TU_12 Sphingobacterium sp. enrichment culture clone GIN723 padj ... TU_1843 Sphingobacterium pakistanense padj = 0.00022029. O .... spiritivorum + sp. 3.
16S rRNA sequencing reveals likely beneficial core microbes within faecal samples of the EU protected slug Geomalacus maculosus Inga Reich, Umer Zeeshan Ijaz, Mike Gormally, Cindy J. Smith
Supplementary Information
Supplementary Table S1. Diversity measures for each sample Sample site
Sample ID
Glanteenassig Forest Glanteenassig Forest Glanteenassig Forest Ballaghbeama Gap Ballaghbeama Gap Ballaghbeama Gap Ballycarbery Ballycarbery Ballycarbery Lough Currane Lough Currane Lough Currane Derrycunnihy Woods Derrycunnihy Woods Derrycunnihy Woods Barraduff Barraduff Barraduff Raferigeen Raferigeen Crookhaven Derreen Forest Derreen Forest Derreen Forest Glengarriff Woods Glengarriff Woods Glengarriff Woods Cloosh Forest Cloosh Forest Cloosh Forest Reared - Lichen Reared - Lichen Reared - Lichen Reared - Oats Reared - Oats Reared - Oats
A1 A4 A5 C2 C3 C5 D1 D2 D3 E1 E4 E5 F1 F4 F5 H2 H3 H4 I1 I2 J1 M2 M3 M4 N1 N2 N5 T1 T2 T4 L1 L2 L3 O2 O3 O5
Observed OTUs 319 269 389 444 445 533 255 364 371 336 319 233 575 315 163 445 523 346 305 189 586 422 406 313 348 560 573 312 389 294 234 335 260 254 204 159
Number Number of Species of doubletons Richness singletons 93 58 290.21 102 51 241.82 124 60 273.16 110 71 357.77 141 90 339.3 103 96 400.65 54 35 193.36 94 69 298.32 116 74 324.62 62 42 285.55 117 37 234.89 87 43 159.09 171 103 421.23 105 59 224.17 50 27 139.69 144 63 344.39 185 100 410.29 85 60 275.54 85 40 210.72 71 25 136.63 125 98 453.7 117 64 331.32 139 58 309.16 113 44 313 116 54 284.7 179 111 417.12 167 112 454.98 114 50 210.56 154 66 278.86 57 48 248.21 69 54 176.63 73 60 271.69 64 49 180.7 77 43 194.52 42 26 160.37 36 21 119.66
Shannon's Diversity Index 3.61 3.36 2.14 4.4 3.65 4.22 3.04 3.5 3.47 3.07 2.17 1.28 4.14 2.2 2.15 3.46 3.67 3.13 3 2.27 3.27 3.97 3.64 3.73 3.02 4.02 4.02 2.61 2.52 2.82 2.82 3.27 2.79 2.72 2.93 2.65
Pielou's Evenness 0.63 0.6 0.36 0.72 0.6 0.67 0.55 0.59 0.59 0.53 0.38 0.23 0.65 0.38 0.42 0.57 0.59 0.53 0.52 0.43 0.51 0.66 0.61 0.65 0.52 0.64 0.63 0.45 0.42 0.5 0.52 0.56 0.5 0.49 0.55 0.52
Supplementary Table S2 Sample weight [g] and DNA amount [μg/ml] of PCR product Sample A1 A4 A5 C2 C3 C5 D1 D2 D3 E1 E4 E5 F1 F4 F5 H2 H3 H4 I1 I2 J1 M2 M3 M4 N1 N2 N5 T1 T2 T4 L1 L2 L3 O2 O3 O5
Weight 0.14 0.08 0.06 0.06 0.07 0.07 0.07 0.1 0.08 0.09 0.15 0.08 0.16 0.12 0.12 0.04 0.03 0.02 0.05 0.05 0.02 0.18 0.17 0.13 0.08 0.02 0.09 0.08 0.05 0.12 n/a n/a n/a n/a n/a n/a
DNA amount 17.7 17.3 14.6 14 21.5 25.6 25.2 17 26.9 36.8 16 20 25.2 23.5 18.9 15.5 22.8 11.2 12.7 27.9 13.1 32.9 21.8 18.3 25.1 33 15.5 17.9 25.8 12.9 15.8 30.1 15.1 23.2 26.3 19.1
Pielou's evenness *
Richness **
Supplementary Figure S1. Pielou's evenness, richness and Shannon's Diversity Index for each sample (dots) grouped by site and arranged by habitat (ES = Exposed Siliceous). The thick horizontal line is the median sample value while the box shows the interquartile range. Statistically significant differences between sites at p < 0.05 are shown above the plot.
Shannon * 7
p= 0.038 *
p= 0.04 *
800
p= 0.015 * p= 0.026 * p= 0.0022 ** p= 0.043 * p= 0.027 * p= 0.034 * p= 0.021 * p= 0.01 ** p= 0.016 * p= 0.014 *
p= 0.0025 ** p= 0.0077 ** p= 0.01 ** p= 0.028 *
1.0
p= 0.016 * p= 0.026 * p= 0.04 * p= 0.0099 ** p= 0.045 * p= 0.0078 ** p= 0.023 * p= 0.027 * p= 0.048 *
0.8
600
p= 0.027 *
p= 0.036 *
p= 0.013 *
p= 0.0011 ** p= 0.0014 ** p= 0.011 * p= 0.032 * p= 0.013 * p= 0.021 * p= 0.038 * p= 0.017 * p= 0.028 * p= 0.0041 ** p= 0.0053 ** p= 0.014 * p= 0.036 *
6
p= 0.011 * p= 0.0018 ** p= 0.012 * p= 0.013 *
p= 0.035 * p= 0.023 *
●
Observed Values
p= 0.04 *
5
p= 0.013 *
Groups
p= 0.027 * ●
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Glanteenassig Forest
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Ballaghbeama Gap
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Barraduff
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Ballycarbery
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Lough Currane
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Derrycunnihy Woods
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Glengarriff Woods
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Raferigeen
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Crookhaven
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Derreen Forest
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Cloosh Forest
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Lichen
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Oats
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0.6
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400
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3
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0.4
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2
200
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Reared
Mixed Forest
Blanket Bog
Wet Grassl.
Heath
Samples
Conifer Plantation
Reared
Mixed Forest
Blanket Bog
Wet Grassl.
Heath
Conifer Plantation
Oats
Lichen
Site T
Site M
Deciduous Forest
Site I
ES Rock
Site J
Site N
Site D
Site H
Site C
Site A
Oats
Lichen
Site T
Site M
Deciduous Forest
Site I
ES Rock
Site J
Site N
Site D
Site H
Site C
Site A
Oats
Lichen
Site T
Conifer Plantation
Site F
Heath
Site M
Site I
Site J
Site N
Site F
Deciduous Forest
Site E
ES Rock
Site F
Wet Grassl.
Site E
Site D
Site H
Blanket Bog
Site E
Mixed Forest
Site C
Site A
0.2
Reared
A1
Bray-Curtis
R2 = 0.45, P = 0.004
B
UW UniFrac
R2 = 0.28, P = 0.001
C
A2
UW UniFrac
R2 = 0.45, P = 0.001
UW UniFrac
D
A3
W UniFrac
R2 = 0.42, P = 0.03
R2 = 0.25, P = 0.008
UW UniFrac
R2 = 0.05, P = 0.006
E1
UW UniFrac
E2
R2 = 0.49, P = 0.001
Supplementary Figure S2.
NMDS
W UniFrac
plots
based
on
Bray-Curtis,
R2 = 0.47, P = 0.001
unweighted
(UW)
and weighted (W) UniFrac distances and colour coded by sample site (A, E), habitat (B), substrate (C) or environment type (D). E1 and E2 also include the reared specimens. Only plots with P < 0.05 are shown.
Type
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
−10.0 ●
Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
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Reared Wild
Reared Wild
Reared Wild
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Reared Wild
−7.5
Reared Wild
Reared Wild
Reared Wild
Log−relative normalised
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OTU_432 Saccharibacteria padj = 0.00089012
OTU_61 Mucilaginibacter sp. JC2901 padj = 0.00051239
OTU_214 Brevundimonas padj = 0.00045497
OTU_3 Microvirga padj = 0.00044284
OTU_76 Bryobacter padj = 0.00042318
OTU_422 Acetobacter padj = 0.00041015
OTU_1063 Granulicella padj = 0.00037436
OTU_35 Opitutae vadinHA64 padj = 0.0003355
OTU_841 alphaI cluster padj = 0.00031005
OTU_1368 Raoultella padj = 0.00028917
OTU_53 Mucilaginibacter padj = 0.00027794
OTU_381 Bacillus padj = 0.00027659
OTU_1558 Rhizobiales padj = 0.00027659
OTU_488 Nosocomiicoccus padj = 0.00022297
OTU_1843 Sphingobacterium pakistanense padj = 0.00022029
OTU_100 Acidobacteriaceae (Subgroup 1) padj = 0.00020363
OTU_866 Serratia padj = 0.00016682
OTU_38 Burkholderia padj = 0.00012858
OTU_373 Marvinbryantia padj = 9.5586e−05
OTU_93 Edaphobacter padj = 9.5586e−05
OTU_51 Singulisphaera padj = 7.3183e−05
OTU_17 Rahnella padj = 6.2109e−05
OTU_1971 Roseomonas padj = 6.2109e−05
OTU_137 Pseudomonas padj = 4.9663e−05
OTU_67 Burkholderia sp. CPA6 padj = 4.8505e−05
OTU_277 Pedobacter sp. padj = 3.997e−05
OTU_2372 Stenotrophomonas padj = 2.7385e−05
OTU_46 Mucilaginibacter padj = 2.7385e−05
OTU_26 Lactococcus padj = 2.3149e−05
OTU_461 alphaI cluster padj = 2.0497e−05
OTU_104 Luteolibacter padj = 1.1032e−05
OTU_558 Dyadobacter padj = 1.0145e−05
OTU_585 Delftia sp. padj = 1.0145e−05
OTU_166 Massilia sp. DR−a8 padj = 1.0145e−05
OTU_27 Chitinolyticbacter meiyuanensis padj = 1.0145e−05
OTU_32 Opitutae vadinHA64 padj = 7.1803e−06
OTU_11 Burkholderia padj = 5.2396e−06
OTU_790 Acidobacteriaceae (Subgroup 1) padj = 4.8811e−06
OTU_196 Flavobacterium padj = 4.3368e−06
OTU_2847 Cedecea sp. padj = 4.1721e−06
OTU_271 Burkholderia sp. IpA−51 padj = 2.4542e−06
OTU_262 Prosthecobacter sp. EBTL04 padj = 1.2141e−06
OTU_216 Serratia padj = 9.543e−07
OTU_2 Rhodococcus padj = 6.0453e−07
OTU_7 Mucilaginibacter sp. RG4−7 padj = 4.5207e−07
OTU_70 Bdellovibrio padj = 3.3593e−07
OTU_1414 Enterobacter padj = 2.2073e−07
OTU_770 Comamonadaceae padj = 6.7626e−08
OTU_342 Raoultella ornithinolytica padj = 2.9368e−08
OTU_3185 Singulisphaera padj = 2.9368e−08
OTU_223 Paenibacillus sp. FSL R5−0345 padj = 2.3795e−08
OTU_3139 Singulisphaera padj = 2.1537e−08
OTU_186 Albidiferax padj = 5.1294e−09
OTU_243 Candidatus Captivus padj = 4.2559e−09
OTU_161 Rohodovulum sp. padj = 3.1418e−09
OTU_47 Beijerinckiaceae padj = 2.9778e−09
OTU_37 Singulisphaera padj = 2.6351e−09
OTU_580 Pseudomonas padj = 1.7224e−09
OTU_9 Flavobacterium padj = 1.4735e−09
OTU_65 Acidobacterium padj = 6.0564e−10
OTU_153 Comamonas padj = 6.0487e−13
OTU_71 Planomicrobium padj = 2.4429e−13
OTU_3075 Escherichia coli 2−156−04_S1_C1 padj = 1.2489e−13
OTU_4 Acidiphilium padj = 1.0964e−13
OTU_224 Enterobacteriaceae padj = 4.952e−14
OTU_5 Ewingella americana ATCC 33852 padj = 3.6346e−15
OTU_106 Comamonas padj = 8.1795e−16
OTU_206 Achromobacter padj = 3.4189e−16
OTU_94 Nubsella zeaxanthinifaciens padj = 2.2016e−19
OTU_3117 Cedecea sp. padj = 3.0311e−22
OTU_40 Flavobacterium lindanitolerans padj = 1.3866e−22
OTU_12 Sphingobacterium sp. enrichment culture clone GIN723 padj = 8.8426e−31
OTU_10 Stenotrophomonas padj = 1.3667e−34
Supplement ary Figure S3. OTUs with log-fold changes between reared and wild specimens (OTUs without any classification not shown).
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−2.5 ● ● ●
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Type
●
Reared
Wild
Supplementary Figure S4. The most common isolation sources of the bacteria from the NCBI website which were identified as matches to the sequences in this study Wild specimens
Soil Sea Sea water Sediment Glacier Biofilm Rhizosphere Forest soil Waste water Coast Others
Reared - Lichen
Soil Rhizosphere Lake Sea water River Wetland Agricultural soil Epilimnion Sea Cave Others
Reared - Oats
Soil Sea water Sea Rhizosphere Plantation Marine sediment Sediment Bay Inlet River Others
Supplementary Figure S5. Bacteria isolated from guts, crops and faeces from other studies investigating terrestrial snails and slugs (only genera mentioned in more than 1 study are included) Authors Organism(s) Method Bacteria isolated from Class Bacteroidetes
Order Bacteriodales Flavobacteriales
Family Bacteroidaceae Flavobacteraceae
Genus Bacteroides Chryseobacterium
Cardoso et al. 2012 Charrier et al. 1980 Joynson et al. 2014 Joynson et al. 2017 Rossing & Hietpas 2007 Stalder et al. 2014 Helix pomatia , Achatina fulica Arion ater Arion fasciatus Arion vulgaris Arion ater Cornu aspersum (land snail) (terrestrial slug) (terrestrial slug) (terrestrial slug) (terrestrial slug) (land snails) Culture Culture based + Culture Culture based Culture based Culture based independent independent independent Gut and crop Gut Gut and crop Gut Gut Faeces
B. sp.
B. sp. + vulgatus C. gleum
Wilkinson 2010 Deroceras reticulatum (terrestrial slug) Culture based + independent Gut
C. sp.
M. paludis
M. sp. Sphingobacteriales Sphingobacteriaceae Mucilaginibacter
Firmicutes
Beta-Proteobacteria
Clostridiales Lactobacillales
Burkholderiales
Clostridiaceae Carnobacteriaceae
Enterococcaceae Streptococcaceae Comamonadaceae Oxalobacteraceae
Sphingobacterium Clostridium Carnobacterium
Enterococcus Lactococcus Comamonas Janthinobacterium
Clostridiaceae
L. sp. C. sp.
C. sp.
Enterobacteriales
Aeromonadaceae
C. leptum C. sp.
E. casseliflavus, malodoratus + raffinosus L. lactis
A. sp. Gamma-Proteobacteria Aeromonadales
E. casseliflavus + sp.
A. hydrohila B. agrestis + noackiae
Enterobacteriaceae Buttiauxella C. sp. Citrobacter
C. freundii
E. amnigenus
Enterobacter sp.
Enterobacter E. amylovora + tasmaniensis Erwinia
K. intermedia
O. proteus P. sp. 57917
Pantoea
P. carotovorum
Pectobacterium Rahnella Raoultella
S. sp.
Serratia
A. sp.
Acinetobacter
P. sp.
5 B. sp.
C. koseri, rodentium, youngae + sp.
C. freundii
C. freundii
A. beijerinckii
B. noackiae C. freundii + sp.
E. cloacae, cancerogenus + sp. E. amylovora, bellingiae, pyrifoliae, tasmaniensis + sp. E. albertii, coli, fergusonii + sp.
E. aerogenes + sp.
7
4
3 E. coli
E.-Shigella
E. sp.
E. americana
K. cryocersens
K. sp.
P. ananatis, vagans + sp. P. carotovorum, wasabiae + sp. R. sp.
3 2 3 5 1 2
R. sp.
S. enterica + typhimorium S. odorifera, proteamaculans + sp.
R. sp. R. sp.
2 3 3 2
S. sp.
A. baumannii, calcoaceticus, haemolyticus, johnsonii, junii, lwoffii, radioresistens + sp. P. aeruginosa, entomophila, fluorescens, mendocina, palustris, putidae, savastanoi, stutzeri, syringae + sp. S. maltophilia
4
E. sp.
K. sp. K. pneumoniae, variicola + sp.
3 4 3 2
A. media + sp.
R. sp.
S. sp.
Pseudamonadaceae Pseudomonas Xanthomonadales Xanthomonadaceae Stenotrophomonas
A. encheleia , hydrophilia , media , popoffii , salmonicida + sobria
R. terrigena
Salmonella
Pseudomonadales Moraxellaceae
J. sp.
K. sp. K. intermedia
Kluyvera Obesumbacterium
L. sp. C. sp.
B. agrestis
C. gillenii + sp.
K. sp.
A.hydrophilia , salmonicida + sp.
Observed studies B. sp. 3 3 C. daeguense + sp. M. calamicampi, mallensis, ximonensis + sp. 3 S. pakistanense, spiritivorum + sp. 3 C. saccharogumia 4 C. sp. 2 E. faecium + sp.
L. lactis C. testosteroni J. sp.
Aeromonas
Escherichia Ewingella Klebsiella
S. sp.
S. spiritivorum
This study Geomalacus maculosus (terrestrial slug) Culture independent Faeces
S. plymuthica + sp.
4
A. indicus + sp.
4
P. tolaasii
S. maltophilia
P. aeruginosa + sp.
S. sp.
P. lutea + sp.
S. sp.
5 4
