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Karst landscapes are characterized by intermittent and sinking streams. The most common method used to study underground hydrological connections in karst is tracing tests. However, a more biologically oriented approach has been suggested: analysis of the genetic structure of aquatic organisms. Biological tracers can be sought among trogloxenes, that is, surface species that occasionally enter caves and groundwater. One such example is the fish genus Phoxinus, which exhibits high genetic diversity and complex phylogeography in the Balkan Peninsula. In the north-western Dinaric Karst, the complex hydrological network was digitalized in 2020. Contemporaneously, Phoxinus lumaireul populations in the Slovenian Dinaric Karst were intensively sampled and analysed for fragments of two mitochondrial genes and one nuclear gene. The derived phylogeographic structure and data on hydrological connections were compared to evaluate support for three alternative scenarios: The genetic structure (1) is a consequence of the ongoing geneflow through underground connections, (2) reflects a previous hydrological network or (3) is an outcome of anthropogenic translocations. The results suggest that the first two scenarios seem to have played a major role, while the third has not had profound effects on the genetic composition. Comparison between the genetic structure of Slovenian Dinaric Karst sampling sites and that of hydrologically isolated reference sampling sites indicated a greater genetic connectivity in the former. Moreover, the range of Adriatic (1a) and Black Sea (1c) haplotypes does not correspond to the Adriatic–Black Sea basin divide but is shifted northwards.

The bloodfeeding leech genus Placobdella is dominated by North American diversity, with only a single nominal species known from Central America and one from the Palearctic region. This is likely due to considerable underestimation of Palearctic biodiversity, but investigations into potential hidden diversity are lacking. To shed light on this, the present study introduces new data for specimens initially identified as Placobdella costata from Ukraine (close to the type locality), Italy, Germany, Latvia, Montenegro, Bulgaria, Slovenia, Turkey, Azerbaijan, Tunisia, and Algeria, and uses both nuclear (Internal Transcribed Spacer [ITS] region) and mitochondrial (cytochrome c oxidase subunit I [COI]) sequence data in phylogenetic and DNA barcoding frameworks, in order to better understand species-level diversity. Seven independent lineages are present in the trees, five of which show adequate separation at the COI locus to suggest their unique species-level status (COI distances between these clades range from 4.86 to 8.10%). However, the ITS data suggest that speciation is recent or incipient in these clades, and that not enough time has passed for clear separation at this locus. We discuss the evolutionary and taxonomic implications of our findings and speculate on dispersal events that may have contributed to shaping this pattern of geographic distribution.

Erpobdellid leeches of the genus Trocheta are among the biggest and most charismatic members of their class. The studies of the group have a long and complicated history due to the lack of reliable...

Karst landscapes are characterized by intermittent and sinking streams. The most common method used to study underground hydrological connections in karst is tracing tests. However, a more biologically oriented approach has been suggested: analysis of the genetic structure of aquatic organisms. Biological tracers can be sought among trogloxenes, that is, surface species that occasionally enter caves and groundwater. One such example is the fish genus

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Deciphering the genetic code of organisms with unusual phenotypes can help answer fundamental biological questions and provide insight into mechanisms relevant to human biomedical research. The cave salamander Proteus anguinus (Urodela: Proteidae), also known as the olm, is an example of a species with unique morphological and physiological adaptations to its subterranean environment, including regenerative abilities, resistance to prolonged starvation, and a life span of more than 100 years. However, the structure and sequence of the olm genome is still largely unknown owing to its enormous size, estimated at nearly 50 gigabases. An international Proteus Genome Research Consortium has been formed to decipher the olm genome. This perspective provides the scientific and biomedical rationale for exploring the olm genome and outlines potential outcomes, challenges, and methodological approaches required to analyze and annotate the genome of this unique amphibian.

The Holarctic leech genus Haemopis currently includes 11 species, all of which are macrophagous, as opposed to their more infamous bloodfeeding counterparts among hirudiniform leeches. In spite of their ecological importance as fish food and predators of freshwater invertebrates, there is a paucity of data regarding morphology and genetic variation that might guide future identification efforts for members of the genus. The lack of detailed descriptions of distinguishing morphological features, coupled with the absence of a robust phylogenetic hypothesis for the genus, have conspired to prevent meaningful inferences on the natural history of the group. In an attempt to remedy this, we present new genetic (using COI, 12S rDNA, 28S rDNA and 18S rDNA) data for the majority of the known species diversity within the genus in order to both infer a phylogenetic hypothesis and to introduce authoritative DNA barcodes for the newly collected species. The potential of these barcodes is increased through rigorous morphological investigations of the specimens, with comparisons to the original literature. Our resulting phylogenetic hypothesis is agnostic as to the geographic origin of the genus, with equal probability afforded to both a Nearctic and Palearctic origin. Beyond this, we show that there is a strong tendency towards a barcoding gap within the genus, but that a distinct gap is lacking due to the relatively high genetic variation found within H. marmorata. Taken together, our results shed light on species delimitation within, and evolutionary history of, this often-neglected group of leeches.

Throughout most of the kingdom Animalia, evolutionary transitions from surface life to a life permanently bound to caves and other subterranean habitats have occurred innumerous times. Not so in tetrapods, where a mere 14 cave-obligate species—all plethodontid and proteid salamanders—are known. We discuss why cave tetrapods are so exceptional and why only salamanders have made the transition. Their evolution follows predictable and convergent, albeit independent pathways. Among the many known changes associated with transitions to subterranean life, eye degeneration, starvation resistance, and longevity are especially relevant to human biomedical research. Recently, sequences of salamander genomes have become available opening up genomic research for cave tetrapods. We discuss new genomic methods that can spur our understanding of the evolutionary mechanisms behind convergent phenotypic change, the relative roles of selective and neutral evolution, cryptic species diversity, and data relevant for conservation such as effective population size and demography.

Synapomorphies are fundamental to phylogenetic systematics as they offer empirical evidence of monophyletic groups. However, no method exists to directly measure synapomorphy. Here, we propose a method that quantifies synapomorphy using the pattern of character state distribution over a cladogram separately for each character and for each clade. We define a fully synapomorphic character state as one shared by all of a clade’s terminal taxa and at the same time completely absent from all terminal taxa outside that clade. The extent to which this condition is met corresponds to the support for the character state being synapomorphic or, in short, support for synapomorphy. It is calculated as the probability of randomly selecting, by multi-stage sampling following the topology of the tree, two terminals from inside a clade sharing the same character state and one terminal from outside the clade bearing a different character state. The method is independent of tree inference and free of transformational assumptions, and so can be applied to any tree and used for any type of discrete character. By measuring synapomorphy, the method offers a potential tool for determining diagnostic character states for taxa on different hierarchical levels, for evaluating alternative systems of character coding, and for evaluating clade support. We show how the method differs from ancestral character state reconstruction methods and goodness-of-fit indices. We demonstrate the behaviour of our method with several hypothetical scenarios and its potential use with two real-life examples.

Adaptive radiations are bursts of evolutionary species diversification that have contributed to much of the species diversity on Earth. An exception is modern Europe, where descendants of ancient adaptive radiations went extinct, and extant adaptive radiations are small, recent and narrowly confined. However, not all legacy of old radiations has been lost. Subterranean environments, which are dark and food-deprived, yet buffered from climate change, have preserved ancient lineages. Here we provide evidence of an entirely subterranean adaptive radiation of the amphipod genus Niphargus, counting hundreds of species. Our modelling of lineage diversification and evolution of morphological and ecological traits using a time-calibrated multilocus phylogeny suggests a major adaptive radiation, comprised of multiple subordinate adaptive radiations. Their spatio-temporal origin coincides with the uplift of carbonate massifs in South-Eastern Europe 15 million years ago. Emerging subterranean environments likely provided unoccupied, predator-free space, constituting ecological opportunity, a key trigger of adaptive radiation. This discovery sheds new light on the biodiversity of Europe., There are relatively few known extant adaptive radiations in Europe that predate the Pleistocene. Here, Borko et al. characterize the diversity and diversification of the subterranean amphipod genus Niphargus, showing evidence for a large adaptive radiation associated with massif uplift 15 million years ago.

The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79–94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context.

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.

Amphipods from the genus Niphargus represent an important part of the Western Palearctic subterranean fauna. The genus is morphologically diverse, comprising several distinct ecomorphs bound to microhabitats in the subterranean environment. The most impressive among them are “lake giants,” a series of massive, large-bodied species. These range from morphologically distinct to morphologically cryptic taxa. We analysed the taxonomic structure of the Niphargus arbiter–Niphargus salonitanus species complex, belonging to “lake giants” from the Dinaric Karst (West Balkans), and assessed their phylogenetic, morphological and ecological diversity. Multilocus phylogeny suggested that the complex is monophyletic and nested within other cave lake ecomorphs. Unilocus and multilocus coalescence species delimitations indicated that the complex totals nine species. These species substantially overlap in morphology and cannot be unambiguously told apart without the use of molecular markers. An analysis of splitting events within a palaeogeological context, and modelling of environmental characteristics on the phylogeny unveiled a complex history of diversification. Part of this diversification might have been influenced by ecological divergence along the altitudinal gradient reaching from the Adriatic coast to inland Dinaric mountain chains and Poljes. Other splits coincide with the marine regression–transgression cycles during Pliocene. We describe Niphargus alpheus sp. n., Niphargus anchialinus sp. n., Niphargus antipodes sp. n., Niphargus arethusa sp. n., Niphargus doli sp. n., Niphargus fjakae sp. n. and Niphargus pincinovae sp. n., and by doing so hope to prompt their further research.

Terrestrial life typically does not occur at depths greater than a few meters. Notable exceptions are massifs of fissured rock with caves and hollow spaces reaching depths of two kilometres and more. Recent biological discoveries from extremely deep caves have been reported as sensations analogous to wondrous deep sea creatures. However, the existence of unique deep terrestrial communities is questionable when caves are understood as integral parts of a bedrock fissure network (BFN) interconnecting all parts of a massif horizontally and vertically. We tested these two opposing hypotheses – unique deep cave fauna vs. BFN – by sampling subterranean communities within the 3D matrix of a typical karst massif. There was no distinction between deep core and shallow upper zone communities. Beta diversity patterns analysed against null models of random distribution were generally congruent with the BFN hypothesis, but suggested gravity-assisted concentration of fauna in deep caves and temperature-dependent horizontal distribution. We propose that the idea of a unique deep terrestrial fauna akin to deep oceanic life is unsupported by data and unwarranted by ecological considerations. Instead, the BFN hypothesis and local ecological and structural factors sufficiently explain the distribution of subterranean terrestrial life even in the deepest karst massifs.

Rapid development of molecular genetics has provided ecologists and wildlife managers with a powerful set of tools for studying and monitoring wildlife. We applied these tools to estimate the size of the brown bear population in Slovenia in 2007. In the years after the estimate was made public, we followed how this estimate affected policy and management actions in Slovenian bear management. We designed and executed a large-scale noninvasive genetic sampling across the range of this species in the country with a network of volunteers and estimated the size of the brown bear population in Slovenia using mark-recapture modeling. In a highly intensive 3-month sampling in autumn 2007, we collected 1057 samples. A total of 931 samples were successfully genotyped, yielding 354 different genotypes. Using mark-recapture and correcting for the edge effect caused by bears moving in and out of the sampling area across the Slovenian-Croatian border, and accounting for detected mortality, we estimated “winter” population size (after annual mortality, before reproduction) at 424 (95% confidence interval 383–458). We also observed an uneven male and female ratio of 0.405 and 0.595, respectively. Using “citizen science,” we managed to conduct a highly intensive large-scale sampling with modest financial resources, something that would be impossible to do otherwise. We produced the first robust, scientifically defensible estimate of the brown bear population size in Slovenia. Although at first reluctantly considered by managers as equivalent to other “traditional” population monitoring data, awareness of the importance of the estimate grew with time. It became the first reference point for understanding population dynamics, a basis to which current and future development of the population is being compared to. As such, we can expect it will profoundly affect Slovenian bear management in the years to come.

The European cave salamander Proteus anguinus is a charismatic amphibian endemic to the concealed and inaccessible subterranean waters of the Dinaric Karst. Despite its exceptional conservation importance not much is known about its ecology and interactions with the groundwater microbiome. The cutaneous microbiota of amphibians is an important driver of metabolic capabilities and immunity, and thus a key factor in their wellbeing and survival. We used high-throughput 16S rRNA gene sequencing based on seven variable regions to examine the bacteriome of the skin of five distinct evolutionary lineages of P. anguinus and in their groundwater environment. The skin bacteriomes turned out to be strongly filtered subsamples of the environmental microbial community. The resident microbiota of the analyzed individuals was dominated by five bacterial taxa. Despite an indicated functional redundancy, the cutaneous bacteriome of P. anguinus presumably provides protection against invading microbes by occupying the niche, and thus could serve as an indicator of health status. Besides conservation implications for P. anguinus, our results provide a baseline for future studies on other endangered neotenic salamanders.

Historical biogeography explains disjunct ranges either by vicariance, that is, the breakage of a formerly continuous range, or by dispersal of individuals across unsuitable areas followed by colonization of a new habitat. In obligate cave species, large and disjunct ranges are especially challenging to explain because animals cannot move beyond the limits of the porous subterranean network within a structural block of carbonate or other cave-bearing rock. Here, the in addition to subterranean dispersal and subterranean vicariance (endogenous processes), processes acting at the surface (exogenous) need to be taken into account. The most important of these are multiple independent invasions of caves by the same ancestral surface species in different geographical areas. Cave invasions do not alter spatial distribution patterns, and are therefore neither vicariant nor dispersalist. Evidence in the form of molecular phylogeography and species delimitation suggests that subterranean dispersal is possible and can produce disjunct ranges, but only within the confines of units of contiguous subterranean habitat. Ranges that encompass larger portions of nonporous ground, or exceed several hundreds of kilometers, either still represent a puzzle or have to be explained by occasional dispersal via the surface during periods of favorable conditions. Vicariance can produce large and highly disjunct ranges of obligate cave species via tectonic plate drifting. This geological process can act endogenously when it disrupts ranges of already formed cave species. It has been well documented at the level of microplate movement, but received inconclusive molecular support at the level of continental plates. Other forms of endogenous vicariance—the breakup of a large, contiguous subterranean range—are very rare. This might be because they are difficult to demonstrate methodologically, or because they require a large and interconnected subterranean range to begin with.

In principle, the distribution of freshwater fauna follows the extent of drainage basins, but various historical and concurrent factors weaken this correspondence. Subterranean karstic drainages can be particularly baffling as their boundaries are decoupled from the surface relief and change with the level of the karstic groundwater. We explored how the distribution and genetic structure of cave fauna correlate to data obtained by conventional groundwater tracers in the Danubian–Adriatic divide in the classical Karst between Postojna and Trieste. Here, waters from the Pivka Polje drain to either basin, depending on the water level. This ambiguity is reflected by the distribution of several aquatic cave species that disperse from one drainage to the other. However, using DNA barcoding methods, we found a clear-cut separation between the closely related subterranean aquatic isopods Asellus aquaticus on the Danubian and Asellus kosswigi on the Adriatic side. The boundary between them agrees with the established hydrology at normal water levels. Further phylogeographic and demographic analyses suggest that the superficial concurrent correspondence has a deeper historical component. We inferred that the Adriatic species did not reach its present boundary by recent range dispersal, but rather has remained there as relict from Pleistocene times. Today, dispersal from the lower part of the Adriatic (Reka River) drainage is impossible because of the large altitudinal difference within. These results demonstrate that biological and particularly genetic groundwater tracing have the advantage of being sensitive to both historical and contemporary groundwater connectivity. Copyright © 2016 John Wiley & Sons, Ltd.

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 response to Živic et al. (Aquat Ecol 49:81–90, 2015. doi: 10.1007/s10452-015-9506-7 ), who dismissed the previously established subdivision of the southern medicinal leech Hirudo verbana into an Eastern and Western phylogroup, we present a re-analysis of their and other available DNA sequence (COI) data. We demonstrate that their data not only perfectly support this subdivision, but also improve the understanding of the boundary between both phylogroups. As predicted previously, it lies on the Balkan Peninsula, roughly following the divide between Danubian and Adriatic drainage basins. This finding is important from a conservation perspective, as medicinal leeches are endangered by over-collecting and habitat destruction.

Shelter-seeking is a vital behavior for stress reduction and survival in a range of animals. It comes at the cost of reduced foraging, mate finding, dispersal and territoriality, and is expected to reflect the trade-off between fitness costs and benefits. One way to test this hypothesis is to compare shelter-seeking behavior in surface habitats and in caves where external threat factors are largely reduced. We did so using the freshwater isopod Asellus aquaticus from the Postojna-Planina Cave System and surrounding surface waters. Animals from two distinct, replicated pairs of surface and cave populations were individually offered a choice between shelter and open area. The thigmotatic sensation of a transparent plastic plate was the only stimulus that could trigger the sheltering behavior. Video recordings showed a clear reduction of shelter-seeking behavior in the cave ecomorph in one population pair (Pivka). There were no changes in the other population pair (Rak), where the behavioral response had a much higher variance. Our results were partly in agreement with the hypothesis that shelter-seeking behavior should be selected against in an environment with reduced external threats. It is nevertheless too early for generalizations as the results in the second population pair were inconclusive. Additionally, we showed that for benthic walkers like A. aquaticus the use of rough substrate is crucial to obtain unbiased behavioral responses. Results of some previous studies using smooth glass or plastic substratum could be affected by unnatural behavior of animals constantly trying to find firm contact with the ground.

Repeated evolution of similar phenotypes is a widespread phenomenon found throughout the living world and it can proceed through the same or different genetic mechanisms. Cave animals with their convergent traits such as eye and pigment loss, as well as elongated appendages, are a striking example of the evolution of similar phenotypes. Yet, few cave species are amenable to genetic crossing and mapping techniques making it challenging to determine the genetic mechanisms causing their similar phenotypes. To address this limitation, we have been developing Asellus aquaticus, a freshwater isopod crustacean, as a genetic model. Many of its cave populations originate from separate colonization events and thus independently evolved their similar cave-related phenotypes which differ from the still existent ancestral-like surface populations. In our prior work, we identified genomic regions responsible for eye and pigment loss in a single cave population from Slovenia. In this study we examined another, independently evolved cave population, also from Slovenia, and asked whether the same or different genomic regions are responsible for eye and pigment loss in the two cave populations. We generated F2 and backcross hybrids with a surface population, genotyped them for the previously identified genomic regions, and performed a complementation test by crossing individuals from the two cave populations. We found out that the same genomic regions are responsible for eye and pigment loss and that at least one of the genes causing pigment loss is the same in both cave populations. Future studies will identify the actual genes and mutations, as well as examine additional cave populations to see if the same genes are commonly associated with eye and pigment loss in this species.

Cave populations have been traditionally perceived as genetically strongly structured and less diverse than related surface populations because of the patchiness of subterranean habitats, low dispersal of cave species, and genetic bottlenecks at colonization. These patterns are at odds with relatively large and/or disjunct ranges of many troglobionts, as well as the countless successful cave colonizations. One way to disentangle those discrepancies is to discriminate between exogenous processes that take place at the surface or during invasion, and endogenous ones that take place in and are governed by the specific conditions of caves. Genetic evidence collected over the past decades suggests that ongoing endogenous processes, endogenous vicariance, and gene flow between allopatric cave populations have less effect on the genetic structure of cave populations than patterns inherited from past exogenous events. Conversely, most known cases of recent migration between non-neighboring cave populations entail the possibility of dispersal via the surface. Gene pools of young cave populations often show strong exogenous imprints such as multiple colonization events and/or recurring gene flow from the surface. This is compatible with the high propagule pressure hypothesis of successful biological invasions, while convincing molecular evidence for the genetic bottleneck hypothesis of cave colonization is sparse.

Caves are long-known examples of evolutionary replications where similar morphologies (troglomorphies) evolve independently as the result of strong natural selection of the extreme environment. Recently, this paradigm has been challenged based on observations that troglomorphies are inconsistent across taxa and different subterranean habitats. We investigated the degree of replicated phenotypic change in two independent cave invasions by the freshwater isopod Asellus aquaticus; the first in a sulphidic aquifer in Romania, the second in a sinking river in the Dinaric Karst in Slovenia. Both ancestral surface populations still live alongside the subterranean ones. Phylogenetic analyses show independence of the two colonization events, and microsatellite analysis shows no evidence of ongoing genetic exchange between surface and subterranean ecomorphs. The overall morphology has changed dramatically at both sites (50 of 62 morphometric traits). The amount of phenotypic change did not reflect differences in genetic diversity between the two ancestral populations. Multivariate analyses revealed divergent evolution in caves, not parallel or convergent as predicted by the current paradigm. Still, 18 traits changed in a parallel fashion, including eye and pigment loss and antennal elongation. These changes might be a consequence of darkness as the only common ecological feature, because Romanian caves are chemoautotrophic and rich in food, whereas Slovenian caves are not. Overall, these results show that morphologically alike surface populations can diverge after invading different subterranean habitats, and that only about one-third of all changing traits behave as troglomorphies in the traditional sense.

Europe’s obligate cave-dwelling amphibian Proteus anguinus inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander’s exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of Proteus using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of Proteus at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of Proteus from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white Proteus eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna.

The pantropical orb web spider family Nephilidae is known for the most extreme sexual size dimorphism among terrestrial animals. Numerous studies have made Nephilidae, particularly Nephila, a model lineage in evolutionary research. However, a poorly understood phylogeny of this lineage, relying only on morphology, has prevented thorough evolutionary syntheses of nephilid biology. We here use three nuclear and five mitochondrial genes for 28 out of 40 nephilid species to provide a more robust nephilid phylogeny and infer clade ages in a fossil-calibrated Bayesian framework. We complement the molecular analyses with total evidence analysis including morphology. All analyses find strong support for nephilid monophyly and exclusivity and the monophyly of the genera Herennia and Clitaetra. The inferred phylogenetic structure within Nephilidae is novel and conflicts with morphological phylogeny and traditional taxonomy. Nephilengys species fall into two clades, one with Australasian species (true Nephilengys) as sister to Herennia, and another with Afrotropical species (Nephilingis Kuntner new genus) as sister to a clade containing Clitaetra plus most currently described Nephila. Surprisingly, Nephila is also diphyletic, with true Nephila containing N. pilipes + N. constricta, and the second clade with all other species sister to Clitaetra; this “Nephila” clade is further split into an Australasian clade that also contains the South American N. sexpunctata and the Eurasian N. clavata, and an African clade that also contains the Panamerican N. clavipes. An approximately unbiased test constraining the monophyly of Nephilengys, Nephila, and Nephilinae (Nephila, Nephilengys, Herennia), respectively, rejected Nephilengys monophyly, but not that of Nephila and Nephilinae. Further data are therefore necessary to robustly test these two new, but inconclusive findings, and also to further test the precise placement of Nephilidae within the Araneoidea. For divergence date estimation we set the minimum bound for the stems of Nephilidae at 40 Ma and of Nephila at 16 Ma to accommodate Palaeonephila from Baltic amber and Dominican Nephila species, respectively. We also calibrated and dated the phylogeny under three different interpretations of the enigmatic 165 Ma fossil Nephila jurassica, which we suspected based on morphology to be misplaced. We found that by treating N. jurassica as stem Nephila or nephilid the inferred clade ages were vastly older, and the mitochondrial substitution rates much slower than expected from other empirical spider data. This suggests that N. jurassica is not a Nephila nor a nephilid, but possibly a stem orbicularian. The estimated nephilid ancestral age (40–60 Ma) rejects a Gondwanan origin of the family as most of the southern continents were already split at that time. The origin of the family is equally likely to be African, Asian, or Australasian, with a global biogeographic history dominated by dispersal events. A reinterpretation of web architecture evolution suggests that a partially arboricolous, asymmetric orb web with a retreat, as exemplified by both groups of “Nephilengys”, is plesiomorphic in Nephilidae, that this architecture was modified into specialized arboricolous webs in Herennia and independently in Clitaetra, and that the web became aerial, gigantic, and golden independently in both “Nephila” groups. The new topology questions previously hypothesized gradual evolution of female size from small to large, and rather suggests a more mosaic evolutionary pattern with independent female size increases from medium to giant in both “Nephila” clades, and two reversals back to medium and small; combined with male size evolution, this pattern will help detect gross evolutionary events leading to extreme sexual size dimorphism, and its morphological and behavioral correlates.

A series of potential pitfalls (fallacies) in estimating subterranean biodiversity are outlined: (1) provincialism—treating different regions differently, especially with respect to new discoveries and undescribed species; (2) equality of described and undescribed species—ignoring the possibility that undescribed species are not really new species; (3) isotropy—assuming all cave regions of similar size have equally rich faunas; (4) scale invariance—ignoring the affect of area on species richness; and (5) misuse of expert opinion—the over-reliance on experts estimates often without comparable estimates for all areas. Some standard procedures are suggested for subterranean biodiversity studies, and the value of such studies is emphasized.

Medicinal leeches (Hirudo spp.) are among the best-studied invertebrates in many aspects of their biology. Yet, relatively little is known about their biogeography, ecology and evolution. Previous studies found vast ranges but suggested low genetic diversity for some species. To examine this apparent contradiction, the phylogeny and phylogeography of the widespread Hirudo verbana, Hirudo medicinalis and Hirudo orientalis were investigated in a comparative manner. Populations from across their ranges in Europe, Asia Minor, the Caucasus and Central Asia, were analyzed by various phylogenetic and population genetic approaches using both mitochondrial (COI and 12S) and nuclear DNA sequences (ITS1, 5.8S and ITS2). The populations showed surprisingly little genetic differentiation despite vast ranges. The only clear structure was observed in H. verbana. This species is subdivided into an Eastern (southern Ukraine, North Caucasus, Turkey and Uzbekistan) and a Western phylogroup (Balkans and Italy). The two phylogroups do not overlap, suggesting distinct postglacial colonization from separate refugia. Leeches supplied by commercial facilities belong to the Eastern phylogroup of H. verbana; they originate from Turkey and the Krasnodar Territory in Russia, two leading areas of leech export. H. verbana and H. medicinalis have experienced recent rapid population growth and range expansion, while isolation by distance has shaped the genetic setup of H. orientalis. The habitat of the latter is patchy and scattered about inhospitable arid and alpine areas of Central Asia and Transcaucasia. Centuries of leech collecting and transport across Europe seem not to have affected the natural distribution of genetic diversity, as the observed patterns can be explained by a combination of historical factors and present day climatic influences.

It has been suggested that both niche-based and neutral mechanisms are important for biological communities to evolve and persist. For communities in extreme and isolated environments such as caves, theoretical and empirical considerations (low species turnover, high stress, strong convergence owing to strong directional selection) predict neutral mechanisms and functional equivalence of species. We tested this prediction using subterranean amphipod communities from caves and interstitial groundwater. Contrary to expectations, functional morphological diversity within communities in both habitats turned out to be significantly higher than the null model of randomly assembled communities. This suggests that even the most extreme, energy-poor environments still maintain the potential for diversification via differentiation of niches.

The effective population size (N(e) ) could be the ideal parameter for monitoring populations of conservation concern as it conveniently summarizes both the evolutionary potential of the population and its sensitivity to genetic stochasticity. However, tracing its change through time is difficult in natural populations. We applied four new methods for estimating N(e) from a single sample of genotypes to trace temporal change in N(e) for bears in the Northern Dinaric Mountains. We genotyped 510 bears using 20 microsatellite loci and determined their age. The samples were organized into cohorts with regard to the year when the animals were born and yearly samples with age categories for every year when they were alive. We used the Estimator by Parentage Assignment (EPA) to directly estimate both N(e) and generation interval for each yearly sample. For cohorts, we estimated the effective number of breeders (N(b) ) using linkage disequilibrium, sibship assignment and approximate Bayesian computation methods and extrapolated these estimates to N(e) using the generation interval. The N(e) estimate by EPA is 276 (183-350 95% CI), meeting the inbreeding-avoidance criterion of N(e) > 50 but short of the long-term minimum viable population goal of N(e) > 500. The results obtained by the other methods are highly consistent with this result, and all indicate a rapid increase in N(e) probably in the late 1990s and early 2000s. The new single-sample approaches to the estimation of N(e) provide efficient means for including N(e) in monitoring frameworks and will be of great importance for future management and conservation.

Understanding the process of evolution is one of the great challenges in biology. Cave animals are one group with immense potential to address the mechanisms of evolutionary change. Amazingly, similar morphological alterations, such as enhancement of sensory systems and the loss of eyes and pigmentation, have evolved multiple times in a diverse assemblage of cave animals. Our goal is to develop an invertebrate model to study cave evolution so that, in combination with a previously established vertebrate cave system, we can address genetic questions concerning evolutionary parallelism and convergence. We chose the isopod crustacean, Asellus aquaticus , and generated a genome-wide linkage map for this species. Our map, composed of 117 markers, of which the majority are associated with genes known to be involved in pigmentation, eye, and appendage development, was used to identify loci of large effect responsible for several pigmentation traits and eye loss. Our study provides support for the prediction that significant morphological change can be mediated through one or a few genes. Surprisingly, we found that within population variability in eye size occurs through multiple mechanisms; eye loss has a different genetic basis than reduced eye size. Similarly, again within a population, the phenotype of albinism can be achieved by two different genetic pathways—either by a recessive genotype at one locus or doubly recessive genotypes at two other loci. Our work shows the potential of Asellus for studying the extremes of parallel and convergent evolution—spanning comparisons within populations to comparisons between vertebrate and arthropod systems.

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.

1. Distribution and status of medicinal leeches were re-considered in the light of the new taxonomy recognizing four Western Palaearctic species: Hirudo medicinalis, Hirudo verbana, Hirudo orientalis and Hirudo troctina. 2. Recent records and new data obtained on expeditions to Ukraine, Russia, Azerbaijan, Kazakhstan, Uzbekistan and the Western Balkans were mapped to obtain an up-to-date overview of the distribution. 3. Three hypotheses explaining the current ranges of all Hirudo species were tested. The ecological hypothesis, suggesting a strong impact of large-scale environmental factors, received the highest support, while anthropogenic influence was minimal, and no historical patterns of refugia and colonization were detected. 4. Mapped localities of all Hirudo species show extensive, belt-shaped ranges extending from east to west. H. medicinalis is distributed from Britain and southern Norway to the southern Urals and probably as far as the Altai Mountains, occupying the deciduous arboreal zone. H. verbana has been recorded from Switzerland and Italy to Turkey and Uzbekistan, which largely corresponds to the Mediterranean and sub-boreal steppe zone. H. orientalis is associated with mountainous areas in the sub-boreal eremial zone and occurs in Transcaucasian countries, Iran and Central Asia. H. troctina has been found in north-western Africa and Spain in the Mediterranean zone. 5. Based on the data gathered, and considering real and potential threats, global IUCN category Near Threatened is proposed for H. medicinalis, H. verbana, and H. orientalis, while H. troctina can only be assigned to category Data Deficient. Copyright © 2010 John Wiley & Sons, Ltd.

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...