The characteristic of the complete chloroplast genome of Lithocarpus konishii (Fagaceae), a rare and endemic species in South China

Abstract Lithocarpus konishii, a rare species endemic to islands in South China, was evaluated as a vulnerable species (VU) by the ‘China Species Red List.’ Here, we first presented the complete chloroplast genome sequence of L. konishii. The chloroplast genome was 161,059 bp in length with 36.76% GC content, containing a small single-copy region (SSC, 18,967 bp), a large single-copy region (LSC, 90,250 bp), and a pair of inverted repeats (IRs, 25,921 bp each). A total of 139 genes were predicted, including 87 protein-coding genes (CDS), 8 rRNAs, and 44 tRNAs. Based on the concatenated shared unique CDS sequence dataset, maximum-likelihood and Bayesian inference methods were used to build the phylogenetic trees of 18 species from the Fagaceae family. Results indicated that L. konishii is closely related to L. longnux and L. pachyphyllus var. fruticosus, and forms a monophyly of the subfamily Castaneoideae with Castanopsis and Castanea. This study provides a theoretical basis for the conservation genomics of this endangered plant.


Introduction
Lithocarpus konishii (Hayata) Hayata 1917 is a small evergreen tree belonging to the Fagaceae family, and is endemic to South China (Huang et al. 1999). It is found sporadically in a few island habitats in Hainan, Zhuhai, Hong Kong, and Taiwan, indicating a typical island disjunctive distribution (Shi et al. 2016). Lithocarpus konishii typically grows between 4 and 9 meters in height, with papery leaf blade, acute to caudate-acuminate apex, 3-6 obtuse teeth leaf margin, depressed globose nut, and discoid cupule ( Figure 1). It blossoms twice a year in April and August, with fruits ripening from July to October (Huang et al. 1999;Hung et al. 2005). L. konishii exhibits exceptional resilience to salt, alkali, drought, and sterile conditions. Additionally, it has robust wind resistance capabilities and plays a vital role in preserving water and soil within island ecosystems (Shi et al. 2016). Furthermore, L. konishii holds significant economic value and shows promising potential for development. For instance, the fruits are consumed by residents in Hainan after cooking, while in Taiwan, it has been cultivated as a landscaping tree and a source for truffle reproduction (You 2021).
However, habitat destruction and excessive deforestation have resulted in grave habitat fragmentation, population decline, and wild germplasm resources reduction of L. konishii. Based on a study utilizing chloroplast DNA atpB-rbcL for the genetic diversity analysis of L. konishii in Taiwan, it was found that the habitat of this species was severely damaged by the 1999 Chi-Chi earthquake, which resulted in a substantial loss of genetic diversity, placing L. konishii on the brink of endangerment in Taiwan. (Hung et al. 2005). Moreover, in the eastern part of Hainan, the population of L. konishii has been strongly affected by human activities. The expansion of roads, farmland, and housing have directly encroached upon its habitat (Shi et al. 2016). Due to the scarcity of wild populations of L. konishii, it has been evaluated as a vulnerable species (VU) and listed in the 'China Species Red List' (Wang and Xie 2004). However, few studies have focused on the maintenance and conservation of this species. This study sequenced the complete chloroplast genome of L. konishii, and explored its phylogenetic relationships with other species in the Fagaceae family, which could be valuable to the effective utilization and protection of this species, as well as the further phylogenetic studies of this family.

Methods
The total genomic DNA was extracted from fresh leaves using a modified cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1987). A genomic library consisting of an insert size of 300 bp was established by using a TruSeq DNA Sample Prep Kit (Illumina, USA) and sequenced on the Illumina Novaseq platform (Guangzhou Jierui Biotech). 5 Gb of raw data of 150 bp paired-end reads were obtained and further assembled using GetOrganelle v.1.7.7.0 (Jin et al. 2020). The GeSeq was used for chloroplast genome annotation (Tillich et al. 2017), whilst CPGAVAS2 was used to correct the annotated genome , after which it was manually checked by comparison against the complete cp genome of Lithocarpus hancei (GenBank accession number: MW375417) using Geneious v.9.0.2 (Biomatters, https://www. geneious.com) (Kearse et al. 2012). The complete chloroplast genome of L. konishii was submitted to GenBank with the accession number ON422319. The chloroplast Genome Viewer (CPGView, www.1kmpg.cn/cpgview/) (Liu et al. 2023) was used to visualize the structural features of L. konishii ( Figure 2).
To further understand the intrageneric phylogenetic relationship of L. konishii, the complete chloroplast genome sequences of 18 species of family Fagaceae and 2 outgroup species (Morella salicifolia and Carpinus laxiflora) from the National Center for Biotechnology Information (NCBI) were aligned using MUSCLE v.3.8.31 (Edgar 2004). Based on the concatenated shared unique CDS sequence dataset, the phylogenetic trees were constructed using the maximum likelihood (ML) method by IQ-TREE v.2.0.3 (Nguyen et al. 2015) and Bayesian Inference (BI) method by Mrbayes v.3.2.6 (Ronquist et al. 2012). For ML analysis, a best-fit model K3Pu þ F þ I was selected, and the reliability of the phylogenetic tree topology was evaluated with 1,000 repeated selfexpanding analyses; while for BI analysis, a best-fit model GTR þ G þ I was estimated by ModelTest-NG v.0.1.7 (Darriba et al. 2020) on the CIPRES Science Gateway (http://www. phylo.org/portal2/), the Markov Chain Monte Carlo (MCMC) was conducted for 5,000,000 generations and sampled every 100 iterations with the first 20% discarded. Branch supports were tested using the ultrafast bootstrap (UFBoot) (Hoang et al. 2018).
Both the ML and BI trees displayed identical topologies, here we presented the ML tree (Figure 3). The phylogenetic analysis shows that L. konishii is closely related to L. longnux and L. pachyphyllus var. fruticosus, and is the sister clade of Castanopsis and Castanea, forming a monophyly of the subfamily Castaneoideae, in agreement with the findings based on the nuclear and chloroplast DNA sequence data by Manos et al. (2001) and the morphological data by Wang and Bo (2004). Fagus represented an early branch within the family, consistent with the previous phylogeny studies of Fagaceae (Manos et al. 2001;Yan 2021;Wang and Bo 2004). These findings can serve as a valuable chloroplast genome resource for genetic research on germplasm resources update and afforestation tree species in the future. The map contains six tracks by default. From the center outward, the first track shows the dispersed repeats, which consist of direct (D) and palindromic (P) repeats, connected with red and green arcs. The second track shows the long tandem repeats as short blue bars. The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors. The colors, the type of repeat they represent, and the description of the repeat types are as follows. Black: c (complex repeat); green: p1 (repeat unit size ¼ 1); yellow: p2 (repeat unit size ¼ 2); purple: p3 (repeat unit size ¼ 3); blue: p4 (repeat unit size ¼ 4); orange: p5 (repeat unit size ¼ 5); red: p6 (repeat unit size ¼ 6). The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown on the fourth track. The GC content along the genome is plotted on the fifth track. The genes are shown on the sixth track. The optional codon usage bias is displayed in the parenthesis after the gene name. Genes are color-coded by their functional classification. The transcription directions for the inner and outer genes are clockwise and anticlockwise, respectively. The bottom left corner shows the functional classifications of the genes.

Ethical approval
This study was ethically approved and received permission for the sample collection from the Guangdong Zhuhai Qi'ao-Dangan Island Provincial Level Nature Reserve (Admission number: 07103300202302110900180053439). The fieldwork of this study was supported and assisted by the staff from Zhuhai Qi'ao-Dangan Island Provincial Level Nature Reserve. Lithocarpus konishii is vulnerable (VU) according to China species red list: Vol. I red list (2004)

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

Funding
This work was supported by the Guangzhou Science and Technology Project (202102021016); the Project of the Educational Commission of Guangdong Province of China (2020KTSCX368); and the Zhi Lan Foundation of Shenzhen of China (2020070181B).

Data availability statement
The data that support the findings of this study are openly available in NCBI at [https://www.ncbi.nlm.nih.gov/], reference number ON422319. The associated "BioProject", "Bio-Sample" and "SRA" numbers are PRJNA838448, SAMN28422863 and SRR19233775 respectively.