Recombination Suppression is Unlikely to Contribute to Speciation in Sympatric Heliconius Butterflies

Mechanisms that suppress recombination are known to help maintain species barriers by preventing the breakup of co-adapted gene combinations. The sympatric butterfly species H. melpomene and H. cydno are separated by many strong barriers, but the species still hybridise infrequently in the wild, with around 40% of the genome influenced by introgression. We tested the hypothesis that genetic barriers between the species are reinforced by inversions or other mechanisms to reduce between-species recombination rate. We constructed fine-scale recombination maps for Panamanian populations of both species and hybrids to directly measure recombination rate between these species, and generated long sequence reads to detect inversions. We find no evidence for a systematic reduction in recombination rates in F1 hybrids, and also no evidence for inversions longer than 50 kb that might be involved in generating or maintaining species barriers. This suggests that mechanisms leading to global or local reduction in recombination do not play a significant role in the maintenance of species barriers between H. melpomene and H. cydno. Author Summary It is now possible to study the process of species formation by sequencing the genomes of multiple closely related species. Heliconius melpomene and Heliconius cydno are two butterfly species that have diverged over the past 2 million years and have different colour patterns, mate preferences and host plants. However, they still hybridise infrequently in the wild and exchange large parts of their genomes. Typically, when genomes are exchanged, chromosomes are recombined and gene combinations are broken up, preventing species from forming. Theory predicts that gene variants that define species might be linked together because of structural differences in their genomes, such as inverted pieces of chromosomes that will not be broken up when the species hybridise. However, in this paper, we use deep sequencing of large crosses of butterflies to show that there are no long chromosome regions that are not broken up during hybridisation, and no long chromosome inversions anywhere between the two genomes. This suggests that hybridisation is rare enough and mate preference is strong enough that inversions are not necessary to maintain the species barrier.


Introduction
It is now widely appreciated that the evolution and maintenance of new species is constrained by genetic as well as ecological factors. One major genetic constraint is that, if populations remain in contact, recombination will break down the associations between alleles that characterise emerging species [1]. A large body of work has invoked genetic mechanisms that couple speciesspecific alleles and so reduce the homogenising effects of gene flow [2,3]. These include assortative mating, one-allele mechanisms, tight physical linkage, pleiotropy and multiple (or 'magic') traits. One mechanism that has been extensively studied is the role of genomic rearrangements such as chromosomal inversions in suppressing recombination in hybrids.
Inversions are widespread in natural populations and are often associated with adaptive traits [4,5,6]. Shifts in frequencies of inversions are often associated with ecological niches and clines in natural populations. This has been widely observed across animal taxa including insects (e.g.

Philomachus pugnax
Several authors have predicted that inversions can enable the formation and maintenance of species barriers in sympatry or parapatry by favouring the accumulation of barrier loci in the presence of gene flow [43,63,64,65]. Drosophila species pairs differing by one or more inversions are typically sympatric, whereas homosequential pairs are typically allopatric [43]; in the particular case of Drosophila pseudoobscura, sterility factors are associated with inversions in a sympatric species pair but with collinear regions in an allopatric pair [66]. In rodents, sympatric sister species typically have more autosomal karyotypic differences than allopatric sister species [67]. Sympatric sister species of passerine birds are significantly more likely to differ by an inversion than allopatric sister species, with the number of inversion differences best explained by whether the species ranges overlap [68].
However, inversions are not the only mechanism by which recombination rate can be modified during speciation, and more recently attention has been drawn to the potential role of genic recombination modifiers [57]. If recombination rate were systematically or locally reduced in hybrids, this could be favoured in a manner similar to an inversion. Nonetheless, the very strong effect of inversions on recombination rate, and the fact that they are completely linked to the locus at which recombination is reduced, means that they are perhaps the most likely mechanism of recombination rate evolution during speciation.
We set out to test the role of recombination rate evolution in the maintenance of species barriers in Heliconius butterflies. The 46 Heliconius species allow for a wide range of comparisons at different points along a putative speciation continuum [69,70,71,72]. Chromosomal inversions are known to play an important role in the maintenance of a complex colour pattern polymorphism in Heliconius numata [37]. However, no other Heliconius inversions are known, due to the lack of resolution in traditional karyotyping [73].
Here, we systematically searched for inversions between sympatric populations of two Heliconius species, H. melpomene rosina and H. cydno chioneus, which are sympatric in the lowland tropical forests of Panama. These species differ by many traits [74] including colour pattern [75], mate preference [76,77,78], host plant choice [79], pollen load and microhabitat [80]. Hybrid colour pattern phenotypes are attacked more frequently than parental forms, indicating disruptive selection against hybrids [81]. Assortative mating between the species is strong, and genetic differences in mate preference are linked to different colour pattern loci [78]. Matings between H.
cydno females and H. melpomene males produce sterile female offspring, but male offspring are fertile, and female offspring of backcrosses show a range of sterility phenotypes [82]. Hybrids are extremely rare in the wild, but many natural hybrids have been documented in museum collections [83] and examination of present day genomic sequences indicate that gene flow has been pervasive, affecting around 40% of the genome [84,85]. Modelling suggests that the species diverged around 1.5 million years ago, with hybridisation rare or absent for one million years, followed by a period of more abundant gene flow in the last half a million years [86,87], suggesting that the species originated in parapatry, but have been broadly sympatric and hybridising during their recent history.
Models predict that inversions, or other modifiers of recombination, can be established during both sympatric speciation and secondary contact [43,55,56,63,88]. Furthermore, the genetic basis for species differences between H. melpomene and H. cydno is well understood and would seem to favour the establishment of inversions. Wing pattern differences are controlled by a few loci of major effect [75], some of which consist of clusters of linked elements. There is also evidence for linkage between genes controlling wing pattern and those underlying assortative mating [78]. The existing evidence for clusters of linked loci of major effect would therefore seem to favour the evolution of mechanisms to reduce recombination between such loci, and hold species differences in tighter association.  Figure 1). We also generated long read sequencing data and new genome assemblies for both species, to test for inversions on smaller scales. This is the first systematic survey of genome structure and recombination at fine scale in a Lepidopteran species, which may be relevant to the study of holocentric chromosomes and species with no recombination in the heterogametic sex (females in Lepidoptera), in addition to exploring the role of structural variations during speciation.

Summary of crosses and improved ordering of Heliconius melpomene assembly
We raised crosses within H. melpomene, within H. cydno and between H. cydno and H. melpomene, generated RAD sequencing data for a total of 963 offspring, and built linkage maps for each sequenced cross ( Table 2, Table S1, Table S2). We also generated Pacific Biosciences long read data for pools of male and female larvae from H. melpomene cross 2 and H. cydno cross 1 (

Differences in recombination rate between species and hybrids
We placed our linkage maps on the new H. melpomene chromosomal assembly to look for evidence of reduced recombination in the hybrids. Figure 2 shows Marey maps [92] for each of the melpomene hybrids, with recombinations from all crosses per species combined (see Figure S1 and

Recombination maps show no major inversions between species
Page of 12 42 We also examined our recombination maps for evidence of inversions between species ( Figure 1).
However, there are no regions of any map with a detectable reversed region in H. cydno or the hybrids with respect to H. melpomene ( Figure 2). This is true for the species maps and for all individual cross maps ( Figure S1). This indicates there are no large fixed inversions between H.
Known or predicted chromosome inversions involved in the maintenance of species barriers are typically megabases long (  Figure   S3, Figure S4). Simulation of random inversions indicates that our existing maps give us power to detect ~98% of 500 kb inversions, ~90% of 250 kb inversions and ~75% of 100 kb inversions ( Figure S5). These sizes are smaller than most inversions known to be associated with adaptive traits or species barriers; however, they are on the order of the sizes of the known inversion in Heliconius numata and others in, for example, sticklebacks (Table 1). Therefore, we cannot rule out the presence of an inversion in any remaining gap between markers within H. melpomene or H.
cydno from the recombination maps alone.

Detection of small inversions with long sequence reads and trio assemblies
To test for the presence of smaller fixed inversions between H. melpomene and H. cydno that were undetectable using our recombination maps, we generated Pacific Biosciences long read sequence data for pools of male and female larvae from one each of the H. melpomene and H.
cydno crosses used to generate recombination maps (Table 3, Figure S6, Figure S7). We called candidate inversions from the long read data using PBHoney to identify reads with clipped alignments, realign the clipped read ends, and detect such split reads with inverted alignments. We also generated Illumina short read assemblies of the maternal and paternal genomes of one offspring from the same crosses used to generate the linkage maps and PBHoney candidates.
These assemblies were constructed using a trio assembly method that separates maternal and paternal reads from one offspring and attempts to construct haplotypic assemblies of each parental genome, providing longer and more accurate contigs compared to standard Illumina assemblies of heterogenous genomes such as those of Heliconius species ( [93]; Table 3). We aligned these trio assemblies to the H. melpomene genome and assessed whether the resulting alignments supported or conflicted with candidate split read inversions.
In total, 1494 raw PBHoney split read candidates were identified across the four samples (two sexes for each of two species; Table 3, Table S3), but many of these were rejected as being incompatible with the linkage maps and trio assemblies. 438 candidates (30%) spanned recombinations in the linkage maps (Table S3), of which 294 (20%) were longer than 1 Mb, with 36 (2.5%) longer than 10 Mb. A further 344 candidates (23%) were removed because the candidate was spanned by a trio scaffold from the same species by 50% of the inversion length on either side (Table S3). These rejected candidates may be false positives; when we simulated PacBio reads directly from the ordered Hmel2 reference genome, PBHoney called 49 'false-positive' inversions.
Alternatively, they may be generated by polymorphic inversions, but as we are only interested in fixed inversions, we have not considered these candidates any further.
A further 199 of the 1494 candidates (13%) were removed because they were shorter than 1 kb (Table S3); while these may be real, they are very unlikely to be necessary for species barriers as there is already above-background linkage disequilibrium between SNPs separated by 1 kb or less in H. melpomene [94]. The remaining 463 split read candidate inversions from the four samples were merged into 185 candidate groups based on their overlaps. The four samples were sequenced with variable coverage, with low coverage for the H. cydno males in particular (Table   3). Given this, 173 additional candidates with less robust support that overlapped with the 185 merged groups were included in the data set (Table S3). Each of the merged groups was then classified based on their presence in either or both species and their support by split read and trio assembly evidence (

Candidate inversions assessed using trio assemblies and population genetics
As the false positive rate for PBHoney is high, we made further use of the trio assemblies to find support for the remaining PBHoney candidate inversion groups (Table 5, Figures S8-14). 13 H.
cydno and 9 H. melpomene groups were also supported by trio scaffolds aligning with inverted hits within inversion breakpoints ( Figure 3, "Split reads and trio assembly"; Figures S8, S10). Of these, are a small number of likely species-specific inversions, but that these are too small to play a role in speciation. Notably, none of these candidate inversions were located near loci known or suspected to determine species differences in wing pattern or any other trait with known locations [90].
We also calculated FST and fd [95] across inverted regions (Figures S8-S14) to test for differences in the level of admixture at the inversion relative to surrounding regions. An inversion acting as a species boundary typically produces a signal of elevated FST and reduced fd [3,33,34,38,96], and an inversion enabling the spread of an adaptive cassette might produce a signal of elevated fd [59].
However, we see very little evidence for increases in these statistics within the handful of  no annotated features, although of course this does not rule out some functional importance of this region.
Some candidates with only split read evidence, many in only one sex, are hundreds of kilobases long (outliers labelled in Figure 3, particularly those marked with circles, where breakpoints are not near contig boundaries), which, if real, may be relevant to speciation. However, given the large number of false positives produced by PBHoney, the lack of supporting evidence from trio assemblies, and the lack of FST and fd signals at these candidates, it is unlikely these candidates, even if they are real, are substantial species barriers. rearrangement, on current scaffold ordering, appears to be an inversion followed by a translocation (for scaffold Herato0214), but given that scaffolds Herato0212, Herato0213 and Herato0214 are all found at the same marker on the linkage map, it may be that these scaffolds need to be reoriented and reordered, so this region could represent a single inversion.
From these comparisons, it is clear that, while some chromosome rearrangements exist, Heliconius species do not feature inversions on the scale of, for example, Drosophila, where there are over 300 known inversions in D. melanogaster and over 50 on the third chromosome alone for D. pseudoobscura and D. persimilis [97].

Discussion
We have systematically tested the hypothesis that reduced recombination rates in hybrids might be favoured during speciation with gene flow [57]. High density linkage maps and high coverage longread sequence data give us considerable power to both measure recombination rate and detect structural rearrangements, but we find no evidence for differences between H. melpomene and H.
cydno at a scale that is likely to influence the speciation process. chromosomes. This suggests that lepidopteran males do not require recombination for meiosis to proceed, which is perhaps consistent with the fact that meiosis in lepidopteran females occurs without recombination; alternatively, recombination could occur at the ends of chromosomes and be undetectable with our current genome assemblies.
We have found no evidence for significantly reduced recombination in hybrids. Larger crosses would also give greater resolution to this test, and might detect smaller regions of reduced recombination. Nevertheless, we can decisively rule out the presence of any multi-megabase rearrangements among these samples.
We complemented the linkage maps with PacBio sequencing and trio assemblies, in order to detect candidate inverted regions at a smaller scale. This approach also has challenges and generated a high rate of false positives. One potential source of difficulties is reliance on alignment to the H. melpomene reference genome assembly. The existing assembly has 25% transposable element content [98] and is likely missing around 6% of true genome sequence, mostly due to collapsed repeats [90]. Inversion breakpoints are typically repeat-rich, which increases the likelihood that reads or scaffolds will not align correctly, and that the breakpoint regions could be misassembled or absent in the reference genome and in the trio assemblies. This problem may be worse for H. cydno, where more divergent sequence may align incorrectly or not align at all, and unique H. cydno sequence will not be present in the reference (an additional ~5% of H. cydno sequence did not map to the H. melpomene genome compared to H. melpomene samples (Table   3)). These issues may explain the high observed rate of false positives in our data.
Nonetheless, many of the candidate inversions found in both species are likely to be real with respect to the reference genome, as they are supported by multiple lines of evidence and fall near contig boundaries. Where these are identified in both species, we hypothesise that they reflect misassemblies relative to the reference assembly. These misassemblies could be due to whole inverted contigs, or to misassembled inverted regions at the ends of contigs, which may be preventing the contigs being joined by spanning reads. These examples of misassemblies demonstrate that our methods are capable of detecting large rearrangements in the sampled reads relative to the genome assembly.
In contrast, candidate species-specific inversions are typically smaller than the candidate misassemblies, and are mostly not supported by multiple lines of evidence. Indeed, we can find no compelling fixed candidate inversions supported by both the split read and trio assembly data sets that also show evidence of an increase in FST, except for the 11.7 kb inversion shown in Figure   S8.4, which is too small to substantially increase linkage across this locus beyond that expected by normal decay of linkage disequilibrium [94]. It is possible that some of the candidates with less robust evidence are genuine, given the limitations described above, but on the existing evidence we cannot robustly identify any inversions that are likely to be involved in maintaining species barriers between H. melpomene and H. cydno.
Although existing models identify situations where chromosome inversions can spread to fixation between two species and maintain a species barrier, the spread of inversions is by no means inevitable during speciation with gene flow. For example, in the model of Feder, Nosil and Flaxman [56], inversions only fix when the strength of selection on the loci captured by the inversion is considerably lower than migration between the species. Similarly, Dagilis and Kirkpatrick [58], modelling the spread of inversions that capture a mate preference locus and one or more epistatic hybrid viability genes, show that inversions are unlikely to spread where pre-and post-zygotic reproductive isolation is already strong. In a recent review, Ortiz-Barrientos et al. [57] also highlight that during reinforcement, assortative mating and recombination modifiers such as inversions are antagonistic; if strong assortative mating arises first, there is only weak selection for reduced recombination.
We considered H. melpomene and H. cydno to be good candidates for the spread of inversions because there are linked loci causing reproductive isolation and hybridisation has been ongoing for much of their history. Comparisons between sympatric and allopatric populations of the two species have shown that almost a third of the genome is admixed in sympatry [84]. Thus, hybridisation has been ongoing for a long time, perhaps at a low rate. Nonetheless, perhaps strong selection on species differences and assortative mating do not provide the conditions that would have promoted the spread of inversions. Aposematic warning patterns are strongly selected [99], with F1 hybrids twice as likely to be attacked as parental phenotypes [81], and pre-zygotic isolation in the form of mate preference is almost complete [76]. Therefore, inversions may not be necessary for divergent loci to accumulate between the species. Perhaps in this case, the evolution of strong assortative mating was favoured by reinforcement selection before inversions or other mechanisms for reducing recombination arose.
Page of 21 42 This conclusion is also consistent with the apparently low background rate of fixation of inversions in Heliconius genomes. H. melpomene and H. erato, which last shared a common ancestor over 10 million years ago, have largely collinear genomes, and there is substantial chromosomal synteny across the Nymphalids (Ahola et al. 2014). This contrasts with the frequent occurrence of inversions in Drosophila genomes, for example, even within single species. The association of multiple inversions with the wing pattern polymorphism in H. numata is all the more remarkable given the low background rate of inversions in these butterflies. In conclusion, we have clearly shown that species barriers can persist during speciation with gene flow without suppression of between-species recombination.

Materials and Methods
Bespoke scripts, data files and further details of methods can be found in the data repository associated with this manuscript. This repository will be submitted to Dryad on acceptance but is currently available on Dropbox at https://www.dropbox.com/sh/aezq5a35f78o03l/AAALDO0kvB1r-YMXH8_1PTiIa?dl=0. and stored at -20ºC. Adults were preserved and stored in the same way after dissecting wings.

Crosses
Adult males were preserved after mating; adult females were preserved when egg laying ceased.
Interspecific crosses were achieved by placing pupae from a H. cydno stock in a cage of wild H. melpomene males. Hybrid individuals used to produce linkage maps were then obtained from backcrosses produced by mating F1 males to females from the H. cydno stock. As with the infraspecific crosses, eggs were collected daily, and larvae were then raised individually; however, unlike the infraspecific crosses, individuals were raised to adults.

Dissection and DNA extraction
All dissections were performed with a new sterile scalpel, fresh Parafilm and tweezers washed in 80% ethanol. For adults, the thorax was dissected away from the head and abdomen and cut in half along the median plane. One half of thorax was used for DNA extraction with the remaining tissue returned to storage. Larvae were cut in half along the median plane and gut contents removed, with one half used for extraction and the other returned to storage. Some larvae were too small to be cut along the median plane and were cut along a transverse plane instead, whereas some halves of large larvae were cut into smaller pieces to fit within kit tissue size limits.  Libraries were A-tailed (add 5uL 10x NEB Buffer 2, 1uL 100mM dATP, 3uL exo-Klenow fragment (NEB E6044) and incubate for 30 minutes at 37°C, then purify as before to 40.5uL eluate) and P2 adapters were ligated (add 3uL 10uM P2 adapter, 5uL 10x NEB Buffer 2, 0.5uL 100mM rATP, 2uL T4 Quick Ligase (NEB M2200L) and leave for 60 minutes at room temperature); samples were purified as above and quantified, to a final yield of 5-20 ng/uL. Libraries were amplified by PCR using P1 and P2 adapter primers for 12-16 cycles depending on initial yield, and using P2 primers with different barcodes for each batch of 16 P1-ligated samples. Each batch was amplified in 8 separate reactions, to increase diversity of initial samples and reduce sequencing of PCR duplicates, and pooled. Amplified samples were purified as above and eluted in EB-Tween 0.1%.  Tables S1 and S2).

Pacific Biosciences library prep and sequencing
Larvae from H. cydno cross 1 and H. melpomene cross 2 were sexed by examining the segregation of the Z chromosome where linkage maps were available at time of sequencing, or the ratio of average autosomal RAD sequencing read coverage to sex chromosome read coverage.
Pools of 12 males and 12 females were constructed, using larvae with the highest DNA yields. 12 individuals were used per pool to achieve sufficient yield for Pacific Biosciences sequencing and to approach even coverage of the four parental genomes (insufficient DNA was available to generate long read sequences from parents directly). Each pool was size selected to fragments greater than 6 kb using the SageELF system. Libraries were prepared following the standard 10 kb protocol (SMRTbell Template Prep Kit 1.0, 100-259-100) but with only two bead clean ups at the end rather than three. Libraries were sequenced on a PacBio RSII machine with P6/C4 reagents for 3-4 hour run times. Subreads were used for analyses.

Linkage mapping: hybrid crosses
Due to more complex cross structure involving F2 populations, smaller cross sizes and lower sequence quality for some crosses, different methods were used to construct linkage maps for the

Genome scaffolding
Hmel2 scaffolds were manually ordered according to the linkage maps for each of the three Heliconius melpomene crosses wherever possible. A small number of misassemblies in Hmel2 were corrected, with scaffolds being split and reoriented where necessary. Not all scaffolds could be ordered based on the linkage maps alone, so Pacific Biosciences reads were also used. PacBio reads were aligned to Hmel2 scaffolds using BWA mem with -x pacbio option [106]. Scaffolds were ordered by manual inspection of spanning reads between scaffolds, identified and summarised by script find_pacbio_scaffold_overlaps.py. Chromosomal positions were assigned by inserting dummy 100 bp gaps between each pair of remaining scaffolds. Although PacBio sequencing could fill gaps between scaffolds, we chose not to do this for these analyses to avoid disrupting Hmel2 linkage map and annotation feature coordinates.

Recombination rate measurement and permutation testing
CentiMorgan values were calculated using the recombination fraction alone, as the maps were sufficiently fine-scale that mapping functions were not necessary [107]. Per-cross maps ( Figure  S1) and map statistics ( Table 2) were calculated for F1 parents within-species and grandparents for hybrids. Recombination rates were calculated in windows of 1 Mb with 100 kb steps.
Chromosomes were tested for reductions in recombination rate in the hybrids compared to H. melpomene or H. cydno using a bootstrapped Kolmogorov-Smirnov test suitable for discrete data with ties such as recombination counts (ks.boot in the R Matching package [108]), using a onetailed test for reduced rates in hybrids with 10 000 bootstrap samples, declaring significance at a 0.05 false discovery rate with control for multiple testing (42 tests, with 2 comparisons for each of 21 chromosomes). 1 Mb windows were tested for differences in recombination rate by calculating null distributions of rate differences by permutation of species labels across all offspring, again testing at a 0.05 false discovery rate over 270 000 permutations, controlling for multiple tests with 3 comparisons for each of 2 633 windows. 95% confidence intervals in Figure S2 were calculated by bootstrap, sampling offspring for each species by replacement 10 000 times and calculating cM values, plotting 2.5% and 97.5% quantiles for each window.

Inversion discovery
PBHoney (in PBSuite version 15.8.24 [109]) was used to call candidate inversions between H. melpomene and H. cydno, using alignments of PacBio data to ordered Hmel2 scaffolds made with BWA mem with -x pacbio option [106], retaining only primary alignments, and accepting alignments with minimum mapping quality of 30 in Honey.py tails, running on each of four samples (H. cydno females, H. cydno males, H. melpomene females, H. melpomene males) separately. Breakpoint candidate sets were compiled together into one file and scaffold positions converted to chromosome positions using script compile_tails.py. PBHoney was run with default options, requiring a minimum of 3 overlapping reads from 3 unique zero-mode waveguides to call a breakpoint candidate. As the H. cydno male sample had low coverage, we also ran PBHoney requiring a minimum of 2 reads from 1 zero-mode waveguide and included these tentative candidates where they overlapped with candidates from other samples called with the default settings.
PBHoney was tested for false positives by simulating PacBio reads with pbsim 1.0.3 [110], generating a sample profile using the H. melpomene female sample and simulating 15 'SMRT cells' at 5x coverage each. Simulated data was then aligned with BWA and inversions called with PBHoney as above.
Trio assemblies were aligned to Hmel2_ordered using NUCmer from the MUMmer suite ( [111]; version 3.23), followed by show-coords with -Tlcd options, to produce tab-separated output including scaffold lengths, percentage identities and directions of hits.
Script detect_inversion_gaps.py was used to integrate the PBHoney inversion candidates with the linkage maps, trio alignments and H. melpomene annotation (from Hmel2). As this data is being used to rule out inversions in regions without recombinations, PBHoney inversion candidates were rejected if at least one recombination for the same species as the candidate was contained within the inversion. PBHoney candidates were also rejected if there was a trio scaffold alignment spanning the candidate inversion, with spanning defined as extending more than half the length of the candidate inversion in either direction. Finally, candidates shorter than 1 kb were rejected, as linkage disequilibrium between SNPs separated by 1 kb or less in H. melpomene rises above background levels [94] and so inversions are unlikely to be required to maintain linkage. The retained inversion candidates were then combined into groups by overlap.
Each group of overlapping inversion candidates was then classified as follows ( Figure 3, Table 5, Figures S8-14): Split reads and trio assembly, group has at least one PBHoney inversion candidate and at least one trio scaffold with forward and reverse alignments either side of an inversion breakpoint; Split reads only, group has at least one PBHoney inversion candidate in at least one sex, but no matching inverted trio scaffolds; Split reads in one species, trio assembly in both, group has at least one PBHoney inversion candidate in at least one sex of only one species, but trio assembly has inverted scaffolds in at least one sex in both species. These classifications do not cover whether there are multiple contigs across the candidate inversion (see Table 5, Figures S8-14) or whether trio scaffolds with alignments that span whole PBHoney inversion candidates or cross candidate breakpoints (see Figures S8-14).

Population genetics statistics
FST and fd were calculated within and around candidate inversions following Martin et al. 2013 [84] and Martin
Each page shows linkage map, split read, trio assembly and population genetics evidence for each candidate inversion group, across a region of the Hmel2 genome assembly. Black lozenge, candidate inverted region (white text in lozenge shows length of region). Genome, contigs from