1
Sample filtration and DNA extraction
2
Details of our water filtration and DNA extraction protocols can be downloaded from the
3
website: http://amarallab.mbl.edu.
4
5
Geochemical analyses
6
For offshore water samples MCR_1 – MCR_4 and MCR_14 – MCR_19, we measured
7
temperature and salinity aboard the R/V Seamans using a Sea-Bird Electronics SBE 19+
8
SEACAT CTD (Sea-Bird Electronics, Bellevue, WA) profiler integrated into a carousel water
9
sampler from which we collected the seawater used for DNA analyses. A Sea-Bird SBE 43
10
served to measure dissolved oxygen. Inshore samples MCR_5 – MCR_9 were collected in an
11
acid washed, Teflon coated, 8-liter Niskin Bottle. Chlorophyll-a concentrations were measured
12
from 1.0 L seawater filtered through either a 0.45 µm Gelman GN-6 filter (SEA cruise) or
13
Whatman GF/F filters digested at -20°C in 90% acetone for 24- 48 hours before analysis on a
14
Turner Designs 10AU fluorometer (Turner Designs, Sunnyvale, CA) (Welschmeyer, 1994). We
15
measured phosphate and nitrate concentrations using standard colorimetric methods (Parsons, et
16
al., 1984) using an Ocean Optics USB-2000 fiber optic spectrometer (Ocean Optics, Dunedin,
17
FL) or via standard flow injection technology on a QuikChem 8000 (Lachat Instruments
18
Division of Zellweger Analytics, Inc.) for inshore samples. Temperature and salinity samples
19
were measured inshore using a SBE 19+ SEACAT CTD. Geochemical data represented by
20
italicized font in Table S1 are estimates that represent data collected through other LTER studies
21
in analogous seasons and stored on the MCR LTER website at the following address:
22
http://mcr.lternet.edu/data/.
23
24
Collector’s curve
25
We calculated OTU accumulation with increasing sequencing effort using the EstimateS
26
program v. 8.0.2 (Colwell, R.K., 2009; http://purl.oclc.org/estimates), implementing the Species
27
Accumulation algorithm, random sequence order and 999 permutations. Bacterial and archaeal
28
OTUs used in this analysis were clustered at the 97% sequence similarity threshold, while
29
eukaryotic sequences (minus those classified as metazoan) were clustered at the 94% sequence
30
similarity threshold.
31
32
References
33
Parsons TR, Maita L, Lalli CM. (1984). A manual of chemical and biological methods for
34
seawater analysis. Pergamon Press: New York.
35
36
Welschmeyer NA. (1994). Fluorometric analysis of chlorophyll a in the presence of chlorophyll
37
b and pheopigments. Limnol Oceanogr 39: 1985-1992.
38
39
Colwell, R. K. (2009). EstimateS: Statistical estimation of species richness and shared species
40
from samples. Version 8.2. User's Guide and application published at:
41
http://purl.oclc.org/estimates.
42
43
Figure legends
44
Figure S1. Depth profiles of environmental data at offshore stations 5 km, 60 km and 130 km
45
north of Moorea. Concurrent biological sequence data collected from surface and 200 m depth.
46
47
Figure S2. Dendrogram of bacterial community similarity among inshore and offshore sample
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stations using: (a) abundant OTUs (those containing >1.0% of total bacterial sequences); and (b)
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rare OTUs (those containing <0.01% of total bacterial sequences). Dendrograms are calculated
50
based on relative abundance-based Bray-Curtis similarity matrices of OTUs among inshore and
51
offshore sample stations.
52
53
Figure S3. Non-metric Multidimensional Scaling ordination of Bray-Curtis bacterial group-
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specific OTUs. Ordination based on samples collected in January 2008 and May 2009.
55
56
Figure S4. Collector’s curves illustrating the number of OTUs observed with increasing
57
addition of sequences to the dataset. Curves based on 999 permutations and random sequence
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addition order.
59