617 Bacterial diversity and biogeography of the cold-water gorgonian Primnoa resedaeformis in Norfolk and Baltimore canyons Christina A. Kellogg* and Michael A. Gray U.S. Geological Survey, St. Petersburg Coastal and Marine Science Center, St. Petersburg, Florida 33701, USA Abstract Much attention has been paid to the bacterial associates of the cold-water reef- forming scleractinian coral, Lophelia pertusa. However, many other cold-water coral species remain microbiological terra incognita, including the locally abundant, habitat-forming Atlantic octocoral Primnoa resedaeformis. During research cruises in 2012 and 2013, we collected samples from 10 individual coral colonies in each of Baltimore and Norfolk Canyons in the western Atlantic Ocean. DNA was extracted from each sample and the V4-V5 variable regions of the 16S ribosomal RNA gene were amplified and then subjected to 454 pyrosequencing. Samples were dominated by Proteobacteria followed by smaller amounts of Firmicutes, Planctomycetes, Bacteroidetes and Actinobacteria. Bacterial community sequences were found to cluster based on submarine canyon of origin. Norfolk Canyon bacterial communities had much higher representation of Gammaproteobacteria, particularly the family Moraxellaceae, compared to Baltimore Canyon communities that were dominated by Alphaproteobacteria, particularly the family Kiloniellaceae. These data provide a first Figure 2. Primnoa resedaeformis, a cold-water octocoral Figure 3. A non-metric multidimensional scaling (NMDS) plot comparing the look at the biogeographic structuring of the bacterial associates of P. resedaeformis bacterial communities of Primnoa resedaeformis from Baltimore and Norfolk likely caused by physical barriers to dispersal imposed by submarine canyons. Canyons to those of P. pacifica collected in the Gulf of Alaska (Pacific Ocean). 1 0.9 0.8 Unclassified;Other;Other;Other;Other Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;Rubritaleaceae Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae 0.7 Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae 0.6 Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Pseudoalteromonadaceae Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Colwelliaceae Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae 0.5 Figure 1. Map showing location of Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Methylobacteriaceae Baltimore and Norfolk Canyons in Bacteria;Proteobacteria;Alphaproteobacteria;Other;Other relation to the rest of the United States 0.4 Bacteria;Planctomycetes;Planctomycetacia;Planctomycetales;Planctomycetaceae of America. Bacteria;Other;Other;Other;Other Bacteria;Firmicutes;Clostridia;Other;Other 0.3 Bacteria;Firmicutes;Clostridia;Clostridiales;Incertae Sedis XI Table 1. Primnoa sample locations and environmental parameters. Note that Atlantic samples are Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae P. resedaeformis and Pacific samples are P. pacifica. Samples of P. pacifica were collected by Bacteria;Chlamydiae;Chlamydiae;Chlamydiales;Simkaniaceae collaborators Bob Stone and Rhian Waller using SCUBA. 0.2 Bacteria;Chlamydiae;Chlamydiae;Chlamydiales;Parachlamydiaceae Bacteria;Bacteroidetes;Other;Other;Other Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae Sample Year Collection Location Ocean Basin Temp (°C) Depth (m) Salinity (psu) 0.1 Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Propionibacteriaceae NF12-9Q7 2012 Baltimore Canyon Atlantic 7.3 494 35.1 NF12-10Q6 2012 Baltimore Canyon Atlantic 7.6 500 35.1 0 NF12-9Q6 2012 Baltimore Canyon Atlantic 7.3 506 35.1 NF12-5Q6 2012 Baltimore Canyon Atlantic 7.4 443 35.1 NF12-5Q7 2012 Baltimore Canyon Atlantic 7.4 443 35.1 NF12-6Q6 2012 Baltimore Canyon Atlantic 7.5 430 35 NF12-1Q6 2012 Baltimore Canyon Atlantic 6.2 450 35.1 Figure 4. Relative diversity of bacterial families associated with Primnoa samples. Only families that comprise 3% or more of at least one sample are shown. Families below 3% are summed under the NF12-2Q6 2012 Baltimore Canyon Atlantic 9 450 35.2 category “Bacteria; Other; Other; Other; Other”. NF12-6Q7 2012 Baltimore Canyon Atlantic 7.5 431 34.9 NF12-12Q6 2012 Norfolk Canyon Atlantic 6.2 535 35 Discussion NF12-12Q7 2012 Norfolk Canyon Atlantic 6.6 523 35.1 ♦ This is a first look at the bacterial communities associated with cold-water corals of the genusPrimnoa. NF12-20Q1 2012 Norfolk Canyon Atlantic 6.3 434 35 RB684Q1 2013 Norfolk Canyon Atlantic 10.79 411 35.47 ♦ The bacterial communities on Baltimore and Norfolk Canyon populations of Primnoa resedaeformis are distinguishable from each other to the same RB684Q2 2013 Norfolk Canyon Atlantic 9 441 35.21 degree that bacterial communities from P. resedaeformis are distinct from those of another species in a different ocean (P. pacifica). RB684Q3 2013 Norfolk Canyon Atlantic 9 441 35.27 ♦ Many P. pacifica samples were dominated by Parachlamydiaceae sequences and one Norfolk Canyon sample was dominated by Simkaniaceae sequences. RB684Q4 2013 Norfolk Canyon Atlantic 6.32 489 35.05 These families are obligate intracellular bacteria of amoebae. Do these associations suggest an infestation of certain gorgonian coral individuals with RB684Q5 2013 Norfolk Canyon Atlantic 6.32 498 35.05 amoebae? Or are our primers just better at picking up this bacterial group when it is present? RB686Q3 2013 Norfolk Canyon Atlantic 6.58 479 35.06 RB687Q2 2013 Norfolk Canyon Atlantic 5.55 576 35.02 ♦ Baltimore Canyon bacterial communities were characterized by having larger numbers of Alphaproteobacterial sequences, the family Xanthomonadaceae AK325 2011 Gulf of Alaska Pacific 4.6 12.8 26.86 (not found in any Norfolk Canyon samples), and more Bacilliaceae sequences. Sequences previously identified as Kiloniellaceae were discarded due to AK342 2011 Gulf of Alaska Pacific 4.6 12.5 26.86 being chimeras or singletons. AKUT1 2011 Gulf of Alaska Pacific 4.6 13.4 26.86 ♦ Norfolk Canyon bacterial communities were characterized by having higher numbers of Moraxellaceae sequences (although also present in Baltimore AKPP1 2012 Gulf of Alaska Pacific 5.02 9.8 30.14 Canyon samples) and Pseudoalteromonadaceae were present in several Norfolk samples but none of the other locations. AKPP2 2012 Gulf of Alaska Pacific 5.02 13.1 30.14 AKPP3 2012 Gulf of Alaska Pacific 5.02 11.6 30.14 AKPP4 2012 Gulf of Alaska Pacific 5.02 16.2 30.14 U.S. Department of the Interior February 2014 U.S. Geological Survey.