DNA barcoding is a valuable tool for fish species identification by food regulators, however, it does not perform well when multiple species are present within the same food product. PCR cloning has high potential to be used in combination with DNA barcoding to overcome this challenge. The objective of this study was to examine the use of PCR cloning combined with DNA barcoding to identify fish in a mixed-species product that cannot be identified with standard DNA barcoding. A total of 15 fish ball mixtures were prepared with known amounts of Nile tilapia (Oreochromis niloticus), Pacific cod (Gadus macrocephalus), and walleye pollock (Gadus chalcogrammus). The fish balls underwent DNA extraction in triplicate, followed by DNA barcoding across the full barcode (655 bp) and SH-E mini-barcode (226 bp) of the cytochrome c oxidase subunit 1 (CO1) region. Samples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. Full barcoding enabled identification of at least one species in 80% of the fish ball mixtures compared to 51% for minibarcoding. The results of PCR cloning with samples that did not pass DNA barcoding showed identification success rates of 61% for clones (54 of 90) that underwent full barcoding and 51% for clones (111 of 220) that underwent mini-barcoding. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock).. The combination of standard full barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 6 of 12 (50%) of mixed-species fish balls. In comparison, the combination of standard mini-barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 9 of 12 (75%) of mixed-species fish balls. Overall, the results of this study show that PCR cloning may be an effective method to identify certain fish in mixed-species products when standard DNA barcoding fails. However, additional research is needed to understand the limitations associated with primer bias.

No description available.

The current method to determine a mosquito specimen to a certain species is by morphological keys basically following the taxonomy developed by Carl Linnaeus in the 1700. Since Watson and Crick presented their model of the double-helix DNA in 1953, a new era of molecular based taxonomic studies have revolutionized the field. The revolution is not in terms of how the classification of species is done but how the biological diversity is seen. However, morphological, ecological and behavioral characteristics are still important and are used together with the information a gene or whole genome can give. DNA barcoding is one of the promising methods for molecular identification. A small segment of a gene, approximately 400-1000 base pairs (bp), are examined by a Polymerase chain reaction (PCR) and sequencing. Like the barcodes in the grocery store these sequences work like unique ID: s for every species. This thesis shows how a fast DNA extraction method could be combined with DNA barcoding to get a 658-bp segment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) from different species of the mosquito family Culicidae. A total of 15 thoraxes or wings, from individual specimen of mosquitoes, were examined and 11 different barcode sequences could be retrieved. Six correspond to already published COI sequences and could therefore be determined to the species level, including a sequence from a new species for Sweden, Aedes (Ochlerotatus) nigrinus. All mosquitoes were collected during the national inventory of species in summer of 2012 in Sweden, ”Myggjakten”, and have been morphological examined by experts at the National Veterinary Institute (SVA) prior to molecular determination. This thesis also highlights the importance of building a reference library of barcode sequences, so DNA barcoding could become an effective diagnostic tool. Inventory projects like “Myggjakten” may, if repeated, provide excellent material for such a library collection of barcode data. / När en stickmyggsart skall artbestämmas är den vanligaste metoden att använda morfologiska nycklar. I princip görs det här efter den taxonomi som Carl von Linne utvecklade på 1700-talet. Men sedan Watson och Crick presenterade sin DNA modell 1953 så har dock en ny era av molykylärt baserade metoder revolutionerat taxonomin. Förändringen består egentligen inte i hur vi klassificerar och använder taxonomin utan mer hur vi ser på den biologiska mångfalden. Morfologiska och ekologiska studier, samt studier av arters beteende, är fortfarande viktiga och komplementerar den molekylära informationen från ett genom eller från en enstaka gen. DNA barcoding är en av de lovande nya molekylära metoderna för artbestämning. Ett litet segment av en gen, på ungefär 400-1000 baspar (bp), undersöks med hjälp av polymeras-kedjereaktion (PCR) och sekvensering. Likt streckkoder i livsmedelsbutiken ger metoden ett unikt ID för varje art. Den här studien visar hur en snabb DNA-extraktionsmetod kan kombineras med DNA barcoding, för att ge en 658-bp lång DNA-sekvens, från den mitokondriella genen cytokrom c oxidas subunit 1 (COI) från olika arter av myggfamiljen Culicidae. I undersökningen ingick 15 mellankroppar eller vingar från individuella stickmyggor och av dessa kunde 11 olika barcode sekvenser utläsas. Sex av dessa stämde överrens med redan publicerade COI-sekvenser och kunde bestämmas till artnivå, varav en av sekvenserna kommer från den nyligen i Sverige funna morfologiskt artbestämda Aedes (Ochlerotatus) nigrinus. Stickmyggorna i detta arbete insamlades av privatpersoner på olika ställen i Sverige under sommaren 2012 i det nationella mygginsamlingsprojektet ”Myggjakten”. Dessa artbestämdes morfologiskt av personal på Statens veterinärmedicinska anstalt (SVA) innan de artbestämdes molekylärt. Det här arbetet belyser även vikten av att bygga upp ett referensbibliotek av barcode sekvenser för att DNA-barcoding ska kunna bli ett effektivt diagnostiskt verktyg vid studier av vektorburna zoonoser. Nationella projekt som Myggjakten kan vara mycket användbara för insamling av sådana data.

The Myristicaceae, or nutmeg family, consists of 21 genera and about 500 species of dioecious canopy to sub canopy trees that are distributed worldwide in tropical rainforests. The Myristicaceae are of considerable ecological and ethnobotanical significance as they are important food for many animals and are harvested by humans for timber, spices, dart/arrow poison, medicine, and a hallucinogenic snuff employed in medico-religious ceremonies. Despite the importance of the Myristicaceae throughout the wet tropics, our taxonomic knowledge of these trees is primarily based on the last revision of the five neotropical genera completed in 1937. The objective of this thesis was to perform a molecular and morphological study of the neotropical genera Compsoneura and Virola. To this end, I generated phylogenetic hypotheses, surveyed morphological and genetic diversity of focal species, and tested the ability of DNA barcodes to distinguish species of wild nutmegs. Morphological and molecular analyses of Compsoneura. indicate a deep divergence between two monophyletic clades corresponding to informal sections Hadrocarpa and Compsoneura. Although 23 loci were tested for DNA variability, only the trnH-psbA intergenic spacer contained enough variation to delimit 11 of 13 species sequenced. A morphological and molecular investigation of Compsoneura capitellata showed little discrete morphological variation among populations but significant genetic structure among populations. Phylogenetic analysis of Virola also revealed a deep molecular divergence between two clades having numerous contrasting morphologies. In contrast to Compsoneura, the trnH-psbA intergenic spacer failed to differentiate the majority of Virola species tested. An infraspecific morphological and molecular study of V. sebifera and V. loretensis showed that each of these species contains morphologically and ecologically discrete sympatric morphotypes that likely represent new species. In total, this investigation found 5 provisional new species from fewer than 600 collections at biological stations in Ecuador and Peru where these new species were among the most abundant trees in the forest. This suggests that much diversity likely remains to be described in the Myristicaceae and other tropical plant families.

Biodiversity across the globe is threatened. Rapid surveying and monitoring techniques are required to understand the origin of the threats to biodiversity and to enable conservation actions to be undertaken. Kuwait is an arid desert country with a small flora of only 402 species. This flora is endangered by environmental factors, overgrazing, and human activities. DNA barcoding the flora and using Next Generation Sequencing (NGS) technologies allowed us to identify plants to species level, conduct a molecular taxonomic revision, and distinguish plant diversity found in soil environmental DNA samples. After investigating the discriminatory power of five commonly used DNA markers from plastid (matK, rbcL, trnH-psbA, trnL) and a nuclear genome (ITS2) on four largest genera of the flora using phylogenetics reconstruction tree based methods, two barcoding markers (rbcL and ITS2) were assigned to build a DNA reference library of the flora. Furthermore, the DNA reference library was tested to identify the plant diversity found below-ground level and comparing it with that above-ground, using environmental soil samples collected from both species rich and poor habitats in Kuwait by applying high-throughput sequencing methods. The DNA database provided in this study could be used as a reference library for the identification process and contribute towards the future of molecular taxonomy, biodiversity and ecological research in Kuwait.

Tropical rain forests around the world suffer from deforestation, which is caused mainly by small-scale farmers. These farmers largely employ slash-and-burn methods to clear the land for agricultural settlement. Agroforestry systems are widely found in the humid tropics, where they could have great potential to increase the productivity of farming systems and sustain continuous crop production and they are also supposed to conserve biodiversity. As a group of freshwater invertebrates, dragonflies (Odonata) are commonly used as ecological indicators of freshwater ecosystems. The main objective of the study is to assess the impact of land use changes on dragonflies (Odonata) species richness and diversity, namely primary and secondary forest, cocoa agroforest and slash-and-burn agriculture in the Tropical Africa. We hypothesize, that the species richness and diversity of dragonflies decrease with disturbance of the ecosystems, along the land-use changes gradient.

Desert environments are characterised by harsh conditions and possess low biodiversity largely caused by abiotic factors such as; low precipitation, soil organic matter, high temperatures, high levels of evapo-transpiration, pH and salinity. These factors significantly reduce primary production, which influences the availability of food resources for deserts organisms. The diversity and the drivers of diversity for below ground invertebrates including Collembola (springtails) are relatively unknown in the Namib Desert. Previous morphological studies have found only five species on the basis of traditional taxonomy. This study assesses the diversity of Namib Desert Collembola and determines the effect of environmental parameters on this diversity, The diversity of Namib Desert Collembola was assessed using DNA Barcoding. The sequence information of the 178 Collembola specimens, taken from mitochondrial barcoding using the Cytochrome-c oxidase subunit I (COI) gene, was analyzed and Molecular Operational Taxonomic Units (MOTUs) were defined. Collembola community responses to soil physicochemical properties were investigated by using Redundancy Analysis (RDA). MOTUs were successfully indentified to family level (Isotomidae, Neanuridae and Sminthuridae). The researcher found a total of 30 MOTUs, most of which showed limited geographical localisation. The mtDNA COI (barcode) locus revealed high levels of previously unreported genetic diversity of Collembola in the Namib Desert. The RDA indicated that none of the soil physicochemical properties significantly drove variation in Collembola community composition. However, total soil nitrogen was shown to be a strong but not significant driver of variation in community composition (p<0,054). The taxonomic identification of the Collembola specimens was also attempted using traditional morphological analysis. A total of 23 individuals, collected from pitfall traps or extracted from soil samples, were selected for identification. Available European keys were used for identification to genus level where possible. A total eight of specimens were identified to genus level (Folsomides sp), 14 to family level (Entomobryidae) and one to order level (Symphypleona). Both Symphypleona and Entomobryidae were previously unreported from the Namib Desert. The Folsomides genus and the family Entomobryidae were the most abundant groups. This research suggests that soil dwelling Collembola in the Namib Desert have a much higher level of diversity than previously known. However, the study also highlighted the need for a more comprehensive database for Namib Collembola that includes COI sequence data as well as the morphological identification of species. / Dissertation (MSc)--University of Pretoria, 2018. / National Research Foundation (NRF) / Genetics / MSc / Unrestricted

The cosmopolitan echiniscid genus Hypechiniscus contains exclusively rare species. In this contribution, by combining statistical morphometry and molecular phylogeny, we present qualitative and quantitative aspects of Hypechiniscus diversity, which remained hidden under the two purportedly cosmopolitan species: H. gladiator and H. exarmatus. A neotype is designated for H. gladiator from Creag Meagaidh (Scotland), and an informal re-description is provided for H. exarmatus based on animals from Creag Meagaidh and the Isle of Skye (Inner Hebrides). Subspecies/forms of H. gladiator are suppressed due to the high developmental variability of the cirrus dorsalis. At the same time, four species of the genus are described: H. daedalus sp. nov. from Roan Mountain and the Great Smoky Mountains (Southern Appalachian Mountains, USA), H. flavus sp. nov. and H. geminus sp. nov. from the Yatsugatake Mountains (Honshu, Japan), and H. cataractus sp. nov. from the Malay Archipelago (Borneo and the Moluccas). Dorsal and ventral sculpturing, together with morphometric traits, are shown to be the key characters that allow for the phenotypic discrimination of species within the genus. Furthermore, the morphology of Hypechiniscus is discussed and compared to that of the most similar genera, Pseudechiniscus and Stellariscus. Finally, a diagnostic key to all recognized Hypechiniscus species is provided.

Am Rebstock werden in der Natur von Agrobacterium vitis, dem Auslöser Wurzelhalsgallenerkrankung, charakteristische Wurzelhalsgallentumore induziert. Virulente Vertreter der Gattung der Agrobacteria schleusen bakterielle DNA in das pflanzliche Genom ein, wodurch die Pflanze Tumore produziert. Die Wurzelhalsgallenerkrankung wird seit einem Jahrhundert als ein Beispiel der Pflanzen-Pathogen-Interaktion untersucht. Die Rolle der bakteriellen Flora im Zusammenhang mit der Wurzelhalsgallenerkrankung beim Rebstock wurde bisher kaum betrachtet. Um dieser Frage nachzugehen, habe ich die endophytische mikrobielle Zusammensetzung von Rebstöcken mit und ohne Wurzelhalsgalle analysiert. Es werden Proben von drei Zeitpunkten einer Wachstumsperiode (Frühling, Sommer und Herbst) und von den Organen der Rebstöcke (Wurzeln, Pfropfstelle und einjährige Triebe) sowie dem Boden in einer Weinanlage bei Himmelstadt in Unterfranken genommen. Die Bakterienflora dieser Umweltproben wird mit kultivierungsabhängigen (Isolierung von Bakterien) und kultivierungsunabhängigen (Hochdurchsatzsequenzierungen) Methoden untersucht. Zudem werden i) die Virulenz der verschiedenen Agrobacterium-Isolate in Tumorassays bestimmt, ii) synthetische Bakteriengemeinschaften von in vitro kultivierten Weinpflänzchen mit Wurzelhalsgallen analysiert, iii) die Genome von einem virulenten und einem nicht-virulenten Agrobacteria-Isolat aus der Wurzelhalsgalle verglichen, iv) erste Interaktionsstudien auf festen Nährmedien durchgeführt und v) virulente Agrobacteria mittels bildgebender Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) in Wurzelhalsgallen lokalisiert. Die Rebstöcke dieser Studie haben eine organspezifische Bakterienflora, die innerhalb einer Wachstumsperiode variiert. Nur die Bakterienflora der Pfropfstelle (mit oder ohne Wurzelhalsgalle) aber nicht die des Bodens, der Wurzeln, und der einjährigen Triebe unterscheidet sich strukturell zwischen gesunden und erkrankten Rebstöcken. Mikroskopisch konnten virulente Agrobacteria punktuell in Interzellularen, sklerenchymatischen Geweben und assoziiert mit Leitgefäßen nachgewiesen werden. Dadurch ist ausreichend Lebensraum vorhanden, der zusätzlich von tumorspezifischen Bakterien besiedelt werden kann. Im Gegensatz zur gesunden Pfropfstelle ist in der Wurzelhalsgalle eine saisonal stabile Kernmikroflora, bestehend aus Vertreter von A. vitis, Pseudomonas, Enterobacteriaceae, Agrobacterium tumefaciens, Gammaproteobacteria und Burkholderiales, vorhanden. Diese Bakterien werden überwiegend aus dem Boden rekrutiert und profitieren von der Nährstoffsituation in der Wurzelhalsgalle. Wurzelhalsgallen enthalten Opine, die nur von der transformierten Pflanzenzelle produziert werden. Interessanterweise hat in dieser Arbeit ein Agrobacterium-Isolat Gene, die zum Opinkatabolismus beitragen und ein Pseudomonas-Isolat kann Opine als einzige Kohlenstoffquelle nutzen. Trotzdem sind beide Isolate weder virulent noch verdrängen sie die virulenten A. vitis, die ebenso Opine nutzen, aus der Wurzelhalsgalle. In synthetischen Bakteriengemeinschaften an in vitro kultivierten Weinpflänzchen konnte gezeigt werden, dass diese und weitere tumorspezifischen Bakterien, neben A. vitis, nicht essentiell zur Entstehung der Wurzelhalsgalle nötig sind aber unterschiedliche Funktionen in der Wurzelhalsgalle übernehmen. Ein Serratia-Isolat hemmt das Wachstum von A. vitis auf festen Nährmedium, andere fördern oder hemmen das Wachstum der Wurzelhalsgalle. Nach Studien in der Literatur erhöhen weitere Bakterien die Resistenz des Rebstocks gegenüber biotischem und abiotischem Stress. Zusammengefasst identifizierten und isolierte ich in dieser Studie unter 150 unterschiedlichen Bakterien in der Wurzelhalsgalle jene Bakterien, die neben A. vitis von der neuen ökologischen Nische profitieren und somit wahrscheinlich Opportunisten mit unterschiedlichen Funktionen sind. In Folge von multiplen Interaktionen in der Wurzelhalsgalle entsteht ein ökologisches Gleichgewicht zwischen den opportunistischen Bakterien, der Wurzelhalsgalle und dem Rebstock, das den Fortbestand des Rebstocks mit Wurzelhalsgalle ermöglicht. / In nature, Agrobacterium vitis is known for the ability to introduce bacterial DNA into the grapevine genome, thereby causing crown gall disease. This plant disease has been studied for a century as a model for plant-pathogen interaction, while the role of the plant microbiota in disease development is not well understood. My study contributes to the understanding of the microbial ecology in crown galls of grapevine, combining culture-dependent with culture-independent high-throughput sequencing techniques. I analysed the structure of the endophytic microbiota by collecting different samples (soil, roots, graft unions and canes) of diseased and non-diseased grapevines from one vine-yard in Franconia, Bavaria, Germany during one growing season (spring, summer, autumn). The characterization of the grapevine-associated bacterial microbiota was completed by (i) detecting the virulence of diverse agrobacterial isolates using a tumour growth assay with in vitro cultivated grapevine plantlets, (ii) microbial analysis of synthetic communities of in vitro cultivated grapevine plantlets with crown galls, (iii) genome sequencing of a virulent and a non-virulent agrobacterial isolate, (iv) in vitro interaction studies on solid medium with bacterial isolates and (v) localisation of virulent A. vitis using Fluorescence Lifetime Imaging Microscopy (FLIM) in tumour tissues. Grapevine plants of this study have an organ-specific bacterial community that varies during one growing season. Healthy and diseased grapevine plants differed in the struc-ture of the bacterial community only in the graft union (with or without a crown gall), but not in the soil, root and one-year old cane. Microscopy revealed that virulent Agrobacteria mainly accumulate in defined spots of sclerenchymatous tissue, intercellular space and tissues associated with vessels. Therefore, there is unoccupied living space in a crown gall, which can be additionally colonized by tumour-specific bacteria. A season-independent stable core bacteria exists in grapevine crown galls in contrast to healthy graft unions, consisting of OTUs assigned to A. vitis, Pseudomonas, Enterobacteriaceae, Agrobacterium tumefaciens, Gammaproteobacteria and Burkholderiales. These bacteria are predominantly recruited from the soil and most likely profit from special nutrients in the crown gall. The crown gall contains opines, exclusively produced by transformed plant cells. Curiously individual isolates of Agrobacteria and Pseudo-monas of this study that are non-virulent do not outcompete virulent A. vitis in the crown gall but harbour, like A. vitis, genes involved in octopin-catabolism or use opines in liquid cultures as a sole nutrient source. Although synthetic bacterial communities revealed that the tumour-specific bacteria are not required for crown gall induction us-ing in vitro grown grapevine plantlets, they may have different functions in crown gall persistence. A Serratia-isolate inhibits the growth of A. vitis on solid medium, others reduce or support crown gall development, while some, according to literature, increase resistance of the grapevine plant against biotic and abiotic stresses. Taken together, among the 150 bacteria found in the crown galls, I identified and isolated bacteria in addition to A. vitis that profit from the new ecological niche suggesting an opportunistic lifestyle with different ecological functions. An ecological equilibrium in a bacterial community that balances crown gall growth will support the existence of grapevine plants with a crown gall in vineyards.

Invasive fire ants (Solenopsis invicta and its hybrid with S. richteri) have been reported from carrion in the southeastern United States and are considered a part of the succession community. Alteration of ecological processes by fire ants could affect forensic interpretation of entomological data; therefore, I conducted studies to investigate the relative roles and interactions of fire ants and blow flies in carrion decomposition. The blow fly species composition in Mississippi has not been studied since 16 species were reported in 1983. Specimens from the Mississippi Entomological Museum were used to update the checklist of the blow flies of Mississippi and produce a photographic key to adults and third instar larvae. A total of 23 species of blow flies are now known or expected to occur in the state. I conducted an experiment whereby portions of the succession fauna were excluded from access to carrion to study the relative effects of fire ants and blow flies on carrion decomposition and their interactions with each other. Fire ants made lesions in and partially buried carcasses, but their exclusion did not affect carrion decomposition rates; slightly affected the succession community; and strongly affected succession of blow flies, specifically. Lastly, I collected fire ants from mounds at set distances from carrion and analyzed their guts for pig and blow fly DNA. The probability of detecting pig or blow fly DNA in ants collected directly from carrion increased with each succeeding day, and the probability of detecting either pig or blow fly DNA in ant guts decreased with increasing distance between carrion and the mound. Probability of detecting pig or blow fly DNA in ant guts from ants collected directly from the carcasses was 42% and 33%, respectively. This study documented that fire ants scavenge on carrion, prey on other members of the succession fauna, and transfer acquired nutrients at least 3 m into the landscape. Thus, fire ants represent a barrier to normal faunal succession patterns on carrion and these delays should be considered by forensic entomologists for postmortem interval estimation.