Population Genetic Structure And Complete Mitochondrial Genome Of Citrus Red Mite Panonychus Citri (Acari: Tetranychidae)

The citrus red mite, Panonychus citri (McGregor) (Arachnida, Acari: Tetranychidae), has a worldwide distribution and is regarded as one of the most important citrus pests in many countries. In China, the distribution range of this mite covers all citrus planting regions. This mite is often difficult to manage because their ability to rapidly develop resistance to various acaricides. Currently, few studies on P. citri focus on the population genetics and evolution, largely because of the lack of efficient genetic markers and the difficulty in DNA extraction from a single mite. Reliable estimates of genetic diversity and population genetic structure of P. citri from different geographical regions are crucial to gain insight into the role of different evolutionary forces and environmental factors in determining population dynamics, and to make a decision on effective pest management strategies. Recently, it has been reported that the resistance of spider mites to the acaricide bifenazate is highly correlated with the remarkable mutations in the mitochondrial cob gene. Therefore, sequencing the complete mitochondrial (mt) genome of P. citri not only increases the amount of Acari mt genomes, prompts the molecular systematics of Acari and other arthropods, but also allows the identification of specific changes of mitochondrial cob and the subsequent development of robust diagnostics, which are essential in resistance management.In this study, we constructed the P. citri microsatellite-enriched libraries, analyzed the genetic structure of the P. citri populations using the mitochondrial coxl gene and ribosomal internal transcribed spacer 1 (ITS1), sequenced the complete mt genome of P. citri and provided a comparison to other Acari. Additionally, we identified several mitochondrial genes as potential markers for population genetics/phylogenetics studies for spider mites, and assessed the utility of complete mt genome sequences as molecular markers for phylogenetic analyses of Acari. The main results are as follows:1. Three microsatellite-enriched libraries of AC-repeat, TC-repeat, and ATG-repeat were constructed for P. citri using microsatellite-enrichment method. A total of 44 unique microsatellite loci, which can be used to design PCR primers, were obtained by sequencing partial positive clones. Sequence analyses showed that a much larger proportion of microsatellite loci of AC library shared the same flanking regions and some were present as multi-copy microsatellite DNA families. Therefore, the microsatellite loci from TC and ATG libraries will be more suitable for the study of population genetic structure of P. citri. The DNA digestion and ligation were performed simultaneously, which enhanced the concentration of enzyme-digested products and the efficiency of microsatellite enrichment.2. The genetic diversity, population differentiation, and gene flow among 15 P. citri populations were investigated using the mitochondrial coxl gene and ITS1 sequence. The main results are:(1) There were 22 haplotypes among 15 geographical populations based on the coxl gene, whereas total 134 haplotypes were found in these populations based on the ITS1 sequence. Comprehensively analyses of these results, we proposed that the P. citri populations may have relatively high genetic diversity, which probably is one of the most important reasons that this mite has the ability to severely infest citrus and rapidly develop resistance to various acaricides. (2) The phylogenetic tree and haplotype network showed that the coxl or ITS1 haplotypes from the same citrus planting regions or populations did not cluster together. AMOVA analyses showed that there was no significant population genetic structure among the P. citri populations examined. However, an AMOVA without Yuxi and Danjiangkou populations found a weak, but significant geographic structuring(coxl). Also, when all populations from citrus belt of upper and middle reaches of Yangtze River (UMYR) and citrus base of Yungui Plateau (YGP) were considered as a large group, a significant population structure was detected at this large scale (ITS1). Additionally, the influence of host plants on the genetic structure of P. citri populations was detected by the AMOVA analysis for five host-related groups based on the ITS1 sequences. On the whole, there was no significant genetic differentiation among most populations, which can be contribute to several factors together, i.e. ongoing gene flow, the retention of ancestral polymorphisms, and natural selection (e.g., host plants, acaricides). (3) Although no significant genetic differentiation among most populations, even there were high levels of gene flow among some geographically far populations (e.g., Meishan and Guangdong populations), the Mantel tests showed that the isolation by distance was a factor responsible for the genetic differentiation. Due to its small body size and wingless, the dispersal ability of P. citri is very limited. Thus, long-distance dispersal and gene flow of P. citri among populations may largely rely on passive dispersal by the movement of plants between populations and other human activities. (4) The results of neutrality tests, mismatch analyses, and star-like network strongly supported that P. citri in China have undergone population expansion in the past. (5) Considering that Chinese P. citri populations have developed resistance to various acaricides and current high gene flow exists between some populations, great attention should be paid to the spread of acaricide-resistance alleles to help gain insight into P. citri resistance management.3. Overcoming the difficulty in the PCR amplification and sequencing resulting from the high A+T content and gene rearrangement, the complete mt genome of P. citri was successfully sequenced using Long-PCR and Sub-PCR techniques. This mt genome has several features:(1) This is the smallest mt genome among arthropods sequenced so far, but it does not lack anyone of 37 genes typical of metazoan mt genomes. (2) Compared to Limulus polyphemus, which is considered as the representative ground pattern for arthropod mt genomes, a series of gene rearrangements have occurred in the evolutionary history of P. citri, and the most striking features are the inversions of two segments containing several protein-coding genes. In addition,24 RNA genes are highly rearranged. (3) The largest non-coding region is only 57 bp long, and is completely comprised of adenines and thymines. and can be folded into stable stem-loop structure, indicating that this region possibly functions as a control region. (4) The mt genome of P. citri has high A+T content, making it the second highest within sequenced Acari. The high A+T content also reflected in the codon usage of protein-coding genes, i.e., codons harbouring A or T in the third position are always overused as compared to other synonymous codons. Among 62 amino-acid encoding codons of invertebrate mitochondrial code, the P. citri mt genome uses 57 codons and never utilizes the five G+C rich codons. The P. citri mt genome is characterized by a positive GC-skew, which is reverse to that of most metazoans mt genomes. (5) The tRNA genes found in the P. citri mt genome are extremely truncated:only three tRNA genes (trnN, trnL2, and trnK) can potentially fold into a typical cloverleaf structure, whereas all the remaining 19 tRNA genes appear to lack the sequence to code the D-or T-arm. Thirteen tRNA genes have 1-3 bp mismatches in the amino acceptor stem, and trnl has eight nucleotides in the anticodon loop. In addition, two tRNA genes(trnM and trnSl) have a single mismatch in the anticodon stem. (6) The two genes encoding the large and small rRNA subunits(rrnL and rrnS) are inverted to the J-strand. The substantial reduction of rrnL and rrnS lead to the loss of several stem-loop structures, as found in the other two mites Leptotrombidium pallidum and Dermatophagoides pteronyssinus.4. The genetic divergence and molecular evolution of mt genomes of spider mites were analyzed from three levels (within family, genus, and species) using comparative genomics and bioinformatics. (1) The mt genomes of spider mites are smallest within the sequenced arthropods, but contain 37 genes typically found in most metazoans. The relatively small size is primarily due to the significant size reduction of PCGs, rrnL, and the putative control region in comparison with other arthropods and Acari. (2) The mt genomes of four spider mites have the same gene order, similarly high A+T content, and strong codon usage bias, but Panonychus had more GC-rich codons never used than Tetranychus. (3) The Panonychus J-strand has a positive GC-skew, which is contrast to that of Tetranychus. Two stem-loop structures of A+T-rich region were found in Panonychus, but only one in Tetranychus. (4) The tRNA genes found in the Tetranychidae mt genomes are extremely short, and 19 tRNA genes lack the D-or T-arm, even some tRNA genes lack the two arms simultaneously. There are several unusual features, such as gene overlap between adjacent tRNA genes, mismatched base at amino acid acceptor stem or anticodon stem,8 nucleotides at anticodon loop of trnI. However, these genes are not likely to be pseudogenes. First of all, their sequences are highly conserved among spider mites, especially for anticodon arm. Secondly, it has been shown that in the nematode Ascaris suum the tRNA genes that lack either the D-or T-arm are functional. Thirdly, stem mismatches and sequence overlap are common for mitochondrial tRNA genes of Acariform mites, and are probably repaired by a posttranscriptional editing process. However, functional tRNA genes that lack both the D-and T-arms have not been found before. Therefore, further experiments are needed to investigate whether these truly tRNA genes lack both D-and T-arms and if so, whether they are functional. (5) The rrnS and rrnL of four Tetranychidae mt genomes have similar stem-loop structures, but the helix H3 appears to only be present in P. citri. Compared to the 5'-end, the 3'-end of rrnL structure is more conserved among Tetranychidae. especially for the helices G16-G20. The most conserved sequences of rrnS among Tetranychidae are found in the helices 19,21,32,33,49, and 50. (6) The genetic divergence of complete mt genome sequences,13 protein-coding genes and 24 RNA genes are high among genera compared to that within genus or species. The average values of P-distance between two Tetranychus species are lower than those between two Panonychus species, but higher than those between the two P. citri strains. The cytochrome oxidase subunits (cox1, cox2) and cytochrome b (cob) are the slowest evolving genes and proteins, making them useful markers for investigating phylogenetic relationships at higher taxonomic levels. In addition, the atp8, nad2, nad6 and nad4L show high P-distance and Ka, implying that they can be used as potential markers to analyze intraspecific relationships within the Tetranychidae species.5. Although there was strong compositional heterogeneity among 28 Acari mt genome sequences, the maximum likelihood (ML) and Bayesian inference (BI) trees based on three data sets (PCG123, PCG12, and PCG2), and the ML trees based on another two data sets (PCG-RNA and PCG1) strongly supported the monophyly of Parasitiformes and Acariformes. The monophyly of Ixodida, Mesostigmata, Trombidiformes, and Sarcoptiformes were recovered:the former two consisted of Parasitiformes, and the latter two consisted of Acariformes. The sister-group relationship between Parasitengona and Eleutherengona was resolved within Trombidiformes. Also, the sister-group between Ixodidae and Argasidae was supported within Ixodida. The supported relationship within Mesostigmata was "(Phytoseioidea+ Dermanyssoidea)+Rhodacaroidea".