Variation in Heat Shock Protein 40kDa relates to divergence in thermotolerance among cryptic rotifer species

doi:10.21203/rs.3.rs-2070566/v1

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Variation in Heat Shock Protein 40kDa relates to divergence in thermotolerance among cryptic rotifer species

https://doi.org/10.21203/rs.3.rs-2070566/v1

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published 31 Dec, 2022

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Genetic divergence and the frequency of hybridization are central for defining species delimitations, especially among cryptic species where morphological differences are merely absent. Rotifers are known for their high cryptic diversity and therefore are ideal model organisms to investigate such patterns. Here, we used the recently resolved Brachionus calyciflorus species complex to investigate whether previously observed between species differences in thermotolerance and gene expression are also reflected in their genomic footprint. We identified a Heat Shock Protein gene (HSP 40kDa) which exhibits cross species pronounced structural variation. This gene exhibits species-specific fixed sites, alleles, and sites putatively under positive selection. These sites are located in protein binding regions involved in chaperoning and may therefore reflect adaptive diversification. By comparing three genetic markers (ITS, COI, HSP 40kDa), we revealed hybridization events between the cryptic species. The low frequency of introgressive haplotypes/alleles suggest a tight, but not fully impermeable boundary between the cryptic species.

Brachionus calyciflorus species complex

HSP 40kDa

temperature adaptation

hybridization

rotifers

SNPs

According to the biological species concept (Mayr, 1942), a species is defined as a group that is reproductively isolated from other species. Reproductive isolation can be maintained either by pre-zygotic isolation, post-zygotic isolation, or a combination of both (Ostevik, 2016). Pre-zygotic isolation occurs before a zygote is formed and involves physiological or systemic barriers that prevent successful mating, such as differences in mating behaviour, habitat preferences or isolation through ecological or geographical barriers (Seehausen et al., 2014). Post-zygotic isolation mechanisms, such as increased zygote mortality or hybrid sterility, occur after zygote formation (Seehausen et al., 2014). Reproductive isolation mechanisms are however often imperfect, so that many closely related species such as cryptic species hybridize (Cheng & Sha, 2017; Papakostas et al., 2016; Zhang & Declerck, 2022a), which counteracts species divergence or may even erode species boundaries (Seehausen et al., 1997).

Cryptic species are species that are morphologically almost indistinguishable and therefore genetic markers are needed to inform species delimitations (Bickford et al., 2007; Gill et al., 2016; Sáez & Lozano, 2005). Such genetically identified species not only call for a revised taxonomy (Fišer et al., 2018; Mills et al., 2017; Struck et al., 2017), but also pose a major challenge to evolutionary and ecological theories. Due to the lack of morphological and physiological differences, high similarity in ecological traits and adaptations is expected (Leibold & McPeek, 2006). When these species occur in sympatry (Gabaldón et al., 2017; Nicholls & Racey, 2006; Ortells et al., 2003; Wellborn & Cothran, 2007), the principle of competitive exclusion (Gause, 1934) is compromised, which further challenges the understanding of co-existence and the niche concept.

Rotifers, with their approximately 2000 described species, belong to a very diverse phylum (Segers, 2008), in which an increasing number of nominal species have been declared cryptic species complexes in the last decades (Fontaneto, 2014). Among them, the monogonont freshwater Brachionus calyciflorus species complex has recently been subdivided into four species using genetics and morphometrics: Brachionus calyciflorus sensu stricto (s.s.), Brachionus dorcas, Brachionus fernandoi, and Brachionus elevatus (Papakostas et al., 2016, Michaloudi et al., 2018). Prior to its delimitation into four species, the Brachionus calyciflorus species complex had been studied with regard to molecular phylogeny (García-Morales & Elias-Gutierrez, 2013; Papakostas et al., 2016; Wang et al., 2013), co-existence (Wen et al., 2016), life history characteristics (Xiang et al., 2016), phylogeography (Xiang et al., 2011a, b), and reproductive isolation (Gilbert et al., 2005; Xiang et al., 2011a, b). More recent studies, which have taken into account the new species classification, focus on ecological processes, e.g., niche differentiation (Zhang et al., 2018), life history traits (Zhang et al., 2019), adaptation and the underlying regulatory mechanisms (Lemmen et al., 2021; Paraskevopoulou et al., 2019, 2020), as well as on the robustness of species boundaries through hybridisation experiments (Zhang & Declerck, 2022a, 2022b). These studies are of great importance to understand not only how these species have diverged, but also which of their adaptations are critical for the co-existence or exclusion of certain species in a specific environment.

A previous lifetable experiment by Paraskevopoulou et al. (2018) has demonstrated diverging temperature optima between Brachionus calyciflorus s.s. (heat-tolerant) and Brachionus fernandoi (heat sensitive). Comparison of transcriptome data from the heat-tolerant B. calyciflorus s.s. and the heat-sensitive B. fernandoi revealed differential expression of Heat Shock Protein (HSP) genes relative to temperature (Paraskevopoulou et al., 2020). HSP gene expression levels correlated with life history traits such that the upregulation of HSP gene expression consistently occurred when the population growth rate was low. The two species were found to exhibit optimal growth at a different temperature and increased their HSP expression outside their optimal temperature range, demonstrating a stress response of the organisms when optimal growth was not feasible (Paraskevopoulou et al., 2020). The pronounced fitness differences at different thermal conditions indicate underlying mechanisms of phenotypes’ responses to environmental conditions that allow them to occur in sympatry, but in different seasons of the year (Paraskevopoulou et al., 2020, Zhang et al., 2018).

Temperature as an environmental variable is one of the most important drivers of evolution and diversification (Takemoto & Akutsu, 2008). Temperature adaptation is particularly interesting in aquatic invertebrates which as ectotherms are stronger influenced by temperature changes than homeotherms (Angilletta, 2009). Increased temperature accelerates ontogeny, shortens the time to maturation and the generation time (Atkinson, 1994; Gillooly et al., 2001; Paraskevopoulou et al., 2020; Walczyńska et al., 2017). The associated faster population growth can foster competitive superiority and accelerate the underlying divergent selection that enforces niche partitioning (Brown & Wilson, 1956; Marrone et al., 2022; Pekkonen et al., 2013). An organism’s adaptation to a particular environment or resource availability may occur at the protein level through the regulation of gene expression (Brawand et al., 2011; Fraser, 2011, 2013; Drummond & Wilke, 2009). This gene expression regulative mechanism can be observed in predator-prey cycles of B. calyciflorus and Chlamydomonas reinhardtii (Becks et al., 2012), or situations of rapid environmental changes (i.e., length of growing seasons) in B. plicatilis s.s. (Franch-Gras et al., 2019; Tarazona et al., 2020), but as well in stress response via HSP upregulation (Smith et al., 2012; Paraskevopoulou et al., 2020), which is a rapid and often the first response mechanism to environmental perturbation. However, a differential regulatory response among genotypes/species to the same environmental cue (genotype x environment (G x E) variation) has to be encoded somewhere in the genome, typically upstream in gene regulatory elements (e.g., promoters, transcription factors; Romero et al., 2012; Alonso & Wilkins, 2005). In addition, a divergent adaptation to different environmental conditions may also yield adaptive differences in structural protein coding genes (as the HSP genes), such that signatures of selection can occur in specific genes or distributed over the whole genome (Franch-Gras et al., 2018; Nowell et al., 2018). Evolution of gene expression and genetic structure do not necessarily occur independently of each other (Pai et al., 2015; Feugeas et al., 2016), and we often find their respective contribution correlating with the progress of time, i.e., altered gene expression typically evolves more rapidly than altered protein structure (Romero et al., 2012). Furthermore, discovering signs of selection in gene sequences between different species allows us to understand the genetic underpinning of the differentiation (Gribble & Welch, 2012; Via, 2009). When put into perspective with the divergence time, the time frame of the adaptation/speciation can be unravelled (Ho & Duchêne, 2014).

In this study, we investigated sequence evolution and divergence in the HSP 40kDa gene, which was inferred by a candidate gene selection for temperature adaptation, both by its temperature-related differential expression (Paraskevopoulou et al., 2020) and by a transcriptome-wide SNP comparison among the heat-tolerant species B. calyciflorus s.s. and the heat-sensitive B. fernandoi. We hypothesise, (I) that the adaptation of B. calyciflorus s.s. and B. fernandoi to different environments is driven by divergent selection in structural genes (here, HSP 40kDa) and (II) that the selection for different temperatures can promote niche partitioning, and hence constitute a pre-zygotic isolation mechanism.

Candidate Gene selection

We assembled 72,165 and 94,884 super-transcripts in B. calyciflorus s.s and B. fernandoi transcriptomes, respectively. The reciprocal best blast hit identified 9,655 putative orthologs which were reduced to 7,976, after predicting open reading frames, locally aligning, and translating into proteins. In the selection tests, out of 7,976 orthologs, 649 were significant (<0.05) in all four pairwise LRT comparisons, hence inferred to be putatively under positive selection. Comparing the orthologous genes under positive selection with the gene expression data, 155 genes were differentially expressed relative to temperature in at least one of the two species (125 in B. calyciflorus s.s., 22 in B. fernandoi, and 8 in both species). Twenty-seven of these genes were assigned to the GO term GO:0006950 (response to stress), of which one gene was annotated to “response to heat” (GO:00009408). This gene (i.e., HSP 40kDa) was differentially expressed in B. calyciflorus s.s. in cultures grown at different temperature, while it did not show a temperature-specific expression in B. fernandoi (figure 1).

HSP 40kDa Sequence Diversity

Comparing HSP 40kDa and Cytochrome Oxidase Subunit I (COI) sequence variation among five Brachionus species (B. calyciflorus s.s., B. fernandoi, B. rubens, B. angularis, and B. diversicornis), we found an overall, higher nucleotide diversity in the mitochondrial COI compared to the HSP 40kDa gene fragment (figure 2). For the HSP 40kDa sequences, the lowest nucleotide diversity was detected between B. angularis and B. diversicornis (0.103), followed by B. calyciflorus s.s. and B. fernandoi (0.127). On the contrary, the highest nucleotide diversity was found between B. fernandoi and B. rubens (0.198). For the COI fragment, the highest nucleotide diversity was detected between B. calyciflorus s.s. and B. diversicornis (0.244) while the lowest was between B. calyciflorus s.s. and B. fernandoi (0.152). The cross-species comparison of synonymous (dS) vs. non-synonymous (dN) substitutions in the HSP 40kDa gene yielded an overall dN/dS: 0.048, with the the highest dN/dS ratio between B. angularis and B. diversicornis (0.085), while the lowest one was found between B. calyciflorus s.s. and B. diversicornis, followed by B. calyciflorus s.s. and B. fernandoi (0.037 and 0.038, respectively) (Supplementary figure 4).

Structural Variation in HSP 40kDA between B. calyciflorus s.s. and B. fernandoi

The translated protein alignment (see Supplementary figure 5) revealed 51 non-synonymous substitutions between the two species in genomic sequences in all specimens (B. calyciflorus s.s., n = 50; B. fernandoi, n = 38) examined by polymerase chain reaction (PCR), among them the 14 found in the transcriptome data. The highest rate dN of non-synonymous substitutions per non-synonymous site among the two species was detected in position 256, followed by position 292 (figure 3).

HSP 40kDa Allele Identification

The network approach based on all 88 phased sequences identified 32 unique HSP 40kDa alleles (corresponding to 27 unique protein sequences) from which 13 alleles were exclusively found in B. fernandoi specimens, and 19 alleles were solely found in B. calyciflorus s.s. specimens (figure 4). Only one allele, which phylogenetically clustered to the B. calyciflorus s.s. HSP 40kDa allele type 18 (A18) was found in a B. fernandoi specimen (2484_4).

Congruence among ITS1, HSP 40kDa, and COI species affinity

The comparison of the nuclear Internal Transcribed Spacer 1 (ITS1), HSP 40kDa, and the mitochondrial COI sequences yielded congruent species assignments for most analysed specimens. However, we identified four descendants of hybridization events between B. calyciflorus s.s. and B. fernandoi (all originating from the same pond; 2484), all of which carry the identical maternally inherited B. calyciflorus s.s. COI haplotype but were assigned to B. fernandoi based on ITS1 sequences (figure 5, Supplementary figure 6,7). Three of these introgressed specimens carried B. fernandoi - specific HSP 40kDa alleles (2484_1, 2484_9, 2484_10), while one individual (2484_4) carried a B. calyciflorus s.s. - specific HSP 40kDa allele, such that only its ITS1 sequence points towards a B. fernandoi affinity. Of the three introgressed specimens carrying B. fernandoi – specific HSP 40kDa alleles, two were heterozygous (2484_1: A11, A1; 2484_10: A12, A2), while one was homozygous for A1 allele (2484_9). The introgressed specimen which carried a B. calyciflorus s.s. specific allele was homozygous for A18.

Inferences on selection and divergence

Four LRT comparisons of the site model test gained significance (table 1). The Bayes Empirical Bayes (BEB) analysis detected six sites under selection (table 1). Visual inspection of these positively selected sites on the protein alignment (Supplementary figure 5) showed a species-specific protein variant pattern at one of these sites (292), while variants were shared at the other sites, but differed in their relative frequencies among the species. The comparison of the positively selected sites with the functional domain description of the HSP 40kDa by Yang et al. (2014) revealed that four out of the six sites are located in functional domains (J-domain and C-terminal domain) (Supplementary figure 5). The branch site model, which was conducted to identify selection and sites under selection in specific species was able to identify selection on the CD (B. calyciflorus s.s. & B. fernandoi) and D branch (B. fernandoi), but did not detect any species-specific sites under selection (BEBs) (table 2). The selection scenario investigation by comparing only B. calyciflorus s.s. and B. fernandoi using a McDonald-Kreitman Test (MKT), calculated a significant Neutrality Index of 12.06, p-value: ≤ 0.001 (i.e., indicative for balancing selection or slightly deleterious mutations), while Tajima’s D (1.538) was not significant (p-value: ≥ 0.10).

The comparison of the fixed species-specific sites revealed 11 fixed sites between B. calyciflorus s.s. and B. fernandoi (table 3). Out of these 11 fixed sites, 9 amino acid substitutions found in B. fernandoi are considered benign in our Polyphen2 analysis, while two (site 63 and 66may change protein function (inferred "possibly damaging", table 3). Additionally, among the six positively selected sites, two amino acid substitutions may influence protein function (inferred "possibly damaging", table 3). The inference of character polarity (ancestral vs. derived) showed a prevalence of B. calyciflorus s.s. to retain the ancestral variant (10/17). For position 292, the status ancestral or derived could not be identified with certainty (table 3).

The estimation of the divergence time between B. calyciflorus s.s. and B. fernandoi, based on mitochondrial cds found to be between 25-29 Million years (Myr), while the split between the freshwater species and the marine species was estimated to be in a range of 41-47 Myr (Supplementary figure 8).

Candidate gene selection pipeline

The utilization of available transcriptomes and different expression patterns of B. calyciflorus s.s. and B. fernandoi allowed us to identify a candidate gene which likely plays a role in the differences in temperature adaptation among the two Brachionus species. Indeed, HSPs have been shown to be involved in the response to various environmental stressors such as thermal stress, toxins, oxidative conditions (Mahmood et al., 2014; Park et al., 2020; Santoro, 1999), and aging (Yang et al., 2014). The importance of HSPs in the process of thermotolerance for invertebrates, particularly HSP 40kDa, HSP 60kDa and HSP 70kDa, was previously shown by Smith et al. (2012) in the monogonont rotifer B. manjavacas.

Genetic variation in the HSP 40kDa gene among Brachionus species

The analysis of the successfully amplified HSP 40kDa gene of the 44 specimens of the B. calyciflorus cryptic species complex (B. calyciflorus s.s. and B. fernandoi) from 14 different locations (Europe, North America) revealed 32 unique alleles, indicating divergent evolution in the HSP 40kDa. All but one allele, which originated from descendants of a hybrid, were species-specific. The high number of alleles and expressed structural differences (27 unique protein sequences) within and between the two species might be related to their cyclical parthenogenesis (Birky & Gilbert, 1971). The dominant asexual mode of reproduction via mitotic eggs from amictic females (i.e., clones) and a less frequent switch to sexual reproduction together with a short generation time (Snell, 2014) strongly reduces genomic recombination, making purifying selection to eliminate slightly deleterious mutations less effective for those organisms, a phenomenon known as Muller’s ratchet (Felsenstein, 1974; Muller, 1932; Muller, 1964).

The comparison between all five analysed species revealed that the species from the B. calyciflorus species complex are more similar (based on nucleotide diversities of the HSP 40kDa and COI) to one another than representatives (B. rubens, B. angularis and B. diversicornis) from outside that species complex, with an overall general higher nucleotide diversity in the COI. The higher diversity in the mitochondrial COI compared to nuclear HSP 40kDa gene is to be expected as mitochondrial DNA in general exhibits a higher mutation rate than nuclear genes (Ballard & Whitlock, 2004), a phenomenon observed as well in rotifers (Zhang et al., 2021).

Pairwise HSP 40kDa gene divergence among Brachionus species generally resembled their phylogenetic affinity and B. fernandoi was closer to B. calyciflorus s.s. compared to other representatives outside of the species complex, has the highest nucleotide divergence. However, for functional genes, the phylogenetic signal may be superimposed by natural selection. Hence, the similarity in HSP 40KDa gene sequences among the not directly related species B. diversicornis and B. calyciflorus s.s. may have arisen from similar environmental parameters that lead to similar selection pressures (Turner et al., 2018). Indeed, a study by Lan et al. (2021) found the occurrence of B. diversicornis to be correlated with warmer water temperatures, which could indicate a higher thermotolerance – as observed in B. calyciflorus s.s. (Paraskevopoulou et al. 2018, 2020) - also in this species.

Selection scenario of the HSP 40kDa

Within the HSP 40KDa gene, six amino acid sites were inferred to be under positive selection. A closer inspection of those sites revealed that four of them are in functional domains of the gene. One site is located in the J-domain, a region known to be important in the chaperone activity of HSP 40kDa and the HSP 70kDa by interacting with the ATP domain of the HSP 70kDa (Pellecchia et al., 1996), which accelerates the ATP hydrolysis (Greene et al., 1998; Wittung-Strafshede et al., 2003) and thus, regulates the ATPase activity (Cintron & Toft, 2006). Three other sites are located in the C-terminal domain of the HSP 40kDa. Although the exact function of these sites is still undetermined, it is known that the C-terminal domain of yeast Saccharomyces cerevisiae contains a peptide binding site (Li et al., 2003; Sha et al., 2000) and is therefore enabled to interact with other proteins. Changes in amino acid composition at these sites can affect folding patterns and thus binding with other proteins (e.g., HSP 70kDa) through suboptimal structures or polarities (Brender & Zhang, 2015; Shortle, 2009). These suboptimal structures can affect the strength of HSP 70kDa ATPase regulation and potentially alter an organism's downstream stress response, which is underlined by the prediction of an impact of amino acid substitutions on protein function at two of the six sites. However, among the inferred sites under positive selection, only one (292, cf. Supplementary figure 4) exhibited species-specific amino acids. One explanation could be that the large geographic range of our study correlated with different local selection pressures, such that most substitutions were not fixed among the entire distribution range of a species. Another explanation could be a low genomic recombination rate (Muller’s ratchet), whereby clonal organisms easily accumulate slightly deleterious mutations that can generate noise in the selection analysis, hindering the identification of species-specific sites under selection (Charlesworth, 2012). The latter is in line with the findings of the MKT where a high Neutrality Index of 12.05 was found between the unique alleles of B. calyciflorus s.s. and B. fernandoi. This is most likely the outcome of an overabundance of replacement-site polymorphisms, which can arise from slightly deleterious polymorphic variants not eliminated by purifying selection (Cutter, 2019), a phenomenon known from clonal organisms (Barraclough et al., 2007).

Because of the potentially compromised power of selection analyses in clonal organisms (Charlesworth, 2012), we inspected all further amino acid substitutions as well. Indeed, among them eleven fixed positions between our species were identified, two of which (63, 66; cf. table 3) with a potential impact on protein function. These sites are not located in known functional domains of the HSP 40kDa, yet they may have an impact on the structure of the protein. The results of the branch site model test indicated positive (directional) selection in B. fernandoi, but not in B. calyciflorus s.s.. This is corroborated by character polarity, i.e., B. fernandoi, while B. calyciflorus s.s. mostly retained the ancestral amino acids. This allows us to draw the conclusion that B. fernandoi diverged from B. calyciflorus s.s. A divergent selection scenario for both species is compatible with the positive Tajima’s D statistics (albeit not significant).

The substantial nucleotide divergence among the two formerly cryptic species B. fernandoi and B. calyciflorus s.s. suggest these lineages to be evolutionary more ancient than one might expect for morphologically hardly distinguishable sister species. We had inferred their divergence time as ~25-29 Myr. The same analysis dated the split between the Brachionus freshwater clade comprising B. angularis, B. fernandoi and B. calyciflorus s.s. and the marine clade comprising B. plicatilis, B. manjavacas, B. rotundiformis and B. koreanus to about 41-47 Myr. This latter estimate is considerably larger than an early estimate of divergence time between the freshwater species B. calyciflorus (species undefined) and the marine B. plicatilis, using COI and 18S as markers (~25 Myr.; Tang et al., 2014). This discrepancy could be due to the increased number of Brachionus species used our analysis and the different genes and mutation rates used in the present study, relative to Tang et al. (2014). Tang et al. (2014) used a mutation rate of 1.76 % Myr^-1 for COI and 0.02 % Myr^-1for 18S, while we used the standard mitochondrial mutation rate for invertebrates (0.0115; Brower, 1994). However, all these rates are derived from distant taxa and – to the best of our knowledge – no rotifer-specific substitution rates are available so far. Hence, these analyses should be repeated when the substitution rates for the mitochondrial genome of our target species (B. calyciflorus s.s. and B. fernandoi) become available. In any case, we note that estimated divergence times within the freshwater B. calyciflorus species complex are in the same order of magnitude as in the marine B. plicatilis species complex (represented by B. plicatilis and B. manjavacas in our analysis; cf. Supplementary figure 8).

Hybridization event

Our study revealed a natural hybridization event between B. fernandoi and B. calyciflorus s.s. individuals originating at one sampling location (a single pond): Among the specimens collected there, four exhibited B. fernandoi ITS1 alleles, but shared the same B. calyciflorus s.s COI haplotype.). This comprises - to the best of our knowledge - the first reported in situ hybridization among these two species. The ability of the species within the B. calyciflorus species complex to hybridize has already been suggested by the widespread occurrence of mitonuclear discordance (Papakostas et al., 2016). However, only recent studies under laboratory conditions observed hybridization between the most closely related species B. calyciflorus s.s. and B. elevatus (Zhang & Declerck, 2022a, 2022b), but with a significantly lower intraspecific fertilization (i.e., pre-zygotic isolation) and higher dormant propagule mortality (i.e., post-zygotic isolation) (Zhang & Declerck, 2022a). Another study by Yang et al. (2022) detected signs of hybridization between B. elevatus and B. dorcas. The comparison of alleles/haplotypes (HSP, ITS1, COI) in our four hybrid specimens with other samples from the same location revealed the hybrids to be F₂ or a subsequent generation and that genes from B. calyciflorus s.s. introgressed into the local B. fernandoi gene pool. This is because allthe introgressed specimens we detected in this study were homozygous for B. fernandoi alleles at the ITS1 locus. Furthermore, three detected hybrids carried B. fernandoi specific HSP 40kDa alleles, while in one case a B. calyciflorus s.s. specific HSP 40kDa allele occurred. This different genetic makeup among the hybrids suggests that they either have emerged from independent hybridization events or that the hybridization was sufficiently ancient to allow for repeated sexual recombination after the hybridization event. In any case, our findings demonstrate that B. calyciflorus s.s. and B. fernandoi can naturally hybridize at sites where they occur in sympatry. It was found in the marine B. plicatilis species complex that niche differentiation of very similar species (e.g., body size, biotic niches, competition abilities) is facilitated by their response to changing physical environments in combination with life and diapause history traits (Gabaldón et al., 2017). In the B. calyciflorus species complex, a study by Zhang et al. (2018) reported strongest pronounced differences in life history traits of B. fernandoi compared to B. calyciflorus s.s., B. elevatus and B. dorcas, such as a prolonged egg and juvenile development times and an overall lower egg production rate and mictic ratio. The adaptation to different temperature optima (Paraskevopoulou et al., 2019), associated with differences in HSP 40Da expression (Paraskevopoulou et al., 2020) and protein structure (this study), may lead to seasonal isolation of the two species. This illustrates the above example that the niche of very similar species can be determined by their response to abiotic environmental conditions. The differential niches (here preferred temperature) occupied by the species reflects divergent adaptation but may at the same time serve as a pre-zygotic isolation mechanism maintaining the species boundaries. This mechanism may however be imperfect under certain environmental conditions, leading to occasional hybridization as observed here.

We used transcriptome and expression data to select a suitable candidate, the HSP 40kDa gene, which may play a role in temperature adaptation in the Brachionus calyciflorus species complex. Species-specific alleles and fixed amino acid substitutions sites as well as positively selected sites located in functional domains of the protein were identified. The high number of detected alleles, the results of the branch site selection test, and the fixed sites indicate a divergent adaptation process with B. calyciflorus s.s. retaining ancestral features, from which B. fernandoi derived by positive directional selection. Furthermore, the study revealed descendants of an in-situ hybridization event of B. calyciflorus s.s. and B. fernandoi. This finding indicates that hybridization between those species is possible. We hypothesize that the temporally isolated niches they populate serve as pre-zygotic isolation mechanisms. This isolation may be imperfect under certain environmental conditions, yet hybridizations seem rare and seasonal niche separation generally prevents species boundaries from becoming blurred.

Candidate gene selection

To identify candidate genes with a sequence divergence pattern related to differential temperature adaptation, previously published transcriptome data (Paraskevopoulou et al., 2020; GenBank accessions number SRA: SRR10426055-76) was analysed using the following customized bioinformatic pipeline (Supplementary figure 1): De novo transcriptome assemblies were used to generate a super transcript for each species using Trinity v. 2.11.0 gene splicer modeler (Grabherr et al., 2011). This super transcript step was integrated to minimize redundancy in the data by inferring original genes and alternative splice forms (Davidson et al., 2017). A reciprocal best blast hit process was performed, using the blastn program of BLAST 2.9.0+ (Altschul et al., 1990) to detect orthologous genes shared by B. calyciflorus s.s. and B. fernandoi. To determine the best blast hit, results were filtered using a combination of E-value, which is a size corrected measure of statistical significance, and the bit score, which is a measure of matches and mismatches between two sequences (Altschul et al., 1990). When E-values of the first two or more hits were the same, the best hit was chosen using the bit score. For each search, a list was created that contained one best hit per query. Orthologous gene pairs were extracted, and open reading frames were predicted using TransDecoder v 5.5.0 (Haas et al., 2013). To calculate ratios of the rates of non-synonymous and synonymous substitutions (dN/dS) from protein-coding regions, sequences were then translated into amino acid sequences using Biopython (Cock et al., 2009) and locally aligned within the Biopython integrated package pairwise2 using a Smith-Waterman algorithm. Locally aligned coding sequences and the corresponding unaligned DNA sequences were used to create codon alignments using pal2nal (Suyama et al., 2006). Selection tests were conducted using all available seven (M0, M1a, M2, M3, M7, M8 and M8a) codeML models (Supplementary table 1), as implemented in PAML v. 4.9 (Yang, 2007). These models where then compared in four (M0 vs. M3, M1a vs. M2, M7 vs. M8, M8 vs. M8a) predefined likelihood-ratio tests (LRT) to decide whether the different null models (i.e., model that does not allow for any codons ω > 1, corresponding to absence of positive selection) can be rejected (Yang, 2007).

Orthologous genes that were significant (< 0.05) in all four LRT comparisons were annotated using Gene Ontology (GO) terms (Ashburner et al., 2000). These terms are hierarchically ordered descriptions of genes or proteins molecular functions, biological processes, and cellular components. The GO term annotation was done on the online Server Argot2.5 and its batch processing function (Lavezzo et al., 2016). To use this function, the longest available amino acid sequence of each protein was used in a blast search against the Uniprot Swiss-Prot database (The UniProt Consortium, 2021) and a hmmer search (Finn et al., 2011) against the Pfam-A database (Finn et al., 2014). In addition, we compared the genes exhibiting positive selection in the selection tests with those found to be differentially expressed relative to species and temperature in Paraskevopoulou et al. (2020). Among those genes functionally related to temperature tolerance (GO:00009408; “response to heat”; GO:00009409; “response to cold”), only one orthologous gene, the HSP 40kDa, exhibited signs of positive selection and was differentially expressed in more than one temperature category, hence chosen for in-depth analysis.

DNA extraction

To extract DNA, single rotifer specimens were collected from laboratory cultures, washed in 96% EtOH and stored in a 1.5mL reaction tube in10µL HPLC-H₂0. In total 56 rotifer individuals (B. calyciflorus s.s., n = 25; B. fernandoi, n = 19; B. rubens, n = 4; B. diversicornis, n = 3; B. angularis, n = 5) were collected from different locations in Germany. For B. calyciflorus s.s., additional samples from USA, Italy and Austria were available (Supplementary figure 2, Supplementary table 2). To prevent that the DNA from single individuals being lost during the extraction process, 3µL carrier RNA [1µg/µL] (QIAGEN, Germany) was added to the sample during the lysis. DNA was extracted using the NucleoSpin®Tissue extraction kit (Macherey-Nagel, Germany) following the manufacturer’s protocol for animal tissue (page 30-33).

Primer design HSP 40kDa

To amplify the HSP 40kDa from all five different Brachionus species, primers were designed using the candidate gene sequences (B. calyciflorus s.s. and B. fernandoi) as well as the published HSP 40kDa sequence of B. calyciflorus (unknown species assignment regarding current taxonomy; Genbank: KC176712.1; Yang et al. 2014). Sequences were aligned in Geneious v. 8.1.9 (Kearse et al., 2012) using ClustalW (Thompson et al., 1994) and the implemented primer3 algorithm (Untergasser et al., 2012) was used to design primer pairs.

Amplification and Sequencing of the ITS1, COI and HSP 40kDa

To determine the species and clonal diversity, both the nuclear internal transcribed spacer (ITS1) and the mitochondrial Cytochrome Oxidase Subunit I (COI) were amplified, using universal invertebrate primers (for ITS1: Palumbi, 1996; for COI: Folmer et al., 1994). PCR conditions, primer sequences, and used PCR chemicals mixes can be found in the Supplementary table 3, 4 and 5. The HSP 40kDa gene of all five Brachionus species was amplified using newly designed primer pairs (Supplementary table 3, 6 and 7). Successfully amplified HSP 40kDa, COI and ITS1 products were purified using an enzymatic (ExoAP) procedure and sequenced on a Sanger sequencing platform (Applied Biosystems™ 3500 Genetic Analyzer). Sequences from all specimens used in this study (Supplementary table 2) were visually inspected using Geneious v. 8.1.9 and heterozygous positions (for ITS1 and HSP 40kDa) were encoded with the standard IUPAC code for ambiguity (Comnish-Bowden, 1985). To reconstruct both alleles of the diploid nuclear DNA, the PHASE algorithm version 2.1 (Stephens et al., 2001; Stephens & Donnelly, 2003) was used, as implemented in DNAsp v. 6 (Rozas et al., 2017).

HSP 40kDa Sequence Diversity

To compare structural variability and the heterozygosity level of the HSP 40kDa gene among different species, consensus sequences per species were generated. In order to avoid any bias which may arise from uneven geographic sampling across species, we only used specimens originating from the same region “Uckermark” for this analysis: B. calyciflorus s.s. (n = 11), B. fernandoi (n = 8), B. rubens (n = 4), B. diversicornis (n = 3) and B. angularis (n = 5). Sequence ambiguities in the consensus sequence were coded as N’s. Pairwise nucleotide diversities of the 1,184bp long HSP 40kDa gene sequence and the 435bp long COI sequences between the five species were calculated and non-synonymous resp. synonymous substitutions in the HSP 40kDa gene were identified using DNAsp v. 6 (Rozas et al., 2017).

Expressed and Structural Variation in the HSP 40kDa gene between B. calyciflorus s.s. and B. fernandoi

Sequences originating from multiple individuals (B. calyciflorus s.s., n = 50; B. fernandoi, n = 38) were translated using the published translation reading frame of the HSP 40kDa gene of B. calyciflorus (Genbank: KC176712.1). As we did not yield the complete sequence for all specimens, the alignment was truncated to 888bp present in all sequences. Number of non-synonymous substitutions per non-synonymous site between the two species were compared using SLAC analysis implemented in Datamonkey (Weaver et al., 2018; Kosakovsky Pond & Frost, 2010).

HSP 40kDa allele Identification

To identify different allele types within the B. calyciflorus species complex, allele networks were generated from the resulting 88 phased sequences (B. calyciflorus s.s. n = 50 and B. fernandoi n = 38) using the program popART version 1.7 (Leigh & Bryant 2015). Subsequently, a Circos plot (Krzywinski et al., 2009) was generated based on allele types identified by popART, species, and specimens’ origin.

HSP 40kDa gene diversity relative to established barcoding markers

Species recognition within the B. calyciflorus complex relies on species-specific ITS1 sequences and clonal lineages are further characterized by their mitochondrial COI haplotype. For 43 specimens (n = 43; B. fernandoi n = 18, B. calyciflorus s.s. n = 22, B. rubens n = 1, B. angularis n = 1, and B. diversicornis n = 1), sequences were available for both these marker genes and the HSP 40kDa gene. For these specimens, congruence in genetic affinity across genetic markers was assessed using a tanglegram approach. Dendrograms and the tanglegrams were generated in R v.4.0.5 with the package dendextend v.1.15.1 (Galili, 2015).

Selection tests and divergence time

To test whether the expressed genetic variation among the different Brachionus species is under selection, a selection test was performed based on the 29 HSP 40kDa species-specific alleles using the Program PAML v.4.9 (Yang, 2007). A specific focus was on the divergence of the two species B. calyciflorus s.s. and B. fernandoi. This analysis needs a phylogeny as input which was based on the 534 bp long ITS1 fragment and calculated using RaxML v.8 (Stamatakis, 2014), performing 1000 bootstrap iterations (Supplementary figure 3). This ITS1 phylogeny as well as the HSP 40kDa unique alleles were used to infer sites under selection according to the codeML site model implemented in PAML, testing all seven different models of selection (table 1) with a subsequent site under selection inference via Bayes Empirical Bayes (BEBs) (for parameter setting see Supplementary table 8). An additional branch site test was performed to identify patterns of selection on three different branches: (I) the shared branch of B. calyciflorus s.s. and B. fernandoi, (II) B. calyciflorus s.s. only and (III) B. fernandoi only (Supplementary figure 3). Subsequently, to further compare the two formerly cryptic species of B. calyciflorus s.s. and B. fernandoi, a codon-based McDonald-Kreitman test (MKT) and Tajima’s D statistics (allele frequency based) were calculated based on inferred the unique alleles using DNAsp v. 6 (Rozas et al., 2017).

Species-specific fixed amino acid substitutions were identified (Supplementary figure 5) and the ancestral amino acid was inferred under parsimony, using sequence information from B. rubens, B. angularis and B. diversicornis and the ITS1 phylogeny (Supplementary figure 3). The functional effects of the specific amino acid (aa) substitutions were predicted with Polypen2 (Adzhubei et al., 2010) using B. calyciflorus s.s. as a reference. Note here that Polyphen2 does not consider the possibility of a positive effect of an aa substitution, such that it ranks any substitution without a predicted change in protein function as "benign" (i.e., no effect), while any predicted change is ranked as "damaging" (i.e., predicted to impact protein function).

To estimate the divergence time between B. calyciflorus s.s. and B. fernandoi the program BEAST v.1.10.4 (Suchard et al., 2018) was used. Whole mtGenome coding sequences (cds) of different rotifer species were downloaded from NCBI: B. calyciflorus s.s. (Kiemel et al., 2022), B. fernandoi Kiemel et al., 2022), B. angularis (Kim et al., 2020), B. manjavacas (Kim et al., 2021), B. plicatilis (Suga et al., 2008), B. koreanus (Hwang et al., 2014), B. rotundiformis (Kim et al., 2017), B. rubens (Choi et al., 2019) and P. similis (Choi et al., 2020) (for Genebank accession numbers cf. Supplementary figure 8). In absence of a published mutation rate for rotifers, the standard divergence rate for invertebrates/insects (2.3% Myr^-1) with a constant substitution rate of 0.0115 per million years per lineages (Brower, 1994) was used to calibrate the phylogeny. Cds were aligned in Geneious v. 8.1.9 using ClustalW and the IQ-Tree webserver (Trifinopoulos et al., 2016) was used to determine the best fitting substitution model. The BEAST run was conducted with 10 million MCMC iterations, with GTR+G4+I chosen as the substitution model. Convergence was checked with Tracer v.1.7.2 (Drummond & Rambaut, 2007), using the first million MCMC iterations as the burn-in value.

Data availability statement

The genome sequence data (ITS1, COI and HSP 40kDa) that supports the findings of this study will be openly available on Genbank of NCBI at http://www.ncbi.nlm.nih.gov/ upon acceptance. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA544636, SRR10426055 - SRR10426076 and SAMN11845726 - SAMN11845747.

Acknowledgement

The authors would like to acknowledge Magdalena Litwin for providing rotifer specimens, Dr. Stefanie Hartmann for her assistance in analysing the transcriptome data, Anja Ernst for her valuable help in the laboratory, and Christina Schirmer, Sabine Donath and Claudia Wahl for their support in cultivating the rotifers.

Author Contributions

K.K., R.T. and G.W. designed the research. K.K. and S.P. collected the samples. K.K. performed laboratory processing of samples with support from K.H. K.K., M.G. and S.P. analysed the data with input from R.T. and G.W. K.K. produced the graphics and wrote the manuscript with the support of R.T. S.P., M.G. and G.W. All authors approved the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by Deutsche Forschungsgemeinschaft (DFG) in the framework of the BioMove Research Training Group (DFG-GRK 2118).

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Table 1

Likelihood ratio tests of seven different site models performed on 296 amino acids (888 bp) of the HSP 40kDa, including 29 of the 32 unique alleles (A11, A12 and A18 were excluded from the analysis, as they only occurred in specimens affected by hybridization/introgression). Underlying phylogeny is based on the ITS1 (534 bp) of B. calyciflorus s.s., B. fernandoi, B. angularis, B. diversicornis and B. rubens. For each likelihood ratio test (LRT), we provide log likelihood (l) values of the compared tests (2∆l), degrees of freedom (df), tp-values, estimated rate of synonymous and non-synonymous sites, and the sites inferred to be under positive selection by BEB, labeled with an * for a posterior probability of > 95% and ** for > 99%.

Site Model	LRT	2∆l	df	p-value	l	ω	BEBs
M0 (one-ratio)	M3 vs. M0	262398852	4	<< 0.001	-3598.034542	0.0718	-
M3 (discrete)					-3466.835116	0.1439
M1a (nearly Neutral)	M2a vs. M1a	11558380	2	<< 0.001	-3480.993265	0.1328	-
M2a (positive selection)					-3475.214075	0.1815
M7 (β)	M7 vs. M8	21480488	2	<< 0.001	-3477.904393	0.1108	-
M8 (β & ω)					-3467.164149	0.1450
M8a (β & ω = 1)	M8a vs. M8	3189007	1	<< 0.001	-3483.109184	0.0769	10; 38; 249*; 256; 286; 292*

Table 2

Likelihood ratio test of branch site model performed on 296 amino acids (888 bp) of the HSP 40kDa including the 29 of the 32 unique alleles (A11, A12 and A18 hybrid alleles are excluded from the analysis) of B. calyciflorus s.s. and B. fernandoi, B. angularis, B. diversicornis and B. rubens. Phylogeny is based on 534 bp of the ITS1. For each branch selection test, we provide log likelihood (l) value of the compared tests (2∆l), degrees of freedom (df), and the p-value. No sites were inferred to be under positive selection by BEB in this test framework.

Branch site model	Branch	2∆l	df	p-value	l	BEBs
selection	CD	66	1	<< 0.001	-3480.993266	-
neutral					-3480.993299
selection	C	2	1	0.1573	-3480.993266	-
neutral					-3480.993265
selection	D	3051170	1	<< 0.001	-3478.544980	-
neutral					-3480.070565

Table 3

Prediction of impact of non-synonymous (fixed or under selection cf. table 2) amino acid substitutions between B. calyciflorus s.s. and B. fernandoi in comparison to B. rubens, B. angularis and B. diversicornis. Predictions were made using Polyphen2 (Abzhubei et al., 2010). The code provides details on the amino acid substitution and the position. Bc-Bf-Br-Ba-Bd provides the information of the respective amino acid of B. calyciflorus s.s. (Bc), B. fernandoi (Bf), B. rubens (Br), B. angularis (Ba) and B. diversicornis (Bd). Ancestral amino acid was inferred using the most parsimonious (i.e., fewest substitutions) scenario, taking the species' phylogeny into account. In case of multiple amino acids (aa) per species, compared aa are bold in the row Code. Asteriks indicate sites under selection inferred by codeML. Note that Polyphen2 does not consider the possibility of a positive effect of an aa substitution, such that it ranks any predicted change in protein function as "damaging".

Site (aa)	Code	*Bc-Bf-Br-Ba-Bd*	Ancestral	Score	Sensitivity	Specificity	Prediction
10*	H10V/I	H-V/I-H-R-R	H	0.002	0.99	0.30	Benign
	H10V/I	H-V/I-H-R-R	H	0.004	0.97	0.59	Benign
27	E27G	E-G-E-E-E	E	0.000	1.00	0.00	Benign
30	F30-	F---F-F-F	F	-	-	-	-
31	S31-	S---S-S-S	S	-	-	-	-
38*	E/K38E/K	E/K-E/K-E-E-E	E	0.231	0.91	0.88	Benign
63	L63M	L-M-L-M-L	L	0.953	0.97	0.93	Possibly damaging
66	T66S/F	T-F-T-F-T	T	0.143	0.92	0.86	Benign
	T66S/F	T-F-T-F-T	T	0.997	0.41	0.98	Possibly damaging
127	T127A	T-A-T-T-T	T	0.019	0.95	0.80	Benign
175	F175V	F-V-V-F-V	V	0.001	0.99	0.15	Benign
177	Y177N	Y-N-Y-Y-Y	Y	0.000	1.00	0.00	Benign
226	R226K	R-K-R-K-R	R	0.000	1.00	0.00	Benign
242	H242L	H-L-I-H-H	H	0.000	1.00	0.00	Benign
249*	P/S249P	P/S-P-P-Q-Q	E	0.116	0.93	0.86	Benign
256*	L/F/N256L	L/F/N-L-L-L-L	L	0.024	0.95	0.81	Benign
	L/F/N256L	L/F/N-L-L-L-L	L	0.094	0.93	0.85	Benign
285	D285E	D-E-D-E-D	E	0.000	1.00	0.00	Benign
286*	I/K286I	I/K-I-I-I-I	I	0.914	0.81	0.94	Possibly damaging
292*	T/I292R	T/I-R-A-S-V	?	0.001	0.99	0.15	Possibly damaging
	T/I292R	T/I-R-A-S-V	?	0.000	1.00	0.00	Benign

No competing interests reported.

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Variation in Heat Shock Protein 40kDa relates to divergence in thermotolerance among cryptic rotifer species

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