The first complete chloroplast genome of Alsophila costularis (Cyatheaceae), a least concerned relict tree fern

Abstract

It is the first report on complete chloroplast genome of Alsophila costularis, a least concerned relict tree fern. The genome size is 156,675 bp, with the typical quadripartite structure containing a pair of inverted repeats (IRs) of 24,356 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region of 86,338 bp and 21,625 bp, respectively. A total of 133 genes are predicted, including 89 protein-coding genes, eight ribosomal RNA genes, 33 tRNA genes, and three pseudogenes. The ML phylogenetic tree revealed A. costularisis as sister to Alsophila spinulosa.

Keywords:

• Alsophila costularis
• a least concerned relict tree fern
• chloroplast genome
• phylogeny

Alsophila costularis Baker, known as Cyathea chinensis Copel, is a relict fern with the accepted name in Cyatheaceae. Its trunk is 1.8–7.5 m tall with loose crown and rhizomes are covered with densely and scaly dark brown scales. Fronds are bipinnate and 1–2 m long. As a tree fern native to Yunnan in China, it occurs in India, Nepal, Myanmar, Laos, and Vietnam as well. The species grows in ravine forests at an altitude of 700–2100 m (Zhang and Nishida 2013). Different from other ‘living fossil’ ferns in Cyatheaceae, A. costularis is least concerned. The current report focuses only on tissue culture (Cheng and Liu 1992), gametophyte development (Wang et al. 2007), and genetic diversity (Li et al. 2010). Due to a second-class protected plant, considerable attention should be paid to the protection of A. costularis based on chloroplast genetic variation. In addition, A. costularis is very critical for the classification and phylogeny of Cyatheaceae (Ching 1978; Wang et al. 2003). Hence, it is necessary to acquire its whole chloroplast (cp) genome sequence for conservation genetics and the chloroplast phylogenomics of Cyatheaceae.

Fairy Lake Botanical Garden, Shenzhen, Chinese Academy of Sciences (22°34′45.69″N, 114°10′7.92″E) provided plant material of A. costularis, which is stored in the Herbarium of Sun Yat-sen University (SYS; voucher: SS Liu 201619). Tiangen Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China) was used to extract total genomic DNA from leaves, which was further broken and used for Illumina library preparation. Genome was sequenced on a Hiseq 2500 platform (Illumina Inc., San Diego, CA) with paired-end reads of 300 bp. We obtained 12,053,058 raw reads. After filtering out and trimming with Trimmomatic v0.32 (Bolger et al. 2014), the remaining 11,109,827 high-quality clean data with an average coverage of 100× were used to de novo assemble the chloroplast genome in Velvet (Zerbino and Birney 2008) by mapping to referenced-based chloroplast genomes of A. spinulosa (NC_012818). Using the same reference, all genes were annotated with the Dual Organellar GenoMe Annotator (DOGMA; Wyman et al. 2004) and tRNAscan-SE programs (Lowe and Eddy 1997). We used RAxML v8.0 (Stamatakis 2014) with 1000 bootstraps to reconstruct a maximum-likelihood (ML) phylogeny of 13 ferns including Diplopterygium glaucum as outgroup.

Alsophila costularis has a total length 156,675 bp with the typical quadripartite structure including a pair of inverted repeats (IRs) of 24,356 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region of 86,338 bp and 21,625 bp, respectively (Genbank accession number: MH684489). The genome contains 133 genes, including 89 protein-coding genes, eight ribosomal RNA genes, 33 tRNA genes, and three pseudogenes. Among them, 15 genes contain one intron, while three genes have two introns. Thirteen genes are duplicated, including four protein-coding genes, five tRNA genes, and four rRNA genes. The overall A/T content was 60%, while the corresponding values of the LSC, SSC and IR regions were 60%, 62% and 57%, respectively. The phylogenetic analysis revealed A. costularis as sister to A. spinulosa with 100% bootstrap support (Figure 1). The complete chloroplast genome of A. costularis lays a solid foundation for further phylogenetic studies.

Figure 1. Maximum likelihood phylogenetic tree of A. costularis with 12 ferns including Diplopterygium glaucum as outgroup based on chloroplast genome sequences.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [31370364, 31570652, 31670200, 31770587, and 31872670], the Natural Science Foundation of Guangdong Province, China [2016A030313320 and 2017A030313122], Science and Technology Planning Project of Guangdong Province, China [2017A030303007], Project of Department of Science and Technology of Shenzhen City, Guangdong, China [JCYJ20160425165447211, JCYJ20170413155402977, and JCYJ20170818155249053], and the Science and Technology Planning Project of Guangzhou City, China [201804010389], Chang Hungta Science Foundation of Sun Yat-sen University, and Undergraduate Training Programs for Innovation and Entrepreneurship of Sun Yat-sen University.