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; Róis 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, Stefanović, & 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-Tévar, 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 (Paspalum vaginatumSwartz) 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 (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, Paspalum vaginatumalso provides the benefits of a genome-enabled model system with
experimental tractability and economic value that facilitate applied
studies. It has a manageably-sized ~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) P. vaginatumgermplasm 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 P. vaginatum and 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 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 P. vaginatum , and lay
the necessary groundwork for future investigations into the genetic
basis of salt tolerance in this genomic model halophytic grass species.