Morpho-Mineralogical and Bio-Geochemical Description of Cave Manganese Stromatolite-Like Patinas (Grotta del Cervo, Central Italy) and Hints on Their Paleohydrological-Driven Genesis

Caves are dark subsurface environments with relatively constant temperatures that allow studying bio-mineralization processes and paleoenvironmental or climate changes in optimal conditions. In the extreme and oligotrophic cave environment, manganese patinas having stromatolite-like features are uncommon. Here we provide the first detailed mineralogical, geochemical, and microbiological investigation of fine-grained and poorly crystalline MnFe stromatolite-like wall patinas formed in a deep-cave environment in Italy. These mineralizations, about 3 mm thick, consist of an alternation of Mn-layers and Fe-lenses. We show that the microbial communities' composition is dominated by Mn-oxidizing bacteria, such as Bacillus, Flavobacterium, and Pseudomonas. Our multidisciplinary investigation, integrating data from different analytical techniques (i.e., optical microscopy, SEM-EDS, μXRF, XRPD, FT-IR, Raman spectroscopy, and DNA sequencing), revealed peculiar chemical, mineralogical, and biological features: 1) A cyclical oscillation of Mn and Fe along the growth of the patinas. We propose that this oscillation represents the shift between oxic and suboxic conditions related to different phases occurring during paleo-flood events; 2) A typical spatial distribution of mineralogy and oxidation state of Mn, bacterial imprints, detrital content, and stromatolite-like morphologies along the Mn-layers. We propose that this distribution is controlled by the local hydraulic regime of the paleo-floods, which, in turn, is directly related to the morphology of the wall surface. Under less turbulent conditions, the combination of clay mineral catalysis and biological oxidation produced vernadite, a poor-crystalline phyllomanganate with a low average oxidation state of Mn, and branched columnar stromatolite-like morphologies. On the other hand, under more turbulent conditions, the sedimentation of clay minerals and microbial communities' development are both inhibited. In this local environment, a lower oxidation rate of Mn2+ favored the formation of todorokite and/or ranciéite, two compounds with a high average oxidation state of Mn, and flat-laminated or columnar stromatolite-like morphologies

Transcriptomic analysis supports similar functional roles for the two thymuses of the tammar wallaby

Background: The thymus plays a critical role in the development and maturation of T-cells. Humans have a single thoracic thymus and presence of a second thymus is considered an anomaly. However, many vertebrates have multiple thymuses. The tammar wallaby has two thymuses: a thoracic thymus (typically found in all mammals) and a dominant cervical thymus. Researchers have known about the presence of the two wallaby thymuses since the 1800s, but no genome-wide research has been carried out into possible functional differences between the two thymic tissues. Here, we used pyrosequencing to compare the transcriptomes of a cervical and thoracic thymus from a single 178 day old tammar wallaby.Results: We show that both the tammar thoracic and the cervical thymuses displayed gene expression profiles consistent with roles in T-cell development. Both thymuses expressed genes that mediate distinct phases of T-cells differentiation, including the initial commitment of blood stem cells to the T-lineage, the generation of T-cell receptor diversity and development of thymic epithelial cells. Crucial immune genes, such as chemokines were also present. Comparable patterns of expression of non-coding RNAs were seen. 67 genes differentially expressed between the two thymuses were detected, and the possible significance of these results are discussed.Conclusion: This is the first study comparing the transcriptomes of two thymuses from a single individual. Our finding supports that both thymuses are functionally equivalent and drive T-cell development. These results are an important first step in the understanding of the genetic processes that govern marsupial immunity, and also allow us to begin to trace the evolution of the mammalian immune system

Potential factors driving the distribution of subterranean invertebrates in karst groundwaters of the Rotolo Cave (southern Italy).

The study of subterranean environments is hindered by the lack of information that does not allow to fully explain the subsurface abiotic and biotic dynamics, especially when considering the 3D interactions in the deep karst. The fragmented knowledge of mechanisms and factors regulating the subsurface environment and its biological features makes it difficult to assess the connections between the various components, and the changes related to anthropogenic impacts. Challenges for data collection in karst are mainly referred to explorations and surveying in caves, because they are difficult to access. Despite the limitation in accessing the subsurface, karst groundwaters offer privileged sites to study ecosystems of high intrinsic value due to their specialized fauna (stygofauna), often comprising rare and endemic species. The aquatic cave-dwelling organisms live in fissures of the rock mass and in larger conduits and channels, resulting in consequential relations with the aquifer hydrogeological dynamics. The extreme vulnerability of karst and the occurrence of low resilient communities therein, requires urgent monitoring and conservation tools to protect the underground ecosystems in their entirety. This work offers the basis for setting practical conservation actions by means of a systematic and long-term scientific program in the Apulian karst. The carbonates of Apulia host significant groundwater resources and over 2000 caves in need of protection. Many efforts have to be made to assess the abiotic dynamics, together with the status of the stygodiversity. A first attempt was carried out at Grave Rotolo, the deepest cave in Apulia, where cavers have reached the groundwater bodies. A preliminary screening of both saturated and unsaturated karst (May-October 2021) allowed us to assess the minimum theoretical number of species living in the system through asymptotic species richness estimators (Chao1, Chao2, Abundance Coverage Estimator, Incidence Coverage Estimator). This karst system harbors a diverse and composite array of species across groundwater habitat types. Indeed, the faunal sampling underestimated the theoretical true baseline richness level of the cave (range 16.77-76.05%). In addition, the sites where the groundwater fauna live in the cave could be related to complex subsurface flow pathways or different recharge areas, as suggested by the differences observed in species compositions and species abundances among different cave water bodies. Therefore, the combined biological and hydrogeological approach could shed light on their mutual information, where the biological component may work as hydrogeological tracer of the karst system. These tools are necessary to select proper monitoring sites, and define the most appropriate sampling and analytic techniques for achieving a better understanding of the groundwater dynamics and species partitioning in deep karst environments

The Influence of the Recording Time in Modelling the Swimming Behaviour of the Freshwater Inbenthic Copepod Bryocamptus pygmaeus

The analysis of copepod behaviour gained an increasing impetus over the past decade thanks to the advent of computer-assisted video analysis tools. Since the automated tracking consists in detecting the animal's position frame by frame and improving signals corrupted by strong background noise, a crucial role is played by the length of the video recording. The aim of this study is to: (i) assess whether the recording time influences the analysis of a suite of movement descriptive parameters; (ii) understand if the recording time influences the outcome of the statistical analyses when hypotheses on the effect of toxicants/chemicals on the freshwater invertebrate behaviour are tested. We investigated trajectory parameters commonly used in behavioural studies-swimming speed, percentage of activity and trajectory convex hull-derived from the trajectories described by the inbenthic-interstitial freshwater copepod Bryocamptus pygmaeus exposed to a sub-lethal concentration of diclofenac. The analyses presented in this work indicate that the recording time did not influence the outcome of the results for the swimming speed and the percentage of activity. For the trajectory convex hull area, our results showed that a recording session lasting at least 3 min provided robust results. However, further investigations are needed to disentangle the role of concurrent factors, such as the behavioural analysis of multiple individuals simultaneously, whether they are of the same or opposite sex and the implications on sexual behaviour, competition for resources and predation

Linking hydrogeology and ecology in karst landscapes: The response of epigean and obligate groundwater copepods (crustacea: Copepoda)

Groundwater invertebrate communities in karst landscapes are known to vary in response to multiple environmental factors. This study aims to explore the invertebrate assemblages’ composition of an Apennine karst system in Italy mainly described by the Rio Gamberale surface stream and the Stiffe Cave. The stream sinks into the carbonate rock and predominantly feeds the saturated karst into the cave. For a minor portion, groundwater flows from the epikarst and the perched aquifer within it. The spatial distribution of the species belonging to the selected target group of the Crustacea Copepoda between the surface stream and the groundwater habitats inside the cave highlighted a different response of surface-water species and obligate groundwater dwellers to the hydrogeological traits of the karst unit. Our results suggest that fast endorheic infiltration routes promoted the drift of epigean species from the surface to groundwater via the sinking stream while most of the obligate groundwater dwellers come from the perched aquifer in the epikarst from diffuse infiltration pathways

Effectiveness of Biomass/Abundance Comparison (ABC) Models in Assessing the Response of Hyporheic Assemblages to Ammonium Contamination

Abundance-Biomass Comparison (ABC) models, first described for marine benthic macrofauna, have been applied successfully to other marine and terrestrial/freshwater fauna but never to ecotonal communities. In particular, to our knowledge, ABC models have not been applied to hyporheic communities. This study represents the first application of ABC models to hyporheic assemblages. We aimed at testing the effectiveness of ABC models in describing the perturbation of hyporheic communities subjected to an existing/known disturbance. To this end, we applied the models to the hyporheic community of an Apennine creek, where the hyporheic waters of the upstream stretch were uncontaminated, whereas those of the downstream stretch were contaminated by ammonium. We also tested separated models for the summer and winter periods to account for potential variability due to season. ABC models provided a satisfactory description of the hyporheic community changes due to ammonium by showing the abundance dominance curve overlying that of the biomass in the downstream stretch contrarily to what was observed in the upstream stretch. However, ABC models did not highlight any significant seasonal effects. Our results showed that ABC models have the potential to be used as assessment tools for ecological quality of hyporheic zones in temperate regions

Regional climate contributes more than geographic distance to beta diversity of copepods (Crustacea Copepoda) between caves of Italy

Despite the study of subterranean biodiversity facing harsh sampling and mapping challenges, the huge diversity of taxa, ecological adaptations and evolutionary trajectories in subterranean environments is gaining increasing attention. Yet, the spatial and environmental factors driving the composition of groundwater communities are still poorly understood. To partially fill this knowledge gap, we collected copepod crustaceans from 12 caves along the Italian peninsula between 2019 and 2022, sampling each cave twice. The resulting presence-absence data were analysed to assess: (i) between-cave taxonomic beta diversity, also partitioning between turnover and nestedness resultant dissimilarity; (ii) the relative weight of geographic distance and climatic differences in shaping observed beta diversity. Seventy-one species of copepods were collected overall. Pairwise beta diversity was high for most pairs of caves, with turnover being the major component. Geographic distance-decay models partially explained total beta diversity and turnover patterns. However, in Generalized Dissimilarity Models (GDM), including surface climatic conditions as predictors, the contribution of seasonal temperature averages was generally higher than that of geographic distance. Further, the explanatory and predictive performance of the GDMs notably increased, along with temperature contribution, when widening the spatial extent from which climate data were gathered. Our results confirmed a high spatial turnover in groundwater copepods’ assemblages and strengthened the link between regional climate and subterranean biodiversit

Coping with the “Racovitzan impediment” in caves: unexpected discoveries open new horizons to the knowledge of groundwater copepods in southern Italy

The stygobitic copepods of southern Italy are known in spots, and species’ records are limited to a few caves and some wells sampled in the past decades. Nevertheless, the groundwater biodiversity of the Apulia region is probably the most well-known in the Italian peninsula. From a biogeographic perspective, the Apulia region constitutes the Apulian Stygoprovince, and its groundwater fauna differs from those of the Apennines and Alpine area. The Apulia Region is the main karstic area of southern Italy. The complex geodynamic history of the area accounts for its litho-stratigraphic succession, the overall geomorphology, and karst evolution. More than 2000 natural caves are present, many of which are along the seashore. This composite paleogeographic scenario may have determined differences in groundwater assemblages, mainly, in terms of species composition. The present study aims at analysing the copepod assemblages of three caves belonging to distinct Apulian hydrogeological districts and characterised by different salinity ranges. Rotolo Cave is a freshgroundwater cave located about 12 km from the Adriatic coastline; Grotta Puntore is a brackish system opening about 500 m far from the Adriatic Sea, and Zinzulusa Cave is an anchialine cave located in the southernmost part of the Salento peninsula. Our investigations confirmed the presence of 7 out of the 17 stygobitic species already known for the Apulian region extending the distribution patterns of the harpacticoids Nitocrella stammeri, Stammericaris orcina, Elaphoidella elaphoides, Troglophonte spelaea, and of the cyclopoids Halicyclops dalmatinus, Metacyclops stammeri and Diacyclops lindae. Remarkably, three new stygobitic species were discovered: a new species of the rare ectinosomatid harpacticoid Pseudectinosoma and a new species of the ameirid harpacticoid Nitokra from Grotta Puntore. Surprisingly, a new species of the cyclopoid genus Hesperocyclops was collected from a single subhabitat in the unsaturated karst of Grotta Rotolo, thus expanding the distribution of this genus in the Palaearctic region. Zinzulusa Cave hosts the highest copepod species richness, 80% composed of stygobites mostly of direct marine origin, followed by Grotta Puntore, where thalassoid species overrule limnicoid ones. The endemicity score of Grotta Puntore was the highest among the three caves in the present study. Grotta Rotolo showed the lowest stygobitic species richness and endemicity score. The three caves showed sharp differences in stygobitic species assemblages, likely reflecting the different paleogeographic events of the three karst areas and, consequently, the different colonisation pathways followed by the epigean ancestral populations to enter groundwaters