Converging forms: an examination of sub-Arctic, circumarctic, and Central Asian Ranunculus auricomus agg. populations.

Introduction: Phenotypic complexity in species complexes and recently radiated lineages has resulted in a diversity of forms that have historically been classified into separate taxa. Increasingly, with the proliferation of high-throughput sequencing methods, additional layers of complexity have been recognized, such as frequent hybridization and reticulation, which may call into question the previous morphological groupings of closely related organisms. Methods: We investigated Northern European, Asian, and Beringian populations of Ranunculus auricomus agg. with phylogenomic analysis of 736 genes and 27,586 SNPs in order to deduce the interrelatedness and hybrid origin of this phenotypically and taxonomically complicated group from Europe characterized by a history of hybridization, polyploidy, apomixis, and recent radiation. The ploidy levels and the reproductive mode of the Northern European populations were assessed via flow cytometric seed screening. In addition, in order to examine the phenotypic plasticity of the dwarf forms previously described as species and summarized as the Ranunculus monophyllus group, we conducted climate chamber experiments under cold (northern) and warm (temperate) conditions. Results: The Northern European populations are tetra- to hexaploid and propagate primarily through apomixis. The complex is characterized by highly reticulate relationships. Genetic differentiation of the main clusters has occurred between the above-mentioned geographical regions. We find evidence for the hybrid origin of the taxa in these areas with differing genomic contributions from the geographically nearest European sexual progenitor species. Furthermore, polyphyly in the taxa of the R. monophyllus group is supported. Experiments show low lability in the traits associated with the R. monophyllus group. Discussion: We conclude that multiple adaptations of hybrids to colder climates and shorter vegetation periods have shaped the phenotypes of the R. monophyllus group, and we suggest a formal classification as nothotaxa within the R. auricomus group.

New Frontiers in Potato Breeding: Tinkering with Reproductive Genes and Apomixis.

Potato is the most important non-cereal crop worldwide, and, yet, genetic gains in potato have been traditionally delayed by the crop's biology, mostly the genetic heterozygosity of autotetraploid cultivars and the intricacies of the reproductive system. Novel site-directed genetic modification techniques provide opportunities for designing climate-smart cultivars, but they also pose new possibilities (and challenges) for breeding potato. As potato species show a remarkable reproductive diversity, and their ovules have a propensity to develop apomixis-like phenotypes, tinkering with reproductive genes in potato is opening new frontiers in potato breeding. Developing diploid varieties instead of tetraploid ones has been proposed as an alternative way to fill the gap in genetic gain, that is being achieved by using gene-edited self-compatible genotypes and inbred lines to exploit hybrid seed technology. In a similar way, modulating the formation of unreduced gametes and synthesizing apomixis in diploid or tetraploid potatoes may help to reinforce the transition to a diploid hybrid crop or enhance introgression schemes and fix highly heterozygous genotypes in tetraploid varieties. In any case, the induction of apomixis-like phenotypes will shorten the time and costs of developing new varieties by allowing the multi-generational propagation through true seeds. In this review, we summarize the current knowledge on potato reproductive phenotypes and underlying genes, discuss the advantages and disadvantages of using potato's natural variability to modulate reproductive steps during seed formation, and consider strategies to synthesize apomixis. However, before we can fully modulate the reproductive phenotypes, we need to understand the genetic basis of such diversity. Finally, we visualize an active, central role for genebanks in this endeavor by phenotyping properly genotyped genebank accessions and new introductions to provide scientists and breeders with reliable data and resources for developing innovations to exploit market opportunities.

Doubled haploid technology and synthetic apomixis: Recent advances and applications in future crop breeding.

Doubled haploid (DH) technology and synthetic apomixis approaches can considerably shorten breeding cycles and enhance breeding efficiency. Compared with traditional breeding methods, DH technology offers the advantage of rapidly generating inbred lines, while synthetic apomixis can effectively fix hybrid vigor. In this review, we focus on (i) recent advances in identifying and characterizing genes responsible for haploid induction (HI), (ii) the molecular mechanisms of HI, (iii) spontaneous haploid genome doubling, and (iv) crop synthetic apomixis. We also discuss the challenges and potential solutions for future crop breeding programs utilizing DH technology and synthetic apomixis. Finally, we provide our perspectives about how to integrate DH and synthetic apomixis for precision breeding and de novo domestication.

An epiQTL underlying asexual seed formation in Arabidopsis.

KEY MESSAGE: The DNA methylation status at an epigenetic quantitative trait locus in the Arabidopsis chromosome 2 is linked to the formation of apomictic-like endosperms. Seed development in most angiosperms is coupled to fertilization of the maternal gametes by two sperm cells. However, apomictic species can reproduce asexually via seeds. This trait is of great agricultural interest, as it would fix complex genotypes and allow for pollen-independent seed production. However, engineering full apomixis requires three independent processes: apomeiosis, parthenogenesis and autonomous endosperm development. While the first two have been successfully engineered in some crops, the formation of autonomous endosperms remains a challenge. Although it is known that this trait is under epigenetic control, such as of DNA methylation, the underlying mechanisms remain mostly undiscovered. Here, using epigenetic recombinant inbred lines, we identified an epigenetic quantitative trait locus in the Arabidopsis chromosome 2, which correlates with permissiveness for the formation of asexual seeds: hypomethylation at this genomic region allows the formation of larger autonomous endosperms. Importantly, the methylation at this locus only correlates with asexual seed size, and not to the size of sexual seeds or that of other organs. With this, we aim to show that screening for epialleles is a promising strategy to uncover loci underlying relevant traits and could pave the way to identifying genes necessary for the engineering of apomixis.

Genome-Wide Identification and Characterization of the RWP-RK Proteins in Zanthoxylum armatum.

Apomixis is a common reproductive characteristic of Zanthoxylum plants, and RWP-RKs are plant-specific transcription factors known to regulate embryonic development. However, the genome-wide analysis and function prediction of RWP-RK family genes in Z. armatum are unclear. In this study, 36 ZaRWP-RK transcription factors were identified in the genome of Z. armatum, among which 15 genes belonged to the RKD subfamily and 21 belonged to the NLP subfamily. Duplication events of ZaRWP-RK genes were mainly segmental duplication, and synteny analysis revealed a close phylogenetic relationship between Z. armatum and Arabidopsis. The analysis of cis-elements indicated that ZaRWP-RK genes may be involved in the regulation of the embryonic development of Z. armatum by responding to plant hormones such as abscisic acid, auxin, and gibberellin. Results of a real-time PCR showed that the expression levels of most ZaRWP-RK genes were significantly increased from flowers to young fruits. Protein-protein interaction network analysis further revealed the potential roles of the ZaRWP-RK proteins in apomixis. Collectively, this study is expected to improve our understanding of ZaRWP-RK transcription factors and provide a theoretical basis for future investigations into the ZaRWP-RK genes and their regulatory mechanisms in the apomixis process of Z. armatum.

An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

Pollination Syndrome, Florivory, and Breeding System of Satyrium nepalense var. ciliatum (Orchidaceae) in Central Yunnan, China.

Research on Satyrium nepalense var. ciliatum (Lindl.) Hook. f. has primarily focused on populations in Northwestern Yunnan, with limited studies on pollination syndromes and insect behavior. In addition, it is geographically limited in its breeding system studies. Here, pollination syndromes, florivory, and breeding systems of S. nepalense var. ciliatum from Liangwang Mountain (Central Yunnan, China) were investigated through field work, microscope, scanning electron microscope (SEM), and parafin section. It was revealed that the pollination syndrome was possessing out-crossing, such as bright color, a developed rostellum, nectar glands in the spur, and food hairs at the lip base. The color and nectar attracted flower visitors, and florivory was observed. Some flower visitors pollinated their companion species. Ants were identified as floral visitors for the first time in Satyrium, although substantial pollination was not observed. Ants might be potential pollinators. S. nepalense var. ciliatum possessed a mixed breeding system, including selfing, out-crossing, and apomixis, with apomixis being predominant in nature. It is suggested that the pollination syndrome, florivory, and pollination competition would contribute to its mixed breeding systems, particularly leading to the occurrence of apomixis.

Effects of species diversity on trait expression of the clonal herb Taraxacum officinale and its relation to genotype diversity and phenotypic plasticity.

Plant species respond to varying plant species diversity and associated changes in their abiotic and biotic environment with changes in their phenotype. However, it is not clear to what degree this phenotypic differentiation is due to genotype diversity within populations or phenotypic plasticity of plant individuals. We studied individuals of 16 populations of the clonal herb Taraxacum officinale grown in plant communities of different species richness in a 17-year-old grassland biodiversity experiment (Jena Experiment). We collected 12 individuals in each population to measure phenotypic traits and identify distinct genotypes using microsatellite DNA markers. Plant species richness did not influence population-level genotype and trait diversity. However, it affected the expression of several phenotypic traits, e.g. leaf and inflorescence number, maximum leaf length and seed mass, which increased with increasing plant species richness. Moreover, population-level trait diversity correlated positively with genotype richness for leaf dry matter content (LDMC) and negatively with inflorescence number. For several traits (i.e. seed mass, germination rate, LDMC, specific leaf area (SLA)), a larger portion of variance was explained by genotype identity, while variance in other traits (i.e. number of inflorescences, leaf nitrogen concentration, leaf number, leaf length) resided within genotypes and thus was mostly due to phenotypic plasticity. Overall, our findings show that plant species richness positively affected the population means of some traits related to whole-plant performance, whose variation was achieved through both phenotypic plasticity and genotype composition of a population.

Sex- versus apomixis-specific polymorphisms in the 5'UTR of APOLLO from Boechera shift gene expression from somatic to reproductive tissues in Arabidopsis.

Introduction: Among candidate genes underlying the control components of apomixis, APOLLO is known for its strong linkage to apomeiosis in the genus Boechera. The gene has "apo alleles," which are characterized by a set of linked apomixis-specific polymorphisms, and "sex alleles." All apomictic Boechera genotypes are heterozygous for the apo/sex alleles, whereas all sexual genotypes are homozygous for sex alleles. Methods: In this study, native and synthetic APOLLO promoters were characterized by detecting the expression level of the ß-glucuronidase (GUS) gene in Arabidopsis. Results: Comparing various flower developmental stages in transgenic lines containing different constructs with 2-kb native transgenic lines revealed that changes to the APOLLO promoter causes shifts in tissue and developmental stage specificity of GUS expression. Importantly, several apomixis-specific polymorphisms in the 5'UTR change the timing and location of GUS activity from somatic to reproductive tissues. Discussion: These synthetic data simulate a plausible evolutionary process, whereby apomixis-specific gene activity can be achieved.

A novel strategy to study apomixis, automixis, and autogamy in plants.

KEY MESSAGE: The combination of a flow cytometric seed screen and genotyping of each single seed offers a cost-effective approach to detecting complex reproductive pathways in flowering plants. Reproduction may be seen as one of the driving forces of evolution. Flow cytometric seed screen and genotyping of parents and progeny are commonly employed techniques to discern various modes of reproduction in flowering plants. Nevertheless, both methods possess limitations constraining their individual capacity to investigate reproductive modes thoroughly. We implemented both methods in a novel manner to analyse reproduction pathways using a carefully selected material of parental individuals and their seed progeny. The significant advantage of this approach lies in its ability to apply both methods to a single seed. The introduced methodology provides valuable insights into discerning the levels of apomixis, sexuality, and selfing in complex Rubus taxa. The results may be explained by the occurrence of automixis in Rubus, which warrants further investigation. The approach showcased its effectiveness in a different apomictic system, specifically in Taraxacum. Our study presents a comprehensive methodological approach for determining the mode of reproduction where flow cytometry loses its potential. It provides a reliable and cost-effective method with significant potential in biosystematics, population genetics, and crop breeding.

Emerging Trends in Wheat (Triticum spp.) Breeding: Implications for the Future.

Wheat (Triticum spp and, particularly, T. aestivum L.) is an essential cereal with increased human and animal nutritional demand. Therefore, there is a need to enhance wheat yield and genetic gain using modern breeding technologies alongside proven methods to achieve the necessary increases in productivity. These modern technologies will allow breeders to develop improved wheat cultivars more quickly and efficiently. This review aims to highlight the emerging technological trends used worldwide in wheat breeding, with a focus on enhancing wheat yield. The key technologies for introducing variation (hybridization among the species, synthetic wheat, and hybridization; genetically modified wheat; transgenic and gene-edited), inbreeding (double haploid (DH) and speed breeding (SB)), selection and evaluation (marker-assisted selection (MAS), genomic selection (GS), and machine learning (ML)) and hybrid wheat are discussed to highlight the current opportunities in wheat breeding and for the development of future wheat cultivars.

Apomixis and the paradox of sex in plants.

BACKGROUND: The predominance of sex in eukaryotes, despite the high costs of meiosis and mating, remains an evolutionary enigma. Many theories have been proposed, none of them being conclusive on its own, and they are, in part, not well applicable to land plants. Sexual reproduction is obligate in embryophytes for the great majority of species. SCOPE: This review compares the main forms of sexual and asexual reproduction in ferns and angiosperms, based on the generation cycling of sporophyte and gametophyte (leaving vegetative propagation aside). The benefits of sexual reproduction for maintenance of genomic integrity in comparison to asexuality are discussed in the light of developmental, evolutionary, genetic and phylogenetic studies. CONCLUSIONS: Asexual reproduction represents modifications of the sexual pathway, with various forms of facultative sexuality. For sexual land plants, meiosis provides direct DNA repair mechanisms for oxidative damage in reproductive tissues. The ploidy alternations of meiosis-syngamy cycles and prolonged multicellular stages in the haploid phase in the gametophytes provide a high efficiency of purifying selection against recessive deleterious mutations. Asexual lineages might buffer effects of such mutations via polyploidy and can purge the mutational load via facultative sexuality. The role of organelle-nuclear genome compatibility for maintenance of genome integrity is not well understood. In plants in general, the costs of mating are low because of predominant hermaphroditism. Phylogenetic patterns in the archaeplastid clade suggest that high frequencies of sexuality in land plants are concomitant with a stepwise increase of intrinsic and extrinsic stress factors. Furthermore, expansion of genome size in land plants would increase the potential mutational load. Sexual reproduction appears to be essential for keeping long-term genomic integrity, and only rare combinations of extrinsic and intrinsic factors allow for shifts to asexuality.

Plant kleptomaniacs: geographical genetic patterns in the amphi-apomictic Rubus ser. Glandulosi (Rosaceae) reveal complex reticulate evolution of Eurasian brambles.

BACKGROUND AND AIMS: Rubus ser. Glandulosi provides a unique model of geographical parthenogenesis on a homoploid (2n = 4x) level. We aim to characterize evolutionary and phylogeographical patterns in this taxon and shed light on the geographical differentiation of apomicts and sexuals. Ultimately, we aim to evaluate the importance of phylogeography in the formation of geographical parthenogenesis. METHODS: Rubus ser. Glandulosi was sampled across its Eurasian range together with other co-occurring Rubus taxa (587 individuals in total). Double-digest restriction site-associated DNA sequencing (ddRADseq) and modelling of suitable climate were used for evolutionary inferences. KEY RESULTS: Six ancestral species were identified that contributed to the contemporary gene pool of R. ser. Glandulosi. Sexuals were introgressed from Rubus dolichocarpus and Rubus moschus in West Asia and from Rubus ulmifolius agg., Rubus canescens and Rubus incanescens in Europe, whereas apomicts were characterized by alleles of Rubus subsect. Rubus. Gene flow between sexuals and apomicts was also detected, as was occasional hybridization with other taxa. CONCLUSIONS: We hypothesize that sexuals survived the last glacial period in several large southern refugia, whereas apomicts were mostly restricted to southern France, whence they quickly recolonized Central and Western Europe. The secondary contact of sexuals and apomicts was probably the principal factor that established geographical parthenogenesis in R. ser. Glandulosi. Sexual populations are not impoverished in genetic diversity along their borderline with apomicts, and maladaptive population genetic processes probably did not shape the geographical patterns.

One-line hybrid rice with high-efficiency synthetic apomixis and near-normal fertility.

KEY MESSAGE: High-frequency clonal seeds and near-normal fertility were obtained by engineering synthetic apomixis in hybrid rice. The one-line strategy, with the advantage of unnecessary seed production, is the final stage for the hybrid rice development and can be achieved through the fixation of heterosis via artificially inducing apomixis. Recently, synthetic apomixis has been generated in rice by combining MiMe (Mitosis instead of Meiosis) with either the ectopic expression of BABY BOOM (BBM1 or BBM4) or mutation of MATRILINEAL (MTL), resulting in over 95.00% of clonal seeds. However, the frequency of clonal seeds was only 29.20% when AtDD45 promoter was used to drive BBM1. In addition, achieving both a high frequency of clonal seeds and near-normal fertility simultaneously had been elusive in earlier strategies. In this study, using AtDD45 promoter to drive BBM1 expression in combination with the MiMe mutant resulted in the apomixis frequency as high as 98.70%. Even more, employing fusion promoters (AtMYB98_AtDD1_OsECA1-like1) to drive WUS expression in combination with pAtDD45:BBM1 and MiMe could produce clonal seeds at rates of up to 98.21%, the highest seed setting rate reached to 83.67%. Multiple-embryos were observed in clonal lines at a frequency ranging from 3.37% to 60.99%. Transmission of the high frequency of apomixis through skipped generations (atavism) was identified in two clonal lines, even though it remained stable in the majority of clonal lines. These findings significantly advance the pursuit of fixed heterosis in rice through synthetic apomixis, edging closer to its agricultural application.

Distinct geographic parthenogenesis in spite of niche conservatism and a single ploidy level: A case of Rubus ser. Glandulosi (Rosaceae).

Asexual organisms often differ in their geographic distributions from their sexual relatives. This phenomenon, termed geographic parthenogenesis, has long been known, but the underlying factors behind its diverse patterns have been under dispute. Particularly problematic is an association between asexuality and polyploidy in most taxa. Here, we present a new system of geographic parthenogenesis on the tetraploid level, promising new insights into this complex topic. We used flow cytometric seed screen and microsatellite genotyping to characterise the patterns of distribution of sexuals and apomicts and genotypic distributions in Rubus ser. Glandulosi across its range. Ecological modelling and local-scale vegetation and soil analyses were used to test for niche differentiation between the reproductive groups. Apomicts were detected only in North-western Europe, sexuals in the rest of the range in Europe and West Asia, with a sharp borderline stretched across Central Europe. Despite that, we found no significant differences in ecological niches. Genotypic richness distributions suggested independence of the reproductive groups and a secondary contact. We argue that unless a niche differentiation (resulting from polyploidy and/or hybridity) evolves, the main factors behind the patterns of geographic parthenogenesis in plants are phylogeographic history and neutral microevolutionary processes, such as clonal turnover.

Clonal reproduction by seed of a cultivated hybrid plant: a new perspective for small-scale rice growers.

Transferring an asexual mode of reproduction by seeds (apomixis) to cultivated plants would enable clonal reproduction of heterozygous genotypes such as F1 hybrids with hybrid vigor (heterosis), facilitating their access and multiplication by small-scale growers. Although sources of apomixis and the genetic loci controlling its constituent elements have been identified in wild species, their transfer by crossing to cultivated species has so far been unsuccessful. Here, we have introduced synthetic apomixis in hybrid rice to produce a high (95-100%) frequency of clonal seeds, via the inactivation of three meiotic genes-resulting in unreduced, non-recombined gametes-and the addition of an egg cell parthenogenesis trigger. The genotype and phenotype, including grain quality, of the F1 hybrid are reproduced identically in the clonal apomictic progenies. These results make synthetic apomixis compatible with future use in agriculture.

Le transfert d'un mode de reproduction clonale asexuée par grain (apomixie) aux plantes cultivées permettrait de reproduire de façon génétiquement identique des génotypes hétérozygotes comme ceux des hybrides F1 dotés d'une vigueur hybride (heterosis), facilitant ainsi leur accès et leur multiplication par les petits cultivateurs. Bien que des sources d'apomixie et les loci génétiques contrôlant ses éléments constitutifs aient été identifiés chez les espèces sauvages, leur transfert par croisement aux espèces cultivées a jusqu'à présent été infructueux. Ici, nous avons introduit chez un riz hybride une apomixie synthétique produisant une haute fréquence de grains clonaux (95­100%), via l'inactivation de trois gènes méiotiques ­ permettant d'obtenir des gamètes non réduits et non recombinés ­ et l'apport d'un déclencheur de la parthénogenèse. Le génotype et le phénotype, incluant la qualité de grain, de l'hybride F1 sont reproduits à l'identique dans les descendances apomictiques clonales. Ces résultats rendent compatible l'apomixie synthétique avec une future utilisation en agriculture.

Differential expression and evolutionary diversification of RNA helicases in Boechera sexual and apomictic reproduction.

In higher plants, sexual reproduction is characterized by meiosis of the first cells of the germlines, and double fertilization of the egg and central cell after gametogenesis. In contrast, in apomicts of the genus Boechera, meiosis is omitted or altered and only the central cell requires fertilization, while the embryo forms parthenogenetically from the egg cell. To deepen the understanding of the transcriptional basis underlying these differences, we applied RNA-seq to compare expression in reproductive tissues of different Boechera accessions. This confirmed previous evidence of an enrichment of RNA helicases in plant germlines. Furthermore, few RNA helicases were differentially expressed in female reproductive ovule tissues harboring mature gametophytes from apomictic and sexual accessions. For some of these genes, we further found evidence for a complex recent evolutionary history. This included a homolog of Arabidopsis thaliana FASCIATED STEM4 (FAS4). In contrast to AtFAS4, which is a single-copy gene, FAS4 is represented by three homologs in Boechera, suggesting a potential for subfunctionalization to modulate reproductive development. To gain first insights into functional roles of FAS4, we studied Arabidopsis lines carrying mutant alleles. This identified the crucial importance of AtFAS4 for reproduction, as we observed developmental defects and arrest during male and female gametogenesis.

Differentially methylated genes involved in reproduction and ploidy levels in recent diploidized and tetraploidized Eragrostis curvula genotypes.

Epigenetics studies changes in gene activity without changes in the DNA sequence. Methylation is an epigenetic mechanism important in many pathways, such as biotic and abiotic stresses, cell division, and reproduction. Eragrostis curvula is a grass species reproducing by apomixis, a clonal reproduction by seeds. This work employed the MCSeEd technique to identify deferentially methylated positions, regions, and genes in the CG, CHG, and CHH contexts in E. curvula genotypes with similar genomic backgrounds but with different reproductive modes and ploidy levels. In this way, we focused the analysis on the cvs. Tanganyika INTA (4x, apomictic), Victoria (2x, sexual), and Bahiense (4x, apomictic). Victoria was obtained from the diploidization of Tanganyika INTA, while Bahiense was produced from the tetraploidization of Victoria. This study showed that polyploid/apomictic genotypes had more differentially methylated positions and regions than the diploid sexual ones. Interestingly, it was possible to observe fewer differentially methylated positions and regions in CG than in the other contexts, meaning CG methylation is conserved across the genotypes regardless of the ploidy level and reproductive mode. In the comparisons between sexual and apomictic genotypes, we identified differentially methylated genes involved in the reproductive pathways, specifically in meiosis, cell division, and fertilization. Another interesting observation was that several differentially methylated genes between the diploid and the original tetraploid genotype recovered their methylation status after tetraploidization, suggesting that methylation is an important mechanism involved in reproduction and ploidy changes.

Phylogenomics of Southern European Taxa in the Ranunculus auricomus Species Complex: The Apple Doesn't Fall Far from the Tree.

The taxonomic status of many Southern European taxa of the Ranunculus auricomus complex remains uncertain despite this region's proximity to the native ranges of the sexual progenitor species of the complex. We investigated whether additional sexual progenitor species are present in the Mediterranean region. Utilizing target enrichment of 736 single-copy nuclear gene regions and flow cytometry, we analyzed phylogenomic relationships, the ploidy level, and the reproductive mode in representatives of 16 populations in Southern Europe, with additional sequence data from herbarium collections. Additionally, phased sequence assemblies from suspected nothotaxa were mapped to previously described sexual progenitor species in order to determine hybrid ancestry. We found the majority of Mediterranean taxa to be tetraploid, with hybrid populations propagating primarily via apomixis. Phylogenomic analysis revealed that except for the progenitor species, the Mediterranean taxa are often polyphyletic. Most apomictic taxa showed evidence of mixed heritage from progenitor species, with certain progenitor genotypes having mapped more to the populations from adjacent geographical regions. Geographical trends were found in phylogenetic distance, roughly following an east-to-west longitudinal demarcation of the complex, with apomicts extending to the southern margins. Additionally, we observed post-hybridization divergence between the western and eastern populations of nothotaxa in Southern Europe. Our results support a classification of apomictic populations as nothotaxa, as previously suggested for Central Europe.

Genetic mapping of the LOSS OF PARTHENOGENESIS locus in Pilosella piloselloides and the evolution of apomixis in the Lactuceae.

Pilosella piloselloides var. praealta (syn. P. praealta; Hieracium praealtum) is a versatile model used to study gametophytic apomixis. In this system apomixis is controlled by three loci: one that controls the avoidance of meiosis (LOA), one that controls the avoidance of fertilization (LOP) and a third that controls autonomous endosperm formation (AutE). Using a unique polyhaploid mapping approach the LOP locus was mapped to a 654 kb genomic interval syntenic to linkage group 8 of Lactuca sativa. Polyhaploids form through the gametophytic action of a dominant determinant at LOP, so the mapped region represents both a functional and a physical domain for LOP in P. piloselloides. Allele sequence divergence (ASD) analysis of the PARTHENOGENESIS (PAR) gene within the LOP locus revealed that dominant PAR alleles in Pilosella remain highly similar across the genus, whilst the recessive alleles are more divergent. A previous report noted that dominant PAR alleles in both Pilosella and Taraxacum are modified by the presence of a class II transposable element (TE) in the promoter of the gene. This observation was confirmed and further extended to the related genus Hieracium. Sufficient differences were noted in the structure and location of the TE elements to conclude that TE insertional events had occurred independently in the three genera. Measures of allele crossover amongst the polyhaploids revealed that P. piloselloides is an autopolyploid species with tetrasomic inheritance. It was also noted that the dominant determinant of LOP in P. piloselloides could transmit via a diploid gamete (pollen or egg) but not via a haploid gamete. Using this information, a model is presented of how gametophytic apomixis may have evolved in several members of the Lactuceae, a tribe of the Asteraceae.