α-diversity of SCC and the relative abundance of taxa in ARMS
A total of 69 MSPs, encompassing 12 phyla, were identified within the sessile cryptobenthic communities that settled in the 15 ARMS at the La Saline site. The Chao estimator predicted a potential species pool of 116.9 MSPs (ChaoSE: ± 6.8). Accumulation curves of MSP detection, plotted against the number of plate faces analysed across immersion times, deployment, and retrieval seasons, approached a plateau (Fig. S2). This suggests that the sampling effort conducted using Coral Point Count was sufficient to capture most species present and allowed reliable comparisons of SCC diversity across immersion times and seasons.
Species richness (α-diversity) increased with immersion time (Fig. S3). On average, ARMS immersed for 6 months supported 24.8 ± 1.9 (mean ± standard deviation) morphospecies, while those immersed for 1 year harboured 27.8 ± 3.4 species. ARMS retrieved after 2 years exhibited the highest richness, with 33.3 ± 5.8 species. Permutational ANOVA revealed a statistically significant effect of immersion time on species richness (Niterations = 5000, p = 0.007). However, pairwise comparisons using permutational tests with Bonferroni correction did not confirm significant differences between immersion times (p6months-1year = 0.276, p1year-2years = 0.326, p6months-2years = 0.077). The observed increase in mean richness between 6-month and 2-year immersions approached but did not reach statistical significance. MSP richness did not vary significantly between ARMS deployed or retrieved during the hot or cool seasons.
The biotic categories occupying the largest surface areas on ARMS plates, in decreasing order of relative abundance, were CCA, foraminifers, annelids, prokaryotic biota (such as biofilms and cyanobacteria), bryozoans, hydrozoans, and ascidians (Fig. 3). While certain categories, such as prokaryotic biota, maintained relatively stable coverage over time, others varied notably with immersion time and deployment season. Sponges, for example, only became a prominent category after 2 years of immersion (RUNA2, Fig. S4a). Among abiotic categories, which accounted for approximately one-third of total surface area, sediment increased after 2 years of immersion, while the proportion of bare plate declined.
Mean percent cover of main benthic categories across ARMS plate faces. Stacked column chart representing the mean percent cover of the 13 main benthic categories across all ARMS plate faces. Categories are ordered by decreasing relative abundance (except for bare plate and sediment). The chart illustrates the distribution of both biotic and abiotic cover types within the ARMS communities. The labels ‘6 mo’, ‘1 yr’, ‘2 yr’ appears in red for triplicates deployed in the hot season and in blue for those deployed in the cool season.
Influence of immersion time and deployment season
PERMANOVA revealed that immersion time was the dominant factor driving variation in SCC composition among ARMS (R2 = 0.45, F = 6.77, p < 0.001). Deployment season also contributed to community differences, though to a lesser extent (R2 = 0.12, F = 3.51, p = 0.013). A significant interaction between immersion time and deployment season (R2 = 0.10, F = 3.15, p = 0.021) indicated that seasonal influence diminished with longer immersion time.
Principal Coordinate Analysis (PCoA) of biotic categories revealed clustering of SCC by immersion time (Fig. 4a). ARMS immersed for 6 months and deployed during the hot season formed a distinct cluster from those deployed in the cool season. Ascidians (both solitary and colonial) and prokaryotic biotas were more abundant in the 6-month ARMS deployed during the hot season, while 6-month ARMS deployed in the cool season (CINA3) exhibited significantly greater colonised surface area, driven by higher abundances of annelids, foraminifers, and bryozoans (Figs. S4a, b). Notably, SCC in CINA3 resembled those from ARMS immersed for 1 year.
Principal Coordinate Analysis (PCoA) based on Bray–Curtis dissimilarity of sessile cryptobenthic communities for: (a) the 13 main cryptobenthic categories (dimensions 1 and 2 explaining 54% and 26% of the variance, respectively); (b) morpho-species (MSPs) contributing up to 95% of dissimilarities among ARMS (dimensions 1 and 2 explaining 19% and 11% of the variance, respectively). Arrows indicate the relative contribution of categories and MSPs to the dissimilarity between ARMS triplicates. Colours represent different immersion times (purple: 6 months, green: 1 year, orange: 2 years) while shapes denote deployment seasons (triangles: hot, circles: cool). Prefixes indicate taxonomic groups: Ascc = Colonial ascidian; Ascs = Solitary ascidian; Bry = Bryozoan; Cni = Cnidarian; For = Foraminiferan; Por = Poriferan.
All ARMS immersed for 1 year (CINA2 and CINA4) clustered in the lower central region of the PCoA projection, indicating reduced seasonal influence compared to 6-month immersions. This aligns with the significant interaction between immersion time and deployment season detected by PERMANOVA, where seasonal effects were pronounced early in succession but diminished over time. After 1 year, SCC were characterised by increased abundances of annelids, bryozoans, and algae such as red erect macroalgae (Fig. 4b).
After 2 years of immersion, ARMS displayed greater coverage of sponges and CCA, along with sediment accumulation. Though bivalves remained rare, they also appeared later in succession. Boxplots of relative category abundances (Figs. S4a, b), supported by ANOVA and t-tests, confirmed the patterns observed in the PCoA analysis.
The finer taxonomic resolution (MSP level, Fig. 4b) detailed the taxa contributions to dissimilarities. ARMS immersed for 6 months and deployed in the hot season (CINA1) were characterised by two colonial ascidian species of the genus Botryllus (B. gregalis and B. tuberatus), a solitary ascidian MSP5, and green biofilms. In contrast, ARMS deployed for 6 months in the cool season (CINA3) hosted two colonial ascidian species of the family Didemnidae (sp2 and sp3) and juvenile foraminifers of the genus Miniacina.
We observed that communities from 1-year ARMS deployed in the cool season (CINA4) tended to exhibit higher abundances of calcareous worm tubes, red erect algae, cyanobacteria, and a dominant crust-forming bryozoan (Watersiporidae sp., Fig. 4b).
ARMS immersed for 2 years (RUNA2) formed a distinct cluster divergent from shorter immersions. PCoA at MSP level revealed compositional differences among the three RUNA2 replicates, a pattern less apparent when analysing broader biotic categories. Key contributors included 5 sponge MSPs, and 4 ascidians (including a solitary species of the genus Polycarpa, three colonial ascidians Eusynstyela sp., Aplidium sp. and an unidentified MSP). These ARMS also showed increased coverage of brown erect algae, CCA, and adult foraminifers of the genus Miniacina.
Effect of immersion time and deployment season on β-diversity components
Analysis of β-diversity indicated that ARMS from the same batch (triplicates) were significantly less dissimilar in SCC composition than ARMS from different batches (with different immersion times and/or deployment seasons, p-value < 0.001, Fig. 5). Jaccard dissimilarity was primarily driven by MSPs turnover (Fig. 5a), reflecting species replacement rather than nestedness.
β-diversity indices and components among ARMS units. Boxplots of β-diversity indices (Bray–Curtis and Jaccard dissimilarity and its components turnover and nestedness) for comparisons between ARMS (a) from the same batch versus different batches; (b) immersed for different time periods; and (c) deployed in the same season versus different seasons. N indicates the number of pairwise comparisons used to compute each index. Asterisks denote significance levels (ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
BC dissimilarity showed no significant change between ARMS immersed for 6 months and 1 year, nor between 1 and 2 years (Fig. 5b), suggesting gradual changes in relative species abundances. In contrast, Jaccard dissimilarity was significantly higher between 1- and 2-year ARMS than between 6-month and 1–year ARMS (Fig. 5b), indicating increased species turnover during later successional stages.
β-diversity decomposition showed that composition differences were largely attributable to species turnover. Therefore, while MSP richness increased over time (Fig. S2), communities from 6-month and 1-year ARMS were not merely subsets of the more diverse communities found in 2-year ARMS.
Consistent with the abundance-based PCoA and PERMANOVA results, BC dissimilarity was significantly higher between ARMS deployed in different seasons than within the same season (Fig. 5c). However, Jaccard dissimilarity did not differ significantly between or within deployment seasons (Fig. 5c), suggesting that seasonality primarily influenced relative abundance of MSPs rather than community membership.
Testing for deterministic and stochastic processes
Distinct patterns in β-diversity were revealed through null models analyses, providing insights into the processes driving SCC assembly. BC dissimilarity was consistently lower than expected under the null model for comparisons between ARMS within the same batch. Most BC values fell within the first quantile range (0%—2.5%), below the red line in Fig. 6. This marked β-diversity shortfall suggests that deterministic processes, such as environmental filtering and competition, played a dominant role in shaping MSPs relative abundances during succession.
Null model projections of β-diversity within ARMS batches. Projection of β-diversity values (Bray–Curtis and Jaccard dissimilarity, and its components turnover and nestedness) for ARMS pairs within the same batch (purple: 6 months, green: 6 months, orange: 2 years) onto the quantile distribution of β-diversity values from 999 null models. Red lines represent significance thresholds at the 2.5% and 97.5% quantiles.
In contrast, Jaccard dissimilarity and species turnover did not significantly deviate from null expectations. Most values were distributed between the 2.5th and 97.5th percentiles (Fig. 6), closely matching the null distribution. This indicates that MSP membership was primarily governed by stochastic processes, including dispersal limitation, random larval settlement, and survival, throughout the successional sequence.
β-diversity among micro-habitats within ARMS along succession
PERMANOVA revealed that microhabitat type was the primary driver of SCC composition at the plate face level (R2 = 0.23, F = 29.21, p < 0.001). Immersion time also contributed to variation in composition, albeit to a lesser extent (R2 = 0.10, F = 19.04, p < 0.001). The interaction between microhabitat and immersion time was statistically significant (R2 = 0.04, F = 2.93, p < 0.001), suggesting that the effect of microhabitat on SCC differentiation changes throughout succession.
Mean BC dissimilarity values between different micro-habitats were significantly higher than those between same micro-habitat types, except for one comparison after 2 years of immersion (Upward/Closed vs. Upward/Open, Fig. 7). This pattern supports the PERMANOVA results, reinforcing the structuring role of micro-habitat characteristics. Community differentiation across micro-habitats was evident at all successional stages. The most pronounced differences in β-diversity were observed between downward-oriented closed plate faces and upward-oriented open plate faces. PCoA indicated that the taxa driving these differences remained relatively consistent between the 6-month and 1-year immersion times (Fig. 7b). Upward-oriented open plate faces were predominantly colonised by foraminifers, CCA and cyanobacteria, reflecting higher light availability and greater water flow. Conversely, downward-oriented closed plate faces were dominated by Didemnidae ascidians and bryozoans.
Micro-habitat β-diversity within ARMS plate faces after 6 months (purple), 1 year (green), and 2 years (orange). (a) Boxplots of Bray–Curtis dissimilarity between ARMS plate faces. D: Downward, U: Upward, O: Open, C: Closed. Thus, ‘DC_UO’ indicates comparisons between micro-habitats DOWNWARD/CLOSED and UPWARD/OPEN); ‘same’ indicates comparisons between the same micro-habitat types. (b) Principal Coordinate Analysis (PCoA) of DC and UO plate faces. Triangles represent UO plate faces, circles represent DC plate faces, while arrows indicate the relative contribution of MSPs to the dissimilarity between these plate faces. Prefixes indicate taxonomic groups: Ascc = Colonial ascidian; Ascs = Solitary ascidian; Bry = Bryozoan; Cni = Cnidarian; For = Foraminiferan; Por = Poriferan.
After 2 years of immersion, SCC composition became more diverse, and certain Porifera species arose as key contributors to micro-habitat differentiation. Sponge MSP4 and MSP15 exhibited higher abundances on downward-oriented closed plate faces, while MSP12 and MSP3 were more prevalent on upward-oriented open plate faces. These observations underscore both the increasing role of sponges in structuring mature SCCs and the importance of micro-habitat complexity in fostering cryptic reef diversity and structuring their communities over time.
