DNA metabarcoding reveals unexpected predator-prey-microbial dynamics in a Southern Ocean predator (Eubalaena australis)

Supplementary information for DNA metabarcoding reveals unexpected predator-prey-microbial dynamics in a Southern Ocean predator (Eubalaena australis) Aashi Parikh1*, Richard O’Rorke2, Emma L. Carroll2, Els Vermeulen3, Robert Harcourt1, Stephanie Plön4, William J. Rayment5, Anthony Chariton1 1School of Natural Sciences, Macquarie University, Wallumattagal Campus, Macquarie Park NSW 2109 Australia 2School of Biological Sciences, Waipapa Taumata Rau – University of Auckland, Auckland 1142 Aotearoa - New Zealand 3Mammal Research Institute Whale Unit, Faculty of Natural and Agricultural Sciences, University of Pretoria, Hatfield 0028 South Africa 4BioConsult SH, Schobüller Str. 36, Husum 25813 Germany 5Department of Marine Science, Ōtākou Whakaihu Waka - University of Otago, Dunedin 9016 Aotearoa – New Zealand *Corresponding author – [email protected]; (m): +61 475 579 168 Table S1. PCR conditions for the three metabarcoding primers used in this study. PCRs were performed in two rounds with conditions for the second round being the same for all three primer sets. PCR Round 1 Amplicon PCR Mixture PCR Conditions 18S Reagent Volume (uL) PCR Stage Temperature Time PCR cycles Mastermix 10 Initial denaturation 95°C 8 min Primer-F (0.5uM) 3.5 Denaturation 95°C 20 s x 35 Primer-R (0.5uM) 3.5 Annealing 58°C 20 s PNA clamp* 1 Elongation 72°C 30 s DNA 2 Final elongation 72°C 8 min Crust16S Mastermix 10 Initial denaturation 95°C 10 min Primer-F (0.5uM) 4 Denaturation 95°C 20 s x 45 Primer-R (0.5uM) 4 Annealing 51°C 30 s DNA 2 Elongation 72°C 45 s Final elongation 72°C 10 min 16S Mastermix 10 Initial denaturation 95°C 10 min Primer-F (0.5uM) 4 Denaturation 95°C 20 s x 30 Primer-R (0.5uM) 4 Annealing 50°C 30 s DNA 2 Elongation 72°C 45 s Final elongation 72°C 5 min PCR Round 2 The second round of PCR for all 3 amplicons added Illumina Unique Dual Indexes (UDIs) from index adapter sets A, B and C. Each PCR reaction was 16 mL, including Q5 High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, USA), 0.2 mM UDI, and 1 mL of product from the first round PCR diluted to 5%. The PCR conditions were: 30s of initial denaturation at 98°C; 14x PCR cycles of 10s denaturation at 98°C, 10s annealing at 57°C and 20s elongation at 72°C; concluding with a 1 min final elongation at 72°C. *Whale blocking PNA clamp: CGACCGTCTTCTCAGC-Lys Table S2. Frequency of prey at finest taxonomic resolution detected at each calving/socialising ground (Algoa Bay, Auckland Islands and Fowlers Bay) and foraging ground (St Helena Bay). Order Species Algoa Bay Auckland Islands Fowlers Bay St Helena Bay Polymorphida Polymorphidae 0 2 0 0 Diplostraca Penilia avirostris 1 1 4 10 Calanoida Calanoida 0 0 1 9 Calanoida Parvocalanus crassirostris 1 0 0 0 Neocopepoda Neocopepoda 1 0 1 10 Amphipoda Cyamus boopis 0 0 0 4 Cumacea Diastylis laevis 0 0 0 12 Decapoda Epilobocera capolongoi 0 0 2 0 Decapoda Goneplax rhomboides 0 0 0 1 Decapoda Hymenosoma geometricum 0 0 0 1 Decapoda Hymenosoma orbiculare 0 0 0 2 Decapoda Jasus 0 0 0 1 Decapoda Monodaeus 0 0 0 5 Decapoda Mursia cristiata 0 0 0 4 Decapoda Neoxanthops quadrilobatus 0 0 1 0 Decapoda Ovalipes trimaculatus 0 0 0 4 Decapoda Penaeoidea 0 0 0 1 Decapoda Pilumnoides perlatus 0 0 2 11 Decapoda Pilumnus 0 0 0 0 Decapoda Plagusia chabrus 1 0 0 1 Decapoda Portunus 1 0 2 4 Decapoda Thia scutellata 0 1 1 5 Decapoda Xanthidae 1 0 0 0 Euphausiacea Euphausia 0 0 0 5 Euphausiacea Euphausia superba 0 0 0 2 Euphausiacea Nyctiphanes australis 1 1 2 0 Euphausiacea Thysanoessa gregaria 0 0 1 2 Euphausiacea Thysanoessa sp. BD2006 0 0 0 3 Isopoda Scutuloidea maculata 0 1 0 0 Stomatopoda Pterygosquilla schizodontia 0 0 0 12 Aphragmophora Sagittidae 0 0 0 2 Phlebobranchia Ciona intestinalis 0 0 0 1 Leptothecata Leptothecata 0 0 0 2 Scyphozoa Scyphozoa 0 0 0 3 Semaeostomeae Chrysaora 0 0 0 11 Semaeostomeae Pelagiidae 0 0 0 2 Euheterodonta Euheterodonta 0 0 1 0 Galeommatida Montacutidae 1 0 1 0 Nudibranchia Jorunna tomentosa 0 0 0 1 Plagiorchiida Plagiorchiida 0 1 0 0 Table S3. Relative abundances of bacterial phyla detected in the gut microbiomes of SRWs from calving/socialising and foraging grounds. Calving/socialising Foraging Phylum Mean relative abundance (%) SE Phylum Mean relative abundance (%) SE Firmicutes 87.06 2.27 Firmicutes 76.22 3.20 Actinobacteria 8.38 1.09 Bacteroidetes 12.59 2.62 Fusobacteria 2.13 2.08 Actinobacteria 5.04 1.06 Bacteroidetes 1.33 0.92 Spirochaetota 3.58 1.36 Proteobacteria 0.48 0.33 Proteobacteria 1.05 0.26 Verrucomicrobiota 0.43 0.16 Verrucomicrobiota 1.02 0.26 Synergistota 0.09 0.05 Euryarchaeota 0.19 0.06 Spirochaetota 0.04 0.02 Mycoplasmatota 0.10 0.04 Euryarchaeota 0.04 0.03 Synergistota 0.10 0.03 Lentisphaerota 0.02 0.01 Lentisphaerota 0.09 0.04 Mycoplasmatota 0.001 0.001 Fusobacteria 0.01 0.01 Cyanobacteriota 0.000 0.000 Cyanobacteriota 0.004 0.002 Planctomycetota 0.000 0.000 Planctomycetota 0.003 0.002 Table S4. Relative abundances of top 10 most abundant bacterial classes (or higher taxonomic levels not resolved to the genus) detected in the gut microbiomes of SRWs from calving/socialising and foraging grounds each. Calving/socialising Foraging Order Mean relative abundance (%) SE Order Mean relative abundance (%) SE Clostridia 63.82 4.01 Clostridia 66.68 3.79 Erysipelotrichia 17.61 4.68 Bacteroidia 12.53 2.62 Coriobacteriia 7.48 1.03 Erysipelotrichia 6.52 2.20 Firmicutes_c 3.00 1.13 Coriobacteriia 3.78 0.77 Bacilli 2.49 2.31 Spirochaetia 3.58 1.36 Fusobacteriia 2.13 2.08 Firmicutes_c 1.92 0.63 Bacteroidia 1.31 0.91 Actinobacteria_c 1.25 0.65 Actinobacteria_c 0.90 0.12 Bacilli 0.63 0.30 Gammaproteobacteria 0.43 0.31 Verrucomicrobiota 0.60 0.20 Verrucomicrobiota_c 0.29 0.13 Betaproteobacteria 0.49 0.18 Table S5. Relative abundances of top 25 most abundant bacterial genera (or higher taxonomic levels not resolved to the genus) detected in the gut microbiomes of SRWs from calving/socialising and foraging grounds each. Calving/socialising Foraging Genus Mean relative abundanc e (%) SE Genus Mean relative abundanc e (%) SE Romboutsia 23.69 5.45 Clostridium sensu stricto 28.68 6.34 Faecalibaculum 16.12 4.79 Phocaeicola 7.68 1.72 Clostridium sensu stricto 8.02 3.47 Oscillospiraceae_g 7.65 1.31 Coriobacteriia_g 5.23 0.80 Romboutsia 4.61 1.77 Ihubacter 4.47 0.69 Vescimonas 3.63 0.92 Oscillospiraceae_g 4.23 0.91 Treponema 3.55 1.35 Peptococcus 3.96 1.02 Terrisporobacter 3.48 1.36 Firmicutes_g 3.00 1.13 Faecalibaculum 2.80 1.68 Mediterraneibacter 2.71 0.77 Faecalitalea 2.75 1.58 Guopingia 2.51 1.47 Flintibacter 2.60 1.28 Carnobacterium 2.30 2.15 Lachnospiraceae_g 2.46 0.81 Cetobacterium 2.13 2.08 Prevotellamassilia 2.16 0.63 Mogibacterium 2.12 0.46 Firmicutes_g 1.92 0.63 Peptacetobacter 1.95 0.76 Ihubacter 1.78 0.58 Gallibacter 1.66 0.86 Coriobacteriia_g 1.58 0.56 Dorea 1.21 0.37 Peptococcus 1.58 0.35 Eubacteriaceae_g 1.17 0.14 Guopingia 1.49 0.88 Lachnospiraceae_g 1.03 0.47 Xylanibacter 1.22 0.37 Eubacteriales_g 0.94 0.21 Curtanaerobium 1.08 0.40 Erysipelotrichaceae_g 0.88 0.15 Bariatricus 0.99 0.43 Atopobiaceae_g 0.81 0.40 Eubacteriales_g 0.93 0.19 Oscillibacter 0.81 0.28 Bifidobacterium 0.89 0.62 Lentihominibacter 0.61 0.15 Oscillibacter 0.88 0.18 Vescimonas 0.55 0.13 Mediterraneibacter 0.85 0.46 Curtanaerobium 0.50 0.15 Dorea 0.70 0.21 Fig. S1. Proportion of gut bacterial classes (or higher taxonomic levels not resolved to class) with >5% total relative abundance, detected in whale faecal samples from calving/socialising and foraging grounds. Only samples where prey was also detected have been included (59% of the whales from calving/socialising grounds and 86% of the whales from the foraging grounds). Fig. S2. Principal Component Analysis (PCA) of Hellinger transformed (a) SRW prey communities and (b) gut bacterial communities, with prey communities grouped by SRW migratory stages: calving/socialising and foraging Fig. S3. Tanglegram comparing the clustering of gut bacterial and prey communities in the sampled SRWs. The straight bars connecting the prey and bacterial communities suggest high similarity in community structure, with significant clusters highlighted in dark pink. Sample names are coloured in light purple for calving/socialising SRWs and dark purple for foraging SRWs. Table S6. ANOVA results for stepwise selection model used to select SRW prey groups significantly associated with gut bacterial communities. Df Variance F Pr(>F) Cumacea 1 0.01556 1.2212 0.203 Decapoda 1 0.01693 1.329 0.19 Euphausiacea 1 0.02977 2.3364 0.025 * Neocopepoda 1 0.03685 2.8919 0.009 ** Polymorphida 1 0.02604 2.0437 0.022 * Semaeostomeae 1 0.07392 5.8009 0.001 *** Stomatopoda 1 0.03317 2.6027 0.001 ***