Dexter G. Pahayo,Charles Anthon E. Cadorna,Marilyn O. Quimado,Jessica D. Rey
The complete chloroplast genome of Calophyllum soulattri Burm. f. (Calophyllaceae)
Calophyllum soulattri,chloroplast genome,next-generation sequencing,phylogenetic analysis,traditional medicine
Abstract Calophyllum soulattri Burm. f. (1768) is an evergreen tree native to Southeast Asia, Australia, and the Solomon Islands. It is known for its medicinal uses and has been utilized in traditional folk medicine. However, genomic resources for this species are still unavailable. In this study, we sequenced and assembled the first complete chloroplast genome of C. soulattri using next-generation sequencing data. The chloroplast genome of C. soulattri is 161,381 bp in length with a total GC content of 36.36%. The chloroplast genome contains a large single copy (LSC) region of 88,680 bp, a small single copy (SSC) region of 17,453 bp, and two inverted repeat (IR) regions of 27,624 bp each. Furthermore, the chloroplast genome has 131 genes, which include 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis indicated that C. soulattri is clustered in the same branch with C. inophyllum and is closely related to Mesua ferrea.
The complete chloroplast genome of Calophyllum soulattri Burm. f. (Calophyllaceae)

Calophyllum soulattri Burm. f. (1768) is an evergreen tree native to Southeast Asia, Australia, and the Solomon Islands. It is known for its medicinal uses and has been utilized in traditional folk medicine. However, genomic resources for this species are still unavailable. In this study, we sequenced and assembled the first complete chloroplast genome of C. soulattri using next-generation sequencing data. The chloroplast genome of C. soulattri is 161,381 bp in length with a total GC content of 36.36%. The chloroplast genome contains a large single copy (LSC) region of 88,680 bp, a small single copy (SSC) region of 17,453 bp, and two inverted repeat (IR) regions of 27,624 bp each. Furthermore, the chloroplast genome has 131 genes, which include 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis indicated that C. soulattri is clustered in the same branch with C. inophyllum and is closely related to Mesua ferrea.

Calophyllum is a genus that belongs to the family Calophyllaceae under the Clusioid clade of the order Malpighiales (Angiosperm Phylogeny Group 2009). Calophyllum species are known for their use in traditional medicine. For example, oils extracted from Calophyllum seeds are used to treat wounds and infections (Léguillier et al. 2015). Furthermore, the genus Calophyllum is a potential source of compounds for drug discovery and development due to the presence of different secondary metabolites in plant tissues such as terpenes (Nigam et al. 1988), xanthones (Mah et al. 2015), coumarins (Ee et al. 2011), phenols, and flavonoids (Hapsari et al. 2022). In fact, coumarins extracted from a Calophyllum species were found to be potent against HIV-1 virus (Spino et al. 1998). Aside from being used in traditional medicine, Calophyllum species are also known for being sources of wood for locals (Rabena and Macandog 2017). Calophyllum soulattri Burm.f. (1768) is a small tree species with opposite leaves that are ovate to elliptical or sub-oblong; it has black or purple fruit and a white inflorescence (Figure 1). Its distribution is limited to mainland Southeast Asia, the Solomon Islands, and northern Australia (Pelser et al. 2011). However, the complete chloroplast genome sequence of C. soulattri has not been reported. This study reports the first complete chloroplast genome sequence of C. soulattri that provides a reference for understanding the phylogenetic relationship and plastome evolution in the Calophyllaceae and the clusioid clade.

Disease-free leaf samples of C. soulattri were collected from the germplasm collection of the Metallophytes Laboratory, Forest Biological Sciences, College of Forestry and Natural Resources, University of the Philippines, Los Baños, Laguna, Philippines (14°9′17″N 121°14′6.25″E). The leaf specimen was submitted at the Jose Vera Santos Memorial Herbarium of the Institute of Biology, University of the Philippines, Diliman (https://biology.science.upd.edu.ph/index.php/jose-vera-santos-memorial-herbarium/, Dr. Edwino S. Fernando, puh.upd.edu@gmail.com; and compared with accession number 14288 (Figure 1). Leaves were thoroughly cleaned and flash frozen using liquid nitrogen and were brought to the Plant Molecular Phylogenetics Laboratory at the Institute of Biology, University of the Philippines, Diliman, Quezon City for DNA extraction.

The total genomic DNA of C. soulattri was extracted using Wizard® HMW DNA Extraction Kit (Promega, USA) with slight modification by adding 1% (w/v) polyvinylpyrrolidone-40 (PVP-40) to the HMW lysis buffer A and submitted to MACROGEN, Inc. (Seoul, South Korea) for next-generation sequencing service. Paired-end reads were sequenced using the Illumina HiSeq 2500 platform (San Diego, California). Approximately, 2.8 Gb of raw data was generated. The quality of raw reads was checked using FastQC v0.11.9 (Andrews 2010) and read error correction was performed using Musket v1.1 (Liu et al. 2013). The plastome of C. soulattri was assembled using GetOrganelle v1.7.6.1 (Jin et al. 2020). Subsequently, the assembled chloroplast genome was annotated using GeSeq (Tillich et al. 2017) and then manually curated. The chloroplast genome map was drawn using the CPGView program (Liu et al. 2023). To validate the phylogenetic position of C. soulattri, the complete chloroplast genome sequences of 22 species in the order Malpighiales were downloaded from NCBI GenBank and aligned using MAFFT v7.490 (Katoh and Standley 2013) on the CIPRES Science Gateway (www.phylo.org) (Miller et al. 2015). The maximum-likelihood (ML) phylogenetic analysis was performed using RaxML-NG v1.1.0 (Kozlov et al. 2019) based on GTR + G nucleotide substitution model on the CIPRES Science Gateway (www.phylo.org) (Miller et al. 2015) with 1,000 bootstrap replicates and Bayesian inference using MrBayes v3.2.7a (Ronquist et al. 2012). Lophira alata (MZ274135) (Mascarello et al. 2021) from the Ochnaceae family and Passiflora edulis (NC_034285) (Cauz-Santos et al. 2017) was used as an outgroup.

The complete chloroplast genome (plastome) of C. Soulattri has a typical circular and quadripartite structure. The plastome is 161,381 bp in length with a total GC content of 36.36% consisting of the large single copy (LSC: 88,680 bp) and small single copy (SSC: 17,453 bp) regions, which are separated by two inverted repeat regions (IRa and IRb: 27,624 bp each). The GC contents of the LSC, SSC, and IR regions were 33.96%, 30.57%, and 42.04%, respectively (Figure 2). The average and minimum read mapping depth of the assembled plastome were 97.3x and 80.3x, respectively (Figure S1). Furthermore, the plastome of C. soulattri has 131 genes including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes.

Our ML phylogenetic analysis revealed two major clades with high bootstrap support (BS = 100%) (Figure 3). Garcinia (Clusiaceae) formed a monophyletic clade with a highly supported bootstrap value (BS = 100%). The family Hypericaceae was related to Podostemaceae. Moreover, the family Calophyllaceae also formed a monophyletic clade (BS = 100%) in which M. ferrea was related to C. soulattri and C. inophyllum indicating that they are more closely related than to other species.

The first complete chloroplast genome (plastome) of C. soulattri was sequenced, assembled, and annotated in this study. The general features of the chloroplast genome of C. soulattri are similar to that of most land plants. Furthermore, our results mostly concur with a recent study which elucidates a generally conserve plastome structure (Trad et al. 2021). Calophyllum soulattri belongs to the tribe Calophylleae of the family Calophyllaceae. There are almost 90 genera and approximately 2,090 species within the family Calophyllaceae. Also, the family Calophyllaceae is monophyletic along with Clusiaceae, Bonnetiacea, Podostemaceae, and Hypericaceae in the order Malpighiales which forms a group typically known as the clusiod clade (Stevens 2001 onwards; Cook and Rutishauser 2007; Angiosperm Phylogeny Group 2009; Wurdack and Davis 2009; Ruhfel et al. 2011; Koi et al. 2012; Nürk et al. 2013). Although the group is species-rich, complete plastome information is still unavailable for most species. Our phylogenetic analysis using available whole chloroplast genomes has suggested a different topology with high bootstrap support than those previously reported. Nevertheless, species groupings are consistent in the clusioid clade as has been suggested in several studies (Ruhfel et al. 2011; 2016; Trad et al. 2021). Furthermore, previous reports support our findings on M. ferrea being closely related to Calophyllum species using plastid, mitochondrial, and nuclear sequences, and even the whole chloroplast genome (Ruhfel et al. 2016; Cabral et al. 2021; Trad et al. 2021). This study provides significant data for understanding species placement in the clusioid clade which would be useful in future analyses. Thus, the complete chloroplast genome of C. soulattri provides a valuable genetic resource for future studies on the phylogeny and population genetics of Calophyllum.

This project has obtained permission to collect plant samples through the Department of Natural Resources with DENR BMB Wildlife Gratuitous Permit No. 299, issued Sept 2019. The plant material collection and experimental research were conducted according to RA 9147 or the Wildlife Resources Conservation and Protection Act of the Philippines.

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