3 Discussion
3.1 Characteristics of SSRs of chloroplast genomes of eight species of Cyatheaceae
The chloroplast genomes of all eight species of Cyatheaceae are similar in structure and gene content, and the types and order of genes are the same. On the phylogenetic background of dividing the eight species of Cyatheaceae into three genera, the characteristics of the chloroplast genome SSRs have genus specificity. The distribution of SSRs is not random, which has an effect on gene regulation, DNA recombination, DNA replication, the cell cycle, and DNA mismatch repair, and the presence of repeat motifs and their repeat number can affect DNA recombination (Li et al., 2002). SSR copy number is an important source of genetic variation and can produce large phenotypic variation (Gemayel et al., 2010; Kashi & King, 2006). Single-nucleotide SSRs are the most abundant, which is a characteristic of eukaryotic genomes (Sharma et al., 2007), and A/T motifs are the most common. The number, relative abundance, relative density, and GC content of SSRs of different unit length are also genus specific. This is especially true for single-nucleotide and dinucleotide SSRs, which may be related to the lower content of SSRs of other unit lengths. The distribution of different repeat types (from single-nucleotide to hexanucleotide) of motifs in coding and noncoding regions, introns, and intergenic regions displays a high degree of taxon specificity, which can be partially explained by the interaction of mutation mechanisms and differential selection (Toth et al., 2000). The SSRs in eukaryotic genomes are mainly located in intergenic and noncoding regions, with a few in exons (Toth et al., 2000; Li et al., 2004), and the results of this study are consistent with this. This phenomenon is related to the higher variability (Nie et al., 2012; Wu et al., 2010) and faster evolutionary rates of the intergenic and noncoding regions of the chloroplast genome, so their sequences can be used to effectively classify low taxonomic and closely related groups and subspecies variant plants. The proportion of SSRs in the IR region was 2-3.3 times the proportion of IR sequences out of the whole genome sequence (Figure 3), indicating that there are fewer SSRs in the IR region. Mismatch repair is the key to the stability of SSRs. SSRs have a high mutation rate, which facilitates the study of the effect of environmental factors on the mutation rate of the genome. The lower distribution of SSRs in the IR region may be related to the lower mutation rate in the IR region (Ellegren, 2004; Li et al., 2016). The lower GC content may be associated with the fact that GC-rich regions are prone to mutations toward AT(Ren et al., 2007). The high GC content is significantly associated with the high recombination rate in meiosis (Tortereau et al., 2012), while AT-rich SSRs may be more conducive to maintaining the stability of the genome structure. The number, relative abundance, relative density, and GC content of SSRs are not proportional to the size of genome, indicating that the abundance of SSRs is related to the genetic characteristics of the species (Li et al., 2014).
3.2 Phylogenetic significance of SSR characteristics of chloroplast genomes of the eight species of Cyatheaceae
The focus of the debate between the Holttum and Edwards (1963) system and the Tryon (1970) system, which classify Cyatheaceae based on morphological characteristics, is the theoretical explanation of the morphological evolution of indusium in this family. In this study, we show that the genus Sphaeropteris , lacking the indusium, is a basal group, supporting Tryon’s hypothesis that the indusium is derived from the tissues or scales on the abaxial side of the leaf, away from the leaf margin, and that the indusium is a derived trait, which is consistent with a phylogenetic analysis based on the chloroplasttrnL intron sequence and the trnL-F intergenic region sequence (Wang et al., 2003). Dong (2018) pointed out thatGymnosphaera and Alsophila were significantly differentiated in morphological traits such as petiole color, the presence or absence of degenerated pinnae at the base, and the presence or absence of indusium and sporogenesis, and advocated the restoration of the hierarchical status of the genus Gymnosphaera to reflect the divergence mechanisms of this group of plants in molecular phylogeny, morphology, and sporogenesis. In this study, eight species of Cyatheaceae were divided into three genera or two genera, in which case SSRs were compared by the Kruskal-Wallis H test or the Mann-Whitney U test, respectively, and the results showed that Gymnosphaera was an independent genus-level taxon under the Cyatheaceae family. The results of the PV clustering analysis of SSRs also indicated thatGymnosphaera should be independent from the genusAlsophila . Phylogenetic trees were constructed using the chloroplast genomes of eight species of Cyatheaceae, and the topology of the phylogenetic trees obtained by the four methods was consistent. Except for the lower support rate of the branches of the Alsophila denticulata and Alsophila gigantea , the support rate for the branches was 100%. This result supports the monophyletic nature ofGymnosphaera , and its sister group is the genus Alsophila .Gymnosphaera , Alsophila , and Cyathea constitute a monophyletic group with a high support rate, while Sphaeropterisis resolved as the basal group of the Cyatheaceae family. In this phylogenetic context, the SSR characteristics have genus specificity.
SSRs play a role in genome-wide regulation. Some definite distribution patterns exist in the genomes of organisms, and the characteristic distribution of SSRs in the genomes of different taxonomic units has a significantly similar pattern (Qi et al., 2015; Wang et al., 2015; Liu et al., 2017; Manee et al., 2019; Srivastava et al., 2019). The SSRs of different groups of genomes have specific distribution patterns, which are related to their common ancestors. Evolutionary trends have been linked to the inclusion of SSRs, which may have been preserved because of their ability to adapt to novel regulatory mechanisms (Srivastava et al., 2019). Unique DNA replication, repair, and recombination mechanisms may play an important role in the evolution of SSRs (Katti et al., 2001). The molecular mechanism of the origin of SSRs is not yet fully understood. The most common mutation mechanism affecting SSRs is slipped replication. Other mechanisms, such as unequal crossing-over, nucleotide substitution, and duplication events, are also responsible for SSR variation(Schlotterer & Tautz, 1992; Hancock, 1999). Codon preference, DNA replication, and mismatch repair systems, as well as the unique structure and function of the genome, may be responsible for the unique SSR distribution pattern in plant genomes. In addition, the length, motif structure, and GC content of genomic SSRs are also factors that influence the evolution of the SSRs (Chakraborty et al., 1997; Anderson et al., 2000; Whittaker et al., 2003). The plants in the three genera of Cyatheaceae have similar phenotypes and specific characteristics, which may be the result of interactions of their common ancestors with similar habitats. The analysis of the characteristics of SSRs provides useful clues for the phylogenetic study of Cyatheaceae and helps to understand the evolution of SSRs in plant genomes.
The chloroplast genome has a simple structure, low molecular weight, and high copy number, and its genes are maternally inherited in ferns with few gene rearrangements, thus facilitating the study of plant phylogeny (Tonti-Filippini et al., 2017). Based on high-throughput sequencing technology, the highly conserved chloroplast genome sequence will make primer design easier. SSR markers in chloroplast genomes can be used for the analysis of multiple chloroplast regions, thereby improving the resolution of phylogenetic studies of target species (Melotto-Passarin et al., 2011). Since the software programs that identify SSRs are limited by their efficiency and parameter settings and may also be affected by the quality of the SSR dataset generated, their accuracy needs to be improved (Ellegren, 2004; Lim et al., 2013). In addition, chloroplast genomes play an important role in dissecting the higher hierarchical phylogenetic relationships of ferns. However, in the process of evolution, plants experience events such as hybridization, polyploidization, introgression, and incomplete lineage sorting, so the evolutionary relationship of plants is essentially reticular. Such reticular relationships are often manifested in the form of gene tree conflicts(Guo & Ge, 2005) .
In this study, the chloroplast genomes of eight species of Cyatheaceae were used to construct phylogenetic trees. This analytical method has some limitations. There are still relatively few studies on phylogenetic relationships in ferns that have analyzed the SSRs of the chloroplast genome. This study provides a new basis for the classification of Cyatheaceae at the levels of species and genus, thus advancing the phylogenetic study of Cyatheaceae. In the future, more genomic and transcriptomic data are needed to validate these results.