The complete chloroplast genome sequence of the water fern Ceratopteris thalictroides (Pteridaceae)

Abstract This work determined and analyzed the complete chloroplast genome sequence of Ceratopteris thalictroides (Linnaeus) Brongniart 1822 (Pteridaceae). The results indicate that the total chloroplast genome size of C. thalictroides is 149,399 bp in length, and the genome contains a large single-copy (LSC) region of 83,580 bp, a small single-copy (SSC) region of 21,241 bp, and a pair of inverted repeat (IR) regions of 22,289 bp. The GC content of C. thalictroides is 36.7%. The genome encodes a total of 131 unique genes, including 82 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The phylogenetic analysis results strongly suggest that C. thalictroides is closely related to C. cornuta.

The water fern Ceratopteris thalictroides (Linnaeus) Brongniart 1822 grows in marshlands and paddy fields and sometimes floats on the water. It was previously classified as a member of Parkeriaceae (Ching 1978) but is now considered a member of Pteridaceae, which is located at the base of the order Polypodiales on the phylogenetic tree (Smith et al. 2006;PPG I 2016;Shen et al. 2018). It is widely distributed worldwide throughout the tropics. Because of the destruction of its habitat and extensive plundering for its edible, ornamental, and medicinal properties, C. thalictroides is treated as a second-class protected wild plant in China (National Forestry Administration of the People's Republic of China & Ministry of Agriculture of the People's Republic of China 2021). C. thalictroides is an excellent model plant because of its short lifespan and diverse mechanisms of reproduction (Li and Wang 1997). To date, no studies on the complete chloroplast genome of C. thalictroides have been published. Therefore, we analyzed the complete chloroplast genome of C. thalictroides in this study for subsequent molecular phylogenetic analysis.
The genomic DNA was extracted from the silica gel dried leaf of C. thalictroides and sequenced on the Illumina HiSeq platform (Shanghai Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China). The plastid genome was assembled using GetOrganelle  with the chloroplast genome of C. cornuta (accession number: MH173068) as the reference sequence. The assembled chloroplast genome was annotated by Geneious Prime (Biomatters Ltd., Auckland, New Zealand) (Kearse et al. 2012). Finally, the complete chloroplast genome of C. thalictroides was obtained; the genome sequence data are openly available in the GenBank database of the National Center for Biotechnology Information (NCBI) at https://www. ncbi.nlm.nih.gov/ under accession number OK524221.
The results indicate that the chloroplast genome size of C. thalictroides (OK524221) is 149,399 bp in length, and the genome contains a large single-copy (LSC) region of 83,580 bp, a small single-copy (SSC) region of 21,241 bp, and a pair of inverted repeat (IR) regions of 22,289 bp. The GC content of C. thalictroides is 36.7%. The genome encodes a total of 131 unique genes, including 82 protein-coding genes, 38 tRNA genes, and 8 rRNA genes.
For the molecular phylogenetic analysis, the complete chloroplast genomes of 15 ferns were downloaded from GenBank to obtain a phylogenetic tree containing C. thalictroides. The sequences were aligned by MAFFT (Katoh and Standley 2013). The phylogenetic tree was constructed using the IQ-TREE (maximum likelihood) method in PhyloSuite, and branch supports were constructed with ultrafast bootstrap approximation (Guindon et al. 2010;Minh et al. 2013;Nguyen et al. 2015;Zhang et al. 2020). The results revealed that C. thalictroides is a sister of C. cornuta MH173068 (Figure 1). This is consistent with previous studies on the Pteridaceae phylogenetic tree.

Ethical approval statement
Research on plant chloroplast genome sequencing does not require ethical approval.

Author contribution statement
Liu Xing-Feng (corresponding author and first author): resources, conceptualization, investigation, and writing-original draft. Zhou Xi-Le (first author): investigation, funding acquisition, resources, supervision, writing-review & editing. Gu Yu-Feng: data curation, methodology, and visualization. Liu Si-Fan: resources, formal analysis, and validation. Yu Jun-Hao: data curation. Zhang Rui: funding acquisition and methodology. Shu Jiang-Ping: software. All authors have read and agreed to the published version of the manuscript.

Plant material collection statement
The species is widely distributed in China; the plant materials were not obtained from nature reserves, and the chloroplast genome sequencing research did not affect the population, so collection licenses were not needed.