Morphology, molecules and fritillaries: approaching a stable phylogeny for (: ) 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). Syst. Evol. 37: 405-418. Copenhagen, December, 2006. ISSN 1399-560X. We examine the phylogenetic relationships among 29 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: , , , , and . 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 in the tribe Since the molecular revolution in systematics, Argynnini of the subfamily . 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 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: , , 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 are in preparation (J. Dunford, pers. Simonsen (2006a). comm.). The only molecule based phylogenetic . – 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 partitions are due to homoplastic were equally weighted and unordered; morpholog- character state transformations, while the combi- ical data were coded as in Simonsen (2006a). nation of the three gene regions adds 1%, and the Heuristic searches were run with 1000 random- addition of the morphological data to the com- addition replicates using TBR branch swapping. bined DNA data adds another 1%. The four datasets were analysed separately and in combination. The effects of adding new data to a published phylogenetic dataset (Simonsen 2006a) Phylogenetic patterns in Argynnini was analysed by sequentially adding the molecular The analyses of each data partition both separate- datasets to the morphological dataset in all combi- ly and combined in various ways revealed stable nations, including a total evidence analysis with patterns of relationships (Figs 1-4). All analyses all four datasets combined in a single analysis. The recover the following clades: Euptoieta, Yramea, molecular data were also combined and analysed Boloria, Brenthis and the clade comprising all without the morphological data. Argynnini except Euptoieta. The only analysis in Robustness of the clades in the resulting clado- which the monophyly of the tribe Argynnini is not grams was evaluated with Bremer support values resolved is when COI is analysed on its own (Fig. (BS) (Bremer 1988; Bremer 1994). The scripting 1B). The monophyly of the subtribe Argynnina is feature of TNT was used to calculate BS values not resolved when morphology and the wingless (see Peña et al. 2006). The contribution of each are analysed separately (Figs 1A, D), nor when data partition to the BS values of the combined these two are combined (Fig. 2C), but the subtribe analyses was assessed using partitioned Bremer is found to be monophyletic in all other analyses. support (Baker & DeSalle 1997; Baker et al. 1998; Monophyly of Issoria is likewise unresolved in the Gatesy et al. 1999) using another script in TNT separate analyses of morphology and wingless (scripts available from N. Wahlberg). Basic se- (Figs 1A, D), but is found to be monophyletic in quence statistics and genetic distances were calcu- the combined analysis of these two partitions (Fig. lated in MEGA2 (Kumar et al., 2001). The distri- 2C), as well as in all other analyses. The genus Ar- bution of homoplasy within and among partitions gynnis (sensu Simonsen, 2006a) is not found to be was examined with the ILD statistic (Mickevich & monophyletic in the analysis of the EF-1 parti- Farris 1981). The Genbank accession codes for a tion (Fig. 1C), and its monophyly is unresolved in the gene sequences from the different species are the analysis of wingless (Fig. 1 D), and in the com- shown in Table 1. bined analyses of morphology+EF-1a and mor- phology+EF-1a+wingless (Figs 2B, F). Results The combined molecular dataset (1 tree, 3318 steps) recovers the Argynnini as monophyletic General (Fig. 3) and only differs from the total evidence Of the 141 morphological characters from Simon- analysis in the internal phylogeny of a subordinate 408 Simonsen, T. J. et al. INSECT SYST. EVOL. 37:4 (2006) with ZMUC: Y090137 Y090133 Y090135 (2006a) DQ922815 DQ922814 DQ922822 DQ922823 DQ922824 DQ922813 DQ922817 DQ922818 DQ922816 DQ922825 DQ922812 DQ922830 DQ922831 AF169921 DQ922810 DQ922811 DQ922829 A A DQ922809 DQ922828 DQ922820 DQ922819 DQ922821 wingless DQ922808 A DQ922838 herlands. Net Simonsen The a wing Y090166 Y090170 Y090168 numbers DQ922879 DQ922878 DQ922886 DQ922887 DQ922888 DQ922877 DQ922881 DQ922882 DQ922880 DQ922889 DQ922876 DQ922894 DQ922895 DQ922873 DQ922874 A DQ922875 A DQ922893 DQ922872 DQ922892 DQ922884 DQ922883 DQ922885 EF-1 DQ922871 A DQ922902 follo Leiden, , nera Accession History subge to Y090200 Y090204 Y090202 Natural DQ922847 DQ922846 DQ922854 DQ922855 DQ922856 DQ922845 DQ922849 DQ922850 DQ922848 DQ922857 DQ922844 DQ922862 DQ922863 DQ922841 DQ922842 A DQ922843 A DQ922861 DQ922840 DQ922860 DQ922839 DQ922852 DQ922851 DQ922853 A Genbank COI DQ922870 listed of taxa Museum Ingroup -BMNH -BMNH -ZMUC -ZMUC -ZMUC -RMNH -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -BMNH R R R R R R R R R R R R R R R R R R R R R R R R R R R + + + + + + + + + + + + + + + + + + + + + + + + + + + = = Museum ======collection = = = National analysis. RMNH: present Malaysia Africa Nigeria Nigeria enezuela V the Assam Romania Spain Japan East India Britain. Brazil in a a v v Denmark. anama, used Great Philippines, Nysaland, British Denmark Estonia Denmark Brazil Sikkim, Denamrk USA France, France, Nepal Nyasaland, Locality Russia, specimen Ja P Denmark China, Cuba, Denmark Kashmir Morphology Japan Ja Denmark Japan Hungary London, Rica Rica specimens A gisia Copenhagen, the anzania anzania anzania T China Russia Australia Finland Costa Costa Finland T USA Spain France Russia Russia Bangladesh Australia Japan Locality Sweden Japan T USA Japan DN specimen Sweden India Denmark Japan Kir of Museum, origin 1862) (Zoology), History and 1775) 1775) Feld., f., 1866 1775) , & f., 1775) 1767) y Denmark 1758) f., Natural species Schif 1763) f., of 1775) 1895) 1847) 1767) 1758) 1758) Schif 1771) & 1847 , 1875) 1758) , the 1831) 1758) (Feld. 1775) 1889 1775) 1773) 1773) Schif 1775) , & Schif 1777) , , of , & g, 1773) & 1775) BMNH: allas, , wes, (Linnaeus, ur list Motschulsk (Butler (Denis (Linnaeus, (P (Gray Museum abricius, , El (Linnaeus, (Moore, a a (Drury (Denis (Drury ersmann, (F abricius, abricius, (Linnaeus, (Linnaeus, (Linnaeus, bold. (Linnaeus, (Denis (Doubleday (Linnaeus, (Cramer eni (F (Denis Ev (Drury (F glaja in anadyomene a abricius, gdifer ruslana laodice (Rottemb History gynnina a haritonia pandor (F hildr adippe niobe kamala c hecale enia gana taxonomic c sinoe s.l Ar phalanta hyperbius paphia cydippe cyane osope cybele vanillae hecate ino daphne sa A ar names hanningtoni lathonia eug smar gynnis pr erymanthis a iulia oup ena 1. eus onome onome Natural aulis yeria briciana briciana briciana ble enthis enthis gyr enthis gynnis gyr gyr indula a a a andoriana a Issoria Br Issoria Issoria Issoria Issoria Childr Damor F Br Ar Spe Brenthis Br Dryas Heliconius Heliconius Ingroup Subtribe Argynnis Ar Ar Ar Agr P V Cethosia Cethosia Nephar Phalanta Outgr Cupha Cupha generic The Species T F F Mesoacidalia INSECT SYST. EVOL. 37:4 (2006) Phylogeny of Argynnini 409

Table 2. Empirical base frequencies (%) for the three molecular data sets in 36 species of Heliconiinae includ- ed in the analysis.

Y090134 Empirical base frequencies (%) DQ922827 DQ922834 A DQ922833 DQ922832 DQ922837 DQ922826 DQ922835 DQ922836 Gene A G C T COI 31.2 13.6 14.5 40.6 EF-1a 26.8 23.5 26.1 23.6 wingless 25.1 28.0 25.2 21.7 Y090167 DQ922897 DQ922896 DQ922891 DQ922901 DQ922890 A DQ922898 DQ922899 DQ922900 group within Argynnis, where A. pandora is found to be the sister taxon of a group comprising A. paphia, A. sagana, A. anadyomene, A. laodice, A. ruslana, A. hyperbius and A. childrena, in which (A. paphia A. sagana) in turn are found to com- Y090201 DQ922865 DQ922864 DQ922859 DQ922869 DQ922858 A DQ922866 DQ922867 DQ922868 prise the sister group of (A. anadyomene (A. lao- dice A. ruslana)). The total evidence analysis yielded one most parsimonious tree of 3724 steps (Fig. 4). Com- pared to the molecules only analysis, the support -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC -ZMUC R R R R R R R R R for almost all nodes in the total evidence tree is + + + + + + + + + ======increased, sometimes substantially (Fig. 5). Add- ing morphological data decreases the BS at only four nodes: BS for the two nodes within the Isso- ria clade is decreased by 3, support for the sister

Cuba relationship of A. hyberbius and A. childreni is

Switzerland decreased by 1, and the support for the sister rela- , Rica,

ay tionship of Argynnina and Bolorina is decreased

gentina by 1. There is a clear trend of increasing BS values Costa Switzerland Denmark USA Ar Peru Denmark Norw Denmark for the major clades (genera and subtribes) as additional characters are added to the combined analyses (Fig. 5). In the total evidence analysis (Fig. 4), the Ar- gynnini are monophyletic with high Bremer sup- Brazil Chile Peru Denmark USA Spain Italy Spain Sweden port (BS 15). The first split within the Argynnini is between Euptoieta and the remaining in-group genera. The latter clade is well supported (BS 20). Euptoieta is very strongly supported with a very high BS (50). Yramea is also very strongly sup- 1775)

1758) ported with a very high BS (87). The sister group f., 1775) of Yramea, a clade comprising Boloria and Ar- 1799) 1908) , f., 1779) 1775) gynnina, is fairly well supported with a BS of 7. Schif , , 1894) , 1773) & Boloria appears very strongly supported with a BS Schif , (Esper (Linnaeus,

& of 18. Within Boloria s.l., the position of B. euno- (Stichel, (Cramer (Cramer mia is unstable to method of analysis, being sister (Denis (Drury osyne to Boloria s. str. (BS 5). Boloria s. str. is strongly (Denis esia eunomia (Staudinger

g supported by a BS value of 37. euphr selene he claudia Euptoietina Boloriina Yrameiina The Argynnina is monophyletic and well sup- s.l. cytheris inca pales aquilonaris ported with a high BS of 10. Within Argynnina, Issoria s.l. is the sister group of a clade compris- oclossiana amea amea Euptoieta Subtribe Euptoieta Euptoieta Subtribe Yramea Yr Yr Pr Clossiana Boloria Clossiana Subtribe Boloria Boloria ing Brenthis and Argynnis. Issoria s.l. is well sup- 410 Simonsen, T. J. et al. INSECT SYST. EVOL. 37:4 (2006)

A B 5 Agraulis vanillae 3 Heliconius hecale 1 Dryas iulia Agraulis vanillae Heliconius charitonia Cethosia cyane 3 Vindula arsinoe Dryas iulia Cethosia cydippe Phalant a phalantha 4 Cupha prosope Phalant a phalantha 2 Vindula arsinoe 12 4 Euptoietina Euptoieta claudia Euptoietina 1 Euptoieta hegesia Issoria hanningtoni 3 42 Yramea cytheris 5 Yramea inca Yrameina 1 Brenthis hecate 37 Boloria selene 1 4 Boloria euphrosyne 2 Issoria lathonia 7 4 Boloria eunomia Boloriina Issoria smaragdifera 21 Boloria pales Issoria eugenia Boloria aquilonaris 5 Yramea cytheris 13 Brenthis hecate Argynnini Yrameina 3 1 1 Yramea inca 2 Brenthis ino Boloria eunomia 2 Brenthis daphne 5 3 Boloria pales 6 Issoria eugenia Boloria aquilonaris Boloriina 3 Issoria lathonia 2 Boloria selene 7 Issoria smaragdifera Boloria euphrosyne Issoria hanningtoni 4 1 Argynnis hyperbius Argynnis anadyomene 3 Argynnis anadyomene Argynnis pandora 9 Argynnis laodice 12 Argynnis paphia 4 Argynnis ruslana Argynnis sagana Argynnina 2 Argynnis childreni 4 Argynnis hyperbius 4 Argynnis aglaja 4 Argynnis childreni 1 Argynnis cybele 13 Argynnis laodice Argynnis pandora Argynnis ruslana 2 3 Argynnis paphia Argynnis aglaja 1 Argynnis sagana Argynnis cybele 7 Argynnis kamala 8 Argynnis kamala 5 Argynnis niobe 22 Argynnis niobe Argynnis adippe Argynnis adippe C D 6 Heliconius hecale 7 Heliconius hecale Agraulis vanillae Agraulis vanillae Dryas iulia Dryas iulia Cethosia cyane Cethosia cyane 3 Vindula arsino Vindula arsinoe 11 Cupha prosope 2 Cupha prosope Phalant a phalantha 1 Phalant a phalantha 3 12 Euptoieta claudia 12 Euptoieta claudia Euptoieta hegesia Euptoietina Euptoieta hegesia Euptoietina 27 Yramea cytheris Yrameina 5 Argynnis laodice 5 Yramea inca Argynnis ruslana Boloria eunomia Issoria lathonia 1 9 Boloria pales Issoria smaragdifera 7 1 Boloria aquilonaris Boloriina 1 Issoria hanningtoni Argynnini 19 Boloria selene Issoria eugenia Boloria euphrosyne Argynnis anadyomene 6 11 Issoria lathonia Argynnis pandora 1 Issoria eugenia Argynnini 1 Brenthis hecate 2 Issoria smaragdifera Brenthis ino Issoria hanningtoni 2 Brenthis daphne Argynnis pandora 1 Argynnis paphia 1 4 Argynnis paphia 1 Argynnis sagana 7 Argynnis hyperbius 2 Argynnis hyperbius 2 Argynnis childreni Argynnis childreni 5 Argynnis laodice Argynnis kamala 4 3 Argynnis ruslana 3 Argynnis niobe 1 Argynnis sagana Argynnina Argynnis adippe Argynnis anadyomene 2 Argynnis aglaja 9 Brenthis hecate Argynnis cybele 2 Brenthis ino 14 Yramea cytheris Yrameina 1 Brenthis daphne 2 Yramea inca Argynnis aglaja Boloria eunomia 7 Argynnis cybele 8 3 Boloria pales 5 Argynnis kamala 2 Boloria aquilonaris Boloriina 4 Argynnis niobe 9 Boloria selene Argynnis adippe Boloria euphrosyne

Fig. 1. Strict consensus trees from the separate analyses of the four datasets. A Morphology (8 trees, each 376 steps with CI = 0.41 and RI = 0.69), B COI (26 trees, each 2127 steps, CI = 0.36, RI =0.43), C EF-1a (4 trees, each 772 steps, CI = 0.52, RI = 0.68), D wingless (6 trees, each 388 steps, CI = 0.51, RI = 0.63). The numbers above the nodes are Bremer support values. ported by a high BS of 26. Within Issoria, I. euge- species I. smaragdifera and I. hanningtoni, a clade nia is the sister group of the remainder of the modestly supported (BS 5). The clade comprising genus, which is modestly supported with a BS of Brenthis and Argynnis has a fairly high BS of 8. 4. I. lathonia is the sister group of the two African Brenthis is well supported (BS 26). INSECT SYST. EVOL. 37:4 (2006) Phylogeny of Argynnini 411

A B 2 Dryas iulia 10 Agraulis vanilla e 1 Heliconius Dryas iulia 1 Agraulis vanilla e Heliconius Cethosia Cethosia Cupha Vindula arsinoe 15 Cupha Vindula arsinoe Phalanta phalantha 23 Euptoieta claudia 1 24 Euptoieta claudia 1 Euptoieta hegesia Euptoietina Euptoieta hegesia Euptoietina 47 Yramea cytheris 30 Yramea cytheris 1 Yramea inca Yrameina 5 Yramea inca Yrameina 36 Boloria selene Boloria eunomia 7 Boloria euphrosyne 5 20 Boloria selene 5 7 Argynnini 6 Boloria eunomia Boloriina Boloria euphrosyne Boloriina 26 Boloria pales Argynnini 11 Boloria pales Boloria aquilonaris Boloria aquilonaris 17 Brenthis hecate 3 15 Issoria hanningtoni 1 2 Brenthis ino 3 Issoria lathonia 1 Brenthis daphne Issoria smaragdifera 5 Issoria eugenia Issoria eugenia 1 Issoria lathonia 3 13 Brenthis hecate 1 Issoria smaragdifera 1 Brenthis ino 3 Issoria hanningtoni Brenthis daphne 6 Argynnis aglaja 7 Argynnis aglaja 1 Argynnis cybele 8 Argynnis cybele 18 Argynnis kamala 6 14 Argynnis kamala 29 Argynnis niobe Argynnina 8 Argynnis niobe Argynnina 10 Argynnis adippe Argynnis adippe 1 Argynnis hyperbius 2 Argynnis pandora 1 Argynnis childreni 6 Argynnis paphia 6 Argynnis anadyomene 2 Argynnis sagana 23 Argynnis laodice 6 Argynnis hyperbius 1 2 Argynnis ruslana Argynnis childreni 2 Argynnis pandora 2 Argynnis anadyomene 18 Argynnis paphia 16 Argynnis laodice C Argynnis sagana D Argynnis ruslana 13 Agraulis vanilla e 9 Agraulis vanilla e 1 Dryas iulia 1 Dryas iulia Heliconius Heliconius Vindula arsinoe Cethosia Cethosia 4 Vindula arsinoe 5 Cupha 18 Cupha Phalanta phalantha Phalanta phalantha 24 Euptoieta claudia 8 35 Euptoieta claudia 5 Euptoieta hegesia Euptoietina Euptoieta hegesia Euptoietina 77 2 Issoria hanningtoni 9 Yramea cytheris 4 Issoria lathonia Yramea inca Yrameina 5 1 Issoria smaragdifera 57 Boloria selene Issoria eugenia 14 8 Boloria euphrosyne 19 Yramea cytheris Argynnini 4 Boloria eunomia Boloriina 2 Yramea inca Yrameina 34 Boloria pales Argynnini 10 Boloria selene Boloria aquilonaris 2 15 Boloria euphrosyne 6 22 Issoria eugenia 1 Boloria eunomia 3 Issoria lathonia 6 Boloria pales Boloriina 5 Issoria smaragdifera Boloria aquilonaris Issoria hanningtoni 5 Brenthis daphne 8 24 Brenthis hecate 1 Brenthis hecate 5 Brenthis ino Brenthis ino Brenthis daphne 9 2 Argynnis hyperbius 5 Argynnis aglaja 1 Argynnis childreni 13 Argynnis cybele 7 Argynnis aglaja 25 Argynnis kamala 5 Argynnis cybele 33 Argynnis niobe Argynnina 3 Argynnis anadyomene 8 Argynnis adippe 9 Argynnis laodice 2 Argynnis pandora Argynnis ruslana 27 Argynnis paphia 5 1 Argynnis pandora Argynnis sagana 5 Argynnis paphia 12 Argynnis hyperbius 1 Argynnis sagana 2 Argynnis childreni 9 Argynnis kamala 7 Argynnis anadyomene E 9 Argynnis niobe F 29 Argynnis laodice Argynnis adippe Argynnis ruslana 9 Dryas iulia 20 Agraulis vanilla e 1 Agraulis vanilla e 4 Dryas iulia Heliconius Heliconius Cethosia Cethosi a Vindula arsinoe Vi ndula arsinoe 4 6 Cupha 5 18 Cupha Phalanta phalantha Phalanta phalantha 36 38 6 Euptoieta claudia 6 Euptoiet a c laudi a Euptoieta hegesia Euptoietina Euptoieta hegesi a Euptoietina 59 Yramea cytheris 44 Yramea cytheri s Yramea inca Yrameina 12 Yramea inca Yrameina 9 44 Boloria selene 28 Boloria selene 16 14 Boloria euphrosyne 14 Boloria euphrosyne 5 Boloria eunomia Boloriina Argynnini 1 Boloria eunomi a Boloriina 29 Boloria pales 14 Boloria pales Argynnini 9 Boloria aquilonaris Boloria aquilonari s Issoria lathonia 3 Issoria hanningtoni 9 18 Issoria eugenia 2 Issoria lathoni a 1 Issoria smaragdifera Issoria smaragdifera Issoria hanningtoni Issoria eugeni a 3 17 Brenthis hecate 3 14 Brenthis hecat e 1 Brenthis ino 1 Brenthi s i no Brenthis daphne Brenthis daphne 7 Argynnis hyperbius 9 Argynnis aglaj a 1 Argynnis childreni 12 Argynnis cybel e 7 1 Argynnis pandora 14 Argynnis kamal a 20 Argynnis paphia Argynnina Argynni s n iobe Argynnina 11 Argynnis sagana Argynnis adippe 5 Argynnis anadyomene 2 Argynnis pandora 23 Argynnis laodice 7 Argynnis paphi a Argynnis ruslana 2 Argynnis sagana 8 Argynnis aglaja 9 Argynnis hyperbius 5 Argynnis cybele 2 Argynnis childreni 18 Argynnis kamala 2 Argynnis anadyomene 32 Argynnis niobe 16 Argynni s l aodice Argynnis adippe Argynnis ruslana Fig. 2. Strict consensus trees from various combinations of the four datasets. A Morphology and COI (2 trees, each 2544 steps, CI = 0.36 and RI = 0.48), B Morphology and EF-1a (12 trees, each 1179 steps, CI = 0.47 and RI = 0.67), C Morphology and wingless (8 trees, each 778 steps, CI = 0.46 and RI = 0.66), D Morphology, COI and EF-1a (1 tree, 3327 steps, CI = 0.40 and RI = 0.53), E Morphology, COI and wingless (6 trees, each 2940 steps, CI = 0.38 and RI = 0.50), F Morphology, EF-1a and wingless (3 trees, each 1573 steps, CI = 0.48 and RI = 0.66). The numbers above the nodes are Bremer support values. 412 Simonsen, T. J. et al. INSECT SYST. EVOL. 37:4 (2006)

12 Agraulis vanilla e -3,11,4 1 Dryas iulia -0.5,1,0.5 Heliconius hecal e Cethosia cyane 2 Vindula a rsinoe 3.5, 14 Cupha prosope 4.5,-6 2,16,-4 Phalant a phalantha 11 34 Euptoieta claudia Euptoietina 6.5,7.5,-3 9,12,13 Euptoieta hegesi a 83 Yramea cytheris 10 41,36,6 Yramea inca Yrameina -1.5,7,4.5 63 Boloria selene 31.5,23, Boloria euphrosyne 15 13 8.5 1.5,2, -1,12,4 3 Boloria eunomia Boloriina Argynnini 9.5 4,0,-1 32 Boloria pales 20,9,3 Boloria aquilonaris 8 22 Issoria eugenia 2,5,1 6,13,3 7 Is soria lathonia 3,3,1 8 Iss oria smaragdifera 6,2,0 Iss oria hanningtoni 6 20 Brenthis hecate 2.5,1.5,2 11,8,1 5 Brenthis ino 1.5,3.5,0 Brenthis daphne Argynnina 4 3 Argynnis aglaja -3,5,2 15 0,0.7,2.3 Argynnis cybele 3,8,4 15 Argynnis kamal a 7,8,0 33 Argynnis niobe 3 25,4,4 5,-2,0 Argynnis adippe Argynnis pandora 2 16 Argynnis hyperbius -2,4,0 5,8,3 Argynnis childreni 1 15 Argynnis paphia -2,3,0 1 10,4,1 Argynnis sagana 0,1.50,-0.50 1 Argynnis anadyomene -2,3,0 19 Argynnis laodice 13,7,-1 Argynnis ruslana Fig. 3. The combined analysis of the three molecular datasets. The single most parsimonious tree (3318 steps, CI = 0.41, RI = 0.52). The numbers above the nodes are Bremer support values, whereas the numbers below the nodes are partitioned Bremer support values yielded by COI, EF-1a and wingless respectively.

Argynnis s. l. as defined by Simonsen (2006a) is Speyeria is well supported and the BS (11) is high. reasonably well supported and has a fairly high BS The subgenus is well supported and (8). The first split within Argynnis is between a the BS (25) is very high. The clade comprising the clade comprising the two subgenera Fabriciana remaining Argynnis is only moderately supported (represented by A. kamala, A. niobe and A. adippe) (BS 5). The basal split within this clade is between and Speyeria (represented by A. aglaja and A. cy- a clade comprising A. pandora, A. sagana and A. bele) and a clade comprising the remaining paphia and a clade comprising A. hyperbius, A. Argynnis. The Fabriciana+Speyeria clade is well childreni, A. anadyomene, A. laodice and A. rus- supported with a very high BS (17). The clade lana. The latter is poorly supported with a low BS comprising the two representatives of subgenus (2). Within this clade, A. hyperbius and A. chil- INSECT SYST. EVOL. 37:4 (2006) Phylogeny of Argynnini 413

16 Agraulis vanillae 6,-8,8,10 2 Dryas iulia 2,-3,3, 0 Heliconius Cethosia Vindula arsinoe 8 20 Cuph a 7,-0.50, -1,0,5, 4 11.5,2 Phalant a phalantha 2 50 Euptoieta claudia 16.5,8, 4,-5,-3, 6 Euptoieta hegesia Euptoietina 9,16.5 87 Yramea cytheris 15 6,40.5, Yramea inca Yrameina 7,-4,2,10 28.5,12 65 Boloria selene 20 18 3,35,19, 8 Bo loria euphrosyne 4,4,1, 9 5 Bo loria eunomia Argynnini 6,-3,7.5,9.5 Boloriina 1,4,0, 0 37 Bo loria pales 5,20,9, 3 Boloria aquilonaris 7 26 Issoria eugenia 0,2,5,0 -2,8,16, 4 4 Issoria lathonia -2,2,3, 1 5 Issoria smaragdifera -3,6,2, 0 Issoria hanningtoni 10 26 Brenthis hecate 4,4,1, 1 5,12,8, 1 5 Brenthis ino 0,2,3, 0 Brenthis daphne 11 Argynnis aglaja 8 Argynnina 17 7,2,0, 2 Argynnis cybele 2,-3,7,2 1,4,8, 4 25 Argynnis kamala 9.5,9.5,6, 0 37 Argynnis niobe 8 4,25,4, 4 Argynnis adippe 3,7,-2, 0 2 Argynnis pandora 5,-1 , 23 Argynnis paphia -2, 0 6.7,13.7, 5 1,1. 7 Argynnis sagan a 1.75,-0.75,4, 0 15 Argynnis hyperbius 2 -2,6,8,3 Argynnis childreni 3,-1,0,0 7 Argynnis anadyomen e 4,4,-1,0 29 Argynnis laodice 10,13,6, 0 Argynnis ruslana

Fig. 4. The combined analysis of all four datasets. The single most parsimonious tree (3724 steps, CI = 0.41, RI = 0.54). The numbers above the nodes are Bremer support values, whereas the numbers below the nodes are partitioned Bremer support values yielded by morphology, COI, EF-1a and wingless respectively. dreni comprise the sister group of A. anadyomene, and A. paphia is well supported with a BS of 24. A. laodice and A. ruslana. A. hyperbius and A. The positive partitioned total support values childreni form a well supported clade with a high show that all datasets contributed significantly to BS (15). The clade comprising A. anadyomene, A. the final result (Table 3). However, the datasets laodice and A. ruslana is well supported and the were conflicting or ambiguous at some nodes (Fig. BS (7) is fairly high. A. laodice and A. ruslana 4). Of 28 nodes, 12 were unanimously supported (subgenus Argyronome) form a strongly supported by all datasets. In 4 cases the three molecular clade (BS 29). The clade comprising A. pandora, datasets unanimously supported the node, whereas A. sagana and A. paphia is weakly supported, with the morphology was in conflict (3 of these nodes a BS of 2, while the clade comprising A. sagana are in the Issoria clade). There was no node for 414 Simonsen, T. J. et al. INSECT SYST. EVOL. 37:4 (2006)

increasing support with sample size

90

80

70

60

50 bremer support 40

30

20

10

0 amea r Y Issoria YBA Euptoieta otal Brenthis T Boloria clade Argynnini number of Argynnina Argynnis mean for 3 Bol+Arg partitions mean for 2 mean for 1 Fig. 5. The development of Bremer support values at major nodes as the number of partitions increase. Bol+Arg = Boloriina+Argynnina, YBA = Yrameina+Boloriina+Argynnina. which the three molecular datasets unanimously study we have shown that increasing the number yielded negative PBS. In one case (the position of of functionally separate datasets also increases the A. pandora as sister to A. paphia and A. sagana) stability and robustness of the resulting phyloge- only morphology supported a node (the molecular netic hypothesis. All four datasets have consider- datasets were either conflicting or ambiguous). able positive impact on the results of the combined The extremely small number of extra steps en- analysis. By sequentially adding the new molecu- tailed by combining the molecular data partitions, lar datasets to the already published morphologi- and subsequently the molecular and morphologi- cal dataset, we have been able to discover which cal partitions (Table 3), also gives a clear indica- clades are very stable (i.e. unlikely to change no tion of the high degree of congruence among these matter how much new data we add), which clades different sources of evidence. tend to stabilise as new data are added (Bremer supports increase substantially in combined analy- Discussion ses) and which clades are still relatively uncertain. When analysed separately, each of the datasets Combining data to increase support show incongruence, both among one another and The question of how much data is needed to arrive with the combined result. However, this is likely to at a robust phylogenetic hypothesis is still debated be caused by the intrinsic properties of each finite (Rokas et al. 2003, Gatesy & Baker 2005). In our dataset, i.e. the number of variable characters is INSECT SYST. EVOL. 37:4 (2006) Phylogeny of Argynnini 415

Table 3. Parameters for the individual and combined data partitions. D homoplasy is the additional steps due to incongruence among data partitions when they are combined. The total support is the sum of PBS values across all branches of the combined tree for a given partition. No. Variable Informative No. of Total Intrinsic D Total Data partition Char. Char. Chars Trees Tree homo- homo- support in length plasy plasy TE tree Morphology 141 140 125 8 376 222 (59%) 123.4 COI 1450 550 421 26 2127 1361 (64%) 202.4 Ef-1a 1240 305 225 4 772 371 (48%) 177.5 Wingless 400 136 112 6 388 190 (49%) 113.7 Comb. DNA 3090 991 233 1 3318 31 (1%) 493.6 Tot. evidence 3231 1131 248 1 3724 30 (1%) 617

limited and homoplasy is high (as in all of our comprises the sister group of the remaining taxa. datasets). Combining the various datasets allows The placement of Yramea as the sister group of the underlying phylogenetic signal to come out Boloria + Argynnina, however, conflicts with the (Gatesy et al. 1999, Baker & Gatesy 2002). In our earlier results. In Simonsen (2006a), Yramea was analyses, this phenomenon is striking in the total sister of Boloria, and these two genera were evidence analysis (Fig. 5). Recent studies have placed in a subtribe named Yrameina. However, suggested that incongruence between molecular according to our current results this name should datasets may be due to real differences in evolu- be reserved only for the genus Yramea. The inter- tionary histories of genes (e.g. Holland et al. 2004, nal phylogeny of Boloria, where B. eunomia is the Jeffroy et al. 2006). Such studies have taken ad- sister group of (B. pales B. aquilonaris) is in con- vantage of very large datasets that are not yet tract- flict with Simonsen (2006a) where B. eunomia able for many groups of organisms. Our study is was placed as the sister group of (B. selene B. typical of most studies today (about 3000 charac- euphrosyne), but is congruent with the morpholo- ters) and we suggest that in such cases a total evi- gy based analysis focused on Boloria by Simonsen dence approach is not only appropriate but also (2005). necessary. It is likely that instances of real incon- This analysis corroborates the hypothesis that gruence will only be reliably inferred with larger the subtribe Argynnina is monophyletic. However, numbers of independent datasets. in contrast to Simonsen (2006a), we found that the In our case, by combining morphological char- genus Issoria s.l. is monophyletic. In part due to acters and sequences from three genes, a more the absence of the ‘rectal plate’ in two African resolved, well-supported phylogenetic hypothesis species (I. baumanni and I. hanningtoni), Simon- is inferred than when any of the data partitions are sen (2006a) found that these two species formed analyzed alone. As shown in Fig. 5, the support for the sister group to the rest of Argynnina and they all higher clades increased as character sampling were thus placed in the genus Prokuekenthaliella. increased. Our study emphasizes the desirability Our results here suggest that the two African of analyzing large datasets to arrive at robust phy- species are nested well within Issoria. The rectal logenetic hypotheses. Single gene studies, particu- plate is a very complex structure associated with larly using short mitochondrial gene regions, the tegumen and uncus in Palaearctic Issoria and should be viewed with circumspection (Brower the African species I. smaragdifera (Simonsen 2006). 2006b), but absent in other Heliconiinae. Given the simple structure of the uncus and tegumen in I. hanningtoni and I. baumanni the present results Phylogeny seem to indicate that these structures (and hence Although the present results are largely in agree- the rectal plate) have been secondarily reduced in ment with those of Simonsen (2006a), some very these two species compared to other Issoria. important differences are obvious and deserve There is agreement that Brenthis form the sister attention. There is agreement that Argynnini are group of a monophyletic Argynnis s.l.. The genus monophyletic and that the subtribe Euptoietina Brenthis is well supported here though the internal 416 Simonsen, T. J. et al. INSECT SYST. EVOL. 37:4 (2006) relationships of the genus differ from Simonsen Simonsen (2006a) there is little justification for (2006a). the large number of generic names traditionally Monophyly of Argynnis s.l. is moderately well applied to various members of that group, and one supported, but the internal phylogeny of the genus large, unified genus Argynnis seems to be the only contradicts the results of Simonsen (2006a), where stable and “natural” solution for this problem. Not a clade comprising (A. hyperbius (A. anadyomne only is Argynnis as defined here well delimited (A. laodice A. ruslana))) is the sister group of the and easily recognized based on the highly special- remaining species in the genus. In our total evi- ized male alar androconials (Barth 1944), it is also dence analysis the first split in Argynnis is between supported in the morphological analysis, the com- a clade comprising the two subgenera Fabriciana bined molecular analysis and the total evidence and Speyeria (including Mesoacidalia) and a clade analysis. Additionally, no alternative division of comprising the remaining subgenera. Though con- Argynnis into two or three genera seems ideal. The tradicting Simonsen (2006a), a close relationship combined analyses do split Argynnis into two between Fabriciana and Speyeria was suggested reciprocally monophyletic groups. However, these by Penz & Peggie (2003) and here the clade is well two groups are contradicted by morphology and supported by all datasets. Although not found by not easily and immediately recognizable as a unit. Simonsen (2006a), the clade comprising the In summary, the tribe Argynnini is a well sup- remaining Argynnis bears some similarities to the ported monophyletic group comprising of the sub- previous results, but also conflicts with these. The tribes Euptoietina, Yrameina, Boloriina and Ar- clade comprising A. pandora, A. sagana and A. gynnina. Six monophyletic, well-supported, robust paphia is supported in both analyses and the sister and morphologically well-defined clades are group relationship between the latter two suggest- termed genera in this study. These are Euptoieta, ed by Simonsen (2006a) is strongly supported Yramea, Boloria, Issoria, Brenthis and Argynnis. here. The sister clade that here comprises A. We feel that this classification of Argynnini will be hyperbius, A. childreni, A. anadyomene, A. laodice stable to the addition of new data based on our and A. ruslana contradicts Simonsen (2006a) analyses in this paper. where A. childreni (and its sister species A. zeno- bia) is placed with the subgenus Speyeria. Correction added in proof However, Penz & Peggie (2003) placed A. chil- Just as the paper was being typeset, the authors became dreni with A. hyperbius. We agree with Simonsen aware that the Boloria euphrosyne specimen from which (2006a) that A. ruslana and A. laodice form a the DNA sequences were obtained was in fact a misiden- strongly supported clade and that their sister group tified, slightly aberrant, Boloria selene (identified by T. is A. anadyomene. J. Simonsen). This does not affect the results and conclu- sions, since the internal relationships of Boloria were outside the scope of the study. Classification Acknowledgements The present results necessitate three changes in the classification proposed by Simonsen (2006a). We are grateful to Dr. Steve Collins (African Research Institute) for providing us with specimens of Since the clade comprising Boloria and Yramea is Issoria hanningtoni and I. smaragdifera and to Toshiya not supported here we suggest that the name Hirowatari, Andrew D. Warren, James C. Dunford, Yrameina should be reserved for a subtribe com- Gerardo Lamas, Torben B. Larsen, Robert K. Robbins, prising only Yramea. Given its phylogenetic posi- and Francesca Vegliante for help in collecting specimens in the field. Rene Glas, University of Leiden, The tion, Boloria should be placed in its own subtribe. Netherlands for help with DNA extractions. Sören The name Boloriina (Warren 1944, Warren et al. Nylin, Carla M. Penz, Lars B. Vilhelmsen, Michael 1946) is available for this genus and should be Braby and Felix A. H. Sperling for reading and com- assigned to it. The inclusion of the re-established menting an earlier version of the manuscript. Niels P. Kristensen, Natural History Museum of Denmark genus Prokuekenthaliella (Simonsen 2006a) in (Zoology), University of Copenhagen, Denmark and Issoria removes the need for retaining Prokueken- Christoph Häuser, Staatsliches Museum fur Naturkunde, thaliella as a separate genus. Stuttgart, Germany for help and comments. Thanks also The fairly well supported clade comprising the to Carlos Peña for helping with the TNT script for doing PBS analyses. The Faculty of Science, University of Argynnis s.l. species supports the unification of all Copenhagen financed a PhD-grant for TJS during which the “larger fritillaries” in one genus. As argued by period most of the work on the manuscript was done, INSECT SYST. EVOL. 37:4 (2006) Phylogeny of Argynnini 417 and a grant from the SYS-RESOURCES program under vey of the Argynninae (Lepidoptera: Nymphalidae). the EU-commission gave him the possibility to visit the American Museum Novitiates 1296: 1-29. Natural History Museum in London and examine mate- Gatesy, J. & Baker, R. H. (2005) Hidden likelihood sup- rial in the collection. AVZB’s research was supported by port in genomic data: can forty-five wrongs make a NSF DEB 0089886 and the Harold and Leona Rice right? Systematic Biology 54: 483-492. Endowment for Systematic Entomology. Gatesy, J., O’Grady, P. & Baker, R. H. (1999) Corrobo- ration among data sets in simultaneous analysis: hid- References den support for phylogenetic relationships among higher level artiodactyl taxa. Cladistics 15: 271-313. Aubert, J., Barascud, B., Descimon, H. & Michel, F. Goloboff, P., Farris, J. & Nixon, K. (2003) T.N.T.: Tree (1996) Molecular systematics of the Argynninae Analysis Using New Technology. Program and docu- (Lepidoptera: Nymphalidae) (in French with English mentation available from the authors, and at summary). Comptes Rendus de l’Academie des Scien- www.zmuc.dk/public/phylogeny. ces Serie III Sciences de la Vie 319: 647-651. Hall, T. A. (1999) BioEdit: a user-friendly biological Baker, R. H. & DeSalle, R. (1997) Multiple sources of sequence alignment editor and analysis program for character information and the phylogeny of Hawaiian Windows 95/98/NT. Nucleic Acids Symposium Series drosophilids. Systematic Biology 46: 654-673. 41: 95-98. Baker, R. & Gatesy, J. (2002) Is morphology still rele- Higgins, L. G. (1975) The Classification of European vant? Pp 163-174 in DeSalle, R., Wheeler, W. & Giri- Butterflies. Collins, St. James’s Place, London. bet, G.: Molecular Systematics and Evolution: Theory Holland, B. R., Huber, K. T., Moulton, V. & Lockhart, P. and Practice. Birkhauser Verlag, Basel. J. (2004) Using consensus networks to visualize con- Baker, R. H., Yu, X. & DeSalle, R. (1998) Assessing the tradictory evidence for species phylogeny. Molecular relative contribution of molecular and morphological Biology and Evolution 21: 1459-1461. characters in simultaneous analysis trees. Molecular Jeffroy, O., Brinkmann, H., Delsuc, F. & Philippe, H. Phylogenetics and Evolution 9: 427-436. (2006) Phylogenomics: the beginning of incongru- Barth, R. (1944) Die männlichen Duftorganen einiger ence? Trends in Genetics 22: 225-231. Argynnis-Arten. Zoologischer Jarhbücher (Anatomie) Liu H, & Beckenbach A. T. (1992) Evolution of the 68: 331-362. mitochondrial Cytochrome Oxidase II gene among Bremer, K. (1988) The limits of amino acid sequence ten orders of . Molecular Phylogenetics and data in angiosperm phylogenetic reconstruction. Evol- Evolution 1: 41-52. ution 42: 795-803. Kluge, A. G. (1989) A concern for evidence and a phy- Bremer, K. (1994) Branch support and tree stability. logenetic hypothesis of relationships among Epicrates Cladistics 10: 295-304. (Boidae, Serpentes). Systematic Zoology 38: 7-25. Brower, A. V. Z. (1994) Phylogeny of Heliconius butter- Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001) flies inferred from mitochondrial DNA sequences. MEGA2: Molecular Evolutionary Genetics Analysis Molecular Phylogenetics and Evolution 3: 159-174. software. Bioinformatics 17: 1244-1245. Brower, A. V. Z. (1997) The evolution of ecologically Mickevich M. F. & Farris J. S. 1981. The implications of important characters in Heliconius butterflies (Lepi- congruence in Menidia. Systematic Zoology 30: 351- doptera: Nymphalidae): a cladistic review. Zoological 370. Journal of the Linnaean Society 119: 457-472. Miller J. S, Brower A. V. Z, & DeSalle R. (1997) Brower A. V. Z. (2006) Problems with DNA barcodes Phylogeny of the neotropical moth tribe Josiini (Noto- for species delimitation: “ten species” of Astraptes dontidae: Dioptinae): comparing and combining evi- fulgerator reassessed (Lepidoptera: Hesperiidae). dence from DNA sequences and morphology. Biolo- Systematics and Biodiversity 4: 127-132. gical Journal of the Linnean Society 60: 297-316. Brower, A. V. Z. & DeSalle, R. (1998) Patterns of mito- Peña, C., N. Wahlberg, E. Weingartner, U. Kodandara- chondrial versus nuclear DNA sequence divergence maiah, S. Nylin, A. V. L. Freitas & A. V. Z. Brower. among nymphalid butterflies: the utility of wingless as 2006. Higher level phylogeny of Satyrinae butterflies a source of characters for phylogenetic inference. In- (Lepidoptera: Nymphalidae) based on DNA sequence sect Molecular Biology 7: 73-82. data. Molecular Phylogenetics and Evolution 40: 29- Brower, A. V. Z. & Egan, M. G. (1997) Cladistic analy- 49. sis of Heliconius butterflies and relatives (Nymphal- Penz, C. M. (1999) Higher level phylogeny for the pas- idae: Heliconiiti): a revised phylogenetic position for sion-vine butterflies (Nymphalidae, Heliconiinae) Eueides based on sequences from mtDNA and a based on early stage and adult morphology. Zoologi- nuclear gene. Proceedings of the Royal Society of cal Journal of the Linnaean Society 127: 277-344. London Series B Biological Sciences 264: 969-977. Penz, C. & Peggie, D. (2003) Phylogenetic relationships DeSalle R. & Brower A. V. Z. 1997. Process partitions, among Heliconiinae genera based on morphology congruence and the independence of characters: (Lepidoptera: Nymphalidae). Systematic Entomology inferring relationships among closely-related Ha- 28: 451-479. waiian Drosophila from multiple gene regions. Syste- Rokas, A., Williams, B. L., King, N. & Carroll, S. B. matic Biology 46: 751-764. (2003) Genome-scale approaches to resolving incon- DeSalle, R., Freedman, T., Prager, E. M. & Wilson, A. C. gruence in molecular phylogenies. Nature 425: 798- (1987) Tempo and mode of sequence evolution in 804. mitochondrial DNA of Hawaiian Drosophila. Journal Shirôzu, T. & Saigusa, T. (1973) A generic classification of Molecular Evolution 26: 157-164. of the genus Argynnis and its allied genera (Lepi- Dos Passos, C. F. & Grey, L. P. (1945): A genitalic sur- doptera: Nymphalidae). Sieboldia 4: 99-104. 418 Simonsen, T. J. et al. INSECT SYST. EVOL. 37:4 (2006) Shirôzu, T. & Saigusa, T. (1975) The systematic position gistic effects of combining morphological and molec- of Argynnis argyrospilata, Kotzsch (Lepidoptera: ular data in resolving the phylogeny of butterflies and Nymphalidae). Sieboldia 6: 115-119. skippers. Proceedings of the Royal Society of London Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. Series B Biological Sciences 272: 1577-1586. & Flook, P. (1994) Evolution, weighting, and phylo- Wahlberg, N. & Nylin, S. (2003) Morphology versus genetic utility of mitochondrial gene sequences and a molecules: resolution of the positions of Nymphalis, compilation of conserved polymerase chain reaction Polygonia and related genera (Lepidoptera: Nymphal- primers. Annals of the Entomological Society of idae). Cladistics 19: 213-223. America 87: 651-701. Wahlberg, N. & Zimmermann, M. (2000) Pattern of Simonsen, T. J. (2005) Boloria phylogeny (Lepidoptera: phylogenetic relationships among members of the Nymphalidae): tentatively reconstructed on the basis tribe Melitaeini (Lepidoptera: Nymphalidae) inferred of male and female genitalic morphology. Systematic from mtDNA sequences. Cladistics 16: 347-363. Entomology 30: 653-655. Warren, B. C. S. (1944): Review of the classification of Simonsen, T. J. (2006a). Fritillary phylogeny, classifica- the Argynnidi: with a systematic revision of the genus tion and larval hostplants: reconstructed mainly on the Boloria (Lepidoptera: Nymphalidae). Transactions of basis of male and female genitalic morphology the Royal Entomological Society of London 94: 1-101. (Lepidoptera: Nymphalidae: Argynnini). Biological Warren, B. C. S. (1955): A revision of the classification Journal of the Linnaean Society 89: 47 pp. of the subfamily Argynninae (Lepidoptera: Nymphal- Simonsen, T. J. (2006b). The male genitalia segments in idae). Part 2. Definition of the Asiatic genera. Trans- fritillary butterflies: Comparative morphology with actions of the Royal Entomological Society of London special reference to the ’rectal plate’ in Issoria (Lepi- 107: 381-391. doptera: Nymphalidae). European Journal of Ento- Warren, B. C. S., Dos Passos, C. F. & Grey, L. P. (1946): mology 103: 425-432. Supplementary notes on the classification of Argynn- Wahlberg, N., Braby, M. F., Brower, A. V. Z., de Jong, inae (Lepidoptera: Nymphalidae). Proceedings of the R., Lee, M.-M., Nylin, S., Pierce, N., Sperling, F. A., Royal Entomological Society of London 15: 71-73. Vila, R., Warren, A. D. & Zakharov, E. (2005) Syner- Accepted for publication September 2006