biogeography, evolution, and diversity of epibionts in ... · none of the epibiont 16s rrna gene...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2004, p. 4821–4830 Vol. 70, No. 80099-2240/04/$08.00�0 DOI: 10.1128/AEM.70.8.4821–4830.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biogeography, Evolution, and Diversity of Epibionts inPhototrophic Consortia

Jens Glaeser† and Jorg Overmann*Bereich Mikrobiologie, Department Biologie I, Ludwig-Maximilians-Universitat Munchen,

D-80638 Munich, Germany

Received 23 December 2003/Accepted 14 April 2004

Motile phototrophic consortia are highly regular associations in which numerous cells of green sulfur bac-teria surround a flagellated colorless �-proteobacterium in the center. To date, seven different morphologicaltypes of such consortia have been described. In addition, two immotile associations involving green sulfurbacteria are known. By employing a culture-independent approach, different types of phototrophic consortiawere mechanically isolated by micromanipulation from 14 freshwater environments, and partial 16S rRNAgene sequences of the green sulfur bacterial epibionts were determined. In the majority of the lakes investi-gated, different types of phototrophic consortia were found to co-occur. In all cases, phototrophic consortia withthe same morphology from the same habitat contained only a single epibiont phylotype. However, morpho-logically indistinguishable phototrophic consortia collected from different lakes contained different epibionts.Overall, 19 different types of epibionts were detected in the present study. Whereas the epibionts within onegeographic region were very similar (Dice coefficient, 0.582), only two types of epibionts were found to occur onboth the European and North American continents (Dice coefficient, 0.190). None of the epibiont 16S rRNAgene sequences have been detected so far in free-living green sulfur bacteria, suggesting that the interactionbetween epibionts and chemotrophic bacteria in the phototrophic consortia is an obligate interaction. Basedon our phylogenetic analysis, the epibiont sequences are not monophyletic. Thus, the ability to form symbioticassociations either arose independently from different ancestors or was present in a common ancestor prior tothe radiation of green sulfur bacteria and the transition to the free-living state in independent lineages. Thepresent study thus demonstrates that there is great diversity and nonrandom geographical distribution ofphototrophic consortia in the natural environment.

Motile phototrophic consortia are associations of bacterialcells in which a flagellated colorless �-proteobacterium is sur-rounded by numerous cells of green sulfur bacteria, the so-called epibionts (42, 44). The barrel-shaped, motile phototro-phic consortia were first described in the early 20th century (7,35). Since their discovery, seven different morphotypes, name-ly, “Chlorochromatium aggregatum,” “Chlorochromatium glebu-lum,” “Chlorochromatium magnum,” “Chlorochromatium luna-tum,” “Pelochromatium roseum,” “Pelochromatium roseo-viride,”and “Pelochromatium selenoides,” have been described on the ba-sis of morphology and the cellular arrangement of the epibionts(42).

The cellular arrangement in phototrophic consortia is alwaysvery regular, and the cell division of the partner bacteria pro-ceeds in a highly coordinated fashion (43). Furthermore, rapidsignal transfer between the epibionts and the central bacteriumhas been demonstrated (19). These observations indicate thatthe two bacterial partners are tightly and specifically associ-ated. Theoretically, such a highly specific association couldhave emerged only once during evolution. Indeed, initial anal-yses of the phylogenetic affiliations of five types of phototro-phic consortia from two lakes indicated that the epibionts form

a single cluster within the green sulfur bacterial radiation,whereas most other free-living members of this phylum aremore distantly related (20). However, the available data arenot sufficient to elucidate whether a change from the symbioticlife style to the independent life style or vice versa has occurredmultiple times during the radiation of the green sulfur bacteria.

Phototrophic consortia have been reported to occur in nu-merous freshwater lakes and ponds worldwide (8, 40, 43).Since consortia have been found to thrive exclusively in thechemocline of freshwater lakes at low concentrations of dis-solved sulfide and low light intensities, they are assumed tooccupy a narrow and well-defined ecological niche (43). Basedon the postulate that bacteria are ubiquitous (2, 4), competitiveexclusion of species with identical ecological niches would beexpected to result in low overall diversity. In contrast, ende-mism of microorganisms would result in significantly greaterglobal diversity (16, 17), since such diversity is maintained bygeographic isolation (48). In the case of phototrophic consor-tia, it is not known whether the identical morphotypes ob-served in lakes on different continents harbor the same types ofbacteria or whether the diversity surmounts the seven morpho-types that are currently recognized.

In the present study, the 16S rRNA gene sequences of pho-totrophic consortia were analyzed in order to (i) obtain moreinformation on the actual diversity of the green sulfur bacterialepibionts, (ii) obtain a broader view of the geographical dis-tribution of different types, and (iii) assess whether the switchfrom the symbiotic life style to the independent life style (orvice versa) occurred once or more frequently during the radi-

* Corresponding author. Mailing address: Bereich Mikrobiologie,Department Biologie I, Ludwig-Maximilians-Universitat Munchen,Maria-Ward-Straße 1a, D-80638 Munich, Germany. Phone: 49-89-21806123. Fax: 49-89-21806125. E-mail: [email protected].

† Present address: Justus-Liebig-Universitat Gießen, Institut furMikro- und Molekularbiologie, 35392 Gießen, Germany.

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ation of green sulfur bacteria. We used a culture-independentapproach (20), in which different types of phototrophic con-sortia are mechanically isolated by micromanipulation and 16SrRNA gene sequences of epibionts are obtained after a highlysensitive group-specific amplification step, followed by separa-tion by denaturing gradient gel electrophoresis (DGGE). Sofar, there is not an amplification method with comparablesensitivity for the 16S rRNA genes of �-proteobacteria. Inaddition, no single 16S rRNA gene sequence of a central bac-terium is available at this time. Therefore, primers specific forcentral bacteria of phototrophic consortia have not been de-veloped so far. Consequently, the present study was limited tothe green sulfur bacterial epibionts.

MATERIALS AND METHODS

Study sites. Fourteen lakes and ponds were selected based on previous studiesand previously published data. The study sites are located in six different geo-graphic regions (Fig. 1) and comprise three lakes in Germany, two lakes in Spain,one pond in Massachusetts, seven lakes in Michigan, and one lake in the state ofWashington (Table 1). In each of the lakes, an anoxic, sulfide-containing hy-polimnion builds up after the onset of summer stratification. Concomitantly,dense accumulations of anoxygenic phototrophic bacteria and phototrophic con-sortia develop in the chemocline (5, 14, 23, 24, 25, 27, 43, 50). The physicochem-ical conditions in the chemoclines during summer stratification were comparablein the lakes (Table 1). In Oyster Pond (Woods Hole, Mass.), the surface sedi-ment usually becomes anoxic during the summer and is subsequently colonizedby anoxygenic phototrophic bacteria. Although not detectable directly by micros-copy in sediment samples, phototrophic consortia can be enriched from thissediment if the samples are incubated under appropriate conditions (Overmann,unpublished data) (Table 1). During the present study, the sampling locationswere visited in the years from 1998 to 2001 between July and October (Table 1).

Water sampling. Water samples were obtained from the deepest parts of thelakes by employing a bilge pump connected to gas-tight isoversinic tubing. Theinlet of the tubing consisted of two polyvinyl chloride cones that were 1 cm apart(34). This device allowed reproducible sampling of different water layers at 5-cmintervals. Sampling depths were chosen based on the vertical distribution ofanoxygenic photosynthetic bacteria as determined by light and phase-contrastmicroscopy.

Samples from European lakes were placed in autoclaved 1-liter glass bottlesand kept in the dark to avoid damage to the phototrophic bacteria, which occursat the high light intensities at the lake surface (43). The bottles were sealed gastight to prevent abiotic oxidation of sulfide, and then they were transported backto the laboratory at the in situ temperature and processed within 8 h aftersampling. Samples obtained from North American lakes were transferred to10-ml gas-tight screw-cap glass tubes. Anoxic conditions were maintained byaddition of 200 �M neutralized (pH 7.3) sulfide solution (45). The glass tubeswere shipped on artificial ice and by courier to the home laboratory, and theywere processed within 48 h.

Isolation of intact phototrophic consortia and molecular fingerprinting ofepibionts. Phototrophic consortia were mechanically separated from the chemo-cline microbial community by using a micromanipulator connected to an inverted

microscope (18, 19). At first, water samples containing phototrophic consortiawere evenly spread on coverslips by squeezing 100 �l of a water sample betweentwo acetone-cleaned coverslips (60 by 20 mm). Excess water was removed with apaper tissue, and the coverslips were separated and air dried aseptically. Afterdrying, different morphotypes of phototrophic consortia could still be distin-guished by bright-field and phase-contrast microscopy based on size and on thecolor, number, and shape of the epibionts.

Batches of 5 to 45 consortia with the same morphology were collected bymicromanipulation and directly subjected to PCR amplification. 16S rRNA genefragments that were 540 bp long were amplified by employing oligonucleotideprimers GC 357f and GSB 840r and the PCR conditions described previously(41). Our method permitted highly sensitive and specific amplification of 16SrRNA gene fragments of the epibionts (20). A particular fragment was chosenbecause it included region V3, which is the largest of the highly variable regionsin the 16S rRNA gene (11). In order to check for the presence of differentepibionts in the same morphotype of phototrophic consortia, the resulting am-plification products were separated by denaturing gradient gel electrophoresis(DGGE) according to melting behavior (39). The 6% (wt/vol) polyacrylamideDGGE gels contained a linear gradient of 35 to 70% denaturing agents. Afterthe gels were stained with ethidium bromide, individual DNA bands were excisedfrom each DGGE gel, recovered by electroelution, reamplified, and sequencedas described previously (41).

Phylogenetic analysis of 16S rRNA gene sequences of the epibionts. Phyloge-netic analysis of 16S rRNA gene sequences of the epibionts was performed byusing the ARB phylogeny package (37). The program Fast Aligner V1.03 wasused for alignment of all 16S rRNA gene sequences of green sulfur bacteriaavailable through the National Center for Biotechnology Information website(1). The sequence of Chloroherpeton thalassium ATCC 35110T was chosen as theoutgroup, since it has been shown to branch at the root of the green sulfurbacteria and significantly deeper than all other known green sulfur bacteria (40).The alignment was manually corrected based on secondary structure informationfor Chlorobium vibrioforme ATCC 6030, and a phylogenetic tree was constructedfrom all sequences longer than 1,300 bp by using the maximum-likelihood pro-gram DNA_ML. After this, the partial 16S rRNA gene sequences of epibiontswere inserted into the phylogenetic tree by using the Parsimony Interactive toolwithout changing the overall tree topology.

Small differences between partial 16S rRNA gene sequences can be caused bysequencing errors and hence can lead to overestimation of the sequence diversityof epibionts. In order to identify such potential sequencing errors, sequence pairswhich differed at only one or two nucleotide positions were reassessed based onsecondary structure information for the 16S rRNA molecule (20). We investi-gated whether nucleotide changes in double-stranded regions of the 16S rRNAmolecule were compensated for by nucleotide changes in the opposite strand.Substitutions which resulted in the loss of Watson-Crick base pairing wereregarded as sequencing errors and not included in the phylogenetic analysis.Based on our assessment, all sequences that differed at only one nucleotideposition were treated as a single sequence type. Only two of the sequences(environmental sequence GSB5 and “P. roseum” PrH [see Fig. 5]) differed at twoconfirmed nucleotide positions. All other 16S rRNA gene sequences analyzed inthe present study differed at three or more nucleotide positions. Consequently, theestimate of microbial diversity presented in this paper is a conservative estimate.

Comparison of epibionts in different geographic regions. The presence orabsence of distinct types of epibionts was used to compare the communities of

FIG. 1. Geographic regions sampled during the present study. The numbers refer to Table 1, which lists the lakes studied and provides detailson environmental parameters.

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phototrophic consortia in lakes within the same geographic area or in lakes indifferent regions.

The binary similarity of communities in different lakes was computed byemploying the coincidence index of Dice (D) (12): D � 2a/(2a � b � c), wherea is the number of epibiont types present in the two environments compared, bis the number of types found exclusively in the first environment, and c is thenumber of epibiont types present in the second environment.

Chemotaxis assays. To date, the chemotactic response of intact phototrophicconsortia has been studied only for specimen from a single lake in easternGermany (19, 28). The close proximity of a laboratory to Lake Siso provided the

opportunity to investigate the chemotactic behavior of the population of con-sortia in this second lake. Chemotaxis was tested by incubation of rectangularcapillaries (length, 50 mm; inside dimensions, 0.1 by 1.0 mm; capacity, 5 �l; VitroDynamics, Rockaway, N.J.) filled with diluted test substances. Sterile 100 mMstock solutions of sulfide, thiosulfate, glycerol, acetate, pyruvate, lactate, propi-onate, citrate, succinate, 2-oxoglutarate, and glycine were prepared anoxically inHungate tubes sealed with butyl rubber septa and flushed with N2. For prepa-ration of organic acids, the corresponding sodium salts were used, and the pH ofeach stock solution was adjusted to 7.3 with NaOH. This pH value was chosenbased on the pH values determined previously for the chemocline water of Lake

TABLE 1. Selected physicochemical parameters and phototrophic consortia in the chemoclines of different lakes

Lake or pond

Characteristic environmental parametersa

Reference(s)Sampling

date(day/mo/yr)

Depth(m) Morphotype Epibiont

phylotypebChemo-cline(m)

Temp(°C) pH

Light intensity(�mol � m�2 �

s�1)

Sulfideconcn(�M)

Geographical region 1c

Dagow 6.0 10 7.35 0.7 10 26 7/7/1998 6.4 “C. aggregatum” M“C. magnum” CmD E“P. roseum” PRDBD7 K“P. latum” PRDBD8 N

Haussee 6.0 13.7 7.5 2.4 — f H.-D. Babenzien, 20/7/1999 6.5 “C. aggregatum” CaH Cpersonal “P. roseum” PrH Gcomunication “P. selenoides” PSH D

Geographical region 2Schleinsee 6 8–10 7.8 0.5–5 30 14 9/9/2001 7 “P. roseum” 25 K

Geographical region 3Siso 2.75 15 7.2 — 200 Unpublished data 23/8/1999 2.75 “C. aggregatum” CaS PCoromina 1.5 20 7.4 0.01 10 24 22/8/1999 2.5 “C. aggregatum” CaC P

Geographical region 4Oyster Pondd NAe 23 7 7 100 Unpublished data 30/6/2000 0.01 “C. aggregatum” CaSp L

Geographical region 5Wintergreen 3.5 17 8.1 0.95 192 24, 25 4/9/2001 4.2 “C. aggregatum” 7b-2 M

“C. aggregatum” 8a-2 N“C. magnum” 13-2 H“P. latum” 29-2 N

Baker 7 6 7.5 2.94 102 24, 25 4/9/2001 6 “C. aggregatum” 11b D“C. magnum” 14-2 H“P. roseum” 1a-2 O“P. latum” 1b-2 N

Sheffer — f — — — — — 4/9/2001 5.8 “C. aggregatum” 9b-2 D“C. magnum” 9a H“P. roseum” 18a O“P. roseum 18c-2 B“P. latum” 18b N“C. vacuolata” D

Mud 4 15 7 0.4 8 24, 25 4/9/2001 3.5 “C. magnum” 52 ALeach 3.5 21 7.2 1.52 87 24, 25 4/9/2001 7 “C. aggregatum” 10b-2 N

“C. magnum” 10a H“P. roseum” 20 O

Round 4.5 12 7.5 3.27 133 24, 25 4/9/2001 4.7 “C. aggregatum” 4b-2 N“C. magnum” 14-2 H“P. latum” 19-2 N

Cassidy 8 5 7.4 0.05 62 24, 25 4/9/2001 6.7 “C. aggregatum” 5-2 D“C. magnum” 10 H“C. lunatum” 12-2 D“P. roseum” 16a-2 O“P. latum” 2b N

Geographical region 6Echo — — — — — — 10/10/1998 10 “C. aggregatum” CaEL J

“C. glebulum” ELGSB5 F“C. magnum” ELGSB2 H“P. roseum” PrEL B

a Data from the literature. See the references cited for geographical coordinates of individual lakes.b See the phylogenetic tree in Fig. 5.c See Fig. 1.d Phototrophic consortia were not detectable in sediment samples, but they were enriched under the conditions provided.e NA, not applicable.f —, no information available.

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Siso (pH 7.2 to 7.4) (Table 1) (27, 43). All compounds were diluted to a finalconcentration of 500 �M with filter-sterilized (cellulose nitrate membrane filters;pore size, 0.2 �m; Sartorius, Gottingen, Germany), anoxic chemocline water.Controls were filled with sterile, anoxic chemocline water only. The capillarieswere immediately sealed with plasticine (Idena, Berlin, Germany) at one end,and then the open end of each capillary was inserted into a sample containingconsortia (19). Incubation lasted for 3 to 5 h.

After incubation, each capillary was recovered and immediately closed withplasticine. The phototrophic consortia were counted directly in each capillary byphase-contrast and dark-field microscopy at magnifications of �100 and �400.

RESULTS AND DISCUSSION

Individual populations of phototrophic consortia as de-tected by molecular fingerprinting. Theoretically, phototro-phic consortia could form randomly from bacterial cells whichencounter each other by chance. Under these conditions, mor-phologically identical consortia would be expected to harbordifferent types of green sulfur bacteria. To test this hypothesis,the epibionts associated with particular phototrophic consortiawere analyzed by 16S rRNA gene sequencing. Different num-bers of “C. magnum” were picked from Lake Cassidy, and the16S rRNA gene fragments of the epibionts were amplified andsubsequently separated by DGGE. Irrespective of the samplesize, all fingerprints exhibited the same melting behavior (Fig.2A). Sequencing confirmed that each band contained the same16S rRNA gene sequence (phylotype). Similarly, identical 16S

rRNA gene sequences were obtained for “P. roseum” isolatedfrom Lake Dagow when batches of 10 and 45 individual con-sortia were used (data not shown). Each other type of consor-tia, if it was obtained from one lake, always yielded a single 16SrRNA gene fingerprint and nucleotide sequence (Fig. 2B and3). It is unlikely that additional types of epibionts were presentin the same consortia but were missed by our approach be-cause of the following considerations. DGGE fingerprintinghas been shown to detect the 16S rRNA gene sequences whichrepresent more than 1% of all sequences present in a DNAsample (39). The mean number of epibionts per consortiumwas determined to be 19.9 � 4.4 in “P. roseum” and 36 � 4.4in “C. magnum” (43). Based on its sensitivity, the DGGEfingerprinting method is therefore suitable for detecting even asingle cell of a second phylotype per phototrophic consortium.Evidently, phototrophic consortia with the same morphologywhich share the same habitat contain only a single epibiontphylotype.

Different results were obtained when morphologically simi-lar phototrophic consortia were obtained from different lakes,however. Phylogenetic fingerprinting revealed that differentgreen sulfur bacterial phylotypes were present in “C. aggrega-tum” from European and North American lakes (Fig. 2B),although the consortia were identical with respect to shape andthe arrangement and color of the epibionts (Fig. 2C). Overall,seven different phylotypes were detected for “C. aggregatum,”three different 16S rRNA sequences were detected for “C.magnum,” and four phylotypes were detected for the brown “P.roseum” consortia (Table 1). We concluded that morphologi-cally indistinguishable consortia which occur in geographicallydistant locations frequently harbor distinct epibionts.

Detection of a novel type of phototrophic consortia. Watersamples from five Michigan lakes and Lake Dagow (easternGermany) contained a previously undescribed morphologicaltype of phototrophic consortium. This type consisted of 44 to

FIG. 2. (A) Epibiont 16S rRNA gene fragments amplified fromdifferent numbers of “C. magnum” consortia obtained from LakeCassidy and separated by DGGE. (B) Epibiont 16S rRNA gene frag-ments amplified from the same “C. aggregatum” morphotype found infour different lakes in North America and one lake in eastern Ger-many. Fragments were separated by DGGE. The gradient of denatur-ant in the gels shown in panels A and B was a gradient from 40% (top)to 60% (bottom). Negative images of ethidium bromide-stained gelsare shown. (C) Phase-contrast photomicrographs of the different “C.aggregatum” consortia corresponding to the DGGE fingerprints fromthe five lakes in panel B. Bar, 5 �m.

FIG. 3. Diversity of epibionts of phototrophic consortia in LakeCassidy. 16S rRNA gene fragments were amplified from all differentmorphotypes of phototrophic consortia present and were subsequentlyseparated by DGGE. A negative image of an ethidium bromide-stained gel is shown.



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58 cells of a brown epibiont which were associated with a singlecentral colorless motile bacterium (Fig. 4A and B). Epibiontswere arranged in several layers around the central rod, result-ing in a more stumpy shape for the intact consortium (Fig. 4A)compared to the shape of “P. roseum” (Fig. 4C and D). Thenovel type of consortium always contained identical 16S rRNAgene sequences in Michigan as well as in German lakes (Table1 and Fig. 5) (phylotype N), and the epibionts differed from allother brown epibionts. Based on these data, the name “Pelo-chromatium latum” is proposed for the newly discovered pho-totrophic consortium. Although we found only one phylotypefor “P. latum,” it is feasible that similar consortia which harborother epibiont phylotypes may be found in the future. Asindicated by the quotation marks, however, “P. latum” has nostanding in nomenclature and, like all previously publisheddesignations of phototrophic consortia, is only used to refer toa particular morphotype.

Diversity of epibionts of phototrophic consortia. To date,seven different morphotypes have been described based onmorphology and the cellular arrangement of their epibionts(20, 40, 42). In “C. aggregatum,” the colorless bacterium issurrounded by green rod-shaped bacteria, while brown epi-bionts are found in “P. roseum.” Consortia of the “C. glebulum”type are bent and contain gas-vacuolated green epibionts. “C.magnum” contains a larger number (�36) of green epibiontsthan “C. aggregatum” and has a globular morphology. “P. roseo-viride” has an inner layer of brown cells and an outer layer con-sisting of green bacteria. Finally, “C. lunatum” and “P. selenoides”harbor epibionts which are half-moon shaped and are greenand brown, respectively. In addition, two immotile associations(“Chloroplana vacuolata” and “Cylindrogloea bacterifera”) havebeen described, and these appear to occur more rarely.

The results presented above demonstrate that the phyloge-netic diversity of the epibionts in phototrophic consortia sig-nificantly surpasses the diversity deduced solely from morphol-

ogy. Our systematic survey of phototrophic consortia from the14 lakes yielded a total of 41 partial 16S rRNA gene sequences(Fig. 5 and Table 1). Among these, 15 distinct sequence types(phylotypes in Fig. 5 and Tables 1 and 2) were detected. Acumulative plot of the phylotypes versus the sample numbersreached saturation (Fig. 6). In the lakes studied, all types ofphototrophic consortia co-occur in the same layer (24) andcould therefore be analyzed from the same water sample.Therefore, our results indicate that the majority of epibiontphylotypes which were present in the lakes sampled were in-deed recovered by our approach.

Further analysis of our data demonstrated that the genomicdiversity of the epibionts is even greater than the phylogeneticdiversity since certain epibionts showed considerable differ-ences in morphology and pigmentation. This was observed forsome “C. aggregatum” consortia, “C. lunatum,” “P. selenoides,”and “C. vacuolata” (phylotype D in Table 2 and Fig. 5). Also,epibionts from another “C. aggregatum” consortium and from“P. latum” were phylogenetically identical (phylotype N in Ta-ble 2 and Fig. 5). Despite their identical 16S rRNA genesequences, these epibiont cells clearly differ from each other.Thus, the epibionts of “C. aggregatum” are green and rod shaped,whereas those of “P. selenoides” are vibrioid and brown (phy-lotype D). The epibionts in “P. latum” are brown, whereas thosein “C. aggregatum” are green (phylotype N). Like other mem-bers of the Chlorobiaceae, green epibionts of phototrophicconsortia harbor bacteriochlorophyll c as a light-harvestingpigment, whereas brown strains and epibionts contain bacteri-ochlorophyll e (19, 26). It is well established that both sets ofpigments are constitutively expressed and that the two sets aremutually exclusive (40). Consequently, the different shapes andpigmentations of epibionts with identical partial 16S rRNAgene sequences indicate that the epibionts with the same phy-lotype differ genetically. Therefore, 16S rRNA gene sequencesalone are not sufficient for classification of the epibionts ofphototrophic consortia and for assessment of their diversity.While sequences of the internal transcribed spacer regions ofthe rrn operon or of functional genes potentially provide higherresolution and may be used to distinguish between the differentepibionts of phototrophic consortia in natural populations, thecurrently available amplification methods do not provide thenecessary sensitivity and specificity. Therefore, only the com-bination of phylogenetic, morphological, and pigment datacurrently provide the means to describe the full diversity ofgreen sulfur bacterial epibionts of phototrophic consortia.

In order to arrive at a more appropriate estimate of the totaldiversity of consortium epibionts, epibionts with identical phy-lotypes but different morphologies were considered separatetypes (D1to D4, N1, and N2 in Table 2). If each unique com-bination of a phylotype and a morphotype (phylomorphotype)was counted separately, a total of 19 different types could berecognized in our data set (Fig. 6). Again, the cumulative plotof the different phylomorphotypes reached saturation, indicat-ing that further sampling in the six geographic regions at bestwould yield only a few, very rare additional types of epibionts.In the individual geographic regions, the highest number ofmorphologically different phototrophic consortia was encoun-tered in Michigan lakes (Table 2). In this region, the 26phototrophic consortia analyzed yielded 10 different typesof epibionts. The repeated detection of identical epibiont

FIG. 4. Comparison of two morphotypes, “P. latum” (A and B) and“P. roseum” (C and D). The images are phase-contrast photomicro-graphs of consortia in the intact state (A and C) and in the disinte-grated state produced by squeezing consortia on agar-coated micro-scope slides (43) (B and D). Bar, 5 �m.



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phylomorphotypes (Fig. 6) indicates that essentially the en-tire diversity of epibionts had been recovered from thisgeographic area. In contrast, eight different types of epi-bionts were detected in 10 analyses of consortia in the Eu-ropean lakes. Consequently, the cumulative plot of epibionttypes did not reach saturation (not shown in Fig. 6), andmore types of epibionts are to be expected to occur in otherEuropean lakes.

The presence of a single type of epibiont in each type ofphototrophic consortium appears to be a general feature of alltypes of phototrophic consortia investigated (Fig. 2 and 3).Since an amplification method with comparable sensitivitydoes not exist for the 16S rRNA genes of the central �-pro-teobacteria, the present study was limited to the green sulfurbacterial epibionts. It remains to be determined whether thesame colorless motile bacterium occurs in different phototro-

FIG. 5. Phylogenetic tree of 16S rRNA gene sequences of green sulfur bacterial epibionts and their relatives. Distinct phylotypes are indicatedby uppercase letters A through P. Bar � 0.1 fixed point mutation per nucleotide position. Pld., Pelodictyon; Chl., Chlorobium; Env., environmental.



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phic consortia or whether each of the different types of epi-bionts is associated with another central bacterium. However,the first evidence for physiological differences between thecentral bacteria of different consortia comes from our chemo-taxis experiments with phototrophic consortia in Lake Siso(Fig. 7). The chemotaxis towards organic carbon compounds islikely to be mediated by the central motile bacterium (28). “C.aggregatum” from Lake Siso was attracted by pyruvate (Fig. 7).By comparison, the morphologically similar “C. aggregatum”from Lake Dagow was reported to respond to 2-oxoglutarate

but not to pyruvate (19), and intact “P. roseum” consortiaexhibited a pronounced chemotaxis towards sulfide and weaklyresponded to 2-oxoglutarate (28).

Implications for the evolution of epibionts. Epibionts of allconsortia investigated to date represent unique 16S rRNAsequence types and so far have not been found in a free-livingstate. Together with the rapid signal transfer demonstratedpreviously (19), our findings support the view that the interac-tion between the green sulfur bacteria and the chemotrophicmotile bacteria is an obligate interaction. Epibionts of pho-

TABLE 2. 16S rRNA signature sequences and biogeographical distribution of the different epibiont types

Phylo-type

Accessionno.

16S rRNA nucleotide signature(s)a

Morphotype Phylo-morphotype

Presence ingeographical regionsb

409 453-454 476 479 613 624 627 638 658 746 748 1 2 3 4 5 6

A AJ578401 “C. magnum” A XB AJ578402 “P. roseum” B X XC AJ578404 “C. aggregatum” C XD AJ578405 “P. selenoides” D1 X

“C. lunatum” D2 X“C. aggregatum” D3 X“C. vacuolata” D4 X

E AJ272094 “C. magnum” E XF AJ272092 G “C. glebulum” F XG AJ578410 “P. roseum” G XH AJ578411 U “C. magnum” H X XJ AJ272091 “C. aggregatum” J XK AJ578417 “P. roseum” K X XL AJ578418 AC A G Cc A U “C. aggregatum” L XM AJ272090 AC A G C A G U “C. aggregatum” M X XN AJ578423 “C. aggregatum” N1 X

“P. latum” N2 X XO AJ578428 C A “P. roseum” O XP AJ578432 A “C. aggregatum” P X

a E. coli numbering.b See Fig. 1. There were 8 phylomorphotypes in Europe and 13 phylomorphotypes in North America. We found 7, 1, 1, 1, 10, and 4 phylomorphotypes in regions

1, 2, 3, 4, 5, and 6, respectively. Two, one, two, one, seven, and one lakes were sampled in regions 1, 2, 3, 4, 5, and 6, respectively, and the numbers of phylomorphotypesper lake in these regions (as calculated from the data in Table 1) were 3.5, 1, 1, 1, 3.7, and 4, respectively.

c Changes are complementary according to the secondary structure (20).

FIG. 6. Comparison of the total diversity of phylotypes of epibionts(solid circles) and of phylotypes with distinct morphologies (phylomor-photypes) (shaded triangles) present in the data set. Also included is acumulative plot for epibiont types found in Michigan lakes (shadeddiamonds). Random numbers were generated and assigned to thestrains (Sample No.), and the sequential detection of different phylo-types and phylomorphotypes was plotted.

FIG. 7. Chemotactic accumulation of “C. aggregatum” from LakeSiso. The bars indicate the numbers of phototrophic consortia countedin capillaries after incubation for 3 to 5 h. Three or four parallelexperiments were performed. The error bars indicate standard devia-tions of the counts. Values that differ from the control value at a highlevel of significance (P 0.001) are indicated by three asterisks.



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totrophic consortia form several distinct phylogenetic clusters(Fig. 5). This suggests either that the ability to form symbioticassociations arose independently from different ancestors or,alternatively, that the common ancestor of the green sulfurbacteria was symbiotic. In the latter case, several descendantlineages would have developed the ability to sustain an inde-pendent life style. In any event, our phylogenetic analysis im-plies that the switch from the symbiotic state to the free-livingstate (or vice versa) occurred more than once during the radi-ation of the green sulfur bacteria.

Biogeography of phototrophic consortia. For the most part itis assumed that physical or geographical barriers do not existfor prokaryotes (36), and it has been suggested that the highpopulation densities of microorganisms drive rapid, large-scaledispersal across physical and geographical barriers (16, 17).Species like Escherichia coli, Salmonella enterica, or Haemophi-lus influenzae consist of a limited number of clones; each,however, has a worldwide distribution (38). For Neisseria men-ingitidis, particular genotypes have been detected on differentcontinents (9). Evidently, dispersal of such human pathogensand commensals is rapid enough to permit widespread geo-graphic distribution not only of bacteria with identical 16SrRNA gene sequences (phylotypes) but even of cells withthe same genotype. For most other free-living or nonhumanpathogens and symbionts investigated, endemicity seems tobe limited to the level of genotypes (strains or subspecies)rather than bacterial species (phylotypes). This is true forvery different bacteria like the fluorescent pseudomonads,the gram-positive organism Renibacterium salmoninarum, orstrains of the symbiotic cyanobacterium Nostoc sp. (10, 30,32).

While studies of marine and freshwater microorganisms in-dicate that most of the species are ubiquitous (3, 13, 15, 16, 17,21, 22, 46, 51), some soil bacteria appear to have a morelimited distribution (10, 47). Limited migration between NorthAmerica and Central America has been shown for Rhizobiumleguminosarum biovar phaseoli (47). Cosmopolitan speciescould not be detected among psychrophilic sea ice bacteriafrom Arctic and Antarctic samples (49). Similarly, 16S rRNAgene sequencing of different populations of Achromatium ox-aliferum suggested a high degree of endemism, since identicalsequences were never recovered from geographically sepa-rated freshwater sediments (29). Hence, endemic species ofbacteria may exist in some rare cases.

Our data permit a first assessment of the biogeography ofphototrophic consortia. A pairwise comparison of the types ofepibionts between the Michigan lakes (excluding Mud Lake,which contained only a single type of epibiont) yielded a meanvalue for the Dice coefficient of 0.582 � 0.162 (n � 15). Thishigh value shows that communities of phototrophic consortiain lakes in this geographic area are highly similar. In contrast,only two types of epibionts (phylomorphotypes M and N2)were found to occur on the two continents investigated (Fig. 5and Table 2). The Dice coefficient for the pairwise comparisonbetween North American and European epibionts was only0.190. Thus, there is a nonrandom biogeographical pattern anda low level of similarity of epibionts between the two conti-nents. Theoretical considerations indicate that a higher level ofsimilarity between North American and European lakes isunlikely. A high level of similarity (i.e., a Dice coefficient of

0.58) between North American and European lakes would bepossible only if at least 20 different types of epibionts occurredin Europe and at least 50% of these types were also present inNorth American lakes. In reality, much less (25%) of theepibiont types detected in Europe were also present in NorthAmerica. While the possibility of greater diversity (�20 epi-biont types) cannot be excluded for European lakes (since thedata set for this continent is incomplete [see above]), it appearshighly unlikely that our random sampling procedure selectivelyexcluded just those epibionts which are common to NorthAmerican and European lakes.

Theoretically, the environmental conditions in North Amer-ican lakes could differ significantly from those in Europeanlakes and thereby select for different epibiont types. In the caseof phototrophic consortia this is highly unlikely for two rea-sons. First, different epibionts were detected in lakes with verysimilar physicochemical conditions if they were located ondifferent continents. For example, Lake Dagow, Schleinsee,and Mud Lake were very similar with respect to environmentalconditions (Table 1). Whereas the phylotype K epibiont ofSchleinsee was also found in Lake Dagow (both Europeanlakes), the unique phylotype A found in Mud Lake (located inNorth America) was not found in the two other lakes. Second,in 9 of the 14 lakes investigated, multiple (i.e., up to six differ-ent) types of consortia occurred simultaneously in the sameenvironment (Tables 1 and 2). The latter finding in particularsupports the conclusion that environmental conditions are notthe selective factor governing the geographic distribution ofthe epibionts of phototrophic consortia.

It has been shown that common soil bacteria, like Curtobac-terium citreum, Bacillus megaterium, Arthrobacter globiformis,Microbacterium spp., Sphingomonas spp., Sinorhizobium spp.,and Paracoccus spp., and fungal pathogens of plants are trans-ported on dust particles and are capable of surviving long-range transport through the atmosphere (6, 31). Transportbetween continents occurs on a time scale of �6 days (33). Inthe case of epibionts of phototrophic consortia, the high sim-ilarity between populations in neighboring lakes and the pro-nounced intercontinental differences suggest that the dispersalof consortia is comparatively slow.

Conclusions. During the present study, 16S rRNA gene se-quences of epibionts of phototrophic consortia were foundexclusively in the associated state. Although we cannot entirelyrule out the possibility that epibionts also occur in the free-living state in other environments, our results strongly suggestthat there is a close association and that there is specific ad-aptation of epibionts to the symbiotic state. Phototrophic con-sortia therefore do not form just by chance. Still, multiplechanges from the symbiotic life style to the independent lifestyle (or vice versa) must have occurred during the evolutionof green sulfur bacteria, as indicated by the phylogenetic anal-ysis.

Our survey of 14 different aquatic environments yielded 15distinct phylotypes of epibionts and, when the differences inmorphology and pigmentation were considered, a total of 19phylomorphotypes. The first data on the chemotactic behaviorof phototrophic consortia suggest that the central chemotro-phic bacteria are also different in different types of consortia.The unexpected diversity of the epibionts of phototrophic con-sortia is distributed in a nonrandom fashion among different



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geographic regions. Phototrophic consortia therefore harbor apreviously unrecognized diversity of green sulfur bacterial epi-bionts which appear to be only slowly dispersed over longgeographic distances.

ACKNOWLEDGMENTS

We thank Carlos Abella (University of Girona, Girona, Spain) forkindly providing samples from Michigan lakes and J. Staley (Universityof Seattle) for providing the water sample from Echo Lake. We areindebted to Ann-Katrin Manske for assistance with construction of thephylogenetic tree and to Kajetan Vogl for providing the epibiont se-quence of “C. vacuolata.” J. Garcia-Gil kindly offered valuable supportin the field and laboratory space at the University of Girona.

This study was supported by grants Ov 20/3-3 and Ov 20/10-1 fromthe Deutsche Forschungsgemeinschaft to J. Overmann.

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