©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at SEM studies on type specimens of two rare Comatricha taxa H. Singer, G. Moreno* & C. Illana Departamento Biologia Vegetal, Universidad de Alcalä, 28871 Alcalä de Henares, Madrid, Spain Singer H., Moreno G. & Illana C. (2006). SEM studies on type specimens of two rare Comatricha taxa - II. Two rare taxa of Comatricha. - Sydowia 58 (2): 300-308. The lectotype of Comatricha brachypus was examined with light and electron microscopy and compared with an isotype of C. tenerrima var. macrospora. Key words: Myxomycota, scanning electron microscopy, taxonomy, type material. The myxomycetes are a peculiar group of organisms forming together with dictyostelid slime molds a single coherent (monophyletic) group, whichs closest relatives are the amoebas (Protozoa), followed by the animals and the fungi (Baldauf et al., 2000). Originally having been studied by botanists and mycologists, they are currently the focus of study of specialised investigators, the " myxomy cetologists ". Myxomycetes usually have small fructifications and very uniform spores with a spore ornamentation that is often very difficult to see clearly by light microscopy (LM). This situation frequently makes identification impossible. Scanning electron microscopy (SEM) with the application of the critical point drying technique makes it possible to observe spores in their native state, thus maintaining their complete spore ornamentation. It is therefore a basic tool in the revision of type material that has been described in years when optical microscopy was imprecise and the original diagnoses were quite incomplete. With SEM we can now obtain very valuable information that can be applied to resolving taxonomic problems in taxa that are difficult to distinguish and only occasionally collected. This is the case for the study described herein, which involved a revision of the type material of Comatricha brachypus (Meyl.) Meyl. * e-mail: gabriel.moreno@uah.es 300 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at Materials and Methods The examined specimens were obtained from the herbaria BR, LAU and AH. Specimens were mounted in Hoyer's medium and studied with a Nikon microscope. SEM micrographs were produced with a Zeiss DSM-950. Spore measurements were made under the oil immersion objective and include surface structures such as spines or warts. Twenty five spores of each specimen have been measured to determine spore size. Two microscopic slides have been examined of each specimen studied, except for type specimens, where only one slide has been examined. Spore diameters are expressed as ranges between smallest measured diameter and largest measured diameter. Values in brackets represent rare measurements (a maximum of two spores of 25 spores measured). For ultramicroscopic studies the material was rehydrated in concentrated ammonium hydroxide (28-30%) for 30 minutes, dehydrated in aqueous ethanol (70%) for 30 minutes, fixed for 2 hours in pure ethylene glycol dimethyl ether (= 1,2-dimethoxymethane) and finally immersed in pure acetone for at least 2 hours followed by critical point drying and sputtering with gold-palladium. Taxonomy Comatricha brachypus (Meyl.) Meyl., Bull. Soc. Vaud. Sei. Nat., 57: 41. 1929.-Figs. 1-5. = Comatricha nigra var. brachypus "brachypes" Meyl., Bull. Soc. Vaud. Sei. Nat., 56: 71. 1925. = Stemonitopsis brachypus (Mey.) Y. Yamam., Myxomycete Biota Japan: 627. 1998. Original description of Comatricha nigra var. brachypus (Meylan 1925). Aspect des formes ovoides et courtes de C. laxa, soit: sporanges ovoides, ä partie large en bas, de 1 ä 1.5 mm de hauteur, portes par un stipe tres court, de 0.2 ä 0.4 mm, bruns. Capillitium de C. nigra. Spores brunes, de 10 ä Hum. La Vaux, 1250 m, septembre 1924. Original description of Comatricha brachypus (Meylan 1929). - Plasmodium blanc laiteux. Sporanges elliptiques ou ovoides de 1 ä 1.5 mm de hauteur sur 0.7 ä 1 mm de largeur, brun fonce, portes par un stipe toujours plus court que le sporange et mesurant de 0.2 ä 0.5 mm. Columelle atteignant en general les % ou les % de la hauteur du sporange. Capillitium dense, forme de filaments sinueux, comme celui de C. nigra. Spores 10-11 urn, finement verruqueuses. Maturite: septembre, octobre. Description: The type material is very well conserved in a matchbox on remains of liverworts growing on dead wood, glued to a piece of cardboard that can be removed from the box. The type material consists of about 50 sporangia. 301 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at Figs. 1 - 5. Comatricha brachypus (holotype): 1. Sporangium (bar = 0.1mm). 2. Detail of capillitium (bar = 30 um). 3. Lid of herbarium box where the type is kept. 4. Spore (bar = 2 urn). 5. Detail of spore ornamentation (bar = 1 urn). 302 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at Sporangia gregarious to crowded, stalked, 1-1.5 mm total height. Sporotheca broadly ellipsoidal to ovoid, 0.5 - 1.2 x 0.5 - 1 mm, lilac brown. Peridium entirely evanescent (i.e. not remaining partly as a basal cup). Stalk 0.2 - 0.5 mm high, smooth, flattened and widening towards the base, blackish brown. Columella occurring as a continuation of the stalk and concolorous, rostrate, reaching almost to the apex of the sporotheca where it sometimes becomes sinuous. Hypothallus membranous, hyaline, common to several sporangia. Capillitium dense, sinuous (resembling that of Comatricha nigra), branched and anastomosed, without main branches, arising from the length of the columella, threads 1-2 urn wide, uniformly reddish brown. S p o r e - m a s s violaceous brown, light violaceous by LM, (9.0-)10.0 - 11.0 urn in diam, with abundant, regularly distributed warts. By SEM spore ornamentation is formed by small and narrow baculae that converge obliquely, forming pyramid or "tripod-like" structures with part of the base more or less hollow, sometimes constituting irregularly crested structures. M a t e r i a l examined. - Comatricha brachypus (Meyl.) Meyl.: SWITZERLAND, Canton Vaud, Föret de La Vaux, near Sainte Croix, 1250 m, on dead wood, Sep 1924, leg. C. Meylan, holotype in LAU (without herbarium number). Comatricha tenerrima (M. A. Curt.) G. Lister var. macrospora Rammeloo, Bull. Jard. Bot. Belg., 53 (1/2): 298. 1983. - Figs. 6 - 10. Latin diagnosis. - Ob sporocystas minores stipitc brcviore et capite globoso obscurioreque, capillitium minus differentiatum atque sporas majores a var. tenerrima removenda. Description: The type material is in very good condition and is attached to a piece of cardboard. The entire collection consists of five peaces of wood, most of which are covered by numerous, welldeveloped sporangia. Sporangia scattered, 1.8-2.2 mm total height. Sporotheca globose to subglobose, 0.5-0.6 mm in diam, blackish brown. Peridium evanescent, leaving a collar at the base of the sporotheca. Stalk 0.5 - 1 mm high, smooth, cylindrical, widening somewhat towards the base, blackish brown. Columella occurring as a continuation of the stalk and concolorous, cylindrical, reaching to the apex of the sporotheca or nearly doing so. Hypothallus membranous, hyaline, discoid. Capillitium dense, sinuous, branched and anastomosed, without main branches, arising from all along the columella, threads 1-2 urn wide, uniformly blackish brown. Spore-mass dark violaceous brown, violaceous brown by LM, 8.0-10.0 (im in diam, with abundant, regularly distributed warts. By SEM spore ornamentation is composed of baculae with the 303 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at Karlslrbl volcar-j, C. siJs, .ii.AU>« alt. : 3670. Inner sl Je of rotting giant lobelia slcrt Figs. 6-10. Comatricha tenerrima var. macrospora (isotype): 6. Sporangium (bar = 0.1 mm). 7. Herbarium box. 8. Detail of capillitium (bar = 30 um). 9. Spore (bar = 2 urn). 10. Detail of spore ornamentation (bar = 1 (.im). 304 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at apex divided into 3 - 4(- 6) lobes giving it a more-or-less stellate appearance. Material examined. - Comatricha tenerrima var. macrospora Rammeloo: RWANDA, Karisimbi volcano, SW side, 3670 m, inner side of a rotting giant Lobelia stem, 11 Oct 1974, leg. J. Rammeloo (JR 4996 in BR 1728, isotype). Discussion Meylan (1925) originally described Comatricha nigra var. brachypes Meyl. from a collection made from dead wood in the forest of La Vaux (Switzerland) at 1250 m elevation. Meylan indicated from this variety to which - somewhat misleading - he refers to as a form: "Cette forme parait si differente des formes connues de C. nigra qu'elle constitue peut-etre une espece autonome. De nouvelles recoltes et observations pourront seules en decider" (translation: The form seems so different from the known forms of C. nigra that it constitutes perhaps an autonomous species). Several years later Meylan (1929) decided to consider this taxon an autonomous species. Subsequent authors such as NannengaBremekamp (1991) and Kowalski (1975) have also considered it as an independent species, although Kowalski noted its similarity to C. pulchella (C. Bab.) Rostaf., "except for spore size". Martin & Alexopoulos (1969) regarded Comatricha brachypus as a synonym of C. subcaespitosa Peck (= Stemonitopsis subcaespitosa (Peck) Nann.-Bremek.) and concluded that there were "no significant differences when compared an authentic specimen of C. brachypus from Meylan with specimens of C. subcaespitosa". Following our detailed study of the type material of Stemonitopsis subcaespitosa, we find this to be a different species due to its capillitium having a more-or-less complete surface net and possessing a spiny spore ornamentation (Moreno et al. 2004). More recently, Yamamoto & Nishikawa (1995) found in Japan a specimen they determined as Comatricha cf. brachypus, and three years later, Yamamoto (1998) made the combination Stemonitopsis brachypus (Meyl.) Y. Yamam., based on this material (the drawings are the same in both publications). The Japanese material shows longly cylindrical sporangia with a short stalk and capillitium threads connected perpendicularly to the columella and forming an internal three dimensional net and a dense, small-meshed, almost complete surface net, very different from the studied type material of Comatricha brachypus (see drawings in Yamamoto, 1998). We do not know further records of this species. When the type of Comatricha brachypus is observed under a magnifying glass, the first impression one has is that he is looking at a specimen of C. pulchella, as already indicated by Kowalski (1975). 305 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at But when its microscopic characters are studied, notable differences between the two taxa are observed, because the capillitium of C. brachypus forms a sinuous and intricate net of filaments without differentiation into primary and secondary branches, while the capillitium of C. pulchella has thicker primary branches arising from the columella at right angles from which once again arise secondary branches in the form of thinner and sinuous threads that form a net (Lizärraga et al. 2003) Comatricha brachypus also reminds one microscopically of C. meandrispora A. Castillo, G. Moreno, & Illana due to its rostrate columella that becomes abruptly narrower at the apex (Castillo etal. 1993). Comatricha brachypus can be distinguished from the afore mentioned species by studying its spore ornamentation by SEM, the latter consisting of small and narrow baculae that converge obliquely forming pyramid or "tripod-like" structures with part of the base more or less hollow, sometimes constituting irregularly crested structures. In C. pulchella the spore surface is covered by long, spiny baculae with excrescences in the middle part, as can be observed in the SEM photographs provided by Rammeloo (1983 a), and in C. meandrispora spore ornamentation is subreticulate to reticulate. At first glance, the spore ornamentation of Comatricha brachypus is similar to that of C. tenerrima (M.A.Curtis) G. Lister. Rammeloo (1983 a) studied the spore ornamentation of C. tenerrima var. tenerrima by SEM and provides a photo that shows spore ornamentation with a surprising resemblance. However, when both types of spore ornamentation are compared thoroughly, they can be distinguished on the basis of several characteristics: While in C. brachypus the ornamentation is formed by fusing baculae that appear as pyramid or "tripod-like" structures, C. tenerrima has solitary baculae with lobed apices (Lizärraga et al. 2005). Both types of spore ornamentation have a star-like appearance when seen from above (Figs. 4, 9). Rammeloo (1983 b) described a macrosporic variety of Comatricha tenerrima, C. tenerrima var. macrospora Rammeloo, basing himself on a single collection from Rwanda, the isotype of which we have studied. Rammeloo (1983 a) gives further records of this variety collected several times in Rwanda, between 2350-3670 m elevation, on bamboo, leaves of Dendrosenecio, and on the inner side of an inflorescence of Lobelia gigantea. Comatricha tenerrima var. macrospora differs from C. tenerrima var. tenerrima by having smaller, more globose and darker sporothecae, a shorter stalk, a less differentiated capillitium and larger spores (8.0- 10.8 urn in diam). Spore ornamentation is formed by baculae with an apex which is frequently divided into 6 lobes, in contrast to those of C. tenerrima var. tenerrima which have 3-4 lobes. 306 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at Comatricha tenerrima var. macrospora shares with C. brachypus a sinuous capillitium without main branches (Sinuaria type), and similar spore diameters, 10.0-11.0 urn in C. brachypus, and 8.0 -10.8 um in C. tenerrima var. macrospora (according to the authors). But C. brachypus can easily be distinguished by its broadly ellipsoidal to ovoid sporotheca, a short stalk that attains approximately V3 of the height of the sporotheca, a rostrate columella, the absence of a collar at the base of the sporotheca and a different spore ornamentation that has been described above. Comatricha tenerrima var. macrospora has globose to subglobose sporothecae, a long stalk that is approximately twice the height of the sporotheca, a more or less cylindrical columella and a collar at the base of the sporotheca as a remnant of the peridium. The differences between the three taxa mentioned above are summarized in Tab. 1. The description of Comatricha tenerrima var. tenerrima follows Rammeloo (1983 a). Tab. 1. - Differences among Comatricha tenerrima var. tenerrima, C. tenerrima var. macrospora and C. brachypus. C. tenerrima C. tenerrima var. tenerrima var. macrospora C. brachypus Sporangia Total height 3 - 4 mm 0.9-1.3 mm Sporotheca Height Form Colour Stalk Spores Height Diameter Ornamentation by SEM ± fusiform or long ovoid light brown, reddish or lilac 2 - 3 mm 5.8-8.2 urn baculae with the apex divided into 3 - 4 lobes 1.5 - 2 mm 0.5-0.6 mm globose to subglobose blackish brown 0.5-1.0 mm 8.0-10.8 urn baculae with the apex divided into 3 - 4(- 6) lobes 1-1.5 mm 0.5- 1.2 x 0.5- 1.0 mm broadly ellipsoidal to ovoid lilac brown 0.2-0.5 mm (9.0 -)10.0- 11.0 urn baculae that converge obliquely forming pyramid or "tripodlike" structures Taxonomic treatment C. tenerrima var. tenerrima 1 Spores 6.0-8.0 urn in diam 2 1* Spores 8.0 - 11.0 urn in diam 2 Stalk higher than sporotheca, collar present at its base. Spore ornamentation (SEM) formed by baculae with lobed apex C. tenerrima var. macrospora Stalk less than half the height of the sporotheca, without collar at its base. Spore ornamentation (SEM) formed by fusing baculae that form pyramid or "tripod-like" structures C. brachypus 307 ©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at Acknowledgements This investigation has been financed in part by the Research Project of the Ministry of Science and Technology, National Plan of Scientific Investigation, Technological Development and Innovation, REN2002-01965. We express our gratitude to Dr. R.P.Korf and D.W. Mitchell for their suggestions of revisions to the manuscript and wish to thank J.A.Perez and A. Priego of the Electron Microscopy Service of the University of Alcalä for their invaluable help with the SEM and the curators of the herbaria BR and LAU, and especially Dr. J. Rejos, curator of the herbarium AH. H. Singer thanks the National Program of the Professorship Formation, Ministry of Education and Culture of Spain for the scholarship that supported his doctoral thesis at the University of Alcalä. References Baldauf S.L., Roger A. J., Wenk-Siefert I., Doolittle W.F. (2000) A kingdom-level phylogeny of Eukaryotes based on combined protein data. Science 290: 972-977. Castillo A., Moreno G., Illanu C. (1993) A new species of Comatricha with incompletely reticulated spores from Spain. Mycotaxon 46: 315-319. Kowalski D.T. (1975) The Myxomycete taxa described by Charles Meylan. Mycologia 67: 448 - 493. Lizärraga M., Moreno G., Singer H., Illana C. (2003) Myxomycetes from Chihuahua, Mexico. Mycotaxon 88: 409 - 424. Lizärraga M., Moreno G., Illana C, Singer H. (2005) Myxomycetes from Chihuahua, Mexico III. Mycotaxon 93: 75 - 88. Martin G.W., Alexopoulos C. J. (1969) The Myxomycetes. University of Iowa Press, Iowa. Meylan C. (1925) Note sur divers Myxomycetes du Jura et des Alpes. Bulletin de la Saddle Vaudoise des Sciences Naturelles 56: 05 - 74. Meylan C. (1929) Recherches sur les Myxomycetes en 1927-28. Bulletin de la Socie'te Vaudoise des Sciences Naturelles 57: 39 - 47. Moreno G., Singer H., Sanchez A., Illana C. (2004) A critical study of some Stemonitales of North American Herbaria and comparison with European nivicolous collection. Boletin de la Sociedad Micolögica de Madrid 28: 21-41. Nannenga-Bremekamp N. E. (1991) A guide to temperate Myxomycetes. Biopress Limited, Bristol. Rammeloo J. (1983 a) Flore illustre'e des Champignons d'Afrique Centrale. Fascicule 11. Jardin Botanique National de Belgique, Meise. Rammeloo J. (1983 b) Taxons nouveaux de Stemonitales (Myxomycetes). Bulleti7i du Jardin Botanique National de Belgique 53: 297-298. YamamotoY., Nishikawa T. (1995) Additions to the Myxomycetes of Hokkaido. Reports of the Taisetsuan Institute of Science 30: 75 - 98. Yamamoto Y. (1998) The Myxomycete biota of Japan. Tokyo Shorin Publishing Co., Tokyo. (Manuscript accepted 24 Oci 2006; Corresponding Editor: U. Peintner) 308 ZOBODAT - www.zobodat.at Zoologisch-Botanische Datenbank/Zoological-Botanical Database Digitale Literatur/Digital Literature Zeitschrift/Journal: Sydowia Jahr/Year: 2006 Band/Volume: 58 Autor(en)/Author(s): Singer H., Moreno G., Illana Carlos Artikel/Article: SEM studies an type specimens of two rare Comatricha taxa. 300-308