Macowanites ammophilus (Russulales) una nueva combinación basada en nuevos
datos . Después de estudiar abundante material procedente de nuevas recolecciones, Gymnomyces ammophilus se transfiere al género Macowanites, basandonos en los nuevos datos morfológicos y
genéticos encontrados. Como consecuencia, se presenta una nueva descripción de este taxon.Macowanites ammophilus (Russulales) a new combination based on new evidence. 
After studying abundant new collections, Gymnomyces ammophilus is transferred to the genus Macowanites based on the emergence of new data on morphological and genetic characters. A
redescription of this taxon is given

Calonge, Francisco de Diego

Martín Esteban, María Paz

Vidal i Frigola, Josep Maria

Revistes Catalanes amb Accés Obert

Revista Catalana de Micologia, vol. 24: 69-74 ; 2002. Homenatge A. Rocabruna
MACOWANITES AMMOPHILUS (RUSSULALES) A NEW
COMBINATION BASED ON NEW EVIDENCE
1.M. VIDAL1, F.D. CALONGE 2 & M.P. MARTIN 2
69
1.- Dept. Biologia Vegetal, Unitat de Botànica, Fac. Biologia, Univ. Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain.
(Personal correspondence: Massaballs 10, 171 18 Sant Sad urní de lH eura, Girona, Spain)
2.- Real Jardín Botanico, CSIC, Plaza de Murillo 2,280 14 Madrid , Spain
Work dedicated to the f riend and colleage
August Rocabruna, in his 80 birthday
ABSTRACT. Macowanites ammophilus tRussulalesï a new combination based on new evidence.
After studying abundant new collections, Gymnomyces ammophilus is transferred to the genus
Macowanites based on the emergence of new data on morphological and genetic characters. A
redescription of this taxon is given.
Key words: Russulales, Macowanites ammophilus, taxonomy, Iberian Peninsula.
RESUMEN. Macowanites ammophilus (Russulales) una nueva combinación basada en nuevos
datos . Después de estudiar abundante material procedente de nuevas recolecciones, Gymnomyces
ammophilus se transfiere al género Macowanites, basandonos en los nuevos datos morfológicos y
genéticos encontrados. Como consecuencia, se presenta una nueva descripción de este taxon.
Palabras clave: Russulales, Macowanites ammophilus, taxonomía, Península Ibérica.
INTRODUCTION
Recent1y, CALONGE & VIDAL (1999) published an article where they proposed Gymnomyces
ammophilus as a new species. That new taxon was based on several collections without any presence
of stipe-columella. As a result of a later foray to Portugal and South of Spain, during November 1999,
new collections of basidiomata in all states of development were obtained, making possible to
distinguish a clear stipe-columella. This fact induced us to study all the material and investigate
carefully both, macro- and microscopic features, complemented with DNA sequencing. The results of
this investigation suggest that our previous collections, named as Gymnomyces ammophilu s, fit better
within the genus Macowanites, following the generic key proposed by SINGER & SMITH (1960).
Thus, a redescription is needed, which compiles all the main taxonomic characters.
MATERIAL AND METHODS
Samples from all the three places were dried up and the spores prepared for SEM, shadowing with
gold and observed following the usual techniques. For the identification of colours we followed
KORNERUP & WANSCHER"s colour guide (1978). Molecular analysis of the internal transcribed
spacer of the rDNA (including the 5.8 S rDNA) were done following the DNA isolation, PCR
amplification and sequencing protocols described by CALONGE & MARTIN (2000). The new
sequences have been logged in the EMBL database with the accession number AJ438038 (MA-Fungi
51165), AJ438037 (MA-Fungi 51166) and AJ438036 (MA-Fungi 51167). These sequences were
compared with the homologous sequences published by CALONGE & MARTIN (2000) and two
new sequences obtained from Macowanites vinaceodorus collections MA-Fungi 46524 (AJ438035)
and MA-Fungi 474 16-2 (AJ438034). Sequence Navigator ' Sequence Comparison for pairwise
comparisons and SEQAPP software for multiple sequences were used to search for the best
alignment. Alignment gaps were marked "-" and unresolved nucleotides or unknown sequences were
70 Revista Catalana de Micologia, vol. 24: 69-74; 2002 . Homenatge A. Rocabrun a
indicated with "N". Par simonium (branch-and-bound search) and maximum likelihood (substitutions
model) analyses were performed using the computer program PAUP 4.0b* (Phylogenetic Program
Using Parsimon y) of SWOFFORD (1996) . Branch robu stness was estimated by bootstrap analys is
(FE LSENSTEIN, 1985) of 10.000 heuri stic repli cates using the fast stepwise-ad dition option.
DESCRIPTION
Macowanites ammophilus (Vidal et Calonge) Vidal et Calonge, comb. nov.
Basion .: Gymn omyces ammophilus Vidal et Calonge, in Ca longe & Vidal, Bo!. Soc. Micol.
Madrid 24: 66 (1999)
Basidioma hypogeous, solitary or gregarious, subglobose to irregular, 1-3 cm diam. , when unmature,
angiocarpo us. During ripening process it becomes semihypogeous and partially gymnoca rpous,
show ing a well-developed stipe with rhizomorph. Pileus 2-7 cm diam., globose to hemisphaerical, not
expanding, depressed to umbili cate, often gibbous. Perid ium smoo th, viscid, with adhere nt granules
of sand, initially attached to stipe and finall y separated frorn it and disintegrated in the per imarginal
zone, orange white (K& W 5A2) at first, becomin g light orange (K&W 5A4) and mac ulated of
brownish orange (K& W 7C8) to dark bro wn (K&W 7F8) finally. Stipe-co lumella percurrent,
prominent, cylindrical, 1-4 x 0.7-2.5 cm, initially white, then tipically puncta te of brownish orange
(K& W 7C8) from the basis, unchan ging when bruished . Co ntext white, initially solid, then cave rnose
in the stipe. Gleba loculate, labyrinthiform, annexed to the stipe-colume lla, the basal chambers later
free and exposed after disintegration of the peridium, orange white (K&W 5A2) to pale orange
(K&W 5A3). Spore-mass in the locules white to pale yellow (K&W 4A2). Odor fruity, taste very
slowly acrid to swee tish.
Spores 7-9 x 5.5 -7.5 ~m (ornamentation exc luded), 7.5 -10 x 6-9 ~m (ornamentation included),
subglobose to broadl y ellipsoidal or subovoid, heterotropi c, with a prominent hilar appendage 2
~ In long. Am yloid ornamentation consisting of isolated warts, sometimes forming short cristae,
0.25-0.75 urn high (Figs . 2-3). Suprahilar plage abse nt. Basidi a clavate, hyalin e in KOH, 35-45 x
8- 11 ~m, 2-4-spored. Macro cystidia abundant, 45 -60 x 6- 1O ~m, lanceolate, with a sinuose
mucronate apex. Subhymenium cellular, with globose cells, 15-25 u rn diam . Hymenophoral trama
heterom erous, with septa te hyph ae, 3- 12 u m diam ., and sphaerocysts 6-35 u m dia m., with
abunda nt oleiferous hyph ae 2.5- 1O urn diam. Context of pileus and stipe-colume lla typ ically
heteromerous. Peridi opelli s thin, 125-200 ~m, suprape llis an intri cate trichoderm of 15-40 x 2-5
~m cylindrica l, sinuose, clav iform, lanceolate or mucronate ele ments, without dermatocystidi a;
mediopelli s an intri cate ixocuti s, compose d of ge latinized repent hyph ae, 2.25-7 .5 ~m diam .,
subpellis an intricate cutis of non -gelatinizated hyphae, 4-1Ou rn diam. , with frequent en large rnents
up to 16-(25) u m, that co nnect with the context. Abundant oleiferous hyph ae are present in the
middl e and internal layers. Stipitipellis 30-100 ~m, co nsis ting in cutis of repent incrustated
hyph ae, 2.5-7.5 ~m diam. , with bundles of long and semierec ted, septate, incru state, yellow to red
co loured hairs, 50 -150 x 2-7.5 ~m.
Th e alignment of the ITS rDNA sequences gives no amb iguous regions and 8 10 charac ters were
included in the analyses. Among them , 257 were parsimony- informat ive. Und er branch-and -bound
sea rch, 90 most parsimoni ou s trees (MPTs) were obtained (tree length = 758, co nsistency index
CI= 0.7784 , retention index RI= 0.7908, rescaled consistency index RC = 0.6155. The strict
consensus tree is show n in Fig. 3. Similar tree topology was generated by maximum likelihood
analysis following FELSENSTEIN (1985) (not shown). Both analyses agree (with a 99 % of
bootstrap ), in placin g in the same group collections described by CALONGE & VIDAL ( 1999) as
Gymnomyces amm ophilu s and collections with a clear stipe-col umella described in this paper.
COLLECTIONS EXAM INED. PORTUGAL: Beira Alta, Castelo Ventoso, road to Alcacer do Sal, coastal dunes, under Pinus
pinea, and Core tna, 26-11-1999, leg. lM. Vidal and FO. Calonge, MA-Fungi 5 1167.-SPAIN: Huelva, Mazagón, coastal
dunes, under P. pin ea, with Halimium and Corema , 27-11-1999, leg. J.M. Vidal and F O. Calonge, MA-Fungi 5 1166.-
Matalascañas, the same ecology, but in the presence or Juniperus phoenicea, 27-11-1999, leg. J.M. Vidal and FO.
Calonge, MA- Fungi 5 1165.
Revista Catalana de Micologia, vol. 24: 69-74; 2üü2. Homenatge A. Rocabrun a 71
Fig. 1. Macowanites ammophilus. Spore showing a warty omamentation forming some crests. Material collected
in Castelo Ventoso, Portugal. MA-Fungi 51167.
Fig. 2. Macowanites ammophilus. Spores showing warty and subreticulate omamentation. Material collected in
Mazagón, Spain . MA-Fun gi 51166.
72 Revista Catalana de Micologia, vol. 24: 69-74 ; 2002. Homenatge A. Rocabruna
DISCUSSIO . Following the work by PEGLER & YOU G (1979) and according to our new data
on this fungus (CALONGE & MARTIN, 2000; CALONGE & VIDAL, 200 1), we consider that
the genus in which our material fits better is Macowanites Kalchbr. In fact, the mature basidiomata
look like a typical Russula at a first sight, and later observations, both under light and scanning
electron microscopy, confirm this idea. Thus, they show a well-developed stipe, the gleba is
exposed at base of peridium, at the margin, after ripening; the hymenophoral trama is
heterornerous and macrocystidia of Ru ssula -type are present. Thus, it can not be maintained in
Gymnomyces, as previously thought (CALONGE & VIDAL, 1999) after the study of only
unmatured material. In consequence, we propose the new combination Ma cowa nites ammophilus.
M. ammophilus seems to be close to the North American M. luteolus A.H. SITI. et Trappe
(SMITH, 1963). The microscopic characters are similar in both species, but the basidioma is
larger in M. ammoph ilus, 2-7 cm, against 1-3 cm in M. luteolu s. Also the brownish orange
coloration of the stipe basis and the peridium in mature specimens are distinctive of
M. ammophilus. Regarding their ecology, M. ammophilu s is a psammophilous species associated
with Pinus p inea , growing in the Atlantic coastal dunes of the South Iberian Peninsula, and
probably it may be also present in similar habitats of the coast of North Africa, and rest of the
Mediterranean littoral. M. luteolu s is common in the Pacific coast of Oregon, Washington and
California, under conifers (Picea and Tsuga) (SMITH, 1963; ARORA, 1986; PHILLIPS , 199 1).
On the other hand, there is some mophological resemblance between M. ammophilus and
M. vinace odorus (CALONGE & VIDAL , 2001), which grows in the same ecological conditions,
but, with the exception of several differences (see Table 1), after the phylogenetic analyses both
species cluster in different cIades (Fig. 3). Moreover, Macowanites seems to be polyphyletic.
MILLER et al. (2001) used sequences of n-LSU (nuclear large subunit rDNA) to infer
phylogenetic relationships among agaricoid, gasteroid and hypogaeous gasteroid taxa included in
Russulales. In their study, Macowanites. Gymnomyces, Cystan gium and Martellia grouped with
Russula cIades; whereas, Zelleromyces / and Arcangiella cluster with Lactarius species, as first
suggested by CALONGE & MARTIN (2000). Moreover, Cystan gium, Gymn omyces and
Ma rtellia , appear to be polyphyletic, too. This is in agreement with our results concerning
M. ammophilus and M. vinace odorus . Fol1owing KRETZER & BRUNS (1997), as many
gasteroid and hypogeous gasteroid genera seem to be polyphyletic, MILLER et al. (200 1)
consider that a synonomisation of these genera into Lactarius and Russula is justifiable.
ACKNOWLEDGEMENTS
We express our gratitute to Prof. Julieta Carranza, for revision of the text, and to Miguel Jerez for
his valuable SEM assistance. One of us (FDC) wishes to acknowledge the financial support
received from the DGES and CSIC under research project no. PB-95-0129-003-01 and to the
ICCTI for the help received to afford the travelling to Portugal, The molecular studies have been
financial1y supported by DGES, under the research project PB-98-0538-C04-0 1 and the
postdoctoral grant from the Comunidad de Madrid to MPM.
BIBLIOGRAPHY
ARORA, D. ( 1986).- Mu shrooms Demystified . Secondedition.Ten SpeedPress.Berkeley.959 pp.
CALONGE, FO. & lM. VIDAL (1999).- Gymn omyces ammophilus Vidal & Calonge, sp. nov. , encontrado en Portugal. Bo!.
Soc. Mico!. Madrid 24: 65-70 .
CALONGE, FO. & LM. VIDAL (200 1).- Ma cowanit es vinaceodo rus sp. nov. (Russulales) a new gasteroid fungus from
coastaldunesofSpain. Myc otaxon 79: 1-6.
CALO GE, FO. & M.P. MARTIN (2000).- Morphological and moleculardata on the taxonomyof Gymnomyces, Mart ellia
and Zell eromyces (Elasmomycetaceae, Russulales). Mycotaxon 76: 9- 15.
FELSENSTEIN, J. (1985).- Confidence limitsonphylogenesis: an approachusingthebootstrap. Evolution 39: 783-79 1.
KORNERUP, A. & J.H. WANSCHER(1978).- Methuen handbook olcotour. Thirdedition. Eyre Methuen. London. 252 pp.
KRE1ZER, A. & T.D.BRUNS( 1997).- Molecularrevisitation ofthegenus Gas trosuillus. Myco logia 89: 586-589.
MILLER, S.L., McCLEAN, T.M, WALKER, i .r . & B. BUYCK(200 I).- Amolecularphylogenyof the Russulales including
agaricoid,gasteroid and pleurotoid taxa. Mycologia 93(2): 344-354.
Revista Catalana de Micologia, vol. 24: 69-74; 2002. Homenatge A. Rocabruna 73
Stri ct
Macowanites vinaceodorus AJ383D34
Macowanites vinaceodorus AJ383D35
100
85
Russula integra AF23D896
Russula postiana AF23D897
Russula tragi/is AF23D898
Russula mairei AF23D899
,...--- Macowanites ammophi/us AJ383D36
99 .....--- Macowanites ammophi/us AJ383D37
Macowanites ammophi/us AJ383D38
82
55
Macowanites ammophiJus AF23D89D
=Gymnomyces arnrnopnnus
Macowanites ammophilus AF23D891
=Gymnomyces ammophilus
Marfellia pi/a AF23D893
MarfelJia pi/a AF23D894
.....--- Russula foetens AF23D895
Lactarius deJiciosus AF23D892
Lactarius deliciosus U8D999
L.._-~--..r----- Zelleromyces giennensis AF23D8DD
Zelleromyces hyspanicus AF231911
ZeJJeromyces hyspanicus AF231912
ZeJJeromyces hyspanicus AF231913
------------ Hygrophorus latitabundus AF23191 S
SuiJJus variegatus AF 231914
100
Fig. 3. Strict consensus tree of 90 MPTs under branch-and-bound algorithm. Percentages from 10.000
replications are given on the branches.
74 Revista Catalana de Mic ologia, vol. 24: 69-74 ; 2002 . Hom enatge A. Rocabruna
PEGLER, D.N. & T.W.K. YOUNG ( 1979).- The gasteroid Russula les. Trans. Br. Myco/. Soc. 72(3): 353-388.
PHILLIPS, R. ( 1991).- Mushrooms ofNorth Ame rica. Little, Brown and Co. Boston . 3 19 pp.
SINGE R, R. & A.H. SMIT H ( 1960).- Studies on seco tiaceo us fungi. IX. The Astrogas traceo us series. Mem. Torrey Bot. Club
2 1: 1-212.
SMIT H, A.H. ( 1963).- New Astrogas traceo us fungi l'rom the Pacific North west. Myco logia 55: 42 1-44 1.
SW OF FORD, D.L. (1996).- PAUP* version 4.0. Sinauer Assoc . Inc., Sund erland, Mass.
Tabl e 1. Comparison between some characters of Ma cowan ites ammophilus and M. vinaceodorus
Ch aracters M. ammophilus M. vinaceodorus
Spore mass White-yellowish Orange-yello wish
Spore ornamentation 0.25-0.75 11m thick 0.7-1.5 11m thick
Suprahilar plage Absent Present
Dennatocystidia Ab sent Present
Gleba Loculate, whiti sh Locul ate-subl amellate, yellow ish
Od our Fruity Vinaceo us
Macowanues anunophilus (Vidal et Calonge) Vidal et Calonge (MA-Fungi S 1167)


Macowanites ammophilus (Russalales) a new combination based on new evidence

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