The complete chloroplast genome sequence of Lithocarpus longinux (Fagaceae).

Lithocarpus longinux (Hu) Chun ex Y.C.Hsu & H.Wei Jen is a Critically Endangered tree distributed in Ma-li-po county in the Southeastern Yunnan Province China. Less than ten individuals have been found since the species was established 70 years ago. In this study, we assembled and annotated the complete chloroplast genome of L. longinux. The complete chloroplast genome of the species is 161,420 bp in length and has a GC content of 36.8%, including one large single-copy region (LSC, 90,409 bp), one small single-copy region (SSC, 19,255 bp), and two copies of inverted repeat regions (IRs, 25,878 bp). A total of 112 unique genes were detected, including 81 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Phylogenetic analysis of 31 representative chloroplast genomes of the Fagales suggests Lithocarpus is monophyletic with strong bootstrap support and that L. longinux is closely related to L. balansae.

Lithocarpus Blume is one of the dominant woody lineages widespread in the East Asian monsoon evergreen broadleaved forests with ca. 400 species (Govaerts and Frodin 1998, Huang et al. 1998, Huang et al. 1999). Many species of the genus are canopy trees in the forests playing significant roles in regional ecosystem functioning and biodiversity conservation (Cannon et al. 2018).

Lithocarpus longinux is a Critically Endangered tree with a restricted distribution in Ma-li-po county of the Southeastern Yunnan Province, China. Its habitat is locateds in mid-altitude montane cloud limestone forests. The species was first described in 1951 (Hu 1951), called Pasania longinux. This species name Lithocarpus longinux was first published in 1976 (Chun et al. 1976). However, according to Flora of China (both Chinese and English versions), the species used to be treated as a synonym of L. areca (Huang et al. 1998, Huang et al. 1999). Fortunately, some researchers found that the entire leaf margin and prominent concentric rings on the cupule of L. longinux differ from L. areca. Therefore, the name L. longinux was reinstated by Zhou Wei et al. (Zhou et al. 2013). Moreover, based on our previous Endangered status assessments on Asian Fagaceae, less than 10 individuals of L. longinux were found. The species urgently requires conservation.

Like other Lithocarpus species, L. longinux is a typical biennial fruiting species. Its flowering time is from March to April, and the fruits fully ripen from September to October of the following year (Zhou et al. 2013). Despite the genus’ significant ecological and economic importance, the genome information on Lithocarpus is limited, particularly in the chloroplast genome. Only three whole chloroplast genomes of the genus have ever been uploaded to Genebank. Meanwhile, the phylogenetic framework on Lithocarpus is still not resolved due to limited sampling and low throughput of SNPs generated by Sanger-based sequencing markers, which cannot provide a robust phylogenetic framework. As a result, the phylogenetic trees on Lithocarpus obtained by previous works only received mediate-low credibility supports at the main topologies (Yang et al. 2018). Therefore, the use of genome data is essential for untangling the genus’ evolutionary history. Compared with nuclear genomes, chloroplast genomes have multiple advantages, e.g. relatively conservative structure, uniparental inheritance, and easy sequencing (Birky et al. 1983). Therefore, the complete chloroplast genome has been widely used in the phylogenetic studies of various plant taxa (Daniell et al. 2016).

In this study, the complete chloroplast genome of L. longinux was sequenced, assembled, and annotated. The phylogenetic tree of Fagaceae was constructed to test the discrimination abilities of the chloroplast genome at different taxonomic ranks. Our study can provide helpful genomic information for conserving this Endangered species and to further explore the chloroplast genome evolution pattern of the genus Lithocarpus.

Fresh leaves of L. longinux were collected from Ma-li-po county, Yunnan Province, China (23.1673 N, 104.8540 E, alt. 2300 m). The voucher specimen was deposited at herbarium of Kunming Institute of Botany (Tao Deng, dengtao@mail.kib.ac.cn) under the voucher number DM22970. Total genomic DNA was extracted from silica-dried leaves based on modified CTAB method (Doyle and Doyle 1987). Whole-genome sequencing was conducted with the paired-end (PE) reads of 2 × 150 bp on the Illumina NovaSeq platform. Raw reads were filtered and trimmed to remove the low quality reads by Fastp (Chen et al. 2018). A total of 60,366,324 clean reads were used for de-novo assembly with GetOrganelle v1.7.2 (Jin et al. 2020). Gene annotation was performed by the pipeline PGA (Qu et al. 2019) and Geneious V10.2.3 (Kearse et al. 2012). Circular mapping of the L. longinux chloroplast genome was performed using the OGDRAW online tool (Lohse et al. 2013). The annotated genome sequence was deposited in GenBank under Accession Number OK181903.

The complete chloroplast genome of L. longinux is 161,420 bp in length. It has a typical quadripartite structure including one large single-copy (LSC) region (90,409 bp), one small single-copy (SSC) region (19,255 bp), and two copies of inverted repeat (IRs) regions (25,878 bp). The overall GC content of this species is approximately 36.8% (LSC, 34.6%; SSC, 31.0%; IRs, 42.7%). A total of 112 unique genes, including four rRNA genes, 29 tRNA genes, and 81 protein-coding genes, were annotated.

To infer the systematics of L. longinux, 31 published complete chloroplast genomes of Fagales were aligned by MAFFT 7.487 (Rozewicki et al. 2019). Finally, a maximum likelihood (ML) tree was constructed using RAxML v.7.2.6 with 1000 bootstraps under the GTRGAMMA model (Stamatakis 2014). The monophyletic status of Lithocarpus with two main clades within the genus was robustly supported (100% boostrap). L. longinux and L. balansae are sister species (Figure 1). This result suggest that complete chloroplast genome data has good potential to resolve species phylogenetic placement in Lithocarpus.

Figure 1.

The phylogenetic tree of Lithocarpus longinux and other 30 species from Fagales (incl. 26 species from family Fagaceae, two species from family Betulaceae, one species from family Myricaceae and family Juglandaceae respectively) were reconstructed based on maximum-likelihood analysis with 1000 bootstraps under the GTR + GAMMA model. The bootstrap value for each clade is provided above.

Author contributions

Min Deng and Chun-Ya Wu were involved in the conception and design, Min Deng and Lin Lin collected the plant materials, Chun-Ya Wu, Kai-ping Yao and Rui-Jie Yang performed experiments, Chun-Ya Wu analyzed the data, Min Deng and Chun-Ya Wu wrote the manuscript.