Inferring on Speleomantes Foraging Behavior from Gut Contents Examination.

We here provide the first comprehensive analysis and discussion on prey consumed by the European cave salamanders of the genus Speleomantes. Our study stems from the need to shed light on the still unknown foraging behavior adopted by Speleomantes cave salamanders. Starting from the published datasets on gut contents from all Speleomantes species (including hybrids), we here discuss additional information (i.e., species ecology, lower taxonomic level), which were systematically omitted from those data sets. We analyzed a data set consisting of 17,630 records from 49 categories of consumed prey recognized from gut contents of 2060 adults and juveniles Speleomantes. Flying prey accounted for more than 58% of the prey items, while elongated crawling prey accounted for no more than 16% of the diet within a single population. Among the total recognized prey items, only three can be surely ascribed to the group of strictly-cave species (i.e., troglobites), meaning that European cave salamanders mostly forage in surface environment, and therefore represent one of the major drivers of allochthonous organic matter in subterranean environments. Some of the consumed prey seemed to be aquatic, allowing us to hypothesize whether Speleomantes are able to catch prey from a shallow body water. Furthermore, European cave salamanders possess the ability to prey upon taxa characterized by particular anti-predator defenses, while morphological constraints seem to be the most important limit to prey consumption. For each specific case, we provide insights and propose hypotheses concerning the foraging behavior that need to be tested to properly understand the foraging behavior of this cryptic salamanders.

Sensory evolution in a cavefish radiation: patterns of neuromast distribution and associated behaviour in Sinocyclocheilus (Cypriniformes: Cyprinidae).

The genus Sinocyclocheilus, comprising a large radiation of freshwater cavefishes, are well known for their presence of regressive features (e.g. variable eye reduction). Fewer constructive features are known, such as the expansion of the lateral line system (LLS), which is involved in detecting water movements. The precise relationship between LLS expansion and cave adaptation is not well understood. Here, we examine morphology and LLS-mediated behaviour in Sinocyclocheilus species characterized by broad variation in eye size, habitat and geographical distribution. Using live-staining techniques and automated behavioural analyses, we examined 26 Sinocyclocheilus species and quantified neuromast organ number, density and asymmetry within a phylogenetic context. We then examined how these morphological features may relate to wall-following, an established cave-associated behaviour mediated by the lateral line. We show that most species demonstrated laterality (i.e. asymmetry) in neuromast organs on the head, often biased to the right. We also found that wall-following behaviour was distinctive, particularly among eyeless species. Patterns of variation in LLS appear to correlate with the degree of eye loss, as well as geographical distribution. This work reveals that constructive LLS evolution is convergent across distant cavefish taxa and may mediate asymmetric behavioural features that enable survival in stark subterranean microenvironments.

Environmental DNA of insects and springtails from caves reveals complex processes of eDNA transfer in soils.

Subterranean environments host a substantial amount of biodiversity, however assessing the distribution of species living underground is still extremely challenging. Environmental DNA (eDNA) metabarcoding is a powerful tool to estimate biodiversity in poorly known environments and has excellent performance for soil organisms. Here, we tested 1) whether eDNA metabarcoding from cave soils/sediments allows to successfully detect springtails (Hexapoda: Collembola) and insects (Hexapoda: Insecta); 2) whether eDNA mostly represents autochthonous (cave-dwelling) organisms or it also incorporates information from species living in surface environments; 3) whether eDNA detection probability changes across taxa with different ecology. Environmental DNA metabarcoding analyses detected a large number of Molecular Operational Taxonomic Units (MOTUs) for both insects and springtails. For springtails, detection probability was high, with a substantial proportion of hypogean species, suggesting that eDNA provides good information on the distribution of these organisms in caves. Conversely, for insects most of MOTUs represented taxa living outside caves, and the majority of them represented taxa/organisms living in freshwater environments (Ephemeroptera, Plecoptera and Trichoptera). The eDNA of freshwater insects was particularly abundant in deep sectors of caves, far from the entrance. Furthermore, average detection probability of insects was significantly lower than the one of springtails. This suggests that cave soils/sediments act as "conveyer belts of biodiversity information", possibly because percolating water lead to the accumulation of eDNA of organisms living in nearby areas. Cave soils hold a complex mix of autochthonous and allochthonous eDNA. eDNA provided unprecedented information on the understudied subterranean cave organisms; analyses of detection probability and occupancy can help teasing apart local eDNA from the eDNA representing spatially-integrated biodiversity for whole landscape.

Capture-mark-recapture data on the strictly protected Speleomantes italicus.

This data set collects capture-mark-recapture data, biometric data, and stomach contents of seven populations of the Italian cave salamander (Speleomantes italicus), one of the strictly protected European plethodontid species endemic to mainland Italy. We monitored six subterranean populations inside caves, and one fully epigean population living in a forest, surveying a total area of >5200 m2 . Data collection was performed 24 times throughout a year for each of the subterranean populations, and seven times in late winter-early spring for the epigean population. Salamanders were individually identified using two different marking methods: subcutaneous injection of visual implant elastomers (VIE) and photographic recognition of the dorsal pattern. Overall, the data set contains information on 1283 captured salamanders, corresponding to 783 different individuals and 500 recapture events. This type of data can be used to assess the species detection probability and to estimate the size of the populations, which are fundamental parameters for the assessment of its conservation status. Captured salamanders were weighed using a digital scale and photographed next to a reference ruler to perform post hoc measurements. This allows to assess the potential variation of the body condition of individuals through the time, and the potential divergences between conspecific populations. Furthermore, repeated measurements of recaptured individuals can allow to evaluate the seasonal growth rates of Speleomantes. Before their release, the salamanders underwent stomach flushing, a non-invasive technique that allows us to investigate the food residues in the salamanders' stomach. In 951 salamanders, we were able to recognize a total of 7077 consumed prey items belonging to 37 different prey categories (i.e., order level or lower), completing the information on the consumed prey for the entire Speleomantes genus. Data on consumed prey can be used to assess potential divergences between populations or between individuals of different ages/sexes, but also to assess the potential trophic specialization of individuals. The distinctiveness of this data set is that, by combining the capture-mark-recapture data with those on the diet of individuals, it allows to perform detailed studies on the consistency of individuals' food preference over time, an analysis that has never been performed on these salamanders. We release the data set into the public domain under Creative Commons Attribution 4.0 International license (CC-BY). When you use this data in your publication, we request that you cite this data paper; if you are using the whole data set related to the entire project, please cite all the related papers. If this data set will be an important part of the data analyzed in your study, you should consider discussing a collaboration with the data set contact person. If you plan to use data from the European plethodontid salamanders trophic niche project, please contact the contact person to find out if similar analyses are already underway or if unpublished updates are available.

Fundamental research questions in subterranean biology.

Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field.

Do Chinese cavefish show intraspecific variability in morphological traits?

Cavefishes represent one of the most bizarre and intriguing life forms inhabiting groundwater environments. One-third of the known cavefishes worldwide is endemic to China, and almost half of those belongs to a single genus, Sinocyclocheilus (Cypriniformes: Cyprinidae). Analyzing the morphometrics of three Sinocyclocheilus species, we aimed to assess whether variability among conspecific populations exists. We predict that populations inhabiting different subterranean habitats (shallow vs. deep) show divergences in specific morphological traits to better cope with the local ecological conditions. Our results showed that the populations showing bigger eyes and reduced humpback were those occurring close to the cave entrance (habitats with light and high food availability), while specimens with smaller eyes and increased humpback were collected from deeper groundwater areas (habitats laying in darkness with food scarcity). This explorative study paves the way for further researches aiming to collect novel data on Chinese cavefishes and highlights the usefulness of these species in evolutionary studies.

Cave morphology, microclimate and abundance of five cave predators from the Monte Albo (Sardinia, Italy).

BACKGROUND: Systematic data collection on species and their exploited environments is of key importance for conservation studies. Within the less-known environments, the subterranean ones are neither easy to be studied, nor to be explored. Subterranean environments house a wide number of specialised organisms, many of which show high sensitivity to habitat alteration. Despite the undeniable importance to monitor the status of the subterranean biodiversity, standardised methodologies to record biotic and abiotic data in these environments are still not fully adopted, impeding therefore the creation of comparable datasets useful for monitoring the ecological condition in the subterranean environments and for conservation assessment of related species. NEW INFORMATION: In this work we describe a methodology allowing the collection of standardised abiotic and biotic data in subterranean environments. To show this, we created a large dataset including information on environmental features (morphology and microclimate) and abundance of five predators (one salamander, three spiders and one snail) occurring in seven caves of the Monte Albo (Sardinia, Italy), an important biodiversity hotspot. We performed 77 surveys on 5,748 m2 of subterranean environments througout a year, recording 1,695 observations of the five cave predators. The fine-scale data collection adopted in our methodology allowed us to record detailed information related to both morphology and microclimate of the cave inner environment. Furthermore, this method allows us to account for species-imperfect detection when recording presence/abundance data.

Seven new species of the spider genus Matta Crosby from caves in the State of Minas Gerais, Brazil (Araneae, Tetrablemmidae).

Seven new species of the spider genus Matta are described from caves in the State of Minas Gerais, Brazil: M. zuiuda sp. n., Matta pititinha sp. n., Matta humhum sp. n., Matta cambito sp. n., Matta humrrum sp. n., Matta nuusga sp. n., and Matta teteia sp. n. Among these seven species, six have morphological features consistent with specialization to the subterranean environment and we hypothesize that they are troglobites. One species, Matta zuiuda sp. n., shares morphology similar to M. hambletoni Crosby and M. angelomachadoi Brescovit which are known from surface populations in the State of Minas Gerais.

Seven new species of the spider genus Matta Crosby from caves in the State of Minas Gerais, Brazil (Araneae, Tetrablemmidae)

Seven new species of the spider genus Matta are described from caves in the State of Minas Gerais, Brazil: M. zuiuda sp. n., Matta pititinha sp. n., Matta humhum sp. n., Matta cambito sp. n., Matta humrrum sp. n., Matta nuusga sp. n., and Matta teteia sp. n. Among these seven species, six have morphological features consistent with specialization to the subterranean environment and we hypothesize that they are troglobites. One species, Matta zuiuda sp. n., shares morphology similar to M. hambletoni Crosby and M. angelomachadoi Brescovit which are known from surface populations in the State of Minas Gerais.

Ecology and life history of Meta bourneti (Araneae: Tetragnathidae) from Monte Albo (Sardinia, Italy).

The orb-weaver spider Meta bourneti Simon 1922 (Araneae: Tetragnathidae) is one of the most common cave predators occurring in the Mediterranean basin. Although the congeneric M. menardi represented the model species in several studies, our knowledge of M. bourneti is only founded on observations performed on a handful of populations. In this study M. bourneti spiders were studied in caves of Monte Albo (Sardinia, Italy) over a year. Generalized Linear Mixed Models were used to analyze spider occupancy inside cave environments, as well as spider abundance. Analyses on M. bourneti occupancy and abundance were also repeated for adults and juveniles separately. Generalized Linear Models, were used to weight species absence based on its detection probability. Linear Mixed Models were used to detect possible divergences in subterranean spatial use between adult and juvenile spiders. Although widespread on the mountain, M. bourneti generally showed low density and low detection probability. Most of the individuals observed were juveniles. The spiders generally occupied cave sectors with high ceilings that were deep enough to show particular microclimatic features. Adults tended to occupy less illuminated areas than juveniles, while the latter were more frequently found in sectors showing high humidity. The abundance of M. bourneti was strongly related to high humidity and the presence of two troglophile species, Hydromantes flavus Wake, Salvador & Alonso-Zarazaga, 2005 (Amphibia: Caudata) and Oxychilus oppressus (Shuttleworth, 1877) (Gastropoda: Panpulmonata). The abundance of juveniles was related to sector temperature and humidity, the presence of H. flavus and O. oppressus and to morphological sector features. However, when only adults were considered, no significant relationships were found. Adult and juvenile spiders did not differ in their spatial distribution inside the caves studied, but a seasonal distribution of the species along cave walls was observed. Microclimate was one of the most important features affecting both the presence and abundance of M. bourneti in subterranean environments. Individuals tended to occupy lower heights during hot seasons.

Cave features, seasonality and subterranean distribution of non-obligate cave dwellers.

Seasonality impacts species distributions through changes of the environmental conditions that affect the presence of individuals at a given place. Although the dynamics of cave microclimates are well known, only a few studies have evaluated the effects of such dynamics on non-strictly cave species. Here we assessed if species exploiting subterranean environments show changes in habitat occupation related to seasonal variation of cave microclimates. We surveyed 16 caves in central Italy every month for one year. Caves were subdivided into longitudinal sectors of three meters. In each sector we measured cave morphology and microclimatic features, assessed the occurrence of eight non-troglobitic taxa (orthopterans, spiders, gastropods and amphibians), and related species distribution to environmental features and sampling periods. The occurrence of most species was related to both cave morphology and microclimatic features. The survey month was the major factor determining the presence of species in cave sectors, indicating that cave-dwelling taxa show strong seasonality in activity and distribution. For multiple species, we detected interactions between sampling period and microclimatic features, suggesting that species may associate with different microhabitats throughout the year. The richest communities were found in sites with specific microclimates (i.e., high humidity, warm temperature and low light) but seasonality for species richness was strong as well, stressing the complexity of interactions between outdoor and subterranean environments.

Cyto-nuclear discordance suggests complex evolutionary history in the cave-dwelling salamander, Eurycea lucifuga.

Our understanding of the evolutionary history and ecology of cave-associated species has been driven historically by studies of morphologically adapted cave-restricted species. Our understanding of the evolutionary history and ecology of nonrestricted cave species, troglophiles, is limited to a few studies, which present differing accounts of troglophiles' relationship with the cave habitat, and its impact on population dynamics. Here, we used phylogenetics, demographic statistics, and population genetic methods to study lineage divergence, dates of divergence, and population structure in the Cave Salamander, Eurycea lucifuga, across its range. In order to perform these analyses, we sampled 233 individuals from 49 populations, using sequence data from three gene loci as well as genotyping data from 19 newly designed microsatellite markers. We find, as in many other species studied in a phylogeographic context, discordance between patterns inferred from mitochondrial relationships and those inferred by nuclear markers indicating a complicated evolutionary history in this species. Our results suggest Pleistocene-based divergence among three main lineages within E. lucifuga corresponding to the western, central, and eastern regions of the range, similar to patterns seen in species separated in multiple refugia during climatic shifts. The conflict between mitochondrial and nuclear patterns is consistent with what we would expect from secondary contact between regional populations following expansion from multiple refugia.

The rise of Astyanax cavefish.

Numerous animals have invaded subterranean caverns and evolved remarkably similar features. These features include loss of vision and pigmentation, and gains in nonvisual sensation. This broad convergence echoes smaller-scale convergence, in which members of the same species repeatedly evolve the same cave-associated phenotypes. The blind Mexican tetra of the Sierra de El Abra region of northeastern Mexico has a complex origin, having recurrently colonized subterranean environments through numerous invasions of surface-dwelling fish. These colonizations likely occurred ∼1-5 MYa. Despite evidence of historical and contemporary gene flow between cave and surface forms, the cave-associated phenotype appears to remain quite stable in nature. This model system has provided insight to the mechanisms of phenotypic regression, the genetic basis for constructive trait evolution, and the origin of behavioral novelties. Here, we document the rise of this model system from its discovery by a Mexican surveyor in 1936, to a powerful system for cave biology and contemporary genetic research. The recently sequenced genome provides exciting opportunities for future research, and will help resolve several long-standing biological problems. Developmental Dynamics 244:1031-1038, 2015. © 2015 Wiley Periodicals, Inc.