The complete chloroplast genome sequence of Bolbitis multipinna (Dryopteridaceae)

Abstract Bolbitis multipinna is an endemic fern found in Yunnan, China. In the case of the chloroplast genome, Illumina Hiseq X-Ten was used, and it revealed a conserved quadripartite structure of 152,437 bp, including a large single-copy (LSC) region of 82,618 bp, a small single-copy (SSC) region of 21,403 bp, and a pair of inverted repeat regions of 24,208 bp (IRa and IRb). The chloroplast genome of B. multipinna contains 132 genes, including 89 protein-coding genes, 35 tRNA genes, and eight rRNA genes. By using the maximum-likelihood method and the Bayesian analysis on the chloroplast genomes of 15 species ferns, the phylogenetic analysis revealed that B. multipinna is closely related to B. heteroclita.

With about 80 species, Bolbitis is a pan-tropical genus mainly found in Asia and Pacific islands (Moran et al. 2010;Zhang et al. 2013;PPG I 2016). Bolbitis multipinna is endemic to Yunnan, China, and was first described by Wang et al. (2008). It presents significant morphological variability, with one to more reticulate veins appearing on simple pinnate leaves (Zhang et al. 2013). Despite recent study of Bolbitis, there is little information on the chloroplast genomes (Chao et al. 2019). An in-depth analysis of chloroplast genomics of this genus will contribute to the development of a phylogenetic framework for morphological and taxonomical studies of the species.
The raw reads of B. multipinna are approximately 6 G, and there are 152,437 bp in the B. multipinna chloroplast genome, including a large single-copy (LSC) region of 82,618 bp, a small single-copy (SSC) region of 21,403 bp, and a pair of inverted repeat regions of 24,208 bp (IRa and IRb). In total, 132 genes are encoded by the B. multipinna chloroplast genome, including 89 protein-coding genes, 35 tRNA genes, and eight rRNA genes ( Figure 2). In the LSC and SSC regions as well as the IRs, the GC content was 41.3%, 38.8%, and 45.3%, respectively.
In order to infer the phylogenetic position of the newly sequenced B. multipinna in Bolbitis, we constructed a phylogeny using this newly sequenced genome in conjunction with 14 published chloroplast genomes in NCBI (Figure 3). The 71 common chloroplast protein-coding genes were extracted from the 15 complete genomes, which were then aligned using MAFFT v7 (Katoh and Standley 2013). By concatenating these alignments, phylogenetic trees were generated using maximum-likelihood (ML) and the Bayesian inference (BI) methods in IQ-TREE v1.4.2 (Nguyen et al. 2015) and MrBayes v3.2.1 (Ronquist et al. 2012). Based on the results, B. multipinna was found to be more closely related to B. heteroclita with strong support (MLBS: 100%, BIPP: 1). Bolbitis was well supported as the sister species of Elaphoglossum (MLBS: 100%, BIPP: 1), which is in agreement with previous studies (Moran 2010;Liu et al. 2016).

Author contributions
The paper was drafted by Xinxin Cheng who assembled and annotated the samples; data analysis and interpretation were performed by Liyun Nie and Yujie Liao; Faguo Wang was responsible for the experimental design and the revision of the paper; all authors are accountable for all aspects of the work.

Ethics statement
Neither the samples used in this study nor the sampling site is listed on either the IUCN Red List of Threatened Species or the List of State-protected Plant Species. Both field and laboratory studies were conducted in accordance with the guidelines formulated by the South China Botanical Garden, Chinese Academy of Sciences.

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

Funding
This study was supported by the grants from the National Natural Science Foundation of China [Grant No. 31870188].