The role of wild boars in spore dispersal of hypogeous fungi FEDERICA PIATTONI1, FRANCESCA ORI2, MARCO MORARA3, MIRCO IOTTI4 and ALESSANDRA ZAMBONELLI5 Dipartimento di Scienze Agrarie, viale Fanin 46, I-40127 Bologna 1federica.piattoni@unibo.it, 2francesca.ori4@studio.unibo.it, 3mamo46it@yahoo.it, 4mirco.iotti2@unibo.it, 5corresponding author: alessandr.zambonelli@unibo.it Piattoni F., Ori F., Morara M., Iotti M., Zambonelli A.: The role of wild boars in spore dispersal of hypogeous fungi. Acta Mycol. 47 (2): 145–153, 2012. Wild boars (Sus scrofa L.) are well-known for soil disturbance in natural and cultivated truffières but their role in spore dispersal is poorly investigated. In the present work we studied the occurrence of hypogeous fungal spores in faecal contents of 14 wild boars randomly hunted in “Parco dei Gessi e Calanchi dell’Abbadessa” Regional Park (North of Italy) where truffle production has been previously investigated for three years. Six methods for spore analysis in faeces were compared and the suspension of faeces in ZnSO4 (70%) solution resulted to be the most reliable. Hypogeous fungal spores, including Tuber magnatum and Tuber aestivum spores, were detected in 9 animals. This result suggests that the detection of fungal spores in faeces of wild boars may provide information on the presence of hypogeous fungi in an area. However, the poor abundance of spores suggests that the wild boar can be considered an opportunistic mycophagist, ingesting truffles only occasionally, as a seasonal source of food. Considering the magnitude of wild boar movements during seasonal migrations, it is possible to speculate that they play a key role in truffle long distance dispersal. Key words: Sus scrofa, truffles, mycophagous, spore dispersal, faeces INTRODUCTION Hypogeous fungi comprise species belonging to several genera of Ascomycota (true truffles), Basidiomycota and Muromycotina (false truffles) (Trappe et al. 2009; Bonito et al. 2013). The hypogeous lifestyle comported several morphological changes, as the loss of the ability to discard spores actively (Trappe, Claridge 2005). This led to develop several survival strategies, as the development of a strong aroma, even typical of any ACTA MYCOLOGICA Vol. 47 (2): 145–153 2012 146 F. Piattoni et al. truffle species (Gioacchini et al. 2005). This aroma is useful to the fungus to be found and eaten by wild animals, mainly rodents and mammals, ensuring spore dispersal into the environment by animal faeces (Cazares et al. 1999; Trappe, Claridge 2005). Indeed, faeces examination of mycophagous rodents, such as the giant white- tailed rat, Uromys caudimaculatus Krefft, the golden mantled ground squirrel, Sper- mophilus saturates Rhoads, and the deer mouse, Peromyscus maniculatus Wagner, demonstrated that truffles represent a consistent part of their diet (Comport, Hume 1998; Cork, Kenagy 1989). Among mammals, wild boars (Sus scrofa L.) are sadly known to damage truf- fle production in natural and cultivated truffières (Ricci 2008; Moreno-Arroyo et al. 2005). The negative impact of wild boars on truffles is attributed to soil distur- bance and ascoma consumption, as we demonstrated for Tuber aestivum in natural truffières in Central Italy (Salerni et al. 2011). However, less is known on the possible ecological role of wild boars in truffle spore dispersal (Genard et al. 1986; Steiner, Fielitz 2009). As far as we know, only the former Authors reported specifically about T. aestivum consumption by wild boars, but the paper lacks in technical information about spore isolation methods. Thus, in the present work we investigated the occurrence of hypogeous fun- gal spores in faecal contents of wild boars hunted in “Parco dei Gessi e Calanchi dell’Abbadessa” Regional Park, an area known for truffle production. MATERIALS AND METHODS Study area. The study was carried out in “Parco dei Gessi Bolognesi e Calanchi dell’Abbadessa” (Ente di gestione per i Parchi e la Biodiversità - Emilia Orientale, 2013) which is located in the South Eastern hilly area of Bologna (Central Italy) in the Municipalities of Bologna, Ozzano dell’Emilia, Pianoro and San Lazzaro di Savena (surface area 4.815,87 ha). In this area, the forests are mainly represented by Ostrya carpinifolia Scop. and Quercus pubescens Willd. (all. Orno Osrtryon Auct. Ital. and all. Ostryo-Carpinion orientalis, Horvat 1954) (Corbetta 1994). The park is characterized by a temperate Mediterranean climate (Köppen-Geiger classification), where the highest and low- est temperatures occur in July-August and December-January, respectively, and the annual average precipitation is 750 mm (Pieri et al. 2011). Truffles, in particular T. aestivum and T. magnatum, are principally found in Q. pubescens mixed forests, in calcareous soils derived from marnous arenaceous rocks, within the Municipalities of Ozzano dell’Emilia, Pianoro and San Lazzaro di Savena. Hypogeous sporoma collection. Fruiting bodies of truffles were collected in Q. pube- scens forests of the Park, using trained dogs, between September and January 2004-2007. Animals. Between October 3rd and December 4th 2011, the faeces of 14 wild boars hunted in the Park area, in compliance with the permitted hunter-kill ratio actions established by Bologna Province, were examined. Animals, randomly chosen regard- less sex, age and weight, were dissected by the “Azienda Agricola S. Uberto” accred- ited slaughtering house (Monterenzio, Bologna) (Provincia di Bologna 2007). Wild boars and truffle spores 147 Preparation of faecal samples for spore detection. Faeces were forced out of the rectum and put into a screw cap vial. In the lab, faecal samples were weighted and diluted 10-fold with sterile distilled water. The suspension was decanted for one hour. The precipitate was sifted through a series of metal sieves of decreasing mesh size (800, 400, 150, 60 and 20 μm). Only the material between 150 μm and 20 μm was in size considered for centrifugation (1500 rpm, 3 min) with sterile distilled water be- cause hypogeous fungal spores (and asci) generally range between these dimensions. The supernatant was discharged and the precipitate was treated with 6 modified so- lutions: 1) NaCl (26.5%); 2) MgSO4 (35%); ZnSO4 (33%); ZnSO4 (70%); 5) sucrose (68%); 6) sucrose gradient (Crede 2007; Gudmundsdottir, Skirnisson 2006; Pet in- formet 2008; Mitosciences 2007). For the latter, 3 different sucrose solutions (35%, 25% and 15%) were sequentially and carefully layered in a 15 ml tube and, finally, 1 ml of faecal precipitate was layered on top. After sucrose gradient centrifugation (1500 rpm, 2 min), three aliquots (100 μl each) were collected from each 1-ml inter- val of the gradient, transferred onto a slide and immediately examined. For the first 5 methods, the faecal precipitate (10 g) was 5-fold diluted with the corresponding solution and centrifuged (1500 rpm, 3min). After centrifugation, new solution was added to the very top of the tube and a cover slip was placed on it for 10-15 min for recovering the floating fungal spores. Cover slips were mounted on a slide and immediately examined under a light microscope. Each sample was treated in triplicate. Among the six methods tested, the ZnSO4 (70%) solution resulted to be the most reliable to isolate spores and thus it was used for analyzing all faecal samples. Morphological identification. Fresh samples of fruiting bodies were preliminary identified on the basis of their macroscopic (colour, surface, smell, etc.), and mi- croscopic characteristics (morphological and biometric characteristics of spores and peridium cells) numbered, dried and stored in the herbarium of the Dipartimento di Scienze Agrarie (CMI-Unibo), University of Bologna (Italy). The spores found in the faeces were identified basing on their external characteristics (shape, dimensions and type of ornamentation). TuberKey (Zambonelli et al. 2000) was used as refer- ence for Tuber spp. identification whereas Montecchi and Sarasini (2000) monogra- phy was used as reference for the species of hypoeous fungi belonging to different genera of ascomycetes and basidiomycetes. RESULTS Hypogeous sporoma collection. During the surveys, only a few ascomata of T. aes- tivum and T. magnatum were found because the area is regularly visited by other truffle hunters, although truffle harvesting within the Park is forbidden. Other hy- pogeous Ascomycetes were common in the park, such as T. excavatum, T. rufum, T. macrosporum T. borchii, T. dryophilum, T. brumale, Balsamia vulgaris, Stephensia bomycina, Genea spp. Basidiomycetes were only represented by the species of the Hymenogaster genus and by Melanogaster ambiguus (Tab. 1). 148 F. Piattoni et al. Detection and identification of faecal spores. The data of the 14 wild boars exam- ined and the characteristics of the truffle spores detected in the corresponding faecal samples are shown in Table 2. No fungal spores were detected in 5 animals whereas in 9 ones spores of hypogeous fungi, including T. magnatum and T. aestivum, were identified (Fig. 1). Other unidentified fungal spores were present in most faecal contents, including several spores of Alternaria spp. (data not shown). Truffle spores were found in both sexes and in the animals from all the Municipalities within the Park. Table 1 Hypogeous fungi found in “Parco dei Gessi Bolognesi e Calanchi dell’Abbadessa” Regional Park (Bologna, Italy) Herbarium n. Species Date Municipality 3382 Balsamia vulgaris Vittad. 30 12 2006 San Lazzaro 3352 23 01 2007 Ozzano dell’Emilia 3357 Genea fragrans (Wallr.) Sacc. 23 01 2007 Ozzano dell’Emilia 2546 15 11 2004 San Lazzaro 3205 08 11 2006 Ozzano dell’Emilia 3375 30 12 2006 San Lazzaro 3372 03 12 2006 San Lazzaro 3351 Genea lespiaultii Corda 23 01 2007 Ozzano dell’Emilia 3373 Genea verrucosa Vittad. 03 12 2006 San Lazzaro 2466 Hymenogaster lycoperdineus Vittad. 27 09 2004 San Lazzaro 3343 24 01 2007 Ozzano dell’Emilia 3353 Melanogaster ambiguus (Vittad.) Tul. & C. Tul. 23 01 2007 Ozzano dell’Emilia 3379 30 02 2007 San Lazzaro 3344 24 01 2007 Ozzano dell’Emilia 3867 27 12 2005 San Lazzaro 2552 15 11 2004 San Lazzaro 2550 15 11 2004 San Lazzaro 2452 11 09 2004 Pianoro 1099 25 09 2007 San Lazzaro 2449 Tuber aestivum Vittad. 11 09 2004 Pianoro 3192 08 11 2006 Ozzano dell’Emilia 3380 30 12 2006 San Lazzaro 3348 Tuber borchii Vittad. 23 01 2007 Ozzano dell’Emilia 3346 23 01 2007 Ozzano dell’Emilia 2544 Tuber brumale Vittad. 15 11 2004 S. Lazzaro 3354 Tuber dryophilum Tul. & C. Tul. 23 01 2007 Ozzano dell’Emilia 1367 27 09 2004 San Lazzaro 1511 Tuber excavatum Vittad. 01 11 2004 Pianoro 2548 15 11 2004 San Lazzaro 3190 08 11 2006 Ozzano dell’Emilia 3197 08 11 2006 Ozzano dell’Emilia 3198 08 11 2006 Ozzano dell’Emilia 3191 08 11 2006 Ozzano dell’Emilia 3189 08 11 2006 Ozzano dell’Emilia 3376 30 12 2006 San Lazzaro 3350 23 01 2007 Ozzano dell’Emilia 3345 23 01 2007 Ozzano dell’Emilia 3185 08 11 2006 Ozzano dell’Emilia 3183 Tuber macrosporum Vittad. 08 11 2006 Ozzano dell’Emilia 3184 08 11 2006 Ozzano dell’Emilia 3186 08 11 2006 Ozzano dell’Emilia 3381 30 12 2006 San Lazzaro 3355 23 01 2007 Ozzano dell’Emilia 2547 15 11 2004 San Lazzaro Wild boars and truffle spores 149 Herbarium n. Species Date Municipality 3194 Tuber magnatum Pico 08 11 2006 Ozzano dell’Emilia 3204 08 11 2006 Ozzano dell’Emilia 3193 08 11 2006 Ozzano dell’Emilia 3374 30 12 2006 San Lazzaro 3241 15 01 2007 Ozzano dell’Emilia 3240 15 01 2007 Ozzano dell’Emilia 3239 15 01 2007 Ozzano dell’Emilia 3242 15 01 2007 Ozzano dell’Emilia 3349 23 01 2007 Ozzano dell’Emilia 3243 15 01 2007 Ozzano dell’Emilia 3255 01 01 2007 Ozzano dell’Emilia 2545 15 11 2004 San Lazzaro 2450 Tuber rufum Pico 11 09 2004 Pianoro 2448 11 10 2004 Pianoro 2549 15 11 2004 San Lazzaro 2469 27 09 2004 San Lazzaro 3206 08 11 2006 Ozzano dell’Emilia 3200 08 11 2006 Ozzano dell’Emilia 3195 08 11 2006 Ozzano dell’Emilia 3377 30 12 2006 San Lazzaro 3378 30 12 2006 San Lazzaro 3347 25 01 2007 Ozzano dell’Emilia 1100 Stephensia bombycina (Vittad.) Tul. 25 09 2007 San Lazzaro Fig. 1. Spores found in wild boar faeces: a) Tuber aestivum, b) Tuber magnatum, c) Stephensia bombycina, d) Hymenogaster lycoperdineus. Scale bars = 10 μm. 150 F. Piattoni et al. DISCUSSION In this study a simple method to detect truffle spores in wild boars’ faeces was per- fected. This method can be used for ecological studies involving the role of wild animals in hypogeous fungal spore dispersal. Examining the faeces of just 14 animals hunted in two months, we were able to detect seven hypogeous fungal species including T. aestivum and T. magnatum, which are the most widespread and economically important species growing in the studied area. These results suggest that the detection of fungal spores in the faeces of wild boars may provide a first rough indication of the presence of hypogeous fungi in an Table 2 Wild boar data and characteristics of the spores detected in the faecal material Animal code Locality (Municipality) Sex Age (months) Weigth (kg) Number of spores Mean dimensions (μm) Attempt of identification 34532 La Croara (San Lazzaro) ♂ 19-22 64 2 20.51 x 23.73 Tuber magnatum Pico 34497 Via Gaibola (Bologna) ♂ 35 68 <10 6 12.32 x 22.58 21.52 x 22.87 Hymenogaster lycoperdineus Vittad. Stephensia bombycina (Vittad.) Tul. 34526 Ozzano dell’Emilia ♂ 8 23 1 1 15.03 x 11.07 19.20 x 33.69 Hymenogaster sp. Tuber sp. 34222 Montecalvo (Pianoro) ♀ 7 26 1 1 19.61 x 26.32 13.38 x 29.43 Tuber rufum Pico Hymenogaster sp. 34241 Settefonti (Ozzano dell’Emilia) ♂ 7 26 - 34233 Settefonti (Ozzano dell’Emilia) ♀ 6 27 - 34530 Settefonti (Ozzano dell’Emilia) ♀ 22 65 - 34223 Settefonti (Ozzano dell’Emilia) ♀ 25 69 2 4 23.78 x 27.12 13.00 x 21.54 Genea verrucosa Vittad. Hymenogaster lycoperdineus Vittad. 54321 Acquafredda (Pianoro) ♂ 10 32 2 21.40 x 31.03 Tuber aestivum Vittad. 34235 Pieve (Ozzano dell’Emilia) ♂ 9 26 - 34236 Sabbioni (Ozzano dell’Emilia) ♂ 8 26 <10 2 23.55 x 33.41 11.96 x 20.81 Tuber aestivum Vittad. Hymenogaster lycoperdineus Vittad. 54337 Acquafredda (Pianoro) ♀ 9 26 1 20.31 x 32.45 Tuber sp. 54389 Ozzano dell’Emilia ♀ 7 21 - 54328 Montecalvo (Pianoro) ♀ 12 31 3 25.07 x 20.03 Tuber magnatum Pico Wild boars and truffle spores 151 unknown area, when trained dogs are not available. This methodology may also be useful in countries were truffle harvesting is forbidden even for scientific purposes. For example, in the Czech Republic and in Slovakia truffles are considered endan- gered species and they are, thus, protected by the law (Grynder et al. 2011). The spores of hypogeous fungi were found in most of the animals analyzed (9/14) regardless of weight, age or sex. In the past, truffles were harvested with female pigs as it was thought that truffle scent resembled that of the male pig’s pheromone (To Tuscany 2012). In fact, the steroid 5α-androst-16-en-3α-ol, which is a major compo- nent of the boar pheromone (Claus et al. 1981) has been detected in the black truffle (Tuber melanosporum). In the studied area, the lack of preference by female boars for truffles could be explained by the presence of truffle species different from T. melanosporum, which probably do not contain this specific pheromone. Although most animals revealed the presence of hypogeous fungal spores in the faeces, the abundance of spores was poor, limited to just one or few spores in the amount of sample analyzed. This suggests that the wild boar may occasionally eat truffles and that the main damages in natural and cultivated truffières are mainly due to soil disturbance caused by excavation with the snout (Moreno-Arroyo et al. 2005; Salerni et al. 2011; Ricci 2008). On the opposite, other small mammals, like the northern flying squirrels or some marsupials, just eat truffles or simply prefer to eat truffles, whose spores accumulate in faecal pellets (Lehmkuhl et al. 2004; Clar- idge, Trappe 2005). According to the Claridge and Trappe (2005) classification of mycophagous animals in obligate, preferential, casual, opportunistic or accidental mycophagists, the wild board can be considered an opportunistic mycophagist, in- gesting truffles only occasionally, as a seasonal source of food. In mycophagous animals, there is an evidence of spore germination stimulation by the passage through the digestive system, although the effect on spore metabolic activities may differ among animals and among hypogeous fungal genera (Trappe, Claridge 2005). In fact, most studies on hypogeus asco- and basidiomycetes report a positive effect on spore germination (Colgan, Claridge 2002; Claridge, Trappe 2005). In contrast Miller (1985) reported that the germination of spores of Tuber spp. was not stimulated by digestive process in rodents. However, all the studies were carried out only on small hydnophagous mammals or marsupials and never considered large mammals like wild boars (Claridge, Trappe 2005). Wild boars have larger movements than small sized animals, ranging between 2 and 15 km in one night and up to 300 km for males and 100 km for females during seasonal migrations (Andrzejewski, Jezierski 1978; Singer et al. 1981; Defra 2005). 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