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Comparative molecular and morphological variation analysis of Siderastrea (Anthozoa, Scleractinia) reveals the presence of Siderastrea stellata in the Gulf of Mexico.

Abstract. The genus Siderastrea exhibits high levels of morphological variability. Some of its species share similar morphological characteristics with congeners, making their identification difficult. Siderastrea stellata has been reported as an intermediary of S. siderea and S. radians in the Brazilian reef ecosystem. In an earlier study conducted in Mexico, we detected Siderastrea colonies with morphological features that were not consistent with some siderastreid species previously reported in the Gulf of Mexico. Thus, we performed a combined morphological and molecular analysis to identify Siderastrea species boundaries from the Gulf of Mexico. Some colonies presented high morphologic variability, with characteristics that corresponded to Siderastrea stellata. Molecular analysis, using the nuclear ITS and ITS2 region, corroborated the morphological results, revealing low genetic variability between S. radians and S. stellata. Since the ITS sequences did not distinguish between Siderastrea species, we used the ITS2 region to differentiate S. stellata from S. radians. This is the first report of Siderastrea stellata and its variability in the Gulf of Mexico that is supported by morphological and molecular analyses.

Introduction

Siderastrea is a coral of the family Siderastreidae (Pallas, 1766; Laborel, 1970; Veron, 1995, 2000). This genus is widespread from the Caribbean to the Atlantic Ocean (Budd, 1980), and is represented by five species. Three species are found in the Tropical Atlantic region (Fig. 1): Siderastrea siderea (Ellis & Solander, 1786), S. radians (Pallas, 1766), and S. stellata Verrill, 1868, the last one reported as being endemic to the Brazil region (Verrill, 1868). These species comprise the "Atlantic Siderastrea Complex" (Pallas, 1766). The other two species, Siderastrea savignyana Milne Edwards and Haime, 1850 and S. glynni Budd and Guzman, 1994, are restricted to the Eastern Pacific. However, there is genetic evidence from the host (Forsman et al., 2005) and symbiont (LaJeunesse et al., 2015) that S. glynni may not be a separate species, but rather S. siderea, introduced from the Caribbean to the Eastern Pacific near the Panama Canal. The theory of S. glynni's unintentional transplantation from the Caribbean to the Eastern Pacific was recently confirmed (Glynn et al., 2016). Colonies of Siderastrea are usually massive, varying between the incrusting (5. stellata, S. radians, S. savignyana) to the hemispherical (S. stellata, S. radians) and spherical (S. siderea, S. glynni) or roller forms (5. radians); they are abundant in coral reef shallow waters (Laborel, 1974).

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Identification of the Siderastrea species is based on the number of septa and septa cycles per corallite, and by the species' mechanisms of reproduction. Siderastrea siderea is hermaphroditic spawning, while S. stellata and S. radians are gonocoric brooding (Laborel, 1970; Budd and Guzman, 1994; Neves et al., 2008). Based on morphological analysis, S. stellata has been reported to be a synonym of S. radians (Yonge, 1935) or an intermediary between S. siderea and S. radians (Laborel, 1970). However, the skeletal morphology of Siderastrea varies among organisms, even those of the same population (Todd et al., 2001); they can modify their shape and structure (morphology) as an adaptation strategy in response to different environmental conditions, (Gattuso et al., 1991). In the Gulf of Mexico, colonies of Siderastrea are variable and have overlapping morphological traits, a fact that poses a challenge to identification and demarcation of species boundaries.

The S. stellata and S. siderea species always exhibit four complete septa cycles, while S. radians presents three complete septa cycles (Neves et al., 2010). Siderastrea siderea forms larger colonies and larger corallites (3-5 mm) than the other species, has numerous septa (44-50), deep and papillose columellae, and thin synapticulae; and the septa alternate between corallites (Yonge, 1935; Laborel, 1970; Budd and Guzman, 1994; Beck, 2005; Menezes et al., 2013). Siderastrea glynni, from the Pacific Ocean, differs from other siderastreids in its small, well-rounded colonies, small corallite size (2.5-3.5 mm), numerous thin septa (40-48 per corallite), shallow fossae, and synapticular meshwork (Laborel, 1970). It is morphologically similar to S. radians, which exhibits small corallites (2.5-3.5 mm) and unattached colonies. Yet S. radians has fewer septa (30-40), deep fossae, and solid columellae (Yonge, 1935; Budd and Guzman, 1994; Beck, 2005). However, differentiation between S. radians and S. stelleta is unclear, due to high morphological variability of these species and their similar morphology (Forsman et al., 2005; Neves et al., 2010).

The nuclear rDNA array, particularly the internal transcribed spacer (ITS) regions, has been used for systematics and for reconstructing phylogenetic relationships in scleractinian corals (Lopez and Knowlton, 1997; Odorico and Miller, 1997; Medina et al., 1999; van Oppen et al., 2000; Diekmann et al., 2001; Chaolun et al., 2004; Frade et al., 2010; Filatov et al., 2013) and Gorgonian corals (Grajales et al., 2007). Genetic analysis of Siderastrea using the ITS region has confirmed that S. stellata and S. radians are very closely related (0.3% [+ or -] 0.2% uncorrected pairwise sequence divergence across the ITS region), yet it is unclear if these genetic differences represent population or species-level variation (Forsman et al., 2005). The ITS region is a multi-copy, non-coding marker that is prone to insertions, deletions, repeats, and inversions, which can make multiple sequence alignment problematic. Furthermore, Sanger sequencing tends to result in double peaks, which can be difficult to interpret (Chaolun et al.. 2004; Forsman et al. 2005). However, in some cases use of the ITS2 region can overcome the lack of differentiation between closely related species that remains when other molecular markers are compared (Chen et al., 2004). The ITS2 region is located between the 5.8S and 28S genes of the rDNA ITS, and is among the most widely used markers for exploring evolutionary patterns in scleractinian corals (Chen et al., 2004; Schultz et al. 2005).

The genus Siderastrea in the Veracruz Reef System National Park (VRSNP), Mexico, is represented by two species: S. siderea and 5. radians (Horta-Puga and Tello-Musi, 2009). However, in two reefs close to the coast some colonies of Siderastrea exhibit high phenotypic variability with morphological characteristics similar to those of S. stellata (N. A. Colin Garcia, pers. obs.). In this study we conducted a morphological and molecular analysis using the ITS and the ITS2 region to evaluate genetic divergence in Siderastrea.

Materials and Methods

Study area

The Veracruz Reef System National Park (VRSNP) is a protected area of about 54,000 Ha, located in the central portion of the continental shelf in the Gulf of Mexico. This park system consists of 28 individual platforms and fringing reefs (Diario Oficial de la Federacion, 2012), divided into two subgroups by the Jamapa river flow. The northern group is located in front of the Port of Veracruz and the southern group is adjacent to the town of Anton Lizardo (Chavez, 2009). The northern subgroup is unique in that the reefs are located 20 km from the coastline. Other reefs that are close to the Port of Veracruz have been impacted by anthropogenic activities (Fukami et al., 2004; Ronquist et al., 2012). Small fragments from 40 colonies of Siderastrea were collected from the north subgroup area: 20 fragments from the "Gallega" reef and 20 from the "Galleguilla" reef, both located near the coastline of the Port of Veracruz (Fig. 2). These reefs were chosen based on observations that a few Siderastrea colonies appeared to exhibit intermediate colony morphology that prevented species identification. We collected fragments from these colonies in order to identify all of the possible morphological variants of Siderastrea. All samples were obtained at a depth of about 1.5 m; fragments were removed using a hammer and chisel. For molecular analysis, we collected coral fragments of ~2-3 [cm.sup.2], and for the morphological analysis we obtained coral fragments of ~9 cm" from the top, middle, and bottom of each colony so as not to have to remove the entire colony. The sampling protocol in the VRSNP was approved by the Comision Nacional de Acuacultura y Pesca (permit no. PPF/DGOPA-072/13). All organisms collected were neither endangered nor protected according to Secretary of the Environment and Natural Resources (SEMARNAT) NOM-059-SEMARNAT-2010.

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Morphological analysis

The coral fragments from the collected colonies were stored in 30-ml plastic tubes for transport to the laboratory. For the morphological analysis, the corallite samples were kept in 0.5% boric acid for 8 h to remove soft tissue from the skeletal structures; they were dried afterward in an oven for two days at 120 [degrees]C. Only mature corallites were examined, that is, those with at least the third well-formed septa cycle. Once samples were dried, they were covered with a conductive platinum film to optimize visibility by scanning electron microscope (SEM). Previous studies have reported six morphological characters to use when classifying Siderastrea species: corallite diameter, columella diameter, number of septa, theca thickness, columellar depth, and distance between individual corallites (Laborel, 1970, 1974; Budd and Guzman, 1994; Budd, 1980; Menezes et al., 2013). In this study we performed a morphological analysis that included macromorphology, specifically the study of corallite form (corallite budding and integration); size and shape of the calice; arrangement of septa (number, length, cycle, and spacing); and columellar structure, using a stereoscope. The micromorphology analysis, particularly examination of the septum wall (shape of teeth, granulation, and columella) was conducted with a SEM (model XL-30 ESEM; Phillips, Amsterdam, the Netherlands), according to methodology established by Budd et al. (2012). The images were taken with a 3-nm resolution, with markers of 1 to 2 mm and acceleration from 10 to 20 kV. Morphometric analysis of each sample was conducted using Image J software (Schneider et al., 2012).

A list of the evaluated characters is shown in Table 1. We did not use gross colony morphology as a character to differentiate between species, because most samples showed high levels of morphological variation.

Statistical analyses

Differences in morphological characters between species and among sites were tested using ANOVA. Discriminant component analysis (DCA) was performed to distinguish the morphological characters between species and those characters that have discriminant functions. The DCA was complemented by ANOVA between species for each variable. The univariate analysis is constructed from a matrix standardized by the covariance between group and the residual covariance matrix. This allows evaluation of the covariance matrices as a function of the group effect and the residual variance-covariance matrices obtained after discounting the group effect, allowing us to construct, from the diagonals of these matrices, the F-value, non-self-correlated, for each variable. ANOVA and DCA analyses were performed using InfoStat software (Di Rienzo et al., 2002).

Molecular analysis

A small coral tissue fragment (2-3 cm) was washed with distilled water and stored in a bottle containing 90% ethanol. DN A was extracted from ~ 10 mg of coral tissue, using the isolation tissue Qiagen kit (Qiagen, Hilden, Germany) protocol. DNA was precipitated with 80% ethanol. Polymerase chain reaction (PCR) amplification was carried out using the following primers ITS-1F: 5'-TCC GTA GGT GAA CCT GCG G-3' and ITS-1R: 5'-TCC TCC GCT TAT TGA TAT GC-3' (White et al., 1990). We used the thermal cycler condition for amplification, as described by Fukami et al. (2004). The nucleotide sequences were obtained with a Genetic Analyzer sequencer of 16 capillaries (model 3130; Applied Biosystems Corp., Foster City, CA) and BigDye Terminator chemistry V3.1 Cycle Sequencing Kit (Thermo Fisher Scientific. Waltham, MA). The electropherograms were analyzed using 4Peaks free software (Nucleobytes, Amsterdam, the Netherlands). Alignment of the sequences was performed with Clustal IX ver. 2.1 (Thompson et al., 1994), using default parameters. Alignment was visually checked with PhyDE ver. 1.0 (Hyde Co., Atlanta, GA). Parsimony informative sites, haplotype diversity (h), and variable sites were calculated with the [D.sub.NA]SP ver. 5.10 program (Librado and Rozas, 2009). The evolutionary distances were computed using the Maximum Composite Likelihood method in MEGA ver. 4 (MEGA4; Tamura et al., 2007); distances are expressed in the units of the number of base substitutions per site; gaps were treated as missing data.

To obtain previously published sequences of the ITS2 region, we performed a BLAST analysis using Geneious ver. 7.0 (Kearsen et al., 2012). In this case, the ITS2 region (from the beginning to the end) from our sequences was obtained. Champuru software (Flot, 2007) was used to input the two sequences (forward and reverse) in order to interpret multiple chromatogram peaks, as expected from Sanger sequencing multiple length variants (Table 1). Sequences obtained from the Champuru software were used for the phylogenetic analyses. A set of sequences of Siderastrea stellata (AY322611), S. radians (AY322609), and S. siderea (AY322603), available in the GenBank database, were included in the phylogenetic analysis. The Siderastrea sequences obtained in this study have been deposited in the GenBank database under accession numbers KT750800 to KT750839.

Phylogenetic trees were inferred using the Bayesian method in MEGA ver. 4. The positions that contain gaps in molecular data can be useful in phylogenetic analyses; all gaps were coded manually using the simple gap coding method. This process codes gaps as a separate character in the data matrix, which is considered in the phylogenetic analysis (Simmons and Ochoterena, 2000). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. We used the HKY85 model of evolution (Hasegawa et al., 1985), according to Akaike Information Criteria (AIC) and Bayesian Information Criteria (BIC); analyses were performed with IModel Test 2 software (Darriba et al., 2012). Additional Bayesian Analysis using the Markov Chain Monte Carlo (MCMC) method was constructed in MrBayes ver. 3.1.2 (Ronquist et al., 2012). To evaluate support trees, we conducted 1000 Bootstrap replicates in MrBayes ver. 3.1.2 (Ronquist et al., 2012).

Haplotype networks

The alignments of the ITS and ITS2 sequences were visually checked using Phyde ver. 1.2 software. The final alignment was imported into [D.sub.NA]SP ver. 5.10 (Librado and Rozas, 2009) and converted to ROI FILE format to perform the network tree with Network 5 software (Bandelt et al., 1999). Haplotype networks illustrate relationships among haplotypes that have a short evolutionary period of time such that ancestral and descendant haplotypes exist simultaneously (Kratysberg et al., 2004). The analysis was performed using the Median-Joining method, which computes a network using all sites in the data matrix and treats gaps or missing data as additional sites (Bandelt et al., 1999).

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Results

Morphological analysis

After evaluating the 20 different morphological characters (Table 1), we characterized those colonies with morphology similar to Siderastrea siderea, S. radians, or S. stellata. All samples exhibited extracalicular budding and the colonies showed massive or encrusting growth. Colonies of S. siderea were round-shaped, with the 4-septa cycle complete most of the time, and an occasional incomplete 4-septa cycle. Septa number showed high variability (34-44 septa per corallite) and deep calice, and all samples alternated septa with corallites (Fig. 3a, d). Siderastrea radians exhibited hemispherical colonies, 3 complete septa cycles, septa numbering between 26-38 per corallite, deep columellar fossae, and thick septa walls and columellae (Fig. 3b, e). Samples identified as S. stellata exhibited both encrusting and hemispherical colonies, with a complete 4-septa cycle, septa numbering between 38-48, always presenting continuous septa between corallites, shallow columellar fossae, and thinner septa walls (Fig. 3c, f). All samples exhibited morphologic variability; the average values of the 15 characters evaluated are shown in Table 2. The rest of the characters (i.e., septa cycle (SC), free septa (SF), fused septa (Sfu), teeth form (TF), and continuous septa or septa discontinuous between corallites (SC/D)) were used as hierarchical data. Values were assigned based on the presence or absence of characters (Table 1).

Morphological characteristics of teeth (n) (TN), septa (n) (SN), septa space (SS), free septa (SF), and distances between centers of calcification (DC) were informative for distinguishing between the three species (Fig. 4; Table 2). Of the Atlantic siderastreids, Siderastrea radians had the fewest septa and fewest teeth per septum, the shortest septum length, and the largest gap between septa (Fig. 4: Table 2). Colonies of S. siderea exhibited the greatest numbers of septa, the longest septa, greatest distance between corallites, and the deepest fossae. Siderastrea radians and 5. stellata had similar fossa depths (Fig. 4; Table 2).

Samples identified as S. stellata showed a similar number of septa as S. siderea and smaller space between septa (Fig. 4; Table 2). Discriminant component analysis showed that septa cycle (F = 33.66; P < 0.0001) was the character that differentiated S. radians (3 complete septa cycles) from S. stellata (a fourth septa cycle always complete). In addition, the continuity of septa between corallites (SC/D) (F = 85.83; P < 0.0001) was a character that allowed differentiation of S. stellata from S. radians (both with continuous septa between corallites). Siderastrea sidera, in contrast, presented discontinuous (alternating) septa between corallites (Fig. 3; Table 3). On the basis of the character analysis, the DCA plot was separated into three subgroups corresponding to the three Siderastrea species (S. radians, S. siderea, and S. stellata). However, most of the colonies exhibited high phenotypic variability, and many colonies shared characters of two species (e.g., colonies with the same number of septa as S. radians; and those that exhibited large septa, small space between septa, and complete 4-septa cycles, as with S. Stellata). The first canonical axis (CA1) showed that 77.77% of the total variation found was mainly related to continuity of septa between corallites. The second canonical axis (CA2) showed that 22.23% of the variation was related mostly to septa cycle (rate of cross-classification error = 0).

Univariate analysis of variance revealed that continuity of septa between corallites, septa cycle, number of teeth per septum, number of septa, septa space, free septa, and distances between centers of calcification were the characters that showed statistically significant differences between species. The remaining characters exhibited high variability and were not useful for differentiating among Siderastrea species (P < 0.05, a statistically significant distance).

Molecular analysis

Forty sequences of ITS regions containing ITS 1, 5.8S, and ITS2 were obtained by PCR. The average fragment length was 589 bp. The ITS 1 region had a length of 216 bp, with 41.1% guanine+cytosine (G+C) content. The ITS2 region had an average length of 193 bp and a 53.8% G+C content. The 5.8S region was 156 bp in length and had 48% G+C content. These obtained sequences were compared with the Reference Sequence Database of the National Center for Biotechnology Information (NCBI) through a BLAST analysis. All of the sequences were very similar to the previously reported sequences for S. stellata (AY322611), S. radians (AY322609), and S. siderea (AY322603). The average haplotype (h) diversity was h = 0.967 [+ or -] 0.013; molecular analyses relied on 26 parsimony informative sites.

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[FIGURE 5 OMITTED]

The phylogenetic tree showed four distinct clades, using the ITS region (Fig. 5a). This result was congruent with the identified clusters in the network analysis (Fig. 5b). The genetic divergence between clades (uncorrected pairwise sequence divergence) was low (Table 4). Clade IV was subdivided into 4 haplotype subclades (Fig. 5a) that were clearly supported: Clade IV-A 0.026%, Clade IV-B 0.033%, Clade IV-C 0.0.023%, and Clade IV-D 0.029% uncorrected pairwise sequence divergence across the ITS.

The phylogenetic tree and the network analysis using the ITS2 region resulted in three significant clades. Clade I had a 0.025% uncorrected pairwise sequence divergence (Table 4; Fig. 6a) and formed a monophyletic group with S. siderea. Clade II had no genetic divergence (0%) and formed a phylogenetic clade with S. radians and S. stellata (AY322612; Foreman et al., 2005) (Table 4; Fig. 6a). Clade EI exhibited 0.010% uncorrected pairwise sequence divergence and formed a monophyletic clade with S. stellata (AB441407; Fukami et al., 2004) (Fig. 6a). The consensus tree of the Bayesian analysis was used to represent the evolutionary history of the taxa analyzed in both regions (Figs. 5, 6). Branches corresponding to partitions reproduced in less than 50%, bootstrap replicates were collapsed. The variations in the sequences of ITS2 region and their morphological variant are represented in Figure 6a, b.

Discussion

Our morphological results revealed the presence of three different morphologies of Siderastrea in the Veracruz Reef System National Park in the Gulf Mexico (VRSNP) (Fig. 3). Those samples that showed three complete septa cycles, had fewer than 34 septa per corallite, deep calices, and thick, solid columellae were identified as Siderastrea radians. Samples that presented four complete cycles, had between 34-42 septa per corallite, deep and shallow calices, and septa alternating between corallites were identified as S. siderea. Samples that showed four complete septa cycles, had more than 36 septa per corallite, medium or deep calices, thin columellae, and continuous septa between corallites were identified as S. stellata. We used previous reports of morphological Siderastrea characters as a reference for identifying species in the VRSNP (Yonge, 1935;Laborel, 1970; Budd and Guzman, 1994;Beck, 2005; Menezes et al., 2013). There were statistically significant differences among the main characters that allowed us to differentiate between the three species of Siderastrea. Number of septa (SN) and number of septa cycles (SC) distinguished S. radians from the other two species; continuous (S. stellata) or alternating (S. sidera) septa between corallites (SC/D) differentiated S. siderea from S. stellata (SC/D) (Fig. 4; Table 3).

Siderastrea siderea alternates septa between corallites while S. stellata has continuous septa (Yonge, 1935; Laborel, 1970; Budd and Guzman, 1994; Menezes et al., 2013). Previous authors performed a systematic analysis between S. radians and S. stellata, and found that differences in these two species were mainly related to number of septa, followed by calice depth (Budd and Guzman, 1994; Menezes et al., 2013). In this study we used number of septa and number of septa cycles to differentiate between S. stellata (> 36 septa per corallite, 4 cycles complete) and S. radians (< 34 septa per corallite, 3 complete septa cycles). However, we found several colonies that showed an overlap of morphological characters. The high phenotypic plasticity that was seen in the analyzed samples could be the consequence of environmental conditions, or a combination of high phenotypic plasticity and their genetic variation (polymorphism) could have induced changes in their morphology (Bruno and Edmunds, 1997; Muko et al., 2000; Todd et al., 2004; Forsman et al., 2005; Todd, 2008). Environmental conditions could induce changes in the morphology of corals as an adaptation strategy increasing phenotypic plasticity (Foster, 1977; Lloyd, 1984; Willis and Ayre, 1985; Miller, 1994; Bruno and Edmonds, 1997; Muko et al., 2000; Todd et al., 2004) in Siderastrea colonies from the VRSNP. Reefs where Siderastrea colonies were sampled were highly impacted by anthropogenic activity. Large quantities of these terrigenous sediments, which have been found in these waters, may have induced changes in their morphology.

The presence of colonies in the VRSNP with similar morphology to S. stellata probably indicates that this species was present in the VRSNP but not reported. Earlier work in this reef system used the traditional classification system established by Vaughan and Wells (1943) to identify the species; this system does not detect all of the variations in the morphology of the Siderastrea species. Thus, inaccurate classification of S. stellata as S. siderea is possible. Nevertheless, the morphological analyses of the corallite structure of Siderastrea allowed us to make the first record of S. stellata in the Gulf of Mexico.

Morphological characters in several colonies exhibited overlapping characteristics. Some colonies presented the same numbers of septa as S. radians, and the same four septa cycles as S. siderea, making it difficult to identify the species even using micro-morphological characters. Therefore, it was necessary to use molecular tools to corroborate identification of these species.

Molecular analysis, using only the ITS2 region, agreed with three distinct Siderastrea species (Fig. 6); high genetic variability and the presence of several haplotypes also were found (Fig. 5). Samples that were identified as S. radians, using morphology (Sra3 and Sra4 in the phylogenetic tree), presented most of the characters that identified S. radians; however, the samples also exhibited discontinuous (alternating) septa between corallites, as in S. siderea. Overlapping morphology and high molecular variability suggested the presence of cryptic species of the Siderastrea genus, which could be playing an important role in evolutionary and ecological processes in the VRSNP. These organisms could be different species that recently diverged, or they may occasionally hybridize. However, we were unable to draw further conclusions, using only the ITS region. Further studies using mitochondrial and nuclear markers are warranted.

Molecular results, using only the ITS2 region, allowed identification of the three species of Siderastrea (Fig. 6). According to ITS2 analysis, some colonies that were classified as S. radians (using morphological characteristics) formed a phylogenetic group with S. siderea. These results confirm the hypothesis of cryptic species having recently diverged in the VRSNP (Veron, 1995; Piggott et al., 2011). Samples identified as S. stellata, using morphology, disagreed with the molecular results; ITS2 revealed that these samples belonged to S. siderea. Nevertheless, sequences of the ITS2 region and predicted secondary structure were used to resolve taxonomic relationships between Siderastrea species from the VRSNP, as previous work has used the ITS2 to resolve relationships among closely related taxa (Harris and Crandall, 2000; Schultz et al., 2005; Coleman, 2007; Grajales et al., 2007).

In the phylogenetic tree of ITS2 (Fig. 6), Group I corresponds to all samples that were genetically similar to S. siderea. Group II represents the colonies that were genetically similar to S. stellata from Brazil and S. radians from Panama, as reported by Forsman et al. (2005). However, Groups I and II showed a genetic divergence of 0.011% with S. stellata from Panama, as reported by Fukami et al. (2004). This could have been due to misidentification of S. stellata by Fukami et al. (2004) or by Forsman et al. (2005), or could be evidence of the presence of different haplotypes of S. stellata with a wider distribution. Samples from Group II presented ITS2 sequences that contained alleles from S. radians and S. stellata. This finding may be the reason these colonies exhibited morphological characteristics of both S. radians and S. stellata. The genetic divergence between S. radians and S. stellata was too low to distinguish between the species using the ITS, but the combination of the ITS2 region and the morphometric approach allowed us to identify S. stellata in the VRSNP for the first time.

The high phenotypic and genotypic variability shown by Siderastrea could be due to environmental differences inducing a response in corals that changes their morphology as an adaptive strategy, increasing species diversity, thus enabling corals to survive in different environments. Coral species can respond to environmental conditions by modifying colony morphology, for example, in response to light or sediment (Todd, 2008). Because corals are also capable of mass spawning, coral species may have opportunities to hybridize, which would increase morphological and genetic variability and promote formation of new hybrid species (Vollmer and Palumbi, 2002).

Our results confirm the presence of three species of Siderastrea in the VRSNP; we also report for the first time the presence of S. stellata in the Gulf of Mexico. The genetic variability between Siderastrea species is very low for the ITS region, yet there are still unique haplotypes of each species that allow their differentiation.

Acknowledgments

We thank LANNBIO CINVESTAV Merida, which supported projects FOMIX-Yucatan 2008-108160 and CONACYT LAB-2009-01 No. 123913; Daniel Aguilar, MSc; Ana Cristobal, Biol.; and Ligia Vilca, PhD, for assistance with photography on SEM. We also thank Ann F. Budd for her help in identification of the specimens, Dra. Rossana Rodriguez Canul, and Calum Campbell for her help in proofreading this manuscript. ZHF would like to thank the Seaver Institute for support. NACG has a postgraduate scholarship sponsored by CONACyT.

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NORBERTO A. COLIN GARCIA (1,*,[dagger]), JORGE E. CAMPOS (2), JOSE L. TELLO MUSI (3), ZAC H. FORSMAN (4), JORGE L. MONTERO MUNOZ (5), ALEJANDRO MONSALVO REYES (2), AND JESUS E. ARIAS GONZALEZ (1)

(1) Laboratorio de Ecologia de Ecosistemas de Arrecifes Coralinos, Centro de Investigation y de Estudios Avanzados, Unidad Merida, Departamento de Recursos del Mar, Km6 Antigua carretera a Progreso, Apartado Postal 73, Colonia Cordemex C.P. 97310, Merida, Yucatan, Mexico; (2) Laboratorio de Bioquimica Molecular, Unidad de Biotecnologia y Prototipos, UNAM, FES Iztacala, Avenida de los Barrios No. 1, Colonia Los Reyes Iztacala, C.P. 54090, Tlalnepantla, Estado de Mexico, Mexico; (3) Laboratorio de Biologia Marina, UNAM, FES Iztacala, Avenida de los Barrios No. 1, Colonia Los Reyes Iztacala, C.P. 54090, Tlalnepantla, Estado de Mexico, Mexico; (4) Hawaii Institute of Marine Biology, P.O. Box 1346, Kane'ohe, Hawaii 96744; and (5) Centro de Investigation y de Estudios Avanzados, Unidad Merida, Departamento de Recursos del Mar, Km6 Antigua carretera a Progreso, Apartado Postal 73, Colonia Cordemex C.P. 97310, Merida, Yucatan, Mexico

Received 25 October 2016; Accepted 2 February 2017; Published online 28 March 2017.

(*) To whom correspondence should be addressed. E-mail: norberto82@gmail.com

([dagger]) Current address: Universidad Nacional Autonoma de Mexico, Parque Cientifico y Tecnologico de Yucatan, Laboratorio de Biologia de Conservation. Carretera Sierra Papacal Chuburna Puerto Km 5,97302 Sierra Papacal, Yucatan, Mexico

Abbreviations: CA, canonical axis; DCA, discriminant component analysis; ITS, internal transcribed spacer; ITS1, ITS2, subunits of ITS; VRSNP, Veracruz Reef System National Park
Table 1
List of characters evaluated in each sample collected from the Veracruz
Reef System National Park

            Character              Abbreviation       Description

Septa (n)                              SN        Septa per corallite
                                                  (n)
Septa cycles                           SC        Septa cycles per
                                                  corallite (n) (1
                                                  =three
                                                  complete and four
                                                  incomplete;
                                                  2=four complete and
                                                  incomplete)
Septa length                           SI,       Length of first set
                                                  of septa per
                                                  corallite
Septa space                            SS        Space between septa
Columella size                         COLL      Length of columella
                                                  per corallite
Corallite length                       CL        Length of corallite
                                                  on larger axis
Corallite width                        CW        Width of corallite
                                                  on small axis
Synapticular rings                     SR        Synapticular rings
                                                  per corallite in)
Distances of calcification             DC        Distances between
                                                  centers of
                                                  calcification in
                                                  first set of septa
Septa width                            SW        Width of first set
                                                  of septa per
                                                  corallite
Theca thickness                        TT        Thickness of theca
                                                  between corallites
Synapticular ring thickness            SRT       Thickness of
                                                  synapticular
                                                  rings per corallite
Size of last septa                     SLt       Length of last set of
                                                  septa per corallite
Teeth (n)                              TN        Teeth in first set of
                                                  septa (n)
Free septa                             SF        Septa cycles form
                                                  those that are
                                                  free (1 = 1st, 2nd
                                                  septa free; 2= 1st,
                                                 2nd, 4th septa free)
Fused septa                            SFu       Septa cycles form
                                                  those that are fused
                                                  (1 = 1st and 3rd
                                                  septa fused;
                                                 2=3rd septa fused to
                                                 2nd septa).
Teeth form                             TF        Form of teeth in septa
                                                  wall (1 =single;
                                                  2 = bidentate; 3 =
                                                  multidentate)
Fossa depth                            FD        Depth of fossa per
                                                  corallite
Septa continuous or discontinuous      SC/D      Continuity of septa
                                                  between corallites
                                                  (1=continuous; 2=
                                                  alternating
                                                 between corallites
Corallite distance                     C-C       Distance between
                                                  columella among
                                                  corallites

Table 2
Average values of 15 characters evaluated in the three species of
Siderastrea

            Siderastrea radians    Siderastrea sidera
Character  Average ([+ or -] SD)  Average ([+ or -] SD)

SN          27.86 [+ or -] 3.17    38.66 [+ or -] 4.54
FD           0.56 [+ or -] 0.23     0.63 [+ or -] 0.18
SL           1.50 [+ or -] 0.32     1.76 [+ or -] 0.23
SS           0.28 [+ or -] 0.07     0.25 [+ or -] 0.05
DC           0.18 [+ or -] 0.04     0.16 [+ or -] 0.05
COLL         0.36 [+ or -] 0.08     0.38 [+ or -] 0.10
C-C          2.71 [+ or -] 0.18     3.12 [+ or -] 0.48
CL            3.6 [+ or -] 0.51     4.48 [+ or -] 0.49
CW           2.64 [+ or -] 0.25     3.19 [+ or -] 0.45
SR           2                      2.65 [+ or -] 0.49
SW           0.15 [+ or -] 0.07     0.17 [+ or -] 0.05
TT           0.16 [+ or -] 0.05     0.13 [+ or -] 0.03
SRT          0.15 [+ or -] 0.03     0.11 [+ or -] 0.02
SLt          0.71 [+ or -] 0.34     0.79 [+ or -] 0.19
TN            7.4 [+ or -] 0.55      9.7 [+ or -] 1.42

           Siderastrea stellata
Character  Average ([+ or -] SD)

SN         36.75 [+ or -] 3.68 (*)
FD          0.56 [+ or -] 0.14 (*)
SL          1.61 [+ or -] 0.27
SS          0.23 [+ or -] 0.05 (*)
DC          0.17 [+ or -] 0.05 (*)
COLL        0.35 [+ or -] 0.10
C-C         2.76 [+ or -] 0.48
CL          3.68 [+ or -] 0.53
CW          2.70 [+ or -] 0.39
SR          2.33 [+ or -] 0.49
SW          0.15 [+ or -] 0.04
TT          0.15 [+ or -] 0.06
SRT         0.14 [+ or -] 0.11
SLt         0.67 [+ or -] 0.23
TN          9.51 [+ or -] 1.34 (*)

(*) Statistically significant differences in discriminant component
analysis (DCA) (P < 0.05).
C-C, distance between corallites; CL, corallite length; COLL.
columella size; CW, corallite width; DC, distances between centers of
calcification; FD, fossa depth; SL. septa length; SLt, size of last
septa; SN, septa (n); SR, synapticular rings: SRT, synapticular ring
thickness; SS, septa space; SW, septa width; TN, teeth (n): TT, theca
thickness.

Table 3
Analyses of variance of the 20 morphological characters evaluated

Character  Sum of squares  Mean square(s) group  Mean square(s) error

SC/D             8.04              4.02                 0.05
SC               6.31              3.15                 0.09
TN              28.92             14.46                 1.56
SN             363.4             181.71                23.6
SS               0.03              0.01                 2.60 (*)
SF               1.63              0.82                 0.23
DC               0.02              0.01                 2.30 (*)
SR               1.13              0.57                 0.24
SLt              0.2               0.1                  0.05
CL               1.44              0.72                 0.4
SRT              0.02              0.01                 4.90 (*)
TT               0.01              2.80 (*)             2.10 (*)
C-C              0.6               0.3                  0.23
TF               1.77              0.88                 0.71
SW               4.70 (*)          2.40 (*)             2.30 (*)
CW               0.36              0.18                 0.23
SL               0.1               0.05                 0.07
SFu              0.17              0.08                 0.24
COLL             4.70 (*)          2.30 (*)             0.01
FD               3.10 (*)          1.60 (*)             0.03

Character  df error  F-value  P-value

SC/D          37      85.83   < 0.0001
SC            37      33.66   < 0.0001
TN            37       9.28   < 0.0005
SN            37       7.7    < 0.0016
SS            37       5.11   < 0.0109
SF            37       3.61   < 0.0369
DC            37       3.45   < 0.0422
SR            37       2.39     0.1055
SLt           37       2.02     0.1473
CL            37       1.81     0.1771
SRT           37       1.61     0.2136
TT            37       1.33     0.2771
C-C           37       1.29     0.286
TF            37       1.25     0.2995
SW            37       1.01     0.3747
CW            37       0.79     0.4635
SL            37       0.68     0.5145
SFu           37       0.35     0.7104
COLL          37       0.24     0.7858
FD            37       0.05     0.9516

(*) E-03 (0.0026).
C-C, corallite distance; CL, corallite length; COLL, columella size;
CW. corallite width; DC, distances between centers of calcification;
df, degrees of freedom; FD, fossa depth; SC, continuity of septa
between corallites; SC/D, continuous septa or septa alternating with
corralites; SF, free septa; SFu, fused septa; SL, septa length; SLt,
size of last septa; SN, septa (n); SR, synapticular rings; SRT,
synapticular ring thickness; SS, septa space; SW, septa width; TF,
teeth form; TN, teeth (n): TT, theca thickness.

Table 4
Genetic divergence of Siderastrea specimens

                    Genetic divergence ITS/ITS2
                        1                 2

Clade I              0% / 0%
Clade II         0.023% / 0.025%        0% / 0%
Clade III        0.024% / 0.031%   0.0115% / 0.010%
Clade IV         0.025%            0.016%
5. siderea       0.043% / 0.0%     0.045% / 0.025%
S. radians       0.011% / 0.025%   0.012% / 0.0%
S. stellata (a)  0.015% / 0.025%   0.019% / 0.0%
S. stellata (b)      NA / 0.031 %      NA / 0.025%

                             Genetic divergence ITS/ITS2
                        3             4         5         6        7

Clade I
Clade II
Clade III            0% / 0%
Clade IV         0.033%            0% / 0%
5. siderea       0.069% / 0.031 %   0.056%   0% / 0%
S. radians       0.035% / 0.025%    0.021%    0.038%   0% / 0%
S. stellata (a)  0.041% / 0.025%    0.027%    0.041%    0.009%  0% / 0%
S. stellata (b)      NA / 0.0%        -         -         -        -

(a) (Forsman et al.. 2005)
(b) (Fukami et al., 2004)
ITS, internal transcribed spacer.
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Publication:The Biological Bulletin
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Date:Feb 1, 2017
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