Identity of the rat louse Hoplopleura sp.
Two blood-sucking louse species (H. kitti and P. insulsa) parasitize in L. edwardsi (http://phthiraptera.info/category/mammal-wilson-reeder/mammals/rodentia/muridae/murinae/leopoldamys/leopoldamys-edwardsi). The Hoplopleura sp. has close morphological and morphometric similarities with H. kitti recovered from the same host (L. edwardsi). The mt cox1 and rrnS genes of Hoplopleura sp. shared 76% and 77.6% identity with previously published sequences of H. kitti (KJ648943) from Berylmys bowersi and H. akanezumi (KJ648928) from Apodemus chevrieri in China, respectively.
General features of the mt genome of the rat louse Hoplopleura sp.
We sequenced the Hoplopleura sp. genome and produced 3 Gb of Illumina short-read sequence data and obtained a total of 6,526,349×2 raw reads from adults of Hoplopleura sp.. After quality filtration, 3,937,826×2 clean reads (2 Gb) were generated for assembly of the mt genome. We assembled these sequence-reads into contigs and identified 37 mt genes typical of bilateral animals (Fig. 2; Table 3). These genes are on 12 minichromosomal; each minichromosome is 1.8-2.7 kb in size and consists of a coding region and a non-coding region (NCR) in a circular organization (Table 3). The coding regions have 1-5 genes each and vary in size from 675 bp to 1,760 bp (Table 3). All genes are transcribed in the same direction except for nad1 gene. The nucleotide sequences of the mt minichromosomes of Hoplopleura sp. were deposited in GenBank under accession numbers MT792483-94.
We sequenced the full-length non-coding regions of all of the 12 mt minichromosomes of the Hoplopleura sp., which range from 935 (H-nad5-F minichromosome) to 1,305 bp (C-nad6-W-L2 minichromosome) (Table 3). The longest non-coding region of Hoplopleura sp. was shorter than the longest non-coding region of other sucking lice known, such as pig lice (2,370 bp)  and horse lice (3,276 bp) . As in the human lice , rat lice  and pig lice , each coding region of Hoplopleura sp. is flanked by a conserved non-coding AT-rich motif (88 bp,71.6%) upstream and a GC-rich motif (39 bp, 79.5%) downstream, indicating functional significance of these motifs in the mt genomes of blood-sucking lice.
The boundaries between protein-coding genes of the mt genome of Hoplopleura sp. were determined by aligning its sequence and identifying translation initiation and termination codons with those of H. kitti and H. akanezumi . Hoplopleura sp. mt genome encoded 13 protein-encoding genes, which had four initiation codons (ATT, ATG, TTG, GTG). Among them, both ATT (nad2, nad4L, nad5, cox3 and cytb) and ATG (nad3, nad4, nad6, atp6 and atp8) are the highest frequency of being used as initiation codons. Moreover, TTG (nad1 and cox2) and GTG (cox1) are used in the mt genome. This mt genome has three termination codons (TAA, TAG, T). Among them, TAG is the most frequently used with five times altogether, by cox1, nad2, nad3, nad4L and cytb. TAA with secondary high rate of recurrence (four times) as termination codons, cox2, atp6, atp8 and nad4, used it in the mt genome of Hoplopleura sp.. Furthermore, cox3, nad1, nad5 and nad6 genes use T as termination codons. Incomplete terminations (TA and T) of protein-coding genes are commonly found in other mt genomes of blood-sucking lice, including H. suis , H. apri , H. asini , H. kitti , P. asiatica , P. spinulosa , P. schaeffi , M. praelongiceps  and P. pubis . In the mt genome of Hoplopleura sp., the sizes of the rrnL and rrnS genes were 1,125 bp and 675 bp, respectively. The 22 tRNA genes ranged from 59 to 71 bp in size. The secondary structures predicted (not shown) were similar to those of H. kitti and H. akanezumi .
Variation in mt minichromosome composition among three rat lice
The complete mt genome sequences of Hoplopleura sp. fragmented into 12 circular minichromosomes. The incomplete mt genomes of H. kitti and H. akanezumi have identified 11 circular minichromosomes . Eleven minichromosomes of the rat louse, Hoplopleura sp., have the same gene content and gene arrangement as their counterparts of the rat louse, H. kitti. Eight of these minichromosomes of the rat lice, Hoplopleura sp. and H. kitti, have the same gene content and gene arrangement as their counterparts of the rat louse, H. akanezumi . The other two minichromosomes of the rat louse Hoplopleura sp., however, are not present in the rat louse H. akanezumi . In the Hoplopleura sp., one of the minichromosomes has four genes, D-Y-cox2-T (Fig. 2). In the H. akanezumi, however, this minichromosome has only three genes, D-Y-cox2. Similarly, another minichromosome of the Hoplopleura sp. has five genes, R-nad4L-P-cox3-A (Fig. 2). In the H. akanezumi, however, this minichromosome has six genes, R-nad4L-P-cox3-A-T. Interestingly, a chimeric minichromosome has found in the H. akanezumi which contains parts of the two rRNA genes, prrnL and prrnS, which are only 5% (51 bp) and 24% (172 bp) of the full-length rrnL and rrnS, respectively . However, this case has unidentified in the H. kitti and Hoplopleura sp..
Comparative mt genomic analyses of Hoplopleura sp. with H. kitti and H. akanezumi
A comparison of the nucleotide and the amino acid sequences of each protein-encoding gene (except for nad1, nad3 and nad5) of the three Hoplopleura species is given in Table 4. Pairwise comparisons of the nucleotide and amino acid sequences revealed identities of 50.6-77.2% and 37.5-90.2% among them, respectively. The greatest nucleotide variation was in the atp8 gene (49.4%), whereas least differences (22.8%) was detected in the cox1 gene (Table 4). The difference across both concatenated nucleotide and amino acid sequences of the ten protein-coding genes was 37.5% and 36.8% between Hoplopleura sp. and H. kitti, 36.7% and 34.7% between Hoplopleura sp. and H. akanezumi, and 34.6% and 33.4% between H. kitti and H. akanezumi.
In the present study, phylogenetic analysis of the concatenated amino acid sequence datasets for eight mt protein-coding genes (Fig. 3) showed that the family Hoplopleuridae (Hoplopleura sp., H. kitti and H. akanezumi) clustered to the exclusion of representatives of the families Polyplacidae (P. asiatica and P. spinulosa), Haematopinidae (H. apri, H. asini and H. suis), Pediculidae (P. humanus corporis, P. humanus capitis and P. schaeffi), Pthiridae (P. pubis), and the family Microthoraciidae (M. praelongiceps) clustered separately with strong nodal support (Bootstrap = 100). Within the family Hoplopleuridae, Hoplopleura sp. and H. akanezumi clustered together with moderate support (Bootstrap value = 73), to the exclusion of H. kitti, and then they formed a monophyletic group (Bootstrap value = 100). The result was also strongly supported by RAxML analysis (Bootstrap value = 100) (Additional file 1: Fig. S1)
The work of Johnson et al. (2018) created robustness and stability in higher systematics within the order Phthiraptera based on analyses of 1,107 single-copy orthologous genes from sequenced genomes of 46 species of lice . Their result has indicated that the genera Hoplopleura and Haematopinus were more closely related than to the genus Pediculus with strong bootstrap value . However, mt genomic phylogenetic relationships deviated from phylogenies derived from the nuclear genome. Shao et al. (2017) performed a phylogenetic analysis with mt genomes, indicating that the genera Haematopinus and Pediculus were more closely related than to the genus Hoplopleura with strong bootstrap value . Our result also showed the genera Haematopinus and Pediculus were more closely related than to the genus Hoplopleura, but was weak bootstrap value (Bootstrap value = 55) (Fig. 3). Although the number of sucking lice mt genome sequences is increasing, so far, mt genomes of many lineages of sucking lice are underrepresented or not represented. Insufficient taxon sampling for the suborder Anoplura mt genomes might be the cause of the discordance between the mt and nuclear phylogenies.
Many studies have indicated that the mt genome sequence is a valuable genetic marker for phylogenetic studies at various taxonomic levels of different organisms [27,28], including lice [14,15]. The fragmentation of the mt genome may have arisen independently in multiple louse clades. Therefore, the mt genome sequences of rat louse Hoplopleura sp. could promote to reassess the systematic relationships of lice within suborder Anoplura using mt genomic datasets. No species from the other genera (Ancistroplax, Ferrisella, Haematopinoides, Paradoxophthirus, Pterophthirus, Schizophthirus and Typhlomyophthirus) within family Hoplopleuridae was included in our analyses. Therefore, more expanding taxa sampling is necessary for future phylogenetic studies of family Hoplopleuridae using mt genomic dataset.