Molecular phylogeny of frogs (Amphibia: Anura) based on complete mitochondrial genomes and partial nuclear genes

Modern anurans (frog and toads) have a long evolutionary history of more than 200 million years, having undergone an extraordinary phylogenetic and ecological diversification, which gave rise to the 6,000 species currently recognized. They exhibit a variety of morphological, ecological and behavioural adaptations that allowed them to inhabit many different environments, across every continent (except Antarctica) and most continental islands. Early studies on morphology made important contributions towards understanding the evolutionary history of frogs. Later on, the analysis of sequence data, and the development of powerful and sophisticated probabilistic methods of inference provided substantial advance in the phylogeny of frogs. In the last decade, the application of molecular phylogenetic techniques allowed resolving some of the controversial issues regarding higher-level relationships among living frogs, and currently, we have a reasonably well-defined portrait of the frog tree of life. Yet, many important issues still remain under debate. Most studies agree on a monophyletic origin of the Anura, and four major lineages are generally recognized (Discoglossoidea, Pipoidea, Pelobatoidea, and Neobatrachia), along with two basal genera (Leiopelma and Ascaphus) of uncertain placement. Here, a sequence data set combining complete mitochondrial genomes and nine nuclear loci was used to estimate a molecular phylogeny of frogs and tackle long-standing contentious questions. First, the relative phylogenetic position of Leiopelma and Ascaphus, two genera that have traditionally been considered basal within Anura, was addressed. Second, new sequence data was generated for Pipoidea, aiming to discriminate among competing hypotheses for the relationship between this group and Discoglossoidea, as well as to elucidate the internal phylogeny of the family Pipidae. Taking advantage of having reconstructed a robust phylogenetic framework for Pipoidea, the unusual sound production mechanism of the poorly known species Pseudhymenochirus merlini was studied by means of behavioural and anatomical observations. Third, the phylogenetic relationships among basal families within Neobatrachia were examined to further understand their extraordinary diversification, as well as the evolution of the specific gene order and higher substitution rates of neobatrachian mitochondrial genomes. The sequence data set combining complete mitochondrial genomes and nine nuclear loci showed good phylogenetic performance in inferring deep level phylogenetic relationships among frogs. Results from phylogenetic analyses pointed to a sister group relationship between Leiopelma and Ascaphus (Amphicoela hypothesis), and their placement together as sister group of all remaining frogs. Remarkably, the mitochondrial genome of Leiopelma archeyi has a gene arrangement that is unique among frogs but convergent with that of other vertebrates. Moreover, comparative data on mitochondrial gene orders from other vertebrates strongly suggested that the 5' end of the control region is a hot spot of gene rearrangement. Further phylogenetic analyses were congruent in supporting the successive branching of the five major lineages of living frogs, as (i) Amphicoela, (ii) Discoglossoidea, (iii) Pipoidea, (iv) Pelobatoidea, and (v) Neobatrachia. Within Pipidae, both mitochondrial and nuclear data were congruent in recovering a deep divergence between an American lineage (Pipa) and an African lineage, in which dactylethrines (Xenopus + Silurana) were the sister group of hymenochirines (Hymenochirus + Pseudhymenochirus). Behavioural observations on Pseudhymenochirus unambiguously showed an air-driven mechanism for sound production, in contrast to all other members of the family, which have a mechanism independent of air. Given the derived phylogenetic position of Pseudhymenochirus, this observation was interpreted as a reversal to the ancestral non-pipid condition, which according to the performed anatomical observations, seems to have evolved constrained under the restrictions imposed by the derived larynx of pipids. New mitogenomic and nuclear data on basal neobatrachian families provided insights into their phylogenetic position. Heleophryne was recovered as the sister group to all other neobatrachians. Lechriodus and Calyptocephalella were recovered as sister taxa, and both as sister to Nobleobatrachia. Within Nobleobatrachia, Duttaphrynus and Telmatobius were sister genera to the exclusion of Hyla. Phylogenetic analyses also suggested a sister group relationship between Sooglossus and Ranoides. The analysis of mitochondrial genomes within a phylogenetic framework provided further information to understand gene rearrangement dynamics and unravel mechanisms of molecular evolution. The reconstructed phylogeny showed that the neobatrachianspecific mitochondrial gene order was already present in the earliest branching living lineage of the group, suggesting that it might represent a molecular synapomorphy for Neobatrachia. Furthermore, mitochondrial substitution rates were found to be accelerated at the origin of Neobatrachia, and were higher in both basal and derived neobatrachian lineages compared to non-neobatrachian frogs. However, no consistent patterns were found among the nine nuclear genes studied. Further examination of mitochondrial protein-coding genes suggests that relaxation of purifying selection might account, at least in part, for the observed rate acceleration at the origin of the Neobatrachia.