Category: Uncategorized

symbioses
Sponges form symbiotic relationships with various organisms, including mutualisms with photosynthetic bacteria/plant cells, and swimming scallops and crabs, and parasitisms on mollusc shells.

NOTE “symbiosis” is a general term used in North America for any two organisms living together. There are several categories of symbiosis described in the scientific literature, but the one most commonly used, commensalism (one partner benefits while the other is unaffected) assumes information about the participants that is usually unavailable. The two symbioses of clearest definition (and most interest) are mutualism, where both partners benefit, and parasitism, where one partner benefits and the other is harmed.

Mutualism

Several west-coast sponges house single-celled plants known as zoochlorellae. The relationship is thought to be mutualistic in that the plants provide products of photosynthesis (glycerol, glucose, amino acids) to the host in return for a place to stay, and the host provides carbon dioxide and ammonia nutrients required for photosynthesis. A common host for zoochlorellae is the “crumb-of-bread” sponge Halichondria panicea which has two growth forms. One is a flat, tough open-coast form that often contains so many zoochlorellae that it appears greenish in colour.

For some reason, the green open-coast Halichondria often has a bad smell. Whether this has something to do with its algal symbionts, or with the presence of defensive chemicals, is not known.

The other form is often found on docks in quiet water conditions, although may occur in open-coast situations. It has a softer consistency and mostly lacks symbionts.

NOTE thought to be similar to green plant cells found in west-coast sea anemones. These are discussed elsewhere in the ODYSSEY: SEA ANEMONES: SYMBIONTS.

What is the explanation for the enhanced growth performance of the nudibranch? Select the best idea from the list below and  see explanations of the answers. Ideas from Knowlton & Highsmith 2005 J Exper Mar Biol Ecol 327: 36.

Nudibranchs eat more of the zoochlorella-containing sponge.

The symbionts increase the density of the sponge tissue; hence, providing more tissue per bite for the nudibranch.

The symbionts increase the nutritional content of the food.

 

 

Introduction

Russulaceae is one of the largest families of ectomycorrhizal fungi that plays a dominant role in many vegetations worldwide. The core of this family consists of two genera, Lactarius and Russula, both described from Europe two centuries ago as clearly recognizable agaricoid genera. However, recent research (Buyck et al., 2008) demonstrated that the traditional classification of Russulaceae into these two genera is not supported. Instead, the family consists of four mainly agaricoid genera and includes some corticioid species. In one of these genera (Multifurca), some rare representatives of the former Lactarius and Russula are united. The rest of Russula is supported as a monophyletic group. Lactarius, notwithstanding its striking feature of exuding milk or latex (thus: the milkcaps) which always seemed a good synapomorphic character, is paraphyletic and is split in two genera: Lactarius and Lactifluus.

Based on morphological and molecular characteristics, Lactifluus seems the most variable out of the four Russulaceae genera. Lactifluus mainly consists of tropical species and recent studies discovered large cryptic diversity within the genus (Stubbe et al., 2010; Van de Putte et al., 2010). Morphologically, this genus is characterized by the complete absence of zonate and viscose to glutinose caps, and it contains many species with veiled and velvety caps and all known annulate species (Buyck et al., 2008). Lactarius on the other hand, has a more temperate distribution, with many cold-adapted species and contains the typical zonate and viscid to glutinose milkcaps. Our present knowledge results in a large genus Lactarius with about 75% of the milkcaps and a smaller genus Lactifluus, but the real number of species in Lactifluus might be higher due to cryptic diversity, which seems to be absent in Lactarius. Our hypothesis is that Lactifluus evolved genetically faster but is morphologically more stable, while morphological variation is more common in the genetically slower evolving genus Lactarius. The speciation-extinction dynamics of both genera are clearly different. These discrepancies have strongly influenced the evolutionary history of both genera. 

Objectives and approach

In current phylogenies, the genus Lactifluus is strongly underrepresented. We want to meet this deficit by collecting more species of this genus. Recent biodiversity studies and expeditions to unexplored areas provided surprising new insights into the phylogeny of Lactifluus (e.g. Van de Putte et al., 2009). Previous research indicated that many valuable sites are highly endangered remnants of original forest. Some of these sites are protected and studied by the Center for Tropical Forest Science (CTFS, part of the Smithsonian Tropical Research Institute). One important benefit of CTFS woodlands is that all tree species are inventoried, so the ectomycorrhizal trees are known, which makes targeted searches possible. We plan expeditions to such selected areas, for searching phylogenetic relevant material (missing links) and to contribute to an inventory of the biodiversity. This might also contribute to the protection of these relict areas, as prioritizing habitats for conservation often relies on estimation of species richness and endemism (Bickford et al., 2007).
A phylogeny of Russulaceae, focusing strongly on Lactifluus, will be constructed based on the following nuclear and mitochondrial genes: nLSU (nuclear ribosomal large subunit 28S region), tef1 (codes for the translation elongation factor 1α), rpb1 and rpb2 (code for the largest and second largest subunit of the RNA polymerase II gene) and mSSU (mitochondrial ribosomal small subunit 16S region).

• Constructing a comprehensive phylogeny is a first step towards obtaining insight into any existing phylogenetic predispositions to certain morphological and ecological character states within these genera. In this second step we will use phylogenies to address general trends and broad historical patterns in the evolution of Russulaceae. We will collect and integrate existing data concerning distribution, morphology and ecology of the different species. Subsequently, computational techniques will be used to study the correlation of the collected data and to compare the evolution of the Lactarius and Lactifluus diversity through time. These phylogenetic comparative analyses will be carried out with the use of stochastic mapping techniques, applying Bayesian approaches. First of all we will check if, besides the L. gerardii and L. volemus species complexes, L. piperatus and L. gymnocarpoides are also cryptic species complexes with a large genetic diversity, and whether cryptic speciation is thus a widespread phenomenon in Lactifluus. The diversification consequences of particular character states will be explored by testing key-innovative hypotheses, in which stochastic models of character evolution and cladogenesis are employed, integrated into a Bayesian framework in which uncertainty in historical inferences (such as phylogenetic relationships) is allowed (Ree, 2005). With these tests we can investigate whether or not a morphological novelty or a switch in host plant family results in a different rate of speciation. We will assess directionality in character state transformations using ancestral state reconstruction methods, and we will investigate the possible correlation of characters (Schmitt et al., 2009). The outcome of these techniques can provide an idea on how the ancestor of Lactarius and Lactifluus may have looked like and how their morphological and ecological characters have evolved. Russulaceae is an ideal model group to apply these new techniques to, because these techniques require comprehensive phylogenies, which will be available for the four genera (Lactarius and Lactifluus in Ghent, Russula and Multifurca in Paris). Furthermore it is a group which apparently unites genera with a very different evolutionary history, a world-wide distribution, host plant specialists and generalists.

References

Bickford D., Lohman D.J., Sodhi N.S., Ng P.K.L., Meier R., Winker K., Ingram K.K. & Das I. (2007). Cryptic species as a window on diversity and conservation. Trends in Ecology and Evolution 22(3): 148–155.

Buyck B., Hofstetter V., Eberhardt U., Verbeken A. & Kauff F. (2008). Walking the thin line between Russula and Lactarius: the dilemma of Russula sect. Ochricompactae. Fungal Diversity 28: 15–40.

Hibbett D.S. & Matheny P.B. (2009). The relative ages of ectomycorrhizal mushrooms and their plant hosts estimated using Bayesian relaxed molecular clock analyses. BMC Biology 7: 13.

Matheny P.B., Aime M.C., Bougher N.L., Buyck B., Desjardin D.E., Horak E., Kropp B.R., Lodge D.J., Soytong K., Trappe J.M. & Hibbett D.S. (2009). Out of the Palaeotropics? Historical biogeography and diversification of the cosmopolitan ectomycorrhizal mushroom family Inocybaceae. Journal of Biogeography 36: 577–592.

Otálora M.A.G., Martínez I., Aragón G. & Molina M.C. (2010). Phylogeography and divergence date estimates of a lichen species complex with a disjunct distribution pattern. American Journal of Botany 97(2): 216–223.

Ree R.H. (2005). Detecting the historical signature of key innovations using Stochastic models of character evolution and cladogenesis. Evolution 59(2): 257–265.

Schmitt I., del Prado R., Grube M. & Lumbsch H.T. (2009). Repeated evolution of closed fruiting bodies is linked to ascoma development in the largest group of lichenized fungi (Lecanoromycetes, Ascomycota). Molecular Phylogenetics and Evolution 52: 34–44.

Stubbe D., Nuytinck J. & Verbeken A. (2010). Critical assessment of the Lactarius gerardii species complex (Russulales). Fungal Biology 114(2-3): 271–283.

Van De Putte K., De Kesel A., Nuytinck J. & Verbeken A. (2009). A new Lactarius species from Togo with an isolated position. Cryptogamie Mycologie 30(1): 1–6.

Van De Putte K., Nuytinck J., Stubbe D., Le H.T. & Verbeken M. (2010). Lactarius volemus sensu lato (Russulales) from northern Thailand: morphological and phylogenetic species concepts explored. Fungal diversity 45(1): 99–130.

Abstract

The genus Cistus comprises a group of about 20 shrub species found in wide areas throughout the whole
Mediterranean region to the Caucasus. Being one of the main constituents of the Mediterranean-type maquis, this
plant genus is peculiar in that it has developed a range of specific adaptations to resist summer drought and frequent
disturbance events, such as fire and grazing. In addition, it can form both ectomycorrhizas and arbuscular mycorrhizas. In this paper, we review the information available on the ectomycorrhizal fungi of Cistus across its entire
geographic range, as gathered and critically sifted from both published literature sources and personal observations. Although the resulting data matrix was based primarily on accounts of sporocarp inventories in the field, existing knowledge of the features of Cistus natural and synthesized ectomycorrhizas was also included and discussed. In total, more than 200 fungal species belonging to 40 genera have been reported so far to be associated with
Cistus. An analysis of the pattern of ectomycorrhizal diversity and host specificity revealed that members of the
Cortinariaceae and Russulaceae make the most of both Cistus-aspecific and Cistus-specific mycobionts. Further
studies are needed to expand our preliminary knowledge of the mycorrhizal ecology and biology of Cistus and its
fungal associates, focusing on topics such as mycobiont diversity, host specificity, fungal succession, mycorrhizal
influence on stress tolerance, and impact of disturbances, while comparing the findings with those from other
ecosystems.

Introduction

Although Europe and adjacent areas are relatively wellknown from the mycological point of view, some ecosystems have received comparatively little attention,especially concerning the role played by mycorrhizal symbiosis. In particular, given the ecological importance of host specificity for plant ectotrophic communities and the
associated mycota, studies describing the specificity patterns occurring in selected ecosystems are of premium
significance, as they can contribute to a better definition of the environmental biotic and abiotic factors that affect
specificity phenomena, and how the specialization of ectomycorrhizal fungi and plant hosts originated and
evolved (Molina et al. 1992; Erland and Taylor 2002; Van der Heijden and Sanders 2002). A significant example
of these “neglected mycorrhizal niches” is the Cistusdominated maquis. Cistus harbors a group of about 20
woody, evergreen or semi-deciduous shrub species found in wide semi-arid areas from Madeira and the Canary
Islands throughout the whole Mediterranean region to the Caucasus (Arrington and Kubitzki 2003). Some species,
such as C. carthaginensis in Spain (Boscaiu and Guemes 2001), have a very limited or even punctiform range and
are severely threatened of extinction.

Cistus species have evolved specific adaptations to resist severe summer
drought stress and to regenerate rapidly after fire, a key ecological factor influencing the evolution and dynamics of
the Mediterranean vegetation. Thanks to their ability as early colonizers after disturbance, Cistus species often
form pure stands in vast areas heavily subjected to fire and/or grazing (for an extensive bibliography on various
aspects of Cistus ecology and biology, see (http://www.cistuspage.org.uk).Cistus may form both ectomycorrhizas and vesicular arbuscular mycorrhizas, the other widespread type of mycorrhizal association (Smith and Read 1997). Although most plant species form only one type of association, the dual mycorrhizal status is not unique of Cistus, but is also present in Populus, Salix, Alnus, and Eucalyptus (Smith and Read 1997), and also in some tropical tree genera such as the myrtaceous Ixora, Syzygium, and Tristania (Reddell et al. 1996; Moyerson et al. 2001). In these hosts,
ectomycorrhizas and arbuscular mycorrhizas can either co-occur at a comparable level in the root system or one
type can be predominant, but it is not yet clear which factors favour dominance by each functional association.
In the case of Cistus and Eucalyptus, both genera inhabiting fire-susceptible ecosystems, it has been suggested that mycorrhizal plasticity may represent an important adaptive trait to the cyclical pattern of accumulation and loss of organic resources due to fire (Smith and Read 1997). At variance with the cistaceous host genus Helianthemum (Yu et al. 2001), no reports of ectendomycorrhizas formation by Cistus species are known to date.To expand current knowledge of mycorrhizal biology of Cistus, a new research program at our institution is focusing on the isolation and full characterization of the ectomycorrhizas formed by the fungal symbionts exclusively or prevalently associated with Cistus spp. (Nuytinck et al. 2004; Rinaldi and Comandini, unpublished observations). While screening published data on Cistus mycorrhizas to select suitable candidates for ectomycorrhizal characterization, we were struck by the fact that information on Cistus-associated mycota is generally widely dispersed in the mycological literature, and no recent
comprehensive accounts exist on the topic. In the present review paper, we attempt to fill this gap by providing an
updated checklist of fungal species reported to establish ectomycorrhizas on Cistus spp. on the basis of field
observations. The cases where the association has been confirmed experimentally, either through direct observation of naturally occurring or synthesized mycorrhizas, and by molecular approaches, have been highlighted. We also
attempt to discuss the ecological significance of Cistus mycorrhizas in relation to what is known on the role played
by ectomycorrhizal diversity in other better-studied ecosystems. Finally, we indicate those we believe are the
main research needs to fully disclose Cistus mycorrhizal ecology.

When more than one host genus was mentioned in the original reference, for example “under Quercus and
Cistus”, or “in Quercus stands with Cistus understory”, the relevant fungal species was generally not considered as
a Cistus symbiont, unless this particular association was confirmed by other sources. When more than one Cistus
species was present, the relevant mycobiont(s) was assigned to all potential hosts. Evidence from studies on
the morpho-anatomical and molecular characterization of ectomycorrhizas formed by taxonomically diverse fungal
species on Cistus spp. were also inserted in the data set, as they support hypotheses from field observations. In
addition to studies concerning naturally-occurring, fieldcollected mycorrhizas, data coming from synthesized
mycorrhizas were also considered.

Despite all efforts to cover as large a number of bibliographic sources as possible, our literature survey
was clearly partial and incomplete, and a number of valuable records may have been missed. However, we are
confident that the assembled data matrix includes the majority of the ecologically relevant information available
on Cistus-associated fungal species, especially when Cistus-specific mycobionts are concerned. As the vast
majority of the data set consists of field observations of sporocarps rather than associations confirmed by direct
inspection of ectomycorrhizas, the limitations inherent to data sources must be emphasized. Indeed, the reports of
putative mycorrhizal relationships based solely on sporocarp associations are obviously subject to an unquantifiable degree of error. Trappe (1962) has explained the criteria that should lead to the exclusion or inclusion of
literature reports when compiling lists of mycorrhizal associations based on sporocarp observations. In compiling
the data set for this study, we strictly observed these guidelines and, also on the basis of our personal experience, discarded or labeled all spurious and/or dubious records. As regards synthesized ectomycorrhizas, it should
be stressed that associations induced in laboratory experiments may not occur under field conditions (Harley and
Smith 1983; Molina et al. 1992). Finally, the identification of some fungi in the references we have considered may
not be correct.

Collecting the data

Data on the association between Cistus spp. and ectomycorrhiza-forming fungi presented in this paper are overwhelmingly based on reports of field observations of sporocarp associations with potential hosts. The data set
contains both information collated from a variety of published sources, including taxonomic monographs of
specific groups of ectomycorrhizal fungal genera and the few previous surveys of fungi associated with Cistus, and
personal collections and observations. Fungal taxa belonging to genera for which the mycorrhizal status is currently
uncertain were not listed. Only records clearly mentioning (potential) Cistus hosts were included in the data matrix

crispus, incanus, ladanifer (often mentioned under the synonym ladaniferus), laurifolius, monspeliensis, populifolius, salvifolius] are explicitly reported in the literature to form specific associations. Most fungal records are referred to common host species occurring over large geographic areas (C. incanus, C. monspeliensis, C.salvifolius), and few records are available for rare or uncommon species such as C. clusii, C. crispus, and C. populifolius.

In most cases, the potential host species was not indicated in the original reference, probably due to the
rather homogeneous morphology of the species within the genus Cistus, with subsequent identification problems for
non-specialists, and to the common occurrence of several Cistus species growing tightly together in a stand, whicmade it difficult, if not impossible, to specify the plant partner in a number of instances. Geographically, the
largest number of records were originally collected in Spain, followed by peninsular and insular Italy and
southern France, and a few in Morocco. Virtually no information is available for other extended regions in North
Africa (e.g., Tunisia and Algeria) and for the eastern side of the Cistus range, i.e., the Balkans, the Aegean Archipelago,
the coasts of Anatolia, the Caucasus, Syria, Lebanon. In Israel, studies on the mycorrhizal status of naturally
occurring C. incanus and C. villosus have been conducted(Litav 1965; Berliner et al. 1986), although the involved
fungal taxa were not identified.

In general, all the larger and ecologically key ectomycorrhizal fungal genera harbor a significant number of
species that are associated with Cistus. Cortinarius (29 species) and Russula (28) are the best represented in the
list, followed by Inocybe (23), Amanita (22), Hygrophorus(13), Lactarius (12, excluding the probable spurious report
of L. deliciosus, see below), Hebeloma (12), Boletus (10),Tuber (10, excluding T. californicum), Tricholoma (10). At
the family level, Cortinariaceae (for a comment on the concept of families in the Agaricales, see notes in Table 1)
and Russulaceae, clearly form the prevalent groups. The association of Cistus with numerous hypogeous ascomycetes (Table 1) seems to be a common feature of the Cistaceae as a whole, as other genera, such as Helianthemum, also show similar mycorrhizal preferences (e.g., Malloch and Thorn 1985).

“Broad host range” fungi, which can form mycorrhizal with host plants from diverse plant families, orders, and
even classes (Molina et al. 1992), occur frequently in the list, and include Amanita vaginata, Hebeloma crustuliniforme, Paxillus involutus, Telephora terrestris, and Tricholoma sulphureum. Many other listed fungal species
are more frequently associated with Quercus spp., and their association with Cistus, when reliably identified, is
probably sporadic and/or occasional. These include, for example, several of the Lactarius species reported here,
such as L. atlanticus, L. chrysorrheus, L. ilicis, L. mairei,and L. zonarius (Basso 1999). However, these records are
important because they provide valuable information concerning the mycorrhizal ecology of selected fungal
species, revealing their tendency to switch hosts when

Diversity and host specificity of Cistus ectomycorrhizal fungi

Current knowledge about Cistus ectomycorrhizal fungal diversity is based mostly on above-ground observations of
fungal fruitbodies (Table 1). About 230 fungal species belonging to 40 genera are listed, belonging to both
Ascomycota and Basidiomycota, a number of Cistus associates considerably larger than previous accounts
(Malloch and Thorn 1985; Lavorato 1991; Ballero et al. 1992; Vila and Llimona 1999, 2002). Early studies
reviewing ectomycorrhizal fungi and relevant plant hosts overlooked Cistus and its mycoflora (e.g., Trappe 1962),
partly due to a focus on forests rather than shrublands. On the plant side, nine different Cistus species [albidus, clusii,

these share the same environment—a feature that has certainly played a major role in the evolution of host
specificity among ectomycorrhizal fungi—and broaden our perspective of Cistus-compatible fungi.
The analysis of the assemblage of the fungal taxa more closely linked to Cistus also reveals an interesting pattern.
Two different groups of mycorrhizal fungi can be discerned here. The first comprises “narrow host range” fungi, i.e.,
fungal species forming mycorrhizas only in association with a single plant genus (Molina et al. 1992), Cistus in this
case. About 35 Cistus-specific fungi are identifiable to date, all of them being epigeous basidiomycetes. These
include Leccinum corsicum, Amanita cistetorum, Hebeloma album, H. cavipes, H. cistophilum, H. erumpens,
Inocybe cistobulbipes, I. rocabrunae, Hygrophorus pseudodiscoideus var. cistophilus, Lactarius cistophilus, L.
cyanopus, L. tesquorum, Russula cistoadelpha, R. monspeliensis, R. tyrrhenica, and a number of Cortinarius
species. Again, a few genera within the Cortinariaceae and,on a minor scale, Russulaceae, seem to comprise almost all
the fungal species highly specialized to Cistus. A second group has those fungal species which although not
associated exclusively with Cistus, commonly establishfunctional symbiosis with this host, and are often a
component of the macromycetous fungal flora in Cistusdominated plant communities. Tuber melanosporum, Amanita muscaria, Hebeloma hiemale, Laccaria laccata fo.pseudobicolor, and L. proxima belong to this group.
Among the macromycetes putatively linked to Cistus spp., it is apparent that some of the claimed associations are
very unlikely. This is the case, for example, of the related Suillus collinitus and Rhizopogon spp., both genera being
currently accepted as specific to conifers (almost exclusively Pinaceae) (Kretzer et al. 1996; Molina et al. 1999).
Also Amphinema byssoides has been reported as being restricted to coniferous tree species (Erland and Taylor
1999). Another clear example of a most likely spurious report is that of the common Pinus-associated Lactarius
deliciosus from Greece (Zervakis et al. 1998) and Spain (Ortega and Esteve-Raventós 1999). It is, thus, possible
that some fungi in the list are in reality not compatible with Cistus, but rather with other hosts present in the same
community, most frequently Quercus spp. (see above) and Pinus spp. The absence of Cenococcum in Table 1 is also
noticeable, as this symbiont is almost ubiquitous on the roots of most ectomycorrhizal hosts in a range of ecological
situations (LoBuglio 1999). Given the little attention devoted so far to the observation of Cistus ectomycorrhizal
morphotypes (see below), however, it cannot be excluded that Cenococcum forms mycorrhizas with Cistus.
In spite of the fairly large number of ectomycorrhizal macromycetes linked to Cistus spp. in Mediterranean-type
ecosystems, information on the features of relevant mycorrhizas is remarkably limited. To date, only very
few accounts of Cistus ectomycorrhizal types exist in the literature. Most studies focused on the ectomycorrhizas
formed by hypogeous ascomycetes, such as Tuber and Terfezia with Cistus spp., both under natural and cultured
conditions. These investigations resulted in the full

characterization of the ectomycorrhizas formed by Tuber spp. on C. incanus (Fontana and Giovannetti 1979;
Giovannetti and Fontana 1982), and in several other preliminary and/or not exhaustive descriptions of mycorrhizal morpho-anatomical details (Chevalier et al. 1975; Fusconi 1983; Leduc et al. 1986; Wenkart et al. 2001;
Roth-Bejerano et al. 2003). For basidiomycetes, descriptions of the mycorrhizas formed by L. laccata and Boletus
rhodoxanthus on C. ladanifer (Torres et al. 1995; Hahn 2001), by Hebeloma sacchariolens on C. salvifolius
(Rosell 1981) and, more recently, by Lactarius tesquorum on Cistus sp. (Nuytinck et al. 2004), are available.
Common host-dependent features of all the ectomycorrhizal types described so far on Cistus spp. are (for the
specific terminology used to characterize ectomycorrhizae, see Agerer 1986, 1987–1998, 1991): simple or monopodial-pinnate ramification systems; small diameter of ectomycorrhizal tips; thin mantle thickness; cortical cells
generally present in two rows, tangentially rectangular, either radially rectangular orientated or square to radially
rectangular orientated; Hartig net generally uniseriate, surrounding one–two rows of cortical cells and rarely
reaching the endodermis (“cortical Hartig net”, for a description of this peculiar structure, see Smith and Read
1997).

In addition to the ectomycorrhizal types mentioned above, very recent observations of Cistus roots excavated
in Sardinia (Italy) have led to the isolation of ectomycorrhizas of Lactarius cistophilus and of a morphotype
unequivocally formed by a sebacinoid mycobiont (Rinaldi and Comandini, unpublished observations). The latter
finding adds to the increasing evidence that members of the Sebacinaceae, a family assigned to the heterobasidiomycetous order Auriculariales, are common symbionts in various ectomycorrhizal communities. Typical ectomycorrhizas formed by these fungi have been recently detected by both molecular and morphological analyses on several temperate deciduous and coniferous trees, including Carpinus, Corylus, Fagus, Tilia, Picea, and Abies (Selosse
et al. 2002; Urban et al. 2003; Comandini, unpublished observations), and also in Australian Eucalyptus Mediterranean-type forests (Glen et al. 2002).

In recent years, the application of a range of molecular tools has greatly enhanced our knowledge of ectomycorrhizal communities, expanding well beyond classic morphotyping our possibilities to track and identify ectomycorrhizas and to compare the above- and belowground mycorrhizal fungal components of ecosystems (Horton and Bruns 2001). Unfortunately, this “revolution” has only marginally impacted research on Cistus ectomycorrhizas. To date, indeed, only the mycorrhizas of Lactarius tesquorum on Cistus sp. have been fully characterized from both a molecular and morphological point of view (Nuytinck et al. 2004). Clearly, the confirmation of many putative Cistus mycobionts and the unambiguous identification of their mycorrhizas await a more general recourse to molecular methods.

Comparing ectomycorrhizal diversity in different ecosystems

In addition to forming extensive patches of pure shrublands, Cistus is also a significant presence in other
vegetation communities. These communities include the mixed Mediterranean maquis or garrigue, where Cistus
occurs together with other sclerophyllous (not-ectomycorrhizal) scrubs, such as Olea, Phyllirea, Pistacia, Erica,
Arbutus, and some Quercus and Pinus-dominated communities, where Cistus is an element of the undercanopy
vegetation and colonizes clearings and open areas. The data gathered in this study indicate that the number of
ectomycorrhizal fungal species associated with Cistus is significant, and support the importance of this symbiosis in
these Mediterranean ecosystems. On the other hand, when compared to other better-known host plants such as
Pseudotsuga menziesii, which associates with some 2000 fungal species in North America (Trappe 1977), the
ectomycorrhizal diversity of Cistus might look rather poor.However, it should be stressed that comparing lists of
fungal associates drafted by different authors might be biased to some extent by personal choices for inclusion or
exclusion of those fungal genera for which the mycorrhizal status is uncertain or not fully confirmed. Focusing on the
information available for other better-studied Mediterranean-type ecosystems, some 660 fungal associates, many
of which are endemic, have been recorded for Eucalyptus in natural environments of Australia (Castellano and
Bougher 1994; Bougher 1995). Eucalyptus is also believed to have the potential to associate with the richest flora of
host genus-specific ectomycorrhizal fungi in the world (Molina et al. 1992), while diversity in its plantations is
considerably lower in both Australian and exotic areas (Lu et al. 1998a,b; Giachini et al. 2000) (an extensive account
of Eucalyptus ectomycorrhizal fungi can be found at http://www.ffp.csiro.au/research/mycorrhiza/eucfungi.html). Eucalyptus and Cistus share a small contingent of associated fungi, included in the genera Pisolithus, Scleroderma, and Laccaria, with the characteristics of “early stage” species.These mycobionts appear in the early phases of fungal
successions and represent, in some cases, pioneer fungi,being often typical of disturbed habitats with young trees
and shrubs, or may occur even in freshly cleared areas and often associate with a diversity of hosts. Some Hebeloma
listed in Table 1 are also well known early stage fungi.

What needs to be done

Our knowledge of Cistus mycorrhizal ecology is too limited to draw any simple conclusions for this host plant
and its associated fungi as for many key aspects mentioned above. However, information acquired from studies of
other ecosystems may help to highlight priority areas for future research. In particular, the pattern and role of host
specificity, possibly for each fungal species, in Cistus ectomycorrhizal communities should be investigated, as
the importance of host specificity in other ectomycorrhizal communities has been underlined (Bruns et al. 2002).

Examining the plant-fungal associations for Cistus would also serve to elucidate mycobiont–host distribution and
mycobiont–host species relationships (Newton and Haigh 1998), to see if they have a general validity or, and
eventually how, they change in specialized environments.Many of the Cistus-specific symbionts listed here, e.g.,
Cortinarius spp. and Inocybe spp., have been identified only very recently, which highlights the need for in-depth
taxonomical surveys of these and other fungal genera in Cistus maquis over the entire host geographic range.
Belowground ectomycorrhizal diversity in Cistus natural communities should also be explored to see if it reflects the
Cortinariaceae and Russulaceae dominance detected aboveground, and the morphological and molecular
characterization of the prevailing ectomycorrhizal types should be performed.

Work aimed at confirming the identity of putative mycobionts and extending their number would certainly be
easier if carried out in pure Cistus stands, rather than in areas where it grows mixed with other ectomycorrhizal
hosts. However, the exploration of diversity and biology of Cistus mycorrhizas in mixed plant communities could
provide key information on the structure of both the plant and fungal components of these communities in Mediterranean-type ecosystems. Doubtlessly, molecular tools will play a major role in tackling these complex issues. As data on the succession of mycorrhizal fungi in Cistus ecosystems are totally lacking, community level studies should
also be conducted to address this important aspect, as well as the impact of mycorrhizal colonization upon the fitness
of Cistus and its resilience to natural and anthropogenic disturbances, such as strong water limitation, fire, overcutting, and grazing.

Considering the ecological niches occupied by Cistus and the intermediate position of this host genus in the
vegetation series leading to evergreen Quercus or Pinus climax forests on one side, and to impoverished pastures
and/or desertified lands on the other, a deeper knowledge of Cistus ectomycorrhizal fungal communities may well
prove to be of wider significance and to contribute to understand the role and dynamics of mycorrhizas in inherently unstable ecosystems, especially if integrated into broader ecological investigations. These studies may also provide valuable tools to help shape future programs of protection and management of natural resources in vast
areas across the Mediterranean basin. In general, Cistus holds the potential to develop into an alternative model to
assess the role of mycorrhizal symbiosis in ecosystem functioning of Mediterranean-climate shrublands throughout the world. Clearly, we are just at the beginning of this research journey.

 Introduction

In the Federal Register of August 13, 1998 (63 FR 43402) (FDA 1998), the FDA announced the availability of a new document entitled “Accidental Radioactive Contamination of Human Food and Animal Feeds: Recommendations to State and Local Agencies” (hereinafter 1998 FDA document) replacing an earlier 1982 FDA document (FDA 1982). The 1998 FDA document provides guidance to State and local agencies to aid in emergency response planning and execution of protective actions associated with the production, processing, distribution, and use of human food and animal feeds accidentally contaminated, e.g., due to a nuclear power plant accident, with radioactive elements called radionuclides. In addition, the 1998 FDA document provides broader and more current information regarding radionuclides in food than the 1982 FDA document.

FDA has adopted guidance levels for radionuclide activity concentration established in the 1998 FDA document in a Compliance Policy Guide (CPG) entitled “Guidance Levels for Radionuclides in Domestic and Imported Foods.” The CPG rescinds and replaces guidance levels for radionuclide activity concentration in food offered for import established in current CPG Sec. 560.750 Radionuclides in Imported Foods — Levels of Concern (CPG 7119.14) with guidance levels established in the 1998 FDA document. FDA has also adopted these same guidance levels for radionuclide activity concentration for domestic food in interstate commerce. Additionally, the FDA has expanded its policy from food accidentally contaminated with radionuclides to food accidentally or intentionally contaminated with radionuclides. The 1998 FDA document remains unaffected by the issuance of the CPG.

FDA uses guidance levels for radionuclide activity concentration in food to help determine whether domestic food in interstate commerce or food offered for import into the United States presents a safety concern. The purpose of this supporting document is to present information taken from the 1998 FDA document that explains the basis for the guidance levels in the CPG and how these guidance levels differ from guidance levels established in 1986, which have been superseded.

Protective Action Guides

The calculation of guidance levels for radionuclide activity concentration in food depends in part on Protective Action Guides (PAGs). PAGs are radiation dose levels to an individual at which protective action should be considered to limit the radiation dose to that individual. PAGs were previously defined in the1982 FDA document as “projected dose commitment values to individuals in the general population that warrant protective action following a release of radioactive material.” In this context, the phrase “dose commitment” refers to the radiation dose received by an individual. The PAGs contained in the 1982 FDA document were developed from the prevailing scientific understanding of the relative risks associated with radiation as described in the 1960 and 1961 reports of the Federal Radiation Council (FRC 1960, 1961). The 1982 FDA guidance document established two levels for PAGs. The lower level, called the Preventative PAG, was a projected dose commitment of 5 mSv to the whole body, active bone marrow, or any other organ except the thyroid, or a projected dose commitment of 15 mSv to the thyroid. The upper level, called the Emergency PAG, was a projected dose commitment of 50 mSv to the whole body, active bone marrow, or any other organ except the thyroid, or a projected dose commitment of 150 mSv to the thyroid.

The 1998 FDA document redefined a PAG as the “committed effective dose equivalent⁽¹⁾ or committed dose equivalent⁽²⁾ to an individual organ or tissue that warrants protective action following a release of radionuclides.” The 1998 FDA document replaced the Preventative and Emergency PAGs with one set of PAGs for the ingestion pathway. The PAGs established in the 1998 FDA document are 5 mSv for committed effective dose equivalent or 50 mSv committed dose equivalent to an individual tissue or organ, whichever is more limiting (i.e., the most limiting PAG results in the lowest level of radionuclide activity concentration in food).

For 5 mSv committed effective dose equivalent (the PAG adopted in the new CPG), the associated lifetime total cancer mortality would be 2.25 x 10^-4 or approximately 1 in 4400. For comparison, the estimate of the normal lifetime total cancer mortality in the United States for the general population, not associated with additional radiation dose from ingestion of food contaminated with radionuclides, is 0.19 or approximately 1 in 5 (CIRRPC 1992). Thus, in a general population of 10,000 individuals, the number of cancer deaths over the lifetime of the individuals would be 1990; if each received a committed effective dose equivalent of 5 mSv, the number of cancer deaths over the lifetime of the individuals could increase in theory by about 2, for a total of 1992 cancer deaths.

The numerical estimate of cancer deaths presented above for the recommended PAG of 5 mSv was obtained by the practice of linear extrapolation from the nominal risk estimate for lifetime total cancer mortality for the general population at 100 mSv dose equivalent in the whole body.⁽³⁾ Other methods of extrapolation to the low-dose region could yield higher or lower numerical estimates of cancer deaths. Studies of human populations exposed at low doses are inadequate to demonstrate the actual magnitude of risk. There is scientific uncertainty about cancer risk in the low-dose region below the range of epidemiological observation, and the possibility of no risk cannot be excluded (CIRRPC 1992).

The term “PAG” is used by FDA and other Federal and state agencies. International organizations use the term “intervention level of dose” (ICRP 1984b). The PAGs established in the 1998 FDA document and adopted in this supporting document are the same as the intervention levels of dose used by international organizations. The 1998 FDA guidance and this supporting document retain use of the term “PAG” for consistency with other U.S. Federal and state agencies.

FDA’s Guidance Levels for Radionuclide Activity Concentrations in Food Established in 1986

Following the Chernobyl nuclear accident in 1986, FDA that same year issued CPG Sec. 560.750 Radionuclides in Imported Foods — Levels of Concern (CPG 7119.14) which established guidance levels referred to as Levels of Concern (LOCs) for radionuclide activity concentration in food offered for import.

The LOCs in CPG 7119.14 were derived from the Preventative PAGs established in the 1982 FDA guidance document and were based on the following assumptions: 1) the entire intake of food would be contaminated, 2) Iodine-131 would be a major source of radiation dose for only 60 days following the accident, and 3) Cesium-134 + Cesium-137 could be a major source of radiation dose for up to one year. The LOCs provided such a large margin of safety that derivation of LOCs for other radionuclides, judged to be of less health significance, was considered unnecessary. The LOCs in CPG 7119.14, established in 1986, are given in Table 1.

FDA’s 1998 Guidance Levels for Radionuclide Activity Concentration in Food Adopted in the CPG

The guidance levels for radionuclide activity concentration in food in the CPG are referred to in the 1998 document as “Derived Intervention Levels” or DILs. DILs are used by scientists internationally to describe the radionuclide activity concentrations at which introduction of protective measures should be considered. The term DILs as used in the CPG replaces the term LOCs used in CPG 7119.14 and allows for consistency in scientific terminology between the CPG and the internationally accepted scientific term. Efforts by international organizations to develop DILs have been extensive. Derivations have been based on consensus values for the intervention levels of dose, called PAGs by FDA, and have been used to establish guidance levels for radionuclides in foods within individual countries and in international trade. In general, food with concentrations below the DILs is permitted to move in international trade without restriction. Food with concentrations at or above the DILs is not normally permitted into international trade.⁽⁴⁾

By definition, a DIL corresponds to the radionuclide activity concentration in food present throughout the relevant period of time that, in the absence of any intervention, could lead to an individual receiving a radiation dose equal to the PAG. The equation given below is the formula that the agency used for calculating recommended DILs.

DILs (Bq/kg) = [PAG (mSv)] / [f x FI (kg) x DC (mSv/Bq)]

Where:

DC = Dose Coefficient; the radiation dose received per unit of radionuclide activity ingested (mSv/Bq)

f = Fraction of the food intake assumed to be contaminated

FI = Food Intake; the quantity of food consumed in an appropriate period of time (kg)

Guidance levels or LOCs contained in CPG 7119.14 addressed only I-131, Cs-134 and Cs-137 because these radionuclides were known at that time to be the principal radionuclides that contribute to radiation dose by ingestion following a nuclear reactor accident. Information gained following the Chernobyl accident determined that Ru-103 and Ru-106 could also contribute to radiation dose and, therefore, these radionuclides were included in the 1998 FDA document. In addition, other radionuclides were included in the 1998 FDA document to address other radiological emergencies where there is a possibility of accidental radioactive contamination of food. This approach provides the flexibility necessary to respond to special circumstances that may be unique to a particular accident. The types of accidents and the principle radionuclides for which DILs were developed are:

• Nuclear reactors (I-131; Cs-134 + Cs-137; Ru-103 + Ru-106)
• Nuclear fuel processing plants (St-90; Cs-137; Pu-238 + Pu-239 + Am-241)
• Nuclear waste storage facilities (Sr-90; Cs-137; Pu-238 + Pu-239 + Am-241)
• Nuclear weapons (i.e., dispersal of nuclear weapon material without nuclear detonation) (Pu-239), and
• Radioisotope thermoelectric generators and radioisotope heater units are used in space vehicles (Pu-238).

The DILs are for radionuclides expected to deliver the major portion of the radiation dose from ingestion during the first year following an accidental episode of radiological food contamination. If there is concern that food will continue to be significantly contaminated beyond the first year, the long-term circumstances need to be evaluated to determine whether the recommended DILs would be appropriate or if other guidance is more applicable.

Detailed information on derivation of DILs is presented in the appendix. The DILs are based upon calculations for nine radionuclides expected to be the predominant contributors to radiation dose through ingestion (Sr-90, I-131, Cs-134, Cs-137, Ru-103, Ru-106, Pu-238, Pu-239, and Am-241). For each radionuclide, DILs were calculated for six age groups using PAGs, dose coefficients relevant to each radionuclide and age group, and dietary intakes relevant to each age group. The age groups include 3 months, 1 year, 5 years, 10 years, 15 years and adult (>17 years). The dose coefficients were adopted by FDA from the International Commission on Radiological Protection Publication 56 (ICRP 1989). The dietary intakes were derived from a 1984 EPA report which presented average daily food intake by age and gender (EPA 1984a, EPA 1984b).

The nine radionuclides listed above comprise five radionuclide groups, each having common characteristics. The five groups are: Strontium-90; Iodine-131; Cesium-134 + Cesium-137; Ruthenium-103 + Ruthenium-106; and Plutonium-238 + Plutonium-239 + Americium-241. An accident could involve more than one of the five groups. A single DIL for each radionuclide group was chosen based on the most limiting PAG and age group for the radionuclide group (i.e., the most limiting PAG and age group result in the lowest DIL). These five DILs are the ones incorporated into the new CPG.

The calculations underlying the DILs are based on the entire diet for each age group, not for individual foods or food groups. Unlike the previous LOCs that assumed 100 percent radionuclide contamination of the diet, DILs assume ten percent radionuclide contamination of the diet which is then multiplied by a factor of three. Use of ten percent of the dietary intake as the portion contaminated is consistent with recommendations made by a group of experts to the Commission of the European Communities (CEC 1986b) and by the Nuclear Energy Agency (NEA) of the Organization for Economic Cooperation and Development (NEA 1989). FDA applied an additional factor of three to account for limited sub-populations that might be more dependent on specific food supplies. Therefore, a value of thirty percent is the fraction of food intake that FDA presumed to be contaminated. For infants, (i.e., the 3-months and 1-year age groups) DILs were calculated assuming 100 percent radionuclide contamination of the infant diet.

With one exception (LOCs for I-131 in non-infant food), guidance levels or DILs for radionuclides established in the 1998 FDA document that FDA has adopted in the CPG are higher than guidance levels or LOCs for those same radionuclides contained in the CPG 7119.14. In deriving guidance levels or DILs contained in the 1998 FDA document, FDA employed updated international consensus values for intervention levels of dose (called PAGs by FDA) as well as updated dose coefficients and food intake estimates. In addition, information gained by FDA and others following the Chernobyl accident determined that the amount of food affected by an accident would be significantly lower than the level originally estimated. For this reason, DILs contained in the 1998 FDA document assume thirty percent of the dietary intake would be contaminated after a nuclear accident, compared to the 100 percent assumption of contamination employed in deriving LOCs. FDA’s decision to reduce the assumption for dietary intake contamination from 100 percent to thirty percent is the main reason that the guidance levels established in the 1998 FDA document and adopted in the CPG are higher than the guidance levels contained in CPG 7119.14.

The 1998 FDA document established guidance levels only for food accidentally contaminated with radionuclides in domestic interstate commerce. In the CPG, FDA has adopted those same guidance levels for food either accidentally or intentionally contaminated with radionuclides, regardless of whether that food is in domestic interstate commerce or offered for import. FDA has taken this action because radionuclides that could be implicated in an event involving the accidental contamination of food could also be implicated in an event involving the intentional contamination of food. The radionuclides addressed in the 1998 FDA document are widely used and available. Furthermore, an incident resulting in release of radionuclides from one of the nuclear facilities addressed in the 1998 FDA document would likely result in the release of the same radionuclides regardless of whether the cause of the release was accidental or intentional. FDA has therefore concluded that the assumptions used in establishing the DILs in the 1998 FDA document and adopted in the CPG are appropriate for both accidental as well as intentional radionuclide contamination of food.

The DILs established in the 1998 FDA document and contained in the CPG for food offered for import and food in domestic interstate commerce are given in Table 2.

Appendix — Derivation of Recommended Derived Intervention Levels

The Derived Intervention Level (DIL) for a specific radionuclide is calculated as follows:

DILs (Bq/kg) = [PAG (mSv)] / [f x FI (kg) x DC (mSv/Bq)]

Where:

DC = Dose Coefficient; the radiation dose received per unit of radionuclide activity ingested (mSv/Bq)

f = Fraction of the food intake assumed to be contaminated

FI = Food Intake; the quantity of food consumed in an appropriate period of time (kg)

The Protective Action Guides (PAGs) used are 5 mSv committed effective dose equivalent, or 50 mSv committed dose equivalent to individual tissues and organs, whichever is more limiting.

Dose coefficients (DCs) are given in Table 3 and food intakes are given in Tables 4 and 5. The fraction of food intake assumed to be contaminated (f) equals 0.3, except for I-131 in infant diets where f equals 1.0.

Radionuclides

Based upon data on radionuclides in human food following the Chernobyl accident, DILs for I-131, Cs-134, Cs-137, Ru-103 and Ru-106 would apply following incidents involving nuclear reactors. For incidents at nuclear fuel reprocessing facilities and nuclear waste storage facilities, DILs for Sr-90, Cs-137, Pu-239, and Am-241 would apply. For nuclear weapons incidents and incidents involving radioisotope thermal generators (RTGs) and radioisotope heater units (RHUs) used in space vehicles, DILs for Pu-239 and Pu-238, respectively, would apply. The selection of these radionuclides as the major contributors to radiation dose through ingestion is consistent with recommendations on DILs published by NEA, WHO, CODEX, and CEC (NEA 1989, WHO 1988, CODEX 1989, CEC 1989a, IAEA 1994).

Age Groups and Dose Coefficients (DCs)

The general population was divided into six age groups ranging from infants to adults and corresponding to the age groups in ICRP Publication 56 (ICRP 1989) for which ICRP has published DCs. The age groups are 3 months, 1 year, 5 years, 10 years, 15 years, and adult. The radionuclides, age groups and dose coefficients used in the calculations are presented in Table 3.

 Food Intake

Food intake included all dietary components including tap water used for drinking, and is the overall quantity consumed in one year, with exceptions in the period of time for I-131 (T_1/2 = 8.04 days) and Ru-103 (T_1/2 = 39.3 days). For these radionuclides, the quantities consumed were for a 60-day period and a 280-day period, respectively, due to the more rapid decay of these radionuclides. The intake periods for I-131 and Ru-103 are the nearest whole number of days for decay of these radionuclides to less than 1% of the initial activities.

Dietary intakes were derived from a 1984 EPA report which presented average daily food intake by age and gender (EPA 1984a, EPA 1984b). The EPA intakes were based on data from the 1977-1978 Nationwide Food Consumption Survey published by the U. S. Department of Agriculture (USDA 1982, USDA 1983). The age groups and annual dietary intakes for various food classes and the total, calculated from data in the EPA report, are given in Table 4. The dietary intakes derived for the ICRP age groups for which DCs are available, using the results in Table 4, are presented in Table 5.

 Fractions of Food Intake Assumed to be Contaminated

For food consumed by most members of the general public, ten percent of the dietary intakes was assumed to be contaminated. This assumption recognizes the ready availability of uncontaminated food from unaffected areas of the United States or through importation from other countries, and also that many factors could reduce or eliminate contamination of local food by the time it reaches the market.

Use of ten percent of the dietary intake as the portion contaminated was consistent with recommendations made by a Group of Experts to the Commission of the European Communities (CEC 1986a) and by the Nuclear Energy Agency (NEA) of the Organization for Economic Cooperation and Development (NEA 1989). The NEA noted that modification of this value would be appropriate if justified by detailed local findings.

FDA applied an additional factor of three to account for the fact that sub-populations might be more dependent on local food supplies. Therefore, during the immediate period after a nuclear accident, a value of 0.3 (i.e., thirty percent) is the fraction of food intake presumed to be contaminated. If, subsequently, there is convincing local information that the actual fraction of food intake that is contaminated (f) is considerably higher or lower, there will be adequate time to determine whether to adjust the value of f (and therefore adjust the values of the DILs) for the affected area.

For infants, (i.e., the 3-months and 1-year age groups) the diet consists of a high percentage of milk and the entire milk intake of some infants over a short period of time might come from supplies directly impacted by an accident. Therefore, f was set equal to 1.0 (100%) for the infant diet.

Selection of Recommended Derived Intervention Levels

DILs are presented in Table 6 for Sr-90, I-131, Cs-134, Cs-137, Ru-103, Ru-106, Pu-238, Pu-239, and Am-241 for six population age groups and applicable PAGs. Two criteria were used in selecting a single DIL for each radionuclide for inclusion in the new CPG.

First, the most limiting DIL for either of the applicable PAGs was selected for each of the nine radionuclides. These DILs are presented in Table 7 for each of the six age groups. In addition, the average DIL is presented for the radionuclide group Pu + Am, composed of Pu-238, Pu-239, and Am-241, and the radionuclide group Cs, composed of Cs-134 + Cs-137. The three radionuclides in the Pu + Am group deposit on the bone surface and are alpha-particle emitters. The radionuclides in the Cs group are deposited throughout the body and are beta-particle and gamma-ray emitters. The average values are used for these groups because the calculated DILs for radionuclides in each group are similar.

The radionuclides Ru-103 and Ru-106 are chemically identical, are deposited throughout the body, and are beta-particle and gamma-ray emitters. However, their widely differing half lives (i.e., 39.3 days and 373 days, respectively) result in markedly differing individual DILs which do not permit simple averaging. Instead, the concentrations of Ru-103 (C3) and Ru-106 (C6) are divided by their respective DILs and are then summed. The DIL for the Ruthenium group is set at less than one.

Therefore, [(C3) / (DIL3)] + [(C6) / (DIL6)] < 1.0   (equation 1)

This assures that the sum of the separate radiation dose contributions from the Ru-103 and Ru-106 concentrations will be less than that contemplated by the Protective Action Guide during the first year after an accident.

Second, there are dietary components which are common to all six age groups. A principal example is fresh milk, for which the consumer of particular supplies cannot be identified in advance. Therefore, the most limiting DIL for all age groups in Table 7, for each radionuclide or radionuclide group, was selected and is applicable to all components of the diet.

These DILs are presented in Table 8 and were rounded to two significant figures (one significant figure for the Pu + Am group). These are the DILs adopted in the new CPG. The DILs in Table 8 apply independently to each radionuclide or radionuclide group, because they apply to different types of incidents, or in the case of a nuclear reactor incident, to different limiting age groups. However, the DILs for Ru-103 and Ru-106 are used in equation 1 to evaluate that criterion for the radionuclide group Ru-103 + Ru-106. The DILs in Table 8 are given in Table 2 in the main text.

 

 

 

 

Introduction: Phosphate as an Essential Mineral

“The story of phosphorus is a long, fascinating one. But we are here interested primarily in knowing about its role in agriculture,” wrote Vincent Sauchelli in “Manual on Phosphates in Agriculture” published in 1942.

“Therefore in order to start at the beginning or our story we shall have to go back to the year 1840 – the year when Justus von Liebig, a German scientist, made an historical address before the British Association of Science in which he for the first time gave a clear, intelligent exposition of the role of minerals in plant growth and laid the ground work for modern agricultural science. He was the first to show that insoluble phosphates such as bone could be made to release their phosphorus in a form more quickly accessible to growing plants if they were caused to react with sulfuric acid. That suggestion stimulated John Bennett Lawes, an Englishman, to treat coprolites, a phosphorus bearing ore fairly abundant in Great Britain, with sulfuric acid and to test the resultant phosphate as a plant nutrient. In 1842 Lawes was given a patent on this idea, which permitted him to establish the first ‘superphosphate’ works. From then on is fertilizer history.

Within 20 years after Lawes got his patent the British were producing 150,000 tons a year of superphosphate. Then occurred the discovery of sources of mineral phosphates – rich deposits of rock phosphate in South Carolina in 1867 and in Florida in 1887. These discoveries gave American industry the opportunity to take the lead in the mining of rock phosphates and the production of superphosphate – a lead which has been maintained ever since.”

In a message to the United States Congress in 1938, President Franklin D. Roosevelt underscored the importance of phosphate to agriculture and people.

“The phosphorus content of our land, following generations of cultivation, has greatly diminished,” President Roosevelt said. “It needs replenishing. I cannot over-emphasize the importance of phosphorus not only to agriculture and soil conservation but also the physical health and economic security of the people of the nation. Many of our soil deposits are deficient in phosphorus, thus causing low yield and poor quality of crops and pastures…”

Why?

Phosphorus (P) is required by every living plant and animal cell. Deficiencies in available P in soils are a major cause of limited crop production. Phosphorus deficiency also is probably the most critical mineral deficiency in grazing livestock, according to “The Effect of Soils and Fertilizers on Human and Animal Nutrition,” U. S. Department of Agriculture (USDA) Information Bulletin No. 378, issued in 1975. When P fertilizers are added to soils deficient in the available form of this element, increased crop and pasture yields ordinarily follow.

Phosphorus is one of the primary nutrients essential for plant growth and crop production. It is a non-renewable resource that must be mined from nature. It cannot be artificially produced. We do not, however, mine phosphorus itself. We mine phosphate minerals.

Phosphorus is highly reactive and is not found in its elemental form in nature. It occurs in nature as phosphate, which is a charged group of atoms, or an ion. It is made up of a phosphorus atom and four oxygen atoms (PO₄) and carries three negative charges. The phosphate ion combines with various atoms and molecules within living organisms to form many different compounds essential to life.

Some examples of phosphate’s role in living matter include:

• Giving shape to DNA (deoxyribonucleic acid), which is a blueprint of genetic information contained in every living cell. A sugar-phosphate backbone forms the helical structure of every DNA molecule.
• Playing a vital role in the way living matter provides energy for biochemical reactions in cells. The compound adenosine triphosphate (ATP) stores energy that living matter gets from food (and sunlight in plants) and releases it when it is required for cellular activity. After the energy, in the form of a high-energy phosphate bond, is released the ATP becomes a lower-energy adenosine diphosphate (ADP) or a still lower-energy adenosine monophosphate (AMP) molecule. These will be replenished to the higher-energy ATP (or ADP) state with the addition of phosphate by various mechanisms in living cells.
• The forming and strengthening of bones and teeth.

Humans get phosphate from the foods they eat. These examples show the amount of phosphorus* (mg/100 grams) in various foods.

• Milk 93
• Lean Beef 204
• Potatoes 56
• Broccoli 72
• Wheat Flour 101
Cheddar Cheese 524

* NOTE: Although phosphorus is not found in elemental form in food, by convention the phosphate content of foods is expressed in terms of its phosphorus content.

Plants get phosphate from the soil along with nitrogen, potassium and a number of other nutrients they need to thrive. Fertilizer is added to nutrient-deficient soil to replenish these vital chemicals. Animals get phosphate from their food.

The bulk of the phosphate we mine – about 90% – is used to produce phosphate fertilizers. Another 5% is used to make animal feed supplements. The remaining 5% goes into making a variety of products from soft drinks to toothpaste to metal coatings.

Phosphate is a limited resource that cannot be replaced. As such, an international group of earth science and mineral resource agencies have designated it a strategic mineral resource. This group includes Australia, Canada, the Federal Republic of Germany, the Republic of South Africa and the United States of America.

“The International Strategic Minerals Inventory Summary Report – Phosphate” (USGS Circular 930-C) is a cooperative effort of this international group and was published in 1984 by the U.S. Geological Survey. It describes Phosphorus as “an important component of the cell tissues of plants and animals; it is necessary for the structure, growth, and propagation of living organisms. Phosphorus enters the organic food chain from the soil through the roots of plants. The human body contains about 1 percent by weight phosphorus, most of it in the bones and teeth. The human body requires a daily intake of 0.6-0.7 g of phosphorus.”

 

 

A cell is the unit of life, as in a tiny space separated from external space by a thin closed membrane system, all necessary apparata for life are equipped and a single nucleus spatially and temporally controls all the functions of life (12).   We have believed that the uninuclear organization and principal functions of a cell, such as DNA replication, protein biosynthesis and respiraion, should almost be the same in mammals and tall trees. However, is this always true for all living things? Or, is it only a naiive oversimplification?

We know, on the otherhand, that there is alternative cellular organization, called coenocyte or apocyte in which a cell (to call “cell” may not be appropriate: sometimes, called acellular structure) contains many nuclei. There are various kinds of coenocytes, widely spreading among phylogenetic branches. Are the celular mechanisms of coenocytes also the same as in the ordinary uninuclear cells? Some coenocytes are very large and easy to handle, and hence used for experimental materials for electrophysiological and cell physiological studies.

Coenocytic cells can be classified into several groups in term of theirposition and status in the individuals (Fig. 1).
a: Xanthophycean alga (Chromista, Stramenopiles), Vaucheria and Zygomycetous fungus, Phycomyces, Oomycetes, etc. are coenocytic throughout their cell cycles, except when they are mechanically injured or at the formation of sex organs or zoosporangiophores.

b: Slime molds, such as Physarum polycepharum . are conocytic only in a limited phase of life cycle. Marine green algae Bryopsis, Caulerpa, Acetabularia, etc. are well known large coenocytes. But their diploid (2n) sporophytes, as well as their gametes and zoosopores are nuninuclear cells.

c: Internodal cells of Chara (Charophyte) are giant coenocytes, whereas their node cells are ordinary uninuclear cells. Since the Chara internodal cells reach 0. 5 mm in diameter and 20 cm in length, they have been suitable materials in cell physiology. Differentiation of sex organs and new internodal cells are only from these node cells. The internodal cell contains numerous giant nuclei which are circulated in the large cell by the protoplasmic streaming.Each giant nucleus contains more than1,000 copies of genome. These giant nuclei divide amitotically, and the internodal cell eventually lacks the ability of differentiation.

d: Cladophora spp. (also Aegagropira linnaei = Marimo), Acrosiphonia (green algae) , and Griffithsia(red algae) are consist of sausage-shaped coenocytic tubes (Fig. 1). The common characteristics which lead coenocytic organization is that cytokinesis does not follow mitotic nuclear division.

Following questions are, however, heretofore asked: How is the coenocytic status maintained? Have their Genes for cytokinesis been lost or disfunctioned?

New questions also arise: Is there any causal relationship between being a coenocyte and being a giant cell? Is coenocytic organization ecologically more profitable? How are the internuclear distances regulated? Which nucleus or a group of nuclei does take leadership in nuclear division and cell cycle rotation? How is the signal for nuclear division transmitted to others in the different region? Are individual nuclei taking part different functions, such as CPUs in a parallel-task computer? Does the function of the nuclei (gene expression) depend on the site of residence? And so on.

We don’tstill have any answer to these questions. However, we are finding some interesting features and functions which are specific to coenocytes, through detailed observation of cytoskeletons and nuclear movement.

We recently found an admirable function of Vaucheria in photo-cytomorphogenesis, which is only possible in coenocytes. The body of Vaucheria (V. terrestris sensu G嗾z =V. frigida) consists of a sparsely branched coenocytic tube of 50-70 ｵm in diameter(Fig. 2). Tips of the branches exhibit typical tip growth andpositive- as well as negative phototropism. Vaucheria has thus been very useful material for the photoresponses of tip-growing cells (1-10).

When a narrow region of the cell was irradiated with blue light(400- 500 nm) of moderate intensity, a new growth center was induced at the center of the irraidated region 4 h (at the earliest) after the onset of light. The initiated growth center then bulged out and developed as a new branch (Fig. 3). Only blue light is effective and yellow-red light is completely inert. This is an experimentally induced branching. But, the similar thing must occurs in natural habitats: Vaucheria grows as a mat on wet soil near the stream. In fall, the mat would be shaded by fallen leaves. In the shaded region no photosyntehsis would take place. The ability to form a branch from the opening would have been evolutionary and ecologically profitable trait.Thus, study of the photocytomorpho-genetic response is very important in photobiology and cell physiology.

We found that the accumulation of nuclei was indispensable for this blue light-induced branching. The accumulation of chloroplasts which starts immediately and completed 1 h after the onset of light was by itself not sufficient (Fig. 4) (9-10). The nuclear accumulation started 30- 40 min after the onset of light. Not only nuclei, but also protoplasm, which also included chlorplasts rushed into the lighted region.

Density of nuclei in the irradiated region reached 200% of that in the adjacent dark region by 5 h. The increase in number of nuclei was never due to induction of nuclear division in the lighted region. This was performed by careful observation with many specimen fixed at intervals of 10 min.

Accumulation of protoplasm continued and after 3 h the central vacuole was occasionally teared off. Then, about 4 h after the onset of blue light, cell wall at the center of the irradiated region was softend, and a bulge developed to an actively growing branchlet.

Surprizingly, it was microtubular bundle that translocated the nuclei. During interphase every nucleus had a long microtubule bundle at its head(Fig. 5) (11, 12). Centrosome (including centrioles) serves as an MTOC (12). Additional 2 (or 3?) short microtubule bundles are extending backward from the centrosome region. Observing the movement of nuclei under optical microscope (DIC optics), we confirmed that the frontal microtubule bundles pulled nuclei in longitudinal direction. If the microtubule bundles are destroyed, nuclei are not accumulated, and hence no branch is induced (11). We are now intending to identify the microtubule associated motor protein and to elucidate the motor mechanism.
　Nuclear accumulation is probaly necessary for producing cellulytic enzymes, ion channel proteins and deliver exocytotic vesicles to the presumptive site of branching. It is not known yet that the accumulated nuclei themselves are the site of blue light reception and that only in those nuclei new expression of genes start. By the way, the present results also suggest that the nuclear control for the morphogenesis is rather a short range, as is the case in uninuclear fern protonema cell.
　Now, let’s compare the Vaucheria system with morphogenesis of multicullular plants. In multicellular plants morphogenesis always requires a local cell division(13). If a high nuclear density is principally necessary for creating a new shape, all multicellular plants must inevitably use nuclear- and cell division. In coenocytic cells, however, the high nuclear density can be achieved by gathering nuclei into desired locus without waiting for the next mitosis. Only coenocytic cells can adopt this principle. But we don’t know yet, whether this is also the case in coenocytic algae other than Vaucheria.
　Then, what on earth the roles of individual nuclei in the coenocytic cell? Is the coenocytic organization, monarchy, anarchy, feudalismic, or republic? what is necessary for maintaining coenocytic organization other than deletion of prevention of cytokinesis? Such exciting questions lead us to a new subdiscipline, CELL ECOLOGY. Recently, we have found in Vaucheria that the nuclear division started mainly at the growing apex and the phase of the mitotis propgated from tip to the base as a mitotic wave (14). In a narrow region (about 200 µm in length) the mitosis occur synchrounously. Probably, the mitotic wave is driven via the production of cyclin- like proteins by the dividing nuclei. This may indicate that the nuclein the apical growing region plays a role as the leaders in the coenocytic continuum. ヾimilar　internucleus communication in a coenocytic cell can be observed in the green alga, Bryopsis plumosa (15,16) aritificially fused protoplasts between gametophyte and sporophyte.

　It is not always guaranteed that a phenomenon or a function found in Arabidopsis is also acting in other plants. Also, common senses in multicellular plants are not usually common sense in unicellular or coenocytic organisms. Also, the reason d’etre of coenocyte cannot be solved by the study of unicellular and multicellular organisms. Instead, from the study of coenocytes a quite new, heretofore overlooked important points on the mechanism of cell division could be found. We started to organize “coenocytes Research Group” a few years ago. 

 

 

In 1985 Ian Hall, a mycologist at the Invermay Agricultural Centre near Dunedin began research on the Périgord black truffle (Tuber melanosporum). He believed that it would be possible to establish truffle growing in New Zealand, with the aim of supplying out-of-season Northern Hemisphere markets. By 1987 Invermay had produced its first batch of Périgord black truffle infected plants and in the Spring of that year two truffières (truffle plantations) were established in North Otago. In 1988, truffières were planted in nine other areas of New Zealand, from North Canterbury to Gisborne. In July 1993 a few Périgord black truffles were found in Alan Hall’s Gisborne truffière 5 years after planting – the first in the Southern Hemisphere. Small numbers of black truffles continued to form in the 0.5 hectare truffière during the following few years as well as up to 100kg of an inferior truffle (Tuber maculatum). However, in March and April 1997, 8.5 years after planting, large numbers of immature Périgord black truffles began to form and by May mature truffles were being harvested. By mid June 1997, the 0.5ha truffière had produced 6kg of Périgord black truffles with one tree producing 1.75 kg. Some of the truffles were particularly large and weighed 750g or more. Alan Hall’s truffière has continued to produce commercial quantities of black truffle, and has been joined by truffieres in the Bay Of Plenty, Kapiti, Nelson, North Canterbury and near Taumarunui. There are now over 100 truffieres of all sizes in New Zealand, the largest being near Christchurch and North of Auckland. Annual production remains small but it is expected to grow rapidly over the next five years as plantations mature, and growers refine their management techniques. Ian Hall’s team has significantly expanded their production of Perigord black truffle inoculated trees in recent years, and introduced two new species of truffle – the Burgundy truffle (T. uncinatum) and the bianchetto (T. borchii). Trial plantations of these species began in 2001.

Note: recent genetic evidence indicates that this species should no longer be considered in the genus Coprinus. The new proposed name is Parasola plicatilis (Fries) Redhead.

Photographer: Nathan Wilson

Location: My front lawn is in Los Angeles, California.

Time: Spring 1995

Habitat: Lawns.

Comments: This delicate species makes up for its small size through numbers. For a while, last spring between 10 and 20 caps would appear on my lawn early each morning only to shrivel up and disappear by 10 am. It is now a year later and caps are continuing to show up, though not in as great numbers, probably because I cut back a bit on my watering – or maybe I’m just sleeping in a bit longer.

Processing: The original image was taken on 35mm Fuji Velvia using a Nikon 55mm macro lens. The image was scanned on a Power Mac 8100/100 using a Microtek ScanMaker 35t slide scanner and Adobe Photoshop.

The original photograph was awarded an honorable mention in the limited pictorial category of the 1995 North American Mycological Society photo contest.

When: 1995-07-15

 

 

Merry Christmas from BioStim

If you are looking for a gardening gift for yourself or a green thumb you know try our store at https://biostim.com.au

abstract

Keystones are defined as relatively low biomass species with a structuring role in their food
webs. Thus, identifying keystone species in a given ecosystem may be formulated as: (1) estimating the impact on the different elements of an ecosystem resulting from a small change to the biomass of the species to be evaluated for its ‘keystoneness’; and (2) deciding on the keystoneness of a given species as a function of both the impact estimated in (1) and its own biomass. Experimental quantification of interaction strength necessarily focus on few
species, and require a priori assumptions on the importance of the interactions, which can
bias the identification of keystone species. Moreover, empirical measurements, although
very important, are expensive and time consuming and, owing to the spatio-temporal heterogeneity of habitats, physical conditions, and densities of organisms, published results tend to be case-specific and context-dependent.

Although models can only represent but a caricature of the complexity of the real world,
the modelling approach can be helpful since it allows overcoming some of the difficulties
mentioned. Here we present an approach for estimating the keystoneness of the functional groups (species or group of species) of food web models. Network mixed trophic impact analysis, based on Leontief’s economic input–output analysis, allows to express the relative change of biomasses in the food web that would result from an infinitesimal increase of the biomass of the observed group, thus identifying its total impact.

The analysis of the mixed trophic impacts presented here was applied to a suite of massbalance models, and the results allow us to rank functional groups by their keystoneness. Overall, we concluded that the straightforward methodology proposed here and the broad use of Ecopath with Ecosim (where mixed trophic impact analysis is implemented) together give a solid empirical basis for identification of keystone functional groups.

Introduction

Keystones are defined as species with a structuring role within ecosystems and the food webs that interconnect in spite of a relatively low biomass and hence food intake (Power et al., 1996). It may be noted that the
low biomass requirement eliminates species that structure ecosystems by virtue of their high biomass, such as

Corresponding author at: Dept. Oceanography, Istituto Nazionale di Oceanografia e di Geofisica Sperimentale—OGS, Borgo Grotta Gigante
42/. 34010 Sgonico (TS), Italy. Tel.: +39 040 2140376; fax: +39 040 2140266.
E-mail addresses: [email protected], [email protected] (S. Libralato).0304-3800/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecolmodel.2005.11.029

trees in terrestrial forests, or seagrass and kelp in coastal ecosystems.Keystone species affect the communities of which they are part in a manner disproportionate to their abundance (Power et al., 1996). Keystone species strongly influence the abundances of other species and the ecosystem dynamic (Piraino et al., 2002). Therefore, it is important to identify keystone species, notably to maintain ecosystem integrity, and biological diversity in the face of exploitation and other stresses (Naeem and Li, 1997; Tilman, 2000). However, it is expected that all species of a given ecosystem rank in a continuum of levels of ‘keystoneness’, with only some designated to be keystone species.Identifying keystone species in a given ecosystem may be thus formulated as

1. Estimating the interaction strength as the impact on the
different elements of an ecosystem resulting from a small
change to the biomass of the species to be evaluated for its
keystoneness.
2. Deciding on the keystoneness value of a given species as a
function of the impact estimated in (1) and its biomass.

Several studies report on field-based, experimental quantification of interactions strength, as evaluated through the
impacts induced by changes in abundance of one species on the other species in a community (Paine, 1992; Wootton, 1994; Wootton et al., 1996; Berlow, 1999). However, these experiments necessarily focused on few species; thus, they required a priori assumptions on the importance of the interactions, in order to exclude the uninteresting species from the experiment, which can bias the identification of keystone species (Wootton, 1994; Bustamante et al., 1995).

Although the importance of indirect interactions is recognized (Wootton, 1993, 1994; Yodzis, 2001), their explicit consideration within a purely experimental approach is difficult. Most indirect interactions are weak, which seemingly justifies their being neglected. However, indirect interactions can also be magnified by cascading effects (Brett and Goldman, 1996; Pace et al., 1999), and thus need to be taken into account.

Moreover, empirical measurements of interaction strengths are usually limited to easily accessible, sessile
or errant macrobenthic species (Paine, 1992, 2002; Wootton, 1994; Wootton et al., 1996; Berlow, 1999), or microscopic
species (Naeem and Li, 1997), because these are easier to manipulate and control than nektonic species (especially
large fish, or marine mammals), for which only indirect evidence is available (Power et al., 1996).

Finally, empirical measurements, although very important,are expensive and time consuming (Ernest and Brown, 2001).Consequently, and owing to the spatio-temporal heterogeneity of habitats, physical conditions, and densities of organisms(Paine, 1994, 2002; Piraino et al., 2002), published results tend to be case-specific and context-dependent (Power et al., 1996).

The modelling approach allows overcoming some of the difficulties of the experimental quantification of keystoneness. Through a model it is possible to estimate the strength of the interactions between model functional groups (here referred to as ‘species’, although often composed of groups of species with similar sizes and feeding habits). Therefore, the modelling approach provides at least a pre-screening analysis and allows for improved planning of subsequent field experiments.

This suggestion is not new. Previous estimates of keystone species from mathematical models exists, based on successive elimination of functional groups from a trophic web and evaluating impacts on the other functional groups using a graph theoretical method (Jordan et al., 1999; Jordan, 2001; Sole and Montoya, 2001), or evaluating changes in the biomass of ecosystem with dynamic models (Okey et al., 2004a).

However, estimating the trophic impact on the functional groups of an ecosystem resulting from a change (increase) of
the biomass of a single group can be achieved more directly and rigorously through the mixed trophic impact matrix, M, as adopted for food webs by Hannon (1973) from input–output analysis of Leontief (1951). Each element of the matrix represents the relative change of biomass that would result from an infinitesimal increase of the biomass of the functional groups in the rows (Ulanowicz and Puccia, 1990). Thus, M can be used to estimate the total effect of one functional group on all the others in a given model.

The analysis of the mixed trophic impacts presented here was applied to a suite of mass-balance models built with the software package Ecopath with Ecosim (http://www.ecopath.org; Christensen and Walters, 2004), allowing the estimation of keystoneness for the functional groups of models representing marine ecosystems spread over
the world. The results allow us to rank groups by their keystoneness, which then can be compared with ranking from
previous studies, and with the ecological experience resulting from previous experimental research.

Also, the use of models constructed using the same approach, i.e., using Ecopath with Ecosim, allows standardizing the evaluation of species’ roles and the quantification of the interaction strengths in various environments, thus providing at least some of the pre-screening alluded to above, and required for the design of experimental studies.
The present paper aims to answer the following questions:

(1) How can we use the matrix M obtained for mass-balance
models to quantify the total impact of one functional
group on the others in the ecosystem?
(2) Do the resulting estimates obtained in this manner make
ecological sense?
(3) How do we ensure that our measure of keystoneness
correctly balances the strength of interactions measured
through the total effects (by and on species) and the effect
that species exert due to their biomass?
(4) What are, in general, the major features of keystone
species identified in a comparative analysis of models representing different ecosystems?

Methods

Ecopath approach

Ecopath is the core routine of the Ecopath with Ecosim (EwE), a software package based on an approach proposed by Polovina (1984) and subsequently upgraded with a variety of ecologi-cal and theoretical approaches (Christensen and Pauly, 1992; Walters et al., 1997, 2000; Pauly et al., 2000; Christensen and Walters, 2004). Ecopath allows construction of a massbalance model of a given trophic network by representing the ecosystem functional groups as interacting by means of feeding relationships and, when necessary, subjected to fishing (Christensen and Walters, 2004). The balance of mass (energy, or nutrients) for any functional group (i) of the network is obtained by setting its production equal to the sum of the consumption components, expressed as

where production, on the left side of the equation, is expressed as the product between the production–biomass ratio (P/Bi) and the biomass (Bi), and the right-hand side terms are the sum of the predation terms, each expressed as the product of the consumption–biomass ratio (Q/Bj), the biomass of the predators (Bj) and the proportion of the prey i in the diet of the predator j (DCij); the net flow trough the boundaries of the system, i.e., dispersal (Ei); the fishing exploitation, represented through the catches (Yi); the accumulation or depletion of biomass (BAi); and non-predation natural mortality, expressed by means of the ecotrophic efficiency (EEi). The resulting system of equations, when solved, provides a snapshot of the flows within a trophic web (numerous examples are reported in Christensen and Pauly, 1993; see also http://www.ecopath.org).

Mixed trophic impact

Given the mass-balance model of a trophic network, the mixed trophic impact is estimated for each pair of functional groups (i, j, interacting directly or not) of the trophic web, by means of the net impact matrix. According to Ulanowicz and Puccia (1990), the net impact of i on j (qij) is given by the difference between positive effects, quantified by the fraction of the prey I in the diet of the predator j (dji), and negative effects, evaluated through the fraction of total consumption of I used by predator j (fij). Therefore the resulting matrix of the net impacts, Q, has elements:

The mixed trophic impact mij is then estimated by the product of all the net impacts qij for all the possible pathways
in the trophic web that link the functional groups i and j. Ulanowicz and Puccia (1990) demonstrated that the matrix of
the mixed trophic impacts, M, can be obtained by the inverse of the matrix Q, as it is calculated in EwE (Christensen et al.2004). Table 1 shows the elements of such matrix M derived from a food web representing the ecosystem off the coast of Newfoundland (Bundy, 2001).

The elements mij of the matrix M quantify the direct and indirect impacts that each (impacting) group i has on
any (impacted) group j of the food web (Ulanowicz and Puccia, 1990). Positive/negative values of mij indicate the increase/decrease of biomass of the group j due to a slight increase of biomass of the impacting group i. Thus, the
mixed trophic impacts represent the first order partial derivatives, in term of biomass, of the Ecopath master equation
(1). This can be illustrated by Ecosim simulations, as shown below.

Moreover, from Eq. (2) one can tell that negative elements of matrix M indicate a prevailing of negative effects, i.e. the
effects of the predator on the prey; analogously, positive elements of M indicate prevailing effects of the prey on the predator. Therefore, negative elements of M can be associated to prevailing top-down effects and positive ones to bottom-up effects.

Ecosim

The dynamic routine of the EwE package, Ecosim, is based on a set of differential equations, derived from the Ecopath’s master equation (1), allowing dynamic representation of the system variables, i.e. biomasses, predation, andproduction (Christensen and Walters, 2004). In order to describe dynamically the predator–prey interaction, Ecosim uses Lotka–Volterra relationships modified to account for foraging arena theory (Walters et al., 1997, 2000), which allow to avoid the unrealistic Lotka–Volterra assumption of uniform and random distribution of interactions, typically assumed with the mass-action functions (Walters and Martell, 2004). In foraging arena theory, rather, the biomass of the prey available to predators is only a vulnerable fraction of total biomass, with exchanges rates between the vulnerable and the invulnerable states calculated using vulnerability coefficients set by the user (Christensen and Walters, 2004).

Comparison between simulation outputs and matrix M

In order to test if the elements of M represent the relative change of biomass that would result from an infinitesimal
increase of the biomass of the functional groups in the rows, a set of Ecosim simulations (covering a period of 100 years) were done. Since biomass and production are multiplicative terms in EwE formulations, changing proportionally one or the other has identical resulting effects. Therefore, since changes in production are easier to implement in Ecosim than biomass changes, simulations were done perturbing production. In each simulation, the initial (Ecopath) production was kept constant for the first 10 years and then perturbed. From year 10 to year 20 of each simulation, indeed, the initial production of the functional group being observed (corresponding to the
group in the row of the M matrix) was decreased linearly to 90% of its initial value, then maintained constant until the end of the simulation and the resulting ecosystem changes observed. Following Christensen and Walters (2004), a forcing function was used, in Ecosim, to represent this production decrease. The perturbation on the observed group produced dynamic changes on the biomass of other groups in the web. The resulting relative changes in the biomass (excluding detritus and the observed group), were compared with the corresponding elements of the row of the matrix M. Therefore, each mij was

 

Estimating total impact

Given that there is agreement between the mij and Sij (see below), it is appropriate to estimate the total impact of one
functional group on the ecosystem through the mixed trophic impact. Since each impact can be either negative or positive, we define our proposed measure of the overall effect of each group as 

in which the effect of the change in biomass on the group itself (i.e., mii) is not included.The normalized flows in Eq. (2) bound the sum of the elements of the rows and columns of matrix Q between the interval −1 to +1 (Ulanowicz and Puccia, 1990), which guarantees that the overall effect, estimated as the sum of the elements of M as in Eq. (4), does not need to be weighted by the numbers of groups used to describe the trophic network.

Accounting for the positive and negative contributions to the overall effect estimated as in Eq. (4), allows evidencing for a given group, respectively, bottom-up and top-down effects contributing to its overall effect.

Identifying an index of keystoneness

Several alternatives for combining overall effect and biomass in our keystoneness index were explored. The biomass component was adequately represented by the contribution of the functional group to the total biomass of the food web (Power et al., 1996), that is:

was strongly influenced by the biomass proportions, attributing high keystoneness to functional groups with low biomass (as required) and low overall effect (which should not be the case; see Fig. 1A).Therefore, in order to balance the two components (overall effect and biomass), we define our index of keystoneness as follows: 

groups such as macrophytes. Moreover, the negative and positive contributions to the overall effect, as outlined above, allow calculating the bottom-up and top-down effects contributing to the keystoneness index of Eq. (7).

The estimation of the total impacts and of the ‘keystoneness’ proposed here (Eq. (7)) was applied to each living functional group (thus excluding detritus groups) of a suite of 33 Ecopath models considered well described and
detailed, i.e., with a minimum of 24 functional groups used for describing the ecosystem. These models represent the
trophic web of marine ecosystems that differ for location, period and type of habitat represented. The proposed analysis was applied also to 9 models representing different upwelling ecosystems in different periods, for a total of 42 models analysed.

Results

The comparison between the observed changes resulting from the Ecosim simulations, Sij, and the changes predicted by means the mixed trophic impact, mij, are given in Figs. 2–4, respectively, for the Prince William Sound, the Gulf of Thailand and the North Pacific model. The comparisons showed, with few exceptions, a high Spearman’s (rank) correlation (Zar,1999) between changes observed by means of the dynamic simulation (abscissa) and the correspondent row of the matrix M (ordinate of each plot of the figures). The bisecting line is also shown, together with the rank correlation coefficient between observed and predicted values. The Prince William Sound functional groups (Fig. 2), showed low rank correlations only for the herbivorous zooplankton and adult salmon (both with R2 = 0.02), while the analysis on the Gulf of Thailand model (Fig. 3) resulted in low correlations for rays (R2 = 0.03), phytoplankton (R2 = 0.05), ‘trashfish’ (R2 = 0.09), Priacanthus spp. (R2 = 0.12) and juveniles Nemipterus spp. (R2 = 0.13). The lowest rank correlation for the North Pacific model (Fig. 4) was R2 = 0.17 for the large blue shark group. However, most of the functional groups show high agreement between simulated biomass change and mixed trophic impacts. Thus the overall rank correlations were 54.8%, 51.5% and 54.5%, respectively, for the Prince William Sound, the Gulf of Thailand and the North Pacific. Results of non-parametric test of correlation significance (Zar, 1999) evidenced that the above reported overall rank correlations are all significant at p < 0.001.

The high agreement between the mij and Sij, show that it is legitimate to use Eq. (4) to draw inferences on total
impacts from the M matrix, and subsequently applying Eq. (7) to calculate the ‘keystoneness’ of its various functional
groups. Fig. 5 represents the estimated keystoneness index for the functional groups of four selected models, representing the ecosystems of Newfoundland (after Bundy, 2001), Eastern Tropical Pacific (Watters et al., 2003), Chesapeake Bay (Baird and Ulanowicz, 1989) and Bolinao reef (Alin˜ o et al., 1993).

As might be seen, the keystoneness index estimated here show a common pattern in different ecosystems, and allows
ranking the groups of each model by decreasing keystoneness. The keystone functional groups are those that have value of the proposed index close to or greater than zero.

Different groups of marine mammals (cetaceans, harp and hooded seals) show high keystoneness in the Newfoundland ecosystem, where capelin, a forage species, ranks second. Toothed whales rank second in the Eastern Pacific, between large and small sharks, which rank first and third, respectively. For the Chesapeake and Bolinao ecosystems, zooplankton has the highest keystoneness.

Tables 2 and 3 lists, for the 42 Ecopath models analysed, the four functional groups that ranked highest in term of their keystoneness. The top-down effect as percentage contributions to the keystoneness for each species, evaluated through the proportion of the negative values contributing to the sum in Eq.(4), is also reported in Tables 2 and 3.

Table 2 contains the results of the analysis applied to 33 models considered well described and detailed on the basis
of the number of groups used to describe the ecosystems (minimum 24 groups, maximum 59 groups). Marine mammals often have high keystoneness, and rank first (Alaska gyre, Azores, Newfoundland, Norwegian Barents Sea models) or second (Easter tropic Pacific models, Floreana, Georgia Strait, Newfoundland 1985–1987) in many models. Sea birds rank third in Lancaster model and fourth in the Prince William Sound model. Sharks and rays have high keystoneness in many ecosystems, ranking first (Biscaya, Easter tropic Pacific, Floreana, Hong Kong, Morocco models) or second (Gulf of Thailand and Western Gulf of Mexico) in many models. All these

functional groups have effects on the other components of the ecosystem mainly via top-down impacts.A few ecosystems (North Coast Central Java, Prince Williams Sound) show high keystoneness for phytoplankton, while in coastal and semi-enclosed marine environments(Bolinao reef, Chesapeake Bay, Georgia Strait, Gulf of Thailand), the zooplankton group has high keystoneness.

Table 3 presents analogous results for the nine models of upwelling ecosystems. The seabirds are important
in the California (both models) and Peru (1960) upwelling ecosystems, while marine mammals rank second and third
in the California models. Mackerel has high keystoneness in California (77–85), Peru and Northwest Africa. The zooplankton and phytoplankton groups rank for keystoneness among the first four in all the upwelling ecosystems analysed. Moreover, models of the same upwelling system in different periods seem to show an increase of keystoneness of intermediate trophic levels concomitant with a decrease of keytoneness of top predators over time: marine mammals are ranking second in California model for 1965–1972.while they rank third in the model for the later period (1977–1985); in Peru 1960 cormorants are ranking first while later periods show high keystoneness for lower TL functional groups (large scombrids in 1964–1971 and horse mackerel in 1973–1981).

Discussion

The high general agreement between the mixed trophic impacts estimated by the mass-balance routine, Ecopath,
and the observed relative changes in the biomasses obtained with long-term Ecosim simulations, allowed use of the mixed trophic impact matrix M as a straightforward basis to quantify the effect one functional group has on all the other groups in the ecosystem. Thus, the method proposed allows estimating the keystoneness of the species or group of species in a model without having to perform the time-dynamic simulations, as performed, e.g. by Okey et al. (2004a), thus avoiding differences induced by different simulation protocols.

The wide use of EwE and the easy implementation of the method proposed here allow standardizing the procedure to
estimate the keystoneness of functional groups in models of different marine ecosystems and of the same ecosystem at different periods or scales. Although the methodology has the potential for ranking groups across models we examined here rankings of keystoneness within models.

Generally, marine mammals ranked high in most ecosystems, but in some, they had low rank; thus, spotted dolphin
and baleen whales rank 24 and 33 in Eastern Pacific model. Similarly, skates and sharks ranked high in the Newfoundland model, but very low in the Eastern tropical Pacific model (dogfish was only 30th). Seabirds appear to have high keystoneness in shallow and very productive environments (upwelling systems), but low keystoneness in open seas, ranking e.g., last in Newfoundland and the Easter Tropic Pacific. In shallow coastal ecosystems, phyto- and zooplankton can have high keystoneness. Indeed, the lower part of the trophic web appears to be very important in these ecosystems, where other benthic groups also tend to have high keystoneness index.

Thus, we concur with Power et al. (1996) and Piraino et al. (2002), that keystones are not straightforwardly predicable.
Certainly, and perhaps surprisingly, there is no general correlation between trophic level and keystoneness.

The index proposed assign low keystoneness to functional groups with high abundance, whether they have high impacts or not, thus allowing us to distinguish between keystone species, and dominant and structuring species—with which keystones must not be confounded (Power et al., 1996). Macrobenthic producers generally have low keystoneness, e.g., in the Bolinao reef model, where seagrasses and seaweeds have low keystoneness index.

Another important result is that keystone species do not always exert their high impact by means of top-down effects,
a feature initially suggested to be a defining characteristic of keystone species (Paine, 1969), and thus proposed for identifying keystones (Davic, 2003). In fact, although results highlight that keystone functional groups exert their effect via topdown in many ecosystems (e.g., Newfoundland and Eastern tropical Pacific), keystoneness via bottom-up effects appears also very important in others (e.g., North Sea, Prince Williams Sound). These results are not contradicting previous works highlighting the high importance of top-down effects in keystoneness (Paine, 1966; Menge et al., 1994; Estes et al., 1998) and confirm that bottom-up influences can also be important
(Bustamante et al., 1995; Menge, 1995). Moreover, upwelling systems show a prevalence of keystone functional groups with intermediate positions in trophic webs. This indicates that these intermediate functional groups contribute to the mixture of top-down and bottom-up control typical of wasp-waist ecosystems (Cury et al., 2000). Moreover, changes over time of keystone functional groups in upwelling systems seem to evidence the increase of keystoneness of intermediate functional groups and the concomitant decrease of keystoneness of high trophic level groups thus suggesting an increase of wasp-waist control over time.

Conclusion

Since the first definition of keystone species by Paine (1969), their importance for conservation purposes has been widely recognized. However, difficulties in experimental approaches and the different roles assumed by species in time and space (Paine, 1994; Menge et al., 1994; Estes et al., 1998) lead to increasing scepticism about the original definition of the keystone species concept and the flourishing of different definitions (Mills et al., 1993; Bond, 2001; Davic, 2003). Therefore, although the importance of the concept is well recognized, a widely accepted approach for quantifying keystoneness is still lacking (Bond, 2001).

The straightforward methodology proposed here may contribute to filling this gap. The mixed trophic matrix upon
which it relies allows including direct and indirect effects of trophic interactions. Moreover, the broad use of EwE will facilitate application of the methodology to the large number of ecosystems for which models exist, thus providing a broad empirical basis for the new approach. In view of the key role experiments must continue to play (Paine, 1966, 1994; Menge et al., 1994; Power et al., 1996) the methodology proposed can also be used for a priori identification of keystone species, thus focusing empirical studies.

Acknowledgements

Simone Libralato gratefully acknowledges the Istituto Centrale per la Ricerca Applicata al Mare-ICRAM (Chioggia) for supporting his visiting period at Fisheries Centre—UBC, during which this work was deeply improved. S. Libralato also acknowledges Fabio Pranovi (Universita Ca’ Foscari, Venezia) for advice and support. Villy Christensen and Daniel Pauly gratefully acknowledge support from the Sea Around Us Project, initiated and funded by the Pew Charitable Trusts, Philadelphia. D. Pauly also acknowledges support from Canada’s Natural Science and Engineering Research Council.

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