Bacterial community associated with T. hoshinota

2 Materials and Methods

3.3 Phylogenetic analysis

3.3.2 Bacterial community associated with T. hoshinota

According to previous result that there were cyanobacteria groups specific to T.

hoshinota and P. lutea samples, we constructed the phylogenetic tree of comparing representative cyanobacteria clones from individual OTU groups in this study with sponge-associated, black band disease related Cyanobacteria obtained from other studies (Steindler, 2005 and Frias-Lopez, 2004) (Fig. 25). The result showed that Cyanobacteria from T. hoshinota was different with other sponges-associated Cyanobacteria and had individual clade. This clade was more close to group V (Chroococcales, Oscillatoriales, Pleurocapsales, and Prochlorales) than group II consisting of four sequences from Dysidea herbacea, Dysidea granulose, Lendenfeldia dendyi, and Aplysina gerardogreeni, originating from Guam, the Mexican Pacific, and Zanzibar. Cyanobacteria related black band disease was close to group II because both of them belonged to filamentous Cyanobacteria. Furthermore, NSI P. lutea associated Cyanobacteria (PH_OTU12, 13) appeared being unrelated to any major group of Cyanobacteria. These sequences might not belong to Cyanobacteria or a new cyanobacterial group.

In PM clone library (Table 12), 76% clones were related (96% similarity) to uncultured Prochloron sp. clone NA-108 which was obligate symbiont in Didemnid

ascidians associated with marine invertebrate. A few other clones were related uncultured bacterium from marine environment.

Table 9 16S rRNA gene sequence analysis of bacterial clones from the non-sponge-infected Porites lutea, based on BLAST analysis

Nearest relative Accession

number Reference

Sequence similarity

(%)

Bacterial group

PH_OTU1 1 872 Uncultured sponge symbiont PAUC38fp AF186414 Sponge Theonella 99% Actinobacteria PH_OTU2 1 810 Uncultured Actinobacterium clone CN81 AM259924 Mediterranean Sponge Chondrilla

nucula 98% Actinobacteria

PH_OTU3 1 789 Uncultured Alphaproteobacterium clone

CD204C11 DQ200432 Caribbean coral Montastraea

annularis 97% Alphaproteobacteria

PH_OTU4 1 820 Roseobacter sp. SPO804 DQ993342 Caribbean coral Montastraea

annularis 99% Alphaproteobacteria

PH_OTU5 2 878 Uncultured bacterium clone B8S-114 EU652589 Marine environment 95% Alphaproteobacteria PH_OTU6 8 848 Uncultured bacterium clone w3uc5 DQ416510 Marine environment 81% Alphaproteobacteria PH_OTU7 1 817 Uncultured bacterium clone DC-3 AY529881 Disease outbreak on corals from

Magnetic Island 92% Alphaproteobacteria PH_OTU8 30 833 Uncultured Gammaproteobacterium clone

CD207G01 DQ200629 Caribbean coral Montastraea

annularis 97% Gammaproteobacteria

PH_OTU9 1 760 Uncultured Bacteroidetes bacterium clone

MPCa6_D05 EF414089 Sponge Microciona

prolifera,Chesapeake Bay 90% Bacteroidetes PH_OTU10 1 790 Microscilla marina strain:IFO 16560 AB078080 Marine environment 95% Bacteroidetes PH_OTU11 19 796 Uncultured Candidate division CAB-I clone

CD205F11 DQ200556 Caribbean coral Montastraea

annularis 98% Candidate division CAB-I PH_OTU12 1 813 Uncultured Cyanobacterium clone DPC025 DQ269091 Marine eukaryotes 93% Cyanobacteria

PH_OTU13 15 859 Uncultured cyanobacterium clone DPC164 DQ269106 Marine eukaryotes 92% Cyanobacteria

PH_OTU14 3 826 Mycoplasma cottewii strain VIS U67945 Goat 81% Firmicutes

PH_OTU15 14 830 Uncultured phototrophic eukaryote clone

CD205F08 DQ200553 Marine environment 88% Plastid uncultured

phototrophic eukaryote

Table 10 16S rRNA gene sequence analysis of bacterial clones from the sponge-infected Porites lutea (far-away sponge, P

H), based on BLAST analysis

Nearest relative Accession

number Reference Sequence

similarity (%) Bacterial group P^dH_OTU1 1 739 Uncultured Alphaproteobacterium clone

BBD216b_13f EF123418 Black band disease microbial mats on

Caribbean Siderastrea siderea 94% Alphaproteobacteria P^dH_OTU2 1 821 Uncultured bacterium clone DC-3 AY529881 Disease outbreak on corals from

Magnetic Island 91% Alphaproteobacteria P^dH_OTU3 1 829 Rhodobacteraceae bacterium MOLA 442 AM990709 Marine environment 99% Alphaproteobacteria

P^dH_OTU4 53 839 Uncultured Gammaproteobacterium clone

CD207G01 DQ200629 Caribbean coral Montastraea annularis 97% Gammaproteobacteria P^dH_OTU5 1 720 Uncultured Gammaproteobacterium clone

CD204G09 DQ200474 Caribbean coral Montastraea annularis 98% Gammaproteobacteria

P^dH_OTU6 1 729 Acinetobacter sp. MUB1 AY273199 Marine environment 93% Gammaproteobacteria

P^dH_OTU7 1 829 Uncultured bacterium clone A8S-109 EU652550 Marine environment 93% Gammaproteobacteria P^dH_OTU8 5 820 Uncultured Gammaproteobacterium clone

Ax29_A1 EF092194 Marine environment 94% Gammaproteobacteria

P^dH_OTU9 2 839 Uncultured Gammaproteobacterium clone

CD207F03 DQ200619 Caribbean coral Montastraea annularis 95% Gammaproteobacteria P^dH_OTU10 1 839 Uncultured bacterium clone P7X3b1C05 EU491022 Marine environment 93% Gammaproteobacteria

P^dH_OTU11 1 779 Bdellovibrio sp. JS5 AF084859 Coastal Marsh 91% Deltaproteobacteria

P^dH_OTU12 1 794 Uncultured Deltaproteobacterium clone

GASP-WC2W3_E07 EF075373 Marine environment 92% Deltaproteobacteria P^dH_OTU13 3 856 Uncultured Bacteroidetes bacterium clone:

pKB3B-27 AB247902 Hydrothermal vents in Kermadec

Volcanic Arc 84% Bacteroidetes

P^dH_OTU14 1 849 Uncultured Bacteroidetes bacterium clone

MPWIC_G09 EF414162 Sponge Microciona prolifera from the

Chesapeake Bay 95% Bacteroidetes P^dH_OTU15 1 821 Uncultured Cytophagaceae bacterium clone

1-13 AY094494 Marine environment 90% Sphingobacteria

P^dH_OTU16 1 822 Uncultured Flexibacteraceae bacterium

clone EC206 DQ889876 Octocoral Erythropodium caribaeorum 90% Sphingobacteria P^dH_OTU17 19 788 Uncultured candidate division CAB-I clone

CD205F11 DQ200556 Caribbean coral Montastraea annularis 98% Candidate division CAB-I P^dH_OTU18 4 749 Uncultured phototrophic eukaryote clone

CD205F08 DQ200553 Marine environment 88%-91% Plastid uncultured phototrophic eukaryote

Table 11 16S rRNA gene sequence analysis of bacterial clones from the sponge-infected Porites lutea (close-to sponge, P

I), based on BLAST analysis

Nearest relative Accession

number

Reference Sequence similarity

(%)

Bacterial group

P^dI_OTU1 1 850 Uncultured Actinobacterium pItb-RF-5 AB294984 Marine environment 96% Actinobacteria P^dI _OTU2 2 878 Uncultured Roseobacter AC2_A2 EF092158 Marine environment 99% Alphaproteobacteria P^dI _OTU3 1 828 Uncultured Ruegeria clone BME18 DQ917819 Bleached and wild-type Muricea elongata 99% Alphaproteobacteria

P^dI _OTU4 3 799 Uncultured Alphaproteobacterium BBD_HS216b_07

DQ644016 Marine environment 99% Alphaproteobacteria

P^dI _OTU5 1 840 Alphaproteobacterium MOLA 143 AM990917 Coastal NW Mediterranean ecosystem 93% Alphaproteobacteria P^dI _OTU6 1 782 Uncultured Alphaproteobacterium LC1-35 DQ289905 Permeable shelf sediments 98% Alphaproteobacteria P^dI _OTU7 3 828 Uncultured Alphaproteobacterium

CD204H01

DQ200478 Caribbean coral Montastraea annularis 97% Alphaproteobacteria

P^dI _OTU8 2 806 Alphaproteobacterium Y3F AF253467 Marine Roseobacter 96% Alphaproteobacteria P^dI _OTU9 1 825 Rhodobacteraceae bacterium DG1297 DQ486508 Marine environment 95% Alphaproteobacteria P^dI _OTU10 1 858 Pseudoruegeria aquimaris SW-255 DQ675021 Isolated from seawater of the East Sea in

Korea

95% Alphaproteobacteria

P^dI _OTU11 1 879 Uncultured Alphaproteobacterium BBD_STXLK_42b

DQ644018 Production of cyanobacterial toxin Microcystin in black band disease of corals

96% Alphaproteobacteria

P^dI _OTU12 60 837 Uncultured Gammaproteobacterium CD207G01

DQ200629 Caribbean coral Montastraea annularis 98% Gammaproteobacteria

P^dI _OTU13 1 865 Uncultured Gammaproteobacterium Ax29_A1

EF092194 Marine environment 94% Gammaproteobacteria

P^dI _OTU14 1 835 Uncultured Gammaproteobacterium CD207F03

DQ200619 Caribbean coral Montastraea annularis 94% Gammaproteobacteria

P^dI _OTU15 1 825 Cycloclasticus sp. E AF093003 Marine environment 86% Gammaproteobacteria

P^dI _OTU16 1 844 Gammaproteobacterium L193 AY371439 Cultured microbial associates of deep-water marine invertebrates

98% Gammaproteobacteria

P^dI _OTU17 1 811 Uncultured Gammaproteobacterium SARG_3

AM238545 Marine dimethylsulfide-consuming bacteria

97% Gammaproteobacteria

P^dI _OTU18 1 860 Uncultured bacterium PDA-OTU12 AY700599 Coral Pocillopora damicornis from the Great Barrier Reef

97% Gammaproteobacteria

P^dI _OTU19 1 855 Uncultured Bacteroidetes MPWIC_G09 EF414162 Sponge Microciona prolifera from the Chesapeake Bay

96% Bacteroidetes

P^dI _OTU20 1 833 Uncultured Flexibacteraceae EC206 DQ889876 Octocoral Erythropodium caribaeorum 90% Bacteroidetes P^dI _OTU21 1 778 Mucus bacterium 86 AY654823 Culturable bacteria from the mucus of

Oculina patagonica

94% Bacteroidetes

P^dI _OTU22 1 876 Uncultured Cytophaga JTB248 AB015263 Sediments of Deepest Cold-Seep Area, the Japan

86% Bacteroidetes

P^dI _OTU23 1 853 Uncultured Bacteroidetes IRD18C04 AY947919 Marine environment 90% Bacteroidetes P^dI _OTU24 1 795 Uncultured candidate division CAB-I

CD205F11

DQ200556 Caribbean coral Montastraea annularis 98% Candidate division CAB-I

P^dI _OTU25 1 820 Leptolyngbya sp. HBC3 EU249121 Cyanobacterial Diversity of a Marine Stromatolite

93% Cyanobacteria

P^dI _OTU26 1 838 Uncultured bacterium PoritesC32clC05 EU636623 Marine environment 99% Lentisphaerales P^dI _OTU27 6 847 Uncultured phototrophic eukaryote clone

CD205F08

DQ200553 Marine environment 88% Plastid uncultured phototrophic eukaryote

Table 12 16S rRNA gene sequence analysis of bacterial clones from Terpios hoshinota (infected Porites lutea), based on BLAST analysis

Nearest relative Accession number

Reference Sequence similarity (%)

Bacterial group

PM_OTU1 2 779 Bacterium s1cb31 DQ416551 Mucus and Tissues of the Coral Oculina patagonica

99% Alphaproteobacterium

PM_OTU2 9 795 Roseobacter sp. Y2 DQ120728 Marine environment 94% Alphaproteobacterium PM_OTU3 2 749 Uncultured bacterium clone

D8S-106

EU652584 Yellow Sea sediment 96% Alphaproteobacterium

PM_OTU4 4 739 Uncultured bacterium clone 1-52 EF040508 Associated with Halichondria sp. 88% Gammaproteobacterium

PM_OTU5 2 802 Uncultured Proteobacterium clone JL-ESNP-H53

AY664193 Eastern subtropical North Pacific 93% Gammaproteobacterium

PM_OTU6 2 799 Uncultured Bacteroidetes bacterium clone: pItb-RF-14

AB294988 Shallow Submarine Hydrothermal System

99% Bacteroidetes

PM_OTU7 76 779 Uncultured Prochloron sp. clone NA-108

DQ357945 Obligate symbiont in didemnid ascidians

96% Cyanobacteria

PM_OTU8 2 780 Uncultured Cyanobacterium clone BB31NT16S-12

EF089520 Black band disease in Red Sea stony corals

90% Cyanobacteria

Fig. 25 Maximum parsimony tree of sponge-associated and black band disease related cyanobacteria based on 16S rRNA.

Bootstrap values above 50% are indicated. Sponge-associated cyanobacteria (which were named following their host species and sampling localities: AU, Australia; BH, Bahamas; GU, Guam; MS, Mediterranean Sea; MX, Mexican Pacific; ZZ, Zanzibar) are indicated in bold. Black band disease related cyanobacteria are indicated using underline. Sequences form this work are indicated by asterisk. The group numbers follow Steindler et al..

VI

VII IV III II VIII

II

V

SW OTU4 Microscilla aggregans catalatica SW OTU14 SW OTU13 PDI OTU23 SW OTU17 Uncultured Bacteroidetes FNE11-29 SW OTU9 Uncultured Bacteroidetes DPC237 PDI OTU22 Uncultured Cytophaga JTB248 Uncultured marine KG_A3_120m182 PDH OTU16

Uncultured candidate division CAB-I CD204B02 PDH OTU17

PM OTU7

Uncultured Prochloron sp. clone NA-108 PM OTU8

Uncultured phototrophic eukaryote clone Cobs2TisH11 PH OTU13

Uncultured delta proteobacterium AKYH967 Chondromyces crocatus PDH OTU11 Bdellovibrio sp. JS3

de lta prote oba c te ria Mycoplasma cottewii strain VIS PDI OTU26

Uncultured Lentisphaera sp. clone EC115 SW OTU3

Uncultured alpha proteobacterium LC1-34

alpha proteobacterium endosymbiont 1b of Inanidrilus makropetalos PDI OTU5

SW OTU6 Erythrobacter sp. JL-378 PDI OTU6

Uncultured Hyphomicrobium JdFBGBact_73 PDI OTU7

Uncultured Phaeobacter Sc70 SW OTU12 Paracoccus sp. MBIC3966 PH OTU4 Mucus bacterium 116 PM OTU1 Mucus bacterium 103 PDH OTU1 alpha ProteobacteriumTH1 Roseobacter sp. TM1038 PDI OTU8 Arctic sea ice ARK9994 SW OTU10 Sulfitobacter sp. BSi20563

Alpha prote oba c te ria

PH OTU6

Carsonella ruddii natural-host Panisopelma PDH OTU8

PDI OTU13

Endosymbiont of Bugula turbinata isolate DP PDH OTU10

PDI OTU16 Amphritea japonica JAMM 1866 PDH OTU6 Acinetobacter calcoaceticus PDH OTU7 PM OTU4

Bacterial community associated with T. hoshinota

2 Materials and Methods

3.3 Phylogenetic analysis

3.3.2 Bacterial community associated with T. hoshinota

Table 9 16S rRNA gene sequence analysis of bacterial clones from the non-sponge-infected Porites lutea, based on BLAST analysis

Table 10 16S rRNA gene sequence analysis of bacterial clones from the sponge-infected Porites lutea (far-away sponge, P

H), based on BLAST analysis

Table 11 16S rRNA gene sequence analysis of bacterial clones from the sponge-infected Porites lutea (close-to sponge, P

I), based on BLAST analysis

Table 12 16S rRNA gene sequence analysis of bacterial clones from Terpios hoshinota (infected Porites lutea), based on BLAST analysis

Fig. 25 Maximum parsimony tree of sponge-associated and black band disease related cyanobacteria based on 16S rRNA.

VI

VII IV III II VIII

II

V

Fig. 26 Neighbor-Joining tree of Porites lutea

-associated and seawater related bacteria based on

16S rRNA Bootstrap values above 50% are

indicated. The labels SW, Chai-Kou sample. KW,

Kun-Guan sample. PH, non sponge-infected Porites

lutea colonies. P

H, sponge-infected Porites lutea

colonies (far-away sponge). P

I, sponge-infected

Porites lutea colonies (close-to sponge).