Species recognition and clinical relevance of the zygomycetous genus Lichtheimia (syn. Absidia pro parte, Mycocladus)

Abstract

The zygomycete genus Lichtheimia (syn. Absidia pro parte, Mycocladus) consists of saprotrophic fungi inhabiting soil or dead plant material. Lichtheimia corymbifera (syn. Absidia corymbifera, Mycocladus corymbifer) and Lichtheimia ramosa (syn. Absidia ramosa, Mycocladus ramosus) may cause fulminant infections in patients with impaired immunity. The present study investigated the species boundaries in Lichtheimia using genealogical concordance phylogenetic species recognition (by comparison of the genealogies of the internal transcribed spacer [ITS] sequence, the D1/D2 region of the large subunit [LSU], and actin), biological species recognition by mating tests, as well as morphological and physiological characteristics. The three molecular markers used were selected by evaluating the polymorphisms and paralogies of several loci, including those for beta-tubulin, translation elongation factor 1alpha, the two largest subunits of the RNA polymerase II (RPB1 and RPB2), the mitochondrial cytochrome c oxidase subunit I (COI), and the mitochondrial small-subunit (mtSSU) rDNA, among four strains belonging to different putative species. Comparing the genealogies of the ITS, LSU, and actin genes, we recognized seven phylogenetic species. However, mating tests did not show intrinsic reproductive barriers for two pairs of the phylogenetic species. Therefore, we regard five species in Lichtheima to be confirmed: Lichtheimia corymbifera, L. ornata comb. nov., L. ramosa, L. hyalospora, and L. sphaerocystis sp. nov. Only the first three species seem to have clinical relevance. Lichtheimia blakesleeana is reduced to a synonym of Lichtheimia hyalospora. We provide a detailed description of Lichtheimia sphaerocystis sp. nov. and a key for the identification of all accepted species identified in the present study on the basis of their morphological traits and growth at different temperatures.

FIG. 1.

Maximum parsimony phylograms of three different markers: ITS (a), actin (b) (the small-scale tree includes outparalog I [blue frame] and outparalog II [red frame]; the large-scale tree represents only outparalog I [blue frame in the small-scale tree]), and the D1/D2 region of the LSU (c). Branches with bootstrap values in the maximum parsimony analysis of 85 or higher are printed in boldface. Branch support values are indicated by the numbers near the branches (maximum parsimony bootstrap proportions/Bayesian posterior probabilities) but are given only for decisive branches. Strains forming a well-supported group in the ITS phylogram are marked in a certain color. The same color is also used for each strain in the LSU and the actin phylograms. In the ITS phylogram, clinical strains are underlined in red; names are given only for type strains. For each strain of L. ramosa, L. corymbifera, and L. ornata, the predominant spore shape is mapped on the ITS phylogram: white/spherical, subspherical to broadly ellipsoidal, subspherical predominant; light gray/broadly ellipsoidal, subspherical to broadly ellipsoidal; medium gray/broadly ellipsoidal, subspherical to broadly ellipsoidal, with broadly ellipsoidal being predominant; dark gray/ellipsoidal, broadly ellipsoidal to ellipsoidal; black/ellipsoidal, ellipsoidal to broadly cylindrical. The respective numbers of clones (c) are given after the strain numbers. Actin sequences belonging to the single-spore cultures obtained in this study are indicated by ss. T, type strain, NT, neotype strain.

FIG. 2.

Results of 73 intraspecific and 95 interspecific matings in Lichtheimia. X, no mating test was performed; white field, no zygospore formation; large gray field, formation of large dark red-brown zygospores, with less than 10 zygospores being formed; small gray field, formation of smaller orange brown zygospores, with less than 10 zygospores being formed; large black field, formation of large dark red-brown zygospores, with more than 10 zygospores being formed; small black field, formation of smaller orange-brown zygospores, with more than 10 zygospores being formed. NT, neotype strain; T, type strain.

FIG. 3.

Zygospores of Lichtheimia. (a to c) Intraspecific matings: L. blakesleeana CBS 100.28 × CBS 102.36 (a), L. ramosa CBS 124198 × CBS 100.24 (b), and L. ramosa CBS 271.65 × CBS 223.78 (c). (d to f) Interspecific matings: L. blakesleeana CBS 100.28 × L. ornata CBS 958.68 (d), L. ramosa CBS 124198 × L. ornata CBS 958.68 (e), and L. blakesleeana CBS 100.36 × L. ramosa CBS 100.49 (f). Scale bar, 50 μm.

FIG. 4.

Spore shape in Lichtheimia corymbifera (a and c) and L. ramosa (b and d): subglobse to broadly ellipsoidal shape of L. corymbifera (strain CBS 429.75, neotype strain) (a), ellipsoidal to cylindrical shape of L. ramosa (strain CBS 582.65, neotype strain) (b), predominantly broadly ellipsoidal shape in L. corymbifera (strain CBS 100.31) (c), and predominantly broadly ellipsoidal shape in L. ramosa (strain CBS 112528) (d).

FIG. 5.

Mean growth rate (mm/h) and standard deviations of Lichtheimia species at eight different temperatures.

FIG. 6.

Lichtheimia sphaerocystis CBS 420.70^T. (a) Aerial hypha-bearing sporangiophores (scale bar, 25 μm); (b to e) sporangiophores with columella (scale bar, 40 μm); (f) sporangiospores (scale bar, 20 μm); (g) mature giant cell; (h) young giant cell, strain CBS 648.78; (i) mature giant cell. (g to i) Scale bar, 40 μm.

FIG. 7.

Lichtheimia sphaerocystis. (a) Colony surface of strain CBS 420.70; (b) colony reverse of CBS 420.70; (c) colony surface of CBS 648.78 (predominant giant cell formation); (d) colony surface of CBS 647.78 (predominant sporangiospore formation); (e to g) CBS 647.78 sporangiophores (scale bar, 50 μm); (h to j) CBS 647.78 sporangiophores; (k to m) CBS 420.70, part of the sporangiophore with mature sporangium; (n to q) CBS 420.70 columella; (r) CBS 420.70 sporangiospores; (s to v) CBS 420.70, young (s and t) and mature (u and v) giant cells; (w) CBS 648.78 mature giant cell. (h to w) Scale bars, 20 μm.

FIG. 8.

Giant cells of Lichtheimia hyalospora strains CBS 102.36 (a) and CBS 100.36 (b) formed in PDA cultures. Scale bars, 100 μm.

FIG. 9.

Giant cells of Lichtheimia ornata formed in 2-week-old YEA cultures: younger giant cell formed by CBS 291.66 (a); mature giant cell formed by CNM-CM4978 (b). Scale bar, 100 μm.