Four in One: Cryptic Diversity in Geoffroy’s Side-Necked Turtle \u3cem\u3ePhrynops geoffroanus\u3c/em\u3e (Schweigger 1812) (Testudines: Pleurodira: Chelidae) in Brazil

Turtles are one of the most threatened groups of vertebrates, with about 60% of species classified at some level of extinction risk. Compounding this extinction crisis are cryptic species and species complexes that are evaluated under a single species epithet but harbor multiple species, each of which needs to be evaluated independently. The Phrynops geoffroanus species group is a classic example. Described first in 1812, it is currently thought to harbor multiple species. To test this hypothesis, we collected mitochondrial and nuclear genomic data, morphometric data, and distribution and associated biome information. We applied statistically rigorous species delimitation analyses, taxonomic hypotheses tests, and fully coalescent phylogenetic reconstruction methods, concluding that the Phrynops geoffroanus species complex comprises four geographically structured species/lineages that diverged during the Pleistocene and are currently geographically structured along the main South American river basins and biomes. These species/lineages show subtle and largely non-significant differences in shape but are characterized by differences in coloration and patterns of marks on the head and plastron. Our results contribute to the understanding of species diversity and diversification of biodiversity in South America and provide an important basis for the conservation of freshwater turtles

História evolutiva de uma espécie de passiflora da Mata Atlântica

A Mata Atlântica está entre um dos ecossistemas mais ricos em biodiversidade do mundo. Os processos históricos e evolutivos que levaram a esta enorme diversidade permanecem pouco entendidos até os dias de hoje, o que suscita muitos estudos sobre a fauna e flora da região na busca de hipóteses que expliquem tamanha riqueza. Nesse contexto, utilizando uma espécie de Passiflora endêmica da Mata Atlântica, cuja distribuição está restrita às bordas de mata em áreas ao nível do mar, o presente estudo teve como objetivo caracterizar os padrões de variabilidade genética da espécie, bem como testar duas hipóteses que buscam explicar as possíveis causas da diversidade encontrada na Mata Atlântica, a Teoria dos Refúgios e a Hipótese de Rios como barreiras. Foram realizadas análises filogeográficas, utilizando marcadores moleculares dos genomas plastidial e nuclear, com amostras coletadas ao longo de toda a distribuição geográfica da espécie. Os resultados obtidos demonstraram que as populações da espécie encontram-se bastante estruturadas em virtude de baixo fluxo gênico, sem sinal de expansão populacional recente e com indícios de isolamento por distância. As análises realizadas com o marcador nuclear obtiveram resultados mais informativos em relação ao marcador plastidial, tendo sido observado um padrão Norte- Sul de distribuição geográfica na espécie. Foram encontrados três filogrupos e sua separação coincidiu com a posição dos rios Jequitinhonha e Doce, corroborando a hipótese de rios como barreiras contra o fluxo gênico para esta espécie. Os maiores índices de diversidade genética foram encontrados na região central da Bahia, região esta já reportada como possível área de refugio durante as oscilações climáticas do Quaternário, e embora nossos resultados apontem para uma diversificação mais antiga da espécie, ocorrida no Terciário, às áreas de refugio podem ter sido importantes na manutenção e preservação da diversidade já existente durante as alterações climáticas mais recentes. Como estratégia de conservação da espécie que habita um ecossistema já bastante fragmentado, sugerimos que as áreas prioritárias sejam as que possuem a maior diversidade genética, considerando-se também a existência de três grupos de diversidade genética distintos, representados pelos filogrupos encontrados. Este trabalho ainda fez uma importante contribuição para o entendimento da classificação taxonômica da espécie, reabilitando sua divisão em duas entidades taxonômicas distintas.The Atlantic Rainforest is one of the most species-rich ecoregions in the world. The factors concerning the historical origin and evolutionary processes that gave rise to such diversification and that promoted speciation in this ecosystem remains poorly understood. Many studies of fauna and flora have been conducted in this region with the aim of explaining such richness. In this context, this study aimed to characterize the patterns of genetic variability in a species of the Passiflora genus endemic to the Atlantic Rainforest and distributed exclusively at the sea level, in forest edges, and test two hypotheses that attempt to explain the possible causes of the diversity found in the Atlantic Rainforest: the refuge theory and riverine barrier hypothesis. The phylogeographic analyzes were performed using molecular markers of nuclear and plastid genomes, with samples collected throughout the geographic distribution of the species. The results showed that populations were structured possibly due to the low gene flow. There is also evidence of demographic stability without recent population expansion and evidence for isolation by distance. The nuclear marker dataset was more informative than the plastidial marker dataset, and showed the existence of a clear phylogeographical structure and the presence of three phylogeographic groups. It was observed that the separation of the three groups coincides to the location of the Jequitinhonha and Doce Rivers, corroborating the riverine barriers acting as barriers against gene flow in this species. The highest levels of genetic diversity were found in central Bahia, a region reported as a possible refugial area for other species during the climatic changes in the Quaternary, and although the results obtained in this study showed that the origin and diversification of this species was older, occurring during the Tertiary, the refugial area should have been very important for the maintenance of an already existing diversity throughout the climatic changes. As a conservation strategy for this species, which inhabits a extremely endangered ecosystem, we suggest that the locations presenting high genetic diversity levels and belonging to each one of the three phylogroups must be given high priority for conservation. In this work we have done an important contribution for the taxonomic classification of this species, recovering the two taxa condition to this geographic area

Die populationsgenetische Struktur des in der Mata Atlântica endemischen Rotkehl-Mückenfressers ( Conopophaga lineata , Passeriformes: conopophagidae) lässt eine Kontaktzone in der Mata Atlântica erkennen

The Rufous Gnateater, Conopophaga lineata, is a small insectivorous understory bird which is endemic to and widely distributed in the tropical and subtropical Atlantic forest of South America. Its distribution makes it ideally suited for testing two major hypotheses for the origin of biodiversity, namely, the riverine barrier and the forest refuge hypotheses. In this study, we sequenced mitochondrial (control region) and nuclear markers (intron 5 of the β-fibrinogen gene) for individuals distributed in the southern Atlantic forest and obtained a strong genetic structure with one clear discontinuity in northern Brazilian state of São Paulo. We consistently detected signals of demographic expansion for both markers, with estimates indicating that expansion started in the Late Pleistocene (250,000 years ago), suggesting that the forest refuge hypothesis potentially explains Rufous Gnateater’s diversification. We also found evidence of gene flow between populations from each side of this discontinuity, with a possible secondary contact zone occurring in the states of Minas Gerais, São Paulo, and Rio de Janeiro.Der Rotkehl-Mückenfresser, Conopophaga lineata, ist ein kleiner insektivorer Vogel, der im Unterholz der tropischen und subtropischen Mata Atlântica in Südamerika endemisch und weit verbreitet ist. Die Verbreitung des Rotkehl-Mückenfressers macht ihn besonders dafür geeignet, zwei Haupthypothesen zum Ursprung von Biodiversität zu testen, die „Flüsse als Barrieren“-Hypothese (“riverine barrier hypothesis”) und die „Wälder als Rückzugsgebiete“-Hypothese (“forest refuges hypothesis”). Wir haben mitochondriale Marker (Kontrollregion) und Zellkernmarker (Intron 5 von β-Fibrinogen) für in der südlichen Mata Atlântica verbreitete Individuen sequenziert und eine ausgeprägte genetische Struktur mit einer klaren Diskontinuität im Norden des Bundesstaates São Paulo gefunden. Für beide Marker haben wir durchweg Signale demographischer Ausbreitung entdeckt, die schätzungsweise im späten Pleistozän (vor 250000 Jahren) begann, was darauf hindeutet, dass die „Wälder als Rückzugsgebiete“-Hypothese einen möglichen Einflussfaktor bei der Diversifikation des Rotkehl-Mückenfressers darstellt. Wir haben auch Hinweise auf Genfluss zwischen Populationen auf beiden Seiten der Diskontinuität gefunden, mit einer möglichen sekundären Kontaktzone in den Bundesstaaten Minas Gerais, São Paulo und Rio de Janeiro.\ud Communicated by J. Fjeldså.This work was supported by FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais 17228), CNPq, FAPESP (BIOTA 2013/50297-0), NSF (DOB 1343578), NASA, CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), and the Research Center on Biodiversity and Computing (BioComp) of the Universidade de São Paulo (USP), supported by the USP Provost’s Office for Research. Gisele Dantas worked under a Post-doctoral grant CAPES/PNPD (2010/52590-8) and CNPq (503145/2009-2). We also thank the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (Brazil), Instituto Florestal de São Paulo (Brazil), and Instituto Estadual de Florestas de Minas Gerais (Brazil) for the permits to collect samples (IBAMA/MMA no 03/2004 IBAMA/MMA: 011/2000, processes 1835/2000; 053/2001, 1835/00-07; 070/2002, 02015.001835/00-07; 207/2003, 02015.023482/98-38). We gratefully acknowledge the improvements in English usage made by Caitlin Stern through the Association of Field Ornithologists’ program of editorial assistance

Examining Pathogenesis and Preventatives in Spontaneous and Staphylococcus-Induced Bacterial Chondronecrosis with Osteomyelitis in Broilers

Bacterial chondronecrosis with osteomyelitis is the most important cause of lameness in broilers. This is important to poultry production, as it poses animal welfare issues, and causes a significant loss in revenue. The remediation of this disease requires the study of its etiology with fitting models and evaluating preventatives. The research reported herein covers genomic virulence analysis of BCO isolates, mainly Staphylococcus aureus, and Escherichia coli retrieved from lame birds. We found that S. aureus isolates were closest to chicken strains in Europe but may have been in the Arkansas area for a decade. Phylogenomics suggest our S. aureus is restricted to poultry, while the E. coli spans various hosts. This dissertation includes the analyses of mainly BCO isolates to determine virulence using the embryo lethality assay. Human S. aureus was the most lethal to layer and broiler embryos. Staphylococcus agnetis 908 that may induce lameness to \u3e50% did not show virulence. This inconsistency among others compelled us to offer that embryo lethality assay may not be an effective tool for estimating the pathogenicity of BCO isolates. Furthermore, research reported herein covers the investigation of the feed additive Availa-ZMC for lameness reduction potential in broilers using litter and wire-flooring models for inducing lameness. Availa-ZMC (a mixture of organic trace minerals) resulted in a reduction of lameness by 20% in the wire-flooring model, and 25% in the challenge on litter flooring model. Finally, this dissertation reports on a Typhoid-Mary experiment in broilers raised on the wire flooring to determine whether broilers challenged with S. agnetis can transmit the bacterium to birds in the same pen thereby spreading BCO and lameness. The Typhoid Mary experiment shows that young broilers exposed to S. agnetis at an early age harbor the bacterium and if mixed with unexposed birds 10 days later can transmit the bacterium to their pen mates

Evolution and biogeography of fire-eye antbirds (genus Pyriglena): Insights from molecules and songs

The importance of climatic and geologic factors as drivers of population differentiation and speciation in the Neotropical region has long been appreciated. However, many questions remain regarding their roles underlying the processes and patterns of diversification. Studies conducted in distinct regions containing a suite of geological and ecological conditions constitute ideal scenarios to assess the role of Pleistocene climatic changes, rivers, and mountain building as historical diversification mechanisms. In chapters 1 and 2, I used an integrative approach combining molecular phylogenetics, phylogeography and population genetics to elucidate the importance of climatic and geological factors as engines of diversification. I focused on the South American fire-eye antbirds (genus Pyriglena), which occur in forested areas in southeastern Amazonian basin, Pantanal basin, Andes and Atlantic Coast. My study suggests that fire-eyes represent a young and rapid diversification in South America. It is conceivable that the origin of the major clades in this group trace back to the formation of the modern course of large rivers in the Amazonian basin and Atlantic Forest, with subsequent diversification fostered by more recent Pleistocene climatic oscillations creating opportunities for range expansion and geographic isolation in the Andes, Pantanal and Atlantic Forest. However, the role of large rivers as historical barriers to dispersal was apparently stronger in the Amazon basin relative to western South America (western lowlands and Andes) and Atlantic Forest. On the other hand, climatic oscillations seemed less important in creating opportunities for geographic differentiation within the Amazon comparative to the other regions. Fire-eyes seemed to have a complex history of diversification, involving large-scale geological and climatic processes acting over regional and continental scales during the last ~ 2.5 Mya. In chapter 3, I examined in detail how songs varied across the range of Atlantic Forest fire-eye antbirds, and I tested several different hypotheses of the origin of song divergence in an attempt to explain their current vocal variation. Genetic differentiation and introgressive hybridization seemed to explain the overall song variation and geographic structuring in fire-eyes better than alternative factors such as body size, bill morphology and ecology

Bacterial Chondronecrosis with Osteomyelitis in broilers: genomics, phylogenomics, and methods to detect specific pathogens during outbreaks.

Lameness is a major issue in animal welfare and the broiler industry. Bacterial chondronecrosis with osteomyelitis (BCO) is one of the main causes of lameness. Many staphylococcal species, including Staphylococcus agnetis isolate 908, have been isolated from the bones and blood of lame broilers at the University of Arkansas. Staphylococcus agnetis is a coagulase-variable, Gram-positive bacterial species that has been previously associated with subclinical or mild clinical cases of mastitis in dairy cattle. The annotated complete genome of hypervirulent strain 908 was published at NCBI. In this study, it has been compared to nine genomes we assembled for hypervirulent isolates in dairy cattle. Phylogenomic analyses of chicken and cattle isolates of S. agnetis and Staphylococcus hyicus suggest a very close relationship between the cattle and chicken isolates. The hypervirulent chicken isolate, 908, clustered with two of the cattle isolates, including strain 1379. A catalogue of gene differences between the cattle and chicken isolates was constructed using reciprocal blast analyses at the nucleotide and polypeptide level. More than 40 genes and 3 plasmids from strain 908 are absent or poorly conserved in any of the cattle S. agnetis isolates. No transformation protocol has been described for S. agnetis. Subsequently, an electroporation procedure has been optimized for DNA transformation of Staphylococcus agnetis. Therefore, we have optimized an electroporation method for DNA transformation so that we regularly obtain 10 to 20 transformants per ng using a Gram+/Gram- shuttle vector. Moreover, among the BCO pathogens isolated from the lame broilers, there are a number of Staphylococcus species, such as, S. agnetis, S. hyicus, S. chromogenes, S. aureus, S. cohnii, S. saprophyticus, S. epidermidis, and S. capitis, which are hard to accurately identify based just on genes like 16S rDNA. Therefore, using pfbA gene, a novel PCR assay was optimized for Staphylococcus species discrimination and strain typing. Moreover, extraction of bacterial DNA for subsequent molecular diagnostic applications remains a costly and time-consuming operation. We developed a technique for rapidly extracting genomic DNA from the BCO pathogens and other environmental bacteria based on sodium hydroxide cell lysis with or without magnetic bead capture. Finally, the BCO pathogens are transmitted via air. Our efficient air sampling system was designed for the quick screening of these airborne BCO pathogens and is transferable to monitor agriculturally important pathogenic bacteria

Evolução morfológica na radiação dos roedores sigmodontíneos : ecologia e história evolutiva

Radiações evolutivas estão entre os eventos mais fascinantes da evolução. Grande parte da diversidade da vida, tanto de espécies como ecológica, surgiu nos breves intervalos temporais de rápida especiação que configuram as radiações. As causas ecológicas e não-ecológicas do surgimento da diversidade em radiações evolutivas, em especial nas radiações adaptativas, são tema de pesquisa há muito tempo, pelo menos desde que Darwin observou a imensa diversidade de um grupo de pássaros nas ilhas Galápagos. Desde então, as ilhas têm sido os ambientes ideais para o estudo desse fenômeno, e foi a partir das observações e experimentos em ilhas que toda a teoria ecológica das radiações evolutivas surgiu. Contudo, as causas ecológicas das radiações explosivas ocorridas em amplas escalas continentais permanecem tema de constante debate. Nesta tese, foram investigados os determinantes ecológicos e não-ecológicos (e.g., geografia, contingências históricas, efeitos filogenéticos) da evolução morfológica dos roedores sigmodontíneos durante sua radiação na região Neotropical, em especial no continente sul-americano. Para isso, foi quantificada a morfologia do crânio e mandíbula de mais de dois mil exemplares do grupo, e foram investigadas variações ecomorfológicas nos níveis interespecífico (I), intraespecífico (II), e entre assembleias de sigmodontíneos (III). Na Parte I da tese, foram investigadas duas predições da teoria da radiação adaptativa, a correlação-fenótipo ambiente (capítulo 1) e a funcionalidade do fenótipo através da força da mordida (capítulo 2), permitindo determinar o papel da divergência ecológica na evolução morfológica das espécies. Na Parte II (capítulo 3), foram investigadas as contribuições relativas de processos determinísticos e neutros sobre a variação morfológica entre populações de uma espécie de roedor sigmodontíneo amplamente distribuída, Akodon cursor. Na Parte III, a influência da variação ambiental e da distribuição espacial das linhagens filogenéticas de sigmodontíneos sobre o tamanho corporal (capítulo 4) e forma do crânio e mandíbula (capítulo 5), foram investigados no contexto biogeográfico da variação no tamanho e forma média entre assembleias de sigmodontíneos. As contribuições originais desta tese foram: (i) mostrar que a radiação evolutiva dos roedores sigmodontíneos foi guiada principalmente por fatores históricos e geográficos ao invés de fatores ecológicos; (ii) sugerir que radiações evolutivas ocorridas em escalas continentais, especialmente de roedores, têm um componente geográfico e histórico mais determinante do que o componente ecológico; (iii) revelar que a força da mordida varia pouco entre roedores sigmodontíneos herbívoros e granívoros, o que provavelmente é resultado do fenótipo generalista desses roedores; (iv) apontar que sigmodontíneos com dieta insetívora têm uma taxa de evolução mais rápida, e parecem estar evoluindo sua forma do crânio/mandíbula e sua força da mordida em uma direção diferente das demais espécies; (v) demonstrar que, dentro de uma espécie de sigmodontíneo (Akodon cursor), fluxo gênico e deriva genética explicam melhor a forma do crânio entre populações, enquanto a variação ambiental explica melhor o tamanho do crânio, indicando que o tamanho é uma característica mais lábil e mais sujeita a pressões ambientais do que a forma do crânio; (vi) mostrar que a variação biogeográfica, tanto do tamanho quanto da forma média do crânio/mandíbula entre assembleias de sigmodontíneos, está sob influência da distribuição diferencial das linhagens filogenéticas ao longo do espaço geográfico, bem como de variáveis ambientais; o que indica conservação filogenética de nicho à nível de metacomunidades. De modo geral, ao investigar as contribuições relativas dos componentes adaptativo e não-adaptativo da evolução morfológica, foram obtidas informações importantes para conhecer as causas da diversificação morfológica em Sigmodontinae, aumentando nosso conhecimento sobre as origens de toda a diversidade biológica.Evolutionary radiations are among the most fascinating phenomena of evolution. Most of the biological diversity on the planet, both in terms of species and ecological diversity, appeared during these brief intervals of rapid speciation. The ecological and non-ecological causes of the emergence of diversity in evolutionary radiations, especially in adaptive radiations, have long been the subject of research, beginning with Darwin and his notice of the astonishing diversity of bird forms in the Galapagos Islands. Islands have since been ideal environments in which to study evolutionary and adaptive radiations, and indeed it was from observations and experiments on islands that all ecological theory of evolutionary radiations arose. However, the ecological causes of explosive radiations occurring on large continental scales are still a matter of debate. In this dissertation, I investigated the ecological and non-ecological (e.g., geography, historical contingencies, phylogenetic effects) determinants of morphological evolution in sigmodontine rodents during their radiation in the Neotropical region, particularly on the South-American continent. The skull and mandible morphology of more than two thousand specimens was quantified, and ecomorphological variation was investigated on three levels: interspecific (I), intraspecific (II), and among sigmodontine assemblages (III). In part I, two predictions from the ecological theory of adaptive radiation were investigated: the phenotype-environment correlation (chapter 1), and the trait utility through the bite force (chapter 2). This approach enabled determination of the role of ecological divergence in species morphological evolution. In part II (chapter 3), I investigated the relative contributions of deterministic and neutral processes to morphological variation among populations of one widely distributed sigmodontine species, Akodon cursor. In part III, I investigated the influence of environmental variation and spatial distribution of phylogenetic lineages on body size (chapter 4) and on shape of the skull and mandible (chapter 5), in the context of biogeographical variation of mean size and shape in sigmodontine assemblages. The original contributions of this dissertation are as follows: (i) to demonstrate that the evolutionary radiation of sigmodontines was driven mainly by historical and geographical factors instead of ecological factors; (ii) to suggest that evolutionary radiations on continental scales, especially rodent radiations, have a more determinant historical and geographical component than an ecological one; (iii) to show small variation in bite force between sigmodontine herbivores and granivores, which is likely a consequence of the generalist phenotype of these rodents; (iv) to highlight that insectivorous sigmodontines have a faster rate of morphological evolution than other diet groups, and that skull and mandible morphology and bite force are evolving in different directions than in other species; (v) to demonstrate that within a sigmodontine species (Akodon cursor), gene flow and genetic drift better explain variation in skull shape among populations, while environmental variation better explains variation in skull size, which suggests that size is more labile feature than shape and thus more prone to change with environmental pressures; and (vi) to show that biogeographical variation in mean body size, mean skull shape, and mean mandible shape across sigmodontine assemblages is influenced by the different distributions of phylogenetic lineages over geographical space, as well by environmental variables, which indicates phylogenetic niche conservatism at the metacommunity level. These results shed light on some of the factors driving morphological diversification in Sigmodontinae. Further, the analytical approach(es) utilized may be useful for general investigations of the relative contributions of adaptive and non-adaptive components of morphological evolution, thereby potentially increasing our knowledge of the origins of all biological diversity

Genetic evidence from mitochondrial DNA corroborates the origin of Tibetan chickens

<div><p>Chicken is the most common poultry species and is important to human societies. Tibetan chicken (<i>Gallus gallus domesticus</i>) is a breed endemic to China that is distributed mainly on the Qinghai-Tibet Plateau. However, its origin has not been well characterized. In the present study, we sequenced partial mitochondrial DNA (mtDNA) control region of 239 and 283 samples from Tibetan and Sichuan indigenous chickens, respectively. Incorporating 1091 published sequences, we constructed the matrilineal genealogy of Tibetan chickens to further document their domestication history. We found that the genetic structure of the mtDNA haplotypes of Tibetan chickens are dominated by seven major haplogroups (A-G). In addition, phylogenetic and network analyses showed that Tibetan chickens are not distinguishable from the indigenous chickens in surrounding areas. Furthermore, some clades of Tibetan chickens may have originated from game fowls. In summary, our results collectively indicated that Tibetan chickens may have diverged from indigenous chickens in the adjacent regions and hybridized with various chickens.</p></div