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.

Evolution of multiple sex-chromosomes associated with dynamic genome reshuffling in Leptidea wood-white butterflies.

Sex-chromosome systems tend to be highly conserved and knowledge about their evolution typically comes from macroevolutionary inference. Rapidly evolving complex sex-chromosome systems represent a rare opportunity to study the mechanisms of sex-chromosome evolution at unprecedented resolution. Three cryptic species of wood-white butterflies-Leptidea juvernica, L. sinapis and L. reali-have each a unique set of multiple sex-chromosomes with 3-4 W and 3-4 Z chromosomes. Using a transcriptome-based microarray for comparative genomic hybridisation (CGH) and a library of bacterial artificial chromosome (BAC) clones, both developed in L. juvernica, we identified Z-linked Leptidea orthologs of Bombyx mori genes and mapped them by fluorescence in situ hybridisation (FISH) with BAC probes on multiple Z chromosomes. In all three species, we determined synteny blocks of autosomal origin and reconstructed the evolution of multiple sex-chromosomes. In addition, we identified W homologues of Z-linked orthologs and characterised their molecular differentiation. Our results suggest that the multiple sex-chromosome system evolved in a common ancestor as a result of dynamic genome reshuffling through repeated rearrangements between the sex chromosomes and autosomes, including translocations, fusions and fissions. Thus, the initial formation of neo-sex chromosomes could not have played a role in reproductive isolation between these Leptidea species. However, the subsequent species-specific fissions of several neo-sex chromosomes could have contributed to their reproductive isolation. Then, significantly increased numbers of Z-linked genes and independent neo-W chromosome degeneration could accelerate the accumulation of genetic incompatibilities between populations and promote their divergence resulting in 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.

Neo-sex chromosomes and adaptive potential in tortricid pests.

Changes in genome architecture often have a significant effect on ecological specialization and speciation. This effect may be further enhanced by involvement of sex chromosomes playing a disproportionate role in reproductive isolation. We have physically mapped the Z chromosome of the major pome fruit pest, the codling moth, Cydia pomonella (Tortricidae), and show that it arose by fusion between an ancestral Z chromosome and an autosome corresponding to chromosome 15 in the Bombyx mori reference genome. We further show that the fusion originated in a common ancestor of the main tortricid subfamilies, Olethreutinae and Tortricinae, comprising almost 700 pest species worldwide. The Z-autosome fusion brought two major genes conferring insecticide resistance and clusters of genes involved in detoxification of plant secondary metabolites under sex-linked inheritance. We suggest that this fusion significantly increased the adaptive potential of tortricid moths and thus contributed to their radiation and subsequent speciation.

Dynamic karyotype evolution and unique sex determination systems in Leptidea wood white butterflies.

BACKGROUND: Chromosomal rearrangements have the potential to limit the rate and pattern of gene flow within and between species and thus play a direct role in promoting and maintaining speciation. Wood white butterflies of the genus Leptidea are excellent models to study the role of chromosome rearrangements in speciation because they show karyotype variability not only among but also within species. In this work, we investigated genome architecture of three cryptic Leptidea species (L. juvernica, L. sinapis and L. reali) by standard and molecular cytogenetic techniques in order to reveal causes of the karyotype variability. RESULTS: Chromosome numbers ranged from 2n = 85 to 91 in L. juvernica and 2n = 69 to 73 in L. sinapis (both from Czech populations) to 2n = 51 to 55 in L. reali (Spanish population). We observed significant differences in chromosome numbers and localization of cytogenetic markers (rDNA and H3 histone genes) within the offspring of individual females. Using FISH with the (TTAGG) n telomeric probe we also documented the presence of multiple chromosome fusions and/or fissions and other complex rearrangements. Thus, the intraspecific karyotype variability is likely due to irregular chromosome segregation of multivalent meiotic configurations. The analysis of female meiotic chromosomes by GISH and CGH revealed multiple sex chromosomes: W1W2W3Z1Z2Z3Z4 in L. juvernica, W1W2W3Z1Z2Z3 in L. sinapis and W1W2W3W4Z1Z2Z3Z4 in L. reali. CONCLUSIONS: Our results suggest a dynamic karyotype evolution and point to the role of chromosomal rearrangements in the speciation of Leptidea butterflies. Moreover, our study revealed a curious sex determination system with 3-4 W and 3-4 Z chromosomes, which is unique in the Lepidoptera and which could also have played a role in the speciation process of the three Leptidea species.

Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella.

BACKGROUND: We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. RESULTS: Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. CONCLUSIONS: We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.

Rapid turnover of the W chromosome in geographical populations of wild silkmoths, Samia cynthia ssp.

Our previous studies revealed a considerably high level of chromosomal polymorphism in wild silkmoths, Samia cynthia ssp. (Lepidoptera: Saturniidae). Geographical populations of this species complex differ in chromosome numbers and show derived sex chromosome systems including Z0/ZZ in S. cynthia ricini (2n = 27/28; Vietnam), neo-Wneo-Z/neo-Zneo-Z in S. cynthia walkeri (2n = 26/26; Sapporo, Hokkaido) and neo-WZ1Z2/Z1Z1Z2Z2 in S. cynthia subsp. indet. (2n = 25/26; Nagano, Honshu). In this study, we collected specimens of S. cynthia pryeri in Japanese islands Kyushu, Shikoku and Honshu, with an ancestral-like karyotype of 2n = 28 in both sexes and a WZ/ZZ sex chromosome system, except for one population, in which females have lost the W chromosome. However, the S. cynthia pryeri W chromosome showed a very unusual morphology: It was composed of a highly heterochromatic body, which remained condensed throughout the whole cell cycle and of a euchromatin-like "tail." We examined molecular composition of the W and neo-W chromosomes in S. cynthia subspecies by comparative genomic hybridisation and fluorescence in situ hybridisation with W chromosome painting probes prepared from laser-microdissected W chromatin of S. cynthia pryeri. These methods revealed that the molecular composition of highly heterochromatic part of the S. cynthia pryeri W chromosome is very different and lacks homology in the genomes of other subspecies, whereas the euchromatin-like part of the W chromosome corresponds to a heterochromatic part of the neo-W chromosomes in S. cynthia walkeri and S. cynthia subsp. indet. Our findings suggest that the curious WZ system of S. cynthia pryeri may represent an ancestral state of the Samia species complex but do not exclude an alternative hypothesis of its derived origin.

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae).

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n = 12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.

Sex Chromosomes of the Iconic Moth Abraxas grossulariata (Lepidoptera, Geometridae) and Its Congener A. sylvata.

The magpie moth, Abraxas grossulariata, is an iconic species in which female heterogamety was discovered at the beginning of the 20th century. However, the sex chromosomes of this species have not yet been cytologically identified. We describe the sex chromosomes of A. grossulariata and its congener, A. sylvata. Although these species split only around 9.5 million years ago, and both species have the expected WZ/ZZ chromosomal system of sex determination and their sex chromosomes share the major ribosomal DNA (rDNA) representing the nucleolar organizer region (NOR), we found major differences between their karyotypes, including between their sex chromosomes. The species differ in chromosome number, which is 2n = 56 in A. grossularita and 2n = 58 in A. sylvata. In addition, A. grossularita autosomes exhibit massive autosomal blocks of heterochromatin, which is a very rare phenomenon in Lepidoptera, whereas the autosomes of A. sylvata are completely devoid of distinct heterochromatin. Their W chromosomes differ greatly. Although they are largely composed of female-specific DNA sequences, as shown by comparative genomic hybridization, cross-species W-chromosome painting revealed considerable sequence differences between them. The results suggest a relatively rapid molecular divergence of Abraxas W chromosomes by the independent spreading of female-specific repetitive sequences.

Insights into the Structure of the Spruce Budworm (Choristoneura fumiferana) Genome, as Revealed by Molecular Cytogenetic Analyses and a High-Density Linkage Map.

Genome structure characterization can contribute to a better understanding of processes such as adaptation, speciation, and karyotype evolution, and can provide useful information for refining genome assemblies. We studied the genome of an important North American boreal forest pest, the spruce budworm, Choristoneura fumiferana, through a combination of molecular cytogenetic analyses and construction of a high-density linkage map based on single nucleotide polymorphism (SNP) markers obtained through a genotyping-by-sequencing (GBS) approach. Cytogenetic analyses using fluorescence in situ hybridization methods confirmed the haploid chromosome number of n = 30 in both sexes of C. fumiferana and showed, for the first time, that this species has a WZ/ZZ sex chromosome system. Synteny analysis based on a comparison of the Bombyx mori genome and the C. fumiferana linkage map revealed the presence of a neo-Z chromosome in the latter species, as previously reported for other tortricid moths. In this neo-Z chromosome, we detected an ABC transporter C2 (ABCC2) gene that has been associated with insecticide resistance. Sex-linkage of the ABCC2 gene provides a genomic context favorable to selection and rapid spread of resistance against Bacillus thuringiensis serotype kurstaki (Btk), the main insecticide used in Canada to control spruce budworm populations. Ultimately, the linkage map we developed, which comprises 3586 SNP markers distributed over 30 linkage groups for a total length of 1720.41 cM, will be a valuable tool for refining our draft assembly of the spruce budworm genome.

Chromosomal evolution in tortricid moths: conserved karyotypes with diverged features.

Moths of the family Tortricidae constitute one of the major microlepidopteran groups in terms of species richness and economic importance. Yet, despite their overall significance, our knowledge of their genome organization is very limited. In order to understand karyotype evolution in the family Tortricidae, we performed detailed cytogenetic analysis of Grapholita molesta, G. funebrana, Lobesia botrana, and Eupoecilia ambiguella, representatives of two main tortricid subfamilies, Olethreutinae and Tortricinae. Besides standard cytogenetic methods, we used fluorescence in situ hybridization for mapping of major rRNA and histone gene clusters and comparative genomic hybridization to determine the level of molecular differentiation of the W and Z sex chromosomes. Our results in combination with available data in the codling moth, Cydia pomonella, and other tortricids allow us a comprehensive reconstruction of chromosomal evolution across the family Tortricidae. The emerging picture is that the karyotype of a common ancestor of Tortricinae and Olethreutinae differentiated from the ancestral lepidopteran chromosome print of n = 31 by a sex chromosome-autosome fusion. This rearrangement resulted in a large neo-sex chromosome pair and a karyotype with n = 30 conserved in most Tortricinae species, which was further reduced to n = 28 observed in Olethreutinae. Comparison of the tortricid neo-W chromosomes showed differences in their structure and composition presumably reflecting stochasticity of molecular degeneration of the autosomal part of the neo-W chromosome. Our analysis also revealed conservative pattern of the histone distribution, which is in contrast with high rDNA mobility. Despite the dynamic evolution of rDNA, we can infer a single NOR-chromosome pair as an ancestral state not only in tortricids but probably in all Lepidoptera. The results greatly expand our knowledge of the genome architecture in tortricids, but also contribute to the understanding of chromosomal evolution in Lepidoptera in general.