The occurrence of at least one species of Erhaia in Bhutan, viz. Erhaia wangchuki sp. n., is confirmed by DNA sequencing. A second unnamed species from Bhutan, that might be congeneric, is known from only a single shell. According to the molecular analysis, E. wangchuki is most closely related to a still undescribed Erhaia species from China. These two species together with E. jianouensis and Akiyoshia kobayashii, both also from China, form a well supported clade. Awaiting additional molecular data, the apparent inconsistency regarding Erhaia versus Akiyoshia is not dealt with here. The extant true sister species of E. wangchuki could be among the four SE Himalayan species from Bhutan and Nepal that are classified with Erhaia on the basis of conchological data only.

Erhaia , Akiyoshia , 16S, taxonomy, distribution, Nepal, Bhutan

The extremely speciose superfamily Rissooidea, with over 400 recent genera ‘one of the largest gastropod families’ (Wilke et al. 2001: 1), encompasses very many species that cannot be identified on the basis of only shell characters, distribution and ecology. Anatomical characters may additionally be used, when the equipment for dissection is available, but even that technically demanding approach does not always bring conclusive results. Therefore, molecular analyses are advisable in cases like this. This has resulted in a more reliable classification allowing these snails to be used as ecological indicator species on the one hand and model organisms in historical biogeography on the other hand.

After its description by Davis and Kuo in Davis et al. (1985), the genus Erhaia turned out to be widespread in Asia. From Nepal, two species of Erhaia are reported by Nesemann et al. (2007) on the basis of shell shape and ecology. Here, we report the occurrence of a species of Erhaia in Bhutan, confirmed by a molecular analysis. The shell of a species that might be congeneric is additionally described but without naming it.

The first author coincidentally collected a minute snail in a marshy source area in Bhutan, in the district Thimphu, W of Geneykha at 2825 m altitude. The locality could not yet be visited again. The specimen got lost after being photographed with a Ricoh WG-4 digital camera, using the extreme macro facility. Some equally small snails were found by Jigme Wangchuk of the Ugyen Wangchuck Institute for Conservation and Environment, Bumthang, in a source in the district Wangdue Phodrang at 2883 m altitude, and preserved in alcohol 70%. One specimen (Fig. 1) of the latter sample, a paratype, was photographed with a digital microscope system (KEYENCE VHX-2000; KEYENCE Corp., Itasca, IL, USA) and then used for a molecular analysis. Another shell, the holotype (Fig. 2), was photographed with a Canon EOS 7D, using a Canon extension tube EF25 and a Sigma DC 18-50 mm lens.

DNA was isolated using standard protocols for molluscs (see Stelbrink et al. 2016 for details). A mitochondrial DNA fragment (16S rRNA) was amplified and sequenced (GenBank accession number: KY798003). In addition, a maximum likelihood tree (Fig. 4) was obtained using RAxML BlackBox (substitution model: GTR+Γ, 100 bootstrap replicates; Stamatakis et al. 2008) by using the 16S rRNA dataset of Liu et al. (2014).

Our molecular analysis resulted in a tree (Fig. 4) with Erhaia wangchuki sp. n. as the sister taxon of an undescribed Erhaia sp. from China, Guangxi, Xiangjiang river at Quanzhou (after Liu et al., 2014: 22). The sister taxon of these two species is unclear, but a clade formed by four species, viz. E. wangchuki, Erhaia spec., Akiyoshia kobayashii Kuroda & Habe, 1958 and E. jianouensis (Liu & Zhang, 1979), is highly supported.

After its introduction by Davis et al. (1985), the classification of the genus Erhaia remained uncertain for some time. Wilke et al. (2000) eventually published maximum likelihood phylogenies based on sequences of both nuclear and mitochondrial markers, viz. 18S, and 16S and COI, and a combination of all three markers. COI supported a clade formed by Erhaia and the combined Amnicola Gould & Haldeman, 1840 and Bythinella Moquin-Tandon, 1856. Both 18S and 16S placed Amnicola next to the combined Erhaia and Moria Kuroda & Habe, 1958. The combined data showed the combined Erhaia and Moria as the sister group of Amnicola, and these three genera together in a sister group relation with Bythinella. Later on, maximum likelihood phylogenies were published using data from 18S and COI and a combination of both (Wilke et al. 2001). According to this study, Erhaia, Amnicola and Marstoniopsis Van Regteren Altena, 1936 together form a clade in the COI and in the combined tree as the sister group of Bithynella; the same three genera cluster together in the 18S-based tree, where the position of Bythinella remains unresolved. Liu et al. (2014), while including other genera in their molecular study, once again confirmed the systematic position of Erhaia together with Akiyoshia Kuroda & Habe, 1954 and Bythinella within the Amnicolidae.

Erhaia is known by several species in China, from the Yangtze River drainage and, by one species, from the Mekong River drainage (Davis and Kang 1995, Davis and Rao 1997). The probability that Erhaia is represented in Nepal indeed (Nesemann et al. 2007) is considerably enhanced by the anatomically confirmed occurrence of E. nainitalensis Davis & Rao, 1997, in Nainital in northern India west of Nepal, and E. wangchuki sp. n. in Bhutan east of Nepal. It may be hypothesized that there is a radiation in Erhaia along the southern border of the Himalaya, far west and northwest of its large range in China. With hardly any spring area in Bhutan searched for micro-snails, additional species may wait for discovery there at least.

We cordially thank Prof. Dr. T. Wilke (Gießen) for valuable information regarding Erhaia and Jigme Wangchuk (Bumthang) for placing interesting specimens at our disposal for research. Damber Bdr Chhetri, forester in Phobjikha, is gratefully acknowledged for providing photographs and detailed data about the source and its environment. We are grateful to Dr. B. Páll-Gergely for constructive comments on the manuscript.