Microsatellite discovery in an insular amphibian (Grandisonia alternans) with comments on cross-species utility and the accuracy of locus identification from unassembled Illumina data

Abstract

The Seychelles archipelago is unique among isolated oceanic islands because it features an endemic radiation of caecilian amphibians (Gymnophiona). In order to develop population genetics resources for this system, we identified microsatellite loci using unassembled Illumina MiSeq data generated from a genomic library of Grandisonia alternans, a species that occurs on multiple islands in the archipelago. Applying a recently described method (PALFINDER) we identified 8001 microsatellite loci that were potentially informative for population genetics analyses. Of these markers, we screened 60 loci using five individuals, directly sequenced several amplicons to confirm their identity, and then used eight loci to score allele sizes in 64 G. alternans individuals originating from five islands. A number of these individuals were sampled using non-lethal methods, demonstrating the efficacy of non-destructive molecular sampling in amphibian research. Although two loci satisfied our criteria as diploid, neutrally evolving loci with the statistical power to detect population structure, our success in identifying reliable loci was very low. Additionally, we discovered some issues with primer redundancy and differences between Illumina and Sanger sequences that suggest some Illumina-inferred loci are invalid. We investigated crossspecies utility for eight loci and found most could be successfully amplified, sequenced and aligned across other species and genera of caecilians from the Seychelles. Thus, our study in part supported the validity of using PALFIN-DER with unassembled reads for microsatellite discovery within and across species, but importantly identified major limitations to applying this approach to small datasets (ca. 1 million reads) and loci with small tandem repeat sizes.

Endemic, endangered and evolutionarily significant: cryptic lineages in Seychelles’ frogs (Anura: Sooglossidae)

“…Recently identified phylogeographic patterns within the archipelago's endemic herpetofauna have revealed a variety of geographic correlations: skinks and geckos broadly differentiate into northern (Praslin and surrounding islands) and southern (Mahé, Silhouette, and surrounding islands) groups (Rocha et al, 2010;Rocha et al, 2011;Valente et al, 2013;Rocha et al, 2016a;Rocha et al, 2016b); while for the nonsooglossid anurans, a distinct lack of variability is shown in the multi-distributed treefrog Tachycnemis seychellensis (Maddock et al, 2014), conflicting with observed structuring in Seychelles endemic caecilians (Adamson et al, 2016;Maddock et al, 2016;Maddock et al, 2017). Our mtDNA analyses appear to confirm the relationship posited by Taylor et al (2012), namely that Silhouette and Praslin populations of So.…”

The roles of vicariance and isolation by distance in shaping biotic diversification across an ancient archipelago: evidence from a Seychelles caecilian amphibian

“…Despite the key attributes of the Seychelles system such as its continental origin, truly oceanic setting, and mosaic of ancient (including Gondwanan relictual) lineages and more recent overwater arrivals that make it attractive for studying evolutionary processes, the Seychelles biota remains poorly studied at the intraspecific level. Greater use of molecular techniques has underpinned a recent increase in the number of studies of phylogeographic patterns in the Seychelles [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48]. These studies have documented some common e.g.…”

The roles of vicariance and isolation by distance in shaping biotic diversification across an ancient archipelago: evidence from a Seychelles caecilian amphibian

“…Despite the key attributes of the Seychelles system such as its continental origin, truly oceanic setting, and mosaic of ancient (including Gondwanan relictual) lineages and more recent overwater arrivals that make it attractive for studying evolutionary processes, Seychelles biota remain poorly studied at the intraspecific level. Recently, through greater use of molecular techniques, studies of phylogeographic patterns in the Seychelles have increased (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). Previous findings have uncovered some common (e.g.…”