Supplemental Material:
Biogeochemical and metagenomic analysis of nitrite accumulation
in the Gulf of Mexico hypoxic zone
5
10
Contributors: Laura A. Bristow, Neha Sarode, John Cartee, Alejandro Caro-Quintero,
Bo Thamdrup, Frank J. Stewart
1
15
Table S1: Summary of sampling depths, molecular analyses, and 15N rate determinations
conducted at each station.
1
stn
1
1
1
2
2
2
2
3
3
3
3
3
4
4
4
5
5
5
6
6
6
6
6
6
6
6
6
6
6
7
7
7
8
8
8
date (2012)
07/24
07/24
07/24
07/25
07/25
07/25
07/25
07/25
07/25
07/25
07/25
07/25
07/26
07/26
07/26
07/27
07/27
07/27
07/29
07/29
07/29
07/31
07/31
07/31
07/31
07/31
07/31
07/31
07/31
07/28
07/28
07/28
07/28
07/28
07/28
6
07/29 to 07/31
(diel sampling)
depth (m) analysis1
2
A; Mg
6
A; Mg
11
A; Mg
2
20
40
63
2
A; Mg
20
45
A; Mg
60
120
A; Mg
2
A; Mg
10
A; Mg
18
2
A; Mg
5
A; Mg
10
A; Mg
2.5
7
15.7
2
A; Mg
3
A; Mg; Mt
5
A; Mg
7
A; Mg
9
A; Mg; Mt
11
A; Mg
13
A; Mg; Mt
15
A; Mg
2
A; Mg
10
A; Mg
21.5
2
A; Mg
7
15
A; Mg
7 and 15
rate expts (15N Additions)
15
NH4+; 15NO215
NH4+; 15NO215
NH4+; 15NO2-
15
15
NH4+; 15NO2NH4+; 15NO2-; 15NO3-
15
NH4+; 15NO215
NH4+; 15NO2-; 15NO3-
15
15
NH4+; 15NO2NH4+; 15NO2-
A
A, indicates 16S rRNA gene amplicon; M, metagenome; T, metatranscriptome
2
Table S2: Sequencing statistics.
16S rRNA gene
Metagenome
stn
depth (m)
reads1
OTU2
Chao13
reads1
PC4
1
2
4,644
106
183
323,449
200,859
1
6
3,094
95
152
4,596,587
3,318,666
1
11
15,205
115
229
1,454,885
1,144,319
3
2
3,642
153
238
2,436,387
1,844,574
3
45
4,842
166
257
3,993,001
2,921,228
3
120
6,138
204
369
3,236,605
2,451,508
4
2
5,780
99
175
245,309
138,696
4
10
6,800
126
255
2,600,017
1,916,125
5
2
661
106
111
765,588
505,165
5
5
5,005
158
269
1,910,220
1,382,951
5
10
7,912
170
322
1,568,301
1,145,446
6
0
14,567
123
244
592,241
360,226
6
3
23,787
118
220
919,528
475,220
6
5
2,372
113
178
1,493,425
940,317
6
7
4,369
114
198
657,500
393,877
6
9
13,327
108
241
1,167,202
819,251
6
11
4,753
126
214
1,198,003
867,194
6
13
2,498
131
210
1,101,495
780,807
6
15
4,271
155
261
1,647,797
1,057,387
7
2
16,983
121
204
340,336
223,766
7
10
4,859
103
153
1,686,304
1,219,568
8
2
377,452
168
349
2,051,412
1,599,501
8
15
13,602
169
223
2,711,399
2,040,456
Metatranscriptome
reads1
PC4
6,518,172
565,136
10,466,363
887,798
2,342,438
129,119
20
1
reads, sequences post quality control filtering and paired end merging
OTU, number of observed OTUs (97% similarity clusters) based on rarified counts (n = 661 sequences)
3
Chao1, number of Chao1 estimated OTUs based on rarified counts (n = 661 sequences)
4
PC, reads with significant (> bit score 50) BLASTX matches to protein-coding genes in NCBI-nr
2
3
25
Supplemental Discussion - 16S rRNA amplicon diversity
Despite high variability in taxonomic composition across sites, some taxonomic groups
exhibited clear depth-specific patterns across sites. Similar to the pattern for the
Thaumarchaeota (described in the main text), sequences matching the uncultured division
SAR406 (Marine Group A) also consistently increased with depth (from an average of
30
1.6 % at depths <10 m to 5% at depths of ≥10 m) and declining oxygen (r2=0.28), with a
vast majority (on average 94% of SAR406 amplicons) affiliated with the subclade
Arctic96B-7. A positive relationship between SAR406 abundance and deoxygenation has
been observed in other OMZ sites in the Eastern Tropical North Pacific (Beman and
Carolan 2013) and North Subarctic Pacific (Allers et al 2012). Recent genomic evidence
35
suggests that this group may be involved in dissimilatory polysulfide reduction to sulfide,
or sulfide oxidation (Wright et al 2014). Lacking rates or targeted genome sequencing,
the functional contributions of SAR406 to Shelf biochemical cycling remain unknown.
Allers, E., J. J. Wright, K. M. Konwar, C. G. Howes, E. Beneze, S. J. Hallam, and M. B.
40
Sullivan. 2012. Diversity and population structure of Marine Group A bacteria in the
Northeast subarctic Pacific Ocean. ISME J. 7: 256-268, doi: 10.1038/ismej.2012.108
Beman, J. M., and M. T. Carolan. 2013. Deoxygenation alters bacterial diversity and
community composition in the ocean's largest oxygen minimum zone. Nat. Commun. 4:
45
2705, doi: 10.1038/ncomms3705
4
Time-series Sampling, Station 6.
50
Figure S1: Time-series data over the period 29-31 July at station 6 (57 hours total;
sampling approximately every 4-6 hours). Panels show salinity (a), fluorescence (µg L-1;
b), oxygen (µmol kg-1; c), nitrite (µM; d), and community taxonomic composition at
intermediate (7 m) and bottom water (15 m) depths, inferred from 16S rRNA gene
amplicons (e). Numbers in parentheses in panel e indicate time of day.
55
60
Repeated sampling (every 4 hrs) of the same site (station 6) over a 57 hr period
revealed significant variation in environmental conditions and microbial community
structure due to water mass advection. Over the 57 hours sampled at station 6, a
consistent layer of low salinity water (< 33) was present in the top 6 to 8 m, with the
halocline deepening during daylight hours (Fig. S1). The oxycline occurred over an
5
65
70
75
80
85
90
95
identical depth range to that of the halocline and also deepened during daylight hours
(Fig. S1). Oxygen concentrations over the vertical gradient were exceptionally dynamic,
with bottom water (15-16 m) concentrations being hypoxic for only the first 21 hours of
sampling and then increased to maximum levels of 100 µmol kg-1 on July 31st at 14:00
(Fig. S1). Bottom water salinity showed an increase from 35.5 at the beginning of the
time series to 36 at the end, correlating well with the increase in oxygen (r2=0.86; p <
0.05). As bottom water oxygen concentrations increased, nitrite concentrations decreased,
from 3.4 to 1.3 µM over the time series. Highest nitrate concentrations (6.4 µM)
coincided with highest nitrite and lowest oxygen concentrations in bottom waters at the
beginning of the time series. As bottom water oxygen increased, nitrate concentrations
dropped to 1.4 µM. Nitrate and nitrite showed a weak positive correlation over the whole
time series (r2 = 0.47; p < 0.05). Chlorophyll fluorescence was highest between 7 and 9 m
during daylight hours, with maximal concentrations of 3.7 µg L-1 observed at 8 m at
14:00 on July 30th (Fig. S1). Fluorescence in the bottom waters remained low throughout
the sampling period. Unfortunately, ammonia and nitrite oxidation rates were only
determined at the beginning of the time series, so we cannot comment on how decoupling
of ammonia and nitrite oxidation was influenced over this period.
Microbial community structure also fluctuated substantially during time series
sampling (Fig. S1). Although clear diel-dependent periodicity was not observed for any
particular taxon, taxon relative abundance varied markedly and unpredictably over the 57
hr period. Notably, the proportional contribution of ammonia-oxidizing Thaumarchaeota
ranged from 0.2% to 52% of total amplicon sequences at 7 m, and from 1% to 33% at
15m. Similarly dramatic swings were observed for cyanobacteria (2% to 43% of
sequences at 15m), and the Actinobacteria (4% to 62% at 15 m). Such shifts are
comparable in magnitude to those distinguishing communities from different stations and
depths (Fig. 3, main text). Given the short time frame of the sampling, these wide
fluctuations likely resulted from water mass shifts, or potentially methodological
variation associated with the amplicon sequencing protocol, rather than population
growth or turnover. These data provide a glimpse into the highly variable nature of the
shelf ecosystem and the potential for changes in water mass circulation to quickly
reshape the distribution of environmental parameters controlling biochemical flux. This
analysis is a reminder that inferences of marine microbial community composition based
on single time-point samples from a single site merely reflect ‘snapshots’ of a system in
flux. Such variability suggests that linkages between community biochemical processes,
including coupling between nitrification steps, are similarly dynamic over short time
scales, and over shallow vertical gradients.
6