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Molecular Evidence that Sclerolinum brattstromi Is Closely Related to Vestimentiferans, not to Frenulate Pogonophorans (Siboglinidae, Annelida).

KENNETH M. HALANYCH [1][*]

ROBERT A. FELDMAN [2]

ROBERT C. VRIJENHOEK [3]

Abstract. Siboglinids, previously referred to as pogonophorans, have typically been divided into two groups, frenulates and vestimentiferans. Adults of these marine protostome worms lack a functional gut and harbor endosymbiotic bacteria. Frenulates usually live in deep, sedimented reducing environments, and vestimentiferans inhabit hydrothermal vents and sulfide-rich hydrocarbon seeps. Taxonomic literature has often treated frenulates and vestimentiferans as sister taxa. Sclerolinum has traditionally been thought to be a basal siboglinid that was originally regarded as a frenulate and later as a third lineage of siboglinids, Monilifera. Evidence from the 18S nuclear rDNA gene and the 16S mitochondrial rDNA gene presented here shows that Sclerolinum is the sister clade to vestimentiferans although it lacks the characteristic morphology (i.e., a vestimentum). The rDNA data confirm the contention that Sclerolinum is different from frenulates, and further supports the idea that siboglinid evolution has been driv en by a trend toward increased habitat specialization. The evidence now available indicates that vestimentiferans lack the molecular diversity expected of a group that has been argued to have Silurian or possibly Cambrian origins.

Introduction

Siboglinids were formerly called pogonophorans and include two groups of marine protostomes, frenulates and vestimentiferans, that are commonly referred to as beard-worms and tubeworms, respectively. Both groups lack a functional gut as adults and rely on endosymbiotic bacteria for nutrition. They have a closed circulatory system and possess a metamerized tail region called the opisthosoma. Vestimentiferans are distinguished from frenulates by the presence of a vestimentum, a winged region near the anterior of the organism. Both taxa occur in reducing environments and typically are found at depths below several hundred meters. Due to the limited availability of samples and the difficulty of retrieving live specimens, several aspects of their biology (e.g., reproduction, physiology) are still poorly understood. Vestimentiferans, in general, have been better studied than frenulates because they are keystone species in eastern Pacific hydrothermal vent habitats and in Pacific and Caribbean seeps.

The taxonomic literature concerning frenulate and vestimentiferan siboglinids has a colorful and confusing history. One taxonomic scheme recognizes frenulates (aka pogonophorans sensu stricto) and vestimentiferans as distinct phyla (Jones, 1985). Alternatively, vestimentiferans have also been recognized as a class within the phylum Pogonophora (Jones, 1981; Ivanov, 1994). Others place frenulates and vestimentiferans within the phylum Annelida (Land and N[phi]rrevang, 1977; Kojima et al., 1993; Bartolomaeus, 1995; McHugh, 1997; Rouse and Fauchald, 1997; also see Southward, 1988). The latter hypothesis has been supported by recent morphological (Rouse and Fauchald, 1995, 1997), embryological (Young et al., 1996; Southward, 1999), and molecular analyses (Kojima et al., 1993; McHugh, 1997; Black et al., 1997; Kojima, 1998; Halanych et al., 1998). To further complicate matters, a ranked classification scheme has produced different names for the same clade of organisms. Vestimentiferans have been called Vestimenti fera (Jones, 1981), Obturata (Jones, 1981; Southward, 1988; Southward and Galkin, 1997), and Afrenulata (Webb, 1969). Frenulates have been called Pogonophora (Jones, 1985), Frenulata (Webb, 1969), Perviata (Southward, 1988), and originally Siboglinidne (Caullery, 1914).

Hereafter we apply the following nomenclature: (1) Vestimentifera are equated with Obturata and Afrenulata; (2) Frenulata are equated with Perviata and Pogonophora (sensu Jones, 1985); (3) Monilifera is a third monogeneric dade that includes Sclerolinum; and (4) Siboglinidae refers to the dade that includes Vestimentifera, Frenulata, and Monilifera. We recognize that the term "Pogonophora" is more commonly used and that rules of priority for nomenclature do not apply to higher taxa. However, we have opted to use the term "Siboglinidae" throughout this manuscript to emphasize that this group of organisms represents derived annelids (McHugh, 1997; Rouse and Fauchald, 1997). We restrict the term "pogonophoran" to common usage.

Even among siboglinids, there has been one group, Sclerolinum, that has been particularly problematic in terms of phylogenetic position. Unlike most frenulates that live in the mud, Sclerolinum species can live on decaying organic material like wood or rope made from natural fibers (Webb, 1964a; Southward, 1972). This taxon was originally considered a member of the frenulate family Polybrachiidae (Southward, 1961), but Webb (1964b), mainly citing differences in the postannular region, argued that Sclerolinum could not be ascribed to either of the two orders (Thecanephria and Athecanephria) of siboglinids recognized at the time (vestimentiferans had not been discovered yet). He erected a new family, Sclerolinidae, that he states should "have order rank." Ivanov (1991) more formally recognized the unique nature of Sclerolinum, and in 1994 he proposed that Frenulata (= Perviata), Monilifera (= Sclerolinidae), and the Vestimentifera be regarded as three taxa with equal rank (i.e., classes within the phylum Pogon ophora). Additionally, Ivanov (1994) further suggested that Monilifera are allied to the Vestimentifera on the basis of the common absence of several characters (e.g., spermatophores, telosomal diaphragm, metasoma preannular and postannular regions) relative to the Frenulata. Southward (1999) suggested that Monilifera might be similar to the ancestral siboglinid form, thus predicting that it should occupy a basal position in siboglinid phylogeny. Distinguishing between these hypotheses on the placement of Sclerolinum will allow us to test the notion of Black et al. (1997) that habitat preference or specificity may be an important factor in siboglinid evolution. If Black et al. are correct, Sclerolinum is expected to occupy a position between frenulates and vestimentiferans (which may be consistent with Ivanov's ideas), and not a position basal to the frenulate-vestimentiferan dade.

To date, molecular studies that include siboglinids have either focused on vestimentiferans (Williams et al., 1993; Black et al., 1997; Kojima et al., 1997; Halanych et al., 1998) or have addressed siboglinid origins (Winnepenninckx et al., 1995a; Kojima et al., 1993; Kojima, 1998; McHugh, 1997). Most studies have included only one frenulate representative. Although Black et al. (1997) included two "frenulate" siboglinids, one of these, the Loihi worm, was undescribed. Additionally, several 18S sequences were reported in a symposium contribution (Halanych et al., 1998) for which page limitations did not permit detailed analyses or explanation. Herein we extend these previous analyses by increasing the sampling of frenulates, including Scierolinum, and using novel 1 8S rDNA and 16S rDNA data. The present findings support the notion that habitat requirements have been important in siboglinid evolution. Additionally, frenulates are sister to a Sclerolinum-vestimentiferan clade, the latter of which showed limite d diversity suggestive of a recent radiation within the clade.

Materials and Methods

Taxa employed

Table 1 lists the species analyzed and GenBank accession numbers for the rDNA sequences used in this study. The frenulate and vestimentiferan operational taxonomic units (OTUs) included in this study represent all of the currently recognized genera available to the authors. The addition of closely related species within a genus would have increased OTUs without increasing the phylogenetic signal for the issues under examination and were therefore excluded. For example, there are no nucleotide differences observed in the 18S rDNA of Escarpia spicata (Guaymas Basin) and E. laminata (Florida Escarpment). Limiting the number of OTUs also reduced computation time, allowing for more thorough analyses. Unless otherwise noted, collection localities correspond to those given in Black et al. (1997). Siboglinum ekmnani, S. fiordicum, and Sclerolinum brattstromi were collected near Bergen, Norway, and identified by Eve Southward, Marine Biological Association of the United Kingdom. Identification of the frenulates Spiro brachia and Polybrachia were made by Eve Southward on the basis of animal and tube morphology. Both specimens were collected by TVGrab from the Aleutian Trench (57[degrees]27.394'N, 148[degrees]00.013'W) at a depth of 4890 m on the German research vessel Sonne.

The non-siboglinid annelid OTUs for the 18S data were chosen to represent a diversity of lineages for which sequences were available. The arthropod (Artemia) sequence was designated as the most distant outgroup for rooting purposes. Based on both morphology (e.g., Eernisse et al., 1992) and molecular studies (e.g., Halanych et al., 1995; Winnepenninckx et al., 1995a; Aguinaldo et al., 1997; Eernisse, 1997), arthropods are clearly outside of the protostome worm radiation. Because siboglinids are not closely related to molluscs and because of rate heterogeneity problems within the Mollusca, only a single representative (the aplacophoran Scutopus) was used. Due to alignment limitations, outgroups employed in the 16S analyses-a leech, an oligochaete, two polynoid polychaetes, and an echiurid-were more limited (see Table 1). Because different investigators collected the data at different times, there was not a 1:1 correspondence in OTUs between data sets. We felt it more important to present all the relevant data rather than trim taxa from the data sets. The aligned data sets are available at the journal's Supplement's page (http:// www.mbl.edu/BiologicalBulletin/VIDEOIBB.video.html) and at TREEBASE (http://phylogeny.harvard.edu/treebase).

Data collection

Total genomic DNA was extracted using a modified hexadecyl-trimethyl-ammonium bromide (CTAB) protocol (Doyle and Dickson, 1987). The entire 18S nuclear rDNA gene was amplified via PCR (polymerase chain reaction), using the universal metazoan oligonucleotide primers 1 8e and 18P (Halanych et al., 1998). A region of the 16S mitochondrial rDNA was amplified using 16Sar-5' and 16Sbr-3' primers (Palumbi, 1996). Each 50 [micro]l reaction consisted of about 50 ng of template DNA, 0.5 [micro]M of each primer, 2.5 mM [MgCl.sub.2] 200 [micro]M dNTPs, 5 [micro]l of manufacturer's 10X reaction buffer, and 1.5 U Taq polymerase (Promega Inc., Wisconsin). Cycling profiles were as follows: 18S--initial denaturation at 95 [degrees]C for 3 min, 35 cycles of amplification (denaturation at 95 [degrees]C for 1 min, annealing at 50 [degrees]C for 2 min, extension at 72 [degrees]C for 2 min 30 s), and a final extension at 72 [degrees]C for 5 min; 16S--initial denaturation at 94 [degrees]C for 2 min, 40 cycles of amplification (den aturation at 94 [degrees]C for 30 s, annealing at 46 [degrees]C for 30 s, extension at 72 [degrees]C for 1 min), and a final extension at 72 [degrees]C for 7 min. PCR products were purified using the QIAEX II gel extraction kit (Qiagen Inc., California). Approximately 60 ng of purified PCR product was used in sequencing reactions according to the manufacturer's instructions (FS Dye Termination Mix or Big Dye, Applied Biosystems Inc., California). The reaction profile was 25 repetitions of denaturation at 94 [degrees]C for 30 s, annealing at 50 [degrees]C for 15 s, and extension at 64 [degrees]C for 4 min. Dye-labeled fragments were separated by electrophoresis on a Perkin Elmer ABI 373A or 377 DNA sequencer. Both strands of the PCR product were sequenced. In addition to the PCR primers, the oligo-nucleotide primers used for sequencing are given in Halanych et al. (1998) or Hillis and Dixon (1991). The sequences were assembled and verified using the AutoAssembler and Sequence Navigator programs (Applied Bio-sy stems Inc., California). The terminal primer regions were not included in the sequences submitted to GenBank or in the phylogenetic analyses.

Phylogenetic analyses

Sequence alignment was produced with a Clustal W program (Thompson et al., 1994) and subsequently corrected by hand using the protostome secondary structure models available through the Ribosomal Database project (http://rdp.cme.msu.edu/html/). Regions that could not be unambiguously aligned (e.g., divergent loop domains) were excluded from analyses. Tree reconstructions were implemented with the PAUP* 4.0b4b2 program (Swofford, 2000), and MacClade 3.06 (Maddison and Maddison, 1992) was used for character and tree analyses. Neighbor-joining (NJ), parsimony, and maximum likelihood (ML) analyses were performed and yielded similar results. In the interest of brevity, results and discussion will focus on ML analyses.

NJ trees were reconstructed under Jukes-Cantor, Kimura-2-parameter, Tamura-Nei, general-time-reversible, and log/det models. All except log/det were examined under equal rates of among-site rate variation using the empirically derived gamma shape parameter, [alpha], of 0.3 (see Swofford et al., 1996, for summary of different assumptions used in these models). A Kishino-Hasegawa (1989) likelihood evaluation of the resulting topologies revealed no significant differences between models for either the 16S or the 18S data. Kishino-Hasegawa evaluations estimated a six-substitution-type rate matrix for which nucleotide base frequencies were set to empirical values and [alpha] was estimated. NJ bootstraps consisted of a log/det correction (model was arbitrarily chosen) with 1000 iterations. Parsimony analyses consisted of heuristic searches with 100 random sequence additions and tree-bisection-reconnection (TBR) branch swapping. Transitions (Ti) and transversions (Tv) were given equal weighting. ML evaluation of pa rsimony topologies was the same as for NJ topologies. One thousand iterations were used for parsimony bootstrap analyses. When using likelihood to search for the "best" tree (as opposed to evaluating given trees), computation time was limiting. Therefore, we used a nucleotide model with two substitution types where the Ti/Tv ratio was set to the value estimated for the best parsimony tree (empirical base frequencies were used). ML searches were heuristic with 10 random sequence-addition replicates. ML bootstraps employed the "Faststep" option with 100 iterations.

Results

The 18S rDNA data set consisted of 26 OTUs and 1935 nucleotide positions. Of the 1614 nucleotide positions that could be unambiguously aligned, 34.6% (559 positions) were variable and 18.7% (303 positions) were parsimony informative. Figure 1 shows the single best likelihood tree (Ln likelihood = -8260.55148) recovered. All search methods in all analyses found a monophyletic siboglinid clade (bootstrap support was [greater than or equal to]98% for all methods). Resolution within the vestimentiferan clade, as well as between annelid groups, was poor, however. The moniliferan Sclerolinum brattstromi falls out with the vestimentiferan taxa in all analyses (bootstrap [greater than or equal to]98%). The remaining frenulates form a distinct sister-clade to the Sclerolinum-vestimentiferan clade with [greater than or equal to]99% bootstrap support.

Resolution among annelid taxa and within the vestimentiferans was poor due to the lack of phylogenetic signal. Because this paper does not focus on the annelid radiation, we did not try to enhance resolution among all annelid taxa. However, we did attempt to boost the signal within the vestimentiferan clade by employing a less inclusive taxonomic alignment. For metazoan 18S sequences, inclusion of broader taxonomic diversity can often create larger regions of ambiguous alignment that should not be included in analyses, due to poor assumptions about positional homology. Thus by reducing the taxonomic breadth examined, the phylogenetic signal can potentially be increased by a "better" alignment (Halanych, 1998). Unfortunately, even when just the siboglinids were aligned, little genetic diversity was observed, and the vestimentiferan taxa were still poorly resolved (not shown). The exception was Lamellibrachia barhami, which was consistently placed as the most basal vestimentiferan. Table 2 shows the logdet/par alinear distances (below diagonal) and absolute distances (above diagonal) for this less-inclusive, siboglinid-only alignment (in which most divergent domains could be unambiguously aligned). Even though the distance values for the siboglinid-only alignment are only slightly greater than the full alignment values, the greatest distance within vestimentiferans was only 0.02 (with a maximum of 25 nucleotide differences), revealing that there was very little 18S genetic diversity within this group.

The 16S rDNA data set consisted of 24 OTUs, each with 497 nucleotide positions. Of the 465 nucleotide positions that could be unambiguously aligned, 60.4% (281 positions) were variable and 47.7% (222 positions) were parsimony informative. The reconstructed topology (Ln likelihood = -3967.21062), Figure 2, was qualitatively similar to the 18S topology. Siboglinids are divided into two major lineages: vestimentiferans plus the moniliferan Sclerolinum brattstromi (bootstrap support 83% for ML and 100% for NJ and parsimony) and a frenulate sister-clade (bootstrap support [greater than or equal to]94% in all analyses). Again, S. brattstromi was basal to the vestimentiferans. In a departure from the 18S analyses, Riftia pachyptila, not Lamellibrachia barhami, often fell out as the most basal vestimentiferan. However, this was never supported by [greater than]54% bootstrap support; ML analyses that excluded the non-siboglinid outgroups revealed that the base of the Vestimentifera was poorly resolved with 16S data. A comparison of genetic divergence values (Table 3) indicates that there was limited genetic Variation within vestimentiferans ([less than or equal to]0.11 log/det distance; a maximum of 47 nucleotide differences).

As for the frenulate clade, neither 185 or 16S supported a monophyletic Siboglinum; but because only two Siboglinum species were examined, additional taxa are needed to verify the status of this frenulate taxon. Additionally, we performed Kishino-Hasegawa (1989) likelihood evaluation for both genes to test the monophyly of the frenulate and vestimentiferan-Sclerolinum clades. To this end, we used the constraints option in PAUP* 4.0b4b2 to conduct parsimony heuristic searches (specifics same as above) to find the best trees that were consistent and inconsistent with the monophyly of these clades. Both the 16S and the 18S data significantly support the monophyly of both groups (18S frenulates--average ML score supporting monophyly = -8244.69, non-monophyly score = -8278.135, P value [less than] 0.01; 16S frenulates--monophyly = -3894.889, non-monophyly = --3927.49, P value [less than] 0.005; 18S vestimentiferan-Sclerolinum--monophyly = --8244.69, non-monophyly = --827 1.922, P value [less than] 0.05; 16S vestim entiferan-Sclerolinum--monophyly = -3894.889, non-monophyly = --3911.802, P value [less than] 0.05).

Discussion

The monophyly of siboglinids (aka, Pogonophora sensu latu) is supported by morphological (Southward, 1988, 1993; Rouse and Fauchald, 1995; Rouse, 2001), embryological (Southward, 1999), and molecular (Winnepenninckx et al., 1995a; Black et al., 1997; McHugh, 1997; Halanych et al., 1998, this study) evidence. Thus, in agreement with others (Southward, 1988, 1999; Ivanov, 1994; McHugh, 1997), we see no support for the recognition of vestimentiferans and frenulates as having fundamentally different body plans (i.e., "phyla" sensu Jones, 1985). The assertion made by Webb (1964b) and later by Ivanov (1991, 1994) that Scierolinum was notably different from frenulates is validated by the present data. Moreover, we found that Sclerolinum brattstromi is closely allied to the vestimentiferans, and does not occupy a position basal to a frenulate-vestimentiferan clade, confirming Ivanov's (1991; 1994; Ivanov and Selivanova, 1992) ideas that moniliferans occupy a position intermediate between vestimentiferans and frenula tes.

Southward (1993) also suggested a possible evolutionary link between Sclerolinum and vestimentiferans. This contention is confirmed by the present analysis, as well as a recent morphological cladistic analysis (Rouse, 2001). Using 44 morphological characters coded for all recognized siboglinid genera, Rouse found support for the monophyly of Frenulata, Vestimentifera, and the Sclerolinum-vestimentiferan clade. However, our use of nomenclature differs from Rouse with regard to the term Monilifera, which he applies to the Sclerolinum-vestimentiferan clade. Because this term was originally (Ivanov and Selivanova, 1992) applied to only Sclerolinum, and because of the morphological differences from vestimentiferans, Rouse's use of the term will inject confusion into the literature. Although we acknowledge that Monilifera, as defined here, is redundant with the generic name Sclerolinum, several aspects of siboglinid evolution and taxonomy are in need of additional study. Thus, we have chosen not to name this clade until more is understood about siboglinid evolution.

The placement of Sclerolinum was especially interesting in the context of the evolution of habitat preference. Previous studies of vestimentiferans (Black et al., 1997), clams (Peek et al., 1997), mussels (Craddock et al., 1995), and shrimp (Shank et al., 1999) reveal that vent-endemic organisms are related to, and possibly derived from, species associated with hydrocarbon seeps that occur near subduction zones and continental margins. Furthermore, recent observations (Feldman et al., 1998; Baco et al., 1999; Distel et al., 2000) reveal that several symbiont-bearing clams, vestimentiferan tubeworms, and mussels can survive on rotting organic material, such as wood or a whale carcass. The moniliferan S. brattstromi and related species (e.g., S. javanicum, S. minor, and S. major) are typically found growing on decaying organic material such as wood or rope (Webb, 1964a, b; Southward, 1972; Ivanov and Selivanova, 1992). Other members of the genus, (e.g., S. sibogae and S. magdalenae) lived buried in mud (Southw ard, 1972). These habitat preferences suggest that affinity for a mud or silt habitat was ancestral in siboglinids, allowing us to speculate that a pattern of evolution from low-oxygen, sedimented habitats to decaying organic material to hydrocarbon seeps to hydrothermal vents has occurred within the Sclerolinumvestimentiferan clade.

Although neither the 18S nor the 16S data clearly resolve relationships within the Vestimentifera, the cytochrome c oxidase subunit I (COI) data of Black et at. (1997) show seep tubeworms to be basal to vent tubeworms (but see Williams et al., 1993). This pattern in the evolution of habitat preference roughly proceeds from less reducing to more reducing (greater sulfide and methane availability) environments. A similar evolutionary trend was observed in bathymodiolid mussels (Craddock et al., 1995; Distel et al., 2000). Examination of additional taxa is needed to verify whether this is a general trend in the evolution of vent and seep taxa.

All molecular studies to date (Williams et al., 1993; Black et al., 1997; and Tables 2 and 3) reveal that vestimentiferans exhibit very limited molecular diversity for a group suggested to be several hundred million years old. This lack of diversity may be due to a slowdown in the rate of molecular change (i.e., nucleotide substitution) in vestimentiferans, a recent common origin for extant vestimentiferans, or possibly both. For the present 18S rDNA sequences, vestimentiferans appear to have experienced a significant molecular slowdown relative to the frenulates or other protostome taxa (Table 4; as judged using an HKY plus gamma correction model in the HyPhy software package distributed by S. Muse, Department of Statistics, North Carolina State University). With 16S data, only 13.4% of tests between frenulates and members of the vestimentiferan-Sclerolinum clade were significant. Although this value is not statistically significant, it is a greater percentage than is found within either group ([sim]-3%), suggesting that a limited rate discrepancy may exist. Similar rate disparities were not observed for COI data (Black et al., 1997), but only one frenulate was used in the comparison. Nonetheless, we concluded that present-day vestimentiferans constitute a young evolutionary group.

In contrast, previous interpretation of Silurian tubeworm fossils (Little et al., 1997) as vestimentiferans suggested that these worms constitute an ancient animal lineage. It is possible that the Silurian tubeworm fossils represent an earlier offshoot from an ancient siboglinid lineage, but this will be impossible to test as the fossils lack the necessary soft-tissue preservation. Additionally, we note that many wormlike invertebrates make tubes. For example, some alvinellid polychaetes observed during our recent expedition to vents along the Southern Eastern Pacific Rise (32[degrees]S, 100[degrees]W) occupied tubes with diameters comparable to the tubes of mature Riftia pachyptila. Many of the alvinellid tubes were partially overgrown by sulfide chimneys, and thus were effectively "fossilized." Although we are not convinced of the interpretation of Silurian fossils as representative of an extant lineage of vestimentiferans, we should point out that specimens from the Cretaceous are convincing (Little et al ., 1999). In contrast, all the hydrothermal vent-endemic taxa that have been examined with appropriate molecular tools appear to be from relatively recent radiations (i.e., [less than]100 MY; Black et al., 1997; Peek et al., 1997; Shank et al., 1999; McArthur and Koop, 1999; but see McArthur and Tunnicliffe, 1998, for possible exceptions).

Acknowledgments

We appreciate thoughtful interactions and support of our colleagues at Rutgers University. We wish to thank the crews and staff of the RIV Altantis/Alvin, the German research vessel Sonne, and the Bergen Marine Station in Espegrend for their help in obtaining organisms. Samples of the Spirobrachia and Polybrachia were provided by R. Lutz (with help from Gyongyver Levai) and identified by Eve Southward, who has been especially generous with information and guidance. The Escarpiid n. sp. was kindly made available by Verena Tunnicliffe and Eve Southward. Material from Norway was collected with aid from the Training and Mobility of Researchers Programme of the European Union, through Contract NO. ERBFMGECT950013 to Eve Southward. Research was supported by an NSF grant, 0CE96-33131 to RCV and R. Lutz. The Richard B. Sellars Endowed Research Fund and The Andrew W. Mellon Foundation Endowed Fund for Innovative Research provided partial support to KMH. This is WHOI contribution number 10443.

(*.) To whom correspondence should be addressed. E-mail: khalanych@whoi.edu

(1.) Biology Department MS 33, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543; (2.) Molecular Dynamics, Inc., part of Amersham Pharmacia Biotech, 928 East Arques Ave., Sunnyvale, California 94086-4250; and (3.) Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, California 95039

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TABLE 1
Taxa used in rDNA analyses
                                 GenBank Accession a
Organism                         18S rDNA             16S rDNA
Pogonophora
  Frenulata
    Galathealinum brachiosum     AF168738             AF315040
    Polybrachia sp.              AF168739             AF315037
    Siboglinum fiordicum GB      X79876 b
    Siboglinum fiordicum         AF315060             AF315039
    Siboglinum ekmani            AF315062             AF315038
    Spirobrachia sp.             AF168740             AF315036
  Vestimentifera
    Escarpia spicata             AF168741             AF315041
    Escarpiid n. sp.                                  AF315053
    Lamellibrachia barhami       AF168742             AF315043
                                                      AF315044
                                                      AF315045
                                                      AF315047
    Oasisia alvinae              AF168743             AF315052
    Ridgeia piscesae             AF168744             AF315048
                                                      AF315051
                                                      AF315054
    Ridgeia piscesae GB          X79877 b
    Riftia pachyptila            AF168745             AF315049
                                                      AF315050
    Tevnia jerichonana           AF168746             AF315042
  Monilifera
    Sclerolinum brattstromi      AF315061             AF315046
Annelida
  Alvinellidae
    Paralvinella palmiformis     AF168747
  Chaetopterida
    Chaetopterus variopedatus    U67324 c
  Hirudinea
    Haemopis sanguisuga          X91401 d
    Hirudo medicinalis                                AF315058
  Oligochaete
    Enchytraeus sp.              Z83750 d
  Phyllodocida
    Glycera americana            U19519 e
  Polynoidea
    Lepidonotopodium funbriatum                       AF315056
    Branchipolynoe symmytilida                        AF315055
  Sabellida
    Sabella pavonina             U67144 c
  Tubificidae
    Tubifex sp.                                       AF315057
Echiura
    Ochetostoma erythrogrammon   X79875 b
    Urechis sp.                                       AF315059
Sipuncula
    Phascolosoma granulatum      X79874 b
Nemertea
    Lineus sp.                   X79878 b
Mollusc
    Scutopus ventrolineatus      X91977 f
Priapulida
    Priapulus caudatus           X80234 g
Arthropod
    Artemia salina               X01723 h
(a) Unless otherwise noted, sequences were obtained in this study.
(b) Sequence from Winnepenninckx et al. (1995a).
(c) Sequence from Nadot and Grant (unpublished).
(d) Sequence from Kim et al. (1996).
(e) Sequence from Halanych et al. (1995).
(f) Sequence from Winnepenninckx et al. (1996)
(g) Sequence from Winnepenninckx et al. (1995b).
(h) Sequence from Nelles at al. (1984).
TABLE 2
Pairwise distances for the siboglinid-only 18S rDNA data set; absolute
distances above diagonal and log/dat distances below diagonal
                              1      2      3       4       5
 1 Spirobrachia              --    109     113      87     132
 2 Polybrachia               0.07    --      9     104     138
 3 Galathealinum             0.07    0.01    --    106     142
 4 Siboglinum ekmnani        0.05    0.06    0.06    --    116
 5 Siboglinum fiordicum      0.08    0.09    0.09    0.07    --
 6 Siboglinum fiordicuin GB  0.08    0.08    0.09    0.07    0.00
 7 Escarpia                  0.08    0.09    0.09    0.07    0.09
 8 Ridgeia                   0.08    0.09    0.09    0.07    0.09
 9 Ridgeia GB                0.08    0.09    0.09    0.07    0.09
10 Oasisia                   0.08    0.09    0.09    0.07    0.09
11 Riftia                    0.08    0.09    0.09    0.07    0.09
12 Tevnia                    0.08    0.09    0.09    0.07    0.09
13 Lamellibrachia            0.07    0.09    0.09    0.07    0.08
14 Sclerolinum               0.08    0.09    0.09    0.07    0.09
                              6       7       8       9       10
 1 Spirobrachia              131     124     122     125     124
 2 Polybrachia               137     139     140     140     140
 3 Galathealinum             141     142     143     143     143
 4 Siboglinum ekmnani        117     113     110     113     110
 5 Siboglinum fiordicum        5     140     136     136     140
 6 Siboglinum fiordicuin GB    --    143     139     139     143
 7 Escarpia                    0.09    --      7      14      10
 8 Ridgeia                     0.09    0.00    --      8       7
 9 Ridgeia GB                  0.09    0.01    0.00    --     14
10 Oasisia                     0.09    0.01    0.00    0.01    --
11 Riftia                      0.09    0.01    0.01    0.01    0.01
12 Tevnia                      0.09    0.00    0.00    0.01    0.00
13 Lamellibrachia              0.09    0.01    0.01    0.01    0.01
14 Sclerolinum                 0.09    0.02    0.02    0.02    0.02
                              11      12      13     14
 1 Spirobrachia              131     125     121     120
 2 Polybrachia               147     142     140     136
 3 Galathealinum             150     145     143     139
 4 Siboglinum ekmnani        121     112     107     112
 5 Siboglinum fiordicum      143     138     134     139
 6 Siboglinum fiordicuin GB  146     141     137     140
 7 Escarpia                   19       6      13      31
 8 Ridgeia                    17       4      12      32
 9 Ridgeia GB                 21      11      19      38
10 Oasisia                    20       7      14      32
11 Riftia                      --     16      25      39
12 Tevnia                      0.01    --     11      30
13 Lamellibrachia              0.01    0.01    --     28
14 Sclerolinum                 0.02    0.02    0.02   --
TABLE 3
Pairwise distances for 16S rDNA data set; absolute distances above
diagonal and log/det distances below diagonal
                          1      2      3      4      5      6
 1 Spirobrachia           --   41     52     65     65     119
 2 Polybrachia           0.09   --     8     68     67     113
 3 Galathealinum         0.12   0.02   --    81     79     118
 4 Siboglinum fiordicum  0.16   0.17   0.21   --    90     129
 5 Siboglinum ekmani     0.15   0.16   0.19   0.23   --    121
 6 Escarpia              0.30   0.30   0.31   0.35   0.33    --
 7 Escarpiid n. sp.      0.29   0.29   0.30   0.34   0.31    0.03
 8 Tevnia                0.30   0.28   0.29   0.33   0.32    0.07
 9 Ridgeia 1             0.30   0.29   0.28   0.33   0.33    0.08
10 Ridgeia 2             0.30   0.29   0.30   0.33   0.32    0.08
11 Ridgeia 3             0.28   0.28   0.30   0.31   0.31    0.08
12 Oasisia               0.30   0.30   0.31   0.32   0.32    0.09
13 Lamellibrachia 1      0.31   0.30   0.32   0.35   0.34    0.08
14 Lamellibrachia 2      0.32   0.29   0.31   0.36   0.34    0.09
15 Lamellibrachia 3      0.32   0.30   0.31   0.35   0.33    0.08
16 Lamellibrachia 4      0.30   0.28   0.31   0.34   0.32    0.08
17 Riftia 1              0.32   0.28   0.27   0.32   0.32    0.08
18 Riftia 2              0.32   0.28   0.29   0.33   0.32    0.09
19 Sclerolinum           0.31   0.31   0.32   0.34   0.32    0.15
20 Branchipolynoe        0.38   0.39   0.41   0.38   0.38    0.39
21 Lepidonotopodium      0.37   0.34   0.38   0.39   0.39    0.35
22 Urechis               0.42   0.38   0.40   0.42   0.43    0.41
23 Tubifex               0.35   0.35   0.40   0.34   0.34    0.35
24 Hirudo                0.43   0.42   0.46   0.44   0.40    0.44
                           7       8       9       10      11
 1 Spirobrachia          116     114     112     116     111
 2 Polybrachia           111     108     108     108     106
 3 Galathealinum         117     113     107     115     114
 4 Siboglinum fiordicum  125     120     116     121     116
 5 Siboglinum ekmani     117     120     116     119     115
 6 Escarpia               12      29      32      35      34
 7 Escarpiid n. sp.        --     30      29      32      27
 8 Tevnia                  0.07    --     16      17      17
 9 Ridgeia 1               0.07    0.04    --      2       2
10 Ridgeia 2               0.08    0.04    0.00    --      5
11 Ridgeia 3               0.06    0.04    0.00    0.01    --
12 Oasisia                 0.08    0.06    0.04    0.05    0.04
13 Lamellibrachia 1        0.08    0.10    0.09    0.09    0.08
14 Lamellibrachia 2        0.08    0.10    0.08    0.10    0.08
15 Lamellibrachia 3        0.07    0.09    0.08    0.08    0.08
16 Lamellibrachia 4        0.07    0.09    0.08    0.09    0.07
17 Riftia 1                0.08    0.08    0.09    0.10    0.10
18 Riftia 2                0.09    0.08    0.09    0.09    0.10
19 Sclerolinum             0.13    0.18    0.19    0.19    0.17
20 Branchipolynoe          0.38    0.37    0.40    0.39    0.38
21 Lepidonotopodium        0.33    0.31    0.32    0.32    0.30
22 Urechis                 0.39    0.42    0.43    0.43    0.41
23 Tubifex                 0.34    0.36    0.37    0.37    0.35
24 Hirudo                  0.42    0.43    0.45    0.44    0.42
                           12      13      14      15      16
 1 Spirobrachia          114     119     121     121     117
 2 Polybrachia           113     114     111     113     109
 3 Galathealinum         118     119     118     119     117
 4 Siboglinum fiordicum  117     128     130     129     125
 5 Siboglinum ekmani     117     123     125     123     119
 6 Escarpia               36      32      36      31      31
 7 Escarpiid n. sp.       34      30      31      29      27
 8 Tevnia                 26      43      43      40      39
 9 Ridgeia 1              17      35      33      32      33
10 Ridgeia 2              21      37      40      34      36
11 Ridgeia 3              16      35      36      34      32
12 Oasisia                 --     38      39      41      41
13 Lamellibrachia 1        0.09    --      7      11      11
14 Lamellibrachia 2        0.09    0.02    --     14      12
15 Lamellibrachia 3        0.10    0.03    0.03   --       4
16 Lamellibrachia 4        0.10    0.03    0.03    0.01    --
17 Riftia 1                0.10    0.09    0.09    0.08    0.07
18 Riftia 2                0.11    0.10    0.10    0.08    0.08
19 Sclerolinum             0.18    0.13    0.13    0.13    0.12
20 Branchipolynoe          0.38    0.39    0.38    0.40    0.39
21 Lepidonotopodium        0.32    0.34    0.34    0.35    0.34
22 Urechis                 0.40    0.40    0.38    0.41    0.40
23 Tubifex                 0.34    0.35    0.36    0.36    0.35
24 Hirudo                  0.43    0.45    0.45    0.46    0.45
                           17      18      19      20      21
 1 Spirobrachia          118     122     120     129     125
 2 Polybrachia           106     106     117     126     114
 3 Galathealinum         107     112     122     134     125
 4 Siboglinum fiordicum  117     122     127     128     129
 5 Siboglinum ekmani     115     120     120     126     129
 6 Escarpia               35      37      58     126     119
 7 Escarpiid n. sp.       35      39      52     124     114
 8 Tevnia                 35      37      70     122     109
 9 Ridgeia 1              38      37      70     123     106
10 Ridgeia 2              40      38      73     127     109
11 Ridgeia 3              40      43      68     124     107
12 Oasisia                43      47      69     123     108
13 Lamellibrachia 1       38      42      51     127     115
14 Lamellibrachia 2       36      43      53     127     116
15 Lamellibrachia 3       34      35      52     130     118
16 Lamellibrachia 4       31      35      50     128     116
17 Riftia 1                --      6      58     122     112
18 Riftia 2                0.02    --     62     129     117
19 Sclerolinum             0.15    0.16    --    120     120
20 Branchipolynoe          0.38    0.39    0.36    --     52
21 Lepidonotopodium        0.34    0.35    0.36    0.13    --
22 Urechis                 0.40    0.42    0.40    0.38    0.38
23 Tubifex                 0.36    0.36    0.36    0.34    0.35
24 Hirudo                  0.47    0.48    0.43    0.43    0.42
                           22      23     24
 1 Spirobrachia          141     123     141
 2 Polybrachia           129     121     134
 3 Galathealinum         134     133     146
 4 Siboglinum fiordicum  142     121     142
 5 Siboglinum ekmani     140     116     133
 6 Escarpia              130     117     144
 7 Escarpiid n. sp.      126     112     139
 8 Tevnia                133     119     139
 9 Ridgeia 1             127     115     138
10 Ridgeia 2             134     120     142
11 Ridgeia 3             131     116     138
12 Oasisia               128     114     139
13 Lamellibrachia 1      128     116     146
14 Lamellibrachia 2      125     118     148
15 Lamellibrachia 3      131     118     150
16 Lamellibrachia 4      129     116     148
17 Riftia 1              125     116     149
18 Riftia 2              133     121     154
19 Sclerolinum           129     119     143
20 Branchipolynoe        126     114     139
21 Lepidonotopodium      126     118     136
22 Urechis                 --    119     150
23 Tubifex                 0.36    --    122
24 Hirudo                  0.46    0.36   --
TABLE 4
Percent of significant tests when comparing relative substitution rates
between the two major siboglinid clades
Test type *               Number   Significant    Percent
                         of tests    results    significant
Between frenulates and
    vestimentiferan-
    Sclerolinum clade
  18S rDNA                 480         331         69.0
  16S rDNA                 350          47         13.4
Within frenulates
  18S rDNA                 150          53         35.3
  16S rDNA                  50           1          2.0
Within vestimentiferan-
    Sclerolinum clade
  18S rDNA                 280          80         28.6
  16S rDNA                 455          13          2.9
Results of relative rates tests based on an HKY plus gamma model in the
HyPhy program. The program is distributed by S. Muse, Department of
Statistics, North Carolina State University.
(*) The 18S comparisons employed all Lophotrochozoan outgroups.
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Author:HALANYCH, KENNETH M.; FELDMAN, ROBERT A.; VRIJENHOEK, ROBERT C.
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Aug 1, 2001
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