Genome ofSpea multiplicata, a Rapidly Developing, Phenotypically Plastic, and Desert-Adapted Spadefoot Toad

Abstract

Frogs and toads (anurans) are widely used to study many biological processes. Yet, few anuran genomes have been sequenced, limiting research on these organisms. Here, we produce a draft genome for the Mexican spadefoot toad, Spea multiplicata, which is a member of an unsequenced anuran clade. Atypically for amphibians, spadefoots inhabit deserts. Consequently, they possess many unique adaptations, including rapid growth and development, prolonged dormancy, phenotypic (developmental) plasticity, and adaptive, interspecies hybridization. We assembled and annotated a 1.07 Gb Sp. multiplicata genome containing 19,639 genes. By comparing this sequence to other available anuran genomes, we found gene amplifications in the gene families of nodal, hyas3, and zp3 in spadefoots, and obtained evidence that anuran genome size differences are partially driven by variability in intergenic DNA content. We also used the genome to identify genes experiencing positive selection and to study gene expression levels in spadefoot hybrids relative to their pure-species parents. Completion of the Sp. multiplicata genome advances efforts to determine the genetic bases of spadefoots' unique adaptations and enhances comparative genomic research in anurans.

Transcriptomic bases of a polyphenism

“…To measure gene expression, we followed the general procedure described by Seidl et al (2019) for 3' RNA‐seq data. We began by trimming the 3' RNA‐seq reads to remove adapter and poly‐A contamination using BBDuk (B. Bushnell, https://sourceforge.net/projects/bbmap) with the parameters: ktrim=r k = 23 mink=11 hdist=1 tpe tbo qtrim=r trimq=6 ftl=12 maq=6.…”

“…We began by trimming the 3' RNA‐seq reads to remove adapter and poly‐A contamination using BBDuk (B. Bushnell, https://sourceforge.net/projects/bbmap) with the parameters: ktrim=r k = 23 mink=11 hdist=1 tpe tbo qtrim=r trimq=6 ftl=12 maq=6. Individual reads were then mapped to the S. multiplicata genome (Seidl et al, 2019) using STAR aligner (Dobin et al, 2013) with default parameters for the Lexogen Quantseq kit. We used bedtools genomecov (Quinlan & Hall, 2010) to generate bed coverage files at all nucleotide positions and performed peak discovery by using the “findpeaks” function in the R package “pracma” (Borchers, 2019) with the following parameters: nups=1, ndowns=1, zero="0," peakpat=NULL, minpeakheight=500,minpeakdistance=3000, threshold=0, sortstr=FALSE.…”

“…We then extracted coverage of each peak ±25 bp for each individual using the bedtools coverage tool and took the average coverage over the 50 bp window surrounding the peak (rounded to the nearest whole number) as our measure of gene expression. Because there is currently insufficient data on 3'UTR location for S. multiplicata genes (Seidl et al, 2019), to assign gene identities to our peaks, we used bedtools closest (Quinlan & Hall, 2010) to match our peak to the nearest known S. multiplicata genes following Seidl et al (2019). The best match resulting from a BLASTx of these S. multiplicata genes against the Uniprot tetrapod database was used as each gene's identifier.…”

Comparative genomics of Glandirana rugosa using unsupervised AI reveals a high CG frequency

“… The phylogenetic relationship of the frogs is derived from the tree on AmphibiaWeb ( https://amphibiaweb.org ), and the divergence time scale shown at the bottom is based on times estimated using TIMETREE ( http://www.timetree.org ). The genome size of each frog was estimated in this study ( Glandirana rugosa ) and in previous studies of other groups: Denton et al (2018) Preprint ( Pyxicephalus adspersu s), Seidl et al (2019) ( Spea multiplicata ), Li et al (2019) ( Leptobrachium leishanense ), Hellsten et al (2010) ( Xenopus tropicalis ), and Session et al (2016) ( Xenopus laevis ). The assembled genome size of Rhinella marina was estimated to be in the range of 1.98–2.38 Gb ( Edwards et al, 2018 ), and the average of the range is shown here.…”

Identification of candidate loci for adaptive phenotypic plasticity in natural populations of spadefoot toads

“…Loci that were identified as significantly different in each contrast (Dryad dataset: https://doi.org/10.5061/dryad.h9w0vt4fj) were then mapped to the S. multiplicata reference genome (Seidl et al., 2019) using the “very sensitive local” setting in Bowtie2 (Langmead & Salzberg, 2012). Following mapping, we identified candidate genes for phenotype and/or plasticity as those for which an outlier locus occurred within the gene itself or where an outlier mapped within 10 kb upstream or downstream of the gene.…”