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under number AF38F6E, in compliance with Brazilian Law No. 13,123/2015
and its regulations.
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