Species and Varieties in the Rhizopus Arrhizus-Rhizopus Oryzae Group As Indicated by Their Dna Complementarity

SPECIES AND VARIETIES IN THE RHIZOPUS ARRHIZUS-RHIZOPUS ORYZAE GROUP AS INDICATED BY THEIR DNA COMPLEMENTARITY

J. J. ELLIS

Northern Regional Research Center, Agricultural Research Service, U.S. Department ofAgriculture, I Peoria, Illinois 61604

ABSTRACT

DNA renaturation experiments gave evidence to conclude that Rhizopus arrhizus, R. OIyzae, R. delemar, Amylomyces rou.xii, R. delemar var. minimus, R. delemar var. multiplici sporus, R. arrhizus var. delemar, R. chungkuoensis var. isqfermentarius, and R. javanicus var. kawasakiensis can be accommodated in three taxa, namely, R. arrhizus var. arrhizus, R. arrhizus var. rozedi, and R. arrhizus var. delemar. Key Words: Rhizopus, DNA renaturation, taxonomy.

Numerous attempts have been made to clarify the species concepts of Rhi zopus, with primary emphasis on a single or relatively few morphological or physiological features (2,3,10, 15). Some workers attempted to rearrange existing data, using relatively newer techniques, with either weighted or nonweighted characteristics to place strains in appropriate species and to show relationships (1). Traditionally, we have weighted heavily characteristics of the sexual stage over other characteristics exhibited by a species. Problems arise when, as is the case for many species of Rhizopus, the sexual stage is unknown or is rarely produced. Thus, we have depended on morphological characteristics ofthe spor angial apparatus and attempted to support our viewpoint with physiological data. The lack ofdefinitive information on the numerous described species ofRhizopus has led to considerable difficulty in determining the extent of variability within a species and how the species can be readily distinguished. In particular, it has been most difficult to attach names to strains belonging in the Rhizopus arrhizus R. oryzae-R. delemar group of species and varieties. To delimit the taxa and the morphological characteristics that will help place strains in those taxa, deoxyri bonucleic acid (DNA) renaturation studies were performed.

MATERIALS AND METHODS Fungal cultures. - The fungal strains (TABLE I) used in this study are maintained in the Agricultural Research Service Culture Collection (NRRL) at the Northern Regional Research Center. Stock cultures were grown on either potato dextrose agar (PDA) or yeast-malt agar (YM). These media are described as M-20 and M-69, respectively, in the Mycological Guidebook (12). For DNA extraction, cultures were grown in YM broth (400 ml per I liter Erlenmeyer flask) for 30 h on a reciprocal shaker at 28 C. Cultures were harvested by filtering with suction and were suspended in the spermidine-spermine-sucrose buffer ofMorris (as cited by Timberlake, 14). DNA purification.-DNA was extracted and purified by modifying Marmur's method (8) with the use ofhydroxylapatite (Bio-Gel RTP, Bio-Rad Laboratories)

I The mention of firm names or trade products does not imply that they are endorsed or rec ommended by the U.S. Department ofAgriculture over other firms or similar products not mentioned. 243 Purchased by U.S. Department of Agriculture for Official Use 244 MYCOLOGIA

TABLE I SOURCE OF STRAINS NRRL 1469 Rhizopus arrhizus Fischer. Received from Blakeslee collection as C 606 in June, 1940. Blakeslee's notes say "lvfucor arrhizus (=Rhiz. arrhizus) evidently fro Cen trastelle." (sic) NRRL 1472 R. delemar (Boid.) Wehmer & Hanz. Received from Blakeslee collection as C 608 in June, 1940. Blakeslee's notes say "prob. fro Centralstelle." NRRL 2871 R. javanicus var. kawasakiensis Takeda & Takamatsu. Received from Institute for Fermentation, Osaka, as IFO 4801 Type, in June, 1960. NRRL 2872 Rhizopus delemar var. minimus Takeda. Received from Institute for Fermentation, Osaka, as Takeda 7301, in June, 1960. Isolated from Indonesian ragi. NRRL 2873 R. chungkuoensis var. isofermentarius Takeda. Received from Institute for Fermen- tation, Osaka, as IFO 4726 Type, in June, 1960. NRRL 2915 R. microsporus v. Tiegh. Received from H. Schoch, University ofBasel, in December, 1960. Originally from Centraalbureau voor Schimmelcultures. NRRL 3133 R. oryzae Went & Geerligs. Received from Centraalbureau voor Schimmelcultures in May, 1964, as Went strain and presumed type strain. NRRL 3142 R. oryzae Went & Geerligs. Isolated from Chinese yeast obtained from the Taiwan Sugar Co. in April, 1964. NRRL 3258 R. delemar var. multiplicisporus Inui, Takeda & Iizuka. Received from H. Iizuka, University ofTokyo, as lAM 6258, in January, 1967. NRRL 5129 R. arrhizus var. delemar Mauvernay, Laboureur & Labrousse. Received from Mu- seum of Nat. Hist. Paris as No. 1916, in September, 1970. No Latin diagnosis in U.S. Patent No. 3,513,073 ofMay 19, 1970. NRRL 5866 Amylomyces rouxii Calmette. Neotype strain isolated January, 1974, from Look Pang (sweet fermented glutinous rice) made in Thailand. chromatography (7). Purity ofDNA preparations was checked by their absorbancy ratios at 2301260 nm and 2601280 nm as well as by their thermal denaturation profiles. DNA renaturation. - The extent of DNA relatedness was measured spectropho tometrically as described by Seidler and Mandel (11) and Kurtzman et al. (4). These workers have shown that results from this method are comparable to relatedness values obtained using radioisotope techniques. The main steps in the spectrophotometric techniques are the following. Purified DNA was sheared by two passages through a French press cell at 10,000 psi. Following filtration through a micropore filter (0.45 ,urn, from Mi1lipore" Corp.), the sheared, double-stranded DNA was dialyzed (Spectrapore membrane tubing with 12,000 mol wt cut-off) at 3-5 C for 3 da against O.OOlx SSC (SSC = standard saline citrate, 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0) with 0.001 M EDTA. The DNA solution was freeze-dried and redissolved in deionized-distilled water. DNA renaturations were monitored in a Gilford 260 recording spectrophotometer equipped with an elec tronically-heated, four-place cuvette holder. The reactions were carried out in 5x SSC-20% DMSO with 75 ,ug DNA per ml for a single strain or a mixture of 37.5 ,ug DNA for each ofthe paired strains, i.e., four fused quartz cuvettes were used: one contained only water, SSC, and DMSO (blank); two contained DNA of one strain each; and the fourth contained the DNA mixture of the two strains. Pre ceding renaturation, the DNA was denatured by raising the temperature to 90 C and holding the reaction mixtures at that temperature for 10 min. Then the temperature was lowered 3 C/min until it reached the incubation temperature of 55 C. This temperature corresponds to T m - 25 C which was determined experi- ELLIS: RHIZOPUS DNA 245

TABLE II REASSOCIATION OF NUCLEAR DNA IN SPECIES OF Rhizoplls

NRRLNo. Name 1472' 3142 3133 1469 5866 2915 b 2915 b R. microsporlls 18 e 26 24 19 25 100 5866 Amylomyces rouxii 65 96 96 95 1469 R. arrhizlIs var. arrhizus 65 97 97 3133 R.oryzae 60 97 3142 R.oryzae 65 • R. delemar var. delemar. b Two different fermentations and extractions of DNA for this strain. e Average per cent of 2-3 reassociations.

mentally in the reaction buffer. The A 260 readings for the four cuvettes were recorded successively at 10 sec intervals for the 4-9 h necessary to reach Cot.s. Two to three determinations were made for each pairing. Per cent renaturation was calculated by Seidler and Mandel's method (11) using the Cot.s values. Determination ofbase composition. - The guanine plus cytosine (G+C) content of the nuclear DNA preparations was calculated from 2-3 buoyant density de terminations in cesium chloride gradient (20 h, 44,000 rpm) (9, 13). Measurements were made in a Spinco model E analytical ultracentrifuge equipped with an elec tronic scanner.

RESULTS AND DISCUSSION A summary ofthe results ofDNA renaturation experiments ofstrains selected as most representative of Rhizopus microsporus, R. arrhizus, the presumed type ofR. Olyzae, a second strain ofR. Olyzae, and the neotype strain ofAmylomyces rouxii are presented in TABLE II. Rhizopus microsporus was chosen to be "the obvious distinct species." The extent of DNA relatedness of this species with other strains listed in TABLE II is quite low, and no pair shows more than 26% complementarity. When DNA from the strains of A. rouxii, R. arrhizus var. arrhizus, and R. Olyzae were interacted, all showed more than 95% relatedness. The first clue that strain NRRL 1472 R. delemar was somewhat different in its DNA make-up is shown by its 60-65% complementarity with the aforementioned strains that gave high relatedness with one another. Further indication is seen in TABLE III, where results are given ofrenaturations between strains ofR. delemar var. delemar and R. arrhizus var. 'arrhizus with strains of varieties thought to oe synonyms ofR. delemar var. delemar on a morphological basis. Indeed, these

TABLE III REASSOCIATION OF NUCLEAR DNA FOR STRAINS OF Rhizoplls delemar

NRRLNo. Name NRRL 1472' NRRL 1469b 2872 R. delemar var. minimus 85 e 71 3258 R. delemar var. mliltiplicisporlis 89 67 5129 R. arrhizlIs var. delemar 100 70 2873 R. chzlIlgklloensis var. isofermentarius 96 73 2871 R. javanicus var. kawasakiensis 95 73 • R. delemar var. delemar. b R. arrhizus var. arrhizus. e Average per cent of 2-3 reassociations. 246 MYCOLOGIA

TABLE IV GUANINE PLUS CYTOSINE CONTENT OF NUCLEAR DNA FROM RhizopliS STRAINS NRRLNo. Name G + C(mo1%) 1469 R. arrhizlIs 36.6 (±O.22)a 3133 R.oryzae 36.7 (±O.21) 1472 R. delemar 36.1 (±O.07) 5866 Amylomyces rozedi 35.5 (±O.12) 2915 R. microsporliS 43.9 (±O.35) a Mean of2-3 determinations (± standard deviation). so-called varieties gave at least 85% complementarity with strain NRRL 1472 R. delemar var. delemar, whereas they gave at least 10% less complementarity with strain NRRL 1469 R. arrhizus var. arrhizus. The results of a determination of G+C content of the DNA from pertinent strains are shown in TABLE IV. The only obvious distinction noted is that for R. microsporus. All other G+C ratios fall within a 1.2 mol% range, consistent with their demonstrated conspecificity. With the exception of R. microsporus, it is evident from the data presented that one is examining the DNA of a single species, if one follows the lead of workers investigating yeasts. Strains of some heterothallic species of yeasts have indicated genetic exchange when complementarity has been as low as 25% (5). This viewpoint is acceptable on a morphological basis in the current investigation because R. arrhizus has quite a wide morphological variation in a number of its characteristics, namely, columellae, rhizoidal, sporangial, sporangiophore, and sporangiospore size and shape. Because of the wide range of variation it seems logical to retain the name R. arrhizus Fischer, even though Fischer (2) stressed the applanate nature of the columella in this species (R. arrhizus, described in 1892, has date precedence over R. Olyzae Went & Geerl., described in 1895). Many strains seen by the author in culture produced at least a few columellae that were more broad than high, al though not consistently so. Also, most recent workers have accepted R. l1odosus as a synonym ofR. Olyzae, yet ithas been described as having applanate columellae (see Lendner, 6, fig. 45). Although the neotype strain of A. rouxii gave a very high percentage of reas sociation with strains of R. arrhizus and should probably be a part of the type variety on this basis, the author proposes to retain the taxon as a variety for convenience because (1) it is propagated primarily by hand in association only with making fermented ragi materials, (2) it has not been isolated from nature insofar as the author can determine, and (3) strains can be readily identified on a morphological basis. The probable type strain of R. delemar, namely NRRL 1472, as well as the strains listed in TABLE III, have sporangiospores distinctly larger than strains of R. arrhizus var. arrhizus. They can, therefore, be relegated to varietal status on a morphological basis. The amount ofcomplementarity ofthese strains with strain NRRL 1472 and with NRRL 1469 R. arrhizus var. arrhizus indicates that the strains listed in TABLE III are synonymous with strain NRRL 1472. They have DNA like R. arrhizus enough to be the same species, yet have lower (ca. 65%) DNA complementarity with representative strains as well as morphological dif ferences to classify them as variety. In conclusion, the author proposes: (1) that the name R. arrhizus var. arrhizus Fischer be retained with NRRL 1469 designated as the neotype strain; (2) that R. oryzae Went & Geerl. be reduced to synonymy under R. arrhizus; (3) that A. ELLIS: RHIZOPUS DNA 247 rouxii be reduced to R. arrhizus var. rmcdi (Calmette) Ellis with the basionym A. rouxii Calmette. Ann. Inst. Pasteur 6: 611, 1892, and (4) that R. delemar be reduced to R. arrhizus var. delemar (Wehmer & Hanz.) Ellis with the basionym R. delemar Wehmer & Hanz., in Hanzawa-Mykol. Zentbl. 1: 77, 1912.

ACKNOWLEDGMENTS I thank C. P. Kurtzman for helpful guidance and suggestions with many aspects ofthe DNA experiments and M. Mahoney and S. Janson for technical assistance.

LITERATURE CITED

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Accepted for publication September 8, 1984