Phylogenetic relationships and diversification processes in Allium subgenus Melanocrommyum Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. Nat.) Vorgelegt der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg von Frau M. Sc Maia Gurushidze geb. am 11.09.1976 in Kvareli, Georgien Gutachter: 1. Prof. Dr. Martin Röser, MLU Halle-Wittenberg 2. Prof. Dr. Joachim W. Kadereit, Universität Mainz 3. Dr. Frank R. Blattner, IPK Gatersleben Halle (Saale), 19.05.2009 Nothing in biology makes sense except in the light of evolution T. Dobzhansky Everything makes a lot more sense in the light of phylogeny J. C. Avise Contents 1. Introduction 5 1.1. Why phylogeny matters 6 1.2. Plant species-level systematics 7 1.3 Phylogenetic inference 7 Distance methods 7 Maximum parsimony methods 8 Maximum likelihood and Bayesian methods 8 Bootstrap confidence for phylogenetic trees 9 Networks – genealogical methods 12 1.4 Molecular clock 13 Nonparametric rate smoothing (NPRS) 13 Penalized likelihood (PL) 13 1.5 Study group – Allium subgenus Melanocrommyum 14 Taxonomic history and systematics 14 Geographical distribution and ecology 17 Phylogenetic relationships within the subgenus 18 Karyology and ploidy level 19 2. Phylogenetic analysis of nuclear rDNA ITS sequences of the subgenus Melanocrommyum infers cryptic species and demands a new sectional classification 21 2.1 Introduction 21 2.2 Materials and methods 21 Plant Material 21 Anatomy of septal nectaries 22 Molecular methods 23 Data analyses 24 2.3 Results 25 Characteristics of the ITS region and phylogenetic analyses 25 RAPD analysis 30 Anatomy of septal nectaries 30 2.4 Discussion 31 Variation at the ITS locus 31 Divergent ITS types within species and individuals 32 RAPD analysis 33 ITS phylogeny and incongruence with taxonomic classification 33 Reasons for the molecular-taxonomical discordance 37 Nectary types 38 2.5 Conclusions 38 1 3. Species level phylogeny of the subgenus Melanocrommyum – phylogenetic and genealogical analyses of noncoding chloroplast DNA 39 3.1 Introduction 39 3.2 Materials and Methods 40 Taxon Sampling 40 Molecular Methods 41 Data analyses: phylogenetic inference and maximum parsimony network 41 3.3 Results 42 Sequence variation and inference of chloroplast haplotypes 42 TrnF duplication 45 Phylogenetic analyses 45 Statistical parsimony network 48 Phylogenetic and taxonomic relationships of chloroplast haplotypes 49 3.4 Discussion 50 Network construction and comparison of different analyses methods 50 Overall congruence with the nuclear rDNA ITS phylogeny 51 Phylogenetic implications, lineage sorting and hybridization 53 Reasons for current chloroplast haplotype distribution 54 TrnF duplication 54 4. Dating diversification events in the genus Allium and its subgenus Melanocrommyum is impeded by low rbcL and extremely high nrDNA ITS substitution rates 57 4.1 Introduction 57 4.2 Materials and Methods 60 Sampling 60 PCR and sequencing 60 Data sets and phylogenetic analyses 61 Estimation of divergence times 62 4.3 Results 63 Phylogenetic analyses of Allium rbcL sequences 63 Estimation of divergence times 65 4.4 Discussion 68 RbcL variation in Allium 68 The timing of diversification events in the genus Allium and subgenus Melanocrommyum 69 Biogeographic implications 71 2 5. Genome size variation and evolution in the subgenus Melanocrommyum 73 5.1 Introduction 73 5.2 Materials and Methods 75 Plant material 75 Nuclear Genome size Estimation 75 Chromosome numbers 76 Data analyses 80 5.3 Results 81 Genome size correlation with phylogenetic clades 81 Genome size variation 84 Ancestral genome size estimation and genome size evolution 84 5.4 Discussion 85 Genome size variation within the subgenus Melanocrommyum 85 Taxonomic relevance of genome size variation 85 Genome size changes in relation to phylogeny 86 6. Summary 89 6.1 Phylogeny and diversification of the subgenus Melanocrommyum 89 6.2 Eurasian – North American disjunct distribution of Allium 90 6.3 Hybrids versus cryptic species 90 6.4 Reticulations within the subgenus Melanocrommyum 91 6.5 Advantages of network-based methods over bifurcating phylogenies 91 6.6 Genome size and Phylogeny 92 Abstract 93 Zusammenfassung 95 Literature cited 99 Abbreviations, Figure legends and Table captions 115 Acknowledgments 119 Curriculum Vitae 121 Eigenständigkeitserklärung 125 Appendices 127 3 4 Introduction 1. Introduction 1.1 Why phylogeny matters Phylogenetic analyses of DNA or protein sequences have become an important tool for studying the evolutionary history of organisms from bacteria to humans. Since the rate of sequence evolution varies extensively with gene or DNA sequences one can study the evolutionary relationships at virtually all hierarchical levels (e.g., kingdoms, phyla, families, genera, species, and intraspecific classifications) by using different genes or DNA segments (Nei and Kumar 2000). The important realization that comparisons of species or gene sequences in a phylogenetic context can provide meaningful insights into different fields of plant biology, including ecology, molecular biology, and physiology, has been illustrated in several reviews (Soltis and Soltis 2000, 2003; Hall et al. 2002; Doyle et al. 2003). Studies of the rate of evolutionary change as well as estimation of the age of diversification pattern of lineages depend directly on knowledge of phylogenetic relationships (Magallon and Sanderson 2001). The phylogenetic analyses of angiosperms have resulted in their reclassification (APG 1998, APGII 2003). This ordinal-level reclassification is perhaps the most dramatic and important change in higher-level angiosperm taxonomy in the past 200 years (Soltis and Soltis 2000). The more a classification reflects evolutionary history (phylogeny) the more useful it will be to point to taxa to look for important features. For example, taxol is a compound known only from the yew family (Taxaceae). If one wishes to look for additional sources of taxol and other taxanes, rather than randomly sample the plant kingdom, one can focus on the nearest relatives as understood from a phylogenetic tree of the seed plants. This in turn leads to investigate the Podocarpaceae because the phylogenetic analyses indicated that this is the sister group to the yew family. Subsequently, taxanes have been reported also in the Podocarpaceae (Stahlhut et al. 1999). Indeed, phylogenies guide the search for plants of potential commercial importance. Revealing the closest relatives of crops could direct comparative studies and subsequent applications in plant breeding. For instance, Hordeum bulbosum, the sister species of cultivated barley (H. vulgare) has been widely used in breeding, namely in producing the completely homozygous double-haplotype lines of barley due to the fact that H. bulbosum chromosomes are completely eliminated from the H. vulgare × H. bulbosum hybrid (von Bothmer et. al. 1995). Besides, knowing the clear picture of phylogenetic relationships of the cultivated species can point to taxa that may serve as sources of novel traits for crops. The value of placing “model organisms” in the appropriate phylogenetic context is another aspect where phylogenies could help answering fundamental evolutionary questions. The most obvious example concerns the model plant Arabidopsis thaliana and its relatives, where phylogenetic studies revealed that the closest relatives of A. thaliana are the representatives of former genus Cardaminopsis (Koch et al. 2001; Clauss and Koch 2006). The accumulated knowledge in the function and evolution of genes that control responses to draught, pathogens, insects and other environmental challenges, as well as 5 Phylogeny of the subgenus Melanocrommyum knowledge about the genes involved in the evolution of breeding system can be transferred from the model organism to the closest relatives to study these long-standing evolutionary questions (Mitchell-Olds 2001; Mitchell-Olds et al. 2005). In addition, a phylogenetic framework has revealed the patterns of evolution of many morphological and chemical characters, including complex pathways such as nitrogen- fixing symbioses, chemical defense mechanisms and mustard oil production (for review see Soltis and Soltis 2000; Daly et al. 2001). For example, mustard oil glucosinolates are known to be produced in at least 15 different plant families. Before the advances in molecular phylogenetics, this biochemical pathway was thought to have arisen and been lost on multiple occasions. Molecular phylogenetic information, however, indicates that all mustard oil producing families with the exception of the genus Drypetes (a member of Malpighiales) are part of the same ordinal clade Brassicales, indicating only two evolutionary origins for mustard glucosinolates (Rodman et al. 1993). Moreover, these mustard oils are derived from two different biosynthetic pathways, thus showing that even though the final product may be the same, the ways they were synthesized are not; hence they are not homologous in an evolutionary sense (Daly et al. 2001). 1.2 Plant species-level systematics As noted above, plant systematics at the generic and higher levels has advanced greatly in the last decades (Chase et al. 1993; Savolainen et al. 2000; Soltis et al. 2000), and the phylogenetic tree of major angiosperm groups is perhaps “better known than any other equivalent group of organisms” (Barraclough and Reeves 2005). These phylogenies have been used to investigate patterns and processes causing