With over 430 species currently described, the amphipod genus Niphargus Schiödte, 1849 is the most species-rich crustacean genus in subterranean waters. Previous phylogenetic studies of this genus have relied mainly on mitochondrial COI and nuclear 28S sequences, which do not resolve all the nodes in its phylogeny. As a first step towards a mitogenome-based phylogeny of niphargids, we present here the first complete mitogenome sequence of Niphargus . To obtain high-accuracy mitogenome sequences and annotations, genome skimming of three individuals of Niphargus dolenianensis Lorenzi, 1898 was performed using both short, accurate reads (Illumina) and long, noisier reads (nanopore). Whereas the direct assembly of Illumina sequences yielded structurally incorrect mitogenome sequences, the assembly of nanopore reads produced highly accurate sequences that were corroborated by the mapping of Illumina reads. Polishing the nanopore consensus using Illumina reads corrected a handful of errors at the homopolymer level. The resulting mitogenome sequences ranged from 14,956 to 15,199 bp and shared the same arrangement of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a putative control region. Phylogenetic analyses based on protein-coding genes confirmed that the Niphargidae family is sister to Pseudoniphargidae, resolving their relationships with other amphipod families. This highlights the utility of mtDNA genome sequences for studying the evolution of this groundwater genus, and the refinement of new methodological approaches, such as nanopore sequencing, is promising for the study of its origin and diversification.

The genus Marmocandona, established in 2012 to encompass species formerly attributed to the Pseudocandona zschokkei species group, currently includes five subterranean species distributed across the Western Palearctic region. The presence of M. zschokkei, the most widely distributed species and the only one with a documented fossil record in the genus, has previously been reported in Italy, albeit without conclusive evidence. This study substantially enhances the known diversity of Marmocandona through the description of three species from northern Italy: two collected from the hyporheic zone of streams and one from a spring, the latter currently left in open nomenclature. Additionally, the occurrence of M. sp. aff. zschokkei has been documented in a cave in Central Italy. Morphological analyses of these newly described taxa, along with previously recognized species, revealed a combination of plesiomorphic and apomorphic traits in their chaetotaxy. This study also offers preliminary insights into the potential timing and mechanisms underlying the colonization of subterranean habitats by this new species.

Groundwater ecosystems host highly specialised and frequently endemic faunas, yet their biodiversity remains poorly explored. Although Italy is recognised as a hotspot of subterranean diversity, knowledge of its stygobitic ostracods (i.e., species that are obligate inhabitants of subterranean waters) remains fragmentary. Here we report new data on ostracods collected from 15 natural caves, a mine and an aquifer across mainland Italy and Sicily between 2009 and 2018, with the aim of documenting new occurrence records from poorly investigated subterranean sites, discussing their taxonomic implications, and reassessing the diversity and endemism of Italian stygobitic ostracods in a broader European context. Nineteen taxa belonging to 12 genera and five families were identified, several of which are regarded as strictly stygobitic. These include representatives of Mixtacandona (Candonidae), among which several putative undescribed species of the laisi-chappuisi group were detected. Particularly noteworthy is the occurrence of Mixtacandona cf. botosaneanui in southern Italy: if its identity with the nominal species is confirmed, this record would considerably extend its known geographical range and provide the first description of the male, thereby adding important taxonomic information for a poorly known subterranean lineage. We also report a new record of Typhlocypris cf. eremita and specimens referable to Pseudolimnocythere, further expanding the known distribution of these genera within Italian subterranean habitats. Although some specimens could be assigned only tentatively because well-preserved adults were scarce, the new records substantially refine current knowledge of Italian groundwater ostracod diversity and distribution patterns at both regional and European scales. An updated checklist raises the number of Italian stygobitic ostracods to more than 30 taxa, representing approximately 30% of the currently known European groundwater ostracod diversity. This proportion is remarkable given the limited extent of Italian territory, and the high frequency of endemic and potentially undescribed species further highlights the Italian peninsula and its islands as important centres of diversification for subterranean ostracods. These results emphasize the need for continued biospeleological surveys and integrative taxonomic approaches combining morphology and molecular data to resolve species boundaries, phylogenetic relationships and colonization histories in subterranean lineages.

Problematic taxa, often referred to as species complexes, are common among freshwater organisms. Understanding their species composition, distribution and history is key to integrating these taxa into many ecological disciplines. We investigated the taxonomy and biogeography of the Proasellus coxalis species complex (PCC), a diversified and functionally important group of asellid isopods (Pancrustacea) in freshwaters of the Mediterranean Basin and central Europe. We used phylogenetic and phylogeographic analyses from specimens collected at 143 localities across the range of the PCC to document its diversity, phylogenetic position and historical biogeography. First, we used molecular species delimitation methods based on the mitochondrial cytochrome oxidase subunit I (COI) gene to identify molecular operational taxonomic units (MOTUs) representing putative species within that complex. Second, we used three additional genes-the 16S mitochondrial rDNA, FASTKD4 nuclear and 28S nuclear rDNA genes-to generate a four-gene dated phylogeny of Asellidae to test the monophyly and phylogenetic position of the PCC. Third, we applied statistical ancestral reconstruction methods to the four-gene dated phylogeny of the PCC to infer its biogeographic history. Fourth, we used Bayesian phylogeographic diffusion models based on the COI and 16S genes to reconstruct the range dynamics of the only widely distributed MOTU in this species complex. We identified >= 25 MOTUs within the PCC. The eastern Mediterranean Basin, including the Pontic-Mediterranean and Italian peninsulas, contained numerous narrowly distributed MOTUs. In contrast, central Europe, northern Italy, the Dinarides, the Iberian peninsula and northwestern Africa were colonised by a single MOTU, which we term P. banyulensis. The four-gene phylogeny of the Asellidae clustered all PCC MOTUs into a well-supported monophyletic group, which we term the P. coxalis clade. Ancestral reconstruction methods indicated that the clade originated during the early Miocene in the southern Hellenides, at that time part of the Balkanian-Anatolian landmass. From there, it dispersed and diverged mainly during the Miocene, through the eastern Mediterranean Basin, including the Italian peninsula. Bayesian phylogeographic diffusion models revealed that central Europe and the western Mediterranean Basin were recently colonised from Italian populations of P. banyulensis. During the Pleistocene, multiple waves of colonisation of that species followed each other from central Italy, the most northerly (Germany, Sweden) and southerly (southern Spain, Morocco and Algeria) regions being colonised last. We resolved major taxonomic, phylogenetic and biogeographic uncertainties that have marked more than a century of research on the PCC. Our updated understanding of this complex is that of a species-rich clade that has long diversified in the eastern Mediterranean Basin, but of which a single species-P. banyulensis-has recently colonised central Europe and the western Mediterranean Basin from Italian refugia. In central Europe, this species adds to two other widespread species-Asellus aquaticus from Pannonian refugia and P. meridianus, most probably from Iberian refugia-to produce mixed asellid communities.

The new Checklist of the Italian Fauna provides an updated inventory of Cladocera species in Italy, encompassing the orders Ctenopoda, Anomopoda, Onychopoda, and Haplopoda. The checklist contains 121 species belonging to 13 families, with Chydoridae and Daphniidae being the most abundant. Seven freshwater species have been added since the previous checklist, while the number of marine species remains unchanged at six. Following recent taxonomic revisions, changes in the nomenclature have been introduced, primarily within the subfamily Aloninae. None of the species is endemic to Italy, but six are considered alien. The presence of species in each macroregion and administrative region is reported, with the number of species per region ranging from 10 in Molise to 77 in Lombardy administrative regions. A total of 165 new regional citations are recorded, with southern regions showing increases exceeding 40% of their total records respect to the previously reported ones in the CKmap database published in 2005. Although the total number of Italian cladoceran species is well known, their taxonomy is still evolving, particularly within Aloninae and Daphniinae. Molecular studies are expected to reveal cryptic species and refine biogeographic patterns.

ABSTRACTMotivationSubterranean biodiversity is increasingly threatened by multiple intertwined anthropogenic impacts, including habitat loss, pollution, overexploitation of resources, biological invasions and climate change. Worryingly, subterranean biodiversity is still poorly represented in conservation agendas, also due to persisting gaps in our knowledge of the organisms thriving in the often‐secluded and difficult‐to‐access subterranean ecosystems. This is even more apparent for small‐sized (body size < 1 mm) groundwater‐dwelling metazoans, among which copepods (Crustacea: Copepoda) represent the dominant group in terms of both species richness and biomass.We present a dataset including 6986 occurrence records of 588 species/subspecies of European obligate groundwater‐dwelling copepods. We curated all records to make their taxonomy consistent with the current systematics of Copepoda, while assessing uncertainty in the geographic coordinates by coupling in‐depth web and literature searches with GIS analyses. We suggest the data provided can be used to explore a range of eco‐evolutionary questions—from the drivers of the distribution of groundwater fauna to the assembly of groundwater communities—as well as to prompt the conservation of groundwater biodiversity and more.Main Types of Variables ContainedOccurrence records of groundwater‐dwelling copepods, with details about specimen taxonomy, source of the record, occurrence locality and habitat type.Spatial Location and GrainGeographical Europe (including western Russian Federation), along with Turkey and Georgia. Occurrence records were assigned projected geographic coordinates (EPSG:3035) at 100 m resolution but with varying spatial uncertainty.Time Period and Grain1907–2017.Major Taxa and Level of MeasurementCrustacea: Copepoda. Most records have species‐level identification, while some of them are identified at the subspecies level.Software FormatComma‐separated values file (.csv) and Excel file (.xlsx), with UTF‐8 encoding and meta‐data provided following the Darwin Core standard.

Niphargus is the most speciose amphipod genus inhabiting groundwater of the Western Palearctic. The genus is renowned for its notorious taxonomy, associated with large within- but often negligible between-species morphological variation. Results of molecular taxonomy suggested that morphological variation captured less than half of its species diversity, calling for a molecular revision of past taxonomic and faunistic works. Molecular assessment of species diversity within Niphargus has been conducted since the 2000s, but it remains spatially incomplete given the genus’ extensive geographic range. Here, we provide the first comprehensive assessment of the diversity of Niphargus in France using molecular markers. We sequenced the COI mitochondrial marker from 436 individuals collected from 234 locations. Using Assemble Species by Automated Partitioning, we identified 99 molecular operational taxonomic units (MOTU), among which 74 could represent potential new species. In turn, we selected one individual per MOTU, amplified nuclear markers 28S and Histone 3, aligned them with an additional 142 MOTUs from across the genus range, and reconstructed their phylogenetic relationships using the Maximum Likelihood method. We found that several nominal species occurring in France are polyphyletic. Furthermore, Niphargus from France comprised lineages derived from deep and terminal splits, some of which showed signs of evolutionary radiation. The phylogenetic relationships are consistent with the hypothesis that Niphargus originated in the western part of its range. This study addressed a knowledge gap in the molecular and spatial coverage of Niphargus, provided new barcodes for monitoring the diversity of the genus, and identified future directions for research.

ABSTRACT Cryptic species are an important part of freshwater biodiversity, yet it remains unclear how these species integrate into communities from local to regional geographic scales. To protect biodiversity, particularly overlooked cryptic species, an accurate understanding of the underlying processes and adequate level of protection is needed. We analysed patterns of syntopies (local co‐occurrences) and sympatries (regional range overlap) to explore how the phylogenetic origin of cryptic species shapes biodiversity patterns. We hypothesised (i) that syntopies were more common among distantly than closely related cryptic species, and (ii) that the existing sympatries were an outcome of phylogenetic relatedness and dispersal. The hypotheses were tested on a polyphyletic species complex of subterranean amphipod species ( Niphargus rhenorhodanensis complex) by deploying molecular species delimitation, time‐calibrated phylogenies, and co‐occurrence analyses with probabilistic and generalised linear models (GLM). The studied complex comprised 37–48 molecular operational taxonomic units (MOTUs) from nine different clades, with syntopies occurring at random or less frequently than expected. GLM indicated age of divergence did not predict species sympatries, although they emerged more frequently among MOTUs from different clades. Sympatries, however, emerged through dispersal, in MOTUs with large geographic ranges. These mostly overlapped at the foothills of the Alps, the Jura and the Central Massif. We conclude that the observed spatial patterns are mainly driven by dispersal and presumably reflect the geographic circumstances of speciation. While species richness on a local scale may be an outcome of competition and dispersal, regional biodiversity patterns presumably arise through a clade‐level cascade of historical events, including orogeny and climatic shifts.

The genus Niphargus is the most diverse subterranean amphipod genus in the western Palearctic region but, owing to the presence of cryptic species and homoplasy, its taxonomy and biogeographic scenarios are complex, making molecular methods essential to understand its evolution. We conducted a study combining dna -based taxonomy with traditional morphotaxonomy to investigate Niphargus bihorensis Schellenberg, 1940, known from the Western Alps and Carpathians. We redescribed the type material of N. bihorensis from Bihor County, Romania, and revealed the presence of a cryptic species, N. absconditus n. sp., in the same karstic area (Pădurea Craiului Mountains). Additionally, the Alpine populations previously attributed to N. bihorensis turned out to belong to a new, not so closely related species, N. tizianoi n. sp. Phylogenetic analyses based on a concatenated dataset of one mitochondrial and two nuclear markers suggest that the N. bihorensis species complex belongs to a strongly supported clade, together with several species distributed from Switzerland to Iran.

Subterranean ecosystems (comprising terrestrial, semi-aquatic, and aquatic components) are increasingly threatened by human activities; however, the current network of surface-protected areas is inadequate to safeguard subterranean biodiversity. Establishing protected areas for subterranean ecosystems is challenging. First, there are technical obstacles in mapping three-dimensional ecosystems with uncertain boundaries. Second, the rarity and endemism of subterranean organisms, combined with a scarcity of taxonomists, delays the accumulation of essential biodiversity knowledge. Third, establishing agreements to preserve subterranean ecosystems requires collaboration among multiple actors with often competing interests. This perspective addresses the challenges of preserving subterranean biodiversity through protected areas. Even in the face of uncertainties, we suggest it is both timely and critical to assess general criteria for subterranean biodiversity protection and implement them based on precautionary principles. To this end, we examine the current status of European protected areas and discuss solutions to improve their coverage of subterranean ecosystems.

The subterranean karst environment (terrestrial, semi-aquatic and aquatic components) is unique containing a high diversity of species with specific morphologic, physiologic, and metabolic adaptations (Moldovan et al., 2018). The fragile nature of this environment poses significant challenges when dealing with biodiversity safeguarding and its habitats protection against increasing threats of human activities (Mammola et al., 2019).

Owing to the “Racovitzan impediment”, the groundwater fauna of most biogeographical regions is currently inadequately known, thus hampering our understanding of subterranean biodiversity and its protection. Based on an extensive bibliographical review accompanied by fieldwork to localize occurrence sites, a checklist of crustacean taxa reported to date from Sicilian groundwater is provided, and their distribution is described. Among the 63 taxa recorded to date, 43 belong to the class Copepoda (orders Cyclopoida and Harpacticoida), 15 to the class Malacostraca (orders Amphipoda, Bathynellacea, Isopoda, and Thermosbaenacea), and 5 to the class Ostracoda (order Podocopida). Conversely, to date, no representatives of the copepod order Calanoida nor species of the class Branchiopoda have been recorded from groundwater habitats on the island. Several taxa require accurate taxonomic revision or are yet to be formally described and are thus at present left in open nomenclature. Finally, the date of publication of several copepod and amphipod taxa is amended. A high incidence of stygobites (i.e., obligate groundwater dwellers) has been observed in malacostracans, whereas nearly half of the recorded copepods were non-stygobites. This pattern is mirrored by the incidence of endemic species, which is higher in malacostracans than in copepods. The only non-stygobitic crustacean species endemic to Sicily observed in the frame of present review is the asellid isopod Proasellus montalentii. The paucity of information currently available on the Sicilian groundwater ostracods prevents us from drawing conclusions regarding this crustacean group. The origin and composition of Sicilian groundwater crustacean fauna can be explained by considering three major faunal assemblages: the presence of ancient paleoendemic taxa, likely of Miocene origin, the colonization of the groundwater of the island during late Pliocene and Pleistocene land connections with peninsular Italy, and the direct colonization of these environments from the sea; no species of African origin have been discovered to date. Based on currently available data, the groundwater of southeastern Sicily hosts the highest species richness and some of the most biogeographically interesting taxa. Unfortunately, a progressive lowering and salinization of the local aquifers possibly due to climate change and its overexploitation threats this fauna, and several taxa are disappearing even before their discovery and description.

In recent decades, medicinal leeches of the genus Hirudo have experienced a sharp decline throughout their distribution range due to their overexploitation for medical use and habitat alteration. Such a phenomenon is not fully understood because of the complex taxonomy of the genus, which remained unsettled until the beginning of the XXI century, when the implementation of DNA taxonomy allowed for a better understanding of the diversity of the genus and the distribution ranges of Hirudo species. Only the Mediterranean medicinal leech Hirudo verbana Carena, 1820 is currently reported to occur in Italy and its major islands, although records of other Hirudo species have been reported in the literature. In this study, we report the occurrence of the Dragon leech Hirudo troctina Johnson, 1816 in Sardinia and provide its molecular characterization. In addition, based on a review of the existing literature and museum collections, we provide an ample synopsis of the available evidence regarding the occurrence of the species on the island. Hirudo troctina proved to be widespread and abundant in Sardinia during the XIX century but became rarer and rarer in the XX and XXI centuries possibly due to its overexploitation for medicinal use and export in mainland Europe, the abandonment of traditional grazing practices, and the decline of wetland habitats and amphibian populations. Currently, the only recently validated occurrence sites of the species are the water bodies of the “Giara di Gesturi”, a basaltic plateau in central-southern Sardinia. No corroborated evidence of the possible occurrence of other Hirudo species on the island was found. A better understanding of Hirudo troctina distribution in Sardinia will allow the amelioration of its protection and management practices, also under the provisions of the European “Habitats Directive”, where the H. medicinalis species complex is listed in Annex V.

This chapter briefly reviews the taxonomy, biology, ecology, and morphological characters of freshwater crustaceans of the class Copepoda (orders Calanoida, Cyclopoida, Harapacticoida) and the parasitic Ichthyostraca of the subclass Branchiura (fish lice, order Arguloida) recorded in the Mediterranean Basin. Methods to properly collect, preserve, and study these crustaceans are reported. The main morphological characters, recorded from the literature and the authors' datasets, are used in an illustrated taxonomic key to the families and genera of the ordered Calanoida (7 families and subfamilies, 25 genera and subgenera), Cyclopoida (5 families and subfamilies, 26 genera and subgenera), and Harpacticoida (10 families, 41 genera and subgenera). The order Arguloida is represented in the study area by one family (Argulidae) and one genus.

The intriguing origin and distribution of subterranean amphipods of the family Niphargidae, particularly in north-western Europe and the British Isles, were heavily influenced by Pleistocene glaciations, which obscured most ancient events. The discovery of a new species in Brittany (described herein as Niphargus quimperensis sp. nov.) that is closely related to two tiny endemic species of the British Isles (one ascribed to the genus Niphargus and the other to Niphargellus), along with further collection of specimens of Niphargellus in Europe, allowed the construction of a comprehensive molecular phylogeny and a new time-calibrated tree of the family Niphargidae.The discovery of Niphargus quimperensis sp. nov. confirmed the position of its clade as sister to all other niphargids. Moreover, phylogenetic analysis demonstrate that the genus Niphargellus is polyphyletic and must be treated as a junior synonym of Niphargus. Finally, we propose that the colonization of continental freshwater by the marine ancestors of Niphargus may have occurred in north-western Europe during the opening of the North Atlantic in the Late Cretaceous, whereas the split of Niphargus quimperensis sp. nov. from the other representatives of its clade may have been related to the isolation of Brittany from the British Isles in the Early Miocene.

The Niphargus stygius species complex is a groundwater group of large-sized, sexually dimorphic species inhabiting mainly caves and, less frequently, wells and springs. According to the taxonomists of the last century, this species complex was supposed to be present in the whole Southern Limestone Alps of Italy as well as in peninsular Italy, Slovenia, and Croatia. Considering the large, presumed distribution area, we tested the contrasting hypotheses of monophyly versus paraphyly of this subterranean species complex, taking in account the presence of putative cryptic species. For this reason, we sampled the type localities of all the described species in the complex present in the Italian Southern Limestone Alps and neighbouring areas, and used nuclear rDNA (28S, ITS region) and mtDNA (COI) markers to assess the phylogenetic relationships and species richness. Phylogenetic analysis confirmed that the Niphargus stygius complex in the Southern Limestone Alps is polyphyletic comprising an eastern clade (the N. julius clade, present in NW Italy, northern Slovenia, and southern Austria) and two western clades (the N. brixianus and N. montellianus clades). These two clades are not closely related to the eastern one but rather form a monophyletic group together with a widely distributed Apennine clade ( N. speziae clade). None of these clades is closely related to typical N. stygius . Three different molecular species delimitation methods applied to COI and rDNA sequences recognized slighlty different numbers of putative species, suggesting that each clade is a species complex. Bayesian time-calibrated phylogeny revealed that most clades began to split up during Miocene and Pliocene, ruling out the effect of Pleistocene glaciations, evidenced only by the COI marker, in explaining their speciation process and justifying the presence of several putative cryptic or pseudocryptic species. ### Competing Interest Statement The authors have declared no competing interest.

Aim Morphologically cryptic species are an important part of global biodiversity, yet it remains unclear how these species contribute to and integrate into communities at different geographic scales. It is especially unclear at which scales they co-occur, and if and how their ranges overlap. To adequately protect biodiversity, an accurate understanding of the underlying processes and adequate level of protection is needed, in particularly for often overlooked cryptic species. Question We analyzed patterns of syntopies (local co-occurrences) and sympatries (range overlap) to test how the evolutionary origin of cryptic species shapes biodiversity patterns at different geographic scales. We hypothesized i) that syntopies are more common among phylogenetically deeply divergent cryptic species than among close relatives, and ii) that sympatries are an outcome of phylogenetic relatedness and dispersal, with range size as a proxy of dispersal ability. Location Western Europe. Taxon Subterranean amphipod crustaceans of the polyphyletic Niphargus rhenorhodanensis species complex. Methods Unilocus species delimitations (PTP, ASAP), calibrated multilocus phylogenetic analyses, co-occurrence analyses using a probabilistic model, generalized linear models (GLM). Results The studied species complex comprises 37–48 molecular operational taxonomic units (MOTUs) from nine different clades. Syntopies are random or less frequent than expected, implying an insufficient between-MOTU differentiation allowing stable coexistence. GLM suggested that age of divergence does not predict species sympatries, although they emerge more frequently among MOTUs from different clades. By contrast, sympatries emerge when at least one MOTU disperses over a large geographic range. Biodiversity rich regions were found at the foothills of the Alps, the Jura and the Central Massif, regardless the inference method. Main conclusions Biodiversity patterns of the herein studied species complex are driven mainly by dispersal and reflect geographic circumstances of speciation. While species richness on a local scale may be the outcome of competition and dispersal, regional biodiversity patterns emerged through biogeographic history on a clade-level.