Characterization of 10 microsatellite markers for the southern torrent salamander ( Rhyacotriton variegatus )
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MICROSATELLITE LETTERS
Characterization of 10 microsatellite markers for the southern torrent salamander (Rhyacotriton variegatus) Sarah L. Emel • Andrew Storfer
Received: 5 May 2014 / Accepted: 19 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Rhyacotriton variegatus is an endemic salamander species with specific habitat requirements and a restricted geographic range in the northwestern United States. Although not currently listed at the state or federal level, R. variegatus is vulnerable to habitat loss from timber harvest. We isolated and characterized 10 polymorphic microsatellite loci among 367 individuals from across the species’ range. The number of alleles per locus ranged from 7 to 16, observed heterozygosity ranged from 0.16 to 0.58, and expected heterozygosity ranged from 0.53 to 0.87. These new loci will assist in examining the genetic diversity and structure of R. variegatus and potentially related species. Keywords Amphibian Rhyacotriton variegatus Microsatellite PCR primers Genetic diversity The southern torrent salamander, Rhyacotriton variegatus, has a restricted geographic range in coastal Oregon and California, United States. Larval and adult R. variegatus are associated with old-growth forests and highly oxygenated headwater streams (Welsh and Lind 1996). These specific requirements make the species vulnerable to habitat loss due to timber harvest. Although attempts to list R. variegatus at the state or federal level have failed largely due to lack of information about population and habitat status, it is considered a Species of Special Concern in Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0230-8) contains supplementary material, which is available to authorized users. S. L. Emel (&) A. Storfer School of Biological Sciences, Washington State University, PO Box 644236, Pullman, WA 99164-4236, USA e-mail: [email protected]
California and denoted as Vulnerable on the Oregon Sensitive Species List (Lannoo 2005). We sent 20 tissue samples of R. variegatus from 12 populations to ecogenics GmbH to generate an enriched microsatellite library and test for polymorphism. Size selected fragments were enriched for microsatellite content using magnetic streptavidin beads and biotin-labeled repeat oligonucleotides (di-, tri-, and tetranucleotide repeats). This enriched library was analyzed on a Roche 454 platform using GS FLX titanium reagents. A total of 63,618 reads had an average length of 167 base pairs. Microsatellite inserts with a tri- or tetranucleotide of at least 6 repeat units or a dinucleotide with at least 10 repeat units were found in 2,050 of these reads. Suitable primer design was possible in 569 reads, of which 61 were tested for polymorphism. These tests revealed 10 readable, polymorphic loci (Table 1). We assessed the variability of the 10 loci in 367 tissue samples collected in the form of tail clips across 19 sampling localities in both the northern and southern portions of the R. variegatus range. DNA was extra
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