A curated DNA barcode reference library for parasitoids of northern European cyclically outbreaking geometrid moths.

Large areas of forests are annually damaged or destroyed by outbreaking insect pests. Understanding the factors that trigger and terminate such population eruptions has become crucially important, as plants, plant-feeding insects, and their natural enemies may respond differentially to the ongoing changes in the global climate. In northernmost Europe, climate-driven range expansions of the geometrid moths Epirrita autumnata and Operophtera brumata have resulted in overlapping and increasingly severe outbreaks. Delayed density-dependent responses of parasitoids are a plausible explanation for the 10-year population cycles of these moth species, but the impact of parasitoids on geometrid outbreak dynamics is unclear due to a lack of knowledge on the host ranges and prevalences of parasitoids attacking the moths in nature. To overcome these problems, we reviewed the literature on parasitism in the focal geometrid species in their outbreak range and then constructed a DNA barcode reference library for all relevant parasitoid species based on reared specimens and sequences obtained from public databases. The combined recorded parasitoid community of E. autumnata and O. brumata consists of 32 hymenopteran species, all of which can be reliably identified based on their barcode sequences. The curated barcode library presented here opens up new opportunities for estimating the abundance and community composition of parasitoids across populations and ecosystems based on mass barcoding and metabarcoding approaches. Such information can be used for elucidating the role of parasitoids in moth population control, possibly also for devising methods for reducing the extent, intensity, and duration of outbreaks.

DNA barcoding reveals different cestode helminth species in northern European marine and freshwater ringed seals.

Three subspecies of the ringed seal (Pusa hispida) are found in northeastern Europe: P. h. botnica in the Baltic Sea, P. h saimensis in Lake Saimaa in Finland, and P. h. ladogensis in Lake Ladoga in Russia. We investigated the poorly-known cestode helminth communities of these closely related but ecologically divergent subspecies using COI barcode data. Our results show that, while cestodes from the Baltic Sea represent Schistocephalus solidus, all worms from the two lakes are identified as Ligula intestinalis, a species that has previously not been reported from seals. The observed shift in cestode communities appears to be driven by differential availability of intermediate fish host species in marine vs. freshwater environments. Both observed cestode species normally infect fish-eating birds, so further work is required to elucidate the health and conservation implications of cestode infections in European ringed seals, whether L. intestinalis occurs also in marine ringed seals, and whether the species is able to reproduce in seal hosts. In addition, a deep barcode divergence found within S. solidus suggests the presence of cryptic diversity under this species name.

Genomes of the willow-galling sawflies Euura lappo and Eupontania aestiva (Hymenoptera: Tenthredinidae): a resource for research on ecological speciation, adaptation, and gall induction.

Hymenoptera is a hyperdiverse insect order represented by over 153,000 different species. As many hymenopteran species perform various crucial roles for our environments, such as pollination, herbivory, and parasitism, they are of high economic and ecological importance. There are 99 hymenopteran genomes in the NCBI database, yet only five are representative of the paraphyletic suborder Symphyta (sawflies, woodwasps, and horntails), while the rest represent the suborder Apocrita (bees, wasps, and ants). Here, using a combination of 10X Genomics linked-read sequencing, Oxford Nanopore long-read technology, and Illumina short-read data, we assembled the genomes of two willow-galling sawflies (Hymenoptera: Tenthredinidae: Nematinae: Euurina): the bud-galling species Euura lappo and the leaf-galling species Eupontania aestiva. The final assembly for E. lappo is 259.85 Mbp in size, with a contig N50 of 209.0 kbp and a BUSCO score of 93.5%. The E. aestiva genome is 222.23 Mbp in size, with a contig N50 of 49.7 kbp and a 90.2% complete BUSCO score. De novo annotation of repetitive elements showed that 27.45% of the genome was composed of repetitive elements in E. lappo and 16.89% in E. aestiva, which is a marked increase compared to previously published hymenopteran genomes. The genomes presented here provide a resource for inferring phylogenetic relationships among basal hymenopterans, comparative studies on host-related genomic adaptation in plant-feeding insects, and research on the mechanisms of plant manipulation by gall-inducing insects.

Author Correction: Spotted phenotypes in horses lost attractiveness in the Middle Ages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic variability and history of a native Finnish horse breed.

BACKGROUND: The Finnhorse was established as a breed more than 110 years ago by combining local Finnish landraces. Since its foundation, the breed has experienced both strong directional selection, especially for size and colour, and severe population bottlenecks that are connected with its initial foundation and subsequent changes in agricultural and forestry practices. Here, we used sequences of the mitochondrial control region and genomic single nucleotide polymorphisms (SNPs) to estimate the genetic diversity and differentiation of the four Finnhorse breeding sections: trotters, pony-sized horses, draught horses and riding horses. Furthermore, we estimated inbreeding and effective population sizes over time to infer the history of this breed. RESULTS: We found a high level of mitochondrial genetic variation and identified 16 of the 18 haplogroups described in present-day horses. Interestingly, one of these detected haplogroups was previously reported only in the Przewalski's horse. Female effective population sizes were in the thousands, but declines were evident at the times when the breed and its breeding sections were founded. By contrast, nuclear variation and effective population sizes were small (approximately 50). Nevertheless, inbreeding in Finnhorses was lower than in many other horse breeds. Based on nuclear SNP data, genetic differentiation among the four breeding sections was strongest between the draught horses and the three other sections (FST = 0.007-0.018), whereas based on mitochondrial DNA data, it was strongest between the trotters and the pony-sized and riding horses (ΦST = 0.054-0.068). CONCLUSIONS: The existence of a Przewalski's horse haplogroup in the Finnhorse provides new insights into the domestication of the horse, and this finding supports previous suggestions of a close relationship between the Finnhorse and eastern primitive breeds. The high level of mitochondrial DNA variation in the Finnhorse supports its domestication from a large number of mares but also reflects that its founding depended on many local landraces. Although inbreeding in Finnhorses was lower than in many other horse breeds, the small nuclear effective population sizes of each of its breeding sections can be considered as a warning sign, which warrants changes in breeding practices.

Targeted PCR Amplification and Multiplex Sequencing of Ancient DNA for SNP Analysis.

The analysis of single-nucleotide polymorphisms (SNPs) has proven to be advantageous for addressing variation within samples of highly degraded or low-quality DNA samples. This is because only short fragments need to be amplified to analyze SNPs, and this can be achieved by multiplex PCR. Here, we present a sensitive method for the targeted sequencing of SNP loci that requires only small amounts of template DNA. The approach combines multiplex amplification of very short fragments covering SNP positions followed by sample barcoding and next-generation sequencing. This method allows generation of data from large sample sets of poorly preserved specimens, such as fossil remains, forensic samples, and museum specimens. The approach is cost-effective, rapid, and applicable to forensics, population genetics, and phylogenetic research questions.

Decline of genetic diversity in ancient domestic stallions in Europe.

Present-day domestic horses are immensely diverse in their maternally inherited mitochondrial DNA, yet they show very little variation on their paternally inherited Y chromosome. Although it has recently been shown that Y chromosomal diversity in domestic horses was higher at least until the Iron Age, when and why this diversity disappeared remain controversial questions. We genotyped 16 recently discovered Y chromosomal single-nucleotide polymorphisms in 96 ancient Eurasian stallions spanning the early domestication stages (Copper and Bronze Age) to the Middle Ages. Using this Y chromosomal time series, which covers nearly the entire history of horse domestication, we reveal how Y chromosomal diversity changed over time. Our results also show that the lack of multiple stallion lineages in the extant domestic population is caused by neither a founder effect nor random demographic effects but instead is the result of artificial selection-initially during the Iron Age by nomadic people from the Eurasian steppes and later during the Roman period. Moreover, the modern domestic haplotype probably derived from another, already advantageous, haplotype, most likely after the beginning of the domestication. In line with recent findings indicating that the Przewalski and domestic horse lineages remained connected by gene flow after they diverged about 45,000 years ago, we present evidence for Y chromosomal introgression of Przewalski horses into the gene pool of European domestic horses at least until medieval times.

Coat colour adaptation of post-glacial horses to increasing forest vegetation.

Wild horses unexpectedly survived terminal Pleistocene megafaunal extinctions until eventual European extirpation in the twentieth century. This survival is tied to either their occurrence in cryptic open habitats or their adaptation to forests. Our niche modelling inferred an increasing presence of horses in post-glacial forests, and our analysis of ancient DNA suggested significant selection for black phenotypes as indicating adaptation to forests.

Genetic Structure and Phylogeography of the Leopard Cat (Prionailurus bengalensis) Inferred from Mitochondrial Genomes.

The Leopard cat Prionailurus bengalensis is a habitat generalist that is widely distributed across Southeast Asia. Based on morphological traits, this species has been subdivided into 12 subspecies. Thus far, there have been few molecular studies investigating intraspecific variation, and those had been limited in geographic scope. For this reason, we aimed to study the genetic structure and evolutionary history of this species across its very large distribution range in Asia. We employed both PCR-based (short mtDNA fragments, 94 samples) and high throughput sequencing based methods (whole mitochondrial genomes, 52 samples) on archival, noninvasively collected and fresh samples to investigate the distribution of intraspecific genetic variation. Our comprehensive sampling coupled with the improved resolution of a mitochondrial genome analyses provided strong support for a deep split between Mainland and Sundaic Leopard cats. Although we identified multiple haplogroups within the species' distribution, we found no matrilineal evidence for the distinction of 12 subspecies. In the context of Leopard cat biogeography, we cautiously recommend a revision of the Prionailurus bengalensis subspecific taxonomy: namely, a reduction to 4 subspecies (2 mainland and 2 Sundaic forms).

Spotted phenotypes in horses lost attractiveness in the Middle Ages.

Horses have been valued for their diversity of coat colour since prehistoric times; this is especially the case since their domestication in the Caspian steppe in ~3,500 BC. Although we can assume that human preferences were not constant, we have only anecdotal information about how domestic horses were influenced by humans. Our results from genotype analyses show a significant increase in spotted coats in early domestic horses (Copper Age to Iron Age). In contrast, medieval horses carried significantly fewer alleles for these phenotypes, whereas solid phenotypes (i.e., chestnut) became dominant. This shift may have been supported because of (i) pleiotropic disadvantages, (ii) a reduced need to separate domestic horses from their wild counterparts, (iii) a lower religious prestige, or (iv) novel developments in weaponry. These scenarios may have acted alone or in combination. However, the dominance of chestnut is a remarkable feature of the medieval horse population.

The origin of ambling horses.

Horseback riding is the most fundamental use of domestic horses and has had a huge influence on the development of human societies for millennia. Over time, riding techniques and the style of riding improved. Therefore, horses with the ability to perform comfortable gaits (e.g. ambling or pacing), so-called 'gaited' horses, have been highly valued by humans, especially for long distance travel. Recently, the causative mutation for gaitedness in horses has been linked to a substitution causing a premature stop codon in the DMRT3 gene (DMRT3_Ser301STOP) [1]. In mice, Dmrt3 is expressed in spinal cord interneurons and plays an important role in the development of limb movement coordination [1]. Genotyping the position in 4396 modern horses from 141 breeds revealed that nowadays the mutated allele is distributed worldwide with an especially high frequency in gaited horses and breeds used for harness racing [2]. Here, we examine historic horse remains for the DMRT3 SNP, tracking the origin of gaitedness to Medieval England between 850 and 900 AD. The presence of the corresponding allele in Icelandic horses (9(th)-11(th) century) strongly suggests that ambling horses were brought from the British Isles to Iceland by Norse people. Considering the high frequency of the ambling allele in early Icelandic horses, we believe that Norse settlers selected for this comfortable mode of horse riding soon after arrival. The absence of the allele in samples from continental Europe (including Scandinavia) at this time implies that ambling horses may have spread from Iceland and maybe also the British Isles across the continent at a later date.