Journal of the American Mosquito Control Association, 16(2):75-.g5,2OOO Copyright @ 20OO by the American Mosquito Control Association, Inc. PHYLOGENETIC RELATIONSHIPS AMONG EIGHTEEN NEOTROPICAL CULICINI SPECIES JUAN-CARLOS NAVARRO aNp JONATHAN LIRIA Instituto de Zoologia Tropical, Facultad de Ciencias, Laboratorio de Biologia de Vectores, Universidad Central de Venezuela, Apartado 47058, Caracas I04I_A, Venezuela ABSTRACT. The subgeneric relationships among 8 generic and infrageneric taxa of the Culicini rribe were examined by cladistic analysis based on 30 larval mouthpart characters (maxillae and mandibles) of7 subgenera of Culex L. and I of Deinocerites Theobald. We analyzed 18 ingroup species as well as Deinocerites melano- phylum Dyar and Knab as a sister group, and Aedes taeniorhynchus Wiedemann as an outgroup. A parsimony analysis using the Nona program resulted in 2 trees each of lO9 steps (consistency and retention indices : 0.88j. The topologies obtained were similar to the current classification of the tribe, based on nonexplicit methods mainly including adult characters, with 2 exceptions. In the present work, the monophyly of the tribe Culicini (Culex * Deinoceites) was supported by 4 synapomorphies. The subgenus Lutzia Theobald formed the most basal clade in the tribe Culicini and the placement of Phenacomyra Harbach and Peyton as a subgenus was validated by its location as a sister group of the subgenus Culex and other subgenera. The subgenus Carrollia Lutz was the most robust taxon, supported by 5 synapomorphies, and was congruent with the infragroups of the current classification. The relationships arnong Deinocerites, Anoedioporpa Dyar, Microculex Theobald, and Melanoconion Theobald were unresolved, but were placed in the most internal clade of the tribe. The lst exception to the accepted classification was the poorly resolved boundary between Anoedioporpa and Microculex The 2nd was the strong support (with 1 I synapomorphies) for the inclusion of Deinocerites as a subgenus of Culex in the Culicini, which is proposed here. KEY \ilORDS cladistics, Culex, Cnlicidae, Deinocerites, larval mouthparts, mandibles, maxillae, phylogeny INTRODUCTION natural classification of the Culicidae. Cladistics us- Since the pioneering work of Edwards (1932) ing molecular characters has been used by Pape (1992) and Dyar (1923, 1928) the taxonomy and classifi- with chromosomal characters in Anopheles (Cellia): (1992) cation of the family Culicidae has been based on Wesson et al. with Aedesa Besansky (1994) classical adult and immature morphologic charac- et al. with Anopheles gambiae, and Miller (1996) ters. Although the use of holomorphology in sys- et al. with the Pipiens Complex. All of these tematics inference (Hennig 1966) is preferred, re- studies have used ribosomal DNA. However, Be- (1997) cent authors have found that larval mouthparts sansky and Fahey estimated phylogenetic provide good morphologic characters for alpha tax- relationships among 14 Culicidae species using the gene. onomy (Harbach and Peyton 1993) and their use in white-eye phylogenetics should be explored. Using classical morphologic characters and cla- (1996) Snodgrass (1959) was the lst to describe the distic analyses, Judd studied the tribe Sa- bethini and (1998) morphology of some mosquito mouthparts and Harbach and Kitching analyzed 34 genera Knight (1971) showed the structural diversity in the of Culicidae. However, despite conclu- mandibles of several genera. Later, Harbach and sions about the higher relationships in the family, (infrageneric) Knight (1977) reported a variety of maxillary struc- the internal relationships remain tures and shapes that offer additional diagnostic largely unresolved. characters for identifi cation. We used larval mouthpart characters to infer Harbach and Peyton (1993) resumed studies with phylogenetic relationships among taxa within the some species of the tribe Sabethini and recognized tribe Culicini. We examined species belonging to 7 the importance of these structures for the identifi- of 8 subgenera reported from Venezuela, and 13 cation of generic taxa; they suggested their use to that occur in the Neotropics. We also evaluated the power achieve more natural classifications. Recently, Pe- and importance of larval mouthparts in the rez and Navarro (1996) reported diagnostic char- cladistic analysis in obtaining a natural classifica- acters for 3 subgenera of Anopheles Meigen, and tion for this medically important taxon. concluded that morphology of mouthparts is an ad- ditional tool for identification of these taxa. MATERIALS AND METHODS Despite extensive alpha taxonomy studies of Cu- licidae, phylogenetic relationships have not been Source of specimens.' We used 4th-stage-larvae intensively studied. The large size of the family belonging to the Mosquito Collection of the La- (3,000 species) (Knight and Stone 1977) represents boratorio de Biologfa de Vectores, Museo de Bio- a challenge. The new taxonomy described by Mun- logfa of the Universidad Central de Venezuela stermann and Conn (1997) represents a powerful (LBY after Guimardes 1997). These specimens approach toward obtaining a more objective and came from a variety of different breeding sites (Ta- JoURNALor rns AuerrcAN MosQUno Coxrnol AssocmrroN Vol. 16,No.2 Table l. List of species examined with data of locality in Venezuela, date of collection, and breeding site. Species Locality Date Breeding site Culex (Carrollia) bihaicolus Loma de Hierro, Aragua State Feb. 1992 Heliconia aurea Cx. (Car.) rausseoi Loma de Hierro, Aragua State Feb.1992 Palm spathes Euterpe sp. Cx. (Car.) iridescens Hacienda Rfo Claro, Zulia State Aug. 1995 Palms spathes Euterpe sp. Cx. (Car.) urichi La Azulita, M€rida State Dec. 1981 Xanthosoma sp. Cx. (P he nac omyia) c orni ge r Panaquire, Miranda State Dec.198l Cacao husks Cx. (Andoedioporpa) bam- Panaquire, Miranda State Feb. 1986 Bamboo internodes borum Cx. (Culex) dolosus La Azulita, Mdrida State March 1995 Discarded tire/rockhole Cx. (Cux.) nigripalpus El Jobo (Sinamaica), Zulia State Ocr. 1997 Tiee hole at ground level Cx. (Cux.) coronator Instituto de Zoologia Tropical UCV, Aug. 1997 Artificial container Caracas, D.E Cx. (Cux.) quinquefosciatus Cementerio del Sur, Caracas, D.E Jan.1981 Artificial container Cx. (Microculex) microphy- Guanay Tepui, Amazonas State Feb. 1995 Brocchinia tatei lus Cx. (Mcx.) chryselatus Sierra de San Luis, Falc6n State Aug. 1993 Guzmania mucronata Cx. (Mcx.) pleuristriatus Cerro Santa Ana. Fa1c6n State Aug. 1993 Aechmea aquilega Cx. (Melanoconion) albi- Hacienda Las Nubes, Zulia State Oct. 1997 Ground pool with aquatic plants nens$ "grapo Cx. (Mel.) atratus" Haciendas Las Nubes/Rfo Claro, Oct. 1997 Ground pools Zulia State Cx. (Mel.) nicceriensis Haciendas Las Nubes/Rfo Claro, Oct. 1997 Ground pools Zulia State Cx. (Lutzia) bigoti Sierra San Luis, Falc6n State April 1994 Discarded tire Deinoce rite s me lanophylum Cayo Borracho, Falc6n State Jan.1983 Crab hole Aedes taeniorhynchus Guajira-Paraguaipoa, Zulia State Oct. 1986 Ground pools in mangroves ble l). A total of 19 species was examined. These species), and also Aedes taeniorhynchus Wiedmann belonged to the subgenera Anoedioporpa Dyar (l and Deinocerites melanophylum Dyar and Knab. species), Microculex Theobald (3 species), Lutaia Mounting techniques and terminology: Larvae Theobald (l species), Culex L. (4 species), Melan- were stored in 8OVaethanol and clarified in a lOTo oconion Theobald (3 species), Carrollia Lltz (4 KOH solution, then processed as described by Har- species), and Phenacomyia Harbach and Peyton (1 bach and Peyton (1993), with the modifications of Perez and Navarro (1996). A total of 30 characters was scored. These consisted of 16 characters as- sociated with mandibles and 14 characters of the maxillae (Appendix 1). Characters were scored based on the nomenclature of Harbach and Knight (1980). The generic and subgeneric abbreviations followed those of Reinert (1975). Drawings of the morphometric characters used are shown in Fig. 1. A schematic of the general morphology from each supraspeciflc taxon examined is shown in Figs. 2a (Lutzia), 2b (Phenacomyia), 3a (Culex), 3b (Car- rollia), 4a (Deinocerites), 4b (Melanoconion), 5a (Anoedioporpa), and 5b (Microculex). Selection of characters and cladistics analysis: Characters were selected based upon the results of previous papers that also used mouthpart structures (Knight 1971, Harbach and Knight 1977, Harbach and Peyton 1993, Perez and Navarro 1996). We also included new characters (character [ch.] 13, PMnL, and ch. 15, ppMAdA) not previously stud- ied based on structural diversity (Appendix 1). Polymorphic (multistate characters) were also in- cluded in the data set. Characters not determined in one or more taxa were treated as missing. Fig. 1. Morphometric measurements for some man- The genus Aedes Meigen (Ae. taeniorhynchus) dibular and maxillary structures. was chosen as outgroup to root trees but without JUNE2000 PHyr-ocnNsrrc RpLarroNsrnpsrr..i CwrcrNr 77 ,ffi, Fig.2. a. Ventral (left) and dorsal (right) view of Cu- lex (Lutzia) bigoti. Mandibles (above) and maxillae (be- low) are shown. b. Ventral (left) and dorsal (righr) view of Culex (Phenacomyia) corniger. Mandibles (above) and maxillae (below) are shown. Fig. 3. a. Ventral (left) and dorsal (right) view of Cu- the intention of exploring the sister relationships of lex (Culex) dolosus. Mandibles (above) and maxillae (be- Culicini. Choice of this taxon as outgroup is sup- low) are shown. b. Ventral (left) and dorsal