Nuclear and mitochondrial population genetics of the Australasian arbovirus vector Culex annulirostris (Skuse) reveals strong geographic structure and cryptic species
Authors: Atherton William , Ambrose Luke , Wisdom James , Lessard Bryan D. , Kurucz Nina , Webb Cameron E. , Beebe Nigel W. 
Published: December 2024

Abstract
Background
The mosquito Culex annulirostris Skuse (Diptera: Culicidae) is an important arbovirus vector in Australasia. It is part of the Culex sitiens subgroup that also includes Cx. palpalis and Cx. sitiens. Single locus mitochondrial and nuclear DNA sequencing studies suggest that Cx. annulirostris consists of a complex of at least two species. We tested this hypothesis by analysing both nuclear microsatellite data and additional mitochondrial sequence data to describe the population genetics of Cx. annulirostris through Australia, Papua New Guinea (PNG) and the Solomon Archipelago.

Methods
Twelve novel microsatellite markers for Cx. annulirostris were developed and used on over 500 individuals identified as Cx. annulirostris by molecular diagnostics. Ten of the 12 microsatellites then used for analysis using Discriminant Analysis of Principal Components, a Bayesian clustering software, STRUCTURE, along with estimates of Jost’s D statistic that is similar to FST but better suited to microsatellite data. Mitochondrial cytochrome oxidase I (COI) DNA sequence were also generated complementing previous work and analysed for sequence diversity (Haplotype diversity, Hd and Pi, π), Tadjima’s D, and pairwise FST between populations. An allele specific molecular diagnostic with an internal control was developed.

Results
We confirm the existence of multiple genetically and geographically restricted populations. Within mainland Australia, our findings show that Cx. annulirostris consists of two genetically and geographically distinct populations. One population extends through northern Australia and into the south-east coast of Queensland and New South Wales (NSW). The second Australian population occurs through inland NSW, Victoria, South Australia, extending west to southern Western Australia. These two Australian populations show evidence of possible admixture in central Australia and far north Queensland. Australia’s Great Dividing Range that runs down southeast Australia presents a strong gene-flow barrier between these two populations which may be driven by climate, elevation or river basins. In PNG we find evidence of reproductive isolation between sympatric cryptic species occurring through PNG and Australia’s northern Cape York Peninsula. A PCR-based molecular diagnostic was developed to distinguish these two cryptic species.

Conclusion
This study adds to the growing body of work suggesting that the taxon presently known as Cx. annulirostris now appears to consist of at least two cryptic species that co-occur in northern Australia and New Guinea and can be distinguished by a ITS1 PCR diagnostic. The Solomon Islands population may also represent a distinct species but in light its geographic isolation and lack of sympatry with other species would require further study. Additionally, the mitochondrial and nuclear DNA evidence of population structure between geographic regions within Australia appears latitudinal and elevational driven and may suggest an additional subspecies in that hybridise where they overlap.

Graphical Abstract