The complete chloroplast genome and phylogenetic analysis of Stewartia sichuanensis (Theaceae), a Chinese endemic tree with narrow distribution

Abstract Stewartia sichuanensis is a rare plant species of Theaceae and is endemic to China. Its distribution area is highly restricted, and genomic information is extremely limited. The present study reports the first complete chloroplast of S. sichuanensis. The chloroplast genome length was 158,903 bp, with a GC content of 37.3%. The chloroplast genome was comprised of an 87,736 bp long large single copy (LSC), an 18,435 bp long small single copy (SSC), and two copies of inverted repeat (IR) regions of 26,366 bp. It contained 129 genes, including 85 encoding, 36 transfer RNA, and eight ribosomal RNA genes. The phylogenetic analysis suggested that S. sichuanensis was closely related to S. laotica and S. pteropetiolata.


Introduction
Tea family (Theaceae) members are of great economic and ecological importance (Zhang et al. 2022). One of the genera, Stewartia L. shows a disjunct distribution between eastern Asia and eastern North America (referred to as the EA-ENA disjuncts; Wen 1999). Hence, the augmenting of Stewartia genomic resources will facilitate a better understanding of the evolutionary history of the EA-ENA disjuncts.
Stewartia sichuanensis (S. Z. Yan) J. Li & T. L. Ming shows a narrow distribution exclusively restricted to Pingshan Co., Sichuan province, China (Li 1996;Ming and Bartholomew 2007). According to Flora of China, S. sichuanensis is an evergreen shrub or small tree with pubescent branchlets and 1-1.5 cm long petioles surrounded by approximately 2 mm wide wings (Ming and Bartholomew 2007). It has 7.5-10 cm Â 3-4.5 cm leathery leaves, which are elliptic or obovate-elliptic. With solitary flowers and orbicular sepals, S. sichuanensis mostly resemble S. micrantha and S. calcicola morphologically (Ming and Bartholomew 2007). Therefore, S. sichuanensis is a rare plant that can play a critical role in deciphering the diversification trajectory of Stewartia. Yet, the genetic information of S. sichuanensis remains extremely poor, and the phylogenetic placement of S. sichuanensis within Stewartia remains elusive.
In this study, we report the first complete chloroplast genome of S. sichuanensis (NCBI GenBank accession number: ON853833) and reveal its phylogenetic relationships to other Stewartia species.
The total genomic DNA was isolated using DNA Plantzol (Invitrogen, Carlsbad, California, USA) following the manufacturer's protocol. The high-throughput sequencing was performed using the Illumina HiSeq 2500 platform (Novogene, Tianjin, China). Raw paired-end reads of 150 bp were processed using SOAPnuke (Chen et al. 2018) to remove adapters and low-quality sequences. Then, the complete chloroplast genome was assembled using GetOrganelle (Jin et al. 2020) and was later annotated using CpGAVAS2 (Shi et al. 2019). Schematic maps of the cis-splicing genes and trans-splicing genes were drawn using CPGView (Liu et al. 2023).

Results
After the quality control, we obtained 3.11 Gb of clean sequencing data. The length of the assembled S. sichuanensis complete chloroplast genome was 158,903 bp, with a GC content of 37.3%. The read coverage depth is sufficient (with an average of 80 Â), indicating the robustness of genome assembly (Supplementary Figure 1). The chloroplast genome showed a conserved circular structure comprising a large single copy (LSC) of 87,736 bp, a small single copy (SSC) of 18,435 bp, and two copies of inverted repeat (IR) regions of 26,366 bp. The complete chloroplast genome contained 129 genes, including 85 encoding, 36 transfer RNA, and eight ribosomal RNA genes (Figure 2). The genome contained the 13 cis-splicing genes and one trans-splicing gene ( Supplementary Figures 2 and 3). The constructed Stewartia phylogeny strongly supported that S. sichuanensis was most affinitive to S. laotica and S. pteropetiolata (Figure 3).

Discussion and conclusion
The constructed Stewartia phylogeny was well-resolved and is highly consistent with a previous report based on plastomic data (Lin et al. 2019). It is the first time that the phylogenetic position of S. sichuanensis being unveiled. It was suggested that S. laotica is sister to S. pteropetiolata, which is congruent with the present results (Lin et al. 2019). To conclude, the release of the chloroplast genome of S. sichuanensis provides valuable genetic and phylogenetic information for future research focusing on both Stewartia and the EA-ENA disjuncts.