Commentary

Do black truffles avoid sexualharassment by linking matingtype and vegetativeincompatibility?

The black Perigord truffle (Tuber melanosporum) is recognizedworldwide as an icon of European gastronomy. Its edible fruitbodyis a hypogeous fleshy structure producing meiospores (Fig. 1). Thisascomycete fungus is ectomycorrhizal, that is, symbioticallyassociates with tree roots. The demand for this highly appreciateddelicacy and the decrease of its production over the twentiethcentury (Savignac et al., 2012) have fuelled intense efforts at itscultivation, and at sequencing its genome (Martin et al., 2010). Inthis issue of New Phytologist, Murat et al. (pp. 000–000)1 analyzeT. melanosporum population genetics in two truffle plantations.They reveal genotypes extending over a few meters, displaying astrong genetic structure at fine scale, with a pattern of isolation-by-distance within the plantation, and a striking spatial segregation ofgenotypes according to their mating type.

‘“Dwarf males” exist in animal and plant species, such as

dioecious mosses … in truffles, their existence remains an

appealing speculation.’

Tuber melanosporum is heterothallic: mating can only occurbetween haploid cells of different mating types, that is, carryingdifferent alleles at the MAT locus (Billiard et al., 2012). Inascomycetes such as T. melanosporum, vegetative hyphae arehaploid, and fruiting first requires mating (Fig. 1). In fact, markersegregation has been observed when extracting DNA frommeiospores in T. melanosporum, confirming that same-clonemating is prevented (Paolocci et al., 2006; Riccioni et al., 2008).Truffle flesh, from which most if not all DNA is extracted bystandard protocols, arises only from the female parent that buildsand feeds the fruitbody. The male genotype can be deduced, bydifference, from the sporal genotype (Rubini et al., 2011).

Spatial segregation of mating types

Murat et al. observed that spatially close individuals differing intheir genotypes, according tomicrosatellitemarkers, carry the same

mating type. Rubini et al. (2011) observed such spatial segregation,and showed that it emerged secondarily: on each nursery-grownseedling, ectomycorrhizas frommultiple individuals with differentmating types initially co-occurred, and the dominance of myceliacarrying the same mating type emerged after several months. Suchspatial segregation may be due to competitive exclusion betweendifferent genotypes, with use of theMAT locus as a marker for self-recognition, probably in addition to other polymorphic loci.

Although the MAT locus is rarely used as a marker for self-recognition in fungi, in Neurospora crassa, Sordaria brevicollis,Ascobolus stercorarius and A. heterothallicus theMAT locus is one ofthe loci controlling vegetative incompatibility (Glass et al., 2000).Vegetative incompatibility is a common phenomenon in filamen-tous fungi that often results in death of the hyphal cells that havefused between individuals carrying different alleles at the lociinvolved in self-recognition (Glass et al., 2000). Vegetativeincompatibility is thought to protect resources within hyphaefrom exploitation by non-kin (Debets & Griffiths, 1998), whereaskin cooperate by sharing space and resources. This is selected forbecause the allele present in an individual controlling for altruismtowards related individuals is often present in these relatedindividuals: the allele controlling altruism therefore benefits fromthe altruism for its transmission to the next generation (Hamilton,1964). The loci controlling vegetative incompatibility often displayhigh degrees of polymorphism and even trans-specific polymor-phism (Debets & Griffiths, 1998; Wu et al., 1998), both of whichare footprints of balancing selection expected at markers used forkin recognition. Competitive exclusion of non-kin is expected infungi, and particularly in symbiotic fungi that would benefit frommonopolizing the host root system, and in fact, vegetativeincompatibility has been reported in plant parasites (L�opez-Villavicencio et al., 2011) and for ectomycorrhizal basidiomycetes(Worrall, 1997). Competitive exclusion of non-kin would havefurther consequences: fewer genetic conflicts are expected in thiscase on a root system, due to kin selection among genets (Buckling& Brockhurst, 2008), which should lead to more cooperativebehaviors, and thereby to more benefits for the symbioticassociation, including the tree – an intriguing prediction thatdeserves testing.

Why would selection recruit the MAT locus for vegetativeincompatibility? Vegetative incompatibility typically involvesmultiple, non-homologous loci, which increases the precision ofkin recognition (Glass et al., 2000), and recruiting MAT wouldincrease the number of discriminating loci. However, the MATlocus is biallelic in ascomycetes (Billiard et al., 2012), and thuspoorly efficient for kin recognition. The coupling of sexual andvegetative incompatibility furthermore complicates mating, wherecell fusion occurs between two different mating types, whilevegetative fusion requires identical alleles at the vegetative incom-patibility loci. Developmental switch must then evolve to

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differentiate the compatibility at the sexual vs vegetative stages, withchanges in gene expression, as described inNeurospora crassa (Shiu&Glass, 1999). One possible evolutionary explanation for the linkbetweenmating type and vegetative incompatibilitymay be that theresulting spatial segregation decreases the probability that compat-ible cells meet and therefore lowers the odds of mating; this isbeneficial under the assumption that sex is costly (Otto &Lenormand, 2002). The best strategy for reproduction is indeedthe clonal propagation of the fittest allelic combinations, with onlysome rare recombination events for purging deleterious mutationsand producing new, possibly beneficial, allelic combinations. If alink between vegetative incompatibility andmating type evolved toavoid ‘sexual harassment’, this would be bad news for truffleproduction!

Looking for a father

However, avoiding sexual harassment may not be simple: the stepstoward truffle fruitbodies remain unknown (Fig. 1), and asrecognized by Murat et al., we ignore what structure plays the roleof themale partner.We therefore ignorewhether gamete limitationactually occurs in truffle grounds. In fact, Linde & Selmes (2012)found no difference in fruitbody production between root systemsdisplaying a single vs both mating types, and Murat et al. did findfruitbodies within large areas dominated by a single mating type.

In related ascomycetes, the male partner can produce adifferentiated hyphal structure, an undifferentiated hypha, or apassively mobile cell, called a ‘microconidium’, or more appropri-ately a spermatium (Fig. 1). In this case, dispersal of spermatia mayalleviate gamete limitation. Urban et al. (2004) and Healy et al.(2012) reported occurrence of candidate spermatia in ectomycor-rhizal Pezizales and Tuber spp., whose failures to germinate on

sterile media or form ectomycorrhizas support a role of spermatia.Their small size (5 lm in diameter), thin cell walls and lack ofreserves are unusual features for asexualmultiplication. If spermatiawere described in T. melanosporum, their dispersal range would beof interest: indeed, Murat et al. and Rubini et al. (2011) failed toidentify the males in the plantations, suggesting a migration fromquite far.

Alternatively, mating may require close contact, either becausespermatia do not disperse, or because direct hyphal contact isrequired (Fig. 1). But even this does not necessarily imply gametelimitation. Indeed, PCR amplifications from soil sometimes detectboth mating types, as reported by Murat et al. and Rubini et al.(2011). Such DNA may issue from the spore bank expected forhypogeous fruitbodies, due to specimens not removed by dispersers(Grubisha et al., 2007; Fig. 1).However, sporeDNA is notoriouslydifficult to extract: for example, DNA extracted from fruitbodiesreveals only maternal alleles and a single mating type. Instead, verysmall mycelia, attached to a few roots, or even nonmycorrhizalgerminations, may exist and act as males (Douhan et al., 2011).They cannot act as female, since they would not have enoughresources to sustain fruitbody growth. ‘Dwarf males’ (Fig. 1) existin animal and plant species, such as dioecious mosses (Heden€as &Bisang, 2011); in truffles, their existence remains an appealingspeculation.

A modern protodomestication

We thus do not control truffle reproduction, and ignore whethersome gamete limitation occurs. This fits the definition ofprotodomestication, where the harvest is enhanced by empiricaltreatments favoring establishment and persistence (without controlof reproduction, which is the hallmark of a true domestication).

?

Mature fruitbody

Mating Meiotic spores

Spore

bank?

Dispersal

by animals

Ectomycorrhizal

and soil mycelium

Unknown receiving

female structure

Germination

Dwarf soil

mycelia?

Spermatia?

Hypha or

‘spermatocyst’

from another

mycelium?

Fig. 1 The life cycle of the black truffle, Tubermelanosporum. Three hypotheses for thepaternal contribution tomatingaredepicted inred (see text for description). Spermatia arereproduced with permission from Urban et al.

(2004; putative T. borchii spermatia arerepresented, since no T. melanosporum

spermatia have been observed so far); picturesof mature fruitbodies and germination werekindly prepared by Fabien Garces and AnnieGuillen, and Franc�ois Le Tacon photographedthe mycorrhiza.

COLOR

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Protodomestication is a difficult step as long as the biology of thetarget species remains unknown, in which some practices can slowdown the emergence of interesting traits. In wheat and barley, forexample, archaeological data show that the emergence of grainindehiscence, a major trait that allows the harvesting of all matureseeds, took more than one millennium (Tanno &Willcox, 2006).This is farmore than requested usingmodern selection. Facingwildpopulations where dehiscence is the rule to allow seed dispersal,early farmers ignored this possibility and harvested cereals beforeripening to avoid seed loss (Tanno & Willcox, 2006), so thatindehiscent mutants were only weakly selected for. Furthermore,selection for smaller grains for sowing vs larger ones for humanfeeding likely delayed the augmentation of grain size.

It is thus still preliminary to recommend practices for truffleproducers and to comment about existing empirical methods. Ashighlighted by Murat et al., European truffle producers oftendisseminate pieces of mature truffles to re-inoculate the soil inalready established plantations.While thismay increase populationdensity and counter-act gamete limitation if any, any use of lower-quality fruitbodies for this purpose, to sell the best ones,may inducea huge genetic load for future generations. Itmay favor genetic traitsdetermining low flavor and/or size, or even high sensitivity topathogens. Similarly, the use of frozen truffles (M-A. S. & E.T.,pers. obs.2 ) found after the harvesting season, may select for slowmaturating or late initiated truffles,maladapted to local conditions.The same applies to fruitbodies used for nursery inoculation.

Since inoculated plantations now produce > 80% of thefruitbodies (Savignac et al., 2012), their genetic quality becomesdeterminant for future production and inoculants. Truffle pro-ducers should avoid inadequate practices reminiscent of those ofearly cereal domestication; the antagonism between selling the besttruffles immediately, and using some of them as inoculants toimprove the truffle grounds in the long term should be explained.

Perspectives

Uncertainties about the truffle life cycle lead us to ancestralproblems in the domestication process. This also calls for morestudies of populations in natural vs planted stands, to check theimpact of the ongoing protodomestication. Tuber melanosporumpopulations from plantations are mostly studied in plantations,where trees had been inoculated in nurseries, and where theobserved patterns are partly of anthropogenic origin. ‘Wild’populations should be compared for genetic diversity, strength ofthe spatial segregation of mating types and other loci, at variousscales – although anthropic genetic disturbance has likely alreadyoccurred. The large diffusion of nursery-inoculated trees may haveimpacted T. melanosporum genetic structure at regional scales inplantations. Many empirical traditions typical of European truffleproduction (Savignac et al., 2012) also await studies to validatethem: for example no genetic study has so far rigorously validatedthe inoculation method by monitoring the persistence of theinoculants in the long term.

Population genetics of ectomycorrhizal ascomycetes remains toorarely investigated (Douhan et al., 2011): Murat et al. and Rubiniet al. (2011) have contributed to change this. The possibility,

already described, of accessing the male genotype raises excitingpossibilities to unravel the range of male gene dispersal, the matechoice, and the nature of themale contribution (Fig. 1). Identifyingthe location of fathers, by increasing sample sizes and spatial scale ofsampling, would also be enlightening. Last, many other ectomy-corrhizal ascomycetes (Healy et al., 2012) also await similarinvestigations.

Marc-Andr�e Selosse1,2*, Elisa Taschen2 and Tatiana Giraud3,4

1Mus�eum national d’Histoire naturelle (UMR 7205 OSEB),CP50, 45 rue Buffon, F-75005, Paris, France;

2Centre d’Ecologie Fonctionnelle et Evolutive,CNRSUMR5175,1919 Route de Mende, F-34293, Montpellier Cedex 5, France;

3Ecologie, Syst�ematique et Evolution, Universit�e Paris-Sud,F-91405,Orsay Cedex, France;

4Ecologie, Syst�ematique et Evolution, CNRS, F-91405,OrsayCedex, France

(*Author for correspondence: tel +33 (0)4 67 61 32 30;emailma.selosse@wanadoo.fr)

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� 2013 The Authors

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Key words: ectomycorrhiza, fruitbodies, mating types, black Perigord truffle,

population genetics, protodomestication, Tuber melanosporum, vegetative

incompatibility.

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