In flowering plants, a distinct post-zygotic hybridization barrier between closely related species can arise during seed maturation, resulting in embryo lethality due to abnormal endosperm development. The endosperm initially works as a nutrient sink, acquiring nutrients from adjacent tissues, but later undergoes cellularization, switching to serve as a nutrient source. In hybrid seeds, this cellularization switch can be hampered if the endosperm genomic ratio is imbalanced. Disruption in the genomic ratio can be caused when species of different ploidy are crossed, but also by crosses between species with identical ploidy, if the effective ploidy differs. One factor proposed to influence effective ploidy is the epigenetic phenomenon genomic imprinting, the parent-of-origin specific expression of alleles inherited either maternally or paternally. It has been proposed that outbreeding species exhibit higher effective ploidy compared to selfing species, as a consequence of parental conflict in resource allocation to the developing progenies. This suggests a low anticipation of endosperm-based post-zygotic hybridization barriers between selfing species of similar ploidy. Here, we show that in crosses between the diploid selfing arctic species Draba fladnizensis , D. nivalis and D. subcapitata , the endosperm-based post-zygotic hybridization barrier is absent, supporting low parental conflict. To investigate parent-of-origin allele specific expression, we conducted a genomic imprinting study in D. nivalis and compared to previous studies in other Brassicaceae species. We report a high number of maternally expressed genes (MEGs) and concomitantly low numbers of paternally expressed genes (PEGs). Our results suggest rapid evolution of MEGs and loss of PEGs in a mating system with low parental conflict, proposing that selfing arctic species may exhibit a generally stronger maternal expression bias as an adaptive mechanism to efficiently cope with an extreme environment.### Competing Interest StatementThe authors have declared no competing interest.

Little is known about the evolution of cold tolerance in polar plant species and how they differ from temperate relatives. To gain insight into their biology and the evolution of cold tolerance, we compared the molecular basis of cold response in three Arctic Brassicaceae species. We conducted a comparative time series experiment to examine transcriptional responses to low temperature. RNA was sampled at 22°C, and after 3, 6, and 24 at 2°C. We then identified sets of genes that were differentially expressed in response to cold and compared them between species, as well as to published data from the temperate Arabidopsis thaliana. Most differentially expressed genes were species-specific, but a significant portion of the cold response was also shared among species. Among thousands of differentially expressed genes, ~200 were shared among the three Arctic species and A. thaliana, while ~100 were exclusively shared among the three Arctic species. Our results show that cold response differs markedly between Arctic Brassicaceae species, but probably builds on a conserved basis found across the family. They also confirm that highly polygenic traits such as cold tolerance may show little repeatability in their patterns of adaptation.

The repeated, rapid and often pronounced patterns of evolutionary divergence observed in insular plants, or the ‘plant island syndrome’, include changes in leaf phenotypes, growth, as well as the acquisition of a perennial lifestyle. Here, we sequence and describe the genome of the critically endangered, Galápagos-endemic species Scalesia atractyloides Arnot., obtaining a chromosome-resolved, 3.2-Gbp assembly containing 43,093 candidate gene models. Using a combination of fossil transposable elements, k -mer spectra analyses and orthologue assignment, we identify the two ancestral genomes, and date their divergence and the polyploidization event, concluding that the ancestor of all extant Scalesia species was an allotetraploid. There are a comparable number of genes and transposable elements across the two subgenomes, and while their synteny has been mostly conserved, we find multiple inversions that may have facilitated adaptation. We identify clear signatures of selection across genes associated with vascular development, growth, adaptation to salinity and flowering time, thus finding compelling evidence for a genomic basis of the island syndrome in one of Darwin’s giant daisies.

Morphologically similar species, that is cryptic species, may be similar or quasi-similar owing to the deceleration of morphological evolution and stasis. While the factors underlying the deceleration of morphological evolution or stasis in cryptic species remain unknown, decades of research in the field of paleontology on punctuated equilibrium have originated clear hypotheses. Species are expected to remain morphologically identical in scenarios of shared genetic variation, such as hybridization and incomplete lineage sorting, or in scenarios where bottlenecks reduce genetic variation and constrain the evolution of morphology. Here, focusing on three morphologically similar Stygocapitella species, we employ a whole-genome amplification method (WGA) coupled with double-digestion restriction-site associated DNA sequencing (ddRAD) to reconstruct the evolutionary history of the species complex. We explore population structure, use population-level statistics to determine the degree of connectivity between populations and species, and determine the most likely demographic scenarios which generally reject for recent hybridization. We find that the combination of WGA and ddRAD allowed us to obtain genomic-level data from microscopic eukaryotes (∼1 millimetre) opening up opportunities for those working with population genomics and phylogenomics in such taxa. The three species share genetic variance, likely from incomplete lineage sorting and ancient admixture. We speculate that the degree of shared variation might underlie morphological similarity in the Atlantic species complex.

The complex nature of species boundaries has been a central topic in evolutionary biology ever since Darwin. Despite numerous separate studies on reproductive isolation and hybridization, their relationship remains underinvestigated. Are the strengths and asymmetries of reproductive barriers reflected in the extent and directionalities of interspecific genetic exchange? We combined field, experimental, and molecular data to quantify strengths and asymmetries of sympatric reproductive barriers and hybridization between florally heteromorphic primroses. We also assessed whether generalist pollinators discriminate between different floral cues and contribute to reproductive isolation, a long-debated topic. Sympatric reproductive isolation is high but incomplete, and most phenotypic intermediates are genetic F1 hybrids, whereas backcrosses are rare, revealing low interspecific gene flow. Species integrity rests on multiple barriers, but ethological isolation is among the strongest, demonstrating that even generalist pollinators crucially contribute to the maintenance of species boundaries. Furthermore, reproductive barriers are weaker for Primula veris and short-styled plants, results corroborated by molecular data. Thus, in florally heteromorphic systems, both species- and morph-dependent asymmetries affect permeability of species boundaries. Our study illustrates how the interactions between complex floral syndromes and pollinators shape species boundaries in unique, previously undescribed ways.

Abstract The Arctic is one of the most extreme terrestrial environments on the planet. Here, we present the first chromosome‐scale genome assembly of a plant adapted to the high Arctic, Draba nivalis (Brassicaceae), an attractive model species for studying plant adaptation to the stresses imposed by this harsh environment. We used an iterative scaffolding strategy with data from short‐reads, single‐molecule long reads, proximity ligation data, and a genetic map to produce a 302 Mb assembly that is highly contiguous with 91.6% assembled into eight chromosomes (the base chromosome number). To identify candidate genes and gene families that may have facilitated adaptation to Arctic environmental stresses, we performed comparative genomic analyses with nine non‐Arctic Brassicaceae species. We show that the D. nivalis genome contains expanded suites of genes associated with drought and cold stress (e.g., related to the maintenance of oxidation‐reduction homeostasis, meiosis, and signaling pathways). The expansions of gene families associated with these functions appear to be driven in part by the activity of transposable elements. Tests of positive selection identify suites of candidate genes associated with meiosis and photoperiodism, as well as cold, drought, and oxidative stress responses. Our results reveal a multifaceted landscape of stress adaptation in the D. nivalis genome, offering avenues for the continued development of this species as an Arctic model plant.

Extreme environments offer powerful opportunities to study how different organisms have adapted to similar selection pressures at the molecular level. Arctic plants have adapted to some of the coldest and driest biomes on Earth and typically possess suites of similar morphological and physiological adaptations to extremes in light and temperature. Here, we compare patterns of molecular evolution in three Brassicaceae species that have independently colonized the Arctic and present some of the first genetic evidence for plant adaptations to the Arctic environment. By testing for positive selection and identifying convergent substitutions in orthologous gene alignments for a total of 15 Brassicaceae species, we find that positive selection has been acting on different genes, but similar functional pathways in the three Arctic lineages. The positively selected gene sets identified in the three Arctic species showed convergent functional profiles associated with extreme abiotic stress characteristic of the Arctic. However, there was little evidence for independently fixed mutations at the same sites and for positive selection acting on the same genes. The three species appear to have evolved similar suites of adaptations by modifying different components in similar stress response pathways, implying that there could be many genetic trajectories for adaptation to the Arctic environment. By identifying candidate genes and functional pathways potentially involved in Arctic adaptation, our results provide a framework for future studies aimed at testing for the existence of a functional syndrome of Arctic adaptation in the Brassicaceae and perhaps flowering plants in general.

Taxonomists have proposed numerous hybrid species in plants, but to gain a better understanding of the role that hybridization may play in plant diversification, such taxonomic hypotheses must be tested using genomic data. In this study, we employ ddRAD sequence data to test taxonomic hypotheses of hybrid origins in Carex salina and C. ramenskii (Carex section Phacocystis). Sequence data from multiple Norwegian and Icelandic populations of the putative hybrid and parental species were generated for hundreds of ddRAD loci. These data were used to estimate geographical structuring of genetic diversity and admixture and to explicitly test for hybrid origins using several analytical approaches. Our results indicate recurrent hybrid origins for the populations of C. salina and C. ramenskii sampled in our study and show that these populations are characterized by high interspecific heterozygosity. Our results support the idea that hybridization may indeed play an important role in the diversification of lineages of Carex and highlight the important role that clonal propagation might play in maintaining hybrid populations. Future studies focusing on a broader geographical sampling would be needed to assess if the genetic structuring in these Nordic populations reflects range-wide patterns in these hybrid lineages.

The island-like distribution of subalpine habitats across mountain ranges can trigger the parallel evolution of locally adapted ecotypes. Such naturally replicated scenarios allow testing hypotheses on how elevational differentiation structures genetic diversity within species. Nevertheless, the parallel colonization of subalpine habitats across different mountain ranges has only rarely been documented with molecular data. We chose Primula elatior (Primulaceae), naturally spanning entire elevation range in multiple mountain regions of central Europe, to test for the origin of its scattered subalpine populations. Nuclear microsatellite variation revealed three genetic groups corresponding with the distinct study regions. We found that genetic differentiation between foothill and subalpine populations within each region was relatively low, suggesting that the colonization of subalpine habitats occurred independently within each mountain range. Furthermore, the strongest differentiation was usually found between the subalpine populations suggesting that mountain ridges may act as migration barriers that can reduce gene flow more strongly than elevational differences between foothill and subalpine populations. Finally, we found that subalpine colonization did not result in a loss of genetic diversity relative to foothill populations in agreement with the high migration rates that we document here between the subalpine and the foothill populations. In summary, our study shows subalpine Primula elatior populations are genetically diverse and distinct results of parallel colonization events from multiple foothill gene pools.

Recently we discussed problems and challenges associated with inconsistent definitions and methods used to identify cryptic species, and how these hamper studies of their evolutionary significance. We proposed a conceptual framework that is focussed on evolutionary processes and advocated for a shift from pattern- to process-driven research concerning cryptic species, in order to circumvent these issues [ 1 Struck T.H. et al. Finding evolutionary processes hidden in cryptic species. Trends Ecol. Evol. 2018; 33: 153-163 Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar ]. In his response, Heethoff [ 2 Heethoff M. Cryptic species – conceptual or terminological chaos? A response to Struck et al.. Trends Ecol. Evol. 2018; 33: 310 Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar ] argued that cryptic species are merely a reflection of the limitations of applied taxonomy. He stated that cryptic species 'represent nothing more than an incompatibility of species "concepts" in applied taxonomy'. As such, he rejected our proposed framework as an approach that 'prioritizes the "evolutionary truth" of genetic over morphological species concepts'. In our opinion Heethoff's conclusions are based on his misconceptions about the proposed framework.

Plant mating systems have profound effects on levels and structuring of genetic variation, and can affect the impact of natural selection. While theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina . We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, both mating system and demography shape the impact of purifying selection on genomic variation in A. alpina . These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies. Significance Intermediate outcrossing rates are theoretically predicted to maintain effective selection against harmful alleles, but few studies have empirically tested this prediction using genomic data. We used whole-genome resequencing data from alpine rock-cress to study how genetic variation and purifying selection vary with mating system. We find that populations with intermediate outcrossing rates have similar levels of genetic diversity as outcrossing populations, and that purifying selection against harmful alleles is efficient in mixed-mating populations. In contrast, self-fertilizing populations from Scandinavia have strongly reduced genetic diversity, and accumulate harmful mutations, likely as a result of demographic effects of postglacial colonization. Our results suggest that mixed-mating populations can avoid the negative evolutionary consequences of high self-fertilization rates.

Premise of research. Self-compatibility is a requirement for reproductive assurance via selfing and may therefore be beneficial in environments with infrequent or unpredictable pollinator service. However, self-compatible plants may spatially separate anthers from stigmatic surface within flowers (herkogamy), potentially preventing autonomous self-pollination while promoting outcrossing. We investigated the effects of herkogamy on patterns of mating in a self-compatible, homostylous alpine herb, Primula halleri, which evolved from obligately outcrossing, heterostylous ancestors. Primula halleri displays diminishing herkogamy during anthesis, affecting its capacity for self-pollination. Specifically, we tested whether higher herkogamy at the end of anthesis (terminal herkogamy) promotes higher outcrossing rates at the cost of lower seed set, while lower terminal herkogamy ensures seed set via self-pollination at the cost of inbreeding.Methodology. We estimated family- and population-level genetic outcrossing rates in four populations using microsatellites and derived an estimate for inbreeding depression. We tested effects of different levels of terminal herkogamy on means and variances of outcrossing rates. We also assessed whether seed set was resource or pollen limited. We interpreted results in light of previously published data on seed set with and without pollinator-exclusion treatment.Pivotal results. Population-level outcrossing rates ranged between 0.5 and 0.8. Outcrossing rates of plants with higher terminal herkogamy had similar means but lower variances than those of plants with lower terminal herkogamy. This result demonstrates that separation between sexual organs larger than 1 mm in mature flowers favors outbreeding, while below this threshold, delayed selfing ensures reproduction.Conclusions. Contrary to long-held notions about the association of homostyly and selfing, we provide new genetic evidence that the homostylous P. halleri adopts a mixed mating system due to variable herkogamy. Moreover, our results support a trade-off between seed number and seed quality effected through variation in herkogamy, illustrating that herkogamy affects multiple aspects of reproductive fitness simultaneously.

Interactions between fungi and plants, including parasitism, mutualism, and saprotrophy, have been invoked as key to their respective macroevolutionary success. Here we evaluate the origins of plant-fungal symbioses and saprotrophy using a time-calibrated phylogenetic framework that reveals linked and drastic shifts in diversification rates of each kingdom. Fungal colonization of land was associated with at least two origins of terrestrial green algae and preceded embryophytes (as evidenced by losses of fungal flagellum, ca. 720 Ma), likely facilitating terrestriality through endomycorrhizal and possibly endophytic symbioses. The largest radiation of fungi (Leotiomyceta), the origin of arbuscular mycorrhizae, and the diversification of extant embryophytes occurred ca. 480 Ma. This was followed by the origin of extant lichens. Saprotrophic mushrooms diversified in the Late Paleozoic as forests of seed plants started to dominate the landscape. The subsequent diversification and explosive radiation of Agaricomycetes, and eventually of ectomycorrhizal mushrooms, were associated with the evolution of Pinaceae in the Mesozoic, and establishment of angiosperm-dominated biomes in the Cretaceous.

Cryptic species could represent a substantial fraction of biodiversity. However, inconsistent definitions and taxonomic treatment of cryptic species prevent informed estimates of their contribution to biodiversity and impede our understanding of their evolutionary and ecological significance. We propose a conceptual framework that recognizes cryptic species based on their low levels of phenotypic (morphological) disparity relative to their degree of genetic differentiation and divergence times as compared with non-cryptic species. We discuss how application of a more rigorous definition of cryptic species in taxonomic practice will lead to more accurate estimates of their prevalence in nature, better understanding of their distribution patterns on the tree of life, and increased abilities to resolve the processes underlying their evolution.

The completion of the genome assembly for the economically important coffee plant Coffea canephora (Rubiaceae) has allowed the use of bioinformatic tools to identify and characterize a diverse array of transposable elements (TEs), which can be used in evolutionary studies of the genus. An overview of the copy number and location within the C. canephora genome of four TEs is presented. These are tested for their use as molecular markers to unravel the evolutionary history of the Millotii Complex, a group of six wild coffee (Coffea) species native to Madagascar. Two TEs from the Gypsy superfamily successfully recovered some species boundaries and geographic structure among samples, whereas a TE from the Copia superfamily did not. Notably, species occurring in evergreen moist forests of eastern and southeastern Madagascar were divergent with respect to species in other habitats and regions. Our results suggest that the peak of transpositional activity of the Gypsy and Copia TEs occurred, respectively, before and after the speciation events of the tested Madagascan species. We conclude that the utilization of active TEs has considerable potential to unravel the evolutionary history and delimitation of closely related Coffea species. However, the selection of TE needs to be experimentally tested, since each element has its own evolutionary history. Different TEs with similar copy number in a given species can render different dendrograms; thus copy number is not a good selection criterion to attain phylogenetic resolution.

Many arctic-alpine plants display a highly disjunct distribution between the Arctic/Boreal regions and the southern Asian mountains. Two main hypotheses have been proposed to explain the origin of this biogeographic pattern: (1) south-to-north migration in the late Pliocene/early Pleistocene, and (2) north-to-south migration during the Miocene. The genus Cassiope is disjunctly distributed between the Arctic/Boreal regions and the Himalayan-Hengduan Mountains (HHM) and was selected to test these hypotheses. We constructed a fossil-calibrated phylogeny of Ericaceae using two plastid regions to estimate the crown group age of Cassiope, and used sequence data from thousands of loci produced by restriction site associated DNA sequencing (RAD-seq) to reconstruct the phylogeny of Cassiope. We also performed Bayesian divergence time analysis and biogeographic analysis. The Cassiope crown group was estimated to have originated in the Miocene, which predates the onset of Northern hemisphere glaciation. All HHM species formed a clade together with one eastern Siberian species, and this clade was sister to all other Arctic/Boreal species. This topology implies a northern origin of Cassiope, which is confirmed by our biogeographic analysis. Our results thus suggest that the ancient north-to-south migration hypothesis is most consistent with the origin of Cassiope.

Hybridization is frequent in the large and ecologically significant genus Carex (Cyperaceae). In four important sections of the northern regions (Ceratocystis, Glareosae, Phacocystis and Vesicariae), the frequent occurrence of hybrids often renders the identification of "pure" species and hybrids difficult. In this study we address the origins and taxonomic rank of two taxa of section Vesicariae: Carex rostrata var. borealis and C. stenolepis. The origin and taxonomic status of C. stenolepis has been the subject of substantial debate over the years, whereas C. rostrata var. borealis has received very little attention in the years since its first description in the 19th century. By performing an extensive sampling of relevant taxa from a broad distribution range, and analyzing data from fifteen microsatellite loci developed specifically for our study together with pollen stainability measures, we resolve the hybrid origins of C. rostrata var. borealis and C. stenolepis and provide new insights into this taxonomically challenging group of sedges. Our results are in accordance with previous findings suggesting that C. stenolepis is a hybrid between C. vesicaria and C. saxatilis. They are also in accordance with a previous proposition that C. rostrata var. borealis is a hybrid between C. rostrata and C. rotundata, and furthermore suggest that both hybrids are the result of multiple, recent (i.e., postglacial) hybridization events. We found little evidence for successful sexual reproduction within C. rostrata var. borealis and C. stenolepis, but conclude that the common and recurrent, largely predictable occurrence of these taxa justifies accepting both hybrids as hybrid species with binomial names. There are, however, complications as to types and priority names, and we therefore choose to address these problems in a separate paper.

Heterostyly is a wide-spread floral adaptation to promote outbreeding, yet its genetic basis and evolutionary origin remain poorly understood. In Primula (primroses), heterostyly is controlled by the S-locus supergene that determines the reciprocal arrangement of reproductive organs and incompatibility between the two morphs. However, the identities of the component genes remain unknown. Here, we identify the Primula CYP734A50 gene, encoding a putative brassinosteroid-degrading enzyme, as the G locus that determines the style-length dimorphism. CYP734A50 is only present on the short-styled S-morph haplotype, it is specifically expressed in S-morph styles, and its loss or inactivation leads to long styles. The gene arose by a duplication specific to the Primulaceae lineage and shows an accelerated rate of molecular evolution. Thus, our results provide a mechanistic explanation for the Primula style-length dimorphism and begin to shed light on the evolution of the S-locus as a prime model for a complex plant supergene.

Background The flowering plant Primula veris is a common spring blooming perennial that is widely cultivated throughout Europe. This species is an established model system in the study of the genetics, evolution, and ecology of heterostylous floral polymorphisms. Despite the long history of research focused on this and related species, the continued development of this system has been restricted due the absence of genomic and transcriptomic resources. Results We present here a de novo draft genome assembly of P. veris covering 301.8 Mb, or approximately 63% of the estimated 479.22 Mb genome, with an N50 contig size of 9.5 Kb, an N50 scaffold size of 164 Kb, and containing an estimated 19,507 genes. The results of a RADseq bulk segregant analysis allow for the confident identification of four genome scaffolds that are linked to the P. veris S -locus. RNAseq data from both P. veris and the closely related species P. vulgaris allow for the characterization of 113 candidate heterostyly genes that show significant floral morph-specific differential expression. One candidate gene of particular interest is a duplicated GLOBOSA homolog that may be unique to Primula ( PveGLO2 ), and is completely silenced in L-morph flowers. Conclusions The P. veris genome represents the first genome assembled from a heterostylous species, and thus provides an immensely important resource for future studies focused on the evolution and genetic dissection of heterostyly. As the first genome assembled from the Primulaceae, the P. veris genome will also facilitate the expanded application of phylogenomic methods in this diverse family and the eudicots as a whole.