ZOOLOGICAL RESEARCH A new genus and three new species of miniaturized microhylid frogs from Indochina (Amphibia: Anura: Microhylidae: Asterophryinae) Nikolay A. Poyarkov, Jr.1,2,*, Chatmongkon Suwannapoom3, Parinya Pawangkhanant3, Akrachai Aksornneam4, Tang Van Duong1,5, Dmitriy V. Korost6, Jing Che7,8 1 Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Moscow 119234, Russia 2 Joint Russian-Vietnamese Tropical Research and Technological Center, Nghia Do, Cau Giay, Hanoi, Vietnam 3 Division of Fishery, School of Agriculture and Natural Resources, University of Phayao, Phayao 56000, Thailand 4 Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 5 Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Hanoi, Vietnam 6 Petroleum Geology Department, Geological Faculty, Lomonosov Moscow State University, Moscow 119234, Russia 7 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China 8 Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin Nay Pyi Taw 05282, Myanmar ABSTRACT of the three new species of Vietnamophryne Gen. nov. is known only from a single specimen; thus, We report on the discovery of a new genus of their distribution, life history, and conservation status microhylid subfamily Asterophryinae from northern require further study. and eastern Indochina, containing three new species. Keywords: Vietnamophryne Gen. nov.; Vietnamophryne Vietnamophryne Gen. nov. are secretive miniaturized inexpectata sp. nov.; Vietnamophryne orlovi frogs (SVL<21 mm) with a mostly semi-fossorial sp. nov.; Vietnamophryne occidentalis sp. lifestyle. To assess phylogenetic relationships, we nov.; Siamophryne; Gastrophrynoides; mtDNA; studied 12S rRNA – 16S rRNA mtDNA fragments micro-CT scanning; Vietnam; Thailand; Herpetofauna; with a final alignment of 2 591 bp for 53 microhylid Amphibia; Biogeography; Taxonomy; Indochina species. Morphological and osteological characters were analyzed using micro-CT scanning and used to describe the new genus. Results of phylogenetic Received: 30 January 2018; Accepted: 03 March 2018; Online: 20 April analyses assigned the new genus into the mainly 2018 Australasian subfamily Asterophryinae as a sister Foundation items: This work was supported by the programs taxon to the genus Siamophryne from southern of the National Natural Science Foundation of China (31501843, Indochina. The three specimens collected from 31622052), Southeast Asia Biodiversity Research Institute, Chinese Gia Lai Province in central Vietnam, Cao Bang Academy of Sciences (CAS) (Y4ZK111B01: 2017CASSEABRIQG002), Province in northern Vietnam, and Chiang Rai Animal Branch of the Germplasm Bank of Wild Species, CAS Province in northern Thailand proved to be separate (Large Research Infrastructure Funding), and Thailand Research Fund species, different both in morphology and genetics (TRF) (DBG6180001). Molecular experiments, phylogenetic analyses, (genetic divergence 3.1%≤P≤5.1%). Our work specimen storage, examination and microCT-analysis were carried out provides further evidence for the “out of Indo-Eurasia” with the financial support of the Russian Science Foundation (RSF scenario for Asterophryinae, indicating that the initial 14-50-00029) cladogenesis and differentiation of this group of frogs *Corresponding author, E-mail: [email protected] occurred in the Indochina Peninsula. To date, each DOI: 10.24272/j.issn.2095-8137.2018.019 130 Science Press Zoological Research 39(3): 130–157, 2018 INTRODUCTION Indochina, we encountered three specimens of miniaturized frogs. Although these frogs were found in different localities Frogs of the family Microhylidae form one of the most speciose in central and northern Vietnam and northern Thailand (Figure groups of amphibians with pantropical distribution. Currently, 1), all three specimens were superficially very similar to each some 642 species are recognized, inhabiting areas from other and found in similar microhabitats — soil or leaf litter under the tropics and subtropics of Africa, Madagascar, Southern large tree logs or among plant roots. They were assigned to and Northern America, and South, East, and Southeast Microhylidae due to the presence of morphological characters Asia to the islands of the Australasian archipelago and diagnostic for the family: namely, lack of mandibular teeth, lack of northernmost Australia (Frost, 2018). The basal split within parotoid glands, firmisternal pectoral girdle with non-overlapping Microhylidae is estimated to have occurred ∼65 Ma, coinciding epicoracoids, well-developed coracoids reaching midline of girdle with the Cretaceous-Paleogene boundary (Feng et al., 2017). and scapulae, large, cartilaginous sternum, and absence of The family Microhylidae is assumed to be of Gondwanan clavicles and omosternum. Further morphological, osteological, origin and is currently divided in 13 subfamilies, each of and molecular analyses demonstrated that each of the three which are associated with a certain landmass derived from specimens represented a new species of a previously unknown the breakup of Gondwana (De Sá et al., 2012; Kurabayashi lineage of frogs, assigned to the subfamily Asterophryinae and et al., 2011; Peloso et al., 2016). Despite significant progress sister taxon to Siamophryne. We describe this new genus and in understanding the evolutionary relationships within the three new species herein. family, the level of congruence between morphology-based and molecular phylogenetic hypotheses is still low and further MATERIALS AND METHODS changes in family- and genus-level taxonomy are required (De Sá et al., 2012; Kurabayashi et al., 2011; Matsui et al., 2011; Sample collection Peloso et al., 2016; Pyron & Wiens, 2011; Rivera et al., 2017). Field work was conducted from 23 May to 2 June 2016 in Kon The subfamily Asterophryinae is the most speciose group Chu Rang Nature Reserve, Gia Lai Province, Tay Nguyen Plateau, within Microhylidae, currently consisting of 327 species central Vietnam (N14.506◦, E108.542◦; elevation 1 000 m a.s.l.); inhabiting the tropical forests of northern Australia, New from 8 to 17 June 2017 in Phia Oac-Phia Den National Park, Guinea, and adjacent Australasian islands westwards to Cao Bang Province, northern Vietnam (N22.600◦, E105.884◦; Sulawesi, southern Philippines, and crossing the Wallace elevation 1 200 m a.s.l.) and from 5 to 15 February and 4 to 8 April line in Bali (Frost, 2018). The original biogeographic 2017 in the environs of Doi Tung Mt., Pong Ngam District, Chaing hypothesis for this subfamily suggested that the common Rai Province, northern Thailand (N20.344◦, E99.830◦; elevations ancestor of Asterophryinae dispersed to Australia via an from 900 to 1 050 m a.s.l.). All fieldwork and collection permits Antarctic land bridge (Hill, 2009; Savage, 1973), where are listed in the Acknowledgements. Geographic coordinates and it diversified and subsequently dispersed to New Guinea elevation were obtained using a Garmin GPSMAP 60CSx (USA) and adjacent Australasian islands. However, based on and recorded in WGS84 datum. In total, three adult specimens multilocus phylogenetic analyses, Kurabayashi et al. (2011) (all males) were collected from three surveyed localities. The demonstrated that the enigmatic genus Gastrophrynoides from specimens were photographed in life and then euthanized using Sundaland (Borneo and Malay Peninsula) belongs to the 20% benzocaine prior to fixation in 96% ethanol and subsequent subfamily Asterophryinae as a sister-lineage with respect to storage in 70% ethanol. Tissue samples for genetic analysis all Australasian taxa, suggesting that the basal split of the were taken prior to preservation and stored in 95% ethanol. subfamily may not have occurred in Gondwana, but instead Specimens and tissues were subsequently deposited in the on the Eurasian mainland. Thus, Kurabayashi et al. (2011) herpetological collections of the Zoological Museum of Moscow proposed an “out of Indo-Eurasia” biogeographic scenario for University (ZMMU, Moscow, Russia) and School of Agriculture and Asterophryinae, suggesting that its colonization route was from Natural Resources, University of Phayao (AUP, Phayao, Thailand). Asia to Australia, and not via Antarctica as suggested earlier. In their work, Kurabayashi et al. (2011:9) predicted, Laboratory methods that the “biogeographic findings on Gastrophrynoides imply Total genomic DNA was extracted from ethanol-preserved the possible occurrence of further microhylid taxa with femoral muscle tissue using standard phenol-chloroform- unexpected evolutionary backgrounds and give a basis for proteinase K (final concentration 1 mg/mL) extraction with future paleontological and biogeographic studies of Asian subsequent isopropanol precipitation (as per Hillis et al., 1996 anurans”. Our more recent work (Suwannapoom et al., 2018) and Sambrook & Russell, 2001). The isolated DNA was reported on the unexpected discovery of Siamophryne — visualized using agarose electrophoresis in the presence of a striking troglophilous microhylid frog found in a limestone ethidium bromide. The resulting DNA concentration in 1 μLwas cave in Tenasserim (southern Thailand) — with phylogenetic measured using a NanoDrop 2000 (Thermo Scientific, USA) analyses placing it as a sister lineage of Gastrophrynoides, and consequently adjusted to 100 ng DNA/μL. further suggesting that mainland Southeast Asia