Phylogenomic analysis of Cladochytriales (Chytridiomycota): a new approach to the zoosporic fungi

Abstract

The traditional morphologic classification of the Chytridiomycota have been changing due to the availability of DNA sequences and investigation of differences in zoospore ultrastructural characters. Currently this phylum contains nine orders, with Rhizophydiales, Chytridiales and Cladochytriales representing the most diverse. The first two were intensively investigated in recent years in relation to the phylogeny of ribosomal genes, systematics and zoospore ultrastructure, for which new families, genera and species were proposed. Contrarily, Cladochytriales has been poorly studied and sparse modifications were performed since its preposition. Recently, a new approach has been used to study the phylogenetic relationships in the Fungi kingdom, which are based on genomic data. It has many advantages compared to the traditional genes methods, which are restricted to small fragments of ribosomal and mitochondrial DNA, since this evaluate the evolutionary relationship based on the complete genome of the organisms. Differently from Ascomycota, Basidiomycota e Zygomycota sensu Moreau, to which the genomic analyses have broad influenced our comprehension of the phylogeny and evolutionary process, this approach was not performed to Chytridiomycota groups. This gap in knowledge of the Chytridiomycota evolution results in a poor comprehension of the homologies of several essential traits in the evolutionary history of Fungi. Considering that, we are proposing to sequence the complete genome of Cladochytriales members deposited in MICH Herbarium, aiming to answer the hypotheses of the original proposal, origin of catenulation and polyphilia of some genera. Thus, 21 taxa deposited in MICH Herbarium were selected to have their genome sequenced since that represent the type species of the genera, possess a wide geographic distribution and/or indefinite phylogenetic placement. Strains of the target species will be reactivated and inoculated in PmTG and CMAg solid culture media to have their genomic DNA extracted. Then, multiple displacement amplifications (MDA) will be prepared with MDA master mix and the successful ones will be diluted for quality control and purity by PCR analysis. Upon confirmation of the target species, each MDA reaction will be prepared as a separate library through Nextera XT Library Prep kit. Independent libraries will be sequenced at the Joint Genome Institute (JGI) and preliminary quality control of the raw readings will be performed with fastqc 0.11.5. The libraries for each individual species will be co-assembled in SPAdes 3.10.0 and contigs less than 1000 bp will be removed using a publicly available script. Considering that assemblies will probably be metagenomes, we will use three approaches to metagenomic sequence binning in order to sort the target genome from potential contaminations. Each assembly will be binned using ESOM 1.1, GC-coverage blob plots, and the combination of relative synonymous codon usage (RSCU) and ClaMS. The final genome will only include contigs if at least two methods identified them as part of the target genome. The final tree will be inferred with RAxML 8.2.8 using the model of evolution determined automatically and bootstrap of 500 pseudoreplicates. A greedy consensus tree will be constructed with ASTRAL 5.6.1 using the individual gene trees. This proposal represents an unprecedented approach to sequence the genome of representatives of this order, extending the genomic data availability for Fungi, in special to the basal lineages. (AU)