Coral-dwelling Caribbean gobies exhibit distinct skin microbiota compared to other sympatric species

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Hendrick, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7390480/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Apr, 2026 Read the published version in Coral Reefs → Version 1 posted 9 You are reading this latest preprint version Abstract Microbial communities fundamentally shape ecosystem function and biodiversity across all biological systems through complex dynamics. In coral reef ecosystems, understanding the dynamics of these microbial communities has become critical for predicting reef responses to environmental stressors. Fish skin microbiota are highly susceptible to environmental changes and may vary significantly across species and geographic locations, yet the extent to which these variations occur remain poorly understood. Here, we compared the skin microbiota of four closely related and sympatric cryptobenthic gobiid species, that exhibit different behavioral ecologies (cleaning vs non-cleaning), ecological niches (water column, coral-, or sand-dwelling) and phylogenetic affinities ( Elacatinus vs Coryphopterus ), yet reside in the same reef patches in St. Croix and Puerto Rico, eastern Caribbean. Coral-dwelling gobies, including cleaning sharknose and non-cleaning peppermint gobies, exhibited significantly lower microbial diversity compared to reef-hovering and sand-dwelling species (both non-cleaning). These coral dwellers showed unique microbial signatures despite having similar alpha diversity levels. Core microbiota analysis also revealed striking differences between coral-dwelling and reef-hovering/sand-dwelling species, with the core microbiome of the former dominated by Vibrio , Pseudoalteromonas , and Alteromonas in the case of cleaning gobies and by Endozoicomonas in the case of peppermint gobies, while reef-hovering and sand-dwelling gobies exhibited diverse core microbiota with greater overlap between species. Ecological niche occupancy and reef habitat selection appear to be primary drivers of skin microbiota composition in gobiid fishes, rather than cleaning behavior and/or host phylogenetic affinities alone, though species-specific skin mucus properties likely also contribute to selective bacterial colonization patterns. skin microbiome cleaner fish cryptobenthic Elacatinus evelynae Coryphopterus spp. goby coral reefs Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Microorganisms represent dynamic and crucial components of biological systems, shaping the structure and function of ecosystems from the molecular to the landscape scale. Microbial communities serve as fundamental drivers of ecosystem function and biodiversity, mediating biogeochemical processes that underpin the stability and productivity of natural systems (Arrigo 2005 ; Rousk and Bengtson 2014 ). Beyond their ecosystem-wide functions, host-microbial associations both influence and are influenced by animal physiology and behavior (Archie and Tung 2015 ; Vuong et al. 2017 ; Sehnal et al. 2021 ). Host-associated microbial communities are shaped by an array of interconnected factors including environmental gradients (Nguyen et al. 2021 ), networks of trophic interactions (Gralka et al. 2020 ), host-microbe interactions, microbe-microbe competition and cooperation, and stochastic processes (Adair and Douglas 2017 ). Coral reefs are among the most productive and biodiverse systems per unit area on Earth (Reaka-Kudla 1997 ; Knowlton et al. 2010 ), with microbes playing a crucial role in their processes (Haas et al. 2011 ; Silveira et al. 2017 ; Rappuoli et al. 2023 ). Understanding microbial dynamics within coral reef ecosystems has emerged as a crucial frontier in marine ecology (Ainsworth et al. 2010 ), as these microbial assemblages facilitate coral homeostasis through symbiotic relationships and pathogen defense, potentially driving coral adaptation to stress and environmental changes (Bourne et al. 2016 ; Peixoto et al. 2017 ; Webster and Reusch 2017 ). While coral microbiomes are increasingly researched (Hernandez-Agreda et al. 2017 ; Dunphy et al. 2019 ; van Oppen and Blackall 2019 ; Vanwonterghem and Webster 2020 ; Mohamed et al. 2023 ), there is still a knowledge gap regarding the larger coral reef ecosystem microbiome. In particular, the microbiomes of resident fish, which may act as promoters of microbial connectivity between different reef environments due to their mobility (Leon-Zayas et al. 2020 ). Seminal research indicates that reef fish microbiota are distinct from bacterioplankton; they are highly variable but species-specific, with host phylogeny and diet influencing fish skin, gill and gut microbial assemblages (Chiarello et al. 2018 ; Pratte et al. 2018 ; Kavazos et al. 2022 ). Additionally, reef fish microbiota are also seemingly influenced by host age (Pratte et al. 2018 ; Xavier et al. 2020 ) and regional/local habitat conditions (Xavier et al. 2020 ; Pereira et al. 2023 ), such as levels of eutrophication (Degregori et al. 2021 ). Among the most abundant yet understudied components of coral reef fish communities are cryptobenthic gobies, which represent a highly variable group of small-bodied fishes that are closely associated with the reef substrate (Brandl et al. 2018 ). Despite their small size and cryptic nature, gobiids exhibit an important ecological diversity across Caribbean reefs, occupying a wide range of microhabitats including live coral heads, sponges, rubble, and sandy areas (Taylor and Hellberg 2005 ; Harborne et al. 2012 ). This ecological diversification is evident across multiple genera, with species from the Elacatinus genus being coral-dwelling cleaners or sponge associates (Taylor and Hellberg 2005 ), while Coryphopterus species demonstrate adaptations to various substrates from coral to rubble and open sand (Beeken et al. 2021 ; Ziebell et al. 2023 ). Obligate or “dedicated” cleaner fish are unique to coral reef systems and have an ecological impact that is disproportional to their abundance (Côté 2000 ; Vaughan et al. 2017 ). Through the activity of removing parasites from other fishes (clients), they attract multiple species and thus have significant impacts on the local abundance and diversity of fishes (Bshary 2003 ; Grutter et al. 2003 ; Brown et al. 2025 ). Because of their daily physical contact with multiple other fishes, as well as contact with the substrate, cleaner fishes are potentially exposed to a much wider variety of microbes than most other reef fishes. Dedicated cleaner fish include two genera from two families: cleaner wrasses of the genus Labroides in the Indo-Pacific, and cleaning gobies of the genus Elacatinus in the Caribbean. While convergent in coloration and overall ecological function (Cheney et al. 2009 ), cleaning gobies are much smaller than wrasses and reside on the substrate, typically live coral or sponge (Côté and Soares 2011 ; Whittey et al. 2021 ; Budd et al. 2024 ), leaving only temporarily to tend to a client before returning. In a comparison of cleaning and sponge-dwelling ecotypes of the broadstripe cleaning goby Elacatinus prochilos , Xavier et al. ( 2019 ) showed that gobies that resided on live coral and engaged in more cleaning had significantly higher skin microbial diversity and higher abundance of potential pathogens than those that seldom cleaned and resided on sponges, leading to the hypothesis that the frequent contact with other fish during cleaning could promote microbial transmission and higher diversity. A prediction of this hypothesis was later tested in a field study by Pereira et al. ( 2023 ), confirming an association between levels of cleaning activity of the Caribbean sharknose cleaning goby, Elacatinus evelynae , and increased variability of their skin microbiome. A more recent study reported that the presence of E. evelynae in the reef directly impacts the microbial diversity of the coral heads where cleaners are stationed, as well as the microbial heterotrophs of water near the cleaning station (Brown et al. 2025 ). Although these effects were context-dependent, they suggest that frequent contact with a cleaner and/or increased local fish traffic can impact reef holobiont and the skin microbiota of fish residents (Brown et al. 2025 ). The intimate association of cryptobenthic gobies with specific reef microhabitats, combined with their limited mobility and high site fidelity, makes them ideal model organisms for understanding how fine-scale habitat differences and behavioral ecology influence host-associated microbial communities in coral reef ecosystems. Here, we report findings of a field study at St. Croix and Puerto Rico, eastern Caribbean, comparing the skin microbiota of the cleaning goby E. evelynae with another three closely related cryptobenthic gobiid species collected from the same reef patches that exhibit different reef habitat associations and/or behavioral ecologies. These included a non-cleaning coral-dwelling species and two non-cleaning species that do not reside on live hard coral. We hypothesized that the microbiomes of the gobies captured in the two islands will show intraspecific differences between islands due to different environmental conditions. Within each island, we expected that the cleaning gobies will have higher microbial diversity when compared to the non-cleaning gobies. We also hypothesized that the microbial composition in gobiid skin could reflect phylogenetic affinities of their hosts, or that it could be driven by the reef habitat where they live. Methods Study species Our study included two reef-hovering goby species ( Coryphopterus hyalinus and Coryphopterus personatus ), one sand-dwelling species ( Coryphopterus tortugae ), and two coral-dwellers ( Coryphopterus lipernes and Elacatinus evelynae ). Goby species are typically segregated in different habitats, however, two sister taxa, C. hyalinus (glass goby) and C. personatus (masked goby) (Thacker and Cole 2002 ; Baldwin and Robertson 2015 ), form heterospecific shoals, which hover above the benthos (Beeken et al. 2021 ; Selwyn et al. 2022 ). These two species feed on plankton (Beeken et al. 2021 ), are morphologically very similar and may occasionally hybridize (Selwyn et al. 2022 ), and therefore will be referred to as C. hyalinus/personatus (glass/masked goby) in our study. C. lipernes (peppermint goby) is sister to the C. hyalinus/personatus clade (Baldwin and Robertson 2015 ) and lives in close physical contact with live corals, where they spend most of their time (Smith and Tyler 1977 ). Data on their diet is scarce, but they have been seen feeding on particulate matter in the water column (Smith and Tyler 1977 ). C. tortugae (patch-reef goby) is the most phylogenetic distant relative of the other Coryphopterus species considered herein (Thacker and Cole 2002 ; Baldwin and Robertson 2015 ), with individuals living in sand patches between coral outcrops and preying upon small benthic invertebrates (Kramer et al. 2009 ). The coral-dwelling E. evelynae (sharknose cleaning goby) is an obligate cleaner that contacts with multiple heterospecific client species, feeding on their ectoparasites and dead skin through cleaning interactions (Côté and Soares 2011 ). Study area and sample collection In June and July 2021, we collected gobies (n = 78) from two localities in the Eastern Caribbean Sea: Cane Bay, along the north shore of St. Croix, US Virgin Islands (17º46’26.8’’N 64º48’45.6’’W) and Media Luna reef, off La Parguera, Puerto Rico (17°56'16.5"N 67°03'10.8"W) from reef habitat at depths from 10–18 m. Within each island, gobiid species were collected from the same reef patches. The number of gobies collected for each species per island is given in Table S1 of the Supplementary Information. We collected gobies individually, using hand nets and without the use of anesthetic. For each collecting dive, we used a different net for each fish in order to avoid cross contamination. Immediately after collection, we placed each fish in individual hermetically sealed plastic bags, which we then placed in a mesh bag for transport back to the shore or boat, respectively. We then transferred each bag into an insulated plastic container to maintain thermal stability until processing. At the lab, we swabbed the entire surface of each fish using a Puritan HydraFlock swab (Guilford, ME), handling each fish with sterile gloves. We placed swab samples in cryovials and then deposited them into a dry shipper, which kept samples cold using liquid nitrogen vapors (St. Croix) or a -80 ºC freezer (Puerto Rico) before being sent to Woods Hole Oceanographic Institution (WHOI) for processing. We returned fish to the site of collection within 24 hours. Water (60 ml) was also collected from the fish collection depth at each site using a syringe. Back at the lab, the water was filtered through 25 mm, 0.2 µm Supor filters (Pall, Port Washington, NY) and the filter deposited into a cryovial and frozen as detailed above. DNA extraction and libraries preparation DNA from the fish skin swabs and water filter was extracted using the DNeasy PowerBiofilm kit (Qiagen, Germantown, MD) following manufacturer’s protocol. Extractions included negative controls (swab, filter, and extraction blank). Libraries were prepared using a standard dual indexing amplification of the V4 hypervariable region of the 16S rRNA gene (Kozich et al. 2013 ) with the primers 515Y (Parada et al. 2016 ) and 806RB (Apprill et al. 2015 ). The Polymerase Chain Reactions (PCRs) were performed in a total volume of 50 µL with 10 µL of GoTaq Flexi colorless buffer, 5 µL of 25 mM MgCl2, 1 µL of each 10 mM forward and reverse primers, 1 µL of 10 mM dNTPs, 0.5 µL GoTaq DNA polymerase (Promega, Madison WI), 29.5 µL of PCR grade water, and 4 µL of DNA template. Each PCR was run with a negative control, and the following PCR conditions were used: of 95 ºC for 2 min, and then 95 ºC (20 s), 55 ºC (15 s), 72 ºC (5 min) for 30–35 cycles, and a final elongation step of 72 ºC for 10 min. PCR products were visualized on a 1.5% agarose gel, and the target amplification product was identified based on a 50 bp ladder and a positive control. Amplified bands were gel excised, cleaned, and purified with the MinElute Gel Purification kit (Qiagen) following manufacturer instructions. Purified products were quantified using the Qubit 2.0 fluorometer (Invitrogen, Waltham, MA). Each purified sample product was added to a final pool with a target concentration of 2 ng/µL. Each pool also contained negative PCR controls using PCR-grade water, and the Microbial Mock Community B (even, low concentration, HM-782D, BEI Resources, ATCC). The pooled libraries were sequenced on a MiSeq (Illumina, San Diego CA) for 250 bp paired-end sequencing in three different runs. Sequence processing The DADA2 pipeline (Callahan et al. 2016 ) (version 1.32.0) was used on the demultiplexed FASTQ files from the combined sequencing runs for quality control, merging paired-end reads, sequencing error rates estimation and quality filtering, using the following parameters: truncLen = c(190, 150), maxN = 0, maxEE = c(2, 2), truncQ = 2. Reads were collapsed into Amplicon Sequence Variants (ASVs) discarding chimeras, and an ASV frequency table was constructed. The mock community of each run was verified against the reference mock and then, removed from the dataset. The SILVA reference database (Quast et al. 2013 ) (release 138) was used to assign the taxonomy, and ASVs that remained unclassified or were classified as belonging to Family mitochondria and Class chloroplast were removed from the dataset. The presence of potential contaminants was checked by calculating the prevalence of taxa of the negative controls (extraction, PCR, and swab blanks) with a probability threshold of 0.1 in all dataset using the package decontam (Davis et al. 2018 ). Moreover, the presence of water-associated microbial taxa in the skin swabs was identified using the water filter samples using the same method. Contaminant and water-associated ASVs were removed from the dataset, as well as control and water samples. Singletons and samples with less than 1000 reads were also removed (n = 1, Glass/Masked goby from St. Croix), and read normalized counts were obtained using the negative binomial distribution implemented in DESeq2 (Love et al. 2014 ). Raw sequence reads were deposited into NCBI’s Short Read Archive under accession PRJNA986111. Microbial diversity analyses Alpha diversity was estimated using Shannon and Fisher indices using the R package phyloseq (McMurdie and Holmes 2013 ), and Faith’s Phylogenetic Diversity (PD) using picante (Kembel et al. 2010 ). For each diversity index, a first linear model was used to test for intraspecific differences between islands, including only cleaning gobies and patch-reef gobies, with the factor goby species nested within island in the model. Peppermint gobies were not used in this analysis because they were sampled only in St. Croix, as well as patch-reef gobies due to insufficient sample size in Puerto Rico (n = 2), which would create unbalanced comparisons between locations. Then, to test the effect of cleaning behavior and reef habitat, two linear models were performed with island as a nested factor: cleaning versus non-cleaning gobies, and coral-dwelling (cleaning and peppermint gobies) versus reef-hovering/sand-dwelling gobies (glass/masked and patch-reef gobies). Beta diversity was estimated using the Bray Curtis dissimilarity and weighted and unweighted Unifrac indices using the R package phyloseq. To test our hypothesis, we performed permutational multivariate analysis of variance (PERMANOVA) tests on the normalized data using similar statistical models to the ones for alpha diversity, with the adonis function of the vegan package (Oksanen et al. 2020 ), with 999 permutations and the option for a sequential test. When significant results were found, pairwise PERMANOVAs were performed using the pairwise.adonis2 wrapper function (Martinez Arbizu 2020 ) with the Bonferroni p-value correction for multiple comparisons. Dissimilarity in microbial structure among all samples was visualized using Principal Coordinates Analysis (PCoA) with the three beta diversity indices previously calculated. The most abundant microbial taxa for each species were assessed by collapsing ASVs to the genus level and defined by ≥ 2% on average of all sequences within location. For each species sampled in St. Croix, the core microbiome was also assessed using the relative abundance transformed data with the function core from the package microbiome (Lahti et al. 2017 ) and a threshold of 90% prevalence. Samples from Puerto Rico were not included in the core analysis due to low sample number for glass/masked gobies (n = 2) and therefore the lack of a representative number of species to allow the comparison between gobies with different types of behavior and reef habitat. Lastly, to infer a phylogenetic signal in the skin microbial composition of gobies, a hierarchical clustering of the Bray Curtis dissimilarity was performed for each species within both islands using the function hclust from the R base package stats , using the ward.D2 method. The resulting dendrogram was compared to the most recent published phylogenies of the Coryphopterus genus (Baldwin and Robertson 2015 ; Forrester et al. 2021 ) to assess whether microbial community clustering patterns reflect known phylogenetic relationships. For all statistical tests, differences were considered significant when p < 0.05. Results A total of 4 041 048 merged reads were recovered after filtering and cleaning the dataset against negative and water samples. The number of reads per individual sample ranged between 7 311 and 221 493, resulting in a total of 11 058 ASVs. A first statistical model was used to compare the skin microbiome of the same fish species collected across both islands. Although no significant differences were found for alpha diversity ( F 0.38; Fig. 1 ; Table 1 ), beta diversity was significantly different ( R 2 > 0.05, p = 0.001; Fig. 1 ; Table 1 ) in gobies between Puerto Rico and St. Croix. The most abundant microbial genera in the skin of gobies sampled at Puerto Rico included Alteromonas , Ekhidna , Mycoplasma , and Vibrio , while in St. Croix the most abundant microbial genera found included Endozoicomonas , Pseudoalteromonas , and Vibrio , although the abundances varied among species (Fig. 2 ). A striking high abundance of Endozoicomonas in the skin of peppermint gobies is noteworthy, with a relative abundance of more than 75% of this genus in 8 out of 16 samples. Comparison between cleaning and non-cleaning gobies To test for differences in the skin microbiome between cleaning and non-cleaning gobies at each location (Puerto Rico and St. Croix), the microbiome of the sharknose cleaning goby was compared with non-cleaning gobies (i.e. peppermint, patch-reef and glass/masked gobies) within locations. No significant differences were found in the alpha diversity between cleaning and non-cleaning gobies ( F 0.13; Fig. 1 ; Table 1 ), although microbial beta diversity differed significantly ( R 2 > 0.04, p = 0.001; Fig. 1 ; Table 1 ), with pairwise comparisons showing differences in both islands with the Bray Curtis and Unweighted Unifrac indices. Table 1 – Differences in the skin microbial alpha and beta diversity between goby species sampled in different islands, between cleaner and non-cleaner gobies and between coral and reef-hovering/sand habitats. Alpha diversity Df Sum Sq Mean Sq F-value Pr(> F) Island comparison Shannon 1 0.32 0.32 0.30 0.58 Fisher 1 1015.00 1014.80 0.77 0.38 PD 1 5.30 5.28 0.02 0.89 Cleaner vs. Non-cleaner Shannon 2 0.49 0.25 0.13 0.88 Fisher 2 1956.00 978.18 0.86 0.43 PD 2 603.60 301.79 1.03 0.36 Coral vs. Reef Habitat Shannon 2 25.49 12.74 8.07 0.0007 Fisher 2 8341.00 4170.50 3.95 0.02 PD 2 2334.60 1167.30 4.31 0.02 Beta diversity Df Sum Sq R 2 F-value Pr(> F) Island comparison Bray Curtis 1 1.51 0.08 3.97 0.001 Weighted Unifrac 1 0.01 0.14 7.44 0.001 Unweigthed Unifrac 1 0.97 0.05 2.34 0.001 Cleaner vs. Non-cleaner Bray Curtis 2 3.14 0.10 4.34 0.001 Weighted Unifrac 2 0.01 0.08 3.92 0.001 Unweigthed Unifrac 2 1.48 0.04 1.75 0.001 Coral vs. Reef Habitat Bray Curtis 2 4.89 0.15 7.22 0.001 Weighted Unifrac 2 0.01 0.11 5.39 0.001 Unweigthed Unifrac 2 2.01 0.06 2.43 0.001 a Alpha diversity results are represented by F-statistics values for the Shannon, Fisher and Faith’s Phylogenetic Diversity (PD) indices obtained in the statistical models. Beta diversity (Bray Curtis and Weighted and Unweighted Unifrac indices) results are represented by R 2 values obtained in the PERMANOVA tests. P-values are in bold when significant differences were found. Examining the effects of reef habitat To examine for differences in the skin microbiome between gobies living in different reef habitats within each island, the microbiome of coral-dwellers (sharknose and peppermint gobies) was compared to the reef/sand dwelling fish (glass/masked and patch-reef gobies). Significant differences were found in alpha diversity ( F > 4.31, p < 0.02; Fig. 1 ; Table 1 ), with pairwise differences only in St. Croix, where the skin microbiome alpha diversity of peppermint gobies is significantly lower when compared to the patch-reef and glass/masked gobies (P = 0.03). Beta diversity was also significantly different ( R 2 > 2.43, p = 0.001; Fig. 1 ; Table 1 ), with compositional differences between reef habitats in both islands with the Bray Curtis and Unweighted Unifrac indices. The species skin core microbiome in St. Croix showed that the core microbiota of the coral-dwellers cleaning and peppermint gobies was comprised of three and two ASVs, respectively (Fig. 3 ), and differed between the two species. For cleaning gobies, the core ASVs were identified as Alteromonas , Pseudoalteromonas , and Vibrio , while for peppermint gobies an unclassified Alphaproteobacteria and Endozoicomonas were identified. On the other hand, in the skin of reef dwelling gobies, a highly diverse core microbiota was found, with 27 ASVs in patch-reef gobies and 26 ASVs in glass/masked gobies, where 18 out of 35 ASVs (51%) are shared between the two species. Testing the effects of phylogeny To test the effect of phylogenetic affinities of gobiid species in microbiome composition, a hierarchical clustering of the Bray Curtis dissimilarity among all species for both locations was performed (Fig. 4 ). In the phylogenetic study by Baldwin and Robertson ( 2015 ) that included species of the Coryphopterus genus, they showed that the peppermint gobies and the glass/masked gobies are more phylogenetically related than patch-reef gobies, which were shown to be within a more distant clade. In our hierarchical clustering dendrogram (Fig. 4 ), almost all samples from reef-dwelling gobies cluster together (patch-reef and glass/masked gobies), while all cleaning gobies’ samples belong to a different cluster, and the majority of the peppermint gobies’ samples are in the most distant cluster of the dendrogram. Discussion In this study, we compared the skin microbiome of four sympatric cryptobenthic gobiids sampled in two locations in the Eastern Caribbean, in relation to their cleaning vs non-cleaning behavior, type of reef habitat, phylogenetic affinities, and intraspecific differences between islands. Our results showed that the microbiome of coral-dwelling gobies consistently clustered and exhibited significantly lower bacterial diversity independently of cleaning behavior compared to reef-hovering and sand-dwelling species. Notably, the core microbiome of reef-hovering and sand-dwelling gobies exhibited higher diversity and greater overlap between species than coral-dwellers, which showed less diverse and more unique core microbiomes. These results suggest that niche occupancy and reef habitat may be stronger drivers of the skin microbiota of gobies, rather than cleaning behavior and/or host phylogeny. Although we found no intraspecific differences in skin microbial alpha diversity within gobies collected in Puerto Rico and St. Croix, there were significant compositional differences (beta diversity) between fish species captured in the two islands. Our hypothesis that intraspecific differences in the alpha diversity of the skin microbiota of gobies would be found at such large spatial scales (between islands), was based on previous general knowledge regarding teleost microbiome, which indicates fish skin microbiota as being highly susceptible to local changes in environmental conditions (e.g., temperature, pH, phosphorus concentration, salinity, reviewed in Xavier et al. 2023 ), together with our previous findings for the same cleaning goby E. evelynae and also findings on the beaugregory damselfish ( Stegastes leucostictus ), collected from different reefs in the U.S. Virgin Islands (Xavier et al. 2020 ; Pereira et al. 2023 ). Specifically, for the skin microbial diversity of the cleaning goby E. evelynae , there were differences between cleaning gobies collected in two sites in St. Thomas, U.S. Virgin Islands and between the gobies collected at St. Thomas sites and St. John’s sites. For S. leucostictus , differences were found in the skin microbiota of fish collected from Brewers Bay and La Parguera, both in Puerto Rico. These differences were attributed to different local environmental conditions influencing the microbial species present in fish skin. Although geographic distance did not affect skin microbial richness of the sampled goby species in the present study, the differences in microbial composition indicated intraspecific diversity at a regional scale, which could also be associated with local differences in environmental conditions. Alternatively, microbial diversity within-host may consist of taxa that are specifically regulated by host factors, while microbial composition (i.e., among-host microbial prevalence and abundance patterns) may be mostly determined by stochastic processes, such as dispersal and ecological drift, acting on the regional pool of microorganisms from which communities are assembled (Adair and Douglas 2017 ). While intraspecific changes in skin microbiota at a larger regional scale may be attributed to differences in environmental conditions, interspecific differences between gobiid species within each capture site may come from differences in species’ ecology, niche occupancy and phylogeny. Cleaning gobies engage in frequent contact with heterospecifics, with the number of clients and client fish genera affecting the bacterial species diversity present in the skin of the studied cleaning goby in the Virgin Islands (Pereira et al. 2023 ). However, in the present study, there was no evidence for increased alpha diversity in the cleaning goby relative to other non-cleaning gobies, indicating that cleaning interactions with heterospecifics do not seem to lead to an inflation of the number of bacterial species present in their skin. However, we did find significant differences in microbial composition when comparing the microbiome of the cleaning against all the non-cleaning gobies grouped together. Although cleaners’ behavior could be driving these differences, we could not discard the possibility that host phylogenetic affinities may also play a role in the differences observed between E. evelynae and Coryphopterus gobies, at least to some degree. While host systematics could drive microbiome composition, hierarchical clustering analysis of microbial composition of specimens of all studied species collected in St. Croix, consistently distributed the coral-dwelling gobies (i.e., cleaning and peppermint gobies) within the same cluster, and the patch-reef and masked/glassed gobies in another. Indeed, statistical analysis also highlighted diversity and compositional differences between the coral-dwelling gobies and the other goby species, with the former exhibiting significantly less microbial diversity than sand-dwelling and reef hovering gobies ( C. tortugae and C. hyalinus/personatus , respectively). These results indicate that the coral substrate or surrounding environment could influence the diversity of bacteria available to colonize goby skin or that skin mucous properties of these fishes are more selective. The skin microbiota of fish is frequently found to differ from bacterioplankton (Berggren et al. 2021 ; Rosado et al. 2021 ; Sehnal et al. 2021 ), showing that skin mucous is a nutrient rich medium that favors growth of microorganisms (e.g., Carda-Dieguez et al. 2017 ), while also being highly selective. Indeed, fish skin mucus has several antimicrobial properties that can protect them from microbial pathogens and parasites (Reverter et al. 2018 ). For example, the skin of some coral-dwelling gobiids from genus Gobiodon produces toxins that are both antiparasitic and also serve as antipredatory defense (Munday et al. 2003 ; Dirnwoeber and Herler 2013 ) and the skin mucous properties ( e.g. , mucin production) of the common carp are known to limit bacterial adhesion in response to high bacterioplankton load (van der Marel et al. 2010 ). In fact, the metabolites (e.g., glucose concentration) in teleost fish mucus respond to acute environmental changes, such as hypoxia, heat stress, disease, food restriction or feed additives (e.g., Micallef et al. 2017 ; de Mercado et al. 2018 ), with some studies showing that they are accompanied by changes to colonizing microbiota (Liu et al. 2025 ). While the skin microbiota of the two coral-dwelling species studied at St. Croix share more similarities than when compared to the other studied gobies, the analysis of the most abundant taxa and core microbiota shows striking differences between the two coral-dwellers. Indeed, bacteria from the genera Vibrio and Pseudoalteromonas were both more prevalent and abundant in cleaning gobies, while Endozoicomonas bacteria largely dominated the skin of the peppermint gobies. Endozoicomonas is a ubiquitous bacterial genus associated with stony corals and a key member of the coral holobiont, while in other marine organisms, such as clams and fish, it is considered a parasite and pathogen (Pogoreutz et al. 2022 ; Pogoreutz and Ziegler 2024 ). Since our study represents the first characterization of the skin microbiome of peppermint gobies, no baseline data are available for comparison. Therefore, it remains unclear whether the dominance of Endozoicomonas in peppermint goby skin at this collection site results from opportunistic colonization due to their close association with corals or represents a common member of the skin microbial community of this species. The diversity of core taxa in coral-dwellers was also considerably lower when compared to the other gobies (2–3 vs 27–28 core taxa). Additionally, the core taxa of both species show no overlap, with cleaning gobies in St. Croix exhibiting three core taxa from Vibrio , Pseudoalteromonas and Alteromonas and the peppermint goby two taxa from Endozoicomonas and an unidentified Alphaproteobacteria . Contrasting to coral-dwellers, both patch-reef and glass/masked gobies from St. Croix (reef-hovering and sand-dwellers), had a more diverse core microbiota and shared a higher number of core taxa. The unique and less diverse core microbiome observed in coral-associated gobies compared to the other studied species may be explained by the selective microbial properties of coral mucus through antimicrobial peptides (Ritchie 2006 ), consequently influencing the microbiome composition of coral-associated organisms. Given these results, we concluded that ecological and reef habitat occupancy differences may be major drivers of the microbial diversity of gobiid species. Our findings suggests that the skin microbiota of sympatric gobiids is structured by an interplay of geographic, ecological, and niche occupancy factors. The distinct microbial signatures of coral-dwelling gobies, characterized by reduced diversity and unique core taxa compared to reef-hovering and sand-dwelling species, underscore how reef habitat occupancy can outweigh phylogenetic patterns in determining skin microbiome composition. These results emphasize the need to consider both environmental context and host ecological traits when predicting microbiome responses to environmental change. As coral reef ecosystems face increasing anthropogenic pressures, the specialized microbial communities of habitat-specific cryptobenthic species may serve as sensitive indicators of ecosystem homeostasis and reef habitat degradation. Declarations Competing interests The authors have no competing interests to declare. Ethical approval Research was performed under permits from Puerto Rico (021-IC-2021, O-VS-PVS15-Sj-01204-17052021 and the USVI (DFW21017U, 2021-23) and experiments were performed in accordance with and approval from the Woods Hole Oceanographic Institution IACUC protocols (25581.00). Author Contribution PCS, RX, AA, MS and AB conceived the study. PCS, MDN, GCH, and AB collected the field samples, AA, AP, MK, and JB processed microbial samples, AA, RX, AB, AP, MK, and JB analyzed the data, AP and RX led the writing, with major input from AA, PCS, and MS, and additional editing/input from MDN, GCH, MK, and JB. Acknowledgement Thanks to S. Russel, T. Hobbs and L. Ma for field work assistance, E. Weil, M. Carlo and the Isla Maqueyes Marine Laboratory for support in Puerto Rico and Sweet Bottom Dive Center, W. Welsh and the Landing Beach Resort for support in U.S. Virgin Islands. Sequencing services were performed by the University of Illinois W.M. 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Fishes 8 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Apr, 2026 Read the published version in Coral Reefs → Version 1 posted Editorial decision: Revision requested 02 Dec, 2025 Reviews received at journal 24 Nov, 2025 Reviews received at journal 30 Oct, 2025 Reviewers agreed at journal 28 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Editor assigned by journal 21 Aug, 2025 Submission checks completed at journal 19 Aug, 2025 First submitted to journal 17 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":29585,"visible":true,"origin":"","legend":"\u003cp\u003eSkin microbial alpha and beta diversity for each goby species and island. For alpha diversity, Shannon, Fisher and Faith’s Phylogenetic Diversity (PD) indices are shown in bloxplots, where the median is represented by a horizontal line, the lower and upper whiskers correspond to the furthest data points that are within 1.5 times the interquartile, and points represent individual samples. For beta diversity, Principal Coordinate Analysis (PCoA) of the Bray Curtis, Weighted Unifrac and Unweighted Unifrac indices are represented. Sharknose gobies are shown in blue, glass/masked gobies in orange, patch-reef gobies in green and peppermint gobies in yellow; samples from Puerto Rico are represented with dots and from St. Croix with triangles in the PCoAs\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7390480/v1/47a8952a5a281cfd484f6dd5.png"},{"id":95499562,"identity":"dfdb5867-504e-4124-a4f4-da087e34b4c5","added_by":"auto","created_at":"2025-11-10 05:15:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8998,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the relative abundance of the most abundant microbial genera at ≥ 2% of average abundance (y-axis) in each fish (x-axis), for each species collected in A) Puerto Rico and B) St. Croix\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7390480/v1/dff95d3c1a4572a96b7e42ed.png"},{"id":95527883,"identity":"8b5b227a-dce0-4d44-9345-37547b9d385b","added_by":"auto","created_at":"2025-11-10 10:15:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11445,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of the core microbiome ASVs at a 90% prevalence (y-axis) for A) the coral-dweller cleaning and peppermint gobies, and B) the sand-dwelling patch-reef and glass/masked gobies in St Croix (x-axis). ASVs were identified to species level when possible, otherwise to the lowest taxonomic level\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7390480/v1/7aaa16de0e260a81abd32ce4.png"},{"id":95499554,"identity":"c21dffac-7f1d-4b64-963b-36b139af0985","added_by":"auto","created_at":"2025-11-10 05:15:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12139,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic effects in microbial structure of gobiid species. Dendrogram of the hierarchical clustering of the Bray Curtis dissimilarity (y-axis) of microbial communities among all studied species (x-axis) is shown. Cleaning gobies are represented in blue, patch-reef gobies in green, glass/masked gobies in orange, and peppermint gobies in yellow, where samples from Puerto Rico are in dotted branches and St. Croix in solid lines\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7390480/v1/e4a45b77b9a1a6053cac9463.png"},{"id":108437912,"identity":"1ccfa554-0fec-497f-b894-e9b2a35ddc74","added_by":"auto","created_at":"2026-05-04 16:04:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":495264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7390480/v1/485b0987-2940-4232-ae8f-f2bcc3fe7b58.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Coral-dwelling Caribbean gobies exhibit distinct skin microbiota compared to other sympatric species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicroorganisms represent dynamic and crucial components of biological systems, shaping the structure and function of ecosystems from the molecular to the landscape scale. Microbial communities serve as fundamental drivers of ecosystem function and biodiversity, mediating biogeochemical processes that underpin the stability and productivity of natural systems (Arrigo \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Rousk and Bengtson \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Beyond their ecosystem-wide functions, host-microbial associations both influence and are influenced by animal physiology and behavior (Archie and Tung \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vuong et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sehnal et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Host-associated microbial communities are shaped by an array of interconnected factors including environmental gradients (Nguyen et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), networks of trophic interactions (Gralka et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), host-microbe interactions, microbe-microbe competition and cooperation, and stochastic processes (Adair and Douglas \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCoral reefs are among the most productive and biodiverse systems per unit area on Earth (Reaka-Kudla \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Knowlton et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), with microbes playing a crucial role in their processes (Haas et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Silveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rappuoli et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Understanding microbial dynamics within coral reef ecosystems has emerged as a crucial frontier in marine ecology (Ainsworth et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), as these microbial assemblages facilitate coral homeostasis through symbiotic relationships and pathogen defense, potentially driving coral adaptation to stress and environmental changes (Bourne et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Peixoto et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Webster and Reusch \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). While coral microbiomes are increasingly researched (Hernandez-Agreda et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dunphy et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; van Oppen and Blackall \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vanwonterghem and Webster \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mohamed et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), there is still a knowledge gap regarding the larger coral reef ecosystem microbiome. In particular, the microbiomes of resident fish, which may act as promoters of microbial connectivity between different reef environments due to their mobility (Leon-Zayas et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Seminal research indicates that reef fish microbiota are distinct from bacterioplankton; they are highly variable but species-specific, with host phylogeny and diet influencing fish skin, gill and gut microbial assemblages (Chiarello et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pratte et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kavazos et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, reef fish microbiota are also seemingly influenced by host age (Pratte et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xavier et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and regional/local habitat conditions (Xavier et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), such as levels of eutrophication (Degregori et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the most abundant yet understudied components of coral reef fish communities are cryptobenthic gobies, which represent a highly variable group of small-bodied fishes that are closely associated with the reef substrate (Brandl et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Despite their small size and cryptic nature, gobiids exhibit an important ecological diversity across Caribbean reefs, occupying a wide range of microhabitats including live coral heads, sponges, rubble, and sandy areas (Taylor and Hellberg \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Harborne et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This ecological diversification is evident across multiple genera, with species from the \u003cem\u003eElacatinus\u003c/em\u003e genus being coral-dwelling cleaners or sponge associates (Taylor and Hellberg \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), while \u003cem\u003eCoryphopterus\u003c/em\u003e species demonstrate adaptations to various substrates from coral to rubble and open sand (Beeken et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ziebell et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eObligate or \u0026ldquo;dedicated\u0026rdquo; cleaner fish are unique to coral reef systems and have an ecological impact that is disproportional to their abundance (C\u0026ocirc;t\u0026eacute; \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Vaughan et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Through the activity of removing parasites from other fishes (clients), they attract multiple species and thus have significant impacts on the local abundance and diversity of fishes (Bshary \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Grutter et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Brown et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Because of their daily physical contact with multiple other fishes, as well as contact with the substrate, cleaner fishes are potentially exposed to a much wider variety of microbes than most other reef fishes. Dedicated cleaner fish include two genera from two families: cleaner wrasses of the genus \u003cem\u003eLabroides\u003c/em\u003e in the Indo-Pacific, and cleaning gobies of the genus \u003cem\u003eElacatinus\u003c/em\u003e in the Caribbean. While convergent in coloration and overall ecological function (Cheney et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), cleaning gobies are much smaller than wrasses and reside on the substrate, typically live coral or sponge (C\u0026ocirc;t\u0026eacute; and Soares \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Whittey et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Budd et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), leaving only temporarily to tend to a client before returning. In a comparison of cleaning and sponge-dwelling ecotypes of the broadstripe cleaning goby \u003cem\u003eElacatinus prochilos\u003c/em\u003e, Xavier et al. (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) showed that gobies that resided on live coral and engaged in more cleaning had significantly higher skin microbial diversity and higher abundance of potential pathogens than those that seldom cleaned and resided on sponges, leading to the hypothesis that the frequent contact with other fish during cleaning could promote microbial transmission and higher diversity. A prediction of this hypothesis was later tested in a field study by Pereira et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), confirming an association between levels of cleaning activity of the Caribbean sharknose cleaning goby, \u003cem\u003eElacatinus evelynae\u003c/em\u003e, and increased variability of their skin microbiome. A more recent study reported that the presence of \u003cem\u003eE. evelynae\u003c/em\u003e in the reef directly impacts the microbial diversity of the coral heads where cleaners are stationed, as well as the microbial heterotrophs of water near the cleaning station (Brown et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Although these effects were context-dependent, they suggest that frequent contact with a cleaner and/or increased local fish traffic can impact reef holobiont and the skin microbiota of fish residents (Brown et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe intimate association of cryptobenthic gobies with specific reef microhabitats, combined with their limited mobility and high site fidelity, makes them ideal model organisms for understanding how fine-scale habitat differences and behavioral ecology influence host-associated microbial communities in coral reef ecosystems. Here, we report findings of a field study at St. Croix and Puerto Rico, eastern Caribbean, comparing the skin microbiota of the cleaning goby \u003cem\u003eE. evelynae\u003c/em\u003e with another three closely related cryptobenthic gobiid species collected from the same reef patches that exhibit different reef habitat associations and/or behavioral ecologies. These included a non-cleaning coral-dwelling species and two non-cleaning species that do not reside on live hard coral. We hypothesized that the microbiomes of the gobies captured in the two islands will show intraspecific differences between islands due to different environmental conditions. Within each island, we expected that the cleaning gobies will have higher microbial diversity when compared to the non-cleaning gobies. We also hypothesized that the microbial composition in gobiid skin could reflect phylogenetic affinities of their hosts, or that it could be driven by the reef habitat where they live.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy species\u003c/h2\u003e\u003cp\u003eOur study included two reef-hovering goby species (\u003cem\u003eCoryphopterus hyalinus\u003c/em\u003e and \u003cem\u003eCoryphopterus personatus\u003c/em\u003e), one sand-dwelling species (\u003cem\u003eCoryphopterus tortugae\u003c/em\u003e), and two coral-dwellers (\u003cem\u003eCoryphopterus lipernes\u003c/em\u003e and \u003cem\u003eElacatinus evelynae\u003c/em\u003e). Goby species are typically segregated in different habitats, however, two sister taxa, \u003cem\u003eC. hyalinus\u003c/em\u003e (glass goby) and \u003cem\u003eC. personatus\u003c/em\u003e (masked goby) (Thacker and Cole \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Baldwin and Robertson \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), form heterospecific shoals, which hover above the benthos (Beeken et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Selwyn et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These two species feed on plankton (Beeken et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), are morphologically very similar and may occasionally hybridize (Selwyn et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and therefore will be referred to as \u003cem\u003eC. hyalinus/personatus\u003c/em\u003e (glass/masked goby) in our study. \u003cem\u003eC. lipernes\u003c/em\u003e (peppermint goby) is sister to the \u003cem\u003eC. hyalinus/personatus\u003c/em\u003e clade (Baldwin and Robertson \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and lives in close physical contact with live corals, where they spend most of their time (Smith and Tyler \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Data on their diet is scarce, but they have been seen feeding on particulate matter in the water column (Smith and Tyler \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). \u003cem\u003eC. tortugae\u003c/em\u003e (patch-reef goby) is the most phylogenetic distant relative of the other \u003cem\u003eCoryphopterus\u003c/em\u003e species considered herein (Thacker and Cole \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Baldwin and Robertson \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), with individuals living in sand patches between coral outcrops and preying upon small benthic invertebrates (Kramer et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The coral-dwelling \u003cem\u003eE. evelynae\u003c/em\u003e (sharknose cleaning goby) is an obligate cleaner that contacts with multiple heterospecific client species, feeding on their ectoparasites and dead skin through cleaning interactions (C\u0026ocirc;t\u0026eacute; and Soares \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStudy area and sample collection\u003c/h3\u003e\n\u003cp\u003eIn June and July 2021, we collected gobies (n\u0026thinsp;=\u0026thinsp;78) from two localities in the Eastern Caribbean Sea: Cane Bay, along the north shore of St. Croix, US Virgin Islands (17\u0026ordm;46\u0026rsquo;26.8\u0026rsquo;\u0026rsquo;N 64\u0026ordm;48\u0026rsquo;45.6\u0026rsquo;\u0026rsquo;W) and Media Luna reef, off La Parguera, Puerto Rico (17\u0026deg;56'16.5\"N 67\u0026deg;03'10.8\"W) from reef habitat at depths from 10\u0026ndash;18 m. Within each island, gobiid species were collected from the same reef patches. The number of gobies collected for each species per island is given in Table S1 of the Supplementary Information. We collected gobies individually, using hand nets and without the use of anesthetic. For each collecting dive, we used a different net for each fish in order to avoid cross contamination. Immediately after collection, we placed each fish in individual hermetically sealed plastic bags, which we then placed in a mesh bag for transport back to the shore or boat, respectively. We then transferred each bag into an insulated plastic container to maintain thermal stability until processing. At the lab, we swabbed the entire surface of each fish using a Puritan HydraFlock swab (Guilford, ME), handling each fish with sterile gloves. We placed swab samples in cryovials and then deposited them into a dry shipper, which kept samples cold using liquid nitrogen vapors (St. Croix) or a -80 \u0026ordm;C freezer (Puerto Rico) before being sent to Woods Hole Oceanographic Institution (WHOI) for processing. We returned fish to the site of collection within 24 hours. Water (60 ml) was also collected from the fish collection depth at each site using a syringe. Back at the lab, the water was filtered through 25 mm, 0.2 \u0026micro;m Supor filters (Pall, Port Washington, NY) and the filter deposited into a cryovial and frozen as detailed above.\u003c/p\u003e\n\u003ch3\u003eDNA extraction and libraries preparation\u003c/h3\u003e\n\u003cp\u003eDNA from the fish skin swabs and water filter was extracted using the DNeasy PowerBiofilm kit (Qiagen, Germantown, MD) following manufacturer\u0026rsquo;s protocol. Extractions included negative controls (swab, filter, and extraction blank). Libraries were prepared using a standard dual indexing amplification of the V4 hypervariable region of the 16S rRNA gene (Kozich et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with the primers 515Y (Parada et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and 806RB (Apprill et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The Polymerase Chain Reactions (PCRs) were performed in a total volume of 50 \u0026micro;L with 10 \u0026micro;L of GoTaq Flexi colorless buffer, 5 \u0026micro;L of 25 mM MgCl2, 1 \u0026micro;L of each 10 mM forward and reverse primers, 1 \u0026micro;L of 10 mM dNTPs, 0.5 \u0026micro;L GoTaq DNA polymerase (Promega, Madison WI), 29.5 \u0026micro;L of PCR grade water, and 4 \u0026micro;L of DNA template. Each PCR was run with a negative control, and the following PCR conditions were used: of 95 \u0026ordm;C for 2 min, and then 95 \u0026ordm;C (20 s), 55 \u0026ordm;C (15 s), 72 \u0026ordm;C (5 min) for 30\u0026ndash;35 cycles, and a final elongation step of 72 \u0026ordm;C for 10 min. PCR products were visualized on a 1.5% agarose gel, and the target amplification product was identified based on a 50 bp ladder and a positive control. Amplified bands were gel excised, cleaned, and purified with the MinElute Gel Purification kit (Qiagen) following manufacturer instructions. Purified products were quantified using the Qubit 2.0 fluorometer (Invitrogen, Waltham, MA). Each purified sample product was added to a final pool with a target concentration of 2 ng/\u0026micro;L. Each pool also contained negative PCR controls using PCR-grade water, and the Microbial Mock Community B (even, low concentration, HM-782D, BEI Resources, ATCC). The pooled libraries were sequenced on a MiSeq (Illumina, San Diego CA) for 250 bp paired-end sequencing in three different runs.\u003c/p\u003e\n\u003ch3\u003eSequence processing\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eDADA2\u003c/em\u003e pipeline (Callahan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) (version 1.32.0) was used on the demultiplexed FASTQ files from the combined sequencing runs for quality control, merging paired-end reads, sequencing error rates estimation and quality filtering, using the following parameters: truncLen\u0026thinsp;=\u0026thinsp;c(190, 150), maxN\u0026thinsp;=\u0026thinsp;0, maxEE\u0026thinsp;=\u0026thinsp;c(2, 2), truncQ\u0026thinsp;=\u0026thinsp;2. Reads were collapsed into Amplicon Sequence Variants (ASVs) discarding chimeras, and an ASV frequency table was constructed. The mock community of each run was verified against the reference mock and then, removed from the dataset. The SILVA reference database (Quast et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) (release 138) was used to assign the taxonomy, and ASVs that remained unclassified or were classified as belonging to Family mitochondria and Class chloroplast were removed from the dataset. The presence of potential contaminants was checked by calculating the prevalence of taxa of the negative controls (extraction, PCR, and swab blanks) with a probability threshold of 0.1 in all dataset using the package \u003cem\u003edecontam\u003c/em\u003e (Davis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, the presence of water-associated microbial taxa in the skin swabs was identified using the water filter samples using the same method. Contaminant and water-associated ASVs were removed from the dataset, as well as control and water samples. Singletons and samples with less than 1000 reads were also removed (n\u0026thinsp;=\u0026thinsp;1, Glass/Masked goby from St. Croix), and read normalized counts were obtained using the negative binomial distribution implemented in DESeq2 (Love et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Raw sequence reads were deposited into NCBI\u0026rsquo;s Short Read Archive under accession PRJNA986111.\u003c/p\u003e\n\u003ch3\u003eMicrobial diversity analyses\u003c/h3\u003e\n\u003cp\u003eAlpha diversity was estimated using Shannon and Fisher indices using the R package \u003cem\u003ephyloseq\u003c/em\u003e (McMurdie and Holmes \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and Faith\u0026rsquo;s Phylogenetic Diversity (PD) using \u003cem\u003epicante\u003c/em\u003e (Kembel et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). For each diversity index, a first linear model was used to test for intraspecific differences between islands, including only cleaning gobies and patch-reef gobies, with the factor goby species nested within island in the model. Peppermint gobies were not used in this analysis because they were sampled only in St. Croix, as well as patch-reef gobies due to insufficient sample size in Puerto Rico (n\u0026thinsp;=\u0026thinsp;2), which would create unbalanced comparisons between locations. Then, to test the effect of cleaning behavior and reef habitat, two linear models were performed with island as a nested factor: cleaning versus non-cleaning gobies, and coral-dwelling (cleaning and peppermint gobies) versus reef-hovering/sand-dwelling gobies (glass/masked and patch-reef gobies). Beta diversity was estimated using the Bray Curtis dissimilarity and weighted and unweighted Unifrac indices using the R package \u003cem\u003ephyloseq.\u003c/em\u003e\u0026nbsp;To test our hypothesis, we performed permutational multivariate analysis of variance (PERMANOVA) tests on the normalized data using similar statistical models to the ones for alpha diversity, with the \u003cem\u003eadonis\u003c/em\u003e function of the \u003cem\u003evegan\u003c/em\u003e package (Oksanen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with 999 permutations and the option for a sequential test. When significant results were found, pairwise PERMANOVAs were performed using the \u003cem\u003epairwise.adonis2\u003c/em\u003e wrapper function (Martinez Arbizu \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with the Bonferroni p-value correction for multiple comparisons. Dissimilarity in microbial structure among all samples was visualized using Principal Coordinates Analysis (PCoA) with the three beta diversity indices previously calculated.\u003c/p\u003e\u003cp\u003eThe most abundant microbial taxa for each species were assessed by collapsing ASVs to the genus level and defined by \u0026ge;\u0026thinsp;2% on average of all sequences within location. For each species sampled in St. Croix, the core microbiome was also assessed using the relative abundance transformed data with the function \u003cem\u003ecore\u003c/em\u003e from the package \u003cem\u003emicrobiome\u003c/em\u003e (Lahti et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and a threshold of 90% prevalence. Samples from Puerto Rico were not included in the core analysis due to low sample number for glass/masked gobies (n\u0026thinsp;=\u0026thinsp;2) and therefore the lack of a representative number of species to allow the comparison between gobies with different types of behavior and reef habitat. Lastly, to infer a phylogenetic signal in the skin microbial composition of gobies, a hierarchical clustering of the Bray Curtis dissimilarity was performed for each species within both islands using the function \u003cem\u003ehclust\u003c/em\u003e from the R base package \u003cem\u003estats\u003c/em\u003e, using the \u003cem\u003eward.D2\u003c/em\u003e method. The resulting dendrogram was compared to the most recent published phylogenies of the \u003cem\u003eCoryphopterus\u003c/em\u003e genus (Baldwin and Robertson \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Forrester et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to assess whether microbial community clustering patterns reflect known phylogenetic relationships.\u003c/p\u003e\u003cp\u003eFor all statistical tests, differences were considered significant when \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 4 041 048 merged reads were recovered after filtering and cleaning the dataset against negative and water samples. The number of reads per individual sample ranged between 7 311 and 221 493, resulting in a total of 11 058 ASVs.\u003c/p\u003e\u003cp\u003eA first statistical model was used to compare the skin microbiome of the same fish species collected across both islands. Although no significant differences were found for alpha diversity (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.38; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), beta diversity was significantly different (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in gobies between Puerto Rico and St. Croix. The most abundant microbial genera in the skin of gobies sampled at Puerto Rico included \u003cem\u003eAlteromonas\u003c/em\u003e, \u003cem\u003eEkhidna\u003c/em\u003e, \u003cem\u003eMycoplasma\u003c/em\u003e, and \u003cem\u003eVibrio\u003c/em\u003e, while in St. Croix the most abundant microbial genera found included \u003cem\u003eEndozoicomonas\u003c/em\u003e, \u003cem\u003ePseudoalteromonas\u003c/em\u003e, and \u003cem\u003eVibrio\u003c/em\u003e, although the abundances varied among species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A striking high abundance of \u003cem\u003eEndozoicomonas\u003c/em\u003e in the skin of peppermint gobies is noteworthy, with a relative abundance of more than 75% of this genus in 8 out of 16 samples.\u003c/p\u003e\n\u003ch3\u003eComparison between cleaning and non-cleaning gobies\u003c/h3\u003e\n\u003cp\u003eTo test for differences in the skin microbiome between cleaning and non-cleaning gobies at each location (Puerto Rico and St. Croix), the microbiome of the sharknose cleaning goby was compared with non-cleaning gobies (i.e. peppermint, patch-reef and glass/masked gobies) within locations. No significant differences were found in the alpha diversity between cleaning and non-cleaning gobies (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.88, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.13; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), although microbial beta diversity differed significantly (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.04, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with pairwise comparisons showing differences in both islands with the Bray Curtis and Unweighted Unifrac indices.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026ndash; Differences in the skin microbial alpha and beta diversity between goby species sampled in different islands, between cleaner and non-cleaner gobies and between coral and reef-hovering/sand habitats.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"12\" rowspan=\"13\"\u003e\u003cp\u003eAlpha diversity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSum Sq\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean Sq\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePr(\u0026gt;\u0026thinsp;F)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIsland comparison\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShannon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFisher\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1015.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1014.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCleaner vs. Non-cleaner\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShannon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFisher\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1956.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e978.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e603.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e301.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCoral vs. Reef Habitat\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShannon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.0007\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFisher\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8341.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4170.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2334.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1167.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"12\" rowspan=\"13\"\u003e\u003cp\u003eBeta diversity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSum Sq\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePr(\u0026gt;\u0026thinsp;F)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIsland comparison\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBray Curtis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeighted Unifrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnweigthed Unifrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCleaner vs. Non-cleaner\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBray Curtis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeighted Unifrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnweigthed Unifrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCoral vs. Reef Habitat\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBray Curtis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeighted Unifrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnweigthed Unifrac\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eAlpha diversity results are represented by F-statistics values for the Shannon, Fisher and Faith\u0026rsquo;s Phylogenetic Diversity (PD) indices obtained in the statistical models. Beta diversity (Bray Curtis and Weighted and Unweighted Unifrac indices) results are represented by R\u003csup\u003e2\u003c/sup\u003e values obtained in the PERMANOVA tests. P-values are in bold when significant differences were found.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eExamining the effects of reef habitat\u003c/h3\u003e\n\u003cp\u003eTo examine for differences in the skin microbiome between gobies living in different reef habitats within each island, the microbiome of coral-dwellers (sharknose and peppermint gobies) was compared to the reef/sand dwelling fish (glass/masked and patch-reef gobies). Significant differences were found in alpha diversity (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;4.31, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.02; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with pairwise differences only in St. Croix, where the skin microbiome alpha diversity of peppermint gobies is significantly lower when compared to the patch-reef and glass/masked gobies (P\u0026thinsp;=\u0026thinsp;0.03). Beta diversity was also significantly different (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2.43, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with compositional differences between reef habitats in both islands with the Bray Curtis and Unweighted Unifrac indices.\u003c/p\u003e\u003cp\u003eThe species skin core microbiome in St. Croix showed that the core microbiota of the coral-dwellers cleaning and peppermint gobies was comprised of three and two ASVs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and differed between the two species. For cleaning gobies, the core ASVs were identified as \u003cem\u003eAlteromonas\u003c/em\u003e, \u003cem\u003ePseudoalteromonas\u003c/em\u003e, and \u003cem\u003eVibrio\u003c/em\u003e, while for peppermint gobies an unclassified \u003cem\u003eAlphaproteobacteria\u003c/em\u003e and \u003cem\u003eEndozoicomonas\u003c/em\u003e were identified. On the other hand, in the skin of reef dwelling gobies, a highly diverse core microbiota was found, with 27 ASVs in patch-reef gobies and 26 ASVs in glass/masked gobies, where 18 out of 35 ASVs (51%) are shared between the two species.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTesting the effects of phylogeny\u003c/h2\u003e\u003cp\u003eTo test the effect of phylogenetic affinities of gobiid species in microbiome composition, a hierarchical clustering of the Bray Curtis dissimilarity among all species for both locations was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the phylogenetic study by Baldwin and Robertson (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) that included species of the \u003cem\u003eCoryphopterus\u003c/em\u003e genus, they showed that the peppermint gobies and the glass/masked gobies are more phylogenetically related than patch-reef gobies, which were shown to be within a more distant clade. In our hierarchical clustering dendrogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), almost all samples from reef-dwelling gobies cluster together (patch-reef and glass/masked gobies), while all cleaning gobies\u0026rsquo; samples belong to a different cluster, and the majority of the peppermint gobies\u0026rsquo; samples are in the most distant cluster of the dendrogram.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we compared the skin microbiome of four sympatric cryptobenthic gobiids sampled in two locations in the Eastern Caribbean, in relation to their cleaning vs non-cleaning behavior, type of reef habitat, phylogenetic affinities, and intraspecific differences between islands. Our results showed that the microbiome of coral-dwelling gobies consistently clustered and exhibited significantly lower bacterial diversity independently of cleaning behavior compared to reef-hovering and sand-dwelling species. Notably, the core microbiome of reef-hovering and sand-dwelling gobies exhibited higher diversity and greater overlap between species than coral-dwellers, which showed less diverse and more unique core microbiomes. These results suggest that niche occupancy and reef habitat may be stronger drivers of the skin microbiota of gobies, rather than cleaning behavior and/or host phylogeny.\u003c/p\u003e\u003cp\u003eAlthough we found no intraspecific differences in skin microbial alpha diversity within gobies collected in Puerto Rico and St. Croix, there were significant compositional differences (beta diversity) between fish species captured in the two islands. Our hypothesis that intraspecific differences in the alpha diversity of the skin microbiota of gobies would be found at such large spatial scales (between islands), was based on previous general knowledge regarding teleost microbiome, which indicates fish skin microbiota as being highly susceptible to local changes in environmental conditions (e.g., temperature, pH, phosphorus concentration, salinity, reviewed in Xavier et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), together with our previous findings for the same cleaning goby \u003cem\u003eE. evelynae\u003c/em\u003e and also findings on the beaugregory damselfish (\u003cem\u003eStegastes leucostictus\u003c/em\u003e), collected from different reefs in the U.S. Virgin Islands (Xavier et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Specifically, for the skin microbial diversity of the cleaning goby \u003cem\u003eE. evelynae\u003c/em\u003e, there were differences between cleaning gobies collected in two sites in St. Thomas, U.S. Virgin Islands and between the gobies collected at St. Thomas sites and St. John\u0026rsquo;s sites. For \u003cem\u003eS. leucostictus\u003c/em\u003e, differences were found in the skin microbiota of fish collected from Brewers Bay and La Parguera, both in Puerto Rico. These differences were attributed to different local environmental conditions influencing the microbial species present in fish skin. Although geographic distance did not affect skin microbial richness of the sampled goby species in the present study, the differences in microbial composition indicated intraspecific diversity at a regional scale, which could also be associated with local differences in environmental conditions. Alternatively, microbial diversity within-host may consist of taxa that are specifically regulated by host factors, while microbial composition (i.e., among-host microbial prevalence and abundance patterns) may be mostly determined by stochastic processes, such as dispersal and ecological drift, acting on the regional pool of microorganisms from which communities are assembled (Adair and Douglas \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile intraspecific changes in skin microbiota at a larger regional scale may be attributed to differences in environmental conditions, interspecific differences between gobiid species within each capture site may come from differences in species\u0026rsquo; ecology, niche occupancy and phylogeny. Cleaning gobies engage in frequent contact with heterospecifics, with the number of clients and client fish genera affecting the bacterial species diversity present in the skin of the studied cleaning goby in the Virgin Islands (Pereira et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, in the present study, there was no evidence for increased alpha diversity in the cleaning goby relative to other non-cleaning gobies, indicating that cleaning interactions with heterospecifics do not seem to lead to an inflation of the number of bacterial species present in their skin. However, we did find significant differences in microbial composition when comparing the microbiome of the cleaning against all the non-cleaning gobies grouped together. Although cleaners\u0026rsquo; behavior could be driving these differences, we could not discard the possibility that host phylogenetic affinities may also play a role in the differences observed between \u003cem\u003eE. evelynae\u003c/em\u003e and \u003cem\u003eCoryphopterus\u003c/em\u003e gobies, at least to some degree. While host systematics could drive microbiome composition, hierarchical clustering analysis of microbial composition of specimens of all studied species collected in St. Croix, consistently distributed the coral-dwelling gobies (i.e., cleaning and peppermint gobies) within the same cluster, and the patch-reef and masked/glassed gobies in another. Indeed, statistical analysis also highlighted diversity and compositional differences between the coral-dwelling gobies and the other goby species, with the former exhibiting significantly less microbial diversity than sand-dwelling and reef hovering gobies (\u003cem\u003eC. tortugae\u003c/em\u003e and \u003cem\u003eC. hyalinus/personatus\u003c/em\u003e, respectively). These results indicate that the coral substrate or surrounding environment could influence the diversity of bacteria available to colonize goby skin or that skin mucous properties of these fishes are more selective. The skin microbiota of fish is frequently found to differ from bacterioplankton (Berggren et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rosado et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sehnal et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), showing that skin mucous is a nutrient rich medium that favors growth of microorganisms (e.g., Carda-Dieguez et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), while also being highly selective. Indeed, fish skin mucus has several antimicrobial properties that can protect them from microbial pathogens and parasites (Reverter et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For example, the skin of some coral-dwelling gobiids from genus \u003cem\u003eGobiodon\u003c/em\u003e produces toxins that are both antiparasitic and also serve as antipredatory defense (Munday et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Dirnwoeber and Herler \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and the skin mucous properties (\u003cem\u003ee.g.\u003c/em\u003e, mucin production) of the common carp are known to limit bacterial adhesion in response to high bacterioplankton load (van der Marel et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In fact, the metabolites (e.g., glucose concentration) in teleost fish mucus respond to acute environmental changes, such as hypoxia, heat stress, disease, food restriction or feed additives (e.g., Micallef et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; de Mercado et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), with some studies showing that they are accompanied by changes to colonizing microbiota (Liu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile the skin microbiota of the two coral-dwelling species studied at St. Croix share more similarities than when compared to the other studied gobies, the analysis of the most abundant taxa and core microbiota shows striking differences between the two coral-dwellers. Indeed, bacteria from the genera \u003cem\u003eVibrio\u003c/em\u003e and \u003cem\u003ePseudoalteromonas\u003c/em\u003e were both more prevalent and abundant in cleaning gobies, while \u003cem\u003eEndozoicomonas\u003c/em\u003e bacteria largely dominated the skin of the peppermint gobies. \u003cem\u003eEndozoicomonas\u003c/em\u003e is a ubiquitous bacterial genus associated with stony corals and a key member of the coral holobiont, while in other marine organisms, such as clams and fish, it is considered a parasite and pathogen (Pogoreutz et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pogoreutz and Ziegler \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Since our study represents the first characterization of the skin microbiome of peppermint gobies, no baseline data are available for comparison. Therefore, it remains unclear whether the dominance of \u003cem\u003eEndozoicomonas\u003c/em\u003e in peppermint goby skin at this collection site results from opportunistic colonization due to their close association with corals or represents a common member of the skin microbial community of this species. The diversity of core taxa in coral-dwellers was also considerably lower when compared to the other gobies (2\u0026ndash;3 vs 27\u0026ndash;28 core taxa). Additionally, the core taxa of both species show no overlap, with cleaning gobies in St. Croix exhibiting three core taxa from \u003cem\u003eVibrio\u003c/em\u003e, \u003cem\u003ePseudoalteromonas\u003c/em\u003e and \u003cem\u003eAlteromonas\u003c/em\u003e and the peppermint goby two taxa from \u003cem\u003eEndozoicomonas\u003c/em\u003e and an unidentified \u003cem\u003eAlphaproteobacteria\u003c/em\u003e. Contrasting to coral-dwellers, both patch-reef and glass/masked gobies from St. Croix (reef-hovering and sand-dwellers), had a more diverse core microbiota and shared a higher number of core taxa. The unique and less diverse core microbiome observed in coral-associated gobies compared to the other studied species may be explained by the selective microbial properties of coral mucus through antimicrobial peptides (Ritchie \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), consequently influencing the microbiome composition of coral-associated organisms. Given these results, we concluded that ecological and reef habitat occupancy differences may be major drivers of the microbial diversity of gobiid species.\u003c/p\u003e\u003cp\u003eOur findings suggests that the skin microbiota of sympatric gobiids is structured by an interplay of geographic, ecological, and niche occupancy factors. The distinct microbial signatures of coral-dwelling gobies, characterized by reduced diversity and unique core taxa compared to reef-hovering and sand-dwelling species, underscore how reef habitat occupancy can outweigh phylogenetic patterns in determining skin microbiome composition. These results emphasize the need to consider both environmental context and host ecological traits when predicting microbiome responses to environmental change. As coral reef ecosystems face increasing anthropogenic pressures, the specialized microbial communities of habitat-specific cryptobenthic species may serve as sensitive indicators of ecosystem homeostasis and reef habitat degradation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e\n\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eResearch was performed under permits from Puerto Rico (021-IC-2021, O-VS-PVS15-Sj-01204-17052021 and the USVI (DFW21017U, 2021-23) and experiments were performed in accordance with and approval from the Woods Hole Oceanographic Institution IACUC protocols (25581.00).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003ePCS, RX, AA, MS and AB conceived the study. PCS, MDN, GCH, and AB collected the field samples, AA, AP, MK, and JB processed microbial samples, AA, RX, AB, AP, MK, and JB analyzed the data, AP and RX led the writing, with major input from AA, PCS, and MS, and additional editing/input from MDN, GCH, MK, and JB.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThanks to S. Russel, T. Hobbs and L. Ma for field work assistance, E. Weil, M. Carlo and the Isla Maqueyes Marine Laboratory for support in Puerto Rico and Sweet Bottom Dive Center, W. Welsh and the Landing Beach Resort for support in U.S. Virgin Islands. Sequencing services were performed by the University of Illinois W.M. Keck Center for Comparative and Functional Genomics.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eRaw sequence reads were deposited into NCBI\u0026rsquo;s Short Read Archive under accession PRJNA986111.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding was provided by National Science Foundation DEB 2231250 to PCS and OCE-2022955 to PCS and AA and Portuguese Science and Technology Foundation grants 2022.00854.CEECIND/CP1601/CT001 and 2021.01458.CEECIND/CP1668/CT0003 to RX and MCS, respectively.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdair KL, Douglas AE (2017) Making a microbiome: the many determinants of host-associated microbial community composition. 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Fish and Fisheries 18:698\u0026ndash;716\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVuong HE, Yano JM, Fung TC, Hsiao EY (2017) The Microbiome and Host Behavior. Annu Rev Neurosci 40:21\u0026ndash;49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWebster NS, Reusch TBH (2017) Microbial contributions to the persistence of coral reefs. ISME J 11:2167\u0026ndash;2174\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWhittey KE, Dunkley K, Young GC, Cable J, Perkins SE (2021) Microhabitats of sharknose goby (\u003cem\u003eElacatinus evelynae\u003c/em\u003e) cleaning stations and their links with cleaning behaviour. Coral Reefs\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXavier R, Severino R, Silva SM (2023) Signatures of dysbiosis in fish microbiomes in the context of aquaculture. Reviews in Aquaculture\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXavier R, Mazzei R, Perez-Losada M, Rosado D, Santos JL, Verissimo A, Soares MC (2019) A Risky Business? Habitat and Social Behavior Impact Skin and Gut Microbiomes in Caribbean Cleaning Gobies. Front Microbiol 10:716\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXavier R, Pereira A, Pagan A, Hendrick GC, Nicholson MD, Rosado D, Soares MC, P\u0026eacute;rez-Losada M, Sikkel PC (2020) The effects of environment and ontogeny on the skin microbiome of two \u003cem\u003eStegastes\u003c/em\u003e damselfishes (Pomacentridae) from the eastern Caribbean Sea. Marine Biology 167\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZiebell A-C, Vogel ML, Ratajczak NK, Hoeksema BW (2023) Habitat Use of Two Coral-Associated Cryptobenthic Gobiid Fishes (Family: Gobiidae) in the Southern Caribbean. Fishes 8\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"skin microbiome, cleaner fish, cryptobenthic, Elacatinus evelynae, Coryphopterus spp., goby, coral reefs","lastPublishedDoi":"10.21203/rs.3.rs-7390480/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7390480/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial communities fundamentally shape ecosystem function and biodiversity across all biological systems through complex dynamics. In coral reef ecosystems, understanding the dynamics of these microbial communities has become critical for predicting reef responses to environmental stressors. Fish skin microbiota are highly susceptible to environmental changes and may vary significantly across species and geographic locations, yet the extent to which these variations occur remain poorly understood. Here, we compared the skin microbiota of four closely related and sympatric cryptobenthic gobiid species, that exhibit different behavioral ecologies (cleaning vs non-cleaning), ecological niches (water column, coral-, or sand-dwelling) and phylogenetic affinities (\u003cem\u003eElacatinus\u003c/em\u003e vs \u003cem\u003eCoryphopterus\u003c/em\u003e), yet reside in the same reef patches in St. Croix and Puerto Rico, eastern Caribbean. Coral-dwelling gobies, including cleaning sharknose and non-cleaning peppermint gobies, exhibited significantly lower microbial diversity compared to reef-hovering and sand-dwelling species (both non-cleaning). These coral dwellers showed unique microbial signatures despite having similar alpha diversity levels. Core microbiota analysis also revealed striking differences between coral-dwelling and reef-hovering/sand-dwelling species, with the core microbiome of the former dominated by \u003cem\u003eVibrio\u003c/em\u003e, \u003cem\u003ePseudoalteromonas\u003c/em\u003e, and \u003cem\u003eAlteromonas\u003c/em\u003e in the case of cleaning gobies and by \u003cem\u003eEndozoicomonas\u003c/em\u003e in the case of peppermint gobies, while reef-hovering and sand-dwelling gobies exhibited diverse core microbiota with greater overlap between species. Ecological niche occupancy and reef habitat selection appear to be primary drivers of skin microbiota composition in gobiid fishes, rather than cleaning behavior and/or host phylogenetic affinities alone, though species-specific skin mucus properties likely also contribute to selective bacterial colonization patterns.\u003c/p\u003e","manuscriptTitle":"Coral-dwelling Caribbean gobies exhibit distinct skin microbiota compared to other sympatric species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 05:15:53","doi":"10.21203/rs.3.rs-7390480/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-02T22:06:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T20:01:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T04:20:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196968821053262698021796547602230595634","date":"2025-10-28T19:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80961653887499022342137709507015956395","date":"2025-10-28T00:33:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T21:24:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-22T01:26:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-20T02:12:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Coral Reefs","date":"2025-08-17T05:00:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"coral-reefs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"core","sideBox":"Learn more about [Coral Reefs](http://link.springer.com/journal/338)","snPcode":"338","submissionUrl":"https://submission.nature.com/new-submission/338/3","title":"Coral Reefs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"862cce75-2094-4f6e-9678-cb0aaca275a8","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:03:39+00:00","versionOfRecord":{"articleIdentity":"rs-7390480","link":"https://doi.org/10.1007/s00338-026-02870-7","journal":{"identity":"coral-reefs","isVorOnly":false,"title":"Coral Reefs"},"publishedOn":"2026-04-28 15:57:18","publishedOnDateReadable":"April 28th, 2026"},"versionCreatedAt":"2025-11-10 05:15:53","video":"","vorDoi":"10.1007/s00338-026-02870-7","vorDoiUrl":"https://doi.org/10.1007/s00338-026-02870-7","workflowStages":[]},"version":"v1","identity":"rs-7390480","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7390480","identity":"rs-7390480","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}