Correct species identification is fundamental to all areas of biology, but particularly the policy related areas of conservation and fisheries management. To enable guidelines to be developed for environmental management and conservation, such identifications need links to studies of the evolutionary history, biological factors and environmental influences driving species divergence and population dynamics for the target species. This study concerns two genera of gadiform fish, Muraenolepis and Macrourus, found in southern temperate and Antarctic waters, with a single species, Macrourus berglax, present in the North Atlantic. With similar distribution patterns to toothfish species, Dissostichus eleginoides and D. mawsoni, they are a major food source and by-catch of the toothfish fishery. Both are slow growing and long lived, with different evolutionary histories, life expectancies and strategies for reproduction. For both genera, the accuracy of morphological keys, number of described species and their distribution is under debate. This study has identified specimens to species level using both morphological and genetic techniques, redefining the range for morphological features and taxonomic keys. For Muraenolepis, this has clarified confusion over Mu. marmoratus and Mu. microps being a single species, confirmed some mis-identification from sexual dimorphism and provided genetic evidence for the recently described species Mu. evseenkoi. For Macrourus, this work has identified a new species, now named Ma. caml, and found that Ma. holotrachys and Ma. berglax are genetically identical, raising the question of bipolar distribution or recent divergence. The low level of genetic variation within both species suggests a recent evolution and expansion into Antarctic waters. Similar geographic species limits imply common processes influencing divergence, with the oceanographic fronts as potential barriers. Further investigation of niche overlap and fine scale population structure are required to fully understand the processes driving speciation and provide the underlying data required for fisheries management.

This diploma thesis is focused on species identification of sand flies belonging to two genera of the subfamily Phlebotominae, genus Phlebotomus and Sergentomyia. Genus Phlebotomus together with the genus Lutzomyia of New World include the only proven vectors of Leishmania parasites and they are also carriers of viral and bacterial infections. Species of the genus Sergentomyia are proven vectors of sister genus Sauroleishmania that infects reptiles, but for several decades there have been speculations about their possible involvement in the transmission of mammalian Leishmania species. These suspicions arise mainly from repeated findings of mammalian Leishmania parasites in their digestive system. Correct species determination of medically significant hematophagous arthropods is very important especially for purposes of epidemiological studies so that efficient vector control may be correctly set. Routine identification of sand flies is based on morphological characters located mainly on their heads and genitalia. However, these characters may be variable within a species, they require certain expertise and in the field samples they may be damaged, making proper species identification impossible. This thesis therefore presents two alternatives of sand fly identification based on molecular...

Barcode identification is a method that uses genetic information to differentiate species. Because of its general versatility it can be applied to contexts from archaeology to the food industry. Atlantic salmon (Salmo salar) is a fish species commonly hunted in modern times and has been found in archaeological settings. However, barcoding requires enough quality DNA for amplification and abiotic exposure tends to degrade it. High temperatures, such as when boiling, can diminish DNA quality. The extent of DNA degradation between fresh and boiled tissues and whether all tissues retain the same amount of DNA is unclear. In this study DNA was extracted from S. salar tissues fins, muscle, bones and scales without treatment and with boiling treatment. DNA concentrations between fresh and boiled bones were not significantly different, nor were comparisons between samples with the same treatments. Muscles had higher DNA concentrations when boiled and fins had higher when fresh. These findings show that regarding certain tissue types can be expected to better retain DNA concentrations after boiling.

A sistemática de Cnidaria Anthozoa foi revisada inúmeras vezes e por vários autores, sendo que os Ceriantharia sempre foram avaliados através de dados clássicos e muito pretéritos. O clado Ceriantharia, uma ordem dentre Hexacorallia, apresenta muitos caracteres dúbios e que poderiam colocar os animais em várias posições sistemáticas dentre Cnidaria. Ainda, as revisões de Ceriantharia são sempre derivadas de análises das mesmas espécies, principalmente do Mar Mediterrâneo. O presente trabalho tem como enfoque a discussão dos caracteres taxonômicos (morfológicos e moleculares) de Ceriantharia, sua posição sistemática, levantamento taxonômico de áreas no Oceano Atlântico praticamente inexploradas para o grupo e aspectos da biologia das espécies encontradas. Os resultados apontam para uma nova posição sistemática de Ceriantharia, levando o grupo à base de Anthozoa e como um ramo independente. A utilização de dados moleculares evidenciou o vigor do emprego de DNA barcoding na zoologia comparada de Ceriantharia. Aspectos de especiação e de revisão sistemática resultaram pelas análises moleculares. Ao mesmo tempo, os dados morfológicos são extremamente confusos e muito do que foi utilizado como caractere de delimitação de espécies pela tradição de se assumir homologias com Actiniaria é, aparentemente, inconsistente. O ciclo de vida de Isarachnanthus noturnus foi verificado, sendo que o seu estágio jovem, planctônico, e de dispersão é de longa duração. Dados sobre regeneração em pólipos também foi verificada e com a constatação de mecanismos divergentes entre as espécies. / The Cnidaria Anthozoa systematics was revised several times by a number of authors, and the Ceriantharia always evaluated using very classical and ancient data. The Ceriantharia clade, an Hexacorallia order, has many dubious characters that could place the animals in various systematic positions within Cnidaria. Also, the Ceriantharia reviews are always derived from analysis of the same species, mainly from the Mediterranean Sea. This work focuses on the discussion of taxonomic characters (morphological and molecular) of Ceriantharia, the systematic position, taxonomic survey of virtually unexplored Atlantic Ocean areas for the group and the biology of the observed species. The results suggest a new systematic position of Ceriantharia placing the group at the basis of Anthozoa and as an independent branch. The molecular data comparison showed the force of DNA barcoding use in Ceriantharia comparative zoology. The molecular data allowed the discussion of several aspects of speciation and systematics. At the same time, the morphological data are extremely confusing and much of it was used as delimiting species character by the tradition of assuming homologies with Actiniaria is, apparently, inconsistent. The life cycle of Isarachnanthus noturnus was observed, and the young, planktonic and dispersal stage is long-lived. Data on regeneration of polyps was also verified with the observation that mechanisms differ between species.

Background. Caryophyllaceae is a large and cosmopolitic flowering plant family, however the systematics of many of its basal groups has been unclear, due to a lack of unambiguous morphology. Some members of Caryophyllaceae are used medicinally, e.g. Corrigiola roots in Morocco. Monitoring the trade in medicinal plants is complicated due to the absence of useful identification characters in plant products such as roots, bark, and powders. This thesis aims at elucidating the systematics and the ethnobotany of some of the basal clades in Caryophyllaceae. Methods. A comprehensive sampling from herbarium as well as market and field collected material was used in systematic studies combining morphological investigation, molecular phylogenetic and molecular dating analyses. Results. The data show that Polycarpon is polyphyletic, that Sphaerocoma is sister to Pollichia and shows some intraspecific variation, that Sanctambrosia falls within the genus Spergularia, and that both Spergula and Spergularia are monophyletic. Preliminary data suggest that Polycarpaea is polyphyletic and should be split into three larger and several smaller genera, that the members of Paronychia subgen. Anaplonychia will need to be transferred to Herniaria to maintain monophyly, and that Caryophyllaceae emerged during the Paleocene. All the major extant lineages originated in the Oligocene and diversified later. Using molecular identification it was possible to identify around 50% of the Moroccan medicinal roots to species level and an additional 30% to genus level. Discussion and conclusions. The polyphyletic Polycarpon needs to be split into at least three separate genera, but no name changes were made pending further research. The two species of Sphaerocoma were merged into one species with two subspecies. The San Ambrosio Island endemic Sanctambrosia, the only tree-like plant in Caryophyllaceae, is probably the result of a long distance dispersal event and its woody habit and gynodioecy are probably caused by inbreeding depression. Sanctambrosia manicata is transferred to Spergularia. Molecular identification put into practice on traded medicinal roots has a somewhat lower success rate than most theoretical studies, indicating that a global barcoding database would need to include reference sequences from a broad range of populations for each species.

The oceans represent a wealth of biological diversity where many species remain to be discovered and described. Among seaweeds, a paucity of morphological features by which to differentiate species means that many genera harbour overlooked or cryptic species. Fucus, Porphyra and Ulva are three common genera of marine intertidal algae and all include species that are particularly difficult to distinguish morphologically. DNA barcoding has been championed as a revolutionary tool for species identification and discovery and applying this tool to algae was a logical step due to the difficulty of morphological identification of many algal species. This thesis is part of a significant initiative aimed at identification and discovery of all species of seaweeds in Canadian waters, using a DNA barcoding approach. The original concept of DNA barcoding relied on comparing the 5’ region of the mitochondrial cytochrome c oxidase 1 (COI-5P) gene among animal species. In this study, DNA barcoding with COI-5P was applied to the brown algal genus Fucus and worked as well as any other marker to assign morphologies to known species. The DNA barcoding results also uncovered substantial phenotypic diversity in Pacific F. distichus. Results were confirmed by comparison with sequences of the nuclear internal transcribed spacer region (ITS). For Porphyra, COI-5P DNA barcoding was compared with species identification using the chloroplast large rubisco subunit (rbcL) and the Universal Plastid Amplicon (UPA) in a floristic survey of Canadian Porphyra species. Two new species were discovered and described (Porphyra corallicola and Porphyra peggicovensis), and P. cuneiformis was synonymized with P. amplissima. The COI-5P emerged as the best marker for species discrimination despite difficulties with primer universality. To aid in choosing a marker for DNA barcoding for green algae, the universality and species discriminatory power of the rubisco large subunit (rbcL) (considering the 5’ and 3’ fragments independently), the UPA, the D2/D3 region of the nuclear large ribosomal subunit (LSU-D2/D3) and the ITS were evaluated. While the rbcL-3P highlighted several cryptic species, and worked well to distinguish Ulva species, more research is needed to recommend a marker for DNA barcoding generally in marine green macroalgae.

Orientador: Sérgio Nascimento Stampar / Resumo: Existe uma considerável abundância de espécies de cnidários no litoral do Brasil e uma das espécies mais intrigantes e pouco conhecidas é a Tamoya haplonema, a qual dentre suas características mais interessantes se destaca a presença de um padrão de bandas nos tentáculos, o que não é comum nos cubozoários, classe à qual pertence. Essa espécie é conhecida desde meados do século XIX, sendo uma das poucas espécies no Atlântico Sul capaz de causar acidentes graves em humanos. Em 2011 alguns registros de espécimes do mesmo gênero para o Mar do Caribe resultaram na descrição de uma nova espécie, Tamoya ohboya. Essa espécie foi delimitada, principalmente, por dados moleculares e devido à disponibilidade de poucos indivíduos a definição é dúbia em vários trechos. Já foi constatado que o cenário taxonômico do gênero Tamoya no Atlântico Ocidental não é consistente. Desta maneira, o principal objetivo deste estudo foi a comparação taxonômica do gênero Tamoya no Atlântico Sul e Mar do Caribe.Para tal, indivíduos do gênero Tamoya do Atlântico Sul foram analisados e comparados com T.ohboya, utilizando dados morfológicos/morfométricos e moleculares, incluindo o estudo da composição dos nematocistos através das medidas e análises comparativas. Os resultados morfológicos, bem como a análise do cnidoma e os resultados moleculares deixam claro que T.haplonema e T.ohboya são na realidade a mesma espécie. / Abstract: There is a considerable abundance of cnidarian species along the Brazilian coast and one of the most intriguing and little known species is the medusa Tamoya haplonema, which among its most interesting characteristics stands out the presence of a pattern of bands in the tentacles, which is not common in the Cubozoa, the class to which it belongs. This species has been known since the mid-nineteenth century, being one of the few species in the Southern Atlantic capable of causing serious stings to humans. In 2011 specimens of the same genus the Caribbean Sea resulting in the description of a new species, Tamoya ohboya. This species was delimited, mainly, by molecular data and due to the availability of few individuals the definition is dubious in several parts. It has already been verified that the taxonomic scenario of the genus Tamoya in the Western Atlantic is not consistent. Thus, the main objective of this study was the taxonomic comparison of the genus Tamoya in the South Atlantic and the Caribbean Sea. Thus individuals of the genus from the South Atlantic were analyzed and compared with T.ohboya, using morphological / morphometric and molecular data, including the study of the nematocysts composition through measurements and comparative analyzes. The morphological results as well as the analysis of the cnidome and molecular results make it clear that T.haplonema and T.ohboya are actually the same species. / Mestre

Cette thèse s'inscrit dans le cadre du traitement des données issues de séquençage haut débit, et en particulier des données produites en metabarcoding ADN. Le metabarcoding ADN consiste à identifier des taxons ou des groupes taxinomiques à partir de l'ADN présent dans des échantillons environnementaux (eau, sol, fèces...). Après extraction de l'ADN, de courtes séquences utilisées comme marqueurs taxinomiques sont amplifiées par PCR puis séquencées en utilisant les nouvelles techniques de séquençage haut débit. De très importants volumes de données sont ainsi générés, le plus souvent, de plusieurs milliers à plusieurs centaines de milliers de séquences par échantillon. L'objectif principal de cette thèse était le développement de méthodes d'analyse de ces séquences. Les méthodes de classification permettent de traiter de nombreuses problématiques en metabarcoding ADN. La classification supervisée est utilisée pour assigner les séquences à des taxons en les comparant aux séquences de bases de données de référence. Les méthodes de classification non supervisée permettent de créer des groupes taxinomiques (MOTU) à partir des séquences, afin de faire des estimations de biodiversité. Ces méthodes sont aussi employées pour identifier les séquences erronées produites par la PCR et le séquençage notamment, où les séquences erronées dérivent souvent des vraies séquences et leur sont très similaires. Les méthodes de classification demandent une méthode de comparaison des séquences qui soit idéalement à la fois très rapide et exacte. Une telle méthode a été développée, en utilisant un algorithme d'alignement global de type Needleman-Wunsch calculant la longueur de la plus longue sous-séquence commune entre les séquences à aligner, associé à un filtre sans perte permettant d'éviter l'alignement de certaines paires de séquences n'ayant aucune chance de présenter une similarité supérieure à un seuil choisi. L'utilisation d'instructions Single Instruction, Multiple Data, de même que le multithreading optionnel des calculs, permettent d'associer rapidité et exactitude. Cette méthode de comparaison est implantée dans SUMATRA, un programme calculant toutes les similarités deux à deux d'un jeu de données ou entre deux jeux de données, avec possibilité de fixer un seuil de similarité en dessous duquel les similarités ne sont pas rapportées. Elle est aussi utilisée dans SUMACLUST. SUMACLUST est un programme regroupant les séquences en utilisant un algorithme de clustering en étoile, où chaque groupe possède une séquence représentative. Il peut être utilisé pour créer des MOTU, ou pour détecter les séquences erronées dérivant de vraies séquences. Plus spécialisé, le programme SUMACLEAN a été développé pour détecter les séquences contenant des erreurs ponctuelles de PCR. Pour cela, des graphes orientés acycliques sont générés, dont la topologie correspond parfaitement aux cascades d'erreurs générées par les erreurs ponctuelles de PCR. Par ailleurs, une réflexion a été menée pour le développement d'une nouvelle approche de classification supervisée pour l'assignation taxinomique des séquences. Aujourd'hui, la plupart des approches d'assignation utilisent des méthodes mal adaptées au polymorphisme important des marqueurs, et ne considèrent pas suffisamment l'incomplétude et les erreurs inhérentes aux bases de données de référence. Une nouvelle approche a été testée, basée sur l'idée d'un départ depuis la racine de l'arbre taxinomique, suivi d'une descente jusqu'à un arrêt possible lorsque descendre à un niveau taxinomique plus précis semble irraisonnable. Cela permettrait en théorie de mieux gérer les problèmes inhérents aux bases de données de référence, mais pose le problème de la représentation des séquences aux différents niveaux de l'arbre, et du modèle de choix du chemin à prendre, pour lesquels aucune solution complètement satisfaisante n'a été trouvée à ce jour. / This thesis positions itself in the context of the processing of high-throughput sequencing data, and specifically DNA metabarcoding data. DNA metabarcoding consists of the identification of taxa or taxonomic groups from DNA extracted from environmental samples (water, soil, animal feces). After extraction of the DNA, short sequences used as taxonomic markers are amplified by PCR, then sequenced using high-throughput sequencing technologies. Important volumes of data are produced that way, usually from several thousands to several hundreds of thousands sequences per sample. This thesis aimed for the development of methods for the analysis of these sequences. Classification methods allow the treatment of numerous problems in DNA metabarcoding. Supervised classification is used for the taxonomic assignment of sequences to taxa, by comparing them to the sequences of a reference database. Unsupervised classification methods are used to create taxonomic groups (MOTUs) from the sequences, in order to estimate biodiversity. They are also used to identify the erroneous sequences generated during the PCR and sequencing steps in particular, where erroneous sequences often derive from true sequences and remain very close to them. Classification approaches used in the context of DNA metabarcoding necessitate a sequence comparison method that should be both fast and exact. Such a method was developed, using a Needleman-Wunsch type global alignment algorithm computing the length of the longest common subsequence between the two sequences being aligned, associated with a lossless filter allowing to avoid the alignment of some pairs of sequences that have no chance to present a similarity superior to a chosen threshold. The use of Single Instruction, Multiple Data instructions, as well as the availability of multithreading speed up the calculations. This comparison method is implanted in SUMATRA, a program computing all the pairwise similarities of a dataset or between two datasets, with the possibility to set a threshold under which similarities are ignored. It is also used in SUMACLUST, a program grouping sequences using a star clustering algorithm, where each cluster possesses a representative sequence. This algorithm can be used to generate MOTUs, or to identify erroneous sequences deriving from true sequences, by using the fact that true sequences tend to end up as the representative sequences of their cluster. More specialized, the SUMACLEAN program was developed to identify sequences containing ponctual PCR errors. To that end, directed acyclic graphs are created, whose topology matches perfectly the successions of errors generated by ponctual errors during PCR. A new approach for the taxonomic assignment of sequences with a supervised classification method was also studied. Nowadays, most taxononomic assignment approaches use methods that are badly suited for the important polymorphism of markers, and don't take in account enough the incompleteness and errors inherent to reference databases. A new approach was tested, based on the idea of a start from the root of the taxonomic tree, and a descent in it with a possible stop before reaching a leaf, if descending to a more precise taxonomic level seems unreasonable. This approach would theoretically allow for a better handling of the problems inherent to reference databases, but poses a few issues, such as the representation of sequences at intermediate tree levels, and the model used to make choices regarding the path to take in the tree, for which no satisfying solutions have been found yet.

Magister Scientiae (Biodiversity and Conservation Biology) / The spread of invasive species is one of the greatest threats to global biodiversity. Alien plant invasions also have serious economic impact in terms of the delivery of ecosystem goods and services. Studies of biological invasions in southern Africa have tended to overlook grasses (family Poaceae), although there are many naturalised species in the region. Only a few of these, all perennials, have been officially categorised as invasive in South Africa, but in the winter rainfall region of the Western Cape, grass invasion especially by Mediterranean European annuals have also been noted. These grasses can be difficult to identify. DNA barcoding has been suggested as an alternative method of identifying grasses in the hope of facilitating identification of existing invaders and preventing future invasions. In this study a list of all known naturalised grasses in South Africa was compiled, and a DNA barcoding reference database was assembled for these naturalised grass species as well as for native southern African grass species. The two official markers for plant DNA barcoding (rbcLa + matK) were used in barcoding and phylogenetic analyses, both individually and in combination. The barcoding data was assessed for identification efficacy using three distance-based metrics and one tree-based metric in the R package SPIDER, both including and excluding singleton data. This study lists 128 naturalised grass species and subspecies found in South Africa. In the DNA barcoding analyses, matK was found to perform better as a single barcode than rbcLa, with identification success rates of up to 84% for matK and 76% for rbcLa, using the most successful metric which was the Nearest Neighbour criterion for both of these markers in the data sets without singletons. The combined rbcLa + matK data set performed better than either of the two individual markers, with identification success rates of up to 91% in the data without singletons, with the most successful metric again being the Nearest Neighbour criterion. The combined rbcLa + matK data would therefore be the recommended DNA barcode for southern African grasses of the three data sets tested, based on the results of this study. Phylogenetic trees were constructed with the DNA barcoding data using Bayesian Inference (BI) and Maximum Parsimony (MP) to assess the usefulness of the data in phylogenetic studies and to confirm the efficacy of this grass DNA barcoding data when using tree-based methods of identification. Both the matK and combined datasets resolved all of the grass tribes represented in this study as monophyletic, but the rbcLa data did not.

abstract: An understanding of diet habits is crucial in implementing proper management strategies for wildlife. Diet analysis, however, remains a challenge for ruminant species. Microhistological analysis, the method most often employed in herbivore diet studies, is tedious and time consuming. In addition, it requires considerable training and an extensive reference plant collection. The development of DNA barcoding (species identification using a standardized DNA sequence) and the availability of recent DNA sequencing techniques offer new possibilities in diet analysis for ungulates. Using fecal material collected from controlled feeding trials on pygmy goats, (Capra hicus), novel DNA barcoding technology using the P6-loop of the chloroplast trnL (UAA) intron was compared with the traditional microhistological technique. At its current stage of technological development, this study demonstrated that DNA barcoding did not enhance the ability to detect plant species in herbivore diets. A higher mean species composition was reported with microhistological analysis (79%) as compared to DNA barcoding (50%). Microhistological analysis consistently reported a higher species presence by forage class. For affect positive species identification, microhistology estimated an average of 89% correct detection in control diets, while DNA barcoding estimated 50% correct detection of species. It was hypothesized that a number of factors, including variation in chloroplast content in feed species and the effect of rumen bacteria on degradation of DNA, influenced the ability to detect plant species in herbivore diets and concluded that while DNA barcoding opens up new possibilities in the study of plant-herbivore interactions, further studies are needed to standardize techniques and for DNA bar-coding in this context. / Dissertation/Thesis / M.S. Applied Biological Sciences 2012