Extremophile at a Glance: Ostracod Crustacean From a Chemoautotrophic Sulphidic Cave Ecosystem

Sulphidic cave ecosystems are remarkable evolutionary hotspots that have witnessed adaptive radiation of their fauna represented by extremophile species having particular traits. Ostracods, a very old group of crustaceans, exhibit specific morphological and ecophysiological features that enable them to thrive in groundwater sulphidic environments. Herein, we report a peculiar new ostracod species Pseudocandona movilaensis sp. nov. thriving in the chemoautotrophic sulphidic groundwater ecosystem of Movile Cave (Romania). The new species displays a set of homoplastic features specific for unrelated stygobitic species, for e.g., triangular carapace in lateral view with reduced postero – dorsal part and simplification of limb chaetotaxy (i.e., loss of some claws and reduction of secondary male sex characteristics), driven by a convergent or parallel evolution during or after colonization of the groundwater realm. P. movilaensis sp. nov. thrives exclusively in sulphidic meso-thermal waters (21°C) with high concentrations of sulphides, methane, and ammonium. Based on the geometric morphometrics-based study of the carapace shape and molecular phylogenetic analyses based on the COI marker (mtDNA), we discuss the phylogenetic

Sulphidic ecosystems, such are thermal vents in deep sea or continental karst springs and caves, are inhabited by some of the most extraordinary extremophile organisms on the planet 1- 3 . In these ecosystems, the absence of light precludes photosynthesis-the chemical process by which green plants draw energy from sunlight to build carbohydrates from water and carbon dioxide. Here, chemoautotrophic bacteria are at the bottom of the food chain and use a different strategy to extract energy by oxidizing hydrogen sulphide as a replacement for radiant energy from sunlight [4][5][6] . They not only survive in this challenging environment but also provide food for organisms of higher trophic levels.
In the last decade, deep sea thermal vents have attracted much attention due to their high diversity of chemoautotrophic bacteria and marine invertebrates that have adapted to wide thermal gradients, high pressure, and chemically extreme environments 7 . Biota in these environments live in association with chemosynthetic microbes, enabling them to cope with toxic waters rich in hydrogen sulphide and methane [8][9] . In contrast, in continental sulphidic cave ecosystems (SCE), which share their highly unusual nature with sulphuric deep-sea hydrothermal vents, fundamental studies on invertebrate biota are still scarce 5,[10][11][12][13][14][15] . An exception is the first discovered cave ecosystem of this type, the Movile Cave in Romania, the microorganism communities of which have been exhaustively studied since its discovery [5][6][16][17] .
Caves are generally considered extreme on their own account, but SCE are even more extreme, characterized by warm waters; high levels of sulphide, methane, and ammonium; heavy metals (iron, zinc, and copper); and low oxygen concentrations up to hypoxia 5, 18-20 . Hydrogen sulphide is significantly high in SCE, being abundant in the ancient, anoxic oceans of the Proterozoic era, and serves as an energy source for early forms of life by being involved in biochemical and physiological processes of organisms 21 . Likewise, in thermal vents, organisms from various phyla have colonized this toxic environment, giving rise to unique ecological communities supported entirely by chemoautotrophic bacteria and particularly by sulphate-reducing bacteria and forming complex trophic networks 1,5,22-24 .
Although sulphide is generally highly toxic to most organisms, SCE host extremophile life forms that show a combination of morpho-ecological traits and metabolic and physiologic adaptations, enabling them to cope with such extreme conditions that are considered lethal for most species. In this regard, SCE and their fauna are considered remarkable 'evolutionary hotspots' 25 , which can be a model for extra-terrestrial life on Mars 26 .
They retain some archetypal features that appeared adaptive to cope with hypoxia and high concentrations of sulphide and methane in a similar way as their marine relatives [27][28][29][30][31] .
Moreover, they show high capacity for long-term anaerobiosis 32,33 and well-developed mechanisms for sulphide detoxification [34][35][36] . However, being subterranean forms, even members of different phyla attain striking similarity 37 . Stygobite species 38 show typical 'regressive' characters, for e.g., reduction or absence of eyes, loss of pigment, impermeability of the cuticle, elongated body shape and appendages, modifications of sensory organs, and slow metabolism 39 .
Among crustaceans, ostracods are only mentioned as being present in SCE 5,11,14 , but no taxonomic descriptions of new species or species exclusively associated with hydrogen sulphide-rich continental groundwaters are currently available. Ostracoda are a primitive class dating back to the Early Ordovician or Late Cambrian period (~505-485 Mya) [40][41] . They are small crustaceans with the body enclosed in a calcified bivalve shell that completely covers the entire animal 41 . They generally feed on aquatic bacteria, fungi, algae, and detritus 42 .
Studies on ostracod evolution and adaptive radiation have a long history. In particular, the functional morphology of calcitic carapaces, which easily fossilize and wherein evolution is often expressed as an adaptive response to environmental conditions, has been studied intensively from empirical and theoretical viewpoints 43 (references cited therein). The shape, ornamentation, and size of the ostracod carapace have often been subjected to evolution in the same direction in distinct and unrelated species but sharing similar environmental pressures [44][45][46][47][48] . Such similarity between organisms or their parts for reasons other than inheritance from a common ancestor is termed homoplasy and is caused by either convergent or parallel evolution [49][50] . Convergence may lead to homeomorphy, which is defined as similarity affecting the whole outer appearance to such a degree that one organism may be mistaken for the other 51 .
Homoplasy (or its special case homeomorphy) is an important issue in ostracod evolutionary biology as homoplastic similarities, particularly occurring in reasonably close phylogenetic groups, can make phylogenetic analysis more challenging 44,48,[52][53][54] . The expression of homoplasy in the design of an external morphological feature in unrelated species is supposed to arise independently, often as a result of a similar functionality of a trait or due to similar environmental constrains 55 . Morphological homoplasy is assumed to act with preference on those structures that have the highest probability to become advantageous for a species living in a certain environment.
Here, we describe a new cave ostracod species thriving in sulphidic waters of the Movile Cave (southeast Romania). We use a) geometric morphometrics related to the carapace shape of the new species in comparison with its closest relatives of the genera Pseudocandona Kaufmann and Typhlocypris Vejdovský as well as b) DNA sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene to infer the phylogenetic relationships of the new species. Sequences originated from these paratypes are deposited at GeneBank (see Table S1 for accession numbers). Pseudocandona ex gr. rostrata, see Table 1) show that the first squared canonical correlation was relatively large (0.904) and indeed the first canonical axis clearly separated the stygobitic species of the genus Typhlocypris, all having triangular valve shape in lateral view. This was the most distinct group, which had 100% allocation success under cross-validation. The other two groups of the genus Pseudocandona (gr. compressa and gr. rostrata) were hardly distinct from one another (Fig. 3), although their allocation success rates were still considerably large (at 80.0% and 85.7%, respectively). The second canonical axis had a much smaller eigenvalue (0.127) and there is actually no separation of the three groups along the second axis. When our new species Pseudocandona movilaensis sp. nov. was introduced to the existing CAP model to classify this species into one of the three existing groups specified above, it was clearly located within the cloud of the triangular Typhlocypris species, close to T. marmonieri ( Fig. 3) with the distance to the centroid of this group 0.029, compared with distances of 0.399 and 0.352 to the centroids of the groups of Pseudocandona gr. compressa and P. gr. rostrata, respectively. Although uneven number of species was included in the three studied groups (Table 1), the distance-based test for homogeneity of multivariate dispersion (PERMDISP) showed no statistically significant differences (F = 2.557; P(perm) = 0.170) in the within-group multivariate dispersion among the three groups. To conclude, based on the valve shape in lateral view Pseudocandona movilaensis sp. nov. resembles to a great extent the stygobitic species of the genus Typhlocypris.

Molecular phylogenetic analysis.
In the NJ tree generated based on the haplotype COI data set (Table 1, Table S1), only the shallow branches were well-resolved (Fig. 4). The deep nodes remained poorly supported as the COI marker is unsuitable for the phylogenetic reconstructions of deep evolutionary histories. Nevertheless, focusing on well-supported terminal branches, our results showed that Pseudocandona movilaensis sp. nov. appeared to be closely affiliated to Pseudocandona species (Fig. 4). Furthermore, the new species is close to the clade formed by species of the Pseudocandona rostrata group (P. marchica and P. hartwigi) with the mean K2P pairwise genetic distances at the level of 0.15 (Table S2). The mean genetic distance between P. movilaensis sp. nov. and the P. compressa species-group (P. albicans and P. compressa) was 0.20, whereas between P. movilaensis sp. nov. and species of the genus Typhlocypris 0.24.
Habitat characteristics, ecology and distribution. Pseudocandona movilaensis sp. nov. was reported from sulfidic thermal groundwaters (21˚C) characterized by slightly alkaline waters (pH of 7.2) and high concentrations of sulfide (0.25 mol/l -3 ), methane (0.02 mol/l -3 ) and ammonia (0.28 mol/l -3 ) 5 . The species is known exclusively from the sulfide waters of the Movile Cave, but empty carapaces have been also found in a hand-dug well 1 km from the cave and assumed to be passively transported with the groundwater flow (Fig. 5). In situ live specimens were observed to move down to the bottom lake (which is almost hypoxic) and return to the surface after few seconds or crawling on the walls. They probably live at the redox interphase between the oxygenated and the cross-formational rising anoxic water, where the chemosynthetic sulfide-oxidizing bacteria thrive. The examination of live and dead specimens immediately after sampling revealed that all living specimens bear bacterial filaments on the shells, whereas none of the carapaces of dead animals presents these attachments.

Discussion
First taxonomic description of an ostracod from a SCE. SCE species are examples of extremophiles dwelling in habitats subjected to high environmental pressure due to water toxicity. Non-marine ostracods reported from sulphidic groundwaters are very rare, although non-marine ostracods generally thrive in a large array of extreme habitats, such as hot springs (with temperatures exceeding 50°C), cold (up to freezing temperature), acidic (with pH as low as 3.4), and hypersaline waters (at salinities in excess of 100‰) as well as in temporary ponds prone to frequent complete drying or in deep groundwaters [55][56][57][58][59] .
As it stands, there are few well-documented SCE continental sites where ostracods are essential contributors to species diversity and an important functional group in the food web network. Among them are Movile Cave in Romania, Frasassi Cave in Italy, Ayalon Cave in Israel, and the more recently discovered Melissotrypa Cave in Greece 5, [10][11]15 . Ostracods in SCE, however, are yet to be taxonomically studied to determine their species-specific adaptations to SCE or to investigate the environmental conditions in sulphidic waters that govern species spatial distribution. This is the first taxonomic description of an endemic nonmarine ostracod species thriving exclusively in sulphidic cave waters, which enables other biological studies and generalization of the conclusions beyond this study species.
P. movilaensis sp. nov. has three distinctive morphological traits, which we consider homoplastic, i.e. gained or lost independently by species representing separate phylogenetic lineages: 1) triangular shape of the carapace and left valve in lateral view, 2) lack of so-called male bristles on the second antenna (setae t2 and t3 are not transformed in males into thick sensory bristles and remain similar to their counterparts in females), and 3) reduction of the posterior claw of the uropodal ramus in both sexes (with stronger reduction in males).
There is a striking and absorbing resemblance in general triangular carapace shape between several subterranean species belonging to various genera of the subfamily Candoninae. This triangular shape is one of the diagnostic traits (coupled with fine valve ornamentation and narrow inner lamellae in both adult and juvenile stages) of the exclusively subterranean genus Typhlocypris 60 , which shares this trait with some species of morphologically distinct genera containing species having mostly different (non-triangular) carapace shapes. Examples include subterranean Fabaeformiscandona aemonae, Mixtacandona tabacarui, Schellencandona triquetra 60 , and Candonopsis mareza but also epigean species living in ancient lakes of Ohrid (e.g., Neglecandona goricensis or N. litoralis 61 ) and Baikal (e.g., Baicalocandona navitarum or B. zenkevichi) 62 . These 'triangular' species, however, can be morphologically easily distinguished from the lineage constituting the genus Typhlocypris based on differential diagnostic characters (of both carapace and limbs) of the genera to which they belong. P. movilaensis sp. nov. also possesses the carapace of triangular shape in lateral view (Fig. 1A, D), which suggests a close affinity with species of the genus Typhlocypris (Fig. 3). The only character of the valve morphology of P. rostrata based on morphological characters may be considered tenuous at best. The phylogenetic placement of P. movilaensis sp. nov. on the COI sequence tree (Fig. 4) and genetic distances with other studied species (Tables S2 and S3), however, supports hypothesis of its closer affinity with the species of the rostrata-group of the genus Pseudocandona, which typically develop carapaces of rectangular shape when viewed laterally. Thus, more distant relationship of P. movilaensis sp. nov. and Typhlocypris implies homoplastic evolution of the triangular carapace shape in the studied ostracods. The only species of the genus Pseudocandona with triangular carapace (except for some Baikalian species) is P. punctata known from lakes in Ohio 63-64 , but this species has an isolated position in the genus (with possible affinities with Baikalian candonids) and differs from P. movilaensis sp. nov. in ornamented valves, shape of male prehensile palps, straight (not curved) h1 seta on the cleaning leg, and the shape of lobes of hemipenis 64 . Although denser species sampling is needed for genetic data (but see position of P. movilaensis sp. nov. in the wider phylogenetic context) 65 , where this species is marked as Pseudocandona sp. nov.), our new findings add to previous morphological evidence for morphological homoplasy of the triangular carapace shape among species of various genera of the subfamily Candoninae, further disassociating a polyphyletic group of 'triangular' Candoninae into different genera.
Typically, in the subfamily Candoninae, the second antenna (A2) is sexually dimorphic. In males, among other dimorphic traits, the penultimate segment is subdivided and bears the so-called male bristles, which play important prehensile and sensorial roles during courtship and copulation. These bristles are believed to be modified setae t2 and t3, which in females remain untransformed and are set on the undivided penultimate segment 66  for the latter genus, to which we assigned our new species, thus far, males of only four species have been known to lack male bristles, viz. European P. insculpta and P. regnisnicolai of the compressa-species group 66,69 , north American P. punctata of the carribeana-species group 64 , and Japanese P. atmeta of the rostrata-species group 70 . All these species (except for P. punctata, see above) have non-triangular carapace shape, clearly distinguishing them from P. movilaensis sp. nov. Our new species differs also from 'triangular' Typhlocypris pretneri (the single species of its genus lacking male bristles) in the morphology of the hemipenis and appearance of a uropod. In any case, the lack of male bristles in different genera (or even tribes) within Candoninae indicates signatures of homoplastic evolution, implying that developmental transformation of t-setae into male bristles may be caused by recurrent mutations across not closely related taxa. This does not, however, preclude that lack of male bristles shared by all species of some genera is a result of a common descent and may indicate synapomorphy.
In the subfamily Candoninae, uropod commonly consists of two rod-shaped rami, each bearing distally two claws and two setae 66,68 . A number of various reductions of this chaetotaxic scheme have been described within separate genera and tribes. The common reductions include the absence of a posterior seta (e.g., some genera of the tribe Candonopsini 54 ) or reduction of size, transformation to seta, or complete lack of a posterior claw Gp (e.g., several subterranean genera endemic to Australia or Meischcandona 68 ; Karanovic 2012). In some species, the uropodal ramus is strongly reduced with only one apical claw or seta (e.g., some genera of the tribe Cabralcandonini 67 of the tribe Namibcypridini 71 ) or even the ramus is reduced to a flagellum without any setae or claws (as in Cabralcandona) 67 . Beyond doubt, simplification of the uropodal ramus has occurred several times within the subfamily Candoninae, and if the similarity in the form of the caudal ramus exists in different lineages, it presents another example of homoplasy, which may create difficulties in phylogenetic analysis. In the genus Pseudocandona and three most closely related genera Typhlocypris, Schellencandona, and Marmocandona, the uropodal ramus is well-developed, with two claws and two setae. To our knowledge, P. movilaensis sp.
nov. is the unique species of its genus with evidently reduced Gp claw in both sexes. The only other species of Pseudocandona with reduced Gp, but only in males, is P. marchica, which can be easily distinguished from our new species by having a non-triangular carapace shape and well-developed male bristles 66 .
Although traditionally homoplasies are considered to be caused by convergence (when arising by different developmental pathways) or parallelism (if similar developmental mechanisms are involved) 72 , some evolutionary biologists argue that convergent and parallel evolution are difficult to distinguish as there is a continuum between these, and thus, propose to use a single term-convergent evolution 73 . Nevertheless, at this stage, it is entirely speculative if the three above-mentioned homoplastic traits have evolved independently in P. movilaensis sp. nov. by changes in the same or different genes using similar of distinct ontogenetic modifications related to those causing similar phenotypes in other species within Candoninae. Stygobitic cavernous crustaceans belonging to different phylogenetic groups evolve independently with similar suits of traits termed troglomorphic [74][75][76] . For example, several amphipods and isopods inhabiting cave waters show increased appendage length or setation and advanced development of chemo-sensorial organs 39 . As presented above, the three homoplastic characters of P. movilaensis sp. nov. can be also considered troglomorphic. We hypothesize that at least two of these traits (lack of male bristles and triangularly-shaped carapace) may have resulted from paedomorphosis, a well-known heterochronic evolutionary process of the retention of youthful ancestral features by adult descendants 77 . There are two distinct processes explaining paedomorphosis: acceleration of sexual maturation relative to the rest of development (progenesis) and retardation of somatic development with respect to the onset of reproductive activity (neoteny). We believe that paedomorphic characters of P. The carapace of ostracods acts as an interface between the organism and environment and is more likely to be subjected to selection pressures and to have an adaptive value [78][79][80][81] .
The adaptive significance of the valve shape among the true subterranean ostracods is still a debated issue [83][84] . If the evolutionary process is not driven by the selective pressure of cave conditions, 'triangularization' of the ostracod carapace shape may start either outside the cave (being already present in the ancestor or gained during the colonization of the cavernous environment) or inside the cave (gained after successful colonization of the subterranean realm). We hypothesize that triangular carapace shape may be an adaptive feature selected under environmental conditions in caves, where underdevelopment of the postero-dorsal section of the carapace may provide energetic solution to the oligotrophic cave conditions (less material needed, see below) coupled with low reproduction rate (less space needed for lower number of eggs).
Engineering construction of the carapace. The carapace in ostracods has functional implications, and it is viewed as an efficient 'engineering construction' adopting a shape and a structure design according to the environmental conditions in which the species lives with the use of minimal amounts of material 85 . The first author advancing the idea that the ostracod carapace 'is a static frame structure with a shape, which, during evolution, can be deformed following specific rules' was Benson 86 . He stated that a hint to understand the solutions adopted by ostracods to obtain the most advantageous carapace shape can be traced by making analogies with the techniques used in architecture constructions. Hence, the ostracod carapace is seen as having similar design to a dome with a double walled cupola with the exterior part being thicker and more resistant and the internal one thin (Fig. 6). Later on, Danielopol 79 advanced the idea that the triangular shape of the ostracod valves is a benefit and fitness solution for species thriving in subterranean environment. The triangular shape of carapace is viewed in a similar way as a tripod, wherein the weight is distributed more efficiently among the three faces (Fig. 6). In agreement with the principles of geometry and mechanics, it is well-known that a triangular shape structure in general has two advantages: 1) deformation is more difficult and is able to balance the stretching and compressive forces inside the structure and 2) is less costly as it requires less material to make the three sides of the triangle.
The triangular shape of the carapace can also have an ecological meaning. In an environment with a high concentration of sulphide and methane, the species must take protective measures against the diffusion of these elements from water into the body 87 . For example, marine ostracods from thermal vents have a waterproof shell 88 . Moreover, an appropriate shell shape can also help the animal to reduce the surface area and volume so that diffusion of toxic elements is minimal. According to Fick's law 89 , a triangle is a geometric shape with the smallest ratio of area to volume compared to a rectangular shape. Hence, the triangular shape remarked in P. movilaensis sp. nov. vs. the typical rectangular shape of the Pseudocandona species from the rostrata-group may offer a selective advantage for the species that developed or retained this solution, which was already present in its ancestor. Conclusions. We assume that the triangular valve shape in the newly described cave species P. movilaensis sp. nov. is a paedomorphic trait, which could be advantageous in the groundwater environment. The new species had a limited range of options to shift into a beneficial trait in the reduced or even lacking environmental fluctuations in the subterranean realm. We further assume that phenotypic similarity in the valve shape of P. movilaensis sp.

Ancientness
nov. with the stygobite species of the genus Typhlocypris (as well as with other triangularshaped stygobitic species of other Candoninae genera) is a homoplasy caused by convergent or parallel evolution, attributable to similar constrains of the subterranean realm as well as a developmental solution to survive in an extreme environment, such as groundwater. The position of the new species within the rostrata group of the genus Pseudocandona, as indicated by the COI phylogeny, confirms the homoplastic nature of also other traits that are shared with species morphologically assigned to separate lineages. These results suggest that some traditional characters used to unite certain non-marine ostracods (such as triangular carapace shape) evolved more than once, indicating extensive morphological homoplasy in groundwater ostracods of the subfamily Candoninae.  (Table 1). The obtained outlines were digitized with TpsDig2 software, version 1.37 for further morphometric analyses 98 . The geometric analyses of the outlines were performed using the Linhart B-spline algorithm in Morphomatica v. 1.6 using 32 control points points [99][100][101] . The obtained Mean Delta Quadrat distances were used as morphological disparities between the obtained valve outlines. The distance matrix was subsequently used to discriminate between species belonging to three groups: A) 13 species of the genus Typhlocypris, B) seven species of the rostrata-group of the genus Pseudocandona, and C) five species of the compressa-group of the genus Pseudocandona (Table 1). We used Canonical Analysis of Principal Coordinates (CAP) implemented in the PERMANOVA+ add-on to PRIMER v7 software 102 to predict the genus/species-group to which individual species belong based on the valve shape and to diagnose misclassification error. Having the CAP model, Pseudocandona movilaensis sp.

Methods
nov. was placed onto the obtained canonical axes to classify this species into one of the three existing groups specified above. In addition, we performed a test of the null hypothesis of no differences in the within-group multivariate dispersion among the three groups by PERMDISP routine in PERMANOVA+ 102 .

Molecular phylogenetic analysis.
Genomic DNA was extracted from 38 specimens, representing nine selected ostracod species of the subfamily Candoninae (Table 1, Table S1).
Details of the DNA extraction, amplification and sequencing procedure were described previously 65 . The DNA barcoding fragment of Cytochrome-c-Oxidase subunit I gene (COI) 103 was amplified using standard primers LCO1490/HCO2198 104 (Table S1).
Mean genetic distances under the Kimura 2-parameter model (K2p) 109 between COI data set obtained from the nine ostracod species were calculated in MEGA X 10.0.3 110 (Tables S2 and   S3). For graphic presentation of the relationships among the studied species, Neighbour-Joining (NJ) tree for COI data was generated using K2P distances with 1000 bootstrap replicates 111 in MEGA.    Neighbour-joining tree of the studied ostracod species based on the COI gene sequences (for species codes see Table 1). The distances were calculated with Kimura 2-parameter method. The numbers in front of the nodes indicate bootstrap support (1000 replicates, only values higher than 50% are presented).

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