Tracing the origins and evolution of nymphalid butterflies (Lepidoptera) in the Atlantic Forest

Understanding the relative roles of diversification and dispersal is key to explaining large-scale biogeographic patterns. Although both processes are known to shape biodiversity, their relative contributions remain understudied in many systems. Here, we examine how these processes have jointly contributed to the exceptional diversity and endemism of Nymphalidae butterflies in South America’s Atlantic Forest, a global biodiversity hotspot. We obtained DNA sequences for 68 Nymphalidae species (43 missing from major phylogenies) and integrated them into published time-calibrated phylogenies. We used Dispersal-extinction- cladogenesis models and Biogeographical Stochastic Mapping to infer historical biogeographic patterns over time, and ClaDS to estimate region-specific diversification rates. We further evaluate whether regional patterns of diversification are associated with occurrence in montane environments or across the north–south biogeographic break within the Atlantic Forest. Our results show that butterfly diversity in the region was driven primarily by recurrent dispersal from Amazonia and the Andes, rather than by elevated in situ diversification, which remained low and stable through time. Although dispersal increased progressively during the Cenozoic, we found no evidence that the Diagonal of open formations acted as a major barrier, indicating that forest corridors probably allowed extensive exchanges between the Atlantic Forest and other Neotropical regions. Southern lineages exhibited slightly higher diversification rates, especially among montane generalist species, but overall diversification contributed little compared to the sustained input of dispersing lineages. Together, these findings highlight the central role of biome connectivity in shaping Atlantic Forest Nymphalidae diversity, while underscoring the importance of jointly considering diversification and dispersal processes to better understand the macroevolutionary dynamics underlying current biodiversity patterns. Tracing the origins and evolution of nymphalid butterflies (Lepidoptera) in the Atlantic Forest Running title: Nymphalidae Evolution in the Atlantic Forest Understanding the relative roles of diversification and dispersal is key to explaining large- scale biogeographic patterns. Although both processes are known to shape biodiversity, their relative contributions remain understudied in many systems. Here, we examine how these processes have jointly contributed to the exceptional diversity and endemism of Nymphalidae butterflies in South America’s Atlantic Forest, a global biodiversity hotspot. We obtained DNA sequences for 68 Nymphalidae species (43 missing from major phylogenies) and integrated them into published time-calibrated phylogenies. We used Dispersal-extinction-cladogenesis 1 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. models and Biogeographical Stochastic Mapping to infer historical biogeographic patterns over time, and ClaDS to estimate region-specific diversification rates. We further evaluate whether regional patterns of diversification are associated with occurrence in montane environments or across the north–south biogeographic break within the Atlantic Forest. Our results show that butterfly diversity in the region was driven primarily by recurrent dispersal from Amazonia and the Andes, rather than by elevated in situ diversification, which remained low and stable through time. Although dispersal increased progressively during the Cenozoic, we found no evidence that the Diagonal of open formations acted as a major barrier, indicating that forest corridors probably allowed extensive exchanges between the Atlantic Forest and other Neotropical regions. Southern lineages exhibited slightly higher diversification rates, especially among montane generalist species, but overall diversification contributed little compared to the sustained input of dispersing lineages. Together, these findings highlight the central role of biome connectivity in shaping Atlantic Forest Nymphalidae diversity, while underscoring the importance of jointly considering diversification and dispersal processes to better understand the macroevolutionary dynamics underlying current biodiversity patterns. KEY WORDS : Atlantic Forest, biogeography, Diagonal of open formations, dispersal, Neotropics, Nymphalidae. 1 Introduction Large-scale historical biogeographic patterns are determined by the interplay between rates of diversification (comprising speciation and extinction) and dispersal (Hubbell, 2001). Traditionally, macroevolutionary explanations for biotic assembly have emphasised two main ideas: (1) that older clades and larger geographic areas accumulate more species — the time- and area-for-speciation hypotheses, or together, as the time- integrated species-area effect(Fine and Ree, 2006; Wallace, 1878); and (2) that regional differences in species richness result from episodic shifts in speciation and extinction rates — the rate-of-diversification hypothesis (Mittelbach et al., 2007). Nevertheless, more recent frameworks also acknowledge the influence of asymmetric dispersal rates between regions (e.g., Goldberg, Roy, Lande, & Jablonski, 2005; Goldberg, Lancaster & Ree, 2011; Kuhnh¨auser et al., 2025). This has led to a better characterisation of the relative importance of time, diversification and dispersal processes in explaining extant regional differences in species diversity (Carrillo et al., 2020; Igea and Tanentzap, 2019; Kuhnh¨auser et al., 2025). However, empirical evidence is still mainly restricted to a few taxonomic groups or is mainly focused on diversification rates, neglecting the contribution of dispersal in explaining large-scale biodiversity patterns. With only a fraction of its original vegetation remaining (8% – 22%), mostly in small, disconnected fragments (Vancine et al., 2024, Joly, Metzger & Tabarelli, 2014), the Atlantic Forest is considered one of the most threatened biodiversity hotspots on Earth (Myers, Mittermeier, Mittermeier, da Fonseca & Kent, 2000; Mar- ques & Grell, 2021). This biogeographical domain harbours high levels of endemism across plants (Martini, Fiaschi, Amorim & Paix˜ ao, 2007; Fiaschi & Pirani, 2009), amphibians (Hadad et al., 2013), birds (Silveira, Olmos & Long, 2003), and butterflies (Brown & Freitas, 2000; Santos et al., 2018), among other groups. Throughout its area, congruent patterns of species richness and endemism across animals and plants are thought to be shaped by contemporary and historical climatic changes and topographical variation. For instance, a large turnover in ecological communities exists between the southern and northern regions (Peres et al., 2020), but the relative contribution of species diversification and dispersal in shaping such patterns remains unknown for most species in the Atlantic Forest (da Silva Oliveira-Silva, Antonelli, Carnaval & Provete, 2024; Peres et al., 2020). Dispersal dynamics between the Atlantic Forest and other Neotropical regions have been linked to paleoen- vironmental changes in the Cenozoic (i.e., the past 66 million years). During the Paleogene (ca. 66 to 23 million years ago, Mya), Amazonia and the Atlantic Forest seem to have been more interconnected than today (Sobral-Souza & Lima Ribeiro, 2017). Yet, the extent of this connectivity remains under debate, and the timing of the Atlantic Forest’s origin is still unclear (Jaramillo & C´ ardenas, 2013; Jaramillo, 2023). Global cooling and the major Andean uplift in the Neogene (ca. 23 to 2.6 Mya) promoted the expansion 2 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. of open habitats in the Neotropics, likely reducing the connectivity between the Atlantic Forest and other Neotropical regions (Hoorn et al., 2010; Werneck, 2011). However, fossil and molecular evidence suggest that intermittent humid corridors maintained connectivity during wet periods during the Pleistocene (Bonaccorso et al., 2006; Batalha-Filho, Fjelds˚ a, Fabre & Miyaki, 2013; Cohelho et al., 2022), but also earlier in the Neo- gene (Batalha-Filho et al., 2013; Marques-Souza et al., 2022). Under this scenario, the increasing isolation of the Atlantic Forest, combined with intermittent reconnections to other regions, may have led to different dispersal dynamics and biogeographical patterns across lineages (Batalha-Filho et al., 2013; Prates et al., 2016; Sobral-Souza & Lima-Ribeiro, 2017; Bocalini, Bol´ ıvar-Leguizam´ on, Silveira & Bravo, 2021). Pleistocene climatic fluctuations have also been major determinants of diversification and community as- sembly within the Atlantic Forest – as initially proposed in the Pleistocene refugia hypothesis (Haffer 1969; Brown 1976) and later including divergent selection in the Vanishing refuge model (Damasceno, Strangas, Carnaval, Rodrigues & Moritz 2014). Both paleoclimatic (Carnaval and Moritz, 2008) and phylogenetic data (Carnaval, Hickerson, Haddad, Rodrigues & Moritz, 2009; Silva, Moraes-Barros, Ribas, Ferrand & Morgante, 2012; Martins et al., 2011) support distinct regional evolutionary trajectories during climatic fluctuations: while the region north of the Doce River (central Bahia) has remained paleoclimatically stable, the southeast underwent dramatic environmental reconfigurations during cool phases, prompting many lineages to retreat to mountain refuges (Carnaval et al., 2009; Peres et al., 2020). These regional differences may have led to different diversification rates through time: constant in the north and increasing in the unstable south during climatic fluctuations (Paz et al., 2021). To offer further insights into the Atlantic Forest’s macroevolutionary history, we focus on the species-rich butterfly family Nymphalidae, with nearly 500 species across the region (Brown & Freitas, 2000; Santos et al., 2018). Their distribution patterns in the Atlantic Forest are well documented (Santos et al., 2018; Shirai, Machado, Mota, Rosa & Freitas, 2019; Freitas et al., in prep.) and similar to other taxa, with the highest species richness in the southeastern montane regions (Iserhard, Romanowski, Richter & Mendon¸ ca, 2017; Santos et al., 2018). We build upon the most comprehensive global phylogeny of Nymphalidae to date (Chazot et al., 2021), and sequenced 68 species, including 43 species previously absent from global time-calibrated butterfly phylogenies (Chazot et al., 2021; Kawahara et al., 2023). Based on compiled distribution data for 1,096 species, we explored whether the diversity of Atlantic Forest Nymphalidae results from (1) a long history of complete biotic isolation, reflected in early dispersal events (i.e., during the Paleogene) followed by in situ diversification (i.e., within the Atlantic Forest); (2) gradual isolation, leading to decreasing interchange with Amazonia and a stronger role of local speciation; or (3) continuous connectivity with other Neotropical biomes, where recurrent dispersal dominates and local diversification plays a minor role. Furthermore, we evaluated whether occurrence in montane regions and along the north–south biogeographic break may explain the extant diversification rates within the Atlantic Forest nymphalids. 2 Material and methods 2.1 Molecular data We targeted Nymphalidae species from the Atlantic Forest that were not previously sequenced in phylogenetic studies. We sampled 68 adult specimens across eastern Brazil, including 43 species newly sequenced for time-calibrated phylogenies (Table S1). We extracted DNA from legs using DNeasy Blood and Tissue Kits (QIAGEN), and libraries were prepared and sequenced on an Illumina NovaSeq 6000 platform by the company Novogene, UK. We checked the reads quality (FastQC v0.12.1, Andrews, 2010), trimmed the adapters (fastp, Chen, 2023) and assembled de novo contigs (SPAdes v3.15.4, Prjibelski, Antipov, Meleshko, Lapidus & Korobeynikov, 2020). Following the BUTTERFLY1.0 probe set (Espeland et al., 2018; Kawahara et al., 2023), we retained 391 loci, including 11 legacy genes traditionally used in butterfly systematics (Wahlberg and Wheat, 2008; Kawahara et al., 2018). We used the SECAPR v2.2.3 environment (Andermann, Cano, Zizka, Bacon & Antonelli, 2018; Ribeiro et al., 2021) from de novo contig assembly to multiple sequence alignments. We focused on monophyletic groups from the Nymphalidae phylogeny of Chazot et al., (2021), with more 3 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. than half of the species diversity occurring in the Neotropics. The function “CladeByTrait()” of the R package ‘speciesgeocodeR’ (T ¨opel et al., 2016) found nine neotropical groups (“Clades for Phylogenetic Inference” in Table 1), representing the clades Danainae, Heliconiinae, Limenitidinae, Biblidini, a clade containing Cyrestinae and Nymphalinae, Charaxinae, a Satyrinae subclade, Euptychiina and Pronophilina. We merged our data with the most comprehensive phylogenies of Nymphalidae (Chazot et al., 2021) and butterflies in general (Kawahara et al., 2023), and conducted multi-species alignments for each of the nine clades using MAFFT v7.520 (Katoh & Standley, 2013), only retaining loci present in [?]10% of the species in that clade. Final alignments were checked in Geneious Prime 2023.1.2, and are reported in Supporting Information (https://doi.org/10.5061/dryad.wm37pvn11). 2.2 Time-calibrated phylogenetic inference We infer phylogenetic relationships for each of the nine clades using IQ-TREE 2.2.0 (Minh et al., 2020) under a concatenated, codon-partitioned scheme (Chernomor, von Haeseler & Minh, 2016), with model selection performed by ModelFinder (Kalyaanamoorthy, Minh, Wong, von Haeseler & Jermiin , 2017). Branch support was calculated using 1,000 ultrafast bootstrap replicates (Minh, Nguyen & von Haeseler, 2013), Shimodaira-Hasegawa approximate likelihood ratio tests, and approximate Bayes test (Anisimova, Gil, Dufayard, Dessimoz & Gascuel, 2011). To time calibrate each of the nine tree-topologies, we used the Bayesian approach implemented in MCMC- Tree from PAML package v4.10.6 (Yang 2007). We used an independent-rates relaxed molecular clock model (Drummond, Ho, Phillips & Rambaut, 2006) and the most complex substitution model available in MCMC- Tree, the HKY85 (Hasegawa, Kishino & Yano, 1985). Due to the limited availability of butterfly fossils, we used eight secondary calibration points, along with the crown age of each clade (Table S2), extracted from the fossil-calibrated phylogeny of Chazot et al., (2021). To ensure convergence, the analysis was run two times independently using random seeds, and similar results were observed (Figure S1). 2.3 Inference of biogeographical history We estimated range evolution using the Dispersal-Extinction-Cladogenesis (DEC) model in the R package ‘BioGeoBEARS v.1.1.3’ (Matzke, 2018), subdividing the nine time-calibrated trees into 14 Neotropical sub- clades to reduce noise and improve computational performance (Table 1 and Figure S2). We subdivided the Neotropics into eight biogeographic regions: (1) Mesoamerica and northwestern lowland Andean slopes, (2) Northern Andes (Ecuador, Colombia, Venezuela), (3) Central Andes (Peru, Bolivia), (4) Amazonia, (5) Diagonal of open formations encompassing the Caatinga, Cerrado and Chaco biomes, (6) Northern Atlantic Forest and (7) Southern Atlantic Forest, both delimited by the Doce river which reflects a strong community turnover of animal and plant diversity, and (8) outside the defined Neotropical areas (Figure 1), largely following the biogeographical regions proposed by Morrone (2014). To avoid unrealistic biogeographical scenarios, we disallowed dispersal events between Mesoamerica and the Northern Andes from/to the Diag- onal of open formations and the Atlantic Forest. This constraint was applied globally across the trees and not stratified temporally. Unconstrained analyses (i.e., without adjacency matrix) were also performed for comparison. Species ranges were compiled from the literature, including original descriptions, databases of approximate range maps (Lepidoptera and some other life forms, Savela M.), geo-referenced occurrences (GBIF and iNaturalist), and experts’ knowledge (distributional data reported in Table S3). To account for divergence time uncertainty, we utilised 50 randomly selected trees from the MCMCtree posterior distribution for each study clade, and to address ancestral geographical state uncertainty, we performed 50 Biogeographical Stochastic Mappings per tree (BSM; Dupin et al., 2017). Dispersal rates were extracted in 1 Myr intervals and scaled by total branch length per bin, following the approach described in Antonelli et al., (2018) and the R scripts from Matos-Maravi et al., (2021). 2.4 Within-region species diversification analysis We used CLaDS (Maliet et al., 2019; Maliet & Morlot, 2022) to estimate branch-specific diversification rates, assuming a constant turnover across the entire phylogeny (one for each of the 14 subclades), and accounting 4 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. for incomplete taxon sampling with a sampling probability vector (Table S4). To calculate diversification rates within geographic regions, we combined the CLaDS outputs with BioGeoBEARS ancestral range esti- mates from BSM. For each branch section, we identified the region(s) it occupied, weighted its diversification rate by the proportion of its length in that region (“occupancy”), and further adjusted by the frequency of occurrence across all BSM replicates. This approach ensured that branches only partially present in a region, or with uncertain regional assignment, contributed proportionally less to the regional diversification estimates. This was done for every 1 Myr time bin, allowing us to extract the diversification rate through time at each region (R scripts in Supporting Information). 2.5 Effect of altitudinal variation on the diversification of Atlantic Forest Nymphalidae We assessed whether environmental variation along altitudinal gradients in the Atlantic Forest were asso- ciated with extant speciation rate heterogeneity among clades (i.e., tip rates). We categorised species into lowland, highland, or generalist, and into northern, southern, or widespread distributions. Speciation rate for each tip was estimated using either the ClaDS estimates or the tip-specific DR-statistic (Redding and Mooers 2006), a non-model-based approach that approximates speciation rates (Jetz et al., 2012), calculated with the R package ‘epm’ (Title, Swiderski & Zelditch, 2022). To test whether geographical occurrence predicted tip speciation rates, we used phylogenetic generalised least-squares regression (PGLS; Martins and Hansen, 1997), as implemented in the R package ‘caper’ (Orme, 2012). We ran the analyses for every subclade separately, using either CLaDS rate or DR as response variables. An extended version of the Methods section and the R scripts used here are available in the supplementary

and Supporting information (https://doi.org/10.5061/dryad.wm37pvn11). 3 Results 3.1 Molecular data and time-calibrated phylogenetic inference The size of our molecular datasets varied between 159,716 bp (Charaxinae clade) and 162,060 bp (Satyrinae subclade), representing 387 and 391 loci, respectively. The Maximum Likelihood tree topologies inferred in IQ-TREE 2.2.0 (Minh et al., 2020) were congruent with other recent published phylogenies focused on specific taxa (e.g., Barbosa, Seraphim, Valencia, Azeredo-Espin & Freitas et al., 2022; Chazot et al., 2021; Cicconardi et al., 2023; Espeland et al., 2023; Kawahara et al., 2023; Silva-Brandao et al., 2008; Yang & Zhang, 2015; Yan et al., 2023). The few differences found were either at the species level or for relationships among lineages that have been previously difficult to resolve: e.g., the genera Brassolis (Brassolini), Methona, Hypothyris, and Hyalyris (Ithomiini), which were paraphyletic in our inferred phylogenies but were also reported as having low phylogenetic support in previous studies (Chazot et al., 2019; Matos-Maravi et al., 2021). Regardless, because such incongruences were very few and mostly at shallow phylogenetic levels, we do not consider them to have biased our macroevolutionary and biogeographical inferences. 3.2 Inference of biogeographical history Our Biogeographical Stochastic Mapping for the whole Neotropical region showed that the highest number of dispersal events were into and out of Amazonia (231 events into and 427 events out), followed by the Andes (336 events into and 423 events out) (Figure 1). Regarding the Atlantic Forest, Amazonia was also the major source (144.78 events from Amazonia to Southern and Northern Atlantic Forest), compared to the 131.3 events from the Andes to the Atlantic Forest. Dispersal into the Atlantic Forest from other Neotropical regions occurred constantly through time until about 10 Mya, when it accelerated toward the present (Figure 2). Overall, Amazonia has been the primary source of lineages dispersing into the Atlantic Forest relatively constant throughout the Neogene, only sur- passed by the Andean dispersal during the Eocene (prior to 33 Mya) and around 15 Mya, during the mid Miocene (Figure 2). Dispersal from the Diagonal of open formations into the Atlantic Forest intensified from 11 Mya toward the present (Figure 2). When the northern and southern regions of the Atlantic Forest were analysed separately, connectivity with other Neotropical regions was higher for the southern region (Figure 5 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. S3). The unconstrained analysis showed a higher connectivity between northern Andes and the Atlantic Forest, rather than central Andes (Figure S4). The first extant lineages to disperse to the Atlantic Forest were Morphini (Satyrinae) which occurred around 47 Mya. This was followed by lineages of Charaxinae ( Memphis and Prepona ) and Biblidini (Biblidinae), during the Eocene, around 43 Mya, and Euptychiina (Satyrinae) around 37 Mya. During the Oligocene (33.9–23 Mya), lineages from the Haeterini (Satyrinae) and Melitaeini (Nymphalinae) clades dispersed to the Atlantic Forest. The remaining Nymphalidae clades dispersed to the Atlantic Forest during the Miocene. Within Charaxinae, two genera (Memphis and Prepona ) showed the highest dispersal rates into the Atlantic Forest, with these rates increasing exponentially since the late Miocene. Another notable clade was the Heliconiini (Heliconiinae), which showed high dispersal rates from Amazonia to the Atlantic Forest around 5 Mya (Figure S5). 3.3 Within-region species diversification analysis The estimates of regional diversification rates obtained by ClaDS and BioGeoBEARS’ BSM suggested that Atlantic Forest lineages had a steady and low diversification rate through time (Figure 3). This trend was observed in both the northern and southern Atlantic Forest lineages, although the latter exhibited a higher rate of diversification. A notable peak in diversification was estimated to have occurred around 15–17 Mya in the southern Atlantic Forest, alongside a decline in diversification rates between 20 and 25 Mya for both the southern and northern Atlantic Forest (Figure 3). When examining which lineages contributed most to the overall increase in speciation rates, the Ithomiini and Adelpha had the most significant impact, showing high speciation rates during the mid-Miocene across the Neotropics (˜15 Mya), and mostly represented in the southern Atlantic Forest. In addition, the speciation rates of Euptychiina also stands out from the rest during the Paleogene, ca 35-30 Mya (Figure S6). 3.4 Effect of altitudinal variation on the diversification of Atlantic Forest Nymphalidae We did not find any phylogenetic correlation between tip-specific speciation rates and the current distri- butional patterns of nymphalid butterflies in the Atlantic Forest, except for the tribe Ithomiini and the genus Actinote (Heliconiinae: Acraeini). For Ithomiini, we found that altitudinal generalists (species that are widespread along elevational gradients) that occur in the southern Atlantic Forest had higher speciation rates compared to other lineages across the Atlantic Forest. Both ClaDS estimates (Estimate = 0.0593, p-value = 0.0065) and DR metrics (Estimate = 0.1768, p-value = 0.0216) produced the same results. A similar correlation was found for Actinote ; however, it was statistically significant only when using ClaDS’ speciation rates (Estimate = 0.0155, p-value = 0.0418) but not with DR metrics estimates (Estimate = - 0.0155, p-value = 0.6624). Although a significant positive effect on speciation by the lowland and widespread state was found using the DR metrics in Haeterini (Estimate = 0.102, p-value = 7.306e-05), the effect was found to be non-significant using ClaDS’ speciation rates (Estimate = 0.0046, p-value = 0.1304). 4 Discussion The Atlantic Forest is regarded as one of the most threatened biodiversity hotspots in the world (Myers et al., 2000). However, the macroevolutionary processes explaining extant biodiversity patterns of many species- rich groups remain unclear. In this study, we inferred the evolutionary history of neotropical nymphalid butterflies and found that the extant species diversity in the Atlantic Forest resulted from a consistently low and steady net diversification rate over time, coupled with continuous biotic influx from other Neotropical regions. Notably, dispersal rates into the Atlantic Forest intensified around 15 million years ago, suggesting that regional species accumulation was primarily shaped by dispersal from other regions —primarily from Amazonia— rather than in situ diversification, in line with the third scenario outlined in the introduction. 4.1 Atlantic Forest dispersal dynamics We observed an overall increase in species dispersal to the Atlantic Forest since the mid-Miocene, 15 million years ago, with an acceleration in the last ˜7 million years. The sources of dispersal into the Atlantic Forest varied over time, which may be partially attributed to major environmental and geological changes. During 6 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. the Neogene, the major Andean uplift and the global cooling trend drastically reconfigured the landscape, precipitation patterns, and climate across the Neotropical region, resulting in the expansion of dry and open formations in northern and central South America (Hoorn et al., 2010; Werneck, 2011). Yet, the precise timing of the establishment of the Diagonal of open formations remains under debate. While molecular phylogenies suggest an early to mid-Miocene split between forest biomes, fossil and molecular evidence from C4 grasses and woody savanna flora indicate that their dominance began in the late Miocene (Azevedo et al., 2020). Nevertheless, there is evidence of open vegetation formations as far back as the Palaeocene (Aguiar et al., 2020). Our findings reveal an exponential-like increase in dispersal from the Diagonal of open formations into the Atlantic Forest around 10 Mya. Even though the underlying drivers of such increase remain elusive, they support the idea of a mid-to-late Miocene reconfiguration of environmental conditions, including the establishment of suitable dispersal corridors for Nymphalid lineages with different ecological characteristics. This increase may also reflect a larger source pool of lineages from northwestern South America, triggered by the rapid speciation of many taxa during that period and in response to orogenic processes and regional climate change (e.g., Antonelli et al., 2018b). 4.1.1 Amazonia as a source of Atlantic Forest diversity As demonstrated by Antonelli et al., (2018), Amazonia played a major role as a source of Neotropical diversity, including the Atlantic Forest. Here we also recovered Amazonia as the main source of Atlantic Forest diversity, with a high and constant dispersal rate through time. Despite the ecological barrier posed by the Diagonal of open formations, biotic corridors—likely formed by episodic wet forests or continuous wet vegetation along rivers—might have enabled the exchange of fauna and flora between Neotropical forest domains. For instance, Prates et al., (2017) suggested that throughout the Miocene, patches of suitable humid habitats may have connected the Atlantic Forest with western South American forests, facilitating species dispersal. This agrees with our findings and supports the idea that the expansion of the Diagonal of open formations did not significantly reduce dispersal between Amazonia and Atlantic Forest nymphalids. We speculate that the Neogene forest corridors allowed for continuous dispersal among forest-adapted Nymphalid butterflies. These connections likely varied regionally, with stronger links to Amazonia through central and northern routes, and weaker connections through southern corridors, including the Andes. Sim- ilar connectivity patterns have been reported previously, although most indicate that the interchange was frequent due to Pleistocene paleoclimatic fluctuations (Costa, 2003; Cohelho et al., 2022; Peres et al., 2020; Sobral-Souza & Lima-Ribeiro, 2017). In contrast, only a few studies have documented older connections during the Neogene (e.g., Marques-Souza et al., 2022), possibly mediated by riparian forests or remnant hu- mid corridors (Trujillo-Arias et al., 2020, Pirani et al., 2020). In line with these studies, our results recover continuous connectivity between the Atlantic Forest and other Neotropical biomes since the early Neogene, likely facilitated by historical forest corridors linking these regions. We acknowledge that long-distance dispersal events, facilitated for instance, by extreme winds, could also explain certain biotic interchange without the necessity of fully connected corridors. Seasonal movements of individual butterflies have been documented over distances of several kilometres in the Atlantic Forest. Additionally, migrations of certain populations and communities can be inferred to occur over many hundreds of kilometres (Brown & Freitas 2000, 2002). However, we consider that such events should have been largely random, adding to the “noise” in our analyses rather than being able to explain the steady increase in dispersals inferred, which are more likely to reflect the conditions of the vegetation matrix over which animal movements could take place. 4.1.2 The role of the Andes on Atlantic Forest diversity The central Andes is the second largest source of Nymphalidae diversity for the Atlantic Forest, as it has also been reported for other taxonomic groups, such as rodents (Vallejos-Garrido et al., 2023), bats (Velazco & Patterson, 2013), birds (Fjeldsa & Rahbek, 2006), and plants (Antonelli & Sanmartin, 2011). The Andes have played an important role in generating species diversity in the Neotropics through high diversification and emigration rates (Rangel et al., 2018; Antonelli et al., 2018c). For instance, the ithomiine genus Pteronymia mainly diversified in the northern Andes, with much of their non-Andean diversity resulting from independent dispersal events out of them (De-Silva et al., 2017). Similarly, tanagers and hummingbirds seem to have 7 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. radiated and dispersed out of the northern Andes, which might have acted as cradles and museums of species diversity (Beckman & Witt, 2015; Sonne et al., 2022). Indeed, a high connectivity between northern Andes, rather than central Andes, and the Atlantic Forest was recovered in our unconstrained biogeographical analyses (see Supplementary Materials), a pattern also recovered in a synthesis of Andean flora biogeography (Perez-Escobar et al., 2022). Unlike Amazonian dispersion –more or less constant–, the Andean dispersal rate has fluctuated over time. During the early Miocene, the connectivity between the Andes and the Atlantic Forest increased, reaching its peak around 15 Mya, when it even surpassed dispersal from Amazonia. This pattern may have been enabled by biotic corridors linking the central Andes to the Atlantic Forest through the Cerrado and Chaco transition zones, as reported for several lineages (Matos-Maravi, Pena, Willmott, Freitas & Wahlberg, 2013; Moreira-Munoz et al., 2020; Pirani et al., 2020; Prates et al., 2017; Trujillo-Arias et al., 2018, 2020). In addition, montane environments of the Atlantic Forest, like Serra do Mar and Serra da Mantiqueira, may have facilitated the settlement and diversification of high-elevation Andean lineages in the region. For instance, the montane genera Actinote diversified in the Andes but, once it colonized the southern Atlantic Forest, achieved its greatest species richness in the mountain forests of the region, with some lineages recolonizing the Andes from the Atlantic Forest (Guerato et al., 2023; Magaldi et al., 2024). In this regard, Brown (1987) identified a high similarity in butterfly lineage composition between the Andes and south-eastern Brazil, whereas Safford (2007) suggested a similar shared evolutionary pattern based on floristic composition analyses. Therefore, the high rates of Andean diversification together with early Miocene dispersal corridors might have promoted an interchange of lineages with the Atlantic Forest. 4.2 Atlantic Forest diversification Within the Neotropical region, a gradual accumulation of Nymphalidae lineages is explained by steady speciation rates and low extinction rates (Chazot et al., 2021), a common pattern found in Neotropical butterflies (Matos-Maravi, 2016) and plants (Meseguer et al., 2022). We found that net diversification rates within the Atlantic Forest have remained low and constant, contrasting with other global tropical biodiversity hotspots characterized by high diversification rates (Igea and Tanentzap, 2019). This slow and gradual accumulation of lineages is consistent with previous reports that considered the Atlantic Forest as a ‘museum of diversity’ for butterflies (Matos-Maravi et al., 2021). Despite the overall constant diversification rate, we find certain regional differences when studying the southern and northern Atlantic Forest individually. In particular, during the Paleogene, the southern region experienced a slight increase in net diversification rate, likely related to the arrival of rapidly diversifying nymphalid groups across the Neotropics, such as Adelpha and Ithomiini (two clades known by their remark- ably high species richness in the Andes) (Chazot et al., 2016; Ebel et al., 2015). Moreover, the complex topography of the south may have further contributed to these regional disparities. We hypothesise that the Atlantic Forest mountain ranges played an important role in the diversification of such lineages. Different mechanisms are known to promote species diversification in the Neotropical mountains. On one hand, mountain tops can act as isolated islands promoting allopatric diversification between scattered populations with restricted elevational ranges (Brown, 1987; Chaves, Freitas, Vasconcelos & Santos 2014, Magaldi et al., 2024). On the other hand, habitat complexity of montane environments can act as species attractors, with higher rates of colonization towards the mountains, as found for the ithomiine subtribe Godyridina in the Andes (Chazot et al., 2016b). Interestingly, a positive correlation emerged between wide altitudinal distribution (i.e., generalists species occurring in highlands and lowlands) and increased speciation rates in ithomiine butterflies of the southern Atlantic Forest. Highland specialists, however, showed no such pattern, contrary to expectations. This pattern may be explained by habitat heterogeneity along mountain ranges that could facilitate population differentiation in widespread lineages and eventually speciation. Indeed, habitat fragmentation driven by the Pleistocene climatic fluctuations might have pushed lineages toward mountain climatic refuges and facilitated allopatric diversification (Brown 1987, Carnaval and Moritz, 2008). Furthermore, the southern Atlantic Forest has higher levels of species richness than the northern region, which is explained not by higher dispersal rates but also higher speciation 8 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. rates, particularly involving mountain lineages in both the Atlantic Forest and the Andes. Both scenarios explain the current species richness in mountain ranges but are driven by different processes. Overall, for nymphalids, Pleistocene climatic fluctuations did not increase species diversification, but Nymphalidae species diversity accumulated gradually in the Atlantic Forest mainly through dispersal. 5 Conclusions Our study shows that the Atlantic Forest Nymphalidae diversity has been shaped under a complex scenario of varying biotic connectivity through time with different biogeographical regions, including the central Andes and Amazonia. Despite the Diagonal of open formations being hypothesised as an important biogeo- graphical barrier for wet forest lineages, the increased late Miocene to Pleistocene dispersal we detected in the Atlantic Forest suggests that corridors with suitable habitats for different Nymphalidae lineages likely maintained connectivity through time, especially between Amazonia and the Atlantic Forest. Overall, our findings underscore the importance of jointly studying diversification and dispersal rates to understand the macroevolutionary processes shaping extant biodiversity patterns in one of the world’s most biodiverse yet threatened regions.

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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. biogeographical analyses and plots, are available in Dryad (temporary link before publication: http://datadryad.org/share/t50cO4uD14GiJzxBQSKO6T1YvaBVWinmjD0syaC00Qw). Tables TABLE 1. Number of species per clade used for phylogenetic inference and biogeographical and diversi- fication analyses. Nine clades were extracted for phylogenetic inference and calibration. The nine clades were then subdivided into fourteen subclades for biogeographical and diversification analyses, in order to minimise the number of species outside the Neotropics. (phylogenetic inference) & Species from Chazot et al. (2021) Species from Kawahara et al. (2023) Species from this study Subclades for Biogeographical Analyses Species number (Biogeographical analyses) Danainae 436 376 60 0 Ithomiini 331 Heliconiinae 192 148 42 2 Actinote 37 Heliconiini 69 Limetidinae 107 88 19 0 Adelpha 67 Biblidini 210 154 42 14 Biblidini 170 Cyrestinae & Nymphalinae 447 380 67 0 Hypanartia 9 Marpesia 13 Melitaeini 174 Charaxinae 265 228 29 8 Memphis 54 Prepona 23 Satyrinae subclade 242 173 59 10 Haeterini 17 Morphini & Brassolini 108 Euptychiina (Satyrini, Satyrinae) 320 201 89 30 Euptychiina subclade 194 Pronophilina (Satyrini, Satyrinae) 136 93 39 4 Pronophilina 111 TOTAL 2355 1841 446 68 1377 Figures FIGURE 1. Nymphalidae interchange (dispersal events) across Neotropical regions. The analyses are based on 1377 Nymphalidae species (see Material and Methods). (a) Map of Neotropical biogeographic regions considered in this study following Morrone (2014), together with the species number per area included in our biogeographical analyses. Letters correspond to: F = Northern Atlantic Forest; S = southern Atlantic Forest; D = Diagonal of open vegetation formations; Z = Amazonia; C = Mesoamerica + NW slopes of Andes; N = Northern Andes; A = Central Andes. (b) Nymphalidae dispersal events from the Biogeographical Stochastic Mapping results. Arrows indicate the direction and number of events; line thickness is proportional to the number of events, with a minimum of 30. (c) Proportion of within-speciation and immigration events among 17 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. regions. Proportions were estimated by dividing the number of immigration events (sum of dispersal events into each region) by the number of extant species included in the analysis per region. Within-speciation events were calculated as the rest of the extant number of species minus the immigration events. FIGURE 2. Dispersal rates through time to the Atlantic Forest overall (A) and (B) from different Neotrop- ical regions. Andes = red; Amazonia = blue; Diagonal of open formations = dark yellow; Mesoamerica and NW slopes of Andes = light sand-coloured. Continuous lines are the median values, and coloured ribbons are the lower and upper quantiles (0.40 and 0.60). Abbreviations: Eoc: Eocene, Plio: Pliocene, Plei: Pleis- tocene, Mya: million years ago. The plots were generated with R (https://cran.r-project.org/) and edited with Inkscape (https://inkscape.org). 18 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. FIGURE 3. Diversification rates from ClaDS results for the entire Atlantic Forest (green), Southern Atlantic Forest (orange) and northern Atlantic Forest (blue). Continuous lines are the median values, and coloured ribbons are the lower and upper quantiles (0.40 and 0.60). Abbreviations: Plio: Pliocene, Plei: Pleistocene, Mya: million years ago. The plots were generated with R (https://cran.r-project.org/) and edited with Inkscape (https://inkscape.org). 19