High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae)
BMC Genomics volume 24, Article number: 443 (2023)
Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics.
The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function.
Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).
The genus Hyles comprises about 30 species, is distributed worldwide and, with an estimated divergence time of 5.7–9.3 million years (Mya) , is rather young from an evolutionary perspective . Some species are still in the process of speciation, and others hybridize because reproductive barriers are still low. These processes are difficult to discern in Hyles euphorbiae, which was previously comprised of five separate species . The Spurge Hawkmoth, Hyles euphorbiae is a charismatic Palearctic species with large, colorful, and polymorphic larvae and camouflaged, heavy adults with strong flight abilities (Fig. 1a). Despite their aposematic coloration, the larvae do not sequester the toxic spurge diterpene esters  from their food plants. Instead, they protect themselves by spewing the plant slurry that has not yet been detoxified. Larval specialization on toxic host plants of the genus Euphorbia has evolved twice independently within the genus Hyles [5, 6]. The impressively high morphological variability of larvae has complicated the taxonomy of H. euphorbiae by contributing to an overestimation of species diversity (overview in Hundsdoerfer et al. ). Similarly, the high intraspecific diversity of mitochondrial marker genes bedeviled the reconstruction of the molecular phylogeny of the formerly five species (synonymized with H. euphorbiae in 2019 ), but provided valuable resolution for phylogeography . Although some larval patterns are correlated with geography  and are thus expected to be based on underlying genetic diversity causing phenotypic variability, others appear to be environmentally driven by phenotypic plasticity.
Early studies have shown that similarity in wing patterns is not based on phylogenetic affiliation in this genus [1, 2]. Seven basic wing patterns are observed in the Central Palearctic Hyles species, which neither correlate with currently defined species, nor reflect phylogenetic relationships within the genus. One very prominent wing pattern shows many white stripes on brown forewings, a set of shared character states  that originally led to the assumption that the Palearctic H. livornica and the Australian H. livornicoides were conspecific with the New World species H. lineata. After their recognition as three species, they were still considered to be a distinct subgenus ‘Danneria’ , but this has been refuted as a clade by previous molecular phylogenetic work . The species group in very different parts of the tree and do not form a tight cluster of cryptic species. Another prominent wing pattern is one of dark brown spots and stripes on a lighter, cream-colored background, corresponding to the typical forewing pattern of H. euphorbiae, and thus the group included numerous species with a similar wing pattern (or slight variations thereof) . However, again, some of these, e.g., H. centralasiae, H. nicaea, and H. euphorbiae were not found to have a close molecular phylogenetic relationship . The species H. vespertilio (Fig. 1b), the genome of which was recently published , has a forewing that lacks the pattern elements described. The forewings of this species almost completely lack a wing pattern , raising the possibility that the gene(s) controlling wing pattern formation are inactive, making genomic comparisons with this species particularly intriguing.
Interspecific differences in forewing patterns within the genus Hyles should be based on detectable genetic differences, as patterns are stable within species in the well-separated, oldest Neotropical (and Nearctic) taxa . In the Palearctic, incomplete lineage sorting and ongoing hybridization impede such insights, justifying ongoing systematic, phylogenetic and taxonomic research (e.g., Patzold et al. ).
Genomic data promise insights into numerous aspects of speciation, including ongoing gene flow, food plant utilization, and the genetic basis for morphological differentiation. Therefore, we sequenced and examined the genome of Hyles euphorbiae. Numerous Sphingidae genomes are currently being published (e.g. Hyles vespertilio, Macroglossinae, by Pippel et al. , Mimas tiliae, Smerinthinae, by Boyes et al. , Laothoe populi, Smerinthinae ). This wealth of data will provide insights into the evolution of wing patterns in hawkmoths. Pioneering studies of the genes underlying the wing pattern of Lepidoptera have been performed on the family Nymphalidae, facilitated by a classical model describing wing-pattern elements (e.g. eyespots, bands) in an idealized ‘nymphalid groundplan’ [16, 17]. The genetic induction of colored scales of pattern elements on Lepidoptera wings has been well-studied in several genera of the family Nymphalidae, namely Bicyclus [18, 19], Heliconius , Junonia , and Vanessa . It has been previously proposed that the evolution of lepidopteran wing pattern stripes occurred through the repeated gain, loss, and modification of only a handful of serially repeated elements, arranged in a modular architecture with narrow stretches of the genome associated with specific differences in color and pattern . In addition, phenotypic plasticity in variation of colors and certain color patterns of species is expected to be primarily driven by biotic and abiotic factors  acting as a selection pressure on individuals with differential expression of genotypes [18, 25]. Strong selection pressure can limit polymorphism, keeping the best-protected wing pattern, but is has been shown that alternative adaptive phenotypes can be maintained by heterozygote genotypes of the wing-pattern P supergene in natural populations of Heliconius numata . The P supergene represents a non-recombining part of chromosome 15 consisting of three stretches with co-adaptive loci (P1-P3 of sizes 400 kb, 200 kb and 1150 kb) that are kept in close linkage by polymorphic inversions .
Optix is a single-exon gene in Heliconius [20, 28] encoding a homeobox transcription factor that regulates the production of ommochrome pigments  and is associated with red and orange forewing patterning in these butterflies. Nearby non-coding regions control its expression in Heliconius wing development  and these are the regions of divergence, while the coding region is a conserved homeobox gene. The forewings of H. euphorbiae are known for their pink flush in some regions of its distribution range (see moths in Fig. 3 of Hundsdoerfer, Lee et al. ), whereas red is not prominent in the grey wings of H. vespertilio.
The WntA signaling ligand is highly conserved at the amino acid level within Heliconius ) and is associated with the forewing black band. Variation is again more likely to be influenced by expression during wing development and is therefore found in the nearby control region .
The wing pattern gene cortex is associated with the yellow patterns on Heliconius wings  and has been suggested to underlie switches in color pattern across Lepidoptera . For example, it has been shown that insertion of a transposable element (TE) into an intron of cortex results in industrial melanism , i.e. black morphs in the Peppered Moth (Biston betularia; Geometridae), corroborating its function across Lepidoptera, due to, e.g., the cortex TE insertion. This insertion , estimated to have occurred around 1819, could only manifest itself in the population through a selection pressure against the light-colored moths showing up on the dark trees and thus experiencing higher predation. In Heliconius numata, cortex lies within the P1 supergene inversion polymorphism and controls color pattern switching among mimicry morphs [26, 31].
Expression patterns of the two genes Distal-less and engrailed  have been postulated to be a homologous developmental mechanism between butterfly and moth eyespots, also studied in saturniid moths of the genera Antheraea and Saturnia . The gene aristaless, known to control wing color variation , has recently been shown to play an additional role in wing appendage formation during butterfly development , showing the seminal possibilities this field of research opens in understanding morphological evolution on the level of gene function. Overall, it is expected that a set of conserved, flexible wing patterning genes, possibly linked in a supergene, has also driven the rapid morphological diversification in the genus Hyles.
In contrast to H. vespertilio , in which the forewings have a naturally occurring knock-out appearance (as if the wing pattern genes were dysfunctional) of near-uniform grey wings lacking high-contrast patterning, H. euphorbiae shows important elements of the typical ground forewing pattern of the genus, which has been reconstructed as the ancestral set of characters for proto-Hyles . By comparing the chromosome-level genomes of H. euphorbiae and H. vespertilio, the present work aims to provide reference genome data for future studies to understand the origin of Hyles wing patterns as phenotypic variability starting with several wing pattern control genes described above (e.g. [20, 23, 30]).
Hyles euphorbiae genome assembly with chromosome-level scaffolding
The H. euphorbiae genome was assembled with Pac-Bio HiFi reads  combined with Hi-C data. Assembly contiguity statistics are summarized in Table 1. The final contig assembly consists of 56 scaffolds with an N50 of 18.2 Mb (321 contigs with an N50 of 2.76 Mb) and a size of 504 Mb and is available at NCBI (SRA accession number SRR17432892, BioProject PRJNA794104, BioSample SAMN24610150 and genome JALBCW000000000, JALBCX000000000, see data availability statement for itemized accession numbers). The annotated circular mitochondrial genome has a length of 15,322 bp. Final assembly statistics are summarized in Table 2.
Chromosome scaffolding with Hyles vespertilio Hi-C data
HiRise  scaffolding of H. vespertilio using more than 132 million read pairs (NCBI SRA accession number SRX14530528) yielded a scaffold N50 of 22.1 Mb. In total, 146 joins and only one break of the input assembly were introduced. The contact map clearly shows 29 well-supported scaffolds representing chromosomes (Fig. 2). Sizes of chromosomes of both Hyles species are of the same order of magnitude, but not identical (Table 3). The assembly of H. vespertilio is almost 147 Mb larger than that of H. euphorbiae.
Chromosome structure in Sphingidae
Comparison of single copy orthologs in the H. euphorbiae genome assembly to those in H. vespertilio shows strong conservation of both synteny and chromosome structure, defined as conservation of gene order. This suggests that the last common ancestor of Hyles had 29 chromosomes that subsequently remained intact (Fig. 3a). These 29 chromosomes correspond to intact Merian elements, the ancestral linkage groups of Lepidoptera , with the exception of three simple fusion events between pairs of Merian elements (M17 + M20, M30 + M9, M24 + M25). Next, the orthologs of H. euphorbiae were compared with those of Deilephila porcellus (Fig. 3b), both of which belong to Macroglossinae, a subfamily of Sphingidae. This also revealed a set of 29 orthologous chromosomes, suggesting that chromosome structure has remained stable throughout the evolution of this subfamily. To determine whether the stability of this set of 29 chromosomes extends throughout Sphingidae, we compared the orthologs of two further species, Manduca sexta (n = 28; Fig. 3c) of the subfamily Sphinginae and Laothoe populi (n = 28; Fig. 3d), which belongs to a third subfamily, Smerinthinae. This revealed that 27 chromosomes of M. sexta are orthologous and highly syntenic compared to H. euphorbiae. The remaining chromosome in M. sexta (CM026259.1) maps to two autosomes in H. euphorbiae (CM041057.1, CM041033.1), thus accounting for the difference in karyotype. This chromosome in M. sexta corresponds to a M17 + 20 + M29 fusion. Similarly, one chromosome of Laothoe populi (HG992147.1) is orthologous to two autosomes in H. euphorbiae (CM041039.1, CM041057.1). This is due to a fusion between M29 and M4. Therefore, two independent fusion events have occurred in L. populi and M. sexta. This would suggest that the Sphingidae originally had a karyotype of n = 29 that has remained largely intact with a limited number of fusion events in extant species. Moreover, the strong conservation of gene order in H. euphorbiae compared with M. sexta illustrates limited intrachromosomal rearrangements since the last common ancestor of the Sphingidae.
The two species of the genus Hyles have one more chromosome than B. mori (n = 28), but the alignment between H. vespertilio and B. mori can still be illustrated (Fig. 4a) to allow comparison by visual inspection. Chromosome proportion values of this alignment are reported in Table S1.
CIRCOS plots of the alignment between the two species show well-preserved synteny, except for some chromosomes that could be involved in chromosome rearrangements. Chromosome 2 (HvChr2) of H. vespertilio corresponds to chromosomes 2 and 26 of B. mori (BmChr2, BmChr26). Chromosome 24 of B. mori, BmChr24, corresponds to parts of H. vespertilio chromosomes 22, 24, and 28 (HvChr22, HvChr24, HvChr28), and chromosome 11 of B. mori (BmChr11) corresponds to parts of H. vespertilio chromosomes 11 and 26 (HvChr11, HvChr26). These chromosome rearrangements between B. mori and H. vespertilio are consistent with those between B. mori and M. sexta [37, 43], corroborating the strong synteny between the two hawkmoth species, H. vespertilio and M. sexta (Fig. 4b). Chromosome 23 of B. mori (BmChr23) is split into H. vespertilio chromosomes 23 and 29 (HvChr23, HvChr29), resulting in an additional chromosome in H. vespertilio (HvChr29). In M. sexta, which has 28 chromosomes, HvChr29 and HvChr15 correspond to MsChr28, thus showing a different combination.
The plot comparing the 29 chromosome sequences of H. vespertilio and H. euphorbiae (Fig. 4c) illustrates the highly conserved synteny within the genus Hyles in the definition of collinearity, the conservation of block order in the two sets of chromosomes. The larger size of the H. vespertilio genome based on the genome size estimation and its longer assembly than that of H. euphorbiae are reflected by the larger size of every chromosome (Fig. 4c). Annotation and methylation profile.
The proportion of the genome covered by the major classes of repetitive elements is 37% and 47% for H. euphorbiae and H. vespertilio, respectively (Table 4, Fig. 5). Thus, H. vespertilio has a much larger genome (651 Mbp to 504 Mbp in H. euphorbiae) and a higher proportion of repetitive DNA than H. euphorbiae. A comprehensive comparison of transposon content in seven species of the family Sphingidae (Fig. 5) demonstrates the dynamics of repetitive elements and the contribution of recent bursts in transposable element families to genome size expansion in H. vespertilio and independently in Laothoe populi.
The number of genes per chromosome is very similar in the two species’ genomes presented here (Fig. 6), but exhibits a wide range from a minimum of 169 genes in both species on chromosome 29 to 826 / 798 genes in H. vespertilio / H. euphorbiae on chromosome 22.
The mitochondrial genome of H. euphorbiae which was assembled with the mitoHifi pipeline [45, 46] based on the closely related species, Theretra oldenlandiae, is illustrated in Fig. S1. It was annotated using MITOS and contains 13 protein coding genes, two ribosomal RNA (rRNA) genes plus 22 transfer RNA (tRNA) sequences and the control region.
The methylation profile of H. euphorbiae (track “HL Methylation” in the genome browser; e.g. Fig. S5a) shows modification probabilities for every site. Base modifications with probability of modification < 50% (hypomethylation) are colored blue and base positions that have a high probability to be methylated and thus not be expressed are marked red (base modification > 50% probability, hypermethylation).
Wing pattern genes
The wing pattern genes optix, WntA, cortex, aristaless and Distal-less (accession numbers of reference sequences are presented in Table S2) were identified with high confidence using BLAT [47, 48]; only one hit was found for each with thresholds of identity above 85% and bit score above 420.
The hits of the Heliconius erato optix protein sequence revealed a position on chromosome 23 in H. euphorbiae (HeChr23, "sc_12: 1,582,862–1,583,680", size: 819 bp) and H. vespertilio (HvChr23, "ScWp86a_31_HRSCAF_41: 20,799,551–20,800,375", size: 825 bp). No repetitive elements were annotated within the area of the coding sequence in either species (Fig. S2). However, upon zooming out 10 × or 100 × a high amount of indels are seen in the genome browser (400 kb up-& downstream of the coding sequence, Fig. S3).
The Heliconius himera WntA amino acid sequence (Table S2) mapped to the Hyles genomes returned a position on chromosome 28 in H. euphorbiae (HeChr28, “sc_25: 5,829,059–5,837,439”, spanning six exons and 8,381 bp; Fig. S4a; annotated by transcript LOC115447391_rna-XM037443222.1.47 of Manduca sexta spanning seven exons and 17,247 bp) and H. vespertilio (HvChr28, “ScWp86a_140_HRSCAF_198: 8,200,403–8,210,485”, spanning six exons and 10,083 base pairs, Fig. S4b; annotated by evm07703.91 spanning seven exons and 14,395 bp). Views of the WntA BLAT results are illustrated in Fig. S4. There are numerous differences in the repeats between the two Hyles species. For example, H. vespertilio contains a 443 bp long DNA repeat insertion (“ScWp86a_140_HRSCAF_198: 8,200,940–8,201,382”; rnd-4_family-526; family TcMar-Tc1, pos. 512–1311 in the repeat) in intron 2 that H. euphorbiae lacks. The genomic interval spanning the exons and introns of WntA is inverted in H. euphorbiae (or H. vespertilio, depending on the polarity definition).
BLAT search of the M. sexta cortex protein sequence (8 exons) resulted in a match corresponding to 7 exons on chromosome 17 in both species [Fig. S5; H. euphorbiae (HeChr17, “sc_9: 7,671,034–7,674,090”) and H. vespertilio (HvChr17, “ScWp86a_210_HRSCAF_302: 14,798,018–14,801,349”)]. Views of the cortex BLAT results are illustrated in Fig. S5. The methylation profile in H. euphorbiae (Fig. S5a) indicates normal transcription activity in this stretch of exons, as the probabilities of base modifications are all < 50% (blue). The position, quality, and quantity of repeats in introns differ between the two species. For example, the first intron in H. euphorbiae contains no repeats, whereas in H. vespertilio it contains two LINEs in addition to a 211 bp stretch (ScWp86a_210_HRSCAF_302: 14,798,399–14,798,609; Fig. S5b) of unknown repeats (named rnd-1_family-40). It overlaps downstream with the LINE of family L2, which shows an irregular simple repeat of 5’ TATT. It is in intron 1 of the cortex X1 sequence of M. sexta (annotated with M. sexta transcript LOC115451467_rna-XM_030179809.2.8). Again, the area surrounding the gene cortex is inverted in H. euphorbiae (or H. vespertilio, depending on the polarity definition). The 100 kb views of the cortex BLAT results are illustrated in Fig. S6 and reveal a high number of repeats in the vicinity of the stretch of gene exons in both species.
The BLAT search of M. sexta aristaless (in 4 variants, Table S2) revealed the gene to occur on chromosome 4 in both species (HeChr4, "sc_6: 4,437,062–4,474,302", spanning 37,241 bp) and H. vespertilio (HvChr4, "ScWp86a_21_HRSCAF_27: 5,766,997–5,884,695", 5 exons within a large stretch of 117,699 bp). The first intron of aristaless contains a repeat motif in H. euphorbiae that is not found in H. vespertilio (Fig. S7). Apart from this difference, the positions of the other repeats within the stretch of exons are very similar in both species, but the stretches of repeats are longer in H. vespertilio (scale of 2 kb in both views of Fig. S7). Upon zooming out, the large diversity in repeats in the vicinity of this gene becomes apparent (Fig. S8), as well as the large stretches of non-coding DNA between the numerous exons.
The BLAT result of Distal-less (Table 2) reveals a position on chromosome 2 in both species (HeChr2, "sc_7: 16,368,465–16,384,630", 2 exons within 16,166 bp) and H. vespertilio (HvChr2, "ScWp86a_159_HRSCAF_226: 4,646,872–4,661,762", 2 exons within 14,891 bp). The single intron between the two exons contains repeats in both species, however very different types in different positions and extents (Fig. S9).
Analysis of male mitotic chromosomes stained by FISH with a telomeric probe (telomere-FISH) showed that the karyotype of H. euphorbiae is composed of 2n = 58 chromosomes (Fig. 7a). As typical for Lepidoptera, the chromosomes are of the holokinetic type, i.e. they lack a primary constriction (centromere) and are morphologically uniform, differing only in size. Chromosome number was confirmed by analysis of meiotic nuclei at the pachytene stage, where homologous chromosomes pair and form elongated bivalents. Pachytene complements, stained by GISH in combination with telomere-FISH, showed a haploid number of 29 bivalents in both sexes (Fig. 7b, c). In addition, GISH identified a WZ sex chromosome bivalent in pachytene oocytes by labeling a large portion of the W chromosome with the female genomic DNA (gDNA) probe (Fig. 7c), whereas no bivalent was identified in pachytene spermatocytes (Fig. 7b). These results clearly show that H. euphorbiae has a WZ/ZZ (female/male) sex chromosome system, which is common in Lepidoptera. It should be noted that the WZ bivalent is relatively long (Fig. 7c), suggesting that the W and Z chromosomes are among the largest chromosomes in the H. euphorbiae karyotype.
Chromosome size estimation from karyotype image data
Chromosome size estimation is based on Fig. 7c, bivalents from a female gonad cell at the pachytene stage. The chromosome size estimates are shown in Fig. 8. They corroborate the WZ bivalent as the largest chromosome. Based on semi-automated image processing, the software package napari-karyotype  relies on threshold-based image segmentation to detect chromosome-related components. Identified chromosomal objects are surrounded by red rectangles and labeled with the estimates.
Genome size estimation
Genome sizes of H. vespertilio and H. euphorbiae were measured using flow cytometry in three replicates, each from a single individual. The average results of 0.575 pg and 0.484 pg showed that the 1C DNA values were 562 Mb and 472 Mb, respectively (1 pg = 978 Mb).
The chromosomal genome assemblies of H. euphorbiae and H. vespertilio presented here are highly contiguous, as demonstrated by the scaffold N50 values exceeding those of the sequenced close relatives M. sexta  and B. mori . The percentage of assembly length that could be placed in chromosome scaffolds is highest in Hyles euphorbiae (Table 2). Both metrics underline the high quality of the two new assemblies for the Hyles genus. Gene content is similarly high in all compared assemblies.
Comparison of the two new Hyles genomes
The large difference in genome size estimates between the two species within the genus Hyles, i.e. H. vespertilio with 562 Mb being ~ 20% larger than that of H. euphorbiae with 472 Mb in the flow cytometry measurements, was unexpected due to the short divergence time between the two species . Hyles lineata is one of the first Hyles species to diverge within the genus  and has an even smaller flow cytometry genome size of 450 Mb (0.46 pg) . The potentially smaller genome size of this third Hyles species suggests that the larger genome of H. vespertilio is the result of a recent proliferation of repetitive elements and is a derived character.
Although the values of genome sizes based on assembly lengths are not directly comparable to genome size values based on flow cytometry due to technical differences in methodology, the relative sizes are of the same order of magnitude. The assembly length of H. vespertilio with 651 Mb is ~ 30% longer than that of H. euphorbiae with 504 MB, although the number of genes is very similar in both species (Fig. 6).
The genome of H. euphorbiae presented in this work has much less repetitive elements, in particular LINEs, than H. vespertilio. The high repeat content in all four species compared, H. euphorbiae, H. vespertilio, M. sexta, and B. mori, is typical for lepidopteran genomes [52, 53]. However, the number of repeats varies between species. Previous research has shown a correlation between the repetitive fraction of the genome and genome size within and between species [54, 55]. Indeed, the genome of H. vespertilio is the largest in our comparison and the genome with the most extensive repeat content, whereas the other sphingid genomes show lower numbers of repeats, correlating with their smaller genome size. Therefore, as described in previous research, the repetitive elements found here are thus likely the driving force for the expansion of genome size, possibly due to a positive feedback loop that allows these elements to spread more easily in large genomes . In addition, the repeatome is thought to play a significant role in genetic innovation and speciation [57,58,59,60,61]. This could explain why H. vespertilio is known to be one of the most isolated species in the genus of hawkmoths with respect to the lack of hybridization with numerous other Hyles species. In contrast, H. euphorbiae is well known for its wide distribution and frequent hybridization with even distantly related species [2, 50, 62].
Since the two species are separated by a p-distance of only 2.1% in the neutral marker genes COI and COII , corresponding to a split timing of only 4.2–5.6 Mya (according to the respective molecular clocks of 1.1–1.2% per million years per lineage, resulting in 2.3% sequence divergence between species pairs per million years of separation ), high synteny of genes between H. vespertilio and H. euphorbiae in the nuclear chromosomes set was expected (Figs. 3, 4). Indeed, the illustrated genome alignments (Figs. 3, 4) show how similar the two Hyles genomes are. The mitochondrial genome of H. euphorbiae (Fig. S1) is also very similar to that of H. vespertilio (see ). Comparison of single copy orthologs in each genome demonstrates conservation of 29 orthologous chromosomes. These chromosomes closely correspond to the 32 ancestral chromosomes of Lepidoptera (Merian elements), with the exception of three fusions, thus accounting for the reduction in chromosome number. Moreover, we observe a high degree of synteny, defined as conservation of gene order within each chromosome, in line with other studies[42, 52, 64, 65]. Extending the comparisons beyond Hyles suggests that these 29 chromosomes have remained stable throughout Sphingidae, with the exception of two independent fusion events in M. sexta and L. populi, which are absent in Hyles and D. porcellus. Such strong conservation of chromosome structure has been previously observed in Nymphalidae . Our analyses presented here suggest that the Sphingidae chromosomes are also remarkably syntenic over long periods of evolution.
Karyotype, chromosome taxonomy
In addition to examining conservation of chromosome structure through comparison of single copy orthologs to Merian elements in each genome, we also performed whole genome alignments. Whole genome alignments between each Hyles genome and that of the well-studied species B. mori were done using positional information of synteny blocks (Table S1), in order to assign homology between B. mori and Hyles chromosomes, enabling chromosome naming based on B. mori. This resulted in the robust chromosome taxonomy based on B. mori (presented for the two Hyles species studied in Fig. 4c).
Given the young age of the genus (the oldest intra-genus split is 10.8–12.4 Mya, based on p-distance ), it can be assumed that the chromosome taxonomy also applies to the entire genus Hyles. We propose our protocol of homology inference based on genome alignments as the gold standard for chromosome taxonomy. The most recent hawkmoth assemblies [14, 39], including that of M. sexta , use only arbitrary numbers according to chromosome size. A table showing homology of chromosomes, accession and scaffold numbers is provided in Table S3. Using this comparative approach as a standard in bombycoid chromosome taxonomy will greatly facilitate discussions based on genomic comparisons for specific research questions, such as the homologies of wing pattern genes in the present study.
In early genetic studies, three chromosomes of B. mori (BmChr11, BmChr23 and BmChr24) were often interpreted to be split in other species, increasing the chromosome number compared to B. mori, e.g. [43, 67, 68]. The recent discovery of 32 Merian elements, the ancestral linkage groups of Lepidoptera, provides a framework within which to understand the chromosomal changes in B. mori relative to other species. The ancestral state of Ditrysia is n = 31 due to an ancient fusion between two Merian elements. Therefore, B. mori has a reduced chromosome number relative to other Ditrysians due to a subsequent three fusions.
The whole genome alignments between B. mori and Hyles demonstrate that these two groups have undergone independent sets of fusions, leading to the reduced chromosome numbers. This is consistent with the previous observation that three B. mori chromosomes are often split in other species.
The H. euphorbiae chromosome images obtained had a sufficient clarity to be annotated with a size estimate by napari-karyotype (Table 3) . However, it should be noted that this was more difficult than expected because the chromosome bivalents at the pachytene stage were touching each other in the image and thus had to be extracted manually. Without this step, the assignment of object vs. background would have been inaccurate. Furthermore, chromosomes are flexible structures and their length depends on the stage, the degree of condensation and also on the preparation methods. Therefore, their measured length does not always correspond to their size, which is especially true for meiotic chromosomes at the pachytene stage . In order to annotate the chromosome images of the karyotype with the chromosome numbers of the B. mori chromosome taxonomy, it will be necessary to implement further in-situ-hybridization with gene-specific fluorochrome-labeled probes following Yasukochi et al.  (their Fig. 2) in the future.
Wing pattern genes
The location of numerous wing pattern genes has been well studied in Nymphalidae. Here, we infer the likely locations of optix, WntA, cortex, aristaless and Distal-less in Hyles based on Heliconius amino acid or the homologous M. sexta nucleotide similarity and characterize their genomic context.
Optix and surrounding genes are highly conserved in Lepidoptera . Zhang et al.  have shown that optix knock-outs exhibit complete replacement of color pigments by melanins, resulting in black and grey butterflies. In contrast, the high percentage of indels found in the alignment up- and downstream of optix between the genomes of H. vespertilio and H. euphorbiae may support the previously proposed hypothesis  that wing patterns are indeed controlled by cis-regulatory elements near the position of optix. As more sequence data from more individuals become available, FST plots can be calculated to corroborate correlations between particular candidate regions. Although Manduca sexta is the reference moth genome most apt in the comparative framework presented here, the wing pattern of this species also consists of black and grey, without colored stripes or spots. Thus, the inversion of optix on chromosome 23 in H. euphorbiae with respect to H. vespertilio and M. sexta, could possibly also be the other way round, in that H. euphorbiae with its cream, olive-brown forewings, sometimes with pink, holds the gene with normal function, and H. vespertilio and M. sexta have a non-functional inversion. The hypothesis if optix is part of a supergene will be studied in more detail by targeted resequencing of phenotypes in comparison. However, the causal relationship between grey wings in H. vespertilio and optix and surrounding gene regions could only be answered by analyzing knock-outs generated using the CRISPR/Cas9 system.
The highly varying amounts, classes, and families of repeats between the two Hyles species sequenced suggest that the function of this gene could have been corrupted in the grey H. vespertilio, as this species has the larger genome and more repeats. Sequence data from more individuals are needed to pinpoint a candidate region and establish a robust correlation.
Another striking difference between the two species near the stretch of 8 cortex exons is the insertion of 229 bp, which was marked as an unknown repeat (Fig. S5b) on chromosome 17 in H. vespertilio. Mapping this fragment to the genome of H. euphorbiae using BLAT yielded 200 hits between 89.8–96.7% identity on each chromosome and 203 hits on the new genome of H. vespertilio (data not shown), indicating the wide distribution of this repeat region across the genomes. It was also found in the very closely related macroglossine species Deilephila porcellus  and Hemaris fuciformis , as well as several hits in the genome of the smerinthine Mimas tiliae , a phylogenetically somewhat more distant species. Again, cortex is found to lie on an inversion in H. euphorbiae (although in this comparative framework, it is possibly the other way round), directing thoughts to a possible connection within a second supergene (since it is on a different chromosome than optix). The two new Hyles reference genomes open doors to investigating these possibilities. Further analyses and comparisons between the genomes presented here and others becoming available are expected to lead to a better understanding of the mechanisms controlling wing patterns in the family Sphingidae.
Aristaless & Distal-less
The two genes appear similarly diverse between the species with respect to the number and type of repeats. The two Hyles moths studied do not show eyespots in their wings, nor do other species of the genus. However, further studies could clarify whether these genes control the simple brown spots on the forewings in H. euphorbiae and many other species of this genus by understanding if the genes are not expressed or possibly silenced in H. vespertilio.
Previous studies had already shown that similarity of wing patterns does not correlate with phylogenetic relationships in the genus Hyles [1, 2, 5], neither as currently defined by morphology nor as reflected by molecular phylogeny. Morphologists have long argued that they must rely on phenotypic characters in the absence of molecular data, and have often included striking differences in wing patterns in their species descriptions. In Hyles, however, the evolution of wing pattern characters does not reflect the evolution of the species. Of course, gene trees are not species trees , so even traditional genetic analyses, e.g. [1, 2, 5], do not necessarily reflect the true tree.
In this study, we present two high-quality annotated chromosome-level assemblies and report the presence, sequence, and location of wing pattern genes. Using reference genomes of H. euphorbiae and H. vespertilio, which have very different forewing patterns and coloration, we open up possibilities for studying the evolution of wing patterns based on a numerically evaluable, objective data source. The genes optix (on chromosome 23), WntA (on chromosome 28), cortex (on chromosome 17), aristaless (on chromosome 4) and Distal-less (on chromosome 2) promise to be of interest in a comparative study of numerous Hyles individuals, as the genomic regions surrounding these genes show high numbers of indels potentially corrupting their function and inversions were detected between the two Hyles species. Since the five genes compared in this study all lie on different chromosomes, much work is still needed until we understand the basis of the changes between e.g. the two prominent Hyles forewing patterns described in the introduction, let alone the additional often unique forewing patterns in other species. The results of our comparative repeatome analysis confirm that repetitive elements are likely the driving force for the expansion of genome size in Hyles vespertilio compared to H. euphorbiae. The chromosome-level genomic data provided in this study for these two species provide reliable references in the family Sphingidae for future studies involving as many species as possible to elucidate the evolution of forewing patterns in this group of Lepidoptera.
We strongly suggest the application of a standardized chromosome naming process for every newly sequenced genome to facilitate comparability and propose using a two-fold comparative genomics approach. The combination of CIRCOS plots based on pairwise alignments in a given system (such as B. mori for Bombycoidea) for chromosome taxonomy with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements, enables more precise evolutionary deductions.
Material and methods
For karyotype analysis, specimens of the H. euphorbiae population from Greece (leg. P. Mazzei, Serifos) were reared in the laboratory (summer 2019). Several larvae and young pupae were used to make chromosome preparations (see below). For genome sequencing, one male specimen of H. euphorbiae (Fig. 1a) was collected near Berbisdorf (Germany) on 27 July 2021 and received the tissue voucher number MTD-TW- 13387 in the Molecular Laboratory of the Senckenberg Naturhistorische Sammlungen Dresden, Museum of Zoology. A second, similar male moth from the same locality was placed as a voucher specimen in the main SNSD Entomology collection. The Hi-C data for H. vespertilio (Fig. 1b) was obtained from frozen (-80 °C) tissue of the same individual from Vallonina, Italy (collected in 2018) that was used for the original genome publication in Pippel et al.. The tissue was moved from the Max Planck Institute of Molecular Cell Biology and Genetics (Dresden), where it had the accession number LG2117, to the SNSD-MTD tissue collection, and received the voucher number MTD-TW-13386.
Spread chromosome preparations were made as described by Yoshido et al. . Mitotic chromosomes were obtained from wing imaginal discs or testes of final instar larvae. Meiotic chromosomes in the pachytene stage of prophase I were obtained either from the testes of last instar larvae or from the ovaries of 3–5-day old pupae. Briefly, tissues were dissected in saline solution, swollen in hypotonic solution (75 mM KCl) for either 5 min (ovaries) or 15 min (testes and wing imaginal discs) and then fixed in Carnoy’s fixative (ethanol, chloroform, acetic acid, 6:3:1) for 10 min. Cells dissociated in 60% acetic acid were spread on a heating plate at 45 °C. All chromosome preparations were passed through a graded ethanol series (70%, 80%, and 100%, 30 s each) and stored at –80 °C.
Fluorescence in situ hybridization (FISH) with the (TTAGG)n telomeric probe and genomic in situ hybridization (GISH) were performed according to the procedure described by Yoshido et al. . (TTAGG)n telomeric sequences were generated by non-template PCR following the protocol of Sahara et al. . Male and female genomic DNAs (gDNAs) of H. euphorbiae were obtained separately from last instar larvae by standard phenol–chloroform extraction. DNA probes were labeled by nick translation using a mixture of DNase I and DNA polymerase I (both Thermo Fisher Scientific, Waltham, MA, USA) with either aminoallyl-dUTP-Cy3 or fluorescein-12-dUTP (both Jena Bioscience, Jena, Germany).
Chromosome preparations were removed from the freezer, passed through the graded ethanol series, air-dried and then denatured in 70% formamide in 2 × SSC for 3.5 min at 70 °C. For one preparation, the probe cocktail contained 500 ng of fluorescein-labeled female gDNA, 100 ng of Cy3-labeled telomeric probe, 3 μg of unlabeled sonicated male gDNA, and 25 μg of sonicated salmon sperm DNA (Sigma‐Aldrich, St. Louis, MO, USA) in 10 μl hybridization buffer (50% formamide, 10% dextran sulfate in 2 × SSC). Denaturation of the probe cocktail was performed for 5 min at 90 °C. The preparations were examined under a Zeiss Axioplan 2 microscope (Carl Zeiss, Jena, Germany). Digital images were captured with an Olympus CCD monochrome camera XM10 equipped with cellSens 1.9 digital imaging software (Olympus Europa Holding, Hamburg, Germany) and processed with Adobe Photoshop CS4.
Karyotype-based automated chromosome annotation and size estimation
The meiotic chromosome image was preprocessed using the image processing software GIMP (version 2.10)  to manually cut out individual chromosome bivalents. The processed image was loaded into the tool napari-karyotype (version c41103e) . The image segmentation threshold, blur factor and genome size were set to 0.13, 0.5 and 504 Mb, respectively.
Genome size estimation
Genome sizes of two hawkmoth species were estimated following the flow cytometry protocol with propidium iodide-stained nuclei as described previously . Neural tissue from frozen (− 80 °C) adult samples of H. vespertilio and H. euphorbiae (from one head each; the same individuals used for the PacBio genome and Hi-C sequencing) and neural tissue from the internal reference standard Acheta domesticus (female, 1C = 2 Gb) were each chopped with a razor blade in a Petri dish containing 2 ml of ice-cold Galbraith buffer. The suspension was filtered through a 42-μm nylon mesh, then stained with the intercalating fluorochrome propidium iodide (PI, Thermo Fisher Scientific) and treated with Rnase A (Sigma-Aldrich), each at a final concentration of 25 μg/ml. The mean red PI fluorescence of the stained nuclei was quantified using a Beckman-Coulter CytoFLEX flow cytometer with a solid-state laser emitting at 488 nm. Fluorescence intensities of 10,000 nuclei per sample were recorded. Subsequently, the nuclei suspensions of H. vespertilio and H. euphorbiae were each mixed with the nuclei suspension of the internal reference standard (see above) and again the fluorescence intensities of 10,000 nuclei per mixed sample were recorded. For the histogram analyses, we used CytExpert 2.3 software. The total amount of DNA in each sample of the two Hyles species was calculated as the ratio of the mean fluorescence signal of the 2C peak of the stained nuclei of the respective species divided by the mean fluorescence signal of the 2C peak of the stained nuclei of the reference standard times the 1C amount of DNA in the reference standard. Three replicates, each from the same individual of H. vespertilio and H. euphorbiae, were measured on three different days to minimize possible random instrumental errors. Genome size was reported as 1C, the mean amount of DNA in Mb in a haploid nucleus.
PacBio genome DNA and sequencing
Head tissue (38 mg) from a single male Hyles euphorbiae specimen (the same single individual used for genome size estimation) was used for high molecular weight DNA extraction using an adaptation of the protocol of Miller et al. . Final purity and concentrations of DNA were measured using the NanoPhotometer (Implen GmbH, Munich, Germany) and the Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA). One SMRTbell library was constructed following the instructions of the SMRTbell Express Prep kit v2.0 with Low DNA Input Protocol (Pacific Biosciences, Menlo Park, CA). The total input DNA for the library was 3 µg. The library was loaded at an on-plate concentration of 80 pM using diffusion loading. One SMRT cell sequencing run was performed on the Sequel System II in CCS mode using 30-h movie time with 2 h of pre-extension and sequencing chemistry V2.0.
Genome assembly of Hyles euphorbiae
We created PacBio CCS reads from the Hyles euphorbiae subreads.bam file using PacBio’s ccs command line tool (version 6.3.0, –all flag was used). We obtained 7.9 Gb of high-quality CCS reads (HiFi reads, rq > 0.99) with an N50 of 11.74 kb. To further increase the read coverage, we applied DeepConsensus (v0.2 with default settings, actc v0.1.1) [79, 80] on all reads of the previous ccs step and obtained a total yield of 8.8 Gb (N50: 11.83 kb). We ran HiFiasm (version 0. 16.1-r375)  to create the contig assembly. Remaining haplotypic duplications in the primary contig set were removed using default parameters in the purge-dups pipeline (v.1.2.3) [81, 82]. The assembly was scaffolded with HiC data using yahs (v 1.1a)  and manually curated with HiGlass . Remaining gaps in the scaffolds were filled by mapping the raw PacBio subreads with pbmm2 (version 1.7.0) , and for alignment piles that fully spanned the gap regions with 1000 anchor bases on both sides, a consensus sequence was produced with gcpp (version 2.0.2) . The consensus sequence was used to fill a gap only if: 1) the complete consensus sequence was covered at least 5x; and 2) the coverage profile of the closed gaps fully supported the consensus sequence (i.e. no alignment breaks or huge repeat alignment piles occurred). Two rounds of error correction were performed by aligning the DeepConsensus reads to the assembly using pbmm2, calling variants using DeepVariant (version 1.3.0)  and correcting homozygous errors using bcftools [88, 89] consensus. The assembly was checked for contamination using blobtoolkit (version 1.1)  and an in-house pipeline that screens several blast databases. Assessment of completeness regarding single copy orthologs was performed via Benchmarking Using Single Copy Orthologues (BUSCO) (version 5.2.2)  together with the lepidoptera_odb10 set and the options “–long –offline”. and merqury (version 1.3) . QV values were generated for the final scaffolds (H. euphorbiae QV = 58.1).
The mitochondrial genome was created using the mitoHifi pipeline (version 2) [45, 46] based on CCS reads and the closely related mitochondrial reference genome of Theretra oldenlandiae (NCBI accession: MN885801.1).
Hi-C sequence data
Dovetail Hi-C libraries for H. vespertilio and H. euphorbiae were prepared from head tissues (52.2 mg and 40.6 mg; the same single individuals used for PacBio genome sequencing and genome size estimation) using the Dovetail Hi-C kit (Dovetail Genomics, Scotts Valley, CA, USA) following the manufacturer’s protocol (version 1.4) for insect samples. Briefly, chromatin was fixed with formaldehyde and then extracted. The fixed chromatin was digested with DpnII, the 5’ overhangs were filled with biotinylated nucleotides and the free blunt ends were ligated. After ligation, the crosslinks were reversed, the associated proteins were degraded and the DNA was purified. DNA was then sheared to a mean fragment size of ~ 350 bp and sequencing libraries were generated using Illumina-compatible adapters. Biotinylated fragments were captured with streptavidin beads before PCR amplification.
The fragment size distribution and concentration of the final PacBio and Dovetail Hi-C libraries were assessed using the TapeStation (Agilent Technologies) and the Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA), respectively. The Hi-C libraries were sequenced on a NovaSeq 6000 platform at Novogene (UK), generating 100 million 2 × 150 bp paired-end reads each with a total volume of 30 Gb.
Scaffolding the assembly of H. vespertilio with HiRise
The H. vespertilio input assembly of Pippel et al.  and the H. vespertilio Dovetail Hi-C library reads were used as input data for HiRise, a software pipeline specifically designed for using proximity ligation data to scaffold genome assemblies . The Dovetail Hi-C library sequences were aligned to the draft input assembly using a modified SNAP read mapper (http://snap.cs.berkeley.edu). The separations of the Hi-C read pairs mapped within draft scaffolds were analyzed using HiRise version 2.1.7 to produce a likelihood model for the genomic distance between the read pairs. The model was used to identify and break putative misjoins, score prospective joins, and make joins above a threshold.
Structural annotation of protein coding genes was performed using TOGA , a method that uses pairwise genome alignment chains between an annotated reference genome (here Manduca sexta) and other query species (here Hyles euphorbiae and H. vespertilio). Briefly, TOGA uses machine learning to infer orthologous loci for each reference transcript, using the concept that orthologous genes display more alignments between intronic and flanking intergenic regions. TOGA then projects each reference transcript to its orthologous query locus using CESAR 2.0 , a hidden Markov model method that takes reading frame and splice site annotation of the reference exons into account. CESAR avoids spurious frameshifts and is able to detect evolutionary splice site shifts and precise intron deletions [94, 95]. Using the CESAR alignment, TOGA determines whether the transcript has inactivating mutations (frameshifting mutations, premature stop codons, splice site disrupting mutations, deletions of entire coding exons).
To generate whole genome alignments as input for TOGA, we aligned the assemblies of H. euphorbiae and H. vespertilio to the Manduca sexta (GCF_014839805.1)  assembly. To compare contiguity between H. euphorbiae, H. vespertilio, L. populi, Hemaris fuciformis, D. porcellus, Mimas tiliae and Manduca sexta, Quast 5.0.2  was used.
Genomes were aligned using LASTZ (version 1.04.03)  with the parameters (K = 2400, L = 3000, Y = 9400, H = 2000 and the lastz default scoring matrix). Then, we used axtChain  (default parameters except linearGap = loose) to compute co-linear alignment chains, RepeatFiller  (default parameters) to capture previously missed alignments between repetitive regions and chainCleaner  (default parameters except minBrokenChainScore = 75,000 and -doPairs) to improve alignment specificity. H. vespertilio was used as reference and H. euphorbiae, B. mori and M. sexta as queries. We used the NCBI annotation (GCF_014839805.1_JHU_Msex_v1.0_genomic.gff.gz) as input for TOGA.
The mitochondrial genome of H. euphorbiae was annotated using the MITOS WebServer  and the result illustrated using shinyCircos . This software was also used for the CIRCOS-Plots of the aligned genomes.
With the aim of naming the chromosomes according to homology to B. mori, nucleotide homology was assessed based on sequence alignment of H. vespertilio, H. euphorbiae and M. sexta contigs via scaffold chaining as the primary tool for discovering similarities using LASTZ . Visualization was carried out using CIRCOS plots (Fig. 4) using positional information of synteny blocks (in the bed files) of the genome alignments as input (c.f. Table S3).
The Hyles euphorbiae and H. vespertilio genome assemblies (with annotations and probabilities of site modification/methylation, and genome alignments, see below) are accessible in the Senckenberg Genome Browser (https://genome.senckenberg.de/cgi-bin/hgTracks?db=HLhylEup1; https://genome.senckenberg.de/cgi-bin/hgTracks?db=HLhylVes2). A genome alignment of H. vespertilio to B. mori (GCF_014905235.1 ) was generated as above and used to postulate chromosome homologies and name chromosomes accordingly. Detailed values of the proportions of homologous regions per chromosome are provided in Table S1.
The genomes of Mimas tiliae , Laothoe populi , Hemaris fuciformis  and Deilephila porcellus  (these are the additional genomes of Sphingidae that were available on 14.10.2022) were included for assembly statistics comparisons and a phylogenetic comparison of repeat evolution.
Analyses of repeat and transposon content
Comparative annotation of repeats for H. vespertilio, H. euphorbiae, D. porcellus, Mimas tiliae, L. populi, H. fuciformis, and Manduca sexta was performed using RepeatModeler (version 2.0.3)  and RepeatMasker (version 4.1.2-p1)  as provided in Dfam TETools docker container (version 1.85) , in combination with rmblastn (version 2.11.0 +). The repeat library for RepeatMasker contains a combination of all repeat families identified with RepeatModeler, combined and made non-redundant using Cdhit (version 4.8.1)  and manual curation of models according to guidelines detailed in . Occurrence of various transposable element families were illustrated on the respective branches of a tree drawn by hand based on Kawahara and Barber , in relative order according to the assembly divergence and sequence amount as provided by a helper script of repeatmasker (calcDivergenceFromAlign.pl). TE families with higher sequence diversity (presumed to represent older transposon bursts) are drawn more left on the branches, TE families shared between taxa (and with high sequence diversity) are drawn on the branch of their common ancestors.
For the methylation profile, the H. euphorbiae PacBio HiFi reads containing 5mC base modification tags were converted to CpG/5mC data using pb-CpG-tools (version 1.1)  and uploaded to the genome browser (s.a.). Sites with a modification probability of < 50% are marked blue and > 50% red.
Chromosome structure analysis
To compare chromosome structure across species, single copy orthologs were inferred using BUCSOs (version 5.4) (‘lepidoptera odb 10’ dataset, metaeuk mode) . Orthologues were filtered to retain those that can be assigned to a Merian element, using the table of orthologues assignments to Merian elements from Wright et al. . Next, unlocalized scaffolds were removed by only retaining scaffolds with three or more single copy orthologs. Finally, synteny between pairs of genomes was then visualized using Oxford plots with using custom code , which plots the relative position of each ortholog along the chromosomes of each genome. In the resulting Oxford plots, orthologues were colored by Merian element identity to identify chromosomes resulting from fusion events. Chromosomes resulting from fusion events were also verified by running https://github.com/charlottewright/lep_fusion_fission_finder with a window size of 17. This identifies chromosomes which are the product of fusion events based on the presence of orthologues that correspond to more than one Merian element within a single chromosome.
Wing pattern genes
The positions of the wing pattern genes optix, WntA, cortex, aristaless and Distal-less (see Table S2 for accession numbers of references) were identified in the two Hyles genomes using the online BLAT tool [47, 48] with default options as implemented in the Senckenberg Genome browser. The BLAT results are presented sorted by alignment length, with the longest selected for each gene.
For the cortex gene, we additionally compared the Hyles data with the sequences of Biston betularia (Geometridae; KT182637), in which the common pale (typica) form was replaced by a previously unknown black (carbonaria) form during the Industrial Revolution, driven by the interaction between bird predation and smoke pollution  and caused by a transposon insertion in a cortex intron [32, 111].
Availability of data and materials
The data generated and analyzed during the current study are available in the NCBI repository, [https://www.ncbi.nlm.nih.gov/genome/?term=hyles]. The SRA accession number for H. euphorbiae PacBio Sequel HiFi is SRX13604162, and for the Dovetail Hi-C data is SRX14310646. The assembled genome is accessible under GCA_023078785.2, and accession numbers are itemized in the following. Mitochondrion: CM041059, chromosome Z: CM041058 and chromosomes 2–29: CM041030-CM041057).
The SRA accession number for the Dovetail Hyles vespertilio Hi-C raw data is SRX14530528. The new assembly presented in this work is accessible under GCA_009982885.2, and accession numbers are itemized in the following. Chromosome Z: CM042833 and chromosomes 2–29: CM042805-CM042832.
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This study benefitted from the sharing of expertise within the DFG priority program SPP 1991 Taxon-Omics. Dovetail Genomics provided HiRise scaffolding service. Thanks go to Alexander Ben Hamadou (LOEWE-TBG) for DNA isolation and library preparations. We thank Ian J. Kitching for language correction and helpful suggestions, as well as Markward H. Fischer for improving numerous figures. Thank you to Paolo Mazzei for providing live H. euphorbiae from Greece.
Open Access funding enabled and organized by Projekt DEAL. This study was supported by grants from the German Research Foundation (DFG) in the framework of the priority program SPP 1991: Taxon-OMICS (HU 1561/5–2).
MP was partially funded by the BMBF (grant 01IS18026C). AY and FM acknowledge support from grant 20-13784S of the Czech Science Foundation. CJW was funded by the Wellcome Trust (grants 206,194 and 218,328). HiRise scaffolding service by Dovetail Genomics for H. vespertilio was funded by LOEWE-TBG.
Ethics approval and consent to participate
The individual of the protected species H. euphorbiae was collected with an exception issued in a permit by “Landratsamt Meißen, Kreisumweltamt, Untere Naturschutzbehörde” (AZ 364.621–377/2009–4026/2017–45577/2021) for the purpose of scientific study.
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The annotated mitochondrial genome of Hyles euphorbiae. Fig. S2. BLAT result of the Heliconius optix protein sequence (KC469894) from the genome browser on chromosome 23 showing the Hyles genome alignment, annotation, and repeat occurrence in the vicinity in a) H. euphorbiae (inverted) and b) H. vespertilio. Fig. S3. BLAT result of Heliconius optix protein sequence (KC469894) from the genome browser on chromosome 23 in a) Hyles euphorbiae (reverse) and b) Hyles vespertilio in a 700 kb view. Fig. S4. BLAT result of the Heliconius WntA protein sequence (AFC75686) from the genome browser showing the Hyles genome alignment, annotation, and repeat occurrence in the vicinity in a) H. euphorbiae (reverse) and b) H. vespertilio. Fig. S5. BLAT result from the genome browser showing the exon/intron structures of the Manduca sexta and Biston betularia cortex protein (Table S2) on chromosome 17 in a) Hyles euphorbiae and b) H. vespertilio. Fig. S6. BLAT result of Manduca sexta mRNA of cortex (Table S2) on chromosome 17 in a) Hyles euphorbiae and b) Hyles vespertilio in a 100 kb view. Fig. S7. BLAT result of Manduca sexta mRNAs of aristaless (Table 2) on chromosome 4 in a) H. euphorbiae and b) H. vespertilio. Fig. S8. BLAT result of Manduca sexta mRNAs of aristaless (Table 2) on chromosome 4 in a) H. euphorbiae 100 kb view, b) H. vespertilio 150 kb view (note the different scales). Fig. S9. BLAT result of Manduca sexta mRNA of Distal-less (AY616435.1) on chromosome 2 in a) Hyles euphorbiae (inverted) and b) Hyles vespertilio. Fig. S10. BLAT result of Manduca sexta mRNA of Distal-less (AY616435.1) on chromosome 2 in a) Hyles euphorbiae (inverted) and b) Hyles vespertilio in a 100 kb view.
Chromosome proportion values of the B. mori – H. vespertilio alignment. Chr. = chromosome.
Accession numbers of reference wing pattern sequences and data type used for blat in the genome browser. Manduca sexta WntA and optix are not displayed on the NCBI web portal.
Chromosome homology, scaffold names and accession numbers of B. mori, M. sexta, H. euphorbiae and H. vespertilio, with chromosome sizes based on assembly lengths obtained via bioinformatics.
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Hundsdoerfer, A.K., Schell, T., Patzold, F. et al. High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae). BMC Genomics 24, 443 (2023). https://doi.org/10.1186/s12864-023-09506-y