Ixodes (Sternalixodes) confusus, Roberts, 1960

Barker, Dayana, Kelava, Samuel, Seeman, Owen D., Shao, Renfu, Seaniger, James R., Jones, Malcolm K., Apanaskevich, Maria A., Nakao, Ryo, Apanaskevich, Dmitry A. & Barker, Stephen C., 2022, 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, International Journal for Parasitology: Parasites and Wildlife 18, pp. 1-11 : 8-9

4.1. Rediscovery of Ixodes (Sternalixodes) confusus in Australia

Before our study, I. confusus was known in Australia from a single specimen collected from a human at Etty Bay in 1949 (Table 1; Roberts 1960, 1970). Thus, it was extremely doubtful that I. c onfusus was endemic to Australia. Indeed, the single female specimen could have been brought to Australia from Papua New Guinea on a human, since humans often travel between Papua New Guinea and Cairns, and not a single adult I. confusus had since been collected in Australia. However, a concerted collection effort (DB, SCB) demonstrated that this species is present in Far North Queensland, where it is probably widespread (Table 1; Fig. 1 ). The host records suggest adults have a preference for macropods (27/35 ticks) but the records from a human, horse and cattle show I. confusus occasionally attaches to other hosts. [We recently found another female in the USNTC from Mossman River Gorge, Mossman, Qld, which was collected during an Archibold Expedition in 1948 (Table 1).]

together with previously published mt genomes of Ixodes , indicate monophyly of the subgenera Sternalixodes and Endopalpiger ( Fig. 7 ). The mitochondrial genomes published for the first time in this paper have been submitted to GenBank database; accession numbers OL614953 to OL614959.

4.2. Mitochondrial (mt) genomes of Ixodes confusus , four of its relatives, and the phylogenetic position of the subgenus Sternalixodes

The live I. confusus provided the first opportunity to investigate the phylogenetic relationships of I. confusus to its relatives with large numbers of nucleotides. Previously, only small numbers of nucleotides have been recovered from museum and other specimens (e.g. Ash et al., 2017; Kwak et al., 2017). Thus, we sequenced the entire mitochondrial genome of I. confusus (five individuals from three localities; refer to Fig. 1 , Appendix 1), and four of its relatives: I. cornuatus , I. hirsti , I. trichosuri , and I. myrmecobii . We presented the first substantial phylogeny of the subgenera of the genus Ixodes ( Fig. 7 ). The subgenera Sternalixodes (6 species) and Ceratixodes Neumann, 1902 ( I. uriae White, 1852 ) were sisters (sister-groups). This is intriguing since I. uriae is exclusively a sea-bird tick whereas the eight known species of Sternalixodes infest mammals in the adult stage, although birds are apparently the main hosts of the nymphs and larvae in at least two species, I. cordifer and I. hirsti ( Barker and Barker 2022) .

It is also intriguing that the subgenus Exopalpiger Schulze, 1935

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( I. fecialis Warburton and Nuttall, 1909 ) was the sister to subgenera Sternalixodes plus Ceratixodes [i.e. the arrangement ( Exopalpiger , ( Sternalixodes , Ceratixodes ))] rather than Exopalpiger being closely related to Endopalpiger ( Fig. 7 ). Indeed, Exopalpiger was well-removed from Endopalpiger in our tree ( Fig. 7 ). So, Camicas and Morel (1977) and Camicas et al. (1998) were mistaken when they subsumed Endopalpiger into Exopalpiger . We can only wonder why Camicas and Morel (1977) and Camicas et al. (1998) made such a major decision without presenting any evidence or argument. 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 ))] ( Fig. 7 ). In other words, Sternalixodes , Ceratixodes and Exopalpiger shared a Most Recent Common Ancestor to the exclusion of Endopalpiger .

The two subgenera in our tree with more than one species, Endopalpiger and Sternalixodes , were monophyletic ( Fig. 7 ). Regarding Sternalixodes , we do not have mt genomes for two of the eight species of this subgenus: I. cordifer from Australia and Papua New Guinea; and I. dendrolagi from Papua New Guinea. We do not expect, however, that mt genomes from these two species will challenge the hypothesis of a monophyletic Sternalixodes since I. confusus , I. cordifer and I. dendrolagi are morphologically similar and thus likely, closely related. Indeed, Wilson (1967) considered I. confusus , I. cordifer and I. dendrolagi to be so closely related that he designated the I. cordifer (species) group for these three species and the associated subspecies of I. cordifer ( I. cordifer cordifer and I. cordifer bibax ).

Finally, we took the opportunity to make a phylogeny from all 27 of the entire mt genomes that are now available for Ixodes (Appendix 2). The subgenera Sternalixodes , Endopalpiger and Ixodes were monophyletic in our tree (Appendix 2).

4.3. Ixodes anatis Chilton, 1904 , the kiwi tick, may be a closely related to the ticks of marsupials of Australia and Papua New Guinea

The present study of I. confusus and its relatives in the subgenus

Sternalixodes led us to consider I. anatis , the kiwi tick, since I. anatis was placed in the subgenus Sternalixodes by Clifford et al. (1973) but considered best removed from Sternalixodes by Kwak and Heath (2018). We made a tree with the cox 1 fragment (674 bp) from I. anatis of Kwak et al. (2017), together with cox 1 sequences from the mt genomes sequenced by us in the present study, and some cox1 sequences from GenBank (Appendix 3). Our trees from this short fragment of cox 1 had I. anatis as a member of the “Australian Ixodes ” clade. These trees, however, were from only 674 bp of one gene, cox 1, of the mt genome. For instance, the unresolved position of I. uriae and I. woyliei in the cox 1 tree is resolved by entire mt genomes ( Fig. 7 ). Thus, conclusions about the affinities and evolutionary history of I. anatis , the kiwi tick, must await entire mt genome sequences.

Figures

Fig. 1. The five known localities in Queensland (Qld) Australia of Ixodes confusus Roberts (1960), indicated by white-with-red dots. Note that there were two sites at Etty Bay: (i) near Etty Bay Caravan Park; and (ii) Etty Bay, in the vicinity of 195 Mourilyan Harbour Rd, Etty Bay. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7. Maximum likelihood (ML) phylogenetic tree from entire mt genomes (15,254 bp alignment). Tip-labels indicate NCBI accession numbers. Numbers above branches show Maximum Likelihood bootstrap support whereas numbers below branches show the Bayesian Posterior Probability support. Ixodes pavlovskyi Pomerantzev, 1946, one of the species of “other Ixodes” clade (sensu Barker et al., 2021), for which an entire mitochondrial (mt) genome was available in GenBank, was set as the outgroup. The scale bar indicates 0.07 nucleotide substitutions per nucleotide site for the 15,254 nucleotide sites in our alignment of theses entire mt genomes. So, for example, there were about 1067 nucleotide substitutions along the branch that leads to I. (Ceratixodes) uriae plus I. (Sternalixodes) holocyclus plus I. (Exopalpiger) fecialis, which is marked with an asterisk (i.e. 0.07 nucleotide substitutions per nucleotide site x 15,254 nucleotide sites (bps) = 1067 nucleotide substitutions). Ticks in bold were sequenced in the present study.