Material analysed, DNA extraction and fragment analysis
DNA has been successfully isolated from various tissues such as megagametophytes (e.g. Mosca et al. 2012b), seedlings (e.g. Semerikov & Lascoux 2003), leaves (e.g. Pluess 2011), buds (Scheepers et al. 2000) and embryogenic mass (L. Pâques, pers. comm.). For isozyme analyses megagametophytes have been commonly used (e.g. Lewandowski & Mejnartowicz 1991a). DNA isolation mostly followed CTAB (Devey et al. 1996; Doyle & Doyle 1990) or manufactured isolation kits such as DNeasy Plant Mini Kit (Qiagen, Gros-Louis et al. 2005) or DNAeasy 96 Plant kit (Pluess 2011).
Organelle markers (mitochondrial and chloroplast DNA)
Organelle markers applicable on Larix decidua have been developed for questions such as phylogeny and species diagnostics. Chloroplast DNA variation has been studied based on sequence variation (Gros-Louis et al. 2005; Qian et al. 1995; Wei & Wang 2003), PCR-RFLPs and microsatellites (Semerikov and Lascoux 2003; Acheré et al. 2004). Mitochondrial DNA variation has been studied using direct PCR, PCR-RFLPs (Acheré et al. 2004; Semerikov & Lascoux 2003; Semerikov & Polezhaeva 2007) and sequencing (Gros-Louis et al. 2005). These studies have identified useful markers and marker combinations for species discrimination. By combining a mitochondrial and a chloroplast marker, L. decidua and L. kaempferi could be discriminated (Acheré et al. 2004; Jagielska 2008). In another study, L. decidua and L. kaempferi could be discriminated from L. sibirica and L. laricina by sequencing four chloroplast regions (matK, trnL-intron, trnT-trnL trnL-trnF) and all four species could be identified by sequencing five mitochondrial introns (cox1-1, matR-1, nad1-b/c, nad3-1 and nad5-1; Gros-Louis et al. 2005). Note that in the same study Gros-Louis et al. (2005) also obtained nuclear markers discriminating the four species (see nuclear markers section below).
Phylogeny, evolutionary history, distinction of species, subspecies and hybrids
The phylogeny of the genus Larix has been reconstructed using various molecular markers (Gernandt & Liston 1999; Qian et al. 1995; Semerikov et al. 2003). All studies showed three monophyletic clades: a North American clade, a South Asian clade and a North Eurasian clade, with L. decidua having a basal position in the Eurasian clade. Special attention has been paid to the var. polonica that has been hypothesized to be a hybrid of L. decidua and L. sibirica larch (Bobrov 1972). Hybrids of European and Japanese larch (L. x eurolepis) have been artificially introduced in the 20th century.
To date conclusions about levels of genetic differentiation in Larix decidua have been drawn from studies focusing on the local and regional scales. On a very local scale (<5km) Pluess (2011) has reported an FST value of 0.014 based on nuclear SSR data. On a broader scale (distance between the populations up to 200km), Mosca et al. (2012a) have obtained an FST value of 0.011 based on nuclear SNP data. Finally, at the scale of the Italian Alpine region (populations located up to 600 km from each other), Mosca et al. (2012a) have reported a mean FST of 0.04, indicating a higher degree of differentiation that can be explained by the subdivision of the studied populations in three major genetic groups, as revealed by Bayesian cluster analysis.
Glacial refugia, biogeographic history
So far, biogeographic history of European larch has not been studied in detail as it is the case for the other European tree species e.g. Quercus (Petit et al. 2002), Fagus sylvatica (Magri et al. 2006) and Pinus sylvestris (Cheddadi et al. 2006) A detailed reconstruction of its history Stand, seed and pollen dispersal, small scale genetic structure Pluess (2011) has performed a landscape-scale analysis of L. decidua along the lateral moraine and the adjacent valley slope of a glacier in the Swiss Alps at 1700–2240 m a.s.l.. Nine SSR markers were used for this purpose. All sampled individuals (N = 730) formed a single genetic cluster indicating homogenizing gene flow despite spatial genetic structure (SGS) up to 80 m. No evidence for selfing or for inbreeding was found in adults or in juveniles (heterozygote deficit was not significantly different from zero). SGS among juveniles was found at up to 30 m in the older sub-population whereas no SGS was found in the younger, recently established sub-population. A maximum likelihood paternity assignment revealed local gene dispersal in the ancient part (2–48 m) and intermediate-to-long distance dispersal into the recently colonized part (115–3132 m), pointing to intensive mixing of the genes in this expanding population and suggesting that genetic diversity can be maintained during rapid population expansion driven by climate warming.
Inference of the number of mother trees from a seed lot by molecular methods
Genetically improved seed lots from clonal seed orchards could be controlled with highly polymorphic markers using parentage analyses. This would allow tracing this material to the seed orchards where it was produced (or possibly ruling out such an origin) and enumerating the number of parental clones involved in the composition of each seed lot. The only 37 reference needed would be one representative of each clone. In principle, such an approach could also be used on unknown seed lots, although more developments would be necessary.
Clone identification, including clonal mixtures
Other than for hybrids of European and Japanese larch there are to our knowledge no commercial plantations with clonal material of European larch. However, clones are used in seed orchards to produce inter- and intraspecific hybrid seeds. Clonal identification with the help of sufficiently variable nuclear markers could help assessing the exact composition of seed orchards to control propagation operations.
Plant material and DNA isolation
In 2010, we collected phloem and needle samples from 18 populations forming a gradient over the natural distribution range (Table 2). Eight had been collected in situ and 10 ex situ in four German provenance trials. In the latter case, each population sample (consisting in 24 individuals) originated from one single trial. Further samples for marker validation as well as for transferability tests were provided by colleagues. These included six progenies (each comprising one female parent and seven offspring) that were collected in a progeny trial (Planches, France). Note that progeny tests in this study only give a rough insight into Mendelian segregation but should help detect null alleles. Originally we planned to start with 12 progenies (12 female parents and 7 offspring) based on seeds. Due to problems with material, we were had to work with the six progenies described above. Furthermore, we obtained DNA samples of another six Larix species. These were L. sibirica (21 individuals from one population), L. kaempferi (12 individuals from 12 populations) and L. gmelinii var. japonica (12 individuals from 12 populations), all from Eurasia. From North America, we obtained samples from L. laricina (10 individuals from 10 populations), L. lyallii (4 individuals from 4 populations), and L. occidentalis (4 individuals from 4 populations). For the samples that we collected ourselves, we mostly relied on phloem as tree height (up to 40m in the trials) made it difficult to collect needles. Phloem was sampled by using a hammer and small leather punch (Ø=1cm, length=10cm). The sampling technique we developed was rapid and easy. The leather punch was positioned between the bark scales (which can be very thick) and with one to three slight hammer strokes a small but sufficient sample (Ø=1cm, depth 1.5cm) was recovered. Damage to the tree was minimal and fast regeneration was ensured by sampling during the growing season. Samples were then put into tea bags that were stored in sealed plastic bags with 10g of silica gel. We isolated DNA from all individuals using 96-well plates. Starting material was mostly phloem (1cm disc 0.5mm thick), but in some cases needles were used (1-3 needles, cut into 2mm pieces). For material disruption, we added two 4mm-tungsten beads to the wells with the starting material. The plates were frozen during 1min in liquid nitrogen before a 1:30 min disruption by a Mixer Mill (Retsch, Germany). This step was repeated once. An Invisorb DNA 96 plant HTS kit (Invitek, Germany) was then used for DNA isolation following the manufacturer protocol. After isolation, DNA quality was evaluated on a 1% (w/v) agarose gel stained with GelRed (Biotium, USA).
Cytonuclear disequilibrium and nuclear admixture analysis
In the combined mito-nuclear analysis we tested for the association between mitochondrial and nuclear lineages. We used a z-test to compare admixture proportions among mitochondrial groups and a one-sided student test to compare q-values among populations with and without indication of recent translocations (cf. Results section).
Translocation frequency, intensity and source areas
We compared observed translocation frequencies with theoretical expectations from the Poisson law and tested if there was a difference with a χ2-test. Existence of regional differences in translocation frequency was tested by comparing proportions of purebred and admixed individuals found in the Alpine and the Central European region using a z-test. To test if translocations often involved multiple sources, we counted the number of different nuclear cluster per population and compared results among populations with and without indication for translocation using a Wilcoxon rank-sum test.
Table of contents :
List of figures
List of tables
CHAPTER 1: General introduction and acknowledgements
Structure of the thesis
Scientific contributions and acknowledgements
CHAPTER 2: Description of the species and review of existing markers
Review of existing genetic markers
Levels of differentiation
Traceability systems in use and future needs
CHAPTER 3: Two highly informative dinucleotide SSR multiplexes for European larch
Materials and methods
Results and Discussion
Conclusions and perspectives
CHAPTER 4: Translocation genetics of European larch
Material and methods
Systematic detection of translocations
The translocation process
Reconstruction of ancient genetic structure
Conclusions and Perspectives
CHAPTER 5: Millennial scale flexibility of European larch populations
Materials and methods
Results and discussion
MIS 5 (~130 – 73.5 ka)
MIS 4 (~73.5 – 59.4 ka)
MIS 3 (~59.4 – 27.8 ka)
MIS 2 (~27.8 – 14.7 ka)
MIS 1 (since ~14.7 ka)
Summary and conclusions
CHAPTER 6: Synthesis and perspectives
Ancient genetic structure
Climatic and anthropogenic impacts