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Abstract
The mountains in the Atlantic Forest domain are environments that harbor a high biodiversity, including species adapted to colder climates that were probably influenced by the climatic variations of the Pleistocene. To understand the phylogeographic pattern and assess the taxonomic boundaries between two sister montane species, a genomic study of the butterflies Actinote mantiqueira  and A. alalia  (Nymphalidae: Acraeini) was conducted. Analyses based on the COI barcode region failed to recover any phylogenetic or genetic structure discriminating the two species or sampling localities. However, SNPs gathered using GBS provided a strong isolation pattern in all analyses (genetic distance, phylogenetic hypothesis, clustering analyses, and FST statistics) that is consistent with morphology, separating all individuals of A. alalia  from all populations of A. mantiqueira . The three sampled mountain ranges whereA. mantiqueira populations occur — Serra do Mar, Serra da Mantiqueira, and Poços de Caldas Plateau — were identified as three isolated clusters. Paleoclimate simulations indicate that both species’ distributions changed according to climatic oscillations in the Pleistocene period, with the two species potentially occurring in areas of lower altitude during glacial periods when compared to the interglacial periods (as the present). Besides, a potential path between their distribution through the Serra do Mar Mountain range was inferred. Therefore, the Pleistocene climatic fluctuation had a significant impact on the speciation process between A. alalia and A. mantiqueira , which was brought on by isolation at different mountain summits during interglacial periods, as shown by the modeled historical distribution and the observed genetic structure.
Keywords
Atlantic Forest, Neotropical, Actinote, Nymphalidae, Population genomics, Speciation
Introduction
Mountain environments are biodiversity hotspots that account for a third of all terrestrial species worldwide (Kohler et al., 2010; Körner et al., 2017). These environments resemble oceanic islands in various aspects, including their limited size, distinct boundaries, isolation, and dispersal restrictions. Due to these features, mountain ranges are often called ’sky islands’ (Gehrke & Linder, 2009; Hughes & Atchison, 2015; Sklenář et al., 2014).
Several intrinsic abiotic factors contribute to generating and maintaining high biodiversity in montane environments, such as topographic variation, heterogeneity of soil types, altitudinal gradients, and climatic variability (Antonelli et al., 2018; Badgley et al., 2017; Contreras-Medina et al., 2003; Fischer et al., 2011; Fjeldså et al., 2012; Körner, 2004; Luebert & Muller, 2015). In general, two main mechanisms were proposed to explain the high diversification in montane areas: 1) the historical fragmentation of previously continuous habitats and dispersal associated with a rough landscape; and 2) the climate fluctuations over geologic time (Badgley, 2010; Knowles & Massatti, 2017; Mayr & Diamond, 1976; Patton & Smith, 1992). These mechanisms may trigger allopatric or parapatric speciation events in montane systems (Moritz et al., 2000; Vuilleumier & Monasterio, 1986), and for instance, several mechanisms of speciation in montane regions were proposed with variable resultant phylogenetic relationships patterns between sister taxa (Willmott et al., 2001).
Pleistocene climatic oscillations are well known for shaping the genetic diversity and geographical distribution of montane taxa (Amaral et al., 2021; Colinvaux et al., 1997; Graham et al., 2014; Hewitt, 2004; Hooghiemstra & Van Der Hammen, 2004; Janzen, 1967; Mutke et al., 2014; Oswald & Steadman, 2015; Pie et al., 2018). Montane species responded to these climate fluctuations by shifting their distributions upward in periods of warm climate (Chen et al., 2009; Flantua et al., 2019; Freeman et al., 2018; Moritz et al., 2008). Accordingly, cold-adapted species often remain isolated on mountain tops during warmer interglacial periods, undergoing genetic drift and divergence induced by natural selection (Brown, 1971; Fjeldså, 1994; Flantua et al., 2019; Ramírez-Barahona & Eguiarte, 2013; Safford, 1999). On the other hand, species range can extend to lower areas during colder periods, connecting previously separate locales, perhaps leading to the mixing of populations and the formation of hybrids (Donoghue et al., 2014; Petit et al., 2003). In the last case, populations can even expand into previously unsuitable regions, triggering diversification through dispersal and settlement in new areas (‘dispersification’; Moore & Donoghue, 2007).
In general, species are expected to either move to follow favorable conditions (i.e., range shifts, both in latitude and elevation, or contractions) or persist in the landscape through evolution to novel environmental conditions (e.g., phenotypic plasticity or adaptation) in response to climatic change (Costello et al., 2022; Waldvogel et al., 2020). Currently, most of the knowledge on montane environments has focused on vertebrates or vascular plants (Wang et al., 2023). Notwithstanding, changes due to climatic change were described for European alpine burnet moth species in the Pyrenees (Dieker et al., 2011). In this sense, invertebrate montane species are especially concerning due to the high degree of specialization that montane species often exhibit within narrow temperature bands. The upslope movements are predicted to result in a reduction in their potential area of occupancy and become more vulnerable to the stochastic extinctions that characterize small populations (Elsen & Tingley, 2015).
The Neotropical region shelters the Andes, the longest mountain range on Earth. It harbors a rich biota, which has been investigated as a paradigm for research on the patterns and processes of montane diversification (Adams, 1985; Bacon et al., 2018; Cadena, 2007; Castroviejo-Fisher et al., 2014; Chazot et al., 2016; Elias et al., 2009; Hall, 2005; Hazzi et al., 2018; Kessler, 2001; Pyrcz & Wojtusiak, 2002; Viloria, 2003; Willmott et al., 2001). In the Atlantic Forest, a highly threatened biome, there are four massifs: the Serra do Mar, the Serra da Mantiqueira, the Serra Geral and the Espinhaço mountain ranges, ranging from sea level to 2,891 m (Moreira & Camelier ,1977). These mountains are located between 15º and 30º South and are mostly covered by tropical or subtropical forests, with climatic variations ranging from no dry season near the coast (ombrophilous dense forests) to the presence of a marked dry season in the interior (semi-deciduous forests with physiological drought and mean temperature below 15°C) (Morellato & Haddad, 2000; Oliveira-Filho & Fontes, 2000; Veloso et al., 1991). During the Late Pleistocene, an expansion of high-altitude grasslands took place in the Montane Atlantic Forest (hereafter MAF), reflecting the colder and drier conditions that became predominant afterward (Behling, 2002; Behling et al., 2007; Behling & Safford, 2010).
The phylogeography of the Atlantic Forest is mainly known vertebrates and plants with low or wide elevational ranges, and points to a clear latitudinal split that isolates the northern and southern diversity, primarily in the central corridor (Peres et al., 2020). However, no common pattern was found for high-altitude species of the MAF (Amaro et al., 2012; Batalha-Filho et al., 2012; Firkowski et al., 2016; Françoso et al., 2016; Peres et al., 2015; Thom et al., 2020). This apparent lack of a common pattern may be the result of various processes that have diverse effects on taxa. For instance, Pleistocene climate oscillations may have shaped the genetic diversity of montane populations for certain taxa within the MAF, such as bees and birds (Amaral et al., 2018; Françoso et al., 2016; Thom et al., 2020). On the other hand, stability in population size during the Pleistocene was observed for other taxa, such as spiders, frogs, and birds (Amaro et al., 2012; Batalha-Filho et al., 2012; Peres et al., 2015). Additionally, population structure of some MAF species show phylogeographic splits that correspond to divergence across river barriers (Amaro et al., 2012; Françoso et al., 2016). Others, however, show a spectrum of phenotypic and genetic divergence across an inter-mountain valley, defined as the São Paulo subtropical gap, around 20ºS (Amaral et al., 2021; Thom et al., 2020). Still, endemic populations showing no genetic structure were observed as well (Batalha-Filho et al., 2012; Françoso et al., 2016; Mota et al., 2020; Peres et al., 2015). Thus, new phylogeographic research on invertebrates can contribute to revealing the processes of diversification that have culminated in this variety of patterns observed for highland species (Peres et al., 2015).
For instance, numerous butterfly species exhibit a restricted or favored distribution at higher elevations, despite the scarcity of knowledge regarding their evolutionary divergence within the MAF (Wang et al. 2023). Butterflies are well-known model organisms for various ecological and evolutionary studies and are especially rich and diversified in the Atlantic Forest, including many endemic taxa (Brown Jr & Freitas, 2000; Santos et al., 2018; Watt & Boggs, 2003). The few studies focusing on the distribution of butterfly diversity in the Atlantic Forest point to a North-South pattern of diversification, following the two bioclimatic regions of the biome (Brown et al., 2020; Pablos et al., 2021; Paz et al., 2021; Seraphim et al., 2016), while virtually nothing is known about the pattern of divergence for montane species.
The nymphalid butterflies of the genus Actinote Hübner [1819] (Heliconiinae: Acraeini) include 38 described species, the vast majority of which are found in the highlands of the Atlantic Forest in southeast Brazil (Freitas et al., 2018, 2020; Lamas, 2004; Paluch, 2006; Paluch & Casagrande, 2006; Gueratto et al. submitted). In the MAF, there are seven known species of Actinote that participate in a Müllerian mimicry ring called “orangish-red mimicry complex” (Francini, 1989; Freitas et al., 2018). The species in this complex are characterized by a dark orange and brown dorsal striped pattern, with a ventral design that is somewhat variable among species. The butterflies of the orangish-red mimicry complex, including Actinote alalia (Felder & Felder, 1860) and A. mantiqueira Freitas, Francini, Paluch & Barbosa, 2018, are remarkably morphologically similar and difficult to distinguish due to the strong similarities in wing pattern, mainly concerning female wing color pattern, and high intraspecific variation (D’Almeida, 1935; Francini, 1989; Francini & Penz, 2006; Freitas et al., 2018; Paluch & Casagrande, 2006). Actinote mantiqueira is a recently described species distributed in the Serra do Mar and Serra da Mantiqueira, at altitudes from 1000 to 2000 m, in sites usually characterized by a well-preserved montane ombrophilous forest (Freitas et al., 2018). The species is geographically separated from its sister species, A. alalia , which occurs in the montane areas of the southernmost Brazil, at altitudes from 800 to 1400 m, associated with preserved montane forest (Freitas et al., 2018). Adults of both sexes ofA. alalia are easily observed in forest edges and areas of contact between forest and high-altitude grasslands (Freitas et al., 2018).
The current geographic distribution of these sister species allows us to test the phylogeographic hypotheses proposed for the MAF species since they are related to distinct paleoclimatic regions of the Atlantic Forest (defined by Ledru et al., 2017) and are distributed in distinct mountain blocks: A. mantiqueira occurs in the Central Atlantic Forest (CAF), between 15° and 23°S, whereas A. alalia inhabits the South Atlantic Forest (SAF), from 23° to 30°S (Fig. 1). This distribution pattern of this sister species (Silva-Brandão et al., 2008) is consistent with the hypothesis that historical climatic changes played a significant role in shaping the current range of these species. The isolation of populations on different mountain ranges due to temperature increases likely contributed to the allopatric speciation observed between montane regions (Wilmott et al. 2001).
To test this hypothesis, a phylogeographic study of the pair of sister species, A. mantiqueira and A. alalia , was conducted to infer their genetic variability and population structure. First, the species limit between these two species was tested using two molecular markers, the COI barcode region (Hebert et al., 2003), which is widely investigated to infer butterflies’ taxonomy (Silva-Brandão et al., 2009), and Single Nucleotide Polymorphisms (SNPs) obtained with the genotyping-by-sequencing technique (GBS) (Elshire et al., 2011). Additionally, the ecological niche of each species was modeled from the present to 800 thousand years ago (kya), including nine glacial-interglacial cycles, to estimate the influence of past climate variation on their distribution and test whether their niche could have expanded under colder conditions. These two species are found predominantly on the top of the Atlantic Forest mountains. Therefore, they represent a high conservation priority globally since mountaintop species stand to face local extinction with upslope range shifts regardless of underlying topography. Using genomic markers, we were able to evaluate the effect of the present disjunct distribution on different mountain ranges in the population genomic structure in both species. Based on the morphological differences of A. mantiqueira andA. alalia and on the results reported for other species of the MAF that present similar distributions, a well-defined genetic structure between these two species is expected, which would also indicate that the São Paulo subtropical gap also functions as geographic barrier for MAF butterflies (Amaral et al., 2018; Freitas et al., 2018; Thom et al., 2020).
Material and Methods