Co-occurring orchid species associated with different low-abundance mycorrhizal fungi from the soil in a high-diversity conservation area in Denmark.

Plant-fungal interactions are ubiquitous across ecosystems and contribute significantly to plant ecology and evolution. All orchids form obligate symbiotic relationships with specific fungi for germination and early growth, and the distribution of terrestrial orchid species has been linked to occurrence and abundance of specific orchid mycorrhizal fungi (OMF) in the soil. The availability of OMF can therefore be a habitat requirement that is relevant to consider when establishing management and conservation strategies for threatened orchid species, but knowledge on the spatial distribution of OMF in soil is limited. We here studied the mycorrhizal associations of three terrestrial orchid species (Anacamptis pyramidalis, Orchis purpurea and Platanthera chlorantha) found in a local orchid diversity hotspot in eastern Denmark, and investigated the abundance of the identified mycorrhizal fungi in the surrounding soil. We applied ITS metabarcoding to samples of orchid roots, rhizosphere soil and bulk soil collected at three localities, supplemented with standard barcoding of root samples with OMF specific primers, and detected 22 Operational Taxonomic Units (OTUs) putatively identified as OMF. The three orchid species displayed different patterns of OMF associations, supporting the theory that association with specific fungi constitutes part of an orchid's ecological niche allowing co-occurrence of many species in orchid-rich habitats. The identified mycorrhizal partners in the basidiomycete families Tulasnellaceae and Ceratobasidiaceae (Cantharallales) were detected in low abundance in rhizosphere soil, and appeared almost absent from bulk soil at the localities. This finding highlights our limited knowledge of the ecology and trophic mode of OMF outside orchid tissues, as well as challenges in the detection of specific OMF with standard methods. Potential implications for management and conservation strategies are discussed.

Local adaptation through genetic differentiation in highly fragmented Tilia cordata populations.

We assessed the level of geographic differentiation of Tilia cordata in Denmark based on tests of 91 trees selected from 12 isolated populations. We used quantitative analysis of spring phenology and population genetic analysis based on SSR markers to infer the likely historical genetic processes within and among populations. High genetic variation within and among populations was observed in spring phenology, which correlated with spring temperatures at the origin of the tested T. cordata trees. The population genetic analysis revealed significant differentiation among the populations, but with no clear sign of isolation by distance. We infer the findings as indications of ongoing fine scale selection in favor of local growth conditions made possible by limited gene flow among the small and fragmented populations. This hypothesis fits well with reports of limited fruiting in the investigated Danish T. cordata populations, while the species is known for its ability to propagate vegetatively by root suckers. Our results suggest that both divergent selection and genetic drift may have played important roles in forming the genetic patterns of T. cordata at its northern distribution limit. However, we also speculate that epigenetic mechanism arising from the original population environment could have created similar patterns in regulating the spring phenology.

Assessing inter- and intraspecific variability of xylem vulnerability to embolism in oaks.

The genus Quercus comprises important species in forestry not only for their productive value but also for their ability to withstand drought. Hence an evaluation of inter- and intraspecific variation in drought tolerance is important for selecting the best adapted species and provenances for future afforestation. The presence of long vessels makes it difficult to assess xylem vulnerability to embolism in oak. Thanks to the development of an in situ flow centrifuge equipped with a large rotor, we quantified (i) the between species variability of embolism resistance in four native and two exotic species of oaks in Europe and (ii) the within species variability in Quercus petraea. Embolism resistance varied significantly among species, with the pressure inducing 50% loss of hydraulic conductivity (P50 ) ranging between - 7.0 and -4.2 MPa. Species native to the Mediterranean region were more resistant than pan-European species. In contrast, intraspecific variability in embolism resistance in Q. petraea was low within provenances and null among provenances. A positive correlation between P50 and vessel diameter among the six oak species indicates that the more embolism resistant species had narrower xylem vessels. However, this tradeoff between hydraulic efficiency and safety was not observed between Q. petraea provenances.

Population genetic structure of the endemic rosewoods Dalbergia cochinchinensis and D. oliveri at a regional scale reflects the Indochinese landscape and life-history traits.

Indochina is a biodiversity hot spot and harbors a high number of endemic species, most of which are poorly studied. This study explores the genetic structure and reproductive system of the threatened endemic timber species Dalbergia cochinchinensis and Dalbergia oliveri using microsatellite data from populations across Indochina and relates it to landscape characteristics and life-history traits. We found that the major water bodies in the region, Mekong and Tonle Sap, represented barriers to gene flow and that higher levels of genetic diversity were found in populations in the center of the distribution area, particularly in Cambodia. We suggest that this pattern is ancient, reflecting the demographic history of the species and possible location of refugia during earlier time periods with limited forest cover, which was supported by signs of old genetic bottlenecks. The D. oliveri populations had generally high levels of genetic diversity (mean He = 0.73), but also strong genetic differentiation among populations (global GST = 0.13), while D. cochinchinensis had a moderate level of genetic diversity (mean He = 0.55), and an even stronger level of differentiation (global GST = 0.25). These differences in genetic structure can be accounted for by a higher level of gene flow in D. oliveri due to a higher dispersal capacity, but also by the broader distribution area for D. oliveri, and the pioneer characteristics of D. cochinchinensis. This study represents the first detailed analysis of landscape genetics for tree species in Indochina, and the found patterns might be common for other species with similar ecology.

Gene flow of common ash (Fraxinus excelsior L.) in a fragmented landscape.

Gene flow dynamics of common ash (Fraxinus excelsior L.) is affected by several human activities in Central Europe, including habitat fragmentation, agroforestry expansion, controlled and uncontrolled transfer of reproductive material, and a recently introduced emerging infectious disease, ash dieback, caused by Hymenoscyphus fraxineus. Habitat fragmentation may alter genetic connectivity and effective population size, leading to loss of genetic diversity and increased inbreeding in ash populations. Gene flow from cultivated trees in landscapes close to their native counterparts may also influence the adaptability of future generations. The devastating effects of ash dieback have already been observed in both natural and managed populations in continental Europe. However, potential long-term effects of genetic bottlenecks depend on gene flow across fragmented landscapes. For this reason, we studied the genetic connectivity of ash trees in an isolated forest patch of a fragmented landscape in Rösenbeck, Germany. We applied two approaches to parentage analysis to estimate gene flow patterns at the study site. We specifically investigated the presence of background pollination at the landscape level and the degree of genetic isolation between native and cultivated trees. Local meteorological data was utilized to understand the effect of wind on the pollen and seed dispersal patterns. Gender information of the adult trees was considered for calculating the dispersal distances. We found that the majority of the studied seeds (55-64%) and seedlings (75-98%) in the forest patch were fathered and mothered by the trees within the same patch. However, we determined a considerable amount of pollen flow (26-45%) from outside of the study site, representing background pollination at the landscape level. Limited pollen flow was observed from neighbouring cultivated trees (2%). Both pollen and seeds were dispersed in all directions in accordance with the local wind directions. Whereas there was no positive correlation between pollen dispersal distance and wind speed, the correlation between seed dispersal distance and wind speed was significant (0.71, p < 0.001), indicating that strong wind favours long-distance dispersal of ash seeds. Finally, we discussed the implications of establishing gene conservation stands and the use of enrichment planting in the face of ash dieback.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Molecular markers for tolerance of European ash (Fraxinus excelsior) to dieback disease identified using Associative Transcriptomics.

Tree disease epidemics are a global problem, impacting food security, biodiversity and national economies. The potential for conservation and breeding in trees is hampered by complex genomes and long lifecycles, with most species lacking genomic resources. The European Ash tree Fraxinus excelsior is being devastated by the fungal pathogen Hymenoscyphus fraxineus, which causes ash dieback disease. Taking this system as an example and utilizing Associative Transcriptomics for the first time in a plant pathology study, we discovered gene sequence and gene expression variants across a genetic diversity panel scored for disease symptoms and identified markers strongly associated with canopy damage in infected trees. Using these markers we predicted phenotypes in a test panel of unrelated trees, successfully identifying individuals with a low level of susceptibility to the disease. Co-expression analysis suggested that pre-priming of defence responses may underlie reduced susceptibility to ash dieback.

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.).

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions.

Microsatellite primers for Parkia biglobosa (Fabaceae: Mimosoideae) reveal that a single plant sires all seeds per pod.

PREMISE OF THE STUDY: Microsatellite primers were developed for an indigenous fruit tree, Parkia biglobosa, as a tool to study reproductive biology and population structure. Here we use the primers to determine the number of fathers per pod. • METHODS AND RESULTS: Microsatellite loci were enriched in a genomic sample and isolated using pyrosequencing. Eleven primer pairs were characterized in two populations of P. biglobosa in Burkina Faso (each with 40 trees). The number of alleles per locus ranged from eight to 15, and one locus had null alleles. We genotyped seeds from 24 open-pollinated pods. The genotypic profiles of seeds per pod suggest that all seeds are outcrossed and that only one pollen donor sires all ovules in a single fruit. • CONCLUSIONS: Ten microsatellite markers were highly polymorphic. All seeds per pod of P. biglobosa were full siblings. The markers will be useful for reproductive and population genetic studies.

Adaptive potential of ash (Fraxinus excelsior) populations against the novel emerging pathogen Hymenoscyphus pseudoalbidus.

An emerging infectious pathogen Hymenoscyphus pseudoalbidus has spread across much of Europe within recent years causing devastating damage on European common ash trees (Fraxinus excelsior) and associated plant communities. The present study demonstrates the presence of additive genetic variation in susceptibility of natural F. excelsior populations to the new invasive disease. We observe high levels of additive variation in the degree of susceptibility with relatively low influence of environmental factors (narrow-sense heritability = 0.37-0.52). Most native trees are found to be highly susceptible, and we estimate that only around 1% has the potential of producing offspring with expected crown damage of <10% under the present disease pressure. The results suggest that the presence of additive genetic diversity in natural F. excelsior populations can confer the species with important ability to recover, but that low resistance within natural European populations is to be expected because of a low frequency of the hypo-sensitive trees. Large effective population sizes will be required to avoid genetic bottlenecks. The role of artificial selection and breeding for protection of the species is discussed based on the findings.