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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
P150-30m
P150-100m
P600-25m
P600-75m
P600-150m
P600-400m
P600-580mP1000-20m
P1000-80m
P1000-100m
P1000-200m
P1000-900m
P850-150m
P850-500mP850-700m
P850-800m
AphoticEuphotic
Depth
Temperature
Density
Oxygen
Chl a
-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
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
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