Phylogenetic analysis of Gigantodax (Diptera: Simuliidae) NELSY ROCÍO PINTO-SÁNCHEZ, DANIEL RAFAEL MIRANDA-ESQUIVEL and PAULINA MUÑOZ DE HOYOS.
Insect Syst.Evol.
Pinto-Sánchez, N. R. et al.: Phylogentic analysis of Gigantodax (Diptera: Simulidae) A study of the monophyly and phylogenetic relationships among the Gigantodax speciesgroups was conducted using all Gigantodax species and six outgroups. This analysis was conducted using 71 characters, 58 of them morphological characters derived from 66 species, and 13 of them cytological characters from 11 species. We conducted two analyses under linear parsimony and implied weights. To define the best concavity value we used the frecuency index. The value that recovered the most of the groups was linear parsimony search. We obtained 2037 equally parsimonious trees of 277 steps (CI=0.37 RI=0.65). The second value to recover most of the groups was implicit weight search with the concavity value of one. Only the igniculus and minor groups were recovered by both analyses (linear parsimony and implicit weights K=1). Under linear parsimony the phylogenetic analysis recovered two species groups (igniculus and cormonsi). The remaining four groups (“brophyi”, “cilicinus”, “minor”, and “wrighti”) are non-monophyletic. Based on the cladogram we suggest the following species groups: igniculus (igniculus group), cormonsi (cormonsi group), minor (containing “minor” + “multifilis”), brophyi (G. antarcticus, G. brophyi, G. femineus, G. rufidulus, and G. trifidus), shannoni (containing G. bettyae, G. incomitatus, G. mariobordai, G. pennipunctus, G. septenarius, and G. shannoni), and rufescens (containing G. aquamarensis, G. arrarteorum, G. basinflatus, G. cervicornis, G. cilicinus, G. clandestinus, G. corniculatus, G. cypellus, G. dryadicaudicis, G. herreri, G. horcotiani, G. impossibilis, G. incapucara, G. nasutus, G. rufescens, and G. wrighti). Some species, previously assigned to “cilicinus”, “wrighti”, and “brophyi” groups are kept as inquerandae. N. R. Pinto-Sánchez. Laboratorio de Biología Reproductiva de Vertebrados, Escuela de Biología, Universidad Industrial de Santander. A. A. 678, Bucaramanga, Colombia. D. R. Miranda-Esquivel*. Professor. Laboratorio de Sistemática & Biogeografía, Escuela de Biología, Universidad Industrial de Santander. A. A. 678, Bucaramanga, Colombia. (
[email protected]) P. Muñoz de Hoyos. Honorary Professor. Instituto de Ciencias Naturales. Facultad de Ciencias. Universidad Nacional de Colombia. (
[email protected]) * Corresponding author
Introduction Gigantodax Enderlein is a Simuliidae genus which has been assigned either to Prosimuliini (sensu Crosskey & Howard 2004) or to Simuliini (sensu Currie 1988; Moulton 2000). Currie (1988) recognized the monophyly of Simuliini, supported by five synapomorphies of the adult morphology: presence of calcipala, radial sector unbranched (or represented by an obscure apical fork), costa vein with dimorphic setae, “strap-like connection” between the paramere and the ventral plate of the male, arising subapically on the anterolateral apodeme of the ventral plate, and the paramere of male apically with spines. Moulton (2000) recog© Insect Systematics & Evolution (Group 6)
nized the monophyly of Simuliini on the basis of molecular information. Wygodzinsky & Coscarón (1989), based on morphological characters, assigned the species of Gigantodax to eight groups: cortesi, igniculus, minor, multifilis, brophyi, cilicinus, cormonsi, and wrighti. Although they considered each species group as monophyletic, they did not present a formal phylogenetic analysis. Subsequently, Coscarón & Miranda-Esquivel (1998) studied the phylogenetic relationships among the species of the cortesi group and the remaining groups of Gigantodax, showing that the cortesi group renders Gigantodax paraphyletic. Therefore, the cortesi
2 Pinto-Sánchez, N. R. et al. group was elevated to the generic rank as Pedrowygomyia, this genus being more related to Prosimulium Roubaud that to Gigantodax, while Gigantodax is more related to Cnesia Edwards, as proposed by Py-Daniel (1990) and Py-Daniel & Moreira (1994). Currently Gigantodax is formed by 62 species assigned to seven species groups (Coscarón & Miranda-Esquivel 1998) distributed from southern USA along the Mesoamerican Mountains through the Andes to Tierra del Fuego. Coscarón & Miranda-Esquivel (1998) defined the Gigantodax genus by the apical shape of the ventral plate strongly bilobed and the lack of the frontal trichomes in the frontoclypeus. The phylogenetic analyses have been based on morphological (Coscarón & Miranda-Esquivel 1998) or cytogenetical information for some species of Gigantodax (Coscarón-Arias 1998), but not on total evidence. There has been only one phylogenetic analysis (Coscarón & MirandaEsquivel 1998) of Gigantodax that has shown the monophyly of this genus and provided a hypothesis of relationship among the species groups. However, that study did not suggest any hypothesis of monophyly of the groups because it was based on the type species for the groups of Gigantodax. The objective of this study is to evaluate the monophyly of Gigantodax species groups and the relationships within these species groups, using morphological and cytogenetical evidence.
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racters 15 to 32), 8 on general adult morphology (characters 33 to 40), 10 on adult female morphology (characters 41 to 50), and 8 on adult male morphology (characters 51 to 58). We obtained the data set from the following literature sources: for Gigantodax groups, Pedrowygomyia, and Cnesia from Wygodzinsky & Coscarón (1989), Coscarón (1991), Muñoz de Hoyos (1995), and Coscarón & Miranda-Esquivel (1998); Simulium metallicum from Coscarón (1987); Simulium (Hearlea) from Coscarón et al. (2004); Gymnopais and Prosimulium from Rubtsov (1989), and Py-Daniel (1990). We also checked additional specimens (listed in appendix 1). The antenna color (character 4), the position of the maxillary palpal sensilla in the first-instar larva (character 6), the hypostomium paralateral teeth (character 7), and the accessory parameral spines (character 57) were coded following Currie (1988); while the apical shape of the ventral plate (character 54) was coded according to Coscarón & Miranda-Esquivel (1998). All measures and the quantitative characters were coded using gap coding (Mickevich & Johnson 1976). Here, we named two types of groups, the groups
Materials and methods Species studied. - We used 66 terminal taxa. 60 of these comprising the ingroup and six as the outgroup terminals (Gymnopais Stone, Prosimulium Robaud, Simulium metallicum Bellardi, Simulium (Hearlea) Vargas, Martínez Palacios & Díaz Nájera, Pedrowygomyia Coscarón & Miranda-Esquivel, and Cnesia Enderlein, leaving Gymnopais as the basal taxon). All the species of Gigantodax were included, except G. adleri Moulton, G. bierigi Vargas & Ramírez-Pérez, G. conviti Ramírez Pérez, G. lazoi Takaoka, Hirai & Tada, and G. willei Vargas & Ramírez-Pérez, because the information about them was not available. Morphological characters. - We used 58 morphological characters, 14 based on larval morphology (characters 1 to 14), 18 on pupal morphology (cha-
Fig. 1. Idiogram comparisons for chromosome I for species included in the chromosomic analysis. CYP: G. cypellus; BAS: G. basinflatus; BRE: G. brevis; OSO: G. osornorum; ORT: G. ortizi. Relative positions of markers are shown. C, centromere and NOR, nucleolar organizer region. Solid brackets on left indicate fixed inversions.
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Phylogeny of Gigantodax 3
Fig. 2. Idiogram comparisons of chromosome I for species included in the chromosomic analysis. PRO: Prosimulium; CNE: Cnesia; MAR: G. marginalis; CHIL: G. chilensis; FUL: G. fulvescens. Relative positions of markers are shown. C, centromere, 3HG, three heavy groups and NOR, nucleolar organizer region. Sexlinked inversions are indicated by dashed lines.
Fig. 3. Idiogram comparisons of chromosome II for species included in the chromosomic analysis. CYP: G. cypellus; BAS: G. basinflatus; BRE: G. brevis; OSO: G. osornorum; ORT: G. ortizi. Relative positions of markers are shown. C, centromere and PB, paraBalbiani. Solid brackets on left indicate fixed inversions.
nominated sensu Wygodzinsky & Coscarón (1989) and the groups nominated by us. The relationship among the groups were named with capital letters, formal definition awaits further research.
(1998); and Simulium metallicum map from Arteaga & Muñoz de Hoyos (1999). Following Dobigny et al. (2004) we considered the chromosomic structural changes as characters and the pattern observed before and after their occurrence, i.e., their presence or absence, as the character states, in order to avoid redundance. Chromosomal nomenclature and mapping conventions follow standard procedures (Gordon 1984). The landmarks used were: the paraBalbiani (PB), the nucleolar organizer (NO), and the three heavy band groups (3HG). (figures 1 to 6). Finally, we considered the multistate characters as non-additive. Characters, character states and codes are shown in Appendix 2, and the data matrix is shown in Appendix 3.
Chromosomal characters. - We used 13 chromosomal characters, three based on Chromosome I (characters 59 to 61), five on chromosome II (characters 62 to 66), three on Chromosome III (characters 67 to 69), and two on sexual differentiation (characters 70 and 71). The chromosomal maps were obtained from different sources: the maps of G. basinflatus Wygodzinsky & Coscarón, G. brevis Wygodzinsky & Coscarón, and G. cypellus Wygodzinsky & Coscarón from Muñoz de Hoyos (unpublished); G. ortizi Wygodzinsky map from Moreno (1990) and Muñoz de Hoyos (unpublished); G. osornorum Muñoz de Hoyos, Martínez, Mejía & Bueno map from Muñoz de Hoyos (1995) and Muñoz de Hoyos (unpublished); G. fulvescens Blanchard, G. chilensis Philippi, G. marginalis Edwards, Cnesia dissimilis, and Prosimulium mixtum maps from Coscarón-Arias
Cladistic analyses. - We conducted two phylogenetic analyses, linear parsimony search using NONA version 2.0 (Goloboff 1998) and implied weights search (Goloboff 1993) with different concavity values from one to six using TNT version 1.0 (Goloboff et al 2004). The tree search strategy, in NONA and TNT, was an heuristic
4 Pinto-Sánchez, N. R. et al.
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Fig. 4. Idiogram comparisons of chromosome II for species included in the chromosomic analysis. PRO: Prosimulium; CNE: Cnesia; SIM: Simulium metallicum; MAR: G. marginalis; CHIL: G. chilensis; FUL: G. fulvescens. Relative positions of markers are shown. C, centromere and PB, paraBalbiani. Solid brackets on left indicate fixed inversions. Sex-linked inversions are indicated by dashed lines.
Fig. 5. Idiogram comparisons of chromosome III for species included in the chromosomic analysis. CYP: G. cypellus; BAS: G. basinflatus; BRE: G. brevis; OSO: G. osornorum; ORT: G. ortizi. Relative positions of markers are shown. C, centromere and 3 HG, three heavy groups. Solid brackets on left indicate fixed inversions.
search using tree bisection reconnection (Swofford & Olsen 1990) and “ratchet” (Nixon 1999), randomizing the addition sequence 1000 times. Zerolength clades were collapsed to eliminate spurious trees (“rule 1”, Coddington & Scharff 1994; Swofford & Begle 1993). With the linear parsimony search, three different analyses were performed in order to evaluate the effect of quantitative and cytogenetic characters, the first with all the data, the second and third excluding the quantitative and cytogenetic data respectively. The number of the shared nodes was calculated as the amount of recovered nodes in regard to the total evidence topology. The unique node number was the number of nodes exclusively recovered by each one of the analyses after eliminating part of the evidence (cytogenetic or quantitative), rescaled to the number of nodes in the analysis with the partial data.
J
Fig. 6. Idiogram comparisons of chromosome III for species included in the chromosomic analysis. MAR: G. marginalis; CHIL: G. chilensis; FUL: G. fulvescens. Relative positions of markers are shown. C, centromere and 3HG, three heavy groups. Solid brackets on left indicate fixed inversions.
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Phylogeny of Gigantodax 5
Fig. 7. The strict consensus of 2037 equally parsimonious trees using linear parsimony search and total evidence. Initial trees of 277 steps (CI=0.37 RI=0.65). The conventions for the groups (abbreviations are given for the groups not genera) in the topology are: I (igniculus), M (minor), MM (multifilis), B (brophyi), Ci (cilicinus), W (wrighti) and Co (cormonsi). The group names used in accordance to Wygodzinsky & Coscarón (1989).Continued on fig. 8.
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Table 1. Average number of recovered nodes based on repeating ten runs, after Jackknife resampling with integer concavity values from one to six under implicit weights and linear parsimony analysis (EW). [* significance p110°) (Fig. 1a, W&C: 1989), (1) narrow (< 90°) (Fig. 17a, W&C:1989). Character # 6 in Coscarón & Miranda-Esquivel (1998:163). Wygodzinsky & Coscarón (1989) considered the state 0 as a diagnostic character for Pedrowygomyia. Coscarón & Miranda-Esquivel (1998) considered the character state 1 as a synapomorphy for Gigantodax + Cnesia. 43. R1, spinules; (0) without spinules (Fig. 2e, W&C: 1989), (1) with spinules (Figs. 24h, 41d, W&C: 1989). Character # 14 in Coscarón (1991:29). 44. Gonapophysis, border shape; (0) approximately straight (Figs. 1l, 41j, W&C:1989), (1) elongated distally (17o, W&C:1989). Character # 15 in Coscarón & Miranda-Esquivel (1998:163). Wygodzinsky & Coscarón (1989) considered the state 1 as a diagnostic character for igniculus group. 45. Spermatheca neck, surface; (0) not sclerotized showing a transluced area on the spermatheca apex (Fig. 17r, W&C:1989), (1) sclerotized extended until the duct base (Figs. 89q, 157s, W&C:1989). 46. Furcasternum mediam arm, diameter; (0) wide (Fig. 8p, W&C:1989), (1) narrow (Fig. 43bb, W&C: 1989). Character # 8 in Coscarón & MirandaEsquivel (1998:163).
22 Pinto-Sánchez, N. R. et al.
47. 48. 49. 50.
Coscarón & Miranda-Esquivel (1998) considered the state 1 as synapomorphy of the monophyly of the Gigantodax + Cnesia clade. Genital fork, length of the apodemes; (0) short (Fig. 5i, Coscarón 1991), (1) long (Fig. 27m, W&C: 1989). Claw, subbasal tooth; (0) absent or reduced (Fig. 1j, W&C:1989), (1) present (Fig. 27j, W&C:1989). Wygodzinsky & Coscarón (1989:8). Claw, transversal sulci of the tooth; (0) absent, (1) present. Wygodzinsky & Coscarón (1989:8) Claw, shape of the subbasal tooth; (0) spine like (Fig. 1j, W&C:1989), (1) hook like (Figs. 17m, 27j, W&C:1989), (2) subtriangular (Figs. 81q, 92d, W&C:1989), (3) subrhomboidal (Fig. 37p, W&C: 1989). Character # 14 in Coscarón & MirandaEsquivel (1998:163). Wygodzinsky & Coscarón (1989) found the state 1 as a shared diagnostic character for minor and igniculus group. According to Wygodzinsky & Coscarón (1989) the subbasal tooth shape could be derived from the subrhomboidal tooth shape present in the most plesiomorphic Neotropical Prosimuliini.
Males 51. Head, curvature. (In the ocellar portion); (0) softly pronounced (Fig. 9b, W&C:1989), (1) strongly pronounced (Fig. 38b, W&C:1989). Character # 20 in Coscarón & Miranda-Esquivel (1998:163). 52. Interocular space; (0) present (Fig. 2a, W&C:1989), (1) absent (Fig. 38a, W&C:1989). Wygodzinsky & Coscarón (1989) presented the character state 0 as diagnostic for Pedrowygomyia. 53. Interocular space, presence of a row of hairs; (0) without hairs (Fig. 38a, W&C:1989), (1) with a row of hairs (Fig. 2a, W&C:1989). Character # 19 in Coscarón & Miranda-Esquivel (1998:163). Coscarón & Miranda-Esquivel (1998) considered the state 1 as a synapomorphic character for Pedrowygomyia. 54. Ventral plate, apical shape; (0) smoothly bilobed (Figs. 81i, 95j, W&C:1989), (1) strongly bilobed (Fig. 38n, W&C:1989), (2) straight (Fig. 2l, W&C:1989). Character # 24 in Coscarón & Miranda-Esquivel (1998:163). Coscarón & Miranda-Esquivel (1998) considered the state 2 as a synapomorphy for Pedrowygomyia. 55. Endoparameres, hooks size; (0) short (Fig. 2q, W&C:1989), (1) elongated (Fig. 19d, W&C:1989). Character # 25 in Coscarón & Miranda-Esquivel (1998:163). Coscarón & Miranda-Esquivel (1998) considered the state 1 as synapomorphy for Gigantodax + Cnesia. 56. Aedeagal membrane; (0) without strong spinules (Fig. 24p, W&C:1989), (1) with strong spinules some of them basally fusioned (Fig. 14j, W&C: 1989). Character # 26 in Coscarón & MirandaEsquivel (1998:163). Wygodzinsky & Coscarón (1989) considered the state 1 as a diagnostic character for the igniculus and
INSECT SYST. EVOL. 36:2 (2005) minor groups. 57. Accessories parameral spines; (0) absent (Fig 16, Currie 1988), (1) present (Figs.19d, g, W&C:1989). Character # 61 in Currie (1989:85). According to Currie (1988) the typical condition for the paramere in Diptera is to be simple posteriorly (without spines), a feature shared by members of Parasimuliinae and Prosimuliini. The presence of parameral spines therefore must be derived; he defined it as a synapomorphy of Simuliini.
Cytogenetical characters Chromosome I 58. NO position; (0) NO in IL, (1) NO in IS, (2) NO in IIS. Character # 2 in Coscarón-Arias (1998:448). According to Moreno (1990) the NO is found in the Chromosome I for the Prosimuliini tribe, while in Simuliini tribe is found in whatever chromosome. 59. “Basal 3” relative position in relation to centromere; (0) proximal 3, (1) proximal 2, (2) medial 3. 60. Capsule (Cp) relative position in IS; (0) proximal 2, (1) proximal 3. 61. Fixed Inversion in IS-1; (0) absent, (1) present. 62. Centromeric region in chromosome I; (0) with synapses regions, (1) without synapses regions
Chromosome II 63. Relative position of RB; (0) proximal 3, (1) distal 1, (2) distal 2, (3) medial 1. Character # 5 in CoscarónArias (1998:448) 64. Relative position of b-RB; (0) centromere-b-RB, (1) centromere-RB-b 65. Relative position of Pb; (0) medial 3, (1) proximal 2, (2) medial 1, (3) proximal 3. Character # 7 in Coscarón-Arias (1998:448) 66. Position of Pb heavy band; (0) proximal to the centromere, (1) distal to the centromere. 67. Fixed inversion in IIS 1.2; (0) absent, (1) present. Character # 6 (Coscarón-Arias 1998:448). 68. Fixed inversion in IIL-1; (0) absent, (1) present. 69. Fixed inversion in IIL-3; (0) absent, (1) present. 70. Centromeric region in Chromosome II; (0) condensed, (1) expanded.
Chromosome III 71. Three heavy groups (3HG), position; (0) IS, (1) IIIL. 72. Fixed inversion in IIIL-1; (0) absent, (1) present. Character #8 (Coscarón-Arias 1998:448) 73. Fixed inversion in IIIL-2; (0) absent, (1) present.
Sexual differentiation 74. Sexual differentiation of males; (0) absent, (1) IL-2, (2) centromere II without synapses region, (3) IIL. Character # 3 in Coscarón-Arias (1998:448) 75. Sexual differentiation of females; (0) absent, (1) IL1, (2) IL-3, (3) IL-4, (4) centromere II with synapses region, (5) IIL. Character # 4 in Coscarón-Arias (1998:448).
Phylogeny of Gigantodax 23
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Appendix 3. Gymnopais Prosimulium Pedrowygomyia Simulium Hearlea Cnesia I_carmenae I_igniculus M_minor M_araucanius M_eremicus M_bolivianus MM_multifilis B_brophyi B_rufidulus B_antarticus B_trifidus B_femineus B_marginalis B_kuscheli B_luispenai B_chilensis B_flabellus B_awa B_paramorum B_vianamartinsi B_ortizi B_multituberculatus B_patihuaycensis B_laevigatus B_zumbahuae B_osornorum Ci_fulvescens Ci_shannoni Ci_destitutus Ci_basinflatus Ci_mariobordai Ci_incomitatus Ci_pennipunctus Ci_arranteorum Ci_clandestinus Ci_cilicinus Co_gracilis Co_misitu Co_brevis Co_wygodzinskyi Co_leonorum Co_abalosi Co_cormonsi Co_praealtus Co_vulcanius Co_siberianus W_horcotiani W_bettyae W_septenarius W_aquamarensis W_cervicornis W_corniculatus W_wrighti W_nasutus W_rufescens W_dryadicaudicis W_incapucara W_herreri W_impossibilis W_cypellus
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23
? 0 0 1 0 1 1 0 1 ? 1 ? 1 1 1 1 1 1 1 1 1 1 ? ? 1 ? 1 1 ? 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
? 0 0 1 0 0 0 0 1 ? 1 ? 1 2 1 1 2 2 1 1 1 1 ? ? 1 ? 1 1 ? 1 1 1 1 1 1 1 ? ? 1 0 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 2 1 0 2 1 0 1 1 1 1 1
? 0 0 0 0 0 0 0 1 ? 1 ? 0 0 0 0 0 0 0 0 0 0 ? ? 0 ? 0 0 ? 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
0 0 1 1 1 1 1 1 1 ? 1 ? 1 1 1 1 1 1 1 1 1 1 ? ? 1 ? 1 1 ? 1 1 ? 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
? ? 0 0 ? ? 0 0 1 ? 1 ? 1 0 0 0 0 ? 0 0 0 0 ? ? 0 ? 0 0 ? 0 0 0 0 1 0 0 ? ? 1 0 0 0 0 0 0 0 0 0 0 ? 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0
0 0 1 1 1 1 1 1 1 ? 1 ? 1 1 1 1 1 1 1 1 1 1 ? ? 1 ? 1 1 ? 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 1 1 1 1 1 1 1 ? 1 ? 1 1 1 1 1 1 1 1 1 1 ? ? 1 ? 1 1 ? 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 [01] 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [01] [01] [01] 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [01] 0 0
? ? 0 1 ? ? ? 1 0 ? ? ? 0 0 0 0 0 0 0 0 0 0 ? ? 0 ? 0 0 ? 0 0 0 0 0 ? 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 1 0 1 0 0 0 1 ? 1 0 ? 1 0 0
0 0 1 0 1 1 0 0 1 ? 1 ? 1 1 1 1 1 1 1 1 1 1 ? ? 1 ? 1 1 ? 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 1 0 1 1 1 0 0 0 ? 0 ? 0 0 0 0 0 0 0 0 0 0 ? ? 0 ? 0 0 ? 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
? ? 1 1 1 0 ? 1 0 ? 0 ? 0 1 1 1 1 1 1 0 1 1 ? ? 1 ? 1 1 ? 1 1 1 1 1 1 1 ? ? 1 0 0 1 1 1 0 0 0 1 1 ? 0 0 1 1 0 0 1 1 1 1 1 0 1 1 ? 1
0 0 1 0 0 0 1 1 1 ? 1 ? 1 1 1 1 1 1 1 1 1 1 ? ? 1 ? 1 1 ? 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 - 0 0 0 1 1 3 1 0 0 1 1 0 0 0 3 1 ? 0 1 1 0 1 0 0 1 ? 3 1 1 0 1 ? ? ? 1 3 1 1 3 1 1 0 1 1 0 1 1 0 1 1 3 1 1 3 1 1 3 1 1 3 1 1 0 1 ? 3 1 ? 3 1 1 3 1 ? 3 1 1 0 1 1 3 1 ? 1 1 1 3 1 1 0 1 1 ? 1 1 3 1 1 1 1 1 3 1 1 1 0 ? ? 0 ? 0 1 1 2 1 1 1 0 1 1 0 1 0 0 1 3 0 1 1 0 1 0 0 1 0 0 1 [01] 0 1 0 0 1 0 0 ? 0 0 1 0 0 1 0 0 1 0 1 1 0 0 1 2 0 1 0 1 1 0 1 1 0 0 1 3 0 1 0 0 1 2 1 0 1 0 1 0 0 1 2 1 1 0 0 1 0 1
0 0 0 0 0 0 0 0 0 0 0 0 2 0 3 0 0 0 - 0 4 0 0 0 0 0 0 0 0 0 0 0 0 ? ? ? 0 0 0 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 0 ? ? 0 2 0 0 2 0 0 2 0 0 2 0 0 0 0 0 0 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0 0 2 0 3 1 0 3 1 0 0 2 1 0 2 0 3 2 0 3 2 0 3 2 0 2 2 0 2 2 0 2 2 0 2 2 0 2 2 0 2 2 0 2 2 0 2 2 0 2 2 0 2 2 0 3 1 0 3 1 1 3 1 1 3 2 1 3 2 1 3 2 1 3 2 0 3 2 0 3 2 0 3 2 1 3 2 0 4 2 [01] 4 2 0 4
0 0 0 0 3 0 4 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4
0 2 3 0 0 2 3 3 0 0 0 ? 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 1 0 0 0 1 1 0 0 0 ? 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 ? 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 0 ? 1 1 1 1
0 0 0 0 0 0 0 0 0 0 0 ? 1 0 0 0 0 0 0 ? 0 0 0 0 1 ? 1 0 0 0 0 1 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 0 0 0 0 1 1 1 1 1 0 1
24 Pinto-Sánchez, N. R. et al.
INSECT SYST. EVOL. 36:2 (2005)
Appendix 3. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Gymnopais Prosimulium Pedrowygomyia Simulium Hearlea Cnesia I_carmenae I_igniculus M_minor M_araucanius M_eremicus M_bolivianus ? MM_multifilis 5 B_brophyi B_rufidulus B_antarticus B_trifidus B_femineus B_marginalis B_kuscheli ? B_luispenai B_chilensis B_flabellus B_awa B_paramorum 0 B_vianamartinsi ? B_ortizi 0 B_multituberculatus B_patihuaycensis B_laevigatus B_zumbahuae B_osornorum 0 Ci_fulvescens Ci_shannoni 0 Ci_destitutus 0 Ci_basinflatus 5 Ci_mariobordai Ci_incomitatus 0 Ci_pennipunctus 0 Ci_arranteorum 1 Ci_clandestinus 1 Ci_cilicinus 1 Co_gracilis 2 Co_misitu 3 Co_brevis 1 Co_wygodzinskyi 3 Co_leonorum 6 Co_abalosi 6 Co_cormonsi 4 Co_praealtus 4 Co_vulcanius Co_siberianus W_horcotiani 0 W_bettyae [02] W_septenarius [02] W_aquamarensis W_cervicornis W_corniculatus W_wrighti W_nasutus 5 W_rufescens 0 W_dryadicaudicis 3 W_incapucara 0 W_herreri 1 W_impossibilis W_cypellus 0
0 0 0 0 0 0 1 1 1 1 1 ? 1 1 1 1 1 1 1 ? 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0 1 1 1 ? 0 0 0 0 0 0 0 ? 0 0 0 0 0 ? 0 0 0 0 0 ? 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
? 0 0 0 2 1 2 1 0 ? 0 ? 1 0 0 0 0 0 0 ? 0 0 1 1 2 ? 1 1 1 1 1 0 ? 1 0 2 ? ? 0 0 0 0 1 1 1 1 1 0 1 ? 2 1 0 0 0 0 0 1 0 0 0 0 1 0 1 0
? ? 0 - ? 0 ? 1 ? ? 0 1 1 3 0 0 2 1 1 ? 1 2 1 1 ? ? ? ? ? 0 0 3 1 ? 0 0 2 1 1 0 ? ? 1 ? 1 ? ? ? ? 1 0 2 ? ? 1 ? ? ? ? ? 0 0 1 ? 1 0 2 1 1 0 0 2 1 1 0 0 3 1 2 0 0 3 1 2 0 0 ? 1 ? 0 0 2 1 1 0 ? ? ? ? ? 0 ? 1 ? 0 0 ? 1 ? 0 0 [02] 1 [01] 0 0 0 1 0 0 0 [02] 1 [01] 0 ? ? ? ? ? 0 2 1 1 0 0 0 1 0 0 0 ? 1 1 0 1 - 0 - 0 0 2 1 1 0 0 ? 1 ? 0 0 1 1 3 0 1 - 1 1 0 0 0 1 0 0 0 0 1 0 0 ? ? 1 1 0 0 2 1 1 0 0 3 1 2 0 0 ? ? ? 0 0 0 1 ? 0 0 3 1 2 0 1 - 1 1 0 0 0 1 0 0 0 1 1 0 0 0 0 1 0 0 0 0 1 0 0 0 ? 1 0 0 0 ? 1 1 0 0 ? 1 1 0 0 0 1 1 0 0 0 1 0 0 0 0 1 0 0 0 2 1 1 0 0 2 1 1 0 0 0 1 0 0 0 0 1 3 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 ? 0 1 - 0 - 0 ? 0 1 0 0 0 0 1 2 0 0 0 1 0 0 0 0 1 0 0
0 0 0 0 0 1 1 1 0 1 [01] 1 0 0 1 0 0 1 0 0 ? 0 0 1 0 0 1 0 0 1 ? ? ? ? ? ? 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 1 0 0 0 0 1 1 0 [01] 0 0 1 0 1 ? 0 0 1 ? ? ? 1 1 1 0 0 1 0 0 1 ? 0 ? 1 0 0 ? ? ? ? ? ? ? 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 1 0 0 1 0 0 [01] 1 0 0 0 ? 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 ? ? 0 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 ? 0 0 ?
0 1 0 1 1 1 ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? ? 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 1 ? 1 0 0 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 ? ? 1 1 ? ? ? ? 1 0 1 ? 1 ? 1 1 1 1 1 ? 1 1 ? ? 1 ? 0 0 1 1 0 0 1 ? 1 ?
0 0 1 0 0 0 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 1 0 0 0 ? 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 ? 0 ? 0 0 0 0 0 ? ? 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 -
0 0 0 0 1 0 0 0 0 1 1 0 0 1 1 1 1 0 ? 1 ? 1 ? 1 1 0 0 1 1 0 ? ? ? 1 1 0 1 1 0 1 0 0 1 1 0 1 0 0 1 0 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 0 1 ? ? 1 1 0 1 ? 0 ? ? ? 1 1 0 1 1 0 ? 1 ? 1 1 0 ? ? ? 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 ? ? ? ? ? ? 1 1 0 1 1 0 1 1 0 ? ? ? 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 [01] 0 1 0 1 1 1 0 1 1 0 1 1 0 1 1 0
? ? 0 0 0 0 ? 0 0 0 ? 1 1 1 0 0 0 0 0 0 0 1 0 0 0 ? 0 0 ? 0 ? 0 1 0 0 1 ? ? 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
0 0 0 1 1 1 ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 ? 1 ? 1 1 1 1 1 ? ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Phylogeny of Gigantodax 25
INSECT SYST. EVOL. 36:2 (2005)
Appendix 3. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 Gymnopais Prosimulium Pedrowygomyia Simulium Hearlea Cnesia I_carmenae I_igniculus M_minor M_araucanius M_eremicus M_bolivianus MM_multifilis B_brophyi B_rufidulus B_antarticus B_trifidus B_femineus B_marginalis B_kuscheli B_luispenai B_chilensis B_flabellus B_awa B_paramorum B_vianamartinsi B_ortizi B_multituberculatus B_patihuaycensis B_laevigatus B_zumbahuae B_osornorum Ci_fulvescens Ci_shannoni Ci_destitutus Ci_basinflatus Ci_mariobordai Ci_incomitatus Ci_pennipunctus Ci_arranteorum Ci_clandestinus Ci_cilicinus Co_gracilis Co_misitu Co_brevis Co_wygodzinskyi Co_leonorum Co_abalosi Co_cormonsi Co_praealtus Co_vulcanius Co_siberianus W_horcotiani W_bettyae W_septenarius W_aquamarensis W_cervicornis W_corniculatus W_wrighti W_nasutus W_rufescens W_dryadicaudicis W_incapucara W_herreri W_impossibilis W_cypellus
0 0 1 0 0 1 ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 ? 1 ? 1 1 1 1 ? ? 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 1 0 0 1 ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 ? 1 ? 1 1 1 1 1 ? ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 1 ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 ? 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 2 ? 1 1 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 ? 3 2 2 2 2 2 ? 2 2 2 ? 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 3 2 2 2 2 2
0 0 0 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 1 0 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 1 1 1 1 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? ? 0 0 ? 1 ? ? 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 ? 2 3 3 0 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 ? ? 0 0 0 0 ? 0 0 0 0 0 1 1 1 1 1 1 1 ? 1 1 0 0 0 ? 0 0 0 ? 0 ? 0 0 0 0
0 ? 0 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 1 1 0 1 1 1 1 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 1 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
? 0 ? 2 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 1 ? ? ? ? 0 ? ? ? ? 0 1 ? ? 0 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? 0 ? 0 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 0 ? ? 0 ? ? ? ? 0 ? ? ? ? 0 0 ? ? 1 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 1
? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0 ? ? 1 ? ? ? ? 0 ? ? ? ? ? 1 ? ? 0 ? ? ? ? ? ? ? ? 1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? ? ? 1 ? 1 ? ? ? ? ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? 1 ? ? ? ? 0 0 ? ? 0 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? 0 ? 0 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 1 ? ? ? ? 0 ? ? ? ? 0 1 ? ? 0 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? 0 ? 0 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 0 ? ? 0 ? ? ? ? 0 ? ? ? ? 0 0 ? ? 1 ? ? ? ? ? ? ? ? 1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? 0 ? 0 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 0 ? ? 0 ? ? ? ? 0 ? ? ? ? 0 0 ? ? 1 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 1
? ? ? 1 ? 1 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 1 ? ? ? ? 0 ? ? ? ? 0 1 ? ? 0 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? 0 ? 1 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 1 ? ? ? ? 1 ? ? ? ? 1 1 ? ? 1 ? ? ? ? ? ? ? ? 1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 1
? 0 ? 0 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 1 ? ? ? ? 0 ? ? ? ? 1 1 ? ? 0 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? 0 ? 0 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 1 ? ? ? ? 0 ? ? ? ? 0 1 ? ? 0 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
? 0 ? 3 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 1 ? ? ? ? 2 ? ? ? ? 2 1 ? ? 2 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
? 0 ? 5 ? 0 ? ? ? ? ? ? ? ? ? ? ? ? 1 ? ? 3 ? ? ? ? 4 ? ? ? ? 4 2 ? ? 4 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
26 Pinto-Sánchez, N. R. et al.
INSECT SYST. EVOL. 36:2 (2005)
