4 DISCUSSION
Based on the comparative genomic analyses of the two strains of F. candida , we found significant differences between them in terms of both genome structure and gene distributions. The genome of FCDK (219.08 M) was 65.18 M (42.4%) larger than that of FCSH (153.90 M); half of this difference consisted of repetitive elements (34.1 M); and each chromosome of FCDK was 25‒54% longer than the corresponding chromosome of FCSH (Figure 1). The seven pairs of chromosomes showed obvious large-scale chromosomal inversions and translocations, and several large distal regions of FCDK chromosomes 1, 3, 4 and 7 did not share any syntenic blocks with FCSH (Figure 2). There were 3,530 more PCGs annotated in FCDK than in FCSH (Table 1). The numbers of FCDK-specific gene families and genes (572/2285) were far greater than the numbers of FCSH-specific sequences (204/820) (Table S7). In addition, FCDK and FCSH showed obvious divergence between their mitochondrial genomes and miRNA distributions. The overall sequence identity of the two mitogenomes was only 78.2%, and the pairwise identity of their standard DNA barcodes (658 bp of COI) was 80.9%, which is much lower than the similarity observed in individuals from the same insect species (Hebert et al., 203). Although 91 common miRNAs of FCDK and FCSH showed conserved synteny on their homologous chromosomes, there were many more strain-specific miRNAs in FCSH (39 miRNAs from 15 predicted families) than in FCDK (9 miRNAs from 6 predicted families). Moreover, most FCDK-specific miRNA families had only one copy, but many FCSH-specific miRNA families showed multiple duplications, which tended to form clusters on chromosome 7 (Figure 6). All of these genetic differences demonstrated that after 10 million years of divergence (Figure 3), FCDK and FCSH have evolved into distinct species.
In F. candida , parthenogenetic strains usually show a wider distribution and stronger environmental adaptability than bisexual strains (Tully & Potapov, 2015). The genomic differences revealed in this study demonstrated obvious evolutionary advantages of the parthenogenetic FCDK genome. Relative to bisexual FCSH, FCDK showed more strain-specific and expanded gene families, many of which were involved in resistance to environmental xenobiotics, such as the ABC, CYP and GST families (Table 2). Interestingly, histone H2A, one of the core histones, showed obvious expansion in FCDK (Figure 3b). The sequences and expression patterns of core histones can profoundly alter chromatin properties, and their posttranslational modifications, such as acetylation, methylation, and ubiquitination, are involved in the regulation of nucleosome dynamics (Lawrence et al., 2016). In particular, variants of histone H2A are associated with DNA repair and gametogenesis (Hanson et al., 2013) as well as environmental responses (Talbert & Henikoff, 2014). The potential roles of expanded H2A genes in FCDK deserve further study. In addition, FCDK genes exhibited longer introns than FCSH genes (Table 1). Introns can provide selective advantages to cells by regulating alternative splicing, gene expression, chromatin assembly, etc. (Jo & Choi, 2015), and they might play roles in determining species-specific characteristics and complexities (Carvunis et al., 2012).
The genetic drivers of species differentiation have always been a hot research topic. Our study reveals several possible factors contributing to the genetic divergence between FCDK and FCSH. First, the increase in transposon copies is a very important driving force leading to the enlargement of the genome (Chalopin et al., 2015; Talla et al., 2017), and transposable elements may contribute significantly to divergent chromatin structures (Diehl et al., 2020). In this study, we found that transposons were significantly enriched in the FCDK-specific regions of Chr1, Chr3, Chr4, and Chr7 (Figure 2), and many repeat families were obviously expanded in FCDK (Figure 3b). Most chromosomal regions with a high transposon density showed a low gene density and high recombination (Figure 1). Second, a large number of genes acquired through HGT located in FCDK-specific chromosome regions probably play important roles in the spread of antibiotic resistance and genome evolution (Faddeeva-Vakhrusheva et al., 2017). Significantly, Wolbachiainfection may induce or accelerate speciation and contribute to reproductive isolation (Werren, 1998), which is probably associated with increased recombination (Singh, 2019). Although we did not find any obvious genomic characteristics related to the differences in reproductive mechanisms between FCDK and FCSH based on comparative genomic analyses, our high-quality genome data provide a basis for further mechanistic studies. In addition, strain-specific miRNAs could drive the adaptive diversification of the genomic regulatory mechanisms of FCDK and FCSH. Novel miRNAs could be gained and lost relatively rapidly across closely related groups, especially when these groups undergo extensive adaptive diversification (Xiong et al., 2019). It would be interesting to further study how miRNA genes evolve and contribute to speciation.
In conclusion, we generated two high-quality chromosome-level genomes of the Folsomia candida DK and SH strains, revealed their genomic differences, and proposed that they have differentiated into two separate species. Our work provides important genomic resources for studying speciation and the effect of Wolbachia on host reproduction and genetic differentiation, as well as the basis for mechanistically understanding soil arthropods in the context of soil ecology, and the evolution links between insects and crustaceans.