Valerija Zakšek

The limits of cryptic diversity in groundwater: phylogeography of the cave shrimp Troglocaris anophthalmus (Crustacea: Decapoda: Atyidae)

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 kilometres. 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 kilometres 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.

Morphologically Cryptic Amphipod Species Are "Ecological Clones" at Regional but Not at Local Scale: A Case Study of Four Niphargus Species

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.

Can Environment Predict Cryptic Diversity? The Case of Niphargus Inhabiting Western Carpathian Groundwater

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.

Evolution of the unique freshwater cave-dwelling tube worm Marifugia cavatica (Annelida: Serpulidae)

Abstract 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 transition to a subterranean environment may have occurred via ancestral marine shallow water to intertidal or estuarine species (like Ficopomatus) that evolved the necessary physiological mechanisms to withstand low salinity and then penetrated into freshwater caves via surface lakes.

Do cryptic species matter in macroecology? Sequencing European groundwater crustaceans yields smaller ranges but does not challenge biodiversity determinants

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.

Taxonomy and biogeography of Niphargus steueri (Crustacea: Amphipoda)

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.

Redescription of two subterranean amphipods Niphargusmolnari Méhely, 1927 and Niphargusgebhardti Schellenberg, 1934 (Amphipoda, Niphargidae) and their phylogenetic position.

Abstract A detailed redescription of two endemic, cave-dwelling niphargid species of the Hungarian Mecsek Mts., Niphargus molnari Méhely, 1927 and Niphargus gebhardti Schellenberg, 1934 is given based on newly collected material. Morphology was studied under light microscopy and with scanning electon microscopy. Morphological descriptions are complemented with mitochondrial cytochrome c oxidase subunit I (COI) sequences as barcodes for both species and with notes on their ecology. Using three independent molecular markers we showed that Niphargus gebhardti belongs to the clade distributed between Central and Eastern Europe, whereas phylogenetic relationship of Niphargus molnari to the rest of Niphargus species is not clear. The two species from the Mecsek Mts. are phylogenetically not closely related. Both species need to be treated as vulnerable according to IUCN Red List of Threatened Species.

Translating Niphargus barcodes from Switzerland into taxonomy with a description of two new species (Amphipoda, Niphargidae)

Abstract 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.