The ISME Journal (2011) 5, 1077–1085 & 2011 International Society for Microbial Ecology All rights reserved 1751-7362/11 www.nature.com/ismej ORIGINAL ARTICLE Co-occurrence patterns for abundant marine archaeal and bacterial lineages in the deep chlorophyll maximum of coastal California J Michael Beman, Joshua A Steele and Jed A Fuhrman Department of Biological Sciences and Wrigley Institute for Environmental Studies, University of Southern California, Los Angeles, CA, USA Microorganisms remineralize and respire half of marine primary production, yet the niches occupied by specific microbial groups, and how these different groups may interact, are poorly understood. In this study, we identify co-occurrence patterns for marine Archaea and specific bacterial groups in the chlorophyll maximum of the Southern California Bight. Quantitative PCR time series of marine group 1 (MG1) Crenarchaeota 16S rRNA genes varied substantially over time but were well- correlated (r2 ¼ 0.94, Po0.001) with ammonia monooxygenase subunit A (amoA) genes, and were more weakly related to 16S rRNA genes for all Archaea (r2 ¼ 0.39), indicating that other archaeal groups (for example, Euryarchaeota) were numerically important. These data sets were compared with variability in bacterial community composition based on automated ribosomal intergenic spacer analysis (ARISA). We found that archaeal amoA gene copies and a SAR11 (or Pelagibacter) group Ib operational taxonomic unit (OTU) displayed strong co-variation through time (r2 ¼ 0.55, Po0.05), and archaeal amoA and MG1 16S rRNA genes also co-occurred with two SAR86 and two Bacteroidetes OTUs. The relative abundance of these groups increased and decreased in synchrony over the course of the time series, and peaked during periods of seasonal transition. By using a combination of quantitative and relative abundance estimates, our findings show that abundant microbial OTUs—including the marine Crenarchaeota, SAR11, SAR86 and the Bacteroidetes— co-occur non-randomly; they consequently have important implications for our understanding of microbial community ecology in the sea. The ISME Journal (2011) 5, 1077–1085; doi:10.1038/ismej.2010.204; published online 13 January 2011 Subject Category: microbial population and community ecology Keywords: Crenarchaeota; SAR11; SAR86; Bacteroidetes; community assembly; time-series Introduction these processes. However, early molecular studies uncovered substantial diversity within marine Microbial members of all three domains of life microbial assemblages (Giovanonni et al., 1990; (Archaea, Bacteria,andEucarya) are ecologically Fuhrman et al., 1992), and recent metagenomic and biogeochemically important in the ocean. studies have revealed even greater diversity in the Microscopic primary producers such as Prochloro- sea (Venter et al., 2004; Sogin et al., 2006). coccus carry out 50% of global primary production Many of the groups identified in these studies (Field et al., 1998), while approximately half of have yet to be assigned to a particular ecological marine primary production is transferred to the niche, although some potential functional roles ‘microbial loop’—within which energy may even- have been identified. For example, the discovery of tually be transferred to other trophic levels or proteorhodopsin (PR; a putative light-driven proton organic material may be broken down and reminer- pump) among the ‘SAR86’ group of alphaproteo- alized (Azam et al., 1983). Certain processes are bacteria (Beja et al., 2000, 2001) indicated that these known to be important in the microbial loop— organisms may use light as a source of energy. particularly heterotrophic respiration and nutrient- SAR86 constitute up to 10% of microbial assem- cycling processes such as nitrification—and a blages in the ocean (Eilers et al., 2000), and PR is number of microbial groups are known to carry out now recognized to be present in a wide range of microbial groups—including the ubiquitous and Correspondence: JM Beman, Department of Biological Sciences abundant SAR11 or Pelagibacter clade (Giovannoni and Wrigley Institute for Environmental Studies, School of et al., 2005a). Pelagibacter spp. respire organic Natural Sciences, University of California, Merced, 5200 North carbon and seem to gain little benefit from light Lake Road, Merced, CA 95343, USA. E-mail: [email protected] in laboratory experiments (Schwalbach et al., Received 20 September 2010; revised 2 December 2010; accepted 2005; Giovannoni et al., 2005a), however, and 2 December 2010; published online 13 January 2011 a paradigm is emerging by which ‘resourceful Abundant marine archaea and bacteria co-occur JM Beman et al 1078 heterotrophs’ such as the SAR11 group may use light regularly made since 1998, and a Microbial to supplement conventional heterotrophy (Moran Observatory program was established for euphotic and Miller, 2007). Some copiotrophs may use PR to zone depths in 2000. Samples were collected at extend survival times during starvation in the light the deep chlorophyll maximum (DCM) on a nearly compared with dark (Gomez-Consarnau et al., 2007), monthly basis from August 2000 to December 2004 or PR may have an array of physiological roles, using 20 L Niskin bottles deployed on a CTD rosette including sensory functions (Fuhrman et al., 2008). from the R/V Sea Watch. Water samples were PR was recently identified within marine group 2 sequentially filtered (Brown et al., 2005) and DNA Euryarchaeota (Frigaard et al., 2006), yet PR phylo- was extracted using previously described protocols geny was inconsistent with 16S rRNA phylogeny, (Fuhrman et al., 1988). Further details of sampling and Euryarchaeota at deeper depths did not seem to are available in Brown et al., 2005; Fuhrman et al., contain PR (Frigaard et al., 2006). 2006 and Beman et al., 2010. In contrast to group 2 Euryarchaeota, marine group 1 (MG1) Crenarchaeota are distributed in large numbers below the euphotic zone, where they QPCR, automated ribosomal intergenic spacer analysis comprise ca. 30% of microbial communities (Karner (ARISA), and local similarity analysis (LSA) et al., 2001). Summed over the volume of the QPCR for the Archaea was performed using primers, deep sea, MG1 Crenarchaeota constitute one the probes, and conditions as reported by Takai and most abundant microbial groups in the ocean, and Horikoshi (2000). MG1 and archaeal amoA QPCR many of these organisms likely oxidize ammonia assays followed established protocols (Beman et al., (reviewed by Francis et al., 2007; Prosser and Nicol 2008, 2010), and all samples from 2000–2004 were 2008; Erguder et al., 2009). Several ammonia- analyzed in triplicate reactions. ARISA (Fisher and oxidizing archaea have been isolated (Ko¨nneke Triplett, 1999) was performed using primers and et al., 2005; de la Torre et al., 2008; Hatzenpichler reaction conditions as previously described (Brown et al., 2008), and with a few exceptions (for example, et al., 2005; Fuhrman et al., 2006, 2008); all samples Agogue et al., 2008; but also see Beman et al. (2010), from 2000 to March 2004 were analyzed. Identifica- Church et al. (2010) and Santoro et al. (2010)), many tion of operational taxonomic units (OTUs) within studies have shown that most marine Crenarchaeota ARISA profiles is based on extensive cloning a have the ability to oxidize ammonia (Francis et al., nd sequencing of the linked 16S-ITS-23S region 2005; Wuchter et al., 2006; Mincer et al., 2007), and detailed in Brown et al. (2005) and Fuhrman et al. that the MG1 Crenarchaeota actively do so in the (2006). Correlations among microbial groups and ocean (Lam et al., 2007, 2009; Beman et al., 2008; with environmental variables were identified over Frias-Lopez et al., 2008; Church et al., 2010; Santoro time using local similarity analysis (LSA) (Ruan et al., 2010). et al., 2006). These and other studies have helped place abundant microbial groups such as SAR86, SAR11, Euryarchaeota and Crenarchaeota into prospective Oceanographic and biogeochemical data ecological niches, but how they may function as a Salinity, temperature, dissolved oxygen, chlorophyll community is poorly understood. Recent results fluorescence and concentrations of different nutri- from the San Pedro Ocean Time series (SPOT) site ents (nitrite, nitrate, phosphate and silicate) were located off the coast of Southern California have measured using standard oceanographic techniques combined automated ribosomal intergenic spacer as discussed in Fuhrman et al. (2006) and Beman et al. (2010). 3H-Thymidine incorporation (Fuhrman analysis (ARISA) to ‘fingerprint’ bacterial commu- 3 nities, with terminal restriction fragment length and Azam, 1982) and H-Leucine incorporation polymorphism patterns for Eukaryotes, and quanti- (Simon and Azam, 1989) were measured using pre- tative PCR (QPCR) assays for archaeal groups to viously published methods (Fuhrman et al., 2006). generate an ecological ‘network’ spanning all three domains of life (J Steele and JA Fuhrman, unpub- Results and discussion lished). In this study, we identify co-occurrence patterns for abundant archaeal and bacterial groups Temporal dynamics of archaeal groups in the DCM in the chlorophyll maximum at SPOT based on Chlorophyll fluorescence at SPOT varied signifi- variation through time. cantly in space, time and magnitude (Figure 1a). Maximum fluorescence values ranged from 0–1.99 mglÀ1, and were typically higher in March– Materials and methods May when upwelling occurs along the CA coast. From 2000–2004, the depth of the DCM varied Sample