Shallow-Water Species Diversity of Common Intertidal Zoantharians (Cnidaria: Hexacorallia: Zoantharia) along the Northeastern Coast of Trinidad, Southern Caribbean

: Zoantharians are colonial cnidarians commonly found in shallow tropical Caribbean coral reefs, and are known to be globally distributed. Common species in genera Zoanthus and Palythoa occur at Toco, Trinidad, where they are more abundant than their Scleractinia counterparts relative to benthic coverage. In this study, distribution, morphological and molecular data were collected to determine species and symbiont identiﬁcation to provide more insight on zoantharians. The Line Intercept Point (LIT) transect method recorded coverage at three sites: Salybia (SB), Pequelle (PB), and Grande L’Anse (GA) Bays along the northeastern coast. Variations in morphology, such as tentacle count, oral disk color and diameter were collected from colonies in situ. All specimens were zooxanthellate, and molecular and phylogenetic analyses were done by sequencing the cytochrome oxidase subunit I (COI) gene, and the internal transcribed spacer (ITS) region for species and symbiont identiﬁcation, respectively. Results showed mean Zoantharia percentage cover was 32.4% ± 5.1 (X ± SE) at SB, 51.3% ± 6.5 (PB), and 72.2% ± 6.1 at GA. Zooxanthellate zoantharians were identiﬁed as Palythoa caribaeorum , Palythoa grandiﬂora , Zoanthus pulchellus , and Zoanthus sociatus . Symbiodiniaceae genera were identiﬁed as Cladocopium and Symbiodinium in Palythoa and Zoanthus spp., respectively. Although this is the ﬁrst molecular examination of zoantharians, and their symbionts in Trinidad, more research is needed to identify and document species distribution and symbiont biodiversity to understand their ecology in these dynamic ecosystems.

Zoantharian coverage on coral reef ecosystems can be extensive [9,10]. For instance, Karlson (1981) noted that two species, Zoanthus sociatus (Ellis, 1768) and Zoanthus solanderi (LeSueur, 1818) had extensive coverage at a northern intertidal area in Jamaica, which he subsequently named the 'Zoanthus zone', as originally named by Tom Goreau in the 1950s [13]. Additionally, Lopez et al. (2018) reported a zoantharian zone located at Cabo Verde Islands, where molecular methods and morphological analysis confirmed the presence of two zoantharian species. Zoantharians, such as Palythoa caribaeorum (Duchassaing and Michelotti, 1860) and Z. sociatus cover large areas in subtidal and intertidal zones [7,9,10,12]. Additionally, many, but not all zoantharian species maintain symbiotic re-Oceans 2021, 2 478 lationships with Symbiodiniaceae (zooxanthellae), hence determining the identity of these holobionts adds more understanding of their ecology and physiological characteristics.
Molecular analyses of zoantharians assist in species identification, where morphological identification is difficult, or impossible. For example, phenotypic plasticity in zoantharians, specifically large variation in morphological characteristics, such as polyp shape, colony shape, size and oral disk color, may cause them to be overlooked in ecological surveys, even though their numbers may be abundant [5,8,14,15]. Given the difficulties in morphological and molecular examination of zoantharians, more priority should be given to determining their distribution and diversity in the Caribbean, especially since zooxanthellate zoantharians play key ecological roles in marine ecosystems [10]. Although success in identifying potential unidentified zoantharian species through morphological characteristics has been proven to be successful [16], the addition of molecular analyses has alleviated issues with morphological ambiguities [16,17]. Additionally, zoantharian species diversity including molecular analyses of zooxanthellate symbionts of family Symbiodiniaceae will continue to add more information about symbiont ecology in light of global climate change [11,18,19].
Although distributed world-wide in tropical and subtropical waters in the Atlantic and Indo-Pacific regions [8,20], zoantharians and Symbiodiniaceae genera identity at the southern-most part of the Caribbean, specifically Trinidad and Tobago, are limited. Two zoantharian genera, Zoanthus and Palythoa spp., are commonly observed occupying shallow waters, and in different intertidal zones along the northeastern coast of Trinidad [9,10,12,14], but relatively few studies have highlighted morphological and molecular details of these species in this part of the southern Caribbean region.
The purpose of this study is to quantitatively assess zoantharian benthic coverage, and use morphological and genetic analyses to identify zoantharians, and confirm Symbiodiniaceae genera identities. Molecular knowledge of these benthic organisms will be recorded here for the first time at this southernmost part of the Caribbean Sea. Consequently, all ecological aspects of identifying the extent of zoantharian benthic coverage provides more understanding of the dynamic coral reef ecosystem. In general, zoantharian distribution in the Caribbean needs to be continually monitored, and species identification confirmed, especially in the face of increased climate change and anthropogenic activities [9].

Study Site and Sampling
The northeastern coast of Trinidad has undefined patch reefs, and a fringing reef (see [12]). Beaches within close vicinity of these reefs are largely defined as sandy, stony, or rocky. The tropical climate has two distinct seasons, with a dry period from January to mid-June and a rainy period that extends the remainder of the year (June-December). Tides are semidiurnal with maximum high tides reaching 2 m (meters) in open water, and extreme low tides can reach 0.2 m.
Study sites in accessible areas along the northeastern coast are located at Salybia Bay (SB) (located between 10 • 50.097 N, 60 • 55.208 W and 10 • 50.100 N, 60 • 55.157 W), which is part of the only fringing reef in Trinidad. This system has been an important site for citizen/volunteer coral reef monitoring focused on cnidarian and invertebrate abundances and distributions, because it is very shallow during spring low tides (~0.2 m) and the intertidal zone extends some 200 m parallel to the shoreline. This fringing reef is affected by sediment discharge from local rivers throughout the year, specifically during the rainy period from June to December.
Pequelle Bay (PB), located to the east of SB (between 10 • 50.111 N, 60 • 55.129 W and 10 • 50.181 N, 60 • 54.954 W), has a mixture of rocks and tide pools along the intertidal zone. During spring low tides, this part of the reef becomes fully exposed for a 3-h period until tides return. Patchy undeveloped reefs are present at Grande L Anse (GA), also known as Toco Bay (between 10 • 50.107 N, 60 • 56.772 W and 10 • 50.266 N, 60 • 56.674 W), which has a mixture of rocky outcrops. This study site has a mixture of sand, stony and rocky beaches with village homes within close vicinity. Spring low tides revealed a rocky patch reef with scattered tide pools interspersed throughout the intertidal zone.
Volunteers marked the study area with GPS points, and quantified an area of 500 m 2 using a 50-m open reel fiberglass measuring tape during extreme low tides (<0.3 m). Each transect was placed within a 500 m 2 study area, and a total area of 1500 m 2 was examined at each site. The Line Intercept Transect (LIT) method [9] was used to examine only the lower intertidal areas at SB, PB, and GA. Transect positions within each 500 m 2 area were marked using a global positioning system (GPS). A 50 m open reel fiberglass measuring tape was placed parallel to the shoreline in each area, and benthic components were recorded at every 0.5 m interval on the measuring tape. Benthic components, such as reef-building corals, zoantharians, macroalgae, coral rubble, and other invertebrates, such as sea urchins, fireworms, and sea cucumbers were recorded if they touched the 0.5 m intervals along the measuring tape. This was repeated three times within each 500 m 2 study area, and for a total of 3 study areas. Water temperature and salinity were measured using a YSI Pro 1030 probe at three random points along each 50 m LIT. Percentage cnidarian and benthic cover was calculated for benthic communities.

Morphological Analyses and Specimen Collection
Morphological data for zoantharian colonies (n = 30) were recorded at three field sites, SB, PB, and GA in June 2019 from the northeastern coast of Toco, Trinidad. A hand-held caliper was used to measure oral disk diameter of 3 opened polyps per colony. Physical characteristics of each polyp, such as tentacle count and color, oral disc color, and polyp form (immersae, intermediate, liberae, see 4, 21 were recorded, together with in situ photographs, which were used to assist in identification (see Table 1). Additional samples were also collected at three other sites between December 2019-February 2020, and were primarily collected for genetic analyses. Samples were collected in areas with high wave action (open habitat exposed to waves), and low wave action (rocky habitat protected from waves). A total of 13 specimens (3-5 polyps per specimen) were collected from SB, PB, and GA. Additionally, specimens were collected at inaccessible areas along the northeastern coast, such as Galera Point (GP) (n = 1 specimen) located just left of the Keshorn Walcott Toco Lighthouse, which is 1 km east of PB, and has a mixture of rocks and tide pools where zoantharians were observed to have extensive coverage. Additionally, samples were collected from Straight Bay (StB) (n = 2 specimens), 1 km west of SB, and from western SB (WSB) (n = 5 specimens), which is an accessible area, but less frequented by local visitors.  Altogether specimens were collected at six sites by snorkeling along the northeast coast of Toco, Trinidad between June 2019 to February 2020. A total of 3-5 closed individual polyps were excised from colonies (specimen) using scalpel and tweezers, and placed in 1.5 mL collection vials with 95% ethanol (Carolina Biological. Burlington, NC, USA), then stored at −20 • C. Specimens are housed at the Andrews Science Building, University of Tennessee-Southern (formerly Martin Methodist College), Pulaski, Tennessee, United States. Specimen catalog nos. are TRIN-2019-001 and TRIN-2020-001.

Phylogenetic Analysis
PCR products for COI gene were sequenced in both directions at Eurofins Genomics (Louisville, KY, USA). DNA sequences were initially inspected by eye and manually edited using Molecular Evolutionary Genetics Analysis (MEGA X, version 7.0 see [26]). Sequences were aligned using CLUSTAL W in MEGA X. Sequences were deposited in GenBank (accession numbers MZ147090-097, MZ150796-807) and were used to align with publicly accessible sequences for other zoantharians (GenBank accession JX119160, JX119164, JX119165, JX119167, JX119157, JX119156, JX119154, JX119159, JX119168, KT454365, AB214177, KF499705, KF499712). All alignments were inspected by eye using MEGA X and errors in nucleotide sequences that were low quality were trimmed prior to phylogenetic analysis. Analysis using maximum likelihood with bootstrap trees (1000 replicates) were incorporated according to the MEGA X protocols.

Specimen Morphological and Molecular Analyses
Morphological analyses and comparisons (see Table 1) showed specimens of Palythoa caribaeorum colonies ( Figure 1A) with various levels of coenenchyme thickness (see description in [4,21]). P. caribaeorum oral disks and tentacle colors varied between brown and green, in comparison to that observed for Zoanthus spp. at all sites, where colors ranged from bright green, orange, grey, blue, and dark green. Maximum mean oral disk diameter for P. caribaeorum was 12.8 mm ± 0.96 (Mean ± SD, n = 15 polyps; 3 polyps per colony),

Specimen Morphological and Molecular Analyses
Morphological analyses and comparisons (see Table 1) showed specimens of Palythoa caribaeorum colonies ( Figure 1A) with various levels of coenenchyme thickness (see description in [4,21]). P. caribaeorum oral disks and tentacle colors varied between brown and green, in comparison to that observed for Zoanthus spp. at all sites, where colors ranged from bright green, orange, grey, blue, and dark green. Maximum mean oral disk diameter for P. caribaeorum was 12.8 mm ± 0.96 (Mean ± SD, n = 15 polyps; 3 polyps per colony), which was larger than for Zoanthus spp. 10.2 mm ± 0.10, n = 15 polyps (Table 1). Although P. caribaeorum and P. grandiflora ( Figure 2B) could easily be identified through morphological analyses using maximum oral disk size, tentacle color, and tentacle numbers, analyses of Zoanthus spp. (Figure 2C-F) were challenging with the aforementioned characteristics. Zoanthus spp. colonies showed variation in coenenchyma thickness.

Discussion
Results showed the lower intertidal zones at Toco, Trinidad, to be dominated by zoantharians, specifically Palythoa and Zoanthus spp., similarly reported by Rabelo et al. [27] further south from Trinidad and Tobago on flat sandstone reefs in northeastern Brazil. As well, zoantharians were common in shallow-water habitats, and Palythoa and Zoanthus spp. were most common in shallow waters (<5 m), as reported at sites along the west coast

Discussion
Results showed the lower intertidal zones at Toco, Trinidad, to be dominated by zoantharians, specifically Palythoa and Zoanthus spp., similarly reported by Rabelo et al. [27] further south from Trinidad and Tobago on flat sandstone reefs in northeastern Brazil. As well, zoantharians were common in shallow-water habitats, and Palythoa and Zoanthus spp. were most common in shallow waters (<5 m), as reported at sites along the west coast of Curaçao [11]. In fact, Belford and Phillip [9,10,12,28] highlighted zoantharians being more abundant than their Scleractinia counterparts at this study's main sites. Lopez et al. [29] ob-served extensive zoantharian coverage for species Zoanthus solanderi and Zoanthus sociatus at a "zoanthid zone" located at Cabo Verde Islands, central eastern Atlantic,. Additionally, Karlson [6] observed similar extensive Zoanthus spp. coverage at Discovery Bay, Jamaica. This study reports a similar presence of a zoantharians covering the majority of the benthic lower intertidal zone, dominated by Palythoa and Zoanthus spp. in the southern-most part of the Caribbean.
Although zoantharian coverage was extensive, identification of zoantharians was difficult, because color morphotypes varied among sites. Similar observations were mentioned by Reimer et al. [20] related to zoantharian identification in the field, and in other studies [30]. Phylogenetic and morphological analyses using the COI marker revealed Palythoa brown and green color morphs were specifically Palythoa caribaeorum and Palythoa grandiflora, respectively, while green, orange, grey, and blue-green were Zoanthus pulchellus and Zoanthus sociatus, respectively. However, it is worth mentioning that caution should be taken since only the COI marker was used in this study. For instance, Sinniger et al. [31] noted that although the COI marker is easily amplifiable with universal primers, and was hence used in this study, the addition of the mitochondrial 16S ribosomal DNA marker is useful for comparison and further species identification. In fact, the addition of 16S sequences adds distinct advantages as they are slightly more variable in zoantharians than COI, thereby adding useful phylogenetic information [31].
Closed polyps are often observed during extreme low tides, where desiccation plays a factor in distribution; however, Rabelo et al. [27] reported Z. sociatus resisting desiccation better than P. caribaeorum. However, in this study, the reverse was observed, where P. caribaeorum had a significantly higher benthic coverage than Zoanthus spp. at lower intertidal zones, nevertheless zoantharian distribution appears to be related to desiccation tolerance [27]. It is not precisely known why the reverse of [27] was observed in this study, however family Zoanthidae, such as Z. pulchellus and Z. sociatus, have generally been known to adapt to different environments [10,19]. Reimer et al. [20] reported variation in Palythoa sp. polyp form and color as a result of variable environments, such as degree of wave action, and benthic type, which were characteristics similarly observed at Toco, Trinidad [9,10,12,28].
Zoantharian color morphotypes were observed in both high and low wave action habitats, especially at the lower intertidal zones where P. caribaeorum carpeted much of the lower intertidal zones. However, Z. pulchellus mainly covered individual rocks, or crevasses, and displayed many colors throughout the intertidal zone. Z. sociatus also was found within crevasses, however it was not observed to be found in extensive coverage at sites in this study. It should be noted that extreme caution should be taken while surveying zoantharian coverage using color morphotypes to identify zoantharians since species may be conspecifics [8,19]. This study successfully used molecular analyses to assist with identification of Zoanthus pulchellus and Zoanthus sociatus. Zoanthus spp. display phenotypic plasticity in both oral disk color and polyp height [27]. Additionally, molecular analyses assisted in identifying P. caribaeorum and P. grandiflora; however it should be noted that these two species can be distinguished from morphological characteristics.
Further phylogenetic analyses of zoantharians confirmed the Symbiodiniaceae genera Cladocopium and Symbiodinium in Palythoa and Zoanthus spp., respectively. These results are consistent to past analyses of both species at different locations in the Caribbean Sea and Atlantic [31][32][33]. Similar results for both Zoanthus species mentioned in this study hosting Cladocopium and Symbiodinium were reported at Cape Verde Archipelagos [30]. As global climate will continue to affect oceanic water temperatures [34], identification and distribution of zooxanthellate zoantharians such as in this study will provide important baseline data for future analyses. Institutional Review Board Statement: Ethical review and approval were waived for this study, because zoantharians were measured in situ, and genetic analyses on polyps did not require colony destruction.
Data Availability Statement: Photographs of zoantharian color morphotypes along the northeastern coast of Toco, Trinidad are available online at the Digital Public Library of America and Martin Methodist College Marine Biology Collection websites: https://dp.la/search?q=martin+methodist+college https: //www.artstor.org/2016/09/26/case-study-going-underwater-with-shared-shelf-commons/ (accessed on 9 June 2021).