A STATISTICAL ANALYSIS OF SPORE SIZE IN THE GENUS * NORMAN F. CONANT, Pa.D. (Received for publication February 24, 1941) The need of a mycologic approach to the problem of identifica- tion and classification of the has long been recog- nized. It became apparent early that Sabouraud's method of establishing genera on the clinical appearance of fungi in lesions of the scalp, and, of establishing species on the type of macroscopic growth produced by these fungi in culture, was not entirely satis- factory. Although dermatophytes when growing in the hair maintained fairly constant patterns which served to distinguish the genera Microsporum and , when invading glab- rous skin they could not be differentiated because they appeared only as fragmented, branching, filamentous forms lacking dis- tinguishing characters. In this respect, a Microsporum could be identified from a lesion on the scalp by the microscopic ap- pearance of the invaded hair, but, from a lesion on the glabrous skin, the would have to be isolated and then identified by the character of the spore forms which it developed in culture. It was also found that the clinical appearance of skin lesions could not be used to separate these fungi. Similar or identical fungi were found to be associated with different types of lesions and diverse fungi were found to be associated with clinically similar lesions. Moreover, Achorion &hoenleini was not always associated with clinical favus, since and Trichophyton violaceum occasionally provoked typical favus "godets" and were identified as Achorion gypseum and Achorion violaceum under these conditions. It became necessary, there- *Fromthe Department of Bacteriology, Duke University School of Medicine, Duke Hospital,' Durham, N. C. 'Aided by a grant from the John and Mary R. Markie Foundation. 265 266 THE JOURNAL OP INVESTIGATIVE DERMATOLOGY fore, to study these fungi critically from a botanical point of view in order to bring together morphologically related forms which had been widely separated on a clinical basis. This type of investigation was undertaken by Ota and Langeron (1), Grigorakis (2), Langeron and Miochevitch (3), and Guiart and Grigorakis (4). These authors made comparative studies of numerous dermatophytes in an attempt to establish a basis for classification on the morphology of the fungi as they grew in cul- ture. Most of the old familiar generic names were abandoned by these authors, however, and the species included in their pro- posed new genera were listed by name without adequate descrip- tion. Certain advances were made, however, when it was noticed that Achorion &hoenleini and Endodermophyton concentricum were morphologically similar to recognized Trichophyton species. This allowed the genera Achorion and Endodermophyton to be discarded. At the present time, therefore, the classification of the dermat- ophytes has been greatly simplified by the recognition of only three genera; namely, Microsporum, Trichophyton, and Epider- mophyton. The mycologic characters of these genera have been clearly defined by Emmons (5) and this grouping was shown to have certain advantages over, and not to be inconsistent with, the five clinical groups proposed by Sabouraud (6). Moreover, species in these three genera have been greatly reduced to simplify identification. In the genus Trichophyton a dozen or more species were reduced to synonymy with T. mentagrophytes by Langeron and Milochevitch (3), and several more were reduced to the single species. T. rubrum, by Ota and Kawatsuré (7). In the genus only one species, E. floccosum, is now recog- nized. The numerous species still included in the genera Trichophyton and Micros porum, however, need accurate descriptions to facili- tate their identification. Size, shape, and genesis of spore forms and other accessory organs to be found in culture should be so treated as to make them important diagnostic characters for specific differentiation. A recent attempt by Conant (8) to describe diagnostic characters for the separation of species of the SPORE SIZE IN GENUS MICROSPORUM 267

genus Microsporum included a cultural, biometric and taxonomic study of several isolations. These studies showed that a marked variability occurred in the gross cultural aspect and production of spore forms when these fungi were grown on agar media. On a medium of polished rice, however, the fungi consistently produced typical macroconidia (fuseaux) which were used, in part, for separating species according to their size, shape, character of wall and color. Because of extreme variability in the size of the macro- conidia of each isolate, several spores were measured, population curves plotted, and differences in spore size obtained by compar- ing the curves. These curves, however, were not treated by statistical methods to determine exact differences between the means, and questions were immediately raised as to true signifi- cance of these differences. The spore measurements from which these curves were derived have, therefore, been subjected to statistical analyses for the purpose of determining the value of spore size as a criterion for differentiating species in the genus Microsporum.

MATERIALS AND METHODS Each isolate or strain of Micros porum was grown on polished rice in 125 cc. Erhlemeyer flasks at room temperature. This medium was made of one part rice and three parts water and sterilized in free steam for one hour on each of three successive days. Bits of the sporulating mycelium from three-week-old cultures (18—21 days) were taken from the flasks and gently teased apart in a drop of lactophenol cotton blue. All measurements of the macro- conidia were made from these preparations by drawing the spores with the aid of a camera lucida accurately calibrated to 1000 x.Thesedrawings could, therefore, be measured with a millimeter rule and sizes directly read in microns. The spore curves for the eight species of Microsporum represent the size (length) of the macroconidia plotted against the number of macroconidia measured for each strain. The size interval included one micron; i.e. those macroconidia were grouped which fell within the length of 28—29 i,30—31 a, etc. For a statistical analysis of these curves the midpoint of these intervals was taken 268 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

(28.5 30.5,32.5 etc.)and Mainland's (9) formulae for standard deviation, standard error of the mean, and "t" were used. Probability for values of "t" was determined from Fisher's (10) tables of "t" or

STATISTICAL ANALYSIS The curves representing the macroconidia of eight species of Microsporum are presented in figure 1. These curves are essen-

130

10

90 Ui 0 U,70

Ui §0 z

30

60 LENGTR IN tially the same as those published previously (8, II) except for the additional number of spores measured for M. pseudolanosum and M. aurantiacum. From this figure the curves for macro- conidia of M. fulvum, M. gypseum and M. canis, with calculated means at 39.56 ,44.96jand73.71 respectively, would seem to indicate three sharply differentiated species. Those curves representing the macroconidia of M. equinum, M. pseudolanosum, and M. aurantiacum, however, not only overlap each other but were overlapped by the spore curve of M. canis. Likewise, the spore curves of M. obesum and M. simiae seriously overlap and SPORE SIZE IN GENUS MICROSPORUM 269 much of their enclosed area falls within that of the spore curve of M. pseudolanosum. Because of the overlapping of these curves, their value for differentiating species is greatly impaired unless a statistical

TABLE I MEASUREMENTOF MACROCONIDIA OF SPECIES PLOTTED IN FIGURE 1

0!4 2 z U

! J 26—29 4 30—31 13 32—33 30 34—35 53 2 36—37 90 8 38—39 129 39 1 40—41 113 69 4 42—43 66 95 3 44—45 41 127 II 46—47 27 107 Ii 2 48—49 19 71 19 5 2 I 50—51 6 53 21 II I 4 52—53 4 23 19 6 12 18 I 54—55 4 6 17 32 14 16 2 I 56—57 I Il 33 17 20 6 4 58—59 15 26 20 29 10 9 60—61 12 25 24 33 18 5 82—63 4 25 35 45 23 21 64—65 8 10 20 49 52 38 66—67 9 10 27 47 45 82 68—69 5 4 IS 47 47 96 70—71 I 0 8 35 42 112 72—73 4 I 2 29 44 129 74—75 I I 2 II 23 lIZ 76—77 I I I II 24 101 78—79 I 3 29 93 80—81 2 7 70 62—83 8 44 84—85 6 36 66—87 6 IS 88—89 5 II 90—91 2 7 92—93 9495 I 96—97 NUMBER 600 600 200 200 200 400 400 1000 MEAN 39.5644.96 55.00 58.29 61.79 64.4770.07 73.71 STANO.DEV.*4.55*3.87 *7.0045.05 *6.51 S.E.MEAN*0.186 *0.381 *0.347

analysis of them could establish significant differences between their means. The measurement of the macroconidia of the species plotted in figure 1 is presented in table I from which an analysis was made of each of the eight species. A comparison 270 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY of the means of these curves (table II) revealed high "t" values which when referred to Fisher's tables of "t" or "x" showed them to be highly significant. These spore curves would seem to have a significant difference which could not be due to sampling fluc- tuations alone and, therefore, might be considered as representing distinct species. Of the species studied, however, only three M. fulvum, M. gypseum and M. canis, were represented by more than one isolate. The spore curves of each of these species and the number of strains studied are presented in figure 2. The curves of M. fulvum and M. gypseum, representing a spore count of 600 macroconidia each, have a calculated mean of 39.56 u and 44.96 respectively. A comparison of these means showed them to be statistically

TABLEii STATISTICAL COMPARISON OF SPECIES TREATED IN TABLE I

M.FULVUM M.OBESUM M.SIMIAE M.EQUINUM M.P$EUOOLANOSUMI M.AURANTIACUMI M. GYPSE%JM M.SIMIAE M. E0UINUM M.PSEUDOLANOSIJM M. AURANTIACUM M.CANIS

t 22.1 4.9 6.6 5.0 11.81 9.03 p > 000.000.001>000,001>000,000,001 > .000.001 > .000,000.001 > .000,000,001 significant (table II). To evaluate properly these curves and the curve of M. canis representing a spore count of 1000 macro- conidia it was necessary to establish the identity of the several strains from which these curves were derived. The measure- ment of the macroconidia of these strains is presented in table III. A comparison of the means of these curves revealed statis- tically significant differences among those strains thought to represent a single species (table IV). In this respect, M. fulvum 24—483 and 483—N. Y. were highly significant. On the other hand, the curves of M. fulvum N. Y. and M. gypseum 12 showed no significant difference and could be considered as representing a single species rather than different species. Likewise, M. gypseum 12—13 were significantly different in contrast with M. gypseum 13—12A which were not significantly different and might FIGURE 2. SPORE CURVES FOR THREE STRAINS OF M. FULVUM. THREE STRAINS OF M.GYPSEUM. AND 5 STRMNS 140 OF M CANIS

120 0

J2 too

I)) M. M.CANIS La M.FULVUM GYPSEUM z 0 0. LI) 0I1

La 0 z

32 36 40 44 48 52 38 42 46 50 54 82 86 90 94 LENGTH IN 272 THEJOURNALOF INVESTIGATIVE DERMATOLOGY be considered as representing a single species. Within these two species, M. fulvum and M. gypseum, therefore, if a statistical analysis of the spore sizes of the different strains should be taken

TABLEIll MEASUREMENT OF MACROCONIDIA OF STRAINS PLOTTED IN FIG. 2

28-29 4 i 30—31 II 2 32—33 22 8 34—35 21 24 8 I I 36—37 45 33 2 4 I 3 38— 39 52 50 27 23 I S 40—41 26 51 36 40 13 6 42—43 II 23 32 41 27 27 44—45 7 6 28 44 48 35 46—47 0 3 24 32 34 41 48—49 I lB S 36 27 50—51 6 6 19 28 52—53 4 I 8 14 54—55 4 2 4 I 56—57 I 3 I 58—59 3 I I 4 60—61 5 4 0 2 4 62—63 9 6 0 I 5 64—65 II 10 B 6 3 66—67 20 9 14 16 13 68—69 IS 20 24 20 14 70—71 25 25 30 19 13 72—73 23 32 3f 28 17 74— 75 24 20 28 9 21 76—77 9 14 27 19 22 78—79 13 9 15 20 26 80—81 14 II 2 17 16 82—83 7 7 5 9 16 64—65 3 5 3 12 13 86—87 2 3 I 4 5 88—89 I I I 6 90—91 2 I 3 3 92—93 I 2 94..95 I 96— 97

NUMBER 200 200 200 200 200 200 200 200 200 200 200 MEAN 37.0238.68 42.97 43.0945.60 46.18 71.97 72.6273.2475.17 75.55 STAND. 0Ev43.65 46.44 *7.28 S.E.MEAN4.258*21 94.3234.2314.262 2.445 4.3484.4714.514 to indicate different species it would suggest that these two species could be further broken down into more species. Likewise, a statistical analysis of the spore size of those strains included in the species M. canis showed significant differences between the means of M. canis 19—27, table IV. While the means SPORE SIZE IN GENUS MICROSPORUM 273 of the strains which closely approximated each other were not significantly different, a comparison of the means of 5—19, 5—27, and 5—33 gave very significant differences as did a comparison of the means of 486—27, 486—33, etc. Because of these differences a test of the variance of the five strains of M. canis was made to determine whether the canis group contained similar or different elements, Table V. In Snedecor's (11) table of F, where n1 =

TABLE IV STATISTICAL COMPARISON OF STRAINS TREATED IN TABLE III

U FULVUM N.Y. U.GYPSEUM M.GYP5EUMI2 Ia_Ia

TABLE V

VARIANCE OF THE 5 STRAtNS OF M. CAN IS

VARIATION SS DF MS

BETWEEN CLASSES I.966.66 4 496.67

WITHIN CLASSES 40,469.14995 40.67

TOTAL 42,455.80999 F 496.67 12.21 40.67

and n2 =4,the 5 per cent level was found to be 5.63 and the 1 per cent level 13.46. Since 12.21 falls between these two figures, and especially as it does not exceed the one per cent level, these five strains might be suspected of including different ele- ments and probably should not be treated as identical. To break up the fulvum, gypseum and canis groups into more species on the evidence of statistically significant differences be- tween the means of the spore curves of several isolates, however, would disregard the possibility of an extreme variation within a 274 TRE JOURNAL OF INVESTIGATIVE DERMATOLOGY single species which might account for these differences. That such extreme variations could be obtained with a single species of Micros porum, when examined under different conditions, was demonstrated by Dowcling and Orr (12). When they cul- tured M. canis on Sabouraud's medium, the mean spore length of a hundred spores was found to be 119 ,but,when cultured on polished rice, to be 90 Furthermore,on a single medium, Sabouraud's agar, the difference in mean spore length was found to be 16 when spores from a 20-day and from a six-week-old culture were compared. Because of such variations on different media or with different aged cultures on the same medium, they based their statistical comparisons of mean spore size of strains TABLEI STATISTICAL COMPARISON OF CANADIAN STRAINS (BASED — ON DOWDING AND ORR) COMPARISON O SPORE WIOTHS COMPARISON OV SPORE LENGTHS Al B B C A C E C C

MEAN 21,19 19.37 16.3? 18.67 21.19 18.87 90.1 102 02

S.D. 2.17 256 2.58 2.05 2 I? 2.05 9.9 11.9 119

t 14.3 7.6 7.8 7.72

p > .300,000.001> .300,000,001 > .300,000,001 ))00.00O,00l of M. canis on material cultured under identical conditions, i.e. from two-week-old cultures on polished rice at room temperature. Although Dowding and Orr (12) claimed no significant differ- ence between the mean widths of three Canadian strains of M. canis, a review of their data showed these widths to be sig- nificantly different, table VI. Likewise, a review of their data concerning the analysis of the mean spore length of their long- spored strains, for which no significant differences were claimed, showed a marked difference between the means of some of these strains,, table VI. These Canadian strains representing the single species M. canis, therefore, even when cultured under iden- tical conditions, showed significant differences between the means of their spore curves as did the strains of M. canis studied in this paper. SPORE SIZE IN GENUS MICROSPORUM 275

The species presented in table I, however, were separated not oniy on the size of their macroconidia but also on such additional characters as the size, character and arrangement of their support- ing structures, the coriidiophores. In the previous study (8, III), for example, it was found possible to associate the small size of the macroconidia of M. equinum with less fiexuous (stouter) and shorter conidiophores than was found in the larger spored M. canis. Such a description was also given by Dowding and Orr for a short-spored Canadian strain whose spore curve closely approximated the spore curve of M. equinum. In spite of this agreement in conidiophore structure and spore size between M. equinum and their short-spored strain, and in spite of the marked difference in mean spore length between these two strains and their long-spored M. canis, they included both of these strains in the species M. canis. Since they found recently isolated long- spored strains to differ 20 .tinmean spore length and found the mean of this short-spored strain to differ 26 from the mean of the smallest long-spored strain, they were probably justified in includ- ing all of these strains in a single species. Such a viewpoint of extreme variability would, therefore, bring M. obesum with a mean spore length of 55 u and all intervening species (M. simiae, M. equinum, M. pseudolanosum and M. aurantiacum) within the species concept of M. canis.

DISCUSSION The statistical analyses of the means of spore curves representa- tive of eight different species of Microsporum have shown the curves to be significantly different. On the other hand, an analy- sis of those species which were represented by more than one strain showed that the means of the spore curves of those strains also were in most cases significantly different. Therefore, if curves representing the length of the macroconiclia were used to separate species in the genus Microsporum on the basis of the statistical significance of their means alone, more species would have to be separated from the fulvum, gypseum and canis groups (tables III, IV). Such a splitting off of species, however, would ob- viously create a multiplicity of new forms and greatly complicate 276 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY the of the genus. The wide variation in spore size might be more logically regarded as an extreme variation of a single species. In the light of what is shown in the present study and with the additional knowledge gained from the Canadian strains, a more conservative view should be taken in regard to specific differentia- tion in the genus Microsporum. Our knowledge of the optimum cultural requirements of this group of fungi, with respect to the best use of nutrient materials under artfficial conditions, appears to be too meager as yet to allow for sufficient stability of mor- phological characters to be used advantageously in separating species. That the gross cultural characteristics of these fungi are too variable for use as differentiating characters has already been noted. To separate species on the variable character that the length of the macroconidia has proven to be, would neces- sitate additional species for each isolate obtained. Whether the type, arrangement, size and character of such structures as the conidiophores will be found to vary as widely as gross cultural characters and spore size will only be determined by as close a study of these structures as has been given to the other characters. It is conceivable, therefore, that the use of the above characters for specific differentiation would result in a limitless number of species which would soon become unmanageable and complicate an otherwise usable taxonomic treatment of the genus. The problem of classification is further complicated by the tendency of isolations of dermatophytes to lose their spore forms and become pleomorphic. Because of this aspect, it is not possible over a long period of time to collect numerous strains which may be used for comparative studies. It would seem, therefore, that only three species of Microsporum should be admitted, namely: M. Audouini, M. canis, and M. gypseurn. All other species described could be considered variations of these three species with the wide differences noted in character of colony, spore size, and supporting morphologic structures. As to the correct terminology of these species a survey of the literature showed M. Audouini Gruby 1843 (13) to be the first described. This species was described, however, from the para- SPORE SIZE IN GENUS MICROSPORUM 277 sitic aspect of the invaded hair and not from a cultural study of the fungus. When Sabouraud (14) later found this fungus in infected scalps, he drew attention to Gruby's publication and described the cultural characteristics of the fungus we now regard as M. Audouini. This species should become the type of the genus. The terminology of the other two species, M. canis and M. gypseum, however, has been greatly confused. Most of the earlier investigators were content to name their isolations Micro- sporum du chein, Microsporum of the cat, etc. and did not desig- nate legitimate binomials for their fungi. In this respect, Fox and Blaxall (15) have been cited often as the authors of Microsporum felineum when the binomial does not appear in their papers Mewborn (16), however, did publish M. felineum as a valid bino- mial but he also referred to Bodin's "Les champignons parasites de l'homme" (Paris, Masson et Cie, 1902) in which the binomial M. canis had previously appeared. Since M. felineum, Mewborn 1902, M. equinum Gueguen 1904, and M. lanosum Sabouraud 1907 may be considered to be the same species, the acceptable name according to the rules of priority should be Microsporum canis Bodin 1902. The species to designate the fulvum-gypeeum group also presented disputable evidence. Microsporum fulvum Uriburu 1907 was cited by Sabouraud (17) as a fungus encoun- tered in Buenos Aires and sent to him for identification. The reference for a publication of this name, previous to Sabouraud's description, however, has been quoted throughout the literature as "Argentina meclica, 1909". Since M. gypseum [Bodin 1907 (18)] Guiart and Grigorakis 1928 (4) is the same fungus and its original description preceded that of M. fulvum (Uriburu 1907— 1909?), Sabouraud 1910 (17), it should become the accepted specific name.

CONCLUSIONS A statistical analysis of spore curves representing several strains of the genus Microsporum has shown the character of spore length to be too variable for specific differentiation. A more conserva- tive viewpoint in the classification of the genus would result in a 278 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY more usable taxonomic treatment by limiting the genus to three species, namely: M. Audouini Gruby 1843, M. canis Bodin 1902 and M. gypseum (Bodin 1907) Guiart and Grigorakis 1928. REFERENCES (1) Om, M., AND LANGSRON, M.: Nouvelle classification des dermatophytes. Ann. de parasitol., 1:305, 1923. (2) GRIGORAKIS, L.: Recherches cytologiques et taxonomiques sur les der- matophytes et quelques autres champignons parasites. Ann. d. sc. nat. bot., 7:165, 1925. (3) LANGERON, M., AND MILOCREVITCH, S.: Morphologic des dermatophytes sur milieux naturels et milieux a base de polysaccharides: Essai de classification. Ann. de parasitol., 8: 465, 1930. (4) GUIART, J., ANDGRIG0RAKI5,L.: La classification botanique des cham- pignons des teignes. Lyon med., 141:369, 1928. (5) EMMON, C. W.: Dermatophytes; natural grouping based on the form of the spores and accessory organs. Arch. Dermat. & Syph., 30: 337, 1934. (6) SABOURAUD, R.: Généralités concernant les dermatophytes. Ann. de dermat. et syph., 10: 236, and 569, 1929. (7) OTA, M., AND KAWATSUR& S.: Sur le Sabouraudites ruber et ses varieties. Ann. de parasitol., 11: 476, 1933. (8) CONANT, N. F.: Studies in the genus Micros p0mm: I. Cultural studies. Arch. IDermat. & Syph., 33: 665, 1936. II. Biometric studies. Ibid., 34: 49, 1936. III. Taxonomic studies. Ibid., 36: 781, 1937. (9) MAINLAND, D.: The treatment of clinical and laboratory data. Oliver and Boyd, London, 1938. (10) FISHER, R. A.: Statistical methods for research workers. Oliver and Boyd, pages 80 and 177, London, 1938. (11) SNEDECOB, G. W.: Statistical methods. Collegiate Press, Inc., Ames, Iowa, 1938. (12) D0wDING, E. S., AND Oaa, H.: The Microsporum lanosum. Mycologia, 31: 76, 1939. (13) GRUnT, M.: Recherches sur la nature, le siege et le development du porrigo decalvans ou phytoalopecie. Compt. rend. Acad. d. sc., 17: 301, 1843. (14) SABOUEAUD, R.: Sur un mycose innominée de l'homme, la teigne tondante spéciale de Gruby, Micros porum Audouini. Ann. Inst. Pasteur, 8: 83, 1894. (15) Fox, T., AND BLAXALL, F. R.: An inquiry into the plurality of fungi causing ringworm in human beings as met with in London. Brit. J. Dermat., 8: 241, 291, 337 and 377, 1896. (16) MEWBORN, A. D.: A case of ringworm of the face, and two of the scalp contracted from a Microsporon of the cat with some observations on the identification of the source of infection in ringworm by means of cultures. New York Med. Jour., 76: 843, 1902. (17) SABOURAUD, R.: Les Teignes, Masson et Cie., page 241, Paris, 1910. (18) BODIN, E.: Sur un nouveau champignon du favus (Achorion gypseum). Ann. de dermat. et syph., 8:585, 1907.