Assessment of intra and interregional genetic variation in the Eastern Red-backed Salamander, Plethodon cinereus, via analysis of novel microsatellite markers

Abstract

The red-backed salamander (Plethodon cinereus) has long-served as a model system in ecology, evolution, and behavior, and studies surveying molecular variation in this species have become increasingly common over the past decade. However, difficulties are commonly encountered when extending microsatellite markers to populations that are unstudied from a genetic perspective due to high levels of genetic differentiation across this species’ range. To ameliorate this issue, we used 454 pyrosequencing to identify hundreds of microsatellite loci. We then screened 40 of our top candidate loci in populations in Virginia, Pennsylvania, and Ohio—including an isolated island population ~ 4.5 km off the shore of Lake Erie (South Bass Island). We identified 25 loci that are polymorphic in a well-studied region of Virginia and 11 of these loci were polymorphic in populations located in the genetically unstudied regions of Ohio and Pennsylvania. Use of these loci to examine patterns of variation within populations revealed that South Bass Island has low diversity in comparison to other sites. However, neither South Bass Island nor isolated populations around Cleveland are inbred. Assessment of variation between populations revealed three well defined genetic clusters corresponding to Virginia, mainland Ohio/Pennsylvania, and South Bass Island. Comparisons of our results to those of others working in various parts of the range are consistent with the idea that differentiation is lower in regions that were once glaciated. However, these comparisons also suggest that well differentiated isolated populations in the formerly glaciated portion of the range are not uncommon. This work provides novel genetic resources that will facilitate population genetic studies in a part of the red-backed salamander’s range that has not previously been studied in this manner. Moreover, this work refines our understanding of how neutral variation is distributed in this ecologically important organism.

Genetic variation in Plethodon cinereus and Plethodon hubrichti from in and around a contact zone

“…In addition, every method we employed indicated that differentiation between populations across the contact zone was greater for P. hubrichti than for P. cinereus . Elsewhere in Virginia, P. cinereus displays much higher levels of genetic diversity in microsatellite alleles and higher differentiation among populations over similar spatial scales (Cabe et al., 2007; Cameron et al., 2017). The comparatively low levels of diversity and differentiation for P. cinereus at our study sites are consistent with the hypothesis that these populations represent a relatively recent range extension of P. cinereus into areas formerly occupied by P. hubrichti .…”

“…The microsatellite loci developed from P. cinereus by Cameron, Anderson, and Page (2017) were screened for cross‐amplification in P. hubrichti , and eight loci (Pc4, Pc7, Pc15, Pc17, Pc20, Pc22, Pc28, and Pc37) that reliably amplified were identified. Genotyping reactions followed the methodology outlined in Cameron et al.…”

Genetic Analysis of a Cryptic Contact Zone between Mitochondrial Clades of the Eastern Red-Backed Salamander, Plethodon cinereus

“…We also amplified 10 microsatellite loci (Cameron et al, 2017). The names of loci and PCR procedures are detailed in Appendixes 3 and 4.…”

Do genetic structure and landscape heterogeneity impact color morph frequency in a polymorphic salamander?

“…Genomic DNA was extracted using Qiagen DNeasy tissue kits following the manufacturer's protocol. We amplified 10 microsatellite loci, with tri-pentanucleotide repeat motifs, developed for P. cinereus (Pc3, Pc7, Pc15-Pc17, Pc25, Pc28, Pc30, Pc34, Pc37; Cameron et al 2017) using PCR. Forward primers were modified with a 5′ florescent tag of 6-FAM, NED, HEX, PET or ATTO 565 (NHS Ester), and were multiplexed in an arrangement of 3-4 loci.…”