Fine‐scale structure among mesophotic populations of the great star coral Montastraea cavernosa revealed by SNP genotyping

Abstract Mesophotic reefs (30‐150 m) have been proposed as potential refugia that facilitate the recovery of degraded shallow reefs following acute disturbances such as coral bleaching and disease. However, because of the technical difficulty of collecting samples, the connectivity of adjacent mesophotic reefs is relatively unknown compared with shallower counterparts. We used genotyping by sequencing to assess fine‐scale genetic structure of Montastraea cavernosa at two sites at Pulley Ridge, a mesophotic coral reef ecosystem in the Gulf of Mexico, and downstream sites along the Florida Reef Tract. We found differentiation between reefs at Pulley Ridge (~68 m) and corals at downstream upper mesophotic depths in the Dry Tortugas (28–36 m) and shallow reefs in the northern Florida Keys (Key Biscayne, ~5 m). The spatial endpoints of our study were distinct, with the Dry Tortugas as a genetic intermediate. Most striking were differences in population structure among northern and southern sites at Pulley Ridge that were separated by just 12km. Unique patterns of clonality and outlier loci allele frequency support these sites as different populations and suggest that the long‐distance horizontal connectivity typical of shallow‐water corals may not be typical for mesophotic systems in Florida and the Gulf of Mexico. We hypothesize that this may be due to the spawning of buoyant gametes, which commits propagules to the surface, resulting in greater dispersal and lower connectivity than typically found between nearby shallow sites. Differences in population structure over small spatial scales suggest that demographic constraints and/or environmental disturbances may be more variable in space and time on mesophotic reefs compared with their shallow‐water counterparts.


| INTRODUC TI ON
Coral reefs are declining worldwide and face a future of increasingly frequent and severe stress. Overfishing, pollution, and changing climate are major drivers of coral decline (Hughes et al., 2017), but their impacts are not spatially homogenous (Van Hooidonk, Maynard, & Planes, 2013). Ecosystems that escape these and other stressors due to depth or remoteness may make an important contribution to the resilience and long-term persistence of reefs (Gilmour, Smith, Heyward, Baird, & Pratchett, 2013).
Mesophotic reefs may contribute to the recovery and persistence of their shallower counterparts primarily through larval subsidy over single or multiple generations (Holstein, Paris, Vaz, & Smith, 2016).
Connectivity can be explored by using genetic markers to infer population structure, which serves as an indicator of the potential for migration across depths. Genetic data tightly correlate with biophysical migration rates in some coral populations (Matz, Treml, Aglyamova, & Bay, 2018), but has little explanatory power in others (Drury, Paris, Kourafalou, & Lirman, 2018), a difference which may be driven by taxonomic and demographic factors (e.g., asexual reproduction, population bottlenecks). Previous work using genetic data to infer connectivity and "reseeding potential" has shown that variable levels of horizontal connectivity between distant shallow reefs and vertical connectivity between reefs along a depth gradient can structure coral populations (Bongaerts et al., 2017;Eckert, Studivan, & Voss, 2019;Hammerman et al., 2017;Serrano et al., 2014Serrano et al., , 2016Studivan & Voss, 2018). The use of next-generation sequencing data to infer migration has also resolved species-specific vertical connectivity within Caribbean coral communities (Bongaerts et al., 2017;Hammerman et al., 2017), suggesting that the reseeding potential of mesophotic reefs may be important for some species assemblages, but is not universal.
Montastraea cavernosa, the Caribbean great star coral, is a suitable candidate for examining these patterns because it is capable of extensive vertical and horizontal connectivity (Nunes, Norris, & Knowlton, 2011). It is a gonochoric broadcast spawning coral which produces large eggs that may allow for longer larval duration (Szmant, 1991) and is also an extreme depth generalist, found between 0.5 and at least 95 m (Goreau & Wells, 1967). Previous work has demonstrated high horizontal connectivity between populations throughout the Caribbean, but these populations are not connected to reefs in West Africa or Brazil (Goodbody-Gringley, Woollacott, & Giribet, 2012;Nunes, Norris, & Knowlton, 2009;Nunes et al., 2011).
Vertical connectivity in this species appears to be site-specific, resolved in some regions of Florida, the Gulf of Mexico, and the Cayman islands, but not in Bermuda, the U.S. Virgin Islands, Belize, or the Bahamas (Brazeau, Lesser, & Slattery, 2013;Eckert et al., 2019;Serrano et al., 2014;Studivan & Voss, 2018).
We take advantage of these life-history traits, using genotyping by sequencing to examine population structure and connectivity in M. cavernosa samples from near the endpoints of the Florida Reef Tract (FRT) and Pulley Ridge (Figure 1). Pulley Ridge is a mesophotic reef in the Gulf of Mexico formed from drowned barrier islands on the West Florida Shelf at >60 m depth; it is considered the deepest known hermatypic reef in the United States (Jarrett et al., 2005 (Jarrett et al., 2005).
Extraction quality was verified on a 1% agarose gel before samples were quantified in triplicate (AccuBlue High-Sensitivity dsDNA Quantitation Kit), and 50 ng of each sample was dried down in a 96well plate and rehydrated in 5 µl of water.
Libraries were prepared as in Drury et al. (2017) using a modified protocol of Elshire et al. (2011) and MseI (New England Biolabs) for digestion. An initial digestion with ApeKI produced inconsistent banding in different populations, likely due to the inhibition of the restriction enzyme. Samples were stored in a single batch of preservative and extracted in a single batch; thus, it is unlikely that preservation or extraction led to this population-specific result. To resolve this, we instead used MseI to digest each library, producing consistent restriction fragment banding patterns across populations.
A 7-9bp 5' barcode unique to each sample and a common adapter were ligated to each library, and postligation product was pooled and bead-purified using 0.65x/1.0x double-sided size selection to select fragments in the 100-250 bp range. Pooled PCR samples were amplified for 18 cycles using primers complimentary to the oligonucleotides used in Illumina sequencing (Elshire et al., 2011), with an additional random 3bp overhang. The overhang decreases the proportion of low-depth and singleton SNPs in the resulting reduced representation library, increasing the efficiency of downstream processing and facilitating analysis using the more frequent cutting restriction enzyme. PCR products were bead-purified and visualized on a 1% agarose gel with a 100 bp DNA ladder to confirm size selection. Libraries were sequenced on an Illumina HiSeq 2500 with 75-bp single-end reads (Elim Biopharmaceuticals Inc.).
We created a combined Symbiodiniaceae reference following Manzello et al. (2019)  Note: Collection locations by region, number of samples, sites, and depths. After initial clustering analysis, all 3 Dry Tortugas sites were assumed to be a single population and 2 Pulley Ridge sites were split by collection site, subsequently called "Pulley Ridge North" and "Pulley Ridge South." Key Biscayne and Dry Tortugas samples are considered "Florida Reef Tract (FRT)" samples. (Bayer et al., 2012), Breviolum (http://sites.bu.edu/davie slab/data-code/), Cladocopium (Davies, Marchetti, Ries, & Castillo, 2016), and Durusdinium (Ladner, Barshis, & Palumbi, 2012). We aligned raw reads using bowtie2.3.5 with local settings, filtered to primary alignments with mapQ > 20, and quantified read count for each genus with SAMtools 1.9. This strategy is mutually exclusive between genera, meaning that a primary alignment to the Cladocopium portion of the combined reference indicates a read was a better fit than for any other genus. Approximately 0.25% of reads mapped to the Symbiodiniaceae references.

| Analysis
The SNP matrix was filtered to include samples with at least 70% of loci called and loci in at least 92% of samples. To select only neutral markers, loci in LD (R 2 > 0.2) and loci not in HWE (p < .01) were identified with VCFtools 0.1.13 (Danecek et al., 2011) and removed. We To examine structure, we used discriminant analysis in the adegenet package (Jombart, 2008) in R to cluster and visualize data using 21 principal components (~n/3) with populations determined by site as in Table 1  with the optimal K defined by the minimum cross-validation error term (Alexander, Novembre, & Lange, 2009). There were small differences between K = 2 and K = 3, so we included all population numbers for comparison ( Figure 4). We used the R package vegan (Oksanen et al., 2010) to test isolation by distance (9,999 permutations for significance) using the identity-by-state matrix described

| Population structure and allelic patterns
Hierarchical clustering of genetic distances based on identity by state  Biscayne is assigned to a distinct additional ancestral group. The separation of Dry Tortugas assignments did not relate to the three "sites" that were pooled based on initial DAPC analysis ( Figure 3a)

| Outlier loci
The

| Symbiosis
Every sample in the dataset had reads that aligned to all four genera of Symbiodiniaceae ( Figure S1). The proportion of reads aligning to Cladocopium was slightly higher in Pulley Ridge corals, which also had fewer reads aligning to Symbiodinium. Low proportions of reads Nucleotide diversity = intrapopulation genetic diversity (Nei & Li, 1979).
The number of samples removed after clonality analysis is N-Ng. All allele frequency calculations were made after clonal replicates were removed.
Abbreviations: H E , average expected heterozygosity; H O , average observed heterozygosity; N, sample size; NA, allelic richness, average number of alleles; Ng, number of genets; Ng/N, genet-toramet ratio; polymorphic sites, percentage of sites with multiple alleles within a population.

F I G U R E 2 Hierarchical Clustering of Samples based on Identity by State (IBS). Hierarchical clustering of identity-by-state pairwise values
for all samples. IBS was calculated from SNP matrix using SNPrelate and used to create a tree using the "complete" method. Samples are color-coded by population as in Figure 1. Dashed horizontal line represents the chosen cutoff for clonality, and nodes below this value with samples from the same site were designated as nodes and are visualized with leaf color. All but one sample were randomly removed from each clonal grouping for downstream analysis  In this case, admixture analysis could be a more comprehensive assessment of structure (i.e., not focused on heterozygosity-based metrics or maximizing discriminant functions); however, the interaction of demography and small sample sizes precludes us from rarefying our results and makes it difficult to determine whether this and other metrics are biased, even after clonal replicates were removed.

| D ISCUSS I ON
Bayesian methods such as STRUCTURE and ADMIXTURE are generally restricted to larger sample sizes when using microsatellites (Porras-Hurtado et al., 2013), but because SNPs are biallelic, as few as four samples are required to obtain accurate allele frequencies (Shi et al., 2010). Conversely, outlier loci suggest that Pulley Ridge is discrete from both FRT sites and there is some differentiation be- sexually (Szmant, 1991), but more recent work has documented common asexual reproduction in massive corals (Manzello et al., 2019), likely due to physical disturbance (Foster et al., 2013). There is some evidence for potential internal fertilization in M. cavernosa (Hagman, Gittings, & Vize, 1998), but as a gonochoric species there is no clear mechanism for creating genetically identical colonies other than parthenogenesis (Combosch & Vollmer, 2013), which is unknown in M.
cavernosa but could be important for reproductive assurance in an isolated population (Yund, 2000). Montastraea cavernosa exhibits a massive or plating morphology (Lesser et al., 2010), and while the physical impacts of hurricanes are less significant at depth , evidence of storm damage has been documented in plating corals > 50m depth (Bongaerts, Muir, Englebert, Bridge, & Hoegh-Guldberg, 2013;Woodley et al., 1981). These impacts suggest that asexual reproduction due to physical fragmentation is possible, but we expect these populations to be mostly nonclonal based on the general rarity of clones as assessed by microsatellites ( (Studivan & Voss, 2018), and this study supports earlier suggestions that horizontal connectivity among mesophotic sites may be more complex and variable than connectivity among shallow sites (Serrano et al., 2014). We hypothesize this is a consequence of the life-history strategy of this species, in which sperm and eggs are synchronously released from male and female colonies and buoyant fertilized eggs (Wyers, Barnes, & Smith, 1991) spend their early development at the surface (Hagman, Gittings, & Deslarzes, 1998). These larvae may be subject to longer time to the surface, contributing to horizontal dispersal prior to the onset of larval motility and settlement at depth. If this is the case, the commitment of these propagules to spending early ontogeny in shallow depths could result in greater population differentiation over smaller spatial scales compared with their shallow counterparts. Biophysical simulations of this process in Orbicella faveolata in the USVI suggest that most propagules are transported to shallower settlement habitat, but a substantial number of propagules from the ~30-50 m depth also settle in that range (Holstein et al., 2016). However, the buoyancy of gametes varies between species and is a critical determinant of this pattern.
The Pulley Ridge ecosystem is approximately 60 km to the west of the Dry Tortugas (Jarrett et al., 2005), which is the westernmost extent of the FRT. This region may be more heavily influenced by oceanographic conditions including the Loop Current, which not only drives circulation in the eastern Gulf of Mexico (Hurlburt & Thompson, 1980;Kourafalou & Kang, 2012), but also produces eddies which stochastically influence larval connectivity in the Florida Keys (Sponaugle, Lee, Kourafalou, & Pinkard, 2005) and exacerbate the highly variable nature of larval connectivity (Hedgecock & Pudovkin, 2011). Biophysical modeling of coral reef fish larvae shows that transport can occur between Pulley Ridge and the Dry Tortugas in as little as 7 days , which is likely within the larval competency period of M. cavernosa (Nunes et al., 2011).
We found equivocal evidence (disagreement between ADMIXTURE and F ST outcomes) that this process is actually occurring in M. cavernosa. Results from simulated migration patterns also indicate common horizontal asymmetry, with more frequent "migration" in the downstream (east and north) direction of the Florida Current except in the Lower Keys, where westward transport is also common due to the complexity of oceanographic patterns in this area (Kourafalou & Kang, 2012;Staaterman, Paris, & Helgers, 2012 (Goulet et al., 2019). The consistent diversity of this community across depths also contrasts with previous results that suggest depth-generalist species that harbor multiple symbiont genera exhibit depth-related partitioning of relative frequencies (reviewed in Ref. (Kahng et al., 2019)). Our analysis cannot precisely determine relative abundance because of variation in reference size (47-99 Mbp), but this study demonstrates the utility of host-focused sequencing data for broad descriptions of symbiosis that may capture more variation than other methods (e.g., DGGE, targeted qPCR).
Vertical and horizontal connectivity may be important for reef recovery, but the contributions of mesophotic ecosystems to shallow reef populations appear to be highly site and species-specific (Bongaerts et al., 2017). Here, we report considerable population structure within adjacent mesophotic systems and across the Florida Reef Tract, highly clonality at Pulley Ridge, and the presence of diverse symbiont assemblages in both shallow and mesophotic M. cavernosa. We demonstrate that the endpoints of the Florida Reef Tract harbor M. cavernosa communities which are genetically distinct, potentially due to long horizontal distances and differences in habitat or environment between shallow and mesophotic reefs. In addition, we find that Pulley Ridge coral communities are isolated and low diversity, with potential selective pressure not found at nearby mesophotic reefs. These trends could be reflective of local adaptation to unique circumstances that are uncommon on other mesophotic reefs, or may reflect a recent mortality or bottleneck event. The role of clonality is also unclear, but suggests the importance of founder effects in these isolated, mesophotic systems. Future work should continue to focus on paired populations and upstream communities which may contribute to this area via larval seeding, better explaining the potential contributions of mesophotic reefs to contemporary reef resilience.
for Sponsored Coastal Ocean Research award NA11NOS4780045 to the University of Miami Cooperative Institute for Marine and Atmospheric Sciences. We are grateful for the helpful suggestions of four anonymous reviewers.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available