Intestinal microbiome richness of coral reef damselfishes (Actinopterygii: Pomacentridae)

doi:10.21203/rs.3.rs-1739757/v1

Fish gastro-intestinal system harbours diverse microbiomes that affect the host’s digestion, nutrition and immunity. Despite the great taxonomic diversity of fish, little is understood about fish microbiome and the factors that determine its structure and composition. Damselfish are important coral reef species that play pivotal roles in determining algae and coral population structures of reefs. Broadly, damselfish belong to either of two trophic guilds based on whether they are planktivorous or algae-farming. In this study, we used 16S rRNA gene sequencing to investigate the intestinal microbiome of five planktivorous and five algae-farming damselfish species (Pomacentridae) from the Great Barrier Reef. We detected Gammaproteobacteria ASVs belonging to the genus Actinobacillus in 80% of sampled individuals across the two trophic guilds, thus, bacteria in this genus can be considered possible core members of pomacentrid microbiomes. Algae-farming damselfish had greater bacterial alpha-diversity, a more diverse core microbiome and shared 35 ± 22 ASVs, whereas planktivorous species shared 7 ± 3 ASVs. Our data also highlight differences in microbiomes associated with both trophic guilds. For instance, algae-farming damselfish were enriched in Pasteurellaceae, whilst planktivorous damselfish in Vibrionaceae. Finally, we show shifts in bacterial community composition along the intestines. ASVs associated with the classes Bacteroidia, Clostridia and Mollicutes bacteria were predominant in the anterior intestinal regions while Gammaproteobacteria abundance was higher in the stomach. Our results suggest that the richness of the intestinal bacterial communities of damselfish reflects host species diet and trophic guild.

General Microbiology

Damselfish

microbiome

gastrointestinal

coral reef

bacteria

Pomacentridae

Fishes represent the greatest taxonomic diversity of vertebrates, and despite our understanding of the importance of intestinal microbiota of terrestrial vertebrates, we still lack an understanding of fish microbiome diversity and functioning [1]. Largely, fish microbiome studies have centred around species with commercial value, including trout, salmon and carp [2]. For example, gastrointestinal fish microbiomes are known to be important in intestinal cell proliferation [3, 4], nutrition [1, 5] and immunity [6-8]. These studies show that the intestines of fishes harbour a large abundance and diversity of bacteria [9] and the regulation of this diversity is important in the maintenance of host health through a complex set of microbe-microbe and microbe-host interactions [10, 11].

There are many factors that affect the structure of fish gastrointestinal microbiomes [1, 2]. These include host-related factors such as genetic attributes, size, age, sex [12-14], host phylogeny [15-17], environmental factors (such as water quality) [16, 18, 19] and host diet [15, 18]. Studies that investigated intestinal microbiome changes have mostly focused on the impact of fish foods on species of aquaculture importance [20, 21], although a few studies have investigated wild fish populations[15, 22]. For instance, bacterial symbionts diversification in wild herbivorous surgeonfish intestines is thought to be an important driver of host niche-partitioning [23, 24], suggesting that intestinal microbiomes can influence the trophic ecology of coral reefs and facilitate resource partitioning in these hyper-diverse ecosystems. However, the involvement of intestinal bacteria in wild fish physiology remains largely unknown.

There is increasing evidence that herbivorous fishes have distinct microbiomes as compared to omnivorous and carnivorous fishes [25]. Herbivorous and carnivorous fish diets are known to cause shifts in intestinal microbiomes; fishes with plant-based diets have intestinal microbiomes dominated by Firmicutes, such as Clostridium, while fishes with fat-based diets have microbiomes dominated by protease producing Proteobacteria [26-28]. In addition, the diversity of herbivorous fish intestinal microbiomes is higher than omnivorous and carnivorous host species under similar environmental conditions [29], suggesting that host feeding behaviour has a significant effect on fish intestinal microbiomes.

Damselfishes (Pomacentridae) are a diverse and abundant group of coral reef fishes [30, 31], and they are among the most widely studied families [32, 33]. Broadly, damselfishes are grouped into either planktivorous or algae-farming trophic guilds, although some herbivorous species may also feed on zooplankton [34]. Planktivorous damselfishes play a key role in transferring energy from the plankton to higher tiers of the food chain, while algae-farming damselfishes influence sediment and algae dynamics on coral reefs and may increase the presence of coral disease-associated pathogens within their territories [33, 35-39]. Algae-farming species can be differentiated based on the algal composition within their territories, and they are divided into several behavioural guilds, including indeterminate grazers, extensive grazers, and intensive grazers [33, 35, 40, 41]. Indeterminate and extensive grazers feed both on macroalgae and turf, while intensive grazers maintain distinct areas of turf algae through selective grazing and weeding of unpalatable algae [40, 42]. Intensive grazing damselfish are also referred to as algae farmers. Research on intensive grazers has focused on competition [43], patterns of co-existence [34, 44, 45], behavioural interactions [46, 47], and their role in structuring algae and coral communities [35, 36, 48-52].

In this study, we investigated and described the intestinal microbial diversity of ten species of planktivorous and algae-farming damselfishes, two guilds that significantly impact coral reef trophic dynamics. We hypothesised that differences in intestinal microbial communities will reflect the differences between these two trophic guilds. Specifically, across the different host species and trophic guilds, we examined (1) differences in bacterial communities across fish species and trophic guilds, (2) core microbial members and (3) changes in microbial community structure along the length of the intestinal tract.

Species collections and dissections

Fishes were collected from the Heron Island lagoon in the southern Great Barrier Reef, Australia (23°26’53”S, 151°56’52”E) in January and February of 2015. Collections occurred at a depth of 1-8 m adjacent to the Heron Island Research Station. Three individuals of ten sympatric damselfish species (Abudefduf sexfasciatus, A.whitleyi, Acanthochromis polyacanthus, Acanthochromis polyacanthus, Chromis atripectoralis, Dischistodus pseudochrysopoecilus, D. perspicillatus, Pomacentrus moluccensis, P. wardi, Stegastes apicalis and S. nigricans) of similar lengths were randomly collected across the two trophic guilds planktivorous and algae-farming. Each trophic guild was represented by five species and 15 individuals. Collections were conducted on SCUBA, and the planktivorous species were collected using a barrier net, while the algae-farming species were collected using a speargun. Following collections, the fishes were immediately placed on ice and transported to Heron Island Research Station. In the laboratory under sterile conditions, fishes were weighed, measured and photographed, then the gastrointestinal tract was removed, and the gut length was recorded and photographed. The entire gut was fixed in 4% DNA/RNA free paraformaldehyde and sterile phosphate-buffered saline for 12 hours, then it was stored in DNA/RNA free water.

DNA extraction, amplification and sequencing

Samples were transported to James Cook University for subsampling along each intestinal tract and DNA extraction. Under sterile conditions, standardized biopsy cores (3x3 mm) were taken from four locations along the intestinal tract: the stomach, the anterior intestine, the mid-intestine, and the posterior intestine. DNA was extracted from tissue biopsies using a QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) following the manufacture's guidelines. A nanodrop was used to record the quality (260/280 ratio) and quantity (ng/μL) of DNA from each extraction.

Amplification of the 16S V1-V3 rRNA gene region was done using the primers 27F (5’-AGRGTTTGATCMTGGCTCAG-3’) [53] and 519R (5’-GTNTTACNGCGGCKGCTG-3’) [54] with barcodes on the forward primer. These 16S rRNA genes were amplified using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Then the pooled and purified PCR products were used to prepare a DNA library by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at the Molecular Research LP (MR DNA; Texas, USA) on a MiSeq V2 System following the manufacturer’s guidelines.

Amplicon sequence data were sorted by the sample and demultiplexed using demux for QIIME 2 (version 2018.11; [55]). Sequences were screened for quality, trimmed at 450 bp after removal of primer sequences and assigned as amplicon sequence variants (ASVs) using DADA2 [56]. Taxonomy of the ASVs was determined using a pre-trained, naїve Bayes classifier [57] and the q2-feature-classifier plugin [58]. The classifier was trained on the target 480 bp region of sequences in the Greengenes 13_8 99% database. ASV clusters were arranged in a phylogenetic tree using FastTree [59] and visualised using Interactive Tree of Life 3.6.1 [60]. The feature table, metadata and taxonomic classifications were exported from QIIME 2 in .biom format and the rooted phylogenetic tree was exported in .nwk format. The closest known sequences and the origin of selected ASVs were identified through a BLASTN-based search against the GenBank nr/nt database.

Statistical analysis

The feature table and phylogenetic tree were imported into R version 3.5.2 and stored as a phyloseq object [61] for downstream analyses. All ASVs not assigned to phylum were filtered from the data, and those designated as chloroplasts or cyanobacteria were removed and stored as a separate object for further analysis. Samples were rarefied to minimum sampling depth for alpha-diversity analyses, which was estimated using the R package vegan [62]. Non-rarefied data were used for generalized linear model (GLM) analysis [63, 64]. Data used for principal component analysis (PCA), betadisper-test and PERMANOVA were computed using centered log-transformed Euclidean distance matrices of the non-rarefied ASV table. Differences in alpha-diversity between trophic guilds were tested via t-test. Multivariate GLM was used to test for significant differences in bacterial communities among host fish species, trophic guild and location along intestines using mvabund in R [65]. PCA, betadisper-test and PERMANOVA were used to test differences in the communities of Proteobacteria, Bacteroidetes and Firmicutes among fish species and between the two trophic guilds. Bacterial taxa were grouped by class when examining microbiome changes along the length of the intestinal tract. Bacterial community data were fitted to negative binomial distributions and tested using log-likelihood ratios (LRT) via 999 simulations using Monte Carlo resampling. A nested analysis of variance (ANOVA) used to test the role of trophic guild and gut location when accounting for species variation. Venn diagrams were produced using the VennDiagram package [66].

A total of 1,254,909 sequences were detected in 119 samples after denoising and removing all chloroplast, mitochondria and uncharacterized sequences. Among these sequences, 3,776 ASVs were detected; 39.4% of which belonged to the phyla Proteobacteria, 26.2% to Bacteroidetes, 13.4% to Firmicutes and 12.6% to Planctomycetes. The 20 most abundant ASVs accounted for 41% of the total number of detected sequences. The most common ASV belonged to the genus Actinobacillus and accounted for 9.9% of the total detected sequences (Table 1). Two unknown species of Mollicutes and Pasteurellacea accounted for 6.9 and 3.8% of sequences, respectively.

Different ASV richness was detected for each fish species with observed ASVs (t = -3.15, p = < 0.01) and Shannon index (t = -3.68, p = < 0.01) differing significantly between the two trophic guilds. The damselfish D. perspicillatus had the greatest mean richness of ASVs, with a total of 322 ± 17 ASVs per individual (Fig. 1). The species with the lowest ASV richness were C. atripectoralis and A. sexfasciatus with 47 ± 21 and 30 ± 8 ASVs per individual, respectively (Fig. 1). Shannon diversity was greatest for two algae-farming species D. perspicillatus and S. apicalis and lowest for the planktivorous species A. polyacanthus and P. moluccensis. PCA biplots, betadisper-test and PERMANOVA revealed that the beta-diversity of Proteobacteria, Bacteroidetes and Firmicutes communities differed among fish species and trophic guilds (Fig. 2; Table 2).

Core Microbiomes

In line with previous studies that investigated the core microbiome of other organisms [67, 68], we choose a minimum threshold of 30% for this metric. Most ASVs occurred in less than 30% of sampled individuals across all fish species (Fig. 3a). A total of 13 bacterial ASVs were found in more than 30% of sampled individuals; therefore, they may represent the 30% core microbiome of pomacentrid investigated in this study (Table 3). The most common ASV in this study belonged to the genus Actinobacillus, which occurred in more than 80% of sampled individuals (Table 3), albeit at a low abundance in many individuals, with the highest abundances in the planktivorous damselfishes A. polyacanthus and P. moluccensis.

The core bacterial assemblages of each fish species (defined as ASVs that were shared between all sampled individuals for each species) were composed of a variable number of ASVs (Fig. 3b). For example, there were 70 bacterial ASVs shared between the three sampled individuals of D. perspicillatus and only two ASVs shared between the three A. sexfasciatus individuals. Core microbiomes within fish species were richer in algae-farming species than planktivorous species (Fig. 3b), with algae-farming species sharing 35 ± 22 ASVs and planktivorous species sharing only 7 ± 3 ASVs (Wilcox test W = 25, p < 0.01).

Core ASVs that occurred in all three individuals of a fish species belonged to the phyla Bacteroidetes, Firmicutes, Tenericutes, Spirochaetes, Planctomycetes, Proteobacteria and Verrucomicrobia. Core ASVs belonging to Coraliomargarita sp. and Verruco-5 (Verrucomicrobia), Pirellulaceae (Planctomycetes) and Desulfovibrionaceae (Deltaproteobacteria) occurred in all three sampled D. perspicillatus individuals (Supplementary Fig. 1). We also detected high diversity of an unknown clade of Gammaproteobacteria in P. moluccensis and P. wardi damselfish. There were 61 core ASVs belonging to the Bacteroidetes, 28 of which occur in S. apicalis and 38 in D. perspicillatus (Supplementary Fig. 2). An unknown clade of Flavobacteriales and a diverse consortium of Rikenellaceae were core members of S. apicalis, while D. perpicillatus had a diverse core assemblage of ASVs belonging to the family Flavobacteriaceae. One ASV belonging to Spirochaetes, Brevinema andersonii, was a core member of S. nigricans and C. atripectoralis, while a Tenericutes ASV belonging to Mollicutes was a core member of all fish species except the planktivorous damselfishes A. polyacanthus and A. sexfasciatus (Supplementary Fig. 3). There was a rich consortium of core Firmicutes ASVs for S. apicales and S. nigricans, which included members of the Erysipelotrichaceae, Ruminococcaceae and Lachnospiraceae families.

Bacterial shifts along the intestinal tract

The interaction between the trophic guild and intestinal region had a significative influence on the gut bacterial community composition (LRT = 152, p = 0.001; Supplementary Table 1). The abundance of nine classes of bacteria changed significantly across the different fish species and locations along the intestinal tract (LRT = -0.0229, p < 0.001; Fig. 4; Supplementary Table 2). Members of Gammaproteobacteria were especially common throughout the planktivorous intestinal tracts, but we also found them along all the intestines regions of the algae-farming species D. perspicillatus, D. pseudochrysopoecilus and P. wardi (Fig. 4). In intestinal regions where Gammaproteobacteria were uncommon, members of Bacteroidia and Clostridia were generally found at higher abundances – especially for algae-farming species (Fig. 4). Members of the Mollicutes and Planctomycetia were more common throughout the intestinal tracts of algae-farming hosts than planktivorous species although their abundances were generally lowest within the stomach region (Fig. 4). The stomach had 286 unique bacterial ASVs, the anterior intestine 753, while 1,139 and 656 ASVs were only found in the mid and posterior intestines, respectively (Fig. 5). Only 19 ASVs were common in the stomach and posterior intestine while 152 ASVs were found throughout the intestine (Fig. 5).

Effect of the trophic guild on microbiomes

There was a significant difference in the microbiome composition between trophic guilds (LRT= -0.021, p < 0.001; Supplementary Table 2). Most bacterial ASVs were unique to either of the trophic guilds, with only 124 ASVs common to both guilds (Fig. 5). A total of 78 bacterial ASVs, belonging to 20 families, were important drivers of this relationship. There were marked differences in abundances of ASVs belonging to Vibrionaceae, Lachnospiraceae and Pasteurellaceae. Two Vibrio sp. (Vibrionaceae) were more common in planktivorous species, and five ASVs of Actinobacillus (Pasteurellaceae) were more abundant in algae-farming species.

Our data show that algae-farming damselfish species have richer microbiomes than planktivorous species (Fig. 1) and this result is also reflected in their core bacterial community (Fig. 3). This result is likely attributable to the specialised feeding behaviour of algae-farming species, which largely consume a narrow range of turf algae species [41, 50], unlike planktivorous species that are adapted to a more opportunistic feeding strategy. These results suggest that the microbiome structure of fish species with specialised feeding behaviour has acquired specific intestinal bacteria and further research is needed to investigate how microbiome specialisation affects host digestion and metabolism. We also note that other processes that were not tested in our study such as host phylogeny and functional traits could influence the composition of damselfish intestinal bacteria and ultimately influence fish physiology.

We found that similar to what was recorded in many other species of marine fish, the damselfish intestinal microbiome was dominated by members of Proteobacteria, Bacteroidetes, Firmicutes and Planctomycetes (Table 1). For example, surgeonfish, parrotfish and rabbitfish intestinal microbiomes from the Red Sea also consist of diverse assemblages of Firmicutes and Proteobacteria [15]. Another dominant ASV in the damselfish microbiome belonging to Mollicutes (Tenericutes) resembled bacteria detected in rabbitfish intestines [22]. The number of highly similar bacterial ASVs shared among pomacentrids, acanthurids and siganids may reflect the similar feeding behaviours of these coral reef fishes. For instance, algae-farming damselfishes may also ingest prey items other than algae, such as zooplankton [34] or other invertebrates [69]. The functional roles of these seemingly important microbial taxa warrant further attention in order to understand the potential consequences on host metabolism and health.

Damselfish microbiomes were largely dominated by the family Pasteurellaceae in the phylum Gammaproteobacteria, with one ASV (b727) occurring in more than 80% of sampled fishes and representing almost 10% of the total detected sequences (Tables 1 and 3). Although this ASV currently represents an unknown species in the Actinobacillus genus, a 98% similar sequence has been retrieved from the intestines of surgeonfishes in Saudi Arabia [24], suggesting that Actinobacillus are common reef fish microbiomes. Members of Pasteurellaceae have also been recorded in high abundances in adult damselfishes and cardinalfishes collected around Lizard Island, Australia [70], and they are deemed as common components of tropical planktivorous fish gut microbiomes [71]. The prevalence of Pasteurellaceae amongst the damselfishes in this study, as well as other reef fishes, provides additional evidence that Pasteurellaceae are likely important members of coral reef-associated fish microbiomes.

Algae-farming damselfishes had more observed ASVs and larger core microbiomes than planktivorous species (Figs. 1 and 3), and these core microbiomes were specific to each host species (Fig. 3). For example, P. wardi and P. moluccensis microbiomes were dominated by different taxa of Gammaproteobacteria, while D. perspicillatus and S. apicalis had large Bacteroidia core communities but were dominated by Flavobacteriaceae and Rikenellaceae, respectively. Different species of algae-farming damselfishes consume different species of algae [50], and the large differences in their specialized microbiomes may reflect these narrow dietary preferences. Conversely, the small core microbiomes of the planktivorous damselfishes may reflect the high variation in consumed plankton of each species, suggesting these fishes have opportunistic feeding behaviours. These results, however, do not support the notion that fish with greater diet variability have more diverse microbiomes [25]. In fact, the damselfish with narrow, algae-farming feeding behaviours tended to have the greatest diversity of intestinal bacteria, suggesting that the host-microbiome interactions may select for specialised bacteria that enhance the digestion and absorption of nutrients from specific algal diets. The richer microbiome of algae-farming fishes could also reflect the necessity of this trophic guild to be associated with a pool of symbionts that facilitate the breakdown of algal cellulose. We also acknowledge that some of the bacteria we retrieved from the damselfish intestine could have been associated with the food recently ingested by the fish and, therefore, not being part of the damselfish microbiome.

Recent evidence suggests a high degree of resource partitioning in fish communities which is a key mechanism that facilitates the high diversity of coral reefs [72, 73]. The largely distinct microbiomes of each host species presented in this study may reflect the high degree of resource partitioning found in coral reef communities, whereby different species of damselfish may be consuming different size classes of zooplankton [73], farm different algal species [50] or occupy different trophic niches [72]. The similarity between closely related host species and microbiomes, such as P. wardi and P. moluccensis, also demonstrates that phylogeny may influence the intestinal microbiomes of damselfishes [15, 16, 18, 74].

Interestingly, Photobacterium damselae, Vibrio harveyi, Vibrio ponticus and other Vibrio sp. were prevalent amongst the damselfishes sampled in this study (Table 3). These bacteria represent potential pathogenic members of Vibrionacaea and have been detected in many fishes of aquaculture importance, including Chromis punctipinnis [75], Lutjanus argentimaculatus [76], Seriola dumerili [77], Scophthalmus maximus [78], Sparus aurata [79], and Solea senegalensis [80]. Although identified as Vibrio harveyi in the GreenGenes database, GenBank revealed there was a high similarity of these sequences to other members of the Harveyi clade, such as Vibrio owensii [77]. It is thought that there are up to 11 species of Vibrio belonging to this clade [81], most of which are pathogens of fish, shrimp and coral [82–84]. Given the apparently healthy state of the sampled fishes and the high abundances of potentially pathogenic Vibrionacaea in the fish guts, we provide support to the idea that these organisms are natural components of healthy fish microbiomes and are opportunistic pathogens in fishes only under specific conditions [76, 85]. Future studies should investigate the involvement of algae-farming damselfish in the spreading of pathogens across reef organisms. For instance, it has recently been reported that the seagrass pathogen Labyrinthula was present in the skeleton of a common coral species [86] and probably infected the abundant endolithic algae living in the coral skeleton [87–89]. It is possible that damselfishes grazing near alive corals were the medium that allowed the pathogen Labyrinthula to infect the corals’ endolithic algae.

The facultative anaerobic bacterial classes Bacteroidia, Clostridia and Mollicutes were generally in higher abundance in the mid and posterior intestinal regions than in the stomach (Fig. 4). Differences in microbiomes along the intestinal tract have been recorded in the rabbitfish Siganus fuscescens [90], with midgut communities more representative of the environmental sources and hindguts hosting a microbiome more specialised to anaerobic conditions and fermentation [91]. The increase in Bacteroidia, Clostridia and Mollicutes along the intestines may be due to some members of these bacterial classes being mutualistic components of the fish gastrointestinal microbiome. Some members of Bacteroidia are known to breakdown polysaccharides and metabolise the derived sugars [92], while members of Clostridium are known to metabolise cellulose [28]. Our results confirm the increased prevalence of anaerobic bacteria in the hindgut of damselfishes, which probably consists of taxa responsible for the fermentation and metabolism of complex molecules before being absorbed by the host [1]. We also note that Actinobacillus sp. that could breakdown cellulose via fermentation [93] were more abundant in the gut of algae-farming damselfish, suggesting that these bacteria could aid the digestion of fish in this trophic guild.

In this study, we show that damselfishes have diverse intestinal microbial communities whereby the bacterial richness of a species reflects diet and trophic guild. We show that algae-farming damselfishes have richer bacterial alpha-diversity and core microbiomes, which may reflect the more specialised diets of this trophic guild. We also provide evidence that damselfish mid and posterior intestines have higher abundances of facultative anaerobic bacteria that are known to play important roles in fermentation and cellulose breakdown. These findings add to a growing body of literature that suggests that host fish feeding behaviour has a strong influence on the composition of intestinal microbiomes.

Ethics approval and consent to participate

All work was authorized by James Cook University, permitting limited impact research under the university’s research accreditation in the Great Barrier Reef Marine Park.

Consent for publication

Not applicable

Availability of data and material

The Illumina MiSeq datasets for each damselfish species are available at the Sequence Read Archive (NCBI) repository under BioProject accession number PRJNA638998, https://www.ncbi.nlm.nih.gov/sra. Data and R-scripts used in this study are available at https://github.com/ChrisKav/WildDamselfishMicrobiomes.

Competing interests

The authors declare that they have no competing interests.

Funding

Not applicable

Authors’ contributions

CRJK analysed and interpreted the amplicon sequence data and was the major contributor to writing the manuscript and preparing figures and tables. FR provided feedback on the data analysis, figures design and manuscript writing. JMC undertook the fieldwork and collected all specimens, performed gut dissections, tissue biopsies and provided feedback on the manuscript. JHC was involved with the initial synthesis and design of this study and provided feedback on the manuscript. WL and TDA were involved with the initial synthesis and design of this study, provided the facilities to undertake laboratory work and provided feedback on the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Philip Munday for his involvement with the synthesis and design of this study. We also thank Simon Brandl, Ben Chapman, Alejandra Hernández Agreda, César Herrera, and the staff at Heron Island Research Station for field and logistical support.

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Table 1: Sequence abundance and taxonomy for each ASVs representing more than 1% of total sequences. Accession numbers for closest GenBank sequences (similarity given in brackets) are supplied.

ASV	Phylum	Lowest taxonomic division	Number of Sequences	Proportion of total (%)	GenBank Accession Number
b727	Proteobacteria	Actinobacillus sp.	124,499	9.9	KT952745 (97.5%)
5647	Tenericutes	Mollicutes	87,057	6.9	HG971018 (96.3%)
94ba	Proteobacteria	Pasteurellacea	47,527	3.8	KT952745 (93.5%)
3023	Firmicutes	Ruminococcaceae	26,355	2.1	MG488771 (98.8%)
6350	Tenericutes	Mycoplasmataceae	24,219	1.9	LN612674 (91.5%)
9b2f	Proteobacteria	Pasteurellacea	24,219	1.9	KT952745 (91.9%)
d532	Proteobacteria	Alteromonadales	23,877	1.9	KT952746 (100.0%)
5a8a	Proteobacteria	Vibrio ponticus	22,112	1.8	MG524941 (100%)
7936	Proteobacteria	Alteromonadales	15,147	1.2	KT952746 (99.8%)
596f	Proteobacteria	Gammaproteobacteria	14,436	1.2	LC121875 (88.4%)
73d1	Proteobacteria	Vibrio sp.	13,977	1.1	KT952854 (98.7%)
6013	Proteobacteria	Pasteurellacea	13,435	1.1	KT952745 (92.3%)
af86	Firmicutes	Clostridium colinum	13,177	1.1	KC993540 (94.2%)

Table 2: Results of betadisper-test and PERMANOVA testing the beta-diversity of Proteobacteria, Bacteroidetes and Firmicutes across fish species and between trophic guilds.

	Fish species		Trophic guild
	betadisper	PERMANOVA	betadisper	PERMANOVA
Proteobacteria	p = 0.084	F = 1.86; p = 0.001***	p = 0.039*	F= 3.52; p = 0.001***
Bacteroidetes	p = 0.269	F= 1.78; p = 0.001***	p = 0.233	F= 2.41; p = 0.001***
Firmicutes	p = 0.001***	F= 2.17; p = 0.001***	p = 0.355	F= 3.92; p = 0.001***

Table 3: Taxonomic composition of core ASVs occurring in more than 30% of sampled

individuals. Accession numbers for closest GenBank sequences (similarity given in brackets) are supplied. Occurrence and relative abundances were generated from rarefied data.

ASV	Phylum	Lowest taxonomic division	Occurrence (%)	Relative Abundance	GenBank Accession Number
b727	Proteobacteria	Actinobacillus sp.	83.3	0.083	KT952745 (97.5%)
94ba	Proteobacteria	Actinobacillus sp.	53.3	0.017	KT952745 (93.5%)
9bd9	Proteobacteria	Photobacterium damselae	43.3	0.013	CP035457 (100%)
5647	Tenericutes	Mollicutes	40.0	0.022	HG971018 (96.3%)
a832	Proteobacteria	Photobacterium damselae	40.0	0.008	CP018297 (100%)
73d1	Proteobacteria	Vibrio sp.	40.0	0.010	KT952854 (98.7%)
9b2f	Proteobacteria	Actinobacillus porcinus	40.0	0.018	KT952745 (91.9%)
6c33	Proteobacteria	Spirobacillales	37.7	0.002	KU578602 (100%)
dc1c	Proteobacteria	Vibrio sp.	37.7	0.004	CP033144 (100%)
5a8a	Proteobacteria	Vibrio ponticus	37.7	0.019	MG524941 (100%)
762a	Bacteroidetes	Lutimonas sp.	30.0	0.001	MG488523 (99.6%)
ca47	Proteobacteria	Vibrio harveyi	30.0	0.009	CP033144 (100%)
6013	Proteobacteria	Pasteurellaceae	30.0	0.007	KT952745 (92.3%)

SupplementaryFigure1v3.tif
Supplementary Figure 1: Phylogenetic tree of core Planctomycetes, Proteobacteria and Verrucomicrobia ASVs. Coloured host species reflect whether the ASV was present in all three sampled individuals of each host fish species. Top five species in the legend represent planktivorous and bottom five represent algae-farming species.
SupplementaryFigure2v3.tif
Supplementary Figure 2: Phylogenetic tree of core Bacteroidetes ASVs. Coloured host species reflect whether the ASV was present in all three sampled individuals of each host fish species. Top five species in the legend represent planktivorous and bottom five represent algae-farming species.
SupplementaryFigure3v3.tif
Supplementary Figure 3: Phylogenetic tree of core Firmicutes, Spirochaetes and Tennericutes ASVs. Coloured host species reflects whether the ASV was present in all three sampled individuals of each host fish species. Top five species in the legend represent planktivorous and bottom five represent algae-farming species.
SupplementaryTables.docx
Supplementary Table 1: ANOVA table summarising multivariate GLM model of gut location nested within fish species. Results based on 999 simulations.Supplementary Table 2: ANOVA table summarising multivariate GLM model of trophic guild and fish species as factors. Results based on 999 simulations.

Intestinal microbiome richness of coral reef damselfishes (Actinopterygii: Pomacentridae)

Status:

Version 1

Abstract

Figures

Background

Methods

Species collections and dissections

DNA extraction, amplification and sequencing

Statistical analysis

Results

Core Microbiomes

Bacterial shifts along the intestinal tract

Effect of the trophic guild on microbiomes

Discussion

Conclusions

Declarations