The loss of the hemoglobin H2S-binding function in annelids from sulfide-free habitats reveals molecular adaptation driven by Darwinian positive selection Xavier Bailly*†‡, Riwanon Leroy†, Susan Carney§, Olivier Collin¶, Franck Zal†, Andre´ Toulmond†, and Didier Jollivet* *Equipe Evolution et Ge´ne´ tique des Populations Marines and †Equipe Ecopyhsiologie, Station Biologique de Roscoff, Unite´Mixte de Recherche 7127, Centre National de la Recherche Scientifique, Universite Pierre et Marie Curie, 29680 Roscoff, France; §Department of Biology, Pennsylvania State University, 208 Mueller Lab, University Park, PA 16802; and ¶Station Biologique de Roscoff, Service Informatique, Centre National de la Recherche Scientifique FR2424, 29680 Roscoff, France Edited by Tomoko Ohta, National Institute of Genetics, Mishima, Japan, and approved March 14, 2003 (received for review December 18, 2002) The hemoglobin of the deep-sea hydrothermal vent vestimentif- original specific function (its disappearance or its maintenance). eran Riftia pachyptila (annelid) is able to bind toxic hydrogen This could occur via diversifying selection when the ancestral sulfide (H2S) to free cysteine residues and to transport it to fuel polymorphism linked to this function is subdivided between endosymbiotic sulfide-oxidising bacteria. The cysteine residues are habitats (4). This can lead to the observation of highly divergent conserved key amino acids in annelid globins living in sulfide-rich variants regarding specific amino acid signatures and can be environments, but are absent in annelid globins from sulfide-free viewed as a positive Darwinian selection event acting on species environments. Synonymous and nonsynonymous substitution that have emerged from this habitat speciation. The estimation analysis from two different sets of orthologous annelid globin of the fixation rate of nonsynonymous and synonymous substi- genes from sulfide rich and sulfide free environments have been tutions along orthologous coding sequences from a cluster of performed to understand how the sulfide-binding function of evolutionarily related taxa appears to be one of the most hemoglobin appeared and has been maintained during the course powerful tools to detect molecular adaptation (5–9). However, of evolution. This study reveals that the sites occupied by free- the signature of molecular adaptation can be cryptic and difficult cysteine residues in annelids living in sulfide-rich environments to extract because an adaptive change (advantageous mutation) and occupied by other amino acids in annelids from sulfide-free may only affect a small number of lineages and only a subset of environments, have undergone positive selection in annelids from sites according to their phylogenetic history (10). The accumu- sulfide-free environments. We assumed that the high reactivity of lation of ancient adaptive mutations is typically the situation cysteine residues became a disadvantage when H2S disappeared encountered in globins, a widespread molecule that is conserved because free cysteines without their natural ligand had the capac- in members of all living kingdoms, including annelids. ity to interact with other blood components, disturb homeostasis, The spatial and environmental distribution of annelids, from reduce fitness and thus could have been counterselected. To our deep-sea hydrothermal vents to terrestrial habitats, is the con- knowledge, we pointed out for the first time a case of function loss sequence of a long history of adaptive strategies since their driven by molecular adaptation rather than genetic drift. If con- radiation (11). One of these adaptations concerns the way by straint relaxation (H2S disappearance) led to the loss of the sulfide- which annelids living in sulfide-rich environments protect them- binding function in modern annelids from sulfide-free environ- selves against or use hydrogen sulfide (H2S). Such a process ments, our work suggests that adaptation to sulfide-rich mainly relies on the occurrence of extracellular hemoglobins that environments is a plesiomorphic feature, and thus that the annelid bind and transport this toxic compound. H2S is toxic to aerobic ancestor could have emerged in a sulfide-rich environment. metabolism, particularly to metalloproteins such as cytochrome c oxidase and hemoglobin (12). This unusual sulfide-binding sulfide binding function ͉ free cysteine ͉ annelid evolution ͉ function of some annelid hemoglobins was first discovered in the loss of function vestimentiferan Riftia pachyptila, a mouthless and gutless organ- ism harboring intracellular chemolithoautotrophic sulfide- oxidizing bacterial symbionts. Riftia is found living close to the mergence of new functions in proteins as a result of a high deep-sea eastern Pacific hydrothermal vents (13). Sulfide bind- Eevolutionary rate after gene duplication has been long ing is enabled by the presence of two highly reactive free cysteine debated in the molecular evolution field. From the neutralist residues that covalently bind H2S (14, 15), each one localized on standpoint, molecular evolution occurs by random drift of two different globin subunits included in extracellular hemoglo- EVOLUTION mutations that are nearly equivalent selectively. In this context, bin complexes found in annelids (see review in ref. 16). The ‘‘it is much more likely, if high rates occur, that they are caused annelid hemoglobin multigenic family is subdivided into two by the removal of a preexisting functional constraint, allowing main gene families, A and B, and four subfamilies. A1, A2, B1, previously harmful mutants to become selectively neutral’’ (1). and B2, that emerged via at least three duplication events (17, For ‘‘selectionist,’’ high evolutionary rates are considered rather 18). These latter authors found that the free cysteine residues ϩ as the result of an acceleration of mutations called positive involved in H2S binding are located at the same positions, Cys Darwinian selection, the likely evolutionary force for the acqui- 1 and Cys ϩ 11 (1 and 11 aa after the well conserved distal sition of new functions after a duplication event (2). According histidine), on globin chains within the B2 and A2 subfamilies to Ohta’s consensual theory (3), positive Darwinian selection is respectively for a set of various annelids living in sulfide-rich needed for the accumulation of favorable mutations that provide habitats. Moreover, other nonsymbiotic annelid polychaetes 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, molec- This paper was submitted directly (Track II) to the PNAS office. ular adaptation and emergence of a new function driven by Abbreviations: SBD, sulfide-binding domain; HCA, hydrophobic cluster analysis. positive Darwinian selection is not always associated with du- Data deposition: The sequences reported in this paper have been deposited in the GenBank plication events. Transition from homogeneous to heteroge- database (accession nos. AY250083–AY250087 and AY273262–AY273265). neous habitats could also play a role in the evolution of an ‡To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.1037686100 PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 5885–5890 Downloaded by guest on September 26, 2021 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 ϩ 1 and Cys ϩ 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 in- ability 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- Fig. 1. Neighbor-joining consensus tree of globin sequences from