Individual genotypes from environmental DNA: Fingerprinting snow tracks of three large carnivore species.

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR), and for brown bear a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear (5/7) and wolf (7/10) than for lynx (4/9), and locus genotyping success was greater for brown bear (0.88). The sex marker was typed in six out of seven brown bear samples. Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success, is needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA-based individual and population-level studies.

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.

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.

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.

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.

Mitochondrial chromosome as a marker of animal migratory routes: DNA barcoding revealed Asian (non-African) origin of a tropical migrant butterfly Junonia orithya in south Israel.

The blue pansy Junonia orithya Linnaeus, 1758 (Lepidoptera, Nymphalidae) is widely distributed along the tropical areas of Africa, Asia and Australia. It is also known as a migrant species in the Levant. Here we record Junonia orithya in south Israel and provide a DNA-barcode-based evidence for its Asian (non-African) origin.

Chromosomal and mitochondrial diversity in Melitaea didyma complex (Lepidoptera, Nymphalidae): eleven deeply diverged DNA barcode groups in one non-monophyletic species?

It is generally accepted that cases of species' polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, and the detected cases reflect misidentifications or/and methodological errors. Here we studied the problem of species' non-monophyly through chromosomal and molecular analysis of butterfly taxa close to Melitaea didyma (Esper, 1779) (Lepidoptera, Nymphalidae). We found absence or low interspecific chromosome number variation and presence of intraspecific variation, therefore we conclude that in this group, chromosome numbers have relatively low value as taxonomic markers. Despite low karyotype variability, the group was found to have unexpectedly high mitochondrial haplotype diversity. These haplotypes were clustered in 23 highly diverged haplogroups. Twelve of these haplogroups are associated with nine traditionally recognized and morphologically distinct species Melitaea chitralensis Moore, 1901, Melitaea deserticola Oberthür, 1909, Melitaea didymoides Eversmann, 1847, Melitaea gina Higgins, 1941, Melitaea interrupta Colenati, 1846, Melitaea latonigena Eversmann, 1847, Melitaea mixta Evans, 1912, Melitaea saxatilis Christoph, 1873 and Melitaea sutschana Staudinger, 1892. The rest of the haplogroups (11 lineages) belong to a well-known west-palaearctic species Melitaea didyma. The last species is particularly unusual in the haplotypes we obtained. First, it is clearly polyphyletic with respect to COI gene. Second, the differentiation in COI gene between these mostly allopatric (but in few cases sympatric) eleven lineages is extremely high (up to 7.4%), i.e. much deeper than the "standard" DNA barcode species threshold (2.7-3%). This level of divergence normally could correspond not even to different species, but to different genera. Despite this divergence, the bearers of these haplogroups were found to be morphologically indistinguishable and, most importantly, to share absolutely the same ecological niches, i.e. demonstrating the pattern which is hardly compatible with hypothesis of multiple cryptic species. Most likely such a profound irregularity in barcodes is caused by reasons other than speciation and represents an extraordinary example of intra-species barcode variability. Given the deep level of genetic differentiation between the lineages, we assume that there was a long period (up to 5.0 My) of allopatric differentiation when the lineages were separated by geographic or/and ecological barriers and evolved in late Pliocene and Pleistocene refugia of north Africa, the Iberian and Balkan Peninsulas, the Middle East and Central Asia. We discuss the refugia-within-refugia concept as a mechanism explaining the presence of additional diverged minor haplogroups within the areas of the major haplogroups. We also provide the first record of Melitaea gina in Azerbaijan and the record of Melitaea didyma turkestanica as a new taxon for Russia and Europe.

DNA barcoding reveals twelve lineages with properties of phylogenetic and biological species within Melitaea didyma sensu lato (Lepidoptera, Nymphalidae).

The complex of butterfly taxa close to Melitaea didyma includes the traditionally recognized species Melitaea didyma, Melitaea didymoides and Melitaea sutschana, the taxa that were recognized as species only relatively recently (Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta) as well as numerous described subspecies and forms with unclear taxonomic status. Here analysis of mitochondrial DNA barcodes is used to demonstrate that this complex is monophyletic group consisting of at least 12 major haplogroups strongly differentiated with respect to the gene COI. Six of these haplogroups are shown to correspond to six of the above-mentioned species (Melitaea didymoides, Melitaea sutschana, Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta). It is hypothesized that each of the remaining six haplogroups also represents a distinct species (Melitaea mauretanica, Melitaea occidentalis, Melitaea didyma, Melitaea neera, Melitaea liliputana and Melitaea turkestanica), since merging these haplogroups would result in a polyphyletic assemblage and the genetic distances between them are comparable with those found between the other six previously recognized species.