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Whilst the variable quality of the assembled genomes being compared complicates synteny analyses (fragmented genomes potentially reducing the apparent degree of synteny detected), this issue did not affect the major conclusions being drawn in the present case. Amongst the species included in the analyses, the Galaxea (C) and Nematostella genome assemblies are represented by the largest numbers of scaffolds, but the N50 for the Nematostella assembly was much longer than in the case of the Galaxea (C) assembly (Additional file 2: Table S3). Therefore, the limited extent of synteny observed in the anemone lineage was not an artefact of assembly quality, and despite accurate estimates of divergence times not being available, the analysis presented here provides compelling evidence that extensive intra- and inter-chromosomal rearrangements have occurred in the sea anemone lineage.
The coral genes implicated in histidine biosynthesis are scattered throughout the genome (i.e. are on different scaffolds) rather than linked and, with the sole exception of that encoding imidazoleglycerol phosphate dehydratase (step 6 in Fig. 5; K01693), all contain introns (Additional file 2: Table S18). Note that the robust coral K01693 sequences were well resolved from bacterial, Symbiodinium, and other K01693 sequences in phylogenetic analyses (Additional file 2: Table S21; Additional file 3: Figure S11 and S12; Additional file 7), ruling out the possibility of contamination or lateral gene transfer. Moreover, examination of the genomic contexts of the robust coral histidine biosynthesis genes revealed synteny between robust and complex corals in the surrounding regions (Additional file 2: Table S19), consistent with gene loss having occurred in the latter (Fig. 7).
Searching the genome assemblies allowed the identification of syntenic blocks of genes surrounding five of the histidine biosynthesis genes in Fungia (R) and Goniastrea (R) (Additional file 2: Table S19), all but one of which (K04486) were not found in complex corals. The corresponding syntenic blocks of genes (but lacking the histidine biosynthesis gene) were identified in at least one complex coral (Additional file 2: Table S19), and some were also found in sea anemones. The genes neighbouring K00765, K01663, K01814, and K14152 in the robust corals can be matched as direct syntenic orthologs in complex coral genomes. K04486 is the only histidine pathway gene which is also found in complex corals, and in this case, synteny around the gene is shared between the robust and complex corals.
The most significant implication of these comparative analyses is that, uniquely amongst animals, robust corals are capable of de novo histidine biosynthesis. Previously, the only known difference between corals with respect to biosynthetic capacity was the lack of the enzyme cystathionine β-synthase (suggesting a requirement for cysteine) in Acropora spp. (C) but not in other corals [17]. Whilst these metabolic differences may play roles in the selection of compatible Symbiodinium strains, experimental support for this idea is presently lacking. Indeed, the robust corals studied here host strains of clade C and clade D Symbiodinium (Additional file 1: Table S1), as do many complex corals, including Acropora and Galaxea. Note, however, that enormous variation exists within the clades (particularly clade C), and few genome data are available, so the possibility of metabolic influences on strain selection cannot be dismissed.
Raw RNA-seq reads were trimmed by the same methods as DNA reads. Trinity c2.0.6 [81] was then applied for de novo assembly (TDN) and genome-guided assembly (TGG). Default parameters were used except for jaccard_clip and strand-specific library type options. Similar TDN transcripts were merged using cd-hit [82, 83] with 90% identity threshold. Because RNA samples from adult tissues are a mixture of coral and Symbiodinium RNA molecules, we applied PSyTrans [84], which is based on support vector machine classification, to separate host (coral) and symbiont (Symbiodinium) transcripts from TDN transcripts. The GC content for the whole transcriptome before and after separation is shown in Additional file 3: Figure S1.
Finally, the MAKER2 [87] annotation pipeline was run for ab initio prediction. The training gene set was used to train AUGUSTUS [92] and SNAP [93]. The resulting parameters were employed by corresponding programs from MAKER. The combined TDN and TGG transcripts were provided as EST evidence, and the proteins downloaded from uniref90 database were taken as external evidence for protein alignment. Finally, putative transposons in the gene model were removed as described above.
Repetitive elements were detected from two analyses for all the genomes compared in this study. Firstly, a de novo repeat library was generated with Repeat-Modeller (Version 1.0.8) [94] with default parameters. This library was combined with RepBase databases [95] and used as input for RepeatMasker [96] to identify repeat categories and locations. A summary of repeat components is presented in Additional file 2: Table S5.
CEGMA software version 2.5 [26] was conducted to assess the completeness of genome assembly and annotated gene models. The download included the reference dataset of 248 ultra-conserved core eukaryotic genes (CEGs). The program was run with default parameters, which define the presence of a CEG in a query sequence if the outcome from the HMM search exceeds a pre-computed minimum alignment score, and the alignment covers over 70% of a CEG.
The first step in this process was the identification of orthologous groups (OGs) from the eight cnidarian species sampled in the present study using OrthoFinder (version 0.2.5) [102] with default parameters. Single-copy orthologous genes were identified from one-to-one relationship OGs, and the results filtered by requiring the same SwissProt gene name match with target coverage greater than 60%. Genes whose predicted protein sequence, when translated from the gene model GFF3 files, did not agree with the downloaded protein sequence were also excluded. This resulted in 687 high-quality single-copy ortholog groups.
A sequential approach was adopted to resolve the branching order of the coral species. Firstly, a four taxa phylogenetic tree was generated for Nematostella, Galaxea (C), A. digitifera (C), and Fungia (R). Nematostella was used as an outgroup to indicate the root position for corals. Secondly, separate five taxa analyses were conducted to infer the position of Goniastrea (R) and Porites (C), relative to the four species in the tree. The results clearly placed Goniastrea (R) with the robust coral Fungia, and Porites (C) with complex corals (Additional file 3: Figure S3). This outcome allowed us to combine the two topologies unambiguously. Finally, Aiptasia was added to the sea anemone group with Nematostella and A. millepora (C) was clustered with A. digitifera (C), to complete the eight taxon phylogenetic tree, from which the branch length from GN was estimated based on the method described in Kaehler et al. [30].
To identify functional residues, we searched the Conserved Domain Database (CDD) using the NCBI Batch Web CD-Search Tool [114, 115]. The query sequences included putative histidine biosynthesis proteins from Fungia, Goniastrea, and their best matching SwissProt proteins. In cases where the matching protein was from a fungal species, the homologous protein from the model organism Saccharomyces cerevisiae was used, on the basis that structure/function relationships have been most extensively studied in this species. The CD search outputs enabled identification of domains shared between reference and coral proteins, and the resulting alignments were manually inspected for the presence of functional residues in the robust coral proteins. In addition, two proteins (Additional file 2: Table S24 (b)) that have functional residue information in the UniProt database [88] were aligned and compared to their corresponding robust coral proteins (Additional file 8).
The sequencing datasets (genome and transcriptome sequencing data) generated by the present study are publicly available at the European Nucleotide Archive (ENA). The accession numbers are PRJEB23333, PRJEB23312, and PRJEB23371 for Galaxea, Fungia, and Goniastrea respectively [116,117,118]. Genome assembly and annotation are publicly accessible through Reefgenomics data repository [119,120,121]. Functional annotations supporting the conclusions of this article are included within the article and its additional files. Protein sequences used for gene phylogeny construction are included in the additional files.
Additional whole genome data used for comparative analyses are available from the following resources. Acropora digitifera data were obtained from the NCBI ftp site [122] with the assembly accession GCF_000222465.1 and annotation release ID 100. The Acropora millepora genome was assembled and annotated by author SF. Genome-related data for this species have been deposited to NCBI under the accession number PRJNA473876 [123]. Porites lutea genome data are publicly available via the Reefgenomics data repository [124]. Nematostella vectensis genome data were downloaded from Ensembl genome metazoan release 29 [125]. Aiptasia genome v1.0 data were obtained from the Reefgenomics data repository [126]. 781b155fdc