Complete mitochondrial genomes of three reef forming Acropora corals (Acroporidae, Scleractinia) from Chagos Archipelago, Indian Ocean

Abstract We present the first mitochondrial genomes from Chagos Archipelago, Indian Ocean, of three putative species of reef forming Acropora (Acropora aff. tenuis, Acropora aff.cytherea and Acropora aff. orbicularis). The circular genome consists respectively of 18,334 bp, 18,353 bp and 18,584 bp. All mitochondrial genomes recovered comprise 13 protein-coding genes, two transfer RNA genes and two ribosomal RNA genes, with an overall GC content ranging from 37.9% to 38.0%. These new genomic data contribute to our increased understanding of genus Acropora and its species boundaries, ultimately aiding species monitoring and conservation efforts.


Introduction
The genus Acropora (Scleractinia, Acroporidae) is a widespread coral, spanning the Indian and Pacific Oceans and the Caribbean Sea (Van Oppen et al. 2001, Veron et al. 2020 and is one of the major reef builders in warm water ecosystems (Fukami et al. 2000). Warm water reef-building corals create some of the most biodiverse ecosystems on the planet and have been estimated to support 830,000 species of multi-cellular plants and animals worldwide (Bellwood and Hughes 2001, Mora et al. 2008, Mora et al. 2011, Fisher et al. 2015, providing a variety of habitats for fish, invertebrates and other taxa in shallow tropical seas (Bellwood and Hughes 2001). Despite the importance of reef-building corals, species' boundaries are considered somewhat blurry and previous investigations on the Acropora genus show that many morphological species in this genus do not correspond to genetically distinct evolutionary units, with examples of intraspecific geographic differences in morphology as large as differences between species (Van Oppen et al. 2001). The Chagos Archipelago's Acropora and Porites dominated reefs (Head et al. 2019) constitute around 2.5% of the world's reefs (Sheppard 1999) and are a potential "stepping stone" for transoceanic species dispersal (Sheppard et al. 2012), so it is a key geographical location for further research.
Systematic research is defined as an interactive process in which taxa are defined or redefined by synthesis of all available information from biological, molecular and other relevant areas of science (WILSON 1985, Wallace andWillis 1994). Systematics attempts to keep pace with developments in these fields so that the most appropriate taxonomic interpretation will facilitate the greatest possible accuracy of research and experimental design (Wallace and Willis 1994). Despite a long history of taxonomic work, Scleractinia systematics is still largely unresolved (Richards et al. 2016) and species identifications in this group have been known to be problematic for more than four decades (Randall 1981, Wallace andWillis 1994). Further issues for Acropora include cases of shared recent ancestry and introgression of loci from ongoing hybridisation (Van Oppen et al. 2001, Willis et al. 2006, Richards et al. 2013, Richards et al. 2016. Species boundaries, currently applied to Acropora, do not stand up to scrutiny; this may in part be methodological, but current species boundaries are also confounded by characteristics of Acropora, such as morphological variability and hybridisation potential (Wallace and Willis 1994). The existence of morphologically cryptic species within recognised "species" of stony corals (Richards et al. 2016), together with evidence of strong and recurring regional genetic differentiation corresponding to the separation of the Indian and Pacific Ocean in Acropora (Richards et al. 2016), support the need for improved sampling across geographically distinct populations.
Mitochondrial DNA (mtDNA) has been used in numerous applications in the past 20 years, ranging from species delimitation (Paz-García et al. 2016) -usually focused on the mitochondrially encoded cytochrome c oxidase I (MT-CO1 or CO1 or COX1) (Hebert et al. 2003) -to phylogeny and molecular evolutionary studies (Curole andKocher 1999, Niu et al. 2020). The mitochondrial genome plays a significant role in studies of phylogenetic reconstruction (Fukami and Knowlton 2005, Arrigoni et al. 2016, Capel et al. 2016, mostly due to a general consensus in the gene order and infrequent mitochondrial genome rearrangements across scleractinians corals (van Oppen et al. 2002, Lin et al. 2012. It typically includes 13 oxidative phosphorylation (OXPHOS) related genes, two rRNAs that encode the two subunits of mitochondrial ribosomes and an array of tRNAs used for translation within the organelle (Niu et al. 2020). Studying the mitochondrial genome could help to further explore Scleractinia's evolutionary process and clarify the evolutionary relationship between Scleractinia and other Hexacorallia members (Lin et al. 2012).

Data resources Data Availability Statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under the accession no. MW773216 -M W773217 -MW773218. The associated **BioProject**, **SRA** and **Bio-Sample** numbers are listed in Table 1

Data Availability Table
Complete mitochondrial genomes of three reef forming Acropora corals (Acroporidae, ... A fragment of coral 2-3 cm in size, containing one or more healthy-looking polyps, was collected during SCUBA surveys and a corresponding photo of the colony was taken (Suppl. materials 1, 2, 3). In order to account for possible clonality, samples were collected at least one metre distance from each other. Upon returning to the ship, the samples were immediately placed in single vials in ethanol (+95%) and labelled with a unique identifier, in addition to collection date and location. The samples were then stored at -20°C until extraction.
Sample identification was performed in the field by eye by a coral expert (Dr Catherine Head  Figure 1.

Map of Peros Banhos and Salomon
Islands with the three collection sites accompanied by location of the larger Chagos Archipelago. The samples were collected respectively at Egmont Mid for Acropora aff. tenuis, Ile Anglaise seaward for Acropora aff. cytherea and Ile du coin Acropora aff. orbicularis.
Subsequently, feature annotations were transferred in Geneious Prime and verified by comparison with alignments of coding regions with the above-mentioned references from GenBank. Inside coding regions, extra bases within a repeated seqeunce were manually removed if they caused a clear and significant frame-shift. Amino acid sequences of 13 concatenated protein-coding genes from our assemblies, together with 20 reference sequences were aligned using Geneious. Aligned sequences were uploaded to the European Galaxy server (Afgan et al. 2018) and subjected to phylogenetic analysis using IQ-TREE, with a 1000 non-parametric bootstrap, automatic model selection and default settings (Minh et al. 2013, Nguyen et al. 2015, Kalyaanamoorthy et al. 2017) and automatic amino acid substitution model selection (Fig. 2). Management, visualisation and annotation of the tree was done through iTOL v.6 (https://itol.embl.de -Letunic and Bork 2021). Maximum Likelihood phylogeny from analysis of concatenated protein-coding genes. Specimens from Chagos Archipelago annotated in blue. Bootstrap support numbers shown at nodes with > 50% support. GenBank accession numbers in parentheses. Outgroup -Isopora palifera NC_024091.
Where available, sequences of species matching the names of our tentative 'aff' assignments were included in the phylogeny; however, our samples did not group with these taxa (Fig. 2), supporting the tentative nature of these identifications and strengthening the case that these are distinct, possibly morphologically cryptic, species.
These new genomic data contribute to our increased understanding of the phylogenetic history and mitochondrial evolution patterns in the Acropora genus, ultimately aiding species monitoring and conservation efforts.  Stop codon TAA  TAG  TAG TAA  TAG  TAG  TAG TAG  TAA TAA TAG  TAA TAG