Vol. 67, Part 1

Trans. Br. mycol. Soc. 67 (1) 1-15 (1976) Printed in Great Brita in

CAROTENOIDS AS ADDITIONAL TAXONOMIC CHARACTERS IN FUNGI: A REVIEW

By L. R. G. VALADON* Department of Botany, Royal Holloway College, University of London, Egham Hill, Egham, Surrey TW20 oEX

The aim of this review is twofold: first to discuss some of the pitfalls of carotenoid identifica­ tion in fungi and secondly to observe to what extent carotenoids may be useful as taxonomic characters. The results suggest that a number of carotenoids have been wrongly identified and that with newer techniques available, new carotenoids will almost certainly be identified which may help taxonomists. In a number of cases carotenoids are very good taxonomic markers. It is well known that in higher plant tissue, caro­ certain photosynthetic purple bacteria had been tenoids are closely tied up with chlorophyll and are identified in Lycogala epidendron, useful during photosynthesis. The carotenoids of (Lederer, 1938) and Neurospora crassa (Haxo, leaves seem to vary little, consisting mainly of 1949) but was later shown to be absent from these fJ-carotene and lutein and of some of their deriva­ organisms (Liaaen-Jensen, 1965). tives. However, flowers and fruits tend to have Liaaen-Iensen (1965) suggests that 3,4-dehydro­ certain carotenoids specific to themselves, e.g, rose lycopene (VI) had been previously mistaken for hips (rubixanthin, I), woody nightshade (lycoxan­ spirilloxanthin (V) in N eurospora crassa and this is thin, II and lycophyll, III) and Gazania rigens more readily understandable in view of the fact flowers (gazaniaxanthin, IV) (Valadon, Sellens & that this carotenoid was rapidly formed in N. crassa Mummery, 1975). In the algae, carotenoids are from more saturated carotenoid precursors (Grob, good taxonomic characters. Valadon (1966) 1958). On the other hand, Haxo (1949) had attempted to show that this may be the case in obtained his pigment as a major component (27 % fungi and concluded that much more information total carotenoid) whereas 3,4-dehydrolycopene was required before any definite conclusions could was only a minor component (2 %) of Liaaen­ be reached. Chemical has interested a Jensen's (1965) cultures. If it were the same com­ great number of workers in the last 10 years or so pound in the two cases then some mutation or a and with more recent studies available it may be different stra in was the cause of such observed possible to take another look as to whether or not differences, since these compounds with similar carotenoids may be good taxonomic markers in absorption spectra were found in different amounts fungi. in the two strains used. From the earlier works of Zopf (1890) and Kohl Another example of mistaken identity was given (1902) it became known that a number of fungi by Valadon & Mummery (197Sb) who showed that contained carotenoids but it was left to later what was identified as neurosporene (VII) by workers to identify these pigments more accu­ Turian (1960) and Fiasson (1968) in Cantharellus rately. As newer methods of identification were infundibuliformis might in fact be an expoxycaro­ used it became obvious that a number of the earlier tenoid (VIII). This compound, which was found works which identified carotenoids, mainly by to make up 72 % total carotenoids (Fiasson, 1968), absorption spectra and by co-chromatography, was also the major component of both Turian's were wrong. There are a number of carotenoids (1960) and Valadon & Mummery's (197Sb) with similar absorption spectra and some of these samples. It had the same adsorption spectrum as may be similarly adsorbed on impregnated paper neurosporene (415, 440 and 470 om in hexane) but or on various columns and may not be separable on the modified cone. HCl-ether test of jungalwala using these techniques, yet they may be different & Cama (1962) it was converted to its furanoid compounds. Spirilloxanthin (V) restricted to oxide (IX) (absorption spectrum 380, 400 and 420 * Lecture deliveredat the meetingon Fungal Secon­ om in hexane). This compound cannot be neuro­ dary Metabolites held at Queen Elizabeth College, sporene and was shown to be an expoxycarotenoid. University of London on 20 March 1975. (Held under A further point worth making is that pathways of the auspices of the Physiology Group of the B.M.S.) carotenoid biosynthesis in fungi seem to have

Vol. 66, Part 3, was issued 13 July 1976

III YC 67 2 Carotenoids as additional taxonomic characters in fungi

Rubixanthin ~R~ (I)

I HO I HOH2C~R~ Lycoxanthin (II)

~~CH20H HOH 2C R I I Lycophyll ~R (III) HO Gazaniaxanthin (IV)

Spirilloxanthin (V)

3.4-Dehydrolycopene (VI)

Neurosporene (VII)

5.6-Epoxy-carotene (VIII)

5,8-Furanoid epoxide (IX)

Key to the chemical formulae (pages 2, 4, 7):

R=

R2=unknown in this particular case

R) and R.=fatty acids

R.= Acyl radical L. R. G. Valadon 3 Phytoene t Phytofluene l , -Carotene Aleuriaxanthin ester + ~eurosrorene~.• Aleuriaxanthin , 2, dehydroplectan- Lycorene iax;nthin ester a -Zeacarotene , 2, dehydro- + Lycoxanthin P-Zeacarotene plectaniaxanthin a-Carotene t + • t Lycophyll r -Carotene--_.~ Plectani- --.. Plectani a-Carotene ~ ~ axanthin axanthinester Torulene p-Carotene ~ Monoepoxy-ji- Lutein• J ~ carotene H Neurospor­ Cryptornthin l Lutein-5, 6­ axanthin epoxide Zeaxanthin Mutatochrome t H Flavoxanthin Neoxanthin - Antheraxanthin Diepoxy-ji­ Monoepoxy­ H carotene a-carotene Violaxanthin t + Aurochrome• Flavochrome Auroxanthin Fig. 1. Suggested pathways of the synthesis of IX-, fl- and y-carotenes and of their derivatives (Fiasson, 1968; Valadon & Mummery, 1975a) evolved along a different line to that of photo­ fied. The following example will illustrate this. synthetic tissues (Fig. 1). No lutein (X) has been Lederer (1938) obtained a dried specimen of found in fungi although in a few cases small Aleuria aurantia (Vienna, Austria) and identified amounts of a-carotene (XI) have been reported. jJ-(XIII), y-carotene (XIV) and possibly ribi­ The identification of a-carotene is not reliable xanthin (I) (458, 490 nm in petroleum ether). because the pigment with absorption spectrum 420, Although Valadon (1964a) was able to obtain eight) 444 and 475 in hexane and weakly absorbed on bands after MgO-Celite (1: 1) v/v column chro­ column chromatography is not always a-carotene. matography, he positively identified only the Arpin et al. (1971) showed that a carotene in same three carotenoids in specimens ofA. aurantia Caloscypha fulgens was, jJ, y-carotene (XII), a obtained in Windsor Great Park, England. Co­ new pigment with a terminal methylene group, chromatography with rubixanthin (I) from rose which could easily be taken for a-carotene. Fiasson hips suggested that a fourth pigment identified as (1968) using the newer methods of identification rubixanthin? by the latter worker was indeed could not identify a-carotene in certain fungi rubixanthin. Liaaen-Jensen (1965) obtained two which were earlier reported to contain it. lots of A. aurantia from two locations in Norway These examples suggest that most fungi will and found only slight differences between their have to be re-investigated using modern tech­ carotenoids (Table 1) and identified a new fungal niques. Certainly new carotenoids will have to be carotenoid, aleuriaxanthin ester (XV), in both. characterized before they become helpful to the The first batch had almost equal quantities of taxonomist. Another aspect which needs consider­ jJ-(XIII) and y-carotene (XIV), minute amounts ing is the fact that different investigators not only of lycopene (XVI) and 3A-dehydrolycopene (VI) extend the range of carotenoids found by previous and 25 % aleuriaxanthin (XV) ester. The second workers but also fail to find those originally identi- batch on the other hand had twice the amount of

1-2 4 Carotenoids as additional taxonomic characters in fungi

,, ·Caroten e (Xl )

p,"I-Carotene (XII )

p·Carotene (XIII)

{'-carotene as p-carotene and no 3>4-dehydrolyco­ carotenoids: a·, P-, {'-carotene, neurosporene, pene, otherwise it was identical to the first batch. lycopene, 3>4-dehydrolycopene, rubixanthin and She found no ribixanthin although 1 %of her total aleuriaxanthin ester. Later on that year Arpin carotenoids was not identified (Table 1). Valadon (1968) obtained 200 g dry weight of specimens of & Mummery (1968) obtained a number of speci­ A. aurantia from Theys (Isere), France, and mens of this from Englefield Green, Surrey, characterized a new pigment, dehydro-a-plecr­ England, and were able to separate twelve bands, aniaxanthin (XVII) and its ester, as well as P-, of which they identified the following eight {'-carotenes, torulene (XVIII) and aleuriaxanthin L. R. G. Va/adon 5

Table 1. Carotenoids found in AIeuria aurantia (Pers. ex Hook.) Fuckel by variousauthors. The results are expressed as percentage total carotenoids A. aurantia A. aurantia A, aurantia A. aurantia A. aurantia A. aurantia from from Windsor from from from from Vienna, Great Park, Jonsvannet, Withelmsyr, EnglefieldGreen, Theys, Austria England Norway Norway England France (Lederer, (Valadon, (Liaaen- (Liaaen- (Valadon & (Arpin, Carotenoids 1938) 1964a) Jensen,1965) Jensen, 1965) Mummery, 1968) 1968) a-Carotene 0'3 p-Carotene + 40 '0 34'0 26'0 43'9 y-Carotene + 27'6 39'0 5°'0 15'5 Rubixanthin ? 28'7 9'9 Neurosporene 4'3 Aleuriaxanthin ester 25'0 22'0 21'5 20'0 Lycopene 0'2 1'0 1'7 3,4-Dehydrolycopene 1'0 0'8 Torulene + Dehydro-z' plectaniaxanthin 0'5 Dehydro-z plectaniaxanthin 4'0 ester Unidentified + 3'7 1'0 1'0 2'1 1'5 Total carotenoids 0,82 1'65 1'50 0·68 1'50 (mg/g dry wt)

(xV) ester. He was able to identify torulene in proposed two subdivisions of the family Crypto­ traces even though this had not previously been coccaceae, one lacking carotenoids (Crypto­ characterized in A. aurantia by other workers. coccoideae) and the other containing carotenoids This is an example whereby carotenoids appearing (Rhodotoruloideae). However, Nakayama, Mac­ in traces could be positively identified because kinney & Phaff (1954) found that whereas certain large amounts of material were available for species of Rhodotorula contained predominantly analysis. He did not obtain rubixanthin or 3,4­ torulene (XVIII) and/or torularhodin (XIX), other dehydrolycopene and suggested that these two species of this and also all species of compounds could have been wrongly identified Cryptococcus studied contained mostly fJ-carotene aleuriaxanthin ester (or an isomer) and/or the ester with minor amounts of y-carotene and lycopene. ofdehydro-zt-plectaniaxanthin. He may have been Temperature was a very important factor con­ right but in the four cases where aleuriaxanthin tributing to the degree of pigmentation and they ester has been identified it made up between 20 concluded that carotenoids were so variable in and 25 % total carotenoids. Valadon & Mummery these fungi that a separation of the two genera on (1968) and Liaaen-Jensen (1965) obtained 3,4­ the basis of manifest carotenoids was vague and dehydrolycopene as well as aleuriaxanthin ester, so arbitrary. Further, Villoutreix (1960) produced a these two are different compounds. What was number of mutants of R. mucilaginosa through identified as rubixanthin (Valadon & Mummery, u.v.virradiation; some of these did not contain 1968) may have been another ester of aleuriaxan­ carotenoids although the wild type did. This was thin having the same chromatographic properties also shown for R. glutinis (Nusbaum-Cassuto, as rubixanthin but not the aleuriaxanthin ester of Villoutreix & Threlfall, 1965). Valadon & Heale Liaaen-jensen (1965) and Arpin (1968), which was (1965) demonstrated another aspect of fungal also characterized by them (Valadon & Mummery, metabolism by producing coloured mutants by 1968). On the other hand, A. aurantia (Liaaen­ u.v.-irradiation of the colourless, wild-type Jensen, 1965; Arpin, 1968) has beenshown to have Verticillium albo-atrum. Some of these mutants two carotenoids (XV, XVII) with hydrolylation on had large amounts of lycopene and/or of torulene positions i ' and 2 ' respectively and it is therefore and of neurosporaxanthin (XX), while the wild­ not unexpected that in some strains the hydroxyla­ type contained only the colourless C,O precursor, tion may be at the 3 position as in rubixanthin (I). phytoene (Fig. 1). This shows that it is possible to This brings us to the question ofdifferent strains produce carotenoid mutants artificially in fungi. and mutation in fungi, In the it Arpin (1968) collected a number of coloured has not been easy to classify the asporogenous strains of Sarcoscypha coccinea and analysed their yeasts but Lodder & Kreger-van Rij (1952) carotenoids content. The results (Table 2) showed 6 Carotenoids as additional taxonomic characters in fungi

Table 2. Carotenoids of different coloured strains of Sarcoscypha coccinea (Jacq. ex S. F. Gray) Lamb. (=Plectania coccinea (Scop. ex Fr.) Fuckel (Arpin, 1968). The results are expressed as percentage total carotenoids Strains

(Normal) Lot 1 Lot 2 Lot 3 Lot 4 Carotenoids red-orange orange orange orange orange Cream p-Carotene 24'0 9°'0 76'0 90'0 75'0 + p-Zeacarotene 8'0 y-Carotene 3'5 Torulene 10'0 ? 12'0 9'0 13'0 Pleetaniaxanthin 3'0 Plectaniaxanthin(monoester) 6'0 Plectaniaxanthin (diester) 47'0 0'5 ? I>ehydro-2'plectaniaxanthin 20'0 Unidentified 1'0 12'0 Total carotenoids (mg/100g) 160'0 70'0 60'4 79'1 75'2 trace ?, Not identifiedwith certainty. that there were differences between the cream, diaceous R. rosea and possibly the mode of origin orange and red-orange strains of this fungus. There in nature of Rhizophlyctis and Blastocladiella was very little carotene' in the cream strain, (Cantino & Hyatt, 1953). between 60 and 80 mg/100 g dry weight in the four It was not until Turian (1960) did some bio­ orange strains and usually about 160 mg/100 gin chemical work on various fungi that the first the normal red-orange strains (several strains were suggestion was made of the possible use of analysed but only one instance given in this case). carotenoids as a taxonomic marker. He showed Not only were there differences between total that the orange-red Guepinia helvelloides did not carotenoids but also among individual carotenoids. contain carotenoids but quite likely anthra­ Plectaniaxanthin (XXI) and its derivatives (plect­ quinones; Lederer (1938) had earlier shown that aniaxanthinmonoester XXII, diester XXIII and Tremella mesentericacontained fJ-carotene. On this dehydro-z/-plectaniaxanthinXVII) were the domi­ basis, Turian (1960) suggested that this is a further nant pigments in the red-orange wild-type, and character which supports the separation of the fJ-carotene in the orange 'mutants'. Differences do tribes; Tremelleae (containing carotenoids) and therefore occur in nature and one must be very Guepineae (containing anthraquinones) among the careful about generalization. large family of Tremellaceae. The genus Guepinia is replaced by Martin (1944) by that of Dacryo­ pinax, a member of the family Dacrymycetaceae TAXONOMIC IMPLICATIONS which also includes Dacrymyces, and Calocera Cantino & Hyatt (1953) were probably the first while Tremella is a member of Tremellaceae. authors to use carotenoids as one of their markers There are therefore other marked differences in suggesting evolutionary relationships between between these two genera. However, it is members Blastocladiella and Rhizophlyctis. Mutants of of the Dacrymycetaceae which on the whole Blastocladiella were obtained which possessed contain carotenoids, e.g. Dacrymyces, Calocera certain characteristics in common with both and Dacryopinax and therefore carotenoids are not Rhizophlyctis rosea and B. emersonii and these good taxonomic characters in this case. This shows results suggested a close relationship between these that we cannot be too careful about deductions two genera. Moreover they found that whereas from data based on only two species. wild-type Blastocladiella possessed a-ketoglutarate Among the lower Ascomycetes a number of oxidase activity and no detectable carotenoids, the fungi have been found, the life cycles of which are two mutants of Blastocladiella and the wild-type relatively difficult to work out (Valadon, 1961). Rhizophlyctis rosea possessed y-carotene and no Species of Protomyces and of Taphrina macro­ detectable a-ketoglutarate oxidase activity. The scopically resemble one another, although T. results they obtained, coupled with the carotenoid deformans causes peach leaf curl and P. inundatus picture, offered direct and undisputable evidence causes galls on Apium nodiflorum. In culture they for the origin, from a blastocladiaceous fungus, of look alike, bud in a yeast-like manner and are an organism which closely resembled the chytri- pigmented. A very simple way of differentiating L. R. G. Valadon 7 OH ~R~

Plectaniax~anthin(XXI) O-C-R3 ~ d ~ R 00

Plectaniaxanthin, mono-ester (XXII)

O-C-R3 ~R~O.~_" Plectaniaxanthin, di-ester (XXIII) 0

~ R ~ Ecl>;n~~(XXIV) ~ ~ R Canthaxanthin (XXV) ~ ~tonnth;n(XXVI) R OH ~ COOH 0l. n, -f Apo-S' -carotenoic acid (XXVII) OH 0 ~R~ OH o Phillipsiaxanthin (XXVIII) ORu o R~ o OH Phillipsiaxanthin, mono-ester (XXIX)

Table 3. Carotenoids in three species ofProtomyces (Valadon, 1963, 1964b) and their absence in two species of Taphrina. The results are expressed as percentage totals carotenoids Total carotenoids Species a-Carotene y-Carotene j1-Carotene Lycopene (pg[g dry wt) Protomyces inundatus Dangeard 8'0 87'0 + 7'4 P. inouyei P. Hennings 4'2 93'7 2'1 4'9 P. pachydermus Thuemen 1'2 95'8 3'0 5'8 Taphrina deformans (Berk.) Tul. T. communis (Sadeb.) Giesenh. 8 Carotenoids as additional taxonomic characters in jungi

Table 4. Carotenoids of certain Phycomycetes fl- b­ y­ /l­ Neuro­ Lyco­ Zea­ Caro­ Caro­ Caro­ Refer­ Phycomycetes sporene pene carotene tene tene tene ences* Chytridiales Rhizophlyctis rosea (de Bary& Woronin)Fischer + +++ 1 Cladochytrium replicatum Kading + +++ 2 Blastocladiales Blastocladiella spp. Matt. +++ 3 Allomyces arbuscula Butler +++ 4 A. macrogynus(Emerson)Emerson & Wilson +++ 4 A. moniliformis Coker& Braxton +++ 4 A. [auanicus Emerson + +++ + 4,5 Mucorales Blakeslea trispora Thaxt. + + + + +++ 6 Choenophora cucurbitarum (Berk, & Rav.) Thaxt, - +++ 7 Mucor hiemalis Wehmer + + + + +++ 8 Phycomyces blakesleanus Burgeff + + + + + +++ 9 P. nitens (Agardh.) Kunze ex Fr. + + + + +++ 10 Pilaira anomala (Ces.) Schroet. +? +++ 11 Pilobus kleinii van Tiegh. + ++ 12 Syzygites megalocarpus Ehrenberg ex Fr. + + +++ 13 + + + indicates the major caroteneand + indicates presence. * 1, Davies (1961); 2, Fuller & Tavares (1960); 3, Arpin (1968); 4, Emerson& Fox (1940); 5, Turian & Haxo (1954); 6, Anderson et al. (1958); 7, Barnett, Lilly & Krause (1956); 8, Herber (1974); 9, Goodwin (1952); 10, Goodwin & Griffiths (1952); 11, Fletcher (1969); 12, Fiasson (1968); 13, Wenger & Lilly (1966). between these two genera in culture is by extracting strengthened their conclusions. Porter & Valadon the pigments. Whereas P. inundatus, P. inouyei and (1975) have shown that S. aggregatum contains the P. pachydermus contains carotenoids, T. communis following carotenoids : a-, j3-carotenes, echinenone and T . deformans do not (Table 3). Therefore one (XXIV), canthaxanthin (XXV) and possibly can easily distinguish between these two genera isocryptoxanthin (XXVI). Very few fungi are (Valadon, 1963, 1964b) although more information known to contain keto-carotenoids, like echinenone on other species of these two genera would be most and canthaxanthin (Fiasson, 1968) and they welcome. certainly have not yet been identified in the Oomy­ Another example where carotenoids have been cetes so far studied. Ketocarotenoids are typical used to differentiate between two genera is given animal carotenoids and are also common among by Arpin (1966). Clitocybe uenustissima which was algae and these results are in agreement with the previously called Hygrophoropsis is a bright orange suggestion that 'there are similarities between Agaric which may be confused with Hygrophoropsis the Thraustochytriaceae and the marine creepy­ aurantiaca, yet j3- and y-carotenes have been crawlie Labyrinthula' (Darley et al. 1973). identified in the former with the complete absence If one takes a closer look at the carotenoids of carotenoids in the latter. This, together with identified in the Phycomycetes, one finds that other morphological data, supports the placing of members of the Chytridiales and Blastocladiales so these two fungi in different genera. far studied contain ')'-carotene as their major pig­ A further case may be mentioned here. The ment, whereas the Mucorales contain j3-carotene Thraustochytriaceae was placed by Sparrow (1943) as the major one (Table 4) so it is possible to as a family of the Saprolegniales (Oomycetes) as separate these three Orders into two groups they are chytrid-like organisms that reproduce by according to the main pigment present. means of biflagellate zoospores. Schizochytrium It is among the Discomycetes that a systematic aggregatum belongs to this family and was recently attack was launched in an attempt to obtain as shown to have very different cell wall character­ much information as possible about carotenoids istics to those of typical Oomycetes (Darley, Porter and taxonomy. Arpin (1968) studied both the & Fuller, 1973). These authors have therefore operculate and the inoperculate Discomycetes. concluded that this family should be dissociated First, a look at Dennis' (1960) taxonomic account from the Oomycetes: carotenoid studies have of British Cup Fungi shows how difficult it is to Table 5. Carotenoids of operculate Discomycetes (Arpin, 1968). See Table z for details of Sarcoscypha spp. which may be part of the subfamily Sarcoscypheae

I .!.t; t:: u_ I .9 <>'" ... I t:: ..c: :s -a... ~ I:: ... ?. :a I:: -e <> I:: • ~ll <> ~ ... <> ~ ... s ~ I:: o~ ~~ 0 ... o » ... ..c:<> ~ ~ go I:: § ~ ~ .~ s ... I:: t:: ~>. ~ ~ .~ .) 0 c, ~0<> ~ .1 5 ... ~ d ... -..c:: "tl 0 I ~~ u~ ta -e e, ~ ::l u U ] 2'0: o ~ll ... » 0 d <>'" ~.~ 'a I ~e ~~ ii::<> ~~ ;J Z ....l ;... E-o ~ ~d ~ ~ I=l.<'" Family Aleuriaceae sensu Arpin Group 1 Sowerb)'ella unicolor (G iil.) Nannf . - - + + + + ++ + S. radiculata )Sowerby ex Fr.) Nannf. - - + - + + +++ ------+ Caloscypha fu :gens (Pers.) Boud, + + + + + - +++ ------+ Anthra cobia melaloma (Alb. & Schw. ex Fr.) Boud, + - + ? -- ++ + ------Pulvinula constellatio (Berk. & Br.) Boud. - - + - - - + ++ - - - + - - - + Group 2 Leucoscypha rutilans (Fr .) Dennis & Rifai. - + + - - - +++ - - + - - - - + ~ Aleur ia rhenana Fuckel - - + + -- + + + ------+ Me/astiza chateri (Smith) Boud. - + - - - + - +++ - + - - + ~ M. greletii Le Gal - + - - - + - +++ - + - - - Octospora calichroa (Boud.) ------+ -- + + +? - - - - O. leucoloma Hedwig ex S. F . Gray - - + --- + -- +++ - - - - - 0 Group 3 ~ Coprobia granulata (Bull. ex Fr.) Boud. - - +++ --- + ------+ Cheilymenia theleboloides (Alb. & Schw.) Boud, - - + ++ ------+ [ C. crucipila (Cooke & l)hi11~s)Le Gal - - + + + ? ------<::l Scutellinia serosa (Nees ex r.) Kuntze - - + -- - ++ + ------;:s S. umbrarum (F r.) Lambotte + - +++ + ------+ S. arenosa (Ve!.) Le Gal - - +++ + ------+ S. superb a (Ve!.) Le Gal - - + + + + ------+ S. scutella var. cervorum (Vel.) Le Gal - - + + + + ------S. ampullaceae (Limm .) Kuntze - - + + + + ------+ S. trechispora (Berk. & Br.) Lambotte -- + + + + ------ (Alb. & Schw. ) Sacc o -- - +++ ------+ + + G. ma ialis (Fr.) Boud. - - + + - - + ------+ + + Family Sarcoscyphaceae Subfamily Sarcoscypheae Pyronema omphalodes (Bell . ex. Fr.) Fuckel ------+ + +? ------+ P. confiuens» Tul. ( = P. omphalodes) - - + + - - + ------+ Pithya vulgaris Fuckel ------+++ -- + + - - - - Cookenia sulcipes (Berk) Kuntze ? + - - - - - + - - - - + + + + + C. tricholoma (Mont .) Kuntze ------+ + + + + + + Phillipsia carminea (Pat.) Le Gal - + ------+ + ++ + - P. subpurpurM Berk. & Dr. - + - -- - - + -- - ++ + - + P . carn icolor Le Gal ------+ + + " Also contains celaxanthin ( = 3-hydroxytorulene). + + + . Major carotenoid; + , carotenoid present.

\0 10 Carotenoids as additional taxonomic characters in fungi differentiate between the various families and the phineae and Pezizineae; the former is divided into tribes therein. The Pezizales which comprise all two families Sarcosomataceae and Sarcoscyphaceae species with operculate asci are divided into six each divided into two tribes. One of the characters families, only two of which contain carotenoids, he uses to separate the Sarcosomataceae from the namely the Humariaceae and the Sarcoscyphaceae. Sarcoscyphaceae is that apothecia of the former The Humariaceae is divided into three tribes: are dark-coloured with melanin-like pigments and Lachneae, Ciliarieae and Aleurieae. ' In Boudier's excipulum devoid of carotenoids; but such pig­ system tribe Lachneae comprises seven genera ments rarely occur in the paraphyses whilst represented in Britain but it must be admitted that apothecia of the latter are bright-coloured, usually several of these are separated on rather ill-defined with carotenoids in the and often also grounds'. Also 'five genera are conventionally in the excipulum. Korf (1973) divides the Sarco­ recognized in the tribe Ciliareae but the distinction scyphaceae into two tribes depending on whether between Scutellinia, Chei/ymenia and Neottiella is all asci within each apothecium mature simul­ not altogether satisfactory. Melastiza may have taneously (Boedijnopezizeae) or asci of various affinities with Aleuria in the other tribe Aleurieae'. ages within a single apothecium mature successive­ The third tribe is Aleurieae 'a residual assemblage ly (Sarcoscypheae). This separates the genus of genera, defined largely by negative characters; Cookeina (Boedijnopezizea) from Phillipsia (Sar­ the absence of a blue tip in iodine, the coscypheae) which were both shown to have the absence of clearly-differentiated hairs and the rare carotenoid phillipsiaxanthin as their major absence of broad asci protruding conspicuously carotenoid and which could be placed together in above the hymeniallevel at maturity'. It is evident the Sarcoscypheae (Table 5). that it is not easy to differentiate not only between Korf (1973) divides the suborder Pezizineae the families but also between the genera. Moreover into five families. The presence and absence of other taxonomists (Le Gal, 1963; Rifai, 1968) are carotenoids are used in two of the families: rather divided in what constitutes tribes and what Ascobolaceae and . The family constitutes families. Arpin (1968) having analysed Ascobolaceae comprises two tribes, Ascoboleae a great number of fungi from these groups for their and Iodophaneae; the latter has five genera, two carotenoids content, and finding no other distinc- of which may be separated by using the character­ tive criterion, felt that on pigmentation character- istics of the apothecia, supplemented by the istics he could group the tribes Ciliarieae and presence and absence of carotenoids. Iodophanus Aleurieae into the family Aleuriaceae Arpin and has lenticular to convex apothecia, almost always the tribes Lachneae and Otideae into the Otidea- with carotenoids, whilst Trecotheus has subconical ceae of Eckblad (1968). He then divided the to cylindrical or turbinate apothecia and is devoid Aleuriaceae into three groups : (1) the first con- of carotenoids. rained p-carotene as the major pigment, e.g. The family Pyronemataceae on the other hand Soaierbyella, Caliscypha; (2) the second p- is divided into five subfamilies: Ascodesmidoideae, carotene and y-carotene and its derivatives (XV, Ascophanoideae, Otideoideae, Scutellinioideae and XXI), e.g. Aleuria, Melastiza; (3) the third y- Pyronematoideae, the first three lacking caro­ carotene, e.g. Coprobia, Scutellinia (Table 5). tenoids altogether. Scutellinioideae and Otideoi- The family Sarcoscyphaceae is not a homogene- deae are easily separable in that the ascospores may ous group and the tribe Sarcoscypheae containing be guttulate in both, yet carotenoids are present in large amounts ofxanthophylls is not related in any the former . The subfamily Scutellinioideae is way to the other tribe Urnuleae which contains divided into three tribes namely Sowerbyelleae, hydrophilic pigments (Arpin, 1968). Some of the Aleurieae and Scutellinieae; the first contains p­ genera of Sarcoscypheae contain large amounts of carotene as the major pigment and carotene P.444 a novel pigment phillipsiaxanthin (XXVIII) and (unknown elsewhere in the Pezizales), the fungus its esters. The monoester is represented as (XXIX) discolouring greenish or red to brownish on where R, is an acyl radical. The presence of this bruising, the second p-carotene or both p- and unusual pigment could be important taxonomic- y-carotenes as major pigments but not discolour­ ally. Although it would simplify matters greatly to ing on bruising, whilst the third contains y­ use certain carotenoids as main taxonomic markers, carotene as the main pigment. These divisions are as was attempted by Arpin (1968), it is not always broadly speaking the same as those of Arpin (1968) possible to do so and other characteristics must who calls them Groups 1, 2 and 3 respectively also be taken into account. The latest classification (Table 5). This is yet another instance where of the Discomycetes incorporates both fungal carotenoids have helped to reinforce morpho­ morphology and some of Arpin's (1968) work. logical characters in fungal taxonomy. Korf (1973) names two suborders : Sarcoscy- Arpin (1968) next looked at pigmentation from L. R. G. Valadon 11

Table 6. Carotenoids of certain y easts: various species of Cryptococcus, Rhodotorula, Sporidiobolus and Sporobolomyces Neuro- Lyco- y- Toru- Toru- fJ- Refer- sporene pene Carotene lene larhodin Carotene encest Cryptococcus flaous (Saito) Phaff & Fell + + +++ 1 (=R.fiava (Saito) Lodder) C. laurentii (Kuff.) Skinner var. flaoescens (Saito) + + +++ 1 Lodder & Kreger-van Rij (=R. aurea (Saito) Lodder) C. laurentii (Kuff.) Skinner var, flauescens (Saito) + + +++ 1 Lodder & Kreger-van Rij (=R. peneaus Phaff, Mrak & Williams) C. laurentii (Kuff.) Skinner var. laurentii + + +++ C. luteolus (Saito) Skinner + + +++ Rhodotorula aurantiaca* (Saito) Lodder + + R. glutinis (Fr.) Harrison + + + +++ + Ri gracilis Rennerfelt (=R.glutinis (Fres.) Harrison) ­ + +++ + R. infirmominiata (Okunuki) Hasegawa& Banno + + + + + (=C. infirmominiatus (Okunuki) Phaff & Fell) R. minuta (Saito) Harrison + +++ + + R. mucilaginosa (Jorg.) Harrison ( =R. rubra + + + + +++ + (Demme) Lodder) R. palida Lodder + +++ + 1 R. rubra (Dernme) Lodder + + + +++ + 1 R. sanniei (Cif. & Red.) Lodder (=R. rubra ? + + +++ + 2 (Demme) Lodder) Sporidiobolus johnsonii Nyland + + + ++ + Sporobolomyces roseus Kluyver & Van Niel + +++ + S. pararoseus Olson & Hammer ( = S. ruber ? + +++ + Yamazaki & Fujii) S. salmonicolor (Fisher & Brebeck) Kluyver & + +++ + Van Niel * The major pigment 'P475 , has not yet been identified (Fiasson, 1968). + + +, Major carotenoid; +, carotenoid present. t 1, Nakayama et al. (1954); 2, Fiasson (1968); 3, Peterson et al. (1958); 4, Bonaly & Villoutreix (1965); 5, Villoutreix (1960); 6, Fiasson (1967); 7, Lederer (1938). an evolutionary viewpoint. Harborne (1967) has van Rij (1952) separate the Cryptococcaceae (no argued that if different compounds can be placed carotenoids) from the Rhodotorulaceae (presence in sequence on a biosynthetic pathway, of carotenoids) but later studies (Nakayama et al, A~B~C~D~E, 1954; Petersen et al. 1958) showed that a number of Cryptococci did contain carotenoids. Lodder then a plant/organism making only A has fewer (1970), following Phaff & Spencer's (1969) enzymes at its disposal than one containing E, i.e. suggestion, separates the two in that Cryptococcus E is more advanced from an evolutionary point of assimilates inositol as a single carbon source and view. Since the inoperculate Discomycetes only Rhodotorula does not. Phaff & Fell (1970) found contained jJ-carotene as their major carotenoid that this division removed all starch positive (Arpin, 1968), they are not considered as advanced species from Rhodotorula and this appeared to as the operculate Discomycetes which have y­ correlate well with the chemical composition of carotene and its oxygenated derivatives as major the capsular polysaccharides of the two genera. pigments (Fig. 1). The same argument presumably Moreover, they could not accept the division of applies to the Phycomyces since the Mucorales Rhodotorula suggested by Hasegawa, Banno & with jJ-carotene as major pigment will be more Yamauchi (1960) into Flavotorula (colonies red­ evolved than either the Chyrtidiales or the dish to pale yellow or pale orange) and Rubro­ Blastocladiales with y-carotene only as their major torula (colonies red to orange) because of the great carotenoid. variability in types and concentration of caroten­ Carotenoids have been implicated as taxo­ oids in different strains and with changes in nomic markers among yeasts. Lodder & Kreger- environmental conditions. However, if conditions 12 Carotenoids as additional taxonomic characters in fungi

Table 7. Carotenoidsof variousspecies ofCantharellus and Craterellus. The resultsareexpressedaspercentage total carotenoids Total carote- y- fJ- Echi- Cantha- noids Neuro- Lyco- Caro- Caro- nen- xan- (jlg!g Refer- Species sporene pene P·444 tene tene one thin Others dry wt) encest Subgenus: Cantharellus Cantharellus cinnabarinus Schw. - + + +++ 30'0 1 Ca.jriesii Quel. + + +++ ++ + + 15'7 1 Ca. cibarius Fr. var. pallidifolius - + + + +++ + 3'0 1 Smith Ca. cibarius Fr. (cream ) ? + + +++ + 4'0 1 Pet. Ca. cibarius Fr. (yellow + + +++ + 10'0 1 spores) Pet. Ca. minor Pk. + +++ 57'0 1 Subgenus: Phaeocantharellus Ca. lutescens Fr. +++ +++ + * 1 Ca.cf lutescens Fr. (No. 3540) + +++ + 3'3 1 Ca.cflutescens Fr. (No.3543) ++ +++ + 0'6 1 Ca. injundibulijormis Fr. +++ + + 20'0 1 Ca. injundibuliformis Fr. +++ + + 10'0 2 Ca. injundibulijormis Fr. + + + + x 457'5 3 Ca. ianthinoxanthus (R. 26 74 18'0 4 Maire) Kuhner Craterellus cornucopioides +++ ++ + 0'6 1 Pers per Fr. Cr.jallax Smith + + +++ + 0'7 1 Cr. odoratus (Schw.) Fr. + + +++ + 1'3 1 Cr. cinereus (Fr.) Quel. 0 1,2 + + +, Major carotenoid. + +, Second major pigment (between 30-45 %). + + x, The major carotenoid is an expoxycarotenoid but NOT neurosporene. *, No amount given. t 1, Fiasson et al. (1970); 2, Turian (1960); 3, Valadon & Mummery (1975b); 4, Fiasson(1973)· are kept constant, carotenoid studies of these two rhodin being the major pigment with smaller genera correlate well with the other above­ amounts of torulene and fl-carotene (Table 6). mentioned parameters (assimilation of inositol and Corner's (1966) treatise on Cantharelloid fungi starch positiveness among Cryptococcus) in that gives one a first-hand insight into the problems of Cryptococcus contains fl-carotene as its major classifying this group of fungi. He divides Can­ carotenoid whilst Rhodotorula have either torulene tharellus into three subgenera: Cantharellus, or its carboxlyated derivative torularhodin (Table Phaeocantharellus and Cantharellotus, and sep­ 6). Since torularhodin is further along the bio­ arates these from the genus Craterellus on the form synthetic pathway than fl-carotene, then Rhodo­ of their fruit bodies, the nature of the hymenial torula will be considered more advanced than folds or ridges and in the short-celled hyphae Cryptococcus. without clamps in the latter. 'Craterellus has been Two other genera of yeasts that may be closely much confused with Cantharellus' he quotes. related are Sporidiobolus and Sporobolomyces, These points are considered further after investi­ both characterized by the presence of ballisto­ gations on the carotenoids of a number of species spores and indistinguishable in the young state in this group (Fiasson, 1973; Fiasson, Petersen, although the latter lack clamp connexions (Phaff, Bouchez & Arpin, 1970) (Table 7). It can be 1970). Fiasson (1967) has supported the close observed from these results that the subgenus relationship between these two genera since Cantharellus contains fl-carotene as the major Sporidiobolus johnsonii, Sporobolomyces roseus and pigment and the subgenus Phaeocantharellus S. salmonicolor have the same carotenoids, torula- either neurosporene or lycopene or both. On this L. R. G. Valadon 13 basis they were in agreement with Corner's (1966) in the new genus Laetiporus (Pegler, 1966). scheme. C. ianthinoxanthus had been placed in the Although laetiporxanthin has not yet been charac­ subgenus Cantharellus but was later found to be terized it may well be ,8-apo-8-carotenoic acid more closely related morphologically to the (XXVII) already synthesized (Isler et al. 1959) Phaeocantharellus group (Fiasson, 1913). The but which has not yet been found in nature. The carotenoids too agreed closely with this since it was presence of a carotenoid in this fungus and its found to contain neurosporene and lycopene. absence in Polyporus grammocephalus, P. luzonenis, Fiasson et al. (1970) conclude as Craterellus is a P. rubidis and P. zonalis (Bose, 1941) shows yet heterogeneous group it is not surprising that some another instance where a biochemical approach species, e.g. Cr. cornucopioides have the same supports evidence obtained on morphological carotenoid composition as the subgenus Phaeo- grounds. cantharellus and others, e.g. C. fallax and C. odoratus the same as the other subgenus Cantha­ CONCLUSIONS rellus. It is obviously time to dispense with the genus Craterellus as such, since the genera con­ Although carotenoids are not universally present taining carotenoids would fit in nicely as Cantha­ in fungi they may still be good taxonomic markers. rellus. On the other hand one might be expected to Other characters being equal, presence and absence keep the genus Craterellus for those where no of these pigments would help to separate two carotenoids are present, e.g, C. cinereus. The point genera. When a systematic approach was insti­ already raised which must be remembered in this tuted on various Discomycetes, Arpin (1968) used context is the true identity of the pigment called the presence of carotenoids to form a new family neurosporene here. It would be interesting to Aleuriaceae; the genera involved were found to reanalyse these various species to ascertain if Ca. have a good affinity with one another. Again among cf lutescens No. 3540 or any of the other Phaeo­ the asporogenous yeasts there has been a certain cantharellus contained neurosporene or the amount of controversyas to what constituted a epoxycarotenoid (Valadon & Mummery, 1975b) genus. With the help of certain other characters which was found in specimens of Ca. infundibuli­ supplemented by carotenoid studies one should be formis. Fiasson et al. (1970) have suggested that the able to separate species of Rhodotorula containing genus Cantharellus may be easily divided into the torulene or torularhodin from those of Crypto­ subgenus Cantharellus (containing the bicyclic coccus which contain ,8-carotene as their major carotenoid ,8-carotene and its ketonic derivatives) pigment. and the subgenus Phaeocantharellus (containing It is possible to use carotenoids in differentiating aliphatic carotenoids like neurosporene and lyco­ the subgenera Cantharellus and Phaeocantharellus pene). The accumulation of large amounts of in that the former contain ,8-carotene as their major neurosporene in nature is unusual and Turian pigment whilst the latter either'neurosporene' or (1960) suggested that C. infundibuliformis might lycopene or both (Fiasson et al. 1970). Further­ contain a natural inhibitor because only in the more it is suggested that since differences are not presence of inhibitors like diphenylamine does always clear cut among the Cantharelloid fungi neurosporene accumulate in large amounts in (Corner, 1966), species of Craterellus which con­ certain micro-organisms. However, if what was tain carotenoids may be absorbed by Cantharellus identified as large amounts of neurosporene in Ca. and only those without carotenoids will keep their lutescens, Ca. infundibuliformis and in Cratellerus same generic status. This would be a helpful cornucopoides is found to be an expoxycarotenoid marker for taxonomists because various species (Valadon & Mummery, 1975b) then Fiasson et al.'s among these two genera are constantly alternating (1970) argument will still be valid provided one between being called Craterellus and Cantharellus. replaces aliphatic carotenoids by epoxycarotenoids Finally, although carotenoids may help the for the subgenus Phaeocantharellus. taxonomist a great deal, it would be worth while Another taxonomic point among the Basidio­ re-investigating the carotenoids of a number of mycetes was raised by Valadon& Mummery (1969) species using new techniques to ascertain the who extracted laetiproxanthin from Laetiporus correct structure of these compounds. This was sulphureus. This fungus was known as Polyporus not always possible in the past. 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(Accepted for publication 20 November 1975)