Relative aging and intraspecific diversity of resilient communities in Belize using microsatellite markers and somatic mutations Senior Honors Thesis

Adele Irwin Dr. Lisa Greer, Research Advisor Dr. Robert Humston, Thesis Committee Advisor Dr. Paul Cabe, Thesis Committee Advisor

Washington and Lee University Biology 2015

Adele Irwin1, Lisa Greer2, Robert Humston1, Meghann Devlin-Durante3, Iliana Baums3, Halard Lescinsky4, Karl Wirth5, Paul Cabe1, H. Allen Curran6 1Department of Biology, Washington & Lee University, Lexington, VA 2Department of Geology, Washington & Lee University, Lexington, VA 3Department of Biology, Pennsylvania State University, University Park, PA 4Department of Biology and Earth Science, Otterbein University, Westerville, OH 5Department of Geology, Macalester College, St. Paul, MN 6Department of Geosciences, Smith College, Northampton, MA

Adele Irwin Biology B.A. Candidate, Washington & Lee University 973-632-9531 [email protected]

Lisa Greer Associate Professor of Geology, Washington & Lee University 540-458-8870 [email protected]

Robert Humston Associate Professor of Biology, Washington & Lee University 540-458-8341 [email protected]

Meghann Devlin-Durante Senior Research Technologist, Pennsylvania State University 814-867-0492 [email protected]

Iliana Baums Associate Professor of Biology, Pennsylvania State University 814-867-0492 [email protected]

Halard Lescinsky Professor of Biology and Earth Science, Otterbein University 614-823-1565 [email protected]

Karl Wirth Associate Professor of Geology, Macalester College 651-696-6449 [email protected]

Paul Cabe Professor of Biology, Washington and Lee University 540-458-8894 [email protected]

H. Allen Curran Kenan Professor Emeritus of Geosciences, Smith College 413-585-3943 [email protected]

1Department of Biology, Washington and Lee University, 204 W. Washington Street, Lexington, VA 24450 2Department of Geology, Washington and Lee University, 204 W. Washington Street, Lexington, VA 24450 3Department of Biology, Pennsylvania State University, 213 Mueller, University Park, PA 16802 4Department of Biology and Earth Science, Otterbein University, 1 S. Grove Street, Westerville, OH 43081 5Department of Geology, Macalester College, 1600 Grand Avenue, Saint Paul, MN 55105 6Department of Geosciences, Smith College, Clark Science Center, 44 College Lane, Northampton, MA 01063

Abstract. Historically and geologically, Acropora palmata and Acropora cervicornis have been key reef framework builders, yet in the Caribbean these species have faced significant mortality over the last few decades. Today, Acropora populations persist in large numbers with high live cover at few Caribbean sites. While many studies have focused on the recent demise of these species, data from areas where Acropora spp. have continued to thrive is less available.

Understanding the genetic diversity, reproductive history, and temporal persistence of healthy populations of Acropora spp. may provide insight into the potential for these populations to continue to thrive. We studied three reef sites with highly abundant Acropora cervicornis,

Acropora palmata, and Acropora prolifera populations offshore of Ambergris Caye, Belize;

Coral Gardens, Manatee Channel, and Rocky Point. Genetic data was collected from all three

Acropora species to determine the clonal composition and relative ages of Acropora colonies.

We used five microsatellite markers to determine 1) genotypic diversity, 2) dominant reproductive style, and 3) minimum and maximum genet age estimates of all three acroporids at these sites. Acropora populations were highly clonal and likely reproduced primarily asexually via fragmentation across all sites. We estimated the ages of 19 Acropora genets. Minimum age estimates from a new genetic aging technique (based on somatic mutation rates) show minimum ages of between 0 and 409 years old for A. cervicornis, between 0 and 561 years for A. palmata, and between 0 and 281 years old for A. prolifera. These results suggest that Acropora abundance and live coral cover are not driven by high genotypic diversity at these sites, as some have suggested for other Acropora-dominated locations. We also show that at least ten of the genets found at these sites are veterans that survived the recent Caribbean-wide Acropora spp. collapse, suggesting that this site is a refugia for Acropora . Introduction The global decline of coral reefs in response to rising sea-surface temperatures, overfishing, rising sea level, disease, eutrophication, sedimentation, and ocean acidification has been well documented in recent years (Hughes 1994; Hughes et al. 2003; Bellwood et al. 2004;

Hoegh-Guldberg et al. 2007; Kuffner et al. 2015). Acropora cervicornis and Acropora palmata

(Figure 1), two of the most important Caribbean reef framework-builders due to their fast growth rates and interlocking branch morphologies, have experienced up to 98% decline in the

Caribbean over just a few decades in response to increasing environmental stress and white-band disease (Gladfelter 1982; Knowlton 1992; Aronson and Precht 2001; Pandolfi 2002; Bowden-

Kerby and Carne 2011). Consequently they were the first two coral species listed as threatened under the US Endangered Species Act (NOAA 2005). Many Acropora-dominated reefs declined during acute die-off events in 1998 and 2005 and during white-band disease prevalence in the

1970s and 1980s (Aronson and Precht 2001; Gardner et al. 2003; Eakin et al. 2010; Randall and van Woesik 2015).

Despite Caribbean-wide declines, recent relatively abundant A. cervicornis and A. palmata populations have been reported in Honduras (Keck et al. 2005; Purkis et al., 2006), the

Dominican Republic (Lirman et al. 2010), Mexico (Larson et al. 2014), Florida (Fort Lauderdale area; Vargas-Angel et al. 2003), and Belize (Peckol et al. 2003; Brown-Saracino et al. 2007;

Macintyre and Toscano 2007). Data from these Acropora sites varies in quantity and character.

Some of these studies have consisted of general habitat surveys, quantification of percent of algal and live coral coverage, Acropora colony sizes, number of Acropora colonies, fish and invertebrate populations, and disease prevalence. While it has been suggested that higher

Acropora population size may increase the probability of sexual reproduction and thus potential genetic diversity (Vargas-Angel et al. 2003; Lirman et al. 2010; Larson et al. 2014), genetic character was not assessed in studies focused on large extant acroporids. Genotypic diversity has been associated with overall habitat health and predicted to have a positive relationship with coral sexual recruitment potential and survival during adverse conditions by providing adequate alleles (Altizer et al. 2003; Reed and Frankham 2003; Vargas-Angel et al. 2003; Reusch et al.

2005, Zubillaga et al. 2008; Lirman et a. 2010; Downing et al. 2012; Larson et al. 2014,

Williams et al. 2014). However, recent findings show that genotypic diversity in Acropora populations does not necessarily increase their sustainability over short time scales as predicted

(Williams et al. 2014). Thus, the role of genotypic diversity in persistence remains unclear and further studies are required to achieve clarification.

Clonal age, or time since a clone’s sexual origin, can be used to determine clonal persistence or newness (Weiher et al. 1999; Eriksson 2000; Ally et al. 2008; deWitte and

Stocklin 2010).

Acropora prolifera is the only known scleractinian hybrid in the Caribbean (Willis et al.

2006). It has not been found in the fossil record (Budd et al., 1994), and has been reported as rare compared to its parents in the past (Lang et al. 1998; Willis et al. 2006; Fogarty 2012) but it has recently been found more frequently and in higher abundance in Florida (FWRI and DTNP

2010), Curcacao, Lesser Antilles (Japaud et al. 2014), and Belize (Fogarty 2010; Fogarty et al.

2012; Richards and Hobbs 2015). The apparent recent increase in A. prolifera may suggest either that hybridization is new or that environmental conditions now better more favorable for A. prolifera and it is exemplifying hybrid vigor and filling the void left behind by diminishing populations of the parent species (Willis et al., 2006). Clonal age data may provide clarification for the history of A. prolifera’s spread and emergence. Both genotypic diversity and clonal age data can be collected from microsatellite DNA from live coral tissue (Baums et al. 2005b;). Calculating clonal age in corals has proved difficult due to lack of feasible methods for recording their histories) developed a new method that uses the number of somatic mutations in the individuals of a clonal genet to estimate minimum and maximum number of years since larval settlement.

We report genetic data from three sites of high A. cervicornis, A. palmata, and A. prolifera abundance offshore of Ambergris Caye, Belize: Coral Gardens, Manatee Channel, and

Rocky Point. This study aimed to determine the clonal structure of Acropora spp. at these sites, and to use a new somatic aging technique (Devlin-Durante to calculate somatic ages of coral clones off Belize. With this information we sought to 1) determine genetic diversity of Acropora spp., 2) characterize the reproductive history of present-day Acropora reefs, 3) estimate the age of Acropora genets, and 3) determine the history and nature of A. prolifera colonization at these sites in Belize.

Materials and Methods

Study Area

The Mesoamerican reefs off Belize are a forereef system with extensive adjacent lagoonal areas dominated by ridges (Macintyre and Aronson 1997).

Rocky Point (18°12'37.95''N, -87°82'60.64"W; Figures 2 and 3). Coral Gardens (17°50'00.36"N,

87°59'32.45"W) is approximately 7 m deep and comprised of patch reefs dominated by thickets of A. cervicornis, large peripheral stands of A. palmata, small A. prolifera colonies. It is surrounded by mixed coral zones dominated by Orbicella, Agaricia, Porities, and Millepora species, separated by sandy bottom. Peckol et al. (2003) and Brown-Saracino et al. (2007) and reported live coral cover nearby greater than forty-three percent, and similar values have been found here (Greer et al. 2015). Coral Gardens has been a primary study site for four years (2010-

2014), where general habitat data was collected, such as percent live coral coverage along five

16-32 m transects (T1-T5). Its live Acropora abundance exceeds 7.5 hectares, making it potentially the largest documented site of its kind in the Caribbean (Busch et al. in prep).Manatee

Channel (17°47'58.08"N, -87°59'45.57"W), is approximately 1-2 m deep, and comprised of patch reefs dominated by thickets of A. prolifera with high live coral cover. Few stands of A. cervicornis and A. palmata were observed and are scattered throughout the site. The thickets of

A. prolifera are surrounded by mixed coral zones dominated by of Orbicella, Agaricia, Porites, and Millepora species, separated by sand. Rocky Point, approximately 2-3 m depth, is dominated by individual colonies and a few small patches of A. prolifera, all with observed high coral cover. Parent Acropora species are absent, and some colonies of Orbicella, Porites, and

Siderastrea spp are present. (Figures 2 and 3).

Sampling

Live 1 cm3 coral samples were collected from Acropora branch tips using bone cutters, preserved in 95% ethanol, and refrigerated prior to DNA extractions. Initial sampling of A. cervicornis at Coral Gardens in 2013 was haphazard (N=60) and every colony of A. palmata

(N=78) and A. prolifera (N=48) was sampled. Additional sampling in 2014 was designed to increase likelihood of sampling clones. At Coral Gardens, A. cervicornis (n=158) samples were collected from 5-m circle plots in which a center point was designated and samples were collected in 4 randomly generated directions every 5 m for a total of 20 samples per plot (Baums et al. 2013). Five previously created transect lines (T1-T5) were used for other general habitat surveys (Greer et al. 2015), and the circle plots were collected along these transects. No plots overlapped. At Manatee Channel A. prolifera samples (n=78) were collected using the same circle plot method, and every observed individual colony of A. cervicornis (N=20) and A. palmata (N=15) were sampled. At Rocky Point A. prolifera was sampled haphazardly (n=32) due to irregular patch shape. See Table 1 for details on sample sizes.

Genotypic Analysis

Tissue DNA was extracted and amplified and evaluated for five following Baums et al. (2005a) were used to distinguish genets. See Appendix 1 for detailed methods.

Somatic Aging

In the past, methods have been used to date clonal species such as Populus tremuloides

(trembling aspen) by considering the number of somatic mutations seen in microsatellite DNA samples, colony growth rates, and somatic mutation rates (Ally et al. 2008). Co-author Meghann

Devlin-Durante applied this somatic aging method () to coral populations to calculate minimum and maximum age estimates of clones. This method is appropriate for coral clones because corals transmit their somatic mutations to both propagule (Shikina et al. 2012) and fragment

(Orive 2001; Schweinsberg et al. 2014) offspring. Unlike higher organisms whose germ cells are acquired at conception and stored for a lifetime, corals produce germ cells yearly with every spawning season (Shikina et al. 2012). This ensures passage of somatic mutations to future gametic pools. When coral spreads via fragmentation, the fragments inherit somatic mutations from their mother colony, in addition to acquiring their own over time (Schweinsberg et al.

2014). Likewise, live coral sitting atop older ancestral coral will likely contain both the somatic mutations acquired from itself and that from which it extends. Microsatellites are a reliable medium of DNA to use in this somatic mutation aging method because microsatellite mutations are more likely to evolve naturally (Ally et al. 2008). They are non-coding and are thought to be adaptively neutral, serving purely as an aging model (Ally et al. 2008). This method has favorable resolution because microsatellite loci have length variants that are easy to score (Schug et al. 1997).

Analyses

Genotypic diversity: Intraspecific genotypic diversity (Go/n), evenness (Go/Ng), and richness

(Ng/N) were calculated within study sites and combined study sites using the methods described in Baums et al. (2006). We compared data from this study against aggregate data from Baums et al. (2006) to make relative inferences about clonal structure of Acropora at our sites compared to others in the Caribbean. Baums et al. (2006) use genotypic diversity calculations to make inferences about the degree of local sexual recruitment, classifying sites with the highest clonality as “asexual” and those with the lowest clonality as “mostly sexual”. We also compare our results against their aggregate data to classify the local sexual recruitment of our sites.

Somatic mutations: Once genets were identified, somatic mutations of individual ramets were distinguished by identifying single- or double-step mutations. When a mutation was found, calls for the mutated allele were verified in GeneMapper. Using Genalex 6.1 in Excel, mutations were relabeled so that the system recognized a given mutation within its original genet (original clonal group). Clones were considered to be identical at all 5 loci, and a mutation was defined as differing from the clones at only one loci. Next we established the datable samples by distinguished clones with at least five ramets. Genetic distance of each of these clonal groups containing at least one mutated individual was calculated using Genalex functions Euclidian distance and π divergence. The genetic distance and the mutation rates established by Devlin-

Durante (lowest=1.43E-05, highest=1.07E-04) were then applied to the equation described in Ally (2008) to obtain minimum and maximum ages of each clone. Mutation rates were developed by comparing published A. palmata growth rates to numbers of somatic mutations of collected samples and the size of the sampled colony. Minimum mutation rates, used to calculate minimum age, were established from a colony with the lowest π divergence (0 mutations) and compared against its size measurements. The maximum mutation rate, used to calculate maximum age, was found by inferring that a Floridian colony with the highest π divergence

(most mutations) was as old as the establishment of reefs in Florida, about 7000 years ago

(Devlin-Durante 2015).

Results

Genotypic diversity

All Acropora populations at all study sites and also when observed together collectively as a

“Northern Belize” population had genotypic characteristics (diversity, Go/Ge; eveness, Go/Ng) classifying them somewhere between Baums et al. (2006) groups “asexual” (Go/Ge=0.05,

Go/Ng=1) and “mostly asexual” (Go/Ge=0.15625, Go/Ng=0.3875; Table 2). A. prolifera at

Manatee Channel was the only population to show complete clonality and thus asexuality

(Go/Ge=0.014, Go/Ng=1). The samples of A. prolifera at Coral Gardens and A. cervicornis and A. palmata at Manatee Channel represent complete censuses (N) of the populations present.

Number of unique clones (Ng) of A. cervicornis were 10 (Coral Gardens) and 8 (Manatee

Channel); of A. palmata 23 (Coral Gardens) and 8 (Manatee Channel); of A. prolifera 9 (Coral

Gardens), 1 (Manatee Channel), and 4 (Rocky Point; Figure 4). A. cervicornis and A. palmata were not present at Rocky Point.

Somatic Ages

From our 328 Acropora samples, we were able to calculate minimum and maximum age estimates for 19 of the 63 clonal populations (those with five or more ramets) (Table 3, Figure

5). Coral Gardens showed a wide range of clonal ages across all Acropora populations. Coral

Gardens A. cervicornis community had 2 new clones (0-62 years), 2 clones 62-460 years, 1 clone

179-1337 years, 1 clone 393-2931 years, and 1 clone 409-3052 years (Figure 6). The A. palmata community had 1 new clone (0-62 years), 1 clone 187-1397 years, 1 clone 219-1636 years, and 1 clone 461-4191 years, and the A. prolifera community had 2 new clones (0-62 years) and 1 clone

281-2096 years. Manatee Channel showed all new clones (0-62 years), with 1 clone aged from each Acropora population. Rocky Point showed a combination of new and old A. prolifera clones, 1 new clone (0-62 years) and 1 clone 156-1164 years. Clones identified as having minimum age estimates of 0 completely lacked somatic mutations.

Discussion

Genotypic diversity is extremely low among A. cervicornis, A. palmata, and A. prolifera at all study sites. In many cases, some patches or entire sites are comprised of a single genotype.

In studies of other abundant sites of Acropora in the Caribbean, it was predicted that high live coral abundance would have a positive relationship with genotypic diversity (Altizer et al. 2003;

Reed and Frankham 2003; Vargas-Angel et al. 2003; Reusch et al. 2005, Zubillaga et al. 2008;

Lirman et a. 2010; Downing et al. 2012; Larson et al. 2014). That is not the case at these three highly abundant and living sites in Northern Belize. From this low genotypic diversity we infer that the local Acropora communities at our study sites are reproducing asexually, with minimal successful sexual gamete fertilization and settlement. The large disparity between Baums et al. (2006) low genotypic diversity values and our values is likely primarily due to variation in sampling method. Our sampling method, however, was extremely thorough as large sample sizes from a wide area of the larger reefs were collected, and complete censuses of colonies were collected when possible (when number of colonies was small enough). Similarly low levels of sexuality seen in a neighboring Caribbean population corroborate the plausibility of our results.

A. palmata from Chinchorro, Mexico (~49 km from our northernmost site, Rocky Point) shows mostly asexual reproductive behavior (Baums et al. 2006). This similarity is also reflected in report of regional genetic isolation throughout the Caribbean, divided into Western and Eastern genetic intermixing populations (Baums et al. 2005b). Our results further validate the Baums et al. (2006) claim that Western Acropora populations exhibit less sexual reproduction than

Eastern.

Studies done at other abundant Acropora sites also predicted that these large populations were critical spawning sources of propagules and might help reestablish genetic diversity and coral coverage at neighboring sites (Keck et al. 2005; Zubillaga et al. 2008; Lirman et al. 2010,

Vargas-Angel et al. 2003). Acropora communities across all of our study sites show a range of asexual to mostly asexual reproductive behavior. This reproductive behavior is also seen when lumping all of our local populations together as a Belize population. The overall trend indicates that fragmentation is the predominant mode of reproduction in this region, and that not much larval recruitment is likely taking place nearby to increase genotypic diversity (Baums et al.

2013). Defining the “local” communities that can be acting as sexual propagule sources is difficult in this case because the regional coastal geography is such that Belize is “cornered” by

Mexico, Guatemala, and Honduras which creates an eddy of circular currents that can be complicated and change seasonally (Cetina et al. 2006; Rousset and Beal 2010; Gygory et al. 2013). The low number of A. prolifera clones observed also indicates that hybridization rates of the parent species A. cervicornis and A. palmata are low. Direct observation of spawning behavior would need to be observed to confirm the claim that Belize Acropora are not largely acting as propagule sources. Due to large surface area of clones at many sites, it remains possible that gamete production occurs but cannot complete fertilization because of self-incompatibility

(Coffroth and Lasker 1998; Baums et al. 2005b; Levitan and McGovern 2005).

Studies done on other dually clonal and sexual species such as tor-grass, piper shrubs,

Baltic fucoids, and pondweed show that even when asexual reproduction is the primary mode of reproduction, usually enough sexual reproduction occurs to prevent the genotypic diversity from becoming threateningly low (Johannesson et al. 2011; Baba et al. 2012; Lasso et al. 2012; Wang et al. 2012). Asexual reproduction is thought to increase short-term success by increasing expanse, while sexual reproduction is thought to increase long-term success by maintaining a larger buffer of alleles to combat future environmental stresses. Thus, even when asexual growth is high, low genotypic diversity can be detrimental to reef populations in the event of continued or worsening environmental conditions or pathogen attack (Schmid 1994; Steinger et al. 1996;

Reusch et al. 2005; Garcia et al. 2008; Williams et al. 2008). The current live coral cover and spread seen at our sites may be stable in the short-term but vulnerable to future compromise.

Inferences about the longevity fitness of these abundant Acropora throughout time can be made when considering our clonal age data. The identified veteran clonal populations predating the Caribbean-wide die-off events, or refugia, may have resisted mortality due to their high compatibility with the environment and their ability to absorb or rebound from disturbances

(Eriksson 2000; Riegl et al. 2009). An interesting note is that some of our oldest clones, such as the A. cervicornis clonal colony at Coral Gardens on Transect 5, have massive volumes exceeding 35 m in continuous length. While clonal stability is not believed to mitigate the need for sexual reproduction (Eriksson 2000), variation within large clonal populations exists and could be responsible for some of the old ages seen at these sites such as this colony of Transect

5. As a clone ages and grows, it accumulates somatic mutations that can produce appreciable internal variation. While mutations in microsatellite markers are considered neutral, it is predicted that they reflect other parts of the genome of adaptive significance (Selkoe and Toonen

2006; Schweinsberg et al. 2015). Competition among individuals of a clone with slight variations may allow for adaptation to an environment within a clone as opposed to among clones (Soane and Watkinson 1979; Hara 1994; van Kleunen et al. 2001; de Witte and Stocklin 2010). A recent study by Schweinsberg et al. (2015) showed that branching scleractinian corals such as Acropora exhibit high degrees of mosaicism, or formation of somatic mutations, that leads to intraclonal genetic variability. They predict that this intraclonal genetic variability may have evolutionary effects such as diversifying the gene pool of consecutive generations and enhancing adaptive ability in the face of external disturbances such as climate change. However, the implications of this diversity remain unclear (Schweinsberg et al. 2015).

While common among clonal species in nature, a positive relationship between age and abundance cannot be assumed in all communities (Hughes and Jackson 1980). In some cases, largely abundant populations can be fairly recent and proliferated quickly due to fast growth rates (Garcia et al. 2008). In order to determine if the large populations observed at our sites are veteran populations or new, fit clones, we turn to the age outputs collected from our somatic mutation data. Coral Gardens shows a wide range of ages across Acropora populations. At

Manatee Channel Acropora populations are all completely new. The single genet of A. prolifera indicates that a founder effect took place in which one parent populated the entire area. At Rocky Point the A. prolifera populations are a combination of relatively new and old. It is difficult to determine if bottleneck events occurred at any of our sites, in which genotypically diverse communities were reduced during an adaptation event such as perhaps the Caribbean-wide decline in the 70s and 80s. Garza and Williamson (2001) established a method to determine the likelihood that a bottleneck event occurred using the mean ratio of the number of alleles to the range in allele size of microsatellite DNA. However, the method requires data from at least 5 microsatellite markers, and A. cervicornis and A. prolifera only amplify at 4 of our 5 markers.

Even though A. palmata amplifies at all 5 of our markers, our sample size was too limited to conduct this test due to high clonality (each clone represents a single individual).

Our results also provide evidence that some A. prolifera genets are older than the hybrids’ recently described expansion in the Caribbean. It has previously been thought that A. prolifera only recently emerged to inhabit substantial surface area (Willis et al. 2006, Fogarty

2010, Richards and Hobbs 2015), but the results here indicate that in Northern Belize it has existed for longer than expected, in locations at Rocky Point for between 0-156 years and Coral

Gardens for between 281-2096 years. Our data suggests that new A. prolifera colonies with the ability to cover large areas exist, but that the hybrid has persisted in some locations for a long time.

Overall, this study provides data suggesting that Acropora populations in Northern Belize sites are largely clonal and reproduce primarily asexually. Furthermore, the ages of these populations range from new to relatively old (>561 years) indicating a combination of refuge populations and new recruits. Colonies of A. prolifera are among some of these older populations indicating that this species has potentially existed in this region longer than previously would have been predicted. While Rocky Point has been designated as a marine protected area (MPA) since the 1996 establishment of the Bacalar Chico National Park and

Marine Reserve, we suggest that Coral Gardens and Manatee Channel be granted the same status. Both sites contain major reef-building corals with massive abundances and high live percentage cover, favorable traits for MPAs (Jameson et al. 2001; Knowlton and Jackson 2001;

McField and Kramer 2007; Cruz et al. 2015). Secondly, we have shown that Coral Gardens may contain veteran or refugia colonies that have exhibited their ability to survive through adverse conditions (Keppel et al. 2012). These veterans demand protection under projected anthropogenic climate change due to their hypothesized potential to expand further (Noss 2001;

Taberlet and Cheddadi 2002; Loarie et al. 2008; Keppel et al. 2012). While Coral Gardens and

Manatee Channel may not be viewed as high priority sites for MPA establishment because of their currently relatively high abundance we think that a proactive approach to preservation may be more effective than a reactive one, in which the integrity of these rare reef spots is maintained. It is our hope that our results will help coastal Caribbean communities and lawmakers to understand better the dynamics of some of the last remaining abundant Acropora reefs and to design preservation efforts accordingly.

Acknowledgments

This work was supported by the National Science Foundation under Grant No. NSF-REU

#1358987 and the Keck Geology Consortium, the Howard Hughes Medical Institute through the

Precollege and Undergraduate Science Education Program, as well as the Washington and Lee

University Department of Geology, Office of the Provost, Johnson Opportunity Grant, and the

Evans Fund for International Student Experiences. We wish to thank Kelly Bezold for laboratory work as well as Ken Mattes and Maureen Gannon at Belize Marine TREC for support of this project. We are grateful for the support of the Belize Fisheries Department and Mr. James

Azueta of the Belize Ecosystems Management Unit for granting permission for this work.

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Methods Appendices Appendix A: DNA Extractions Extractions were performed using Qiagen DNeasy Blood and Tissue Kits. The following instructions are from the extraction kit manual with some modifications.

1. Tissue: Cut tissue (3-5 polyps) into small pieces, and place in a 1.5 ml microcentrifuge tube.

Add 180 μl Buffer ATL. Add 20 μl proteinase K, mix by vortexing, and incubate at 56°C overnight in a stationary water bath. Vortex 15 seconds directly before proceeding to step 2.

2. Add 200 μl Buffer AL. Mix thoroughly by vortexing. Incubate blood samples at 56°C for 10 minutes in a stationary water bath.

3. Add 200 μl ethanol (96-100%). Mix thoroughly by vortexing.

4. Pipet the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at ≥ 6000 x g (8000 rpm) for 1 minute. Discard the flow-through and collection tube.

5. Place the spin column in a new 2 ml collection tube. Add 500 μl Buffer AW1. Centrifuge for 1 minute at ≥ 6000 x g. Discard the flow-through and collection tube.

6. Place the spin column in a new 2 ml collection tube, add 500 μl Buffer AW2, and centrifuge for 3 minutes at 20,000 x g (14,000 rpm). Discard the flow-through and collection tube.

7. Transfer the spin column to a new 1.5 ml microcentrifuge tube.

8. Elute the DNA by adding 200 μl Buffer AE to the center of the spin column membrane.

Incubate for 1 minute at room temperature (15-25°C). Centrifuge for 1 minute at ≥ 6000 x g.

9. Determine DNA concentration of each extraction using a spectrophotometer. Concentrations of ≈20 ng/μl are sufficient. 10. Store extracted DNA in freezer.

Appendix B: DNA Amplification and Genotyping

1. Dilute DNA extractions in a 1/1 ratio with diH2O.

2. Load PCR plates with 1 μl of diluted DNA, centrifuge, and store in freezer.

3. Create master mixes for coral (166-vic, 192-6fam & 181-ned; 207-pet & 182-6fam) and zooxanthellae (ZA3_18, ZA3_28, ZA3_31, ZA3_27, ZA3_41, ZA3_32, ZA3_9, ZA3_7,

ZA3_48, ZA3_1, ZA3_3, ZA3_08, ZA3_02) plexus. See Figure 1 for plexus recipes.

4. Load master mixes for each plexus onto individual, DNA loaded PCR plates.

5. Run the plexus/DNA loaded PCR plate in its respective PCR cycle in a thermocycler. See

Figure 1 for PCR cycles.

6. Run 3 μl of each sample from one column of each finished PCR plate under electrophoresis on a gel comprised of 1.5 g agarose, 75 ml of TAE buffer, and 3.75 μl GelStar. Mix DNA with 1 μl of Hyperladder IV dye. Run on gel for 35 minutes at 100 volts. Store PCR plate in freezer.

7. Upon completion of electrophoresis expose gel under trans-UV for imaging. If image shows

PCR success, proceed to step 8. Otherwise, rerun PCR of the plexus.

8. Load finished PCR products confirmed successful into gene-sequencing plates. Co-load the following plexus plates: zoox 18, zoox 1, zoox 28, zoox 27; zoox 41, zoox 31, zoox 32; zoox 9, zoox 7, zoox 3; ZA3_48, ZA3_08, ZA3_02. Load the remaining plexus plates individually.

Submit gene-sequencing plates for analysis.

9. Call alleles using electropherograms in the program GeneMapper, and then export the alleles to database.