The complete chloroplast genome of Calophyllum soulattri Burm. f. (Calophyllaceae)

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.

Keywords:

• Calophyllum soulattri
• chloroplast genome
• next-generation sequencing
• phylogenetic analysis
• traditional medicine

Introduction

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.

Figure 1. The specimen and morphology of C. soulattri. (A) The specimen of C. soulattri (accession number: 14288); (B) The fruit and (C) the inflorescence and leaves of C. soulattri. (Photo credits: Jessica Rey).

Materials

Plant Materials

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, [email protected]; 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.

Methods

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.

Results

General characteristic of the chloroplast genome

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.

Figure 2. The chloroplast genome (plastome) map of C. soulattri. The plastome map is divided into six circles with different representations. Starting from the inner circle, the first circle shows the dispersed repeats connected by red (forward repeat) and green (palindromic repeat) arcs. The second circle shows long tandem repeats in short blue bars. The third circle presents simple sequence repeats (SSRs) that are color coded based on repeat unit size (RUS) (Black, complex repeat; Green, RUS = 1; Yellow, RUS = 2; and Blue, RUS = 4). The fourth circle displays the four regions of the plastome (LSC, SSC, IRa, and IRb) with their respective sizes. The fifth circle provides the GC content along the genome. The sixth circle provides the genes with their codon usage bias in parentheses and are color-coded, indicating their respective functional group. The functional groups can be found at the bottom left corner. Genes that are found in the inner and outer circle are transcribed clockwise and counterclockwise, respectively.

Phylogenetic analysis of C. soulattri in the Clusioid clade

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.

Figure 3. Maximum likelihood (ML) phylogenetic tree and Bayesian inference tree of C. soulattri and other Clusioid species from the order Malpighiales based on the whole chloroplast genome sequence with L. alata and P. edulis as outgroup. Values on the right represents bootstrap support from Maximum Likelihood analysis while on the left represents Bayesian posterior probabilites. The species shown in bold font is newly sequenced in this study. The following sequences were used: C. inophyllum OL679531, M. ferrea MN052680 (Wang et al. 2019), Bonnetia paniculata MK995182 (Jin et al. 2020), Garcinia mangostana KX822787 (Jo et al. 2017), G. paucinervis MT501656 (Wang et al. 2021), G. oblongifolia MT726019 (Ma et al. 2020), G. anomala MW582313 (Yue and Shi 2021), Caraipa exstipulata MW853790 (Trad et al. 2021), Kielmeyera coriacea MW853789 (Trad et al. 2021), C. heterocarpa MW853787 (Trad et al. 2021), Apinagia riedelii MN165812 (Bedoya et al. 2019), Marathrum utile MN165814 (Bedoya et al. 2020), M. capillaceum MN165813 (Bedoya et al. 2020), Paracladopus chiangmaiensis MZ645928 (Wu et al. 2022), Hypericum ascyron MZ424306 (Claude et al. 2022), Triadenum breviflorum MZ714016 (Sudmoon et al. 2022), H. hookerianum MZ714015 (Sudmoon et al. 2022), Cratoxylum pruniflorum MZ703416 (Sudmoon et al. 2022), C. cochinchinense MT424754 (Huang et al. 2019), Lophira alata MZ274135 (Mascarello et al. 2021), and NC_034285 Passiflora edulis (Cauz-Santos et al. 2017). NCBI accession numbers are given for Genbank sequences.

Discussion and conclusion

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.

Ethical approval

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.

Authors’ contributions

JRey was involved in conceptualization and design, analysis, and interpretation of the data; revising it critically for intellectual content; and the final approval of the version to be published while DPahayo and CACadorna were involved in the design, analysis, and interpretation of the data; the drafting of the paper and revising it critically for intellectual content; MOQuimado provided plant tissue samples and drafting and finalization of manuscript and that all four authors agree to be accountable for all aspects of the work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The chloroplast genome sequence data that support the findings of this study are openly available in GenBank of NCBI database at (https://www.ncbi.nlm.nih.gov/) under the accession no. OP573228. The associated BioProject, SRA, and Biosample numbers of C. soulattri are PRJNA891016, SRR22031315, and SAMN31427486, respectively.

Additional information

Funding

This work was supported by the Department of Science and Technology - Philippine Council Industry, Energy and Emerging Technology Research and Development (DOST-PCIEERD 04339) under the project: Smart Plant Production in Controlled Environments. Dexter G. Pahayo is supported under the ASTHRDP scholarship grant of the Department of Science and Technology.