Molecular footprints of the Holocene retreat of dwarf birch in Britain.

Past reproductive interactions among incompletely isolated species may leave behind a trail of introgressed alleles, shedding light on historical range movements. Betula pubescens is a widespread native tetraploid tree species in Britain, occupying habitats intermediate to those of its native diploid relatives, B. pendula and B. nana. Genotyping 1134 trees from the three species at 12 microsatellite loci, we found evidence of introgression from both diploid species into B. pubescens, despite the ploidy difference. Surprisingly, introgression from B. nana, a dwarf species whose present range is highly restricted in northern, high-altitude peat bogs, was greater than introgression from B. pendula, which is morphologically similar to B. pubescens and has a substantially overlapping range. A cline of introgression from B. nana was found extending into B. pubescens populations far to the south of the current B. nana range. We suggest that this genetic pattern is a footprint of a historical decline and/or northwards shift in the range of B. nana populations due to climate warming in the Holocene. This is consistent with pollen records that show a broader, more southerly distribution of B. nana in the past. Ecological niche modelling predicts that B. nana is adapted to a larger range than it currently occupies, suggesting additional factors such as grazing and hybridization may have exacerbated its decline. We found very little introgression between B. nana and B. pendula, despite both being diploid, perhaps because their distributions in the past have rarely overlapped. Future conservation of B. nana may partly depend on minimization of hybridization with B. pubescens, and avoidance of planting B. pendula near B. nana populations.

Genome sequence of dwarf birch (Betula nana) and cross-species RAD markers.

New sequencing technologies allow development of genome-wide markers for any genus of ecological interest, including plant genera such as Betula (birch) that have previously proved difficult to study due to widespread polyploidy and hybridization. We present a de novo reference genome sequence assembly, from 66× short read coverage, of Betula nana (dwarf birch) - a diploid that is the keystone woody species of subarctic scrub communities but of conservation concern in Britain. We also present 100 bp PstI RAD markers for B. nana and closely related Betula tree species. Assembly of RAD markers in 15 individuals by alignment to the reference B. nana genome yielded 44-86k RAD loci per individual, whereas de novo RAD assembly yielded 64-121k loci per individual. Of the loci assembled by the de novo method, 3k homologous loci were found in all 15 individuals studied, and 35k in 10 or more individuals. Matching of RAD loci to RAD locus catalogues from the B. nana individual used for the reference genome showed similar numbers of matches from both methods of RAD locus assembly but indicated that the de novo RAD assembly method may overassemble some paralogous loci. In 12 individuals hetero-specific to B. nana 37-47k RAD loci matched a catalogue of RAD loci from the B. nana individual used for the reference genome, whereas 44-60k RAD loci aligned to the B. nana reference genome itself. We present a preliminary study of allele sharing among species, demonstrating the utility of the data for introgression studies and for the identification of species-specific alleles.

Phytophthora cinnamomi and other fine root pathogens in north temperate pine forests.

A number of fine root pathogens, including Phytophthora cinnamomi, Pythium ultimum var. ultimum, Pythium undulatum, Pythium violae, Fusarium sp., and two incompletely identified Verticillium species, were isolated from soils taken from under Scots pine trees at five sites in north Scotland, including semi-natural forests and plantations. At least two root pathogens were recovered from each forest. Morphological and molecular data supported the identification of Phytophthora cinnamomi from three of the sites investigated. Isolates of Phytophthora cinnamomi, Pythium ultimum var. ultimum and an incompletely identified Fusarium sp. caused growth reductions of Scots pine seedlings, as determined by dry weight; the most virulent species were Phytophthora cinnamomi and Fusarium sp. The most severe disease symptoms were caused by a mixed inoculum containing Phytophthora cinnamomi, Pythium ultimum var. ultimum and Fusarium sp., or by the Fusarium isolate alone. These nonspecific pathogens may persist on the roots of understorey and herbaceous plants in the pine forests.

Physical and chemical responses of Sitka spruce (Picea sitchensis) clones to colonization by Heterobasidion annosum as potential markers for relative host susceptibility.

Forty-one 2-year-old clones of Picea sitchensis (Bong.) Carr. from three full-sib families (14 clones from each of two families and 13 clones from a third family) were either wounded and inoculated with an isolate of Heterobasidion annosum (Fr.) Bref. or wounded without inoculation. Lesion lengths on the inner bark from the point of inoculation varied among clones 35 days after treatment. There was no relationship between lesion length and relatedness within families. Two clones (21342 and 25202) with the shortest lesions, tentatively designated as less susceptible to H. annosum, and two clones (21176 and 27166) with the longest lesions, designated more susceptible, were selected for comparison of host anatomical and chemical responses to infection. The position and structure of the ligno-suberized boundary zone (LSZ) in the bark of the clones suggested that the less susceptible clones formed thicker layers of suberized cells in the LSZ following wounding plus inoculation. No LSZ was observed in two ramets of the more susceptible Clone 27166 following wounding and inoculation with H. annosum. Compared with more susceptible genotypes, clones of P. sitchensis with low susceptibility to H. annosum had high relative proportions of (+)-alpha-pinene, (-)-beta-pinene and one unidentified terpene constituent (Unknown-15) in cortical resin sampled 25 cm from the lesions. In contrast, more susceptible clones had higher relative proportions of (-)-limonene, Unknown-16, Unknown-18 and Unknown-19. In the secondary resin produced in bark tissues surrounding the lesions, proportions of several monoterpene constituents varied; these changes included a decrease in the relative amount of beta-phellandrene and corresponding small increases in some minor constituents. The concentrations of the monoterpenes, except a few minor constituents, increased in the infected tissues. Wounding plus inoculation with H. annosum resulted in varied monoterpene responses, with distinct differences between less susceptible and more susceptible clones. In less susceptible clones, Unknown-19 increased following wounding plus inoculation, whereas in more susceptible clones, concentrations of delta-3-carene and Unknown-13 and Unknown-16 increased. Differences in both constitutive and induced resin monoterpene profiles may provide useful markers for resistance to H. annosum in selection and breeding programs.

Multiplex real-time PCR detection of pathogen colonization in the bark and wood of Picea sitchensis clones differing in resistance to Heterobasidion annosum.

A quantitative multiplex real-time polymerase chain reaction (PCR) procedure was developed to assess the extent of Heterobasidion annosum (Fr.) Bref. growth in Sitka spruce (Picea sitchensis (Bong.) Carr.) bark and wood and to determine correlations between lesion length and fungal colonization. Based on lesion length and real-time PCR, the responses of four 3-year-old Sitka spruce clones to inoculation with H. annosum were characterized as showing either resistance or susceptibility to the pathogen. In susceptible clones, the extent of bark colonization did not differ from the visible length of the bark lesion, whereas lesions were longer than the extent of fungal colonization in resistant clones. The resistant clones contained considerably less fungal DNA than the susceptible clones, relative to the amount of host DNA in both the bark and the wood, indicating less resistance and more host cell death in the susceptible clones following inoculation. In both resistant and susceptible clones, fungal colonization in the wood extended beyond the visible necrotic lesion in the bark, indicating that host defense responses are weaker in wood than in bark. The spread of the pathogen in both bark and wood was less in the resistant clones than in the susceptible clones, indicating that defenses in both bark and wood of the resistant clones were superior to those in the susceptible clones.