Biol. Lett. (2008) 4, 274–278 Wheat et al. 2007), but the broader implications of doi:10.1098/rsbl.2008.0062 this to the evolutionary history of butterflies are just Published online 25 March 2008 beginning to be explored (Wheat et al. 2007). Evolutionary biology Butterflies in Satyrinae feed on monocotyledonous plants (Ackery 1988) and the bulk of species (tribe Satyrini, approx. 2200 species) almost exclusively use Prehistorical climate grasses (Pen˜a et al. 2006). Grasses are known to have been involved in coevolutionary relationships with change increased grazing vertebrates (MacFadden 2005; Prasad et al. 2005) that have developed adaptations for coping diversification of a group with the high levels of silica in grass leaves, which of butterflies increases their abrasiveness. However, the interactions between grasses and the megadiverse insects are Carlos Pen˜ a1,* and Niklas Wahlberg1,2 not well studied. Although some groups of insects 1Department of Zoology, Stockholm University, 106 91 Stockholm, that feed on grasses are diverse, these are mainly Sweden sap sucking (Dietrich et al. 1997), and there are very 2Laboratory of Genetics, Department of Biology, University of Turku, few cases of insects grazing on grasses. It is known 20014 Turku, Finland that silica content wears out the mandibles of lepi- *Author for correspondence ([email protected]). dopteran larvae (Drave´ & Lauge´ 1978) and silica Satyrinae butterflies (Lepidoptera: Nymphali- ingestion impairs absorption of nitrogen, affecting dae) and grasses (Poaceae) are very diverse and growth and fitness (Van Soest & Jones 1968; Smith distributed worldwide. Most Satyrinae use et al. 1971; Massey et al. 2006). Larvae of satyrine grasses as host plants, but the temporal scale of butterflies are all external grazers of their host plants, this tight association is not known. Here, we and most species feed exclusively on grasses. Any link present a phylogenetic study of Satyrinae butter- flies and related groups, based on 5.1 kilobases between satyrine butterflies and grasses can only be from six gene regions and 238 morphological understood by placing a robust phylogenetic characters for all major lineages in the ‘satyrine hypothesis of the butterfly subfamily in a temporal clade’. Estimates of divergence times calibrated framework and comparing this with the time frame of using a fossil from the Late Oligocene indicate the diversification of grasses. Dating butterfly lineages that the species-rich tribe Satyrini diversified to has been recently attempted for some groups with the its current 2200 species simultaneously with the aid of molecular techniques (Braby et al. 2006; expansion and radiation of grasses during the Wahlberg 2006; Wheat et al. 2007), but nothing is dramatic cooling and drying up of the Earth in known about ages of diversification in Satyrinae. theOligocene.Wesuggestthattheadaptive Here, we present insights into the diversification of radiation of grass feeders in Satyrini has been facilitated by the ubiquitousness of grasses since Satyrinae butterflies by employing an estimate of 25 Myr ago, which was triggered by a change in divergence times of Satyrinae butterflies based on a global climate. robust phylogeny calibrated using a fossil from the Late Oligocene. Keywords: climate change; host plants; butterfly; evolutionary history 2. MATERIAL AND METHODS In order to obtain a phylogenetic hypothesis for Satyrinae butterflies and related subfamilies, estimate the dates of diversification and 1. INTRODUCTION explore the role of grasses in its patterns of evolution, we analysed The highly diverse butterfly subfamily Satyrinae data from 238 morphological characters and 5143 base pairs of DNA sequences from five nuclear genes and one mitochondrial (Nymphalidae) comprises approximately 2500 species gene, for 79 Satyrinae taxa and out-groups representing all three that are distributed worldwide and dominate butterfly extant subfamilies and all 15 extant tribes of the ‘satyrine clade’ communities in several habitats (Pyrcz & Wojtusiak (Wahlberg et al. 2003; Pen˜a et al. 2006; Wahlberg & Wheat in press; see electronic supplementary material). Within the most 2002). The evolutionary history of Satyrinae is closely diverse tribe Satyrini, all 13 subtribes are represented. We tied to the evolutionary history of grasses (Poaceae) performed a maximum-parsimony analysis of the complete dataset on which the majority of species are specialized. using unordered and equally weighted characters. We also analysed Grasses are globally distributed with more than the combined dataset using Bayesian inference (see electronic supplementary material). We used the resulting phylogenetic 10 000 species and are important components of hypotheses to estimate divergence times using the rate smoothing ecosystems providing livelihood for a variety of organ- method of penalized likelihood (PL; Sanderson 2002), with a fossil isms including humans (e.g. cereals and sugarcane; from the Late Oligocene (Nel et al. 1993) as a fixed calibration Osborne & Beerling 2006). However, the investi- point of 25 Myr ago (figure 1). gation of the origin and times of diversification of butterflies, such as Satyrinae, is hindered by the lack 3. RESULTS of higher level phylogenies and scarce fossil record Phylogenetic analyses using diverse methods (see (Braby et al. 2006). These issues have prevented the electronic supplementary material) resulted in a well- study of key chronological events in the evolutionary resolved phylogeny, in which the three subfamilies history and biogeography of the group (Wheat et al. are strongly supported as independent lineages, with 2007). Recent studies suggest that the origin of Calinaginae being sister to CharaxinaeCSatyrinae butterflies dates back to beyond the Cretaceous– (figure 1). Within Satyrinae, our current results largely Tertiary boundary (Braby et al. 2006; Wahlberg 2006; corroborate those of a previous study based on three Electronic supplementary material is available at http://dx.doi.org/ gene sequences (Pen˜a et al. 2006), i.e. the traditional 10.1098/rsbl.2008.0062 or via http://journals.royalsociety.org. concept of Satyrinae is polyphyletic without the Received 1 February 2008 274 This journal is q 2008 The Royal Society Accepted 4 March 2008 Biol. Lett. Calinaga Agatasa Prothoe Polyura (2008) Charaxes 5 Euxanthe Archaeoprepona Agrias Prepona Anaeomorpha 6 Palla Charaxinae Hypna Siderone (350) Zaretis Coenophlebia Anaea Polygrapha Consul Memphis Antirrhea Caerois Morphini (42) Morpho 4 Bia Narope Opoptera Caligo Dasyophthalma Brassolini Dynastor Brassolis (94) Catoblepia Opsiphanes 3 Hyantis Melanitis Melanitini Manataria Aeropetes +Dirini (48) Diversification of Satyrinae butterflies Dira Xanthotaenia Ethope Zethera Neorina Zetherini (22) Penthema Elymnias Discophora Elymniini (46) 2 Faunis Taenaris Stichophthalma Amathusiini Thauria Amathusia (110) Amathuxidia Zeuxidia Haetera Ragadia Haeterini (21) Pararge Lethe corbieri (calibration point 25 Mya) Mycalesis Lethe 1 Satyrini: 36.6 5.1 Mya Neope Zipaetis Erites 2 Amathusiini: 48.6 5 Mya Orsotriaena Heteronympha 3 Melanitini: 52.6 4.6 Mya Coenonympha 1 Hypocysta 4 Morphini: 56.1 6.3 Mya Oressinoma Melanargia Satyrini Paralasa 5 Charaxinae: 51.7 5.7 Mya C. Pen Taygetis (ca. 2200) Euptychia 6 Satyrine clade: 80.5 4.9 Mya Ypthima Manerebia Pampasatyrus Pedaliodes ˜ Pronophila a & N. Wahlberg 275 Brintesia Satyrus Erebia Hyponephele K/T Maniola boundary Paleocene Eoccene Oligocene Miocene Pliocene 100 80 60 40 20 0 Myr ago Figure 1. (Caption opposite.) 276 C. Pen˜a & N. Wahlberg Diversification of Satyrinae butterflies Figure 1. (Opposite.) Estimated times of divergence by PL using the topology of the Bayesian tree. Relative ages were calibrated using the age of Lethe corbieri (25 Myr ago as minimum age) as the split between Lethe and Neope. Numbers in parentheses indicate the number of extant species for the higher level taxa. Libythea Danaus Calinagini Charaxinae 12 2,4,5,7 6 Brassolini Morphini 15 Melanitini 16 1 1 Dirini Zetherinii 6 3,8,9,10,11,17 Amathusiini larval host plants 16 dicot feeders Elymniini Poaceae feeders 4 Arecaceae feeders 15 5,7 Haeterini Poales (12,15,16) 13,14 18 Arecaceae (1) Satyrini Zingiberales (2,4,5,6,7) other monocots (3,8,9,10,11,13,14,17) Lycopodiopsida and mosses (18) loss of host plants Figure 2. Optimization of larval host plant clades mapped onto ‘satyrine’ tribal level phylogeny reduced from the Bayesian tree. Host plant characters: 1, Arecaceae; 2, Cannaceae; 3, Smilacaceae; 4, Heliconiaceae; 5, Marantaceae; 6, Musaceae; 7, Zingiberaceae; 8, Agavaceae; 9, Liliaceae; 10, Orchidaceae; 11, Pandanaceae; 12, Bromeliaceae; 13, Restionaceae; 14, Xyridaceae; 15, Cyperaceae; 16, Poaceae; 17, Flagellariaceae; 18, lower plants (Lycopodiopsida and mosses). inclusion of the tribes Morphini, Brassolini and diversification, taking place as recently as 36 Myr ago, Amathusiini. The clade that comprises the species-rich after the appearance of grasses, which took place tribe Satyrini diversified after a relatively long branch between the Late Cretaceous and the Early Tertiary (figure 1). (65 and 55 Myr ago). We also identified five major Our results provide evidence for an age of origin plant colonization events by the major lineages of butterflies older than the 70 Myr ago frontier of butterflies in the satyrine clade, which took (Vane-Wright 2004), in agreement with an implied place considerably after the main diversification and age obtained from molecular dating of butterflies in radiation of angiosperms