Phylogeography, flowering phenology and cytonuclear interactions of Populus alba and P. tremula

Während der langen Eiszeiten des Quartärs waren die Europäischen Wälder stärker eingeengt als heutzutage, wegen dem Mittelmeer im Süden und der unwirtlichen Umweltbedingungen im Norden. Zwei ökologisch wichtige Baumarten der Gattung Populus: Weisspappel (Populus alba, L.) und Zitterpappel (Populus tremula, L.), sind entlang von Europäischen Auwaldlandschaften beziehungsweise in angrenzenden höher gelegenen Wäldern verbreitet. Beide haben ihr natürliches Verbreitungsgebiet nach der letzten Eiszeit auf ihr heutiges Areal ausgedehnt. Fehlende Pollendaten für Pappeln erschwert die Suche nach Glazialrefugien – diese Refugien werden in Südeuropa vermutet, jedoch fehlen bislang überzeugende Beweise für diese Hypothese. Das Hauptziel des ersten Kapitels war die Klärung der postglazialen Rückwanderung nach der letzten Eiszeit und die Rekonstruktion von Refugien von P. alba und P. tremula im Licht von Hybridisierung der beiden Arten. Ich untersuchte 26 Populationen aus Mittel- und Südosteuopa mithilfe von mütterlich vererbten Chloroplastenmarkern (cpDNA). Phylogeografische Strukturen wurden für P. alba gefunden, mit niedriger Diversität in Osteuropa und hoher Diversität in Italien und Mitteleuropa. Für P. tremula wurde keine Struktur gefunden, was bei einer borealen Baumart durchaus erwartet werden kann. Zwei Hauptrefugien können demnach für P. alba in Italien und Rumänien vermutet werden, hingegen hat P. tremula ihre heutigen Standorte in Mitteleuropa von mehreren Refugien nahe am früheren Eisschild zurückerobert. Ich vermute deshalb gesonderte Refugien für P. alba und P. tremula und schlage ein Immigrationsszenario vor, das die Vermischung der Wanderungswege und interspezifische Introgression für das beobachtete Muster verantwortlich macht. Um zu testen, ob dieses Muster geeignet ist, um Individuen einer bestimmten Population zuzuordnen, wurde die genetische Diversität von P. alba auf Malta untersucht. Insgesamt wurden 38 Proben von den beiden benachbarten Inseln und Nachbarregionen innerhalb des Mittelmeergebietes getestet. Die Untersuchung mittels Kernmikrosatelliten (nuSSR) ergab, daß die 28 untersuchten Bäume von Malta einen Klon darstellten. Chloroplastenmarker deuten auf die Verwandtschaft des Klons mit italienischen P. alba Proben hin. Allerdings stammt väterlicher Anteil vermutlich auch von Pollen aus Nordafrikanischen Populationen. P. alba gibt es auf Malta vermutlich seit dem16. Jh. Wahrscheinlich sind menschliche Aktivitäten und die Funktion des Baumes als Zierpflanze für dessen Ursprung und Verbreitung auf Malta verantwortlich. Um einen formalen Weg zu finden, Individuen den Arten und dem Hybriden bei Beprobungen in Hybridzonen zuzuordnen, wurden morphologische Merkmale getestet. Hierbei wurde versucht abzuschätzen, inwiefern blattmorphologische Merkmale für die Artzuordnung brauchbar waren, zugleich aber sollten es einfache Merkmale sein, die auch im Freiland gemessen werden können. Ich stelle eine Methode vor, die P. alba, P. ×canescens (Aiton, Sm) und P. tremula in einer Hybridzone in Österreich unterscheiden kann. Dabei wird die „Gelapptheit“ der Blätter gemessen, und unterstützt von zwei zusätzlichen Merkmalen identifizieren diese den Hybriden P. ×canescens. Die Anmerkung in der Literatur, daß “Gelapptheit” ein ausgeprägtes Merkmal für die Weißpappel ist, kann nun über den HF-index und das L1/L2 Verhältnis gemessen werden. Darüber hinaus konnte gezeigt werden, daß die zweistufige Vorgehensweise zur Probensammlung erfolgreich war, um die Heterophylie zu behandeln. Hier wurden die nützlichsten Zweigtypen für Morphometrie in den untersuchten Arten identifiziert. Für die Hybridsierung von zwei Pflanzenarten müssen diese in ihren Blütezeiten überlappen. P. alba und P. tremula sind diözische Bäume, die mehrere Hybridzonen in Europa bilden. Hier verwendete ich einen kleinräumigen Ansatz, um präzygotische Barrieren für den Genfluß zu studieren, der auf blühphänologischen Beobachtungen basiert. Die Beobachtungen dazu wurden in zwei aufeinanderfolgenden Jahren auf drei Standorten und insgesamt an 27 Bäumen durchgeführt. Die Gesamtblühdauer variierte zwischen Individuen und Jahren. Jedoch blühten in beiden Jahren die männlichen Bäume vor den weiblichen in beiden Arten und auf allen Standorten (sog. „Protandrie“). Lokales Klima und unterschiedliche Standortseigenschaften ermöglichten ein Überlappen der Blühzeiten der beiden Arten. Insgesamt wurden keine sehr starken Barrieren gegenüber Genfluß beobachtet, jedoch beeinflussen jährliche Kimavariationen dieses Muster. Um postzygotische Barrieren gegenüber Genfluß zu testen, untersuchte ich die Interaktion zwischen zytoplasmatischen und nuklearen Genmarkern. In dieser Studie wurde cpDNA und Kern-DNA von insgesamt 541 Individuen aus 23 Populationen von P. alba und P. tremula aus Mitteleuropa untersucht. Innerhalb von P. alba wurden stärkere geografische Strukturen gefunden, als für P. tremula, vermutlich aufgrund von Wiedereinwanderung aus unterschiedlichen Glazialrefugien. Im Gegensatz dazu war die Variation der cp-DNA einheitlicher über die untersuchten Populationen verteilt. Hybridisierung wurde in beide Richtungen in den Kontaktzonen der beiden Arten beobachtet und die meisten Hybriden waren aus der F1 oder F2 Generation. Signifikante zytonukleare Interaktionen wurden innerhalb der Hybride und in P. alba an sechs beziehungsweise vier Kernmarkern festgestellt. Obwohl Faktoren wie nicht-zufällige Paarung und Migration eine Rolle spielen können, wirken diese eher auf alle Genorte gleichzeitig. Daher sind wahrscheinlich selektive Mechanismen eine bessere Erklärung für die beobachteten Muster. Kopplung von Mikrosatelliten mit bestimmten Genen, die in zytonukleare Interaktionen involviert sind, könnten das beobachtete Ungleichgewicht erzeugt haben; speziell in Hybriden der ersten Generationen, die relativ große Chromosomenblöcke enthalten, die sie von ihren Elternarten geerbt haben.During the long glacial periods of the Quaternary, European forests were considerably more restricted than today because of the Mediterranean Sea in the south and unsuitable environment in the north. Two ecologically important tree species of the genus Populus: the white poplar (Populus alba, L.) and the European Aspen (Populus tremula, L.), are distributed along European floodplain areas and adjacent upland forests, respectively. Both have extended their natural ranges to the present area after the last glaciation. Lacking fossil pollen data for poplar hampers the search for glacial refugia – these refugia are assumed in South-eastern Europe, but there is no convincing proof of this hypothesis yet. The central aim of the first chapter was to clarify the picture of postglacial recolonisation and the reconstruction of refugia of P. alba and P. tremula in the light of hybridisation of the two species. I investigated 26 populations located in Central and South-eastern Europe using maternally inherited chloroplast (cp) markers. Phylogeographic structure was found for P. alba with low diversity in Eastern Europe versus high diversity in Italy and Central Europe. In contrast, lack of phylogeographic structure seems to prevail for P. tremula, as can be expected for a boreal forest tree. Two main refugia were identified for P. alba in Italy and Romania, but in contrast P. tremula probably recolonised its present habitats in Central Europe from several refugia near the former ice cap. I assume separate disconnected refugia for P. alba and P. tremula and suggest an immigration scenario involving the mixing of colonization routes and interspecific introgression to be responsible for the observed patterns. In order to test whether these patterns can be useful in assigning individuals to populations, the genetic diversity of P. alba of Malta was assessed. Altogether 38 samples from the two different, although neighbouring islands of Malta and adjacent regions within the Mediterranean were analysed. Nuclear microsatellite analysis (nuSSR) revealed that the 28 sampled trees of Malta belonged to one clone. Chloroplast data suggested relatedness of the Maltese clone to Italian P. alba samples. However, nuclear data revealed additional admixture through pollen from North Africa. Human activities for ornamental purposes can be considered the main cause for introducing this clone in the 16th century. In order to find a more formal way of assigning individuals to species and hybrids when sampling individuals within hybrid zones, morphological traits were tested. The main purpose of assessing the utility of leaf morphological parameters for species assignment was to establish an easy measurable trait, which can be applied in the field. I present a method of separating P. alba, P. ×canescens (Aiton, Sm) and P. tremula in an Austrian hybrid zone using the degree of "lobedness" of the leaves, supported by two additional parameters, which identify P. ×canescens, the hybrid taxon. The statement in the literature that “lobedness” is a distinctive character in white poplar is now measurable by the HF index and the L1/L2 ratio. Furthermore, the two-step sampling approach was successful in dealing with heterophylly and identifying the most valuable type of shoot for morphometry in the studied taxa. Another aspect of hybridisation in nature is that plant species have to overlap in their flowering time in order to hybridise. P. alba and P. tremula are dioecious trees occurring in several hybrid zones across Europe. Here I used a small scale approach to study prezygotic barriers to gene flow based on flowering phenological observations. Flowering assessment was carried out in two consecutive years in three study plots and a total of 27 trees. Overall flowering period varied between individuals and years. However, in both years male trees started flowering earlier than females of both species on each site (protandry). Here, overlapping flowering times due to local climatic and site conditions between species on different sites facilitated hybridisation. In fact, I observed not very strong barriers to gene flow, although year to year climatic variation can influence this pattern. In order to test for postzygotic barriers to gene flow I studied the interaction between cytoplasmic and nuclear gene markers. Chloroplast and nuclear DNA of altogether 541 individuals in 23 populations of P. alba and P. tremula in Central Europe were investigated. Within P. alba, stronger geographic structuring was found compared to P. tremula based on cpDNA and twenty nuSSRs, presumably due to recolonisation from disconnected glacial refugia. Conversely, in P. tremula genetic variation was more evenly distributed across the studied populations. Hybridisation was observed in both directions in zones of contact. Significant cytonuclear interactions were detected within hybrids (P. ×canescens) and P. alba at six and four nuclear loci, respectively. Although factors like assortative mating and migration may play a role in their origin, those are likely to affect all loci. Thus, selective mechanisms are a more likely explanation for these patterns. Linkage of microsatellite markers to certain genes involved in cytonuclear processes might cause the observed disequilibria, especially in early generation hybrids carrying relatively large chromosome blocks inherited from each parental species

Identification of varieties and gene flow in Douglasfir exemplified in artificially established stands in Germany

Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] is an economicallyvaluable non-native tree species in Germany and is considered verypromising in view of global climate change. Therefore, the genetic characterization of Douglas-fir populations and seed stands in Germany is essential.We studied coastal and interior Douglas-fir varieties, both presentin Germany, by using eleven isoenzyme and four microsatellite loci. Byanalyzing eight reference populations of known origin we were able toidentify the two varieties on the population level using Bayesian and distance based methods. Seven populations present in Bavaria were then successfully assigned to one of the two varieties. Within varieties we found stronger grouping within the interior variety than within the coastal one. Despite lower differences within coastal Douglas-fir we have first indications for the origin of two populations. For two Bavarian populations, natural regeneration was included and genetic data revealed no significant genetic difference between adults and offspring. The parentage analysis for one of the studied stands revealed that a large proportion of adults took part in the reproduction, but some trees were more successful than othersin transferring their genes to the next generation. Our study was ableto improve variety identification of Douglas-fir using isoenzyme markers and nuclear microsatellites and study reproductive patterns, both are important issues for the management of Douglas-fir stands in Bavaria

Exploring the Gene Pool of Silver Fir in Southern Germany on the Search for Climate-Smart Seed Sources

Central European populations of silver fir (Abies alba Mill.) grow under a relatively wide amplitude of environmental conditions. Assuming that forest tree stands are locally adapted, the use of forest reproductive material from sites with limited water availability is expected to increase drought tolerance in future forests. At the same time, genetic diversity is of utmost importance as the basis of adaptation to a changing environment. Currently, a selection of potential sources for climate-smart reproductive material of silver fir is under way in Southern Germany. It is based on a multidisciplinary approach elucidating the genetic variation, site conditions, as well as tree resilience based on a dendroecological approach. The analysis encompasses a large number of stands representing the whole range of the species’ ecological requirements. The population genetic analysis based on molecular markers (nuclear microsatellites) provided important information concerning the gene pool of the species in Southern Germany. On one hand, it revealed genetic differentiation and transition zones between western and eastern clusters. On the other hand, the results indicated gradients and regional variation of genetic diversity. These patterns can be explained by post glacial recolonization and the peripheral character of the species at the northern limit of its distribution. Together with the outcomes of the site condition and dendroecological approaches, the results of the genetic analysis will be used to inform future provenance recommendations

Towards the dynamic conservation of Serbian spruce (Picea omorika) western populations

Key message High levels of genetic diversity, pronounced genetic structure and limitations to gene flow in Serbian spruce, a rare and endangered tree species from the refugial Balkan region, point towards a "one population-one unit" strategy for assembling a network of Genetic Conservation Units (GCUs) for its dynamic conservation. On the other hand, genetic information also permits to prioritize populations for conservation based on their contribution to genetic diversity and differentiation. Context Serbian spruce, Picea omorika (Panc.) Purk., is a rare, IUCN red-listed European conifer endemic to the Balkan region. Its current rigid conservation (without any intervention allowed in similar to 30 remnant populations) and the extant network of Genetic Conservation Units (four natural populations and three planted stands from the western part of the species range, in the Republic of Srpska, Bosnia and Herzegovina, RS-BH) might be ineffective in preserving the species' genetic diversity. Aims To facilitate implementation of dynamic conservation of Serbian spruce by re-assessing the number and size of remnant populations in RS-BH and updating genetic knowledge on these understudied western Serbian spruce populations. Methods Comprehensive field survey in RS-BH, genotyping 689 individuals from 14 western populations with ten highly informative nuclear EST-SSRs and analytical methods for prioritizing populations for conservation based on their contribution to the geographical structuring of genetic diversity. Results The genetic diversity of western Serbian spruce populations (Ae = 2.524, H-E = 0.451) is comparable with what was found for eastern ones; they are highly genetically differentiated (Hedrick's G'(ST) = 0.186; Jost's D = 0.097) and comprise ten distinct gene pools. Effective population size is often gt = 15. As much as 14% of alleles is not preserved in the extant GCUs established in natural populations. Eight populations positively contribute to within-population genetic diversity, four to genetic differentiation, and two are globally important in terms of diversity and differentiation. Although wildfires may contribute to admixture of different gene pools, re-establishment from seeds from extirpated populations has likely prevailed in studied populations. Conclusions A larger network of GCUs is required for the dynamic conservation of western Serbian spruce populations. A "one population-one unit" strategy, with 14 GCUs, would represent the safest approach to conserve species extant genetic variation in this part of the species range. Nonetheless, a strategy to prioritize populations for conservation based on their contribution to allelic diversity has been put forward. Given the rapid global warming and peculiarities of Serbian spruce distribution, habitat and life history traits, conservation measures based on a rigorously designed GCU network are urgent for its rescue and survival

Monitoring of species’ genetic diversity in Europe varies greatly and overlooks potential climate change impacts

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species’ joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union’s Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity

On the use of the L1 in beginner’s English and French language classrooms

Die Verwendung der Muttersprache im Fremdsprachenunterricht ist ein viel diskutiertes Thema in der Sprachdidaktik. Im Laufe des 20. Jahrhunderts wurde die Rolle der Muttersprache größtenteils ignoriert, was implizierte, dass die exklusive Verwendung der Zielsprache die beste Methode ist, um Fremdsprachen zu unterrichten. Allerdings wird dieses Prinzip von Einsprachigkeit zunehmend kritisiert und immer mehr ForscherInnen betonen, dass die Muttersprache ein wertvolles Werkzeug für das Lehren und Lernen von Fremdsprachen sein kann. Diese Fallstudie erforscht Englisch- und Französisch-Anfängerunterricht in Österreich und untersucht die Verwendung der Muttersprache im Unterricht und die Ansichten gegenüber dieser bei FremdsprachenlehrerInnen. Daten wurden in LehrerInneninterviews und Unterrichtshospitationen gesammelt und mit Hilfe der qualitativen Inhaltsanalyse ausgewertet. Die Resultate zeigen, dass die FremdsprachenlehrerInnen in dieser Studie an die Notwendigkeit einer größtmöglichen Verwendung der Zielsprache glauben, damit Spracherwerb stattfinden kann. LehrerInnen verwenden die Muttersprache vor allem, um Grammatik zu unterrichten, um eine persönliche Beziehung zu den SchülerInnen aufzubauen, um SchülerInnen zu disziplinieren und um über Themen zu sprechen, die nicht direkt den Fremdsprachenunterricht betreffen. Das Ziel, welches LehrerInnen mit ihrer Sprachwahl verfolgen, ist zuallererst das Verständnis bei den SchülerInnen zu sichern. Weitere Gründe für die Verwendung der Muttersprache sind Zeitersparnis und die Schaffung einer guten Lernumgebung. Zusätzlich werden in dieser Arbeit interne und externe Faktoren aufgezeigt, die die LehrerInnen in ihren Entscheidungen beeinflussen. Die Natur der Zielsprache kann als Einflussfaktor für den Sprachgebrauch bei LehrerInnen ausgeschlossen werden. Das Können der SchülerInnen und Ansichten gegenüber erfolgreichem Spracherwerb haben einen deutlichen größeren Einfluss auf die Verwendung der Muttersprache bei LehrerInnen.The use of the first language in the foreign language classroom is a controversial issue in language teaching methodology. Throughout the 20th century, the role of the mother tongue was largely ignored, suggesting that target language exclusivity is the preferable way of teaching a foreign language. This monolingual principle is increasingly challenged, and it is suggested that the first language is a valuable resource beneficial for foreign language teaching and learning. This case study explores beginners’ English and French language classrooms and investigates Austrian teachers’ beliefs and practices of first language use. The analysis of teacher interviews and classroom observations shows that the first language serves pedagogical and communicative functions. The results demonstrate that language teachers of this study believe in the need for maximal use of the target language to allow for successful language acquisition. The first language is predominantly used by teachers for teaching grammar, establishing a personal relationship, disciplining students, and discussing non-school related issues. The goal teachers pursue with their language choice is primarily to ensure comprehension, in addition to saving time, and creating a supportive learning environment. Various internal and external factors have been identified to influence teachers. The nature of the target language can be eliminated as a variable. Students’ proficiency and teachers’ beliefs about second language acquisition have a much greater impact on teachers’ use of the first language

Phylogeography of Populus alba (L.) and Populus tremula (L.) in Central Europe: secondary contact and hybridisation during recolonisation from disconnected refugia

The central aim of this paper is to clarify the picture of postglacial recolonisation and the reconstruction of refugia of Populus alba (L.) and Populus tremula (L.) in the light of hybridisation of the two species. We focussed our study on Central and Southeastern Europe including reference samples from Spain, Sweden and Northern Africa.We investigated 414 individuals of 26 populations using restriction fragment length polymorphisms (PCR-RFLPs) in six maternally inherited chloroplast markers. Altogether, 57 haplotypes were analysed of which four indicated hybridisation events in the past. Phylogeographic structure was found for P. alba with low diversity in Eastern Europe versus high diversity in Italy and Central Europe. A lack of phylogeographic structure was assessed for P. tremula as expected for a boreal forest tree, and diversity was evenly distributed in the studied populations. Two main refugia were identified for P. alba in Italy and Romania. A previously described hybrid zone between species in Central Europe turned out also to be a zone of contact between southern and eastern chloroplast lineages in P. alba. In contrast, P. tremula recolonised its present habitats in Central Europe from several refugia near the former ice cap. We assume separate disconnected refugia for P. alba and P. tremula and suggest an immigration scenario involving the mixing of colonisation routes and interspecific introgression to be responsible for the observed patterns

Genetic variability of pedunculate oak (Quercus robur L.) at the Mediterranean margin of the distribution range

In Bosnia and Herzegovina, pedunculate oak (Quercus robur L.) occurs at the southern margin of its distribution range, close to the glacial refugia of this species. To assess the patterns of genetic diversity distribution at the rear edge of the Holocene colonization, we studied genetic variation in 20 pedunculate oak populations using 14 allozyme loci. Despite considerable differences among populations, neither the numbers of alleles nor genetic diversity showed any geographical trend within the studied area, although small isolated populations showed generally lower allelic richness. The Bayesian analysis of population structure indicated a kind of geographical pattern. We identified no signs of a recent bottleneck. The proximity to multiple glacial refugia explains the outcomes