Taxonomy and Molecular Genetics of Culex spiculothorax and Lutzia species (Diptera: Culicidae) Mosquitoes

Abstract

Culex (Culiciomyia) spiculothorax was first described from larval stage on Doi Inthanon, Chiang Mai, Thailand based on the presence of spiculation on the thorax of larvae. Adult females are characterized but are indistinguishable from those of related species, such as Cx. pallidothorax. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunit I (COI) sequences revealed that specimens identified as Cx. spiculothorax from Thailand, Japan and Bhutan form a single clade with Cx. sasai from Japan (Kimura 2-parameter genetic distances 0–0.9%) that is clearly distinct from clades comprised of other species of subgenus Culiciomyia. In this study, attempts to collect Cx. sasai from several locations in Japan were unsuccessful – only larvae with thoracic vesicular-like spicules identified as Cx. spiculothorax were collected. Careful examination of specimens collected near the type locality of Cx. sasai revealed the presence of spicules on the thorax. Based on these findings, Cx. spiculothorax is formally synonymized with Cx. sasai, which replaces the former as the species present in Thailand and is a new country record for Bhutan. We found a species of Lutzia Theobald (1903) (Diptera: Culicidae) in Chiang Mai Province and other provinces in northern Thailand which bears morphological differences from the three species of Lutzia, subgenus Metalutzia Tanaka, previously recorded from Thailand, namely Lt. fuscana (Wiedemann), Lt. halifaxii (Theobald) and Lt. vorax Edwards. The adults of the Chiang Mai form (CM form) have abdominal banding patterns similar to those of Lt. vorax from Japan (which includes the type locality of this species), but differ in having the mediocubital crossvein usually positioned before rather than beyond the radiomedial crossvein. The thoracic and abdominal integument of Lt. vorax larvae is covered with relatively short pointed spicules whereas it is covered by denser, longer and more acutely pointed spicules in the CM form. Some differences are also found in the development of thoracic seta 1-M, and abdominal setae 8-II and 8-III. The pupa of the CM form clearly differs from the pupa of Lt. vorax in having setae 1 and 5 of abdominal segments V and VI branched rather than single. The characters of the wing of mosquito, larva and pupa of the CM form are similar to those of Lt. fuscana and Lt. halifaxii. However, whereas the phallosome of males of the CM form is similar to males of Lt. vorax and Lt. halifaxii, it is distinct from males of Lt. fuscana. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunits I and II (COI and COII) and D3 expansion segment (D3) of the 28S ribosomal RNA sequences revealed that the CM form falls outside a clade comprised of specimens of Lt. vorax from Japan, Korea, Thailand and Bhutan (Kimura 2-parameter, K2P, genetic distances of COI, COII and D3 3.9–5.6, 5.1–6.6, and 1.2%, respectively). However, the two mitochondrial gene sequences of the CM form are not clearly distinct from clades comprised of sequences from specimens of Lt. fuscana and Lt. halifaxii (K2P 0.2–2.4%). From D3 sequences, the CM form and Lt. fuscana form a single clade and their sequences are identical. However, based on the combination of morphological and molecular data, the current study provides evidence that the CM form is a previously unrecognized species of the genus Lutzia.