marine bacterial diversity - Prirodoslovno...of RV ″Naše more” for their help during the...

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Uncovering marine bacterial diversity in the southern Adriatic Sea: from surface to seabed I. Babić (1), M. Mejdandžić (1), I. Petrić (2), S. Bosak S (1), H. Mihanović (3), I. Vilibić (3), I. Dupčić Radić (4), I. Cetinić (5,6), Z. Ljubešić (1) (1) Department of Biology, Faculty of Science, University of Zagreb, Croatia (2) Division for Marine and Environmental Research, Ruđer Bošković Institute, Croatia (3) Physical Oceanography Laboratory, Institute for Oceanography and Fisheries, Croatia (4) Institute for Marine and Coastal Research, University of Dubrovnik, Croatia (5) GESTAR/Universities Space Research Association, 7178 Columbia Gateway Drive, Columbia, USA (6) NASA Goddard Space Flight Space Center, Greenbelt, USA COLLECTION, FILTRATION, DNA ISOLATION CTD PROFILES 1 NISKIN BOTTLES 2 20µm 0.2µm 3 1L Massana et al. 1997 Raw reades were depleted of barcodes and adapters and joined by MrDNA. Joined reads were quality checked with FastQC ver. 0.11.5. (Andrews 2010). split_libraries.py Sequences >250bp, q value >25, no ambiguity and homopolymers of length <6bp were selected. Total no. after quality checking: 1.126,708 sequences (length = min. 250 bp, max. 569 bp and avg. 506 bp) pick_open_reference_otus.py sequences were clustered into OTUs at 97 % similarity and chimeras were removed using usearch_qf. pick_rep_set.py, assign_taxonomy.py Represent sequences (most abundant per OTU) were defined and taxonomic assignments were carried out using SILVA 123 QIIME compatible database. alpha_diversity.py - Chao1, observed OTUs, Simpson and Shannon SIMPER analysis NMDS (Bray-Curtis) cluster dendrogram barplots (relative abundances of identified OTUs and 16S rRNA sequences copy number (per mL of seawater) of total and targeted bacterial taxonomic groups. Figure 1. Map of the BIOTA 2016 cruise study area along transect from station P100 to station P1000. MAJOR RESULTS The investigated area showed an unusual general circulation that was characterized by mixed layer down up to 200 m, which differed from the usual winter convection event, typical for middle-altitude ecosystems and important for seasonal picoplankton dynamics in the South Adriatic Sea (Figs 2, 3). Apparent oxygen utilization (AOU) had positive values indicating the respiration as a main process in the southern Adriatic Sea (Fig. 4). The AOU increased as the POC decreased and the best fit had a slope of 1.5 indicating that the respiration is mainly result of heterotrophic bacteria (Fig. 4). The bacterial community was dominated with Alphaproteobacteria accounted for the largest fraction (42.31 % of the total) - mainly represented by the SAR11 clade (90.84 %) and Marinimicrobia (18.44 % of the total) represented with the clade SAR406 (Fig 5). The bacterial community differed between euphotic and aphotic samples and the highest dissimilarity contribution had OTUs from class Deltaproteobacteria, Bacteroidetes, and Cyanobacteria (Fig. 6). Highest abundances of targeted bacterial populations were recorded for Alphaproteobacteria, followed by Gammaproteobacteria and Bacteriodetes. Abundances were found to vary between different sampling points and sampling depths, 3 5 4 5 with values ranging from 8.7x10 to 9.13 x 10 genes/mL for Alphaproteobacteria, from 1.44 x 10 to 5.11 x 10 genes/mL for 3 5 Gammaproteobacteria and from 1.8x10 to 2.4x10 genes/mL for Bacteriodetes (Fig. 6C). NMDS analysis clearly showed grouping of aphotic and euphotic samples, showing correlation of aphotic samples with temperature , POC, oxygen Chl a and nitrite, while aphotic samples were correlated to density, depth and nitrate (Fig. 7). -0.2 0.0 0.2 0.4 -0.1 0.0 0.1 0.2 NMDS1 P150-30m P150-100m P600-25m P600-75m P600-150m P600-400m P600-580m P1000-20m P1000-80m P1000-100m P1000-200m P1000-900m P850-150m P850-500m P850-700m P850-800m Aphotic Euphotic Depth Temperature Density Oxygen Chl a - NO 2 2- NO 3 NMDS2 Others (4.96%) Bacteriodetes (10.62%) Cytophagales Flavobacteriales Actinobactera (1.96%) Alphaproteobacteria (42.31%) Rhodospirillales Rickettsiales SAR11 Chloroflexi (3.14%) Cyanobacteria (7.46%) Deltaproteobacteria (7.86%) Gammaproteobacteria (13.21%) Oceanospirillales Pseudomonadales Marinimicrobia (18.44%) SAR406 Figure 2. Stations P150 and P600. Vertical profiles of (A, C) temperature, salinity and oxygen (B, D) POC and Chl a. Figure 3. Stations P850 and P1000. Vertical profiles of (A, C) temperature, salinity and oxygen (B, D) POC and Chl a. Figure 4. Apparent oxygen utilization (AOU) vs. POC colored by depth. The best fit (dashed black line) had a slope of 1.5, consistent with that expected for a respiratory quotient arising from bacterial metabolization. AOU is defined as the difference between the saturation oxygen (at measure temperature, salinity and surface pressure) and the measured oxygen. Figure 5. Taxonomic distribution of all sequences retrieved during this study (n = 1.126,708). “Others” comprises taxa accounting for less than 1 % of the total. 1.0 0.8 0.6 0.4 0.2 0.0 P600-400m P600-580m P850-500m P1000-80m P850-800m P850-700m P1000-900m P1000-200m P850-150m P1000-100m P600-75m P600-150m P600-25m P1000-20m P150-30m P150-100m Bray-Curtis Dissimilarity Aphotic Euphotic A Figure 6 A-C. Beta-diversity analysis and taxonomic composition of Southern Adriatic samples collected on the P transect during BIOTA 2016 winter cruise. A: dendogram representing the Bray- Curtis dissimilarity between the composition of 16 bacterial communities from deep (Aphotic, orange color) and surface (Euphotic, blue color) water samples. B: relative abundances of bacterial taxa. C: 16S rRNA sequences copy number (per mL of seawater) of total and targeted bacterial taxonomic groups (Alphaproteobacteria, GammaProteobacteria and Bacteriodetes) determined by RT-PCR in samples collected on the P transect during BIOTA 2016 cruise. Letters (a, b, ab) assigned to each value represent groups appointed by the Kruskal–Wallis statistical analysis (p < 0.05). Values in the same group are not significantly different from each other. P1000-80 m marked with asterix represents sample from the euphotic zone. Figure 7. NMDS analysis of BIOTA 2016 stations according to Bray–Curtis distance between bacterial taxa assemblages, with fitted statistically significant (adjusted P value < 0.05) physico-chemical parameters. Samples that belong deep (Aphotic) and surface (Euphotic) assemblage have been colored according to the orange and blue color, respectively). NMDS stress value: 00013. P600-400m P600-580m P850-500m P1000-80m P850-800m P850-700m P1000-900m P1000-200m P850-150m P1000-100m P600-75m P600-150m P600-25m P1000-20m P150-30m P150-100m Relative abundance (%) 0 10 20 30 40 50 60 70 80 90 100 Archaea Acidobacteria Actinobacteria Bcteriodetes Chloroflexi Cyanobacteria Deinococcus-Thermus Firmicutes Fusobacteria Gemmatimonadetes Gracilibacteria Hydrogenedentes Lentisphaerae Marinimicrobia Nitrospirae PAUC34f Parcubacteria Planctomycetes Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Gammaproteobacteria Other Proteobacteria Tenericutes Verrucomicrobia Other Bacteria B ACKNOWLEDGMENTS This work was fully supported by Croatian Science Foundation under the project BIOTA [UIP-2013-11-6433]. Authors want to thank the crew of RV ″Naše more” for their help during the fieldwork. 15. Symposium on Aquatic Microbial Ecology, September 3-8, Zagreb, Croatia WHY? To determine bacterial diversity and quantify the most represented bacterial groups in the southern Adriatic Sea. WHERE? WITH? The samples were collected from surface to seabed on total of 16 different depths at four stations in the southern Adriatic Sea, during the late winter BIOTA cruise conducted in March 2016 (Fig. 1) Sequencing: MiSeq platform Data analysis: QIIME 1.9.1. following Kuczynski et al. 2012 pipeline. Real-time PCR quantification of: α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, and Bacteriodetes. QIIME PIPELINE 1,E+03 1,E+04 1,E+05 1,E+06 1,E+07 P600-580m P850-500m P1000-80m P850-800m P850-700m P1000-200m P600-150m P600-25m P1000-20m P150-30m P150-100m 16S rRNA abundance (copy number/mL) total Bacteria Alphaproteobacteria Gammaproteobacteria Bacteriodetes * C ab ab ab b ab b ab ab b ab b b ab b ab ab ab ab ab a a a a ab a ab ab ab ab a ab ab ab ab ab ab ab ab SEAWATER

Transcript of marine bacterial diversity - Prirodoslovno...of RV ″Naše more” for their help during the...

Page 1: marine bacterial diversity - Prirodoslovno...of RV ″Naše more” for their help during the fieldwork. 15. Symposium on Aquatic Microbial Ecology, September 3-8, Zagreb, Croatia

Uncovering

marine bacterial diversityin the southern Adriatic Sea: from surface to seabed I. Babić (1), M. Mejdandžić (1), I. Petrić (2), S. Bosak S (1), H. Mihanović (3), I. Vilibić (3), I. Dupčić Radić (4), I. Cetinić (5,6), Z. Ljubešić (1)(1) Department of Biology, Faculty of Science, University of Zagreb, Croatia(2) Division for Marine and Environmental Research, Ruđer Bošković Institute, Croatia(3) Physical Oceanography Laboratory, Institute for Oceanography and Fisheries, Croatia(4) Institute for Marine and Coastal Research, University of Dubrovnik, Croatia(5) GESTAR/Universities Space Research Association, 7178 Columbia Gateway Drive, Columbia, USA(6) NASA Goddard Space Flight Space Center, Greenbelt, USA

COLLECTION, FILTRATION, DNA ISOLATION

CTD PROFILES

1

NISKIN BOTTLES

2

20µm

0.2µm

3

1L Mas

sana

et a

l. 19

97

Raw reades were depleted of barcodes and adapters and joined by MrDNA.

Joined reads were quality checked with FastQC ver. 0.11.5. (Andrews 2010).

split_libraries.pySequences >250bp, q value >25, no ambiguity and homopolymers of length <6bp were selected.

Total no. after quality checking: 1.126,708 sequences (length = min. 250 bp, max. 569 bp and avg. 506 bp)

pick_open_reference_otus.pysequences were clustered into OTUs at 97 % similarity and chimeras were removed using usearch_qf.

pick_rep_set.py, assign_taxonomy.pyRepresent sequences (most abundant per OTU) were defined and taxonomic assignments were carried out using SILVA 123 QIIME compatible database.

alpha_diversity.py - Chao1, observed OTUs, Simpson and ShannonSIMPER analysisNMDS (Bray-Curtis)cluster dendrogrambarplots (relative abundances of identified OTUs and 16S rRNA sequences copy number (per mL of seawater) of total and targeted bacterial taxonomic groups.

Figure 1. Map of the BIOTA 2016 cruise study area along transect from station P100 to station P1000.

MAJOR RESULTS The investigated area showed an unusual general circulation that was characterized by mixed layer down up to 200 m, which differed from the usual winter convection event, typical for middle-altitude ecosystems and important for seasonal picoplankton dynamics in the South Adriatic Sea (Figs 2, 3). Apparent oxygen utilization (AOU) had positive values indicating the respiration as a main process in the southern Adriatic Sea (Fig. 4). The AOU increased as the POC decreased and the best fit had a slope of 1.5 indicating that the respiration is mainly result of heterotrophic bacteria (Fig. 4). The bacterial community was dominated with Alphaproteobacteria accounted for the largest fraction (42.31 % of the total) - mainly represented by the SAR11 clade (90.84 %) and Marinimicrobia (18.44 % of the total) represented with the clade SAR406 (Fig 5). The bacterial community differed between euphotic and aphotic samples and the highest dissimilarity contribution had OTUs from class Deltaproteobacteria, Bacteroidetes, and Cyanobacteria (Fig. 6). Highest abundances of targeted bacterial populations were recorded for Alphaproteobacteria, followed by Gammaproteobacteria and Bacteriodetes. Abundances were found to vary between different sampling points and sampling depths,

3 5 4 5with values ranging from 8.7x10 to 9.13 x 10 genes/mL for Alphaproteobacteria, from 1.44 x 10 to 5.11 x 10 genes/mL for 3 5Gammaproteobacteria and from 1.8x10 to 2.4x10 genes/mL for Bacteriodetes (Fig. 6C). NMDS analysis clearly showed grouping of aphotic

and euphotic samples, showing correlation of aphotic samples with temperature , POC, oxygen Chl a and nitrite, while aphotic samples were correlated to density, depth and nitrate (Fig. 7).

-0.2 0.0 0.2 0.4

-0.1

0.0

0.1

0.2

NMDS1

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P600-25m

P600-75m

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P600-580mP1000-20m

P1000-80m

P1000-100m

P1000-200m

P1000-900m

P850-150m

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P850-800m

AphoticEuphotic

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-NO2

2-NO3

NM

DS

2

Others(4.96%)

Bacteriodetes(10.62%)

CytophagalesFlavobacteriales

Actinobactera(1.96%)

Alphaproteobacteria(42.31%)

RhodospirillalesRickettsiales

SAR11

Chloroflexi(3.14%)

Cyanobacteria(7.46%)

Deltaproteobacteria(7.86%)

Gammaproteobacteria(13.21%)

OceanospirillalesPseudomonadales

Marinimicrobia(18.44%)SAR406

Figure 2. Stations P150 and P600. Vertical profiles of (A, C) temperature, salinity and oxygen (B, D) POC and Chl a.

Figure 3. Stations P850 and P1000. Vertical profiles of (A, C) temperature, salinity and oxygen (B, D) POC and Chl a.

Figure 4. Apparent oxygen utilization (AOU) vs. POC colored by depth. The best fit (dashed black line) had a slope of 1.5, consistent with that expected for a respiratory quotient arising from bacterial metabolization. AOU is defined as the difference between the saturation oxygen (at measure temperature, salinity and surface pressure) and the measured oxygen.

Figure 5. Taxonomic distribution of all sequences retrieved during this study (n = 1.126,708). “Others” comprises taxa accounting for less than 1 % of the total.

1.0 0.8 0.6 0.4 0.2 0.0

P600-400m

P600-580m

P850-500m

P1000-80m

P850-800m

P850-700m

P1000-900m

P1000-200m

P850-150m

P1000-100m

P600-75m

P600-150m

P600-25m

P1000-20m

P150-30m

P150-100m

Bray-Curtis Dissimilarity

AphoticEuphotic

A

Figure 6 A-C. Beta-diversity analysis and taxonomic composition of Southern Adriatic samples collected on the P transect during BIOTA 2016 winter cruise. A: dendogram representing the Bray-Curtis dissimilarity between the composition of 16 bacterial communities from deep (Aphotic, orange color) and surface (Euphotic, blue color) water samples. B: relative abundances of bacterial taxa. C: 16S rRNA sequences copy number (per mL of seawater) of total and targeted bacterial taxonomic groups (Alphaproteobacteria, GammaProteobacteria and Bacteriodetes) determined by RT-PCR in samples collected on the P transect during BIOTA 2016 cruise. Letters (a, b, ab) assigned to each value represent groups appointed by the Kruskal–Wallis statistical analysis (p < 0.05). Values in the same group are not significantly different from each other. P1000-80 m marked with asterix represents sample from the euphotic zone.

Figure 7. NMDS analysis of BIOTA 2016 stations according to Bray–Curtis distance between bacterial taxa assemblages, with fitted statistically significant (adjusted P value < 0.05) physico-chemical parameters. Samples that belong deep (Aphotic) and surface (Euphotic) assemblage have been colored according to the orange and blue color, respectively). NMDS stress value: 00013.

P600-400m

P600-580m

P850-500m

P1000-80m

P850-800m

P850-700m

P1000-900m

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P600-75m

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P150-100m

Relative abundance (%)

0 10 20 30 40 50 60 70 80 90 100

ArchaeaAcidobacteriaActinobacteriaBcteriodetesChloroflexiCyanobacteriaDeinococcus-ThermusFirmicutesFusobacteriaGemmatimonadetesGracilibacteriaHydrogenedentesLentisphaeraeMarinimicrobiaNitrospiraePAUC34fParcubacteriaPlanctomycetesAlphaproteobacteriaBetaproteobacteriaDeltaproteobacteriaGammaproteobacteriaOther ProteobacteriaTenericutesVerrucomicrobiaOther Bacteria

B

ACKNOWLEDGMENTSThis work was fully supported by Croatian Science Foundation under the project BIOTA [UIP-2013-11-6433]. Authors want to thank the crew of RV ″Naše more” for their help during the fieldwork.

15. Symposium on Aquatic Microbial Ecology, September 3-8, Zagreb, Croatia

WHY?To determine

bacterial diversityand quantify the most represented

bacterial groups in the southern Adriatic Sea.

WHERE?

WITH?

The samples were collected

from surface to seabedon total of 16 different depths at four stations in the

southern Adriatic Sea,

during the late winter

BIOTA cruise conducted in March 2016 (Fig. 1)

Sequencing: MiSeq platformData analysis: QIIME 1.9.1. following Kuczynski et al. 2012 pipeline.

Real-time PCR quantification of:

α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, and Bacteriodetes.

QIIME PIPELINE

1,E+03 1,E+04 1,E+05 1,E+06 1,E+07

P600-580m

P850-500m

P1000-80m

P850-800m

P850-700m

P1000-200m

P600-150m

P600-25m

P1000-20m

P150-30m

P150-100m

16S rRNA abundance (copy number/mL)

total Bacteria

Alphaproteobacteria

Gammaproteobacteria

Bacteriodetes

*

C

aba

bab

b

abb

aba

bb

abbba

b

b

abab

aba

bab

a

a a

a

abaa

bab a

babaa

b aba

bab a

bababab

SEAWATER