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

Abstract

The Chytridiomycota representatives have been occurred on the planet for at least 400 million years, according the fossil data. They have cosmopolitan distribution and a crucial role in decomposition and parasitism process in the aquatic and terrestrial ecosystems, where they are frequently observed as parasites of algae, cyanobacteria, macrophytes, invertebrates, amphibians, fungi and oomycetes. The classification and phylogenetic relationships of its representatives have been undergone deep restructuration due to the availability of gene sequence data and differences in the zoospore ultrastructure. Currently, the phyllum Chytridiomycota contains nine orders being Rhizophydiales, Chytridiales and Cladochytriales the more representatives considering the number of species. Rhizophydiales and Chytridiales were intensively studied in recent years through analyses of ribosomal genes and zoospore ultrastructure, for which new families, genera and species were proposed. On the other hand, Cladochytriales has been poorly studied phylogenetically and sparse modifications have been performed since its preposition. A new approach, based on genomic data, has been used to study the phylogenetic relationships within Fungi kingdom. This approach has been transformed us understand of the phylogenetic relationships and evolutionary processes, specially to the Ascomycota, Basidiomycota and Zygomycota sensu Moreau. However, in Chytridiomycota only a few taxa have the genome sequenced. Considering that, the aim of this project is to sequence the complete genome of Cladochytriales representatives in order to test the hypotheses proposed. In addition, it represents a new approach to Chytridiomycota, contributing to expand the information about genomic data. Twenty-six taxa, deposited in the University of Michigan, University of Alabama and "Instituto de Botânica" culture collections, were selected for phylogenomic analysis based on its position as type species of the genera included within the order, present a wide geographic distribution and/or indefinite phylogenetic positioning. During the first six months, samples of water and soil will be collect monthly in the "Parque Estadual das Fontes do Ipiranga (PEFI)" and "Reserva Biológica de Paranapiacaba", in order to expand the availability of Cladochytriales taxa that could be potentially included in this study. Cladochytriales. The cultures selected as well as the species that will potentially be isolated in the study areas, will be reactivated and inoculated in broth culture media for genomic DNA extraction. After this procedure, multiple displacement amplification reactions (MDA) will be prepared with "MDA master mix" and the successful reactions will be diluted and checked about its quality and purity through PCR analysis. Each MDA reaction will be prepared as a separate library through the "Nextera XT Library Prep" kit. Independent libraries will be sequenced at the Joint Genome Institute (JGI) and assembled in SPAdes 3.10.0. As the data sets will be probably metagenomes, we will use three approach criteria for sequence categorization, in order to separate the target genome from possible contaminations. The gene trees will be inferred in RAxML 8.2.8, which will be manually traced in search of paralleling regions as well as possible contaminations. Markers will be concatenated using custom scripts and the final tree will be inferred with RAxML 8.2.8. Finally, a consensus tree will be constructed with ASTRAL 5.6.1, using the individual gene trees.