The effect of substrate type on microscopic epibiont community diversity: A shoreline study from the Sea of Japan at Niigata, Japan

The effect of substrate type on microscopic epibiont community diversity: A shoreline study from the Sea of Japan at Niigata, Japan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The effect of substrate type on microscopic epibiont community diversity: A shoreline study from the Sea of Japan at Niigata, Japan Nandakumar Kanavillil, Y Takada This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3411120/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microscopic epibionts are an important component of intertidal ecosystems. Shortage of space drives these organisms to settle and grow on body/shell surfaces of intertidal organisms. In this study we test the hypothesis that epibiont communities vary significantly between shells belonging to different invertebrate species, and between natural shells and plastic surfaces. Epibiont community variations are caused by differences in shell size, roughness and mobility of the host organisms. Epibionts growing on fifteen different species belonging to Gastropoda, Bivalvia, Polyplacophora and Echinoidea, as well as plastic objects were collected from a rocky shore of the Sea of Japan at Niigata. Most epibionts collected were diatoms, and the highest epibiont density was recorded on the surfaces of the limpet Cellana toreuma . The results showed epibiont species richness, diversity and density varied significantly with the host characteristics such as mobility, shell size and shell roughness. The shells of sessile organisms with large and smooth surfaces had a higher epibiont density, species richness and diversity than did shells of mobile organisms with small and rough shell surfaces. To conclude, epibiont species composition varies significantly between basibiont species, shell morphology, shell size, shell roughness, month of sampling and the mobility of the host. epibionts basibionts gastropod shells Sea of Japan intertidal organisms diatoms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The marine intertidal zone, the coastal strip lying between high and low watermarks, is home to organisms belonging to various trophic levels. In the intertidal zone, primary producers are well exposed to sunlight, tidal cycles, continuous water movement, predation and competition and can support a diverse community of heterotrophs (Poore et al. 2012 ). Space tends to be a limiting factor, so many organisms settle and grow on any hard substrate that is available, including the body surfaces of other living organisms (Dayton 1971 ). Epibiosis or ‘living on’, is the term applied to organisms that live, either temporarily or permanently on the hard surfaces of other organisms called ‘basibionts’ (Walker and Miller 1992 ; Wahl 2009 ). Among other benefits, epibionts gain: the protection on the host’s growing surface, better access to resources such as light and nutrients; and, if the host is mobile, a means of dispersal (Romagnoli et al. 2007 ; Totti et al. 2010 ; Falasco et al. 2018 ). In most cases, this association is not considered harmful to the basibionts; indeed, they can also benefit from their epibionts. For example, the growth of epibionts such as biofilms composed of bacteria, microalgae and fungi, also termed ‘periphyton’, can protect the host’s surfaces from ultraviolet radiations, fluctuations in increased temperature (Patil and Anil 2000 ). The slimy surface may deter predators, thus functioning as a protective layer (Wahl et al. 2012 ; Patil and Anil 2000 ). Marine organisms with hard shells (e.g., hard shelled molluscs) are of special importance here because even after their death, their empty shells provide additional settlement surfaces for attached organisms (Gutierrez et al. 2003 ). However, this association can sometimes have negative impacts on the host. If the epibionts become large macrophytes, they can restrict host movement making the host vulnerable to predators (Patil and Anil 2000 ). The epibionts may also cause shell deterioration (boring) of mollusks, coral reefs, Foraminifera, etc. (cf. Golubic and Schneider 2003 ; Tribollet et al. 2006; Barillé et al. 2017 ). Molluscs are a major group of organisms that contributes to the biodiversity of marine coastal areas. Most secretes calcified shells that are widespread in coastal habitats (Asakura and Suzuki 1987 ). These hard-shell surfaces allow the attachment and growth of microscopic epibiotic communities (Thyrring et al. 2015 , Barillé et al. 2017 ). Intertidal organisms, especially sessile ones, compete for limited settlement space, nutrients and light availability (for plants) (Coates 2002 ). Since there is clear zonation in the intertidal zone, from deeper to shallower areas, there are distinctive bands of communities in terms of the distribution of intertidal flora and fauna (Benedetti-Cecchi and Cinelli 1997 ). In turn, the surfaces of these organisms will be used as settlement surfaces by various macrobenthic (metazoan and macroalgae) and microbenthic (bacteria, diatoms, cyanobacteria and microalgae) organisms. Previous studies on the growth of epibiont communities have focused on the species of the host: for example, on bivalve and gastropod shells (D’Alelio et al. ( 2010 ), Gillan and Cadee (2000), Thyrring et al. ( 2015 ), Barillé et al. ( 2017 ), Kagawa et al. ( 2020 )). Other studies looked at epibionts on Bryozoans (Wuchter et al., 2003 ), Bivalvia (Barillé et al., 2017 ), hermit crabs (Stachowitsch 1980 ), barnacles (Gutierrez and Palomo 2016), crayfish (Falasco et al., 2018 ) and green turtles (Majewska et al. 2017 ). However, to this point, there have been no studies on epibiont communities living on basibionts of different phyla living in the same part of intertidal zone. This information is critical to an understanding of the role of basibionts determining epibiont community composition. Phototrophic epibionts (photosynthetic algae and bacteria) play important roles in the dynamics of aquatic ecosystems by performing carbon and nutrient uptake and producing oxygen essential to the survival of fish and marine invertebrates (Mac Intyre et al. 1996; Penhale and Smith 1997). Among the various groups of epibionts, diatoms are the most intensely studied group with more than 10,000 species described (Mann 1999 ; D’Alelio et al. 2010 ; Majewska et al. 2017 ; Falasco et al. 2018 ). The growth of epibionts on basibionts depends on various factors, both biotic and abiotic such as the epibiont’s growth forms (attached or unattached) and growth characteristics (solitary or colonial) (D’Alelio et al. 2010 ). Other factors that will affect epibionts are: host behaviour (Creed 2000 ; Gribben et al. 2009 ), host shell morphology and surface characteristics (Wahl, 2009 ; Thyrring et al., 2013 ), the host’s developmental stage (Dougherty and Russell 2005 ), host secretion of repellents (antifouling properties, Wahl 1989 ), habitat (Reiss et al. 2003 ), water flow rate and wave action (Wahl 1996 ; Wilson 2021 ), grazing pressure and competition (Rosemond et al. 1993; Burgmer et al. 2010 ), and season (Cabral et al. 2010 ). Taken together these factors suggest that members of the epibiont community are likely to show high host specificity (Majewska et al. 2012 ) and therefore it is important to see how much variation is observed among epibiont communities growing on different substrates in the same marine intertidal habitats. The goal of the present study is to characterize the epibiont communities found on shell surfaces of intertidal organisms belonging to different taxa (Gastropoda, Bivalvia, Polyplacophora and Echinoidea), and to compare these communities with those found on inert materials such as plastic surfaces. This will allow us to compare the niche preferences of these microflora on a finer scale, i.e., in terms of the roughness of the substratum surface. Although epibiont species have a common pool of propagules (e.g., planktonic organisms) in a coastal area, they can be specialists, growing only on a particular substrate, and/or generalists that grow on various surfaces. Therefore, the objectives of the study were to compare epibiont communities on (1) shells of different intertidal organisms; (2) shells of different roughness (smooth vs rough); (3) natural (shells) and artificial (plastic) surfaces; (4) mobile vs non-mobile substrates; and (5) different months of sampling on the epibiont community. We hypothesise that epibiont communities will differ significantly between shells of different invertebrates, between natural (shells) and artificial substrates (plastic) and between mobile and non-mobile substrates. We also hypothesise that epibiont community will favour non-mobile, large and rough shells compared to mobile, small and smooth shell surfaces. Materials and Methods Study site The study was conducted on a natural/artificial rocky shore of the Sea of Japan, behind the Japan Sea National Fisheries Research Institute (JSNFRI), Niigata prefecture, Japan (37 \(^\circ\) 55’N, 139°02’E; Fig. 1 ). The shore here is a man-made seawall consisting of natural rocks and concrete tetrapods constructed several decades ago to prevent the beach erosion to which this area is historically prone (Uda 2017 ). The shore is exposed to tidal fluctuations (0.3 m) and is being colonized by various intertidal organisms. To study epibionts, basibionts from the intertidal zone belong to Gastropoda, Bivalvia, Polyplacophora and Echinoidea were collected three times during early and late spring (April 5, April 10, and May 16, 2019). Care was taken not to cause any damage to the organisms and their epibionts while collections were made. After collection, individuals belonging to different species were put in different plastic bags with seawater collected from the sampling site to prevent drying. Along with the host species, several plastic objects (~ 500 mL (> 25 cm long) smooth surface plastic bottles) exposed to seawater were also collected for epibiont analysis. The tidal fluctuation in this area during sample collection was 0.3 m. The major surface seawater (~ 10 cm below surface) parameters measured during every sampling day varied between 10.8 o C (April) to 16.7 o C (May), pH 8.49 (May) to 8.63 (April), and salinity 18.4‰ (May) to 22.2‰ (April). A portable probe (YSI Model 63, YSI, Yellow Springs, OH) was used to measure pH, water temperature and salinity. Figure 1. Map showing sampling sites. Epibiont extraction After having been brought back to the laboratory, samples were immediately identified to the species level with the help of identification keys (Okutani 2017 ). After identification the heights and widths of individuals were measured with a Vernier caliper with an accuracy of 0.1 mm. Next, the epibionts were scraped from the shell with a clean toothbrush and a known quantity of aged seawater filtered through Whatman No. 1 filter paper). This was done by brushing and rinsing the shell surfaces four times to collect all epibionts growing on the shell surfaces. This method was standardized by running preliminary experiments to test the efficacy of epibiont removal by repeated brushing and rinsing and observing the shell surfaces and the filtrate for the presence of epibionts. Four brushings followed by rinsing with 2.5 mL of filtered aged seawater (at least six months in the refrigerator) each time was found to be an adequate technique to remove most of the epibionts from the shell surfaces. Where larger surfaces were encountered (for example, shells of Magallana sp. or Mytilus sp.; plastic surfaces), a known area of 20 x 20 mm from the edges was scraped to remove the epibionts. After collecting the whole extracts (10 mL/shell), they were properly labelled and kept in the refrigerator for epibiont analysis. The epibiont samples were analyzed immediately after scraping. The samples were concentrated by centrifuging the samples (~ 2000 rpm for 5 min) followed by careful removal of the supernatant. Subsamples were drawn from the concentrated samples after thorough mixing for observation in a haemocytometer at 200x, 400x and 1000x magnifications under a compound microscope (Olympus, Japan); epibionts were thus counted and identified. In this study, the major groups of epibionts examined include diatoms, unicellular and filamentous green algae, cyanobacteria and red algae. When protozoans were present, they were counted but not identified. Each sample was observed eight times and an average was taken to calculate their cell density. The density of each epibiont is expressed as No. mm-2 area and these values were used for the statistical analysis. Altogether 87 samples (shells and plastics) were observed during this study. The epibionts were identified with the help of several identification keys (Round et al. 1990 ; Prescot 1978 ; Yamaji 1984 ; Chihara and Murano 1997 ). Data analysis Basibiont categories To understand the importance of various physical and biological characteristics of the host species for epibiont community composition, the data were divided (categorised) as below before carrying out the statistical analysis. The basibionts/hosts were arbitrarily divided according to their shell size (small and large) and shell morphology (roughness, judged by finger touch arbitrarily divided into two categories, smooth and rough). Accordingly, organisms were categorized into small (organisms with shell size < 48 mm height and width) and large (shell size ≥ 48 mm height and width), and those with smooth (e.g., Mytilus galloprovincialis , Chitons and plastic surfaces) and rough shells (gastropods e.g., Monodonta ) (Table 1 ). In addition, the basibionts were also categorized into mobile (moves on the rock surface, e.g., gastropods and hermit crabs) and non-mobile (bivalves). Basibionts were also grouped into live (shells with live organism – henceforth “live”) and “dead” (only shells) at the time of collection. Finally, they were also grouped into natural (shell) and artificial (plastic) surfaces. Table 1 Types of substrata (shells of live and dead basibionts and plastic) sampled from Sekiya rocky shore during the three sampling episodes (April 05,10 and May 16). Gastropoda, Polyplacophora, and Echinoidea were mobile substrata, while Bivalvia and Plastic were non-mobile ones. Substrata/Intertidal organisms Apr-05 Apr-10 May-16 No. of shells/surfaces Roughness Size range (mm)*** Gastropoda Cellana grata (A. Gould) X X 8 Rough 26.7-L Cellana toreuma (Reeve) X X 7 Rough 21.9–29.9 Tegula rugata (A. Gould) X X 8 Rough 22.2–31.3 Lottia kogamogai Sasaki & Okutani X 1 Rough 17.5 Monodonta confusa Tapparone Canefri X X X 12 Rough 15.0-24.4 Tegula nigerrima (Gmelin) X X 6 (1)* Rough 19.2–22.6 Tegula pfeifferi carpenteri (Dunker) X 1 Rough 24.0 Tegula rustica (Gmelin) X X 9 (3)* Rough 18.9–23.1 Reishia bronni (Dunker) X 1 Rough 26.4 Siphonaria japonica (Donovan) X X X 9 Rough 4.2–20.2 Bivavalvia Magallana gigas (Thunberg) X 5 Rough 24.9-L Mytilus galloprovincialis Lamarck X X 8 Smooth 12.7-L Saccostrea kegaki Torigoe & Inaba ** X 4 Rough L Polyplacophora (Chitons) Onithochiton hirasei Pilsbry X X 2 Smooth 13.2–34.4 Rhyssoplax kurodai (Is. Taki & Iw. Taki) X 1 Smooth 12.7 Echinoidea Stronlyocentrotus intermedius (A. Agassiz) X 1 Rough 21.2 Plastic Plastic X 4 Smooth L * shells occupied by hermit crabs ** dead shells *** L: size larger than 48 mm Statistical analysis Epibiont assemblages observed on basibionts and plastic surfaces were analyzed using multivariate statistical techniques. Abundance data of the epibiont taxonomical units (species) were used to estimate dissimilarity values of assemblages between all pairs of the samples (between the host categories of natural (shells) and artificial (plastics) in both the months of sampling (April and May)) by calculating the Bray-Curtis dissimilarity values (Jost et al. 2011 ). Based on the dissimilarity values, ordinations in reduced spaces were generated by nonmetric multidimensional scaling (nMDS). The data were log (x + 1) transferred before running the analysis. The assemblages were clustered into several groups by K-medoids (Borcard et al., 2011 ) and the average silhouette values were calculated to decide the appropriate number of clusters. The indicator species of the assemblages (clusters) were selected by using the indicator value (IndVal) (Dufrêne and Legendre 1997). The significance of a species as an indicator of an estimated cluster of assemblages was evaluated by using a randomization procedure (n = 9999). The composition of epibiont species on natural substrates (i.e., excluding plastics) was also compared by using PERMANOVA (Anderson 2001 ; McArdle and Anderson 2001 ). To clearly understand the effect of sampling time (April – early spring vs May – late spring), one-way PERMANOVA was analyzed with month as a fixed factor, assuming a large effect of month on the epiphyton community. Then the epibiont data were further divided into those found on natural surfaces (all basibionts) vs artificial surfaces (plastics); small vs large shells (size); and smooth vs rough shells (roughness), and two-way PERMANOVAs were carried out with month and the substrate characteristics (nature of substrates, size and roughness) as fixed factors. In addition, because plastics and hermit crabs were obtained only in the month of May, the epibiont data were compared between plastic and natural (shells) substrates, and between shells of live gastropods ( Tegula nigerrima and T. rustica ) and the dead (ones inhabited by hermit crabs) were done in May by one-way fixed-effect PERMANOVA. For PERMANOVA, the Bray-Curtis dissimilarity with log(x + 1) transformed abundance data were used. Effects of month, mobility, size, and roughness on species richness, Simpson diversity index (inverse Simpson), and density of epibionts (all taxa combined) on the natural substrates were tested by ANOVA. Assuming a large effect of month, as for the PERMANOVA mentioned above, two-way fixed-effects ANOVAs with the factors of month and each of the other three factors (size, mobility and roughness) were performed. When the ANOVA showed significant differences (p < 0.05), Tukey-HSD tests were done for a post-hoc comparison. Similar to PERMANOVA, the effects of plastic and hermit crabs on species richness, Simpson diversity index, and density of epibionts were tested by one-way fixed-effect ANOVA using the samples obtained in May. In addition, within sample diversity (alpha diversity), total diversity (gamma diversity), and between sample diversity (beta diversity which is defined as multiplicative beta diversity (Jost 2007 )), were calculated for epibiont species richness and Simpson diversity indices. Before these analyses, the species richness and the Simpson diversity index were transformed to square-rooted values, and the density was transformed to log values to reduce heteroscedasticity between samples and tested by the Levene test. The establishment of an epibiont community, especially diatoms, depends on their growth characteristics (solitary or colonial) and growth forms (attached or non-attached). In order to study the significance of the growth forms and characteristics of diatoms (comprising > 87% of the epibionts) in the community composition of epibionts, they were grouped (placed in one of four groups): as attached or unattached, and as solitary or colony forming (Round et al. 1990 ; Prescot 1978 ; and Spaulding et al. 2021 ). The species richness and densities of diatoms in the K-medoid clusters were studied with respect to their growth forms and characteristics as mentioned above to understand the role played by these factors in shaping community composition. The analyses were performed using R 4.2.1 software and packages cluster, labdsv, and vegan (R Core Team 2022 ). Results Basibionts The basibionts collected include 10 Gastropoda species, 3 Bivalvia species, 2 Polyplacophora species and one Echinoidea species. In addition, plastic objects found along the rocks and submerged in the water were also collected for the epibiont analysis. The list of host substrates along with their dates of collection are given in Table 1 . Epibiont composition The list of epibionts found on different host substrates during the three sampling dates are given in Supplemental Table S1. The dominant group of epibionts was diatoms, followed by cyanobacteria, Chlorophyta and Rhodophyta. The total number of epibiont species observed on April 5, 10 and May 16 were 28, 50 and 62 species, respectively. Diatoms comprised more than 90% of the total epibiont species encountered. Among the substrates examined, Cellana toreuma recorded the highest density of epibionts (6080.4 ± 10008 cells mm-2) followed by Saccostrea kegaki (1726.6 ± 1521.5 cells mm-2) and Mytilus galloprovincialis (1646.1 ± 491.4 cells mm-2). The major species of epibionts based on the average density found among the substrates were Licmophora abbreviata (135 ± 201.3 cells mm-2) and Navicula membranacea (717 ± 1035.8 cells mm-2) in April 2019 and Fragilaria oceanica (146 ± 2194 cells mm-2) and N. membranacea (141 ± 126.8 cells mm-2) in May 2019, respectively. The overall mean epibiont species richness during this study was 10.06 ± 3.27 (n = 87, alpha diversity = 9.79) with a minimum of min 3spp. on Siphonaria japonica in Apr, and max 24 spp. on Mytilus galloprovincialis in May. In total, 96 species (gamma diversity) were observed across 87 samples. The beta diversity (between samples) of species richness was 9.80. As for Simpson diversity, the alpha, beta, and gamma diversities were 3.17, 1.88, and 5.96, respectively. The epibiont density and species diversity (Simpson diversity) did not show significant difference between natural substrates (shells) and the plastics, and between hermit crabs and live gastropod shells (Fig. 2, Supplemental Table S2). However, in May the species richness (number of species) varied significantly between the natural substrates and plastics with plastics showing lower species richness. The species richness did not vary between hermit crabs and live Gastropod shells (Fig. 2, Supplemental Table S2). Figure 2. Mean (± SD) of epibiont species richness, diversity (Simpson diversity index), and density between selected substrata in May. Differences between natural substrata (shells) and plastics are in the left row, and differences between live gastropods Tegula spp. ( T. nigerrima and T. rustica ) and shells occupied by Hermit crabs are in the right row. Results of one-way ANOVA (*: p < 0.05, ns: not significant) are also shown on the top of each figure. The results of one-way ANOVA on overall epibiont species richness, Simpson diversity and epibiont density on natural substrates (shells of various host species) with month as the fixed factor showed significant variation only in species diversity (F = 31.48, df = 1, p = < 0.001 (Simpson diversity (Supplemental Table S2). Furthermore, species richness, species diversity and epibiont density showed significant variation with the substrate characteristics such as mobility of the host, size of the shells and shell roughness (Fig. 3). Generally, the results indicated shells of non-mobile hosts, hosts with large shells and shells with smooth surfaces had higher epibiont density, species richness and species diversity than shells of mobile hosts, hosts with small shells and hosts with rough shell surfaces, respectively (Fig. 3). The variation was more evident in May than in April (Fig. 3). The epibiont density, species richness and species diversity on non-mobile hosts and on larger shells in May were found to be significantly different from the non-mobile, mobile and small shelled host species in the month of April (Fig. 3). In addition, the effect of interaction of month of sampling and mobility of the hosts, month and size of the host shells and month and shell roughness resulted in significant variation of species richness, species diversity and epibiont density (Fig. 3, Supplemental Table S2). Figure 3. Mean (± SD) of epibiont species richness, diversity (Simpson diversity index), and density between natural substrata (shells). Differences between mobile (Gastropoda, Polyplacophora, and Echinoidea) and non-mobile (Bivalvia), and between large (shell size ≥ 48mm) and small (< 48mm), and between smooth surface and rough surface (Table 1 ) are compared with the interaction of month (April and May). Different letters (a, b, and c) at the top-right of the bars in each figure indicate significant differences (p < 0.05) detected by Tukey-HSD tests and two-way ANOVA. Analysis of variation of epibiont assemblages on natural substrate (shells of invertebrates) using two-way PERMANOVA to study the influence of host characteristics such as mobility, shell size and shell roughness with months as a fixed factor showed highly significant variations (Table 2 ). Community composition was found to vary significantly with respect to the interaction of factors such as months and mobility, months and shell size and months and roughness. In addition, in the month of May, the one-way PERMANONVA results showed significant variations of eipibiont assemblages on hermit crab/gastropods, and plastics/natural shell substrates (Table 2 ). Table 2 Results of two-way PERMANOVA (9999 permutations) for epibiont assemblages on natural substrates with month as a fixed factor (April and May), and other host characteristics such as mobility (mobile/non-mobile), shell size (small/large), and shell roughness (smooth/rough). Also included are results of one-way PERMANOVA to analyze effects of plastics and Hermit crabs on epibiont assemblages in May. df Sum of squares Pseudo-F P Mobility Month 1 4.18 24.37 < 0.001 Mobility 1 0.97 5.68 < 0.001 Month X mobility 1 0.43 2.49 0.012 Shell size Month 1 4.18 24.18 < 0.001 Size 1 0.70 4.06 < 0.001 Month X size 1 0.60 3.44 0.001 Roughness Month 1 4.18 23.52 < 0.001 Roughness 1 0.58 3.29 0.002 Month X Roughness 1 0.33 1.85 0.046 Others Plastics/shells 1 0.437 2.21 0.012 Hermit crabs/gastropods 1 0.320 2.17 0.021 The two-dimensional ordination plot of nMDS using the Bray-Curtis dissimilarity (stress = 0.192) showed a usable representation of the variation of epibionts found on the shells and the plastic surfaces (Fig. 4). Generally, the assemblages were separated by the sampling month (April or May) and the nature of the substrates i.e., plastics or natural surfaces. Thus, the assemblages on plastics were found to be located at the peripheral area of the plot with large variation between each surface studied. Six groups of assemblages based on the dissimilarity values were identified by the K-medoid cluster analysis (Fig. 4 and Table 3 ). These clusters are generally well separated with each other except clusters 3 and 4 which were considerably overlapped (Fig. 4). Medoid samples of each cluster were generally located at the center of the points of the cluster (Fig. 4). The number of samples and their average silhouette values of K-medoid clusters for each assemblage on live invertebrate shells, hermit crabs and plastics in April and May are given in Table 3 . Table 3 Number of samples and average silhouette values of K-medoid clusters for assemblages on invertebrate shells, Hermit crabs and plastics in April and May Cluster ID 1 2 3 4 5 6 April Gastropoda (limpets) 2 1 11 0 0 0 Gastropoda (others) 11 0 3 5 0 0 Bivalvia 0 0 5 4 0 0 Polyplacophora 0 0 1 0 0 0 Echinoidea 0 0 0 0 0 0 plastics 0 0 0 0 0 0 May Gastropoda (limpets) 0 3 0 0 6 2 Gastropoda (others) 0 9 (1) 0 0 4 (1) 5 (2) Bivalvia 0 0 0 0 8 0 Polyplacophora 0 0 0 0 2 0 Echinoidea 0 1 0 0 0 0 plastics 0 2 1 0 0 1 Number of samples 13 16 21 9 20 8 Silhouette value 0.0871 0.06851 0.1240 0.1231 0.1302 0.15132 Numbers in parentheses show Gastropod shells inhabited by Hermit crabs Figure 4. Ordination by nonmetric multidimensional scaling (nMDS) of the assemblages of epibionts (99 species or taxonomic units). Open symbols are samples in April and closed symbols are those in May. Circles are natural substrates and triangles are plastics. Colors indicates 6 groups identified by the k-medoid cluster analysis: cluster 1 - black, cluster 2 - red, cluster 3 - green, cluster 4 - dark blue, cluster 5 - light blue, and cluster 6 - yellow. Six symbols with asterisk on the upper-right indicate the medoid samples of the cluster. All samples belonging to clusters 1 and 4 and most samples of cluster 3 were from April (Table 3 ). On the other hand, all samples belonging to clusters 5 and 6 and most samples of cluster 2 were from May. The Bivalvia, Polyplacophora, and limpet shaped Gastropoda samples were mainly categorized in cluster 3 (in April) or cluster 5 (in May) while the remaining Gastropoda samples were found in other clusters. Table 4 also shows the indicator species of each cluster with their indicator values (IndVal). The higher the value, the stronger is their presence in the cluster. Thus, several species can be detected as strong indicator species of each cluster (Table 4 , Fig. 5). The membership of each cluster also showed several facts; for example, Meuniera membranaceae showed significant presence in all the medoid samples of the six clusters (Fig. 5). On the other hand, there are species which occupies high percentage in one medoid sample of the cluster (e.g., Cocconeis pseudomarginata in cluster 5, IndVal 0.558; Thalassionema.frauenfeldii , in cluster 6, IndVal 0.422) while several other genera can be seen in more than one cluster (Table 4 , Fig. 5). Table 4 Indicator taxonomic units with their indicator value (IndVal) for the six clusters (permutation p < 0.05) – cluster 2 does not have any indicator species Indicator taxonomic units Cluster IndVal Probability Coscinodiscus radiatus 1 0.231 0.0028 Cocconeis placentula 1 0.204 0.034 Amphora lanceolata 1 0.154 0.032 Biddulphia spp. 1 0.154 0.0328 Stephanopyxis palmeriana 1 0.154 0.0326 Melosira nummuloides 3 0.596 0.0002 Licmophora abbreviata 3 0.485 0.002 Gomphonema exiguum 3 0.392 0.0034 Coscinodiscus spp. 4 0.736 0.0002 Cocconeis spp. 4 0.608 0.0002 Melosira hyperborea 4 0.599 0.0002 Coscinodiscus plicatus 4 0.557 0.0002 Nitzschia vitrea 4 0.442 0.0004 Pinnularia spp. 4 0.429 0.0008 Nitzschia spp. 4 0.419 0.0004 Fragilaria spp. 4 0.372 0.0002 Pinnunavis elegans 4 0.316 0.0254 Cyanobacteria sp.1 4 0.259 0.008 Amphora spp. 4 0.252 0.0364 Biddulphia pulchella 4 0.227 0.048 Paralia sulcata 4 0.222 0.0172 Cocconeis pediculus 4 0.167 0.047 Navicula salinarum 5 0.651 0.0002 Cocconeis pseudomarginata 5 0.558 0.0002 Fragilaria oceanica 5 0.461 0.003 Coscinodiscus megalomma 5 0.355 0.0018 Tabularia parva 5 0.327 0.0042 Azpeitia nodulifera 5 0.276 0.0194 Amphora ovalis 5 0.259 0.0174 Grammatophora marina 5 0.250 0.0094 Nitzschia pubens 5 0.229 0.0154 Rhabdonema arcuatum 5 0.227 0.0204 Amphora longiceps 5 0.215 0.0224 Bacillaria paxillifera 6 0.467 0.0002 Thalassionema frauenfeldii 6 0.422 0.0004 Figure 5. The percentage composition of epibiont species observed in the medoid sample of the 6 clusters as identified in the nMDS plot. Diatoms were labeled into attached (63 spp.)/unattached (24 spp.) and solitary (45 spp.)/colony (42 spp.) species. Some substrates did not have unattached or colonial diatoms. So, comparisons to the original (all epibionts, 96 species) assemblage by Mantel correlation and nMDS ordinations were done only for the attached and solitary diatoms. The attached diatoms (63 species) showed a higher correlation (Mantel Pearson, 0.945, log (+ 1) transformed Bray-Curtis) with the original epibionts than the solitary diatoms (47 species) (Mantel Pearson, 0.799). The correlation between attached diatoms and solitary diatoms was 0.825 (Mantel Pearson), where only 31 species were common. The nMDS identified 6 clusters based on K-medoid values using attached diatoms. The species richness of attached colonial diatoms showed a different pattern from the overall microalgae, partly because of the small sample size. Species richness of unattached diatoms in May was significantly higher than in April, while no difference was detected for unattached diatoms. The species richness and density of attached/unattached and solitary/colonial diatoms in the 6 clusters identified in nMDS is shown in Fig. 6 . Figure 6. Mean (±SD) of species richness and density of diatoms in the epibiont community. Diatoms are divided into attached and unattached, and solitary and colony forming species. The species richness of attached diatoms was higher than unattached diatoms in all 6 clusters (Fig. 6 ). The lowest species richness of unattached forms was in cluster 1 while the highest was in cluster 5. The species richness of diatoms among the solitary and colonial forms did not differ much in any of the 6 clusters. Similarly, the species density of attached forms was generally higher than the unattached ones in all the 6 clusters (Fig. 6 ). The lowest species density of the unattached forms was observed in cluster 1. The species densities remained the same in solitary and colonial forms in all 6 clusters. Discussion In this study, 16 natural substrates (gastropods, bivalves, Polyplacophora and Echinoidea) and plastic objects were analyzed for microscopic epibiont community composition. This study provides a comprehensive picture of the epibiont community composition on a variety of intertidal organisms and plastic surfaces. Epibionts, some of them macroscopic, have been studied on various aquatic organisms previously. However, each of these studies involved only a few substrates such as seagrass (De Stefano et al. 2000 ), Porifera (McClintock et al. 2005 ); Hydrozoa (Romagnoli et al. 2007 ); Bryozoans (Wuchter et al. 2003 ); gastropods (Gillan and Cadee 2000; D’Alelio et al. 2010 ; Thyrring et al. 2015 ; Barillé et al. 2017 ; Kagawa et al. 2020 ); hermit crabs (Stachowitsch 1980 ); mud snail, Hydrobia ulvae (Gillan and Cadee 2000); Scallops, Oysters and Mussels (Barillé et al. 2017 ); Crustaceans (Gutierrez and Palomo 2016); Cephalopod and Neogastropod egg shells (Lim et al. 2007 ); crayfish (Falasco et al. 2018 ); turtles (Majewska et al. 2017 ); and whales (Denys 1997 ). This study revealed the composition of epibiont communities on several different substrates (basibionts) in the intertidal zone of Niigata, as well as its alpha (within each basibiont and plastics), beta (between these basibionts), and gamma (total of all these basibionts) diversities. The epibiont species richness can vary from individual to individual of the same basibiont species depending on the microenvironment they are in. For example, individuals belonging to the same species found on exposed rock surfaces may have a different epibiont community from those found in crevices. Thus, it is important to know the alpha diversity to get an overall picture of the epibiont community variation on a certain species. In this study, the alpha values for the epibionts were found to be much smaller than the gamma (overall) diversity values of epibionts across all the species, resulting the large beta diversity. This is expected as varying factors such as basibiont mobility, roughness and size, habitat selection, and the environmental factors in which they are living can alter the epibiont species diversity on the basibionts. As expected, the diatoms formed the major component of the epibionts found on all types of substrates. This finding agrees with the results observed in the previous studies (Totti et al., 2010 ). However, among diatoms, the species that contributed higher densities include Licmophora abbreviata (mostly attached and colonial), Meuniera membranacea (mostly unattached (sometimes prostrate) and single) and Fragilaria oceanica (mostly attached and colonial). This indicates attached and colonial forms along with unattached and single cells equally colonized the substrates. It is noteworthy that the density and species diversity of epibionts did not show significant variations between natural shell surfaces and plastics and hermit crab and gastropods (in the month of May). However, epibiont species richness was significantly higher on natural shell surfaces compared to plastics. Plastics, even though they are inert surfaces, may be prone to abrasion or toxic to several organisms (Worm et al. 2017 ). Epibiont community compositions vary widely between basibionts/substratum surfaces. Under the assumption that epibionts species have a common pool for recruitment, the characteristics of basibionts, environmental factors, and interactions of these and epibionts can play a role in this variation. Specifically, some of the reasons attributed to this variation are the structures of basibiont shells (Wahl 2009 ; Thyrring et al. 2013 ); shell size (D’Alelio et al. 2011); presence/absence of grazers in the environment (Thyrring et al. 2015 ); secretion of antifouling compounds by the basibionts (Whal 1989); and the environmental parameters such as water quality (D’Alelio et al. 2011), water movement (Wahl 1996 ; Wilson 2021 ), atmospheric exposure of the shells among many others (D’Alelio et al. 2011). Substratum surface roughness has been described as an important surface characteristic that can influence the settlement and growth of diatoms on wet surfaces (Sweat and Johnson 2013 ). This study results showed no variation in epibiont density between smooth plastic surfaces and rough shell surfaces (Supplemental Table S2) nor between rough and smooth shell surfaces (Fig. 3). In fact, the results showed a higher epibiont density on smooth shell surfaces (such as the shell of sessile Bivalves, Mytilus galloprovincialis and Chitons) than on the rough shell surfaces of mobile and non-mobile gastropods (Fig. 3). A similar pattern of diatom settlement was reported by Sweat and Johnson ( 2013 ) where they observed higher diatom settlement on smooth surfaces than the rough ones. Scardino et al. ( 2006 ) with the help of attachment point theory explained the higher diatom settlement on smooth surfaces. The smooth surfaces according to Scardino et al. (2013) offer more points of contact for diatoms leading to higher number of diatom settlement. Furthermore, species diversity and richness were found to be higher on smooth shell surfaces than the rough gastropod shell surfaces. However, one important fact to consider here is the variation of basibiont species composition on which the epibiont compositions were studied. Generally, smooth shells belonged to bivalves and chitons (not all species observed in this study, please see Table 1 ) while rough ones were a characteristic of gastropods. Thus, the observed trend might be due to many reasons such as the difference in secreted shells roughness, non-toxic chemical (repellent) production, behavioural changes of basibionts etc. (Wahl et al. 2009 and the references there in). Unfortunately, there are no data available on the role of shell morphology of bivalves and gastropods on the epibiont community formation. The results of this study suggest that the basibiont characteristics such as the shell size and mobility played a significant role in the community compositions of epibionts. Generally, larger shell surfaces had higher species density, diversity and richness (MacArthur and Wilson 1963 ; Relyea 2021 ). This phenomenon can be more clearly perceived in May than in April (Fig. 3). In an aquatic environment this is an expected result as larger shells provide more surface stability compared to smaller ones thereby leading to a higher density, diversity and richness of epibionts (Martins et al. 2014 ). The alternative explanation that larger shells provide a longer period for settlement and development for epibiont during shell development can be rejected because of the larger variations observed between samples in May and April. Similarly, with respect to the mobility of the shells (substratum surfaces), the non-mobile ones harboured higher species density, diversity and richness of epibionts than on the mobile ones (Fig. 3) suggesting that the epibionts prefer stable substratum surfaces than the unstable ones for their attachment and growth. With respect to the plastic surfaces several factors such as the stability (surfaces exposed for a long period by being trapped in crevices vs objects just brought to shore by waves) and nature of the surface (large, smooth, or rough) play an important role in the epibiont community formation. The month of sample collection played a significant role in the composition of epibiont communities. The PERMANOVA analysis on the epibiont assemblages on substratum surfaces (basibionts + plastics) showed a significant variation between months, with May showing a higher density than April. This result is expected as the seawater temperature and salinity went up from April to May (10.8 o C (April) to 16.7 o C (May), pH 8.49 (May) to 8.63 (April), and salinity 18.4‰ (May) to 22.2‰ (April)). Warmer water temperatures and the arrival of water runoff from agriculture fields with higher nutrient concentration may result in a change in an epibiont community (Martins et al. 2014 ; Bulleri et al. 2020 ). A slightly different picture emerged when the density, diversity and species richness of epibionts on the natural substrate (shells) were analyzed with month as a factor. The two-way ANOVA showed epibiont density, diversity and richness varied significantly with the mobility, size and roughness of the shells (Supplemental Table S2). The results once again show the importance of shell characteristics of basibionts on the epibiont community composition. The results of two-dimensional ordination plot of nMDS using the Bray-Curtis dissimilarity showed variation of epibionts found on the shells and the plastic surfaces (Fig. 4) demonstrating the role of substratum surfaces on epibiont community composition. The assemblages on plastics were found to be located at the peripheral area of the nMDS plot with large variation between each surface studied. In the context of the increased presence of plastic objects floating around in aquatic systems, this observation is important. The microbial biota settling over the plastic objects not only find these surfaces for attachment but may also lead to their disintegration/breaking down into smaller objects leading to increased pollution problems (Worm et al. 2017 ). The large variations of epibiont community observed on plastics may partly be due to the variation among the chemical properties of their surfaces, elasticity, history of plastics on the shore (e.g., drifted height on the shore), stability (frequency of overturning by wave and wind), frequency of wave and tidal splash during the growth of epibionts. In comparison to the non-mobile plastics, gastropods generally exhibit a cyclic movement synchronized with tidal and diurnal cycles (Iwasaki 1994 ; Takada 2001 ). This difference may affect the variation of the epibiont communities on these surfaces.The results also showed separation of epibiont assemblages by the sampling month (April or May) as observed in the results of ANOVA. Six groups of assemblages based on the dissimilarity values were identified by the K-medoid cluster analysis (Fig. 4 and Table 3 ). They are generally well separated with each other except clusters 3 and 4 which were considerably overlapped on the two-dimensional plot thus showing the relationship between the clusters, month, and basibionts (Fig. 4). It is interesting to see the key species having the largest IndVal changes between each of the clusters showing the variation of species composition with months and substratum surfaces. On a closer observation, the leading members of these clusters possess their own ecological properties representing the occupied space and time. For example, the species with the highest IndVal Melosira nummuloides (cluster 3, April) is a benthic diatom exhibiting euryhaline and eurithermal properties that help them colonize the sea-land transition zones (Prelle et al. 2021 ). Similarly, Licmophora abbreviata , the other diatom with high IndVal (cluster 3, April) is reported to survive well at low seawater temperatures, typically in spring (between 10–17 o C) (Jung et al. 2017 ) and Coscinodiscus radiata is known to survive in neritic marine water with high light availability (Medvedeva et al. 2009 ). Bacillaria paxillifera (cluster 6, May) a motile euribiontic diatom found living in both freshwater and marine water, is capable of surviving in turbid waters – another character that distinguishes it from the rest (Ussing et al. 2005 ). Community composition also can be influenced by the growth forms (e.g., colonial vs unicellular), growth characteristics (e.g., attached vs free floating), and the type of epibionts, namely, micro algae, diatoms, cyanobacteria, etc. (D’Alelio et al. 2010 ). The ability of diatoms to colonize a surface depends on their attachment properties, such as the type of attachment, namely prostrate vs stalked or unattached. Additionally, the mobility or immobility of diatoms also have roles to play in the colonization process (D’Alelio et al. 2010 ). This study showed diatoms as the major component of the epibionts. Among diatoms the attached and solitary forms were the dominant ones that outnumbered the colonial and unattached ones. This is an expected result as the attachment property helps in the colonization and growth of epibionts. A similar trend was observed in case of diatom species richness as well; the diatom species richness of the attached forms was more than that of unattached ones. The results thus show the importance of attachment properties and growth forms of diatoms in the epibionts community and the role of basibionts on the overall epibiont community composition on a seashore. The basibiont-epibiont community dynamics demonstrates the conceptual context of facilitation cascade via habitat formation and modification as outlined by Thomsen et al. ( 2010 ). Here, the shells of invertebrates in the intertidal habitats act as primary habitat-former while the epibionts as the secondary habitat-formers leading to higher species diversity and abundance in the habitat as noted in other habitats/ecosystems such as forest, seagrass meadows, salt marshes and seaweed beds. In conclusion, the epibiont species composition showed a significant variation between the basibiont species, shell roughness, nature of substratum surface (artificial or natural), months of sampling, size of the basibiont shells and between mobile and non-mobile substratum surfaces. This agrees well with the hypothesis tested during this study. Finally, this study demonstrates the consequences of attachment properties and the growth forms of diatoms for the composition of epibiont communities. Declarations Funding: This work was supported by Japanese Society for Promotion of Science Invitational Fellowship to Dr. Kanavillil (2019, #S19014) that provided financial support for the study, and Japan Sea National Fisheries Research Institute, Niigata for the research facilities. Competing interests: There are no relevant financial or non-financial interests to disclose. Author contributions: Both the authors of the manuscript together planned and conducted the study. Both authors are involved in data analysis and manuscript preparation. Data Availability: The data generated used during the current study are not publicly available] but are available from the corresponding author on request . Ethics approval: This is an observational study involving intertidal organisms and planktonic epibionts and therefore did not require any ethical approval. Consent to participate: Since there are no human subjects involved in the study not consent was required Consent to publish: This was not required as there were no human subjects involved in the study. Compliance with Ethical Standards: The funding for the research was provided by Japanese Society for Promotion of Science (JSPS) in the form of invitational fellowship to Dr. Kanavillil. We declare that we have no conflict of interest and have followed all the guidelines including the ethical guidelines while samples were collected. Acknowledgements Dr. Kanavillil acknowledges Japanese Society for Promotion of Science Invitational Fellowship (2019, #S19014) that provided financial support for the study, and Japan Sea National Fisheries Research Institute, Niigata for the research facilities. Dr. Todd Stubbs and Dr. Tim Kaiser of Lakehead University, Canada and Dr. Lesley Lovett-Doust, Nippising University, Canda are thanked for copy editing the manuscript. 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Hoikusha Publishing Company Ltd, Osaka, Japan Supplementary Files TableS1S2.docx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3411120","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":239279869,"identity":"8924dbbe-a1e4-46a3-805b-41c697ba76f4","order_by":0,"name":"Nandakumar Kanavillil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACHjApIcPPzsDMwNgAZB8gUguPZDOJWhh4DA4Tq8Wc5/CzBz/bLHiMDzM/Nvi4w0ae70YC44cfeLRY9raZG/a2SfCYHWYzTpx5Js1w5o0EZskePFoMzjOYSfCcAWlhMD7M23aYccONBDaYc3FoYf8m+QeoxbiZ/fPhv23/7UFaGP/g03K2x0yap0KCx4CZxziZse1AIkgLM15bzpwpk5YBapE4zFMM9FRy8swzD5ulZfBqSd8m+cagTo6/vX2zxM82O9u+48kHP77BowUbAMfOKBgFo2AUjAJKAAAiJ0t4ffR36QAAAABJRU5ErkJggg==","orcid":"","institution":"Lakehead University Faculty of Science and Environmental Studies","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nandakumar","middleName":"","lastName":"Kanavillil","suffix":""},{"id":239279870,"identity":"68b84506-f9f3-4643-8d43-c3798b876338","order_by":1,"name":"Y Takada","email":"","orcid":"","institution":"Japan Fisheries Research and Education Agency: Kokuritsu Kenkyu Kaihatsu Hojin Suisan Kenkyu Kyoiku Kiko","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Y","middleName":"","lastName":"Takada","suffix":""}],"badges":[],"createdAt":"2023-10-04 17:01:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3411120/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3411120/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44604622,"identity":"64684cab-6377-4c3c-be62-cc0856e017a2","added_by":"auto","created_at":"2023-10-13 22:15:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127020,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing sampling sites.\u003c/p\u003e","description":"","filename":"Fig1MB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/587b6dda640e151f769c6848.jpg"},{"id":44604627,"identity":"ed926fb8-2747-4669-94f4-a0d37c413ed7","added_by":"auto","created_at":"2023-10-13 22:15:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182858,"visible":true,"origin":"","legend":"\u003cp\u003eMean (±SD) of epibiont species richness, diversity (Simpson diversity index), and density between selected substrata in May. Differences between natural substrata (shells) and plastics are in the left row, and differences between live gastropods \u003cem\u003eTegula\u003c/em\u003e spp. (\u003cem\u003eT. nigerrima\u003c/em\u003e and \u003cem\u003eT. rustica\u003c/em\u003e) and shells occupied by Hermit crabs are in the right row. Results of one-way ANOVA (*: p \u0026lt; 0.05, ns: not significant) are also shown on the top of each figure.\u003c/p\u003e","description":"","filename":"Fig2MB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/23c54854471fa48e655a75a6.jpg"},{"id":44604621,"identity":"503d5b74-c4c9-4266-9c5f-61a170508691","added_by":"auto","created_at":"2023-10-13 22:15:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287249,"visible":true,"origin":"","legend":"\u003cp\u003eMean (±SD) of epibiont species richness, diversity (Simpson diversity index), and density between natural substrata (shells). Differences between mobile (Gastropoda, Polyplacophora, and Echinoidea) and non-mobile (Bivalvia), and between large (shell size ≥ 48mm) and small (\u0026lt; 48mm), and between smooth surface and rough surface (Table 1) are compared with the interaction of month (April and May). Different letters (a, b, and c) at the top-right of the bars in each figure indicate significant differences (p \u0026lt; 0.05) detected by Tukey-HSD tests and two-way ANOVA.\u003c/p\u003e","description":"","filename":"Fig3MB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/eaaff1bcd64a352a642404f7.jpg"},{"id":44604625,"identity":"5b027bb0-3080-453e-b1ce-a0f0193d0f3a","added_by":"auto","created_at":"2023-10-13 22:15:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105539,"visible":true,"origin":"","legend":"\u003cp\u003eOrdination by nonmetric multidimensional scaling (nMDS) of the assemblages of epibionts (99 species or taxonomic units). Open symbols are samples in April and closed symbols are those in May. Circles are natural substrates and triangles are plastics. Colors indicates 6 groups identified by the k-medoid cluster analysis: cluster 1 - black, cluster 2 - red, cluster 3 - green, cluster 4 - dark blue, cluster 5 - light blue, and cluster 6 - yellow. Six symbols with asterisk on the upper-right indicate the medoid samples of the cluster.\u003c/p\u003e","description":"","filename":"Fig4MB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/19df842318dd32f4aacb367f.jpg"},{"id":44604626,"identity":"403fc9a7-7be2-4a58-84b0-c7e0b500100e","added_by":"auto","created_at":"2023-10-13 22:15:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":203998,"visible":true,"origin":"","legend":"\u003cp\u003eThe percentage composition of epibiont species observed in the medoid sample of the 6 clusters as identified in the nMDS plot.\u003c/p\u003e","description":"","filename":"Fig5MB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/529659e378aba70cec679f40.jpg"},{"id":44604628,"identity":"94c54dc6-37a3-40a5-8e38-a99ec123b74c","added_by":"auto","created_at":"2023-10-13 22:15:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":225687,"visible":true,"origin":"","legend":"\u003cp\u003eMean (±SD) of species richness and density of diatoms in the epibiont community. Diatoms are divided into attached and unattached, and solitary and colony forming species.\u003c/p\u003e","description":"","filename":"Fig6MB.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/e38e9d34c0ed0893fc6acb27.jpg"},{"id":49552855,"identity":"359e4f80-1945-4eee-b01f-d81d26dbec6f","added_by":"auto","created_at":"2024-01-12 22:59:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":906213,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/48e6451c-c375-470f-b780-0e0b6dfb865c.pdf"},{"id":44604623,"identity":"3b48738d-9e0c-45dc-a3cd-1075deceab3f","added_by":"auto","created_at":"2023-10-13 22:15:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37124,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1S2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3411120/v1/d0e312e4d95800412542998b.docx"}],"financialInterests":"","formattedTitle":"The effect of substrate type on microscopic epibiont community diversity: A shoreline study from the Sea of Japan at Niigata, Japan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe marine intertidal zone, the coastal strip lying between high and low watermarks, is home to organisms belonging to various trophic levels. In the intertidal zone, primary producers are well exposed to sunlight, tidal cycles, continuous water movement, predation and competition and can support a diverse community of heterotrophs (Poore et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Space tends to be a limiting factor, so many organisms settle and grow on any hard substrate that is available, including the body surfaces of other living organisms (Dayton \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1971\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEpibiosis or \u0026lsquo;living on\u0026rsquo;, is the term applied to organisms that live, either temporarily or permanently on the hard surfaces of other organisms called \u0026lsquo;basibionts\u0026rsquo; (Walker and Miller \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Wahl \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Among other benefits, epibionts gain: the protection on the host\u0026rsquo;s growing surface, better access to resources such as light and nutrients; and, if the host is mobile, a means of dispersal (Romagnoli et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Totti et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Falasco et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In most cases, this association is not considered harmful to the basibionts; indeed, they can also benefit from their epibionts. For example, the growth of epibionts such as biofilms composed of bacteria, microalgae and fungi, also termed \u0026lsquo;periphyton\u0026rsquo;, can protect the host\u0026rsquo;s surfaces from ultraviolet radiations, fluctuations in increased temperature (Patil and Anil \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The slimy surface may deter predators, thus functioning as a protective layer (Wahl et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Patil and Anil \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Marine organisms with hard shells (e.g., hard shelled molluscs) are of special importance here because even after their death, their empty shells provide additional settlement surfaces for attached organisms (Gutierrez et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, this association can sometimes have negative impacts on the host. If the epibionts become large macrophytes, they can restrict host movement making the host vulnerable to predators (Patil and Anil \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The epibionts may also cause shell deterioration (boring) of mollusks, coral reefs, Foraminifera, etc. (cf. Golubic and Schneider \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Tribollet et al. 2006; Barill\u0026eacute; et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMolluscs are a major group of organisms that contributes to the biodiversity of marine coastal areas. Most secretes calcified shells that are widespread in coastal habitats (Asakura and Suzuki \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). These hard-shell surfaces allow the attachment and growth of microscopic epibiotic communities (Thyrring et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Barill\u0026eacute; et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Intertidal organisms, especially sessile ones, compete for limited settlement space, nutrients and light availability (for plants) (Coates \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Since there is clear zonation in the intertidal zone, from deeper to shallower areas, there are distinctive bands of communities in terms of the distribution of intertidal flora and fauna (Benedetti-Cecchi and Cinelli \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). In turn, the surfaces of these organisms will be used as settlement surfaces by various macrobenthic (metazoan and macroalgae) and microbenthic (bacteria, diatoms, cyanobacteria and microalgae) organisms. Previous studies on the growth of epibiont communities have focused on the species of the host: for example, on bivalve and gastropod shells (D\u0026rsquo;Alelio et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), Gillan and Cadee (2000), Thyrring et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), Barill\u0026eacute; et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), Kagawa et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)). Other studies looked at epibionts on Bryozoans (Wuchter et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), Bivalvia (Barill\u0026eacute; et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), hermit crabs (Stachowitsch \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1980\u003c/span\u003e), barnacles (Gutierrez and Palomo 2016), crayfish (Falasco et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and green turtles (Majewska et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, to this point, there have been no studies on epibiont communities living on basibionts of different phyla living in the same part of intertidal zone. This information is critical to an understanding of the role of basibionts determining epibiont community composition.\u003c/p\u003e \u003cp\u003ePhototrophic epibionts (photosynthetic algae and bacteria) play important roles in the dynamics of aquatic ecosystems by performing carbon and nutrient uptake and producing oxygen essential to the survival of fish and marine invertebrates (Mac Intyre et al. 1996; Penhale and Smith 1997). Among the various groups of epibionts, diatoms are the most intensely studied group with more than 10,000 species described (Mann \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; D\u0026rsquo;Alelio et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Majewska et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Falasco et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The growth of epibionts on basibionts depends on various factors, both biotic and abiotic such as the epibiont\u0026rsquo;s growth forms (attached or unattached) and growth characteristics (solitary or colonial) (D\u0026rsquo;Alelio et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Other factors that will affect epibionts are: host behaviour (Creed \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Gribben et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), host shell morphology and surface characteristics (Wahl, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Thyrring et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), the host\u0026rsquo;s developmental stage (Dougherty and Russell \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), host secretion of repellents (antifouling properties, Wahl \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), habitat (Reiss et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), water flow rate and wave action (Wahl \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Wilson \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), grazing pressure and competition (Rosemond et al. 1993; Burgmer et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and season (Cabral et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Taken together these factors suggest that members of the epibiont community are likely to show high host specificity (Majewska et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and therefore it is important to see how much variation is observed among epibiont communities growing on different substrates in the same marine intertidal habitats.\u003c/p\u003e \u003cp\u003eThe goal of the present study is to characterize the epibiont communities found on shell surfaces of intertidal organisms belonging to different taxa (Gastropoda, Bivalvia, Polyplacophora and Echinoidea), and to compare these communities with those found on inert materials such as plastic surfaces. This will allow us to compare the niche preferences of these microflora on a finer scale, i.e., in terms of the roughness of the substratum surface. Although epibiont species have a common pool of propagules (e.g., planktonic organisms) in a coastal area, they can be specialists, growing only on a particular substrate, and/or generalists that grow on various surfaces. Therefore, the objectives of the study were to compare epibiont communities on (1) shells of different intertidal organisms; (2) shells of different roughness (smooth vs rough); (3) natural (shells) and artificial (plastic) surfaces; (4) mobile vs non-mobile substrates; and (5) different months of sampling on the epibiont community. We hypothesise that epibiont communities will differ significantly between shells of different invertebrates, between natural (shells) and artificial substrates (plastic) and between mobile and non-mobile substrates. We also hypothesise that epibiont community will favour non-mobile, large and rough shells compared to mobile, small and smooth shell surfaces.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eStudy site\u003c/p\u003e \u003cp\u003eThe study was conducted on a natural/artificial rocky shore of the Sea of Japan, behind the Japan Sea National Fisheries Research Institute (JSNFRI), Niigata prefecture, Japan (37\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e55\u0026rsquo;N, 139\u0026deg;02\u0026rsquo;E; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The shore here is a man-made seawall consisting of natural rocks and concrete tetrapods constructed several decades ago to prevent the beach erosion to which this area is historically prone (Uda \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The shore is exposed to tidal fluctuations (0.3 m) and is being colonized by various intertidal organisms. To study epibionts, basibionts from the intertidal zone belong to Gastropoda, Bivalvia, Polyplacophora and Echinoidea were collected three times during early and late spring (April 5, April 10, and May 16, 2019). Care was taken not to cause any damage to the organisms and their epibionts while collections were made. After collection, individuals belonging to different species were put in different plastic bags with seawater collected from the sampling site to prevent drying. Along with the host species, several plastic objects (~\u0026thinsp;500 mL (\u0026gt;\u0026thinsp;25 cm long) smooth surface plastic bottles) exposed to seawater were also collected for epibiont analysis. The tidal fluctuation in this area during sample collection was 0.3 m. The major surface seawater (~\u0026thinsp;10 cm below surface) parameters measured during every sampling day varied between 10.8 \u003csup\u003eo\u003c/sup\u003eC (April) to 16.7 \u003csup\u003eo\u003c/sup\u003eC (May), pH 8.49 (May) to 8.63 (April), and salinity 18.4\u0026permil; (May) to 22.2\u0026permil; (April). A portable probe (YSI Model 63, YSI, Yellow Springs, OH) was used to measure pH, water temperature and salinity.\u003c/p\u003e \u003cp\u003eFigure 1. Map showing sampling sites.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e \u003cp\u003eEpibiont extraction\u003c/p\u003e \u003cp\u003eAfter having been brought back to the laboratory, samples were immediately identified to the species level with the help of identification keys (Okutani \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). After identification the heights and widths of individuals were measured with a Vernier caliper with an accuracy of 0.1 mm. Next, the epibionts were scraped from the shell with a clean toothbrush and a known quantity of aged seawater filtered through Whatman No. 1 filter paper). This was done by brushing and rinsing the shell surfaces four times to collect all epibionts growing on the shell surfaces. This method was standardized by running preliminary experiments to test the efficacy of epibiont removal by repeated brushing and rinsing and observing the shell surfaces and the filtrate for the presence of epibionts. Four brushings followed by rinsing with 2.5 mL of filtered aged seawater (at least six months in the refrigerator) each time was found to be an adequate technique to remove most of the epibionts from the shell surfaces. Where larger surfaces were encountered (for example, shells of \u003cem\u003eMagallana\u003c/em\u003e sp. or \u003cem\u003eMytilus\u003c/em\u003e sp.; plastic surfaces), a known area of 20 x 20 mm from the edges was scraped to remove the epibionts. After collecting the whole extracts (10 mL/shell), they were properly labelled and kept in the refrigerator for epibiont analysis. The epibiont samples were analyzed immediately after scraping. The samples were concentrated by centrifuging the samples (~\u0026thinsp;2000 rpm for 5 min) followed by careful removal of the supernatant. Subsamples were drawn from the concentrated samples after thorough mixing for observation in a haemocytometer at 200x, 400x and 1000x magnifications under a compound microscope (Olympus, Japan); epibionts were thus counted and identified. In this study, the major groups of epibionts examined include diatoms, unicellular and filamentous green algae, cyanobacteria and red algae. When protozoans were present, they were counted but not identified. Each sample was observed eight times and an average was taken to calculate their cell density. The density of each epibiont is expressed as No. mm-2 area and these values were used for the statistical analysis. Altogether 87 samples (shells and plastics) were observed during this study. The epibionts were identified with the help of several identification keys (Round et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Prescot \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Yamaji \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Chihara and Murano \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eBasibiont categories\u003c/p\u003e \u003cp\u003eTo understand the importance of various physical and biological characteristics of the host species for epibiont community composition, the data were divided (categorised) as below before carrying out the statistical analysis. The basibionts/hosts were arbitrarily divided according to their shell size (small and large) and shell morphology (roughness, judged by finger touch arbitrarily divided into two categories, smooth and rough). Accordingly, organisms were categorized into small (organisms with shell size\u0026thinsp;\u0026lt;\u0026thinsp;48 mm height and width) and large (shell size\u0026thinsp;\u0026ge;\u0026thinsp;48 mm height and width), and those with smooth (e.g., \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e, Chitons and plastic surfaces) and rough shells (gastropods e.g., \u003cem\u003eMonodonta\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, the basibionts were also categorized into mobile (moves on the rock surface, e.g., gastropods and hermit crabs) and non-mobile (bivalves). Basibionts were also grouped into live (shells with live organism \u0026ndash; henceforth \u0026ldquo;live\u0026rdquo;) and \u0026ldquo;dead\u0026rdquo; (only shells) at the time of collection. Finally, they were also grouped into natural (shell) and artificial (plastic) surfaces.\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\u003eTypes of substrata (shells of live and dead basibionts and plastic) sampled from Sekiya rocky shore during the three sampling episodes (April 05,10 and May 16). Gastropoda, Polyplacophora, and Echinoidea were mobile substrata, while Bivalvia and Plastic were non-mobile ones.\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrata/Intertidal organisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApr-05\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eApr-10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay-16\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo. of shells/surfaces\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRoughness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSize range (mm)***\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGastropoda\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCellana grata\u003c/em\u003e (A. Gould)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.7-L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCellana toreuma\u003c/em\u003e (Reeve)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.9\u0026ndash;29.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTegula rugata\u003c/em\u003e (A. Gould)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.2\u0026ndash;31.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLottia kogamogai\u003c/em\u003e Sasaki \u0026amp; Okutani\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\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 \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMonodonta confusa\u003c/em\u003e Tapparone Canefri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.0-24.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTegula nigerrima\u003c/em\u003e (Gmelin)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (1)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19.2\u0026ndash;22.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTegula pfeifferi carpenteri\u003c/em\u003e (Dunker)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTegula rustica\u003c/em\u003e (Gmelin)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 (3)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18.9\u0026ndash;23.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eReishia bronni\u003c/em\u003e (Dunker)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSiphonaria japonica\u003c/em\u003e (Donovan)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.2\u0026ndash;20.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBivavalvia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMagallana gigas\u003c/em\u003e (Thunberg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.9-L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMytilus galloprovincialis\u003c/em\u003e Lamarck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.7-L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSaccostrea kegaki\u003c/em\u003e Torigoe \u0026amp; Inaba\u003cem\u003e**\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePolyplacophora (Chitons)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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=\"c1\"\u003e \u003cp\u003e\u003cem\u003eOnithochiton hirasei\u003c/em\u003e Pilsbry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.2\u0026ndash;34.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRhyssoplax kurodai\u003c/em\u003e (Is. Taki \u0026amp; Iw. Taki)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEchinoidea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStronlyocentrotus intermedius\u003c/em\u003e (A. Agassiz)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePlastic\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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=\"c1\"\u003e \u003cp\u003ePlastic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmooth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e* shells occupied by hermit crabs\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e** dead shells\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e*** L: size larger than 48 mm\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eEpibiont assemblages observed on basibionts and plastic surfaces were analyzed using multivariate statistical techniques. Abundance data of the epibiont taxonomical units (species) were used to estimate dissimilarity values of assemblages between all pairs of the samples (between the host categories of natural (shells) and artificial (plastics) in both the months of sampling (April and May)) by calculating the Bray-Curtis dissimilarity values (Jost et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Based on the dissimilarity values, ordinations in reduced spaces were generated by nonmetric multidimensional scaling (nMDS). The data were log (x\u0026thinsp;+\u0026thinsp;1) transferred before running the analysis. The assemblages were clustered into several groups by K-medoids (Borcard et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and the average silhouette values were calculated to decide the appropriate number of clusters. The indicator species of the assemblages (clusters) were selected by using the indicator value (IndVal) (Dufr\u0026ecirc;ne and Legendre 1997). The significance of a species as an indicator of an estimated cluster of assemblages was evaluated by using a randomization procedure (n\u0026thinsp;=\u0026thinsp;9999).\u003c/p\u003e \u003cp\u003eThe composition of epibiont species on natural substrates (i.e., excluding plastics) was also compared by using PERMANOVA (Anderson \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; McArdle and Anderson \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). To clearly understand the effect of sampling time (April \u0026ndash; early spring vs May \u0026ndash; late spring), one-way PERMANOVA was analyzed with month as a fixed factor, assuming a large effect of month on the epiphyton community. Then the epibiont data were further divided into those found on natural surfaces (all basibionts) vs artificial surfaces (plastics); small vs large shells (size); and smooth vs rough shells (roughness), and two-way PERMANOVAs were carried out with month and the substrate characteristics (nature of substrates, size and roughness) as fixed factors. In addition, because plastics and hermit crabs were obtained only in the month of May, the epibiont data were compared between plastic and natural (shells) substrates, and between shells of live gastropods (\u003cem\u003eTegula nigerrima\u003c/em\u003e and \u003cem\u003eT. rustica\u003c/em\u003e) and the dead (ones inhabited by hermit crabs) were done in May by one-way fixed-effect PERMANOVA. For PERMANOVA, the Bray-Curtis dissimilarity with log(x\u0026thinsp;+\u0026thinsp;1) transformed abundance data were used.\u003c/p\u003e \u003cp\u003eEffects of month, mobility, size, and roughness on species richness, Simpson diversity index (inverse Simpson), and density of epibionts (all taxa combined) on the natural substrates were tested by ANOVA. Assuming a large effect of month, as for the PERMANOVA mentioned above, two-way fixed-effects ANOVAs with the factors of month and each of the other three factors (size, mobility and roughness) were performed. When the ANOVA showed significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Tukey-HSD tests were done for a post-hoc comparison. Similar to PERMANOVA, the effects of plastic and hermit crabs on species richness, Simpson diversity index, and density of epibionts were tested by one-way fixed-effect ANOVA using the samples obtained in May. In addition, within sample diversity (alpha diversity), total diversity (gamma diversity), and between sample diversity (beta diversity which is defined as multiplicative beta diversity (Jost \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e)), were calculated for epibiont species richness and Simpson diversity indices. Before these analyses, the species richness and the Simpson diversity index were transformed to square-rooted values, and the density was transformed to log values to reduce heteroscedasticity between samples and tested by the Levene test.\u003c/p\u003e \u003cp\u003eThe establishment of an epibiont community, especially diatoms, depends on their growth characteristics (solitary or colonial) and growth forms (attached or non-attached). In order to study the significance of the growth forms and characteristics of diatoms (comprising\u0026thinsp;\u0026gt;\u0026thinsp;87% of the epibionts) in the community composition of epibionts, they were grouped (placed in one of four groups): as attached or unattached, and as solitary or colony forming (Round et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Prescot \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; and Spaulding et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The species richness and densities of diatoms in the K-medoid clusters were studied with respect to their growth forms and characteristics as mentioned above to understand the role played by these factors in shaping community composition. The analyses were performed using R 4.2.1 software and packages cluster, labdsv, and vegan (R Core Team \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eBasibionts\u003c/p\u003e \u003cp\u003eThe basibionts collected include 10 Gastropoda species, 3 Bivalvia species, 2 Polyplacophora species and one Echinoidea species. In addition, plastic objects found along the rocks and submerged in the water were also collected for the epibiont analysis. The list of host substrates along with their dates of collection are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eEpibiont composition\u003c/p\u003e \u003cp\u003eThe list of epibionts found on different host substrates during the three sampling dates are given in Supplemental Table S1. The dominant group of epibionts was diatoms, followed by cyanobacteria, Chlorophyta and Rhodophyta. The total number of epibiont species observed on April 5, 10 and May 16 were 28, 50 and 62 species, respectively. Diatoms comprised more than 90% of the total epibiont species encountered. Among the substrates examined, \u003cem\u003eCellana toreuma\u003c/em\u003e recorded the highest density of epibionts (6080.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10008 cells mm-2) followed by \u003cem\u003eSaccostrea kegaki\u003c/em\u003e (1726.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1521.5 cells mm-2) and \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e (1646.1\u0026thinsp;\u0026plusmn;\u0026thinsp;491.4 cells mm-2). The major species of epibionts based on the average density found among the substrates were \u003cem\u003eLicmophora abbreviata\u003c/em\u003e (135\u0026thinsp;\u0026plusmn;\u0026thinsp;201.3 cells mm-2) and \u003cem\u003eNavicula membranacea\u003c/em\u003e (717\u0026thinsp;\u0026plusmn;\u0026thinsp;1035.8 cells mm-2) in April 2019 and \u003cem\u003eFragilaria oceanica\u003c/em\u003e (146\u0026thinsp;\u0026plusmn;\u0026thinsp;2194 cells mm-2) and \u003cem\u003eN. membranacea\u003c/em\u003e (141\u0026thinsp;\u0026plusmn;\u0026thinsp;126.8 cells mm-2) in May 2019, respectively.\u003c/p\u003e \u003cp\u003e The overall mean epibiont species richness during this study was 10.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.27 (n\u0026thinsp;=\u0026thinsp;87, alpha diversity\u0026thinsp;=\u0026thinsp;9.79) with a minimum of min 3spp. on \u003cem\u003eSiphonaria japonica\u003c/em\u003e in Apr, and max 24 spp. on \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e in May. In total, 96 species (gamma diversity) were observed across 87 samples. The beta diversity (between samples) of species richness was 9.80. As for Simpson diversity, the alpha, beta, and gamma diversities were 3.17, 1.88, and 5.96, respectively. The epibiont density and species diversity (Simpson diversity) did not show significant difference between natural substrates (shells) and the plastics, and between hermit crabs and live gastropod shells (Fig.\u0026nbsp;2, Supplemental Table S2). However, in May the species richness (number of species) varied significantly between the natural substrates and plastics with plastics showing lower species richness. The species richness did not vary between hermit crabs and live Gastropod shells (Fig.\u0026nbsp;2, Supplemental Table S2).\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;2. Mean (\u0026plusmn;\u0026thinsp;SD) of epibiont species richness, diversity (Simpson diversity index), and density between selected substrata in May. Differences between natural substrata (shells) and plastics are in the left row, and differences between live gastropods \u003cem\u003eTegula\u003c/em\u003e spp. (\u003cem\u003eT. nigerrima\u003c/em\u003e and \u003cem\u003eT. rustica\u003c/em\u003e) and shells occupied by Hermit crabs are in the right row. Results of one-way ANOVA (*: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ns: not significant) are also shown on the top of each figure.\u003c/p\u003e\u003cp\u003eThe results of one-way ANOVA on overall epibiont species richness, Simpson diversity and epibiont density on natural substrates (shells of various host species) with month as the fixed factor showed significant variation only in species diversity (F\u0026thinsp;=\u0026thinsp;31.48, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (Simpson diversity (Supplemental Table S2). Furthermore, species richness, species diversity and epibiont density showed significant variation with the substrate characteristics such as mobility of the host, size of the shells and shell roughness (Fig.\u0026nbsp;3). Generally, the results indicated shells of non-mobile hosts, hosts with large shells and shells with smooth surfaces had higher epibiont density, species richness and species diversity than shells of mobile hosts, hosts with small shells and hosts with rough shell surfaces, respectively (Fig.\u0026nbsp;3). The variation was more evident in May than in April (Fig.\u0026nbsp;3). The epibiont density, species richness and species diversity on non-mobile hosts and on larger shells in May were found to be significantly different from the non-mobile, mobile and small shelled host species in the month of April (Fig.\u0026nbsp;3). In addition, the effect of interaction of month of sampling and mobility of the hosts, month and size of the host shells and month and shell roughness resulted in significant variation of species richness, species diversity and epibiont density (Fig.\u0026nbsp;3, Supplemental Table S2).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;3. Mean (\u0026plusmn;\u0026thinsp;SD) of epibiont species richness, diversity (Simpson diversity index), and density between natural substrata (shells). Differences between mobile (Gastropoda, Polyplacophora, and Echinoidea) and non-mobile (Bivalvia), and between large (shell size\u0026thinsp;\u0026ge;\u0026thinsp;48mm) and small (\u0026lt;\u0026thinsp;48mm), and between smooth surface and rough surface (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are compared with the interaction of month (April and May). Different letters (a, b, and c) at the top-right of the bars in each figure indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) detected by Tukey-HSD tests and two-way ANOVA.\u003c/p\u003e \u003cp\u003eAnalysis of variation of epibiont assemblages on natural substrate (shells of invertebrates) using two-way PERMANOVA to study the influence of host characteristics such as mobility, shell size and shell roughness with months as a fixed factor showed highly significant variations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Community composition was found to vary significantly with respect to the interaction of factors such as months and mobility, months and shell size and months and roughness. In addition, in the month of May, the one-way PERMANONVA results showed significant variations of eipibiont assemblages on hermit crab/gastropods, and plastics/natural shell substrates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of two-way PERMANOVA (9999 permutations) for epibiont assemblages on natural substrates with month as a fixed factor (April and May), and other host characteristics such as mobility (mobile/non-mobile), shell size (small/large), and shell roughness (smooth/rough). Also included are results of one-way PERMANOVA to analyze effects of plastics and Hermit crabs on epibiont assemblages in May.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePseudo-F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMobility\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMobility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth X mobility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShell size\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth X size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRoughness\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRoughness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth X Roughness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOthers\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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 \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlastics/shells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHermit crabs/gastropods\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.021\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 \u003c/p\u003e \u003cp\u003eThe two-dimensional ordination plot of nMDS using the Bray-Curtis dissimilarity (stress\u0026thinsp;=\u0026thinsp;0.192) showed a usable representation of the variation of epibionts found on the shells and the plastic surfaces (Fig.\u0026nbsp;4). Generally, the assemblages were separated by the sampling month (April or May) and the nature of the substrates i.e., plastics or natural surfaces. Thus, the assemblages on plastics were found to be located at the peripheral area of the plot with large variation between each surface studied. Six groups of assemblages based on the dissimilarity values were identified by the K-medoid cluster analysis (Fig.\u0026nbsp;4 and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These clusters are generally well separated with each other except clusters 3 and 4 which were considerably overlapped (Fig.\u0026nbsp;4). Medoid samples of each cluster were generally located at the center of the points of the cluster (Fig.\u0026nbsp;4). The number of samples and their average silhouette values of K-medoid clusters for each assemblage on live invertebrate shells, hermit crabs and plastics in April and May are given in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of samples and average silhouette values of K-medoid clusters for assemblages on invertebrate shells, Hermit crabs and plastics in April and May\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApril\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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=\"c1\"\u003e \u003cp\u003eGastropoda (limpets)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\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\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGastropoda (others)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBivalvia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolyplacophora\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEchinoidea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eplastics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\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=\"c1\"\u003e \u003cp\u003eGastropoda (limpets)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGastropoda (others)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBivalvia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolyplacophora\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEchinoidea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\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\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eplastics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\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\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSilhouette value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0871\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.15132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNumbers in parentheses show Gastropod shells inhabited by Hermit crabs\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;4. Ordination by nonmetric multidimensional scaling (nMDS) of the assemblages of epibionts (99 species or taxonomic units). Open symbols are samples in April and closed symbols are those in May. Circles are natural substrates and triangles are plastics. Colors indicates 6 groups identified by the k-medoid cluster analysis: cluster 1 - black, cluster 2 - red, cluster 3 - green, cluster 4 - dark blue, cluster 5 - light blue, and cluster 6 - yellow. Six symbols with asterisk on the upper-right indicate the medoid samples of the cluster.\u003c/p\u003e \u003cp\u003eAll samples belonging to clusters 1 and 4 and most samples of cluster 3 were from April (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On the other hand, all samples belonging to clusters 5 and 6 and most samples of cluster 2 were from May. The Bivalvia, Polyplacophora, and limpet shaped Gastropoda samples were mainly categorized in cluster 3 (in April) or cluster 5 (in May) while the remaining Gastropoda samples were found in other clusters.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e also shows the indicator species of each cluster with their indicator values (IndVal). The higher the value, the stronger is their presence in the cluster. Thus, several species can be detected as strong indicator species of each cluster (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;5). The membership of each cluster also showed several facts; for example, \u003cem\u003eMeuniera membranaceae\u003c/em\u003e showed significant presence in all the medoid samples of the six clusters (Fig.\u0026nbsp;5). On the other hand, there are species which occupies high percentage in one medoid sample of the cluster (e.g., \u003cem\u003eCocconeis pseudomarginata\u003c/em\u003e in cluster 5, IndVal 0.558; \u003cem\u003eThalassionema.frauenfeldii\u003c/em\u003e, in cluster 6, IndVal 0.422) while several other genera can be seen in more than one cluster (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIndicator taxonomic units with their indicator value (IndVal) for the six clusters (permutation p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u0026ndash; cluster 2 does not have any indicator species\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndicator taxonomic units\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCluster\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIndVal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProbability\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCoscinodiscus radiatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCocconeis placentula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.034\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphora lanceolata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBiddulphia\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0328\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStephanopyxis palmeriana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0326\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMelosira nummuloides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLicmophora abbreviata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.485\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGomphonema exiguum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0034\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCoscinodiscus\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCocconeis\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMelosira hyperborea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCoscinodiscus plicatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNitzschia vitrea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePinnularia\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNitzschia\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFragilaria\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePinnunavis elegans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0254\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCyanobacteria\u003c/em\u003e sp.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphora\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0364\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBiddulphia pulchella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.048\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParalia sulcata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0172\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCocconeis pediculus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNavicula salinarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.651\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCocconeis pseudomarginata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFragilaria oceanica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCoscinodiscus megalomma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTabularia parva\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0042\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAzpeitia nodulifera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphora ovalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0174\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGrammatophora marina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0094\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNitzschia pubens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0154\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRhabdonema arcuatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0204\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphora longiceps\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0224\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillaria paxillifera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eThalassionema frauenfeldii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0004\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 \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;5. The percentage composition of epibiont species observed in the medoid sample of the 6 clusters as identified in the nMDS plot.\u003c/p\u003e \u003cp\u003eDiatoms were labeled into attached (63 spp.)/unattached (24 spp.) and solitary (45 spp.)/colony (42 spp.) species. Some substrates did not have unattached or colonial diatoms. So, comparisons to the original (all epibionts, 96 species) assemblage by Mantel correlation and nMDS ordinations were done only for the attached and solitary diatoms. The attached diatoms (63 species) showed a higher correlation (Mantel Pearson, 0.945, log (+\u0026thinsp;1) transformed Bray-Curtis) with the original epibionts than the solitary diatoms (47 species) (Mantel Pearson, 0.799). The correlation between attached diatoms and solitary diatoms was 0.825 (Mantel Pearson), where only 31 species were common. The nMDS identified 6 clusters based on K-medoid values using attached diatoms. The species richness of attached colonial diatoms showed a different pattern from the overall microalgae, partly because of the small sample size. Species richness of unattached diatoms in May was significantly higher than in April, while no difference was detected for unattached diatoms. The species richness and density of attached/unattached and solitary/colonial diatoms in the 6 clusters identified in nMDS is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFigure 6. Mean (±SD) of species richness and density of diatoms in the epibiont community. Diatoms are divided into attached and unattached, and solitary and colony forming species. \u003c/p\u003e \u003cp\u003eThe species richness of attached diatoms was higher than unattached diatoms in all 6 clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The lowest species richness of unattached forms was in cluster 1 while the highest was in cluster 5. The species richness of diatoms among the solitary and colonial forms did not differ much in any of the 6 clusters. Similarly, the species density of attached forms was generally higher than the unattached ones in all the 6 clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The lowest species density of the unattached forms was observed in cluster 1. The species densities remained the same in solitary and colonial forms in all 6 clusters.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, 16 natural substrates (gastropods, bivalves, Polyplacophora and Echinoidea) and plastic objects were analyzed for microscopic epibiont community composition. This study provides a comprehensive picture of the epibiont community composition on a variety of intertidal organisms and plastic surfaces. Epibionts, some of them macroscopic, have been studied on various aquatic organisms previously. However, each of these studies involved only a few substrates such as seagrass (De Stefano et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), Porifera (McClintock et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e); Hydrozoa (Romagnoli et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2007\u003c/span\u003e); Bryozoans (Wuchter et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2003\u003c/span\u003e); gastropods (Gillan and Cadee 2000; D\u0026rsquo;Alelio et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Thyrring et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Barill\u0026eacute; et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kagawa et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); hermit crabs (Stachowitsch \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1980\u003c/span\u003e); mud snail, \u003cem\u003eHydrobia ulvae\u003c/em\u003e (Gillan and Cadee 2000); Scallops, Oysters and Mussels (Barill\u0026eacute; et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e); Crustaceans (Gutierrez and Palomo 2016); Cephalopod and Neogastropod egg shells (Lim et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e); crayfish (Falasco et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); turtles (Majewska et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e); and whales (Denys \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). This study revealed the composition of epibiont communities on several different substrates (basibionts) in the intertidal zone of Niigata, as well as its alpha (within each basibiont and plastics), beta (between these basibionts), and gamma (total of all these basibionts) diversities.\u003c/p\u003e \u003cp\u003eThe epibiont species richness can vary from individual to individual of the same basibiont species depending on the microenvironment they are in. For example, individuals belonging to the same species found on exposed rock surfaces may have a different epibiont community from those found in crevices. Thus, it is important to know the alpha diversity to get an overall picture of the epibiont community variation on a certain species. In this study, the alpha values for the epibionts were found to be much smaller than the gamma (overall) diversity values of epibionts across all the species, resulting the large beta diversity. This is expected as varying factors such as basibiont mobility, roughness and size, habitat selection, and the environmental factors in which they are living can alter the epibiont species diversity on the basibionts.\u003c/p\u003e \u003cp\u003eAs expected, the diatoms formed the major component of the epibionts found on all types of substrates. This finding agrees with the results observed in the previous studies (Totti et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, among diatoms, the species that contributed higher densities include \u003cem\u003eLicmophora abbreviata\u003c/em\u003e (mostly attached and colonial), \u003cem\u003eMeuniera membranacea\u003c/em\u003e (mostly unattached (sometimes prostrate) and single) and \u003cem\u003eFragilaria oceanica\u003c/em\u003e (mostly attached and colonial). This indicates attached and colonial forms along with unattached and single cells equally colonized the substrates. It is noteworthy that the density and species diversity of epibionts did not show significant variations between natural shell surfaces and plastics and hermit crab and gastropods (in the month of May). However, epibiont species richness was significantly higher on natural shell surfaces compared to plastics. Plastics, even though they are inert surfaces, may be prone to abrasion or toxic to several organisms (Worm et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEpibiont community compositions vary widely between basibionts/substratum surfaces. Under the assumption that epibionts species have a common pool for recruitment, the characteristics of basibionts, environmental factors, and interactions of these and epibionts can play a role in this variation. Specifically, some of the reasons attributed to this variation are the structures of basibiont shells (Wahl \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Thyrring et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e); shell size (D\u0026rsquo;Alelio et al. 2011); presence/absence of grazers in the environment (Thyrring et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); secretion of antifouling compounds by the basibionts (Whal 1989); and the environmental parameters such as water quality (D\u0026rsquo;Alelio et al. 2011), water movement (Wahl \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Wilson \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), atmospheric exposure of the shells among many others (D\u0026rsquo;Alelio et al. 2011). Substratum surface roughness has been described as an important surface characteristic that can influence the settlement and growth of diatoms on wet surfaces (Sweat and Johnson \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This study results showed no variation in epibiont density between smooth plastic surfaces and rough shell surfaces (Supplemental Table S2) nor between rough and smooth shell surfaces (Fig.\u0026nbsp;3). In fact, the results showed a higher epibiont density on smooth shell surfaces (such as the shell of sessile Bivalves, \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e and Chitons) than on the rough shell surfaces of mobile and non-mobile gastropods (Fig.\u0026nbsp;3). A similar pattern of diatom settlement was reported by Sweat and Johnson (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) where they observed higher diatom settlement on smooth surfaces than the rough ones. Scardino et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) with the help of attachment point theory explained the higher diatom settlement on smooth surfaces. The smooth surfaces according to Scardino et al. (2013) offer more points of contact for diatoms leading to higher number of diatom settlement. Furthermore, species diversity and richness were found to be higher on smooth shell surfaces than the rough gastropod shell surfaces. However, one important fact to consider here is the variation of basibiont species composition on which the epibiont compositions were studied. Generally, smooth shells belonged to bivalves and chitons (not all species observed in this study, please see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) while rough ones were a characteristic of gastropods. Thus, the observed trend might be due to many reasons such as the difference in secreted shells roughness, non-toxic chemical (repellent) production, behavioural changes of basibionts etc. (Wahl et al. 2009 and the references there in). Unfortunately, there are no data available on the role of shell morphology of bivalves and gastropods on the epibiont community formation.\u003c/p\u003e \u003cp\u003eThe results of this study suggest that the basibiont characteristics such as the shell size and mobility played a significant role in the community compositions of epibionts. Generally, larger shell surfaces had higher species density, diversity and richness (MacArthur and Wilson \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; Relyea \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This phenomenon can be more clearly perceived in May than in April (Fig.\u0026nbsp;3). In an aquatic environment this is an expected result as larger shells provide more surface stability compared to smaller ones thereby leading to a higher density, diversity and richness of epibionts (Martins et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The alternative explanation that larger shells provide a longer period for settlement and development for epibiont during shell development can be rejected because of the larger variations observed between samples in May and April. Similarly, with respect to the mobility of the shells (substratum surfaces), the non-mobile ones harboured higher species density, diversity and richness of epibionts than on the mobile ones (Fig.\u0026nbsp;3) suggesting that the epibionts prefer stable substratum surfaces than the unstable ones for their attachment and growth. With respect to the plastic surfaces several factors such as the stability (surfaces exposed for a long period by being trapped in crevices vs objects just brought to shore by waves) and nature of the surface (large, smooth, or rough) play an important role in the epibiont community formation.\u003c/p\u003e \u003cp\u003eThe month of sample collection played a significant role in the composition of epibiont communities. The PERMANOVA analysis on the epibiont assemblages on substratum surfaces (basibionts\u0026thinsp;+\u0026thinsp;plastics) showed a significant variation between months, with May showing a higher density than April. This result is expected as the seawater temperature and salinity went up from April to May (10.8 \u003csup\u003eo\u003c/sup\u003eC (April) to 16.7 \u003csup\u003eo\u003c/sup\u003eC (May), pH 8.49 (May) to 8.63 (April), and salinity 18.4\u0026permil; (May) to 22.2\u0026permil; (April)). Warmer water temperatures and the arrival of water runoff from agriculture fields with higher nutrient concentration may result in a change in an epibiont community (Martins et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Bulleri et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A slightly different picture emerged when the density, diversity and species richness of epibionts on the natural substrate (shells) were analyzed with month as a factor. The two-way ANOVA showed epibiont density, diversity and richness varied significantly with the mobility, size and roughness of the shells (Supplemental Table S2). The results once again show the importance of shell characteristics of basibionts on the epibiont community composition.\u003c/p\u003e \u003cp\u003eThe results of two-dimensional ordination plot of nMDS using the Bray-Curtis dissimilarity showed variation of epibionts found on the shells and the plastic surfaces (Fig.\u0026nbsp;4) demonstrating the role of substratum surfaces on epibiont community composition. The assemblages on plastics were found to be located at the peripheral area of the nMDS plot with large variation between each surface studied. In the context of the increased presence of plastic objects floating around in aquatic systems, this observation is important. The microbial biota settling over the plastic objects not only find these surfaces for attachment but may also lead to their disintegration/breaking down into smaller objects leading to increased pollution problems (Worm et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The large variations of epibiont community observed on plastics may partly be due to the variation among the chemical properties of their surfaces, elasticity, history of plastics on the shore (e.g., drifted height on the shore), stability (frequency of overturning by wave and wind), frequency of wave and tidal splash during the growth of epibionts. In comparison to the non-mobile plastics, gastropods generally exhibit a cyclic movement synchronized with tidal and diurnal cycles (Iwasaki \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Takada \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). This difference may affect the variation of the epibiont communities on these surfaces.The results also showed separation of epibiont assemblages by the sampling month (April or May) as observed in the results of ANOVA. Six groups of assemblages based on the dissimilarity values were identified by the K-medoid cluster analysis (Fig.\u0026nbsp;4 and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). They are generally well separated with each other except clusters 3 and 4 which were considerably overlapped on the two-dimensional plot thus showing the relationship between the clusters, month, and basibionts (Fig.\u0026nbsp;4). It is interesting to see the key species having the largest IndVal changes between each of the clusters showing the variation of species composition with months and substratum surfaces. On a closer observation, the leading members of these clusters possess their own ecological properties representing the occupied space and time. For example, the species with the highest IndVal \u003cem\u003eMelosira nummuloides\u003c/em\u003e (cluster 3, April) is a benthic diatom exhibiting euryhaline and eurithermal properties that help them colonize the sea-land transition zones (Prelle et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, \u003cem\u003eLicmophora abbreviata\u003c/em\u003e, the other diatom with high IndVal (cluster 3, April) is reported to survive well at low seawater temperatures, typically in spring (between 10\u0026ndash;17 \u003csup\u003eo\u003c/sup\u003eC) (Jung et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and \u003cem\u003eCoscinodiscus radiata\u003c/em\u003e is known to survive in neritic marine water with high light availability (Medvedeva et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). \u003cem\u003eBacillaria paxillifera\u003c/em\u003e (cluster 6, May) a motile euribiontic diatom found living in both freshwater and marine water, is capable of surviving in turbid waters \u0026ndash; another character that distinguishes it from the rest (Ussing et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCommunity composition also can be influenced by the growth forms (e.g., colonial vs unicellular), growth characteristics (e.g., attached vs free floating), and the type of epibionts, namely, micro algae, diatoms, cyanobacteria, etc. (D\u0026rsquo;Alelio et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The ability of diatoms to colonize a surface depends on their attachment properties, such as the type of attachment, namely prostrate vs stalked or unattached. Additionally, the mobility or immobility of diatoms also have roles to play in the colonization process (D\u0026rsquo;Alelio et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This study showed diatoms as the major component of the epibionts. Among diatoms the attached and solitary forms were the dominant ones that outnumbered the colonial and unattached ones. This is an expected result as the attachment property helps in the colonization and growth of epibionts. A similar trend was observed in case of diatom species richness as well; the diatom species richness of the attached forms was more than that of unattached ones. The results thus show the importance of attachment properties and growth forms of diatoms in the epibionts community and the role of basibionts on the overall epibiont community composition on a seashore.\u003c/p\u003e \u003cp\u003eThe basibiont-epibiont community dynamics demonstrates the conceptual context of facilitation cascade via habitat formation and modification as outlined by Thomsen et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Here, the shells of invertebrates in the intertidal habitats act as primary habitat-former while the epibionts as the secondary habitat-formers leading to higher species diversity and abundance in the habitat as noted in other habitats/ecosystems such as forest, seagrass meadows, salt marshes and seaweed beds.\u003c/p\u003e \u003cp\u003eIn conclusion, the epibiont species composition showed a significant variation between the basibiont species, shell roughness, nature of substratum surface (artificial or natural), months of sampling, size of the basibiont shells and between mobile and non-mobile substratum surfaces. This agrees well with the hypothesis tested during this study. Finally, this study demonstrates the consequences of attachment properties and the growth forms of diatoms for the composition of epibiont communities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Japanese Society for Promotion of Science Invitational Fellowship to Dr. Kanavillil (2019, #S19014) that provided financial support for the study, and Japan Sea National Fisheries Research Institute, Niigata for the research facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThere are no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBoth the authors of the manuscript together planned and conducted the study. Both authors are involved in data analysis and manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe data generated used during the current study are not publicly available] but are available from the corresponding author on request\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is an observational study involving intertidal organisms and planktonic epibionts and therefore did not require any ethical approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince there are no human subjects involved in the study not consent was required\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was not required as there were no human subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe funding for the research was provided by Japanese Society for Promotion of Science (JSPS) in the form of invitational fellowship to Dr. Kanavillil. We declare that we have no conflict of interest and have followed all the guidelines including the ethical guidelines while samples were collected. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Kanavillil acknowledges Japanese Society for Promotion of Science Invitational Fellowship (2019, #S19014) that provided financial support for the study, and Japan Sea National Fisheries Research Institute, Niigata for the research facilities. Dr. Todd Stubbs and Dr. Tim Kaiser of Lakehead University, Canada and Dr. Lesley Lovett-Doust, Nippising University, Canda are thanked for copy editing the manuscript. Comments from the two anonymous referees helped to improve the manuscript. Lakehead University, Canada is also acknowledged for all the support and encouragements.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAsakura A, Suzuki H (1987) Zoogeographical aspects of rocky-intertidal molluscan fauna of the Pacific coasts of Japan. Mar Biol 95:75\u0026ndash;81\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson MJ (2001) A new method for non-parametric multivariate analysis of variance. Austral Ecol 26:32\u0026ndash;46\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarill\u0026eacute; L, Le Bris A, M\u0026eacute;l\u0026eacute;der V, Launeau P, Robin M, Louvrou I, Ribeiro L (2017) Photosynthetic epibionts and endobionts of Pacific oyster shells from oyster reefs in rocky versus mudflat shores. 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Hoikusha Publishing Company Ltd, Osaka, Japan\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"epibionts, basibionts, gastropod shells, Sea of Japan, intertidal organisms, diatoms","lastPublishedDoi":"10.21203/rs.3.rs-3411120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3411120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroscopic epibionts are an important component of intertidal ecosystems. Shortage of space drives these organisms to settle and grow on body/shell surfaces of intertidal organisms. In this study we test the hypothesis that epibiont communities vary significantly between shells belonging to different invertebrate species, and between natural shells and plastic surfaces. Epibiont community variations are caused by differences in shell size, roughness and mobility of the host organisms. Epibionts growing on fifteen different species belonging to Gastropoda, Bivalvia, Polyplacophora and Echinoidea, as well as plastic objects were collected from a rocky shore of the Sea of Japan at Niigata. Most epibionts collected were diatoms, and the highest epibiont density was recorded on the surfaces of the limpet \u003cem\u003eCellana toreuma\u003c/em\u003e. The results showed epibiont species richness, diversity and density varied significantly with the host characteristics such as mobility, shell size and shell roughness. The shells of sessile organisms with large and smooth surfaces had a higher epibiont density, species richness and diversity than did shells of mobile organisms with small and rough shell surfaces. To conclude, epibiont species composition varies significantly between basibiont species, shell morphology, shell size, shell roughness, month of sampling and the mobility of the host.\u003c/p\u003e","manuscriptTitle":"The effect of substrate type on microscopic epibiont community diversity: A shoreline study from the Sea of Japan at Niigata, Japan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-13 22:15:27","doi":"10.21203/rs.3.rs-3411120/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17ec4f37-f573-4665-8516-43f63b8ab319","owner":[],"postedDate":"October 13th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-12T22:51:24+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-13 22:15:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3411120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3411120","identity":"rs-3411120","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}