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Environmental Microbiology (2014)

doi:10.1111/1462-2920.12366

Amplicon pyrosequencing reveals spatial and temporal consistency in diazotroph assemblages of the Acropora millepora microbiome

Kimberley A. Lema,1,2,3 Bette L. Willis1,3 and David G. Bourne2,3* 1 ARC Centre of Excellence for Coral Reef Studies and School of Marine and Tropical Biology, James Cook University, Townsville, Qld 4811, Australia. 2 Centre for Marine Microbiology and Genetics, Australian Institute of Marine Science, Townsville, Qld 4810, Australia. 3 AIMS@JCU, James Cook University, Townsville, Qld 4811, Australia. Summary Diazotrophic bacteria potentially play an important functional role in supplying fixed nitrogen to the coral holobiont, but the value of such a partnership depends on the stability of the association. Here we evaluate the composition of diazotroph assemblages associated with the coral Acropora millepora throughout four seasons and at two reefs, an inshore and an offshore (mid-shelf) reef on the Great Barrier Reef, Australia. Amplicon pyrosequencing of the nifH gene revealed that diazotrophs are ubiquitous members of the bacterial community associated with A. millepora. Rhizobia (65% of the overall nifH sequences retrieved) and particularly Bradyrhizobia sp.-affiliated sequences (> 50% of rhizobia sequences) dominated diazotrophic assemblages across all coral samples from the two sites throughout the year. In contrast to this consistency in the spatial and temporal patterns of occurrence of diazotroph assemblages, the overall coral-associated bacterial community, assessed through amplicon sequencing of the general bacterial 16S ribosomal RNA gene, differed between inshore and mid-shelf reef locations. Sequences associated with the Oceanospirillales family, particularly with Endozoicomonas sp., dominated bacterial communities associated with inshore corals. Although rhizobia represented a variable and generally small fraction of the overall bacterial comReceived 26 August, 2013; revised 2 December, 2013; accepted 14 December, 2013. *For correspondence. E-mail d.bourne @aims.gov.au; Tel. 61 (07) 47534139; Fax 61 (07) 47725852.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd

munity associated with A. millepora, consistency in the structure of these diazotrophic assemblages suggests that they have a functional role in the coral holobiont. Introduction Efficient recycling of nitrogen, the primary limiting nutrient in coral reef ecosystems (D’Elia and Wiebe, 1990; Capone et al., 1992; Shashar et al., 1994), has ensured the evolutionary success of corals in oligotrophic oceans (Capone et al., 1992; Charpy-Roubaud and Larkum, 2005). Recent evidence suggests that nitrogen-fixing bacteria (i.e. diazotrophs), the only organisms capable of fixing and converting gaseous nitrogen (N2) to biologically available forms like ammonia (NH3) (Scanlan and Post, 2008), are closely associated with corals and potentially contribute an important source of nitrogen directly to the coral holobiont (Williams et al., 1987; Shashar et al., 1994; Lesser et al., 2004; 2007). Although early studies using acetylene reduction assays demonstrated that active nitrogen fixation occurs in corals (Williams et al., 1987; Shashar et al., 1994), it has only recently been shown that both endosymbiotic algae and coral hosts possess enzymes enabling ammonium assimilation (Leggat et al., 2007; Yellowlees et al., 2008; Stambler, 2011). Nano-scale secondary ion mass spectrometry studies have further confirmed that both coral cells and their symbiotic dinoflagellates (Symbiodinium) have the capacity to rapidly assimilate ammonia from the surrounding seawater (Pernice et al., 2012) and that bacteria play a role in fixing nitrogen taken up by Symbiodinium cells within coral larvae (Ceh et al., 2013). Thus, both corals and their Symbiodinium symbionts stand to benefit from associations with nitrogen-fixing bacteria. Molecular approaches targeting the nifH gene have revealed that diverse diazotrophic assemblages occur in association with coral tissues (Olson et al., 2009; Lema et al., 2012), as well as with various other marine organisms and environments (Hewson et al., 2007; Mohamed et al., 2008; Dang et al., 2009). The nifH gene is the marker of choice because it encodes a conserved subunit of the dinitrogenase iron protein responsible for nitrogen fixation, and it is conserved in all known diazotrophic

2 K. A. Lema, B. L. Willis and D. G. Bourne bacteria (Young, 1992). Recently, we reported that the dominant groups of diazotrophic bacteria within tissues of three coral species from the Great Barrier Reef (GBR, Australia) are closely affiliated with rhizobia, a group of diazotrophs that can only accomplish fixation when in symbiosis with their plant host, and moreover, diazotrophic assemblages were species-specific across three similar mid-shelf reefs (Lema et al., 2012). Knowledge of the stability of these coral-associated diazotrophic assemblages across seasons and sites varying in nutrient regimes would help to further evaluate the importance of these communities to the coral host, but such knowledge is currently lacking. Amplicon gene-targeted sequencing approaches are now commonly applied to investigate coral microbial landscapes (Sunagawa et al., 2009; Ceh et al., 2011; Chen et al., 2011; Lee et al., 2012; McKew et al., 2012) and have documented high bacterial diversity and complex community patterns, varying potentially through season (Ceh et al., 2011; Chen et al., 2011) and location (Lee et al., 2012; McKew et al., 2012). To date, however, amplicon pyrosequencing has not been used to target functional genes, such as nifH, to explore the diversity of microbial communities with specific functional roles in corals. In this study, we investigate seasonal and inshoreoffshore patterns in diazotrophic assemblages, in combination with patterns in overall bacterial communities associated with the coral Acropora millepora, using amplicon pyrosequencing to target the nifH functional gene and the general bacterial 16S ribosomal RNA (rRNA) gene. Sampling A. millepora from a mid-shelf reef subject to little anthropogenic impact and an inshore reef exposed to coastal run-off throughout a year-long study enabled us to investigate the stability of coral-diazotroph associations in response to seasonal environmental and potential water quality variation to better characterize the association of nitrogen-fixing bacteria with reef-building corals.

Results Spatial and temporal patterns of coral-associated diazotrophic assemblages A total of ∼9900 high-quality nifH gene sequences were retrieved from fragments of the coral A. millepora that were sampled from the inshore Cattle Bay Reef and the mid-shelf Trunk Reef at four seasonal time points. Comparative diversity analysis (550 sequences per sample) identified a total of 102 OPUs (operational protein units; at a 90% protein distance threshold) across all samples (Table 1). The number of OPUs per sample was relatively low, ranging between 14 and 39. Species richness estimations (Chao1) were similarly low, with gene diversity

associated with the coral samples ranging between 21 and 52 predicted OPUs (Supporting Information Fig. S1; Table 1). The majority of nifH sequences from all coral samples fell within the Alphaproteobacteria class, representing more than 90% of the diazotrophs associated with some of the corals that were sampled from both Cattle Bay and Trunk Reefs (Fig. 1). All Alphaproteobacteria-affiliated sequences were identified as members of the Rhizobiales order (Figs 1, 2 and 3; Table 1). One Rhizobiales group, OPU 1, was dominant across all samples, representing 5472 of the 9900 sequences analysed (∼55% of all sequences), and was most closely affiliated with the nifH amino acid sequence of Bradyrhizobium sp. (99% similarity) (Figs 2 and 3). OPU2 (680 sequences) and OPU4 (259 sequences) were also members of the Rhizobiales order and affiliated with nifH sequences from Sinorhizobium-related species (98% amino acid sequence similarity) and Bradyrhizobium-related species (98% similarity) respectively (Figs 2 and 3). The remaining, less abundant nifH rhizobia sequences (i.e. OPUs 8, 10, 18, 19 and 20) were most closely affiliated with the methanotroph (type I) Methylocystis echinoides (Figs 2 and 3). In total, rhizobia were the dominant group and represented 65% of all nifH sequences retrieved, comprising from 41% to 93% of the total sequences within individual coral samples (Fig. 2; Table 1). Other dominant nifH sequences retrieved from coral samples fell within the Gammaproteobacteria and Deltaproteobacteria, and a few within the Cyanobacteria class (Fig. 1). Within the Gammaproteobacteria, three OPUs were identified (OPU3, OPU5 and OPU7) and constituted ∼9% of the total sequences recovered. Two dominant groups, OPU3 and OPU7, which represented ∼7% of the total sequences recovered, both affiliated with Vibrio species, specifically Vibrio diazotrophicus (99% similarity) and Vibrio natriegens (98% similarity) respectively (Figs 2 and 3). OPU5 was closest to Klebsiella pneumoniae (94% similarity). These Gammaproteobacteria OPUs were generally present in low relative abundance, but were detected in samples from both reefs (Trunk Reef and Cattle Bay) and in all four seasons. Two exceptions to this pattern involved an increase to 265 sequences falling within OPU3 recovered from a spring sample at Trunk Reef, and 228 sequences that grouped into OPU5 in an autumn sample from Trunk Reef (Fig. 2). NifH sequences within the Deltaproteobacteria class were relatively abundant in only a limited selection of coral samples (Fig. 1), and all were affiliated with anaerobic sulphate-reducing bacteria (Figs 2 and 3). Only OPU12, which was dominant in one sample from Cattle Bay, was affiliated with Cyanobacteria (Figs 1 and 2). The remaining sequences represented only 14% of all nifH sequences, thus they were allocated to a ‘minor groups’ category and were not

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

Winter

Autumn

Summer

Spring

Winter

Autumn

Summer

Spring

1 2 3 1 2 3 1 3 1 2 3 4 5 6 4 5 4 5 4 5 6

446 1853 2491 809 1296 2438 646 2755 1300 2952 2768 1083 354 / 375 370 1749 407 1056 805 2538

16S rRNA/nifH 817 / 1661 2593 865 572 2804 1450 2672 2114 / 1404 1149 550 2197 / 2463 1423 2666 1564 998

350 350 350 350 350 350 350 350 350 350 350 350 350 / 350 350 350 350 350 350 350

550 / 550 550 550 550 550 550 550 550 / 550 550 550 550 / 550 550 550 550 550

16S rRNA/nifH

Rarified sequences

The solidus symbol (/) indicates samples for which no amplicon sequence information was recovered.

Trunk Reef (mid-shelf)

Cattle Bay (inshore)

Colony

Initial number of sequences

51 29 22 25 40 39 35 32 22 24 21 25 41 / 49 28 27 41 37 38 106

16 / 14 18 21 17 18 23 17 17 / 18 26 39 17 / 15 20 20 27 28

16S rRNA/nifH

N (OTUs/OPUs)

82 40 52 34 60 81 52 59 28 50 51 47 56 / 63 36 30 58 70 62 187

21 / 23 23 23 22 27 36 28 31 / 35 52 47 24 / 29 35 21 33 30

16S rRNA/nifH

Chao1

8 0 0 0 0 0 4 0 0 0 0 62 0 / 2 1 0 1 1 0 0

93 / 79 89 45 67 79 62 94 68 / 65 45 59 93 / 94 43 88 41 44

16S rRNA/nifH

Percentage (%) of rhizobia in:

Table 1. Sequence information for both 16S rRNA (cut-off 0.03) and nifH (cut-off 0.1) genes datasets from replicate (n = 3) A. millepora samples collected from Cattle Bay (inshore) and Trunk Reef (mid-shelf) throughout seasons. Additionally, percentage of sequences affiliated to rhizobia in 16S rRNA and nifH is presented for each sample.

Diazotroph communities associated with the coral Acropora millepora 3

4

K. A. Lema, B. L. Willis and D. G. Bourne

Australia

Trunk Reef

Orpheus Island Cattle Bay

10 km

Fig. 1. Sampling locations on the Great Barrier Reef, Australia; Cattle Bay, Orpheus Island (inshore reef); and Trunk Reef (mid-shelf reef).

further explored because of their very low relative abundance across samples. Principal coordinates analysis (PCoA) demonstrated that patterns in diazotrophic assemblages associated with corals did not vary in a consistent manner, either spatially (inshore versus offshore) or temporally (sampling season) (Supporting Information Fig. S2). Samples from Cattle Bay and Trunk Reef were not grouped by sampling location but scattered throughout the ordination plot, which explained 46% of the variation (Supporting Information Fig. S2). Statistical analysis [analysis of similarity (ANOSIM)] confirmed that diazotrophic assemblages were not correlated with site (R = 0.04; P = 0.28) nor with sampling season (R = 0.08; P = 0.15) (Table 2). Distribution of the samples on the PCoA ordination was generally correlated with the most abundant OPUs present in each sample (Supporting Information Fig. S2). For example, the spring sample of colony 5 was strongly correlated with OPU3 (Vibrio sp.) because of the high relative abundance of this sequence in this specific sample (Fig. 2).

Spatial and temporal patterns of coral-associated bacterial communities A total of ∼7000 high-quality 16S rRNA gene sequences were retrieved from fragments of the coral A. millepora sampled across two reefs and four seasons. To enable analyses of comparative bacterial diversity and prevent potential bias caused by differences in sequencing effort, the sequence dataset was normalized to 350 sequences per sample. This resulted in a total of 475 operational

taxonomic units (OTUs) (grouped based on 97% similarity) being identified across all samples. The number of OTUs observed was variable, ranging from 22 to 106 OTUs for an individual sample (Table 1). Estimates of sequence diversity (Chao1) indicated that the predicted species diversity was not reached for most of the samples (Table 1; Supporting Information Fig. S1). Comparison of bacterial assemblages associated with A. millepora corals indicated a strong location (i.e. reef) fingerprint for the diversity of coral bacterial communities. All corals sampled from the inshore Cattle Bay Reef were dominated by Gammaproteobacteria, which represented more than 65% of retrieved sequences for inshore samples (Fig. 1). The majority of these sequences were affiliated with the Oceanospirillales family, with the three most dominant ribotypes (OTU1, 2 and 3) affiliated with Endozoicomonas-related species at the genus level and comprising up to 94% of sequences recovered from some samples (Fig. 2). These sequences were most closely affiliated with sequences previously retrieved from corals, with OTU2 and 3 most similar to sequences derived from acroporid corals collected from reefs near the current study site at Orpheus Island in the central GBR (Figs 2 and 4). From the 20 dominant ribotypes recovered, 7 (OTU1, 2, 3, 5, 8, 14 and 19) were closely affiliated to sequences previously recovered from corals, and 15 (out of the 20 recovered) were affiliated with marine organisms (Fig. 2). In contrast to Cattle Bay samples, bacterial communities associated with Trunk Reef corals were more variable. For example, although Gammaproteobacteria were still prevalent in the bacterial assemblages associated with corals from Trunk Reef, sequences from the Bacilli, Alphaproteobacteria, Betaproteobacteria, Actinobacteria, Cyanobacteria and Clostridia were generally more abundant (Fig. 1). Sequences affiliated with Endozoicomonas groups (OTU1, 2 and 3) were present in Trunk Reef corals; however their relative abundance across most samples was lower than that of Cattle Bay corals. Two dominant ribotypes affiliated with a Salsuginibacillus sp. (91% similarity) were found in almost all Trunk Reef samples (OTU4 and OTU18, ∼800 sequences), but were absent in Cattle Bay samples (Fig. 2). Other sequences, such as OTU6, 8, 10, 11 and 13, which were related to Bradyrhizobia sp., Roseobacter sp., Franscisella sp., Endomicrobia sp., and Pseudovibrio sp. respectively, were only found in samples from Trunk Reef. Moreover, their relative abundance in samples was variable, further highlighting less consistent patterns across samples but possibly higher bacterial diversity in these mid-shelf reef samples. For example, Bradyrhizobia sp.-affiliated (OTU6) and Roseobacter sp.-affiliated (OTU8) sequences displayed high relative abundance only in one coral colony (colony 4) in spring and autumn (Fig. 2). Similarly, Clostridaeceae sp.-related (OTU15)

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

Diazotroph communities associated with the coral Acropora millepora

5

Trunk reef (mid-shelf reef )

Cattle Bay (inshore reef )

(A)

Spring

1 3

Summer

1 2 3

Autumn

1 3

Winter

1 2

Spring

4 5 6

Summer

4

Autumn

4 5

Winter

4 5 6 0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Trunk reef (mid-shelf reef )

Cattle Bay (inshore reef )

(B)

Spring

1 2 3

Summer

1 2 3

Autumn

1 3

Winter

1 2 3

Spring

4 5

Summer

4 5

Autumn

4 5

Winter

4 5 6 0%

Alphaproteobacteria (

Rhizobiales )

Deltaproteobacteria Gammaproteobacteria

Bacilli

Actinobacteria

Clostridia

Betaproteobacteria

Cyanobacteria

Minor groups

Fig. 2. Bacterial class affiliations of sequences retrieved from colonies of A. millepora (1, 2, 3, 4, 5 and 6) sampled in four seasons over 1 year and at two reefs, Cattle Bay, Orpheus Island (inshore reef), and Trunk Reef (mid-shelf reef) for (A) NifH gene protein sequences (550 sequences per sample; OPUs defined at a 90% protein sequences similarity) and (B) 16S rRNA gene sequences (350 sequences per sample; OTUs defined at the 97% nucleotide sequence similarity). The proportion of the orderRhizobiales has been highlighted in red.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

194 6 131 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0

1 74 3 0 140 0 43 0 1 0 0 0 0 16 0 0 0 0 0 0

38 12 26 0 108 0 58 0 1 0 0 0 0 8 0 0 0 0 0 0

222 9 8 17 0 191 3 0 1 0 0 0 0 0 0 0 0 0 0 0

178 5 147 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0

361 1 1 11 0 0 0 19 0 30 0 0 0 0 0 38 0 9 7 4

22 280 9 0 11 0 6 0 0 0 0 0 0 1 0 0 0 0 0 0

88 93 63 0 29 0 7 0 0 0 0 1 0 2 0 0 0 0 14 0

227 119 9 24 0 0 3 0 130 0 0 0 0 0 0 0 0 0 0 0

131 57 86 0 7 0 9 0 0 0 0 0 0 3 0 0 0 0 16 0

175 7 16 5 0 0 15 46 0 74 0 0 0 0 0 0 0 19 17 11

51 238 29 0 0 0 0 11 1 0 0 0 1 0 0 0 0 0 3 0

/ / / / / / / / / / / / /

/ / / / / / /

1 0 0 52 0 214 0 0 12 0 0 8 0 0 0 0 7 4 0 0

330 1 46 24 0 0 58 0 0 0 0 0 0 0 0 0 0 0 0 0

0 4 27 85 0 0 0 0 36 0 0 2 0 0 0 0 10 2 0 0

490 9 4 12 0 0 2 0 0 0 0 0 0 0 0 0 1 0 0 0

11 2 3 88 0 0 0 113 0 2 0 14 60 1 0 0 1 3 0 0

396 90 1 30 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

133 0 265 11 0 0 36 0 0 0 0 0 0 0 0 0 0 0 0 0 / / / / / / / / / / / / /

/ / / / / / /

164 56 0 15 228 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

176 35 1 8 0 0 2 0 0 0 0 1 70 0 48 0 7 0 0 0

14 0 6 13 73 4 94 0 0 3 13 0 16 1 64 49 132 232 90 31 0 0 0 2 0 0 0 0 0 0 22 0 0 1 0 5 0 0 3 0 14 7 0 1 1 63 5 11 0 0 0 0 0 78 0 0 0 0 0 27 0 0 0 0 0 3 0 0 3 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 2 3 17 9 1 0 0 0 0 4 0 0 0 0 0

374 84 8 9 0 0 2 0 0 0 0 0 0 0 0 0 16 0 0 0

195 36 5 6 0 0 4 0 0 0 96 0 0 55 0 0 6 0 0 0

0 1 0 0 0 0 0 0 0 0 0 0 0 0 49 46 0 0 0 0

153 0 38 13 0 0 28 123 0 21 0 0 0 0 0 0 0 7 5 12

/ / / / / / / / / / / / / / / / / / / /

OTU1 OTU2 OTU3 OTU4 OTU5 OTU6 OTU7 OTU8 OTU9 OTU10 OTU11 OTU12 OTU13 OTU14 OTU15 OTU16 OTU17 OTU18 OTU19 OTU20 214 160 133 90 81 78 69 61 52 49 46 44 41 37 33

* 1223 * 949 * 835 * 759 314

Spongiobacter sp. (DQ917877-96%) Gammaproteobacteria Endozoicomonas sp. (FJ489763-96%) Gammaproteobacteria Endozoicomonas sp. (FJ809593-97%) Gammaproteobacteria Salsuginibacillus sp. (HE663395-91%) Bacilli Oceanospirillales sp. (EU636648-95%) Gammaproteobacteria Bradyrhizobia sp. (GU477345-98%) Alphaproteobacteria Oceanospirillales sp. (NR_041264-97%) Gammaproteobacteria Roseobacter sp. (JN796464-98%) Alphaproteobacteria Synechococcus sp. (KC425530-99%) Cyanobacteria Francisella sp. (FM242232-96%) Gammaproteobacteria Endomicrobia sp. (AB192274-94%) Endomicrobia Actinomycetales sp. (JF235418-98%) Actinobacteria Pseudovibrio sp. (NR_074229-99%) Alphaproteobacteria Endozoicomonas sp. (FJ809460-99%) Gammaproteobacteria Clostridiaceae sp. (JF541382-98%) Clostridia Clostridia Ruminococcus sp. (JQ110748-98%) Stenotrophomonas sp. (JN986195-100%) Gammaproteobacteria Salsuginibacillus sp. (HE663395-91%) Bacilli Endozoicomonas sp. (FJ809533-97%) Gammaproteobacteria Cyanobacteria sp. (EF629792-97%) Cyanobacteria

Number of sequences

X > 400 200 < X < 400 100 < X < 200 60 < X < 100 20 < X < 60 1 < X < 20 0

Octocoral (unpublished) Scleractinian corals GBR (Littman et al., 2009) Scleractinian corals GBR (Raina et al., 2009) Mollusc (Duperron et al., 2013) Sclearctinian coral carribean (Garren et al., 2008) Soil agriculture (unpublished) Sea slug (Kurahashi and Yokota, 2007) Octocoral (unpublished) Seawater (Yeo et al., 2013) Coastal sediment (Paisse et al., 2010) Temite (Hongoh et al., 2005) Human skin (Kong et al., 2013) Seawater (unpublished) Scleractinian corals GBR (Raina et al., 2009) Sediment (unpublished) Sediment (unpublished) Seawater (Yin et al., 2013) Mollusc (Dupperon et al., 2013) Scleractinian corals GBR (Raina et al., 2009) Marine sponge (Mohamed et al., 2008)

16Sr RNA taxonomic affiliation

Bradyrhizobium sp. (AEC13430-99%) Alphaproteobacteria Roots of Acacia (Fabaceae) (Menna and Hungria, 2011) Sinorhizobium sp. (AEY75285-98%) Alphaproteobacteria Roots of legume (Fabaceae) (Mnasri et al., 2012) Vibrio sp. (AAD55588-99%) Gammaproteobacteria Seawater (unpublished) Bradyrhizobium sp. (AFR69099-98%) Alphaproteobacteria Roots of Acacia (Fabaceae) (unpublished) Klebsiella sp. (AAK11560-94%) Gammaproteobacteria Sea sediment (unpublished) Deltaproteobacteria sp. (ACD87562-91%) Deltaproteobacteria Scleractinian corals Pacifique (Olson et al., 2009) Vibrio sp. (AAA65435-98%) Gammaproteobacteria Cyanobacterial mat (Zher et al., 1995) Methylocystis sp. (CAD91843-96%) Alphaproteobacteria Soil (Dedysh et al., 2004) Deltaproteobacteria sp. (ABQ50695-97%) Deltaproteobacteria Seawater (Man-Aharonovich et al., 2007) Methylocystis sp. (CAD91843-95%) Alphaproteobacteria Soil (Dedysh et al., 2004) Desulfovibrio sp. (YP_002437020-93%) Deltaproteobacteria Sea sediment (unpublished) Cyanobacteria sp. (ABM66745-97%) Cyanobacteria Bacteriooplankton(Hewson et al., 2007) Deltaproteobacteria sp. (ACI26110-96%) Deltaproteobacteria Soil (Hsu and Buckley, 2008) Desulfovibrio sp. (YP_002437020-94%) Deltaproteobacteria Sea sediment (unpublished) Deltaproteobacteria sp. (AGE45133-94%) Deltaproteobacteria Hot spring (unpublished) Methylocystis sp. (CAD91843-98%) Alphaproteobacteria Soil (Dedysh et al., 2004) Mesorhizobium sp. (AFR69097-97%) Alphaproteobacteria Roots of Acacia (Fabaceae) (unpublished) Methylocystis sp. (CAD91843-97%) Alphaproteobacteria Soil (Dedysh et al., 2004) Methylocystis sp. (AEU12171-96%) Alphaproteobacteria Scleractinian corals GBR (Lema et al., 2012) Methylocystis sp. (AEU12171-95%) Alphaproteobacteria Scleractinian corals GBR (Lema et al., 2012)

n (sequences) total = 7000

228 191 189 188 131 125 96 92 73 55 48 38 36 35 29 27

* 5472 * 680 * 459 259

NifH taxonomic affiliation Nearest realtive (Acc.number-% similarity)-biological source

Fig. 3. Heatmap of protein nifH (90% sequence identity cut-off) and 16S rRNA (97% sequence identity cut-off) sequence abundances from the coral A. millepora; samples from two reefs and four seasons. The 20 most abundant OTUs (16S rRNA) and OPUs (nifH ) are represented with their closest sequence match determined from Genebank BLAST (BLASTN for 16S rRNA and BLASTP option for nifH ), its corresponding accession number, its taxonomic affiliation and the biological source of their closest match (with reference publication if any). Dominant groups(> 400 sequences) are highlighted with an asterisk (*). Samples for which no sequences were retrieved are represented with a bar (/).

125 41 70 0 2 0 1 0 0 0 0 5 0 1 0 0 8 0 0 33

/ / / / / / / / / / / / /

/ / / / / / /

179 2 99 0 5 0 2 0 0 0 0 1 0 13 0 0 11 0 0 0

500 3 0 13 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

98 33 48 0 10 0 11 1 16 0 0 9 0 0 0 0 0 0 0 0

413 17 42 7 0 0 26 0 0 0 0 0 2 0 0 0 0 0 0 0

n (sequences) total = 9900

OPU1 OPU2 OPU3 OPU4 OPU5 OPU6 OPU7 OPU8 OPU9 OPU10 OPU11 OPU12 OPU13 OPU14 OPU15 OPU16 OPU17 OPU18 OPU19 OPU20

Colony 6

245 183 1 2 0 0 0 0 0 0 0 91 0 0 0 0 6 0 0 0

Colony 5

426 19 11 42 0 0 7 0 0 0 0 0 1 0 0 0 0 0 0 0

Colony 4

Trunk Reef (mid-shelf reef )

492 11 3 10 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

Colony 3

Spring Summer Autumn Winter Spring Summer Winter

Colony 2

Cattle Bay (inshore reef)

Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn Winter Spring Winter

Colony 1

6 K. A. Lema, B. L. Willis and D. G. Bourne

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

Diazotroph communities associated with the coral Acropora millepora Table 2. Statistical test (ANOSIMs) for changes in community structure, showing reef sites and seasons as sources of variance in A. millepora 16S rRNA and nifH gene samples. nifH sequences

Reefs Seasons

16S rRNA sequences

R-value

P-value

R-value

P-value

0.04 0.08

0.28 0.15

0.65 0.0135

< 0.0001* 0.52

Test was run using the Bray–Curtis algorithm (n = 1000 replications). Significant differences are signalled with an asterisk (*).

and Ruminococcus sp.-related (OTU16) sequences were highly abundant in colony 6, but this bacterial community assemblage was not observed in any other sample (Fig. 2). Season did not influence the bacterial communities associated with corals sampled from either Cattle Bay or Trunk Reef (R = 0.0135; P = 0.52). Instead, PCoA confirmed that samples grouped according to reef sites, regardless of the season (Fig. 5), and this pattern of bacterial community structures differing between

reefs was statistically significant (ANOSIM) (R = 0.65; P < 0.0001*) (Table 2). Although rhizobia were identified within all corals sampled across spatial and temporal scales when the nifH gene was targeted, they represented only a small relative proportion of the overall coral-associated bacterial community (∼4% of total 16S rRNA sequences). On the inshore reef, rhizobia were only detected in one colony (colony 1), representing 8% of 16S rRNA genes in spring and 4% in autumn samples (Fig. 1; Table 1). In samples from Trunk Reef, rhizobia sequences were more prevalent, being observed in five out of nine samples (Fig. 1; Table 1) and retrieved from at least one replicate colony at each seasonal sampling time. During spring, Rhizobiales sequences in colony 4 represented 62% of the total 16S rRNA sequences (Fig. 1; Table 1), with this OTU (OTU6) being closely affiliated (98% similarity) with a Bradyrhizobia sp. (Fig. 2). Other diazotrophic bacteria found in nifH sequence analyses, such as Vibrio sp. and Deltaproteobacteria, represented only minor components of the 16S rRNA gene sequences. Importantly, they did not overlap with diazotrophic phylotypes at the species

27.98%

Winter-1 OTU1

Winter-2 Summer-1

OTU3

Summer-4

Winter-5

Autumn-4 Summer-5

Spring1 Autumn-1

Autumn-3

OTU9

Spring-1

OTU8

OTU4

Autumn-5

OTU6

Winter-4

OTU10

29.65%

Winter-6

OTU7

Summer-3

OTU5

Spring-5

Summer-2 Winter-3 Trunk Reef (mid-shelf) Cattle Bay (inshore)

7

OTU2

Spring-3 Spring-2

Fig. 4. PCoA of 16S rRNA gene sequences (97% sequence identity cut-off) from samples of A. millepora (1, 2, 3, 4, 5, 6) from Cattle Bay (inshore) and Trunk Reef (mid-shelf); colonies sampled through four seasons of the year. The 10 most abundant OTUs (16S rRNA) are represented by vectors and represent differences among samples.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

Crocosphaera watsonii (AAP48976) Cylindrospermopsis raciborskii (ZP 06309412) Corals GBR (AEU12169) Corals GBR (AEU12167)

100% 52%

Xenococcus sp. (AAB37312)

92%

OPU12 (KF307342- 92 sequences)

78% 71% 56%

uncultured microorganism (ABM66745) Mesorhizobium sp.(AFR69097) OPU17 (KF307347- 36 sequences)

94%

Sinorhizobium americanum (AEY75285) OPU2 (KF307332- 680 sequences)*

Methylocystis echinoides (CAD91843)

57% 44% 77%

48% 97%

OPU16 (KF307346- 38 sequences) OPU10 (KF307340- 125 sequences) OPU19 (KF307349- 29 sequences) OPU8 (KF307338- 188 sequences) OPu18 (KF307348- 35 sequences) OPU20 (KF307350- 27 sequences) Corals GBR (AEU12171) Corals GBR (AEU12172)

Bradyrhizobium sp. (AEC13430)

92% 67%

OPU1 (KF307331- 5472 sequences)* OPU4 (KF307334- 259 sequences)

Bradyrhizobium sp. (AFR69099) Halorhodospira halophila (BAD93282) Corals GBR (AEU12177) Corals Hawaii (ACD87603)

65%

Vibrio diazotrophicus(AAA65435) OPU7 (KF307337- 189 sequences) OPU3 (KF307333- 459 sequences)*

72%

Vibrio natriegens (AAD55588) Corals Hawaii (ACD87600)

94% 54%

Corals GBR (AEU12176)

Dickeya dadantii(YP 002986124) OPU5 (KF307335- 228 sequences)

90%

Gammaproteobacteria

83%

nifH cluster 1

50%

Cyanobacteria

K. A. Lema, B. L. Willis and D. G. Bourne

Alphaproteobacteria

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Klebsiella pneumoniae (AAK11560 Corals GBR (AEU12178)

Geobacter sulfurreducens (AAR36215) uncultured soil bacterium (ACI26110) 60%

Desulfobacter latus (AAP48977) Corals GBR (AEU12179) OPU6 (KF307336- 191 sequences) Corals Hawaii (ACD87621) Corals Hawaii (ACD87562)

97%

99%

Desulfovibrio gigas (AAB09059) uncultured microorganism (ABQ50695)

nifH cluster 3

uncultured bacterium (AGE45133) OPU15 (KF307345- 48 sequences)

Deltaproteobacteria

OPU13 (KF307343- 73 sequences) 75%

OPU9 (KF307339- 131 sequences)

Desulfovibrio sp. (YP 002437020)

68% 98%

OPU11 (KF307341- 96 sequences) OPU14 (KF307344- 55 sequences)

Methanosarcina acetivorans (NP 616152) 0.10

Fig. 5. Neighbour-joining phylogenetic tree (generated in MEGA v. 5 and includes bootstrap support of 1000 iterations) of the 20 dominant nifH gene OPUs (90% sequence identity cut-off). The tree was rooted with the nifH protein sequence of an Archaea (Methanosarcina acetivorans). OPUs from this study are indicated in bold with the number of sequence in each group. NifH sequences retrieved from corals in previous studies are indicated in black. OPUs representing over a 450 sequences are signalled with an asterisk (*). Classes of bacteria are indicated to the far right of tree and NifH Clusters indicated with different dashed lines.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

Diazotroph communities associated with the coral Acropora millepora level, so we could not define them as nitrogen-fixing bacteria. Cyanobacteria were detected in a few 16S rRNA samples and represented a small fraction (∼1.5%) of the overall sequences recovered. Discussion Diazotrophs are ubiquitous members of the coral microbiome Speculation that diazotrophs represent an important component of coral-associated microbial communities because of their capacity to provide additional sources of nitrogen to the coral holobiont has been increasing in recent years (Lesser et al., 2004; Olson et al., 2009; Lema et al., 2012). Using an amplicon tag sequencing approach targeting the nifH subunit of the nitrogenase iron gene complex, this study showed that phylotypes closely affiliated with the Rhizobiales order are dominant and consistent within diazotrophic assemblages associated with the common GBR coral A. millepora across two sites and four seasons. One rhizobial group (OPU1), which was most closely affiliated with Bradyrhizobium sp. (99% amino acid sequence similarity), dominated all samples of A. millepora. This dominant phylotype represented up to 93% of the total nifH sequences in some samples and approximately 55% of all sequences recovered from all samples. Two other dominant phylotypes (OPU2 and 4) were also affiliated with the rhizobia group and were found in all samples. Rhizobia are best known for their critical role in nitrogen fixation when associated with legumes, a role they are capable of performing only when established in a symbiotic relationship with a plant (Fred et al., 1932; Cullimore and Dénarié, 2003). In a previous study, we similarly found that the Rhizobiales was the dominant diazotrophic group associated with two species of Acropora (i.e. A. millepora and A. muricata) and a pocilloporid at two other mid-shelf reefs (Lema et al., 2012), providing further evidence that this particular diazotrophic group has potential functional significance for corals. Additionally, this group is also dominant across early life stages of A. millepora, from 4-day-old larvae to 1-year-old juveniles (K.A. Lema, D.G. Bourne and B.L. Willis, unpubl. data), suggesting that they are a continuous component of the coral microbiome. Taken together, nifH gene sequencing results provide evidence that coral rhizobia are ubiquitous members of the coral microbiome, suggesting that their potential to provide additional sources of fixed nitrogen to the coral holobiont may be functionally important. The second most abundant group of diazotrophs detected by our nifH-based gene assay comprised Vibrioaffiliated sequences (OPU3 and 7; ∼7% of the total sequences recovered), which were also found in all samples from both reefs and in all four seasons. A number

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of previous studies have also identified vibrios as consistent members of diazotrophic assemblages associated with corals. For example, members of the Vibrio genus dominated diazotrophic bacterial communities associated with Hawaiian corals in the genus Montipora (Olson et al., 2009) and were the most prevalent isolates from the Brazilian coral Mussismilia hispida (Chimetto et al., 2008). Recently, a Vibrio sp., closely related to V. diazotrophicus and similar to sequences retrieved in this study, was found to dominate isolates from A. millepora in nitrogen-free media (K.A. Lema, P.L. Clode, R. Thornton, B.L. Willis and D.G. Bourne, unpubl. data), suggesting that this group is important under N2 limitation. Overall, these studies highlight that this group is also potentially important within diazotrophic assemblages associated with corals. NifH phylotypes affiliated with the methanotrophic (type II) rhizobial species M. echinoides (OPUs 8,10,16,18,19 and 20) were detected in samples derived from both inshore and mid-shelf reefs and comprised 4% of nifH phylotypes. Sequences related to this methanotroph were also abundant in our previous study of A. millepora (Lema et al., 2012), and genes involved in methane oxidation have been described in other corals (Siboni et al., 2008; Kimes et al., 2010). Anaerobic sulphate-reducing bacteria that possess the nifH gene were also commonly identified from coral samples, both in this study (representing 6% of nifH sequences) and in previous studies of coralassociated microbial diazotrophic communities (Olson et al., 2009; Lema et al., 2012). The role of these organisms in methane and sulphur cycling within the coral holobiont requires further investigation. Overall, rhizobial assemblages represented only a small and variable component of the bacterial communities associated with A. millepora. Although the majority of 16S rRNA sequences identified as diazotrophs were affiliated with rhizobia, rhizobia-affiliated sequences were recovered from only 7 of the 20 samples and represented only 0–8% of the sequences recovered. The relatively low abundance of rhizobia in the total bacterial community, coupled with the lower diversity coverage of the 16S rRNA samples (as low as 69% for one sample), may explain why they were not recovered from all samples in the 16S rRNA assay despite being present in all nifH assays. Intraand inter-colony variability in coral bacterial composition through 16S rRNA sequencing is a common artefact in coral studies that could influence the amplification of less abundant groups such as rhizobia. Indeed, in most coral microbial studies, dominant bacterial groups are relatively consistent across replicate samples, although other less abundant bacterial groups vary among replicate samples (e.g. Rohwer et al., 2002; Bourne and Munn, 2005; Hansson et al., 2009; Ainsworth et al., 2009; Lee et al., 2012). NifH gene analyses revealed the irregular occurrence of some groups (e.g. overrepresentation of OPU5 in

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

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K. A. Lema, B. L. Willis and D. G. Bourne

one autumn sample), which may have represented interor intra-colony variation in coral bacterial composition. A coral colony is a complex habitat, and microenvironmental variability can strongly influence the abundance of associated microbial communities (Rohwer et al., 2002; Ainsworth et al., 2009). This may also explain why in the current study, a Bradyrhizobia sp.-affiliated sequence (OTU6: 98% similarity) was overrepresented (214 sequences and ∼62% of total sequences) in one Trunk Reef sample (colony 4) sampled in spring. Recovery of sequences affiliated with Bradyrhizobia sp. in both nifH and 16S rRNA gene datasets, and from both adult tissues in this study and coral larvae in a concurrent study (K.A. Lema, D.G. Bourne and B.L. Willis, unpubl. data), demonstrate some consistency between both analyses. We suggest that while sequencing 16S rRNA genes to explore coral microbial diversity provides important baselines, sequencing functional genes provides an important step forward in understanding functional members of the coral microbiome. Dominant bacterial assemblages associated with GBR corals The dominance of the Oceanospirillales family, which represented seven of our 20 most abundant 16S rRNA OTUs, and of the genus Endozoicomonas in particular, with which five of these OTUs were affiliated (Fig. 2), suggests that bacteria in this family play an important functional role in the coral holobiont. Gammaproteobacteria are consistently found associated with corals globally (Rohwer et al., 2001; Bourne and Munn, 2005; Bourne et al., 2008; 2013; Hong et al., 2009; Littman et al., 2009; Raina et al., 2009; Kvennefors et al., 2010; Cardenas et al., 2012; Lee et al., 2012; McKew et al., 2012), particularly within the overall bacterial communities of Indo-Pacific corals [reviewed in Hong and colleagues (2009)]. Moreover, the Oceanospirillales group has been identified as a dominant ribotype in many coral diversity studies (Rohwer et al., 2002; Bourne et al., 2008; Littman et al., 2009; Kvennefors et al., 2010; Mouchka et al., 2010; Cardenas et al., 2012; Ceh et al., 2012; Lee et al., 2012), with Endozoicomonas-related species proposed to form a particularly intimate association with corals (Bayer et al., 2013b); thus, this genus may potentially constitute a useful indicator of coral health (Bourne et al., 2008). This genus is also implicated in the degradation of dimethylsulphoniopropionate (Seymour et al., 2010), a central molecule in the marine sulphur cycle that is produced by both the photosymbiont Symbiodionium and the coral itself (Raina et al., 2009; 2010; 2013; Seymour et al., 2010; Bourne et al., 2013). Dominance of all Cattle Bay samples by Endozoicomonas sp. OTUs (1, 2 and 3), which represented 69% of the total sequences recovered

from corals on this reef, combined with their presence in all samples from the offshore Trunk Reef, although at a lower relative abundance (representing only ∼11% of the total sequences recovered), lends further support to this genus being a characteristic associate of healthy corals. Moreover, these three dominant OTUs were also closely related to sequences previously retrieved from octocorals (OTU 1) and acroporid corals (OTUs 2 and 3) from Orpheus Island (Littman et al., 2009; Raina et al., 2009), demonstrating that they have been a consistent member of a range of coral microbiomes in this inshore group of reefs. Endozoicomonas is also found in other aposymbiotic invertebrates (Bayer et al., 2013a; Bourne et al., 2013); thus, their ubiquitous presence in marine invertebrates suggests that the functional role(s) they play are important for their hosts. Comparison of coral-associated bacterial communities between inshore and mid-shelf reefs Nitrogen loads on near-shore fringing reefs within the GBR, including our inshore Cattle Bay site, differ between seasons, with mean particulate nitrogen approximately 33% greater during wet seasons compared with dry seasons (averaged over 5 years; Schaffelke et al., 2012). In contrast, mean particulate nitrogen is consistently 30% lower on more oligotrophic mid-shelf reefs like Trunk Reef (averaged over 10 years; De’ath et al., 2009). Despite differences in nitrogen loads between reef locations and seasons, the diazotrophic assemblages (based on the nifH gene) associated with corals did not differ significantly between inshore and mid-shelf reef samples (ANOSIMs; P = 0.28). Although diazotrophs were more commonly identified in the 16S rRNA gene dataset at the offshore site (55% of samples from Trunk Reef versus 18% of samples from the near-shore Cattle Bay Reef), the presence and abundance of these diazotroph-related sequences were highly variable between samples and seasons. Although our results provide no evidence that nutrients, or nitrogen availability in particular, influence the structure of diazotroph assemblages associated with corals, further quantitative analyses of abundance data (e.g. quantitative polymerase chain reaction) would shed light on the potential significance of shifts in dominant groups that were detected. This study did not measure nitrogenase activity; thus, further studies in this area may indicate if nitrogen fixation activities of communities associated with corals differ under varying water nutrient conditions. In contrast to diazotroph assemblages, strong and significant differences in overall bacterial diversity were observed between inshore and mid-shelf reefs (ANOSIMs; P < 0.0001). These differences were due to higher relative abundance of sequences from the

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

Diazotroph communities associated with the coral Acropora millepora families Bacilli, Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Cyanobacteria, along with the lower relative abundance of Gammaproteobacteria at Trunk Reef in comparison with Cattle Bay. Recent studies have also found that bacterial communities associated with corals of the same species varied among different locations (Hong et al., 2009; Littman et al., 2009; Kvennefors et al., 2010; Lee et al., 2012). Similarly, bacterial communities associated with A. millepora differed significantly between two nearby inshore sites (Orpheus Island and Magnetic Island), although communities were stable across winter and summer samples (Littman et al., 2009). Differences in nutrient loads between the inshore and mid-shelf sites may influence overall diversity of coral-associated bacterial communities, but there was no apparent seasonal change in the bacterial diversity of corals at the inshore Cattle Bay site that could be linked to high nutrient loads delivered by rivers during summer wet seasons. Therefore, differences in the overall bacterial community diversity at the inshore and mid-shelf reefs may be caused by alternative factors, including other water quality or environmental parameters. A priority for future studies should be to identify environmental variables contributing to these shifts in coral bacterial communities and to determine how they influence health of the coral host.

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2009 (spring), 8 March 2010 (summer), 27 May 2010 (autumn) and 28 August 2010 (winter). Three colonies were located in Cattle Bay (18°34′347″S, 146°29′004″E), an inshore fringing reef in the Palm Island group, and the other three were located at Trunk Reef (18°22′233″ S, 146°46′505″E), a mid-shelf platform reef in the central GBR, Australia (Fig. 4). The inshore site is often exposed to nutrient-enriched river run-off from the Herbert and the Burdekin rivers (an intensive cane sugar agricultural region (Brodie et al., 2012), especially during summer when tropical cyclones and monsoonal rainfall occur that can cause flood events carrying high nutrient loads. The average particulate nitrogen concentrations increase by approximately 30% between dry and wet seasons, and also across mid-shelf to inshore reefs [average calculated over the 6 and 10 years monitoring programs, respectively, in the Burdekin region on the GBR; see supplementary Tables S1 and S2 (De’ath et al., 2009; Schaffelke et al., 2012)]. All colonies sampled were apparently healthy (i.e. no signs of physical damage, bleaching or disease), located on the reef flat at a similar depth (2–4 m) and were separated from other colonies at the same reef by 5−10 m. Random coral branches, 6–8 cm in length, were collected from the centre of each colony and placed in plastic bags underwater. At the surface, branches were thoroughly rinsed with autoclaved artificial seawater (ASW) to remove loosely associated bacteria, placed in new sterile plastic bags and frozen in liquid nitrogen for transport. Samples were kept at −80°C until DNA extraction.

DNA extraction and amplicon tag pyrosequencing Conclusions

Experimental procedures

Frozen coral branches were placed in separate sterile falcon tubes and autoclaved ASW added to cover the length of each coral branch. Falcon tubes were vortexed at maximum speed for approximately 10 min until all tissues were removed from branch skeletons. Skeletons were removed and the tissue slurry was then homogenized, centrifuged at 13 000 r.p.m. and the supernatant decanted. Total DNA was extracted from the pelleted coral tissue using the PowerPlant DNA extraction kit (MoBio Laboratories, Carlsbad, CA, USA) as per the manufacturer’s instructions. Extracted DNA was quantified using the GeneQuant Pro spectrophotometer (Amersham Pharmacia Biotech, Amersham, Bucks, UK) and aliquoted to the same concentration (20 ng μl−1) for all samples (n = 24 samples in total; three replicates for each colony at each season). Aliquots were dried and sent to the Research and Testing Laboratory (Lubbock, TX, USA) for gene-specific amplicon pyrosequencing. Two genes were amplified for each sample, targeting: (i) diazotrophic bacterial populations identified by the variable region (360 bp) of the nitrogenase Fe protein gene (nifH ) (Zehr and McReynolds, 1989) (primers: mnifHF ‘TGYGAYCCNAARGCNGA’, mnifHR‘ADNGCCATC ATYTCNCC’); and (ii) the general bacterial community identified by the variable region V1–3 of the 16S rRNA gene (universal Eubacterial primers: 28F‘GAGTTTGATCNTGG CTCAG’, 519R ‘GTNTTACNGCGGCKGCTG’).

Sampling collection and processing

Sequence analyses

Six healthy colonies of the coral A. millepora were tagged and sampled every 3–4 months for 1 year: on 16 November

Analyses of both 16S rRNA and nifH gene tag-sequencing data were performed using MOTHUR [version v.1.28.0.;

This study reveals that diazotrophs represent a small but consistent member of the microbiome of the coral A. millepora and identifies rhizobial species related to Bradyrhizobia sp. as the dominant group likely to play a functionally significant role in the coral holobiont. Results of our amplicon sequencing approach, targeting both the general bacterial 16S rRNA and nifH genes, are supported by those of our previous study based on nifH gene clone libraries. Interestingly, no shifts in diazotrophic assemblages were observed between an inshore, high nutrient fringing reef and a mid-shelf, oligotrophic reef throughout seasonal sampling over 1 year. In contrast, the overall microbial community associated with corals differed between the two sites, although no shifts were detected with season. Our results highlight that, although overall bacterial communities associated with corals are diverse and dynamic, bacteria with a specific functional role, such as nitrogen fixation, are consistent and can be characterized through sequencing of functional genes.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology

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K. A. Lema, B. L. Willis and D. G. Bourne

Department of Microbiology and Immunology, The University of Michigan (http://www.mothur.org/)], following the pipeline and recommendations from Schloss and colleagues (2011). For both amplified and sequenced genes, initial barcoding, primer removal and quality filtering were done using the ‘trim.seqs’ command, which removed short (< 200 bp) and poor (< 25) sequences (Huse et al., 2007). In MOTHUR, 16S rRNA gene sequences were aligned, using the SILVA (v1.08) database (Pruesse et al., 2007) as a reference alignment. ‘Pre.cluster (diffs = 2)’ was used for further error reduction, and UCHIME (Edgar et al., 2011) ‘chimera.uchime’ was used for de novo removal of chimeric reads. Screening for contaminant sequences (mitochondria, chloroplasts and Eukarya) was applied using the ‘remove.lineage’ command. Chimera-free and error-free sequences were built into a pairwise distance matrix through the ‘dist.seqs’ and ‘cluster’ commands. To compare bacterial diversity among coral samples, sequences were then randomly subsampled (1000 permutations, 350 sequences per sample) and assigned to OTUs at the 97% similarity level through clustering. Where possible, OTUs were identified to genus using the SILVA reference taxonomy. Species-level identifications for the 20 most abundant OTUs were assigned based on GenBank searches using the online BLASTN function (Altschul et al., 1997). DNA nifH sequences were analysed using the MOTHUR pipeline for initial alignment and for chimera and error checks, similarly to 16S rRNA sequences processing described above. A reference alignment was built using sequences from the ARB nifH database [Marine Microbiology, University of California (http://www.es.ucsc.edu/∼wwwzehr/research/ database)], and our sequences aligned against this reference alignment (1000 sequences). Chimeric sequences were identified and removed using PERSEUS (‘chimera.perseus’) (Quince et al., 2011). Cleaned unique sequences were then translated into amino acids (120 bp) in MEGA (v.5, Tamura et al., 2011). Sequences having in-frame stop codon(s) were manually removed. Error-free sequences were then randomly subsampled (1000 permutations, 550 sequences) to enable comparisons of diazotroph diversity among coral samples. Protein sequences were then aligned and built into a Phylip formatted distance matrix using the CLUSTAL OMEGA (v 1.0.3, Sievers et al., 2011) source code pipeline. The distance matrix was analysed in MOTHUR to cluster sequences into OPUs at the 90% similarity level (Zher et al., 1998; Lema et al., 2012). The 20 most abundant OPUs (which represented 85% of the total sequences) were checked against the closest related sequence in GenBank (http://www.ncbi .nlm.nih.gov/GenBank/index.html) using the online BLASTP function. A neighbour-joining nifH phylogenetic tree was built, including bootstrap support of 1000 replicates (in MEGA, v.5), and incorporating sequences from previous nifH studies (Olson et al., 2009; Lema et al., 2012) for further taxonomic assignment. The 20 most abundant 16S rRNA and nifH gene sequences from the representative OTUs and OPUs identified in this study have been deposited in GenBank under nucleotide accession numbers (KF111249 to KF111268) for 16S rRNA sequences and under protein accession numbers (KF307331-KF307350) for nifH gene sequences. The raw amplicon sequence datasets for both genes have been

uploaded to the short read archive under bioproject accession SRP026283.

Statistical analysis Alpha and beta diversity statistics were calculated using the sets of random subsamples to ensure that sequencing effort did not affect the diversity values calculated, and analysed using MOTHUR (v.1.28.0), for both DNA 16S rRNA and protein nifH gene sequences. Total observed and predicted Chao1 (Chao, 1984) species (OTUs and OPUs) were calculated and plotted. To identify patterns emerging from beta diversity community structure, a Phylip-formatted distance matrix, which describes dissimilarity among multiple groups, was generated using the Bray–Curtis similarity coefficient. Changes in community structure were then visualized through a PCoA, and ANOSIMs, based on 10 000 permutations, was used to determine if spatial separation between reefs and seasons was statistically significant.

Acknowledgments We thank Geoff Millar for his kind help with installation and operation of MOTHUR. The Australian Institute of Marine Sciences (AIMS), AIMS@JCU and the ARC Centre of Excellence for Coral Reef (James Cook University) are thanked for their financial contributions to this research. Finally Consejo Nacional de Ciencia y Tecnología, Mexico (CONACYT) (Mexico) is thanked for giving financial support to A. K. Lema.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Alpha diversity plot: number of operational taxonomic units observed and predicted (Chao1) for protein

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nifH (90% sequence identity cut-off) (OPUs) and 16S rRNA (97% sequence identity cut-off) (OTUs) in colonies of A. millepora (1, 2, 3, 4, 5 and 6) from Cattle Bay (inshore) and Trunk Reef (mid-shelf) sampled through four seasons of 1 year. Fig. S2. Principal coordinates analysis (PCoA) plot of nifH gene protein sequences (90% sequence identity cut-off) from colonies of A. millepora (1, 2, 3, 4, 5 and 6) from Cattle Bay (Inshore) and Trunk Reef (mid-shelf) sampled through four seasons of 1 year. The 10 most abundant OPUs (nifH operational taxonomic units) are represented by vectors and represent differences among samples. Table S1. Summary statistics of direct water sampling from inshore reefs lagoon sites of Pelorus Island, Burdekin region, Queensland, Australia, from August 2005 to February 2011. Data extracted from the supplementary materials of Schaffelke et al. (2012) and the AIMS GBR water quality monitoring programme report 2011. Total = all sampling occasions; Wet = wet season data (November–April); Dry = dry season data (May–October). N = number of sampling occasions. Fifth and 95th percentiles denote the boundaries in which 90% of the data are found. Data are in μM. The asterisk (*) represents mean values that had a fold increase > 2 between dry and wet seasons. Table S2. Mean annual values of nutrients (μg l−1) at the Burdekin region (GBR, Australia) calculated with data collected from water quality monitoring programmes from 1998 to 2008, across sampling sites from inshore and mid-shelf reefs, including sampling sites from the present study. Data are extracted from the Great Barrier Reef Marine Park Authority water quality report 2008 (De’ath and Fabricius, 2008). The asterisk (*) represents statistical significant differences between inshore and mid-shelf reefs highlighted for these values in De’ath and Fabricius (2008).

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