Mycol. Res. 103 (5) : 527–541 (1999) 527 Printed in the United Kingdom Taxonomy of the Penicillium miczynskii group based on morphology and secondary metabolites M A R T H A C H R I S T E N S EN1, J. C. F R I S V A D2 A N D D O R O T H Y T U T H I L L1 " Department of Botany, University of Wyoming, Laramie, WY 82071, U.S.A. # Department of Biotechnology, Building 221, Technical University of Denmark, DK-2800 Lyngby, Denmark Multivariate analyses (cluster and correspondence) were used to assess the taxonomic structure of 34 isolates of Penicillium miczynskii scored for 95 binary characters. Six isolates of P. raistrickii and P. rolfsii were added solely for the purpose of comparison. The characters used were 38 micromorphological, 15 cultural and 42 secondary metabolite characters. With just one exception, allocation of isolates to clusters was equal in the separate analyses using morphological and secondary metabolite data. The combined data set, using 95 characters, resulted in clearly-defined clusters interpretable as species on the basis of the distribution of ex-type cultures. Four species were accepted : P. miczynskii, P. manginii, P. soppii and P. atrosanguineum. Two additional species, P. syriacum and P. chrzaszczii, represented only by ex-type cultures, were interpreted as a nomen ambiguum and a form of uncertain taxonomic status, respectively. Brief descriptions, illustrations and a synoptic key are included to aid identification of the accepted species. Following his isolation of a distinctive Penicillium from the soil of a Picea forest in Poland, Zaleski described P. miczynskii in 1927 and he or Biourge distributed cultures in 1928 (Zaleski, 1927 ; Raper & Thom, 1949). Both Thom (1930) and Raper & Thom (1949) accepted P. miczynskii as valid and unique and provided full descriptions based upon detailed examination of derivatives of the Zaleski isolates, now represented by NRRL 1077 (¯ CBS 220.28). Pitt (1979) placed P. chrzaszczii, P. matris-meae and P. soppii, also described by Zaleski in 1927, and four additional species (P. atrosanguineum B. X. Dong, P. manginii Duche! & R. Heim, P. pedemontanum Luppi Mosca & A. Fontana, P. syriacum Baghd.) into synonymy with P. miczynskii and accordingly expanded the species circumscription. The stimulation for this study was our discovery of an exact correspondence (in macro- and micromorphology, and secondary metabolites) between NRRL 1077 and two recent isolates from conifer forest soils in the United States. Other isolates in our collections were in essential agreement with the published descriptions of P. pedemontanum, P. soppii and P. atrosanguineum. We have compared recent isolates in the P. miczynskii complex to one another and to 11 ex-type cultures. With a single exception (the ex-types of P. syriacum), the isolates are similar in having relatively tall, erect conidiophores that are divaricately branched, and display metulae and phialides only in any single penicillus. P. miczynskii, often sclerotial, has narrow phialides variable in length and with slender, elongate necks ; it thus resembles both the P. janthinellum and the P. raistrickii series sensu Raper & Thom (1949). On the basis of conidial aggregation (chains mostly separate, in plumes or loose columns), general appearance of the conidiophore and phialide shape, the other isolates except those of P. chrzaszczii and P. syriacum resemble P. brasilianum Bat., P. raistrickii G.Sm. and related species and are unlike members in the P. janthinellum, P. canescens and P. janczewskii (P. nigricans) series sensu Raper & Thom (1949) and Ramirez (1982). Our goal was to examine objectively the validity of P. miczynskii sensu orig. through qualitative and quantitative observations of many features in a variety of cultures and application of multivariate analytical techniques. By involving ex-types of the proposed synonyms of P. miczynskii (Pitt, 1979), we hoped also to clarify the taxonomy of other species. In prior studies, other workers have found a remarkable correspondence between secondary metabolite profiles and species in terverticillate penicillia (Svendsen & Frisvad, 1994 ; Larsen & Frisvad, 1995 b). Fassatiova! & Kubatova! (1990), in a study of divaricately branched penicillia primarily from soil, reported consistency at the species level in any array of morphological features similar to those used here. Species identifiable as constellations of phenetically similar isolates have been reported in numerous multivariate taxonomic studies (e.g. Al Musallam, 1980 ; Mueller, 1985 ; Frisvad, 1992 ; Zambino & Harrington, 1992 ; Svendsen & Frisvad, 1994 ; Taylor, Patterson & Harrod, 1994 ; Larsen & Frisvad, 1995 b). MATERIALS AND METHODS The 40 cultures examined and compared, hereinafter referred to as operational taxonomic units (OTUs), are listed in Table Penicillium miczynskii group 1. They are listed under the species names determined to be correct in the present study. All OTUs isolated after 1959 were lyophilized or placed on silica gel within one year of isolation unless otherwise noted. It can be assumed that the seven ex-types isolated between 1905 and 1931 were maintained on agar media for many years prior to lyophilization. Representative cultures of P. raistrickii and P. rolfsii Thom, both well-defined species that somewhat resemble P. soppii and P. manginii in sclerotial features, penicillus organization and phialide shape, were included to broaden the comparison. The six members of those species (Table 1) agree in all respects with existing descriptions (Raper & Thom, 1949 ; Pitt, 1979 ; Ramirez, 1982). Morphological characterization Media and cultural methods, listed below, were similar to those used by Raper & Thom (1949), Pitt (1979) and Ramirez (1982). Following the recommendation of Larsen & Frisvad (1995 a), however, trace amounts of Cu (5 ppm CuSO \5 % H O final concentration) and Zn (10 ppm ZnSO \7 H O final # % # concentration) were added to four of the five media. In all cases, fresh or day-old agar surfaces were inoculated with a suspension of conidia in water agar (0±4 % agar) using a transfer loop touched at three points (MEA) or at a single point (all other media). The media, in three glass Petri plates per isolate, and growth regimes were : (1) Malt extract agar (Pitt, 1979) amended with Cu and Zn (MEA) ; incubation in diffuse daylight at 24–26 °C for 4–10 d. (2) Four additional agar media, distributed to the wells of a quartered Petri plate ; a single plate per isolate ; incubation in diffuse daylight at 24–26° for 7 d. The four media were : (a) Czapek’s medium made according to Ramirez (1982) but amended with Cu and Zn (Cz) ; (b) Czapek yeast extract agar (Pitt, 1979 ; Ramirez, 1982) amended with Cu and Zn (CYA) ; (c) yeast extract sucrose agar (Filtenborg, Frisvad & Svendsen, 1983) amended with Cu and Zn (YES) ; and (d) creatinesucrose agar (CREA) (Frisvad, 1985). (3) CYA ; a single Petri plate which can be inoculated with three or four different OTUs ; incubation at 37° for 7 d. Micromorphological characterization was done exclusively from colonies grown on MEA for 3–10 d. Conidial surface features were determined by inspecting conidia more than 10 d old, both in liquid and in adjacent air bubbles, using oil immersion (1600¬) and interference contrast microscopy (ICT). Macromorphological features were recorded using colonies grown on the MEA medium for 10 d and on the other media for 7 d at room temperature (24–26°). Analysis of secondary metabolites All isolates listed in Table 1 were analyzed for secondary metabolites after one or two weeks of growth on CYA, MEA and YES agars at 25° in the dark, using TLC (Filtenborg et al., 1995) and HPLC (Frisvad & Thrane, 1987). Multivariate analysis In the computer-based comparison of OTUs by micro- and 528 macromorphology, the selection of characters, so that all salient features of all OTUs could be entered in the system, was a critical task. The classes of characters in the final formulation are shown in Table 2. Within each class, specific characters were written to encompass the variety of expressions in the OTUs under study. Thus, the characters in the second class (Table 2) were : (1) chains in tight clusters, single metulate, ¬10–15 µm ; (2) chains tangled, in loose columns ; and (3) chains straight, separate, divergent, in plumes or loose columns. The characters in the four subclasses under ‘ metular features ’ (Table 2) describe shape of the metulae, number per whorl, length, and evenness within the whorl. Character presence or absence, scored for each OTU, resulted in a binary matrix. Similarity coefficients for all pairs of OTUs were computed using 53 morphological characters, 42 secondary metabolite characters, and the 95 unweighted characters in combination (Sørensen, 1948 ; Rohlf, 1993). Cluster analysis by unweighted pair-group method, arithmetic average (UPGMA) and correspondence analysis, recommended by Frisvad (1992), were accomplished using the software package NTSYS-pc version 1.80 (Rohlf, 1993). The comparisons used 14–23 morphological characters and 2–11 secondary metabolite characters per OTU (averages 17 and 6 respectively). The OTUs per character were 1–38 for the morphological characters (av. 13) and 1–16 for the secondary metabolite characters (av. 6). RESULTS Comparisons among the isolates using (1) 38 micromorphological and 15 cultural characters, (2) 42 secondary metabolites and (3) the 95 characters in combination, resulted in three dendrograms (Figs 1–3). On the basis of these trees, three of the OTUs (24, 25, and 26) were interpreted as separate entities, unrelated to one another and to all other cultures in the comparison. All other OTUs are members of clearly defined clusters. Within-group similarity ranged from 58 to 96 % in the matrix using combined characters (av. 82±5 %). In contrast, average similarity for OTUs in different clusters was approximately 41 %. Several isolates in the soppii cluster were closely similar to one another : there were four coefficients of 94–96 % among isolates from Wyoming, Wisconsin and Denmark in various combinations. Comparison of the dendrograms based on morphological characters and secondary metabolite characters (Figs 1, 2) revealed : (1) Distinctive secondary metabolite patterns in P. rolfsii, P. atrosanguineum and P. manginii, and each of those clusters with equal or less intra-specific variation than was seen in the morphological comparisons. In the comparison by secondary metabolites, several pairs of OTUs were 100 % similar but in no case were OTUs identical in all morphological characters. (2) Somewhat variable secondary metabolite patterns in the P. miczynskii, P. soppii and P. raistrickii isolates, but distinctive and consistent morphologies. Indeed, OTU 2 failed to cluster with the other P. miczynskii isolates on the basis of secondary metabolites, though it clearly nested within that group on the basis of morphology. Martha Christensen, J. C. Frisvad and Dorothy Tuthill 529 Table 1. Sources of cultures and reference numbers in Figs 1–4. Many additional isolates, not included in the multivariate analyses, were analyzed for secondary metabolites Accepted name (this study) and isolate no." P. miczynskii NRRL 1077 (T)# RMF 7771 RMF 8752 RMF 8754 RMF A-105 RMF A-138 IBT 15504 P. astrosanguineum NRRL 5891 (T) IBT 11155 IBT 12875 IBT 13452 IBT 17346 P. chrzaszczii NRRL 903 (T) P. manginii ATCC 18334 (T., P. pedemontanum NRRL 2134 (T) NRRL 3555 (¯ CBS 378.65) CBS 108.66 RMF 8771 RMF A-146 RMF A-152 IBT 15336 P. raistrickii NRRL 1044 (T) RMF G44 (sclerotial) RMF G50 (non-sclerotial) WSF 5225 P. rolfsii NRRL 1078 (T) WSF 3891 P. soppii NRRL 701 NRRL 912 (T., P. matris-meae) NRRL 2023 (T) RMF 205 Source and date of isolation Conifer forest soil, Poland, ca 1927 Pinus contorta forest soil, Wyoming U.S.A., 1977, lyophilized in 1986 Conifer forest soil, (Andrews LTER$), Oregon U.S.A., 1988 Conifer forest soil, (Andrews LTER), Oregon U.S.A., 1988 Conifer forest debris, Wyoming U.S.A., 1994 Pinus forest soil, Wyoming U.S.A., 1994 Heathland soil, Jutland Denmark, 1994 Computer code 2 RMF A-102 RMF A-135 4 WSF 2397 5 IBT 15641 6 IBT K10 7 8 Picea forest soil, Poland, ca 1927 24 Fagus mycorrhizae, Italy, ca 1963, lyophilized in 1968 Ficaria community soil, France, ca 1931 Soil, Zaire, ca 1965, lyophilized in 1969 Soil, Zaire, ca 1965 Conifer forest soil (Andrews LTER), Oregon U.S.A., 1988 Forest soil, Costa Rica, 1994 Forest soil, Costa Rica, 1994 Wheat, United Kingdom, ca 1992 27 Mount Desert Island, Maine U.S.A. (unknown source, E. Melin, 1927) Pinus forest soil, Poland, ca 1927 Pinus forest soil, Poland, ca 1927 Pinus contorta forest soil, Wyoming U.S.A., 1964, lyophilized in 1972 RMF 8829 RMF 9021 35 Pineapple fruit, Florida, U.S.A., 1905 Sphagnum bog peat, Wisconsin U.S.A., 1961 Accepted name (this study) and isolate no." 1 Stored wheat, Czechoslovakia, 1971, lyophilized in 1974 Factory air, Denmark, 1983 Cheese factory air, Hjørring, Denmark, 1992 Oats, Denmark, 1992 Layercake, Stege, Denmark, 1995 Mouldy cotton yarn, United Kingdom, ca 1928 Artemisia-grassland soil, Wyoming U.S.A., 1978 Artemisia-grassland soil, Wyoming U.S.A., 1978 Populus-Salix forest soil, Wisconsin U.S.A., 1964 Table 1. (cont.) 36 37 38 39 28 29 30 31 32 33 34 40 41 42 P. syriacum ATCC 34971 (T) (near P. corylophilum) CBS 418.69 (T) (near P. citreonigrum) Source and date of isolation Computer code Deciduous forest soil (Coweeta LTER), North Carolina U.S.A., 1988 Quercus savanna soil (Cedar Creek LTER), Minnesota U.S.A., 1988 Pine cone, Wyoming U.S.A., 1994 Alpine-conifer soil, Wyoming U.S.A., 1994 Deciduous forest soil, Wisconsin U.S.A., 1960 Heathland soil, Jutland Denmark, 1994 Heathland soil, Jutland Denmark, 1994 13 Soil, Syria, 1963, lyophilized in 1977 Soil, Syria, 1963, lyophilized in 1969 14 19 20 21 22 23 25 26 " Prefixes are acronyms for culture collections : ATCC, American Type Culture Collection ; CBS, Centraalbureau voor Schimmelcultures ; IBT, Institute of Biotechnology, Technical University, Lyngby, Denmark ; NRRL, Northern Regional Research Laboratory, Peoria, Illinois, U.S.A. ; RMF, Rocky Mountain Fungi, Department of Botany, University of Wyoming, Laramie, Wyoming U.S.A. ; WSF, Wisconsin Soil Fungi, University of Wyoming, Laramie, Wyoming U.S.A. # Ex-type culture. $ LTER sites are Long-term Ecological Research areas funded through the National Science Foundation. Table 2. Features used in the calculations of similarity among 40 OTUs 1. 2. 3. 4. 5. 6. 7. 8. 9. Conidium size, shape, surface (5) Conidial aggregation (3) Phialide morphology (3) Metular features (11) Rami presence}absence (2) Stipe surface, length, origin (10) Sclerotia : presence}absence, colour (4) Colony features, MEA (6) Colony features, Cz, CYA, YES ; acid production on CREA ; growth at 37° (9) 10. Secondary metabolites (42) Figures in parentheses indicate number of characters in each class. Total characters used in the combined analysis (Figs 3, 4) was 95 (38 micromorphological, 15 cultural, 42 secondary metabolite characters). 43 44 45 9 10 11 12 (3) Distinctly separate suites of characters in both comparisons for the three cultures representing P. chrzaszczii and P. syriacum (24, 25, 26) (Figs 1, 2). The finding of a relatively low intra-specific similarity for the ex-type cultures in contrast to the higher values among recent isolates in the same species cluster (Table 1 ; Fig. 3) was noted also by Frisvad (1992) and interpreted as the consequence of long maintenance on agar media prior to lyophilization. Within any given species, however, there was no consistent evidence of a correlation between phenetic similarity and geographic proximity (Table 1 ; Figs 3, 4). Overall, the remarkable finding in this study has been that, although individual linkages among OTUs in the clusters Penicillium miczynskii group 0·2 0·4 0·6 530 0·8 1·0 + + + + * 1E 8E 2E 6E 4E 5E 7E 24 9+ 22+ 12+ 23+ 13+ 21+ 19+ 20+ 14+ 11+ 10+ 44D 45D 27_ 31_ 32_ 33_ 34_ 29_ 30_ 28_ 35* 36* 38* 37* 39* 40 41 43 42 25^ 26^ P. miczynskii P. chrzaszczii P. soppii P. rolfsii P. manginii P. atrosanguineum P. raistrickii P. syriacum Fig. 1. Cluster analysis (UPGMA) using 53 morphological characters (see text). Isolates here identified by OTU number are listed in Table 1. Scale is Sørensen – Dice similarity. Cophenetic correlation value ¯ 0±88. 0·00 0·25 0·50 0·75 1·00 + + + + * 1E 4E 5E 6E 7E 8E 26^ 27_ 34_ 32_ 33_ 29_ 30_ 31_ 28_ 2E 24 35* 36* 38* 37* 39* 9+ 12+ 15+ 20+ 21+ 22+ 13+ 14+ 11+ 10+ 23+ 40 41 42 43 44D 45D 25^ P. miczynskii P. syriacum P. manginii P. miczynskii P. chrzaszczii P. atrosanguineum P. soppii P. raistrickii P. rolfsii P. syriacum Fig. 2. Cluster analysis (UPGMA) using 42 secondary metabolite characters. Cophenetic correlation value ¯ 0±96. defined by morphological and secondary metabolite data commonly differed (Figs 1, 2), there was agreement in OTU membership in the clusters (with one exception among 40 OTUs) and definitions of the clusters themselves in the two phenograms. Furthermore, integration of the two sets of data clarified species circumscriptions. Thus, the combined data yielded more sharply defined clusters than those obtained with either morphological or secondary metabolite data alone (Figs 1–3). The pattern of linkages in Fig. 3 and the ordination in Fig. 4 clearly support recognition of P. miczynskii and three of the species treated by Pitt (1979) as synonyms of P. miczynskii (P. atrosanguineum, P. manginii, and P. soppii), each represented by five or more isolates. Further, they are distinct from the two species included for comparison (P. raistrickii and P. rolfsii). The single isolate of P. chrzaszczii and the two isolates of P. syriacum are unlike each other and the other species. DISCUSSION Table 3 summarizes the treatment, by several authorities in the last 50 years, of the eight species interpreted as synonyms by Pitt (1979). Species circumscriptions in the present study, based on many more isolates than were seen by Raper & Thom (1949), are in essential agreement with the latter authors’ concepts for three of the five species described prior to 1949. Our interpretations also agree with those of Stolk & Samson (1983) and Frisvad & Filtenborg (1990) with three exceptions : (1) Penicillium atrosanguineum was listed as a synonym of P. manginii by both Stolk & Samson (1983) and Frisvad & Filtenborg (1990), although the former authors noted that the conidia of P. atrosanguineum are ‘… less ellipsoidal than those of the other strains of P. manginii.’ (2) P. chrzaszczii was treated as a synonym of P. westlingii by Frisvad & Filtenborg (1990) and their placement for the extype culture (NRRL 903) was accepted at CBS (Anonymous, 1996). We agree that the species are closely related, but have chosen to defer a declaration of synonymy pending a thorough review of the apparently common but relatively unstudied P. westlingii-P. turolense complex (Frisvad et al., 1990 ; Gams, 1993). (3) P. syriacum was treated as a synonym of P. dierckxii by Stolk & Samson (1983) and of P. manginii by Frisvad & Filtenborg (1990). Those reports of synonymy with strikingly different species are consistent with dissimilar ex-types at CBS and ATCC. Although we used only NRRL 1077 (ex-type of P. miczynskii) and NRRL 2134 (ex-type of P. manginii) in our cluster analysis, we examined two other cultures mentioned in Raper & Thom’s (1949) discussion of P. miczynskii, NRRL 1024 and NRRL 2133. Those authors based their description of P. miczynskii solely upon NRRL 1077, and prefaced their account of NRRL 2133 with the comment that P. miczynskii ‘ is approximated ’ by NRRL 2133. NRRL 2133 and NRRL 1024, presumably from the same original stock (Raper & Thom, 1949), came from Biourge in 1924 and 1929 ; Biourge considered the culture to be representative of P. sulfureum Sopp. Our conclusion that NRRL 2134 (ex-type of P. manginii), Martha Christensen, J. C. Frisvad and Dorothy Tuthill 0·2 0·4 0·6 531 0·8 1·0 P. miczynskii P. chrzaszczii P. manginii P. soppii P. rolfsii P. atrosanguineum + + + + * 1E 4E 5E 6E 7E 8E 2E 24 27_ 31_ 32_ 33_ 34_ 29_ 30_ 28_ 9+ 12+ 19+ 20+ 21+ 13+ 14+ 22+ 23+ 11+ 10+ 44D 45D 35* 36* 38* 37* 39* 40 41 43 42 25^ 26^ P. raistrickii P. syriacum Fig. 3. Cluster analysis (UPGMA) using combined morphological and secondary metabolite characters. Ex-type cultures are shown in boldface type. Cophenetic correlation value ¯ 0±93. P. raistrickii 41 P. rolfsii 40 44 45 42 43 P. manginii 32 8 28 4 20 P. miczynskii P. atrosanguineum P. chrzaszczii 1 4 6 87 5 39 35 24 37 36 2 10 9 11 23 13 12 21 24 P. soppii Fig. 4. Correspondence analysis using combined morphological and secondary metabolite characters. The two ex-types of P. syriacum have been omitted. Isolates are identified by OTU number (Table 1). NRRL 1024 and NRRL 2133 represent the same taxon is based upon virtual identity in conidial features and a close similarity in cultural features. Stolk & Samson (1983) also interpreted NRRL 2133 and NRRL 2134 as taxonomically equivalent and accepted P. manginii as a valid species (Table 3). With no authentic material of P. sulfureum available, that name must remain a nomen dubium (Pitt, 1979). Raper & Thom (1949) reported that NRRL 2134 ‘ duplicates ’ NRRL 2133 ‘ almost exactly ’, but interpreted both P. manginii (NRRL 2134) and Biourge’s P. sulfureum (NRRL 1024 and NRRL 2133) as probable synonyms of P. miczynskii. Our comparison of the two ex-types cultures with recent isolates supports recognition of P. miczynskii and P. manginii as distinct and valid species. On the basis of just two isolates, Raper & Thom (1949) and Ramirez (1982) described P. soppii as lacking true sclerotia but with small masses of thick-walled cells up to 50–60 µm diameter. Until recently, all representatives of P. soppii in the RMF and WSF collections were catalogued as ‘ P. raistrickii type 2 ’ because of an overall similarity to that species including production of gritty, pale pink sclerotia up to 240–345 µm diam. Our isolates differed from cultures clearly representative of P. raistrickii in producing (1) slightly larger conidia (2±5–3±3 µm diam.) that varied in size in a single slide Penicillium miczynskii group 532 Table 3. Treatment, in major publications, of P. miczynskii and species interpreted by Pitt (1979) as synonyms of that species Raper & Thom, 1949 P. atrosanguineum P. chrzaszczii P. manginii P. matris-meae P. miczynskii P. pedemontanum P. soppii P. syriacum Stolk & Samson, 1983 Frisvad & Filtenborg, 1990 CBS List of cultures, 1994, 1996 Synonym of P. manginii — Synonym of P. manginii Synonym of P. westlingii Accepted sensu Duche! & Heim Accepted sensu Dong Synonym of P. westlingii Synonym of P. atrosanguineum Accepted sensu Duche! & Heim Possible synonym of P. soppii Accepted sensu Zaleski Synonym of P. manginii Synonym of P. soppii Synonym of P. soppii Accepted sensu Zaleski Synonym of P. atrosanguineum Accepted sensu Zaleski Synonym of P. manginii Accepted ; expanded to include NRRL 701 (Raper & Thom 1949, p. 282) Nomen ambiguum Pitt, 1979 Ramirez, 1982 Synonym of P. miczynskii Synonym of P. miczynskii Synonym of P. miczynskii Accepted sensu Dong Synonym of P. jensenii Synonym of P. miczynskii Accepted sensu Duche! & Heim Synonym of P. miczynskii Synonym of P. soppii Synonym of P. soppii Accepted sensu Zaleski — Accepted ; expanded Synonym of P. miczynskii Accepted sensu Zaleski Synonym of P. manginii Accepted sensu Zaleski Synonym of P. miczynskii Accepted sensu Zaleski Accepted sensu Mosca & Fontana Accepted sensu Zaleski — Synonym of P. miczynskii Accepted sensu Baghdadi — Synonym of P. jensenii Probable synonym of P. miczynskii Synonym of P. soppii preparation and (2) stipes that were smooth or very delicately roughened in contrast to the coarsely roughened stipes of our primary assemblage of P. raistrickii isolates and all prior descriptions of stipes in that species (Raper & Thom, 1949 ; Ramirez, 1982 ; Stolk & Samson, 1983 under P. raciborskii Zaleski). Comparison of the ‘ P. raistrickii type 2 ’ isolates with NRRL 701, described by Raper & Thom as essentially similar to NRRL 2023 (ex-type P. soppii) but producing abundant hard sclerotia, revealed essential identity among those isolates and an acceptable taxonomic equivalency to the ex-type of P. soppii. Accordingly, we have broadened the description of P. soppii by noting that in recent isolates sclerotia are abundant, firm to gritty, and up to 345 µm diam. Penicillium chrzaszczii, known only from the ex-type culture (NRRL 903), was described by Pitt (1979) as possessing the ‘ diminutive phialides and small, smooth walled conidia characteristic of P. miczynskii ’. In the present study, as in some earlier studies (Raper & Thom, 1949 ; Ramirez, 1982), conidia in P. chrzaszczii (NRRL 903) were seen to be finely or conspicuously roughed in contrast to their being smooth or nearly so in P. miczynskii (Fig. 13) and in the present study, neither species was characterized as developing diminutive phialides. The use in this study of many isolates and detailed morphological, cultural and biochemical characterizations, has confirmed and clarified the diagnostic features of five species in Penicillium subg Furcatum that produce sclerotia : P. manginii, P. miczynskii, P. soppii, P. raistrickii and P. rolfsii. Penicillium soppii and P. miczynskii may be commonly occurring soil species, especially in conifer forest soils in North America and Europe. All of the confirmed isolates of P. miczynskii are from north temperate conifer forest soil or litter, or heathland soil, Accepted as anamorph of Eupen. shearii ; expanded Accepted sensu Stolk & Samson Accepted sensu Zaleski Synonym of P. dierckxii Synonym of P. manginii Synonym of P. citreonigrum Present study Accepted sensu Dong cf. P. westlingii and 14 of 16 isolates of P. soppii are from north temperate forest or heathland soils or conifer litter. The taxonomic approach taken in this study requires both multiple isolates and characterizations by analysis of many features. The former requirement can be met through a programme of systematic isolation from nature. The second requirement, in tandem with the first, is essential to taxonomic credibility. In Penicillium, as in other organisms, species are dynamic hypotheses that can be reinforced only through a continuing discovery of concordance in morphological, biochemical, physiological and molecular features. SYNOPTIC KEY TO SPECIES The following key is offered as an aid to identification of the species listed above. Prior to use of the key and examination of our technical descriptions, isolates must be grown as described in Methods. Features 1–5 under Morphology are features determined from a careful examination of conidiogenous structures developing on MEA at 3–10 d. The colour standard notations under Colony features are those of Ridgway (1912) ; other standards can be translated to that system using Christensen, Miller & Tuthill (1994). Species names, indicated in the key by number, are : 1. P. atrosanguineum ; 2. P. chrzaszczii ; 3. P. manginii ; 4. P. miczynskii ; 5. P. raistrickii ; 6. P. rolfsii ; 7. P. soppii ; 8. P. syriacum ; 8 A. ATCC ex-type ; 8 C CBS ex-type. Descriptions and notes on species in the P. miczynskii complex The following descriptions of Penicillium atrosanguineum, P. miczynskii, P. manginii and P. soppii are based upon our detailed Martha Christensen, J. C. Frisvad and Dorothy Tuthill 533 examinations of recent isolates, ex-types cultures, and NRRL cultures referred to by Raper & Thom (1949) and deposited by those authors at the Northern Regional Research Laboratory, USDA, Peoria, Illinois U.S.A. (Table 1). In contrast to the above species, which are distinct (Figs 1–4) and undoubtedly will prove to be abundant in specific habitats, P. chrzaszczii and P. syriacum are known only from ex-type cultures. We included them in the study but, as explained below in our brief notes, we recommend against use of those names until additional isolates and comparative data are available. Descriptions of P. raistrickii and P. rolfsii can be found in Raper & Thom (1949), Pitt (1979) and Ramirez (1982). Morphology 1. Conidial aggregation. Chains in tight columns 10–15 µm diam . . . . . . . . . . Chains separate, divergent ; in flaring plumes or loose columns . . . . . . 2. Conidia. Smooth or nearly so, usually becoming delicately roughened in age . . . . . Heavy-walled and conspicuously roughened with short, low ridges . . . . . Uniform in size and shape, globose-subglobose, 2±4–3 µm diam . . . . . . Uniform in size and shape, ellipsoidal with acute or rounded apices, 2±7–4¬2±2–2±7 µm . Variable in size and shape, globose-elongate, 2±3–3±5 µm in longest dimension . . . 3. Metulae. Absent ; phialides borne on an unbranched stipe or on branch elements arising singly or in pairs Present ; in clusters of three or more per whorl . . . . . . . . . 3 A Metular shape. Cylindrical, ending in a bulbous vesicle 4–5 µm or more diam . . . . . . Cylindrical or continuously broadening ; without a bulbous vesicle . . . . . 3 B Metular length. Short, mostly 8–16 µm long . . . . . . . . . . . Longer, commonly up to 18–35 µm long . . . . . . . . . 4. Surface of stipe Smooth, or with sparse delicate roughening . . . . . . . . . Uniformly finely roughened . . . . . . . . . . . . Roughened with coarse, granular incrustations . . . . . . . . . 5. Stipe length. Less than 50 µm . . . . . . . . . . . . . . Up to 100–500 µm or more . . . . . . . . . . . . 6. Sclerotia on MEA. Pale yellow to straw-coloured . . . . . . . . . . . White becoming buff to pale pink or coral pink . . . . . . . . Pale pink with brown flecks or bright brown . . . . . . . . . Yellow-brown to dull brown within a pale-coloured hyphal weft . . . . . Not observed . . . . . . . . . . . . . . . . . . . . . . 1, 5, 8 A . 2, 3, 4, 6, 7, 8 C . . . . . . . . . . . . . . . . 1, 3, 4, 5, 6, 7, 8 . . . 2 . 1, 2, 4, 5, 8 C . . . 3, 6 . . 7, 8 A . . . . . . . . . . . . . . . . . 1, 2, 3, 4 1, 2, 3, 5, 6, 7 . . . . . . . . 1, 2, 3, 5, 6, 7 . 1, 2, 4 . . . . . . . . . . . . . 2, 4, 6, 7, 8 . . 1, 3 . 1, 5, 6 . . . . . . . . . . 8C . 1–7, 8 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3, 4, 5, 6, 7, 8 C . . 1, 2, 8 A . . 1, 3, (5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . 3 . . 7, 8 A . . . 4 . . 1, 5, 8 C . 2, 3, 4, 6, 7, 8 A . . . 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 . 1–7 . 4 . 5, 6 . 7 . 3 . 1–8 Colony features 1. Colour of conidia in mass on MEA at 7–14 d. Light-coloured (Ridgway f, d, b) . . . . . . . . . . . Dark-coloured (Ridgway i, k, m) . . . . . . . . . . . 2. Colony reverse on MEA, 10–12 d, red. . . . . . . . . . 3. Other striking cultural features on Cz, CYA, YES or CREA. Colony reverse on YES dark maroon to black . . . . . . . . Colony reverse on Cz and CYA deep yellow to gold . . . . . . . Colony reverses uncoloured to pale grey or tan on all media at 7 d . . . . Colony reverse yellow-tinged or bright, clear yellow on most media . . . . Abundant acid production on CREA (agar yellow at 7 d) . . . . . . CREA medium remaining purple at 7 d or with very slight yellowing beneath colony only Rapid growth at 37° : 30 mm diam. or more, CYA 7 d . . . . . . . Production of secondary metabolites 1. Citrinin . . 2. Citreoviridin . 3. Citreomontanin . 4. Phoenicin . 5. Terrein . . 6. Pseurotins . 7. Asperentins . 8. Griseofulvin . 9. Benzomalvins . 10. Fumagillin 11. Tryptoquivalins 12. Gregatins . 13. Penicillic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2, (3) . 3, 4, 8 C . . 3 . . 1, 3 . 2, (4), (7) . . 7 . . 7 . 5, (7) . . 7 . . (7) . . 1 . . (1) . . 5, 6 Penicillium miczynskii group 534 7 5 µm 20 µm 5 µm 8 5 6 5 µm Figs 5–8. Penicillium atrosanguineum. Fig. 5. Habit sketch, IBT 11155. Fig. 6. Penicillus, NRRL 5891. Fig. 7. Conidia, NRRL 5891. Fig. 8. Phialides, NRRL 5891. Penicillium atrosanguineum B. X. Dong (Figs 5–9) CeskaU Mykologie 27 : 174–176 with 1 figure (1973) ; Ramirez, Manual and Atlas of the Penicillia, pp. 322–325 (1982). Conidiophores on MEA arising directly from the agar and from trailing hyphae, with stipes moderately tall and rough when viewed directly at low magnification, bearing 2–5 separate columns of conidia (1 per metula) 10–15 µm diam. or columns occasionally loosely merged and 25–30 µm wide. Stipes coarsely roughened, 70–200 (360)¬3–4 µm, occasionally bearing secondary fruiting structures that arise low on the stipe ; rami lacking or infrequent, 15–123 µm long ; metulae compactly arranged, mostly 3–5 per verticil, cylindrical, 10–21¬2±5–3±5 µm, non-vesiculate or with a slight vesicle 3±2–4 µm diam, even to uneven in length within the whorl (length differences up to 7–8 µm) ; phialides 8–9¬2±6–2±7 µm, cylindrical with a slight upward expansion then tapering abruptly at the tip ; conidia globose to subglobose, lightly pigmented, 2±4–3 µm and 3–3±1¬2±6 µm, delicately echinulate when viewed by ICT at 1600¬. The examined cultures are from stored wheat, oats, mouldy layercake and air in Czechoslovakia and Denmark ; to our knowledge, the species is not known from soil. The outstanding features of this species are : (1) production of a deep red to black pigment on YES agar and often a deep red pigment (phoenicin) on MEA ; (2) conspicuously roughened stipes ending in whorls of up to five cylindrical metulae ; (3) compact groups of abruptly tapered phialides bearing conidia in narrow columns, 1 per metula ; (4) globose to subglobose conidia mostly 2±5–2±8 µm diam. ; and (5) production of phoenicin and a series of unknown yellow pigments consistently, and in fresh isolates, production of tryptoquivalins and gregatins. Penicillium atrosanguineum differs from P. manginii, undoubtedly its closest relative, in its secondary metabolites and in forming shorter, conspicuously granular-roughened stipes that bear globose to subglobose conidia in narrow columns. Penicillium chrzaszczii K. M. Zalessky (Fig. 13) Bulletin International de l ’acadeUmie Polonaise des sciences et des lettres. Sci., Math. et Nat. Ser. B, pp. 464–466 (1927) ; Raper & Thom, A Manual of the Penicillia, pp. 464–466 (1949). Known only from the ex-type culture, NRRL 903 (¯ CBS 217.28). The outstanding features of NRRL 903 are : Production of citrinin, terrein and some unknown secondary metabolites ; Martha Christensen, J. C. Frisvad and Dorothy Tuthill 535 Figs 9–12. Penicilli of the four principal species. Bars ¯ 10 µm. Fig. 9. Penicillium atrosanguineum, NRRL 5891. Fig. 10. P. manginii, RMF A-146. Fig. 11. P. miczynskii, NRRL 1077. Fig. 12. P. soppii, RMF A-102. conidial chains in plumes or loose columns ; stipes smooth or rarely with slight, sparse roughening, commonly 350–700¬2±5–3 µm ; rami infrequent, short ; metulae in terminal whorls of 2–5 members, uneven within the whorl, 9–21¬3–3±5 µm, cylindrical, vesiculate ; phialides relatively short, 7–9¬2±3–2±8 µm ; conidia uniform, globose, (2±4) 2±6–2±8 (3) µm diam., conspicuously roughened with short ridges. When more cultures are available, this species should Penicillium miczynskii group Figs 13, 14. Conidia. Bar ¯ 5 µm. Fig. 13. P. chrzaszczii, NRRL 903. Fig. 14. P. miczynskii, NRRL 1077. be compared carefully with the ex-types and recent isolates of P. godlewskii K. M. Zalessky, P. jensenii K. M. Zalessky, P. turolense A. T. Martı! nez & C. Ramı! rez, P. waksmanii K. M. Zalessky and P. westlingii K. M. Zalessky. Penicillium manginii Duche! & R. Heim (Figs 10, 15–18) Travaux Cryptogamiques deUdieUs a Louis Mangin, MuseUum d ’Histoire Naturelle, Paris, pp. 450–454 with five figures (1931) ; Stolk & Samson, Studies in Mycology (Baarn) 23, pp. 48–50 (1983). Conidiophores on MEA tall, arising from the agar surface, commonly ending in a compact whorl of many metulae and a plume of straight but slightly divergent conidial chains, and in most isolates extending above a more conspicuous development of buff-coloured sclerotia that are produced adjacent to a strongly red-pigmented agar substrate. Stipes uniformly finely echinulate or in a few strains almost smooth, (225) 300–550 (775)¬2±8–3±8 µm, occasionally bearing secondary fruiting structures as long, irregular branches that arise low on the stipe ; rami lacking or infrequent, 13–19 µm long ; metulae mostly 5 or 6 per verticil, cylindrical or nearly so, (9) 10–14 (19) ¬2±9–3±7 µm, non-vesiculate or slightly expanded at the tip to 3±6–5 µm, even in length within the whorl or rarely uneven ; phialides 7±5–10¬2–2±7 µm, narrowly cylindrical tapering abruptly at the tip to near 1±3 µm ; conidia broadly ellipsoidal, 2±7–3±5¬2±2–2±7 µm (mostly 3–3±2¬ 2±4–2±6 µm), lightly pigmented, delicately echinulate when viewed by interference contrast at 1600¬. Sclerotia, present in most but not all isolates, at first loosely enveloped in pale yellow hyphae, subglobose to elongate mostly 200–360 µm 536 but up to 500 µm diam, yellow-brown to dull brown (17§d to 13¨o,) on all media, composed of polygonal cells 7–15 µm diam. with walls up to 4 µm thick. The examined isolates are closely similar morphologically and differ only slightly in cultural features. The ex-type culture of P. manginii, NRRL 2134 (¯ CBS 251.31), no longer produces sclerotia although they were described and illustrated as abundant in Duche! and Heim’s original observations. Except for its production of just 2–4 metulae per cluster and only sparsely roughened stipe, however, NRRL 2134 agrees in all morphological details with the above account and differs from our recent isolates culturally only in its failure to develop a deep red reverse pigment on MEA. Duche! & Heim’s isolate was from the sandy soil of a Ficaria ranunculoides community on the Cotentin peninsula of northwestern France. The recent isolates are from soils collected in Norway, Italy, North America, Africa and Costa Rica. The outstanding features of this species are : (1) production in recent isolates of a deep red pigment, phoenicin, on MEA, a gold-coloured reverse on Cz and CYA, and commonly an abundance of sclerotia on most media ; (2) conidiophore stipes with walls finely echinulate, ca 500 µm tall, with single tight whorls of metulae that are cylindrical, non-vesiculate and even in length ; (3) finely echinulate, broadly ellipsoidal, lightlypigmented conidia mostly 3–3±2¬2±4–2±6 µm that accumulate in plumes of straight, slightly divergent chains ; and (4) consistent production of citreoviridin, and of phoenicin and citrinin in most isolates. Twenty-two cultures not listed in Table 1 produce both phoenicin and citreoviridin ; isolates that do not produce red reverse colours in MEA and YES have lost the ability to produce phoenicin (NRRL 2134, NRRL 1024). P. manginii differs from P. miczynskii in conidium size and shape ; metular length, shape and evenness ; ornamentation of the stipe wall ; production of phoenicin ; and in many striking cultural features. Both species produce citreoviridin. We agree with Stolk & Samson’s (1983) interpretation of P. miczynskii and P. manginii as species that resemble one another but are distinct (Fig. 4). On the basis of conidial identity and other morphological and cultural similarities, we also agree with their opinion that P. sulfereum Sopp sensu Biourge as represented by NRRL 1024 and NRRL 2133 probably is a degenerated example of P. manginii. P. manginii differs from P. atrosanguineum in that the latter species has never been reported to produce sclerotia or citreoviridin, has globose to subglobose conidia, bears the conidial chains in columns rather than plumes, and is primarily associated with stored feeds. It differs also in cultural features : notably, colony reverses on Cz and CYA lack the characteristic yellow to gold pigmentation of P. manginii, there is production of acid on creatine agar (transformation to yellow at 7 d), and reverse colour on YES agar is maroon (3 k, m) to black. Penicillium miczynskii K. M. Zalessky (Figs 11, 14, 19–22) Bulletin International de l ’acadeUmie Polonaise des sciences et des lettres. Sci., Math. et Nat. Ser. B, pp. 482–484 (1927) ; Raper & Thom, A Manual of the Penicillia, pp. 309–312 (1949). Conidiophores on MEA mostly arising directly from the substrate, erect, tall, characteristically with neither rami nor Martha Christensen, J. C. Frisvad and Dorothy Tuthill 537 5 µm 17 5 µm 18 16 20 µm 5 15 5 µm Figs 15–18. Penicillium manginii. Fig. 15. Habit sketch, ATCC 18334. Fig. 16. Penicillus, ATCC 18334. Fig. 17. Conidia ATCC 18334. Fig. 18. Phialides, RMF 8771. metulae below the terminal whorl of metulae, bearing straight chains of conidia in plumes or loose columns ; monoverticillate structures occasional in some isolates. Stipes smooth or with a very sparse roughening, (90) 300–700 (1070)¬2±5–3±2 µm ; rami mostly lacking or infrequent, 7–150 µm long ; metulae in clusters of 2–8 (often 4–6), cylindrical, 2±5–2±8 µm diam. or broadening slightly to 3±2–3±5 µm, commonly ending in a vesicle 3±5–6 µm diam., (8) 11–17 (25)¬2±5–3±5 µm, uneven in length within a single whorl (differences in length can be 4–7 µm or more) ; phialides (7±5) 9–11 (12)¬2±2–2±8 µm, cylindrical, variable in length within the cluster and often with slender, elongate necks ; conidia thin-walled, lightly pigmented, at first appearing smooth or nearly so but in most strains becoming delicately roughened in age when viewed by interference contrast at 1600¬, globose to subglobose, mostly 2±5–2±8 µm diam. (range 2±4–3¬2±2–2±7 µm), uniform in size and shape. Sclerotia often present in recently isolated cultures, at first inconspicuous, more abundant at 15° than at room temperature, evident and at maximum size in several isolates after growth for 15–20 days, globose to elongate, (150) 170–310 (450) µm diam., cream-coloured to buff or pale orange in age and in some isolates flecked with dark colour as short segments of external hyphae develop brown to maroon pigment in age, firm but not gritty, constructed of thickwalled, polygonal cells mostly 10–18 µm diam. The examined isolates are from conifer forest soils in Poland, Wyoming and Oregon in the U.S.A., and from a heathland soil in Denmark. The outstanding features of this species are : (1) pale bluegreen sporulation and inconspicuous straw-coloured sclerotia in recent isolates ; (2) colony reverses yellow-tinged or bright, clear yellow in recent isolates on most media ; (3) stipes tall Penicillium miczynskii group 538 21 5 µm 5 µm 20 19 5 µm 20 µm 22 Figs 19–22. Penicillium miczynskii. Fig. 19. Habit sketch, RMF 8752. Fig. 20. Penicillus, RMF 8752. Fig. 21. Conidia, NRRL 1077. Fig. 22. Phialides, NRRL 1077. (commonly 400–700 µm), smooth or nearly so, mostly lacking rami, ending in whorls of metulae bearing conidial chains in plumes or loose columns ; (4) metulae to 19–25 µm long, vesiculate, uneven in length in a single whorl, and (5) conidia 2±5–2±8 µm diam., globose-subglobose, smooth or very finely prickled, and (6) production of citreoviridin in all isolates and terrein in most isolates. Penicillium miczynskii differs from P. soppii and P. manginii in that both of the latter species have even-lengthed metulae, mostly 10–14 µm long, and both produce subglobose or broadly elliptical conidia that are slightly larger than those in P. miczynskii. Additionally, P. miczynskii differs from P. soppii in colony reverse colour especially on Cz and CYA (yellow, yellow-gold or yellow-brown in contrast to pale grey or vinaceous grey in P. soppii), and from P. manginii in ornamentation of the stipe wall. Penicillium soppii K. M. Zalessky (Figs 12, 23–26) Bulletin International de l ’acadeUmie Polonaise des sciences et des lettres. Sci., Math. et Nat. Ser. B, pp. 476–477 (1927) ; Raper & Thom, A Manual of the Penicillia, pp. 279–282 (1949). Conidiophores on MEA arising directly from the agar surface, unbranched or rarely with a basal branch, ending in a whorl of metulae and a plume of straight conidial chains (occasionally tangled) in loose or flaring columns near 40–50 µm wide ; pale pink sclerotia 200–300 µm diam. abundant in most fresh isolates. Stipes smooth to faintly roughened with low puncta especially in fresh isolates, often appearing sparsely roughened when viewed directly, (200) 300–450 (900) µm¬2±5–4 µm ; rami mostly lacking or infrequent ; metulae compactly arranged, 3–7 per verticil, (9) 10–14 (22) by 2±7–3±5 µm occasionally expanding to 4 µm but more often non-vesiculate and with Martha Christensen, J. C. Frisvad and Dorothy Tuthill 539 5 µm 5 µm 25 5 µm 20 µm 26 23 24 Figs 23–26. Penicillium soppii. Fig. 23. Habit sketch, RMF 8829. Fig. 24. Penicillus, RMF 8829. Fig. 25. Conidia, RMF 8829. Fig. 26. Phialides, NRRL 701. little or no apical broadening, even in length within the whorl ; phialides parallel in the cluster, 8–10¬2±3–3 µm with short, tapered necks ; conidia variable in size and shape, from globose-subglobose, 2±3–3±3 µm diam., to broadly ellipsoidal, 3–3±5¬2±3–3 µm mostly 3–3±2¬2±4–2±8 µm, lightly pigmented, smooth, thin-walled, often becoming faintly roughened or prickled in age. Sclerotia globose to subglobose, to 70 µm diam. in the ex-type culture but commonly up to 240–345 µm diam. and abundant in recent isolates, uncoloured becoming pale pink with sparse brown flecks, firm to gritty, constructed of polygonal cells 11–23 µm diam. with walls up to 4 µm thick. Penicillium soppii produces a wide array of secondary metabolites, many of which were produced consistently in all fresh isolates. Terrein is produced by all isolates except the three old cultures that have also partially lost the ability to produce hard sclerotia : NRRL 2023, NRRL 912 and CBS 752.74. Terrein also is produced by most P. miczynskii isolates and P. chrzaczszii. Asperentin (¯ cladosporin) and 5«-hydroxyasperentin are produced by all isolates of P. soppii (except NRRL 2023). Pseurotin A is produced by all isolates except NRRL 701 and CBS 752.74. The benzomalvins are produced by all isolates examined except NRRL 912 and IBT K10. Griseofulvin is produced by most isolates, but in much smaller amounts than in isolates of P. raistrickii. Fumagillin could be detected in approximately one-half of the isolates. The examined isolates (Table 1) are from a pine forest soil in Poland, conifer or mixed forest soils in the U.S.A. 540 5 µm Penicillium miczynskii group 5 µm 28 29 30 20 µm 20 µm 27 31 15 µm Figs 27–31. Penicillium syriacum. Fig. 27. Habit sketch, CBS 418.69. Fig. 28. Conidiophores, CBS 418.69. Fig. 29. Tip of conidiophore, ATCC 140343. Fig. 30. Habit sketch, ATCC 140343. Fig. 31. Tips of conidiophores, after the drawings by Baghdadi (1968). Figs 28, 29 and 31 are at equal magnification. (Minnesota, North Carolina, Wisconsin, Wyoming), conifer litter in Wyoming and heathland soils in Denmark. The outstanding characteristics of P. soppii are : (1) soft green sporulation and recent isolates with pale pink sclerotia on MEA, Cz, CYA and YES agars ; (2) stipes smooth or basally finely roughened, mostly 300–450 µm tall bearing single whorls of 5–7 non-vesiculate, even-lengthed metulae ; (3) conidia variable in size and shape, mostly 2±8–3±2 µm in longest dimension, smooth or nearly so ; (4) colony reverses uncoloured to bland on all media at 7 d, and (5) production of terrein, asperentin, 5«-hydroxyasperentin and pseurotin A. This species differs from P. miczynskii in metular and conidial features, and in numerous cultural characteristics including colour of the sclerotia and reverse colouration on MEA, Cz, CYA and YES. Both Zaleski (1927) and Raper & Thom (1949) also noted relatively even-lengthed metulae up to 14 µm long in P. soppii in contrast to the unequal and longer metulae of P. miczynskii. It differs from P. manginii in that both the stipe and the conidia are essentially smooth rather than finely echinulate, and differs further in size and colour of the sclerotia. It differs from P. raistrickii primarily in that the latter species has coarsely roughened stipes, smaller conidia, and is a strong producer of acid on CREA. NRRL 701, characterized by Raper & Thom (1949) as a probable representative of P. soppii, is in excellent condition and resembles our recent isolates more closely than does the ex-type, NRRL 2023. Sclerotia up to 345 µm diam. are abundant in NRRL 701 and the recent isolates, whereas much smaller sclerotia are produced in the ex-type culture (Raper & Thom, 1949 ; Pitt, 1979 ; Ramirez, 1982). In agreement with other workers (Frisvad, Samson & Stolk, 1990 ; Anon., 1996) our examination of the ex-type cultures of Martha Christensen, J. C. Frisvad and Dorothy Tuthill P. matris-meae K. M. Zalessky, P. michaelis Quintan, P. rolfsii Thom var. sclerotiale Novobr. and P. severskii Schechovtsov has indicated that those names are synonyms of P. soppii. Penicillium syriacum Baghd. (Figs 27–31) Novosti Systematika Nizshikh Rastenii 7, 111–112 (1968). The illustration and description of P. syriacum and our examination of ex-type cultures (Table 1) indicate a mixed culture, and the species therefore is a nomen ambiguum. ATCC 34971, a pure culture, approximates P. corylophilum Dierckx and resembles Baghdadi’s description and drawing of his P. syriacum. The CBS ex-type culture, CBS 418.69, also is a pure culture but is strikingly different from ATCC 34971. We agree with others in interpreting CBS 418.69 as a form related to P. fellutanum Biourge sensu Raper & Thom, P. charlesii G.Sm. and P. citreonigrum Dierckx (Stolk & Samson, 1983 ; Gams, 1993 ; Anon., 1996). IMI 140343, a third ex-type culture, produced a mixed growth of the CBS and ATCC strains. Until additional material becomes available, P. syriacum is a nomen ambiguum. This study has been supported in part by two grants from the United States National Science Foundations (RII-8610680 and DEB 9632880). We thank Ellen Kirstine Lyhne and Terri Johnston for valuable technical assistance. REFERENCES Al-Musallam, A. (1980). Revision of the black Aspergillus species. Thesis. Utrecht University. Anon. (1996). Centraalbureau voor Schimmelcultures List of Cultures : Fungi and Yeasts. 34th Edition. CBS : Baarn-Delft, The Netherlands. Baghdadi, V. Ch. (1968). De speciebus novis Penicillii Fr. et Aspergilli Fr. e terris Syriae isolatis notula. Novosti Systematica Nizschikh Rastenii 7, 96–114. Brummett, R. K. & Powell, C. E. (1992). Authors of Plant Names. Royal Botanic Gardens : Kew. Christensen, M., Miller, S. L. & Tuthill, D. (1994). Colour standards : a review and evaluation in relation to Penicillium taxonomy. Mycological Research 98, 635–644. Fassatiova! , O. & Kubatova! , A. (1990). Evaluation of the diagnostic features of some species of Penicillium section Divaricatum. In Modern Concepts in Penicillium and Aspergillus Classification (ed. R. A. Samson & J. I. Pitt), pp. 149–157. Plenum Press : New York. Filtenborg, O., Frisvad, J. C. & Svendsen, J. A. (1983). Simple screening method for toxigenic moulds producing intracellular mycotoxins in pure cultures. Applied Environmental Microbiology 45, 581–585. Filtenborg, O., Frisvad, J. C., Thrane, U. & Lund, F. (1995). Screening methods for secondary metabolites produced by fungi in pure culture. In Introduction to Food-Borne Fungi (ed. R. A. Samson, E. S. Hoekstra, J. C. Frisvad & O. Filtenborg), pp. 270–274. Centraalbureau voor Schimmelcultures : Baarn. (Accepted 28 July 1998) 541 Frisvad, J. C. (1985). Creatine sucrose agar, a differential medium for mycotoxin producing terverticillate Penicillium species. Letters in Applied Microbiology 1, 109–113. Frisvad, J. C. (1992). Chemometrics and chemotaxonomy : a comparison of multivariate statistical methods for the evaluation of binary fungal secondary metabolite data. Chemometrics and Intelligent Laboratory Systems 14, 253–269. Frisvad, J. C. & Filtenborg, O. (1990). Revision of Penicillium subgenus Furcatum based on secondary metabolites and conventional characters. In Modern Concepts in Penicillium and Aspergillus Classification (ed. R. A. Samson & J. I. Pitt), pp. 159–172. Plenum Press : New York. Frisvad, J. C. & Thrane, U. (1987). Standardized high-performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone indicates and UV-VIS spectra (diode array detection). Journal of Chromatography 404, 195–214. Frisvad, J. C., Samson, R. A. & Stolk, A. C. (1990). Disposition of recently described species of Penicillium. Persoonia 14, 209–232. Gams, W. (1993). Supplement and Corrigendum to the Compendium of Soil Fungi, IHW-Verlag : Eching. Larsen, T. O. & Frisvad, J. C. (1995 a). Characterization of volatile metabolites from 47 Penicillium taxa. Mycological Research 99, 1153–1166. Larsen, T. O. & Frisvad, J. C. (1995 b). Chemosystematics of Penicillium based on profiles of volatile metabolites. Mycological Research 99, 1167–1174. Mueller, G. M. (1985). Numerical taxonomic analyses on Laccaria (Agricales). Mycologia 77, 121–129. Pitt, J. I. (1979). The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromyces. Academic Press : London. Ramirez, C. (1982). Manual and Atlas of the Penicillia. Elsevier Biomedical Press : Amsterdam. Raper, K. B. & Thom, C. (1949). A Manual of the Penicillia. Williams & Wilkins Company : Baltimore. Ridgway, R. (1912). Color Standards and Color Nomenclature. Ridgway : Washington D.C. Rohlf, F. J. (1993). NTSYS-pc : Numerical Taxonomy and Multivariate Analysis System, Version 1-80. Exeter Software : Setauket, NY. Sørensen, T. (1948). A method for establishing groups of equal magnitude in plant sociology based on similarity of species content. Kongelige Danske Videnskabernes Selskab Biologiscke Skrifter 5, 1–34. Stolk, A. C. & Samson, R. A. (1983). The ascomycete genus Eupenicillium and related Penicillium anamorphs. Studies in Mycology, 23, 1–149. Svendsen, A. & Frisvad, J. C. (1994). A chemotaxonomic study of the terverticillate penicillia based on high performance liquid chromatography of secondary metabolites. Mycological Research 98, 1317–1328. Taylor, R. J., Patterson, T. F. & Harrod, R. J. (1994). Systematics of Mexican spruce – revisited. Systematic Botany 19, 47–59. Thom, C. (1930). The Penicillia. William & Wilkins Company : Baltimore. Zaleski, K. (1927). U$ ber die in Polen gefundenen Arten der Gruppe Penicillium Link. I, II and III Teil. Bulletin International de l ’acadeU mie Polonaise des sciences et de lettres. Classe des sciences matheU matiques et naturelles SeU ries B : Sciences Naturelles, 417–563. Zambino, P. J. & Harrington, T. C. (1992). Correspondence of isozyme characterization with morphology in the asexual Leptographium and taxonomic implications. Mycologia 84, 12–25.