* Correspondence:
Yingjuan Su, Xingang West Road, Guangzhou, Guangdong, IA 510275, China.
Email: suyj@mail.sysu.edu.cn
Ting Wang, Wushan Road, Guangzhou, Guangdong, IA 510640, China.
Email: tingwang@scau.edu.cn
Abstract Recent taxonomic and molecular phylogenetic studies have shown that Gymnosphaera should be recognized as an independent taxonomic unit at the genus level under the family Cyatheaceae. In this study, the complete chloroplast genomes of the eight species of Cyatheaceae were sequenced, and their phylogenetic relationships were reconstructed using the maximum likelihood, Bayesian inference, maximum parsimony, and neighbor-joining methods, and the characteristics of their simple sequence repeats (SSRs) were compared and analyzed for the first time. The results showed that when Cyatheaceae was divided into three genera,the number, relative abundance, relative density, and GC content of all SSRs and of SSRs of certain unit lengths in the chloroplast genomes of the eight species of Cyatheaceae were genus specific in the whole chloroplast genomes and in their different regions (large single-copy, small single-copy, inverted repeat, intergenic spacer, intron, rRNA gene, and coding sequence regions). The SSRs overall and the single-nucleotide SSRs had significant differences in number, relative abundance, relative density, and GC content between the chloroplast genomes, their intergenic regions, and large single-copy regions. When Cyatheaceae was divided into two genera, only the difference in GC content was significant. Therefore, our results support the restoration of the hierarchical status of Gymnosphaera . This study provides an important basis for the identification of the phylogenetic relationship of Cyatheaceae plants.
Keywords Cyatheaceae, Phylogeny, SSR, Gymnosphaera
Also known as microsatellites, simple sequence repeats (SSRs) are short tandem repeat sequences with a motif length of 1-6 bp that are characterized by high variability, high repeatability, codominant inheritance, and interspecies generality. SSRs have been widely used in species identification, genetic diversity, and phylogenetic relationships(Chmielewski et al., 2015; Dashnow et al., 2015). SSRs are caused by slipped strand mispairing and subsequent errors during DNA replication, repair, and recombination (Levinson & Gutman, 1987). They are widespread in the genomes of prokaryotes, eukaryotes, and some viruses(Mrazek et al., 2007; Toth et al., 2000; Zhao et al., 2011). SSRs are mainly found in intergenic and noncoding regions, with a few in introns (Toth et al., 2000; Li et al., 2004 ). Under selection, the number of genomic SSRs is significantly higher than that under random accumulation(Ellegren, 2004). The characteristics of genomic SSRs in different taxa (such as their distribution pattern) accurately reflect their phylogenetic relationships (Srivastava et al., 2019). SSRs are highly mutagenic and are an important source of genetic evolution, and they can play an important role in genome evolution by generating and maintaining quantitative genetic variation (Chaoui et al., 2012; Gemayel et al., 2010; Kashi & King, 2006). Therefore, the identification and characterization of SSRs can contribute to the study of population variation, germline identification, and genetic diversity.
Cyatheaceae belong to the phylum Pteridophyta, the class Polypodiopsida, and the order Cyatheales, with more than 600 species present today. Cyatheaceae account for the vast majority of known tree ferns and are mainly distributed in warm and humid tropical and subtropical regions (Kramer, 1990; Korall et al., 2006; Smith et al., 2006; PPG I, 2016). The classification of the genera in Cyatheaceae has always been a difficult problem. In the early classification system, Christensen (1906) classified Cyatheaceae into Cyathea Sm. (with a complete cup-shaped indusium), Hemitelia R. Br. (with a partial indusium), and Alsophila R. Br. (without an indusium) based on the characteristics of indusium. Holttum (1963) treated Cyatheaceae plants as a genus (Cyathea ) and divided it into two subgenera,Cyathea subg. Cyathea (marginate scales) andCyathea subg. Sphaeropteris (Bernh.) Holttum (conform scales), based on the structural characteristics of the scales at the base of the petiole. Based on the study of the American Cyatheaceae plants, Tryon (1970) delineated three evolutionary paths and six genera, namely, (1) Sphaeropteris Bernh (with conform scales); (2)Alsophila and Nephelea R. M. Tryon (with marginate scales and apical setae); and (3) Trichipteris C. Presl, Cyatheaand Cnemidaria C. Presl (with marginate scales but without apical setae). Ching (1978) classified the Cyatheaceae plants that grow in China into three genera: Sphaeropteris , Gymnosphaera andAlsophila . On this basis, Xia (1989) reduced the generaAlsophila and Alsophila to subgenera, respectively, and combined them into Alsophila .
Recent molecular phylogenetic studies have shown that Cyatheaceae includes four monophyletic groups, namely, Alsophila ,Cyathea , Gymnosphaera , and Sphaeropteris (Dong & Zuo, 2018; Janssen & Rakotondrainibe, 2008; Korall et al., 2007; Korall & Pryer, 2014). The morphological characteristics ofGymnosphaera , such as marginate scales, a slightly black rachis, and the lack of an indusium, and molecular evidence suggest that it is different from other Cyatheaceae plants, and particularly obvious is its different sporogenesis from Alsophila (Gymnosphaeraproduces 64 spores per sac, Alsophila produces 16 spores per sac) (Dong & Zuo, 2018). Still, some scholars classify Gymnosphaerainto the genus Alsophila (Xia 1989; Zhang and Nishida, 2013; PPG I, 2016).
In this study, the chloroplast genomes of eight species of Cyatheaceae were sequenced, and the chloroplast genome, the SSRs of different unit lengths in the chloroplast genome, and the number, relative abundance, relative density, and GC content of all SSRs and SSRs of specific unit lengths in the intergenic spacer regions (IGS), intron regions, and protein-coding sequence regions (CDS) of the chloroplast genome were compared and analyzed to reveal the phylogenetic significance of the SSR characteristics of the chloroplast genome of Cyatheaceae plants.