Appendix two

Although Ithomiini (98) and Danaini (101) have long been considered close relatives (Bates, 1862), few unambiguous characters have been discovered to support the grouping (de Jong et al. 1996, Freitas & Brown 2004). Brower’s (2000) analysis based on wg sequences was the first study to provide explicit empirical support for a sister group relationship between Ithomiini and Danaini, although the result was not stable under successive approximations weighting. The monophyly of Ithomiini with respect to Danaini is strongly corroborated by this analysis (BS 8; PCI 7.88) (monophyly of Danainae and the close relationship of Tellervini, Danaini and Ithomini was supported by molecular data in Brower (2000) and morphological characters in Freitas and Brown (2004) and will be addressed in greater detail elsewhere).

In general, the results presented here imply a very similar pattern of relationships to those presented by Brown (1985, 1987) and Brown & Freitas (1994), which were based on morphology of adult and immature stages. However, there are some notable differences, particularly in the branching order near the base of the cladogram.

Prior classifications have implied that the subtribe Tithoreina is the sister group to all other ithomiines, based on larval host preference for Apocynaceae and aposematic larvae with “danaoid” thoracic filaments (Brown, 1985, 1987; Ackery, 1988; Motta, 2003) (Freitas & Brown 2004). Brown & Freitas (1994) found Athesis + Patricia (and then Tithoreina) to be sister taxa to the remaining ithomiines. The molecular data imply that Melinaeina (97), composed of the genera Melinaea, Athyrtis, Olyras and Paititia, is the sister group to all other ithomiines. Known larval stages (from Melinaea, Athyrtis and Olyras) share with Tithoreina the putatively plesiomorphic “danaoid” features, but feed on Solanaceae (Brown & Freitas 1994).

The composition of the subtribe Melinaeina (97) implied by the molecular data is the same found by the combined morphological analysis of Brown & Freitas (1994; see Fig. 1A), with Paititia as the sister taxon of Olyras (96: BS 18; PCI 17.94). This result differs from the suggestion of Lamas (1979), who described Paititia as “very close to Thyridia” and placed the genus in Mechanitina, although he noted its similarity in a number of morphological structures to Olyras.

Aside from the exclusion of Thyridia + Methona (see below), the constitution of the well-supported subtribe Mechanitina (92: BS 13; PCI 13) implied by the molecular data (Mechanitis, Forbestra, Scada, Sais) is fairly traditional. Forbestra (87: BS 17; PCI 16.47)) is strongly supported as sister group to Mechanitis, but the support for monophyly of Mechanitis (89: BS1; PCI 1) is weak. This may represent an artifact of missing data or insufficient sampling, although Fox (1967) stated that they are closely-related when he removed the latter from the former. The subtribe Mechanitini including Thyridia is easily recognized by morphological characters of both adults and immatures, and the exclusion of Thyridia based on the present results should be examined in further detail.

Tithoreina is a well-supported clade (4: BS 8; PCI 8) composed of the genera Tithorea, Elzunia and Aeria. Fox (1956) placed Aeria in Oleriini, but it has since been recognized as belonging in a separate tribe based on larval morphology and host associations (Ackery, 1988; Brown & Freitas, 1994). Keith Brown (pers. comm.) has described Aeria eurimedia as “just a tiny Tithorea,” and the molecular results support his view.

The sister relationship of Methona and Thyridia has not been proposed before, although Brown & Freitas (1994), Motta (2003) and Lamas (2004) placed both genera in the Mechanitina clade. The two genera are so superficially similar in appearance that their nomenclature has been confused in the past (Fox (1956) and others called Methona “Thyridia” and Thyridia “Xanthocleis”). The sister relationship implied by these data (8) is fairly well-supported by the mtDNA (BS 5; PCI 0.47), although quite strongly contradicted by the nuclear genes. The pupae of Methona and Thyridia illustrated in Brown & Freitas (1994) appear to be dorsoventrally longer and less curvaceous than many other ithomiine pupae, which tend to have compressed abdomens and be bent at the thorax-abdomen junction. At the present, no morphological characters are known to support this clade, but if this sister-grouping is correct then the wing patterns, which have been considered as independently derived via mimetic convergence, may actually represent a synapomorphy. Another novel pattern suggested by these data but not supported by morphological evidence to date is the association of Methona + Thyridia with Tithoreina (9: BS 5; PCI 1.3), which is moderately well-supported, but again driven by the mtDNA and opposed by the nuclear genes.

Ithomiina is also a well-supported monophyletic group (82: BS 19; PCI 18.26), and is well-supported as the sister group of Napeogenina (83: BS 8; PCI 6.87), which is also well-supported (71: BS 5; PCI 3.56). Ithomiina consists of the genera Ithomia, Pagyris and Placidina. The latter two genera are strongly supported as sister taxa (81: BS 20; PCI 19.69) with Pagyris weakly paraphyletic with respect to Placidina. Thus, despite its morphological autapomorphies, the separation of Placidina into its own tribe does not seem warranted. A recent study of Ithomia (Mallarino et al., 2005, from which Ithomia eleonora and I. iphianassa were drawn for this study) provides much more detailed sampling within the genus, but does not address relationships between Ithomia and other members of the subtribe. Napeogenina is moderately well-supported: branches 62 (Epityches + “Rhodussa” cantobrica) and 65 (Epityches + Hyalyris + Hypothyris + “Rhodussa”) have among the lowest PCI scores (-9.8; -8.86, respectively) of any in the entire tree, with signal from mtDNA contradicted strongly by both nuclear genes. If Rhodussa is included in Hypothyris, as preferred by Lamas (2004), then this cladogram implies that Hypothyris is paraphlyletic with respect to both Hyalyris and Epityches. Future work will increase sampling in this part of the tree with the hope that denser representation of taxa will provide better support for the subtribe and its constituent genera.

Oleriina is a well-supported monophyletic group (60: BS 14; PCI 13.67) composed of the genera Hyposcada, Oleria, and the recently-described Megoleria and Ollantaya (even more recently synonymized; Lamas, 2004). Species of both of these newer genera were formerly placed in Oleria, but the current analysis implies that monophyly of Oleria is achieved by the removal, at least, of Megoleria. A more intensive exemplar study of Oleriina is in preparation (Whinnett et al., in press).

Dircennina in the broad sense is well-supported (53: BS 13; PCI 12.85), and although there is a basal split reflecting the separate tribes (Dircennina in the narrow sense and Ceratiniina) hypothesized by Harvey (1991), these are weakly supported as distinct from one another (37: BS 1; PCI –5, and 52: BS 1; PCI 1). All genera with more than one representative sampled are recovered as monophyletic except Ceratinia and Episcada, which are paraphyletic with respect to one another. Dircennina was not recovered as a monophyletic group in the morphological analyses of Brown & Freitas (1994).

Fox (1945) erected the genus Veladyris, which he placed in Godyridina, by removing a single species from its former position in the dircennine genus Velamysta. The current analysis supports Lamas’ recent classification (2004), implying that these two genera are sister taxa (28) and that both form a sister taxon to the remaining Godyridina (26). The latter divides subsequently into three well-supported groups: Heterosais + Hypoleria + Brevioleria + Mcclungia (15: BS 8, PCI 8), Godyris (16: BS 26, PCI 25.64), and the Greta (including “Hypomenitis”) + Pseudoscada complex (24: BS 7, PCI 7). Mcclungia is a genus comprising a single geographically variable species that our analysis implies is nested within a paraphyletic Hypoleria and should be synonymized. Brevioleria, another small genus recently erected by Lamas (2004) for species removed from Hypoleria, also renders Hypoleria paraphyletic. The relationships among Greta and Pseudoscada in Fig. 2 are complex, with neither genus appearing as monophyletic with respect to the other. Lamas (2004) has synonymized Hypomenitis into Greta, and this is another component of the tree that we intend to assess with increased taxon sampling to attempt to resolve relationships in greater detail. The genotype of Greta, G. diaphana (Drury), which is the only ithomiine species found in the Greater Antilles, has not yet been sampled.

Evolution of larval host plant affinities

The molecular data presented here provide a quite robust hypothesis of relationships for Ithomiini that provoke reconsideration of prior hypotheses of host shifts in ithomiines. Traditionally, Apocynaceae-feeding Tithoreina have been considered the sister taxon to the rest of Ithomiini, suggesting that the host shift from Apocynaceae to Solanaceae occurred once within the tribe (Brown, 1987; Brown et al., 1991; Brown & Freitas, 1994; Ackery, 1988). The current results imply that the use of Apocynaceae by Tithoreina is an independent shift back to the danaine host family, which is a hypothesis that warrants additional scrutiny in comparison with morphological evidence. By contrast, the aposematic coloration of larvae in Melinaeina, Athesitina (known from Athesis and Patricia; Brown & Freitas, 1994; Freitas, unpubl.) and Tithoreina appears to be a plesiomorphy shared with Danaini and Tellervini, and the shift to cryptic larvae lacking thoracic tubercles appears to be a synapomorphy for the “derived” subtribes (branch 70). We predict that the larvae of Eutresis and Athyrtis, when discovered, will also display aposematic coloration.

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