Persistence and space preemption explain species‐specific founder effects on the organization of marine sessile communities

Abstract Community assembly may not follow predictable successional stages, with a large fraction of the species pool constituted by potential pioneering species and successful founders defined through lottery. In such systems, priority effects may be relevant in the determination of trajectories of developing communities and hence diversity and assemblage structure at later advanced states. In order to assess how different founder species may trigger variable community trajectories and structures, we conducted an experimental study using subtidal sessile assemblages as model. We manipulated the identity of functionally different founders and initial colony size (a proxy of the time lag before the arrival of later species), and followed trajectories. We did not observe any effects of colony size on response variables, suggesting that priority effects take place even when the time lag between the establishment of pioneering species and late colonizers is very short. Late community structure at experimental panels that started either with the colonial ascidian Botrylloides nigrum, or the arborescent bryozoan Bugula neritina, was similar to control panels allowed natural assembling. In spite of high potential for fast space domination, and hence negative priority effects, B. nigrum suffered high mortality and did not persist throughout succession. Bugula neritina provided complex physical microhabitats through conspecific clustering that have enhanced larval settlement of late species arrivals, but no apparent facilitation was observed. Differently, panels founded by the encrusting bryozoan Schizoporella errata led to different and less diverse communities compared to naturally assembled panels, evidencing strong negative priority effects through higher persistence and space preemption. Schizoporella errata founder colonies inhibited further conspecific settlement, which may greatly relax intraspecific competition, allowing resource allocation to colony growth and space domination, thus reducing the chances for the establishment of other species.

While historical contingencies can restrict our ability to forecast patterns of species assembling (Dickie, Fukami, Wilkie, Allen, & Buchanan, 2012;Sutherland, 1974), life history traits of some early colonizers may help predicting the consequences of priority effects (Cifuentes, Krueger, Dumont, Lenz, & Thiel, 2010;Cleland, Esch, & McKinney, 2015;Sutherland, 1978). Depending on traits of the early colonizers, priority effects may occur either by negative or positive species interactions, largely dictated by inhibition or facilitation during early stages of the development of communities (Fukami, 2015;Gerla & Mooij, 2014;Weslien et al., 2011). The best known inhibitory effects occur through niche preemption, when early founders monopolize important resources that would otherwise be available to other species (De Meester et al., 2016;Fukami, 2015;Sutherland, 1978), or when first colonizers act as ecosystem engineers, modifying their habitat and preventing the establishment of other organisms (Bonnici, Evans, Borg, & Schembri, 2012;Jones, Lawton, & Shachak, 1994). Depending on intrinsic species traits, however, ecosystem engineering may otherwise play an opposite role and facilitate species arriving later (Fukami, 2015;Jones et al., 1994), for example, by providing tridimensional substrates and therefore increasing settlement grounds (Russ, 1980), or through the mitigation of abiotic stress by supplying more benign microhabitats (Jurgens & Gaylord, 2016;Perea & Gil, 2014;Vogt et al., 2014), ultimately leading to species coexistence and an increase of biodiversity.
The extent to which founders affect the trajectory of developing communities may depend on how conspecifics of early-colonizer species interact. In the case of species with complex life cycles, the effects of adult cues to propagules or juveniles are particularly important, because they may deeply affect local abundance and spatial distribution patterns of individuals (Lara-Romero, Cruz, Escribano-Ávila, García-Fernández, & Iriondo, 2016). Attraction through chemical cues may constitute an efficient way to promote aggregation in suitable habitats (Pawlik, 1992;Robinson, Larsen, & Kerr, 2011;Silva-Filho, Bailez, & Viana-Bailez, 2012), indirectly increasing the strength of densitydependent regulation mechanisms of founder populations. Positive density-dependent interactions include the mitigation of abiotic stress in crowding intertidal invertebrates (Jurgens & Gaylord, 2016;Minchinton, 1997), caterpillars (Klok & Chown, 1999), and plants (Vogt et al., 2014); enhanced reproduction, such as fruit dispersal in plants (Blendinger, Loiselle, & Blake, 2008) and fertilization in marine invertebrates (Kent, Hawkins, & Doncaster, 2003;Levitan, Sewell, & Chia, 1992) and terrestrial woodlice (Broly, Deneubourg, & Devigne, 2013); and diminishing of predation risk in invertebrates (Denno & Benrey, 1997;Turchin & Kareiva, 1989) and vertebrates (Blumstein & Daniel, 2003;Carrascal, Alonso, & Alonso, 1990). Negative interactions usually lie in some sort of intraspecific competition, which may reach unsustainable levels under conditions of very high-population density (Branch, 1975;Chisholm & Muller-Landau, 2011;Gerla & Mooij, 2014;Hart & Marshall, 2009;Robins & Reid, 1997). Therefore, the potential for propagule dispersal and the strength of intraspecific competitive interactions after establishment may ultimately have shaped the selection of chemical responses to conspecific cues, either positively or negatively. To establish such a link, there is a need to assess conspecific responses in light of species-specific strategies on resource use and competitive hierarchy. In this sense, we could expect species in which individuals grow fast and tend to monopolize resources to repel conspecifics (and thus reduce intraspecific competition), and species in which isolated individuals are poor competitors, and rely on the attraction of conspecifics to form more resistant aggregations (Hart & Marshall, 2009;Porensky, Vaughn, & Young, 2012).
Careful experimental manipulation of founder assemblages, followed by the examination of community trajectories, may help understanding how priority effects modulate species diversity and assemblage structure at advanced successional states (Chang & Marshall, 2017;Cifuentes et al., 2010;Edmunds, 2014). Such methodology, however, has been used majorly for simple organisms, and effects measured in terms of individual counts for species that contributed the most to contrasts between treatments (Geange, Poulos, Stier, & McCormick, 2017;Irving et al., 2007;Jiang et al., 2011). Probably because manipulating the abundance of more complex organisms in natural conditions is more challenging, effects on natural assemblages dominated by clonal or colonial plants or animals are less documented (De Meester et al., 2016;Fukami, 2015). For decades, the construction of marine facilities, such as harbors and marinas, has promoted several modifications in coastal ecosystems, increasing the availability of hard substrata, which may support a diverse community of sessile organisms (Bulleri & Chapman, 2010). While some studies show a clear competition-colonization trade-off, and thus, sessile community succession being determined by niche-based processes (Buss and Jackson, 1979;Edwards and Stachowicz, 2010) suggested that nontransitive competitive relationships among species may explain alternative community states across or within habitats under similar conditions. Under these circumstances, priority effects may be important, because common species cannot be classified a priori as either early or late successional species in any obvious way, and because propagules of several species belonging to distinct functional groups may be available anytime.
The importance of this historical contingency has been extensively discussed in descriptive studies (Osman, 1977;Sebens, 1986), but as long as we know this is one of the few studies addressing experimentally in the field how the identity and survivor of founder species affect community assembling and diversity. To do so, we triggered different history contingencies by manipulating both the identity of early colonizers and the time lag between the founder arrival and the subsequent establishment of other species (Urban & De Meester, 2009) in sessile communities from shallow subtidal artificial substrates. We expected to find (i) founder-specific priority effects, which would be (ii) more intense when the time elapsed between first colonization and the arrival of later species arrivals was longer. We also predicted (iii) lower diversity of advanced assemblages first colonized by strong competitors for bare space.

| Study site
The study was carried out from April to September of 2013 at the Yacht Club Ilhabela (23°46′26.95″S/45°21′21.26″W), in the São Sebastião Channel, Southeastern Atlantic Coast of Brazil ( Figure 1). This site is a recreational marina with a diverse fouling system growing on the sides of the floating blocks. Previous studies showed that the sessile community is dominated mostly by colonial ascidians and encrusting and arborescent bryozoans, but also sponges, barnacles, bivalves, polychaetes, and hydrozoans Oricchio, Pastro et al., 2016;).

| Target founder species
We selected three invertebrate species that are common in the study area since early colonization to test how historical contingency related to variable colonization and eventual priority effects promoted by size advantage influences the development and structure of sessile communities: the flat soft-bodied colonial ascidian Botrylloides nigrum, the flat calcified encrusting bryozoan Schizoporella errata, and the erect arborescent bryozoan Bugula neritina. Both flat founders are good space monopolizers (Kay & Keough, 1981;Nandakumar, 1996;Vieira, Duarte, & Dias, 2012;Vieira et al., 2016), but while the ascidian is less resistant to predation when not having an escape window that enables the colony to attain large sizes Osman & Whitlatch, 2004;Vieira et al., 2012Vieira et al., , 2016, the hard mineralized body of the bryozoan confers resistance against predators since early stages (Lidgard, 2008). Conversely, the arborescent founder does not effectively monopolize space (Walters & Wethey, 1996) and could F I G U R E 1 Yacht Club Ilhabela (YCI) study site at São Sebastião Island, southeastern Brazil (23°46′S/45°21′W). Modified from Vieira et al. (2016) potentially facilitate the establishment of other organisms due to its tridimensional structure (Breitburg, 1985;Russ, 1980). Although we were not able to estimate all differences in life history traits among the species, we assumed that traits related to space preemption/provision and vulnerability to predation are of paramount importance to community dynamics, as long as space is a limiting resource. Previous observations in the same area showed that all the three above-mentioned species are frequent during early stages (first 30 days) at experimental panels deployed year-round, with Schizoporella errata being the most frequent species, occurring in 90% of all panels, followed by Bugula neritina in 80% and Botrylloides nigrum in 50% ( Figure S1). Therefore, we assume that results from our experiments may generally reflect natural processes at the study area throughout the year.

| Priority effects through manipulation of initial size
Besides the manipulation of founder identity, we also tested the effects of founder size. This latter variable was used as a proxy of the time lag between first colonization and later species arrivals. Founders given more time to develop without the interference of other individuals would probably be prone to monopolize resources or modulate recruitment and survivor of later species by any other means.
To test this idea, we started experimental panels with either younger small colonies (10-15 days old), given little advantage over later settling species, or older large colonies (20-30 days old), given large advantage over species coming next. Both time lags simulated natural stages during early colonization: The 10-to 15-day lag represented a moment when bare space is still abundant and most of the colonies are still growing with no restriction imposed by border contacts with others, while the 20-to 30-day lag represented a moment when more than 50% of the space is already covered, colonies may still find empty space to expand but contact interactions become common. Lags over 30 days were not considered as they would simulate an unrealistic scenario [i.e., colonies older than a month would hardly ever found isolated in nature (Dias, Delboni, & Duarte, 2008;Oricchio, Pastro et al., 2016;Vieira et al., 2012Vieira et al., , 2016]. Small and large ascidian colonies or encrusting bryozoans occupied an area around 0.5-1.0 and 1.5-2.0 cm 2 , respectively, while small and large erected bryozoan recruits bear 2-3 and 5-7 bifurcations, over an area of 1.0-1.5 and 3.0-3.5 mm 2 , respectively. Although the size of large colonies was near the double of small ones, the area they covered was still negligible compared to the overall area of experimental plates (around 5%, see below). Therefore, any effects of founding colony size would be an outcome of any advantages conferred to more advanced recruits (e.g., priority effects through improved conditions to resist environmental stress or predation, intra-or interspecific competition, etc.), and not because of differences in initial space preemption.

| Experimental setup
Inventory panels covered by sanded acetate sheets were deployed along the pier docks 2 months prior to the experiment in order to collect a sufficient supply of colonies of the target founder species within the needed size range. The selected settlers were then cut off from acetate sheets of inventory panels and glued with a cyanoacrylate adhesive on experimental sanded PVC panels (15.0 × 20.0 × 0.5 cm; Hart & Marshall, 2009). We cut the colonies off the acetate sheet keeping a free acetate border of as much or twice the size of the colony. This procedure was conducted underwater avoiding touching the colony and did not take longer than 2-3 min. To attach the acetate piece with the settled colony to the panel surface, we used a very small portion of glue, just sufficient to ensure attachment, avoiding leaking or contact with the colony. This needed to be a very fast procedure, no more than few seconds, to ensure no harm to colonies. Each experimental panel received four colonies of the same species and size around the center, leaving approximately 4 cm of distance among them. Twelve replicate panels were prepared for each combination of founder species (B. nigrum, B. neritina, and S. errata) and colony size (small and large, corresponding to the time lag between the foundation and the next arrival, i.e., 10-15 and 20-30 day, respectively) making up 72 panels. Treatment combinations were randomly interspersed along the docks, maintaining a distance of 2 m between neighboring panels. Panels were suspended at ~1.5 m depth, allowing a distance of 1.5 m from the sea bottom to avoid the negative impacts of suspended sediments for filter feeders. For each treatment combination, four panels were retrieved at 1 (early), 3 (mid), and 5 months (advanced).
The early stage is characterized by plenty of bare space with no contact interactions among colonies and thus no competition for space. The mid stage is characterized by increased competition for space, with most colonies already contacting neighbor individuals, and the occurrence of both dead individuals and overgrowth. The advanced stage is characterized by almost no bare spaces and intense competitive interactions. At this stage, overgrowth by strong competitors was common.
In order to better describe the natural assembling and put into context any comparisons among experimental treatments, we deployed 12 extra sanded PVC panels over the same period without any interventions. These were also retrieved in three replicate groups (n = 4) at 1, 3, and 5 months after deployment.

| Sampling
As persistence, space occupation, and density of founders can all affect community succession, we estimated survival rates and coverage for each founder species, besides testing whether their presence had an influence on early total and conspecific recruitment. For panels started with all founding species and colony sizes, we examined images obtained from experimental panels retrieved at 1 month to estimate (i) early survival of founding colonies (percentage of founding colonies alive), (ii) space preemption (as percentage coverage of surviving founder colonies, using a 100 intersection grid), (iii) the number of recruits of all fouling species, and (iv) the number of recruits of each tested founder species. For the latter, we assumed conspecific aggregation if more new colonies of species "a" were found in panels started with species "a," compared to panels started with species "b" or "c." The opposite result would indicate inhibition of conspecific.

| Data analyses
Survival data, coverage, total recruitment per panel and per available bare space were analyzed using a two-way ANOVA, with fixed factors "founder identity" (three levels) and "colony size" (two levels). Conspecific recruitment rates were analyzed separately for each founder species, using the same orthogonal model. We also compared total recruitment per panel and per available bare space of experimental founder treatments (B. nigrum, B. neritina, and S. errata) with panels prone to natural assembling using one-way ANOVA. "Colony size" was not included here because it did not apply to natural plates and also because it was not significant in any case. For all analyses, the Kolmogorov-Smirnov test was performed to check normality, and the Levene test was performed to check homogeneity of variances. Pairwise Tukey post hoc testing was used to further examine significant sources of variation.

| Sampling procedure
After 1 (early), 3 (mid), and 5 months (advanced stage), sets of four replicate panels of all seven treatments (the six combinations of species and colony sizes, plus a set of replicate panels exposed to natural assembling) were retrieved from the field, placed in coolers filled with sea water, and transported to the laboratory. All panels were scanned under a dissecting microscope to identify species to the lowest possible taxonomic level, and photographed to estimate the absolute percentage of area covered by the main taxa as a measure of abundance (following Vieira et al., 2016Vieira et al., , 2012. Analyses were limited to the central 13 × 13 cm area to avoid marginal areas exposed to manipulation.

| Data analyses
Taxa richness estimates were compared using a three-way ANOVA model, testing for "founder identity," "colony size," and "stage" (also a fixed factor, with three levels: early, mid, and advanced). As for total recruitment, we performed an additional analysis comparing richness of experimental founder treatments with panels exposed to natural assembling. Assumptions checking and post hoc comparisons were carried out as above.
The absolute cover area (%), an indicator of abundance of all the invertebrate groups identified, was used to compare community structure among founder treatments and over time, using the same model described above (i.e., testing the effects of "founder identity," "colony size," and "stage"). We built resemblance matrices using Bray-Curtis distances on square-root transformed data, and performed respective PERMANOVA tests using 999 permutations (Anderson, 2001). PERMDISP tests for dispersion homogeneity were carried out for each successional stage (Anderson, 2001). Pairwise post hoc comparisons within significant sources of variation were used to test for specific contrasts. Species contributing the most to between-group differences were identified using the SIMPER procedure (Clarke, 1993). We compared community structure of experimental founder panels with those left to natural assembling following the same multivariate procedures described above.

| Survival and coverage
Regardless of colony size, both survival and coverage differed among founder species (Table 1). Colonies of the bryozoans S. errata and B. neritina survived more, respectively 90% and 70%, than colonies of B. nigrum which suffered 80% mortality after 30 days (Figure 2a).
At that time, S. errata founders covered around 40% of the available space, which corresponded to the double of the area covered by B. neritina founders and five times the area covered by B. nigrum founders (Table 1, Figure 2b).

| Effects on recruitment
Overall recruitment rates depended solely on the identity of founder species, with no detectable effects of initial colony size (Table 2,

| Taxa richness
The number of taxa was variable over time and dependent on founder identity, but not colony size. Communities founded by B. nigrum and B. neritina supported remarkably similar richness (Tukey test: p = .904), around 10 species, during the whole experiment, while communities started by S. errata were on average substantially less speciose, with less than seven species (Tukey test: p < .001). General differences throughout the experiment were the result of a significant richness increase from months 3 to 5 (Tukey test: p = .010; Table 4). The number of species in control panels exposed to natural dynamics did not differ from richness estimates in communities founded by B. nigrum and B. neritina (Tukey test: p = .999 and p = .996, respectively), but was significantly higher than the number of species observed in panels founded by S. errata (Tukey test: p = .001; Table 5, Figure 5).

| Community structure
The differences in community structure among panels founded by different species were variable over time, regardless of colony size (Table 4). After 1 month, each founder species led to a different community structure (Tukey test; p < .05 for all pairwisecomparisons). Panels founded by B. nigrum were dominated by ascidians, panels founded by B. neritina were dominated by arborescent bryozoans, and panels founded by S. errata were dominated by encrusting bryozoans (Figure 6a). The species that most contributed to these differences were the founders themselves and opportunistic species that colonize vacant space during colonization as hydrozoans and filamentous algae (Appendix:  Figure 6).

| DISCUSSION
As predicted, our study indicates that the identity of founder species has an important role in the subsequent organization of sessile communities. However, contrasting to our predictions, significant departures from the natural assembling process, in which chances of priority effects were not increased by any means, were only evident for only one founding species, the space-monopolizing species Schizoporella errata, regardless of initial colony size. When arriving first, S. errata decreased the chances of later species to settle nearby by preempting space, resulting in less diverse communities over time,  do not monopolizes space, and Botrylloides nigrum, that is potentially able to fast space preemption but did not persist longer, led to similar richness and structure compared to natural untouched communities, restraining growth rates of clonal species and ensuring baseline species coexistence. Negative priority effects of S. errata were shown to be pervasive over time, possibly owing to this species resistance to predators (Oricchio, Pastro et al., 2016), and long colony duration (Sutherland, 1978;Sutherland & Karlson, 1977), and should only relax when aged colonies eventually detach, creating new space available for the establishment of other species.
Our results differ from those reported in other studies that suggest larger priority effects when the founding population is ei-   (Poulos & McCormick, 2014). We ensured a differential chance of advantage for arriving first by allowing founding colonies of two size categories (a proxy of time lag) to more (for long-lag/large colonies) or less (for short-lag/small colonies) relaxed competition for resources before the establishment of later species (Urban & De Meester, 2009). Although we expected that colonies of different sizes would show different potential of monopolizing or providing resources, we surprisingly did not observe differences on the extent of outcomes between the two lags tested. This means that priority effects, such as the ones found for S. errata, take place very fast, probably right after settlement (<15 days) when colonies are just a few zooids large.
In spite of its potential to rapidly spread over flat surfaces (Kay & Keough, 1981;Nandakumar, 1996), and thus to impose priority effects through resource preemption when arriving first (De Meester et al., 2016;Fukami, 2015), we observed no effect for the founding colonial ascidian B. nigrum on community structure. This species exhibited the lowest persistence, likely as a result of low-survival rate and the lack of conspecific cues leading to colony clusters and thus the formation of larger and more resistant patches. Low resistance to predation, as previously observed for ascidians in the study area Vieira et al., 2012Vieira et al., , 2016 and elsewhere (Jurgens, Freestone, Ruiz, & Torchin, 2017;Osman & Whitlatch, 2004) (Jackson & Hughes, 1985;Kay & Keough, 1981), which face a high risk of being dislodged or overgrown (Nandakumar, 1996;Russ, 1982). The high-survival rate observed here coupled with the wellknown resistance against predators (Lidgard, 2008;Oricchio, Pastro et al., 2016) resulted in remarkable persistence of S. errata growing colonies, rendering pervasive effects on community structure, which may last until any disturbance event results in the detachment of aged colonies.
The capacity of Schizoporella to efficiently monopolize space and inhibit the arrival of other species was previously suggested in classic studies during the 70's, based on both observational and experimental approaches (Sutherland, 1978;Sutherland & Karlson, 1977). By forcing sets of experimental assemblages to be founded by functionally different sessile invertebrates, we provide here independent and novel evidence for the unique role of Schizoporella species worldwide in the determination of benthic community dynamics. In our study area, S. errata seems to be the strongest competitor in artificial hard substrates, owing both to its fast growth and resistance to predators Oricchio, Pastro et al., 2016). Differently from earlier studies showing later Schizoporella die-off and subsequent species replacement after 1 year (Sutherland, 1978;Sutherland & Karlson, 1977),  (Fukami, 2015). Our results do support overall settlement facilitation, and previous studies had indicated that intricate branches of B. neritina can provide settlers of different species valuable refuge from predation (Breitburg, 1985;Russ, 1980;Walters & Wethey, 1996). However, contrary to expectations, we found no priority effects in panels started with this species. In spite of creating physically complex substrates that could provide settlement secondary habitat and shelter from predators to an array of other species, the overall abundance of recruits in B. neritina plots did not differ from control ones. It is possible that gregarious settlement has led to exceedingly high levels of intraspecific competition (Chisholm & Muller-Landau, 2011;Gerla & Mooij, 2014), ultimately decreasing founder persistence and thus any eventual long-term positive effects on species diversity. Despite of showing a higher survival rate after 30 days when compared to B. nigrum, B. neritina did not persist in advanced community stages, allowing S. errata to colonize and occupy substantial space in panels. This nullified any possible priority effects, and shifted communities to states similar to the ones observed in naturally assembled panels.
Overall, our results highlight that priority effects caused by species capable of fast resource monopolization and that persist through time, such as S. errata, may change recruitment patterns and consequently further community assembly. Schizoporella errata remarkably precluded the establishment of several other species through space preemption, leading to relatively impoverished assemblages. While our results do not support avoidance as a mechanism underlying poor recruitment of the whole suite of species, they do strongly suggest the inhibition of conspecific recruitment near developing S. errata colonies. Such a response may relax intraspecific competition, allowing the allocation of resources to clonal growth resulting in fast resource monopolization. In contrast, poor competitors such as B. neritina may potentially create novel habitat for recruits of many different species, but still not cause longer-term effects on community assembling because of their low persistence in the community. We conclude that founder effects ultimately depend on life history strategies of pioneering species, with density-dependent effects on benthic stages playing a crucial role.

ACKNOWLEDGMENTS
The authors thank the staff at the Center for Marine Biology (CEBIMar-USP) and the Yacht Club Ilhabela (YCI) for field assistance.

CONFLICT OF INTEREST
None declared.

AUTHORS' CONTRIBUTIONS
EAV, AAVF, and GMD conceived the ideas and designed methodology. EAV collected the data. EAV, AAVF, and GMD analyzed the data.
EAV, AAVF, and GMD wrote the manuscript. All authors contributed critically to early drafts and approved its submission for publication.