Systematics and evolutionary history of butterflies in the “Taygetis clade” (Nymphalidae: Satyrinae: Euptychiina): Towards a better understanding of Neotropical biogeography

Molecular Phylogenetics and Evolution 66 (2013) 54–68

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Systematics and evolutionary history of butterflies in the ‘‘Taygetis clade’’ (Nymphalidae: Satyrinae: Euptychiina): Towards a better understanding of Neotropical biogeography Pável F. Matos-Maraví a,⇑, Carlos Peña a, Keith R. Willmott b, André V.L. Freitas c, Niklas Wahlberg a a b c

Laboratory of Genetics, Department of Biology, University of Turku, 20014 Turku, Finland McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, FL 32611-2710, USA Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, PO Box 6109, 13083-970 Campinas, SP, Brazil

a r t i c l e

i n f o

Article history: Received 29 February 2012 Revised 29 August 2012 Accepted 6 September 2012 Available online 18 September 2012 Keywords: Historical biogeography Lepidoptera Molecular phylogeny Neogene Neotropics Quaternary

a b s t r a c t The so-called ‘‘Taygetis clade’’ is a group of exclusively Neotropical butterflies classified within Euptychiina, one of the largest subtribes in the subfamily Satyrinae. Since the distribution of the ten genera belonging to this group ranges throughout the entire Neotropics, from lowlands to lower montane habitats, it offers a remarkable opportunity to study the region’s biogeographic history as well as different scenarios for speciation in upland areas. We inferred a robust and well-sampled phylogeny using DNA sequences from four genes (4035 bp in total) using maximum parsimony and Bayesian inference. We estimated divergence times using the Bayesian relaxed clock method calibrated with node ages from previous studies. Ancestral ranges of distribution were estimated using the dispersal–extinction–cladogenesis (DEC) model as implemented in the program Lagrange. We propose several taxonomic changes and recognize nine well-supported natural genera within the ‘‘Taygetis clade’’: Forsterinaria (subsuming Guaianaza syn. nov.), Parataygetis, Posttaygetis, Harjesia (excluding Harjesia griseola and Harjesia oreba), Pseudodebis (including Taygetomorpha syn. nov.,), Taygetina (subsuming Coeruleotaygetis syn. nov., Harjesia oreba comb. nov., Taygetis weymeri comb. nov. and Taygetis kerea comb. nov.), Taygetis (excluding Taygetis ypthima, Taygetis rectifascia, Taygetis kerea and Taygetis weymeri), and two new genera, one containing Harjesia griseola, and the other Taygetis ypthima and Taygetis rectifascia. The group diversified mainly during late Miocene to Pliocene, coinciding with the period of drastic changes in landscape configuration in the Neotropics. Major dispersals inferred from the Amazon basin towards northwestern South America, the Atlantic forests and the eastern slope of the Andes have mostly shaped the evolution and diversification of the group. Furthermore, expansion of larval dietary repertoire might have aided net diversification in the two largest genera in the clade, Forsterinaria and Taygetis. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Geological processes and climate changes during the Neogene and the Quaternary have dramatically shaped the evolution of Neotropical landscapes and biota (e.g. Whitmore and Prance, 1987; Jackson et al., 1996; Gregory-Wodzicki, 2000; Hoorn and Wesselingh, 2010). Biogeographic hypotheses concerning the origin and distribution of species in the Neotropics often involve geographic and environmental disturbances, for example the accelerating uplift of the Andes cordillera during the Miocene–Pliocene (starting at 23 million years ago (Mya) and intensifying at 12–4.5 Mya) (Hooghiemstra and Van der Hammen, 1998; Bush and De Oliveira, 2006; Hoorn et al., 2010), the dynamic lacustrine–fluvial systems and the establishment of the Amazon river ⇑ Corresponding author. Fax: +358 2 333 6680. E-mail address: [email protected] (P.F. Matos-Maraví). 1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.09.005

by 7 Mya in South America (Räsänen et al., 1995; Lundberg et al., 1998; Nores, 1999; Wesselingh, 2008), the closure of the Isthmus of Panama around 3 Mya followed by the ‘‘Great American Biotic Interchange’’ (GABI) (Coates and Obando, 1996; Cox and Moore, 2000; Kirby et al., 2008), and the severe palaeoclimatic fluctuations (glacial/interglacial cycles) during the Quaternary (the last 2.5 Mya) (Haffer, 1969; Hooghiemstra and Van der Hammen, 1998). Increased diversification in lineages coincident with such changes in the environment might be a result of either increased speciation rates or reduced extinction rates (Wiens and Donoghue, 2004; Rabosky, 2009). Sudden changes in diversification can potentially be recognized using well-sampled and dated phylogenies as well as models designed to identify significant turnover in diversification rates (Ricklefs, 2007; Alfaro et al., 2009; McInnes et al., 2011). Evidence from studies dealing with molecular data (Rull, 2008) or the fossil record (Jaramillo et al., 2006) of Neotropical taxa

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suggests that the main diversification events at genus and species levels of animals and plants date back to the Miocene–Pliocene epochs. Similarly, not only the tempo but also the mode of diversification has been discussed. For instance, the Andean uplift may have played a role in promoting diversification (Antonelli et al., 2009) by providing new habitats and opportunities for adaptive radiation (Willmott et al., 2001; Elias et al., 2009), or stability and therefore lower extinction rates thus permitting species accumulation (Fjeldså, 1994) or by providing opportunities for allo- or parapatric speciation (Adams, 1985; Willmott et al., 2001; Sedano and Burns, 2010). Allopatric speciation as a consequence of climate variation has also been suggested as being the result of forest contractions and expansions during the Pleistocene: the forest refugia hypothesis (Haffer, 1969). Studies on Neotropical Lepidoptera have focused on a number of these hypotheses. For example, Willmott et al. (2001) found speciation within elevational zones and montane regions, suggestive of adaptive radiation, to be the most common mode of speciation in Hypanartia butterflies. In contrast, Hall (2005) found upward or ‘‘vertical’’ parapatric speciation to be more important in montane riodinid butterflies. Alternatively, early butterfly lineages might have pre-adapted to cooler regions at higher latitudes such as the mountain ranges in southeastern Brazil, before spreading and colonizing more tropical montane habitats such as the eastern slope of the Andes (Willmott et al., 2001; Viloria, 2003; Hall, 2005). However, little has been discussed about the potential role of other ecological processes on butterfly diversification (e.g. fluctuations in larval dietary breadth and geographic range size – the ‘‘oscillation hypothesis’’ (Janz and Nylin, 2008; see also Jorge et al., 2011)) in the Neotropics, mainly due to the low number of well-sampled phylogenies published to date and typically a poor knowledge of the natural history of those butterflies (but see Jiggins et al., 2006; Elias et al., 2009). Nevertheless, regardless of the main factors driving speciation, most dated molecular phylogenies of Neotropical Lepidoptera suggest a pre-Pleistocene origin of both highland and lowland species, influenced by the major uplift of the Northern Andes during the Miocene (Wahlberg and Freitas, 2007; Elias et al., 2009; Peña et al., 2010; Casner and Pyrcz, 2010; Strutzenberger and Fiedler, 2011; but see Mullen et al., 2011) while only infraspecific differentiation is likely to have happened during the Pleistocene (Whinnett et al., 2005). The ‘‘Taygetis clade’’ (sensu Murray and Prowell, 2005; Peña et al., 2010) is one of five major groups in the highly diverse subtribe Euptychiina (Nymphalidae, Satyrinae, Satyrini). The 10 genera composing the clade are grouped into a single and robust monophyletic entity based on molecular data (Peña et al., 2010). However, the phylogenetic relationships among these genera are yet poorly defined and some genera are likely to not be monophyletic. Morphological traits are in general highly similar across closely related species, providing few informative characters for phylogenetic studies (Lamas, 2004; Murray and Prowell, 2005; Peña and Lamas, 2005; Freitas and Peña, 2006; Brown et al., 2007; Freitas, 2007; Pulido and Andrade, 2008; Peña et al., 2010). The clade is confined to the Neotropics (Peña et al., 2010) and mainly restricted to lowlands, although some members of the genus Taygetis and all species of Forsterinaria occur in lower Andean and/or southeast Brazilian montane habitats. Larvae feed largely on diverse genera of bambusoid plants (Poaceae) (Miller, 1978; Young, 1984; Ackery, 1988; Murray, 2001; Peña and Lamas, 2005; Freitas and Peña, 2006; Beccaloni et al., 2008). Based on a calibrated phylogeny using a relaxed molecular clock method (Peña et al., 2010, 2011), the ‘‘Taygetis clade’’ appears to have originated around the mid-Miocene, while the main diversification of genera and species happened before the Pleistocene in tropical South America (Peña et al., 2010). However, both the tempo and mode of diversification remain to be studied in the ‘‘Taygetis clade’’, a group of particular interest for

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testing a variety of Neotropical biogeographic hypotheses, since its members inhabit both lowland and lower montane environments across the entire Neotropical realm. In the present study, we investigate the systematics and historical biogeography of the ‘‘Taygetis clade’’ as an attempt to elucidate the factors that might have promoted the diversification of these butterflies. We inferred a well-sampled and robust molecular phylogeny of the ‘‘Taygetis clade’’ using DNA sequence data of four genes and estimated the times of major diversification events using the relaxed clock method. Additionally, we used a parametric-based approach to reconstruct the ancestral range distributions of extant taxa to identify the main historical biogeographic events. Finally, we suggest significant taxonomic rearrangements of the ‘‘Taygetis clade’’ based on strongly supported phylogenetic relationships. 2. Material and methods 2.1. Taxon sampling Butterflies of about 52 species within the ‘‘Taygetis clade’’ (sensu Peña et al., 2010) were sampled from several localities across the Neotropical region (Table A1). This sample contained about 75% of the described species for the group (Lamas, 2004; Peña and Lamas, 2005), representing 10 genera and including all generic type species. For some species, more than one individual was included and analyses were carried out to investigate whether individuals cluster together following the DNA barcoding approach (Hebert et al., 2003). Most butterflies were identified based on external morphological characters before sequencing. Some exemplars were labeled as ‘‘nr’’ if species identity was uncertain, and might potentially represent undescribed species. In addition, sequences of seven outgroup taxa from the closely related clades ‘‘Hermeuptychia’’, ‘‘Pareuptychia’’ and ‘‘Splendeuptychia’’ (sensu Peña et al., 2010) were taken from a previous study (Peña et al., 2010) to root the phylogeny. Photographs are available for most voucher specimens in the Nymphalidae Systematics Group (NSG) database (http://nymphalidae.utu.fi/db.php). 2.2. Laboratory procedures Total DNA from two legs preserved either dry or in 96% ethanol was extracted by using QIAGEN’s DNeasyÒ extraction kit and following the manufacturer’s instructions. We amplified four standard gene regions used in previous studies on nymphalid butterflies which have been shown to provide useful phylogenetic resolution at lower taxonomic levels (Kodandaramaiah and Wahlberg, 2007, 2009; Leneveu et al., 2009; Aduse-Poku et al., 2009; Peña et al., 2010, 2011; Müller et al., 2010). The primer pairs used to amplify the mitochondrial gene COI (1487 base pairs (bp)) and the nuclear genes EF-1a (1240 bp), GAPDH (691 bp) and RpS5 (617 bp) are listed in Wahlberg and Wheat (2008). We carried out PCRs in a total volume of 20 ll containing 1 PCR buffer, 2.5 mM MgCl2, 0.5 lM of each primer, 0.2 mM dNTPs, 0.5U of AmpliTaq GoldÒ polymerase and 1 ll of the total extracted DNA. The thermocycling profile used to amplify the segments containing the COI and the Al/EFrcM4 (second half of the EF-1a segment) primer pairs was 95 °C for 5 min, 40 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min 30 s followed by a final extension period of 72 °C for 10 min. For the remaining primer pairs, the program was set as before but with annealing temperature at 55 °C. Successful PCRs were sequenced by the Macrogen company (South Korea) and the resulting chromatograms and DNA sequences were checked, edited and aligned manually using the BioEdit v7.0.5 program (Hall, 1999). We followed the IUPAC nomenclature code when heterozygous sites on nuclear genes were found. DNA

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sequences and voucher data were organized in the software VoSeq (Peña and Malm, 2012). Datasets for phylogenetic analyses as well as FASTA files for submission to GenBank were created in VoSeq. The DNA sequences were deposited in the GenBank database (see Table A1 for accession numbers). 2.3. Data characterization We performed data characterization analyses in order to evaluate the ability of the dataset to recover congruent phylogenetic hypotheses by detecting potential artifacts such as nucleotide bias and nucleotide substitution saturation on each gene partition. We estimated the nucleotide composition and substitution saturation by gene and by codon position using the MEGA4 program (Tamura et al., 2007); in addition, we investigated the robustness of unexpected arrangements by excluding the third codon position of each gene sequence in further analyses and comparing those to fulldataset trees. Furthermore, we found the GTR + G to be the best model of evolution that explains our dataset based on log-likelihood values and Akaike Information Criteria (AIC) as implemented in FindModel (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). 2.4. Phylogenetic analyses We conducted maximum parsimony (MP) and Bayesian inference (BI) analyses using single- and combined-gene datasets. MP analyses as implemented in TNT v1.1 (Goloboff et al., 2003) were executed utilizing heuristic searches and the ‘‘New Technology Search’’ algorithms (Goloboff, 1999; Nixon, 1999) with 100 random additions. Clade support values were measured by using the resampling-based bootstrap approach (Felsenstein, 1985) performing 1000 pseudo-replicates and 10 independent replicates. The congruence among partitions in the combined analyses was evaluated with the Partitioned Bremer Support (PBS) (Baker and DeSalle, 1997) using the strict consensus tree recovered by the MP analysis and a script written by Carlos Peña (in Peña et al., 2006; available at http://bit.ly/xfZMa3). BI analyses were carried out in MrBayes v3.1 (Ronquist and Huelsenbeck, 2003) on the CIPRES Science Gateway portal v3.1 (http://www.phylo.org/sub_sections/portal/). In addition to gene partitions, we set partitions by codon position, which has been found to be informative for BI (Rota, 2011). The calculations were run twice for 10 million generations each so that the final average standard deviation of split frequencies was below 0.05, sampling trees every 1000 generations and using four simultaneous chains (one cold and three hot) in each run. The parameters and the model of evolution GTR + G were unlinked across character partitions. We discarded as burn-in the first one thousand sampled trees that were not within the stationary distribution of log-likelihoods. Trees and posterior probabilities were summarized using the ‘‘50%-majority rule’’ consensus method. 2.5. Time of divergence estimates We used the BEAST v1.5.2 program (Rambaut and Drummond, 2007) through the Bioportal site, University of Oslo (http:// www.bioportal.uio.no/). We imposed the GTR + G model and the uncorrelated log–normal distribution under the relaxed clock model independently for each gene partition. Priors were left as default except for the ‘‘tree prior’’ which was set to the Birth–Death Speciation option. Two non-independent calibration points were taken from a previous work based on a satyrine fossil age (Peña et al., 2010) to constrain the basal crown age of divergence of the ‘‘Taygetis clade’’ (sensu Peña et al., 2010) at 15 ± 4 Mya (normal prior distribution) and the split between the species Taygetis ypthi-

ma and Taygetis rectifascia between 10 and 4 Mya (uniform prior distribution). The analysis was run for 10 million generations and the log parameter estimates sampled every 1000 generations with an initial burn-in of 100,000 generations. We conducted independent replicates four times using the same parameters. Trees were combined and summarized using LogCombiner v1.5.2 and TreeAnnotator v1.5.2 respectively (both software are included along with the BEAST package) after verifying the credibility of each parameter estimate by the Effective Sample Sized (ESS) values in Tracer v1.5 (included in the BEAST package). The estimated divergence times on each node were summarized as mean heights on the maximum clade credibility tree. A Lineage-Through Time (LTT) plot was constructed using the ape package (Paradis, 2004) in R (R Development Core Team, 2010) from an ultrametric tree displaying one single terminal per species (built in BEAST using the same settings as described above). As estimating diversification rates and potential turnover during the evolution of the ‘‘Taygetis clade’’ is beyond the scope of this study, we only used the shape of the LTT curve to visualize the span of time when the extant diversity originated as well as to identify possible sudden changes against a constant net diversification rate background. In addition, we ran simulations in R (ape, geiger and laser packages) in order to detect any significant deviation from a pure birth null hypothesis (constant speciation (k) and zero extinction (l) rates). We approximated k from our ultrametric phylogeny and used it to replicate 1000 trees of the same root depth (crown age) and same number of terminals as our phylogeny. We assigned confidence intervals based on the distribution of our simulation (null hypothesis) and plotted the actual LTT curve plot against this background (Fig. 3). In order to account for incomplete taxon sampling, we additionally ran the tree replications under the same parameters as described above, but with 69 extant terminals (total number of ‘‘Taygetis clade’’ described species (Lamas, 2004; Peña and Lamas, 2005)) (Fig. A1). 2.6. Optimization of ancestral geographic areas of distribution The Neotropical realm was subdivided into eight major geographical zones based on general patterns of extant butterfly and insect distributions (Lamas, 1982; Viloria, 2003; Morrone, 2006) as well as accounting for the palaeogeographic history of each region throughout the Neogene-Quaternary (Hulka et al., 2006; Kirby et al., 2008; Hoorn and Wesselingh, 2010). Fig. 4 depicts the regions: A – Mesoamerican lowlands; B – Mesoamerican montane areas; C – North and northwestern South America, including the Orinoco basin, the tepuis of the Guiana Shield and northern and western Colombian lowlands; D – the Amazon basin, including both western and eastern Amazonia; E – the Chacoan region, including the Brazilian Cerrado; F – Atlantic forests in southeastern Brazil; G – Lower montane areas on the eastern slope of the Andes; and H – Lower montane areas on the western slope of the Andes. As montane areas, we considered those habitats where the elevations are higher than 500 m. Distribution of species was defined as binary code of presence-absence for a particular biogeographic region. Taxa might thus have more than one distributional or elevational area based on information from the literature (e.g. references listed at ftp://ftp.funet.fi/pub/sci/bio/life/intro.html), sampling data from specimens kept at the Natural History Museum in Lima and in the NSG collection, and the authors’ personal observations. We used the parametric-based method as implemented in Lagrange v.C++ (Ree and Smith, 2008) to infer the biogeographic processes that might have shaped the evolution and distribution of the ‘‘Taygetis clade’’. This method is based on dispersal– extinction–cladogenesis (DEC) models and requires information coming from a single ultrametric dated phylogeny (we used the

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same as for the LTT plot) and distributional information of extant species (see above). A first analysis (L1) was set to unconstrained ‘‘maxareas’’, thus allowing every combination of geographic areas on the ancestral ranges reconstruction at every node. A second analysis (L2) accounting for area connectivity and probabilities for dispersal through time was run by incorporating the main palaeogeographic events that have occurred during the past 25 Mya, dividing the entire span of evolution of the ‘‘Taygetis clade’’ into four major periods (Figs. 4 and 5). We arbitrarily set the different probability values to dispersal between geographic areas in order to recreate the connectivity among the areas during the four periods of time (see Buerki et al., 2011). Dispersal rates were set to 0.5 when two areas were contiguous, to 0.1 when two areas were separated by another one, and to 0.05 when dispersal between two areas necessarily spanned two additional areas. Dispersals spanning more than two additional areas were not allowed. In addition, rates were set to 10 4 or multiplied by 10 1 for dispersals from/towards Mesoamerica during the periods between 24.0–11.0 and 7.0–11.0 Mya respectively in order to recreate the Isthmus of Panama uplift history. Furthermore, rates were multiplied by 10 1 for dispersals involving shifts in elevational ranges (i.e. from lowlands to montane habitats and vice versa) as an attempt to include physiological constraints for dispersal. We believe that reductions by orders of magnitude of dispersal rates, although arbitrary, provide a very conservative counterpart to L1 analysis (null hypothesis with no stratification). All probability values are shown in the matrix presented in the Fig. 5. 3. Results 3.1. Nucleotide sequence characteristics The entire dataset consists of 4035 bp of which 1086 bp (27%) were phylogenetically informative sites. Base frequency estimations show a strong AT bias in the COI gene sequence (72%), strikingly at its third codon position (94%). A similar pattern was reported in other studies of euptychiine butterflies (Murray, 2001; Murray and Prowell, 2005). Nonetheless, phylogenetic trees inferred from analyses excluding the third codon position (phylogenies not shown) are largely congruent to those including the fullcharacter datasets, recovering major groups in the ‘‘Taygetis clade’’ but less resolved due to missing informative sites. All three nuclear genes display similar levels of variation and base composition without any significant nucleotide bias. Estimated mutation rates and nucleotide frequency values for the models used by gene region in the Bayesian inference are given in Table 1. 3.2. ‘‘Taygetis clade’’ phylogenetic relationships DNA barcodes appear to successfully identify the sampled species by clustering their COI sequences in accordance with our designations. In many cases each specimen was also correctly identified at the species level by the BOLD Identification System (www.boldsystems.org). We provided new barcode records for species that are not represented in public databases. The monophyly of the group was previously reported based on molecular characters (Murray and Prowell, 2005; Peña et al., 2010).

In this study, we propose a robust phylogenetic hypothesis for all major lineages within the clade (Figs. 1 and 2) including most of the described species (Lamas, 2004; Peña and Lamas, 2005). Single gene datasets (phylogenies not shown) recovered similar topologies to the combined-gene analyses. Additionally, the level of conflict between different gene partitions estimated using the partitioned Bremer support was fairly low (Fig. 1). The resulting phylogenetic hypothesis recovers the genus Harjesia as a polyphyletic entity, regardless of method used. Harjesia griseola is sister to the remaining taxa in the ‘‘Taygetis clade’’ while Harjesia oreba is phylogenetically distant from other Harjesia species as it is placed within the group composed of the monotypic Taygetina banghaasi and Coeruleotaygetis peribaea as well as the species Taygetis weymeri and Taygetis kerea (hereafter the group is called the Taygetina subclade) (Figs. 1 and 2). Furthermore, Harjesia blanda and Harjesia obscura form a clade closely related to an unresolved group including the genera Parataygetis, Posttaygetis, Guaianaza and Forsterinaria. The lack of resolution in the latter group, however, is not due to a conflicting signal amongst partitions (evidenced by the positive partitioned Bremer support values) but it might be related to a rapid split of lineages shown by the short branches close to the crown node of the group in both single- and combined-dataset Bayesian trees (Figs. 2 and 3). The monotypic genus Guaianaza, on the other hand, is subsumed within Forsterinaria consistently in all analyses (Figs. 1–3) while the weakly-supported position of Forsterinaria quantius does not allow confirmation or rejection of the monophyly of Forsterinaria. Nevertheless, the lower montane Andean Forsterinaria are recovered as a monophyletic group that is highly resolved and congruent among different datasets and among the trees recovered by MP and BI (Figs. 1 and 2). Moreover, these Andean species are the sister group of {Forsterinaria necys + Guaianaza}, a clade which is restricted to the Paraná basin (southeastern Brazil, Paraguay and northern Argentina) and the Brazilian Cerrado. The genus Pseudodebis was recovered as paraphyletic, including also the members of Taygetomorpha. The close relationship between Taygetomorpha and Pseudodebis marpessa is confirmed in all analyses, with high support values. The clade composed by Taygetis ypthima and Taygetis rectifascia appears to be sister to the group formed by {Pseudodebis + Taygetomorpha}; altogether these taxa constitute a monophyletic group (hereafter the Pseudodebis subclade) sister to the Taygetina subclade and the genus Taygetis. The genus Taygetis, on the other hand, is defined as a strongly-supported monophyletic entity including all of the remaining valid Taygetis species (Lamas, 2004) except those discussed before (i.e. T. kerea, T. weymeri, T. rectifascia and T. ypthima). Three highly supported and congruent subgroups within the genus Taygetis are recognized, hereafter named the ‘‘mermeria-group’’, the ‘‘virgiliagroup’’ and the ‘‘laches-group’’ (Figs. 1 and 2). The phylogenetic position of T. angulosa and T. leuctra was not clearly elucidated within the genus Taygetis but they apparently do not belong to any of the subgroups defined above. 3.3. Divergence time estimates The tree topology recovered by the analyses in BEAST is congruent with the topologies found by the MP and BI analyses (Fig. 3).

Table 1 Estimated parameters by gene region using the Bayesian inference method. Gene

TL (all)

r(A M C)

r(A M G)

r(A M T)

r(C M G)

r(C M T)

r(G M T)

pi(A)

pi(C)

pi(G)

pi(T)

alpha

m

COI EF-1a GAPDH RpS5

2.81

0.084 0.048 0.079 0.077

0.121 0.251 0.267 0.25

0.057 0.115 0.122 0.152

0.012 0.039 0.059 0.043

0.717 0.488 0.422 0.414

0.009 0.06 0.052 0.064

0.318 0.273 0.285 0.255

0.093 0.25 0.21 0.226

0.132 0.225 0.186 0.233

0.457 0.252 0.318 0.286

0.23 0.214 0.179 0.175

1.823 0.51 0.501 0.56

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Fig. 1. Strict consensus phylogeny of the 22 equally parsimonious trees (length 6750) inferred from the maximum parsimony analysis of the combined dataset. Numbers above each branch are the bootstrap and Bremer support values, and numbers below are the partitioned Bremer support values for COI, EF-1a, GAPDH and RpS5 datasets, respectively, for the node to the right of the numbers. Branches are colored by genus (Lamas, 2004). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Consensus phylogeny from the Bayesian inference analysis of the combined dataset. Numbers next to the nodes are their respective posterior probabilities, and branch lengths represent expected substitutions per site. Branches are colored by genus (Lamas, 2004). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Times of divergence estimates for the ‘‘Taygetis clade’’. (A) Ultrametric tree scaled in Mya. Numbers and horizontal bars on nodes represent posterior probability values and 95% credibility intervals. Available larval host plant data (see text) are mapped onto the phylogeny, next to each terminal. (B) LTT plot of extant lineages (log) vs. time (Mya). Confidence intervals from a pure birth diversification hypothesis of ‘‘Taygetis clade’’ are shown as the distribution of 1000 simulated trees (see text).

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Credibility intervals for age estimates of each node are rather wide, particularly those near to the root which should be expected when using secondary calibrations (Graur and Martin, 2004). Thus, when taking into account the credibility intervals over nodes on the tree, we found the diversification of the ‘‘Taygetis clade’’ to have likely started during the mid-Miocene while all extant genera originated during the middle to late Miocene (Fig. 3). Most of the species originated through the late Miocene to the middle to late Pliocene except for a very few taxa that originated during the Pleistocene

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(Fig. 3). The diversification pattern does not apparently follow a pure birth model during the early Pliocene as the LTT curve lies just outside of the 99% confidence interval of the null hypothesis distribution (Fig. 3B). However, accounting for missing taxa in our simulations made the replicated tree distribution slightly more convex and thus our estimated LTT curve fits better with the pure birth hypothesis (Fig. A1). Moreover, the taxa not included in the study are likely to be randomly scattered throughout the phylogeny based on the wing pattern similarity to the included species; thus

Fig. 4. Stratified ancestral areas of distribution (analysis L2). The analysis was run dividing the entire time span of evolution of the ‘‘Taygetis clade’’ in four periods (a. 24– 11 Mya; b. 11–7 Mya; c. 7–2.5 Mya; and d. 2.5 Mya – present). Pie charts on each node depict the relative probabilities of ancestral ranges (states). Those reconstructions whose probabilities were lower than 0.2 where combined in the ‘‘remainder’’ category (black sections of the pie charts). A: Mesoamerica; B: Montane Mesoamerica; C: Northern South America; D: Amazon basin; E: Chaco and Cerrado; F: Atlantic forests; G: Lower eastern montane Andes; H: Lower western montane Andes. Distributions of extant species are represented by colored squares next to each terminal in the tree corresponding to the colors on the map. Time axis (in Mya) is annotated with major geologic events (green boxes) and the time slices used in the analysis (gray boxes and red lines across the phylogeny) at the top of the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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it is expected that the shape of the LTT curve would not change significantly with complete taxon sampling. Nonetheless, changes in diversification rates in some groups within the ‘‘Taygetis clade’’ cannot be ruled out as LTT plots in general have been shown to be susceptible to missing taxa due to extinction and might even mislead interpretations on diversification turnover (Wiens and Donoghue, 2004; Rabosky, 2009, 2010). Those species whose origins were likely to be in the Pleistocene are distributed in three clades with species-complexes of highly similar cryptic taxa (including several undescribed species), for instance (1) the laches-complex (Murray, 2001) including the species Taygetis laches, T. thamyra, T. sosis, T. echo and T. uncinata, (2) the morphologically very similar species in the Taygetis virgilia and T. rufomarginata complex, and (3) the clade formed by the members of the boliviana-group (sensu Peña and Lamas, 2005) and three other species within Forsterinaria whose genitalia characteristics are rather homogeneous (Peña and Lamas, 2005). The rapid diversification of Harjesia blanda, H. obscura, Parataygetis, Posttaygetis and Forsterinaria is evidenced by the low posterior probabilities on nodes supporting those lineage groups and the short internal branches. The genus Forsterinaria began to diversify during the late Miocene and most of the taxa represented by the lower montane Andean species diversified throughout the Pliocene and early Pleistocene. The phylogenetic relationships be-

tween the Pseudodebis subclade, the Taygetina subclade and the genus Taygetis are well defined, and the split between them happened during the mid- to late Miocene while the diversification of extant species within the two defined subclades occurred during the late Miocene but before the Pliocene epoch. The relatively speciose genus Taygetis went through a process of apparent ‘‘continuous’’ diversification during the late Miocene, Pliocene and early Pleistocene, although confidence intervals in some cases suggest an even later divergence, clearly seen within the ‘‘laches-group’’. 3.4. Ancestral distribution reconstruction In general, there is a high correspondence between the outcomes of the two analyses performed in Lagrange (L1 and L2). On certain nodes, however, there was evidence of conflict in both the level of relative probabilities for the same ancestral areas and the distinct inferred ancestral ranges, particularly evidenced on those nodes where phylogenetic support values were moderate to low. Incorporating a correlation between dispersal rates through time and the time of divergences in Lagrange L2 (Fig. 4), on the other hand, increases the probabilities of ancestral areas on certain nodes compared to L1, though the general biogeographic patterns were recovered similarly to the unconstrained Lagrange analysis L1 (Fig. A2).

Fig. 5. The four periods used in the stratified biogeographic analysis in Lagrange and the respective dispersal rates across areas A–H used for each time slice in the analysis. Maps are modified from Morrone (2006), Kirby et al. (2008) and Hoorn et al. (2010). Time slice (TS) 1: Origin of the ‘‘Taygetis clade’’ in the middle Miocene in South American rainforests. TS 2: Major dispersal towards northern and southeastern South America followed by local diversification during the late Miocene. TS 3: Establishment of the Amazon River and colonization of the eastern slope of the Andes, which favored speciation in the Pliocene. TS 4: The Isthmus of Panama fully emerged, promoting dispersal in the Quaternary. Assigned dispersal rates by time period are shown in the tables: rates in red are the product of multiplication by 10 1 for dispersals involving elevational shifts (see text).

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The biogeographic reconstructions (Fig. 5) suggest unambiguously that the area of origin of the ‘‘Taygetis-clade’’ was the Amazon basin. Further dispersal into southern and temperate regions such as the Chacoan and the Atlantic Forest in southeastern Brazil early in the evolution of the ‘‘Taygetis-clade’’ was recovered by both analyses in Lagrange (Figs. 4 and A2). Notably, the geographic origin of Forsterinaria appears to include the regions in the southeastern mountains of Brazil, while the Andean lineage spread and colonized the eastern slope of the Andes later, diversifying mostly in lower tropical montane habitats. Similarly, ancestors of the Pseudodebis subclade were distributed throughout Amazonia and the Atlantic forest regions, whereas Taygetis ypthima and Taygetis rectifascia have distributions restricted to the Atlantic forests in southeastern Brazil, Argentina and Paraguay and the southern portions of the cerrados. The genus Taygetomorpha, on the other hand, expanded its range of distribution towards most of the regions included in the analyses. Similarly, the ancestral lineages of the Taygetina subclade might have spread northwards, colonizing tropical lower montane landscapes and the Chacoan and Atlantic forest regions later. Uncertainties are shown regarding the ancestral distributions of Harjesia, Posttaygetis and Parataygetis, although it is deduced that those taxa have expanded their geographical ranges from the Amazon basin further into southeastern Brazil, as in the case of Harjesia and Posttaygetis, and to lower montane forests on the eastern slope of the Andes and in Mesoamerica in the case of Parataygetis. 4. Discussion 4.1. Systematics of the ‘‘Taygetis clade’’ Our inferred phylogenetic hypothesis is robust and suggests some taxonomic changes. The ‘‘Taygetis clade’’ can be subdivided into nine highly supported and natural subgroups recovering to some extent the current taxonomic arrangements at genus level (Lamas, 2004): Forsterinaria (subsuming Guaianaza syn. nov.), Parataygetis, Posttaygetis, Harjesia griseola (whose taxonomic status should be revised, see below), Harjesia (excluding Harjesia griseola and Harjesia oreba, see below), Pseudodebis (including Taygetomorpha syn. nov.), Taygetina (subsuming Coeruleotaygetis syn. nov., Euptychia oreba Butler 1870 comb. nov., Taygetis weymeri Draudt 1912 comb. nov. and Taygetis kerea Butler 1869 comb. nov.), Taygetis (excluding Taygetis ypthima, Taygetis rectifascia, Taygetis kerea and Taygetis weymeri) and one new genus containing Taygetis ypthima and Taygetis rectifascia (see below) (Table 2). Harjesia griseola is sister to the rest of the ‘‘Taygetis clade’’ thus representing an independent lineage which should be ranked as a new genus. This interpretation from the molecular results is corroborated by other morphological data, for instance the genital structure of H. griseola does not display a particularly close resemblance to any other taxon in the clade (A.V.L. Freitas, unpubl. data). Relationships between Harjesia sensu stricto, Forsterinaria, Parataygetis and Posttaygetis are not fully resolved in the molecular phylogeny, although the monophyly of each genus is well-supported and corroborated by other morphological and behavioral traits (Peña and Lamas, 2005; Murray, 2001). A closer relationship between the species Harjesia blanda to the genus Forsterinaria has been suggested based on morphological similarities (Peña and Lamas, 2005), while a close relationship between the genera Parataygetis and Posttaygetis is moderately supported by molecular characters (see also Peña et al., 2010). Furthermore, as previously suggested (Peña et al., 2010), the monotypic genus Guaianaza should be subsumed within Forsterinaria as the former is consistently placed sister to Forsterinaria necys, both of them endemic to southeast Brazil and both displaying certain common morphological features (Freitas and Peña, 2006). Moreover, those taxa

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within Forsterinaria that are distributed along montane habitats in both Central America and the Andes cordillera are recovered as a monophyletic entity whose relationships are resolved with good support values. It has been proposed that the genus Taygetomorpha is closely related to Forsterinaria (L.D. Miller in Lamas (2004)) based on adult characters. Nonetheless, the molecular data consistently place Taygetomorpha within the genus Pseudodebis thus causing the latter to be a paraphyletic entity. Therefore, Taygetomorpha should be considered as a synonym of Pseudodebis in concordance with the results presented here, as well as previous studies on larval morphology and behavior (Murray, 2001), and molecular information (Murray and Prowell, 2005; Peña et al., 2010). Furthermore, both Taygetis ypthima and Taygetis rectifascia are consistently found to be sister to {Pseudodebis + Taygetomorpha} in the Bayesian trees, although with low support in the MP tree. However, these two Taygetis species are highly divergent from their congeneric taxa; thus, they might be classified separately within either Pseudodebis or a new genus closely related to the latter. We believe that T. ypthima and T. rectifascia should be classified within a new genus until further analyses confirm/reject such a hypothesis since Pseudodebis is a rather homogeneous genus, while considerably distinct from the former two species regarding morphology. Karyological data further support a profound divergence in chromosome structure and number between T. ypthima (rather small chromosomes) and other Pseudodebis and Taygetis taxa (Brown et al., 2007). Brown et al. (2007) showed that Taygetis is a particularly variable genus within Euptychiina regarding haploid chromosome number, from n = 8 up to n = 40, as well as with a high degree of variation within certain species. Taygetis ypthima (n = 31, 39) and T. larua (n = 40) are the species with the highest chromosome number in the ‘‘Taygetis clade’’ (the study included 19 species across 5 genera, modal n = 14, and most of the taxa with n = 12–16). However, taken together at the generic level, chromosome number becomes an informative character in this particular group that reinforces the separation of T. ypthima from any other lineage in the clade as shown in our phylogenetic hypothesis. The highly supported Taygetina subclade is formed by five species classified into four different genera. Two of these genera are monotypic (Taygetina and Coeruleotaygetis), and two belong to polyphyletic assemblages (Harjesia and Taygetis). Little is known about the natural history of these five species described from Central America and western Colombia, although some taxa are also found on the eastern slope of the Andes (e.g. H. oreba, T. kerea and T. banghaasi) (Lamas, 2004). Furthermore, the two monotypic genera Taygetina Forster 1964 and Coeruleotaygetis Forster 1964 are shown to be closely related, as suggested by a phylogenetic hypothesis based on morphological characters (Marín et al., 2011). Since this is a well-supported and robust clade, we feel that all species can be included in one single genus, which in this case is Taygetina. We choose Taygetina based on the principle of ‘‘Determination by the First Reviser’’ (ICZN, 1999; article 24). Based on molecular data, the genus Taygetis is a well-supported and robust monophyletic group when T. ypthima, T. rectifascia, T. kerea and T. weymeri are excluded (hereafter Taygetis sensu stricto). There appear to be three consistently recovered groups (the mermeria-, virgilia- and laches-groups) as well as two independent lineages (T. leuctra and T. angulosa). The mermeria-group is sister to the rest of the genus and comprises three species of relatively large satyrines, T. mermeria, T. larua and Taygetis sp. (specimens labeled as ‘‘T. imperator MS’’ in some collections). The virgilia-group contains at least five species of which T. virgilia and T. rufomarginata are highly similar as adults, but distinct as larvae (Murray, 2001). Based on our results, these two species are non-monophyletic, and at least two new species might need to be described after additional morphological work. As an example, the T. virgilia clade

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Table 2 Revised checklist of genera in the subtribe Euptychiina and species within ‘‘Taygetis clade’’. Lamas, 2004 genus name Amphidecta Butler, 1867 Archeuptychia Forster, 1964 Caenoptychia Le, Cerf, 1919 Caeruleuptychia Forster, 1964 Capronnieria Forster, 1964 Cepheuptychia Forster, 1964 Cercyeuptychia Miller and Emmel, 1971 Chloreuptychia Forster, 1964 Cissia Doubleday, 1848 Cyllopsis Felder, 1869 Erichthodes Forster, 1964 Euptychia Hübner, 1818 Euptychoides Forster, 1964 Forsterinaria Gray, 1973

Godartiana Forster, 1964 Harjesia Forster, 1964

Hermeuptychia Forster, 1964 Magneuptychia Forster, 1964 Megeuptychia Forster, 1964 Megisto Hübner, [1819] Moneuptychia Forster, 1964 Neonympha Hübner, 1818 Palaeonympha Butler, 1871 Paramacera Butler, 1868 Parataygetis Forster, 1964 Pareuptychia Forster, 1964 Paryphthimoides Forster, 1964 Pharneuptychia Forster, 1964 Pindis Felder, 1869 Posttaygetis Forster, 1964 Praefaunula Forster, 1964 Prenda Freitas et al., 2011 Pseudeuptychia Forster, 1964 Pseudodebis Forster, 1964

Rareuptychia Forster, 1964 Satyrotaygetis Forster, 1964 Splendeuptychia Forster, 1964 Taydebis Freitas, 2003 Taygetina Forster, 1964

Proposed included species (for ‘‘Taygetis clade’’)

anophthalma (C. Felder and R. Felder, 1867) antje Peña and Lamas, 2005 boliviana (Godman, 1905) coipa Peña and Lamas, 2005 difficilis (Forster, 1964) enjuerma Peña and Lamas, 2005 falcata Peña and Lamas, 2005 guaniloi Peña and Lamas, 2005 inornata (C. Felder and R. Felder, 1867) itatiaia Peña and Lamas, 2005 necys (Godart, [1824]) neonympha (C. Felder and R. Felder, 1867) pallida Peña and Lamas, 2005 pichita Peña and Lamas, 2005 pilosa Peña and Lamas, 2005 pronophila (Butler, 1867), comb. n. proxima (Hayward, 1957) pseudinornata (Forster, 1964) punctata Peña and Lamas, 2005 pyrczi Peña and Lamas, 2005 quantius (Godart, [1824]) rotunda Peña and Lamas, 2005 rustica (Butler, 1868) stella (Hayward, 1957)

Former generic placement

Guaianaza Freitas and Peña, 2006, syn. n.

blanda (Möschler, 1877) obscura (Butler, 1867) vrazi (Kheil, 1896)

albinotata (Butler, 1867) lineata (Godman and Salvin, 1880)

penelea (Cramer, 1777)

celia (Cramer, 1779), comb. n. dubiosa Forster, 1964 euptychidia (Butler, 1868) marpessa (Hewitson, 1862) puritana (A.G. Weeks, 1902), comb. n. valentina (Cramer, 1779) zimri (Butler, 1869)

banghaasi (Weymer, 1910) kerea (Butler, 1869), comb. n. oreba (Butler, 1870), comb. n. peribaea (Godman & Salvin, 1880), comb. n.

Taygetomorpha L.D. Miller, 2004, syn. n.

Taygetomorpha L.D. Miller, 2004, syn. n.

Taygetis Hübner, [1819] Harjesia Forster, 1964 Coeruleotaygetis Forster, 1964, syn. n.

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Yphthimoides Forster, 1964 Zischkaia Forster, 1964 [new genus 1] [new genus 2]

Proposed included species (for ‘‘Taygetis clade’’)

Former generic placement

weymeri (Draudt, 1912), comb. n. acuta Weymer, 1910 angulosa Weymer, 1907 asterie Weymer, 1910 chiquitana Forster, 1964 chrysogone Doubleday, [1849] cleopatra C. Felder and R. Felder, 1867 echo (Cramer, 1775) elegia Weymer, 1910 [n. sp.] L.D. Miller, MS inambari L.D. Miller and Lamas, 1999 inconspicua Daudt, 1931 laches (Fabricius, 1793) [n. sp.] L.D. Miller, MS larua C. Felder and R. Felder, 1867 leuctra Butler, 1870 mermeria (Cramer, 1776) rufomarginata Staudinger, 1888 sosis Hopffer, 1874 sylvia H.W. Bates, 1866 thamyra (Cramer, 1779) tripunctata Weymer, 1907 uncinata Weymer, 1907 uzza Butler, 1869 virgilia (Cramer, 1776) zippora Butler, 1869

Taygetis Hübner, [1819]

griseola (Weymer, 1911) rectifascia Weymer, 1907 ypthima Hübner, [1821]

Harjesia Forster, 1964 Taygetis Hübner, [1819] Taygetis Hübner, [1819]

which is sister to T. tripunctata includes atypically small individuals which might represent a distinct species. Finally, the lachesgroup is composed of several taxa whose divergence events occurred relatively recently, and their species boundaries based on genetic distances are rather unclear. Based on larval traits and host plant use (including several native and exotic grasses that might have promoted the colonization of disturbed and fragmented habitats due to a broader dietary repertoire compared to other conspecifics), T. laches and T. thamyra have been placed in the ‘‘laches species complex’’ by Murray (2001). The complex is here expanded to also include T. sosis, T. uncinata, T. echo and at least two potentially undescribed species (Figs. 1–3). 4.2. Patterns of diversification and biogeographic implications The implementation of DEC models in Lagrange permits a sophisticated biogeographic analysis as biogeographic events can be assigned different probabilities depending on the time period and the geological history of the region. However, for some of the nodes in the phylogeny, the uncertainty in dating implied that the estimated age of the node overlapped adjacent time periods (Buerki et al., 2011). Nevertheless, both analyses L1 and L2 recovered a single general pattern of diversification and distribution of species in the ‘‘Taygetis clade’’ that can be used for inferring the evolutionary history of those taxa, regardless of the assignment of different dispersal probabilities through time periods. The origin and diversification of extant genera and species within the ‘‘Taygetis clade’’ appear to have begun around the mid to late Miocene, during the period of time when several dramatic changes occurred in Neotropical landscapes (Garzione et al., 2008; Kirby et al., 2008). The major uplift of the central Andes occurred geologically rapidly through the late Miocene until the early Pliocene (Whitmore and Prance, 1987; Gregory-Wodzicki, 2000), which might have affected the dispersal of several taxa, and potentially induced differentiation of populations by creation of barriers between and along the

eastern and western slopes of the Andes (Elias et al., 2009). Furthermore, the disappearance of putative barriers between the tropical Andean, Guyanan and Brazilian Atlantic regions during the middle to late Miocene, such as the disappearance of the extensive long-lived Lake Pebas and marine incursions such as the Paranense Sea in southeastern South America (Hoorn et al., 1995; Räsänen et al., 1995; Hulka et al., 2006; Wesselingh, 2008), might have facilitated dispersal to and colonization of new available habitats over the extant western Amazonia (Hoorn et al., 2010). Based on our estimates of times of divergence, the origin of the genera within the ‘‘Taygetis clade’’ not only coincides with the ending of such geographical barriers (Figs. 3 and 5), but it appears to be strongly related to the time of major dispersal into northwestern Colombia (Taygetina), the Chacoan and Atlantic forests regions (Forsterinaria, Pseudodebis) as well as into montane habitats on the eastern slope of the tropical Andes (Parataygetis) (Figs. 4 and 5). Phylogenetic and biogeographic inferences regarding the genus Forsterinaria involve the colonization of lower Andean habitats and southern regions of humid subtropical climate early in the evolution of the group. The extant Andean lineage might have dispersed from southeastern South America towards the eastern slope of the Andes through the currently warmer tropical and subtropical lowlands (Chacoan region) favored by a global climatic cooling during the late Miocene–early Pliocene (Harris and Mix, 2002; Ravelo et al., 2004). Once ancestral Forsterinaria taxa reached the Andes, they greatly diversified and colonized distinct habitats along the Andes range, as well as spreading towards Central American localities, possibly favored by the colonization of a larval host plant that mostly occurs at higher altitudes (Chusquea) (Fig. 3). Conversely, Parataygetis also occurs in lower montane habitats but has not diversified to the same extent as Forsterinaria. Causes for these apparent disparate diversification rates cannot be identified due to scarce knowledge of the natural history of Parataygetis (e.g. its larval host plant has simply been indicated as Poaceae (Beccaloni et al., 2008)). Future research is clearly needed on the life histories

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of Forsterinaria and its sister clades as well as species-level phylogenies incorporating key taxa from both southeastern Brazil and the lower Andes. Although major diversification events occurred before the Pleistocene, infraspecific differentiation during glacial/ interglacial cycles through this epoch is exemplified by the three subspecies of F. rustica whose disjunct distribution is currently reinforced by deep river valleys which apparently keep the three populations isolated (Peña and Lamas, 2005). Clearly, however, these barriers could have been overcome during periods of cooler past temperatures and the establishment of suitable habitats at lower elevations. The genus Taygetis sensu stricto, on the other hand, has colonized almost every region in the Neotropics, occurring not only in lowlands but with some species also in lower montane habitats. The split between Taygetis and its sister lineage Taygetina occurred around the late Miocene. The biogeographic analyses inferred that the ancestors of Taygetina dispersed towards northern and western Colombia (Area C – Orinoco basin – in Fig. 4), coinciding with the rise of the Vaupés Arch which ultimately separated the Orinoco from the Amazon basin around the late Miocene (Hoorn and Wesselingh, 2010). The Taygetina subclade may have later diversified favored by the uplift acceleration of the northern Andes, mostly along Colombia, which created new montane habitats that were colonized by some taxa within the clade during the late Miocene to Pliocene epochs (Whitmore and Prance, 1987; Gregory-Wodzicki, 2000). On the other hand, there is evidence of recent expansion of larval diet breadth in the ‘‘laches complex’’ in the genus Taygetis. These butterflies have expanded their preferences not only to woody bamboos (typical Taygetis host plants (Beccaloni et al., 2008)) but also to herbaceous bamboos (e.g. Olyra, Pariana (Murray, 2001)) and Paniceae grasses (e.g. Andropogon (Murray, 2001), Acroceras, Setaria (Janzen and Hallwachs, 2009), Panicum (Freitas pers. obs.)), which might have given more opportunities to colonize other habitats (Fig. 3). Host plant shifts, furthermore, have been shown to be a rather important trigger of butterfly speciation (e.g. Janz et al., 2006; Janz and Nylin, 2008; Peña and Wahlberg, 2008; Fordyce, 2010; Jorge et al., 2011), which would ultimately increase the lineage accumulation through time in a phylogeny, thus varying the diversification rates in a particular group (Wiens and Donoghue, 2004). Even though at least five described species in the ‘‘laches complex’’ originated during the past 2.5 Mya (a relatively fast lineage origination compared to the whole ‘‘Taygetis clade’’), it is not possible to accurately identify any sudden shift in diversification promoted by dietary expansion based on our higher level phylogeny. Thus, in order to test such speculation, a population level study using rapidly evolving molecular markers might reveal the phylogeographic structure across populations, giving insights into the relation between host plant – geographic range expansions and ecological speciation. Nevertheless, it would be interesting to investigate whether diversification of the ‘‘laches group’’ was driven by host plant shifts and geographic expansion throughout the Neotropical realm rather than by allopatric differentiation linked to climatic oscillations as proposed by the ‘‘Pleistocene refugia hypothesis’’ (Haffer, 1969). Two taxa included in the study are distributed only in Mexico and Central America (Taygetina weymeri and Taygetis uncinata) (Lamas, 2004). The closure of the Isthmus of Panama by 3 Mya (Coates and Obando, 1996; Kirby et al., 2008) allowed faunal exchange between the North and South American biota through a hypothetical savanna corridor (Jackson et al., 1996; Cox and Moore, 2000). The origin of T. uncinata at around the early Pleistocene occurred during the time of such land bridge uplift. Moreover, its sister taxon is inferred to have originated in South America. Thus, it appears that this bridge allowed the dispersal of the ancestor of T. uncinata from South to Central America. On the other hand, the split between T. weymeri and its sister lineage within the Taygetina subc-

lade, at least 3 million years earlier than the appearance of the Isthmus, suggests two possible scenarios: (1) an endemic distribution in northwestern Colombia during most of the Pliocene followed by dispersal towards Central America after the emergence of the Isthmus of Panama and extinction of populations in northwestern Colombia (at least there are no reports of the species in Colombia to date); or (2) a dispersal event of the ancestors of the Taygetina subclade during the late Miocene–early Pliocene through the Atrato seaway into the hypothesized Central America peninsula which possibly existed as early as 19 Mya (Kirby et al., 2008) (Fig. 5), a route also hypothesized for the Adelpha butterflies (Mullen et al., 2011). The ancestral distribution of taxa within the Taygetina subclade appears to be restricted to northern South America and/or Central America (analysis L2, Fig. 4), in agreement with the second hypothesis. However, the non-stratified analysis L1 (Fig. A2) did not recover a Central American ancestral distribution of the Taygetina subclade Although the arbitrary dispersal rates assigned to the stratified analysis L2 may have been decisive in the optimization, dispersal between northern South America and Central America was imposed as one order of magnitude less during the late Miocene than when the Isthmus of Panama appeared. Furthermore, an early dispersal to Central America of the ancestors of the Taygetina subclade is reinforced by the endemic distribution of two old species (originated in the late Miocene): T. weymeri in Mexico and T. peribaea in western Colombia, a region which although belonging to northern South America, has habitats rather similar to those in Panama than those in the Orinoco basin. We believe that the non-stratified analysis L1 (Fig. A2) did not recover an ancient dispersal of the clade to Central America because it does not incorporate closeness (connectivity) of areas through time, being difficult to assign likeliness of dispersal between contiguous regions. Nonetheless, a separate non-stratified analysis (not shown) including the western Colombia T. peribaea in the Mesoamerica area, based on similarities of its habitat to those in Panama, recovered an ancestral Central American distribution of the Taygetina subclade before the emergence of the Isthmus of Panama. The colonization of lower montane habitats on the western slope of the Andes might have happened during the accelerated uplift of the northern Andes during the Pliocene and continued through the Pleistocene by species of Forsterinaria (Peña and Lamas, 2005). Moreover, those taxa that were able to reach the western slope of the Andes are included within the boliviana group (sensu Peña and Lamas, 2005), F. rustica and those species whose genitalia largely resemble the boliviana group (i.e. F. antje, F. neonympha and F. pseudinornata) (Peña and Lamas, 2005). Speciation events through the Pleistocene epoch were probably promoted by the colonization of new landscapes such as the western slope of the Andes, the lower Mesoamerican montane habitats (F. neonympha) or the Venezuelan ‘‘tepuis’’ (F. inornata) (Peña and Lamas, 2005), possibly facilitated by cooler temperatures and dispersal towards ‘‘optimum habitats’’ which might have favored the geographic expansion of such taxa to contiguous mountain systems. In summary, in this study we show that the ‘‘Taygetis clade’’ needs significant taxonomic rearrangement based on molecular data, corroborating suggestions based on previous less comprehensive morphological and behavioral studies. Furthermore, the diversification of these Neotropical butterflies has probably been promoted by the accelerating uplift of the Andes which rebuilt landscapes, altered the climate and created new habitats and niches. The two largest genera in the clade, Forsterinaria and Taygetis, went through a series of successful range expansions and adaptation to these new habitats, while changes in their dietary repertoire might have increased the likelihood of lineage origination (or decreased lineage extinction rates) in both genera.

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Acknowledgments The present study was funded by a grant awarded to NW from the Academy of Finland and the Kone Foundation. PM-M acknowledges the grants awarded by Turun yliopistosäätiö and the Oskar Öflunds Foundation. We are very grateful to Mario A. Marín, Andrew D. Warren, Phil J. DeVries, Carlos Eduardo Giraldo, Danilo B. Ribeiro, Gerardo Lamas, Juan José Ramirez and Liliana Arango for kindly providing the specimens used in this study. AVLF thanks ‘‘Fundação de Amparo à Pesquisa do Estado de São Paulo’’ (FAPESP Grant 04/05269-9, and Biota-Fapesp Grant 11/50225-3), the Brazilian Research Council – CNPq (Fellowship 300282/2008-7, and SISBIOTA-Brasil/CNPq 563332/ 2010-7), and the RedeLep ‘‘Rede Nacional de Pesquisa e Conservação de Lepidópteros’’. KRW thanks the Darwin Initiative and National Science Foundation (DEB0639861) for support. We thank Andrew Neild, Armando Luis Martínez, Blanca Huertas, Jorge Llorente and Mauro Costa for discussion about taxa in the ‘‘Taygetis clade’’. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2012. 09.005. References Ackery, P.R., 1988. Hostplants and classification: a review of nymphalid butterflies. Biol. J. Linn. Soc. 33, 95–203. Adams, M.J., 1985. Speciation in the Pronophiline butterflies (Satyridae) of the Northern Andes. J. Res. Lepido. Suppl., 33–49. Aduse-Poku, K., Vingerhoedt, E., Wahlberg, N., 2009. Out-of-Africa again: a phylogenetic hypothesis of the genus Charaxes (Lepidoptera: Nymphalidae) based on five gene regions. Mol. Phylogenet. Evol. 53, 463–478. Alfaro, M.E., Santini, F., Brock, C., Alamillo, H., Dornburg, A., Rabosky, D.L., Carnevale, G., Harmon, L.J., 2009. Nine exceptional radiations plus high turnover explain species diversity in jawed vertebrates. Proc. Natl. Acad. Sci. USA 106, 13410– 13414. Antonelli, A., Nylander, J.A.A., Persson, C., Sanmartín, I., 2009. Tracing the impact of the Andean uplift on Neotropical plant evolution. Proc. Natl. Acad. Sci. USA 106, 9749–9754. Baker, R.H., DeSalle, R., 1997. Multiple sources of character information and the phylogeny of Hawaiian drosophilids. Syst. Biol. 46, 654–673. Beccaloni, G.W., Viloria, A.L., Hall, S.K., Robinson, G.S., 2008. Catalogue of the Hostplants of the Neotropical Butterflies. Monografías Tercer Milenio, first ed., vol. 8. Sociedad Entomológica Aragonesa, Zaragozam, 536 pp. Brown, K.S., Freitas, A.V.L., Von Schoultz, B., Saura, A.O., Saura, A., 2007. Chromosomal evolution of South American frugivorous butterflies in the Satyroid clade (Nympalidae: Charaxinae, Morphinae and Satyrinae). Biol. J. Linn. Soc. 92, 467–481. Buerki, S., Forest, F., Alvarez, N., Nylander, J.A.A., Arrigo, N., Sanmartín, I., 2011. An evaluation of new parsimony-based versus parametric inference methods in biogeography: a case study using the globally distributed plant family Sapindaceae. J. Biogeogr. 38, 531–550. Bush, M.B., De Oliveira, P.E., 2006. The rise and fall of the Refugial hypothesis of Amazonian speciation: a paleoecological perspective. Biota, Neotropica 6. Casner, K.L., Pyrcz, T.W., 2010. Patterns and timing of diversification in a tropical montane butterfly genus Lymanopoda (Nymphalidae, Satyrinae). Ecography 33, 251–259. Coates, A.G., Obando, J.A., 1996. The geologic evolution of the Central American Isthmus. In: Jackson, J.B.C., Budd, A.F., Coates, A.G. (Eds.), Evolution and Environment in Tropical America. The University of Chicago Press, Chicago 60637, pp 21–56. Cox, C.B., Moore, P.D. (Eds.), 2000. Biogeography: An Ecological and Evolutionary Approach, sixth ed. Blackwell Publishing, Oxford, 428 pp. Elias, M., Joron, M., Willmott, K., Silva-Brandão, K.L., Kaiser, V., Arias, C.F., Gomez Piñerez, L.M., Uribe, S., Brower, A.V.Z., Freitas, A.V.L., Jiggins, C.D., 2009. Out of the Andes: patterns of diversification in clearwing butterflies. Mol. Ecol. 18, 1716–1729. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Fjeldså, J., 1994. Geographical patterns for relict and young species of birds in Africa and South America and implications for conservation priorities. Biodivers. Conserv. 3, 207–226. Fordyce, J.A., 2010. Host shifts and evolutionary radiations of butterflies. Proc. R. Soc. B: Biol. Sci. 277, 3735–3743.

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