Embed Size (px)
Citation preview
J Zool Syst Evol Res. 2020;00:1–14. wileyonlinelibrary.com/journal/jzs | 1© 2020 Blackwell Verlag GmbH
1 | INTRODUC TION
Given alarming rates of biodiversity loss during the Anthropocene, understanding how diversity is generated and maintained has
become an urgent task in order to better preserve it (Dirzo et al., 2014; Wheeler et al., 2012). Speciation processes manifest as a con-tinuum of divergence, during which recognizing boundaries between species or among populations diverging at various grades may be a difficult matter (Hendry, 2009; Hendry, Bolnick, Berner, & Peichel, 2009; Mallet, Beltrán, Neukirchen, & Linares, 2007; Nosil, Harmon, & Seehausen, 2009; Seehausen, 2009; Shaw & Mullen, 2014).
Received: 13 April 2019 | Revised: 9 October 2019 | Accepted: 22 October 2019
DOI: 10.1111/jzs.12356
O R I G I N A L A R T I C L E
Integrative analyses on Western Palearctic Lasiommata reveal a mosaic of nascent butterfly species
Leonardo Platania1 | Raluca Vodă2 | Vlad Dincă3 | Gerard Talavera1 | Roger Vila1 | Leonardo Dapporto4
Contributing authors: Raluca Vodă ([email protected]), Vlad Dincă ([email protected]), Gerard Talavera ([email protected]), Roger Vila ([email protected]), Leonardo Dapporto ([email protected]).
1Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona, Spain2Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy3Ecology and Genetics Research Unit, University of Oulu, Oulu, Finland4Dipartimento di Biologia dell'Università di Firenze, Firenze, Italy
CorrespondenceLeonardo Platania, Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona, Spain.Email: [email protected]
Funding informationMinisterio de Economía y Competitividad, Grant/Award Number: CGL2013-48277-P and CGL2016-76322-; Università degli Studi di Firenze; H2020 Marie Skłodowska-Curie Actions, Grant/Award Number: 609402-2020 and 625997
AbstractSatyrinae butterflies occurring in the Mediterranean apparently have reduced gene flow over sea straits, and for several species, recent wide-scale biodiversity sur-veys indicate the existence of divergent mitochondrial lineages. Here, we apply an integrative approach and examine the phylogeography of the genus Lasiommata in the Western Palearctic. Our research comprised molecular analyses (mitochondrial and nuclear DNA) and geometric morphometrics (wings and genitalia) for two main species groups, and a comparative GMYC analysis, based on COI, of all the tribes within Satyrinae from this region. The GMYC approach revealed a particularly fast coalescence rate in the Parargina subtribe. The Lasiommata group was divided into 12 evolutionary significant units: six clades for the L. maera species group, five for the L. megera species group, and one for L. petropolitana, with divergences of about 1%. The patterns of COI were mirrored by ITS2 in L. maera, but the two markers were generally inconsistent in L. megera. On the contrary, morphological differences were coherent with the results of COI for L. megera, but less clearly so for L. maera. L. paramegaera and L. meadewaldoi were considerably differentiated for all the ana-lyzed markers and likely proceeded faster in the process of speciation because of geographic isolation and reduced effective population size, rendering the rest para-phyletic. Our study illustrates the continuous nature of speciation and the difficulties of delimiting species. In Lasiommata, the recognition of taxa as diverging lineages or distinct, possibly paraphyletic species, mostly depends on the criteria adopted by different species concepts.
K E Y W O R D S
COI, ITS2, Lasiommata, morphometrics, speciation
2 | PLATANIA eT AL.
This is attested by the existence of a high number of species con-cepts, which sometimes disagree in recognizing if particular taxa should be considered as distinct species (de Queiroz, 2005, 2007).
In the speciation continuum, a large number of genes, expressing a wide array of functional/phenotypic traits, are expected to diverge at varied rates, producing discrepancies of traits between popula-tions (Hendry, 2009; Hendry et al., 2009). According to observed geographic patterns, speciation events have been traditionally cat-egorized as sympatric, allopatric or parapatric (Butlin, Galindo, & Grahame, 2008; Coyne & Orr, 2004; Gavrilets, Li, & Vose, 2000; Mayr, 1963; Via, 2001). Sympatric speciation is characterized by the absence of a physical separation between emerging taxa, which di-versify uniquely as a consequence of genetic changes affecting eco-logical and behavioral adaptations (Barluenga, Stölting, Salzburger, Muschick, & Meyer, 2006; Via, 2001). Allopatric speciation, consid-ered as the main mechanism generating biodiversity, occurs in virtu-ally complete geographic separation between populations because physical barriers produced by geological or climatic transformations or after events of exceptional dispersal and colonization of remote areas (Coyne & Orr, 2004; Hewitt, 1996, 2000, 2004; Mittelbach & Schemske, 2015). Following allopatric speciation, sister species may disperse and get into secondary contact (Pigot & Tobias, 2014), but full secondary sympatry is frequently delayed due to density-de-pendent phenomena or competition for resources (Waters, 2011; Waters, Fraser, & Hewitt, 2013). As a result, most sister, cryptic and sibling species have relatively narrow contact zones (Hewitt, 2004; Waters, 2011; Waters et al., 2013) even in the case of highly mobile organisms such as butterflies (Vodă, Dapporto, Dincă, & Vila, 2015a, 2015b). Secondary contacts can also occur before the speciation process is completed, but the genetic lineages still tend to have very narrow boundaries (Waters, 2011; Waters et al., 2013). In these cases, either lineage fusion or parapatric speciation can eventually take place (Gavrilets et al., 2000).
The analysis of genetic and phenotypic traits diverging among populations represents a fundamental resource to understand the speciation process and the mechanisms underlying it. In addition, the results need to be translated into taxonomic hypotheses in order to categorize biodiversity, a necessity for many applications. Integrative taxonomy, the classification of organisms by integrating information from different features such as morphology, behavior, cytology, DNA sequences, or ecology, allows a deeper view of the visible and hidden layers of diversity, providing researchers the means to explore and investigate groups that were difficult to study with classical taxonomy and to refine strategies for species delimi-tation (Padial, Miralles, De la Riva, & Vences, 2010; Schlick-Steiner et al., 2010).
Butterflies (Lepidoptera: Papilionoidea) represent one of the best-known groups of invertebrates and are well studied in terms of taxonomy, distribution, population dynamics, ecology, and biogeog-raphy (Kudrna et al., 2011; Settele, Shreeve, Konvička, & van Dyck, 2009; van Swaay et al., 2010). In recent years, several studies that DNA-barcoded the butterfly species occurring in the Mediterranean and in other regions of Europe highlighted the presence of deep
mitochondrial lineages within generally accepted species, suggest-ing ongoing differentiation or even the presence of cryptic species (Dincă, Dapporto, & Vila, 2011; Dincă, Lukhtanov, & Talavera, 2011; Dincă et al., 2015; Dincă, Zakharov, Hebert, & Vila, 2011; Hausmann et al., 2011). Some of these cases have been subsequently studied in more detail by using integrative approaches and occasionally re-vealed that species diversity is higher than expected even in inten-sively studied areas such as Europe (Dapporto, Vodă, Dincă, & Vila, 2014; Dincă, Zakharov, et al., 2011; Habel et al., 2017; Hernández-Roldán et al., 2016).
In this paper, we analyze the Western Palearctic taxa of the genus Lasiommata Westwood, 1841, belonging to the Parargina subtribe, for which recent wide-scale biodiversity surveys indicate the existence of several divergent mitochondrial lineages in the Mediterranean area (Dapporto et al., 2017; Dincă et al., 2015; Vodă et al., 2016; Weingartner, Wahlberg, & Nylin, 2006). We hypothe-sized that (a) the Lasiommata genus, and the Parargina subtribe in general, have shorter coalescence rates compared with other sub-tribes within the Satyrinae occurring in the Palearctic region. To test for this hypothesis, we compared the results of a series of GMYC analyses among Satyrinae subtribes based on COI data; (b) the main lineages within this species group are likely in an advanced stage of an unusually fast speciation process. To test for this hypothesis and clarify the systematic relationships within these taxa, we applied an integrative approach, using molecular and morphological traits. We sequenced mitochondrial (cytochrome c oxidase subunit I—COI) and nuclear DNA (internal transcribed spacer 2—ITS2) and analyzed phenotypic markers consisting of wing shape (presumably evolv-ing under strong environmental selection) and genitalia structures (mostly evolving under sexual selection) (Habel et al., 2017; Hebert, Cywinska, & Ball, 2003; Hernández-Roldán et al., 2016; Nazari, Hagen, & Bozano, 2010; Zinetti et al., 2013).
2 | MATERIAL & METHODS
2.1 | Studied species
In Europe and North Africa, the butterfly genus Lasiommata is rep-resented by five generally recognized species: Lasiommata megera (Linnaeus, 1767), Lasiommata maera (Linnaeus, 1758), Lasiommata petropolitana (Fabricius, 1787), Lasiommata paramegaera (Hübner, [1824]), and Lasiommata meadewaldoi (Rothschild, 1917), although the taxonomic status of the latter two is still debated. The wall brown L. megera is distributed from North Africa, across a large part of Europe to Western Asia. The northern European border of its range passes through Ireland, Scotland, southern Scandinavia, and the Baltic coun-tries. In the Mediterranean basin, it represents one of the most wide-spread species, being virtually present in all mainland areas and islands. The populations from Corsica, Sardinia, and some surrounding islands have been attributed to a vicariant sibling species, L. paramegaera. The taxonomic relationship between these two species has been controver-sial for a long time, and L. paramegaera has been considered either as a
| 3PLATANIA eT AL.
subspecies of L. megera (Chinery, 1989; Higgins & Riley, 1970; Leraut, 1980) or as a distinct species based on morphological, biochemical, and genetic evidence (Balletto & Cassulo, 1995; Biermann & Eitschberger, 1996; Dapporto, 2008; Jutzeler, 1998; Kudrna et al., 2011).
The general distribution of L. maera is relatively similar to that of L. megera, but the former is absent from the British Isles, it occurs fur-ther north in Scandinavia and further east to Central Siberia, it has a more limited distribution in North Africa, and lacks from several Mediterranean islands (Kudrna et al., 2011; Tarrier & Delacre, 2008; Tshikolovets, 2011). In the Moroccan High Atlas, the endemic taxon L. meadewaldoi has a very restricted distribution, limited to high parts of the Toubkal Massif. Some authors classified it as a subspecies of L. maera (Higgins & Riley, 1970; Tolman & Lewington, 2008), while oth-ers consider it as a distinct species, although there is very little infor-mation about the ecology of this species to be compared with L. maera (Tarrier & Delacre, 2008; Tennent, 1996; Tshikolovets, 2011).
Lastly, L. petropolitana is a specialist of colder habitats and inhab-its the main mountain chains of Southern Europe and Turkey and the lowland areas of Scandinavia and Siberia (Bozano, 1999; Tolman & Lewington, 2008; Tshikolovets, 2011).
2.2 | Molecular analyses
We obtained 372 COI sequences from samples stored in the collec-tion of the Butterfly Diversity and Evolution Lab at the Institute of Evolutionary Biology (CSIC-UPF) in Barcelona, Spain. Other 196 COI sequences were mined from BOLD. ITS2 was sequenced from 150 specimens that were selected to represent the main COI lineages and spatial distribution (Data S1). Instances of intraindividual nuclear variation were coded as ambiguities and gaps were treated as miss-ing data in the ITS2 alignment. All new sequences obtained for this study have been deposited in GenBank and are also available in the data set DS-Lasio (BOLD process ID in Data S1) from the Barcode of Life Data System (http://www.bolds ystems.org/), where additional information for the specimens is also available. The DNA extraction, amplification, sequencing, and alignment protocols are described in Data S2 (Method S1, Table S1).
Phylogenetic relationships were inferred using Bayesian Inference (BI) through the CIPRES Science Gateway (Miller, Pfeiffer, & Schwartz, 2010). BI analyses were done separately for COI (based on 107 haplotypes plus three Pararge aegeria haplotypes used as outgroup and inferred as described below) and ITS2 (for this marker, one analysis included only L. maera and L. meadewaldoi, while an-other included L. megera and L. paramegaera). Both BI analyses and the estimation of node ages (done for COI) were run in BEAST 1.8.0 (Drummond & Rambaut, 2007). The substitution models (GTR+G for COI and ITS2-L. maera and HKY+I+G for ITS2-L. megera) were chosen according to the values of the Akaike information criterion (AIC) ob-tained with JMODELTEST 2.1.3 (Darriba, Taboada, Doallo, & Posada, 2012). For all three analyses, base frequencies were estimated, six gamma rate categories were selected, and a randomly generated ini-tial tree was used.
For COI, rough estimates of node ages were obtained by ap-plying a strict clock and a normal prior distribution centered on the mean between two widely used substitution rates (1.5% un-corrected pairwise distance per million years (Quek, Davies, Itino, & Pierce, 2004), and 2.3% (Brower, 1994)) and with a standard deviation tuned so that the 95% confidence interval of the poste-rior density coincided with the 1.5% and 2.3% rates. For both COI and ITS2 analyses, parameters were estimated using two indepen-dent runs of 20 million generations (for COI) and 30 million gen-erations (for both ITS2 analyses), and convergence was checked using the program TRACER 1.6 (Rambaut, Drummond, Xie, Baele, & Suchard, 2018).
Maximum parsimony haplotype networks for each species group were inferred with the program TCS 1.21 (Clement, Posada, & Crandall, 2000) and then graphically improved with tcsBU (Múrias dos Santos, Cabezas, Tavares, Xavier, & Branco, 2015) and Adobe Illustrator CC2015.
To investigate putative species boundaries, we applied the general mixed Yule-coalescent model (GMYC, Fujisawa & Barraclough, 2013; Pons et al., 2006) to the COI data. The addi-tion of Yule and coalescent signal into the data set (e.g., incor-porating related outgroup taxa with some extent of population structure) has been demonstrated to be beneficial for the GMYC performance (Talavera, Dincă, & Vila, 2013). Thus, we obtained ultrametric phylogenetic trees based on 1,343 unique COI hap-lotypes of Satyrinae sequences retrieved from GenBank and the Lasiommata COI sequences produced for this study. In order to highlight possible differences in coalescence time among tribes, phylogenetic trees and GMYC analyses were also inferred sepa-rately on the Satyrini subtribes counting more than five species in the Palaearctic area (Coenonymphina, Erebiina, Maniolina, Melanargina, Parargina, Satyrina, following systematics by Peña et al. (2006)). Bayesian trees were constructed using BEAST as described above, and single-threshold GMYC was evaluated using the R package SPLITS with default settings.
2.3 | Morphological analyses
A total of 262 males of the L. megera group (European n = 98, African n = 22, and Asian mainland n = 1, Sardinia n = 17, Corsica n = 13, Montecristo n = 9, Capraia n = 7, and other smaller Mediterranean islands n = 95) and 49 males of the L. maera group (European n = 33, African n = 8, and Asian mainland n = 6, Sicily = 1, Lesvos = 1) were examined for genitalia morphology (Data S2: Figure S1). Genitalia were dissected using standard procedures: boiling abdomens in 10% potassium hydroxide, subsequently cleaning the chitinous structures, and removing the aedeagus. The lateral side of tegumen and valvae and the dorsal side of the aedeagus were photographed under a stereomicroscope.
A combination of landmarks and sliding semilandmarks (Bookstein, 1997) was applied to the outlines of tegumen (13 semi-landmarks and 4 landmarks), brachia (10 semilandmarks and 3
4 | PLATANIA eT AL.
landmarks), and valva (17 semilandmarks and 3 landmarks). We con-sidered as landmarks points that could be precisely identified, while the semilandmarks were allowed to slide along the outline trajectory (Data S2: Figure S1).
Wing shape was analyzed from a total of 189 males. Samples of the L. maera group originated from European n = 41, African n = 8, and Asian mainland n = 5, Sicily n = 3, and Lesvos n = 1. Samples of L. megera group originated from European (n = 61), African (n = 17), and Asian mainland (n = 1), Sardinia n = 8, Corsica n = 5, Montecristo n = 4, Capraia n = 3, and other smaller Mediterranean islands n = 31. Thirteen homologous landmarks were identified, representing wing vein intersections or points where the veins meet the edges of the wing (Data S2: Figure S2).
Landmarks of both genitalia and wings were digitized with the program TPS-DIG 2.32 (Rohlf, 2008). In order to remove non-shape variation and to superimpose the objects in a common coordinate system, a generalized procrustes analysis (GPA) was applied to the landmark data (Adams, Rohlf, & Slice, 2004). Partial warps were cal-culated using the shape residuals from GPA. By applying principal component analyses (PCA) to partial warps, relative warps (PCs) were obtained and used as variables in subsequent analyses (Bookstein, 1997). For the aedeagus, we calculated the ratio between the length of the lateral cornuti and the length of the aedeagus.
2.4 | Analysis of configurations for genetic and phenotypic markers
We performed a series of exploratory analyses to understand the main variation patterns of the genetic and phenotypic markers and their degree of concordance. The p-distance dissimilarity matrices for COI and ITS2 were projected in two dimensions by Principal Coordinate Analysis (PCoA) using the “cmdscale” R function. Bidimensional representations were also provided for the two phe-notypic characters (morphology of wings and genitalia) by applying Partial Least Square Discriminant Analyses (PLSDA) to the relative warps (and the ratio of cornuti/aedeagus for genitalia). As a grouping variable, we aggregated specimens into geographic clusters either on the basis of the continent (Europe, Africa, Asia) or island where they occur. For continents and large islands (Sardinia, Corsica, Sicily, Great Britain), specimens were pooled when they belonged to the same square of 2 × 2 degrees of latitude and longitude. To facilitate a direct comparison of the patterns of different markers, we elimi-nated the effect of location and rotation among bidimensional repre-sentations with procrustes analyses, using the COI configuration as reference. Because different markers were represented by different sets of specimens, we used the “recluster.procrustes” function of the recluster R package, which maximizes similarities among configura-tions on the basis of partially overlapping data sets (Dapporto, Vodă, et al., 2014). After procrustes, the aligned bidimensional configura-tions for specimens were projected in the RGB color space using the same package (Dapporto, Fattorini, Vodă, Dincă, & Vila, 2014). Specimens were grouped according to the geographic areas defined
above, and their individual RGB colors were plotted on a map using pie charts.
In a second phase, we tested the existence of a signature of diver-sification in genitalia and wing shape among the clades highlighted by COI (Hernández-Roldán et al., 2016). We carried a series of PLSDA with shape variables (PCs) and the hypothesis for clade attribution as grouping variable. As relative warps can be particularly numerous (2 × number of landmarks-4), overfitting had to be avoided. We thus applied a sparse PLSDA (Lê Cao, Boitard, & Besse, 2011) by includ-ing five shape variables in each component. Finally, to evaluate the degree of diversification as a percentage of cases that can be blindly attributed to their group, we applied a jackknife (leave-one-out) algo-rithm and individually classified each specimen. These analyses were carried out with the “splsda” and “predict” functions in the mixOmics R package (Lê Cao et al., 2011). R scripts are available in Data S3.
3 | RESULTS
3.1 | Molecular analyses
3.1.1 | GMYC assessment
GMYC analyses returned different values for the Yule-coalescent threshold among the tested trees of the subtribes and all Satyrini (the trees are provided in Data S2: Figures S3-S9). The threshold for evolutionary significant units (ESU) was higher when all Satyrinae were analyzed together (1.091%), and lower for individual sub-tribes, the Parargina being the lowest (Parargina 0.612%, Maniolina 0.696%, Erebiina 0.782%, Satyrina 0.833%, Coenonymphina 0.865%, Melanargina 0.918%).
3.1.2 | Phylogenetic results
According to the overall analysis of Satyrini, GMYC highlighted four main COI lineages in the L. megera group, which occur in parapatry along the geographic barriers of the region: (a) a continental clade (European clade) occurring from Spain to the Black Sea, reaching the extreme northern European area of distribution of the species, and stopping in the Danube valleys in Croatia, Hungary, and Romania, without reaching the Balkan highlands (the maps of the entire sam-pled distribution area are provided in Data S2: Figures S10-S13). This lineage is further divided into an Italian lineage showing an abrupt boundary of distribution in the northwest of the peninsula (Italian–European subclade; Figures 1a, 3a and Data S2: Figure S14); (b) the lineage represented by L. paramegaera, restricted to Sardinia, Corsica, and the smaller Capraia and Montecristo islands (L. paramegaera clade) which is sister to the first (Figures 1a, 3a and Data S2: Figure S14); and two sister lineages represented by (c) an African lineage present exclusively in the Maghreb and in Lampedusa and Pantelleria islands (African clade) and (d) a lineage occurring in the Balkans, Turkey, Iran and Ukraine (Balkan–Oriental clade). The lineages differ
| 5PLATANIA eT AL.
from each other by 6–9 mutations (around 1%) (Figures 1a, 3a and Data S2: Figure S14).
The nuclear marker ITS2 did not reveal any clear geographic pat-tern within L. megera (Figure 1b), but L. paramegaera was recovered as a well-supported clade (posterior probability, pp = 1) sister to all L. megera (Figures 1b, 4a and Data S2: Figure S15). However, two specimens concordant in morphology with L. paramegaera from the island of Capraia clustered for ITS2 with L. megera, suggesting the presence of introgression (Figure 1b).
Regarding L. maera, GMYC identified six main COI clades (Figures 2a, 3c): (a) A clade confined to Spain and southwestern France (western European), which in the southwestern part of France is sympatric with the (b) Alpine–Italian clade. This clade occupies the Alps, the Italian Peninsula and Sicily (Italian clade). (c) The north–central–eastern clade widely distributed from central-east Europe and Scandinavia, the Balkan Peninsula to Kazakhstan (the map x of the entire sampled distribution area are provided in Data S2: Figures S16-S19). This group also reaches northeastern Italy, where it is sympatric with the Italian clade; (d–e). Two clades represented by a few specimens, one belonging to Lesvos island and West Turkey, and the other one presents in Romania with one sample, eastern Turkey and Russia (Oriental clades) and (f) a clade represented by Moroccan specimens (African clade). This clade grouped together specimens attributable to L. maera and to L. meadewaldoi, although the two taxa are separated by a minimum of five COI substitutions (Figure 3c). The distances among clades are similar to those found
for L. megera, and the relationships among clades are complex to interpret (Figure 3c).
The Bayesian analysis based on ITS2 highlighted six well-sup-ported main lineages (pp values of one; Figure 4b and Data S2: Figure S20) partially corresponding to those highlighted by GMYC based on COI (Figure 4b): (a) A western European clade confined to Spain and France; (b) An Alpine–Italian clade occupying the Alps, the Italian Peninsula and Sicily; (c) A clade widely distributed from central-east-ern Europe (Romania) and Scandinavia to Russia and Kazakhstan; (d) One clade represented by a few specimens from Greece and Turkey; (e) A cluster with north African L. maera and (f) A clade represented by L. meadewaldoi (Figure 3c, Figure 4b and Data S2: Figure S20).
Lasiommata petropolitana showed a single COI haplogroup in Europe and Asia resulting in a single GMYC entity (Figure 3b). For this reason, this species has not been investigated for other markers.
3.2 | Morphological analyses
3.2.1 | Male genitalia
The analysis of the genitalia pattern resulted in 88 relative warps (PCs) (30 for tegumen, 22 for brachia and 36 for valva). The popu-lations of L. megera/paramegaera and L. maera are geographically well structured for this marker as shown in Figures 1c and 2c. For
F I G U R E 1 Results of the analyses for COI (a), ITS2 (b), genitalia (c), and wings (d) for the L. megera group. The specimens have been projected in the RGB color space after a Principal Coordinates Analysis for COI and ITS2, and a Partial Least Squares Discriminant Analysis for genitalia and wing shape. The resulting colors were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares
6 | PLATANIA eT AL.
L. megera, the only groups showing incongruence with the GMYC assessment are the African and central European clades, which, de-spite the differentiation in COI, do not show any clear diversifica-tion in genitalia shape (Figure 1c, Table 1). The jackknife PLSDA showed that L. paramegaera and the Balkan clade of L. megera could be blindly attributed to their group with more than 70% accuracy (Table 1). Within L. maera/meadewaldoi, the specimens belonging to the western European, the eastern and the Italian clades were cor-rectly attributed in more than 80% of cases, while L. meadewaldoi received a correct attribution in 100% of the cases (Table 2). The relationship between phylogenetic structure in COI and ITS2 and the morphology of genitalia and wings, shown as thin plate spline representations, is presented in Figure 4a, b.
3.2.2 | Wing shape
The morphometric analysis of the forewings resulted in 22 relative warps (PCs) in both L. megera and L. maera groups. The geographic structure is relatively well defined in both cases (Figures 1d, 2d). The jackknife PLSDA for the L. megera group revealed that more than 75% of specimens belonging to the Balkan–Oriental clade and the taxon paramegaera could be blindly attributed to their group (Table 1). Differences in shape among groups are shown as thin plate splines representations in Figure 4a.
Regarding L. maera/meadewaldoi, the specimens belonging to the western European, Italian and the African L. meadewaldoi clade, were correctly assigned to the three groups in more than 75% of the cases (Table 2).
4 | DISCUSSION
Previous studies demonstrated that butterflies in the Satyrinae subfamily have experienced a strong radiation during the Oligocene (Peña, Nylin, & Wahlberg, 2011; Peña & Wahlberg, 2008). Our comparative GMYC analysis revealed a particularly fast coalescence rate in the Parargina subtribe with respect to the other Satyrinae. The tendency of European Parargina to have a strong or fast divergence is reflected by the presence of several species and ESU found in various regions, notably in the Mediterranean and Macaronesian islands and might be partly due to an acceleration of molecular evolution in small island popula-tions (Dapporto, 2008, 2010; Weingartner et al., 2006). Our GMYC approach based on COI revealed that the European Lasiommata has 12 ESU, divided into six clades for L. maera/meadewaldoi, five for L. megera/paramegaera, and one for L. petropolitana. These ESU display divergences among them of about 1% and also show varied degrees of diversification in morphological and nuclear DNA traits (Tables 1 and 2).
F I G U R E 2 Results of the analyses for COI (a), ITS2 (b), genitalia (c), and wings (d) for the L. maera group. The specimens have been projected in the RGB color space after a Principal Coordinates Analysis for COI and ITS2, and a Partial Least Squares Discriminant Analysis for genitalia and wing shape. The resulting colors were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares
| 7PLATANIA eT AL.
F I G U R E 3 Maximum parsimony haplotype networks based on the COI gene for the three taxa under study
8 | PLATANIA eT AL.
The 11 ESU recovered by GMYC for L. megera and L. maera could be regarded as potentially emerging species (Dincă et al., 2015; Pons et al., 2006). However, as reported in Sukumaran and Knowles (2017), the ESU provided by multispecies coalescent analyses are not necessarily taxa diversified at species level. Because of the grad-ual nature of the speciation process, discriminating between struc-ture due to population isolation and structure due to speciation is not always easy. The fact that GMYC is frequently based on a single marker, weakens the reliability of the results produced and may lead to an inflation of species (Sukumaran & Knowles, 2017; Talavera et al., 2013). Thus, as done in previous studies where the existence of cryptic taxa has been documented (Dincă, Dapporto, et al., 2011; Hernández-Roldán et al., 2016; Nazari et al., 2010; Zinetti et al., 2013), we used the GMYC approach to recognize units that were later tested using nuclear DNA sequences and morphological data.
Our results show a clear parapatric/allopatric distribution of the different L. maera and L. megera COI lineages. Their populations gener-ally have little variability within regions but experience abrupt changes
along suture zones often corresponding to geographic barriers (sea straits or mountain chains, notably the sea channels separating North Africa and Sardinia–Corsica from Europe, or the Alps and the Pyrenees). The tendency for sister taxa and COI lineages to be highly segregated in space with narrow contact areas is an increasingly known phenom-enon for Mediterranean butterflies (Cesaroni, Lucarelli, Allori, Russo, & Sbordoni, 1994; Habel et al., 2017; Vodă, Dapporto, Dincă, & Vila, 2015a, 2015b). The same barriers segregating genetic lineages also seem to separate zoogeographic regions identified by the beta-diver-sity patterns displayed by butterfly communities (Dapporto, Fattorini, et al., 2014). The segregation among lineages (and species) and their apparent inability to disperse across relatively narrow sea straits and mainland barriers represent a seeming incongruence, given the gen-erally high dispersal capacity of butterflies and their ability to track suitable areas (Devictor et al., 2012). However, not all butterfly species have the same dispersal capability over sea, as reflected in the patterns of genetic diversification in western Mediterranean islands, which are largely deterministic and depend on environmental constraints (e.g.,
F I G U R E 4 Comparison of the topology of the COI and of the ITS2 trees obtained for L. megera (a) and L. maera (b). Lasiommata meadewaldoi, monophyletic according to COI but not recognized as an evolutionary significant unit, is also included. The thin plate splines of the average configurations of landmarks of the specimens belonging to each lineage for tegumen and wings in L. megera and tegumen and brachia in L. maera are also shown. Support for nodes is provided in Data S1.
| 9PLATANIA eT AL.
winds, environmental suitability) and intrinsic species traits (abun-dance, mobility, specialization, phenology) (Dapporto & Dennis, 2008, 2009; Dapporto, Bruschini, Dincă, Vila, & Dennis, 2012; Dapporto et al., 2017). The high dispersal ability of L. megera is unequivocal since this species has colonized virtually every island in the Mediterranean and possesses the typical species traits characterizing island dispers-ers, such as a high frequency on mainland and a long flight period (Dapporto & Dennis, 2008, 2009). Nevertheless, genetic segregation, reduced gene flow, and delayed secondary sympatry (Pigot & Tobias, 2014) may be maintained across relatively weak physical barriers by mechanisms other than reduced dispersal, such as a combination of competitive exclusion, local adaptation, and incompatibility between different nuclear and mitochondrial DNA variants (Hewitt, 1996, 2000, 2004; Waters, 2011; Waters et al., 2013). Mitochondrial pat-terns in insects may also be determined by infection with Wolbachia. These bacteria can manipulate the reproductive dynamics of species through phenomena such as male-killing and cytoplasmic incompati-bility and can play a role in speciation (Hernández-Roldán et al., 2016; Kodandaramaiah, Simonsen, Bromilow, Wahlberg, & Sperling, 2013). Whatever the mechanisms determining the apparent reduced gene flow at suture zones, this phenomenon is widespread in Mediterranean Satyrinae (Cesaroni et al., 1994; Dapporto, Habel, Dennis, & Schmitt, 2011; Dapporto, Vodă, et al., 2014; Nazari et al., 2010; Vodă et al., 2016) and it can be at the basis of the relatively quick accumulation of genetic differences among populations which, in turn, can promote fast speciation processes.
It must be noted that mtDNA, being maternally transmitted, only reflects the matriline distribution. In many butterfly species, although it is not always the case, males are more active than females, thus more prone to dispersal (Bennett, Pack, Smith, & Betts, 2013; Sielezniew et al., 2011). Accordingly, in an experiment that marked and recaptured specimens of L. megera in different habitat patches, it has been found that males are more prone to disperse to suboptimal patches than
females, probably as an evasive reaction to high population densities in optimal habitats (Elligsen, Beinlich, & Plachter, 1997). It is also plau-sible that the different lineages of Lasiommata are not (completely) reproductively isolated as shown for another Parargina butterfly, P. aegeria where a high vitality of F1 and F2 crosses among lineages has been demonstrated (Livraghi et al., 2018). With these premises, it is possible that male-biased dispersal could maintain gene flow in nu-clear loci across the relatively fixed boundaries of mtDNA lineages and produce discrepancies among nuclear and mitochondrial markers (Sielezniew et al., 2011). The occurrence of male-biased dispersal is suggested by the existence of L. megera ITS2 in some specimens of L. paramegaera from Capraia. Apart from this case, the nuclear DNA marker revealed patterns that are relatively consistent with those highlighted by mtDNA in L. maera and largely inconsistent in L. megera. The four GMYC lineages of L. megera belonging to European, Asiatic, and African mainland were not mirrored by the topology of the ITS2 tree, which did not reveal any clear geographic pattern (Figure 1b). Besides the aforementioned introgression in Capraia, the only GMYC lineage recovered with good support by ITS2 is L. paramegaera.
In L. maera, all the clades identified as ESU by GMYC were also recovered as well-supported clades by ITS2, including the clade cor-responding to L. meadewaldoi. However, COI and ITS2 recovered different relationships among these clades as revealed by the com-parison of the phylogenetic trees (Figure 4b and Figures S15, S18, S20, Data S2) and this can be due to past events of introgression among lineages or even to stochastic fixation events, which could have produced the observed incongruences.
Morphological traits showed a reversed pattern compared to ITS2 in L. megera and L. maera. Even though in many cases speci-mens belonging to different mitochondrial lineages can be blindly attributed to their group based on wing and, especially, geni-talic morphology, the genitalia of L. megera appear much more diversified than those of L. maera. Previous papers (Biermann
TA B L E 1 The correct attribution by Jackknife analysis of specimens to their clade based on wing and genitalia shape, the existence of distinct COI and ITS2 clades, and the recognition of COI clades as independent evolutionary units by GMYC for L. megera group.
Clade Wings Genitalia COI clade ITS2 clade GMYC
African clade 0.619 0.444 Yes No Yes
Balkan–Oriental clade 0.760 0.750 Yes No Yes
European clade 0.297 0.236 Yes No Yes
L. paramegaera clade 0.800 0.739 Yes Yes Yes
TA B L E 2 Correct attribution by the Jackknife analysis of specimens to their clade based on wing and genitalia shape, the existence of distinct COI and ITS2 clades, and the recognition of COI clades as independent evolutionary units by GMYC for the L. maera group.
Clade Wings Genitalia COI clade ITS2 clade GMYC
African clade NA NA Yes No Yes
W European clade 0.769 0.8 Yes Yes Yes
Oriental clades 0 0.6 Yes Yes Yes
L. meadewaldoi clade 0.857 1 Yes Yes With African
N-C-Eastern clade 0.5 0.8 Yes Yes Yes
Italian clade 0.866 0.857 Yes Yes Yes
10 | PLATANIA eT AL.
& Eitschberger, 1996; Dapporto, 2008; Dapporto et al., 2012) highlighted clear differences in genitalia morphology among three groups of L. megera in the study area: (a) the European and Maghreb group, (b) the Sardinian–Corsican group (L. paramegaera), and (c) the Balkan–Eastern group. In this study, we confirm these strong differences in genitalic morphology, which proved to have an almost perfect spatial congruence with the distribution of the mitochondrial lineages, including in the mainland contact zones between the eastern Alps, Romania, and Bulgaria. Lasiommata paramegaera also has a clear difference in wing shape (Figure 1d), in addition to the well-known difference in wing pattern (Biermann & Eitschberger, 1996).
Conversely, although differences in genitalic and wing morphol-ogy among L. maera, mtDNA clades can be retrieved based on a nu-merical approach, upon a visual inspection they appear only slightly different (see thin plate spline representations in Figure 4b). A clear exception is the wing shape and, even more so, the wing pattern of L. meadewaldoi, based on which it has been recognized as a distinct species (Tarrier & Delacre, 2008).
The differences among lineages in morphological structures are encoded in nuclear DNA (Liu et al., 1996) and probably mirror a long-term isolation of European butterfly populations, experienced during the cold periods of the Pleistocene. Throughout most of the Pleistocene, nearly all the central and northern parts of Europe were inhospitable for butterflies, and therefore, they were mainly confined to refugia in the Iberian, Italian, and Balkan Peninsulas and on islands (Hewitt, 1996, 2000, 2004; Schmitt, 2007). The genetic and morpho-logical variation we found are highly concordant with this hypothesis, and an average divergence of 1% in mtDNA fits accurately with a diversification that occurred in Pleistocene climatic pulses. The fact that L. megera did not show a diversification in the ITS2 marker does not necessarily mean that there are no differences in many other nu-clear loci, and further research based on a high number of nuclear DNA markers could provide definitive evidence in this direction.
Lasiommata petropolitana showed a completely different pattern, but still congruent with the hypothesis of Pleistocene differentiation of this genus. Among the three taxa analyzed here, L. petropolitana inhabits the coldest regions and is limited to Scandinavia and southern European mountain areas. It also has very limited diversification with a single haplogroup (five closely related haplotypes) across the spe-cies´ range (Figure 3c). Species that live at high latitudes actually tend to show an exiguous genetic differentiation after the fast postglacial colonization processes, probably due to a wider distribution during the long cold phases of the Pleistocene with a consequent higher gene flow among populations (Hewitt, 2000; Wallis & Arntzen, 1989).
Based on genetic, morphological, and distributional patterns, we confirm that L. paramegaera and L. meadewaldoi represent distinct groups for all the markers under study. Although both taxa appear to generate paraphyly for L. megera and L. maera, respectively, for at least one marker (COI in L. paramegaera and ITS2 and COI for L. meade-waldoi), we do not exclude the possibility of paraphyletic speciation. Considering that paraphyletic groups are commonly formed during the evolutionary process and are conspicuous particularly in cases of
recent divergence (Hörandl & Stuessy, 2010), it is not surprising that paraphyly is detected in the speciation process of Lasiommata but-terflies. They show a divergence coherent with phenomena of insu-larity and segregation of different southern refugia during repeated climatic pulses in the Pleistocene (Schmitt, 2007) and produce a mosaic of lineages separated by similar genetic distances (Figure 3). The diversification in COI within both taxa is not extremely high, al-though there are other cases of Satyrinae species characterized by low diversification in mtDNA that show a clear distinction at the spe-cific level in morphological traits (e.g., Dincă et al., 2015; Habel et al., 2017; Kreuzinger, Fiedler, Letsch, & Grill, 2015; Nazari et al., 2010). Both L. paramegaera and L. meadewaldoi occur in isolated areas, the first in Capraia, Montecristo, Corsica, Sardinia, and circum-Sardinian islands, which are well-known areas of endemism for European but-terflies (Dennis, Williams, & Shreeve, 1991), and the second in the High Atlas, about 130 km far from the closest L. maera population (according to Tarrier & Delacre, 2008). Reduced population size and isolation could have accelerated the speciation process compared to larger populations showing parapatric distributions on the mainland. The occurrence of introgression in Capraia located at an intermedi-ate distance between Corsica (where L. paramegaera occurs) and Elba (where L. megera occurs), does not necessarily disprove the species status of L. paramegaera, and can be rather attributed to a historical introgression event. Indeed, although two of the three specimens from Capraia show L. megera ITS2, their COI and morphology are identical to the other L. paramegaera populations (Dapporto, 2007, 2008). It must be noted that about 12.4% of European butterfly spe-cies are known to produce fertile hybrids (Mallet, 2005). Similar evi-dence in many other taxonomic groups led to the formulation of the differential fitness species concept according to which some genes can be exchanged between species provided that there is a series of peculiar “speciation genes,” such as those involved in the repro-ductive output (e.g., in shaping the male genitalia), which cannot be exchanged without fitness costs (Hausdorf, 2011).
The taxonomic status of the other lineages of L. megera and L. maera is less clear. None of the three L. megera COI lineages (not counting here L. paramegaera) showed a diversification in ITS2; al-though at the contact between the European and the Balkan lin-eages, there is a congruent morphology-COI pattern. An in-depth study of the contact zone and the analysis of more nuclear genes could provide definitive evidence confirming or disproving the exis-tence of two (or three, including the North African lineage) mainland species in the L. megera group. Until further data become available, we consider the mainland COI haplogroups of L. megera as highly di-verged parapatric lineages.
The decision is even more difficult for the lineages of L. maera, for which, based on concordant patterns of COI, ITS2, and morphology, five groups occur, in addition to L. meadewaldoi. However, the differ-ences in wing and genitalic morphology, although detectable with nu-meric analyses, are not evident and would indicate the existence of five cryptic taxa. Definitive identification of cryptic butterfly populations as distinct species requires more in-depth analyses, ideally assessing a higher number of nuclear markers and cytological characteristics,
| 11PLATANIA eT AL.
Wolbachia infection, possible host plant specialization, mate recogni-tion during courtship, and the comparison of different characters in areas of contact (Dincă, Dapporto, et al., 2011; Dincă, Lukhtanov, et al., 2011; Dincă et al., 2013; Hernández-Roldán et al., 2016; Zinetti et al., 2013). For this reason, in this case as well, we prefer to postpone a definitive recognition of L. maera lineages as species. Nevertheless, all these lineages at least seem to represent cases of incipient speciation.
In conclusion, our study exemplifies the continuous nature of evolutionary processes leading to speciation and the difficulties of delimiting species. Through the comparison of nuclear, mitochon-drial, and morphological markers at a relatively high spatial resolu-tion, we showed that L. megera and L. maera groups have experienced a fast divergence in different Mediterranean areas during the long cold periods of the Pleistocene. Undoubtedly, we are dealing with a fast diverging group, composed by a high number of evolutionary units located in the gray zone of an advanced speciation process. The recognition of taxa as diverging lineages or distinct, possibly paraphyletic, species, mostly depends on the criteria adopted by dif-ferent species concepts.
ACKNOWLEDG EMENTSFunding for this research came from the Spanish MINECO and AEI/FEDER, UE (CGL2013-48277-P and CGL2016-76322-P to RVi), Marie Skłodowska-Curie Train2Move to RVo (grant 609402–2020), Marie Sklodowska-Curie IOF grant (project 625997) to VD, from Tuscan Archipelago National Park and Florence University to LD and from the project “Barcoding-Italian-Butterflies.”
ORCIDLeonardo Platania https://orcid.org/0000-0002-1289-5564 Raluca Vodă https://orcid.org/0000-0003-1970-5628 Vlad Dincă https://orcid.org/0000-0003-1791-2148 Gerard Talavera https://orcid.org/0000-0003-1112-1345 Roger Vila https://orcid.org/0000-0002-2447-4388 Leonardo Dapporto https://orcid.org/0000-0001-7129-4526
R E FE R E N C E SAdams, D. C., Rohlf, F. J., & Slice, D. E. (2004). Geometric morphomet-
rics: Ten years of progress following the ‘revolution’. Italian Journal of Zoology, 71(1), 5–16. https ://doi.org/10.1080/11250 00040 9356545
Balletto, E., & Cassulo, L. A. (1995). Lepidoptera Hesperioidea, Papilionoidea. Edizione Calderini.
Barluenga, M., Stölting, K. N., Salzburger, W., Muschick, M., & Meyer, A. (2006). Sympatric speciation in Nicaraguan crater lake cichlid fish. Nature, 439(7077), 719–723. https ://doi.org/10.1038/natur e04325
Bennett, V. J., Pack, S. M., Smith, W. P., & Betts, M. G. (2013). Sex-biased dispersal in a rare butterfly and the implications for its conser-vation. Journal of Insect Conservation, 17(5), 949–958. https ://doi.org/10.1007/s10841-013-9577-5
Biermann, H., & Eitschberger, U. (1996). Bemerkungen zu Lasiommata megera (Linnaeus, 1767) und Lasiommata paramegaera (Hübner 1824)(Lepidoptera Saryridae). Atalanta, 27(1/2), 253–319.
Bookstein, F. L. (1997). Morphometric tools for landmark data: Geometry and biology. Cambridge, UK: Cambridge University Press.
Bozano, G. C. (1999): Satyridae part I. In: G. C. Bozano (Ed.), Guide to the butterflies of the Palearctic region. (58 pp). Milano, Italy: Omnes Artes.
Brower, A. (1994). Rapid morphological radiation and convergence among races of the butterfly Heliconius erato inferred from pat-terns of mitochondrial DNA evolution. Proceedings of the National Academy of Sciences, 91(14), 6491–6495. https ://doi.org/10.1073/pnas.91.14.6491
Butlin, R. K., Galindo, J., & Grahame, J. W. (2008). Sympatric, parapa-tric or allopatric: The most important way to classify speciation? Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1506), 2997–3007. https ://doi.org/10.1098/rstb.2008.0076
Cesaroni, D., Lucarelli, M., Allori, P., Russo, F., & Sbordoni, V. (1994). Patterns of evolution and multidimensional systematics in graylings (Lepidoptera: Hipparchia). Biological Journal of the Linnean Society, 52(2), 101–119. https ://doi.org/10.1111/j.1095-8312.1994.tb009 82.x
Chinery, M. (1989). Butterflies and day-flying moths of Britain and Europe. London, UK: Collins Professional and Technical Books.
Clement, M., Posada, D., & Crandall, K. A. (2000). TCS: A computer pro-gram to estimate gene genealogies. Molecular Ecology, 9(10), 1657–1659. https ://doi.org/10.1046/j.1365-294x.2000.01020.x
Coyne, J. A., & Orr, H. A. (2004). Speciation. Sunderland, MA: Sinauer Associates.
Dapporto, L. (2007). Cuticular lipid diversification in Lasiommata megera and Lasiommata paramegaera: The influence of species, sex, and pop-ulation (Lepidoptera: Nymphalidae): Cuticular mixture diversification in l. Megera and l. Paramegaera. Biological Journal of the Linnean Society, 91(4), 703–710. https ://doi.org/10.1111/j.1095-8312.2007.00833.x
Dapporto, L. (2008). Geometric morphometrics reveal male geni-talia differences in the Lasiommata megera / paramegaera com-plex (Lepidoptera, Nymphalidae) and the lack of a predicted hy-bridization area in the Tuscan Archipelago. Journal of Zoological Systematics and Evolutionary Research, 46(3), 224–230. https ://doi.org/10.1111/j.1439-0469.2007.00453.x
Dapporto, L. (2010). Satyrinae butterflies from Sardinia and Corsica show a kaleidoscopic intraspecific biogeography (Lepidoptera, Nymphlidae). Biological Journal of the Linnean Society, 100(1), 195–212. https ://doi.org/10.1111/j.1095-8312.2010.01434.x
Dapporto, L., Bruschini, C., Dincă, V., Vila, R., & Dennis, R. L. H. (2012). Identifying zones of phenetic compression in West Mediterranean butterflies (Satyrinae): Refugia, invasion and hy-bridization: Phenetic ‘compression’ in Western Mediterranean. Diversity and Distributions, 18(11), 1066–1076. https ://doi.org/10.1111/j.1472-4642.2012.00903.x
Dapporto, L., Cini, A., Menchetti, M., Vodă, R., Bonelli, S., Casacci, L. P., … Vila, R. (2017). Rise and fall of island butterfly diversity: Understanding genetic differentiation and extinction in a highly diverse archipelago. Diversity and Distributions, 23(10), 1169–1181. https ://doi.org/10.1111/ddi.12610
Dapporto, L., & Dennis, R. L. H. (2008). Species richness, rarity and en-demicity on Italian offshore islands: Complementary signals from is-land-focused and species-focused analyses. Journal of Biogeography, 35(4), 664–674. https ://doi.org/10.1111/j.1365-2699.2007.01812.x
Dapporto, L., & Dennis, R. L. H. (2009). Conservation biogeography of large Mediterranean islands. Butterfly impoverishment, conservation pri-orities and inferences for an ecological “island paradigm”. Ecography, 32(1), 169–179. https ://doi.org/10.1111/j.1600-0587.2008.05600.x
Dapporto, L., Fattorini, S., Vodă, R., Dincă, V., & Vila, R. (2014). Biogeography of western Mediterranean butterflies: Combining turnover and nestedness components of faunal dissimilarity. Journal of Biogeography, 41(9), 1639–1650. https ://doi.org/10.1111/jbi.12315
Dapporto, L., Habel, J. C., Dennis, R. L. H., & Schmitt, T. (2011). The bio-geography of the western Mediterranean: Elucidating contradictory distribution patterns of differentiation in Maniola jurtina (Lepidoptera: Nymphalidae): BIOGEOGRAPHY OF MEDITERRANEAN BUTTERFLIES. Biological Journal of the Linnean Society, 103(3), 571–577. https ://doi.org/10.1111/j.1095-8312.2011.01639.x
12 | PLATANIA eT AL.
Dapporto, L., Vodă, R., Dincă, V., & Vila, R. (2014). Comparing popu-lation patterns for genetic and morphological markers with un-even sample sizes. An example for the butterfly Maniola jurtina. Methods in Ecology and. Evolution, 5(8), 834–843. https ://doi.org/10.1111/2041-210X.12220
Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). jModelT-est 2: More models, new heuristics and parallel computing. Nature Methods, 9(8), 772. https ://doi.org/10.1038/nmeth.2109
de Queiroz, K. (2005). Ernst Mayr and the modern concept of species. Proceedings of the National Academy of Sciences, 102(Supplement 1), 6600–6607. https ://doi.org/10.1073/pnas.05020 30102
De Queiroz, K. (2007). Species Concepts and Species Delimitation. Systematic Biology, 56(6), 879–886. https ://doi.org/10.1080/10635 15070 1701083
Dennis, R. L. H., Williams, W. R., & Shreeve, T. G. (1991). A mul-tivariate approach to the determination of faunal structures among European butterfly species (Lepidoptera: Rhopalocera). Zoological Journal of the Linnean Society, 101(1), 1–49. https ://doi.org/10.1111/j.1096-3642.1991.tb008 84.x
Devictor, V., van Swaay, C., Brereton, T., Brotons, L., Chamberlain, D., Heliölä, J., … Jiguet, F. (2012). Differences in the climatic debts of birds and butterflies at a continental scale. Nature Climate Change, 2(2), 121–124. https ://doi.org/10.1038/nclim ate1347
Dincă, V., Dapporto, L., & Vila, R. (2011). A combined genetic-mor-phometric analysis unravels the complex biogeographical his-tory of Polyommatus icarus and Polyommatus celina Common Blue butterflies. Molecular Ecology, 20(18), 3921–3935. https ://doi.org/10.1111/j.1365-294X.2011.05223.x
Dincă, V., Lukhtanov, V. A., Talavera, G., & Vila, R. (2011). Unexpected layers of cryptic diversity in wood white Leptidea butterflies. Nature Communications, 2, 324. https ://doi.org/10.1038/ncomm s1329
Dincă, V., Montagud, S., Talavera, G., Hernández-Roldán, J., Munguira, M. L., García-Barros, E., … Vila, R. (2015). DNA barcode reference library for Iberian butterflies enables a continental-scale preview of potential cryptic diversity. Scientific Reports, 5, 12395. https ://doi.org/10.1038/srep1 2395
Dincă, V., Wiklund, C., Lukhtanov, V. A., Kodandaramaiah, U., Norén, K., Dapporto, L., … Friberg, M. (2013). Reproductive isolation and pat-terns of genetic differentiation in a cryptic butterfly species com-plex. Journal of Evolutionary Biology, 26(10), 2095–2106. https ://doi.org/10.1111/jeb.12211
Dincă, V., Zakharov, E. V., Hebert, P. D. N., & Vila, R. (2011). Complete DNA barcode reference library for a country’s butterfly fauna re-veals high performance for temperate Europe. Proceedings of the Royal Society B: Biological Sciences, 278(1704), 347–355. https ://doi.org/10.1098/rspb.2010.1089
Dirzo, R., Young, H. S., Galetti, M., Ceballos, G., Isaac, N. J., & Collen, B. (2014). Defaunation in the Anthropocene. Science, 345(6195), 401–406.
Drummond, A. J., & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7(1), 214. https ://doi.org/10.1186/1471-2148-7-214
Elligsen, H., Beinlich, B., & Plachter, H. (1997). Effects of large-scale cattle grazing on populations of Coenonympha glycerion and Lasiommata megera (Lepidoptera: Satyridae). Journal of Insect Conservation, 1(1), 13–23.
Fujisawa, T., & Barraclough, T. G. (2013). Delimiting Species Using Single-Locus Data and the Generalized Mixed Yule Coalescent Approach: A Revised Method and Evaluation on Simulated Data Sets. Systematic Biology, 62(5), 707–724. https ://doi.org/10.1093/sysbi o/syt033
Gavrilets, S., Li, H., & Vose, M. D. (2000). Patterns of parapatric speciation. Evolution, 54(4), 1126–1134. https ://doi.org/10.1111/j.0014-3820.2000.tb005 48.x
Habel, J. C., Vila, R., Vodă, R., Husemann, M., Schmitt, T., & Dapporto, L. (2017). Differentiation in the marbled white butterfly species com-plex driven by multiple evolutionary forces. Journal of Biogeography, 44(2), 433–445. https ://doi.org/10.1111/jbi.12868
Hausdorf, B. (2011). Progress toward a general species concept. Evolution, 65(4), 923–931. https ://doi.org/10.1111/j.1558-5646.2011.01231.x
Hausmann, A., Haszprunar, G., Segerer, A. H., Speidel, W., Behounek, G., & Hebert, P. D. N. (2011). Now DNA-barcoded: The butterflies and larger moths of Germany. Spixiana, 34(1), 47–58.
Hebert, P. D., Cywinska, A., & Ball, S. L. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B: Biological Sciences, 270(1512), 313–321.
Hendry, A. P. (2009). Ecological speciation! Or the lack thereof? Canadian Journal of Fisheries and Aquatic Sciences, 66(8), 1383–1398. https ://doi.org/10.1139/F09-074
Hendry, A. P., Bolnick, D. I., Berner, D., & Peichel, C. L. (2009). Along the speciation continuum in sticklebacks. Journal of Fish Biology, 75(8), 2000–2036. https ://doi.org/10.1111/j.1095-8649.2009.02419.x
Hernández-Roldán, J. L., Dapporto, L., Dincă, V., Vicente, J. C., Hornett, E. A., Šíchová, J., … Vila, R. (2016). Integrative analyses unveil specia-tion linked to host plant shift in Spialia butterflies. Molecular Ecology, 25(17), 4267–4284. https ://doi.org/10.1111/mec.13756
Hewitt, G. M. (1996). Some genetic consequences of ice ages, and their role in divergence and speciation. Biological Journal of the Linnean Society, 58(3), 247–276. https ://doi.org/10.1111/j.1095-8312.1996.tb014 34.x
Hewitt, G. M. (2000). The genetic legacy of the Quaternary ice ages. Nature, 405(6789), 907–913. https ://doi.org/10.1038/35016000
Hewitt, G. M. (2004). Genetic consequences of climatic oscillations in the Quaternary. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 359(1442), 183–195. https ://doi.org/10.1098/rstb.2003.1388
Higgins, L. G., & Riley, N. D. (1970). A field guide to the butterflies of Britain and Europe. Collins, London.
Hörandl, E., & Stuessy, T. F. (2010). Paraphyletic groups as natural units of biological classification. Taxon, 59(6), 1641–1653. https ://doi.org/10.1002/tax.596001
Jutzeler, D. (1998). Lasiommata paramegaera (Huebner, 1824) of the Tyrrhenian isles: A good species (Lepidoptera: Nymphalidae, Satyrinae). Linneana Belgica (Belgium), 16(7), 267–276.
Kodandaramaiah, U., Simonsen, T. J., Bromilow, S., Wahlberg, N., & Sperling, F. (2013). Deceptive single-locus taxonomy and phylogeography: Wolbachia-associated divergence in mitochondrial DNA is not reflected in morphology and nuclear markers in a butterfly species. Ecology and Evolution, 3(16), 5167–5176. https ://doi.org/10.1002/ece3.886
Kreuzinger, A. J., Fiedler, K., Letsch, H., & Grill, A. (2015). Tracing the ra-diation of Maniola (Nymphalidae) butterflies: New insights from phy-logeography hint at one single incompletely differentiated species complex. Ecology and Evolution, 5(1), 46–58.
Kudrna, O., Harpke, A., Lux, K., Pennerstorfer, J., Schweiger, O., Settele, J., & Wiemers, M. (2011). Distribution atlas of butterflies in Europe. Gesellschaft für Schmetterlingsschutz eV. Halle:
Lê Cao, K.-A., Boitard, S., & Besse, P. (2011). Sparse PLS discriminant analysis: Biologically relevant feature selection and graphical dis-plays for multiclass problems. BMC Bioinformatics, 12(1), 253. https ://doi.org/10.1186/1471-2105-12-253
Leraut, P. (1980). Liste systématique et synonymique des Lépidoptères de France, Belgique et Corse. Paris: Alexanor.
Liu, J., Mercer, J. M., Stam, L. F., Gibson, G. C., Zeng, Z. B., & Laurie, C. C. (1996). Genetic analysis of a morphological shape difference in the male genitalia of Drosophila simulans and D. mauritiana. Genetics, 142(4), 1129–1145.
Livraghi, L., Vodă, R., Evans, L. C., Gibbs, M., Dincă, V., Holland, P. W., … Breuker, C. J. (2018). Historical and current patterns of gene flow in
| 13PLATANIA eT AL.
the butterfly Pararge aegeria. Journal of Biogeography, 45(7), 1628–1639. https ://doi.org/10.1111/jbi.13354
Mallet, J. (2005). Hybridization as an invasion of the genome. Trends in Ecology & Evolution, 20(5), 229–237. https ://doi.org/10.1016/j.tree.2005.02.010
Mallet, J., Beltrán, M., Neukirchen, W., & Linares, M. (2007). Natural hybridization in heliconiine butterflies: The species boundary as a continuum. BMC Evolutionary Biology, 7(1), 28. https ://doi.org/10.1186/1471-2148-7-28
Mayr, E. (1963). Animal Species and Evolution. Massachusetts Google Scholar: Belknap Press of Harvard University Press Cambridge.
Miller, M. A., Pfeiffer, W., & Schwartz, T. (2010). Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In 2010 gateway computing environments workshop (GCE) (pp. 1–8). IEEE. https ://doi.org/10.1109/GCE.2010.5676129
Mittelbach, G. G., & Schemske, D. W. (2015). Ecological and evo-lutionary perspectives on community assembly. Trends in Ecology & Evolution, 30(5), 241–247. https ://doi.org/10.1016/j.tree.2015.02.008
Múrias dos Santos, A., Cabezas, M. P., Tavares, A. I., Xavier, R., & Branco, M. (2015). tcsBU: A tool to extend TCS network layout and visualiza-tion. Bioinformatics, 32(4), 627–628. https ://doi.org/10.1093/bioin forma tics/btv636
Nazari, V., Hagen, W. T., & Bozano, G. C. (2010). Molecular systematics and phylogeny of the ‘Marbled Whites’ (Lepidoptera: Nymphalidae, Satyrinae, Melanargia Meigen). Systematic Entomology, 35(1), 132–147. https ://doi.org/10.1111/j.1365-3113.2009.00493.x
Nosil, P., Harmon, L. J., & Seehausen, O. (2009). Ecological explanations for (incomplete) speciation. Trends in Ecology & Evolution, 24(3), 145–156. https ://doi.org/10.1016/j.tree.2008.10.011
Padial, J. M., Miralles, A., De la Riva, I., & Vences, M. (2010). The inte-grative future of taxonomy. Frontiers in Zoology, 7(1), 16. https ://doi.org/10.1186/1742-9994-7-16
Peña, C., Nylin, S., & Wahlberg, N. (2011). The radiation of Satyrini but-terflies (Nymphalidae: Satyrinae): A challenge for phylogenetic meth-ods. Zoological Journal of the Linnean Society, 161(1), 64–87. https ://doi.org/10.1111/j.1096-3642.2009.00627.x
Peña, C., & Wahlberg, N. (2008). Prehistorical climate change increased diversification of a group of butterflies. Biology Letters, 4(3), 274–278. https ://doi.org/10.1098/rsbl.2008.0062
Peña, C., Wahlberg, N., Weingartner, E., Kodandaramaiah, U., Nylin, S., Freitas, A. V., & Brower, A. V. (2006). Higher level phylogeny of Satyrinae butterflies (Lepidoptera: Nymphalidae) based on DNA sequence data. Molecular Phylogenetics and Evolution, 40(1), 29–49. https ://doi.org/10.1016/j.ympev.2006.02.007
Pigot, A. L., & Tobias, J. A. (2014). Dispersal and the transition to sympatry in vertebrates. Proceedings of the Royal Society B: Biological Sciences, 282(1799), 20141929. https ://doi.org/10.1098/rspb.2014.1929
Pons, J., Barraclough, T. G., Gomez-Zurita, J., Cardoso, A., Duran, D. P., Hazell, S., … Vogler, A. P. (2006). Sequence-Based Species Delimitation for the DNA Taxonomy of Undescribed Insects. Systematic Biology, 55(4), 595–609. https ://doi.org/10.1080/10635 15060 0852011
Quek, S., Davies, S. J., Itino, T., & Pierce, N. E. (2004). Codiversification in an ant-plant mutualism: Stem texture and the evolution of host use in Crematogaster (Formicidae: Myrmicinae) inhabitants of Macaranga (Euphorbiaceae). Evolution, 58(3), 554–570. https ://doi.org/10.1111/j.0014-3820.2004.tb016 78.x
Rambaut, A., Drummond, A. J., Xie, D., Baele, G., & Suchard, M. A. (2018). Posterior Summarization in Bayesian Phylogenetics Using T racer 1.7. Systematic Biology., 67(5), 901–904.
Rohlf, F. (2008). Tpsdig, version 2.12; tpsrelw, version 1.46. Stony Brook, NY: State University of New York at Stony Brook.
Schlick-Steiner, B. C., Steiner, F. M., Seifert, B., Stauffer, C., Christian, E., & Crozier, R. H. (2010). Integrative Taxonomy: A Multisource Approach
to Exploring Biodiversity. Annual Review of Entomology, 55(1), 421–438. https ://doi.org/10.1146/annur ev-ento-112408-085432
Schmitt, T. (2007). Molecular biogeography of Europe: Pleistocene cy-cles and postglacial trends. Frontiers in Zoology, 4(1), 11. https ://doi.org/10.1186/1742-9994-4-11
Seehausen, O. (2009). Progressive levels of trait divergence along a ’spe-ciation transect’ in the Lake Victoria cichlid fish Pundamilia. In R. K. Butlin, J. R. Bridle, & D. Schluter (Eds.), Speciation and Patterns of Diversity (pp. 155–176). Cambridge, MA: Cambridge University Press.
Settele, J., Shreeve, T., Konvička, M., & van Dyck, H. (2009). Ecology of butterflies in Europe. Cambridge, MA: Cambridge University Press.
Shaw, K. L., & Mullen, S. P. (2014). Speciation Continuum. Journal of Heredity, 105(S1), 741–742. https ://doi.org/10.1093/jhere d/esu060
Sielezniew, M., Ponikwicka-Tyszko, D., Ratkiewicz, M., Dziekanska, I., Kostro-Ambroziak, A., & Rutkowski, R. (2011). Divergent pat-terns in the mitochondrial and nuclear diversity of the special-ized butterfly Plebejus argus (Lepidoptera: Lycaenidae). European Journal of Entomology, 108(4), 537–545. https ://doi.org/10.14411/ eje.2011.069.
Sukumaran, J., & Knowles, L. L. (2017). Multispecies coalescent delim-its structure, not species. Proceedings of the National Academy of Sciences, 114(7), 1607–1612. https ://doi.org/10.1073/pnas.16079 21114
Talavera, G., Dincă, V., & Vila, R. (2013). Factors affecting species de-limitations with the GMYC model: Insights from a butterfly survey. Methods in Ecology and Evolution, 4(12), 1101–1110. https ://doi.org/10.1111/2041-210X.12107
Tarrier, M., & Delacre, J. (2008). Les Papillons de jour du Maroc. Mèze: Biotope.
Tennent, J. (1996). The butterflies of Morocco. Co: Algeria and Tunisia. Gem Pub.
Tolman, T., & Lewington, R. (2008). Butterflies of Europe. London, UK: Harper Collins.
Tshikolovets, V. V. (2011). Butterflies of Europe & the Mediterranean area. Pardubice: Tshikolovets Publications.
van Swaay, C., Cuttelod, A., Collins, S., Maes, D., Munguira, M. L., Šašić, M., … Warren, M. (2010). European red list of butterflies. Publications office of the. European Union.
Via, S. (2001). Sympatric speciation in animals: The ugly duckling grows up. Trends in Ecology & Evolution, 16(7), 381–390. https ://doi.org/10.1016/S0169-5347(01)02188-7
Vodă, R., Dapporto, L., Dincă, V., Shreeve, T. G., Khaldi, M., Barech, G., … Vila, R. (2016). Historical and contemporary factors generate unique butterfly communities on islands. Scientific Reports, 6(1), 28828. https ://doi.org/10.1038/srep2 8828
Vodă, R., Dapporto, L., Dincă, V., & Vila, R. (2015a). Cryptic mat-ters: Overlooked species generate most butterfly beta-diversity. Ecography, 38(4), 405–409. https ://doi.org/10.1111/ecog.00762
Vodă, R., Dapporto, L., Dincă, V., & Vila, R. (2015b). Why Do Cryptic Species Tend Not to Co-Occur? A Case Study on Two Cryptic Pairs of Butterflies. PLoS ONE, 10(2), e0117802. https ://doi.org/10.1371/journ al.pone.0117802
Wallis, G. P., & Arntzen, J. W. (1989). Mitochondrial-DNA varia-tion in the crested newt superspecies: Limited cytoplasmic gene flow among species. Evolution, 43(1), 88–104. https ://doi.org/10.1111/j.1558-5646.1989.tb042 09.x
Waters, J. M. (2011). Competitive exclusion: Phylogeography’s ‘elephant in the room’? Molecular Ecology, 20(21), 4388–4394. https ://doi.org/10.1111/j.1365-294X.2011.05286.x
Waters, J. M., Fraser, C. I., & Hewitt, G. M. (2013). Founder takes all: Density-dependent processes structure biodiversity. Trends in Ecology & Evolution, 28(2), 78–85. https ://doi.org/10.1016/j.tree.2012.08.024
Weingartner, E., Wahlberg, N., & Nylin, S. (2006). Speciation in Pararge (Satyrinae: Nymphalidae) butterflies–North Africa is the source of
14 | PLATANIA eT AL.
ancestral populations of all Pararge species. Systematic Entomology, 31(4), 621–632.
Wheeler, Q. D., Knapp, S., Stevenson, D. W., Stevenson, J., Blum, S. D., Boom, B. M., … Woolley, J. B. (2012). Mapping the biosphere: Exploring species to understand the origin, organization and sustain-ability of biodiversity. Systematics and Biodiversity, 10(1), 1–20. https ://doi.org/10.1080/14772 000.2012.665095
Zinetti, F., Dapporto, L., Vovlas, A., Chelazzi, G., Bonelli, S., Balletto, E., & Ciofi, C. (2013). When the rule Becomes the Exception. No Evidence of Gene Flow between Two Zerynthia Cryptic Butterflies Suggests the Emergence of a New Model Group. PLoS ONE, 8(6), e65746. https ://doi.org/10.1371/journ al.pone.006574
SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section at the end of the article.
Data S1. Supporting Information Specimen List.Data S2. Supporting Information.Data S3. Supporting Information R Script.R.Figure S1. Landmarks (empty circles) and sliding semilandmarks (co-loured circles) used for the analysis of the different structures of Lasiommata spp. male genitalia.Figure S2. Landmarks (white circles) used for the analysis of Lasiommata spp. wings.Figure S3. Phylogenetic tree based on COI for Coenonymphina group and results for GMYC solution (red clades).Figure S4. Phylogenetic tree based on COI for Erebiina group and results for GMYC solution (red clades).Figure S5. Phylogenetic tree based on COI for Maniolina group and results for GMYC solution (red clades).Figure S6. Phylogenetic tree based on COI for Melanargina group and results for GMYC solution (red clades).Figure S7. Phylogenetic tree based on COI for Parargina group and results for GMYC solution (red clades).Figure S8. Phylogenetic tree based on COI for Satyrina group and results for GMYC solution (red clades).Figure S9. Phylogenetic tree based on COI for Satyrinae subfamily and results for GMYC solution (red clades).Figure S10. Uncut version of map for COI in L. megera group. The specimens have been projected in RGB colour space after Principal Coordinate analysis and the resulting colours were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares.Figure S11. Uncut version of map for ITS2 in L. megera group. The specimens have been projected in RGB colour space after Principal Coordinate analysis and the resulting colours were plotted in pie charts grouping specimens from the same island or continent within
2 degrees latitude–longitude squares.Figure S12. Uncut version of map for wings in L. megera group. The specimens have been projected in RGB colour space after Partial Least Squares Discriminant Analysis and the resulting colors were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares.Figure S13. Uncut version of map for genitalia in L. megera group. The specimens have been projected in RGB colour space after Partial Least Squares Discriminant Analysis and the resulting colors were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares.Figure S14. Phylogenetic tree based on COI for Western Palearctic Lasiommata spp.Figure S15. Phylogenetic tree based on ITS2 for L. megera/paramegaera.Figure S16. Uncut version of map for COI in L. maera group. The specimens have been projected in RGB colour space after Principal Coordinate analysis and the resulting colours were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares.Figure S17. Uncut version of map for ITS2 in L. maera group. The specimens have been projected in RGB colour space after Principal Coordinate analysis and the resulting colours were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares.Figure S18. Uncut version of map for wings in L. maera group. The specimens have been projected in RGB colour space after Partial Least Squares Discriminant Analysis and the resulting colors were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude square.Figure S19. Uncut version of map for genitalia in L. maera group. The specimens have been projected in RGB colour space after Partial Least Squares Discriminant Analysis and the resulting colors were plotted in pie charts grouping specimens from the same island or continent within 2 degrees latitude–longitude squares.Figure S20. Phylogenetic tree based on ITS2 for L. maera/meadewaldoi.
How to cite this article: Platania L, Vodă R, Dincă V, Talavera G, Vila R, Dapporto L. Integrative analyses on Western Palearctic Lasiommata reveal a mosaic of nascent butterfly species. J Zool Syst Evol Res. 2020;00:1–14. https ://doi.org/10.1111/jzs.12356
