Morphology, molecules and fritillaries: approaching a stable phylogeny for Argynnini (Lepidoptera: Nymphalidae) THOMAS J. SIMONSEN, NIKLAS WAHLBERG, ANDREW V. Z. BROWER and RIENK de JONG Insect Syst.Evol. Simonsen, T. J., Wahlberg, N. , Brower, A. V. Z. & de Jong, R.: Morphology, molecules and fritillaries: approaching a stable phylogeny for Argynnini (Lepidoptera: Nymphalidae). Insect Syst. Evol. 37: 405-418. Copenhagen, December, 2006. ISSN 1399-560X. We examine the phylogenetic relationships among 29 species of Argynnini based on 141 pre- viously published morphological characters and new data from the mitochondrial gene COI and the two nuclear genes EF-1a and wingless. We investigate the stability and robustness of the resulting phylogenetic hypotheses through various combinations of the 4 functionally sep- arate datasets. Increasing the number of datasets in combined analyses led to increased sup- port for clades, sometimes substantially. We find that the tribe Argynnini is a well-supported, robust, monophyletic clade with the following internal subtribal structure: (Euptoietina (Yrameina (Boloriina Argynnina))). Our analyses support the classification of argynnine species into six robust and stable genera: Euptoieta, Yramea, Boloria, Issoria, Brenthis and Argynnis. We suggest that for moderate amounts of data, a total evidence approach is always best. Thomas J. Simonsen, Department of Entomology, Zoological Museum, University of Copen- hagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark* Niklas Wahlberg, Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden Andrew V. Z. Brower, Department of Biology, Middle Tennessee State University, Murfrees- boro, TN 37132, USA Rienk de Jong, National Museum of Natural History, Department of Entomology, PO Box 9517, 2300 RA Leiden, The Netherlands *Corresponding author. Email: [email protected] Introduction bining different kinds of data into a single analysis Researchers working on the evolutionary history is still debated, but it is now understood that sepa- of a group of species are interested in generating rate analysis of biologically delimited data sets is robust phylogenetic hypotheses for their taxon. A a way of investigating the strength of the phyloge- robust phylogenetic hypothesis can be seen as one netic signal apparent in the total evidence analysis that does not change when new characters are (DeSalle & Brower 1997; Gatesy et al. 1999). added to the data matrix. When inferring phyloge- Here we investigate the relative contributions of netic hypotheses, the combined analysis of data morphological characters and DNA sequences from various sources is commonly considered to from three gene regions to the pattern of phyloge- lead to the most robust hypothesis (Kluge 1989). netic relationships among butterflies in the tribe Since the molecular revolution in systematics, Argynnini of the subfamily Heliconiinae. We are large amounts of new data have been generated by specifically interested in the effects of adding new finding and sequencing new gene regions, al- molecular data to an already published morpho- though positive effects of combining morphologi- logical dataset (Simonsen 2006a). Do we really cal data with molecular data have been reported need to add data from 20 gene regions, as suggest- (Miller et al. 1997, Baker & Gatesy 2002, Wahl- ed by Rokas et al. (2003), to arrive at a robust phy- berg & Nylin 2003, Wahlberg et al. 2005). Com- logenetic hypothesis for this tribe of butterflies? © Insect Systematics & Evolution (Group 3 and 11) 406 Simonsen, T. J. et al. INSECT SYST. EVOL. 37:4 (2006) Phylogenetic assessments of relationships sified in 12 genera, and found that the enigmatic among various clades in the nymphalid subfamily genus Euptoieta was nested within the tribe Ar- Heliconiinae have been increasing over the last gynnini. Simonsen (2006a) found Euptoieta to be few years. The work of Penz & Peggie (2003) the sister-group to the rest of the Argynnini, and established a clear hypothesis for the higher level that there were additionally two distinct clades, diversification and relationships of the major line- which were termed the subtribes Yramiina and ages in the subfamily, although problems still exist. Argynnina. Based on a large morphological data set, Penz & In this contribution we present a phylogenetic Peggie (2003) divided Heliconiinae into four hypothesis of the Argynnini at the species group tribes: Acraeini, Heliconiini, Vagrantini and Ar- level, based on the adult genitalia and wing mor- gynnini. Although these clades did not receive phology from Simonsen (2006a) as well as the strong bootstrap support, they do conform to the mitochondrial gene cytochrome oxidase subunit I intuitive groupings used by many authors prior to (COI) and the nuclear genes elongation factor-1a that study. (EF-1a) and wingless. Most recent studies of relationships of taxa in Heliconiinae have concentrated on the tribe Heli- Material and methods coniini (Brower 1994; Brower 1997; Brower & Egan 1997; Penz 1999), but lately some attention Taxon sampling has been given to the mainly Holarctic tribe Ar- 29 ingroup taxa representing all major groups gynnini. Since the precladistic works of Warren within the tribe (17 “genera”) and 7 heliconiine (1944, 1955), Dos Passos & Grey (1945) and outgroup taxa used by Simonsen (2006a) are Warren et al. (1946) and the early cladistic or sys- included in the analysis of the morphological data- tematic works of Shirôzu & Saigusa (1973, 1975) set. A complete list of the species including speci- and Higgins (1975), only Aubert et al. (1996) have men data is given in Table 1. The morphological dealt with the group in a modern cladistic way, character matrix and character list can be found in using an outgroup and computer analyses. Simonsen (2006a). However, two morphology based phylogenetic studies of the tribe Argynnini and its subtribes Preparation and terminology have recently been concluded (Simonsen 2005, 2006a), and both morphology and DNA-based Morphological characters. – Preparation tech- phylogenetic studies of the Nearctic subgenus niques for morphological characters are found in Speyeria are in preparation (J. Dunford, pers. Simonsen (2006a). comm.). The only molecule based phylogenetic Taxonomy. – We initially adopt the argynnine study of the group so far (Aubert et al. 1996) was classification proposed by Simonsen (2006a) but limited to western Palaearctic taxa, and the results propose some changes based upon the current should be considered preliminary (H. Descimon, results. pers. comm.). No attempt to combine morphologi- cal and molecular data has hitherto been published Molecular characters. – We extracted DNA either for the tribe Argynnini. from one or two legs or from the thorax muscula- The Argynnini comprise 100+ species, histori- ture of freshly frozen, dried or alcohol conserved cally placed in up to 19 genera, although currently butterflies using QIAgen’s DNEasy extraction kit. placed in 6 genera (Simonsen 2006a). Almost all For each of the 36 species we sequenced COI, species are found in temperate, arctic and/or alpine EF-1a and wingless. Primers for COI were taken areas, mainly in the Palaearctic and Nearctic bio- from Wahlberg & Zimmermann (2000), for EF-1a mes. A few species are found in the high Andes of from Peña et al. (2006), and for wingless from South America, or the mountains of East Africa, Brower and DeSalle (1998). We performed all and a single species (Argynnis hyperbius) occurs PCRs in a 20-ml reaction volume. The cycling widely from Japan to Australia to Eastern Africa. profile for both COI and wingless was 95°C for 5 There are two previously published studies with min, 35 cycles of 94°C for 30 s, 47°C for 30 s, taxon sampling relevant to our study, both based 72°C for 1 min 30 s, and a final extension period on morphological data. Penz & Peggie (2003) of 72°C for 10 min. For EF-1a the cycling profile sampled 18 species of Argynnini which they clas- was 95°C for 7 min, 35 cycles 95°C for 1 min, INSECT SYST. EVOL. 37:4 (2006) Phylogeny of Argynnini 407 55°C for 1 min, 72°C for 2 min and a final exten- sen (2006a), 125 were parsimony informative with sion period of 72°C for 10 min. For all three gene- the current taxon sampling. regions the PCR primers were also used for 1450 bp from COI, 1240 bp from EF-1a and sequencing. Sequencing was done with a Beck- 400 bp from wingless were sequenced for all man-Coulter CEQ8000 capillary sequencer (Stock- species. The nucleotide base frequencies for each holm University) or an ABI Prism 377 DNA se- gene are given in Table 2. The AT bias for COI is quencer (University of Leiden). The resulting in accordance with the general AT bias for insect chromatograms were verified using the program mt genes (DeSalle et al., 1987; Liu & Beckenbach BioEdit (Hall 1999) and the sequences were 1992, Simon et al. 1994). The tree statistics for aligned by eye. The sequences are available on each individual data partition and the combined GenBank (Accession numbers in Table 1). sets (including tree lengths, number of trees and total PBS contribution) are given in Table 3. Phylogenetic analyses Almost all homoplasy is intrinsic to individual data partitions, rather than due to incongruence Phylogenetic analyses were carried out in TNT 1.0 among partitions when they are combined. Be- (Goloboff et al. 2003) using maximum parsimony tween 48% and 67% of tree lengths for the analy- and a heuristic search algorithm. Molecular data ses of individual