- Email: [email protected]
Melanin as the spore wall pigment of some myxomycetes
Mycol. Res. 92 (3), 286-292 (1988)
286
Printed in Great Britain
Melanin as the spore wall pigment of some myxomycetes
P. LOGANATHAN, P. PARAMASIV AN AND INDIRA KAL Y ANASUNDARAM Centre for Advanced Study in Botany, University of Madras, Guindy Campus, Madras 600 025, India
Melanin as the spore wall pigment of some myxomycetes. Mycological Research 92 (3): 286-292 (1989). Spore wall pigments from twelve species of myxomycetes, belonging to the orders Physarales (6), Stemonitales (5) and Liceales (1) were extracted with 1 M-KOH and identified as melanin on the basis of various physico-chemical tests, ultraviolet and infrared spectra. While this was the only pigment in the physaraceous and stemonitoid species, the liceaceous species, i.e. Reticularia lycoperdon, had an additional ethanol-extractable yellow-green pigment. The uv and i.r. spectra of melanins of the Physarales and the Stemonitales were more similar to each other than to those of Reticularia melanin. Key words: Melanin, Myxomycetes, Spore wall pigment.
In myxomycetes, the dark pigment of the spores of the Physarales and Stemonitales is assumed to be melanin (Aldrich & Blackwell, 1976), although this has been verified only in the much-investigated Physarum polycephalum (McCormick et a!', 1970; Chet & Huettermann, 1977) and in FuNgo septica (Chapman, Nelson & Orlowski, 1983). To our knowledge, there is no report on the nature of spore pigment in the Stemonitales, an order in which we in this laboratory have long been interested. A comparative study of the nature of spore wall pigments in a few members of the Stemonitales and
Physarales (taxonomically separated from the remaining orders of the subclass Myxogastromycetideae on the basis of spore colour, which is usually some shade of purple-brown) was made. A brown-spored species of the Liceales, namely Reticularia lycoperdon, was also included for comparison.
MATERIALS AND METHODS Myxomycete species The material studied is listed in Table 1.
Table 1. Species studied, herbarium accessions and sources. Accession number Physarales Physarum melleum (Berk. & Broome) Massee P. echinosporum A. Lister Didymium minus (A. Lister) Morgan Diderma effusum (Schwein.) Morgan Fuligo intermedia Macbr. Craterium leueoeephalum (Pers.) Ditmar
MUBL/IK/MH 91 MUBL/IK/FC 23 MUBL/IK/FC 48 MUBL/IK/FC 25 MUBL/IK/FC 98 MUBL/IK/FC 83
Stemonitales Stemonitis fusca Roth' 5. splendens Rost.t 5. herbatica Peck Comatricha aequalis Peck' Lamproderma seintillans (Berk. & Broome) Morgan
MUBL/IK/TNL 1 MUBL/IK/RS 1 MUBL/IK/SH 5 MUBL/IK/TNL 3 MUBL/IK/MH 82
Liceales Reticularia lycoperdon Bull. a
MUBL/IK/23
, Supplied by Professor T. N. Lakhanpal from Shimla. t Supplied by Dr Rajesh Sharma from Punjab. a Supplied by Professor Lilian Hawker from Bristol. U.K. The remaining species were collected locally by the authors.
287
P. Loganathan, P. Paramasivan and Indira Kalyanasundaram
Extraction and purification Initial attempts at extraction of pigment was made with Stemonitis herbatica using various organic solvents, including acetone, methanol, ethyl acetate, chloroform, hexane, tertiary butyl alcohol, benzene and petroleum ether, either singly or in various combinations. Since no pigment was extracted from whole spores in any of these solvents, the process was repeated using empty spore cases separated by sedimentation after germination of spores, so that the inner surface of the spore walls was also exposed to the action of the solvent. As the pigment was not extracted by any of the above procedures, a melanin extraction procedure (Ellis & Griffiths, 1974; Chet & Huettermann, 1977) was followed, using 1'0 M potassium hydroxide solution. Spores were boiled in this solution for 2 h, cooled, and centrifuged at 32°C at 2000 rev. min-I. After collecting the brown supernatant, the residue was re-extracted with 0'1 M KOH and the process was repeated until the supernatant was clear. The extract thus collected was blackish brown, and from this the dark brown pigment was precipitated out by acidification with 1 N HCl up to pH 2. The pigment was washed in three changes of distilled water and dried overnight at 21° in a dehumidified atmosphere. Purification was done by hydrolysing the dried powder in a sealed gas vial with 5 ml of 7 M HCl for 2 h at 100°. After cooling, it was washed several times with distilled water and dried as before.
Identification of pigment The purified pigment was subjected to various physical and chemical tests (Thomas, 1955) as detailed in Table 2. For the spectra in the uv and visible range, known amounts of powder were dissolved in 1'0 M KOH and read for absorption in a double-beam Hitachi 150--20 spectrophotometer at 200--500 nm. For i.I. spectra, the purified pigment was ground with potassium bromide, pressed into disks under vacuum and the spectra recorded in a Perkin-Elmer PE 781 IR spectrophotometer at 4000-600 em-I.
Residual pigment Microscopy of the spores or spore cases after melanin extraction showed them still to have a pale reddish colour. It was therefore suspected that a second pigment may be present, and attempts were made to extract this using all the organic solvents named earlier, but it was not extractible. Further attempts to understand the nature of this pigment were made through histochemical and other tests (Durrell, 1964; Tracey, 1955). The procedures described above were repeated with the remaining eleven species, with slight modifications where necessary, except the last-mentioned histochemical and other tests for residual pigment, which were made only with Stemonitis herbatica.
RESULTS All twelve species had a pigment that could be extracted with 1 M KOH, giving a brown or purple-brown solution. Precipitation of the pigment occurred readily when these solutions 21
Table 2. Diagnostic tests for melanin.
Species
Colour of solution in KOH
Physarum melleum P. echinosporum Didymium minus Diderma e/fusum Fuligo intermedia Craterium leucocephalum Stemonitis Fusca S. splendens S. herbatica Comatricha aequalis Lamproderma scintillans Reticularia lycoperdon
Light brown Light brown Light brown Light brown Dark brown Light brown Reddish brown Dark brown Brown Brown Light brown Brown
All species had the same reactions to the following tests: Reaction with H 2 0 2 Solubility in water Decolourized Insoluble Reaction with Solubility in organic solvents (acetone, ethanoL chloroform) sodium dithionite and potassium Insoluble ferricyanide Decolourize first and tum brown when potassium ferricyanide is added Reaction with Solubility in 1 m-KOH (100 0 ammoniacal silver for 2 h) nitrate Soluble solution Form a greycoloured silver precipitate lining the sides of the test tube Dialysis through CelPrecipitation in 3N-HCl lophane membrane Precipitate readily, forming floccose precipitates 27/32 Does not pass Reaction for polyphenol tests through membrane (FeCl 3 test) Forms a reddish orange test precipitate
were acidified to pH 2 with 3 N HC!. The pigments from all the species dissolved completely when placed in 1 M KOH at 100° for 2 h.
Physical and chemical properties The physical and chemical properties are summarized in Table 2. The pigments from all 12 species had similar properties. They were insoluble in water; not extracted with organic solvents like acetone, chloroform, ethanol, or a mixture of these solvents; decolourized with hydrogen peroxide, and gave a positive reaction for polyphenols, producing a reddish orange precipitate with 1 % FeCi a solution. The pigment solutions were reduced with 1 % sodium dithionite solution, MYC 92
Melanin in myxomycete spore walls
288
Fig. 1. Ultraviolet and visible spectra of the melanins extracted from members of the Physarales. 3
3
Craterium leucocephalum
Didymium minus
3
j
Fuligo intermedia
Physarum echinosporum
-<
3
Diderma ejJusum
Physarum melleum
250
350
450
250
350
450
Wavelength (nm)
becoming colourless, but turning brown by oxidation after 1 % potassium ferricyanide was added. Bleaching took place when the solution was treated with ammoniacal 1 % silver nitrate solution, and a grey-coloured silver precipitate lined the inside of the test tubes on boiling. The pigments appeared to be of high molecular weight because of their inability to dialyze through cellophane membranes from alkaline solutions. From these properties, the pigments were identified as melanin in all cases.
Ultraviolet spectrum The absorption spectra showed characteristic absorption peaks in the uv regions of wavelengths ranging from 200 to 250 nm, but none in the visible region. The overall uv spectra were similar in the three orders, except for small interspecific differences. All the physaraceous species showed characteristic absorption peaks at 240 nm, except P. echinosporum and D. minus in which the peaks occurred at 220 nm (Fig. 1).
In Stemonitales the characteristic bands occurred at 220 nm in all species. In S. herbatica, however, additional bands appeared at 230 and 240 nm (Fig. 2). Because of this dissimilarity with the remaining species of the order, the test was repeated using two additional specimens, obtained in culture from the original material with and without illumination. The melanin extracted from spores of these specimens also showed a uv spectrum identical with that of the natural one. The melanin solutions of R. lycoperdon also gave absorption peaks at 220 and 240 nm, and these were very sharp, in contrast to the somewhat diffuse bands of the Physarales and Stemonitales (Fig. 2). The graphs obtained by plotting the log absorbance against wavelength (200-500 nm) showed similar negative slopes for most of the species, decreasing gradually from 300 to 400 nm but moving slightly upwards at 500 nm suggesting increased absorbance.
P. Loganathan, P. Paramasivan and Indira Kalyanasundaram
289
Fig. 2. Ultraviolet and visible spectra of the melanins extracted from members of the Stemonitales and from Reticularia /ycoperdon. 3
Stemonitis herbatica
Reticularia Iycoperdon
3
3
Stemonitis splendens
i
Lamproderma scintillans
«
3
3
Stemonitis fusca
250
350
Comatricha aequalis
450
250
350
450
Wavelength (nm)
Infrared spectrum
The Lr. spectra of the melanin from members of the Physarales and Stemonitales were broadly similar. A characteristic curve in the range of 9'7 to 10'2 ~m, a very sharp band at 7'2 ~m, and a broad band in the range of 6' I to 6'3 ~m were the salient features in all species. Less marked absorption bands occurred at 4'3 ~ and at 3'4 ~m. The latter was rather marked in S. herbatica and in R. lycoperdon. Stemonitis herbatica also showed another sharp band at 5'9 J..lm, which was faint in the remaining species (Figs 3, 4). In R. lycoperdon, as in other species, the sharpest band was seen at 7'2 ~m, and less marked ones at 6'2 and 4'3 ~m. The most characteristic feature, however, was a prominent band occurring at 9'2 ~m, which was not clear in the remaining species. Residual pigments
The spore residues of Physarales and Stemonitales were not extractable in organic solvents such as acetone, ethanol,
chloroform or a mixture of these solvents. However, a greenish yellow pigment was extracted from R. lycoperdon with ethanol, but not with chloroform or acetone. The residue of S. herbatica gave a positive reaction to the Nile Blue test for melanin. When the residue was treated with a saturated solution of KOH at 160 0 to digest most components other than tensile wall substances, the pigment was not dissolved but remained in the insoluble residue.
DISCUSSION The identification of the spore wall pigments of the Physarales and the Stemonitales as melanin supports the general presumption that the purple-brown pigments in these two orders are this substance (Aldrich & Blackwell, 1976). In the eleven species studied, after extracting the pigment with I M KOH, the residue was still coloured but no further extraction was possible with either KOH or organic solvents. The results of experiments with S. herbatica suggest that the 11-1
Melanin in myxomycete spore walls
290
Fig. 3. Infrared spectra of melanins extracted from physaraceous species. Wavelength (pm)
3·5
4·0
4·5 5·0
6·0
7·0
8·0
9·0 10
12 14
Physarum melleum
3000
2500
2000
1800
1600
1400
1000
800
Wave number (em-I)
residual pigment is melanin, which occurs in a bound form with some tensile component of the cell wall and hence is not extractable. While it may be reasonably assumed that melanin is universal in the Physarales and Stemonitales, it is not restricted to these orders. Some of the traditionally 'brightspored' groups also contain melanin, as seen from R. lycoperdon in this study. In these, however, it occurs as an additional pigment. It would be of interest to study the spore wall pigments of the Echinosteliales, which have been separated from the Stemonitales on the basis of plasmodial type and sporangial size (Martin, 1960), to see if they are common to the two orders. From the ultraviolet and infrared spectra, the melanins from the six physaraceous and five stemonitoid species are fairly similar, and may belong to the same chemical group. They cannot. however, be characterized at this stage as belonging
to anyone of the four types of fungal melanin described in the literature (Bell & Wheeler, 1986). The only attempt to characterize myxomycete melanin is that of Ward & Havir (1957) whose results suggest that it is neither DOPA-melanin nor catechol-melanin, but are still inconclusive. In the present study, the Physarales and the Stemonitales showed slight differences in uv spectra, in that the Physarales usually had an absorption band at 240 nm and the Stemonitales, at 220 nm. However, the few exceptions, having a band at 220 nm in the Physarales and at 240 nm in the Stemonitales, suggest an integradation of the two orders. Small differences in absorbance within a narrow range of wavelength, occurring between species in the uv as well as i.r. spectra, might be explained on the basis of some heterogeneity in the complex melanin molecule. Stemonitis herbatica. however, showed several additional peaks both in the uv and i.r. spectra.
P. Loganathan, P. Paramasivan and Indira Kalyanasundaram
291
Fig. 4. Infrared spectra of melanins extracted from stemonitoid species and from Reticularia Iycoperdon. Wavelength (pm)
3·5
4·0
5·0
6·0
7·0
8·0
9·0 10
12 14
Comatricha aequalis
3000
2500
2000
1800
1600
1400
1000
800
Wave number (em-I)
The melanin from spores of this species obtained from different environmental conditions, did not show any intraspecific differences. The soil-inhabiting nature of this species, contrasting with the wood-inhabiting nature of most species of Stemonitis (Indira, 1968), might also suggest an evolutionary divergence. In R. lycoperdon, the Lr. spectrum showed a prominent band at 9'2 IJm, which was not clear in the physaraceous or stemonitoid species. In fact the Lr. spectrum in this species showed a strong resemblance to that of Agaricus bisporus melanin described by Rast et al. (1981) as being characteristic of GDHB (y-glutaminyl-3A-dihydroxybenzene) melanin, and might belong to this group. The separation of the Stemonitales into a separate subclass, Stemonitomycetidae, on the basis of differences in sporangial morphogenesis, with endogenous stalk development (Ross,
1973), has been supported by Alexopoulos (1978). On the other hand, Kalyanasundaram (1978) states that stalk and capillitial development in Stemonitis is basically similar to that in other Myxomycetes. Considering the similarity between the spore wall pigments of the Stemonitales and the Physarales it is suggested that these two orders are more closely related to each other than to the Liceales or the Trichiales. In the Eumycota, there seems to be some relationship between taxonomy and melanin structure (Bell & Wheeler, 1986). Elucidation of the significance of spore wall pigments in phylogenetic detenninations of myxomycetes, however, should await more extensive studies. We thank the Director of this Centre for facilities, and those mentioned earlier for supplying material of the Stemonitales and Liceales which are not frequently seen in tropical climates.
Melanin in myxomycete spore walls The second author thanks the University Grant Commission, New Delhi, for the award of a Junior Research Fellowship.
REFERENCES ALDRICH, H. C. & BLACKWELL, M. (1976). Resistant structures in the Myxomycetes. In The Fungal Spore: Form and Function (ed. D. J. Weber & W. M. Hess), pp. 413-461. New York: John Wiley. ALEXOPOULOS, C. J. (1978). The evolution of the taxonomy of the Myxomycetes. In Taxonomy of Fungi I (Proceedings of the International Symposium on Taxonomy of Fungi, 1973, ed. C. V. Subramanian), pp. 1-8. Madras, India: University of Madras. BELL, A. A. & WHEELER. M. H. (1986). Biosynthesis and functions of fungal melanins. Annual Review of Phytopathology 24, 411-451. CHAPMAN, C. P., NELSON R. K. & ORLOWSKI, M. (1983). Chemical composition of the spore case of the acellular slime mold Fuligo septica. Experimental Mycology 7, 57--65. CHET, l. & HUETTERMANN, A. (1977). Melanin biosynthesis during differentiation of Physarum polycephalum. Biochimica et Biophysica Acta 499, 148-155. DURRELL, L. W. (1964). The composition and structure of walls in dark fungus spores. Mycopathologia et Mycologia applicata 23, 339-345.
ELUS, D. H. & GRIFFITHS, D. A. (1974). The location and analysis of melanins in the cell walls of some soil fungi. Canadian Journal of Microbiology 20, 1379-1386. (Received for publication 5 May 1988)
292
INDIRA, P. U. (1968). Some slime-molds from Southern India. IX. Distribution, habitat and variation. journal of the Indian Botanical Society 67, 330-341. KALYANASUNDARAM, l. (1978). Capiliitial development in Stemonitis. In Taxonomy of Fungi I (Proceedings of the International Symposium on Taxonomy of the Fungi, 1973, ed. C. V. Subramanian), pp. 9-13. Madras, India: University of Madras. MARTIN, G. W. (1960). The systematic position of Myxomycetes. Mycologia 52, 119-129. McCORMICK, J. 1.. BLOMQUIST, J. S. & RUSCH, H. P. (1970). Isolation and characterization of a galactosamine wall from spores and spherules of Physarum polycephalum. journal of Bacteriology 104, 1119-1125.
RAST, D. M, STUSSI, H.. HAGNAUER. H. & NYHLEN, L. E. (1981). Melanins. In The Fungal Spore: Morphogenetic Controls (ed. C. Turian & H. R. Hohl), pp. 507-531. New York: Academic Press. ROSS, l. K. (1973). The Stemonitomycetidae, a new subclass of Myxomycetes. Mycologia 65, 477-485. THOMAS, M. (1955). Melanins. In Modern Methods of Plant Analysis 4 (ed. K. Paech & M. V. Tracey), pp. 661--675. Berlin: SpringerVerlag. TRACEY, M. V. (1955). Chitin. In Modern Methods of Plant Analysis, 2 (ed. K. Paech & M. V. Tracey), pp. 264-274. Berlin: SpringerVerlag. WARD, J. M & HAVIR, E. A. (1957). The role of 3:4 dihydroxytoluene, sulfhydril groups and cresolase during melanin formation in a slime mould. Biochimica et Biophysica Acta 25, 440-442.
286
Printed in Great Britain
Melanin as the spore wall pigment of some myxomycetes
P. LOGANATHAN, P. PARAMASIV AN AND INDIRA KAL Y ANASUNDARAM Centre for Advanced Study in Botany, University of Madras, Guindy Campus, Madras 600 025, India
Melanin as the spore wall pigment of some myxomycetes. Mycological Research 92 (3): 286-292 (1989). Spore wall pigments from twelve species of myxomycetes, belonging to the orders Physarales (6), Stemonitales (5) and Liceales (1) were extracted with 1 M-KOH and identified as melanin on the basis of various physico-chemical tests, ultraviolet and infrared spectra. While this was the only pigment in the physaraceous and stemonitoid species, the liceaceous species, i.e. Reticularia lycoperdon, had an additional ethanol-extractable yellow-green pigment. The uv and i.r. spectra of melanins of the Physarales and the Stemonitales were more similar to each other than to those of Reticularia melanin. Key words: Melanin, Myxomycetes, Spore wall pigment.
In myxomycetes, the dark pigment of the spores of the Physarales and Stemonitales is assumed to be melanin (Aldrich & Blackwell, 1976), although this has been verified only in the much-investigated Physarum polycephalum (McCormick et a!', 1970; Chet & Huettermann, 1977) and in FuNgo septica (Chapman, Nelson & Orlowski, 1983). To our knowledge, there is no report on the nature of spore pigment in the Stemonitales, an order in which we in this laboratory have long been interested. A comparative study of the nature of spore wall pigments in a few members of the Stemonitales and
Physarales (taxonomically separated from the remaining orders of the subclass Myxogastromycetideae on the basis of spore colour, which is usually some shade of purple-brown) was made. A brown-spored species of the Liceales, namely Reticularia lycoperdon, was also included for comparison.
MATERIALS AND METHODS Myxomycete species The material studied is listed in Table 1.
Table 1. Species studied, herbarium accessions and sources. Accession number Physarales Physarum melleum (Berk. & Broome) Massee P. echinosporum A. Lister Didymium minus (A. Lister) Morgan Diderma effusum (Schwein.) Morgan Fuligo intermedia Macbr. Craterium leueoeephalum (Pers.) Ditmar
MUBL/IK/MH 91 MUBL/IK/FC 23 MUBL/IK/FC 48 MUBL/IK/FC 25 MUBL/IK/FC 98 MUBL/IK/FC 83
Stemonitales Stemonitis fusca Roth' 5. splendens Rost.t 5. herbatica Peck Comatricha aequalis Peck' Lamproderma seintillans (Berk. & Broome) Morgan
MUBL/IK/TNL 1 MUBL/IK/RS 1 MUBL/IK/SH 5 MUBL/IK/TNL 3 MUBL/IK/MH 82
Liceales Reticularia lycoperdon Bull. a
MUBL/IK/23
, Supplied by Professor T. N. Lakhanpal from Shimla. t Supplied by Dr Rajesh Sharma from Punjab. a Supplied by Professor Lilian Hawker from Bristol. U.K. The remaining species were collected locally by the authors.
287
P. Loganathan, P. Paramasivan and Indira Kalyanasundaram
Extraction and purification Initial attempts at extraction of pigment was made with Stemonitis herbatica using various organic solvents, including acetone, methanol, ethyl acetate, chloroform, hexane, tertiary butyl alcohol, benzene and petroleum ether, either singly or in various combinations. Since no pigment was extracted from whole spores in any of these solvents, the process was repeated using empty spore cases separated by sedimentation after germination of spores, so that the inner surface of the spore walls was also exposed to the action of the solvent. As the pigment was not extracted by any of the above procedures, a melanin extraction procedure (Ellis & Griffiths, 1974; Chet & Huettermann, 1977) was followed, using 1'0 M potassium hydroxide solution. Spores were boiled in this solution for 2 h, cooled, and centrifuged at 32°C at 2000 rev. min-I. After collecting the brown supernatant, the residue was re-extracted with 0'1 M KOH and the process was repeated until the supernatant was clear. The extract thus collected was blackish brown, and from this the dark brown pigment was precipitated out by acidification with 1 N HCl up to pH 2. The pigment was washed in three changes of distilled water and dried overnight at 21° in a dehumidified atmosphere. Purification was done by hydrolysing the dried powder in a sealed gas vial with 5 ml of 7 M HCl for 2 h at 100°. After cooling, it was washed several times with distilled water and dried as before.
Identification of pigment The purified pigment was subjected to various physical and chemical tests (Thomas, 1955) as detailed in Table 2. For the spectra in the uv and visible range, known amounts of powder were dissolved in 1'0 M KOH and read for absorption in a double-beam Hitachi 150--20 spectrophotometer at 200--500 nm. For i.I. spectra, the purified pigment was ground with potassium bromide, pressed into disks under vacuum and the spectra recorded in a Perkin-Elmer PE 781 IR spectrophotometer at 4000-600 em-I.
Residual pigment Microscopy of the spores or spore cases after melanin extraction showed them still to have a pale reddish colour. It was therefore suspected that a second pigment may be present, and attempts were made to extract this using all the organic solvents named earlier, but it was not extractible. Further attempts to understand the nature of this pigment were made through histochemical and other tests (Durrell, 1964; Tracey, 1955). The procedures described above were repeated with the remaining eleven species, with slight modifications where necessary, except the last-mentioned histochemical and other tests for residual pigment, which were made only with Stemonitis herbatica.
RESULTS All twelve species had a pigment that could be extracted with 1 M KOH, giving a brown or purple-brown solution. Precipitation of the pigment occurred readily when these solutions 21
Table 2. Diagnostic tests for melanin.
Species
Colour of solution in KOH
Physarum melleum P. echinosporum Didymium minus Diderma e/fusum Fuligo intermedia Craterium leucocephalum Stemonitis Fusca S. splendens S. herbatica Comatricha aequalis Lamproderma scintillans Reticularia lycoperdon
Light brown Light brown Light brown Light brown Dark brown Light brown Reddish brown Dark brown Brown Brown Light brown Brown
All species had the same reactions to the following tests: Reaction with H 2 0 2 Solubility in water Decolourized Insoluble Reaction with Solubility in organic solvents (acetone, ethanoL chloroform) sodium dithionite and potassium Insoluble ferricyanide Decolourize first and tum brown when potassium ferricyanide is added Reaction with Solubility in 1 m-KOH (100 0 ammoniacal silver for 2 h) nitrate Soluble solution Form a greycoloured silver precipitate lining the sides of the test tube Dialysis through CelPrecipitation in 3N-HCl lophane membrane Precipitate readily, forming floccose precipitates 27/32 Does not pass Reaction for polyphenol tests through membrane (FeCl 3 test) Forms a reddish orange test precipitate
were acidified to pH 2 with 3 N HC!. The pigments from all the species dissolved completely when placed in 1 M KOH at 100° for 2 h.
Physical and chemical properties The physical and chemical properties are summarized in Table 2. The pigments from all 12 species had similar properties. They were insoluble in water; not extracted with organic solvents like acetone, chloroform, ethanol, or a mixture of these solvents; decolourized with hydrogen peroxide, and gave a positive reaction for polyphenols, producing a reddish orange precipitate with 1 % FeCi a solution. The pigment solutions were reduced with 1 % sodium dithionite solution, MYC 92
Melanin in myxomycete spore walls
288
Fig. 1. Ultraviolet and visible spectra of the melanins extracted from members of the Physarales. 3
3
Craterium leucocephalum
Didymium minus
3
j
Fuligo intermedia
Physarum echinosporum
-<
3
Diderma ejJusum
Physarum melleum
250
350
450
250
350
450
Wavelength (nm)
becoming colourless, but turning brown by oxidation after 1 % potassium ferricyanide was added. Bleaching took place when the solution was treated with ammoniacal 1 % silver nitrate solution, and a grey-coloured silver precipitate lined the inside of the test tubes on boiling. The pigments appeared to be of high molecular weight because of their inability to dialyze through cellophane membranes from alkaline solutions. From these properties, the pigments were identified as melanin in all cases.
Ultraviolet spectrum The absorption spectra showed characteristic absorption peaks in the uv regions of wavelengths ranging from 200 to 250 nm, but none in the visible region. The overall uv spectra were similar in the three orders, except for small interspecific differences. All the physaraceous species showed characteristic absorption peaks at 240 nm, except P. echinosporum and D. minus in which the peaks occurred at 220 nm (Fig. 1).
In Stemonitales the characteristic bands occurred at 220 nm in all species. In S. herbatica, however, additional bands appeared at 230 and 240 nm (Fig. 2). Because of this dissimilarity with the remaining species of the order, the test was repeated using two additional specimens, obtained in culture from the original material with and without illumination. The melanin extracted from spores of these specimens also showed a uv spectrum identical with that of the natural one. The melanin solutions of R. lycoperdon also gave absorption peaks at 220 and 240 nm, and these were very sharp, in contrast to the somewhat diffuse bands of the Physarales and Stemonitales (Fig. 2). The graphs obtained by plotting the log absorbance against wavelength (200-500 nm) showed similar negative slopes for most of the species, decreasing gradually from 300 to 400 nm but moving slightly upwards at 500 nm suggesting increased absorbance.
P. Loganathan, P. Paramasivan and Indira Kalyanasundaram
289
Fig. 2. Ultraviolet and visible spectra of the melanins extracted from members of the Stemonitales and from Reticularia /ycoperdon. 3
Stemonitis herbatica
Reticularia Iycoperdon
3
3
Stemonitis splendens
i
Lamproderma scintillans
«
3
3
Stemonitis fusca
250
350
Comatricha aequalis
450
250
350
450
Wavelength (nm)
Infrared spectrum
The Lr. spectra of the melanin from members of the Physarales and Stemonitales were broadly similar. A characteristic curve in the range of 9'7 to 10'2 ~m, a very sharp band at 7'2 ~m, and a broad band in the range of 6' I to 6'3 ~m were the salient features in all species. Less marked absorption bands occurred at 4'3 ~ and at 3'4 ~m. The latter was rather marked in S. herbatica and in R. lycoperdon. Stemonitis herbatica also showed another sharp band at 5'9 J..lm, which was faint in the remaining species (Figs 3, 4). In R. lycoperdon, as in other species, the sharpest band was seen at 7'2 ~m, and less marked ones at 6'2 and 4'3 ~m. The most characteristic feature, however, was a prominent band occurring at 9'2 ~m, which was not clear in the remaining species. Residual pigments
The spore residues of Physarales and Stemonitales were not extractable in organic solvents such as acetone, ethanol,
chloroform or a mixture of these solvents. However, a greenish yellow pigment was extracted from R. lycoperdon with ethanol, but not with chloroform or acetone. The residue of S. herbatica gave a positive reaction to the Nile Blue test for melanin. When the residue was treated with a saturated solution of KOH at 160 0 to digest most components other than tensile wall substances, the pigment was not dissolved but remained in the insoluble residue.
DISCUSSION The identification of the spore wall pigments of the Physarales and the Stemonitales as melanin supports the general presumption that the purple-brown pigments in these two orders are this substance (Aldrich & Blackwell, 1976). In the eleven species studied, after extracting the pigment with I M KOH, the residue was still coloured but no further extraction was possible with either KOH or organic solvents. The results of experiments with S. herbatica suggest that the 11-1
Melanin in myxomycete spore walls
290
Fig. 3. Infrared spectra of melanins extracted from physaraceous species. Wavelength (pm)
3·5
4·0
4·5 5·0
6·0
7·0
8·0
9·0 10
12 14
Physarum melleum
3000
2500
2000
1800
1600
1400
1000
800
Wave number (em-I)
residual pigment is melanin, which occurs in a bound form with some tensile component of the cell wall and hence is not extractable. While it may be reasonably assumed that melanin is universal in the Physarales and Stemonitales, it is not restricted to these orders. Some of the traditionally 'brightspored' groups also contain melanin, as seen from R. lycoperdon in this study. In these, however, it occurs as an additional pigment. It would be of interest to study the spore wall pigments of the Echinosteliales, which have been separated from the Stemonitales on the basis of plasmodial type and sporangial size (Martin, 1960), to see if they are common to the two orders. From the ultraviolet and infrared spectra, the melanins from the six physaraceous and five stemonitoid species are fairly similar, and may belong to the same chemical group. They cannot. however, be characterized at this stage as belonging
to anyone of the four types of fungal melanin described in the literature (Bell & Wheeler, 1986). The only attempt to characterize myxomycete melanin is that of Ward & Havir (1957) whose results suggest that it is neither DOPA-melanin nor catechol-melanin, but are still inconclusive. In the present study, the Physarales and the Stemonitales showed slight differences in uv spectra, in that the Physarales usually had an absorption band at 240 nm and the Stemonitales, at 220 nm. However, the few exceptions, having a band at 220 nm in the Physarales and at 240 nm in the Stemonitales, suggest an integradation of the two orders. Small differences in absorbance within a narrow range of wavelength, occurring between species in the uv as well as i.r. spectra, might be explained on the basis of some heterogeneity in the complex melanin molecule. Stemonitis herbatica. however, showed several additional peaks both in the uv and i.r. spectra.
P. Loganathan, P. Paramasivan and Indira Kalyanasundaram
291
Fig. 4. Infrared spectra of melanins extracted from stemonitoid species and from Reticularia Iycoperdon. Wavelength (pm)
3·5
4·0
5·0
6·0
7·0
8·0
9·0 10
12 14
Comatricha aequalis
3000
2500
2000
1800
1600
1400
1000
800
Wave number (em-I)
The melanin from spores of this species obtained from different environmental conditions, did not show any intraspecific differences. The soil-inhabiting nature of this species, contrasting with the wood-inhabiting nature of most species of Stemonitis (Indira, 1968), might also suggest an evolutionary divergence. In R. lycoperdon, the Lr. spectrum showed a prominent band at 9'2 IJm, which was not clear in the physaraceous or stemonitoid species. In fact the Lr. spectrum in this species showed a strong resemblance to that of Agaricus bisporus melanin described by Rast et al. (1981) as being characteristic of GDHB (y-glutaminyl-3A-dihydroxybenzene) melanin, and might belong to this group. The separation of the Stemonitales into a separate subclass, Stemonitomycetidae, on the basis of differences in sporangial morphogenesis, with endogenous stalk development (Ross,
1973), has been supported by Alexopoulos (1978). On the other hand, Kalyanasundaram (1978) states that stalk and capillitial development in Stemonitis is basically similar to that in other Myxomycetes. Considering the similarity between the spore wall pigments of the Stemonitales and the Physarales it is suggested that these two orders are more closely related to each other than to the Liceales or the Trichiales. In the Eumycota, there seems to be some relationship between taxonomy and melanin structure (Bell & Wheeler, 1986). Elucidation of the significance of spore wall pigments in phylogenetic detenninations of myxomycetes, however, should await more extensive studies. We thank the Director of this Centre for facilities, and those mentioned earlier for supplying material of the Stemonitales and Liceales which are not frequently seen in tropical climates.
Melanin in myxomycete spore walls The second author thanks the University Grant Commission, New Delhi, for the award of a Junior Research Fellowship.
REFERENCES ALDRICH, H. C. & BLACKWELL, M. (1976). Resistant structures in the Myxomycetes. In The Fungal Spore: Form and Function (ed. D. J. Weber & W. M. Hess), pp. 413-461. New York: John Wiley. ALEXOPOULOS, C. J. (1978). The evolution of the taxonomy of the Myxomycetes. In Taxonomy of Fungi I (Proceedings of the International Symposium on Taxonomy of Fungi, 1973, ed. C. V. Subramanian), pp. 1-8. Madras, India: University of Madras. BELL, A. A. & WHEELER. M. H. (1986). Biosynthesis and functions of fungal melanins. Annual Review of Phytopathology 24, 411-451. CHAPMAN, C. P., NELSON R. K. & ORLOWSKI, M. (1983). Chemical composition of the spore case of the acellular slime mold Fuligo septica. Experimental Mycology 7, 57--65. CHET, l. & HUETTERMANN, A. (1977). Melanin biosynthesis during differentiation of Physarum polycephalum. Biochimica et Biophysica Acta 499, 148-155. DURRELL, L. W. (1964). The composition and structure of walls in dark fungus spores. Mycopathologia et Mycologia applicata 23, 339-345.
ELUS, D. H. & GRIFFITHS, D. A. (1974). The location and analysis of melanins in the cell walls of some soil fungi. Canadian Journal of Microbiology 20, 1379-1386. (Received for publication 5 May 1988)
292
INDIRA, P. U. (1968). Some slime-molds from Southern India. IX. Distribution, habitat and variation. journal of the Indian Botanical Society 67, 330-341. KALYANASUNDARAM, l. (1978). Capiliitial development in Stemonitis. In Taxonomy of Fungi I (Proceedings of the International Symposium on Taxonomy of the Fungi, 1973, ed. C. V. Subramanian), pp. 9-13. Madras, India: University of Madras. MARTIN, G. W. (1960). The systematic position of Myxomycetes. Mycologia 52, 119-129. McCORMICK, J. 1.. BLOMQUIST, J. S. & RUSCH, H. P. (1970). Isolation and characterization of a galactosamine wall from spores and spherules of Physarum polycephalum. journal of Bacteriology 104, 1119-1125.
RAST, D. M, STUSSI, H.. HAGNAUER. H. & NYHLEN, L. E. (1981). Melanins. In The Fungal Spore: Morphogenetic Controls (ed. C. Turian & H. R. Hohl), pp. 507-531. New York: Academic Press. ROSS, l. K. (1973). The Stemonitomycetidae, a new subclass of Myxomycetes. Mycologia 65, 477-485. THOMAS, M. (1955). Melanins. In Modern Methods of Plant Analysis 4 (ed. K. Paech & M. V. Tracey), pp. 661--675. Berlin: SpringerVerlag. TRACEY, M. V. (1955). Chitin. In Modern Methods of Plant Analysis, 2 (ed. K. Paech & M. V. Tracey), pp. 264-274. Berlin: SpringerVerlag. WARD, J. M & HAVIR, E. A. (1957). The role of 3:4 dihydroxytoluene, sulfhydril groups and cresolase during melanin formation in a slime mould. Biochimica et Biophysica Acta 25, 440-442.