Gene, Genome und Sequenzen auf der einen Seite, Algorithmen, Computer und Informatik auf der anderen - sie üben Faszination aus, halten aber viele Interessierte auf respektvolle Distanz. Die Schnittstelle der Bereiche ist mit dem modernen Begriff Bioinformatik belegt. In der Tat hat die Synthese von zwei unabhängigen Disziplinen selten so viele faszinierende neue Einsichten geliefert. Eine spannende Teildisziplin der Bioinformatik ist die Molekulare Phylogenetik, deren Ziel die Rekonstruktion von Stammbäumen aus molekularen Daten ist: Computer, moderne Molekularbiologie und Kladistik haben der etwas angestaubten biologischen Systematik und Taxonomie eine ungeahnte Renaissance verschafft. Der Einstieg in beide Welten gleichzeitig - Molekularbiologie und Phylogenetik - war nicht unbedingt einfach. Hier schloss „Gene und Stammbäume“ 2006 eine Lücke. Die zweite Auflage behält das bewährte Konzept bei, ist aber inhaltlich um zwei Kapitel erweitert, die den neuesten Trends unter anderem bei Bayesianischen Ansätzen Rechnung tragen. Einführende Kapitel über Molekularbiologie, Evolution, Taxonomie und Kladistik ermöglichen je nach Wissenshintergrund einen leichten Zugang zur Molekularen Phylogenetik. Den besonders schnellen Einstieg erlaubt ein spezielles Kapitel über den Weg von der Sequenz zum Stammbaum ohne Umwege oder Details. Wer es genauer wissen will, bekommt detaillierte Einführungen in die wichtigen methodischen Ansätze: Parsimonie, Distanzverfahren, Maximum Likelihood und Bayesianische Verfahren. Speziellere Kapitel widmen sich neuen Methoden für stammbaumbasierte statistische Tests, Supertrees, Analysen von Substitutionsraten, molekularer Datierung und vielem mehr. Alles wird hands on anhand von nachvollziehbaren Beispielen mit der gängigen Software besprochen, die aus dem Internet bezogen werden kann. Das Buch bietet so eine ideale Balance zwischen Theorie und Praxis. Es hat zahlreiche Illustrationen, bietet am Ende der Kapitel Hinweise zum Weiterlesen und schließt mit einem Glossar und einem umfangreichen Index.

Pinguicula chuquisacensis is described and illustrated as a new species from the department of Chuquisaca of Bolivia. Data on distribution, ecology and a key for identification among other central Andean species are given. DNA sequence data are presented for this new taxon and related Andean species of Pinguicula for the first time. The plastid tree reveals a lineage of central Andean species (within the clade of tropical growth type species), whereas the northern Andean (Colombia, Venezuela) P. elongata appears distantly related and sister to all remaining taxa of the Mexican-Central American-Caribbean clade of Pinguicula.

Sensitivity analysis provides a way to measure robustness of clades in sequence-based phylogenetic analyses to variation in alignment parameters rather than measuring their branch support. We compared three different approaches to multiple sequence alignment in the context of sensitivity analysis: progressive pairwise alignment, as implemented in MUSCLE; simultaneous multiple alignment of sequence fragments, as implemented in DCA; and direct optimization followed by generation of the implied alignment(s), as implemented in POY. We set out to determine the relative sensitivity of these three alignment methods using rDNA sequences and randomly generated sequences. A total of 36 parameter sets were used to create the alignments, varying the transition, transversion, and gap costs. Tree searches were performed using four different character-coding and weighting approaches: the cost function used for alignment or equally weighted parsimony with gap positions treated as missing data, separate characters, or as fifth states. POY was found to be as sensitive, or more sensitive, to variation in alignment parameters than DCA and MUSCLE fo the three empirical datasets, and POY was found to be more sensitive than MUSCLE, which in turn was found to be as sensitive, or more sensitive, than DCA when applied to the randomly generated sequences when sensitivity was measured using the averaged jackknife values. When significant differences in relative sensitivity were found between the different ways of weighting characterstate changes, equally weighted parsimony, for all three ways of treating gapped positions, was less sensitive than applying the sam cost function used in alignment for phylogenetic analysis. When branch support is incorporated into the sensitivity criterion, our results favour the use of simultaneous alignment and progressive pairwise alignment using the similarity criterion over direct optimization followed by using the implied alignment(s) to calculate branch support.

Given the frequent genomic recombinations in plant mitochondrial DNA, intergenic regions of this organelle genome had so far not been considered as loci of potential phylogentic information. Based on the recent evidence that an evolutionary ancient mitochondrial nad5-nad4 gene continuum is conserved in the bryophytes we have compiled a dataset for a phylogenetically wide sampling of 50 mosses covering this intergenic region. The length of the intergenic region was generally in the range of 585 bp (Diphyscium) to 646 bp (Tomentypnum) with rare exceptions, for example all Polytrichales taxa showing a 200 bp deletion as an apparent synapomorphy of this order. Phylogenetic information in the novel marker sequence was compared with that of a nad5 gene region containing a group I intron and a nad2 gene region containing a group II intron as well as with two widely sampled chloroplast data sets, rbcL and rps4. Indel evolution in the three types of non-coding mitochondrial sequences is obviously more taxon-dependent than locus-dependent, indicating lineage-specific insertion/deletion rates. For example, larger sequence deletions are a general feature in Schistostega and Tetraphis. Although confidence for particular nodes in the phylogeny was found to vary among data sets, gene trees were essentially without conflict with respect to well supported ones and add up in information towards a reasonably well resolved moss phylogeny. However, while a consensus on the latter is clearly emerging, sufficient confidence is still lacking for the first dichotomies among the arthrodontous mosses leading into subclasses Bryidae, Dicranidae and Funariidae and the relative placement of nematodontous mosses (Polytrichales and Tetraphidales) on the backbone of early moss phylogeny.

The PlantTribes database (http://fgp.huck.psu.edu.ezaccess.libraries.psu.edu/tribe.html) is a plant gene family database based on the inferred proteomes of five sequenced plant species: Arabidopsis thaliana, Carica papaya, Medicago truncatula, Oryza sativa and Populus trichocarpa. We used the graph-based clustering algorithm MCL [Van Dongen (Technical Report INS-R0010 2000) and Enright et al. (Nucleic Acids Res. 2002; 30: 15751584)] to classify all of these species protein-coding genes into putative gene families, called tribes, using three clustering stringencies (low, medium and high). For all tribes, we have generated protein and DNA alignments and maximum-likelihood phylogenetic trees. A parallel database of microarray experimental results is linked to the genes, which lets researchers identify groups of related genes and their expression patterns. Unified nomenclatures were developed, and tribes can be related to traditional gene families and conserved domain identifiers. SuperTribes, constructed through a second iteration of MCL clustering, connect distant, but potentially related gene clusters. The global classification of nearly 200 000 plant proteins was used as a scaffold for sorting similar to 4 million additional cDNA sequences from over 200 plant species. All data and analyses are accessible through a flexible interface allowing users to explore the classification, to place query sequences within the classification, and to download results for further study.

Angiosperms are the largest and most successful clade of land plants with >250,000 species distributed in nearly every terrestrial habitat. Many phylogenetic studies have been based on DNA sequences of one to several genes, but, despite decades of intensive efforts, relationships among early diverging lineages and several of the major clades remain either incompletely resolved or weakly supported. We performed phylogenetic analyses of 81 plastid genes in 64 sequenced genomes, including 13 new genomes, to estimate relationships among the major angiosperm clades, and the resulting trees are used to examine the evolution of gene and intron content. Phylogenetic trees from multiple methods, including model-based approaches, provide strong support for the position of Amborella as the earliest diverging lineage of flowering plants, followed by Nymphaeales and Austrobaileyales. The plastid genome trees also provide strong support for a sister relationship between eudicots and monocots, and this group is sister to a clade that includes Chloranthales and magnoliids. Resolution of relationships among the major clades of angiosperms provides the necessary framework for addressing numerous evolutionary questions regarding the rapid diversification of angiosperms. Gene and intron content are highly conserved among the early diverging angiosperms and basal eudicots, but 62 independent gene and intron losses are limited to the more derived monocot and eudicot clades. Moreover, a lineage-specific correlation was detected between rates of nucleotide substitutions, indels, and genomic rearrangements.

Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families -S15a, L7 and P2 - contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL713, RPL7C and RPL7D has hornologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protem genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.

We used simulations to compare the performance of 10 approaches that have been used for treating unambiguously aligned gaps in phylogenetic analyses. We examined how these approaches perform under the ideal conditions of correct alignments, as well as how robust they are to errors caused by use of inferred alignments. Our results indicate that 5th-state coding dramatically outperformed all other coding methods, which in turn all outperformed treating gaps as missing data or excluding gapped positions. Simple indel coding (SIC) and modified complex indel coding (MCIC) performed about the same, and generally outperformed the other indel-coding methods. The high performance of 5th-state coding was largely found to be a weighting artifact. We suggest that MCIC-coded gap characters be scored for all unambiguously aligned gaps in parsimony-based molecular phylogenetic analyses. When the number of terminals sampled precludes the use of MCIC, SIC may be used as an effective substitute.

AFLPs (and to a lesser extent ISSRs and RAPDs) are increasingly being used for phylogenetic inference among closely related species. Presence/absence characters for each AFLP allele treat all absences as homologous to one another. With three or more alleles, terminals are grouped by their shared absence of alleles in character-based phylogenetic-inference methods in a manner that is not redundant with their shared presence of an alternative allele. We conducted simulations to quantify how severe the negative effect of using presence/absence characters of individual bands is for phylogenetic inference relative to standard multistate characters. We examined alternative tree topologies, relative branch lengths, numbers of characters, rates of evolution, and numbers of alternative alleles, using both parsimony and Nei-and-Li distance analyses. Multistate parsimony generally outperformed presence/absence parsimony, which in turn outperformed Nei-and-Li distance. Increasing the character-state space (i.e., the number of alternative character states available) was found to be advantageous for all three methods of analysis examined, but was most advantageous for multistate parsimony. However, the advantage of multistate parsimony relative to Nei-and-Li distance decreased when applied to more divergent characters. More parsimony-in formative variation generally alleviated the problem associated with scoring multistate characters as presence/absence characters. The ensemble consistency index was lower for presence/absence characters relative to multistate characters.

With the growing number of phylogenetic studies that use length variable DNA sequences, incorporating information from length-mutational events into phylogenetic analysis is becoming increasingly important. A new method, modified complex indel coding is described that aims at maximizing the phylogenetic information retained from unambiguously aligned sequence regions or regions where the principal relative position of gaps to one another can be safely established. An algorithm is described that allows application of the method to all theoretically possible gap-nucleotide patterns. A platform-independent computer program is introduced that automates the new method as well as several previously published coding schemes. Differences to previously published indel coding approaches as well as to the integration of ambiguously aligned regions into phylogenetic analysis are discussed.

Carnivorous plants have emerged as model systems for addressing many ecological and evolutionary questions, and since Lentibulariaceae comprise more than half of all known carnivorous species (325 spp.), they are of particular interest. Studies using various molecular markers have established that Lentibulariaceae and their three genera are monophyletic with Pinguicula being sister to a Genlisea-Utricularia-clade, while the closest relatives of the family remain uncertain. Character states of the carnivorous syndrome in related proto-carnivorous lamialean families apparently emerged independently. In Utricularia, the terrestrial habit has been reconstructed as plesiomorphic, and an extension of subgenus Polypompholyx is warranted. In the protozoan-attracting Genlisea, subgenus Tayloria is revealed as basal lineage. In Pinguicula, the six major lineages found reflect radiations in clearly defined geographic regions, whereas most previously recognized subgeneric taxa are non-monophyletic. Genlisea and Utricularia exhibit substitutional rates that rank among the highest in angiosperms for the molecular markers analyzed. One possible explanation for this lies in selective constraints on a wide range of genomic regions that may have been lowered due to the use of an alternative mode of acquiring nutrients.

The prevailing view in molecular systematics is that relationships among distantly related taxa should be inferred using DNA segments with low rates of evolution. However, recent analyses of sequences from the rapidly evolving matK and trnT-trnF regions yielded well resolved and highly supported trees for early diverging angiosperms. We compare here the phylogenetic structure in matK, trnT-F, and rbcL datasets for the same 42, primarily basal angiosperm taxa. Phylogenetic trees based on matK or trnT-F are far more robust than those based on rbcL. Combined analysis of the rapidly evolving regions provides support for higher-level relationships stronger than that derived from analyses of multi-gene datasets of up to several fold the number of characters analyzed here. In addition to displaying a higher percentage of parsimony-informative characters, the average phylogenetic signal per informative character is significantly higher in the datasets from rapidly evolving DNA than in the more slowly evolving rbcL, as detected using resampling of identical numbers of parsimony-informative characters from the data matrices and subjecting different statistics for overall tree robustness and phylogenetic signal to significance tests. Automated via a set of scripts, the method used here should be easily extendable to comparisons of a broader range of genomic regions for varying taxon samplings. The relative performance of markers correlates not only with a lower mean homoplasy in matK and trnT-trnF compared to rbcL, but in particular correlates negatively with the percentage of sites exhibiting maximum or close to maximum homoplasy. A likelihood ratio test confirms that the rapidly evolving gene matK evolves significantly closer to neutrality, which may be one of the underlying factors for lower levels of overall homoplasy. Our results are in line with evidence from simulation studies suggesting that the deleterious effect of multiple hits in using rapidly evolving DNA at rather deep phylogenetic levels may have been overestimated, and thus promote extending the use of rapidly evolving DNA to deeper phylogenetic levels.

Gene, Genome und Sequenzen auf der einen Seite, Algorithmen, Computer und Informatik auf der anderen - beide üben Faszination aus, halten aber viele Interessenten auf respektvolle Distanz. Die Schnittstelle der Bereiche ist mit dem modernen Begriff Bioinformatik belegt. In der Tat hat die Synthese von zwei unabhängigen Disziplinen selten so viele faszinierende neue Einsichten geliefert. Eine spannende Teildisziplin der Bioinformatik ist die Molekulare Phylogenetik, deren Ziel die Rekonstruktion von Stammbäumen aus molekularen Daten ist: Computer, moderne Molekularbiologie und Kladistik haben der etwas angestaubten biologischen Systematik und Taxonomie eine ungeahnte Renaissance verschafft. Der Einstieg in beide Welten gleichzeitig - Molekularbiologie und Phylogenetik - ist nicht unbedingt einfach. Hier will das neue Buch eine Lücke schließen. Die molekularen Datenbanken sind öffentlich und die notwendige Software fast immer kostenlos - man braucht nur PC und das Internet. Einführende Kapitel in die Molekularbiologie, in Evolution, Taxonomie und Kladistik, in Computerprogramme zur Sequenzverwaltung und für molekulare Phylogenetik ermöglichen je nach Wissenshintergrund den schnellen Einstieg.Wer schon etwas weiß, dem wird in einem speziellen Kapitel der direkte Weg zum Stammbaum aufgezeigt. Und wer es genau wissen will, bekommt detaillierte Einführungen in die wichtigen methodischen Ansätze: Parsimonie, Distanzverfahren, Likelihood und natürlich auch die neuen Bayesianischen Verfahren. Alles wird hands on anhand von nachvollziehbaren Beispielen mit der gängigen Software besprochen, die aus dem Internet bezogen werden kann (BioEdit, Clustal, MEGA, MrBayes, PAML, PAUP, SplitsTree oder TreePuzzle). Das neue Buch bietet so eine ideale Balance zwischen Theorie und Praxis. Es hat zahlreiche Illustrationen, bietet am Ende der Kapitel Hinweise zum Weiterlesen und schließt mit einem Glossar und einem umfangreichen Index.

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. in the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1 C-value known from species of Lentibulariaceae was found in Genfisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genfisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clacle, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.

The Lamiales are one of the largest orders of angiosperms, with about 22000 species. The Scrophulariaceae, as one of their most important families, has recently been shown to be polyphyletic. As a consequence, this family was re-classified and several groups of former scrophulariaceous genera now belong to different families, such as the Calceolariaceae, Plantaginaceae, or Phrymaceae. In the present study, relationships of the genera Craterostigma, Lindemia and its allies, hitherto classified within the Scrophulariaceae, were analyzed. Sequences of the chloroplast trnK intron and the matK gene (similar to2.5 kb) were generated for representatives of all major lineages of the Lamiales and the former Scrophulariaceae. Bayesian and parsimony analyses revealed two isolated lineages, one of which consists of Lindemia and its allies, the other of Gratiola and allies. Gratiolo was previously assumed to be related to Lindernia and was therefore included here. It is proposed to treat the two clades as separate families, Linderniaceae and Gratiolaceae. For the Linderniaceae, several morphological synapomorphies exist in addition to molecular data, such as conspicuous club-shaped stamen appendages.

Aristolochiaceae, a family of worldwide distribution comprising about 500 species, is a member of Piperales. Although Piperales is clearly monophyletic, the precise relationship within the order is ambiguous due to inconsistent placement of Lactoris fernandeziana. The appearance in some studies of Lactoris within Aristolochiaceae and the incongruence in generic treatments have also raised questions about the infrastructure of the family. This study addresses the overall generic relationships in Aristolochiaceae and its position in Piperales based on dense taxon sampling and sequence data from the plastid trnL-F region. The study resolved Piperales consisting of two major clades (Piperaceae plus Saururaceae and Lactoridaceae plus Aristolochiaceae) and Lactoris nested within Aristolochlaceae but with low support. The concept of two subfamilies in Aristolochiaceae, Asaroideae and Aristolochioideae, gains maximum statistical support. A generic treatment of Aristolochiaceae based on trnL-F is proposed which is congruent with recent analyses based on morphological characters.

Choosing and designing primers based on available DNA sequence data and statistical contrasting of domains or structural features is a common routine among molecular biologists. Currently available, free software tools were found to lack desirable features related to these tasks. This was the motivation for developing a new program, SeqState. SeqState locates regions that remain to be sequenced in phylogenetic DNA datasets, evaluates user-provided primers and selects primers best suited to fill gaps in the sequences. If the primers provided by the user are unsuitable, new primers are designed. Primers can be loaded from a primer database, be supplied as part of the alignment or be entered manually. The position of internal primers is automatically localised in the loaded data file. Primers can be edited, and changes and new primers can be saved to the database. Primer sheets allow the user to view internal dimers, complements to a second primer, mismatches to all loaded sequences, and other primer characteristics. Calculation of various sequence statistics can be requested for the whole dataset or parts thereof (character sets), with standard errors estimated by bootstrapping. Insertion-deletion events can be evaluated statistically and encoded for subsequent phylogenetic analysis according to several published coding principles. Availability: SeqState runs on all major computer platforms and is downloadable for free from http://www.nees.uni-bonn.de/downloads/SeqState, together with documentation and sample data files, or can be requested from the author.

Stellate pore ornamentation is an unusual feature of angiosperm pollen, so far it is known in only ten genera of Amaranthaceae. The pollen grains of these plants have apertures with large hook-shaped ektexinous bodies that are stellately arranged. Previous studies interpreted this character complex as a synapomorphy in consideration of its strong specialization. By reconstructing the evolution of stellate pore ornamentation based on phylogenetic trees of Amaranthaceae obtained by parsimony, likelihood, and Bayesian analysis of chloroplast trnK/matK DNA sequences, clear evidence is provided for several independent origins and reversals to less specialized aperture types. In addition to the gomphrenoid genus Pseudoplantago, stellate pore ornamentation evolved several times among achyranthoid genera, which have an African distribution. The most derived apertures, with 5 - 6 large protruding hooks, appear independently in Centemopsis on the one hand, and in Psilotrichum sericeum on the other. In an effort to break down the complex character syndrome of stellate pore ornamentation, we delimited a set of six pollen morphological characters that could be independently traced on the phylogeny. It appears that stellate pore ornamentation was independently derived from apertures with equally spread ektexinous bodies that became hook-shaped, reduced in number, and symmetrically arranged. Likewise the symmetrically arranged, rectangular ektexinous bodies in Marcelliopsis represent an independent specialization. In view of this pattern of morphological changes, functional significance in the context of specialized insect pollination syndromes and positive selection for stellate pore ornamentation is hypothesized. Stellate pore ornamentation provides an example of a specialized pollen character complex with adaptive significance, and underlines the need for a dense taxon sampling for analyses of character evolution.

Amaranthaceae and Chenopodiaceae together represent the most species-rich monophyletic group in the angiosperm order Caryophyllales. So far, phylogenetic relationships between Amaranthaceae and Chenopodiaceae have remained unclear. Previous morphological and molecular studies have indicated that most of the currently accepted infrafamilial taxa in Amaranthaceae do not reflect natural groups. With the aim to provide a robust phylogenetic framework for analyzing the evolution Of pollen and other phenotypic characters within the family, we conducted maximum parsimony, maximum likelihood, and Bayesian analyses using chloroplast matK/trnK DNA sequence data. The Amaranthaceae-Chenopodiaceae lineage was resolved as monophyletic, with Achatocarpaceae and Caryophyllaceae being Successive sisters. Within the monophyletic Amaranthaceae, a basal grade of Bosea L. (Macaronesian islands, Cyprus, Himalaya), followed by Charpentiera Gaudich. (endemic to Hawaii and the Australian Ridge) receives high support. Celosieae appear as sister to Amaranthus L. + Chamissoa Kunth. While Celosieae were Supported as natural. most other currently recognized infrafamiliar taxa were revealed to be para- or polyphyletic. Among former Aervinae, an Achyranthoid and an Aervoid clade were newly recovered. Psendoplantago Suess.., sharing some morphological features with Amaranthoideae, was found within Gomphrenoideae rather than being in a position linking both subfamilies. The evolution of morphological characters previously used for classification and of some pollen characters was analyzed based on the matK/trnK tree. The recently described metareticulate pollen architecture is reconstructed to have evolved once in the ancestor of a highly supported core Gomphrenoid clade that comprises the traditional Gomphrenoideae but excludes Iresine P. Browne. in contrast, the complex stellate pore ornamentation was reconstructed as having evolved at least twice in parallel. Unlike other morphological characters discussed in this paper, pollen characters in general represent morphological synapomorphies circumscribing many clades recovered with molecular data. In addition. the present paper provides a brief survey of the taxonomic history and the current state of research on systematics and evolution in Amaranthaceae.

The bladderworts (Utricularia, Lentibulariaceae) are the most diverse carnivorous plant genus, with a nearly worldwide distribution. In the present study, chloroplast DNA sequences of the trnK intron were used to reconstruct phylogenetic relationships within the genus. Parsimony, likelihood, and Bayesian analyses resulted in highly congruent and well-resolved trees. The phylogenetic signal provided by the noncoding trnK intron partition of the dataset is similar to that of the matK coding region, although the latter is twice as long. Within matK, indels appeared in multiples of three except very close to the 3-prime end of the gene. Substitutions were found to result in or eliminate stop codons, thus creating a length variable gene end. Indels in both trnK and matK exhibit low degrees of homoplasy, irrespective of their size. A tree based on indels alone is largely congruent to the substitution-based trees but less resolved. Three major clades found within Utricularia are classified as subgen. Utricularia, subgen. Bivalvia, and subgen. Polypompholyx. The immediate common ancestor of Utricularia is suggested to have been a terrestrial plant whereas epiphytic and aquatic habits evolved later in terminal clades.

Background: For parsimony analyses, the most common way to estimate confidence is by resampling plans (nonparametric bootstrap, jackknife), and Bremer support (Decay indices). The recent literature reveals that parameter settings that are quite commonly employed are not those that are recommended by theoretical considerations and by previous empirical studies. The optimal search strategy to be applied during resampling was previously addressed solely via standard search strategies available in PAUP. The question of a compromise between search extensiveness and improved support accuracy for Bremer support received even less attention. A set of experiments was conducted on different datasets to find an empirical cut-off point at which increased search extensiveness does not significantly change Bremer support and jackknife or bootstrap proportions any more. - Results: For the number of replicates needed for accurate estimates of support in resampling plans, a diagram is provided that helps to address the question whether apparently different support values really differ significantly. It is shown that the use of random addition cycles and parsimony ratchet iterations during bootstrapping does not translate into higher support, nor does any extension of the search extensiveness beyond the rather moderate effort of TBR (tree bisection and reconnection branch swapping) plus saving one tree per replicate. Instead, in case of very large matrices, saving more than one shortest tree per iteration and using a strict consensus tree of these yields decreased support compared to saving only one tree. This can be interpreted as a small risk of overestimating support but should be more than compensated by other factors that counteract an enhanced type I error. With regard to Bremer support, a rule of thumb can be derived stating that not much is gained relative to the surplus computational effort when searches are extended beyond 20 ratchet iterations per constrained node, at least not for datasets that fall within the size range found in the current literature. - Conclusion: In view of these results, calculating bootstrap or jackknife proportions with narrow confidence intervals even for very large datasets can be achieved with less expense than often thought. In particular, iterated bootstrap methods that aim at reducing statistical bias inherent to these proportions are more feasible when the individual bootstrap searches require less time.

The genus Pinguicula is one of the three genera of the carnivorous Lentibulariaceae, comprising approximately 80 species. Phylogeny inference using nucleotide sequences of the chloroplast gene matK and the trnK group II intron, as well as a set of 32 morphological characters revealed five well-supported, major lineages within the genus. These lineages largely reflect radiations in clearly defined geographic regions, whereas most previously recognized sections of the genus are shown to be para- or polyphyletic. A species-rich Mexican-Central American-Caribbean clade has the Eurasian P. alpina and an East Asian clade as successive sisters. All three are characterized by a production of flower buds on winter-resting plants, a specific corolla hair structure and a very large corolla lower central lobe. Another diverse clade is composed of species with primarily European distribution including the widespread type species P. vulgaris. For this clade, vegetative reproduction during dormancy is synapomorphic. Species native to SE North America and the South American Andes and a group of Mediterranean and NE Atlantic coast species together appear in a fifth well-supported clade, that is characterized by a tropical-type growth habit. It is the only clade that has reached temperate zones of the southern hemisphere.

The prevailing view in molecular phylogenetics is that relationships at deeper levels, for example among major angiosperm lineages, should be inferred using rather conserved genes. Rapidly evolving DNA has been deemed unsuitable because of putative high levels of homoplasy caused by multiple substitutions and frequent microstructural mutations resulting in their non-alignability. Recent analyses of rapidly evolving spacers and introns in the chloroplast genome from basal angiosperms show that extreme sequence variability is confined to certain mutational hotspots. These hotspots correlate with structural elements (stem loops), where selective pressure is low. Phylogenetic trees of basal angiosperms inferred from rapidly evolving genomic regions are well resolved and highly supported statistically. Reasons are not only greater amounts of informative sites in rapidly evolving DNA, but also differing levels of homoplasy that can result in better phylogenetic signal per informative site. Based on these findings, perspectives for further utilizing non-coding DNA in angiosperm phylogenetic studies are explored.

The trnL group I intron and the intergenic spacer between trnL and trnF are among the most widely utilized noncoding DNA regions in plant systematics. The trnL-F region is probably cotranscribed and located upstream of trnTUGU in the large single copy region of the chloroplast genome. Whereas in algal chloroplast genomes the three tRNA genes are widely scattered, they are found in tandem in land plants. This unique arrangement, therefore, seems to be a synapomorphy for land plants. Secondary structure analysis of the group I trnLUAA intron shows that high sequence variability is confined to certain stem-loop regions (P6, P8), whereas elements of the catalytic core (P, Q, R, S) are highly conserved across land plants. Considerable length differences, particularly in the stem loop P8, as well as in the trnT-L and trnL-F intergenic spacers, were observed among the diverse land plant lineages. Sequences found within mosses and liverworts were generally shorter than in most other lineages. It appears that certain elements in the trnLF spacer and in the most variable P8 stem-loop region of the trnL intron have originated by independent nucleotide additions. These elements do not share a common evolutionary history across all land plant lineages below seed plants because internally repeated sequence segments have further diversified, actually being internal paralogous stretches. Most of the length variation in the trnL-F spacer occurs between the putative sigma70-type bacterial promoter motif upstream of trnF and the trnL 39 exon. Apart from leptosporangiate ferns, lycophytes, and Gnetales this conserved promoter motif is present in most of the land plant lineages sampled as well as in Chaetosphaeridium. Structural data were used to assess the effect that compensating base pair changes (CBC) in helical elements of the intron have for phylogenetic reconstructions. Such changes significantly contribute to the phylogenetic structure of the data set. Although the inclusion of CBCs in general violates the requirement of character independence, their effect is rather comparable to weighting characters. The contribution of CBCs to the total amount of phylogenetic signal is found to be less significant within terminal land plant lineages, such as pleurocarpous mosses or angiosperms, because helical elements are conserved in their primary sequence and variation is lower within these terminal lineages.

The Bremer support (BS, Bremer, 1988, 1994;Källersjö et al., 1992), also known as decay index (Olmstead et al., 1993), length difference (Faith1991), or support index (SI, Davis, 1993; Kluge and Farris, 1969), is a measure of branch support frequently seen in the recent phylogenetic literature. The strengths and limits of this index have lately been examined (DeBry, 2001; Oxelman et al., 1999) and it was generally concluded that care must be taken when interpreting the values. In particular, the calculation of BS becomes problematic for intermediate to large data sets. Either, the resulting support values will be sometimes drastic overestimates of support (Bremer, 1994) or, if more thorough search strategies are invoked to assess support of all the individual branches, the process will become a very time consuming task (Oxelman et al., 1999). Various procedures have been suggested for computationally more demanding data sets (Davis, 1995; Morgan, 1997), and the reverse constraint method has emerged as most effective (Morgan, 1997). The power of all approaches, however, strongly depends on the ability of the applied heuristic search strategy to find shortest trees. With the common search strategies and the currently available processor speed, this ability shrinks rapidly as matrix sizes grow to more than 100-150 taxa (although strongly depending on the data set).......

As a basis for analysing the evolution of the carnivorous syndrome in Lentibulariaceae (Lamiales), phylogenetic reconstructions were conducted based on coding and non-coding chloroplast DNA (matK gene and flanking trnK intron sequences, totalling about 2.4 kb). A dense taxon sampling including all other major lineages of Lamiales was needed since the closest relatives of Lentibulariaceae and the position of proto-carnivores were unknown. Tree inference using maximum parsimony, maximum likelihood, and Bayesian approaches resulted in fully congruent topologies within Lentibulariaceae, whereas relationships among the different lineages of Lamiales were only congruent between likelihood and Bayesian optimizations. Lentibulariaceae and their three genera (Pinguicula, Genlisea, and Utricularia) are monophyletic, with Pinguicula being sister to a Genlisea-Utricularia clade. Likelihood and Bayesian trees converge on Bignoniaceae as sister to Lentibulariaceae, albeit lacking good support. The proto-carnivores (Byblidaceae, Martyniaceae) are found in different positions among other Lamiales but not as sister to the carnivorous Lentibulariaceae, which is also supported by Khishino-Hasegawa tests. This implies that carnivory and its preliminary stages (proto-carnivores) independently evolved more than once among Lanniales. Ancestral states of structural characters connected to the carnivorous syndrome are reconstructed using the molecular tree, and a hypothesis on the evolutionary pathway of the carnivorous syndrome in Lentibulariaceae is presented. Extreme DNA mutational rates found in Utricularia and Genlisea are shown to correspond to their unusual nutritional specialization, thereby hinting at a marked degree of carnivory in these two genera.

TREEGRAPH assists in producing complex ready-to-publish figures of phylogenetic trees. The TGF format used by the program automates formatting of several different statistical support value types (confidence estimates) per tree node. Moreover, internal text and graphical labels are automatically arranged at the nodes as are annotations for clades or groups of terminals. TREEGRAPH imports NEXUS trees and related file formats. Beyond common tree edit operations, simultaneous pruning of subtrees (simplification of the tree to higher order clades) and saving of subtrees is possible. TREEGRAPH exports to the standard vector graphics formats Scalable Vector Graphics and PostScript.

The phylogeny of three pleurocarpous moss families, Meteoriaceae, Brachytheciaceae, and Lembophyllaceae, was studied using both morphology and molecular data. Phylogenetic analyses using different approaches revealed very similar, well-resolved topologies wherein the Meteoriaceae and Brachytheciaceae are monophyletic and sister to each other. The Lembophyllaceae were found not to be closely related to those families, but to the Neckeraceae. Results suggest several independent origins of epiphytism within these families. Character states that are commonly regarded as adaptations to epiphytism are concentrated on those clades with epiphytic species. The evolution of certain characters, such as peristomes that open under humid conditions, seta length, and spore size, is clearly correlated with an epiphytic habit. In phylogenetic analyses, this kind of morphological character could be suspected to lead to biased results if species are grouped based on shared ecological conditions instead of common ancestry. Our study revealed that despite correlated evolution with epiphytism, these characters do not significantly deteriorate the robustness of the hypotheses on relationships between families. However, there may be negative effects on the phylogenetic reconstruction by increasing homoplasy, as well as incongruence between data partitions, and decreasing the phylogenetic structure obtained with morphological data.

The tandemly arranged genes psbT, psbN, and psbH code for proteins of photosystem II and are located in the large single copy region (LSC) of the chloroplast genome, downstream of psbB. So far, most of the studies dealing with this region have been interested in the organization and transcription of the psbB operon, while less is known about the transcription of psbN or the phylogenetic utility of this region. In the current study we discuss a sigma(70)-type bacterial promoter motif upstream of psbN and present its consensus sequence for bryophytes. An analysis of the 3-prime flanking inverted repeat sequences revealed a dyad symmetrical element, which is able to form a stable stem-loop structure. This hairpin structure is characterised for land plants, with an emphasis on bryophytes. Furthermore, we observed an inversion of up to 9 bases in the loop region of the hairpin structure, which occasionally occurred in different unrelated bryophyte families, orders and classes. Small inversions are frequently obscured in the alignment since, during automatic alignment, many gap-weighting schemes may not introduce gaps, nor does a manual insertion of gaps always seem needed at first glance. In subsequent phylogenetic analyses, minute inversion may overweight a particular mutation by interpreting the single inversion event as multiple apomorphic substitutions, which is particularly problematic since such inversion events are known to often be highly homoplastic. In the present study we use the psbT-N spacer as an example to quantify the effect of such small inversions, contrasting the phylogenetic structure obtained with and without information from the hairpin loop. We show that obscured minute inversions can highly significantly reduce the robustness of the phylogenetic hypothesis inferred from the data set.

Plastid matK gene sequences for 374 genera representing all angiosperm orders and 12 genera of gymnosperms were analyzed using parsimony (MP) and Bayesian inference (BI) approaches. Traditionally, slowly evolving genomic regions have been preferred for deep-level phylogenetic inference in angiosperms. The matK gene evolves approximately three times faster than the widely used plastid genes rbcL and atpB. The MP and BI trees are highly congruent. The robustness of the strict consensus tree supercedes all individual gene analyses and is comparable only to multigene-based phylogenies. Of the 385 nodes resolved, 79% are supported by high jackknife values, averaging 88%. Amborella is sister to the remaining angiosperms, followed by a grade of Nymphaeaceae and Austrobaileyales. Bayesian inference resolves Amborella + Nymphaeaceae as sister to the rest, but with weak (0.42) posterior probability. The MP analysis shows a trichotomy sister to the Austrobaileyales representing eumagnoliids, monocots + Chloranthales, and Ceratophyllum + eudicots. The matK gene produces the highest internal support yet for basal eudicots and, within core eudicots, resolves a crown group comprising Berberidopsidaceae/Aextoxicaceae, Santalales, and Caryophyllales + asterids. Moreover, matK sequences provide good resolution within many angiosperm orders. Combined analyses of matK and other rapidly evolving DNA regions with available multigene data sets have strong potential to enhance resolution and internal support in deep level angiosperm phylogenetics and provide additional insights into angiosperm evolution.

The rosids represent one of the major angiosperm clades, constituting about one third of all flowering plants and about 40% of the eudicots (more than 70,000 species). Despite recent advances, knowledge about relationships among rosids still remains limited. This is particularly true for rapid radiations within rosids, such as Malpighiales. Our Eudicot Evolutionary Research team analysed sequence data from 197 taxa of the rapidly evolving matK gene, the group II trnK intron, the group I trnL intron and the trnL-F spacer from nearly all families of the rosids, comprising all major lineages, as well as Vitales and Saxifragales and an outgroup of basal eudicots in a combined matrix. The dataset comprised >13,000 positions of aligned sequence data plus a separate binary indel matrix adding another 2000 characters. Compared to previous analyses, the combined dataset yields significantly increased resolution and support in MP, Bayesian and Likelihood inferences. This is particularly obvious within Fabids and Malvids, including the order Huerteales as recently newly circumscribed by our group. Further, the isolated clades of Geraniales, Myrtales and Crossosomatales can be placed with statistical support. In order to understand the high performance of the spacer and both introns, as well as matK we analysed the phylogenetic structure within and among data partitions in comparison to more conserved genes (e.g. rbcL) using a PERL pipeline. Results underscore that the phylogenetic structure is highest in the spacer and group I intron, followed by group II intron and matK sequences, independently from the tree inference method. Reconstructions of intron secondary structures are addressed as a framework to understand molecular evolutionary patterns governing the performance as phylogenetic markers.

Applying rapidly evolving molecular markers for phylogenetic reconstructions is not as straightforward as using rather slowly evolving genes displaying low degrees of microstructural change. Extensive length mutations and inversions considerably complicate the homology assessment. Using alignment approaches based on repeat recognition (possibly guided by secondary structures) and an enhanced understanding of molecular evolutionary mechanisms might promote the utilization of more complexly evolving regions such as spacers and introns. It appears that inversions are almost always associated with hairpins and display high degrees of homoplasy, unlike other kinds of microstructural changes (e.g., simple sequence repeats). A detailed investigation of differences in mutational patterns observed in the inverted as opposed to the non-inverted motif points towards multiple independent occurrences of such inversions among lineages. Current automated alignment algorithms by design do not take into account inversions and, therefore, frequently create erroneous alignments. Similarly, motif recognition has not yet found its way into major alignment software packages, despite the commonness of simple sequence repeats (SSRs) mediated by slipped-strand-mispairing, a mechanism suggested 25 years ago. A modified slipped-strand-mispairing mechanism for SSRs provides a tool to differentiate between the original motif and its repeated variant and thus facilitates homology assessment.

Multiple sequence alignment is typically the first step estimating phylogenetic trees, with the assumption being that as alignments improve, so will phylogenetic reconstructions. Over the last decade or so, new multiple sequence alignment methods have been developed to improve comparative analyses of protein structure, but these new methods are often not used in phylogenetic analyses. In this talk, we report on a simulation study that we performed to evaluate the consequences of using these new multiple sequence alignment methods in terms of the resultant phylogenetic reconstruction. We find the surprising result that although the new alignment methods do indeed produce improved estimations of the true alignment, slight improvements in alignment accuracy have little consequence for phylogenetic accuracy. Several examples drawn from analyses of whole chloroplast genomes and plant gene families, provide some support for this finding, but also identify situations where alignment improvements can enhance alignment realism, phylogenetic resolution, and phylogenetic accuracy.

Sensitivity analysis provides a way to measure robustness of clades in sequence-based phylogenetic analyses to variation in alignment parameters rather than measuring their branch support. We compared three alternative approaches to multiple sequence alignment in the context of sensitivity analysis: progressive pairwise alignment, as implemented in MUSCLE; simultaneous multiple alignment of sequence fragments, as implemented in DCA; and direct optimization followed by generation of the implied alignment(s), as implemented in POY. We set out to determine the relative sensitivity of these three alignment methods using rDNA sequences and randomly generated sequences. A total of 36 parameter sets were used to create the alignments, varying the transition, transversion, and gap costs. Tree searches were performed using four alternative character-coding and weighting approaches: the cost function used for alignment or equally weighted parsimony with gap positions treated as missing data, separate characters, or as fifth states. POY was found to be as sensitive, or more sensitive, to variation in alignment parameters than DCA and MUSCLE for the three empirical datasets, and POY was found to be more sensitive than MUSCLE, which in turn was found to be as sensitive, or more sensitive, than DCA when applied to the randomly generated sequences when sensitivity was measured using the averaged jackknife values. When significant differences in relative sensitivity were found between the different ways of weighting character-state changes, equally weighted parsimony, for all three ways of treating gapped positions, was less sensitive than applying the same cost function used in alignment for phylogenetic analysis. When branch support is incorporated into the sensitivity criterion, our results favor the use of simultaneous alignment and progressive pairwise alignment using the similarity criterion over direct optimization followed by using the implied alignment(s) to calculate branch support.

Sensitivity analysis provides a way to measure stability of clades in sequence-based phylogenetic analyses to variation in alignment parameters rather than support, as with the bootstrap and jackknife. It has been asserted that stability of clades may be more important than their support. We compared three alternative approaches to sequence alignment in the context of sensitivity analysis: progressive pairwise alignment, as implemented in MUSCLE; simultaneous multiple alignment of sequence fragments, as implemented in DCA; and direct optimization, as implemented in POY. We set out to determine the relative sensitivity of these three alignment methods using both empirical and simulated sequences. A total of 36 parameter sets were used to create the alignments, varying the transition, transversion, and gap costs. Tree searches were performed using four alternative character-coding approaches: the cost matrices used for alignment or equally weighted parsimony with gap positions treated as missing data, separate characters, or as fifth states.

Sensitivity analysis provides a way to measure stability of clades in sequence-based phylogenetic analyses to variation in alignment parameters rather than support, as with the bootstrap and jackknife. It has been asserted that stability of clades may be more important than their support. We compared three alternative approaches to sequence alignment in the context of sensitivity analysis: progressive pairwise alignment, as implemented in MUSCLE; simultaneous multiple alignment of sequence fragments, as implemented in DCA; and direct optimization, as implemented in POY. We set out to determine the relative sensitivity of these three alignment methods using both empirical and simulated sequences. A total of 36 parameter sets were used to create the alignments, varying the transition, transversion, and gap costs. Tree searches were performed using four alternative character-coding approaches: the cost matrices used for alignment or equally weighted parsimony with gap positions treated as missing data, separate characters, or as fifth states.

The number of sequenced organelle genomes is increasing exponentially and is providing rapidly growing opportunities for whole-genome phylogenetic and evolutionary analyses. It has created a huge practical challenge for researchers attempting to assemble genome data into accurately aligned, useable datasets. Here we present a pipeline that fully integrates crucial steps including: i) easy identification and compilation of target data, ii) sequence alignment, iii) automated assessment of alignment quality, iv) coding of structural mutation events, and v) phylogenetic analysis. The pipeline is integrated with other informatic tools previously developed for ChloroplastDB (http://chloroplast.cbio.psu.edu/), an interactive, web-based database for fully sequenced plastid genomes which includes pages for genomic, protein, DNA, and RNA sequences, RNA-editing sites, unified annotations, and homologous protein sets generated using TribeMCL. Alignments are built from user-specified gene and taxon sets, including amino acid alignments, nucleotide alignments, and nucleotide datasets forced onto protein alignments. Taxon specific low scoring alignment sections can be identified, controlled via a set of parameters, visualized, and optionally masked prior to further analysis. Insertion/deletion events are coded using various methods, taking alignment quality into account. The user can implement any subset of these functions to make custom batch files for phylogenetic analyses. We report on first comparisons between analyses that make use of identifying low scoring alignment sections and/or indel coding versus standard analyses that exclude large portions of the data (e.g., all gapped positions) and/or retain questionable alignment sections inside or outside the gapped regions. In addition to promoting phylogenetic analyses of complete plastid genomes, this alignment pipeline is helpful in the analysis of any large dataset. AlignMate greatly facilitates high throughput comparative analyses of changes in gene content, gene regulation, structure-function relationships, and context-dependent substitution processes.