Insights from entire mitochondrial genome sequences into the phylogeny of ticks of the genera Haemaphysalis and Archaeocroton with the elevation of the subgenus Alloceraea Schulze, 1919 back to the status of a genus.

We used entire mitochondrial (mt) genome sequences (14.5-15 kbp) to resolve the phylogeny of the four main lineages of the Haematobothrion ticks: Alloceraea, Archaeocroton, Bothriocroton and Haemaphysalis. In our phylogenetic trees, Alloceraea was the sister to Archaeocroton sphenodonti, a tick of an archetypal reptile, the tuatara, from New Zealand, to the exclusion of the rest of the species of Haemaphysalis. The mt genomes of all four of the Alloceraea species that have been sequenced so far had a substantial insert, 132-312 bp, between the tRNA-Glu (E) gene and the nad1 gene in their mt genomes. This insert was not found in any of the other eight subgenera of Haemaphysalis. The mt genomes of 13 species of Haemaphysalis from NCBI GenBank were added to the most recent data set on Haemaphysalis and its close relatives to help resolve the phylogeny of Haemaphysalis, including five new subgenera of Haemaphysalis not previously considered by other authors: Allophysalis (structurally primitive), Aboimisalis (structurally primitive), Herpetobia (structurally intermediate), Ornithophysalis (structurally advanced) and Segalia (structurally advanced). We elevated Alloceraea Schulze, 1919 to the status of genus because Alloceraea Schulze, 1919 is phylogenetically distinct from the other subgenera of Haemaphysalis. Moreover, we propose that the subgenus Allophysalis is the sister to the rest of the Haemaphysalis (14 subgenera) and that the 'structurally primitive' subgenera Hoogstraal and Kim comprise early diverging lineages. Our matrices of the pairwise genetic difference (percent) of mt genomes and partial 16S rRNA sequences indicated that the mt genome sequence of Al. kitaokai (gb# OM368280) may not be Al. kitaokai Hoogstraal, 1969 but rather another species of Alloceraea. In a similar way, the mt genome sequence of H. (Herpetobia) nepalensis Hoogstraal, 1962 (gb# NC_064124) was only 2% genetically different to that of H. (Allophysalis) tibetensis Hoogstraal, 1965 (gb# OM368293): this indicates to us that they are the same species. Alloceraea cretacea may be better placed in a genus other than Alloceraea Schulze, 1919. Reptiles may have been the host to the most recent common ancestor of Archaeocroton and Alloceraea.

New insights into the molecular phylogeny, biogeographical history, and diversification of Amblyomma ticks (Acari: Ixodidae) based on mitogenomes and nuclear sequences.

BACKGROUND: Amblyomma is the third most diversified genus of Ixodidae that is distributed across the Indomalayan, Afrotropical, Australasian (IAA), Nearctic and Neotropical biogeographic ecoregions, reaching in the Neotropic its highest diversity. There have been hints in previously published phylogenetic trees from mitochondrial genome, nuclear rRNA, from combinations of both and morphology that the Australasian Amblyomma or the Australasian Amblyomma plus the Amblyomma species from the southern cone of South America, might be sister-group to the Amblyomma of the rest of the world. However, a stable phylogenetic framework of Amblyomma for a better understanding of the biogeographic patterns underpinning its diversification is lacking. METHODS: We used genomic techniques to sequence complete and nearly complete mitochondrial genomes -ca. 15 kbp- as well as the nuclear ribosomal cluster -ca. 8 kbp- for 17 Amblyomma ticks in order to study the phylogeny and biogeographic pattern of the genus Amblyomma, with particular emphasis on the Neotropical region. The new genomic information generated here together with genomic information available on 43 ticks (22 other Amblyomma species and 21 other hard ticks-as outgroup-) were used to perform probabilistic methods of phylogenetic and biogeographic inferences and time-tree estimation using biogeographic dates. RESULTS: In the present paper, we present the strongest evidence yet that Australasian Amblyomma may indeed be the sister-group to the Amblyomma of the rest of the world (species that occur mainly in the Neotropical and Afrotropical zoogeographic regions). Our results showed that all Amblyomma subgenera (Cernyomma, Anastosiella, Xiphiastor, Adenopleura, Aponomma and Dermiomma) are not monophyletic, except for Walkeriana and Amblyomma. Likewise, our best biogeographic scenario supports the origin of Amblyomma and its posterior diversification in the southern hemisphere at 47.8 and 36.8 Mya, respectively. This diversification could be associated with the end of the connection of Australasia and Neotropical ecoregions by the Antarctic land bridge. Also, the biogeographic analyses let us see the colonization patterns of some neotropical Amblyomma species to the Nearctic. CONCLUSIONS: We found strong evidence that the main theater of diversification of Amblyomma was the southern hemisphere, potentially driven by the Antarctic Bridge's intermittent connection in the late Eocene. In addition, the subgeneric classification of Amblyomma lacks evolutionary support. Future studies using denser taxonomic sampling may lead to new findings on the phylogenetic relationships and biogeographic history of Amblyomma genus.

The first cryptic genus of Ixodida, Cryptocroton n. gen. for Amblyomma papuanum Hirst, 1914: a tick of North Queensland, Australia, and Papua New Guinea.

We describe a new genus Cryptocroton n. gen. for Amblyomma papuanum Hirst, 1914, a tick of North Queensland, Australia, and Papua New Guinea.

Seventy-eight entire mitochondrial genomes and nuclear rRNA genes provide insight into the phylogeny of the hard ticks, particularly the Haemaphysalis species, Africaniella transversale and Robertsicus elaphensis.

Hoogstraal and Kim (1985) proposed from morphology, three groups of Haemaphysalis subgenera: (i) the "structurally advanced"; (ii) the "structurally intermediate"; and (iii) the "structurally primitive" subgenera. Nuclear gene phylogenies, however, did not indicate monophyly of these morphological groups but alas, only two mitochondrial (mt) genomes from the "structurally intermediate" subgenera had been sequenced. The phylogeny of Haemaphysalis has not yet been resolved. We aimed to resolve the phylogeny of the genus Haemaphysalis, with respect to the subgenus Alloceraea. We presented 15 newly sequenced and annotated mt genomes from 15 species of ticks, five species of which have not been sequenced before, and four new 18S rRNA and 28S rRNA nuclear gene sequences. Our datasets were constructed from 10 mt protein-coding genes, cox1, and the 18S and 28S nuclear rRNA genes. We found a 132-bp insertion between tRNA-Glu (E) gene and the nad1 gene in the mt genome of Haemaphysalis (Alloceraea) inermis that resembles insertions in H. (Alloceraea) kitaokai and Rhipicephalus (Boophilus) geigyi. Our mt phylogenies had the three species of Amblyomma (Aponomma) we sequenced embedded in the main clade of Amblyomma: Am. (Aponomma) fimbriatum, Am. (Aponomma) gervaisi and Am. (Aponomma) latum. This is further support for the hypothesis that the evolution of eyes appears to have occurred in the most-recent-common-ancestor of Amblyocephalus (i.e. Amblyomminae plus Rhipicephalinae) and that eyes were subsequently lost in the most-recent-common-ancestor of the subgenus Am. (Aponomma). Either Africaniella transversale or Robertsicus elaphensis, or perhaps Af. transversale plus Ro. elaphensis, appear to be the sister-group to the rest of the metastriate Ixodida. Our cox1 phylogenies did not indicate monophyly of the "structurally primitive", "structurally intermediate" nor the "structurally advanced" groups of Haemaphysalis subgenera. Indeed, the subgenus Alloceraea may be the only monophyletic subgenus of the genus Haemaphysalis sequenced thus far. All of our mt genome and cox1 phylogenies had the subgenus Alloceraea in a clade that was separate from the rest of the Haemaphysalis ticks. If Alloceraea is indeed the sister to the rest of the Haemaphysalis subgenera this would resonate with the argument of Hoogstraal and Kim (1985), that Alloceraea was a subgenus of "primitive" Haemaphysalis. Alectorobius capensis from Japan had a higher genetic-identity to A. sawaii, which was also from Japan, than to the A. capensis from South Africa. This indicates that A. capensis from Japan may be a cryptic species with respect to the A. capensis from South Africa.

Description of the female, nymph and larva and mitochondrial genome, and redescription of the male of Ixodes barkeri Barker, 2019 (Acari: Ixodidae), from the short-beaked echidna, Tachyglossus aculeatus, with a consideration of the most suitable subgenus for this tick.

BACKGROUND: Ixodes barkeri, a tick with a distinctive ventrolateral horn-like projection on palpal segment 1, was described in 2019 from two male ticks from the Wet Tropics of Far North Queensland, Australia. However, females lie at the core of the taxonomy and subgenus classification of Ixodes; hence, we sought specimens of female ticks, successfully recovering females, plus nymphs and larvae. Mitochondrial genomes are also desirable additions to the descriptions of species of ticks particularly regarding subgenus systematics. So, we sequenced the mt genomes of I. barkeri Barker, 2019, and the possible relatives of I. barkeri that were available to us (I. australiensis Neumann, 1904, I. fecialis Warburton & Nuttall, 1909, and I. woyliei Ash et al. 2017) with a view to discovering which if any of the subgenera of Ixodes would be most suitable for I. barkeri Barker, 2019. RESULTS: The female, nymph, larva and mitochondrial genome of Ixodes barkeri Barker, 2019, are described for the first time and the male of I. barkeri is redescribed in greater detail than previously. So far, I. barkeri is known only from a monotreme, the short-beaked echidna, Tachyglossus aculeatus (Shaw, 1792), from the highland rainforests of the Wet Tropics of Far North Queensland, Australia. CONCLUSIONS: Our phylogeny from entire mitochondrial genomes indicated that I. barkeri and indeed I. woyliei Ash et al., 2017, another tick that was described recently, are best placed in the subgenus Endopalpiger Schulze, 1935.

Rediscovery of Ixodes confusus in Australia with the first description of the male from Australia, a redescription of the female and the mitochondrial (mt) genomes of five species of Ixodes.

We: (i) report the rediscovery of Ixodes (Sternalixodes) confusus Roberts, 1960 in Australia; (ii) redescribe the male and female of I. confusus; (iii) describe the mitochondrial (mt) genome of I. confusus from five ticks from four localities in Far North Queensland; and (iv) present the first substantial phylogeny of the subgenera of the Ixodes. The mt genomes of I. confusus, I. cornuatus, I. hirsti, I. myrmecobii and I. trichosuri are presented here for the first time. In our phylogeny from entire mt genomes (ca. 15 kb), the subgenus Endopalpiger was the sister-group to subgenera Sternalixodes plus Ceratixodes plus Exopalpiger whereas Exopalpiger was the sister to Sternalixodes plus Ceratixodes. [i.e. ((Endopalpiger) (Sternalixodes, Ceratixodes and Exopalpiger))]. Finally, we show that Ixodes anatis, the kiwi tick, may be closely related to the ticks of marsupials of Australia and Papua New Guinea.

Reconstruction of mitochondrial genomes from raw sequencing data provides insights on the phylogeny of Ixodes ticks and cautions for species misidentification.

High-throughput sequencing (HTS) technology has profoundly been involved in sequencing whole genomes of several organisms in a fast and cost-effective manner. Although HTS provides an alternative biomonitoring method to the time-consuming and taxonomy-expertise dependent morphological approach, still we cannot rule out the possibility of the impediment and misidentification biases. In this article we aim to retrieve whole mitochondrial genome (mitogenome) sequences from publicly available raw sequencing data for phylogenetic comparison of Ixodes persulcatus. For this comparison, we sequenced whole mitogenomes of four I. persulcatus ticks from Japan and constructed mitogenomes from raw sequencing data of 74 I. persulcatus ticks from China. Bayesian phylogenetic trees were inferred by the concatenated fifteen mitochondrial genes. We further tested our results by the phylogenetic analysis of cytochrome c oxidase subunit 1 (cox1) gene and internal transcribed spacer 2 (ITS2) sequences. Our findings showed that 70 constructed mitogenomes from China were clustered with the sequenced four mitogenomes of I. persulcatus from Japan. We also revealed that mitogenome sequences retrieved from two data sets CRR142297 and CRR142298 were clustered with Ixodes nipponensis. Moreover, other two mitogenome sequences from CRR142310 and CRR142311 formed a clade with Ixodes pavlovskyi. The phylogenetic analysis of cox1 gene and ITS2 sequences confirmed the identification errors of these four samples. The overall phylogenetics in our study concluded that accurate morphological identification is necessary before implementing HTS to avoid any misidentification biases.

Climatic requirements of the southern paralysis tick, Ixodes cornuatus, with a consideration of its host, Vombatus ursinus, and the possible geographic range of the tick up to 2090.

The southern paralysis tick, Ixodes cornuatus, is a tick of veterinary and medical importance in Australia. We use two methods, CLIMEX, and an envelope-model approach which we name the 'climatic-range method' to study the climatic requirements of I. cornuatus and thus to attempt to account for the geographic distribution of I. cornuatus. CLIMEX and our climatic-range method allowed us to account for 94% and 97% of the records of I. cornuatus respectively. We also studied the host preferences of I. cornuatus which we subsequently used in conjunction with our species distribution methods to account for the presence and the absences of I. cornuatus across Australia. Our findings indicate that the actual geographic distribution of I. cornuatus is smaller than the potential geographic range of this tick, and thus, that there are regions in Australia which may be suitable for I. cornuatus where this tick has not been recorded. Although our findings indicate that I. cornuatus might be able to persist in these currently unoccupied regions, our findings also indicate that the potential geographic range of I. cornuatus may shrink by 51 to 76% by 2090, depending on which climate change scenario comes to pass.

Nuclear (18S-28S rRNA) and mitochondrial genome markers of Carios (Carios) vespertilionis (Argasidae) support Carios Latreille, 1796 as a lineage embedded in the Ornithodorinae: re-classification of the Carios sensu Klompen and Oliver (1993) clade into its respective subgenera.

Argasid systematics remains controversial with widespread adherence to the Hoogstraal (1985) classification scheme, even though it does not reflect evolutionary relationships and results in paraphyly for the main genera of soft ticks (Argasidae), namely Argas and Ornithodoros. The alternative classification scheme, proposed by Klompen and Oliver (1993), has problems of its own: most notably paraphyly of the subgenus Pavlovskyella and the controversial grouping together of the subgenera Alectorobius, Antricola, Carios, Chiropterargas, Nothoaspis, Parantricola, Reticulinasus and Subparmatus into the genus Carios. Recent phylogenetic analyses of 18S/28S rRNA sequences and mitochondrial genomes agree with the scheme of Klompen and Oliver (1993), with regard to the paraphyly of Pavlovskyella, placement of Alveonasus, Ogadenus, Proknekalia and Secretargas in the Argasinae and placement of Carios and Chiropterargas in the Ornithodorinae (Mans et al., 2019). The Carios clade and its constituent subgenera remain controversial, since the phylogenetic position of its type species Carios (Carios) vespertilionis Latreille, 1796 (formerly Argas vespertilionis) has not been determined with confidence. The current study aimed to resolve Carios sensu lato Klompen and Oliver, 1993, and Carios sensu stricto Hoogstraal, 1985, by determining and analysing phylogenetic nuclear and mitochondrial markers for C. (C.) vespertilionis. Both the nuclear and mitochondrial markers support placement of Carios s.s. within the subfamily Ornithodorinae, but to the exclusion of the clade that includes the 6 other subgenera that are part of Carios s.l. Klompen and Oliver (1993), namely Alectorobius, Antricola, Nothoaspis, Parantricola, Reticulinasus and Subparmatus. These 6 subgenera form a monophyletic clade that might be placed as new subgenera within the genus Alectorobius, or elevated to genera. Given the substantial differences in biology among these subgenera, we propose that these 6 subgenera be elevated to genera. Thus, we propose to modify the classification scheme of Mans et al. (2019) so that the subfamily Argasinae now has six genera, Alveonasus, Argas (subgenera Argas and Persicargas), Navis, Ogadenus, Proknekalia and Secretargas, and the subfamily Ornithodorinae has nine genera, Alectorobius, Antricola (subgenera Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (subgenera Microargas, Ornamentum, Ornithodoros, Pavlovskyella and Theriodoros), Otobius, Reticulinasus and Subparmatus (genera indicated in bold).

Climatic requirements of the eastern paralysis tick, Ixodes holocyclus, with a consideration of its possible geographic range up to 2090.

The eastern paralysis tick, Ixodes holocyclus, is an ectoparasite of medical and veterinary importance in Australia. The feeding of I. holocyclus is associated with an ascending flaccid paralysis which kills many dogs and cats each year, with the development of mammalian meat allergy in some humans, and with the transmission of Rickettsia australis (Australian scrub typhus) to humans. Although I. holocyclus has been well studied, it is still not known exactly why this tick cannot establish outside of its present geographic distribution. Here, we aim to account for the presence as well as the absence of I. holocyclus in regions of Australia. We modelled the climatic requirements of I. holocyclus with two methods, CLIMEX, and a new envelope-model approach which we name the 'climatic-range method'. These methods allowed us to account for 93% and 96% of the geographic distribution of I. holocyclus, respectively. Our analyses indicated that the geographic range of I. holocyclus may not only shift south towards Melbourne, but may also expand in the future, depending on which climate-change scenario comes to pass.

Phylogenies from mitochondrial genomes of 120 species of ticks: Insights into the evolution of the families of ticks and of the genus Amblyomma.

The evolution and phylogenetic relationships of the ticks at both the family and genus levels are contested. The genus Amblyomma and its subgenera are in a state of flux; moreover, the relationships among the three tick families are controversial due to conflicting phylogenetic support for different arrangements of the three families of living ticks. With 18 newly sequenced mitochondrial (mt) genomes of ticks included, we executed the largest mt genome phylogenetic study of ticks so far. Phylogenetic trees were inferred from one sea spider mt genome, one horseshoe crab, five mite mt genomes and 146 tick mt genomes from 120 species: 153 mt genomes in total. Sixteen phylogenetic trees were inferred from 10 datasets using both maximum likelihood and Bayesian inference methods. We describe the first novel mt gene-arrangement for the metastriate Ixodidae in Amblyomma (Africaniella) transversale. Also, three unusual partial 16S rRNA gene inserts were found in the mt genome of Haemaphysalis (Alloceraea) kitaokai: we consider the possible role of past genome translocation events in the formation of these inserts. Our phylogenies revealed evidence that: (i) the genus Amblyomma is polyphyletic with respect to Amblyomma (Africaniella) transversale; (ii) the subgenus Aponomma is apparently embedded in the genus Amblyomma; (iii) Haemaphysalis (Segalia) parva and Haemaphysalis (Alloceraea) kitaokai form a clade to the exclusion of other Haemaphysalis species; and (iv) the phylogenetic position of the family Nuttalliellidae is unstable among phylogenies from different datasets.

Full-length genome sequence of segmented RNA virus from ticks was obtained using small RNA sequencing data.

BACKGROUND: In 2014, a novel tick-borne virus of the Flaviviridae family was first reported in the Mogiana region of Brazil and named the Mogiana tick virus (MGTV). Thereafter, the Jingmen tick virus (JMTV), Kindia tick virus (KITV), and Guangxi tick virus (GXTV)-evolutionarily related to MGTV-were reported. RESULTS: In the present study, we used small RNA sequencing (sRNA-seq) to detect viruses in ticks and discovered a new MGTV strain in Amblyomma testudinarium ticks collected in China's Yunnan Province in 2016. We obtained the full-length genome sequence of this MGTV strain Yunnan2016 (GenBank: MT080097, MT080098, MT080099 and MT080100) and recommended it for its inclusion in the NCBI RefSeq database for future studies on MGTV, JMTV, KITV and GXTV. Phylogenetic analysis showed that MGTV, JMTV, KITV and GXTV are monophyletic and belong to a MGTV group. Furthermore, this MGTV group of viruses may be phylogenetically related to geographical regions that were formerly part of the supercontinents Gondwana and Laurasia. CONCLUSIONS: To the best of our knowledge, this is the first study in which 5' and 3' sRNAs were used to generate full-length genome sequences of, but not limited to, RNA viruses. We also demonstrated the feasibility of using the sRNA-seq based method for the detection of viruses in pooled two and even possible one small ticks. MGTV may preserve the characteristic of ancient RNA viruses, which can be used to study the origin and evolution of RNA viruses. In addition, MGTV can be used as novel species for studies in phylogeography.

Precise annotation of tick mitochondrial genomes reveals multiple copy number variation of short tandem repeats and one transposon-like element.

BACKGROUND: In the present study, we used long-PCR amplification coupled with Next-Generation Sequencing (NGS) to obtain complete mitochondrial (mt) genomes of individual ticks and unprecedently performed precise annotation of these mt genomes. We aimed to: (1) develop a simple, cost-effective and accurate method for the study of extremely high AT-content mt genomes within an individual animal (e.g. Dermacentor silvarum) containing miniscule DNA; (2) provide a high-quality reference genome for D. silvarum with precise annotation and also for future studies of other tick mt genomes; and (3) detect and analyze mt DNA variation within an individual tick. RESULTS: These annotations were confirmed by the PacBio full-length transcriptome data to cover both entire strands of the mitochondrial genomes without any gaps or overlaps. Moreover, two new and important findings were reported for the first time, contributing fundamental knowledge to mt biology. The first was the discovery of a transposon-like element that may eventually reveal much about mechanisms of gene rearrangements in mt genomes. Another finding was that Copy Number Variation (CNV) of Short Tandem Repeats (STRs) account for mitochondrial sequence diversity (heterogeneity) within an individual tick, insect, mouse or human, whereas SNPs were not detected. The CNV of STRs in the protein-coding genes resulted in frameshift mutations in the proteins, which can cause deleterious effects. Mitochondria containing these deleterious STR mutations accumulate in cells and can produce deleterious proteins. CONCLUSIONS: We proposed that the accumulation of CNV of STRs in mitochondria may cause aging or diseases. Future tests of the CNV of STRs hypothesis help to ultimately reveal the genetic basis of mitochondrial DNA variation and its consequences (e.g., aging and diseases) in animals. Our study will lead to the reconsideration of the importance of STRs and a unified study of CNV of STRs with longer and shorter repeat units (particularly polynucleotides) in both nuclear and mt genomes.