title: Phylogenetic analysis of phenotypically characterized Cryptococcus laurentii isolates reveals high frequency of cryptic species.
abstract: BACKGROUND
  Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States.
  
  
  METHODS
  In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region.
  
  
  RESULTS
  BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99-100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified.
  
  
  CONCLUSIONS
  Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.
authors.affiliation: Department of Infectious and Parasitic Diseases, Triangulo Mineiro Federal University, Uberaba, Minas Gerais, Brazil.
authors.forename: Kennio
authors.initials: K
authors.lastname: Ferreira-Paim
authors.affiliation: Department of Infectious and Parasitic Diseases, Triangulo Mineiro Federal University, Uberaba, Minas Gerais, Brazil.
authors.forename: Thatiana Bragine
authors.initials: TB
authors.lastname: Ferreira
authors.affiliation: Department of Infectious and Parasitic Diseases, Triangulo Mineiro Federal University, Uberaba, Minas Gerais, Brazil.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Andrade-Silva
authors.affiliation: Department of Infectious and Parasitic Diseases, Triangulo Mineiro Federal University, Uberaba, Minas Gerais, Brazil.
authors.forename: Delio Jose
authors.initials: DJ
authors.lastname: Mora
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Springer
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America; Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America; Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Heitman
authors.affiliation: Department of Infectious and Parasitic Diseases, Triangulo Mineiro Federal University, Uberaba, Minas Gerais, Brazil.
authors.forename: Fernanda Machado
authors.initials: FM
authors.lastname: Fonseca
authors.affiliation: Public Health Reference Laboratory, Adolfo Lutz Institute, São Paulo, São Paulo, Brazil.
authors.forename: Dulcilena
authors.initials: D
authors.lastname: Matos
authors.affiliation: Public Health Reference Laboratory, Adolfo Lutz Institute, São Paulo, São Paulo, Brazil.
authors.forename: Márcia Souza Carvalho
authors.initials: MS
authors.lastname: Melhem
authors.affiliation: Department of Infectious and Parasitic Diseases, Triangulo Mineiro Federal University, Uberaba, Minas Gerais, Brazil.
authors.forename: Mario León
authors.initials: ML
authors.lastname: Silva-Vergara
mesh_terms: D003454:Cryptococcus / Q000145:classification*; D006239:Haplotypes; D010802:Phylogeny; D013045:Species Specificity