Impact of Sideways and Bottom-Up Control Factors on Bacterial Community Succession Over a Tidal Cycle

Impact of sideways and bottom-up control factors on bacterial community succession over a tidal cycle

Ashvini Chauhan1, Jennifer Cherrier, and Henry N. Williams

Environmental Sciences Institute, Florida A&M University, Frederick S. Humphries Science Research Building, Suite 305-D, Tallahassee, FL 32307

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved January 22, 2009 (received for review October 21, 2008) In aquatic systems, bacterial community succession is a function of tandem, top-down factors shape bacterial community structure top-down and bottom-up factors, but little information exists on through a variety of processes, including protistan grazing (10) and ‘‘sideways’’ controls, such as bacterial predation by Bdellovibrio- viral lysis (11). Therefore, in all likelihood at any given time, like organisms (BLOs), which likely impacts nutrient cycling within combinations of these factors drive bacterial community succession the microbial loop and eventual export to higher trophic groups. in aquatic ecosystems (12–14). Here we report transient response of estuarine microbiota and BLO Recently, Mou et al. (2) proposed that in marine systems, spp. to tidal-associated dissolved organic matter supply in a river- transient changes in the dissolved organic carbon (DOC) pool are dominated estuary, Apalachicola Bay, Florida. Both dissolved or- less critical in structuring bacterial communities than those that ganic carbon and dissolved organic nitrogen concentrations oscil- result from viral lysis, protistan grazing, or even physicochemical lated over the course of the tidal cycle with relatively higher conditions. Both grazing and viral lyses are selective, such that concentrations observed at low tide. Concurrent with the shift in factors including nonsusceptibility, morphology, size, and motility dissolved organic matter (DOM) supply at low tide, a synchronous offer protection to certain bacterial groups (12). However, other increase in numbers of bacteria and predatorial BLOs were ob- ‘‘sideways’’ factors, which are only beginning to be understood, also likely contribute to shifts in the bacterial composition through served. PCR-restriction fragment length polymorphism of small processes that exert both positive (syntrophy) and/or negative subunit rDNA, cloning, and sequence analyses revealed distinct (allelopathy) effects (15). In this context, one of the largely ignored shifts such that, at low tide, significantly higher phylotype abun- trophic links within the microbial loop processes is obligate and ␥ ␦ ECOLOGY dances were observed from -Proteobacteria, -Proteobacteria, relatively nonspecific predation by Bdellovibrio-like organisms ؉ Bacteroidetes, and high G C Gram-positive bacteria. Conversely, (BLOs), resulting in potential structural and functional successions diversity of ␣-Proteobacteria, ␤-Proteobacteria, and Chlamydiales- of susceptible prey microbiota. Verrucomicrobia group increased at high tides. To identify meta- BLOs can lyse a variety of Gram-negative bacteria (16, 17) and bolically active BLO guilds, tidal microcosms were spiked with six are characterized by a motile free-living attack form and an 13C-labeled bacteria as potential prey and studied using an adap- intraperiplasmic growth phase. Our recent study indicated that tation of stable isotope probing. At low tide, representative of BLOs are more diverse than previously thought (18); most marine higher DOM and increased prey but lower salinity, BLO community bacteria are susceptible to lysis by these predators (16, 18) and also shifted such that mesohaline clusters I and VI were more hence their sideways trophic interactions would likely result in active; with an increased salinity at high tide, halotolerant clusters successions within the microbial food web processes. III, V, and X were predominant. Eventually, 13C label was identified This study was conducted in Apalachicola Bay, a river-dominated from higher micropredators, indicating that trophic interactions subtropical estuary located in the Florida Panhandle (Fig. S1A). A within the estuarine microbial food web are potentially far more combination of riverine discharge, gulf tides, and winds keep this complex than previously thought. system well mixed, likely resulting in dynamic cycling of DOM and inorganic nutrients from both allochthanous (river, wetlands) and Bdellovibrio-like organisms (BLOs) ͉ dissolved organic matter ͉ autochthanous (in situ production) sources. The overall goal of the predator-prey interactions ͉ stable isotope probing ͉ tidal microbiota work presented here was to evaluate how transient changes in both bottom-up factors (supply of bulk DOM, i.e., both DOC and dissolved organic nitrogen [DON]) and sideways factors transiently arine dissolved organic matter (DOM) is one of the largest influence bacterial community composition and associated func- Mactive reservoirs of reduced carbon at the earth’s surface and, tional changes within the predacious BLO guilds. An improved to a large extent, as the primary consumers of this DOM, bacteria understanding of these tightly coupled predator-prey interactions control its fate via assimilation and/or remineralization processes (1, and the effects of DOM supply will lead to a better understanding 2). The fate of DOM is a also a function of physiologic status and of the trophic links within the microbial loop and recycling of taxonomic composition of the autochthanous microbiota as well as nutrients in coastal systems. the relative DOM lability supplied to the system, all of which vary both spatially and temporally in response to physiochemical con- Results ditions (1, 3, 4). DOM that is assimilated into bacterial biomass is Environmental Parameters and Nutrients. Salinity at our study site potentially available for trophic transfer via the microbial loop (5) indicated vertical stratification of water at both high tides but was and as such must be accounted for in estimates of marine carbon flux. Bacterial groups that mineralize DOM are taxonomically diverse Author contributions: A.C., J.C., and H.N.W. designed research; A.C. and J.C. performed research; A.C. contributed new reagents/analytic tools; A.C. and J.C. analyzed data; and (2, 3, 6), which is often a function of niche variability (1–3). A.C., J.C., and H.N.W. wrote the paper. Specifically, estuarine systems exhibit high spatiotemporal and The authors declare no conflict of interest. physiochemical variability, often resulting in short-lived blooms of some bacterial spp. (7). Among other factors, salinity has been This article is a PNAS Direct Submission. found to typically drive bacterial succession in estuarine systems (3) Data deposition: The 16S rRNA gene sequences reported in this paper have been deposited ␣ in GenBank under accession numbers FJ160298–FJ160358 (bacteria) and FJ160359– such that in Chesapeake Bay, -Proteobacteria were predominant in FJ160412 (BLOs). 13C prey bacteria are included under FJ160294–FJ160297, M59161, and the saltwater regions and ␤-Proteobacteria in the freshwater regions DQ912807. (8). Bacterial succession is also a function of bottom-up substrate 1To whom correspondence should be addressed. E-mail: [email protected]. supply (i.e., dissolved and particulate organic and inorganic nutri- This article contains supporting information online at www.pnas.org/cgi/content/full/ ents) from autochthanous and allochthanous sources (4, 9). In 0809671106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809671106 PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4301–4306 Downloaded by guest on September 27, 2021 193.00 12.00 Table 1. Relative bacterial and BLO phylotype abundances from

191.00 samples collected over the tidal cycle at Dry Bar in Apalachicola

10.00 Bay, Fl, in December 2006 189.00

M) Closest phylogenetic HT-I OT LT IT HT-II

187.00 μ M) ( μ n

8.00 io taxa/specie from NCBI (%) (%) (%) (%) (%) at r

185.00 t n Bacterial (prey) Communitya: 183.00 6.00 ␣-Proteobacteria

181.00 DON Conce DOC Concentration ( Uncultured Alpha-proteobacteria 12 3 6 0 0 179.00 4.00 Pelagibacter sp. 45 35 29 42 54 SAR116- like sp. 8 18 0 0 2 177.00 Uncultured Rhodobacteraceae sp. 6 0 4 17 0 175.00 2.00 Uncultured Roseobacter sp. 8 5 11 0 15 23:20 2:15 3:50 5:20 8:18 10:50 12:20 14:20 Rhizobium sp. 0 0 0 6 0 Fig. 1. Changes in DOC (diamonds) and DON (triangles) concentrations vs. ␤-Proteobacteria time over the course of the 12-h tidal cycle at Dry Bar in Apalachicola Bay, FL. Uncultured Comamonadaceae 8 2 0 0 0 Time points taken at 2320, 0350, 0818, 1220, and 1420 represent HT-I, OT, LT, Burkholderia sp. 0 0 0 0 2 IT, and HT-II, respectively. Error bars represent Ϯ1 SD of duplicate samples. Limnobacter sp.00060 Ralstonia sp. 3 0 0 0 8 ␥-Proteobacteria uniformly mixed due to strong wind mixing at incoming tide (IT) Uncultured Gamma-proteobacteria 3 18 22 21 4 and low tide (LT), respectively (Fig. S1 B and C). Small changes in Oleiphilus sp. 1 0 3 0 0 salinity (19.5–21.8 ppt) were observed at 0.5 m over the course of ␦-Proteobacteria the tidal cycle (Fig. S1C) and, for the most part, closely followed the Uncultured Desulfobulbus sp. 00400 changes in tide with the sharpest increase observed between LT and Bacteroidetes HT-II, when the winds subsided and the water became stratified Uncultured Flavobacterium sp. 4 12 7 4 0 again. No significant changes in temperature (15.4–16.5 °C) or Uncultured Bacteroides sp. 0 5 12 4 11 dissolved oxygen (8.7–9.4 mg/L) were observed. Chlamydiales-Verrucomicrobia group DOC and DON concentrations oscillated over the course of the Uncultured Verrucomicrobia sp. 2 2 0 0 4 tidal cycle with relatively higher concentrations at LT (187 Ϯ 0.2 ϩ ␮ Ϯ ␮ High- G C Gram-positive bacteria: M C and 10 0.4 M N) than at outgoing tide (OT) or IT (Fig. Uncultured Actinobacterium sp. 0 0 2 0 0 1). NO3 was between 2 and 5 ␮M with lowest at low tide. NH4 concentrations remained below detection limit (data not shown). Predator (BLO) Communityb: The DOC:TDN remained fairly constant over the 12-h sampling ␦-Proteobacteria period between 15 and 17. The DOC:DON [or C:Norg], however, Bacteriovorax Cluster III 28 0 0 0 0 showed more variability, being appreciably lower at LT [19] than Bacteriovorax Cluster V 0 0 0 0 76 that observed at high tide [average of first and second high tides Bdellovibrio Cluster VI 0 0 100 36 24 [HT-I and HT-II) ϭ 24], OT [26], and IT [23]. Bacteriovorax Cluster X 72 100 0 16 0 Bacteriovorax Cluster XII 0 0 0 48 0

Most Probable Number Estimates. Estimation of total bacteria and aForty-eight bacterial clones with positive inserts were compared from each BLOs indicated that bacteria in LT were Ϸ20-fold greater than library. those at other time points (Fig. S1C). BLO community showed the bTwenty-ﬁve BLO clones with positive inserts were compared from each same trend, such that higher numbers were observed at low tide. library. All libraries were subjected to Rarefaction analyses to establish that sufﬁcient Phylogenetic Analyses of Microbiota over the Tidal Cycle. Microbial numbers of clones were sequenced to represent microbial diversity over the community structure over the course of the tidal cycle was assessed tidal cycle. SSU rDNA from at least two representatives of each phylotype were by polymerase chain reaction-restriction fragment length polymor- sequenced and compared to the closest cultivable phylogenetic relative from NCBI database. phism (PCR-RFLP) of the 16S rDNA. RFLP phylotypes were grouped into operational taxonomic units (OTUs) based on restriction patterns; 12 OTUs were identified at HT-I, 14 at OT, 15 crease, albeit at low levels, was also shown by ␤-Proteobacteria at LT, and 10 each at IT and HT-II, respectively. BLO clone and Chlamydiales-Verrucomicrobia groups at HT. At LT, an libraries consisted of 2 OTUs in HT-1, 1 each in OT and LT, 3 in increased representation of ␥-Proteobacteria (25%) and Bacte- IT, and 2 in HT-II. Rarefaction curves indicated that sufficient roidetes (19%) were observed with decline in species belonging numbers of clones were sequenced to represent bacterial diversity to ␣-Proteobacteria (50%); ␤-Proteobacteria remained undetect- (data not shown). Relative distribution of sequences within indi- able at this time. Unique groups identified only at LT clustered vidual tidal clone libraries is presented in Table 1. with ␦-Proteobacteria (uncultured Desulfobulbus spp., 4%), and At least 2 clones from each OTU were sequenced and taxonom- ϩ ically characterized by Basic Local Alignment Search Tool high G C Gram-positive bacteria (uncultured Actinobacterium (BLAST). Dynamic bacterial community shifts were observed over spp., 2%); Chlamydiales-Verrucomicrobia group was absent. the course of the tidal cycle mainly within ␣-, ␤-, ␥-, and ␦- Community shifts were also evident within the predacious BLOs Proteobacteria, Bacteroidetes, Chlamydiales-Verrucomicrobia group over the tidal cycle (Table 1), such that clusters III (28%) and X and Gram-positive bacteria, as shown in Fig. 2. Phylogenetic tree of (72%) were identified in HT-I, cluster X (100%) in OT, and cluster microbiota identified over the tidal cycle is shown in Fig. S2. VI (100%) at LT. At incoming tide, BLOs were more variable, with Among Proteobacteria, the most dominant group was ␣- representations from clusters VI (36%), X (16%), and XII (48%). Proteobacteria that contributed from 50% to 79% of the total HT-II consisted of clusters V (76%) and VI (24%), respectively. bacterial compositional makeup (Fig. 2 and Table 1). ␣- Proteobacteria significantly increased at both high tides; species Identification of Functionally Active BLO Guilds. Simultaneous to identified clustered mostly with Pelagibacter/uncultured SAR116 assessing the bacterial diversity, tidal microcosms were set up from clade, Roseobacter spp., and Rhodobacteraceae family. An in- HT-I, OT, LT, IT, and HT-II and spiked with six 13C-labeled

4302 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809671106 Chauhan et al. Downloaded by guest on September 27, 2021 Alpha-proteobacteria Beta-proteobacteria 60 Bacteriovorax sp. MNZ4 100 Bdellovibrio sp. SJ Gamma-proteobacteria Delta-proteobacteria HT-I-8 FJ160360 HT-II-Labeled1 FJ160361 Bacteroidetes Chlamydiales-Verrucomicrobia Group HT-II-Labeled2 FJ160362 Cluster III High G+C Gram-positive Bacteria HT-I-19 FJ160367 70 IT-8 FJ160396 IT-16 FJ160399 40 14 65 HT1-100 FJ160392 HT-II-Labeled17 FJ160400 HT-II-101 FJ160359 35 12 HT-II-Labeled4 FJ160365 HT-II-Labeled5 FJ160366

ribution HT-II-118 FJ160364

st 30 i 10 100 HT-II-115 FJ160363 D HT-II-6 FJ160405 Cluster V HT-II-Labeled19 25 FJ160409 HT-II-15 FJ160406 8 HT-II-Labeled3 other Bacterial Groups other Bacterial 100 FJ160407 Phylotype 20 LT-Labeled7 FJ160408 al Bacteriovorax sp. PS23S 6 of ion Bdellovibrio sp. JS10 15 OT-1 FJ160368 obacteri HT-1-95 FJ160370

ote 4 r 75 OT-14 FJ160371

P 10

pe Distribut pe OT-17 FJ160373 y

pha- OT-3 FJ160369 Cluster X l 2 lot y A 5 HT-1-90 FJ160372 h HT-II-Labeled7 P BacteriovoraxFJ160412 sp. GSL41 100 IT-14 FJ160398 0 0 100 HT-II-Labeled100 FJ160393 23:30 (HT-I) 4:00 (OT) 8:30 (LT) 11:30 (IT) 14:30 (HT-II) IT-3 FJ160394 100 Bdellovibrio sp. clone YE-3E 95 Bacteriovorax sp. OC91 Bdellovibrio sp. clone CA-1F Cluster IV Fig. 2. Bacterial community shifts representing major phyla/taxa identiﬁed LT-Labeled5 FJ160383 LT-8 FJ160382 LTLabeled6 FJ160384 over the course of the 12-h tidal cycle at Dry Bar in Apalachicola Bay, FL, LT-1 FJ160374 LT-10 FJ160387 analyzed by PCR-RFLP of small subunit rDNA followed by sequence analyses. 80 LT-Labeled17 FJ160389 IT-6 FJ160395 Time points at 2320, 0350, 0818, 1220, and 1420 represent HT-I, OT, LT, IT, and IT-12 FJ160397 90 LT-2 FJ160375 HT-II, respectively. LT-Labeled18 FJ160390 100 LT-4 FJ160378 HT-II-1 FJ160402 Cluster VI HT-II-2 FJ160403 100 HT-II-Labeled6 FJ160404 LT-Labeled8 FJ160377 ␥ LT-3 FJ160376 -Proteobacterial species as potential prey to identify metabolically LT-Labeled9 FJ160385 LT-9 FJ160386 active predatory BLOs over the tidal cycle. Prior to establishment LT-14 FJ160388 Bacteriovorax sp. EEC 100 Bacteriovorax sp. ETC of 13C-microcosms, labeled and unlabeled prey bacteria were 100 Bacteriovorax sp. F2 LT-Labeled3 FJ160410 Cluster II separately diluted to extinction and DNA analyzed by ultracen- LT-Labeled4 FJ160411 LT-Labeled2 100 FJ160380 Cluster I Bdellovibrio sp. Gunpowder trifugation to confirm sufficient labeling of cells had occurred such 100 LT-Labeled1 FJ160379 IT-19 FJ160401 that DNA from terminally diluted series of each labeled prey was 100 IT-1 FJ160391 Cluster XII ECOLOGY Bacteriovorax sp. 16SPR3 identified in the heavier bands but not in the lighter bands (data not Geobacter metallireducens shown). Species from ␥-Proteobacteria were chosen as prey for 10 changes stable isotope probing (SIP) studies due in part to being preferred ␥ Fig. 3. Phylogenetic tree of partial 16S rRNA gene sequences of predatory by Bacteriovorax (Bx) spp. (16, 18, 19). Moreover, -Proteobacterial Bdellovibrio-like organisms (BLOs)/Bx spp., over the course of the 12-h tidal numbers were also found to be significantly higher at LT (Fig. 2 and cycle at Dry Bar in Apalachicola Bay, FL. The phylotree was constructed with Table 1). Other prey tested included Vibrio, Pseudoalteromonas, PAUP v. 4.0b8 using maximum parsimony algorithm. Clones marked in bold- and Marinomonas, identified as potential prey from our previous face represent species that were identiﬁed from the ‘‘heavier’’ DNA from the studies in Apalachicola Bay (19), and 3 laboratory strains— 13C prey studies. Numbers at nodes represent bootstrap values (100ϫ resam- Pseudomonas sp., E. coli sp., and Vibrio parahaemolyticus P5. pling analysis); only values Ͼ50 are presented. Geobacter metallireducens Initial numbers and viability of the combined 13C-labeled prey in was used as outgroup. In this tree, BLO clusters I and VI represent the each microcosm was Ϸ1.5 ϫ 104/mL, which was within the range freshwater/terrestrial BLO species, and clusters III, IV, V, X, and XII represent measured in the tidal samples (Fig. S1C). Most probable numbers marine/estuarine relatives. Recently, clusters I and VII have been proposed to belong to Peredibacteraceae family. (MPNs) measured every 24 h from the 13C-microcosms indicated rapid predation such that numbers of prey steadily declined with concomitant increase in BLO numbers (data not shown). Samples 30.29% of the variability, with a cumulative percentage of 76.23%, collected from 24 to 144 h were studied by SIP. Concomitant with indicating a statistical distinction of microbiota as a function of tidal 13 the depletion of RFLPs representing each of the C-labeled prey cycle. Further, significant differences (P Ͻ 0.001) of clone library spp., ‘‘new’’ OTUs were observed in the ‘‘heavier’’ clone libraries, sequences for both bacteria and BLOs indicated that predator-prey with significant differences shown by LT and HT-II microcosms dynamics were significantly different at LT. (Fig. S3 A and B). Sequencing of the new OTUs led to identification of BLOs that had predated and assimilated DNA from the 13C- Discussion labeled prey, providing clues on both BLO structure and function In aquatic systems, both top-down (protistan grazing, viral lyses) along the tidal cycle. Specifically, in LT samples, BLO clusters I, II, and bottom-up (nutrient supply) factors significantly influence IV, V, and VI were found in the labeled fraction; HT-II samples bacterial succession. Little information exists, however, on trophic contained clusters III, V, VI, and X (Fig. 3). Further, cluster VI in interactions of predacious Bdellovibrio-like organisms, resulting in LT and cluster V in HT-II were the most metabolically actively BLOs based on the dominance of their specific RFLPs (Fig. S3 A a potential sideways control on the structure and functions of the and B). Moreover, this adaptation of SIP confirmed that BLOs predated microbiota. In all likelihood, biotic factors work in tandem likely have feeding preferences for certain bacteria, as discussed with abiotic factors such as salinity, temperature, water resident hereafter. time, and hydrology, resulting in bacterial successions in aquatic systems (4, 8, 11–15). However, thus far, studies have not accounted Statistical Comparisons of the Tidal Microbiota. Principal coordinates for the cumulative influences of transient physicochemical changes analyses (PCA) was performed on bacterial and BLO species along with bottom-up and sideways controls on short-term bacterial identified from HT, OT, LT, IT, and HT-II to determine statistical community succession. To this end, we conducted a 12-h tidal study differences. Bacteria identified from HT-I, OT, and IT clustered on in Apalachicola Bay, FL, a river-dominated estuary. the same axis, whereas LT and HT-II microbiota separated out on Estuarine systems are unique such that coexistence between different axes (Fig. S4A). Similarly, BLOs from HT-I, IT, and LT freshwater and marine bacterial ecotypes has been reported (3, 8), clustered on different axes, but OT and HT-II appeared on the with dramatic shifts within these assemblages (8, 20). Bacterial same axis (Fig. S4B). For bacteria, PCA axis 1 explained 46.93%, groups identified at our study site during the 12-h period clustered and axis 2, 24.33% of variability, with a cumulative percentage of with Proteobacteria (␣-, ␤-, ␥-, and ␦-Proteobacteria), Bacteroidetes, 71.26%. For BLOs, PCA axis 1 explained 45.94%, and axis 2, Verrucomicrobia, and high GϩC Gram-positive bacteria (Fig. 2 and

Chauhan et al. PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4303 Downloaded by guest on September 27, 2021 Table 1). For the most part, various groups did coexist throughout low-tide allochthanous). This change in DOM substrate supply is the course of the tidal cycle, but clear shifts in relative contribution also likely to influence the bacterial community composition shifts of each group(s) to the compositional makeup of the community observed at LT. Because DOM produced autochthanously via in were observed concurrent with oscillations in DOM supply, and situ plankton production is generally thought to be more bioavail- salinity, to a lesser extent. We did not directly measure protistan able than that originating from terrestrial sources, due in part to its grazing and viral lyses but, obviously, both factors also likely higher nitrogen content as well as its lower degree of complexity contributed to some of the bacterial shifts observed over the tidal (28), changes in the carbon-to-nitrogen ratio of ambient DOM, cycle. specifically the C:Norg, could potentially be indicative of shifts in the This study has potential implications on short-term processing of relative contribution of the autochthanous and allochthanous carbon, through the microbial food web in estuarine systems. Data DOM source terms over the course of a tidal cycle. collected from other stations and seasons in Apalachicola Bay In addition to evaluating the C:Norg of the ambient DOM over (Carrabelle River, St. Vincent Sound, and Platform Bar) supported the course of the tidal cycle, we also evaluated the C:Norg of DOM the conclusions drawn from this study such that regardless of for the marine and freshwater DOM source terms to Apalachicola spatiotemporal effects, lower tides consisted of substantially higher Bay. For the marine source terms, samples were collected from bacterial numbers and diversity, indicated by RFLP analyses (data West Pass (Fig. S1A), one of the primary conduits of exchange not shown). Of major interest to our findings were the distinct between Gulf of Mexico and Apalachicola Bay waters, both at differences of bacterial communities observed at LT (Table 1, Fig. incoming tide (i.e., marine end-member, C:Norg ϭ 18) and outgoing 2, and Fig. S4A). At low tide, an increased representation of species tide (i.e., Apalachicola Bay signature, C:Norg ϭ 25). For the belonging to ␥-Proteobacteria (25%) and Bacteroidetes (19%) were freshwater source term, a water sample was collected upriver and observed with significant decline of species representing ␣-Pro- adjacent to the Apalachicola marsh system (Fig. S1A), and this teobacteria (50%). Surprisingly, at LT, ␤-Proteobacteria, known to DOM was found to be essentially deplete in nitrogen (C:Norg ϭ dominate in freshwater end-members of estuarine systems (8, 9), 338). The average C:Norg of DOM at HT was 24, which was were absent. The wind mixing and subsequent loss of vertical approximately equal to that observed at West Pass during outgoing stratification at LT (Fig. S1B) may have diminished the ␤-Pro- tide ϭ 25, and, as would be expected in an estuarine system, is teobacterial species; we would have otherwise observed had the indicative of some degree of mixing between autochthanous water column not been well mixed. Conversely, at both high tidal (C:Norg ϭ 18) and allochthanous (C:Norg ϭ 338) DOM source points, ␣-Proteobacteria was the dominant group, comprising terms. However, if the DOM shaping bacterial community struc- greater than 70% of the community concomitant with increased ture at low tide was coming primarily from a freshwater source (i.e., salinity levels (Fig. 2 and Fig. S1B). Our data are in good agreement river and marsh outwelling), then we would expect the LT C:Norg with previous findings that ␣-Proteobacteria have a propensity to of the DOM to be at least higher than the average HT C:Norg of 24 thrive in lower-nutrient conditions, as indicative in the higher-tide rather than lower as was observed (LTC:Norg ϭ 19). Furthermore, events. Conversely, ␥-Proteobacteria species, being opportunistic, the taxonomic composition of the bacterial community during LT, can rapidly use pulses of nutrients such as those after bloom events though distinctly different from other tidal points, was not neces- (21, 22) and the low tidal event in this study (Figs. 1 and 2). sarily exclusively indicative of a freshwater source, which would be Additionally, at LT, Bacteroidetes and high GϩC Gram-positive expected to be dominated by ␤-Proteobacteria (1). bacteria also increased (Table 1 and Fig. 2); Bacteroidetes are also In contrast, C:Norg of the sediment porewater (0–10 cm from known to respond rapidly and remineralize complex and labile data collected from a previous study) from the same study site was DOM (1, 6, 23). Low phylotype abundances of Desulfobulbus sp., found to be 15. The observed C:Norg decrease of DOM from 26 at belonging to ␦-Proteobacteria were also observed at LT (Fig. 2 and IT to 19 at LT and the increased representation of the ␥-Proteobac- Table 1). Wind-mixing likely resulted in vertical immigration of teria, ␦-Proteobacteria, Bacteroidetes, and high GϩC Gram-positive Desulfobulbus sp. from the sediments rather than allochthanously bacteria identified at this time (Fig. 2 and Table 1) might therefore from the river plume because sediments are well-known reservoirs be due to, among other factors, the combined effects of winds and of such anaerobes (24). Bacterial species identified from HT tidal pumping and mixing of N-rich sediment DOM into the samples clustered mainly with Pelagibacter sp./SAR11 clade, which overlying water column. Therefore, bottom-up processes are likely are one of the most abundant bacteria in marine systems (25). fueled by cumulative impacts at low tide in shallow estuarine Interestingly, Roseobacter spp., because of their propensity to systems, which appear to be significant drivers for surface-water respond rapidly to patchiness of nutrient pulses in coastal environ- bacterial productivity and community composition. The same ments (25, 26), showed very tight coupling to the concentrations of might not hold true, however, for a stratified water column where oscillating DOC at high tides (Fig. 1 and Table 1). freshwater allochthanous DOM sources may be more important. Further, concurrent with the distinct bacterial community shifts, In light of the recent evidence from marine systems that top- we also measured a 7- to 9-␮M C net increase of N-rich DOM over down factors (predation/grazing) are significant drivers of bacterial OT and IT, which likely led to a 20-fold increase in bacterial succession, as opposed to the bottom-up factors (transient changes numbers at LT (Fig. S1C). DOM quantity alone, however, is not in carbon pool) (2), we next sought to study the sideways trophic sufficient to explain the bacterial response, as LT values (187 Ϯ 0.2 interactions of predacious BLO guilds on tidal microbiota. Specif- ␮MC) were not significantly different to those observed at HT-I or ically, at low tide, an increase in the numbers and diversity of HT-II (187 Ϯ 4.4 and 184 Ϯ 0.1 ␮MC, respectively). Therefore, in potential prey bacteria resulted in a surprisingly synchronous re- all likelihood, the observed bacterial community response is also a sponse in BLO numbers (Fig. S1C). Higher bacterial and virio- function of both the quantity and quality of DOM supply to the Bay. plankton abundance in lower tides compared with high tides has This is further supported by previous studies where ␤-Proteobacteria previously been reported (29), and our study suggests that a similar and Bacteriodetes were predominantly identified from waters with relationship exists between bacterial predators and prey. An in- variable DOM concentrations and complexity (1, 27). Conversly creased predatorial response at LT more than likely is a function of ␣-Proteobacteria dominated in both less complex and lower con- increased numbers of prey species, especially because BLOs thrive centrations of DOM, such as those from algal-derived substrates (1, at higher prey densities of 105Ϫ106/mL (16). Although BLO 9). Further, in estuarine systems, several source terms, both au- numbers represent only those guilds able to predate V. parahae- tochthanous and allochthanous, can contribute to the ambient molyticus P5 used in our assay, it did indicate an active sideways DOM pool. Over the short term (i.e., on the order of hours), control mechanism over the tidal cycle, which was further studied. however, the relative contribution of each of these sources will As expected in an estuary, BLO community also varied from a change as a function of tidal stage (i.e., high-tide autochthanous, combination of saltwater/halotolerant clusters that were identified

4304 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809671106 Chauhan et al. Downloaded by guest on September 27, 2021 at other tidal points to a single freshwater cluster in low tide soils and wetland sediments (34, 35); it is possible that these groups concomitant with a salinity drop (Fig. S1B, Fig. 3, and Table 1). represent a secondary trophic tier of predatory bacteria within the However, using an adaptation of SIP, a better correlation of the microbial loop that rely on gliding mechanisms for predation. structure to the function of predacious BLO community over the Rigorous confirmation must wait before the role(s) of such sec- tidal cycle was achieved such that BLO sequences from the ondary predators can be established in marine systems. ‘‘heavier’’ clone library were significantly more diverse than those Because BLOs are host dependent and cannot replicate in identified directly from the environmental DNA (Fig. 3 and Table extracellular environments, we do not expect cross-feeding on 1). Because the numbers of native BLO populations in the envi- labeled by-products or dead labeled biomass, resulting in labeling of ronment are low (30, 31), most previous studies on BLO diversity nonactive BLOs as a limitation to this adaptation of SIP. However, using a culture based method or environmental DNA are likely a priori selection of prey is likely a limitation, and results must be flawed. This study therefore successfully demonstrated the use of an interpreted cautiously. Additionally, the latter incubations may adaptation of SIP to trace the trophic flow of carbon from prey into represent enrichments on the 13C biomass. Our attempts to avoid metabolically active bacterial predator guilds in the estuarine these limitations included spiking 13C biomass that was well within microbial food web. the range estimated over the tidal cycle and establishment of several To date, taxonomy of BLOs remains to be fully resolved. For the SIP microcosms that resulted in a time-dependent identification of most part, clusters III, IV, V, IX, X, XI, XII, and XIII have been metabolically active BLO/Bx guilds over the tidal cycle. assigned to marine/estuarine Bx species, and clusters I, II, VI, VII, Collectively, the findings presented here suggest a dynamic and VIII to the freshwater/terrestrial BLOs with 3 outlier isolates interplay between short-term changes to both bottom-up and (GSL371, NZ7, and IP1) (18, 30); recently, clusters I and VII have sideways trophic interactions within the estuarine microbial food been proposed to belong to Peredibacteraceae (32). Using SIP, we webs, complexities of which are only beginning to be understood. observed significant differences between metabolically active BLO clusters at LT and HT-II (Fig. S3 A and B), such that at lower Materials and Methods salinity, a mix of freshwater clusters I and VI were found in the Site Description and Sample Collection. The sampling site (Dry Bar, 29° 40.425Ј labeled fraction along with halotolerant clusters II, IV, and V (Fig. N, 85° 03.406Ј W), is located within Apalachicola River’s hydrologic discharge 3). Concomitant with an increase in salinity at HT-II, BLO com- channel just southwest of the river mouth (Fig. S1A). The total water-column munity was representative of saltwater/halotolerant Bx clusters III, depth at the study site was 3 m. At each tidal time point, temperature, salinity,

V, and X along with the freshwater cluster VI. Further, time- and dissolved oxygen were measured by YSI probe (YSI Inc.) at 0.25-m depth ECOLOGY dependent analyses indicated that most abundant OTU in LT intervals to evaluate the degree of stratification. Simultaneously, discrete and heavier library was the freshwater cluster VI, whereas in HT-II replicate samples were collected using a Geotech peristaltic pump with acid- saltwater Bx cluster V predominated, which has thus far been leached (10% HCl) Teflon tubing from 0.5 m depth to monitor for changes in recovered only from low-salinity regions of Chesapeake Bay and dissolved organic carbon and nitrogen, nitrate, nitrite, and ammonium. For Apalachicola Bay systems (16, 18, 19). Therefore, functional suc- microbial analyses,2Lofsample was collected over the tidal cycle, and 50-mL ␮ cessions within the predatorial guilds must be driven by specific samples were processed through 0.8- m filters for MPNs. Further analyses are described in SI Text. niches, including diversity of prey community. In all likelihood, an increase in the preferred ␥-Proteobacterial prey during lower tide Biogeochemical Methods. Dissolved organic carbon (DOC) concentrations were resulted in the synchronous increase in BLO diversity. This was measured using a Shimadzu TOC-VCPH with modifications (36), as described in SI further supported statistically; both bacteria (prey) and BLO com- Text. Total dissolved nitrogen (TDN) was measured using a Shimadzu TNM-1, as munities identified were significantly different at LT (P Ͻ 0.001; described (37). DOC and TDN results were referenced against the materials Fig. S4). obtained from the Rosenstiel School of Marine and Atmospheric Sciences (SI The degree to which BLO predation rates may have been Text). Ammonium concentrations were determined colorimetrically (38). Dis- influenced by protistan grazing or viral lysis over the tidal cycle solved organic nitrogen (DON) was determined by subtracting the sum of inor- remains tentative. Among a suite of predatory arsenals, motility ganic nitrogen constituents from TDN. speeds of up to 160 ␮m/s and small size of Ϸ0.2–0.5 wide, 0.5–2 ␮m long (16) should lead to feeding failure by protists (10, 12). Also, Most Probable Numbers (MPNs) of Total Bacteria and BLOs. Three-tube dilution unlike phage/protist predation, where prey size and physiology can MPNs assays were performed as described (SI Text). influence mortality efficiency, BLO predation appears to be rather nonspecific (16, 18, 30), with the caveat that smaller prey yield lower DNA Extraction, Purification, and PCR Amplifications. DNA was extracted using numbers of progeny cells (16, 17). Thus BLO predatory rates are UltraClean kit (Mo Bio), with final elution in sterile PCR-grade water (SI Text). Primers 27F (5Ј-AGAGTTTGATCCTGGCTCAG-3Ј)-1492R (5Ј- GGCTACCTTGTTAC- more likely to be a function of prey cell numbers, as high metabolic GACTT -3Ј) (39) were used to amplify total bacteria; Bac676F (5Ј-ATTTCGCATG- activity of free-living BLOs results in rapid starvation if preys are TAGGGGTA-3Ј)-Bac1442R (5Ј-GCCACGGCTTCAGGTAAG-3Ј) and Bd529Fd (5Ј- not encountered. GGTAAGACGAGGGATCCT-3Ј)-Bd1007R (5Ј-TCTTCCAGTACATGTCAAG-3Ј) were The SIP studies also corroborate previous culture-based stud- used for BLO diversity (30). For samples that failed after PCR or gave weak ies that BLOs have predatorial preferences for Vibrio spp. (16, amplicons, a seminested approach was followed (SI Text). 18) based on the comparatively rapid decline of Vibrio RFLPs, even more so for Vibrio sp. isolated from the bay (Fig. S3 A and Cloning of 16S rDNA and RFLP Analyses. Cloning of the 16S rDNA was performed B), indicating that autochthanous bacteria may serve as more using fresh PCR products ligated into pCRII-TOPO vector and transformed into E. lucrative prey than laboratory strains. Sequencing performed on coli TOP10FЈ (Invitrogen) (refs. 19 and 35; SI Text). RFLPs consisted of separately the RFLP phylotypes further confirmed the taxonomic affilia- digesting 10 ␮L of amplicons using HhaI, MsPI, and HaeIII as previously reported tions of depleting prey and OTUs belonging to predacious BLOs. (30) and confirmed by in silico analysis using CloneMap v2.11 software (CGC Scientific Inc.). RFLPs were run in 2.5% agarose gels. Clone libraries were analyzed It appears that cell wall surfaces of susceptible prey may contain ϳ motifs or receptor sites that are recognized by BLOs early in the by aRarefactWin 1.3 (http://www.uga.edu/ strata/software/) to confirm that sufficient clones were sequenced to identify tidal cycle diversity. predatorial response. Conversely, Marinomonas remained resis- tant to predation, likely due to mechanisms such as phenotypic Establishment of 13C Microcosms. Six potential prey included Vibrio sp., plasticity of prey (33). Pseudoalteromonas sp., Marinomonas sp., Pseudomonas putida, E. coli ML35, Notably, after a week, Myxococcales sp., Bacteroides sp., and and Vibrio parahaemolyticus P5. Prey cells were isotopically labeled by growth in 13 Stenotrophomonas sp., were also found in the C-DNA, albeit at ISOGRO-13C Powder-Growth Medium 99 atom % 13C (Isotec). For estuarine prey, lower numbers (data not shown). These genera have been identified media was formulated at a salinity of 15 ppt, which was within the range as bacterial micropredators in previous SIP studies carried out with measured in the samples. 13C-labeled prey were harvested at late log phase,

Chauhan et al. PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4305 Downloaded by guest on September 27, 2021 washed with ASW (18), diluted, and immediately spiked into microcosms at DNA Sequencing and Phylogenetic Analysis. RFLPs were tentatively assigned to Ϸ2.5 ϫ 103/mL (direct counts and viability confirmed by MPNs) containing sam- operational taxonomic units (OTUs); 2 clones from each OTU were sequenced at ples collected at HT-I, OT, IT, LT, and HT-II. All incubations were at ambient Florida State University with 27F/Bac676F/Bd529Fd primers. Chimera evaluation temperature. At 0, 24, 48, 72, 96, 120, and 144 h, MPNs were performed to confirm was performed via Bellerophon (40). Sequences were compared by BLAST (41) depletion of prey and increase of BLOs. Sample (50 mL) was also collected at every and aligned with ClustalX v. 1.8 (42). Evolutionary relationships among taxa were 24 h and studied by SIP. inferred using maximum parsimony by PAUP v. 4.0b8 (Sinauer Associates). Boot- strap resampling analysis for 100 replicates was done to estimate confidence of Separation of 13C-DNA from 12C-DNA. To negate the possibility of 12C-DNA tree topologies. carryover into the 13C-DNA following ultracentrifugation, 200 ng of unlabeled archaeal DNA isolated from M. thermophila TM-1 (ATCC 43570) was added to the Statistical Analyses. Bacterial and BLO sequences generated from HT, OT, LT, IT, environmental DNA and subjected to CsCl-ethidium bromide density gradient and HT-II were statistically analyzed using UniFrac (ref. 43 and SI Text). Compar- centrifugation in a VTI 65.2 rotor at 55,000 rpm for 18 h at 20 °C, as previously ative analyses were run to test which environments significantly differed using P described (34, 35). Bands were visualized with UV lamp (365 nm); separation was test, UniFrac metric test, and PCA with the scatter plot option. observed between lighter and heavier DNA bands. The lower bands were extracted and recentrifuged for additional purification. CsCl/EtBr were removed by ACKNOWLEDGMENTS. We thank G. Fortenberry and P. Jasrotia for technical standard methods; DNA was concentrated by Centricon (Millipore Corp.) and assistance; Apalachicola National Estuarine Research Reserve (ANERR) staff for help with sample collection; and Dr. A. Ogram (University of Florida, Gainesville) resuspended in 100 ␮L of PCR grade water. Purity of labeled DNA was checked by for M. thermophila TM-1. This study was funded by Grants HRD-0531523 (HBCU- the presence of spiked archaeal DNA, which was not detected in the heavier RISE) from the National Science Foundation and NA17AE1624 (EPP) from the 13C-DNA fractions, but detected in all of the lighter 12C-DNA fractions (data not National Oceanographic and Atmospheric Administration with partial support shown), indicating purity of labeled DNA. from the Title III Program at FAMU.

1. Cottrell MT, Kirchman DL (2003) Contribution of major bacterial groups to bacterial 23. Bauer M, et al. (2006) Whole genome analysis of the marine Bacteroidetes ’Gramella biomass production (thymidine and leucine incorporation) in the Delaware estuary. forsetii’ reveals adaptations to degradation of polymeric organic matter. Environ Limnol Oceanogr 48:168–178. Microbiol 8:2201–2213. 2. Mou X, Sun S, Edwards RA, Hodson RE, Moran MA (2008) Bacterial carbon processing 24. Purdy KJ, Embley TM, Nedwell DB (2002) The distribution and activity of sulphate by generalist species in the coastal ocean. Nature 451(7179):708–711. reducing bacteria in estuarine and coastal marine sediments. Antonie Van Leeuwen- 3. Rappe´ MS, Vergin K, Giovannoni SJ (2000) Phylogenetic comparisons of a coastal hoek 81:181–187. bacterioplankton community with its counterparts in open ocean and freshwater 25. Alonso C, Pernthaler J (2006) Roseobacter and SAR11 dominate microbial glucose systems. FEMS Microbiol Ecol 33:219–232. uptake in coastal North Sea waters. Environ Microbiol 8:2022–2030. 4. Cherrier J, Bauer JE (2004) Bacterial utilization of transient plankton-derived dissolved 26. Moran MA, et al. (2007) Ecological genomics of marine roseobacters. Appl Environ organic carbon and nitrogen inputs in surface ocean waters. Aquat Microb Ecol Microbiol 73:4559–4569. 35:229–241. 27. Eiler A, Langenheder S, Bertilsson S, Tranvik LJ (2003) Heterotrophic bacterial growth 5. Azam F (1998) Microbial control of oceanic carbon flux: The plot thickens. Science efficiency and community structure at different natural organic carbon concentra- 280:694–696. tions. Appl Environ Microbiol 69:3701–3709. 6. Cottrell MT, Kirchman DL (2000) Natural assemblages of marine proteobacteria and 28. Benner R (2003) Molecular indicators of the bioavailability of dissolved organic matter. members of the Cytophaga-Flavobacter cluster consuming low- and high-molecular- Aquatic Ecosystems: Interactivity of Dissolved Organic Matter, eds Findlay SEG, weight dissolved organic matter. Appl Environ Microbiol 66:1692–1697. Sinsabaugh RL (Academic, New York), pp 121–137. 7. Piccini C, Conde D, Alonso C, Sommaruga R, Pernthaler J (2006) Blooms of single 29. Almeida MA, Cunha MA, Alcantara F (2001) Loss of estuarine bacteria by viral infection bacterial species in a coastal lagoon of the southwestern Atlantic Ocean. Appl Environ and predation in microcosm conditions. Microb Ecol 42:562–571. Microbiol 72:6560–6568. 30. Davidov Y, Friedjung A, Jurkevitch E (2006) Structure analysis of a soil community of 8. Bouvier TC, del Giorgio PA (2002) Compositional changes in free-living bacterial predatory bacteria using culture-dependent and culture-independent methods re- communities along a salinity gradient in two temperate estuaries. Limnol Oceanogr veals a hitherto undetected diversity of Bdellovibrio-and-like organisms. Environ 47:453–470. Microbiol 8:1667–1673. 9. Kirchman DL, Dittel AI, Findlay SEG, Fisher D (2004) Changes in bacterial activity and 31. Zheng G, Wang C, Williams HN, Pineiro SA (2008) Development and evaluation of a community structure in response to dissolved organic matter in the Hudson River, New quantitative real-time PCR assay for the detection of saltwater Bacteriovorax. Environ York. Aquat Microb Ecol 35:243–257. Microbiol 10:2515–2526. 10. Ju¨rgens K, Matz C (2002) Predation as a shaping force for the phenotypic and genotypic 32. Pin˜eiro SA, Williams HN, Stine OC (2008) Phylogenetic relationships amongst the 435 composition of planktonic bacteria. Antonie Van Leeuwenhoek 81:413–434. saltwater members of the genus Bacteriovorax using rpoB sequences and reclassifica- 11. Bouvier T, del Giorgio PA (2007) Key role of selective viral-induced mortality in tion of 436 Bacteriovorax stolpii as Bacteriolyticum stolpii gen. nov., comb.nov. Int J determining marine bacterial community composition. Environ Microbiol 9:287–297. Syst Evol Microbiol 58:1203–1209. 12. Pernthaler J (2005) Predation on prokaryotes in the water column and its ecological 33. Shemesh Y, Jurkevitch E (2004) Plastic phenotypic resistance to predation by Bdellov- implications. Nat Rev Microbiol 3:537–546. ibrio and like organisms in bacterial prey. Environ Microbiol 6:12–18. 13. Zhang R, Weinbauer MG, Qian PY (2007) Viruses and flagellates sustain apparent 34. Lueders T, Kindler R, Miltner A, Friedrich MW, Kaestner M (2006) Identification of richness and reduce biomass accumulation of bacterioplankton in coastal marine bacterial micropredators distinctively active in a soil microbial food web. Appl Environ waters. Environ Microbiol 9:3008–3018. Microbiol 72:5342–5348. 14. Strom SL (2008) Microbial ecology of ocean biogeochemistry: A community perspec- 35. Chauhan A, Ogram A (2006) Phylogeny of acetate-utilizing microorganisms in soils tive. Science 320:1043–1045. along a nutrient gradient in the Florida Everglades. Appl Environ Microbiol 72:6837– 15. Fuhrman JA, Hagstrom A (2008) Bacterial and archaeal community structure and its 6840. pattern. Microbial Ecology of the Oceans, ed Kirchman DL (Wiley, New York), 2nd Ed. 36. Suzuki Y, Tanoue E, Ito H (1992) A high-temperature catalytic oxidation method for the 16. Williams HN, Pin˜eiro S (2006) Ecology of the predatory Bdellovibrio and like organisms. determination of dissolved organic carbon in seawater: Analysis and improvement. Predatory Prokaryotes, ed Jurkevitch E (Springer, New York), pp 214–244. Deep Sea Res 39:185–198. 17. Jurkevitch E (2000) The genus Bdellovibrio. The Prokaryotes: An Evolving Electronic 37. Braman RS, Hendrix SA (1989) Nanogram nitrite and nitrate determination in envi- Resource for the Microbiological Community, ed Dworkin M, et al. (Springer, New ronmental and biological materials by vanadium (iii) reduction with chemilumines- York), 3rd Ed, release 3.7, November 2, 2001. cence detection. Anal Chem 61:2715–2718. 18. Pin˜eiro SA, et al. (2007) Global survey of diversity among environmental saltwater 38. Solorzano L (1969) Determination of ammonia in natural waters by the phenolhypo- Bacteriovoracaceae. Environ Microbiol 9:2441–2450. chlorite method. Limnol Oceanogr 14:799–801. 19. Chauhan A, Williams HN (2006) Response of Bdellovibrio and like organisms (BALOs) 39. Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial System- to the migration of naturally occurring bacteria to chemo attractants. Curr Microbiol atics, eds Stackebrandt E, Goodfellow M (Wiley, New York), pp 115–175. 53:516–522. 40. Cole JR, et al. (2003) The Ribosomal Database Project (RDP-II): Previewing a new 20. Kisand V, Andersson N, Wikner J (2005) Bacterial freshwater species successfully autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic immigrate to the brackish water environment in the northern Baltic. Limnol Oceanogr Acids Res 31:442–443. 50:945–956. 41. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search 21. Fandino LB, Riemann L, Steward GF, Long RA, Azam F (2001) Variations in bacterial tool. J Mol Biol 215:403–410. community structure during a dinoflagellate bloom analyzed by DGGE and 16S rDNA 42. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX sequencing. Aquatic Microbial Ecology 23:119–130. windows interface: Flexible strategies for multiple sequence alignment aided by 22. Stoica E, Herndl GJ (2007) Bacterioplankton community composition in nearshore quality analysis tools. Nucleic Acids Res 24:4876–4882. waters of the NW Black Sea during consecutive diatom and coccolithophorid blooms. 43. Lozupone C, Knight R (2005) UniFrac: A new phylogenetic method for comparing Aquat Sci 69:413–418. microbial communities. Appl Environ Microbiol 71:8228–8235.

4306 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809671106 Chauhan et al. Downloaded by guest on September 27, 2021