Fig 2: Pulsed-field gel electrophoresis (PFGE) analysis of samples from patients in Hong Kong showing 3 Elizabethkingia anophelis strains compared with reference Elizabethkingia isolates. A) PFGE performed by using CHEF Mapper XA system (Bio-Rad, Hercules, CA, USA) and restriction endonuclease XbaI shows that isolates from patient 2 and patient 3 are indistinguishable, wheras isolates from patient 1 possess distinct PFGE patterns. Lane 1, E. anophelis strain HKU37 from uterine swab specimen of patient 2; lane 2, placental swab specimen from patient 2; lane 3, E. anophelis strain HKU38 from blood of patient 3; lane 4, cerebrospinal fluid from patient 3; lane 5, E. anophelis strain HKU36 from blood of patient 1; lane 6, cerebrospinal fluid from patient 1; lane 7, E. anophelis type strain R26T; lane 8, E. meningoseptica type strain ATCC 13253T; lane 9, E. miricola type strain LMG22470T. B) Dendrogram constructed with PFGE data by similarity and clustering analysis using the Dice coefficient (1% tolerance and 0.5% optimization) and the unweighted pair-group method using average linkages with GelCompar II (Applied Maths, Sint-Martens-Latem, Belgium).

Fig 3: PFGE analysis of the yeast mtDNAs. The whole-cell DNA samples were separated by PFGE using a CHEF Mapper XA Chiller System (Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1—C. viswanathii CBS 4024; lane 2—C. sojae CBS 7871; lane 3—C. maltosa CBS 5611; lane 4—C. neerlandica NRRL Y-27057; lane 5—C. alai NRRL Y-27739; lane 6—C. labiduridarum NRRL Y-27940; lane 7—C. frijolesensis NRRL Y-48060; lane 8—C. subhashii CBS 10753; lane 9—C. jiufengensis CBS 10846; lane 10—C. albicans CBS 562. Note that three discrete bands migrating in the region <50 kb represent three linear mitochondrial chromosomes in C. labiduridarum and C. frijolesensis (lanes 6 and 7). In contrast, four bands in C. alai (lane 5) do not hybridize with mtDNA probes and correspond to linear DNA plasmids (data not shown).

Fig 4: (A) Nuclear chromosomes of C. verbasci, colored based on their homology with C. parapsilosis strain CLIB214 (17). Local alignments between the two genomes were computed using LAST v.830 (lastal with option -E1e-10, followed by last-split) (18) and visualized using the ggplot2 v.3.3.6 library in R (19). (B) Electrophoretic karyotype of C. verbasci (C.v.), analyzed using a CHEF Mapper XA Chiller system (Bio-Rad), with a 120° angle between the electric fields, at the following settings: 120-s pulses for 20 h, followed by 240-s pulses for 28 h at 4 V/cm in a 0.8% agarose gel and 0.5× TBE buffer (45 mM Tris-borate, 1 mM EDTA) at 14°C. The chromosomes of C. parapsilosis strain CLIB214 (C.p.) were used as size markers (for more details, see reference 17). Asterisks indicate the chromosomes containing rDNA repeats. mtDNA, mitochondrial DNA. (C) Phylogenetic tree of the Lodderomyces clade species, constructed using the BUSCO_phylogenomics pipeline (20) from 2,120 single-copy orthologs present in at least 50% of the genome assemblies (options: –psc 50 –supermatrix) identified using BUSCO v.5.1.2 (21). The G+C contents of the nuclear and mitochondrial DNAs are shown. Asterisks indicate that the nuclear and mitochondrial DNA sequences were obtained from different strains of the species; n/a indicates that the sequence of mitochondrial DNA is not available. The inset illustrates a colony of C. verbasci grown for 7 days on a YPD plate.