Abstract
The alsodid ground frogs of the Eupsophus genus are divided into two groups, the roseus (2n = 30) and vertebralis (2n = 28), which are distributed throughout the temperate Nothofagus forests of South America. Currently, the roseus group is composed by four species, while the vertebralis group consists of two. Phylogenetic relationships and species delimitation within each group are controversial. In fact, previous analyses considered that the roseus group was composed of between four to nine species. In this work, we evaluated phylogenetic relationships, diversification times, and species delimitation within the roseus group using a multi-locus dataset. For this purpose, mitochondrial (D-loop, Cyt b, and COI) and nuclear (POMC and CRYBA1) partial sequences from 164 individuals were amplified, representing all species. Maximum Likelihood (ML) and Bayesian approaches were used to reconstruct phylogenetic relationships. Species tree was estimated using BEAST and singular value decomposition scores for species quartets (SVDquartets). Species limits were evaluated with six coalescent approaches. Diversification times were estimated using mitochondrial and nuclear rates with LogNormal relaxed clock in BEAST. Nine well-supported monophyletic lineages were recovered in Bayesian, ML, and SVDquartets, including eight named species and a lineage composed by specimens from the Villarrica population (Bootstrap:>70, PP:> 0.99). Single-locus species delimitation analyses overestimated the species number in E. migueli, E. calcaratus, and E. roseus lineages, while multi-locus analyses recovered as species the nine lineages observed in phylogenetic analyses (Ctax = 0.69). It is hypothesized that Eupsophus diversification occurred during Mid-Pleistocene (0.42–0.14 Mya), with most species having originated after the Last Southern Patagonian Glaciation (0.18 Mya). Our results revitalize the hypothesis that the E. roseus group is composed of eight species and support the Villarrica lineage as a new putative species.
Highlights
From an operational point of view, the notion of biodiversity encompasses several different levels of biological organization, from the genetic make up of the species to ecosystems and landscapes, in which the species is the most significant unit
Phylogenetic analyses using concatenated mitochondrial and nuclear sequences recovered three main well-supported clades corresponding to Clade A, Clade B (E. roseus), and Clade C (E. calcaratus) (Fig 2)
Gaps in morphological, geographic, cytogenetic, bioacoustic, and behavioral information prevent us to carry out a protocol for integrative taxonomy, our molecular approach is concordantly with integrative studies available for some species as E. altor [81]
Summary
From an operational point of view, the notion of biodiversity encompasses several different levels of biological organization, from the genetic make up of the species to ecosystems and landscapes, in which the species is the most significant unit. Despite the importance of the species concepts debate [7,8], and since the species as taxonomic hierarchy is considered a fundamental topic in biology [9], it is broadly accepted that species are best conceptualized as dynamic entities connected by "grey zones" where their delimitation will remain inherently ambiguous [4,10] Under this perspective, species delimitation, i.e. the act of identifying biological diversity at species-level [11], is challenging in actively radiating groups composed of recently diverged lineages. The difficulty lies in the fact that recently separated species are less likely to possess all or even many of the diagnosable characters such as phenetic distinctiveness, intrinsic reproductive incompatibility, ecological uniqueness, or reciprocal monophyly, that constitute operational criteria for their delimitation [4,12] This becomes more complex when hybridization and introgression among related species are considered common and major contributors to speciation and diversification [13]. Hypotheses of the boundaries of recently diverged species may remain unclear due to incomplete lineage sorting, introgression, complex of cryptic species that cannot be distinguished by morphology alone, sampling deficiencies, or different taxonomic practices [2,4]
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