Medical Mycology Advance Access published February 11, 2016

Medical Mycology, 2016, 0, 1–15 doi: 10.1093/mmy/myw004 Advance Access Publication Date: 0 2016 Original Article

Original Article Phylogenetic analysis of species using DNA sequence polymorphism in calmodulin gene Bahram Ahmadi1,2, Hossein Mirhendi3,∗, Koichi Makimura4, G. Sybren de Hoog5, Mohammad Reza Shidfar2, 6 2 Sadegh Nouripour-Sisakht and Niloofar Jalalizand Downloaded from

1Department of Microbiology and Parasitology, School of Para-Medicine, Bushehr University of Medical Sciences, Bushehr, Iran, 2Departments of Medical Parasitology & Mycology, School of Public Health; National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran, 3Departments

of Medical Parasitology & Mycology, School of Medicine, Isfahan University of Medical Sciences, http://mmy.oxfordjournals.org/ Isfahan, Iran, 4Teikyo University Institute of Medical Mycology and Genome Research Center, Tokyo, Japan, 5Fungal Biodiversity Center, Institute of the Royal Netherlands, Academy of Arts and Sciences, Centraalbureau voor Schimmelcultures-KNAW, Utrecht, The Netherlands and 6Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran

∗To whom correspondence should be addressed. Hossein Mirhendi, Professor, Department of Medical Parasitology and Mycology, School of Medicine; Isfahan University of Medical Sciences, Isfahan, Iran. Tel/Fax: +00982188951392; E-mail: [email protected]. by guest on February 12, 2016 Received 23 October 2015; Revised 23 December 2015; Accepted 5 January 2016

Abstract Use of phylogenetic species concepts based on rDNA internal transcribe spacer (ITS) regions have improved the of dermatophyte species; however, confirmation and refinement using other genes are needed. Since the calmodulin gene has not been systematically used in dermatophyte taxonomy, we evaluated its intra- and interspecies sequence variation as well as its application in identification, phylogenetic analysis, and taxonomy of 202 strains of 29 dermatophyte species. A set of primers was designed and optimized to amplify the target followed by bilateral sequencing. Using pairwise nu- cleotide comparisons, a mean similarity of 81% was observed among 29 dermatophyte species, with inter-species diversity ranging from 0 to 200 nucleotides (nt). Intraspecies nt differences were found within strains of interdigitale, Arthroderma simii, T. rubrum and A. vanbreuseghemii, while T. tonsurans, T. violaceum, floccosum, canis, M. audouinii, M. cookei, M. racemosum, M. gypseum, T. mentagrophytes, T schoenleinii, and A. benhamiae were conserved. Strains of E. floc- cosum/M. racemosum/M. cookei, A. obtosum/A. gertleri, T. tonsurans/T. equinum and a genotype of T. interdigitale had identical calmodulin sequences. For the majority of the species, tree topology obtained for calmodulin gene showed a congruence with coding and non-coding regions including ITS, BT2,andTef-1α. Compared with the phylogenetic

C The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. 1 All rights reserved. For permissions, please e-mail: [email protected] 2 Medical Mycology, 2016, Vol. 00, No. 00 tree derived from ITS, BT2,andTef-1α genes, some species such as E. floccosum and A. gertleri took relatively remote positions. Here, characterization and obtained dendro- gram of calmodulin gene on a broad range of dermatophyte species provide a basis for further discovery of relationships between species. Studies of other loci are necessary to confirm the results.

Key words: , Calmodulin gene, Phylogenetics.

Introduction and phenotypically separated Trichophyton species.20,32,33 Annually, millions of humans and animals are infected Moreover, current advances in molecular taxonomy and in- by superficial fungal infections. As specialized filamentous sights into mating discovered that Trichophyton mentagro- fungi, dermatophytes have a specific ability to digest and phytes is a complex of anthropophilic and zoophilic species grow on keratinized host structures such as skin, nails, that produce different teleomorphs, leading to a confusion 34,35 and hair, causing the vast majority of mycotic infections.1,2 with regard to species denomination. Based on their major natural predilection sites, dermato- Regardless of the various advantages of ITS as the pri- phyte species are classified into anthropophilic, geophilic, mary genetic marker of dermatophyte identification, ad- Downloaded from and zoophilic groups. The asexual forms (anamorphs) of ditional verification and refinement using other genes is dermatophytes are classified into Trichophyton, Epidermo- critical to organizing sequence-based and classical species phyton,andMicrosporum, and their sexual forms (teleo- concepts. Since the calmodulin gene has not been systemati- morphs) are members of the genus Arthroderma in the sub- cally used in dermatophyte taxonomy, in this study, our aim http://mmy.oxfordjournals.org/ phylum Ascomycotina.1,3 was to evaluate nucleotide sequence analysis of calmodulin Diagnosis of dermatophyte infections is essential for ap- gene as a new genetic marker for a subset of dermatophyte propriate therapy because of the length of treat- species, as well as to assess its application with regard to ment, potential side effects of the drugs, and their high identification, phylogenetic analysis, and taxonomy stud- cost. Moreover, having information on zoophilic or an- ies based on intra- and interspecies variation. Several ref- thropophilic sources of the causative dermatophyte agent erence strains and clinical isolates including a wide range may allow prophylactic measures such as treatment of both of common and rare pathogenic species were used for this 4 purpose. animal or human reservoirs. Precise definition and clas- by guest on February 12, 2016 sification of microorganisms including bacteria, parasites, viruses, and fungi have always been of great relevance to Materials and methods taxonomic identity, phylogenetic analysis, epidemiology, and clinical microbiology.5 Reference strains and clinical isolates To resolve the evolutionary relationships between A total of 202 strains of 29 dermatophyte species com- dermatophytes, nucleic acid-based methods have been prising 60 reference strains and 142 clinical isolates were used since early 1980 s. Consequently, several ge- used for sequence analysis of partial calmodulin gene nomic and molecular researches for characterization (Table 1). The reference strains were obtained from Cen- of dermatophytes has revealed a homogeneous group traalbureau voor Schimmelcultures (CBS), Utrecht, the of species with very low genetic diversity in com- Netherlands, and Teikyo University, Institute of Medical parison with overall high phenotypic heterogeneity in Mycology (TIMM), Tokyo, Japan. The clinical isolates dermatophytes.5–8 were collected from a variety of specimens, including skin, The following DNA fragments have been noticed as nail, and hair submitted to two medical mycology labo- the main dermatophyte genetic markers: ribosomal DNA ratories in Tehran, Iran. The clinical isolates were identi- (rDNA) regions,9–13 chitin synthase 1 (CHS1),14–17 DNA fied to species level by an already described PCR-restriction topoisomerase II (TOP-II),18,19 beta tubulin (BT2),20,21 fragment length polymorphism (PCR-RFLP) method9 and and translation elongation factor 1-α (Tef-1α) genes.20,22 in some cases based on ITS-sequencing. Species names Phylogenetic analysis and identification of dermatophytes were determined according to Graser et al.35 Hence, T. based on sequencing of the internal transcribed spacer (ITS) mentagrophytes strains CBS 435.73, NBRC 5809, CBS regions of rDNA has proved to be useful as a gold standard 119445, and NBRC5974, were all identified as T. interdig- method.23–31 Only a small number of nucleotide differences itale by ITS-PCR-RFLP analysis. Also, A. gypseum strain in the ITS regions have been observed in several ecologically CBS 161.69 were identified as A. incurvatum. Ahmadi et al. 3

Ta b l e 1 . Reference and clinical strains of dermatophytes used in this study for partial sequence analysis of the calmodulin gene. GenBank accession numbers, fragment size, and the range of intraspecies variations within the species, are shown.

Species (total number Length of calmodulin Range of intra-species of tested strains) Strains (accession numbers) sequence (bp) variation

A. benhamiae (4) Ci1947 (KM678214), Ji362 (KP781967), Ji363 (KP781968), 517 0 Ji364 (KP781969) T. concentricum (1) CBS 563.83 (KM387107) 517 – T. erinacei (1) CBS 344.79 (KM387155) 518 – T. eriotrephon (1) CBS 220.25 (KM387109) 518 – T. verrucosum (1) CBS 554.84 (KM387123) 518 – M. audouinii (2) Ji324 (KP781965), Ji325 (KP781966) 488 0 M. canis (9) CBS 130922 (KM387125), CBS 131110 (KM387126), CBS 488 0 130818 (KM387127) Ci 622 (KM387145), Ci 1335 (KM387146), Ci 675 (KM387147), Ci 9219 (KM387148), Ci987 (KM387149), Ci 9938 (KM387150) Downloaded from M. ferrugineum (1) TIMM 445.51 (KP781964) 488 – A. grubyi (1) CBS 243.66 (KM387098) 525 – A. simii (3) CBS 417.65 (KM387128), Ji 77009 (KP781974), Ji 77010 519 0–2

(KP781975) http://mmy.oxfordjournals.org/ T. mentagrophytes (2) CBS 318.56 (KM387110), CBS 101546 (KM387113) 518 0 T. schoenleinii (4) CBS 434.63 (KM387117), CBS 564.94 (KM387118), NBRC 518 0 8192 (KM387119), CBS 130812 (KM387262) A. vanbreuseghmii (3) Ji 405 (KP781970), TIMM 20066 (KP781971), Ji 1116 518 0–1 (KP781972) T. equinum (1) NBRC 31610 (KM387108) 518 – T. interdigitale (43) CBS 130816 (KM387156), CBS 130923 (KM387158), Ci 518–519 0–7 by guest on February 12, 2016 1188 (KM387159), Ci 1330 (KM387160), Ci 1425 (KM387161), Ci 1435 (KM387162), Ci 2076 (KM387163), Ci 2131 (KM387164), Ci 2412 (KM387165), Ci 2764 (KM387166), Ci 2824 (KM387167), Ci 283 (KM387168), Ci 2890 (KM387169), Ci 448 (KM387170), Ci 712 (KM387171), Ci 855 (KM387172), Ci 1184 (KM387173), Ci 1517 (KM387174), Ci 1542 (KM387175), Ci 1553 (KM387176), Ci 1603 (KM387177), Ci 1895 (KM387178), Ci 1904 (KM387179), Ci 1935 (KM387180), Ci 2185 (KM387181), Ci 2197 (KM387182), Ci 2223 (KM387183), Ci 2303 (KM387184), Ci 2311 (KM387185), Ci 2332 (KM387186), Ci 2382 (KM387187), Ci 2386 (KM387188), Ci 2519 (KM387189), Ci 3186 (KM387190), Ci 2142 (KM387191), Ci 2266 (KM387192), Ci 166 (KM387266), Ci 1746 (KM387277), Ci 1849 (KM387278), CBS 435.73 (KM387111), NBRC 5809 (KM387112), CBS 119445 (KM387114), NBRC 5974 (KM387157) T. tonsurans (12) CBS 120.65 (KM387120), CBS 270.66 (KM387121), CBS 518 0 109035 (KM387122), CBS 130924 (KM387263), NBRC 5928 (KM387264), CBS 130814 (KM387265), Ci 1852 (KM387267), Ci 2430 (KM387268), Ci 1186 (KM387269), Ci 2346 (KM387270), Ci 2347 (KM387271), Ci 2518 (KM387272) T. eboreum (1) CBS 117155 (KM678213) 530 – T. ajelloi (1) IFM 5326 (KP781973) 519 – M. fulvum (1) CBS 130934 (KM213524) 524 – 4 Medical Mycology, 2016, Vol. 00, No. 00

Ta b l e 1 . (Continued.)

Species (total number Length of calmodulin Range of intra-species of tested strains) Strains (accession numbers) sequence (bp) variation

M. gypseum (5) CBS 130820 (KM213525), CBS 258.61 (KM387151), NBRC 523 0 5948 (KM387152), CBS 130939 (KM387153), NBRC 8228 (KM387154) A. incurvatum (1) CBS 161.69 (KM387099) 511 – M. persicolor (1) NBRC 5975 (KM387104) 532 – A. obtusum (2) CBS 321.61 (KP781976), CBS 322.61 (KP781977) 520 0 A. gertleri (1) CBS 665.77 (KM387097) 520 – E. floccosum (20) Ci 1777 (KM213523), CBS 358.93 (KM387100), CBS 767.73 525 0 (KM387101), Ci 1804 (KM387129),Ci 2118 (KM387130), Ci 3094 (KM387131), Ci 625 (KM387132), Ci 629 (KM387133), Ci 679 (KM387134), Ci 699 (KM387135), Ci 943 (KM387136), Ci 1453 (KM387137),Ci 1789 (KM387138), Ci 1964 (KM387139), Ci 2086 (KM387140), Downloaded from Ci 2253 (KM387141), Ci 2300 (KM387142), Ci 2321 (KM387143), Ci 2340 (KM387144), Ci 1876 (KM387280) A. racemosum (2) CBS 423.74 (KM387105), CBS 130935 (KM387106) 525 0 A. cajetanum (2) CBS 228.58 (KM387096), NBRC 7862 (KM387103) 525 0

T. rubrum (72) CBS 288.86 (KM387115), CBS 100237 (KM387116), CBS 518–519 0–1 http://mmy.oxfordjournals.org/ 130825 (KM387193), NBRC 5467 (KM387194), NBRC 5808 (KM387195), CBS 130817 (KM387196) CBS 130808 (KM387197), CBS 130933 (KM387198), Ci 1199 (KM387199), Ci 1218 (KM387200), Ci 1297 (KM387201), Ci 1334 (KM387202), Ci 1388 (KM387203), Ci 1393 (KM387204), Ci 1452 (KM387205), Ci 1572 (KM387206), Ci 2034 (KM387207), Ci 2070 (KM387208), Ci 209 (KM387209), Ci 2106 (KM387210), Ci 2133 by guest on February 12, 2016 (KM387211), Ci 2191 (KM387212), Ci 262 (KM387213), Ci 2856 (KM387214), Ci 2880 (KM387215), Ci 2885 (KM387216), Ci 2887 (KM387217), Ci 2974 (KM387218), Ci 2984 (KM387219), Ci 529 (KM387220), Ci 664 (KM387221), Ci 9541 (KM387222), Ci 9609 (KM387223), Ci 9652 (KM387224), Ci 9965 (KM387225), Ci 1060 (KM387226), Ci 1432 (KM387227), Ci 1525 (KM387228), Ci 1782 (KM387229), Ci 1850 (KM387230), Ci 1951 (KM387231), Ci 1963 (KM387232), Ci 2019 (KM387233), Ci 2032 (KM387234), Ci 2077 (KM387235), Ci 2097 (KM387236), Ci 2170 (KM387237), Ci 2180 (KM387238), Ci 2183 (KM387239), Ci 2215 (KM387240), Ci 2216 (KM387241),Ci 2218 (KM387242), Ci 2219 (KM387243), Ci 2236 (KM387244), Ci 2241 (KM387245), Ci 2251 (KM387246),Ci 2254 (KM387247), Ci 2258 (KM387248), Ci 2262 (KM387249), Ci 2293 (KM387250), Ci 2308 (KM387251), Ci 2314 (KM387252),Ci 2454 (KM387253),Ci 304 (KM387254), Ci 3168 (KM387255), Ci 3173 (KM387256), Ci 3185 (KM387257), Ci 769 (KM387258), Ci 777 (KM387259), Ci 966 (KM387260), Ci 9744 (KM387261), Ci 1860 (KM387279) T. violaceum (4) NBRC 31064 (KM387273), CBS 319.31 (KM387274), CBS 518 0 459.61 (KM387275), Ci 2033 (KM387276)

Note: CBS, Centraalbureau voor Schimmelcultures; Ci, clinical isolate; Ji, Japanese isolate; NBRC, NITE Biological Resource Center, TIMM, Teikyo University Institute of Medical Mycology Ahmadi et al. 5

DNA extraction MEGA638 and Geneious (http://www.geneious.com) soft-  All fungal strains were cultured on Mycobiotic agar (Difco, wares as follows: CF1 5 -TGTCCGAGTACAAGGAAGC-    Detroit, MI, USA) and incubated at 27◦C for 7 days. 3 and CF2 5 -TTACAATCAATTCTGCCGTC-3 . PCR re- μ DNA was extracted and purified from fungal colonies actions contained 12.5 l of premix (Ampliqon, Denmark), μ μ as previously described36. Briefly, a small amount of the 2 L of DNA template, 0.5 M of each primer, and enough μ colonies was allocated to a 1.5-ml tube containing 300 μl water up to reach a final reaction volume of 25 l. Two neg- of lysis buffer (100 mM Tris-HCl, pH 7.5, 10 mM ative controls (water instead of fungal DNA) were added EDTA, 0.5% w/v SDS, 100 mM NaCl), 300 μl of phe- to each PCR. The reaction mixture was initially dena- ◦ nol/chloroform (1:1), and 300 μl of glass beads (0.5 mm in tured at 95 C for 5 min, followed by 35 cycles of 30 s ◦ ◦ ◦ diameter), vortexed for 5 min and centrifuged at 5,000 rpm at 94 C, 45 s at 58 C,and45sat72C, and a ter- ◦ for 5 min; the supernatant was transferred to a new tube minal extension step of 72 C for 5 min. For the strains and re-extracted with chloroform. DNA was precipitated that failed to amplify, a nested PCR was set up for suc- with an equal volume of 2-propanol and 0.1 volume of cessful amplification of the gene by using the degenerate   3 M sodium acetate (pH 5.2), kept at −20◦C for 20 min, primers CF1 5 -GCCGACTCTTTGACYGARGAR-3 and  39 and centrifuged at 10,000 rpm for 10 min. The pellet was CF4 5 -TTTYTGCATCATRAGYTGGAC-3 for the first washed with 300 μl of 70% ethanol, air dried, and eventu- round PCR and the above mentioned primers for the second Downloaded from ally, the DNA was resuspended in 50 μL of sterile distilled round. One microliter of the first PCR product was diluted μ water. 1:50, and 1 l of the dilution was added to reaction mixture as template. PCR products were separated by electrophore- sis on 1.5% agarose gels and visualized by staining with ethidium bromide (0.5 μg/ml) and photographed under UV Species identification by PCR-RFLP http://mmy.oxfordjournals.org/ irradiation. All 202 clinical and reference isolates were sub- jected to PCR amplification using the primers ITS1    (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 - Sequencing and phylogenetic analysis TCCTCCGCTTATTGATATGC-3).37 PCR reactions were For purification of PCR products, 50 μl ethanol was added prepared in final volumes of 25 μL, containing 12.5 μLof to 20 μl of each product, kept at -20◦C for 30 min, and cen- premix (Ampliqon, Denmark), 2 μl of DNA template, and trifuged at 12,000 rpm for 10 min. The pellet was air-dried μ

0.5 M of each forward and reverse primer. PCR cycling by guest on February 12, 2016 and resuspended in 25 μl of distilled water. PCR products conditions were as follows: 6 min initial pre-incubation were sequenced bilaterally using the primers CF1 and CF2 at 94◦C, followed by 35 cycles consisting of denaturation and the ABI PRISM BigDye Terminator Cycle Sequencing at 94◦C for 30 s, annealing at 58◦C for 30 s, and exten- Ready Reaction Kit (Applied Biosystems, Foster City, CA, sion at 72◦C for 1 min, with a final extension at 72◦Cfor USA), on an automated DNA sequencer (ABI PrismTM 10 min. All strains were identified to species level by PCR- 3730 Genetic Analyzer, Applied Biosystems), according to RFLP analysis as previously described.9 Digestion of PCR the manufacturer’s instructions. products was performed by incubating 8 μL of PCR product Forward and reverse sequences of each sample were sub- with 0.5 μl of the MvaI Fast digest (Fermentas Life jected to ClustalW pairwise alignment using Geneious and Sciences, Lithuania), 1.5 μl of 10X buffer, and 5 μlofwa- MEGA638 softwares and edited manually to improve align- ter at 37◦C for 20 min. PCR amplicons and RFLP-products ment accuracy. The consensus nucleotide sequence data were analyzed by agarose gel electrophoresis in TBE buffer determined in this study were deposited in the GenBank, (Tris 0.09 M, Boric acid 0.09 M, EDTA 2 mM) at 100 V under the accession numbers KM213523 - KM213525, for approximately 60 min using 1.5% and 2% agarose gels, KM387096 - KM387280, and KP781963 - KP781977 respectively. For species identification, the size of fragments (Table 1). generated by enzymatic digestion were compared with ref- Based on the quality of the sequencing, consensus se- erences RFLP profiles.9 quences derived from the forward and reverse sequences had different lengths, therefore, the common first and last points of the entire sequence were determined, and PCR for calmodulin gene amplification poorly aligned DNA sequences were removed. Final- For calmodulin gene amplification, after alignment and ized sequences were subject to BioEdit software version analysis of calmodulin gene sequences, a novel set of 7.0.540 for pairwise comparisons and multiple alignment pan-dermatophyte primers was designed manually with to determine similarities and differences in nucleotides. 6 Medical Mycology, 2016, Vol. 00, No. 00

Pairwise similarity values were calculated by dividing the calmodulin genotypes, T. interdigitale exhibited most intra- number of matching nucleotides by the total length of the species variability. The variation between M. gypseum and alignment. A. incurvatum was 56 nt, while the diversity within five Phylogenetic trees were constructed with unambiguously strains of M. gypseum was0nt. aligned sequences using the neighbor-joining (NJ) method A phylogenetic tree constructed for 29 representative with the Tamura-Nei parameter as substitution model as dermatophytes species is presented in Figure 2. A prelim- implemented in the MEGA6 program.38 The reliability of inary analysis using an alignment including all sequences internal branches was assessed using the bootstrap method made in this study gave a similar overall species topology with 1000 replicates. as in Figure 2 (results not shown). Sequence analysis of dermatophyte species revealed eight complexes: 1) Arthroderma vanbreuseghemii Complex, 2) Results Arthroderma simii Complex, 3) Arthroderma benhamiae To evaluate the applicability of using calmodulin gene se- Complex, 4) Arthroderma otae Complex, 5) Trichophyton quences for differentiation and phylogenetic studies of der- rubrum Complex, 6) M. fulvum and M. gypseum Com- matophyte species, a part of the gene was successfully am- plex, 7) M. racemosum and M. cookei Complex, and 8) A. plified for 202 strains. The sizes of the region ranged from uncinatum complex. Downloaded from 488 to 532 nucleotides (nt). The smallest size was found Closely related species in different groups formed well- in M. ferrugineum, M. canis, and M. audouinii, comprising supported clades in the calmodulin gene tree (Fig. 2). Ex- only 488 nt, and the longest in M. persicolor, with 532 nt. amples are T. interdigitale, T. tonsurans,andT. equinum Most Trichophyton species had identical sizes, 518 nt (bootstrap value of 100%); A. simii, T. mentagrophytes, and T. schoenleinii (bootstrap value of 100%); T. rubrum (Table 1). http://mmy.oxfordjournals.org/ Multiple alignment of the sequences indicated the pres- and T. violaceum (bootstrap value of 100%); members of ence of significant diversity and differences within the sec- the A. benhamiae complex (bootstrap value of 96%); M. tions and species of dermatophytes. Figure 1 shows the racemosum, M. cookei, and E. floccosum (bootstrap value multiple DNA sequence alignment of calmodulin gene se- of 100%); and T. ajelloi and T. eboreum (bootstrap value quences in the most common pathogenic dermatophytes of 99%). and confirms that the nucleotide regions 75–89, 184–309, Calmodulin gene tree topologies of dermatophyte α and 438–541 are evolutionarily conserved, which could be species were similar to ITS, BT2 and Tef-1 regions (data useful for robust pan-dermatophyte primer and probe de- not shown), except some species such as E. floccosum and by guest on February 12, 2016 sign, while genetic variability is typically limited to frag- A. gertleri which segregated to relatively remote positions. ments 1–74, 90–183, and 310–437. Bioinformatic anal- The phylogenetic tree of calmodulin sequences revealed a ysis showed that these parts are in fact introns in the cluster consisting anthropophilic and zoophilic Trichophy- gene. ton species, in which members of the Trichophyton were Pairwise nucleotide alignment of calmodulin gene se- classified into four groups (Fig. 2). Based on the nucleotide quences in tested dermatophytes indicated a mean similar- sequences, the members of A. vanbreuseghemii, T. rubrum, ity of 81% between the species. Table 2 shows pairwise A. simii, and A. benhamiae complexes were found in this comparisons between dermatophyte strains as the number cluster, which branched far away from all other complexes of differences in the nucleotide sequences. Sequence differ- of geophilic, zoophilic, and anthropophilic Microsporum ences among the 29 dermatophyte species ranged from 0 species and geophilic Trichophyton species. to 200 nt; the largest distance was observed between M. Phylogenetic analysis showed that E. floccosum is closely ferrugineum and T. eboreum. The nucleotide sequences of related to M. cookei, M. racemosum,andA. obtosum /A. the following species were identical: E. floccosum/M. race- gertleri with identical calmodulin sequences, forming a sin- mosum/M. cookei, A. obtosum/A. gertleri, T. tonsurans/T. gle group with good bootstrap support. equinum, and the strains belonging to one genotype of T. interdigitale. Meanwhile, intra-species differences were found within strains of T. interdigitale, A. simii, T. rubrum, Discussion and A. vanbreuseghemii by 0–7, 0–2, 0–1, and 0–1 nt, is of primary public health concern caus- respectively (Table 1); however, strains of T. tonsurans, ing morbidity and significant costs to the society because of T. violaceum, E. floccosum, M. canis, M. audouinii, M. their chronic nature and longlasting therapy.41 For many cookei, M. racemosum, M. gypseum, T mentagrophytes, T years, morphological analysis and physiologic character- schoenleinii, and A. benhamiae were invariant. Having four istics have been used for dermatophyte identification, but Ahmadi et al. 7 Downloaded from http://mmy.oxfordjournals.org/ by guest on February 12, 2016

Figure 1. Multiple sequence alignment of partial calmodulin gene sequences from common dermatophyte species. A dot indicates an identical nucleotide with respect to the top sequence; a dash indicates an insertion/deletion (indel) event. 8 Medical Mycology, 2016, Vol. 00, No. 00 Downloaded from http://mmy.oxfordjournals.org/ by guest on February 12, 2016 2ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 96 98 96 96 95 99 100 94 94 94 95 90 94 93 161 160 163 ID ID 22 23 23 22 22 26 27 28 28 28 29 5 1 ID 27 2927 28 29 27 28 27 27 32 27 33 32 33 0 0 ID 0 ID 23 24 24 23 23 26 27 29 29 29 30 6 ID 96 9896 96 98 96 96 95 96 99 95 10096 99 94 100 98 94 94 9589 94 94 94 91 95 94 93 91 90 95 97 90 94 90 98 91 93 94 91 161 99 93 160 91 163 98 161 91 160 0 90 163 92 90 0 ID 96 90 96 0 91 95 ID 95 160 160 95 162 94 83 159 83 158 160 83 85 73 85 77 85 79 65 ID 70 72 72 0 ID 12 1412 14 1 3 ID 30 3127 2 31 29 ID 30 28 30 27 27 1 32 ID 33 ID 24 25 25 24 24 27 28 29 29 29 30 ID 89 91 91 90 91 99 98 90 90 90 91 95 95 94 159 158 160 85 85 85 65 70 72 72 ID 13 15 ID 29 30 30 29 29 ID 27 29 29 28 28 33 34 1 1 1 ID 164 166 164 163 164 167 168 166 166101 166 103 167 101 161 100 162 101 162 103 5 102 101 101 7 101 102 ID 102 102 101 156 155 158 78 78 78 ID 100 102 100104 100 106 106 99 105 107 104 106 108 100 107 100 106 100 106 101 106 102 107 102 103 101 102 160 102 159 156 160 154 158 82 91 82 91 82 91 46 50 ID 58 ID 191 193 193 192 193 197 196 188 188 188 188 195 197 196 197 197 200 196 196 196 184 193 178 174 182 182 181 124 ID 162 164 162161 161 163 162 161 165 160 166 161 164 164 164 165 164 163 165 163 159 163 160 164 160 158 159 ID 159 4 ID 136 138 139180 138 182 139 180 136 179 137 180 134 183 134 184 134 180 135 180 134 180 131 181 130 182 109 185 110 184 112 170 134 173 134 174 134 181 128 181 131 181 130 175 127 180 128 159 128 162 176 ID 176 158 ID Sequence differences based on pairwise sequence comparison of calmodulin gene between dermatophyte species investigated in this study. T. tonsurans CBS 120.65 A. vanbreuseghmi A. simii CBS 417.65 T. mentagrophytes CBS T. schoenleinii CBS 130812 M. audouinii Ji 325 M. canis CBS 130922 M. ferrugineum TIMM 445.51 E. floccosum CBS 358.93 A. cajetanum CBS228.58 A. racemosum CBS 423.74 M. fulvum CBS 130934 101 M. gypseum NBRC 8228 A. incurvatum CBS161.69 M. persicolor NBRC 5975 A. gertleri CBS 665.77 A. obtusum CBS 322.61 A. grubyi CBS 243.66 T. ajelloi IFM 5326 183 T. eboreum CBS 117155 A. benhamiae Ci1947 T. concentricum CBS 563.83 T. erinacei CBS 344.79 T.eriotrephon CBS220.25 T. verrucosum CBS 554.84 T. rubrum CBS 288.86 T. violaceum NBRC 31064 T. equinum NBRC 31610 T. interdigitale CBS 130816 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Ta b l e 2 . Row Species 1 2 3 4 5 6 7 8 9 Ahmadi et al. 9 Downloaded from http://mmy.oxfordjournals.org/ by guest on February 12, 2016

Figure 2. Phylogenetic tree of 29 representative dermatophyte species based on analysis of calmodulin gene sequences. The evolutionary history was inferred using the neighbor–joining (NJ) method based on the Tamura–Nei model. misidentification caused by phenotypic variations among could lead to significant decreases of taxa. In comparison, strains necessitates molecular identification methods rely- other authors believe that despite their high genetic sim- ing on stable genetic characteristics.42 ilarity, main dermatophyte taxa should remain separate The lack of correlation between phenotypic observations species.24,43,44 and data provided by molecular techniques has been ob- Molecular biological surveys of fungal phylogeny served for most dermatophyte taxa. Therefore some au- by different methods such as the GC content of thors suggest taxonomic revision of dermatophytes, which chromosomal DNA,45 total DNA homology,46 random 10 Medical Mycology, 2016, Vol. 00, No. 00 amplification of polymorphic DNA,47–49 restriction frag- Phylogenetic relationships inferred from calmodulin ment length polymorphism (RFLP) analysis of mito- gene analysis were in accordance with those derived from chondrial DNA (mtDNA),8,43,50 and determination of analysis of ITS,23 BT2,21 and Tef-1α22 regions, resulting nucleotide sequences28,51–53 have shown low genetic di- in the recognition and separation of Epidermophyton, Tri- versity in dermatophytes, indicating a homogeneous group chophyton and Microsporum species and segregation of of species contrasting the high phenotypic heterogeneity. geophilic species away from zoophilic and anthropophilic Nevertheless, a few gene targets including large riboso- species, while phylogenetic analysis of LSU51 sequences mal RNA subunit (LSU),51 nuclear ribosomal internal tran- does not permit distinction of the three anamorphic gen- scribed spacers (ITS),23,44 CHS1,15,54 TOP-II,55 and re- era of dermatophytes from each other. cently BT221 and Tef- 1α22 have been used as genetic The length of calmodulin sequences across the dif- markers for dermatophyte species. ferent strains varied from 488 to 523 bp. Given that The use of the ITS region as a target for phylogenetic protein-coding genes are usually conserved between differ- analysis and molecular species identification has not only ent species, the differences in sequence length between the led to a revision of dermatophyte taxonomy, but also pro- different dermatophytes are mainly due to length variation vided better understanding of the evolution and insights in the intron regions. into the identification, taxonomy, and epidemiology of the The phylogenetic analysis (Fig. 2) showed a cluster species.15,23,24,27,35,56,57 However, there are still some ar- consisting of anthropophilic and zoophilic Trichophyton Downloaded from eas of conflict.58–60 Depending on genetic marker, owing to species, supported by a bootstrap value of 100%. Mem- low nucleotide differences, discrimination of some closely bers of this cluster were classified into four groups, includ- related species such as T. tonsurans/T. equinum,20 M. ca- ing A. vanbreuseghemii, T. rubrum, A. simii, and A. ben- nis/M. ferrugineum,33 T. mentagrophytes sensu stricto/T. hamiae complexes. The calmodulin sequence homology of http://mmy.oxfordjournals.org/ schoenleinii,22 A. benhamiae/T. concentricum,21 T. schoen- the strains of these complexes was observed to be more leinii/A. simii,21 and T. violaceum/T. rubrum21 can be than 87%, and an interspecies variation rate of 0–34 bp difficult. Some species such as E. floccosum and M. gal- (Table 2) was observed between the taxa. The lengths of linae lack stable positions in phylogenetic trees derived calmodulin sequences of the different strains in this cluster, from ITS,61 BT2,21 and Tef-1α22 genes. Therefore, se- ranged from 517 to 520 bp (Table 1), indicating that these quence analysis of more robust targets is necessary for species are very closely related. Phylogenetic analyses of correct identification and phylogenetic analyses of dermato- actin (ACT),60 ITS,24,60,68 CHS-1,69 b-tubulin60 and man- 68 phytes. A target gene with adequate resolution may po- ganese containing superoxide dismutase (MnSOD) genes by guest on February 12, 2016 tentially enhance the definition of closely related species showed that anthropophilic/zoophilic Trichophyton species or those with atypical morphological features in culture.62 might be divided into two lineages, namely the T. rubrum Therefore, further progress in molecular diagnosis of der- and the T. mentagrophytes/T. tonsurans complexes, and matophytosis requires investigation of additional molecular that the latter two species complexes can be divided into A. markers. benhamiae and A. simii /A. vanbreuseghemii complexes. In The calmodulin gene expressed in all eukaryotic cells, this cluster, intra-species variation in the calmodulin gene is shown to be highly conserved both functionally and was observed in some species, such as T. rubrum, A. van- structurally, and the protein encoded plays a crucial role breuseghemii, A. simii, and T. interdigitale. The ITS,21,70 in proliferation, motility, and cell cycle development.63,64 BT221 and Tef-1α22 sequences so far failed to reveal intra- The target has been well used in phylogenetic analysis and species variation in T. rubrum, while calmodulin sequences identification of species of Aspergillus,39,65 Penicillium,66 have two different genotypes for T. rubrum, based on the and Fusarium.67 insertion in position 18. Therefore, this genetic marker could be a candidate for Pairwise sequence comparison of calmodulin in the eco- genetic analysis of dermatophytes. In this study, the calmod- logically differentiated species T. tonsurans and T. equinum ulin gene was analyzed in a wide range of Trichophyton, showed 100% (0 nt difference) similarity, which was found Microsporum,andEpidermophyton species to determine to be significantly lower than that observed in other loci the nucleotide sequences and phylogeny of dermatophytes. such as ITS (1 nt),32 BT2 (1 nt),21 and Tef-1α (14 nt),22 and Molecular data on this gene could provide insight into the our data suggest that calmodulin is not useful for species molecular basis and genetic relationships of different der- discrimination of T. tonsurans and T. equinum. matophytes. In addition, the availability of nucleotide se- Phylogeny data obtained from partial sequencing of the quence data obtained from distinct DNA fragments will calmodulin gene showed T. eriotrephon, T. verrucosum, help to design specific primers for individual dermatophytes and T. erinacei on the same internode (bootstrap value, species. 99%) together with T. concentricum and A. benhamiae Ahmadi et al. 11 with a separate internode (bootstrap value, 100%) were Trichophyton ajelloei and Trichophyton eboreum as formed A. benhamiae complex. Phylogenic analysis of this geophilic species, and with morphologic, physiologic, and complex by BT2,21 TOP-II, ACT,andTef-1α genes indi- genetic structures unambiguously differing from all previ- cates that all these targets have similar genealogical species ously described Trichophyton species, grouped in a separate relationships.21,22,71 cluster, far away from the other complexes (Fig. 2). Phylo- The interspecies variation rate of 1–15 bp between A. genetic analyses based on ITS,23,29 CHS-1,15 and LSU51 benhamiae complex strains shows that, similar to ITS,21 revealed that geophilic T. ajelloi and T. terrestre were well BT2,21 and Tef-1α,22 calmodulin is able to discriminate all separated from the anthropophilic/zoophilic Trichophyton species in the complex. However, BT2,21 ACT,andTOP- species, being most distant in all trees analyzed. Previous II71 gene sequencing does not enable distinction between phylogenetic studies revealed a division between anthro- A. benhamiae and T. concentricum. pophilic and geophilic species of Trichophyton, suggesting The present study showed that the dermatophytes that ecology is a particularly strong driver of dermatophyte species pathogenic to humans, T. rubrum and T. violaceum, evolution.35 Therefore, the soil environment may have pro- are members of the T. rubrum complex. In addition to es- vided an early ecological niche for all dermatophyte species tablishing the significance of calmodulin gene analysis from prior to more recent adaptation to specialized hosts, in- a taxonomic standpoint, the calmodulin DNA sequence cluding animals and humans; this is supported by the fact database (based on interspecies variation ranging from 1 to that Microsporum, as well as zoophilic and anthropophilic Downloaded from 2 nucleotides) was used to identify these important species. Trichophyton species, evolved from a geophilic member of Calmodulin intraspecific sequence variation was de- Trichophyton.23 tected in T. rubrum, which may prove useful for typing Calmodulin sequence analysis indicated that M. canis purposes, while intra-species variation was not observed and the anthropophilic species M. audouinii and M. ferrug- http://mmy.oxfordjournals.org/ in T. violaceum sequences, indicating that this species is ineum comprised the A. otae complex alongside M. galli- genetically homogeneous. nae as a paraphyletic branch. Phylogenetic analysis showed Graser et al.72 investigated the genetic variation that the anthropophilic species M. audouinii and M. ferrug- in T. rubrum using random amplified monomorphilic ineum were more related to the zoophilic species M. canis DNA (RAMD), single-strand conformation polymorphism and M. gallinae than to M. gypseum, M. fulvum, and M. (SSCP), and RFLP, and found that all of these methods cookei as the other geophilic species. Similar to calmodulin, failed to show any polymorphism across T. rubrum strains, other genetic markers such as ITS,21,23 BT221 and Tef-1α,22

indicating a strictly clonal and genetically homogeneous enables the separation of M. canis and M. audouinii and M. by guest on February 12, 2016 population, while Jackson et al.10 showed that genetic poly- ferrugineum into different clades. These results were also morphism in T. rubrum exists in the NTS region rather than partly confirmed by analysis of CH-173 sequences, as well in other genetic markers such as ITS, BT2,andTef-1α re- as by phylogenetic analyses of 13 DNA markers, including gions. microsatellite and non-microsatellite regions, all supporting The present study showed that the closely related species separate grouping of M. canis from M. audouinii and M. T. simii, T. mentagrophytes,andT. schoenleinii formed a ferrugineum.27 well-supported clade with a bootstrap value of 100% in the No intraspecific variation was observed in the calmod- calmodulin tree, including these as members of the A. simii ulin locus of our M. canis strains. This finding was in con- complex (Fig. 2). This finding is in accordance with results cordance with Brilhante et al.74, who used RAPD and PCR- inferred from ITS,23 actin,60 Tef-1α,22 and BT221 genes and RFLP targeting ITS regions to show that all human and indicate that A. simii is closely related to T. mentagrophytes animals isolates of M. canis were genetically identical in and T. schoenleinii. spite of differing morphological characteristics. Similarly, Comparison of calmodulin sequences showed a varia- no specific fragment patterns were observed among strains tion of one nt between T. mentagrophytes and T. schoen- of M. canis using repetitive sequence PCR-based DNA fin- leinii, while Nishio et al.43 and Harmsen et al.53,based gerprinting, as demonstrated by Pounder et al.75 on mtDNA and 18S rRNA investigation, failed to identify Microsporum canis and M. ferrugineum were distin- heterogeneity between T. mentagrophytes and T. schoen- guished by 2 bp in ITS2,33 while the two species differed by leinii; moreover, Tef-1α22 sequence analysis did not enable 7 nt on calmodulin gene analysis, suggesting that calmod- differentiation between strains of the two taxa. This find- ulin is more useful than ITS for species delineation of these ing together with analyses based on BT221 and ACT60 two taxa. Nonetheless, BT221 and Tef-1α22 genes (12 and genes support the theory that T. schoenleinii originated 10 nt differences, respectively) appear to better resolve phy- from camels. logenetic relationships between the species. 12 Medical Mycology, 2016, Vol. 00, No. 00

The average pairwise sequence divergence between M. Nevertheless, the resolution of the calmodulin gene was canis, M, audouinii, and M. ferrugineum was5.33ntfor inadequate to separate M. nanum and A. gertleri strains. both calmodulin and ITS21; therefore, the calmodulin gene However, due to the low number of tested species, further is just as useful as ITS for differentiation of the three species, studies on more isolates are required to confirm the results. while the discriminatory power of Tef-1α22 and BT221 was In conclusion, in the present study the phylogenetic found to be higher than that of calmodulin and ITS in this positions of dermatophytes, which are fungi pathogenic complex. to humans, were assessed based on nucleotide sequences In agreement with analysis of BT221 and Tef-1α22 genes, of the calmodulin gene as a new genetic marker. The calmodulin gene sequence analysis sees M. gallinae located data reported here provide a basis for further discov- at a separate position, next to the members of M. canis ery of relationships between species. Dermatophyte tree complex. topologies were almost in concordance with those observed E. floccosum along with M. cookei and M. racemosum for other loci, such as ITS, BT2,andTef-1α.Thefol- formed a monophyletic group, supported by a 100% boot- lowing taxa with the same sequences are so closely re- strap value (Fig. 2). The geophilic dermatophytes M. cookei lated that they are probably conspecific: E. floccosum/ M. and M. racemosum (anamorphs of Arthroderma) have been cookei/M. racemosum and A. gertleri/A. obtosum. There- reported to cause superficial infection of animals and occa- fore, studies of other loci as well as more isolates of zo- sionally of humans, while E. floccosum is a common an- ologic and geophilic species are necessary to confirm the Downloaded from thropophilic agent involved in human infections.76 Inter- results. estingly, these three species had identical sequences (100% similarity), being indistinguishable from each other based on calmodulin gene analysis. Likewise, Kawasaki et al.8 Acknowledgements http://mmy.oxfordjournals.org/ used RFLP analysis of mitochondrial DNA and concluded This work was financially supported by Tehran University of Medical that Epidermophyton could not be separated from the gen- Sciences (grant no. 92-01-27-21559), Tehran, Iran. We thank all era Trichophyton and Microsporum. On the other hand, E. personnel in Molecular Biology Laboratory in TIMM and in the reference collection of Centraalbureau voor Schimmelcultures (CBS), floccosum is separated from the other mentioned species by The Netherlands. ITS,23 BT2,21 and Tef-1α.22 In contrast, based on ITS analy- sis, Graser et al.23 concluded that E. floccosum is somewhat related to the anthropophilic Trichophyton, evidenced by a Declaration of interest long paraphyletic branch. by guest on February 12, 2016 The authors report no conflicts of interest. The authors alone are Since calmodulin sequences were identical for M. cookei responsible for the content and the writing of the paper. and M. racemosum, it appears that they could be incorpo- rated into one species. These findings agree with the low res- olution inferred from Tef-1α, ITS and BT2 data sets.21,22,77 References Calmodulin sequence analysis showed that the geophilic 1. Grumbt M, Monod M, Staib P. 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