
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

39229226
10.1101/2024.08.21.608299
preprint
1
Article
Genomes of the Caribbean reef-building corals Colpophyllia natans, Dendrogyra cylindrus, and Siderastrea siderea
http://orcid.org/0000-0001-7850-5015
Locatelli Nicolas S 1
http://orcid.org/0000-0001-6463-7308
Baums Iliana B 1234*
1 Department of Biology, The Pennsylvania State University, University Park, PA, USA
2 Helmholtz Institute for Functional Marine Biodiversity at the University of Oldenburg (HIFMB), Ammerländer, Heerstraße 231, 26129 Oldenburg, Germany
3 Alfred Wegener Institute, Helmholtz-Centre for Polar and Marine Research (AWI), Am Handelshafen Bremerhaven, Germany
4 Institute for Chemistry and Biology of the Marine Environment (ICBM), School of Mathematics and Science, Carl von Ossietzky Universität Oldenburg, Ammerländer Heerstraße 114-118, 26129 Oldenburg, Germany
Author addresses: Nicolas Locatelli, 208 Mueller Laboratory, Department of Biology, University Park, PA, 16802 locatelli@psu.edu

* Corresponding author: Iliana Baums, Marine Conservation, Helmholtz Institute for Functional Marine Biodiversity, Ammerländer Heerstraße 231, 26129 Oldenburg, Germany, Iliana.baums@hifmb.de
22 8 2024
2024.08.21.608299https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
nihpp-2024.08.21.608299.pdf
Corals populations worldwide are declining rapidly due to elevated ocean temperatures and other human impacts. The Caribbean harbors a high number of threatened, endangered, and critically endangered coral species compared to reefs of the larger Indo-Pacific. The reef corals of the Caribbean are also long diverged from their Pacific counterparts and may have evolved different survival strategies. Most genomic resources have been developed for Pacific coral species which may impede our ability to study the changes in genetic composition of Caribbean reef communities in response to global change. To help fill the gap in genomic resources, we used PacBio HiFi sequencing to generate the first genome assemblies for three Caribbean, reef-building corals, Colpophyllia natans, Dendrogyra cylindrus, and Siderastrea siderea. We also explore the genomic novelties that shape scleractinian genomes. Notably, we find abundant gene duplications of all classes (e.g., tandem and segmental), especially in S. siderea. This species has one of the largest genomes of any scleractinian coral (822Mb) which seems to be driven by repetitive content and gene family expansion and diversification. As the genome size of S. siderea was double the size expected of stony corals, we also evaluated the possibility of an ancient whole genome duplication using Ks tests and found no evidence of such an event in the species. By presenting these genome assemblies, we hope to develop a better understanding of coral evolution as a whole and to enable researchers to further investigate the population genetics and diversity of these three species.

genome
coral
reef
gene family expansion
duplication
orthogroups
Siderastrea sidereal
Dendrogyra cylindrus
Colpophyllia natans
This research was funded by the Revive and Restore Advanced Coral Toolkit Program funding to IBB. NSL was supported by CBIOS (NIH T32 Kirschstein-NRSA: Computation, Bioinformatics, and Statistics) training program at The Pennsylvania State University (#T32GM102057). The findings and conclusions do not necessarily reflect the view of the funding agencies.
==== Body
pmcIntroduction

Genomic resources are increasingly available for Pacific reef-building corals (e.g. Fuller et al. 2020; Stephens et al. 2022), yet most Caribbean coral species still lack them despite genetic management of populations becoming necessary (Baums et al. 2022). Caribbean reefs represent ecosystems long diverged from Pacific counterparts. During the mid-Miocene, the Mediterranean closed off at both ends and the eastern connection of the Caribbean with the Indo-Pacific basin was severed (Wallace and Rosen 2006). The Isthmus of Panama to the west of the Caribbean remained open until roughly 3 million years ago, after which ocean circulation drastically changed and Caribbean reefs were isolated from Pacific reefs (Burton et al. 1997; O’Dea et al. 2016).

Cnidarians diverged early in metazoan evolution roughly 700 Mya (Park et al. 2012) and the three species discussed here represent the two major scleractinian lineages, complex (Siderastrea siderea) and robust (Colpophyllia natans and Dendrogyra cylindrus). Dendrogyra cylindrus is a rare Caribbean coral (Hunter and Jones 1996) that has declined sharply in the past two decades due anthropogenic stressors and a highly infectious disease called stony coral tissue loss disease (SCTLD, Brandt et al. 2021). Dendrogyra cylindrus is extinct in the wild in Florida and is considered critically endangered (Neely et al. 2021; Cavada-Blanco et al. 2022). Siderastrea siderea and C. natans were common reef-building corals that have also experienced significant declines in response to disease and anthropogenic impacts. Siderastrea siderea is now listed as critically endangered (Rodriguez-Martinez et al. 2022) and under threat due to acidification, ocean warming (Horvath et al. 2016), and SCTLD (Brandt et al. 2021). Colpophyllia natans is also in decline due to SCTLD (Vermeij and Goergen 2022; Williamson et al. 2022). Despite their ecological and evolutionary importance, genomic resources are not yet available for these species.

Coral genomes are variable in size (e.g., Stephens et al. 2022), but have highly conserved gene order (Ying et al. 2018; Locatelli et al. 2023). Anthozoan genomes contain between 13.57% and 52.2% repetitive content (e.g., Shinzato et al. 2011; Bongaerts et al. 2021) and contain DNA and retrotransposons that are still active (Chapman et al. 2010; Huang et al. 2012), which can result in gene duplication and movement of genes to disparate regions of the genome. Accumulation of somatic mutations in long-lived coral colonies represents another mechanism by which coral genomes gain heterozygosity (Devlin-Durante et al. 2016; López and Palumbi 2020) and some of these mutations can be passed on to their sexually produced offspring (Vasquez Kuntz et al. 2022). Development of genomic resources allows for further study of these complex evolutionary mechanisms in metazoans as a whole (Reusch et al. 2021).

To help bridge the gap in genomic resources for Caribbean corals, we present novel PacBio HiFi-derived assemblies for Colpophyllia natans, Dendrogyra cylindrus, and Siderastrea siderea. With these references, we hope to foster an understanding of how corals will respond to environmental change (Bove et al. 2022) and population decline (Cramer et al. 2020), and how the response of Caribbean corals may differ from Indo-Pacific species.

Methods

Tissue sampling

Tissue of Colpophyllia natans ([12.1095, −68.95497], database ID 22254) was collected from the Water Factory reef in Curaçao on August 6th, 2022 using a hammer and chisel. Dendrogyra cylindrus ([12.0837, −68.89447], database ID 22255) and Siderastrea siderea ([12.0839, −68.8944], database ID 22256) were collected from the Sea Aquarium reef in Curacao on August 12th and 13th, 2022 using hammer and chisel. All collections were made under Curaçao Governmental Permit 2012/48584. All fragments were ca. 12cm2 in size and were kept alive in coolers filled with seawater during transit prior to being preserved in DNA/RNA Shield (Zymo Research, CA, USA). Samples were stored at −20°C or at −80°C until extraction.

Nucleic acid extraction and sequencing

For all species, DNA was extracted from tissue preserved in DNA/RNA Shield (Zymo Research, CA, USA) using the Qiagen (MD, USA) MagAttract HMW DNA kit, following manufacturer protocols. Following initial extraction, DNA was further purified using a 0.9X AMPure XP (Beckman Coulter, CA, USA) bead cleanup. Purified DNA was then size selected using a Pacific Biosciences (formerly Circulomics) SRE size selection kit. The SRE standard kit selects for DNA predominantly >25kb and a near total depletion of fragments <10kb. Barcoded templates were generated and sequenced by the Huck Institutes of the Life Sciences Genomics Core Facility at Penn State University using a Pacific Biosciences (Menlo Park, CA, USA) Sequel IIe across a total of three SMRTcells (further described below).

As RNAseq data was not available for Dendrogyra cylindrus or any close relatives for the purposes of gene prediction, RNA was extracted from the same DNA/RNA Shield (Zymo Research, CA, USA) preserved samples as described above using a TriZol and a Qiagen (MD, USA) RNeasy Mini Kit (as in https://openwetware.org/wiki/Haynes:TRIzol_RNeasy). Compared with the RNA sequence data obtained from NCBI SRA for C. natans and S. siderea (described below in “Gene prediction and functional annotation”), the RNA sample for D. cylindrus was of an untreated colony growing in the wild rather than experimental samples exposed to heat and disease-stress. From the extracted total RNA, libraries were prepared and sequenced by the Oklahoma Medical Research Foundation Clinical Genomics Center using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs Inc., MA, USA), Swift Rapid RNA Library Kit (Swift Biosciences, MI, USA), and 150M read pairs of 2×150bp chemistry on an Illumina (San Diego, CA, USA) NovaSeq 6000 machine.

Genome assembly

A PacBio library was generated by pooling the barcoded templates for each of the three species in equal proportions and was initially sequenced on two SMRTcells. Prior to genome assembly, k-mer (31-mer) counting was performed on PacBio HiFi data for each species using Jellyfish v2.2.10 (Marçais and Kingsford 2011) for the purpose of haploid genome size estimation. Genome size was estimated from 31-mer histograms using GenomeScope2 (Ranallo-Benavidez et al. 2020). With the data from these two initial SMRTcells, a preliminary assembly was performed using hifiasm_meta v0.2 (Feng et al. 2022) to assess assembly size and to determine whether the pool balance needed to be adjusted for the third and final SMRTcell run.

Because the preliminary assembly and genome size estimate from GenomeScope2 of S. siderea was larger than the remaining two species, the final SMRTcell was run with a pool balance of 25:25:50 Colpophyllia:Dendrogyra:Siderastrea to provide additional coverage on the larger Siderastrea genome. Prior to all stages of data delivery, the sequencing facility used PacBio lima to demultiplex and remove adapters and unbarcoded sequences. Across all SMRTcells, total sequence yield was 26Gb across 2.8M reads in Colpophyllia natans, 25Gb across 2.7M reads in Dendrogyra cylindrus, and 32Gb across 3.4M reads in Siderastrea siderea. Further breakdown of PacBio yield and read lengths per species per sequencing run can be found in Table S1. Utilizing all data, a new set of primary assemblies was generated using hifiasm_meta.

Assembly decontamination, haplotig purging, and repeat annotation

HiFi reads were then mapped to the assembly using minimap2 v2.24 (Li 2018) and BAM files were sorted using samtools v0.1.19 (Danecek et al. 2021). Using blastn v2.14.0 (Camacho et al. 2009), assemblies were searched against a custom database comprised of NCBI’s ref_euk_rep_genomes, ref_prok_rep_genomes, ref_viroids_rep_genomes, and ref_viruses_rep_genomes databases combined with dinoflagellate and Chlorella genomes (Shoguchi et al. 2013, 2018, 2021; Hamada et al. 2018; Beedessee et al. 2020). All NCBI RefSeq databases were downloaded on March 28th, 2023. Using the mapping and blastn hits files, blobtools v1.1.1 (Laetsch and Blaxter 2017) was used to identify and isolate non-cnidarian contigs. To better identify symbionts within the metagenome assemblies, blastn (Camacho et al. 2009) was used to query putative Symbiodiniaceae contigs against a curated nuclear ribosomal Internal Transcribed Spacer-2 (ITS2) database (Hume et al. 2019). With all non-cnidarian contigs excluded, a repeat database was modeled using RepeatModeler2 v2.0.2a (Flynn et al. 2020). Purge_dups v1.2.6 (Guan et al. 2020) was utilized to identify and remove any remaining putative haplotigs in the respective assemblies. Following haplotig purging, repeats were soft-masked using a filtered repeat library in RepeatMasker4 v4.1.2.p1 (Smit et al.), following recommendations from the Blaxter Lab (https://blaxter-lab-documentation.readthedocs.io/en/latest/filter-repeatmodeler-library.html). Protein references from Orbicella faveolata (Prada et al. 2016) and Fungia sp. (Ying et al. 2018) were used to filter repeat libraries for the two robust species (C. natans and D. cylindrus). Protein references from Acropora millepora (Fuller et al. 2020), Montipora capitata (Stephens et al. 2022), and Galaxea fascicularis (Ying et al. 2018) were used to filter repeat libraries for S. siderea.

Gene prediction and functional annotation

Prior to gene prediction, the hifiasm_meta assemblies were scanned for mitochondrial contamination using MitoFinder v1.4.1 (Allio et al. 2020) and contigs of mitochondrial origin were removed from the assemblies. Nuclear assemblies were annotated using RNAseq data in funannotate v1.8.13 (Palmer and Stajich 2020). Colpophyllia natans and Siderastrea siderea were annotated using all RNAseq data available on NCBI SRA for the respective species at the time of assembly (see Table S2). As no RNAseq data is publicly available for Dendrogyra cylindrus or its close relatives, RNA was extracted as previously described and included within the funannotate annotation process. All RNAseq data was adapter- and quality-trimmed using TrimGalore v0.6.7 (Krueger et al. 2021).

Briefly, funannotate train was run for all assemblies with a --max_intronlen of 100000. Funannotate train is a wrapper that utilizes Trinity (Grabherr et al. 2011) and PASA (Haas et al. 2008) for transcript assembly. Upon completion of training, funannotate predict was run to generate initial gene predictions using the arguments --repeats2evm, --organism other, and --max_intronlen 100000. Funannotate predict is a wrapper that runs AUGUSTUS (Stanke et al. 2006) and GeneMark (Brůna et al. 2020) for gene prediction and EvidenceModeler (Haas et al. 2008) to combine gene models. Funannotate update was run to update annotations to be in compliance with NCBI formatting. For problematic gene models, funannotate fix was run to drop problematic IDs from the annotations. Finally, functional annotation was performed using funannotate annotate which annotates proteins using PFAM (Bateman et al. 2004), InterPro (Hunter et al. 2009), EggNog (Huerta-Cepas et al. 2019), UniProtKB (Boutet et al. 2016), MEROPS (Rawlings et al. 2009), CAZyme (Huang et al. 2018), and GO (Harris et al. 2004). For all genes not functionally annotated with gene ontology (GO) terms by funannotate, a single network of ProteInfer (Sanderson et al. 2023) was used to infer functional attributes of genes using pre-trained models.

Mitochondrial genome assembly

To assemble mitochondrial genomes for each samples, MitoHiFi v2.2 (Gabriel et al. 2023) was used on all available HiFi data for each species. For Siderastrea siderea, Colpophyllia natans, and Dendrogyra cylindrus, accessions NC_008167.1, NC_008162.1, and DQ643832.1 (whole mitogenomes for Siderastrea radians, Colpophyllia natans, and Astrangia poculata), were used as seed sequences for mitochondrial assembly, respectively. For all assemblies, the arguments -a animal and -o 5 were used to indicate that the organism type was an animal and the organism genetic code was invertebrate.

Duplication and orthogroup analysis

To assess the origin of gene duplications, whole genome duplication pipeline and orthogroup analyses were used. The wgd pipeline v1.1 (Zwaenepoel and Van De Peer 2019) was used to investigate duplication and divergence at the whole paranome and anchor-pair levels. The longest, coding CDS transcript of each gene was used as input for wgd. The wgd pipeline acts as a wrapper for a number of programs, and in the case of the analysis here the following programs were run through wgd: blastp (Altschul et al. 1997), MCL (Markov Cluster Process, Hazewinkel and Van Eijck 2000), PAML (Yang 2007), MAFFT (Katoh and Standley 2013), FastTree (Price et al. 2010), and i-ADHoRe 3.0 (Proost et al. 2012). In addition to wgd, OrthoFinder v2.5.4 (Emms and Kelly 2019) was run to discover orthologous groups unique to each species and shared between species. For OrthoFinder analyses, the longest peptide isoform for each gene was used as input. A full list of taxa included in OrthoFinder and doubletrouble analyses (described below) can be found in Table S3.

CAFE5 v5.1.0 (Mendes et al. 2021) was used to discover hierarchical orthogroups from OrthoFinder undergoing phylogenetically significant gene family expansions or contractions. To begin, r8s v1.81 (Sanderson 2003) was used to time-calibrate the phylogeny from OrthoFinder using fossil priors obtained from the PaleoBioDB fossil record (Peters and McClennen 2016). Priors for Acropora palmata (5.3Mya, Budd et al. 1999), Porites compressa (2.588Mya, Faichney et al. 2011), Acropora (59Mya, Vecsei and Moussavian 1997), Faviina (247Mya, Qi 1984), and Scleractinia (268Mya, Gregorio 1930), were used as calibration points. With significantly expanding or contracting hierarchical orthogroups identified by CAFE5, GO terms for expanding and contracting gene families were extracted and compared to the whole genome background to test for enrichment. Enrichment analyses were performed using GOAtools (Klopfenstein et al. 2018). To reduce false discovery, only terms with a Benjamini-Hochberg adjusted p-value < 0.05, depth > 2, and terms present in 5 or more study orthogroups were preserved.

To classify stony coral (Scleractinia) paralogs into duplication types, doubletrouble v1.3.6 (Almeida-Silva and Peer 2024) was run using the longest peptide isoform for each gene and default arguments. Briefly, doubletrouble classifies genes into segmental (SD), tandem (TD), proximal (PD), transposon-derived (TRD), and dispersed duplications (DD) based on collinearity, intron content, and phylogenetic position of paralogs. For instance, duplications are classified as tandem if two paralogs are separated by fewer than ten genes. If the distance between genes is >10, paralogs are classified as proximal duplications. Dispersed duplications (DD) are considered any duplication that is not otherwise classifiable into more specific categories. For all doubletrouble analyses, Amplexidiscus fenestrafer (Wang et al. 2017), a member of the naked corals, Corallimorpharia, was used as an outgroup. Not all gene annotations were compatible with the “full” scheme, where transposon-derived duplications are further classified into retrotransposon-derived (rTRD) and DNA transposon-derived (dTRD). As such, the “full” scheme was only utilized for the focal study species here, Colpophyllia natans, Dendrogyra cylindrus, and Siderastrea siderea. All other species were run using the “extended” scheme.

Results and Discussion

Assembly contiguity, completeness, and heterozygosity

All assemblies exhibit high contiguity (Table 1) and are gap-free. The S. siderea genome is roughly two times larger than observed in other corals species, with an assembly size of 822M, compared with 526Mb and 399Mb for D. cylindrus and C. natans, respectively. The assembly size of S. siderea is larger than most publicly available coral genome assemblies – only two species have larger assemblies, Pachyseris speciosa (Bongaerts et al. 2021) and Platygyra sinensis (Pootakham et al. 2021). However, the Platygyra sinensis assembly likely contains considerable haplotig duplication, leaving only Pachyseris speciosa as a comparable assembly. In addition to being the largest of the three assemblies presented here, the S. siderea assembly is the most contiguous assembly (N50=9.1Mb), likely due to the larger read N50 of SMRTcell 3 (see Table S1). The genomes of C. natans and D. cylindrus have N50s of 4.647Mb and 4.902Mb, respectively. Further scaffolding with Hi-C data could help elevate these three references to chromosome-level. Genome-wide GC content is similar across all three species, with 39.81% for S. siderea, 38.87% for C. natans, and 39.29% for D. cylindrus. GC estimates are similar to other published stony coral genomes (e.g. Bongaerts et al. 2021).

K-mer duplicity plots from GenomeScope2 (Fig. 1) suggest that all species here are diploid in nature, unlike the recent findings in Hawaiian corals (Stephens et al. 2022). All three assemblies exhibited high completeness as determined by BUSCO Metazoa (Manni et al. 2021), with C. natans, D. cylindrus, and S. siderea showing 96.1%, 95.1%, and 96.2% completeness, respectively (Table 1). In terms of core BUSCO genes, S. siderea has the highest number of duplicated genes, with 2.1% of metazoan genes being duplicated. Additionally, all assemblies are similar to their GenomeScope2 k-mer-based size estimates (Fig. 1 and Table 1). Taken together, these results suggest that the majority of all three genomes were successfully captured in our assemblies with little remaining haplotig duplication.

Genome-wide heterozygosity in corals typically ranges from 1.07% to 1.96% (Shinzato et al. 2021; Yu et al. 2022; Stephens et al. 2022; Young et al. 2024). Genome-wide estimates of heterozygosity in GenomeScope2 suggest that Dendrogyra cylindrus has the lowest heterozygosity of the three species discussed here (0.799%) and among the lowest in any coral species for which genomic resources are available (Shinzato et al. 2021; Yu et al. 2022; Stephens et al. 2022; Young et al. 2024). Dendrogyra cylindrus is extinct in the wild in Florida and all remaining genets exist in land-based collections at the Florida Aquarium (Neely et al. 2021). The species has been rare throughout history (Hunter and Jones 1996; Modys et al. 2023) but with high local abundances in some locations (e.g. St. Thomas in the U.S. Virgin Islands). Recent catastrophic declines due to stony coral tissue loss disease (Neely et al. 2021; Alvarez-Filip et al. 2022) have led to the listing of the species as critically endangered by the Internation Union for Conservation of Nature (IUCN, Cavada-Blanco et al. 2022). In Florida, all genets are now in captivity and captive-based spawning efforts are burgeoning (Craggs et al. 2017; O’Neil et al. 2021) to recover the species. The very low heterozygosity estimate provided here highlights the need for carefully managed breeding (Marhaver et al. 2015) to ensure the persistence of the remaining standing genetic variation and adaptive potential of D. cylindrus (Barrett and Schluter 2008; Kardos et al. 2021). Of the three species, Siderastrea siderea has the highest genome-wide heterozygosity estimate of 1.59% and C. natans is intermediate with 0.862%. Colpophyllia natans also has low genome-wide heterozygosity compared to other coral species and may require genetic management in the future. However, these genome-wide heterozygosity estimates are generated from singular genets and may not accurately represent the heterozygosity of the wider populations of each species. Colpophyllia natans is the only species discussed here that does not have range-wide population genetic information available. As such, further genetic characterization of the species is clearly warranted due to population declines caused by infectious diseases (Alvarez-Filip et al. 2022) and the heterozygosity estimates provided here.

Repetitive content and transposable elements

The proportion of repeats assigned to each repeat category in RepeatMasker was similar across all three species assembled here (Table 2). Repeat content across all three species was very high, with S. siderea, D. cylindrus, and C. natans consisting of 47.80%, 40.40%, and 23.62% repetitive content, respectively. The majority of repeats were interspersed, with unclassified repeats being most abundant in all three species (31.91%, 25.57%, and 12.22%). In all species, the most abundant classifiable category was the Maverick DNA transposons, accounting for 5.20%, 6.74%, and 1.41% of the S. siderea, D. cylindrus, and C. natans genomes, respectively. Compared with other cnidarians, these assemblies contain similar levels of repetitive content to jellyfish species such as members of Clytia, Aurelia, and Chrysaora containing 39–49.5% (Gold et al. 2019; Leclère et al. 2019; Xia et al. 2020). Repetitive content in Colpophyllia natans was similar to the highly speciose genus Acropora (ranging from 13.57–19.62%, Shinzato et al. 2011; Cooke et al. 2020; Locatelli et al. 2023) which has similar genome sizes. Siderastrea siderea and D. cylindrus exhibit similar repetitive content to coral species with larger genome sizes (e.g., Bongaerts et al. 2021; Stephens et al. 2022; Kim et al. 2022; Young et al. 2024), suggesting that repeat expansion is also important in driving genome size disparities across evolutionary time in corals.

Gene prediction

S. siderea is unique amongst the assembled genomes not just for its size and contiguity, but also its gene content. Gene prediction in funannotate identified 61,712 gene models, roughly double the number of genes discovered for D. cylindrus and C. natans (39,739 and 34,139, respectively; Table 1), and compared to other publicly available coral genome assemblies (e.g., Prada et al. 2016; Fuller et al. 2020). Of these gene models, 52,473, 34,738, and 29,090 were predicted to be protein-coding for S. siderea, D. cylindrus, and C. natans, respectively. Dendrogyra cylindrus and C. natans fall within the expectations for stony corals in terms of protein-coding gene content. The gene content of S. siderea is higher than expected, only comparable to Montipora capitata amongst published genomes (Stephens et al. 2022). Of the protein-coding gene models, 1,515, 297, and 287 models in S. siderea, D. cylindrus, and C. natans contained >=90% repeat-masked bases, suggesting that these models may be derived from repetitive DNA and transposition-related events.

Because of the doubling in overall size and gene content present in the S. siderea, Ks tests were performed to test for an ancient whole genome duplication in the evolution of the species. Ks distributions in species having experienced whole genome duplication events exhibit characteristic distributions with a hump (as in Zwaenepoel and Van De Peer 2019), where many gene pairs are derived from a simultaneous duplication event and have all experienced a similar number of synonymous substitutions per synonymous site. Whole genome duplication analyses in wgd did not find Ks ratios indicative of ancient whole genome duplication in any of the species assembled here (Fig. S1), suggesting that other processes may be responsible for gain in genome size. Orthofinder analyses found 21,970 orthogroups in S. siderea, with 1,004 orthogroups private to the species (Fig. 2). An additional 17,286 genes could not be binned into orthogroups by Orthofinder, suggesting that gene duplication and subsequent diversification is prominent in the lineage. Siderastrea siderea harbors three distinct genetic lineages (Aichelman et al. 2024) of which only one was sequenced here. Additional genome assemblies of the other two lineages may shed light on the taxonomic status of these lineages and what role gene duplication and diversification may have played in their evolution.

Mitochondrial genomes

Mitochondrial genomes were successfully assembled for all three species discussed here using MitoHiFi (Gabriel et al. 2023). Both D. cylindrus and C. natans were of similar size with lengths of 17,299bp and 17,104bp, respectively. S. siderea is considerably larger, with a total length of 19,387bp (Fig. 1). The S. siderea mitogenome is among the largest of all stony coral (Scleractinia). Of all sequenced scleractinians, the mitogenome of S. siderea is exceeded in length only by the solitary coral species Polymyces wellsi (Flabellidae, NC_082103.1, 19,924bp), Deltocyathus magnificus (Deltocyathidae, OR625187.1, 19,736bp), and Rhombopsammia niphada (Micrabaciidae, MT706034.1, 19,654bp), and colony-forming species Pseudosiderastrea formosa and P. tayami (Siderastreidae, NC_026530.1 and NC_026531.1, 19,475bp). In terms of gene structure, all three mitochondrial genome assemblies consist of thirteen protein-coding genes and two ribosomal RNA (rRNA, rrnL and rrnS) genes with highly conserved gene order (ND5, ATP8, COX1, rrnL, ND1, CYTB, ND2, ND6, ATP6, ND4, rrnS, COX3, COX2, ND4L, and ND3). Both D. cylindrus and C. natans contain twelve transfer RNA (tRNA) genes while S. siderea contains eleven.

Gene family expansion and duplication

Gene ontology (GO) enrichment analyses of gene families undergoing phylogenetically significant expansion (as identified by OrthoFinder and CAFE5) may point to the importance of specific functional attributes in the evolution of each of the taxa assembled here (Fig. 3). In Siderastrea siderea, fertilization (GO:0009566) is the most enriched GO term in gene families that are significantly expanding (Fig. 3). In dioecious (gonochoric) plants, sex-specific selection has been documented (Yu et al. 2011; Barbot et al. 2023). Further, competition between pollen arriving on stigma has been documented as an evolutionary driver of female-biased sex ratios in dioecious plants as pollen containing male sex chromosomes were less competitive than those containing female sex chromosomes (Taylor et al. 1999; Stehlik and Barrett 2005; Delph 2019). Similar to this observation, S. siderea exhibits highly female-biased sex ratios in Florida (St. Gelais et al. 2016). Given the documented female-biased sex ratios and the enrichment of fertilization-related GO terms in S. siderea, competition between sperm may be driving the expansion of fertilization-related gene families in this species. However, any genetic basis for sex-determination in S. siderea has not yet been discovered and further work is required to explore this hypothesis.

The remainder of the top 10 enriched GO terms in S. siderea are important in the interactions of the coral host and its eukaryotic and prokaryotic symbionts. Coral hosts actively regulate the population size of their algal symbionts via indirect, nutritional control (Falkowski et al. 1993; Xiang et al. 2020; Cui et al. 2022) and may similarly regulate prokaryotic symbionts with secondary compounds (Rivera-Ortega and Thomé 2018; Vilas Bhagwat et al. 2023). The enrichment analysis (Fig. 3) points to additional direct control of other organisms via killing in S. siderea (enriched GO terms include GO:0031640; killing of cells of another organism, GO:0141061; disruption of cell in another organism, GO:0141060; disruption of anatomical structure in another organism, GO:0001906; cell killing, GO:0046649; lymphocyte activation, and GO:0001906; cytolysis). The specific organism target (i.e., eukaryotic vs. prokaryotic symbionts) of these killing genes is unknown and requires further investigation.

The GO term with the highest fold enrichment in Dendrogyra cylindrus is cell recognition (GO:0008037), followed by cell population proliferation (GO:0006325) and chromatin organization (GO:0006325). The themes of symbiont interaction, chromatin remodeling and stress response echoed in the remainder of the top 10 enriched terms (GO:0006955; immune response, GO:0071824; protein-DNA complex organization, GO:0006338; chromatin remodeling, GO:0044403; biological processes involved in symbiotic interaction, GO:0009607; response to biotic stimulus, GO:0080134; regulation of response to stress, and GO:0051707; response to other organism). Dendrogyra cylindrus is a long-lived species and even colonies in early development with no vertical pillar formation may be older than 30 years (Neely et al. 2021). This longevity may explain the enrichment in processes that enable plastic responses of these sessile organisms to changing environments.

Compared with other species in the analysis, gene families most expanded in Colpophyllia natans were functionally classified as related to DNA integration (GO:0015074), followed by biological processes involved in symbiotic interaction (GO:0044403), and regulation of cell population proliferation (GO:0042127). The remainder of the expanded gene families were also involved in processes of cell proliferation, stress response, and symbiont interactions (Fig. 3) as in S. siderea and D. cylindrus.

The functional enrichment analyses of the three scleractinian coral species assembled in this study were conducted relative to eighteen other symbiotic, reef-building corals and two outgroup Corallimorpharia species (Table S4). The mutual enrichment of symbiosis-related processes in all three focal species suggests that there are likely species-specific patterns of gene family expansion lumped into these broad functional categories. For instance, C. natans and D. cylindrus differ in their symbiont specificity. Colpophyllia natans hosts a diversity of symbiont species and strains (Bongaerts et al. 2015; Cunning et al. 2024). Conversely, D. cylindrus exhibits strong symbiont specificity, predominantly hosting a co-evolved symbiont, Breviolum dendrogyrum (Lewis et al. 2019b, 2019a). It is possible that these opposite life history strategies may have driven the mutual enrichment of the symbiosis-related GO term (GO:0044403; biological process involved in symbiotic interaction). Thus, more in-depth, gene family-specific analysis is warranted.

Subsequent analysis of paralogs using doubletrouble found that proximal duplications (locally duplicated with paralogs separated by ten or more genes) were the most prominent form of classifiable gene duplications in Siderastrea siderea (Fig. S2 and Table S4). Previous studies have suggested that tandem duplications drive Scleractinian (stony coral) evolution (Noel et al. 2023). Indeed, tandem duplications appeared to be more abundant in S. siderea in comparison with many of the evaluated taxa (Fig. S2 and Table S4). However, duplicate classification is inherently challenging as the order of genes can be the result of many different potential processes. For instance, tandem duplications can be broken apart by dispersed duplications being copied between tandem paralogs. These would resemble proximal duplications according to doubletrouble’s classification schema, despite being the result of two separate duplication processes. Additionally, analyses comparing species are somewhat reliant on similarly high-quality annotation and assembly across analyzed taxa. Several of the assemblies evaluated in our duplication analyses are of low contiguity and filled with short-read derived gaps, which could reduce the ability to detect certain forms of duplication. For example, Orbicella faveolata (Prada et al. 2016) contains no segmental duplications (Table S4), potentially because the detection of collinear, duplicated blocks of genes is less likely when the genome is highly fragmented. Further, it may not be possible to assign duplicates as transposon-derived (TRD) with assemblies derived from Nanopore or PacBio CLR data (e.g., Acropora cervicornis, Locatelli et al. 2023). Even polished long read assemblies may contain enough error in repetitive proteins such that a single copy of the gene cannot be assigned as ancestral – a requirement for paralogs to be classified as TRDs.

Despite the expansion of duplicated genes in Scleractinian species with larger genome sizes (e.g., Siderastrea siderea and Montipora capitata, Fig. S2), tandemly duplicated genes do not appear to have a disproportionate impact on genome size or gene content as suggested previously (Noel et al. 2023). When all duplicates are scaled to a value of 1 (Fig. S3), no singular duplication category appears to be most important in governing coral genome size. Instead, the proportion of paralogs assigned to each duplication type is similar across all species (an average of 22.0% tandem, 14.5% proximal, 2.3% segmental, 19.0% transposon-related, and 42.2% dispersed, Table S4). This suggests that all duplication types are expanding in synchrony to result in the genome size disparities we see across the phylogeny of Scleractinia. Further expansion of duplication analyses to include assemblies from upcoming efforts of large database projects (e.g., Reef Genomics, Liew et al. 2016; Aquatic Symbiosis Genomics Project, McKenna et al. 2021) could help elucidate more fine-scale, lineage-specific duplication processes that we have been unable to capture here.

Symbiont contigs

As metagenome assemblers were utilized in the assembly of the host species, symbiont data was also co-assembled and was of sufficient coverage to identify the prominent symbiont present to at least the genus-level. Both C. natans and D. cylindrus contained Breviolum, with D. cylindrus most likely containing B. dendrogyrum, as described in (Lewis et al. 2019a). However, the top ITS2 hits (determined by e-value, followed by percent identity) for both species do not closely match formally named strains/species in the curated ITS2 database (C. natans top symbiont hit B4, 89.89%, e-value 3.33e-24; D. cylindrus top hit B1, 97.98%, e-value 2.21e-42). It is possible that the symbionts contained in the genome assembly samples of C. natans and D. cylindrus are not yet represented in this database.

In the initial separation of host and symbiont contigs using BlobTools, the S. siderea genet assembled here was found to be associated with Cladocopium, but comparison of contigs with the ITS2 database did not reveal any more specific hits. The psbA region is a more reliable marker for symbiont strain identification than ITS2 (LaJeunesse and Thornhill 2011). However, symbiont reference sequences for psbA are not currently as extensive as ITS2 in strain coverage. As the ITS2 and psbA databases continue to grow, symbiont contigs assembled here could be identified with greater taxonomic resolution.

In addition to eukaryotic algal symbionts, one notable prokaryotic symbiont was recovered. Within the assembly for C. natans, a 2.13Mb contig was identified as most closely related to Prosthecochloris aestuarii. This bacterium has been proposed as a putatively symbiotic microbe living within coral skeletons (Cai et al. 2017; Chen et al. 2021). Coral metagenomes contain a wealth of symbionts with important functions for the holobiont (Bourne et al. 2009; Thompson et al. 2015; Boilard et al. 2020; Garrido et al. 2021). Further exploration of coral associated microbial communities may identify novel associations that are critical for the survival of the coral host.

Summary

Here, we generated novel genome assemblies for key Caribbean reef-building corals, all of which are listed as vulnerable or critically endangered by the IUCN. All genome assemblies are highly complete (>95% BUSCO Metazoa) and contiguous (N50 > 4.6Mb). The genomes of Dendrogyra cylindrus and Colpophyllia natans fall within nominal expectations of size and gene content based on other published coral genomes. Siderastrea siderea is roughly two times larger than expected with twice the number of predicted gene models, despite no evidence for a whole genome duplication event. Repeat and gene family expansions seem to be drivers of the larger S. siderea genome size. These results align with and expand upon previously published literature which implicated gene duplications as a driving factor of stony coral evolution (Noel et al. 2023). Given the importance of duplications in speciation across corals, further work should explore intraspecific structural polymorphisms (such as copy number variants, CNVs) to understand how structural variation plays a role in structure and adaptation at the population level.

These assemblies will help aid the broader research community by enabling high resolution genomic analyses that explore trait variation within species and potentially provide restoration practitioners with useful information to implement in restoration initiatives. As coral populations continue their decline, it is crucial that we develop a thorough understanding of the genomic processes that have driven coral evolution and have allowed them to overcome past extinction events and global stressors. These reference assemblies provide a key stepping stone towards this goal.

Supplementary Material

Supplement 1

Acknowledgements

The authors wish to thank Kelly Gomez-Campo and C. Cornelia Osborne for field assistance in collection of genome samples. The authors would also like to acknowledge the Huck Institutes’ Genomics Core Facility (RRID:SCR_023645) for use of the PacBio Sequel IIe sequencing platform.

Funder Information

This research was funded by the Revive and Restore Advanced Coral Toolkit Program funding to IBB. NSL was supported by CBIOS (NIH T32 Kirschstein-NRSA: Computation, Bioinformatics, and Statistics) training program at The Pennsylvania State University (#T32GM102057). The findings and conclusions do not necessarily reflect the view of the funding agencies.

Data Availability Statement

Raw sequencing data and assemblies generated for this project are available on the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA982825. These Whole Genome Shotgun projects (assemblies) have been deposited at DDBJ/ENA/GenBank under the accessions JBGLOB000000000, JBGLOC000000000, and JBGLOD000000000, for Dendrogyra cylindrus, Colpophyllia natans, and Siderastrea siderea, respectively. For review purposes and public access, all assemblies, annotations, and associated assembly and analysis scripts and files are publicly available on Zenodo at https://zenodo.org/doi/10.5281/zenodo.13323697.

Figure 1: K-mer multiplicity plots (left panes) from GenomeScope2 (Ranallo-Benavidez et al. 2020) for a kmer size of 31 for A) Siderastrea siderea, B) Dendrogyra cylindrus, and C) Colpophyllia natans. Mitochondrial genome gene order (right panes) in Siderastrea siderea, Dendrogyra cylindrus, and Colpophyllia natans. Mitogenomes assembled using MitoHiFi (Gabriel et al. 2023).

Figure 2: Upset plot describing unique and shared orthogroups across scleractinian corals and an outgroup, Corallimorpharia. Gene models were assigned to orthogroups using OrthoFinder (Emms and Kelly 2019). All included taxa are listed in Table S3. The focal taxa assembled in the present study are indicated by bold font and asterisks (*).

Figure 3: Top 10 gene ontology (GO) terms enriched in orthogroups undergoing phylogenetically significant expansion in Siderastrea siderea, Dendrogyra cylindrus, and Colpophyllia natans. Orthogroups were assigned using OrthoFinder (Emms and Kelly 2019). Gene families undergoing phylogenetically significant expansion were identified using CAFE5 (Mendes et al. 2021). GO enrichment analyses were performed in GOATools (Klopfenstein et al. 2018).

Table 1: Assembly summary statistics for Colpophyllia natans, Dendrogyra cylindrus, and Siderastrea siderea.

	Siderastrea siderea	Dendrogyra cylindrus	Colpophyllia natans	
Contig Total (Mb)	822.514	526.444	398.943	
Gap Percentage	0%	0%	0%	
Number of contigs	265	301	174	
Contig N50	9.1Mb	4.647Mb	4.902Mb	
Largest contig	25.215Mb	21.044Mb	14.745Mb	
GC Content (%)	39.81	39.29	38.87	
% of k-mer estimate recovered	105.61	105.99	103.47	
BUSCO Metazoa, complete (%)	96.2	95.1	96.1	
Single copy	94.1	94.1	95.4	
Duplicated	2.1	1	0.7	
Fragmented (%)	1.3	1.9	2	
Missing (%)	2.5	3	1.9	
Gene models	61,712	39,739	34,139	
Protein-coding gene models	52,473	34,738	29,090	

Table 2: Repetitive content and transposable elements identified by RepeatMasker (Smit et al.) across Siderastrea siderea, Dendrogyra cylindrus, and Colpophyllia natans. The top three repeat families (e.g. Maverick) within each major repeat class (DNA, LINE, LTR, SINE, and RNA repeats) are presented in this table.

		Siderastrea siderea	Dendrogyra cylindrus	Colpophyllia natans	
DNA	Total	138,779	68,819,835	8.39%	82,872	47,945,358	9.09%	68,722	17,971,690	4.52%	
Maverick	22,353	42,786,635	5.20%	13,811	35,496,289	6.74%	5,091	5,626,678	1.41%	
Sola-3	17,315	6,171,939	0.75%	4,051	1,802,393	0.34%	3,249	1,019,211	0.26%	
PIF-Harbinger	9,247	1,246,387	0.15%	9,092	1,764,101	0.34%	5,361	821,524	0.21%	
Academ-1	4,662	1,873,178	0.23%	2,578	872,091	0.17%	1,886	777,963	0.20%	
LINE	Total	104,185	32,929,736	4.02%	53,305	17,696,634	3.36%	46,166	15,626,622	3.92%	
Penelope	38,138	11,157,756	1.36%	16,584	5,217,438	0.99%	21,897	5,971,804	1.50%	
L1-Tx1	13,255	8,443,143	1.03%	9,745	5,273,753	1.00%	6,122	3,626,933	0.91%	
L2	29,218	6,994,614	0.85%	16,810	3,452,973	0.66%	11,440	3,468,236	0.87%	
RTE-BovB	4,796	946,003	0.12%	2,582	1,414,380	0.27%	2,717	1,144,012	0.29%	
LTR	Total	36,888	17,315,718	2.09%	11,593	7,626,581	1.44%	10,698	7,645,820	1.92%	
Gypsy	14,784	5,919,375	0.72%	4,918	2,999,690	0.57%	5,407	4,098,188	1.03%	
Pao	4,227	4,683,913	0.57%	3,053	2,945,104	0.56%	1,809	1,501,851	0.38%	
DIRS	3,419	2,371,017	0.29%	1,323	882,015	0.17%	2,095	1,383,508	0.35%	
Ngaro	7,195	3,053,083	0.37%	869	469,818	0.09%	1,057	496,392	0.12%	
SINE	Total	16,023	2,146,747	0.26%	4,485	541,212	0.10%	5,616	674,504	0.17%	
tRNA-V	3,912	567,988	0.07%	2,623	350,202	0.07%	3,166	512,682	0.13%	
MIR	8,147	1,173,564	0.14%	0	0	0.00%	0	0	0.00%	
tRNA-RTE	1,612	150,650	0.02%	1,010	100,831	0.02%	0	0	0.00%	
Alu	1,485	131,235	0.02%	0	0	0.00%	0	0	0.00%	
Low complexity		455	76,303	0.01%	282	53,301	0.01%	123	26,366	0.01%	
Retroposon	L1-dep	175	32,571	0.00%	0	0	0.00%	0	0	0.00%	
Rolling circle	Helitron	2,701	1,113,967	0.14%	837	186,063	0.04%	5,529	2,007,025	0.50%	
Satellites		476	226,250	0.03%	1,120	113,510	0.02%	1,795	189,861	0.05%	
Simple repeats		17,276	2,670,182	0.32%	11,761	1,849,810	0.35%	6,765	1,167,479	0.29%	
RNA repeats	Total	24,191	5,349,132	0.65%	21,009	2,054,088	0.39%	727	135,302	0.03%	
tRNA	23,498	5,198,251	0.63%	20,541	1,915,611	0.36%	395	44,379	0.01%	
rRNA	693	150,881	0.02%	468	138,477	0.03%	260	82,379	0.02%	
snRNA	0	0	0.00%	0	0	0.00%	72	8,544	0.00%	
Unclassified		1,000,623	262,395,404	31.91%	635,176	134,635,802	25.57%	262,664	48,767,859	12.22%	
Total		1,341,772	393,075,845	47.80%	822,440	212,702,359	40.40%	408,805	94,212,528	23.62%	

Conflict of Interest

The authors declare no conflict of interest.
==== Refs
Literature Cited

Aichelman H. E. , Benson B. E. , Gomez-Campo K. , Martinez-Rugerio M. I. , Fifer J. E. , 2024 Cryptic diversity shapes coral symbioses, physiology, and response to thermal challenge. bioRxiv 2024.07.09.602709.
Allio R. , Schomaker-Bastos A. , Romiguier J. , Prosdocimi F. , Nabholz B. , 2020 MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics. Molecular Ecology Resources 20 : 892–905.32243090
Almeida-Silva F. , and de Peer Y. V. , 2024 doubletrouble: an R/Bioconductor package for the identification, classification, and analysis of gene and genome duplications. 2024.02.27.582236.
Altschul S. F. , Madden T. L. , Schäffer A. A. , Zhang J. , Zhang Z. , 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25 : 3389–3402.9254694
Alvarez-Filip L. , González-Barrios F. J. , Pérez-Cervantes E. , Molina-Hernández A. , and Estrada-Saldívar N. , 2022 Stony coral tissue loss disease decimated Caribbean coral populations and reshaped reef functionality. Communications Biology 2022 5 :1 5: 1–10.
Barbot E. , Dufaÿ M. , and De Cauwer I. , 2023 Sex-specific selection patterns in a dioecious insect-pollinated plant. Evolution 77 : 1578–1590.37094807
Barrett R. D. H. , and Schluter D. , 2008 Adaptation from standing genetic variation. Trends in Ecology & Evolution 23 : 38–44.18006185
Bateman A. , Coin L. , Durbin R. , Finn R. D. , Hollich V. , 2004 The Pfam protein families database. Nucleic Acids Research 32 : D138–D141.14681378
Baums I. B. , Chamberland V. F. , Locatelli N. S. , and Conn T. , 2022 Maximizing Genetic Diversity in Coral Restoration Projects, pp. 35–53 in Coral Reef Conservation and Restoration in the Omics Age, edited by Van Oppen M. J. H. and Lastra M. Aranda . Coral Reefs of the World, Springer International Publishing, Cham.
Beedessee G. , Kubota T. , Arimoto A. , Nishitsuji K. , Waller R. F. , 2020 Integrated omics unveil the secondary metabolic landscape of a basal dinoflagellate. BMC Biology 18 : 1–16.31898513
Boilard A. , Dubé C. E. , Gruet C. , Mercière A. , Hernandez-Agreda A. , 2020 Defining coral bleaching as a microbial dysbiosis within the coral holobiont. Microorganisms 8 : 1682.33138319
Bongaerts P. , Carmichael M. , Hay K. B. , Tonk L. , Frade P. R. , 2015 Prevalent endosymbiont zonation shapes the depth distributions of scleractinian coral species. Royal Society Open Science 2 : 140297.26064597
Bongaerts P. , Cooke I. R. , Ying H. , Wels D. , den Haan S. , 2021 Morphological stasis masks ecologically divergent coral species on tropical reefs. Current Biology 31 : 2286–2298.e8.33811819
Bourne D. G. , Garren M. , Work T. M. , Rosenberg E. , Smith G. W. , 2009 Microbial disease and the coral holobiont. Trends in Microbiology 17 : 554–562.19822428
Boutet E. , Lieberherr D. , Tognolli M. , Schneider M. , Bansal P. , 2016 Uniprotkb/swiss-prot, the manually annotated section of the uniprot knowledgebase, pp. 23–54 in Methods in Molecular Biology,.
Bove C. B. , Mudge L. , and Bruno J. F. , 2022 A century of warming on Caribbean reefs. PLOS Climate 1 : e0000002.
Brandt M. E. , Ennis R. S. , Meiling S. S. , Townsend J. , Cobleigh K. , 2021 The Emergence and Initial Impact of Stony Coral Tissue Loss Disease (SCTLD) in the United States Virgin Islands. Frontiers in Marine Science 8 :.
Brůna T. , Lomsadze A. , and Borodovsky M. , 2020 GeneMark-EP+: Eukaryotic gene prediction with self-training in the space of genes and proteins. NAR Genomics and Bioinformatics 2 :.
Budd A. F. , Johnson K. G. , Stemann T. A. , and Tompkins B. , 1999 Pliocene to Pleistocene reef coral assemblages in the Limon Group of Costa Rica. Bulletins of American Paleontology 113 : 119–158.
Buitrago-López C. , Mariappan K. G. , Cárdenas A. , Gegner H. M. , and Voolstra C. R. , 2020 The Genome of the Cauliflower Coral Pocillopora verrucosa. Genome Biology and Evolution 12 : 1911–1917.32857844
Burton K. W. , Ling H.-F. , and O’Nions R. K. , 1997 Closure of the Central American Isthmus and its effect on deep-water formation in the North Atlantic. Nature 386 : 382–385.
Cai L. , Zhou G. , Tian R.-M. , Tong H. , Zhang W. , 2017 Metagenomic analysis reveals a green sulfur bacterium as a potential coral symbiont. Sci Rep 7 : 9320.28839161
Camacho C. , Coulouris G. , Avagyan V. , Ma N. , Papadopoulos J. , 2009 BLAST+: Architecture and applications. BMC Bioinformatics 10 : 1–9.19118496
Cavada-Blanco F. , Croquer A. , Vermeij M. , Goergen L. , and Rodriguez-Martinez R. , 2022 Dendrogyra cylindrus. IUCN Red List of Threatened Species.
Chapman J. A. , Kirkness E. F. , Simakov O. , Hampson S. E. , Mitros T. , 2010 The dynamic genome of Hydra. Nature 464 : 592–596.20228792
Chen Y.-H. , Yang S.-H. , Tandon K. , Lu C.-Y. , Chen H.-J. , 2021 Potential syntrophic relationship between coral-associated Prosthecochloris and its companion sulfate-reducing bacterium unveiled by genomic analysis. Microb Genom 7 : 000574.33952388
Cooke I. , Ying H. , Forêt S. , Bongaerts P. , Strugnell J. M. , 2020 Genomic signatures in the coral holobiont reveal host adaptations driven by Holocene climate change and reef specific symbionts. Sci Adv 6 : eabc6318.33246955
Craggs J. , Guest J. R. , Davis M. , Simmons J. , Dashti E. , 2017 Inducing broadcast coral spawning ex situ: Closed system mesocosm design and husbandry protocol. Ecology and Evolution 7 : 11066–11078.29299282
Cramer K. L. , Jackson J. B. C. , Donovan M. K. , Greenstein B. J. , Korpanty C. A. , 2020 Widespread loss of Caribbean acroporid corals was underway before coral bleaching and disease outbreaks. Science Advances 6 :.
Cui G. , Liew Y. J. , Konciute M. K. , Zhan Y. , Hung S.-H. , 2022 Nutritional control regulates symbiont proliferation and life history in coral-dinoflagellate symbiosis. BMC Biology 20 : 103.35549698
Cunning R. , Lenz E. A. , and Edmunds P. J. , 2024 Measuring multi-year changes in the Symbiodiniaceae algae in Caribbean corals on coral-depleted reefs. PeerJ 12 : e17358.38827291
Danecek P. , Bonfield J. K. , Liddle J. , Marshall J. , Ohan V. , 2021 Twelve years of SAMtools and BCFtools. GigaScience 10 : 1–4.
Delph L. F. , 2019 Pollen competition is the mechanism underlying a variety of evolutionary phenomena in dioecious plants. New Phytol 224 : 1075–1079.31009082
Devlin-Durante M. K. , Miller M. W. , Group C. A. R. , Precht W. F. , and Baums I. B. , 2016 How old are you? Genet age estimates in a clonal animal. Molecular Ecology 25 : 5628–5646.27671533
Emms D. M. , and Kelly S. , 2019 OrthoFinder: Phylogenetic orthology inference for comparative genomics. Genome Biology 20 : 1–14.30606230
Faichney I. D. E. , Webster J. M. , Clague D. A. , Braga J. C. , Renema W. , 2011 The impact of the Mid-Pleistocene Transition on the composition of submerged reefs of the Maui Nui Complex, Hawaii. Palaeogeography, Palaeoclimatology, Palaeoecology 299 : 493–506.
Falkowski P. G. , Dubinsky Z. , Muscatine L. , and McCloskey L. , 1993 Population Control in Symbiotic Corals: Ammonium ions and organic materials maintain the density of zooxanthellae. BioScience 43 : 606–611.
Feng X. , Cheng H. , Portik D. , and Li H. , 2022 Metagenome assembly of high-fidelity long reads with hifiasm-meta. Nature Methods 19 : 671–674.35534630
Flynn J. M. , Hubley R. , Goubert C. , Rosen J. , Clark A. G. , 2020 RepeatModeler2 for automated genomic discovery of transposable element families. Proceedings of the National Academy of Sciences of the United States of America 117 : 9451–9457.32300014
Fuller Z. L. , Mocellin V. J. L. , Morris L. A. , Cantin N. , Shepherd J. , 2020 Population genetics of the coral Acropora millepora: Toward genomic prediction of bleaching. Science 369 :.
Gabriel J. , Ferreira R. N. , Krasheninnikova K. , Uliano-Silva D. M. , Formenti G. , 2023 MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio High Fidelity reads. bioRxiv 2022.12.23.521667.
Garrido A. G. , Machado L. F. , Zilberberg C. , and Leite D. C. de A. , 2021 Insights into ‘Symbiodiniaceae phycosphere’ in a coral holobiont. Symbiosis 83 : 25–39.
Gold D. A. , Katsuki T. , Li Y. , Yan X. , Regulski M. , 2019 The genome of the jellyfish Aurelia and the evolution of animal complexity. Nat Ecol Evol 3 : 96–104.30510179
Grabherr M. G. , Haas B. J. , Yassour M. , Levin J. Z. , Thompson D. A. , 2011 Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29 : 644–652.
Gregorio A. de , 1930 Sul Permiano di Sicilia (Fossili del calcare con Fusulina di palazzo adriano non descritti del Prof. G. Gemmellaro conservati nel mio private Gabinetto). Annales Géol. Paléontol 52 : 18–32.
Guan D. , Guan D. , McCarthy S. A. , Wood J. , Howe K. , 2020 Identifying and removing haplotypic duplication in primary genome assemblies. Bioinformatics 36 : 2896–2898.31971576
Haas B. J. , Salzberg S. L. , Zhu W. , Pertea M. , Allen J. E. , 2008 Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biology 9 : 1–22.
Hamada M. , Schröder K. , Bathia J. , Kürn U. , Fraune S. , 2018 Metabolic co-dependence drives the evolutionarily ancient Hydra–Chlorella symbiosis. eLife 7 :.
Harris M. A. , Clark J. , Ireland A. , Lomax J. , Ashburner M. , 2004 The Gene Oncology (GO) database and informatics resource. Nucleic Acids Research 32 : D258–D261.14681407
Hazewinkel M. , and Van Eijck J. , 2000 Graph clustering by flow simulation. University of Utrecht Dissertation.
Helmkampf M. , Bellinger M. R. , Geib S. M. , Sim S. B. , and Takabayashi M. , 2019 Draft Genome of the Rice Coral Montipora capitata Obtained from Linked-Read Sequencing. Genome Biology and Evolution 11 : 2045–2054.31243452
Herrera S. , and Cordes E. E. , 2023 Genome assembly of the deep-sea coral Lophelia pertusa. Gigabyte 2023 : 1–12.37732134
Horvath K. M. , Castillo K. D. , Armstrong P. , Westfield I. T. , Courtney T. , 2016 Next-century ocean acidification and warming both reduce calcification rate, but only acidification alters skeletal morphology of reef-building coral Siderastrea siderea. Sci Rep 6 : 29613.27470426
Huang C. R. L. , Burns K. H. , and Boeke J. D. , 2012 Active Transposition in Genomes. Annu Rev Genet 46 : 651–675.23145912
Huang L. , Zhang H. , Wu P. , Entwistle S. , Li X. , 2018 DbCAN-seq: A database of carbohydrate-active enzyme (CAZyme) sequence and annotation. Nucleic Acids Research 46 : D516–D521.30053267
Huerta-Cepas J. , Szklarczyk D. , Heller D. , Hernández-Plaza A. , Forslund S. K. , 2019 EggNOG 5.0: A hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Research 47 : D309–D314.30418610
Hume B. C. C. , Smith E. G. , Ziegler M. , Warrington H. J. M. , Burt J. A. , 2019 SymPortal: A novel analytical framework and platform for coral algal symbiont next-generation sequencing ITS2 profiling. Molecular Ecology Resources 19 : 1063–1080.30740899
Hunter S. , Apweiler R. , Attwood T. K. , Bairoch A. , Bateman A. , 2009 InterPro: The integrative protein signature database. Nucleic Acids Research 37 : D211–D215.18940856
Hunter I. G. , and Jones B. , 1996 Coral associations of the Pleistocene Ironshore Formation, Grand Cayman. Coral Reefs 15 : 249–267.
Kardos M. , Armstrong E. E. , Fitzpatrick S. W. , Hauser S. , Hedrick P. W. , 2021 The crucial role of genome-wide genetic variation in conservation. Proceedings of the National Academy of Sciences 118 : e2104642118.
Katoh K. , and Standley D. M. , 2013 MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30 : 772–780.23329690
Kim J. , Choi J. P. , Kim M. S. , Jo Y. , Min W. G. , 2022 Comparative Genome and Evolution Analyses of an Endangered Stony Coral Species Dendrophyllia cribrosa Near Dokdo Islands in the East Sea. Genome Biology and Evolution 14 : evac132.36017802
Klopfenstein D. V. , Zhang L. , Pedersen B. S. , Ramírez F. , Warwick Vesztrocy A. , 2018 GOATOOLS: A Python library for Gene Ontology analyses. Sci Rep 8 : 10872.30022098
Krueger F. , James F. , Ewels P. , Afyounian E. , and Schuster-Boeckler B. , 2021 FelixKrueger/TrimGalore: v0.6.7.
Laetsch D. R. , and Blaxter M. L. , 2017 BlobTools: Interrogation of genome assemblies. F1000Research 2017 6 :1287 6: 1287.
LaJeunesse T. C. , and Thornhill D. J. , 2011 Improved Resolution of Reef-Coral Endosymbiont (Symbiodinium) Species Diversity, Ecology, and Evolution through psbA Non-Coding Region Genotyping. PLOS ONE 6 : e29013.22216157
Leclère L. , Horin C. , Chevalier S. , Lapébie P. , Dru P. , 2019 The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle. Nat Ecol Evol 3 : 801–810.30858591
Lewis A. M. , Chan A. N. , and LaJeunesse T. C. , 2019a New Species of Closely Related Endosymbiotic Dinoflagellates in the Greater Caribbean have Niches Corresponding to Host Coral Phylogeny. Journal of Eukaryotic Microbiology 66 : 469–482.30281867
Lewis C. , Neely K. , and Rodriguez-Lanetty M. , 2019b Recurring Episodes of Thermal Stress Shift the Balance From a Dominant Host-Specialist to a Background Host-Generalist Zooxanthella in the Threatened Pillar Coral, Dendrogyra cylindrus. Front. Mar. Sci. 6 :.
Li H. , 2018 Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics 34 : 3094–3100.29750242
Liew Y. J. , Aranda M. , and Voolstra C. R. , 2016 Reefgenomics.Org - a repository for marine genomics data. Database.
Locatelli N. S. , Kitchen S. A. , Stankiewicz K. H. , Osborne C. C. , Dellaert Z. , 2023 Genome assemblies and genetic maps highlight chromosome-scale macrosynteny in Atlantic acroporids. 2023.12.22.573044.
López E. H. , and Palumbi S. R. , 2020 Somatic Mutations and Genome Stability Maintenance in Clonal Coral Colonies. Molecular Biology and Evolution 37 : 828–838.31722397
Manni M. , Berkeley M. R. , Seppey M. , Sim~ Ao F. A. , and Zdobnov E. M. , 2021 BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Molecular Biology and Evolution 38 : 4647–4654.34320186
Marçais G. , and Kingsford C. , 2011 A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics 27 : 764–770.21217122
Marhaver K. L. , Vermeij M. J. A. , and Medina M. M. , 2015 Reproductive natural history and successful juvenile propagation of the threatened Caribbean Pillar CoralDendrogyra cylindrus. BMC Ecology 15 :.
McKenna V. , Archibald J. M. , Beinart R. , Dawson M. N. , Hentschel U. , 2021 The Aquatic Symbiosis Genomics Project: probing the evolution of symbiosis across the tree of life. Wellcome Open Research 6 : 254.
Mendes F. K. , Vanderpool D. , Fulton B. , and Hahn M. W. , 2021 CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics 36 : 5516–5518.33325502
Modys A. B. , Toth L. T. , Mortlock R. A. , Oleinik A. E. , and Precht W. F. , 2023 Discovery of a rare pillar coral (Dendrogyra cylindrus) death assemblage off southeast Florida reveals multi-century persistence during the late Holocene. Coral Reefs 42 : 801–807.
Neely K. L. , Lewis C. L. , Lunz K. S. , and Kabay L. , 2021 Rapid Population Decline of the Pillar Coral Dendrogyra cylindrus Along the Florida Reef Tract. Frontiers in Marine Science 8 : 434.
Noel B. , Denoeud F. , Rouan A. , Buitrago-López C. , Capasso L. , 2023 Pervasive tandem duplications and convergent evolution shape coral genomes. Genome Biology 24 : 123.37264421
O’Dea A. , Lessios H. A. , Coates A. G. , Eytan R. I. , Restrepo-Moreno S. A. , 2016 Formation of the Isthmus of Panama. Science Advances 2 : e1600883.27540590
O’Neil K. L. , Serafin R. M. , Patterson J. T. , and Craggs J. R. K. , 2021 Repeated ex situ Spawning in Two Highly Disease Susceptible Corals in the Family Meandrinidae. Front. Mar. Sci. 8 :.
Palmer J. M. , and Stajich J. , 2020 nextgenusfs/funannotate: funannotate v1.8.13 (Version 1.8.13). Zenodo.
Park E. , Hwang D.-S. , Lee J.-S. , Song J.-I. , Seo T.-K. , 2012 Estimation of divergence times in cnidarian evolution based on mitochondrial protein-coding genes and the fossil record. Mol Phylogenet Evol 62 : 329–345.22040765
Peters S. E. , and McClennen M. , 2016 The Paleobiology Database application programming interface. Paleobiology 42 : 1–7.
Pootakham W. , Sonthirod C. , Naktang C. , Kongjandtre N. , Putchim L. , 2021 De novo Assembly of the Brain Coral Platygyra sinensis Genome. Frontiers in Marine Science 8 :.
Prada C. , Hanna B. , Budd A. F. , Woodley C. M. , Schmutz J. , 2016 Empty Niches after Extinctions Increase Population Sizes of Modern Corals. Current Biology 26 : 3190–3194.27866895
Price M. N. , Dehal P. S. , and Arkin A. P. , 2010 FastTree 2 - Approximately maximum-likelihood trees for large alignments. PLoS ONE 5 : e9490.20224823
Proost S. , Fostier J. , De Witte D. , Dhoedt B. , Demeester P. , 2012 i-ADHoRe 3.0-fast and sensitive detection of genomic homology in extremely large data sets. Nucleic Acids Research 40 :.
Qi W. , 1984 An Anisian coral fauna in Guizhou, South China. Palaeontographica Americana 54 : 187–190.
Ranallo-Benavidez T. R. , Jaron K. S. , and Schatz M. C. , 2020 GenomeScope 2.0 and Smudgeplot for reference-free profiling of polyploid genomes. Nat Commun 11 : 1432.32188846
Rawlings N. D. , Barrett A. J. , and Bateman A. , 2009 MEROPS: The peptidase database. Nucleic Acids Research 38 : D227–D233.19892822
Reusch T. B. H. , Baums I. B. , and Werner B. , 2021 Evolution via somatic genetic variation in modular species. Trends in Ecology & Evolution 36 : 1083–1092.34538501
Rivera-Ortega J. , and Thomé P. E. , 2018 Contrasting Antibacterial Capabilities of the Surface Mucus Layer From Three Symbiotic Cnidarians. Front. Mar. Sci. 5 :.
Rodriguez-Martinez R. , Vermeij M. , Kitahara M. V. , and Alvarez-Filip L. , 2022 Siderastrea siderea. IUCN Red List of Threatened Species.
Salazar O. R. , Prasanna N A. , Cui G. , Bay L. K. , van Oppen M. J. H. , 2022 The coral Acropora loripes genome reveals an alternative pathway for cysteine biosynthesis in animals. Science Advances 8 : 304.
Sanderson M. J. , 2003 r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock. Bioinformatics 19 : 301–302.12538260
Sanderson T. , Bileschi M. L. , Belanger D. , and Colwell L. J. , 2023 ProteInfer, deep neural networks for protein functional inference (Dötsch V. & Staller M. V. , Eds.). eLife 12 : e80942.36847334
Shinzato C. , Shoguchi E. , Kawashima T. , Hamada M. , Hisata K. , 2011 Using the Acropora digitifera genome to understand coral responses to environmental change. Nature 476 : 320–323.21785439
Shinzato C. , Takeuchi T. , Yoshioka Y. , Tada I. , Kanda M. , 2021 Whole-Genome Sequencing Highlights Conservative Genomic Strategies of a Stress-Tolerant, Long-Lived Scleractinian Coral, Porites australiensis Vaughan, 1918. Genome Biology and Evolution 13 : evab270.34878117
Shoguchi E. , Beedessee G. , Hisata K. , Tada I. , Narisoko H. , 2021 A New Dinoflagellate Genome Illuminates a Conserved Gene Cluster Involved in Sunscreen Biosynthesis. Genome biology and evolution 13 :.
Shoguchi E. , Beedessee G. , Tada I. , Hisata K. , Kawashima T. , 2018 Two divergent Symbiodinium genomes reveal conservation of a gene cluster for sunscreen biosynthesis and recently lost genes. BMC Genomics 19 : 1–11.29291715
Shoguchi E. , Shinzato C. , Kawashima T. , Gyoja F. , Mungpakdee S. , 2013 Draft assembly of the symbiodinium minutum nuclear genome reveals dinoflagellate gene structure. Current Biology 23 : 1399–1408.23850284
Smit A. , Hubley R. , and Green P. RepeatMasker Open-4.0.
Gelais A. T. St. , Chaves-Fonnegra A. , Brownlee A. S. , Kosmynin V. N. , Moulding A. L. , 2016 Fecundity and sexual maturity of the coral Siderastrea siderea at high latitude along the Florida Reef Tract, USA. Invertebrate Biology 135 : 46–57.
Stanke M. , Keller O. , Gunduz I. , Hayes A. , Waack S. , 2006 AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Research 34 : W435–W439.16845043
Stankiewicz K. H. , Guiglielmoni N. , Kitchen S. A. , Flot J.-F. , Barott K. L. , 2023 Genomic comparison of the temperate coral Astrangia poculata with tropical corals yields insights into winter quiescence, innate immunity, and sexual reproduction. 2023.09.22.558704.
Stehlik I. , and Barrett S. C. H. , 2005 Mechanisms Governing Sex-Ratio Variation in Dioecious Rumex Nivals. Evolution 59 : 814–825.15926691
Stephens T. G. , Lee J. , Jeong Y. , Yoon H. S. , Putnam H. M. , 2022 High-quality genome assemblies from key Hawaiian coral species. GigaScience 11 : giac098.36352542
Taylor D. R. , Saur M. J. , and Adams E. , 1999 POLLEN PERFORMANCE AND SEX-RATIO EVOLUTION IN A DIOECIOUS PLANT. Evolution 53 : 1028–1036.28565519
Thompson J. R. , Rivera H. E. , Closek C. J. , and Medina M. , 2015 Microbes in the coral holobiont: partners through evolution, development, and ecological interactions. Frontiers in Cellular and Infection Microbiology 4 : 1–20.
Vasquez Kuntz K. L. , Kitchen S. A. , Conn T. L. , Vohsen S. A. , Chan A. N. , 2022 Inheritance of somatic mutations by animal offspring. Sci Adv 8 : eabn0707.36044584
Vecsei A. , and Moussavian E. , 1997 Paleocene reefs on the Maiella Platform margin, Italy: An example of the effects of the Cretaceous/Tertiary boundary events on reefs and carbonate platforms. Facies 123–140.
Vermeij M. , and Goergen L. , 2022 Colpophyllia natans. IUCN Red List of Threatened Species.
Vilas Bhagwat P. , Ravindran C. , and Irudayarajan L. , 2023 Characterization of the defense properties of healthy and diseased coral mucus. Journal of Invertebrate Pathology 201 : 108001.37838065
Voolstra C. R. , Li Y. , Liew Y. J. , Baumgarten S. , Zoccola D. , 2017 Comparative analysis of the genomes of Stylophora pistillata and Acropora digitifera provides evidence for extensive differences between species of corals. Sci Rep 7 : 17583.29242500
Wallace C. C. , and Rosen B. R. , 2006 Diverse staghorn corals (Acropora) in high-latitude Eocene assemblages: implications for the evolution of modern diversity patterns of reef corals. Proc Biol Sci 273 : 975–982.16627283
Wang X. , Liew Y. J. , Li Y. , Zoccola D. , Tambutte S. , 2017 Draft genomes of the corallimorpharians Amplexidiscus fenestrafer and Discosoma sp. Molecular Ecology Resources 17 : e187–e195.28407448
Williamson O. M. , Dennison C. E. , O’Neil K. L. , and Baker A. C. , 2022 Susceptibility of Caribbean Brain Coral Recruits to Stony Coral Tissue Loss Disease (SCTLD). Frontiers in Marine Science 9 :.
Xia W. , Li H. , Cheng W. , Li H. , Mi Y. , 2020 High-Quality Genome Assembly of Chrysaora quinquecirrha Provides Insights Into the Adaptive Evolution of Jellyfish. Front. Genet. 11 :.
Xiang T. , Lehnert E. , Jinkerson R. E. , Clowez S. , Kim R. G. , 2020 Symbiont population control by host-symbiont metabolic interaction in Symbiodiniaceae-cnidarian associations. Nat Commun 11 : 108.31913264
Yang Z. , 2007 PAML 4: Phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution 24 : 1586–1591.17483113
Ying H. , Cooke I. , Sprungala S. , Wang W. , Hayward D. C. , 2018 Comparative genomics reveals the distinct evolutionary trajectories of the robust and complex coral lineages. Genome Biology 19 : 175.30384840
Young B. D. , Williamson O. M. , Kron N. S. , Andrade Rodriguez N. , Isma L. M. , 2024 Annotated genome and transcriptome of the endangered Caribbean mountainous star coral (Orbicella faveolata) using PacBio long-read sequencing. BMC Genomics 25 : 226.38424480
Yu Q. , Ellen E. D. , Wade M. J. , and Delph L. F. , 2011 Genetic differences among populations in sexual dimorphism: evidence for selection on males in a dioecious plant. Journal of Evolutionary Biology 24 : 1120–1127.21401772
Yu Y. , Nong W. , So W. L. , Xie Y. , Yip H. Y. , 2022 Genome of elegance coral Catalaphyllia jardinei (Euphylliidae). Frontiers in Marine Science 9 :.
Zwaenepoel A. , and Van De Peer Y. , 2019 Wgd-simple command line tools for the analysis of ancient whole-genome duplications. Bioinformatics 35 : 2153–2155.30398564
