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Frogs of the genus Microhyla include some of the world's smallest amphibians and represent the largest radiation of Asian microhylids, currently encompassing 50 species, distributed across the Oriental biogeographic region. The genus Microhyla remains one of the taxonomically most challenging groups of Asian frogs and was found to be paraphyletic with respect to large-sized fossorial Glyphoglossus . In this study we present a time-calibrated phylogeny for frogs in the genus Microhyla , and discuss taxonomy, historical biogeography, and morphological evolution of these frogs. Our updated phylogeny of the genus with nearly complete taxon sampling includes 48 nominal Microhyla species and several undescribed candidate species. Phylogenetic analyses of 3,207 bp of combined mtDNA and nuDNA data recovered three well-supported groups: the Glyphoglossus clade, Southeast Asian Microhyla II clade (includes M. annectens species group), and a diverse Microhyla I clade including all other species. Within the largest major clade of Microhyla are seven well-supported subclades that we identify as the M. achatina , M. fissipes , M. berdmorei , M. superciliaris , M. ornata , M. butleri , and M. palmipes species groups. The phylogenetic position of 12 poorly known Microhyla species is clarified for the first time. These phylogenetic results, along with molecular clock and ancestral area analyses, show the Microhyla-Glyphoglossus assemblage to have originated in Southeast Asia in the middle Eocene just after the first hypothesized land connections between the Indian Plate and the Asian mainland. While Glyphoglossus and Microhyla II remained within their ancestral ranges, Microhyla I expanded its distribution generally east to west, colonizing and diversifying through the Cenozoic. The Indian Subcontinent was colonized by members of five Microhyla species groups independently, starting with the end Oligocene-early Miocene that coincides with an onset of seasonally dry climates in South Asia. Body size evolution modeling suggests that four groups of Microhyla have independently achieved extreme miniaturization with adult body size below 15 mm. Three of the five smallest Microhyla species are obligate phytotelm-breeders and we argue that their peculiar reproductive biology may be a factor involved in miniaturization. Body size increases in Microhyla-Glyphoglossus seem to be associated with a burrowing adaptation to seasonally dry habitats. Species delimitation analyses suggest a vast underestimation of species richness and diversity in Microhyla and reveal 15-33 undescribed species. We revalidate M. nepenthicola , synonymize M. pulverata with M. marmorata , and provide insights on taxonomic statuses of a number of poorly known species. Further integrative studies, combining evidence from phylogeny, morphology, advertisement calls, and behavior will result in a better systematic understanding of this morphologically cryptic radiation of Asian frogs., Competing Interests: Nikolay A. Poyarkov is an Academic Editor for PeerJ. Suranjan Karunarathna is employed by Nature Explorations and Education Team (Sri Lanka). Luan Thanh Nguyen is employed by Asian Turtle Program—Indo-Myanmar Conservation (Vietnam). Other authors have declared that no competing interests exist., (© 2020 Gorin et al.)

We examine the phylogenetic structure and morphological differentiation within the Hynobius kimurae-H. boulengeri species complex-a widely-distributed group of stream-breeding hynobiid salamanders, inhabiting montane areas of western, central and eastern parts of Honshu Island, Japan. Phylogenetic relationships were assessed based on analyses of mitochondrial (mtDNA) and nuclear (nuDNA) gene fragments for a total of 51 samples representing 23 localities covering the entire range of the species complex. Morphological study included one-way analysis of variance (ANOVA) and principal components analysis (PCA) analyses of 26 morphometric and six meristic characters for 38 adult specimens of H. kimurae and three adult specimens of H. boulengeri . MtDNA genealogy supported monophyly of the H. kimurae-H. boulengeri complex, which is sister to all other Hynobius except H. retardatus . The complex is subdivided into three major clades, corresponding to the Eastern (Clade I) and Western (Clade II) populations of H. kimurae sensu lato, and to H. boulengeri (Clade III). Monophyly of H. kimurae sensu lato is only moderately supported by mtDNA, while nuDNA suggested that the Western form of H. kimurae is closer to H. boulengeri than to the eastern form. The time of the split of the H. kimurae-H. boulengeri complex is estimated as late Miocene and coincides with intensive crust movement in western Japan. Divergence between Clades I and II took place in early Pliocene and was likely influenced by the uplift of Central Japanese Highlands. All three clades were found to be different in a number of morphological characters, allowing us to describe the eastern form of H. kimurae as a new species, Hynobius fossigenus sp. nov ., Competing Interests: The authors declare that they have no competing interests.