Different ways to play it cool: Transcriptomic analysis sheds light on different activity patterns of three amphipod species under long‐term cold exposure

Species of littoral freshwater environments in regions with continental climate experience pronounced seasonal temperature changes. Coping with long cold winters and hot summers requires specific physiological and behavioural adaptations. Endemic amphipods of Lake Baikal, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, show high metabolic activity throughout the year; E. verrucosus even reproduces in winter. In contrast, the widespread Holarctic amphipod Gammarus lacustris overwinters in torpor. This study investigated the transcriptomic hallmarks of E. verrucosus, E. cyaneus and G. lacustris exposed to low water temperatures. Amphipods were exposed to 1.5°C and 12°C (corresponding to the mean winter and summer water temperatures, respectively, in the Baikal littoral) for one month. At 1.5°C, G. lacustris showed upregulation of ribosome biogenesis and mRNA processing genes, as well as downregulation of genes related to growth, reproduction and locomotor activity, indicating enhanced energy allocation to somatic maintenance. Our results suggest that the mitogen‑activated protein kinase (MAPK) signalling pathway is involved in the preparation for hibernation; downregulation of the actin cytoskeleton pathway genes could relate to the observed low locomotor activity of G. lacustris at 1.5°C. The differences between the transcriptomes of E. verrucosus and E. cyaneus from the 1.5°C and 12°C exposures were considerably smaller than for G. lacustris. In E. verrucosus, cold‐exposure triggered reproductive activity was indicated by upregulation of respective genes, whereas in E. cyaneus, genes related to mitochondria functioning were upregulated, indicating cold compensation in this species. Our data elucidate the molecular characteristics behind the different adaptations of amphipod species from the Lake Baikal area to winter conditions.


| INTRODUC TI ON
Water temperature is a pervasive abiotic factor for aquatic ectotherms whose body temperature is determined by the ambient temperature. The thermal window's lower boundary is expanded in cold-adapted species, allowing them to thrive in low-temperature environments. Cold adaptation involves several cellular processes and adjustments, enabling long-term survival at low temperatures.
Depending on the overwintering strategy, ectotherms show various metabolic activity states at low temperatures, ranging from metabolic depression to high activity, including even reproduction (Elgmork, 1991;Graham & Hop, 1995;Schaefer, 1977). The metabolic activity state of aquatic ectotherms at cold temperatures also depends on other parameters, such as availability of food and water oxygen levels (Somero et al., 2017). To lower the energy demand in the cold season, ectotherms often decrease their physiological activity; metabolic rates can be depressed to various degrees, which may lead to semi-torpid or torpid stages (Jackson & Ultsch, 2010;Radzikowski, 2013;Ultsch, 1989;Walsh et al., 1983). In aquatic systems, apart from low temperature, reduction in metabolic activity can be due to shortage in food supply, as well as hypoxia or anoxia as a result of ice cover and decreased photosynthetic activity of water plants. Some ectotherms remain metabolically active at low temperatures during the winter by compensating the biological effects of the cold. This is facilitated by mechanisms of cold adaptation: membrane fluidity adjustments (Bowler, 2018); an increase in mitochondrial density and enzyme capacities (Fangue et al., 2009;Lucassen et al., 2006;Pörtner, 2002); synthesis of cold-adapted enzymes (Fields & Somero, 1998;Genicot et al., 1996); expression of ice-binding proteins (Duman, 2015;Kristiansen & Zachariassen, 2005); and synthesis of polyol cryoprotectants (Joanisse & Storey, 1994;Storey et al., 1991). Most ectotherms in regions with cold winters reproduce in spring. This strategy is widely thought to be selected for so that the initial life phases of the free-living offspring take place under maximally favorable conditions with regard to temperature and food availability (Ultsch, 1989). Reproduction involves high energy consumption. Thus, reproduction during the winter is exceptional among ectotherms from regions with high seasonal temperature changes (Elgmork, 1991;Schaefer, 1977). Lake Baikal is a UNESCO world heritage site inhabited by one of the richest freshwater faunas in the world. More than 80% of the animal species are endemic  and adapted to the specific conditions of the lake, such as low mineralization and high oxygen content of the water and ice cover for several months per year (Cabello-Yeves et al., 2018;Kenny et al., 2019;Sideleva, 1996;Zerbst-Boroffka, 1999). The water temperature in the littoral zone of Lake Baikal varies significantly depending on the location, water depth, and time of the year. In the winter-spring period, when the water is covered by ice, the temperature is close to 0°C; in summer and autumn, it ranges from 5°C to up to 18°C (Figure 1), with occasional substantial temperature shifts within hours or a few days due to storm and upwelling events. Lake Baikal hosts over 350 endemic amphipod species and subspecies that make up approximately 90% of its benthic biomass (Takhteev, 2019). Eulimnogammarus verrucosus (Gerstfeldt, 1858) and Eulimnogammarus cyaneus (Dybowsky, 1874) studied here are endemic amphipod species of Lake Baikal's littoral zone. Gammarus lacustris Sars, 1863, a widespread representative of the Holarctic fauna, is found in ponds in close vicinity to Lake Baikal and in isolated bays of the lake but almost never at sites inhabited by the endemic Baikal fauna (Takhteev, 2019).
The amphipods studied here differ with regard to their physiological upper thermal limits Jakob et al., 2016) and the temperatures of the water at which they preferentially linger (Timofeyev et al., 2001; Table 1). Eulimnogammarus verrucosus is a cold-loving species found in water depths of 0-15 m; it migrates to deeper waters when water temperatures rise in the upper littoral in summer Weinberg & Kamaltynov, 1998   and E. verrucosus was found to reproduce during winter (Gavrilov, 1949). Gammarus lacustris has a slightly higher upper thermal limit than the Baikal endemic E. cyaneus. It is a cold-resistant ectotherm of inland water ecosystems of the Holarctic that prepares for the cold season by the formation of cryoprotective biomolecules (Karanova & Andreev, 2010) and shows hypometabolism and consequently cold-exposure triggered reproductive activity was indicated by upregulation of respective genes, whereas in E. cyaneus, genes related to mitochondria functioning were upregulated, indicating cold compensation in this species. Our data elucidate the molecular characteristics behind the different adaptations of amphipod species from the Lake Baikal area to winter conditions.

K E Y W O R D S
cold adaptation, Eulimnogammarus cyaneus, Eulimnogammarus verrucosus, freshwater lake, Gammarus lacustris, Lake Baikal, transcriptome sequencing drastically reduced locomotor activity upon long-term cold exposure . Gammarus lacustris can be found inactive in leaf litter in winter and spring (own field observations).
The molecular bases of the major activity changes in G. lacustris in hibernation mode have so far not been explored.
Reference-free transcriptome assemblies from next-generation sequencing data enable acquisition of information about the transcriptome activity of non-model organisms (Cahais et al., 2012). In this study, we analysed transcriptomes of the two Baikal amphipods E. verrucosus and E. cyaneus and the Holarctic G. lacustris exposed to 12°C, the mean water temperature of the Baikal littoral at 1 m depth in summer, and 1.5°C, the mean temperature in winter ( Figure 1). We hypothesized that the transcriptomic profiles in each species will mirror the speciesspecific metabolic activity patterns in those temperature scenarios.
Here, we aimed to identify hallmarks of (1) metabolic depression observed at this temperature in G. lacustris; (2) the high metabolic activity of the Eulimnogammarus species kept at this temperature; and (3) the onset of reproductive processes in E. verrucosus in the transcriptomic responses in the animals upon exposure to low temperature (1.5°C).
Gammarus lacustris Sars, 1863 was sampled in a former gold mining pond ("Lake №14"; 51°55′14.39″N, 105°4′19.48″E) in about 2 km distance from the Biological Station and Lake Baikal. There are no obvious signs of toxic contamination of this pond, for example with heavy metals, as it is inhabited by various biota, such as G. lacustris and aquatic insects (Odonata, Diptera, Coleoptera, and Hemiptera) that are highly abundant. Fresh water constantly seeps into the pond from the underground. The temperature of the water at the bottom of the pond (1-1.5 m water depth) is rather stable at 7-8°C during the summer (own observations).

F I G U R E 1
Water temperature regimes in the habitat of the amphipods E. verrucosus and E. cyaneus from Lake Baikal. The black dots represent the mean daily water temperatures in the Baikal littoral zone at 1 m water depth from July 2016 until the beginning of June 2017. For better visualization, the dots were connected with a black line. Temperatures were monitored every 0.5 to 3 h with a data logger (no. DS1922L, iButton, Maxim Integrated, CA, U.S.) mounted to a wooden pillar at the lake shore at Bolshie Koty (south-west Baikal Prior to the exposures to 1.5°C and 12°C, the animals were acclimated to laboratory conditions at 6°C, the mean annual water temperature in the Baikal littoral (Falkner et al., 1991;Weiss et al.,1991;Yoshioka et al., 2002) for two weeks. The water temperature in the tanks with the animals was gradually adjusted to 6°C at a rate of 1-

| Read quality control and de novo transcriptome assembly
Raw Illumina reads were examined using the quality control tool FastQC (Andrews, 2017) version 0.11.4, and the results were summarized with MultiQC (Ewels et al., 2016) v1.8. Adapters were removed using Trim Galore! version 0.6.5 (Krueger, 2019). The FastQC analysis revealed that up to 16% and 5% of the overrepresented sequences were ribosomal RNA (rRNA) in the whole body (ScriptSeq) and muscle (NEBnext) libraries, respectively. rRNA was removed from the data set with read aligner bowtie2 (Langmead & Salzberg, 2012) version 2.2.6 using a custom database of gammarid rRNA sequences manually extracted from the National Centre for Biotechnology Information (NCBI) Nucleotide database. Rcorrector (Song & Florea, 2015) was then applied to fix random sequencing errors in Illumina RNA-seq reads. Subsequently, de novo transcriptome assembly was performed using Trinity (Grabherr et al., 2013)
Misassembled or incomplete contigs were filtered out from nonredundant assemblies using TransRate and the assembly quality was then assessed using bowtie2 and the busco tool (Figures S1 and S2A).
Homology searches of filtered assemblies were performed using diamond (Buchfink et al., 2015) version 0.9.24.125 with its sensitive mode against the NCBI nonredundant protein sequence database of 2 March 2020. diamond provides taxonomic assignment of the annotated contigs and only the contigs assigned to metazoan species were selected.
Downstream analyses were performed on the filtered contigs.

| Transcript abundance and differential expression analysis
Transcript abundances in the transcriptomes were analysed with the align_and_estimate_abundance.pl script from the Trinity toolkit.
Alignment-free quantification was conducted with the abundance estimation tool salmon (Patro et al., 2017)  busco results revealed that more than 90% of the 1066 nearuniversal Arthropoda orthologues were present in the nonfiltered transcriptome assemblies ( Figure S2A). However, only about 30% of the orthologues were identified as complete single copies. The filtering of the assemblies led to an almost two-fold increase in complete single-copy busco groups. The rest of the duplicated genes (~10%-30%) are likely to result from multiple haplotypes, alleles or isoforms.
Amphipods are known to host numerous symbionts, commensals, and pathogens and serve as intermediate hosts in the life cycles of metazoan parasites, such as, for example, trematodes and nematodes (Bojko & Ovcharenko, 2019). Infestations of Baikal endemic amphipods with nematodes, cestodes, acanthocephala and other metazoans were reported . However, the sampled animals appeared all healthy and macroscopically showed no infestations with metazoans. Although the presence of metazoan symbionts, commensals, and parasites cannot be excluded, the overall tissue amount and thus the amount of extracted RNA from such organisms in relation to the extracted gammarid RNA can be considered as negligible. In the transcriptomes, the abundance of metazoan nongammarid transcripts was therefore assumed to only be minor in relation to gammarid transcripts. Metazoan sequences in the transcriptomes were thus generally assumed to be from the gammarids, even if assigned to other taxonomic groups.
The transcriptome of E. verrucosus muscle tissue served as reference presumably free of any nongammarid transcripts. From 29,083 contigs obtained from de novo assembled muscle tissue mRNA sequencing reads, 87.2% were assigned to metazoan species, most of the other contigs to bacteria and archaea and a small proportion to unicellular eukaryotes, plants, and fungi ( Figure S2B). It is assumed that the prokaryotic sequences in the muscle tissue transcriptome are from bacteria contamination that occurred during dissection.  (Figure 3). Accordingly, sample-to-sample distance analysis showed more pronounced differences between G. lacustris individuals from the 1.5°C and 12°C exposures than for the Eulimnogammarus species ( Figure S3).

| DE genes in E. verrucosus
In E. verrucosus, individuals exposed to 1.5°C, 63 genes were upregulated (>2-fold change) compared to individuals from the 12°C treatment; for 44 genes, expression changes were >4-fold. Downregulation was seen for 56 genes in individuals from 1.5°C versus 12°C treatments; expression decreases were >4-fold for 32 genes (Figure 3). The heatmap in Figure 4a shows

| DE genes in E. cyaneus
From the studied species, E. cyaneus from the 1.5°C versus 12°C treatments showed the smallest number of DE genes. Expression was >2-fold increased for 29 genes, among which 11 genes were >4-fold upregulated. Expression levels of 43 genes were decreased >2-fold and of five genes >4-fold (Figure 3). Upregulation in 1.5°C Gene set enrichment analysis with functionally annotated DE genes was performed to identify overrepresented gene ontology (GO) terms in G. lacustris upon exposure to 1.5°C. Compared to animals exposed to 12°C, G. lacustris from the 1.5°C treatment showed upregulation of genes from biological process terms associated with events preceding translation: ribosome biogenesis (GO:0042254),   (Figure 5b). Genes assigned to the KEGG pathway ribosome biogenesis (ko03008) were accordingly found to be upregulated in 1.5°C-exposed G. lacustris ( Figure S5).
Furthermore, the GO term cellular response to cold (GO:0070417) was overrepresented (Figure 5a), comprising transcripts encoding a member of the antioxidant system, peroxiredoxin (PRDX4), a subunit of the deubiquitinating enzyme BRISC (FAM175B) and the E3 ubiquitin-protein ligase (RFFL).
Certain DEAD-box RNA helicases were previously shown to be enriched in prokaryotes and eukaryotes at cold stress (Gracey et al., 2004;Guan et al., 2013;Hunger et al., 2006;Yang et al., 2014); transcriptomes of G. lacustris from the 1.5°C treatment were therefore examined for DE DEAD-box RNA helicase transcripts.

| DISCUSS ION
This study investigated the transcriptomes of amphipods from the Lake Baikal area exposed to the mean water temperatures during summer (12°C) and winter (1.5°C) (Figure 1). In winter, the species show marked differences in their metabolic activities and behaviours. We aimed to identify the transcriptomic expression patterns underlying the different species-specific physiological activities at the winter-specific cold temperature regimes. These comprise strongly reduced metabolic and locomotor activity in G. lacustris but the maintenance of high activities in the Baikal-endemic

| Cold exposure induces the onset of hibernation in G. lacustris
Gammarus lacustris from the 1.5°C treatment showed a pronounced transcriptomic response, along with low locomotor and feeding activity. At temperatures close to 0°C, this species drastically decreases its metabolic activity and falls into torpor Karanova & Andreev, 2010). A gradual decrease in temperature from 10.5°C to 1.5°C at a rate of −0.5°C per day leads to majorly decreased metabolic rates that deviated from those that would be expected according to the Q 10 -rule (Q 10 = 6; Hegarty, 1973;Jakob et al., personal communication). It may thus be assumed that the strong transcriptomic response of G. lacustris is driven by its physiological state rather than being a direct effect of low temperature; the transcriptomic response pattern may thus be related to the species' high seasonal changes in locomotor activity. The strategy of G. lacustris to cope with seasonal temperature changes is based on profound changes in locomotor and metabolic activity. In summer, the species' physiological activity level is comparatively high: G. lacustris was found to have more powerful mitochondria than the studied Eulimnogammarus species Vereshchagina et al., 2021). However, high-performing mitochondria are energetically more costly; correspondingly, at the low temperatures in winter G. lacustris decreases its metabolic activity levels to a high degree to prevent energy depletion. The three overarching tasks, (1) finding food, (2) escaping predators, and (3) reproducing drive natural selection of the most beneficial locomotor activity (Visser 2007). In this context, various habitat-specific parameters in winter, such as oxygen levels, food availability, abundance of potential predators and the presence of overwintering habitats, enable but also require the diverging metabolic states of G. lacustris overwintering in a torpid stage and of the Baikal amphipod species maintaining high metabolic activity.
According to the dynamic energy budget model, organisms allocate available energy primarily to growth, activity, reproduction, and basal maintenance (Sokolova et al., 2012). In order to survive F I G U R E 5 Functional categories of DE genes of G. lacustris exposed to 1.5°C versus 12°C. (a) GO terms of G. lacustris enriched in the transcripts upregulated upon exposure to 1.5°C compared to 12°C (FDR <0.05). Only GO terms with an enrichment p-value <.002 were considered significantly enriched; (b) DE genes (fold change >4x) assigned to ribosome biogenesis (GO:0042254), RNA metabolic process (GO:0016070), mRNA processing (GO:0006396); (c) GO terms of G. lacustris enriched in the transcripts down-regulated under exposure to 1.5°C compared to 12°C (FDR <0.05). Only GO terms with an enrichment p-value <.05 were considered significantly enriched; (d) DE genes (fold change >8x, FDR <0.001) assigned to cellular component assembly (GO:0022607), microtubule-based process (GO:0007017), cell projection organization (GO:0030030), cilium or flagellum-dependent cell motility (GO:0001539), and sperm motility (GO:0097722); Gene ratio is a proportion of the genes related to a GO term from the total number of DE genes F I G U R E 6 DEAD-box RNA helicases differentially expressed in G. lacustris exposed to 1.5°C and 12°C (FDR <0.001) [Colour figure can be viewed at wileyonlinelibrary.com] in the cold during hibernation, energy is entirely allocated to basal maintenance (Wilsterman et al., 2021). Accordingly, we found a high proportion of suppressed genes related to activity, growth, and reproduction in cold-exposed G. lacustris. Moreover, cold exposure induced an enrichment in genes involved in the MAPK signalling pathway, which was shown to play an essential role in the onset of hibernation in vertebrates and invertebrates (Biggar et al., 2015;Childers et al., 2019;Fujiwara et al., 2006;MacDonald & Storey, 2005;Michaelidis et al., 2009;Tessier et al., 2017;Wijenayake et al., 2018;. The observed low locomotor activity of cold-exposed G. lacustris might be due to decreased expression of genes related to the regulation of the actin cytoskeleton pathway, as cytoskeleton stabilization was shown to play a central role for locomotor activity in the cold for a wide variety of organisms (Kim & Denlinger, 2009;Königer & Grath, 2018;Lee et al., 1998). The detected decreased expression levels of transcripts associated here to cilia motility-related GO terms ( Figure 4d) may concern so-called sensilla, cilia on sensory cells of crustaceans, functioning as chemoreceptors (Hessler & Elofsson, 2013) or mechanoreceptors (lacinia mobilis, a hair sensillum on mandibles; Geiselbrecht & Melzer, 2013).
There is no epithelium with motile cilia reported for the amphipods.
In this context, it is noticeable that transcripts encoding axonemal dyneins were detected in the G. lacustris transcriptome (Figure 5d).
Upon exposure to low temperature, a number of genes showed enhanced expression. Up-regulation of protein expression can compensate for a temperature-related decrease in enzymatic activity (Gracey et al., 2004;Li et al., 2019;Sonna et al., 2002;Wang et al., 2013). Increased expression of genes involved in ribosome biogenesis and mRNA processing may be related additionally to the compensation of reduced enzyme activity rates at low temperatures as the respective gene products stabilize the protein machinery for basal maintenance. Previously, the upregulation of ribosomal biogenesis in ectotherms in response to cold was found (Chen et al., 2008;Long et al., 2013). Short-term cold exposure of porcelain crabs induces expression of genes encoding proteins involved in DNA and RNA binding, their modification, and in the regulation of transcription and translation (Ronges et al., 2012). Upregulation of DEAD-box RNAhelicases in gammarids from the 1.5°C exposure ( Figure 5) could be related to reduced integrity of nucleic acids due to low temperature (Tinoco & Bustamante, 1999). Thus, DEAD-box RNA helicases act as enzymes unwinding nucleic acid strands, mitigating stress effects on nucleic acid integrity (Yadav & Tuteja, 2019).

| Baikal endemic amphipods -maintenance of high metabolic activity in the cold
When organisms were long-term acclimated to certain conditions, the magnitude of their transcriptomic response to a stress factor is suggested to reflect the differences in whole animal performance in stress versus no-stress conditions (Windisch et al., 2014). A weak long-term response to thermal changes in an organism thus indicates that an exposure temperature was within a species' thermal tolerance window, within which acclimation had taken place. Along those lines, the comparatively weak transcriptomic reaction of the studied Baikal amphipod species to 1.5°C exposure, as well as their high locomotor and feeding activities at this temperature indicate that 1.5°C is within their thermal window for survival and activity. However, cold acclimation can also occur on the levels of cellular organization that do not become evident on the transcriptome level, concerning, for example, post-translational events and protein structure.
Although the two Eulimnogammarus species showed little transcriptomic changes upon cold exposure, their transcriptomic profiles show differences related to the ecology of the species.
Reproduction of E. verrucosus starts in November to January in conditions of stable cold temperatures in the littoral of Lake Baikal (Gavrilov, 1949). Accordingly, we found an increase in the expression activity of genes related to translation, fertilization and embryonic development, probably indicating the beginning of reproductive processes in E. verrucosus (Figure 6Cc). The physiological performance of the cold-loving E. verrucosus indicated slight heat stress when exposed to 12°C, this temperature being beyond the upper pejus (Latin for "getting worse") temperature of E. verrucosus . Upregulation of cellular and metabolic stress response genes in E. verrucosus from the 12°C treatment could therefore be anticipated and, indeed, higher levels of transcripts of Hsps, other molecular chaperones and protein degrading enzymes were found in animals from this treatment. Although this indicates a cellular stress response in this species at 12°C, the comparatively small number of DE genes indicate a rather weak stress response.
In E. cyaneus acclimated to 1.5°C, several genes related to mitochondria functioning were activated, a common feature of cold compensation observed both in cold-adapted species and coldacclimated individuals. Thus, cold-adapted species show an increase in mitochondrial enzyme capacities and in mitochondria density (Fangue et al., 2009;Lucassen et al., 2006;Pörtner, 2002).
Eulimnogammarus cyaneus was shown to have lower mass-specific metabolic rates than G. lacustris and lower cytochrome c oxidase/ citrate synthase (COX/CS) enzyme activity ratios .
This may indicate high mitochondrial densities with relatively low capacities of individual mitochondria (compared to G. lacustris), which is a feature of metabolically active cold-adapted animals (Clarke & Johnston, 1999).
In E. cyaneus, we also detected upregulation of genes involved in homeostatic processes, including protein degradation. Again, this may indicate some level of cold compensation of this basal process, but may also suggest an increase in protein misfolding events in a low energy (i.e., low temperature) environment. Similarly, activation of the proteasome degradation pathway was found in Antarctic fish upon long-term cold exposure (Windisch et al., 2014). Moreover, enhanced expression of acyl-CoA desaturase transcript (SCD; Figure 6f) at 1.5°C may reflect the compensation of decreased membrane fluidity (Kates et al., 1984) in the frame of homeoviscous adaptation (Hazel, 1995). Strikingly, indications for homeoviscous adaptation processes at low temperature were only obtained for E. cyaneus, not for the other species.
The gene encoding Vg, a precursor of the egg yolk protein, was significantly upregulated in E. cyaneus from the 1.5°C treatment (Figure 4b), which may not be expected as E. cyaneus is a summer reproducing species. However, the Vg encoding gene was also shown to be increasingly expressed in bee larvae exposed to cold stress (Ramirez et al., 2017), suggesting that Vg might also play a protective role in stress conditions.
Both studied Baikal species show reduced growth rates in winter (Govorukhina, 2005). Due to the prolonged period of embryonic development of about 6-7 months in E. verrucosus, this species needs to allocate a major share of the available energy to reproduction in the winter months (Gavrilov, 1949;Govorukhina, 2005). In contrast, E. cyaneus starts reproducing in May; embryos develop in about one month during the summer (Govorukhina, 2005), and the reduced growth rates in cold conditions are probably due to increased energy demands for maintaining homeostasis.

The transcriptomic responses of the two Baikal endemic
Eulimnogammarus species to the low temperature treatment were remarkably little. This may be due to a particularly high tolerance of the species' metabolic and physiological functions within the studied thermal range and certain post-transcriptional and translational processes, including those of protein modification and protein stability/turnover. This may enable the species to cope with strongly fluctuating water temperatures that are characteristic for the species' habitat. Thus, water temperatures frequently change rapidly by up to 0.6°C/h over a range of up to 10°C during the summer months ( Figure 1). By having evolved eurytolerant proteins with stable ligandbinding capacity over the entire range of the encountered habitat temperatures (Somero & Low, 1977) endemic inhabitants of the littoral of the unique Baikal ecosystem may have acquired a competitive advantage over ubiquitous Holarctic species, such as G. lacustris, that may be more sensitive to rapid temperature changes. This may thus be a key parameter of the "species immiscibility barrier" (Timoshkin, 2001) preventing Holarctic, non-Baikal species from establishing stable populations in regions with typical Lake Baikal fauna.

ACK N OWLED G EM ENTS
The authors would like to thank Dmitry Karnaukhov and Renat Adelshin for installing the data loggers in the field and Stephan Schreiber for technical assistance with RNA sequencing. This work was funded by the Helmholtz-RSF Joint Research Groups programme from the Helmholtz Association and the Russian Science

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated during the current study have been submitted to the NCBI database under the BioProject PRJNA660769 (Bedulina et al., 2020a), including the raw RNA sequencing data in the SRA database. The transcriptome assemblies have been submitted to the GenBank database under the accession numbers GIUS00000000, GIUW00000000, GIUX00000000, GJDV00000000 (Lipaeva et al., 2021a(Lipaeva et al., , 2021b(Lipaeva et al., , 2021c(Lipaeva et al., , 2021d