{"paper_id":"01cb7746-16ec-4588-afb3-6aba9b70c072","body_text":"Posted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nTracing the origins and evolution of nymphalid butterflies\n(Lepidoptera) in the Atlantic Forest\nMar Repull´ es1, Nicolas Chazot 2, Leidys Murillo-Ramos 3, Marianne Espeland 4, Alexandre\nAntonelli5, Andr´ e Freitas6, and P´ avel Matos-Marav´ ı1\n1Biology Centre CAS Institute of Entomology\n2Swedish University of Agricultural Sciences\n3University of Sucre\n4Leibniz Institute for the Analysis of Biodiversity Change\n5University of Gothenburg\n6State University of Campinas\nSeptember 15, 2025\nAbstract\nUnderstanding the relative roles of diversification and dispersal is key to explaining large-scale biogeographic patterns. Although\nboth processes are known to shape biodiversity, their relative contributions remain understudied in many systems. Here, we\nexamine how these processes have jointly contributed to the exceptional diversity and endemism of Nymphalidae butterflies in\nSouth America’s Atlantic Forest, a global biodiversity hotspot. We obtained DNA sequences for 68 Nymphalidae species (43\nmissing from major phylogenies) and integrated them into published time-calibrated phylogenies. We used Dispersal-extinction-\ncladogenesis models and Biogeographical Stochastic Mapping to infer historical biogeographic patterns over time, and ClaDS to\nestimate region-specific diversification rates. We further evaluate whether regional patterns of diversification are associated with\noccurrence in montane environments or across the north–south biogeographic break within the Atlantic Forest. Our results show\nthat butterfly diversity in the region was driven primarily by recurrent dispersal from Amazonia and the Andes, rather than\nby elevated in situ diversification, which remained low and stable through time. Although dispersal increased progressively\nduring the Cenozoic, we found no evidence that the Diagonal of open formations acted as a major barrier, indicating that\nforest corridors probably allowed extensive exchanges between the Atlantic Forest and other Neotropical regions. Southern\nlineages exhibited slightly higher diversification rates, especially among montane generalist species, but overall diversification\ncontributed little compared to the sustained input of dispersing lineages. Together, these findings highlight the central role of\nbiome connectivity in shaping Atlantic Forest Nymphalidae diversity, while underscoring the importance of jointly considering\ndiversification and dispersal processes to better understand the macroevolutionary dynamics underlying current biodiversity\npatterns.\nTracing the origins and evolution of nymphalid butterflies (Lepidoptera) in the Atlantic Forest\nRunning title: Nymphalidae Evolution in the Atlantic Forest\nUnderstanding the relative roles of diversification and dispersal is key to explaining large-\nscale biogeographic patterns. Although both processes are known to shape biodiversity, their\nrelative contributions remain understudied in many systems. Here, we examine how these\nprocesses have jointly contributed to the exceptional diversity and endemism of Nymphalidae\nbutterflies in South America’s Atlantic Forest, a global biodiversity hotspot. We obtained DNA\nsequences for 68 Nymphalidae species (43 missing from major phylogenies) and integrated\nthem into published time-calibrated phylogenies. We used Dispersal-extinction-cladogenesis\n1\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nmodels and Biogeographical Stochastic Mapping to infer historical biogeographic patterns over\ntime, and ClaDS to estimate region-specific diversification rates. We further evaluate whether\nregional patterns of diversification are associated with occurrence in montane environments or\nacross the north–south biogeographic break within the Atlantic Forest. Our results show that\nbutterfly diversity in the region was driven primarily by recurrent dispersal from Amazonia\nand the Andes, rather than by elevated in situ diversification, which remained low and stable\nthrough time. Although dispersal increased progressively during the Cenozoic, we found\nno evidence that the Diagonal of open formations acted as a major barrier, indicating that\nforest corridors probably allowed extensive exchanges between the Atlantic Forest and other\nNeotropical regions. Southern lineages exhibited slightly higher diversification rates, especially\namong montane generalist species, but overall diversification contributed little compared to\nthe sustained input of dispersing lineages. Together, these findings highlight the central role of\nbiome connectivity in shaping Atlantic Forest Nymphalidae diversity, while underscoring the\nimportance of jointly considering diversification and dispersal processes to better understand\nthe macroevolutionary dynamics underlying current biodiversity patterns.\nKEY WORDS : Atlantic Forest, biogeography, Diagonal of open formations, dispersal, Neotropics,\nNymphalidae.\n1 Introduction\nLarge-scale historical biogeographic patterns are determined by the interplay between rates of diversification\n(comprising speciation and extinction) and dispersal (Hubbell, 2001). Traditionally, macroevolutionary\nexplanations for biotic assembly have emphasised two main ideas: (1) that older clades and larger geographic\nareas accumulate more species — the time- and area-for-speciation hypotheses, or together, as the time-\nintegrated species-area effect(Fine and Ree, 2006; Wallace, 1878); and (2) that regional differences in species\nrichness result from episodic shifts in speciation and extinction rates — the rate-of-diversification hypothesis\n(Mittelbach et al., 2007). Nevertheless, more recent frameworks also acknowledge the influence of asymmetric\ndispersal rates between regions (e.g., Goldberg, Roy, Lande, & Jablonski, 2005; Goldberg, Lancaster & Ree,\n2011; Kuhnh¨auser et al., 2025). This has led to a better characterisation of the relative importance of time,\ndiversification and dispersal processes in explaining extant regional differences in species diversity (Carrillo\net al., 2020; Igea and Tanentzap, 2019; Kuhnh¨auser et al., 2025). However, empirical evidence is still mainly\nrestricted to a few taxonomic groups or is mainly focused on diversification rates, neglecting the contribution\nof dispersal in explaining large-scale biodiversity patterns.\nWith only a fraction of its original vegetation remaining (8% – 22%), mostly in small, disconnected fragments\n(Vancine et al., 2024, Joly, Metzger & Tabarelli, 2014), the Atlantic Forest is considered one of the most\nthreatened biodiversity hotspots on Earth (Myers, Mittermeier, Mittermeier, da Fonseca & Kent, 2000; Mar-\nques & Grell, 2021). This biogeographical domain harbours high levels of endemism across plants (Martini,\nFiaschi, Amorim & Paix˜ ao, 2007; Fiaschi & Pirani, 2009), amphibians (Hadad et al., 2013), birds (Silveira,\nOlmos & Long, 2003), and butterflies (Brown & Freitas, 2000; Santos et al., 2018), among other groups.\nThroughout its area, congruent patterns of species richness and endemism across animals and plants are\nthought to be shaped by contemporary and historical climatic changes and topographical variation. For\ninstance, a large turnover in ecological communities exists between the southern and northern regions (Peres\net al., 2020), but the relative contribution of species diversification and dispersal in shaping such patterns\nremains unknown for most species in the Atlantic Forest (da Silva Oliveira-Silva, Antonelli, Carnaval &\nProvete, 2024; Peres et al., 2020).\nDispersal dynamics between the Atlantic Forest and other Neotropical regions have been linked to paleoen-\nvironmental changes in the Cenozoic (i.e., the past 66 million years). During the Paleogene (ca. 66 to 23\nmillion years ago, Mya), Amazonia and the Atlantic Forest seem to have been more interconnected than\ntoday (Sobral-Souza & Lima Ribeiro, 2017). Yet, the extent of this connectivity remains under debate, and\nthe timing of the Atlantic Forest’s origin is still unclear (Jaramillo & C´ ardenas, 2013; Jaramillo, 2023).\nGlobal cooling and the major Andean uplift in the Neogene (ca. 23 to 2.6 Mya) promoted the expansion\n2\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nof open habitats in the Neotropics, likely reducing the connectivity between the Atlantic Forest and other\nNeotropical regions (Hoorn et al., 2010; Werneck, 2011). However, fossil and molecular evidence suggest that\nintermittent humid corridors maintained connectivity during wet periods during the Pleistocene (Bonaccorso\net al., 2006; Batalha-Filho, Fjelds˚ a, Fabre & Miyaki, 2013; Cohelho et al., 2022), but also earlier in the Neo-\ngene (Batalha-Filho et al., 2013; Marques-Souza et al., 2022). Under this scenario, the increasing isolation\nof the Atlantic Forest, combined with intermittent reconnections to other regions, may have led to different\ndispersal dynamics and biogeographical patterns across lineages (Batalha-Filho et al., 2013; Prates et al.,\n2016; Sobral-Souza & Lima-Ribeiro, 2017; Bocalini, Bol´ ıvar-Leguizam´ on, Silveira & Bravo, 2021).\nPleistocene climatic fluctuations have also been major determinants of diversification and community as-\nsembly within the Atlantic Forest – as initially proposed in the Pleistocene refugia hypothesis (Haffer 1969;\nBrown 1976) and later including divergent selection in the Vanishing refuge model (Damasceno, Strangas,\nCarnaval, Rodrigues & Moritz 2014). Both paleoclimatic (Carnaval and Moritz, 2008) and phylogenetic data\n(Carnaval, Hickerson, Haddad, Rodrigues & Moritz, 2009; Silva, Moraes-Barros, Ribas, Ferrand & Morgante,\n2012; Martins et al., 2011) support distinct regional evolutionary trajectories during climatic fluctuations:\nwhile the region north of the Doce River (central Bahia) has remained paleoclimatically stable, the southeast\nunderwent dramatic environmental reconfigurations during cool phases, prompting many lineages to retreat\nto mountain refuges (Carnaval et al., 2009; Peres et al., 2020). These regional differences may have led to\ndifferent diversification rates through time: constant in the north and increasing in the unstable south during\nclimatic fluctuations (Paz et al., 2021).\nTo offer further insights into the Atlantic Forest’s macroevolutionary history, we focus on the species-rich\nbutterfly family Nymphalidae, with nearly 500 species across the region (Brown & Freitas, 2000; Santos et\nal., 2018). Their distribution patterns in the Atlantic Forest are well documented (Santos et al., 2018; Shirai,\nMachado, Mota, Rosa & Freitas, 2019; Freitas et al., in prep.) and similar to other taxa, with the highest\nspecies richness in the southeastern montane regions (Iserhard, Romanowski, Richter & Mendon¸ ca, 2017;\nSantos et al., 2018). We build upon the most comprehensive global phylogeny of Nymphalidae to date (Chazot\net al., 2021), and sequenced 68 species, including 43 species previously absent from global time-calibrated\nbutterfly phylogenies (Chazot et al., 2021; Kawahara et al., 2023). Based on compiled distribution data for\n1,096 species, we explored whether the diversity of Atlantic Forest Nymphalidae results from (1) a long\nhistory of complete biotic isolation, reflected in early dispersal events (i.e., during the Paleogene) followed by\nin situ diversification (i.e., within the Atlantic Forest); (2) gradual isolation, leading to decreasing interchange\nwith Amazonia and a stronger role of local speciation; or (3) continuous connectivity with other Neotropical\nbiomes, where recurrent dispersal dominates and local diversification plays a minor role. Furthermore, we\nevaluated whether occurrence in montane regions and along the north–south biogeographic break may explain\nthe extant diversification rates within the Atlantic Forest nymphalids.\n2 Material and methods\n2.1 Molecular data\nWe targeted Nymphalidae species from the Atlantic Forest that were not previously sequenced in phylogenetic\nstudies. We sampled 68 adult specimens across eastern Brazil, including 43 species newly sequenced for\ntime-calibrated phylogenies (Table S1). We extracted DNA from legs using DNeasy Blood and Tissue Kits\n(QIAGEN), and libraries were prepared and sequenced on an Illumina NovaSeq 6000 platform by the company\nNovogene, UK. We checked the reads quality (FastQC v0.12.1, Andrews, 2010), trimmed the adapters (fastp,\nChen, 2023) and assembled de novo contigs (SPAdes v3.15.4, Prjibelski, Antipov, Meleshko, Lapidus &\nKorobeynikov, 2020). Following the BUTTERFLY1.0 probe set (Espeland et al., 2018; Kawahara et al.,\n2023), we retained 391 loci, including 11 legacy genes traditionally used in butterfly systematics (Wahlberg\nand Wheat, 2008; Kawahara et al., 2018). We used the SECAPR v2.2.3 environment (Andermann, Cano,\nZizka, Bacon & Antonelli, 2018; Ribeiro et al., 2021) from de novo contig assembly to multiple sequence\nalignments.\nWe focused on monophyletic groups from the Nymphalidae phylogeny of Chazot et al., (2021), with more\n3\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nthan half of the species diversity occurring in the Neotropics. The function “CladeByTrait()” of the R package\n‘speciesgeocodeR’ (T ¨opel et al., 2016) found nine neotropical groups (“Clades for Phylogenetic Inference”\nin Table 1), representing the clades Danainae, Heliconiinae, Limenitidinae, Biblidini, a clade containing\nCyrestinae and Nymphalinae, Charaxinae, a Satyrinae subclade, Euptychiina and Pronophilina. We merged\nour data with the most comprehensive phylogenies of Nymphalidae (Chazot et al., 2021) and butterflies in\ngeneral (Kawahara et al., 2023), and conducted multi-species alignments for each of the nine clades using\nMAFFT v7.520 (Katoh & Standley, 2013), only retaining loci present in [?]10% of the species in that clade.\nFinal alignments were checked in Geneious Prime 2023.1.2, and are reported in Supporting Information\n(https://doi.org/10.5061/dryad.wm37pvn11).\n2.2 Time-calibrated phylogenetic inference\nWe infer phylogenetic relationships for each of the nine clades using IQ-TREE 2.2.0 (Minh et al., 2020)\nunder a concatenated, codon-partitioned scheme (Chernomor, von Haeseler & Minh, 2016), with model\nselection performed by ModelFinder (Kalyaanamoorthy, Minh, Wong, von Haeseler & Jermiin , 2017).\nBranch support was calculated using 1,000 ultrafast bootstrap replicates (Minh, Nguyen & von Haeseler,\n2013), Shimodaira-Hasegawa approximate likelihood ratio tests, and approximate Bayes test (Anisimova,\nGil, Dufayard, Dessimoz & Gascuel, 2011).\nTo time calibrate each of the nine tree-topologies, we used the Bayesian approach implemented in MCMC-\nTree from PAML package v4.10.6 (Yang 2007). We used an independent-rates relaxed molecular clock model\n(Drummond, Ho, Phillips & Rambaut, 2006) and the most complex substitution model available in MCMC-\nTree, the HKY85 (Hasegawa, Kishino & Yano, 1985). Due to the limited availability of butterfly fossils, we\nused eight secondary calibration points, along with the crown age of each clade (Table S2), extracted from\nthe fossil-calibrated phylogeny of Chazot et al., (2021). To ensure convergence, the analysis was run two\ntimes independently using random seeds, and similar results were observed (Figure S1).\n2.3 Inference of biogeographical history\nWe estimated range evolution using the Dispersal-Extinction-Cladogenesis (DEC) model in the R package\n‘BioGeoBEARS v.1.1.3’ (Matzke, 2018), subdividing the nine time-calibrated trees into 14 Neotropical sub-\nclades to reduce noise and improve computational performance (Table 1 and Figure S2). We subdivided\nthe Neotropics into eight biogeographic regions: (1) Mesoamerica and northwestern lowland Andean slopes,\n(2) Northern Andes (Ecuador, Colombia, Venezuela), (3) Central Andes (Peru, Bolivia), (4) Amazonia, (5)\nDiagonal of open formations encompassing the Caatinga, Cerrado and Chaco biomes, (6) Northern Atlantic\nForest and (7) Southern Atlantic Forest, both delimited by the Doce river which reflects a strong community\nturnover of animal and plant diversity, and (8) outside the defined Neotropical areas (Figure 1), largely\nfollowing the biogeographical regions proposed by Morrone (2014). To avoid unrealistic biogeographical\nscenarios, we disallowed dispersal events between Mesoamerica and the Northern Andes from/to the Diag-\nonal of open formations and the Atlantic Forest. This constraint was applied globally across the trees and\nnot stratified temporally. Unconstrained analyses (i.e., without adjacency matrix) were also performed for\ncomparison. Species ranges were compiled from the literature, including original descriptions, databases of\napproximate range maps (Lepidoptera and some other life forms, Savela M.), geo-referenced occurrences\n(GBIF and iNaturalist), and experts’ knowledge (distributional data reported in Table S3).\nTo account for divergence time uncertainty, we utilised 50 randomly selected trees from the MCMCtree\nposterior distribution for each study clade, and to address ancestral geographical state uncertainty, we\nperformed 50 Biogeographical Stochastic Mappings per tree (BSM; Dupin et al., 2017). Dispersal rates were\nextracted in 1 Myr intervals and scaled by total branch length per bin, following the approach described in\nAntonelli et al., (2018) and the R scripts from Matos-Maravi et al., (2021).\n2.4 Within-region species diversification analysis\nWe used CLaDS (Maliet et al., 2019; Maliet & Morlot, 2022) to estimate branch-specific diversification rates,\nassuming a constant turnover across the entire phylogeny (one for each of the 14 subclades), and accounting\n4\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nfor incomplete taxon sampling with a sampling probability vector (Table S4). To calculate diversification\nrates within geographic regions, we combined the CLaDS outputs with BioGeoBEARS ancestral range esti-\nmates from BSM. For each branch section, we identified the region(s) it occupied, weighted its diversification\nrate by the proportion of its length in that region (“occupancy”), and further adjusted by the frequency\nof occurrence across all BSM replicates. This approach ensured that branches only partially present in a\nregion, or with uncertain regional assignment, contributed proportionally less to the regional diversification\nestimates. This was done for every 1 Myr time bin, allowing us to extract the diversification rate through\ntime at each region (R scripts in Supporting Information).\n2.5 Effect of altitudinal variation on the diversification of Atlantic Forest Nymphalidae\nWe assessed whether environmental variation along altitudinal gradients in the Atlantic Forest were asso-\nciated with extant speciation rate heterogeneity among clades (i.e., tip rates). We categorised species into\nlowland, highland, or generalist, and into northern, southern, or widespread distributions. Speciation rate\nfor each tip was estimated using either the ClaDS estimates or the tip-specific DR-statistic (Redding and\nMooers 2006), a non-model-based approach that approximates speciation rates (Jetz et al., 2012), calculated\nwith the R package ‘epm’ (Title, Swiderski & Zelditch, 2022). To test whether geographical occurrence\npredicted tip speciation rates, we used phylogenetic generalised least-squares regression (PGLS; Martins\nand Hansen, 1997), as implemented in the R package ‘caper’ (Orme, 2012). We ran the analyses for every\nsubclade separately, using either CLaDS rate or DR as response variables.\nAn extended version of the Methods section and the R scripts used here are available in the supplementary\nmaterial and Supporting information (https://doi.org/10.5061/dryad.wm37pvn11).\n3 Results\n3.1 Molecular data and time-calibrated phylogenetic inference\nThe size of our molecular datasets varied between 159,716 bp (Charaxinae clade) and 162,060 bp (Satyrinae\nsubclade), representing 387 and 391 loci, respectively. The Maximum Likelihood tree topologies inferred in\nIQ-TREE 2.2.0 (Minh et al., 2020) were congruent with other recent published phylogenies focused on specific\ntaxa (e.g., Barbosa, Seraphim, Valencia, Azeredo-Espin & Freitas et al., 2022; Chazot et al., 2021; Cicconardi\net al., 2023; Espeland et al., 2023; Kawahara et al., 2023; Silva-Brandao et al., 2008; Yang & Zhang, 2015;\nYan et al., 2023). The few differences found were either at the species level or for relationships among\nlineages that have been previously difficult to resolve: e.g., the genera Brassolis (Brassolini), Methona,\nHypothyris, and Hyalyris (Ithomiini), which were paraphyletic in our inferred phylogenies but were also\nreported as having low phylogenetic support in previous studies (Chazot et al., 2019; Matos-Maravi et al.,\n2021). Regardless, because such incongruences were very few and mostly at shallow phylogenetic levels, we\ndo not consider them to have biased our macroevolutionary and biogeographical inferences.\n3.2 Inference of biogeographical history\nOur Biogeographical Stochastic Mapping for the whole Neotropical region showed that the highest number\nof dispersal events were into and out of Amazonia (231 events into and 427 events out), followed by the\nAndes (336 events into and 423 events out) (Figure 1). Regarding the Atlantic Forest, Amazonia was also\nthe major source (144.78 events from Amazonia to Southern and Northern Atlantic Forest), compared to\nthe 131.3 events from the Andes to the Atlantic Forest.\nDispersal into the Atlantic Forest from other Neotropical regions occurred constantly through time until\nabout 10 Mya, when it accelerated toward the present (Figure 2). Overall, Amazonia has been the primary\nsource of lineages dispersing into the Atlantic Forest relatively constant throughout the Neogene, only sur-\npassed by the Andean dispersal during the Eocene (prior to 33 Mya) and around 15 Mya, during the mid\nMiocene (Figure 2). Dispersal from the Diagonal of open formations into the Atlantic Forest intensified from\n11 Mya toward the present (Figure 2). When the northern and southern regions of the Atlantic Forest were\nanalysed separately, connectivity with other Neotropical regions was higher for the southern region (Figure\n5\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nS3). The unconstrained analysis showed a higher connectivity between northern Andes and the Atlantic\nForest, rather than central Andes (Figure S4).\nThe first extant lineages to disperse to the Atlantic Forest were Morphini (Satyrinae) which occurred around\n47 Mya. This was followed by lineages of Charaxinae ( Memphis and Prepona ) and Biblidini (Biblidinae),\nduring the Eocene, around 43 Mya, and Euptychiina (Satyrinae) around 37 Mya. During the Oligocene\n(33.9–23 Mya), lineages from the Haeterini (Satyrinae) and Melitaeini (Nymphalinae) clades dispersed to\nthe Atlantic Forest. The remaining Nymphalidae clades dispersed to the Atlantic Forest during the Miocene.\nWithin Charaxinae, two genera (Memphis and Prepona ) showed the highest dispersal rates into the Atlantic\nForest, with these rates increasing exponentially since the late Miocene. Another notable clade was the\nHeliconiini (Heliconiinae), which showed high dispersal rates from Amazonia to the Atlantic Forest around\n5 Mya (Figure S5).\n3.3 Within-region species diversification analysis\nThe estimates of regional diversification rates obtained by ClaDS and BioGeoBEARS’ BSM suggested that\nAtlantic Forest lineages had a steady and low diversification rate through time (Figure 3). This trend was\nobserved in both the northern and southern Atlantic Forest lineages, although the latter exhibited a higher\nrate of diversification. A notable peak in diversification was estimated to have occurred around 15–17 Mya\nin the southern Atlantic Forest, alongside a decline in diversification rates between 20 and 25 Mya for both\nthe southern and northern Atlantic Forest (Figure 3). When examining which lineages contributed most to\nthe overall increase in speciation rates, the Ithomiini and Adelpha had the most significant impact, showing\nhigh speciation rates during the mid-Miocene across the Neotropics (˜15 Mya), and mostly represented in\nthe southern Atlantic Forest. In addition, the speciation rates of Euptychiina also stands out from the rest\nduring the Paleogene, ca 35-30 Mya (Figure S6).\n3.4 Effect of altitudinal variation on the diversification of Atlantic Forest Nymphalidae\nWe did not find any phylogenetic correlation between tip-specific speciation rates and the current distri-\nbutional patterns of nymphalid butterflies in the Atlantic Forest, except for the tribe Ithomiini and the\ngenus Actinote (Heliconiinae: Acraeini). For Ithomiini, we found that altitudinal generalists (species that\nare widespread along elevational gradients) that occur in the southern Atlantic Forest had higher speciation\nrates compared to other lineages across the Atlantic Forest. Both ClaDS estimates (Estimate = 0.0593,\np-value = 0.0065) and DR metrics (Estimate = 0.1768, p-value = 0.0216) produced the same results. A\nsimilar correlation was found for Actinote ; however, it was statistically significant only when using ClaDS’\nspeciation rates (Estimate = 0.0155, p-value = 0.0418) but not with DR metrics estimates (Estimate = -\n0.0155, p-value = 0.6624). Although a significant positive effect on speciation by the lowland and widespread\nstate was found using the DR metrics in Haeterini (Estimate = 0.102, p-value = 7.306e-05), the effect was\nfound to be non-significant using ClaDS’ speciation rates (Estimate = 0.0046, p-value = 0.1304).\n4 Discussion\nThe Atlantic Forest is regarded as one of the most threatened biodiversity hotspots in the world (Myers et al.,\n2000). However, the macroevolutionary processes explaining extant biodiversity patterns of many species-\nrich groups remain unclear. In this study, we inferred the evolutionary history of neotropical nymphalid\nbutterflies and found that the extant species diversity in the Atlantic Forest resulted from a consistently low\nand steady net diversification rate over time, coupled with continuous biotic influx from other Neotropical\nregions. Notably, dispersal rates into the Atlantic Forest intensified around 15 million years ago, suggesting\nthat regional species accumulation was primarily shaped by dispersal from other regions —primarily from\nAmazonia— rather than in situ diversification, in line with the third scenario outlined in the introduction.\n4.1 Atlantic Forest dispersal dynamics\nWe observed an overall increase in species dispersal to the Atlantic Forest since the mid-Miocene, 15 million\nyears ago, with an acceleration in the last ˜7 million years. The sources of dispersal into the Atlantic Forest\nvaried over time, which may be partially attributed to major environmental and geological changes. During\n6\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nthe Neogene, the major Andean uplift and the global cooling trend drastically reconfigured the landscape,\nprecipitation patterns, and climate across the Neotropical region, resulting in the expansion of dry and open\nformations in northern and central South America (Hoorn et al., 2010; Werneck, 2011). Yet, the precise\ntiming of the establishment of the Diagonal of open formations remains under debate. While molecular\nphylogenies suggest an early to mid-Miocene split between forest biomes, fossil and molecular evidence from\nC4 grasses and woody savanna flora indicate that their dominance began in the late Miocene (Azevedo et al.,\n2020). Nevertheless, there is evidence of open vegetation formations as far back as the Palaeocene (Aguiar et\nal., 2020). Our findings reveal an exponential-like increase in dispersal from the Diagonal of open formations\ninto the Atlantic Forest around 10 Mya. Even though the underlying drivers of such increase remain elusive,\nthey support the idea of a mid-to-late Miocene reconfiguration of environmental conditions, including the\nestablishment of suitable dispersal corridors for Nymphalid lineages with different ecological characteristics.\nThis increase may also reflect a larger source pool of lineages from northwestern South America, triggered\nby the rapid speciation of many taxa during that period and in response to orogenic processes and regional\nclimate change (e.g., Antonelli et al., 2018b).\n4.1.1 Amazonia as a source of Atlantic Forest diversity\nAs demonstrated by Antonelli et al., (2018), Amazonia played a major role as a source of Neotropical\ndiversity, including the Atlantic Forest. Here we also recovered Amazonia as the main source of Atlantic\nForest diversity, with a high and constant dispersal rate through time. Despite the ecological barrier posed\nby the Diagonal of open formations, biotic corridors—likely formed by episodic wet forests or continuous\nwet vegetation along rivers—might have enabled the exchange of fauna and flora between Neotropical forest\ndomains. For instance, Prates et al., (2017) suggested that throughout the Miocene, patches of suitable\nhumid habitats may have connected the Atlantic Forest with western South American forests, facilitating\nspecies dispersal. This agrees with our findings and supports the idea that the expansion of the Diagonal of\nopen formations did not significantly reduce dispersal between Amazonia and Atlantic Forest nymphalids.\nWe speculate that the Neogene forest corridors allowed for continuous dispersal among forest-adapted\nNymphalid butterflies. These connections likely varied regionally, with stronger links to Amazonia through\ncentral and northern routes, and weaker connections through southern corridors, including the Andes. Sim-\nilar connectivity patterns have been reported previously, although most indicate that the interchange was\nfrequent due to Pleistocene paleoclimatic fluctuations (Costa, 2003; Cohelho et al., 2022; Peres et al., 2020;\nSobral-Souza & Lima-Ribeiro, 2017). In contrast, only a few studies have documented older connections\nduring the Neogene (e.g., Marques-Souza et al., 2022), possibly mediated by riparian forests or remnant hu-\nmid corridors (Trujillo-Arias et al., 2020, Pirani et al., 2020). In line with these studies, our results recover\ncontinuous connectivity between the Atlantic Forest and other Neotropical biomes since the early Neogene,\nlikely facilitated by historical forest corridors linking these regions. We acknowledge that long-distance\ndispersal events, facilitated for instance, by extreme winds, could also explain certain biotic interchange\nwithout the necessity of fully connected corridors. Seasonal movements of individual butterflies have been\ndocumented over distances of several kilometres in the Atlantic Forest. Additionally, migrations of certain\npopulations and communities can be inferred to occur over many hundreds of kilometres (Brown & Freitas\n2000, 2002). However, we consider that such events should have been largely random, adding to the “noise”\nin our analyses rather than being able to explain the steady increase in dispersals inferred, which are more\nlikely to reflect the conditions of the vegetation matrix over which animal movements could take place.\n4.1.2 The role of the Andes on Atlantic Forest diversity\nThe central Andes is the second largest source of Nymphalidae diversity for the Atlantic Forest, as it has also\nbeen reported for other taxonomic groups, such as rodents (Vallejos-Garrido et al., 2023), bats (Velazco &\nPatterson, 2013), birds (Fjeldsa & Rahbek, 2006), and plants (Antonelli & Sanmartin, 2011). The Andes have\nplayed an important role in generating species diversity in the Neotropics through high diversification and\nemigration rates (Rangel et al., 2018; Antonelli et al., 2018c). For instance, the ithomiine genus Pteronymia\nmainly diversified in the northern Andes, with much of their non-Andean diversity resulting from independent\ndispersal events out of them (De-Silva et al., 2017). Similarly, tanagers and hummingbirds seem to have\n7\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nradiated and dispersed out of the northern Andes, which might have acted as cradles and museums of\nspecies diversity (Beckman & Witt, 2015; Sonne et al., 2022). Indeed, a high connectivity between northern\nAndes, rather than central Andes, and the Atlantic Forest was recovered in our unconstrained biogeographical\nanalyses (see Supplementary Materials), a pattern also recovered in a synthesis of Andean flora biogeography\n(Perez-Escobar et al., 2022).\nUnlike Amazonian dispersion –more or less constant–, the Andean dispersal rate has fluctuated over time.\nDuring the early Miocene, the connectivity between the Andes and the Atlantic Forest increased, reaching\nits peak around 15 Mya, when it even surpassed dispersal from Amazonia. This pattern may have been\nenabled by biotic corridors linking the central Andes to the Atlantic Forest through the Cerrado and Chaco\ntransition zones, as reported for several lineages (Matos-Maravi, Pena, Willmott, Freitas & Wahlberg, 2013;\nMoreira-Munoz et al., 2020; Pirani et al., 2020; Prates et al., 2017; Trujillo-Arias et al., 2018, 2020). In\naddition, montane environments of the Atlantic Forest, like Serra do Mar and Serra da Mantiqueira, may have\nfacilitated the settlement and diversification of high-elevation Andean lineages in the region. For instance,\nthe montane genera Actinote diversified in the Andes but, once it colonized the southern Atlantic Forest,\nachieved its greatest species richness in the mountain forests of the region, with some lineages recolonizing\nthe Andes from the Atlantic Forest (Guerato et al., 2023; Magaldi et al., 2024). In this regard, Brown\n(1987) identified a high similarity in butterfly lineage composition between the Andes and south-eastern\nBrazil, whereas Safford (2007) suggested a similar shared evolutionary pattern based on floristic composition\nanalyses. Therefore, the high rates of Andean diversification together with early Miocene dispersal corridors\nmight have promoted an interchange of lineages with the Atlantic Forest.\n4.2 Atlantic Forest diversification\nWithin the Neotropical region, a gradual accumulation of Nymphalidae lineages is explained by steady\nspeciation rates and low extinction rates (Chazot et al., 2021), a common pattern found in Neotropical\nbutterflies (Matos-Maravi, 2016) and plants (Meseguer et al., 2022). We found that net diversification\nrates within the Atlantic Forest have remained low and constant, contrasting with other global tropical\nbiodiversity hotspots characterized by high diversification rates (Igea and Tanentzap, 2019). This slow and\ngradual accumulation of lineages is consistent with previous reports that considered the Atlantic Forest as a\n‘museum of diversity’ for butterflies (Matos-Maravi et al., 2021).\nDespite the overall constant diversification rate, we find certain regional differences when studying the\nsouthern and northern Atlantic Forest individually. In particular, during the Paleogene, the southern region\nexperienced a slight increase in net diversification rate, likely related to the arrival of rapidly diversifying\nnymphalid groups across the Neotropics, such as Adelpha and Ithomiini (two clades known by their remark-\nably high species richness in the Andes) (Chazot et al., 2016; Ebel et al., 2015). Moreover, the complex\ntopography of the south may have further contributed to these regional disparities.\nWe hypothesise that the Atlantic Forest mountain ranges played an important role in the diversification\nof such lineages. Different mechanisms are known to promote species diversification in the Neotropical\nmountains. On one hand, mountain tops can act as isolated islands promoting allopatric diversification\nbetween scattered populations with restricted elevational ranges (Brown, 1987; Chaves, Freitas, Vasconcelos\n& Santos 2014, Magaldi et al., 2024). On the other hand, habitat complexity of montane environments\ncan act as species attractors, with higher rates of colonization towards the mountains, as found for the\nithomiine subtribe Godyridina in the Andes (Chazot et al., 2016b). Interestingly, a positive correlation\nemerged between wide altitudinal distribution (i.e., generalists species occurring in highlands and lowlands)\nand increased speciation rates in ithomiine butterflies of the southern Atlantic Forest. Highland specialists,\nhowever, showed no such pattern, contrary to expectations. This pattern may be explained by habitat\nheterogeneity along mountain ranges that could facilitate population differentiation in widespread lineages\nand eventually speciation. Indeed, habitat fragmentation driven by the Pleistocene climatic fluctuations\nmight have pushed lineages toward mountain climatic refuges and facilitated allopatric diversification (Brown\n1987, Carnaval and Moritz, 2008). Furthermore, the southern Atlantic Forest has higher levels of species\nrichness than the northern region, which is explained not by higher dispersal rates but also higher speciation\n8\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nrates, particularly involving mountain lineages in both the Atlantic Forest and the Andes. Both scenarios\nexplain the current species richness in mountain ranges but are driven by different processes. Overall,\nfor nymphalids, Pleistocene climatic fluctuations did not increase species diversification, but Nymphalidae\nspecies diversity accumulated gradually in the Atlantic Forest mainly through dispersal.\n5 Conclusions\nOur study shows that the Atlantic Forest Nymphalidae diversity has been shaped under a complex scenario\nof varying biotic connectivity through time with different biogeographical regions, including the central\nAndes and Amazonia. Despite the Diagonal of open formations being hypothesised as an important biogeo-\ngraphical barrier for wet forest lineages, the increased late Miocene to Pleistocene dispersal we detected in\nthe Atlantic Forest suggests that corridors with suitable habitats for different Nymphalidae lineages likely\nmaintained connectivity through time, especially between Amazonia and the Atlantic Forest. Overall, our\nfindings underscore the importance of jointly studying diversification and dispersal rates to understand the\nmacroevolutionary processes shaping extant biodiversity patterns in one of the world’s most biodiverse yet\nthreatened regions.\nReferences\nAguiar, A. J. C., Melo, G. A. R., Vasconcelos, T. N. C., Goncalves, R. B., Giugliano, L., & Mar-\ntins, A. C. (2020). Biogeography and early diversification of Tapinotaspidini oil-bees support pres-\nence of Paleocene savannas in South America. Molecular Phylogenetics and Evolution , 143 , 106692.\nhttps://doi.org/10.1016/j.ympev.2019.106692\nAndermann, T., Cano, A., Zizka, A., Bacon, C., & Antonelli, A. (2018). SECAPR-a bioinformatics pipeline\nfor the rapid and user-friendly processing of targeted enriched Illumina sequences, from raw reads to align-\nments. PeerJ , 6 , e5175. https://doi.org/10.7717/peerj.5175\nAndrews, S. (2010). FastQC: a quality control tool for high throughput sequence data, Available online at:\nhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc\nAnisimova, M., Gil, M., Dufayard, J.-F., Dessimoz, C., & Gascuel, O. (2011). Survey of Branch Sup-\nport Methods Demonstrates Accuracy, Power, and Robustness of Fast Likelihood-based Approximation\nSchemes.Systematic Biology , 60 (5), 685–699. https://doi.org/10.1093/sysbio/syr041\nAntonelli A, Ariza M, Albert J, Andermann T, Azevedo J, Bacon C, . . . Edwards SV. 2018. Conceptual and\nempirical advances in Neotropical biodiversity research. PeerJ 6:e5644 https://doi.org/10.7717/peerj.5644\nAntonelli, A., Kissling, W. D., Flantua, S. G. A., Bermudez, M. A., Mulch, A., Muellner-Riehl, . . . Hoorn, C.\n(2018). Geological and climatic influences on mountain biodiversity. Nature Geoscience ,11 (10), 718–725.\nhttps://doi.org/10.1038/s41561-018-0236-z\nAntonelli, A., & Sanmartin, I. (2011). Why are there so many plant species in the Neotropics? TAXON ,\n60 (2), 403–414. https://doi.org/10.1002/tax.602010\nAntonelli, A., Zizka, A., Carvalho, F. A., Scharn, R., Bacon, C. D., Silvestro, D., & Condamine, F. L. (2018).\nAmazonia is the primary source of Neotropical biodiversity. Proceedings of the National Academy of Sciences\n, 115 (23), 6034–6039. https://doi.org/10.1073/pnas.1713819115\nAzevedo, J. A. R., Collevatti, R. G., Jaramillo, C. A., Stromberg, C. A. E., Guedes, T. B., Matos-Maravi,\nP., . . . Antonelli, A. (2020). On the Young Savannas in the Land of Ancient Forests. In V. Rull & A. C.\nCarnaval (Eds.), Neotropical Diversification: Patterns and Processes (pp. 271–298). Springer International\nPublishing. https://doi.org/10.1007/978-3-030-31167-4 12\nBarbosa, E. P., Seraphim, N., Valencia, G., Azeredo-Espin, A. M. L., & Freitas, A. V. L. (2022).\nPhylogenetic systematics of Yphthimoides Forster, 1964 and related taxa, with notes on the biogeo-\ngraphical history of Yphthimoides species. Molecular Phylogenetics and Evolution , 168 , 107390.\nhttps://doi.org/10.1016/j.ympev.2022.107390\n9\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nBatalha-Filho, H., Fjeldsa, J., Fabre, P.-H., & Miyaki, C. Y. (2013). Connections between the Atlantic and\nthe Amazonian forest avifaunas represent distinct historical events. Journal of Ornithology ,154 (1), 41–50.\nhttps://doi.org/10.1007/s10336-012-0866-7\nBeckman, E. J., & Witt, C. C. (2015). Phylogeny and biogeography of the New World siskins and goldfinches:\nRapid, recent diversification in the Central Andes. Molecular Phylogenetics and Evolution , 87 , 28–45.\nhttps://doi.org/10.1016/j.ympev.2015.03.005\nBocalini, F., Bolivar-Leguizamon, S. D., Silveira, L. F., & Bravo, G. A. (2021). Comparative phylogeographic\nand demographic analyses reveal a congruent pattern of sister relationships between bird populations of\nthe northern and south-central Atlantic Forest. Molecular Phylogenetics and Evolution , 154 , 106973.\nhttps://doi.org/10.1016/j.ympev.2020.106973\nBrown JR, K. S. (1976). Geographical patterns of evolution in Neotropical Lepidoptera. Systematics and\nderivation of known and new Heliconiini (Nymphalidae: Nymphalinae). Journal of Entomology Series B,\nTaxonomy , 44 (3), 201–242. https://doi.org/10.1111/j.1365-3113.1976.tb00014.x\nBrown, Jr, K.S. (1987) Biogeography and evolution of Neotropical butterflies; Conclusions, synthesis, and\nalternative hypotheses. In Biogeography and Quaternary History in Tropical America (T.C. Whitmore and\nG.T. Prance, eds) pp.66–104, 175–96. Oxford: Clarendon.\nBrown, K. S., & Freitas, A. V. L. (2000). Atlantic Forest Butterflies: Indicators for Landscape Conservation\n. 23.\nCarnaval, A. C., Hickerson, M. J., Haddad, C. F. B., Rodrigues, M. T., & Moritz, C. (2009). Stabil-\nity Predicts Genetic Diversity in the Brazilian Atlantic Forest Hotspot. Science , 323 (5915), 785–789.\nhttps://doi.org/10.1126/science.1166955\nCarnaval, A. C., & Moritz, C. (2008). Historical climate modelling predicts patterns of current biodiversity in\nthe Brazilian Atlantic forest. Journal of Biogeography , 35 (7), 1187–1201. https://doi.org/10.1111/j.1365-\n2699.2007.01870.x\nCarrillo, J. D., Faurby, S., Silvestro, D., Zizka, A., Jaramillo, C., Bacon, C. D., & Antonelli, A.\n(2020). Disproportionate extinction of South American mammals drove the asymmetry of the Great\nAmerican Biotic Interchange. Proceedings of the National Academy of Sciences ,117 (42), 26281–26287.\nhttps://doi.org/10.1073/pnas.2009397117\nChaves, A. V., Freitas, G. H. S., Vasconcelos, M. F., & Santos, F. R. (2014). Biogeographic patterns,\norigin and speciation of the endemic birds from eastern Brazilian mountaintops: A review. Systematics and\nBiodiversity , 13 (1), 1–16. https://doi.org/10.1080/14772000.2014.972477\nChazot, N., Condamine, F. L., Dudas, G., Pena, C., Kodandaramaiah, U., Matos-Maravi, P., . . .\nWahlberg, N. (2021). Conserved ancestral tropical niche but different continental histories explain\nthe latitudinal diversity gradient in brush-footed butterflies. Nature Communications , 12 (1), 5717.\nhttps://doi.org/10.1038/s41467-021-25906-8\nChazot, N., Willmott, K. R., Condamine, F. L., De-Silva, D. L., Freitas, A. V. L., Lamas, G., . . . Elias, M.\n(2016). Into the Andes: Multiple independent colonizations drive montane diversity in the Neotropical clear-\nwing butterflies Godyridina. Molecular Ecology ,25 (22), 5765–5784. https://doi.org/10.1111/mec.13773\nChazot, N., Willmott, K. R., Freitas, A. V. L., de Silva, D. L., Pellens, R., & Elias, M. (2016). Pat-\nterns of Species, Phylogenetic and Mimicry Diversity of Clearwing Butterflies in the Neotropics. In\nR. Pellens & P. Grandcolas (Eds.), Biodiversity Conservation and Phylogenetic Systematics: Preserv-\ning our evolutionary heritage in an extinction crisis (pp. 333–354). Springer International Publishing.\nhttps://doi.org/10.1007/978-3-319-22461-9 17\nChazot, N., Willmott, K. R., Lamas, G., Freitas, A. V. L., Piron-Prunier, F., Arias, C. F., . . . Elias, M.\n(2019). Renewed diversification following Miocene landscape turnover in a Neotropical butterfly radiation.\n10\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nGlobal Ecology and Biogeography ,28 (8), 1118–1132. https://doi.org/10.1111/geb.12919\nChen, S. (2023). Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using\nfastp. iMeta , 2 (2), e107. https://doi.org/10.1002/imt2.107\nChernomor, O., von Haeseler, A., & Minh, B. Q. (2016). Terrace Aware Data Structure for Phylogenomic\nInference from Supermatrices.Systematic Biology , 65 (6), 997–1008. https://doi.org/10.1093/sysbio/syw037\nCicconardi, F., Milanetti, E., Pinheiro De Castro, E. C., Mazo-Vargas, A., Van Belleghem, S. M., Ruggieri,\nA. A., . . . Montgomery, S. H. (2023). Evolutionary dynamics of genome size and content during the adaptive\nradiation of Heliconiini butterflies. Nature Communications , 14 (1), 5620. https://doi.org/10.1038/s41467-\n023-41412-5\nCoelho, F. E. A., Camurugi, F., Marques, R., Magalhaes, F. D. M., Werneck, F. P., & Garda, A.\nA. (2022). Historical connections between Atlantic Forest and Amazonia drove genetic and ecolog-\nical diversity in Lithobates palmipes (Anura, Ranidae). Systematics and Biodiversity , 20 (1), 1–19.\nhttps://doi.org/10.1080/14772000.2022.2046657\nCosta, L. P. (2003). The historical bridge between the Amazon and the Atlantic Forest of Brazil: A\nstudy of molecular phylogeography with small mammals: Phylogeography of Neotropical forest small mam-\nmals.Journal of Biogeography , 30 (1), 71–86. https://doi.org/10.1046/j.1365-2699.2003.00792.x\nDamasceno, R., Strangas, M. L., Carnaval, A. C., Rodrigues, M. T., & Moritz, C. (2014).\nRevisiting the vanishing refuge model of diversification. Frontiers in Genetics , 5 , 353.\nhttps://doi.org/10.3389/fgene.2014.00353\nda Silva, F. R., Oliveira-Silva, A. E. de, Antonelli, A., Carnaval, A. C., & Provete, D. B. (2024). Zoogeo-\ngraphical regions in the Atlantic Forest: Patterns and potential drivers. Journal of Biogeography ,51 (10),\n1852–1863. https://doi.org/10.1111/jbi.14859\nDe Mello Martins, F. (2011). Historical biogeography of the Brazilian Atlantic forest and the Carnaval-Moritz\nmodel of Pleistocene refugia: What do phylogeographical studies tell us?: History of the Atlantic Forest. Bio-\nlogical Journal of the Linnean Society , 104 (3), 499–509. https://doi.org/10.1111/j.1095-8312.2011.01745.x\nde Moraes Magaldi, L., Gueratto, P. E., Ortega-Abboud, E., Sobral-Souza, T., Joron, M., de Souza, A. P.,\nFreitas, A. V. L., & Silva-Brandao, K. L. (2024). Montane diversification as a mechanism of speciation in\nneotropical butterflies. Ecology and Evolution , 14 (7), e11704. https://doi.org/10.1002/ece3.11704\nDe-Silva, D. L., Mota, L. L., Chazot, N., Mallarino, R., Silva-Brandao, K. L., Pinerez, L. M. G., . . . Elias,\nM. (2017). North Andean origin and diversification of the largest ithomiine butterfly genus.Scientific Reports\n, 7 (1), 45966. https://doi.org/10.1038/srep45966\nDrummond, A. J., Ho, S. Y. W., Phillips, M. J., & Rambaut, A. (2006). Relaxed Phylogenetics and Dating\nwith Confidence. PLOS Biology ,4 (5), e88. https://doi.org/10.1371/journal.pbio.0040088\nDupin, J., Matzke, N. J., Sarkinen, T., Knapp, S., Olmstead, R. G., Bohs, L., & Smith, S. D. (2017).\nBayesian estimation of the global biogeographical history of the Solanaceae. Journal of Biogeography , 44\n(4), 887–899. https://doi.org/10.1111/jbi.12898\nEbel, E. R., DaCosta, J. M., Sorenson, M. D., Hill, R. I., Briscoe, A. D., Willmott, K. R., & Mullen, S.\nP. (2015). Rapid diversification associated with ecological specialization in Neotropical Adelpha butterflies.\nMolecular Ecology , 24 (10), 2392–2405. https://doi.org/10.1111/mec.13168\nEspeland, M., Breinholt, J., Willmott, K. R., Warren, A. D., Vila, R., Toussaint, E. F. A., . . . Kawahara,\nA. Y. (2018). A Comprehensive and Dated Phylogenomic Analysis of Butterflies. Current Biology ,28 (5),\n770-778.e5. https://doi.org/10.1016/j.cub.2018.01.061\nEspeland, M., Nakahara, S., Zacca, T., Barbosa, E. P., Huertas, B., Marin, M. A., . . . Willmott, K. R.\n(2023). Combining target enrichment and Sanger sequencing data to clarify the systematics of the diverse\n11\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nNeotropical butterfly subtribe Euptychiina (Nymphalidae, Satyrinae). Systematic Entomology , 48 (4),\n498–570. https://doi.org/10.1111/syen.12590\nFiaschi, P., & Pirani, J. R. (2009). Review of plant biogeographic studies in Brazil. Journal of Systematics\nand Evolution ,47 (5), 477–496. https://doi.org/10.1111/j.1759-6831.2009.00046.x\nFine, P. V. A., & Ree, R. H. (2006). Evidence for a Time-Integrated Species-Area Effect on the Latitudinal\nGradient in Tree Diversity.The American Naturalist , 168 (6), 796–804. https://doi.org/10.1086/508635\nFjeldsa, J., & Rahbek, C. (2006). Diversification of tanagers, a species rich bird group, from low-\nlands to montane regions of South America. Integrative and Comparative Biology , 46 (1), 72–81.\nhttps://doi.org/10.1093/icb/icj009\nGBIF: The Global Biodiversity Information Facility (year) What is GBIF? . Available from\nhttps://www.gbif.org/what-is-gbif [13 January 2020]\nGoldberg, E. E., Lancaster, L. T., & Ree, R. H. (2011). Phylogenetic Inference of Reciprocal Ef-\nfects between Geographic Range Evolution and Diversification. Systematic Biology , 60 (4), 451–465.\nhttps://doi.org/10.1093/sysbio/syr046\nGoldberg, E. E., Roy, K., Lande, R., & Jablonski, D. (2005). Diversity, Endemism, and Age\nDistributions in Macroevolutionary Sources and Sinks. The American Naturalist , 165 (6), 623–633.\nhttps://doi.org/10.1086/430012\nGueratto, P. E. (2023). Neotropical butterflies: A historical approach to understand biogeographic patterns.\n[Doctoral dissertation, Universidade Estadual de Campinas]. Repositorio da Producao Cientifica e Intelectual\nda Unicamp. 105 p. rpc.oai.1361979\nHadad, C., Toledo, L. F., Prado, C., Loebmann, D., Gasparini, J., & Sazima, I. (2013). Guia dos anfibios\nda Mata Atlantica: Diversidade e biologia / Guide to the amphibians of the Atlantic Forest: Diversity and\nbiology.\nHaffer, J. (1969). Speciation in Amazonian Forest Birds. In Science (Vol. 165, Issue 3889, pp. 131–137).\nHasegawa, M., Kishino, H., & Yano, T. (1985). Dating of the human-ape splitting by a molecular clock of\nmitochondrial DNA. Journal of Molecular Evolution , 22 (2), 160–174. https://doi.org/10.1007/BF02101694\nHoorn, C., Wesselingh, F. P., Ter Steege, H., Bermudez, M. A., Mora, A., Sevink, J., . . . Antonelli, A. (2010).\nAmazonia Through Time: Andean Uplift, Climate Change, Landscape Evolution, and Biodiversity. Science\n, 330 (6006), 927–931. https://doi.org/10.1126/science.1194585\nHubbel, S. P. (2001). The Unified Neutral Theory of Biodiversity and Biogeography (MPB-32) . Princeton\nUniversity Press; JSTOR. http://www.jstor.org/stable/j.ctt7rj8w\nIgea, J., & Tanentzap, A. J. (2019). Multiple macroevolutionary routes to becoming a biodiversity hotspot.\nScience Advances ,5 (2), eaau8067. https://doi.org/10.1126/sciadv.aau8067\nIserhard, C. A., Romanowski, H. P., Richter, A., & Mendonca, M. de S., Jr. (2017). Monitoring Tem-\nporal Variation to Assess Changes in the Structure of Subtropical Atlantic Forest Butterfly Communi-\nties.Environmental Entomology , 46 (4), 804–813. https://doi.org/10.1093/ee/nvx115\nJaramillo, C. (2023). The evolution of extant South American tropical biomes. New Phytologist , 239 (2),\n477–493. https://doi.org/10.1111/nph.18931\nJaramillo, C., & Cardenas, A. (2013). Global Warming and Neotropical Rainforests: A Historical Perspec-\ntive. Annual Review of Earth and Planetary Sciences , 41 (1), 741–766. https://doi.org/10.1146/annurev-\nearth-042711-105403\nJetz, W., Thomas, G. H., Joy, J. B., Hartmann, K., & Mooers, A. O. (2012). The global diversity of birds\nin space and time. Nature ,491 (7424), 444–448. https://doi.org/10.1038/nature11631\n12\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nJoly, C. A., Metzger, J. P., & Tabarelli, M. (2014). Experiences from the Brazilian Atlantic Forest: Ecological\nfindings and conservation initiatives. New Phytologist , 204 (3), 459–473. https://doi.org/10.1111/nph.12989\nKalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., & Jermiin, L. S. (2017). Mod-\nelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods , 14 (6), 587–589.\nhttps://doi.org/10.1038/nmeth.4285\nKatoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version\n7: Improvements in performance and usability. Molecular Biology and Evolution , 30 (4), 772–780.\nhttps://doi.org/10.1093/molbev/mst010\nKawahara, A. Y., Breinholt, J. W., Espeland, M., Storer, C., Plotkin, D., Dexter, K. M., . . .\nLohman, D. J. (2018). Phylogenetics of moth-like butterflies (Papilionoidea: Hedylidae) based on\na new 13-locus target capture probe set. Molecular Phylogenetics and Evolution ,127 , 600–605.\nhttps://doi.org/10.1016/j.ympev.2018.06.002\nKawahara, A. Y., Storer, C., Carvalho, A. P. S., Plotkin, D. M., Condamine, F. L., Braga, M. P., . . .\nLohman, D. J. (2023). A global phylogeny of butterflies reveals their evolutionary history, ancestral hosts\nand biogeographic origins. Nature Ecology & Evolution ,7 (6), 903–913. https://doi.org/10.1038/s41559-\n023-02041-9\nKuhnhauser, B. G., Bates, C. D., Dransfield, J., Geri, C., Henderson, A., Julia, S., Lim, J. Y., . . . Baker,\nW. J. (2025). Island geography drives evolution of rattan palms in tropical Asian rainforests. Science , 387\n(6739), 1204–1209. https://doi.org/10.1126/science.adp3437\nMaliet, O., Hartig, F., & Morlon, H. (2019). A model with many small shifts for estimating species-specific\ndiversification rates.Nature Ecology & Evolution , 3 (7), 1086–1092. https://doi.org/10.1038/s41559-019-\n0908-0\nMaliet, O., & Morlon, H. (2022). Fast and Accurate Estimation of Species-Specific Diversification Rates\nUsing Data Augmentation.Systematic Biology , 71 (2), 353–366. https://doi.org/10.1093/sysbio/syab055\nMarques, M. C. M., & Grelle, C. E. V. (Eds.). (2021). The Atlantic Forest: History, Biodiversity, Threats and\nOpportunities of the Mega-diverse Forest . Springer International Publishing. https://doi.org/10.1007/978-\n3-030-55322-7\nMarques-Souza, S., Pellegrino, K. C. M., Brunes, T. O., Rojas-Runjaic, F. J. M., & Rodrigues, M. T. (2022).\nA molecular perspective on the systematics and distribution of Loxopholis lizards in South and Central\nAmerica, with advances on the biogeography of the tribe Ecpleopodini (Gymnophthalmidae: Squamata).\nSystematics and Biodiversity , 20 (1), 1–14. https://doi.org/10.1080/14772000.2022.2119295\nMartini, A. M. Z., Fiaschi, P., Amorim, A., & Paixao, J. (2007). A hot-point within a hot-spot:\nA high diversity site in Brazil’s Atlantic Forest. Biodiversity and Conservation , 16 , 3111–3128.\nhttps://doi.org/10.1007/s10531-007-9166-6\nMartins, E., & Hansen, T. (1997). Phylogenies and the Comparative Method: A General Approach to\nIncorporating Phylogenetic Information into the Analysis of Interspecific Data.American Naturalist - AMER\nNATURALIST , 149 . https://doi.org/10.1086/286013\nMatos-Maravi, P. (2016). Investigating the timing of origin and evolutionary processes shaping regional\nspecies diversity: Insights from simulated data and neotropical butterfly diversification rates. Evolution , 70\n(7), 1638–1650. https://doi.org/10.1111/evo.12960\nMatos-Maravi, P. F., Pena, C., Willmott, K. R., Freitas, A. V. L., & Wahlberg, N. (2013). Systematics and\nevolutionary history of butterflies in the “Taygetis clade” (Nymphalidae: Satyrinae: Euptychiina): Towards\na better understanding of Neotropical biogeography. Molecular Phylogenetics and Evolution , 66 (1), 54–68.\nhttps://doi.org/10.1016/j.ympev.2012.09.005\n13\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nMatos-Maravi, P., Wahlberg, N., Freitas, A. V. L., Devries, P., Antonelli, A., & Penz, C. M. (2021).\nMesoamerica is a cradle and the Atlantic Forest is a museum of Neotropical butterfly diversity: Insights\nfrom the evolution and biogeography of Brassolini (Lepidoptera: Nymphalidae). Biological Journal of the\nLinnean Society , 21.\nMatzke, N. J. (2018). Nmatzke/BioGeoBEARS: BioGeoBEARS: BioGeography with Bayesian (and\nlikelihood) Evolutionary Analysis with R Scripts (Version v1.1.1) [Computer software]. Zenodo.\nhttps://doi.org/10.5281/zenodo.1478250\nMeseguer, A. S., Michel, A., Fabre, P.-H., Perez Escobar, O. A., Chomicki, G., Riina, R., . . . Condamine,\nF. L. (2022). Diversification dynamics in the Neotropics through time, clades, and biogeographic regions.\neLife , 11 , e74503. https://doi.org/10.7554/eLife.74503\nMinh, B. Q., Nguyen, M. A. T., & von Haeseler, A. (2013). Ultrafast Approximation for Phylogenetic\nBootstrap. Molecular Biology and Evolution , 30 (5), 1188–1195. https://doi.org/10.1093/molbev/mst024\nMinh, B. Q., Schmidt, H. A., Chernomor, O., Schrempf, D., Woodhams, M. D., von Haeseler, A., & Lanfear,\nR. (2020). IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic\nEra.Molecular Biology and Evolution , 37 (5), 1530–1534. https://doi.org/10.1093/molbev/msaa015\nMittelbach, G. G., Schemske, D. W., Cornell, H. V., Allen, A. P., Brown, J. M., Bush, M. B., . . . Turelli, M.\n(2007). Evolution and the latitudinal diversity gradient: Speciation, extinction and biogeography. Ecology\nLetters , 10 (4), 315–331. https://doi.org/10.1111/j.1461-0248.2007.01020.x\nMoreira-Munoz, A., Scherson, R. A., Luebert, F., Roman, M. J., Monge, M., Diazgranados, M., & Silva, H.\n(2020). Biogeography, phylogenetic relationships and morphological analyses of the South American genus\nMutisia L.f. (Asteraceae) shows early connections of two disjunct biodiversity hotspots. Organisms Diversity\n& Evolution ,20 (4), 639–656. https://doi.org/10.1007/s13127-020-00454-z\nMorrone, J. J. (2014). Biogeographical regionalisation of the Neotropical region. Zootaxa , 3782 (1), 1.\nhttps://doi.org/10.11646/zootaxa.3782.1.1\nMyers, N., Mittermeier, R. A., Mittermeier, C. G., da Fonseca, G. A. B., & Kent, J. (2000). Biodiversity\nhotspots for conservation priorities. Nature , 403 (6772), 853–858. https://doi.org/10.1038/35002501\nOrme, D., Freckleton, R., Thomas, G., Petzoldt, T., Fritz, S., Isaac, N., & Pearse, W. (2012). CAPER:\nComparative Analyses of Phylogenetics and Evolution in R .\nPaz, A., Brown, J. L., Cordeiro, C. L. O., Aguirre-Santoro, J., Assis, C., Amaro, R. C., . . . Carnaval, A. C.\n(2021). Environmental correlates of taxonomic and phylogenetic diversity in the Atlantic Forest. Journal of\nBiogeography , 48 (6), 1377–1391. https://doi.org/10.1111/jbi.14083\nPeres, E. A., Pinto-da-Rocha, R., Lohmann, L. G., Michelangeli, F. A., Miyaki, C. Y., & Carnaval, A. C.\n(2020). Patterns of Species and Lineage Diversity in the Atlantic Rainforest of Brazil. In V. Rull & A. C.\nCarnaval (Eds.), Neotropical Diversification: Patterns and Processes (pp. 415–447). Springer International\nPublishing. https://doi.org/10.1007/978-3-030-31167-4 16\nPerez-Escobar, O. A., Zizka, A., Bermudez, M. A., Meseguer, A. S., Condamine, F. L., Hoorn, C., . . .\nChomicki, G. (2022). The Andes through time: Evolution and distribution of Andean floras. Trends in\nPlant Science , 27 (4), 364–378. https://doi.org/10.1016/j.tplants.2021.09.010\nPirani, R. M., Peloso, P. L. V., Prado, J. R., Polo, E. M., Knowles, L. L., Ron, S. R., . . . Wer-\nneck, F. P. (2020). Diversification history of clown tree frogs in Neotropical rainforests (Anura, Hyl-\nidae, Dendropsophus leucophyllatus group). Molecular Phylogenetics and Evolution , 150 , 106877.\nhttps://doi.org/10.1016/j.ympev.2020.106877\nPrates, I., Melo-Sampaio, P. R., Drummond, L. D. O., Teixeira, M., Rodrigues, M. T., & Carnaval, A. C.\n(2017). Biogeographic links between southern Atlantic Forest and western South America: Rediscovery,\n14\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nre-description, and phylogenetic relationships of two rare montane anole lizards from Brazil. Molecular\nPhylogenetics and Evolution ,113 , 49–58. https://doi.org/10.1016/j.ympev.2017.05.009\nPrates, I., Xue, A. T., Brown, J. L., Alvarado-Serrano, D. F., Rodrigues, M. T., Hickerson, M.\nJ., & Carnaval, A. C. (2016). Inferring responses to climate dynamics from historical demography\nin neotropical forest lizards. Proceedings of the National Academy of Sciences ,113 (29), 7978–7985.\nhttps://doi.org/10.1073/pnas.1601063113\nPrjibelski, A., Antipov, D., Meleshko, D., Lapidus, A., & Korobeynikov, A. (2020). Using SPAdes De Novo\nAssembler. Current Protocols in Bioinformatics , 70 (1), e102. https://doi.org/10.1002/cpbi.102\nR Core Team (2024). R: A Language and Environment for Statistical Computing. R Foundation for\nStatistical Computing, Vienna, Austria. Available at https://www.R-project.org/.\nRangel, T. F., Edwards, N. R., Holden, P. B., Diniz-Filho, J. A. F., Gosling, W. D., Coelho, M. T. P.,\n. . . Colwell, R. K. (2018). Modeling the ecology and evolution of biodiversity: Biogeographical cradles,\nmuseums, and graves. Science , 361 (6399), eaar5452. https://doi.org/10.1126/science.aar5452\nRedding, D. W., & Mooers, A. O. (2006). Incorporating Evolutionary Measures into Conservation Prioriti-\nzation. Conservation Biology ,20 (6), 1670–1678. https://doi.org/10.1111/j.1523-1739.2006.00555.x\nRibeiro, P. G., Torres Jimenez, M. F., Andermann, T., Antonelli, A., Bacon, C. D., & Matos-Maravi, P.\n(2021). A bioinformatic platform to integrate target capture and whole genome sequences of various read\ndepths for phylogenomics. Molecular Ecology , 30 (23), 6021–6035. https://doi.org/10.1111/mec.16240\nSafford, H. D. (2007). Brazilian Paramos IV. Phytogeography of the campos de altitude. Journal of Bio-\ngeography , 34 (10), 1701–1722. https://doi.org/10.1111/j.1365-2699.2007.01732.x\nSantos, J. P., Santos, D., Victor, A. E., Freitas, L., Spalding Brown, K., Yasmin, J., . . . Ribeiro, M. C.\n(2018). Atlantic butterflies: A data set of fruit-feeding butterfly communities from the Atlantic forests.\nEcology , 99 (12), 2875. https://doi.org/10.1002/ecy.2507/suppinfo\nShirai, L. T., Machado, P. A., Mota, L. L., Rosa, A. H. B., & Freitas, A. V. L. (2019). DnB, the Database\nof Nymphalids in Brazil, with a Checklist for Standardized Species Lists. The Journal of the Lepidopterists’\nSociety , 73 (2), 93. https://doi.org/10.18473/lepi.73i2.a4\nSilva, S. M., Moraes-Barros, N., Ribas, C. C., Ferrand, N., & Morgante, J. S. (2012). Divide to conquer: A\ncomplex pattern of biodiversity depicted by vertebrate components in the Brazilian Atlantic Forest: VER-\nTEBRATE COMPONENTS IN THE BRAZILIAN ATLANTIC FOREST.Biological Journal of the Linnean\nSociety , 107 (1), 39–55. https://doi.org/10.1111/j.1095-8312.2012.01919.x\nSilva-Brandao, K. L., Wahlberg, N., Francini, R. B., Azeredo-Espin, A. M. L., Brown, K. S., Paluch, M.,\nLees, D. C., & Freitas, A. V. L. (2008). Phylogenetic relationships of butterflies of the tribe Acraeini\n(Lepidoptera, Nymphalidae, Heliconiinae) and the evolution of host plant use. Molecular Phylogenetics and\nEvolution , 46 (2), 515–531. https://doi.org/10.1016/j.ympev.2007.11.024\nSilveira, L., Olmos, F., & Long, A. (2003). Birds in Atlantic Forest fragments in north-east Brazil. Cotinga\n, 20 , 32–46.\nSobral-Souza, T., & Lima-Ribeiro, M. S. (2017). De volta ao passado: revisitando a\nhistoria biogeografica das florestas neotropicais umidas. Oecologia Australis , 21 (02), 93–107.\nhttps://doi.org/10.4257/oeco.2017.2102.01\nSonne, J., Dalsgaard, B., Borregaard, M. K., Kennedy, J., Fjeldsa, J., & Rahbek, C. (2022). Biodiversity\ncradles and museums segregating within hotspots of endemism. Proceedings of the Royal Society B: Biological\nSciences , 289 (1981), 20221102. https://doi.org/10.1098/rspb.2022.1102\nTitle, P. O., Swiderski, D. L., & Zelditch, M. L. (2022). EcoPhyloMapper: An r package for integrating\ngeographical ranges, phylogeny and morphology. Methods in Ecology and Evolution ,13 (9), 1912–1922.\n15\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nhttps://doi.org/10.1111/2041-210X.13914\nTopel, M., Zizka, A., Calio, M. F., Scharn, R., Silvestro, D., & Antonelli, A. (2016). SpeciesGeoCoder: Fast\nCategorization of Species Occurrences for Analyses of Biodiversity, Biogeography, Ecology, and Evolution.\nSystematic Biology , 66 , syw064. https://doi.org/10.1093/sysbio/syw064\nTrujillo-Arias, N., Calderon, L., Santos, F. R., Miyaki, C. Y., Aleixo, A., Witt, C. C., Tubaro, P. L., &\nCabanne, G. S. (2018). Forest corridors between the central Andes and the southern Atlantic Forest enabled\ndispersal and peripatric diversification without niche divergence in a passerine. Molecular Phylogenetics and\nEvolution ,128 , 221–232. https://doi.org/10.1016/j.ympev.2018.08.005\nTrujillo-Arias, N., Rodriguez-Cajarville, M. J., Sari, E., Miyaki, C. Y., Santos, F. R., Witt, . . . Cabanne,\nG. S. (2020). Evolution between forest macrorefugia is linked to discordance between genetic and mor-\nphological variation in Neotropical passerines. Molecular Phylogenetics and Evolution , 149 , 106849.\nhttps://doi.org/10.1016/j.ympev.2020.106849\nVallejos-Garrido, P., Pino, K., Espinoza-Aravena, N., Pari, A., Inostroza-Michael, O., Toledo-Munoz, M., . . .\nRodriguez-Serrano, E. (2023). The importance of the Andes in the evolutionary radiation of Sigmodontinae\n(Rodentia, Cricetidae), the most diverse group of mammals in the Neotropics. Scientific Reports , 13 (1),\n2207. https://doi.org/10.1038/s41598-023-28497-0\nVancine, M. H., Muylaert, R. L., Niebuhr, B. B., Oshima, J. E. de F., Tonetti, V., Bernardo, . . . Ribeiro, M.\nC. (2024). The Atlantic Forest of South America: Spatiotemporal dynamics of the vegetation and implica-\ntions for conservation. Biological Conservation ,291 , 110499. https://doi.org/10.1016/j.biocon.2024.110499\nVelazco, P. M., & Patterson, B. D. (2013). Diversification of the Yellow-shouldered bats, Genus Sturnira\n(Chiroptera, Phyllostomidae), in the New World tropics. Molecular Phylogenetics and Evolution ,68 (3),\n683–698. https://doi.org/10.1016/j.ympev.2013.04.016\nWahlberg, N., & Wheat, C. W. (2008). Genomic Outposts Serve the Phylogenomic Pioneers: Designing\nNovel Nuclear Markers for Genomic DNA Extractions of Lepidoptera. Systematic Biology , 57 (2), 231–242.\nhttps://doi.org/10.1080/10635150802033006\nWallace, Alfred Russel. (1878). Tropical nature, and other essays . London, Macmillan and co, 1878.\nhttps://www.biodiversitylibrary.org/item/16104\nWerneck, F. P. (2011). The diversification of eastern South American open vegetation biomes:\nHistorical biogeography and perspectives. Quaternary Science Reviews , 30 (13–14), 1630–1648.\nhttps://doi.org/10.1016/j.quascirev.2011.03.009\nWickham H (2016). ggplot2: Elegant Graphics for Data Analysis . Springer-Verlag New York. ISBN\n978-3-319-24277-4, https://ggplot2.tidyverse.org\nYan, Z.-T., Fan, Z.-H., He, S.-L., Wang, X.-Q., Chen, B., & Luo, S.-T. (2023). Mitogenomes of\nEight Nymphalidae Butterfly Species and Reconstructed Phylogeny of Nymphalidae (Nymphalidae: Lepi-\ndoptera).Genes , 14 (5). https://doi.org/10.3390/genes14051018\nYang, M., & Zhang, Y. (2015). Molecular phylogeny of the butterfly tribe Satyrini (Nymphalidae: Satyrinae)\nwith emphasis on the utility of ribosomal mitochondrial genes 16s rDNA and nuclear 28s rDNA. Zootaxa ,\n3985 (1), 125. https://doi.org/10.11646/zootaxa.3985.1.7\nYang, Z. (2007). PAML 4: Phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution ,\n24 (8), 1586–1591. https://doi.org/10.1093/molbev/msm088\nData Accessibility Statement\nThe raw data sequences generated in this study are available in NCBI assigned to the Bio-\nProject PRJNA1297423. Alignments, selected partitions, tree files and R scripts used for the\n16\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nbiogeographical analyses and plots, are available in Dryad (temporary link before publication:\nhttp://datadryad.org/share/t50cO4uD14GiJzxBQSKO6T1YvaBVWinmjD0syaC00Qw).\nTables\nTABLE 1. Number of species per clade used for phylogenetic inference and biogeographical and diversi-\nfication analyses. Nine clades were extracted for phylogenetic inference and calibration. The nine clades\nwere then subdivided into fourteen subclades for biogeographical and diversification analyses, in order to\nminimise the number of species outside the Neotropics.\n(phylogenetic inference)\n& Species from Chazot et al. (2021) Species from Kawahara et al. (2023) Species from this\nstudy Subclades for Biogeographical Analyses Species number (Biogeographical analyses) Danainae\n436 376 60 0 Ithomiini 331 Heliconiinae 192 148 42 2 Actinote 37 Heliconiini 69 Limetidinae 107 88 19\n0 Adelpha 67 Biblidini 210 154 42 14 Biblidini 170 Cyrestinae & Nymphalinae 447 380 67 0 Hypanartia 9\nMarpesia 13 Melitaeini 174 Charaxinae 265 228 29 8 Memphis 54 Prepona 23 Satyrinae subclade\n242 173 59 10 Haeterini 17 Morphini & Brassolini 108 Euptychiina (Satyrini, Satyrinae) 320 201 89 30\nEuptychiina subclade 194 Pronophilina (Satyrini, Satyrinae) 136 93 39 4 Pronophilina 111 TOTAL 2355\n1841 446 68 1377\nFigures\nFIGURE 1. Nymphalidae interchange (dispersal events) across Neotropical regions. The analyses are based\non 1377 Nymphalidae species (see Material and Methods). (a) Map of Neotropical biogeographic regions\nconsidered in this study following Morrone (2014), together with the species number per area included in our\nbiogeographical analyses. Letters correspond to: F = Northern Atlantic Forest; S = southern Atlantic Forest;\nD = Diagonal of open vegetation formations; Z = Amazonia; C = Mesoamerica + NW slopes of Andes; N =\nNorthern Andes; A = Central Andes. (b) Nymphalidae dispersal events from the Biogeographical Stochastic\nMapping results. Arrows indicate the direction and number of events; line thickness is proportional to the\nnumber of events, with a minimum of 30. (c) Proportion of within-speciation and immigration events among\n17\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nregions. Proportions were estimated by dividing the number of immigration events (sum of dispersal events\ninto each region) by the number of extant species included in the analysis per region. Within-speciation\nevents were calculated as the rest of the extant number of species minus the immigration events.\nFIGURE 2. Dispersal rates through time to the Atlantic Forest overall (A) and (B) from different Neotrop-\nical regions. Andes = red; Amazonia = blue; Diagonal of open formations = dark yellow; Mesoamerica and\nNW slopes of Andes = light sand-coloured. Continuous lines are the median values, and coloured ribbons\nare the lower and upper quantiles (0.40 and 0.60). Abbreviations: Eoc: Eocene, Plio: Pliocene, Plei: Pleis-\ntocene, Mya: million years ago. The plots were generated with R (https://cran.r-project.org/) and edited\nwith Inkscape (https://inkscape.org).\n18\n\nPosted on 15 Sep 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.175795178.89475040/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary.\nFIGURE 3. Diversification rates from ClaDS results for the entire Atlantic Forest (green), Southern\nAtlantic Forest (orange) and northern Atlantic Forest (blue). Continuous lines are the median values, and\ncoloured ribbons are the lower and upper quantiles (0.40 and 0.60). Abbreviations: Plio: Pliocene, Plei:\nPleistocene, Mya: million years ago. The plots were generated with R (https://cran.r-project.org/) and\nedited with Inkscape (https://inkscape.org).\n19","source_license":"CC-BY-4.0","license_restricted":false}