Thesis (MSc (Botany and Zoology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The comparative phylogeography of the host-parasite relationship of the southern African endemic dark shyshark, Haploblepharus pictus and its nematode parasite, Proleptus obtusus was investigated. To date, no studies have been conducted on the population structure of catsharks and their species specific parasites and little is known about the population dynamics of these species. A total of 116 catsharks and 201 parasites were analysed from seven South African localities. The mitochondrial marker COI was used and species specific primers were designed for both the host and parasite. Haplotype networks were constructed and no strong geographically structured groupings were found for either species. Pairwise st values for the parasite and host found Gansbaai to be significantly differentiated from the other sites. Fu’s Fs were significantly negative for both host and parasite indicating population disequilibrium. Proleptus obtusus displayed a pattern of population expansion which was confirmed by the mismatch distribution. Mismatch distributions failed to indicate population expansion for the sharks. Other factors such as selection, migration or genetic drift are likely the cause of the population disequilibrium detected. Interestingly, no barrier to gene flow was found around Cape Point, a known break for other species such as the clinid, Clinus cottoides and the caridean shrimp Palaemon peringueyi. The outcome of this study suggests that levels of gene flow in H. pictus are high enough to suggest that the documented site fidelity is not as strong as originally proposed. The parasite, being dependent on the host, shows a similarly high level of gene flow among sampling sites. / AFRIKAANSE OPSOMMING: Die vergelykende filogeografie van die gasheer-parasiet verhouding tussen die endemiese suider-Afrikaanse donker skaamhaai, Haploblepharus pictus en sy nematode parasiet, Proleptus obtusus is ondersoek. Huidiglik is daar nog geen ander studies uitgevoer met betrekking tot die populasie struktuur van skaamhaaie en hul spesies-spesefieke parasiete nie en min is bekend oor die populasie dinamiek van hierdie spesies. In hierdies studie is ‘n totaal van 116 skaamhaaie en 201 parasiete vanaf sewe lokaliteite geanaliseer. Die mitikondriale merker COI is hiervoor gebruik en spesie spesefieke inleiers is vir beide gasheer en parasiet ontwerp. Haplotipe netwerke is saamgestel vir beide spesies en het geen duidelike geografies gestruktureerde groepe aangedui nie. Paarsgewyse st waardes van beide parasiet en gasheer het daarop gedui dat Gansbaai geneties gedifferensieerd is van alle ander lokaliteite. Fu se Fs was statisties betekenisvol met ‘n negatiewe waarde vir beide spesies, wat dui op populasie disekwilibrium. Proleptus obtusus het ‘n patroon van populasie groei getoon, wat deur Fu se Fs en die misparing verspreiding bevestig is. Die misparing verspreiding het nie populasie toename vir die skaamhaaie aangedui nie. Die waargeneemde populasie disekwilibrium is waarskynlik die gevolg van seleksie, migrasie of genetiese drywing. Geen genetiese breuk is by Kaap Punt, wat ‘n genetiese breuk vir verskeie ander spesies soos Clinus cottoides en Palaemon peringueyi is, gevind nie. Die uitkomstes van hierdie studie stel voor dat vlakke van geen vloei in H. picuts hoog genoeg is om ‘n patroon van genetiese vermenging tussen lokaliteite, op die mitokondriale DNS vlak, tot gevolg te hê. Dit beteken moontlik dat die gedokumenteerde gebied gebondenheid van hierdie spesie nie so sterk, soos oorspronklik voorgestel, is nie. Die parasiet, waarskynlik aangesien hy van sy gasheer afhanklik is, toon ‘n soortgelyke hoë vlak van geen vloei tussen lokaliteite. Dus toon beide spesies ‘n algehele afwesigheid van genetiese struktuur, met die isolasie van Gansbaai van alle ander lokaliteite.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Within southern Africa, the widely distributed four-striped mouse Rhabdomys is parasitized by, amongst others, the host-specific ectoparasitic sucking louse, Polyplax arvicanthis. The present study investigated this parasite-host association from a phylogenetic and phylogeographic perspective utilizing mitochondrial and nuclear DNA markers. The findings support the existence of four species within Rhabdomys (three distinct lineages within the previously recognized arid-adapted R. pumilio and the mesic-adapted R. dilectus). These species have distinct geographic distributions across vegetational biomes with two documented areas of sympatry at biome boundaries. Ecological niche modelling supports a strong correlation between regional biomes and the distribution of distinct evolutionary lineages of Rhabdomys. A Bayesian relaxed molecular clock suggests that cladogenesis within the genus coincides with paleoclimatic changes (and the establishment of the biomes) at the Miocene-Pliocene boundary. Strong evidence was also found that the sucking louse P. arvicanthis consists of two genetically divergent lineages, which probably represent distinct species. The two lineages have sympatric distributions throughout most of the sampled range across the various host species and also occasionally occur sympatrically on the same host individual. Further, the absence of clear morphological differences among these parasitic lineages suggests cryptic speciation. Limited phylogeographic congruence was observed among the two P. arvicanthis lineages and the various Rhabdomys species and co-phylogenetic analyses indicated limited co-divergence with several episodes of host-switching, despite the documented host-specificity and several other traits predicted to favour congruence and co-divergence. Also, despite the comparatively smaller effective population sizes and elevated mutational rates found for P. arvicanthis, spatial genetic structure was not more pronounced in the parasite lineages compared to the hosts. These findings may be partly attributed to high vagility and social behaviour of Rhabdomys, which probably promoted parasite dispersal among hosts through frequent inter-host contact. Further, the complex biogeographic history of Rhabdomys, which involved cyclic range contractions and expansions, may have facilitated parasite divergence during periods of host allopatry, and host-switching during periods of host sympatry. Intermittent contact among Rhabdomys lineages could also have prevented adaptation of P. arvicanthis to specific host lineages, thus explaining the lack of host-specificity observed in areas of host sympatry. It is thus evident that the association between Polyplax arvicanthis and Rhabdomys has been shaped by the synergistic effects of parasite traits, biogeography, and host-related factors over evolutionary time. / AFRIKAANSE OPSOMMING: Binne suidelike-Afrika word die wyd-verspreide gestreepte veldmuis, Rhabdomys, onder andere deur die gasheer-spesifieke ektoparasitiese luis, Polyplax arvicanthis, geparasitiseer. Die huidige studie het hierdie parasiet-gasheer interaksie vanuit ‘n filogenetiese en filogeografiese oogpunt ondersoek deur van beide mitokondriale en nukluêre merkers gebruik te maak. Die bevindinge dui op die bestaan van vier spesies binne Rhabdomys, waaronder drie nuwe genetiese groepe binne die voorheen erkende R. pumilio asook R. dilectus. Hierdie spesies het nie-oorvleulende geografiese verspreidings binne spesefieke plantegroei biome met twee geidentifiseerde areas van simpatriese voorkoms by bioom grense. Ekologiese nis modellering ondersteun ‘n sterk korrelasie tussen biome en die verspreiding van die evolusionêre groepe binne Rhabdomys. ‘n Bayesiaanse verslapte molekulêre klok dui daarop dat kladoginese binne die genus gedurende paleoklimatiese veranderinge, wat tot die totstandkoming van die huidige biome gelei het, by die Mioseen-Plioseen grens plaasgevind het. Sterk bewyse is ook gevind dat die parasitiese luis P. arvicanthis uit twee geneties verskillende groepe, wat heel moontlik afsonderlike spesies verteenwoordig, bestaan. Hierdie genetiese groepe het simpatriese verspreidings oor meeste van die gebestudeerde geografiese area op die verskeie gasheer spesies en mag ook soms simpatries op dieselfde gasheer individu voorkom. Verder dui die afwesigheid van duidelike morfologiese verskille tusssen die parasiet genetiese groepe op moontlike kriptiese spesiasie. Beperkte filogeografiese ooreenstemming is tussen die P. arvicanthis genetiese groepe en die Rhabdomys spesies waargeneem en die vergelykende-filogenetiese analises het aangedui dat daar beperkte gesementlike-divergensie plaasgevind het met verskeie episodes van gasheer-wisseling, ten spyte van die gasheer-spesifieke aard van die parasiete asook verskeie ander kenmerke wat veronderstel is om filogeografiese ooreenstemming en gesementlike-divergensie te bevorder. Ten spyte van die vergelykbaar kleiner effektiewe bevolking groottes en verhoogde mutasie tempo wat vir P. arvicanthis gevind is, is die geografiese genetiese struktuur nie meer gedifferensieёrd in die parasiet groepe as in die gasheer nie. Hierdie bevindinge mag deels verklaar word deur die hoё beweeglikheid asook die sosiale gedrag van Rhabdomys, wat waarskynlik parasiet beweging tussen gashere bevorder deur gereelde tussen-gasheer kontak. Die komplekse biogeografiese geskiedenis van Rhabdomys, wat sikliese inkrimping en uitsetting van die geografiese verspreiding behels het, het heel moontlik parasiet divergensie tydens tydperke van gasheer allopatrie asook gasheer-wisseling tydens tydperke van gasheer simpatrie, gefasiliteer. Tussentydse kontak tussen Rhabdomys genetiese groepe kon aanpassing van P. arvicanthis tot sekere gasheer genetiese groepe verhoed het en verklaar dus die afwesigheid van waargenome gasheer-spesifisiteit in areas van gasheer simpatrie. Dit is dus duidelik dat die assosiasie tussen P. arvicanthis en Rhabdomys deur die sinergistiese uitwerking van parasiet kenmerke, biogeografie, asook gasheer-verwante faktore oor evolusionêre tyd gevorm is. / National Research Foundation (NRF)

A Filogeografia é um dos campos mais multidisciplinares da Biologia, e agrega diferentes áreas como sistemática filogenética, genética de populações, geologia, modelos paleogeográficos e paleoclimáticos, demografia e conservação. Assim, além da Filogeografia comparada, apresento resultados importantes para a conservação das espécies estudadas e a descrição de uma espécie críptica. Bombus morio e B. pauloensis são espécies simpátricas que ocupam uma grande área em dois importantes biomas brasileiros, Mata Atlântica e Cerrado. Exceto pelas diferenças na dispersão, maior em B. morio, possuem comportamento e nicho ecológico semelhantes. Os resultados gerados a partir de marcadores moleculares e modelagem de distribuição sugerem que as alterações climáticas do final do Pleistoceno influenciaram a estrutura populacional das duas espécies, e que a maior capacidade de dispersão foi responsável pela ausência de estruturação em B. morio. O leste do estado de São Paulo, no qual foram encontradas diferentes quebras filogeográficas para vários organismos, mostrou-se mais uma vez complexo e com mais um diferente cenário filogeográfico. Além disso, essa região por ser o centro da diversidade genética em B. pauloensis e ter sido estável ao longo da mudanças climáticas para ambas espécies, é prioritária para a conservação das mesmas / Phylogeography is one of the most multidisciplinary fields in Biology and joins different areas such as phylogenetic systematics, population genetics, geology, paleogeographic and paleoclimatic models, demography, and conservation. Thus, besides comparative Phylogeography, I also present important results concerning the conservation of the species studied and the description of a cryptic species. Bombus morio and B. pauloensis are sympatric species, occupying a large area in two important Brazilian biomes, Atlantic forest and Brazilian savanna. Except for differences in dispersal, which is greater in B. morio, both species have similar behavior and ecological niches. The results obtained by molecular data and distribution models suggest that climatic oscillations in the late Pleistocene influenced the population structure of both species, and that a greater dispersal capacity was responsible for the absence of genetic structure in B. morio. Eastern São Paulo state, in which different phylogeographic breaks have been found for many organisms, seems to be complex and to have a new phylogeographic scenario. Furthermore, this region, because it is the center of genetic diversity for B. pauloensis and it was stable throughout periods of climatic change for both species, is a priority for their conservation

A Filogeografia é um dos campos mais multidisciplinares da Biologia, e agrega diferentes áreas como sistemática filogenética, genética de populações, geologia, modelos paleogeográficos e paleoclimáticos, demografia e conservação. Assim, além da Filogeografia comparada, apresento resultados importantes para a conservação das espécies estudadas e a descrição de uma espécie críptica. Bombus morio e B. pauloensis são espécies simpátricas que ocupam uma grande área em dois importantes biomas brasileiros, Mata Atlântica e Cerrado. Exceto pelas diferenças na dispersão, maior em B. morio, possuem comportamento e nicho ecológico semelhantes. Os resultados gerados a partir de marcadores moleculares e modelagem de distribuição sugerem que as alterações climáticas do final do Pleistoceno influenciaram a estrutura populacional das duas espécies, e que a maior capacidade de dispersão foi responsável pela ausência de estruturação em B. morio. O leste do estado de São Paulo, no qual foram encontradas diferentes quebras filogeográficas para vários organismos, mostrou-se mais uma vez complexo e com mais um diferente cenário filogeográfico. Além disso, essa região por ser o centro da diversidade genética em B. pauloensis e ter sido estável ao longo da mudanças climáticas para ambas espécies, é prioritária para a conservação das mesmas / Phylogeography is one of the most multidisciplinary fields in Biology and joins different areas such as phylogenetic systematics, population genetics, geology, paleogeographic and paleoclimatic models, demography, and conservation. Thus, besides comparative Phylogeography, I also present important results concerning the conservation of the species studied and the description of a cryptic species. Bombus morio and B. pauloensis are sympatric species, occupying a large area in two important Brazilian biomes, Atlantic forest and Brazilian savanna. Except for differences in dispersal, which is greater in B. morio, both species have similar behavior and ecological niches. The results obtained by molecular data and distribution models suggest that climatic oscillations in the late Pleistocene influenced the population structure of both species, and that a greater dispersal capacity was responsible for the absence of genetic structure in B. morio. Eastern São Paulo state, in which different phylogeographic breaks have been found for many organisms, seems to be complex and to have a new phylogeographic scenario. Furthermore, this region, because it is the center of genetic diversity for B. pauloensis and it was stable throughout periods of climatic change for both species, is a priority for their conservation

There are a number of common distributional patterns that have provided the foundations of our current knowledge of Neotropical biogeography. A distinctive pattern is the so-called \"circum-Amazonian distribution\", which expands across the forested lowlands south and east of Amazonia, the Andean foothills, the Venezuelan Coastal Range, and the Tepuis. To date, there is no clear understanding of the processes giving rise to this distribution. To understand the evolutionary history of taxa exhibiting this pattern it is necessary to test biogeographic hypotheses offering mechanistic explanations. Comparative phylogeography allows more accurate phylogeographic hypotheses for these taxa, as well as better population genetic parameters. Comprehensive comparative studies aiming at unraveling the evolutionary and biogeographic mechanisms underlying the circum-Amazonian distribution have not been conducted yet, and only scarce descriptive information has been published. Therefore, the objective of this work was to elucidate the historical and biogeographic mechanisms underpinning circum-Amazonian distribution by performing comparative genomic analyses of a group of Suboscine passerines. Ultraconserved Elements (UCEs) were obtained for eight taxonomic groups to estimate population parameters and genealogical trees. For the Thamnophilidae species were inferred demographic histories with momi2. The best models of each taxon were analyzed in a comparative framework to relate them with previously proposed biogeographic hypotheses for the Neotropics and to propose plausible biogeographical scenarios for the circum-Amazonian pattern. The circum-Amazonian distributional pattern has two main phylogeographic units: an Andean (plus Central America region) and an eastern-forested region (Atlantic Forest ecoregion, forested areas around southeast of Amazonia), interconnected by a northern and southern corridor, allowing biotic interchanges between them (mainly from the southern) and hybridization. Species-tree analyses recovered (a) an Andean clade with two Andean subgroups in the northern Peru and central Andes, and (b) an eastern-forested clade including northern and central/southern Atlantic Forest subgroups. The demographic histories of the Thamnophilidae taxa suggest that diversification of the circum-Amazonian taxa have a strong influence of climatic fluctuations during the Pleistocene, with interconnected refugia allowing phenotypic/genetic differentiation but maintaining a considerable level of gene flow during varying dry/cool and warm/humid periods. In addition, the results of this work opened interesting taxonomic questions about some taxa that could be covered in the future (T. ruficapillus/torquatus complex, Xiphocolaptes complex). / Existe um número de padrões de distribuição comuns que forneceram os fundamentos do nosso atual conhecimento da Biogeografia Neotropical. Um padrão distintivo é o chamado padrão de distribuição circum-Amazônico, apresentado por grupos filogeneticamente relacionados habitando as florestas de baixada ao sul-leste da Amazônia, as encostas úmidas dos Andes, a área costeira da Venezuela e os Tepuis. Atualmente não existe um entendimento claro dos processos que deram surgimento a este padrão de distribuição. Para compreender a história evolutiva dos táxons exibindo este tipo de padrão é necessário testar hipóteses biogeográficas que ofereçam explicações mecanicistas. A Genômica comparativa permite hipóteses filogeográficas mais exatas para estes táxons, assim como melhores parâmetros demográficos. Estudos comparativos abrangentes visando em esclarecer os mecanismos evolutivos e biogeográficos relacionados a distribuição circum-Amazônica não tem sido elaborados ainda, e só informação descritiva escassa tem sido publicada. Portanto, os objetivo fundamental do projeto foi elucidar os mecanismos históricos e biogeográficos subjacentes à distribuição circum-Amazônica desenvolvendo analises genômicos comparativos de um grupo de Passeriformes Suboscines. Dados do gene ND2 e de Elementos Ultraconservados (UCEs) foram obtidos de oito grupos taxonômicos para estimar parâmetros populacionais e arvores genealógicas. Histórias demográficas foram inferidas só para as espécies da família Thamnophilidae usando momi2. Os melhores modelos de cada táxon foram analisados num marco comparativo para relaciona-os ás hipóteses biogeográficas propostas para o Neotrópico e propor cenários possíveis para a distribuição circum-Amazônica. O padrão de distribuição circum-Amazônico possui duas unidades filogeográficas principais: uma unidade Andina (incluindo a região de Centro América) e uma segunda unidade incluindo as regiões florestais do leste (Mata Atlântica, áreas florestais ao sudeste da Amazônia). Estas unidades estão interconectadas por corredores ao norte e sul da distribuição, permitindo intercâmbios de biota entre elas (principalmente pelo corredor sul). SNAPP identificou o clado Andino subdividido em norte do Peru e central Andes, e um segundo clado das Florestas do Leste incluindo dois subgrupos, um do norte e outro do centro-sul da Mata Atlântica. As histórias demográficas dos Thamnophilidae sugerem que a diversificação na distribuição circum-Amazônica foi altamente influenciada pelas flutuações climáticas durante o Pleistoceno, com refúgios interconectados gerando diferenciação fenotípica/genética mas mantendo certo grau de fluxo gênico nos períodos secos/frios e quentes/húmidos. Adicionalmente, algumas questões taxonômicas sobre alguns táxons estudados que poderiam ser estudadas no em futuros estudos (o complexo T. ruficapillus/torquatus e o gênero Xiphocolaptes).

<p>Because species respond individually to climate change, understanding community assembly requires examination of multiple species from a diversity of forest niches. I present the post-glacial phylogeographic history of an understory, parasitic herb (<italic>Epifagus virginiana</italic>, beechdrop) that has an obligate and host specific relationship with a common eastern North American hardwood tree (<italic>Fagus grandifolia</italic>, American beech). The migration histories of the host and parasite are compared to elucidate potential limits on the parasite's range and to understand their responses to shared climate change. Two chloroplast DNA regions were sequenced and 9 microsatellite loci genotyped from parasite specimens collected throughout the host's range. These data were compared with available cpDNA sequences from the host (McLachlan et al. 2005) and host fossil pollen records from the last 21,000 years (Williams et al. 2004). Analyses of genetic diversity reveal high population differentiation in the parasite's southern range, a possible result of long term isolation within multiple southern glacial refuges. Estimates of migration rates and divergence times using Bayesian coalescent methods show the parasite initiating its post-glacial range expansion by migrating northward into the northeast from southern areas, then westward into the midwest, a pattern consistent with the development of high density beech forests. This result is strongly confirmed through spatial linear regression models, which show host density plays a significant role in structuring parasite populations, while the initial migration routes of the host are irrelevant to parasite colonization patterns. Host density is then used as a proxy for the parasite's habitat quality in an effort to identify the geographic locations of its migration corridors. Habitat cost models are parameterized through use of the parasite's genetic data, and landscape path analyses based on the habitat map show a major migration corridor south of the Great Lakes connecting the northeast and midwest. Host density was the major determinant controlling the parasite's range expansion, suggesting a lag time between host and parasite colonization of new territory. Parasites and other highly specialized species may generally migrate slower due to their complex landscape requirements, resulting in disassociation of forest assemblages during these times. From these results, the low migration capacities of highly specialized species may be insufficient to outrun extirpation from their current ranges.</p> / Dissertation

Phylogeography and molecular phylogenetics have proven remarkably useful for understanding the patterns and processes influencing historical diversification of biotic lineages at and below the species level, as well as delimiting morphologically cryptic species. In this dissertation, I used an integrative approach coupling comparative phylogeography and coalescent-based species delimitation to improve our understanding of the biogeography and species limits of Central American freshwater fishes. In Chapter 1, I conducted a literature review of the contributions of phylogeography to understanding the origins and maintenance of lower Central American biodiversity, in light of the geological and ecological setting. I highlighted emerging phylogeographic patterns, along with the need for improving regional historical biogeographical inference and conservation efforts through statistical and comparative phylogeographic studies. In Chapter 2, I compared mitochondrial phylogeographic patterns among three species of livebearing fishes (Poeciliidae) codistributed in the lower Nicaraguan depression and proximate uplands. I found evidence for mixed spatial and temporal divergences, indicating phylogeographic “pseudocongruence” suggesting that multiple evolutionary responses to historical processes have shaped population structuring of regional freshwater biota, possibly linked to recent community assembly and/or the effects of ecological differences among species on their responses to late Cenozoic environmental events. In Chapter 3, I used coalescent-based species tree and species delimitation analyses of a multilocus dataset to delimit species and infer their evolutionary relationships in the Poecilia sphenops species complex (Poeciliidae), a widespread but morphologically conserved group of fishes. Results indicated that diversity is underestimated and overestimated in different clades by c. ±15% (including candidate species); that lineages diversified since the Miocene; and that some evidence exists for a more probable role of hybridization, rather than incomplete lineage sorting, in shaping observed gene tree discordances. Last, in Chapter 4, I used a comparative phylogeographical analysis of eight codistributed species/genera of freshwater fishes to test for shared evolutionary responses predicted by four drainage-based hypotheses of Neotropical fish diversification. Integrating phylogeographic analyses with paleodistribution modeling revealed incongruent genetic structuring among lineages despite overlapping ancestral Pleistocene distributions, suggesting multiple routes to community assembly. Hypotheses tests using the latest approximate Bayesian computation model averaging methods also supported one pulse of diversification in two lineages diverged in the San Carlos River, but multiple divergences of three lineages across the Sixaola River basin, Costa Rica, correlated to Neogene sea level events and continental shelf width. Results supported complex biogeographical patterns illustrating how species responses to historical drainage-controlling processes have influenced Neotropical fish diversification.

The Guiana Shield is a sub-region of Amazonia, one of the richest areas on earth in terms of species number. It is also one of the most pristine areas and is still largely unexplored. Species number, distribution, boundaries and their evolutionary histories remain at least unclear but most of the time largely unknown. This is the case for most Anurans, a group which is recognized as threatened globally and is disappearing even from pristine tropical forests. Given the pace of forest destruction and the growing concerns about climate change it is urgently necessary to obtain a better estimate of regional biodiversity in Amazonian frogs as well as a better understanding of the origin and distribution of Anuran diversity. Furthermore, given their sensitivity to climatic conditions, amphibians are a good model to investigate the influence of paleoclimatic events on Neotropical diversification which was supposedly the driving force on biotic evolution during Pleistocene in the Guiana Shield. I first test species boundaries in two species Scinax ruber and Rhinella margaritifera. These species are widely distributed, abundant and largely recognized as species complexes. I used an original species delineation method based on the combined use of mitochondrial and nuclear DNA in phylogenetic and phylogeographic analyses. Phylogenetic analyses demonstrated the polyphyly of Scinax ruber and Rhinella margaritifera. These species consist of multiple lineages that may all merit species status. Conflicting signals of mitochondrial and nuclear markers indicated the possibility of ongoing hybridization processes. Phylogeographic analyses added further information in support of the specific status of these lineages. Our results highlight the utility of combining phylogenetic and phylogeographic methods, as well as the use of both mitochondrial and nuclear markers within one study. This approach helped to better understand the evolutionary history of taxonomically complex groups of species. The assessment of the geographic distribution of genetic diversity in tropical amphibian communities can lead to conclusions that differ strongly from prior analyses based on the occurrence of currently recognized species alone. Such studies, therefore, hold the potential to contribute to a more objective assessment of amphibian conservation priorities in tropical areas. Subsequently, I tested if these first results on cryptic species are generalisable, questioning what would potentially be a minimum estimate of the number of cryptic frog species in Amazonia and the Guiana Shield, using mtDNA with multiple complementary approaches. I also combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs. In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contra to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher genetic distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species. Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas. As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised. The Rhinella margaritifera complex is characterisized by the presence of many cryptic species throughout its wide distribution, ranging from Panama to Bolivia and almost entire Amazonia. French Guiana has long been thought to harbor two species of this group, though molecular data analysed in previous chapters indicated as many as five lineages. I tested whether morphological measurements are correlated or not with genetic data using discriminant analysis and if diagnostic characteristics among the previously determined lineages can be used to describe these new species. This is a novel integrative method which can lead to a facilitation of the description of cryptic species that have been detected by phylogenetic and/or phylogeographic studies. These analyses, combined with published data of other Rhinella species, indicated that two of these lineages represent previously unnamed species. Two of the remaining are allocable to R. margaritifera while the status of the fifth is still unclear because so far it is morphologically indistinguishable from R. castaneotica. Determining if codistributed species responded to climate change in an independent or concerted manner is a basic objective of comparative phylogeography. Species boundaries, histories, ecologies and their geographical ranges are still to be explored in the Guiana Shield. According to the refugia hypothesis this region was supposed to host a forest refugium during climatic oscillations of the Pleistocene but the causes and timing for this have been criticized. We investigated patterns of genetic structure within 18 frog species in the eastern Guiana Shield to explore species boundaries and their evolutionary history. We used mtDNA and nuclear DNA and complementary methods to compare the genetic diversity spatially and temporally. With one exception all the species studied diversified repeatedly within the eastern Guiana Shield during the last 4 million years. Instead of one Pleistocene forest refugium the Guiana Shield has probably hosted multiple refugia during late Pliocene and Pleistocene. Most of these Pleistocene refugia were probably situated on the coast of French Guiana, Amapà, Suriname and Guyana. This diversification likely resulted from forest fragmentation. Many species deserve taxonomic revisions and their ranges to be reconsidered. The local endemism of the Anuran fauna of the Guiana Shield is likely to be much higher and some areas consequently deserve more conservation efforts. Specifically I questioned whether major intraspecific diversification started before the Pleistocene and occurred within the Guiana Shield or ex situ. According to ecological characteristics of the species involved I will test different diversification hypotheses. The consequences on the diversity and the endemism of the Guiana Shield will be explored. My results demonstrate that we have been grossly underestimating local biological diversity in the Guiana Shield but also in Amazonia in general. The order of magnitude for potential species richness means that the eastern Guiana Shield hosts one of the richest frog fauna on earth. In most of the species studied high levels of mtDNA differentiation between populations call for a reassessment of the taxonomic status of what is being recognised as single species. Most species display deep divergence between eastern Guiana Shield populations and Amazonian ones. This emphasizes that the local endemism in the Guiana Shield of these zones is higher than previously recognized and must be prioritised elements taken into account in conservation planning. Nevertheless, a few other species appear widely distributed showing that widespread species do exist. This underlines the fact that some species have efficient dispersal abilities and that the frog fauna of the eastern Guiana Shield is a mixture of old Guianan endemic lineages that diversified in situ mostly during late Pliocene and Pleistocene and more recently exchanged lineages with the rest of Amazonia. Recognizing this strong historical component is necessary and timely for local conservation as these zones are likely to be irremediably modified in the near future.

The Guiana Shield is a sub-region of Amazonia, one of the richest areas on earth in terms of species number. It is also one of the most pristine areas and is still largely unexplored. Species number, distribution, boundaries and their evolutionary histories remain at least unclear but most of the time largely unknown. This is the case for most Anurans, a group which is recognized as threatened globally and is disappearing even from pristine tropical forests. Given the pace of forest destruction and the growing concerns about climate change it is urgently necessary to obtain a better estimate of regional biodiversity in Amazonian frogs as well as a better understanding of the origin and distribution of Anuran diversity. Furthermore, given their sensitivity to climatic conditions, amphibians are a good model to investigate the influence of paleoclimatic events on Neotropical diversification which was supposedly the driving force on biotic evolution during Pleistocene in the Guiana Shield. I first test species boundaries in two species Scinax ruber and Rhinella margaritifera. These species are widely distributed, abundant and largely recognized as species complexes. I used an original species delineation method based on the combined use of mitochondrial and nuclear DNA in phylogenetic and phylogeographic analyses. Phylogenetic analyses demonstrated the polyphyly of Scinax ruber and Rhinella margaritifera. These species consist of multiple lineages that may all merit species status. Conflicting signals of mitochondrial and nuclear markers indicated the possibility of ongoing hybridization processes. Phylogeographic analyses added further information in support of the specific status of these lineages. Our results highlight the utility of combining phylogenetic and phylogeographic methods, as well as the use of both mitochondrial and nuclear markers within one study. This approach helped to better understand the evolutionary history of taxonomically complex groups of species. The assessment of the geographic distribution of genetic diversity in tropical amphibian communities can lead to conclusions that differ strongly from prior analyses based on the occurrence of currently recognized species alone. Such studies, therefore, hold the potential to contribute to a more objective assessment of amphibian conservation priorities in tropical areas. Subsequently, I tested if these first results on cryptic species are generalisable, questioning what would potentially be a minimum estimate of the number of cryptic frog species in Amazonia and the Guiana Shield, using mtDNA with multiple complementary approaches. I also combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs. In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contra to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher genetic distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species. Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas. As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised. The Rhinella margaritifera complex is characterisized by the presence of many cryptic species throughout its wide distribution, ranging from Panama to Bolivia and almost entire Amazonia. French Guiana has long been thought to harbor two species of this group, though molecular data analysed in previous chapters indicated as many as five lineages. I tested whether morphological measurements are correlated or not with genetic data using discriminant analysis and if diagnostic characteristics among the previously determined lineages can be used to describe these new species. This is a novel integrative method which can lead to a facilitation of the description of cryptic species that have been detected by phylogenetic and/or phylogeographic studies. These analyses, combined with published data of other Rhinella species, indicated that two of these lineages represent previously unnamed species. Two of the remaining are allocable to R. margaritifera while the status of the fifth is still unclear because so far it is morphologically indistinguishable from R. castaneotica. Determining if codistributed species responded to climate change in an independent or concerted manner is a basic objective of comparative phylogeography. Species boundaries, histories, ecologies and their geographical ranges are still to be explored in the Guiana Shield. According to the refugia hypothesis this region was supposed to host a forest refugium during climatic oscillations of the Pleistocene but the causes and timing for this have been criticized. We investigated patterns of genetic structure within 18 frog species in the eastern Guiana Shield to explore species boundaries and their evolutionary history. We used mtDNA and nuclear DNA and complementary methods to compare the genetic diversity spatially and temporally. With one exception all the species studied diversified repeatedly within the eastern Guiana Shield during the last 4 million years. Instead of one Pleistocene forest refugium the Guiana Shield has probably hosted multiple refugia during late Pliocene and Pleistocene. Most of these Pleistocene refugia were probably situated on the coast of French Guiana, Amapà, Suriname and Guyana. This diversification likely resulted from forest fragmentation. Many species deserve taxonomic revisions and their ranges to be reconsidered. The local endemism of the Anuran fauna of the Guiana Shield is likely to be much higher and some areas consequently deserve more conservation efforts. Specifically I questioned whether major intraspecific diversification started before the Pleistocene and occurred within the Guiana Shield or ex situ. According to ecological characteristics of the species involved I will test different diversification hypotheses. The consequences on the diversity and the endemism of the Guiana Shield will be explored. My results demonstrate that we have been grossly underestimating local biological diversity in the Guiana Shield but also in Amazonia in general. The order of magnitude for potential species richness means that the eastern Guiana Shield hosts one of the richest frog fauna on earth. In most of the species studied high levels of mtDNA differentiation between populations call for a reassessment of the taxonomic status of what is being recognised as single species. Most species display deep divergence between eastern Guiana Shield populations and Amazonian ones. This emphasizes that the local endemism in the Guiana Shield of these zones is higher than previously recognized and must be prioritised elements taken into account in conservation planning. Nevertheless, a few other species appear widely distributed showing that widespread species do exist. This underlines the fact that some species have efficient dispersal abilities and that the frog fauna of the eastern Guiana Shield is a mixture of old Guianan endemic lineages that diversified in situ mostly during late Pliocene and Pleistocene and more recently exchanged lineages with the rest of Amazonia. Recognizing this strong historical component is necessary and timely for local conservation as these zones are likely to be irremediably modified in the near future.

The Quaternary climate has known dramatic global variations oscillating between long glacial and shorter interglacial periods of approximately 100 000 and 20 000 years, respectively with the last succession as an example. During the glacial episodes the continental ice sheet expansion and sea level drop, in turn, have locally disturbed the environment (at least in the northern hemisphere). These climatic and environmental disturbances caused changes in the geographic distributions of animal and plant species. For each species, the changes possibly took the form of demographic events like population extinction or fragmentation associated with genetic bottlenecks (loss in genetic diversity) and, inversely, population expansions sometimes with inherent founder effects (stochastic sampling of the source genetic diversity) and/or contact between diverged groups (secondary contact zone) resulting in genetic diversity gradients through the geographic range of the species. We therefore understand that the demographic history of a species can be reconstruct through the investigation of genetic signature(s) it possibly left and that are still observed in the genome of that species. For European taxa, phylogeographic studies taking advantage of these signatures have mainly focused their attention on temperate species; pieces of knowledge for species adapted to cold environments are too scarce although their response to climate change could not only simply follow an inverse tendency compare to temperate climate species.<p><p>As a whole, this thesis project intended to study the evolutionary history of four sister species of cold-adapted leaf beetles investigating their response to past global climatic changes. The four species share many traits (life cycle, dispersal capacity, morphology, feeding behavior.) but their geographic distributions differ, further calling for interest in the factors that shaped them. Furthermore, leaf beetles are specialist insect herbivores each feeding only on one or a few different plant species. This host plant specialization offers an additional dimension to the study of climatic change impacts to understand the evolution of the insect-host relationship. The study of this species complex thus also aimed at understanding processes like speciation possibly driven through diet specialization. The project connects three main axes briefly described here after.<p><p>The first axe of the project allowed us to gain sufficient knowledge of the four species sub-genus Goniomena (Chrysomelidae, Gonioctena). We have defined some important barriers separating each four species among them. The analysis of five independent molecular markers sequences obtained for many individuals sampled through the entire species ranges allowed us to characterize four genetically distinct groups corresponding to the four species, to precisely identify their host plant(s) and their geographic distribution. The second axe was realized on the same multi-locus dataset and aimed at exploiting an array of methods to reconstruct the demographic history of the four species; the methods consist of some commonly used and some promising ones used in synergy hoping to strengthen our interpretations on the species history. For this purpose, we combined the species distribution modeling (SDM) techniques to infer current and past geographic range for the leaf beetles and for their host(s) in order to generate the most realistic historical hypotheses. Subsequently, the different hypotheses were evaluated with two different complementary approaches, Approximate Bayesian Computation (ABC) and a spatial coalescence simulation-based method, allowing for outputs comparison. Finally, the third axe focus on the study of a commonly used program designed for demographic parameters inference (including divergence times, effective populations sizes, migration rates). We created non-empiric datasets obtained with three largely used simulation programs to investigate the inference performances of the program.<p><p>We hope this project will help to better understand the way species currently present in cold environments in Europe responded to climatic changes. It certainly demonstrated and allowed to isolate some specific character of such respons while suggesting certain common patterns. Our findings are rich and varied; the current distribution of genetic diversity in the four sister species of leaf beetles involves processes like introgression and hybridization, competition and invasion, allopatric and possibly sympatric speciation, dispersal limitation and response differentiation against climatic changes. In the light of our results, further investigations are encouraged; the mechanisms driving or underlying the different speciation settings, the host plant specialization, the niche differentiation and the hybridization in secondary contact zones are planned to be investigated with biological material and analytic resources we already own. Practically, we explored promising analyses procedures using the resources of our multilocus multispecies dataset. All along, we emphasize on the need to work with multispecies empiric datasets at least equivalent to the one in this project (number of molecular markers investigated and sample sizes) if the aim is to study the evolutionary history of a species. We also caution on certain methodological limitations that need to be considered to enlighten a study project from experimental design to result interpretation.<p>/Durant le Quaternaire, le climat de la Terre entière a été marqué par des oscillations importantes entre périodes glaciaires relativement longues et interglaciaires plus courtes d’environ 100 000 et 20 000 ans, respectivement, si l’on prend l’exemple de la dernière succession. Pendant les épisodes glaciaires, la calotte glaciaire continentale et la baisse du niveau des mers et des océans ont, à leur tour, affecté localement les conditions environnementales, au moins en ce qui concerne l’hémisphère nord. Ces perturbations climatiques et environnementales ont provoqué des changements dans la distribution géographique des espèces animales et végétales. Spécifiquement, ces changements ont pris la forme d’événements démographiques tels que l’extinction ou la fragmentation des populations associées à des goulots d’étranglements (réduction de la diversité génétique) ou, à l’inverse, des expansions de populations parfois accompagnées d’un effet fondateur (échantillonnage aléatoire à partir de la diversité génétique source) et/ou de la rencontre subséquente entre des groupes génétiquement différenciés (zone de contact secondaire) résultant en des gradients de diversité génétique à travers toute la distribution actuelle de l’espèce. On comprend dès lors que l’histoire démographique d’une espèce peut être reconstruite en étudiant les signatures qu’une telle histoire a pu laisser dans son génome. En ce qui concerne les taxa européens, les études phylogéographiques, qui utilisent ces signatures, se sont principalement intéressées aux espèces des régions tempérées; les connaissances acquises dans le domaine pour les espèces adaptées aux environnements plus froids sont plus rares bien que leur réponse envers les changements climatiques pourrait ne pas simplement avoir une tendance inverse par rapport à celle des espèces tempérées.<p><p>Dans son ensemble, l’objectif du présent projet de thèse est d’étudier l’histoire évolutive d’un groupe de quatre espèces sœurs de chrysomèles adaptées à un environnement froid dans le but de comprendre leur réponse face aux changements climatiques passés. Les quatre espèces partagent de nombreux traits en commun (cycle de vie, capacité de dispersion, morphologie générale) mais présentent des distributions géographiques qui se différencient incitant encore plus à s’intéresser aux facteurs qui ont pu les structurer. De plus, les chrysomèles sont des insectes herbivores très spécialisés, chaque espèce d’insecte ne se nourrissant que d’une ou quelques espèces de plantes bien précises. Cette spécialisation ajoute une dimension supplémentaire à l’étude de l’impact des changements climatiques pour comprendre l’évolution de la relation insecte-plante hôte. L’étude de ce complexe d’espèces avait donc également pour but de comprendre des processus tels que ceux de spéciation qui a pu être induite via une spécialisation alimentaire ou de séparation géographique suite à un changement des distributions. Le projet s’articule autour de trois axes décrits ci-dessous.<p><p>Le premier axe du projet a permis d’acquérir une connaissance plus fine des quatre espèces de chrysomèle appartenant au sous-genre Goniomena (Chrysomelidae, Gonioctena). Nous avons défini les barrières importantes séparant les quatre espèces. L’analyse des séquences d’ADN de cinq marqueurs moléculaires indépendants obtenues pour un grand nombre d’individus échantillonnés sur toute l’aire de répartition de chacune des quatre espèces nous a permis de définir quatre groupes génétiquement distincts, d’identifier précisément leur(s) espèce(s) de plante(s) hôte(s) ainsi que leur distribution géographique. Le second axe a été réalisé sur le même jeu de données multilocus et avait pour but d’exploiter un éventail de méthodes communément utilisées ainsi que d’autres prometteuses en étude phylogéographique dont on exploite la synergie afin de renforcer les interprétations concernant l’histoire de chaque espèce. Pour cela, nous avons combiné la modélisation des distributions passées et présentes des quatre espèces et de leur(s) plante(s) hôte(s) pour générer des hypothèses les plus réalistes possibles. Ensuite, les différentes hypothèses historiques ont été évaluées via deux approches différentes, une méthode d’approximation de calcul bayésien approché (Approximate Bayesian Computation, ABC) et une autre basée sur des simulations spatiales de coalescence, permettant d’ensuite comparer les résultats. Enfin, avec le troisième axe, nous nous sommes intéressés à un programme d’inférence de paramètres démographiques (temps de divergence, taille effective des populations, taux de migration) communément utilisé. Nous avons créés des jeux de données non empiriques, construits avec trois simulateurs également très répandus dans les analyses de génétique des populations, pour explorer les performances d’inférence du programme.<p><p>Nous espérons que ce projet va aider à mieux comprendre la façon dont les espèces adaptées à un climat froid en Europe répondent aux changements climatiques. Il a démontré et permis d’isoler des traits spécifiques dans ces réponses tout en suggérant néanmoins certains schémas communs. Nos résultats sont riches et variés; la distribution contemporaine de la diversité génétique chez les quatre espèces soeurs de chrysomèle implique des processus tels que l’introgression et l’hybridation, la compétition et l’invasion, la spéciation allopatrique et sympatrique, le potentiel de dispersion et la différentiation des réponses aux changements climatiques. Au vu de ces résultats, d’autres recherches sont envisagées; les mécanismes donnant lieu ou sous-jacents aux différents types de spéciation, à la spécialisation alimentaire, à la différentiation de niche et à l’hybridation dans les zones de contact secondaire seront explorés à l’aide de matériel biologique et de ressources analytiques déjà acquises. En pratique, nous avons réalisé notre étude en explorant des procédures d’analyses prometteuses tout en exploitant les ressources d’un jeu de données multi-locus et multi-espèces. Tout au long du projet, nous mettons l’accent sur la nécessité de travailler avec des jeux de données empiriques multi-espèces au moins équivalents à celui de ce projet (en termes de nombre de marqueurs, de taille d’échantillons) si on vise à réaliser une telle étude sur l’histoire évolutive d’une espèce. Nous mettons également en garde sur certaines limites méthodologiques qui doivent être considérées pour la conception d’un projet d’étude allant de la mise en œuvre expérimentale jusqu’à l’interprétation des résultats.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished