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Endosymbionts of Siboglinum fiordicum and the Phylogeny of Bacterial Endosymbionts in Siboglinidae (Annelida)

¹ Department of Biological Sciences, 101 Rouse Life Sciences Building, Auburn University, Auburn, Alabama 36849
² Department of Animal Sciences, Cellular & Molecular Biosciences Program, 234 Upchurch Hall, Auburn University, Auburn, Alabama 36849
³ Departments of Animal Sciences and Anatomy, Physiology & Pharmacology, Cellular & Molecular Biosciences Program, 236 Upchurch Hall, Auburn University, Auburn, Alabama 36849
⁴ Department of Marine Science, 340 Chapman Hall CB#3300, University of North Carolina—Chapel Hill, Chapel Hill, North Carolina 27599

^* To whom correspondence should be addressed. E-mail: thornhill.dan{at}gmail.com or ken{at}auburn.edu

	Abstract

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Siboglinid worms are a group of gutless marine annelids that are nutritionally dependent upon endosymbiotic bacteria. Four major groups of siboglinids are known—vestimentiferans, moniliferans, Osedax spp. and frenulates. Although endosymbionts of vestimentiferans and Osedax spp. have been previously characterized, little is currently known about endosymbiotic bacteria associated with frenulate and moniliferan siboglinids. This is particularly surprising given that frenulates are the most diverse and widely distributed group of siboglinids. Here, we molecularly characterize endosymbiotic bacteria associated with the frenulate siboglinid Siboglinum fiordicum by using 16S rDNA ribotyping in concert with laser-capture microdissection (LCM). Phylogenetic analysis indicates that at least three major clades of endosymbiotic -proteobacteria associate with siboglinid annelids, with each clade corresponding to a major siboglinid group. S. fiordicum endosymbionts are a group of –proteobacteria that are divergent from bacteria associated with vestimentiferan or Osedax hosts. Interestingly, symbionts of S. fiordicum, from Norway, are most closely related to symbionts of the frenulate Oligobrachia mashikoi from Japan, suggesting that symbionts of frenulates may share common evolutionary history or metabolic features.

	Introduction

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Siboglinids, previously known as the phyla Pogonophora and Vestimentifera, are now recognized as a monophyletic clade within annelid worms (McHugh, 1997; Black et al., 1997; Halanych et al., 2001; Rouse et al., 2004; Struck et al., 2007). As adults, they lack a mouth, gut, and anus and instead have evolved a storage organ for microbial endosymbionts (Southward et al., 2005). This organ, called a trophosome, is perfused with a blood-vascular system to facilitate transport of nutrients to and from endosymbionts (Southward et al., 2005). As currently recognized, siboglinids comprise four major lineages and encompass more than 150 recognized species (Rouse et al., 2004; Glover et al., 2005; Halanych, 2005). Vestimentifera, the best-known lineage, includes large (up to 2 m in length) tubeworms from hydrothermal vents (e.g., Riftia pachyptila) and hydrocarbon cold-seeps (e.g., Lamellibrachia and Escarpia). Other groups include moniliferans, Sclerolinum spp., which live on decaying wood and other organic matter (Halanych et al., 2001), and the recently discovered whale-bone-eating worms, Osedax spp. (Rouse et al., 2004; Glover et al., 2005). Last are Frenulata (a.k.a. perviates), composing about 75% of all recognized siboglinids. They are found throughout the world's oceans, along continental margins, continental slopes, fjords, trenches, at least one mud volcano, and the periphery of vents and seeps (e.g., Ivanov, 1963; Webb, 1963; Southward, 1979, 1988, 1991; Southward et al., 1981; Schmaljohann and Flügel, 1987). In comparison to vestimentiferans, frenulates are typically diminutive, about the thickness of a coarse human hair, and more than 10–20 cm long (Southward et al., 2005). Surprisingly, the symbiotic biology of frenulates, which are found in many more marine habitats than vestimentiferans, has been largely ignored.

Most siboglinids are thought to host a single species of chemosynthetic endosymbiont, which they obligately depend upon for organic nutrition (Distel et al., 1988; but see Chao et al., 2007; Vrijenhoek et al., 2007). Despite this obligate association, there is no evidence for vertical transmission of symbionts from parents to offspring in Siboglinidae. For those hosts examined to date (mainly vestimentiferans), no bacteria have been found in the gonadal tissues, sperm, eggs, or early-stage larvae (Cavanaugh et al., 1981; Cary et al., 1989, 1993), and phylogenies of the host and the symbiont are not congruent (Feldman et al., 1997; Di Meo et al., 2000; Nelson and Fisher, 2000). If symbionts were transmitted from parent to offspring, a tightly coevolved phylogeny would be predicted (i.e., Clark et al., 2000; Degnan et al., 2004), as has been observed in species of the vesicomyid clam Calyptogena (Peek et al., 1998). Furthermore, symbionts of the vestimentiferan R. pachyptila possess an apparently functional flagellin gene; this may indicate a motile, and therefore nonsymbiotic, stage of this endosymbiont's life cycle (Millikan et al., 1999). Although no molecular studies have identified free-living siboglinid symbionts thus far (Southward et al., 2005), bacteria of similar ultrastructure and size have been identified from both the trophosome of Siboglinum poseidoni and the external environment (Schmaljohann and Flügel, 1987). Symbionts are apparently newly acquired from the environment each generation.

Siboglinid endosymbionts are typically thought to fall into one of three general metabolic categories: (1) thiotrophic symbionts that oxidize sulfide or other reduced inorganic compounds and fix CO₂ by the Calvin-Benson cycle, (2) methanotrophic symbionts that use methane as both the source of energy for metabolism and the carbon for assimilation, or (3) heterotrophic symbionts found in the bone-eating worms, Osedax spp. (Fisher, 1990; Stewart et al., 2005; Goffredi et al., 2005, 2007). However, R. pachyptila symbionts also have enzymes for both the energy-generating tricarboxylic acid (TCA) cycle and an alternative carbon fixation pathway via the reverse TCA cycle, indicating that the metabolic mode of these symbionts is more complicated than previously believed (Markert et al., 2007). In theory, the R. pachyptila symbiont community could switch between RuBisCO-autotrophy, heterotrophic oxidation of carbon storage compounds by the TCA cycle, and autotrophic growth by the reverse TCA cycle (Markert et al., 2007). Regardless of the metabolic mode, the host is primarily dependent upon endosymbionts for its nutritional and energetic requirements (Southward et al., 1986), although some host species may also absorb organic materials from the surrounding environment (Southward and Southward, 1970). The host provides a stable environment and access to necessary substrates (e.g., sulfide, methane, oxygen, carbon dioxide) for bacterial metabolism, while the bacteria provide energy via the release of small nutritive molecules (Felbeck and Jarchow, 1998) or possibly by digestion of symbionts (Bright et al., 2000; Goffredi et al., 2005).

Studies to date have focused on endosymbionts associated with the larger vestimentiferans (reviewed in McMullin et al., 2003) and Osedax spp. (Goffredi et al., 2005, 2007), with few molecular studies addressing frenulate endosymbiont diversity (i.e., Oligobrachia mashikoi [Kimura et al., 2003a; Kubota et al., 2007] and Oligobrachia "sp. JT-1" [Naganuma et al., 2005]). Molecular analyses based on 16S rDNA place the majority of known vestimentiferan and Osedax spp. endosymbionts within the –proteobacteria (e.g., McMullin et al., 2003; Stewart et al., 2005; Goffredi et al., 2007). Other bacterial groups have been detected in hospite with vestimentiferans as well, such as , β, and –proteobacteria in Lamellibrachia spp. (Naganuma et al., 1997a, b; Elsaied and Naganuma, 2001; Elsaied et al., 2002; Kimura et al., 2003b) and –proteobacteria and Cytophaga-Flavobacterium-Bacteroides in Ridgeia piscesae (Chao et al., 2007). However, the stability and persistence of these non--proteobacteria associations is not clear at this time (Naganuma et al., 2005; Stewart et al., 2005; Chao et al., 2007). Species of Osedax symbiotically associate with Oceanospirillales-like -proteobacteria endosymbionts (Goffredi et al., 2005, 2007). Currently, there are no studies of endosymbiont diversity in moniliferans.

Because the symbiosis of frenulates is poorly understood, we molecularly characterized bacteria associated with the frenulate siboglinid Siboglinum fiordicum Webb 1963. To this end, we used standard molecular and phylogenetic approaches with laser-capture microdissection (LCM). S. fiordicum endosymbionts are a distinct group of –proteobacteria not associated with the endosymbionts of vestimentiferan or Osedax hosts.

	Materials and Methods

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Data collection
In September 1998, Siboglinum fiordicum was collected with a Smith-MacIntyre benthic grab sampler near Bergen, Norway. Table 1 lists the localities of S. fiordicum used in this study. For six worms, samples were divided into two subsamples and processed as follows. For the first subsample, worms were carefully removed from their tubes and rinsed in seawater. Genomic DNA was extracted from trophosome tissue, using a modified CTAB protocol (Doyle and Dickson, 1987). The 1458-bp fragment of the bacterial 16S rRNA gene was amplified using bacterial 16S rRNA primers GM3F 5'-AGAGTTTGATCMTGGC-3’ and GM4R 5'-TACCTTGTTACGACTT-3’ as described in Muyzer et al. (1995). All PCR cycling conditions were as follows: initial denaturation, 94 °C, 3 min; 30 cycles of denaturation, 94 °C, 45 s; annealing, 50 °C, 1 min; extension, 72 °C, 1.5 min; final extension, 72 °C, 5 min. PCR products were verified by agarose-gel electrophoresis, purified using spin columns (Promega, Inc.), and cloned using the pGEM-Teasy cloning kit (Promega, Inc.). The primers used for amplification and sequencing are described in Muyzer et al. (1995). Dye-labeled automated sequencing was carried out on either a Licor 4000 using the Excel Seqitherm II kit (Epicentre Inc.) or on a Beckman CEQ 8000 using Genome Lab Quick Start mix (Beckman Coulter). About three clones from each individual were completely sequenced in both directions.

Phylogenetic analysis
Taxa used in the phylogenetic analysis and their GenBank accession numbers are listed in Tables 1 and 2. Employed taxa represent a range of -subdivision organisms, but due to the endosymbiotic nature of the bacteria housed in S. fiordicum, this analysis focused on symbiotic organisms. Furthermore, because initial BLAST (Altschul et al., 1997) results returned -subdivision sequences, the taxonomic breadth of the analysis was restricted to this subdivision. Taxa choice was, in part, based on the previous analysis of Teske et al. (1999) and on the most similar (i.e., BLASTN) sequences available from GenBank (www.ncbi.org). 16S rRNA gene sequences of Thiobacillus spp. bacteria from the border of the β- and - proteobacteria subdivisions were used as outgroups (Teske et al., 1999).

	Results

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Phylogenetic analysis
The data set consisted of 1458 nucleotide positions, of which 1376 could be unambiguously aligned. Of these, 618 positions (44.9%) were variable and 443 (32.2%) were parsimony informative. The nucleotide composition of recovered sequences was 26.0% adenines, 22.0% cytosines, 31.5% guanines, and 20.5% thymines and does not appear to have exhibited a strong bias. The aligned data set is available from TREEBASE (http://www.Treebase.org study accession number S2007; matrix accession number M3746).

The topology produced by Bayesian inference is shown in Figure 1. Most Siboglinum fiordicum 16S endosymbiont sequences clustered as a well-supported (posterior probability = 1.0), monophyletic clade. This clade was most closely related to the endosymbionts of the frenulate Oligobrachia mashikoi and more distantly related to an uncultured -proteobacterium, thiotrophic Thiomicrospira spp. bacteria (represented here by T. crunogena), methanotrophic Bathymodiolus spp. endosymbionts, and methanotrophic Methylosarcinalacus sp. bacteria. Siboglinum fiordicum endosymbiont 16S sequences did not cluster with known vestimentiferan or Osedax spp. endosymbionts. Only sequences derived from individual 41 from Raunefjorden fell outside of this S. fiordicum endosymbiont lineage. These particular sequences were also not detected in extractions taken by LCM (see below). Instead, bacteria from individual 41 clustered with E. coli and other nonsymbiotic -proteobacteria. Therefore, we interpret the individual 41 sequences cautiously, as they possibly represent contaminant bacteria from the surrounding environment or the external surface of the host, and not host-associated endosymbionts. Alternatively, these bacteria could represent an additional lineage of mutualistic endosymbionts. Further investigation is warranted to determine the nature of the bacterial lineage associated with individual 41.

View larger version (40K): [in this window] [in a new window]   Figure 1. Bayesian inference phylogeny of known -proteobacteria siboglinid endosymbionts based on 1458-bp 16S rRNA gene sequences. Posterior probability values indicated next to the relevant node. Important taxa are labeled for siboglinid endosymbionts and other -proteobacteria of interest. Unusual Siboglinum fiordicum "endosymbionts" not recovered by laser-capture microdissection are designated by a black triangle. Numbers next to the Siboglinum branches indicate individual and clone numbers (separated by a decimal).

Laser-capture microdissection
Figure 2 illustrates a representative example of trophosome and other internal host tissues extracted via LCM without contamination from the external environment or host epidermis. Excised cells were PCR amplified, sequenced, and compared to sequences obtained from the six extractions from the rinsed host organism. Sequences obtained from LCM-extracted cells were 100% identical to sequences from the corresponding whole-organism preparation (except individual 41, see above), indicating that recovered 16S sequences originated from trophosome tissue.

View larger version (98K): [in this window] [in a new window]   Figure 2. Laser microdissection and capture of endosymbiotic bacteria from Siboglinum fiordicum. Note that preservation in 70% ethanol resulted in dehydration of tissues, potentially altering the worm's internal morphology. Original magnification 20x. (a) Cross section of S. fiordicum including bacteriocytes within the host trophosome. Epidermis and external protein/chitin tube labeled for reference. (b) Region after laser excision of worm tissue including bacteriocytes containing numerous bacteria (note grey excision lines surrounding targeted cells; arrow). (c) Region after excision and capture of targeted bacteriocytes (arrow). (d) Excised and captured bacteriocytes prior to nucleotide extraction.

	Discussion

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Within the frenulate endosymbiont clade, two well-supported subgroups were identified: one group associated with S. fiordicum from Norwegian fjords and a second group associated with O. mashikoi from the coastal waters of Japan. Whether these subgroups are separated on the basis of specificity at the level of host species, geographic isolation, or both of these factors is not clear at this time. On the basis of electron microscopy studies, E. C. Southward suggested that each species of frenulate may harbor unique endosymbionts (Southward, 1982). In comparison, vestimentiferan endosymbionts show little specificity at the level of the host species. For example, site-specific symbiont phylotypes were found in multiple host species, including Riftia pachyptila, R. piscesae, Oasisia alvinae, and Tevnia jerichonana (Feldman et al., 1997; Di Meo et al., 2000; Nelson and Fisher, 2000) and Lamellibrachia spp. and Escarpia spp. (Nelson and Fisher, 2000; Vrijenhoek et al. 2007). Instead, habitat, geography, and host specificity to vent versus seep taxa (or a combination of these) appear to govern symbiotic associations in vestimentiferans. Although vestimentiferan symbionts form one large clade, this clade is divided into several significant subgroups, or phylotypes. The deepest division in the vestimentiferan endosymbiont clade occurs between symbionts found in vents and those found in seep hosts or environments (Feldman et al., 1997; Nelson and Fisher, 2000; Di Meo et al., 2000; Vriejenhoek et al., 2007). This pattern holds true even from geographically proximate vent and seep species, such as at the Middle Valley segment of Juan de Fuca Ridge in the northeast Pacific, where R. piscesae (a vent species) and L. barhami (a seep species) occur in proximity but harbor different symbionts (Nelson and Fisher, 2000). Among seep vestimentiferan endosymbionts, three well-supported subgroups have been identified (Nelson and Fisher, 2000; McMullin et al., 2003). All three groups are found in symbioses with Lamellibrachia and Escarpia spp. and are distinguished by their biogeographic or bathymetric distribution (reviewed in McMullin et al., 2003). Although it seems likely that habitat and geography similarly govern symbiotic associations of frenulate endosymbionts, including those of S. fiordicum, it is entirely possible that different patterns, such as specificity to different host species, have evolved in this group. Additional information from other frenulate endosymbionts will be required to test such hypotheses.

Within a broad bacterial tree spanning five subdivisions, Naganuma et al. (2005) placed a new endosymbiont from an undescribed species of frenulate siboglinid, Oligobrachia sp. JT1, as closely related to vestimentiferan endosymbionts. In contrast, our endosymbiont topology agrees in most respects with those reported in previous studies (e.g., McMullin et al., 2003; Goffredi et al., 2007), and we find the Oligobrachia sp. JT1 endosymbiont to be more basal and unassociated with vestimentiferan endosymbionts. The discrepancy in these findings may result from the breadth of bacterial types included or the rooting with E. coli (within -proteobacteria) in the phylogeny proposed by Naganuma et al. Additionally, the Oligobrachia sp. JT1 endosymbiont was not localized to host trophosome, and therefore it is unclear whether this bacterium represents a novel endosymbiont lineage or a bacterium from the external environment.

Phylogenetic proximity of S. fiordicum's endosymbiont to known thiotrophs, including Thiomicrospira crunogena (Scott et al., 2006), suggests thiotrophy as a likely metabolic mode for this bacteria. Previous studies similarly indicated thiotrophy in S. fiordicum's endosymbiont. For instance, Southward et al. (1986) detected enzymatic activity consistent with thiotrophy, including RuBisCO, adenylylsulphate reductase, and sulphate adenylyltransferase, in the trophosome region of S. fiordicum. Fixation of CO₂ was also enhanced in the presence of sulfide and thiosulfate but not methane (Southward et al., 1986). Similarly, Dando et al. (1986) detected sulfur-oxidizing enzyme activity in S. fiordicum, suggesting probable thiotrophy. However, Kimura et al. (2003a) interpreted the closely related endosymbiont of O. mashikoi as a possible methanotroph on the basis of phylogenetic affinity (also seen in our proposed phylogeny) to known methanotrophs, Methylosarcina sp. and symbionts of Bathymodiolus japonicus and B. platifrons (Fujiwara et al., 2000). Despite this, further evidence for methanotrophy, including methane monooxygenase genes (pmoA and mmoX) and intracytoplasmic-stacked membranes (Cavanaugh et al., 1992; Lidstrom, 1992), was not detected in this endosymbiont (Kimura et al., 2003a; Deguchi et al., 2007). Additionally, a RuBisCO gene was detected in O. mashikoi's trophosome, consonant with a thiotrophic endosymbiont (Kimura et al., 2003a).

Consistent with the suggestion that each species harbors a single type of bacterial symbiont (Southward, 1982), multiple endosymbiont lineages were not detected within an individual S. fiordicum sample. However, our sampling regime was directed only at determining the numerically dominant symbiont in these hosts. Admittedly, with only a few clones sequenced per host, it is possible that other bacterial lineages went undetected in this survey. Other siboglinids appear to harbor multiple bacterial species in the trophosome of a single host. In vestimentiferans, both substrate oxidation and TEM observations suggest the presence of mixed symbiont communities (Fisher and Childress, 1984; Southward, 1988; de Burgh et al., 1989). Multiple metabolic pathways detected in the proteomics study by Markert et al. (2007) could also be interpreted as the presence of multiple symbionts. Most definitively, molecular studies indicate that E. spicata, L. barhami, the undescribed species Lamellibrachia sp. L1, and R. piscesae all host multiple endosymbionts (Elsaied et al., 2002; Kimura et al., 2003b; Chao et al., 2007; Vrijenhoek et al., 2007).

Herein we characterized the dominant bacterial endosymbiont associated with the frenulate siboglinid S. fiordicum as a group of –proteobacteria. Bayesian analysis indicated that these bacteria are closely related to endosymbionts of O. mashikoi, the only previously characterized frenulate endosymbiont. Both phylogenetic affinity and results from previous studies also suggest thiotrophy as the metabolic mode in these endosymbiotic bacteria. Using laser-capture microdissection (LCM), we were able to definitively localize putative symbionts to host trophosome. To our knowledge, this is the first application of LCM to marine symbiosis research. The successful targeting of nucleic acids from specific tissue regions by LCM may be further applicable in future investigations of marine symbioses, such as tissue-specific studies of symbiotic gene expression or microhabitat niche partitioning among different symbionts within a host. Much remains to be discovered about the biology of endosymbionts in the globally distributed and diverse Frenulata and Monilifera siboglinids. Additional studies of their genetic and metabolic diversity will greatly improve our understanding of the ecology and evolution of these important marine symbioses.

	Acknowledgments

 
We are grateful to the staff of the Espeland Marine Biological Station in Norway. E. C. Southward was instrumental in the collection of the study organisms. Access to the MMI-CellCut LCM workstation was made possible through the AU-CMB/CECST with NSF support (NSF EPS-0447675). This work was supported by NSF grants EAR-0120646 (WormNet) and OCE-0425060 to KMH. Finally, we thank two anonymous reviewers for their insightful comments and suggestions on this manuscript. This work is Auburn University Marine Biology Program contribution #34.

	Footnotes

 
Received 21 August 2007; accepted 28 December 2007.

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