Francine Tremblay

Glacial vicariance in Eurasia: mitochondrial DNA evidence from Scots pine for a complex heritage involving genetically distinct refugia at mid-northern latitudes and in Asia Minor

BackgroundAt the last glacial maximum, Fennoscandia was covered by an ice sheet while the tundra occupied most of the rest of northern Eurasia. More or less disjunct refugial populations of plants were dispersed in southern Europe, often trapped between mountain ranges and seas. Genetic and paleobotanical evidences indicate that these populations have contributed much to Holocene recolonization of more northern latitudes. Less supportive evidence has been found for the existence of glacial populations located closer to the ice margin. Scots pine (Pinus sylvestris L.) is a nordic conifer with a wide natural range covering much of Eurasia. Fractures in its extant genetic structure might be indicative of glacial vicariance and how different refugia contributed to the current distribution at the continental level. The population structure of Scots pine was investigated on much of its Eurasian natural range using maternally inherited mitochondrial DNA polymorphisms.ResultsA novel polymorphic region of the Scots pine mitochondrial genome has been identified, the intron 1 of nad7, with three variants caused by insertions-deletions. From 986 trees distributed among 54 populations, four distinct multi-locus mitochondrial haplotypes (mitotypes) were detected based on the three nad7 intron 1 haplotypes and two previously reported size variants for nad1 intron B/C. Population differentiation was high (GST = 0.657) and the distribution of the mitotypes was geographically highly structured, suggesting at least four genetically distinct ancestral lineages. A cosmopolitan lineage was widely distributed in much of Europe throughout eastern Asia. A previously reported lineage limited to the Iberian Peninsula was confirmed. A new geographically restricted lineage was found confined to Asia Minor. A new lineage was restricted to more northern latitudes in northeastern Europe and the Baltic region.ConclusionThe contribution of the various ancestral lineages to the current distribution of Scots pine was asymmetric and extant endemism reflected the presence of large geographic barriers to migration. The results suggest a complex biogeographical history with glacial refugia shared with temperate plant species in southern European Peninsulas and Asia Minor, and a genetically distinct glacial population located more North. These results confirm recent observations for cold tolerant species about the possible existence of refugial populations at mid-northern latitudes contributing significantly to the recolonization of northern Europe. Thus, Eurasian populations of nordic plant species might not be as genetically homogenous as assumed by simply considering them as offsets of glacial populations located in southern peninsulas. As such, they might have evolved distinctive genetic adaptations during glacial vicariance, worth evaluating and considering for conservation.

Clonal and spatial genetic structures of aspen (Populus tremuloides Michx.)

To portray aspen clonal and spatial genetic structures, we mapped and genotyped trees in two 1‐ha plots, each containing three aspen cohorts originating from fire or subsequent secondary disturbances. We used four microsatellite loci to identify aspen clones and increment core analysis to determine tree age. Clonal dimensions were measured by the maximum distance between two ramets and the number of ramets per genet. Standard normal deviate (SND) was used to assess the spatial distribution of aspen genets and cohorts, and multivariate spatial genetic autocorrelations to assess the spatial distribution of aspen genetic variation. Most aspen genets consisted of only one ramet (> 75%). Median clonal dimensions were 19 and 29 m (maxima: 104 and 72 m in the two plots). No segregation was observed between clones. Aspen cohorts were spatially segregated but trees were spatially aggregated within old and medium‐aged cohorts. In contrast, trees were more randomly distributed within the youngest cohorts. This coincided with a spatial genetic autocorrelation at small scales (up to 30 m) in the older cohorts and a more random genetic distribution in the youngest ones. Our results suggest that aspen spatial genetic structuring reflects the spatial patterns produced by the regeneration of discrete cohorts at different stages of succession. Vegetative reproduction leads to aspen genetic spatial structuring at small scales (few metres) until midsuccession. However, as the stand gets older, the spatial distribution of aspen trees and genetic structure evolve from a structured pattern to a more random one under a gap disturbances regime.

Molecular and dendrochronological analysis of natural root grafting in Populus tremuloides (Salicaceae)

Trembling aspen (Populus tremuloides) is a clonal tree species, which regenerates mostly through root suckering. In spite of vegetative propagation, aspen maintains high levels of clonal diversity. We hypothesized that the maintenance of clonal diversity in this species can be facilitated by integrating different clones through natural root grafts into aspens communal root system. To verify this hypothesis, we analyzed root systems of three pure aspen stands where clones had been delineated with the help of molecular markers. Grafting between roots was frequent regardless of their genotypes. Root system excavations revealed that many roots were still living below trees that had been dead for several years. Some of these roots had no root connections other than grafts to living ramets of different clones. The uncovered root systems did not include any unique genotypes that would not occur among stems. Nevertheless, acquiring roots of dead trees helps to maintain extensive root systems, which increases the chances of clone survival. Substantial interconnectivity within clones as well as between clones via interclonal grafts results in formation of large genetically diverse physiological units. Such a clonal structure can significantly affect interpretations of diverse ecophysiological processes in forests of trembling aspen.

In situ Comparison of Tree-Ring Responses to Climate and Population Genetics: The Need to Control for Local Climate and Site Variables

Tree species responses to climate change will be greatly influenced by their evolutionary potential and their phenotypic plasticity. Investigating tree-rings responses to climate and population genetics at the regional scale is therefore crucial in assessing the tree behaviour to climate change. This study combined in situ dendroclimatology and population genetics over a latitudinal gradient and compared the variations between the two at the intra- and inter-population levels. This approach was applied on the northern marginal populations of Thuja occidentalis (eastern white-cedar) in the Canadian boreal forest. We aimed first to assess the radial growth variability (response functional trait) within populations across the gradient and to compare it with the genetic diversity (microsatellites). Second, we investigated the variability in the growth response to climate at the regional scale through the radial growth-climate relationships, and tested its correlation with environmental variables and population genetic structure. Model selection based on the Akaike Information Criteria revealed that the growth synchronicity between pairs of trees of a population covariates with both the genetic diversity of this population and the amount of precipitation (inverse correlation), although these variables only explained a small fraction of the observed variance. At the regional scale, variance partitioning and partial redundancy analysis indicate that the growth response to climate was greatly modulated by stand environmental variables, suggesting predominant plastic variations in growth-response to climate. Combining in situ dendroclimatology and population genetics is a promising way to investigate species’ response capacity to climate change in natural stands. We stress the need to control for local climate and site conditions effects on dendroclimatic response to climate to avoid misleading conclusions regarding the associations with genetic variables.

Herbivore-simulated induction of defenses in clonal networks of trembling aspen (Populus tremuloides)

Trembling aspen (Populus tremuloides Michx.) as a clonal tree species possesses a complex root system through which trees of the same or different clones are connected. Root connections have been studied with respect to resource sharing, but the nature, quantities or extent of what is shared between trees is relatively unknown. In this study, we posed the hypothesis that systemic defense induction signals could also spread through these root networks and trigger defenses in neighboring ramets before arrival of pests. Temporal expression pattern of Kunitz trypsin inhibitor (KTI) and dihydroflavonol reductase (DFR) genes, two markers of poplar defense, was followed by quantitative real-time polymerase chain reaction. The expression was quantified in systemic leaves of wounded and healthy plants that shared the same parental root and in untreated controls grown in separate pots. Untreated interconnected plants did not show induced resistance upon herbivore-simulated attack. Although wound-treated ramets induced defense genes, untreated interconnected plants produced an expression pattern similar to non-connected controls. Root connections do not automatically lead to induction of defensive traits that are expressed in plants directly under damage thought to simulate herbivory. Rather, it seems that other communication means such as airborne volatiles can serve as signal transmission pathways among neighboring plants.

Development of polymorphic nuclear microsatellite markers in sugar maple (Acer saccharum Marsh.) using cross-species transfer and SSR-enriched shotgun pyrosequencing

A set of 23 polymorphic microsatellites was successfully tested for Acer saccharum. Eighteen of these new polymorphic markers were developed using next-generation sequencing and five were transferred from other Acer species. A total of 68 individuals from two populations were genotyped at all markers using a multiplex approach. Mean allelic diversity was 7.7, polymorphism ranged from 2 to 16 alleles per locus. Overall heterozygosity was high, with an average observed heterozygosity of 0.644. Two loci that were obtained from cross-species transfer and one from shotgun pyrosequencing showed heterozygote deficiencies, deviations from Hardy–Weinberg equilibrium, and high frequencies of null alleles for one or both populations. No evidence for linkage disequilibrium was detected. Finally, 20 of the 90 markers could be used in future population genetics studies of A. saccharum.

Genetic consequences of fragmentation in “arbor vitae,” eastern white cedar (Thuja occidentalis L.), toward the northern limit of its distribution range

We tested the hypothesis that marginal fragmented populations of eastern white cedar (EWC) are genetically isolated due to reduced pollen and gene flow. In accordance with the central-marginal model, we predicted a decrease in population genetic diversity and an increase in differentiation along the latitudinal gradient from the boreal mixed-wood to northern coniferous forest. A total of 24 eastern white cedar populations were sampled along the north-south latitudinal gradient for microsatellite genotyping analysis. Positive Fis values and heterozygote deficiency were observed in populations from the marginal (Fis = 0.244; PHW = 0.0042) and discontinuous zones (Fis = 0.166; PHW = 0.0042). However, populations from the continuous zone were in HW equilibrium (Fis = −0.007; PHW = 0.3625). There were no significant latitudinal effects on gene diversity (Hs), allelic richness (AR), or population differentiation (Fst). Bayesian and NJT (neighbor-joining tree) analyses demonstrated the presence of a population structure that was partly consistent with the geographic origins of the populations. The impact of population fragmentation on the genetic structure of EWC is to create a positive inbreeding coefficient, which was two to three times higher on average than that of a population from the continuous zone. This result indicated a higher occurrence of selfing within fragmented EWC populations coupled with a higher degree of gene exchange among near-neighbor relatives, thereby leading to significant inbreeding. Increased population isolation was apparently not correlated with a detectable effect on genetic diversity. Overall, the fragmented populations of EWC appear well-buffered against effects of inbreeding on genetic erosion.

Evidence of Somaclonal Variation in Somatic Embryo-Derived Plantlets of White Spruce (Picea Glauca (Moench) Voss

Unlike angiosperms (especially crop plant species), conifers are considered to be genetically stable following somatic embryogenesis. However, we have been able to identify four different variegata phenotypes among 2270 somatic embryo-derived white spruces. The four types of variegated plants differ from each other with respect to the extent and distribution of their chlorophyll-deficient needles. Microscopy shows that certain leaves of a selected variant are formed of a chimeral mixture of green and white cells. Cells in completely white needles of this variegated plant are characterized by large nuclei with predominant euchromatin, absence of large cytoplasmic vacuoles, and vacuolized plastids with aberrant morphologies. Various observations suggest that the recovered variegata phenotypes reflect some kind of genetic instability of either chloroplastic or nuclear genomes. Molecular approaches, including the use of RAPD markers, are currently employed to find out whether DNA rearrangements are involved in conferring these variegata phenotypes.

Temporal variation in quaking aspen (Populus tremuloides) genetic and clonal structures in the mixedwood boreal forest of eastern Canada

ABSTRACT Two sites that burned in 1847 (H) and 1823 (I) in the mixedwood boreal forest in Québec were selected to follow aspen genetic and clonal diversity over time. At each site, three cohorts were identified by core dating, and about 30 trees per cohort were randomly selected to compare tree genotypes using four microsatellite loci. The first cohorts were of post-fire origin (large disturbance), while the second and third cohorts were promoted by gap disturbances. These gaps were created by the natural mortality of post-fire aspen trees and a spruce budworm outbreak that attacked the coniferous species. Expected heterozygosity ranged from 0.37 to 0.72 across cohorts and averaged 0.66 and 0.54 in H and I, respectively. More than 99% and 96%of the genetic variability existed within cohorts, respectively. Genotypic diversity was high in all cohorts, and most genets were unique. Only two clones suckered for three successive cohorts, indicating little selection for specific genets to dominate aspen stands with time. High genetic and clonal diversity changed slightly between post-fire and gap disturbance cohorts. Apical dominance might have favoured the suckering of genets that existed in the post-fire cohorts but that were later eliminated by natural mortality.

Somatic Embryogenesis in Red Spruce (Picea Rubens Sarg.)

Picea rubens Sarg. (red spruce) is a member of the family Pinaceae, the largest family of conifers. Like the other spruce species, Picea rubens has long straight trunks with a scaly bark, and dense narrow branches that can extend to the ground in open-grown trees (Hosie, 1980). The root system is shallow and the trees are not usually wind-firm. Picea rubens can grow to approximately 21 to 24 m in height and 30 to 60 cm in diameter (Fig. 1). Good cone production usually begins after the tree is 30 years old, with good seed crops every 3 to 8 years (Fowells, 1965).