Due to their peculiar and sometimes bizarre morphology, cave fauna (across invertebrates and vertebrates from both aquatic and terrestrial cave habitats) have fascinated researchers throughout history. Despite their success in colonizing most marine ecosystems, the adaptations of cave brittle stars (Ophiuroidea) to a stygobiotic lifestyle have been scarcely examined. Employing comparative methods on a data set of two species belonging to the genus Ophionereis, this study addresses whether a cave-dwelling species from Cozumel exhibited similar troglomorphic traits as those of other taxa inhabiting caves. Our work demonstrated that some characters representing potential morphological cave adaptations in O. commutabilis were: bigger sizes, elongation of arms and tube feet and the presence of traits potentially paedomorphic. In addition, an element of ophiuroid’s photoreceptor system, as well as pigmentation, was observed to be peculiar in this stygobiotic species, plausibly as a result of inhabiting a low light-energy environment. Finally, we add evidence to the statement that O. commutabilis is a cave endemic species, already supported by demography, distribution and origin of this species, and now by a typical array of troglomorphisms.

adaptation, Aerolito, anchialine system, cave, Cozumel, ophiuroid, stygobiotic

Several traits are often associated with cave-dwellers, which are also known as troglomorphisms, defined as a morphological modification in cave fauna (Culver et al. 1995; Romero 2009). These modifications or morphological traits could be expressed as reductive or constructive traits, such as the lack of photoreceptors or depigmentation in the case of the former and as the hypertrophy of appendages for the latter (Mejía-Ortíz et al. 2006, Gonzalez et al. 2018). All these traits allow organisms to survive in caves, environments that constitute isolated and harsh habitats (Culver and Pipan 2009).

According to Bishop et al. (2015), anchialine caves are “a tidally-influenced subterranean estuary located within crevicular and cavernous karst and volcanic terrains that extends inland to the limit of seawater penetration”. These environments are thought to be food-limited, with detritus and dissolved organic matter being the most important nutritional sources (Mejía-Ortíz et al. 2013). However, several caves have an important contribution of food from bacterial activity (Sarbu et al. 1996; Pohlman et al. 1997; Brankovits et al. 2017). The limitation of food is related to the fact that caves are completely devoid of sunlight and lack of sources of primary productivity (Culver et al. 1995; Gonzalez et al. 2018).

Although cave fauna is mainly composed by crustaceans, several studies have reported organisms of other taxa inhabiting these ecosystems (Sket 1996; Gibert and Deharveng 2002; Calderón-Gutiérrez et al. 2017). Concerning echinoderms, 58 species have been reported inhabiting cave environments, distributed into the five classes of the phylum: Crinoidea (2 species), Asteroidea (8), Ophiuroidea (39), Echinoidea (5) and Holothuroidea (4) (Martínez García et al. 2009; Pomory et al. 2011; Bribiesca-Contreras et al. 2013; Tan et al. 2014; Okanishi and Fujita 2019; Okanishi et al. 2019). However, only five of these species have been described as stygobiotic species (restricted aquatic cave-dwelling organism), the starfish Copidaster cavernicola Solís-Marín & Laguarda-Figueras, 2010 and the ophiuroids Amphicutis stygobita Pomory, Carpenter & Winter, 2011, Ophiozonella cavernalis Okanishi & Fujita, 2018, Ophionereis commutabilis Bribiesca-Contreras et al., 2019 and Ophiopsila xmasilluminans Okanishi, Oba & Fujita, 2019. This is surprising considering that there are over 7,500 species of echinoderms that live in a wide variety of habitats (Zhang 2011). Also striking is the fact that although Ophiuroidea is the largest class of echinoderms (about 2100 described species according to O’Hara et al. (2018)) and are generally photonegative (Stöhr et al. 2012; O’Hara et al. 2018), only a few (39) species have been found in caves.

Troglomorphism has been investigated mainly on arthropods and vertebrates from both aquatic and terrestrial caves (Poulson and White 1969; Mejía-Ortíz and Hartnoll 2005; White and Culver 2012; Mejía-Ortíz et al. 2013; Rizzato and Bichuette 2017). Despite the existence of some studies about troglomorphism on other taxa (White and Culver 2012; Gonzalez et al. 2018), no extensive study has been conducted on stygobiotic echinoderms. Instead, only a few remarks have been made on the morphology of species of echinoderms and its relation with the cave-habitat (Mejía-Ortíz et al. 2007; Solís-Marín and Laguarda-Figueras 2010; Pomory et al. 2011; Brom et al. 2015; Carpenter 2016; Okanishi and Fujita 2018; Bribiesca-Contreras et al. 2019).

Thus, the aim of this study is the identification of morphological adaptations of O. commutabilis. To test the hypothesis that O. commutabilis shows troglomorphisms, we carried out a comparative study between O. commutabilis and its epigean congener O. reticulata. A comparison with other epigean congeners for several traits, as well as the similarities among cave-dwelling brittle stars are discussed. As this is one of the first studies to investigate troglomorphism in brittle stars, our assumptions are mainly based on the analogy to previously identified troglomorphic traits throughout other stygobiotic taxa. Implications for troglomorphism are discussed with respect to concepts of cave biology.

Using a comparative morphological approach, we provide evidence of cave adaptation in O. commutabilis. This study quantified the main morphological differences observed between O. commutabilis and its epigean congener O. reticulata. Our findings were similar to those known in arthropods and are characteristic of changes considered as troglomorphism (Culver et al. 1995; Pérez-Moreno et al. 2016; Gonzalez et al. 2018).

Arm length is the most conspicuous trait of O. commutabilis, here we provide, for the first time, direct and quantitative evidence of this. As previously mentioned, elongation of body appendages is well documented in cave fauna as a troglomorphic trait, hence, arm elongation represents a potential morphological cave adaptation in O. commutabilis. The larger number of segments in O. commutabilis suggests that elongation is the result of having more segments than its epigean congener. Each segment has two tentacle pores, thus cave-dwelling individuals have more tentacle pores and therefore more tube feet, as well as more spines. It has been proposed that appendage elongation increases the ability of cave fauna to locate food, avoid predation or improve sensory capability (Iliffe and Kornicker 2009; Mejía-Ortíz et al. 2006, 2013). Given that both spines and tube feet are important structures in sensory reception of ophiuroids (Hajduk 1992; Zueva et al. 2018), our results are relevant in giving a plausible explanation about the origin and function of adapted elongated arms. Additionally, these results confirm quantitively the first report of a species of Ophionereididae having arms that long (Stöhr et al. 2012; Bribiesca-Contreras et al. 2019) in average and as maximum (Table 5). This is valid not only for Caribbean species but also for species from the Pacific Ocean (e.g. Ophionereis variegata with an arm length average of five times the size of the disc or O. amoyensis with arms ten times the size of the disc) (Clark 1953).

Distal tube feet play an important role on detritus and suspension feeding (Warner 1982), which could explain O. commutabilis having longer distal tube feet than O. reticulata, suggesting that detritus and suspended particles are also (Mejía-Ortíz et al. 2013) an important source of food in the cave El Aerolito. On the other hand, oral tentacle, as well as proximal and middle tube feet, are commonly less specialized (Keogh and Keegan 2006), which provides an explanation for cave species not having longer structures than epigean congeners.

The tube feet, as well as their relation with arms in cave ophiuroids, need to be studied more deeply, as these structures probably play an important role in the evolution of cave ophiuroids. This is suggested by the striking differences in length and function among cave species and their epigean congeners demonstrated in A. stygobita (Carpenter 2016; Pomory et al. 2011), O. xmasilluminans (Okanishi et al. 2019) and O. commutabilis (Table 5), and between the cave species itself. Concerning arm length, the results obtained in this study are similar to that observed in O. xmasilluminans (Okanishi et al. 2019) and contrast with those observed in A. stygobita which have shorter arms (Pomory et al. 2011). Given that abiotic conditions are expected to be similar in the three caves, these differences could support the theory that elongation is associated with food-finding abilities or colonization times rather than specific abiotic factors (Delić et al. 2016).

Our results allowed us to confirm that O. commutabilis is distinguished by reaching big sizes in comparison to other Caribbean species of the genus (Bribiesca-Contreras et al. 2019) (Table 5). This is striking on a cave species, given that usually this fauna has a reduction in size as a response to a diminished food supply or quality (Culver et al. 1995; Culver and Pipan 2009). This could respond to the low predation rate expected for a cave environment (Gibert and Deharveng 2002) and the high population density of O. commutabilis in El Aerolito (Calderón-Gutiérrez and Solís-Marín 2014), factors that have been proposed to favor the survival of bigger organisms (Gage 1990; Bishop and Iliffe 2009). Larger bodies could also be advantageous in terms of energy economy and fecundity, important aspects in a cave (Trontelj et al. 2012). Interestingly, two species inhabiting similar environments show opposite patterns of population densities (low in A. stygobita and high in O. commutabilis) (Carpenter 2016) and relative sizes (small in A. stygobita and big in O. commutabilis) with respect to their epigean congeners.

The contrasting results of this study (O. commutabilis) compared with those obtained by Pomory et al. (2011) in A. stygobita point out the need for more studies on stygobiotic echinoderms. This will improve our understanding of troglomorphisms in the phylum and it would provide useful information to understand the evolution of marine organisms in caves.

Regeneration frequency was lower in O. commutabilis (32% of specimens and 14% of arms) than O. reticulata (70% and 35%). O. commutabilis shows similar percentages to A. stygobita (35% and 14%) (Carpenter 2016), another cave species. On the other hand, the regeneration frequency of O. reticulata is similar to that observed in other populations of this species and other epigean species (Lawrence and Vasquez 1996; Yokoyama and Amaral 2010). Therefore, the low incidence of damage in O. commutabilis indicates that sub-lethal predation is low inside El Aerolito as expected. This could be related with specimens inside the cave being usually found exposed rather than hidden (Bribiesca-Contreras et al. 2019: supplementary video 3), corresponding to the reduced escape behaviour observed on cave fauna (White and Culver 2012).

Stereom organization and porosity is the most conspicuous trait potentially considered as paedomorphic, being more porous in all the analysed ossicles of the cave species. Similar patterns have been proposed as an adaptive trait for deep-sea and cave brittle stars, improving feeding and gas exchange mechanisms and enhancing chemo and mechanoreception in harsh environments (Pomory et al. 2011; Carpenter 2016; Stöhr and Martynov 2016). We suggest that tentacle scales, DAP and VAP shape of O. commutabilis are paedomorphic traits based on the resemblance between mature cave specimen’s ossicles and that of juvenile O. reticulata. Moreover, Bribiesca-Contreras et al. (2019) described conspicuous primary plates as a usual trait of O. commutabilis, which has been suggested as a paedomorphic trait in ophiuroids (Pomory et al. 2011; Martynov et al. 2015). Therefore, it seems evident that like cave brittle stars from Bahamas (Pomory et al. 2011), O. commutabilis specimens show paedomorphic traits on skeletal plates, as previously reported for the size in O. olivacea (Byrne 1991). It must be noted that O. commutabilis does not show some typical paedomorphic traits such as segment length-to-width ratio close to 10 (Stöhr and Martynov 2016). Moreover, some of the traits here reported as paedomorphic could be the result of other factors affecting the biology of this stygobiotic species (e.g. stereom development could be explained by abiotic factors in the cave). However, it must be considered that there are different levels of heterochronic changes affecting ophiuroids, which implies different levels of difficulty to recognize this characters, as well as different evolutionary effects on the species (Stöhr and Martynov 2016). The resemblance in DAP and VAP shape between O. commutabilis and the other Caribbean species (Table 5) should be studied more deeply to understand the evolutionary patterns of this structures in the group as well as the impact of a harsh environment such as caves in this. More species should be compared concerning the potentially paedomorphic traits presented in this work to define whether O. commutabilis present paedomorphosis.

The number of hydropores is equal among species of highly different geographical distribution, but inhabiting similar environments as O. reticulata and O. schayeri (Ezhova et al. 2016). However, that number is very different between closely related species as O. reticulata and O. commutabilis, which inhabit very dissimilar environments. This is consistent with the hypothesis claiming that madreporites are ecologically informative (Ezhova et al. 2016). The differences among species in the type of teeth are relevant if we consider that teeth types have been used as an indicator of feeding preferences (uniform teeth for macrophagous feeders and compound for microphagous) (Medeiros-Bergen 1996; Brogger et al. 2015). Five ophionereidids have been reported to possess only compound teeth (Medeiros-Bergen 1996), while O. commutabilis shows both compound and uniform teeth, which could represent an adaptation, increasing the importance of omnivorous feeding in this cave system (as suggested by Bribiesca-Contreras et al. (2019) for other food sources). An alternative explanation for the presence of uniform teeth in O. commutabilis could be that they represent underdeveloped teeth, either for being ventral (the youngest according to Hendler (2018) or for the slow growth rate of cave organisms (Carpenter 2016). Though all the teeth were obtained from the ventral and dorsal portions of specimens of similar size, this explanation (being underdeveloped teeth) must be considered in further studies.

The presence of EPTs on DAP is conspicuous across the genus Ophionereis, not only in Caribbean species like O. reticulata but also in the Indo-Pacific species O. porrecta and O. degeneri (figures 15c and 16c in: Stöhr (2011)). Furthermore, based on figures from available literature, ophionereidids have higher EPT densities than species of the genus Ophiocoma (e.g. 15 vs 498) (Hendler and Byrne 1987) and Ophiopsila (e.g. 96 vs 498) (Deheyn et al. 2015). Though O. commutabilis shows expanded peripheral trabeculae on dorsal arm plates, these structures show a different pattern that decreases in density in contrast to its epigean congeners. These differences could suggest the effects of low energy and darkness typical of caves, in the photoreceptor system of this species. Moreover, the cave specimens DAP show a disorganized inner mesh, further suggesting stereom organization being reduced as an expensive or dispensable trait, as proposed for cave-dwelling taxa (Klaus et al. 2013). Concerning inner stereom it is especially relevant since it affects chromatophore activity (Hendler and Byrne 1987; Sumner-Rooney et al. 2018), suggesting an important impact of darkness in O. commutabilis life as previously discussed for the diversity of colouration of this species (Bribiesca-Contreras et al. 2019).

Stereom of cave specimens are similar to that of deep-sea ophiuroids in having less defined expanded peripheral trabeculae (Hendler and Byrne 1987), both environments having similar conditions (e.g. absence of light and low energy). On the other hand, agglomeration of EPT has been reported for bioluminescent species, particularly conspicuous in Ophiopsila californica (Deheyn et al. 2015). In addition, transmittance differences (due to whether the light beam passes through EPT first or last) on DAP of the cave species are similar to differences observed among bioluminescent (Ophiopsila californica and Amphipholis squamata) and non-bioluminescent (Amphipholis pugetana) species (Deheyn et al. 2015). Therefore, our results suggest that both photoreception and bioluminescence processes have affected the evolution of this species and confirm that differences in morphology of DAP between both species have an impact on light transmittance.

The lower transmittance peak observed in the two analyzed species corresponds to wavelengths that activate phototaxis in ophiuroids (Delroisse et al. 2016). This adds evidence to the hypothesis that this species diverged from a shallow water lineage (Bribiesca-Contreras et al. 2019). On the other hand, the highest peak is usual in ophiuroids, since it corresponds to wavelengths attenuated on the first 10-20 cm depth (Deheyn et al. 2015). Finally, these results highlight the necessity of more studies to understand if the phototaxis in ophiuroids is related to their relative success in caves (in comparison with the other classes of the phylum).

Cave fauna shows particular morphological traits that could be considered to be troglomorphisms if they allow organisms to successfully colonize these harsh environments (Culver and Pipan 2009; Gonzalez et al. 2018). This paper demonstrates that brittle stars inhabiting an anchialine cave in Cozumel show troglomorphic traits such as arm (through the adding of segments) and distal tube feet elongation, increase in size, and possibly paedomorphic traits. This is, to our knowledge, the second study investigating troglomorphic adaptations in a group of echinoderms; our work confirms that O. commutabilis can be characterized as a stygobiotic species as demography, distribution and origin of this species suggested (Bribiesca-Contreras et al. 2013, 2019).

In conclusion, troglomorphic traits of Ophionereis commutabilis include elongation of arms (as a result of the addition of segments) and increased sizes, similar to those observed for other cave fauna. Additionally, potentially paedomorphic traits are reported for an ophionereidid. Finally, the morphology of O. commutabilis confirms it as a stygobiotic species as demography, distribution and origin of this species previously suggested.

The authors have declared that no competing interests exist.

We are grateful to Alicia Duran for the loan of specimens and Berenit Mendoza for taking the SEM photographs; Susana Guzmán for technical assistance in photography. Thanks to Arodi Farrera for help on geometric morphometrics analysis. To Juan Hernández and Amado Velazquez for assistance with the optical proofs. Thanks to Jill Yager, Laura Arroyo and Tania Pineda for their helpful comments and corrections that greatly improved the manuscript. We are grateful to Guadalupe Bribiesca-Contreras and an additional anonymous reviewer for providing valuable comments that improved considerably the content of this contribution. This work was funded by CONACYT (746189).