Genetic bias in repeated evolution of pigment loss in cave populations of the Asellus aquaticus species complex.

Similar phenotypes can evolve repeatedly under the same evolutionary pressures. A compelling example is the evolution of pigment loss and eye loss in cave-dwelling animals. While specific genomic regions or genes associated with these phenotypes have been identified in model species, it remains uncertain whether a bias towards particular genetic mechanisms exists. An isopod crustacean, Asellus aquaticus, is an ideal model organism to investigate this phenomenon. It inhabits surface freshwaters throughout Europe but has colonized groundwater on multiple independent occasions and evolved several cave populations with distinct ecomorphology. Previous studies have demonstrated that three different cave populations utilized common genetic regions, potentially the same genes, in the evolution of pigment and eye loss. Expanding on this, we conducted analysis on two additional cave populations, distinct either phylogenetically or biogeographically from those previously examined. We generated F2 hybrids from cave × surface crosses and tested phenotype-genotype associations, as well as conducted complementation tests by crossing individuals from different cave populations. Our findings revealed that pigment loss and orange eye pigment in additional cave populations were associated with the same genomic regions as observed in the three previously tested cave populations. Moreover, the lack of complementation across all cross combinations suggests that the same gene likely drives pigment loss. These results substantiate a genetic bias in the recurrent evolution of pigment loss in this model system. Future investigations should focus on the cause behind this bias, possibly arising from allele recruitment from ancestral surface populations' genetic variation or advantageous allele effects via pleiotropy.

Wolf genetic diversity compared across Europe using the yardstick method.

Integrating data across studies with traditional microsatellite genetic markers requires careful calibration and represents an obstacle for investigation of wide-ranging species where populations require transboundary management. We used the "yardstick" method to compare results published across Europe since 2002 and new wolf (Canis lupus) genetic profiles from the Carpathian Mountains in Central Europe and the Dinaric Mountains in Southeastern Europe, with the latter as our reference population. We compared each population with Dinaric wolves, considering only shared markers (range 4-17). For each population, we calculated standard genetic diversity indices plus calibrated heterozygosity (Hec) and allelic richness (Ac). Hec and Ac in Dinaric (0.704 and 9.394) and Carpathian wolves (0.695 and 7.023) were comparable to those observed in other large and mid-sized European populations, but smaller than those of northeastern Europe. Major discrepancies in marker choices among some studies made comparisons more difficult. However, the yardstick method, including the new measures of Hec and Ac, provided a direct comparison of genetic diversity values among wolf populations and an intuitive interpretation of the results. The yardstick method thus permitted the integration of diverse sources of publicly available microsatellite data for spatiotemporal genetic monitoring of evolutionary potential.

Phylogenetic relationships and species delimitation in Haemopis (Annelida: Hirudinea: Haemopidae).

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.

The minnow Phoxinus lumaireul (Leuciscidae) shifts the Adriatic-Black Sea basin divide in the north-western Dinaric Karst region.

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.

From Cave Dragons to Genomics: Advancements in the Study of Subterranean Tetrapods.

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.

Toward the massive genome of Proteus anguinus-illuminating longevity, regeneration, convergent evolution, and metabolic disorders.

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.

A subterranean adaptive radiation of amphipods in Europe.

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.

Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants.

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.

Testing the uniqueness of deep terrestrial life.

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.

No need to hide in caves: shelter-seeking behavior of surface and cave ecomorphs of Asellus aquaticus (Isopoda: Crustacea).

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.

Composition of the cutaneous bacterial community of a cave amphibian, Proteus anguinus.

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.

Common Genetic Basis of Eye and Pigment Loss in Two Distinct Cave Populations of the Isopod Crustacean Asellus aquaticus.

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.

The importance of naming cryptic species and the conservation of endemic subterranean amphipods.

Molecular taxonomy often uncovers cryptic species, reminding us that taxonomic incompleteness is even more severe than previous thought. The importance of cryptic species for conservation is poorly understood. Although some cryptic species may be seriously threatened or otherwise important, they are rarely included in conservation programs as most of them remain undescribed. We analysed the importance of cryptic species in conservation by scrutinizing the South European cryptic complex of the subterranean amphipod Niphargus stygius sensu lato. Using uni- and multilocus delineation methods we show that it consists of 15 parapatric and sympatric species, which we describe using molecular diagnoses. The new species are not mere "taxonomic inflation" as they originate from several distinct branches within the genus and coexist with no evidence of lineage sharing. They are as evolutionarily distinct as average nominal species of the same genus. Ignoring these cryptic species will underestimate the number of subterranean endemics in Slovenia by 12 and in Croatia by four species, although alpha diversity of single caves remains unchanged. The new taxonomy renders national Red Lists largely obsolete, as they list mostly large-ranged species but omit critically endangered single-site endemics. Formal naming of cryptic species is critical for them to be included in conservation policies and faunal listings.

Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus.

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.

Morphological evolution of coexisting amphipod species pairs from sulfidic caves suggests competitive interactions and character displacement, but no environmental filtering and convergence.

Phenotypically similar species coexisting in extreme environments like sulfidic water are subject to two opposing eco-evolutionary processes: those favoring similarity of environment-specific traits, and those promoting differences of traits related to resource use. The former group of processes includes ecological filtering and convergent or parallel evolution, the latter competitive exclusion, character displacement and divergent evolution. We used a unique eco-evolutionary study system composed of two independent pairs of coexisting amphipod species (genus Niphargus) from the sulfidic caves Movile in Romania and Frasassi in Italy to study the relative contribution and interaction of both processes. We looked at the shape of the multifunctional ventral channel as a trait ostensibly related to oxygenation and sulfide detoxification, and at body size as a resource-related trait. Phylogenetic analysis suggests that the sulfidic caves were colonized separately by ancestors of each species. Species within pairs were more dissimilar in their morphology than expected according to a null model based on regional species pool. This might indicate competitive interactions shaping the morphology of these amphipod species. Moreover, our results suggest that the shape of the ventral channel is not subject to long-term convergent selection or to the process of environmental filtering, and as such probably does not play a role in sulfide tolerance. Nevertheless, the ancestral conditions reconstructed using the comparative method tended to be more similar than null-model expectations. This shift in patterns may reflect a temporal hierarchy of eco-evolutionary processes, in which initial environmental filtering became later on superseded by character displacement or other competition-driven divergent evolutionary processes.

A molecular phylogeny of nephilid spiders: evolutionary history of a model lineage.

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.

Ecomorphological convergence of cave communities.

Extreme selective environments are commonly believed to funnel evolution toward a few predictable outcomes. Caves are well-known extreme environments with characteristically adapted faunas that are similar in appearance, physiology, and behavior all over the world, even if not closely related. Morphological diversity between closely related cave species has been explained by difference in time since colonization and different ecological influence from the surface. Here, we tested a more classical hypothesis: morphological diversity is niche-based, and different morphologies reflect properties of microhabitats within caves. We analyzed seven communities with altogether 30 species of the subterranean amphipod (crustacean) genus Niphargus using multivariate morphometrics, multinomial logit models cross-validation, and phylogenetic reconstruction. Species clustered into four distinct ecomorph classes-small pore, cave stream, cave lake, and lake giants-associated with specific cave microhabitats and of multiple independent phylogenetic origins. Traits commonly regarded as adaptations to caves, such as antenna length, were shown to be related to microhabitat parameters, such as flow velocity. These results demonstrate that under the selection pressure of extreme environment, the ecomorphological structure of communities can converge. Thus, morphological diversity does not result from adaptive response to temporal and ecological gradients, but from fine-level niche partitioning.

Niche-based mechanisms operating within extreme habitats: a case study of subterranean amphipod communities.

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.