Sponge prokaryote communities in Taiwanese coral reef and shallow hydrothermal vent ecosystems
Coelho FJRC1, Cleary DFR1, Gomes NCM1, ARM Pólonia1*, YM Huang2, 4, L-L Liu3, NJ de Voogd4
1Department of Biology & CESAM, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
2Department of Marine Recreation, University of Science and Technology, Penghu, Taiwan
3Department of Oceanography, National Sun Yet-Sen University, Kaohsiung, Taiwan
4Naturalis Biodiversity Center, Leiden, the Netherlands
* Present address: Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal
Predictive gene enrichment
To predict the metagenome of each sample, we used PICRUSt (http://picrust.github.com/picrust/ - Version 1.0) [1]. The script can be used to predict the metagenome. Detail explanations can be found in http://picrust.github.io/picrust/tutorials/metagenome_prediction.html#metagenome-prediction-tutorial.
#Pick OTUs for use in PICRUSt (QIIME)
echo "pick_otus:enable_rev_strand_match True" > $PWD/otu_picking_params_97.txt
echo "pick_otus:similarity 0.97" > $PWD/otu_picking_params_97.txt
pick_closed_reference_otus.py -i $PWD/seqs.fna -o $PWD/ucrC97/ -p $PWD/otu_picking_params_97.txt -r $PWD/gg_13_5_otus/rep_set/97_otus.fasta -t $PWD/gg_13_5_otus/taxonomy/97_otu_taxonomy.txt
#Normalize OTU table
normalize_by_copy_number.py
-i otu_table.biom
-o normalized_otus.biom
#Predict function for metagenome
predict_metagenomes.py
-i normalized_otus.biom
-o metagenome_predictions.biom
-a nsti_per_sample.tab
# Collapse predictions into pathways (third level)
Categorize_by_function.py -i metagenome_predictions.biom -c KEGG_Pathways -l 3 -o predicted_metagenomes.L3.biom
BLAST analysis
Sequence Identifiers of closely related taxa of selected OTUs (≥ 1500 sequences) were downloaded using the NCBI Basic Local Alignment Search Tool (BLAST) command line 'blastn' tool with the -db argument set to nt [2]. BLAST identifies locally similar regions between sequences, compares sequences to extant databases and assesses the significance of matches; functional and evolutionary relationships can subsequently be inferred. Each run produces a list of hits based on significant similarity between pairs of sequences, i.e., the target sequence and taxa present in the database (or no hits if no significantly similar sequences are found). A discussion of how significance is determined can be found at http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html.
We used the blastn command line tool in a Linux environment to query representative sequences of selected taxa including all of the most abundant prokaryote (≥ 1500 sequences) OTUs against the online NCBI nucleotide database. We then generated vectors containing sequence identifiers (GI's) of the ten top hits of all representative sequences and used the Entrez.efetch function in BioPython [3] with the rettype argument set to 'gb' to download genbank information of aforementioned top hits including the isolation source of the organism and the host if relevant.
Figure S1: Phylogenetic tree of the Alphaprotobacterial 16S rRNA gene sequences recovered from coral reef and hydrothermal vent habitat. 16S rRNA gene reference sequences of related culture strains representatives (Nisaea nitritireducens, Rhodospirillum rubrum strain and Pelagibacter ubique) were included. Rhizobium ciceri and Pelagibacter unique were also included as representatives of Rhizobiales and Rickettsiales order. Sequences assigned to Kiloniellales from Cleary et al. (2013) and sequences assigned to Alphaproteobacteria from were also included Weigel and Erwin (2016). The number of each OTU and classification at order level is indicated as are GenInfo sequence identifiers of sequences obtained using BLAST. For organisms found using BLAST, we include the host and/or habitat from which the organism was isolated as well as the geographical locality where the organism was isolated.
Figure S2: Ordination showing the first two axes of the principal coordinates analysis (PCO) of KEGG orthologs (KOs) predictions using 16S data with PICRUSt. Only sponges with a minimum of three replicates were analysed. Aa - Acanthella cavernosa, Ea - Echinodictyum asperum, Js - Jaspis splendens, Ps - Ptilocaulis sp., and Hy - Hymeniacidon sp. and Wt – water.
hylogenetic tree of the bacterial 16S
rRNA gene sequenc es recovered from
Suberites diversicolor and Cinachyrella
australiensis hosts in lake Kakaban, Haji Buang
lake, Maratua and open habitat; bootstrap
values lower than 50% were omitted. The
number of each OTU is indicated as are
GenInfo sequence identifiers of sequences
obtained using
BLAST. Classes of bacteria are
indicated. OTUs are assigned to the following
clusters: Sub: found in Suberites diversicolor
hosts, Cin-Opn: found in C. australiensis hosts
in open habitat, Cin-Lak: found in
C. australiensis hosts in lake habitat, Ubq:
ubiquitous, found in all host individuals. For
organisms found using BLAST, we include the
host and/or habitat from which the organism
was isolated as well as the geographical
locality where the organism was isolated.
Table S1: List of most abundant bacterial OTUs (≥ 1500 sequences). The table includes the OTU-numbers (OTU); taxonomic assignment generated by QIIME; Acession (Acc), sequence similarity of the closest matches with our representative OTU sequences (Seq. Sim.) and the source of these organisms (Source/Context).
OTU / Abund / Phylum / Class / Order / Family / Genus / Acc / Seq. Sim (%) / Source/Context (Sponges genus previously found to include LMA sponges are highlighted in bold) / Reference that identifies sponge genus as LMA1 / 62053 / Proteobacteria / Alphaproteobacteria / Unclassified / Unclassified / Unclassified / GU981955 / 97.64 / Tedania ignis|Bahamas: Sweetings Cay, Mangrove / [5]
3 / 25726 / Proteobacteria / Alphaproteobacteria / Kiloniellales / Unclassified / Unclassified / FJ358860 / 98.12 / Marine reef sandy sediment
5 / 13783 / Cyanobacteria / Synechococcophycideae / Synechococcales / Synechococcaceae / Synechococcus / KT731849 / 100 / surface seawater from Changing estuary
6 / 12833 / Proteobacteria / Deltaproteobacteria / [Entotheonellales] / [Entotheonellaceae] / Unclassified / JF809701 / 95.1 / Medea hypersaline basin, Mediterranean Sea
7 / 12039 / Proteobacteria / Betaproteobacteria / EC94 / Unclassified / Unclassified / HQ241773 / 95.32 / Tsitsikamma favus|South Africa: Alagoa Bay
9 / 12296 / Proteobacteria / Gammaproteobacteria / Chromatiales / Unclassified / Unclassified / GU982100 / 95.98 / Aplysina fulva|Bahamas:Sweetings Cay
10 / 13510 / Cyanobacteria / Synechococcophycideae / Synechococcales / Synechococcaceae / Prochlorococcus / KR857588 / 99.76 / Genomic DNA|Orca Basin North hypersaline sediment
11 / 7336 / Actinobacteria / Acidimicrobiia / Acidimicrobiales / Unclassified / Unclassified / KF597097 / 94.37 / Poecillastra compressa / [6]
12 / 7631 / Proteobacteria / Gammaproteobacteria / Chromatiales / Unclassified / Unclassified / JX455272 / 95.76 / Cymbastella coralliophila|Great Barrier Reef / [7]
14 / 17885 / Proteobacteria / Gammaproteobacteria / Unclassified / Unclassified / Unclassified / JN850859 / 95.11 / Raspailia topsenti|marine sponge from a temperate shelf and sea biome, 8-10m depth / [8]
15 / 7629 / Proteobacteria / Betaproteobacteria / EC94 / Unclassified / Unclassified / KF373187 / 95.98 / Haliclona sp.|coral reef, India Gulf of Mannar / [9]
16 / 7032 / Proteobacteria / Gammaproteobacteria / Chromatiales / Unclassified / Unclassified / KJ007847 / 99.11 / Axinella sp.|China / [10]
19 / 6682 / Proteobacteria / Gammaproteobacteria / Chromatiales / Unclassified / Unclassified / KC925758 / 92.86 / Oujiang river
22 / 4556 / Crenarchaeota / Thaumarchaeota / Cenarchaeales / Cenarchaeaceae / Unclassified / KJ504349 / 99.51 / Mycale_laxissima|Key Largo USA / [11]
23 / 5191 / Spirochaetes / Spirochaetes / Unclassified / Unclassified / Unclassified / HQ241785 / 93.54 / Tsitsikamma favus|South Africa: Algoa Bay
24 / 4530 / Proteobacteria / Gammaproteobacteria / Thiohalorhabdales / Unclassified / Unclassified / HQ877729 / 97.99 / Phakellia fusca|South China sea / [12]
31 / 6799 / Proteobacteria / Alphaproteobacteria / Rickettsiales / Pelagibacteraceae / Unclassified / EU802926 / 99.76 / Northeast of Colon, Panama
33 / 3706 / Actinobacteria / Acidimicrobiia / Acidimicrobiales / OCS155 / Unclassified / KU243315 / 99.76 / Scleractinian coral
39 / 3032 / Nitrospirae / Nitrospira / Nitrospirales / Nitrospiraceae / Unclassified / FJ215390 / 99.54 / Axinella corrugata / [10]
41 / 2906 / Proteobacteria / Betaproteobacteria / EC94 / Unclassified / Unclassified / JQ062689 / 91.54 / Callyspongia vaginalis / [7]
42 / 2474 / Chloroflexi / SAR202 / Unclassified / Unclassified / Unclassified / AY942767 / 97.64 / Cymbastela concentrica / [7]
45 / 2176 / Bacteroidetes / Flavobacteriia / Flavobacteriales / Flavobacteriaceae / Unclassified / KX936618 / 99.77 / surface seawater Changjiang estuary
46 / 1961 / Crenarchaeota / Thaumarchaeota / Cenarchaeales / Cenarchaeaceae / Cenarchaeum / JX262712 / 99.75 / Axinella carteri / [10]
53 / 2729 / Proteobacteria / Betaproteobacteria / EC94 / Unclassified / Unclassified / JN850798 / 92.2 / Raspailia topsenti|marine sponge from a temperate shelf and sea biome, 8-10m depth / [13]
54 / 4179 / Bacteroidetes / Flavobacteriia / Flavobacteriales / Cryomorphaceae / Bacteroidetes / JQ195628 / 99.77 / seawater
60 / 1562 / Bacteroidetes / Flavobacteriia / Flavobacteriales / Flavobacteriaceae / Unclassified / KU937395 / 99.77 / surface seawater from Changjiang estuary and adjacent areas
61 / 2053 / Proteobacteria / Alphaproteobacteria / Unclassified / Unclassified / Unclassified / KU937415 / 100 / North Atlantic sedimentary abyssal plain
62 / 1571 / Proteobacteria / Deltaproteobacteria / NB1-j / NB1-i / Unclassified / JQ062836 / 99.55 / Stylissa cartieri|Saudi Arabia / [13]
83 / 1528 / Proteobacteria / Gammaproteobacteria / Oceanospirillales / Halomonadaceae / Candidatus Portiera / KU578402 / 99.77 / Ocean seawater
88 / 1766 / Proteobacteria / Gammaproteobacteria / Oceanospirillales / Endozoicimonaceae / Unclassified / JN388027 / 98.67 / Ianthella basta
90 / 1886 / Proteobacteria / Alphaproteobacteria / Rhodobacterales / Rhodobacteraceae / Unclassified / KX890262 / 99.76 / water sample from Arabian Sea-Shelf
126 / 2343 / Proteobacteria / Alphaproteobacteria / Rickettsiales / Unclassified / Unclassified / KX427591 / 99.53 / porewater near yellow vent off Kueishan Island
747 / 2070 / Proteobacteria / Alphaproteobacteria / Unclassified / Unclassified / Unclassified / JQ435978 / 98.11 / Seawater Hymeniacidon perlevis
6735 / 14116 / Proteobacteria / Alphaproteobacteria / Unclassified / Unclassified / Unclassified / JQ435978 / 98.35 / Seawater Hymeniacidon perlevis
6896 / 2737 / Proteobacteria / Alphaproteobacteria / Unclassified / Unclassified / Unclassified / KJ007952 / 98.11 / Terpios hoshinota|China
11947 / 3003 / Proteobacteria / Alphaproteobacteria / Unclassified / Unclassified / Unclassified / JQ435978 / 97.64 / Seawater Hymeniacidon perlevis
12777 / 1734 / Proteobacteria / Alphaproteobacteria / Rickettsiales / Pelagibacteraceae / Unclassified / AY664069 / 99.76 / West Pacific Gyre
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