First Large-Scale DNA Barcoding Assessment of Reptiles in the Biodiversity Hotspot of Madagascar, Based on Newly Designed COI Primers

Abstract

BackgroundDNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar.Methodology/Principal FindingsRepresentatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7–100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41–48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages. Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family.Conclusions/SignificanceThe new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge.

DNA barcoding assessment of genetic variation in two widespread skinks from Madagascar, Trachylepis elegans and T. gravenhorstii (Squamata: Scincidae)

“…We do not suggest interpreting mitochondrial non-monophyly of T. gravenhorstii and T. elegans as indicative of these taxa containing cryptic species, given that the multi-gene phylogeny of Lima et al (2013) identified each of these two species as monophyletic, in a multi-gene analysis that included four nuclear genes. In fact, our results highlight the need for caution when relying on pure DNA barcode analysis, as the p-distances calculated between the main subclades of these species (ranging from 6.3-14.5%) are well above the species-level threshold established for scincid species (6.1%, Nagy et al 2012), but it is unlikely that each represents a distinct species.…”

“…We used the reptile-specific primers RepCOI-F (TNT TMT CAA CNA ACC ACA AAG A) and RepCOI-R (ACT TCT GGR TGK CCA AAR AAT CA) of Nagy et al (2012) to amplify a COI fragment of 612 bp. The thermocycling profile comprised an initial denaturation at 94°C for 2:20 min, followed by 35 cycles of denaturation (94°C for 30 s), annealing (45-49°C for 45 s), elongation (72°C for 90 s), and a final elongation step at 72°C for 10 min.…”

The conservation status of the world’s reptiles

“…A total of 9,084 species of reptiles have been described so far (Uetz, 2010), and new molecular evidence continues to unearth numerous cryptic species that had not previously been detected by morphological analyses (e.g., Adalsteinsson et al, 2009;Nagy et al, 2012;Oliver et al, 2009). Yet as a group, reptiles are currently poorly-represented on the IUCN Red List of Threatened Species, with only 35% of described species evaluated, and those that are evaluated were done so in a non-systematic manner (IUCN, 2011a).…”

A new species of nocturnal gecko (Paroedura) from karstic limestone in northern Madagascar

“…To obtain molecular comparisons of the new species with previously unstudied nominal Paroedura species, we sequenced a fragment of the mitochondrial gene for cytochrome oxidase subunit I (cox1) with primers and protocols defined by Nagy et al (2012) for several Paroedura samples from Tsingy de Namoroka (corresponding voucher specimens to be catalogued in MNHN). These samples have become available through a recent herpetological survey by one of us (II) and include topotypical P. karstophila, previously unstudied from a molecular perspective.…”

“…These samples have become available through a recent herpetological survey by one of us (II) and include topotypical P. karstophila, previously unstudied from a molecular perspective. The resulting sequences were combined with those of Nagy et al (2012) and Koubová et al (2014) in MEGA version 5 (Tamura et al 2011) to yield an alignment of 664 bp. We performed phylogenetic inference in MEGA under the Maximum Likelihood optimality criterion, with a general time-reversible substitution model with gamma-distributed rates and invariant sites, NNI branch swapping, and assessing robustness of nodes with 500 bootstrap replicates.…”