Highly dynamic evolution of the chemosensory system driven by gene gain and loss across subterranean beetles.

Chemical cues in subterranean habitats differ highly from those on the surface due to the contrasting environmental conditions, such as absolute darkness, high humidity or food scarcity. Subterranean animals underwent changes to their sensory systems to facilitate the perception of essential stimuli for underground lifestyles. Despite representing unique systems to understand biological adaptation, the genomic basis of chemosensation across cave-dwelling species remains unexplored from a macroevolutionary perspective. Here, we explore the evolution of chemoreception in three beetle tribes that underwent at least six independent transitions to the underground, through a phylogenomics spyglass. Our findings suggest that the chemosensory gene repertoire varies dramatically between species. Overall, no parallel changes in the net rate of evolution of chemosensory gene families were detected prior, during, or after the habitat shift among subterranean lineages. Contrarily, we found evidence of lineage-specific changes within surface and subterranean lineages. However, our results reveal key duplications and losses shared between some of the lineages transitioning to the underground, including the loss of sugar receptors and gene duplications of the highly conserved ionotropic receptors IR25a and IR8a, involved in thermal and humidity sensing among other olfactory roles in insects. These duplications were detected both in independent subterranean lineages and their surface relatives, suggesting parallel evolution of these genes across lineages giving rise to cave-dwelling species. Overall, our results shed light on the genomic basis of chemoreception in subterranean beetles and contribute to our understanding of the genomic underpinnings of adaptation to the subterranean lifestyle at a macroevolutionary scale.

Parallel and convergent genomic changes underlie independent subterranean colonization across beetles.

Adaptation to life in caves is often accompanied by dramatically convergent changes across distantly related taxa, epitomized by the loss or reduction of eyes and pigmentation. Nevertheless, the genomic underpinnings underlying cave-related phenotypes are largely unexplored from a macroevolutionary perspective. Here we investigate genome-wide gene evolutionary dynamics in three distantly related beetle tribes with at least six instances of independent colonization of subterranean habitats, inhabiting both aquatic and terrestrial underground systems. Our results indicate that remarkable gene repertoire changes mainly driven by gene family expansions occurred prior to underground colonization in the three tribes, suggesting that genomic exaptation may have facilitated a strict subterranean lifestyle parallelly across beetle lineages. The three tribes experienced both parallel and convergent changes in the evolutionary dynamics of their gene repertoires. These findings pave the way towards a deeper understanding of the evolution of the genomic toolkit in hypogean fauna.

Parallel duplication and loss of aquaporin-coding genes during the "out of the sea" transition as potential key drivers of animal terrestrialization.

One of the most important physiological challenges animals had to overcome during terrestrialization (i.e., the transition from sea to land) was water loss, which alters their osmotic and hydric homeostasis. Aquaporins are a superfamily of membrane water transporters heavily involved in osmoregulatory processes. Their diversity and evolutionary dynamics in most animal lineages remain unknown, hampering our understanding of their role in marine-terrestrial transitions. Here, we interrogated aquaporin gene repertoire evolution across the main terrestrial animal lineages. We annotated aquaporin-coding genes in genomic data from 458 species from seven animal phyla where terrestrialization episodes occurred. We then explored aquaporin gene evolutionary dynamics to assess differences between terrestrial and aquatic species through phylogenomics and phylogenetic comparative methods. Our results revealed parallel aquaporin-coding gene duplications during the ecological transition from marine to nonmarine environments (e.g., brackish, freshwater and terrestrial), rather than from aquatic to terrestrial ones, with some notable duplications in ancient lineages. In contrast, we also recovered a significantly lower number of superaquaporin genes in terrestrial arthropods, suggesting that more efficient oxygen homeostasis in land arthropods might be linked to a reduction in this type of aquaporin. Our results thus indicate that aquaporin-coding gene duplication and loss might have been one of the key steps towards the evolution of osmoregulation across animals, facilitating the "out of the sea" transition and ultimately the colonization of land.

Smelling in the dark: Phylogenomic insights into the chemosensory system of a subterranean beetle.

The chemosensory system has experienced relevant changes in subterranean animals, facilitating the perception of specific chemical signals critical to survival in their particular environment. However, the genomic basis of chemoreception in cave-dwelling fauna has been largely unexplored. We generated de novo transcriptomes for antennae and body samples of the troglobitic beetle Speonomus longicornis (whose characters suggest an extreme adaptation to a deep subterranean environment) in order to investigate the evolutionary origin and diversification of the chemosensory gene repertoire across coleopterans through a phylogenomic approach. Our results suggested a diminished diversity of odourant and gustatory gene repertoires compared to polyphagous beetles that inhabit surface habitats. Moreover, S. longicornis showed a large diversity of odourant-binding proteins, suggesting an important role of these proteins in capturing airborne chemical cues. We identified a gene duplication of the ionotropic coreceptor IR25a, a highly conserved single-copy gene in protostomes involved in thermal and humidity sensing. In addition, no homologous genes to sugar receptors or the ionotropic receptor IR41a were detected. Our findings suggest that the chemosensory gene repertoire of this cave beetle may result from adaptation to the highly specific ecological niche it occupies, and that gene duplication and loss may have played an important role in the evolution of gene families involved in chemoreception. Altogether, our results shed light on the genomic basis of chemoreception in a cave-dwelling invertebrate and pave the road towards understanding the genomic underpinnings of adaptation to the subterranean lifestyle at a deeper level.

Beyond survival experiments: using biomarkers of oxidative stress and neurotoxicity to assess vulnerability of subterranean fauna to climate change.

Accurate assessments of species vulnerability to climate change need to consider the physiological capacity of organisms to deal with temperature changes and identify early signs of thermally induced stress. Oxidative stress biomarkers and acetylcholinesterase activity are useful proxies of stress at the cellular and nervous system level. Such responses are especially relevant for poor dispersal organisms with limited capacity for behavioural thermoregulation, like deep subterranean species. We combined experimental measurements of upper lethal thermal limits, acclimation capacity and biomarkers of oxidative stress and neurotoxicity to assess the impact of heat stress (20°C) at different exposure times (2 and 7 days) on the Iberian endemic subterranean beetle Parvospeonomus canyellesi. Survival response (7 days of exposure) was similar to that reported for other subterranean specialist beetles (high survival up to 20°C but no above 23°C). However, a low physiological plasticity (i.e. incapacity to increase heat tolerance via acclimation) and signs of impairment at the cellular and nervous system level were observed after 7 days of exposure at 20°C. Such sublethal effects were identified by significant differences in total antioxidant capacity, glutathione S-transferase activity, the ratio of reduced to oxidized forms of glutathione and acetylcholinesterase activity between the control (cave temperature) and 20°C treatment. At 2 days of exposure, most biomarker values indicated some degree of oxidative stress in both the control and high-temperature treatment, likely reflecting an initial altered physiological status associated to factors other than temperature. Considering these integrated responses and the predicted increase in temperature in its unique locality, P. canyellesi would have a narrower thermal safety margin to face climate change than that obtained considering only survival experiments. Our results highlight the importance of exploring thermally sensitive processes at different levels of biological organization to obtain more accurate estimates of the species capacity to face climate change.