Larix

Retrospective studies focussing on forest dynamics using fossil and genetic data can provide important keys to prepare forests for the future. In this study we analyse the impact of past climate and anthropogenic changes on Larix decidua Mill. (European larch) populations based on a new range-wide fossil compilation encompassing the last 130 ka and on recently produced genetic data (nuclear, mitochondrial). Results demonstrate that during the last 130 ka L. decidua persisted close to its current distribution range and colonized vast areas outside this range during the first two early Weichselian interstadials (c. 87-109 ka and c. 83-78 ka), reaching a distributional maxima in the north-central European lowlands. Some fossil sites point to notably rapid responses to some abrupt climate events DansgaardeOeschger cycles and Heinrich Events). Combined fossil and genetic data identify at least six MIS 2 refuges and postglacial recolonization pathways. The establishment of extant L. decidua forests dates back to the first two millennia of the Holocene (c. 11.5-9.5 ka) and the onset of anthropogenic impact was inferred since the late Neolithic (c. 6 ka), with major changes occurring since the Bronze Age (c. 4 ka). During the last 300 years human-induced translocations resulted in recent admixture of populations originating from separate refuges. Altogether, the results of this study provide valuable clues for developing sustainable conservation and management strategies targeting ancient genetic lineages and for studying evolutionary issues.

Study of lipophilic and hydrophilic extractives in heartwood, sapwood and knots of 39 industrially important conifer species: 14 pines (Pinus), 7 spruces (Picea), 9 firs (Abies), 5 larches (Larix), 3 hemlocks (Tsuga) and Douglas-fir (Pseudotsuga).

The wood samples were sequentially extracted, and the amount and composition of resin acids, fatty acids, sterols, steryl esters, acylglycerols, juvabiones, lignans, oligolignans, flavonoids and stilbenes were analysed by gas chromatography (GC) and GC-mass spectrometry.

Tree-ring stable isotopes, providing insight into drought-induced eco-physiological mechanisms, are frequently used to reconstruct past changes in growing season temperature and precipitation. Their climatic response is, however, still not fully understood, particularly for data originating from non-extreme, mid-latitude environments with differing ecological conditions. Here, we assess the response of δ(13)C, δ(18)O and tree-ring width (TRW) from a temperate mountain forest in the Austrian pre-Alps to climate and specific drought events. Variations in stem growth and isotopic composition of Norway spruce, common beech and European larch from dry, medium and moist sites are compared with records of sunshine, temperature, moisture, precipitation and cloud cover. Results indicate uniform year-to-year variations in δ(13)C and δ(18)O across sites and species, but distinct differences in TRW according to habitat and species. While the climate sensitivity of TRW is overall weak, the δ(13)...

CHAPTER 1
Phylogeography of Larix sukaczewii Dyl. and L. sibirica L. inferred from nucleotide variation of nuclear genes.

The phylogeography of Larix sukaczewii and L. sibirica was investigated using nucleotide variation at the four following nuclear gene regions: 5.8S rDNA (including two internal transcribed spacers (ITS)), glyoxysomal malate dehydrogenase (gMDH), cinnamyl alcohol dehydrogenase (CAD) and phytochrome-O (PHYO). Sequences of the 4-coumarate: coenzyme A ligase (4CL) gene region obtained in a recent study were also included. CAD and PHYO showed very low nucleotide variation, but ITS, 4CL and gMDH had levels of variation similar to those reported for other conifers. Neutrality tests showed significant deviations at the gMDH region. Namely, positive values of Tajima’s D, Fu and Li’s D and F (with and without outgroup) were observed in all but one population of L. sukaczewii, but negative values were observed in all populations of L. sibirica.
Pleistocene refugia have been hypothesized to exist in the southern Urals and south-central Siberia,
where four out of nine of the investigated populations occur. Moderate to high levels of population
differentiation were found in some pair-wise comparisons suggesting limited gene flow and
independent evolution of some refugial populations. In L. sukaczewii, low levels of differentiation
were found among populations from areas glaciated during the Pleistocene, indicating their recent
origin. Results of this study also suggest these populations were created by migrants from multiple,
genetically distinct refugia. Furthermore, some haplotypes observed in populations from previously
glaciated areas were not found in putative refugial ones, suggesting these populations might have
contributed little to the extant populations created after the Last Glacial Maximum (LGM). Some
authors regard L. sukaczewii and L. sibirica as a single species, while others consider them as separate species. The observed conspicuous differences in haplotype composition and distribution
between L. sukaczewii and L. sibirica, together with high values of FST between populations of the
two species appear to support the latter classification.

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CHAPTER 2

Genetic structure of Dipterocarpus alatus Roxb. populations from Thailand
revealed by nuclear microsatellites.

Four populations of Dipterocarpus alatus from Thailand using nuclear microsatellite loci were investigated. Two populations were from mainland Thailand, one from Samui Island and one from Malay Peninsula. Nine pairs of primers originally designed for the related species Shorea curtisii were tested. However, only four loci were successfully amplified; and only two of them (Shc02 and Shc07) were polymorphic. Null alleles appeared to be present at the Shc07 locus, but significant deviations from HWE was found only in Samui population at this locus. Levels of genetic variation observed in D. alatus were similar to those revealed in other studies on tropical trees using microsatellites. The levels of population differentiation (FST and RST) for microsatellites were lower than those observed for isozymes in another study on D. alatus that included three out of four populations investigated in this study. Genetic distances; however, were generally consistent between microsatellites and isozymes in pair-wise comparisons, except between Kuphrakona (mainland) and Hat Yai (Malay Peninsula), where microsatellite results suggested these two geographically distant populations were relatively close to each other, but isozymes suggested the opposite. This discrepancy could have been caused by homoplasy at microsatellite loci in these two populations.
The genetic distances showed that Hat Yai was the most isolated population indicating it has been isolated for a relatively long period of time. The two mainland populations were closer to each other and the population from Samui had an intermediate position between mainland and Hat Yai, in genetic distances, which is consistent with its geographical position. Therefore, gene flow could have occurred between Samui and other populations in the past.

The chances for sympatric speciation are improved if ecological divergence leads to assortative mating as a by-product. This effect is known in parasites that find mates using host cues, but studies of larch- and pine-feeding races of the larch budmoth (Zeiraphera diniana, Lepidoptera: Tortricidae) suggest it may also occur when mate attraction is via sex pheromones that are independent of habitat. We have previously shown that females releasing pheromones on or near their own host attract more males of their own race than if placed on the alternative host. This host effect would enhance assortative mating provided adults preferentially alight on their native hosts. Here we investigate alighting preferences in natural mixed forest using a novel likelihood analysis of genotypic clusters based on three semidiagnostic allozyme loci. Both larch and pine females show a realized alighting preference for their own host of 86%. The equivalent preferences of males were 79% for the larch race and 85% for the pine race. These preferences are also detectable in small-scale laboratory experiments, where alighting preferences of larch and pine races towards their own hosts were, respectively, 67 and 66% in females and 69 and 63% in males. Pure larch race moths reared in the laboratory had alighting choice similar to moths from natural populations, while hybrids were intermediate, showing that alighting preferences were heritable and approximately additive. The field estimates of alighting preference, coupled with earlier work on mate choice, yield an estimated rate of natural hybridization between sympatric host races of 2.2-3.8% per generation. Divergent alighting choice enhances pheromone-mediated assortative mating today, and is likely to have been an important cause of assortative mating during initial divergence in host use. Because resources are normally 'coarse-grained' in space and time, assortative mating due to ecological divergence may be a more important catalyst of sympatric speciation than generally realized.

Hybrids between European and Japanese larches combine the properties of both parental species (drought resistance, canker resistance, stem straightness) and exhibit a fast growth rate. They are produced in seed orchards, generally by natural pollination. Seeds are collected and used for afforestation as interspecific hybrids. However, there are no convenient tests to assess the interspecific hybrid proportion. In the present study, we developed diagnostic molecular markers suitable for the individual identification of hybrids, whatever their developmental stage. Our strategy involved testing a combination of maternally inherited markers from the mitochondrial genome (mtDNA) and paternally inherited markers from the chloroplast genome (cpDNA). Hybrids were then identified by the presence of a mitochondrial sequence inherited from one parental species and a chloroplast sequence inherited from the other parental species. To achieve this aim, markers discriminating both parental species were first sought. Amplifications of mitochondrial and chloroplast sequences were performed using specific PCR primers. After testing 33 primer pairs in combination with nine restriction enzymes, we detected one mitochondrial marker, f13 which was amplified in Japanese larch and absent in European larch, and one chloroplast marker, ll- TaqI which showed different restriction patterns depending on the species. A restriction fragment of 601 bp was obtained in Japanese larch while two fragments of 120 bp and 481 bp were observed in European larch. These patterns were found in all 197 individuals tested from the two pure species. These markers were then used for the evaluation of the hybrid proportion in a seed lot produced from seed orchards; this was assessed as between 43% and 53% depending on the parental species. The male and female parental species could be determined for each progeny.

The Kangchenjunga Conservation Area in Nepal is a potential World Heritage Site because of its unique characteristics and strategic location. The exceptional altitudinal range (from 1,200 m to 8,586 m at Mt.Kangchenjunga, the world’s third-highest peak) within an area of only 2,035 km2 has created pristine habitats for flora and fauna. The local people practice a variety of livelihoods, including agriculture, pastoralism, forestry, and trade, resulting in a vibrant cultural tapestry. The human settlements within the area are some of the highest altitude settlements known in the world that adapt traditional systems for coping with a harsh environment. The area holds both challenges and opportunities from conservation and development perspectives. In order to translate challenges into opportunities, there is a need for a more integrated and coordinated approach with multiple-stakeholder participation. The recognition of the area as a World Heritage property can provide a platform to b...

Larch (Larix Mill.) is one of the most widely distributed tree genera in Eurasia. To determine population structure and to verify classification of five species and three varieties of the Eurasian Larix species, we investigated levels and patterns of nucleotide variation of two nuclear gene regions: the 4-coumarate coenzyme A ligase (4CL) and the coumarate 3-hydroxylase (C3H). In the 4CL region nucleotide diversity at silent sites (π sil) varied between 0.0020 in L. gmelinii to 0.0116 in L. gmelinii var. japonica and in the C3H region between 0.0019 in L. kaempferi to 0.0066 in L. gmelinii var. japonica. In both gene regions statistically significant population differentiation (F ST) was detected among adjacent refugial populations of some species suggesting limited gene flow and/or long time isolation of some refugial populations. On the other hand, populations of L. sukaczewii from northwestern Russia, which was glaciated 20,000 years ago showed no differentiation. This result is consistent with recent postglacial origin of these populations. Haplotype composition of some of the investigated Eurasian Larix species suggested that they are considerably diverged. Some haplotypes were unique to individual species. Our results indicate that more intensive sampling especially from known refugial regions is necessary for inferring correct classification of Eurasian Larix species and inferring their postglacial migration.

Ozone (O3) pollution and soil infertility may negatively affect boreal forests across the Northern Hemisphere. Impacts to economically and ecologically important larches (Larix sp., Pinacaeae) are particularly concerning. Using a free air O3 enrichment (FACE) system, we investigated the effect of 2-year elevated O3 exposure (≈66 nmol mol(-1)) on Japanese larch (L. kaempferi) and its hybrid larch F1 (L. gmelinii var. japonica × L. kaempferi) planted directly into either fertile brown forest soil (BF) or BF mixed with infertile volcanic ash soil (VA). Overall, photosynthetic pigmentation and the growth performance of the stem and crown were reduced in both taxa exposed to elevated O3. Furthermore, hybrid larch, in both O3 treatments, performed better than Japanese larch. This finding contradicts findings of prior experiments with potential experimental artifacts of O3 exposure facilities and root restrictions. Elevated O3 also disproportionately inhibited stem diameter growth and caus...

With the rapid industrial development and modern agricultural practices, increasing nitrogen (N) deposition can cause nutrient imbalance in immature volcanic ash soil commonly found in Japan. Larch species, widely distributed in northeast Eurasia, are associated with ectomycorrhizal (ECM) fungi which play a critical role in nutrient acquisition for their hosts. In this study, we investigated species richness and diversity of ECM fungi associated with a hybrid larch (F1) and its parents, Dahurian larch (Larix gmelinii var. japonica) and Japanese larch (L. kaempferi), under simulated N deposition (0 and 100kgha-1yr-1) with/without phosphorous (P) (0 and 50kgha-1yr-1). Seedlings planted in immature volcanic ash with low nutrient availability were subjected to the N and P treatments for fifteen months. We found that response of ECM community structure to the increased nutrient availability depended on host genotypes. Nutrient addition significantly affected ECM structure in Japanese lar...

Several deciduous broad-leaved tree species, differing in leaf phenology, invade larch (Larix kaempferii (Lamb.) Carrière) plantations in Japan. The understory light environment of larch forests changes drastically between the leafy and leafless periods. To determine how the invading seedlings exploit the changing light environment, and if phenological differences reflect the light- and nitrogen-use traits of the seedlings, we measured leaf phenology, seasonal changes in light-saturated photosynthetic rate (P(sat)), leaf nitrogen (N) content (N(area)), chlorophyll/nitrogen ratio (Chl/N), specific leaf area (SLA) and N remobilization rate (NRMR) over 3 years. The mid-successional or gap-phase species, Magnolia hypoleuca Siebold & Zucc., had a short leafy period and high P(sat) and NRMR. In contrast, two late-successional tree species, Prunus ssiori Friedr. Schmidt, which undergoes leaf flush before larch, and Carpinus cordata Blume, which maintains green leaves until frost, both had ...

Forest management under a changing climate requires assessing the effects of climate warming and disturbance on the composition, age structure, and spatial patterns of tree species. We investigated these effects on a boreal forest in northeastern China using a factorial experimental design and simulation modeling. We used a spatially explicit forest landscape model (LANDIS) to evaluate the effects of three independent variables: climate (current and expected future), fire regime (current and increased fire), and timber harvesting (no harvest and legal harvest). Simulations indicate that this forested landscape would be significantly impacted under a changing climate. Climate warming would significantly increase the abundance of most trees, especially broadleaf species (aspen, poplar, and willow). However, climate warming would have less impact on the abundance of conifers, diversity of forest age structure, and variation in spatial landscape structure than burning and harvesting. Bu...

Larch (Larix Mill.) is one of the most widely distributed tree genera in Eurasia. To determine population structure and to verify classification of five species and three varieties of the Eurasian Larix species, we investigated levels and patterns of nucleotide variation of two nuclear gene regions: the 4-coumarate coenzyme A ligase (4CL) and the coumarate 3-hydroxylase (C3H). In the 4CL region nucleotide diversity at silent sites (πsil) varied between 0.0020 in L. gmelinii to 0.0116 in L. gmelinii var. japonica and in the C3H region between 0.0019 in L. kaempferi to 0.0066 in L. gmelinii var. japonica. In both gene regions statistically significant population differentiation (FST) was detected among adjacent refugial populations of some species suggesting limited gene flow and/or long time isolation of some refugial populations. On the other hand, populations of L. sukaczewii from northwestern Russia, which was glaciated 20,000 years ago showed no differentiation. This result is co...