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Low-coverage whole genome shotgun sequencing (or genome skimming) has emerged as a cost-effective method for acquiring genomic data in nonmodel organisms. This method provides sequence information on chloroplast genome (cpDNA), mitochondrial genome (mtDNA) and nuclear ribosomal regions (rDNA), which are over-represented within cells. However, numerous bioinformatic challenges remain to accurately and rapidly obtain such data in organisms with complex genomic structures and rearrangements, in particular for mtDNA in plants or for cpDNA in some plant families. Here we introduce the pipeline ORTHOSKIM, which performs in silico capture of targeted sequences from genomic and transcriptomic libraries without assembling whole organelle genomes. ORTHOSKIM proceeds in three steps: (i) global sequence assembly, (ii) mapping against reference sequences and (iii) target sequence extraction; importantly it also includes a range of quality control tests. Different modes are implemented to capture both coding and noncoding regions of cpDNA, mtDNA and rDNA sequences, along with predefined nuclear sequences (e.g., ultraconserved elements) or collections of single-copy orthologue genes. Moreover, aligned DNA matrices are produced for phylogenetic reconstructions, by performing multiple alignments of the captured sequences. While ORTHOSKIM is suitable for any eukaryote, a case study is presented here, using 114 genome-skimming libraries and four RNA sequencing libraries obtained for two plant families, Primulaceae and Ericaceae, the latter being a well-known problematic family for cpDNA assemblies. ORTHOSKIM recovered with high success rates cpDNA, mtDNA and rDNA sequences, well suited to accurately infer evolutionary relationships within these families. ORTHOSKIM is released under a GPL-3 licence and is available at: https://github.com/cpouchon/ORTHOSKIM.

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Climatic history and ecology are considered the most important factors moulding the spatial pattern of genetic diversity. With the advent of molecular markers, species' historical fates have been widely explored. However, it has remained speculative what role ecological factors have played in shaping spatial genetic structures within species. With an unprecedented, dense large-scale sampling and genome-screening, we tested how ecological factors have influenced the spatial genetic structures in Alpine plants. Here, we show that species growing on similar substrate types, largely determined by the nature of bedrock, displayed highly congruent spatial genetic structures. As the heterogeneous and disjunctive distribution of bedrock types in the Alps, decisive for refugial survival during the ice ages, is temporally stable, concerted post-glacial migration routes emerged. Our multispecies study demonstrates the relevance of particular ecological factors in shaping genetic patterns, which should be considered when modelling species projective distributions under climate change scenarios.

International audience; The alpine sedge Carex curvula ssp. curvula is a clonal, dominant graminoid found in the European Alps, the Carpathians, the Pyrenees and in some of the Balkan Mountains. It is a late-successional species of acidophilous alpine meadows that occurs on sites that were covered by ice during the last glacial maximum (LGM). By applying the amplified fragment length polymorphism (AFLP) fingerprinting and chloroplast DNA (cpDNA) sequencing, we attempted to identify the recolonization routes followed by the species after the last ice retreat. We relied on the genetic diversity of 37 populations covering the entire distributional range of the species. As a wind-pollinated species, C. curvula is characterized by a low level of population genetic differentiation. Nuclear and chloroplast data both support the hypothesis of a long-term separation of Eastern (Balkans and Carpathians) and Western (Alps and Pyrenees) lineages. In the Alps, a continuum of genetic depauperation from the east to the west may be related to a recolonization wave originating in the eastern-most parts of the chain, where the main glacial refugium was likely located. The Pyrenean populations are nested within the western Alps group and show a low level of genetic diversity, probably due to recent long-distance colonization. In contrast to the Alps, we found no phylogeographical structure in the Carpathians. The combination of reduced ice extension during the Würm period and the presence of large areas of siliceous substrate at suitable elevation suggest that in contrast to populations in the Alps, the species in the Carpathians underwent a local vertical migration rather than extinction and recolonization over long distance.

International audience; Aim The range of the subalpine species Hypochaeris uniflora covers the Alps, Carpathians and Sudetes Mountains. Whilst the genetic structure and post-glacial history of many high-mountain plant taxa of the Alps is relatively well documented, the Carpathian populations have often been neglected in phylogeographical studies. The aim of the present study is to compare the genetic variation of the species in two major European mountain systems - the Alps and the Carpathians. Locations Alps and Carpathians. Methods The genetic variation of 77 populations, each consisting of three plants, was studied using amplified fragment length polymorphism (AFLP). Results Neighbour joining and principal coordinate analyses revealed three well-supported phylogeographical groups of populations corresponding to three disjunct geographical regions - the Alps and the western and south-eastern Carpathians. Moreover, two further clusters could be distinguished within the latter mountain range, one consisting of populations from the eastern Carpathians and the second consisting of populations from the southern Carpathians. Populations from the Apuseni Mountains had an intermediate position between the eastern and southern Carpathians. The genetic clustering of populations into four groups was also supported by an analysis of molecular variance, which showed that most genetic variation (almost 46%) was found among these four groups. By far the highest within-population variation was found in the eastern Carpathians, followed by populations from the southern and western Carpathians. Generally, the populations from the Alps were considerably less variable and displayed substantially fewer region-diagnostic markers than those from the south-eastern Carpathians. Although no clear geographical structure was found within the Alps, based on neighbour joining or principal coordinate analyses, some trends were obvious: populations from the easternmost part were genetically more variable and, together with those from the south-western part, exhibited a higher proportion of rare AFLP fragments than populations in other areas. Moreover, the total number of AFLP fragments per population, the percentage of polymorphic loci and the proportion of rare AFLP fragments significantly decreased from east to west. Main conclusions Deep infraspecific phylogeographical gaps between the populations from the Alps and the western and south-eastern Carpathians suggest the survival of H. uniflora in three separate refugia during the last glaciation. Our AFLP data provide molecular evidence for a long-term geographical disjunction between the eastern and western Carpathians, previously suggested from the floristic composition at the end of 19th century. It is likely that Alpine populations survived the Last Glacial in the eastern part of the Alps, from where they rapidly colonized the rest of the Alps after the ice sheet retreated. Multiple founder effects may explain a gradual loss of genetic variation during westward colonization of the Alps.

This report presents a survey of previous and more recent ultrastructural and cytochemical investigations of disease development in elm, caused by Ophiostoma novo-ulmi, with results of some comparative studies of other wilt diseases caused by f.spp., of Fusarium oxysporum and of Verticillium sp. For cytochemical studies, probes complexed to colloidal gold to detect cellulose, pectin, chitin, and DNA were used. Thus, the formation of tyloses, pit membrane alterations, and the disease effect on parenchyma cells in mature as well as in young tissue were characterized. Vessel lumina in these plants, in diverse situations, contained heterogeneous matter, among which occurred masses of opaque matter and in certain instances pectin fibrils secreted by parenchyma cells and tyloses. Numerous globoid, opaque bodies of regular sizes, which have been mostly overlooked previously, abounded in vessel elements of U. americana and U. glabra, including trees injected with glucose solutions prior to inoculation. Coarser fibrillar material was also noted in vessel lumina, but mostly shortly after inoculation. Other peculiar forms of O. novo-ulmi are also described. The possible role of the components under study is discussed, and a model for DED is proposed in which hindrance to vessel invasion, including downward spread of the pathogen, and reactions of parenchyma cells are complementary and considered to be conducive to defence mechanisms, including compartmentalization of the invaded xylem., En este informe se presenta una revisión de los más recientes estudios sobre la ultraestructura y la citoquímica del desarrollo infectivo causado por Ophiostoma novo-ulmi en el olmo, y su comparación con otras enfermedades vasculares causada por hongos como Fusarium oxysporum y Verticillium sp. En los estudios citoquímicos se usaron inmunolocalizadores de oro coloidal para la detección de celulosa, pectina, quitina y ADN. De esta manera se caracterizó la formación de tilosas, las alteraciones en las punteaduras, y los efectos de la infección en las células parenquimáticas, tanto en tejidos adultos como jóvenes. En estas plantas, el interior de los elementos conductores contiene, en diversas ocasiones, sustancias heterogéneas en las que aparecen masas de sustancias opacas, así como, en ciertas ocasiones, fibrillas de pectina segregadas por células parenquimáticas y por tilosas. Numerosos cuerpos opacos globulares de tamaño medio, que previamente habían pasado en gran medida inadvertidos, eran abundantes en los elementos conductores de U. americana y U. glabra, incluyendo árboles a los que se inyectó soluciones de glucosa antes de la inoculación. También se detectó la presencia, en el interior de los vasos, de material fibrilar de mayor diámetro, sobre todo al poco de la inoculación. También se describen otras formas peculiares de O. novo-ulmi. Se discute el posible papel de los componentes en estudio, y se propone un modelo para la grafiosis en el cual los obstáculos para la invasión del vaso, incluidas la transmisión corriente abajo del patógeno y la reacción de células parenquimáticas, son complementarios, considerándose que son conducentes a la aparición de mecanismos de defensa, incluida la compartimentación del xilema invadido.

In elms infected with Dutch elm disease, alveolar networks, demarcated by filamentous-like bands and confluent with similar matter (the coating) accumulating on vessel walls, occurred regularly in vessel elements. Similar material lined vessel walls in inoculated, sterilized, thin elm wood sections fixed by high pressure freezing. The coating was observed to connect with fungal cells and occasionally contained small opaque particles, the size of ribosomes, membranous and vesicular structures, and, following incubation of wood chips taken from diseased samples incubated on an agar medium, it still displayed similar matter. Coating and alveolar bands increased in thickness by confluence of other bands or membranous structures. Similar matter and structures also occurred in other plants affected by similar fungal wilt diseases. In all systems, the compact coating did not label for chitin, cellulose and pectin. In staghorn sumac, the probe for DNA attached to the coating. Altogether, in the light of these data, it appears that the coating and alveolar networks are not inert components, a fact which indicates their primordial probable pathogen origin. It is proposed that these elements might be important not only in the initial infection stages but also in older or recurrent infections at a time when host resistance mechanisms are ineffective., En olmos afectados por la grafiosis, la red alveolar, demarcada por bandas filamentosas, y confluente con acumulaciones de la misma sustancia (cubrición) presentes en las paredes de los vasos, aparece regularmente en los elementos conductores. Sustancias similares tapizan las paredes de los vasos en secciones de madera delgada de olmo inoculada y esterilizada, y posteriormente criofijadas a altas presiones. Se observó que la cubrición se conecta con las células del micelio y que ocasionalmente contenía pequeñas partículas opacas del tamaño de los ribosomas, estructuras membranosas y vesiculares, así como que, tras la incubación de astillas leñosas cogidas de muestras enfermas incubadas en agar, aún se presentaba una sustancia similar. La cubrición y las bandas alveolares aumentaron su espesor en la confluencia con otras bandas o estructuras membranosas. Estructuras y sustancias similares aparecieron también en otras plantas afectadas por enfermedades similares originadas por hongos que producen marchitamiento. En todos los sistemas, la cubrición compacta no pudo ser marcada como quitina, celulosa ni pectina. En zumaque (Rhus typhina), la sonda de ADN se pegó a la cubrición. En resumen, a la vista de estos datos, parece ser que la cubrición y la red alveolar no están formados por componentes inertes, un hecho que indica su probable origen patogénico. Se sugiere que esos elementos podrían ser importantes no sólo en las fases iniciales de la infección, sino también, en infecciones más desarrolladas o recurrentes, en el momento en que los mecanismos de resistencia del hospedante no son efectivos.

In elms infected with Dutch elm disease, alveolar networks, demarcated by filamentous-like bands and confluent with similar matter (the coating) accumulating on vessel walls, occurred regularly in vessel elements. Similar material lined vessel walls in inoculated, sterilized, thin elm wood sections fixed by high pressure freezing. The coating was observed to connect with fungal cells and occasionally contained small opaque particles, the size of ribosomes, membranous and vesicular structures, and, following incubation of wood chips taken from diseased samples incubated on an agar medium, it still displayed similar matter. Coating and alveolar bands increased in thickness by confluence of other bands or membranous structures. Similar matter and structures also occurred in other plants affected by similar fungal wilt diseases. In all systems, the compact coating did not label for chitin, cellulose and pectin. In staghorn sumac, the probe for DNA attached to the coating. Altogether, in the light of these data, it appears that the coating and alveolar networks are not inert components, a fact which indicates their primordial probable pathogen origin. It is proposed that these elements might be important not only in the initial infection stages but also in older or recurrent infections at a time when host resistance mechanisms are ineffective. En olmos afectados por la grafiosis, la red alveolar, demarcada por bandas filamentosas, y confluente con acumulaciones de la misma sustancia (cubrición) presentes en las paredes de los vasos, aparece regularmente en los elementos conductores. Sustancias similares tapizan las paredes de los vasos en secciones de madera delgada de olmo inoculada y esterilizada, y posteriormente criofijadas a altas presiones. Se observó que la cubrición se conecta con las células del micelio y que ocasionalmente contenía pequeñas partículas opacas del tamaño de los ribosomas, estructuras membranosas y vesiculares, así como que, tras la incubación de astillas leñosas cogidas de muestras enfermas incubadas en agar, aún se presentaba una sustancia similar. La cubrición y las bandas alveolares aumentaron su espesor en la confluencia con otras bandas o estructuras membranosas. Estructuras y sustancias similares aparecieron también en otras plantas afectadas por enfermedades similares originadas por hongos que producen marchitamiento. En todos los sistemas, la cubrición compacta no pudo ser marcada como quitina, celulosa ni pectina. En zumaque (Rhus typhina), la sonda de ADN se pegó a la cubrición. En resumen, a la vista de estos datos, parece ser que la cubrición y la red alveolar no están formados por componentes inertes, un hecho que indica su probable origen patogénico. Se sugiere que esos elementos podrían ser importantes no sólo en las fases iniciales de la infección, sino también, en infecciones más desarrolladas o recurrentes, en el momento en que los mecanismos de resistencia del hospedante no son efectivos.

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