Invasion biology of Australian ectomycorrhizal fungi introduced with eucalypt plantations into the Iberian Peninsula

Abstract

In the last two centuries, several species of Australian eucalypts (e.g. Eucalyptus camaldulensis and E.␣globulus) were introduced into the Iberian Peninsula for the production of paper pulp. The effects of the introduction of exotic root-symbitotic
fungi together with the eucalypts have received little attention. During the past years, we have investigated the biology
of ectomycorrhizal fungi in eucalypt plantations in the Iberian Peninsula. In the plantations studied, we found fruit bodies
of several Australian ectomycorrhizal fungi and identified their ectomycorrhizas with DNA molecular markers. The most frequent
species were Hydnangium carneum, Hymenogaster albus, Hysterangium inflatum, Labyrinthomyces donkii, Laccaria fraterna, Pisolithus albus, P. microcarpus, Rhulandiella berolinensis, Setchelliogaster rheophyllus, and Tricholoma eucalypticum. These fungi were likely brought from Australia together with the eucalypts, and they seem to have facilitated the establishment
of eucalypt plantations and their naturalization. The dispersion of Australian fungal propagules may be facilitating the spread
of eucalypts along watercourses in semiarid regions increasing the water lost. Because ectomycorrhizal fungi are obligate
symbionts, their capacity to persist after eradication of eucalypt stands, and/or to extend beyond forest plantations, would
rely on the possibility to find compatible native host trees, and to outcompete the native ectomycorrhizal fungi. Here we
illustrate the case of the Australasian species Laccaria fraterna, which fruits in Mediterranean shrublands of ectomycorrhizal species of Cistus (rockroses). We need to know which other Australasian fungi extend to the native ecosystems, if we are to predict environmental␣risks
associated with the introduction of Australasian ectomycorrhizal fungi into the Iberian Peninsula.

Full text

Invasion biology of Australian ectomycorrhizal fungi introduced with
eucalypt plantations into the Iberian Peninsula
Jesu´ sDı
´
ez
Departamento Biologı
´
a Vegetal, Universidad de Alcala
´
, 28871 Alcala
´
de Henares (Madrid), Spain
(e-mail: diez_muriel@yahoo.com; fax: +34-91-885 5066)
Received 4 June 2003; accepted in revised form 30 March 2004
Key words: ectomycorrhizal fungi, Eucalyptus, exotic fungi, forest plantations, invasion ecology
Abstract
In the last two centuries, severa l species of Australian eucalypts (e.g. Eucalyptus camaldulensis and
E. globulus) were introduced into the Iberian Peninsula for the production of paper pulp. The effects of
the introduction of exotic root-symbitotic fungi together with the eucalypts have received little atten-
tion. During the past years, we have investigated the biology of ectomycorrhizal fun gi in eucalypt plan-
tations in the Iberian Peninsula. In the plantations studied, we found fruit bodies of several Australian
ectomycorrhizal fungi and identified their ectomycorrhizas with DNA molecular markers. The most fre-
quent species were Hydnangium carneum, Hymenogaster albus, Hysterangium inflatum, Labyrinthomyces
donkii, Laccaria fraterna, Pisolithus albus, P. microcarpus, Rhulandiella berolinensis, Setchelliogaster
rheophyllus,andTricholoma eucalypticum. These fungi were likely brought from Australia together with
the eucalypts, and they seem to have facilitated the establishment of eucalypt plantations and their nat-
uralization. The dispersion of Australian fungal propagules may be facilitating the spread of eucalypts
along watercourses in semiarid regions increasing the water lost. Because ectomycorrhizal fungi are obli-
gate symbionts, their capacity to persist after eradication of eucalypt stands, and/or to extend beyond
forest plantations, would rely on the possibility to find compatible native host trees, and to outcompete
the native ectomycorrhizal fungi. Here we illustrate the case of the Australasian species Laccaria frater-
na, which fruits in Mediterranean shrublands of ectom ycorrhizal species of Cistus (rockroses). We need
to know whi ch other Australasian fungi extend to the native ecosystems, if we are to predict environ-
mental risks associated with the introduction of Australasian ectomycorrhizal fungi into the Iberian
Peninsula.
Introduction
Alien plants often require mutualistic partners to
overcome barriers to establishment in foreign
environments. Mutualisms that facilitate inva-
sions occur at several phases of the life cycle of
alien invading plants. However, even when wind
and native generalist animals mediate flower pol-
lination and seed spread, the lack of compatible
mycorrhizal symbionts can limit the spread of
alien plants (Richardson et al. 2000a).
Mycorrhizal fungi are essential in plant nutri-
tion in terrestrial ecosystems, and terrestrial
plants present different types of obligate mycor-
rhizal symbioses. Herbs and shrubs form arbuscu-
lar mycorrhizas (AM) with glomales (Smith and
Read 1997). The low specificity of AM relat ion-
ships and the easy acquisition of mutualistic sym-
bionts by herbs and shrubs in any ecosystems are
important reasons for so many ecosystems being
susceptible to invasion by alien plants. As a con-
sequence, the introduction of AM fungi does not
Biological Invasions (2005) 7: 3–15 Ó Springer 2005

seem to play a major role in mediating plant inva-
sions, except on some remote islands that are
poor in AM fungi (Richardson et al. 2000a).
Most forest trees, however, associate with a group
of basidiomycetes and ascomycetes forming ecto-
mycorrhizas (ECM) (Newman and Reddell 1987).
Ectomycorrhizal symbioses present a range of
host–fungus specificities. For this reason, exotic
forestry has often needed the introduction of
compatible ectomycorrhizal fungal symbionts
(Grove and Le Tacon 1993). For many non-
native trees, notably for pines and eucalypts, the
lack of symbionts was a major barrier to estab-
lishment and invasion in the southern hemisphere,
before the build-up of inoculums through human
activity (Armstrong and Hensbergen 1996; Davis
et al. 1996; Richardson et al. 2000a). The role of
ECM fungi in facilitating the establishment and
invasion of alien trees was claimed by Richardson
et al. (1994) to explain the invasion patterns of
pines in South Africa. Little investigation, how-
ever, has been done on the role played by the
ECM fungi in the naturalization of eucalypts
beyond their natural range.
This paper provides a framework for thinking
about the effects of the introduction of exotic
fungi with plantations of exotic forest trees. Dif-
ferent sections will deal with aspects of the inva-
sion biology of eucalypts and their ECM fungi.
We first lay the groundwork for our paper by
briefly introducing the history of the exotic for-
estry and several terms used in studies of biologi-
cal invasions. The paper will be illustrated with a
study conducted on exotic plantations of eucalyp-
ts in the Iberian Peninsula. We will describe ecto-
mycorrhizal commun ities of Australasian fungi
present in these plantations. We will next analyze
the role that ECM fungi plays in promoting (or
limiting) invasion rates of the eucalypts intro-
duced. The central core of the paper will try to
explain the lags between the introduction and the
spread of the eucalypts, as a factor that depends
on the dispersion of propagules of introduced
ECM fungi. We report host shifts of Australian
fungi to native ectomycorrhizal plants detected to
date. We discuss whether the introduce d fungi
could threaten natural communities of ECM
fungi by out-competing the native ECM fungi
from their natural hosts. This work includes an
analysis of the potential effects of the invasion of
these exotic fungi in the nutrient cyclin g of Med i-
terranean forests. To the best of our knowledge,
this is the first investigation dealing wi th the inva-
sion ecology of exotic ECM fungi.
Exotic forestry: invasions of alien trees
Origin of exotic forestry
There is evidence of large-scale forestation in the
ancient Mediterranean basin, where timber- and
crop-producing trees were planted as long ago as
255 B.C. (Zobel et al. 1987). Inspite of its long
history, the scale of forestry remained small until
recently. Large-scale forestry was not widespread
until the second half of the 20th century (Zobel
et al. 1987), when pines and eucalypts were
widely planted outside their natural ranges.
Pines, which comprise only Holarctic species,
were planted in South America, South Africa
and Australia. The Australasian eucalypts were
planted worl dwide. In particular, the need for
increased wood production to improve living
conditions made the genus Eucalyptus one of the
most widely planted silvicultural crops. In addi-
tion, in many damaged ecosystems, afforestation
with alien eucalypts was driven by the belief that
such plantings wer e beneficial to the environment
(Zobel et al. 1987).
Naturalization and invasion of pines and eucalypts
In this paper, we will use the following three con-
cepts: introduction, naturalization and invasion,
as defined by Richardson et al. (2000b). A tree
introduction takes place when humans transport
a tree across a geographical barrier to a new area.
Naturalization refers to the species establishing
new self-perpetuating populations and becoming
incorporated within the native flora. The natural-
ized trees regenerate freely, but mainly under their
own canopies. In contrast, invasive species recruit
seedlings, often in very large numbers, at long dis-
tances from parent plants (often more than
100 m). Only some of the naturalized plants
become invasive, producing important environ-
mental or economical damages.
All trees that are widely planted in alien envi-
ronments can become invasive and spread under
certain conditions (Richardson 1998). Conse-
quently, the use of exotic trees has often caused
4

environmental damages in different parts of the
world (Binggeli 1996). The species that cause the
greatest problems are generally those planted
most widely and for the longest time (Pryor
1991; Rejma
´
nek and Richardson 1996). Accord-
ing to Higgins and Richardson (1998), at least 19
Pinus species are invaders of natural ecosystems
in the southern hemisphere; four of the most
widespread invasive pines are P. halepensis, P. pa-
tula, P. pinaster and P. radiata (Higgins and
Richardson 1998; Richardson et al. 1990).
The genus Eucalyptus L’He
´
rit (Myrtaceae) com-
prises evergreen woody plants, including shrubs
and forest trees (nearly 600 species), which are
confined in natural occurrence entirely to the Aus-
tralasian region, Papua, New Guinea and Timor
(Pryor 1976). More than 43 species of eucalypt
trees are planted outside their natural geographic
distribution. Eucalypts are planted on a large scale
to provide a short rotation crop yielding wood
and paper pulp for industrial use (Eldridge et al.
1994). Although less invasive than pines, several
species of eucalypts already caused problems as
invaders in South Africa (Richardson 1998;
Richardson et al. 2000a). Eucalypts are repre-
sented on many weed lists from other parts of the
world, including California (Warner 1999) and
peninsular Spain (Sanz-Elorza et al. 2001). Signifi-
cant impacts might result from the introduction of
eucalypts into Spain and some transformation on
various ecosystem properties; specially changes in
grasslands and scrubland habitats. Ecology and
environmental politics dictate the desirability of
maintaining Mediterranean grasslands and scrub-
lands due to their high diversity in endemic plants
and because these ecosystems are the natural habi-
tats for many local wildlife. Eucalypt invasions
can cause shifts in life-form dominance, reduced
diversity, disruption of prevailing vegetation
dynamics, and changing nutrient cycling patterns.
Hence, eucalypt plantations are increasingly caus-
ing major conflicts between Spanish foresters, pol-
iticians and conservationists.
Role of ectomycorrhizal fungi in exotic forestry
Mycorrhizas of herbs, shrubs and forest trees
Mutualistic interactions between fungi and plant
roots are common in the plant kingdom, includ-
ing mycorrhizal symbioses. In most mycorrhizal
symbioses, the fungal partner supplies nutrients
to the host plant in exchange for photosynthetic
carbon, and may offer protection against patho-
gens, toxins and drought (Smith and Read 1997).
The main types of mycorrhizal symbioses differ
in the anatomy of the mycorrhiza, which is a
mixed root-fungus structure at which nutrient
interchanges take place (Smith and Read 1997).
Most Mediterranean plants are mycorrhizal with
different types of fungi. Herbs and shrubs mainly
associate with glomales to form arbuscular
mycorrhizas (AM), which is by far the most com-
mon mycorrhizal symbiosis in the Mediterranean
terrestrial ecosystems (Dı
´
ez 1998). A restricted
group of plants form particular types of mycor-
rhizas with a range of particular ascomycetes and
basidiomycetes, such as the Ericales and Orchi-
dales. However, most forest trees form ectomy-
corrhizas (EM) with a polyphyletic group of
basidiomycetes and ascomycetes (Smith and
Read 1997; Hibbett et al. 2000).
Forest trees are obligate ectomycorrhizal plants
Pines and eucalypts are obligate ectomycorrhizal
trees, which depend on these mutualistic symbio-
ses for nutrient uptake in natural conditions
(Smith and Read 1997). Ectomycorrhizal symbio-
ses are essential in the mobilization of nutrients in
soil forests (Fahey 1992; Read and Pe
´
rez-Moreno
2003). In natural conditions, these forest trees rely
on the ectomycorrhizal fungi, which colonize the
root cortex and form a nutrient-gathering ‘organ’
called ectomycorrhiza (Smith and Read 1997). Ec-
tomycorrhizas are almost exclusive to forest trees.
In the Mediterranean, only a restricted group of
shrubs in the Cistaceae (Cistus spp., called rock-
roses) forms EM, in which are involved a range of
endemic ascomycetes and basidiomycetes (Dı
´
ez
1998). Ectomycorrhizas are characterized struc-
turally by the presence of a dense mass of fungal
mycelium surrounding the short lateral roo ts (the
mantle). The mantle originates from the attach-
ment of the fungal hyphae onto epidermal cells,
and the multiplication of hyphae to form a series
of hyphal layers. The fungal mycelium also grows
among the cortical cells, forming the Hartig net.
The Hartig net is the structural and functional
interface between fungal and roots cells. The fun-
gal mantle is connected with a highly extended
5

network of mycelium prospecting the soil and
gathering nutrients. The extra-radical mycelium is
responsible for mobilizing soil nutrients, nutrient
(and water) uptake and transfer to the ectomy-
corrhiza (Peterson and Bonfante 1994). Sclerotia
(and sclerotia-like bodies) are vegetative balls of
hyphae formed by a few species of ECM fungi.
Finally, fruiting bodies arise from discrete points
of the extra-radical mycelium to ensure the sexual
reproduction and the dispersal of the fungal part-
ner (Allen 1991).
The seedling s of forest trees ne ed to be colo-
nized by ectomycorrhizal fungi. In a given bio-
tope, the seedling survival depends on the
presence of the propagules of compatible ectomy-
corrhizal fungi in the soil (spores, sclerotia, or
soil mycelium). Hyphae arising from fungal prop-
agules have a limited capacity to grow and die
unless they come in contact with a root tip of a
compatible host. In the forest, seedlings grow
near mycorrh izal trees and may thus become col-
onized by pre-existing mycorrhizal mycelia of the
living roots of mature trees (Onguene and
Kuyper 2002). In open areas, the fungal hyphae
and then the sclerotia disappear in the long term
in the absence of compatible ECM plants
(Brundrett and Abbott 1995). Thus, the coloniza-
tion of the root seedlings in a new biotope neces-
sarily depends on the spore dispersion from close
forests (Allen 1991). Spores of epigeous (fruiting
aboveground) fungi are mainly dispersed by
wind, whereas mycophagous animals are impor-
tant vectors for disper sing spores of hypogeous
(fruiting below ground) fungi.
Success of exotic forest plantations: the
occurrence of compatible ectomycorrhizal fungi
In the AM symbiosis, the levels of specificity
among host plants and fungal species are low,
and many glomales have a cosmopolitan distri-
bution (Smith and Read 1997). Due to this low
specificity, most invading herbs and shrubs have
no problems to form mycorrhizas with the fungi
of the target habita t (Richardson et al. 2000a). In
contrast, the ECM fungi present different levels
of specificity. A given species of ectomycorrhizal
fungus is usually only able to establish mutualis-
tic symbiosis with a number of species from the
same biogeographic realm, and even more highly
specific interactions occur (Molina et al. 1992).
Specially, Australian ectomycorrhizal plants (e.g.
Eucalyptus, Acacias spp.) have evolved in isola-
tion from the ECM fungal flora associated with
Pinus (and Quercus) in the northern hemisphere
(Halling 2001). This could explain why many (if
not almost all) native fungi from the northern
hemisphere do not associate with eucalypts
in silva, and vice versa.
When a compatible ectomycorrhizal biota is
absent on the plantation site, there is a barrier
for the success of exotic plantations. For this rea-
son, mycorrhizal inoculation of pine and eucalypt
seedlings with forest soil, spores or mycelium of
compatible ECM fungi was often necessary for
the success of exotic plantations of pines (Perry
et al. 1987; Grove and Le Tacon 1993). In the
Southern Hemisphere, pine forestry was de layed
by the lack of suitable ECM fungi until a num-
ber of Holarctic ECM fungi were introduced
with the pin es (Dunstan et al. 1998). Some of
these exotic pines eventually invaded a wide
range of systems with the introduction of such
Holartic ECM fungi (Richardson et al. 2000a).
Study areas and methods used to identify the
ectomycorrhizal fungi
Plantations of eucalypts in Spain
Since the last century, the river red gum Eucalyp-
tus camaldulensis Dehnh. and the blue gu m
E. globulus Labill. have been used in extensive
plantations in the Iberian Peninsula. Nowadays,
there are around 550,000 ha of eucalypts planta-
tions in Spain, 320,000 ha of E. globulus and
about 180,000 ha of E. camaldulensis. Eucalyptus
camaldulensis is planted mainly in southwestern
Spain (Huelva, Cadiz, Badajoz and Seville), and
E. globulus in the northern regions (Galicia,
Asturias and Santander). Eucalyptus gomphocep-
hala DC is used on basic soils in Murcia and
Almerı
´
a (southeastern Spain). These eucalypts
are used also in afforestation and agroforestry
(windbreaks, shelter trees, and intercropping of
trees and arable crops). In the Iberian Peninsula,
the inoculation with ectomyc orrhizal fungi was
not necessary to ensure the success of many euca-
lypt plantations. Two hypotheses might account
for the lack of need of inoculation: (i) whether
there were native ectomycorrhizal fungal species
6

compatible with the eucalypts, (ii) or a range of
ectomycorrhizal fungi native from Australasia
were brought toget her with the eucalypt seed-
lings. To resolve this question, we have been
investigating the origin of ectomycorrhizal fungi
present in the Iberian plantations of eucalypts.
Study area and sampling strategy
The study area is located in the region of Ex-
tremadura, which is formed by the provinces of
Ca
´
ceres and Badajoz, where extensive plantations
with eucalypts took place between 1955 and 1977.
Eucalyptus camaldulens is was by far the most pre-
dominant species, followed by E. globulus. Ex-
tremadura stands out with 14% of the totality of
Spanish plantations of eucalypts. We studied eu-
calypts stands , shelterbelts and road verges. We
sampled watercourses and riparian stands in
which eucalypt s became naturalized and invasive.
We sampled fruit bodies and ectomycorrhizal
root tips. The fruit bodies are identified using
morphological features. In some cases, the use of
molecular and phylogenetic methods is necessary
to discriminate among cryptic species (Dı
´
ez et al.
2001). We collected ectomycorrhizal roots,
because many ectomycorrhizal species do not
fruit, and fruiting patterns do not truly reflect the
belowground community of ECM fungi (Gardes
and Bruns 1996; Horton and Bruns 2001).
Ectomycorrhiza identification: morphological and
molecular methods
Despite the general organization, ectomycorrhi-
zas differ among species in colour, mycelium den-
sity, size, form s and biochemical composition.
Morphological typing of ectomycorrhizas enables
us to identify fungi that seldom or never produce
fruiting bodies. Methods for the morphological
characterization of ectomycorrhizas are described
in Ingleby et al. (1990) and Agerer (1997), who
provided descriptions of ectomycorrhizas and cri-
teria to discriminate species based on morpholog-
ical features and chemical tests. Typing
ectomycorrhizas with morphological methods is
time consuming; and in many cases, it is not con-
clusive, because the ectom ycorrhizas have not
been described for many ECM fungi. In addition,
the ectomycorrhizas of one fungal species can
also show different morphologies according to
the host, physiological and environmental condi-
tions (Egger 1995). With such a morphological
approach, we can classify the ectomycorrhizal
tips as much as in morphotypes.
To overcome the problems of morphological
typing, we use molecular methods for the identifi-
cation of ectomycorrhizas, as described in Martı
´
n
et al. (2000). Such methods are based on the
restriction fragment length polymorphism
(RFLP) of the internal transcribed sequences
(ITS) of the nuclear rDNA. The IT S regions is
amplified for ectomycorrhizal root tips with the
polymerase reaction technique (PCR ), using a
thermostable DNA polymerase and primer pairs
annealing at conserved regions of the 18S and
28S ribosomal genes (White et al. 1990). Such
PCR-based methods are of great value in these
kinds of studies (Glen et al. 2001a, b). We
amplify the ITS regions from DNA obtained
from ECM root tips, using fungal-specific prim-
ers to avoid the amplification of plant DNA
(Gardes et al. 1991; Gardes and Bruns 1993).
After cutting the PCR-amplified ITS with restric-
tion enzymes, we obtain RFLP patterns.
To identify the different ectomycorrhizas, we
are compiling a database of RFLP profiles of
fruit bodies, so that we can compare them with
those obtained from ECM roots. This PCR-
RFLP database is of great help in the identifica-
tion of unknown ectomycorrhizas. Most fungal
species sho w a unique RFLP pattern, and their
ectomycorrhizas can be identified by their PCR-
RFLP profiles. For RFLP patterns not associated
with any of the fruit bodies, direct sequencing of
the ITS regions followe d by a ‘Blast research’
(Altschul et al. 1997) in the National Centre for
Biotechnology Information (http://www2.ncbi.
nlm.nih.gov/) enable us to identify the ectomy-
corrhizal morphotypes, with luck, even at the
species level.
Ectomycorrhizal fungi of eucalypt plantations in
the Iberian Peninsula
Species of introduced Australian ectomycorrhizal
fungi
Over our surveys in Iberian plantations of euca-
lypts, we found fungi known only from Australian
forests and eucalypts plantations worldwide
7

(Table 1). Our analyses of ECM roots confirmed
the presence of Australian ECM fungi in the euca-
lypt roots of Spanish plantations. Surveys of fruit
body proved that fungi fruiting in the Iberian
plantation of eucalypts are of Australian origin.
The most frequent ly occurring species in the
Spanish plantations are Hydnangium carneum
Wallr., Hymenosgaster albus (Klotzsch) Berkeley
and Broome, Hysterangium inflatum Rod. (= H.
pterosporum Donadini and Riousset), Labyrinth-
omyces donkii Malen c¸ . Pisolithus albus (Cke and
Mass.) M.J. Priest, P. microcarpus (Cke and
Mass.) Cunn., Ruhlandiella berolinensis (Henn.)
Diss. and Korf, Laccaria fraterna (Cooke and
Mass.) Sacc. (L. lateritia Malenc¸ ), Setchelliogas-
ter rheophyllus (Ber. and Malenc¸ .) G. Moreno
and Kreisel, and Tricholoma eucalypticum Pear-
son. Most of these Australian fungi are able to
fruit under the climatic conditions of the Iberian
Peninsula. Additional ECM fungi, likely unable
to fruit outside their natural range, were detected
belowground during the molecular typing of ec-
tomycorrhizal roots. We identified ectomycorrhi-
zas of Cenococcum geophilum Fr. and several
species of Sebacina and Thelephora. Whether the
C. geophilum, Sebacinia and Thelephora strains
infecting eucalypts in the Iberian plantations are
natives or Australian deserves further studies,
because these species are known to be native to
the area studied.
In Extremadura, the soil dries up in summer,
and most fungi in the eucalypt plantations fruit
in late winter and spring. Some of these species
are secotioids or truffle-like (sequestrate) fungi,
most of them adapted to fruit belowground
(hypogeous fungi). Hydnangium carneum is one
of the most common truffle-like fungi in the
eucalypt stands; which is a gastroid relative of
the agaricoid genus Laccaria; due to its hypoge-
ous habit, this Australian fungus is well adapted
to the conditions of the Mediterranean climate.
We also found the Australian false truffle Hyme-
nogaster albus, which is common in plantations
near the Monfragu
¨
e Natural Park. Hysterangium
inflatum is hypogeous relative to Phallus, very
common in the Monfragu
¨
e region. Setchelliogas-
ter rheophyllus is another secotioid fungus pres-
ent in eucalypt plantations near Monfragu
¨
e and
Badajoz. Rhulandiella berol inensis is considered
specific to Australasian ectomycorrhizal plants,
and is very common in riparian stands of Euca-
lyptus camaldulensis near Me
´
rida (Badajoz). The
main epigeous fungus (fruiting aboveground) was
Laccaria fraterna, which is an agaricoid species
native to Australia and introduced into the Medi-
terranean with eucalypts. To the best of our
knowledge, our collections of Tricholoma euca-
lypticum are among the first records of this fun-
gus outside Australia.
Growing on debris of eucalypts and humus of
eucalypt plantations, we found the Australasian
species Urnula rhytidia (Berk.) Cooke. (Pezizales)
and Discinella terrestris (Berk. and Br.) Dennis
(Leotiales); these two species are regarded as
characteristic of sclerophyllous eucalypt forests
of Australia and Tasmania. There is no reliable
information on whether these fungi are sapro-
phytic or facultatively ectomycorrhizal as many
other pezizales.
In the studied plantations of eucalypts in Ex-
tremadura, we found strains of the Australasian
species Pisolithus albus and P. microcarpus,as
proved with molecular analyses of the ITS
sequences of the nuclear rDNA (Dı
´
ez et al.
2001). For many years, the name Pisolithus ar-
rhizus (Pers.) Rauscher (synonym of P. tinctorius
[Pers.] Coker and Couch) have been used for all
Pisolithus strains occurring in eucalypt and pine
plantations worldwide, regardless of the host
plant (Cairney 2002). This misunderstanding
occurred because many researchers considered
Table 1. Twelve frequent Australasian fungi that have been
introduced in the Iberian Peninsula together with the euca-
lypts.
Taxonomic
group
Species Habit
Basidiomycetes Laccaria fraterna
a
Epigeous
Hydnangium carneum
a
Hypogeous
Hymenogaster albus
a
Hypogeous
Hysterangium inflatum
a
Hypogeous
Pisolithus albus
a
Epigeous
Pisolithus microcarpus
a
Epigeous
Setchelliogaster rheophyllus
a
Semi-hypogeous
Tricholoma eucalypticum
a
Epigeous
Ascomycetes Labyrinthomyces donkii
a
Hypogeous
Rhulandiella berolinensis
a
Hypogeous
Discinella terrestris
c
Epigeous
Urnula rhytidia
b
Epigeous
The table also includes a saprophytic fungus and a possible
facultatively ectomycorrhizal fungus of Australasian origin.
a
Ectomycorrhyzal.
b
Saprophytic, and probably facultatively ectomycorrhizal.
c
Saprophytic.
8

Pisolithus as a monospecific genus, and P. arhizus
as a fungus with a wide host range (Chambers
and Cairney 1999). However, our molecular anal-
yses proved that the genus Pisolithus comprises
several phylogenetic species, and that each spe-
cies of Pisolithus is confined to hosts from one
single biogeographic realm. Our study also
proved that Pisolithus arhizus is rest ricted to Hol-
arctic host plants (e.g. Quercus and Pinus spp.)
and does not occur in eucalypt plantations
(Martin et al. 2002). In a previous work (Dı
´
ez
et al. 2001), we showed that Pisolithus albus and
P. microcarpus fruit in litter and on open ground
and at the edges of eucalyptus plantations, and
on dry an d disturbed sites such as gravelly road-
sides in Morocco and Spain. Endemic to Austra-
lia, P. albus and P. microparpus are not restricted
to eucalypts and form ectomycorrhizas with
other Australasian plants; we have found these
two species in association with Australasian aca-
cias in Portugal (Muriel and Dı
´
ez, unpublished).
The species of Pisolithus native to the Iberian
Peninsula correspond to P. tinctorius, and two
unnamed species, one basophilic species and
another Cistus-specific Pisolithus species (Dı
´
ez
et al. 2001); these three Holarctic species of Pisol-
ithus never occur in association with eucalypts
(Dı
´
ez et al. 2001; Martin et al. 2002).
A group of Australasi an ectomycorrhizal fungi
were introduced with the eucalypts
The fungi we found in the Iberi an plantations of
eucalypts are of Australian origin. These exotic
fungi were likely introduced with eucalypt seed-
lings brought into peninsular Spain before plant
quarantine restrictions were observed. In Austra-
lia, gum seedlings are container grown in nur ser-
ies, which are naturally colonized by a limited
number of ectomycorrhizal fungi. Most of these
ECM fungal species may persist during the first
years of eucalypt plantations (Lu et al. 1999).
Foresters probably dispersed these exotic ECM
fungi in soil or eucalypt seedlings worl dwide.
Our results are in agreement with investigations
by other mycologists, and there is a growing con-
sensus in a worldwide dispersal of a number of
Australasian ECM fungi toget her with the euca-
lypts (Giachini et al. 2000). Saprophytic and even
pathogenic fungi seem to have spread worldwide
as well with the eucalypts. In this regard, it has
been suggested that dissemination of the basidio-
mycetous yeast Cryptococcus neoformans,a
human pathogen associated with Eucalyptus
leaves in southern California and India resulted
from the introduction of eucalypts (Casadevall
and Perfect 1998; Chakrabarti et al. 1997).
Do native fungi infect exotic eucaly pts in sylva?
In the Iberian Peninsula, there are many native
ECM fungi in association with pines, oaks, and a
restricted number of ectomycorrhizal shrubs (i.e.
Cistus spp.) (Dı
´
ez 1998). In our surveys, we did
not find Holarctic ECM fungi in the eucalypt
plantations, though propagules of native fungi
are often present on planting sites. We do not
know any reliable evidence of Eur opean fungi
forming ectomycorrhizas with eucalypts in natu-
ral conditions in Spain. Because eucalypts hav e
evolved in isolation from the ectomycorrhizal
mycobiota associ ated with Pinus and Quercus in
the Holarctic Realm, the native fungi (e.g. Pisoli-
thus tinctorius) might be unable to associate with
eucalypts in silva. Even within each biogeograph-
ic realm, there would exist some level of host-
symbiont specificity. Parlade
´
et al. (1996)
described, in pure culture syntheses, the ability of
native Iberian fungi to colonize several North
American conifers planted in northern Spain.
Such an ability can be easily explained by the
similarities between the fungal floras of North
America and the Iberian Peninsula (Halling
2001), as these two regions belong to the Holarc-
tic Realm. However, some host specificity might
exist, because in tree nurseries and experimental
plantations in northern Spain, native ECM fungi
do not seem to outcompete exotic North Ameri-
can fungi inoculated (or that accident ally infect
seedlings) in tree nurseries (Pera et al. 1999).
Role of the introduction of Australasian
ectomycorrhizal fungi in promoting eucalypt
invasiveness
We now have evidence on the introduction of
Australian ectomycorrhizal fungi with the planta-
tions of eucalypts in the Iberian Peninsula. Such
introductions seem to mediate the naturalization
of eucalypts, because once dense tree plantations
are established, new seedlings can become
9

ectomycorrhizal very rapidly through infection
from the established fungal network. In many
cases, the introduced eucalypts compete with
native species, and the eradication of the eucalyp-
ts is difficult, especially in areas with a long hi-
story of large and extensive plantations. In
Spain, the regional and national governments are
promoting the eradication of eucalypts from nat-
ural and national parks.
An example is the National Park of Don
˜
ana.
Don
˜
ana is well known for its variety of species
of birds, either permanent residents, winter visi-
tors from north and central Europe, or summer
visitors from Africa, such as numerous types of
geese and colourful colonies of flamingo. Do-
n
˜
ana’s configuration is the result of its past as
the estuary of the Guadalquivir river, and mainly
consists of beaches, coastal mobile dune s,
marshes and lakes. Fauna in Don
˜
ana is rich and
some in danger of extinction, such as the Iberian
lynx, the Egyptian mongoose and the imperial
eagle. The local wildlife of Don
˜
ana depends on
the water level. Autumn rains brought life back
to the marshes and filled the lagoons after the
dry summer. Gradually, the water attains a uni-
form depth of 30–60 cm over vast areas, and the
resulting marshes attract flocks of water birds of
the most varied kind. Among other causes, the
level of the freshwater of the marshes is in dan-
ger as a result of intensive plantations of eucalyp-
ts (Sacks et al. 1992). In the Don
˜
ana National
Park (southern Spain), the eradication of euca-
lypts is necessary to reduce the water loss and
the conservation of the local wildlife (e.g. Iberian
lynx, Felix pardina) (Palomares et al. 1991).
The eucalypts introduced are competitive with
native species, and their control and eradication
are difficult. These eucalypts and the associated
exotic ECM fungi can regenerate from root frag-
ments. We know as well that ectomycorrhizal
spores can remain dormant in soil for long peri-
ods, and might colonize eucalypt seedlings grown
from remaining eucalypt seeds Moreover, ecto-
mycorrhizal eucalypt seedlings often efficiently
compete for soil nutrients with the planted young
trees of native Quercus species (i.e. Q. ilex L.,
Q. pyrenaica Willd. and Q. suber L) (Muriel and
Dı
´
ez, unpublished). These reasons would account
for the naturalization of eucalypts in Peninsular
Spain (Vila et al. 2001) and in other regions of
the Mediterranean Basin (Le Floc’h 1991).
In the checklist of alien species in Spain, Sanz-
Elorza et al. (2001) classified the eucalypts as
alien plants with a clear invasive behaviour, and
as dangerous (causing ecological damage and
alterations) for natural ecosystems. To date, the
invasion behaviour is limited to areas with a long
history of large and extensive plantations. In
Spain, the red river gum often invades along the
watercourse, which is its natural habitat in Aus-
tralasia. In these areas, the plantations of euca-
lyptus have a great impact on lowering water
tables, and have a devastating ecological impact,
reducing soil quality and the habitat of local
wildlife. The invasive tree species have a predict-
able set of life-history attributes, including low
seed mass and short juvenile periods , and a short
interval between seed crops (Rejma
´
nek and
Rhichardson 1996). Many eucalypts present
many of these characteristics and produce large
quantities of small seeds easily propagated by
wind or/and animals (Pryor 1976). In addition,
the pollination and seed dispersal do not limit
the eucalypt invasiveness. Most eucalypts species
are facultative outbreeders and are pollinated by
a variety of generalist insects, in natural forest
and exotic plantations (Pryor 1976). Therefore,
the limited success of eucalypts as efficient invad-
ers in the Iberian Peninsula can be puzzling. One
can expect eucalypts to be successful as invaders,
because these species are likely to be different in
their resource utilization, easily escaping competi-
tion with native species (Richardson 1998).
To date, the difficulties of the eucalypts to find
compatible fungal partners wi thin the Iberian
fungal biota seem to restrict their spread in the
Iberian Peninsula to areas close to large planta-
tions. The expansion of the invasion of the euca-
lypts from the plantation sites is likely to be
hindered by the lack of compatible ectomycorrhi-
zal fungi at potential seedling recruitment places.
Lack of, or low extent of, colonization by com-
patible ectomycorrhizal fungi may be an impor-
tant factor preventing or reducing seedling
establishment of these alien trees. Eucalypt inva-
sions would often result from the dispersal and
propagules of Aust ralian fungi. The importance
of eucalypts as invaders correlates with the extent
and the duration of planting, which relates to the
dispersal rates of ECM propagules. The spread
of propagules will facilitate a successful seedling
establishment in new biotopes. As stressed above,
10

the spread of ECM fungi in new biotopes is often
by air spores, which is slow (Brundrett and
Abbott 1995). This could explain why eucalypts
might exist in plantations for many years, before
they start to invade indigenous vegetation. A fac-
tor contributing to this lag is that compatible ec-
tomycorrhizal propagules , in the form of spores
(often air-born spores), needed time to accumu-
late in the soil (spore bank) before eucalypts can
establish and proliferate. However, it is only a
matter of time before the fungal spore bank
reaches sufficiently high levels to allow ectomy-
corrhizal eucalypts to spread everywhere in com-
patible environments.
Invasiveness of Australasian ectomycorrhizal fungi
Awareness by politicians of the negative effects
of exotic trees on natural biodiversity has led to
an attempt to eradicate eucalypts in natural
parks in Spain. But do Aust ralasian ectomycor-
rhizal fungi persist after eucalypt eradication?
After the removal of eucalypts, there will persist
a high level of inoculums (spores, mycelium) of
Australasian ectomycorrhizal fungi, which might
colonize the roots of native trees that are planted
on old eucalypt plantation sites. We have little
information of the ability of the introduced Aus-
tralian fungi to infect plants native to the Iberi an
Peninsula. It would be necessary to investigate
whether these Australian fungi colonize roots of
the Mediterranea n trees planted on former euca-
lypt plantations. We do not know whether these
exotic fungi spread beyond the plantations and
colonize native ECM flora.
ECM fungi need to live in association with the
tree roots, and their spread beyond the planta-
tions will depend on (i) their compatibility with
any native ectomycorrhizal plants, and (ii) their
ability to exclude the indigenous ECM fungi. In
our survey, we did not find Australian fungi in
association with native ectomycorrhizal plants,
except Laccaria fraterna, which naturally occurs
in association with eucalypts in Australia and
worldwide plantations. We found this Australian
fungus in shrublands of the ectomycorrhizal
shrub Cistus ladanifer L. near plantations of eu-
calypts. In two sites near the Monfragu
¨
e Natural
Park, fruiting bodies of L. fraterna were found
500 and 700 m far from the nearest eucalypt tree,
and no eucalypt seedlings were found near the
fruiting bodies of L. fraterna. Molecular typing
of the ectomycorrhizal root tips identified L. fra-
terna on roots of Cistus ladanifer (Muriel and
Dı
´
ez, unpublished). We do not know whet her
these native woody plants that are associated
with exotic ectomycorrhizal fungi (i.e. C. ladanif-
er) ha ve access to resources that these native
plants normally cannot tap, which would modify
the nutrient cycling and affect the ecosystem
functioning.
In our study, we detected the Australian fungi
Urnula rhytidia and Discinella terrestis, whi ch are
considered as saprophytic (or facultatively ecto-
mycorrhizal). These fungi should have come with
the eucalypts. Urnula rhytidia was also found on
fallen leaves of Quercus ilex L., in oak wood-
lands near eucalypt plantations in Badajoz. These
data suggest that many other saprophytic fungi
could be introduced with the eucalypts and might
spread to native ecosystems. Due to their sapro-
phytic life style, these exotic fungi might persist
for a long time after the eradication of the euca-
lypts, altering nutrient cycling in the soil.
Limiting furt her introduction of exotic EM
Australian forests have one of the most privative
and rich mycobiota of the world (Castellano and
Bougher 1994; Bougher and Syme 1998), but
only a few Australian ECM fungi seem to have
been introduced into the Iberian Peninsula. The
limited functional and genetic diversity of intro-
duced ECM species should be determining the
environment range that eucalypts are able to
invade. However, we continue to move soil and
microbes around the world to establish new plan-
tations, which favours the introduction of more
and more Australian ECM fungi. Particular
ECM fungal species might confer unique advan-
tages for obtaining nutrients in potential habitats
or to use particular nutrient resources (Buscot
et al. 2000). An increased diversity of introduced
Australian ECM fungi might increase the ability
of the eucalypts to invade new habitats in the
Iberian Peninsula.
Some authors propose to increase the diversity
of the introduced ECM fungi to improve the
productivity of exotic eucalypt plantations (Dell
et al. 2002). This may involve selecting hardy
11

ECM strains tolerating a wide range of edaphic
and environmental conditions and strains that
elicit growth responses of eucalypt plantations
(Neves-Machado 199 5). Among these inoculums,
there may be highly competitive strains, which
could make the natural ecosystems more vulnera-
ble to invasion by eucalypts. This would include
strains coping with extremely harsh or toxic soil
conditions. Rare and ecologically sensitive eco-
systems, such as serpentine communities, may be
particularly vulnerable to the introduction of
Australasian ECM fungi with broad environmen-
tal tolerances.
Elevated levels of atmospheric carbon dioxide
(global climate change) create an increasing con-
cern. Exotic plantations are now promoted for
their presumed capacity to provide a net sink of
atmospheric carbon, and mycorrhizal fungi may
play a critical role in terrestrial carbon exchange
processes (Staddon et al. 2002). Chapela et al.
(2001) described how exotic ectomycorrhi zal fungi
induce soil carbon depletion in pine plantations in
Central America. We have no information on the
impact the Australasian fungi could have on the
cycles of nutrients in the Iberian soils.
Several methods for the transformation of ec-
tomycorrhizal fungi are already available (Hanif
et al. 2002; Pardo et al. 2002). Some scientists
propose to use strains genetically manipulated to
form better symbiotic systems, including ECM
fungi which are more efficient in mobilizing
nutrients from soils. This might result in eucalypt
species that do not invade at present but become
invasive if associated with such selected (or
genetically modified) fungal strains.
Conclusions
Several conclusions can be drawn from our stud-
ies on the ectomycorrhizas of eucalypt planta-
tions in the Iberian Peninsula:
(1) In the Iberian Peninsula, a number of Aus-
tralasian ECM fungi were introduced together
with the eucalypts. The reduced number of Aus-
tralian ECM fungal species, together with the
low ability of Iberian fungi to colonize eucalypt
roots, would explain the low diversity of ECM
fungal communities in exotic stands of eucalypts.
(2) The introduction of these Australasian
ECM fungi appears to be one of the main factors
accounting for the successful establishment of
eucalypt plantations in the Iberian Peninsula,
their naturalization, and invasive behaviour.
Consequently, a deeper knowledge on the ecto-
mycorrhizas of eucalypt stands in the Iberian
plantations will help to refine our ability to pre-
dict the invasiveness of the eucalypts introduced.
The knowledge generated may be crucial for
determining potential endangerment and to sug-
gest strategies for protecting the diversity of the
Mediterranean ecosystems.
(3) It will be necessary to investigate potential
host shifts of Australian fungi to native hosts,
and their effects on the native ECM fungal com-
munities and on the functioning of Mediterra-
nean ecosystems. For these reasons, it is urgent
to investigate the ectomycorrhizas of native trees
planted in former plantation sites, and roots of
indigenous ectomycorrhizal plants growing near
eucalypt plantations.
(4) The present investigation highlights the
need to regulate the translocation of ectomycor-
rhizal fungi for forest inoculations. Quarantine
measures would be necessary to control any
future introduction of ECM fungi of Australian
origin.
(5) Before introducing beneficial Australasian
strains of ECM fungi, we would recommend
screening their ability to improve eucalypt inva-
siveness and to infect roots of native ECM
plants. Screening the invasiveness of introduced
strains will help to prevent negative effects on
Iberian natural ecosystems.
Acknowledgements
The author is indebted to A. Muriel for her help
during the fruiting body surveys, ectomycorrhi za
sampling, and for her stimulating discussions.
Special thanks are due to Dr J. Garbaye (INRA-
Nancy, France) for critical comments on an early
version of this manuscript. I sincerely thank Dr
G. Moreno for his help with the identification of
fruiting bodies of fungi. The assistance of Dr P.
Rubio (Unit of Molecular Biology, University of
Alcala
´
) with the DNA sequencing during the
molecular identification of ectomycorrhizas is
gratefully acknowledged. This research is sup-
ported by a ‘Ramo
´
n y Cajal’ researcher contract
from the MCyT (Spain) (RC2002/2091), and a
12

research grant funded by the University of Al-
cala
´
; ‘Ectomycorrhizal fungi of eucalypt planta-
tions in the Mediterranean – native or exotic
fungi?’ (Contract UAH2002/049).
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