Chromosomal conservatism vs chromosomal megaevolution: enigma of karyotypic evolution in Lepidoptera.

In the evolution of many organisms, periods of slow genome reorganization (= chromosomal conservatism) are interrupted by bursts of numerous chromosomal changes (= chromosomal megaevolution). Using comparative analysis of chromosome-level genome assemblies, we investigated these processes in blue butterflies (Lycaenidae). We demonstrate that the phase of chromosome number conservatism is characterized by the stability of most autosomes and dynamic evolution of the sex chromosome Z, resulting in multiple variants of NeoZ chromosomes due to autosome-sex chromosome fusions. In contrast during the phase of rapid chromosomal evolution, the explosive increase in chromosome number occurs mainly through simple chromosomal fissions. We show that chromosomal megaevolution is a highly non-random canalized process, and in two phylogenetically independent Lysandra lineages, the drastic parallel increase in number of fragmented chromosomes was achieved, at least partially, through reuse of the same ancestral chromosomal breakpoints. In species showing chromosome number doubling, we found no blocks of duplicated sequences or duplicated chromosomes, thus refuting the hypothesis of polyploidy. In the studied taxa, long blocks of interstitial telomere sequences (ITSs) consist of (TTAGG)n arrays interspersed with telomere-specific retrotransposons. ITSs are sporadically present in rapidly evolving Lysandra karyotypes, but not in the species with ancestral chromosome number. Therefore, we hypothesize that the transposition of telomeric sequences may be triggers of the rapid chromosome number increase. Finally, we discuss the hypothetical genomic and population mechanisms of chromosomal megaevolution and argue that the disproportionally high evolutionary role of the Z sex chromosome can be additionally reinforced by sex chromosome-autosome fusions and Z-chromosome inversions.

Genomic introgression from a distant congener in the Levant fritillary butterfly, Melitaea acentria.

Introgressive hybridization is more common in nature than previously thought, and its role and creative power in evolution is hotly discussed but not completely understood. Introgression occurs more frequently in sympatry between recently diverged taxa, or when the speciation process has not yet been completed. However, there are relatively few documented cases of hybridization that erodes reproductive barriers between distantly related species. Here, we use whole genome and mitochondrial data to examine how introgression from a distant congener affects pattern of genetic differentiation in the Levant fritillary butterfly Melitaea acentria. We show that this local taxon has evolved as a peripatric geographic isolate of the widespread Melitaea persea, and that there has been significant unidirectional gene flow from the sympatric, nonclosely related Melitaea didyma to M. acentria. We found direct evidence of ongoing sporadic hybridization between M. didyma and M. acentria, which are separated by at least 5 million years of independent evolution. Elevated differentiation and lower level of introgression on the sex Z chromosome compared to autosomes suggest that the Z chromosome has accumulated loci acting as intrinsic postzygotic barriers. Our results show that introgression from M. didyma has been an additional source of nucleotide diversity in the M. acentria population, providing material for drift and selection.

Versatility of multivalent orientation, inverted meiosis, and rescued fitness in holocentric chromosomal hybrids.

Chromosomal rearrangements (e.g., fusions/fissions) have the potential to drive speciation. However, their accumulation in a population is generally viewed as unlikely, because chromosomal heterozygosity should lead to meiotic problems and aneuploid gametes. Canonical meiosis involves segregation of homologous chromosomes in meiosis I and sister chromatid segregation during meiosis II. In organisms with holocentric chromosomes, which are characterized by kinetic activity distributed along almost the entire chromosome length, this order may be inverted depending on their metaphase I orientation. Here we analyzed the evolutionary role of this intrinsic versatility of holocentric chromosomes, which is not available to monocentric ones, by studying F1 to F4 hybrids between two chromosomal races of the Wood White butterfly (Leptidea sinapis), separated by at least 24 chromosomal fusions/fissions. We found that these chromosomal rearrangements resulted in multiple meiotic multivalents, and, contrary to the theoretical prediction, the hybrids displayed relatively high reproductive fitness (42% of that of the control lines) and regular behavior of meiotic chromosomes. In the hybrids, we also discovered inverted meiosis, in which the first and critical stage of chromosome number reduction was replaced by the less risky stage of sister chromatid separation. We hypothesize that the ability to invert the order of the main meiotic events facilitates proper chromosome segregation and hence rescues fertility and viability in chromosomal hybrids, potentially promoting dynamic karyotype evolution and chromosomal speciation.

Integrative analyses unveil speciation linked to host plant shift in Spialia butterflies.

Discovering cryptic species in well-studied areas and taxonomic groups can have profound implications in understanding eco-evolutionary processes and in nature conservation because such groups often involve research models and act as flagship taxa for nature management. In this study, we use an array of techniques to study the butterflies in the Spialia sertorius species group (Lepidoptera, Hesperiidae). The integration of genetic, chemical, cytogenetic, morphological, ecological and microbiological data indicates that the sertorius species complex includes at least five species that differentiated during the last three million years. As a result, we propose the restitution of the species status for two taxa often treated as subspecies, Spialia ali (Oberthür, 1881) stat. rest. and Spialia therapne (Rambur, 1832) stat. rest., and describe a new cryptic species Spialia rosae Hernández-Roldán, Dapporto, Dinca, Vicente & Vila sp. nov. Spialia sertorius (Hoffmannsegg, 1804) and S. rosae are sympatric and synmorphic, but show constant differences in mitochondrial DNA, chemical profiles and ecology, suggesting that S. rosae represents a case of ecological speciation involving larval host plant and altitudinal shift, and apparently associated with Wolbachia infection. This study exemplifies how a multidisciplinary approach can reveal elusive cases of hidden diversity.

Homoploid hybrid speciation and genome evolution via chromosome sorting.

Genomes of numerous diploid plant and animal species possess traces of interspecific crosses, and many researches consider them as support for homoploid hybrid speciation (HHS), a process by which a new reproductively isolated species arises through hybridization and combination of parts of the parental genomes, but without an increase in ploidy. However, convincing evidence for a creative role of hybridization in the origin of reproductive isolation between hybrid and parental forms is extremely limited. Here, through studying Agrodiaetus butterflies, we provide proof of a previously unknown mode of HHS based on the formation of post-zygotic reproductive isolation via hybridization of chromosomally divergent parental species and subsequent fixation of a novel combination of chromosome fusions/fissions in hybrid descendants. We show that meiotic segregation, operating in the hybrid lineage, resulted in the formation of a new diploid genome, drastically rearranged in terms of chromosome number. We also demonstrate that during the heterozygous stage of the hybrid species formation, recombination was limited between rearranged chromosomes of different parental origin, representing evidence that the reproductive isolation was a direct consequence of hybridization.

Taxonomic Position and Status of Arctic Gynaephora and Dicallomera Moths (Lepidoptera, Erebidae, Lymantriinae).

We use analysis of mitochondrial DNA barcodes in combination with published data on morphology to rearrange the taxonomy of two arctic species, Gynaephora groenlandica and G. rossii. We demonstrate that (1) the taxon lugens Kozhanchikov, 1948 originally described as a distinct species is a subspecies of Gynaephora rossii, and (2) the taxon kusnezovi Lukhtanov et Khruliova, 1989 originally described as a distinct species in the genus Dicallomera is a subspecies of Gynaephora groenlandica. We also provide the first evidence for the occurrence of G. groenlandica in the Palearctic region (Wrangel Island).

In the shadow of phylogenetic uncertainty: the recent diversification of Lysandra butterflies through chromosomal change.

The phylogeny of the butterfly genus Lysandra (Lycaenidae, Polyommatinae) has been intractable using both molecular and morphological characters, which could be a result of speciation due to karyotype instability. Here we reconstruct the phylogeny of the group using multi-locus coalescent-based methods on seven independent genetic markers. While the genus is ca. 4.9 Mya old, the diversification of the extant lineages was extremely recent (ca. 1.5 Mya) and involved multiple chromosomal rearrangements. We find that relationships are uncertain due to both incomplete lineage sorting and hybridization. Minimizing the impact of reticulation in inferring the species tree by testing for mitochondrial introgression events yields a partially resolved tree with three main supported clades: L. punctifera+L. bellargus, the corydonius taxa, and L. coridon+the Iberian taxa, plus three independent lineages without apparently close relatives (L. ossmar, L. syriaca and L. dezina). Based on these results and new karyotypic data, we propose a rearrangement recognizing ten species within the genus. Finally, we hypothesize that chromosomal instability may have played a crucial role in the Lysandra recent diversification. New chromosome rearrangements might be fixed in populations after severe bottlenecks, which at the same time might promote rapid sorting of neutral molecular markers. We argue that population bottlenecks might be a prerequisite for chromosomal speciation in this group.

Establishing criteria for higher-level classification using molecular data: the systematics of Polyommatus blue butterflies (Lepidoptera, Lycaenidae).

Most taxonomists agree on the need to adapt current classifications to recognize monophyletic units. However, delineations between higher taxonomic units can be based on the relative ages of different lineages and/or the level of morphological differentiation. In this paper, we address these issues in considering the species-rich Polyommatus section, a group of butterflies whose taxonomy has been highly controversial. We propose a taxonomy-friendly, flexible temporal scheme for higher-level classification. Using molecular data from nine markers (6666 bp) for 104 representatives of the Polyommatus section, representing all but two of the 81 described genera/subgenera and five outgroups, we obtained a complete and well resolved phylogeny for this clade. We use this to revise the systematics of the Polyommatus blues, and to define criteria that best accommodate the described genera within a phylogenetic framework. First, we normalize the concept of section (Polyommatus) and propose the use of subtribe (Polyommatina) instead. To preserve taxonomic stability and traditionally recognized taxa, we designate an age interval (4-5 Myr) instead of a fixed minimum age to define genera. The application of these criteria results in the retention of 31 genera of the 81 formally described generic names, and necessitates the description of one new genus (Rueckbeilia gen. nov.). We note that while classifications should be based on phylogenetic data, applying a rigid universal scheme is rarely feasible. Ideally, taxon age limits should be applied according to the particularities and pre-existing taxonomy of each group. We demonstrate that the concept of a morphological gap may be misleading at the genus level and can produce polyphyletic genera, and we propose that recognition of the existence of cryptic genera may be useful in taxonomy.

Whole-Genome Analysis Reveals the Dynamic Evolution of Holocentric Chromosomes in Satyrine Butterflies.

Butterfly chromosomes are holocentric, i.e., lacking a localized centromere. Potentially, this can lead to rapid karyotypic evolution through chromosome fissions and fusions, since fragmented chromosomes retain kinetic activity, while fused chromosomes are not dicentric. However, the actual mechanisms of butterfly genome evolution are poorly understood. Here, we analyzed chromosome-scale genome assemblies to identify structural rearrangements between karyotypes of satyrine butterfly species. For the species pair Erebia ligea-Maniola jurtina, sharing the ancestral diploid karyotype 2n = 56 + ZW, we demonstrate a high level of chromosomal macrosynteny and nine inversions separating these species. We show that the formation of a karyotype with a low number of chromosomes (2n = 36 + ZW) in Erebia aethiops was based on ten fusions, including one autosome-sex chromosome fusion, resulting in a neo-Z chromosome. We also detected inversions on the Z sex chromosome that were differentially fixed between the species. We conclude that chromosomal evolution is dynamic in the satyrines, even in the lineage that preserves the ancestral chromosome number. We hypothesize that the exceptional role of Z chromosomes in speciation may be further enhanced by inversions and sex chromosome-autosome fusions. We argue that not only fusions/fissions but also inversions are drivers of the holocentromere-mediated mode of chromosomal speciation.

Taxonomic Structure and Wing Pattern Evolution in the Parnassius mnemosyne Species Complex (Lepidoptera, Papilionidae).

In our study, using the analysis of DNA barcodes and morphology (wing color, male genitalia, and female sphragis shape), we show that the group of species close to P. mnemosyne comprises the western and eastern phylogenetic lineages. The eastern lineage includes P. stubbendorfii, P. glacialis, and P. hoenei. The western lineage includes three morphologically similar species: P. mnemosyne (Western Eurasia), P. turatii (southwestern Europe), and P. nubilosusstat. nov. (Turkmenistan and NE Iran), as well as the morphologically differentiated P. ariadne (Altai). The latter species differs from the rest of the group in the presence of red spots on the wings. Parnassius mnemosyne s.s. is represented by four differentiated mitochondrial clusters that show clear association with specific geographic regions. We propose to interpret them as subspecies: P. mnemosyne mnemosyne (Central and Eastern Europe, N Caucasus, N Turkey), P. mnemosyne adolphi (the Middle East), P. mnemosyne falsa (Tian Shan), and P. mnemosyne gigantea (Gissar-Alai in Central Asia). We demonstrate that in P. ariadne, the red spots on the wing evolved as a reversion to the ancestral wing pattern. This reversion is observed in Altai, where the distribution areas of the western lineage, represented by P. ariadne, and the eastern lineage, represented by P. stubbendorfii, overlap. These two species hybridize in Altai, and we hypothesize that the color change in P. ariadne is the result of reinforcement of prezygotic isolation in the contact zone. The lectotype of Parnassius mnemosyne var. nubilosus Christoph, 1873, is designated.

Phylogenetic Structure Revealed through Combining DNA Barcodes with Multi-Gene Data for Agrodiaetus Blue Butterflies (Lepidoptera, Lycaenidae).

The need for multi-gene analysis in evolutionary and taxonomic studies is generally accepted. However, the sequencing of multiple genes is not always possible. For various reasons, short mitochondrial DNA barcodes are the only source of molecular information for some species in many genera, although multi-locus data are available for other species of the same genera. In particular, such situation exists in the species-rich butterfly subgenus Polyommatus (Agrodiaetus). Here, we analyzed the partitioning of this subgenus into species groups by using three data sets. The first data set was represented by short mitochondrial DNA barcodes for all analyzed samples. The second and third data sets were represented by a combination of short mitochondrial DNA barcodes for part of the taxa with longer mitochondrial sequences COI + tRNA-Leu + COII (data set 2) and with longer mitochondrial COI + tRNA-Leu + COII and nuclear 5.8S rDNA + ITS2 + 28S rDNA sequences (data set 3) for the remaining species. We showed that the DNA barcoding approach (data set 1) failed to reveal the phylogenetic structure, resulting in numerous polytomies in the tree obtained. Combined analysis of the mitochondrial and nuclear sequences (data sets 2 and 3) revealed the species groups and the position within these species groups, even for the taxa for which only short DNA barcodes were available.

Investigations into the Melitaea ornata species complex in the Levant: M. telona and the newly erected species Melitaea klili Benyamini, 2021 (Lepidoptera: Nymphalidae).

Melitaea klili Benyamini, 2021 was recently described from Israel as a species closely related to M. telona Fruhstorfer, 1908, but different in phenology, ecological preferences and with an allopatric distribution. Here, based on comparative examinations of mitochondrial DNA-barcodes, male genitalia and larval behaviour under laboratory conditions, we synonymize M. klili with M. telona. The COI barcodes of M. klili were found to be identical to those of M. telona. Analysis of 658 bp fragment of the mitochondrial gene COI demonstrated that the minimum uncorrected p-distance between M. ornata and M. telona was 1.98%. This value is remarkably less than the 3% threshold traditionally accepted as a species boundary in DNA barcoding studies. The morphological differences between these taxa are minimal. In fact, M. ornata and M. telona represent two phylogenetic lineages, the taxonomic status of which (separate species or subspecies of the same species) is intermediate and debatable.

More hidden diversity in a cryptic species complex: a new subspecies of Leptideasinapis (Lepidoptera, Pieridae) from Northern Iran.

A new subspecies of Leptideasinapis from Northern Iran, discovered by means of DNA barcoding, is described as Leptideasinapistabarestanassp. nov. The new subspecies is allopatric with respect to other populations of L.sinapis and is genetically distinct, appearing as a well-supported sister clade to all other populations in COI-based phylogenetic reconstructions. Details on karyotype, genitalia, ecology and behaviour for the new subspecies are given and a biogeographical speciation scenario is proposed.

Molecular Phylogeny and Taxonomy of the Butterfly Subtribe Scolitantidina with Special Focus on the Genera Pseudophilotes, Glaucopsyche and Iolana (Lepidoptera, Lycaenidae).

The Palearctic blue butterfly genus Pseudophilotes Beuret, 1958 is not homogenous regarding the morphology of its genital structures. For this reason, some of its species have been considered to be representatives of other genera of the subtribe Scolitantidina (subfamily Polyommatinae). Here, we address these taxonomic problems by analyzing the phylogenetic relationships between the genera, subgenera, and species of this subtribe inferred via the analysis of five nuclear and two mitochondrial DNA sequences. We demonstrate that the enigmatic Asian species P. panope (Eversmann, 1851) belongs to the genus Pseudophilotes but not to Praephilotes Forster, 1938 or Palaeophilotes Forster, 1938 and does not represent the independent genus Inderskia Korshunov, 2000, as hypothesized previously. We synonymize P. svetlana Yakovlev, 2003 (syn. nov.) and P. marina Zhdanko, 2004 (syn. nov.) with P. panope. We demonstrate a deep genetic divergence between lineages that were previously considered as subspecies of the single species Iolana iolas (Ochsenheimer, 1816). As a result, we confirm the multispecies concept of the genus Iolana Bethune-Baker, 1914. We show that the Holarctic genus Glaucopsyche can be divided into four subgenera: Glaucopsyche Scudder, 1872 (=Shijimiaeoides Beuret, 1958), Apelles Hemming, 1931, Bajluana Korshunov and Ivonin, 1990, and Phaedrotes Scudder, 1876.

Reassessment of the status of some European and Asian Melitaea taxa described as subspecies of Melitaea phoebe ([Denis amp; Schiffermller], 1775), with designations of lectotypes where appropriate (Lepidoptera: Nymphalidae).

Morphological characters of value in distinguishing Melitaea phoebe from M. ornata are exemplified from photographs of specimens from sympatric and partially synchronic populations in the Russian Federation and Slovenia. Type material of a number of subspecies classified as M. phoebe have been examined and confirmed as being subspecies of M. phoebe or as subspecies of either M. ornata or M. telona. Larval and adult morphology of M. telona is very similar to those of M. ornata, making morphological separation difficult. Distribution of M. telona forms a major criterion for separation. Lectotypes are designated for amanica Rebel, 1917 and dorae Graves, 1925. The name enoch Higgins, 1941 is recognised as a distinct species and the names abbas Gross Ebert, 1975 and zagrosi Tth Varga, 2011 synonymised with Melitaea enoch.

Unprecedented within-species chromosome number cline in the Wood White butterfly Leptidea sinapis and its significance for karyotype evolution and speciation.

BACKGROUND: Species generally have a fixed number of chromosomes in the cell nuclei while between-species differences are common and often pronounced. These differences could have evolved through multiple speciation events, each involving the fixation of a single chromosomal rearrangement. Alternatively, marked changes in the karyotype may be the consequence of within-species accumulation of multiple chromosomal fissions/fusions, resulting in highly polymorphic systems with the subsequent extinction of intermediate karyomorphs. Although this mechanism of chromosome number evolution is possible in theory, it has not been well documented. RESULTS: We present the discovery of exceptional intraspecific variability in the karyotype of the widespread Eurasian butterfly Leptidea sinapis. We show that within this species the diploid chromosome number gradually decreases from 2n = 106 in Spain to 2n = 56 in eastern Kazakhstan, resulting in a 6000 km-wide cline that originated recently (8,500 to 31,000 years ago). Remarkably, intrapopulational chromosome number polymorphism exists, the chromosome number range overlaps between some populations separated by hundreds of kilometers, and chromosomal heterozygotes are abundant. We demonstrate that this karyotypic variability is intraspecific because in L. sinapis a broad geographical distribution is coupled with a homogenous morphological and genetic structure. CONCLUSIONS: The discovered system represents the first clearly documented case of explosive chromosome number evolution through intraspecific and intrapopulation accumulation of multiple chromosomal changes. Leptidea sinapis may be used as a model system for studying speciation by means of chromosomally-based suppressed recombination mechanisms, as well as clinal speciation, a process that is theoretically possible but difficult to document. The discovered cline seems to represent a narrow time-window of the very first steps of species formation linked to multiple chromosomal changes that have occurred explosively. This case offers a rare opportunity to study this process before drift, dispersal, selection, extinction and speciation erase the traces of microevolutionary events and just leave the final picture of a pronounced interspecific chromosomal difference.

Reinforcement of pre-zygotic isolation and karyotype evolution in Agrodiaetus butterflies.

The reinforcement model of evolution argues that natural selection enhances pre-zygotic isolation between divergent populations or species by selecting against unfit hybrids or costly interspecific matings. Reinforcement is distinguished from other models that consider the formation of reproductive isolation to be a by-product of divergent evolution. Although theory has shown that reinforcement is a possible mechanism that can lead to speciation, empirical evidence has been sufficiently scarce to raise doubts about the importance of reinforcement in nature. Agrodiaetus butterflies (Lepidoptera: Lycaenidae) exhibit unusual variability in chromosome number. Whereas their genitalia and other morphological characteristics are largely uniform, different species vary considerably in male wing colour, and provide a model system to study the role of reinforcement in speciation. Using comparative phylogenetic methods, we show that the sympatric distribution of 15 relatively young sister taxa of Agrodiaetus strongly correlates with differences in male wing colour, and that this pattern is most likely the result of reinforcement. We find little evidence supporting sympatric speciation: rather, in Agrodiaetus, karyotypic changes accumulate gradually in allopatry, prompting reinforcement when karyotypically divergent races come into contact.

Chromosomal and DNA barcode analysis of the Polyommatus (Agrodiaetus) damone (Eversmann, 1841) species complex (Lepidoptera, Lycaenidae).

The Polyommatus (Agrodiaetus) damone (Eversmann, 1841) species complex comprises from 5 to 8 species distributed in southeastern Europe and southern Siberia. Here we used chromosomal and DNA-barcode markers in order to test the taxonomic hypotheses previously suggested for this complex. We revealed that all taxa within this group demonstrate chromosomal stasis and share the same or very similar haploid chromosome number (n = 66 or n = 67). This finding is unexpected since the karyotypes are known to be very diverse and species-specific within the other taxa of the subgenus Agrodiaetus Hübner, 1822. Analysis of the mitochondrial gene COI revealed six diverged clusters of individuals within the complex. Each cluster has a specific geographic distribution and is characterized by distinct morphological features in the wing pattern. The clusters mostly (but not always) correlate with traditionally recognized species. As a result of our study, we describe a new subspecies P. (A.) iphigenides zarmitanussubsp. nov. from Uzbekistan and Tajikistan and show that the taxon originally described as Lycaena kindermanni var. melania Staudinger, 1886 represents a subspecies P. (A.) iphigenides melanius (Staudinger, 1886). Polyommatus (A.) samusi Korb, 2017 (syn. nov.) and P. (A.) melanius komarovi Korb, 2017 (syn. nov.) are considered here as junior subjective synonyms of P. (A.) iphigenides iphigenides (Staudinger, 1886).

Chromosomal and DNA barcode analysis of the Melitaea ala Staudinger, 1881 species complex (Lepidoptera, Nymphalidae).

The species of the Melitaea ala Staudinger, 1881 complex are distributed in Central Asia. Here we show that this complex is a monophyletic group including the species, M. ala, M. kotshubeji Sheljuzhko, 1929 and M. enarea Fruhstorfer, 1917. The haploid chromosome number n=29 is found in M. ala and M. kotshubeji and is, most likely, a symplesiomorphy of the M. ala complex. We show that M. ala consists of four subspecies: M. ala zaisana Lukhtanov, 1999 (=M. ala irtyshica Lukhtanov, 1999, syn. nov.) (South Altai, Zaisan Lake valley), M. ala ala (Dzhungarian Alatau), M. ala bicolor Seitz, 1908 (North, East, Central and West Tian-Shan) and M. ala determinata Bryk, 1940 (described from "Fu-Shu-Shi", China). We demonstrate that M. kotshubeji kotshubeji (Peter the Great Mts in Tajikistan) and M. kotshubeji bundeli Kolesnichenko, 1999 (Alai Mts in Tajikistan and Kyrgyzstan) are distinct taxa despite their geographic proximity in East Tajikistan. Melitaea enarea is widely distributed in the southern part of Central Asia and is sympatric with M. kotshubeji.