Long-term effects of ocean warming on the prokaryotic community: evidence
from the vibrios
**ISMEJ-11-00087OA REVISED2**
Luigi Vezzulli1*, Ingrid Brettar2, Elisabetta Pezzati3, Philip C. Reid 4,5,6, Rita R. Colwell7,
Manfred G. Höfle2, Carla Pruzzo1
REVISED 2
Supplementary Information
The file contains Supplementary Tables 1-2; Supplementary Figures 1-3
The Supplementary Table 1 provides information on sampling sites where CPR samples were
collected. The Supplementary Table 2 reports total bacteria and Vibrio spp. concentrations assessed
by Real-Time PCR in CPR samples. The Supplementary Figures 1-2 provide information on the
development and validation of the Vibrio Abundance Index (VAI). Supplementary Figure 3
provides information on read sequences showing >95% similarities to Vibrio cholerae.
Table S1.
CPR samples used in the study
Area
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
tow ID
27LR
91LR
156LR
171LR
199LR
226LR
309R321R345R125LG
371R152LG
401R413R425R447R220LG
473R485R498R307C319C332C344C356C368C381C404C415C213LR
436C465C485C253LR
265LR
277LR
289LR
301LR
313LR
60HE
336LR
346LR
360LR
372LR
383LR
sample ID
27
27
31
31
27
33
7
7
7
1
7
1
7
7
7
7
1
7
8
8
1
1
1
1
1
1
1
3
1
17
1
1
1
1
21
21
21
21
21
1
21
21
21
21
21
latitude
52.1
52.2
52.2
52.4
52.1
52.2
52.1
51.9
52.1
52.2
52.1
52.2
52.1
52.1
52.1
52.1
51.9
52.1
52.2
52.3
53.6
53.6
53.6
53.6
53.7
53.7
53.7
53.9
53.7
54.0
53.6
53.7
53.9
53.7
53.8
53.8
53.8
53.9
53.8
53.6
53.8
53.9
53.9
53.9
53.9
longitude
3.5
3.3
3.7
3.6
3.4
3.6
3.5
3.3
3.5
4.0
3.5
3.6
3.5
3.5
3.5
3.6
3.4
3.5
3.7
3.8
0.3
0.4
0.5
0.3
0.5
0.6
0.5
0.9
0.5
0.9
0.4
0.3
0.6
0.3
0.2
0.2
0.2
0.1
0.2
0.5
0.2
0.3
0.1
0.1
0.2
date
11-august
24- august
15- august
26- august
6- august
14- august
18- august
16- august
8- august
20- august
7- august
28- august
8- august
6- august
8- august
14- august
16- august
12- august
9- august
15- august
13- august
13- august
12- august
9- august
9- august
7- august
16- august
15- august
14- august
16- august
13- august
21- august
14- august
7- august
6- august
12- august
18- august
2- august
8- august
25- august
13- august
4- august
17- august
9- august
15- august
year
1961
1966
1971
1972
1974
1976
1989
1990
1992
1993
1994
1996
1997
1998
1999
2001
2002
2003
2004
2005
1965
1966
1967
1968
1969
1970
1971
1973
1974
1975
1976
1979
1981
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
190LE
141HE
545C428LR
437LR
190HE
199HE
212HE
615C236HE
1
1
1
21
1
1
1
1
1
1
53.7
53.6
53.7
53.9
53.8
53.6
53.6
53.6
53.7
53.6
0.7
0.4
0.5
0.1
0.2
0.4
0.4
0.6
0.5
0.6
11- august
9- august
18- august
17- august
13- august
16- august
8- august
9- august
13- august
9- august
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
Table S2.
Total bacteria and Vibrio spp. counts (average  SD from five independent replicate
measurements) (CV<15%) assessed by Real-Time PCR in CPR samples collected in the North
Sea from 1961 to 2005. Vibrio spp. and total bacterial concentrations were expressed as
number of cells per square centimetre of the CPR sample (cells/cm2) by dividing the total
16SrDNA copy number by the average 16SrDNA copy number in vibrios (n=9, Acinas et al.,
2004) and proteobacteria (n=3.5, Kormas, 2011), respectively.
Area
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Rhine
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
year
1961
1966
1971
1972
1974
1976
1989
1990
1992
1993
1994
1996
1997
1998
1999
2001
2002
2003
2004
2005
1965
1966
1967
1968
1969
1970
1971
1973
1974
1975
1976
sample ID
27
27
31
31
27
33
7
7
7
1
7
1
7
7
7
7
1
7
8
8
1
1
1
1
1
1
1
3
1
17
1
Total bacteria
(cell x 105/cm2)
2.1  0.3
2.4  0.3
0.6  0.1
1.2  0.1
1.0  0.1
1.4  0.2
0.1  0.01
0.7  0.1
0.5  0.1
2.0  0.2
0.7  0.08
1.3  0.1
1.2  0.1
1.5  0.2
15.8  1.9
1.1  0.1
0.9  0.1
0.7  0.1
3.9  0.4
0.1  0.01
6.7  0.4
6.8  0.8
6.3  0.3
6.8  1.1
8.1  1.0
11.4  1.4
21.3  2.6
18.7  2.2
4.0  0.5
4.9  0.6
4.9  0.6
Vibrio spp.
(cell x 105/cm2)
0.2  0.03
0.01  0.0
0.02  0.0
0.1  0.01
0.2  0.03
0.2  0.03
0.1  0.01
0.3  0.04
0.1  0.02
0.8  0.12
0.3  0.04
0.8  0.09
0.5  0.06
0.6  0.07
13.5  2.8
0.3  0.04
0.7  0.08
0.6  0.1
1.8  0.1
0.1  0.01
0.2  0.02
0.2  0.03
0.2  0.01
0.2  0.01
0.1  0.01
0.2  0.02
0.2  0.03
0.2  0.02
0.2  0.03
0.3  0.02
0.2  0.01
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
Humber
1979
1981
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
1
1
1
21
21
21
21
21
1
21
21
21
21
21
1
1
1
21
1
1
1
1
1
1
1.8  0.2
8.0  0.5
5.3  0.6
0.5  0.1
0.2  0.01
0.1  0.01
0.1  0.03
0.1  0.04
0.9  0.1
11.4  1.3
2.2  0.3
2.7  0.2
7.4  0.6
0.2  0.02
8.1  0.9
3.5  0.4
5.2  0.3
2.3  0.3
0.7  0.1
8.7  0.8
3.7  0.5
9.5  1.1
1.3  0.2
3.5  0.1
0.1  0.01
0.2  0.02
0.2  0.02
0.04  0.01
0.02  0.00
0.01  0.00
0.01  0.00
0.01  0.00
0.1  0.01
0.3  0.05
0.1  0.01
0.1  0.01
0.2  0.02
0.01  0.00
0.2  0.02
0.1  0.01
0.2  0.03
0.1  0.01
0.1  0.01
0.1  0.01
0.2  0.02
0.2  0.03
0.03  0.00
0.1  0.01
Figure S1
DNA fragmentation analysis
Electropherogram summaries obtained by fragmentation analysis of genomic DNA extracted from a
subset of selected CPR samples (1961, 1976, 1998, 2004) that were collected off the Rhine Estuary
in the North Sea.
Figure S2
Validation of VAI index using historic plankton samples
Correlation analysis between culturable Vibrio counts (CFU/10L) associated with small plankton
samples collected in the Adriatic Sea from April 1996 to March 1997 (Montanari et al., 1999) and
VAI index (average  STD of 5 replicate measurements) calculated 14 years later on the same
samples.
Figure S3
Analysis of Vibrio cholerae sequences
BLASTn alignment against the V6RefDB database of a consensus sequence (Query) calculated
from a group of 51 sequences that clustered together phylogenetically in 1998 (total of 22
sequences) and 2004 (total of 29 sequences) CPR samples showing >95% identity to V. cholerae