Standing genetic variation as a potential mechanism of novel cave phenotype evolution in the freshwater isopod, Asellus aquaticus

Novel phenotypes can come about through a variety of mechanisms including standing genetic variation from a founding population. Cave animals are an excellent system in which to study the evolution of novel phenotypes such as loss of pigmentation and eyes. Asellus aquaticus is a freshwater isopod crustacean found in Europe and has both a surface and a cave ecomorph which vary in multiple phenotypic traits. An orange eye phenotype was previously revealed by F2 crosses and backcrosses to the cave parent within two examined Slovenian cave populations. Complete loss of pigmentation, both in eye and body, is epistatic to the orange eye phenotype and therefore the orange eye phenotype is hidden within the cave populations. Our goal was to investigate the origin of the orange eye alleles within the Slovenian cave populations by examining A. aquaticus individuals from Slovenian and Romanian surface populations and Asellus aquaticus infernus individuals from a Romanian cave population. We found orange eye individuals present in lab raised surface populations of A. aquaticus from both Slovenia and Romania. Using a mapping approach with crosses between individuals of two surface populations, we found that the region known to be responsible for the orange eye phenotype within the two previously examined Slovenian cave populations was also responsible within both the Slovenian and the Romanian surface populations. Complementation crosses between orange eye Slovenian and orange eye Romanian surface individuals suggest that the same gene is responsible for the orange eye phenotype in both surface populations. Additionally, we observed a low frequency phenotype of eye loss in crosses generated between the two surface populations and also in the Romanian surface population. Finally, in a cave population from Romania, A. aquaticus infernus, we found that the same region is also responsible for the orange eye phenotype as the Slovenian cave populations and the Slovenian and Romanian surface populations. Therefore, we present evidence that variation present in the cave populations could originate from standing variation present in the surface populations and/or transgressive hybridization of different surface phylogenetic lineages rather than de novo mutations

Fundamental research questions in subterranean biology

Five decades ago, a landmark paper inSciencetitledThe Cave Environmentheralded 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.Peer reviewe

Avoidance behaviour toxicity tests should account for animal gregariousness: a case study on the terrestrial isopod Porcellio scaber

Avoidance behaviour enables woodlice to escape suboptimal environmental conditions and to mitigate harmful effects of pollutants. However, several studies have shown that at least in some woodlice species the tendency to aggregate can lead to suboptimal responses as the between-conspecific attraction can outweigh the aversive stimuli. The present study evaluated the influence of gregariousness on the behaviour of Porcellio scaber in a heterogeneously polluted environment. The hypothesis was that the tendency for aggregation outweighs the tendency for exploratory activity, therefore animals in groups will be less active. Consequently, this will affect their avoidance of polluted environmental patches. To test this hypothesis, isolated individuals or pairs of individuals were monitored in free-choice arenas where animals could select between uncontaminated and pyrethrin-contaminated soils. Animals were video recorded for 3 h in darkness using infrared light and analysed for avoidance behaviour and locomotor activity. In general, isolated animals were more locomotory active and avoided the contaminated soil more than paired animals. It can be concluded that aggregation behaviour suppresses exploratory behaviour and consequently also the avoidance of polluted environments. This should be accounted for when interpreting results of avoidance tests with groups of gregarious animals, which may underestimate the effect of pollutants

Avoidance behaviour toxicity tests should account for animal gregariousness: a case study on the terrestrial isopod Porcellio scaber

Avoidance behaviour enables woodlice to escape suboptimal environmental conditions and to mitigate harmful effects of pollutants. However, several studies have shown that at least in some woodlice species the tendency to aggregate can lead to suboptimal responses as the between-conspecific attraction can outweigh the aversive stimuli. The present study evaluated the influence of gregariousness on the behaviour of Porcellio scaber in a heterogeneously polluted environment. The hypothesis was that the tendency for aggregation outweighs the tendency for exploratory activity, therefore animals in groups will be less active. Consequently, this will affect their avoidance of polluted environmental patches. To test this hypothesis, isolated individuals or pairs of individuals were monitored in free-choice arenas where animals could select between uncontaminated and pyrethrin-contaminated soils. Animals were video recorded for 3 h in darkness using infrared light and analysed for avoidance behaviour and locomotor activity. In general, isolated animals were more locomotory active and avoided the contaminated soil more than paired animals. It can be concluded that aggregation behaviour suppresses exploratory behaviour and consequently also the avoidance of polluted environments. This should be accounted for when interpreting results of avoidance tests with groups of gregarious animals, which may underestimate the effect of pollutants

Morphologically cryptic amphipod species are “ecological clones” at regional but not at local scale: a case study of four niphargus species

Recent studies indicate that morphologically cryptic species may be ecologically more different than would be predicted from their morphological similarity and phylogenetic relatedness. However, in biodiversity research it often remains unclear whether cryptic species should be treated as ecologically equivalent, or whether detected differences have ecological significance. In this study, we assessed the ecological equivalence of four morphologically cryptic species of the amphipod genus Niphargus. All species live in a small, isolated area on the Istrian Peninsula in the NW Balkans. The distributional ranges of the species are partially overlapping and all species are living in springs. We reconstructed their ecological niches using morphological traits related to feeding, bioclimatic niche envelope and species’ preference for epihypogean habitats. The ecological meaning of differences in niches was evaluated using distributional data and co-occurrence frequencies. We show that the species comprise two pairs of sister species. All species differ from each other and the degree of differentiation is not related to phylogenetic relatedness. Moreover, low co-occurrence frequencies in sympatric zones imply present or past interspecific competition. This pattern suggests that species are not differentiated enough to reduce interspecific competition, nor ecologically equivalent to co-exist via neutral dynamics. We tentatively conclude that the question of ecological equivalence relates to the scale of the study: at a fine scale, species’ differences may influence dynamics in a local community, whereas at the regional level these species likely play roughly similar ecological roles

The morphology of the colonizers of the springs from the down-under

Transition from surface to subterranean environment generally results in easily detectable morphological changes, generally referred as troglomophism. Although the cave colonisation history of species can differ considerably, in certain cases there is a possibility to directly examine the cave-induced morphological and life history changes, as some of the troglobiont and stygobiont species still have closely related surface species. A less common and therefore less known phenomenon is the invasion in the opposite direction, when cave-adapted species establish stable populations (and later, new species) in surface or surface-connected (ecotonal) habitats. A great model taxon to study if morphological changes occur in such cases is the Niphargus genus, which primarily comprises subterranean species, although some species successfully colonised surface or ecotonal habitats.To get insight of morphological changes and whether these changes were affected by sex, we measured 15 functional morphological traits on 488 individuals from both sex of eight Niphargus species, out of which four inhabit ecotonal (spring) habitats, while the other four are exclusively subterranean. We analysed our data trait-by-trait with linear mixed models. We found that colonising the “new” habitat did not demonstrably affect morphology. In contrast habitat dependent sexual dimorphism was found in case of the 2nd gnathopods’ coxa. Besides the aforementioned habitat dependent sexual dimorphism, we found sexual dimorphism in case of 11 out of the 15 traits. Based on our result we can assume that morphological changes might happen in the course of the colonisation of the novel habitat, but they are not as unambiguous as when surface species colonise caves. It is also possible that although we consider springs as ecotone or even surface habitats, they might be feasible for subterranean species without striking morphological changes as they do not meet selection forces strikingly different from those present in caves

Evaluating different conservation approaches to protection of subterranean fauna in Slovenia

Due to increasing human activities with negative impacts on wildlife and natural habitats, it has become necessary to establish conservation policies protecting at least parts of natural diversity. Subterranean fauna presents a challenge for conservation as species with small ranges or single site occurrences are common. In addition to the high level of strict endemism, the concealed lifestyles in habitats that are difficult to access make it challenging to establish the conservation status of subterranean species. Conservation approaches have different practical implications, but they should: i) include a sufficient number of important sites or proportion of species populations, and ii) involve monitoring of target species and habitats to check the effectiveness of conservation efforts. We evaluated both aspects in two study cases on subterranean fauna from Slovenia, which is known as one of the global biodiversity hotspots in the Western Balkans. In the first case, we investigated to what extent current conservation schemes cover single site species. In the second case, we studied a region in Southern Slovenia and evaluated to what extent the EU legislation, namely EU Habitats directive, has been successfully implemented. In the frame of the latter, Sites of Community Importance were designated also due to the presence of important cave habitats and subterranean species. Our results suggest that the inclusion of important subterranean sites in conservation schemes is considerable. But, the apparent lack of research and monitoring of both subterranean habitats and species hampers the conclusions on effectiveness of different conservation approaches