Background: Lake Baikal is one of the oldest freshwater lakes and has constituted a stable environment for millions of years, in stark contrast to small, transient bodies of water in its immediate vicinity. A highly diverse endemic endemic amphipod fauna is found in one, but not the other habitat. We ask here whether differences in stress response can explain the immiscibility barrier between Lake Baikal and non-Baikal faunas. To this end, we conducted exposure experiments to increased temperature and the toxic heavy metal cadmium as stressors.Results: Here we obtained high-quality de novo transcriptome assemblies, covering mutiple conditions, of three amphipod species, and compared their transcriptomic stress responses. Two of these species,Eulimnogammarus verrucosusandE. cyaneus, are endemic to Lake Baikal, while the HolarcticGammarus lacustrisis a potential invader.Conclusions: Both Baikal species possess intact stress response systems and respond to elevated temperature with relatively similar changes in their expression profiles.G. lacustrisreacts less strongly to the same stressors, possibly because its transcriptome is already perturbed by acclimation conditions.

Ancient lakes are known speciation hotspots. One of the most speciose groups in the ancient Lake Baikal are gammaroid amphipods (Crustacea: Amphipoda: Gammaroidea). There are over 350 morphological species and subspecies of amphipods in Baikal, but the extent of cryptic variation is still unclear. One of the most common species in the littoral zone of the lake, Eulimnogammarus verrucosus (Gerstfeldt, 1858), was recently found to comprise at least three (pseudo)cryptic species based on molecular data. Here, we further explored these species by analyzing their mitogenome-based phylogeny, genome sizes with flow cytometry, and their reproductive compatibility. We found divergent times of millions of years and different genome sizes in the three species (6.1, 6.9 and 8 pg), further confirming their genetic separation. Experimental crossing of the western and southern species, which are morphologically indistinguishable and have adjacent ranges, showed their separation with a post-zygotic reproductive barrier, as hybrid embryos stopped developing roughly at the onset of gastrulation. Thus, the previously applied barcoding approach effectively indicated the separate biological species within E. verrucosus. These results provide new data for investigating genome evolution and highlight the need for precise tracking of the sample origin in any studies in this morphospecies.

Lake Baikal is the only freshwater reservoir inhabited by deep-water fauna, which originated mostly from shallow-water ancestors. Ommatogammarus flavus and O. albinus are endemic scavenger amphipods (Amphipoda, Crustacea) dwelling in wide depth ranges of the lake covering over 1300 m. O. flavus had been previously collected close to the surface, while O. albinus has never been found above the depth of 47 m. Since O. albinus is a promising model species for various research, here we tested whether O. albinus is less metabolically adapted to atmospheric pressure than O. flavus. We analyzed a number of energy-related traits (contents of glucose, glycogen and adenylates, as well as lactate dehydrogenase activity) and oxidative stress markers (activities of antioxidant enzymes and levels of lipid peroxidation products) after sampling from different depths and after both species’ acclimation to atmospheric pressure. The analyses were repeated in two independent sampling campaigns. We found no consistent signs of metabolic disturbances or oxidative stress in both species right after lifting. Despite O. flavus surviving slightly better in laboratory conditions, during long-term acclimation, both species showed comparable reactions without critical changes. Thus, the obtained data favor using O. albinus along with O. flavus for physiological research under laboratory conditions.

Although deep-water environments make up the world’s largest ecosystem, they are poorly characterized. Lake Baikal, the only freshwater reservoir possessing rich deep-water fauna, offers unique opportunities to examine the evolutionary processes that occurred independently and concurrently with adaptation to these environments in the ocean. Here, we focus on amphipods as one of the dominant elements of Baikal deep-water fauna. This study examines the genetic diversity across broad vertical (~1 km) and horizontal (~500 km) ranges, as well as depth-related traits in two deep-water scavengers, Ommatogammarus flavus (Dybowsky, 1874) and Ommatogammarus albinus (Dybowsky, 1874). Our results revealed low intraspecific diversity of the cytochrome c oxidase subunit I gene marker fragment across locations and depths, indicating the absence of significant barriers in the distribution of either species and a bottleneck event in their evolutionary histories. At the same time, we found depth-related stratification in carotenoid-based body coloration and eye shape in O. flavus, as well as in eye color for both species. These findings suggest partial isolation between vertically stratified populations and help to characterize the ecological differences between the two studied species.

Reproductive barriers to gene flow play a key role in speciation. However, as it is not always feasible to study them directly, most studies rely on genetic divergence to infer species delimitation. In order to correlate genetic distances to reproductive incompatibilities, compact groups of closely related species are needed. In this work, we explored a species complex of Baikal amphipods (Crustacea: Amphipoda: Gammaroidea), Eulimnogammarus verrucosus. Three biological species (W, S, and E), geographically isolated in Baikal, had been found to have interspecific differences exceeding the patristic distance threshold of 0.16, and a postzygotic incompatibility had been confirmed for the closest pair, W and S. Here, we expanded our knowledge on geographical distribution of the species, discovering that secondary contact between the W and S species already occurs in natural conditions near the source of the Angara River. Our experiments have shown that the three species within the E. verrucosus species complex are separated by both prezygotic and postzygotic barriers. While neither of these barriers is absolute, their combination can ensure reproductive isolation upon secondary contact of the species. The experimental system we have developed in this and previous works can provide support for testing species delimitation hypotheses based on sequencing data and further extend these results to related species for which such experiments are unfeasible.

Deep-water environments constitute the largest ecosystem on Earth and are insufficiently characterized. Lake Baikal, the only freshwater reservoir possessing rich deep-water fauna, provides unique opportunities to investigate the evolutionary processes that happened to such fauna independently and in parallel to adaptation to these environments in the ocean. Here, we focus on amphipods as one of the dominant elements of Baikal deep-water fauna and study the genetic diversity across a wide vertical (~1 km) and horizontal (~500 km) ranges, as well as depth-related traits in two deep-water scavengers Ommatogammarus flavus (Dybowsky, 1874) and Ommatogammarus albinus (Dybowsky, 1874). Our data revealed low intraspecific diversity of the cytochrome c oxidase subunit I gene marker fragment, at both vertical and horizontal scales, indicating no significant distribution barriers and a bottleneck event in the evolutionary history of both species. At the same time, we found depth-related stratification in carotenoid-based body coloration and eye shape in O. flavus and eye color over stable opsin expression in both species. These findings might indicate partial isolation between vertically stratified populations and characterize the ecological distinctions between the two studied species.