- Research article
- Open Access
Diversification and adaptive sequence evolution of Caenorhabditislysozymes (Nematoda: Rhabditidae)
© Schulenburg and Boehnisch; licensee BioMed Central Ltd. 2008
- Received: 22 May 2007
- Accepted: 19 April 2008
- Published: 19 April 2008
Lysozymes are important model enzymes in biomedical research with a ubiquitous taxonomic distribution ranging from phages up to plants and animals. Their main function appears to be defence against pathogens, although some of them have also been implicated in digestion. Whereas most organisms have only few lysozyme genes, nematodes of the genus Caenorhabditis possess a surprisingly large repertoire of up to 15 genes.
We used phylogenetic inference and sequence analysis tools to assess the evolution of lysozymes from three congeneric nematode species, Caenorhabditis elegans, C. briggsae, and C. remanei. Their lysozymes fall into three distinct clades, one belonging to the invertebrate-type and the other two to the protist-type lysozymes. Their diversification is characterised by (i) ancestral gene duplications preceding species separation followed by maintenance of genes, (ii) ancestral duplications followed by gene loss in some of the species, and (iii) recent duplications after divergence of species. Both ancestral and recent gene duplications are associated in several cases with signatures of adaptive sequence evolution, indicating that diversifying selection contributed to lysozyme differentiation. Current data strongly suggests that genetic diversity translates into functional diversity.
Gene duplications are a major source of evolutionary innovation. Our analysis provides an evolutionary framework for understanding the diversification of lysozymes through gene duplication and subsequent differentiation. This information is expected to be of major value in future analysis of lysozyme function and in studies of the dynamics of evolution by gene duplication.
- Gene Duplication
- Lysozyme Gene
- Recent Gene Duplication
- Caenorhabditis Species
Since their discovery by Ian Fleming, lysozymes have become an important model system in molecular biology, biochemistry, and structural biology with major biomedical importance . They are ubiquitous enzymes known from almost all groups of organisms including phages, bacteria, protists, fungi, animals, and plants [2–7]. Several distinct lysozyme types are recognised, including the chicken-type, goose-type, invertebrate-type, or amoeba lysozymes [2, 7, 8]. Because of their ability to break up peptidoglycan (an important component of bacterial cell walls) and their induced expression upon pathogen exposure, their original function was suggested to be defence against bacterial infections. At the same time, some lysozymes are involved in digestion. This function is found in vertebrate and insect taxa, which obtain nutrition from microorganisms involved in decomposing organic matter, e.g. the vertebrate foregut fermenters like ruminant artiodactyls, leaf-eating monkeys, the bird hoatzin, and the Drosophila and Musca flies [5, 9–11].
Lysozymes have additionally become an important model in studies of molecular evolution. The origin of a digestive function in the leaf-eating monkeys was found to show the characteristic signature of adaptive sequence evolution, i.e. the non-synonymous substitution rate was significantly larger than the synonymous substitution rate, strongly indicating that amino acid-changing mutations were favoured by natural selection [12, 13]. Gene duplication appears to play an important role in lysozyme evolution. Impressive examples include the ruminant artiodactyls with at least seven genes per genome , Drosophila fruitlies with at least eleven loci [5, 14], and the mosquito Anopheles gambiae with at least nine lysozymes . In these examples, some lysozymes have a digestive function. Functional diversification is further indicated by variation in gene expression pattern (e.g., timing, tissue, expression level) and several biochemical characteristics. For instance, the digestive lysozymes differ from the antimicrobial lysozymes by an increased expression in the gut, their resistance to protease degradation, an acidic isoelectric point and pH optimum [5, 9]. Taken together, these patterns are consistent with the specific role of gene duplication as a source of evolutionary innovation , as known for diverse gene families like the animal hox and the vertebrate MHC genes [17, 18].
An unexpected diversity of lysozymes is found in nematodes of the genus Caenorhabditis. They contain up to 15 different lysozymes of two distinct types [19, 20]: the invertebrate-type and another distinct type that is characterized by lysozymes from various protist taxa (hereafter termed protist-type lysozymes). Although the exact function of these enzymes has not as yet been assessed systematically, some of them are involved in pathogen defence [19–21]. In the current paper, we provide a framework for understanding diversification of the Caenorhabditis lysozymes. In particular, we explore the lysozyme genealogy and test the hypothesis that gene duplications associate with diversifying selection, as expected for a role in immunity against the usually rapidly evolving repertoire of pathogens. Lysozyme sequences are considered from the three Caenorhabditis species with completely sequenced genomes, i.e. C. elegans, C. briggsae, and C. remanei . Their genealogies are reconstructed at both protein and DNA sequence level with the help of maximum likelihood (ML) tree inference methods . Signatures of positive selection are assessed across branches of the inferred genealogy and across the aligned sequences with the help of the maximum likelihood approach developed by Ziheng Yang and co-workers [24, 25]. The results are related to the current data on lysozyme function.
Overview and general phylogenetic position of the Caenorhabditislysozymes
Information on the invertebrate-type lysozymes
Gene name/Protein ID
Information on the protist-type lysozymes
Gene name/Protein ID
For the more detailed phylogenetic analyses, we examined the two lysozyme types separately. For this purpose, we generated two new alignments and several subsets of these (see methods and below).
Evolution of invertebrate-type lysozymes
Results of the analysis of adaptive sequence evolution for individual branches of the invertebrate-type lysozyme tree.
d N /d S
d N /d S
Evolution of protist-type lysozymes
The protist-type lysozymes are present with either seven (both C. briggsae and C. remanei) or ten genes (C. elegans; Table 2). Synteny is found for the genes lys-1, lys-2, and lys-3, which are clustered in all three species – in both C. elegans and C. briggsae on chromosome V and in C. remanei on supercontig 13 (Table 2; Fig. 1B). The gene lys-1 is always found in opposite orientation to the other two. In C. remanei, the lys-3 homologue is separated from the other two genes by approximately 10,000 nucleotides (and four open-reading frames) in contrast to both C. elegans and C. briggsae, where the three genes are directly adjacent to each other. In C. elegans, the lys-7 gene is additionally found on chromosome V, but in a different location than the three clustered genes (Fig. 1B). C. elegans contains a second well-defined cluster of protist-type lysozymes on chromosome IV, including Cel-lys-4, Cel-lys-5, and Cel-lys-6. In this case, there is no synteny in the other species. Interestingly, however, the C. briggsae chromosome IV contains a cluster that combines genes from the above C. elegans cluster (in this case the C. briggsae genes Cbr-lys-6.1 and Cbr-lys-6.2) with the gene Cbr-lys-10. The C. elegans orthologue of the latter gene, Cel-lys-10, is similarly present on chromosome IV but in a different location than the cluster (Fig. 1A). In C. remanei, two additional genes are found in relatively close physical proximity to each other: Cre-lys-8.1 and Cre-lys-8.2 are located on supercontig 9 (Table 2) separated by approximately 200,000 nucleotides and 56 open-reading frames.
(i) The protist-type lysozymes fall into two distinct clades (clade 1 and 2), which diverged before separation of the three species (Fig. 6A).
(ii) Within clade 1, four distinct phylogenetic groups are identified (Fig. 7A). Three of them contain one orthologue per species, indicating duplication of genes before species separation. The fourth group includes one gene for C. briggsae, two monophyletic genes for C. elegans, and two monophyletic genes for C. remanei.
(iii) The inferred clade 2 topology shows less hierarchical structure than the clade 1 topology (Fig. 8A). Here, the lys-10 orthologues form a monophyletic group, which is most closely related to Cel-lys-4. The remainder of this clade shows a single gene from C. remanei, two monophyletic genes from C. elegans, and two monophyletic genes from C. briggsae.
The analysis of positive selection across sequence alignments yielded a single significant result. In particular, for the aligned clade 1 coding sequences (alignment 7, see methods and Fig. 5) model M8 differed significantly from both model 7 (LRT, 2ΔL = 8.786, P = 0.012) and model 8a (LRT, 2ΔL = 6.589, adjusted P = 0.005). A single alignment position was found to be subject to adaptive sequence evolution according to the Bayes empirical Bayes method (P = 0.99). The alignment position is found in the middle of the genes and it is highlighted in Fig. 5. For the other data sets, the comparisons were all insignificant
Results of the analysis of adaptive sequence evolution for individual branches of the whole protist-type lysozyme tree.
d N /d S
d N /d S
Results of the analysis of adaptive sequence evolution for individual branches of the clade 1 protist-type lysozyme tree.
d N /d S
d N /d S
Results of the analysis of adaptive sequence evolution for individual branches of the clade 2 protist-type lysozyme tree.
d N /d S
d N /d S
Characteristics and function of the different lysozymes
Differences in the characteristics of the three main lysozyme clades
1: p-lys clade 1
289.93 ± 2.83
31.73 ± 0.44
6.17 ± 0.23
-2.36 ± 1.21
0.011 ± 0.03
2: p-lys clade 2
216.11 ± 1.75
23.66 ± 0.28
7.07 ± 0.27
-0.11 ± 0.56
0.018 ± 0.03
155.60 ± 9.89
17.33 ± 1.21
7.29 ± 1.16
1.50 ± 1.27
-0.297 ± 0.04
1↔2, 1↔3, 2↔3
1↔2, 1↔3, 2↔3
Information on the function of the C. elegans lysozymes
p-lys clade 1
I, ILN, HN
SEK-1, NSY-1, TIR-1, DBL-1
BT, EC, EF, PA, PL, SM
[20, 41, 43, 45, 76–80]
SEK-1, PMK-1§, ELT-2
[43, 45, 76–79]
BT, EC, MN, PA
[19, 41, 43, 78, 79]
EF, MN*, SM
[19, 20, 40, 41, 76, 79–81]
I, PB, PG
DBL-1, SMA-2, SEK-1, NSY-1, DAF-16
[19, 20, 40, 42–44, 76, 79, 80]
p-lys clade 2
[43, 76, 77, 79]
[43, 76, 79]
[19, 45, 76, 79]
[19, 40, 45, 77, 79]
[41, 43, 77]
Evolution of Caenorhabditislysozymes
Caenorhabditis nematodes are among the organisms with the highest number and the most extreme diversity of lysozyme genes. Their lysozymes fall into three distinct clades, one being part of the invertebrate-type and the other two of the evolutionary very distant protist-type lysozymes. Moreover, the Cel-lys-9 gene from C. elegans, which undoubtedly belongs to the protist-type lysozymes (Fig. 2), shows only limited similarities to the other nematode genes and it may thus represent a class of its own. To date, it is impossible to say whether the invertebrate-type and the protist-type lysozymes evolved from a common ancestor or not. In the latter case, their general similarity as lysozymes would be a consequence of convergent evolution towards a similar function in defence or digestion. Additional data from more basal nematode as well as metazoan taxa (e.g. cnidarians, poriferans, platyhelminths) is required to distinguish between these alternatives.
Some of the Caenorhabditis lysozyme genes are found in clusters within the genome, as known for about one fifth of the protein-coding genes of C. elegans and apparently characteristic for genes involved in interactions with the environment . Thus, lysozymes may be subject to similar evolutionary dynamics recently described for several of the clustered gene families . These clustered gene families are most likely shaped by concerted molecular evolution. They are characterized by species-specific clades of the gene clusters, the presence of inverted genes that have been proposed to stabilize concerted evolution of clusters over time, and strong purifying selection . However, the inferred evolutionary history of lysozyme clearly contrasts with such patterns. Genes in close genomic proximity do not form species-specific phylogenetic clades. None of the genomic lysozyme clusters contain "stabilizing" genes with inverted orientation in the middle of the cluster. Furthermore, although the majority of genes appears to be subject to purifying selection, we did obtain a strong indication for several episodes of diversifying selection.
We conclude that the lysozymes follow a different evolutionary trajectory. Our analysis reveals three main patterns.
(i) Gene duplication prior to species separation and maintenance of the duplicated genes. This scenario is most evident where lysozyme orthologues are monophyletic and distributed in synteny across genomes in all three taxa, e.g. the protist-type lys-1, lys-2, and lys-3 genes. Other likely cases are the protist-type lys-6, lys-8, lys-10, and the invertebrate-type ilys-4 and ilys-5 genes, for which corresponding orthologues fall into monophyletic clades. In all these cases, the orthologous genes must have an age of at least three million years, which is the minimum time since the last most common ancestor of the three Caenorhabditis species . Their maintenance across time suggests an important conserved biological role for each group of orthologues. In this case, their original divergence after gene duplication may have been favoured by diversifying selection and thus, it may associate with signatures of adaptive sequence evolution. Such a signature is indeed found for the clade 1 protist-type lysozymes (including lys-1 to lys-3, lys-8, and orthologues).
(ii) Recent gene duplication and diversification. Phylogenetic analysis revealed five cases of lineage-specific duplication events (Figs. 4, 7, and 8). One of these cases (Cre-ilys-4.1 and Cre-ilys4.2) is associated with a significant signature of adaptive sequence evolution, suggesting that diversifying selection favoured lysozyme differentiation upon duplication. The other four cases (Cre-lys-8.1 and Cre-lys-8.2; Cbr-lys-6.1 and Cbr-lys-6.2; Cel-lys-5 and Cel-lys-6; Cel-lys-7 and Cel-lys-8) appear to be subject to purifying selection. This pattern indicates strong selection for maintenance of gene function after the duplication event.
(iii) Gene duplication prior to species separation followed by differential gene loss. This scenario appears to apply to the Cel-ilys-1, Cel-ilys-2, Cel-ilys-3, and Cel-lys-4 genes, which are each present in only one of the species and diverge from internal nodes, some of them along long branches indicative of old evolutionary age. Loss of genes after duplication events in the other Caenorhabditis species may then suggest redundant functions of lysozymes in these taxa. As above under (i), their original diversification may have been driven by diversifying selection. Indeed, two episodes of adaptive sequence evolution were found to associate with these genes (Figs. 4A, 8A).
Phylogenetic inferences can only yield an approximation of the past and thus come with some uncertainty. Considering that the inferred relationships are generally supported by high bootstrap values and that they are based on the maximum likelihood approach, which was shown in the past to be less susceptible to biases (e.g. long-branch attraction) than other tree reconstruction methods , our results should provide a realistic image of Caenorhabditis lysozyme evolution. Taken together, their lysozyme repertoire is shaped by both ancestral and recent gene duplications. Sequence evolution is to a large extent determined by purifying selection. Yet, it also includes several episodes of diversifying selection, which associate with ancient as well as recent duplications. To our knowledge, similar evolutionary dynamics have not as yet been inferred for the lysozymes from other taxa.
It is worth noting that we did not find an indication for adaptive sequence evolution between the two main protist-type clades (Fig. 6, Table 4). Two explanations are conceivable. On the one hand, differentiation of the two clades was not subject to diversifying selection. On the other hand, diversifying selection was important but could not be detected due to a lack of power of the analysis, which had to be based on a reduced data set including only the conserved sequence regions that could be reliably aligned across the different genes and taxa. At the same time, this specific result (as well as all other cases of comparatively long branches with d N /d S rate ratios below 1) strongly suggests that our analysis is not compromised by a possible saturation of synonymous substitutions along long branches, which could have led to underestimated d S rates and thus artificially high d N /d S rate ratios. It is also worth noting that only a single alignment site was inferred to be under positive selection in our analyses. This is unusual because in most immunity gene data sets associated with adaptive sequence evolution a larger number of positively selected sites is identified, e.g. in MHC class I receptors [30, 31]. A possible reason is that the different evolutionary lineages vary as to the position of the positively selected sites or that only few lineages are subject to positive selection on specific sites. In both cases, the method employed would hinder detection of these positively selected sites because it assumes the same pattern of selection across all lineages . We did not attempt to perform an analysis, in which d N /d S ratios are allowed to vary simultaneously across sites and lineages, because these types of analyses may be liable to higher error rates [32, 33]. The single site, which we identified to be under positive selection, is thus predicted to be of main – albeit currently unknown – functional importance.
Gene duplications are likely to be one of the main sources of evolutionary innovation . The duplicated genes may acquire new functions (neo-functionalisation) or they may partition the multiple functions of the ancestral gene (sub-functionalisation) . The relevance of either alternative as well as additional scenarios is a topic of intense current debate [34–38]. Importantly, in all cases the genetic diversity of duplicates is predicted to translate into functional diversity. Such a pattern is found in the ruminantia, which possess at least five different lysozyme types: the stomach, tracheal, intestinal, kidney, and milk lysozymes . The first type is involved in digestion, whereas the others may function as antibacterial enzymes in immunity . A similar pattern is observed for the at least eleven different Drosophila lysozymes. Most of them have a digestive role and show specialisation as to their time and site of expression [5, 14]. A recent study additionally suggested an anti-fungal immune function for some of the genes (Lys B, C, D, E and CG16756) . A further example includes the nine lysozymes of the mosquito Anopheles gambiae, which vary as to their role in immunity and digestion and also as to their time and location of expression .
The Caenorhabditis lysozymes show clear signatures of functional diversification. Pronounced differences between the three main clades and also within each of the clades are observed for molecular characteristics of the genes, their pathogen-induced expression, and also their regulation by the immune system. Based on the current data, it appears that the protist-type clade 1 lysozymes play an important role in immunity: They are all induced upon pathogen exposure. Most of them are under positive control of immunity pathways, including components of the insulin-like signalling cascade (DAF-16) [40–42], the p38 mitogen-activated protein kinase (MAPK) pathway (SEK-1 and PMK-1) , the TGF-β pathway (DBL-1, SMA-2) , or the GATA transcription factor ELT-2 . Most interestingly, the different genes from this clade vary in their response to pathogens and immunity pathways. This variation may contribute to high immune specificity, as has recently been identified phenomenologically for invertebrates [46–48] and which is consistent with highly specific C. elegans-pathogen interactions . Although the underlying molecular mechanisms are currently unknown, they are likely to be based on the genetic diversification of pathogen recognition receptors and/or immune effectors such as the lysozymes [21, 50, 51]. They may also include the synergistic interaction between different components of the immune system , as generally known for lysozymes and antimicrobial peptides [5, 7, 52, 53]. In C. elegans, the immune function has been tested for two genes of the clade 1 protist-type lysozymes. Overexpression of Cel-lys-1 enhances resistance against S. marcescens , whereas silencing of Cel-lys-7 increases susceptibility to M. nematophilum . The importance of lysozyme diversification for immunity in general and also for immune specificity clearly warrants further investigation.
The role of the invertebrate-type and also the clade 2 protist-type lysozymes is as yet unclear. The only exception may be Cel-ilys-3. Its silencing enhances susceptibility to M. nematophilum . In the same study, no effect was observed after Cel-ilys-2 knock-down . In general, both invertebrate-type and clade 2 protist-type lysozymes are less often activated by pathogens than the clade 1 protist-type lysozymes. At the same time, several of the genes are downregulated by pathogens and by known immunity pathways. The latter observation may suggest that their main function somehow interferes with the immune response. A similar finding was made for some of the digestive lysozymes from D. melanogaster, which are also downregulated upon immune challenge . This particular similarity may indicate that the primary function of these nematode lysozymes is also digestion. The information on their molecular characteristics (e.g. isoelectric point) or the localization of gene expression is consistent with a role in both immunity and digestion. Unfortunately, the nematode's intestines are the main location for bacterial digestion and at the same time immune defence against pathogens that are easily taken up during feeding . Therefore, lysozymes are expected to have similar characteristics (e.g. regarding pH optimum) even if they vary in function. Future analyses should thus be performed with either exclusive food bacteria or exclusive pathogens, in order to distinguish between the alternative functions.
Our study provides an evolutionary framework for understanding lysozyme diversification in Caenorhabditis nematodes. The comprehensive lysozyme repertoire falls into three distinct clades and it is shaped by both purifying selection and several episodes of adaptive sequence evolution. The genetic diversification appears to translate into functional differentiation. The information obtained should prove useful as a primer for future analysis of lysozyme function in digestion and immunity. The Caenorhabditis lysozymes may further serve as an example of the importance of evolution by gene duplication in invertebrate immune systems.
For the three considered Caenorhabditis species, protein and DNA sequences of annotated genes with similarities to known lysozymes were obtained from wormbase . Three main alignments were generated (alignments 1, 2, and 4; see below). The first one of these, alignment 1, served to infer the general phylogenetic relationship of the C. elegans lysozymes to those from other taxa. We specifically considered taxa, which were included in similar lysozyme phylogenetic analyses in the past [8, 56, 57], thus allowing comparison between our results and those from previous studies. The alignment was produced with the hierarchical method, implemented in the programme CLUSTALW  and using the default settings. The resulting alignment contained substantial sequence variation. Moreover, variations of the programme settings (gap open, gap extension, and gap distance penalties) resulted in differences among generated alignments. Therefore, positional homology across alignment 1 may not be entirely reliable. Since it is based on the hierarchical alignment method (i.e. similar sequences are aligned first, followed by subsequent addition of less similar sequences), it should still be informative as to the general phylogenetic position of the Caenorhabditis lysozymes in comparison to those from other taxa.
The more detailed analysis of Caenorhabditis lysozyme evolution was based on the main alignments 2 and 4. Six additional alignments were extracted from these two alignments (see below, alignments 3, 5–9). In particular, alignments 2 and 3 served to analyse the invertebrate-type lysozymes. The overall phylogeny of the protist-type lysozymes from nematodes and one outgroup taxon was examined with alignment 4. Five additional alignments were extracted from alignment 4 for the detailed analysis of lysozyme evolution (alignments 5–9). Here, we excluded highly variable sequence regions from alignments, if these could not be recovered in identical form under alternative settings of the alignment programme (only relevant for alignments 5–7), in order to ensure a high likelihood of positional homology and thus a reduced risk of homoplasy. Alignments 4–9 are subsets of each other with identical position of indels as indicated in Fig. 5.
(i) Alignment 1 included protein sequences of all lysozyme genes from C. elegans but none of the other Caenorhabditis species. The C. elegans genes were combined with lysozymes from various taxa that have previously been considered in similar phylogenetic analyses [8, 56, 57], including the chicken-type lysozyme from chicken (Gallus gallus, accession number CAA23711), one of the chicken-type lysozymes from mice (Mus musculus, AAA39473), the goose-type lysozyme from chicken (NP_001001470), two chicken-type and one invertebrate-type lysozymes from D. melanogaster (NP_523882 [previously AAF47448], NP_476828 [previously AAF47452], and CAA21317), one chicken-type and one invertebrate-type lysozyme from the mosquito Anopheles gambiae (AAC47326, AAT51799), the invertebrate-type lysozymes from the cestode Tapes japonica (BAB33389), the molluscs Mytilus edulis (AAN16207), Chlamys islandica (CAB63451), and Calyptogena sp. 1 (AF334666), the leech Hirudo medicinalis (AAA96144) and the sea star Asterias rubens (AAR29291), and four protist-type lysozymes from Dictyestelium discoideum (XP_644284, AAM08434, AAB06786, XP_643993), two from Entamoeba histolytica (AAC67235, Q27650), and one from Tetrahymena thermophila (XP_001008528), and one lysozyme from a T4 entobacteria phage (1LYD).
(ii) Alignment 2 contained protein sequences of all invertebrate-type lysozymes from the three Caenorhabditis species (Fig. 3).
(iii) Alignment 3 is the DNA version of alignment 2.
(iv) Alignment 4 has all protein sequences of the Caenorhabditis protist-type lysozymes and also one from D. discoideum (XP_644284).
(v) Alignment 5 is the modified DNA version of alignment 4. Here, we excluded the taxon D. discoideum and additionally one 5' and one 3' end region, which could not be aligned reliably at the DNA sequence level. The excluded region at the 5' end corresponds to positions 1 to 149 and that at the 3' end to positions 301 to 345 of the protein sequence alignment (see Fig. 5).
(vi) Alignment 6 represents a subset of alignment 4. It contains the Caenorhabditis protein sequences of the clade 1 protist-type lysozymes, whereby we excluded a fragment at the 5' end (positions 1 to 67; Fig. 5), another fragment towards the 3' end (positions 314 to 331; Fig. 5), and a small region at the 3' end (positions 339 to 345; Fig. 5).
(vii) Alignment 7 is the DNA version of alignment 6.
(viii) Alignment 8 is again a subset of alignment 4. It includes all complete Caenorhabditis protein sequences of the clade 2 protist-type lysozymes (Fig. 5).
(ix) Alignment 9 is the DNA version of alignment 8.
General properties of the different lysozymes were inferred with the help of the ProtParam tool of the ExPASy server [59, 60], including protein length, molecular weight, isoelectric point (pI), charge, and also the grand average of hydrophobicity. Differences in these traits between lysozyme clades were assessed with an analysis of variance (ANOVA) and posthoc Tukey-Kramer comparisons, using the program JMP IN 5.1.2 (SAS Institute Inc.). The presence and position of a signal peptide was inferred with the SIGNALP 3.0 server . Further information on the genomic location, the function and also regulation of the C. elegans lysozymes were taken from wormbase  and the current literature.
Phylogenetic tree inference
Phylogenies were reconstructed using the maximum likelihood (ML) optimality criterium . For protein sequence alignments, the optimal substitution model was first inferred using the program ProtTest version 1.3  and the Akaike information criterion, following the recommended approach [63, 64]. The optimal substitution model was employed for a heuristic tree search with the help of the program PhyML [65, 66] using default settings. The robustness of the inferred tree topology was evaluated via non-parametric bootstrapping  based on 200 replicate data sets.
For DNA sequence alignments, the optimal substitution model was found using the same strategy as above and as implemented in the program ModelTest version 3.7 [63, 64, 68]. The phylogenetic tree was then inferred with the help of the ML option of the program PAUP* 4.0b10 , using the optimal substitution model, a heuristic tree search based on branch-swapping by tree bisection and reconnection (TBR), the random addition of sequences, which was repeated ten times, and otherwise default settings. The robustness of the tree topology was assessed with non-parametric bootstrapping using 500 replicates.
Analysis of adaptive sequence evolution
The presence of positive selection (i.e. a d N /d S rate ratio larger than 1) along branches of the different tree topologies or along the sequence alignments was assessed using the ML approach implemented in the program CODEML of the PAML package version 3.15 . DNA sequences of the coding regions were used as input data files (alignments 3, 5, 7, and 9) and the inferred unrooted ML tree topologies as input tree files. Positive selection along sequence alignments was inferred following recommendations [71, 72]. In particular, likelihood ratio tests (LRT) were used to compare the NS sites model 8 (8 rate categories across sequences, one of which was allowed to have a d N /d S rate ratio larger than 1) with either NS sites model 7 (8 rate categories, none above 1) or NS sites model 8a (8 rate categories, whereby one was set to exactly 1). The significance of the comparison between models 8 and 8a was assessed by dividing the inferred LRT probability by 2, as recommended previously . If both comparisons were significant, then individual sites under positive selection were identified using the Bayes empirical Bayes method .
The presence of positive selection along branches was assessed using two approaches. On the one hand, we compared a model, in which all branches were forced to have the same d N /d S rate ratio (1-ratio model), with a model, in which one branch was allowed to differ whereas all others were forced to be identical (2-ratio model). Using this approach, we tested each individual branch of a given tree topology. The significance of the comparison was evaluated with a likelihood ratio test . We corrected for multiple testing by adjusting the significance level according to Bonferroni  and the false discovery rate (FDR) [74, 75]. If a particular comparison was significant and if the individual branch, which was allowed to differ, had a d N /d S rate ratio larger than 1, then this was taken as an indication for positive selection along this branch.
On the other hand, we assessed the significance of positive selection along branches using non-parametric bootstrapping. In particular, we first used the free-ratio model, in which all branches were allowed to vary, in order to calculate d N /d S rate ratios for each individual branch of the topology. The significance of a value either above or below 1 was tested by repeating the calculations on 100 bootstrapped data sets. Non-parametric bootstrapping of the data was performed in consideration of the coding structure of the genes using the program CODEML of the PAML package . If a specific branch had a d N /d S rate ratio larger 1 and bootstrap support of more than 50, then this was taken as an indication of positive selection.
We are grateful to the members of the Department of Animal Evolutionary Ecology in Tübingen for helpful discussions, the German Science Foundation for financial support (grant SCHU 1415/3), the Wissenschaftskolleg zu Berlin for a fellowship to HS, and three anonymous reviewers for valuable comments on the manuscript.
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