Supplementary Information
Environmental distribution of coral-associated relatives of apicomplexan parasites
Jan Janouškovec1, Aleš Horák1,2, Katie L. Barott3, Forest L. Rohwer3, and Patrick J. Keeling1
Supplementary Materials and Methods:
Sequence data availability: All sequences used in this study and their accession numbers were
described previously (Janouskovec et al, 2012, Curr Biol; see Table S1 within) with the exception of
three studies additionally identified here: V6 tags of ARL-V from a survey of seven coral species in the
Caribbean (Sunagawa et al, 2010; data available at http://vamps.mbl.edu), V6 tags of ARL-V from a
Isopora palifera survey on Taiwan (Chen et al, 2011; data available at http://140.109.29.21/~tanglab/)
and a single sequence of ARL-V from a Stylopora pistillata survey (data available at
http://140.109.29.21/~tanglab/).
Coral-macroalgal transects (Figure 1A, Supplementary Table 1): Microbial surveys by Barott et al.,
2011 and Barott et al., 2012 obtained 16S ribosomal RNA sequence data using general bacterial
primers, and analysed the majority of bacterial and non-ARL plastid sequence. Here we focused on
previously overlooked presence of ARLs in this data and compared their distribution to the other
already identified plastid lineages. The two studies sampled coral reef in southern coast of Curacao,
Caribbean. Four transects were designed to span between multiple specimen of Montastraea annularis
and the four following algae: Halimeda opuntia, Dictyota bartayresiana, crustose corraline algae, and
turf algae. Within each transect, five different zones were sampled: coral tissue/surface from the centre
of the colony 5-10 cm away from coral-algal interface, CAI (Zone 1); coral tissue/surface immediately
adjacent to CAI (Zone 2), CAI, the contact zone between coral and algae (Zone 3), algal tissue/surface
immediately adjacent to CAI (Zone 4), and algal tissue/surface 5-10 cm away from CAI (Zone 5).
Zones 2 and 4 were principally similar to Zones 1 and 5 respectively, however, their proximity to the
point of coral-algal contact (Zone 3) suggested they might differ in their microbial composition. All 20
samples (4 transects x 5 zones) were obtained in 5 replicates. All replicates were independently PCRamplified and pooled before sequencing. Plastid sequences in the resulting sequence pools (containing
mostly bacteria) were identified using BLASTn searches and phylogenies, as described in Barott et al,
2011. In this study, we concentrated on five groups of unicellular microalgae (diatoms, pelagophytes,
haptophytes, unicellular red and green algae), in addition to newly idetified ARLs (ARL-I, ARL-III,
ARL-V). We excluded the following plastid sequences of macroalgae, the representatives of which
were innate part of the transects: phaeophytes (Dictyota), florideophytes (crustose corraline algae), and
ulvophytes (Halimeda, turf algae). Absolute and relative occurrences of sequences from selected plastid
lineages for all zones were summarized in Supplementary Table 1. To display general trends in plastid
abundance between corals and dominant reef macroalgae as a whole, median relative occurrences for
across all four transects were plotted for all five zones and eight microalgal groups (Figure 1A).
Medians rather than means were used to plot these trends in order to mediate the effect of several
outliers in the data (none of the outliers belonged to ARL-V). However, the alternative use of means
provided a picture consistent with the use of medians (Supplementary Table 2). A reference sample of
M. annularis tissue/surface equivalent to Zone 1 (marked ‘WP (ref.)’ in Supplementary Table 1) was
taken from northern-west tip of Curacao (approx. 37 km aerial distance) using the same sampling
method (Barott et al, 2011).
Seasonal distribution of ARL-V: Chen et al. sampled Isopora palifera colonies (three replicates) in
the southern tip of Taiwan during February, April, May, June, July, August and November 2008, and
obtained microbial 16S ribosomal RNA sequence for all samples. We identified 117 plastid sequences
of ARL-V in this data, all of which were derived from February and April-August samples (none were
derived from November samples).
ARL-V abundance within 7 coral species and reef water (Figure 1B): This data is derived from a
study conducted near Bocas Del Toro, Panama, Caribbean. Seven coral species were sampled in five
replicates using hammer and chisel and coral fragments 1–4 cm2 in size crushed using the same
method. Five liters of reef water were collected in the vicinity of corals and filtered using 0.22 mm
filter. DNA extractions were done from equivalent amount of homogenized corals (~50 mg). For all
samples, equivalent amounts of DNA were used for 16S rDNA amplication, and all amplification
reactions were run in triplicates, before they were pooled and sequenced. Parallel approach in sequence
generation has made these samples suitable for mutual comparisons. We measured relative sequence
occurrence of ARL-V in each sample and plotted the result in Figure 1B.
Note: All supplementary references are covered in the main text.
Supplementary Table 1 Legend:
Occurrence of ARLs and other plastids in coral-macroalgal transects. Values were extracted from
sequence surveys by Barott et al., 2011 and Barott et al., 2012. Absolute sequence counts and relative
sequence occurrence (note: x10-5 units) for plastid groups in question (left column) are shown for four
coral-algal transects (Hal, Dict, CCA, Turf), five zones within each transect (Z1-Z5), and a reference
Z1 site (WP (ref.)). The transects cover natural associations between the reef coral Montastraea
annularis and four types of macroalgae: Hal = Halimeda opuntia, Dict = Dictyota bartayresiana, CCA
= crustose corraline algae, Turf = turf algae. Five zones were sampled within each transect as follows:
Z1=coral tissue and surface (T/S) 5-10 cm away from coral-alga interface (CAI), Z2=coral T/S
immediately adjacent to CAI, Z3=coral and algal T/S at CAI, Z4=algal T/S immediately adjacent to
CAI, Z5=algal T/S 5-10 cm away from CAI. Sample marked as WP (ref.) is Montastraea annularis T/S
collected a different site on Curacao providing a reference observation for Z1 (see ‘Coral-macroalgal
transects’ section above for details). Number of total sequences generated for each transect/zone are at
the bottom of the table (the majority of these are bacterial sequences; the focus of the two surveys
above).
Supplementary Table 2 Legend:
Statistical values for relative occurrence of ARLs and other plastids in coral-algal associations. Four
observations for each zone were obtained as described in the ‘Coral-macroalgal transects; section
above. Medians, means, 25% and 75% percentiles, statistical errors, and 95% confidence intervals for
medians were calculated.
Supplementary Table 3 Legend:
Distribution of all ARL-V sequence tags in coral species. Sequence counts are presented for 20 coral
species from three distinct clades. Long reads were retrieved from NCBI database. 454-pyrosequence
tags were generated in three studies (next three columns). Three dashes (---) indicate absence of
sampling for a particular species. The last column indicates the ability for each coral species to form
symbiotic associations with zooxanthellae, the dinoflagellates from the genus Symbiodinium.
Supplementary Table 1: Occurence of ARLs and other plastids in coral-macroalgal transects
absolute abundance
relative abundance (10E-5)
Group
Association
Z1
Z2
Z3
Z4
Z5
Total
Z1
Z2
Z3
Z4
Z5
WP
30
67.8656
n.a.
n.a.
Hal
7
23
4
5
0
39 12.2463 51.321 7.84314 7.52627
0
Dict
10
19
0
0
0
29 30.0039 22.1818
0
0
0
ARL-V
CCA
45
0
1
0
0
46 47.0997
0 1.41225
0
0
Turf
5
7
0
0
0
12 7.89378 6.70395
0
0
0
Total
97
49
5
5
0
156 33.0407 15.3447 2.16273 2.23142
0
WP
0
0
n.a.
n.a.
Hal
0
0
0
31
43
74
0
0
0 46.6629 77.4105
ARL-I
Dict
0
0
7
0
0
7
0
0 11.3638
0
0
Vitrella
CCA
0
1
1
1
0
3
0 1.18426 1.41225 2.08238
0
clade
Turf
0
0
16
3
16
35
0
0 33.4861 7.55934 23.0302
Total
0
1
24
35
59
119
0 0.31316 10.3811 15.6199 21.0394
WP
0
0
n.a.
n.a.
Hal
0
0
0
1
3
4
0
0
0 1.50525 5.40073
ARL-III
Dict
0
0
1
0
0
1
0
0 1.6234
0
0
Chromera
CCA
0
0
0
8
4
12
0
0
0 16.659 8.42336
clade
Turf
0
0
0
0
18
18
0
0
0
0 25.909
Total
0
0
1
9
25
35
0
0 0.43255 4.01655 8.91501
WP
2
4.52438
n.a.
n.a.
Hal
0
2
6
8467
3383
11858
0 4.46269 11.7647
12745 6090.23
Dict
10
3
145
867
1986
3011 30.0039 3.50238 235.393 1239.79 1840.3
Diatoms
CCA
5
4
5
47
245
306 5.2333 4.73704 7.06125 97.8718 515.931
Turf
1
1
39
140
237
418 1.57876 0.95771 81.6224 352.769 341.135
Total
18
10
195
9521
5851
15595 6.13127 3.13157 84.3466 4249.06 2086.47
WP
0
0
n.a.
n.a.
Hal
0
1
0
12
27
40
0 2.23135
0 18.063 48.6066
Dict
0
0
1
2
13
16
0
0 1.6234 2.85996 12.0463
HaptoCCA
0
0
0
3
0
3
0
0
0 6.24714
0
phytes
Turf
0
0
1
2
13
16
0
0 2.09288 5.03956 18.712
Total
0
1
2
19
53
75
0 0.31316 0.86509 8.47938 18.8998
WP
1
2.26219
n.a.
n.a.
Hal
0
0
0
102
184
286
0
0
0 153.536 331.245
0
0
13
80
21
114
0
0 21.1042 114.398 19.4594
Pelago- Dict
CCA
0
7
133
190
359
689
0 8.28981 187.829 395.652 755.996
phytes
Turf
0
0
30
275
1149
1454
0
0 62.7865 692.94 1653.86
Total
1
7
176
647
1713
2544 0.34063 2.1921 76.1282 288.745 610.856
WP
0
0
n.a.
n.a.
Hal
0
0
0
10
0
10
0
0
0 15.0525
0
0
0
7
4
0
11
0
0 11.3638 5.71992
0
Unicellular Dict
0
0
0
2
12
14
0
0
0 4.16476 25.2701
red algae CCA
Turf
1
0
5
369
119
494 1.57876
0 10.4644 929.799 171.287
Total
1
0
12
385
131
529 0.34063
0 5.19056 171.819 46.7146
WP
0
0
n.a.
n.a.
Hal
13
0
3
40
45
101 22.7432
0 5.88235 60.2101 81.011
Unicellular Dict
0
0
12
0
2
14
0
0 19.4808
0 1.85328
green
CCA
0
0
3
0
10
13
0
0 4.23675
0 21.0584
algae
Turf
0
0
0
34
19
53
0
0
0 85.6725 27.3484
Total
13
0
18
74
76
181 4.42814
0 7.78584 33.025 27.1016
WP
44205
n.a.
n.a.
Hal
57160
44816
51000
66434
55548 274958
n.a.
Dict
33329
85656
61599
69931 107917 358432
n.a.
All
95542
84441
70809
48022
47487 346301
sequence CCA
n.a.
Turf
63341 104416
47781
39686
69474 324698
n.a.
Total
293577 319329 231189 224073 280426 1348594
n.a.
Total
14.184
8.0908
13.2832
3.69574
11.5676
26.9132
1.95295
0.8663
10.7792
8.824
1.45477
0.27899
3.46519
5.54361
2.5953
4312.66
840.048
88.3624
128.735
1156.39
14.5477
4.46389
0.8663
4.92766
5.56135
104.016
31.8052
198.96
447.801
188.641
3.63692
3.06892
4.04273
152.141
39.226
36.7329
3.9059
3.75396
16.3229
13.4214
Supplementary Table 2: Statistical values for relative occurence of ARLs and other plastids in coral-algal associations
Zone
1
2
3
4
5
Statistical values
Observations
Minimum
25% Percentile
Median
75% Percentile
Maximum
Mean
Standard Deviation
Standard Error of Mean
Lower 95% CI of Median
Upper 95% CI of Median
Observations
Minimum
25% Percentile
Median
75% Percentile
Maximum
Mean
Standard Deviation
Standard Error of Mean
Lower 95% CI of Median
Upper 95% CI of Median
Observations
Minimum
25% Percentile
Median
75% Percentile
Maximum
Mean
Standard Deviation
Standard Error of Mean
Lower 95% CI of Median
Upper 95% CI of Median
Observations
Minimum
25% Percentile
Median
75% Percentile
Maximum
Mean
Standard Deviation
Standard Error of Mean
Lower 95% CI of Median
Upper 95% CI of Median
Observations
Minimum
25% Percentile
Median
75% Percentile
Maximum
Mean
Standard Deviation
Standard Error of Mean
Lower 95% CI of Median
Upper 95% CI of Median
ARL-V
4
7.894
8.982
21.13
42.83
47.1
24.31
17.95
8.976
7.894
47.1
4
0
1.751
14.44
44.04
51.32
20.08
22.79
11.4
0
51.32
4
0
0
0.7561
6.235
7.843
2.364
3.705
1.852
0
7.843
4
0
0
0
5.67
7.526
1.957
3.713
1.857
0
7.526
4
0
0
0
0
0
0
0
0
0
0
Vitrella
4
0
0
0
0
0
0
0
0
0
0
4
0
0
0
0.9132
1.184
0.3711
0.5421
0.2711
0
1.184
4
0
0.4281
6.388
27.96
33.49
11.59
15.44
7.72
0
33.49
4
0
0.5956
4.821
36.89
46.66
14.1
21.94
10.97
0
46.66
4
0
0
11.57
63.82
77.41
25.16
36.47
18.24
0
77.41
Chromera
4
0
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
4
0
0
0
1.243
1.623
0.4809
0.7617
0.3809
0
1.623
4
0
0
0.8026
12.87
16.66
4.591
8.073
4.036
0
16.66
4
0
1.425
6.912
21.54
25.91
9.958
11.18
5.588
0
25.91
Diatoms
4
0
0.4697
3.406
23.81
30
9.229
14.02
7.008
0
30
4
0.9577
1.594
3.983
4.668
4.737
3.415
1.722
0.8608
0.9577
4.737
4
7.061
8.237
46.69
197
235.4
83.96
106.6
53.28
7.061
235.4
4
97.87
161.6
796.3
9869
12745
3609
6110
3055
97.87
12745
4
341.1
384.8
1178
5028
6090
2197
2680
1340
341.1
6090
Haptophytes
4
0
0
0
0
0
0
0
0
0
0
4
0
0
0
1.699
2.231
0.6328
1.066
0.5328
0
2.231
4
0
0
0.8617
1.976
2.093
0.9791
1.033
0.5165
0
2.093
4
2.86
3.405
5.643
15.11
18.06
8.052
6.819
3.41
2.86
18.06
4
0
3.087
15.38
41.13
48.61
19.87
20.65
10.32
0
48.61
Pelagophytes
4
0
0
0
0
0
0
0
0
0
0
4
0
0
0
6.242
8.29
2.147
4.095
2.047
0
8.29
4
0
5.351
41.95
156.6
187.8
67.95
84.06
42.03
0
187.8
4
114.4
124.2
274.6
618.6
692.9
339.1
266.7
133.3
114.4
692.9
4
19.46
97.41
543.6
1429
1654
690
709.9
354.9
19.46
1654
Unicellular
red algae
4
0
0
0
1.209
1.579
0.4697
0.7394
0.3697
0
1.579
4
0
0
0
0
0
0
0
0
0
0
4
0
0
5.282
11.14
11.36
5.507
6.254
3.127
0
11.36
4
4.165
4.554
10.39
701.1
929.8
238.7
460.8
230.4
4.165
929.8
4
0
0
12.69
134.8
171.3
49.19
82.26
41.13
0
171.3
Unicellular
green algae
4
0
0
0
17.08
22.74
5.761
11.32
5.661
0
22.74
4
0
0
0
0
0
0
0
0
0
0
4
0
1.134
5.06
16.08
19.48
7.425
8.397
4.199
0
19.48
4
0
0
306
79.31
85.67
36.52
43.32
21.66
0
85.67
4
1.853
6.655
24.2
67.6
81.01
32.82
33.91
16.95
1.853
81.01
Supplementary Table 3: Distribution of all ARL-V sequence tags in coral species
Coral group
Species
Long reads
Sunagawa et Barott et al., Chen et al.,
Total
Zooxanthellae coral
(NCBI)
al., 2010
2011, 2012
2011
sequences
(hosts Symbiodinium)?
Diploria strigosa
--2
----2
YES
Hexacorals,
Scleractinians,
Favites sp.
21
------21
YES
Robust clade
Fungia scutaria
2
------2
YES
Montastraea annularis
67
--156
--223
YES
Montastraea faveolata
1
17
----18
YES
Montastraea franksi
5
118
----123
YES
Mussismilia braziliensis
2
------2
YES
Pocillopora meandrina
1
------1
YES
Stylopora pistillata
1
------1
YES
Acropora
cervicornis
--10
----10
YES
Hexacorals,
Scleractinians,
Acropora palmata
--21
----21
YES
Complex clade
Galaxea fascicularis
3
------3
YES
Isopora palifera
------117
117
YES
Pavona cactus
1
------1
YES
Porites astreoides
5
11
----16
YES
Porites compressa
1
------1
YES
Porites cylindrica
1
------1
YES
Porites lobata
1
------1
YES
Porites lutea
3
------3
YES
Octocorals, Gorgonians Gorgonia ventalina
21
536
----557
YES
3 distinct coral lineages 20 coral species
136
715
156
117
1124
Only found in
zooxanthellate corals