Cavefish Research Articles

Gross morphology of the brain and some sense organs of subterranean pencil catfishes of the genus Ituglanis Costa and Bockmann, 1993 (Siluriformes, Trichomycteridae), with a discussion on sensory compensation versus preadaptation in subterranean fishes.

Subterranean organisms provide excellent opportunities to investigate morphological evolution, especially of sensory organs and structures and their processing areas in the central nervous system. We describe the gross morphology of the brain and some cephalic sensory organs (olfactory organ, eye, semicircular canals of the inner ear) and the swim bladder (a non-sensory accessory structure) of subterranean species of pencil catfishes of the genus Ituglanis Costa and Bockmann, 1993 (Siluriformes, Trichomycteridae) and compare them with an epigean species of the genus, Ituglanis goya Datovo, Aquino and Langeani, 2016. We compared qualitatively the size of the different brain regions and sense organs of the subterranean species with those of the epigean one, searching for modifications possibly associated with living in the subterranean environment. Our findings suggest that species of Ituglanis exhibit sensory characteristics that are preadaptive for the subterranean life, as only slight modifications were observed in the brains and sense organs of the subterranean species of the genus when compared with the epigean one. Because most subterranean fish species belong to lineages putatively preadapted for subterranean life, our results, discussed in the context of available information on the brain and sense organs of other subterranean species, help identify general trends for the evolution of the brain and sensory organs of subterranean fishes in general.

Sinocyclocheilusxiejiahuai (Cypriniformes, Cyprinidae), a new cave fish with extremely small population size from western Guizhou, China.

This study describes a new species, Sinocyclocheilusxiejiahuai sp. nov., discovered within a cave located in Hongguo Town, Panzhou City, Guizhou Province, southwestern China, with the type locality in the Nanpanjiang River basin. Phylogenetic trees reconstructed based on mitochondrial genes show that the new species represents an independent evolutionary lineage with large genetic differences, 1.9%-13.8% in mitochondrial Cyt b, from congeners. Morphologically, this species can be differentiated from the 79 species currently classified under the genus Sinocyclocheilus by several characteristics: absence of horn-like structures and indistinct elevation at the head-dorsal junction, absence of irregular black markings on the body lateral and scaleless, eyes large, eye diameter 13% of head length, dorsal-fin rays, iii, 6½, last unbranched ray strong, with serrations along posterior margin, pectoral-fin rays, i, 13, anal-fin rays, iii, 5, pelvic-fin rays, i, 7, lateral line pores 74, gill rakers well developed, nine on first gill arch, pectoral fins short, tip not reaching to pelvic-fin origin. The number of Sinocyclocheilus species has been increased from 79 to 80 since the description of this new species.

Four new hypogean species of the genus Triplophysa (Osteichthyes, Cypriniformes, Nemacheilidae) from Guizhou Province, Southwest China, based on molecular and morphological data

Recently described cave species of the genus Triplophysa have been discovered in southwestern China, suggesting that the diversity of the genus is severely underestimated and that there may be many undescribed species. In this work, four new species of the genus Triplophysa are described from southwestern Guizhou Province, China, namely Triplophysa cehengensis Luo, Mao, Zhao, Xiao & Zhou, sp. nov. and Triplophysa rongduensis Mao, Zhao, Yu, Xiao & Zhou, sp. nov. from Rongdu Town, Ceheng County, Guizhou, Triplophysa panzhouensis Yu, Luo, Lan, Xiao & Zhou, sp. nov. from Hongguo Town, Panzhou City, Guizhou, and Triplophysa anlongensis Song, Luo, Lan, Zhao, Xiao & Zhou, sp. nov. from Xinglong Town, Anlong County, Guizhou. These four new species can be distinguished from all recognized congeners by a combination of morphological characteristics and significant genetic divergences. The discovery of these species increases the number of known cave species within the genus Triplophysa to 39, making the genus the second most diverse group of cave fishes in China after the golden-line fish genus Sinocyclocheilus. Based on the non-monophyletic relationships of the different watershed systems in the phylogenetic tree, this study also discusses the use of cave species of the genus Triplophysa to determine the possible historical connectivity of river systems.

The biomimetic potential of novel adaptations in subterranean animals

Subterranean habitats can be considered harsh conditions with lack of light, low nutrients levels and constant high humidity. To thrive under such conditions, cave-adapted species have evolved a range of novel morphological, physiological and behavioural adaptations. These adaptations might have significant biomimetic potential. Biomimetics or biologically inspired design is a relatively new interdisciplinary field that aims to harness the processes and mechanisms in nature that have been optimised over millions of years’ evolution to improve our own technology. There are two main approaches to biologically inspired design—the problem-driven approach starting with an engineering problem and searching through biological equivalents and the solution-driven approach, which starts with a biological example or solution followed by the identification of a suitable engineering application. While the former approach is the most popular and is favoured by engineers, the latter remains the most successful and is typically driven by fundamental biological research. However, few biomimetic solutions or concepts have so far been described from subterranean habitats despite the rich potential. In this review paper, I first outline the theory behind biologically inspired design before I review the few biomimetic related studies of cave adapted organisms mainly based on the exceptional lateral line systems in blind cave fish. However, the main body of the review focuses on identifying and discussing subterranean adaptations with a particular strong potential including biomimetics sensors, adhesion in high humidity and wet conditions and biomaterials

Selection Maintains the Phenotypic Divergence of Cave and Surface Fish.

AbstractGenetic divergence in the presence of gene flow has been well documented, but there is little information on the specific factors maintaining divergence. The present study investigates this in the Mexican tetra (Astyanax mexicanus), an excellent model for studying this question because surface and cave populations differ markedly in phenotype and genotype but are interfertile. Previous population studies documented significant gene flow among cave and surface populations, but they focused on analyses of neutral markers whose evolutionary dynamics likely differ from those of genes involved in cave adaptation. The present study advances our understanding of this question by focusing specifically on the genetics responsible for eye and pigmentation reduction, signature traits of cave populations. Direct observations of two cave populations over the course of 63 years verify that surface fish frequently move into the caves and even hybridize with the cave fish. Importantly, however, historical records show that surface alleles for pigmentation and eye size do not persist but are rapidly eliminated from the cave gene pool. It has been argued that regression of eyes and pigmentation was driven by drift, but the results of this study suggest that strong selection actively eliminates surface alleles from the cave populations.

Evidence for rapid divergence of sensory systems between Texas populations of the Mexican tetra (Astyanax mexicanus)

Population divergence is often quantified using phenotypic variation. However, because sensory abilities are more difficult to discern, we have little information on the plasticity and rate of sensory change between different environments. The Mexican tetra (Astyanax mexicanus) is a fish distributed throughout Southern Texas and Northern Mexico and has evolved troglomorphic phenotypes, such as vestigial eyes and reduced pigmentation, when surface ancestors invaded caves in the past several hundred thousand years. In the early 1900s, surface A. mexicanus were introduced to the karstic Edwards-Trinity Aquifer in Texas. Subsequent cave colonization of subterranean environments resulted in fish with phenotypic and behavioral divergence from their surface counterparts, allowing examination of how new environments lead to sensory changes. We hypothesized that recently introduced cave populations would be more sensitive to light and sound when compared to their surface counterparts. We quantified divergence using auditory evoked potentials (AEPs) and particle acceleration levels (PALs) to measure differences in sound sensitivity, and electroretinography (ERGs) to measure light sensitivity. We also compared these results to measurements taken from native populations and lab-born individuals of the introduced populations. Honey Creek Cave fish were significantly more sensitive than proximate Honey Creek surface fish to sound pressure levels between 0.6 and 0.8 kHz and particle acceleration levels between 0.4 and 0.8 kHz. Pairwise differences were found between San Antonio Zoo surface and the facultative subterranean San Pedro Springs and Blue Hole populations, which exhibited more sensitivity to particle acceleration levels between 0.5 and 0.7 kHz. Electroretinography results indicate no significant differences between populations, although Honey Creek Cave fish may be trending toward reduced visual sensitivity. Auditory thresholds between wild-caught and lab-raised populations of recently invaded fish show significant differences in sensitivity, suggesting that these traits are plastic. Collectively, while these results may point to the rapid divergence of A. mexicanus in cave habitats, it also highlights the responsive plasticity of A. mexicanus auditory system to disparate environments.

The world’s largest cave fish from Meghalaya, Northeast India, is a new species, Neolissochilus pnar (Cyprinidae, Torinae)

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.

Draft genome assembly for the colombian freshwater bocachico fish, Prochilodus magdalenae.

We report the first draft genome assembly for Prochilodus magdalenae, the leading representative species of the Prochilodontidae family in Colombia. This 1.2-Gb assembly, with a GC content of 42.0% and a repetitive content of around 31.0%, is in the range of previously reported characid species genomes. Annotation identified 34,725 nuclear genes, and BUSCO completeness value was 94.9%. Gene ontology and primary metabolic pathway annotations indicate similar gene profiles for P. magdalenae and the closest species with annotated genomes: blind cave fish (Astyanax mexicanus) and red piranha (Pygocentrus nattereri). A comparative analysis showed similar genome traits to other characid species. The fully sequenced and annotated mitochondrial genome reproduces the taxonomic classification of P. magdalenae and confirms the low mitochondrial genetic divergence inside the Prochilodus genus. Phylogenomic analysis, using nuclear single-copy orthologous genes, also confirmed the evolutionary position of the species. This genome assembly provides a high-resolution genetic resource for sustainable P. magdalenae management in Colombia and, as the first genome assembly for the Prochilodontidae family, will contribute to fish genomics throughout South America.

Sinocyclocheiluslongicornus (Cypriniformes, Cyprinidae), a new species of microphthalmic hypogean fish from Guizhou, Southwest China.

Sinocyclocheiluslongicornussp. nov. is described from the Pearl River basin in Hongguo Town, Panzhou City, Guizhou Province, Southwest China. Based on the presence of the long horn-like structure on the back of the head, Sinocyclocheiluslongicornussp. nov. is assigned to the Sinocyclocheilusangularis species group. Sinocyclocheiluslongicornussp. nov. is distinguished from its congeners by a combination of morphological characters: (1) presence of a single, relatively long horn-like structure on the back of the head; (2) pigmentation absent; (3) reduced eyes; (4) dorsal-fin rays, ii, 7; (5) pectoral-fin rays, i, 13; (6) anal-fin rays, iii, 5; (7) pelvic-fin rays, i, 7; (8) lateral line pores 38-49; (9) gill rakers well developed, nine on first gill arch; and (10) tip of adpressed pelvic fin not reaching anus.

A Review on Hidden Subterranean (Hypogean) Ichthyofaunal Resources of India along with IUCN Status, Threats and Its Conservation Strategies

Subterranean habitats are one of the most peculiar and mysterious ecosystems on earth, yet we still have very less information about their biodiversity. These animals often live in extremely nutrient-limited environments. Consequently, most of the subterranean fish species are relatively small in size to survive on limited food resources. We present here a systematic checklist along with IUCN conservation status and a comprehensive account of the diversity, distribution, threats and suggest conservation measures for the Indian subterranean fishes. A total of 18 species under 5 orders, 8 families and 7 genera are listed from the secondary data. According to the IUCN Red List status, nine subterranean fish fauna are listed under the Not Evaluated (50%) category, five Data Deficient (27.78%), two endangered (11.11%), and only one species come under the Critically Endangered (5.56%) and Least Concern category (5.56%). Several anthropogenic stressors including agriculture, overexploitation, deforestation, habitat disturbance, hydroelectrically plans, invasive species, human-fish conflicts, climate change, sand mining and un-ecofriendly tourism are threatening these underground fishes. Nevertheless, there have been only a few studies on subterranean fish fauna, partly because many subterranean occur in tropical regions, sometimes in caves of difficult access, where there are few local researchers. It is necessary to promote throughout society the importance and urgency of studying and conserving subterranean resources to preserve their ecosystem services.

Geology, climate, and hydrochemistry shape the spatial patterns of multiple diversity facets and functional traits of fishes

AbstractIn a large river with significant environmental variations in space (i.e., microhabitat) or over time (i.e., seasonality and/or long‐term trend), discovering the spatial changes in fish diversity, distributions, and functional traits would provide an important foundation for the understanding of ecosystem structure and function. In the Pearl River in China, three datasets as fish distribution, species functional traits, and environmental variables were constructed and sorted to (1) test the relationships between and congruence of taxonomic diversity, functional diversity, and phylogenetic diversity by a randomisation procedure, (2) map the spatial pattern of diversity and the correlation to environmental variables by maximal information coefficient, and (3) determine the fish traits tendency under environmental changes by the fourth‐corner method. The Pearl River possesses the highest fish richness among all the rivers in China, with 438 freshwater fish species including 119 cave species. At basin scale, phylogenetic diversity showed faster change than functional diversity, suggesting a little divergence of functional traits despite high phenotypic or genetic diversity across species, as parallel evolution. However, at a small scale, when only considering cave fishes, the variation rate of species traits was faster than that of species increase, as evolutionary radiation in a variety of microhabitats. For all three aspects of diversity, random permutation tests confirmed that congruence values in each grid cell were significantly different from the random expectations. Besides widely accepted variables such as altitude, precipitation, and slope concerned with fish biogeography, hydrochemical variables showed influential impacts on the morphological and growth traits of fishes in the Pearl River characterised by the dominance of the karst habitat.

Mutagenesis alters sperm swimming velocity in Astyanax cave fish

We investigated the hypothesis that intra ejaculate sperm competition screens against the transmission of deleterious alleles, including new mutants, from male parent to offspring. Recent investigations have established that sperm haploid genotypes can have major effects on sperm traits such as cellular robustness, longevity, and fertilization success. However, there is no evidence that new mutations can meaningfully affect sperm phenotypes. We tested this directly by comparing sperm from mutagenized and non-mutagenized control males in Astyanax fish. We used N-ethyl-N-nitrosourea (ENU) to induce single base substitutions in spermatogonial stem cells. We looked at swimming velocity, an important factor contributing to fertilization success, and flagellar length. Variability in swimming velocity was significantly higher in sperm from mutagenized males than in control sperm, reflecting their increased allelic diversity. In contrast, flagellar length, which is fixed during diploid stages of spermatogenesis, was unaffected by ENU treatment. We briefly discuss the implications of intra-ejaculate screening for maintenance of anisogamy and for outcomes of assisted reproductive technology.

Color Cost: Cave fish trade their pigment for a thriftier metabolism.

Color Cost: Cave fish trade their pigment for a thriftier metabolism.

A New Locality for the Blind Loach, Eidinemacheilus smithi (Teleostei: Nemacheilidae) in Iranian Zagros: A Morpho-Molecular Approach

Four obligate cave fish species have been recorded from Zagros in Iran: Garra typhlops (Bruun and Kaiser, 1944), G. lorestanensis (Mousavi-Sabet and Eagderi, 2016), G. tashanensis (Mousavi-Sabet, Vatandoust, Fatemi and Eagderi, 2016) and Eidinemacheilus smithi (Greenwood, 1976). So far, the only known locality of the latter has been the Loven cave. Here, we extend the known range of the Zagros blind loach (E. smithi) by reporting a new locality, the Tuveh spring, located 31 km south of Loven. We combine morphological evidence with the calculated K2P genetic divergences of 1.39% between Tuveh and Loven to confirm that these populations are conspecific. Our discovery of the second E. smithi population has implications for the conservation of this rare and vulnerable species.

Environmental Hypoxia is a Driver of Sonic Hedgehog-dependent Troglomorphic Evolution in Astyanax Cavefish

Astyanax mexicanus cavefish (CF) have evolved numerous troglomorphic traits since their divergence from surface fish (SF) ancestors about 20,000 years ago. A large number of these traits are under the control of Sonic Hedgehog (Shh), a signaling morphogen that is overexpressed along the midline in CF relative to SF embryos. The Shh-dependent traits include eye degeneration and increases in the olfactory lobes, hypothalamus, jaws, and oral taste buds. Eye degeneration and tastebud enhancement are antagonistic tradeoffs governed by the pleiotropic shh gene. CF have also evolved multiple changes in circulatory system development, highlighted by larger hematopoietic domains, more erythrocytes, and reversals in normal heart asymmetry, which are also controlled by Shh signaling. Although Shh signaling is an important factor in CF troglomorphism, the shh gene is not associated with any of the many QTL identified by genetic analysis, suggesting that upstream processes, rather than mutation of the shh locus itself, may be important in Shh regulation. Due to the absence of primary productivity, cave pools harboring Astyanax CF are deficient in dissolved oxygen. Accordingly, the Hypoxia Inducing Factor (HIF) transcription factors and their downstream targets are constitutively active during CF development. To determine if oxygen deficiency is an upstream regulator of Shh signaling, we subjected early SF embryos to 8 or 16 hours of hypoxia in the laboratory and assayed the effects on shh expression, erythrocyte formation, heart asymmetry, and eye development. The results indicated that hypoxia can cause shh overexpression, increase erythropoiesis, reverse heart asymmetry, and induce eye degeneration in SF embryos. We conclude that environmental hypoxia may be responsible for the evolution of troglomorphic traits in Astyanax CF through hyperactivation of the Shh signaling pathway.

Maximized Hydrodynamic Stimulation Strategy for Placement of Differential Pressure and Velocity Sensors in Artificial Lateral Line Systems

Fish can perceive the surrounding flow field using their lateral line systems, consisting of canal neuromasts (CNs) for flow pressure gradient perception and superficial neuromasts (SNs) for flow velocity detection. Although various artificial lateral line (ALL) systems have been developed inspired by the lateral line of fish, few studies have combined both pressure and velocity sensors for hydrodynamic perception, and how to optimize sensor placement remains unanswered, particularly on three-dimensional models. Herein we proposed a maximized hydrodynamic stimulation strategy for sensor placement optimization. We built a fish model and designed an ALL system by mimicking the geometry of a blind cavefish, <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Sinocyclocheilus tianlinensis</i> . Differential pressure sensors and hot-film flow velocity sensors were used to mimic the CNs and SNs, respectively, with optimized sensor placement. The simulation and experimental results on yaw angle detection suggested that both sensor placement of our ALL system and distribution of the biological lateral line showed good agreement with the maximized hydrodynamic stimulation strategy. Our ALL system could detect a wall within a distance of 0.1 body length, comparable to the perception ability of cave fish. The new sensor placement strategy can be used to equip ALL systems on bionic robotic fish and other underwater vehicles.

Two ecological gradients drive phenotypic differentiation of a cave fish over a few hundred metres

AbstractEcological gradients can drive adaptive phenotypic diversification, but the extent of local adaptation depends on the strength, temporal stability and spatial dimensions of selective forces at play. We examined diversification in morphology and life histories of a live-bearing fish (Poecilia mexicana) that has adapted to two concomitant gradients: differential light regimes and toxicity levels in and around a hydrogen sulphide-rich cave. Even though typical ranging distances of P. mexicana exceed the spatial dimensions of both gradients (few hundred metres), clines in head size, body depth, male and female lean mass, offspring size, fecundity and maternal provisioning strategies tracked both gradients. Comparison of two life-history samplings from 2007/2008 and 2014 revealed remarkable temporal stability, even though some degree of temporal variation was seen in fat content and investment into reproduction. Our study demonstrates how patterns of phenotypic gradient evolution can emerge when spatially and temporally stable, strong selection forces alter selective landscapes, resulting in alternate fitness optima of different locally adapted genotypes and impeding individuals’ movement even at a spatial scale far below the species’ potential lifetime ranging distance. This is further evidence for the role of active colonization and gradual adaptation to cave environments in the evolution of some cave organisms.

Molecular phylogeny and historical biogeography of the cave fish genus Sinocyclocheilus (Cypriniformes: Cyprinidae) in southwest China.

Modern accumulations of genetic data offer unprecedented opportunities for understanding the systematic classification and origins of specific groups of organisms. The genus Sinocyclocheilus is among the most cave abundant genera in Cyprinidae, with 76 recognized species, belonging to 4 species groups. Recent phylogenetic studies have shown that the classification of species groups within the genus Sinocyclocheilus remains controversial. In this study, we constructed a sequence supermatrix of 26 species from 4 species groups of the genus Sinocyclocheilus using the mitochondrial genome to reveal phylogenetic relationships, historical biogeography and patterns of species diversification in the genus Sinocyclocheilus. Phylogenetic analysis strongly supports the monophyletic groups of the 3 species groups (S. jii, S. cyphotergous, and S. tingi groups) except the S. angularis group. Phylogenetic analysis showed that S. anshuiensis and S. microphthalmus, which were recognized as numbers of S. angularis group, formed a strongly supported independent clade. Therefore, we propose a new species group, the S. microphthalmus group, which contains S. anshuiensis and S. microphthalmus. Biogeographic reconstruction suggests that the living Sinocyclocheilus may have originated in north-central Guangxi at the late Eocene and dispersed outward after a vicariance at 32.31 Million years ago (Ma). Early diversification is focused on the late Oligocene (ca. 25 Ma), which is related to the second uplift of the Qinghai-Tibetan Plateau and the lateral extrusion of the Indochina at the Oligocene/Miocene boundary. Our results suggest that 2 uplifts of the Qinghai-Tibetan Plateau and climate change in the Miocene may have influenced the diversification of the Sinocyclocheilus lineage.

EDNA metabarcoding as a means to assess distribution of subterranean fish communities: Iranian blind cave fishes as a case study

AbstractOne of the most important steps in conservation of the subterranean life forms is to decipher their distribution and ecology, which is challenging using traditional approaches. Development of an environmental DNA (eDNA) assay provides an efficient means for discovering and monitoring subterranean life forms. In this study, the distribution of three Iranian blind cave fish species (blind Iran cave barb Garra typhlops, blind Lorestan cave barb Garra lorestanensis, and blind cave loach Eidinemacheilus smithi) was assessed using 12S rRNA eDNA metabarcoding performed using MiFish‐U PCR primers and preliminary species distribution modeling (SDM) using bioclimatic data. The majority of sampling localities with positive detection of cave barb eDNA fall within suitable habitats in the Zagros Mountains of Iran. Our results revealed that Lorestan and Iran cave barbs have differential distribution patterns, with some extent of habitat overlap in the vicinity of the originally discovered cave barb locality. According to the observed distribution patterns, the blind Lorestan cave barb and cave loach are mostly distributed in habitats close to the Sezar River (Dez River drainage, Iran), and the blind Iran cave barb is distributed towards the west and probably in a few springs in the Karkheh River drainage. Our data support the previously proposed distribution pattern for the cave barbs, in which the species show partial niche separation and reproductive isolation, with the Lorestan cave barb being a water flow‐dependent species and the Iran cave barb being a generalist species preferring variable flow rates. We showed eDNA metabarcoding to be a useful approach for ecological surveys of subterranean fish biodiversity with implications for conservation.

A first qualitative and quantitative study of marine cave fish assemblages of intracave cavities

This study aims to describe for the first time the diversity and abundance of “cave within cave” fish species assemblage and to study the effects of habitat variables on these assemblage patterns. The term “cave within cave” is used to describe small, enclosed cavities and other enclosed spaces regularly present on the inner surface of large marine caves. The quantitative study was conducted from October to November 2019, in a large marine cave under Lučice Bay, on the south side of Brač Island in the eastern Adriatic Sea. In total 48 one-square-meter surface squares were sampled, extending 40m inside from the cave entrance, and down to a depth of 26m. A total of 18 fish species were found in the studied cave, of which 15 were recorded on the square frames. Nine species were found in intracave cavities, including five species which occurred exclusively in this highly cryptic habitat. The three strictly “cave within cave” fish species (Corcyrogobius liechtensteini, Didogobius splechtnai and Zebrus zebrus) prevailed by abundance and by frequency of occurrence over other twelve species recorded on the squares. The total fish abundance significantly increased with increased light intensity, with steeper surface and from the bottom to the top of the cave, while the total biomass and species richness significantly increased only with the increased light intensity. The assemblage structure of “cave within cave” fishes differed significantly with light intensity, among different surface inclinations and among different cave positions. The “cave within cave“ fish species differ in habitat preferences, some species inhabit the entrance of the cave with high light conditions and rich zoocover, and others inhabit further inside the cave with low light conditions.