A user-friendly method to get automated pollen analysis from environmental samples.

Automated pollen analysis is not yet efficient on environmental samples containing many pollen taxa and debris, which are typical in most pollen-based studies. Contrary to classification, detection remains overlooked although it is the first step from which errors can propagate. Here, we investigated a simple but efficient method to automate pollen detection for environmental samples, optimizing workload and performance. We applied the YOLOv5 algorithm on samples containing debris and c. 40 Mediterranean plant taxa, designed and tested several strategies for annotation, and analyzed variation in detection errors. About 5% of pollen grains were left undetected, while 5% of debris were falsely detected as pollen. Undetected pollen was mainly in poor-quality images, or of rare and irregular morphology. Pollen detection remained effective when applied to samples never seen by the algorithm, and was not improved by spending time to provide taxonomic details. Pollen detection of a single model taxon reduced annotation workload, but was only efficient for morphologically differentiated taxa. We offer guidelines to plant scientists to analyze automatically any pollen sample, providing sound criteria to apply for detection while using common and user-friendly tools. Our method contributes to enhance the efficiency and replicability of pollen-based studies.

Disentangling the origins of viticulture in the western Mediterranean.

We present direct evidence of early grape domestication in southern Italy via a multidisciplinary study of pip assemblage from one site, shedding new light on the spread of viticulture in the western Mediterranean during the Bronze Age. This consist of 55 waterlogged pips from Grotta di Pertosa, a Middle Bronze Age settlement in the south of the Italian peninsula. Direct radiocarbon dating of pips was carried out, confirming the chronological consistency of the samples with their archaeological contexts (ca. 1450-1200 BCE). The extraordinary state of conservation of the sample allowed to perform geometric morphometric (GMM) and paleogenetic analyses (aDNA) at the same time. The combination of the two methods has irrefutably shown the presence of domestic grapevines, together with wild ones, in Southern Italy during the Middle/Late Bronze Age. The results converge towards an oriental origin of the domestic grapes, most likely arriving from the Aegean area through the Mycenaeans. A parent/offspring kinship was also recognised between a domestic/wild hybrid individual and a domestic clonal group. This data point out a little known aspect of the diffusion of the first viticulture in Italy, and therefore in the western Mediterranean, which involved the hybridization between imported domestic varieties with, likely local, wild vines.

Barley systematics and taxonomy foreseen by seed morphometric variation.

Since its Neolithic domestication in the Fertile Crescent, barley has spread to all continents and represents a major cereal in many modern agrarian systems. Current barley diversity includes thousands of varieties divided into four main categories corresponding to 2-row and 6-row subspecies and naked and hulled types, each of them with winter and spring varieties. This diversity is associated to different uses and allow cultivation in diverse environments. We used a large dataset of 58 varieties of French origin, (1) to assess the taxonomic signal in barley grain measurements comparing 2-row and 6-row subspecies, and naked and hulled types; (2) to test the impact of the sowing period and interannual variation on the grains size and shape; (3) to investigate the existence of morphological differences between winter and spring types; and finally (4) to contrast the relationship between the morphometric and genetic proximity. Size and shape of 1980 modern barley caryopses were quantified through elliptic Fourier Transforms and traditional size measurements. Our results indicate that barley grains record morphological diversity of the ear (89.3% classification accuracy between 2-row/6-row subspecies; 85.2% between hulled and naked type), sowing time of the grains (from 65.6% to 73.3% within barley groups), and environmental conditions during its cultivation and varietal diversity. This study opens perspectives for studying archaeological barley seeds and tracing the barley diversity and evolution since the Neolithic.

Agro-morphological traits assessment of Tunisian male date palms (Phœnix dactylifera L.) for preservation and sustainable utilization of local germplasm.

Date palm (Phœnix dactylifera L.) like other crop species in the arid Mediterranean region is being threatened by genetic erosion and climate change. Therefore, the understanding and assessment of the diversity extent of this species is a primary requisite for preserving these crop resources. This study was designed to quantify the potential of Tunisian male date palms using a set of agro-morphological characteristics i.e. flowering traits, inflorescence morphology and pollen quality. The flowering time traits exhibited a trend of precocious phenotype at emergence spathe trait and the dominance of the full-season phenotype at the opening date. At inflorescence morphology, all observed traits expressed wide ranges which reflected the broad variability of the evaluated male genotypes. Significant difference was recorded for the majority of the examined traits with a high significant variation in the tree quantitative traits: Spathe Total Length, Spathe Maximum Width and Length to the brunched part. Pollen viability ranged from 51.10% to 98.75% while the germination rate was between 0.90% and 70.50%. Our phenotypic investigation has allowed the identification of males with desirable agronomic traits which have been genotyped using 18 nuclear SSR markers and a chloroplast minisatellite for preservation and effective utilization purposes.

Pip shape echoes grapevine domestication history.

The pip, as the most common grapevine archaeological remain, is extensively used to document past viticulture dynamics. This paper uses state of the art morphological analyses to analyse the largest reference collection of modern pips to date, representative of the present-day diversity of the domesticated grapevine from Western Eurasia. We tested for a costructure between the form of the modern pips and the: destination use (table/wine), geographical origins, and populational labels obtained through two molecular approaches. Significant structuring is demonstrated for each of these cofactors and for the first time it is possible to infer properties of varieties without going through the parallel with modern varieties. These results provide a unique tool that can be applied to archaeological pips in order to reconstruct the spatio-temporal dynamics of grape diversity on a large scale and to better understand viticulture history. The models obtained were then used to infer the affiliations with archaeobotanical remains recovered in Mas de Vignoles XIV (Nîmes, France). The results show a twofold shift between the Late Iron Age and the Middle Ages, from table to wine grape varieties and from eastern to western origins which correlates with previous palaeogenomic results.

Resisting Aridification: Adaptation of Sap Conduction Performance in Moroccan Wild Olive Subspecies Distributed Over an Aridity Gradient.

In the current context of global change, the increasing frequency and the length of drought periods are testing the resistance capacities of plants of dry habitats. However, although the adaptation of plants to drought has been widely studied, the anatomical features of wood influencing the functional responses of plants to drought are still lacking at the intraspecific level, especially for species with a wide geographical distribution. As a result, we have studied the variation of wood anatomical traits related to sap conduction (i.e., vessel surface area, vessel density, and number of vessels joined by radial file) in two wild olive subspecies distributed in Morocco (i.e., Olea europaea subsp. europaea. var. sylvestris and Olea europaea subsp. maroccana), in relation to various drought conditions. This functional study, based on wood trait measurements of 351 samples from 130 trees and 13 populations, explores potential sap conduction in relation to environmental parameters and as a result, strategies to resist water stress. We found that (1) branch diameter (BD) captured 78% of total wood trait variation, (2) vessel size (SVS) expressed 32% of intraspecific variation according to cambium age, and (3) the positive relationship between SVS and BD could be explained by climate type, vegetation cover changes, and therefore available water resources. Taking into consideration the diameter of the branch as the main factor of anatomical variation, established reaction norms (linear models) at the intrapopulation scale of vessel lumen area according to aridity show for the first time how the functioning of the cambium modulates and controls sap conduction, according to aridity and thus available water resources. They pinpoint the risks incurred by the wild olive tree in the perspective of a dramatic increase in aridity, in particular, the inability of the cambium to produce large enough vessels to efficiently transport sap and irrigate the leaves. Finally, this study opens new and interesting avenues for studying at a Mediterranean scale, the resistance and the vulnerability of wild forms and cultivated varieties of olive to heterogeneous and changing environmental conditions.

Understanding anatomical plasticity of Argan wood features at local geographical scale in ecological and archaeobotanical perspectives.

The emergence of the Argan tree as an agricultural, pastoral, cultural, economic and ecological keystone species in Southern Morocco is considered to be linked to the settlement of agropastoral communities that favored its expansion. Nevertheless, the use and exploitation of Argan tree is documented by both few medieval written sources and archaeobotanical studies, from a single location, Îgîlîz (Toughmart, Morocco), a famous medieval site of the Anti-Atlas Mountains. Therefore, data remain scarce regarding the type of Argan communities exploited at this period. In order to document this question, a quantitative eco-anatomical approach aiming to understand variations of wood characters involved in sap conduction and reserve storage, is developed from modern samples collected in the area of Îgîlîz. Results show that diameter of branches and environmental factors are the major parameters explaining plasticity of wood anatomical characters. Quantitative eco-anatomical features of Argan archaeological charcoal confronted to two statistical models, allow assessing both the diameter of the branches from which it derives and the agro-ecological conditions of tree growth and development. This preliminary study may be considered as a relevant and pioneering work for the understanding of the eco-history of the Argan tree, and of its use and exploitation during the past.

A morphometric approach to track opium poppy domestication.

Opium poppy (Papaver somniferum L. subsp. somniferum) was likely domesticated in the Western Mediterranean, where its putative wild ancestor is indigenous, and then spread to central and northern Europe. While opium poppy seeds are regularly identified in archaeobotanical studies, the absence of morphological criteria to distinguish the seeds of wild and domestic forms prevents the documentation of their respective historical and geographical occurrences and of the process of opium domestication as a whole. To fill this gap and better understand the status of this crop in the Neolithic, we combined seed outline analyses, namely elliptic Fourier transforms, with other morphometric descriptors to describe and identify Papaver setigerum, Papaver somniferum and other Papaver taxa. The combination of all measured parameters gives the most precise predictions for the identification of all seven taxa. We finally provide a case study on a Neolithic assemblage from a pile-dwelling site in Switzerland (Zurich-Parkhaus Opéra, ca. 3170 BC). Our results indicate the presence of mixed populations of domestic and wild seeds belonging to the P. somniferum group, suggesting that the plant was already in the process of domestication at the end of 4th millennium BC. Altogether, these results pave the way to understand the geography and history of the poppy domestication and its spread into Europe.

Seed morphology uncovers 1500 years of vine agrobiodiversity before the advent of the Champagne wine.

A crucial aspect of viticulture is finally unveiled as the historical dynamics of its agrobiodiversity are described in the Champagne region for the first time. Outline analyses were carried out to compare the morphology of archaeological grape seeds from Troyes and Reims (first c. AD to fifteenth c. AD) with that of a reference collection of modern seeds, including wild vines and traditional grape varieties, believed to be ancient and characteristic of the French vine heritage. This allows us to document the chronological dynamics of the use of the wild Vitis type and of the diversity of the varieties used, based on morphological disparity. After showing the existence of morphological types corresponding to geographical groups, we highlight a geochronological dynamic. Our results show that the wild type is used throughout the series, up to the Middle Ages. In addition, domestic forms, morphologically related to southern varietal groups, are very early involved in the Champagne grape agrodiversity. The groups corresponding to the typical grape varieties of today do not appear until the second millennium. These previously unsuspected dynamics are discussed in light of the social, societal and climatic changes documented for the period.

Eco-evo-devo implications and archaeobiological perspectives of trait covariance in fruits of wild and domesticated grapevines.

The phenotypic changes that occurred during the domestication and diversification of grapevine are well known, particularly changes in seed morphology, but the functional causes and consequences behind these variations are poorly understood. Wild and domesticate grapes differ, among others, in the form of their pips: wild grapes produce roundish pips with short stalks and cultivated varieties have more elongated pips with longer stalks. Such variations of form are of first importance for archaeobotany since the pip form is, most often, the only remaining information in archaeological settings. This study aims to enlighten archaeobotanical record and grapevine pip development by better understanding how size and shape (co)variates between pip and berry in both wild and domesticated Vitis vinifera. The covariation of berry size, number of seeds per berry ("piposity"), pip size and pip shape were explored on 49 grapevine accessions sampled among Euro-Mediterranean traditional cultivars and wild grapevines. We show that for wild grapevine, the higher the piposity, the bigger the berry and the more elongated the pip. For both wild and domesticated grapevine, the longer is the pip, the more it has a "domesticated" shape. Consequences for archaeobotanical studies are tested and discussed, and these covariations allowed the inference of berry dimensions from archaeological pips from a Southern France Roman site. This systematic exploration sheds light on new aspects of pip-berry relationship, in both size and shape, on grapevine eco-evo-devo changes during domestication, and invites to explore further the functional ecology of grapevine pip and berry and notably the impact of cultivation practices and human selection on grapevine morphology.

On the necessity of combining ethnobotany and genetics to assess agrobiodiversity and its evolution in crops: A case study on date palms (Phoenix dactylifera L.) in Siwa Oasis, Egypt.

Crop diversity is shaped by biological and social processes interacting at different spatiotemporal scales. Here, we combined population genetics and ethnobotany to investigate date palm (Phoenix dactylifera L.) diversity in Siwa Oasis, Egypt. Based on interviews with farmers and observation of practices in the field, we collected 149 date palms from Siwa Oasis and 27 uncultivated date palms from abandoned oases in the surrounding desert. Using genotyping data from 18 nuclear and plastid microsatellite loci, we confirmed that some named types each constitute a clonal line, that is, a true-to-type cultivar. We also found that others are collections of clonal lines, that is, ethnovarieties, or even unrelated samples, that is, local categories. This alters current assessments of agrobiodiversity, which are visibly underestimated, and uncovers the impact of low-intensity, but highly effective, farming practices on biodiversity. These hardly observable practices, hypothesized by ethnographic survey and confirmed by genetic analysis, are enabled by the way Isiwans conceive and classify living beings in their oasis, which do not quite match the way biologists do: a classic disparity of etic versus. emic categorizations. In addition, we established that Siwa date palms represent a unique and highly diverse genetic cluster, rather than a subset of North African and Middle Eastern palm diversity. As previously shown, North African date palms display evidence of introgression by the wild relative Phoenix theophrasti, and we found that the uncultivated date palms from the abandoned oases share even more alleles with this species than cultivated palms in this region. The study of Siwa date palms could hence be a key to the understanding of date palm diversification in North Africa. Integration of ethnography and population genetics promoted the understanding of the interplay between diversity management in the oasis (short-time scale), and the origins and dynamic of diversity through domestication and diversification (long-time scale).

Origins and insights into the historic Judean date palm based on genetic analysis of germinated ancient seeds and morphometric studies.

Germination of 2000-year-old seeds of Phoenix dactylifera from Judean desert archaeological sites provides a unique opportunity to study the Judean date palm, described in antiquity for the quality, size, and medicinal properties of its fruit, but lost for centuries. Microsatellite genotyping of germinated seeds indicates that exchanges of genetic material occurred between the Middle East (eastern) and North Africa (western) date palm gene pools, with older seeds exhibiting a more eastern nuclear genome on a gradient from east to west of genetic contributions. Ancient seeds were significantly longer and wider than modern varieties, supporting historical records of the large size of the Judean date. These findings, in accord with the region's location between east and west date palm gene pools, suggest that sophisticated agricultural practices may have contributed to the Judean date's historical reputation. Given its exceptional storage potentialities, the date palm is a remarkable model for seed longevity research.

The Discovery of Wild Date Palms in Oman Reveals a Complex Domestication History Involving Centers in the Middle East and Africa.

For many crops, wild relatives constitute an extraordinary resource for cultivar improvement [1, 2] and also help to better understand the history of their domestication [3]. However, the wild ancestor species of several perennial crops have not yet been identified. Perennial crops generally present a weak domestication syndrome allowing cultivated individuals to establish feral populations difficult to distinguish from truly wild populations, and there is frequently ongoing gene flow between wild relatives and the crop that might erode most genetic differences [4]. Here we report the discovery of populations of the wild ancestor species of the date palm (Phoenix dactylifera L.), one of the oldest and most important cultivated fruit plants in hot and arid regions of the Old World. We discovered these wild individuals in remote and isolated mountainous locations of Oman. They are genetically more diverse than and distinct from a representative sample of Middle Eastern cultivated date palms and exhibit rounded seed shapes resembling those of a close sister species and archeological samples, but not modern cultivars. Whole-genome sequencing of several wild and cultivated individuals revealed a complex domestication history involving the contribution of at least two wild sources to African cultivated date palms. The discovery of wild date palms offers a unique chance to further elucidate the history of this iconic crop that has constituted the cornerstone of traditional oasis polyculture systems for several thousand years [5].

The Domestication Syndrome in Phoenix dactylifera Seeds: Toward the Identification of Wild Date Palm Populations.

Investigating crop origins is a priority to understand the evolution of plants under domestication, develop strategies for conservation and valorization of agrobiodiversity and acquire fundamental knowledge for cultivar improvement. The date palm (Phoenix dactylifera L.) belongs to the genus Phoenix, which comprises 14 species morphologically very close, sometimes hardly distinguishable. It has been cultivated for millennia in the Middle East and in North Africa and constitutes the keystone of oasis agriculture. Yet, its origins remain poorly understood as no wild populations are identified. Uncultivated populations have been described but they might represent feral, i.e. formerly cultivated, abandoned forms rather than truly wild populations. In this context, this study based on morphometrics applied to 1625 Phoenix seeds aims to (1) differentiate Phoenix species and (2) depict the domestication syndrome observed in cultivated date palm seeds using other Phoenix species as a "wild" reference. This will help discriminate truly wild from feral forms, thus providing new insights into the evolutionary history of this species. Seed size was evaluated using four parameters: length, width, thickness and dorsal view surface. Seed shape was quantified using outline analyses based on the Elliptic Fourier Transform method. The size and shape of seeds allowed an accurate differentiation of Phoenix species. The cultivated date palm shows distinctive size and shape features, compared to other Phoenix species: seeds are longer and elongated. This morphological shift may be interpreted as a domestication syndrome, resulting from the long-term history of cultivation, selection and human-mediated dispersion. Based on seed attributes, some uncultivated date palms from Oman may be identified as wild. This opens new prospects regarding the possible existence and characterization of relict wild populations and consequently for the understanding of the date palm origins. Finally, we here describe a pipeline for the identification of the domestication syndrome in seeds that could be used in other crops.

A small XY chromosomal region explains sex determination in wild dioecious V. vinifera and the reversal to hermaphroditism in domesticated grapevines.

BACKGROUND: In Vitis vinifera L., domestication induced a dramatic change in flower morphology: the wild sylvestris subspecies is dioecious while hermaphroditism is largely predominant in the domesticated subsp. V. v. vinifera. The characterisation of polymorphisms in genes underlying the sex-determining chromosomal region may help clarify the history of domestication in grapevine and the evolution of sex chromosomes in plants. In the genus Vitis, sex determination is putatively controlled by one major locus with three alleles, male M, hermaphrodite H and female F, with an allelic dominance M > H > F. Previous genetic studies located the sex locus on chromosome 2. We used DNA polymorphisms of geographically diverse V. vinifera genotypes to confirm the position of this locus, to characterise the genetic diversity and traces of selection in candidate genes, and to explore the origin of hermaphroditism. RESULTS: In V. v. sylvestris, a sex-determining region of 154.8 kb, also present in other Vitis species, spans less than 1% of chromosome 2. It displays haplotype diversity, linkage disequilibrium and differentiation that typically correspond to a small XY sex-determining region with XY males and XX females. In male alleles, traces of purifying selection were found for a trehalose phosphatase, an exostosin and a WRKY transcription factor, with strikingly low polymorphism levels between distant geographic regions. Both diversity and network analysis revealed that H alleles are more closely related to M than to F alleles. CONCLUSIONS: Hermaphrodite alleles appear to derive from male alleles of wild grapevines, with successive recombination events allowing import of diversity from the X into the Y chromosomal region and slowing down the expansion of the region into a full heteromorphic chromosome. Our data are consistent with multiple domestication events and show traces of introgression from other Asian Vitis species into the cultivated grapevine gene pool.

Bioarchaeological insights into the process of domestication of grapevine (Vitis vinifera L.) during Roman times in Southern France.

Grapevine (Vitis vinifera), one of the most important fruit species in the Classical Mediterranean world, is thought to have been domesticated first in South-Western Asia, during the Neolithic. However, the domestication process remains largely unknown. Crucial unanswered questions concern the duration of the process (rapid or slow?) and the related geographical area (single or multiple-origins?). Seeds from domesticated grapevine and from its wild ancestor are reported to differ according to shape. Our work aims, first, to confirm this difference and secondly to identify the extent of domestication in the grapes cultivated by Romans in Southern France during the period 50 BCE-500 CE. We had the opportunity to analyze uncharred waterlogged grape pips from 17 archaeological sites. Based on an extended reference sample of modern wild grapevines and cultivars our work shows that both subspecies can be discriminated using simple measurements. The elongation gradient of the pip's body and stalk may be regarded as an indicator of the strength of the selection pressures undergone by domesticated grapes. Grapevines cultivated during the Roman period included a mix of morphotypes comprising wild, intermediate and moderately selected domesticated forms. Our data point to a relative shift towards more selected types during the Roman period. Domestication of the grapevine appears to have been a slow process. This could result from the recurrent incorporation into cultivation of plants originating from sexual reproduction, when grape cultivation essentially relies on vegetative propagation.

Evolution and history of grapevine (Vitis vinifera) under domestication: new morphometric perspectives to understand seed domestication syndrome and reveal origins of ancient European cultivars.

BACKGROUND AND AIMS: In spite of the abundance of archaeological, bio-archaeological, historical and genetic data, the origins, historical biogeography, identity of ancient grapevine cultivars and mechanisms of domestication are still largely unknown. Here, analysis of variation in seed morphology aims to provide accurate criteria for the discrimination between wild grapes and modern cultivars and to understand changes in functional traits in relation to the domestication process. This approach is also used to quantify the phenotypic diversity in the wild and cultivated compartments and to provide a starting point for comparing well-preserved archaeological material, in order to elucidate the history of grapevine varieties. METHODS: Geometrical analysis (elliptic Fourier transform method) was applied to grapevine seed outlines from modern wild individuals, cultivars and well-preserved archaeological material from southern France, dating back to the first to second centuries. KEY RESULTS AND CONCLUSIONS: Significant relationships between seed shape and taxonomic status, geographical origin (country or region) of accessions and parentage of varieties are highlighted, as previously noted based on genetic approaches. The combination of the analysis of modern reference material and well-preserved archaeological seeds provides original data about the history of ancient cultivated forms, some of them morphologically close to the current 'Clairette' and 'Mondeuse blanche' cultivars. Archaeobiological records seem to confirm the complexity of human contact, exchanges and migrations which spread grapevine cultivation in Europe and in Mediterranean areas, and argue in favour of the existence of local domestication in the Languedoc (southern France) region during Antiquity.