Phylogeny of Genus Wyeomyia (Diptera:Culicidae) Inferred from Morphological and Allozyme Data

591 Phylogeny of genus Wyeomyia (Diptera: Culicidae) inferred from morphological and allozyme data

Monique Albuquerque Motta,1 Ricardo Lourenço-de-Oliveira Departamento de Entomologia, Instituto Oswaldo Cruz, Avenida Brasil 4365, C.E.P. 21045- 900, Rio de Janeiro, Brazil Maria Anice Mureb Sallum Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, Avenida Dr. Arnaldo 715, C.E.P. 01246-904, São Paulo, Brazil

Abstract—Phylogenetic relationships within the genus Wyeomyia Theobald are presented, based on a cladistic analysis of 88 morphological characters (from adults, larvae, and pupae) of 38 named species and 1 unnamed species and 46 allozyme markers from a subset of 19 of these species. Two taxa are used as outgroup (Sabethes aurescens Lutz and Limatus durhami Theobald). The analysis indicates that, as currently defined, the genus Wyeomyia is not a monophyletic lineage: firstly, the genus Onirion Peyton and Harbach is nested within the genus Wyeomyia, and secondly, the subgenus Phoniomyia Theobald is a monophyletic lineage outside the genus Wyeomyia. Our results also demonstrate that the subgenera Cruzmyia Lane and Cerqueira, Decamyia Dyar, Dendromyia Theobald, Spilonympha Motta and Lourenço-de- Oliveira, and Prosopolepis Lutz are monophyletic lineages nested within the genus Wyeomyia. Triamyia Dyar is resurrected as a subgenus of Wyeomyia to include W. aporonoma Dyar and Knab and W. staminifera Lourenço-de-Oliveira, Motta, and Castro. The subgenus Miamyia Dyar is resurrected to include seven species: W. codiocampa Dyar and Knab, W. lutzi (Costa Lima), W. limai Lane and Cerqueira, W. serrata (Lutz), W. hosautos Dyar and Knab, W. oblita (Lutz), and W. sabethea Lane and Cerqueira.

Résumé—Nous présentons les relations phylogénétiques au sein du genre Wyeomyia Theobald d’après une analyse cladistique de 88 caractères morphologiques (des adultes, des larves et des nymphes) chez 38 espèces nominales et une espèce inédite, ainsi que de 46 marqueurs allozymes chez un sous-ensemble de 19 de ces espèces. Deux taxons (Sabethes aurescens Lutz et Limatus durhami Theobald) servent de groupes externes. L’analyse indique que, dans sa définition cou- rante, le genre Wyeomyia ne forme pas une lignée monophylétique: (a) le genre Onirion Peyton et Harbach s’emboîte dans le genre Wyeomyia et (b) le sous-genre Phoniomyia Theobald est une lignée monophylétique qui se situe hors du genre Wyeomyia. Nos résultats démontrent aussi que les sous-genres Cruzmyia Lane et Cerqueira, Decamyia Dyar, Dendromyia Theobald, Spilonym- pha Motta et Lourenço-de-Oliveira et Prosopolepis Lutz sont des lignées monophylétiques au sein du genre Wyeomyia. Nous tirons de la synonymie le nom de Triamyia Dyar pour servir de sous-genre de Wyeomyia et regrouper W. aporonoma Dyar et Knab et W. staminifera Lourenço- de-Oliveira, Motta et Castro, ainsi que le nom du sous-genre Miamyia Dyar pour inclure 7 espè- ces: W. codiocampa Dyar et Knab, W. lutzi (Costa Lima), W. limai Lane et Cerqueira, W. serrata (Lutz), W. hosautos Dyar et Knab, W. oblita (Lutz) et W. sabethea Lane et Cerqueira.

[Traduit par la Rédaction]

Motta627 et al. Introduction Neotropical Diptera: Culicidae, but is also the group that is most diverse in morphology and The tribe Sabethini not only contains some of behavior. The tribe comprises 13 genera: 8 are the most striking mosquitoes among the restricted to the Neotropical region, 1 is found

Received 2 November 2006. Accepted 19 April 2007. 1Corresponding author (e-mail: [email protected]).

Can. Entomol. 139: 591–627 (2007) © 2007 Entomological Society of Canada 592 Can. Entomol. Vol. 139, 2007 in both the Neotropical and Neartic regions, 2 and Peyton 1992), Prosopolepis (Lourenço-de- are from the Australian and Oriental regions, 1 Oliveira et al. 1999), Dendromyia (Motta and is restricted to the Australian region, and 1 is Lourenço-de-Oliveira 2000), Spilonympha (Motta found in the Ethiopian/Oriental region. A recent and Lourenço-de-Oliveira 2005), and Hystato- investigation of cladistic relationships within myia (Judd 1998a). However, the subgeneric the Sabethini was done by Judd (1996), who boundary of Wyeomyia, the largest subgenus, re- demonstrated that the Sabethini was a mono- mains unclear because there are conflicting defi- phyletic group but the genera Wyeomyia Theo- nitions, owing to the use of inconsistent bald, Tripteroides Giles, and Runchomyia morphological characters. Moreover, numerous Theobald were not. In an extensive morpholog- species previously included in Dendromyia are ical cladistic analysis within Culicidae, Harbach currently without subgeneric placement (Motta and Kitching (1998) corroborated the mono- and Lourenço-de-Oliveira 2000). Except for phyly of the Sabethini. Judd’s study on the Sabethini (Judd 1996, 1998a, Wyeomyia is the largest of all the Neotropical 1998b), nearly all hypotheses concerning subgen- genera of Sabethini. It includes species that are era of Wyeomyia have been formulated without sylvatic and characterized by phytotelmatic im- the support of a phylogenetic analysis: neither the mature stages. Mosquitoes belonging to the ge- monophyly of the genus nor relationships among nus Wyeomyia collected in tropical rain forests subgenera have been tested using phylogenetic have been found to be infected with several methodology. Interestingly, the results of Judd’s arboviruses: Iaco and Maguari viruses were iso- (1996) cladistic analysis suggest that the genus lated from Wyeomyia sp., Taiassui and Tucun- Wyeomyia, as traditionally defined, is paraphyletic duba viruses from Wyeomyia sp. and Wyeomyia because it does not include the genera Phoniomyia aporonoma, and Una and Triniti viruses from Theobald and Limatus Theobald; the author recov- Wyeomyia sp. (Hervé et al. 1986; Vasconcelos ered these two genera nested within the Wyeomyia et al. 2001). clade. The main objectives of the present study are The genus Wyeomyia includes more than 100 to test the monophyly of the genus Wyeomyia as species, currently comprising 15 subgenera currently defined by Judd (1998a) and Harbach (Knight and Stone 1977; Judd 1998a; Motta and Kitching (1998) and to estimate the phylogen- and Lourenço-de-Oliveira 2005). However, few etic relationships within the genus. mosquito taxa are as poorly studied as this genus. Until recently, the internal classification of Materials and methods Wyeomyia was based mainly on the revision of Lane and Cerqueira (1942). The name Wyeo- Selection of taxa myia was proposed by Theobald (1901) to include 7 species; however, this description was Morphological analysis based on adult females only and was conse- Using morphology, phylogenetic analyses quently brief and inconsistent. Dyar (1928) hy- were conducted for 41 species (Table 1). The pothesized a new classification for Wyeomyia, ingroup consisted of 38 named species and 1 proposing four genera and 19 subgenera that in- unnamed species of the genus Wyeomyia, repre- cluded 66 species. Subsequently, Edwards senting 11 of 15 currently recognized subgen- (1932) restricted Dyar’s four genera to era. Nine species are without subgeneric Wyeomyia, which was subdivided into 4 sub- assignment. We selected at least 2 species of genera. Later, Lane (1953) proposed that each of the following subgenera: Cruzmyia, De- Wyeomyia comprised 7 subgenera. camyia, Dendromyia, Phoniomyia, Spilonym- The scant knowledge of immature stages and pha, and Wyeomyia. We examined only 1 the absence of reliable characters in the adult species of the subgenera Dodecamyia, Meno- stage confuse the classification of Wyeomyia. lepis, and Prosopolepis because they are mono- Belkin et al. (1970) noted that the organization of typic, and the subgenus Exallomyia for which the genus was unnatural and recommended an ex- only 1 species is available. Considering the haustive study of the immature stages to reveal availability of the three life stages (fourth-instar monophyletic lines. Recent studies employing larva, pupa, and adult) for the subgenus Antun- morphological characters from all life stages es- esmyia, W. alani was included because it is the tablished some small and better defined groups, only species for which the immature stages are the subgenera Caenomyiella and Decamyia (Har- described. The outgroup included Sabethes bach and Peyton 1990a), Exallomyia (Harbach aurescens and Limatus durhami, which were

Table 1. The 41 species examined in the morphological analysis. Genus Subgenus Species Wyeomyia Spilonympha W. aningae* Motta and Lourenço–de–Oliveira W. mystes* Dyar W. bourrouli* (Lutz) W. forcipenis Lourenço-de-Oliveira and Silva W. airosai* Lane and Cerqueira W. finlayi* Lane and Cerqueira W. howardi Lane and Cerqueira Dendromyia W. ypsipola* Dyar W. testei* Sevenet and Abonnenc W. luteoventralis Theobald W. jocosa (Dyar and Knab) W. complosa (Dyar) Decamyia W. ulocoma (Theobald) W. felicia (Dyar and Nunez-Tovar) Antunesmyia W. alani Lane and Cerqueira Prosopolepis W. confusa (Lutz) Dodecamyia W. aphobema* (Dyar) Cruzmyia W. forattinii Clastrier W. dyari Lane and Cerqueira Exallomyia W. tarsata Lane and Cerqueira Wyeomyia W. lutzi* (Costa Lima) W. sabethea Lane and Cerqueira W. oblita* (Lutz) W. arthrostigma* (Lutz) W. medioalbipes* Lutz W. limai Lane and Cerqueira Menolepis W. leucostigma* Lutz Without subgeneric placement W. melanocephala* Dyar and Knab W. surinamensis Bruijning W. flui Bonne-Wepster and Bonne Wyeomyia sp. 4 W. argenteorostris Bonne-Wepster and Bonne W. aporonoma* Dyar and Knab W. staminifera Lourenço-de-Oliveira, Motta, and Castro W. negrensis* Gordon and Evans W. chalcocephala Dyar and Knab Phoniomyia W. palmata* Lane and Cerqueira W. edwardsi Lane and Cerqueira Onirion O. personatum* (Lutz) Sabethes S. aurescens Theobald Limatus L. durhami Theobald Note: Species for which data from both morphological and allozyme analyses were available are indicated by an asterisk. reconstructed as sister groups of the genus Saúde Pública, Universidade de São Paulo, São Wyeomyia in Harbach and Kitching’s (1998) Paulo, Brazil; and the National Museum of Nat- study. Onirion personatum was also included in ural History, Washington, D.C., United States the analysis to evaluate its phylogenetic rela- of America. tionship with the genus Wyeomyia. The specimens examined for this study were Allozyme analysis from the collections of the Instituto Oswaldo An allozyme study was conducted for 19 spe- Cruz, Rio de Janeiro, Brazil; the Faculdade de cies (Table 1) because fresh specimens were not

Table 2. Data matrix for 41 taxa and 134 characters used in this analysis. Species/Character 123456789101112131415 S. aurescens 311101100113000 L. durhami 301000100012000 W. edwardsi 101113000001101 W. howardi 200100110011000 W. luteoventralis 301100100011000 W. jocosa 301100100011000 W. complosa 301100100011000 W. ulocoma 200100100?11000 W. felicia 201100100011000 W. alani 200104110012000 W. confusa 00———2—01011000 W. forattinii 101100100011000 W. dyari 201100100011000 W. tarsata 101103120011010 W. sabethea 301104110011000 W. limai 200104120011000 W. surinamensis 201101100011000 W. flui 201101100011000 W. sp. 4 201100100011000 W. argenteorostris 201100100011000 W. staminifera 201100110011000 W. chalcocephala 201100100011000 W. palmata 101113000001101 O. personatum 201100100110000 W. aningae 301100100011000 W. mystes 301100100011000 W. bourrouli 201100100011000 W. forcipenis 201100100011000 W. airosai 201100100011000 W. finlayi 201100100011000 W. ypsipola 301100100011000 W. testei 301100100011000 W. aphobema 101103100011000 W. lutzi 200104120011000 W. oblita 201104120011000 W. arthrostigma 201101100011000 W. medioalbipes 201101100011000 W. leucostigma 101100100011000 W. melanocephala 201100100011000 W. aporonoma 201100110011000 W. negrensis 201100100011000 available for all the 41 species used in the mor- interpretable and reproducible bands. The enzymes phological analysis. All mosquitoes examined tested were malate dehydrogenase (MDH), EC were collected as either larvae or adults in sepa- 1.1.1.37; malic enzyme (ME), EC 1.1.1.40; rate localities in the Brazilian states of Rondônia, phosphoglucose isomerase (PGI), EC 5.3.1.9; Amazonas, Mato Grosso, Bahia, and Rio de Ja- phosphoglucomutase (GM), EC 5.4.2.2; isocitrate neiro. We included in the analysis only the fe- dehydrogenase (IDH), EC 1.1.1.42; hexokinase (K), male adult stage, and the number of assayed EC 2.7.1.1; adenylate kinase (AK), EC 2.7.4.3; individuals varied among species (Table 4). glycerol-3-phosphate dehydrogenase (αPGD), EC Agarose gels were stained for 23 different en- 1.1.1.8; fumarate hydratase (FUM), EC 4.2.1.2; and zymes, of which only 10 (coded by 11 loci) gave 6-phosphogluconate dehydrogenase (PGDH), EC

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 0 11112010000010101111 1 0000101001001?011001 1 000011?0100000001001 1 1111101111000?011100 0 11111010000000111002 0 11111010000000111002 1 11111010000000111001 1 10011010111001010001 1 11111010110001010001 1 1001101101101?011000 1 11110000000011011001 1 0000101111000?110000 1 1000101101000?011000 1 0000101011011?011001 0 0000202000001?011001 2 000020211?100?01?101 1 11112000110010011001 1 11112000110010011001 1 11111010110000011001 1 10001010001001011001 1 1000101000000?011001 1 11111010110000011001 1 00001110100000001001 1 10010000010010010111 1 11111011110001011000 1 11111011110001011000 1 1111101111000?011100 1 1111101111000?011100 1 1111101111000?011100 1 1111101111000?011100 0 11111010000000111002 0 11111010000000111002 1 000010?0110001011001 2 0000202111100?011101 2 0000202111100?111001 1 0001101101000?011001 1 0000101001000?011001 1 11111010110011010001 1 111100000100/110010101 1 1000101000000?011001 1 11111010110011011001 1.1.1.44. Enzyme electrophoresis was carried out in characters were coded as binary and 14 as agarose gels following Hjerten’s (1961) protocol multistate. All characters were initially treated with the modifications proposed by Salles et al. as unweighted and unordered. Fourteen autapo- (1986) and Rosa-Freitas et al. (1990). morphies, which are parsimony non- informative, were excluded from all cladistic Morphology analyses; consequently they did not contribute Eighty-eight characters from three life stages to tree statistics. All 14 autapomorphies are in- were included in the analysis, 39 from the dicated with an asterisk (*) in the character list. fourth-instar larva, 15 from the pupa, and 34 When more than one character state was ob- from the adult male and female. Seventy-four served in a single taxon, these are scored in the

Table 2 (continued). Species/Character 37 38 39 40 41 42 43 44 45 46 47 48 49 50 S. aurescens 00001001101110 L. durhami 00001101101011 W. edwardsi 01111000101011 W. howardi 10010001101011 W. luteoventralis 0001000110100? W. jocosa 0??1000110100? W. complosa 0001000110100? W. ulocoma 0??10001101011 W. felicia 0??10011101010 W. alani 0??1000110110? W. confusa 00010001101010 W. forattinii 0??10001001011 W. dyari 0??10011001011 W. tarsata 00010001101011 W. sabethea 0001000110110? W. limai 0??1001110110? W. surinamensis 0??10011111011 W. flui 00010011111011 W. sp.4 0011000110100? W. argenteorostris 00010001101011 W. staminifera 00010001101011 W. chalcocephala 0??10001101011 W. palmata 01111000101011 O. personatum 00010001100011 W. aningae 10010001100011 W. mystes 10010001100011 W. bourrouli 10010001101011 W. forcipenis 10010001101011 W. airosai 10010001101011 W. finlayi 10010001101011 W. ypsipola 0001000110100? W. testei 0001000110100? W. aphobema 10010001101011 W. lutzi 0001001100110? W. oblita 0001001100110? W. arthrostigma 00010001001111 W. medioalbipes 10010001001111 W. leucostigma 00011001100011 W. melanocephala 00010001101011 W. aporonoma 00010001101011 W. negrensis 0001000110100? data matrix as polymorphic. Missing values are Characters listed by Judd (1996), Harbach represented by a question mark (?) and were and Kitching (1998), and Sallum et al. (2000) employed when the character-state assignments were included or reinterpreted. Additional were not applicable or the condition of the characters, particularly those distinguishing available specimens precluded observation of subgenera, were also used. Except for designa- that character. A dash (—) indicates that the tions of gonostylar lobes (Belkin et al. 1970), character-state homology could not be estab- the morphological terminology follows that of lished. Harbach and Knight (1980).

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 000100111000100010102 001000110100100012100 0000?21011101000?0000 000110011000110011101 010111111000100011101 01011111100011001?111 010111111000100011111 0001001?1?01100010101 01010011110111001?101 1001101110101?001?101 010101111000110010111 0001001111001?0000100 0001?01111001?0010100 000100111000100011101 1101?0111100101011101 120100111110001011101 000210111100110010121 000210111100110010121 000010111100110010101 020110111100100012101 000110111000100010101 0002001?11?0110010101 000001101110100012000 000102111000110110100 000210011100110011101 000210011100110011101 000110011110110011101 000110011110110011101 000110011100110011101 000110011110110011101 010111111000100011101 010111111000100011101 000112111100110010101 120100111110001011101 110100111100001011101 100110111100100011101 020110111100100011101 0001021110001?0011101 000100111110110010101 000111111000100010101 010101111000100010101

Cladistic analysis morphological (1–88) or allozyme (89–134). Two Morphological and allozyme data were com- sets of taxa were determined in MacClade: group bined in a single matrix and analyzed using the 1 contained 22 species from which we collected parsimony criterion in PAUP version 4.0 only morphological data and group 2 contained (Swofford 2002). 19 species with both morphological and allozyme The data matrix was constructed using data. For the allozyme data, individual alleles MacClade version 4 (Maddison and Maddison were scored as characters for presence and ab- 2002), and characters were identified as sence as alternative states (binary character).

Table 2 (continued). Species/Character 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 S. aurescens 001011 100100000 L. durhami 100110 11?001130 W. edwardsi 110101 101?00000 W. howardi 111101 001000130 W. luteoventralis 111101 001000110 W. jocosa 111101 011?10110 W. complosa 111101 011010110 W. ulocoma 111101 001010110 W. felicia 111111 001010110 W. alani 111?11 001000000 W. confusa 111101 001000130 W. forattinii 110101 001101000 W. dyari 100101 001100000 W. tarsata 111111 001100000 W. sabethea 111111 001101130 W. limai 111101 001101130 W. surinamensis 111101 021010110 W. flui 111101 021010110 W. sp.4 111101 021010110 W. argenteorostris 111101 001000110 W. staminifera 111101 001000110 W. chalcocephala 111101 011010110 W. palmata 110101 101100000 O. personatum 111101 001010110 W. aningae 111101 011000110 W. mystes 111101 011000110 W. bourrouli 111101 001000130 W. forcipenis 111101 001000130 W. airosai 111101 001000130 W. finlayi 111101 001000130 W. ypsipola 111101 011000110 W. testei 111101 011000110 W. aphobema 101101 001101000 W. lutzi 111111 001101130 W. oblita 111111 001101130 W. arthrostigma 101111 010101130 W. medioalbipes 111101 001101130 W. leucostigma 100101 021000130 W. melanocephala 101111 001000110 W. aporonoma 111101 001000110 W. negrensis 111111 021100121

The data sets were analyzed as follows. Ini- the monophyly of the ingroup, the outgroup tially the morphology data set with 41 taxa and was not initially used to root the trees. After 88 characters (Table 2) was analyzed under the that, the same heuristic search criterion was parsimony method, with 74 parsimony- applied to the set of equally parsimonious re- informative characters treated as unordered sult trees, with unweighted, unordered search and unweighted. Tree searches were conducted and 10 random-addition replicates per search, using the heuristic search option, tree bisection and five rounds of successive approximations and reconnection branch-swapping, and 10 000 character weighting (Farris 1969; Carpenter random stepwise addition replicates. To test 1994). Character weights were based on the

87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 10?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 10?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0??? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0??? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0??? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0??? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 01?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 01?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 01?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 00?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 01?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0?11 0 0 1 0 1 0 0 0 0 0 1 0 0 1 1010 0 0 0 0 1 0 1 1 0 1 1 0 0 0 0010 1 0 0 0 1 0 0 0 0 1 0 1 0 0 0010 0 0 0 0 1 0 0 0 0 1 0 1 0 0 0010 1 1 0 0 0 0 0 0 1 0 0 1 0 0 0010 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0010 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0001 0 0 0 1 1 0 0 0 0 0 0 1 0 1 0001 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0001 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0001 0 0 1 0 0 0 0 1 0 0 0 0 1 0 1001 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0001 0 0 0 1 0 1 0 1 0 1 0 1 0 0 1001 0 0 1 0 1 0 0 1 1 1 0 0 0 0 0001 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0001 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0000 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0000 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0101 0 0 0 0 0 0 1 0 0 0 0 1 1 0 maximum value of the rescaled consistency in- To evaluate the relative robustness of clades re- dex (CI) (Farris 1989). covered in the parsimony analyses, bootstrap The combined morphological and allozyme (Felsenstein 1985) values were calculated in PAUP. character data set (Table 2) for the 19 species Bootstrap analyses consisted of 1000 pseudo- (group 2) was analyzed under the parsimony crite- replicates and employed heuristic search with tree rion with 95 parsimony-informative characters bisection and reconnection branch-swapping and 10 treated as unordered and unweighted. Tree random-addition replicates per bootstrap searches were conducted using the method de- pseudoreplicate. Bremer support values (Bremer scribed above. 1994) for clades were also estimated using PAUP.

Table 2 (concluded).

Species/Character 105 106 107 108 109 110 111 112 113 114 115 116 117 118 S. aurescens ?????????????? L. durhami ?????????????? W. edwardsi ?????????????? W. howardi ?????????????? W. luteoventralis ?????????????? W. jocosa ?????????????? W. complosa ?????????????? W. ulocoma ?????????????? W. felicia ?????????????? W. alani ?????????????? W. confusa ?????????????? W. forattinii ?????????????? W. dyari ?????????????? W. tarsata ?????????????? W. sabethea ?????????????? W. limai ?????????????? W. surinamensis ?????????????? W. flui ?????????????? W. sp. 4 ? ? ? ? ? ? ? ? ? ? ? ? ? ? W. argenteorostris ?????????????? W. staminifera ?????????????? W. chalcocephala ?????????????? W. palmata 00100001010000 O. personatum 10001000000101 W. aningae 10000010010010 W. mystes 00010010010010 W. bourrouli 00100010110001 W. forcipenis 00100010110001 W. airosai 00100010010000 W. finlayi 00000010110001 W. ypsipola 00100100011000 W. testei 00100100000000 W. aphobema 10000100010000 W. lutzi 00110010000000 W. oblita 00010010010000 W. arthrostigma 00010001001000 W. medioalbipes 01001010100000 W. leucostigma 10001000100000 W. melanocephala 00101000000001 W. aporonoma 00000000000100 W. negrensis 01000000110000

Allozyme Character descriptions Individual genotype data were used to estimate gene frequencies for 19 ingroup taxa. We Morphological characters also estimated pairwise genetic identities and distances (Nei 1978). Identity values were used Larva to construct a UPGMA phenetic dendrogram (1) Maxilla, laciniarastrum 1 (CI = 0.375): (Sneath and Sokal 1973) with the BIOSYS-1 absent (0) (Fig. 2a); spiculate with flexible program, version 2.0 (Swofford and Selander apex (1) (Fig. 2b); spiculate with stiff apex 1981) (Fig. 1). (2) (Fig. 2c); denticular (3) (Fig. 2d).

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? ?????????? ? ? ? ? ? ? 0100000000 0 0 0 1 0 0 0000100100 1 0 1 0 0 0 0001000100 1 1 0 0 0 0 0010000100 1 1 0 0 0 0 1010000101 0 0 0 1 1 0 1010000101 0 1 0 1 1 0 1000100010 1 1 0 1 0 0 1010100100 1 0 0 1 0 0 1001001101 0 0 0 1 0 0 1001000100 1 0 0 1 0 0 0001101100 0 0 0 0 0 0 1000101100 1 0 0 0 1 0 1000110100 1 0 0 0 1 0 1000010100 1 0 0 0 1 0 1000100100 1 0 0 0 1 0 1001000100 1 0 0 0 0 1 0000000100 1 0 1 0 0 0 0000010100 1 0 0 0 1 0 0100000010 1 0 0 0 0 1

A denticular laciniarastrum (3) occurs in the possess a maxillary palpus with 3 apical setae. outgroup, Dendromyia, two species of Spilonym- Condition 1 is an autapomorphy for Sabethes, and pha, and W. sabethea Lane and Cerqueira. Judd thus it is uninformative for the analysis. (1995) suggested that the denticular condition is (3) Seta 1-Mx, position (CI = 0.250): apical primitive within the Sabethini. (0); displaced slightly basally (1). (2) Maxillary palpus (*): with 3 apical setae (0); Apical position was considered only when with 4 apical setae (1). Only members of the genus seta 1-Mx was inserted on the anterior margin Sabethes show a maxillary palpus with 4 apical of the maxillary body; all other basal positions setae. All other species, including those in Limatus, were assigned to condition 1.

Fig. 1. UPGMA phenetic dendrogram generated using Nei’s (1978) pairwise unbiased genetic identities.

(4) Seta 1-Mx, development (*): elongate (0); species possess condition 1. According to Judd short (1). (1996, 1998a), Phoniomyia and Hystatomyia Among all taxa examined, Limatus is the have seta 3-Mx arising from this notch. only taxon in which seta 1-Mx is elongate. Be- (6) Seta 4-Mx, development (CI = 0.571): cause condition (0) is an autapomorphy for single, pointed (0); single, truncate (1); spine- Limatus , character 4 was excluded from the like (2); brush-tipped (3); forked (4) (Fig. 2c). analysis. (5) Maxilla, lateral notch containing seta 3- A forked seta 4-Mx occurs in the majority of Mx (CI = 1.000): absent (0); present (1) species of the subgenus Wyeomyia.InPhonio- (Fig. 2e). myia species, seta 4-Mx is brush-tipped, a con- In some species, seta 3-Mx arises from the dition shared with Dodecamyia and Exallomyia. outer margin of the maxilla in a cuplike exten- Seta 4-Mx is slightly stronger in Exallomyia sion of the cuticle called the maxilla lateral species than in Dodecamyia and Phoniomyia notch. In the present study, only Phoniomyia species.

Fig. 2. Maxillae of fourth-instar larvae of Wyeomyia confusa (a), W. palmata (b), W. lutzi, showing maxillary setae 4-Mx and 6-Mx (c), W. ypsipola (d), W. palmata (e); and anterior margin of the head of W. palmata (f). The arrows indicate some character states. Scale bars = 0.1 mm.

(7) Seta 4-Mx, position (CI = 1.000): on but is stellate in some species of the subgenus anterolateral margin (0); posterior (1). Wyeomyia. Judd (1995) observed that a simple, In Phoniomyia, seta 4-Mx is located on the unbranched seta 6-Mx is present in most anterolateral margin of the maxillary body, di- sabethines. rectly lateral to the maxillary brush. In the re- (9) Dorsomentum, position (*): arising poste- maining species examined, seta 4-Mx arises riorly, beyond middle of head (0); arising ante- posteriorly on the maxillary body. riorly, beyond middle of head (1). (8) Seta 6-Mx, development (CI = 0.400): In W. confusa, in which the larva displays single (0); branched (1); stellate (2) (Fig. 2c). predatory behavior, the dorsomentum is located Seta 6-Mx is produced as a single seta in the well to the anterior. Condition 1 is an autapo- majority of species included in this analysis, morphy for W. confusa.

(10) Anterior edge of dorsomentum (CI = hypostomal ridge, close to posterior end of hy- 0.500): median tooth prominent, lateral teeth postomal suture (2). smaller (0); median and lateral teeth prominent Except for W. complosa, all Dendromyia spe- (1). cies have seta 15-C between the anterior border The dorsomentum with prominent median of the labiogula and the termination of the and lateral teeth occurs only in Sabethes and hypostomal ridge. Only W. lutzi, W. oblita, and Onirion. All other species possess only a prom- W. limai have seta 15-C arising close to the pos- inent median tooth. terior end of the hypostomal suture. (11) Anterior margin of head (CI = 1.000): (17) Seta 4-P, development (CI = 0.250): somewhat triangular (0) (Fig. 2f); rounded (1). aciculate, aciculae similar in development to Larvae of Phoniomyia have a unique feature those of seta 7-P (0); strongly aciculate, acicu- that distinguishes them from all other lae considerably more developed than those of sabethines. The mesal anterior part of the head seta 7-P (1). is pronounced and thus the anterior area of the Because seta 7-P is nearly identical in the head is triangular. taxa included in the study, it was used as a (12) Occipital foramen (CI = 0.750): circu- landmark to define the development of seta 4-P. lar (0) (Fig. 3c); transverse, slit-like, extending In the majority of Wyeomyia species seta 4-P is anterolaterally (1) (Fig. 3d); transverse, not ex- strongly aciculate, mainly in the middle of the tending laterally (2) (Fig. 3a); transverse, slit- branches, and thus different from the other pro- like, extending dorsolaterally (3) (Fig. 3b). thoracic setae. The occipital foramen in Onirion species is (18) Seta 11-P (CI = 0.250): hairlike (0); circular, whereas in the remaining species ex- spiniform (1). amined it is transverse. In L. durhami and (19) Seta 11-M, development (CI = 0.250): W. alani, the occipital foramen is transverse, hairlike (0); spiniform (1). not extending laterally, whereas in the remain- (20) Seta 11-T, development (CI = 0.250): ing Wyeomyia and Phoniomyia species it is slit- hairlike (0); spiniform (1). like, extending anterolaterally, not toward the ventral or dorsal surface as in Sabethes. Seta 11-T is spiniform in the majority of the Wyeomyia (13) Seta 1-A, development (CI = 1.000): species. flexible (0); stiff (1). (21) Seta 13-I, development (CI = 0.500): In Phoniomyia, antennal seta 1-A consists of single (0); branched, with flexible branches (1); a branched stiff seta, whereas in all other spe- stellate (2). cies examined seta 1-A is normally single and Wyeomyia melanocephala and W. confusa always flexible. share condition 1 (seta 13-I single) with (14) Seta 1-A, placement (*): apical (0); Onirion. In the majority of Wyeomyia species, midlength (1). seta 13-I is branched and flexible, although Seta 1-A arises from the apical third of the stellate setae occur within the subgenus antenna in all species examined; only in Wyeomyia and in the outgroup taxa. Exallomyia was seta 1-A placed in the middle (22) Setae 6,7-III position (CI = 1.000): in- of the antennal shaft, as assigned by Harbach serted on separate plates (0); inserted on same and Peyton (1992). Condition 1 is an plate (1). autapomorphy for W. tarsata. Except for Phoniomyia species, all species (15) Seta 4-C, position (CI = 1.000): anterior, examined have setae 6,7-III arising from sepa- on dorsal apotome, slightly posterior to median rate plates. labral plate (0); posterior, on dorsal apotome, (23) Seta 1-VIII, development (CI = 0.667): directly mesal to antennal prominence (1). branched, short (0); branched (2); stellate (2). In Phoniomyia, seta 4-C arises more posteri- The length of the comb scales was used as a orly, directly mesal to the antennal prominence. landmark to determine the length of seta 1-VIII. In all other species examined, seta 4-C is ante- Consequently, in those species in which seta 1- rior, arising near to the median labral plate. VIII was longer than the comb scales it was (16) Seta 15-C, position (CI = 0.400): be- scored as state 1. In both states (0; 1) the setae tween anterior border of labiogula and termina- were not stellate. According to Harbach and tion of hypostomal ridge (0); posterior to Knight (1980), a stellate seta has numerous stiff termination of hypostomal ridge at middle of branches projecting at various angles from a labiogula (1); well posterior to termination of single base.

Fig. 3. Heads of fourth-instar larvae of Limatus durhami (a) Sabethes aurescens (b), Onirion personatum (c), and Wyeomyia airosai (d), sclerotized plate on segment VIII of W. airosai (e), and siphon apex of W. palmata (f). The arrows indicate some character states. Scale bars = 0.1 mm.

(24) Segment VIII, sclerotized plate (CI = and W. negrensis, the comb scales arise in sev- 0.250): absent (0); present (1) (Fig 3e). eral rows from the integument, not from a plate. All Spilonympha species have a sclerotized (26) Segment X, saddle (CI = 0.167): cover- plate on segment VIII, although this condition ing dorsal half of segment X (0); covering more also occurs in members of the subgenera Cruz- than dorsal half of segment X (1). myia, Wyeomyia, and Antunesmyia. In some species, the saddle is small and cov- (25) Comb scales (CI = 0.167): in a single ers only the dorsal half of segment X. This con- row (0); in several rows (1). dition is observed in all Dendromyia species. The scales arising from a plate were assigned (27) Spines on posterolateral margin of sad- as condition 1. In Phoniomyia, Decamyia, Do- dle (CI = 0.250): absent (0); present (1). decamyia, Exallomyia, and Menolepis, W. sur- Based on the outgroup taxa, the primitive inamensis, Wyeomyia sp. 4, W. chalcocephala, condition is the absence of spines on the

© 2007 Entomological Society of Canada 606 Can. Entomol. Vol. 139, 2007 posterolateral margin and this condition is ob- Seta 2-S has a spiculate margin only in served in the majority of species examined. The Onirion and Sabethes. All Wyeomyia species saddle has spines on the posterolateral margin have seta 2-S with a smooth margin. in species of the subgenera Wyeomyia, Antunes- (36) Siphon (CI = 0.400): without seta 2a-S myia, and Decamyia and in W. argenteorostris. (0); with hairlike seta 2a-S (1); with strong, (28) Seta 4-X, development (*): branched stout and sclerotized seta 2a-S (2). (0); single (1). Harbach and Peyton (1990a, 1992) consid- A single seta 4-X is observed only in ered 2a-S to be the seta that arises in a similar W. tarsata and W. melanocephala. The poly- position on the dorsal surface of the siphon. morphic condition for seta 4-X was observed in Only in Dendromyia species is seta 2a-S strong, W. melanocephala. stout and sclerotized (1); it is absent in (29) Seta 4-X, development (CI = 0.125): Spilonympha species. less than 0.5 of seta 1-X length (0); more than (37) Numerous accessory setae on siphon 0.5 of seta 1-X length (1). (CI = 0.333): absent (0); present (1) (Fig. 4a) The majority of Wyeomyia species have a Condition 1 was defined as the presence of short seta 4-X. Based on the outgroup compari- numerous setae dispersed on all surfaces of the son, the primitive condition is a long seta 4-X. siphon. (30) Pecten spines, development (CI = (38) Feeding behavior (CI = 1.000): ventral 0.250): a single row (0); more than one row (1). side down (0); ventral side up (1). Missing values were assigned for this charac- Feeding ventral side up is characteristic of ter in species where pecten spines are absent. Phoniomyia species and until now has not been (31) Spicules on siphon (CI = 0.250): absent observed in any other Neotropical Sabethini. (0); present (1). (39) Larval pigmentation (CI = 0.500): ab- All Dendromyia species possess spicules on sent (0); present (1). W. forattinii the siphon; with the exception of The occurrence of pigmentation in larvae of W. oblita and , the remaining species have no Wyeomyia species is rare, at least in larvae conspicuous spicules on the siphon. In the reared under laboratory conditions; reddish pig- Sabethes outgroup taxa, only possesses spines mentation was observed only in the unnamed on the siphon. Wyeomyia sp. 4. The larvae of Phoniomyia spe- (32) Siphon, apex (CI = 0.500): narrow, as cies are always colored. wide as seta 2-S length (0) (Fig. 3f); broad, wider than seta 2-S length (1). Seta 2-S was used as a landmark to define Pupa the width of the siphon apex. Seta 2-S varies in (40) Seta 1-CT, development (CI = 1.000): length in some species, but only Phoniomyia straight (0) (Fig. 4d); sigmoidal (1) (Fig. 4c). and Sabethes species have the siphon apex as Harbach and Peyton (1990a) pointed out the wide as seta 2-S length. sigmoidal development of seta 1-CT in (33) Seta 1-S, development (CI = 0.250): sin- Wyeomyia species. All Wyeomyia species exam- gle (0); branched (1). ined for this study have a sigmoidal seta 1-CT. The majority of species examined have seta (41) Apex of seta 1-CT (CI = 0.500): hooked 1-S branched; only W. ulocoma, W. felicia, (0); straight (1). W. forattinii, W. leucostigma, and W. melano- This character was used by Judd (1996) for cephala have seta 1-S single. the Sabethini, taking into consideration the de- (34) Seta 1-S, position (CI = 0.250): arising gree to which the apex is hooked. However, in basally (0); arising beyond basal 0.3 (1). Wyeomyia species the apex varies, so it is diffi- The position of seta 1-S varies among the cult to determine the degree to which it is Wyeomyia species. The basal margin of the si- hooked. We therefore considered only two phon was used as a landmark for the position of states for the apex of seta 1-CT. seta 1-S. Only setae that arise from the base of (42) Seta 5-CT, development (*): strong, more the siphon were considered to exhibit condition developed than seta 4-CT (0); hairlike, similar in 0. The majority of Wyeomyia species have seta degree of development to seta 4-CT (1). 1-S arising basally. In L. durhami, seta 5-CT is hairlike, whereas (35) Seta 2-S, anterior margin (CI = 0.500): in all other species examined for this study, seta smooth (0); spiculate (1) (Fig. 4b). 5-CT is strongly developed.

Fig. 4. Siphonal accessory setae of W. airosai (a), seta 2-S of the fourth-instar larva of O. personatum (b), pupal seta 1-CT of W. felicia (c), pupal seta 1-CT of S. aurescens (d), pupal seta 2-V of W. oblita (e), and posterolateral angle of segment VIII of the pupa of W. oblita ( f ). The arrows indicate the character states. Scale bars = 0.1 mm.

(43) Setae 10,11-CT, placement (CI = 0.250): In species in the subgenera Cruzmyia and mesal to seta 11-CT (0); directly posterior to Wyeomyia, seta 2-V is placed anteriorly. In all seta 11-CT (1). other species, seta 2-V occurs roughly at the (44) Seta 5-IV, position (CI = 1.000): arising level of setae 1 and 5-V. close to setae 1 and 2-IV (0); arising far from (46) Seta 6-V,VI, development (CI = 1.000): setae 1 and 2-IV (1). normal, similar to that of seta 3-V,VI (0); In Phoniomyia species, seta 5-IV arises strongly dissimilar to that of seta 3-V,VI (1). mesally, close to setae 1 and 2-IV. Seta 6-V,VI similar in aspect to seta 3-V,VI (45) Seta 2-V, position (CI = 0.200): dis- was considered normal (condition 0). Only placed well anterior to seta 1-V (0) (Fig. 4e); W. flui and W. surinamensis have seta 6-V,VI not displaced anterior to seta 1-V (1). strong.

(47) Seta 5-VI, development (CI = 0.333): All conditions of this character are observed shorter than tergum VI (0); longer than tergum in Wyeomyia species. However, in the majority VI (1). of species examined for the study, the The majority of Wyeomyia species have a gonostylus is branched at the apex (with stem), long seta 5-VI. which is also the condition in the outgroup (48) Seta 9-VI, development (CI = 0.500): taxa. hairlike (0); stout (1). (57) Lobe C of gonostylus, development All members of the subgenera Wyeomyia and (CI = 1.000): ladle-like (0); not ladle-like (1). Antunesmyia possess a stout seta 9-VI. In the The development of lobe C of the gonostylus outgroup taxon S. aurescens, seta 9-VI is multi- varies among Wyeomyia species, but in ply branched but equally strong. Spilonympha, lobe C is distinguished by its (49) Seta 6-VII, position (CI = 0.333): ven- unique ladle-like shape. tral (0); dorsal (1). (58) Aedeagus (CI = 1.000): elongate (0); This character varies among the Wyeomyia rounded (1). species; however, in all Spilonympha species The shape of the aedeagus is determined by seta 6-VII arises dorsally, whereas in all the projection of the median sternal plate. Only Dendromyia species it is ventral. Phoniomyia species possess an elongate aede- (50) Seta 6-VII, position (CI = 0.333): ante- agus. rior to seta 9-VII (0); posterior to seta 9-VII (59) Apical tergal arm of aedeagus, develop- (1). ment (*): with tooth-like projection on mesal Seta 9-VII was used as a landmark to define margin (0); without tooth-like projection (1). the position of seta 6-VII. Taxa in which seta 6- A tooth-like projection on the mesal margin VII arises ventrally were scored as not applica- of the apical tergal arm is an autapomorphy for ble. Limatus. (51) Posterolateral angle of segment VIII, de- (60) Apical tergal arm of aedeagus, develop- velopment (CI = 1.000): not produced (0); pro- ment (CI = 0.111): fused mesally to arm of op- duced as a posterior lobe (1) (Fig. 4f). posite side (0); separate (1). In taxa where segment VIII does not possess Apical tergal arms fused mesally in an apical a posterior lobe, the posterior margin of seg- tergal bridge was scored as condition 0. All ment VIII is slightly projected. In Antunesmyia Dendromyia species have fused apical tergal and Wyeomyia species (except for arms, but this character varies within the subge- W. medioalbipes), segment VIII forms a distinct nus Wyeomyia. lobe from which seta 9-VIII arises. (52) Seta 9-VIII, position (CI = 0.286): at (61) Aedeagal median sternal plate (CI = posterolateral angle (0); directly mesal to pos- 0.200): barely extending beyond tergal arm (0); terolateral angle (1); directly anterior to pos- extending well beyond tergal arm (1). terolateral angle (2). The majority of the species examined in this (53) Paddle, outer margin, position (*): in study have the aedeagal median sternal plate line with insertion of seta 9-VIII (0); displaced barely extending beyond the tergal arm. well mesal to insertion of seta 9-VIII (1). (62) Basal plate (CI = 1.000): without leaf- (54) Paddle apex (CI = 0.400): rounded (0); like seta (0); with leaf-like seta (1) (Fig. 5b). triangular (1); truncate (2). Only Decamyia species (W. ulocoma and A paddle with a rounded apex was observed W. felicia) have a basal plate with leaf-like in the outgroup taxon Limatus,inPhoniomyia setae. species, and in Wyeomyia sp. 4. Except for five (63) Tergum VIII, thick and sclerotized setae species that have a truncate apex, all Wyeomyia on posterolateral margin (CI = 1.000): present species have a paddle with a somewhat triangu- (0) (Fig. 5c); absent (1) (Fig. 5d). lar apex. Thick and sclerotized setae on the (55) Male genital lobe, distal margin (CI = posterolateral margin of tergum VIII is a dis- 0.143): without fingerlike projection (0); with tinct feature present in three of the six species fingerlike projection (1) (Fig. 5a). of the subgenus Wyeomyia examined (W. lutzi, W. oblita, and W. limai). Wyeomyia Adult arthrostigma has posterolateral setae that are (56) Gonostylus (CI = 0.250): branched at not developed as in the above species, therefore apex (0); branched at base (1); simple (2). this was scored as condition 1.

Fig. 5. Genital lobe of the pupa of W. airosai (a), basal plate of aedeagus of W. felicia (b), tergum VIII of male genitalia of W. lutzi (c), tergum VIII of W. ypsipola (d), proctiger of W. lutzi (e), and proctiger of W. bourrouli ( f ). The arrow indicates the character state. Scales = 0.1 mm.

(64) Tergum VIII, posterolateral lobe (CI = Among the species examined, only Onirion 0.250): present (0); absent (1). possesses a paraproct with exceptionally long (65) Proctiger (CI = 1.000): without filamen- cercal setae, so this is an autapomorphy for tous projection (0) (Fig. 5f); with filamentous Onirion. Harbach and Peyton (2000) assigned projection (1) (Fig. 5e) condition 1 as a distinctive state for Onirion. A proctiger with a filamentous projection (67) Semi-erect forked scales on head (*): occurs only in W. lutzi, W. sabethea, W. oblita, absent (0); present (1). and W. limai.InW. oblita, this filamentous The majority of the Wyeomyia species possess projection forms a strong structure, fused ba- short, semi-erect, usually pale, forked scales dis- sally. posed in a single transverse row on the occiput. (66) Cercal setae (*): normal, short (0); ex- These scales are absent only in Wyeomyia ceptionally long (1). (Cruzmyia) forattinii (autapomorphy).

(68) Interocular space (CI = 0.286): narrow (75) Scutal scales, condition (*): broad, over- (0); broad (1); very broad (2). lapping scales (0); moderately broad, slightly Spilonympha, Dendromyia, Exallomyia, overlapping scales (1). and Menolepis species possess a broad Sabethes species are unique in having broad, interocular space, whereas the other species flat, overlapping scales on the scutum, whereas have a narrow interocular space, though all other species possess moderately broad, W. palmata, W. argenteorostris, and Limatus slightly overlapping scales. Condition 0 is an have a broader interocular space than the autapomorphy for Sabethes. other species examined. (76) Paratergite (CI = 0.143): bare (0); with (69) Proboscis (CI = 1.000): very long (0); scales (1). short (1). Harbach and Kitching (1998) pointed out that A proboscis more than twice the antennal no sabethine has paratergal scales or setae. length was considered very long. Only However, we observed paratergal scales in the Phoniomyia species possess a very long probos- outgroup taxa, as well as in the subgenera cis. Although Cruzmyia species possess a long Antunesmyia, Decamyia, Wyeomyia, and proboscis, it is not similar in proportion. Exallomyia, and in W. melanocephala and (70) Clypeus (CI = 0.667): bare (0); with W. negrensis. scales on dorsal surface (1); with scales on dis- (77) Prespiracular area (*): with scales (0); tal margin of dorsal surface (2). with setae (1). Scales on the clypeus was subdivided into The presence of prespiracular scales is an two states determined by the distribution of autapomorphy for Limatus. All other species scales. W. jocosa, W. complosa, and W. confusa examined possess prespiracular seta. have scales on the dorsal surface, whereas (78) Metallic-colored scutal scales (CI = W. flui and W. surinamensis have scales only on 1.000): absent (0); present (1). the distal margin of the dorsal surface. The This may not be a strong character in itself, clypeus of the remaining species is bare. but considering the overall observations from (71) Antepronota (CI = 0.750): more widely other studies on Wyeomyia species that are separated (0); moderately approximated (1); ap- known for their dull scutal scaling, it would be proximated (2). valid. Color is usually distinctive for sabethine The antepronota are more approximated in species (Dyar 1919; Lane and Cerqueira 1942). Sabethes, in which they nearly touch dorsally. Harbach and Peyton (1990b) took it into con- These structures are separate in Limatus and sideration when they transferred a species of Phoniomyia, whereas they are somewhat sepa- Wyeomyia to the genus Sabethes. Belkin et al. rated but still close together in all Wyeomyia (1970) also distinguished adults of Wyeomyia species. species from those of other sabethines by the (72) Antepronotum (*): with a conspicuous dull appearance of the scutal scales. Harbach apicomesal angle (0); without a conspicuous (1991) suggested that dull scutal scales was a apicomesal angle (1). plesiomorphic condition among sabethine species. Furthermore, in their cladistic analysis of Huang (2002) pointed out the development of the Culicidae, Harbach and Kitching (1998) re- the antepronotum in Sabethes, which possesses stored the relationship between Limatus and an apicomesal angle. Condition 0 is an Sabethes because of the strong metallic sheen autapomorphy for Sabethes. of the scutelar scales. (73) Postpronotal scales (CI = 0.167): contin- (79) Mesopostnotal area (CI = 0.250): with- uous (0); discontinuous (1). out scales (0); with scales intermixed with setae Continuous postpronotal scales resemble a (1); with scales anterior to mesopostnotal setae unique cover of fused scales. The outgroup taxa (2). and few Wyeomyia species exhibit condition 0. The presence of scales was scored as two (74) Scutum, profile in lateral view (CI = states: one state with only a few scales inter- 0.333): somewhat round anteriorly (0); some- mixed with mesopostnotal setae and a second what flat anteriorly (1). for those species that possess a tuft of scales lo- A slightly convex scutum is a synapomorphy cated anterior to the mesopostnotal setae. Con- for Limatus, Phoniomyia, Cruzmyia, and dition 2 occurs in W. negrensis, W. leucostigma, Menolepis. W. flui, and W. surinamensis.

(80) Mesokatepisternal scales (CI = 0.500): (88) Number of spermathecal capsules (CI = covering entire mesokatepisternum (0); cover- 0.500): 3 (0); 1 (1). ing posterior 0.5 of mesokatepisternum (1). Female mosquitoes possess either one or (81) Lower mesokatepisternal setae (CI = three spermathecal capsules (Harbach and 0.250): extending above dorsal margin of Kitching 1998). Three spermathecal capsules mesomeron (0); not extending above dorsal were observed in the outgroup taxa and also in margin of mesomeron (1). the majority of Wyeomyia species. One Lower mesokatepisternal setae not extending spermathecal capsule was observed in W. flui, above the dorsal margin of the mesomeron was W. surinamensis, W. chalcocephala, Wyeomyia used by Dyar (1928) as a diagnostic character sp. 4, and W. negrensis. Judd (1996) observed for species of the subgenus Wyeomyia. In our one spermathecal capsule in W. albosquamata analysis, condition 1 was observed in members and W. phroso. Within the Sabethini, of the subgenera Wyeomyia, Dodecamyia, Tripteroides and Topomyia (Oriental and Cruzmyia, and Exallomyia. Australasian) possess one spermathecal capsule. (82) Wing, upper calypter (CI = 0.333): without marginal setae (0); with marginal setae (1). Results and discussion In the Sabethini, marginal setae on the upper calypter are usually represented by two setae. The discussion about monophyletic lineages Condition 1 was observed in Onirion, within the genus Wyeomyia is based on the Dendromyia, Decamyia, W. surinamensis, strict-consensus topology of 31 equally parsi- W. flui, Wyeomyia sp. 4, and W. chalcocephala. monious trees (length = 267 steps, CI = 0.355, and retention index (RI) = 0.644) obtained in (83) Vein Rs (CI = 0.200): without proximal spur (0); with proximal spur (1). the unweighted, unordered character data analy- A proximal spur is present in the subgenera sis of 41 species of Sabethini. The strict- Wyeomyia, Dodecamyia, and Cruzmyia. consensus tree of those trees is shown in Figure 6. Using the successive approximations (84) Vein M, anterior scales on proximal 0.5 character weighting method, the character of dorsal surface (CI = 0.333): decumbent (0); weights stabilized after three rounds and identi- anteriorly projected (1). fied a set of four equally parsimonious trees Scales on the proximal 0.5 of the dorsal sur- with a weighted length of 61.69326. These four face of vein M can arise parallel to the vein on trees are not a subset of those 30 unweighted the anterior side. The majority of Wyeomyia trees, and the strict consensus is shown in species have scales projecting anteriorly to the Figure 7. longitudinal axis of vein M. The analysis of the combined morphological (85) Condition of scales on dorsal surface of and allozyme data for the 19 species included proximal 0.5 of vein M (CI = 0.375): narrow 95 parsimony-informative characters. The un- spatulate (0); moderately broad spatulate (1); weighted parsimony analysis of combined data broad spatulate (2); ligulate (3). sets resulted in a single topology of 250 steps Species of the subgenera Wyeomyia, (CI = 0.440, RI = 0.588). In the successive ap- Prosopolepis, and Spilonympha have narrow, proximations weighting analyses, character elongate, spatulate scales on the dorsal surface weights stabilized in the first round resulting in of the proximal 0.5 of vein M. The states mod- one successive approximations character erately broad spatulate, broad spatulate, and weighting tree with a weighted length of narrow spatulate are defined according to Fig- 64.347 62. The unweighted and weighted topol- ure 82 I, l/m, k in Harbach and Knight (1980, ogies are identical (Fig. 8). page 321). The results of all cladistic analyses were (86) Condition of posterior scales on dorsal largely topologically congruent. Minor differ- surface of proximal 0.5 of vein M (*): ences were observed in the phylogenetic posi- decumbent (0); posteriorly projected (1). tions of W. negrensis, W. leucostigma Among the species examined for the study, (W. aporonoma, W. staminifera), W. argent- only W. negrensis has the posterior scales on eorostris, and W. confusa. Similarly, relation- the proximal half of vein M projecting posteri- ships among Dodecamyia and Exallomyia orly. species show variable arrangements, with weak (87) Crossvein mcu (CI = 0.250): proximal to bootstrap support values in any method and crossvein rm (0); adjacent to rm (1). data set adopted. Parsimony bootstrap analysis

Fig. 6. Strict-consensus topology of the 30 most parsimonious trees recovered in the unweighted, unordered analysis of morphological data for 41 species. The number in boldface type above each branch indicates the percentage of bootstrap pseudoreplicates in which that node is supported, and the number below indicates the Bremer support value. The shaded areas denote monophyletic groups within the genus Wyeomyia, the former genus Phoniomyia, and Wyeomyia.

of the combined morphological and allozyme Parsimony character optimization (Table 3) data sets (Fig. 8) better supported the was done in a topology recovered in the un- monophyletic groups found in the morphologi- weighted, unordered analysis shown in Fig- cal analysis. ure 9.

Fig. 7. Strict-consensus topology of four equally parsimonious trees recovered in the successive approximations weighting analyses using the rescaled consistency index to down-weight homoplastic morphological characters based on morphological data for 41 species. The shaded areas denote monophyletic subgenera of Wyeomyia, except for the “flui group”.

The results of all analyses did not recover the the genera Wyeomyia and Limatus and the genus Wyeomyia in the way it is currently de- subgenus Phoniomyia. Judd (1996) found that the fined, as a monophyletic lineage, because the ge- genus Wyeomyia was a paraphyletic assemblage, nus Onirion is embedded within Wyeomyia as the with Limatus and Phoniomyia embedded within sister of W. melanocephala. Also, the results par- the clade formed by Wyeomyia species. One of tially contradict those of Judd (1996) relative to three synapomorphies for Wyeomyia, Phoniomyia,

Fig. 8. The single most parsimonious tree recovered in the unweighted, unordered analysis of morphological and allozymic data for 19 species. The number in boldface type above each branch indicates the percentage of the bootstrap pseudoreplicates in which that node is supported, and the number below indicates the Bremer support value. The shaded areas denote monophyletic subgenera of Wyeomyia.

and Limatus (Judd 1996) was herein redefined and a second synapomorphy was employed for all and tested using the present data set (seta 14,15-C 41 species as defined by that author. However, located midway between the anterior margin of phylogenetic signal was lost following the inclu- the labial sclerite and the posterior tentorial pit), sion of additional species because these characters

Table 3. Optimization of character states using the Table 3 (concluded). tree shown in Figure 9 generated in the unweighted, unordered analysis (length = 267, consistency Node Character CI Stage change index (CI) = 0.3558, retention index = 0.6614*). 32 1 0.375 2÷3 47 0.333 1÷0 Node Character CI Stage change 54 0.400 1÷2 3 6 0.500 3÷0 79 0.286 0÷1 24 0.250 0÷1 33 34 0.250 0÷1 36 0.400 1÷0 85 0.375 1÷2 45 0.200 1÷0 34 61 0.200 0÷1 10 16 0.400 1÷0 35 5 1.000 0÷1 20 0.250 1÷0 7 1.000 1÷0 21 0.500 1÷2 11 1.000 1÷0 23 0.667 1÷2 13 1.000 0÷1 52 0.250 0÷1 15 1.000 0÷1 55 0.200 1÷0 22 1.000 0÷1 65 1.000 0÷1 38 1.000 0÷1 11 8 0.400 1÷2 39 0.500 0÷1 16 0.400 0÷2 44 1.000 1÷0 25 0.167 0÷1 56 0.250 0÷1 29 0.167 1÷0 58 1.000 1÷0 43 0.250 0÷1 61 0.200 0÷1 45 0.200 1÷0 69 1.000 1÷0 63 1.000 1÷0 *Includes only nodes that are supported by $50% boot- 12 3 0.250 1÷0 strap proportion. 34 0.250 0÷1 52 0.250 1÷2 61 0.200 0÷1 were inconsistent within Wyeomyia. Our results 15 8 0.400 0÷1 show the genus Limatus and the subgenus 60 0.143 1÷0 Phoniomyia as separate lineages arising outside 22 1 0.375 2÷3 the genus Wyeomyia, although the bootstrap sup- 16 0.400 1÷0 port is weak (<50%). 26 0.167 1÷0 Phoniomyia was proposed as a genus by 30 0.250 1÷0 Theobald (1903). Its taxonomic position 31 0.250 0÷1 changed several times (Bonne-Wepster and 36 0.400 1÷2 Bonne 1921; Dyar 1924; Edwards 1932) and fi- 49 0.250 1÷0 nally Lane and Cerqueira (1942) defined it as a 68 0.250 0÷1 genus. The taxonomic instability of the group 27 30 0.250 1÷0 has been caused mainly by the fact that most of 54 0.400 1÷2 the species descriptions were based on the adult 79 0.286 0÷1 stages with no association with immature 88 0.500 0÷1 stages, despite adult characters being generally 29 6 0.500 0÷1 conservative in all Phoniomyia species. Judd 21 0.500 1÷2 (1998a) recovered the genus Phoniomyia as the 23 0.667 1÷0 sister of the subgenus Hystatomyia. Thus, the 29 0.167 0÷1 genus Wyeomyia, as it was defined by Lane and 43 0.250 0÷1 Cerqueira (1942), was a paraphyletic lineage. 46 1.000 0÷1 As a result, Judd (1998a) reduced Phoniomyia 70 0.667 0÷2 to a subgenus of Wyeomyia. Because no speci- 31 24 0.250 0÷1 men of the subgenus Hystatomyia was available 36 0.400 1÷0 for the present study, the sister-group relation- 37 0.333 0÷1 ship between Phoniomyia and Hystatomyia 57 1.000 0÷1 could not be tested using the present morpho- 68 0.250 0÷1 logical data set. It is important to point out that the placement of Phoniomyia outside Wyeomyia

Fig. 9. One of the most parsimonious unweighted, unordered trees generated using the morphological data for 41 species. This topology was employed for parsimony character optimization (Table 3) and shows the nodes. The shaded areas denote monophyletic subgenera of Wyeomyia, except for the “flui group”.

may also be a result of not having any 4-Mx on the anterolateral margin, directly Hystatomyia species in the data set. However, lateral to the maxillary brush (character 7); an- Phoniomyia as either a genus or subgenus of terior margin of the head somewhat triangular Wyeomyia is a distinct phylogenetic lineage, (character 11); seta 1-A stiff (character 13); seta which is well supported by 100% bootstrap and 4-C arising posteriorly on the dorsal apotome a Bremer support value of 12. The monophyly directly mesal to the antennal prominence of Phoniomyia is supported by 10 consistent (character 15); setae 6,7-III inserted on the synapomorphies: maxilla with a lateral notch same plate (character 22); larva feeds ventral containing seta 3-Mx (character 5); larval seta side up (character 38); pupal seta 5-IV inserted

© 2007 Entomological Society of Canada Motta et al. 617 close to seta 1-IV (character 44); aedeagus Antunesmyia was proposed as a subgenus of elongate (character 58); and proboscis twice as Wyeomyia by Lane and Cerqueira (1942) to in- long as antenna (character 69). clude three species. While the fourth-instar Within Wyeomyia, the basal clade (node 3) larva and pupa of W. alani are described, the (Fig. 9) leads to Cruzmyia species (W. forattinii two other species were defined on the basis of and W. dyari). Four synapomorphies support the adult female characters. Some larval and pupal Cruzmyia clade: larval seta 4-Mx single, features of W. alani are similar to those of spe- pointed (character 6); presence of a sclerotized cies of the subgenus Wyeomyia. Members of plate on segment VIII (character 24); absence both Antunesmyia and Wyeomyia inhabit of seta 2a-S (character 36); and pupal seta 2-V bamboo-internode habitats. displaced well anterior to seta 1-V (character The clade formed by W. lutzi, W. limai, 45). Judd (1996) recovered Cruzmyia and W. oblita, and W. sabethea (node 10) is mono- Limatus as sister groups. Cruzmyia was pro- phyletic and supported by six synapomorphies: posed as a subgenus of Wyeomyia by Lane and larval seta 15-C arises well posterior to the Cerqueira (1942), based mainly on adult char- hypostomal ridge, close to the posterior end of acters (wing and proboscis features), and the re- the hypostomal suture (character 16); seta 11-T sults of the present study corroborate this hairlike (character 20); larval seta 13-I stellate hypothesis, being supported by a 66% bootstrap with stiff branches (character 21); pupal seta 1- proportion and a Bremer support value of 2. VIII stellate (character 23); seta 9-VIII anterior to posterolateral angle of tergum VIII (character Nodes 4 and 5 show the subgenera Dode- 52); male genital lobe of pupa with distal mar- camyia (W. aphobema) and Exallomyia (W. gin without a fingerlike projection (character tarsata) as sister taxa of a major clade (node 6). 55) and proctiger with filamentous projections In the topology generated using the successive (character 65). Parsimony bootstrap support for approximations weighting method (Fig. 7), node 10 is moderate (82%), with a Bremer sup- Dodecamyia and Exallomyia were recovered as port value of 2. Dyar (1928) defined the subge- sister groups, supported by a single synapo- nus Miamyia to include W. codiocampa Dyar morphy (larval seta 4-Mx brush-tipped (charac- and Knab, W. serrata (Lutz), and W. hosautos ter 6)). The bootstrap proportion for the Dyar and Knab. However, Miamyia has been Dodecamyia plus Exallomyia clade is <50%. treated as a junior synonym of the subgenus Monophyly of the nominal subgenus Wyeomyia since Edwards (1932). Wyeomyia Wyeomyia was not corroborated by the results lutzi, W. limai, W. codiocampa, and W. serrata of the present analysis (node 7) because the were also included in the “serrata series” within subgenus Antunesmyia (W. alani) arises among the subgenus Wyeomyia by Lane and Cerqueira members of the subgenus Wyeomyia. Placement (1942). Recently, Judd (1996) found W. of Antunesmyia within the subgenus Wyeomyia codiocampa in a sister relationship with corroborates Judd’s (1996) hypothesis that the W. oblita. Our results corroborate Judd’s (1995, subgenus Wyeomyia is a polyphyletic lineage. 1996) hypotheses and support the recognition Antunesmyia plus Wyeomyia (node 8) is the sis- of Miamyia as a subgenus of the genus ter to a clade leading to (Antunesmyia, W. lutzi, Wyeomyia. We therefore formally resurrect the W. limai, W. oblita, W. sabethea). The relation- subgenus Miamyia from synonymy with the ship between Antunesmyia and Wyeomyia is subgenus Wyeomyia. The species comprising supported by five synapomorphies: fourth-instar the subgenus Miamyia are W. codiocampa, larvae with seta 4-Mx forked (character 6); seta W. serrata, W. hosautos, W. lutzi, W. limai, 6-Mx stellate (character 8); presence of spines W. oblita, and W. sabethea. on the posterolateral margin of the saddle (char- A major clade (node 13) is formed mainly by acter 27); larval seta 4-X longer than 0.5 of seta species that are placed in the subgenera 1-X length (character 29); pupa with seta 2-V Dendromyia (sensu Lane and Cerqueira 1942) displaced well anterior to seta 1-V (character and Menolepis. Within this major clade there is a 45), and seta 6-VII ventral (character 49). How- secondary group defined by node 14, which in- ever, these characters are not entirely consis- cludes W. aporonoma plus W. staminifera as the tent, so the Wyeomyia plus Antunesmyia sister of W. argenteorostris. This arrangement is arrangement is only weakly supported by a supported by a synapomorphy, larva with saddle <50% bootstrap proportion. covering the dorsal half of segment X (character

26), and supported by a bootstrap proportion of to W. confusa and W. melanocephala plus <50%. In contrast, node 15 leading to O. personatum in the successive weighting W. aporonoma and W. staminifera is supported analysis; however, this arrangement is poorly by a 64% bootstrap value, a Bremer support supported. value of 1, and two synapomorphies: seta 6-Mx The subgenus Prosopolepis (W. confusa) branched (character 8); and apical tergal arm of was recovered as a sister lineage of the aedeagus fused mesally (character 60). A hy- W. melanocephala plus O. personatum (node pothesis about the monophyly and definition of a 20). This arrangement is supported by three group formed by W. aporonoma and synapo-morphies: larval seta 13-I single W. staminifera became evident to us during a (character 21); seta 1-VIII branched (charac- study that included the description of ter 23); pupa with posterolateral lobe of W. staminifera (Lourenço-de-Oliveira et al. tergum VIII present (character 64); parsimony 1992). Because the results of the present analysis bootstrap support <50%. Prosopolepis was corroborate the monophyly of the group formed proposed as a genus of the Sabethini by Lutz by W. aporonoma and W. staminifera, and the (1905) to include a single species named split leading to it is moderately supported, we Prosopolepis confusus. Dyar (1928) included consider that the group may be defined as a sub- this species and three others in the genus genus within the genus Wyeomyia. Dyar (1928) Dendromyia Theobald. Subsequently, Edwards defined the subgenus Triamyia to include (1932) recognized Dendromyia as a subgenus W. aporonoma and W. personata (Lutz), based of genus Wyeomyia and Lane (1953) retained on adult female and male genitalia characters. this change of status. As a consequence of the Later, Edwards (1932) included these species in revision of the subgenus Dendromyia (Motta the subgenus Dendromyia. Here we propose to and Lourenço-de-Oliveira 2000), W. confusa resurrect Triamyia as a subgenus of genus was removed from the subgenus and left with- Wyeomyia to include W. aporonoma and out subgeneric placement in the genus W. staminifera. Wyeomyia. Later, Lourenço-de-Oliveira et al. Node 18 defines a clade formed by (1999) resurrected Prosopolepis from synon- W. leucostigma plus W. negrensis as the sister ymy with the subgenus Dendromyia to ingroup of W. melanocephala plus O. personatum clude W. confusa. as the sister group of W. confusa. Node 18 is The clade formed by O. personatum and supported by larval seta 4-X longer than 0.5 of W. melanocephala (node 21) is supported by six seta 1-X length (character 29); male genital synapomorphies: larva with pecten spines dis- lobe without a fingerlike projection (character posed in a single row (character 30); seta 1-S 55); anterior scales on dorsal surface of proxi- single (character 33); seta 1-S arising beyond mal 0.5 of vein M elongate and ligulate (char- the basal 0.3 (character 34); pupal seta 9-VIII acter 85). arising from posterolateral angle of tergum VIII Node 19 leads to a clade formed by (character 52); male genitalia with gonostylus W. negrensis and W. (Menolepis) leucostigma. simple (character 56); dorsal surface of proxi- Two homoplastic synapomorphies support the mal 0.5 of vein M with moderately broad split: larval comb scales inserted in several spatulate scales (character 85). Bootstrap sup- rows (character 25) and mesopostnotal area port for the relationship between O. personatum with scales anterior to mesopostnotal setae and W. melanocephala is weak. (character 79). In the successive weighting In the present analysis, the branch leading to analysis (Fig. 7), W. negrenis was recovered as Onirion plus Wyeomyia (node 2) is supported the sister of W. chalcocephala, W. surinamensis, by six synapomorphies: larva with pecten W. flui, and Wyeomyia sp. 4 (“flui group”). In spines disposed in one row (character 30); si- the analysis with combined morphology and phon with a broad apex, wider than length of allozyme data from 19 species (Fig. 8), we ob- seta 2-S (character 32); segment X with saddle served the arrangement W. negrensis plus covering more than dorsal half of segment W. leucostigma, but the bootstrap value was low (character 26); apex of pupal seta 1-CT hooked (<50%). The phylogenetic position of (character 41); apex of paddle triangular (char- W. negrensis remains unresolved, mainly be- acter 54); scutal scales without metallic sheen cause its position varies in the topologies gener- (character 78). This arrangement disagrees with ated using different methods of analysis. that of Harbach and Peyton (2000) regarding Wyeomyia leucostigma also appears as the sister the placement of the genus Onirion, which was

© 2007 Entomological Society of Canada Motta et al. 619 recovered embedded within the genus detailed studies are necessary. As a conse- Wyeomyia in this study, whereas in Harbach quence, we choose to maintain the generic status and Peyton (2000), the taxon was placed out- of Onirion and the subgeneric status of all side it in an unresolved relationship with genera monophyletic lineages recovered in the study. Isostomyia Coquillett plus Shannoniana Lane Furthermore, elevation to generic rank and ex- and Cerqueira and Wyeomyia plus Limatus and cessive splitting of tribe could cause taxonomic Sabethes. The following morphological charac- instability. ters were used to distinguish Onirion from The subgenus Dendromyia (node 22) was other Sabethini: larva with circular occipital fo- confirmed as a monophyletic group within ramen; concentration of aciculae on one side of Wyeomyia, supported by eight synapomorphies: the branches of setae 4-P and 7-T; stout seta larva with maxilla with a denticular 11-T with multiple apical spikes; strong devel- laciniarastrum 1 (character 1); seta 15-C in- opment and placement of seta 13-IV,V lateral serted between the anterior border of the to setae 11 and 12; presence of a dense filamen- labiogula and the termination of the hypostomal tous pecten extending the length of the siphon; ridge (character 16); segment X with the saddle the presence of punctures on abdominal seg- covering half of segment X (character 26); ments III and IV of the pupa; and strong devel- pecten spines disposed in a single row (charac- opment of the cercal setae on the male genitalia ter 30); presence of spicules on the siphon (Harbach and Peyton 2000). In the cladistic (character 31); siphon with seta 2a-S very analysis, we scored two characters, the circular strong, stout, and sclerotized (character 36); pu- occipital foramen of the fourth-instar larva and pal seta 6-VII ventral (character 49); and a development of the cercal setae of the male broad interocular space (character 68). Boot- genitalia; however, because they were strap support for the Dendromyia clade is 95% autapomorphies for Onirion they were excluded and the Bremer support value is 6. The subge- from all analyses. Although the autapomorphies nus Dendromyia was redefined by Motta and are not informative in terms of resolving rela- Lourenço-de-Oliveira (1995, 2000) on the basis tionships, they are arrayed as unique characters of morphological and allozyme data (Motta et that are not contradicted, and thus give a clear al. 1998), but its phylogenetic position within indication of the degree of morphological diver- Wyeomyia has not been tested until now. gence. The presence of a circular occipital fora- Node 26 defines a clade formed by men in Onirion is shared with other genera of (W. chalcocephala, W. surinamensis, W. flui, Sabethini, including Shannoniana and and Wyeomyia sp. 4) plus (W. ulocoma and Trichoprosopon Theobald. However, it is a dis- W. felicia), supported by two synapomorphies: tinctive character for Onirion in comparison male genital lobe without a fingerlike projec- with other Wyeomyia species. None of the taxa tion (character 65) and upper calypter with mar- with a circular occipital foramen were used as ginal setae (character 82). The clade (node 27) outgroup in our analysis and this character composed of W. chalcocephala, W. surina- could have affected the relationship of Onirion mensis, W. flui, and Wyeomyia sp. 4 (“flui with Wyeomyia. Similarly, O. personatum was group”) is supported by four synapomorphies: placed in an unresolved relationship in the larva with pecten spines disposed in a single cladistic analysis of the Sabethini by Judd row (character 30); paddle apex truncate (char- (1996). This taxon appears as a separate lineage acter 54); adult with mesopostnotal scales ante- arising between W. melanocephala and a group rior to mesopostnotal setae (character 79); and comprised of Limatus, Phoniomyia, and presence of one spermathecal capsule (character Wyeomyia species. 88). Bootstrap support for the split leading to The phylogenetic position of Onirion within this group is 59%. Although bootstrap support the Sabethini is ambiguous. Consequently, it is is not strong, the results of the study suggest suggestive of two scenarios. According to the that W. chalcocephala, W. surinamensis, W. flui, principle of equivalent rank, Onirion can be re- and Wyeomyia sp. 4 belong to a phylogenetic tained as a genus of the Sabethini, and thus cer- lineage that can be defined as a new, unnamed tain subgenera of Wyeomyia might be elevated subgenus, which will be defined in a future to generic rank. An alternative scenario would publication (M.A. Motta, in preparation). be to regard Onirion as a subgenus of The subgenus Decamyia (W. ulocoma and Wyeomyia. However, we consider that it is pre- W. felicia) forms a clade (node 30) supported mature to make any taxonomic change, as more by two synapomorphies (larval seta 1-S single

Table 4. Allele frequencies for 11 loci in 19 species studied. Population Locus 1 2 3 4 5 6 7 8 9 10 MDH n 29 27 23 19 7 1 26 16 29 15 A 0.000 0.000 0.457 0.000 0.000 0.000 0.000 0.000 0.000 B 1.000 1.000 0.543 0.000 1.000 1.000 0.000 0.000 0.000 0.000 C 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 0.000 0.000 0.000 1.000 0.000 0.000 1.000 1.000 1.000 1.000 E 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 F 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PGM n 24 22 27 20 7 2 29 16 26 17 A 0.146 0.000 0.000 0.000 0.000 0.250 0.000 0.000 0.000 0.000 B 0.542 0.205 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.750 0.324 D 0.313 0.795 0.000 0.175 0.000 0.000 0.086 0.750 0.000 0.000 E 0.000 0.000 1.000 0.825 1.000 0.750 0.000 0.000 0.250 0.265 F 0.000 0.000 0.000 0.000 0.000 0.000 0.810 0.250 0.000 0.000 G 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 H 0.000 0.000 0.000 0.000 0.000 0.000 0.103 0.000 0.000 0.412 I 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 IDH-1 n 28 29 27 20 5 2 31 17 29 18 A 0.054 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.028 B 0.000 0.000 0.500 0.000 0.000 0.250 0.161 0.029 0.000 0.972 C 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 0.946 0.931 0.500 1.000 1.000 0.750 0.839 0.971 0.000 0.000 E 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 F 0.000 0.069 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 IDH-2 n 28 27 2 21 7 1 31 17 30 19 A 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 B 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 C 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 D 1.000 1.000 0.000 0.000 1.000 0.000 0.000 1.000 0.000 0.000 E 0.000 0.000 1.000 0.000 0.000 0.000 1.000 0.000 0.000 1.000 F 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 FUM n 25 22 24 14 7 2 25 17 20 18 A 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 B 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 0.000 0.000 0.000 0.214 0.000 0.000 0.000 0.000 0.000 0.000 E 1.000 1.000 1.000 0.786 1.000 1.000 1.000 1.000 1.000 0.000 F 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 GPI n 32 27 30 21 7 2 27 16 29 14 A 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 B 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 C 0.172 0.315 0.000 0.833 0.000 0.000 0.000 0.000 0.190 0.000 D 0.828 0.685 1.000 0.167 1.000 1.000 0.000 0.000 0.810 0.000 E 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000

11 12 13 14 15 16 17 18 19

26 26 26 29 12 28 24 29 31 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.952 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 0.938 1.000 0.048 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.063 0.000 0.000

25 28 26 30 12 28 28 29 28 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.107 0.000 0.339 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.880 0.214 0.000 0.667 0.292 0.036 0.000 0.017 0.661 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.120 0.786 0.058 0.333 0.708 0.929 0.000 0.879 0.000 0.000 0.000 0.942 0.000 0.000 0.036 0.893 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.103 0.000

27 28 29 26 12 29 29 30 29 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.083 0.000 0.000 0.000 0.000 0.000 0.000 0.017 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.083 0.983 0.000 0.000 1.000 0.000 0.000 0.603 1.000 0.000 0.000 0.000 0.917 0.000 0.000 0.000 0.000 0.000 0.917 0.000 0.000 0.000 0.000 1.000 0.000 0.397 0.000 0.000 0.000 1.000 0.000 0.000

26 28 25 27 12 29 29 20 28 0.000 0.000 0.000 0.000 0.000 0.000 0.000 .000 0.161 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 1.000 0.000 0.000 0.000 0.839 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000

26 24 20 28 12 24 24 26 24 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 1.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000

26 22 25 25 12 25 30 25 27 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.040 0.000 0.000 0.000 0.000 1.000 0.000 0.712 0.000 0.960 0.000 0.000 0.000 1.000 0.000 1.000 0.288 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Table 4 (concluded). Population Locus 1 2 3 4 5 6 7 8 9 10 F 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 G 0.000 0.000 0.000 0.000 0.000 0.000 0.870 0.000 0.000 0.000 H 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 I 0.000 0.000 0.000 0.000 0.000 0.000 0.130 0.000 0.000 0.000 6PG n 17 17 29 14 1 2 32 18 32 14 A 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 B 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C 0.000 0.000 1.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 D 0.559 0.559 0.000 0.500 0.000 0.000 0.000 0.000 0.000 0.000 E 0.441 0.441 0.000 0.500 1.000 0.000 1.000 1.000 1.000 1.000 F 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 G 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 AK n 22 22 19 17 7 2 13 9 14 16 A 0.000 0.000 0.000 0.000 0.214 0.000 0.000 0.000 0.000 0.000 B 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C 1.000 1.000 1.000 0.882 0.000 0.000 0.000 0.000 0.000 0.000 D 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 E 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 F 0.000 0.000 0.000 0.118 0.786 0.000 0.923 1.000 1.000 0.000 G 0.000 0.000 0.000 0.000 0.000 0.000 0.077 0.000 0.000 1.000 GPD n 28 30 32 20 7 2 32 18 31 19 A 0.000 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 B 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.056 0.000 0.000 C 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 1.000 1.000 1.000 0.850 0.000 1.000 1.000 0.944 1.000 1.000 E 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 HK n 31 30 28 20 7 2 28 14 25 18 A 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 B 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C 0.000 0.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 D 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 ME n 23 22 28 20 6 2 26 12 26 17 A 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 B 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 000 D 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 E 0.000 0.091 1.000 0.000 0.167 1.000 0.000 0.000 0.000 0.000 F 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 G 0.739 0.659 0.000 1.000 0.833 0.000 0.000 0.000 0.000 0.000 H 0.261 0.250 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 I 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 J 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Note: n is the sample size per locus. Alleles shown in boldface type were not considered for cladistic analysis.

11 12 13 14 15 16 17 18 19 0.000 0.000 0.000 1.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

13 20 17 15 1 21 14 20 15 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000

21 17 15 22 12 19 23 20 21 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.478 1.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.522 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000

28 30 28 31 13 29 30 29 29 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.911 0.000 0.000 0.000 0.000 0.000 0.033 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.089 1.000 0.000 1.000 1.000 1.000 0.967 1.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000

25 28 24 28 13 28 28 24 22 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 1.000 .000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000

25 22 21 20 13 22 29 16 28 0.000 0.000 0.000 0.000 0.000 0.000 0.310 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.690 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.911 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.089 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.810 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.190 0.000 0.000 0.000 0.000 0.000 0.000

Table 5. Estimates of Neis (1978) unbiased genetic identity (above the diagonal) and genetic distance (below Population 123456789 1: bourr ***** 0.981 0.508 0.555 0.601 0.479 0.369 0.492 0.352 2: forci 0.019 ***** 0.503 0.565 0.580 0.469 0.371 0.527 0.349 3: myste 0.677 0.688 ***** 0.527 0.513 0.780 0.450 0.337 0.400 4: finla 0.589 0.572 0.641 ***** 0.538 0.438 0.535 0.545 0.492 5: airos 0.509 0.544 0.668 0.621 ***** 0.557 0.449 0.554 0.470 6: aning 0.735 0.756 0.248 0.825 0.584 ***** 0.367 0.360 0.394 7: oblit 0.997 0.991 0.798 0.626 0.802 1.003 ***** 0.778 0.681 8: lutzi 0.709 0.640 1.087 0.606 0.591 1.022 0.252 ***** 0.669 9: medio 1.045 1.053 0.915 0.710 0.756 0.932 0.384 0.401 ***** 10: arthr 1.736 1.742 0.993 0.995 1.515 1.423 0.480 0.734 661 11: ypsip 0.915 0.944 1.792 1.034 0.842 1.412 1.577 1.192 0.970 12: teste 1.114 1.155 1.539 0.775 0.953 1.184 0.949 0.758 950 13: negre 1.939 2.024 1.486 1.225 1.049 1.422 1.347 1.375 0.887 14: aporo 1.117 1.124 0.901 0.849 1.046 0.882 0.738 0.759 0.917 15: melan 1.336 1.335 1.493 1.322 1.510 1.526 1.630 1.265 1.609 16: perso 1.356 1.355 1.391 1.378 1.322 0.948 1.268 1.284 1.256 17: aphob 1.654 1.695 1.609 1.550 1.959 1.049 1.379 1.443 0.839 18: leuco 1.376 1.326 1.426 0.652 1.273 0.785 0.772 0.779 0.914 19: palma 1.293 1.348 1.524 2.336 1.081 1.408 5.332 2.444 2.072

(character 33) and male genitalia with a basal frequencies of 11 loci in the 19 species studied plate with a leaf-like seta (character 62)) and is (Table 4) produced genetic identity values (I) supported by a bootstrap value of <50%. that were used to construct the UPGMA Decamyia was proposed as a genus by Dyar dendrogram. This analysis resulted in an ar- (1919) and has been treated as a synonym of rangement similar to that of Spilonympha, Dendromyia since Edwards (1932) until Wyeomyia, Dendromyia, Phoniomyia, and the Harbach and Peyton (1990a) resurrected it as a genus Onirion in the cladistic analysis. In the subgenus of Wyeomyia. Although bootstrap allozyme analysis W. palmata and support for the Decamyia clade is weak W. aphobema appeared to be the most divergent (<50%), it is supported by a non-homoplastic species in the group, which partially agrees synapomorphy (basal plate with a leaf-like with the results of the morphological cladistic seta). Also, the results of the present analysis analysis. The I values between W. palmata and corroborate the monophyly of Decamyia and its other Wyeomyia species were low (0.005– placement as a subgenus of Wyeomyia. 0.339). As is noted in Table 5, the limits of I The current analysis also supports the values varied from one group to another; conse- monophyly of the subgenus Spilonympha (node quently, it is difficult to fix a range for each 31), corroborating the previous taxonomic sta- taxon. However, in reviews of the relationships tus of this group (Motta and Lourenço-de- of I values in the systematic diversity of differ- Oliveira 2005). The subgenus Spilonympha is ent groups, Thorpe (1982) and Thorpe and supported by four synapomorphies: larval seg- Solé-Cava (1994) concluded that among conge- ment VIII with a sclerotized plate (character neric species the usual range is 0.30–0.85. 24); absence of seta 2a-S (character 36); pres- Thus, the I values obtained for species of the ence of numerous accessory setae on the siphon subgenera Phoniomyia and Wyeomyia suggest (character 37); lobe C of the gonostylus some- that they belong to distinct groups. what ladle-like (character 57); and a broad Onirion personatum formed a cluster with interocular space (character 68). This arrange- W. melanocephala in the allozyme analysis, and ment is supported by a bootstrap value of 81%. the levels of identity observed between them Analysis of allozyme data from 19 species by were similar to those defined for congeneric means of the BIOSYS program yielded the species (I = 0.54). The internal organization of dendrogram shown in Figure 1. Allele Spilonympha shows that W. mystes is more

10 11 12 13 14 15 16 17 18 19 0.176 0.400 0.328 0.144 0.327 0.263 0.258 0.191 0.253 0.274 0.175 0.389 0.315 0.132 0.325 0.263 0.258 0.184 0.266 0.260 0.370 0.167 0.215 0.226 0.406 0.225 0.249 0.200 0.240 0.218 0.370 0.356 0.461 0.294 0.428 0.267 0.252 0.212 0.521 0.097 0.220 0.431 0.386 0.350 0.351 0.221 0.267 0.141 0.280 0.339 0.241 0.244 0.306 0.241 0.414 0.217 0.387 0.350 0.456 0.245 0.619 0.207 0.387 0.260 0.478 0.196 0.281 0.252 0.462 0.005 0.480 0.304 0.469 0.253 0.468 0.282 0.277 0.236 0.459 0.087 0.516 0.379 0.387 0.412 0.400 0.200 0.285 0.432 0.401 0.126 ***** 0.255 0.390 0.236 0.401 0.201 0.194 0.231 0.384 0.131 1.366 ***** 0.611 0.207 0.069 0.139 0.022 0.427 0.308 0.216 0.940 0.492 ***** 0.198 0.227 0.344 0.255 0.333 0.543 0.100 1.446 1.577 1.619 ***** 0.158 0.102 0.104 0.319 0.335 0.201 0.913 2.673 1.484 1.846 ***** 0.232 0.312 0.096 0.304 0.043 1.605 1.974 1.068 2.283 1.461 ***** 0.542 0.096 0.345 0.109 1.642 3.813 1.367 2.259 1.165 0.613 ***** 0.246 0.449 0.188 1.464 0.850 1.099 1.144 2.347 2.340 1.401 **** 0.334 0.110 0.958 1.177 0.611 1.094 1.190 1.064 0.800 1.098 ***** 0.006 2.029 1.533 2.306 1.606 3.150 2.214 1.671 2.208 5.150 ***** closely related to W. aningae (Fig. 1), as was ob- homoplasy and masks phylogenetic relation- served in the cladistic analysis (Fig. 6), and the ships. In the sabethines, we observed that level of identity among Spilonympha species var- those species that develop in bromeliads have ied from 0.43 to 0.98. Species of the subgenus similar morphological characteristics. Wyeomyia are included in a distinct cluster that is Phoniomyia species, which inhabit bromeli- separate from the remaining species included in ads, possess morphological features in the lar- the present study. The most closely related spe- val and pupal stages similar to those of cies were W. oblita and W. lutzi, which corrobo- bromeliad-inhabiting Wyeomyia species (e.g., rates the results of the cladistic analysis (Fig. 6). the subgenus Hystatomyia). Analysis of the allozyme data failed to resolve Our results also suggest that Cruzmyia, De- relationships among W. aporonoma, W. apho- camyia, Dendromyia, Spilonympha, and bema, W. leucostigma, and the remaining species Prosopolepis are monophyletic lineages included in the study, probably because of sam- within the genus Wyeomyia. In the present ple sizes. study, hypotheses concerning phylogenetic re- As has been observed by several authors (Dyar lationships within the genus Wyeomyia have and Knab 1906; Dyar 1919, 1928; Belkin et al. been proposed on the basis of synapomorphies 1970; Judd 1998b), knowledge of all life stages, that are unique or have the least amount of primarily the larva, is of considerable value for homoplasy. Based on the results of morpho- making phylogenetic inferences concerning taxa. logical and allozyme analyses, this study indi- However, problems with life-stage incongruence cates that, as currently defined, the genus have been observed since Dyar and Knab (1906). Wyeomyia is not a monophyletic lineage. We Judd (1998b) discussed this problem. Different propose two main changes in the current clas- life stages are subject to different environmental sification of the genus: (1) resurrection of sub- selection pressures that can account for observed genus Triamyia Dyar to include morphological differences (Judd 1998b); such as W. aporonoma and W. staminifera; (2) resur- in the sabethines, which are restricted to develop- rection of the subgenus Miamyia Dyar to in- ing and ovipositing in one type of habitat. clude seven species, W. codiocampa, W. lutzi, Significant structural modifications occur W. limai, W. serrata, W. hosautos, W. oblita, mainly in the larval stage and are probably influ- and W. sabethea. We also suggest that W. flui, enced by adaptation. This is a possible source of W. surinamensis, W. chalcoceplala, and

Wyeomyia sp. 4 belong to a distinct unnamed Journal of the New York Entomological Society, subgenus. The relationships of W. negrensis 14: 169–230. and W. argenteorostris varied, and for this rea- Dyar, H.G., and Shannon, R.C. 1924. Entomology: son they will continue to be without subgeneric the subfamilies, tribes, and genera of American assignment in the genus Wyeomyia. Culicidae. Journal of the Washington Academy of The present analysis of Wyeomyia is a start Sciences, 14: 472–486. toward resolving the evolutionary history of the Edwards, F.W. 1932. Diptera fam. Culicidae. In Gen- era Insectorum. Vol. 94. Edited by P. Wytsman. P. genus, a first step in establishing a natural clas- Wytsman Press, Desmet-Verteneuil, Brussels, Bel- sification. Moreover, there are numerous par- gium. tially described species without subgeneric Farris, J.S. 1969. A successive approximations ap- placement and several undescribed species. proach to character weighting. Systematic Zool- Also,better knowledge of the genus Wyeomyia ogy, 18: 374–385. will provide a footing for establishing relation- Farris, J.P. 1989. The retention index and the ships within the Sabethini. rescaled consistency index. Cladistics, 5: 417– 419. Felsenstein, J. 1985. Confidence limits on phylo- Acknowledgements genies: an approach using the bootstrap. Evolu- tion, 39: 783–791. The authors are grateful to Rodrigo Méxas of Harbach, R.E. 1991. A new subgenus of the genus the Instituto Oswaldo Cruz for technical sup- Sabethes (Diptera: Culicidae). Mosquito System- port with photography and to Instituto atics, 23: 1–9. Brasileiro do Meio Ambiente e dos Recursos Harbach, R.E., and Kitching, I.J. 1998. Phylogeny Naturais Renováveis for fieldwork authorization and classification of the Culicidae (Diptera). Sys- in several National Parks. M.A.M.S. is finan- tematic Entomology, 23: 327–370. cially supported by Fundação de Amparo à Harbach, R.E., and Knight, K.L. 1980. Taxonomist’s Pesquisa do Estado de São Paulo (grant glossary of mosquito anatomy. Plexus Press, 05/53973-0) and Conselho Nacional de Marlton, New Jersey. Desenvolvimento Cientifico e Tecnológico Harbach, R.E., and Peyton, E.L. 1990a. A new sub- (grant 472485/2006-7). Our thanks also go to genus in Wyeomyia (Diptera: Culicidae), with the the anonymous referees for their critical re- reclassification and redescription of the type species, Sabethes fernandezyepezi. Mosquito System- views. atics, 22: 15–23. Harbach, R.E., and Peyton, E.L. 1990b. Transfer of the subgenus Davismyia from Wyeomyia to References Sabethes and description of the type species, Belkin, J.N., Heinemann, S.J., and Page, W.A. 1970. Miamyia petrocchiae (Diptera: Culicidae). Mos- Mosquito studies (Diptera, Culicidae). XXI. The quito Systematics, 22: 149–159. Culicidae of Jamaica. Contributions of the Ameri- Harbach, R.E., and Peyton, E.L. 1992. A new subge- can Entomological Institute (Ann Arbor), 6:1– nus of Wyeomyia (Diptera: Culicidae), with the re- 458. classification and redescription of Wyeomyia Bonne-Wepster, J., and Bonne, C. 1921. Notes on (Davismyia) arborea, Wyeomyia (Dendromyia) South American mosquitoes in the British Mu- tarsata and Sabethes (Sabethes) carrilloi. Mos- seum. Insecutor Inscitiae Menstruus, 9: 1–26. quito Systematics, 23: 92–109. Bremer, K. 1994. Branch support and tree stability. Harbach, R.E., and Peyton, E.L. 2000. Systematics Cladistics, 10: 295–304. of Oniriun, a new genus of Sabethini (Diptera: Carpenter, J.M. 1994. Successive weighting, Culicidae) from the Neotropical region. Bulletin reability and evidence. Cladistics, 10: 215–220. of the Natural History Museum (Entomology), 69: Dyar, H.G. 1919. A revision of the American 115–169. Sabethini of the Sabethes group by the male geni- Hervé, J.P., Dégallier, N., Travassos da Rosa, A.P.A., talia. Insecutor Inscitiae Menstruus, 7: 114–142. Pinheiro, F.P., and Sá Filho, G.C. 1986. Dyar, H.G. 1924. Phoniomyia and Dendromyia Arboviroses — aspectos ecológicos. In Instituto Theobald (Diptera, Culicidae). Insecutor Inscitiae Evandro Chagas — 50 anos de contribuição às Menstruus, 12: 107–113. ciências biológicaseàmedicinatropical. Dyar, H.G. 1928. The mosquitoes of the Americas. Fundação Serviço de Saúde Pública, Belém, Part I. Carnegie Institution of Washington, Wash- Brazil. pp. 409–437. ington, D.C. Hjerten, S. 1961. Agarose as an anticonventional Dyar, H.G., and Knab, F. 1906. The larvae of agent in zone eletrophoresis. Biochemical Bio- Culicidae classified as independent organisms. physical Acta, 53: 514–517.

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