Evolution of the Sulfide-Binding Function Within the Globin Multigenic Family of the Deep-Sea Hydrothermal Vent Tubeworm Riftia pachyptila

Xavier Bailly*, Didier Jollivet*, Stephano Vanin{dagger}, Jean Deutsch{ddagger}, Franck Zal*, François Lallier* and André Toulmond*

*Station Biologique de Roscoff, UPR 9042 CNRS-UPMC-INSU, Laboratoire Ecophysiologie, Roscoff, France;
{dagger}Universita di Padova, Department of Biology, Padova, Italy;
{ddagger}Biologie du Développement, UMR 7622, CNRS et Université P & M CURIE, Paris


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The giant extracellular hexagonal bilayer hemoglobin (HBL-Hb) of the deep-sea hydrothermal vent tube worm Riftia pachyptila is able to transport simultaneously O2 and H2S in the blood from the gills to a specific organ: the trophosome that harbors sulfide-oxidizing endosymbionts. This vascular HBL-Hb is made of 144 globins from which four globin types (A1, A2, B1, and B2) coevolve. The H2S is bound at a specific location (not on the heme site) onto two of these globin types. In order to understand how such a function emerged and evolved in vestimentiferans and other related annelids, six partial cDNAs corresponding to the six globins known to compose the multigenic family of R. pachyptila have been identified and sequenced. These partial sequences (ca. 120 amino acids, i.e., 80% of the entire protein) were used to reconstruct molecular phylogenies in order to trace duplication events that have led to the family organization of these globins and to locate the position of the free cysteine residues known to bind H2S. From these sequences, only two free cysteine residues have been found to occur, at positions Cys + 1 (i.e., 1 a.a. from the well-conserved distal histidine) and Cys + 11 (i.e., 11 a.a. from the same histidine) in globins B2 and A2, respectively. These two positions are well conserved in annelids, vestimentiferans, and pogonophorans, which live in sulfidic environments. The structural comparison of the hydrophobic environment that surrounds these cysteine residues (the sulfide-binding domain) using hydrophobic cluster analysis plots, together with the cysteine positions in paralogous strains, suggests that the sulfide-binding function might have emerged before the annelid radiation in order to detoxify this toxic compound. Moreover, globin evolutionary rates are highly different between paralogous strains. This suggests that either the two globin subfamilies involved in the sulfide-binding function (A2 and B2) have evolved under strong directional selective constraints (negative selection) and that the two other globins (A1 and B1) have accumulated more substitutions through positive selection or have evolved neutrally after a relaxation of selection pressures. A likely scenario on the evolution of this multigenic family is proposed and discussed from this data set.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
From the earliest studies of evolutionary processes at a molecular level, hemoglobin has been one of the favorite molecules for modeling the evolution of multigenic families (Rossi-Fanelli, Cavallini, and De Marco 1955Citation ; Zuckerkandl, Jones, and Pauling 1960Citation ). Ingram (1961)Citation stressed the process of gene duplication to explain human hemoglobin subunits' emergence and different levels of selection to maintain such proteins through the course of evolution. Numerous amino acid- and nucleotide-based phylogenies of vertebrate globins have been carried out to reconstruct the most appropriate scenario of the evolution of the paralogous genes encoding these molecules (Goodman et al. 1971Citation ; Goodman, Moore, and Matsuda 1975Citation ). Invertebrate globins have also been investigated. Their progressive identification and characterization at a gene level allowed several authors to relate the huge hemoglobin diversity found in invertebrates to their evolutionary history (Goodman et al. 1988Citation ; Hardison 1998Citation ).

The description of the complete multigenic globin family of the vestimentiferan Riftia pachyptila Jones (1981)Citation 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 1987Citation ). 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 1992Citation ). 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 1975Citation ). 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 1991Citation ; Vismann 1991Citation ; Grieshaber and Volkel 1998Citation ). One of these is the use of extracellular hemoglobins via the occurrence of free cysteine residues (Zal et al. 1998Citation ). 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 1980Citation ), which is only found in the Annelida and Vestimentifera (Weber and Vinogradov 2001Citation ). 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. 1996aCitation , 1996bCitation , 1997bCitation ). The free cysteine residues only occur in two globin strains, one per globin. As suspected by Suzuki et al. (1989)Citation , these free cysteine residues are involved in the binding of H2S (Zal et al. 1997bCitation , 1998Citation ). 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. 1991Citation ), the symbiotic pogonophoran Oligobrachia mashikoi (Yuasa, Green, and Takagi 1996Citation ), and the nonsymbiotic polychaetes Sabella spallanzanii (Pallavicini et al. 2001Citation ) and Sabellastarte indica (Suzuki, Hirao, and Vinogradov 1995Citation ). 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 1999Citation ). 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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Biological Material
Juvenile specimens of the hydrothermal vent tube worm R. pachyptila (3–5 cm length) were collected twice at one single vent site from the ridge segment 9°50'N on the EPR (Riftia field: 9°50.75'N, 104°17.57'W) at a depth of about 2,500 m, during the French oceanographic cruise HOT 96 and the American cruise LARVE99. The worms were sampled using the telemanipulated arms of the submersibles Nautile and Alvin, brought back alive to the surface inside an insulated basket, and immediately frozen and stored in liquid nitrogen after their recovery on board.

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. 1991Citation ], A2 [accession number: P15469], B1, and B2 [Takagi et al. 1991Citation ]). 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.



View larger version (95K):
[in this window]
[in a new window]
 
Fig. 1.—Multiple alignment of partial amino acid globin sequences of S. spallanzanii (Sab), O. mashikoi (Oli), Lamellibrachia sp. (Lam), T. heterochaetus (Tyl), L. t. terrestris (Lum), and R. pachyptila(Rif). Amino acids in Lam sequences correspond to the portions used to design the following specific subfamily degenerated primers: A1 forward, 5'-TGG GCN RAR GCN TAY GG-3'; A1 reverse, 5'-CCA NGC NGG NAR RTC RAA-3'; A2 forward, 5'-TGG GCN RAR GCN TAY GG-3'; A2 reverse, 5'-YTG CCA NGC RTC YTK RTC-3'; B1 forward, 5'-GTN AYH AAR CAG TGG VA-3'; B1 reverse, 5'-CCA NGC RTC NGG NTT RAA-3'; B2 forward, 5'-RAT GCA GYT NAT GTG GG-3'; and B2 reverse, 5'-NGC RTC NGG RTT RAA RCA-3'. The new globin sequences of R. pachyptila are shaded. (*) indicates the position of the well-conserved amino acids in the extracellular globins of annelids according to Vinogradov et al. (1993)Citation . (#) indicates the location of the free cysteine residues ({dotsquare}) involved in the sulfide-binding function, and C indicates the cysteine residues involved in the globin dissulfide bridge intra- or interglobin chain. Bold and underlined R. pachyptila globin sequences correspond to the restricted portion of the contiguous most conserved amino acids obtained using Gblock software between the six extracellular globins of R. pachyptila. The boxed multiple-alignment portion corresponds to the amino acid portion of the second exon selected for the phylogeny reconstruction according to Gblock

 
Total RNA Extraction and cDNA Synthesis
Entire juveniles of R. pachyptila were crushed in liquid nitrogen. Total RNAs were then recovered and extracted using the RNAble® buffer (Eurobio). The mRNA purification was performed from the total RNAs using the oligodT resin of the mRNA Purification Kit® (Pharmacia Biotech). Reverse transcription was initiated with an anchored oligodT CTC CTC TCC TCT CCT CTT(16) primer.

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 10–50 ng cDNA target, 50–100 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)Citation and Fushitani, Matsuura, and Riggs (1988)Citation , Tylorrhynchus heterochaetus (accession numbers: P02219, P02220, P09966, and P13578) of Suzuki and Gotoh (1986)Citation , Lamellibrachia sp. (accession number: S08284) of Suzuki, Takagi, and Ohta (1990)Citation , O. mashikoi (accession numbers: S72251, S72252, and S72253) of Yuasa, Green, and Takagi (1996)Citation , and S. spallanzanii (accession numbers: CAC37411 and CAC37412) of Negrisolo et al. (2001)Citation . DNA or amino acid sequences were aligned with Clustal X software (Thompson et al. 1997Citation ) and using the hidden Markov model procedures and the SAM system (Karplus et al. 1997Citation ) (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 2000Citation ). 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 position—8, minimum number of sequences for a flank position—12, maximum number of contiguous nonconserved positions—8, minimum length of an initial block—15, and minimum length of a block after gap cleaning—10.

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 1996Citation ), PAUP version 3.1.1 (Swofford 1993Citation ), and Puzzle version 4.0 (Strimmer and von Haeseler 1996Citation ). Phylogenetic distances were computed from observed levels of divergence and from that expected under the Dayhoff's PAM matrix (Dayhoff, Schwartz, and Orcutt 1978Citation ). 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 1992Citation ), 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 1998Citation ), which was originally proposed as an alternative to root the universal tree of life for which outgroups are not available (Iwabe et al. 1989Citation ), 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)Citation 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)Citation , 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. 1997Citation ) (http://www.lmcp.jussieu.fr/~soyer/www-hca/hca-form.html).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Identification and Characterization of the Complete Globin Multigenic Family of R. pachyptila
Six specific globin sequences (accession numbers: AJ439732, AJ439733, AJ439734, AJ439735, AJ439736, and AJ439737) were identified after cloning and sequencing, in agreement with the number of subunits previously described by mass spectrometry (Zal et al. 1996aCitation , 1996bCitation ). The six globins were then aligned and compared with the four extracellular globins of Lamellibrachia sp. designated A1, A2, B1, and B2 following the nomenclature of Suzuki, Takagi, and Ohta (1993)Citation and with the globin sequences of O. mashikoi, S. spallanzanii, L. terrestris, and T. heterochaetus (fig. 1 ). Among the six R. pachyptila sequences, three were unambiguously identified as being orthologous to the A1, A2, and B2 globins of Lamellibrachia sp. The last three R. pachyptila sequences were much more variable. However, they share residues with the B family (fig. 1 ): a serine (S) at position 6, present in a majority of B globins; and an asparagine (N) at position 66, which is absent from the A globins. In addition, they share two indels with the B globin: an insertion of one residue at position 89 and a deletion of two residues at positions 113–114. Furthermore, a three-residues motif, PQV, at positions 108–110, specific to the B globins, was well conserved. These characters allowed us to allocate these three sequences to the B family without ambiguity. Furthermore, we propose that these three latter globins fall within the B1 group on the following basis: B1a, B1b, and B1c shared a four-residues motif, VNVA, at positions 40–43 and a methionine (M) at position 99 with the globin B1 of Lamellibrachia sp. B1a and B1b also shared a valine (V) at position 102 with Lamellibrachia sp B1 (fig. 1 ). The high amino acid identity between R. pachyptila and Lamellibrachia sp. globin subfamilies (72%, 81%, 51%, and 82% for the A1, A2, B1, and B2 globin chains, respectively) and the occurrence of well-conserved amino acid motifs typifying the extracellular HBL-Hb of annelids (Vinogradov et al. 1993Citation ) strongly sustain the orthological relationship of R. pachyptila globin subfamilies with other annelid globin subfamilies described so far. The well-conserved distal histidine at position 53 (fig. 1 ), known to be involved in fine-tuning the ligant affinities of hemoglobin (Olson et al. 1988Citation ), is substituted by a glutamine in the B1c globin.

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



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.—The ML consensus tree obtained from the six R. pachyptila globins using 100,000 puzzling steps. Clade A was used as a root to refine the topology of clade B. Bold values at each node correspond to the bootstrap confidence level and values above the branches to branch lengths

 
Globin Molecular Phylogeny of Vestimentiferans, Pogonophorans, and Closely Related Polychaetes Living in Sulfidic Environments
Globins from several additional closely related annelid species were added to the R. pachyptila globin sequences to test the phylogenetical signal within each set of orthologous sequences, provided these species possess at least the four main paralogous globin types, namely A1, A2, B1, and B2. Following Gblock and tree reconstruction methods similar to those described previously, we constructed a restricted alignment, of the 42 most informative contiguous amino acids, that corresponds to a portion of the well-described second exon of the extracellular globin gene (see boxed alignment in fig. 1 ). Irrespective of the method used (NJ, MP, and ML), the data gave identical tree topologies (fig. 3 ).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.—The NJ consensus tree obtained using the observed distances (branch length above the branches) estimated from the Gblock-selected portion of globin sequences and 1,000 bootstrap replicates (values in bold). Rooting is performed according to the duplicate rooting procedure. The first dichotomy separates the A and B families and the second the A1, A2, B1, and B2 subfamilies. S. spallanzanii (Sab), O. mashikoi (Oli), Lamellibrachia sp. (Lam), and R. pachyptila (Rif)

 
The ancestral duplications that have led to the emergence of the A and B globin families and then to the A1, A2, B1, and B2 subfamilies are clearly identified, i.e., sequences from each orthologous set of genes are clustered together in a similar fashion (fig. 3 ). MP gave a slightly different result, with two aberrant branchings in the tree. To produce a coherent phylogeny with this method, we examined the alignment, character by character, and were able to detect and thus eliminate two homoplasic positions (i.e., positions 23 and 40) involved in the unexpected topology. This restored a tree topology similar to that obtained with the NJ and ML methods for the four paralogous sets of globins.

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).


View this table:
[in this window]
[in a new window]
 
Table 1 Maximum Likelihood Estimates of the Expected Number of Amino Acid Substitutions Per Site (below the diagonal) and the Observed Distances (above the diagonal)

 

View this table:
[in this window]
[in a new window]
 
Table 2 Relative Rate Test Values (r) Obtained for the Globin Subfamilies A1, A2, and B2

 
Evolutionary Rates Between Paralogous Globins
Genetic distances were recalculated over the whole range of globin amino acid sequences between the vestimentiferans R. pachyptila and Lamellibrachia sp. for the four paralogous globin types (i.e., amino acids occurring between the conserved COOH- and NH2-terminal tryptophans: table 3 ). First, genetic distances differ between the four subfamilies. Second, genetic distances obtained from pairwise comparisons of R. pachyptila and Lamellibrachia sp. globins from partial sequences generated with Gblock (74 a.a. and 42 a.a., respectively) produced the same trend. This result clearly indicates that A2 and B2, which possess the free cysteine residues at positions Cys + 11 and Cys + 1, display a lower evolutionary rate as opposed to the A1 and B1 globins.


View this table:
[in this window]
[in a new window]
 
Table 3 Maximum Likelihood Estimates of the Expected Number of Amino Acid Substitutions per Site (genetic distance, [d]) Between Orthologous Sequences of Riftia pachyptila and Lamellibrachia sp. for each Globin Subfamily

 
HCA of R. pachyptila Globins
The secondary structure of globins in R. pachyptila, Lamellibrachia sp., O. mashikoi, and S. Spallanzanii were deduced from HCA plots and compared according to the tree topology of figure 4 . The analysis focused on the restricted sulfide-binding domain (SBD) that flanks the free cysteine residues, i.e., the specific local HCA shape flanking the Cys + 1 and the Cys + 11. It is clear that two different hydrophobic motifs are conserved in annelid species (fig. 4 ). Another interesting finding is of a B2-like structure in O. mashikoi A1, with a free cysteine residue, and an A2 and B2–like structure, but with no cysteine residue, in Lamellibrachia sp B1. Hence, there is either a typical SBD structure or a degenerated fingerprint of such a structure in almost all the HBL-Hb globins. This motif persistence may indicate that the SBD is ancestral.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 4.—The NJ tree (similar to fig. 3 ) showing phylogenetical relationships between the 2-D HCA plots of the SBDs (from positions 49 to 69) from the four extracellular globin subfamilies. SBDs are similar and well conserved in shape for both the A2 and B2 subfamilies and remain as fingerprints in the A1 and B1 subfamilies. The maintenance of such a hydrophobic pattern even for globins that lack free cysteine residues (data not shown) suggests a putative ancestral sulfide-binding function

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Rooting the Globin Family and Extracting the Most Informative Amino Acid Sequences
Ingram (1961)Citation implicitly suggested that the hemoglobin gene is paralogous to the gene of myoglobin and arose by gene duplication. Although the concept of a universal myoglobin-like ancestor has been recently discussed (Suzuki and Imai 1998Citation ), these authors, together with Goodman et al. (1988)Citation and Hardison (1998)Citation , proposed the existence of a common ancestral globin gene before the invertebrate-vertebrate divergence, which is paralogous to the ancestral myoglobin gene. As a consequence, a closely related annelid myoglobin should have been used to root our extracellular paralogous globin trees. However, the available Annelida myoglobins were too genetically distant from our extracellular annelid globins to provide good outgroups. To overcome this rooting procedure, the R. pachyptila multigenic globin tree was rooted by using the globin A family against the globin B family and vice versa because the A and B Annelida extracellular globin genes are paralogous and arose from a single ancient duplication event. This allowed us to reconstruct trees that fit the recent annelid phylogeny of Rouse and Fauchald (1995Citation , 1997)Citation .

Typically, the nonvertebrate globins exhibit a substantially greater amount of variation than that found in vertebrate globins (Kapp et al. 1995Citation ). 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 1981Citation ; Go 1981Citation ). The second exon encodes for the well-conserved and ubiquitous heme-binding fold in vertebrates (Craik, Buchman, and Beychok 1981Citation ) 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 1988Citation ; 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 2000Citation ), leading to the emergence of polychaetes, achaetes, oligochaetes, vestimentiferans, and pogonophorans. Moreover, Rouse (2001)Citation 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)Citation , Suzuki, Takagi, and Ohta (1993)Citation , 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. 1996Citation ; Yuasa, Green, and Takagi 1996Citation ; Zal et al. 1997bCitation ; Negrisolo et al. 2001Citation ). 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. 1997Citation ; Halanych, Lutz, and Vrijenhoek 1998Citation ; Rouse 2001Citation ). 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)Citation 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)Citation , 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 1995Citation ). 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)Citation 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. 2002Citation ) together with a phylogenetic tree for the vestimentiferans and the pogonophorans for the mitochondrial COI marker (Black et al. 1997; Halanych, Lutz, and Vrijenhoek 1998Citation ). 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 1987Citation ; Kini and Chan 1999Citation ) and can be caused by the relaxation of selective pressures (Kimura 1981Citation ) or positive Darwinian selection (Zhang, Rosenberg, and Nei 1998Citation ). 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 1987Citation ). 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. (1997aCitation ) and Zal, Gotoh, and Toulmond (1999)Citation 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-Hb–bearing 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).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.—Speculative evolutionary history of the sulfide-binding function through duplications that led to the actual HBL-Hbs in worms living in sulfidic environments. This evolutionary scenario is based on (1) our present knowledge of the positions of free cysteine residues in vestimentiferans, (2) the occurrence of a cysteine residue at position Cys + 1 in the A1 subfamily of O. mashikoi, and (3) knowledge of the positions of free cysteine residues in other polychaete species. Arrows symbolize selective pressures acting on extracellular globins. The scenario proposes an asymmetrical loss of the free cysteine residues before annelid radiation, the Cys + 1 residue being maintained in the A strain before annelid radiation and subsequently lost in the A1 subfamily after the annelid radiation, depending on the taxa (*: lost in Vestimentifera)

 
Is Sulfide-Binding Function via Free Cysteine a Plesiomorphic Innovation in Annelid Evolution?
H2S has been a widespread molecule on the earth's surface since prebiotic times until the present and is found at high concentrations in some places such as hydrothermal vents which have been proposed to mimic the primary earth conditions (Clark, Dowling, and Huang 1998Citation ). Tunnicliffe (1992)Citation and Tunnicliffe and Fowler (1996)Citation pointed out that hydrothermal vents have existed from Phanerozoic and possibly Archean times. In addition, Haymon, Koski, and Sinclair (1984)Citation , Banks (1985)Citation , and Little et al. (1997)Citation have described tube worm and wormlike fossils in ophiolites of 350 MYA or more (Carboniferous and Silurian). It is therefore likely that hydrothermal vent species may have adapted to sulfidic conditions since the beginning of metazoan life. Recent studies regarding organismal sulfide resistance strategies (Zierenberg, Adams, and Arp 2000Citation ) clearly indicate the fundamental contribution of H2S concentrations to species ecology. In that respect, one can strongly suspect the necessity of maintenance of the sulfide-binding function by a directional selection process (negative selection) acting on genes encoding A2 and B2 globins. Despite very few annelid hemoglobins having been studied so far, many nonsymbiotic and symbiotic polychaetes and oligochaetes colonize sulfidic environments, such as mangrove swamps, anoxic basins, marine sediments, deep-sea groundwater seeps, and hydrothermal vents (Vetter and Powell 1991Citation ; Arp, Menon, and Julian 1995Citation ; Volkel 1995Citation ; Hahlbeck, Arndt, and Schiedek 2000Citation ). So far, all the worms studied living in sulfidic environments possess (1) intracellular hemoglobins with free cysteine residues (Garey and Riggs 1986Citation ) or (2) extracellular hemoglobins with free cysteine residues (Zal et al. 1997bCitation ). Both intra- and extracellular hemoglobins are thus involved in sulfide binding. Chlorocruorins of some of these worms possess free cysteine residues as well, but no evidence for a sulfide-binding function has yet been provided. In conclusion, the SBD resemblance and the conserved positions of free cysteine residues strongly suggest that the sulfide-binding function is a widespread adaptation in annelid, vestimentiferan, and pogonophoran extracellular globins which emerged before the annelid radiation and may also have concerned some intracellular globins in the same phylum. It is interesting to note that annelids display an H2S covalent mechanism, whereas molluscs living in sulfide-rich environments bind H2S via amino acid electrostatic interactions (Nguyen et al. 1998Citation ). This suggests a functional convergence that involves different binding processes and that H2S binding via free cysteine is an annelid innovation.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
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, F. Gaill (UPMC, Paris, France), L. S. Mullineaux (WHOI, Woods Hole, Mass.), H. Felbeck (Scripps Institute, San Diego, Calif.), and the chief scientists of the HOT96 and LARVE99 cruises. We wish to thank Dr. Dugald McGlashan for helping us with the correction of this manuscript and two anonymous referees for their useful comments. This work was supported by the Conseil Régional de Bretagne and the Ministère de l'Education Nationale et de la Recherche (ACC-SV3).


    Footnotes
 
Pierre Capy, Reviewing Editor

Keywords: hexagonal bilayer hemoglobin duplication selection H2S sulfide-binding domain Riftia pachyptila Back

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 Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Arp A. J., J. J. Childress, R. D. Vetter, 1987 The sulfide-binding protein in the blood of the vestimentiferan tube-worm Riftia pachyptila, is the extracellular hemoglobin J. Exp. Biol 128:139-158[ISI]

    Arp A. J., J. G. Menon, D. Julian, 1995 Multiple mechanisms provide tolerance to environmental sulfide in Urechis caupo Am. Zool 35:132-144[ISI]

    Banks D. A., 1985 A fossil hydrothermal worm assemblage from the Tynagh lead-zinc deposit in Ireland Nature 313:128-131[ISI]

    Black M. B., K. M. Halanych, P. A. Y. Maas, W. R. Hoeh, J. Hashimoto, D. Desbruyères, R. A. Lutz, R. C. Vrijenhoek, 1997 Molecular systematics of vestimentiferan tubeworms from hydrothermal vents and cold-water seeps Mar. Biol 130:141-149[ISI]

    Blake C. C., 1981 Exons and the structure, function and evolution of haemoglobin Nature 291:616.[ISI][Medline]

    Callebaut I., G. Labesse, P. Durand, A. Poupon, L. Canard, J. Chomilier, B. Henrissat, J. P. Mornon, 1997 Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives Cell Mol. Life Sci 53:621-645[ISI][Medline]

    Castresana J., 2000 Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis Mol. Biol. Evol 17:540-552[Abstract/Free Full Text]

    Chevaldonné P., D. Jollivet, D. Desbruyères, R. D. Lutz, R. C. Vrijenhoek, 2002 Sibling species of eastern Pacific hydrothermal-vent worms (Ampharetidae, Alvinellidae, Vestimentifera) provide new mitochondrial COI clock calibration Cah. Biol. Mar. (in press).

    Childress J. J., C. R. Fischer, 1992 The biology of hydrothermal vent animals: physiology, biochemistry and autotrophic symbioses Oceanogr. Mar. Biol. Annu. Rev 30:337-441

    Clark P. D., N. I. Dowling, M. Huang, 1998 Comments on the role of H2S in the chemistry of Earth's early atmosphere and in prebiotic synthesis J. Mol. Evol 47:127-132[ISI][Medline]

    Craik C. S., S. R. Buchman, S. Beychok, 1981 O2 binding properties of the product of the central exon of beta-globin gene Nature 291:87-90[ISI][Medline]

    Dayhoff M. O., R. M. Schwartz, B. C. Orcutt, 1978 A model of evolutionary change in protein Pp. 354–352 in M. O. Dayhoff, ed. Atlas of protein sequence structure, Vol. 5., Suppl. 3. National Biomedical Research Foundation, Washington, D.C.

    Dewilde S., M. Blaxter, M. L. Van Hauwaert, J. Vanfleteren, E. L. Esmans, M. Marden, N. Griffon, L. Moens, 1996 Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata J. Biol. Chem 271:19865-19870[Abstract/Free Full Text]

    Donoghue M. J., S. Mathews, 1998 Duplicate genes and the root of angiosperms, with an example using phytochrome sequences Mol. Phylogenet. Evol 9:489-500[ISI][Medline]

    Fitch W. M., E. Markowitz, 1970 An improved method for determining codon variability in a gene and its application to the rate of fixation of mutations in evolution Biochem. Genet 4:579-593[ISI][Medline]

    Fushitani K., M. S. Matsuura, A. F. Riggs, 1988 The amino acid sequences of chains a, b and c that form the trimer subunit of the extracellular hemoglobin from Lumbricus terrestris terrestris J. Biol. Chem 263:6502-6517[Abstract/Free Full Text]

    Galtier N., M. Gouy, C. Gautier, 1996 SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny Comput. Appl. Biosci 12:543-548[Abstract]

    Garey J. R., Riggs A. F., 1986 The hemoglobin of Urechis caupo. The cDNA-derived amino acid sequence J. Biol. Chem 261:16446-16450[Abstract/Free Full Text]

    Go M., 1981 Correlation of DNA exonic regions with protein structural units in haemoglobin Nature 291:90-92[ISI][Medline]

    Goodman M., J. Barnabas, G. Matsuda, G. W. Moore, 1971 Molecular evolution in the descent of man Nature 233:604-613[ISI][Medline]

    Goodman M., G. W. Moore, G. Matsuda, 1975 Darwinian evolution in the genealogy of hemoglobin Nature 253:603-608[ISI][Medline]

    Goodman M., J. Pedwaydon, J. Czelusniak, T. Suzuki, T. Gotoh, L. Moens, F. Shishikura, D. Walz, S. Vinogradov, 1988 An evolutionary tree for invertebrate globin sequences J. Mol. Evol 27:236-249[ISI][Medline]

    Gotoh T., F. Shishikura, J. W. Snow, K. I. Ereifej, S. N. Vinogradov, D. A. Walz, 1987 Two globin strains in the giant annelid extracellular haemoglobins Biochem. J 241:441-445[ISI][Medline]

    Graur D., 1985 Amino acid composition and the evolutionary rates of protein-coding genes J. Mol. Evol 22:53-62[ISI][Medline]

    Grieshaber M. K., S. Volkel, 1998 Animal adaptations for tolerance and exploitation of poisonous sulfide Annu. Rev. Physiol 60:33-53[ISI][Medline]

    Hahlbeck E., C. Arndt, D. Schiedek, 2000 Sulfide detoxification in Hediste diversicolor and Marenzelleria viridis, two dominant polychaete worms within the shallow coastal waters of the southern Baltic Sea Comp. Biochem. Physiol. B 125:457-471[ISI][Medline]

    Halanych K. M., R. A. Lutz, R. C. Vrijenhoek, 1998 Evolutionary origins and age of vestimentiferan tube-worms Cah. Biol. Mar 39:355-358[ISI]

    Hardison R., 1998 Hemoglobins from bacteria to man: evolution of different patterns of gene expression J. Exp. Biol 201:1099-1117[Abstract/Free Full Text]

    Haymon R. M., R. A. Koski, C. Sinclair, 1984 Fossils of hydrothermal vent worms from cretaceous sulfide ores of the Samail Ophiolites, Oman Science 223:1407-1409[ISI]

    Hill R. E., N. D. Hastie, 1987 Accelerated evolution in the reactive centre regions of serine protease inhibitors Nature 326:96-99[ISI][Medline]

    Ingram V., 1961 Gene volution and the haemoglobin Nature 189:704-708[ISI][Medline]

    Iwabe N., K. Kuma, M. Hasegawa, S. Osawa, T. Miyata, 1989 Evolutionary relationship of archaebacteria, eubacteria, and eukaryotes inferred from phylogenetic trees of duplicated genes Proc. Natl. Acad. Sci. USA 86:9355-9359[Abstract]

    Jhiang M. S., J. R. Garey, A. F. Riggs, 1988 Exon-intron organization in genes of earthworm and vertebrate globins Science 240:334-336[ISI][Medline]

    Jones D. T., W. R. Taylor, J. M. Thornton, 1992 The rapid generation of mutation data matrices from protein sequences CABIOS 8:275-282[Abstract]

    Jones M. L., 1981 Riftia pachyptila Jones: observations on the vestimentiferan worm from the Galapagos Rift Science 213:333-336[ISI]

    Kapp O. H., L. Moens, J. Vanfleteren, C. N. A. Trotman, T. Suzuki, S. N. Vinogradov, 1995 Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume Protein Sci 4:2179-2190[Abstract/Free Full Text]

    Karplus K., K. Sjolander, C. Barrett, M. Cline, D. Haussler, R. Hughey, L. Holm, C. Sander, 1997 Predicting protein structure using hidden Markov models Proteins Struct. Funct. Genet. (Suppl. 1):134–139.

    Kimura M., 1981 Was globin evolution very rapid in its early stages? A dubious case against the rate-constancy hypothesis J. Mol. Evol 17:110-113[ISI][Medline]

    Kini R. M., Y. M. Chan, 1999 Accelerated evolution and molecular surface of venom phospholipase A2 enzymes J. Mol. Evol 48:125-132[ISI][Medline]

    Little C. T. S., R. J. Herrington, V. V. Maslennikov, N. J. Morris, V. V. Zaykov, 1997 Silurian hydrothermal-vent community from the southern Urals, Russia Nature 385:146-148[ISI]

    McHugh D., 2000 Molecular phylogeny of the Annelida Can. J. Zool 78:1873-1884[ISI]

    Miyamoto M. M., W. M. Fitch, 1995 Testing the covarion hypothesis of molecular evolution Mol. Biol. Evol 12:503-513[Abstract]

    Negrisolo E., A. Pallavicini, R. Barbato, S. Dewilde, A. Ghiretti-Magaldi, L. Moens, G. Lanfranchi, 2001 The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans J. Biol. Chem 276:26391-26397[Abstract/Free Full Text]

    Nguyen B. D., X. Zhao, K. Vyas, G. N. La Mar, R. A. Lile, E. A. Brucker, G. N. Phillips Jr.,, J. S. Olson, J. B. Wittenberg, 1998 Solution and crystal structures of a sperm whale myoglobin triple mutant that mimics the sulfide-binding hemoglobin from Lucina pectinata J. Biol. Chem 273:9517-9526.[Abstract/Free Full Text]

    Nicholls P., 1975 The effect of sulfide on cytochrome aa3. Isosteric and allosteric shifts of the reduced alpha-peak Biochim. Biophys. Acta 396:24-35[ISI][Medline]

    Ohta T., 2000 Mechanisms of molecular evolution Philos. Trans. R. Soc. Lond. B 355:1623-1626[ISI][Medline]

    Olson J. S., A. J. Mathews, R. J. Rohlfs, B. A. Springer, K. D. Egeberg, S. G. Sligar, J. Tame, J. P. Renaud, K. Nagai, 1988 The role of the distal histidine in myoglobin and haemoglobin Nature 336:265-266[ISI][Medline]

    Pallavicini A., E. Negrisolo, R. Barbato, S. Dewilde, A. Ghiretti-Magaldi, L. Moens, G. Lanfranchi, 2001 The primary structure of globin and linker chains from the chlorocruorin of the polychaete Sabella spallanzanii J. Biol. Chem 276:26384-26390[Abstract/Free Full Text]

    Rossi-Fanelli A., D. Cavallini, C. De Marco, 1955 Amino-acid composition of human crystallized myoglobin and haemoglobin Nature 17:377-381

    Rouse G. W., 2001 A cladistic analysis of Siboglinidae Caullrey, 1914 (Polychaeta, Annelida): formerly the phyla Pogonophora and Vestimentifera Zool. J. Linn. Soc 132:55-80[ISI]

    Rouse G. W., K. Fauchald, 1995 The articulation of annelids Zool. Scripta 24:269-301[ISI]

    ———. 1997 Cladistics and polychaetes Zool. Scripta 26:139-204[ISI]

    Sarich V. M., A. C. Wilson, 1973 Generation time and genomic evolution in primates Science 179:1144-1147[ISI][Medline]

    Shishikura F., J. W. Snow, T. Gotoh, S. M. Vinogradov, D. A. Waltz, 1987 Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris terrestris J. Biol. Chem 262:3123-3131[Abstract/Free Full Text]

    Strimmer K., A. von Haeseler, 1996 Quartet Puzzling: a quartet maximum-likelihood method for reconstructing tree topologies Mol. Biol. Evol 13:964-969[Free Full Text]

    Suzuki T., T. Gotoh, 1986 The complete amino acid sequence of giant multisubunit hemoglobin from the polychaete Tylorrhynchus heterochaetus J. Biol. Chem 261:9257-9267[Abstract/Free Full Text]

    Suzuki T., Y. Hirao, S. N. Vinogradov, 1995 Primary structure of a constituent polypeptide chain of the chlorocruorin from Sabellastarte indica Biochim. Biophys. Acta 1252:189-193[ISI][Medline]

    Suzuki T., K. Imai, 1998 Evolution of myoglobin Cell Mol. Life Sci 54:979-1004[ISI][Medline]

    Suzuki T., T. Takagi, S. Ohta, 1990 Primary structure of a constituent polypeptide chain (AIII) of the giant haemoglobin from the deep-sea tube worm Lamellibrachiasp. A possible H2S-binding site Biochem. J 266:221-225[ISI][Medline]

    ———. 1993 N-terminal amino acid sequence of 440 kDa haemoglobins of deep-sea tube worms, Lamellibrachia sp.1, Lamellibrachia sp.2 and slender vestimentifera gen.sp.1. Evolutionary relationship with annelid hemoglobins Zool. Sci 10:141-146[ISI][Medline]

    Suzuki T., T. Takagi, K. Okuda, T. Furukohri, S. Ohta, 1989 The deep-sea tube worm hemoglobin: subunit structure and phylogenetic relationship with annelid hemoglobin Zool. Sci 6:915-926[ISI]

    Swofford D. L., 1993 PAUP: phylogenetic analysis using parsimony. Version 3.1.1 Illinois Natural History Survey, Champaign

    Takagi T., H. Iwaasa, S. Ohta, T. Suzuki, 1991 Primary structure of 440 kDa hemoglobin from the deep sea tubeworm Lamellibrachia Pp. 245–249 in S. N. Vinogradov and O. H. Kapp, eds. Structure and function of invertebrate oxygen carriers. Springer, New York

    Terwilliger R. C., N. B. Terwilliger, E. Schabtach, 1980 The structure of hemoglobin from an unusual deep sea worm (Vestimentifera) Comp. Biochem. Physiol 65B:531-535[ISI]

    Thompson J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, 1997 The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25:4876-4882[Abstract/Free Full Text]

    Tunnicliffe V., 1992 The nature and origin of the modern hydrothermal vent fauna Palaios 7:338-350

    Tunnicliffe V., C. M. R. Fowler, 1996 Influence of sea-floor spreading on the global hydrothermal vent fauna Nature 379:531-533[ISI]

    Vetter R. D., G. N. Powell, 1991 Metazoan adaptation to hydrogen sulfide Pp. 109–128 in C. Bryant, ed. Metazoan life without oxygen. Chapman and Hall, London

    Vinogradov S. N., D. A. Walz, B. Pohajdak, L. Moens, O. H. Kapp, T. Suzuki, C. N. A. Trotman, 1993 Adventitious variability? The amino acid sequences of nonvertebrate globins Comp. Biochem. Physiol 106B:1-26

    Vismann B., 1991 Sulfide tolerance: physiological mechanisms and ecological implications Ophelia 34:1-27[ISI]

    Volkel S., 1995 Sulfide tolerance and detoxification in Arenicola marina and Sipunculus nudus Am. Zool 35:145-153[ISI]

    Weber R. E., S. N. Vinogradov, 2001 Nonvertebrate hemoglobins: functions and molecular adaptations Physiol. Rev 81:569-628[Abstract/Free Full Text]

    Yuasa H. J., B. N. Green, T. Takagi, 1996 Electrospray ionization mass spectrometic composition of the 400 kDa hemoglobin from the pogonophoran Oligobrachia mashikoi and the primary structures of the three major globin chain Biochim. Biophys. Acta 1296:235-244[ISI][Medline]

    Zal F., T. Gotoh, A. Toulmond, 1999 The novel function of giant hemoglobins from tubeworms and annelids P. S16. Fifth International Congress of Comparative Physiology and Biochemistry, 124A, Calgary, Alberta, Canada

    Zal F., B. N. Green, F. H. Lallier, S. N. Vinogradov, A. Toulmond, 1997a. Quaternary structure of the extracellular haemoglobin of the lugworm Arenicola marina: a multi-angle-laser-light-scattering and electrospray-ionisation-mass-spectrometry analysis Eur. J. Biochem 243:85-92[Abstract]

    Zal F., F. H. Lallier, B. N. Green, S. N. Vinogradov, A. Toulmond, 1996a. The multi-hemoglobin system of hydrothermal vent tube worm Riftia pachyptila: II—Complete polypeptide chain composition investigated by Maximum Entropy analysis of mass spectra J. Biol. Chem 271:8875-8881[Abstract/Free Full Text]

    Zal F., F. H. Lallier, J. S. Wall, S. N. Vinogradov, A. Toulmond, 1996b. The multi-hemoglobin system of hydrothermal vent tube worm Riftia pachyptila: I—Reexamination of the number and masses of its constituents J. Biol. Chem 271:8869-8874[Abstract/Free Full Text]

    Zal F., E. Leize, F. H. Lallier, A. Toulmond, A. Van Dorsselaer, J. J. Childress, 1998 S-sulfohemoglobin and disulfide exchange: the mechanisms of sulfide binding by Riftia pachyptila hemoglobins Proc. Natl. Acad. Sci. USA 95:8997-9002[Abstract/Free Full Text]

    Zal F., T. Suzuki, Y. Kawasaki, J. J. Childress, F. H. Lallier, A. Toulmond, 1997b. Primary structure of the common polypeptide chain b from the multi-haemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: an insight on the sulfide binding-site Proteins Struct. Funct. Genet 29:562-574[ISI][Medline]

    Zhang J., H. F. Rosenberg, M. Nei, 1998 Positive Darwinian selection after gene duplication in primate ribonuclease genes Proc. Natl. Acad. Sci. USA 95:3708-3713[Abstract/Free Full Text]

    Zierenberg R. A., M. W. Adams, A. J. Arp, 2000 Life in extreme environments: hydrothermal vents Proc. Natl. Acad. Sci. USA 97:12961-12962[Free Full Text]

    Zuckerkandl E., R. T. Jones, L. Pauling, 1960 A comparison of animal hemoglobin by triptic peptide pattern analysis Proc. Natl. Acad. Sci. USA 46:1349-1360[ISI]

Accepted for publication April 12, 2002.