molecular typing of spanish species of amanita by restriction analysis of the its region of the dna
TRANSCRIPT
903
Vicente GONZA; LEZ1†, Francisco ARENAL1†, Gonzalo PLATAS1,
Fernando ESTEVE-RAVENTO; S2 and Fernando PELA; EZ*
"Centro de InvestigacioU n BaU sica. Merck, Sharp & Dohme de Espanh a, S.A., Calle Josefa ValcaU rcel 38, E-28027 Madrid,
Spain.
#Department of Plant Biology, AlcalaU University, E-28871 AlcalaU de Henares, Spain.
E-mail : fernandojpelaez!merck.com
Received 11 July 1999; accepted 22 May 2002.
The genetic relatedness among 29 collections belonging to 20 of the most common species of genus Amanita in the
Iberian Peninsula have been studied by means of restriction analysis of the amplified ITS1-5±8S-ITS2 region of rDNA.
Sixty restriction fragments were considered for the analysis. The main conclusion is that this technique could be useful
to identify and characterize isolates from Amanita species. Although ARDRA did not show an appropriate level of
discrimination to unambiguously infer phylogenetic relationships at or below the section level, some general trends
could be outlined when the different haplotypes generated were compared by means of neighbour joining analysis.
Thus, members from the same sections were frequently grouped together.
INTRODUCTION
Amanita is one of the most studied and well-known
agaric genera. It is characterized mainly by its terrestrial,
mostly ectomycorrhizogenous and fleshy basidiomes
with a lamellate hymenophore, bilateral trama, and a
universal veil. Spore-prints are white, and the spores
are smooth, amyloid or inamyloid. These characters
provide clear limits that distinguish this genus from
other closely related genera, such as Limacella.
Amanita can be considered cosmopolitan, with
numerous representatives in all continents except
Antarctica. Singer (1986) records at least 100 species,
although subsequent studies suggest that this could
be underestimated. A large amount of morphology-
based taxonomic literature concerning this genus has
been published, with numerous and well-known mono-
graphic studies especially in Europe, North America
and tropical Africa (e.g. Bas 1969, Jenkins 1986,
Fraiture 1993).
This genus has been traditionally included, together
with Limacella, in the Amanitaceae (Singer 1986). Some
authors (e.g. Moser 1983), also consider Torrendia,
formerly placed among Gasteromycetes, as a genuine
* Corresponding author.† Current address : Departamento de Biotecnologı!a Microbiana,
Centro Nacional de Biotecnologı!a (CSIC-UAM), campus Canto-blanco, Universidad Auto! noma de Madrid, E-28049 Madrid, Spain.
member of the Amanitaceae but, at present Torrendia is
generally placed in the Torrendiaceae (Agaricales).
Two major systematic schemes have been widely
accepted for the genus. Singer (1986) subdivided the
genus into two subgenera, Amanita and Lepidella, on
the basis of the amyloidity of the basidiospores. Five
sections were defined within subgenus Lepidella
(Amidellae, Mappae, Phalloideae, Roanokenses, and
Validae), and four within subgenus Amanita (Amanita,
Ovigerae, Vaginatae, and Caesareae). An alternative
scheme was proposed by Corner & Bas (1962), and later
modified by Bas (1969) ; this also divides the genus into
the same subgenera, but Lepidella was divided into four
sections (Amidella, Lepidella, Phalloideae, and Validae),
and subgenus Amanita into two (Amanita and
Vaginatae). The Vaginatae includes most of the species
formerly placed in sections Ovigerae and Caesareae by
Singer (1986).
Since the invention and spread of the PCR, different
DNA fingerprinting methods have been introduced for
the assessment of the genetic relationships among
taxonomically related organisms. We used Amplified
Ribosomal DNA Restriction Analysis (ARDRA) for
the analysis of the phenetic relationships among several
Spanish Amanita species. This technique is based on the
comparison of the electrophoretic profiles obtained
after the digestion of the ITS1-5.8S-ITS2 region with
different restriction endonucleases. Nuclear rDNA
Mycol. Res. 106 (8) : 903–910 (August 2002). # The British Mycological Society
DOI: 10.1017}S0953756202006305 Printed in the United Kingdom.
Molecular typing of Spanish species of Amanita by restriction
analysis of the ITS region of the DNA
ARDRA of Amanita species 904
Table 1. Identity and origin of the Amanita specimens used for the study. The classification system follows Bas (1969).
Specimens Collection* Site Province Habitat Date
Subgen. Amanita
Sect. Amanita
A. gemmata AH19200 Casavieja Avila Pinus pinaster 5 Nov. 1995
A. gemmata AH11951 Guadarrama Madrid P. pinaster 17 May 1975
A. gemmata AH9364 Cercedilla Madrid P. sylvestris 22 May 1983
A. muscaria AH12840 Cantalojas Guadalajara P. sylvestris 12 Oct. 1989
A. pantherina AH20722 Casavieja Avila P. pinaster 8 Dec. 1995
A. pantherina AH12766 Ciruelos del Pinar Guadalajara P. pinaster 3 Oct. 1990
Sect. Vaginatae
A. caesarea AH11854 Galda! cano Alava Quercus spp. 18 Oct. 1975
A. lividopallescens CFB73 Pto. Somosierra Madrid Q. petraea 30 Oct. 1995
A. malleata AH16833 Can4 amero Ca! ceres Populus nigra 12 May 1995
A. malleata CFB15 San Martı!n de Valdeiglesias Madrid Pinus pinea 24 Sept. 1995
A. oblongospora AH12942 La Cabrera Madrid Q. ilex ssp. ballota 30 Sept. 1990
A. pachyvolvata AH16840 Can4 amero Ca! ceres Castanea sativa 12 May 1994
Subgen. Lepidella
Sect. Phalloideae
A. citrina AH19342 Casavieja Avila Pinus pinaster 19 Nov. 1995
A. phalloides AH19119 Casavieja Avila C. sativa 29 Oct. 1995
A. phalloides AH12835 Ciruelos del Pinar Guadalajara P. pinaster 3 Oct. 1990
A. phalloides var. verna AH20314 La Iglesuela Toledo Q. ilex ssp. ballota 5 April 1996
A. porphyria AH16831 Can4 amero Ca! ceres C. sativa 12 May 1994
Sect. Amidella
A. curtipes CFB136 Riaza Segovia Q. pyrenaica 3 May 1996
A. ponderosa AH98 Carmonitas Badajoz Q. suber 30 April 1976
A. ovoidea AH12772 Baides Guadalajara Q. ilex ssp. ballota 30 Sept. 1990
Sect. Validae
A. franchetii AH11876 Sant Grau Gerona Q. suber 20 Oct. 1988
A. rubescens AH19175 Casavieja Avila C. sativa 19 Nov. 1995
A. rubescens AH12821 Ciruelos del Pinar Guadalajara P. pinaster 3 Oct. 1990
A. spissa AH3081 Montejo de la Sierra Madrid Fagus sylvatica 26 June 1983
Sect. Lepidella
A. codinae AH10364 Alcala! de Henares Madrid Grassland 19 Oct. 1987
A. strobiliformis AH108 Galda! cano Alava Quercus spp. 18 Oct. 1975
A. vittadini AH2522 Madrid Madrid Quercus spp. 18 April 1982
Genus Torrendia
Torrendia pulchella AH18581 Monfragu$ e Ca! ceres P. pinaster 24 Sept. 1992
Torrendia pulchella AH20469 La Iglesuela Toledo Q. ilex ssp. ballota 8 Dec. 1995
* AH, herbarium Alcala! de Henares University. CFB, herbarium of CIBE-MSD.
genes have been widely employed for inferring taxo-
nomic and phylogenetic relationships on a wide range
of organisms, recently including genus Amanita
(Drehmel, Moncalvo & Vilgalys 1999, Moncalvo,
Drehmel & Vilgalys 2000, Oda, Tanaka & Tsuda 1999,
Weiss, Yang & Oberwinkler 1998). Although most of
molecular systematic studies are currently performed
by rDNA sequence analysis, ARDRA has been applied
to the study of the phenetic relationships in several
fungal genera, such as Coprinus (Hopple & Vilgalys
1994), Phellinus (Fischer 1996) or Rhizoctonia (Cubeta,
Vilgalys & Gonza! lez 1996). It has been suggested that
the comparison of a significant number of restriction
sites data could be used to estimate genetic distances
and generate characters for phylogenetic recon-
structions (Cubeta et al. 1996).
Castro (1998) recorded 45 taxa of Amanita for the
Iberian Peninsula. The present paper is focused on the
study of 29 collections belonging to 20 Amanita species,
representing most of the best known species of the
genus reported in Spain. In addition, two isolates of
Torrendia pulchella were also included in the analysis,
to test if ARDRA was able to detect genetic affinities
between both genera.
MATERIALS AND METHODS
Material studied
The samples used in this study consisted of dried fungal
herbarium specimens (Table 1). Notes on the date and
place, as well as brief descriptions of the habitats are
provided for each specimen.
DNA extraction
The protocol used is a modification of Bruns, Fogel &
Taylor (1990). The dry herbarium material was frag-
mented and suspended in 1 ml of a lysis buffer (50 m
EDTA pH 8±5, 0±2% SDS) and heat-shock treated at
75 °C for 30 min. After centrifugation in a Biofuge 13
(Heraeus, Hanau, Germany) at 15000 g, the super-
V. Gonza! lez and others 905
natant was adjusted to a final concentration of 0±3
potassium acetate and chilled for 1 h in ice. After a
second centrifugation, the supernatant was mixed with
2 vols of absolute ethanol to precipitate the DNA,
which was dissolved in TE buffer. The DNA con-
centration obtained using this procedure ranged from
0±01 to 0±1 µg ml−".
Amplification reactions
Reactions were performed in a volume of 50 µl, under
the conditions recommended by the manufacturer
(Appligene-Oncor, Illkirch, France) : 0±2 m each of
the four dNTPs (GeneAmp dNTPS Perkin Elmer,
Norwalk, CT), 0±4 µ final concentration of the primer
or combination of primers, 1 µl of the DNA solution
and 2 units of Taq polymerase (Appligene) with its
appropriate reaction buffer. Amplification was per-
formed in a Perkin Elmer Cetus DNA Thermal cycler
480. The primers ITS1F and ITS4 (White et al. 1990,
Gardes & Bruns 1993) were used for the amplification
of ITS1-5.8S-ITS2 of the rDNA. Standard PCR
reaction conditions consisting of 40 cycles of 30 s at
93 °, 30 s at 53 °, 2 min at 72 °, ending by 10 min at 72 °were employed.
ARDRA
The amplification products were digested with 5–10
units of six restriction enzymes independently (AluI,
EcoRI, HinfI, MspI, and TaqI from Pharmacia Biotech,
San Francisco, CA; and HhaI from Amersham, Bucks),
following the conditions recommended by the manu-
facturer. The amplification products and restriction
fragmentswere separated by electrophoresis in VisigelTM
separation matrix (Stratagene, La Jolla, CA), and
visualized by staining with ethidium bromide.
Phenetic analysis
Each strain was assigned a composite rDNA haplotype
defined by the combination of the patterns obtained
with the six restriction endonucleases. The presence or
absence of each restriction fragment obtained by each
enzyme was scored for all taxa. This resulted in a binary
1}0 matrix that was used to estimate the genetic
relationships among Amanita species. Jaccard similarity
indexes (Sneath & Sokal 1973) were calculated for all
pairs of strains, using SYN-TAX pc 5.0 statistical
package (Podani 1990). A dendrogram was generated
from the similarity matrix using the neighbour-joining
algorithm of the TreeView application of PHYLIP 3.5
(Felsenstein 1993).
RESULTS AND DISCUSSION
The samples used in this study (Table 1) represent the
most frequent members of genus Amanita in Spain,
according to Bas (1969), Fraiture (1993) and Castro
(1998). Origins of the samples are diverse ; collections of
both Mediterranean and Atlantic areas are represented,
and in the case of specimens belonging to the same
species, they were collected in different locations. DNA
from all the herbarium samples was successfully
amplified, even when some of the material was more
than 20 yr old. Dried fungal herbarium specimens have
proved to be an important resource for obtaining
genetic data and for molecular systematic studies (Bruns
et al. 1990).
The gel electrophoresis revealed a remarkable het-
erogeneity in the size of the amplified ITS1-5.8S-ITS2
PCR products, which ranged from roughly 600 to
750 bp (Table 2). Interestingly, all the species with the
smaller sizes (600–650 bp) belonged to section Vaginata,
whereas the larger sizes corresponded to species from
section Amanita and subgenus Lepidella. Torrendia
pulchella also gave a 600 bp product.
As a rule, all the restriction enzymes processed the
amplification products into two or three fragments
(Table 2), although they showed quite different abilities
to digest the PCR products. Thus, HinfI and TaqI were
able to cut the PCR products from all the species tested.
HhaI worked for all the species but one and EcoRI for
all but three. On the other hand, AluI digested DNA
from eleven species and MspI only from six species.
Generally the addition of the sizes of the restriction
fragments matched with the size of the undigested
amplified fragments : in 93% of the cases, those sums
were in the range of ³50 bp with respect to the
complete fragment. This is well within the limits of the
method used to estimate the size of the fragments. Only
in six cases, the difference was larger, being the sums
75–125 bp lower than expected. This was observed
with HinfI on A. lividopallescens, A. malleata and
A. pantherina, and with TaqI on A. caesarea, A. ovoidea
and A. rubescens. This observation has been reported in
other studies of this type (Fischer 1996), and could be
attributed to small size fragments not detected in the
gels.
Sixty different restriction fragments were considered
in the analysis. In general terms, the digestion patterns
observed among different species of Amanita were quite
dissimilar, and therefore the Jaccard indexes obtained
from the comparison of those restriction patterns were
usually low. However, the restriction patterns observed
in samples from the same species were identical (data
not shown), even when the specimens were collected
from geographically distant locations (Table 1). This
suggests a low level of intraspecific variability in the
ITSs sequence of these species, as it has been already
described for other ectomycorrhizogenous basidio-
mycetes (Ka/ re!n et al. 1997). This would suggest that
this technique could be used for the identification of
specimens from the field, although the degree of
infraspecific variability should be addressed more
broadly by testing a larger number of specimens.
Four main clusters were apparent in the dendrogram
built by neighbour-joining analysis from the matrix of
AR
DR
Aof
Am
anita
species
906
Table 2. Data matrix used for the phenetic analysis. Data based on presence (1) or absence (0) of restriction bands. The indicated sizes of the PCR products and the digested fragments are only
approximate, they were estimated according to their proximity to the closest marker in a 100 bp ladder.
Fragment
size CAEb CIT COD CUR FRA GEM LIV MAL MUS OBL OVO PAC PAN PHA PHV PON POR RUB SPI STR VIT TPU
Size amplified
fragment
650 750 750 750 750 750 600 600 700 650 750 650 700 700 700 750 700 750 700 700 750 600
HinfI 400 bp 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 0
375 bp 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
350 bp 0 0 0 1 0 0 0 0 1 0 1 0 0 1a 1 1 0 0 0 0 1 0
325 bp 0 1 0 0 1 0 1 1 1 1 0 1 0 0 1 0 0 1 0 1 0 0
275 bp 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0
250 bp 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
225 bp 0 0 0 0 0 0 0 0 0 0 1 0 1a 0 0 0 0 0 0 0 0 0
200 bp 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 1 0 0 0 0 0
175 bp 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1
150 bp 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0
125 bp 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
100 bp 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Fragment
size sum
675 725 775 750 725 775 525 525 675 600 775 600 600 700 675 750 750 725 725 700 750 550
EcoRI Undigested 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0
400 bp 1 0 1a 1a 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0
375 bp 0 1 0 0 0 0 0 0 1a 0 0 0 0 0 1a 1 0 0 0 0 1a 0
350 bp 0 1 0 0 1 1 0 0 1 0 1a 0 1a 1a 0 0 1 1 1 1a 0 0
300 bp 1 0 0 0 0 0 0 0 0 0 0 1a 0 0 0 0 0 0 0 0 0 1a
Fragment
size sum
700 725 800 800 750 750 725 700 600 700 700 750 775 750 750 750 700 750 600
HhaI Undigested 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 0
450 bp 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 0
400 bp 0 0 0 0 0 1 0 0 1 0 0 0 1 1 1 0 0 0 0 0 0 0
375 bp 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
350 bp 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1
325 bp 0 0 1 1 0 0 0 0 1 0 0 0 1 1 1 1 0 0 0 0 1 0
300 bp 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0
275 bp 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1
250 bp 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
225 bp 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
175 bp 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
V.G
onza !lez
and
oth
ers907
Table 2. (cont.)
Fragment
size sum
650 750 775 775 750 775 600 600 725 650 725 725 725 775 750 750 750 775 625
TaqI 400 bp 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
375 bp 0 1 1 1 1 0 0 0 1 1 0 0 1 1 0 1 1 0 1 1 1 0
350 bp 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0
325 bp 0 0 0 1 1 1 0 0 1 1 1* 0 1 1 0 1 1 0 1 1 0 0
300 bp 1 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0
225 bp 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
200 bp 0 0 0 0 0 0 1 0 0 0 0 1 0 0 1a 0 0 0 0 0 0 1a
175 bp 0 0 0 0 0 0 1a 1a 0 0 0 1 0 0 0 0 0 0 0 0 0 0
150 bp 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
125 bp 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
Fragment
size sum
600 725 725 700 700 725 625 675 700 700 650 700 700 700 800 700 700 675 700 700 725 625
MspI Undigested 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1
550 bp 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
450 bp 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0
225 bp 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
200 bp 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0
Fragment
size sum
775 750 650 650 750 650
AluI Undigested 1 1 0 0 1 0 0 0 1 1 1 1 0 1 1 0 0 1 0 1 0 0
525 bp 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
500 bp 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0
425 bp 0 0 0 0 0 1 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 1
250 bp 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0
225 bp 0 0 1 1 0 1 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0
175 bp 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1
100 bp 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Fragment
size sum
725 750 750 600 600 650 750 750 750 725 600
a Double bands of very similar size.
b CAE, Amanita caesarea ; CIT, A. citrina ; COD, A. codinae ; CUR, A. curtipes ; FRA, A. franchetii ; GE, A. gemmata ; LIV, A. lividopallescens ; MAL, A. malleata ; MUS, A. muscaria ; OBL, A.
oblongospora ; OVO, A. ovoidea ; PAC, A. pachyvolvata ; PAN, A. pantherina ; PHA, A. phalloides ; PHV, A. phalloides var. verna ; PON, A. ponderosa ; POR, A. porphyria ; RUB, A. rubescens ; STR, A.
strobiliformis ; VIT, A. vittadini ; SPI, A. spissa ; TPU, Torrendia pulchella.
ARDRA of Amanita species 908
A. rubescens
A. rubescens
A. franchetii
A. spissa
A. porthyriaA. citrina
A. codinae
A. vittadini
A. ponderosa
A. curtipes
A. pantherina
A. pantherina
A. gemmata
A. gemmata
A. gemmata
A. muscaria
A. phalloides
A. phalloides
A. phalloides var. verna
A. ovoidea
A. strobiliformis
A. oblongospora
A. caesarea
A. pachyvolvata
A. lividopallescensA. malleata
A. malleata
T. pulchellaT. pulchella
Vaginatae Vaginatae
Vaginatae Caesareae
Vaginatae
Roanokenses
Vaginatae
Lepidella
Amidella Amidella
Phalloideae Phalloideae
Amanita Amanita
Amidella Amidella
RoanokensesLepidella
Phalloideae Mappae
Validae Validae
Bas 1969 Singer 1986
Fig. 1. Dendrogram showing the relationships among Amanita species based on ARDRA. The sections to which the
species are ascribed, according to Bas (1969) or Singer (1986), are shown in the columns to the right.
similarity indexes generated as described above (Fig. 1).
The first clade in the tree from top to bottom contained
those species with amplification products ranging from
700 to 750 bp, members from subgenus Lepidella. This
cluster was divided in two subclusters, one with taxa
from sections Validae and Mappae (sensu Singer 1986)
and the other including members of sections Amidella
and Lepidella. The second clade arranged taxa from
subgenus Amanita sect. Amanita. The third cluster
contained the isolates of A. phalloides (sect. Phalloideae,
subgen. Lepidella), which were arranged together with
A. muscaria (sect. Amanita). These three main clades
were grouped together and separated from a fourth
cluster, which grouped all the species classified in sect.
Vaginatae (sensu Bas 1969) which provided ampli-
fication products of 600–650 bp, together with T.
pulchella. In addition, two isolates from sections
Amidella and Lepidella (A. strobiliformis and A. ovoidea)
formed a sister group to this fourth cluster, rooted at
the base of the tree. These results suggest that ARDRA
is able to recognize only partially the sections from
genus Amanita, although a trend to group together
members from the same section was observed.
Asmentioned in the Introduction, molecularmethods
have been already applied to the study of genus
Amanita. Some studies (Kreuzinger et al. 1996, Haudek
et al. 1996, Ka/ re!n et al. 1997) have focused on the
identification of ectomycorrhizal basidiomycetes using
PCR-based fingerprinting techniques, but with very few
Amanita species and no attempt to assess phylogenetic
relationships. More recently, Weiss et al. (1998) and
Drehmel et al. (1999) have reported molecular phylo-
genies of Amanita based on partial sequencing of the
nuclear 28S rRNA gene, and Oda et al. (1999) published
a phylogenetic reconstruction of Japanese Amanita’s,
based on ITS sequences. Finally, Moncalvo et al. (2000)
compared the phylogeny based on the nuclear 28S
rRNA gene with that obtained with the mitochondrial
12S rRNA gene. In general, all those studies found
more support for the division of the genus into
subgenera and sections than our analysis. This can be
related to the lower resolution power of ARDRA
compared with DNA sequencing. It might also be
significant that we examined species that are mostly
different to those used in other studies (only ten of the
species analyzed had been studied previously).
Two subclades could be observed within the cluster
containing only Lepidella species. One contained species
V. Gonza! lez and others 909
from sections Validae and Mappae, while the other
included species from sections Lepidella and Amidella.
Within the Validae-Mappae cluster, a narrow con-
nection between A. rubescens and A. franchetii was
apparent. These two species have been traditionally
considered as closely related, and old, washed and non-
colour-changing specimens from A. rubescens (specially
those from var. annulosulfurea) can be mistaken for A.
franchetii. The Jaccard index (0±64) suggests that they
are two different species, although closely related. The
highest similarity observed between any pair of species
in this analysis (0±86) corresponded to the pair A. spissa
– A. porphyria, currently allocated to different sections
(Validae and Mappae respectively). Sect. Mappae has
been traditionally considered to be closely related to
Phalloideae, based on the structure and shape of the
volva, to such an extent that both sections were merged
by Corner & Bas (1962). Nevertheless, the ARDRA
results suggest that sect. Mappae is more akin to sect.
Validae than to sect. Phalloideae, in agreement with
other rDNA sequence-based studies (Drehmel et al.
1999, Moncalvo et al. 2000, Oda et al. 1999, Weiss et al.
1998).
With respect to the other subgroup recognized within
this cluster, a close relationship among four species
from sections Lepidella (A. codinae, A. vittadini) and
Amidella (A. ponderosa, A. curtipes), was observed.
These four species are usually referred in the literature
as a group of Mediterranean amanitas collected from
thermophilous habitats, and live in sclerophyllous
evergreen oak forests (Malenc: on & Bertault 1970–75,
Chevassut 1985). Our results would be consistent with
this homogeneity of habitats and geographical dis-
tribution. In addition, within subgenus Lepidella,
sections Amidella and Lepidella are morphologically
related, sharing features such as the whitish colour of
the basidiomata and the spore shape. The remaining
species studied from these two sections (A. strobiliformis
and A. ovoidea) did not cluster together with the
Mediterranean taxa, but as an independent clade. A.
strobiliformis was also found to be excluded from the
clade containing species from sect. Lepidella by Weiss
et al. (1998).
The next cluster in the tree arranged the species
studied from section Amanita, with the exception of A.
muscaria. Other molecular phylogenetic reconstructions
based on the 28S rRNA gene sequence (Weiss et al.
1998, Drehmel et al. 1999), have found a well supported
monophyletic origin for this section. However, this was
not supported by ITS (Oda et al., 1999) nor by the
mitochondrial small rRNA subunit sequencing
(Moncalvo et al. 2000). Interestingly, ARDRA did not
recognize the extremely close relationship between A.
muscaria and A. pantherina at the ITS level described by
Oda et al. (1999). This relationship was not observed by
Weiss et al. (1998) at the 28S rRNA level.
Amanita phalloides var. verna is considered either as
a separate species (e.g. Jenkins 1986) or as a variety of
A. phalloides (e.g. Jaquetant 1992). The sample analyzed
here of A. phalloides var. verna was not grouped
together with the other A. phalloides specimens, and the
similarity index between them was low (0±31). This
could suggest that this variety is not genetically as close
to A. phalloides as some authors considered, and
probably it should be regarded as a different species.
Concerning section Vaginatae, our results support
the hypothesis of Bas (1969, 1976), Fraiture (1993) and
others, who include A. caesarea in sect. Vaginatae.
Corner & Bas (1962) indicated the existence of
intermediate forms between the type species of the
section, A. vaginata, a fragile species without a ring,
and A. caesarea, a robust and ringed species. Moreover,
the North American collections of this last taxon range
from slender specimens with a rather thin-fleshed cap,
a slender stem and not conspicuously heavy volva,
strongly resembling a typical ‘Vaginatae ’ habit (e.g. A.
caesareoides or A. hemibapha. Sacc), to some robust
ones (e.g. A. jacksonii). Consistently with these obser-
vations, A. caesarea appeared to be included within
sect. Vaginatae in our tree. However, other works based
on rDNA sequence analysis have yielded contradictory
results. Thus, Drehmel et al. (1999) reported some
phylogenetic relationship between sections Caesareae
and Vaginatae, and the first section was proposed as a
subsection included in Vaginatae, but other reports did
not find any relation between these two sections (Oda et
al. 1999, Weiss et al. 1998). Our results also suggest a
close relationship between A. malleata and A. livido-
pallescens. Whether these two species should be
considered as members of a species complex, as
suggested by several authors (e.g. Fraiture 1993), needs
further studies.
The secotioid genus Torrendia has been traditionally
considered by some authors (e.g. Miller & Horak, 1992)
as a gasteromycete. Our results show that, in agreement
with Malenc: on (1955), Bas (1975), and Fraiture (1993),
Torrendia could be related to Amanita. Bas (1975)
considered sect. Amidella as the closest group to
Torrendia, whereas our results suggest a closer re-
lationship between Torrendia and the A. malleata –
lividopallescens clade, within section Vaginatae. The
affinity of T. pulchella with Amanita species has been
confirmed by partial sequencing of the 28S rRNA gene
(J. M. Moncalvo, pers. comm.).
In summary, our study shows that ARDRA may be
a useful molecular fingerprinting method to investigate
genetic relationships among species of Amanita, and as
such, it is relevant to mycologists working on this
group. The differences between the results obtained
with this technique and those reported from DNA
sequencing are most likely related to the lower
resolution power of restriction analysis, since the
number of characters studied is much lower. Also, it
may be important that many of the species analyzed are
different to those used in other studies. The technology
employed in this work has been widely applied for the
molecular typing of ectomycorrhizal fungi (including
Amanita species) isolated in the field (Ka/ re!n et al.
ARDRA of Amanita species 910
1997), as well as for rapid detection and characterization
of wood-decay basidiomycetes (Jalasavich, Ostrofsky
& Jellison 2000). Likewise, it is conceivable that it could
be used for the fast identification of Amanita specimens,
avoiding the need of more expensive sequencing
technologies.
ACKNOWLEDGEMENTS
We are grateful to Javier Rejos, curator of the Alcala! de Henares
Herbaria (AH), and Manuel Villarreal (Plant Biology Department,
Universidad de Alcala! ) for the loan of specimens.
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