The world’s largest subterranean fish was discovered in 2019, and was tentatively identified as a troglomorphic form of the golden mahseer, Tor putitora. Detailed analyses of its morphometric and meristic data, and results from molecular analyses now reveal that it is a new species of the genus Neolissochilus, the sister taxon of Tor. We formally describe the new species as Neolissochilus pnar, honouring the tribal communities of East Jaintia hills in Meghalaya, Northeast India, from where it was discovered. Neolissochilus pnar possesses a number of characters unique among species of Neolissochilus, with the exception of the similarly subterranean N. subterraneus from Thailand. The unique characters that diagnose N. pnar from all epigean congeners comprise highly reduced eye size to complete absence of externally visible eyes, complete lack of pigmentation, long maxillary barbels, long pectoral-fin rays, and scalation pattern. Neolissochilus pnar is distinguished from the hypogean N. subterraneus, the type locality of which is a limestone cave ~2000 kms away in Central Thailand, by a lesser pre-pelvic length (47.8–49.4 vs. 50.5–55.3 %SL), a shorter caudal peduncle (16.1–16.8 vs. 17.8–23.7 %SL), and shorter dorsal fin (17.4–20.8 vs. 21.5–26.3 %SL). In addition, Neolissochilus pnar is also genetically and morphologically distinct from its close congeners with a raw genetic divergence of 1.1–2.7% in the COI gene with putative topotype of N. hexastichus and 2.1–2.6% with putative topotype of N. hexagonolepis.

Eastern Himalaya, limestone cave, mahseer, new species, subterranean fishes

Roughly 1.6% (293 species) of all known (~18,000) freshwater fish species live their whole lives either in caves, or in groundwater aquifers (Proudlove 2023). These ‘troglobiotic’ or ‘stygobiotic’ fishes occur in 36 countries across six continents, with China harbouring close to one-third (96 species) of the global diversity, followed by Brazil (43 species), Mexico and India (18 species each) (Proudlove 2023). A number of these species are evolutionary relics of an ancient fauna, often with long-term isolation in these high-stress environments (Gibert and Deharveng 2002). Such evolutionarily relictual lineages include among others, the Aenigmachannidae – a “living fossil” with a putative Jurassic origin (Britz et al. 2020), the enigmatic Kryptoglanidae (Britz et al. 2014), the cistern catfish Phreatobius (Muriel-Cunha and de Pinna 2005), and the blind aquifer-dwelling Horaglanis (Raghavan et al. 2023). However, the large majority of cavefishes represent “young” lineages that have much more recently invaded subterranean habitats – examples include the European cave loach, Barbatula sp., and the Mexican blind swamp eel, Ophisternon infernale (Behrmann-Godel et al. 2017; Mar-Silva et al. 2022).

Most subterranean fishes have evolved a small-sized body plan to meet the limitations in food resources, light availability and space in underground habitats. The mean size of subterranean fish species is 85.5 mm, with most species below 130 mm (Harries et al. 2019), with at least eight species reaching sizes in excess of 200 mm (Table 1). The largest-known subterranean fish until now is the Australian blind cave eel Ophisternon candidum Mees (family Synbranchidae) which measures 385 mm in total length (Moore et al. 2018).

Stories of a ‘white cavefish’ from the Siju Caves in the Garo Hills of Meghalaya, Northeast India have been documented for 100 years, but were suggested to be slightly decolorized specimens of Neolissochilus hexastichus (M’Clelland) that appeared almost white when observed inside the water, under the light of a torch (Kemp and Chopra 1924). Only in the 1990s, a large, pale, cyprinid fish was observed in the limestone caves of the Jaintia Hills of Meghalaya (Harries et al. 2008), individuals of which were eventually photographed and collected in 2019 (Harries et al. 2019), and made available for detailed scientific studies. The largest individual observed in the cave exceeded 400 mm in standard length, which makes it the largest known individual of any subterranean fish in the world (Harries et al. 2019). Preliminary morphological studies based on two, medium-sized specimens, revealed morphometric and meristic data similar with the golden mahseer, Tor putitora Hamilton (member of the cyprinid sub-family Torinae). However, they also showed significantly different characters including a complete lack of pigmentation and a reduction of the eye, which is small in juveniles, and completely invisible externally in adults (Harries et al. 2019).

The availability of additional fresh specimens of this unique cyprinid fish has now enabled us to study its morphological characters in more detail and to include it in a molecular genetic analysis. This combined evidence reveals that the world’s largest cavefish is an undescribed species of the cyprinid genus Neolissochilus, for which we make a name available below.

The limestone caves of Meghalaya, in northeastern India harbour a remarkable diversity of subterranean taxa (Harries et al. 2008), including several enigmatic fish species. The region is one of the two hotspots of subterranean fish diversity and endemism on the Indian subcontinent, the other being the lateritic aquifers of Kerala (Raghavan et al. 2021). At least three species of subterranean nemacheilid loaches, Schistura sijuensis Menon, S. papulifera Kottelat, Harries & Proudlove, and S. larketensis Choudhury, Mukhim, Basumatary, Warbah & Sarma are already known from this region (Proudlove 2022). Neolissochilus pnar, the largest cave fish described until now, is a remarkable addition to this cave ichthyofauna of the Eastern Himalayan region.

The genus Neolissochilus represents a poorly-known group of medium- to large-sized cyprinids, with currently 31 species, distributed across South and Southeast Asia (Fricke et al. 2023). Despite its cultural and commercial importance, there have been no comprehensive studies on the taxonomy or systematics of this group since the description of the genus (Rainboth 1985). As a result, the identity and distribution of the majority of Neolissochilus species remains unclear. Even the identity of commercially-valuable species of Northeast India, such as N. hexastichus and N. hexagonolepis, on which much research has been carried out, have been considered to be confusing (Rainboth 1985). The advent of molecular taxonomy, has nevertheless resulted in the proliferation of large numbers of genetic sequences representing various species of Neolissochilus, but only very few sequences are linked to morphological data and/or voucher specimens. Our phylogenetic analysis reveals clearly the extent of this chaos related to the misidentifications of Neolissochilus species in GenBank, with currently available sequences forming distinct monophyletic clades, despite being identified and lodged under the same name (Fig. 5).

Laskar et al. (2013) clarified the identity of both N. hexagonolepis and N. hexastichus using an integrative taxonomic approach using topotypic specimens. For the sake of the present study, we consider the clades that includes sequences used by Laskar et al. (2013) for N. hexagonolepis and N. hexastichus to represent these species (marked as sensu Laskar et al. 2013 in Figs 4 and 5). Neolissochilus pnar, and the putative topotypes of N. hexastichus sensu Laskar et al. (2013) show very low genetic divergence – a raw genetic distance of 1.1–2.7% in the COI gene. However, the two species are clearly, morphologically distinct. Based on the original description (M’Clelland 1839, p. 269, pl. 39, fig. 2), N. pnar differs from N. hexastichus in having more lateral line scales (30–32 vs. 25), more dorsal-fin rays (13 vs. 11), more transverse scale rows (8 vs. 6) and a longer maxillary barbel that reaches the anterior margin of the opercle (vs. shorter, not reaching anterior margin of opercle). Similarly, Neolissochilus pnar and putative topotypes of N. hexagonolepis sensu Laskar et al. (2013) are separated by a raw genetic distance of 2.1 to 2.6% in the COI gene. Based on the original description of N. hexagonolepis (M’Clelland 1839, p. 270, pl. 41, fig. 3), N. pnar is distinct in having more lateral line scales (30–32 vs. 27) and anal-fin rays (8 vs. 7).

The sister taxon of Neolissochilus pnar (Fig. 5) is likely to be an epigean congener that is currently misidentified both in the literature, and in GenBank as ‘N. hexastichus’. This sister group includes specimens from the Brahmaputra River basin in the neighboring states of Assam and Nagaland, as opposed to the type locality of N. pnar that drains into the Meghna River basin. The fish identified as N. hexastichus in the Siju Cave, Garo Hills, Meghalaya (Hora 1924; Kemp and Chopra 1924), and the unidentified pale cyprinids in the same cave in February 2019 (Harries et al. 2020) could also likely belong to this group, but this needs to be confirmed. Though N. pnar forms a reciprocally monophyletic clade distinct from this sister taxon, and supported additionally by the ASAP-based species delimitation, the two groups are separated by a low raw genetic distance of 0.5–0.8% in the COI gene. The low genetic divergence between the valid species of Neolissochilus have also been documented previously (Laskar et al. 2013; Lalramliana et al. 2019). However, further studies using multiple genes, supported by morphological and skeletal anatomical observations are required to conclusively understand this interesting sister-taxon relationship, and also the genetic diversity and its correlation with morphological diversity in members of the genus Neolissochilus.

One of the paratypes (KUFOS.F.2022.703) was collected from about 1.2 km inside the adjacent Krem Chympe cave, had a slightly different appearance including distinctly larger eyes and scalation pattern than fish of a similar size from those in the Krem Um Ladaw. Although the paratype of Neolissochilus pnar from the Krem Chympe cave was identical to the COI barcoding region and partial 16S genes of holotype and paratype from Krem Um Ladaw, there is a 1.4% raw genetic distance in the cyt b gene between the two populations.

The eye size in individuals of the Krem Um Ladaw population reduces as fish size increases. The smallest individuals have distinct, but atrophied eyes, which then become less distinct in larger individuals, and appear to be entirely absent in the largest individuals. This pattern appears consistent over all individuals photographed and videoed in the Krem Um Ladaw.

Genetic data for Neolissochilus subterraneus, the only other known subterranean species of Neolissochilus described from Tham Phra Wang Daeng cave in Thailand (Vidthayanon and Kottelat 2003), are not available. However, N. pnar is morphologically distinct from N. subterraneus, and the two species form distinct clusters (PERMANOVA, 9999 permutations, F = 7.572, p = 0.0084) in multivariate morphometric space (Fig. 6). It is also highly unlikely that these two species inhabiting two distinct biogeographic regions – N. pnar in the Eastern Himalaya and N. subterraneus in Indo Burma, separated by the 950km long Arakan Mountains, are conspecific.

Figure 6. 

Principal component analysis biplot of factor scores and factor loadings of morphometric data of the two subterranean species of Neolissochilus. Factor scores are shown as scatter of points and factor loadings are shown as arrows. Percentage variation, out of total variation in the data, explained by each principal component, are provided in parenthesis.

Neolissochilus pnar is the largest known troglobitic species by a considerable margin. There has been the view that troglobitic adaptations are a consequence of the limited food availability in cave habitats. The need to locate sparse food reserves is thought to drive the development of the enhanced chemosensory capabilities typical of troglobites (Wilkens and Strecker 2017: 91–94). Among troglobitic fish, the limited food availability is also thought to constrain the body size of fish that can develop in the cave environment (Volkoff 2016). So, the occurrence of N. pnar, a cave fish with both a relatively large size and striking troglomorphies from the Um Ladaw requires further study.

Despite the ichthyofaunal richness in aquifers and caves on the Indian subcontinent, there is only a limited number of studies dealing with their diversity and distribution. Recent descriptions of not only new species (Choudhury et al. 2017; Anoop et al. 2019; Britz et al. 2019; Sundar et al. 2022; Raghavan et al. 2023), but also new genera (Vincent and Thomas 2011; Britz et al. 2019, 2021) and even family level taxa (Britz et al. 2014; 2020) of freshwater fishes from the subterranean waters of India suggests major knowledge gaps in our understanding of these largely inaccessible habitats of the Indian subcontinent. Given that these habitats are also the most vulnerable to a number of anthropogenic activities (Raghavan et al. 2021), there is an immediate need to explore and understand the hidden diversity of subterranean realms in the region. Description of the world’s largest subterranean fish Neolissochilus pnar is therefore likely to drive further explorations and understanding of this unique habitat and its remarkable fauna.

The authors thank Daniel Harries, Heriot Watt University, Edinburgh, UK for his generous support and help for this study. Studies of the Meghalayan cave biota would not have been possible without the support of the Meghalayan Adventurers Association (led by Brian Kharpran-Daly), which has supported international teams of cave explorers under the banner of the ‘Caving in the Abode of the Clouds Project’ for the last thirty years. All participants in this project deserve acknowledgement, but are too numerous to mention. Key individuals include Thomas Arbenz (Switzerland) and Simon Brooks (UK). Duwaki Rangad is grateful to the Shri S.M. Sahai IFS, Principal Chief Conservator of Forests (Bio-Diversity & Wildlife) and Chief Wildlife Warden, Government of Meghalaya, Shillong for necessary permissions for survey and collection of specimens (FWC/Research/128). Rajeev Raghavan thanks Mithun Sukumaran (Department of Aquatic Biology, University of Kerala, Thiruvananthapuram, India), Arya Sidharthan (KUFOS, Kochi) and Arjun C.P (MARC, Kannur) for their help and support during the study; Rohan Pethiyagoda (Australian Museum, Sydney) and Ralf Britz (Senckenberg Collections, Dresden, Germany) for useful discussions, guidance and support. Lukas Rüber (Natural History Museum, Bern, Switzerland), an anonymous reviewer, and the handling editor provided constructive comments and useful suggestions that greatly improved the paper.