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Applied and Environmental Microbiology, February 2000, p. 651-658, Vol. 66, No. 2
Graduate College of Marine Studies, University of Delaware,
Lewes, Delaware 199581; Florida Marine
Research Institute, St. Petersburg, Florida
337012; Division of Biological Sciences,
The University of Montana, Missoula, Montana
59812-10023; Molecular Dynamics,
Sunnyvale, California 94086-45204; and
Center for Theoretical and Applied Genetics, Cook College,
Rutgers University, New Brunswick, New Jersey
08903-02315
Received 29 July 1999/Accepted 30 November 1999
Vestimentiferan tubeworms thriving in sulfidic deep-sea
hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent
and seep habitats form two distinct clades with little variation within
each clade. In the present study, we used two different approaches to
assess the genetic variation among biogeographically distinct
vestimentiferan symbionts. DNA sequences were obtained for the
noncoding, internal transcribed spacer (ITS) regions of the rRNA
operons of symbionts associated with six different genera of
vestimentiferan tubeworms. ITS sequences from endosymbionts of host
genera collected from different habitats and widely distributed vent
sites were surprisingly conserved. Because the ITS region was not
sufficient for distinguishing endosymbionts from different habitats or
locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the
bacterial endosymbionts. Most of the endosymbionts displayed unique
REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors,
including host genera, geographic location, and bottom type.
Invertebrates that are endemic to
the highly sulfidic, reducing environments at deep-sea hydrothermal
vents and cold seeps are commonly associated with chemosynthetic
endosymbiotic bacteria (7, 18, 19). These bacteria oxidize
the reduced sulfur compounds that are abundant in hydrothermal fluid
(8, 36, 46). The resultant energy produced by the
endosymbiont is coupled to the production of carbon sources that
support the growth and maintenance of the invertebrate hosts (6,
8, 37, 48). The metabolic link between the invertebrate hosts and
their endosymbionts has obvious implications for the dispersal and
colonization strategies employed by the host organisms.
Vent endemic host organisms may employ one of three different
mechanisms for transmission of their endosymbionts to the next generation. Previous studies have shown that the Vesicomyid bivalves form species-specific associations with endosymbiotic bacteria (12, 13, 16). Host specificity is maintained in these
associations by vertical transmission of the bacteria through the egg
from the parent to the offspring (3, 35). The endosymbiont
and host phylogenies are congruent, which is consistent with a vertical mode of symbiont transmission (3, 4, 12, 35). In contrast, results from several studies suggest that endosymbiont transmission in
vestimentiferan tubeworms occurs horizontally (5, 17), possibly through ingestion of bacteria upon larval settlement (28,
42). It is also possible that horizontal transmission in
vestimentiferan-bacterial symbioses could occur through infection of
new recruits with bacteria previously associated with established congeners (20), although potential mechanisms for this
transfer have not been determined. If horizontal transmission is indeed the mechanism for bacterial acquisition in vestimentiferans, we expect
that biogeographic variation would exist among symbionts acquired from
the same host species collected in geographically isolated vent and
seep sites. Conversely, if vestimentiferan larvae settle in the same
locations where their symbionts are acquired, we expect that
vestimentiferans living at the same site would harbor identical symbionts.
In a recent study, genetic variation in vestimentiferan endosymbionts
was investigated by comparing sequences of the 16S rRNA genes of
bacteria collected from various species of hydrothermal vent and cold
seep tubeworms (20). The results showed a marked phylogenetic distinction between vestimentiferan endosymbionts collected from hydrothermal vents and soft-bottom, cold seep
environments. This study suggested that vestimentiferans acquire one of
two distinct species of free-living bacteria depending on whether they
settle on basaltic, hydrothermal vent sites or sedimented, cold seep
sites. A third, unique bacterial species was discovered in a tubeworm
that inhabited a sedimented whale fall. Significant genetic variation
was not found among symbionts found within each of the habitat types.
Symbionts from three different host genera collected from five
different hydrothermal vent sites along the East Pacific Rise (EPR) had
identical 16S rRNA gene sequences.
The observation of so little sequence variation among
endosymbiont 16S rRNA genes suggests that all tubeworm
endosymbionts may belong to a single species. However, the
conservative nature of the 16S rRNA gene often renders it
inadequate for distinguishing among conspecific strains of bacteria
(50). Therefore, it is not surprising that Feldman et al.
(20) did not find significant genetic variation within the
habitat clades. It is possible that significant genetic variation
exists between endosymbionts of geographically distributed
vestimentiferan hosts but that these species- or strain-level
differences have not been detected by approaches that have been
employed to date.
The goal of our study was to resolve the question of genetic variation
among widely distributed vestimentiferan endosymbionts by utilizing two
molecular techniques that have historically proven useful for comparing
closely related strains of bacteria. Our first approach entailed
sequencing of the internal transcribed spacer (ITS) region, a noncoding
region within the rRNA that often contains genetic variation sufficient
for differentiating species of prokaryotes (2). Our second
approach involved using a DNA fingerprinting technique that utilizes
repetitive extragenic palindromes (REPs) as priming sites to generate
diagnostic banding patterns from prokaryotic genomic DNA (25,
45). The REP-PCR technique is particularly useful for
investigations of bacterial symbiosis because REP sequences have been
found only in bacterial DNA; phage and eukaryotes do not contain REP
elements (25). By virtue of these repetitive elements,
symbiont DNA can be selectively amplified from a mixed population of
symbiont and host DNA to allow analysis of bacterial population
diversity. Our results provide information on the extent of genetic
variation among the symbionts, as well as further evidence to support a
horizontal mode of symbiont acquisition among the vestimentiferan tubeworms.
Sample collection.
Vestimentiferans were collected with the
aid of submersibles from several locations, including two sites in the
Western Pacific Ocean, four sites along the EPR, two sites in the
Pacific Northwest, one site along the Galapagos Rift (GR), two sites
along the western coast of California and Mexico, and two locations in
the Gulf of Mexico (GOM) (Table 1).
Samples included in this study were chosen to represent the entire
range of host vestimentiferan distribution. Habitat types included
hydrothermal vent sites with a basaltic substrate, cold seep habitats
with a sedimented bottom, and one sedimented site associated with a
whale carcass (Table 1). All samples were carried to the surface in
chilled seawater (0.5 to 2°C).
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Genetic Variation among Endosymbionts of Widely
Distributed Vestimentiferan Tubeworms
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
Collection sites of vestimentiferan symbionts included in
this studya
DNA extraction. The symbiont-containing trophosome tissue from each vestimentiferan host was aseptically removed, and a small portion of it was homogenized in 5 M guanidinium isothiocyanate. Prior to DNA extraction, 25 µl of a 20% Chelex 100 (47) solution was added to the homogenate and incubated for 1 h at 4°C. The samples were briefly spun to remove the Chelex 100. Total bacterial DNA was extracted from 100 µl of guanidinium isothiocyanate homogenate using the IsoQuick DNA purification kit (Orca, Bothell, Wash.) according to the manufacturer's instructions and quantified spectrophotometrically. In addition to symbiont DNA, host DNA from three different genera was extracted from vestimentum tissue (non-symbiont-containing tissue) using the same extraction protocol to serve as controls. A number of the DNA samples used in this study (e.g., 18°S Riftia pachyptila, Guaymas Vent and Seep [GY-V and GY-S, respectively] Escarpia spicata, whale E. spicata, Gorda Lamellibrachia sp., 21°N Oasisia alvinae, and GY-V R. pachyptila) were prepared separately at Rutgers University according to previously published protocols (20).
16S rRNA gene characterization. The methods used to PCR amplify, purify, and sequence the 16S rRNA symbiont genes were as described in the work of Feldman et al. (20). DNA sequence alignments were initially constructed using PileUp and then optimized (minimizing overall alignment differences) by eye in the SeqLab environment (Genetics Computing Group). Insertions and deletions were eliminated from the alignment before phylogenetic trees were determined. Phylogenetic trees for the symbiont 16S rRNA genes were computed using fastDNAml and bootstrapped 100 times using fastDNAml boot (21, 34). For the maximum likelihood analyses, operational taxonomic unit input order was randomized and global rearrangements were performed at each bootstrap replication.
ITS region characterization. PCRs amplified the ITS region of the symbiont ribosomal DNA (rDNA) using two ITS-specific primers, ITS16F-G1 (5'-GAAGTCGTAACAAGG-3') (27) and ITS23R-L1 (5'-CAAGGCATCCACCGT-3') (27). ITS16F-G1 is nested about 30 to 40 nucleotides upstream from the spacer boundary in the 3' end of the 16S rRNA gene (positions 1491 to 1505, Escherichia coli 16S rRNA gene). ITS23R-L1 is nested in the 5' end of the 23S rRNA gene, approximately 20 bases downstream from the spacer boundary (positions 21 to 35, E. coli 23S rRNA gene). PCR mixtures (50-µl total volume) contained final concentrations of the following: 50 ng of symbiont DNA, 1× PCR buffer, 0.2 mM (each) deoxynucleoside triphosphates, 10 pmol of each primer, 1.5 mM MgCl2, 5% (vol/vol) acetamide, and 1.25 U of Taq polymerase (Promega). PCR conditions were as follows: 35 cycles, each consisting of denaturation at 92°C for 1 min, hybridization at 55°C for 2 min, and elongation at 72°C for 2 min with 5 s added to each extension per cycle. A hot start (9) with denaturation at 95°C for 2 min was used at the beginning of the reaction to heighten reaction specificity. In addition, acetamide was added to the PCR to encourage more efficient amplification of GC-rich templates (38). All PCRs were performed on an MJ-Minicycler (MJ Research, Inc., Watertown, Mass.).
Because our attempts to directly sequence the smaller ITS PCR amplicon yielded poor results, amplified ITS regions were cloned to facilitate sequencing. Amplification products were pooled from three separate PCRs and cloned directly using the TA cloning kit with the pCR-II or the pCR-2.1-TOPO cloning vectors (Invitrogen, San Diego, Calif.) according to the manufacturer's instructions. Plasmid preparations were made using a standard alkaline-lysis preparation (39). The sizes of the inserts were verified before sequencing by restriction analysis. The plasmid DNA was further purified for sequencing using the Plasmid MiniKit (Qiagen, Inc., Valencia, Calif.) according to the manufacturer's instructions. Two clones from each amplified ITS region were cycle sequenced using the Perkin-Elmer (Foster City, Calif.) ABI BigDye dye termination cycle sequencing ready reaction kit with Ampli Taq DNA polymerase FS according to the manufacturer's instructions. Sequencing was performed on an ABI PRISM 310 genetic analyzer. The ITS regions were bidirectionally sequenced and confirmed prior to their alignment using the Sequence Navigator and AutoAssembler programs (Applied Biosystems, Inc.). Only sequences that overlapped with 0% ambiguity were included in the analysis. Final alignments were generated in the Genetic Data Environment, version 3.2. Phylogenetic relationships were determined from these aligned sequences using PHYLIP version 3.572 (22). A Kimura two-parameter distance matrix was constructed, from which neighbor-joining trees with 100 bootstrap replications were generated. A second distance matrix, based on pairwise differences in the geographic distribution between samples, was generated from latitude-longitude information for each sample site using GeoDist version 3.01e, R package (32). A third distance matrix, based on pairwise comparisons of the sample habitat types, was created by assigning "0" values to pairs of samples that were taken from similar habitat types (both vent or both seep) and "1" values to pairs of samples that were taken from different habitat types. Mantel correlation tests were performed to compare ITS sequence distance with geographic and habitat distance (14, 33). Two- and three-way Mantel tests were performed using different combinations of the three distance matrices to assess whether ITS genetic distance significantly correlates with the geographic distances between endosymbiont collection sites or their habitat types (41). These Mantel tests were performed using the R package (32).REP elements.
REP sequences were amplified from each of the
vestimentiferan symbionts with PCR using two universal REP primers,
REPIR-I (5'-IIIICGICGICATCIGGC-3') (45) and REP2-I
(5'-ICGICTTATCIGGCCTAC-3') (44). Each 25-µl REP-PCR
mixture contained 50 ng of symbiont or host DNA and final
concentrations of 1× Gitschier buffer [16.6 mM
(NH4)2SO4, 67 mM Tris-HCl, 6.7 mM
MgCl2, 6.7 µM EDTA, 30 mM 
-mercaptoethanol
(29)], 160 µg of DNA-grade bovine serum albumin per ml,
10% dimethyl sulfoxide, 1.25 mM (each) four deoxynucleoside triphosphates, 10 pmol of each REP primer, and 2 U of Taq
polymerase (Gibco). One cycle of 95°C for 7 min was performed to
denature the genomic DNA, followed by 30 cycles of 94°C for 1 min,
44°C for 1 min, and 65°C for 8 min, and a final extension at 65°C
for 15 min. PCRs were performed with an MJ-Minicycler (MJ Research, Inc.). Reaction products (7.5 µl) were analyzed via agarose gel electrophoresis.
Nucleotide sequence accession numbers. GenBank accession numbers for the new ITS sequences reported in this paper are as follows: AF076795, Ridgeia piscesae endosymbiont, Juan de Fuca Ridge (JDF); AF076796, Escarpia laminata endosymbiont, West Florida Escarpment (WFE); AF076797, Lamellibrachia sp. endosymbiont, GOM-10; AF076798, E. spicata endosymbiont, GY-S; AF076799, R. pachyptila endosymbiont, GY-V; AF076800, Tevnia jerichonana endosymbiont, 9N; AF076801, R. pachyptila endosymbiont, 9N; AF076802, T. jerichonana endosymbiont, 13N; AF076803, R. pachyptila endosymbiont, 13N; AF076804, Lamellibrachia sp. endosymbiont, Gorda; AF076805, R. pachyptila endosymbiont, GR; AF076806, R. pachyptila endosymbiont, 18S; AF076807, O. alvinae endosymbiont, 21N; AF076808, E. spicata endosymbiont, whale; AF076809, undescribed species endosymbiont, Nikko Seamount; AF076810, L. columna endosymbiont, Lau-Fiji; AF076811, Lamellibrachia sp. endosymbiont, GOM-12; AF076812, E. spicata endosymbiont, GY-V; AF076813, Lucina floridana endosymbiont. Accession numbers for the three 16S rRNA sequences generated for this study are as follows: AF165909, E. spicata endosymbiont, GY-V; AF165907, undescribed species endosymbiont, Nikko Seamount; and AF165908, E. spicata endosymbiont, GY-S.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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16S rDNA sequence variation among vestimentiferan endosymbionts. In order to maintain a comparable data set, many of the endosymbiont DNA samples used in our ITS and REP-PCR analyses are the same DNA samples used in the previous 16S rDNA study of geographically distinct endosymbionts (20). Our study also includes additional samples to expand the representation of symbiont distribution. In concordance with our expanded data set, a new 16S rDNA phylogenetic tree was generated to include three of the endosymbionts that were added: GY-V E. spicata, GY-S E. spicata, and the undescribed species from the Nikko Seamount (Fig. 1A). In this new analysis of 16S rDNA sequences, the symbionts continue to cluster into two main groups: group I includes all of the symbionts from seep- or sediment-dwelling host organisms, while group II includes all of the symbionts from vent-dwelling host fauna. These relationships are in accordance with our previous findings (20). Interestingly, the difference between vent and seep symbionts is maintained between symbionts from the same host, E. spicata, collected from vent (GY-V) and seep (GY-S) habitats at a single site in Guaymas Basin. Likewise, symbionts from two different vestimentiferan hosts are closely related when collected from the same habitat type (e.g., 9N R. pachyptila and T. jerichonana symbionts, both from vent habitats).
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ITS sequence variation among vestimentiferan symbionts. ITS amplifications of vestimentiferan symbionts were generally 550 bp in length, although some individuals yielded a second, larger PCR product of roughly 800 bp in length (data not shown). The larger amplicon did not match any ITS sequences in the database, and we found that its intensity in the total PCR product was reduced by the use of higher annealing temperatures. For these reasons, we believe that the larger product represented an artifact of the PCR rather than rRNA operon heterogeneity. To separate these two distinct amplicons, we cloned the ITS PCR amplification products and used only the smaller amplicon from each symbiont for sequence analysis. As in many bacteria, all of the endosymbiont ITS regions contained putative sequences for the tRNA genes for alanine and isoleucine (Fig. 2). Five of the symbionts contained prominent insertion sequences (approximately base position 312 in ITS region) that totaled approximately 50 bp (Fig. 2).
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50% were reported. The ITS cladogram showed a prominent
break within the symbionts: group I included the symbionts of E. spicata from the whale carcass, L. columna from the
Lau-Fiji basin, the undescribed species from a vent site at the Nikko
Seamount, one of the two Lamellibrachia spp. collected from
a seep site in the GOM (GOM-12), and E. laminata from the seep site at Guaymas Basin; group II included the rest of the symbionts
(Fig. 1B). Apart from differing in sequence from the members of group
II, the symbionts in group I contained prominent insertion sequences
that totaled approximately 50 bp (Fig. 2). The sequences of these
insertions were nearly identical in all of the symbionts in which they
were found. Groups I and II in the 16S rDNA cladogram (Fig. 1A)
correlate well with groups I and II in the ITS cladogram (Fig. 1B),
with one exception: E. laminata symbiont, WFE, appears in
group I according to its 16S rDNA sequence and in group II according to
its ITS sequence (Fig. 1).
To determine if there was a significant correlation between ITS
sequence diversity and either geography or habitat type, we performed
two- and three-way Mantel tests to compare distance matrices generated
from pairwise differences between ITS sequences, geographic distances,
or habitat types. The results of these Mantel tests are shown in Table
2. Despite the remarkable similarity among the ITS sequences, the genetic distances among ITS regions correlate significantly with geographic distances. Although none of the
correlations were particularly strong (r 
0.334 for
all tests), P values from two different Mantel tests
indicated that ITS sequence similarity and geography were significantly
correlated (P = 0.0349, test 3; P = 0.0480, test 4). In contrast, P values for a two-way
Mantel test of ITS sequence similarity versus habitat type alone (test
1) or a three-way test versus habitat type with geography factored out
(test 5) were not statistically significant (P = 0.146
and 0.102, respectively). Thus, ITS sequence similarity was
significantly correlated with broad-scale endosymbiont geography (P 0.05) but not significantly correlated with
habitat type. In light of the fact that the ITS sequences for 9°N,
18°S, and Guaymas R. pachyptila were identical, our
results suggest that the ITS region is variable enough to show
broad-scale biogeographic variation but is not variable enough to
distinguish among symbionts located along the same ridge axis. These
results are in contrast to the results of Feldman et al.
(20) and the new analysis of 16S rDNA relationships
presented in this study, which suggest that symbiont sequence distances
correlate with habitat type (basaltic, vent versus sedimented, seep
substrates). Although our data suggest that the geographic distribution
of the hosts may be a dominant influence in determining genetic
variation of the endosymbionts, it is clear that several factors affect
endosymbiont diversity.
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REP-PCR fingerprints of the vestimentiferan symbionts. The REP-PCR technique is particularly well suited for investigations of bacterial symbiosis, in that it allows investigators to survey the symbiont genome in the presence of host DNA because the REP priming sites are exclusively bacterial (25). To confirm that REP sequences were not present in the genomes of the vestimentiferan hosts, we performed REP-PCR amplifications on DNA that was aseptically acquired from vestimentum (non-symbiont-containing) tissue from three different host genera (Escarpia, Riftia, and Tevnia). Most of these samples failed to amplify at all (data not shown). The exception was E. laminata, which produced a single band that did not correspond with any of the bands seen in the REP fingerprint of its endosymbiont.
Fingerprint patterns of the vestimentiferan endosymbiont DNA displayed remarkable variation (Fig. 3). Relationships based upon the number of shared bands among the fingerprint patterns were compared using GelCompar software and depicted as a cladogram with a corresponding, computer-generated gel image (Fig. 4). All of the symbionts included in the REP analysis displayed unique fingerprints, with the exception of 9°N R. pachyptila, and GR R. pachyptila, which exhibited indistinguishable patterns (Fig. 3 and 4). The REP analysis seems to divide the symbionts into four distinct groups, or clades (Fig. 4). Group I includes both Lamellibrachia symbionts from the GOM and the symbiont from Ridgeia piscesae from the JDF. Group II includes the R. pachyptila symbionts and the symbiont from T. jerichonana. Group III includes all of the Escarpia symbionts, from both E. spicata and E. laminata. Group IV includes the endosymbionts from the undescribed species at Nikko Seamount and L. columna from the Lau-Fiji Basin, two distantly located sites in the western Pacific Ocean.
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Implications for symbiont relatedness and acquisition. Previous studies have suggested that vestimentiferans acquire their symbionts through ingestion of free-living bacteria upon larval settlement (28, 42). If this mechanism alone is employed, one would expect that symbionts from two different vestimentiferan host species living at the same location would be identical or related more closely to each other than they are to other endosymbionts collected from distant vent sites. This, in fact, was not the case, as symbionts from 9°N T. jerichonana and 9°N R. pachyptila (both collected from the same rock) did not display identical ITS sequences or REP-PCR fingerprints. Instead, the symbionts grouped according to their respective host species as revealed through comparisons of shared bands in their REP-PCR fingerprints.
One explanation for this is that larvae could acquire their symbionts prior to attachment, either from a different substrate than where they eventually colonize or from the water column. This could enable two vestimentiferans that reside in the same site to harbor different symbionts. Alternatively, symbiont acquisition may be related to ecological succession. T. jerichonana is the first colonizer of new hydrothermal vent fields, in areas of the most intense diffuse flow (40). Presumably, this is because T. jerichonana is more tolerant (than R. pachyptila) of the high temperatures and elevated concentrations of H2S that are present in newly formed vent sites. It is only after T. jerichonana has become established and the levels of reduced chemicals have attenuated that R. pachyptila begins to colonize a new hydrothermal vent site (40). The T. jerichonana and R. pachyptila hosts may simply acquire a different subset of a free-living chemoautotrophic community due to temporal and/or spatial consequences of ecological succession. Specific host recognition mechanisms may exist that allow certain strains of endosymbiont bacteria to survive in specific vestimentiferan hosts. Although these mechanisms have not been demonstrated in vestimentiferans, they could explain how two different hosts living on the same rock could harbor different symbionts. Alternatively, the larvae may acquire bacteria that have been released from congeners residing at the site of larval settlement, potentially through host decomposition. These mechanisms might yield REP-PCR fingerprint patterns that imply close relationships between endosymbionts of the same host genus. Because only one individual from each vestimentiferan genus was sampled from most of the locations, we cannot determine the extent of ITS sequence variation among endosymbionts at each site. The extent of vestimentiferan symbiont relatedness and the host transmission mechanisms used to maintain these symbioses have remained ongoing questions. The results presented here demonstrate the existence of significant strain-level variation between endosymbionts of different vestimentiferan hosts collected from geographically separated areas. Further work to pinpoint the stage during vestimentiferan development when the symbiosis is first established will shed light on symbiont transmission and help to explain the genetic diversity among symbiont populations.| |
ACKNOWLEDGMENTS |
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We thank the crews and pilots of the R/V Atlantis II and the deep-submergence vehicle Alvin, the Nautile, and the U.S. Navy Advanced Tethered Vehicle for their assistance in sample collection. Samples of L. columna from the Lau-Fiji Basin were generously provided by A. M. Alayse. Samples of Lamellibrachia sp. from Green Canyon, GOM, were graciously supplied by Craig Young and Adele Pile. ITS primers and sequences for 13°N Tevnia and Riftia, Gorda Lamellibrachia, and Galapagos Riftia were contributed by Jeff Stein. We thank Barbara Campbell for assistance with REP-PCR fingerprinting and Kathy Coyne for critically reviewing the manuscript. We also thank Ken Halanych for thoughtful discussion. The manuscript was greatly improved by the helpful comments of two anonymous reviewers.
This work was supported by two NSF grants to S.C.C. (OCE-9314595 and OCE-9596082). The whale carcass E. spicata sample was collected on a cruise led by Craig Smith and funded by an NSF grant to R.C.V. (OCE-9633131).
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FOOTNOTES |
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* Corresponding author. Mailing address: Graduate College of Marine Studies, University of Delaware, Cannon Lab, 700 Pilottown Rd., Lewes, DE 19958. Phone: (302) 645-4078. Fax: (302) 645-4007. E-mail: caryc{at}udel.edu.
Present address: Monterey Bay Aquarium Research Institute, Moss
Landing, CA 95039-0628.
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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