Critical re-examination of known purported fossil Bombycoidea (Lepidoptera).

We critically re-examine 17 records of fossils currently assigned to the lepidopteran superfamily Bombycoidea, which includes the silk moths, emperor moths and hawk moths. These records include subfossils, compression and impression fossils, permineralizations and ichnofossils. We assess whether observable morphological features warrant their confident assignment to the superfamily. None of the examined fossils displays characters that allow unequivocal identification as Sphingidae, but three fossils and a subfossil (Mioclanis shanwangiana Zhang, Sun and Zhang, 1994, two fossil larvae, and a proboscis in asphaltum) have combinations of diagnostic features that support placement in the family. The identification of a fossil pupa as Bunaeini (Saturniidae) is well supported. The other fossils that we evaluate lack definitive bombycoid and, in several cases, even lepidopteran characters. Some of these dubious fossils have been used as calibration points in earlier studies casting doubt on the resulting age estimates. All fossil specimens reliably assigned to Bombycoidea are relatively young, the earliest fossil evidence of the superfamily dating to the middle Miocene.

High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae).

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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).

Cryptic diversity in a vagile Hawaiian moth group suggests complex factors drive diversification.

Allopatric speciation should be the dominant model of diversification across archipelagos because islands naturally promote isolation. It also follows that ecologically similar, vagile species should be more resistant to this kind of isolation due to dispersal and unifying selection. In a closely-related group of endemic Hawaiian hawkmoths, we found confounding patterns of inter-island connectivity and speciation that did not correlate with vagility, ecological specialization, or island age. Speciation occurred both in allopatric and sympatric taxa, with only the oldest and youngest islands fostering single-island endemic species. The intermediately-sized, central islands supported a combination of endemic and more widely-occurring lineages, suggesting no clear pattern leading to the current diversity in Hawaii. While some species are relatively common, others are apparently extinct or very rare, even on the same island. Further research into the specific mechanisms for these patterns in Hyles may prove broadly informative for understanding both cladogenesis and improving conservation planning. Our study identifies one new species endemic to Kauai and unique mitochondrial lineages in H. perkinsi, which may prove to be new species.

Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens.

Here we present and justify an approach for minimal-destructive DNA extraction from historic insect specimens for next generation sequencing applications. An increasing number of studies use insects from museum collections for biodiversity research. However, the availability of specimens for molecular analyses has been limited by the degraded nature of the DNA gained from century-old museum material and the consumptive nature of most DNA extraction procedures. The method described in this manuscript enabled us to successfully extract DNA from specimens as old as 241 years using a minimal-destructive approach. The direct comparison of the DNeasy extraction Kit and the Monarch® PCR & DNA Clean-up Kit showed a significant increase of 17.3-fold higher DNA yield extracted with the Monarch Oligo protocol on average. By using an extraction protocol originally designed for oligonucleotide clean-up, we were able to combine overcoming the restrictions by target fragment size and strand state, with minimising time consumption and labour-intensity. The type specimens used for the minimal-destructive DNA extraction exhibited no significant external change or post-extraction damage, while sufficient DNA was retrieved for analyses.

A highly contiguous genome assembly of the bat hawkmoth Hyles vespertilio (Lepidoptera: Sphingidae).

BACKGROUND: Adapted to different ecological niches, moth species belonging to the Hyles genus exhibit a spectacular diversity of larval color patterns. These species diverged ∼7.5 million years ago, making this rather young genus an interesting system to study a wide range of questions including the process of speciation, ecological adaptation, and adaptive radiation. RESULTS: Here we present a high-quality genome assembly of the bat hawkmoth Hyles vespertilio, the first reference genome of a member of the Hyles genus. We generated 51× Pacific Biosciences long reads with an average read length of 8.9 kb. Pacific Biosciences reads longer than 4 kb were assembled into contigs, resulting in a 651.4-Mb assembly consisting of 530 contigs with an N50 value of 7.5 Mb. The circular mitochondrial contig has a length of 15,303 bp. The H. vespertilio genome is very repeat-rich and exhibits a higher repeat content (50.3%) than other Bombycoidea species such as Bombyx mori (45.7%) and Manduca sexta (27.5%). We developed a comprehensive gene annotation workflow to obtain consensus gene models from different evidence including gene projections, protein homology, transcriptome data, and ab initio predictions. The resulting gene annotation is highly complete with 94.5% of BUSCO genes being completely present, which is higher than the BUSCO completeness of the B. mori (92.2%) and M. sexta (90%) annotations. CONCLUSIONS: Our gene annotation strategy has general applicability to other genomes, and the H. vespertilio genome provides a valuable molecular resource to study a range of questions in this genus, including phylogeny, incomplete lineage sorting, speciation, and hybridization. A genome browser displaying the genome, alignments, and annotations is available at https://genome-public.pks.mpg.de/cgi-bin/hgTracks?db=HLhylVes1.

Genome-wide SNP Data Reveal an Overestimation of Species Diversity in a Group of Hawkmoths.

The interface between populations and evolving young species continues to generate much contemporary debate in systematics depending on the species concept(s) applied but which ultimately reduces to the fundamental question of "when do nondiscrete entities become distinct, mutually exclusive evolutionary units"? Species are perceived as critical biological entities, and the discovery and naming of new species is perceived by many authors as a major research aim for assessing current biodiversity before much of it becomes extinct. However, less attention is given to determining whether these names represent valid biological entities because this is perceived as both a laborious chore and an undesirable research outcome. The charismatic spurge hawkmoths (Hyles euphorbiae complex, HEC) offer an opportunity to study this less fashionable aspect of systematics. To elucidate this intriguing systematic challenge, we analyzed over 10,000 ddRAD single nucleotide polymorphisms from 62 individuals using coalescent-based and population genomic methodology. These genome-wide data reveal a clear overestimation of (sub)species-level diversity and demonstrate that the HEC taxonomy has been seriously oversplit. We conclude that only one valid species name should be retained for the entire HEC, namely Hyles euphorbiae, and we do not recognize any formal subspecies or other taxonomic subdivisions within it. Although the adoption of genetic tools has frequently revealed morphologically cryptic diversity, the converse, taxonomic oversplitting of species, is generally (and wrongly in our opinion) accepted as rare. Furthermore, taxonomic oversplitting is most likely to have taken place in intensively studied popular and charismatic organisms such as the HEC.

Functional characterization of the Hyles euphorbiae hawkmoth transcriptome reveals strong expression of phorbol ester detoxification and seasonal cold hardiness genes.

BACKGROUND: The European spurge hawkmoth, Hyles euphorbiae (Lepidoptera, Sphingidae), has been intensively studied as a model organism for insect chemical ecology, cold hardiness and evolution of species delineation. To understand species isolation mechanisms at a molecular level, this study aims at determining genetic factors underlying two adaptive ecological trait candidates, phorbol ester (TPA) detoxification and seasonal cold acclimation. METHOD: A draft transcriptome of H. euphorbiae was generated using Illumina sequencing, providing the first genomic resource for the hawkmoth subfamily Macroglossinae. RNA expression levels in tissues of experimental TPA feeding larvae and cooled pupae was compared to levels in control larvae and pupae using 26 bp RNA sequence tag libraries (DeepSuperSAGE). Differential gene expression was assessed by homology searches of the tags in the transcriptome. RESULTS: In total, 389 and 605 differentially expressed transcripts for detoxification and cold hardiness, respectively, could be identified and annotated with proteins. The majority (22 of 28) of differentially expressed detox transcripts of the four 'drug metabolism' enzyme groups (cytochrome P450 (CYP), carboxylesterases (CES), glutathione S-transferases (GST) and lipases) are up-regulated. Triacylglycerol lipase was significantly over proportionally annotated among up-regulated detox transcripts. We record several up-regulated lipases, GSTe2, two CESs, CYP9A21, CYP6BD6 and CYP9A17 as candidate genes for further H. euphorbiae TPA detoxification analyses. Differential gene expression of the cold acclimation treatment is marked by metabolic depression with enriched Gene Ontology terms among down-regulated transcripts almost exclusively comprising metabolism, aerobic respiration and dissimilative functions. Down-regulated transcripts include energy expensive respiratory proteins like NADH dehydrogenase, cytochrome oxidase and ATP synthase. Gene expression patterns show shifts in carbohydrate metabolism towards cryoprotectant production. The Glycolysis enzymes, G1Pase, A1e, Gpi and an Akr isoform are up-regulated. Glycerol, an osmolyte which lowers the body liquid supercooling point, appears to be the predominant polyol cryoprotectant in H. euphorbiae diapause pupae. Several protein candidates involved in glucose, glycerol, myo-inositol and potentially sorbitol and trehalose synthesis were identified. CONCLUSIONS: A majority of differently expressed transcripts unique for either detoxification or cold hardiness indicates highly specialized functional adaptation which may have evolved from general cell metabolism and stress response.The transcriptome and extracted candidate biomarkers provide a basis for further gene expression studies of physiological processes and adaptive traits in H. euphorbiae.

Endemics or strangers? The integrative re-appraisal of taxonomy and phylogeny of the Greenland Lymnaeidae (Mollusca: Gastropoda).

The Lymnaeidae constitute a significant part of the freshwater molluscan diversity of Greenland. Since 1842, not less than 10 nominal taxa of the species and variety rank were described to organize the diversity of the Greenland lymnaeid snails. All previous attempts to revise these taxa were systematically based on morphological evidence only. Here, we provide a molecular analysis of the phylogenetic affinity and systematic status of three alleged species of the Greenland Lymnaeidae: Lymnaea vahlii (Møller, 1842), L. holboellii (Møller, 1842), and L. pingelii (Møller, 1842). We examined the newly collected material and inspected the type series of the three species. Our results show a very tight relationship between the Greenland snails and the Nearctic species Ladislavella catascopium (Say, 1817) s. lato. From the genetic point of view, the Greenland populations should be classified within L. catascopium, albeit probably with the merit of a subspecies status. The three nominal species of lymnaeids described by Møller (1842) are apparently synonyms of each other. Our findings assume a rather recent colonization of Greenland by snails arriving from the North American mainland, which is compatible with the so-called "tabula rasa" hypothesis, proposed to explain the currently observed taxonomic diversity of continental animals and plants of the North Atlantic islands. No lymnaeid species endemic to Greenland is thus revealed.

Historic DNA for taxonomy and conservation: A case-study of a century-old Hawaiian hawkmoth type (Lepidoptera: Sphingidae).

Analysing historic DNA from museum specimens offers the unique opportunity to study the molecular systematics and phylogenetics of rare and possibly extinct taxa. In the Hawaiian fauna, the hawkmoth, Hyles calida calida, occurs on several of the main islands and is quite frequent, whereas Hyles c. hawaiiensis is restricted to the Island of Hawaii where it appears to be very rare. Analysis of mitochondrial DNA sequences shows that Hyles c. hawaiiensis differs from the nominotypical subspecies by an average p-distance of 2.8%, which is of a similar order of magnitude to that found between other species of Hyles, suggesting that Hyles c. hawaiiensis should perhaps be awarded species status, although more data are required for a formal taxonomic revision. Given the rarity of this taxon, these analyses should be undertaken urgently so that conservation measures can be implemented before it becomes extinct.

Molecular phylogeny, morphology and taxonomy of Moroccan Triops granarius (Lucas, 1864) (Crustacea: Notostraca), with the description of two new species.

We used three molecular markers to investigate populations of Triops granarius from a study area in western Morocco that had a north-south span of approx. 434 km, the most distant populations situated at more than 470 km distance from each other. Previous studies had already investigated two Triops granarius populations from this region and revealed their affiliation to the major phylogenetic lineage that includes Triops cancriformis. By contrast, based on the geographic position of the type locality and the morphology of the type, Triops granarius s.s. likely belongs to a clade that forms the sister group to American and Australian Triops, i.e. including Triops longicaudatus and Triops australiensis. In the present study a second, hitherto unknown phylogenetic lineage was discovered among Moroccan populations of Triops granarius s.l. Our phylogenetic analyses show that both Moroccan lineages of Triops granarius s.l. represent a pair of genetically and morphologically well differentiated sister species that should be separated from Triops granarius. We therefore formally describe them as two new species, Triops maximus sp. nov. and Triops multifidus sp. nov. The early larval stages of both species show a peculiar morphology with 10 to 15 setae on the exopodite of the 2nd antenna. The number of these setae was generally thought to span five to seven in Notostraca. Despite the fact that the antennal setae form a central part of the main locomotory organ in early instars, we found their number to vary by up to two between body-sides of single individuals.

A comprehensive phylogeography of the Hyles euphorbiae complex (Lepidoptera: Sphingidae) indicates a 'glacial refuge belt'.

We test the morphology based hypothesis that the Western Palaearctic spurge hawkmoths represent two species, the Eurasian H. euphorbiae and Afro-Macaronesian H. tithymali. It has been suggested that these species merged into several hybrid swarm populations, although a mitochondrial phylogeography revealed substructure with local differentiation. We analysed a three-gene mt-dataset (889 individuals) and 12 microsatellite loci (892 individuals). Microsatellite analyses revealed an overall weak differentiation and corroborated the superordinate division into two clusters. The data indicate that the populations studied belong to only one species according to the biological species concept, refuting the opening hypothesis. A future taxonomic revision appears necessary to reflect the division into two subgroups. Ancestral mitochondrial polymorphisms are retained in H. euphorbiae, indicating gene flow within a broad 'glacial refuge belt' and ongoing postglacial gene flow. Diverse patterns of extensive mito-nuclear discordance in the Mediterranean and the Middle East presumably evolved by more recent processes. This discordance indicates introgression of H. tithymali-related mitochondrial haplogroups, accompanied (to a lesser degree) by nuclear alleles, into Italian and Aegean H. euphorbiae populations as recently as the late Holocene. The complex mosaic of divergence and reintegration is assumed to have been influenced by locally differing environmental barriers to gene flow.

Molecular phylogeny of the Notostraca.

We used a combined analysis of one nuclear (28S rDNA) and three mitochondrial markers (COI, 12S rDNA, 16S rDNA) to infer the molecular phylogeny of the Notostraca, represented by samples from the six continents that are inhabited by this group of branchiopod crustaceans. Our results confirm the monophyly of both extant notostracan genera Triops and Lepidurus with good support in model based and maximum parsimony analyses. We used branchiopod fossils as a calibration to infer divergence times among notostracan lineages and accounted for rate heterogeneity among lineages by applying relaxed-clock models. Our divergence date estimates indicate an initial diversification into the genera Triops and Lepidurus in the Mesozoic, most likely at a minimum age of 152.3-233.5 Ma, i.e., in the Triassic or Jurassic. Implications for the interpretation of fossils and the evolution of notostracan morphology are discussed. We further use the divergence date estimates to formulate a biogeographic hypothesis that explains distributions of extant lineages predominantly by overland dispersal routes. We identified an additional hitherto unrecognised highly diverged lineage within Lepidurus apus lubbocki and three additional previously unknown major lineages within Triops. Within T. granarius we found deep differentiation, with representatives distributed among three major phylogenetic lineages. One of these major lineages comprises T. cancriformis, the T. mauritanicus species group and two hitherto unrecognised T. granarius lineages. Samples that were morphologically identified as T. granarius diverged from the most basal nodes within this major lineage, and divergence dates suggested an approximate age of 23.7-49.6 Ma for T. cancriformis, indicating the need for a taxonomic revision of Triassic and Permian fossils that are currently attributed to the extant T. cancriformis. We thus elevate T. cancriformis minor to full species status as Triops minorTrusheim, 1938 and include in this species the additional Upper Triassic samples that were attributed to T. cancriformis. We further elevate T. cancriformis permiensis to full species status as Triops permiensisGand et al., 1997.

Mitochondrial lineage sorting in action--historical biogeography of the Hyles euphorbiae complex (Sphingidae, Lepidoptera) in Italy.

BACKGROUND: Mitochondrial genes are among the most commonly used markers in studies of species' phylogeography and to draw conclusions about taxonomy. The Hyles euphorbiae complex (HEC) comprises six distinct mitochondrial lineages in the Mediterranean region, of which one exhibits a cryptic disjunct distribution. The predominant mitochondrial lineage in most of Europe, euphorbiae, is also present on Malta; however, it is nowadays strangely absent from Southern Italy and Sicily, where it is replaced by 'italica'. A separate biological entity in Italy is further corroborated by larval colour patterns with a congruent, confined suture zone along the Northern Apennines. By means of historic DNA extracted from museum specimens, we aimed to investigate the evolution of the mitochondrial demographic structure of the HEC in Italy and Malta throughout the Twentieth Century. RESULTS: At the beginning of the Twentieth Century, the European mainland lineages were also present at a moderate frequency in Southern Italy and Sicily. The proportion of 'italica' then steadily increased in this area from below 60 percent to near fixation in about 120 years. Thus, geographical sorting of mitochondrial lineages in the HEC was not as complete then as the current demography suggests. The pattern of an integral 'italica' core region and a disjunct euphorbiae distribution evolved very recently. To explain these strong demographic changes, we propose genetic drift due to anthropogenic habitat loss and fragmentation in combination with an impact from recent climate warming that favoured the spreading of the potentially better adapted 'italica' populations. CONCLUSIONS: The pattern of geographically separated mitochondrial lineages is commonly interpreted as representing long term separated entities. However, our results indicate that such a pattern can emerge surprisingly quickly, even in a widespread and rather common taxon. We thus caution against drawing hasty taxonomic conclusions from biogeographical patterns of mitochondrial markers derived from modern sampling alone.

Overlooked cryptic endemism in copepods: systematics and natural history of the calanoid subgenus Occidodiaptomus Borutzky 1991 (Copepoda, Calanoida, Diaptomidae).

Our comprehension of the phylogeny and diversity of most inland-water crustaceans is currently hampered by their pronounced morphological bradytely, which contributed to the affirmation of the "Cosmopolitanism Paradigm" of freshwater taxa. However, growing evidence of the existence of cryptic diversity and molecular regionalism is available for calanoid copepods, thus stressing the need for careful morphological and molecular studies in order to soundly investigate the systematics, diversity and distribution patterns of the group. Diaptomid copepods were here chosen as model taxa, and the morphological and molecular diversity of the species belonging to the west-Mediterranean diaptomid subgenus Occidodiaptomus were investigated with the aim of comparing the patterns of morphological and molecular evolution in freshwater copepods. Three species currently lumped under the binomen Hemidiaptomus (Occidodiaptomus) ingens and two highly divergent clades within H. (O.) roubaui were distinguished, thus showing an apparent discordance between the molecular distances recorded and Occidodiaptomus morphological homogeneity, and highlighting a noteworthy decoupling between the morphological and molecular diversity in the subgenus. Current Occidodiaptomus diversity pattern is ascribed to a combined effect of ancient vicariance and recent dispersal events. It is stressed that the lack of sound calibration points for the molecular clock makes it difficult to soundly temporally frame the diversification events of interest in the taxon studied, and thus to asses the role of morphological bradytely and of accelerated molecular evolutionary rates in shaping the current diversity of the group.

A revised molecular phylogeny of the globally distributed hawkmoth genus Hyles (Lepidoptera: Sphingidae), based on mitochondrial and nuclear DNA sequences.

The hawkmoth genus Hyles comprises some 29 species with a global distribution. In this study, we augment the previous taxon sampling with more species and add sequences from a nuclear gene to produce a refined phylogenetic hypothesis. A total evidence reconstruction based on Bayesian analysis of the combined mitochondrial (COI, t-RNA-Leu, COII; 2284 bp) and nuclear (EF1alpha; 773 bp) sequences is discussed and compared with the results from separate analyses of the two genes. The total evidence phylogeny corroborates many of the phylogenetic relationships previously postulated within the genus. In addition, the hitherto unsampled enigmatic species Hyles biguttata from Madagascar appears as sister group to Hyles livornicoides from Australia, although support for the relationship is relatively weak. The high level of differentiation of Hyles perkinsi from H. calida (both Hawaii), and the status of these two as sister species, is corroborated by both sources of sequence data. However, their phylogenetic position when mt DNA sequences alone are considered differs markedly from that under total evidence. The previously postulated relationships within the Hyles euphorbiae complex (HEC) s.s. are largely corroborated, but H. dahlii is now more closely related and the HEC s.l. is redefined to include H. zygophylli and H. stroehlei (two species that had not been studied previously using molecular data) and to exclude H. siehei and H. hippophaes. The nuclear sequences alone are insufficiently variable to fully resolve all lineages and the phylogeny suggests that nuclear gene swapping and incomplete lineage sorting have occurred implying recent divergence. The results from the total evidence analysis provide a phylogenetic hypothesis that both corroborates and complements the previous biogeographic scenario, and provides new insights into the origins of several of the included taxa.

New source of genetic polymorphisms in Lepidoptera?

The variability level of the ISSR (inter-simple sequences repeat) primer (GACA)4 was examined in the three Lepidoptera families Pyralidae, Sphingidae and Pieridae. Our study shows that the tetra-repeat (GACA)n is evidently present in sufficient numbers in these butterflies to provide informative DNA fingerprints. The variability is mostly rather high, but within a comparable range to other ISSR studies. Although less polymorphisms may be encountered in some butterfly families, this study indicates that high variability of this marker may be a common characteristic of Lepidoptera genomes. An appeal for a minimal level of standardization of ISSR-PCR data analysis is formulated to enable an exact comparison between the groups of organisms studied with this fingerprint technique.

A molecular phylogeny of the hawkmoth genus Hyles (Lepidoptera: Sphingidae, Macroglossinae).

The hawkmoth genus Hyles is one of 15 genera in the subtribe Choerocampina of the subfamily Macroglossinae. Due to a remarkable uniformity, morphological characters usually used to identify and classify Lepidoptera at the species level cannot be used in this genus. Instead, we used DNA sequences comprising about 2300 bp derived from the mitochondrial genes COX I, COX II, and tRNA-leucine to elucidate the phylogeny of Hyles. The results corroborate the monophyly of Hyles but conflict with previous internal classifications of the genus based on morphology. Hyles seems to have evolved in the Neotropics during the Oligocene/Eocene epochs and the molecular data (which evolved clock-like) confirm the hypothesis that it is a very young genus that radiated on a global scale rather quickly. We hypothesize its sister group to be one of the genera Deilephila, Theretra or Xylophanes. The Nearctic may have been colonized rapidly by Hyles once the land bridge formed during the Pliocene, since within this same Epoch, the invasion of the Palaearctic appears to have proceeded from the East, via the Bering route. The colonization of Australia appears to have occurred rather early in Hyles radiation, although the route is not clear. We propose that the radiation of the Hyles euphorbiae-complex s. str. (HEC) occurred as recently as the Pliocene/Pleistocene boundary and that its roots can still be reconstructed in Asia. Hyles dahlii is closely related to the HEC, but a sister group relationship to the HEC s. str. cannot be corroborated unequivocally. HEC population ranges appear to have tracked climate oscillations during the Pleistocene Ice Ages, resulting in hybridization around the Mediterranean Sea as they repeatedly intermingled. Comparison of the phylogeny with food plant affiliations leads us to hypothesize that Euphorbia monophagy evolved at least two times independently within Hyles.