A genomic view on the diversification of Neotropical frogs

Editor's Notes

• #3: A genomic view on the diversification of neotropical frogs
• #4: The theory of evolution is based on the idea that all species are related and gradually change over the time. The accumulation of small changes over the time can produce that some populations of the same species get enough differences that they will be “reproductively isolated” and so they will no be able to mate between them anymore. We call this “speciation event”.
• #5: What is speciation? The formation of new and distinct species in the course of evolution. There are several ways in which a species can evolve to become two (or more) distinct species. These ways are: isolation, mutation, recombination, natural selection, genetic drift, hybridization and polyploidy.
• #6: The study of speciation by using genetic resources has been mainly applied on model organisms, such as human & mouse. However, nowadays we can use high-throughput sequencing techniques (such as illumina) to study the speciation in non-model organisms too. We call this “genomics speciation”.
• #7: Before, with Sanger sequencing (and other similar approaches) we had to pre-select some markers and we had to “believe” that this markers were showing us “the truth”. For this reason it was necessary to use multiple individuals from several populations in order to know if ”the truth that we saw” was actually “the right truth”. Nowadays, by using high-throughput sequencing techniques, we can obtain thousands of orthologous loci, and so this allow us to use less individuals. So, “speciation genomics” mainly refers to: 1) finding genes involved in speciation, 2) finding genes that are homogenized by gene-flow, and 3) finding genes that are related to adaptation processes. But genomics is not only about finding new genes, we can also use it to reconstruct evolutionary and demographic histories.
• #8: Neotropical amphibians are the 50% of amphibians in the world. Many new species are being described every year. At the same time that many others become extinct too. However, not many studies have applied genomics to study the speciation in Neotropical frogs. This means that we still know little about the patterns and processes that produced this huge diversity on the tropical regions.
• #9: Our study model is a genus of Neotropical frogs named Oreobates. To the present date, this genus includes 24 species that are distributed across a wide range of habitats and altitudes. All of them are found in South America (as you can see in the map). It’s quite interesting that Oreobates species can be mainly found up to four types of habitat: cloud forests, montane forests, lowland forests, and even dry forests.
• #10: Oreobates is a very interesting genus of frogs not only because they live in a wide variety of habitats but also because little is know about them. More than half of the 24-named species have been described in the last ten years. And, the phylogenetic relationships among them are not clear. Some ”trees” have been published (using few molecular markers) but still the evolutionary history is not clear either (as you can see).
• #11: We think that Oreobates are one of the best non-model species in the world. They are extremely difficult to find. Also, there are very few museums that have Oreobates samples. And if you want to get samples from them, there is always problems with permits. Moreover, there are some species that have been only found once. Luckily our collaborators were the ones collecting it. And that’s probably another big reason why we choose Oreobates.
• #12: Our collaborators (José Manuel Padial and Ignacio de la Riva, from Madrid) they probably are the ones who know the most about these frogs. As you can see “in blue” most of the Oreobates species have been described by them. And “in red” you can see which species are available in the MNCN (Madrid). As you can see, we have access to almost all the species. And this quite good, because it means that nobody can steal us this work.
• #13: So, the main goal of my PhD project is to study the evolution rates, demographic history and adaptation patterns on the frogs of the genus Oreobates (using genomic data). As you can see, we have four different goals (phylogenomics, evolutionary history, demographic history and adaptation). I’m going to explain them in more detail in the following slides.
• #14: First stage: phylogenomics. Our goal is to reconstruct a highly supported tree among Oreobates species (using genomic data). This will give us a very good idea about the relationships among Oreobates. We will use this tree not only for downstream analysis but also to address questions related to the phylogeny. We know that there are Oreobates species living on both highlands and lowlands. It has been hypothesized that Oreobates emerged on the Andes highlands. This is quite weird, since usually it’s the contrary (species first emerge on the lowlands and the colonize the highlands). We want to know WHEN the genus Oreobates was originated and HOW many colonization events to lowland rainforest have occurred. We will use a time-calibrated phylogenomics tree for looking at this.
• #15: Second stage: evolutionary history. Here our goal is to study the variation in the evolution rate of the different Oreobates species. Previous studies (by Alvaro Dugo-Cota) have shown that glass-frogs living in the highlands are subjected to a reduction in the rate of evolution. In this study, the people from our group studied over 100 species of frogs, but using few molecular markers. The results were crystal clear specially for mitochondrial markers but not so clear for nuclear. They hypothesized that is because ectotherm metabolism is reduced at low temperatures. In our case, using thousands of molecular markers, we want to test if Oroebates species from the highlands also show a lower rate of evolution compared to the species living on the lowlands.
• #16: Third stage: demographic history. Our goal is to study the effect of the past environmental conditions on the Oreobates demography. The Andes formation was about 20 to 30 MYA and so the origin of Oreobates too. As you may know, about 2.5 MYA (on the Pleistocene) the Earth faced repeated glacial cycles. And so species living in the highlands are expected to suffer populations increasing and reductions following climatic changes. We hypothesized that species with similar habitat requirements will show parallel demographic changes during the Pleistocene glaciations. And so we want to know if highland species show different demographic trends compared to lowland. But also, we want to use genomics to study hybridization as a speciation mechanisms between diverging lineages that inhabit the lowlands.
• #17: Fourth stage: study of adaptation. Our goal is to study the genomics regions that could be associated with adaptation. We know that some Oreobates species inhabit a wide diversity of habitats. For example, Oreobates cruralis (which is the species on the picture here) is usually found on the Amazonian lowland forests, but the same species can also be found in montane forests and even on dry forests. There are some other species that have found living on both highlands and lowlands environments. So we want to study if there are any genes related to adaptation to altitude or genes related to adaptation to dry environmental conditions.
• #18: Regarding the methods, since we don’t have a closely related genome of reference, our initial idea was to (first) sequence the transcriptome for one species and (then) do whole genome sequencing for the other species. Our idea was to use (then) an exome assembly to address our hypothesis. However, we realized that this was quite expensive and also a big waste of resources (since –depending on the genome size– we would be throwing away about 95% of the data). For this reason, we decided to first try to find out the genome size for Oreobates species.
• #19: Actually I spent a lot of time try to develop a method for estimating genome size based on real-time PCR. However, I’m not going into details since this work was not conclusive enough. Anyway, amphibians have a very large variation in the genome size (as you can see) compared to other vertebrates such as birds, reptiles or mammals. For this reason we decided to change our initial approach, and use a small representation of the genome.
• #20: In my PhD project, we will use the transcriptome as a reduced representation of the genome
• #21: How to do it? The main problem is that we don’t have RNA for all the Oreobates species. As you may know, RNA is very instable at room temperature and so it’s very difficult to preserve it. In fact, we only had RNA available for one single Oreobates species (O. cruralis). So, we extracted the RNA for the three tissues that we had (liver, intestine and spleen) and did transcriptome sequencing. Then, I spent 4 months in Sweden, doing fancy bioinformatics analysis to reconstruct a de-novo transcriptome assembly for one species. Don’t’ worry I’m going to explain you this a little bit more in detail in the next slide. Using this transcriptome as a reference, now we are working on a transcriptome-based exon capture (which, by the way, is a very novel technique) that will allow us to obtain the transcriptome for all the others Oreobates species. I will also explain this on the last slide. And so finally, once we get all the data, we will be able to test the initial hyphotesis.
• #24: Pfam: database of protein families SwissProt: curated protein sequence database Xenopus: protein database of Xenopus tropicalis GO_Pfam: Gene Ontology (using only Pfam results) GO_SwissProt: Gene Ontology (using only SwissProt results) GO_SwissProt_Pfam: Gene Ontology (using SwissProt + Pfam results) Kegg: Kyoto Encyclopedia of Genes and Genomes eggNOG: Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups
• #27: In brief, using the transcriptome of one species, we will synthesize thousands of short nucleic acid probes that will specifically hybridize to genomic DNA of other closely related species (Maricic et al. 2010). Thereby, we will be able to retain and enrich homologous sequence-based loci across our target species (that is, transcriptome enrichment). Our main goal is to use this transcriptomic information to assess divergence patterns between Oreobates species. As this genus includes species adapted to highland and lowland environments, it could be interesting to know if highlands frogs have differences in gene expression compared to lowland frogs.
• #29: My thesis is about diversification so…
• #30: My thesis is about diversification so…
• #31: My thesis is about diversification so…