Species delimitation in frogs from South American temperate forests: The case of Eupsophus, a taxonomically complex genus with high phenotypic variation

Abstract

One of the most characteristic and abundant amphibian taxa of South American temperate forests is Eupsophus. The ten currently recognized species of the genus have been divided in two species groups, roseus and vertebralis, but most of them, eight, belong to the roseus group. Recent phylogeographic and phylogenetic studies have suggested that species diversity of the roseus group could be underestimated. An examination of the literature shows that species of the roseus group exhibit high levels of variation in their external characteristics, particularly those used as diagnostic characters, which compromises their taxonomy and hinders their field recognition. High levels of variation were also observed in several new populations of the roseus group discovered in southern Chile (36°-40°S), which could not be identified to the species level by their external characteristics. On the other hand, the literature reveals a scarse karyotype differentiation and a high bioacoustic uniformity among the species of the roseus group. We performed a Bayesian phylogenetic analysis using mitochondrial and nuclear genes to reevaluate the species diversity of the roseus group, including all the nominal species of Eupsophus and new populations. This analysis was complemented with three species delimitation approaches, General Mixed Yule Coalescent, multi-rate Poisson Tree Process and Automatic Barcode Gap Discovery. We favored a conservative delimitation of only four species for the roseus group, a result more consistent with the distribution of pairwise genetic distances, and the available chromosome and bioacoustic evidence. The four recognized lineages, which have nearly completely allopatric distributions, are named after the earliest nominal species that they include, but because high levels of phenotypic variation, they are not diagnosable by consistent differences in external morphology. We discuss the implications of this new proposal for the taxonomy and conservation of the genus, and the possible causes of the difficulty to estimate its species diversity.

Speciation in a biodiversity hotspot: phylogenetic relationships, species delimitation, and divergence times of the Patagonian ground frogs ofEupsophus roseusgroup (Alsodidae)

“…Our delimitation results were not agreed with a recent hypothesis [35], which would be related to use of different molecular markers and species delimitation analyses. Three of our markers were found to be highly variables (Cyt b , COI, D-loop), while two were conserved (POMC and CRYBA1; see S2 table).…”

“…Nevertheless, comparisons with some scenarios including that of nine-species were around four, which indicate non-strong or decisive support to the best model (S5 Table). Other possible scenarios, including that proposed by Correa et al [35] (scenario 3), were lowly supported for all multi-locus analyses (Fig 4).…”

“…Thus we use at least three strong markers (sequences with many polymorphic sites), a key aspect to carried out coalescent analyses when less than ten markers are used [86]. On the other hand, we used several multi-locus coalescent methods to delimitate species (BPP, STEM, R2, and BFD), while Correa et al [35] based its inferences in single-locus analyses (GMYC, mPTP, and Automatic Barcode Gap Discovery, ABGD). In this sense, mPTP (using mitochondrial data set) and ABGD (using mitochondrial + nuclear data set) recovered to the two groups of sinonimized species as two species [35].…”

“…All of them with 30 chromosomes, and whose individuals have a body size of 34-42 mm (snout-vent distance) [42]; and 2) the vertebralis group, composed of E. vertebralis and E. emiliopugini , both species with 28 chromosomes and individuals with a body size of 50-59 mm (snout-vent distance) [42]. Nevertheless, recently molecular analyses within roseus group synonymized E. altor with E. migueli as well as E. contulmoensis, E. septentrionalis , and E. nahuelbutensis with E. roseus [35]. Therefore, currently the roseus group is composed by four species: E. migueli, E. insularis, E. roseus and E. calcaratus [33].…”

“…Here, we present phylogenetic and species delimitation of the roseus group, using 164 new samples from all species covering most of their distribution range. We used three mitochondrial and two nuclear markers, three of them are different to those used by Blotto et al [34] and Correa et al [35] [Control Region (D-loop), Propiomelanocortin (POMC), and β Crystallin A1 (CRYBA1)]. These molecular dataset are used to carry out phylogenetic reconstructions and an extensive number of single- and multi-locus species delimitation methods.…”