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The surface ancestors of subterranean species are often hypothesized to possess pre-adaptations that enable them to successfully initiate colonization of caves. Nocturnal habits and the use of microhabitats such as the underside of rocks or leaf litter are two simple examples. However, there are many exceptions, and a mechanistic explanation for the evolution of a whole range of troglomorphic adaptations has yet to be found. To shed light on the phenotypic and molecular changes that accompany the early stages of cave colonization, we experimentally exposed the most closely related surface species or population of several cave-dwelling species including fish and crustaceans, from the earliest embryonic stage possible, to constant darkness, the single environmental condition characteristic of all subterranean habitats. Our results show that darkness induced a surprisingly large number of phenotypic changes, many of which resembled troglomorphic adaptations found in their respective cave-dwelling relatives (for example changes in fat content). However, some changes were contrary to what is considered adaptive in darkness, such as increased body pigmentation and enhancement in visual system, opposite to the two iconic features of troglomorphy. Overall, we conclude that phenotypic plasticity is an important mechanism for the rapid initiation of changes that may be favored or targeted by natural selection during critical initial steps in the evolution of cave-dwelling species.

Objective obstacles such as high water levels that prevent access to caves at certain times of the year or the need to use complex tools and skills such as cave diving make biological research in caves extremely complex and occasionally dangerous. Moreover, to study physiological and behavioral adaptations of cave animals, they must be kept under controlled laboratory conditions. For this reason, we have established an animal facility in our laboratory. The available literature on invertebrate setup, operation, and care was sparse and unsuitable for the species we were studying. Fortunately, many cave biologists provided us with advice and support during the initial phase. Here we present our experiences to provide some guidelines for other researchers undertaking similar efforts. Among other animal groups, we are culturing several morphotypes of the cave snail Physella sp. and two cave and one surface species pair of bivalves: Congeria spp. and Dreissena polymorpha. We discuss good and bad practices in mollusk care - housing, water treatment, different types of food, and small aquarium design. Special attention is given to problems we have encountered with our colonies and changes we have made to address these problems, both successful and unsuccessful. We also address the general protocols required for keeping multiple species in a single facility, including procedures for decontamination of equipment and tools, and quarantine.

Albinism, or loss of pigment, is a typical adaptation to living in caves and occurs in numerous cave dwellers. To understand the evolution of this trait, one must identify the type of biological pigment present in the phylogenetically closest surface relatives. The absence of pigmentation has been studied primarily in terms of melanin deficiency, while the exact mechanism of ommochrome loss in any cave dwellers is largely unknown. Ommochromes are tryptophan derivatives and generally belong to a less studied class of biological pigments previously described in protostomes such as isopod crustaceans. Using a simple method for extraction and detection of ommochromes in conjunction with liquid chromatography coupled with mass spectrometry (UPLC-MS), we characterized the nature of pigments in surface isopods. We detected, for the first time and to the best of our knowledge, ommochromes in three groups of surface isopods: the families Sphaeromatidae (Flabellifera) and Trichoniscidae (Oniscidea) and the genus Proasellus (Asellota). To determine which step of the ommochrome production pathway is disrupted in cave isopods, we quantified the precursors of ommochrome synthesis in closely related surface and cave species. Our results suggest that the disruption of ommochrome synthesis is preferentially located at the beginning of this anabolic cascade, as we observed an accumulation of the precursor tryptophan in albino species. To address this problem at the molecular level, we are trying to determine the exact genes and mutations that may be involved in the loss of ommochromes. We performed RNA-seq on two representatives of surface and cave species of the genus Proasellus and analyzed the data for expression changes in candidate genes.

In 1962, Milan Meštrov defined the hypotelminorheic as “ constitué par les sols humides des montagnes, riches en mati&egrave ; res organiques et traverses par des filets d’ eau courante.” On the basis of studies of Medvednica Mountain (Croatia), Nanos Mountain (Slovenia), and the lower Potomac River drainage (U.S.A.) we refine the definition as (1) a persistent wet spot in a slight depression in an area of low to moderate slope (2) rich in organic matter (3) with a drainage area of typically less than 10, 000 m2 (4) underlain by a clay layer typically from 5 and 50 cm beneath the surface (5) with a characteristic black color derived from decaying leaves which are usually not skeletonized and (6) occurring in a wide variety of geologic settings. Water chemistry (temperature, pH, conductivity, and O2) varied considerably depending on the geologic setting. The presence of clay is likely key to the survival of the fauna as clay retains considerable moisture during periods of drying, and animals can burrow into the clay to escape desiccation. The macroscopic fauna is dominated by amphipods. The combination of high organic matter and relatively large spaces between the leaves may make this a less extreme environment than cave (with low food) or interstitial habitats (with small spaces). It has characteristics of an ecotone, and because of this and its environmental characteristics, it may be an important invasion path into the subsurface.