**1. Introduction**

Explaining mechanisms for the variation in community structure on multiple spatial scales is one of the fundamental problems in marine ecology. Physical factors such as water flow [1–4], larval supply [5–9], substratum inclination [10–13], wave exposure [1,14–16], disturbance [17], upwelling [18,19], and salinity [20,21] have all been shown to affect the distribution and abundance of organisms on rocky shores, singly or in combination. One variable that has received relatively little attention, however, is the direct effect of bedrock type on the settlement and development of epibenthic organisms [22–25]. Rock types vary in physical and chemical characteristics that might influence the settlement and survival of sessile organisms and thereby influence community structure. For example, the amount of quartz in substrates can influence the settlement of epibenthic and infaunal

**Citation:** Ambrose, W.G., Jr.; Renaud, P.E.; Adler, D.C.; Vadas, R.L. Naturally Occurring Rock Type Influences the Settlement of *Fucus spiralis* L. zygotes. *J. Mar. Sci. Eng.* **2021**, *9*, 927. https://doi.org/ 10.3390/jmse9090927

Academic Editor: Francesca Cima

Received: 18 July 2021 Accepted: 24 August 2021 Published: 26 August 2021

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organisms [26,27]. If substrate minerology is important in the recruitment of marine biota, it would offer considerable insight into explaining the patterns in spatial distribution in these communities.

Physical characteristics of substrates such as substrate roughness, microtopography, refs. [28–41] and mineralogy [23,27] are known to affect the settlement and attachment of marine organisms. Bedrock type can certainly influence these characteristics. It is difficult, however, to compare the results of these studies because the scale of substratum heterogeneity in each study varies widely. Further, little distinction is made between surface heterogeneity on a scale smaller than the size of the propagule ('texture'), and that on a scale larger than the size of the propagule ('contour') [2,35]. The size of features relative to the size of the settling propagule is known to be important in determining the success of settlement [42].

The chemical influence of natural substrates on the adhesion of marine propagules has been open to debate [36,43]. There is increasing evidence that the attachment success of macroalgae is directly linked to the chemical characteristics of the adhesives involved and their interaction with the physical–chemical characteristics of the surface to which they bond [43–46]. The presence of biofilms, which develop rapidly on immersed material, however, can make the identification of causal mechanisms challenging.

Studies of the adhesion of zygotes and larvae to artificial and natural surfaces are not only of academic interest. The need to develop nontoxic coatings to prevent the adhesion of marine fouling organisms has revived this area of research (see reviews by [46,47]). This knowledge has implications for developing materials with anti-fouling properties and building artificial reefs and eco-friendly structures. Furthermore, in areas where coastal geologic formations are highly variable, understanding the effect of rock types on the settlement and attachment of sessile organisms may help explain the variability in successful invasion by nonnative species.

Fucoids are common members of intertidal and subtidal hard substrate communities worldwide, ranging from the Arctic to the tropics [48]. Studies have addressed the importance of surface roughness [49,50], water flow and wave action [16,49,51], and substrate type and surface properties [43,44,52] on the settlement and early development of fucalean algae. No studies that we are aware of, however, have addressed differences in settlement among different bedrock types where these algae regularly occur.

We tested the effects of four naturally occurring rock types and their surface contour on the initial attachment of zygotes of the brown alga *Fucus spiralis* Linnaeus 1753. To determine the effect of contour (surface heterogeneity on a scale larger than the size of the settling zygote), adhesion was tested on rock plates that were prepared with both natural surfaces and smooth-cut surfaces. We addressed the following questions in a series of laboratory settling experiments: (1) Are there differences in the primary adhesion of *Fucus spiralis* zygotes on limestone, schist, basalt, and granite substrates? (2) Do differences in the surface contour of these rock types affect the primary adhesion of zygotes? (3) Does exposure to a wave alter the initial settlement patterns? As there were differences in attachment success to different substrata that were independent of surface contour, we explored other physical and chemical characteristics of natural substrata to explain our results.

#### **2. Materials and Methods**

#### *2.1. Rock Types*

The four rock types we used were selected based on their varying physical appearance and geological composition, and because of their close juxtaposition in the intertidal zone (Supplemental Figure S1). All rock types occurred within 4 km of each other in mid-coast Maine, USA. Basalt and schist were collected from the southeast coast of Bailey Island (43◦43030" N, 69◦59040" W); limestone was collected from the southwest shore of South Harpswell at an outcrop across from Bar Island (43◦44025" N, 70◦00005" W); and granite was collected from the southernmost tip of Bethel Point (43◦47030" N, 69◦54040" W). For a detailed lithologic description of these rock units, see [53].

*Basalt:* This was the youngest rock type used in our study (Triassic age, 195–230 myo). Basalt was collected from an intrusive dike located at Bailey Island, which fills a 5–7 m fissure in heavily folded metasedimentary schist. The mineralogy of the basalt is quite uniform (because parent magma was uniform), and the rock is undeformed and unmetamorphosed (because of its young age). The grain size of this rock is very fine, but not as fine as the Spurwink Limestone used in this study. The homogeneity of basalt and its dark color result in rapid desiccation and quick thermal regulation to the environment [7].

*Granite:* The granite we used has been identified as two-mica granite on the Orrs Island 7 1/20 United States Geographic Society quadrangle [53]. This is an intrusive rock of middle to late Devonian age (about 345–370 myo). Minerals include garnet, biotite and muscovite mica flakes, potassium feldspar (which gives the rock its yellow appearance), and significant quartz content. The two-mica granite has a large grain size (relative to the other rocks in this study), and a heterogeneous composition.

*Schist:* The schist used in this study is a metasedimentary rock of the Cape Elizabeth formation, and is estimated to be of Ordovician, Silurian, or Devonian age (345–500 myo) [53]. Such stratified metasedimentary rocks were laid down in a deep basin environment as fine clays. These layers were then compressed and deformed by the formation of the Atlantic Ocean, which resulted in their metamorphism. The clay component of this and the granite has come from the similar chemical weathering of crustal rocks, and thus, the schist and two-mica granite have similar mineralogy. The main chemical difference between the granite and schist is the presence of aluminum (from the fine clays) in schist. Because of the parallel foliation of the dominant mica flakes in this schist, the microheterogeneity (<500 µ) of the surface can vary significantly as a result of the exposure of different minerals. Large-scale surface topography (on the scale of meters) is also extremely variable in this rock formation because of the intense folding that the unit has undergone.

*Limestone:* The limestone we used belongs to the Spurwink Formation [53] and has a very similar tectonic history as the schist (Creasy, pers. comm.). This unit crops out as a thin exposure in the high intertidal zone of a sandflat on the west side of South Harpswell. Due to the thin exposure of this limestone unit (15–30 m), it was difficult to locate in the local study area (future workers are directed to limestone outcrops near Rockport Harbor, Maine). The Spurwink Limestone is a laminated rock that has undergone severe deformation. The dark component resembles the schist in composition and derivation, containing biotite, muscovite, quartz, and feldspar. The lighter colored member is very fine-grained gray limestone that contains 90% calcium carbonate (CaCO3) by definition. Both members were equally represented in the experimental plates due to the highly mixed nature of the rock. The most significant difference between this rock type and others used in this study is the extremely small grain size, and the high levels of CaCO3.

#### *2.2. Fucus Spiralis*

Zygotes of *Fucus spiralis,* a brown alga common to the high intertidal zone, were selected for study because of their relatively simple fertilization process and well documented attachment strategy. The *Fucus* zygote is an excellent model system for bioadhesion studies because it is unicellular, develops synchronously, and adheres rapidly to the substrate after fertilization [54]. *F. spiralis* was specifically chosen because it is monoecious (male and female reproductive structures on the same thallus), has a wide temporal range of reproductive activity, and is well studied [55]. Attachment of the *Fucus* zygote to the substrate is a multistage process. Shortly after fertilization, the negatively buoyant zygote drops to the substrate surface [3], and then adheres initially via an extracellular mucilaginous material of unknown chemical composition. After approximately 9 to 12 h, primary rhizoid development is initiated, and the zygote becomes permanently attached to the substrate [54].

#### *2.3. Preparation of Settling Plates*

Settlement plates were prepared from samples of basalt, granite, schist, and limestone collected from bedrock using a sledgehammer. No cobbles or boulders were used. Plates with natural settling surfaces were prepared by first cutting a 1 cm slab from the surface of a bedrock sample using a slab saw. Smooth surfaces were prepared by cutting 1 cm slabs from the interior of the bedrock samples. The angle of cuts on all samples was dictated by the foliation, cleavage, and mineral veins in the rocks, which affected the structural integrity of the samples. As the blade of the slab saw was lubricated with oil, cut slabs were vigorously washed to eliminate any possible effect on the adhesion of zygotes (cf. [56]). In order to remove microorganisms and eliminate the formation of a biofilm, the slabs were scrubbed in a hot solution of water, dilute hydrochloric acid, and detergent (Ajax), and then rinsed with fresh water. Slabs were then cut into 5 cm × 5 cm (+/−1 mm plates) with a rock saw (blade water-lubricated). All plates were rinsed in running fresh water for 24 h. Slab thicknesses of between 7 and 12 mm for the natural plates and 8 and 10 mm for the smooth plates were maintained to ensure minimal differences in surface properties (see [4]). Ten plates of each surface type of each rock type were prepared, resulting in a total of 80 plates (40 natural and 40 smooth).

#### *2.4. Preparation of Zygote Solution*

We conducted two settlement trials, one in February 1995 ('winter') and one in August 1995 ('summer'). We collected receptacles of *Fucus spiralis* near Gun Point, Maine (69◦56055" W 43◦45055" N) at low tide on 14 February 1995, and 13 August 1995, respectively. We only collected receptacles that were visibly producing gametes, and these were sealed in a plastic bag with absorbent tissue as a desiccant. Receptacles were stored at 5 ◦C for ten days after the February 14 collection, and two days after the August 13 collection until gamete release was induced in the laboratory. To obtain gametes, we placed receptacles in a freshwater-ice bath for five minutes. The receptacles were then desiccated in direct sunlight until they had swelled and emitted gametes (ca. 10 min), and then soaked in seawater until a sufficient gamete release had been observed (ca. 1 h in winter and 45 min in summer). After release, we manually agitated receptacles to shake off any remaining gametes. This solution was then placed in a growth chamber at 20 ◦C for 15 min on a magnetic stirring plate to keep the gametes in suspension and promote fertilization.

### *2.5. Settlement of Zygotes on Plates*

Settlement plates were randomly arranged in two shallow metal trays (natural vs. smooth surfaces) in a 4 × 4 grid consisting of 40 plates (10 of each rock type). Natural and smooth plates were placed in different trays to minimize the variation in profile heights of the plates and thereby reduce the possibility that uneven plate heights would modify the water flow and influence settlement. There were no gaps left between plates, and the trays were larger than the 4 × 10 plate grid, leaving a 5 cm perimeter around settling plates. Fifteen minutes prior to zygote addition, artificial seawater (Instant Ocean, 32 psu, 5 ◦C) was added to the trays so that all plates were covered by ca. 2 cm of water. After fertilization (15 min), we poured 250 mL of the zygote solution (in suspension) over the settlement plates in a constant flow in a grid pattern to homogenize zygote densities over each plate. The settlement trays were left undisturbed under diffuse fluorescent light at room temperature (14–16 ◦C) for 1.5 h. The surface water was then carefully siphoned out of the trays with a plastic tube (6 mm inside diameter) placed at least 5 cm from any plate. The flow caused by siphoning, estimated by recording the time required for zygotes on the bottom surface of the tray to pass by 2 plates (10 cm), was approximately 1 cm/s.

Five plates of each rock type were chosen at random, using a table of random numbers, from the settlement-plate array for the wave treatment, and the remaining 'control' plates were sprayed with a fine mist of Instant Ocean (32 psu, 5 ◦C). These control plates were transferred to individual plastic containers with airtight lids and moist towels underneath and maintained in a horizontal position at 5 ◦C until zygotes were counted under a

dissecting scope. From the remaining plates, one plate of each rock type to be tested for wave-treatment effects was placed in random order in the track of a wave tank (see [16,57] for description). The direction of water flow was recorded for each plate and the surface types (natural vs. smooth) were tested separately. A single 3 L wave was then released and allowed to wash over the plates and drain through holes behind the plates. We allowed the water to drain off the plates (ca. 30 s), and then the plates were carefully transferred to plastic containers with air-tight lids and handled as the control plates described above. The wave treatment experiment was repeated 4 times with rock plates randomly assigned to positions in the flume each time, leading to 5 individuals of each rock type exposed to a wave ('survival' after a single wave treatment), and 5 replicate plates of each rock type serving as controls (initial settlement only).

#### *2.6. Counting Methodology*

We counted zygotes using a dissecting microscope at 40<sup>×</sup> magnification. The 25 cm<sup>2</sup> surface of the plates was divided into nine evenly spaced 1 cm<sup>2</sup> quadrats. The peripheral 1 cm of the plates was avoided (to reduce edge effects). A humidity chamber to prevent desiccation was created by placing a moist towel beneath the plate and covering the plate with a gridded petri dish during counting. In order to reduce processing time, we randomly selected six of the nine 1 cm<sup>2</sup> areas. We counted the zygotes in each quadrat and calculated a mean number of zygotes per cm<sup>2</sup> .

#### *2.7. Statistical Analyses*

Mean zygote densities per square centimeter from the initial attachment trial were compared using a three-way ANOVA with season, rock type, and wave exposure as main factors. Variances were not homogenous until the data were log10-transformed (F-max test, [58]). Separate 3-way ANOVAs were conducted for natural and smooth plates because surface types were tested independently. We used Tukey's HSD post hoc test to compare differences among rock types because there were no significant interactions in either of the ANOVAs.

Zygote densities at initial attachment were significantly different among rock types (Figure 1). It was not possible, therefore, to simply compare zygote densities after the wave treatment plates to reveal how successfully zygotes remained attached to different rock types. In order to examine the success of the initial attachment of zygotes to plates of both natural and smooth surfaces of different rock types following the wave treatment, we calculated zygote 'survivorship' [16]. This was calculated by dividing the mean zygote density found on each surface type and rock type following the wave treatment by the mean zygote density for each plate in each control. The resultant dataset consisted of 8 groups (4 rock types x 2 wave treatments) of 5 (number of replicates) percentages for each experiment (summer and winter). These percentages were then arcsine-transformed to make the variances homogenous [59]. The transformed data were analyzed using a twoway ANOVA with season and rock type as main factors. As with the analysis of patterns of initial attachment, we analyzed survivorship data for surface type (natural, smooth) using separate ANOVAs. The interaction term in these ANOVAs was not significant, so as with the 3-way ANOVAs, we used Tukey's HSD post hoc test to compare differences among rock types. We used the terms 'survival' and 'survivorship' to represent the percentage of attached zygotes persisting after the wave treatment, not in the biological sense of surviving for days beyond the treatment.

**Figure 1.** Mean density (zygotes per cm2 + 1SE, N = 5) of *Fucus spiralis* zygotes on 4 rock types after 1.5 h of settlement (control) and 1.5 h of settlement followed by one wave in summer and winter experiments. A three-way ANOVA compared zygote density as a function of season, rock type, and wave treatment (wave or no wave) for each surface type: (**a**) natural and (**b**) smooth. The zygote density was higher in winter than summer on smooth plates (*p* < 0.001), but there was no significant difference (*p* > 0.05) between seasons on natural plates. The density was always significantly lower (*p* < 0.0001) regardless of rock type on plates subjected to a wave compared to control plates. The mean density among rock types was compared using Tukey's HSD post hoc test. For both natural and smooth plates, the density was significantly higher (*p* < 0.0001) on limestone than on basalt and schist, which were not significantly different from each other (*p* > 0.05); granite had a significantly (*p* < 0.0001) lower density than all other rock types. **Figure 1.** Mean density (zygotes per cm<sup>2</sup> + 1SE, N = 5) of *Fucus spiralis* zygotes on 4 rock types after 1.5 h of settlement (control) and 1.5 h of settlement followed by one wave in summer and winter experiments. A three-way ANOVA compared zygote density as a function of season, rock type, and wave treatment (wave or no wave) for each surface type: (**a**) natural and (**b**) smooth. The zygote density was higher in winter than summer on smooth plates (*p* < 0.001), but there was no significant difference (*p* > 0.05) between seasons on natural plates. The density was always significantly lower (*p* < 0.0001) regardless of rock type on plates subjected to a wave compared to control plates. The mean density among rock types was compared using Tukey's HSD post hoc test. For both natural and smooth plates, the density was significantly higher (*p* < 0.0001) on limestone than on basalt and schist, which were not significantly different from each other (*p* > 0.05); granite had a significantly (*p* < 0.0001) lower density than all other rock types.

#### **3. Results**  *3.1. Initial Attachment*  **3. Results**

#### The mean number of zygotes per square centimeter that settled on plates in the win-*3.1. Initial Attachment*

ter ranged from 0.67 (natural granite receiving a wave treatment) to 14.2 (smooth limestone control) and in summer from 0.4 (natural granite receiving a wave treatment) to 13.4 (smooth and natural limestone control). There was a significant difference in mean zygote density among rock types and between wave treatments for both natural and smooth surfaces, but there was only a significant difference between seasons on the smooth surface substrate (Table 1). None of the interactions between factors were significant for either surface. Plates subjected to a wave always had lower zygote densities, between 61% (limestone, smooth, winter) and 91% (granite, natural, summer), than the control plates. On the The mean number of zygotes per square centimeter that settled on plates in the winter ranged from 0.67 (natural granite receiving a wave treatment) to 14.2 (smooth limestone control) and in summer from 0.4 (natural granite receiving a wave treatment) to 13.4 (smooth and natural limestone control). There was a significant difference in mean zygote density among rock types and between wave treatments for both natural and smooth surfaces, but there was only a significant difference between seasons on the smooth surface substrate (Table 1). None of the interactions between factors were significant for either surface. Plates subjected to a wave always had lower zygote densities, between 61% (limestone, smooth, winter) and 91% (granite, natural, summer), than the control plates. On

the smooth surface, the mean initial attachment was significantly greater (*p* < 0.0001) in the winter (5.1 zygotes per cm<sup>2</sup> , SE = 1.3) than in the summer experiment (3.5 zygotes per cm<sup>2</sup> , SE = 1.0) (Figure 1). For both natural and smooth plates, the density was significantly higher (*p* < 0.0001) on limestone than on basalt and schist, which were not significantly different from each other (*p* > 0.05); granite had a significantly (*p* < 0.0001) lower density than all other rock types. The pattern was the same before and after the wave treatment.

**Table 1.** Results of 3-way ANOVA analyzing the effect of season (summer and winter), rock type (limestone, schist, basalt, and granite), and wave or no wave on the density of zygotes recorded on settlement plates of natural and smooth surfaces. There were 5 replicate plates of each rock type and flow combination. Data were log10-transformed before analysis.


#### *3.2. Survivorship*

Across all treatments, survivorship averaged 27.3% (SE = 3.2) in the winter and 21.4% (SE = 2.5) in the summer. The results of the two-way ANOVAs comparing the effects of season and rock type on the survivorship of zygotes after a wave treatment were the same for natural and smooth surfaces (Table 2). The mean survivorship of zygotes was significantly affected by rock type, but not season, and there were no significant interactions in the ANOVAs. On natural rock, there was no significant difference (*p* > 0.05) in mean percent survival of zygotes among limestone, basalt, and schist treatments, but survival was significantly (*p* < 0.01) higher on these rock types than on granite (Figure 2). The survival of zygotes was similar on smooth and natural rock surfaces except that there was no significant difference (*p* > 0.05) in mean percent survival between basalt and granite.

(b)

**Figure 2.** Mean percent survival (+1SE, N = 5) of *Fucus spiralis* zygotes after one-wave treatment in experiments with natural (**a**) and smooth (**b**) rock surfaces. Percentages are based on mean densities of no-wave treatment for each rock type. Arcsine-transformed proportions were compared between seasons and among rock types for each experiment using a two-way ANOVA. The season x rock type interaction was not significant in either ANOVA (*p* > 0.05). Means (by rock type) with a common letter over the bar are not significantly different from each other (Tukey's HSD post hoc test, *p* < 0.05).
