Estimation of contemporary effective population size in plant populations: Limitations of genomic datasets.

Effective population size (N e) is a pivotal evolutionary parameter with crucial implications in conservation practice and policy. Genetic methods to estimate N e have been preferred over demographic methods because they rely on genetic data rather than time-consuming ecological monitoring. Methods based on linkage disequilibrium (LD), in particular, have become popular in conservation as they require a single sampling and provide estimates that refer to recent generations. A software program based on the LD method, GONE, looks particularly promising to estimate contemporary and recent-historical N e (up to 200 generations in the past). Genomic datasets from non-model species, especially plants, may present some constraints to the use of GONE, as linkage maps and reference genomes are seldom available, and SNP genotyping is usually based on reduced-representation methods. In this study, we use empirical datasets from four plant species to explore the limitations of plant genomic datasets when estimating N e using the algorithm implemented in GONE, in addition to exploring some typical biological limitations that may affect N e estimation using the LD method, such as the occurrence of population structure. We show how accuracy and precision of N e estimates potentially change with the following factors: occurrence of missing data, limited number of SNPs/individuals sampled, and lack of information about the location of SNPs on chromosomes, with the latter producing a significant bias, previously unexplored with empirical data. We finally compare the N e estimates obtained with GONE for the last generations with the contemporary N e estimates obtained with the programs currentNe and NeEstimator.

Effective population size in a partially clonal plant is not predicted by the number of genetic individuals.

Estimating effective population size (N e) is important for theoretical and practical applications in evolutionary biology and conservation. Nevertheless, estimates of N e in organisms with complex life-history traits remain scarce because of the challenges associated with estimation methods. Partially clonal plants capable of both vegetative (clonal) growth and sexual reproduction are a common group of organisms for which the discrepancy between the apparent number of individuals (ramets) and the number of genetic individuals (genets) can be striking, and it is unclear how this discrepancy relates to N e. In this study, we analysed two populations of the orchid Cypripedium calceolus to understand how the rate of clonal versus sexual reproduction affected N e. We genotyped >1000 ramets at microsatellite and SNP loci, and estimated contemporary N e with the linkage disequilibrium method, starting from the theoretical expectation that variance in reproductive success among individuals caused by clonal reproduction and by constraints on sexual reproduction would lower N e. We considered factors potentially affecting our estimates, including different marker types and sampling strategies, and the influence of pseudoreplication in genomic data sets on N e confidence intervals. The magnitude of N e/N ramets and N e/N genets ratios we provide may be used as reference points for other species with similar life-history traits. Our findings demonstrate that N e in partially clonal plants cannot be predicted based on the number of genets generated by sexual reproduction, because demographic changes over time can strongly influence N e. This is especially relevant in species of conservation concern in which population declines may not be detected by only ascertaining the number of genets.

Microsatellites and petal morphology reveal new patterns of admixture in Orchis hybrid zones.

PREMISE: The genetic structure of hybrid zones provides insight into the potential for gene flow to occur between plant taxa. Four closely related European orchid species (Orchis anthropophora, O. militaris, O. purpurea, and O. simia) hybridize when they co-occur. We aimed to characterize patterns of hybridization in O. militaris-O. purpurea, O. purpurea-O. simia, and O. anthropophora-O. simia hybrid zones using molecular and morphological data. METHODS: We used 11 newly isolated nuclear microsatellites to genotype 695 individuals collected from seven hybrid zones and six allopatric parental populations in France. Geometric morphometric analysis was conducted using 15 labellum landmarks to capture the main aspects of petal shape. RESULTS: Backcrossing was asymmetric toward O. militaris in multiple O. militaris-O. purpurea hybrid zones. Hybrids in O. purpurea-O. simia and O. anthropophora-O. simia hybrid zones were largely limited to F1 and F2 generations, but further admixture had occurred. These patterns were reflected in labellum geometric morphometric data, which correlated strongly with nuclear microsatellite data in all three species combinations. CONCLUSIONS: The coexistence of parental and admixed individuals in these Orchis hybrid zones implies they are likely to be tension zones being maintained by a balance between gene flow into the hybrid zone and selection acting against admixed individuals. The pattern of admixture in the three species combinations suggests intrinsic selection acting on the hybrids is weaker in more closely related taxa.

Effective double-digest RAD sequencing and genotyping despite large genome size.

Obtaining informative data is the ambition of any genomic project, but in nonmodel species with very large genomes, pursuing such a goal requires surmounting a series of analytical challenges. Double-digest RAD sequencing is routinely used in nonmodel organisms and offers some control over the volume of data obtained. However, the volume of data recovered is not always an indication of the reliability of data sets, and quality checks are necessary to ensure that true and artefactual information is set apart. In the present study, we aim to fill the gap existing between the known applicability of RAD sequencing methods in plants with large genomes and the use of the retrieved loci for population genetic inference. By analysing two populations of Cypripedium calceolus, a nonmodel orchid species with a large genome size (1C ~ 31.6 Gbp), we provide a complete workflow from library preparation to bioinformatic filtering and inference of genetic diversity and differentiation. We show how filtering strategies to dismiss potentially misleading data need to be explored and adapted to data set-specific features. Moreover, we suggest that the occurrence of organellar sequences in libraries should not be neglected when planning the experiment and analysing the results. Finally, we explain how, in the absence of prior information about the genome of the species, seeking high standards of quality during library preparation and sequencing can provide an insurance against unpredicted technical or biological constraints.

Epipactis tremolsii Seed Diversity in Two Close but Extremely Different Populations: Just a Case of Intraspecific Variability?

Analysis of the seed morphology is a widely used approach in ecological and taxonomic studies. In this context, intraspecific variability with respect to seed morphology (size, weight, and density) was assessed in two close Epipactis tremolsii Pau. populations sharing the same ecological conditions, except for the soil pollution distinguishing one of them. Larger and heavier seeds were found in plants growing on the heavy metal polluted site, while no differences in seed density were detected between seeds produced by plants growing on the contaminated and the control site. Moreover, seed coats and embryos varying together in their dimensions were described in the control population, while coats varying in their size independently from embryos were described in plants growing on the polluted site. Seeds from the two studied populations significantly differed in several parameters suggesting that intraspecific seed variability occurred in the case study.

Lost and Found: Coffea stenophylla and C. affinis, the Forgotten Coffee Crop Species of West Africa.

Coffea arabica (Arabica) and C. canephora (robusta) almost entirely dominate global coffee production. Various challenges at the production (farm) level, including the increasing prevalence and severity of disease and pests and climate change, indicate that the coffee crop portfolio needs to be substantially diversified in order to ensure resilience and sustainability. In this study, we use a multidisciplinary approach (herbarium and literature review, fieldwork and DNA sequencing) to elucidate the identity, whereabouts, and potential attributes, of two poorly known coffee crop species: C. affinis and C. stenophylla. We show that despite widespread (albeit small-scale) use as a coffee crop species across Upper West Africa and further afield more than 100 years ago, these species are now extremely rare in the wild and are not being farmed. Fieldwork enabled us to rediscover C. stenophylla in Sierra Leone, which previously had not been recorded in the wild there since 1954. We confirm that C. stenophylla is an indigenous species in Guinea, Sierra Leone, and Ivory Coast. Coffea affinis was discovered in the wild in Sierra Leone for the first time, having previously been found only in Guinea and Ivory Coast. Prior to our rediscovery, C. affinis was last seen in the wild in 1941, although sampling of an unidentified herbarium specimen reveals that it was collected in Guinea-Conakry in 2015. DNA sequencing using plastid and ITS markers was used to: (1) confirm the identity of museum and field collected samples of C. stenophylla; (2) identify new accessions of C. affinis; (3) refute hybrid status for C. affinis; (4) identify accessions confused with C. affinis; (5) show that C. affinis and C. stenophylla are closely related, and possibly a single species; (6) substantiate the hybrid C. stenophylla × C. liberica; (7) demonstrate the use of plastid and nuclear markers as a simple means of identifying F1 and early-generation interspecific hybrids in Coffea; (8) infer that C. liberica is not monophyletic; and (9) show that hybridization is possible across all the major groups of key Africa Coffea species (Coffee Crop Wild Relative Priority Groups I and II). Coffea affinis and C. stenophylla may possess useful traits for coffee crop plant development, including taste differentiation, disease resistance, and climate resilience. These attributes would be best accessed via breeding programs, although the species may have niche-market potential via minimal domestication.

Conservation of the Threatened Species, Pulsatilla vulgaris Mill. (Pasqueflower), is Aided by Reproductive System and Polyploidy.

Population loss due to habitat disturbance is a major concern in biodiversity conservation. Here we investigate the genetic causes of the demographic decline observed in English populations of Pulsatilla vulgaris and the consequences for conservation. Using 10 nuclear microsatellite markers, we compare genetic variation in wild populations with restored and seed-regenerated populations (674 samples). Emergence of genetic structure and loss of allelic variation in natural populations are not as evident as expected from demographic trends. Restored populations show genetic variation comparable to their source populations and, in general, to the wild ones. Genetic homogeneity is observed in regeneration trials, although some alleles not captured in source populations are detected. We infer that polyploidy, longevity, and clonal reproduction have provided P. vulgaris with the standing genetic variation necessary to make the species resilient to the effects of demographic decline, suggesting that the use of multiple sources for reintroduction may be beneficial to mimic natural gene flow and the availability of multiple allele copies typical of polyploid species.

A molecular survey concerning the origin of Cyperus esculentus (Cyperaceae, Poales): two sides of the same coin (weed vs. crop).

BACKGROUND AND AIMS: Cyperus esculentus is widespread in tropical and temperate zones and is also present in cooler regions. It is used as a crop plant, but it also occurs in the wild and as a weed. As a consequence of its ecological plasticity, C. esculentus has remarkable variability, with several morphotypes. Four wild-type varieties are presently recognized, in addition to the cultivated form. This study investigates the phylogenetic position and biogeography of C. esculentus with the objective of contributing new data to increase the understanding of its evolutionary history. METHODS: Genealogical relationships among genotypes were inferred by using plastid DNA haplotype and nuclear ribosomal (nr) DNA ribotype sequences for 70 specimens either collected in the field or obtained from herbaria. Statistical dispersal-vicariance (S-DIVA) and Bayesian binary method (BBM) analyses were used to reconstruct the possible ancestral ranges of C. esculentus. In order to determine the age of C. esculentus, a time-measured phylogenetic analysis was performed. KEY RESULTS: Considerable variation between the chosen nuclear and plastid markers was detected (27 ribotypes vs. six haplotypes). No geographical structure was displayed among the haplotypes, but information on the dispersal pattern may be deduced. Two types of ribotypes were detected in nrDNA, with an evident geographical segregation into an Old World group and a polymorphic New World group. Both S-DIVA and BBM analyses suggested a biogeographical history in which dispersal from the African region has been crucial in shaping the current distribution pattern of C. esculentus. The most recent common ancestor between C. esculentus races has an age of 5.1 million years (95 % highest posterior density 2.5-10.2). CONCLUSIONS: The molecular analysis provides novel insights into the evolutionary history of C. esculentus. The results have various taxonomic and phylogenetic implications, including a hypothesis on the origin and phylogeography of this species, which probably originated in the late Cenozoic in Africa, and reached the Americas repeatedly, independently of Columbian exchanges.