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Novel chloroplast markers for the study of intraspecific variation and hybridisation in the Lepidosperma costale species complex (Cyperaceae)

DOI:10.1007/s12686-010-9361-8
Authors:
Mark J Wallace at Australian National University
Mark J Wallace
Matthew D. Barrett at James Cook University
Matthew D. Barrett
Russell L Barrett at Royal Botanic Gardens
Russell L Barrett
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Abstract

To investigate intraspecific variation and hybridisation in the Lepidosperma costale species complex, forty-one primers to amplify regions containing chloroplast microsatellites and insertions/deletions were designed using de novo sequencing. Twenty-six loci were polymorphic within three diploid populations, with 2 to 9 alleles per locus. A further 15 loci exhibited fixed size differences between the diploid individuals and a population of putative allopolyploid origin. These markers will assist studies of genetic diversity and hybridisation in Lepidosperma, help inform the conservation of taxa in the Lepidosperma costale species complex, and may have utility across the Cyperaceae. KeywordsChloroplast microsatellite–cpSSR–Schoeneae
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TECHNICAL NOTE
Novel chloroplast markers for the study of intraspecific variation
and hybridisation in the Lepidosperma costale species complex
(Cyperaceae)
Mark J. Wallace •Matthew D. Barrett •
Russell L. Barrett
Received: 20 November 2010 / Accepted: 25 November 2010 / Published online: 12 December 2010
ÓSpringer Science+Business Media B.V. 2010
Abstract To investigate intraspecific variation and
hybridisation in the Lepidosperma costale species com-
plex, forty-one primers to amplify regions containing
chloroplast microsatellites and insertions/deletions were
designed using de novo sequencing. Twenty-six loci were
polymorphic within three diploid populations, with 2 to 9
alleles per locus. A further 15 loci exhibited fixed size
differences between the diploid individuals and a popula-
tion of putative allopolyploid origin. These markers will
assist studies of genetic diversity and hybridisation in
Lepidosperma, help inform the conservation of taxa in the
Lepidosperma costale species complex, and may have
utility across the Cyperaceae.
Keywords Chloroplast microsatellite cpSSR
Schoeneae
Chloroplast DNA sequences are useful in evolutionary
studies of plants because they are generally uniparentally
inherited and non-recombinant (Provan et al. 2001); how-
ever, observed nucleotide substitution rates are too low for
most intraspecific studies (Shaw et al. 2007). Conse-
quently, investigations of within-species variation rely on
highly polymorphic regions such as chloroplast microsat-
ellite markers, also known as simple sequence repeats
(cpSSRs). These markers, which usually consist of mono-
nucleotide repeats, have shown utility in investigations of
population genetics and hybridisation (Provan et al. 2001).
Chloroplast microsatellite markers are currently enjoying
a revival, in part due to the many whole-chloroplast
sequences now available on GenBank (156 at the time of
writing; www.ncbi.nlm.nih.gov/genbank), which simplify
cpSSR discovery (Ebert and Peakall 2009a). For little-stud-
ied wild species without commercial congeners or pre-
existing sequence information, however, de novo sequencing
is still the most efficient way to develop cpSSRs (Ebert and
Peakall 2009a). Recently, a set of universal primers to
amplify noncoding regions of chloroplast DNA was pub-
lished (Ebert and Peakall 2009b). Here, we use these uni-
versal primers to identify and characterise polymorphic
chloroplast markers for the Lepidosperma costale Nees
species complex, a group of sedges of ecological and con-
servation importance in Western Australia.
All members of the genus Lepidosperma Labill. (Schoe-
neae: Cyperaceae) are long-lived, wind-pollinated sedges.
The Lepidosperma costale species complex currently
includes three named species: L. costale,L. benthamianum
C.B.Clarke, and L. gibsonii R.L.Barrett; however, precise
species boundaries are still unclear. The species complex
occurs in southwest Australia, an area recognized as a world
biodiversity hotspot due to the high number of endemic
species in an area suffering considerable habitat loss (Myers
et al. 2000). Habitat loss is particularly acute for populations
that occur on banded ironstone formations because several of
these formations will be largely removed for mining in the
near future (Gibson et al. 2007); L. gibsonii, which occurs
almost exclusively on a single banded ironstone formation, is
currently recognised as vulnerable to extinction under the
guidelines of the IUCN (2001), and is listed as Declared Rare
M. J. Wallace (&)M. D. Barrett R. L. Barrett
Botanic Gardens and Parks Authority, Kings Park and Botanic
Garden, West Perth, WA 6005, Australia
e-mail: mark.wallace@bgpa.wa.gov.au
M. J. Wallace M. D. Barrett R. L. Barrett
School of Plant Biology, University of Western Australia,
Nedlands, WA 6009, Australia
123
Conservation Genet Resour (2011) 3:355–360
DOI 10.1007/s12686-010-9361-8
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... ). Although not yet formally described, these two species are placed in different phylogenetic clades and are clearly differentiated by leaf morphology (Barrett, Barrett & Wallace, 2010). There have been no empirical studies of their ecology or mating systems to date and we did not observe any viable seed set in the two years of this study to enable our own investigations. ...
... We trialled chloroplast microsatellite markers developed for Lepidosperma costale (Wallace, Barrett & Barrett, 2010) and identified six loci (LepCt02a, 02b, 07a, 23c, 24a, 27a) that were transferable to both species in this study. Amplification was performed in 15 μL reactions containing 50 mM KCl, 20 mM Tris- HCl (pH 8.4), 0.2 mM each dNTP, 2.5 mM MgCl2, 0.53 mM fluorescently labelled forward primer, 0.53 mM reverse primer, 0.5 U Taq DNA polymerase and 10 ng template DNA. ...
... Other explanations for the maintenance of genetic diversity seem unlikely. Hybridization between the two study species (Ellis et al., 2006) is unlikely, given that they occur in different phylogenetic clades (Barrett et al., 2010) and we found no chloroplast haplotypes in common; however, we cannot rule out hybridization with other sympatric Lepidosperma. Recent rarity (Ellis et al., 2006) is also unlikely given that we found no evidence for recent bottlenecks in the nuclear data and, as discussed below, patterns of chloroplast diversity suggest a long history of rarity for these species. ...
Not all rare species are the same: Contrasting patterns of genetic diversity and population structure in two narrow-range endemic sedges
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The many spatial and temporal configurations in which species can be classified as rare may result in various genetic signatures, despite a persistent generalization that populations of rare species are genetically depauperate and highly differentiated. We assessed genetic diversity and differentiation in two narrow endemics with contrasting geographical distributions using 12 nuclear and six chloroplast microsatellite loci. Consistent with both marker types, the smaller, more isolated Lepidosperma sp. Parker Range populations were characterized by lower diversity and stronger divergence, relative to higher diversity and extensive connectivity among the geographically clustered L. sp. Mt Caudan populations. However, neither species exhibited low diversity, despite high inbreeding. Together, our results suggest that these species are naturally rare and have long persisted in this landscape while maintaining genetic diversity and tolerating considerable inbreeding. Their resilience is probably due to large population sizes and the flexibility afforded by employing sexual and asexual reproduction. Their contrasting genetic dynamics demonstrate that not all rare species share generalized patterns, even within the same rarity category. Moreover, these patterns were better predicted by fine-scale descriptors of rarity, population size and distribution, rather than the more typically used geographical range. This study highlights the complex dynamics of rare species and cautions against using broad assumptions to classify and manage rare species. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ●●, ●●–●●.
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... .wa.gov.au). Although not yet formally described, these two species are placed in different phylogenetic clades and are clearly differentiated by leaf morphology (Barrett, Barrett & Wallace, 2010). There have been no empirical studies of their ecology or mating systems to date and we did not observe any viable seed set in the two years of this study to enable our own investigations. ...
... We trialled chloroplast microsatellite markers developed for Lepidosperma costale (Wallace, Barrett & Barrett, 2010) and identified six loci (LepCt02a, 02b, 07a, 23c, 24a, 27a) that were transferable to both species in this study. Amplification was performed in 15 μL reactions containing 50 mM KCl, 20 mM ...
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For plants with mixed reproductive capabilities, asexual reproduction is more frequent in rare species and is considered a strategy for persistence when sexual recruitment is limited. We investigate whether asexual reproduction contributes to the persistence of two co-occurring, rare sedges that both experience irregular seed set and if their differing geographic distributions have a role in the relative contribution of clonality. Genotypic richness was high (R=0.889±0.02) across the clustered populations of Lepidosperma sp. Mt Caudan and, where detected, clonal patches were small, both in ramet numbers (⩽3 ramets/genet) and physical size (1.3±0.1 m). In contrast, genotypic richness was lower in the isolated L. sp. Parker Range populations, albeit more variable (R=0.437±0.13), with genets as large as 17 ramets and up to 5.8 m in size. Aggregated clonal growth generated significant fine-scale genetic structure in both species but to a greater spatial extent and with additional genet-level structure in L. sp. Parker Range that is likely due to restricted seed dispersal. Despite both species being rare, asexual reproduction clearly has a more important role in the persistence of L. sp. Parker Range than L. sp. Mt Caudan. This is consistent with our prediction that limitations to sexual reproduction, via geographic isolation to effective gene exchange, can lead to greater contributions of asexual reproduction. These results demonstrate the role of population isolation in affecting the balance of alternate reproductive modes and the contextual nature of asexual reproduction in rare species.Heredity advance online publication, 15 April 2015; doi:10.1038/hdy.2015.32.
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... Although the present study has provided a useful guide to where germplasm of equivalent ploidy can be sourced, we recommend screening germplasm using the methods described herein, to ensure that the targeted ploidy is collected. We are continuing to study the evolutionary history of the L. costale complex using nuclear and chloroplast markers (Wallace et al. 2011;, to explore patterns of genetic diversity, and guide conservation and restoration. ...
DNA ploidy variation and distribution in the Lepidosperma costale complex (Cyperaceae): Implications for conservation and restoration in a biodiversity hotspot
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Intraspecific ploidy variation is an important component of angiosperm biodiversity; however, this variation is rarely considered in conservation programs. This is of particular concern when conservation activities include augmentation, reintroduction or ecological restoration because there are potentially negative consequences when ploidy variants are unintentionally mixed within populations. We surveyed regional ploidy variation in the Lepidosperma costale Nees species complex (Schoeneae: Cyperaceae) in the South West Australian Floristic Region, an international biodiversity hotspot. Several L. costale sensu lato populations are threatened by iron-ore extraction, including the rare L. gibsonii R.L.Barrett, and these populations are the subject of ecological restoration programs. The DNA ploidy of 2384 individuals from 28 populations across the range of the species complex was determined and four DNA ploidy levels were discovered, namely, diploid, triploid, tetraploid and pentaploid. Diploids and tetraploids were the most common cytotypes and were largely geographically segregated, even at an exhaustively studied contact zone. Triploids were found at a low frequency in two populations. The rarity of triploids suggests substantial interploidy sterility, and that mixing of ploidy variants should, therefore, be avoided when restoring L. costale s.l. populations. These data provide a guide for L. costale s.l. germplasm collection and suggest that polyploidy may be an important driver of diversification in these sedges.
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... The name L. costale Nees has been applied to a large number of distinct taxa in Western Australia. Even when restricted to the complex of species related to the type specimen, complex genetic processes, including polyploidy and hybridisation, make matching the type specimen as closely as possible to an extant population critical (Wallace et al. 2011;Wallace et al. in prep.). ...
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Many species of Lepidosperma Labill. have been poorly circumscribed, resulting in significant nomenclatural confusion. Full descriptions of six species based solely on type specimens held in the Webb Herbarium in Florence (FI-W) and the Preiss Herbarium in Lund (LD) are presented here in order to facilitate accurate application of the names involved. Descriptions are provided for Lepidosperma costale Nees, L. gladiatum Labill., L. fimbriatum Nees, L. humile (Nees) Boeck., L. longitudinale Labill. and L. squamatum Labill., as well as notes about these species.
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... The name L. costale Nees has been applied to a large number of distinct taxa in Western Australia. Even when restricted to the complex of species related to the type specimen, complex genetic processes, including polyploidy and hybridisation, make matching the type specimen as closely as possible to an extant population critical (Wallace et al. 2011; Wallace et al. in prep.). Two names, L. fimbriatum Nees and L. humile (Nees) Boeck. ...
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... The 10 microsatellite loci reported here are highly variable and are currently being used to investigate population diversity, clone size, and dispersal in L. gibsonii and other Lepidosperma species in southwestern Australia. The successful amplifi cation of markers in sampled Lepidosperma species indicate that these markers can be usefully applied across the genus and will be used in concert with chloroplast microsatellite markers we have recently developed ( Wallace et al., 2011 ). ...
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