Phylogeny of spiny frogs Nanorana (Anura: Dicroglossidae) supports a Tibetan origin of a Himalayan species group

Abstract

Recent advances in the understanding of the evolution of the Asian continent challenge the long‐held belief of a faunal immigration into the Himalaya. Spiny frogs of the genus Nanorana are a characteristic faunal group of the Himalaya–Tibet orogen (HTO). We examine the phylogeny of these frogs to explore alternative biogeographic scenarios for their origin in the Greater Himalaya, namely, immigration, South Tibetan origin, strict vicariance. We sequenced 150 Nanorana samples from 62 localities for three mitochondrial (1,524 bp) and three nuclear markers (2,043 bp) and complemented the data with sequence data available from GenBank. We reconstructed a gene tree, phylogenetic networks, and ancestral areas. Based on the nuDNA, we also generated a time‐calibrated species tree. The results revealed two major clades (Nanorana and Quasipaa), which originated in the Lower Miocene from eastern China and subsequently spread into the HTO (Nanorana). Five well‐supported subclades are found within Nanorana: from the East, Central, and Northwest Himalaya, the Tibetan Plateau, and the southeastern Plateau margin. The latter subclade represents the most basal group (subgenus Chaparana), the Plateau group (Nanorana) represents the sister clade to all species of the Greater Himalaya (Paa). We found no evidence for an east–west range expansion of Paa along the Himalaya, nor clear support for a strict vicariance model. Diversification in each of the three Himalayan subclades has probably occurred in distinct areas. Specimens from the NW Himalaya are placed basally relative to the highly diverse Central Himalayan group, while the lineage from the Tibetan Plateau is placed within a more terminal clade. Our data indicate a Tibetan origin of Himalayan Nanorana and support a previous hypothesis, which implies that a significant part of the Himalayan biodiversity results from primary diversification of the species groups in South Tibet before this part of the HTO was uplifted to its recent heights.

Relict groups of spiny frogs indicate Late Paleogene-Early Neogene trans-Tibet dispersal of thermophile faunal elements

“…The Paini tribe is currently composed of the genus Nanorana Günther, 1896 (around 30 species), Quasipaa Dubois, 1992 (11 species), Allopaa Ohler and Dubois, 2006 (possibly two species), and the monotypic genus Chrysopaa Ohler and Dubois, 2006, with one species, C. sternosignata (Murray, 1885). Following Che et al (Che et al 2010) and our own findings (Hofmann et al 2019), Nanorana can be subdivided into three subgenera ( Nanorana , Paa , and Chaparana ). However, the phylogenetic and mostly taxonomic relationships among Paini are not completely resolved with several taxonomic changes during the last decade including taxa descriptions (Che et al 2009; Frost 2021; Huang et al 2016; Jiang et al 2005; Pyron & Wiens 2011).…”

“…We used sequence data of the 16S ribosomal, COI mitochondrial and Rag1 nuclear region available from our previous study (Hofmann et al 2019) and complemented the data with a newly generated sequences for these three gene regions from Allopaa hazarensis (Dubois and Khan, 1979) (n = 6; Pakistan, including the type locality of the species – Datta, Manshera District, Hazera Division; for details see Fig. 1 and supplemental Tab.…”

“…We also included 16S rRNA and COI sequence data of Chrysopaa sternosignata from the Hindu Kush available in NCBI GenBank (accession numbers: MG700155 and MG699938). Our Nanorana samples from Himachal Pradesh, which were previously referred to as “sp.” (Hofmann et al 2019), were identified as Nanorana vicina based on morphological characters (Boulenger 1920; Stoliczka 1872); for photos of live specimens Fig. S1).…”

“…Genomic DNA was extracted from ethanol-muscle tissues using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands) following the manufacturer’s protocol. Approximately 571 bp of the ribosomal RNA (rRNA) 16S, 539 bp of the COI, and a sequence segment of 1207 bp of Rag1 gene were amplified via the polymerase chain reaction (PCR) using primers and PCR conditions as previously described (Hofmann et al 2019). PCR products were purified using the mi-PCR Purification Kit (Metabion, Planegg, Germany) and the ExoSAP-IT enzymatic clean-up (USB Europe GmbH, Staufen, Germany; manufacturer’s protocol) or directly purified by Eurofins Genomics (Germany) with in-house protocols.…”

“…Previous studies proposed contrasting hypotheses to explain the current distributional and phylogenetic patterns of spiny frogs in the HTO. While a strict vicariance driven scenario suggests species formation among major lineages when the species were “trapped” in the mountain mass and become separated when it uplifted (Che et al 2010), a more recent study found no clear support for this model but indications for a Paleo-Tibetan origin of Himalayan spiny frogs (Hofmann et al 2019), matching modern hypotheses for the past topographic surfaces of the southern parts of the HTO. This Tibetan-origin scenario (Schmidt et al 2012) assumes that the ancestral lineages of Himalayan spiny frogs had adapted to the high-altitude environment in South Tibet, prior to the final uplift of the Greater Himalaya.…”