Study site and sample collection

Carrizales Reef is located in Carrizales Bay (19°05´42” N, 104°26’21” W), just southwest of the coast of Manzanillo, Colima, Mexico (Fig. 1). On 13 February 2017, an expedition to Carrizales Reef was carried out using autonomous diving equipment, during which a total of 36 fragments of the corals P. cf. capitata, P. cf. damicornis, and P. cf. verrucosa were collected from 12 colonies on the east and west margins of the reef (n = 6). To reduce the effects of environmental variation, colonies that were sympatrically distributed at 3 m depth were selected. Each colony was identified by the typical morphological characteristics (following ^{Veron 2000}). However, since colony morphology is generally incongruous with genetics (^{Pinzón and LaJeunesse 2011}), there is a possibility that the colonies belong to homogeneous genetic groups.

Figure 1 Location of Carrizales Reef on the coast of Colima, Mexico. 

After the expedition, biological samples were divided into 3 groups of 12 fragments, which were immediately processed in the field and protected from light during transportation. Two of the groups were separately preserved in plastic vials, one with 10 mL of 10% formaldehyde and the other with 10 mL of cold RNAlater solution. Afterwards, samples in formaldehyde were refrigerated and samples in RNAlater were frozen at -80 °C until laboratory analysis. The remaining group of samples were initially transferred into microtubes with 2 mL of 100% methanol, subsequently stored at 4 °C for 24 h in total darkness, and lastly analyzed in the laboratory.

In addition, 6 samples of 1.0 L of seawater were collected at 1.0 m depth at each reef sampling site (east and west), at approximately 1-h intervals throughout the sampling duration (~4 h). Chlorophyll a (Chla) concentration in these samples was determined with a Jenway 6500 spectrophotometer (Chelmsford, Essex, UK), according to the methodologies of ^{Strickland and Parsons (1972)} and ^{Grasshoff et al. (1999)}. The results are shown as Chla concentration per volume of seawater (mg·m^-3). Owing to the technical difficulties in continuously recording light and temperature data during the expedition on 13 February, a second expedition was conducted on 27 May 2017 to obtain a new log. To do this, Onset HOBO Pendant UA-002-08 sensors (Onset Computer Corporation; Bourne, MA, USA) were used. Prior bi-monthly monitoring showed that seasonal variability in the reef occurs every 4 months (^{Muñiz-Aguiano et al. 2017}). So even though these data do not correspond to the conditions on 13 February, we believe the values obtained on 27 May do allow for a reliable comparison of the spatial variation between the east and west sides of Carrizales Reef.

Laboratory analysis

The density of endosymbiont dinoflagellates was quantified from the fragments in formaldehyde by employing a modification to the protocol proposed by ^{Zamoum and Furla (2012)}. Briefly, coral tissue was removed from the skeleton by incubation in 10 mL of 4 M NaOH at 65.0 °C for 1 h. The homogenate was then kept overnight at 37.5 °C and Symbiodiniaceae cell density was later quantified from a 10-μL aliquot using a Neubauer hemocytometer (n = 8 replicates).

Pigment concentration was determined from the fragments that were preserved in 100% methanol at 4.0 °C. After storing for 24 h, the homogenate was sonicated for 15 s and then centrifuged at 1,500 × g for 5 min at 4 °C; the resulting extracts were protected from the light and immediately used to measure absorbance by scanning wavelengths from 300 to 750 nm every 1 nm. Duplicate measurements were performed in a Spectronic Genesys 5 spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA) using 96-well microplates. Pigment content was calculated from the extinction coefficients for the β-carotene (145.0 L·g^-1·cm^-1) and perinidine (132.5 L·g^-1·cm^-1) carotenoids (^{Jeffrey and Haxo 1968}) and for the diadinoxanthin (223.0 L·g^-1·cm^-1) and dinoxanthin (135.0 L·g^-1·cm^-1) xanthophylls (^{Vernon 1960}), and total chlorophyll concentration (Chla and Chlc) was calculated according to the equations by ^{Jeffrey and Humphrey (1975)}, with the recommended turbidity correction.

Nucleic acids and total proteins were extracted from the fragments in RNAlater after thawing. First, RNAlater excess was removed with a Kimwipes tissue (Kimberly-Clark; Roswell, GA, EUA). Then, samples were ground in a mortar, and approximately 100 mg of tissue and skeleton were transferred into 1.5-mL microcentrifuge tubes with 1.0 mL of TRIzol reagent (Life Technologies); samples were then homogenized using a mechanical disruptor (FastPrep24, MP Biomedicals; Santa Ana, CA, USA). The extractions were made following the specifications of the manufacturer with the following modifications. Briefly, up to 2 μL of 6M HCl were added to the homogenate to prevent the neutralization reaction that occurs between the calcium carbonate in the skeleton and the acidic TRIzol (^{Anderson et al. 2016}). RNA was precipitated by adding isopropanol and incubating overnight at -20 °C, and the solution was then centrifuged to precipitate the RNA, which was eluted in 50 μL of ribonuclease-free water (RNase). DNA was recovered from the rest of the phenol solution following the recommendations by ^{Mayfield et al. (2012)}. Briefly, a back extraction buffer (4M guanidine thiocyanate, 50 mM sodium citrate, and 1M Tris free base) was used. Equal volumes of back extraction buffer and phenol solution were mixed, and the mixture was subsequently centrifuged to obtain an aqueous phase, which was transferred into a new tube. DNA was precipitated and eluted in 200 μL deoxyribonuclease-free water (DNase), with an additional 10-min incubation at 55 °C. Lastly, proteins were precipitated from the remaining phenol solution by modifying the time of incubation and final solubilization (40 min at 50 °C in 1% SDS). The quantity and quality of nucleic acids and proteins were analyzed in a Nanodrop spectrophotometer and the samples were stored at -80 °C.

For the gene expression analysis, all RNA samples were thawed and purified by treatment with DNase I enzyme (without RNase) following the specifications of the manufacturer (Invitrogen, Thermo Fisher Scientific). With the purified RNA, 300 ng were reverse transcribed to the first strand of complementary DNA (cDNA) using the high-capacity reverse transcription kit and following the recommendations of the manufacturer (Applied Biosystems, Life Technologies; CA, USA). Aliquots of 20 ng·μL^-1 of cDNA were prepared and then stored at -20 °C.

Relative mRNA expression was done by amplifying target genes (CA, CuZnSOD, GFP-like cp, and HSP70) and an internal control fragment (18 ribosomal RNA) to calibrate the complementary DNA template (Table 1). Eight independent real-time quantitative PCR (q-PCR) assays were performed, in a total volume of 20.0 μL with 6.3 μL of 2X SYBR Green Master Mix (Applied Biosystems), 0.6 μL of each primer for the target gene and the internal control gene (10 mmol·μL^-1), 5.0 μL of the cDNA sample (20 ng·μL^-1), and 6.9 μL of RNase/DNase-free water. Q-PCR assays were carried out in a StepOnePlus thermocycler (Applied Biosystems) with the following thermocycling conditions: (1) “hot start” at 95 °C for 10 min, (2) amplification of 40 cycles at 95 °C for 15 s followed by 60 s at 60 °C, and (3) fusion at 95 °C for 15 s followed by 60 s at 60 °C and 15 s at 95 °C. Results are given in terms of relative mRNA expression as calculated with the 2^-ΔΔCt method (^{Schmittgen and Livak 2008}).

Statistical analysis

Statistical analyses were performed using R software. All data were analyzed with the Shapiro-Wilk normality test and the Bartlett test to determine homoscedasticity (95% confidence intervals). For the environmental parameters, an unpaired 2-sample t-test was used. For biological parameters, 2-way analysis of variance (ANOVA) was used, followed by multiple comparisons (Tukey’s test) to assess significant differences (P < 0.05). In some cases, the bidirectional non-parametric Friedman test and the paired sign test were used. All data were presented as mean values ± standard error of the mean (n = 6).

Results

The expression of CA in P. cf. capitata was higher in the western (P < 0.05) than in the eastern reef colonies (Fig. 2). The GFP-like cp expression was higher in the P. cf. verrucosa colonies from the west side (P < 0.05) than in the colonies from the east side (Fig. 3). On the other hand, the CuZnSOD expression in P. cf. capitata was also higher in the western colonies (P < 0.05) compared to eastern reef colonies. Contrary to CA and GFP-like cp expressions, the expression of the CuZnSOD enzyme was lower in the P. cf. verrucosa colonies (P < 0.05) in relation to the P. cf. capitata colonies (Fig. 4). Finally, the HSP70 expression did not show significant differences between the east and the west; nevertheless, all mean values of the HSP70 expression were consistently higher in western corals (Fig. 5).

Figure 2 Expression levels of carbonic anhydrase (CA) mRNA in Pocillopora morphospecies on the east and west sides of Carrizales reef. Values are the mean ± standard error of the mean (n = 6). Letters denote significant differences between the data (P < 0.05). 

Figure 3 Expression levels of green fluorescent protein-like chromoprotein(GFP-likecp) mRNAin Pocillopora morphospecies on the east and west sides of Carrizales reef. Values are the mean ± standard error of the mean (n = 6). Letters denote significant differences between the data (P < 0.05). 

Figure 4 Expression levels of copper,zinc-superoxide dismutase (CuZnSOD) mRNA in Pocillopora morphospecies on the east and west sides of Carrizales reef. Values are the mean ± standard error of the mean (n = 6). Letters denote significant differences between the data (P < 0.05). 

Figure 5 Expression levels of heat shock protein (HSP70) mRNA in Pocillopora morphospecies on the east and west sides of Carrizales reef. Values are the mean ± standard error of the mean (n = 6). Letters denote significant differences between the data (P < 0.05). 

In the joint analysis of Chla and Chlc, carotenoids, xanthophylls, endosymbiont density, mycosporine-like amino acids, and the RNA/DNA, protein/DNA, and RNA/ protein ratios, significant differences were observed in the colonies of P. cf. capitata and P. cf. verrucosa from the east and west sides of the reef, whereas in the P. cf. damicornis colonies no significant differences were observed in any marker (Table 2). The environmental values for the Carrizales Reef area showed Chla concentrations of 0.18-0.99 mg·m^-3 throughout the sampling. The highest values occurred on the west side of the reef (0.99 ± 0.17 mg·m^-3) compared to the east side (0.42 ± 0.10 mg·m^-3). Finally, the light values that were recorded months after sampling at Carrizales Reef were higher for the west (63.60 ± 7.91 W·m^-2·S^-1) in comparison with the light values for the east (44.42 ± 5.26 W·m^-2·S^-1). On the other hand, temperature data were the same at both sites (25 ± 0.10 °C).