Synthesis of geological and comparative phylogeographic data point to climate, not mountain uplift, as driver of divergence across the Eastern Andean Cordillera, bioRxiv, 2020-01-15

AbstractAimTo evaluate the potential role of the orogeny of the Eastern Cordillera (EC) of the Colombian Andes and the Mérida Andes (MA) of Venezuela as drivers of vicariance between populations of 37 tetrapod lineages co-distributed on both flanks, through geological reconstruction and comparative phylogeographic analyses.LocationNorthwestern South AmericaMethodsWe first reviewed and synthesized published geological data on the timing of uplift for the EC-MA. We then combined newly generated mitochondrial DNA sequence data with published datasets to create a comparative phylogeographic dataset for 37 independent tetrapod lineages. We reconstructed time-calibrated molecular phylogenies for each lineage under Bayesian inference to estimate divergence times between lineages located East and West of the Andes. We performed a comparative phylogeographic analysis of all lineages within each class of tetrapod using hierarchical approximate Bayesian computation (hABC) to test for synchronous vicariance across the EC-MA. To evaluate the potential role of life history in explaining variation in divergence times among lineages, we evaluated 13 general linear models (GLM) containing up to six variables each (maximum elevation, range size, body length, thermoregulation, type of dispersal, and taxonomic class).ResultsOur synthesis of geological evidence suggested that the EC-MA reached significant heights by 38–33 million years ago (Ma) along most of its length, and we reject the oft-cited date of 2–5 Ma. Based on mtDNA divergence from 37 lineages, however, the median estimated divergence time across the EC-MA was 3.26 Ma (SE = 2.84) in amphibians, 2.58 Ma (SE = 1.81) in birds, 2.99 Ma (SE = 4.68) in reptiles and 1.43 Ma (SE = 1.23) in mammals. Using Bayes Factors, the hypothesis for a single temporal divergence interval containing synchronous divergence events was supported for mammals and but not supported for amphibians, non-avian reptiles, or birds. Among the six life-history variables tested, only thermoregulation successfully explained variation in divergence times (minimum AICc, R2 0.10), with homeotherms showing more recent divergence relative to poikilotherms.Main conclusionsOur results reject the hypothesis of the rise Andean Cordillera as driver of vicariance of lowland population because divergence dates are too recent and too asynchronous. We discuss alternative explanations, including dispersal through mountain passes, and suggest that changes in the climatic conditions during the Pliocene and Pleistocene interacted with tetrapod physiology, promoting older divergences in amphibians and reptiles relative to mammals and birds on an already established orogen.

biorxiv evolutionary-biology 0-100-users 2020

CRISPR-Cas13d induces efficient mRNA knock-down in animal embryos, bioRxiv, 2020-01-14

AbstractEarly embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to induce RNA degradation in yeast, plants and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-Cas13d is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that both zygotically-expressed and maternally-provided transcripts are efficiently targeted, resulting in an 80% average decrease in transcript level and the recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish and mouse embryos. Altogether our results demonstrate that CRISPR-Cas13d is an efficient knock-down platform to interrogate gene function in animal embryos.

biorxiv developmental-biology 100-200-users 2020

URMAP, an ultra-fast read mapper, bioRxiv, 2020-01-14

AbstractMapping of reads to reference sequences is an essential step in a wide range of biological studies. The large size of datasets generated with next-generation sequencing technologies motivates the development of fast mapping software. Here, I describe URMAP, a new read mapping algorithm. URMAP is an order of magnitude faster than BWA and Bowtie2 with comparable accuracy on a benchmark test using simulated paired 150nt reads of a well-studied human genome. Software is freely available at <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsdrive5.comurmap>httpsdrive5.comurmap<jatsext-link>.

biorxiv bioinformatics 100-200-users 2020

Ferrosomes are iron storage organelles formed by broadly conserved gene clusters in bacteria and archaea, bioRxiv, 2020-01-12

Cellular iron homeostasis is vital and maintained through tight regulation of iron import, efflux, storage, and detoxification1–3. The most common modes of iron storage employ proteinaceous compartments that are composed of ferritin or related proteins4,5. While lipid-bounded iron compartments have also been described, the basis for their formation and function remains unknown. Here, we focus on one such compartment, the ferrosome, which had been previously observed in the anaerobic bacterium Desulfovibrio magneticus6. We identify three ferrosome-associated (Fez) proteins, encoded by a putative operon, that are associated with and responsible for forming ferrosomes in D. magneticus. Fez proteins include FezB, a P1B-6-ATPase found in phylogenetically and metabolically diverse species of bacteria and archaea with anaerobic lifestyles. In the majority of these species, two to ten genes define a cluster that encodes FezB. We show that two other species, Rhodopseudomonas palustris and Shewanella putrefaciens, make ferrosomes in anaerobic conditions through the action of their six-gene fez operon. Additionally, we find that the S. putrefaciens fez operon is sufficient for ferrosome formation in Escherichia coli. Using S. putrefaciens as a model, we find that ferrosomes likely play a role in the anaerobic adaptation to iron starvation. Overall, this work establishes ferrosomes as a new class of lipid-bounded iron storage organelles and sets the stage for studying ferrosome formation and structure in diverse microorganisms.

biorxiv microbiology 100-200-users 2020

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific lineages of the blast fungus, bioRxiv, 2020-01-12

AbstractSupernumerary mini-chromosomes–a unique type of genomic structural variation–have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae, mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the polyketide synthase Ace1 and the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes independently emerge from structural rearrangements of core-chromosomes and might contribute to adaptive evolution of the blast fungus.Author summaryThe genomes of plant pathogens often exhibit an architecture that facilitates high rates of dynamic rearrangements and genetic diversification in virulence associated regions. These regions, which tend to be gene sparse and repeat rich, are thought to serve as a cradle for adaptive evolution. Supernumerary chromosomes, i.e. chromosomes that are only present in some but not all individuals of a species, are a special type of structural variation that have been observed in plants, animals, and fungi. Here we identified and studied supernumerary mini-chromosomes in the blast fungus Magnaporthe oryzae, a pathogen that causes some of the most destructive plant diseases. We found that rice, foxtail millet and goosegrass isolates of this pathogen contain mini-chromosomes with distinct sequence composition. All mini-chromosomes are rich in repetitive genetic elements and have lower gene densities than core-chromosomes. Further, we identified virulence-related genes on the mini-chromosome of the rice isolate. We observed large-scale genomic rearrangements around these loci, indicative of a role of mini-chromosomes in facilitating genome dynamics. Taken together, our results indicate that mini-chromosomes facilitate genome rearrangements and possibly adaptive evolution of the blast fungus.

biorxiv genomics 100-200-users 2020

Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2, bioRxiv, 2020-01-12

AbstractSingle cell transcriptome analysis elucidates facets of cell biology that have been previously out of reach. However, the high-throughput analysis of thousands of single cell transcriptomes has been limited by sample preparation and sequencing technology. High-throughput single cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5’ or 3’ end. These cDNA pools are currently analyzed by short read Illumina sequencing which can identify the cellular origin of a transcript and what gene it was transcribed from. However, these methods fail to retrieve isoform information. In principle, cDNA pools prepared using these approaches can be analyzed with Pacific Biosciences and Oxford Nanopore long-read sequencers to retrieve isoform information but all current implementations rely heavily on Illumina short-reads for the analysis in addition to long reads. Here, we used R2C2 to sequence and demultiplex 9 million full-length cDNA molecules generated by the 10X Chromium platform from ∼3000 peripheral blood mononuclear cells (PBMCs). We used these reads to – independent from Illumina data – cluster cells into B cells, T cells, and Monocytes and generate isoform-level transcriptomes for these cell-types. We also generated isoform-level transcriptomes for all single cells and used this information to identify a wide range of isoform diversity between genes. Finally, we also designed a computational workflow to extract paired adaptive immune receptor – T cell receptor and B cell receptor (TCR and BCR) –sequences unique to each T and B cell. This work represents a new, simple, and powerful approach that –using a single sequencing method – can extract an unprecedented amount of information from thousands of single cells.

biorxiv genomics 100-200-users 2020