Novel approaches for large-scale phylogenetics and applications in the context of the amphibian tree of life

During this thesis, I addressed some problems associated with large-scale phylogenetic analyses by tackling issues related to missing data and careful handling and addition of novel data in large-scale reconstructions, presenting an application of this approach in the context of amphibian phylogenetics. I developed a method (called “Concatabominations”) building on the original Safe Taxonomic Reduction method (Wilkinson 1995) as an alternative approach to the issue of identifying rogue taxa. The safe removal of rogue taxa due to missing data can potentially reduce the terraces in tree space search and improve resolution in the final consensus tree. In a pragmatic point of view, the new method can help in targeting taxa that require further sampling during a research design. Novel sequence data for the rediscovered Ericabatrachus baleensis allowed to explore its placement in the Amphibian tree of life. I tested the inclusion of novel data using a backbone alignment from a previous work (de novo analysis) and a backbone phylogenetic tree (constrained analysis), after careful curation of gene partitions to include in an analysis. I found that the use of a constrained phylogenetic inference using a previous accepted tree seems to be a practical solution to the rapid phylogenetic placement of a taxon in cases of well-supported relationships. However, a de novo analysis might ensure an optimal alignment and avoid risks introduced when adding new data. Finally, I investigated the evolutionary relationships of the three lineages of the extant amphibians (Anura, Caudata and Gymnophiona) using an independent source of evidence: miRNAs, recently used to help resolve difficult phylogenetic problems. The analyses yielded a high number of shared miRNAs using the Xenopus tropicalis genome, contrasting with a lower number of miRNAs discovered using the Axolotl transcriptome. This suggests that not using genomic data is not ideal to validate miRNAs. Nevertheless, in spite of the limitations, I was able to find two potential novel miRNAs: one supporting the monophyly of Lissamphibia, and another supporting the Batrachia hypothesis. Overall, I hope the work developed in this thesis contributes with new insights into large-scale phylogenetics and in particular to amphibian phylogenetics

Evaluación de la diversidad críptica en anfibios de amplia distribución en la Amazonía peruana

Publicación a texto completo no autorizada por el autorEvalúa la diversidad críptica de especies de anfibios de amplia distribución en la Amazonia de Perú, usando los marcadores genéticos más utilizados en el código de barras de anfibios (16S rRNA y COI), y tomando como modelo grupos taxonómicos para los cuales hay antecedentes de diferenciación morfológica y de divergencia genética. El enfoque geográfico del estudio estuvo principalmente definido en ocho localidades de la Amazonia de Perú: Jenaro Herrera, Gueppí, Curupa y Redondococha (en el Departamento de Loreto, norte de Perú); Panguana (Departamento de Huánuco, centro de Perú); Los Amigos, Inkaterra y Tambopata (las tres en el Departamento de Madre de Dios, sureste de Perú), abarcando una distancia geográfica de 1000 km de norte a centro y 790 km de centro a sur. Se enfocó el análisis en algunas especies nominales para cinco géneros: Ameerega, Oreobates, Pristimantis, Hypsiboas y Scinax. Se obtuvo un total de 183 secuencias con el gen 16S rRNA y 181 secuencias con el gen COI en el presente trabajo, las cuales fueron completadas con 407 secuencias del marcador 16S rRNA y 49 secuencias del marcador COI de la literatura. De los análisis filogenéticos se determinó que existe al menos dos linajes divergentes en Ameerega, un linaje divergente en Oreobates, dos linajes divergentes en Pristimantis, ocho en Hypsiboas y tres en Scinax. En el caso de Ameerega, las divergencias entre linajes con el gen 16S rRNA fueron bajas (entre 2% - 3%), mientras que en los linajes obtenidos para Oreobates, Pristimantis, Hypsiboas y Scinax, las divergencias tuvieron valores más altos (entre 4% - 8%). Se utilizó los lineamientos de la taxonomía integrativa y el criterio para clasificar linajes divergentes en especies candidatas confirmadas o no confirmadas. Los resultados de los análisis realizados muestran que hay linajes divergentes que podrían constituir especies candidatas, así se encontró un total de siete especies candidatas confirmadas (una del género Ameerega, una del género Oreobates, y cinco del género Hypsiboas) y diez especies candidatas sin confirmar (una del género Ameerega, dos del género Pristimantis, tres del género Hypsiboas y cuatro del género Scinax). Los resultados del presente estudio generan nueva e importante información que ayudará a incrementar el conocimiento de la diversidad en anfibios de Perú. Para lograr este fin, será necesario complementar con estudios a futuro que provean más datos de morfología y otros aspectos ecológicos y reproductivos que permitan entender la divergencia entre los grupos estudiados. De esta forma, se podría conseguir mejorar el conocimiento del estado taxonómico de dichas especies nominales

Concatabominations:Identifying Unstable Taxa in Morphological Phylogenetics using a Heuristic Extension to Safe Taxonomic Reduction

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Variação morfológica em Telmatobius atahualpai (Anura: Telmatobiidae), com comentários sobre suas relações filogenéticas e sinapomorfias para o gênero.

Telmatobius atahualpai foi descrita com base em uma fêmea subadulta e três juvenis. Apresentamos aqui uma nova diagnose e uma descrição do esqueleto do adulto feita a partir de espécimes de duas localidades. Tecemos ainda comentários sobre sinapomorfias do gênero e sobre a possível relação filogenética de T. atahualpai com outras espécies congenéricas.Telmatobius atahualpai was described on the basis of a subadult female and three juveniles. A new diagnosis and a description of the adult skeleton of specimens from two localities are provided. We also comment on synapomorphies of the genus and on possible phylogenetic relationships of T. atahualpai with other members of the genus.Telmatobius atahualpai fue descrita en base a una hembra subadulta y tres juveniles. Se proporciona una nueva diagnosis y una descripción del esqueleto adulto de especímenes que proceden de dos localidades. También hacemos comentariossobre sinapomorfías del género y la posible relación filogenética de T. atahualpai con otros miembros del género

Two New Harlequin Frogs (Anura: \u3ci\u3eAtelopus\u3c/i\u3e) from the Andes of Northern Peru

Two new species of Atelopus (harlequin frogs) are described from the Andes of northern Peru, one from the northern Cordillera de Huancabamba (Departamento de Piura), 2,950 m elevation, and the other from an inter-Andean valley of the upper Marañon basin, Cordillera Central (Departamento de La Libertad), 2,620 m elevation. We also suggest the possible link between infection by the chytrid fungus and mortality of harlequin frogs in the upper Marañon basin in 999

Reptilia, Squamata, Iguanidae, Enyalioides touzeti Torres-Carvajal, Almendáriz, Valencia, Yañez-Muñoz and Reyes, 2008: distribution extension and first country record for Peru

The current article provides the first country record of Enyalioides touzeti in Peru. This new record extends the known species distribution ca. 52 Km SW from the southernmost record at the Province of El Oro, Ecuador, and increases the number of Enyalioides species in Peru to six

MORFOMETRIA DE TESTÍCULOS Y CELULAS GERMINALES DE ALLOBATES FEMORALIS (BOULENGER 1883) (DENDROBATIDAE: ANURA: AMPHIBIA)

El presente estudio describe la morfometría del testículo y la línea espermatogénica de especímenes adultos (N=8) de Allobates femoralis (Boulenger, 1883) colectados en la Estación Biológica Panguana (9°37'S 74°56'W), 250 msnm, 4 – 5 km rio arriba del Rio Pachitea, Distrito de Yuyapichis, provincia de Puerto Inca, Huánuco, Perú. Los anfibios (longitud 25,99±0,97 mm) fueron diseccionados y aislados los testículos con el propósito de obtener datos biométricos, posteriormente se realizaron cortes histológicos, coloreándose con hematoxilina y eosina; los estadios germinales observados fueron descritos y luego medidos con ayuda de un ocular micrométrico. La biometría obtenida de los testículos fue: eje mayor = 2,254 mm (DE = 0,282); eje menor = 1,343 mm (DE = 0,181); ancho = 0,971 mm (DE = 0,097); a nivel microscópico encontramos una capa albugínea (6,51±1,74 mì) constituida por tejido conectivo y músculo liso. Los túbulos seminíferos (162.56±47.61mì) están delimitados por una delgada capa de tejido conectivo. En relación a los aspectos reproductivos, se observó en A. femoralis que la espermatogenesis ocurre en los túbulos seminíferos donde los elementos celulares están organizados en espermatocistos. Cada cisto contiene células en el mismo estadio de desarrollo. La caracterización de cada tipo celular permitió la identificación de las células del linaje germinal: 1) Espermatogonias, son células aisladas o en grupos más voluminosas del linaje espermático (7,07±1,187 mì), tienen núcleo irregular, presenta granulaciones cromatínicas y se encuentran por lo general muy cerca de la pared tubular. 2) Espermatocito I, con tamaño menor que las espermatogonia (6,95±1,21mì), 3) Espermátides; redondeadas, con cromatina fina; a medida que avanzan en su desarrollo, éstas se elongan y se organizan en “atados” cuyas cabezas están sujetas entre si. 4). Los espermatozoides tienen forma alargada siendo su flagelo menos teñido, el núcleo es compactado y el citoplasma se reduce, están distribuidos hacia el lumen del túbulo seminífero. Se reporta por primera vez la descripción macroscópica y microscopia del testículo de A. femoralis para el Perú

Inadvertent paralog inclusion drives artifactual topologies and timetree estimates in phylogenomics

Increasingly, large phylogenomic datasets include transcriptomic data from non-model organisms. This has allowed controversial and unexplored evolutionary relationships in the tree of life to be addressed but also increases the risk of inadvertent inclusion of paralogs in the analysis. While this may be expected to result in decreased phylogenetic support it is not clear if it could also drive highly supported artefactual relationships. Many groups, including the hyper-diverse Lissamphibia, are especially susceptible to these issues due to ancient gene duplication events, small numbers of sequenced genomes and because transcriptomes are increasingly applied to resolve historically conflicting taxonomic hypotheses. We tested the potential impact of paralog inclusion on the topologies and timetree estimates of the Lissamphibia using published and de novo sequencing data including 18 amphibian species, from which 2,656 single-copy gene families were identified. A novel paralog filtering approach resulted in four differently curated datasets, which were used for phylogenetic reconstructions using Bayesian inference, maximum likelihood and quartet-based supertrees. We found that paralogs drive strongly supported conflicting hypotheses within the Lissamphibia (Batrachia and Procera) and older divergence time estimates even within groups where no variation in topology was observed. All investigated methods, except Bayesian inference with the CAT-GTR model, were found to be sensitive to paralogs, but with filtering convergence to the same answer (Batrachia) was observed. This is the first large-scale study to address the impact of orthology selection using transcriptomic data and emphasises the importance of quality over quantity particularly for understanding relationships of poorly sampled taxa

Evolutionary Relationships of the Critically Endangered Frog \u3cem\u3eEricabatrachus baleensis\u3c/em\u3e Largen, 1991 with Notes on Incorporating Previously Unsampled Taxa into Large-scale Phylogenetic Analyses

Background: The phylogenetic relationships of many taxa remain poorly known because of a lack of appropriate data and/or analyses. Despite substantial recent advances, amphibian phylogeny remains poorly resolved in many instances. The phylogenetic relationships of the Ethiopian endemic monotypic genus Ericabatrachus has been addressed thus far only with phenotypic data and remains contentious. Results: We obtained fresh samples of the now rare and Critically Endangered Ericabatrachus baleensis and generated DNA sequences for two mitochondrial and four nuclear genes. Analyses of these new data using de novo and constrained-tree phylogenetic reconstructions strongly support a close relationship between Ericabatrachus and Petropedetes, and allow us to reject previously proposed alternative hypotheses of a close relationship with cacosternines or Phrynobatrachus. Conclusions: We discuss the implications of our results for the taxonomy, biogeography and conservation of E. baleensis, and suggest a two-tiered approach to the inclusion and analyses of new data in order to assess the phylogenetic relationships of previously unsampled taxa. Such approaches will be important in the future given the increasing availability of relevant mega-alignments and potential framework phylogenies

CowPI::A rumen microbiome focussed version of the PICRUSt functional inference software

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and over 500 fully sequenced microbial genomes from this environment. Using this information we have developed ?CowPI? a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and captures the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiomepublishersversionPeer reviewe