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APPENDIX TWO
PHYLOGENETIC RELATIONSHIPS AMONG THE ITHOMIINI (LEPIDOPTERA: NYMPHALIDAE) INFERRED FROM ONE MITOCHONDRIAL AND TWO NUCLEAR GENE REGIONS
Abstract

A phylogenetic hypothesis for the tribe Ithomiini (Lepidoptera: Nymphalidae: Danainae) is presented, based on sequences of the mitochondrial COI-COII region and regions of the nuclear genes wingless and Elongation factor I-alpha. The monophyly of the clade is strongly supported, as are many of the traditionally-recognized subtribes and genera. The data imply paraphyly of some genera and tribes, but largely support recent classifications and phylogenetic hypotheses based on morphological characters.

Appendix two has been accepted by Systematic Entomology.

Introduction

The Ithomiini are a diverse neotropical butterfly clade comprising more than 300 species in some 50 genera (Motta, 2003; Lamas, 2004). The group has been treated historically as a subfamily of Nymphalidae or a family in its own right, but it is currently considered (and is treated here) as a tribe within the nymphalid subfamily Danainae (Ackery et al. 1999), with concomitant reduction of traditional ithomiine and danaine tribes to subtribal status. The diagnostic synapomorphy for the group is the presence in adult males of patches of elongated androconial scales on the anterior margin of the hindwing. The monophyly of ithomiines with respect to other Nymphalidae was further supported by phylogenetic analysis of a short region of the wingless gene (Brower, 2000), and by three additional homoplastic morphological characters (extensive reflective areas on the pupa, relatively long antennae, saccus relatively long; Freitas & Brown, 2004). Danainae is also supported as monophyletic in the higher-level molecular study of Nymphalidae by Wahlberg et al. (2003).

As far as is known, ithomiine butterflies are chemically defended; most species acquire pyrrolizidine alkaloids from the nectar of Eupatorium (Asteraceae), Heliotropum (Boraginaceae) and other flowers, and do not sequester toxins from larval food plants, (Brower, 1984; Trigo et al., 1996; but see Freitas et al., 1996). Adult ithomiine butterflies are slow-flying and are generally considered to be aposematic (but see Kassarov, 2004). All members of the group are engaged in Müllerian mimicry rings with other ithomiines, danaines, heliconiines, day flying moths and various other insects (Bates, 1862; Brown, 1979; Beccaloni, 1997). The remarkable convergence of appearances between unrelated forms at a given locality, combined with geographical polymorphism within individual species, has resulted in a complex and confusing taxonomy, with over 1200 ostensibly valid described and undescribed subspecies, not to mention hundreds of additional synonyms, infrasubspecific names and nomina nuda (Lamas, 2004).

The phylogenetic position of Ithomiini within the Nymphalidae has been controversial. Prior to Bates (1862), all long-winged neotropical nymphalids (and even some Pieridae) were usually placed in “Heliconidae” a holdover from the Linnean subgeneric group “Heliconii” (Linné, 1758; Godart, 1819; Boisduval, 1836; Doubleday, [1847]). Recognizing that adaptive convergence and not phylogenetic affinity explained the similar wing patterns and body forms of “danaoid Heliconidae” and “acraeoid Heliconidae” (which included true Heliconius), Bates (1862) separated ithomiines from heliconiines and placed them in the “Danaidae.” At the same time, Felder & Felder (1862) independently discovered that the anastomosis of the first and second anal veins of the forewing unites Danaini and Ithomiini, a character that may represent one of the few morphological synapomorphies uniting the Danainae, albeit with several convergent origins in other nymphalid clades (Ackery & Vane-Wright, 1984). Harvey (1991) made note of several larval chaetotaxy characters that suggested an affinity between Danaini and Ithomiini (including one discovered by Müller (1886)), but had very limited material available for comparison. Ackery et al. (1999) list a behavioral trait, “adults imbibe pyrrolizidine alkaloids from damaged or withered plants, or from nectar, and use them to make sex pheromones and/or for defence” as the synapomorphy uniting Danainae in their analysis. Freitas & Brown (2004) improved significantly on this, listing four adult and ten larval/pupal morphological characters the define the Danainae (Ithomiini + Tellervini + Danaini),

R. M. Fox (1956) considered Ithomiini to be one of the most primitive nymphalid groups, and to be more closely-related to Satyrinae than Danaini, with particular affinity to Haeterini. Fox established the basis for the currently-accepted classification of Ithomiini, and engaged in comprehensive monography of a number of genera (Fox, 1949, 1956, 1960, 1967; Fox & Real 1971). Table 1 shows Fox’s (1956) and subsequent classifications of ithomiine genera. A few new genera have been split out of old larger genera in recent times (e. g., Brown & Freitas, 1994; Constantino, 1999), but the generic classification has remained relatively stable. Relationships among genera as implied by these classifications are much less stable, with some genera, such as Methona, Placidina, Epityches and Aeria assigned to different tribes in almost every different scheme.

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There are several recent hypotheses of relationships among ithomiine genera based on phylogenetic analyses of morphological features. Brown & Freitas (1994) published an exemplar study of 41 genera represented by characters from all life stages, rooted with the Australasian genus Tellervo. They obtained a well-resolved tree (Fig. 1A) that largely corroborates traditional tribal classification. An analysis by Motta (2003; Fig. 1B) based on morphology of first instar larvae bears some similarity to the Brown & Freitas hypothesis and to traditional classifications, but close scrutiny reveals that the two topologies share only three of a possible 22 components in common. Again, among the peripatetic taxa are Methona, Placidula and Aeria.

Molecular data may provide a valuable complement to morphological evidence, particularly in situations where groups are weakly supported or unstable in traditional analyses (Miller et al., 1997). In this paper, we analyze sequences of three gene regions to assess the relationships among ithomiine genera and test the monophyly of the various hypothesized subtribes. Sampling is quite complete, with only three small and rarely-collected genera (Eutresis, Aremfoxia, Haenschia) not represented. The resultant cladogram is well-supported and provides a robust framework for studying evolutionary patterns and processes in this intriguing group of butterflies. The relationship among Ithomiini, Danaini and Tellervini and the position of Danainae within Nymphalidae will be addressed in a separate paper.
Figure 1. Phylogenetic hypotheses for Ithomiini redrawn from (A, on left) Brown & Freitas (1994, Fig. 1c) and (B, on right) Motta (2003 fig. 19.5). The thick branches are the components shared in common between the two trees.



Materials and methods

Taxon sampling

The taxa examined in the current analysis are listed in Table 2. Ninety nine exemplars representing 41 ithomiine genera (and several recently synonymized genera, such as Rhodussa, Hypomenitis and Prittwitzia) and 81 species are included, as well as five outgroup genera representing the other two danaine tribes, Tellervini and Danaini. The three unsampled genera, Eutresis, Aremfoxia and Haenschia, are all small and considered to be closely-related to sampled genera (Athesis, Hypothyris, and Episcada, respectively). It is doubtful that their absence from the current analysis will have a major impact on the implied pattern of relationships, but we are sustaining our efforts to obtain representatives of as many taxa as possible.

Adult butterflies were netted in the field by the authors and numerous colleagues. Specimens were preserved either dry or in 95-100% EtOH (with wings removed and preserved dry, to preserve pigmentation). Voucher wings and abdomens were prepared as in Brower (1996) and are maintained by the first author (for ultimate deposition in the American Museum of Natural History), except as noted in Table 2. Sequences for six samples published by Wahlberg et al. (2003) and Mallarino et al. (2005) were downloaded from Genbank.
DNA extraction, PCR and sequencing

DNA was extracted from individual butterflies. In most cases, the head and thorax were ground up, while the abdomen and appendages were preserved as voucher material. In a few instances (rare species that are poorly represented in collections), DNA was extracted from two dried legs so the voucher specimen could be retained as a relatively complete pinned specimen. If the opportunity is available, we prefer to extract as much DNA as possible and preserve it indefinitely (frozen at –20° C). The first author retains DNA samples, but they will ultimately be deposited at the AMNH. DNA was purified using either SDS-phenol-chloroform extraction (see Brower, 1994) or DNAeasy kits (Qiagen), according to the manufacturer’s instructions, with an initial 3 hour incubation at 55C, and a final elution volume of 300 l.


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