Most ancestry inference methods rely on putatively pure reference panels to define ancestry informative variants. This approach is often unrealistic and can bias inference. The genome polarization algorithm diem, introduced previously by Baird et al., avoids reference panels by jointly inferring the polarity of common allelic states and quantifying variant diagnosticity via an expectation-maximization procedure. Importantly, we use "polarization" strictly to mean the assignment of alleles to opposing sides of a barrier to gene flow, rather than the assignment of ancestral versus derived states. Here, we present diempy, an efficient python implementation of diem coupled with tools that turn polarized calls into analysis-ready outputs. diempy offers lossless VCF-to-diem BED conversion; ploidy-aware handling of individuals and chromosomes; flexible masking of sites, regions, and individuals; and interactive visualization of polarized genomes, hybrid indices, clines, and ternary plots. Postprocessing functions include thresholding via the diagnostic index, kernel smoothing, and automatic detection and run-length encoding of contiguous ancestry tracts. BED-based I/O facilitates integration with population-genomic workflows (e.g. filtering by annotation or ploidy). These features make reference-panel-free genome polarization with diempy practical and reproducible for studies of population structure, admixture and species barriers.

Most ancestry-assignment methods rely on putatively pure reference panels, which are often unrealistic and bias inference. The genome polarisation algorithm diem , introduced previously, avoids reference panels by jointly inferring the polarity of common allelic states and quantifying variant diagnosticity via an expectation–maximisation procedure. Here we present diempy , an efficient python implementation of diem coupled with tools that turn polarised calls into analysis-ready outputs. diempy offers lossless VCF-to- diem BED conversion; ploidy-aware handling of individuals and chromosomes; flexible masking of sites, regions and individuals; and interactive visualisation of polarised genomes, hybrid indices, clines and ternary plots. Post-processing functions include DI thresholding, kernel smoothing, and automatic detection and run-length encoding of contiguous ancestry tracts. BED-based I/O facilitates integration with population-genomic workflows (e.g. filtering by annotation or ploidy). These features make reference-free genome polarisation with diempy practical and reproducible for studies of population structure, admixture and species barriers.

Parasites are expected to hybridise in similar ways to free-living organisms, although this may be modified by their reduced genome architecture. Recombinant strains and taxa of hybrid origin can be studied in nature where hosts come into secondary contact. Here we apply genome-wide analysis to parasites from a contact previously characterised for many individuals using classical markers. The host contact is the European house mouse hybrid zone; the parasites are lung fungus Pneumocystis and gut pinworm Syphacia. The genomic (many loci) and classical (many-individual) results are broadly consistent in scale and centring of transitions across the host hybrid zone. Whole mitogenome comparisons confirm earlier suggestions that parasite divergence is low compared to their hosts, perhaps due to reduced-genome stabilising selection. In the recombining genome, we are able to show blocks of the parasite genome of alternating host origin, including one Pneumocystis strain which appears to be an F3+ cross and one recombinant Syphacia strain found over multiple localities. Functional analyses of introgressing genes show enrichment for genes likely important for parasitic lifestyle. Our work confirms that evolutionary models of hybridisation apply equally to hosts and their parasites.

Species barriers are shaped by variation in recombination coupled with natural selection. Canids lost the Prdm9 gene 40-60 million years ago, stabilising low recombination at the centromeric ends of acrocentric autosomes despite overall high recombination rates. We predict canid recombination architecture will tend to maintain barriers to gene flow at one end of autosomes while eroding them at the other. Polarization of 31 million single nucleotide variants (SNVs) across 980 canid genomes reveals elevated barriers to gene flow at pericentromeric regions between grey wolves, coyotes and golden jackals. Highly diagnostic SNVs are six-fold enriched in pericentromeric regions. The exception is the barrier to gene flow between dogs and wolves, which accumulated divergence differently from other comparisons: genomes of domesticated dogs preserve ancient genetic diversity lost in contemporary grey wolf populations, positioning them as reservoirs of ancestral alleles. Our results show that genome architecture modulates permeability of barriers to gene flow, pointing to pericentromeric regions as targets for reinforcement selection that may maintain canid taxa. ### Competing Interest Statement The authors have declared no competing interest.

Many closely related species continue to hybridise after millions of generations of divergence. However, the extent to which current patterning in hybrid zones connects back to the speciation process remains unclear: does evidence for current multilocus barriers support the hypothesis of speciation due to multilocus divergence? We analyse whole-genome sequencing data to investigate the speciation history of the scarce swallowtails Iphiclidespodalirius and I . feisthamelii, which abut at a narrow ( ∼ 25 km) contact zone north of the Pyrenees. We first quantify the heterogeneity of effective migration rate under a model of isolation with migration, using genomes sampled across the range to identify long-term barriers to gene flow. Secondly, we investigate the recent ancestry of individuals from the hybrid zone using genome polarisation and estimate the coupling coefficient under a model of a multilocus barrier. We infer a low rate of long-term gene flow from I . feisthamelii into I . podalirius - the direction of which matches the admixture across the hybrid zone - and complete reproductive isolation across ≈ 33% of the genome. Our contrast of recent and long-term gene flow shows that regions of low recent hybridisation are indeed enriched for long-term barriers which maintain divergence between these hybridising sister species. This finding paves the way for future analysis of the evolution of reproductive isolation along the speciation continuum.

The house mouse is the best-studied mammal species after humans, yet our understanding of its evolutionary history remains incomplete. Here, we focused on the colonisation of Europe by two subspecies and formation of a hybrid zone between them. We carried out a large-scale study of similar to 7000 mice sampled across an area embracing an similar to 900 km long portion of the zone, supplemented with cytochrome b and D-loop sequencing of similar to 1200 individuals collected worldwide. We demonstrate that the course of the mtDNA contact front is intricate and highly discordant with the consensus front for autosomal and X-linked markers, suggesting that local snapshots of mtDNA introgression may be misleading when treated in isolation. While multiple unrelated M. m. domesticus haplotypes occur in the zone area, the diversity of M. m. musculus haplotypes is limited. Moreover, we uncovered a vast region of domesticus introgression into musculus territory in northern areas and showed this introgression is unlikely to be driven by positive selection. We consider two previously published hypotheses explaining this phenomenon and put forth an alternative scenario assuming associations between mice and humans in northern Europe during the Nordic Bronze Age and subsequent zone movement associated with the expansion of Balto-Slavic peoples.

A remarkable gene copy number (CN) arms race system has recently been described in laboratory mice, where Slx;Slxl1 and Sly genes compete over transmission by altering the fertilization success of X and Y chromosome-bearing sperm, respectively. Here, we focus on this system in nature, where natural selection can counter CN/gene product escalation. Our model is house mouse subspecies hybridizing in Europe. In some regions, Y chromosomes of the Eastern subspecies have introgressed onto Western genomic backgrounds, accompanied by sex ratio distortion in favor of males, consistent with the inbred lines suggested mechanism: Overabundance of SLY protein expressed by invading Y chromosomes. We take Slx as representative of the X side of this arms race and measure Slx|Sly CN and expression across an "Invasion" transect where Ys introgress and a "Control" transect with negligible introgression. Since we found similar Slx|Sly ratios in both transects, SLY overabundance is unlikely to explain the introgression. However, Slx CN is relatively low in the introgression area, suggesting that Slx is less able to combat Sly effects here. Furthermore, deterministic changes in Slx;Sly expression proportions versus CN proportions suggest standing variation for trans regulation of Slx|Sly is being co-opted in nature where their arms race reduces population fitness.

Hybrid zones are important windows into the evolutionary dynamics of populations, revealing how processes like introgression and adaptation structure population genomic variation. Importantly, they are useful for understanding speciation and how species respond to their environments. Here, we investigate two closely related sea star species, Asterias rubens and A. forbesi, distributed along rocky European and North American coastlines of the North Atlantic, and use genome-wide molecular markers to infer the distribution of genomic variation within and between species in this group. Using genomic data and environmental niche modelling, we document hybridization occurring between northern New England and the southern Canadian Maritimes. We investigate the factors that maintain this hybrid zone, as well as the environmental variables that putatively drive selection within and between species. We find that the two species differ in their environmental niche breadth; Asterias forbesi displays a relatively narrow environmental niche while conversely, A. rubens has a wider niche breadth. Species distribution models accurately predict hybrids to occur within environmental niche overlap, thereby suggesting environmental selection plays an important role in the maintenance of the hybrid zone. Our results imply that the distribution of genomic variation in North Atlantic sea stars is influenced by the environment, which will be crucial to consider as the climate changes.

Mastomys natalensis-borne mammarenaviruses appear specific to subspecific M. natalensis taxa rather than to the whole species. Yet mammarenaviruses carried by M. natalensis are known to spill over and jump hosts in northern sub-Saharan Africa. Phylogeographic studies increasingly show that, like M. natalensis, small mammals in sub-Saharan Africa are often genetically structured into several subspecific taxa. Other mammarenaviruses may thus also form virus-subspecific host taxon associations. To investigate this, and if mammarenaviruses carried by M. natalensis in southern Africa are less prone to spill-over, we screened 1225 non-M. natalensis samples from Tanzania where many small mammal taxa meet. We found mammarenavirus RNA in 6 samples. Genetic/genomic characterisation confirmed they were not spill-over from M. natalensis. We detected host jumps among rodent tribe members and an association between mammarenaviruses and subspecific taxa of Mus minutoides and Grammomys surdaster, indicating host genetic structure may be crucial to understand virus distribution and host specificity.

Semi-permeable barriers to geneflow in principle allow distantly related organisms to capture and exchange pre-adapted genes potentially speeding adaptation. However, describing barriers to geneflow on a genomic scale is non-trivial. We extend classic diagnostic allele counting measures of geneflow across a barrier to the case of genome-scale data. Diagnostic index expectation maximisation (diem) polarises the labelling of bistate markers with respect to the sides of a barrier. An initial state of ignorance is enforced by starting with randomly generated marker polarisations. This means there is no prior on population or taxon membership of the genomes concerned. Using a deterministic data labelling, small numbers of classic diagnostic markers can be replaced by large numbers of markers, each with a diagnostic index. Individuals' hybrid indices (genome admixture proportions) are then calculated genome wide conditioned on marker diagnosticity; within diploid, haplodiploid and/or haploid genome compartments; or indeed over any subset of markers, allowing classical cline width/barrier strength comparisons along genomes. Along-genome barrier strength heterogeneity allows for barrier regions to be identified. Furthermore, blocks of genetic material that have introgressed across a barrier are easily identified with high power. diem indicates panmixis among Myotis myotis bat genomes, with a barrier separating low data quality outliers. In a Mus musculus domesticus/Mus spretus system, diem adds multiple introgressions of olfactory (and vomeronasal) gene clusters in one direction to previous demonstrations of a pesticide resistance gene introgressing in the opposite direction across a strong species barrier. diem is a genome analysis solution, which scales over reduced representation genomics of thousands of markers to treatment of all variant sites in large genomes. While the method lends itself to visualisation, its output of markers with barrier-informative annotation will fuel research in population genetics, phylogenetics and association studies. diem can equip such downstream applications with millions of informative markers.

The Natal multimammate mouse (Mastomys natalensis) is the most widespread rodent species in sub-Saharan Africa, often studied as an agricultural pest and reservoir of viruses. Its mitochondrial (Mt) phylogeny revealed six major lineages parapatrically distributed across open habitats of sub-Saharan Africa. In this study we used 1949 sequences of the mitochondrial cytochrome b gene to elaborate on distribution and evolutionary history of three Mt lineages inhabiting the open habitats of the Zambezian region (corresponding roughly to the African savannas south of the Equator). We describe in more detail contact zones between the lineages-their location and extent of co-occurrence within localities-and infer past population trends. The estimates are interpreted in the light of climatic niche models. The lineages underwent reduction in effective population size during the last glacial, but they spread widely after that: two of them after the last glacial maximum and the last one in mid-Holocene. The centers of expansion, i.e., possible long-term savanna refugia, were estimated to lie close to the Eastern Arc Mountains and lakes of the Great African Rift, geomorphological structures likely to have had long-term influence on geographical distribution of the lineages. Environmental niche modeling shows climate could also affect the broad scale distribution of the lineages but is unlikely to explain the narrow width of the contact zones. The intraspecific Mt differentiation of M. natalensis echoes phylogeographic patterns observed in multiple co-distributed mammal species, which suggests the mammal communities in the region are shaped by the same long-term processes.

The Livingstone Mountains (LM; also known as the Kipengere Range) found in south-western Tanzania at the northern end of Lake Nyasa are an important region for understanding the biogeography of Eastern Africa. The two branches of the East African Rift Valley meet here and the mountains might represent stepping stones for colonization and migration between different parts of the Eastern Afromontane Biodiversity Hotspot (especially the link between the Eastern Arc Mountains, EAM, and the Southern Rift Mountains, SRM), as well as an efficient barrier to gene flow for taxa living in drier savannahs in lower elevations. Here we combine new mitochondrial sequence data from 610 recently sampled rodents and shrews with available georeferenced genetic data (3538 specimens) from southern Tanzania, northern Malawi/Zambia and northern Mozambique and compare the spatial genetic structure among different taxa. There is no universal phylogeographic pattern in taxa preferring humid montane habitats. For some of them, the Makambako Gap acts as a barrier between the SRM and the EAM, but other taxa can bridge this gap. Barriers within the EAM (frequently) and within the SRM (sometimes) appear more important. The Rukwa rift between the SRM and the ARM is an important barrier that perhaps can only be crossed by taxa that are not that strictly tied to humid montane environments. For mammals living in lower-elevation savannah-like habitats, the LM can act as a strict barrier to gene flow, and together with the Ufipa Plateau, Lake Nyasa and the EAM create a very similar phylogeographic pattern with three recognizable genetic groups in most savannah-dwellers. The Livingstone Mountains thus appear to be one of the most important biogeographic crossroads in Eastern Africa.

Paternal genome elimination-a type of reproduction in which males inherit but fail to pass on their father's genome-evolved independently in 6-8 arthropod clades. Thousands of species, including several important for agriculture, reproduce via this mode of reproduction. While paternal genome elimination is well established in some of the clades, the evidence in globular springtails (Symphypleona) remains elusive, even though they represent the oldest and most species-rich clade putatively reproducing via paternal genome elimination. We sequenced genomic DNA from whole bodies of Allacma fusca males with high fractions (>27.5%) of sperm to conclusively confirm that all the sperm carry 1 parental haplotype only. Although it is suggestive that the single haplotype present in sperm is maternally inherited, definitive genetic proof of the parent of origin is still needed. The genomic approach we developed allows for the detection of genotypic differences between germline and soma in all species with sufficiently high fraction of germline in their bodies. This opens new opportunities for scans of reproductive modes in small organisms.

Genomic analysis of hybrid zones offers unique insights into emerging reproductive isolation and the dynamics of introgression. Because hybrid genomes consist of blocks inherited from one or the other parental taxon, linkage information is essential. In most cases, the spectrum of local ancestry tracts can be efficiently uncovered from dense linkage maps. Here, we report the development of such a map for the hybridizing toads, Bombina bombina and Bombina variegata (Anura: Bombinatoridae). Faced with the challenge of a large (7-10 Gb), repetitive genome, we set out to identify a large number of Mendelian markers in the nonrepetitive portion of the genome that report B. bombina vs B. variegata ancestry with appropriately quantified statistical support. Bait sequences for targeted enrichment were selected from a draft genome assembly, after filtering highly repetitive sequences. We developed a novel approach to infer the most likely diplotype per sample and locus from the raw read mapping data, which is robust to over-merging and obviates arbitrary filtering thresholds. Validation of the resulting map with 4755 markers underscored the large-scale synteny between Bombina and Xenopus tropicalis. By assessing the sex of late-stage F2 tadpoles from histological sections, we identified the sex-determining region in the Bombina genome to 7 cM on LG5, which is homologous to X. tropicalis chromosome 5, and inferred male heterogamety. Interestingly, chromosome 5 has been repeatedly recruited as a sex chromosome in anurans with XY sex determination.