The fragmented mitochondrial genomes of two Linognathus lice reveal active minichromosomal recombination and recombination hotspots

Summary Evidence for recombination between mitochondrial (mt) minichromosomes has been reported in sucking lice, but it is still not clear how frequent mt minichromosomal recombination occurs. We sequenced the mt genomes of the cattle louse Linognathus vituli and the goat louse L. africanus. Both Linognathus species have 10 mt minichromosomes, and seven of them have the same gene content and gene arrangement. Comparison of mt karyotypes revealed numerous inter-minichromosomal recombination events in the evolution of Linognathus species. Minichromosome merger, gene duplication and gene translocation occurred in the lineage leading to Linognathus lice. After the divergence of L. vituli and L. africanus, duplication, degeneration, deletion and translocation of genes also occurred independently in each species. Most of the recombination events in the Linognathus species occurred upstream of either cox3 or nad2, indicating these two locations were hotspots for inter-minichromosomal recombination. Our results provide an important perspective on mt genome evolution in metazoans.


INTRODUCTION
Mitochondrial (mt) genomes have been extensively explored in evolutionary studies of metazoans due to their high mutation rates and maternal inheritance of these genomes. 1 Metazoan mt genomes typically have a single chromosome with 36-37 genes (including 12-13 protein-coding genes, 22 tRNA genes, and two rRNA genes), a single large non-coding region (NCR), and often highly conserved gene arrangement. 1 Sucking lice (Anoplura), however, have an unusual, fragmented mt genome organization. These lice feed on host blood and are of medical and veterinary importance as ectoparasites and vectors of diseasecausing microorganisms. 2,3 An extremely fragmented mt genome with 20 minichromosomes was found first in the human body louse, Pediculus humanus humanus. 4,5 To date, 21 species of sucking lice from eight families have been sequenced; all these species have fragmented mt genomes with nine to 20 minichromosomes in each species. [4][5][6][7][8][9][10][11][12][13][14][15][16] The mt karyotypes, i.e., number of minichromosomes, gene content and gene arrangement in each minichromosome, 17 vary substantially between families, between genera, and even between congeneric species of sucking lice. 5,12,13,16,17 Recombination between mt minichromosomes has been proposed as one of the mechanisms that lead to the highly variable mt karyotypes among sucking lice. 9 Shao et al., showed stretches of identical sequences shared between mt genes on different minichromosomes as unequivocal evidence for inter-minichromosomal recombination. 4,5 This type of evidence has been found in 13 species of sucking lice; the shared stretches of identical sequences are up to 133 bp long and are much longer than expected by chance. [4][5][6][8][9][10][11][12]14,16 Furthermore, Shao and Barker reported eight types of chimeric mt minichromosomes in the human body louse and presented evidence for both homologous recombination and non-homologous recombination between mt minichromosomes in this louse. 18 Inter-minichromosomal recombination explains well the highly variable mt karyotypes observed in sucking lice, in particular gene translocation between different minichromosomes and merger of minichromosomes. 9,11,12,14, 16 It is still not clear, however, how frequent mt minichromosomal recombination occurs in sucking lice, and whether recombination tends to occur more often at any particular minichromosomal locations.
The mt genome organizations of sucking lice in seven families (Enderleinellidae, Linognathidae, Hamophthiriidae, Hybophthiridae, Neolinognathidae, Pecaroecidae, and Ratemiidae) remain unstudied. In the current study, we sequenced the complete mt genomes of two Linognathus species from the family Linognathidae. Linognathus species are among the most common ectoparasites of ruminants, 19 causing major economic losses to livestock industry worldwide. 20 We compared the mt karyotypes between these two Linognathus species and with the inferred ancestral mt karyotype of sucking lice to further understand inter-minichromosomal recombination in sucking lice.

RESULTS
Mitochondrial genomes of the cattle louse L. vituli and goat louse L. africanus We obtained a large number of clean reads for L. vituli using Miseq paired-end sequencing: 6,894,874 pairs from the genomic DNA, and 158,040,342 pairs from the PCR amplicons of individual minichromosomes (Table 1). We assembled these sequence-reads into contigs and identified the typical 37 mt genes in L. vituli (13 protein-coding genes, 22 tRNA genes, and two rRNA genes), and a duplicate trnL 1 gene, two pseudo rrnS genes, a pseudo rrnL gene, and a pseudo cox2 gene ( Figure 1; Table 1). These genes and pseudo genes are on 10 minichromosomes; each minichromosome is 2,086 to 3,052 bp in size and consists of a region with genes and a large NCR in a circular organization ( Figure 1; Table 1). Each minichromosome has two to eight genes clustered in a region, 893 bp to 1,887 bp in size (Table 1). All genes are transcribed in the same direction except for trnT-nad1-trnQ gene cluster (genes underlined have opposite orientation of transcription to other genes) ( Figure 1). We sequenced the full-length large NCRs of all of the 10 mt minichromosomes of L. vituli, which were 686 bp to 1,296 bp in size (Table 1). A conserved AT-rich motif (45 bp, 100% A and T) is present in every minichromosome in the large NCR upstream of the gene cluster, and a conserved GC-rich motif (78 bp, 60% C and G) is downstream of the gene cluster ( Figure 1). The nucleotide sequences of the mt minichromosomes of L. vituli have been deposited in GenBank under accession numbers OL677823-32.
We obtained 7,378,362 pairs of clean reads from the genomic DNA and 194,422,436 pairs of clean reads from the PCR amplicons of individual minichromosomes of L. africanus using Miseq paired-end sequencing ( Table 2). Additional to the typical 37 mt genes, three duplicate tRNA genes (trnL 1 , trnH, and trnM) and a pseudo atp8 gene were also identified in L. africanus ( Figure 2; Table 2). These genes and pseudo gene are on 10 circular minichromosomes, 3,102 bp to 3,915 bp in size ( Figure 2; Table 2). Each minichromosome has two to eight genes clustered in a region, 965 bp to 1,874 bp in size ( Table 2). All genes are transcribed in the same direction except for trnT-nad1-trnQ gene cluster ( Figure 2). The large NCRs of L. africanus were 1,892 bp to 2,519 bp in size ( Table 2). A conserved AT-rich motif (60 bp, 75% A and T) is present in every large NCR upstream of the gene cluster and a conserved GC-rich motif (38 bp, 76.3% C and G) is downstream of the gene cluster ( Figure 2). The nucleotide sequences of the mt minichromosomes of L. africanus were deposited in GenBank under accession numbers OP948897-906.
Duplicate genes and pseudo genes in the mitochondrial genomes of Linognathus species Four duplicate tRNA genes and five pseudo genes in total were found in the mt genomes of L. vituli and L. africanus. All of the duplicate genes and pseudo genes are located in different minichromsomes from iScience Article their corresponding original gene copies or the full-length genes (Figures 1 and 2). In both species, the original copy of trnL 1 is retained at its inferred ancestral location upstream of rrnL whereas a duplicate trnL 1 is present in a different minichromosome upstream of nad2 (Figures 1 and 2). The two copies of trnL 1 of each Linognathus species are identical in sequence and length, while between the two Linognathus species their trnL 1 genes differ by 19.4% in sequence ( Figure 3A). Similarly, the other two duplicate tRNA genes (trnH and trnM) of L. africanus also have identical sequences and lengths as their corresponding original copy of tRNA genes ( Figure 3B), which are retained at their inferred ancestral locations on different minichromosomes ( Figure 2). Between the two Linognathus species, their trnH genes differ by 36.8% and their trnM genes differ by 18.8% ( Figure 3C).
There are two pseudo rrnS genes, a pseudo rrnL gene and a pseudo cox2 gene in L. vituli (Figure 1), and a pseudo atp8 gene in L. africanus ( Figure 2). The pseudo genes of L. vituli are 198 bp to 252 bp in size and are much shorter than their corresponding full-length genes (Figures S1-S3). The pseudo genes of L. vituli, however, are identical in sequence or 99.5% similar to a portion of their corresponding full-length genes (Figures S1-S3). In contrast, the pseudo atp8 gene of L. africanus is very similar in length (7 bp shorter) to the full-length atp8 but has much lower nucleotide sequence similarity (44.6%) with the full-length atp8 ( Figure S4). The pseudo atp8 gene of L. africanus does not have a start codon, nor a stop codon, nor the highly conserved sequence motif at the 5 0 end for MPQ amino acid sequence ( Figures S4  and S5). In both Linognathus species, atp8 is on a minichromosome with cytb, nad4L, and five tRNA genes (Figures 1 and 2). The pseudo atp8 of L. africanus is upstream of atp6 in the inferred ancestral location of atp8 ( Figure 2); no pseudo atp8 gene can be found in L. vituli ( Figure 1). Linognathus vituli and L. africanus are distinct from each other in mt karyotype although both species have 10 minichromosomes (Figures 1 and 2). Seven of their 10 minichromosomes have the same gene content and gene arrangement between the two Linognathus species; the other three minichromosomes, however, differ in gene content and gene arrangement between the two species. In L. vituli, one of the minichromosomes has three genes in a cluster, trnY-cox2-nad6; in L. africanus, however, the minichromosome that has this gene cluster also has trnD upstream of trnY (Figures 1 and 2). Another minichromosome of L. vituli has three genes, a pseudo rrnS and a pseudo rrnL, i.e., prrnS 1 -prrnL-cox3-trnW-trnA. In L. africanus, however, the minichromosome that has cox3-trnW-trnA gene cluster does not have any pseudo genes but has a duplicate trnH upstream of cox3 (Figures 1 and 2). Furthermore, in L. vituli, one minichromosome has three genes, a pseudo rrnS and a pseudo cox2, i.e., prrnS 2 -pcox2-trnD-trnL 1 -nad2. In L. africanus, however, the corresponding minichromosome has only three genes, trnM-trnL 1 -nad2 (Figures 1 and 2).
Phylogenetic relationships of Linognathus species to other sucking lice

DISCUSSION
Linognathus species are distinct in mitochondrial karyotype from other sucking lice and the inferred most recent common ancestor of sucking lice (Anoplura) The current study reports the complete mt genomes of two Linognathus species from the family Linognathidae for the first time. Like the 21 species of sucking lice from other eight families studied previously, 4-16 the two Linognathus species also have fragmented mt genomes. The Linognathus species, however, differ substantially in mt karyotype from other species of sucking lice including Hoplopleura species which are most closely related to Linognathus species (Figures 4 and S6) and from the inferred most recent common ancestor (MRCA) of sucking lice. 11 There are five differences in mt karyotype between the Linognathus species and the MRCA of sucking lice. First, the Linognathus species have 10 minichromosomes, which are one less than the MRCA of sucking lice (Figures 1 and 2; Tables 1 and 2). 11 iScience Article Second, as detailed previously, the two Linognathus species have four duplicate tRNA genes and five pseudo genes in total, none of which is in the MRCA of sucking lice. 11 Third, rrnS and atp6, which are on two different minichromosomes in the MRCA of sucking lice, are on the same minichromosome in both Linognathus species. Fourth, trnL 2 and trnW of both Linognathus species and trnD of L. vituli are on different minichromosomes relative to their corresponding tRNA genes of the MRCA of sucking lice. Fifth, cytb, nad4L, and atp8, which are on three different minichromosomes in the MRCA of sucking lice, are on the same minichromosome in both Linognathus species. The separation of atp8 and atp6 into different minichromosomes is unique for the Linognathus species; all other sucking louse species sequenced to date have the atp8-atp6 gene cluster on a minichromosome of their own and this is inferred to be ancestral to all sucking lice. [4][5][6][7][8][9][10][11][12][13][14][15][16] Indeed, atp8-atp6 gene cluster is present in the mt genomes of most animals. [21][22][23] Furthermore, a pseudo atp8 gene is present in L. africanus ( Figure 2). Apparently, duplication of atp8 occurred in the MRCA of the two Linognathus species, thus adding a copy of atp8 to the minichromosome that contained cytb, nad4L, and five tRNA genes ( Figure 5). Subsequently, the original copy of atp8 upstream of atp6 degenerated to become a pseudo atp8 in L. africanus but was deleted in L. vituli ( Figure 5).

Mitochondrial minichromosome recombination is highly active in Linognathus lice
As introduced, evidence for recombination between mt minichromosomes has been found in many sucking louse species. [4][5][6][7][8][9][10][11][12]14,16 Identical sequences up to 133 bp long shared between genes on different minichromosomes were reported as unequivocal evidence for inter-minichromosomal recombination. [4][5][6][8][9][10][11][12]14,16 Evidence for both homologous recombination and non-homologous recombination between mt minichromosomes is found in the human body louse based on the observation of eight types of chimeric mt minichromosomes. 18 The Linognathus lice investigated in the current study provided different type of evidence for inter-minichromosomes recombination not seen previously in other sucking lice. Four observations lead us to suggest that inter-minichromosomal recombination is highly active in iScience Article Linognathus lice. First, in the lineage leading to Linognathus lice, two ancestral minichromosomes of sucking lice (rrnS-trnC and atp8-atp6-trnN) merged as one; five genes, i.e., trnL 2 , trnR-nad4L-trnP, and trnW, translocated from their inferred ancestral minichromosomes to other minichromosomes; and atp8 and trnL 1 were duplicated leading to two copies of each gene on different minichromosomes ( Figure 5). Second, after the divergence of L. africanus from L. vituli, trnH, and trnM were duplicated in L. africanus leading to two copies of both trnH and trnM on different minichromosomes (Figures 2  and 5). Third, after the divergence of L. vituli from L. africanus, portions of cox2, rrnL, and rrnS were duplicated in L. vituli, leading to a pseudo cox2 gene, two pseudo rrnS genes and a pseudo rrnL gene on different minichromosomes from those of their corresponding full-length genes (Figures 1 and 5). Fourth, in both Linognathus species, the duplicate genes and pseudo genes (except pseudo atp8 gene) are identical or 99.5% similar in sequence to their corresponding full-length genes or a portion of their corresponding full-length genes ( Figures S1-S3). There is no evidence for mt minichromosome split in Linognathus lice. Selection may maintain sequence similarities but cannot produce duplicate genes or pseudo genes in the first place. Thus, recent homologous recombination between minichromosomes is probably the most plausible explanation for such high sequence similarity between the duplicate tRNA genes and between the pseudo genes and their corresponding full-length genes. The observation of such high sequence similarity simultaneously in four duplicate tRNA genes and four pseudo genes indicates rather frequent homologous recombination between mt minichromosomes in the two Linognathus species. Ten of the 11 mt minichromosomes of the MRCA of sucking lice 11 were involved in one or more recombination events in the evolution of Linognathus species, either minichromosome merger or tRNA translocation or gene duplication ( Figure 5). trnK-nad4 minichromosome is the only one that is not involved in any recombination events and remains unchanged in gene content and gene order ( Figure 5). Of all the mt minichromosomal locations, the location upstream of cox3 and the location upstream of nad2 had the most frequent recombination events ( Figure 5). The gene cluster R-nad4L-P upstream of cox3 was translocated to trnE-cytb-trnS 1 -trnS 2 minichromosome in the lineage leading to Linognathus species ( Figure 5). The translocation of trnR-nad4L-trnP would require at least one recombination event between trnR-nad4L-trnP-cox3-trnA minichromosome and trnE-cytb-trnS 1 -trnS 2 minichromosome ( Figure 5), both ancestral to sucking lice. 11 Subsequently, two pseudo genes, prrnS 1 and prrnL, were inserted upstream of cox3 in L. vituli, whereas in L. africanus a duplicate copy of trnH gene was inserted in this location (Figures 1, 2, and 5). The insertion of prrnS 1 , prrnL, and duplicate trnH would require at least three recombination events as rrnS, rrnL, and trnH were on three different minichromosomes in the MRCA of sucking lice ( Figure 5). 11 There was no gene upstream of nad2 in the MRCA of sucking lice ( Figure 5). In the lineage leading to Linognathus species, a duplicate copy of trnL 1 gene was inserted upstream of nad2 (Figures 1 and 2). The insertion of duplicate trnL 1 would require one recombination event between nad2 minichromosome and trnM-trnL 1 -rrnL-trnV minichromosome, both ancestral to sucking lice ( Figure 5). 11 Subsequently, trnD was translocated upstream of nad2-trnL 1 and two pseudo genes, prrnS 2 and pcox2, were inserted upstream of nad2-trnL 1 -trnD in L. vituli; whereas in L. africanus a duplicate copy of trnM gene was inserted upstream of nad2-trnL 1 (Figures 1 and 2). The translocation of trnD and the insertion of prrnS 2 , pcox2, and duplicate trnM would require at least three recombination events as trnD, rrnS, cox2, and trnM were on three different minichromosomes in the MRCA of sucking lice, or possibly four recombination events as trnD translocation and pcox2 insertion might occur separately although trnD and cox2 were on the same minichromosome in the MRCA of sucking lice ( Figure 5). 11 As discussed previously, all of the duplicate genes and pseudo genes upstream of cox3 and nad2 in both Linognathus species have identical or near identical sequences as their corresponding original copy of iScience Article genes or a portion of their corresponding full-length genes ( Figures S1-S3). Retention of multiple duplicate or pseudo genes in the highly compact mt genomes of animals is uncommon 1 ; redundant genes often become degenerated and deleted. Recent homologous recombination that involved the minichromosomal locations upstream of cox3 and nad2 is probably the most plausible explanation for the extremely high sequence similarity between the duplicate genes and their corresponding original copy of genes, and between the pseudo genes and their corresponding full-length genes in the Linognathus species.

Limitations of the study
Sucking lice (Anoplura) parasitize members of 12 of the 29 mammalian orders and $840 mammalian species. 19 To date, more than 540 species of sucking lice have been described and assigned to 50 genera in 15 families. 24,25 The genus Linognathus has 51 species that parasitize a range of mammals in four families (Bovidae, Cervidae, Giraffidae, and Canidae) of two orders (Artiodactyla and Carnivora). 24,26 The hosts of the two Linognathus species we sequenced in the current study are both in the family Bovidae. To verify the findings of the current study, Linognathus species that parasitize mammals in the Cervidae, Giraffidae, and Canidae should be sequenced and compared in future studies. Furthermore, the current study used a comparative mt genomics approach, to understand minichromosomal recombination in the mt genomes of parasitic lice, biochemical experimental approaches are also needed.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:   d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Louse samples
All procedures involving animals in the present study were approved and this study was approved by the Animal Ethics Committee of Hunan Agricultural University (No. 201703386). The adult cattle louse L. vituli samples were collected from the body surface of a bull Bos taurus slaughtered in Hunan province, China. The adult goat louse L. africanus samples were collected from the body surface of a healthy male goat Capra hircus in Khyber Pakhtunkhwa province, Pakistan. The animals' entire body was inspected visually for the presence of lice for three minutes. All louse specimens were removed with the aid of a forceps and stored in centrifuge tubes. Morphological analysis of lice were performed using a stereoscopic microscope (Nikon SMZ18, Tokyo, Japan). 25 Cattle louse is 2.0-3.0 mm long, and goat louse is 2.1-2.9 mm in length. In addition, the sex was identified based on morphological characteristics: male with basal apodeme slender and pseudopenis elongate or poorly sclerotized; parameres well developed; female with subgenital plate variously shaped, gonopods VIll well developed; gonopod IX well developed and prolonged posteriorly, with either a spiniform genital seta or pointed apical process; spermatheca not strongly sclerotized. All louse specimens were then washed five times in physiological saline solution, and stored in 100% (v/v) ethanol at À40 C.

DNA extraction
For each louse species, total genomic DNA was extracted from 50 individual lice (25 females and 25 males) using QIAampâ DNA Micro Kit (QIAGEN, Hilden, Germany) according to the manufacturer's recommendations. The species identity of each specimen was verified by sequencing regions of mt cox1 and rrnS genes. 11, 38 The cox1 and rrnS gene sequences of our L. vituli specimens have 97% and 100% similarity with that of L. vituli from B. taurus in Australia (accession numbers HM241900 and HM241899, respectively). The cox1 gene sequences of our L. africanus specimens have 99.7% similarity with that of L. africanus from C. hircus in Peru (accession no. EU375760).

Sequencing and assembling
Genomic DNA concentration was determined using Qubit 4.0 (Invitrogen, Carlsbad, USA); DNA integrity was analysed with agarose-gel electrophoresis. Genomic DNA library (530-bp inserts) was constructed for high-throughput sequencing with Miseq (Illumina, San Diego, CA, USA); paired-end raw reads (300 bp each) were exported in FASTQ format. Raw reads were trimmed and filtered using Trimmomatic v.0.36. 27 For each Linognathus louse species, 2Gb of high-quality clean reads were obtained after removing adaptor sequences, highly redundant sequences, reads containing more than 10% 'N' ('N' representing ambiguous bases in reads), and reads containing more than 50% bases with Q-value %20.
We used the Map-to-Reference(s) option in Geneious 11.1.5 28 to assemble mt genomes. The cox1 and rrnS sequences of the two Linognathus lice were used respectively as the initial references to assemble the Miseq sequence reads. The assembly parameters were minimum overlap identity 99% and minimum overlap 200 bp. The assemblies were iterated until cox1 and rrnS minichromosomes were assembled in full length for the gene-containing region and from each end of this region, extended $500 bp into the large non-coding regions (NCRs). Previous studies showed that the large NCRs are highly conserved among the mt minichromosomes of each sucking louse species 11-13 or chewing louse species. 39 The conserved large NCR sequences were identified between the cox1 and rrnS minichromosomes and were used as references to align the Miseq sequence reads. This allowed us to extract sequence reads derived from the two ends of the gene-containing regions of all other mt minichromosomes. We then assembled these minichromosomes individually in full length for the gene-containing region and extended the contigs into the large NCRs as we did above for cox1 and rrnS minichromosomes.

Verification and sequencing of individual mitochondrial minichromosomes
The size and circular structure of each mt minichromosome of L. vituli and L. africanus were verified by PCR using specific primers targeting gene-containing regions (Tables S1 and S2). The forward primer and reverse primer in each pair were next to each other with a small gap less than 10 bp. PCR with these primers ll OPEN ACCESS