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.