A north-south hemispheric migratory divide in the butterfly Vanessa cardui

Domain: biology
Published: 2025-12-30
Editorial outcome: major_revision

Abstract
Reversed seasonality and distinct navigation cues in the Earth’s two hemispheres may shape the evolution of migratory behaviour in animals. Migratory divides—contact zones where populations have evolved alternative migratory strategies—are well-documented in birds and typically occur longitudinally. We hypothesise that insect migratory divides are less likely to emerge longitudinally, but may exist latitudinally, driven by hemisphere-specific sensory adaptations that lead to spatial and temporal isolation. Here, we examine this hypothesis in the cosmopolitan painted lady butterfly (Vanessa cardui), whose Southern Hemisphere dynamics remain unexplored. Investigating the genomes of 300 individuals across Africa and Europe, we identify a 9 Mb chromosomal inversion on chromosome 8, which exhibits strong haplotype structure aligned with hemispheric origin, with a few potential heterozygotes near the equator. The inversion harbours 336 genes, including several directly relevant to migration. Notably, one inversion breakpoint intersects the gene encoding the GABA-B receptor, which responds to the neuropeptide γ-aminobutyric acid (GABA), crucial for insect navigation. Our findings provide genomic evidence of a migratory divide in insects and highlight the role of inverted seasonality in the two hemispheres and genomic rearrangements as isolating barriers for highly mobile species.

Introduction
Movement plays a fundamental role in the evolution of life. Among various forms of movement, migration stands out as a particularly specialised and multifaceted behaviour – a choreography of finely tuned responses to fluctuating environmental conditions1. The evolutionary and molecular underpinnings of this complex behaviour remain largely unknown, as do the impacts of migration on species diversification2,3.

Seasonality is crucial in triggering migratory behaviour, as animals rely on environmental signals such as changes in photoperiod, plant phenology, and temperature to initiate, maintain, and terminate migration. These cyclical signals are thought to modulate transcriptional and epigenetic pathways that regulate migratory responses4,5. Once migration begins, navigational adaptations become essential for maintaining directional movement towards favourable destinations. Celestial cues, both diurnal and nocturnal, along with Earth’s magnetic field, influence navigation. These signals are integrated into neural networks that form an internal compass, guiding movement across vast distances6,7.

Seasonality patterns are generally consistent across longitude (east-west) but vary latitudinally (north-south), driving animal migratory movements predominantly in a north-south direction8. Invertebrates, migratory divides – zones where partially sympatric migratory populations adopt distinct migratory strategies – have been widely described, particularly on philopatric species migrating north-south, where divides occur longitudinally9,10. These divides are often characterised by a clear separation between breeding and non-breeding seasons and areas, as well as by behavioural mechanisms such as learning and cultural transmission11.

The emergence of migratory divides can have important implications for early speciation processes in birds and other vertebrates12–16 by exposing sympatric populations to different selective pressures, such as different parasites, predator and climate regimes9,12,17. In addition to selection, assortative mating can also contribute to reproductive isolation12,15,18. Efforts to unravel the genetic basis of population differentiation in contrasting migratory phenotypes have primarily identified single-nucleotide polymorphisms (SNPs)19–22. However, with the acquisition of contiguous genomes, large genomic alterations such as structural variants have been found to underlie the genetic architecture of migratory divides. Recent studies in fish and birds highlight the importance of chromosomal inversions23–27, deletions28, and long repetitive-rich regions of transposable elements (TEs)29. These major structural variants can suppress recombination over large genomic regions, facilitating the formation of islands of differentiation even in the presence of ongoing gene flow30,31. However, the conditions under which these genomic islands might expand to generalised genome-wide differentiation are not fully understood32.

In insects, long-range cyclical migrations typically span multiple overlapping generations, with each generation completing only one leg of the migratory cycle, and where individuals rarely learn their routes from experience33. Each moving generation involves vast population numbers shaping complex reticular movements, formed by individuals travelling varying distances34–37. Moreover, because individuals tend to migrate and then reproduce within relatively short lifespans, breeding can take place in any suitable habitat along their migratory routes. Only a few well-known insect species, such as the monarch butterfly, seem to migrate to non-breeding destinations as part of their annual cycle. These dynamics disfavour the evolution of strict philopatry, instead resulting in broad north-south seasonal migratory movements that span extensive geographical ranges34–37. As a result, the formation of longitudinal migratory divides in insects seems unlikely, except for scenarios where major geographic barriers hinder gene flow37. Indeed, no migratory divides supported by genetic evidence has been identified in insects to date. Nevertheless, the possibility of latitudinal migratory divides with a low temporal overlap in the equator exists for species whose range extends to both hemispheres.

In this study, we investigate how living in the Northern or Southern Hemisphere may influence the evolution of insect migratory strategies – a question that remains largely unexplored. Most insights into migratory patterns in animals come from species from the Northern Hemisphere, while the Southern Hemisphere has been relatively neglected and rarely compared mechanistically. Although independent migratory circuits in each hemisphere are observed in some migratory vertebrates, such as certain birds38–41 and whales42–44, the potential role of hemispheric barriers in driving these patterns has not yet been proposed as a unifying hypothesis. Given that butterflies and moths possess magnetoreceptor organs that may aid in navigation45–47, we hypothesise that reversed seasonality, a necessarily opposite response to the magnetic field, and distinct celestial cues in the two hemispheres of Earth may shape the evolution of contrasting migratory strategies. Since environmental rhythmicity displays inverted pulses between hemispheres, populations of migratory insects may adjust their biological clocks, either through genetic changes or phenotypic plasticity, and respond differently to hemisphere-specific seasonal cues, potentially leading to spatial and temporal isolation between populations. We use genomics to address the hypothesis of a migratory divide aligned with hemispheric effects in a highly migratory insect species. The painted lady butterfly, Vanessa cardui, provides an ideal system to examine these dynamics, since its distribution in Africa encompasses the Northern and Southern hemispheres. In both hemispheres, painted ladies are well-known seasonal migrants, often among the most common butterflies in a wide diversity of habitats8. Recent studies have characterised ecological and genetic connectivity between Europe and equatorial Africa35–37,48–51, yet migratory patterns of populations south of the equator remain unknown.

Results
Observational data suggest independent movement dynamics in the two hemispheres
Insects migrate in multigenerational waves, resulting in a latitudinal progression of the species distribution through time. Earlier studies of V. cardui revealed two annual peaks of abundance in equatorial latitudes corresponding to the wet seasons, while only one major demographic peak is observed in the European part of the distribution48,50,52. Our analysis of population latitudinal progression throughout the year, based on 760,973 compiled observations of V. cardui in Europe and Africa (Supplementary Data S1), reveals similar patterns, with successive generations tracking locations with optimal conditions (Fig. 1A). As temperatures drop in the boreal autumn, V. cardui populations migrate from the Palaearctic to Africa, reaching as far south as the equatorial latitudes before migrating northwards again in early spring48,50,51. However, another peak of abundance is observed at equatorial latitudes from May to July (Fig. 1A). These individuals are unlikely to belong to the Northern population that has migrated to Europe during this period. We argue that they are part of a Southern African population expanding northward to avoid the austral winter and the progressive desiccation of subtropical zones (Fig. 1B). While these two populations might rarely overlap in time, they inhabit equatorial latitudes during the two different wet seasons. Overall, observational data suggest that individuals in the Southern Hemisphere constitute an independent migratory circuit.

A chromosomal inversion segregates populations in the two hemispheres
To investigate whether V. cardui individuals in the Southern Hemisphere form an independent migratory population from that found in the Palaearctic-African region, we examined genome-wide variation in 290 specimens collected along the entire latitudinal extent of Africa and Europe (Supplementary Data S2, Fig. 2A) using a reduced representation approach (see Methods). Genetic differentiation (fixation index, FST) was minimal across most of the genome, consistent with patterns observed in other migratory insects53–56. However, a single large and contiguous 9 Mb region in chromosome 8 stood out with a sharp increase in genetic differentiation between northern and southern individuals (Table S1, Fig. 3A). This region, spanning about 60% of chromosome 8, also showed pronounced divergence (dXY) and low diversity (π) (Table S1), suggesting the presence of a chromosomal inversion. A PCA based on this region showed two clusters, each corresponding to a different hemisphere (Fig. 2C). This signal persisted after linkage disequilibrium (LD) pruning (Fig. S3).

Further evidence of population differentiation was provided by a co-ancestry Bayesian clustering analysis (fineRADstructure57), which showed higher levels of co-ancestry within individuals in each hemisphere (Fig. 2B), a pattern not observed when analysing the rest of the genome (Fig. S4). Phylogenetic analyses also support this hemispheric differentiation: while a tree inferred from the inverted region grouped individuals into well-supported clades corresponding to their hemisphere of origin, no phylogenetic structure following latitude arose from a tree inferred from the remainder of the genome (Fig. S5). Remarkably, phylogenetic divergence across the Northern clade, spanning a vast longitudinal range across the Holarctic, is lower than the divergence observed between hemispheres (Figs. 3B, S5), underscoring the pronounced role of the inversion and hemispheric barriers in shaping population structure.

To investigate in greater detail whether this region is an inversion, we analysed read mapping information (BreakDancer58) from a northern individual (reference genome assembly59) and a southern individual using previously published whole-genome data34 (sample 15D327, Namibia). This analysis identified inversion breakpoint coordinates at positions 4,402,570 and 13,379,476, which coincide with the region highlighted by the population genetic summary statistics. Additionally, several smaller inversions (<50 kb) were detected within the larger inverted region (Table S2), potentially reflecting artifacts introduced by mapping uncertainties60,61. However, two nested larger inversions of 3.2 Mb and 1.2 Mb (Table S2) suggest a complex history of structural rearrangements in this genomic region.

Interestingly, the PCA of this region on chromosome 8 revealed that some individuals do not cluster within the northern or the southern genotype groups (Fig. 2C), suggesting that these individuals are heterozygous for the inversion. While northern homozygous individuals formed a cohesive group at one extreme of the PCA, some individuals collected in the south and near the equator were located between this cluster and the southern homozygous type. Heterozygosity in the inverted region of these individuals was significantly higher than for the northern and southern homozygous groups (H=0.056, Kruskal-Wallis χ2(2)=64.15, P-value<0.001, df=2), but not significantly different in the rest of the genome (Kruskal-Wallis χ2(2)=3.66, p-value=0.160, df = 2). Most of these individuals were sampled from regions around equatorial latitudes in East Africa (e.g., Ethiopia, Kenya, Zambia), and outside peak abundance seasons. These findings suggest this area is a contact zone where gene flow can occur between alternative inversion haplotypes.

Chromosomal inversions lead to reduced recombination rates within the inverted region30,62. However, if genetic exchange between inversion variants occurs within an inversion via double crossovers or gene conversion, a U-shaped divergence profile is expected, with stronger divergence near the inversion breakpoints and less pronounced divergence towards the centre25,63,64. Consistent with this prediction, we observed the highest sequence divergence (dXY) between northern and southern populations near the breakpoints (Fig. 3A), supporting marginal gene exchange between migratory ranges.

Altogether, the existence of a single highly divergent genomic region between Northern and Southern Hemispheres, coupled with ecological observations, strongly indicates the presence of a migratory divide between populations of V. cardui butterflies. The lack of population differentiation outside the inversion and the U-shaped divergence within the inversion suggests that gene flow occurs extensively between populations on either side of the divide. This finding highlights the role of chromosomal inversions in maintaining sets of locally adapted alleles through recombination suppression65, acting as a key mechanism leading to local adaptation in highly mobile species66,67.

Recent divergence between hemispheres
We estimated a divergence time for the inversion of 0.55–0.69 mya, although this value is likely underestimated due to gene flow between the inverted and non-inverted haplotypes. In line with a recent split or ongoing gene flow between populations, none of the 32,719 SNPs were fixed between the haplotypes. These findings, combined with the fact that the divergence between V. cardui and its sister species, V. kershawi, is estimated to be much older – around 7 mya68 – suggest that the inversion arose in V. cardui after the split between these two species of Vanessa.

Our analysis based on paired-end read mapping information revealed no chromosomal inversions between V. kershawi and the Northern V. cardui reference genome that match the coordinates and length of the inversion detected with population data between V. cardui of northern and southern origins. Therefore, V. kershawi shares collinearity with the Northern haplotype, supporting the Southern haplotype as the derived form. Under this scenario, the emergence of a rearranged haplotype, likely in southern Africa, may have facilitated the expansion of the ancestral haplotype across the Palaearctic and subsequent diversification in the Nearctic37, an interpretation that aligns with our phylogenomic analysis (Fig. 3B). A southern origin of the inversion is also supported by the asymmetrical distribution of haplotypes: both haplotypes as well as individuals presumably heterozygous for the inversion occur in the South, whereas only the Northern haplotype is found in the North (Fig. 2A). This pattern indicates that the inversion is still segregating in the Southern population. Furthermore, the lower heterozygosity observed in Southern individuals homozygous for the inversion, compared to their Northern counterparts (Fig. 3C), may result from a reduction in genetic diversity during the emergence of the Southern haplotype. Positive selection acting on the derived haplotype could also reinforce this pattern, and the higher heterozygosity in the Northern haplotype (apparently fixed) may be maintained through intra-population recombination.

Functional significance of the inversion polymorphism: role in navigation and mating behaviour
Inversion polymorphisms can affect gene expression and subsequently phenotypes through various mechanisms. Primarily, breakpoints can directly affect fitness by disrupting coding and regulatory sequences or altering their positions in the genome69. Additionally, reduced recombination rates associated with chromosomal inversions facilitate faster accumulation of advantageous allele combinations and thus promote local adaptation, often leading to divergent behavioural phenotypes (e.g., refs. 70–75). Inversions have been linked to differences in migratory phenotypes in fish such as the rainbow trout76 and the Atlantic cod23,25, and birds such as the common quail26 and the willow warbler27,29,77, where an inversion and an expansion of a transposable element underlies a migratory divide in Europe.

Notably, here we found that both breakpoints overlapped with protein-coding genes. The distal breakpoint intersected the last intron of the subunit 2 of the type B receptor of the γ-aminobutyric acid (GABA) gene (GABAB2). GABA is a neurotransmitter involved in learning and memory that plays a key role in the neural network processing skylight information during navigation78. This network, extensively studied in migratory species like the Monarch butterfly6,7 and the desert locust79, integrates signals in the brain’s central complex to form an internal compass necessary for determining heading-direction during flight80–82. The neurons forming this network are GABAergic83–85, suggesting that alterations in GABA-B receptor patterns of expression might impact the interpretation of celestial cues. Interestingly, these neurons also receive circadian signals in fruit flies86. The proximal breakpoint was located in an uncharacterised protein from a leucine-rich repeat (LRR) family (Table S3), pointing at another intriguing candidate for future investigation. The fact that the estimated breakpoints of the inversion intersect with genes that are fully annotated and presumably intact in the Northern reference genome implies that these genes are likely disrupted in the Southern haplotype, providing additional support for the Southern haplotype being the derived form.

While the precise environmental cues triggering butterfly migration are not fully understood, synergistic factors like photoperiod, temperature shifts, and host plant availability likely play crucial roles87–90. To investigate whether the inverted region is rich in genes potentially involved in migratory responses, we first conducted a Gene Ontology (GO) term analysis. The analysis, encompassing the 336 genes within the inverted region, revealed enriched functions related to morphogenesis of locomotion-related tissues, fatty acid metabolism, sensory perception and mating behaviour (Figs. S6, S7).

Secondly, we investigated the association of highly differentiated SNPs within the inverted region with potentially altered gene functions. The inverted region contains 3342 highly differentiated SNPs (FST > 0.25) between southern and northern migratory ranges. Among these, 857 SNPs are located within 48 annotated protein-coding genes of varying degrees of putative impact in protein sequence and function (synonymous and non-synonymous substitutions) (Table S3). Below, we focus on functional roles of the candidate genes within the inverted region. However, we also report 10 genes containing isolated FST outlier SNPs outside the inversion in Table S4 to provide a complete overview and potential follow-up research, as some are associated with neural activity and metabolism. Among the genes containing FST outliers, several are involved in neuroendocrine regulation and behavioural control. This set comprises receptors regulating the main excitatory neuropeptides, such as the Vesicular glutamate transporter 1 and Sodium-dependent dopamine transporter. We also identified hormonal modulators of interest, like the Pituitary homeobox homologue (Ptx1), a transcription factor that activates the promoters of most pituitary hormone-coding genes91, the Juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme in the biosynthesis of juvenile hormone and repeatedly pinpointed by insect migration studies7,92–94 including V. cardui89,90, and the Pyrokinin-1 receptor (PK1R) of the diapause hormone, involved in pheromone biosynthesis95. The acyl-CoA desaturase Delta-9 gene, which produces the main component of the Spodoptera littoralis moth’s sex pheromone96, was also found in this gene set. In fact, several highly differentiated variants were identified in genes related to mating. Of particular interest are the dsf and siwi genes, which are encoding the dissatisfaction protein (DSF) affecting courtship behaviour, successful copulation and fertile egg-laying in Drosophila97, and the Siwi protein, which plays an essential role during spermatogenesis/oogenesis by repressing the mobility of transposable elements98. These genes are promising candidates for investigating premating mechanisms that may contribute to reproductive isolation67,99,100 and assortative mating between hemispheres.

We also found highly differentiated variants in genes related to energy metabolism, such as the main triglyceride-lipase in insect fat bodies (Phospholipase A1101), and the facilitated trehalose transporter (Tret1). Trehalose is the primary sugar in insect haemolymph, and its differential expression has been linked to migratory behaviour90,102,103. Additionally, we detected highly differentiated SNPs in some ABC transporter genes and Argonaute2 (AGO2) in the 3’ and 5’ UTR, respectively, which are associated with xenobiotic resistance. The protein AGO2 is crucial for antiviral defence by targeting and degrading viral RNAs104,105, and variation on its expression could contribute to differing innate immunity signatures across hemispheres, potentially due to variation in microbial community compositions.

Regulatory regions play a critical role in the evolution of novel traits, with most trait-associated loci falling in non-coding regions106. We found 1203 highly differentiated SNPs (FST > 0.25) in the vicinity of 120 genes (5kb flanking regions), accounting for 36% of the total variants identified. However, our ability to detect genetic differentiation from RAD sequenced variants is limited, as the genes covered in our RAD sequences represent only 21% of the genes encoded within the 9 Mb inverted region. Comprehensive population analyses using whole-genome sequencing (WGS) data could elucidate the full extent of the diverged gene sequences.

Hemispheric migratory divides: a speciation driver in migratory species?
Migratory divides have been proposed as a driving force in speciation by exposing sympatric or parapatric populations to divergent ecological conditions or by limiting genetic exchange through assortative mating or selection against hybridization12–18,21,107. These divides represent boundaries between adjoined populations with locally adapted migratory traits, such as migration direction or length, often leading to geographic segregation and genetic differentiation. While this phenomenon is well-documented in many vertebrate species (despite its genetic basis being known only in a handful of species), evidence for similar patterns in migratory insects has remained elusive. Indeed, no genetic differentiation has been found between differentially migrating insect populations such as those of monarch butterflies108,109 (reviewed in 110), the Bogong moth111 or the fall armyworm54.

However, our study provides evidence from Vanessa cardui suggesting that migratory divides may also play a significant role in insect speciation, despite the substantial differences between vertebrate and insect migration dynamics34. A key distinction in our findings is the identification of a latitudinal migratory divide spanning two hemispheres – a major departure from previously described longitudinal migratory divides that often arose from secondary contact following post-glacial colonisation from separate glacial refugia9. The biogeographic distributions of many migratory butterflies appear restricted to either hemisphere, with some species pairs showing evidence of speciation across hemispheric divides. An example is found in Danaus butterflies: D. plexippus (the monarch) is native to Central and North America, while its sister species, D. erippus, is confined to South America, engaging in extensive parallel migrations. Similarly, in Vanessa butterflies, the sister species V. carye and V. annabella complete their migratory cycles exclusively in South and North America, respectively, while V. kershawi, the painted lady sister species, migrates within Australia and New Zealand, where V. cardui is absent. While these cases may represent instances involving complete speciation, other systems may shed light on earlier stages of the speciation continuum. Several migratory butterfly species have distributional ranges spanning both hemispheres, such as Ascia monuste, Aphrissa statira, Phoebis sennae in the Americas, and Belenois aurota and Catopsilia florella in the Afrotropical region. Although the migratory cycles and phylogeographic structures of most migratory insect species are still to be fully characterised, we hypothesise that gene flow across such extensive migratory ranges might be constrained by reversed seasonality and differential navigational cues in each hemisphere, eventually leading to adaptation to hemisphere-specific biological rhythms and cues.

This pattern likely extends beyond insects. For example, long-distance bird migrants (excluding shorebirds) breeding in the Northern Hemisphere and wintering in the Southern Hemisphere are proportionally a minority, and few Southern Hemisphere breeders are found to migrate north of the Equator38–41. Similarly, populations of humpback, fin whales, and several marine migratory fish species circulate almost exclusively within the Northern or Southern hemisphere, and rarely interact42–44.

These eco-evolutionary patterns suggest that hemispheric divides could significantly influence the speciation of widespread migratory insect species. The existence of hemispheric divides and their segregating genetic diversity should be investigated across animal migratory lineages as a potentially overlooked pattern for speciation.

Methods
Observational and field data
Observational occurrences of V. cardui used to infer phenological patterns of presence and absence by latitude were retrieved from (1) the Global Biodiversity Information Facility (GBIF112, https://www.gbif.org/es/), (2) the African butterfly and moth mapping project (LepiMAP113, http://vmus.adu.org.za/), both accessed the 23rd of May 2023, and (3) the authors’ own field expeditions from 2006 to 2023 (e.g. ref. 50), including 802 observations from Africa. We excluded the downloaded observations recorded on the 1st of January, as this date appears to be disproportionately used and is likely a placeholder for unknown collection dates within a given year. We retrieved 69,134 observations in total throughout Europe and Africa, dating from 1800 to 2023 (Supplementary Data S1). Monthly data were compiled, and relative abundances for established latitudinal divisions were plotted using ggplot2114 R package (Fig. 1).

We calculated the maximum and minimum yearly photoperiod relative to each latitude using the R packages geosphere115 and terra116. Photoperiod amplitude was estimated as the subtraction of the maximum and minimum yearly day length. The temperature range was estimated as the subtraction of the lowest monthly average temperature from the highest monthly average temperature within each year, where monthly temperature averages were computed for a series of 20 years (2001–2021) using data from the Climate Research Unit (CRU v4.06) database117. Analyses were conducted using R v4.1.2, and R code is provided at the GitHub repository https://github.com/GTlabIBB/MigratoryDivide118. Both photoperiod and temperature amplitudes were projected to a map using QGIS v3.34.4119 to generate Fig. 1A.

Sampling
Painted ladies (V. cardui) are among the world’s most widespread butterfly species. We gathered 300 individuals across 38 countries in Europe and Africa (Fig. 1; Supplementary Data S2), as well as five individuals of V. kershawi, its sister species, which inhabits Australia and New Zealand (Supplementary Data S2). These specimens are deposited in the collections of the Botanical Institute of Barcelona (IBB, CSIC, Spain), the Institute of Evolutionary Biology (IBE, CSIC-Universitat Pompeu Fabra, Spain), and the Museum of Comparative Zoology (Harvard University, USA).

Sequencing and preprocessing of ddRAD data
For population genetic analysis, we prepared ddRAD genomic libraries for the 304 collected samples (Supplementary Data S2) following the protocol of Peterson et al.120. We used EcoRI and BfaI restriction enzymes and size selection of 300 bp using a Pippin Prep instrument and 2% agarose cassettes (Sage Science, Beverly, MA, USA). Libraries were sequenced in 150 bp paired-end reads on Illumina HiSeq 2500 at the Harvard University Bauer Core Facility. Sequencing reads are deposited in the ENA (European Nucleotide Archive) database under project accession no. PRJEB80315. A total of 287,916,481 reads were generated, which were demultiplexed and assembled using ipyrad v0.9.8121 by mapping on the Vanessa cardui chromosome-level reference genome59 (GCA_905220365.1). Adapters were filtered using the cutadapt software integrated into the filter_adapters parameter of ipyrad. The clustering threshold was set to 0.85, resulting in a total of 188,408 loci across all 300 individuals. We used ipyrad default filtering parameters except for the minimum number of samples per locus, which was set to 100, i.e., one third of the samples (min_samples_locus parameter). After applying the filters, the number of assembled loci was 10,672. For downstream analysis, we subsequently filtered by variant missing data in VCFtools v0.1.16122, keeping SNPs that were present in at least 80% of the individuals (--max-missing parameter) and by minimum allele count, keeping alleles present in at least 2 individuals (--mac parameter). Last, in order to keep high-quality samples, we filtered out 3 individuals for which less than 40% of the total loci were recovered using the prop_typed_ind function in adegenet v2.1.5 R package123, and removed 7 individuals that shared particularly high levels of coancestry according to fineRADstructure57, and thus were potentially related. These samples were indeed collected as larvae in the same locality, further reinforcing this inference. These filters resulted in a dataset, named dataset1, comprising 290 samples and 32,719 SNPs. For local analysis of the inverted region on chromosome 8, we selected 976 SNPs located within this region using BCFtools v1.20124.

We constructed a second dataset for fineRADstructure analysis, dataset2, using a reduced set of high quality samples. We selected the first 30 samples from each hemisphere with the lowest levels of missing data. We ran the ipyrad reference-aligned pipeline with default filtering parameters, but the minimum number of samples per locus was set to 30 (i.e., half of the dataset). Additionally, we filtered for missing data with VCFtools, retaining SNPs that were present in at least 80% of the individuals (--max-missing parameter). Initially, we recovered 109,614 loci and 546,458 SNPs across 60 individuals; after filtering, the dataset was reduced to 13,571 loci and 169,590 SNPs. For local analysis on chromosome 8, we selected 2855 SNPs.

A third dataset, named dataset3, was generated for phylogenetic inference using the 60 samples from dataset2, along with five additional samples from the sister species Vanessa kershawi as outgroup. The ipyrad assembly parameters and filtering steps were identical to those used for dataset2. We recovered 117,546 loci across 65 samples, which were reduced to 12,973 loci and 569,090 SNPs after filtering steps. Summary statistics resulting from all ipyrad assembly steps for dataset1, 2 and 3 are detailed in Supplementary Data S3.

Genome-wide genetic differentiation analysis
To assess population structure, we first performed a PCA analysis using the adegenet v2.1.5 R package123 on all samples (dataset1, 290 samples, Supplementary Data S3) across three genomic partitions: (i) the whole genome (Fig. S1), (ii) the inverted region on chromosome 8 (Fig. 2B), and (iii) the whole genome excluding the inverted region (Fig. S2). Additional PCAs were performed in LD-pruned versions of these datasets to assess for potential linkage effects, using SNP pairwise comparisons in PLINK with r2 < 0.2 and r2 < 0.05 (Fig. S3).

We then used fineRADstructure to infer the number of genetic clusters and recent shared ancestry of individuals on each side of the migratory divide in dataset2, both in the entire genome (60 samples, 169,590 SNPs, Fig. S1) and in the inverted region on chromosome 8 (Fig. 2A). In both analyses, sex chromosomes were excluded. Additionally, repetitive regions were filtered out using the annotation from Shipilina et al.125 with BEDTools v2.29.2126. No LD filtering was applied for fineRADstructure analyses, as the method specifically uses haplotype linkage information from RAD data to infer recent ancestry57.

We next explored patterns of genetic differentiation by calculating window-based estimates of FST, π and dXY along the genome for dataset2. To account for the fragmented coverage of RAD loci we used Pixy v1.2.5.beta1127, which handles missing data by excluding regions lacking mapped reads, thus excluding regions without RAD loci from genome-wide calculations. In order to accommodate this strategy, we generated an All Sites VCF file from the dataset2 alignments obtained from ipyrad. This variant calling step was implemented in SAMtools mpileup followed by BCFtools call. Additionally, we filtered the resulting file for variant missing data in VCFtools, retaining SNPs present in at least 80% of the individuals (--max-missing parameter). The resulting All Sites VCF contained 1,136,244 variant sites and 2,788,714 invariant sites. Window size was set to 100 bp for FST to enable dot plot visualisation, while 1 Mb was used for π and dXY for curve visualisation, for which no smoothing algorithm was used. Results were plotted using ggplot2.

Sequencing and preprocessing of WGS data
Short read data were used for phylogenetic inference and detection of inversion breakpoints. We used five V. cardui samples from a previous study34, collected in Ethiopia (14U392; 9°23′54.096′′ N, 38°49′25.323′′ E;), Namibia (15D327; 17°52′16.086′′ N, 19°24′22.611′′ E), Nepal (07F575; 28°25′0.635′′ N, 83°49′2.114′′ E), California, USA (15A205; 38°34′30.414′′ N, 121°34′38.117′′ E), and Spain (15A710; 40°15′29.16′′ N, 1°36′20.879′′ E). A Vanessa kershawi specimen, from Australia (AAM97U335; 34°19′30.63′′ S, 117°52′36.735′′ E), was sequenced for this study. A 350 bp insert paired-end library was prepared following the Illumina TruSeq DNA PCR-Free protocol. The library was sequenced at the Swedish National Genomics Infrastructure (NGI) facilities at 150 bp from both ends using a NovaSeq 6000 S4 sequencing platform, producing 140,660,532 reads (Table S5). Sequences are available in the ENA (European Nucleotide Archive) database under project accession no. PRJEB80315.

Reads were mapped against the reference V. cardui genome59 (GCA_905220365.1) with BWA v0.7.12128, except for the V. kershawi specimen, where reads were mapped using Stampy v1.0.32129 and using an estimated substitution rate of 0.0406 to account for ~7 my of divergence with V. cardui68. We obtained an average of 194 million mapped reads per sample (Table S5), which were sorted using SAMtools. The consensus sequences were obtained using SAMtools mpileup followed by BCFtools Call. The final fasta files were obtained with vcfutils vcf2fq122 command using a minimum coverage of 6x. The arthropod BUSCO v3.0.2130 analysis of completeness performed in the resulting assemblies using gvolante131 yielded an average of 98.63% completeness.

Identification of inversion breakpoints
To identify the inversion breakpoints, we used paired-end read mapping information from the southern V. cardui individual (Namibia) reads mapped to the northern V. cardui reference genome59 (GCA_905220365.1). The resulting alignment had a mean mapping coverage of 32.34x and mapping quality of 71.3 (Table S5), as calculated with Qualimap v2.2.1132. Structural variant detection was performed using BreakDancerMax, a pipeline to identify breakpoints implemented in the software BreakDancer58. BreakDancer identifies anomalous read pairs in order to predict structural variants, and estimates a confidence score for each variant based on a Poisson model that takes into consideration the number of supporting anomalous read-pairs, the size of the anchoring regions, and the coverage of the genome58. With the highest probability score, BreakDancer identified 14 paired-end reads with abnormally large insert sizes and inverted orientation, pinpointing the inversion breakpoints at coordinates 4,402,570 and 13,379,476 (Table S2). We used the established coordinates to extract the inverted region for all analyses.

Additionally, to investigate the synteny between V. cardui and its sister species, Vanessa kershawi on chromosome 8, we repeated the same procedure using WGS data from the sequenced V. kershawi specimen. The alignment had a mean coverage of 40.24x and a mean mapping quality of 43.61 (Table S5). No chromosomal inversions corresponding to the identified breakpoints with population data in V. cardui (positions 4,402,570–13,379,476) were detected in V. kershawi using read mapping information.

Divergence time estimation of the inverted region
We calculated the divergence time between the inverted and non-inverted haplotypes using the estimated absolute divergence (dXY) from Pixy. Absolute divergence was not consistent across the inversion length. Despite reduced crossover frequency, regions within large inversions can still pair in heterozygotes, allowing for gene conversion63,64. Stronger divergence near the inversion breakpoints and less pronounced divergence toward their centres are expected, showing a U-shaped profile25,26. Therefore, we focused on the first and last Mb of the inversion where recombination is lower, and which therefore provide the most reliable estimate of accumulated divergence since the origin of the inversion. We averaged the values obtained from the first and last Mb. Then, following the approach of Sánchez-Doñoso et al.26, we converted this divergence estimate to an estimate of divergence time using the equation133:

T = dXY / 2 * μ * g

Where T is the divergence time in years, dXY is the absolute divergence, μ is the mutation rate in mutations per site per generation, and g is the number of generations per year. We used the estimated mutation rate for Heliconius melpomene134 (2.9 × 10−9), and considered a range of 7 to 10 generations per year50. Note that the divergence equation assumes a molecular clock, which implies neutral sequence divergence and a constant mutation rate. However, these regions are likely not neutral and might have complex demographic histories, so the time of divergence could be underestimated. Thus, it should be interpreted as an approximate indicator of the inversion’s origin and the onset of independent evolutionary trajectories between the arrangements.

Heterozygosity estimates
Heterozygosity was estimated both for the inverted region on chromosome 8 and along the genome (excluding the inverted region) in dataset1. We calculated heterozygosity as the number of variant sites over the total number of genotyped sites for each sample. We determined the North group as the specimens clustered in the negative values of PC1, the South group as the specimens clustered at the other side of the PC1 with values > 6, and the specimens scattered between these two thresholds were considered as potential heterozygotes. The obtained heterozygosity estimates did not follow a normal Gaussian distribution (Shapiro-Wilk normality test; inverted region: W=0.98641, p-value=0.008075 and excluding inverted region: W=0.98409, p-value=0.002754). Therefore, we statistically searched for differences in heterozygosity between North, South, and potential heterozygous groups within each region using Kruskal-Wallis one-way analysis of variance.

Phylogenetic analyses
We investigated the phylogenetic history of the inverted region using whole-genome data from 5 specimens of Vanessa cardui across its worldwide distribution and one Vanessa kershawi specimen as an outgroup. The V. cardui and V. kershawi reference-based assemblies were aligned using MUSCLE135, implemented in Geneious Prime 2023.0.4136 (https://www.geneious.com). Gblocks v0.91b137 was then applied on the multiple sequence alignment to remove potentially ambiguously aligned regions using default parameters, generating concatenated alignments of 4,319,737 bp for the inverted region and 330,406,868 bp for the rest of the genome. Phylogenetic relationships were then inferred through a maximum likelihood (ML) analysis using RAxML-NG v1.1.0138, with a GTR+I model of nucleotide evolution. Nodal support was evaluated by 200 bootstrap iterations using the transfer bootstrap expectation (TBE).

Phylogenetic relationships were also inferred for RAD data using dataset3. A total of 420 RAD loci alignments were obtained for the inverted region and 12,550 for the rest of the genome using the ipyrad assembly pipeline. We used Gblocks with default parameters in each alignment to discard highly ambiguously aligned regions. Sequences with more than 90% of unmapped or uncalled bases (“Ns”) were removed from the alignments using cutadapt v5.0139 --max-n. These two steps resulted in a total of 397 and 11,935 filtered RAD loci alignments for the inverted region and for the rest of the genome, which were concatenated into 88,704 bp and 2,677,821 bp alignments, respectively. We inferred phylogenetic trees with IQ-TREE v2.0140, using ModelFinder to select the best-fit evolutionary model for each alignment based on the Bayesian Information Criterion (BIC). The resulting phylogenetic trees were plotted using the ggtree141 R package.

Functional analysis
Enrichment of functional categories in the genes located within the inverted region was analysed using the Bioconductor package topGO v2.44.0142. The enrichment test was performed with Fisher’s exact test using the default algorithm (“weight01”), which accounts for the hierarchical structure of the GO-terms. The annotated geneset from Shipilina et al.125 was used as the functional database. We adjusted the p-values of the resulting tests using the Benjamini-Hochberg method143 to correct for multiple testing.

We explored whether SNPs showing the highest differentiation between southern and northern migratory ranges were located within protein-coding genes. We selected SNPs with FST > 0.25 (93.8th percentile) in dataset 2 and predicted effect strength and position in relation to known genes using SnpEff v5.2c144. We created the custom gene database in SnpEff based on the annotation version from Lohse et al.59. Information regarding the known biological functions of these proteins and functional effects is presented in Table S3 (inside the inversion) and Table S4 (outside the inversion).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Demultiplexed and adapter-trimmed RAD-sequencing reads, and whole genome sequencing reads generated in this study have been deposited in the European Nucleotide Archive under project accession number PRJEB80315. All accession numbers for samples used in this study are listed in Supplementary Data 2, including newly generated sequences and those from García-Berro et al.34 and Suchan et al.37 Data underlying Figs. 1–3 are available in Supplementary Data 1 and in the OSF repository at the following URL: https://osf.io/6u32k/ (https://doi.org/10.17605/OSF.IO/6U32K).

Code availability
Scripts used to process the data are available at: https://github.com/GTlabIBB/MigratoryDivide (https://doi.org/10.5281/zenodo.17113173).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a novel and well written manuscript that I would like to see published. In places, the manuscript needs clarifications\nby the authors and there are some conflicting issues with reported SNP’s for the various analyses and samples. Overall, it is\na great study that will attract a broad readership.\nDetailed comments\nLine 29. “insect migratory divides more likely to form latitudinally”. I would say that they are less likely to form longitudinally\nwithin hemispheres than in birds (for reasons you bring up), but latitudinally between hemispheres, I’m not sure it’s more\nlikely than in birds.\nLine 58. The citation to Moller et al is a strange one for the claim “migratory movements predominantly in a sorth-south\ndirection”. Find a better one.\nLine 83. Bring up already here that there are a few examples from birds (you cite them in the discussion) that migrates like\nthe here proposed lady butterfly, i.e. forming a latitudinal migratory divide between the hemispheres.\nLines 130-137 & Table S2. The identification of the inversion needs to be better explained. One way of doing this is to\nexpand the legend of Table S2 and help the reader what to look for. The first column is redundant (looks to be the same\nchromosome for all entries). Why should we ignore all entries except the last? Mark the two entries proposing inversions\nwithin inversions. How can Size (bp) take negative values in 5 cases? Explain in simple text how you interpret the results\nand may also include a brief summary of it in the main text.\nLines 181-182. Without a reference genome of a close relative to the lady butterfly I agree it will be difficult to separate which\nof the haplotypes that are ancestral versus derived. However you say elsewhere in the manuscript that the breakpoints\nintersect two genes (GABA on one side and an uncharacterized protein on the other). My guess would be that the haplotype\nthat breaks (the function?) these would correspond to the inverted (derived) haplotype.\nLine 221 “GABAb” Why “b”, GABA reported above?\nLine 287. I have not read the reference to the Bogong moth but the title of the paper suggests that they do report on genetic\ndifferences related to migration direction. Please check\nLine 315. “bird migrants breeding in the Northern Hemisphere are relative rare to winter in the Southern Hemisphere”. I\nwould not say rare. I took a quick look in a field guide of birds of Southern Africa and found > 3 dozens of species\n(shorebirds excluded) that are regular winter visitors from the Palearctic.\nLine 333. Not at all clear why records of 1st January were filtered?\nLines 368 & 377. That you have three times as many SNP’s as loci suggests that many SNP’s are physically linked. Is that a\nproblem for the analyses?\nLines 388-389. Same potential problem as above but more extreme, 50 times more SNP’s than loci.\nFig 1A. Why two occurrence peaks (March-May, Sept-Nov) in southern Africa? Worth a comment?\nFig 2A. It is difficult to see the “potential heterozygotes” on the map. Try other colors.\nFig 2 legend. On line 532 you say that the analysis is based on 546,458 SNPs belonging to the region with high Fst on chr\n8. But according to the methods (line 389) that’s the full data set of dataset 2 (whole genome)? And the same inconsistency\nseems to be for panel C which report all SNPs for dataset 1 but says it only include the region on chr 8.\nFig 3. Line 543. The reported number of SNP’s for this analysis does not correspond to dataset 2.\nSuppl Fig S1 & S2. Number of SNP’s correspond to the complete dataset 1 but says it only analyses those in the divergent\nregion of chr 8\nSuppl Fig S3. Maybe consider to show the origin of the individuals by different colors for Europe and Africa.", "reviewer_2": "I really enjoyed reading the manuscript and discovering this peculiar behavioural evolution. The reported evidences are\nrobust and the conclusions drawn are generally supported but I recommend a number of corrections before publication. In\nparticular, more details on the origin of the inversions and differential evolution of migration in the Vanessa clade should be\nprovided. It would also be useful to compare with the evolution of divergent migration behaviour in other clades where\nmigration occur within the same generation, and discussing the potential consequences for speciation.\nMain comments\nLine 28 and throughout the manuscript: I am not comfortable with the term ‘strategy’. It conveys the misleading idea that\nevolution could have an aim, rather than that it occurs through a series of random mutations, selected or not. Perhaps even\nmore importantly here, understanding how a divergent migration behaviour may arise is not straightforward: can a single\nmutational event trigger such a divergent evolution? Or does it request a series of genetic changes, and in that case, what\nwas the first event and how much does the first change facilitate the evolution of the following ones? This leads to important\nquestion on the expected genetic architecture underlying the migration syndromes: how much are we expecting a\nsupergene architecture? How do inversions may facilitate supergene evolution? I would thus recommend avoiding using\nthe term ‘strategy’ throughout the manuscript and clarifying the expectations in term of genetic architecture underlying such\ndivergence in migration syndromes.\nFor instance, line 83, you seem to hypothesize that the reversed seasonality may have evolved in response to the evolution\nof an opposite behaviour triggered by the magnetic field cue. This needs to be clearly justified.\nLine 46-47 : In line with my previous comment, more details on the genetic architecture controlling variation in migration\nbehaviours in different clades are necessary here.\nLine 52: The term ‘optimizing’ is also misleading : several migratory routes can allow survival and reproduction, and\nselection is likely to favour the best ones among the different genetic variants within the population, but not necessary the\noptimal ones. I think here it would be useful to clearly pinpoint the traits, linked to migration, targeted by selection, e.g. genes\ninvolved in the perception of different environmental cues (photoperiodic cues triggering departure, magnetic cues guiding\ndirection…).\nLine 61 : One of the major difference between butterflies as compared to birds, fishes or cetaceae relies on the multiple\ngenerations requested to complete the migration cycle. This has probably strong consequences on the speciation process,\nbecause in one out of two generations, reproduction occurs in well-separated areas (Northern vs. Southern populations of\nV. cardui) with zero possibility of gene flow.\nLine 62-63 : This notion of reticular movements and consequences for the lack of philopatry is unclear to me. Independently\nfrom the generational effect, there is individual variations on the migration behaviour in birds and fishes as well. The\nevolution of philopatry could be driven by individual experiences that are unlikely to occur in butterflies because different\nindividuals take the north-South vs. South-North journey. This should be made clearer.\nLine 28-29 and 66: Could you precise the proportion of latitudinal vs. Longitudinal divides documents in different clades ?\nWhy do you expect latitudinal divide to be more prevalent in insects ?\nLine 84-85 : evidence for the use of magnetic field and celestial cues in Lepidoptera (e.g. moth) should be detailed here (e.g.\nDreyer, D., Frost, B., Mouritsen, H., Günther, A., Green, K., Whitehouse, M., ... & Warrant, E. (2018). The Earth’s magnetic\nfield and visual landmarks steer migratory flight behavior in the nocturnal Australian Bogong moth. Current Biology, 28(13),\n2160-2166.)\nLine 85-86 : the term ‘adjust’ is unclear : behavioural adjustment could be plastic. You need to clarify whether you expect (1)\nan evolution of behaviour triggered by genetic change, selected in different evolutionary environment or (2) an individual\nbehavioural variation triggered by the environment encountered by the individual itself.\nLine 120-121: are the collection dates known for the different samples? I could not find it in the Sup. Mat 1, it would be nice\nalso to explore the genetic variations associated with the sampling dates, especially in the equatorial regions.\nLine 146-147 : Is there any evidence that the Southern haplotype is dominant over the Northern one ? This would explain\nthe heterozygotes observed within the Southern population? Since the newly emerged haplotype is expected to be\ndominant over the ancestral one, this could also provide a hypothesis on the ancestral vs. derived state (see Llaurens, V.,\nWhibley, A., & Joron, M. (2017). Genetic architecture and balancing selection: the life and death of differentiated variants.\nMolecular ecology, 26(9), 2430-2448.)\nLine 172 : What is the ancestral gene order ? Does the gene order observed in sister species of V. cardui (or in other\nbutterfly genomes) fit with the Northern or the Southern haplotype?\nLine 176-177 : I am not entirely convinced by the argument, introgressions have been found to happen between highly\ndivergent species : even if the hybridization events are rare between divergent species, if selection on the introgressed\nregion is very high, this might allow the introgression to stay within the genome (see Edelman, N. B., & Mallet, J. (2021).\nPrevalence and adaptive impact of introgression. Annual Review of Genetics, 55(1), 265-283.)\nLine 182 : how much is genome-wide pi smaller in the Northern vs. Southern populations ? Could you provide population\ngenomics arguments for this bottleneck using genomic variations outside the inverted region ?\nLine 196-197 : This section in unclear, here you refer to as the genome-wide heterozygosity ? Could you provide a statistical\ncomparison of genome-wide heterozygosity between the three genotypes (fig. 3C, left-panel). Given the low difference in\ngenome wide heterozygosity between the Southern vs. Northern populations, I am not convinced that the difference in\neffective population sizes can explain the difference in heterozygosity observed in the inverted regions in the Northern vs.\nSouthern populations.\nLine 252-257 : I would discard this highly speculative section and more generally, I would not focus too much on the\npotential candidate genes at this point: as mentioned by the authors, RAD-sequencing data do not allow to precisely\ncharacterize the whole region (272-274). This part of the discussion could thus be reduced.\nLine 297-302 : It would be nice to have a phylogeny of the different Vanessa species with information on (1) the gene order\n(when available) in the region where the inversion is observed in V. cardui and (2) the migration behaviour. The within\nsouthern-hemisphere migration of V. kershawi could indeed potentially point at an ancestral Southern migration behaviour?\nLine 300: did you mean ‘WITHin South and North America respectively’ ?\nLine 3014-319 : One of the main difference between the V. cardui butterflies and the cited shorebird and whale examples is\nthe single vs. multiple generation patterns of migration which is likely to have consequences on population divergence and\nspeciation, as mentioned in one of my previous comments. I think it is worth discussing this specific point.\nFigure 3B is very nice, but here we would like to know what is the gene order in V. kershawi in the inverted region? Could\nyou also provide trees based on your RAD-sequencing data within and outside the inverted regions for all your samples to\nvisualize how much the evolutionary history of this specific region departs from the genome wide history ?\nMinor comments :\nLine 29 : I am not sure longitudinally is that easy to understand, I would rather use an East/West vs. North/West terminology\nLine 61 : replace ‘one leg of the migratory cycle’ by ‘one step of the migratory cycle’\nLine 101 : ‘species distribution THROUGH TIME’\nLine 106 : Again, evolution has no aim, you may replace ‘tracking seasonality in search for optimal conditions’ by ‘exhibiting\ndifferent seasonal pattern favouring their occurrence in suitable habitats’.\nLine 221 : replace ‘transcripts’ by ‘patterns of expression’\nLine 238 : replace ‘protein behaviour’ by ‘protein sequence’", "reviewer_3": "Garcia-Berro et al 2024, Nat Comm\nThis is a wonderful manuscript that shows genetic evidence for a migratory divide in painted lady butterflies, and I greatly\nenjoyed reading it. The premise of this study is that migratory divides are pretty common in vertebrates, but that they have\nnot been found in insects, despite extensive studies looking for them. As the authors point out, however, many of these\nstudies have been done on insect populations that are separated longitudinally, with predominantly western and eastern\npopulations. While such populations may experience different environmental conditions, they experience similar seasonality\nand magnetic fields, two factors that underlie many migratory behaviors. In the painted lady however, populations are also\ndivided latitudinally, with populations migrating either in the northern or southern hemisphere. Analyzing a massive data set\non abundance, the authors convincingly show temporal separation in the abundance of northern and southern painted\nladies around the equator. Using a genome-wide RADseq analysis, they then demonstrate significant genetic differentiation\nbetween these populations, and even find a chromosomal inversion that is tied to this north-south separation. Within this\ninversion, they also discover many genes that are known to be involved in insect migration, navigation and mating behavior,\nthereby providing potential mechanistic explanations for differences in migration and reproductive isolation. By showing this\nhemispheric divide, the authors provide a novel way of thinking of migratory divides, with applications to many different\nspecies (including mammals and birds) and strengthening the hypothesized mechanisms of navigation and migration based\non seasonality and magnetism. I would personally add polarized light and solar azimuths as well, which are also likely to\nvary based on latitude.\nI only have a few comments.\n1. Before diving into the chromosomal inversion, I feel that the authors could emphasize the genome-wide genetic\ndifferentiation more. The data in Fig. 3A clearly show overall levels of FST and dxy much higher than those found in other\ninsects where similar studies have been done. Moreover, there are quite a few locations in the genome that show high FST\nvalues of 0.10 and higher, and it may be worth investigating if these chromosomal regions include other genes that could\nprovide insight into navigation, reproductive isolation etc.\n2. I was wondering why a demographic analysis was not carried out. When studying the phylogenetic trees in Fig. 3B, it is\nindeed tempting to conclude directionality of evolution, with northern painted ladies deriving from southern painted ladies,\nbut the tree is rather limited and the observed patterns on the basis of five samples may be driven by specific samples. It\nwould have been helpful to add additional southern samples to confirm that they cluster with the Namibian sample rather\nthan with the northern samples.\n3. Also with respect to the phylogenetic tree, I think it would be helpful to emphasize that the northern samples clustering\ntogether derive from a very wide longitudinal gradient, from the USA all the way to Nepal. That in itself provides an internal\ncomparison of the east-west lack of divide as observed in many other systems, while emphasizing the north-south divide as\nobserved here."}, "author_response": "[RESPONSE]: Thank you very much for reviewing our manuscript and for your insightful\ncomments. Please, see point by point comments below.\nDetailed comments\nLine 29. “insect migratory divides more likely to form latitudinally”. I would say that they are less\nlikely to form longitudinally within hemispheres than in birds (for reasons you bring up), but\nlatitudinally between hemispheres, I’m not sure it’s more likely than in birds.\n[RESPONSE]: We have rewritten this sentence, which now reads: “We hypothesise that insect\nmigratory divides are less likely to emerge longitudinally, but they may exist latitudinally, driven\nby hemisphere-specific sensory adaptations that lead to spatial and temporal isolation”\nLine 58. The citation to Moller et al is a strange one for the claim “migratory movements\npredominantly in a sorth-south direction”. Find a better one.\n[RESPONSE]: We have changed this reference.\nLine 83. Bring up already here that there are a few examples from birds (you cite them in the\ndiscussion) that migrates like the here proposed lady butterfly, i.e. forming a latitudinal migratory\ndivide between the hemispheres.\n[RESPONSE]: We have now added the following sentence: “Although independent migratory\ncircuits at each hemisphere are observed in some migratory vertebrates, such as certain birds26-\n29 and whales30-32, the potential role of hemispheric barriers in driving these patterns has not yet\nbeen proposed as a unifying hypothesis”.\nLines 130-137 & Table S2. The identification of the inversion needs to be better explained. One\nway of doing this is to expand the legend of Table S2 and help the reader what to look for. The\nfirst column is redundant (looks to be the same chromosome for all entries). Why should we\nignore all entries except the last? Mark the two entries proposing inversions within inversions.\nHow can Size (bp) take negative values in 5 cases? Explain in simple text how you interpret the\nresults and may also include a brief summary of it in the main text.\n[RESPONSE]: Thank you for these thoughtful suggestions. We focused the discussion on the\nlarger inversions because their size makes them more likely to have functional consequences\ndue to the suppression of recombination across multiple genes. In contrast, small inversions are\ncommon and typically have a limited impact on population differentiation, particularly in V.\ncardui, a species characterized by high levels of recombination (Shipilina et al. 2022).\nAdditionally, inversions smaller than 10kb identified by BreakDancer and similar pipelines based\non paired-end mapping information are more prone to be false positives, often arising from\nmapping ambiguities or low-complexity regions (Alkan et al., 2011, Chen et al., 2019, Kosugi et\nal., 2019). We have now expanded on these points in the main text (lines 153-156).\nRegarding the negative values reported in size (bp), they do not have biological meaning and\nare a product of Breakdancer’s output (when read 1 maps to a higher coordinate than read 2).\nWe now report the absolute values for the inversion sizes to avoid confusion.\nWe have also updated Table S2 to make it more intuitive and informative, including the following\nchanges: 1) the first column (chromosome ID) has been removed to avoid redundancy; 2) we\nhave added a more detailed explanatory text to the legend to guide the reader to interpret the\ntable; 3) we have highlighted the two larger inversions nested within the primary inversion,\nwhich suggest a complex history of structural rearrangements.\nLines 181-182. Without a reference genome of a close relative to the lady butterfly I agree it will\nbe difficult to separate which of the haplotypes that are ancestral versus derived. However you\nsay elsewhere in the manuscript that the breakpoints intersect two genes (GABA on one side\nand an uncharacterized protein on the other). My guess would be that the haplotype that breaks\n(the function?) these would correspond to the inverted (derived) haplotype.\n[RESPONSE]: This is a very good point and we agree that this rationale strengthens the\ninference that the Northern haplotype is ancestral. Since the protein-coding genes at the\nbreakpoints (GABA receptor and an uncharacterized protein) are intact in the reference genome\nrepresenting the Northern haplotype, the Southern haplotype—where these genes are disrupted\n—is likely derived. We have expanded on this point in the discussion (lines 240-244) to\nemphasize its significance.\nLine 221 “GABAb” Why “b”, GABA reported above?\n[RESPONSE]: The b refers to the fact that the intersected gene is subunit 2 of the type B GABA\nreceptor (GABA-B-R2). This distinction may not have been sufficiently clear in the text, so we\nhave now revised the text to improve clarity and maintain consistency in terminology throughout.\nLine 287. I have not read the reference to the Bogong moth but the title of the paper suggests\nthat they do report on genetic differences related to migration direction. Please check\n[RESPONSE]: This study reports panmixia and low migratory connectivity (meaning unprecise\nreturn to specific regions where they hatched), using a GWAS approach. Five populations within\nthe spring hatching regions were analysed in two groups: western and northern. Genotypes\nwere also obtained for aestivation sites. The study reports mixed genotype proportions at the\naestivation sites from the western and northern spring groups and concludes that there is no\npopulation structure. However, they do report 4 variants as candidates to be associated either\nwith the western or the northern spring group (geographic location), and by inference, with\nmigratory direction. The reasons why this may happen are discussed in the paper, but they\nmainly refer to differing local selection pressures at the western and northern spring sites, rather\nthan to an adaptive migratory divide. Thus, we are convinced that our statement is supported by\nreferencing this study.\nLine 315. “bird migrants breeding in the Northern Hemisphere are relative rare to winter in the\nSouthern Hemisphere”. I would not say rare. I took a quick look in a field guide of birds of\nSouthern Africa and found > 3 dozens of species (shorebirds excluded) that are regular winter\nvisitors from the Palearctic.\n[RESPONSE]: Thank you for your comment. We have rephrased the text to make clear we refer\nto a minority within the migratory birds, and not for that meaning exceptional or infrequent. The\nsentence now reads:\n“For example, long-distance bird migrants (excluding shorebirds) breeding in the Northern\nHemisphere and wintering in the Southern Hemisphere are proportionally a minority, and few\nSouthern Hemisphere breeders are found to migrate north of the Equator”\nLine 333. Not at all clear why records of 1st January were filtered?\n[RESPONSE]: We have found this filter to be important, particularly when using data to\nunderstand annual phenological patterns. When searching in GBIF, there is often a\ndisproportionately high number of entries assigned to January 1st, many of which correspond to\nolder entries from museum collections. It appears to be common practice to assign January 1st\nas a placeholder date when the exact collection date within a specific year is unknown. This\npractice can introduce a significant error, as these observations could have been made in any\nmonth of that year. Removing these January 1st entries minimizes the noise introduced by such\nplaceholder assignments, even if it means excluding a small number of legitimate observations\nmade on that date. In our analysis, we filtered out a total of 9426 observations assigned to\nJanuary 1st from a total of 10306 observations recorded for the entire month of January,\nmeaning that 92% of January entries were assigned to January 1st.\nFor clarity we have revised the sentence, which now reads: “We excluded the downloaded\nobservations recorded on January 1st, as this date appears to be disproportionately used and is\nlikely a placeholder for unknown collection dates within a given year”.\nLines 368 & 377. That you have three times as many SNP’s as loci suggests that many SNP’s\nare physically linked. Is that a problem for the analyses?\nLines 388-389. Same potential problem as above but more extreme, 50 times more SNP’s than\nloci.\n[RESPONSE]: We agree that this point needs further attention. Some of the methodologies\nused in this study, such as fineRADstructure (discussed in detail below), are inherently sensitive\nto linkage and designed to utilize it. For other analyses, such as PCA, we have applied\nadditional filtering steps, which are now described and justified in the revised manuscript.\nBy leveraging linkage within RAD loci, fineRADstructure treats them as non-recombining\nhaplotypes rather than analyzing individual SNPs within a locus in isolation (Malinsky et al.,\n2018). These haplotypes are then assembled into a haplotype matrix and used to infer nearest-\nneighbor relationships among individuals. Given the characteristics of our data—namely high\nheterozygosity and high recombination rates—we deliberately chose this haplotype-based\napproach over traditional SNP-based methods such as STRUCTURE or ADMIXTURE.\nTo investigate the potential effects of physically linked SNPs in our conclusions, we now provide\nPCA plots for SNPs using an LD pruned VCF file (Figure S2), both genome-wide and for the\ninverted region. We used PLINK to compute pairwise linkage disequilibrium, a measure based\non observed allele frequencies and genotype correlations. We retained only those markers with\nr2<0.2 and r2<0.05. These thresholds resulted in an average of 17 SNPs and 10 SNPs per\nlocus respectively, an expected density given the high heterozygosity of the species and the\nlarge size of our dataset (290 samples). While these markers are indeed in close proximity\nwithin the genome, their low levels of LD suggest that they are not strongly linked. This\nobservation aligns with the high recombination rates reported for Vanessa cardui (approximately\n4 cM/Mb) (Shipilina et al. 2022), which reduces the likelihood of strong LD among closely\nlocated markers. The methods have been updated to include these analyses (lines 431-433), as\nwell the results (lines 134-135). These show concordance with the original analyses using all\nSNPs.\nFig 1A. Why two occurrence peaks (March-May, Sept-Nov) in southern Africa? Worth a\ncomment?\n[RESPONSE]: That is an excellent observation. The climate in the southernmost part of Africa\n(Fynbos and the coastal belt) is characterized by a temperate climate, unlike the rest of the\nAfrican southern region, where the climate is tropical and driven by seasonal rainfall. This\ntemperate climate results in two distinct seasons of high habitat suitability for butterflies - spring\nand fall - leading to the observed double occurrence peaks. It is not surprising that most\nbutterfly observations, including those of V. cardui, are concentrated during these periods.\nMigration in insects, particularly in V. cardui is largely driven by seasonality, as it opens up\nsuitable habitats over space. Therefore, the butterflies expand and contract their ranges to\nexploit regions when they become favorable. The peak observed around September-October\n(spring) is likely associated with a decline in the equatorial region, as the intermediate regions\n(represented in the figure between latitudes -11º to -28º) typically experience a single rainy\nseason around April. Butterflies observed in Southern Africa are likely migrants from the\nequatorial zone.\nThe peak observed around April (autumn) follows a decline during the warmest part of the\nsummer, which likely causes plant desiccation in the southern region. During this time, habitat\nsuitability begins to reopen in the intermediate latitudes of the southern hemisphere after the\nrainy season, as well as in the southernmost part of the range. While the general trend is for\nbutterflies to start expanding northward, it is not unusual to see significant population growth\nacross this entire portion of their range during this period.\nNevertheless, when interpreting Figure 1A, it is important to consider that: i) the extent of the\nsouthern population’s range is relatively limited and contiguous, unlike the northern population.\nThe northern range is significantly larger, with most of the population migrating to a different\ncontinent, and where the range is further discontinued by a large barrier, the Sahara; ii) the\nrepresentation in Figure 1A is necessarily simplistic, and does not account for factors such as\naltitudinal migrations, which we believe are particularly relevant across mountain ranges in\nEastern and Southeastern Africa. Additionally, the latitudinal divisions used in Figure 1A are\nartificial. While further subdivisions -both latitudinal and longitudinal- could reveal clearer\npatterns of presence-absence transitions, the current observational data is insufficient for such\ndetailed analyses at the African continental scale.\nWe think that expanding this topic for this article would require a substantially deep dive into\nhabitat suitability patterns, likely involving ecological niche modelling analyses and deviating\nfrom the main focus of the study. We plan to address movements within Africa in greater detail\nin a future article.\nFig 2A. It is difficult to see the “potential heterozygotes” on the map. Try other colors.\n[RESPONSE]: We are now representing these in dark blue.\nFig 2 legend. On line 532 you say that the analysis is based on 546,458 SNPs belonging to the\nregion with high Fst on chr 8. But according to the methods (line 389) that’s the full data set of\ndataset 2 (whole genome)? And the same inconsistency seems to be for panel C which report\nall SNPs for dataset 1 but says it only include the region on chr 8.\n[RESPONSE]: Thanks a lot for pointing this out. The reviewer is right, this is an error. This has\nbeen corrected in the Figure 2 legend with the correct number of SNPs for the inverted region.\nFig 3. Line 543. The reported number of SNP’s for this analysis does not correspond to dataset\n2.\n[RESPONSE]: Thanks again for highlighting this mistake. We have corrected the number of\nSNPs accordingly.\nSuppl Fig S1 & S2. Number of SNP’s correspond to the complete dataset 1 but says it only\nanalyses those in the divergent region of chr 8\n[RESPONSE]: We apologize for this recurrent error. We have corrected these values in the\nlegends of Suppl. Figures S1 and S3, and have carefully reviewed the whole manuscript so no\nother inconsistencies might have occurred.\nSuppl Fig S3. Maybe consider to show the origin of the individuals by different colors for Europe\nand Africa.\n[RESPONSE]: That is an excellent idea, as the font is too small and does not provide immediate\ninformation. We have coloured the origin of each individual according to the two groups and\nslightly increased the font size.\nReviewer #2 (Remarks to the Author):\nI really enjoyed reading the manuscript and discovering this peculiar behavioural evolution. The\nreported evidences are robust and the conclusions drawn are generally supported but I\nrecommend a number of corrections before publication. In particular, more details on the origin\nof the inversions and differential evolution of migration in the Vanessa clade should be provided.\nIt would also be useful to compare with the evolution of divergent migration behaviour in other\nclades where migration occur within the same generation, and discussing the potential\nconsequences for speciation.\n[RESPONSE]: We truly appreciate the positive take and the thorough revision of our\nmanuscript. Please find below point-by-point responses to your comments.\nMain comments\nLine 28 and throughout the manuscript: I am not comfortable with the term ‘strategy’. It conveys\nthe misleading idea that evolution could have an aim, rather than that it occurs through a series\nof random mutations, selected or not. Perhaps even more importantly here, understanding how\na divergent migration behaviour may arise is not straightforward: can a single mutational event\ntrigger such a divergent evolution? Or does it request a series of genetic changes, and in that\ncase, what was the first event and how much does the first change facilitate the evolution of the\nfollowing ones? This leads to important question on the expected genetic architecture\nunderlying the migration syndromes: how much are we expecting a supergene architecture?\nHow do inversions may facilitate supergene evolution? I would thus recommend avoiding using\nthe term ‘strategy’ throughout the manuscript and clarifying the expectations in term of genetic\narchitecture underlying such divergence in migration syndromes.\nFor instance, line 83, you seem to hypothesize that the reversed seasonality may have evolved\nin response to the evolution of an opposite behaviour triggered by the magnetic field cue. This\nneeds to be clearly justified.\n[RESPONSE]: We sincerely appreciate the reviewer’s thoughtful feedback and the important\nquestions raised. We recognize the complexity of unraveling the genomic basis of behavior, and\nagree that understanding the genetic architecture of divergent migratory behaviors remains an\nopen and challenging area of research. In lines 70-77, we provide an overview, supported by\nextensive citations, of what is currently known about the genetic underpinnings of migratory\ndivides in animals.\nIt is important to keep in mind that research into insect migration, particularly about its genetic\narchitecture, lags significantly behind studies in other systems, such as vertebrates. With the\ndescription of genomic features such as those in this manuscript, we take a significant step\ntowards advancing the basic understanding of potential genetic mechanisms driving migration in\ninsects. These mechanisms may be diverse, ranging from highly epistatic interactions and\nstructural rearrangements to single mutations affecting specific components of the migratory\nphenotype. In case of inversion polymorphisms, beneficial mutations that arise within genes or\nregulatory regions inside an inversion may be subject to selection and remain in strong linkage\ndue to suppressed recombination in the inverted region. In this context, a supergene\narchitecture facilitated by a chromosomal inversion is plausible and one of the expected\nmechanisms underlying divergent migratory behaviours. This is because of 1) inversions can\npreserve adaptive combinations of mutations that influence multiple functionally connected traits\nand are thus good candidates to underlie divergent selection to polygenic traits, and 2) the\nreduction of recombination by inversions can promote the evolution of barriers in species with\nvery high mobility and panmixia, where differentiation in collinear regions is typically eroded by\ngene flow.\nOur findings provide critical groundwork into these broader questions, reporting the first\ngenomic signal correlated with a population-level migratory pattern in insects. While our study\ndoes not aim to mechanistically explain the genetic basis of specific migratory behaviors, our\nfindings allow us to contribute legitimate hypotheses. We acknowledge the reviewer’s concerns\nabout the scope of certain questions raised and we see them as natural follow-ups rather than\nissues that can be fully resolved within this study.\nRegarding the specific comments, we have edited line 83 (now line 86) and incorporated new\ncitations, which now reads: “Given that butterflies and moths possess magnetoreceptor organs\nthat may determine orientation (Guerra et al., 2014, Dreyer et al., 2018, Wan et al., 2021), we\nhypothesize that reversed seasonality, a necessarily opposite response to the magnetic field,\nand distinct celestial cues in the two hemispheres of Earth may shape contrasting migratory\nstrategies”.\nRegarding the use of the term strategy, we note that this is a well-established term across\nanimal migration literature, commonly used to describe migratory behavior at ecological and\npopulation scales. In other words, although evolution is a mechanistic process that uses no\nstrategies, an ecological strategy can arise because of natural selection. While we agree that\ncare should be taken when applying it to evolutionary mechanisms or individual decision-\nmaking, its usage in our manuscript aligns with standard terminology in the field and ecological\nscales, thus ensuring consistency with the broader body of research. We have ensured that it is\nemployed with appropriate context to avoid misinterpretation.\nGuerra, P. A., Gegear, R. J., & Reppert, S. M. (2014). A magnetic compass aids monarch\nbutterfly migration. Nature communications, 5(1), 1-8.\nDreyer, D., Frost, B., Mouritsen, H., Günther, A., Green, K., Whitehouse, M., ... & Warrant, E.\n(2018). The Earth’s magnetic field and visual landmarks steer migratory flight behavior in the\nnocturnal Australian Bogong moth. Current Biology, 28(13), 2160-2166.\nWan, G., Hayden, A. N., Iiams, S. E., & Merlin, C. (2021). Cryptochrome 1 mediates light-\ndependent inclination magnetosensing in monarch butterflies. Nature communications, 12(1),\n771.\nLine 46-47 : In line with my previous comment, more details on the genetic architecture\ncontrolling variation in migration behaviours in different clades are necessary here.\n[RESPONSE]: This is the first paragraph of the introduction, where we introduce the role of\nseasonality in triggering migratory responses. Details on the genetic architecture determining\nvariation in migratory behavior are developed soon after, in the introduction lines 70-77.\nLine 52: The term ‘optimizing’ is also misleading : several migratory routes can allow survival\nand reproduction, and selection is likely to favour the best ones among the different genetic\nvariants within the population, but not necessary the optimal ones. I think here it would be useful\nto clearly pinpoint the traits, linked to migration, targeted by selection, e.g. genes involved in the\nperception of different environmental cues (photoperiodic cues triggering departure, magnetic\ncues guiding direction…).\n[RESPONSE]: We agree with the reviewer, the term optimising is not the most adequate. We\nhave changed the term “optimising” by “regulating”. This paragraph aims to introduce the role of\nenvironmental rhythmicity in regulating migratory behavior, but not to elaborate on migratory\ntraits or “migratory genes”, which are developed in the discussion.\nLine 61 : One of the major difference between butterflies as compared to birds, fishes or\ncetaceae relies on the multiple generations requested to complete the migration cycle. This has\nprobably strong consequences on the speciation process, because in one out of two\ngenerations, reproduction occurs in well-separated areas (Northern vs. Southern populations of\nV. cardui) with zero possibility of gene flow.\n[RESPONSE]: Indeed, in this sentence we introduce the concept of multigenerational migratory\ncycles in insects. In Vanessa cardui, the migratory cycle involves not just two, but up to ten\ngenerations per year. Our study highlights minimal spatio-temporal overlap between northern\nand southern populations: when the northern population reaches its European breeding\ngrounds, the southern population is typically located near equatorial Africa—the only shared\nportion of both migratory ranges. Indeed, V. cardui butterflies are present year-round in Eastern\nAfrican highlands, albeit with significant demographic fluctuations (Talavera et al 2023). In this\ngeographic region, as a result, genetic exchange likely occurs marginally. This overlap is only\npossible for a low proportion of generations and is likely both temporally and spatially limited.\nSuch constraints may promote reproductive isolation over time, especially if reinforced by\ninversion polymorphisms.\nLine 62-63 : This notion of reticular movements and consequences for the lack of philopatry is\nunclear to me. Independently from the generational effect, there is individual variations on the\nmigration behaviour in birds and fishes as well. The evolution of philopatry could be driven by\nindividual experiences that are unlikely to occur in butterflies because different individuals take\nthe north-South vs. South-North journey. This should be made clearer.\n[RESPONSE]: We totally agree with the reviewer's thoughts, and these concepts were\nextensively explored in a previous study by our team (García-Berro et al 2022), which is cited in\nthis section. To improve clarity, we have rewritten the sentence as follows: “In insects, long-\nrange cyclical migrations typically span multiple overlapping generations, with each generation\ncompleting only one leg of the migratory cycle, and where individuals rarely learn their routes\nfrom experience.”\nPlease note that there may be cases in insects where individuals may learn from experience,\nsuch as the Monarch butterfly or the Bogong moth, where overwintering and aestivating\nindividuals undertake return migrations.\nGarcía‐Berro, A., Talla, V., Vila, R., Wai, H. K., Shipilina, D., Chan, K. G., ... & Talavera, G.\n(2023). Migratory behaviour is positively associated with genetic diversity in butterflies.\nMolecular Ecology, 32(3), 560-574.\nLine 28-29 and 66: Could you precise the proportion of latitudinal vs. Longitudinal divides\ndocuments in different clades ? Why do you expect latitudinal divide to be more prevalent in\ninsects ?\n[RESPONSE]: The expectation of latitudinal divides is outlined in lines 28-29: “driven by\nhemisphere-specific sensory adaptations that lead to spatial and temporal isolation”. Since this\nstatement appears in the abstract, we cannot elaborate further because of length restrictions,\nbut note that we do so shortly after, in the introduction.\nOn the other hand, as this is the first documented case of a migratory divide in insects, it is\ncurrently impossible to quantify or predict the proportion of longitudinal vs latitudinal divides\nacross different clades. Nevertheless, we believe that the arguments and references provided in\nthe introduction allow us to present this hypothesis.\nLine 84-85 : evidence for the use of magnetic field and celestial cues in Lepidoptera (e.g. moth)\nshould be detailed here (e.g. Dreyer, D., Frost, B., Mouritsen, H., Günther, A., Green, K.,\nWhitehouse, M., ... & Warrant, E. (2018). The Earth’s magnetic field and visual landmarks steer\nmigratory flight behavior in the nocturnal Australian Bogong moth. Current Biology, 28(13),\n2160-2166.)\n[RESPONSE]: We have now included three citations referring to the use of magnetic field and\ncelestial cues:\nGuerra, P. A., Gegear, R. J., & Reppert, S. M. (2014). A magnetic compass aids monarch\nbutterfly migration. Nature communications, 5(1), 1-8.\nDreyer, D., Frost, B., Mouritsen, H., Günther, A., Green, K., Whitehouse, M., ... & Warrant, E.\n(2018). The Earth’s magnetic field and visual landmarks steer migratory flight behavior in the\nnocturnal Australian Bogong moth. Current Biology, 28(13), 2160-2166.\nWan, G., Hayden, A. N., Iiams, S. E., & Merlin, C. (2021). Cryptochrome 1 mediates light-\ndependent inclination magnetosensing in monarch butterflies. Nature communications, 12(1),\n771.\nLine 85-86 : the term ‘adjust’ is unclear : behavioural adjustment could be plastic. You need to\nclarify whether you expect (1) an evolution of behaviour triggered by genetic change, selected in\ndifferent evolutionary environment or (2) an individual behavioural variation triggered by the\nenvironment encountered by the individual itself.\n[RESPONSE]: Indeed, both scenarios are possible in theory. We have revised the sentence as\nfollows:\n“Since environmental rhythmicity displays inverted pulses between hemispheres, populations of\nmigratory insects may adjust biological clocks, either through genetic changes or plastic\nbehavioral responses, and respond differently to hemispheric-specific seasonal cues, potentially\nleading to spatial and temporal isolation between populations.”\nLine 120-121: are the collection dates known for the different samples? I could not find it in the\nSup. Mat 1, it would be nice also to explore the genetic variations associated with the sampling\ndates, especially in the equatorial regions.\n[RESPONSE]: We have now included the collection dates in the supplementary material.\nHowever, the temporal span of the sampling in the equatorial regions is too limited to deeply\nexamine temporary population genetic patterns.\nLine 146-147 : Is there any evidence that the Southern haplotype is dominant over the Northern\none ? This would explain the heterozygotes observed within the Southern population? Since the\nnewly emerged haplotype is expected to be dominant over the ancestral one, this could also\nprovide a hypothesis on the ancestral vs. derived state (see Llaurens, V., Whibley, A., & Joron,\nM. (2017). Genetic architecture and balancing selection: the life and death of differentiated\nvariants. Molecular ecology, 26(9), 2430-2448.)\n[RESPONSE]: Thank you for the comment. We cannot infer dominance with the current study\ndesign and genomic data only, but this is indeed a strong hypothesis. However, in the revised\nversion we have expanded on the discussion on the ancestral and derived states.\nLine 172 : What is the ancestral gene order ? Does the gene order observed in sister species of\nV. cardui (or in other butterfly genomes) fit with the Northern or the Southern haplotype?\n[RESPONSE]: As the reviewer suggests, by investigating the relative gene order of the sister\nspecies (Vanessa kershawi) we will gain further insights into the evolutionary history of the\ninversion haplotypes. The two species diverged around 7 mya, and the estimated age of the\ninversion is <1 mya. Based on these estimations, we hypothesize that the inversion arose after\nthe split between V. cardui and V. kershawi, and thus we expect to find synteny in chromosome\n8 between V. kershawi and the ancestral V. cardui population.\nUnfortunately, there is no assembled genome and annotation yet for V. kershawi. Our data\nincludes one assembled specimen for the North population of V. cardui (the reference genome),\nand one specimen of short-read data for V. kershawi. To investigate the synteny between the\ntwo species in chromosome 8, we mapped V. kershawi’s short-reads against the North\nhaplotype of V. cardui, and used mapping information in the Breakdancer pipeline to identify\npotential inversions. Breakdancer uses inconsistent orientation or unexpected insert size in\nmapped read-pairs to identify structural variants.\nWe did not find any chromosomal inversions between V. kershawi and the North V. cardui\nhaplotype matching the breakpoint coordinates (4,402,570 and 13,379,476) and length (9Mb) of\nthe inversion between North and South V. cardui populations. This result indicates that the gene\norder of the sister species is collinear with the North haplotype, and agrees with the V. cardui\ninversion haplotype present in the North being the ancestral form. As suggested by reviewer 1,\nwe can also argue that the haplotype where both ends of the inversion directly affect the\ncontiguity of genes and likely their function (the southern population) should be the derived\nstate. We have added this piece of information and adapted the manuscript accordingly in lines\n197-200 and 495-500.\nLine 176-177 : I am not entirely convinced by the argument, introgressions have been found to\nhappen between highly divergent species : even if the hybridization events are rare between\ndivergent species, if selection on the introgressed region is very high, this might allow the\nintrogression to stay within the genome (see Edelman, N. B., & Mallet, J. (2021). Prevalence\nand adaptive impact of introgression. Annual Review of Genetics, 55(1), 265-283.)\n[RESPONSE]: We agree with the reviewer, so have removed any reference to introgression\nevents. The sentence now reads: “These findings, combined with the fact that the divergence\nbetween V. cardui and its sister species, V. kershawi, is estimated to be much older - around 7\nmya57- suggest that the inversion arose in V. cardui after the split between these two species of\nVanessa.”\nLine 182 : how much is genome-wide pi smaller in the Northern vs. Southern populations ?\nCould you provide population genomics arguments for this bottleneck using genomic variations\noutside the inverted region ?\n[RESPONSE]: The difference in π between the Northern (avg=0.016) and Southern\npopulations (avg=0.017) is rather small (0.001). The difference measured in 1Mb windows was\nstatistically significant when using the non-parametric test for mean comparison (Mann-Whitney\nU-test, p-value<0.001 ), as well as when computing p-values by resampling using permutations\n(p-value<0.001). However, the significance could result from the large sample size effect (π\nwas estimated in 383 windows) and may not be as biologically relevant. Provided that this\nargument might not be very strong, we have decided to remove this point from the discussion\nand instead focus on the clearer signal of reduced heterozygosity within the inverted region.\nLine 196-197 : This section in unclear, here you refer to as the genome-wide heterozygosity ?\nCould you provide a statistical comparison of genome-wide heterozygosity between the three\ngenotypes (fig. 3C, left-panel). Given the low difference in genome wide heterozygosity between\nthe Southern vs. Northern populations, I am not convinced that the difference in effective\npopulation sizes can explain the difference in heterozygosity observed in the inverted regions in\nthe Northern vs. Southern populations.\nThank you for pointing this out, we have now added the statistical tests for the genome-wide\nestimates of heterozygosity between populations. Please see details reported in lines 163-166\nof the Results & Discussion section and lines 527-537 of the Methods section.\nWe refer to the genomic heterozygosity in the inverted region, not the genome-wide estimates\nof heterozygosity. We have rephrased the second sentence to clarify that we still refer to the\nsame within-inversion heterozygosity estimate throughout the paragraph.\nDifferences in heterozygosity between populations inside the inverted region are indeed large.\nWe now consider other possibilities explaining the lower heterozygosity in the South, such as a\nlow population size effect during the emergence of the derived haplotype, and the result of\npositive selection acting on this region. Last, we consider an additional possible effect: the fact\nthat the Northern haplotype seems to be largely fixed. Intra-population recombination could be\nplaying a role in restoring diversity in the homozygous (and therefore collinear) inverted region\nin the North. We have expanded our reflections in the main text.\nLine 252-257 : I would discard this highly speculative section and more generally, I would not\nfocus too much on the potential candidate genes at this point: as mentioned by the authors,\nRAD-sequencing data do not allow to precisely characterize the whole region (272-274). This\npart of the discussion could thus be reduced.\n[RESPONSE]: We have slightly reduced this section, while retaining the list of genes detected\nwithin the inversion that exhibit high F values. We believe this information remains valuable to\nST\nmany readers, as also indicated by Reviewer 3, because it provides insights into potential\ncandidate genes involved in the observed genetic differentiation. While we acknowledge that\nRADseq data do not allow for a full characterization of the entire region, the genes identified still\nrepresent noteworthy findings worth reporting.\nLine 297-302 : It would be nice to have a phylogeny of the different Vanessa species with\ninformation on (1) the gene order (when available) in the region where the inversion is observed\nin V. cardui and (2) the migration behaviour. The within southern-hemisphere migration of V.\nkershawi could indeed potentially point at an ancestral Southern migration behaviour?\n[RESPONSE]: While we agree with the reviewer, we are afraid that assemblies are not available\nfor most Vanessa species to report gene order. The study of the evolution of hemisphere-\nspecific behaviors at a phylogenetic scale is a compelling avenue for future research, and we\nfully agree that this represents a logical next step for follow-up studies.\nLine 300: did you mean ‘WITHin South and North America respectively’ ?\n[RESPONSE]: Thanks for pointing this out, this has been corrected.\nLine 3014-319 : One of the main difference between the V. cardui butterflies and the cited\nshorebird and whale examples is the single vs. multiple generation patterns of migration which\nis likely to have consequences on population divergence and speciation, as mentioned in one of\nmy previous comments. I think it is worth discussing this specific point.\n[RESPONSE]: We explain this in the introduction. The purpose of this particular paragraph is to\nprovide a comparative framework, highlighting potential hemispheric migratory divides across\ndifferent animal taxa, rather than to delve into population dynamics. The evolutionary and\ndemographic implications of multi-generational migration in insects were extensively explored in\na previous study by our team (García-Berro et al 2022), which we cite in the text.\nFigure 3B is very nice, but here we would like to know what is the gene order in V. kershawi in\nthe inverted region? Could you also provide trees based on your RAD-sequencing data within\nand outside the inverted regions for all your samples to visualize how much the evolutionary\nhistory of this specific region departs from the genome wide history ?\n[RESPONSE]: Unfortunately, there is no genome assembly and annotation available for\nVanessa kershawi. Therefore, identifying the breakpoints using our short-read data is the\nclosest we can get to this suggestion, as we report and explain in our answer to your previous\ncomment. We now report no apparent inversion in V. kershawi compared to the northern\npopulation of V. cardui and based on a BreakDancer analysis. This suggests that the inversion\nevent is unique to the southern population of V. cardui.\nIn addition, we have now inferred phylogenetic trees based on RAD data (dataset 3), for both\nwithin and outside the inverted region. The inferred trees are presented as a new\nSupplementary File (S3). The resulting tree for the inverted region shows well-supported clades\nfor the Northern and Southern populations, whereas a phylogeny for the rest of the genome\ndoes not reflect biogeographic structure.\nMinor comments\nLine 29 : I am not sure longitudinally is that easy to understand, I would rather use an East/West\nvs. North/West terminology\n[RESPONSE]: We appreciate the reviewer’s suggestion. We prefer the use of longitudinal and\nlatitudinal terminology, as it avoids implying specific directionality (East-West vs West-East, or\nNorth-South vs South-North). However, to ensure clarity, we have now explicitly stated the\nequivalence between longitudinal (East-West) and latitudinal (North-South) the first time these\nterms appear in the text.\nLine 61 : replace ‘one leg of the migratory cycle’ by ‘one step of the migratory cycle’\n[RESPONSE]: The word \"leg\" is commonly used to describe a distinct segment or stage of a\njourney, especially in migration contexts. It implies a defined portion of the overall cycle, such as\na specific route or phase of movement. \"One step of the migratory cycle\" is less precise, as\n\"step\" typically refers to smaller, incremental progressions rather than major, structured\nsegments of a journey.\nLine 101 : ‘species distribution THROUGH TIME’\n[RESPONSE]: Done.\nLine 106 : Again, evolution has no aim, you may replace ‘tracking seasonality in search for\noptimal conditions’ by ‘exhibiting different seasonal pattern favouring their occurrence in suitable\nhabitats’.\n[RESPONSE]: This sentence describes an ecological pattern rather than an evolutionary\nprocess, where population movements align with vegetation phenology - similar to the adaptive\nphenological match between butterflies and their host plants. To clarify, we have revised the\nsentence from “with successive generations tracking seasonality in search of optimal conditions”\nto “with successive generations tracking seasonality that offers optimal conditions”.\nLine 221 : replace ‘transcripts’ by ‘patterns of expression’\n[RESPONSE]: Done.\nLine 238 : replace ‘protein behaviour’ by ‘protein sequence’\n[RESPONSE]: Done.\nReviewer #3 (Remarks to the Author):\nGarcia-Berro et al 2024, Nat Comm\nThis is a wonderful manuscript that shows genetic evidence for a migratory divide in painted\nlady butterflies, and I greatly enjoyed reading it. The premise of this study is that migratory\ndivides are pretty common in vertebrates, but that they have not been found in insects, despite\nextensive studies looking for them. As the authors point out, however, many of these\nstudies have been done on insect populations that are separated longitudinally, with predominantly\nwestern and eastern populations. While such populations may experience different\nenvironmental conditions, they experience similar seasonality and magnetic fields, two factors\nthat underlie many migratory behaviors. In the painted lady however, populations are also\ndivided latitudinally, with populations migrating either in the northern or southern hemisphere.\nAnalyzing a massive data set on abundance, the authors convincingly show temporal\nseparation in the abundance of northern and southern painted ladies around the equator. Using\na genome-wide RADseq analysis, they then demonstrate significant genetic differentiation\nbetween these populations, and even find a chromosomal inversion that is tied to this north-\nsouth separation. Within this inversion, they also discover many genes that are known to be\ninvolved in insect migration, navigation and mating behavior, thereby providing potential\nmechanistic explanations for differences in migration and reproductive isolation. By showing this\nhemispheric divide, the authors provide a novel way of thinking of migratory divides, with\napplications to many different species (including mammals and birds) and strengthening the\nhypothesized mechanisms of navigation and migration based on seasonality and magnetism. I\nwould personally add polarized light and solar azimuths as well, which are also likely to\nvary based on latitude.\n[RESPONSE]: We truly appreciate the reviewer’s comments and positive take on our\nmanuscript. As suggested, we now refer to polarized light and solar azimuths as potential\nfactors involved in differential navigation mechanisms.\nI only have a few comments.\n1. Before diving into the chromosomal inversion, I feel that the authors could emphasize the\ngenome-wide genetic differentiation more. The data in Fig. 3A clearly show overall levels of FST\nand dxy much higher than those found in other insects where similar studies have been done.\nMoreover, there are quite a few locations in the genome that show high FST values of 0.10 and\nhigher, and it may be worth investigating if these chromosomal regions include other genes that\ncould provide insight into navigation, reproductive isolation etc.\n[RESPONSE]: This is a good point. Although genome-wide F values may seem high from a\nST\nvisual inspection in Figure 3A, average F outside the inversion is indeed very low: 0.009\nST\n(Table S1). Compared to other similar studies using genomic data, this value is in the same\norder of magnitude of F variation than in the cotton bollworm populations (Helicoverpa\nST\narmigera, Jin et al., 2023), the fall armyworm populations (Spodoptera frugiperda, Schlum et al.,\n2021), the diamondback moth populations (Plutella xylostella, Chen et al., 2021), but one order\nof magnitude smaller than in the bogong moth populations (Agrotis infusa, Ahrens et al., 2025).\nWe now add a sentence comparing the genome-wide F levels of V. cardui to the above-\nST\nmentioned insect species and emphasizing the genome-wide differentiation more (lines 127-\n129).\nHowever, we refrain from further elaboration on the comparison of F estimates across studies\nST\nsince differences could result from several factors, such as sampling design (the geographic\nscale, and the number of samples per population, ultimately affecting within-population diversity)\nand filtering parameters, particularly thresholds for minor allele frequencies (MAF), all of which\ncan greatly influence F estimates. Moreover, F is a measure of differentiation relative to the\nST ST\ndiversity of the species (Bathia et al., 2013) and inherently agnostic to the underlying\ndemographic processes, so causal differences between F values in similar insect studies may\nST\nrange from different evolutionary scenarios to inherent study characteristics.\nOn the other hand, we now provide a new table in the supplementary material with a list of\ngenes with high differentiation (F >0.25) located outside the inversion.\nST\nJin, M., North, H. L., Peng, Y., Liu, H., Liu, B., Pan, R., ... & Xiao, Y. (2023). Adaptive evolution\nto the natural and anthropogenic environment in a global invasive crop pest, the cotton\nbollworm. The Innovation, 4(4).\nSchlum, K. A., Lamour, K., de Bortoli, C. P., Banerjee, R., Meagher, R., Pereira, E., ... & Jurat-\nFuentes, J. L. (2021). Whole genome comparisons reveal panmixia among fall armyworm\n(Spodoptera frugiperda) from diverse locations. BMC genomics, 22, 1-12.\nChen, M. Z., Cao, L. J., Li, B. Y., Chen, J. C., Gong, Y. J., Yang, Q., ... & Wei, S. J. (2021).\nMigration trajectories of the diamondback moth Plutella xylostella in China inferred from\npopulation genomic variation. Pest Management Science, 77(4), 1683-1693.\nAhrens, C. W., Slavenko, A., Kriesner, P., van Rooyen, A., Heinze, D., Bartonicek, N., & Weeks,\nA. R. (2025). Stochastic wind-driven migration likely maintains panmixia in the endangered\nbogong moth, Agrotis infusa. Biological Conservation, 302, 110993.\nBhatia, G., Patterson, N., Sankararaman, S., & Price, A. L. (2013). Estimating and interpreting\nFST: the impact of rare variants. Genome research, 23(9), 1514-1521.\n2. I was wondering why a demographic analysis was not carried out. When studying the\nphylogenetic trees in Fig. 3B, it is indeed tempting to conclude directionality of evolution, with\nnorthern painted ladies deriving from southern painted ladies, but the tree is rather limited and\nthe observed patterns on the basis of five samples may be driven by specific samples. It would\nhave been helpful to add additional southern samples to confirm that they cluster with the\nNamibian sample rather than with the northern samples.\n[RESPONSE]: As developed in paragraph 197-214, we discuss that the most likely explanation\nis that the southern haplotype is the derived state where the rearrangement occurred, but there\nare other possibilities that can’t be fully excluded. Including additional WGS data for both\npopulations would indeed help providing a more complete phylogenetic view, and we plan to do\nthis in the future. However, we now include a new phylogenetic analysis based on our RAD\ndataset (dataset 3), and five V. kershawi specimens as outgroups, thus increasing sampling\nsize for each population. The resulting phylogeny for the inverted region also shows well-\nsupported clades for the Northern and Southern populations, whereas the phylogenetic tree for\nthe rest of the genome remains unstructured.\nRegarding a demographic analysis, we did perform a PSMC analysis in a previous study\n(García-Berro et al 2022), showing similar demographic trends through time in populations from\nboth hemispheres, although with slightly lower Ne for the southern population.\n3. Also with respect to the phylogenetic tree, I think it would be helpful to emphasize that the\nnorthern samples clustering together derive from a very wide longitudinal gradient, from the\nUSA all the way to Nepal. That in itself provides an internal\ncomparison of the east-west lack of divide as observed in many other systems, while emphasizing the north-south divide as\nobserved here.\n[RESPONSE]: That is a good observation. We now include this idea in the text (lines 142-146),\nwhich reads: “Remarkably, phylogenetic divergence across the Northern clade, spanning a vast\nlongitudinal range across the Holarctic, is lower than the divergence observed between\nhemispheres (Figure 3B, Figure S5), underscoring the pronounced role of the inversion and\nhemispheric barriers in shaping population structure”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have clarified all concerns in the revision and response letter. It is a very nice piece of work that I’m looking\nforward to seeing printed.", "reviewer_2": "While the authors answered to most of my previous comments, two important points still need to be addressed before\npublication in my opinion.\n(1) The link between migratory divide and speciation is not evidenced in the manuscript, and more precise discussion\nshould be brought on the lack of genetic differentiation between Northern and Southern populations, outside the locus of\ninterest.\nLines 64-65 : In response to my previous comment, this section which is central to the manuscript was improved, but there\nare still some imprecision. In particular, migratory divide is defined as ‘sympatric populations with divergent migration\nstrategies’, and the putative effect of such divide in driving population divergence and ultimately speciation is highlighted.\nWhat is still unclear in this section is the level of spatial, and more importantly temporal sympatry observed in cases where\ndivergent migration behaviours actually fuel speciation. Another point that needs to be clarified is the location and timing of\nmating events with respect to the migration events, because only temporal and/or spatial divergence of population during the\nreproductive period can fuel genetic divergence between populations. Those are key elements needed for the comparisons\n(1) between longitudinal and latitudinal divide and (2) between birds where a single reproduction phase occurs per year and\ninsects where several generations can be observed within a year.\nLine 111-122 : Given you observational data, can you infer the putative timing of reproduction events in Northern and\nSouthern populations ? Do you have data on both adult and caterpillar stages? This would be relevant to draw some\nputative scenarios to discuss on the putative effect of migratory divide on population divergence.\nLine 117 : ‘inhabit equatorial latitude’ : could you estimate the level of spatial overlap between the two waves of populations\narriving in these equatorial latitudes ? This is also important to estimate the level of reproductive opportunities between\nindividuals from the northern and southern populations.\nLine 180 : “gene flow occurs to some extent’: In my opinion the gene flow is very large between the southern and northern\npopulations, as highlighted by the very low Fst throughout the genome (except for the inversion region) (fig.3A)\nLine 260-287 : Providing a list of candidate genes is potentially useful but I would advise to limit the overinterpretation,\nunless there is ecological data supporting the hypothesis of divergent evolution on other traits in the Northern vs. southern\npopulations. For instance, is there any evidence for divergence in host-plant use between the northern vs. southern\npopulations ? Or for divergence in mate preferences ?\nLines 275-278 : Given the lack of population differentiation (fig.3A and see my comment on low level of Fst above), I do not\nsee the relevance of discussion about potential DBM incompatibilities here.\n(2) If the authors want to explore the putative candidate genes involved in the evolution of divergent migration behaviours,\nconsidering both the effect of positive selection acting on adaptive mutations and purifying selection acting on deleterious\nmutations would be useful. dN/dS analyses can potentially mix up the two effects, you may consider alternative methods\naccounting for pN/pS approaches for instance.\nLine 250-258: Investigating the selective regime acting on the gene located within the locus of interest is highly relevant, but\nsince none of the 32,719 SNPs were fixed between the haplotypes (as stated line 189), this means that there is probably no\nfixed non-synonymous substitutions between your haplotypes? As a consequence, signatures of selection may rely more on\npN/pS rather than dN/dS.\nLine 152-153 : Is there any differences in size between the two haplotypes within your locus of interests? If so, what is the\norigin of such difference in size (gene duplications ? TE enrichment ?).\nSpecific comments :\nLine 41: ‘movements play a fundamental role in the evolution of life’ is too general and vague to bring sufficient relevance to\nthe manuscript, did you mean that migration likely plays a key role in species diversification ? This should be made more\nprecise and referenced, helping the reader to immediately connect to the research questions addressed in your manuscript.\nLine 68 : I am wondering whether non-migratory populations of Monarch could be considered as a form of migratory divide ?\n(see for instance : Hilburn, D. J. (1989). A Non-Migratory, Non-Diapausing Population of the Monarch Butterfly, Danas\nPlexippus (Lepidoptera: Danaidae), in Bermuda. Florida Entomologist, 494-499.)\nLine 88 : It is very important to distinguish (1) proximal factors (e.g. temperature encountered by an individual) acting on the\nmigration behaviour of that given individuals from (2) selective factors (seasonal variation in temperature in the geographic\nrange of a given species) acting on the evolution of migration behaviour within this species. Here for instance, did you mean\n‘shape THE EVOLUTION of contrasting migratory strategies’ ?\nLine 109 : ‘tracking LOCATIONS with optimal conditions’\nLine 110 : ‘V. cardui POPULATIONS MOVE from the Paleartic…’ One of the challenges of the observational data provided\nhere is that it does not allow to track individual migration behaviour.", "reviewer_3": "Thank you for the edits. I have no further comments, and will reiterate that I think this is a great manuscript."}, "author_response": "Response to referees (second revision) for\nA north-south hemispheric migratory divide in butterflies\nAurora García-Berro†*, Daria Shipilina†, Niclas Backström, Tomasz Suchan, Aleix Palahí,\nSteve C. Collins, Dino J. Martins, Naomi E. Pierce, Roger Vila, Gerard Talavera*\n*Correspondence to: aurora.garcia@ibb.csic.es; gerard.talavera@csic.es\nReviewer #1 (Remarks to the Author)\nThe authors have clarified all concerns in the revision and response letter. It is a very nice piece\nof work that I’m looking forward to seeing printed.\nRESPONSE: Thank you very much for your insightful and helpful review that greatly improved\nthe manuscript.\nReviewer #2 (Remarks to the Author)\nWhile the authors answered to most of my previous comments, two important points still need to\nbe addressed before publication in my opinion. (1) The link between migratory divide and\nspeciation is not evidenced in the manuscript, and more precise discussion should be brought\non the lack of genetic differentiation between Northern and Southern populations, outside the\nlocus of interest.\nLines 64-65 : In response to my previous comment, this section which is central to the\nmanuscript was improved, but there are still some imprecision. In particular, migratory divide is\ndefined as ‘sympatric populations with divergent migration strategies’, and the putative effect of\nsuch divide in driving population divergence and ultimately speciation is highlighted. What is still\nunclear in this section is the level of spatial, and more importantly temporal sympatry observed in\ncases where divergent migration behaviours actually fuel speciation. Another point that needs to\nbe clarified is the location and timing of mating events with respect to the migration events,\nbecause only temporal and/or spatial divergence of population during the reproductive period\ncan fuel genetic divergence between populations. Those are key elements needed for the\ncomparisons (1) between longitudinal and latitudinal divide and (2) between birds where a\nsingle reproduction phase occurs per year and insects where several generations can be\nobserved within a year.\nRESPONSE: We appreciate the reviewer’s thoughtful and constructive feedback. We\nacknowledge the importance of clearly articulating the link between migratory divides and\nspeciation in vertebrates. We have carefully revised the introduction to elaborate on the\nmechanisms by which migratory divides are thought to contribute to speciation in vertebrates,\nand their main contrasts with insects (lines 58-68, 76-78). We would like to stress that this is the\nfirst migratory divide described in insects. Consequently, the broader framework for\nunderstanding the relationship between migratory divides and speciation in insects is\nunderdeveloped. Our study focuses on providing integrative evidence for this phenomenon,\ndrawing on behavioral, ecological, and genomic data, and to lay the foundation for further\ndiscussion, while formulating hypotheses for future research, including those explicitly focused\non speciation. Regarding the exact location and timing of mating and migration events in V.\ncardui, we have extensively reviewed the current state of knowledge, much of which originates\nfrom our own recent work. As noted, the breeding phenology across the Afrotropical range has\nbeen recently described (Talavera et al., 2023, PNAS), and we refer to this work throughout the\nmanuscript where relevant. Migration and breeding are not temporally or spatially discrete\nevents in multigenerational migrating insects, particularly in V. cardui. This inherent complexity\nposes a significant challenge when attempting to compare migratory divides across taxa,\nespecially between organisms with a single annual reproductive cycle (e.g., many birds) and\nthose with multiple overlapping generations and more flexible breeding strategy.\nLine 111-122 : Given you observational data, can you infer the putative timing of reproduction\nevents in Northern and Southern populations ? Do you have data on both adult and caterpillar\nstages? This would be relevant to draw some putative scenarios to discuss on the putative\neffect of migratory divide on population divergence.\nRESPONSE: We thank the reviewer for pointing out how important and relevant this information\nis for understanding this phenomenon. In the manuscript, we summarize available data for\nVanessa cardui supporting our interpretation, which was already published in Talavera et al.,\n2023, Menchetti et al., 2019, and Talavera & Vila 2017. Our observations include both adult\nbutterflies and immature stages, allowing us to infer breeding activity.\nIn equatorial regions such as Kenya and Cameroon, monitoring efforts have revealed two\ndistinct peaks of breeding abundance, which correspond to the bimodal rainfall pattern typical of\nthese areas. In contrast, sites located further north—such as Ethiopia, Benin, or Senegal—show\na single peak of breeding activity per year. These spatial and temporal patterns suggest a\nscenario where one breeding peak (November–January) likely corresponds to individuals\nmigrating from or destined for northern latitudes, while the other peak (around June) likely\ncorresponds to southern populations. Worth noting, V. cardui populations are formed by many\nmillions of individuals and these demographic peaks have tails that seem to overlap. While\nfurther data are needed to better delineate the geographical area and characteristics of the\n“contact zone” of the migratory divide, the general patterns for the bulk of the population allow\nus to generate hypotheses about its potential role in shaping population divergence.\nLine 117 : ‘inhabit equatorial latitude’ : could you estimate the level of spatial overlap between\nthe two waves of populations arriving in these equatorial latitudes ? This is also important to\nestimate the level of reproductive opportunities between individuals from the northern and\nsouthern populations.\nRESPONSE: Unfortunately, we don’t see how we could estimate such overlap given the current\ndataset, which is limited in both spatial resolution and temporal genomic sampling. We\nrecognize the importance of this question and hope to address it more directly in future work\nthrough expanded temporal sampling and higher-resolution genomic data.\nLine 180 : “gene flow occurs to some extent’: In my opinion the gene flow is very large between the southern and northern\npopulations, as highlighted by the very low Fst throughout the genome (except for the inversion region) (fig.3A)\nRESPONSE: We have changed “gene flow occurs to some extent” by “gene flow occurs\nextensively” (line 200).\nLine 260-287 : Providing a list of candidate genes is potentially useful but I would advise to limit\nthe overinterpretation, unless there is ecological data supporting the hypothesis of divergent\nevolution on other traits in the Northern vs. southern populations. For instance, is there any\nevidence for divergence in host-plant use between the northern vs. southern populations ? Or\nfor divergence in mate preferences ?\nRESPONSE: Thanks for making this point – we are in complete agreement, and we have tried\nto limit our discussion of the possible significance of candidate genes that differ between the\npopulations. So far, we could find no evidence for ecological divergence between northern and\nsouthern populations in host plants used. While further study may show subtle differences in\nsome natural history traits, the current evidence suggests this is a case of divergent evolution\nlinked to contrasting behavioral differences in movement.\nLines 275-278 : Given the lack of population differentiation (fig.3A and see my comment on low level of Fst above), I do not\nsee the relevance of discussion about potential DBM incompatibilities here.\nRESPONSE: Thank you for your point. We agree that the DBM model does not provide a good\nexplanation for isolation in this case. We have deleted these lines.\n(2) If the authors want to explore the putative candidate genes involved in the evolution of\ndivergent migration behaviours, considering both the effect of positive selection acting on\nadaptive mutations and purifying selection acting on deleterious mutations would be useful.\ndN/dS analyses can potentially mix up the two effects, you may consider alternative methods\naccounting for pN/pS approaches for instance.\nRESPONSE: We appreciate the reviewer’s suggestion to assess effects of positive selection.\nHowever, several limitations of our current dataset prevent us from performing such analyses at\nthis stage. Although it is in theory possible to test for effects of positive selection using RADdata,\nour RAD data only covers a limited subset of the genome, including just a fraction of the genes\nlocated within the inverted region. Specifically, only 21% of the inversion are represented in their\nentirety. In addition, unfortunately, detailed selection analyses lie beyond the scope of this study,\nwhich is primarily focused on documenting genomic differentiation associated with divergent\npatterns of migration. As we mentioned in our previous responses and in the manuscript, we\nfully agree that identifying candidate genes under selection is a natural next research step. Our\nmanuscript opens a research line of study of selection, structural and behavioral/phenotypic\naspects at a deeper level, and we plan to pursue these questions with more comprehensive\nwhole genome data in future work.\nLine 250-258: Investigating the selective regime acting on the gene located within the locus of\ninterest is highly relevant, but since none of the 32,719 SNPs were fixed between the\nhaplotypes (as stated line 189), this means that there is probably no fixed non-synonymous\nsubstitutions between your haplotypes? As a consequence, signatures of selection may rely\nmore on pN/pS rather than dN/dS.\nRESPONSE: The reviewer is right and we appreciate these comments. We believe that a\nproper assessment of selection deserves the use of WGS data.\nLine 152-153 : Is there any differences in size between the two haplotypes within your locus of\ninterests? If so, what is the origin of such difference in size (gene duplications ? TE enrichment\n?).\nRESPONSE: Unfortunately, the nature of our RAD dataset does not allow us to identify and\nanalyze gene duplications and TE enrichment.\nSpecific comments :\nLine 41: ‘movements play a fundamental role in the evolution of life’ is too general and vague to\nbring sufficient relevance to the manuscript, did you mean that migration likely plays a key role\nin species diversification ? This should be made more precise and referenced, helping the\nreader to immediately connect to the research questions addressed in your manuscript.\nRESPONSE: Our opening sentence is intended to be an engaging introduction to the reader of\nthe direction of the article. We then focus on the topic and provide more specific information and\nreferences in the following sentences.\nLine 68 : I am wondering whether non-migratory populations of Monarch could be considered as\na form of migratory divide ? (see for instance : Hilburn, D. J. (1989). A Non-Migratory,\nNon-Diapausing Population of the Monarch Butterfly, Danas Plexippus (Lepidoptera: Danaidae),\nin Bermuda. Florida Entomologist, 494-499.)\nRESPONSE: We take the reviewer’s point that becoming sedentary also represents a change in\nthe movement patterns of an insect. However, our primary interest is the coexistence of\ndivergent strategies that each involve directional movement, so our interpretation of “migratory\ndivide” is that it implies the coexistence of divergent migratory strategies among sympatric\npopulations that still migrate. This definition also matches with that of literature on vertebrates.\nLine 88 : It is very important to distinguish (1) proximal factors (e.g. temperature encountered by\nan individual) acting on the migration behaviour of that given individuals from (2) selective\nfactors (seasonal variation in temperature in the geographic range of a given species) acting on\nthe evolution of migration behaviour within this species. Here for instance, did you mean ‘shape\nTHE EVOLUTION of contrasting migratory strategies’ ?\nRESPONSE: Thanks for this helpful observation; we have rewritten the sentence as suggested.\nLine 109 : ‘tracking LOCATIONS with optimal conditions’\nRESPONSE: We have adapted the sentence as suggested.\nLine 110 : ‘V. cardui POPULATIONS MOVE from the Paleartic...’ One of the challenges of the\nobservational data provided here is that it does not allow to track individual migration behaviour.\nRESPONSE: Thanks for pointing this out; we have rewritten the sentence as suggested.\nReviewer #3 (Remarks to the Author):\nThank you for the edits. I have no further comments, and will reiterate that I think this is a great\nmanuscript.\nRESPONSE: Thank you very much for your helpful and dedicated review."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The revised version now answers all my previous comments, I would like to particularly congratulate the authors for this\noriginal work with high scientific quality."}, "author_response": ""}]

Decision letter