Highly Rearranged Mitochondrial Genome in Falcolipeurus lice (Phthiraptera: Philopteridae) from Endangered Eagles

doi:10.21203/rs.3.rs-249932/v1

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Highly Rearranged Mitochondrial Genome in Falcolipeurus lice (Phthiraptera: Philopteridae) from Endangered Eagles

https://doi.org/10.21203/rs.3.rs-249932/v1

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published 20 May, 2021

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Background: Fragmented mitochondrial (mt) genomes and extensive mt gene rearrangements have been frequently reported from parasitic lice (Insecta: Phthiraptera). However, relatively little is available about the mt genomes from the family Philopteridae that is the most species-rich family within the suborder Ischnocera.

Methods: Herein, we use next-generation sequencing to decode the mt genome sequences of Falcolipeurus suturalis and compared it with the mt genome sequences of F. quadripustulatus. Phylogenetic relationship of the concatenated amino acid sequence data for 13 protein-coding genes of the two Falcolipeurus lice and selected members of the family Philopteridae was evaluated using Bayesian inference (BI).

Results: The complete mt genome of F. suturalis is a circular double-stranded DNA molecule of 16,659 bp, and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, as well as three putative non-coding regions. The gene order in F. suturalis mt genome was rearranged compared with that of F. quadripustulatus, and they were radical different from other louse species and the ancestral insect. Phylogenetic analyses revealed that the clear genetic distinctiveness between F. suturalis and F. quadripustulatus (Bayesian posterior probabilities=1.0), and the genus Falcolipeurus is more closely related to the genus Ibidoecus than to other genera (Bayesian posterior probabilities=1.0).

Conclusions: These novel datasets will help to better understand the gene rearrangements and phylogenetic position of Falcolipeurus and provide useful genetic markers for systematics and phylogenetic studies of bird lice.

Parasitology

Bird lice

Mitochondrial genome

Gene rearrangement

Phylogenetic analyses

The typical insect mitochondrial (mt) genome is a circular double stranded DNA molecule of about 12–20 kb in length, which commonly contains 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA) [1, 2]. However, in some lineages of parasitic lice (Insecta: Phthiraptera) are notable exceptions. For example, the following groups show extensively fragmented mt genomes, with the 37 genes separated onto multiple circular chromosomes: the families Haematopinidae [3], Hoplopleuridae [4, 5], Menoponidae [6], Pediculidae [7–9], and Polyplacidae [10], Trichodectidae [11]. Parasitic lice are currently divided into chewing lice (including three suborders Ischnocera, Amblycera and Rhynchophthirina) and sucking lice (the suborder Anoplura) by their mouthparts.

Chewing lice are permanent, obligate and host-specific ectoparasites commonly found on birds and mammals. The suborder Ischnocera (about 3,120 species) are currently divided into two families Philopteridae (about 2,600 species) and Trichodectidae. The Philopteridae is a large family within the suborder Ischnocera [12], but the complete mt genomes of only a limited number of species have been sequenced, including Bothriometopus macrocnemis [13], Campanulotes bidentatus compar [14], Campanulotes compar [11], Coloceras sp. SLC-2011 [15], Falcolipeurus quadripustulatus [11], Ibidoecus bisignatus [15], Columbina picui, Columbina cruziana, Columbicola columbae [16]. These results showed extensive gene rearrangements in their mt genomes. Very surprisingly, a recently report has demonstrated that highly-fragmented, mt minicircles are present in four species of the genus Columbicola within the Philopteridae [16], indicated fragmented mt genomes are more prevalent in parasitic lice than previously assumed [11]. However, our knowledge on the structure of mt genomes of bird lice from this family is far from comprehensive. Therefore, there is a need to obtain more mt genome data from this family. Such data would help to better understand the pattern and mechanisms of fragmented mt genomes and gene rearrangements in bird lice within the Philopteridae.

Owing to maternal inheritance, relatively high evolution rate, conserved gene components and low rate of recombination, mt genome has been widely used as a genetic marker for comparative, evolutionary and phylogenetic analysis at different taxonomic levels [5, 17, 18]. Herein, we (i) decoded and characterized the complete mt genome sequences of F. suturalis from the tawny eagle; (ii) compared it with that of F. quadripustulatus from the vulture; and (iii) assessed the phylogenetic positions in combination with all mt genomic datasets available for representative bird lice from Philopteridae in public databases.

Sample collection and DNA extraction

Adult samples of F. suturali were taken from Aquila rapax from Beijing Wildlife Rescue Center, China. These parasitic lice were washed twice with sterile physiological saline solution (0.85%), and initial identification as F. suturali based on morphological characteristics and host species (Fig. 1) [19], and then stored in 95% (v/v) ethanol at -40°C. The total genomic DNA was extracted from 60 individual bird lice (30 females and 30 males) using DNeasy Tissue Kit (Promega, Madison, USA) following the manufacturer's instructions. The molecular identity of each bird louse was also confirmed by PCR using previously reported method [20] and then sequenced directly. The both cox1 and rrnS sequences had 86.0% and 84.5% similarity to previously published sequences of F. quadripustulatus from China, respectively (GenBank accession nos. NC_039529.1), indicating that these samples of bird lice belong to the genus Falcolipeurus.

Sequencing, assembling and verification

The quality of the extracted genomic DNA was tested using by agarose-gel electrophoresis [21]. The genomic DNA concentration was quantified on the Quibt 2.0 Fluorometer (Thermo Scientific). Genomic DNA library (350 bp inserts) was prepared and sequenced in Novogene Bioinformatics Technology Co. Ltd. (Tianjin, China). The library was sequenced using Illumina HiSeq 2500 (250 bp pair-end reads). Raw reads were filtered and cleaned with Prinseq [22]. The obtained cox1 and rrnS sequences of F. suturali were used as the initial references to assemble the clean reads with Geneious 11.1.5 (minimum overlap identity = 99%, minimum overlap = 150 bp, maximal gap size = 5 bp) [23]. The size and circular organization of the mt genome assembly were further verified by long PCR using four pairs of designed primers (Table S1; Fig. S1).

Annotation and sequence analysis

13 protein-coding genes and two rRNA (rrnL and rrnS) genes were identified by alignment with homologous genes of previously sequenced mt genome of the vulture louse F. quadripustulatus [11] using the MAFFT 7.122 software [24]. The 22 tRNA genes were verified using ARWEN [25] and the program tRNAscan-SE [26] with manual adjustment. Correctly annotated mt genomes were illustrated using the visualize module of MitoZ [27]. Nucleotide composition, amino acid sequences of each protein-coding genes and codon usage were mainly analyzed using MEGA 6.0 [28]. Asymmetry of base composition was calculated as the following formula: AT-skew = (A-T)/ (A + T), GC-skew = (G-C)/(G + C) [29].

Phylogenetic analysis

Total of 10 mt genomes of the family Philopteridae were used for phylogenetic analysis, using one rat louse, Hoplopleura sp. (GenBank: MT792483-94) as an outgroup [5]. Each amino acid sequences were aligned individually using MAFFT algorithm. We concatenated the alignments of the individual genes and obtained a single dataset. The ambiguous areas of alignment were removed by Gblocks 0.91b with the options for a less stringent selection [30], and then subjected to phylogenetic analyses under Bayesian inference (BI). BI was conducted with four independent Markov chains run for 1,000,000 metropolis-coupled MCMC generations, sampling a tree every 100 generations in MrBayes 3.1.1 [31]. The initial 25% (2,500) trees of each MCMC were treated as the burn-in and the majority-rule consensus tree were used to calculate Bayesian posterior probabilities (BPP). Phylograms were drawn using FigTree v.1.31.

Genome organization

A total of 3.7 Gb data (about 20-fold coverage) was obtained from Illumina HiSeq 2500 platform which produced 15,178,382×2 raw reads. Extracted reads were cleaned and 9,630,532×2 clean reads were obtained for the assembly of the mt genome. The longest contig is 16,659 bp in size that represented the complete mt genome of F. suturalis (GenBank accession: MI456908). We identified and annotated all of the 37 mt genes typical of metazoan mt genomes (Fig. 2; Table 1). This mt genome contains 13 protein-coding genes, 22 tRNA genes, two rRNA genes and three non-coding (AT-rich) regions (Fig. 2; Table 1). The mt gene arrangement and distribution of genes are distinct from those of F. quadripustulatus [11] and I. bisignatus [15]. The overall nucleotide composition was: A = 27.8%, T = 44.8%, C = 11.1%, G = 16.3%. All mt genes were encoded on the heavy strand, which is similar to the other bird louse species [11, 13]. The three pairs of overlapping regions in the mt genome of F. suturalis were observed among nad4L/nad1, tRNA-His/tRNA-Asp and tRNA-Asp/tRNA-Arg. The overlapping regions ranged from − 4 bp to − 8bp (Table 1). Besides, 22 intergenic regions were observed in this mt genome, ranging from 1 bp to 180 bp in size. The longest space was found between tRNA-S₂ and tRNA-G genes (Table 1).

Table 1

The organization of the mt genome of *F. suturalis*.
Gene/Region	Positions	Size (bp)	Number of aa^a	Ini/Ter codons^b	Anticodon^c	In
cox1	34-1557	1524	507	ATA/TAA		+ 33
tRNA-Met (M)	1574–1637	64			CAT	+ 16
tRNA-Gln (Q)	1639–1705	67			TTG	+ 1
tRNA-Glu (E)	1706–1770	65			TTC	0
atp6	1774–2445	672	223	ATA/TAA		+ 3
tRNA-Asn (N)	2451–2517	67			GTT	+ 5
rrnS	2518–3243	726				0
rrnL	3244–4318	1075				0
tRNA-Ala (A)	4319–4382	64			TGC	0
nad6	4385–4858	474	157	ATG/TAA		+ 2
tRNA-Val (V)	4861–4922	62			TAC	+ 2
cox3	4977–5726	750	249	ATA/TAA		+ 54
tRNA-Lys (K)	5746–5808	63			TTT	+ 19
nad4	5843–7156	1314	437	ATT/TAG		+ 34
AT-loop region	7157–7985	829
tRNA-LeuUUR (L₂)	7986–8047	62			TAA	0
tRNA-Pro (P)	8064–8124	61			TGG	+ 16
nad2	8130–9101	972	323	ATG/TAA		+ 5
tRNA-Thr (T)	9171–9235	65			TGT	+ 69
tRNA-Tyr (Y)	9249–9313	65			GTA	+ 13
cox2	9314–9991	678	225	ATA/TAA		0
AT-loop region	9992–10713	722
nad5	10714–123889	1676	558	ATG/TA		0
tRNA-Phe (F)	12390–12456	67			GAA	0
tRNA-Cys (C)	12477–12543	67			GCA	+ 20
atp8	12565–12765	201	66	ATG/TAA		+ 21
tRNA-SerUCN (S₂)	12772–12840	69			TGA	+ 6
tRNA-Gly (G)	13021–13091	71			TCC	+ 180
AT-loop region	13092–13516	425
nad3	13517–13903	387	128	ATT/TAG		0
tRNA-LeuCUN (L₁)	13905–13966	62			TAG	+ 1
nad4L	13992–14264	273	90	ATT/TAA		+ 25
nad1	14257–15163	907	302	ATG/T		-8
tRNA-SerAGN (S₁)	15164–15231	68			TCT	0
cytb	15232–16323	1092	363	TTG/TAG		0
tRNA-Trp (W)	16330–16396	67			TCA	+ 6
tRNA-His (H)	16398–16460	63			GTG	+ 1
tRNA-Asp (D)	16457–16524	68			GTC	-4
tRNA-Arg (R)	16516–16585	70			ACG	-8
tRNA-Ile (I)	16593–16659	67			GAT	+ 6
^aThe inferred length of amino acid (aa) sequence of 13 protein-coding genes; ^bIni/Ter codons: initiation and termination codons;
^cIn: Intergenic nucleotides.

The observed total A + T and G + C content of the complete mt genome were 73.0% and 27.0%, respectively, which were consistent with those of previous studies [11, 15] (Table 2). A negative AT skew (-23.3) and a positive GC skew (18.9) were calculated in this my genome (Table 2), which are common features of ectoparasites mt genome [11, 15]. All bird lice from Philopteridae reported to date and in the present study show strand asymmetry (GC skew between 6.3% and 38.1%) (Table 2).

Table 2

Nucleotide composition of the mt genomes of Philopteridae species, including that of *Falcolipeurus suturalis.*
Species	Nucleotide frequency (%)				Whole genome sequence
Species	A	T	G	C	A + T%	AT skew	GC skew
Bothriometopus macrocnemis	32.1	38.7	15.5	13.8	70.8	-9.2	6.1
Campanulotes bidentatus compar	26.5	43.7	20.67	9.77	70.1	-24.5	38.1
Campanulotes compar	26	44.5	20.4	9.1	70.5	-26.3	38.1
Coloceras sp. SLC-2011	27.5	42.9	19.9	9.6	70.4	-21.8	35.1
Ibidoecus bisignatus	35.5	40.6	13.2	10.8	76	-6.7	10.2
Columbicola columbae	39.1	29.2	16.3	15.4	68.2	14.6	2.8
Columbina picui	33.5	31.6	18.3	16.6	65.1	2.9	5
Columbina cruziana	32.9	31.4	19	16.7	64.3	2.4	6.3
Falcolipeurus quadripustulatus	26.3	45.5	16.9	11.3	71.8	-26.8	20.1
Falcolipeurus suturalis	28	45	16.4	11.2	73	-23.3	18.9

Annotation

As the mt genomes of parasitic lice can contain non-standard initiation codons [1, 5, 13], the identification of initiation codons can sometimes be challenging. In this mt genome, all protein-coding genes had ATA or ATG or ATT or TTG as their initiation codon. 4 genes (cox1, atp6, cox3 and cox2) start with ATA, 5 genes (nad6, nad2, nad5, atp8 and nad1) start with ATG, 3 genes (nad3, nad4L and nad4) start with ATT and 1 gene (cytb) use TTG (Table 1). All protein-coding genes had TAA or TAG or TA or T as their termination codon (Table 1). 8 genes (cox1, atp6, nad6, cox3, nad2, cox2, atp8 and nad4L) stop with TAA, 3 genes (nad4, nad3, and cytb) stop with TAG, nad1 gene stop with TA and nad5 gene use T (Table 1). Incomplete termination codons (TA or T) were identified in nad1 and nad5 genes, which is consistent with studies of some other bird lice, including B. macrocnemis (nad1), F. quadripustulatus (nad5, nad6 and nad1). In F. suturalis mt genome, the rrnL genes was located between rrnS and tRNA-Ala genes, and rrnS genes was between tRNA-Asn and rrnL genes (Fig. 2; Table 1). The lengths of the rrnS and rrnL genes were 726 bp and 1075 bp, respectively. The 22 tRNA genes length varied from 61 to 71 bp (Table 1). All 22 tRNA genes can fold into cloverleaf structure (Fig. 3), which were consistent with those of previous studies [32, 33]. Non-coding region (NC1) (829 bp), located between nad4 and tRNA-L₂, has the highest A + T content of 75.4%. Non-coding region (NC2) (722 bp; A + T = 74.4%) located between cox2 and nad5 and Non-coding region (NC3) (425bp; A + T = 75.1%) located between tRNA-G and nad3 (Table 1).

Comparative analyses between F. suturalis and F. quadripustulatus

The entire mt genome of F. suturalis is 537 bp longer than that of F. quadripustulatus [11]. A comparison of the nucleotide and the amino acid sequences of each protein-coding gene of the two Falcolipeurus species is given in Table 3. Nucleotide sequence difference across the entire mt genome was 31.4%. The magnitude of nucleotide sequence variation in each gene between F. suturalis and F. quadripustulatus ranged from 13.2–27.5%. The greatest variation was observed in the atp8 gene (27.5%), whereas least differences (13.2%) were found in the cytb genes (Table 3). For the rrnL and rrnS genes, sequence difference was 28.4% and 14.6% between F. suturalis and F. quadripustulatus, respectively (Table 3). Amino acid sequences inferred from individual mt protein genes of F. suturalis were compared with those of F. quadripustulatus. The amino acid sequence differences ranged from 4.5%-41.2%, with cox1 being the most conserved protein, and atp8 the least conserved (Table 3). This level of amino acid difference is very high. Previous studies of other lice have detected high level difference in protein sequences. For example, difference in amino acid sequences of the 13 protein-coding genes between C. picui and C. cruziana was 5.5–50% [16], and C. bidentatus compar and C. compar was 0-37.3% [11, 14]. Taken together, the molecular evidence presented here supports that F. suturalis and F. quadripustulatus represent distinct louse species.

Table 3

Nucleotide (nt) and/or predicted amino acid (aa) sequence differences in mitochondrial genes among *Falcolipeurus quadripustulatus* (FQ) and *Falcolipeurus suturalis* (FS) upon pairwise comparison
Gene/region	Nt sequence length		Nt diference (%)	Number of aa		aa diference (%)
Gene/region	FS	FQ	FS/FQ	FS	FQ	FS/FQ
cox1	1524	1554	15.3	507	517	4.5
atp6	672	675	18.5	223	224	16.1
rrnS	726	610	28.4
rrnL	1075	1084	14.6
nad6	474	478	22	157	159	25.8
cox3	750	789	21.2	249	265	16.2
nad4	1314	1305	24	437	434	27.2
nad2	972	972	27	323	323	32.5
cox2	678	675	14.4	225	224	7.5
nad5	1676	1711	19.6	558	570	21
atp8	201	204	27.5	66	67	41.2
nad3	387	354	27.4	128	117	34.4
nad4L	273	288	20.8	90	95	21.1
nad1	907	848	20	302	282	12.6
cytb	1092	1092	13.2	363	363	9

Gene rearrangement

The mt genome arrangement of two Falcolipeurus species substantially differs from those of other bird louse species within the family Philopteridae and from the inferred typical gene arrangement of ancestral insect mt genome (Fig. 4). Only two gene blocks are shared between B. macrocnemis and the ancestral insect pattern: G-nad3 and atp8-atp6 [13], and one gene block is shared between Campanulotes species and the ancestral insect: atp8-atp6 [11, 14, 34]. However, no derived mt gene arrangements are shared between the two Falcolipeurus species. In addition, three gene blocks, V-cox3, Y-cox2 and L₁-nad4L, are shared by Falcolipeurus and Ibidoecus [11]. Such a lack of conserved gene arrangement in the mt genome of bird lice precludes the accurate reconstruction and identification of the rearrangement events and model [13].

Usually, the gene arrangement in the mt genome is very conserved within the same genus of ectoparasites [11, 14, 35]. Gene arrangement events between F. suturalis and F. quadripustulatus were also analyzed (Fig. 5); at least one translocation could be inferred. The nad3 gene is located between cox2 and tRNA-Thr genes in F. quadripustulatus, but was found between tRNA-Gly and tRNA-Leu^CUN in F. suturalis (Fig. 5). The gene arrangement in the mt genomes of two Falcolipeurus species indicated that the rate of change in the arrangement of mt genes may vary substantially among closely related groups of lice [36].

One tRNA gene (tRNA-Gly) was lacking and the duplication of three genes (tRNA-Thr, tRNA-Tyr and cox2) was detected in the F. quadripustulatus mt genome [11]. However, 37 genes have been identified in the F. suturalis mt genome. Gene duplication have been also reported in mt genomes of several families of the class Insecta, such as Brontostoma colossus [37], Phalantus geniculatus [38] and Reduvius tenebrosus [39]. In addition, tRNA loss was also found in the mt genome of several families of the class Insecta [11, 40], and this case can be explained by the tandemduplication-random loss (TDRL) model.

Phylogenetic relationships

Phylogenetic analysis showed the clear genetic distinctiveness between F. suturalis and F. quadripustulatus (Bayesian posterior probabilities = 1.0). The branch leading to the two Falcolipeurus species is much longer than the branch of two Columbina species (looking at branch lengths in tree). The genus Falcolipeurus is more closely related to the genus Ibidoecus than to other genera (Bayesian posterior probabilities = 1.0) (Fig. 6), which was consistent with that of a previous study [11].

Mt genome sequences are effective molecular markers to study the phylogenetic and systematic relationships at various taxonomic ranks across the phylum Arthropoda, including ectoparasites [41–46]. DNA sequencing provides the opportunity to further evaluate the phylogenetic relationships of the Philopteridae. For examples, Cruickshank et al. analyzed nuclear elongation factor-1 alpha (EF1α) sequences of 127 species from the four suborders and showed the Philopteridae to be paraphyletic [41]. Yoshizawa and Johnson 2003 analyzed mt 12S and 16S rDNA sequences of 18 species and showed the Philopteridae to be paraphyletic [42]. However, Johnson et al. analyzed 1107 single-copy orthologous genes of 46 species and showed that the Philopteridae to be monophyletic [43]. de Moya et al., analyzed 2,370 orthologous genes and showed that the Philopteridae to be monophyletic [44]. To date, the phylogenetic position of the Philopteridae in deep-level relationships within the order Phthiraptera could not be confidently determined. Although mt genomic data have been proven to be useful genetic marker to explore the phylogenetic relationships among several major lineages of parasitic lice [7, 9, 11], mt genome sequences of many lineages of the family Philopteridae are underrepresented or not represented. Therefore, more complete mt genomes of bird louse species representing this families that have not yet been decoded should be included in future analysis to resolve the phylogenetic position of the family Philopteridae within the order Phthiraptera.

The present study presents the entire mt genome sequences of F. suturalis and compared it with the mt genome sequences of F. quadripustulatus. Gene order is rearranged and represents a new pattern within the order Phthiraptera. These novel datasets will help to better understand the gene rearrangements and phylogenetic position of Falcolipeurus and provide useful genetic markers for systematics and phylogenetic studies of bird lice.

mt: mitochondrial; rDNA: ribosomal DNA; BI: Bayesian inference; nad2: NADH dehydrogenase subunit 2; cox1: cytochrome c oxidase subunit 1; cox2: cytochrome c oxidase subunit 2; ATP6: ATP synthase F0 subunit 6; cox3: cytochrome c oxidase subunit 3; nad3: NADH dehydrogenase subunit 3; nad5: NADH dehydrogenase subunit 5; nad4: NADH dehydrogenase subunit 4; nad4L: NADH dehydrogenase subunit 4L; nad6: NADH dehydrogenase subunit 6; cytb: cytochrome b; atp8: ATP synthase F0 subunit 8; nad1: NADH dehydrogenase subunit 1; tRNA: transfer RNA; rrnL: large subunit of rRNA; rrnS: small subunit of rRNA

Acknowledgements

Not applicable.

Authors’ contributions

GHL and YT conceived and designed the study, and critically revised the manuscript. YN performed the experiments. YN YTF and GHL analyzed the data. YN and YTF drafted the manuscript. YZ YPD helped in study design, study implementation, and manuscript preparation. All authors read and approved the final manuscript.

Funding

This study was supported by the Planned Programme of Hunan Province Science and Technology Innovation (Grant no. 2018RS3085) and the Training Programme for Excellent Young Innovators of Changsha (Grant No. KH2002001).

Availability of data and materials

The complete mitochondrial genome sequences of Falcolipeurus suturalis have been deposited in the GenBank database under the accession number MI456908.

Ethics approval and consent to participate

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. 43321503).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan Province 410128, China. ²Beijing Wildlife Rescue Center, Beijing 101300, China.

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Fig.S1.tif
Fig. S1: PCR amplicons from the mitochondrial genome of Falcolipeurus suturalis. Amplicons generated with the F. suturalis primers. M: DL8000 DNA marker, 1: Validation_01, 2: Validation_02, 3: Validation_03, 4: Validation_04, 5: Validation_05, 6: Negative control.
TableS1.docx
Table S1 Primers used for assembly validation.
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Highly Rearranged Mitochondrial Genome in Falcolipeurus lice (Phthiraptera: Philopteridae) from Endangered Eagles

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Abstract

Figures

Background

Methods

Sample collection and DNA extraction

Sequencing, assembling and verification

Annotation and sequence analysis

Phylogenetic analysis

Results And Discussion

Genome organization

Annotation

Gene rearrangement

Phylogenetic relationships

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1