A reinvestigation of phylogeny and divergence times of Hynobiidae (Amphibia, Caudata) based on 29 nuclear genes

• 29 nuclear genes (a total number of 29,232 bp) were used in this study.
• Concatenation and specie-tree analyses yielded congruent and robust results.
• The phylogenetic positions for Pachyhynobius and Salamandrella are determined.
• Previous time estimates for hynobiids based on mtDNAs are overestimated.

Although several recent studies have investigated the major phylogenetic relationships within Hynobiidae, their evolutionary history remains partially resolved and the phylogenetic positions of some genera, particularly Pachyhynobius and Salamandrella are still disputed. Notably, previous studies relied primarily on mitochondrial DNA data and concatenated analyses; thus, a new investigation based on multiple nuclear genes and species-tree inference is needed. Here, we provide an in-depth phylogenetic analysis, based on 29 nuclear genes comprising 29,232 bp of data from a comprehensive taxonomic sampling (24 hynobiids and 7 outgroups), using both concatenated and species-tree methods. Our results robustly resolved most genus-level relationships within Hynobiidae, including the placement of Salamandrella as the sister group to a clade containing Batrachuperus, Liua and Pseudohynobius, and the placement of Pachyhynobius as the sister group to a clade containing all hynobiids excluding Onychodactylus, Paradactylodon and Ranodon. Time estimates based on our data suggest that the major group of living hynobiids (excluding Onychodactylus) originated approximately 40 Ma, ∼50% younger than estimates from mtDNA data (62.5 Ma) but 10% older than estimates from three nuclear genes (36 Ma). Our results highlight the benefits of using a large number of nuclear loci to infer both phylogeny and time for relatively old lineages.

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