Phylogenetic and Phylogenomic Definition of Rhizopus Species

Abstract

Phylogenomic approaches have the potential to improve confidence about the inter-relationships of species in the order Mucorales within the fungal tree of life. Rhizopus species are especially important as plant and animal pathogens and bioindustrial fermenters for food and metabolite production. A dataset of 192 orthologous genes was used to construct a phylogenetic tree of 21 Rhizopus strains, classified into four species isolated from habitats of industrial, medical and environmental importance. The phylogeny indicates that the genus Rhizopus consists of three major clades, with R. microsporus as the basal species and the sister lineage to R. stolonifer and two closely related species R. arrhizus and R. delemar A comparative analysis of the mating type locus across Rhizopus reveals that its structure is flexible even between different species in the same genus, but shows similarities between Rhizopus and other mucoralean fungi. The topology of single-gene phylogenies built for two genes involved in mating is similar to the phylogenomic tree. Comparison of the total length of the genome assemblies showed that genome size varies by as much as threefold within a species and is driven by changes in transposable element copy numbers and genome duplications.

Keywords: genome duplication; orthologs; sexual reproduction; transposons; zygomycete.

Figure 1

Morphology of Rhizopus species. (A) R. delemar CBS 390.34 colony on MEA after three days of cultivation at 30 °C. (B) Intact and germinating sporangiospores of R. delemar CBS 390.34. Arrows indicate spores of different sizes. Scale bar = 10 μm. (C) Zygospores with unequal suspensors. C1, C2, and C3 show R. microsporus CBS 344.29 azygospores; these were formed in the absence of a mating partner and are morphologically different from typical zygospores because they are smaller in size and have a single suspensor. Scale bar = 10 μm. (D) R. microsporus CBS 700.68 sporangiophore with columella. Scale bar = 10 μm. (E) R. arrhizus var. arrhizus CBS 330.53 sporangiospore release and columella. Scale bar = 10 μm. (F) Sporangiophore, rhizoids, and pigmented hyphae of R. arrhizus var. arrhizus CBS 330.53. Scale bar = 10 μm, (G) R. stolonifer CBS 926.87 stolons. Scale bar = 50 μm. (H) R. stolonifer CBS 926.87 empty sporangiophore. Scale bar = 50 μm.

Figure 2

Genome-based maximum likelihood phylogeny and parsimony phylogeny based on non-molecular characters. (A) Rooted maximum likelihood tree of the genus Rhizopus based on 192 orthologous genes. Misidentified strains are indicated in quotes: “Mucor racemosus” B9645 = R. microsporus B9645 and “Mucor ramosissimus” 97-1192 = R. arrhizus 97-1192. Genome size is indicated in bold after the strain name. (B) Unrooted parsimony tree of 16 non-molecular (14 micromorphological and two ecological) characters. Morphological and physiological data for different strains of the same species are consolidated in the tree except for those strains that differ in at least one character. Thick branches denote statistically significant bootstrap values.

Figure 3

Genome size and repeat content in Rhizopus genomes. Colored boxes are used to highlight the species identity of each strain.

Figure 4

Number of transposons with ORFs typical of LTR/LINE/DNA/Helitron elements. Colored boxes are used to highlight the species identity of each strain.

Figure 5

The structure of the mating type (sex) loci in representative strains of the four Rhizopus species and in the outgroup Mucor circinelloides (NCBI sequence accession numbers are HQ450311-12, HQ450315-16 (R. arrhizus), HQ450313 (R. delemar), MG967658 (R. stolonifer), MG967659-60 (R. microsporus var. azygosporus), HM565940-41 (M. circinelloides). Note that the structure of the mating type locus is shown for R. arrhizus and R. delemar together; these two closely related species share a similar arrangement in the mating type locus. The color-coding for each gene is listed above the M. circinelloides homologs, except for arbA (which is listed above the R. stolonifer graphic). Red arrows indicate sexP and orange arrows indicate sexM genes. Genes depicted in white are genes that were not previously found physically linked with the sex loci in Mucorales species. The gray bars above the diagrams indicate the idiomorphic regions that differ between (+) and (–) strains. Genome sequence is available for only a (+) strain of R. stolonifer, so the extent of the idiomorphic region, and the nature of the (–) form are unknown. There is a remnant of a transposable element (ψTn) between the arbA and sexP genes in R. stolonifer. For R. azygosporus, both sexM and sexP idiomorphic sequences are found in the same strain. Dashes indicate spacing of 1 kb.