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Antonie van Leeuwenhoek DOI 10.1007/s10482-006-9090-x

ORIGINAL PAPER

Microbial diversity in natural environments: focusing on fundamental questions

David M. Ward

Received: 11 May 2006 / Accepted: 11 May 2006 Springer Science+Business Media B.V. 2006

Abstract Interactions with Gijs Kuenen and communities, and for investigating how such units other Dutch scientists have led my lab to funda- coordinate the physiological activities within mental insights into the composition, structure guilds of the community. and function of a cyanobacterial mat community that should influence our thinking Keywords Microbial mats Æ Molecular analysis Æ about all microbial communities. By focusing on Æ the distribution of molecular sequence variants of predominant mat phototrophs, we have discov- Abbreviations ered that small-scale sequence variation can be GNSB green nonsulfur or nonsulfur-like ecologically meaningful. By applying novel culti- vation approaches, we have been able to obtain DGGE denaturing gradient gel electrophoresis genetically relevant community members and thus to test the hypothesis that closely related sequence variants arose via adaptive evolutionary Introduction . By applying the analytical tools of organic geochemistry we have gained insight into It is a pleasure to submit this contribution in the of major phototrophic members honor of Gijs Kuenen, a long- friend and of the mat community as well as interactions colleague in microbial , as well as between phototrophic guilds. These observations other Dutch scientists, many of whom are challenge traditional paradigms about prokary- also associated with the Delft School of otic species and cause us to consider evolutionary , who have had a major influence ecology theory as we develop -based on my research through the years. As I methods for high-resolution analysis of the spe- contemplate the impact Gijs has had on my cies-like fundamental units comprising microbial program, I think back to a backpacking trip we made together in the early 1990s to the Edith Baldy Basin in the high country of Montana. We were en route between Hidden Lake and D. M. Ward (&) Edith Lake, when Gijs was inquiring as to what Department of Land Resources and Environmental my plans were following our initial molecular Sciences, Montana State University, Bozeman, MT 59715-3120, USA analyses of well studied hot spring cyanobacte- e-mail: [email protected] rial mat communities. I don’t recall my specific

123 answer, but it was something along the lines of Methods continuing our analysis deeper and deeper into the molecular diversity of of the The variety of methods used to obtain the results community, as if on a quest to discover all the presented in this review have been detailed in the community members through their molecular primary peer-reviewed papers cited below. signatures. I also don’t recall Gijs’ specific reaction, but it was something along the lines of how silly an idea that was. He encouraged me Results to focus on the predominant and easily detected members of the community, asking fundamental An example of the communities in question, questions about their diversity and ecology, microbial mats in alkaline siliceous hot springs including their contribution to community (Octopus Spring and Mushroom Spring,Yellow- function. In the intervening years, my students stone National Park) is shown in Fig. 1. Before and I have followed this advice and I believe it my students and I began studying this system as has led to much more important insights, as a model for microbial community ecology in Gijs predicted. In this paper, I would like to 1977, Tom Brock (my mentor), Richard highlight our progress, paying special attention Castenholz and their students had studied the to ways in which Gijs and other Dutch scientists system for a number of years (Brock 1978; were involved. Ward and Castenholz 2000). The mat seemed to

Fig. 1 Landscape view of Octopus Spring, an alkaline in the summer of 1991. The top green layer of a mat siliceous hot spring in Yellowstone National Park. sample collected at ~ 60C (inset at lower left) is comprised Cyanobacterial mat is visible beneath the inset (upper of sausage-shaped and filamentous right) showing Alyson Ruff-Roberts, Gijs Kuenen, Eric Roseiflexus cells (inset to right of mat image) Kopczynski and Dave Ward conducting fieldwork in this

123 Antonie van Leeuwenhoek be a relatively stable and simple system, con- variants were observed, all distantly related structed by a single morphotype of unicellular to the S. lividus 16S rRNA sequence, cyanobacterium, Synechococcus, and a single with variants falling into two (A and B morphotype of green nonsulfur bacterium clades in Fig. 2A). Similarly, several closely (GNSB), Chloroflexus. The cultivation from hot related GNSB-like 16S rRNA sequence variants spring mats such as these of S. lividus (Meeks were observed, all distantly related to the 16S and Castenholz 1971; Kallas and Castenholz rRNA sequence of C. aurantiacus (C-like 1982) and C. aurantiacus strains (Bauld and sequences in Fig. 2B). The GNSB sequences Brock, 1973; Pierson and Castenholz 1974) were later recognized as closer relatives of showing little genetic difference reinforced the Roseiflexus castenholzii, a bacteriochlorophyll notion of a simple community composition a-rich filamentous anoxygenic phototroph (Ferris et al. 1996b). By the early 1990s, we had (Nu¨ bel et al. 2002). This paper will focus on developed 16S rRNA-based cloning and the in-depth study of these phototrophic sequencing methods for molecular analysis of community members, which appeared to be the community and had already discovered that predominant based on the frequency of recov- the system was more complex than originally ery of sequence variants of these types and on thought (Ward et al. 1990, 1992). Several closely enrichments on highly diluted samples (see related Synechococcus 16S rRNA sequence below).

Fig. 2 16S rRNA-based phylogenetic trees of (A) cyano- predominant in situ (lower shaded regions) bacterial and (B) green nonsulfur-like bacterial diversity in and those readily cultivated (higher shaded regions). the Octopus Spring (OS, thick lines in A) or Mushroom Isolate C1 in (A) has a 16S rRNA sequence identical to Spring (MS, boxed entries in B) cyanobacterial mats that of S. lividus. [Ward et al. 1998 and Nu¨ bel et al. 2002] relative to pure-cultured organisms. Shading contrasts

123 Antonie van Leeuwenhoek

Community composition and structure Shortly thereafter, my lab had the good fortune of collaborating with another Dutch scientist, Patterns lead to hypotheses about their causes Gerard Muyzer, who later joined Gijs’ department in Delft. Gerard had introduced to microbial We hypothesized that distribution along environ- ecology a new and powerful method, denaturing mental gradients might give clues to the signifi- gradient gel electrophoresis (DGGE), which sep- gance of closely related 16S rRNA sequence arates similarly sized molecules that differ in variants in the community. Our first approach to sequence (Muyzer et al. 1993). DGGE offers distribution analysis was oligonucleotide probing of greater resolution of closely related sequence the 16S rRNAs of both cultivated and uncultivated variants and is particularly useful for simulta- (S. lividus and genotypes A, B) and neously observing multiple 16S rRNA sequences filamentous anoxygenic phototrophs (C. aurantia- in a single sample. Gerard visited my lab to teach cus and genotype C) that were known to us at the my student Mike Ferris DGGE technology. The time (Ruff-Roberts et al. 1994). Gijs was visiting resulting papers (Ferris et al. 1996a; Ferris and my lab in the summer of 1991, helping my student, Ward 1997) revealed the presence of even more Alyson Ruff-Roberts design and use such probes. closely related variants of Synechococcus (A¢,A¢¢ This work gave us the first insights that these and B¢)andRoseiflexus (C¢,C¢¢) 16S rRNA se- closely related genotypes (e.g, type-A and type-B quences (Figs. 2, 3B and 4A). Importantly, DGGE Synechococcus) were contributed by ecologi- analysis sharpened our focus on how these se- cally distinct community members, since distinct quence variants distribute along thermal (Ferris genotypes were found at different temperatures and Ward 1997) and vertical (Ramsing et al. 2000) (Fig. 3A) and sometimes at different . gradients. It became clear that very closely related 16S rRNA variants (Fig. 4B) exhibited distinct ecological distributions (Fig. 4A). Such - ary and ecological patterning lead us to hypothesize that, like many and species, adaptive evolutionary radiation might explain the origin of the diversity of closely related, yet ecologically distinct populations we were seeing (Ward et al. 1998)(Fig. 4C).

Cultivating the as yet uncultivated

To test the hypothesis that these closely related 16S rRNA sequence variants were from repre- sentatives of populations with different ecological , it was necessary to first cultivate these novel Synechococcus and Roseiflexus strains. Mi- crobiologists often use the word ‘‘uncultivatable’’ to describe the microbes recognized by molecular analysis, but not yet in culture. I prefer the phrase Fig. 3 Patterns suggesting of Octopus Spring ‘‘as yet uncultivated’’ to reflect the optimism cyanobacteria to different temperatures. (A) Response of oligonucleotide hybridization probes targeting 16S rRNA (typical of the Delft School of Microbiology) sequences of A-like and B-like Synechococcus with mat that we will eventually understand how to RNA extracted from different temperature sites [redrawn cultivate such organisms. The extincting dilution from data of Ruff-Roberts et al. 1994]. (B) DGGE gel of enrichment culture approach (i.e., dilution of 16S rRNA segments PCR-amplified from DNA extracted from mat from different temperature sites; inoculum before liquid enrichment to avoid labeled bands were purified and sequenced [from Ferris overgrowth by rare, but rapidly growing, commu- et al. 1996a] nity members) gave some success in cultivating

123 Antonie van Leeuwenhoek

Fig. 4 Patterns suggesting that A/B-type Octopus Spring ancestor. (C) Adaptive radiation of Galapagos finches Synechococcus populations evolved via adaptive evolu- from recent common ancestors into ecologically specialized tionary radiation. (A) DGGE gel showing progression of B, populations [from Begon et al. 1996]. (D) Relationships B¢,A,A¢ and A¢¢ sequence variants from 48–49C to 64– between temperature and growth rate for Synechococcus 72C temperature sites. [lanes within each grouping isolates with B, B¢ and A 16S rRNA genotypes that confirm are replicate samples from Ferris and Ward 1997] the hypothesis of adaptive differences [from Allewalt et al. (B) Phylogenetic tree shown in Fig. 2A highlighting the 2006] descent of these populations from a recent common genetically relevant mat community members, as Holland, Sjila Santegoeds, whom Gijs had shown for mat phototrophs by Ferris et al. (1996b) encouraged to visit my lab (Santegoeds et al. and Ward et al. (1997), and for mat 1996). As shown in Fig. 5A, organisms contribut- (Fig. 5A) by a visiting undergraduate student from ing DGGE bands 1–3 were outnumbered by

123 Antonie van Leeuwenhoek organisms contributing DGGE bands 4, 5 and 6 bacteria, by using direct counting it was pos- but competitively excluded the more dominant sible to determine growth rates of Synechococcus community members unless first removed by isolates corresponding to these genotypes dilution to extinction. However, our success in (Fig. 4D) and thus to note thermal adaptations cultivating strains of both major types of photo- consistent with environmental distributions. For trophs that are genetically relevant compared to instance, the Synechococcus B, B¢ and A isolates natural populations came as a result of utilizing a are optimized for growth at 50C, 50–55C and unique approach introduced by de Bruyn et al., 55–60C and were not observed to grow above (1990) in Gijs’ department to cultivate iron-oxi- 55C, 60Cand65C, respectively, similar to the dizing bacteria. This method involves dilution of a distributions of these genotypes in situ (Fig. 4A). natural sample and filtration onto a nucleopore Miller and Castenholz (2000) showed similar filter, which is then incubated above a support filter results for Oregon Synechococcus strains, includ- saturated with culture medium (Fig. 5B). The idea ing an A¢-like isolate that showed yet higher tem- is that physical separation of cells prevents over- perature adaptation. growth by less numerically relevant cells with In collaboration with Mike Madigan (Southern greater fitness for growth on the culture medium. Illinois University) and myself another Dutch The approach also eliminates the problem that student, Marcel van Der Meer, used the filter medium solidifying agents may contain toxic cultivation approach to obtain strains of Roseifl- materials. In the case of Synechococcus, discrete exus that are genetically relevant to the mat (van green colonies formed on low-dilution filters der Meer et al., in prep.). It is interesting that (Fig. 5B) and these colonies were successfully these Roseiflexus strains did not form discrete subcultured and purified to unicyanobacterial colonies, but rather red-colored growth that cultures (Allewalt et al. 2006). Representatives of developed slowly between colonies. This material genotypes B, B¢ and A were obtained. Although was subcultured and purified to obtain cultures were contaminated by small rod-shaped cultures. The 16S rRNA sequences of these

Fig. 5 Cultivation of predominant mat populations. (A) DGGE analysis of enrichment cultures for mat heterotrophic microorganisms inoculated with various 10-fold dilutions of mat sample [from Santegoeds et al. 1996]. (B) Colonies of Synechococcus growing on floating filters

123 Antonie van Leeuwenhoek strains were nearly identical to Roseiflexus geno- research in our NSF Frontiers in Integrative types discovered by Uli Nu¨ bel, a student of Research program, see http://www.lan- Gerard’s who later became a postdoc in my lab dresources.montana.edu/FIBR/), and a Yellow- (Nu¨ bel et al. 2002). Uli used fluorescent in situ stone Roseiflexus strain (unpublished results of oligonucleotide hybridization probing to demon- Don Bryant, Pennsylvania State University). strate that Roseiflexus is a dominant filamentous These databases are guiding us to yet higher of the mat. resolution studies designed to reveal the full complement of ecologically Entering the genomics era specialized populations in the mat community.

We have recently obtained the genomic sequences Community function of Synechococcus strains with genotypes B¢ and A as well as metagenomic sequences obtained Together with Dutch organic geochemists, Jan de directly from the mat communities (ongoing Leeuw, and Jaap Sinninghe-Damste´ (formerly of

A midday evening night morning

CO2 CO2 Sunlight CO2 Sunlight

. Polyglucose Polyglucose Polyglucose Storage Synechococcus Fermentation Fermentation Acetate (+ Propionate) Acetate (+ Propionate) Glycolate + + Photo- Photo- Photo- heterotrophy heterotrophy heterotrophy H2 H2

Photo- autotrophy GNSB Photo- autotrophy H2S H2S Sulfate reduction Sulfate reduction

B Synechococcus C Synechococcus GNSB 300.0 2450 normal 250.0 dark pre-incubated 1950 dark pre-incubated + H2 -2 200.0 dark pre-incubated + S

C 1450 T1 C

150.0 13 T2 13 T3 δ δ GNSB T3+Gly/Ac 100.0 950

50.0 450 0.0

-50 -50.0 FA FA WE WE WE FA FA WE WE WE WE WE 18 33 35 32 alkane 16 18 32 16 34 35 34 33 C C C C alkane C 17 C C C C C C C C 17 C

13 Fig. 6 Physiological processes in alkaline siliceous hot GNSB biomarkers with CO2 and subsequent overnight spring microbial mats. (A) Model for carbon flow during (T2) and morning (T3) redistribution of 13C in the 13 a diel cycle. (B) Incorporation of CO2 into Synecho- absence or presence (T3 + Gly/Ac) of a mixture of and GNSB biomarkers during a morning incuba- unlabeled glycolate and acetate. GNSB, green nonsul- tion period under different incubation conditions. (C) fur-like bacteria WE, wax esters. [from or redrawn from Afternoon (T1) pulse-labeling of Synechococcus and van der Meer et al. 2005]

123 Antonie van Leeuwenhoek

TU Delft) and Stefan Schouten (all now of the GNSB biomarkers were isotopically much hea- Royal Netherlands Institute for Sea Research, vier (–16.4 & to À18:9 &) (van der Meer et al. Texel) we have been able to gain fundamental 2000, 2003). One explanation for this apparent insights into how the major phototrophic com- anomaly is photoautotrophic munity members function in the mat (Fig. 6A). involving CO2 fixation by the 3-hydroxypropio- Since mats are considered modern analog of nate pathway, which has been described in , ’s predominant of the C. aurantiacus (Herter et al. 2002). This pathway Precambrian Era, our aim is to investigate how discriminates less between 12C and 13C than the complex reality of mat community members does the Calvin Cycle and thus leads to pro- and their interactions may influence geochemical duction of organic with a heavier 13C signals that might survive in ancient mats and signature (Holo and Sireva˚g 1986; van der Meer sediments. Past work had suggested that the fila- et al. 2001). We were able to find evidence mentous anoxygenic phototrophs (i.e., GNSB-like suggesting the potential for autotrophy through 13 organisms, such as Roseiflexus and Chloroflexus) incorporation of CO2 into wax esters during of these mats, which grow especially well morning hours, when H2S (and possibly H2) photoheterotrophically in culture, were doing so accumulate as products of fermentation and in situ as well, consuming low molecular weight sulfate reduction in the mat photic zone (van organic materials produced and excreted by der Meer et al. 2005). This activity required Synechococcus. This hypothesis was supported by preincubation to allow labeled substrate to dif- observing light-stimulated uptake of fermentation fuse throughout the sample, and was stimulated products (Sandbeck and Ward 1981; Anderson by addition of H2 and H2S (Fig. 6B). et al., 1987) and glycolate (Bateson and Ward 1988) by filaments in the mat. Photoheterotrophy by green nonsulfur bacteria-like community members Photoautotrophy by green nonsulfur bacteria-like community members We have recently been exploring a second possi- ble explanation for the heavier d13C signatures of Marcel first screened C. aurantiacus, R. cast- Roseiflexus biomarkers. My NIOZ colleagues enholzii and Yellowstone Roseiflexus isolates were aware that sugars produced by cyanobacteria (van der Meer et al., 1999, 2002, in prep.) to have heavier d13C signatures than do lipids (van discover diagnostic lipid biomarkers that could Dongen et al. 2001). We had discovered in earlier be used to study the physiology of Roseiflexus work that polyglucose is the main photosynthetic Synechococcus 14 independently of . All of these product in the mats (~84% of fixed CO ), and GNSB-like organisms produce long-chain wax 2 that this polyglucose is fermented primarily to esters, but wax esters of Yellowstone Roseiflexus acetate and CO2 under dark anaerobic conditions isolates most closely matched those found in the (Nold and Ward 1996)1. Furthermore, as men- mat (van der Meer et al., in prep.). He then tioned above, we knew that acetate is photoin- used isotope ratio monitoring gas chromatogra- corporated into filamentous cells in the mat. We phy-mass spectrometery to study the natural hypothesized that transfer of isotopically heavy abundance of 13C in biomarkers from these sugar fermentation products, especially acetate, organisms and cyanobacterial mat inhabitants between Synechococus and GNSB could explain (van der Meer et al. 2000). Based on the argu- the heavier d13C of GNSB lipid biomarkers. We ment that ‘‘you are what you eat’’, we expected 13 first demonstrated that the d C of sugars in the GNSB lipid biomarkers to have d13C signatures mat was heavy enough ( À11‰) to explain the similar to those of Synechococcus lipid biomar- kers. As expected, cyanobacterial biomarkers (e.g., nC alkane) showed d13C signatures 17 1 While pursuing a Ph.D. in my lab, Steve Nold mentored typical of autotrophs using the Calvin Cycle for Sjila Santegoeds; after receiving his degree he did a post- CO2 fixation (e.g., –29.6 to À34:1). However, doctoral in Holland with Riks Laanbroek.

123 Antonie van Leeuwenhoek heavy GNSB lipids (–16.4 & to À18:9 &) (van der Discussion Meer et al. 2003). We then attempted to track the flux of carbon between Synechococcus and GNSB, The advice of Gijs Kuenen to focus on questions as follows (Fig. 6C). We (i) conducted an after- of fundamental importance, and on major players/ 13 noon labeling of mat samples with CO2, which dominant organisms, has led us to new insights appeared to label mainly Synechococcus lipids into the composition, structure and function of (Fig. 6C, T1) and, based on the earlier 14C labeling microbial communities. Clearly, the development studies, should have resulted primarily in pro- of techniques for molecular analysis of microbial duction of 13C-labeled polyglucose, (ii) removed diversity has revolutionized . 13 unreacted CO2 and (iii) allowed the samples to Molecular analysis has revealed a vastly different incubate overnight. 13C appeared in GNSB wax view of microbial community ecology than was esters by morning (Fig. 6C, T2) and the extent of provided by traditional methods (e.g., microscopy labeling increased through the morning hours and cultivation). More importantly, it has enabled (Fig. 6C, T3), suggesting transfer from Synecho- us in recent years to begin to take a population- coccus. Interestingly, addition of an unlabeled based view of microbial community ecology sim- acetate/glycolate mixture (intended to trap sus- ilar to the view that plant and animal ecologists pected labeled intermediates) prevented the in- have developed. creased labeling during morning hours (Fig. 6C, As a community ecologist I view the most T3 + gly/ac), suggesting that acetate and/or gly- pressing concerns as being (i) which microorgan- colate may be specifically involved as intermedi- isms comprise a community, (ii) how they are ates in the exchange of 13C between structured within the community and (iii) how Synechococcus and GNSB. Overall, these results they control the functioning of the community. are consistent with the hypothesized night-time Molecular approaches have identified the popu- fermentative metabolism of Synechococcus and lations that are truly dominant in microbial photoheterotrophic incorporation of fermentation communities and have revealed the vast genetic products by GNSB in the morning. differences between these and the more readily The relative importance of these two mecha- cultivated microorganisms. In our system, in both nisms has not yet been resolved. Nearly equiva- photosynthetic guilds (oxygenic and anoxygenic), lent rates of C uptake via autotrophy and microscopy oversimplified diversity and cultiva- heterotrophy (estimated from 13C-acetate incor- tion methods reinforced this oversimplified view. poration experiments) were observed, however, Furthermore, more often than not, sets of closely these estimates are subject to many experimental related molecular variants are observed in limitations (van der Meer et al. 2005). Further- molecular analysis of microbial communities. This more, the durations of photoautotrophic and raises the obvious question of the importance of photoheterotrophic metabolisms during the diel small-scale molecular diversity. cycle are unknown. New approaches enabled by To truly understand community composition genomic data are now being taken to attempt to and structure we need to know whether, and if so learn more about the timing of these processes how, molecular variation is linked to species during a diel cycle. Steunou et al., (2006) have variation. I use the word ‘‘species’’ intentionally demonstrated that in situ expression of Synecho- for two reasons. First, biologists studying com- coccus involved in photosynthesis and munities of and consider species as fermentation follows a pattern that is entirely the fundamental units of which communities are consistent with a shift from daytime photosyn- comprised, the populations that will uniquely rise thesis to night-time cyanobacterial fermentation. and fall when the environment changes because We plan to conduct parallel studies of Roseiflexus they are adapted to distinct niches (Mayr 1982) , enabled by the new genome Second, use of the word species evokes debate sequence that should reveal when its photoauto- about the subject, which I believe is healthy, trophic and photoheterotrophic metabolisms especially in microbiology today. I have argued occur in the mat as well. against traditional prokaryotic species concepts

123 Antonie van Leeuwenhoek and for more natural species concepts (Ward silversword plants, see Ward et al. 2002). A 1998, 2006). This argument was first based on popular view in microbiology is that we can only evolutionary and ecological patterns that reveal be certain that populations whose 16S rRNA that closely related 16S rRNA variants of Syn- sequences are at least 2–3% different are distinct echococcus represent distinct ecological popula- species (Stackebrant and Goebel 1994; Goodfel- tions. As I have suggested in a current review low et al. 1997; Venter et al. 2004). However, this (Ward 2006), such patterns have been commonly does not seem reasonable, given the obvious observed in molecular microbiology studies of adaptive differences of populations exhibiting various microbial communities. Here, I highlight < 1% 16S rRNA difference, and, in some cases, of the work of Uli Nu¨ bel, Gerard Muyzer and col- populations with identical 16S rRNA sequence, leagues (Nu¨ bel et al. 2000), which demonstrated but apparently unique (Ferris et al. that members of evolutionary clusters of cyano- 2003). It is important to realize that such molec- bacterial 16S rRNA variants detected in hypers- ular cutoff’s result from quantification of the aline microbial mats exhibit unique distributions genetic differences among strains of named pro- along a salinity gradient, at least in part due to karyotic species, hence, reinforcing our faith that adaptations to salinity (Fig. 7). As mentioned named species are true species (Cohan 2002). above, in botany and zoology such patterns have In a current review (Ward 2006) I present the been interpreted as adaptive evolutionary radia- argument that there is much to be gained by tions of species (e.g., Galapagos finches, Hawaian considering how macrobiologists think about

Fig. 7 Patterns suggesting evolutionary radiation of cy- to highlight 5 major phylogenetic clades. (B) Proportional s d anobacteria with different salinity adaptations in Guerrero abundances ( , microscopic counts; , DGGE band Negro, Baja California Sur, Mexico salt evaporation pond intensity) of the members of each highlighted in microbial mats [redrawn from Nu¨ bel et al. 2000]. (A) (A) as a function of salinity of different ponds. Dark and Cyanobacterial phylogenetic tree based on 16S rRNA hashed horizontal bars above each distribution plot exhibit sequence variation, showing relationship of sequences ranges of optimal and suboptimal growth rate ranges, retrieved as DGGE bands from ponds of different salinity respectively, for isolates from each clade. Abundance (boxed entries with pond designations P1–P6) to diagrams in (B) are highlighted using the same intensity sequences of representative cyanobacterial cultures. Shad- scheme used in (A) ing of decreasing intensity (top to bottom of tree) is used

123 Antonie van Leeuwenhoek species. The argument is based in large part on A B A le l lle s le interactions with Fred Cohan (Wesleyan Univer- A 1 et le et sity) with whom I spent a recent sabbatical leave. s 2 His course ‘‘Origins of Bacterial Diversity’’ (available at http://www.landresources.montana. edu/FIBR/ and which makes a wonderful gradu- ate-level tutorial) and approaches (e.g., Cohan, 2002; Gevers et al. 2005) organized my thinking about evolutionary ecology theory. Periodic selection theory is based on the premise that species are populations whose individual members The Actual History Molecular Phylogenetic of Organismic Lineages Analysis exhibit variation, but occupy the same niche (Fig. 8A). Occasionally, natural selection (more Fig. 8 Periodic selection model for the evolution of precisely periodic selection) favors a most-fit species populations. (A) proceeding upward from the base of the tree, a blue fan of diversity is variant, squashing diversity within the population; squashed (dashed lines indicate extinction of individuals) the survivor founds a new population that, with due to the selection of a single most-fit variant (solid line), time, redevelops diversity as neutral mutations which diversifies again (2nd fan); a series of periodic accumulate within new individuals. A series of selection events provides a cohesive force holding the population together. The divergence of a new population periodic selection events provides the cohesive (green) occurs when a variant acquires the ability to force holding the ecological population together occupy a new niche and is thus immune from the periodic as it evolves within the niche. The evolution of a selection events affecting the parental (blue) population. variant that is capable of occupying a new niche, Eventually, the two populations diverge into two distinct clusters corresponding to two distinct ecological popula- leads to the evolution of a new ecological popu- tions. (B) Modern phylogenetic tree showing distinct lation that is free to diverge from the parent sequence clusters with unique sets of allelic variants population because periodic selection affects only the organisms within a single niche. Given suffi- cient time, the new population diverges through evolutionary ecology theory to help us predict its own series of periodic selection events to form how individual molecular variants group into a new species. Periodic selection theory thus pre- clades. Cohan is developing theory-based evolu- dicts the evolution of terminal evolutionary clades tionary simulations to do this. One method that unite individual variants into species-like (Cohan 2002), is based on multi-locus sequence ecologically specialized populations, matching the typing, a high-resolution molecular approach empirical observations we (Ward and Cohan used by microbial population geneticists to dem- 2005) and others (Ward 2006; Nu¨ bel et al. 2000) onstrate the existence of many species-like pop- have made. Interestingly, both empiricists and ulations within named bacterial species (Maiden theorists have used the word ‘‘ecotype’’ to de- et al. 1998). Cohan and I, together with other scribe these species-like populations (Ward and collaborators are currently working to develop Cohan 2005). other approaches, based on genomic analysis As I see it, the detection of species in a (http://www.landresources.montana.edu/FIBR/). microbial community will require development of These approaches will be used to test various methods with sufficient molecular resolution to models of speciation (Ward and Cohan 2005; observe variation among individuals within ter- Gevers et al. 2005), including models that con- minal evolutionary clades. Cohan has pointed out sider horizontal gene flow ‘‘rare, but promiscu- that the neutral mutations that accumulate ous’’ (e.g., periodic selection model) or so uniquely in the genes of diverging populations frequent that it would prevent the formation of may provide a means of detecting unique eco- species through the divergence of ecologically logical populations (Fig. 8B), at least those that specialized populations, as suggested by Fig. 8. I have diverged sufficiently to accumulate enough find it hard to imagine how a high-frequency unique neutral mutations. We must then rely on horizontal gene flow model of species could be

123 Antonie van Leeuwenhoek reconciled with the frequent observations of perhaps nongrowing) physiological state (Nold evolutionary and ecological patterning referred to and Ward 1996). In such a high-density popula- above. If horizontal gene flow erased the genetic tion (i.e., ~1010 Synechococcus cells/ml) it is easy signatures of ecologically distinct populations as to speculate that this phenotype might be under they form, why would such signals ever be left in control. As opposed to balanced the phylogenetic record? macromolecular synthesis, daytime photosynthe- This goal of understanding community struc- sis leads mainly to polyglucose accumulation and ture depends on identification of the fundamental polyglucose is fermented at night. The diel species-like units of which a community is com- rhythm of photosynthesis and fermentation in prised. However, we are already gaining insight Synechococcus may well be under circadian con- through studies of conserved molecular markers. trol (ongoing research in our NSF Frontiers in At present, we may be probing sets of species-like Integrative Biology Research program). The units rather than the individual species-like units routing of carbon via sugar has an themselves, but we can at least begin to observe interesting effect of the 13C signatures of organ- the kinds of adaptations (e.g., to temperature, isms in the mat, since sugar biosynthesis exhibits a light, pH and salinity) that help us understand the lower selection of 12C over 13C than does lipid diversity we see. Given the enormous diversity biosynthesis. This heavier 13C signature may be that has already been revealed by studying slowly transferred from oxygenic photoautotrophs to evolving molecular markers (e.g., 16S rRNA), I photoheterotrophically metabolizing filamentous marvel at the multitude of adaptations that might anoxygenic phototrophs that assimilate isotopi- have to be invoked to explain the full ecological cally heavy fermentation products. Since GNSB diversity of microorganisms in . I see as one cell components appear to be more persistent of the big challenges for the future unraveling the than cyanobacterial cell components (van der features of the abiotic and biotic environment Meer et al. 2000), this signal may have a potential that have influenced the evolutionary trajectories to survive and become a part of the organic of species-like populations. This will not be a geochemical record. Roseiflexus and Chloroflexus simple problem to resolve. We must keep in mind are best known for their photoheterotrophic Hutchinson’s concept of niche as n-dimensional metabolism and, in fact, only Chloroflexus has hypervolume (Hutchinson 1957). Possible first been grown photoautotrophically (Holo and examples of the complexity of niche defnition Sireva˚g 1986; van der Meer et al. 2001; Hanada appear to be emerging from comparative genomic et al. 2002; van der Meer et al., in prep.). Nev- analysis of closely related cyanobacterial species- ertheless, it appears that photoautotrophic like populations. For instance, marine Prochlo- metabolism is possible in morning (and perhaps rococcus populations that exhibit distinct evening) hours, when anaerobic processes lead to adaptations to light and hot spring mat Synecho- the accumulation of possible electron donors in coccus populations that exhibit distinct tempera- the dimly lit, anoxic photic zone. Clearly, we have ture adaptations, have also been found to exhibit much to learn about the physiology of these ma- differences in ability to acquire and store inor- jor mat phototrophs (e.g., can Roseiflexus conduct ganic nutrients, consistent with possible spatial photoautotrophic metabolism?), but genomics is differences in nutrient availability in marine and beginning to provide a more global appreciation geothermal (Rocap et al. 2003; unpub- for possible metabolisms. A striking example is lished research in our NSF Frontiers in Integra- the discovery that Synechococcus A and B¢ iso- tive Biology Research program). lates possess and express (Steunou et al. 2006) nif The main insight gained to date from our genes in situ in concert with the occurrence of studies of phototroph function in the mats may be nitrogen fixation. This would not be such a sur- to ‘‘expect the unexpected’’. While we had prise were it not for the fact that previous workers thought of native Synechococcus populations as failed to detect nitrogen fixation in these mats (at actively growing and dividing, it appears that they these temperatures; Stewart, 1970; Wickstrom are instead in an active, but slowly growing (or 1980), perhaps because their incubations did not

123 Antonie van Leeuwenhoek take into consideration that the mat environment gene expression studies in our mat system and is anaerobic at night (Revsbech and Ward 1984). we are aiming at ecotype-specific gene expres- Our research team is conducting targeted gene sion studies in future work. This would put us in expression studies of specific Synechococcus and a position to evaluate how distinct ecotype Roseiflexus metabolisms and is developing populations express genes spatiotemporally in microarrays with the aim to determine in situ the community. spatiotemporal patterns of expression of all genes It should be clear that Dutch scientists, many in these organisms. associated with the Delft School of Microbiology, By combining a population-based analysis of have had a significant impact on research in my molecular microbial diversity with functional laboratory, and, for this I am very grateful. I look analysis, we are beginning to understand the forward to continued collaborations. I have often value of diverse ecological populations to micro- wondered how , the first head bial communities. In the mat systems we study, of the Delft School of Microbiology, would have Michael Ku¨ hl (Marine Biological Laboratory, reacted to the kind of research we have done University of Copenhagen) has observed together. Gijs Kuenen, the retiring head of the evidence that multiple, co-existing cyanobacterial Delft School of Microbiology, thought he would populations, differently adapted to temperature, have enjoyed it, as evidenced by his comments in might increase the resilience of oxygenic photo- a copy of Verzamelde Geschriften van M.W. synthesis to variations in temperature (see Fig. 6 Beijerinck (van Iterson et al. 1940) he presented in Ward et al. 1998). This suggests that intra-guild to my lab after his visit in 1991. diversity may stabilize guild activities and pro- When Beijerinck retired, he said, ‘‘Gelukkig zij vides a means of organizing our thinking on how die nu beginnen’’: ‘‘Happy those who are begin- community structure is linked to community ning now.’’ Clearly, at that time (1921) Beijerinck function. could see the tremendous new possibilities for new The very sophisticated tools of organic research which had come up and was envying the geochemistry are beginning to help us see that youngsters, who were just starting off, when he was the complex realities of microbial community forced to retire. No doubt, today he would have composition, structure and function do matter to said and felt the same, and I am sure that Beijer- the resulting geochemical traces that might pro- inck would have liked to do the kind of research vide relicts that could be used to unravel the you are doing here in Bozeman on DNA and chemical record of microorganisms in the RNA probing of creatures in Nature. Precambrian Era. However, even these tech- For me the benchwork in Bozeman has been niques only allow us to evaluate guild processes, both a happy beginning and returning to DNA and rather than the activities of the individual spe- RNA work that I started when I majored in cies-like populations whose ensemble activities biochemistry 25 years ago. It also has opened new comprise guild activities. Our ultimate goal is to possibilities for future research in Delft. understand the unique functional contributions Now, on the occasion of Gijs’ retirement, this of each species-like population in a microbial comment has relevance for Gijs himself. He must community. Here, evolutionary ecology may have considered himself a youngster in 1991 and suggest a new way of proceeding. As mentioned indeed carried molecular methods into the above (Fig. 8B) when distinct ecological popu- department in Delft. Though retiring, I’m certain lations diverge, all genes should be subject to that he will continue to be young at heart, sharing neutral mutational (including recombinational) his insight and wisdom long into the future. I’m changes that lead to distinctive allelic variants in looking forward to how his ideas influence my each ecological population. The distinction of future science. I’m also looking forward to his allelic variants in different ecological populations continuing friendship, as he has led the Delft may provide a means of analyzing ecotype-spe- School of Microbiology by showing us how the cific gene expression. Emerging results (Steunou combination of good science and good nature can et al. 2006) demonstrate the feasibility of in situ be both beneficial and rewarding.

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Acknowledgements I am very grateful to Jerry Ensign Ferris MJ, Ward DM (1997) Seasonal distributions of (University of Wisconsin-Madison) for teaching me dominant 16S rRNA-defined populations in a hot microbiology in the tradition of the Delft School of spring examined by denaturing gradient Microbiology in his graduate class on Microbial Diversity. gel electrophoresis. Appl Environ Microbiol I thank Tony Scotti for providing Fig. 4B, Marcel van der 63:1375–1381 Meer for providing Figs. 6B and C and Fred Cohan for Ferris MJ, Ku¨ hl M, Wieland A, Ward DM (2003) Differ- providing the concept for Fig. 8. I appreciate the long term ent light-adapted ecotypes in a 68C Synechococcus support of the U.S. National Science Foundation (Ecology mat community revealed by analysis of 16S-23S and Frontiers in Integrative Biology Research programs), intervening transcribed spacer variation. Appl the U.S. National Aeronautics and Administration Environ Microbiol 69:2893–2898 (Exobiology and Astrobiology programs), the Montana Gevers D, Cohan FM, Lawrence JG, Spratt BG, Coenye State University Thermal Biology Institute and the Rotary T, Feil EJ, Stackebrandt E., van de Peer Y, Vand- Foundation of Rotary International, who supported Sjila amme P, Thompson FL, Swings J (2005) Reevaluating Santegoeds’ visit to my lab. I also appreciate the support of prokaryotic species. Nature Rev Microbiol 3:1–7 the U.S. National Park Service for permission to collect in Goodfellow M, Manfio GP, Chun J (1997) Towards a Yellowstone National Park and for invaluable assistance practical species concept for cultivable bacteria. In: over the years. Claridge MF, Dawah HA, Wison MR (eds) Species: the units of . 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Ward DM, Cohan FM (2005) Microbial diversity in hot Ward DM, Santegoeds CM, Nold SC, Ramsing NB, Ferris spring cyanobacterial mats: pattern and prediction.In: MJ, Bateson MM (1997) Biodiversity within hot Inskeep WP, McDermott TR (eds) Geothermal biol- spring microbial mat communities: molecular moni- ogy and geochemistry in Yellowstone National Park. toring of enrichment cultures. Antonie van Leeu- Montana State University Thermal Biology Inst., wenhoek 71:143–150 Bozeman, MT, pp 185–202 Ward DM, Weller R, Bateson MM (1990) 16S rRNA se- Ward DM, Papke RT, Nu¨ bel U, McKitrick MC (2002) quences reveal numerous uncultured microorganisms Natural history of microorganisms inhabiting hot in a natural community. Nature 344:63–65 spring microbial mat communities: clues to the ori- Wickstrom CE (1980) Distribution and physiological gin of microbial diversity and implications for mi- determinants of blue-breen algal nitrogen fixation cro- and macro-biology. In: Staley JT, Reysenbach along a thermogradient. J Phycol 16:436–443 A-L (eds) Biodiversity of microbial life: foundation of earth’s . John Wiley and Sons, NY, pp 25–48

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