*Station Biologique de Roscoff, UPR 9042 CNRS-UPMC-INSU, Laboratoire Ecophysiologie, Roscoff, France;
Universita di Padova, Department of Biology, Padova, Italy;
Biologie du Développement, UMR 7622, CNRS et Université P & M CURIE, Paris
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Abstract |
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Introduction |
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The description of the complete multigenic globin family of the vestimentiferan Riftia pachyptila Jones (1981)
provides an additional original model of evolution in which globins exhibit different ligant specificities, raising questions about the subfunctionalization of duplicated genes. Living in a sulfide-rich environment at hydrothermal vents along the East Pacific Rise (EPR) at a depth of 2,500 m, the tube worm R. pachyptila possesses hemoglobins that are able to bind simultaneously and reversibly O2 and a highly toxic molecule, hydrogen sulfide (H2S) (Arp, Childress, and Vetter 1987
). Endosymbiotic sulfo-oxidizing bacteria harbored within a specific organ of the worm, the trophosome, use H2S to synthesize organic compounds which are used by the host (Childress and Fischer 1992
). Adaptation to H2S is of prime importance for this worm, which has to cope with its nutritional requirements and the toxicity of this reduced compound, the latter inhibiting the mitochondrial oxidative chain reaction and the transport of oxygen (Nicholls 1975
). Life in sulfidic habitats has been widely studied, and several kinds of adaptive mechanisms have been selected in organisms to detoxify H2S (Vetter and Powell 1991
; Vismann 1991
; Grieshaber and Volkel 1998
). One of these is the use of extracellular hemoglobins via the occurrence of free cysteine residues (Zal et al. 1998
). Hemoglobins of R. pachyptila are extracellular, two are vascular, and one is coelomic. One of the two vascular hemoglobins possesses a hexagonal-bilayer hemoglobin (HBL-Hb) quaternary structure (Terwilliger, Terwilliger, and Schabtach 1980
), which is only found in the Annelida and Vestimentifera (Weber and Vinogradov 2001
). This complex multimeric hemoglobin is made of 144 globin chains and structural nonglobin chains, the linkers, which enable such a typical shape. Six different globin subunits and their associated molecular weights and free cysteine residues (i.e., cysteine residues not involved in intra- or interchain dissulfide bridges) have been described previously from these hemoglobins by mass spectrometry and amino acid sequencing of the A2 chain (Zal et al. 1996a
, 1996b
, 1997b
). The free cysteine residues only occur in two globin strains, one per globin. As suspected by Suzuki et al. (1989)
, these free cysteine residues are involved in the binding of H2S (Zal et al. 1997b
, 1998
). Free cysteine residues were also found in other globins of worms living in transient or permanent sulfide-rich or polluted environments, such as the symbiotic vestimentiferan Lamellibrachia sp. (Takagi et al. 1991
), the symbiotic pogonophoran Oligobrachia mashikoi (Yuasa, Green, and Takagi 1996
), and the nonsymbiotic polychaetes Sabella spallanzanii (Pallavicini et al. 2001
) and Sabellastarte indica (Suzuki, Hirao, and Vinogradov 1995
). One might consider that these species all have the ability to bind sulfides via their free cysteine residues by analogy to the sulfide-binding function of both the vestimentiferans Lamellibrachia sp. and R. pachyptila, simply because such a binding process has been demonstrated in other polychaetes such as the lugworm Arenicola marina (Zal, Gotoh, and Toulmond 1999
). This article addresses a presentation of a new and complete multigenic family of extracellular globins in R. pachyptila and a possible scenario of duplication events that explains the evolution of such a peculiar respiratory pigment. It also focuses on the sulfide-binding function and its possible role in the evolution of extracellular globins in annelids, vestimentiferans, and pogonophorans living in sulfidic environments. HBL-Hbs carry an original function involved in the binding and transport of H2S, which indeed raises two fundamental questions about (1) how selection acts to maintain free cysteine residues solely in two globin subfamilies after several duplication events at the multigenic family level and (2) what the likely evolutionary history of such a detoxifying mechanism is since its appearance in annelids.
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Materials and Methods |
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Characterization of Extracellular Globin Genes
Globin-Specific Primer Design
All degenerate forward and reverse primers were designed according to the amino acid sequences of the four extracellular globin subfamilies that form the HBL-Hb of Lamellibrachia sp. (i.e., subfamilies A1 [Takagi et al. 1991
], A2 [accession number: P15469], B1, and B2 [Takagi et al. 1991
]). Primer designs were performed using Oligo 4.0 software according to the following criteria: (1) the corresponding amino acid domains used must be close to either the NH2-terminal end (forward primer) or the COOH-terminal end (reverse primer: legend of fig. 1
), (2) they must contain a large proportion of twofold or threefold degenerated amino acids, and (3) they must be specific to a single globin subfamily.
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Globin Amplification and Sequencing
Each partial globin cDNA was amplified by PCR using a set of degenerate primers on a Perkin-Elmer GenAmp PCR System 2400®. PCR steps were performed identically for each globin subfamily amplification, with the same annealing temperature (50°C). PCR conditions were as follows: initial denaturation at 96°C for 5 min, 35 cycles consisting of 96°C for 50 s, 50°C for 50 s, and 72°C for 50 s. The reaction was completed by an elongation step of 10 min at 72°C. Amplifications were carried out in 25-µl reaction mixtures containing 1050 ng cDNA target, 50100 ng of each degenerate primer, 200 µM dNTPs, 2.5 mM MgCl2, and 1 unit of Taq DNA polymerase (Promega). PCR products were visualized on a 1% agarose gel under UV radiation. Gel slices containing DNA fragments of the expected size were collected and subsequently purified onto Ultrafree®-DA. All PCR products were then cloned using a TOPO-TA Cloning® Kit (Invitrogen). Purified plasmids containing the globin insert were used in a dye-primer cycle sequencing reaction, using either the labeled Texas Red universal primer T7 (5'-GTA ATA CGA CTC ACT ATA GGG C-3') or the M13 reverse (5'-GGA AAC AGC TAT GAC CAT G-3') and the Thermo SequenaseTM premixed cycle sequencing kit from AmershamTM. PCR products were subsequently run on a Vistra automated DNA Sequencer 725.
Molecular Phylogenetic Analysis
Analyses were performed on different sets of data: (1) paralogous cDNA sequences of the globin family of R. pachyptila and (2) orthologous amino acid sequences of globins from R. pachyptila and closely related annelids sharing similar HBL-Hb extracellular respiratory pigments. These globin sequences belong to Lumbricus terrestris (accession numbers: A29134, A28151, B28151, and C28151) of Shishikura et al. (1987)
and Fushitani, Matsuura, and Riggs (1988)
, Tylorrhynchus heterochaetus (accession numbers: P02219, P02220, P09966, and P13578) of Suzuki and Gotoh (1986)
, Lamellibrachia sp. (accession number: S08284) of Suzuki, Takagi, and Ohta (1990)
, O. mashikoi (accession numbers: S72251, S72252, and S72253) of Yuasa, Green, and Takagi (1996)
, and S. spallanzanii (accession numbers: CAC37411 and CAC37412) of Negrisolo et al. (2001)
. DNA or amino acid sequences were aligned with Clustal X software (Thompson et al. 1997
) and using the hidden Markov model procedures and the SAM system (Karplus et al. 1997
) (http://www.cse.ucsc.edu/research/compbio/) in order to assess the congruence of alignments for both paralogous and orthologous data sets.
Informative Sites
The more informative and conserved blocks of amino acids were selected for molecular phylogenetic analysis from a multiple alignment of globin sequences using Gblock software (Castresana 2000
). Final conserved blocks of contiguous amino acids were obtained applying the following settings in option b (blocks parameters): minimum number of sequences for a conserved position8, minimum number of sequences for a flank position12, maximum number of contiguous nonconserved positions8, minimum length of an initial block15, and minimum length of a block after gap cleaning10.
Molecular Phylogeny
Molecular phylogenetic trees were constructed using Neighbor-Joining (NJ), Maximum Parsimony (MP), and Maximum Likelihood (ML) methods. NJ, MP, and ML were, respectively, computed using the Phylo_win package (Galtier, Gouy, and Gautier 1996
), PAUP version 3.1.1 (Swofford 1993
), and Puzzle version 4.0 (Strimmer and von Haeseler 1996
). Phylogenetic distances were computed from observed levels of divergence and from that expected under the Dayhoff's PAM matrix (Dayhoff, Schwartz, and Orcutt 1978
). MP trees were obtained with a heuristic search using the tree bisection-reconnection (TBR) branch swapping option, stepwise addition (closest taxa addition), and the collapse option for zero-length branches. ML analysis was performed according to the Jones, Taylor, and Thornton (JTT) model of amino acid substitutions (Jones, Taylor, and Thornton 1992
), with and without a gamma distribution of substitution rates among sites, and using the quartet puzzling method. Bootstrap proportions were obtained from 1,000 resampling sequence alignments for NJ and parsimony trees and from 100,000 puzzling steps in the particular case of the likelihood method. The duplicate gene rooting procedure (Donoghue and Mathews 1998
), which was originally proposed as an alternative to root the universal tree of life for which outgroups are not available (Iwabe et al. 1989
), was used to root our paralogous globin trees.
Evolutionary Rates
To test whether subfamilies of vestimentiferan globins may evolve at different evolutionary rates, genetic distances between R. pachyptila and Lamellibrachia sp. orthologous amino acid sequences of each subfamily, namely A1, A2, B1, and B2, were calculated according to the Jones, Taylor, and Thornton (1992)
matrix using the ML procedure of Puzzle. In this particular case, the four genetic distances obtained between the two taxa relate directly to the evolutionary rate because the evolutionary time is identical between the four paralogous genes. The occurrence of a global molecular clock was tested on three orthologous sets of globins, namely A1, A2, and B2, using the relative rate test comparing substitution rates between sequences, according to the absolute difference |d1 - d2| of Sarich and Wilson (1973)
, where d1 and d2 represent the ML distances of species 1 and species 2 to a third, more divergent reference species.
Two-Dimensional Cysteine Structural Domains
A Hydrophobic Cluster Analysis (HCA plot) was performed to predict the secondary structure of primary globin amino acid sequences to assess the molecular environment of free cysteine residues that are involved in the sulfide-binding function. These plots were obtained using DrawHCA software (Callebaut et al. 1997
) (http://www.lmcp.jussieu.fr/
soyer/www-hca/hca-form.html).
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Results |
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Cysteine Residues in R. pachyptila Globins
The R. pachyptila globin sequences also display free cysteine residues (i.e., not involved in dissulfide bridges within or between the globin polypeptide chains), whose positions correspond to those previously described in Lamellibrachia sp. (fig. 1
). These free cysteine residues are only located in the A2 and B2 subfamilies of R. pachyptila. These cysteine residues occur at position 1 after the distal histidine in B2 and at position 11 after the homologous histidine in A2. Cysteine residues in similar positions are also found in Lamellibrachia sp., O. mashikoi, and S. spallanzanii A2 and B2 globins, species living in sulfide-rich (or sporadically rich) environments, whereas no such free cysteine residues are found in the A2 and B2 globins of L. terrestris and T. heterochaetus, two species living in H2S-free environments. Another free cysteine residue is also found in the globin A1 sequence of O. mashikoi (fig. 1
). We called these two free cysteine residues Cys + 1 (i.e., one a.a. from the well-conserved distal histidine) and Cys + 11 (i.e., eleven a.a. from the well-conserved distal histidine). Other cysteine residues are also found in R. pachyptila globin sequences. However, these residues are always involved in dissulfide bridges in the HBL-Hb of both R. pachyptila and the other annelids investigated so far. Cysteine residues involved in intrachain dissulfide bridges were not detected because of the incomplete sequencing of our clones at both COOH- and NH2-ending sides. However, cysteine residues involved in dissulfide bridges between globins A1 and B2 occur at positions 115 and 17, respectively.
Phylogenetic Reconstruction of the R. pachyptila Globin Multigenic Family
Because most sites of the second and the third codon positions were saturated between the six paralogous sequences (data not shown), a computed selection of conserved blocks of amino acids obtained by the elimination of poorly aligned positions and divergent regions (Gblock software) was used to generate a restricted alignment of 74 amino acids (fig. 1
). The trees were constructed subsequently, using NJ, MP, and ML methods, and compared. All methods gave identical topologies; consequently we only present the ML consensus tree (fig. 2
). Tree topologies clearly indicate that this multigenic family emerged from an initial duplication event and then led to four duplication events that produced the actual multigenic family. These duplications appear to be asymmetrical between families A and B and have led to the recent emergence of the three B1 globins
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Such superimposable species clustering between the four sets of orthologous sequences led us to test the occurrence of a molecular clock in order to investigate whether our molecular data set follows a nearly neutral rate of evolution. To test this hypothesis, genetic distances were calculated from pairwise combinations of globins from the same subfamily using Puzzle (ML), the Jones, Taylor and Thornton matrix, and 10,000 puzzling steps (table 1 ). Then, a relative rate test was applied on the A1, A2, and B2 subfamilies, but not for B1, for which no ancestral reference has been obtained (i.e., a third ancestral species). The results are presented in table 2 . The global mean of distance differences (d1 - d2) is 0.145 ± 0.113. We used a t-test for paired comparisons in order to test the null hypothesis of identical evolutionary rates, i.e., if the difference does not significantly depart from zero, then we cannot reject the hypothesis of a molecular clock. The analysis indicated that the taxa evolved at the same rate within each set of orthologous sequences at a threshold of 1% but not of 5% (ddl = 4, t = 3.85).
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Discussion |
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Typically, the nonvertebrate globins exhibit a substantially greater amount of variation than that found in vertebrate globins (Kapp et al. 1995
). As such, analyses must be performed on a carefully selected group of sequences to recover the less biased phylogenetic signal from this new set of globin sequences. We assume that the central exonic region of the extracellular globins from annelids is more appropriate for reconstructing the molecular evolutionary history. Indeed, there is a positive correlation between the gene organization of the globin and its structural features (Blake 1981
; Go 1981
). The second exon encodes for the well-conserved and ubiquitous heme-binding fold in vertebrates (Craik, Buchman, and Beychok 1981
) and thus is less likely to differentiate with time. We assume that the second exon of extracellular globins encodes for the same structural features because intron positions are conserved between annelids and the intracellular globins of vertebrates (Jhiang, Garey, and Riggs 1988
; X. Bailly, unpublished data). Interestingly, the second exon also corresponds to the amino acid portion selected by GBlock and is the only set of data allowing the reconstruction of congruent phylogenies. Moreover, this amino acid portion of the globin sequence also displays the two free cysteine residues involved in the sulfide-binding function, allowing us to parallel the globin phylogeny and the molecular history of the unusual sulfide uptake in annelids living in sulfidic environments.
Globin Assignment to A1, A2, B1, and B2 Subfamilies: Are Subfamilies True Paralogous Genes?
One important consideration in phylogeny reconstruction from gene families is to discriminate between orthologous and paralogous genes when the time of divergence between some observed species is important or if gene duplication is ancient (i.e., paralogous genes under saturation). The annelid radiation took place about 600 MYA (McHugh 2000
), leading to the emergence of polychaetes, achaetes, oligochaetes, vestimentiferans, and pogonophorans. Moreover, Rouse (2001)
argued that pogonophorans and vestimentiferans are specialized Siboglinidae polychaetes that separated from the Sabellidae even more recently. The extracellular HBL-Hb is found in various annelid taxa, including achaetes, oligochaetes, polychaetes, and vestimentiferans. Because such a molecule is a complex edifice made of at least 144 globin chains and additional linkers (structural chains), it is unlikely that this hemoglobin could have appeared in different lineages independently and thus have evolved subsequently through convergent evolution. As a consequence, because four main types of extracellular globins are present in the oligochaete L. terrestris, the vestimentiferans Lamellibrachia sp. and R. pachyptila, and the polychaete T. heterochaetus, this strongly suggests that the HBL-Hb emerged before the annelid radiation via two subsequent duplications. At least, it follows that one annelid ancestor possessed the A and B family globins as stated by Gotoh et al. (1987)
, Suzuki, Takagi, and Ohta (1993)
, and other authors dealing with annelid globin gene organization and evolution. By restricting our analysis to extracellular globins from vestimentiferans and closely related polychaete relatives (e.g., Sabella) and by sorting sequences according to family-specific amino acid motifs and two dimensional (2-D) structure, we were able to demonstrate for the first time an unequivocal discrimination between paralogous and orthologous annelid globins and that the four main globin strains A1, A2, B1, and B2 coevolved separately. In this respect, our work reveals that previous studies using extracellular globins produced unresolved tree topologies with incorrect subfamily assignment (Dewilde et al. 1996
; Yuasa, Green, and Takagi 1996
; Zal et al. 1997b
; Negrisolo et al. 2001
). Our results, therefore, confirm the hypothesis that the HBL-Hb arose before the annelid radiation from two ancient subsequent duplications, other duplications being more recent and probably species-specific. The present molecular phylogeny displays an identical species clustering for the four paralogous clades in which the species branching order is in agreement with other annelid phylogenies which involve vestimentiferans (Black et al. 1997
; Halanych, Lutz, and Vrijenhoek 1998
; Rouse 2001
). This finding demonstrates that extracellular globins could be used to test the molecular clock hypothesis in multigenic families of invertebrates when uninformative portions of the globin sequence are removed.
Different Evolutionary Rates Between Paralogous Genes Despite a Molecular Clock
The evolution of the four main paralogous globin clades, i.e., the similar species branching order (in agreement with other gene phylogenies), allowed us to test the hypothesis of a molecular clock using the relative rate test. Our results suggest the occurrence of a globin-specific and domain-specific molecular clock between annelid species. Interestingly, this clock seems to be detectable only within a portion of the well-conserved second exon of the globin sequence. This raises questions about how a clock, which translates a neutral or nearly neutral molecular evolutionary rate, could be detected in such a highly conserved structural domain (i.e., the second exon is prone to strong selective pressures). One explanation is to consider that only few amino acids (those responsible for the globin fold) are under selection, others (the highest fraction) evolving neutrally, i.e., being free to vary. Indeed, despite the common widespread idea that selective processes mainly act on the active catalytic site, Graur (1985)
showed that the lowest rate of amino acid changes is not focused in the active parts of the molecule. An alternative explanation is that extracellular globins fit the covarion (concomitant variable codon) model of Fitch and Markowitz (1970)
, which asserts that only a small fraction of the amino acid positions of a protein is free to vary (covarions) at a given time in its evolutionary history because of functional constraints. The present situation may illustrate "how protein sequences can differ considerably among distant taxa even though only a limited number of positions are free to vary within a particular lineage" (Miyamoto and Fitch 1995
). The joint phylogenetic analysis of the four paralogous sets of extracellular globins allows us to discriminate between these two possible explanations. Although the relative rate test is unable to ascertain a molecular clock in extracellular globins of Sabellidae, Vestimentifera, and Pogonophora, our results suggest that (1) the four globin subfamilies evolve at different rates (i.e., branch lengths differ between paralogous clades) and (2) each rate appears to be constant between species for the four orthologous clades which exhibit the same species branching order. This finding, therefore, seems to reject the first hypothesis simply because one might expect the four subfamilies to evolve at a very similar rate if neutral (similar branch lengths between clades). Fractions of amino acids free-to-vary are thus probably different between the four subfamilies, hence the different evolutionary rates between subfamilies, but could explain why species follow the same pattern of clustering and hence the neutral evolution in orthologous globins. Thus, each subfamily displays a specific molecular clock because natural selection acted differentially on the four globin subfamilies. However, the fact that selective pressures acted differently on paralogous strains but homogeneously on orthologous strains suggests that the constraints of the whole environment did not change since the radiation of both Siboglinidae and Sabellidae. Such an assumption seems likely because these organisms are all found in sulfidic environments. Thus, our results could be viewed as a strong insight for underlining the joint action of selective pressure and (nearly) neutral evolution claimed by Ohta (2000)
to reconcile selectionist and neutralist partisans. However, even if the selected amino acid portion used here to reconstruct this molecular phylogeny could be refined, it cannot be extended to highly divergent taxa such as the oligochaete L. terrestris or the polychaete T. heterochaetus, although they both possess an HBL-Hb and thus the four types of extracellular globins. In this case, one can argue that such a loss of signal is because of saturation. But an alternative explanation would be to also consider that selective pressures may have been altered because these two highly divergent species are also known to inhabit very well oxygenated environments. These observations suggest that extracellular globins are not informative markers for performing phylogeny analyses over the whole Annelida phylum. However, they are powerful tools for studying the molecular evolution of paralogous genes within closely related species.
Lower Evolutionary Rates Correlate with the Occurrence of Free Cysteine Residues and SBD
To test whether the differing evolutionary rates between R. pachyptila globin subfamilies could be caused by the action of selective pressures, genetic distances between the orthologous sequences of two closely related species of Vestimentifera (R. pachyptila and Lamellibrachia sp.) were calculated for the four paralogous subfamilies, given that both species are endosymbiotic, live in the same ecological niche, and are separated by less than 50 Myr. Indeed, a recent clock calibration has been obtained for the vent annelids (rate = 0.2% per Myr: Chevaldonné et al. 2002
) together with a phylogenetic tree for the vestimentiferans and the pogonophorans for the mitochondrial COI marker (Black et al. 1997; Halanych, Lutz, and Vrijenhoek 1998
). These results allowed us to estimate the divergence time between Lamellibrachia sp. and R. pachyptila to be around 46 Myr (no divergence time is available between Sabellidae and Siboglinidae). In other words, because genetic distances measure the expected accumulation of mutations since speciation, an attempt was made to establish whether A1, A2, B1, and B2 evolved at the same evolutionary speed. It is clearly seen that A2 and B2 subfamilies display the lowest evolutionary rates. Interestingly, the A2 and B2 globin chains are both involved in sulfide binding via their respective free cysteine residues (Cys + 1 and Cys + 11), and one could obviously assume that this rate decrease may be the result of a strong directional selection toward the maintenance of this unusual globin function. Genetic distances from the orthologous R. pachyptila and Lamellibrachia sp. globin without the portion corresponding to the SBD (i.e., 21 a.a. surrounding the free cysteine) provide the same trend. Thus the sulfide-binding function associated with the A2 and B2 globin chains could have been strongly selected by the limitation of amino acid changes during the course of evolution. In addition, such an assumption is well supported by (1) the maintenance of a typical A2 and B2 hydrophobic secondary structure associated with the free cysteine residues in most lineages, (2) the remains of a putative SBD in A1 and B1, these globins being devoid of free cysteine residues in species living in sulfidic environments, and (3) the noticeable absence of homologous free cysteine residues in A2 and B2 globins of annelids living in well-oxygenated environments despite the persistence of a more or less degenerated SBD fingerprint.
A nonexclusive additional explanation might be that A1 and B1 globin subfamilies have accumulated more mutations than A2 and B2 have just after the duplication event. This is the acceleration of evolution by the duplication effect (Hill and Hastie 1987
; Kini and Chan 1999
) and can be caused by the relaxation of selective pressures (Kimura 1981
) or positive Darwinian selection (Zhang, Rosenberg, and Nei 1998
). However, one cannot exclude a selective structural effect of the multimeric hemoglobin on monomers A2 and B2 relative to A1 and B1 according to their location (allosteric effect). Further information concerning the hemoglobin structure of R. pachyptila is required.
Because both L. terrestris and T. heterochaetus do not exhibit free cysteine residues and the ability to bind H2S, it may be assumed that sulfide binding is a derived function in the Siboglinodae. However, from our data, we demonstrated that the positions Cys + 1 and Cys + 11 involved in the sulfide-binding function were both well conserved in the A2 and B2 strains, for at least vestimentiferans, pogonophorans, and Sabellid worms. Moreover, a free cysteine residue was also found in the A1 strain in O. mashikoi at the Cys + 1 position with a similar superimposable SBD. Because free cysteine residues were found at the same location on different paralogous strains, the cysteines at positions Cys + 1 and Cys + 11 might have arisen before the first duplication that led to the emergence of the A and B globin families. These data allowed us to refute the hypothesis that H2S detoxification-transport via a reactive cysteine acquisition process may be derived or gained independently in different annelid lineages. It is more parsimonious to propose that the first annelids inhabiting sulfidic environments lost the ability to bind sulfides in well-oxygenated environments. These findings indicate that free cysteine residues have been conserved since the radiation of the Siboglinidae and more so before the radiation of annelids. This hypothesis is also well supported by the remnants of a putative SBD of annelids living in well-oxygenated environments (T. heterochaetus and L. terrestris). A parsimonious evolutionary scenario is presented in figure 5
; it relies on the assumption that the vestimentiferan nonsymbiotic ancestors lived in contact with high sulfide concentrations and had a hemoglobin involved in a H2S detoxification process. Indeed, if A2 and B2 strains are both involved in sulfide binding via their respective free cysteine residues, one could assume that this property must have been maintained during the annelid evolution. Therefore, this function has then evolved to be a H2S detoxification-transport function in vestimentiferans as a means of fueling their endosymbionts during the symbiosis acquisition process (Arp, Childress, and Vetter 1987
). This evolutionary scheme is well supported by the occurrence of free cysteine residues also involved in the sulfide-binding function in other nonsymbiotic polychaetes living in sulfidic environments. Zal et al. (1997a
) and Zal, Gotoh, and Toulmond (1999)
have described a HBL-Hb which binds sulfides via free cysteine residues (positions unknown) in the shallow-water polychaete A. marina and the hydrothermal vent polychaete Alvinella pompejana. Thus, the presence of a degenerated SBD in globins that do not possess free cysteine residues in HBL-Hbbearing annelids living in well-oxygenated habitats (L. terrestris and T. heterochaetus) is an insight into the probable loss of these free cysteine residues by mutation-drift when selection is relaxed (nearly neutral evolution).
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Acknowledgements |
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Footnotes |
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Keywords: hexagonal bilayer hemoglobin
duplication
selection
H2S
sulfide-binding domain
Riftia pachyptila
Address for correspondence and reprints: Xavier Bailly, Station Biologique de Roscoff, UPR 9042 CNRS-UPMC-INSU, Laboratoire Ecophysiologie, BP. 74, Place Georges Teissier, 29682 Roscoff cedex, France. E-mail: bailly{at}sb-roscoff.fr
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