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Proc Natl Acad Sci U S A. May 13, 2003; 100(10): 5885–5890.
Published online Apr 29, 2003. doi:  10.1073/pnas.1037686100
PMCID: PMC156296
Evolution

The loss of the hemoglobin H2S-binding function in annelids from sulfide-free habitats reveals molecular adaptation driven by Darwinian positive selection

Abstract

The hemoglobin of the deep-sea hydrothermal vent vestimentiferan Riftia pachyptila (annelid) is able to bind toxic hydrogen sulfide (H2S) to free cysteine residues and to transport it to fuel endosymbiotic sulfide-oxidising bacteria. The cysteine residues are conserved key amino acids in annelid globins living in sulfide-rich environments, but are absent in annelid globins from sulfide-free environments. Synonymous and nonsynonymous substitution analysis from two different sets of orthologous annelid globin genes from sulfide rich and sulfide free environments have been performed to understand how the sulfide-binding function of hemoglobin appeared and has been maintained during the course of evolution. This study reveals that the sites occupied by free-cysteine residues in annelids living in sulfide-rich environments and occupied by other amino acids in annelids from sulfide-free environments, have undergone positive selection in annelids from sulfide-free environments. We assumed that the high reactivity of cysteine residues became a disadvantage when H2S disappeared because free cysteines without their natural ligand had the capacity to interact with other blood components, disturb homeostasis, reduce fitness and thus could have been counterselected. To our knowledge, we pointed out for the first time a case of function loss driven by molecular adaptation rather than genetic drift. If constraint relaxation (H2S disappearance) led to the loss of the sulfide-binding function in modern annelids from sulfide-free environments, our work suggests that adaptation to sulfide-rich environments is a plesiomorphic feature, and thus that the annelid ancestor could have emerged in a sulfide-rich environment.

Keywords: sulfide binding function‖free cysteine‖annelid evolution‖ loss of function

Emergence of new functions in proteins as a result of a high evolutionary rate after gene duplication has been long debated in the molecular evolution field. From the neutralist standpoint, molecular evolution occurs by random drift of mutations that are nearly equivalent selectively. In this context, “it is much more likely, if high rates occur, that they are caused by the removal of a preexisting functional constraint, allowing previously harmful mutants to become selectively neutral” (1). For “selectionist,” high evolutionary rates are considered rather as the result of an acceleration of mutations called positive Darwinian selection, the likely evolutionary force for the acquisition of new functions after a duplication event (2). According to Ohta's consensual theory (3), positive Darwinian selection is needed for the accumulation of favorable mutations that provide a new function or a modified function to a (duplicated) gene, whereas a gene whose function has been fixed for a long time evolves mostly through random genetic drift. However, molecular adaptation and emergence of a new function driven by positive Darwinian selection is not always associated with duplication events. Transition from homogeneous to heterogeneous habitats could also play a role in the evolution of an original specific function (its disappearance or its maintenance). This could occur via diversifying selection when the ancestral polymorphism linked to this function is subdivided between habitats (4). This can lead to the observation of highly divergent variants regarding specific amino acid signatures and can be viewed as a positive Darwinian selection event acting on species that have emerged from this habitat speciation. The estimation of the fixation rate of nonsynonymous and synonymous substitutions along orthologous coding sequences from a cluster of evolutionarily related taxa appears to be one of the most powerful tools to detect molecular adaptation (59). However, the signature of molecular adaptation can be cryptic and difficult to extract because an adaptive change (advantageous mutation) may only affect a small number of lineages and only a subset of sites according to their phylogenetic history (10). The accumulation of ancient adaptive mutations is typically the situation encountered in globins, a widespread molecule that is conserved in members of all living kingdoms, including annelids.

The spatial and environmental distribution of annelids, from deep-sea hydrothermal vents to terrestrial habitats, is the consequence of a long history of adaptive strategies since their radiation (11). One of these adaptations concerns the way by which annelids living in sulfide-rich environments protect themselves against or use hydrogen sulfide (H2S). Such a process mainly relies on the occurrence of extracellular hemoglobins that bind and transport this toxic compound. H2S is toxic to aerobic metabolism, particularly to metalloproteins such as cytochrome c oxidase and hemoglobin (12). This unusual sulfide-binding function of some annelid hemoglobins was first discovered in the vestimentiferan Riftia pachyptila, a mouthless and gutless organism harboring intracellular chemolithoautotrophic sulfide-oxidizing bacterial symbionts. Riftia is found living close to the deep-sea eastern Pacific hydrothermal vents (13). Sulfide binding is enabled by the presence of two highly reactive free cysteine residues that covalently bind H2S (14, 15), each one localized on two different globin subunits included in extracellular hemoglobin complexes found in annelids (see review in ref. 16). The annelid hemoglobin multigenic family is subdivided into two main gene families, A and B, and four subfamilies. A1, A2, B1, and B2, that emerged via at least three duplication events (17, 18). These latter authors found that the free cysteine residues involved in H2S binding are located at the same positions, Cys + 1 and Cys + 11 (1 and 11 aa after the well conserved distal histidine), on globin chains within the B2 and A2 subfamilies respectively for a set of various annelids living in sulfide-rich habitats. Moreover, other nonsymbiotic annelid polychaetes living in sulfide-rich habitats such as Alvinella pompejana and Arenicola marina also possess hemoglobins that display a H2S-binding capability via free cysteines residues (19, 20, ) for which positions are unknown (no sequence available).

Many habitats that display high sulfide concentrations are known to occur on the Earth's surface. Environmental sulfide may have either geothermal (hydrothermal vents, sulfurous springs) or biogenic (cold seeps, marine sediments, mangroves) origins, including anthropogenic deposits in polluted marine or brackish areas. We postulated that species from sulfide-rich environment exhibiting free cysteine residues at positions Cys + 1 and Cys + 11 are able to bind sulfide by analogy to the mechanism used by both of the vestimentiferans Lamellibrachia sp. and R. pachyptila. Such H2S-binding function appears to be absent in annelids from sulfide-free environments such as the oligochaete Lumbricus terrestris (earthworm) and the polychaete Tylorrhynchus heterochaetus which lack these residues. The inability of Lumbricus terrestris hemoglobin to bind sulfide was confirmed by Zal et al. (15) using specific cysteine inhibitors. It was found that H2S-binding A2 and B2 globins exhibit a lower evolutionary rate than the O2-binding A1 and B1 globins, which do not possess free cysteines (18). Such evolutionary rates suggest that A2 and B2 globins and their H2S-binding function are strongly selected. As a consequence, the authors proposed an evolutionary scenario regarding the evolution of the hemoglobin H2S-binding function in symbiotic and nonsymbiotic annelids living in sulfide-rich habitats and suggested that the H2S-binding function via a free cysteine residue was (i) an innovation in Phylum Annelida and (ii) lost by the relaxation of selective constraint (neutral evolution) in the annelid ancestors that colonized the newly emerging sulfide-free habitats.

Starting with these assumptions, we focused our attention here on the A2 and B2 homologous free cysteine sites that are located in a well-conserved secondary structure region called the sulfide-binding domain (SBD) (18). Recent maximum likelihood models of synonymous and nonsynonymous substitution estimation called the branch site specific models (22) were used to investigate the functional evolution of free cysteines in annelids from sulfide-rich and sulfide-free habitats.

We present here a case of molecular adaptation (replacement of the free cysteines) due to the relaxation of selective constraints (decline in H2S concentrations). In other words, the loss of a function (H2S binding function in sulfide-free habitats) can be also driven by positive Darwinian selection. The dramatic changes of environmental conditions during the course of evolution and the associated physiological modifications in annelids from well oxygenated emerging habitats are pointed out to explain such a loss of function by molecular adaptation.

Materials and Methods

Biological Materials.

Juvenile specimens of Lamellibrachia nov sp. were collected around cold-seeps from mud volcanoes in the Mediterranean Sea during the French oceanographic cruise Medinaut (Kazan site: 35°25.88′ N, 24°33.56′ E) at a depth of ≈1,705 m. Juvenile specimens of the hydrothermal-vent tubeworms Riftia pachyptila, Oasisia alvinae, and Tevnia jerichonana (up to 3–5 cm length) were collected at one single vent site from the ridge segment 9°50′N on the East Pacific Rise (Riftia field: 9°50.75′ N, 104°17.57′ W) at a depth of 2,500 m during the French oceanographic cruise HOT 96 and the American cruise LARVE98. Hydrothermal vent tubeworms Ridgeia piscesae were collected at the Endeavour Segment of the Juan de Fuca Ridge (Clam Bed, 47° 57′ N, 129° 05′ W) at a depth of 2197 m during the Canadian–American Hi-Rise cruise 2001. Worms were sampled by using the telemanipulated arms of the submersibles “Nautile,” “Alvin,” and “ROPOS,” brought back alive to the surface inside an insulated box, and immediately frozen and stored in liquid nitrogen after their recovery on board.

Identification and Characterization of Novel Extracellular Globins.

In the present paper, globin primer design, total RNA extraction, cDNA synthesis, RT-PCR amplification, PCR-product cloning, and sequencing were performed as described in Bailly et al. (18). This protocol was applied to the three hydrothermal vent annelid species; (encircled in Fig. Fig.1)1) Ridgeia piscesae (Ridg), Oasisia alvinae (Oas), and Tevnia jerichonana (Tevnia), and to the Mediterranean cold seep annelid species Lamellibrachia nov sp. (LaM) (see Fig. Fig.1).1).

Figure 1
Neighbor-joining consensus tree of globin sequences from annelids living in sulfide-rich habitats with percentage bootstrap support (1,000 replicates). Rooting is performed according to the “duplicate rooting procedure” (21) using clade ...

Multiple Alignments and Tree Reconstructions.

Unrooted tree topologies from multiple alignments of the A2 and B2 amino acid globin sequences were obtained by using the neighbor joining method computed by using phylip program (23) with 1,000 bootstrap resamplings of the data (Fig. (Fig.2).2).

Figure 2
Orthologous globin A2 and B2 topologies from nucleotide sequences of Riftia pachyptila (Riftia), Sabella spallanzanii (Sabspal), Sabellastarte indica (Sabindica), Lamellibrachia nov sp. (LaM), Tevnia jerichonana (Tevnia), Oasisia alvinae (Oasisia), Ridgeia ...

The orthologous A2 globin set (Fig. 3, which is published as supporting information on the PNAS web site, www.pnas.org) was composed of 213 bp of DNA sequence (71 codons) from the terrestrial (sulfide-free) oligochaete Lumbricus rubellus (GenBank accession no. BF422675), the littoral (transitory sulfide-rich) polychaete Sabella spallanzanii (GenBank accession no. AJ131285), the Mediterranean cold-seep (sulfide-rich) vestimentiferan Lamellibrachia nov sp. (GenBank accession no. AY250084) and the three East Pacific Rise hydrothermal vent (sulfide-rich) vestimentiferans Oasisia alvinae (GenBank accession no. AY250087), Tevnia jerichonana (GenBank accession no. AY250086) and Riftia pachyptila (GenBank accession no. AJ439733). The orthologous B2 globin set (Fig. 4, which is published as supporting information on the PNAS web site) was composed of 213 bp of DNA sequence (71 codons) from Lumbricus rubellus (GenBank accession no. BF422540), the two littoral polychaetes Sabella spallanzanii (GenBank accession no. AJ131283) and Sabellastarte indica (GenBank accession no. D58418), the Mediterranean cold-seep vestimentiferan Lamellibrachia nov sp. (GenBank accession no. AY250085), the East Pacific Rise hydrothermal vent vestimentiferans Riftia pachyptila (GenBank accession no. AJ439737) and Juan de Fuca Ridge Ridgeia piscesae (GenBank accession no. AY250083).

The secondary structure of the molecular domain (SBD) surrounding the free cysteine residues involved in the sulfide-binding function was predicted by using a hydrophobic cluster analysis (HCA) from amino acid globin sequences and plotted according to the drawhca software (24). These plots were of prime importance to deduce the level of conservation of the sulfide-binding domain between the globin subfamilies (Fig. (Fig.11).

Search for Darwinian Positive Selection from dN/dS Ratios.

To detect Darwinian positive selection acting on extracellular globins, an approach based on the maximum likelihood estimation of the nonsynonymous/synonymous substitution rate ratio (dN/dS = ω; ref. 25) was applied by using our sets of coding sequences. The ratio ω provides a sensitive measure of selective pressures acting at the protein level, with ω values of <1, = 1, and >1 indicating negative selection, neutral evolution, and positive Darwinian selection, respectively.

The branch site-specific models (22) were considered to conduct selection pressure analysis for both the A2 and B2 globin subfamilies. These models use maximum likelihood methods for estimating the parameters of a transition matrix describing the substitution rate between pairs of codons, including dN/dS ratios (ω), transition/transversion ratios and branch lengths. We compared nested models (a null and an alternative hypothesis) with the likelihood ratio test (LRT) following the formula: 2δL = 2(L1 − L0), where L1 is the alternative hypothesis and L0 is the null hypothesis. The log likelihood difference between the two models is expected to be χ2 distributed with the number of degrees of freedom equal to the difference in the number of parameters between the models. The branch site-specific model is the combination of a lineage-specific model (5) and a site-specific model (26). This program provides an interesting tool to test for positive selection at each amino acid site within a prespecified lineage of the phylogeny (foreground branch) as opposed to the rest of the lineages (background branches). In other words, these models allow testing the assumption that some orthologous amino acid sites have undergone positive selection only in some evolutionary strains of a given phylogeny. These models, called Bm2 and Bm3, can be respectively compared for LRT with some site-specific models M1 and M3 (26). The M1 model only assumes two sites in which ω0 = 0 (any mutation is deleterious at a given site) and ω1 = 1 (any mutation is neutral at a given site) and for which the proportions p0 and p1 could be estimated over the whole protein. The model M3 uses a general discrete distribution of the ω ratios among sites with two site classes for which the proportions p0 and p1 are estimated. For branch site-specific models the Bayes theorem is (automatically) used to calculate posterior probabilities of site classes for each site. Sites displaying ω > 1 and a high posterior probability can be suspected to be under positive selection. All analyses were performed by using the codeml program of the paml package (27).

Results

New Globin Sequences from Hydrothermal Vent and Cold Seep Species.

Five partial sequences of both the A2 and B2 globins have been obtained for Tevnia jerichonana (A2: 88 aa), Oasisia alvinae (A2: 76 aa and B2: 53 aa) and Ridgeia piscesae (B2: 71 aa). Globin assignment was based on sequence homology, specific amino acid patterns and the presence of free cysteine residues Cys + 1 and Cys + 11 (18) without ambiguity. A complete set of the globin subfamilies A1, A2, B1, and B2 was also sequenced from Lamellibrachia nov sp. (see Appendix). As above, globin sequences were unambiguously assigned to the right paralogous subfamily by the reconstruction of molecular phylogenies in which the well-defined globin subfamilies of R. pachyptila and Lamellibrachia sp. from Japan (14, 18, 28) were inserted (Fig. (Fig.1).1). All of the A2 and B2 globins of vestimentiferan tubeworms displayed free cysteine residues at positions Cys + 11 and Cys + 1, respectively, and similar SBD amino acid patterns.

A2 and B2 Globin Subfamilies dN/dS Ratio Analyses.

The dN/dS ratio analyses were performed solely on the two globin subfamilies A2 and B2 because they are both involved in the sulfide-binding function via the free cysteine residues in position Cys + 11 and Cys + 1 in annelids living in sulfide-rich habitats. All model parameters (fixed or estimated), likelihood ratio tests and the putative positively selected sites are reported in Table Table1.1.

Table 1
Likelihood values (L), parameter estimates, and LTR obtained for the branch-site models

Results obtained from the Bm2 and Bm3 models indicate that both the A2 and B2 globin subfamilies of the Lumbricus rubellus (foreground) lineage, representing annelids from terrestrial sulfide-free habitats, had undergone selection in a different way than lineages representing annelids living in various sulfide-rich habitats (background; see Table Table11 and Fig. Fig.2).2). The models Bm2 and Bm3 provide significantly better likelihood values than site specific models M1 and M3, and they have detected positively selected sites in the foreground lineage. It is worth noting that a part of the positively selected sites fall into the SBD for the Lumbricus B2 and A2 globin subfamilies, respectively (Table (Table1).1). The Bm3 model detected more positively selected sites than the Bm2 model for both A2 and B2 globins, suggesting that this former model is more appropriate to detect positive selection. In both models and both subfamilies, the key sites for H2S-binding (i.e., the free cysteine 32C in B2 and 42C in A2 from Table Table1,1, respectively positions Cys + 1 and Cys + 11) are subjected to positive selection with a posterior probability of 99% in A2 (with the Bm3 model) and B2 (with the Bm2 and Bm3 models) and 88% in A2 (with the Bm2 model). Other analyses using Bm2 and Bm3 with different fixed foreground lineages were also performed, but they yielded low and insignificant likelihood scores and no positively selected amino acid residues within the SBD (data not shown).

Discussion

Sulfide-Binding Function: A Widespread Function in Annelids Living in Sulfide-Rich Habitats That May Have Been Lost in Annelids from Sulfide-Free Habitats.

Bailly et al. (18) suspected that the A2 and B2 globin subfamilies of the hemoglobin of annelids have undergone strong directional selective constraints driven by high levels of H2S concentrations in taxa living in sulfide-rich habitats. This would explain the maintenance of the free cysteine residues at the conserved positions Cys + 1 and Cys + 11, and the maintenance of a conserved SBD secondary structure in two sets of highly divergent paralogous strains (A2 and B2). The presence of homologous free cysteine residues at positions Cys + 1 and Cys + 11 exclusively found in A2 and B2 globins from the Mediterranean cold seep Lamellibrachia nov sp., and the eastern Pacific hydrothermal vent Oasisia alvinae, Tevnia jerichonana, and Ridgeia piscesae demonstrate the widespread occurrence and the conservation of these residues in vestimentiferans. Moreover, some globins of nonsymbiotic polychaetes, such as Sabellastarte indica (29) and Sabella spallenzani (30), and the Branchipolynoe sp. (31) also exhibit such free cysteines in the same position. Bailly et al. (18) proposed that the occurrence of free cysteines at positions Cys + 1 and Cys + 11 is a plesiomorphic state already present in the annelid ancestor, rather than an apomorphic one, occasionally acquired before annelid radiation from sulfur-rich environment. The absence of free cysteine residues in globins from polychaetes living in free-sulfur environment such as Tylorrhynchus heterochaetus (32) or oligochaetes such as Lumbricus terrestris (33) has been interpreted as a loss. Despite the absence of the free cysteine residues in the latter species, the nonfunctional SBD is still more or less conserved in all globin subfamilies with an obvious degenerated signature (18). Such a conserved structure in common paralogous globins, which emerged before radiation of annelids, reinforces the assumption of a loss of the free cysteines as well as the inference that the SBD, and therefore its original function, represents the primitive condition in annelid hemoglobins. These molecular data form a set of insights that allow us to assume that annelid ancestors initially lived in sulfide-rich conditions. Another argument in favor of the loss of free cysteine residues and which sustains the previous postulate is that adaptation to sulfide did not only require cysteine acquisition in respiratory pigments but implied the establishment of a long biochemical pathway by which animals also acquired various adaptive physiological detoxification mechanisms, at the levels of molecules, cells, tissues, and up to the whole organism. It is unlikely that this complicated metabolic process was the result of a preadapted pathway that abruptly shifted to sulfide utilization from using a molecule that possessed similar detoxifying and energy carrier potentials for the purpose of oxidizing a different metabolic compound. One must also keep in mind that the symbiotic worms from sulfidic environments use H2S to fuel their endosymbionts, a situation not encountered in nonsymbiotic worms from similar habitats. It is likely that a detoxification function in nonsymbiotic worms (a plesiomorphic trait) could have evolved in symbiotic worms into a H2S transport function (an apomorphic trait). This means that the H2S-binding capacity is an intricate mechanism that acts differentially according to the location of the cysteines and that the heterotrophic H2S-binding pathway which has preceded the symbiotic H2S-binding pathway was probably present early in annelid evolution, even before annelid radiation.

It is thus more reasonable to support the hypothesis of the loss of sulfide-binding function in nonsymbiotic worms in sulfide-free environments rather than a repeated gain of this function in different sulfur-rich environments in various annelid lineages.

Seeking for Positively Selected Sites in Well Conserved Orthologous Sequences.

Hemoglobin is an ubiquitous molecule found “from bacteria to man” (34, 35) for which the three-dimensional structure (globin fold) (36) is well conserved among highly divergent evolutionary lineages. This structural universality demonstrates that globin is a strongly selected molecule, due in part to its ability to bind and transport oxygen. Despite their great evolutionary distance in terms of common ancestry (two duplication events), the A2 and B2 subunits share an obvious secondary structure conservation of the SBD and the maintenance of functional free cysteines in annelids living in sulfide-rich conditions, two noteworthy insights of a functional coevolution between these duplicated genes. Thus, it is more appropriate to search for selected sites in such a well conserved structure than to try to detect cryptic adaptive evolution over the whole set of globin sequences. Historically two programs were performed to detect positive selection: the lineage-specific program, which averages the dN/dS ratio over all sites of a given coding sequence (5), and site-specific models, which average the dN/dS ratio for each homologous site over all coding sequences (26). But because they average either all sites or all lineages they are not sensitive enough to detect selective pressures at a specific amino acid of a given lineage. This lack of power was already pointed out by in ref. 10, when episodic positive selection has occurred only on a few amino acids of a strongly negatively selected molecule. The absence of apparent positive selection in the A2 and B2 globin strains by using these two programs (data not shown) may reflect either that these globins are highly negatively selected even in sulfide-free lineages or that the accumulation of positively selected amino acid sites occurred in ancient times before the annelid radiation, leading to this unusual, complex, extracellular hemoglobin. This may explain why most examples of positive selection come from xenobiotic recognition molecules or genes associated with male reproduction that have undergone recent adaptive changes such as primate lyzozymes (5, 37), HIV membrane proteins (6, 7) or the salmon gamete recognition system (9).

Positively Selected Sites in Sulfide-Free Lineages: Why the Loss of a Function Could Also Be Considered as a Molecular Adaptation Rather than a Relaxation of Constraints (Neutral Evolution).

Bailly et al. (18) proposed a possible relaxation of selective constraints on the annelid globins from sulfide-free environments to explain the absence of free cysteine residues on the A2 and B2 subunits. H2S was one of the most abundant molecules on the Earth's surface during the prebiotic era and its concentration greatly decreased during the course of evolution, remaining only at some specific places (e.g., hydrothermal vents) as a relic of the primitive conditions (38, 39). Thus, the acquisition and the maintenance of H2S binding functions were of a prime importance during the beginning of life and were probably subjected to purifying selection in the ancient annelid lineages living in sulfide-rich habitats. To test whether the SBD and especially the free cysteine residues in globins from annelids in sulfide-free environments were subjected to ancient diversifying selection, we used the recent branch-site set of models implemented in paml (22). These more intricate and realistic models simultaneously allow a combination of different ω ratios among sites and lineages. These models relies on a priori evolutionary hypotheses within a prespecified lineage that is suspected of a specific history: in our case, the hypothetical loss (versus the gain) of free cysteine residues in sulfide-free lineages was combined with the way that they were lost during evolution (neutral relaxation or Darwinian positive selection). For both the A2 and B2 globins, the significantly lowest likelihood values were obtained when we assumed a different dN/dS ratio in the sulfide-free lineages as opposed to lineages from the other habitats (hydrothermal vents, cold-seeps, coastal or intertidal hypoxic sediments). In only this case (all branch combinations have been tested; results not shown), the sites Cys + 1 and Cys + 11 were clearly subjected to positive selection with a strong posterior probability together with other residues of the SBD. These positively selected sites, found only within the SBD, are in agreement with the loss or the degeneracy of the hydrophobic secondary structure surrounding the free cysteine residues in annelids from terrestrial sulfide-free habitats (18).

The high posterior probabilities obtained for both the Cys + 1 and Cys + 11 sites are associated with a dN/dS >1. This result rules out the assumption that the loss of the free cysteine residues have followed a neutral pattern of evolution. Free cysteine residues are key amino acids in biochemical reactions because of their well-known highly reactive lateral chain. To our knowledge, all free cysteines reported from protein analysis display an active and diversified functional role. These residues are involved in the binding of some catalytic cofactors (40), pH-dependent oligomerization (41), macromolecular complexation, and many other molecular detoxifying functions. However, in the case of specific genetic diseases or metabolic disorders, free cysteines can bind atypical ligands. Examples of such ligands include benzoquinone, a known carcinogenic metabolite, in rodent globin (42), mercury ions inhibiting enzymatic reactions (43), or oligomeric complexation by abnormal aggregation with subsequent physiological modification in mice mutant hemoglobin (44). These three examples among others illustrate the deleterious role of unexpected free cysteines in mutant organisms and indicate that the disappearance of a specific ligand (e.g., H2S) can drastically reduce organismal fitness when conditions have changed.

We assumed that when sulfide concentrations decreased or when annelid ancestors moved from sulfide-rich to sulfide-free habitats, the presence of free cysteine residues could have induced irreversible deleterious effects and homeostatic disorders. Thus, it is not surprising to find such sites to have undergone molecular adaptation. In addition, the loss of free cysteines on both the A2 and B2 globin subunits in annelids from sulfur free environments confirms their implication in sulfide binding in annelids from sulfur rich environments. This event has probably occurred during a short period as, because of their biochemical reactivity, free cysteines would have been rapidly recruited for alternative biochemical uses via a cooperative process in a manner similar to the occurrence of San Marco spandrels in Venice (45). The loss of globin genes in human and apes (46) and the loss of oxygen carrier function in Antarctic fishes (4749) have already been reported, but our study is the first to demonstrate that the loss of a molecular function (capacity to bind H2S) could be driven by positive Darwinian selection.

The use of dN/dS ratio analyses shows that the sulfide-binding function has been secondarily lost by positive selection (i.e., molecular adaptation) in annelid lineages from sulfide-free environments. In other words, we propose that the loss of sulfide-binding function is a disadaptation process (50) that was a prerequisite to avoid the detrimental physiological effects associated with the collateral activity of free cysteine residues regarding other blood compounds.

Supplementary Material

Supporting Information:

Acknowledgments

We gratefully acknowledge the captains and crews of the NO L'Atalante and the RV Atlantis II, the pilots and teams of the submersibles Nautile and Alvin, and F. Gaill (Université Pierre et Marie Curie, Paris), L. S. Mullineaux (Woods Hole Oceanographic Institute, Woods Hole, MA), and H. Felbeck (Scripps Research Institute, La Jolla, CA), chief scientists of the HOT96 and LARVE98 cruises. We thank C. Fisher (Pennsylvania State University, State College) for friendly collaboration, and Myriam Sibuet (Institut Français de Recherche pour L'Exploitation de la Mer) who provided us with Lamellibrachia specimens from Mediterranean Sea. We are particularly indebted to Z. Yang (University College London, London) and M. J. Ford (Northwest Fisheries Science Center, Seattle) who have helped us in the use of the paml software. We thank two anonymous referees for their precious comments and advice. This work was supported by the Conseil Régional de Bretagne, the Ministère de l'Education Nationale et de la Recherche (ACC-SV3), and the Institut National des Sciences de l'Univers and National Oceanic and Atmospheric Administration/National Undersea Research Program Grant UAF01-0042.

Abbreviations

SBD
sulfide-binding domain
HCA
hydrophobic cluster analysis

Footnotes

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