A hemizygous supergene controls homomorphic and heteromorphic self-incompatibility systems in Oleaceae.

Self-incompatibility (SI) has evolved independently multiple times and prevents self-fertilization in hermaphrodite angiosperms. Several groups of Oleaceae such as jasmines exhibit distylous flowers, with two compatibility groups each associated with a specific floral morph.1 Other Oleaceae species in the olive tribe have two compatibility groups without associated morphological variation.2,3,4,5 The genetic basis of both homomorphic and dimorphic SI systems in Oleaceae is unknown. By comparing genomic sequences of three olive subspecies (Olea europaea) belonging to the two compatibility groups, we first locate the genetic determinants of SI within a 700-kb hemizygous region present only in one compatibility group. We then demonstrate that the homologous hemizygous region also controls distyly in jasmine. Phylogenetic analyses support a common origin of both systems, following a segmental genomic duplication in a common ancestor. Examination of the gene content of the hemizygous region in different jasmine and olive species suggests that the mechanisms determining compatibility groups and floral phenotypes (whether homomorphic or dimorphic) in Oleaceae rely on the presence/absence of two genes involved in gibberellin and brassinosteroid regulation.

African bushpigs exhibit porous species boundaries and appeared in Madagascar concurrently with human arrival.

Several African mammals exhibit a phylogeographic pattern where closely related taxa are split between West/Central and East/Southern Africa, but their evolutionary relationships and histories remain controversial. Bushpigs (Potamochoerus larvatus) and red river hogs (P. porcus) are recognised as separate species due to morphological distinctions, a perceived lack of interbreeding at contact, and putatively old divergence times, but historically, they were considered conspecific. Moreover, the presence of Malagasy bushpigs as the sole large terrestrial mammal shared with the African mainland raises intriguing questions about its origin and arrival in Madagascar. Analyses of 67 whole genomes revealed a genetic continuum between the two species, with putative signatures of historical gene flow, variable FST values, and a recent divergence time (<500,000 years). Thus, our study challenges key arguments for splitting Potamochoerus into two species and suggests their speciation might be incomplete. Our findings also indicate that Malagasy bushpigs diverged from southern African populations and underwent a limited bottleneck 1000-5000 years ago, concurrent with human arrival in Madagascar. These results shed light on the evolutionary history of an iconic and widespread African mammal and provide insight into the longstanding biogeographic puzzle surrounding the bushpig's presence in Madagascar.

Climate change threatens olive oil production in the Levant.

The olive tree (Olea europaea L.) is one of the species best adapted to a Mediterranean-type climate1-8. Nonetheless, the Mediterranean Basin is deemed to be a climate change 'hotspot' by the Intergovernmental Panel on Climate Change9,10 because future model projections suggest considerable warming and drying11,12. Within this context, new environmental challenges will arise in the coming decades, which will both weaken and threaten olive-growing areas, leading to a loss of productivity and changes in fruit and oil quality13-15. Olive growing, a core of the Mediterranean economy, might soon be under stress. To probe the link between climate and olive trees, we here report 5,400 years of olive tree dynamics from the ancient city of Tyre, Lebanon. We show that optimal fruiting scales closely with temperature. Present-day and palaeo data define an optimal annual average temperature of 16.9 ± 0.3 °C for olive flowering that has existed at least since the Neolithic period. According to our projections, during the second half of the twenty-first century, temperature increases in Lebanon will have detrimental consequences on olive tree growth and olive oil production, especially in the country's southern regions, which will become too hot for optimal flowering and fruiting. These data provide a template to understand present and future thresholds of olive production under climate change.

How ancient forest fragmentation and riparian connectivity generate high levels of genetic diversity in a microendemic Malagasy tree.

Understanding landscape changes is central to predicting evolutionary trajectories and defining conservation practices. While human-driven deforestation is intense throughout Madagascar, exceptions in areas such as the Loky-Manambato region (north) raise questions regarding the causes and age of forest fragmentation. The Loky-Manambato region also harbours a rich and endemic flora, whose evolutionary origin remains poorly understood. We assessed the genetic diversity of an endangered microendemic Malagasy olive species (Noronhia spinifolia Hong-Wa) to better understand the vegetation dynamics in the Loky-Manambato region and its influence on past evolutionary processes. We characterized 72 individuals sampled across eight forests through nuclear and mitochondrial restriction-associated DNA sequencing data and chloroplast microsatellites. Combined population and landscape genetics analyses indicate that N. spinifolia diversity is largely explained by the current forest cover, highlighting a long-standing habitat mosaic in the region. This sustains a major and long-term role of riparian corridors in maintaining connectivity across these antique mosaic habitats, calling for the study of organismal interactions that promote gene flow.

Hybridization boosts dispersal of two contrasted ecotypes in a grass species.

Genetic exchanges between closely related groups of organisms with different adaptations have well-documented beneficial and detrimental consequences. In plants, pollen-mediated exchanges affect the sorting of alleles across physical landscapes and influence rates of hybridization. How these dynamics affect the emergence and spread of novel phenotypes remains only partially understood. Here, we use phylogenomics and population genomics to retrace the origin and spread of two geographically overlapping ecotypes of the African grass Alloteropsis angusta. In addition to an ecotype inhabiting wetlands, we report the existence of a previously undescribed ecotype inhabiting Miombo woodlands and grasslands. The two ecotypes are consistently associated with different nuclear groups, which represent an advanced stage of divergence with secondary low-level gene flow. However, the seed-transported chloroplast genomes are consistently shared by distinct ecotypes inhabiting the same region. These patterns suggest that the nuclear genome of one ecotype can enter the seeds of the other via occasional pollen movements with sorting of nuclear groups in subsequent generations. The contrasting ecotypes of A. angusta can thus use each other as a gateway to new locations across a large part of Africa, showing that hybridization can facilitate the geographical dispersal of distinct ecotypes of the same grass species.

Genome sequence of the coffee root-knot nematode Meloidogyne exigua.

Root-knot nematodes (Meloidogyne spp.) cause serious damages on most crops. Here, we report a high-quality genome sequence of Meloidogyne exigua (population Mex1, Costa Rica), a major pathogen of coffee. Its mitogenome (20,974 bp) was first assembled and annotated. The nuclear genome was then constructed consisting of 206 contigs, with an N50 length of 1.89 Mb and a total assembly length of 42.1 Mb.

Genome skims analysis of betel palms (Areca spp., Arecaceae) and development of a profiling method to assess their plastome diversity.

The betel nut (Areca catechu L., Arecaceae) is a monoecious cultivated palm tree that is widespread in tropical regions. It is mainly cultivated for producing areca nuts, from which seeds are extracted and chewed by local populations principally in the Indo-Pacific region. Seeds contain alkaloids which are central nervous system stimulants and are highly addictive. Wild relatives of the betel nut are distributed in South Asia and Australasia, with ca. 40-50 Areca species currently recognized. The geographic origin(s) of the betel nut and its subsequent diffusion and diversification remains poorly documented. Here, a genome skimming approach was applied to screen nucleotidic variation in the most abundant genomic regions. Low coverage sequencing data allowed us to assemble full plastomes, mitochondrial regions (either full mitogenomes or the full set of mitochondrial genes) and the nuclear ribosomal DNA cluster for nine representatives of the Areca genus collected in the field and herbarium collections (including a 182-years old specimen collected during the Dumont d'Urville's expedition). These three genomic compartments provided similar phylogenetic signals, and revealed very low genomic diversity in our sample of cultivated betel nut. We finally developed a genotyping method targeting 34 plastid DNA microsatellites. This plastome profiling approach is useful for tracing the spread of matrilineages, and in combination with nuclear genomic data, can resolve the history of the betel nut. Our method also proves to be efficient for analyzing herbarium specimens, even those collected >100 years ago.

Contrasting Genetic Footprints among Saharan Olive Populations: Potential Causes and Conservation Implications.

The Laperrine's olive is endemic to the Saharan Mountains. Adapted to arid environments, it may constitute a valuable genetic resource to improve water-stress tolerance in the cultivated olive. However, limited natural regeneration coupled with human pressures make it locally endangered in Central Sahara. Understanding past population dynamics is thus crucial to define management strategies. Nucleotide sequence diversity was first investigated on five nuclear genes and compared to the Mediterranean and African olives. These data confirm that the Laperrine's olive has a strong affinity with the Mediterranean olive, but it shows lower nucleotide diversity than other continental taxa. To investigate gene flows mediated by seeds and pollen, polymorphisms from nuclear and plastid microsatellites from 383 individuals from four Saharan massifs were analyzed. A higher genetic diversity in Ahaggar (Hoggar, Algeria) suggests that this population has maintained over the long term a larger number of individuals than other massifs. High-to-moderate genetic differentiation between massifs confirms the role of desert barriers in limiting gene flow. Yet contrasting patterns of isolation by distance were observed within massifs, and also between plastid and nuclear markers, stressing the role of local factors (e.g., habitat fragmentation, historical range shift) in seed and pollen dispersal. Implications of these results in the management of the Laperrine's olive genetic resources are discussed.

Utility of the Mitochondrial Genome in Plant Taxonomic Studies.

Size, structure, and sequence content lability of plant mitochondrial genome (mtDNA) across species has sharply limited its use in taxonomic studies. Historically, mtDNA variation has been first investigated with RFLPs, while the development of universal primers then allowed studying sequence polymorphisms within short genomic regions (<3 kb). The recent advent of NGS technologies now offers new opportunities by greatly facilitating the assembly of longer mtDNA regions, and even full mitogenomes. Phylogenetic works aiming at comparing signals from different genomic compartments (i.e., nucleus, chloroplast, and mitochondria) have been developed on a few plant lineages, and have been shown especially relevant in groups with contrasted inheritance of organelle genomes. This chapter first reviews the main characteristics of mtDNA and the application offered in taxonomic studies. It then presents tips for best sequencing protocol based on NGS data to be routinely used in mtDNA-based phylogenetic studies.

Resolving the Phylogeny of the Olive Family (Oleaceae): Confronting Information from Organellar and Nuclear Genomes.

The olive family, Oleaceae, is a group of woody plants comprising 28 genera and ca. 700 species, distributed on all continents (except Antarctica) in both temperate and tropical environments. It includes several genera of major economic and ecological importance such as olives, ash trees, jasmines, forsythias, osmanthuses, privets and lilacs. The natural history of the group is not completely understood yet, but its diversification seems to be associated with polyploidisation events and the evolution of various reproductive and dispersal strategies. In addition, some taxonomical issues still need to be resolved, particularly in the paleopolyploid tribe Oleeae. Reconstructing a robust phylogenetic hypothesis is thus an important step toward a better comprehension of Oleaceae's diversity. Here, we reconstructed phylogenies of the olive family using 80 plastid coding sequences, 37 mitochondrial genes, the complete nuclear ribosomal cluster and a small multigene family encoding phytochromes (phyB and phyE) of 61 representative species. Tribes and subtribes were strongly supported by all phylogenetic reconstructions, while a few Oleeae genera are still polyphyletic (Chionanthus, Olea, Osmanthus, Nestegis) or paraphyletic (Schrebera, Syringa). Some phylogenetic relationships among tribes remain poorly resolved with conflicts between topologies reconstructed from different genomic regions. The use of nuclear data remains an important challenge especially in a group with ploidy changes (both paleo- and neo-polyploids). This work provides new genomic datasets that will assist the study of the biogeography and taxonomy of the whole Oleaceae.

Genome structure and content of the rice root-knot nematode (Meloidogyne graminicola).

Discovered in the 1960s, Meloidogyne graminicola is a root-knot nematode species considered as a major threat to rice production. Yet, its origin, genomic structure, and intraspecific diversity are poorly understood. So far, such studies have been limited by the unavailability of a sufficiently complete and well-assembled genome. In this study, using a combination of Oxford Nanopore Technologies and Illumina sequencing data, we generated a highly contiguous reference genome (283 scaffolds with an N50 length of 294 kb, totaling 41.5 Mb). The completeness scores of our assembly are among the highest currently published for Meloidogyne genomes. We predicted 10,284 protein-coding genes spanning 75.5% of the genome. Among them, 67 are identified as possibly originating from horizontal gene transfers (mostly from bacteria), which supposedly contribute to nematode infection, nutrient processing, and plant defense manipulation. Besides, we detected 575 canonical transposable elements (TEs) belonging to seven orders and spanning 2.61% of the genome. These TEs might promote genomic plasticity putatively related to the evolution of M. graminicola parasitism. This high-quality genome assembly constitutes a major improvement regarding previously available versions and represents a valuable molecular resource for future phylogenomic studies of Meloidogyne species. In particular, this will foster comparative genomic studies to trace back the evolutionary history of M. graminicola and its closest relatives.

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species.

C4 photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C4 and non-C4 populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C4 lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C4 phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C4 physiology away from its region of origin.

Genetic origins and diversity of bushpigs from Madagascar (Potamochoerus larvatus, family Suidae).

The island of Madagascar, situated off the southeast coast of Africa, shows the first evidence of human presence ~ 10,000 years ago; however, other archaeological data indicates a settlement of the modern peoples of the island distinctly more recent, perhaps > 1500 years ago. Bushpigs of the genus Potamochoerus (family Suidae), are today widely distributed in Madagascar and presumed to have been introduced from Africa at some stage by human immigrants to the island. However, disparities about their origins in Madagascar have been presented in the literature, including the possibility of endemic subspecies, and few empirical data are available. Furthermore, the separation of bushpigs in Madagascar from their mainland relatives may have favoured the evolution of a different repertoire of immune genes first due to a founder effect and then as a response to distinct pathogens compared to their ancestors. Molecular analysis confirmed the species status of the bushpig in Madagascar as P. larvatus, likely introduced from the central region of southern Africa, with no genetic evidence for the recognition of eastern and western subspecies as suggested from previous cranial morphology examination. Investigation of the immunologically important SLA-DQB1 peptide-binding region showed a different immune repertoire of bushpigs in Madagascar compared to those on the African mainland, with seventeen exon-2 haplotypes unique to bushpigs in Madagascar (2/28 haplotypes shared). This suggests that the MHC diversity of the Madagascar populations may have enabled Malagasy bushpigs to adapt to new environments.

A simple method for high molecular-weight genomic DNA extraction suitable for long-read sequencing from spores of an obligate biotroph oomycete.

Long-read sequencing technologies are having a major impact on our approaches to studying non-model organisms and microbial communities. By significantly reducing the cost and facilitating the genome assembly pipelines, any laboratory can now develop its own genomics program regardless of the complexity of the genome studied. The most crucial current challenge is to develop efficient protocols for extracting genomic DNA (gDNA) with high quality and integrity adapted to the organism of interest. This can be particularly complex for obligate pathogens that must maintain intimate interactions inside infected host tissues. Here we propose a simple and cost-effective method for high molecular weight gDNA extraction from spores of Plasmopara halstedii, an obligate biotroph oomycete pathogen responsible for downy mildew in sunflower. We optimized the yield, the quality and the integrity of the extracted gDNA by fine-tuning three critical parameters, the grinding, the lysis temperature and the lysis duration. We obtained gDNA with a fragment size distribution reaching a peak ranging from 79 to 145 kb. More than half of the extracted gDNA consisted of DNA fragments larger than 42 kb, with 23% of fragments larger than 100 kb. We then demonstrated the relevance of this protocol for long-read sequencing using PacBio RSII technology. With this protocol, we were able to obtain a mean read length of 9.3 kb, a max read length of 71 kb and an N50 of 13.3 kb. The development of such DNA extraction protocols is an essential prerequisite for fully exploiting technologies requiring high molecular weight gDNA (e.g. long-read sequencing or optical mapping). These technological advances will help generate data to answer questions such as the role of newly duplicated gene clusters, repeated regions, genomic structural variations or to define number of chromosomes that still remains undefined in many species of pathogenic fungi and oomycetes.

Phylogenomics indicates the "living fossil" Isoetes diversified in the Cenozoic.

The fossil record provides an invaluable insight into the temporal origins of extant lineages of organisms. However, establishing the relationships between fossils and extant lineages can be difficult in groups with low rates of morphological change over time. Molecular dating can potentially circumvent this issue by allowing distant fossils to act as calibration points, but rate variation across large evolutionary scales can bias such analyses. In this study, we apply multiple dating methods to genome-wide datasets to infer the origin of extant species of Isoetes, a group of mostly aquatic and semi-aquatic isoetalean lycopsids, which closely resemble fossil forms dating back to the Triassic. Rate variation observed in chloroplast genomes hampers accurate dating, but genome-wide nuclear markers place the origin of extant diversity within this group in the mid-Paleogene, 45-60 million years ago. Our genomic analyses coupled with a careful evaluation of the fossil record indicate that despite resembling forms from the Triassic, extant Isoetes species do not represent the remnants of an ancient and widespread group, but instead have spread around the globe in the relatively recent past.

SILVOLIVE, a Germplasm Collection of Wild Subspecies With High Genetic Variability as a Source of Rootstocks and Resistance Genes for Olive Breeding.

Wild subspecies of Olea europaea constitute a source of genetic variability with huge potential for olive breeding to face global changes in Mediterranean-climate regions. We intend to identify wild olive genotypes with optimal adaptability to different environmental conditions to serve as a source of rootstocks and resistance genes for olive breeding. The SILVOLIVE collection includes 146 wild genotypes representative of the six O. europaea subspecies and early-generations hybrids. These genotypes came either from olive germplasm collections or from direct prospection in Spain, continental Africa and the Macaronesian archipelago. The collection was genotyped with plastid and nuclear markers, confirming the origin of the genotypes and their high genetic variability. Morphological and architectural parameters were quantified in 103 genotypes allowing the identification of three major groups of correlative traits including vigor, branching habits and the belowground-to-aboveground ratio. The occurrence of strong phenotypic variability in these traits within the germplasm collection has been shown. Furthermore, wild olive relatives are of great significance to be used as rootstocks for olive cultivation. Thus, as a proof of concept, different wild genotypes used as rootstocks were shown to regulate vigor parameters of the grafted cultivar "Picual" scion, which could improve the productivity of high-density hedgerow orchards.

Paternity tests support a diallelic self-incompatibility system in a wild olive (Olea europaea subsp. laperrinei, Oleaceae).

Self-incompatibility (SI) is the main mechanism that favors outcrossing in plants. By limiting compatible matings, SI interferes in fruit production and breeding of new cultivars. In the Oleeae tribe (Oleaceae), an unusual diallelic SI system (DSI) has been proposed for three distantly related species including the olive (Olea europaea), but empirical evidence has remained controversial for this latter. The olive domestication is a complex process with multiple origins. As a consequence, the mixing of S-alleles from two distinct taxa, the possible artificial selection of self-compatible mutants and the large phenological variation of blooming may constitute obstacles for deciphering SI in olive. Here, we investigate cross-genotype compatibilities in the Saharan wild olive (O. e. subsp. laperrinei). As this taxon was geographically isolated for thousands of years, SI should not be affected by human selection. A population of 37 mature individuals maintained in a collection was investigated. Several embryos per mother were genotyped with microsatellites in order to identify compatible fathers that contributed to fertilization. While the pollination was limited by distance inside the collection, our results strongly support the DSI hypothesis, and all individuals were assigned to two incompatibility groups (G1 and G2). No self-fertilization was observed in our conditions. In contrast, crosses between full or half siblings were frequent (ca. 45%), which is likely due to a nonrandom assortment of related trees in the collection. Finally, implications of our results for orchard management and the conservation of olive genetic resources are discussed.

Continued Adaptation of C4 Photosynthesis After an Initial Burst of Changes in the Andropogoneae Grasses.

C$_{4}$ photosynthesis is a complex trait that sustains fast growth and high productivity in tropical and subtropical conditions and evolved repeatedly in flowering plants. One of the major C$_{4}$ lineages is Andropogoneae, a group of $\sim $1200 grass species that includes some of the world's most important crops and species dominating tropical and some temperate grasslands. Previous efforts to understand C$_{4}$ evolution in the group have compared a few model C$_{4}$ plants to distantly related C$_{3}$ species so that changes directly responsible for the transition to C$_{4}$ could not be distinguished from those that preceded or followed it. In this study, we analyze the genomes of 66 grass species, capturing the earliest diversification within Andropogoneae as well as their C$_{3}$ relatives. Phylogenomics combined with molecular dating and analyses of protein evolution show that many changes linked to the evolution of C$_{4}$ photosynthesis in Andropogoneae happened in the Early Miocene, between 21 and 18 Ma, after the split from its C$_{3}$ sister lineage, and before the diversification of the group. This initial burst of changes was followed by an extended period of modifications to leaf anatomy and biochemistry during the diversification of Andropogoneae, so that a single C$_{4}$ origin gave birth to a diversity of C$_{4}$ phenotypes during 18 million years of speciation events and migration across geographic and ecological spaces. Our comprehensive approach and broad sampling of the diversity in the group reveals that one key transition can lead to a plethora of phenotypes following sustained adaptation of the ancestral state. [Adaptive evolution; complex traits; herbarium genomics; Jansenelleae; leaf anatomy; Poaceae; phylogenomics.].

Duplication history and molecular evolution of the rbcS multigene family in angiosperms.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is considered to be the main enzyme determining the rate of photosynthesis. The small subunit of the protein, encoded by the rbcS gene, has been shown to influence the catalytic efficiency, CO2 specificity, assembly, activity, and stability of RuBisCO. However, the evolution of the rbcS gene remains poorly studied. We inferred the phylogenetic tree of the rbcS gene in angiosperms using the nucleotide sequences and found that it is composed of two lineages that may have existed before the divergence of land plants. Although almost all species sampled carry at least one copy of lineage 1, genes of lineage 2 were lost in most angiosperm species. We found the specific residues that have undergone positive selection during the evolution of the rbcS gene. We detected intensive coevolution between each rbcS gene copy and the rbcL gene encoding the large subunit of RuBisCO. We tested the role played by each rbcS gene copy on the stability of the RuBisCO protein through homology modelling. Our results showed that this evolutionary constraint could limit the level of divergence seen in the rbcS gene, which leads to the similarity among the rbcS gene copies of lineage 1 within species.

Evolutionary transcriptomics reveals the origins of olives and the genomic changes associated with their domestication.

The olive (Olea europaea L. subsp. europaea) is one of the oldest and most socio-economically important cultivated perennial crop in the Mediterranean region. Yet, its origins are still under debate and the genetic bases of the phenotypic changes associated with its domestication are unknown. We generated RNA-sequencing data for 68 wild and cultivated olive trees to study the genetic diversity and structure both at the transcription and sequence levels. To localize putative genes or expression pathways targeted by artificial selection during domestication, we employed a two-step approach in which we identified differentially expressed genes and screened the transcriptome for signatures of selection. Our analyses support a major domestication event in the eastern part of the Mediterranean basin followed by dispersion towards the West and subsequent admixture with western wild olives. While we found large changes in gene expression when comparing cultivated and wild olives, we found no major signature of selection on coding variants and weak signals primarily affected transcription factors. Our results indicated that the domestication of olives resulted in only moderate genomic consequences and that the domestication syndrome is mainly related to changes in gene expression, consistent with its evolutionary history and life history traits.