Genomic analysis of novel Yarrowia-like yeast symbionts associated with the carrion-feeding burying beetle Nicrophorus vespilloides

Abstract

Background: Mutualistic interactions with microbes can help insects adapt to extreme environments and unusual diets. An intriguing example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate carcasses. Its fungal microbiome is dominated by yeasts that potentially facilitate carcass utilization by producing digestive enzymes, eliminating cadaver-associated toxic volatiles (that would otherwise attract competitors), and releasing antimicrobials to sanitize the microenvironment. Some of these yeasts are closely related to the biotechnologically important species Yarrowia lipolytica.

Results: To investigate the roles of these Yarrowia-like yeast (YLY) strains in more detail, we selected five strains from two different phylogenetic clades for third-generation sequencing and genome analysis. The first clade, represented by strain B02, has a 20-Mb genome containing ~ 6400 predicted protein-coding genes. The second clade, represented by strain C11, has a 25-Mb genome containing ~ 6300 predicted protein-coding genes, and extensive intraspecific variability within the ITS-D1/D2 rDNA region commonly used for species assignments. Phenotypic microarray analysis revealed that both YLY strains were able to utilize a diverse range of carbon and nitrogen sources (including microbial metabolites associated with putrefaction), and can grow in environments with extreme pH and salt concentrations.

Conclusions: The genomic characterization of five yeast strains isolated from N. vespilloides resulted in the identification of strains potentially representing new YLY species. Given their abundance in the beetle hindgut, and dominant growth on beetle-prepared carcasses, the analysis of these strains has revealed the genetic basis of a potential symbiotic relationship between yeasts and burying beetles that facilitates carcass digestion and preservation.

Keywords: Carrion beetles; Detoxification; Digestion; Ephemeral resources; Metabolic profiling; Nicrophorus vespilloides; Transcriptomics; Yarrowia; rDNA variability.

Fig. 1

Sequence alignment of Yarrowia-like yeast genomes. Homologous regions are depicted in the same colors with connecting lines. Contig ends are indicated by black vertical lines. a Clade I genomes of strains C11 and E02. The comparison shows major sequence rearrangements between the two genomes. However, both genomes generally comprise the same building blocks. b Clade II genomes of strains B02, F05 and H10. All three genomes have an overall similar structure. No sequence rearrangements or additional sequences were detected

Fig. 2

Number of reciprocal best BLAST hits between subsets of the five Yarrowia-like yeast genomes. Plots were visualized using R package UpSetR [28] The core genome of all five YLY strains includes 5784 genes (90.3%). Clade I genomes share 410 genes and the clade II genomes share 581 genes. Aside from the core genome, no other subset of the diagram includes as many genes as the clade specific subsets. This indicates that genes in the clade I genomes C11 and E02 are more closely related to each other than the other genomes, which is also true for the three clade II genomes B02, F05 and H10. Furthermore, this comparison shows that clade I genomes (C11, E02) have more strain-specific genes than the clade II genomes (B02, F05, H10). For a detailed analysis of subsets of different YLY genomes also see Additional File: Fig. S3

Fig. 3

Intraspecific variable base positions within the ITS–D1/D2 regions of the single Yarrowia-like yeast genomes. Assemblies of the five genomes analyzed in this study contain different copy numbers of the ITS–D1/D2 regions (B02 = 3, C11 = 9, E02 = 9, F05 = 8, H10 = 1). To identify intraspecific variability among the rRNA copies, the genomes were analyzed separately without cross-species comparisons. Strains C11 and E02 showed high intraspecific variability of the assembled rRNA copies. In contrast, of the clade II strains B02, F05 and H10, only B02 displayed any intraspecific variability with three variable positions. Blue bars represent the sequence alignments of the ITS–D1/D2 region for each strain separately with the variable sequence positions marked as white bars. Numbers beneath the white bars are the numbers of adjacent variable positions. Positions of the primers that determine the ITS–D1/D2 region are shown as black arrows

Fig. 4

Phylogenetic tree of Yarrowia-like yeasts. The tree was constructed in EDGAR [31] based on the amino acid sequences of the “Yarrowia core genome” using FastTree 2.8.1 with default settings. The core genome includes the common set of genes shared by Y. yakushimensis, Y. porcina, Y. phangngaensis, Y. osloensis, Y. hollandica, Y. galli, Y. divulgata, Y. deformans, Y. bubula, Y. alimentaria, Yarrowia sp. JCM 30695, Yarrowia sp. JCM 30696, Yarrowia sp. JCM 30694, Y. keelungensis and 2 Y. lipolytica strains. This gene set is composed of 3542 genes. The five yeast strains we investigated are highlighted in bold. The scale displays substitutions per site. Local branch support values were computed with the Shimodaira-Hasegawa test [32]

Fig. 5

Schematic maps of Yarrowia-like yeast mitochondrial genomes. Blue arrows represent rRNA genes; red arrows represent protein-coding genes. Red rectangles and arrows connected by gray bars indicate genes with an exon-intron structure, where introns are gray. Shaded red arrows represent open reading frames not present in any of the other mitochondrial genomes. White arrows with red outline represent regions homologous to genes, but due to indels or SNPs these genes are not functional (marked with *). The end of each mitochondrial contig is indicated by a black bar. In case of splice variants, the longest version is displayed. Phylogenetic relation was calculated on the amino acid level based on the mitochondrial core genome using EDGAR and is indicated on the left. Lengths of the branches in this tree have no significance

Fig. 6

Substrate utilization assays using Biolog Phenotype Microarrays for strains C11 and B02. Black shading indicates utilization of the substrate based on threshold score values whereas gray shading indicates lack of utilization. Carbon sources (MicroPlates PM1 and PM2A) are indicated in red, nitrogen sources (MicroPlates PM3B) are indicated in blue, osmolytes (MicroPlates PM9 9) and pH conditions (MicroPlates PM10) are indicated in green

Fig. 7

Metabolic pathways of putrescine degradation and uric acid recycling in strains C11 and B02. Green boxes represent EC numbers of enzymes annotated for strains C11, B02 and Y. lipolytica. White boxes represent EC numbers of enzymes that are not annotated. EC 6.3.4.5* is annotated for strains C11 and B02 but is not present in Y. lipolytica. This figure was constructed using information provided by KEGG pathway maps 00220, 00230, 00330 and 00410 [38]