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Regular Article |
a Laboratoire de Physiologie Générale et Comparée, UMR 8572 CNRS, Muséum National d'Histoire Naturelle, 75231 Paris Cedex 05, France
ABSTRACT
The ß subunits of the two gonadotropins (GTH1 and GTH2) and of the thyroid-stimulating hormone (TSH) of a chondrostean fish, Acipenser baeri, were cloned. These new sequences and selected representative members of ß subunits of vertebrate glycoprotein hormones, including tetrapod follicle-stimulating hormones (FSH) and luteinizing hormones (LH), allowed us to infer the phylogenetic relationships within this family. Both distance matrix and maximum parsimony methods were used on both nucleotide and amino acid sequences, with bootstrapping evaluation over 1000 replicates. The four trees obtained had highly similar topologies. In each case, three monophylogenetic lineages, TSH, GTH1-FSH, and GTH2-LH were clearly identified. The three monophylogenetic lineages were supported by 21–23 specific characters at the amino acid level, out of a total of 121 characters. The resolved topologies within each monophyletic hormone cluster were congruent with the known phylogenetic relationships between the related species. The inferred parental relationships within gonadotropins are in agreement with data concerning their biological functions. The present study demonstrates that GTH1 and GTH2 are the actinopterygian homologues of tetrapod FSH and LH, respectively.
anterior pituitary, FSH, LH, pituitary, TSH
INTRODUCTION
Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH), are members of the vertebrate glycoprotein hormone family [1–4]. These hormones are secreted from pituitary cells as heterodimers composed of an 
and a ß subunit. In a given species, the subunit is common to the three hormones, whereas the ß subunit is specific. In primates, the glycoprotein hormone family also comprises a chorionic gonadotropin, the ß subunit of which arose from a duplication of the LHß gene [5]. In equidaes, the single LHß gene has acquired the capacity to be expressed in the placenta, as well as in the pituitary [6]. The glycoprotein hormones have been characterized to the amino acid or nucleotide sequence level only in tetrapods and in teleost fish. In teleosts, in addition to the TSH, two pituitary gonadotropins have been described [4]. They were originally named GTH I (or GTH1) and GTH II (GTH2) according to the order of elution on chromatography [7]. On the basis of their biological action, secretion pattern during the reproductive cycle, or sequential expression during development, GTH1 and GTH2 were proposed to represent the functional counterparts of FSH and LH, respectively [8, 9]. The phylogenetic trees obtained from aligned sequences also clearly showed that GTH2 is directly related to tetrapod LH [4, 10]. The phylogenetic origin of GTH1 was, however, more difficult to ascertain from inferred phylogenetic reconstructions. This was due to the rapid evolution of the GTH1ß subunit in teleosts that provokes distortions in the phylogenetic trees constructed from ß subunits alone [4, 10]. In a recent paper, Li and Ford [11] proposed a phylogenetic tree from a data set in which the ß subunits were aligned together with the common subunits. Actually, although conceivable, there is no evidence that the subunit and the ß subunits are derived from a common ancestor gene. In addition, only five amino acids (four cysteines and one glycine) were present in positions of more than 150 amino acids in the alignment produced by Li and Ford. The phylogenetic reconstruction based on this alignment is thus biased. Considering the ß subunits alone, the high rate of evolution of GTH1 ß subunits in teleosts still represents a major difficulty. In an attempt to address this problem, we cloned the three ß subunits of the glycoprotein hormones of a chondrostean fish, the sturgeon, Acipenser baeri. The chondrosteans separated early on from the growing actinopterygian branch, before the appearance of the first teleosts. As a result, their hormones should have characteristics closer to those from the common ancestors of Actinopterygies and Sarcopterygies (including the tetrapods) than those of teleosts. Consequently, these data should be helpful for the phylogenetic inference.
MATERIALS AND METHODS
Animals
Sturgeons (A. baeri) were reared at the Moulin des Logeries (CREA-CEMAGREF, France). Mature female sturgeons were killed. The pituitary glands were removed and kept in liquid nitrogen.
RNA Extraction and cDNA Synthesis
Total RNA was extracted from 4 pooled, frozen pituitary glands by using RNA-Plus solution (Bioprobe Systems, Montreuil, France). Poly(A) RNA was purified by using Dynabeads Oligo(dT)25 mRNA purification kit (Dynal AS, Oslo, Norway), from 125 µg of total pituitary RNA. A 0.5-µg quantity of poly(A)+ RNA was reverse transcribed into cDNA using the Marathon cDNA Amplification kit (Clontech Laboratories, Inc., Palo Alto, CA). The blunt-ended, double-stranded cDNAs were ligated to Marathon adaptors in order to perform 5'- and 3'-rapid amplification of cDNA ends (RACE).
Cloning Procedure
All polymerase chain reactions (PCRs) were performed in 25 µl of a solution containing 50 mM Tris pH 8.3, 0.25 mg/ml bovine serum albumin, 3 mM MgCl2, 0.2 mM dNTP, 0.2–1 µM of each primer, and 0.5 units of Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). Sequences of primers other than Marathon adaptor primers (MAP) are given in Table 1. They were purchased from Eurogentec. PCR mixtures were loaded into 30-µl capillary tubes, the ends of which were flame sealed. Capillaries were cycled in the 1605 Rapid Cycler (Idaho Technology, Idaho Falls, ID) by the following program: 1 min at 94°C, then 35–45 cycles of 5–10 sec at 94°C, 8–10 sec at 55–60°C, and 20–30 sec at 72°C. A last step of 20–30 min at 72°C was performed in order to optimize the 3'A tailing. Amplified fragments were eluted from agarose gel or, if free from contaminating bands, directly ligated to pGEM-T Easy vector (Promega Corporation, Madison, WI) and cloned in JM109 high-efficiency competent cells (Promega). Sequencing of the recombinant plasmids were performed on both strands with an ABI Prism 377 DNA sequencer with ABI Prism Big Dye Primer and Amplitaq DNA pol, FS (Perkin Elmer Applied Biosystem, Foster City, CA).
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For the GTH2 ß subunit, two overlapping fragments were obtained using LH5 and LH55 with LH3 (Table 1) that were purified after further amplification using LH33 as a nested primer (all four primers designed from the GTH2 ß subunit peptide sequence of Acipenser stellatus). The 5' extension was obtained using MAP1 and LH3, followed by a nested PCR using MAP2 and LH33. On the basis of the sequences obtained, two specific primers (LHs5 and LHs55) were designed and used together with MAP1 and MAP2 in two successive rounds of amplification to generate the 3' extension. In total, five partially overlapping fragments from independent PCR reactions were sequenced on both strands.
From the first round of amplification for the GTH1 ß subunit using FSHd5 and GlHd3 (designed from conserved regions), a fragment of the expected length—180 base pairs (bp)—was obtained and sequenced. Using the same GlHd3 and MAP 1, two bands, of 500 and 350 bp, were obtained and purified. The two bands were subjected to analytical PCR using oligonucleotides corresponding to the different types of ß-subunits. The longest one appeared to correspond to GTH1, whereas the shortest was putatively identified as a fragment of the TSH ß subunit. Two different clones corresponding to the longest (GTH1-type) fragment were sequenced. The 3' end was obtained using two specific primers (FSHs5 and FSHs55; Table 1) and the MAP1 and MAP2 in two successive rounds of amplification. In total, four overlapping fragments were sequenced.
For the TSH ß subunit, a 200-bp fragment was first obtained using TSHd5 (based on a conserved region) and GlHd3 and sequenced. The 5' extension was generated by nested PCR on the 350-bp (MAP 1/Glhd3) fragment (see above) as template, using MAP 2 and TSHd3. The 3' extension was generated by two successive rounds of amplification, using TSHd5/MAP 1 then TSHd55/MAP 2. Three overlapping fragments were sequenced.
Sequence Data and Phylogenetic Analysis
The nomenclature GTH1 and GTH2 was applied for teleost gonadotropins type I and type II. The same nomenclature was used for chondrostean gonadotropins. Most of the sequences were extracted from EMBL or SwissProt data banks (Table 2) or copied from the literature [12–16]. A. stellatus GTH2ß sequence was a personal communication (from S. Khilko and M. Govoroun). Whenever possible and depending on the available sequences (77 nucleotide sequences and 96 amino acid sequences in total), the following guidelines were used for the establishment of sequence data sets: two representative sequences for each taxonomic subgroup were used (i.e., two rodents or two salmonids) in order to distinguish real synapomorphies for the subgroup (evolved characters shared by the subgroup) from apomorphies for the chosen species (evolved characters specific to the species); the mammalian sequences were represented by marsupials, rodents, carnivora, and artiodactyles; primate and equidae sequences were omitted because of the recent evolutionary events specific to these groups in the LH lineage, and the sequences from modern fish (Percomorphs) were not used because of the rapid evolution of teleost GTH1 that causes saturations (a character at a given position results from numerous successive evolutionary substitutions) and thus distortion of the trees [17, 18]. The phylogenetic analysis was performed using both maximum parsimony and distance matrix methods on both amino acid (46) and nucleotide (40) sequences. The same set of data was used for maximum parsimony and distance matrix methods. Sequences (121 amino acids, or 363 nucleotides, long) were aligned by hand (see Fig. 1 for the alignment of representative sequences) between the eighth amino acid left of the first conserved cysteine (i.e., corresponding to the first amino acid of mammalian LH and thus including signal peptide sequences from others) and the seventh amino acid to the right of the 12th cysteine (i.e., corresponding to the last amino acid of mammalian FSH). All characters were assigned the same weight. A bootstrapping procedure [19] was used to evaluate the robustness of the topologies. This consists of constructing a large number of data sets, each one using randomly picked-up characters (amino acids or nucleotides) from the aligned sequences, with replacement, and inferring phylogenetic trees for each resampling data set. A consensus tree is computed that represents the majority topology with, for each node, the value corresponding to the number of times this particular node has been obtained, expressed as a percentage. These bootstrap values reflect the robustness of the nodes obtained from the original data set. They are to be taken into account with caution because even false nodes can be supported by high bootstrap values [20]. PAUP, version 3.1.1. (Phylogenetic Analysis Using Parsimony; Illinois Natural History Survey, Champaign, IL), was used for the maximum parsimony method. Bootstrap sampling using a heuristic search was performed over nonexcluded/nonignored characters only (104 informative amino acids out of 121, 305 informative over 363 nucleotides). The following settings were used: 1000 replicates, simple sequence addition, TBR branch swapping, no steepest descent, no topological constraints. The outgroup consisted of the TSH sequences (known as monophyletic because of the two inserted amino acids shared by actinopterygians and sarcopterygians). The trees were unrooted. For the distance matrix method, different programs from the PHYLIP package, version 3.57c were used: SEQBOOT (Bootstrap) to generate 1000 data sets, DNADIST (Kimura two-parameter type) or PROTDIST (Kimura's distance) for the construction of the distance matrices, NEIGHBOR (Neighbor-Joining) for the generation of 1000 phylogenetic trees, and CONSENSE for the computation of the consensus tree. The trees were also unrooted. In order to trace characters, the trees were transferred to the MacClade software program 3.08a version (Sinauer associates, Sunderland, MA).
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RESULTS
Acipenser baeri GTH2 ß Subunit
Two types of clones were obtained from the pooled cDNAs (EMBL accession numbers AJ251656 and AJ251657; because of space limitations, the nucleotide sequences are not presented). The full-length cDNA of the complete one was 570 bp long without the poly(A) tail. It encoded a protein of 137 amino acids, comprising a signal peptide of 22 amino acids (Fig. 1). The position of the signal peptide cleavage site was determined by comparison with the GTH2 sequence from A. stellatus. The two types of clone differed mainly by a single deletion (just after the seventh cysteine codon), followed by a single insertion 18 nucleotides downstream, allowing recovery of the reading frame. Only the sequence from the longest one, the one given in Figure 1, was used for the following studies. The highest degree of similarity (Fig. 1 and Table 3) was found with teleost GTH2 (59%–64% and 65%–68% at the amino acid and nucleotide levels, respectively) and Rana LH (56% at the amino acid level). The similarity with other hormones was in the ranges of 44%–49% and 54%–56% at the amino acid and nucleotide levels, respectively.
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Acipenser baeri GTH1 ß Subunit
Two types of clone were obtained that differed from each other in the length of the 5' untranslated region (one was five nucleotides shorter and had a further deletion of four nucleotides) and in having a single silent mutation on the first cysteine codon of the mature peptide. The complete cDNA of the longest clone (EMBL accession number AJ251658) was 1051 bp long. It encoded a 128-amino acid-long protein including a signal peptide of 20 amino acids, the first amino acid of the mature peptide being determined by analogy with mammalian FSH (Fig. 1). As for FSH from tetrapods or GTH1 from the Japanese eel (a primitive teleost), the sturgeon GTH1 presented two potential N-linked glycosylation sites in conserved positions. The highest degree of similarity (Fig. 1 and Table 3) was found with Rana FSH (56% at the amino acid level) and Anguilla GTH1 (54% and 64% at the amino acid and nucleotide levels, respectively). The similarity with other hormones was in the ranges 46%–50% and 53%–58% at the amino acid and nucleotide levels, respectively.
Acipenser baeri TSH ß Subunit
The complete cDNA was 1010 bp long (EMBL accession number AJ251659). It encoded a protein of 143 amino acids including a 20-amino acid-long signal peptide as deduced from comparison with mammalian or teleost TSHs (Fig. 1). Like other TSHs, sturgeon TSH ß subunit presented two amino acids inserted between the fifth and sixth cysteines as compared with the case of gonadotropin ß subunits. An unusual potential glycosylation site was identified a few amino acids downstream of the seventh cysteine. The highest degree of similarity (Fig. 1 and Table 3) was found with avian and mammalian TSH (59%–60% at the amino acid level and 61%–64% at the nucleotide level). The similarity with amphibian and teleost TSH was lower (48%–54% and 58%–61% at the amino acid and nucleotide levels, respectively). The similarity with other hormone ß subunits ranged 38%–44% at the amino acid level and ranged 50%–56% at the nucleotide level.
Phylogenetic Analysis
The alignment used for the phylogenetic analysis is presented for a sample of representative sequences in Figure 1. A similarity matrix (number of identical characters divided by the total number of aligned characters for each couple of sequences) is presented for the selected sequences on Table 3. Both distance matrix and maximum parsimony methods were used on both nucleotide and amino acid sequences. For the maximum parsimony method, the heuristic search on nonexcluded/nonignored characters yielded the following lengths and fit measures: length, 950; consistency index, 0.642 for amino acid sequences; length, 2113; consistency index 0.351 for nucleotide sequences. The 50% majority-rule consensus trees are presented (the nodes that are present in less than 50% of the trees are depicted as polytomies [multifurcations] at the lower rank; Fig. 2). The four topologies obtained were almost identical, the differences being mainly restricted to the bootstrap values of certain nodes. In each case, the glycoprotein hormone ß subunits were separated into three monophyletic groups composed of the TSH, the sarcopterygian LH clustered with the actinopterygian GTH2, and the FSH clustered with the GTH1.
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The robustness of the LH-GTH2 cluster was rather strong (up to 94%), except with the parsimony method on nucleotide sequences, for which the bootstrap value was only 54% (Fig. 2). In each case, the Acipenser GTH2 branched out at the basis of teleost GTH2 sequences to form a monophyletic actinopterygian GTH2 cluster that was quite strongly supported by bootstrap values (70–95%), reflecting the high similarity between teleost and chondrostean GTH2. The position of the avian LHs was not resolved because of their numerous autapomorphies (characters that are specific to avian LH; Fig. 1) and to their higher degree of similarity with teleost GTH2 than with mammalian LH (Fig. 1 and Table 3). The position of amphibian LH was also unsolved, probably because of the highest overall similarity with teleost GTH2 that are equilibrated by the largest number of autapomorphies with mammalian LH. Apart from these unsolved positions, the topologies of the subunit trees were congruent with the known phyletic relationships between the related species. An exception was the mispositioning of salmonid (Oncorhynchus and Coregonus) GTH2 in the neighbor tree of nucleotide sequences, which could not be explained. With MacClade, 77 amino acid characters out of 121 characters were traced down to the root of the LH-GTH2 lineage (Fig. 1). These plesiomorphic characters were thus probably present in the LH-GTH2 ß subunit of the common ancestor to the primitive Actinopterygies and Sarcopterygies. Out of these 77 characters, 23 were autapomorphies (evolved characters that are specific to the LH-GTH2 lineage) or, at least, were not plesiomorphic for the other lineages.
The less strongly supported group was that of FSH-GTH1 (Fig. 2), with bootstrap values of 61%–68%. This was due to the rapid evolution of teleost GTH1, even in the absence of percomorph species sequences: 51%–62% similarity between selected species, as compared with 73%–79% similarity for the GTH2 sequences from the same species and with 57%–73% for the TSH sequences (Table 3). Except for the distance matrix method on nucleotide sequences (Fig. 2B), the Acipenser GTH1 was clustered with tetrapod FSH (because of its better overall similarity to amphibian FSH than to teleost GTH1), but the bootstrap values were always below 50%. Seventy-five plesiomorphic characters were tentatively identified, 21 of them being autapomorphic for this FSH-GTH1 lineage or, at least, not plesiomorphic for the other lineages (Fig. 1).
The TSH group was supported by the highest bootstrap value in each case (97–100%). This was mainly due to the two TSH specific insertions. The Acipenser TSH was always clustered with tetrapod TSH, but except for the case of using the distance matrix method on amino acid sequences (Fig. 2A), these nodes were not supported by high bootstrap values. This was due to the stability of tetrapod TSH, to a better similarity score of Acipenser TSH with mammalian TSH, and to a relatively high rate of evolution of teleost TSH (Fig. 1 and Table 3). Seventy-two plesiomorphic characters were identified, from which 21 were not plesiomorphic for the other lineages (Fig. 1).
Since the trees were unrooted, the total number of symplesiomorphies (plesiomorph characters for the three lineages together) could not be traced. However, 46 of them could be identified that were not dependent on the position of the root (Fig. 1). They corresponded to characters that were either symplesiomorph or plesiomorph for one or two lineages and were present in at least two different phyletic groups in the other lineage(s) (e.g., in mammals and birds or in mammals and teleosts). Three different positions for the rooting are theoretically possible. Written according to the New Hampshire format, the three possible rooted trees would be as follows:
None of these was strongly supported by the respective numbers of inferent synapomorphies (evolved characters common to two lineages). However, eight synapomorphies (the two amino acid insertions of TSH were considered as one single evolutionary event) were in support of a direct ancestor for the two gonadotropin lineages (the first proposed tree), six supported the second hypothesis, and four were in support of the third hypothesis (Fig. 1).
DISCUSSION
The sequence of the ß subunit of the three glycoprotein hormones from A. baeri was obtained. Two types of clone were obtained for the GTH1 and the GTH2 ß subunits, whereas all the TSH ß subunit clones presented the same sequence. No differences in the encoded peptides were seen between the two GTH1 ß subunit clones, whereas the two GTH2 ß subunit clones encoded peptides with marked differences. The main difference was due to an alteration in the open reading frame. Acipenser baeri was shown to be tetraploid and has as many as 250 chromosomes (2n) [21]. Whether the two types of clones were encoded by two expressed gene copies generated through polyploidization or resulted from allelic heterogeneity of a single expressed gene (cDNAs were from pooled pituitary glands collected from four animals) was not determined.
Acipenser GTH2 sequence was closer to that of primitive teleosts than to the amphibian Rana LH sequence. In contrast, Acipenser GTH1 and TSH sequences had a higher similarity score with amphibian FSH and TSH sequences than with teleost GTH1 and TSH sequences. Chondrostean fish are closer to teleosts (they both belong to the actinopterygian branch that diverged from the sarcopterygian branch some 400 million years ago) than to amphibians (a taxonomic group that diverged from the main sarcopterygian branch some 250 million years ago). The discrepancies between the relative similarity scores reflect the acceleration in the rate of evolution of the GTH1 and TSH ß subunit lineages in teleosts in comparison to that of GTH2.
Using these new sequences and applying restricting rules for the constitution of the sampling data set, phylogenetic trees could be inferred. Three monophyletic lineages were clearly identified: TSH, GTH1-FSH, and GTH2-LH. The reliability of the inferences leans on the fact that the four topologies obtained, using both distance matrix and maximum parsimony methods on both nucleotide and amino acid sequences, were very close if not identical. In addition, the three monophylogenetic lineages were supported by more than 20 autapomorphies (at the amino acid level). Also, the topologies within each monophyletic hormone cluster (when resolved according to the 50% majority-rule consensus) were congruent with the known phyletic relationships between the related species, indicating that the sampling data set did not generate misleading distortions. Finally, the structural relationships between GTH1 and FSH and between GTH2 and LH are in good agreement with functional data, on the basis of GTH1 and GTH2 synthesis and secretion patterns, of steroidogenic actions [8, 9], or of receptor tissue distribution during the reproductive cycle [9], as compared with those of FSH and LH.
Once the three lineages are definitely identified, the next task is to determine the order of apparition of these lineages from the common ancestral gene. An outgroup corresponding to sequences of products related to the common ancestral gene is necessary. It has long been proposed that the subunit and the ß subunits derived from a common ancestral gene. This hypothesis was first proposed on the basis of a loose similarity between mammalian and ß subunits [22], even before the tertiary structure of both subunits was discerned [23, 24]. The and ß subunits belong to the cystine-knot superfamily of peptides, a family that also contains several growth factors and a variety of other peptides [25, 26]. These various protein families share a common overall topology but have very little sequence homology. In other words, their structural resemblance does not necessary mean that they share a common origin. It could actually be a structural convergence. In any case, the alignment of sequences between and ß subunits (and possibly other members of the cystine-knot family) for a phylogenetic reconstruction should be based on the cystine-knot backbone that is the common structural constraint of the family. This was not used by Li and Ford [11] in their phylogenetic analysis of glycoprotein hormone family, in which two out of the six cysteines that constitute the backbone were not aligned between the and the ß subunits. The phylogenetic inferences from this alignment are thus biased. More data are needed to assign a common origin for the and the ß subunits of glycoprotein hormones and to contribute to a reliable alignment of their sequences. In the absence of peptide sequence definitely recognized as directly related to the glycoprotein hormone ß subunit ancestral sequence, the order of apparition of the three lineages could not be determined. Theoretically, three rooted trees are possible. In the first hypothetical tree, the gonadotropins are monophyletic (the two gonadotropin lineages would derive from a common direct ancestor), whereas in the two others, the gonadotropins are paraphyletic (the gonadotropin lineages would have arisen independently). The different hypothesis can be tentatively evaluated by the number of supporting synapomorphies; the overall distances cannot be used because of the differences in the rate of evolution in the three lineages. The first hypothetical tree (monophyly of gonadotropins) seems to be more realistic. It is the one supported by the highest number of synapomorphies. It is also tempting to assume that the hormone-receptor specificities within a given species will be more or less affected by the degree of parental relationships between orthologous hormones. In that sense, it was shown that LH was able to bind to FSH receptor and to efficiently displace FSH binding in the amphibian Rana catesbeiana [27]. Similar results were obtained in salmonids with GTH1 and GTH2 [28, 29]. These observations plead for a recent (relative to the emergence of amphibians and teleosts and hence, to the split between Sarcopterygians and Actinopterygians) individualization of gonadotropin specificities and thus for the first rooting hypothesis, (TSH, (LH-GTH2, FSH-GTH1)).
In conclusion, this study demonstrates that actinopterygian GTH1 and GTH2 are structurally related to tetrapod FSH and LH, respectively. Because functional data are consistent with this conclusion, the nomenclature of actinopterygian gonadotropins should be revised, and FSH and LH naming should be adopted in place of GTH1 and GTH2.
ACKNOWLEDGMENTS
We are indebted to Pierre Elie, Patrick Williot, and Thierry Rouault (CEMAGREF) for helping us to collect pituitary glands from the sturgeons. The amino acid sequence of A. stellatus LH-GTH2 was kindly provided by Marina Govorun (INRA, Rennes, France) and Sergeï Khilko, then at the NIAID (Immunology Lab, Bethesda), now deceased. It was sequenced in 1992 by Mariana Gawinowicz (Dept of Medicine, Columbia University, New York). This sequence has not yet been published. We would like to thank Jean-Pierre Furet and Corine Giraut-Deville (INRA) for the sequencing. Many thanks to Guillaume Lecointre (Ichthyology Lab, MNHN, Paris) for helpful discussions about phylogenetic reconstructions.
FOOTNOTES
First decision: 13 January 2000.
1 This work was supported in part by the GDR 1005 (CNRS). 
2 Correspondence: Bruno Quérat, Laboratoire de Physiologie Générale et Comparée, UMR 8572 CNRS, Muséum National d'Histoire Naturelle, 7, rue Cuvier, 75231 Paris Cedex 05, France. FAX: 33 1 4079 3618; querat{at}mnhn.fr
Accepted: February 29, 2000.
Received: December 7, 1999.
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