Erwan Guichoux

Species relative abundance and direction of introgression in oaks

Successful hybridisation and subsequent introgression lead to the transfer of genetic material across species boundaries. In this process, species relative abundance can play a significant role. If one species is less abundant than the other, its females will receive many heterospecific gametes, increasing mate‐recognition errors and thus hybridisation rate. Moreover, first‐generation hybrids will also more likely mate with the more abundant species, leading to asymmetric introgression. These predictions have important fundamental consequences, especially during biological invasions or when a rare species threatened by extinction is surrounded by individuals from a related species. However, experimental tests in nature of the importance of the relative abundance of each species on hybridisation dynamics remain scarce. We assess here the impact of species relative abundance on hybridisation dynamics among four species from the European white oak species complex. A total of 2107 oak trees were genotyped at 10 microsatellite markers and Bayesian clustering methods were used to identify reference trees of each species. We then used these reference trees to simulate purebred and hybrid genotypes to determine optimal threshold for genetic assignment. With this approach, we found widespread evidence of hybridisation between all studied oak species, with high occurrence of hybrids, varying from 11% to 31% according to stand and sampling strategies. This finding suggests that hybridisation is a common phenomenon that plays a significant role in evolution of this oak species complex. In addition, we demonstrate a strong impact of species abundance on both hybridisation rate and introgression directionality.

Two highly validated multiplexes (12‐plex and 8‐plex) for species delimitation and parentage analysis in oaks (Quercus spp.)

Multiplex PCR is a fast and cost‐effective technique allowing increased genotyping throughput of microsatellites. We developed two multiplexes for Quercus petraea and Q. robur, a 12‐plex of EST‐SSRs (eSSRs) and an 8‐plex of genomic SSRs (gSSRs). We studied the origin of allele calling errors at the human reader and software levels. We showed that the robustness of allele identification can be improved by binning on raw peak sizes prior to genetic data analysis. We checked through simulation the power of these markers for species delimitation and hybrid detection. The resolution achieved with all 20 markers was greatly improved compared to that of previous studies based on a subset of the markers. Preliminary PCR tests suggest that these multiplexes might be useful to study other oak species as well. The strategy used for multiplex microsatellite development (from PCR conditions to the definition of allele calling rules) should be broadly applicable.

Biogeography of Oenococcus oeni Reveals Distinctive but Nonspecific Populations in Wine-Producing Regions

ABSTRACT Understanding the mechanisms behind the typicity of regional wines inevitably brings attention to microorganisms associated with their production. Oenococcus oeni is the main bacterial species involved in wine and cider making. It develops after the yeast-driven alcoholic fermentation and performs the malolactic fermentation, which improves the taste and aromatic complexity of most wines. Here, we have evaluated the diversity and specificity of O. oeni strains in six regions. A total of 235 wines and ciders were collected during spontaneous malolactic fermentations and used to isolate 3,212 bacterial colonies. They were typed by multilocus variable analysis, which disclosed a total of 514 O. oeni strains. Their phylogenetic relationships were evaluated by a second typing method based on single nucleotide polymorphism (SNP) analysis. Taken together, the results indicate that each region holds a high diversity of strains that constitute a unique population. However, strains present in each region belong to diverse phylogenetic groups, and the same groups can be detected in different regions, indicating that strains are not genetically adapted to regions. In contrast, greater strain identity was seen for cider, white wine, or red wine of Burgundy, suggesting that genetic adaptation to these products occurred. IMPORTANCE This study reports the isolation, genotyping, and geographic distribution analysis of the largest collection of O. oeni strains performed to date. It reveals that there is very high diversity of strains in each region, the majority of them being detected in a single region. The study also reports the development of an SNP genotyping method that is useful for analyzing the distribution of O. oeni phylogroups. The results show that strains are not genetically adapted to regions but to specific types of wines. They reveal new phylogroups of strains, particularly two phylogroups associated with white wines and red wines of Burgundy. Taken together, the results shed light on the diversity and specificity of wild strains of O. oeni, which is crucial for understanding their real contribution to the unique properties of wines.

Oak genome reveals facets of long lifespan

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.Oaks can live hundreds of years. Comparative genomics using a high-quality genome sequence provides new insights that may explain tree longevity. Samples from branches and corresponding acorns also help quantify heritable somatic mutations.

Dealing with paralogy in RADseq data: in silico detection and single nucleotide polymorphism validation in Robinia pseudoacacia L.

Abstract The RADseq technology allows researchers to efficiently develop thousands of polymorphic loci across multiple individuals with little or no prior information on the genome. However, many questions remain about the biases inherent to this technology. Notably, sequence misalignments arising from paralogy may affect the development of single nucleotide polymorphism (SNP) markers and the estimation of genetic diversity. We evaluated the impact of putative paralog loci on genetic diversity estimation during the development of SNPs from a RADseq dataset for the nonmodel tree species Robinia pseudoacacia L. We sequenced nine genotypes and analyzed the frequency of putative paralogous RAD loci as a function of both the depth of coverage and the mismatch threshold allowed between loci. Putative paralogy was detected in a very variable number of loci, from 1% to more than 20%, with the depth of coverage having a major influence on the result. Putative paralogy artificially increased the observed degree of polymorphism and resulting estimates of diversity. The choice of the depth of coverage also affected diversity estimation and SNP validation: A low threshold decreased the chances of detecting minor alleles while a high threshold increased allelic dropout. SNP validation was better for the low threshold (4×) than for the high threshold (18×) we tested. Using the strategy developed here, we were able to validate more than 80% of the SNPs tested by means of individual genotyping, resulting in a readily usable set of 330 SNPs, suitable for use in population genetics applications.

Development of nuclear SNP markers for genetic tracking of Iroko, Milicia excelsa and Milicia regia

Restriction associated DNA sequencing was conducted on two genetically independent individuals of Iroko, Milicia excelsa, for the discovery of nuclear SNPs. Ninety-four samples, well-distributed over the natural range and including timber DNA, were screened at 138 loci on a MassARRAY iPLEX system. Amplification success was high and 77 loci were selected to design a set of markers for genetic timber tracking purposes.

A nuclear DNA barcode for eastern North American oaks and application to a study of hybridization in an Arboretum setting

Abstract DNA barcoding has proved difficult in a number of woody plant genera, including the ecologically important oak genus Quercus. In this study, we utilized restrictionsite‐associated DNA sequencing (RAD‐seq) to develop an economical single nucleotide polymorphism (SNP) DNA barcoding system that suffices to distinguish eight common, sympatric eastern North American white oak species. Two de novo clustering pipelines, PyRAD and Stacks, were used in combination with postclustering bioinformatic tools to generate a list of 291 potential SNPs, 80 of which were included in a barcoding toolkit that is easily implemented using MassARRAY mass spectrometry technology. As a proof‐of‐concept, we used the genotyping toolkit to infer potential hybridization between North American white oaks transplanted outside of their native range (Q. michauxii, Q. montana, Q muehlenbergii/Q. prinoides, and Q. stellata) into a horticultural collection surrounded by natural forests of locally native trees (Q. alba and Q. macrocarpa) in the living collection at The Morton Arboretum (Lisle, IL, USA). Phylogenetic and clustering analyses suggested low rates of hybridization between cultivated and native species, with the exception of one Q. michauxii mother tree, the acorns of which exhibited high admixture from either Q. alba or Q. stellata and Q. macrocarpa, and a hybrid between Q. stellata that appears to have backcrossed almost exclusively to Q. alba. Together, RAD‐seq and MassARRAY technologies allow for efficient development and implementation of a multispecies barcode for one of the more challenging forest tree genera.

Development of nuclear SNP markers for the timber tracking of the African tree species Sapelli, Entandrophragma cylindricum

We describe the development of new nuclear SNP markers for the genetic timber tracking of the geographical origin of Sapelli, Entandrophragma cylindricum (Meliaceae). Restriction associated DNA sequencing (RADseq) of two reference individuals yielded 1131 putative SNPs. Among those, 131 were selected to design four MassARRAY multiplexes and screened at 178 individuals. Seventy-two loci were selected for further use in genetic tracking.

Genetic divergence within the monotypic tree genus Platycarya (Juglandaceae) and its implications for species’ past dynamics in subtropical China

Subtropical East Asia harbours a large plant diversity that is often attributed to allopatric speciation in this topographically complex region characterized by a relative climate stability. Here, we use observations of Platycarya, a widespread subtropical Asian tree genus, to explore the consequences of past climate stability on species’ evolutionary history in subtropical China. This genus has a controversial taxonomy: while it is now prevailingly treated as monotypic, two species have been originally described, Platycarya strobilacea and P. longipes. Previous information from species distribution models, fossil pollen data and genetic data based on chloroplast DNA (cpDNA) were integrated with newly obtained genetic data from the two putative species. We used both cpDNA (psbA-trnH and trnL-F intergenic spacers, including a partial trnL gene sequence) and nuclear markers. The latter included sequences of the internal transcribed spacer region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA and random genomic single nucleotide polymorphisms. Using these nuclear genetic markers, we found interspecific genetic divergence fitting with the ‘two species’ scenario and geographically structured intraspecific variation. Using cpDNA markers, we also found geographically structured intraspecific variation. Despite deep inter- and intraspecific genetic divergence, we detected genetic admixture in southwest China. Overall, our findings of genetic divergence within Platycarya support the hypothesis of allopatric speciation. However, episodes of population interconnection were identified, at least in southwest China, suggesting that the genus has had a dynamic population history.

The use and domestication of Theobroma cacao during the mid-Holocene in the upper Amazon

Cacao (Theobroma cacao L.) is an important economic crop, yet studies of its domestication history and early uses are limited. Traditionally, cacao is thought to have been first domesticated in Mesoamerica. However, genomic research shows that T. cacao’s greatest diversity is in the upper Amazon region of northwest South America, pointing to this region as its centre of origin. Here, we report cacao use identified by three independent lines of archaeological evidence—cacao starch grains, absorbed theobromine residues and ancient DNA—dating from approximately 5,300 years ago recovered from the Santa Ana-La Florida (SALF) site in southeast Ecuador. To our knowledge, these findings constitute the earliest evidence of T. cacao use in the Americas and the first unequivocal archaeological example of its pre-Columbian use in South America. They also reveal the upper Amazon region as the oldest centre of cacao domestication yet identified.Evidence from starch grains, theobromine residues and ancient DNA demonstrate cacao use in the upper Amazon circa 5,300 years ago. This is earlier than previous evidence for cacao domestication in Mesoamerica.