The Radiation of Satyrini Butterflies (Nymphalidae: Satyrinae): A

Home , Altopedaliodes, Amathusia (butterfly), Amphidecta, Aphantopus, Arethusana, Argynnina (Satyrinae)

Zoological Journal of the Linnean Society, 2011, 161, 64–87. With 8 ﬁgures

The radiation of Satyrini butterﬂies (Nymphalidae: Satyrinae): a challenge for phylogenetic methods

CARLOS PEÑA1,2*, SÖREN NYLIN1 and NIKLAS WAHLBERG1,3

1Department of Zoology, Stockholm University, 106 91 Stockholm, Sweden 2Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Av. Arenales 1256, Apartado 14-0434, Lima-14, Peru 3Laboratory of Genetics, Department of Biology, University of Turku, 20014 Turku, Finland

Received 24 February 2009; accepted for publication 1 September 2009

We have inferred the most comprehensive phylogenetic hypothesis to date of butterflies in the tribe Satyrini. In order to obtain a hypothesis of relationships, we used maximum parsimony and model-based methods with 4435 bp of DNA sequences from mitochondrial and nuclear genes for 179 taxa (130 genera and eight out-groups). We estimated dates of origin and diversification for major clades, and performed a biogeographic analysis using a dispersal–vicariance framework, in order to infer a scenario of the biogeographical history of the group. We found long-branch taxa that affected the accuracy of all three methods. Moreover, different methods produced incongruent phylogenies. We found that Satyrini appeared around 42 Mya in either the Neotropical or the Eastern Palaearctic, Oriental, and/or Indo-Australian regions, and underwent a quick radiation between 32 and 24 Mya, during which time most of its component subtribes originated. Several factors might have been important for the diversification of Satyrini: the ability to feed on grasses; early habitat shift into open, non-forest habitats; and geographic bridges, which permitted dispersal over marine barriers, enabling the geographic expansions of ancestors to new environments that provided opportunities for geographic differentiation, and diversification.

‘To a man with a hammer, everything looks like a nail’. Mark Twain

ADDITIONAL KEYWORDS: Bayesian – biogeography – diversity – grasses – habitat shift – host plants – likelihood – parsimony.

INTRODUCTION Wahlberg & Freitas, 2007; Peña & Wahlberg, 2008), Papilionidae (Braby, Trueman & Eastwood, 2005; The evolutionary history of butterflies (Hesperioidea Nazari et al., 2007), and Pieridae (Braby, Vila & and Papilionoidea) has been largely a mystery. The Pierce, 2006; Wheat et al., 2007). Butterflies in the lack of robust phylogenetic hypotheses and a tempo- subfamily Satyrinae include some 2500 species of ral framework (Vane-Wright, 2004) has inhibited the worldwide distribution (Ackery, de Jong & Vane- study of aspects of the evolution of butterflies, such as Wright, 1999). Despite the high number of species biogeographical events and evolution of adaptive this group has been largely neglected: in particular, traits. It is only recently that studies using molecular there have been very few phylogenetic studies. The methods have provided time estimates for the origin only phylogenetic hypothesis available for the group and diversification of butterflies in the Nymphalidae as a whole reveals that Satyrinae as traditionally (Wahlberg, 2006; Kodandaramaiah & Wahlberg, 2007; construed is a polyphyletic entity in need of taxonomic revision (Peña et al., 2006). Peña et al. (2006) *Corresponding author. E-mail: [email protected] and Peña & Wahlberg (2008) show that the bulk of

Satyrinae species are included in one clade, the tribe Holarctic, Palaearctic (Dennis & Eales, 1997), and Satyrini, that encompasses approximately 2200 Oriental regions (Kodandaramaiah & Wahlberg, species. 2009), and in tropical habitats in Indo-Australia The species of Satyrini, now distributed worldwide, (Braby, 1993; Kodandaramaiah et al., 2009). Members began to diversify about 36 Mya, in the Late Eocene, of the subtribe Parargina occur in woodland, wooded almost simultaneously with the rise and spread of savannahs, and forests of Europe, North Africa, and grasses (Peña & Wahlberg, 2008). In a previous study temperate Asia (van Swaay, Warren & Loïs, 2006; (Peña & Wahlberg, 2008), we proposed that ancestral Konvicka et al., 2008). Maniolina is found in moist Satyrinae inhabited the ubiquitous dicotyledonous- meadows and forest edges (Billeter, Sedivy & Dieköt- dominated forests of the Paleocene and Eocene, ter, 2003) in the Palaeartics and Asia. The only genus feeding on early monocots and basal Poales. We in Melanargiina, Melanargia, has a Palaearctic dis- speculated that the mostly grass-feeding tribe tribution, inhabiting grasslands from Western Europe Satyrini was able to diversify, and spread throughout to Asia and North Africa (Vandewoestijne et al., 2004). the world, after shifting habitats from dicotyledonous The subtribe Mycalesina occurs in the understory of forests to the grasslands and savannahs (Peña & marginal forests and secondary vegetation in Africa Wahlberg, 2008) that have replaced vast areas of and Indo-Australia, and some taxa extend into tem- forest since the Oligocene (33–26 Mya) (Willis & perate Asia. Erebiina can be found in the Holarctic McElwain, 2002). Peña & Wahlberg (2008) did not region, mostly in alpine or arctic areas (Albre et al., draw major conclusions on the evolution of the lin- 2008). Satyrina is found in grassland habitats (van eages in the Satyrini because of limited taxon sam- Swaay et al., 2006) in Europe, Asia, and North pling: we only included 33 Satyrini species out of America, and some species are found in North Africa. 2200. In order to investigate the diversification of this Members of the Lethina (sensu Peña et al., 2006) are diverse and interesting group of butterflies, a denser found in woodland areas (Ide, 2002) and grasslands of taxon sampling is necessary to discover which factors Europe, Africa, Asia, and Indonesia, and some taxa are important in the spectacular radiation of Satyrini. are found in North America. The Ypthimina occurs in open area habitats such savannahs (Sourakov & Emmel, 1997) in Asia, New Caledonia, Africa, and DISTRIBUTION PATTERNS Indonesia. Satyrini butterflies are distributed worldwide, with the highest diversity found in tropical regions. The 13 Satyrini subtribes include 209 genera, with some HISTORICAL BIOGEOGRAPHY subtribes being almost entirely restricted to single The global distribution of Satyrini, combined with the biogeographical regions. These butterflies inhabit more restricted nature of most of the subtribes, sug- temperate and tropical habitats around the world: gests that a biogeographic study of the group could be occuring in most habitats from lowland savannahs informative for understanding the evolution and and rainforests to high-elevation cloud forests and radiation of other butterfly and insect groups. grasslands (páramos and puna; Viloria, 2003), alpine Miller (1968), in his important taxonomic revision and arctic areas in the Holarctic (Albre, Gers & Legal, of the entire Satyrinae, proposed an evolutionary tree 2008), tropical lowland habitats in the Oriental and for the higher taxa in the Satyrinae, and detailed a Australian regions (Braby, 1994), and grasslands and biogeographic scenario for the evolution of the group. woodlands in Africa (Fitzherbert et al., 2006). In a phylogenetic study of Pronophilina and the New The Pronophilina inhabits Andean cloud forest Zealand endemic Argyrophenga antipodum Double- environments from Venezuela to Bolivia, from day, 1845, Viloria (2003, 2007b) stated that his data 1400 m a.s.l. up to the border of the páramos at set supported a close relationship between southern 3200–3400 m a.s.l. (Pyrcz & Wojtusiak, 2002). The temperate pronophilines and Argyrophenga. Viloria subtribe Euptychiina was thought to be entirely (2003, 2007b) proposed that subtribes in the Satyrini restricted to the Americas (Miller, 1968; Murray & originated in Gondwana, and that after the break-up Prowell, 2005); however, there is mounting evidence of that land mass (c. 60 Mya) some members of Eup- that the Oriental Palaeonympha opalina Butler, 1871 tychiina and Hypocystina remained in South belongs to this subtribe, as suggested by morphologi- America. Later on, the Pronophilina diverged from cal characters (Miller, 1968) and molecular data the Hypocystina and colonized Mesoamerica and the (Peña et al., 2006). The euptychiines are distributed Caribbean Islands by 10–3 Mya. Unfortunately, in lowland and cloud forest habitats from central USA Viloria (2003, 2007b) based his biogeographical con- to Argentina (Murray & Prowell, 2005). The clusions on erroneous interpretations of his phyloge- Coenonymphina (formerly Hypocystina) includes rep- netic trees. In the caption of his figure 1, Viloria resentatives that inhabit oligotrophic mires in the (2003: 248) writes: ‘New Zealand Argyrophenga

© 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 161, 64–87 66 C. PEÑA ET AL. antipodum is included as the out-group, and its Satyrini genera (out of 209) from all subtribes in the closest species is the Chilean endemic Argyrophorus Satyrini, following the classifications of Miller (1968) argenteus’ (his tree had A. argenteus as the top-most and Peña et al. (2006). As there is no phylogenetic species of a monophyletic neotropical clade, simply hypothesis available for all genera in Satyrini, we appearing adjacent to A. antipodum). However, tried to include all currently recognized subtribes as a several observations suggest another interpretation. way to sample as many potential Satyrini lineages as All members of a monophyletic group are more closely possible. We also included eight Satyrinae taxa that related to one another than any member is to a taxon represent major lineages in the ‘satyrine’ clade as outside of the group. It would be true to state that the out-groups (sensu Wahlberg, Weingartner & Nylin, out-group at the root is most closely related if all 2003; Peña et al., 2006). All sequences have been possible out-groups were included, but presumably deposited in GenBank. Table 1 shows the current they were not. Moreover, the taxon at the root can be classification of sampled species and the GenBank arbitrarily replaced with any other taxon. This accession numbers. hypothesis would gain support if Satyrini fossils older than 65 Myr were found in current continents that used to be part of Gondwana. However, the scant MOLECULAR CHARACTERS fossil record of butterflies is not of much help, as only We extracted DNA from two butterfly legs, dried or four fossil species are assigned to the Satyrinae, and freshly conserved in 96% alcohol, using QIAGEN’s the oldest specimen is thought to be around 25 Myr in DNeasy extraction kit. For all species, we sequenced age (Grimaldi & Engel, 2005). One way to test the 1487 bp of the cytochrome oxidase subunit I gene Gondwanan hypothesis is by estimating the diver- (COI) from the mitochondrial genome, 1240 bp of the gence times of the Satyrini lineages by using inferred elongation factor 1a gene (EF-1a), 400 bp of the wing- molecular rates of character state change of extant less gene, 691 bp of glyceraldehyde-3-phosphate dehy- taxa. drogenase (GAPDH), and 617 bp of ribosomal protein By contrast, based on a molecular clock, the study S5 (RpS5) from the nuclear genome. We used the of Peña & Wahlberg (2008) hypothesized a post- hybrid primers for PCR amplification and sequencing Gondwanan origin for the Satyrini, at 36 Mya. Thus, from Wahlberg & Wheat (2008). Sequencing and the ancestor that eventually gave rise to the globally sequence alignment was performed following proto- distributed Satyrini inhabited an unknown drifting cols in Peña & Wahlberg (2008). The complete data fragment of either Gondwana or Laurasia. Although set consisted of 179 taxa and 4435 aligned nucleotide it is not known where Satyrini originated, its inferred sites. Of the 1487 bp sequenced for COI, 851 sites age suggests that its current intercontinental distri- were variable, and of these 665 were parsimony infor- bution is best explained in part by dispersal events. mative. The respective numbers for the other frag- Moreover, evidence is accumulating that dispersal ments were: EF-1a, 1240 bp, with 612 bp variable has been a prominent factor in the historical bioge- and 470 bp parsimony informative; wingless, 400 bp, ography of butterflies (Wahlberg, 2006; Kodandara- with 354 bp variable and 319 bp parsimony informa- maiah & Wahlberg, 2007). tive; GAPDH, 691 bp, with 318 bp variable and In this study, we use extensive taxon sampling of 272 bp parsimony informative; RpS5, 617 bp, 298 bp Satyrini subtribes, and related taxa as out-groups, to variable and 250 bp parsimony informative. generate a phylogenetic hypothesis for the subtribes in the Satyrini. We discuss the incongruent topologies retrieved by three phylogenetic methods, and choose a PHYLOGENETIC ANALYSES preferred hypothesis of relationships. We use the pre- We performed a maximum parsimony analysis, treat- ferred tree to study the evolution of habitat use, ing all characters as unordered and equally weighted. estimate dates of origin and divergence for major We performed heuristic searches with the software Satyrini clades, and perform a biogeographical analy- TNT 1.1 (Goloboff, Farris & Nixon, 2003), using a sis using a dispersal–vicariance analysis (DIVA), in level of search 10, followed by branch swapping of order to reconstruct the biogeographical history of the resulting trees, with up to 10 000 trees held during group. each step. The searches were performed using the New Technology Search algorithms of TNT. We ini- tially rooted the maximum parsimony analyses with Haetera piera (Linnaeus, 1758) (Haeterini) because it MATERIAL AND METHODS appeared to be sister of Satyrini in our recent study of TAXON SAMPLING Satyrinae (Peña & Wahlberg, 2008). However, we Our data set consists of 179 terminal taxa, including found long-branch attraction (LBA) artifacts between 171 Satyrini species encompassing 130 representative Euptychia and this out-group (see results). We

© 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 161, 64–87 01TeLnenSceyo London, of Society Linnean The 2011 © Table 1. Information of specimens used for molecular studies

Hostplant use, Subfamily Tribe Subtribe Species Code Source of Specimen COI EF-1a GAPDH RpS5 Wingless character state source

Morphinae Morphini Morpho helenor NW66-5 UK: London Pupae Supplies AY090210 AY090176 EU141507 EU141407 AY090143 0 Morphinae Amathusiini Amathusia phidippus NW114–17 INDONESIA: Bali DQ018956 DQ018923 EU141488 EU141384 DQ018894 0 Morphinae Brassolini Brassolis sophorae NW122-21 BRAZIL: São Paulo, EU528314 EU528291 GQ357384 EU528425 EU528270 0 Campinas Satyrinae Dirini Dirina Dira clytus CP15-04 S. AFRICA: W Cape EU528319 EU528296 EU528388 EU528432 EU528275 1 1 Paradise Coast Satyrinae Elymniini Elymniina Elymnias casiphone NW121-20 INDONESIA: Bali DQ338760 DQ338900 – EU141388 DQ338627 0 2 Satyrinae Haeterini Haetera piera CP01-84 PERU: Madre de Dios DQ018959 DQ018926 EU141475 EU141371 DQ018897 0 Satyrinae Melanitini Melanitis leda NW66-6 AUSTRALIA: Cairns, AY090207 AY090173 EU141508 EU141408 AY090140 0 2 Queensland Satyrinae Zetherini Zetherina Zethera incerta NW106-10 INDONESIA: Sulawesi DQ338776 DQ338918 EU141483 EU141379 DQ338635 0 Satyrinae Satyrini Parargina Chonala miyatai NW142-16 CHINA: N Sichuan, GQ357183 GQ357251 GQ357385 GQ357513 GQ357317 ? olgclJunlo h ina Society Linnean the of Journal Zoological Songpan env. Satyrinae Satyrini Parargina Kirinia climene CP10-08 IRAN: Lorestan GQ357184 GQ357252 GQ357386 GQ357514 GQ357318 1 3 Satyrinae Satyrini Parargina Kirinia roxelana CP10-09 IRAN: Lorestan DQ338767 DQ338908 GQ357387 GQ357515 DQ338615 1 3 Satyrinae Satyrini Parargina Lasiommata megera EW24-23 FRANCE: Languedoc DQ176351 GQ357253 GQ357388 GQ357516 DQ176326 1 3 Satyrinae Satyrini Parargina Lopinga achine EW3-6 SWEDEN DQ338769 DQ338910 GQ357389 GQ357517 DQ338617 1 3 Satyrinae Satyrini Parargina Pararge aegeria EW1-1 FRANCE: Carcassonne DQ176379 DQ338913 EU141476 EU141372 DQ338620 0 3 Satyrinae Satyrini Parargina Rhaphicera dumicola NW142-23 CHINA: N Sichuan, GQ357185 GQ357254 GQ357390 GQ357518 GQ357319 ? Songpan env. Satyrinae Satyrini Parargina Tatinga thibetana CP16-05 CHINA: N Sichuan, GQ357186 GQ357255 GQ357391 GQ357519 GQ357320 ? Songpan env.

Satyrinae Satyrini Mycalesina Bicyclus anynana EW10-5 ZIMBABWE: Harare AY218238 AY218258 EU141478 EU141374 AY218276 1 4 BUTTERFLIES SATYRINI OF RADIATION THE Satyrinae Satyrini Mycalesina Hallelesis halyma CP10-05 GHANA DQ338763 DQ338903 GQ357392 GQ357520 DQ338630 1 5 Satyrinae Satyrini Mycalesina Mycalesis terminus EW18-8 AUSTRALIA: Cairns DQ338765 DQ338905 EU528400 EU528446 DQ338632 1 6 Satyrinae Satyrini Lethina Aphysoneura NW117-22 TANZANIA: Lulando GQ357187 GQ357256 – GQ357521 GQ357321 1 pigmentaria Satyrinae Satyrini Lethina Enodia anthedon NW166-9 USA: VA, Chantilly GQ357188 GQ357257 GQ357393 GQ357522 GQ357322 1 Satyrinae Satyrini Lethina Lethe minerva NW121-17 INDONESIA: Bali DQ338768 DQ338909 EU141492 EU141387 DQ338616 1 Satyrinae Satyrini Lethina Neope bremeri EW25-23 TAIWAN: Hsiaokuehu DQ338770 DQ338911 EU528402 EU528448 DQ338618 0 7 Satyrinae Satyrini Lethina Ninguta schrenkii NW140-8 RUSSIA: Vladivostok distr. GQ357189 GQ357258 GQ357394 GQ357523 GQ357323 1 8 2011, , Satyrinae Satyrini Lethina Satyrodes eurydice CP11-01 USA GQ357190 GQ357259 GQ357395 GQ357524 GQ357324 1 Satyrinae Satyrini Coenonymphina Coenonympha myops CP16-22 IRAN: Golestan EU920741 EU920774 GQ357396 GQ357525 EU920805 1 Satyrinae Satyrini Coenonymphina Coenonympha pamphilus EW7-3 SWEDEN: Öland DQ338777 DQ338920 EU528385 EU528428 DQ338637 1 6

161 Satyrinae Satyrini Coenonymphina Coenonympha phryne CP16-21 RUSSIA: SW Siberia EU920739 EU920773 GQ357397 GQ357526 EU920804 1 8 Satyrinae Satyrini Coenonymphina Coenonympha saadi NW150-14 ARMENIA: Armavir marz, EU920758 EU920791 GQ357398 GQ357527 EU920819 1 8 64–87 , Vanand Satyrinae Satyrini Coenonymphina Coenonympha thyrsis UK4-2 GREECE: Psyloritis EU920761 EU920794 – – EU920822 1 3 Moutains Satyrinae Satyrini Coenonymphina Sinonympha amoena NW161-17 CHINA: Sichuan GQ357191 GQ357260 GQ357399 GQ357528 GQ357325 ? Satyrinae Satyrini Coenonymphina Altiapa decolor NW136-13 PAPUA NEW GUINEA: EU920737 EU920771 – GQ357529 EU920803 1 2 Simbu Prov. Satyrinae Satyrini Coenonymphina Altiapa klossi NW161-1 PAPUA NEW GUINEA: GQ357192 GQ357261 GQ357400 GQ357530 GQ357326 1 2 Kandep, Enga Prov Satyrinae Satyrini Coenonymphina Argynnina cyrila NW124-24 AUSTRALIA GQ357193 GQ357262 GQ357401 GQ357531 GQ357327 1 6 Satyrinae Satyrini Coenonymphina Argyronympha gracilipes NW136-1 SOLOMON ISLANDS: DQ338816 GQ357263 GQ357402 GQ357532 DQ338676 0 2 Guadalcanal Satyrinae Satyrini Coenonymphina Argyronympha ugiensis NW136-2 SOLOMON ISLANDS: San DQ338819 DQ338966 GQ357403 GQ357533 DQ338679 0 2 Cristobal Satyrinae Satyrini Coenonymphina Argyrophenga NW123-18 NEW ZEALAND DQ338821 DQ338968 GQ357404 GQ357534 DQ338686 1 antipodium 67 Table 1. Continued 68

Hostplant use, PEÑA C. Subfamily Tribe Subtribe Species Code Source of Specimen COI EF-1a GAPDH RpS5 Wingless character state source

Satyrinae Satyrini Coenonymphina Dodonidia helmsi NW123-15 NEW ZEALAND DQ338822 DQ338970 GQ357405 GQ357535 DQ338688 1 Satyrinae Satyrini Coenonymphina Erebiola butleri NW123-16 NEW ZEALAND DQ338823 DQ338971 GQ357406 GQ357536 DQ338689 1 TAL. ET Satyrinae Satyrini Coenonymphina Erycinidia gracilis NW161-2 PAPUA NEW GUINEA: Mt. GQ357194 GQ357264 GQ357407 GQ357537 GQ357328 1 2 Hagen Satyrinae Satyrini Coenonymphina Erycinidia virgo NW161-3 PAPUA NEW GUINEA: GQ357195 GQ357265 GQ357408 GQ357538 GQ357329 1 2 Kandep 01TeLnenSceyo London, of Society Linnean The 2011 © Satyrinae Satyrini Coenonymphina Geitoneura klugii RA64 AUSTRALIA: Tasmania GQ357196 GQ357266 GQ357409 GQ357539 GQ357330 1 6 Satyrinae Satyrini Coenonymphina Geitoneura minyas UK1-2 AUSTRALIA: WA, Perth GQ357197 GQ357267 GQ357410 GQ357540 GQ357331 1 6 Satyrinae Satyrini Coenonymphina Harsiesis hygea NW136-11 PAPUA NEW GUINEA: GQ357198 GQ357268 GQ357411 GQ357541 GQ357332 1 2 Morobe Prov. Satyrinae Satyrini Coenonymphina Hypocysta adiante KB339 AUSTRALIA: WA, EU920738 EU920772 GQ357412 GQ357542 – 1 6 Kimberley Satyrinae Satyrini Coenonymphina Hypocysta pseudirius NW123-5 AUSTRALIA: Newcastle DQ338826 DQ338974 GQ357413 EU528440 – 0,1 6 Satyrinae Satyrini Coenonymphina Nesoxenica leprea RA61 AUSTRALIA: Tasmania DQ338587 DQ338976 GQ357414 GQ357543 DQ338692 0 6 Satyrinae Satyrini Coenonymphina Oreixenica latialis UK1-20 AUSTRALIA: NSW, GQ357199 GQ357269 GQ357415 GQ357544 GQ357333 1 6 Tinderry mountians Satyrinae Satyrini Coenonymphina Oreixenica lathoniella UK1-6 AUSTRALIA: ACT, Mt. GQ357200 GQ357270 GQ357416 GQ357545 GQ357334 1 6 Gingeria Satyrinae Satyrini Coenonymphina Paratisiphone lyrnessa NW162-1 NEW CALEDONIA: Monis GQ357201 GQ357271 GQ357417 GQ357546 GQ357335 1 des Khogis Satyrinae Satyrini Coenonymphina Percnodaimon merula NW123-17 NEW ZEALAND DQ338829 DQ338978 GQ357418 GQ357547 DQ338694 1 Satyrinae Satyrini Coenonymphina Platypthima homochroa NW136-10 PAPUA NEW GUINEA: GQ357202 GQ357272 GQ357419 GQ357548 GQ357336 1 2 Morobe Prov. Satyrinae Satyrini Coenonymphina Platypthima ornata NW161-4 PAPUA NEW GUINEA: Mt. GQ357203 GQ357273 GQ357420 GQ357549 GQ357337 1 2 olgclJunlo h ina Society Linnean the of Journal Zoological Hagen Satyrinae Satyrini Coenonymphina Tisiphone abeona NW124-21 AUSTRALIA: Kulnura DQ338830 DQ338980 GQ357421 GQ357550 DQ338695 1 6 Satyrinae Satyrini Erebiina Erebia epiphron EW24-3 FRANCE: Languedoc DQ338778 DQ338921 – – DQ338638 1 6 Satyrinae Satyrini Erebiina Erebia ligea EW5-19 SWEDEN: Brottby, DQ338779 DQ338922 – – DQ338639 1 6 Vallentuna Satyrinae Satyrini Erebiina Erebia oeme EW24-7 FRANCE: Languedoc DQ338780 DQ338923 EU141479 EU141375 DQ338640 1 6 Satyrinae Satyrini Erebiina Erebia palarica EW9-4 SPAIN: Serra do Couvel AY090212 AY090178 GQ357422 GQ357551 AY090145 1 6 Satyrinae Satyrini Erebiina Erebia triaria EW9-1 SPAIN: Serra de Ancares DQ338782 DQ338925 – – DQ338642 1 6 Satyrinae Satyrini Euptychiina Amphidecta calliomma NW126-21 BRAZIL: Mato Grosso DQ338879 DQ339037 GQ357423 GQ357552 DQ338745 0,1 9 Satyrinae Satyrini Euptychiina Caeruleuptychia lobelia CP01-67 PERU: Madre de Dios DQ338788 DQ338930 GQ357424 GQ357553 DQ338648 1 Satyrinae Satyrini Euptychiina Cepheuptychia sp. nov. CP01-31 PERU: Madre de Dios DQ338789 DQ338931 GQ357425 GQ357554 DQ338649 0 Satyrinae Satyrini Euptychiina Chloreuptychia herseis CP01-72 PERU: Madre de Dios DQ338790 DQ338932 GQ357426 GQ357555 DQ338650 0 Satyrinae Satyrini Euptychiina Cissia myncea NW108-6 BRAZIL: São Paulo DQ338581 DQ338933 GQ357427 GQ357556 DQ338651 1 Satyrinae Satyrini Euptychiina Cyllopsis pertepida NW165-3 MEXICO: Guanajuato GQ357204 GQ357274 GQ357428 GQ357557 GQ357338 1 Satyrinae Satyrini Euptychiina Erichthodes antonina CP02-24 PERU: Madre de Dios DQ338792 DQ338935 GQ357429 GQ357558 DQ338653 0 Satyrinae Satyrini Euptychiina Euptychia enyo CP06-73 PERU: Cordillera del GQ357205 GQ357275 GQ357430 GQ357559 GQ357339 0 Cóndor Satyrinae Satyrini Euptychiina Euptychia sp. nov. 2 CP01-33 PERU: Madre de Dios DQ338794 DQ338937 EU528392 EU528437 DQ338654 0 Satyrinae Satyrini Euptychiina Euptychia sp. nov. 5 CP01-53 PERU: Madre de Dios DQ338795 DQ338938 GQ357431 GQ357560 DQ338655 0 2011, , Satyrinae Satyrini Euptychiina Euptychia sp. nov. 6 CP04-55 PERU: Mina Pichita DQ338796 DQ338939 GQ357432 GQ357561 DQ338656 0 Satyrinae Satyrini Euptychiina Euptychia sp. nov. 7 CP02-58 PERU: Quebrada Siete GQ357206 DQ338940 GQ357433 GQ357562 DQ338657 0 Jeringas

161 Satyrinae Satyrini Euptychiina Euptychoides castrensis NW126-9 BRAZIL: Ribeirão das DQ338798 DQ338942 GQ357434 GQ357563 DQ338659 1 Pedras 64–87 , Satyrinae Satyrini Euptychiina Forsterinaria boliviana CP04-88 PERU: Quebrada Siete DQ338799 DQ338943 GQ357435 GQ357564 DQ338660 0 10 Jeringas Satyrinae Satyrini Euptychiina Harjesia blanda CP01-13 PERU: Madre de Dios DQ338800 DQ338945 GQ357436 GQ357565 DQ338662 0 01TeLnenSceyo London, of Society Linnean The 2011 ©

Satyrinae Satyrini Euptychiina Hermeuptychia hermes NW127-16 BRAZIL: Extrema, MG DQ338583 DQ338946 GQ357437 GQ357566 DQ338663 1 11 Satyrinae Satyrini Euptychiina Hermeuptychia hermes CP01-07 PERU: Madre de Dios GQ357207 GQ357276 GQ357438 GQ357567 GQ357340 1 11 Satyrinae Satyrini Euptychiina Magneuptychia sp. nov. CP01-91 PERU: Madre de Dios DQ338584 DQ338947 – GQ357568 DQ338664 0 4 Satyrinae Satyrini Euptychiina Megisto cymela CP21-04 USA: Valley Falls GQ357208 GQ357277 GQ357439 GQ357569 GQ357341 1 11 Satyrinae Satyrini Euptychiina Oressinoma sorata CP06-89 PERU: Oxapampa GQ357209 GQ357278 GQ357440 GQ357570 GQ357342 0 Satyrinae Satyrini Euptychiina Oressinoma typhla CP07-71 PERU: Junín DQ338802 DQ338949 GQ357441 EU528452 DQ338666 0 12 Satyrinae Satyrini Euptychiina Paramacera xicaque CP15-08 MEXICO: Distrito Federal GQ357210 GQ357279 GQ357442 GQ357571 GQ357343 1 11 Satyrinae Satyrini Euptychiina Parataygetis albinotata CP04-53 PERU: Mina Pichita DQ338804 DQ338950 GQ357443 GQ357572 DQ338668 0 Satyrinae Satyrini Euptychiina Pareuptychia hesionides CP01-66 PERU: Madre de Dios DQ338805 DQ338951 GQ357444 GQ357573 DQ338669 0 Satyrinae Satyrini Euptychiina Pindis squamistriga NW165-5 MEXICO: GTO: Mpio. GQ357211 GQ357280 GQ357445 GQ357574 GQ357344 0 13 Penjamo Satyrinae Satyrini Euptychiina Posttaygetis penelea NW126-13 BRAZIL DQ338813 DQ338959 GQ357446 GQ357575 DQ338682 1 Satyrinae Satyrini Euptychiina Splendeuptychia itonis CP02-44 PERU: Madre de Dios DQ338811 DQ338957 GQ357447 GQ357576 DQ338684 1 Satyrinae Satyrini Euptychiina Taygetis virgilia NW108-3 BRAZIL: São Paulo DQ338812 DQ338958 EU141487 EU141383 DQ338683 0 12 Satyrinae Satyrini Euptychiina Yphthimoides cipoensis CP10-02 BRAZIL: Serra Do Cipó DQ338814 DQ338961 GQ357448 GQ357577 DQ338681 1 14 Satyrinae Satyrini Ypthimina Callerebia polyphemus CP16-19 CHINA: N Sichuan, GQ357212 GQ357281 GQ357449 GQ357578 GQ357345 1 olgclJunlo h ina Society Linnean the of Journal Zoological Songpan env. Satyrinae Satyrini Ypthimina Cassionympha cassius NW144-2 SOUTH AFRICA: W. Cape GQ357213 GQ357282 GQ357450 GQ357579 GQ357346 1 1 Satyrinae Satyrini Ypthimina Loxerebia saxicola CP16-06 CHINA: Shanxi, Tshingling GQ357214 GQ357283 GQ357451 GQ357580 GQ357347 1 Mts. Satyrinae Satyrini Ypthimina Neocoenyra petersi NW91-5 TANZANIA DQ338874 DQ339032 GQ357452 GQ357581 DQ338741 1 1 Satyrinae Satyrini Ypthimina Paralasa hades NW139-13 TADZHIKISTAN: GQ357215 – GQ357453 GQ357582 GQ357348 1 8 Turkestan Mt. Rng. Satyrinae Satyrini Ypthimina Paralasa jordana CP-AC23-35RUSSIA: Karasu DQ338597 DQ339027 EU532176 EU528455 DQ338736 1 8 Satyrinae Satyrini Ypthimina Paralasa styx CP11-05 UZBEKISTAN: W GQ357216 GQ357284 GQ357454 GQ357583 GQ357349 1 8 Tian-Shan

Satyrinae Satyrini Ypthimina Pseudonympha magus NW144-1 SOUTH AFRICA: E. Cape GQ357217 GQ357285 GQ357455 GQ357584 GQ357350 1 1 BUTTERFLIES SATYRINI OF RADIATION THE Satyrinae Satyrini Ypthimina Stygionympha vigilans NW144-5 SOUTH AFRICA: E. Cape GQ357218 GQ357286 GQ357456 GQ357585 GQ357351 1 Satyrinae Satyrini Ypthimina Ypthima baldus NW98-5 INDONESIA: Central DQ338875 DQ339033 EU528416 EU528469 DQ338742 1 Sulawesi Satyrinae Satyrini Ypthimina Ypthimomorpha itonia NW117-23 ZAMBIA: NW, Ikelenge DQ338878 DQ339036 GQ357457 GQ357586 DQ338744 1 5 Satyrinae Satyrini Maniolina Aphantopus arvensis NW148-16 CHINA: N Sichuan GQ357219 GQ357287 – GQ357587 GQ357352 1 Satyrinae Satyrini Maniolina Aphantopus hyperantus EW2-1 SWEDEN: Stockholm AY090211 AY090177 GQ357458 GQ357588 AY090144 1 3 Satyrinae Satyrini Maniolina Maniola jurtina EW4-5 SPAIN: Sant Climent, N AY090214 AY090180 EU141481 EU141376 AY090147 1 3 Spain 2011, , Satyrinae Satyrini Maniolina Maniola telmesia CP10-14 GQ357220 – GQ357459 GQ357589 – 1 3 Satyrinae Satyrini Maniolina Proterebia afra NW143-7 GREECE: Askion Mt GQ357221 GQ357288 GQ357460 GQ357590 GQ357353 1 3 Satyrinae Satyrini Maniolina Pyronia cecilia EW4-2 SPAIN: Sant Climent, N DQ338842 DQ338992 GQ357461 GQ357591 DQ338705 1 3

161 Spain Satyrinae Satyrini Melanargiina Melanargia galathea EW24-17 FRANCE: Languedoc DQ338843 DQ338993 EU528398 EU528444 DQ338706 1 3 64–87 , Satyrinae Satyrini Melanargiina Melanargia hylata CP10-10 IRAN: Ardabil DQ338844 DQ338994 GQ357462 GQ357592 DQ338707 1 Satyrinae Satyrini Melanargiina Melanargia lachesis NW149-3 FRANCE: Languedoc GQ357222 GQ357289 GQ357463 GQ357593 GQ357354 1 15 Satyrinae Satyrini Pronophilina Altopedaliodes sp. CP07-86 PERU: Cerro de Pasco GQ357223 GQ357290 GQ357464 – GQ357355 1 Satyrinae Satyrini Pronophilina Apexacuta astoreth CP09-78 PERU: S.N. Ampay DQ338846 DQ338996 GQ357465 GQ357594 DQ338709 1 Satyrinae Satyrini Pronophilina Argyrophorus sp. CP-C04 PERU GQ357224 GQ357291 GQ357466 GQ357595 GQ357356 1 Satyrinae Satyrini Pronophilina Auca barrosi RV-03-V39 CHILE: Céspedes DQ338832 DQ338982 – – DQ338697 0 16 Satyrinae Satyrini Pronophilina Auca coctei RV-03-V13 CHILE: Céspedes DQ338833 DQ338983 – – DQ338698 0 16 Satyrinae Satyrini Pronophilina Calisto pulchella DR003 DOMINICAN REPUBLIC: GQ357225 GQ357292 GQ357467 GQ357596 GQ357357 1 17 Puerto Plata Satyrinae Satyrini Pronophilina Cheimas opalinus CP17-06 GQ357226 GQ357293 GQ357468 GQ357597 GQ357358 0 18 Satyrinae Satyrini Pronophilina Chillanella stelligera CH-24A-1 CHILE: Termas de Chillán DQ338589 DQ338984 – – DQ338699 1 Satyrinae Satyrini Pronophilina Corades enyo CP04-06 PERU: Quebrada Siete GQ357227 GQ357294 GQ357469 GQ357598 GQ357359 0 Jeringas Satyrinae Satyrini Pronophilina Cosmosatyrus CH-15-5 CHILE: Cordillera DQ338834 DQ338985 –––1 69 leptoneuroides Nahuelbuta 70 .PEÑA C.

Table 1. Continued

Hostplant use, Subfamily Tribe Subtribe Species Code Source of Specimen COI EF-1a GAPDH RpS5 Wingless character state source AL. ET

Satyrinae Satyrini Pronophilina Daedalma sp. CP13-05 ECUADOR: Prov. DQ338848 DQ338998 GQ357470 GQ357599 GQ357360 0 19

01TeLnenSceyo London, of Society Linnean The 2011 © Tungurahua Satyrinae Satyrini Pronophilina Diaphanos curvignathos CP17-03 GQ357228 GQ357295 GQ357471 GQ357600 GQ357361 1 20 Satyrinae Satyrini Pronophilina Elina montrolii CH-25-1 CHILE: Ñuble, Cueva DQ338835 DQ338986 –––1 Pincheira Satyrinae Satyrini Pronophilina Eretris sp. nov. 8 CP08-04 PERU: La Solitaria GQ357229 GQ357296 GQ357472 GQ357601 GQ357362 0 Satyrinae Satyrini Pronophilina Etcheverrius chiliensis CH-30-4 CHILE: Los Andes, Portillo DQ338836 DQ338987 – – DQ338700 1 Satyrinae Satyrini Pronophilina Eteona tisiphone NW127-21 BRAZIL: Extrema, MG DQ338849 DQ338999 GQ357473 GQ357602 DQ338711 0 21 Satyrinae Satyrini Pronophilina Faunula leucoglene CH-30-5 CHILE GQ357230 – – – GQ357363 1 Satyrinae Satyrini Pronophilina Foetterleia schreineri NW127-19 BRAZIL: Extrema, MG DQ338590 DQ339000 GQ357474 GQ357603 DQ338712 0 22 Satyrinae Satyrini Pronophilina Haywardella edmondsii CP14-04 GQ357231 GQ357297 GQ357475 GQ357604 GQ357364 1 Satyrinae Satyrini Pronophilina Junea dorinda CP06-94 PERU: La Antena DQ338850 DQ339001 GQ357476 GQ357605 DQ338713 0 Satyrinae Satyrini Pronophilina Lasiophila cirta CP04-36 PERU: Quebrada Malambo DQ338851 DQ339002 GQ357477 GQ357606 DQ338714 0 Satyrinae Satyrini Pronophilina Lymanopoda caudalis CP04-22 PERU: Pampa Hermosa GQ357232 GQ357298 GQ357478 GQ357607 GQ357365 0 Satyrinae Satyrini Pronophilina Lymanopoda rana CP03-33 PERU: Pampa Hermosa DQ338853 DQ339004 GQ357479 GQ357608 DQ338715 0 Satyrinae Satyrini Pronophilina Manerebia lisa CP04-23 PERU: Quebrada Malambo GQ357233 GQ357299 GQ357480 GQ357609 GQ357366 0 Satyrinae Satyrini Pronophilina Mygona irmina CP17-04 GQ357234 GQ357300 GQ357481 GQ357610 GQ357367 0 23 Satyrinae Satyrini Pronophilina Nelia nemyroides CH-8A-2 CHILE: Los Lagos AY508562 AY509088 –––0 16 Satyrinae Satyrini Pronophilina Oxeoschistus leucospilos CP04-67 PERU: Quebrada Siete DQ338854 DQ339005 GQ357482 GQ357611 DQ338716 0

olgclJunlo h ina Society Linnean the of Journal Zoological Jeringas Satyrinae Satyrini Pronophilina Oxeoschistus pronax CP07-73 PERU: La Solitaria GQ357235 GQ357301 GQ357483 GQ357612 GQ357368 0 Satyrinae Satyrini Pronophilina Pampasatyrus glaucope NW149-7 BRAZIL: São Paulo GQ357236 GQ357302 GQ357484 GQ357613 GQ357369 1 Satyrinae Satyrini Pronophilina Pampasatyrus gyrtone NW126-12 BRAZIL: Campos do Jordão, DQ338837 DQ338988 EU528406 EU528454 DQ338701 1 SP Satyrinae Satyrini Pronophilina Pampasatyrus reticulata CP17-09 BRAZIL: Campos do Jordão, GQ357237 GQ357303 GQ357485 GQ357614 GQ357370 1 SP Satyrinae Satyrini Pronophilina Panyapedaliodes drymaea CP09-53 PERU: S.N. de Ampay DQ338855 DQ339006 GQ357486 GQ357615 DQ338717 1 Satyrinae Satyrini Pronophilina Parapedaliodes parepa CP07-51 PERU: Lima DQ338591 DQ339007 GQ357487 GQ357616 DQ338718 1 Satyrinae Satyrini Pronophilina Pedaliodes phrasiclea CP03-35 PERU: Quebrada Siete GQ357238 GQ357304 GQ357488 GQ357617 GQ357371 0 Jeringas Satyrinae Satyrini Pronophilina Pedaliodes sp. nov. 26 CP09-90 PERU: Ampay GQ357239 GQ357305 GQ357489 GQ357618 GQ357372 1 Satyrinae Satyrini Pronophilina Pedaliodes sp. nov. 117 CP09-66 PERU: S.N. de Ampay DQ338856 DQ339008 EU528407 EU528456 DQ338719 1 Satyrinae Satyrini Pronophilina Proboscis propylea CP07-15 PERU: La Antena DQ338858 DQ339011 GQ357490 GQ357619 DQ338722 0 Satyrinae Satyrini Pronophilina Pronophila thelebe CP03-70 PERU: Quebrada Siete DQ338859 DQ339012 EU528410 EU528461 DQ338723 0 Jeringas Satyrinae Satyrini Pronophilina Pseudomaniola loxo CP13-13 COLOMBIA: Prov. Antioquia DQ338860 DQ339013 GQ357491 GQ357620 – 0 23 Satyrinae Satyrini Pronophilina Pseudomaniola phaselis CP04-01 PERU: Quebrada Siete DQ338593 DQ339014 GQ357492 GQ357621 DQ338724 0 Jeringas

2011, , Satyrinae Satyrini Pronophilina Punapedaliodes CP07-87 PERU: Cerro de Pasco DQ338861 DQ339015 GQ357493 – DQ338725 1 ﬂavopunctata Satyrinae Satyrini Pronophilina Punargentus sp. CP08-51 PERU: Junín-Pachacayo GQ357240 GQ357306 GQ357494 GQ357622 GQ357373 1 Satyrinae Satyrini Pronophilina Punargentus sp. CP08-75 PERU: Junín-Pachacayo GQ357241 GQ357307 GQ357495 GQ357623 GQ357374 1 161 Satyrinae Satyrini Pronophilina Punargentus sp. CP09-39 PERU: Junín-Pachacayo GQ357242 GQ357308 GQ357496 GQ357624 GQ357375 1

64–87 , Satyrinae Satyrini Pronophilina Quilaphoetosus monachus CH-12-1 CHILE: Valdivia DQ338838 DQ338979 –––1 Satyrinae Satyrini Pronophilina Redonda empetrus CP17-02 GQ357243 GQ357309 GQ357497 – GQ357376 1 24 Satyrinae Satyrini Pronophilina Steremnia umbracina CP07-89 PERU: La Unión DQ338862 DQ339016 GQ357498 – DQ338726 1 01TeLnenSceyo London, of Society Linnean The 2011 ©

Satyrinae Satyrini Pronophilina Steromapedaliodes CP17-01 GQ357244 GQ357310 GQ357499 – GQ357377 1 20 albonotata Satyrinae Satyrini Pronophilina Thiemeia phoronea CP13-08 VENEZUELA: P.N. Avila GQ357245 GQ357311 – GQ357625 GQ357378 0 19 Gavilan Satyrinae Satyrini Satyrina Arethusana arethusa CP11-06 SPAIN: La Aldea (Navarra) DQ338863 DQ339018 GQ357500 GQ357626 DQ338728 1 3 Satyrinae Satyrini Satyrina Berberia lambessanus EW26-29 MOROCCO: Moyen Atlas DQ338864 DQ339019 GQ357501 GQ357627 GQ357379 1 3 central Satyrinae Satyrini Satyrina Brintesia circe CP-B01 FRANCE: Aude, Villegly DQ338865 DQ339020 EU141474 EU141370 DQ338729 1 Satyrinae Satyrini Satyrina Chazara briseis EW26-19 MOROCCO: Rif oriental DQ338866 DQ339021 GQ357502 GQ357628 DQ338730 1 3 olgclJunlo h ina Society Linnean the of Journal Zoological Satyrinae Satyrini Satyrina Hipparchia statilinus EW25-24 GREECE: Peloponessos near DQ338596 DQ339024 GQ357503 GQ357629 DQ338733 1 3 Patras Satyrinae Satyrini Satyrina Karanasa bolorica NW166-10 RUSSIA: E Pamir, Karateke GQ357246 GQ357312 GQ357504 GQ357630 GQ357380 1 8 distr Satyrinae Satyrini Satyrina Karanasa pamira CP-AC23-32 RUSSIA: Vanch DQ338869 DQ339025 GQ357505 GQ357631 DQ338734 1 8 Satyrinae Satyrini Satyrina Neominois ridingsii CD-1-1 USA: Colorado DQ338870 DQ339026 – – DQ338735 1 11 Satyrinae Satyrini Satyrina Oeneis jutta EW4-1 SWEDEN DQ018958 DQ018925 GQ357506 GQ357632 DQ018896 1 3 Satyrinae Satyrini Satyrina Pseudochazara mamurra CP10-11 IRAN: Isfahan DQ338598 DQ339028 GQ357507 GQ357633 DQ338737 1 3 Satyrinae Satyrini Satyrina Satyrus actaea EW20-12 FRANCE: Carcassonne DQ338871 DQ339029 EU528412 EU528463 DQ338738 1 3 Satyrinae Satyrini Satyrina Satyrus iranicus CP10-12 IRAN DQ338873 DQ339031 GQ357508 GQ357634 DQ338740 1 H AITO FSTRN BUTTERFLIES SATYRINI OF RADIATION THE Satyrinae Satyrini Eritina Coelites euptychioides CP16-14 INDONESIA: Kalimantan GQ357247 GQ357313 GQ357509 GQ357635 GQ357381 0 25 Satyrinae Satyrini Eritina Erites argentina CP16-13 INDONESIA: Kalimantan EU528321 EU528298 EU528390 EU528435 EU528277 0 Satyrinae Satyrini Eritina Orsotriaena medus EW25-17 BANGLADESH: Sylhet Div. DQ338766 DQ338906 EU528405 EU528453 DQ338633 1 6 Satyrinae Satyrini Eritina Zipaetis saitis D30 INDIA DQ338831 DQ338981 EU528418 EU528472 DQ338696 0 26 Satyrinae Satyrini Ragadiina Acrophtalmia leuce CP16-16 INDONESIA: Central GQ357248 GQ357314 GQ357510 GQ357636 GQ357382 0 7 Sulawesi Satyrinae Satyrini Ragadiina Ragadia makuta CP16-09 INDONESIA: Kalimantan GQ357249 GQ357315 EU532177 EU532178 – 0 Satyrinae Satyrini uncertain Cercyonis meadii CP15-09 USA: Colorado, Douglas Co. GQ357250 GQ357316 GQ357511 GQ357637 GQ357383 1 11

2011, , Satyrinae Satyrini uncertain Cercyonis pegala EW8-1 USA: OR, Benton Co. AY218239 AY218259 – – AY218277 1 11 Satyrinae Satyrini uncertain Hyponephele cadusia CP10-07 IRAN: Hamadan DQ338839 DQ338989 EU528395 EU528441 DQ338702 1 Satyrinae Satyrini uncertain Hyponephele shirazica CP10-13 IRAN: Bakhtiari DQ338840 DQ338990 GQ357512 GQ357638 DQ338703 1 161 1van Son, 1955; 2Parsons, 1999; 3Tolman & Lewington, 1997; 4Pijpe, 2007; 5Larsen, 2005; 6Braby, 2000; 7Igarashi & Fukuda, 2000; 8Tuzov, 1997; 9Freitas, 2004a; 64–87 , 10Peña & Lamas, 2005; 11Scott, 1986; 12DeVries, 1987; 13Luis & Llorente, 1993; 14Freitas, 2004b; 15Habel et al. 2005; 16Concha & Parra, 2006; 17Sourakov, 1996; 18Viloria, 2000; 19Pyrcz, 2004b; 20Pyrcz, 2004a; 21Freitas, 2002; 22Viloria, 2007; 23Henao, 2005; 24Viloria et al., 2003; 25Tangah et al. 2004; 26Nguyen et al. 2002. 71 72 C. PEÑA ET AL. decided to add several taxa to our out-group selection in order to break the attraction of in-group taxa to the out-group species (Bergsten, 2005). Additionally, we performed long-branch extractions (sensu Siddall & Whiting, 1999; see Discussion) in order to identify other taxa also suffering LBA. Thus, we included related species in the Brassolini and Morphini, and rooted the resulting networks from our analyses with Morpho helenor (Cramer, 1776). We tested whether the branching order of the out-groups had any effect on the tree topology by rooting the networks with different out-group taxa. We evaluated clade robust- ness by using Bremer support (Bremer, 1988) and the partitioned congruence index (PCI) (Brower, 2006). The PCI was drawn from partitioned Bremer support (PBS) values (Gatesy, O’Grady & Baker, 1999) obtained using the scripting feature of TNT (script pbsup.run taken from http://www.zmuc.dk/public/ phylogeny/TNT/scripts/). For maximum likelihood (ML) analyses we used the software RaxML v7.0.3 (randomized ‘axelerated’ maximum likelihood for high-performance computing; T) pi(A) pi(C) pi(G) pi(T) alpha pinvar Stamatakis, Ludwig & Meier, 2005; Stamatakis, ↔ (G

Hoover & Rougemont, 2008) on the BlackBox cluster of r the Vital-IT Unit of the Swiss Institute of Bioinformat- ics (http://phylobench.vital-it.ch/raxml-bb/index.php). T) We used the software MrBayes 3.1.2 (Ronquist & ↔ Huelsenbeck, 2003) for Bayesian inference. We mod- (C elled the evolution of sequences according to the r GTR +G model. Parameter values were estimated

separately for each gene region (Table 2). The analy- G)

sis was run twice for 20 million generations, with ↔

every 1000th tree sampled, and with the ﬁrst 80 000 (C r sampled generations discarded as burn-in (based on a visual inspection of the log likelihood reaching sta- tionarity). We ran the analyses on an AMD 64 dual- T) core twin processor workstation using LAM/MPI ↔ (A technology for parallel computing (http://www. r lam-mpi.org/). G)

TIMES OF DIVERGENCE ↔ (A We used the Bayesian analysis software BEAST 1.4.7 r (Drummond & Rambaut, 2007) under a log-normal

relaxed molecular clock and a Yule birth model of C)

speciation to model the rate of molecular evolution ↔

along the Satyrini phylogenetic trees. The DNA (A r 0.050.07 0.29 0.28 0.09 0.12 0.06 0.03 0.46 0.42 0.06 0.08 0.31 0.16 0.23 0.37 0.21 0.34 0.26 0.13 0.58 0.59 0.38 0.30 sequences were divided into ﬁve data sets (one for each gene), with parameter values estimated independently. The data set was analysed under the GTR +Gmodel with a relaxed clock, allowing branch lengths to vary following an uncorrelated log-normal 20.81 0.05 0.32 0.03 0.07 0.50 0.04 0.41 0.09 0.03 0.47 0.40 0.54 distribution (Drummond et al., 2006). The analysis Parameter values estimated using Bayesian phylogenetic methods was run twice for 19 million generations (with a a pre-run burn-in of 800 000 generations) with sampled Table 2. Gene TL (all) trees every 2000 generations, and the results were COI EF-1 wingless RpS5GAPDH 0.08 0.09 0.30 0.29 0.12 0.12 0.05 0.05 0.38 0.40 0.07 0.05 0.28 0.23 0.24 0.22 0.15 0.25 0.33 0.31 0.65 0.65 0.46 0.48

© 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 161, 64–87 THE RADIATION OF SATYRINI BUTTERFLIES 73 compiled using both runs. The tree priors were set to biogeographic patterns and hypotheses of area rela- a Yule speciation process and all other priors were left tionships are not taken into account by DIVA. Dispers- to the default values in BEAST. als and extinctions are assigned a cost of one, whereas As we want to test whether major events in the vicariance and within-area speciation are assigned a evolutionary history of Satyrini are correlated with cost of zero. The least-cost ancestral area reconstruc- geological events, we cannot calibrate our phylogram tion is derived from this cost matrix (Ronquist, 1997). using geological events (Braby et al., 2005). Thus, the As a result, DIVA favours speciation by vicariance and hypothesis of a Gondwanan origin for Satyrini was minimizes the impact of dispersal on biogeographic independently tested in our previous study (Peña reconstructions. Although recent studies suggest that & Wahlberg, 2008) by our calibration with the dispersal might have been an important factor shaping fossil Lethe corbieri Nel, Nel & Balmer, 1993 the current distributions of butterflies (e.g. Wahlberg (25 ± 1.0 Mya). & Freitas, 2007; Kodandaramaiah & Wahlberg, 2009), In order to obtain absolute times of divergence, we the satyrines show a remarkable conservatism in used four calibration points. We fixed the root (= sub- global distributions, where several subtribes are family Satyrinae) at 60.99 Mya with a standard restricted to major biogeograhic regions. This implies deviation of 6.1 Myr, as inferred from our previous that, although dispersals might have been important study (Peña & Wahlberg, 2008). We used an age of in the evolutionary history of the group, dispersal 36.6 ± 5.1 Myr for Satyrini (from Peña & Wahlberg, events did not occur too frequently to obliterate the 2008). We used the age of 25±1Myrforthesatyrine biogeographical signal. fossil L. corbieri from the Late Oligocene (Nel et al., We divided the world into eight biogeographical 1993), and fixed the clade (Satyrodes, Lethe, and regions largely reflecting those of Sclater (1858; Enodia)attheminimumageof25±1Myr,andwith Fig. 1). When replacing our terminal taxa with their 4.3 ± 0.5 Mya for the split of Coenonympha pamphi- distributions, we included the distributions of all lus (Linneaus, 1758) and Coenonympha thyrsis member species of our sampled genera, so that our (Freyer, 1845), based on results by Kodandaramaiah dispersal–vicariance analysis would not be affected by & Wahlberg (2009). Nel et al. (1993) disscuss the taxon sampling (as, in some cases, we sampled only morphological characters in the venation of fore- and one species per genus for our data matrix). DIVA was hindwings of the fossil L. corbieri [shape of discal not able to cope with all of our terminals, so we cells, swelling of subcosta (Sc) and anal veins, and collapsed part of the Coenonymphina and the Prono- position of media 1–3 (M1–M3) and cubital veins], philina. This did not have any effect on the inference and state that this specimen shares the potential of ancestral areas of distribution because all the synapomorphies of Miller’s (1968) Lethe series (sub- pruned taxa are distributed in the same broad bio- tribe Lethina). Nel et al. (1993) considered that the geographical region as demarcated in Figure 1 (Aus- ornamentation and venation of the fossil’s wings are tralia for some Coenonymphina and the Neotropics similar enough to members of the extant genus Lethe for the Pronophilina). For the out-groups, we used the to be included in this genus. topology from Peña & Wahlberg (2008) in order to Convergence was analysed with Tracer 1.3 and avoid interference in the ancestral distributions of the trees were summarized with TreeAnnotator 1.4.7, in-group. Ancestral distributions were inferred using which are distributed along with the BEAST package. default costs in the software DIVA (Ronquist, 1996): The standard deviation of the primary calibration vicariance events cost zero, dispersal and extinction points is intended to take into account the uncer- events cost one per unit area. tainty of estimated ages (Graur & Martin, 2004). We analysed the data by constraining the maximum ancestral areas to three (‘maxareas = 3’), in order to improve the resolution of the analysis when BIOGEOGRAPHICAL ANALYSIS estimating the most likely ancestral distribution of We decided to follow the topology from the BEAST the nodes (Ronquist, 1997). analysis for the biogeographic analysis, because this software also estimates the times of origin and diversification for the nodes of interest in the evolution of PATTERNS OF BUTTERFLY–HABITAT ASSOCIATIONS Satyrini. We examined the evolution of habitat use in Satyrini We investigated the biogeographical history of by the optimization of data gathered from the litera- Satyrini butterflies by analysing our preferred phylo- ture on butterfly habitats, from van Son (1955); Scott genetic hypothesis (see Discussion) under DIVA (Ron- (1986), DeVries (1987), Luis & Llorente (1993), Soura- quist, 1997). DIVA infers ancestral distributions in a kov (1996), Tolman & Lewington (1997), Tuzov (1997), phylogeny using a three-dimensional cost matrix Parsons (1999), Braby (2000), Igarashi & Fukuda derived from a simple biogeographic model. General (2000), Viloria (2000), Freitas (2002), Nguyen et al.

Figure 1. The eight different biogeographical regions used in this study for the dispersal–vicariance analysis (DIVA). A, Western Palaearctic. B, Eastern Palaearctic. C, Southeast Asia. D, Africa. E, Neotropics. F, Nearctic. G, Central America. H, Australia.

(2002), Viloria et al. (2003), Freitas (2004a, b), Pyrcz or not (Bergsten, 2005), we analysed different subsets (2004a, b), Tangah et al. (2004), Habel, Schmitt & of our data excluding each of the taxa grouping with Müller (2005), Larsen (2005), Concha & Parra (2006), Euptychia (long-branch extraction). During this exer- Pijpe (2007), Viloria (2007a), and C. Peña (unpubl. cise, we found that all these taxa continued to be data), and coded the habitat use by the sampled attracted to each other except when Euptychia was Satyrini species as a single binary character with the absent in the analysis. In the latter case, Calisto and states ‘forest habitat’ and ‘non-forest habitat’. We Eretris appear as sisters, Ragadia groups with coded 0 for forest and 1 for non-forest habitats. The Coelites, Acrophtalmia, and Loxerebia, whereas character coding for each species is given in Table 1. Ypthima and Ypthimomorpha group with the other Character states were optimized under maximum Ypthimina (Stygionympha, Cassionympha, Neo- parsimony using ACCTRAN in WinClada 1.00.0.8 coenyra, Callerebia, Paralasa, and Pseudonympha, (Nixon, 2002) on the phylogenetic tree used for and surprisingly with Proterebia). When we analysed showing the estimated times of divergence for our data set without all other LBA taxa, MP recovers Satyrini lineages. Euptychia as sister to all other Euptychiina (Fig. 2). Consistent with Peña et al. (2006) and Peña & Wahlberg (2008), Satyrini is a strongly supported RESULTS monophyletic tribe with a Bremer support (BS) value MAXIMUM PARSIMONY ANALYSIS of 21. There is a very low BS value for several deep When using only H. piera as the out-group, the genus nodes that deﬁne some subtribes. The only subtribes Euptychia (represented by Euptychia enyo and Eupty- with good to moderate support are Satyrina (BS 38), chia sp. nov. 2, E. sp. nov. 5, E. sp. nov. 6 and E. sp. nov. Maniolina (BS 33), Eritina (BS 13), and Mycalesina 7) was pulled towards the root, appearing as a sister to (BS 31) (Figs 2, 3). These strongly supported nodes all other Satyrini. Bergsten (2005) suggested that the are very robust, as shown by the high PCI values LBA between the out-group and long-terminal (Fig. 2). in-group branches can be broken by having a more Not surprisingly there is only a low BS value of 1 intensive sampling of out-groups. When we included for an Euptychiina including Euptychia. Murray & members of the Morphini and Brassolini as out-groups Prowell (2005) also found the position of Euptychia to and rooted the trees with M. helenor, we found that be unstable, where the exclusion of Euptychia from Euptychia no longer appears to be attracted to the root. Euptychiina was strongly supported based on two However, its position within the in-group is unstable, gene regions (COI and EF-1a). Coenonymphina appearing attracted to Ragadia, and to taxa in the (including the Australian ‘hypocystines’) is strongly Pronophilina and Ypthimina (Calisto, Eretris, Callere- supported (BS 9), and our results suggest that the bia, Proterebia, Ypthima, and Ypthimomorpha), with genus Argyronympha is sister to the rest of the these taxa forming a clade. As maximum parsimony genera in this subtribe. Pronophilina has two clearly (MP) can group long branches whether they are related deﬁned clades, one of them being mainly southern

Figure 2. Strict consensus of 12 equally parsimonious trees (29 647 steps; consistency index, CI = 0.13; retention index, RI = 0.44) from the maximum parsimony analysis (MP). Numbers given above branches are Bremer support values and the numbers below the branches are partitioned congruence index (PCI) values for the node to the right of the number.

Figure 3. Reduced cladograms from (A) maximum parsimony and (B) model-based methods (Bayesian inference and maximum likelihood), showing the incongruent hypotheses of relationships for the subtribes in the Satyrini. pronophilines, although also including some northern sister to the Coenonymphina. Melanargiina is sister genera – Steremnia, Manerebia, and Lymanopoda. to the Satyrina, with strong support. There is mod- The pronophilines Calisto and Eretris appear as sister erate support for Ypthimina without Paralasa –the taxa, and do not group with other Pronophilina. This latter taxon appears as a sister to Melanargiina + is caused in part by the long-branch artifacts and the Satyrina. Hyponephele and Cercyonis are recovered as weak support of basal nodes. It appears that our ﬁve a strongly supported clade (bootstrap 100), in agree- genes do not carry enough phylogenetic signal to ment with Peña et al. (2006). As in the MP analysis, resolve unambiguously the relationships among sub- Pronophilina are recovered in two strongly supported tribes. Ypthimina is monophyletic if Proterebia is clades. However, there is weak support for these included, although the support is weak (BS 1); two clades being sister groups (bootstrap 19). currently, the genus Proterebia is classiﬁed under This was also found in the MP analysis where Pro- Erebiina. nophilina appeared as polyphyletic when we performed the long-branch extraction exercise. Although several nodes are weakly supported by bootstrap MAXIMUM LIKELIHOOD values (Fig. 4), some clades are recovered as being For the full data set (including all other out-groups in robust, i.e. (Parargina (Mycalesina + Lethina)) and addition to Haetera), the analysis in RAxML was also ((Hyponephele + Cercyonis) Maniolina). A pruned cla- disturbed by long-branch taxa. As in the parsimony dogram of the relationships of the Satyrini subtribes analysis, the pronophiline Calisto appears attracted is shown in Figure 3. to Euptychia, although this relationship is not strongly supported (bootstrap value 20). The pronophiline Eretris, although weakly supported, appears as sister to all other Euptychiina (which has strong BAYESIAN INFERENCE support, with a bootstrap value of 89). The majority-rule cladogram is completely resolved In the analyses without long branches, it is possible (Fig. 5) and entirely congruent with the majority-rule to identify a strongly supported clade containing cladogram obtained in the ML analysis, which is not Euptychiina, Ypthimina (Paralasa (Melanargiina + surprising, given that the two analyses employed the Satyrina)), Pronophilina, Erebiina, Maniolina, and same model. The Bayesian inference (BI) analysis (Hyponephele + Cercyonis) (Fig. 4). This clade was recovered two major clades with good support: a clade also strongly supported in the Bayesian analysis comprising (Eritina + Coenonymphina), sister to the (see below). The genera Coelites, Loxerebia, and same clade from ML (Parargina (Mycalesina + Acrophtalmia form a clade, and appear as sisters to Lethina)); and all other subtribes included in a robust all other Satyrini taxa (Fig. 4). As well as in the MP clade (posterior probability = 0.98). The posterior analysis, Zipaetis, Erites, and Orsotriaena group probability values are similar to the bootstrap values together, forming a cohesive Eritina that appears as a obtained in the ML analysis.

Figure 4. ‘Bipartitions tree’ obtained from the Maximum Likelihood (ML) analysis in RaxML. Numbers at the branches are bootstrap values for the node to the right of the number.

Figure 5. Majority-rule cladogram based on Bayesian inference (BI), modelled with a GTR +Gmodel. Numbers at the branches are posterior probability values for the node to the right of the number.

TIMES OF DIVERGENCE the DIVA analysis in order to avoid ambiguity. Thus, The phylogram obtained by the BEAST analysis we were unable to estimate its ancestral area of (Fig. 6) is not completely congruent with the MP and origin and test whether it has any influence on our other model-based methods (ML and BI). Our BEAST biogeographical analysis. In any case Satyrini analysis showed fairly wide intervals of confidence for appears to have originated in the Old World and/or most of the estimated times of divergence, despite Indo-Australia. using four calibration points (Fig. 6). However, it is Whereas Coenonymphina and Ragadiina evolved in possible to identify some patterns in the origin and Southeastern Asia and Indo-Australia, the Parargina, diversification of Satyrini lineages. Our results Mycalesina, and Lethina clade originated in the indicate that Satyrini originated around 42 Mya Eastern Palaearctic, and dispersals into Africa and (41.8±6Mya), during the Eocene. It seems that the North America resulted in some taxa in the subtribes Satyrini underwent a quick diversification phase in a Mycalesina and Lethina, respectively. If Satyrini relatively short span of time – virtually all of the originated in the Palaeartic and/or Oriental regions, subtribes in the Satyrini appeared during the Oli- it would imply that the clades including the bulk of gocene, between 32 and 24 Mya (Fig. 6). The clade Satyrini species, the mainly Neotropical Euptychiina formed by Parargina, Mycalesina, Lethina, Eritina, and Pronophilina, are the product of a dispersal event and Coenonymphina originated early in the evolution followed by a dramatic diversification from the Palae- of Satyrini (40 Mya). The Coenonymphina and arctic into the New World. Similarly, a New World Eritina are recovered as the oldest subtribes, diverg- origin for the group implies that dispersal was an ing at 39 Mya. Melanargiina appears to be the young- important factor in the evolution of Satyrini. est subtribe in Satyrini, diversifying at 4 ± 2.5 Mya. It will be necessary to recover unambiguously the most basal relationships in Satyrini in order to con- fidently identify the centre of origin of this highly DISPERSAL–VICARIANCE ANALYSIS diverse group of butterflies. According to our DIVA analysis, the estimated area of origin for Satyrini is ambiguous. The analysis showed EVOLUTION OF HABITAT USE that Satyrini appeared in any combination of the following biogeographical regions: Eastern Palaearc- We obtained data on habitat use for 162 species. We tic, Oriental, Indo-Australia and Neotropical (areas B, could not confirm records for Chonala miyatai C, E, and H in Fig. 7). Satyrini is recovered as two (Koiwaya, 1996), Tatinga thibetana (Oberthür, 1876), major sister clades. The clade formed by Parargina, Rhaphicera dumicola (Oberthür, 1876), and Sinonym- Mycalesina, Lethina, Eritina, and Coenonymphina pha amoena (Lee, 1974), whereas Hypocysta pseudi- appeared either in the Eastern Palaearctic + Oriental rius (Butler, 1875) and Amphidecta calliomma (F. & region (BC) or Eastern Palaearctic and Indo- F., 1862) were recorded using both habitats. The Australian regions (BCH) – DIVA shows a third pos- evolution of habitat use is shown in Figure 8. Our sibility being the Eastern Palaearctic + Australian results show that the habitat shift from forests into region (BH). DIVA resolved the ancestral distribution open habitats occurred with the origin of the tribe of the other major clade of Satyrini (including sub- Satyrini. Although open habitats are exploited by tribes Euptychiina, Pronophilina, Ypthimina, Mela- most species, there have been several shifts back to nargiina, Satyrina, Erebiina, and Maniolina) to be the the ancestral forest habitats. Some clades in the Eup- Neotropical region (area E; Fig. 7). Thus, we are tychiina and Pronophilina are almost entirely com- unable to identify whether the Satyrini originated in posed of species inhabiting non-forests. either the Neotropical or Palaeartic + Oriental regions. Such disjunct distribution of Satyrini sister DISCUSSION clades might suggest that the common ancestor was distributed in a land mass connecting the New and COMPETING PHYLOGENETIC METHODS the Old World, placing the origin of Satyrini in In this study, we compare the results from three Pangaea; however, this possibility is ruled out accord- phylogenetic methods, MP, ML, and BI, in an effort to ing to the dating estimates for the origin of the group, obtain a phylogenetic hypothesis for the diverse tribe which is much younger that the break-up of Pangaea Satyrini. We found long-branch taxa that affected the and Gondwana. accuracy of all three methods. Moreover, different The position of the clade Coelites + Acrophtalmia + methods produced incongruent phylogenies. The Loxerebia is ambiguous – it is either sister to all degree of uncertainty for some nodes remained after Satyrini (as found by the analyses in ML and BI; efforts to remove long-branch taxa from the analyses. Figs 4, 5) or groups in a clade with Eritina (MP However, some clades were consistently recovered by analysis; Fig. 2). We did not include these genera in all three methods. These clades were backed by high

Eocene Oligocene Miocene Pliocene Pleistocene

Elymnias casiphone 0.61 Brassolis sophorae 0.99 Amathusia phidippus 0.45 Zethera incerta 0.55 Haetera piera 0.36 Morpho helenor 0.5 Melanitis leda 1 Dira clytus Kirinia roxelana 1 Kirinia climene 1 Lasiommata megera 0.89 Pararge aegeria PARARGINA Tatinga thibetana 0.98 1 1 Lopinga achine 0.9 Chonala miyatai Bicyclus anynana 1 Hallelesis halyma MYCALESINA 1 1 Mycalesis terminus Rhaphicera dumicola 0.62 0.99 Neope bremeri Ninguta schrenkii LETHINA 0.79 0.75 Lethe minerva 1 Satyrodes eurydice 1 Enodia anthedon Ragadia makuta 0.98 Zipaetis saitis 1 1 Erites argentina ERITINA 1 Orsotriaena medus Argyronympha gracilipes 0.98 1 Argyronympha ugiensis Oreixenica latialis 1 Erycinidia virgo 1 Erycinidia gracilis 0.6 Oressinoma sorata 1 1 0.98 Oressinoma typhla Sinonympha amoena 1 Coenonympha myops 0.96 Coenonympha phryne 1 1 Coenonympha saadi 1 Coenonympha pamphilus 1 Coenonympha thyrsis Paratisiphone lyrnessa 1 Tisiphone abeona COENONYMPHINA Platypthima ornata 1 Harsiesis hygea 0.97 0.68 1 Platypthima homochroa Altiapa decolor 1 1 Altiapa klossi Hypocysta adiante 1 0.8 Hypocysta pseudirius Dodonidia helmsi 1 1 Erebiola butleri 1 Percnodaimon merula 0.47 1 Argyrophenga antipodium Nesoxenica leprea 0.32 Argynnina cyrila 1 Geitoneura klugii 1 Geitoneura minyas Paramacera xicaque 1 Cyllopsis pertepida Megisto cymela 1 1 Euptychoides castrensis 1 1 Yphthimoides cipoensis Amphidecta calliomma Pindis squamistriga 0.78 1 Hermeuptychia hermes 1 Hermeuptychia hermes 0.67 Splendeuptychia itonis Parataygetis albinotata 1 EUPTYCHIINA 0.86 1 Posttaygetis penelea Taygetis virgilia 0.44 Forsterinaria boliviana 0.99 1 Harjesia blanda Erichthodes antonina 1 1 Pareuptychia hesionides Chloreuptychia herseis 0.5 Cepheuptychia sp. n. 1 Cissia myncea 1 Caeruleuptychia lobelia 1 Magneuptychia sp. n. 4 Loxerebia saxicola 1 1 Callerebia polyphemus 1 Proterebia afra 0.81 Paralasa styx 1 Paralasa hades 0.98 Paralasa jordana YPTHIMINA 0.78 Ypthimomorpha itonia 1 1 Ypthima baldus Stygionympha vigilans 1 Cassionympha cassius 1 Neocoenyra petersi 1 Pseudonympha magus Erebia oeme 1 Erebia epiphron 1 Erebia triaria EREBIINA 0.95 Erebia palarica 1 0.99 Erebia ligea Maniola jurtina 1 Pyronia cecilia MANIOLINA 0.72 Aphantopus arvensis 1 1 Aphantopus hyperantus 0.96 Hyponephele shirazica 1 1 Hyponephele cadusia 0.58 Cercyonis pegala 1 Cercyonis meadii Melanargia hylata 1 Melanargia galathea MELANARGIINA 1 Melanargia lachesis 1 Berberia lambessanus 1 Hipparchia statilinus Neominois ridingsii 1 1 1 Oeneis jutta Karanasa pamira 1 1 Karanasa bolorica SATYRINA Arethusana arethusa 1 Brintesia circe 1 Chazara briseis 1 1 Pseudochazara mamurra Satyrus actaea 1 0.55 Satyrus iranicus Steremnia umbracina 0.69 Manerebia lisa 1 Diaphanos curvignathos 1 Lymanopoda caudalis 1 1 Lymanopoda rana Nelia nemyroides Elina montrolii 1 Quilaphoetosus monachus 1 1 1 Auca barrosi Auca coctei 1 1 Chillanella stelligera 1 Cosmosatyrus leptoneuroides Haywardella edmondsii 1 Pampasatyrus gyrtone 1 Pampasatyrus reticulata 1 1 Pampasatyrus glaucope 0.96 1 Etcheverrius chiliensis Argyrophorus sp. 1 Punargentus sp. 1 1 Punargentus sp. Punargentus sp. Panyapedaliodes drymaea 0.91 Redonda empetrus 1 Steromapedaliodes albonotata PRONOPHILINA 1 Parapedaliodes parepa 1 Punapedaliodes flavopunctata 1 0.89 Altopedaliodes sp. Pedaliodes phrasiclea 1 1 1 Pedaliodes sp. n. 26 Pedaliodes sp. n. 117 Cheimas opalinus 0.97 Corades enyo Pronophila thelebe 1 1 Junea dorinda 0.89 Foetterleia schreineri 1 Eteona tisiphone 5.0 0.78 Daedalma sp. Thiemeia phoronea 0.99 1 Apexacuta astoreth 1 Pseudomaniola loxo 1 1 Pseudomaniola phaselis Lasiophila cirta 1 Mygona irmina 1 Proboscis propylea 0.83 Oxeoschistus pronax 1 Oxeoschistus leucospilos

45 40 35 30 25 20 15 10 5 0

Figure 6. Chronogram derived from the BEAST analysis with associated posterior credibility intervals. Numbers at the branches are posterior probability values for the node to the left of the number.

Figure 7. Results of a dispersal–vicariance analysis (DIVA), using three as the maximum number of ancestral areas in the DIVA. The topology of relationships for the out-groups are taken from Peña & Wahlberg (2008). Many terminals that belong to the same subtribe, and are distributed in the same biogeographical area of Figure 1, appear in one leaf: other Hypocystina, Pronophilina clade 1 and Pronophilina clade 2.

Figure 8. Optimization of habitat use (forest and non-forest) onto the phylogenetic tree from the BEAST analysis.

BS values in the MP analysis and appeared resolved Satyrini (Wahlberg & Wheat, 2008). It may be too in the ML analysis (majority-rule tree), whereas they naive to assume that our current tools are sophisti- were recovered with high posterior probability in BI. cated enough to uncover the evolutionary history of It is notable that the deep internal nodes leading to such a diverse group of butterflies – that might have the subtribes are mostly very short branches, and are been even more diverse in the past – that started supported by very low bootstrap values in ML and BI evolving around 42 Mya somewhere in the New or analyses (Figs 4, 5). Our timing estimates show that Old World, and managed to spread all over the most of the subtribes in Satyrini appeared between 32 world, radiating in a short span of time, and, possi- and 24 Mya (Fig. 6). This pattern is compatible with a bly, suffering many instances of extinctions. In a ‘rapid radiation’ scenario (Whitfield & Lockhart, way, it is gratifying that not all patterns are laid 2007), in agreement with the narrative scheme bare in a single stroke, and that work remains to be described by Miller (1968). If indeed this group under- done to clarify relationships among the basal clades went a quick succession of cladogenesis events, it is of the tribe. possible that complete lineage sorting was not Although it appears that these methods are incon- achieved by the five genes in our data set, and addi- gruent when dealing with the basal nodes, where tional gene sequences might not be able to resolve Satyrini underwent rapid radiation, each has its own unambiguously these relationships (Rokas, Krüger & merits. This does not mean that we claim that the Carroll, 2005; Hallström & Janke, 2008; but see methods are completely worthless either (Ebach et al., Wahlberg & Wheat, 2008). 2008). Analyses of ‘easy and clean’ data sets are likely The model-based methods (BI and ML) and MP are to be resolved identically by all three methods incongruent in resolving the positions of several (Brooks et al., 2007). At least for Satyrini butterflies Satyrini clades, most remarkably regarding the these methods are unable to provide an unambigu- ‘Coelites clade’ – (Coelites (Acrophtalmia, Loxerebia)). ously supported hypothesis of phylogenetic relation- In BI and ML analyses, this clade appears as sister to ships. Nevertheless, the methods were able to all other Satyrini species (Figs 4, 5), whereas it uncover interesting patterns of relationships. These groups with the Ragadiina in the MP analysis relationships were consistently recovered by all three (Fig. 2). In our model-based trees, the genus Paralasa methods, and are strongly supported by bootstrap and is sister to Melanargiina + Satyrina, whereas MP BS values. recovers Paralasa as a member of the subtribe We argue that all phylogenetic methods are mere Ypthimina. More interesting is the fact that although tools, and are not panacean, because they only the MP analysis was not able to resolve the relation- perform satisfactorily when certain criteria are met ships of Maniolina, Erebiina (Melanargiina and Saty- (e.g. when long branches are not included), and rina), and Pronophilina unambiguously, these should be used depending on the question the relationships correspond to very short branches in researcher needs to answer. ML and BI analyses that are supported by very low bootstrap values. It is tempting to infer that this reflects the incapability of the methods to find suffi- HISTORICAL BIOGEOGRAPHY OF SATYRINI cient phylogenetic signal from our matrix of charac- The hypothesis of a Gondwanan origin of Satyrini or ters to uncover the phylogenetic relationships. MP any of its subtribes is not tenable because the tribe analysis reflects this ambiguity by producing a set of diversified around 42 Mya (somewhat earlier than our different most-parsimonious trees, whereas BI and previous estimate in Peña & Wahlberg, 2008), after ML analyses do it by showing very low support the break-up of Gondwana. Thus, the current global values. Even by using DNA sequences from five distribution of Satyrinae must have arisen via at genes, these methods could not deal with the probable least some intercontinental dispersal events. The rapid radiation of the most speciose clade of the Satyrini might have originated either in the Neotro- Satyrini, and failed to produce a uniformly robust pical region or the Palaearctic + Oriental + Indo- hypothesis of relationships. Although each method Australia regions. One of the equally parsimonious may have its own shortcomings and be affected by origins of the group is consistent with Miller’s (1968) artifacts of the data, it is fair to acknowledge that suggestion that Satyrini originated in the Eastern part of the problem is the nature of the study group. Palaearctic, Oriental, or Indo-Australian regions, It seems very likely that at least some of the genes refining the date estimate to around 42 Mya. It was in in the genome of Satyrini butterflies underwent com- these regions where the ancestor of the Parargina, plete lineage sorting, and retained enough phyloge- Mycalesina, Lethina, Eritina, and Coenonymphina netic signal to reveal the phylogenetic patterns. If might have originated. The Mycalesina appeared this is true, a phylogenomic approach might be able between 27 and 15 Mya, when its ancestor dispersed to resolve unambiguously the phylogeny of the from the Palaearctic into Africa. A subclade of the

Lethina originated as a result of ancestral incursion becomes clear. According to our results, a split in the from the Palaearctic into North America (at around Satyrini lineage (at around 31 Mya) originated with 25 Mya). The evolution of the Eritina and the early Euptychiina that colonized South America Coenonymphina started around 38 Mya in Southeast- (see above), whereas the other lineage remained in ern Asia and Indo-Australia (areas CH; Fig. 7). It the Palaearctic giving rise to Ypthimina (with some seems that the early evolution of Coenonymphina dispersals into Africa at 32 Mya), and another lineage took place in the Australian region, and that one colonized North America (probably through Beringia), lineage dispersed into the Palaearctic, giving rise to and evolved into several subtribes: Erebiina, Manio- the genera Coenonympha and Sinonympha at around lina, Melanargiina, Satyrina, and Pronophilina. 20 Mya. However, although the Pronophilina dispersed south- If Satyrini indeed originated in the Old World, wards, the other lineages remained in the Holarctic dispersals from either Europe or Asia into the region. Americas permitted the origin of the subtribes con- We recognize that although the expansion and taining the bulk of Satyrini species: the Euptychiina radiation of grasses during the Oligocene (Willis & and Pronophilina. Our results indicate that this McElwain, 2002) permitted the spread and diversifi- migration happened at around 37 Mya. It is known cation of Satyrini throughout the world (Peña & that around this time there was a continuous belt of Wahlberg, 2008), the trait ‘feeding on grasses’ was not forest that extended from Asia through North a key factor that triggered the radiation within America across Beringia (Beringian Bridge I in San- Satyrini per se. It is an inherited character from the martín, Enghoff & Ronquist, 2001) that facilitated the common ancestor of Satyrinae s.s. + Morphini + exchange of flora and fauna between these continents. Brassolini (Peña & Wahlberg, 2008). However, our We suggest that this route was used by the ancestors analysis of the evolution of habitat use reveals that of Euptychiina and related subtribes to invade the the combination of two factors might have facilitated New World. It appears that the ancestor(s) of the the remarkable diversification of Satyrini: (1) the Euptychiina and Pronophilina migrated into the New inherited ability to use grasses as host plants, coupled World almost simultaneously, around 32 Mya (Fig. 6). with (2) an early habitat shift, by the common ances- This route might have been used by ancestors of the tor of Satyrini, from forested environments to open, highly diverse butterfly genus Adelpha (Willmott, non-forest habitats, where grasses are diverse, eco- 2003), which has its centre of diversity in South logically dominant, and therefore abundant as a America. It is interesting to note that some ‘basal’ larval food resource. It is possible that these two Euptychiina are distributed in North and Central factors facilitated the colonization of new habitats, America (Paramacera, Cyllopsis, and Megisto), which invasion to novel continents, and expansion of distri- suggests that these genera were ‘left behind’ during bution ranges, which provided opportunities for the dispersal process. Moreover, the ‘long-branch’ genetic differentiation and diversification by geo- pronophiline genus Calisto, which is endemic to the graphic isolation (Janz, Nylin & Wahlberg, 2006) and Caribbean islands, might be a relict genus that vicariant events (Peña & Wahlberg, 2008). evolved during the south-bound colonization route of the ancestors of Pronophilina from North America CONCLUSION into South America. As Central America did not connect North and South America at 32 Mya, we Our data set proved difficult to analyse. Our results argue that the temporal land connection between the imply that there are long-branch taxa and basal Greater Antilles and north-western South America nodes that are very short and weakly supported, during the Eocene and Oligocene (35–33 Mya), known perhaps reflecting the rapid radiation undergone by as the GAARlandia land span (Iturralde & MacPhee, the Satyrini butterflies (see Discussion). Thus, it is 1999), permitted the migration of early lineages in not surprising that the phylogenetic methods (model- Pronophilina towards South America. It has been based and MP) produced incongruent results. We hypothesized that this land bridge was also important argue that any one particular method should not be in the evolution of Phyciodina butterflies (Nymphal- relied on to solve every phylogenetic problem, but idae) (Wahlberg & Freitas, 2007) and plants in the that certain methods are more appropiate for certain coffee family (Rubiaceae) (Antonelli et al., 2009). types of data. As a result of the short branches and low support Although none of the three methods performed sat- for the nodes leading to the subtribes Ypthimina, isfactorily with our Satyrini data, each has its merits. Erebiina, Maniolina, Melanargiina, Satyrina, and We argue that these methods should be considered as Pronophilina (see Results), our estimation of ances- mere tools, useful to tackle different sets of phyloge- tral areas of distribution might need to be revised netic problems. We believe that phylogeneticists pre- once the pattern of relationships in this clade ferring only one ‘superior’ method over the others are

© 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 161, 64–87 THE RADIATION OF SATYRINI BUTTERFLIES 85 in danger of suffering the ‘man with a hammer’ syn- Erebia tyndarus (Lepidoptera, Rhopalocera, Nymphalidae, drome, when every data set of every taxonomic group Satyrinae) species group combining CoxII and ND5 mito- are treated equally, just as another nail. chondrial genes: a case study of a recent radiation. Molecu- Our results show evidence of the effect of past lar Phylogenetics and Evolution 47: 196–210. dispersal events on the current distribution of Antonelli A, Nylander JAA, Persson C, Sanmartín I. Satyrini butterflies. Satyrini might have originated in 2009. Tracing the impact of the Andean uplift on Neotropi- either the Neotropical or Palaearctic + Oriental cal plant evolution. Proceedings of the National Academy of regions. If Satyrini originated in the Old World, it is Sciences 106: 9749–9754. Bergsten J. 2005. A review of long-branch attraction. Cla- plausible that the bulk of Satyrinae species, in the distics 21: 163–193. subtribes Euptychiina and Pronophilina, are the Billeter R, Sedivy I, Diekötter T. 2003. Distribution and result of dispersal events from the Old World, prob- dispersal patterns of the ringlet butterfly (Aphantopus hype- ably via North America, followed by a radiation in rantus) in an agricultural landscape. Bulletin of the Geobo- South America. The remarkable rarity of euptychiines tanical Institute ETH 69: 45–55. and pronophilines in North America would be attrib- Braby MF. 1993. Early stages, biology and taxonomic status uted to extinction, and some Central American prono- of Tisiphone helena (Olliff) (Lepidoptera: Nymphalidae: philines such as the genus Calisto could be relict taxa Satyrinae). Journal of the Australian Entomological Society of past colonization events. 32: 273–282. We show that a combination of factors might have Braby MF. 1994. Morphology of the early stages of Mycalesis facilitated the spread and diversification of Satyrini Hübner (Lepidoptera: Nymphalidae: Satyrinae) from north- butterflies: ability to feed on grasses (Peña & Wahl- eastern Australia. Journal of the Australian Entomological berg, 2008); an early habitat shift into open, non- Society 33: 289–294. forest habitats; and, geographic bridges (the forest Braby MF. 2000. Butterflies of Australia: their identification, belt across Beringia and the GAARlandia land span) biology and distribution. Victoria, Australia: CSIRO. that permitted geographic expansions of ancestors to Braby MF, Trueman JWH, Eastwood R. 2005. When and new environments, which provided opportunities for where did troidine butterflies (Lepidoptera: Papilionidae) geographic differentiation and diversification. evolve? Phylogenetic and biogeographic evidence suggests and origin in remnant Gondwana in the Late Cretaceous. Invertebrate Systematics 19: 113–143. ACKNOWLEDGEMENTS Braby MF, Vila R, Pierce NE. 2006. Molecular phylogeny This work has been supported in part by a Synthesys and systematics of the Pieridae (Lepidoptera: Papilion- grant to visit the Hungarian Museum of Natural oidea): higher classification and biogeography. Zoological History, with funding from Amazon Conservation Journal of the Linnean Society 147: 239–275. Association and IDEA WILD to CP, from the Swedish Bremer K. 1988. The limits of amino acid sequence data in Research Council to SN and NW, as well as from the angiosperm phylogenetic reconstruction. Evolution 42: 795– Academy of Finland to NW (grant number 118369). 803. Brooks DR, Bilewitch J, Condy C, Evans DC, Folinsbee We are grateful to Alex Grkovich, André Freitas, KE, Fröbisch J, Halas D, Hill S, McLennan DA, Andrew Brower, Andrew Warren, Angélico Asenjo, Mattern M, Tsuji LA, Ward JL, Wahlberg N, Zamparo Carol Castillo, Chris Müller, Christian Schulze, D, Zanatta D. 2007. Quantitative phylogenetic analysis in Darrell Kemp, Dave Edge, Elisabet Weingartner, the 21 century. Revista Mexicana de Biodiversidad 78: 225– Fabrice Caulson, George Gibbs, Gerardo Lamas, John 252. Tennent, José Böttger, Juan Grados, Kjell Arne Brower AVZ. 2006. The how and why of branch support and Johanson, Marta Vila, Michael Braby, Michel Tarrier, partitioned branch support, with a new index to assess Minna Miettinen, Roger Grund, Tim Davenport, partition incongruence. Cladistics 22: 378–386. Tomasz Pyrcz, Tony Nagypal, Torben Larsen, and Concha I, Parra LE. 2006. Análisis cualitativo y cuantitativo Williams Paredes for providing specimens and DNA de la diversidad de mariposas de la estación biológica Senda sequences used in this study. We thank Andrew Darwin, Chiloe, X Región, Chile. Guyana 70: 186–194. Brower, Keith Willmott, and Adam Porter for provid- Dennis RLH, Eales HT. 1997. Patch occupancy in ing useful comments on the manuscript. 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