Lentodium squamulosum has been shown to be tetrapolarly heterothallic. The 4 predicted mating reactions can be recognized by gross characters on Potato dextrose agar, but not on other media tested. Three sectors were found, 2 of which do not seem to dikaryotize with any of the 4 established mating types. Malt extract would appear to yield more consistent fruiting than Oat flake agar

Robinson, Albert D.

Rosinski, Martin A.

University of Northern Iowa

Proceedings of the Iowa Academy of Science Volume 73 Annual Issue Article 6 1967 Tetrapolar Heterothallism in the Basidiomycete, Lentodium Squamulosum Albert D. Robinson University of Iowa Martin A. Rosinski University of Iowa Let us know how access to this document benefits you Copyright ©1966 Iowa Academy of Science, Inc. Follow this and additional works at: https://scholarworks.uni.edu/pias Recommended Citation Robinson, Albert D. and Rosinski, Martin A. (1967) "Tetrapolar Heterothallism in the Basidiomycete, Lentodium Squamulosum," Proceedings of the Iowa Academy of Science, 73(1), 15-22. Available at: https://scholarworks.uni.edu/pias/vol73/iss1/6 This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu. Tetrapolar Heterothallism in the Basidiomycete, Lentodium Squamulosum ALBERT D. RomNso:-J AND MARTIN A. Rosrnsxr1 Abstract: Lentodium squamulosum has been shown to be tetrapolarly heterothallic. The 4 predicted mating reactions can be recognized by gross characters on Potato dextrose' agar, but not on other media tested. Three sectors were found, 2 of which do not seem to dikaryotize with any of the 4 estab-lished mating types. Malt extract would appear to yield more consistent fruiting than Oat flake agar. Lentodium squamulosum is a species of Basidiomycetes oc-curring on decorticated logs of deciduous species. It is commonly collected in Iowa and has been routinely collected in the Iowa City area during each of the past 3 years. It is curious in being very similar to Lentinus tigrinus, a typical member of the Agri-caceae, in both habit and morphology except that L. squamulo-su1n produces gills which anastomose ve1y early in development, giving rise to enclosed locules which bear basidia and basidio-spores. For a detailed account of development of L. squamulo-swn the reader is referred to Lyman ( 1907) and Bobbitt ( 1965). Singer ( 1949) considers L. squamulosum to be merely a sterile form of L. tigrinus. Martin ( 1956) takes issue with Singer em-phasizing that typical basidia and basidiospores are produced on sporophores of L. squamulosum and concludes that "It is not a teratological form, still less an abnormality or monstrosity, but a common, easily recognized, constant entity, with a wide dis-tribution extending at least from Massachusetts to Iowa. In south-western Ohio and in eastern Iowa it is more abw1clant than L. tigrinus, from which it is supposed, with good reason, to have derived." Martin considers the species to be an excel-lent example of an agaric showing a distinct approach to a Gas-teromycete habit. Bobbitt ( 1965) carried out a developmental study of basidiocarps of L. squamulosum under pure culture conditions on agar. This work expanded on the previous devel-opment study by Lyman ( 1907) and the observations by Martin ( 1956) in showing that under the more moist conditions existing on agar media in test tubes, gill structure was readily apparent and that spores were discharged externally. Bobbitt found that the tendency toward gill production in culture was extremely variable. Though he worked with only 2 different dikaryons and though he observed a wide range of variability in the tendency toward gills, it seemed that some work should be done to deter-mine how genetic variability might influence sporophore mor-phology as well as to attempt crosses between L. squamulosum and L. tigrinus. 1 Department of Botany, University of Iowa, Iowa City, Iowa 15 1Robinson and Rosinski: Tetrapolar Heterothallism in the Basidiomycete, Lentodium SquamulPublished by UNI ScholarWorks, 196616 IOWA ACADEMY OF SCIENCE [Vol. 73 L. tigrinus is known to be tetrapolarly heterothallic (Quin-tanillia et. al. 1941) and any likelihood of mating between the 2 species would undoubtedly depend upon their both having the same basic compatibility structure. Since preliminary work by one of us had indicated that L. squamulosum is heterothallic, it was deemed advisable to determine at the outset if L. squamu-losum is also tetrapolarly heterothallic. The question of tetra-polar heterolthallism is the principal subject of this paper. METHODS AND MATERIALS Fruiting bodies were obtained in pure culture on Oat flake agar {see Martin, 1956) from a compatible cross of 2 presumed rnonokaryotic mycelia, each of which had been derived from single basidospores obtained from sporophore collected in 1962. Potato dextrose agar (Difeo) was routinely used for germinating the spores and for making the initial crosses. Two other media, which were used by Raper and San Antonio ( 1954) in their work on Schizophyllum commune, were used later in crosses for comparative purposes. These media are quite similar except that one contains 3 g yeast extract in addition to the following basic formula: 20.0 g glucose, 2.0 g "Bacto-peptone", 0.46 g KH2P0 4 , 1 g K2HP0 4 , 0.5 g MgS04, trace of FeCl3 and 20.0 g "Bacto Agar" to I liter of distilled water. The medium with the yeast extrnct added was used by Raper and San Antonio to obtain fruits of S. commune. In all cases the sterile media were poured into sterile petri dishes to a depth of 2-3 mm. The presence of clamp connections indicates compatible mat-ings. To detect the clamp connections, samples of mycelia were mounted and stained with cotton blue in lacto-phenol. To isolate single spore colonies, a mature basidocarp was transferred aseptically to a 125 ml erlenmeyer flask containing sterile distilled water. The pileus was then cut into several pieces by reaching into the flask with a sterile scalpel. The flask and contents were agitated, until the water became cloudy with the liberated spores. Ten ml of this suspension was used to obtain a dilution series of 1:10, 1:100, 1:1000 etc, up to l:l,000,000,000 in distilled water. One ml samples from each of the above dilu-tions were placed aseptically in separate sterile petri dishes and mixed with warm, melted agar. The plates were swirled to dis-tribute the spores evenly. After 2 days the spores had germinated and had produced hyphal colonies which could be seen under a dissecting microscope. Discreet colonies were removed with a sterile dissecting needle and transferred to fresh plates of Potato dextrose agar. Particular care was taken to select only those colonies which had not touched other colonies. The mono-s pore isolates thus obtained were labeled 101-114. One isolate, 102, was later discarded as it was suspected of having been dikaryotized. The crosses were made by placing 5 mm diameter discs of agar and mycelia from two different isolates on fresh 2Proceedings of the Iowa Academy of Science, Vol. 73 [1966], No. 1, Art. 6https://scholarworks.uni.edu/pias/vol73/iss1/61966] TETRAPOLAR HETEROTHALLISM 17 agar media in perti dishes. The 2 plugs were placed 1 cm apart. The 13 isolates were :paired with each other in all possible com-binations, and consequently 78 different crosses were made. RESULTS AND DISCUSSIONS After 10 days, examination of hyphae from the zone of con-tact between certain of the matings showed numerous and con-spicuous clamps. Fig. 1 shows all the possible combinations be-tween the 13 isolates and which combinations produced clamps. Fig. 2 shows a separation of the four groups obtained on the basis of their mating reactions with all other strains. The only discrepancies ( 101 X 112 and 103 X 108) are cases where clamps were not seen and should have been. A repeat of these doubtful crosses yielded clamps. On the basis of these observations, it seems clear that L. squamulosum fits Whitehouse's (1949) concept of a tetrapolarly heterothallic species in which compatibility is dependent upon heterozygosity at 2 independent loci, and for this reason the 4 mating types herein described are designated AB, Ab, aB, and ab. Since some workers studying tetrapolar heterothallism in other species of Basidiomycetes have been able to distinguish between the 4 mating types on the mode of interaction between them (Papazian, 1950), and since we had previously observed that compatible matings in L. squamulosum produced a dikaryotic mycelium conspicuously different in appearance from the my-celia developed in non-compatible crosses, we attempted to determine differences between the appearance of mycelia in the various incompatible combinations. Mycelia produced from in-compatible combinations have been described as "flat" (homo-zygous at the "a" locus) "barrage" (homozygous at the "b" lo-cus) and "overgrowth" (common factors1 at both "a" and "b" loci) by Papazian for S. commune. By referring to fig. 2, it can be seen that numbering the groups from top to bottom as first, second, etc. that the first group is compatible with the third and that the second group is compati-ble with the fourth. We arbitrarily assigned group 1 as ab, and therefore group 3 as AB. In order to determine which of the mating interactions might correspond to Papazian's "common a" and which might correspond to "common b", we re-ecxamined some of the matings for the presence of incomplete clamp con-nections, "pseudoclamps". Some workers (see Fincham and Day 1963) have shown that pseudoclamps occur in the common b heterokaryon but not in the common a heterokaryon. Pseudo-clamps were observed in crosses between groups 1 and 2 and between groups 3 and 4. On this basis group 2 was disignated as Ab and group 4 as aB. 1 Mycelia having both compatibility factors the same do not usually anastomose, and hence no heterokaryon is formed. 3Robinson and Rosinski: Tetrapolar Heterothallism in the Basidiomycete, Lentodium SquamulP blished by UNI ScholarWorks, 196618 IOWA ACADEMY OF SCIENCE [Vol. 73 103 104 105 106 107 108 109 IlO lll 112 113 114 IOI - + -103 - + + 104 + - + 105 - + + - + 106 - + + 107 - + 108 - + - + All possible matings of 109 - - + single spore isolates. uo + - + +indicates presence of lll -clamp connections. 112 - + 113 -CD !01 103 104 105 106 107 108 109 110 lll 112 113 m fOl - + 104 - + + alt I07 - + + 109 - + + II4 - + + 103 - - + -· + -jb lil - + - + + -Il3 - + - + + -115 + + - + - + + All Ill - + - + + + l!li - + + + ill lOIJ - + + no - + + + 0 Four mating types. + indicates presence of clamp connections. With the mating types so designated, the macroscopic detec-tion of different mating reactions could be attempted. Repre-sentatives of each mating reaction are shown in fig. 3-6. The 4Proceedings of the Iowa Academy of Science, Vol. 73 [1966], No. 1, Art. 6https://scholarworks.uni.edu/pias/vol73/iss1/61966] TETRAPOLAR HETEROTHALISM 19 Fig. 3-6. Four mating reactions on Potato dextrose agar. Fig. 3. compatible reaction. Fig. 4. reaction where both a and bfactors common. Fig. 5. common a. Fig. 6. common b. compatible reaction results in a streaky appearance with the streaks radiating from the inoculation points (fig. 3). Fig. 4 is the reaction between mating types where both the a and b fac-tors are common (Ab X Ab etc. ) . It is characterized by the lack of a distinct reaction line coupled with a uniformly aerial mat of myceliwn. Both common a reactions (AB X Ab and aB X ab) are characterized by depressed flat growth and a thin line of aerial mycelium along the line of original contact between the two parent mycelia (fig. 5). The common b reactions (AB X aB and Ab X ab) show a marked line of aerial mycelium at the point of juncture of the two parental strains with a narrow de-pressed area on either side (fig. 6). The main body of the my-celium however remains fluffy. Our next step was to determine whether these reactions varied greatly on different kinds of media. The two media used by Raper and San Antonio ( 1954) were selcted for this test. Again all possible combinations were tried using all 13 isolates. None of 5Robinson and Rosinski: Tetrapolar Heterothallism in the Basidiomycete, Lentodium SquamulPublished by UNI ScholarWorks, 196620 IOWA ACADEMY OF SCIENCE [Vol. 73 Fig. 7-10. Matings of sector isolated from the 105 side of (105 " 114) with a representative from each oi the 4 established mating types on Potato dextrose agar. In all cases the sector is on the left. Fig. 7. with ab. Fig. 8. with Ab. Fig. 9. with AB. Fig. 10. with aB. Fig. 11-14. Matings oi sector i9olated from the midline oi ( 106 z 113) with a representative from e!""h of the four establishtd mating types on Potato denrose agar. In all cases the s~tor JS on the left. Fig. 11. with a b.Fig. 1.2, with AB. Fig. 13. with Ab. Fig. 14. with aB. 6Proceedings of the Iowa Academy of Science, Vol. 73 [1966], No. 1, Art. 6https://scholarworks.uni.edu/pias/vol73/iss1/61966] TETRAPOLAR HETEROTHALISM 21 the anticipated reactions were observed on these 2 media though they were again apparent on a Potato dextrose agar control. The basal medium with yeast extract added did produce 3 in-teresting sectors from 3 different matings. Two of these matings were compatible and 1 was common b. One sector which came from the 105 side of a cross between 105 and 114 (compatible) was isolated and then mated with a representative of each of the 4 mating types. These 4 test matings were done on Potato dex-trose agar in hopes that they would yield reactions to indicate the mating type of the sector. Figs. 7-10 show these 4 matings. No differences between the 4 combinations are observable, and the reaction line is different from anything seen previously. An examination for clamps or pseudoclamps proved negative in all 4 combinations. The sector side of the crosses developed a mound of white smooth mycelium along the reaction: line. This ri1ound was very tough, making it difficult to remove a sample for micrscopic examination. A second sector from the reaction line of 106 X 113 (compatible) was isolated and mated with all 4 of the mating types on Potato dextrose agar. These reactions are shown in figs. 11-14. Again no differences are observable between the 4 test matings. The reaction of this sector, however, is different from that previously mentioned, as is the morpholo-gy of the sector itself. This sector has a "powdery" appearance. As before, no clamps were observable in any of the 4 com-binations. Prelimina1y experiments with the third sector which originated on the 103 side of a 103 X 109 mating indicates that it acts as a donor in crosses with the 4 mating types, dikaryotiz-ing unilaterally. Observations on the nature of these sectors, mating type, effect on sporophore production, and ability to recombine and to segregate with other factors are presently be-ing planned. In addition, we have preliminary evidence that 2% malt extract agar is a better medium for consistent fruiting than is Oat flake agar. Since it is desirable from the standpoint of studying gene-tic variability to have a medium which can be uniformly pro-duced and which will consistently produce basidiocarpsj this would represent an important contribution to the continued study of the genetics of L. squamul,osuni. We are currently eval-uating various media and their effect on fruiting. Literature Cited Bobbitt, T. F. 1965. A developmental study of Lentodium squamulosum Morgan. Thesis. University of Iowa, Iowa City, Iowa Fincham, J. R. S. and Day, P. R. 1963. Fungal Genetics F. A. Davis Co. Phila. Lyman, G. R. 1907. Proc. Boston Soc. Nat. Hist. 33: 125-209. Martin, G. W. 1956. Proc. Iowa Acad. Sci. 63: 280-286. Papazian, H. P. 1950. Bot. Caz. 112: 143-163. 7Robinson and Rosinski: Tetrapolar Heterothallism in the Basidiomycete, Lentodium SquamulPublished by UNI ScholarWorks, 196622 IOWA ACADEMY OF SCIENCE [Vol. 73 Quintanilha, A., Quintanilha, L. and Vasermanis, A. 1941. Rev. Mycol. 6: 3-48. Raper, J. R. and San Antonio, J. P. 1954. Am. Jour. Bot. 41: 69-86. Singer, R. 1949. Lilloa 22: 1-832. Whitehouse, K. L. K. 1949. Bio. Reviews 24: 411-449. The Vascular Plants of Beirry Woods DONALD G. DELISLE1 Abstract. A list of the genera and species of vascular plants occurring in Berry Woods is presented. This 40 acre mixed hardwood forest, located in central Warren County, Iowa, is owned and maintained as a natural preserve by The Nature Conservancy. Berry Woods, a 40 acre mixed hardwood forest, is located about two miles northwest of Indianola, Iowa in Warren County, (T-76N, R-24W, S.W. section 2). It was given to The Nature Conservancy in 1961 by Mr. and Mrs. Don L. Berry of Indianola as a memorial to Captain B. C. Berry and Senator \V. H. Berry. The woods covers a portion of the bluffs overlooking the flood plain of Middle River, in an area of Shelby loam with some Tama loam soils along the western edges. A small creek runs diagonally through the woods dividing it into predominantly eastern and western slopes. The area is completely fenced and is bordered on the west by a county road, on the south and east by agricultural land and on the north by cut-over woodland which is rapidly being cleared. Since the woodland is now preserved for scientific study and teaching it was deemed advisable to make a thorough survey of the vascular flora for future references. Voucher collectiolllS made in Berry Woods during the past four years are maintained in the herbarium of Simpson College. Identification and nomen-clature has followed the manuals of Gleason and Gronquist ( 1963) and Steyermark ( 1963). A total of 105 genera and 134 species of vascular plants have been collected and identified! from this area. The dominant tree species include Quercus alba, Th"lia americana, Carya ovata and Ostrya virginiana. A typical pre-vernal flora is present during April an:d May and includes such forms as Dicentra cucullaria, Claytonia virginica, Sanguinaria canadensis Asarmn canadense, Hepatica acutiloba, Dentaria laciniata, Erythronium albidum, lsopyrum biternatum and Uvul-aria grandiflora. 'Department of Biology, Simpson College, Indianola, Iowa 8Proceedings of the Iowa Academy of Science, Vol. 73 [1966], No. 1, Art. 6https://scholarworks.uni.edu/pias/vol73/iss1/6

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