Dissertations / Theses on the topic 'Gene flow'

Ecological interactions such as predation, competition, and mutualism are important forces that influence species evolution. Coevolution is defined as reciprocal evolutionary change in interacting species. The Geographic Mosaic Theory of Coevolution (GMTC) provides a theoretical framework to explain how collections of populations should coevolve across space. Two fundamental aspects of the GMTC are gene flow among populations and the presence of selection mosaics, which are collections of localities with particular selection regimes. Several studies have explored how phenotypic trait matching between species evolves in pairs or small groups of species. However, ecological interactions frequently form large networks that connect dozens of species present in a given community. In networks of mutualisms, for instance, the organization of interactions may affect ecological and evolutionary processes. A next step in understanding the coevolutionary process is to investigate how aspects of the GMTC affect the evolution of species embedded in interaction networks. In this dissertation, we tried to fill this gap using a mathematical model of coevolution, complex networks tools, and information on empirical mutualistic networks. Our numerical simulations of the coevolutionary model allow us to draw three main conclusions. First, gene flow affects trait patterns generated by coevolution and may favor the emergence of trait matching depending on the selection mosaic. Second, the organization of mutualistic networks influences coevolution, but this effect may vanish when gene flow favors trait matching. Intimate mutualisms, such as protection of host plants by ants, form small and compartmentalized networks that generate higher trait matching than large and nested networks typical of mutualisms among free-living species, such as pollination. Third, habitat fragmentation resulting in the disruption of gene flow should reduce the reciprocal adaptations between interacting species and at the same time promote adaptations to the local abiotic environment. In conclusion, we show that a complex interplay between gene flow, the geographic structure of selection, and the organization of ecological networks shapes the evolution of large groups of species. Our results therefore allow predictions of how environmental impacts such as habitat fragmentation will modify the evolution of species interactions<br>Interações ecológicas como predação, competição e mutualismo são importantes forças que influenciam a evolução de espécies. Chamamos de coevolução a mudança evolutiva recíproca em espécies que interagem. A Teoria do Mosaico Geográfico da Coevolução (TMGC) fornece um arcabouço teórico para entender como conjuntos de populações coevoluem ao longo do espaço. Dois aspectos fundamentais da TMGC são o fluxo gênico entre populações e a presença de mosaicos de seleção, isto é, conjuntos de locais com regimes de seleção particulares. Diversos estudos exploraram como o acoplamento entre fenótipos de diferentes espécies evolui em pares ou pequenos grupos de espécies. Entretanto, interações ecológicas frequentemente formam grandes redes que conectam dezenas de espécies presentes em uma comunidade. Em redes de mutualismos, por exemplo, a organização das interações pode influenciar processos ecológicos e evolutivos. Um próximo passo para a compreensão do processo coevolutivo consiste em investigar como aspectos da TMGC influenciam a evolução de espécies em redes de interações. Nesta dissertação, tentamos preencher esta lacuna usando um modelo matemático de coevolução, ferramentas de redes complexas e informação sobre redes mutualistas empíricas. Nossas simulações numéricas do modelo coevolutivo apontam para três principais conclusões. Primeiro, o fluxo gênico influencia os padrões fenotípicos gerados por coevolução e pode favorecer a emergência de acoplamento fenotípico entre espécies dependendo do mosaico de seleção. Segundo, a organização de redes mutualistas influencia a coevolução, mas este efeito pode desaparecer quando o fluxo gênico favorece acoplamento fenotípico. Mutualismos íntimos, como proteção de plantas hospedeiras por formigas, formam redes pequenas e compartimentalizadas que geram um maior acoplamento fenotípico do que as redes grandes e aninhadas típicas de mutualismos entre espécies de vida livre, como polinização. Por fim, a fragmentação de habitat, ao extinguir o fluxo gênico, pode reduzir as adaptações recíprocas entre espécies e ao mesmo tempo tornar cada espécie mais adaptada ao seu ambiente abiótico local. Em suma, mostramos que interações complexas entre fluxo gênico, estrutura geográfica da seleção e organização de redes ecológicas moldam a evolução de grandes grupos de espécies. Dessa forma, podemos traçar previsões sobre como impactos ambientais como a fragmentação de habitat irão alterar a evolução de interações ecológicas

La metagenomica ha permesso lo studio della diversità degli organismi non coltivabili e il loro ruolo nei vari ecosistemi tra cui suoli e sedimenti marini. acque interne e di oceano aperto. L’analisi della diversità attraverso gli approcci metagenomici è basata sull’estrazione del DNA genomico assumendo che questo sia completamente associato a biomassa vivente. Tuttavia, recenti studi hanno dimostrato che il metagenoma completo di ogni campione ambientale è costituito da pool differenti, tra cui: ,quello dei virus, quello associato a biomassa microbica e quello associato a biomassa non vivente (i.e., DNA extracellulare). Le procedure comunemente utilizzate per isolare il DNA da campioni ambientali non discriminano fra i diversi pool di DNA, influenzando quindi i risultati delle indagini effettuate. I viromi sono metagenomi contenenti DNA virale. I virus non sono solo le più abbondanti entità biologiche negli oceani del mondo, ma attraverso le loro infezioni controllano sia l'abbondanza e la diversità procariotica, sia cicli biogeochimici importanti a livello dell’ecosistema marino. L’approccio metagenomico applicato alla componente virale dei sistemi marini ha permesso di scoprire che i virus potenti possono essere importanti agenti di trasferimento genico: infatti , attraverso la ricombinazione e integrazione, i virus possono sia prelevare porzioni di genomi dei loro ospiti sia trasferirli ad altri ospiti. Nonostante la loro importanza, la diversità virale negli ecosistemi bentonici marini profondi è ancora del tutto sconosciuta e la metagenomica sembra essere l'approccio più efficace per analizzarla. Finora, diversi strumenti bioinformatici sono stati utilizzati per analizzare le sequenze virali in campioni ambientali, ma la maggior parte di questi strumenti non sono stati specificamente progettati per l'analisi delle sequenze virali e confronti per verificare la loro validità non esistono o sono troppo limitati. In questo studio abbiamo sviluppato una procedura specifica per il recupero selettivo del DNA virale da sedimenti marini profondi. Il DNA virale è stato sequenziato, attraverso tecniche di pirosequenziamento, e analizzato confrontando tre pipeline di annotazione di sequenze metagenomiche (MG-RAST, VMGAP, MetaVir). Questo al fine di testare la loro efficienza nell'analisi della diversità virale, sia utilizzando i dati di sequenziamento sia quelli derivanti da metagenomi virali ottenuti in silico a partire da sequenze depositate nelle banche dati. Queste analisi indicano che la diversità tassonomica e funzionale dei virus varia in funzione della pipeline utilizzata. MetaVir è risultata la pipeline più affidabile per l’annotazione tassonomica della diversità virale. Tuttavia, tale pipeline, non essendo stata progettata per annotazioni di tipo funzionale, deve essere necessariamente integrata con altre come VMGAP. Pertanto, questo studio evidenzia la necessità di sviluppare una piattaforma bioinformatica completa per un’annotazione efficiente sia tassonomica sia funzionale, al fine di .far luce sull'enorme diversità genetica contenuta nei virus presenti nel più grande ecosistema della Terra. La diversità tassonomica virale è stata esplorata in campioni di sedimento marino raccolti in differenti aree oceaniche del Globo, da 2000 a 10000 m di profondità. I risultati di questa analisi hanno permesso di evidenziare, per la prima volta, che la diversità virale negli ecosistemi bentonici profondi non solo è molto elevata, ma anche che alcune famiglie virali sono molto diffuse, nonostante le differenze ambientali ed ecologiche degli ecosistemi analizzati. Le similarità tra i campioni analizzati in questo studio e la maggior parte dei viromi ad oggi pubblicati suggeriscono che diversi fattori contribuiscono a modellare la diversità dell’assemblage virale. In aggiunta, tutti i viromi presentano un’elevata diversità funzionale putativa e contengono anche funzioni derivanti dai loro ospiti, tra cui funzioni metaboliche chiave. I metagenomi microbici sono costituiti da DNA associato a biomassa vivente, che nei sedimenti marini profondi è rappresentata prevalentemente da procarioti (Batteri e Archaea). La possibilità di studiare le comunità microbiche attraverso gli approcci metagenomici ha permesso di capire meglio il loro ruolo nell'ambiente marino anche negli ambienti marini profondi, che sono molto difficili da raggiungere, consentendo anche la scoperta di nuovi enzimi e vie metaboliche, spesso utili per applicazioni industriali o biotecnologiche. Il DNA extracellulare ha un ruolo chiave negli ecosistemi marini, sia come fonte di nutrienti che come fonte di geni. Può essere sia rilasciato dalla comunità procariotica durante la crescita sia attraverso la lisi cellulare (a causa di infezione virale o per morte cellulare naturale). Sedimenti e suoli possono anche preservare questo DNA rilasciato, che può rimanere adeso alle particelle minerali e organiche. Questo pool extracellulare conservato può essere incorporato da cellule naturalmente competenti, che possono andare incontro a processi di trasformazione naturale. In questo studio l'analisi contestuale dei metagenomi microbici ed extracellulari nei diversi ecosistemi bentonici profondi, non solo ha fornito informazioni riguardanti la composizione specifica di ciascun pool, ma ha anche rivelato che il DNA extracellulare contiene una diversità genetica elevata che ad oggi non era mai stata considerata. Tale diversità genetica costituisce una frazione rilevante della diversità genetica associata all’intero metagenoma. La maggior parte della diversità genetica del DNA totale è rappresentata da geni afferenti al DNA extracellulare e, in media, circa il 50% delle «specie» contenute nel DNA extracellulare è condiviso con il microbioma. Inoltre, dal confronto tra tutti i metagenomi risulta che sequenze virali sono presenti non solo, come atteso, nei viromi ma anche nei microbiomi e nei metagenomi extracellulari. L’analisi bioinformatica comparativa tra tutti i metagenomi ha rivelato la presenza di funzioni putative coinvolte in differenti processi di trasferimento genico orizzontale. Di particolare rilevanza sono le funzioni relative all’uptake del DNA e la sua mobilizzazione e quelle relative ad elementi genetici mobili come i gene transfer agents (GTAs) ed i profagi. Esperimenti di laboratorio condotti su sedimenti marini profondi dimostrano anche che il DNA extracellulare può essere una risorsa genetica importante per la comunità microbica dal momento che fino al 6% delle cellule procariotiche risultano essere competenti e in grado di acquisire nuove funzioni. Nel loro insieme, questi risultati suggeriscono che gli ecosistemi bentonici profondi hanno un elevato potenziale di trasferimento genico che può avvenire attraverso meccanismi multipli.<br>Metagenomics has allowed the study of the diversity of organisms which can’t be cultivated and their role in various ecosystems, including soils and marine sediments, inland waters and the open ocean. The analysis of diversity through metagenomic approaches is based on the extraction of genomic DNA, assuming that it is completely associated with living biomass. However, recent studies have shown that the whole metagenome of each environmental sample is composed of different pools, including that of viruses, that associated with microbial biomass and that associated with non-living biomass (i.e, extracellular DNA). The procedures commonly used to isolate DNA from environmental samples do not discriminate between the various pools of DNA, thus affecting the results of investigations carried out. Viromes are metagenomes containing viral DNA. Viruses are not only the most abundant biological entities in the World’s oceans, but through their infection they control both prokaryotic abundance and diversity and important biogeochemical cycles of the marine ecosystem. The metagenomic approach applied to the viral component of marine systems has led to the discovery that viruses can be important agents of gene transfer: in fact, through recombination and integration viruses can either excide portions of the genomes of their hosts and transfer them to other hosts. Despite their importance, viral diversity in the deep-sea benthic ecosystems is still completely unknown and metagenomics seems to be the most effective approach to analyze it. So far, several bioinformatics tools were used to analyze viral sequences in environmental samples, but most of these tools have not been specifically designed for the analysis of viral sequences and comparisons to test for their validity do not exist or are too limited. In this study, we developed a specific procedure for the selective recovery of viral DNA from deepsea sediments. Viral DNA was sequenced, through pyrosequencing techniques, and analyzed comparing three annotation pipelines for metagenomic sequences (MG-RAST, VMGAP, MetaVir). To test their efficiency in the analysis of viral diversity we used both the sequencing data derived from viral metagenomes and those obtained in silico from sequences deposited in public databases. These analyses indicate that the taxonomic and functional diversity of viruses varies depending on the pipeline used. MetaVir proved to be the most reliable pipeline for the annotation of viral taxonomic diversity. However, since this pipeline was not designed for functional annotations of viral sequences, its results must necessarily be integrated with those obtained by other pipelines such as VMGAP. Therefore, this study highlights the need to develop a comprehensive bioinformatics platform for efficient functional and taxonomic annotation of viromes to shed light on the enormous genetic diversity contained in the viruses present in the largest ecosystem on Earth. The viral taxonomic diversity has been explored in marine sediment samples collected in different ocean areas of the globe, ranging from 2000 to 10000 m depth. The results of this analysis have revealed, for the first time, that viral diversity in benthic deep-sea ecosystems, is not only very high but also that some viral families are widespread, despite the environmental and ecological differences in the ecosystems analyzed. The similarity between the samples analyzed in this study and the majority of viromes published to date suggests that several factors contribute to shape the diversity of the viral assemblage. In addition, all viromes have a high functional diversity and also contain putative functions derived from their hosts, including key metabolic functions. The microbial metagenomes are made of DNA associated with living biomass and in deep-sea sediments they are mainly represented by prokaryotes (Bacteria and Archaea). The possibility of studying the microbial communities through metagenomic approaches has allowed us to better understand their role in the marine environment in deep marine environments, which are very difficult to reach, enabling the discovery of new enzymes and metabolic pathways, often useful for industrial or biotechnological applications. The extracellular DNA plays a key role in marine ecosystems, both as a source of nutrients and as a source of genes. It can be released from prokaryotic community during growth and through cell lysis (due to viral infection or to natural cell death). Sediments and soils can also preserve this released DNA, which can be adsorbed onto mineral and organic particles. This preserved extracellular pool can be incorporated into naturally-competent cells, which may undergo natural transformation processes. In this study, the contextual analysis of extracellular and microbial metagenomes in different benthic deep-sea ecosystems, has not only provided information on the specific composition of each pool, but also has revealed that the extracellular DNA contains a high genetic diversity, which to date has never been considered. This genetic diversity represents a major fraction of the genetic diversity associated with the whole metagenome. Most of the genetic diversity of the total DNA is represented by genes related to the extracellular DNA and, on average, about 50 % of the "species" contained in the extracellular DNA is shared with the microbiome. Moreover, the comparison among all the metagenomes showed that viral sequences are present not only in viromes, as expected, but also in microbiomes and extracellular metagenomes. The comparative bioinformatic analysis between all metagenomes has revealed the presence of putative functions involved in different processes of horizontal gene transfer. Of particular relevance are the functions for DNA uptake and mobilization and those related to mobile genetic elements, such as gene transfer agents (GTAs) and prophages. Laboratory experiments conducted in deep-sea sediments also show that the extracellular DNA may be an important genetic resource for the microbial community, since up to 6% of prokaryotic cells appear to be competent and able to acquire new functions. Taken together, these results suggest that deep benthic ecosystems have a high potential for gene transfer that can occur through multiple mechanisms.

Begonia L. is one of the largest plant genera, comprising over 1500 species. Weak species cohesion, and the rapid evolution of reproductive barriers in allopatry, are two processes that have been postulated to explain the generation of such hyper-diversity of taxa within a single genus of plants. The aim of this thesis is to investigate whether these factors are likely to have been important contributors to the diversity of species found in Central American Begonia. Species cohesion was analysed in the widespread Central American species Begonia heracleifolia and B. nelumbiifolia. Interpopulation seed flow was estimated with seven plastid microsatellites. Breeding system estimates and measures of genetic differentiation at nine nuclear microsatellites were used to infer levels of interpopulation pollen flow. Controlled crosses were employed to assess the strength of reproductive barriers both between populations within species, and between species differing in ecology. The potential for gene flow between species in the wild was assessed in natural hybrid zones using molecular markers. Finally a quantitative trait locus (QTL) approach was employed to investigate the genetic basis of reproductive traits that differ between species. No plastid polymorphisms were found in B. nelumbiifolia, suggesting it has been through a recent population bottleneck. In contrast, B. heracleifolia possessed many plastid haplotypes that were strongly differentiated between populations (G’ST = 0.829). Nuclear microsatellites showed high genetic differentiation within species, and both species were self-compatible and self-fertilize at a moderate rate (B. heracleifolia F’ST = 0.506, FIS = 0.249; B. nelumbiifolia F’ST = 0.439, FIS = 0.380). F1s between ecologically similar B. heracleifolia and B. sericoneura were partly fertile (2-5% seed set), and F1s and early generation backcrosses were found in a hybrid swarm. F1s between B. heracleifolia and the ecologically contrasting B. nelumbiifolia were pollen sterile, and 3 hybrid swarms showed no evidence of hybrids beyond the F1 generation. Seven QTL were found for reproductive traits, including: sex ratio, pollen sterility and stamen number. The population biology of Begonia, with limited seed and pollen dispersal, small population sizes and frequent self-fertilization predisposes them to genetic isolation, increasing the chances that reproductive barriers evolve. These characteristics may underlie the large number of endemics in Begonia.

The main goal of this thesis has been to study the population structure of a marine sponge species (<i>Crambe crambe</i>) at different geographic levels using different molecular markers.<br/><br/>In many organisms, particularly terrestrials, molecular markers have been used to answer questions about the structure and connectivity of populations, the potential of dispersal or the history of species, and these data were of crucial help in conservation and management of species. Most of these studies used allozymes and, more recently, mitochondrial or nuclear sequences with success. Also, microsatellite markers are being developed and applied with terrific success in many species. Unfortunately, application of molecular markers in marine environments lags much behind, especially for invertebrate organisms, where population genetic studies are still scarce and most of them involve only allozymes. Thus, this study attempts to be contribute to the development and application of molecular markers for ecologic and population genetic studies in marine invertebrates.<br/> Most marine invertebrate organisms are benthic, and many of them sessile, with larvae as the main form of dispersal. Because of the intrinsic difficulty on following larval movements in natural environments, molecular markers appear as the perfect tool to track and characterize populations, and even individuals. <br/>Hence, in this thesis several molecular markers have been applied in a sponge species, and with the complementary help of previous ecological and biological knowledge of the species, the present-day population structure of this sponge has been studied, trying to uncover which factors are affecting and might have affected in the past the structure found, while comparing the resolution and the "snapshot" information recovered with each of the markers used.

Many populations seem adapted to their environment, through a match between their phenotype and habitat or resources. However, significant maladaptation is widespread, perhaps partly due to gene flow between populations, which prevents tracking a local optimum, by introducing alleles adapted to alternative conditions. Threespine stickleback show significant adaptive divergence over a variety of phenotypic traits, and the divergent lake-stream pairs of Vancouver Island illustrate the balance between selection and gene flow that exists in natural populations. In my thesis, I use the threespine stickleback stream populations of the Misty system, where the gene flow constraint in the outlet stream is high, to explore maladaptation. To determine whether gene flow is reducing adaptive divergence, I use measurement of selection to detect the gene flow constraint. My primary hypothesis is that selection should be higher in the outlet due to a higher deviation from the optimal stream phenotype. Using an individual tagging mark-recapture experiment over two years, I estimate natural selection on body shape, using several metrics of viability selection. I estimate total intensity of selection, and examine specific traits, such as body depth, that show important differences in divergence between the two sites. Using multivariate distances, I estimate both multivariate selection intensity and determine the direction of selection with reference to the inlet population. Results show inconsistent selection on body depth and overall body shape, suggesting that the pattern of selection is temporally dynamic, changing from one selection model to another. Different selection models may apply if the causality between divergence and gene flow is decoupled in each site. My project gives indications that to better answer the question of the role of gene flow in adaptation, it is necessary to quantify consequences for populations at both the trait level and the fitness level. Future attemps would also benefit from examining the role of sexual dimorphism, as well as using a broader suite of traits and fitness components.<br>Les populations naturelles sont généralement adaptées à leur environnement, et il existe une correspondance entre la morphologie et l'habitat ou les ressources. Pourtant, la maladaptation significative et elle aussi présente. Elle peut-être causé par flux génétique entre populations. Ce flux génétique, empêche la population d'atteindre leur optimum local, dû fait de l'introduction d'allèles adaptés aux conditions alternatives. Dans ma thèse, pour investiguer la maladaptation , j'utilise des populations d'épinoche à trois épines du système Misty, où la contrainte causée par le flux génétique est élevée. Pour séparer la causalité entre le flux génétique et la divergence adaptative, j'utilise la méthode de mesurer la sélection naturelle pour estimer la contrainte du flux génétique. L'épinoche à trois épines montre une divergence adaptative importante dans une variété de traits phénotypiques. Les paires lac-rivière de l'île de Vancouver illustrent bien l'équilibre entre la sélection et le flux génétique qu'il existe entre les populations des lacs et des rivières. En effectuant une expérience de capture marquage recapture individuelle sur deux ans, j'ai pu estimer la sélection naturelle sur la forme des poissons, en utilisant plusieurs mesures de sélection. J'ai estimé l'intensité totale de la sélection et ses effets sur des traits spécifiques, comme la profondeur du corps, qui montrent une divergence entre les deux sites. En utilisant des distances multivariées, j'ai estimé l'intensité de sélection multivariée et j'ai déterminé la direction de sélection en référence a la population du inlet. Mon hypothèse principale est que la sélection devrait être plus élevée dans le outlet du fait de son plus grande déviation du phénotype riverain typique. Les résultats indiquent une sélection variable sur la profondeur du corps, et sur la forme générale; ce qui suggère que le patron de sélection a une dynamique temporelle, changeant d'un modèle de sélection à un autre dans le temps. Les différents modèles de sélection ne s'appliqueront pas, si la causalité entre la divergence et le flux génétique est renversé dans les deux sites. Mes résultats indiquent que pour mieux répondre à la question du rôle du flux génétique dans l'adaptation, il est nécessaire de quantifier les conséquences pour les populations au niveau des traits et du fitness. Les travaux futurs intégreront aussi le rôle du dimorphisme sexuel en utilisant aussi une plus large gamme de traits et de composants de fitness.

Albatrosses and petrels (Order Procellariiformes) are renowned for the huge distances they can cover at sea, and since the advent of tracking technology their pelagic lifestyles are generally well studied. However, tropical species are under-represented in the literature, and may be particularly flexible in their behaviour since tropical oceans are oligotrophic and prey availability is often patchily distributed. Round Island petrels breed in such an environment off the coast of Mauritius in the south-western Indian Ocean. Whilst originally identified as Trindade petrels (Pteromdroma arminjoniana), it has recently been revealed that this population is in fact a mixed, hybridizing population with at least two additional species, namely the Kermadec and Herald petrels (P. neglecta and P. heraldica). However, to date no research has been conducted on the colony-based at-sea distribution of these petrels, or how their mixed ancestry may influence their distribution at sea. In this thesis I firstly explore the possibility that Round Island may not be the only point of contact between these species and find that migration and introgression between wide-ranging Pterodroma may be more common than previously thought. I go on to develop a novel data cleaning method to enable the analysis of geolocation data from Round Island petrels, and use that data to describe for the first time their at-sea distribution and the extensive within-population variation in these patterns. Finally, I use a combination of tracking and microsatellite genotype data to ultimately weigh the influence of individual genetic background and the wider seasonal environment on distribution variability around the breeding colony. The Round Island petrel population is a stronghold where seabird populations globally are in decline. This thesis adds to the limited literature on ecology of tropical petrel species, and highlights the importance of considering behavioural and genetic diversity in future conservation plans.

Diversifying selection and gene flow were traditionally viewed as antagonistic forces, with diversifying selection promoting adaptation and speciation, and gene flow opposing them. However, their roles are far more complex than this. While gene flow can prevent speciation or initiate despeciation, it can also generate new hybrid species. Similarly, while adaptive divergence can be wiped out by gene flow, new adaptive variation can be introduced via hybridisation. The relative frequency of these outcomes, and indeed the frequency of gene flow and diversifying selection in general are largely unknown. This thesis illuminates these questions through evolutionary genomic analyses focussed on a recently diverged group of ragworts (Senecio). The Mediterranean Senecio species-complex contains several cases of hybrid speciation, as well as two species, S. aethnensis and S. chrysanthemifolius, which are a potential case of ecological speciation with gene flow, having adapted to high and low altitude habitats respectively on Mount Etna. However, their demography was previously un-studied. I first show that S. aethnensis and S. chrysanthemifolius diverged recently, at a time that coincides with the growth of Mount Etna to the altitudes which separate the species today, have experienced significant gene flow following their split, and are likely to be sister species, bolstering the hypothesis of ecological speciation. I further demonstrate that gene flow is common in the wider clade, and pinpoint multiple cases of gene flow amongst them. Finally, I identify several genes under positive selection in the clade, and show that the proportion of genes under selection is high relative to many other plant genera. The results further establish Senecio species as an invaluable model system for the study of diversification with gene flow, and suggest that high levels of gene flow and selection are features of their evolution, a situation which may prove to be common in plants.

The aim of this project is to study the fragmentation in broad-leaved forests, so the species selected were mainly linked to this ecosystem. Almost all amphibian species are model candidates for studies of fragmentation effects on connectivity: in fact most amphibian species occur as metapopulations; in addition, they are a taxon particularly susceptible to isolation, as they generally have low dispersal capabilities and are rather philopatric to breeding sites. These characteristics often lead to high genetic differentiation, even at restricted scales. In particular we choose the Fire Salamander (Salamandra salamandra, AMPHIBIA, URODELA) that is strongly linked to broad-leaved forest ecosystems, depends on ecosystem processes (such as those that allow the development of an adequate litter structure); is strongly affected by specific resources (such as hydrology, some chemical and physical water parameters); and it is also limited by a low dispersal capability. However not only amphibian species are affected by habitat fragmentation, also arboreal mammals, such tree squirrels and dormice, are often more threatened than other species due to their low dispersal capability in absence of structural connection between habitat patches. In European regions, various single-species studies have been carried out, suggesting similar responses in this guild of mammals. Among them, the Hazel Dormouse (Muscardinus avellanarius, RODENTIA, GLIRIDAE) is an arboreal rodent living in woodland and hedgerows, whose presence is negatively affected by fragmentation. For its poor conservation status and negative long-term trend in almost all Europe, the species is included in the Annex IV of the 92/43/ECC Directive (Habitat Directive), concerning species that require a strict protection regime that must be applied across their entire natural range within the EU. For this reason we sampled also the Hazel Dormouse that can be defined as a focal species sensu Lambeck (1997) at least for three out four ecological traits (they are limited by resources, dispersal capability, and ecological processes). We choose two different areas of study, both affected by the habitat fragmentation: Lombardy Region (Northern Italy, where we worked on the Fire Salamander), and Latium Region (central Italy, where we sampled the Dormouse). In both regions we sampled a fragmented forest area and a continuous one, our control.

Roads are a pervasive cause of habitat fragmentation around the world. Roads can present barriers to movement through direct mortality, behavioral avoidance, or by acting as an impassable physical object in the landscape. The barrier effect of roads has been demonstrated for species from multiple taxa. Species inhabiting the interior of forests may be particularly sensitive to roads because of their inability or disinclination to traverse gaps in forest cover. We combined telemetry and molecular genetic techniques to examine the effects of a high-speed, high-volume highway on the movement and population genetic structure of northern flying squirrels (Glaucomys sabrinus) in the Cascade Mountains of Washington, USA. During 2009 and 2010, we trapped and radio-tracked flying squirrels (n = 17) to gather data on movement within their home ranges and to detect movement across the highway. Additionally, we tested for effects of the highway on genetic variation in the study area using DNA extracted from cheek cells of 59 squirrels and genotyped at 11 microsatellite loci. Seven of the 17 radio-tracked squirrels crossed the highway at least once during their nightly movements. The width of the gap between forest edges across the highway appeared to negatively influence crossing rates and no crossings were observed at a site where the average gap width exceeded 80 meters. Genetic analysis provided no evidence that either geographic distance or the presence of the highway was associated with genetic differences between sites at the landscape scale. Results suggest that populations on either side of the highway are well connected demographically and genetically, and that connectivity can be maintained if gaps in forest canopy associated with the highway are kept to a width within the gliding range of flying squirrels.

Fig trees (Ficus spp., Moraceae) are widely known as one of the most important plant genera in terms of providing food resources for wildlife. They also have an obligatory mutualism with fig wasps (Agaonidae), which makes fig trees a good subject to investigate coevolution between plants and animals. Many fig tree species are found in high humidity riparian areas, where some display adaptations for seed dispersal by water. However, the majority of research on fig tree seed dispersal has focused on big monoecious strangler fig tree species (Subgenus Urostigma), with few studies of smaller shrubby dioecious fig trees. This thesis aims to examine the vegetative and reproductive phenology of riparian fig tree species in Kanchanaburi Province, western Thailand including their interactions with terrestrial and aquatic frugivores, the function of a jelly-like substance produced by one species, and how gene flow has influenced the genetic structure of a second riparian fig tree. Also, current knowledge about global fig tree species and their interactions with vertebrate frugivores, based on published articles, is updated. The four riparian fig tree species studied are all evergreen. Figs of the dioecious F. oligodon, F. ischnopoda and F. montana were produced seasonally, with less seasonality of fig production in the monoecious F. racemosa. Fig production was likely to be influenced by temperature. Figs of F. racemosa were mostly produced towards the canopy level (more than 5 m above the ground), but the other three species tended to produce figs near to ground level. Most fig crops of the three dioecious species, which produced relatively smaller crop sizes than F. racemosa, were not visited by any frugivores during observation periods, and uneaten figs mainly fell to the ground or water. Where crops were visited, bulbuls (Pycnonotidae) were the most frequent frugivores visiting the small shrubs F. ischnopoda and F. montana and the Grey-bellied squirrel (Callosciurus caniceps, Sciuridae) were frequent visitors to the larger fig tree species (F. oligodon and F. racemosa). Figs that fell onto the water were consumed quickly by a fish, Blue mahseer (Neolissochilus stracheyi, Cyprinidae). However, seeds of F. montana ingested by this and another cyprinid fish (Barbonymus altus) were mostly destroyed, suggesting their roles are as seed predators rather than dispersers. A mucilaginous jelly covering F. oligodon seeds did not attract any ant species. Seeds covered with this jelly did not germinate, but could germinate rapidly once it was removed, suggesting its function is seed germination suppression and probably to protect the seeds from pathogens. Most genetic variation was within rather than between populations of F. montana, but extensive gene flow between populations was detected, generated mostly by pollen flow, because gene flow by seed dispersal was limited. This is consistent with the observations that very few frugivores interacted with this fig tree species. The global review of interactions between vertebrate frugivores and fig trees, which included data from this thesis, showed that the major frugivorous bird families that interact with fig trees are mynahs (Sturnidae), pigeons (Columbidae), bulbuls (Pycnonotidae) and parrots (Psittasidae), and the major families of frugivorous mammals were Old World monkeys (Cercopithecidae), Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae). Strangler figs had the highest number records of frugivore species interactions, and few small dioecious species had any records of what eats their fruits. The limitations of the relevant literature outlined in an earlier (2001) review are unchanged, with recent records adding to the number of species records, but generally failing to add to a detailed understanding of how fig trees interact with frugivores.

Thesis (MSc)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: Limited dispersal, territoriality and the occupation of patchy habitats; characters that typify most African rock-dwelling (saxicolous or rupicolous) species, often result in structured genetic patterns with little or no gene-flow among populations (e.g., velvet worms, net-winged midges, elephant shrews, red rock rabbits and a variety of lizards and agamas). In an attempt to deepen our understanding of taxa that inhabit these “terrestrial islands” the distribution of genetic variation was studied at several spatial scales in the rock hyrax, Procavia capensis. This species has a polygynous social system that is unusual among taxa with similar ecological requirements, and a morphology that would intuitively be associated with poor dispersal capabilities (short limbs and a squat, heavy body). Possibly as a consequence of these considerations, few studies have attempted to determine the distance of migration by rock hyraxes and the influence that their social system and the surrounding landscape has on dispersal success. This investigation therefore tests hypotheses of how the ecology, distribution, social structure and the connectivity of the surrounding landscape have contributed to shaping the structure of rock hyrax genetic variation across the Namaqualand and western Fynbos regions. To do so, mitochondrial and microsatellite markers were used to document gene-flow at a fine spatial scale (an isolated population comprising 5 koppies), an intermediate spatial scale (across known geographic barriers to saxicolous taxa - the Cape Flats and Knersvlakte), and a regional spatial scale (across the Namaqualand/western Fynbos regions of South Africa - regions exhibiting contrasting landscape connectivity). In addition the genetic diversity, spatial clustering, sex-biased dispersal and relatedness (fine-scale) of colonies is described and the major genetic breaks detected in the investigation dated using a relaxed molecular clock approach. Finally, these results were compared to other studies that identified the Cape Flats and Knersvlakte as phylogeographic disruptors. The genetic patterns at a fine spatial scale were complex: Gene-flow was restricted by the social structure of the rock hyrax rather than geographic distance, dispersal was female-biased and there was significant genetic structure. Genetic structure was also evident at the intermediate and regional spatial scales. In the Hottentots Holland Mountains and Cape Peninsula gene-flow was restricted (in both data sets) in comparison to localities that traversed the Cape Flats. In contrast, gene-flow across the Knersvlakte was restricted in the mitochondrial DNA data set but not so with microsatellites. A similar pattern was observed at a regional scale pointing to male-biased dispersal within this species - a result of its polygynous social structure. In addition to sex-biased dispersal, landscape connectivity also influenced gene-flow on a regional spatial scale as the Namaqualand region, which has greater intermediate suitable habitat compared to the western Fynbos region, displayed significantly higher levels of gene-flow between sampling localities. Consequently, colonies in Namaqualand were genetically more diverse compared to those of the western Fynbos region. Two major matrilineal clades were evident on both side of the Knersvlakte - one to the north of this biogeographic break (Namaqualand), and the other to the south (western Fynbos). This was not, however, evident from the microsatellite data (reflecting the influence of male dispersal) where seven nuclear clusters were found. In keeping with other studies on saxicolous vertebrate taxa straddling the same region, this area of low connectivity has acted (and probably still does) as a barrier to gene-flow. Importantly, unlike in many other (admittedly invertebrate) species, no evidence of a genetic break was detected among hyrax populations across the Cape Flats. Colonies across the Hottentots Holland Mountains and Cape Peninsula regions may have been subject to founder-events and breeding isolation. This investigation demonstrated the importance of using a well-structured sampling regime that included both mitochondrial and nuclear markers and it underscores the need to apply appropriate statistical programmes for inferring genetic patterns. It shows that landscape genetics may be useful in a conservation context and should be taken into account when planning conservation initiatives that include the implementation of corridors. In brief, the information contained in this study advances our knowledge of the dispersal capability and genetic diversity of contemporary rock hyrax populations.<br>AFRIKAANSE OPSOMMING: ‘n Beperkte spreidingsvermoë, territorialiteit en die bewoning van yl-verspreide habitat is kenmerkend van die meeste klip-bewonende spesies in Afrika en dit veroorsaak gereeld gestruktureerde genetiese patrone met min of geen genevloei tussen populasies (bv., die velvetwurms, net-vlerk muggies, klipklaasneuse, klipkonyne en ‘n verskeidenheid akkedisse en koggelmanders). In ‘n poging om kennis oor taksa wat hierdie “terrestriële eilande” bewoon te verdiep, het ons die die verspreiding van genetiese variasie bestudeer oor verskeie ruimtelike skale in die klipdassie, Procavia capensis. Hierdie spesie het ‘n veelwywige sosiale sisteem, wat vreemd is onder taksa met soortgelyke ekologiese vereistes, en ‘n morfologie wat intuïtief verbind kan word met swak spreidingsvermoëns (kort bene en ‘n kort, dik liggaam). As ‘n moontlike resultaat van hierdie oorwegings het min studies tot dusver daarop gefokus om die migrasie-afstand van klipdassies en die invloede van hulle sosiale sisteem en die omliggende landskap op spreidings-sukses te bepaal. Hierdie studie toets daarom hipoteses oor hoe die ekologie, verspreiding, sosiale struktuur en die konnektiwiteit (verbindheid) van die omliggende landskap bydra om die struktuur van genetiese variasie in klipdassies oor die Namakwaland en westelike Fynbos streke te beïnvloed. Derhalwe is mitochondriale en mikrosatelliet merkers gebruik om genevloei te bepaal op ‘n fyn ruimtelike skaal (‘n geïsoleerde populasie bestaande uit 5 koppies), ‘n gemiddelde ruimtelike skaal (oor bekende geografiese grense vir klipbewonende taksa - die Kaapse Vlakte en die Knersvlakte), en op ‘n streeks (groot) ruimtelike skaal (oor die Namakwaland/westelike Fynbos streke van Suid-Afrika - streke met kontrasterende konnektiwiteit van die landskap). Bykomend is die genetiese diversiteit, ruimtelike groepering, seksuele eensydigheid in spreiding en genetiese verwantskappe (fyn skaal) van kolonies beskryf en die hoof genetiese skeiding gedateer deur gebruik te maak van ‘n ontspanne molekulêre klok. Laastens het is die resultate van hierdie studie vergelyk met dié van ander studies wat die Kaapse Vlakte en Knersvlakte as filogeografiese skeidings gevind het. Die genetiese patrone op ‘n fyn ruimtelike skaal was kompleks: Genevloei is beperk deur die sosiale struktuur van die klipdassie eerder as geografiese afstand, migrasie was wyfie-spesifiek en daar was beduidende genetiese struktuur tussen kolonies. Genevloei was beperk in die Hottentots Holland berge en die Kaapse Skiereiland (in beide datastelle) in vergelyking met lokaliteite oor die Kaapse Vlakte. In kontras was genevloei oor die Knersvlakte beperk in die mitochondriale DNA, maar nie in die mikrosatelliete nie. ‘n Soortgelyke patroon is waargeneem op ‘n streeks skaal wat dui op mannetjie-spesifieke spreiding in hiérdie spesie - ‘n resultaat van die veelwywige sosiale struktuur. Bykomend, saam met geslag-spesifieke spreiding, het landskaps konnektiwiteit ook genevloei beïnvloed op ‘n streeks skaal omdat die Namakwaland streek, wat meer tussenleggende geskikte habitat bevat in vergelyking met die westelike Fynbos streek, beduidende hoër vlakke van genevloei tussen lokaliteite getoon het. Gevolglik was kolonies in Namakwaland geneties meer divers in vergelyking met dié van die westelike Fynbos streek. Twee hoof moederlike genetiese groepe is waargeneem op elke kant van die Knersvlakte - een aan die noorde van hierdie biogeografiese skeiding (Namakwaland) en een in die suide (westelike Fynbos). Dieselfde patroon was egter nie waarneembaar in die mikrosatelliet data nie (wat die invloed van mannetjie-spesifieke spreiding toon) waar sewe nukluêre groepe gevind is. In ag genome ander studies op klipbewonende gewerwelde taksa oor dieselfde verspreiding, het hierdie area van lae konnektiwiteit histories (en heelmoontlik ook huidiglik) as ‘n grens vir genevloei gedien. Belangrik, anders as in ander (hoewel ongewerwelde) spesies, kon ons geen bewyse verskaf van ‘n genetiese skeiding tussen klipdassie populasies oor die Kaapse Vlakte nie. Kolonies in die Hottentots Holland berge en Kaapse Skiereiland is dus onderhewig aan moontlike vestigings-effekte en telings-isolasie. Hiérdie studie demonstreer die belang van die gebruik van ‘n goed-gestruktureerde monsternemingskema, die insluiting van beide mitochondriale en nukluêre merkers en dit beklemtoon ook die noodsaaklikheid van die gebruik van toepaslike statistiese programme vir gevolgtrekkings oor genetiese patrone. Dit toon ook dat landskapsgenetika nuttig mag wees in ‘n bewaringskonteks en in ag geneem moet word in die beplanning van bewarings inisiatiewe wat die implementering van korridors insluit. Kortliks, die informasie in hierdie studie bevorder ons kennis oor die spreidingsvermoë en genetiese diversiteit van kontemporêre klipdassie populasies.

Australia’s largest and most important waterway- the Murray-Darling Basin (MDB) - is under threat owing to predicted increases in temperature extremes and reduction in rainfall - runoff in the coming decades. Management strategies are required that incorporate an understanding of dispersal patterns of the MDB fauna and flora. Patterns of dispersal have typically been studied through direct organismal studies but genetic approaches, in which the movement of genes in the landscape is used as a correlate of species dispersal, can provide a more comprehensive view by investigating at a much larger temporal and spatial scale. Genetic connectivity (dispersal) is influenced by the biology of the species, and by flow regime and the dendritic pattern of the network in riverine landscapes. An understanding of the relative influence of each on connectivity is required to deliver informed management strategies. Decisions regarding whether management for conservation is necessary also require an understanding of a species susceptibility to a changing environment. Species already exhibiting deleterious trajectories under current flow regimes in the basin may require more drastic measures than those that have remained unaffected.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Griffith School of Environment<br>Science, Environment, Engineering and Technology<br>Full Text

Die intrazellulären Parasiten der Bakteriengattung Wolbachia sind weit verbreitet im Phylum der Arthropoden. In vielen Wirten lösen sie eine Paarungsinkompatibilität zwischen nicht infizierten Weibchen und infizierten Männchen aus. Die mögliche Rolle dieser zytoplasmatischen Inkompatibilität in Artbildungsprozessen der Wirtsorganismen wird seit langer Zeit diskutiert. In dieser Arbeit analysieren wir häufig angeführte Kritikpunkte einer solchen Rolle mit Hilfe von mathematischen Modellen, in denen Infektionsdynamik von Wolbachia und Populationsgenetik der Wirte kombiniert werden. Die einzelnen Teile befassen sich mit dem Folgenden: (i) Wir untersuchen die Stabilität von Infektionsmustern in Wirts-Metapopulationen, indem wir kritische Migrationsraten herleiten. (ii) Zur Abschätzung des Einflusses der zytoplasmatischen Inkompatibilität auf den Genfluss zwischen Populationen berechnen wir effektive Migrationsraten. (iii) Wir bestimmen die Bedingungen, die die Verstärkung von Reproduktionsbarrieren durch die Evolution von weiblichen Paarungspräferenzen begünstigen. Schließlich (iv) wenden wir unsere Modelle auf einen realen Artbildungsprozess zweier Drosophila-Arten in Nordamerika an, diskutieren auftretende Probleme und unterbreiten Vorschläge für weiterführende Forschung. Zusammenfassend implizieren unsere Ergebnisse, dass Wolbachien häufig mit der Entstehung neuer Wirtsarten verknüpft sein können, allerdings in den meisten Fällen nur, indem sie als einer von mehreren Faktoren zur reproduktiven Isolation beitragen. Eine Verstärkung sexueller Isolation wird nur unter speziellen Bedingungen bewirkt.<br>The intracellular bacterial parasites of the genus Wolbachia are widespread among arthropod species. In many hosts, they induce a reproductive incompatibility between uninfected females and infected males. The potential role of this cytoplasmic incompatibility in speciation processes of the bacteria''s hosts has long been debated. In this thesis, we analyze common criticisms of such a role by means of mathematical models, combining Wolbachia infection dynamics and host population genetics. In particular, we are concerned with the following: (i) In order to measure the stability of infection patterns within host metapopulations, we derive critical migration rates. (ii) We evaluate the impact of cytoplasmic incompatibility on gene flow between populations by calculating effective migration rates. (iii) We determine the conditions that favor the evolution of female mating preferences through reinforcement. Finally, (iv) we apply our models to a particular real-world speciation process of two sibling Drosophila species in North America, discuss emerging problems, and suggest future directions of research. In summary, our results implicate that Wolbachia might be a frequent factor in host speciation, but usually only by contributing to overall reproductive isolation among other factors. Reinforcement of premating isolation is selected for only under stringent conditions.

Natural hybrid zones provide a great opportunity to study the evolutionary relationships between closely related species. I have combined ten microsatellites (SSR) and 311 single nucleotide polymorphism (SNP) markers with quantitative data to investigate the genetic structure, interspecific gene flow and adaptation of the economically and ecologically important Picea glauca (white spruce) x P. engelmannii (Engelmann spruce) hybrid zone in western North America. Climate modelling and paleoclimate analysis was used to study the historical evolutionary relationships between hybridizing species; and to predict future patterns of genetic variation in the zone. This modelling suggests these species may have been in contact for as long as 21,000 years. Current levels of admixture and introgression are extensive, as suggested by both the SSR and SNP analyses, with populations showing elevational and latitudinal unimodal clines in admixture. Hybrids occupy intermediate environments in the zone and show a higher genetic contribution from Engelmann spruce than from white spruce on average. Despite a long history of interspecific gene flow, pure species and hybrids are adapted to different environments. Results of the quantitative analysis based on long-term data on growth and survival, as well as bud phenology and cold hardiness, indicate that the white x Engelmann spruce hybrid zone is maintained by adaptation to the length of growing seasons and the persistence of the snowpack (exogenous selection), in which hybrids are fitter than pure species in intermediate environments, fitting the "Bounded hybrid superiority" model of hybrid zone maintenance. I identified 12 outlier SNPs among the 311 SNPs; these were genes responsible for carbohydrate metabolism, signal transduction and transcription factors. These results have significant implications for forest management and breeding of spruce species in British Columbia, where this species complex is managed as one species without considering the complexity in population structure and adaptive differences between pure species and hybrids.

I here explore the dual roles of gene flow in determining evolutionary and demographic processes in the Misty Lake threespine stickleback (Gasterosteus aculeatus L.). In the Misty watershed, the lake fish have streamlined bodies and numerous gill rakers whereas the inlet stream fish have deeper bodies and reduced number of gill rakers, differences that are adaptive for lake and stream environments respectively. The outlet stream population, however, is morphologically intermediate between the lake and inlet populations as a result of high gene flow from the lake preventing adaptation to the stream environment. First, I quantify the constraining effect of gene flow on adaptive divergence in the Misty outlet using two complementary approaches. By comparing phenotypic values and environmental differences between the three habitats (i.e. lake, inlet and outlet), I estimate that the constraint imposed by gene flow on phenotypic divergence is in the order of 80%, i.e. the outlet population only achieves 20% of the phenotypic divergence expected in the absence of gene flow. Parameterization of a quantitative genetic model confirms this value is possible given a biologically realistic range of parameter values. Second, I demonstrate that this constraint imposed by gene flow on adaptation likely contributes to an observed reduction in abundances along the outlet stream. I do so using a transplant experiment and a three-year selection experiment. Quantification of the amount of dispersal suggests that the negative influence of gene flow offsets the positive demographic influence of the immigration of individuals. In summary, gene flow has profound consequences for both evolutionary and demographic processes taking place in the Misty system.

Aedes aegypti mosquito is the principle vector for dengue virus transmission. It is the day-biting mosquito which natually breeds in different water containers in and around houses. Dengue/dengue haemorrhagic fever (DF/DHF) is a re-emerging public health problem with > 2.5 billkm people at risk in th world with the greatest disease bilrden in Southeast Asia and the c. An understanding of genetic population structure and dispersal ability in vector populations is important basic Knowledge for effective vector control using both inventional methods and novel strategies such as genetic modification of the vectors.

Thesis: S.M., Massachusetts Institute of Technology, Department of Biology, 2014.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 24-25).<br>In this thesis, I present a technique for profiling gene expression of specific cell types in the central nervous system (CNS), called fluorescence activated nuclei sorting (FANS). FANS utilizes flow cytometry to isolate cell nuclei from CNS subtypes and microarray analysis of nuclear mRNA. When compared to an existing technique, translating ribosome affinity purification, it was found that FANS was able to detect differentially expressed genes between two types of medium spiny neurons in the striatum with similar or higher sensitivity, using transgenic mice with fluorescence proteins labeling the desired cell types. Immunofluorescence experiments were also performed to label cell nuclei isolated from wild type mouse CNS for FANS analysis. It was found that our staining method successfully labeled neuronal nuclei using a NeuN antibody, but did not label enriched markers of medium spiny neurons. Further studies are needed to increase the signal-to-back-ground ratio of these stainings, which would allow FANS to be applied to wild type animals.<br>by Tewei Luo.<br>S.M.

Thesis (Ph.D.)--Biomedical Engineering, Georgia Institute of Technology, 2008.<br>Committee Chair: Jo, Hanjoong; Committee Member: Griendling, Kathy; Committee Member: Harrison, David; Committee Member: Wang, May; Committee Member: Yoganathan, Ajit.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005.<br>The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (July 18, 2006) Vita. Includes bibliographical references.

Phylogeography is a field that has been constantly developing. Many methods exist that can be used to tease apart the demographic processes and events that affect a species evolution. One method in particular, Nested Clade Phylogeographic Analysis (NCPA), has become a popular method for doing this. It claims that the distance statistics it uses can discriminate between multiple demographic scenarios. However, a major limitation of NCPA is that it does not provide estimates of error. Lack of automation of two stages in NCPA has meant few datasets can be simulated. A fully automated package, ANeCA, is developed, tested and applied to published datasets to recover published inferences. Many inferences are recovered showing that ANeCA is representative of a typical application of NCPA. ANeCA is also applied to data simulated under a model of panmixia. Results show on average 76% of datasets gave rise to at least one inference of an event or process when there was no geographic association. This is due to the generation of multiple statistics per clade, and multiple clades per dataset. It is also shown that the statistics are not independent of each other, making it difficult to correct the multiple tests performed. ANeCA is also applied to simulated data under three models of gene flow; an island model; a stepping stone model; and a lat- , tice model with some long distance dispersal. The results show that NCPA has a tendency to infer Restricted Gene Flow with Isolation By Distance, and Contiguous Range Expansion, under panmixia and the three gene flow models. Unlike the panmictic model where other inferences were rare, many inferences are found in higher frequencies irrespective of the underlying gene flow model. Comparison between the island and stepping stone models show that NCPA is unable to discriminate between long and short distance movements. Three classical summary statistics; AMOVAj Fu's FSj and the Mantel test, were also calculated on each dataset. Results show false positives rates to be around 5% for the AMOVA method and Mantel test, and 3% for Fu's Fs at the deme level. In general the AMOVA method is more sensitive to population structure than NCPA, and the Mantel test is more sensitive to isolation by distance than NCPA. The results that have been obtained in this study suggest that previous publications that have used NCPA have most likely made false inferences and it is recommended that classical, and most probably model-based, methods of inference should be used in preference to NCPA.