Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20–40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

AbstractApplication of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20 to 40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence, quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

Rear-edge populations represent reservoirs of potentially unique genetic diversity but are particularly vulnerable to global changes. While continental-scale phylogeographic studies usually do not cover these populations, more focused local scale study of rear-edge populations should help better understand both past evolutionary history and its consequences for the persistence and conservation of these potentially unique populations. We studied molecular variation at 36 sequenced nuclear microsatellites in 11 rear-edge Quercus faginea and Q. canariensis populations across Algeria to shed light on taxonomic relationship, population past evolutionary history and recent demographic trajectory. We used descriptive approach and simulation-based inference to assess the information content and complementarity of linked microsatellite and flanking sequence variations. Genetic differentiation among populations classified into eight well-defined genetic clusters do not allow to unambiguously delineate two species. Instead, continuous level of genetic differentiation indicates interspecific gene flow or drift in isolation. Whereas the analysis of microsatellite variation allowed inferring recent interspecific gene flow, additional nucleotide variation in flanking sequences, by reducing homoplasy, pointed towards ancient interspecific gene flow followed by drift in isolation. The assessment of the weight of each polymorphism in the inference demonstrates the value of linked variation with contrasted mutational mechanisms and rates to refine historical demographic inference. Past population size decline inferred in some of these oak populations as well as low contemporary effective population size for most populations is a concern for the persistence of these populations of high evolutionary significance and conservation value.

The Portuguese Oak in Algeria (Quercus faginea, Fagaceae): germination potential and variability of acorn and seedling morphology Background and aims – The Portuguese Oak, Quercus faginea Lam. (Fagaceae) forms part of iconic Mediterranean forest communities and is of great ecological and taxonomic value in Algeria. Unfortunately, the species is under threat and its populations remain poorly characterized. The aim of this paper is to study the variability of acorn morphology and germination potential of Q. faginea in Algeria, to examine variability in the growth of seedlings and to evaluate the influence of physical treatments on germination capacity and the subsequent development of seedlings at the intra- and inter-provenance levels.Methods – Mature acorns from four Q. faginea provenances of the species in Algeria were used for this work. In each provenance, ten trees were selected, and 100 acorns collected from each tree. Prior to germination, phenotypic seed traits were measured and a set of physical treatments was performed. The germinated seedlings were transplanted into polyethylene bags and several characteristics of plant growth were measured.Key results – The comparative analysis on acorn morphology and germination features revealed heavier acorns in the Baloul provenance and smaller and lighter leaves in the Chelia provenance, in comparison with other provenances. Germination features and seedling growth showed that the size of the acorns had no influence on seedling development.Conclusions – Our results show a morphological variability of leaves and acorns of the Portuguese Oak at the inter-provenance level. This diversity is probably a consequence of genetic variation and / or variable environmental conditions of the provenances. The study of the germination and seedling development reveals a considerable potential for germination and growth. Given all the factors that threaten the persistence of Portuguese Oak in Algeria, we recommend conservation action for the species through reforestation, ex situ plantations and protective fences to avoid genuine and irreversible population regression, which would most likely lead to its disappearance.   Contexte et buts visés – Le chêne faginé, Quercus faginea Lam. (Fagaceae), représente un intérêt patrimonial, écologique et taxonomique remarquable en Algérie, mais malheureusement l’espèce est menacée et peu étudiée. L’objectif de ce travail est d’étudier la variabilité morphologique et le potentiel germinatif des glands de Q. faginea en Algérie, d’examiner la variabilité de la croissance des semis et d’évaluer l’influence des traitements physiques sur le potentiel germinatif et le développement ultérieur des semis, tant au niveau intra- qu’inter-stationnel.Méthodes – Des glands mûrs de chêne faginé récoltés sur quatre stations de l’espèce en Algérie ont été utilisés. Sur chaque station, dix arbres ont été sélectionnés, représentés par 100 glands chacun. Une analyse de traits phénotypiques a été réalisée sur les glands ; ensuite un ensemble de traitements physiques ont été effectués afin de tester leur effet sur la germination. Les semis issus de la germination ont été transplantés dans des sachets en polyéthylène et plusieurs caractères de croissance ont été mesurés. Résultats-clés – L’étude comparative morphologique et germinative révèle des glands plus longs et plus lourds à la station de Baloul, et des feuilles plus petites et plus légères à la station de Chélia, en comparaison avec les autres stations. Un effet significatif des traitements sur la germination a été montré dans toutes les stations. La taille des glands n’a aucune influence sur le développement des jeunes semis.Conclusions – Nos résultats montrent une variabilité morphologique inter-stationnelle des feuilles et des glands du chêne faginé. Cette diversité est probablement la conséquence de variations génétiques et/ou des conditions environnementales des stations. L’étude de la germination et de l’évolution de plants révèle un potentiel germinatif et de croissance considérables. Compte tenu de l’ensemble des facteurs qui menacent la pérennité du chêne faginé en Algérie, nous recommandons des actions pour la conservation de l’espèce telles que des reboisements, des plantations ex situ et des clôtures de protection pour éviter une régression réelle et irréversible des peuplements qui conduirait très probablement à sa disparition.

La systématique de Quercus faginea Lam. (s.l) (Fagaceae) reste encore indéfinie, d’autant plus qu’elle s’hybride avec d’autres espèces et qu’elle est polymorphe. Quatre sous-espèces ont été identifiées et présentement reconnues en péninsule ibérique et au Maroc, or, les données disponibles ne permettent pas de trancher entre les taxons présents en Algérie. Pour remédier au manque de données nécessaires, une analyse morphologique et taxonomique de treize populations du complexe dans le pays a été effectuée. Dans chacune, vingt feuilles et vingt bourgeons sur dix arbres matures ont été prélevés pour une description détaillée de 23 traits morphologiques. L’ACM (analyse des correspondances multiples) et la CAH (classification ascendante hiérarchique) ont été utilisées pour analyser les données et identifier des groupes homogènes. Une variabilité significative intra et inter-populationnelle est remarquable, avec une taille des feuilles qui tend à se diminuer en allant de l’est vers l’ouest et du nord au sud du pays. Cette variabilité ne permet pas, à elle seule, de différentier entre les populations étudiées, en revanche, les traits liés à la forme du limbe et aux types du tomentum sur la feuille s’avèrent plus utiles. Cette étude confirme que le complexe Q. faginea Lam. est représenté en Algérie par deux espèces : Quercus canariensis Willd. et Q. faginea Lam., lui-même représenté par deux taxons, subsp. faginea et subsp. broteroi. The systematics of Quercus faginea Lam. (s.l) (Fagaceae) remains obscure, as it tends to crossbreed with other species and show signs of polymorphism. Four subspecies were identified and currently recognized in the Iberian Peninsula and Morocco. However, the literature to date does not allow the distinction between the taxa present in Algeria. To remedy the lack of required data, morphological and taxonomic analysis of thirteen tree populations has been carried out in the country. In the process, twenty leaves and twenty buds from ten mature trees were collected at each population to produce a more detailed description of 23 particular morphological traits. MCA (multiple correspondence analysis) and AHC (agglomerative hierarchical clustering) were used to analyze the data and to identify homogeneous groups. The results obtained show high intra and inter-population variability, with leaf size diminishing from east to west and from north to south. Though the findings were remarkable, they alone could not be used to differentiate between populations under examination; the traits relating to limb shape and indumentum type on the leaf prove to be more useful. It is clear from this study that the Q. faginea Lam. group in Algeria is represented by two species: Quercus canariensis Willd. and Q. faginea Lam., the latter being represented by two taxa: subsp. faginea and subsp. broteroi