Medical Mycology, 2016, 0, 1–15 doi: 10.1093/mmy/myw004 Advance Access Publication Date: 0 2016 Original Article Original Article Phylogenetic analysis of dermatophyte species using DNA sequence polymorphism in calmodulin gene Bahram Ahmadi 1, 2 , Hossein Mirhendi 3, ∗ , Koichi Makimura 4 , G. Sybren de Hoog 5 , Mohammad Reza Shidfar 2 , Sadegh Nouripour-Sisakht 6 and Niloofar Jalalizand 2 1 Department of Microbiology and Parasitology, School of Para-Medicine, Bushehr University of Medical Sciences, Bushehr, Iran, 2 Departments of Medical Parasitology & Mycology, School of Public Health; National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran, 3 Departments of Medical Parasitology & Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran, 4 Teikyo University Institute of Medical Mycology and Genome Research Center, Tokyo, Japan, 5 Fungal Biodiversity Center, Institute of the Royal Netherlands, Academy of Arts and Sciences, Centraalbureau voor Schimmelcultures-KNAW, Utrecht, The Netherlands and 6 Cellular 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]. 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 taxonomy 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 Trichophyton interdigitale, Arthroderma simii, T. rubrum and A. vanbreuseghemii, while T. tonsurans, T. violaceum, Epidermophyton floccosum, Microsporum 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, and Tef-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. All rights reserved. For permissions, please e-mail: [email protected]1 Medical Mycology Advance Access published February 11, 2016 by guest on February 12, 2016 http://mmy.oxfordjournals.org/ Downloaded from
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Medical Mycology, 2016, 0, 1–15doi: 10.1093/mmy/myw004
Sadegh Nouripour-Sisakht6 and Niloofar Jalalizand2
1Department of Microbiology and Parasitology, School of Para-Medicine, Bushehr University of MedicalSciences, Bushehr, Iran, 2Departments of Medical Parasitology & Mycology, School of Public Health;National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran, 3Departmentsof Medical Parasitology & Mycology, School of Medicine, Isfahan University of Medical Sciences,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 MolecularResearch Center, Yasuj University of Medical Sciences, Yasuj, Iran∗To whom correspondence should be addressed. Hossein Mirhendi, Professor, Department of Medical Parasitology andMycology, School of Medicine; Isfahan University of Medical Sciences, Isfahan, Iran. Tel/Fax: +00982188951392;E-mail: [email protected].
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 taxonomy of dermatophyte species; however, confirmationand refinement using other genes are needed. Since the calmodulin gene has not beensystematically used in dermatophyte taxonomy, we evaluated its intra- and interspeciessequence variation as well as its application in identification, phylogenetic analysis, andtaxonomy of 202 strains of 29 dermatophyte species. A set of primers was designedand optimized to amplify the target followed by bilateral sequencing. Using pairwise nu-cleotide comparisons, a mean similarity of 81% was observed among 29 dermatophytespecies, with inter-species diversity ranging from 0 to 200 nucleotides (nt). Intraspeciesnt differences were found within strains of Trichophyton interdigitale, Arthroderma simii,T. rubrum and A. vanbreuseghemii, while T. tonsurans, T. violaceum, Epidermophytonfloccosum, Microsporum 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 agenotype of T. interdigitale had identical calmodulin sequences. For the majority of thespecies, tree topology obtained for calmodulin gene showed a congruence with codingand non-coding regions including ITS, BT2, and Tef-1α. Compared with the phylogenetic
tree derived from ITS, BT2, and Tef-1α genes, some species such as E. floccosum andA. gertleri took relatively remote positions. Here, characterization and obtained dendro-gram of calmodulin gene on a broad range of dermatophyte species provide a basis forfurther discovery of relationships between species. Studies of other loci are necessary toconfirm the results.
Annually, millions of humans and animals are infectedby superficial fungal infections. As specialized filamentousfungi, dermatophytes have a specific ability to digest andgrow on keratinized host structures such as skin, nails,and hair, causing the vast majority of mycotic infections.1,2
Based on their major natural predilection sites, dermato-phyte species are classified into anthropophilic, geophilic,and zoophilic groups. The asexual forms (anamorphs) ofdermatophytes are classified into Trichophyton, Epidermo-phyton, and Microsporum, and their sexual forms (teleo-morphs) are members of the genus Arthroderma in the sub-phylum Ascomycotina.1,3
Diagnosis of dermatophyte infections is essential for ap-propriate antifungal therapy because of the length of treat-ment, potential side effects of the drugs, and their highcost. Moreover, having information on zoophilic or an-thropophilic sources of the causative dermatophyte agentmay allow prophylactic measures such as treatment of bothanimal or human reservoirs.4 Precise definition and clas-sification of microorganisms including bacteria, parasites,viruses, and fungi have always been of great relevance totaxonomic identity, phylogenetic analysis, epidemiology,and clinical microbiology.5
To resolve the evolutionary relationships betweendermatophytes, nucleic acid-based methods have beenused since early 1980 s. Consequently, several ge-nomic and molecular researches for characterizationof dermatophytes has revealed a homogeneous groupof species with very low genetic diversity in com-parison with overall high phenotypic heterogeneity indermatophytes.5–8
The following DNA fragments have been noticed asthe main dermatophyte genetic markers: ribosomal DNA(rDNA) regions,9–13 chitin synthase 1 (CHS1),14–17 DNAtopoisomerase II (TOP-II),18,19 beta tubulin (BT2),20,21
and translation elongation factor 1-α (Tef-1α) genes.20,22
Phylogenetic analysis and identification of dermatophytesbased on sequencing of the internal transcribed spacer (ITS)regions of rDNA has proved to be useful as a gold standardmethod.23–31 Only a small number of nucleotide differencesin the ITS regions have been observed in several ecologically
and phenotypically separated Trichophyton species.20,32,33
Moreover, current advances in molecular taxonomy and in-sights into mating discovered that Trichophyton mentagro-phytes is a complex of anthropophilic and zoophilic speciesthat produce different teleomorphs, leading to a confusionwith regard to species denomination.34,35
Regardless of the various advantages of ITS as the pri-mary genetic marker of dermatophyte identification, ad-ditional verification and refinement using other genes iscritical to organizing sequence-based and classical speciesconcepts. Since the calmodulin gene has not been systemati-cally used in dermatophyte taxonomy, in this study, our aimwas to evaluate nucleotide sequence analysis of calmodulingene as a new genetic marker for a subset of dermatophytespecies, as well as to assess its application with regard toidentification, phylogenetic analysis, and taxonomy stud-ies based on intra- and interspecies variation. Several ref-erence strains and clinical isolates including a wide rangeof common and rare pathogenic species were used for thispurpose.
Materials and methods
Reference strains and clinical isolates
A total of 202 strains of 29 dermatophyte species com-prising 60 reference strains and 142 clinical isolates wereused for sequence analysis of partial calmodulin gene(Table 1). The reference strains were obtained from Cen-traalbureau voor Schimmelcultures (CBS), Utrecht, theNetherlands, and Teikyo University, Institute of MedicalMycology (TIMM), Tokyo, Japan. The clinical isolateswere collected from a variety of specimens, including skin,nail, and hair submitted to two medical mycology labo-ratories in Tehran, Iran. The clinical isolates were identi-fied to species level by an already described PCR-restrictionfragment length polymorphism (PCR-RFLP) method9 andin some cases based on ITS-sequencing. Species nameswere determined according to Graser et al.35 Hence, T.mentagrophytes strains CBS 435.73, NBRC 5809, CBS119445, and NBRC5974, were all identified as T. interdig-itale by ITS-PCR-RFLP analysis. Also, A. gypseum strainCBS 161.69 were identified as A. incurvatum.
Table 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-speciesof tested strains) Strains (accession numbers) sequence (bp) variation
A. benhamiae (4) Ci1947 (KM678214), Ji362 (KP781967), Ji363 (KP781968),Ji364 (KP781969)
517 0
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), CBS130818 (KM387127)
488 0
Ci 622 (KM387145), Ci 1335 (KM387146), Ci 675(KM387147), Ci 9219 (KM387148), Ci987 (KM387149), Ci9938 (KM387150)
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(KP781975)
519 0–2
T. mentagrophytes (2) CBS 318.56 (KM387110), CBS 101546 (KM387113) 518 0
T. schoenleinii (4) CBS 434.63 (KM387117), CBS 564.94 (KM387118), NBRC8192 (KM387119), CBS 130812 (KM387262)
518 0
A. vanbreuseghmii (3) Ji 405 (KP781970), TIMM 20066 (KP781971), Ji 1116(KP781972)
518 0–1
T. equinum (1) NBRC 31610 (KM387108) 518 –
T. interdigitale (43) CBS 130816 (KM387156), CBS 130923 (KM387158), Ci1188 (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), Ci2890 (KM387169), Ci 448 (KM387170), Ci 712(KM387171), Ci 855 (KM387172), Ci 1184 (KM387173), Ci1517 (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), Ci1746 (KM387277), Ci 1849 (KM387278), CBS 435.73(KM387111), NBRC 5809 (KM387112), CBS 119445(KM387114), NBRC 5974 (KM387157)
518–519 0–7
T. tonsurans (12) CBS 120.65 (KM387120), CBS 270.66 (KM387121), CBS109035 (KM387122), CBS 130924 (KM387263), NBRC5928 (KM387264), CBS 130814 (KM387265), Ci 1852(KM387267), Ci 2430 (KM387268), Ci 1186 (KM387269),Ci 2346 (KM387270), Ci 2347 (KM387271), Ci 2518(KM387272)
Species (total number Length of calmodulin Range of intra-speciesof tested strains) Strains (accession numbers) sequence (bp) variation
M. gypseum (5) CBS 130820 (KM213525), CBS 258.61 (KM387151), NBRC5948 (KM387152), CBS 130939 (KM387153), NBRC 8228(KM387154)
523 0
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(KM387101), Ci 1804 (KM387129),Ci 2118 (KM387130),Ci 3094 (KM387131), Ci 625 (KM387132), Ci 629(KM387133), Ci 679 (KM387134), Ci 699 (KM387135), Ci943 (KM387136), Ci 1453 (KM387137),Ci 1789(KM387138), Ci 1964 (KM387139), Ci 2086 (KM387140),Ci 2253 (KM387141), Ci 2300 (KM387142), Ci 2321(KM387143), Ci 2340 (KM387144), Ci 1876 (KM387280)
525 0
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), CBS130825 (KM387193), NBRC 5467 (KM387194), NBRC5808 (KM387195), CBS 130817 (KM387196)
518–519 0–1
CBS 130808 (KM387197), CBS 130933 (KM387198), Ci1199 (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(KM387211), Ci 2191 (KM387212), Ci 262 (KM387213), Ci2856 (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),Ci304 (KM387254), Ci 3168 (KM387255), Ci 3173(KM387256), Ci 3185 (KM387257), Ci 769 (KM387258), Ci777 (KM387259), Ci 966 (KM387260), Ci 9744(KM387261), Ci 1860 (KM387279)
T. violaceum (4) NBRC 31064 (KM387273), CBS 319.31 (KM387274), CBS459.61 (KM387275), Ci 2033 (KM387276)
518 0
Note: CBS, Centraalbureau voor Schimmelcultures; Ci, clinical isolate; Ji, Japanese isolate; NBRC, NITE Biological Resource Center, TIMM, Teikyo UniversityInstitute of Medical Mycology
All fungal strains were cultured on Mycobiotic agar (Difco,Detroit, MI, USA) and incubated at 27◦C for 7 days.DNA was extracted and purified from fungal coloniesas previously described36. Briefly, a small amount of thecolonies was allocated to a 1.5-ml tube containing 300 μlof lysis buffer (100 mM Tris-HCl, pH 7.5, 10 mMEDTA, 0.5% w/v SDS, 100 mM NaCl), 300 μl of phe-nol/chloroform (1:1), and 300 μl of glass beads (0.5 mm indiameter), vortexed for 5 min and centrifuged at 5,000 rpmfor 5 min; the supernatant was transferred to a new tubeand re-extracted with chloroform. DNA was precipitatedwith an equal volume of 2-propanol and 0.1 volume of3 M sodium acetate (pH 5.2), kept at −20◦C for 20 min,and centrifuged at 10,000 rpm for 10 min. The pellet waswashed with 300 μl of 70% ethanol, air dried, and eventu-ally, the DNA was resuspended in 50 μL of sterile distilledwater.
Species identification by PCR-RFLP
All 202 clinical and reference isolates were sub-jected to PCR amplification using the primers ITS1(5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′).37 PCR reactions wereprepared in final volumes of 25 μL, containing 12.5 μL ofpremix (Ampliqon, Denmark), 2 μl of DNA template, and0.5 μM of each forward and reverse primer. PCR cyclingconditions were as follows: 6 min initial pre-incubationat 94◦C, followed by 35 cycles consisting of denaturationat 94◦C for 30 s, annealing at 58◦C for 30 s, and exten-sion at 72◦C for 1 min, with a final extension at 72◦C for10 min. All strains were identified to species level by PCR-RFLP analysis as previously described.9 Digestion of PCRproducts was performed by incubating 8 μL of PCR productwith 0.5 μl of the enzyme MvaI Fast digest (Fermentas LifeSciences, Lithuania), 1.5 μl of 10X buffer, and 5 μl of wa-ter at 37◦C for 20 min. PCR amplicons and RFLP-productswere analyzed by agarose gel electrophoresis in TBE buffer(Tris 0.09 M, Boric acid 0.09 M, EDTA 2 mM) at 100 Vfor approximately 60 min using 1.5% and 2% agarose gels,respectively. For species identification, the size of fragmentsgenerated by enzymatic digestion were compared with ref-erences RFLP profiles.9
PCR for calmodulin gene amplification
For calmodulin gene amplification, after alignment andanalysis of calmodulin gene sequences, a novel set ofpan-dermatophyte primers was designed manually with
MEGA638 and Geneious (http://www.geneious.com) soft-wares as follows: CF1 5′-TGTCCGAGTACAAGGAAGC-3′ and CF2 5′-TTACAATCAATTCTGCCGTC-3′. PCR re-actions contained 12.5 μl of premix (Ampliqon, Denmark),2 μL of DNA template, 0.5 μM of each primer, and enoughwater up to reach a final reaction volume of 25 μl. Two neg-ative controls (water instead of fungal DNA) were addedto each PCR. The reaction mixture was initially dena-tured at 95◦C for 5 min, followed by 35 cycles of 30 sat 94◦C, 45 s at 58◦C, and 45 s at 72◦C, and a ter-minal extension step of 72◦C for 5 min. For the strainsthat failed to amplify, a nested PCR was set up for suc-cessful amplification of the gene by using the degenerateprimers CF1 5′-GCCGACTCTTTGACYGARGAR-3′ andCF4 5′-TTTYTGCATCATRAGYTGGAC-3′39 for the firstround PCR and the above mentioned primers for the secondround. One microliter of the first PCR product was diluted1:50, and 1 μl of the dilution was added to reaction mixtureas template. PCR products were separated by electrophore-sis on 1.5% agarose gels and visualized by staining withethidium bromide (0.5 μg/ml) and photographed under UVirradiation.
Sequencing and phylogenetic analysis
For purification of PCR products, 50 μl ethanol was addedto 20 μl of each product, kept at -20◦C for 30 min, and cen-trifuged at 12,000 rpm for 10 min. The pellet was air-driedand resuspended in 25 μl of distilled water. PCR productswere sequenced bilaterally using the primers CF1 and CF2and the ABI PRISM BigDye Terminator Cycle SequencingReady Reaction Kit (Applied Biosystems, Foster City, CA,USA), on an automated DNA sequencer (ABI PrismTM3730 Genetic Analyzer, Applied Biosystems), according tothe manufacturer’s instructions.
Forward and reverse sequences of each sample were sub-jected to ClustalW pairwise alignment using Geneious andMEGA638 softwares and edited manually to improve align-ment accuracy. The consensus nucleotide sequence datadetermined in this study were deposited in the GenBank,under the accession numbers KM213523 - KM213525,KM387096 - KM387280, and KP781963 - KP781977(Table 1).
Based on the quality of the sequencing, consensus se-quences derived from the forward and reverse sequenceshad different lengths, therefore, the common first andlast points of the entire sequence were determined, andpoorly aligned DNA sequences were removed. Final-ized sequences were subject to BioEdit software version7.0.540 for pairwise comparisons and multiple alignmentto determine similarities and differences in nucleotides.
Pairwise similarity values were calculated by dividing thenumber of matching nucleotides by the total length of thealignment.
Phylogenetic trees were constructed with unambiguouslyaligned sequences using the neighbor-joining (NJ) methodwith the Tamura-Nei parameter as substitution model asimplemented in the MEGA6 program.38 The reliability ofinternal branches was assessed using the bootstrap methodwith 1000 replicates.
Results
To evaluate the applicability of using calmodulin gene se-quences for differentiation and phylogenetic studies of der-matophyte species, a part of the gene was successfully am-plified for 202 strains. The sizes of the region ranged from488 to 532 nucleotides (nt). The smallest size was foundin M. ferrugineum, M. canis, and M. audouinii, comprisingonly 488 nt, and the longest in M. persicolor, with 532 nt.Most Trichophyton species had identical sizes, 518 nt(Table 1).
Multiple alignment of the sequences indicated the pres-ence of significant diversity and differences within the sec-tions and species of dermatophytes. Figure 1 shows themultiple DNA sequence alignment of calmodulin gene se-quences in the most common pathogenic dermatophytesand confirms that the nucleotide regions 75–89, 184–309,and 438–541 are evolutionarily conserved, which could beuseful for robust pan-dermatophyte primer and probe de-sign, while genetic variability is typically limited to frag-ments 1–74, 90–183, and 310–437. Bioinformatic anal-ysis showed that these parts are in fact introns in thegene.
Pairwise nucleotide alignment of calmodulin gene se-quences in tested dermatophytes indicated a mean similar-ity of 81% between the species. Table 2 shows pairwisecomparisons between dermatophyte strains as the numberof differences in the nucleotide sequences. Sequence differ-ences among the 29 dermatophyte species ranged from 0to 200 nt; the largest distance was observed between M.ferrugineum and T. eboreum. The nucleotide sequences ofthe following species were identical: E. floccosum/M. race-mosum/M. cookei, A. obtosum/A. gertleri, T. tonsurans/T.equinum, and the strains belonging to one genotype ofT. interdigitale. Meanwhile, intra-species differences werefound within strains of T. interdigitale, A. simii, T. rubrum,and A. vanbreuseghemii by 0–7, 0–2, 0–1, and 0–1 nt,respectively (Table 1); however, strains of T. tonsurans,T. violaceum, E. floccosum, M. canis, M. audouinii, M.cookei, M. racemosum, M. gypseum, T mentagrophytes, Tschoenleinii, and A. benhamiae were invariant. Having four
calmodulin genotypes, T. interdigitale exhibited most intra-species variability. The variation between M. gypseum andA. incurvatum was 56 nt, while the diversity within fivestrains of M. gypseum was 0 nt.
A phylogenetic tree constructed for 29 representativedermatophytes species is presented in Figure 2. A prelim-inary analysis using an alignment including all sequencesmade in this study gave a similar overall species topologyas in Figure 2 (results not shown).
Sequence analysis of dermatophyte species revealed eightcomplexes: 1) Arthroderma vanbreuseghemii Complex, 2)Arthroderma simii Complex, 3) Arthroderma benhamiaeComplex, 4) Arthroderma otae Complex, 5) Trichophytonrubrum Complex, 6) M. fulvum and M. gypseum Com-plex, 7) M. racemosum and M. cookei Complex, and 8) A.uncinatum complex.
Closely related species in different groups formed well-supported clades in the calmodulin gene tree (Fig. 2). Ex-amples are T. interdigitale, T. tonsurans, and T. equinum(bootstrap value of 100%); A. simii, T. mentagrophytes,and T. schoenleinii (bootstrap value of 100%); T. rubrumand T. violaceum (bootstrap value of 100%); members ofthe A. benhamiae complex (bootstrap value of 96%); M.racemosum, M. cookei, and E. floccosum (bootstrap valueof 100%); and T. ajelloi and T. eboreum (bootstrap valueof 99%).
Calmodulin gene tree topologies of dermatophytespecies were similar to ITS, BT2 and Tef-1α regions (datanot shown), except some species such as E. floccosum andA. gertleri which segregated to relatively remote positions.
The phylogenetic tree of calmodulin sequences revealed acluster consisting anthropophilic and zoophilic Trichophy-ton species, in which members of the Trichophyton wereclassified into four groups (Fig. 2). Based on the nucleotidesequences, the members of A. vanbreuseghemii, T. rubrum,A. simii, and A. benhamiae complexes were found in thiscluster, which branched far away from all other complexesof geophilic, zoophilic, and anthropophilic Microsporumspecies and geophilic Trichophyton species.
Phylogenetic analysis showed that E. floccosum is closelyrelated to M. cookei, M. racemosum, and A. obtosum /A.gertleri with identical calmodulin sequences, forming a sin-gle group with good bootstrap support.
Discussion
Dermatophytosis is of primary public health concern caus-ing morbidity and significant costs to the society because oftheir chronic nature and longlasting therapy.41 For manyyears, morphological analysis and physiologic character-istics have been used for dermatophyte identification, but
Figure 1. Multiple sequence alignment of partial calmodulin gene sequences from common dermatophyte species. A dot indicates an identicalnucleotide with respect to the top sequence; a dash indicates an insertion/deletion (indel) event.
Figure 2. Phylogenetic tree of 29 representative dermatophyte species based on analysis of calmodulin gene sequences. The evolutionary historywas inferred using the neighbor–joining (NJ) method based on the Tamura–Nei model.
misidentification caused by phenotypic variations amongstrains necessitates molecular identification methods rely-ing on stable genetic characteristics.42
The lack of correlation between phenotypic observationsand data provided by molecular techniques has been ob-served for most dermatophyte taxa. Therefore some au-thors suggest taxonomic revision of dermatophytes, which
could lead to significant decreases of taxa. In comparison,other authors believe that despite their high genetic sim-ilarity, main dermatophyte taxa should remain separatespecies.24,43,44
Molecular biological surveys of fungal phylogenyby different methods such as the GC content ofchromosomal DNA,45 total DNA homology,46 random
amplification of polymorphic DNA,47–49 restriction frag-ment length polymorphism (RFLP) analysis of mito-chondrial DNA (mtDNA),8,43,50 and determination ofnucleotide sequences28,51–53 have shown low genetic di-versity in dermatophytes, indicating a homogeneous groupof species contrasting the high phenotypic heterogeneity.Nevertheless, a few gene targets including large riboso-mal RNA subunit (LSU),51 nuclear ribosomal internal tran-scribed spacers (ITS),23,44 CHS1,15,54 TOP-II,55 and re-cently BT221 and Tef- 1α22 have been used as geneticmarkers for dermatophyte species.
The use of the ITS region as a target for phylogeneticanalysis and molecular species identification has not onlyled to a revision of dermatophyte taxonomy, but also pro-vided better understanding of the evolution and insightsinto the identification, taxonomy, and epidemiology of thespecies.15,23,24,27,35,56,57 However, there are still some ar-eas of conflict.58–60 Depending on genetic marker, owing tolow nucleotide differences, discrimination of some closelyrelated species such as T. tonsurans/T. equinum,20 M. ca-nis/M. ferrugineum,33 T. mentagrophytes sensu stricto/T.schoenleinii,22 A. benhamiae/T. concentricum,21 T. schoen-leinii/A. simii,21 and T. violaceum/T. rubrum21 can bedifficult. Some species such as E. floccosum and M. gal-linae lack stable positions in phylogenetic trees derivedfrom ITS,61 BT2,21 and Tef-1α22 genes. Therefore, se-quence analysis of more robust targets is necessary forcorrect identification and phylogenetic analyses of dermato-phytes. A target gene with adequate resolution may po-tentially enhance the definition of closely related speciesor those with atypical morphological features in culture.62
Therefore, further progress in molecular diagnosis of der-matophytosis requires investigation of additional molecularmarkers.
The calmodulin gene expressed in all eukaryotic cells,is shown to be highly conserved both functionally andstructurally, and the protein encoded plays a crucial rolein proliferation, motility, and cell cycle development.63,64
The target has been well used in phylogenetic analysis andidentification of species of Aspergillus,39,65 Penicillium,66
and Fusarium.67
Therefore, this genetic marker could be a candidate forgenetic analysis of dermatophytes. In this study, the calmod-ulin gene was analyzed in a wide range of Trichophyton,Microsporum, and Epidermophyton species to determinethe nucleotide sequences and phylogeny of dermatophytes.Molecular data on this gene could provide insight into themolecular basis and genetic relationships of different der-matophytes. In addition, the availability of nucleotide se-quence data obtained from distinct DNA fragments willhelp to design specific primers for individual dermatophytesspecies.
Phylogenetic relationships inferred from calmodulingene analysis were in accordance with those derived fromanalysis of ITS,23 BT2,21 and Tef-1α22 regions, resultingin the recognition and separation of Epidermophyton, Tri-chophyton and Microsporum species and segregation ofgeophilic species away from zoophilic and anthropophilicspecies, while phylogenetic analysis of LSU51 sequencesdoes not permit distinction of the three anamorphic gen-era of dermatophytes from each other.
The length of calmodulin sequences across the dif-ferent strains varied from 488 to 523 bp. Given thatprotein-coding genes are usually conserved between differ-ent species, the differences in sequence length between thedifferent dermatophytes are mainly due to length variationin the intron regions.
The phylogenetic analysis (Fig. 2) showed a clusterconsisting of anthropophilic and zoophilic Trichophytonspecies, supported by a bootstrap value of 100%. Mem-bers of this cluster were classified into four groups, includ-ing A. vanbreuseghemii, T. rubrum, A. simii, and A. ben-hamiae complexes. The calmodulin sequence homology ofthe strains of these complexes was observed to be morethan 87%, and an interspecies variation rate of 0–34 bp(Table 2) was observed between the taxa. The lengths ofcalmodulin sequences of the different strains in this cluster,ranged from 517 to 520 bp (Table 1), indicating that thesespecies are very closely related. Phylogenetic analyses ofactin (ACT),60 ITS,24,60,68 CHS-1,69 b-tubulin60 and man-ganese containing superoxide dismutase (MnSOD)68 genesshowed that anthropophilic/zoophilic Trichophyton speciesmight be divided into two lineages, namely the T. rubrumand the T. mentagrophytes/T. tonsurans complexes, andthat the latter two species complexes can be divided into A.benhamiae and A. simii /A. vanbreuseghemii complexes. Inthis cluster, intra-species variation in the calmodulin genewas observed in some species, such as T. rubrum, A. van-breuseghemii, A. simii, and T. interdigitale. The ITS,21,70
BT221 and Tef-1α22 sequences so far failed to reveal intra-species variation in T. rubrum, while calmodulin sequenceshave two different genotypes for T. rubrum, based on theinsertion in position 18.
Pairwise sequence comparison of calmodulin in the eco-logically differentiated species T. tonsurans and T. equinumshowed 100% (0 nt difference) similarity, which was foundto be significantly lower than that observed in other locisuch as ITS (1 nt),32 BT2 (1 nt),21 and Tef-1α (14 nt),22 andour data suggest that calmodulin is not useful for speciesdiscrimination of T. tonsurans and T. equinum.
Phylogeny data obtained from partial sequencing of thecalmodulin gene showed T. eriotrephon, T. verrucosum,and T. erinacei on the same internode (bootstrap value,99%) together with T. concentricum and A. benhamiae
with a separate internode (bootstrap value, 100%) wereformed A. benhamiae complex. Phylogenic analysis of thiscomplex by BT2,21 TOP-II, ACT, and Tef-1α genes indi-cates that all these targets have similar genealogical speciesrelationships.21,22,71
The interspecies variation rate of 1–15 bp between A.benhamiae complex strains shows that, similar to ITS,21
BT2,21 and Tef-1α,22 calmodulin is able to discriminate allspecies in the complex. However, BT2,21 ACT, and TOP-II71 gene sequencing does not enable distinction betweenA. benhamiae and T. concentricum.
The present study showed that the dermatophytesspecies pathogenic to humans, T. rubrum and T. violaceum,are members of the T. rubrum complex. In addition to es-tablishing the significance of calmodulin gene analysis froma taxonomic standpoint, the calmodulin DNA sequencedatabase (based on interspecies variation ranging from 1 to2 nucleotides) was used to identify these important species.
Calmodulin intraspecific sequence variation was de-tected in T. rubrum, which may prove useful for typingpurposes, while intra-species variation was not observedin T. violaceum sequences, indicating that this species isgenetically homogeneous.
Graser et al.72 investigated the genetic variationin T. rubrum using random amplified monomorphilicDNA (RAMD), single-strand conformation polymorphism(SSCP), and RFLP, and found that all of these methodsfailed to show any polymorphism across T. rubrum strains,indicating a strictly clonal and genetically homogeneouspopulation, while Jackson et al.10 showed that genetic poly-morphism in T. rubrum exists in the NTS region rather thanin other genetic markers such as ITS, BT2, and Tef-1α re-gions.
The present study showed that the closely related speciesT. simii, T. mentagrophytes, and T. schoenleinii formed awell-supported clade with a bootstrap value of 100% in thecalmodulin tree, including these as members of the A. simiicomplex (Fig. 2). This finding is in accordance with resultsinferred from ITS,23 actin,60 Tef-1α,22 and BT221 genes andindicate that A. simii is closely related to T. mentagrophytesand T. schoenleinii.
Comparison of calmodulin sequences showed a varia-tion of one nt between T. mentagrophytes and T. schoen-leinii, while Nishio et al.43 and Harmsen et al.53, basedon mtDNA and 18S rRNA investigation, failed to identifyheterogeneity between T. mentagrophytes and T. schoen-leinii; moreover, Tef-1α22 sequence analysis did not enabledifferentiation between strains of the two taxa. This find-ing together with analyses based on BT221 and ACT60
genes support the theory that T. schoenleinii originatedfrom camels.
Trichophyton ajelloei and Trichophyton eboreum asgeophilic species, and with morphologic, physiologic, andgenetic structures unambiguously differing from all previ-ously described Trichophyton species, grouped in a separatecluster, far away from the other complexes (Fig. 2). Phylo-genetic analyses based on ITS,23,29 CHS-1,15 and LSU51
revealed that geophilic T. ajelloi and T. terrestre were wellseparated from the anthropophilic/zoophilic Trichophytonspecies, being most distant in all trees analyzed. Previousphylogenetic studies revealed a division between anthro-pophilic and geophilic species of Trichophyton, suggestingthat ecology is a particularly strong driver of dermatophyteevolution.35 Therefore, the soil environment may have pro-vided an early ecological niche for all dermatophyte speciesprior to more recent adaptation to specialized hosts, in-cluding animals and humans; this is supported by the factthat Microsporum, as well as zoophilic and anthropophilicTrichophyton species, evolved from a geophilic member ofTrichophyton.23
Calmodulin sequence analysis indicated that M. canisand the anthropophilic species M. audouinii and M. ferrug-ineum comprised the A. otae complex alongside M. galli-nae as a paraphyletic branch. Phylogenetic analysis showedthat the anthropophilic species M. audouinii and M. ferrug-ineum were more related to the zoophilic species M. canisand M. gallinae than to M. gypseum, M. fulvum, and M.cookei as the other geophilic species. Similar to calmodulin,other genetic markers such as ITS,21,23 BT221 and Tef-1α,22
enables the separation of M. canis and M. audouinii and M.ferrugineum into different clades. These results were alsopartly confirmed by analysis of CH-173 sequences, as wellas by phylogenetic analyses of 13 DNA markers, includingmicrosatellite and non-microsatellite regions, all supportingseparate grouping of M. canis from M. audouinii and M.ferrugineum.27
No intraspecific variation was observed in the calmod-ulin locus of our M. canis strains. This finding was in con-cordance with Brilhante et al.74, who used RAPD and PCR-RFLP targeting ITS regions to show that all human andanimals isolates of M. canis were genetically identical inspite of differing morphological characteristics. Similarly,no specific fragment patterns were observed among strainsof M. canis using repetitive sequence PCR-based DNA fin-gerprinting, as demonstrated by Pounder et al.75
Microsporum canis and M. ferrugineum were distin-guished by 2 bp in ITS2,33 while the two species differed by7 nt on calmodulin gene analysis, suggesting that calmod-ulin is more useful than ITS for species delineation of thesetwo taxa. Nonetheless, BT221 and Tef-1α22 genes (12 and10 nt differences, respectively) appear to better resolve phy-logenetic relationships between the species.
The average pairwise sequence divergence between M.canis, M, audouinii, and M. ferrugineum was 5.33 nt forboth calmodulin and ITS21; therefore, the calmodulin geneis just as useful as ITS for differentiation of the three species,while the discriminatory power of Tef-1α22 and BT221 wasfound to be higher than that of calmodulin and ITS in thiscomplex.
In agreement with analysis of BT221 and Tef-1α22 genes,calmodulin gene sequence analysis sees M. gallinae locatedat a separate position, next to the members of M. caniscomplex.
E. floccosum along with M. cookei and M. racemosumformed a monophyletic group, supported by a 100% boot-strap value (Fig. 2). The geophilic dermatophytes M. cookeiand M. racemosum (anamorphs of Arthroderma) have beenreported to cause superficial infection of animals and occa-sionally of humans, while E. floccosum is a common an-thropophilic agent involved in human infections.76 Inter-estingly, these three species had identical sequences (100%similarity), being indistinguishable from each other basedon calmodulin gene analysis. Likewise, Kawasaki et al.8
used RFLP analysis of mitochondrial DNA and concludedthat Epidermophyton could not be separated from the gen-era Trichophyton and Microsporum. On the other hand, E.floccosum is separated from the other mentioned species byITS,23 BT2,21 and Tef-1α.22 In contrast, based on ITS analy-sis, Graser et al.23 concluded that E. floccosum is somewhatrelated to the anthropophilic Trichophyton, evidenced by along paraphyletic branch.
Since calmodulin sequences were identical for M. cookeiand 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
Calmodulin sequence analysis showed that the geophilicstrains M. fulvum, M. gypseum, and A. incurvatum alongwith the less human-pathogenic strains such as M. nanum,A. gertleri, and M. persicolor formed a distinct group, sep-arated from the other geophilic species. The close relation-ship between the strains of all these species in the phyloge-netic tree is also supported by ITS,23 BT2,21 and Tef-1α22
sequence data.Based on phenotypic methods, M. fulvum and M. gyp-
seum are similar in macro- and micromorphological as-pects,76,78 but these two species are genetically distinct,having different calmodulin, mitochondrial DNA,79 ITS-rDNA,80 BT2,21 and Tef-1α22 sequences, with the last geneexhibiting highest resolution.
Microsporum gypseum, the most common geophilicspecies implicated in human dermatophytosis, is linked totwo teleomorphs, A. gypseum and A. incurvatum. Despitemany morphological similarities, these two teleomorphsdiffered by a total 56 bp across the calmodulin gene.
Nevertheless, the resolution of the calmodulin gene wasinadequate to separate M. nanum and A. gertleri strains.However, due to the low number of tested species, furtherstudies on more isolates are required to confirm the results.
In conclusion, in the present study the phylogeneticpositions of dermatophytes, which are fungi pathogenicto humans, were assessed based on nucleotide sequencesof the calmodulin gene as a new genetic marker. Thedata reported here provide a basis for further discov-ery of relationships between species. Dermatophyte treetopologies were almost in concordance with those observedfor other loci, such as ITS, BT2, and Tef-1α. The fol-lowing taxa with the same sequences are so closely re-lated that they are probably conspecific: E. floccosum/ M.cookei/M. racemosum and A. gertleri/A. obtosum. There-fore, studies of other loci as well as more isolates of zo-ologic and geophilic species are necessary to confirm theresults.
Acknowledgements
This work was financially supported by Tehran University of MedicalSciences (grant no. 92-01-27-21559), Tehran, Iran. We thank allpersonnel in Molecular Biology Laboratory in TIMM and in thereference collection of Centraalbureau voor Schimmelcultures (CBS),The Netherlands.
Declaration of interest
The authors report no conflicts of interest. The authors alone areresponsible for the content and the writing of the paper.
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