The chloroplast matK gene has been identified as a rapidly evolving gene at nucleotide and corresponding amino acid levels. The high number of nucleotide substitutions and length mutations in matK has provided a strong phylogenetic signal for resolving plant phylogenies at various taxonomic levels. However, these same features have raised questions as to whether matK produces a functional protein product. matK is the only proposed chloroplast-encoded group II intron maturase. There are 15 genes in the chloroplast that would require a maturase for RNA splicing. Six of these genes have introns that are not excised by a nuclear imported maturase, leaving MatK as the only candidate for processing introns in these genes. Very little research has been conducted concerning the expression and function of this important gene and its protein product. It has become crucial to understand matK expression in light of its significance in RNA processing and plant systematics. In this study, we examined the expression, function and evolution of MatK using a combination of molecular and genetic methods. Our findings indicate that matK RNA and protein is expressed in a variety of plant species, and expression of MatK protein is regulated by development. In addition, matK RNA levels are affected by light. Furthermore, genetic analysis has revealed that although MatK has a high rate of amino acid substitution, these substitutions are not random but are constrained to maintain overall chemical structure and stability in this protein. We have also identified an alternate start codon for matK in some plant species that buffers indels (insertions and deletions) in the open reading frame (ORF) that are not in multiples of three in the gene sequence. Usually, indels not in multiples of three result in frame shifts that destroy the reading frame. Our results indicate that an out-of-frame matK start codon in some orchids compensates for these otherwise deleterious indels. This research represents the first in-depth analysis of matK gene expression and contributes to several fields of biology including plant systematics, genetics and gene expression. / Ph. D.

Astilbe (Saxifragaceae) is a genus of herbaceous perennials widely cultivated for their ornamental value. The genus is considered taxonomically complex because of its geographic distribution, variation within species, and the lack of adequate morphological characters to delineate taxa. To date, an inclusive investigation of the genus has not been conducted. This study was undertaken to (a) develop a well-resolved phylogeny of the genus Astilbe using an expanded morphological data set and sequences from the plastid gene matK, (b) use single nucleotide polymorphisms to determine the lineages of cultivated varieties, and (c) successfully culture Astilbe in vitro and evaluate potential somaclonal variation of resulting Astilbe microshoots. Phylogenetic trees generated from a morphological character matrix of 28 character states divided Astilbe into three distinct clades. Relationships were well resolved among the taxa, though only a few branches had greater than 50% bootstrap support. There is evidence from the phylogeny that some described species may actually represent variation within populations of species. From our analysis I propose an Astilbe genus with 13 to 15 species and offer a key for distinguishing species and varieties. There was little matK sequence variation among taxa of Astilbe. Phylogeny of Astilbe generated from the maximum parsimony and maximum likelihood analysis of matK sequences resulted in a polytomy of seven Astilbe species, with relationships within the genus poorly resolved. A second phylogeny of 21 taxa of Astilbe was more informative, aligning cultivated varieties near species from which they were derived. The matK sequence variation for Astilbe taxa was aligned to reveal DNA polymorphisms. Closely related taxa retained polymorphisms at the same sites within the gene sequence. These polymorphic sites could potentially be utilized to confirm the lineage of popular cultivated Astilbe varieties. Propagation of Astilbe seedlings in tissue culture gave rise to various numbers of microshoots from each of 15 seedlings. Multivariate and cluster analysis of morphological characters from 138 plants derived from 15 seedlings revealed potential somaclonal variants. These variants were characterized by one or more of the following traits: dwarf habit, dark green leaves (high chlorophyll content), increased flowering, or larger plant size. Somaclonal variants with desirable phenotypes may be valuable for cultivar development. / Ph. D.

ABSTRACT GENETIC DIFFERENTIATION OF LIQUIDAMBAR ORIENTALIS MILL. VARIETIES WITH RESPECT TO matK REGION OF CHLOROPLAST GENOME &Ouml / ZD&amp / #272 / LEK, Asli M.S., Department of Biology Supervisor: Prof. Dr. Zeki Kaya August 2007, 87 pages Liquidambar L. genus is represented with mainly 4 species in the world and one of these species, Turkish sweet gum (Liquidambar orientalis Mill.) which is a relictendemic species is naturally found in only southwestern Turkey, mainly in Mugla Province. The limited distribution of species with two disputed varieties (var. integriloba Fiori and var. orientalis) and increased anthropogenic threats to its genetic resources signify the importance of studying genetic diversity in the species to have better conservation and management programs. For this purpose, 18 different populations were sampled throughout the species range and matK region of chloroplast DNA (cpDNA) was sequenced to assess the genetic structure of the species. Turkish Liquidambar orientalis populations were evaluated at two categories: variety level and geographic level. Also, two sectors of matK region were examined to assess which part of the region was more variable. All molecular analysis was conducted in this study by using MEGA version 3.1 and Arlequin 2.000 softwares. v Moleculer diversity analysis indicated that the population located in Fethiye- G&uuml / nl&uuml / kbasi district has the highest number of polymorphic sites. This population is also genetically the most distant from the others (average genetic distance 0.0038). Among the studied varieties, the average genetic distance within var. integriloba (0.0016) which also includes population Fethiye-G&uuml / nl&uuml / kbasi was the greatest. Among the geographic regions, Mugla-1 including Fethiye-K&ouml / ycegiz-Aydin district as well as population Fethiye-G&uuml / nl&uuml / kbasi showed the highest average genetic distances within the region with a value of 0.0015. According to the molecular variance results, among varieties and among geographic regions, there was no significant differentiation, but great amount of total variation was found (~86%) within Turkish sweet gum populations. With respect to the Fst values among varieties, the highest genetic differentiation was observed between var. orientalis and unknown group (0.040). Furthermore, based on the results of phylogenetic analysis, Turkish populations of L. orientalis have genetically closer to USA relative (L. styraciflua L.) than Chinese relatives (L. acalycina H.T Chang and L. formosana Hance). In conclusion, 10 Turkish sweet gum populations were found to be important for conservation issues. Furthermore, eight of these located in Mugla province and sixth of them belong to var. integriloba. Especially Fethiye-G&uuml / nl&uuml / kbasi, Marmaris&Ccedil / etibeli and Mugla-Kiyra populations should be included in either insitu or exsitu or in both conservation programs in the future.

The recent call to reconstruct a detailed picture of the tree of life for all organisms has forever changed the field of molecular phylogenetics. Sequencing technology has improved to the point that scientics can now routinely sequence complete plastid/mitochondrial genomes and thus, vast amounts of data can be used to reconstruct phylogenies. These data are accumulating in DNA sequence repositories, such as GenBank, where everyone can benefit from the vast growth of information. The trend of generating genomic-region rich datasets has far outpaced the expasion of datasets by sampling a broader array of taxa. We show here that expanding a dataset both by increasing genomic regions and species sampled using GenBank data, despite the inherent missing DNA that comes with GenBank data, can provide a robust phylogeny for the plant order Caryophyllales (angiosperms). We also investigate the utility of trnK intron in phylogeny reconstruction at relativley deep evolutionary history (the caryophyllid order) by comparing it with rapidly evolving matK. We show that trnK intron is comparable to matK in terms of the proportion of variable sites, parsimony informative sites, the distribution of those sites among rate classes, and phylogenetic informativness across the history of the order. This is especailly useful since trnK intron is often sequenced concurrently with matK which saves on time and resources by increasing the phylogenetic utility of a single genomic region (rapidly evolving matK/trnK). Finally, we show that the inclusion of RNA edited sites in datasets for phylogeny reconstruction did not appear to impact resolution or support in the Gnetales indicating that edited sites in such low proportions do not need to be a consideration when building datasets. We also propose an alternate start codon for matK in Ephedra based on the presense of a 38 base pair indel in several species that otherwise result in pre-mature stop codons, and present 20 RNA edited sites in two Zamiaceae and three Pinaceae species. / Ph. D.

Evolution of Piperales – matK gene and trnK intron sequence data reveal lineage specific resolution contrast. Piperales are one of the largest basal angiosperm orders with a nearly worldwide distribution. The order includes three species rich genera, Piper (ca. 1,000 species), Peperomia (ca. 1,500-1,700 species), and Aristolochia s. l. (ca. 500 species). Sequences of the matK gene and the non-coding trnK group II intron are analysed for a dense set of 105 taxa representing all families (except Hydnoraceae) and all generic segregates (except Euglypha within Aristolochiaceae) of Piperales. A large number of highly informative indels are found in the Piperales trnK/matK dataset. Within a narrow region approximately 500 nt downstream in the matK coding region (CDS), a length variable simple sequence repeat (SSR) expansion segment occurs, in which insertions and deletions have led to short frame-shifts. These are corrected shortly afterwards, resulting in a maximum of 6 amino acids being affected. Furthermore, additional non-functional matK copies were found in Zippelia begoniifolia, which can easily be discriminated from the functional open reading frame (ORF). The trnK/matK sequence data fully resolve relationships within Peperomia, whereas they are not effective within Piper. The resolution contrast is correlated with the rate heterogenity between those lineages. Parsimony, Bayesian and likelihood analyses result in virtually the same topology, and converge on the monophyly of Piperaceae and Saururaceae. Lactoris gains high support as sister to Aristolochiaceae subf. Aristolochioideae, but the different tree inference methods yield conflicting results with respect to the relationships of subfam. Asaroideae. In Piperaceae, a clade formed by the monotypic genus Zippelia and the small genus Manekia (=Sarcorhachis) is sister to the two large genera Piper and Peperomia. Systematics of pipevines – Combining morphological and fast-evolving molecular characters to investigate the relationships within subfamily Aristolochioideae (Aristolochiaceae) A combined phylogenetic analysis of the Aristolochioideae was conducted based on 72 morphological characters and molecular datasets (matK gene, trnK intron, trnL intron, trnL-trnF spacer). The analysis sampled 33 species as the ingroup, including two species of Thottea and 30 species of Aristolochia and the monotypic genus Euglypha, which represent all the infrageneric taxa formally described; Saruma henryi and Asarum caudatum were used as the outgroup. The results corroborate a sister-group relationship between Thottea and Aristolochia, and the paraphyly of Aristolochia with respect to Euglypha that consequently should be included into Aristolochia. Two of the three subgenera within Aristolochia (Isotrema and Pararistolochia) are shown to be monophyletic, whereas the signal obtained from the different datasets about the relationships within subg. Aristolochia is low and conflicting, resulting in collapsed or unsupported branches. The relationship between the New World and the Old World species of subgenus Aristolochia is conflictive because morphological data support these two groups as monophyletic, whereas molecular data show the monophyletic Old World species of Aristolochia nested within the New World species. A sister group relationship is proposed between A. lindneri and pentandrous species, which suggests that a group of five species from central and southern South America (including A. lindneri) could be monophyletic and sister to Aristolochia subsection Pentandrae, a monophyletic taxon consisting of about 35 species from southern USA, Mesoamerica, and the West Indies. Colonisation, phylogeography and evolution of endemism in Mediterranean Aristolochia (Aristolochiaceae). This study provides evidence for a multiple colonisation of the western Old World from Asian ancestors within Aristolochia section Diplolobus (subsection Aristolochia and Podanthemum). Within subsection Podanthemum it is assumed, that the colonisation of the African continent happened at least two times independently. In contrast, for subsection Aristolochia, a rapid morphological radiation in the Near East (or close to this area) with subsequent star like colonisation of the different current distribution areas, which is not paralleled on the molecular level, appears to be more likely. Phylogenetic tree reconstruction is unsupported for these clades, but most clades are highly supported as monophyletic. Interestingly the Mediterranean and temperate Eurasian species, which are morphologically distinct (A. pistolochia, A. clematitis) are not clustering within the main clades, but are independent lineages. Analogue, A. rigida a species from Somalia is well-supported sister to the subsection Aristolochia. Within subsection Podanthemum the colonisation event from an Asian ancestor is clearly traceable, whereas in subsection Aristolochia the path is not traceable, since the ancestors are extinct or not present in the connecting areas. Within the Mediterranean, Near East and Caucasian species of subsection Aristolochia two morphologically and biogeographically well supported groups can be identified: the Near East/Caucasian species and the West Mediterranean species. The previous groupings for the latter, based on morphological characters, could be substantiated only partly by our results. This study provides the first phylogeny of all West Mediterranean species. In addition an independent complex is established including some micro endemic species. The phylogenetic results are discussed with respect to biogeography, and morphology, to give a first insight into the radiation and colonisation of the genus Aristolochia in the Mediterranean region. Universal primers for a large cryptically simple cpDNA microsatellite region in Aristolochia. We provide a new and valuable marker to study species relationships and population genetics in order to trace evolutionary, ecological, and conservational aspects in the genus Aristolochia. Universal primers for amplification and subsequent sequencing of a chloroplast microsatellite locus inside the trnK intron are presented. Utility of the primers has been tested in 32 species representing all clades of Aristolochia, including population studies within the A. pallida complex, A. clusii and A. rotunda. The microsatellite region is characterized as a (AnTm)k repeat of 22–438 bp containing a combination of different repeats arranged as ‘cryptically simple’. Trapped! Pollination of Aristolochia pallida Willd. in the Mediterranean A first study of the pollination biology of a Mediterranean Aristolochia species in its natural habitat is presented. 183 flowers of Aristolochia pallida were investigated, which in total contained 73 arthropods, dominated by two groups of Diptera, Sciaridae (37%) and Phoridae (19%). However, only Phoridae are regarded as potential pollinators, since pollen has been found exclusively on the body surfaces of these insects. All Phoridae belong to the genus Megaselia and are recognised as four undescribed species. The measurements of flower and insect dimensions suggest that size is an important constrain for successful pollination: 1) the insects must have a definitive size for being able to enter the flower and 2) must be able to get in touch with the pollen. Only very few insect groups found in Aristolochia pallida fulfil these size requirements. However, size alone is not a sufficient constrain as too many fly species of the same size might be trapped but not function as pollinators. Instead, specific attraction is required as otherwise pollen is lost. Since all trapped Phoridae are males, a chemical attraction (pheromones) is proposed as an additional constrain. Since A. pallida flowers are protogynous, the record of Megaselia loaded with pollen found in a flower during its female stage proves that this insect must have been visited at least one different flower during its male stage before. Further on, this observation provides strong evidence that the flowers are cross-pollinated. All these factors indicate a highly specialised pollination of Aristolochia pallida by Megaselia species.

This work attempts to offer an insight into the problematic of the genus Elettariopsis Baker, the last unrevised genus in the subfamily Alpinioideae (Zingiberaceae). Phylogenetic analyses are performed on ITS, matK and DCS sequence data and correlated with absolute genome size and biogeographical distribution of the samples. Elettariopsis as a genus is found to be weakly supported and strongly supported only with the addition of some species of Amomum Roxb., including the type species A. subulatum. The absolute genome size in this group is greater than in the outgroup represented by members of the Zingiberoideae subfamily. The evidence given by sequence data further suggests that Elettariopsis is divided into two well-supported groups, the "E. curtisii" group and the "E. triloba/E. unifolia" group, each of which contains several well-supported clades. In the analysis of absolute genome size it is shown that the absolute genome size in the "E. triloba/E.unifolia" group is higher than in the "E. curtisii" group. These two groups also differ slightly in their biogeographical distribution, the group G being distributed in only in Vietnam, Laos, and Thailand, while members of group H are also occurring in Singapore and Indonesia (Borneo). Keywords: Zingiberaceae, Elettariopsis, South-East Asia, ITS,...

Pineacea is the largest family of conifers that includes 51 species of Abies which is the second largest genus after Pinus. There are six native taxa in Turkey belonging to this genus. Four of these taxa (Abies cilicica subsp. isaurica, Abies nordmanniana subsp. bornm&uuml / lleriana, Abies nordmanniana subsp. equi-trojani, Abies x olcayana) are endemic and considered as low risk (LR) species according to the IUCN criteria. To determine the phylogenetic relationship in Abies spp. in Turkey, 18 populations of different taxa were collected from their natural distribution areas in Turkey. The matK gene regions of chloroplast DNA (cpDNA) were studied comparatively to reveal the genetic relationship among Turkish fir species. The available sequences from the NCBI database for the matK region of the other Abies species in the world were also investigated v comparatively with the sequences from Turkish firs. With the matK sequence data, a phylogenetic tree was constructed for the fir species and the molecular diversity parameters such as conserved and variable sites, nucleotide diversity, and evolutionary divergence were estimated using the MEGA software. The results indicated that there are no variable sites among Turkish firs with regard to matK regions of cpDNA. It appears that the matK region of cpDNA for Turkish firs is highly conserved. Since sequence data for all matK regions were not available from the NCBI data base, the phylogentic analysis with the sequence data of matK were comparatively analyzed in all firs including Turkish firs. According to matK1 region, the results showed that there were three major clades. One of the clades included all Turkish fir taxa and one species from European firs, A.numidica / however, A.holopylla, A.firma, A.veitchii, A.sachalinensis, A.nephrolepis, A.lasiocarpa, A.koreana, A.homolephis, A.fraseri, A.fargesii, A.sibirica and A.fabri formed in another clade. In addition to this, A.mariesii, A.hidalgensis, A.bracteata, A.alba formed in different major clade. According to matK1 region the results showed that Turkish firs and European firs are closer to each other. Furthermore, based on matK2 region, the results indicated that Turkish firs formed a monophyletic group.The other fir species, with respect to matK2 regions, formed different clades from Turkish firs. The results based on matK region suggest that all Turkish firs may have evolved from single ancestral fir species and the matK gene region appears to be highly conserved region in fir species.

The genus Picea is located from temperate to Taiga (boreal) regions of northern hemisphere from subtropical to high altitude with 34 species. Picea orientalis is endemic to Eastern Black Sea Mountainous region of Turkey and Western Caucasus. To determine the genetic relatedness within Picea orientalis populations, as well as the relationship between other Pinaceae species from database, populations were sampled from 15 different locations within the natural range of species and grouped into 5 depending on several criteria. In order to evaluate the genetic structure of the taxon, non-coding trn and matK regions of choloroplast DNA (cpDNA) were sequenced. According to genetic diversity analysis of 15 Picea orientalis populations with respect to trn and matK regions, there is not much variation among populations. Among 3 non-coding trn and the matK region, there is only one variable site which was parsimony informative. The results indicated that the populations from Artvin had the highest divergence. In this study, the genetic divergence of Picea orientalis from other Pinaceae species were also observed. According to the results obtained from trnV region the studied Picea orientalis observed to display a close relationship with Larix and distinct from other Pinaceae especially Pinus genus. This result is unrepresentative due to the results of other studies. Moreover, as a result of analysis with trncd-ef region, the studied Picea orientalis populations possessed close relationship with species from clade Picea. Moreover, based on molecular clock estimations the studied Picea orientalis populations had close relationships with the species form Asia. Finally, the relationship of Picea orientalis with other Picea species were analyzed with respect to matK region. The result is consistent with the results of trncd-ef region and also with other studies.

Variability in the genetic information DNA tracking individuals carry is easy to detect using molecular markers. In the thesis we examined related Dracaena species. For the study of genetic diversity in the genus Dracaena are used mainly noncoding regions of cpDNA, spacer trnH -- psbA, regions trnL -- trnF and trnS -- trnG -- trnG, which are more variable than coding regions. Also used in this work were coding regions of matK and rbcL. In the genus Dracaena belongs xerophytic species that are characterized by typical shaped treetop. 14 representatives of species were used in thesis growing in tropical regions of the African continent and adjacent islands and the southeastern part of the Arabian peninsula. They are monocotyledonous trees with atypical abilities of secondary thickness of trunk, which I find interesting. The massive trunk is very strong and there is potencial to used it for wood. Trees are very significant for their red plant sap which flowing from demaged trunk. The sap is very precious resource which is used in many areas of industry, for example pharmacy, traditional medicine, dye making etc. Determination of genetic affinity was based on an amplification of cpDNA template of individual Dracaena samples with primers for the studied regions. Data was obtained and evaluated by Multiple alignment program ClustalX and BioEdit after their sequencing. Evalueted data was used to create dendograms affinity. According the resulting phylogenetic tree we find out similarities and identified relationship of the monitored species of the genus Dracaena. The main purpose of research was to get answers to understand phylogenetic relationship between group of Dracaena forestry used trees. The thesis was made in cooperation of Department of Forest Botany, Dendrology and Geobiocoenology, Faculty of Forestry and Wood Technology of Mendel University in Brno.

As técnicas de DNA barcoding (código de barras de DNA) têm como objetivo principal a identificação taxonômica de organismos através da amplificação e análise de sequências de DNA curtas, padronizadas e previamente definidas. Apesar do sucesso relativo desta abordagem em animais usando um único locus, a aplicação deste método em plantas apresenta menor capacidade de identificar espécies usando uma única região gênica, levando a necessidade de utilização de múltiplos loci. Além disso, ainda há certo debate sobre qual região gênica seria mais apropriada para o DNA barcoding em plantas, embora as regiões plastidiais rbcL, matK e o espaçador intergênico trnH-psbA juntamente com o espaçador intergênico nuclear do RNA ribossomal (ITS) sejam as mais comumente utilizadas até então. As tribos Sisyrinchieae e Tigridieae da família Iridaceae foram testadas de acordo com diferentes métodos e marcadores indicados para o DNA barcoding em plantas. Os resultados indicaram uma alta universalidade para membros da tribo Sisyrinchieae, mas também revelaram uma capacidade de identificação de espécies considerada baixa. Apesar disto, os espaçadores ITS foram indicados como a melhor sequência para DNA barcoding em Sisyrinchieae. Em Tigridieae, problemas inerentes ao sequenciamento impediram a utilização dos ITS em nossas análises. Assim, apenas marcadores plastidiais foram utilizados na tentativa de identificar espécies, apresentando novamente resultados modestos. A região gênica que atingiu maior capacidade de identificação em Tigridieae foi o gene matK. A incapacidade de se alcançar maiores taxas de identificação provavelmente está relacionada à complexa história evolutiva apresentada pelos grupos em análise. Este trabalho forneceu o primeiro conjunto significativo de dados de DNA barcoding aplicados a dois importantes grupos de Iridaceae de considerável biodiversidade no Brasil. As tribos em análise apresentam espécies consideradas filogeneticamente próximas e são de difícil identificação devido a sua morfologia homogênea, principalmente em estado vegetativo, justificando plenamente o uso de métodos molecularespara a identificação taxonômica. / The main objective of DNA barcoding methods is the taxonomic identification of organisms by amplifying and analyzing short, standardized and previously defined DNA sequences. In spite of the relative success of this approach in animals using a single locus, the application of this method in plants has less ability to identify species using a single gene region, leading to the need of using multiple loci. Furthermore, there is still some debate concerning which gene region would be more suitable for DNA barcoding in plants, although the plastid regions rbcL, matK and the trnH-psbA intergenic spacer along with the nuclear intergenic spacer of ribossomal DNA (ITS) are the most commonly regions used thus far. The tribes Sisyrinchieae and Tigridieae of the family Iridaceae were tested according to different methods and markers used for DNA barcoding in plants. The results indicated a great universality for members of tribe Sisyrinchieae, but also showed a low ability to identify species. Nevertheless, ITS was imputed as the best sequence for DNA barcoding in Sisyrinchieae. In Tigridieae, problems inherent of ITS sequencing prevented its use in our analysis. Thus, only plastid markers were used in an attempt to identify species, showing modest results once again. The gene region that reached higher identification ability in Tigridieae was matK. The inability to achieve higher identification levels is probably related to the complex evolutionary history presented by the groups in question. This work provided the first large data set of DNA barcoding applied to two important groups of Iridaceae with significant biodiversity in Brazil. The tribes in question present species considered phylogenetically related and are difficult to identify due to their homogeneous morphology, especially in vegetative stage, fully justifying the use of molecular methods for taxonomic identification.