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Transition to novel environments, such as groundwater colonization by surface organisms, provides an excellent research ground to study phenotypic evolution. However, interspecific comparative studies on evolution to groundwater life are few because of the challenge in assembling large ecological and molecular resources for species-rich taxa comprised of surface and subterranean species. Here, we make available to the scientific community an operational set of working tools and resources for the Asellidae, a family of freshwater isopods containing hundreds of surface and subterranean species. First, we release the World Asellidae database (WAD) and its web application, a sustainable and FAIR solution to producing and sharing data and biological material. WAD provides access to thousands of species occurrences, specimens, DNA extracts and DNA sequences with rich metadata ensuring full scientific traceability. Second, we perform a large-scale dated phylogenetic reconstruction of Asellidae to support phylogenetic comparative analyses. Of 424 terminal branches, we identify 34 pairs of surface and subterranean species representing independent replicates of the transition from surface water to groundwater. Third, we exemplify the usefulness of WAD for documenting phenotypic shifts associated with colonization of subterranean habitats. We provide the first phylogenetically controlled evidence that body size of males decreases relative to that of females upon groundwater colonization, suggesting competition for rare receptive females selects for smaller, more agile males in groundwater. By making these tools and resources widely accessible, we open up new opportunities for exploring how phenotypic traits evolve in response to changes in selective pressures and trade-offs during groundwater colonization.

A group of four amphoras found in the anchialine cave of Vodeni Rat in the Croatian island of Sveti Klement, on a rocky ledge at 24 m water depth, indicates that a freshwater source was exploited at the bottom of this karstic pit by islanders and/or passing-by sailors from the late Roman Republican Period to the Early Medieval Period. In other words, prior to the 4th–7th century CE, Vodeni Rat was not an anchialine cave but a Pleistocene karstic pit with a freshwater pool at the bottom. Seawater started to infiltrate this cavity via newly opened fissures in the surrounding limestone after the 4th–7th century CE, as the archeometric age of the youngest intact amphora found down in the pit suggests. At present, the 29-m-deep water body is stratified, with a saline water layer at the bottom, a freshwater layer at the surface, and a transitional brackish water layer in the middle. Our study of the physical and biological characteristics of the cave water column revealed diversified and highly partitioned populations of stygobitic crustaceans including ostracods, copepods, amphipods, and thermosbaenaceans. Some species known to be exclusively endemic of highly saline habitats were collected from the bottom saline layer, whereas others known to be of freshwater origin were found in the upper water layer. This suggests that the freshwater dwellers were already present in this cave prior to the early medieval event that caused seepage of seawater into this karstic pit.

Subterranean fauna is an important contributor to the global fauna, but it is still understudied and a large part of its taxonomy is not yet resolved. One species complex with unresolved taxonomy is the groundwater amphipod Niphargus ruffoi, endemic to the Alpine chain. Here, we used new samples from across the Alpine arc to review the taxonomic status of the entire clade, including the species N. ruffoi and Niphargus arolaensis. We sequenced four genetic markers from the collected specimens, assessed the phylogenetic position of N. ruffoi within the genus, and studied the structure of this species complex using four molecular species delimitation methods. We tested for recombination using the alignments of the concatenated nuclear rDNA genes. The phylogenetic analyses revealed high support for the monophyly of the studied species complex, defining two lineages (i.e., N. arolaensis and N. ruffoi) within the clade. Molecular species delimitation methods suggested that N. arolaensis is a single species, while N. ruffoi should be considered as a species complex of three (using ITS) to eight (using COI) putative species. Moreover, we found a discrepancy between the different nuclear ribosomal DNA markers, indicating a possible recombination with fragments of 28S DNA of N. ruffoi s. lat. present in the genome of N. arolaensis. For the above-mentioned reasons, the internal phylogenetic structure of N. ruffoi s. lat. could not be fully resolved. Moreover, no clear morphological evidence supported the molecular species delimitation. Consequently, no taxonomic changes were proposed. We postulate that this complex scenario was influenced by Pleistocene climate oscillations with subsequent fragmentation events and secondary contacts, making this an interesting study system to investigate the evolution and biogeography of Alpine clades.

Historically, groundwater ecosystems were thought to harbor few species and their conservation was mainly as an exploitable water resource. In recent decades, it has become clear that many groundwater ecosystems are biodiverse, contain species absent from surface habitats, and carry out many different ecological processes that provide crucial ecosystem services beyond simply being a water source. After briefly reviewing the evolution of concepts about groundwater biodiversity, this chapter describes some approaches now being used to aid groundwater conservation, including molecular methods such as environmental DNA and metabarcoding. Data from these can be used with remotely sensed data to enhance vulnerability mapping and systematic conservation planning approaches. Future advances in groundwater conservation will include educating people about groundwater ecosystems and their often-rich troves of species that are potentially threatened by overexploitation of groundwater and by surface activities that harm the quality or quantity of recharge., SCOPUS: ch.b, info:eu-repo/semantics/published

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., SCOPUS: ch.b, info:eu-repo/semantics/published

The unsaturated karst represents a transitional zone connecting the land surface to the water table of the underlying saturated zone through percolating water and small perched aquifers. It constitutes a significant groundwater reservoir and hosts a high biodiversity of specialized and endemic species. Whereas investigating this ecotone can shed light on the invertebrate diversity of the whole karst system, ecological studies in the unsaturated karst have been hampered by access, mapping, and sampling challenges. We studied the unsaturated zone of 52 caves in the Lessinian Massif (NE-Italy), which is dominated by carbonate rocks interspersed with spot areas composed mainly of igneous rocks. Our aim was to understand which ecological and historical factors may have shaped the distribution of the groundwater crustacean copepods at a regional spatial scale in the Lessinian unsaturated karst. We collected 33 species belonging to the orders Harpacticoida and Cyclopoida. Species richness estimators and related species accumulation curves suggest an exhaustive sampling effort. A hydrologic analysis performed in a Geographic Information System environment divided the study area in three distinct drainage basins: Eastern Lessini, Lessinian Core, and Western Lessini. Regression analyses indicated that richness of non-stygobitic copepods (i.e. surface-water species which exploit groundwater for different purposes) was mainly driven by inter-basin differences. Richness of stygobitic copepods (i.e. obligate groundwater dwellers) was instead strongly related to lithological features and responded negatively to surface thermal variability. Next, we used a kernel density estimation to spatially explore diversity patterns in the area. The highest density of stygobitic harpacticoids was identified in the Eastern Lessinian basin, which is lithologically more heterogeneous and surrounded by practically non-aquiferous igneous rocks that may limit dispersal. Conversely, the Western Lessi, SCOPUS: ar.j, info:eu-repo/semantics/published

Groundwater ecosystems host a rich and unique, but still largely unexplored and undescribed, biodiversity. Several lineages of ostracod crustaceans have subterranean representatives or are exclusively living in groundwaters. The stygobitic genus Pseudolimnocythere Klie, 1938 has a West Palearctic distribution, and includes few living and fossil species of marine origin. Through a comprehensive literature review and the description of the two new living species, Pseudolimnocythere abdita sp. nov. and Pseudolimnocythere sofiae sp. nov. from springs in the Northern Apennines, Italy, a morphological analysis was carried out with the aim of comparing the valve morphology of living and fossil species, and to discuss previous hypotheses about time and mode of colonization of inland waters. Pseudolimnocythere species show a low variability in valve morphology, with a remarkable stasis over geological times. The distribution of extant and fossil species is consistent with a scenario of multiple and independent events of colonization of continental habitats linked to sea level variations starting from Middle Miocene in the Paratethys and, later, in the Mediterranean. The most common colonization routes of inland waters have taken place through karst formations along ancient coastlines, although we cannot exclude some minor active migration through the hyporheic zone of streams. Available distribution data suggest a poor dispersal ability of Pseudolimnocythere species after they had colonized continental waters., SCOPUS: ar.j, info:eu-repo/semantics/published

The Scientific Committee for the Italian Fauna is managing the new checklist of the animal species of Italy. The previous checklist was published in 1993-1995 and included both protozoans and Metazoa (more than 57,000 species); the new project, which includes only Metazoa, started in 2020 and is aimed at updating the former checklist (with more than 60,000 expected species) by on-line datasets and data papers. The new checklist includes marine species recorded in the Italian seas, divided into nine marine sectors, within the Italian Economic Exclusive Zone, and terrestrial and freshwater species recorded in administrative regions, as well as in the three macro-regions (northern continental, southern peninsular and insular - Sicily and Sardinia - macro-regions). Records from geopolitical units biogeographically related to Italy (i.e. Canton Ticino, CH; Corsica, F; San Marino Republic and Vatican City) are also included. Over 180 Italian and foreign taxonomists have so far participated to the first phase of this new project, providing datasets for taxa at different hierarchical level, from phyla to subfamilies and tribes. The list is intended to be a fundamental instrument not only for the faunistic knowledge of Italy, but also for biodiversity conservation strategies in the country and in the European Union. The new Checklist of the Italian fauna will be available from the LifeWatch Italy platform, and it will be progressively updated. Furthermore, data papers for taxa at different hierarchical level could be published with continuity in a special section of the journal Biogeographia – The Journal of Integrative Biogeography., SCOPUS: ar.j, info:eu-repo/semantics/published

This chapter introduces the structure and ecology of karstic subterranean habitats that support a diverse and specialized fauna from tropical to sub-polar zones. These karstic habitats are distinguished in four main functional vertical layers, i.e. the epikarst, vadose, epiphreatic and phreatic zones, which are controlled by local climate conditions, particularly temperature, precipitation, and subsurface hydrology, alongside vegetation. All these factors have been affected by human activity to varying degrees in the past and present. Water enters the karstic subterranean environment through rain and snowmelt, which infiltrates vertically through the four zones, or enters a particular zone as temporary or permanent streams and rivers. Outlets of karstic habitats are temporary or permanent springs. Ecological conditions, especially living space, hydrology and the amount of available organic material determine the community structure within a given karstic zone. The structure of stygofauna, both functional (body size, feeding habits) and compositional (species diversity), is in close relation with habitat structure and environmental conditions., SCOPUS: ch.b, info:eu-repo/semantics/published