. vaginatum} and \emph{P. distichum} (Fig. 3, Fig.
S3), although we did identify limited gene flow between the coarse- and
fine-textured ecotypes of \emph{P. vaginatum} (Fig. S4)\emph{.} It is
possible that ploidy differences between \emph{P. distichum} (5x and 6x)
and \emph{P. vaginatum} (2x and 3x) represent a sufficient reproductive
barrier to prevent gene flow.

While we did not find evidence for interspecific hybridization in the
wild between \emph{P. distichum} and \emph{P. vaginatum} , both the
coarse-textured and \emph{P. distichum} groups appear to have been
formed from interspecific hybridization between fine-textured \emph{P.
vaginatum} and other \emph{Paspalum} species. Interestingly,
coarse-textured \emph{P. vaginatum} and \emph{P. distichum} do not
appear to share the same non-\emph{P. vaginatum} subgenome (Fig. S5).
This difference in subgenome composition is thus likely the result of
hybridization between \emph{P. vaginatum} and at least two
different\emph{Paspalum} species.

Niche differentiation between these two hybrid groups could be due to
the genomic contribution of these non-\emph{P. vaginatum} parents. To
our knowledge, no \emph{Paspalum} species is as salt-tolerant as\emph{P.
vaginatum} ; therefore, it is likely that the non-\emph{P. vaginatum}
parental species of both groups are not halophytic. Despite this,
coarse-textured \emph{P. vaginatum} largely maintained its high salt
tolerance while \emph{P. distichum} shows adaptations for freshwater
environments. Hypotheses for why this might have occurred include lower
expression of the \emph{P. vaginatum} subgenome in\emph{P. distichum}
relative to coarse-textured \emph{P. vaginatum} , differences in salt
tolerance between the non-\emph{P. vaginatum}parents, and
post-hybridization selection for freshwater adaptations in\emph{P.
distichum} .

\textbf{Subgenome composition but not ploidy is associated with local
ocean salinity}

Studies in other systems indicate that an increase in ploidy tends to be
associated with increased salt tolerance both within and between closely
related species (Chao et al., 2013; Wu et al., 2019; Yang et al., 2014).
However, when ploidy is considered alone, there is no detectable
difference in local ocean salinity between diploid and triploid \emph{P.
vaginatum} (Fig. 5). Instead, copy number of the \emph{P.
vaginatum}subgenome may be the driving factor. Gene dosage effects can
have a dramatic influence on phenotypes and increased copy number of
salt tolerance genes has been implicated in the evolution of salt
tolerance in halophytes (Oh, Dassanayake, Bohnert, \& Cheeseman, 2012).
Variation in gene copy number can also occur on a whole genome level in
populations of hybrids that vary in the number of subgenomes they
received from each parental species. Other studies have investigated
reciprocal allotriploid hybrids (e.g. 1:2 vs 2:1), similar to those we
describe here, which are often more similar to the parent from which
they received two copies (Betto-Colliard, Hofmann, Sermier, Perrin, \&
St\selectlanguage{ngerman}öck, 2018; Harvey, Fjelldal, Solberg, Hansen, \& Glover, 2017; Tan et
al., 2016; Yao, Gray, Auger, \& Birchler, 2013).

Our results support the hypothesis that the other unknown subgenome
present in the coarse-textured ecotype is not as adapted for saline
environments as is \emph{P. vaginatum} (Fig 5). Plants with two copies
of the \emph{P. vaginatum} subgenome (fine-textured, and 2:1 triploids)
were found in locations where the local ocean salinity was on average
\textasciitilde{}15\% higher than those with only one copy (1:1 diploids
and 1:2 triploids). The effect of increasing counts of the unknown
subgenome was weaker and in the opposite direction (slightly less ocean
salinity with each copy). We cannot say for certain whether this weaker
effect is due to increased ploidy or subgenome dosage effects, or both.
If it is a ploidy effect then it is the opposite of predictions that
polyploids should be more salt tolerant.

The large effect of the number of \emph{P. vaginatum} subgenomes is
consistent with our finding that coarse-textured hybrids are likely
``general-purpose genotypes'' because their local ocean salinity is not
only lower, but also more variable. Furthermore, unlike most other
studies of reciprocal triploids, we can rule out maternal subgenome
effects because all of the coarse-textured hybrids carry the same cpDNA
haplotype, implying the same maternal parent species for each cross.

Although the trend we observe between \emph{P. vaginatum} subgenome copy
number and local salinity is striking, environmental associations are
necessarily correlational in nature and must be confirmed with
controlled salt tolerance assays and transcriptomics. It is also
possible that unknown demographic or introduction history could
contribute to these patterns. Additionally, our sea surface salinity
dataset does not have the resolution to identify the microhabitat
variation likely experienced by plants in the wild (for example, in an
estuary with a steep salinity gradient). While higher resolution
salinity data exist for some coastal areas, temporal and methodological
variation between datasets make them unsuited for comparison across the
geographic scale of this study.

\textbf{Implications for plant breeding}

Most of our knowledge of the genetic and physiological underpinnings of
salt tolerance comes from crop species and plant model species that are
generally not salt tolerant, with limited studies of highly
salt-tolerant plant species. Additionally, studies on the genetic basis
of salt tolerance in halophytes have been limited to a small number of
species, few of which are grasses (Fan, 2020; Mishra \& Tanna, 2017).
This is problematic because soil salinity affects agricultural fields
worldwide, and breeding for increased tolerance in cereal crops, which
are all grasses, has been slow. Understanding the mechanisms and genes
involved in salt tolerance in halophytic grass species may allow us to
transfer that knowledge to develop more salt tolerant cereals. Our
finding that salt tolerance in the wild may be associated with
whole-genome dosage effects indicates that a single gene approach may be
insufficient and that changes to copy number or expression of multiple
interacting genes and pathways may be the key to improving crop salt
tolerance.

Breeding programs for seashore paspalum turfgrass have been hampered by
the lack of genetic diversity in publicly available germplasm, as
confirmed in this study. The apparent presence of natural crosses
between fine and coarse-textured ecotypes points to the possibility of
introgression of useful traits from coarse-textured populations. This
could provide a valuable source of genetic diversity in turf breeding
programs for the genetically depauperate, but more economically
relevant, fine-textured ecotypes. Future work to identify these traits
and genetic markers associated with them in the coarse-textured ecotypes
will be required.

\textbf{Acknowledgments}

We thank Mike Dyer, Sally Fabbri, Aileen Wolk and the rest of the
greenhouse staff at WUSTL and the DDPSC for providing plant care; Paul
Raymer (University of Georgia Griffin) for providing collection advice;
Doug Eudy (Bayer Crop Science) for useful discussions in the early
stages of the project; Pablo Speranza for sending tissue from Uruguay;
Jeremey Schmutz and the assembly team at JGI for providing the reference
genome; and, Linda Small for performing cpDNA amplification. This work
was funded by the United States Golf Association (project 2016-35-605).
DMG has been supported by the William H. Danforth Plant Science
Fellowship.

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\textbf{Data Accessibility}

Demultiplex and trimmed reads will be submitted to NCBI SRA before
publication. The \emph{rpl16} cpDNA sequences are available on GenBank
(accessions MT361650- MT361665). SNP datasets and snapshots of code used
in the project will be submitted to dryad. Future development of scripts
will occur at https://github.com/david-goad/paspalum-hybridization/.

\textbf{Author Contributions}

DMG collected plants, performed lab work, analyzed data, generated
figures, and drafted the manuscript. KMO aligned cpDNA sequences. All
authors contributed to designing the study, data analysis and
interpretation, writing grant proposals and writing the manuscript.



\textbf{Supporting Information}

Additional supporting information may be found in the on-line version of
this article.

\textbf{Table S1} Information for all samples including collection
location, taxonomic and genotype labels, and data used to infer ploidy

\textbf{Table S2} . Polymorphic sites in the intron of the chloroplast
gene \emph{rpl16} . Positions are based on alignment to GenBank
accession KF853027 (Scataglini et al. 2014).

\textbf{Figure S1} Histogram of pairwise genetic distances between
samples within (\textbf{A} ) fine-textured \emph{P. vaginatum} ,
(\textbf{B} ) Coarse-textured \emph{P. vaginatum} , and (\textbf{C}
)\emph{P. distichum} . Dotted red line indicates distance threshold for
identifying clonal genotypes.

\textbf{Figure S2} (\textbf{A} ) The basic workflow of
triploid\_comp.py. (\textbf{B} ) An illustration of how
triploid\_comp.py counts reads at a locus for a hypothetical triploid
genotype. Because GBS reads start at the same location due to the
restriction cut site, reads representing each allele at a locus are
expected to be identical to each other. Therefore, reads not identical
to the most common two read types are considered to be sequencing errors
and ignored (i.e. not included in calculating ratios or percentages of
reference reads). Due to the \textasciitilde{}2:1 ratio of reference
reads this hypothetical example could represent a triploid with two
haploid copies of \emph{the P. vaginatum} subgenome and one from the
unknown progenitor; however, a single locus could have a skewed ratio
due to chance so it is necessary to look at the trend genome wide to
make a definitive assessment.

\textbf{Figure S3} Principal component analysis (PCA) showing the
genetic separation of all sampled \emph{Paspalum} subgroup Disticha
accessions into three well defined groups as well as three admixed
individuals (two of which are clonal and overlap). The color of each
individual indicates its genome-wide heterozygosity rate (reported as
the individual inbreeding coefficient F). Darker shades of blue indicate
lower heterozygosity.

\textbf{Figure S4} ADMIXTURE plots showing population assignment of 33
unique diploid \emph{P. vaginatum} genotypes at K=2 (\textbf{A} ) and
K=3 (\textbf{B} ). Putative fine-coarse admixed individuals are
indicated with stars. (\textbf{C} ) Cross-validation error for K = 1-7.

\textbf{Figure S5} Number of SNP markers where heterozygous calls are
unique to given group or shared between groups. The relatively small
number of loci that are heterozygous in both coarse-textured \emph{P.
vaginatum} and \emph{P. distichum} but not fine-texture
\emph{P.vaginatu} m (light blue region) indicates that \emph{P.
distichum}and coarse-textured \emph{P. vaginatum} likely do not share a
non-\emph{P. vaginatum} subgenome.

\textbf{Figure S6} The raw counts of the two most common reads (where
one is identical to the reference and the other is alternate) covering
heterozygous SNPs in four coarse-textured \emph{P. vaginatum} samples
representing the most common diploid hybrid genotype (coarse\_03,
represented by DG076) (\textbf{A} ) and the three triploid genotypes:
coarse\_01(accession DG117) (\textbf{B)} , coarse\_02 (accession DG107)
(\textbf{C} ) and coarse\_04 (accession DG120) (\textbf{D)} . Red lines
indicate expected ratio of reference to alternate reads for diploids at
1:1 and reciprocal triploids at 1:2 and 2:1. A bias toward either 1:2 or
2:1 in the triploids reflects having one or two haploid copies of
the\emph{P} . \emph{vaginatum} subgenome respectively.

\textbf{Tables and Figures}

\textbf{Table 1} Inferred clonality and ploidy variation for
sampled\emph{Paspalum} accessions. Relative genome size estimates were
calculated assuming a diploid genome size of 2x = 1.0 for the \emph{P.
vaginatum} reference genome (accession PI 509022); values in brackets
indicate relative genome size estimates for \emph{P. distichum} assuming
a basal genome size that is 11\% smaller than the \emph{P.
vaginatum}reference genome.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Species} & \textbf{Ecotype} & \textbf{Esti-mated ploidy} &
\textbf{No. accessions sampled} & \textbf{No. unique geno-types} &
\textbf{Relative genome size (N\textsuperscript{+})} & \textbf{Average
F\textsuperscript{++}}\tabularnewline
\midrule
\endhead
\emph{P. vaginatum} & & & & & &\tabularnewline
& Fine-textured & 2x & 55 & 24 & 1.01 (2) & 0.541\tabularnewline
& Coarse-textured & 2x\selectlanguage{ngerman}\textsuperscript{§} & 32 & 9 & 1.04 (9) &
-0.278\tabularnewline
& & 3x & 116 & 3 & 1.56 (3) & -0.520\tabularnewline
\emph{P. distichum} & & 5x & 9 & 2 & 2.25 {[}2.53{]} (2) &
-1.241\tabularnewline
& & 6x & 6 & 2 & 2.69 {[}3.03{]} (2) & -1.323\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{+} N = Number of accessions measured with three
replicates per accession

\textsuperscript{++} F = individual inbreeding coefficient (Purcell et
al. 2007); more negative values indicate higher genome-wide
heterozygosity.

\selectlanguage{ngerman}\textsuperscript{§}. Includes three putative coarse/fine-textured
admixed samples (see Fig. 2 and Table S1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} (\textbf{A} ) Map of US collections of
\emph{Paspalum}group Disticha accessions in the southeastern United
States. Colors indicate major genetic subgroups and ploidy variants.
Shapes indicate geographical locations of the most common clonal
genotypes (detected in 6 or more accessions). (\textbf{B} ) From left to
right: \emph{P. distichum,} fine-textured \emph{P. vaginatum} and
coarse-textured

\emph{P. vaginatum.}

\emph{}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/351065/articles/475745-hybridization-polyploidy-and-clonality-influence-geographic-patterns-of-diversity-and-salt-tolerance-in-the-model-halophyte-seashore-paspalum-paspalum-vaginatum}

\textbf{Figure 2} Distributions of pairwise geographic (\textbf{A} ) and
environmental (\textbf{B} ) distances between collection locations of
clones from each clonally propagating \emph{P. vaginatum} genotype. A
single dot indicates that there is no variation in pairwise distance for
that genotype. Unique genotype IDs indicate whether a genotype is
coarse-textured or fine-textured. The number of each genotype collected
in the wild (with GPS coordinates) is indicated by n. Color indicates
ploidy level.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/351065/articles/475745-hybridization-polyploidy-and-clonality-influence-geographic-patterns-of-diversity-and-salt-tolerance-in-the-model-halophyte-seashore-paspalum-paspalum-vaginatum}

\textbf{Figure 3} Principal component analysis (PCA) of
\emph{Paspalum}group Disticha accessions based on a single sample from
each clonal group. Three well defined groups are formed as well as two
putatively admixed individuals. The color of each individual indicates
its genome-wide heterozygosity rate (reported as the individual
inbreeding coefficient F). Darker shades of blue indicate lower
heterozygosity.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/351065/articles/475745-hybridization-polyploidy-and-clonality-influence-geographic-patterns-of-diversity-and-salt-tolerance-in-the-model-halophyte-seashore-paspalum-paspalum-vaginatum}

\textbf{Figure 4} Distribution of the percent of reads that are
identical to the \emph{P. vaginatum} reference genome at heterozygous
loci in four coarse-textured \emph{P. vaginatum} samples representing
the most common diploid hybrid genotype (coarse\_03, represented by
DG076) (\textbf{A} ) and the three triploid genotypes: coarse\_01
(accession DG117) (\textbf{B)} , coarse\_02 (accession DG107)
(\textbf{C} ) and coarse\_04 (accession DG120) (\textbf{D)} . Bars in
the top right corner indicate the number of haploid subgenomes from
\emph{P. vaginatum} (black) or an unidentified progenitor species
(grey).

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/351065/articles/475745-hybridization-polyploidy-and-clonality-influence-geographic-patterns-of-diversity-and-salt-tolerance-in-the-model-halophyte-seashore-paspalum-paspalum-vaginatum}

\textbf{Figure 5} Local ocean salinity in practical salinity units (PSU)
for each of our wild \emph{P. vaginatum} collections based on subgenome
composition. Black and grey bars represent copy number of haploid
subgenomes from \emph{P. vaginatum} (N\textsubscript{Pv}) and the
unknown progenitor species (N\textsubscript{other}) respectively. Boxes
indicate mean and standard error. Color indicates ploidy.\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Fig1/Fig1}

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\includegraphics[width=0.70\columnwidth]{figures/Fig2/Fig2}

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\includegraphics[width=0.70\columnwidth]{figures/Fig4/Fig4}

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