Abstract
An important aspect of evaluating biodiversity in a region, starting a monitoring program or informing conservation management decisions is having a good understanding of the taxonomy of local species. However, identification to the species-level can be challenging. A combination of DNA-based and phenotypic character analysis can provide a preliminary species list and help identify diagnostic features for taxonomically difficult groups such as the Neotropical leaf litter frogs (Craugastor spp.). We used 16S and COI marker sequences to assess the number of phylogenetic Craugastor species present in Cusuco National Park, Honduras. We conducted a linear discriminant analysis to determine if phenotypic characteristics could validate identified monophyletic species. Subsequently, we evaluated the efficacy of the Automatic Barcode Gap Discovery (ABGD) algorithm, a DNA sequence similarity-based tool, for species delineation within Neotropical amphibians. Phylogenetic analyses conducted on sequences derived from 194 specimens produced concordant results between both loci, with reciprocal monophyly of mitochondrial DNA haplotypes for all clades, revealing the presence of four Craugastor species: C. rostralis, C. chac, C. laticeps and C. c.f. charadra. Iris colouration was discovered to be a diagnostic character and the ABGD algorithm accurately identified all four monophyletic species within the phylogenetic and phenotypic analyses. A further three species have been reported from Cusuco National Park including C. milesi, C. laevissimus and C. coffeus. These species are more readily identifiable than the cryptic species we examined, but they have yet to be confirmed using molecular analyses. We demonstrate that the use of molecular tools, in conjunction with the post hoc evaluation of phenotypic variation, can aid with the delineation of cryptic biological diversity and with the discovery of key diagnostic features for accurate species recognition in the field.
Highlights
An accurate assessment of species richness is a critical first step toward informed conservation and management decisions at a regional level
The phylogenetic species concept distinguishes a species as the smallest biological unit that is monophyletic and supported by unique apomorphic characters [32]
The 16S and c oxidase I (COI) phylogenetic analyses distinguished three well supported monophyletic clades identified as C. rostralis, C. chac, and C. cf. charadra
Summary
An accurate assessment of species richness is a critical first step toward informed conservation and management decisions at a regional level. Molecular tools are highly valuable in the discovery and identification of species, when traditional taxonomic identification is difficult to apply, such as cases of cryptic diversity [5,6]. The Automatic Barcode Gap Discovery (ABGD) algorithm has been a useful tool because it allows multiple thresholds to be evaluated as opposed to a single standard limit [11]. It statistically infers the potential threshold value from a set of input sequences, partitions the data based on that threshold, and recursively applies the same procedure to the newly obtained groups of sequences. ABGD has previously been successful in species demarcation and cryptic species identification, within taxonomically difficult amphibian groups [12,13]
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