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Subterranean ecosystems are among the most widespread environments on Earth, yet we still have poor knowledge of their biodiversity. To raise awareness of subterranean ecosystems, the essential services they provide, and their unique conservation challenges, 2021 and 2022 were designated International Years of Caves and Karst. As these ecosystems have traditionally been overlooked in global conservation agendas and multilateral agreements, a quantitative assessment of solution-based approaches to safeguard subterranean biota and associated habitats is timely. This assessment allows researchers and practitioners to understand the progress made and research needs in subterranean ecology and management. We conducted a systematic review of peer-reviewed and grey literature focused on subterranean ecosystems globally (terrestrial, freshwater, and saltwater systems), to quantify the available evidence-base for the effectiveness of conservation interventions. We selected 708 publications from the years 1964 to 2021 that discussed, recommended, or implemented 1,954 conservation interventions in subterranean ecosystems. We noted a steep increase in the number of studies from the 2000s while, surprisingly, the proportion of studies quantifying the impact of conservation interventions has steadily and significantly decreased in recent years. The effectiveness of 31% of conservation interventions has been tested statistically. We further highlight that 64% of the reported research occurred in the Palearctic and Nearctic biogeographic regions. Assessments of the effectiveness of conservation interventions were heavily biased towards indirect measures (monitoring and risk assessment), a limited sample of organisms (mostly arthropods and bats), and more accessible systems (terrestrial caves). Our results indicate that most conservation science in the field of subterranean biology does not apply a rigorous quantitative approach, resulting in sparse evidence for the effectiveness of interventions. This raises the important question of how to make conservation efforts more feasible to implement, cost-effective, and long-lasting. Although there is no single remedy, we propose a suite of potential solutions to focus our efforts better towards increasing statistical testing and stress the importance of standardising study reporting to facilitate metaanalytical exercises. We also provide a database summarising the available literature, which will help to build quantitative knowledge about interventions likely to yield the greatest impacts depending upon the subterranean species and habitats of interest. We view this as a starting point to shift away from the widespread tendency of recommending conservation interventions based on anecdotal and expert-based information rather than scientific evidence, without quantitatively testing their effectiveness., This study is funded by the European Commission via the Marie Sklodowska-Curie Individual Fellowships program (H2020-MSCA-IF-2019; project number 882221), awarded to S.M. Additional support is provided by the PRIN SHOWCAVE “A multidisciplinary research project to study, classify and mitigate the environmental impact in tourist caves” (project number 2017HTXT2R; funded by the Italian Ministry of Education, University and Research). M.B.M. acknowledges support from the Kone Foundation (project number 202007611). T.D., C.F., V.Z., and M.Z. were supported by the Slovenian Research Agency, through core programme P1-0184 and P6-0119. O.T.M. was supported by a grant of the Romanian Ministry of Research, Innovation and Digitization, CNCS/CCCDI— UEFISCDI, project number 2/2019 (DARKFOOD), within PNCDI III. S.I.S. acknowledges funding by MEMOBIC (EU Operational Programme Research, Development and Education No. CZ.02.2.69/0.0/ 0.0/16_027/0008357), and by the Ministry of Education, Youth and Sports of the Czech Republic (grant number CZ.02.1.01/0.0/0.0/16 025/0007417). E.P. is supported by the PON “Research and Innovation” Programme (Axis IV “Education and Research for recovery” – Action IV.6 “Research contracts on Green themes”).

The new troglobiotic shrimp Ficticaris serbica, gen. nov., sp. nov., Atyidae, was found in northern part of the Balkan Peninsula within a vast territory between the Dinaric Karst (on the north-western part of the Balkan Peninsula) and the Caucasus, a disjunct gap of Troglocaris distribution. No other epigean or hypogean decapod shrimps were known from the area. A combined morphological and molecular data were used for its description from the only locality in central Eastern Serbia (Vrelo Krupaja). This new cave shrimp species with a short unarmed rostrum lacks eye or body pigmentation, and can be easily distinguished from any other atyid genus and species by a combination of unique morphological characters, among which (1) the concave posterior margin of the telson, (2) maxilliped I exopodite with no trace of a flagellum, (3) maxilliped III and (4) all pereopods without exopodites, are the most striking. The taxonomic status of the genus within the family Atyidae together with the unique taxonomical traits in Ficticaris are discussed. Among these traits, the telson that has been neglected until now, has shown some taxonomically important characters within Atyidae.

Sex allocation theory predicts that the proportion of daughters to sons will evolve in response to ecological conditions that determine the costs and benefits of producing each sex. All else being equal, the adult sex ratio (ASR) should also vary with ecological conditions. Many studies of subterranean species reported female-biased ASR, but no systematic study has yet been conducted. We test the hypothesis that the ASR becomes more female-biased with increased isolation from the surface. We compiled a data set of ASRs of 35 species in the subterranean amphipod Niphargus, each living in one of three distinct habitats (surface-subterranean boundary, cave streams, phreatic lakes) representing an environmental gradient of increased isolation underground. The ASR was female-biased in 27 of 35 species; the bias was statistically significant in 12 species. We found a significant difference in the ASR among habitats after correction for phylogeny. It is most weakly female-biased at the surface-subterranean boundary and most strongly female-biased in phreatic lakes. Additional modelling suggests that the ASR has evolved towards a single value for both surface-subterranean boundary and cave stream-dwelling species, and another value for 9 of 11 phreatic lake dwellers. We suggest that a history of inbreeding in subterranean populations might lower inbreeding depression such that kin selection favours mating with siblings. This could select for a female-biased offspring sex ratio due to local mate competition among brothers. The observed patterns in sex ratios in subterranean species make them a group worthy of more attention from those interested in sex allocation theory.

We characterized taxonomically, ecologically, and phylogenetically the amphipod community of Melissotrypa Cave (Central Greece), which comprises both freshwater and sulphidic lakes. We found four amphipod species: Niphargus jovanovici, Niphargus lindbergi, Niphargus gammariformis sp. nov. and an unknown species of Bogidiella. The three Niphargus species form a well-supported monophylum but differ in their ecology and morphology: N. jovanovici is a small and slender species inhabiting small freshwater voids, N. lindbergi is a large and stout species living in freshwater lakes, whereas N. gammariformis sp. nov. is a small and stout species found predominantly in a sulphidic lake. Available evidence suggests that diversification may have happened in a geographically restricted area and was driven by ecological differentiation. Niphargus gammariformis sp. nov. shows morphological convergences in diagnostic traits with two species hitherto classified into the genus Pontoniphargus. As molecular phylogenies show Pontoniphargus nested within Niphargus, we synonymize here Pontoniphargus with Niphargus. The species originally named Pontoniphargus ruffoi needed to be renamed into Niphargus pontoruffoi.http://www.zoobank.org/urn:lsid:zoobank.org:pub:4F5EED3B-DEB4-4402-BCE8-6AE2B65CCC3A., info:eu-repo/semantics/published

Acanthocephalus balkanicus Batchvarov et Combes, 1974 was incompletely described from the northern crested newt, Triturus cristatus (Laurenti) (Amphibia: Salamandridae), a possible synonym of the Balkan crested newt, Triturus ivanbureschi Arntzen et Wielstra, from a pond in village of Pesnopoy, southern Bulgaria. We provide a full description of adult males and females of the same taxon from the olm, Proteus anguinus Laurenti (Amphibia: Proteidae), the only exclusively aquatic cave-dwelling vertebrate in Europe, captured in Postojna-Planina Cave System in Slovenia. Cystacanths were also collected from the cave ecomorph of Asellus aquaticus (Linnaeus) (Crustacea: Asellidae) in the same location. Molecular analysis of specimens from Slovenia revealed that they are genetically almost identical to those of Acanthocephalus anguillae (Müller, 1780), a common parasite of European freshwater fishes. We propose to recognise the morphological and host differences by describing A. balkanicus as a new subspecies of A. anguillae. Acanthocephalus anguillae balkanicus is rather small and cylindrical with cylindrical proboscis having 10 rows of 6 hooks with simple roots each, long neck, large balloon-shaped lemnisci, small spherical anterior testis, and 6 club-shaped cement glands in 3 pairs. SEM images reveal more morphological details and the X-ray scans of gallium cut hooks shows considerably higher levels of phosphorus and calcium in adult hooks than in cystacanth hooks, especially in basal areas. Sulfur levels were higher in the arch and basal area of cystacanth hooks than adult hooks. Considering that both definitive and intermediate hosts of the Slovenian population of this acanthocephalan are bound to cave life, it is possible that its entire life cycle is uniquely completed underground.

The European cave salamander, Proteus anguinus, or proteus, is the largest obligate cave animal in the world. It is an endangered and charismatic species of high conservation importance for subterranean waters. Conservation genetic studies are hampered by the extreme size and repetitiveness of its nuclear genome. The aim of the study was to develop and characterize the first microsatellite markers for proteus, and test their informativeness at the level of individuals, populations and between populations in the Postojna and Planina caves in Slovenia. Twenty-three novel polymorphic microsatellite markers were amplified in 201 individuals from both caves using three multiplex reactions. The number of alleles per locus varied from three to nine. The loci are largely unlinked and conform to Hardy–Weinberg genotype frequencies. Genetic equilibrium and an FST value of 0.0024 suggest a nearly panmictic population in both caves separated by some 10 km of subterranean river course, while Bayesian clustering detected weak genetic structure. The microsatellites described fill the gap of urgently needed nuclear markers in Proteus that can be applied in genetic mark–recapture studies, population monitoring and identification of management units to assist conservation efforts.

Ecologists increasingly rely on molecular delimitation methods (MMs) to identify species boundaries, thereby potentially increasing the number of putative species because of the presence of morphologically cryptic species. It has been argued that cryptic species could challenge our understanding of what determine large-scale biodiversity patterns which have traditionally been documented from morphology alone. Here, we used morphology and three MMs to derive four different sets of putative species among the European groundwater crustaceans. Then, we used regression models to compare the relative importance of spatial heterogeneity, productivity and historical climates, in shaping species richness and range size patterns across sets of putative species. We tested three predictions. First, MMs would yield many more putative species than morphology because groundwater is a constraining environment allowing little morphological changes. Second, for species richness, MMs would increase the importance of spatial heterogeneity because cryptic species are more likely along physical barriers separating ecologically similar regions than along resource gradients promoting ecologically-based divergent selection. Third, for range size, MMs would increase the importance of historical climates because of reduced and asymmetrical fragmentation of large morphological species ranges at northern latitudes. MMs yielded twice more putative species than morphology and decreased by 10-fold the average species range size. Yet, MMs strengthened the mid-latitude ridge of high species richness and the Rapoport effect of increasing range size at higher latitudes. Species richness predictors did not vary between morphology and MMs but the latter increased the proportion of variance in range size explained by historical climates. These findings demonstrate that our knowledge of groundwater biodiversity determinants is robust to overlooked cryptic species because the latter are homogeneously distributed along environmental gradients. Yet, our findings call for incorporating multiple species delimitation methods into the analysis of large-scale biodiversity patterns across a range of taxa and ecosystems., SCOPUS: ar.j, info:eu-repo/semantics/published

Subterranean animals are known for their highly evolved phenotypes. They are eyeless, depigmented and possess elongated appendages compared to their surface relatives. Increased antenna and leg length of cave species has traditionally been explained as a consequence of selection for non-visual senses and increased food-finding ability in an environment low in energy. Variation in appendage length between cave species is usually thought to result from differences in time since the colonization of the subterranean habitat. In this study, we analyzed appendage length variation in the Dinaric amphipod species Niphargus croaticus. Relative length of appendages varied substantially among populations. Using multilocus phylogenetic analysis, we showed that the species is nested within highly specialized N. steueri species complex and rejected the time hypothesis. Next, we explored the effects and strength of two environmental factors, water flow and presence of a competing species, N. subtypicus. Populations in caves with flowing water had shorter appendages than populations in cave lakes. Presence of the competing sister species did lead to longer appendages in stagnant water, but had no effect in flowing water. Abiotic factors had a stronger effect than biotic factors, but their relative strength differed among appendage pairs. High variation in appendage length between adjacent population shows that the morphology of cave arthropods is changing quickly and therefore cannot be used to predict species age. Rather than being a general adaptation to cave life, long appendages seem to be associated with the absence of water flow as well as character displacement when in sympatry with ecologically similar competing species.

In an ever more artificially illuminated world, common moth behaviour, flight-to-light, causes declines in their abundance and diversity that can have severe impacts on ecosystems. To test if it is possible to reduce the number of moths attracted to artificially illuminated objects, the original lighting of 15 cultural heritage buildings in Slovenia was substituted with blue or yellow lighting. These three illumination types differed in the amount of luminance, percentage of UV and short-wavelength light which are known to affect flight-to-light of moths. During our three-year field study approximately 20% of all known moth species in Slovenia were recorded. The blue and yellow illumination type attracted up to six times less specimens and up to four times less species compared to the original illumination type. This was true for all detected moths as well as within separate moth groups. This gives our study a high conservation value: usage of alternative, environmentally more acceptable illumination can greatly reduce the number of moths attracted to artificially illuminated objects.

The amphipod genus Niphargus (Amphipoda: Niphargidae Bousfield, 1977) is the most species-rich genus of freshwater amphipods in the World. Species of this genus, which live almost exclusively in subterranean water, offer an interesting model system for basic and applied biodiversity science. Their use, however, is often limited due to the hitherto unresolved taxonomy within the whole genus. As a comprehensive taxonomic revision of the currently >425 Niphargus species is too demanding, it has been suggested that the taxonomy of the genus could be advanced in smaller steps, by reviewing regional faunas, that would eventually integrate into a global revision. In this study, we provide such a revision of Niphargus in Switzerland. First, we molecularly delimited, morphologically diagnosed, and formally described two new species, namely Niphargus luchoffmanni sp. n. and Niphargus tonywhitteni sp. n. Second, we updated and revised a checklist of Niphargus in Switzerland with new findings, and prepared a list of reference sequences for routine molecular identification, available at BOLD and GenBank. All available specimens of 22 known species from the area were morphologically examined, and their morphological variation was compiled in a data file of DEscription Language for TAxonomy, which can be used for automated generation of dichotomous or interactive keys. The data file is freely available at the World Amphipoda Database. Together, the checklist, the library of reference sequences, the DELTA file, but also a list of hitherto unresolved aspects are an important step towards a complete revision of the genus within a well-defined and biogeographically interesting area in Central Europe.

The amphipod genusNiphargus(Amphipoda: Niphargidae Bousfield, 1977) is the most species-rich genus of freshwater amphipods in the World. Species of this genus, which live almost exclusively in subterranean water, offer an interesting model system for basic and applied biodiversity science. Their use, however, is often limited due to the hitherto unresolved taxonomy within the whole genus. As a comprehensive taxonomic revision of the currently >425Niphargusspecies is too demanding, it has been suggested that the taxonomy of the genus could be advanced in smaller steps, by reviewing regional faunas, that would eventually integrate into a global revision. In this study, we provide such a revision ofNiphargusin Switzerland. First, we molecularly delimited, morphologically diagnosed, and formally described two new species, namelyNiphargusluchoffmannisp. n.andNiphargustonywhittenisp. n.Second, we updated and revised a checklist ofNiphargusin Switzerland with new findings, and prepared a list of reference sequences for routine molecular identification, available at BOLD and GenBank. All available specimens of 22 known species from the area were morphologically examined, and their morphological variation was compiled in a data file of DEscription Language for TAxonomy, which can be used for automated generation of dichotomous or interactive keys. The data file is freely available at the World Amphipoda Database. Together, the checklist, the library of reference sequences, the DELTA file, but also a list of hitherto unresolved aspects are an important step towards a complete revision of the genus within a well-defined and biogeographically interesting area in Central Europe.

Three new freshwater Amphipods of the family Niphargidae G. Karaman, 1962 are described from subterranean waters of Greece, Niphargus aitolosi sp. nov., Niphargus karkabounasi sp. nov. and Niphargus koukourasi sp. nov. Phylogenetic analysis based on 28S rDNA and H3 genes suggests that N. aitolosi sp. nov. belongs to N. longicaudatus species-complex. Species from that complex are distributed along both sides of Adriatic Sea and share roughly similar morphology, including the newly described species from Greece. Niphargus karkabounasi sp. nov., the second new species, belongs to a clade distributed broadly across Europe and the Middle East. Many species of this group, including N. karkabounasi, are small and likely live in small crevices. The third species, Niphargus koukourasi sp.nov. is phylogenetically related and morphologically similar to N. sanctinaumi and N. maximus from Lake Ohrid on the border of the Former Yugoslav Republic of Macedonia and Albania. In addition, this group of species may be related to species collected from Iran.

Subterranean species show a distinct morphology, yet the adaptive significance of some traits, like body size and shape, is poorly understood and cannot be explained solely by distinct environmental conditions (darkness, less food). We predicted that in females some morphological changes may have co-evolved with life history traits, and that co-evolving life history traits provide at least part of the explanation for evolutionary changes of morphology. Using museum material we tested this prediction on the subterranean amphipod genus Niphargus. We studied six species found in springs and eight species found in cave lakes. We treated them as two ecologically distinct groups, and the major ecological differences between them were the availability of nutrients and the water currents. Cave species were found to be larger and stouter (as inferred from the shape of coxal plates, which are part of the marsupium), they had larger eggs and lower reproductive effort per brood, whereas the egg number and brood volume if corrected for the body size were not different. Using phylogenetic independent contrasts, we found a positive correlation between body shape and egg volume, a positive correlation between body size and egg volume, and a negative correlation between body size and reproductive effort per brood. We tentatively conclude that evolutions of morphology and life histories are functionally connected and that co-evolving traits contribute to overall selective regime.

Of the approximately 350 described species of serpulid polychaetes, only Marifugia cavatica inhabits fresh water. It is distributed in ground waters of the Dinaric Karst in northeastern Italy, Slovenia, Croatia, and Bosnia and Hercegovina. Five other serpulid species, comprising the genus Ficopomatus, are found in brackish water locations worldwide; otherwise serpulids are all marine organisms. We re‐describe M. cavatica and examine the fine structure of its chaetae with SEM as well as summarise its distribution. The morphology of Marifugia provides an ambiguous indication of its phylogenetic relationships, thus DNA sequence data was also used. Phylogenetic analysis of nuclear rDNA 18S and 28S sequences using maximum parsimony, maximum likelihood and Bayesian analyses places Marifugia as a sister group to a clade of brackish‐water Ficopomatus species. Osmoconformity and penetration into non‐marine waters hence appears to have taken place once in the evolutionary history of Serpulidae. The transit...

SUMMARY 1. Various groundwater habitats have exceptionally high levels of endemism caused by strong hydrographical isolation and low dispersal abilities of their inhabitants. More than 10% of macro-stygobiotic species nevertheless occupy relatively large ranges, measuring from some hundred to over 2000 km in length. These species represent a challenge because their distributions disregard hydrographical boundaries, and their means to disperse and maintain long-term gene flow are unknown. 2. Based on mitochondrial and nuclear gene sequences, we examined the phylogeographic structure of six formally recognised stygobiotic species (Niphargus virei, N. rhenorhodanensis, Troglocaris anophthalmus, T. hercegovinensis, Spelaeocaris pretneri, Proteus anguinus) and searched for cryptic lineage diversity in a genus-wide phylogeny of Niphargus. Using treebased criteria as well as comparative divergence measures, we identified cryptic lineages, which may tentatively be equated with cryptic species. 3. Fourteen analysed nominal stygobiotic species with large ranges emerged as highly diversified, splitting into 51 tentative cryptic lineages. The degree of divergence was within the range or larger than the divergence of other related pairs of sister species. A substantial part (94%) of the cryptic lineages had ranges

Endemic atyids of southern Europe have been ascribed to Dugastella, and to subterranean Typhlatya and Troglocaris: Dugastella is epigean, and Typhlatya and Troglocaris are subterranean. An extensive collection from all centres of distribution in southern Europe (excepting the Caucasus) was morphologically examined. A taxonomic redefinition of the group, at different levels, is based on recently published and newly generated molecular phylogeny, whereas newly established taxa have also been morphologically defined. The accordance between the phylogenetic tree and the geographical distributions suggested that a re-evaluation of some traditionally used morphological characters should generate the most parsimonious solution: this enabled a novel taxonomic division. Gallocaris gen. nov. is erected for the French Troglocaris inermis Fage, 1937, which is more closely related to the epigean Dugastella valentina (Ferrer Galdiano, 1924) than to its supposed congeners. Both western Mediterranean Typhlatya species are closely related to their Caribbean congeners. All other European cave shrimps constitute a monophylum, Troglocaris, which is divisible into subgenera (already with available names): the holo-Dinaric Troglocaris (Troglocaris) Dormitzer, 1853, south-eastern mero-Dinaric Troglocaris (Troglocaridella) Babic, 1922, and Troglocaris (Spelaeocaris) Matjasic, 1956, and the Caucasian Troglocaris (Xiphocaridinella) Sadovsky, 1930. Four new species are described: Troglocaris (Troglocaris) bosnica sp. nov., Troglocaris (Spelaeocaris) prasence sp. nov., Troglocaris (Spelaeocaris) kapelana sp. nov., and Troglocaris (Spelaeocaris) neglecta sp. nov. The distribution of all established species is shown, and the value of the morphological characters is discussed. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155, 786–818.

Recent studies have revealed high local diversity and endemism in groundwaters, and showed that species with large ranges are extremely rare. One of such species is the cave shrimp Troglocaris anophthalmus from the Dinaric Karst on the western Balkan Peninsula, apparently uniform across a range of more than 500 kilometers. As such it contradicts the paradigm that subterranean organisms form localized, long-term stable populations that cannot disperse over long distances. We tested it for possible cryptic diversity and/or unexpected evolutionary processes, analysing mitochondrial (COI, 16S rRNA) and nuclear (ITS2) genes of 232 specimens from the entire range. The results of an array of phylogeographical procedures congruently suggested that the picture of a widespread, continuously distributed and homogenous T. anophthalmus was wrong. The taxon is composed of four or possibly five monophyletic, geographically defined phylogroups that meet several species delimitation criteria, two of them showing evidence of biological reproductive isolation in sympatry. COI genetic distances between phylogroups turned out to be a poor predictor, as they were much lower than the sometimes suggested crustacean threshold value of 0.16 substitutions per site. Most results confirmed the nondispersal hypothesis of subterranean fauna, but the southern Adriatic phylogroup displayed a paradoxical pattern of recent dispersal across 300 kilometers of hydrographically fragmented karst terrain. We suggest a model of migration under extreme water-level conditions, when flooded poljes could act as stepping-stones. In the north of the range (Slovenia), the results confirmed the existence of a zone of unique biogeographical conflict, where surface fauna is concordant with the current watershed, and subterranean fauna is not.

Niphargus steueri (Niphargidae) comprises a complex of four subspecies (N. s. steueri, N. s. kolombatovici, N. s. subtypicus, and N. s. liburnicus), the morphology and distribution of which have been poorly known until now. New diagnostic characters of the species and its four subspecies are presented and illustrated. The species is distributed along the major part of the Dinaric Karst, between Slovenia in the northwest and Bosnia and Herzegovina in the southeast. The distribution of the four subspecies approximately resembles the distribution of the evolutionary lineages of the subterranean amphibian Proteus anguinus and the Dinaric lineage of the cave shrimp Troglocaris agg. anophthalmus. Niphargus s. steueri is restricted to the Istran Peninsula; N. s. subtypicus is distributed in southeast Slovenia and northwest Croatia; N. s. liburnicus is known from two disjunctive localities, one on the island of Krk (Croatia) and the other in Gorizia (Italy); and N. s. kolombatovici is restricted to Dalmacija and Herzegovina. The somewhat variable putative synapomorphies of N. steueri probably suggest that the group is old and that the present distribution pattern is a result of historical events, possibly events in the Miocene Dinaride Lake system. Two populations of N. s. kolombatovici and one population of N. s. subtypicus deviate from the general distributional pattern and may belong to cryptic taxa that cannot be distinguished on the basis of morphology. Both hypotheses corroborate with the estimated times of divergence and with the number of independent lineages in the similarly distributed but unrelated stygobionts Proteus and Troglocaris.

The remarkably discontinuous distribution of the cave shrimp genus Troglocaris in South France, West Balkans, and West Caucasus has long been considered a biogeographic enigma. To solve it, its phylogeny was reconstructed by analyzing sequences from two mitochondrial (cytochrome oxidase I and 16S rRNA) and one nuclear gene (28S rRNA) using maximum likelihood, parsimony and Bayesian inference. The genus was found to be polyphyletic because the French taxon T. inermis had no direct common ancestry with other Troglocaris taxa but was sister to the epigean freshwater atyid Dugastella valentina. All other Troglocaris species constituted a well-supported monophylum, the second cave shrimp genus Spelaeocaris nested within. The monophylum had a well-defined structure: (1) a clade restricted to the Dinaric area of the Western Balkans containing the type species T. anophthalmus along with some unnamed species, and (2) a geographically mixed clade split between the Caucasian T. kutaissiana species complex on one, and T. hercegovinensis, S. pretneri, plus an unnamed taxon on the other side. It was surprising to find the dichotomy between the Caucasian and one of the West-Balkan lineages so low in the phylogenetic hierarchy of the genus. Taking into account molecular rates of other decapods, we tentatively dated this split at 6-11 Myr. This time is in agreement with the brackish and freshwater phase of the Paratethys thus allowing for a freshwater common ancestor of Caucasian and Dinaric cave shrimps. This would weaken the marine relicts hypothesis that has often been invoked to explain the distribution of freshwater cave species with close marine relatives.

Using a comparative phylogeographic approach and different independent molecular clocks we propose a timescale for the evolution of troglobionts in the Dinaric Karst that is relatively consistent over a wide taxonomic range. Keystone events seem to belong to two age classes. (1) Major splits within holodinaric taxa are from the mid-Miocene. They present the potential upper limit for the age of cave invasions. (2) Regional differentiation, including speciation, which can at least in part be associated with a subterranean phase, took place from early Pliocene to mid-Pleistocene. We suggest two to five million years as the time when most of the analyzed lineages started invading the Dinaric Karst underground.

Recent studies indicate that morphologically cryptic species may be ecologically more different than would be predicted from their morphological similarity and phylogenetic relatedness. However, in biodiversity research it often remains unclear whether cryptic species should be treated as ecologically equivalent, or whether detected differences have ecological significance. In this study, we assessed the ecological equivalence of four morphologically cryptic species of the amphipod genus Niphargus. All species live in a small, isolated area on the Istrian Peninsula in the NW Balkans. The distributional ranges of the species are partially overlapping and all species are living in springs. We reconstructed their ecological niches using morphological traits related to feeding, bioclimatic niche envelope and species’ preference for epi-hypogean habitats. The ecological meaning of differences in niches was evaluated using distributional data and co-occurrence frequencies. We show that the species comprise two pairs of sister species. All species differ from each other and the degree of differentiation is not related to phylogenetic relatedness. Moreover, low co-occurrence frequencies in sympatric zones imply present or past interspecific competition. This pattern suggests that species are not differentiated enough to reduce interspecific competition, nor ecologically equivalent to co-exist via neutral dynamics. We tentatively conclude that the question of ecological equivalence relates to the scale of the study: at a fine scale, species’ differences may influence dynamics in a local community, whereas at the regional level these species likely play roughly similar ecological roles.

In the last decade, several studies have shown that subterranean aquatic habitats harbor cryptic species with restricted geographic ranges, frequently occurring as isolated populations. Previous studies on aquatic subterranean species have implied that habitat heterogeneity can promote speciation and that speciation events can be predicted from species' distributions. We tested the prediction that species distributed across different drainage systems and karst sectors comprise sets of distinct species. Amphipods from the genus Niphargus from 11 caves distributed along the Western Carpathians (Romania) were investigated using three independent molecular markers (COI, H3 and 28S). The results showed that: 1) the studied populations belong to eight different species that derive from two phylogenetically unrelated Niphargus clades; 2) narrow endemic species in fact comprise complexes of morphologically similar species that are indistinguishable without using a molecular approach. The concept of monophyly, concordance between mitochondrial and nuclear DNA, and the value of patristic distances were used as species delimitation criteria. The concept of cryptic species is discussed within the framework of the present work and the contribution of these species to regional biodiversity is also addressed.