Data scarcity and knowledge shortfalls are two of the most important impediments to our ability to understand and conserve life on Earth (Hortal et al. 2015). Despite more than three centuries of natural history exploration and research, we continue to lack fundamental information on the diversity (the ‘Linnean shortfall’) and distribution (the ‘Wallacean shortfall’) of many plant and animal groups (Hortal et al. 2015). Such impediments to our knowledge of the living world are even more acute in the case of organisms inhabiting hidden or inaccessible environments, such as caves and subterranean waters (i.e., the Racovitzan shortfall) (Ficetola et al. 2019), which are at the same time increasingly subjected to anthropogenic threats (Mammola et al. 2019).

Subterranean aquatic habitats often harbour unique assemblages of fishes with a high proportion of ‘point endemics’ or relic lineages, with no close relatives in surface waters (Galassi et al. 2014). This is especially true for bony fishes, with 289 valid species currently known from subterranean aquatic habitats on every continent except Antarctica (Proudlove 2022). These include some spectacular radiations of cave-adapted species (Mao et al. 2022), as well as lineages of ‘living fossils’ (Britz et al. 2020). These unusual fish species have been aptly called the ‘wrecks of ancient life’ (Darwin 1809-1822) and ‘ghosts in the water’ (Niemiller et al. 2019); many of them enjoy unusual scientific names (e.g., Satan eurystomus Hubbs & Bailey, Aenigmachanna gollum Britz, Anoop, Dahanukar & Raghavan), or have emerged as laboratory models (e.g., Astyanax mexicanus (de Filippi)) for understanding evolution, development, behaviour, and human health (Krishnan and Rohner 2017; McGaugh et al 2020).

Notwithstanding their interesting and often extraordinary fauna, subterranean aquatic habitats are good examples of biodiversity shortfalls (Ficetola et al. 2019), primarily because they are inaccessible and their inhabitants are rarely collected. Encounters with these subterranean animals are therefore often serendipitous, or happen when the gateways to the underground water-world are scrutinized – for example in the case of dug-out wells that are drained for maintenance (Ohara et al. 2016; Anoop et al. 2019). As a result of their unique habitat, information on diversity and distribution for most subterranean fish species is either highly incomplete or even absent, with most species known only from type material. Local communities interested in natural history, often the first or sometimes the only people to encounter these species (Ohara et al. 2016; Anoop et al. 2019), are thus potentially able to play a significant role in improving scientific knowledge of this unusual fauna.

A special area of subterranean fish diversity is the lateritic landscape in the southern Indian state of Kerala (Raghavan et al. 2021). It is recognized as a global hotspot for subterranean fishes, presently numbering 10 endemic species in five genera (Aenigmachanna, Horaglanis, Kryptoglanis, Pangio and Rakthamichthys) and two monotypic families (Aenigmachannidae and Kryptoglanidae) (Raghavan et al. 2021; Britz et al. 2022). Some of these fishes exhibit unusual morphological characters such as the absence of eyes and body pigments (Horaglanis spp., and Rakthamichthys spp.), as well as the absence of dorsal- (Kryptoglanis shajii) or pelvic-fins (Aenigmachanna gollum), or even both these fins (Pangio bhujia Anoop, Britz, Arjun, Dahanukar & Raghavan and P. pathala Sundar, Arjun, Sidharthan, Dahanukar & Raghavan).

Horaglanis (Fig. 1A) is a genus of catfishes, remarkable for their bizarre appearance (blind, pigmentless and of blood-red coloration), tiny size (< 35mm), occurrence in a unique habitat (lateritic aquifers) (Fig. 1B), rarity (appearing only occasionally in dug-out wells, Fig. 1C), paucity of museum specimens (known until recently just from a handful of examples), and unresolved phylogenetic and biogeographic affinities (Menon 1951; de Pinna 1993). Though three species are currently known (Horaglanis krishnai Menon, 1951, H. alikunhii Subhash Babu & Nair, 2004, H. abdulkalami Subhash Babu, 2012), the latter two were poorly described; the taxonomic and geographic boundaries between the three species have thus remained unclear. Fewer than ten published records of Horaglanis backed by voucher specimens are available (Fig. 1D), and to date no studies have attempted to understand the distribution and genetic diversity of the genus.

A six-year exploratory and citizen science-backed survey across the lateritic landscape of Kerala has resulted in an extensive biogeographic and molecular dataset – the largest ever assembled for Horaglanis. Utilizing new information derived from these samples, we significantly advance knowledge of, and reduce key biodiversity shortfalls for, these enigmatic catfishes. In particular, we unravel range sizes and boundaries, highlight their deep genetic divergence alongside remarkable morphological stasis, and describe a new cryptic species, recovered as the sister taxon of the three previously described members of the genus.

Aquifers are unique subterranean microhabitats owing to their strong hydrographical isolation, limited connectivity with surface waters (mostly through springs, small pools and dug-out wells), and reduced possibilities for long-range dispersal (Trontelj et al. 2009; Juan and Emerson 2010; Galassi et al. 2014; Segherloo et al. 2018). Life in these microhabitats is constrained by ecological conditions including darkness, reduced concentration of nutrients, carbon and dissolved oxygen, and highly restricted free space (Hancock et al. 2005). Of the 289 known subterranean fish species, which include 53 catfishes (Proudlove 2022), fewer than 10% reside in aquifers. Examples include the enigmatic blind catfishes Trogloglanis pattersoni Eigenmann and Satan eurystomus of the Edwards Aquifer in Texas (Langecker and Longley 1983), the Phreatobius catfishes of South America (Muriel-Cunha and de Pinna 2005), the blind gobies of the genus Typhleotris from southwestern Madagascar (Vences et al. 2018), and the blind species of Garra from the Zagros mountains of Iran (Vatandoust et al. 2019). Horaglanis represents the only example of a genus of stygobitic fishes associated exclusively with lateritic aquifers – all other aquifer-dwelling fishes inhabit limestone formations.

Interestingly, no aquifers elsewhere in the world appear to have evolved as diverse a fish fauna as that associated with the laterite soil formations in the coastal area of southwest peninsular India. With the discovery and description of Horaglanis populi, four species of this catfish genus, as well as three species of the swamp-eel genus Rakthamichthys and two species of the eel loach genus Pangio have been discovered from aquifer-fed wells. All these show the typical troglomorphies associated with life underground (Raghavan et al. 2021). Two additional subterranean species, Aenigmachanna gollum and Kryptoglanis shajii Vincent & Thomas, collected also from rice paddies and adjoining wetlands, show no obvious troglomorphies: they may not be strictly aquifer-dwelling species (Raghavan et al. 2021). Overall, the diversity of subterranean fishes in southern peninsular India is rivalled only by the radiation of cave fishes of the genus Sinocyclocheilus in the karstic regions of southwest China (Zhou et al. 2022).

Much like the subterranean habitat in which the genus is found, Horaglanis has received very little scientific attention, despite being arguably one of the most unusual genera of catfishes known. Of the three nominal species previously known, only Horaglanis krishnai has been studied in any detail (Menon 1952; Mercy et al. 1982; Mercy and Pillai 1985; Mercy et al. 2001; Mercy and Pillai 2001). The remaining two are known only from their original descriptions (Babu and Nayar 2004; Babu 2012). Our extensive dataset of 47 new location records and 65 new genetic sequences shows that Horaglanis is endemic to the part of Kerala State south of the Palghat Gap (Fig. 1E), a well-known biogeographic barrier. With the exception of the species pair H. abdulkalami and H. alikunhii, the species of Horaglanis occur in allopatry. The genus is restricted to lateritic aquifers and has been encountered solely in groundwater-fed wells. While we have no information on the number, size and extent of the aquifers populated by Horaglanis, we found that the northern extent of its range is limited by the Bharathapuzha (the second largest river basin in the region), as well as a wide zone of rock formations in which laterite rock is absent. This zone (i.e., the northern extremity of the distribution of the genus) coincides with the Palghat Gap. Similarly, the major barrier separating the southern H. krishnai and H. populi from the other two species is likely the Periyar-Chalakudy River basin (the largest river basin in the region). However, some populations of H. abdulkalami, though substantially divergent genetically from those close to the type locality of this species, occur south of the Periyar.

Some stygobitic fishes are known to have large distribution ranges, such as the catfish Prietella phreatophila Carranza, whose northern and southernmost populations in Mexico are separated by a span of 750 km (Hendrickson et al. 2001), or the blind, subterranean cave eel, Ophisternon candidum (Mees), a north-western Australian endemic (>400 km) (Moore et al. 2018). This vast distribution range could be attributed to the ‘interstitial highway’ hypothesis (Ward and Palmer 1994), i.e., the presence of an extensive, continuous hypogean habitat. Compared to P. phreatophila and O. candidum, the distribution ranges of all four species of Horaglanis lie within a north-south span of only 150 km. The current distribution of the various species of Horaglanis is likely the result of vicariance events, or the traditional low-dispersal (movement within the aquifers) hypotheses (Trontelj et al. 2009) associated with most subterranean taxa – for example, the blind cave fishes of Iran (Segherloo et al. 2022). Though Horaglanis populations are able to move through the narrow pores of aquifers, they are likely confined by barriers such as the ones mentioned above, which limit longer distance movements. Another potential vicariance barrier may have resulted from historical changes of eustatic sea level. These have occurred frequently since the late Miocene and continued into the Pleistocene (Miller et al. 2005, 2020); they would have led to prolonged marine transgression of the coastal areas of Kerala, to which Horaglanis is endemic. These marine transgressions may also have played a role in forming distribution barriers leading to vicariant speciation (discussed for the case of Sri Lanka in Pethiyagoda & Sudasinghe 2021). The interesting and complex distribution pattern of Horaglanis is thus likely linked to successive isolation and reconnection events (Devitt et al. 2019), that can be further unraveled through integrative phylogenomic and hydrological studies.

By generating the first multi-gene phylogeny of Horaglanis, we discovered that H. populi comprises the sister group of the clade containing its congeners, from which it is separated by a genetic distance (in the barcoding region of COI) of 13.8–17.4%. With interspecific divergences of 7.0-17.4%, the four species of Horaglanis were unambiguously delimited into distinct species based on both the barcode gap analysis and the Poisson tree process. This large interspecific genetic distance in Horaglanis is in sharp contrast to the lower genetic divergence in the COI gene (3.8%) between the morphologically distinct Garra typhlops Bruun & Kaiser and G. lorestanensis Mousavi-Sabet & Eagderi, two sympatric, blind Iranian cave barbs (Segherloo et al. 2012), but comparable to the three obligate cave-dwelling gobies of the Malagasy genus Typhleotris (Vences et al. 2018), in which it is 6.3-9.8%. Although the data on genetic divergence in subterranean catfishes are limited, both the maximum intraspecific and minimum interspecific genetic divergence in Horaglanis is higher than those for most surface-dwelling catfishes (see for example, Anjos et al. 2020; Bhattacharjee et al. 2012; Hashimoto et al. 2020; Zou et al. 2020).

While the descriptions of H. abdulkalami and H. alikunhii (Babu and Nayar 2004; Babu 2012), which were based on four specimens and a single specimen, respectively, provided several characters to distinguish them from H. krishnai, our study, based on larger series of specimens, indicates that all four species of Horaglanis are indistinguishable in external morphology. The meristic data showed strong overlap in the character states among the four species. Though the morphometric data were available only for three of these (Table 6, Fig. 4), the limited data available on H. alikunhii from its original description (Subhash Babu and Nayar 2004) suggests that the species is not morphometrically different from its three congeners. Horaglanis thus provides a case of extreme morphological stasis, similar to that seen in the African freshwater butterfly fish Pantodon (Lavoué et al. 2010), the Lake Tanganyikan cichlid Tropheus (Sturmbauer and Meyer 1992), and the subterranean catfishes of the genera Rhamdiospsis and Trichomycterus (Trajano 2021). Morphological stasis is often attributed to stabilizing selection (Sturmbauer and Meyer 1992; Parson 1994) influenced by factors such as lack of interspecific competition (Sturmbauer and Meyer 1992; Trajano 2021), the high energetic costs of life in harsh environments that preclude major evolutionary change (Turner 1986), low metabolic rates leading to low fecundity as an adaptation to survive in stressful and low-energy environments that restrict further morphological changes (Howarth 1993), and to life in stable phreatic environments over a long period (Trajano 2021). We suspect that morphological stasis in Horaglanis may be the result of a combination of several of these factors. The small pore size of the lateritic rocks in the aquifers restricts access to this habitat for other subterranean predators such as Aenigmachanna (Britz et al. 2020), resulting in a predator-free environment for Horaglanis, thus severely limiting interspecific competition. The low number of just 25 to 30 comparatively large eggs in Horaglanis (Mercy 1981) may be a response to living in a nutrient poor habitat. Compared to other aquatic systems, which are influenced by numerous external factors, lateritic aquifers would have provided a stable and ecologically homogenous environment for Horaglanis over a long period of time, likely the most important factor in stabilizing its external morphology despite speciation and significant diversification at the genetic level.

Our decision to describe a new species of Horaglanis based exclusively, at this point, on differences in the COI barcoding gene has not been taken lightly. We were faced with the decision to either (1) synonymize H. alikunhii and H. abdulkalami with H. krishnai, given the lack of external diagnostic characters, and also include the southernmost Horaglanis, we here refer to H. populi, in this taxon, or (2) to make a name available for the latter. In view of the substantial genetic divergence of this southern Horaglanis from its already named congeners, a divergence otherwise not even encountered between genera of catfishes (see for example, Bhattacharjee et al. 2012; Zou et al. 2020), we decided to describe a new species based solely on molecular characters. Lending further support to our decision is the fact that the four species form reciprocally monophyletic clades in the multigene phylogeny. This is confirmed by genetic species delimitation based on two independent methods: genetic barcode gap analysis (using ASAP) and Poisson tree process (using PTP, bPTP and mPTP). It remains to be investigated whether the morphological stasis among species of Horaglanis that we encountered in relation to external characters applies also to internal anatomical characters.

Part of the reason why Horaglanis has received only cursory scientific attention in the past is the rarity of the occasions on which these fishes have been collected. The dearth of specimens has rendered detailed studies on their anatomy, ecology and life history impossible. Our ongoing ‘citizen science’ campaign has helped raise awareness of the subterranean fauna of southern India, and this has in turn dramatically increased the number of occurrence reports of these interesting fishes. It has also led to more specimens becoming available for research. Interested citizen naturalists have, no doubt, been at the forefront of aiding improvement of our knowledge of Horaglanis, especially through making available rare observations, photographs, videos and specimens. Our Horaglanis project is an excellent example of how the involvement of the general public can substantially increase our knowledge of rarely collected organisms that live in relatively inaccessible habitats, through multiplying eyes and ears of researchers by several orders of magnitude (Tricario 2022).

Cryptic species and evolutionarily distinct lineages with small distribution ranges are highly vulnerable to extinction, particularly if residing in groundwater and subterranean habitats (Niemiller et al. 2013). The species of Horaglanis have received little or no protection through local or regional legislation, and their habitats are embedded within densely populated human landscapes. The entrances (often as dug-out wells) to the lateritic aquifers inhabited by Horaglanis have hitherto been reported entirely from privately owned lands, where groundwater is extracted at high levels for both household and agricultural purposes, and laterite soil is extensively mined for developmental activities (Raghavan et al. 2021). Given that many localities in which Horaglanis occurs are within 30 km of the coast, an additional threat is the intrusion of seawater into these aquifer systems, from which water extraction is both substantial and unregulated (see Prusty and Farooq 2020). Ensuring the security of these enigmatic stygobitic catfishes in the lateritic aquifers of Kerala will therefore require a landscape-level planning and implementation approach involving a variety of stakeholders. These will have to include local communities that have played the most important role in helping bridge biodiversity knowledge shortfalls.