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Objectives: • Quantitate species richness and relative abundance of ectomycorrhizal fungi on roots that 1) form hypogeous or secotioid sporocarps 2) are most commonly dispersed by small mammals Literature cited: Kennedy, P.G., A.D. Izzo, and T.D. Bruns. in press. High potential for common mycorrhizal networks between understory and canopy trees in a mixed evergreen forest. J.Ecol. Meyer, M. Forests, fungi, and small mammals: the impact of thinning and burning on a tri-trophic mutualism.. Ph.D. dissertation, University of California, Davis North, M. 2002. Seasonality and abundance of truffles from oak woodlands to red fir forests. USDA Forest Service Gen. Tech. Rep. PSW-GTR-183. 91-97 North, M., J. Trappe, and J. Franklin. 1997. Standing crop and animal consumption of fungal sporocarps in Pacific Northwest forests. Ecology 78:1543-1554. Smith, J.E., R. Molina, M.M.P. Huso, D.L. Luoma, D. McKay, M.A. Castellano, T. Lebel, and Y. Valachovic. 2002. Species richness, abundance, and composition of hypogeous and epigeous ectomycorrhizal fungal sporocarps in young, rotation-age, and old-growth stands of Douglas-fir (Pseudotsuga menziesii) in the Cascade Range of Oregon, U.S.A. Can. J. Bot. 80:186-204 Stendell, E.R., T.R. Horton, and T.D. Bruns. 1999. Early effects of prescribed fire on the structure of the ectomycorrhizal fungus community in a Sierra Nevada ponderosa pine forest. Mycol. Res. 103:1353-1359. Acknowledgements: Special thanks to many Bruns lab members for aid in collection of root samples. This research was supported by funding from the USDA Hypogeous ectomycorrhizal fungi on Abies spp. roots and in small mammal diet in a Sierra Nevada mixed-conifer forest Antonio D. Izzo 1 , Marc Meyer 2 , James M. Trappe 3 , Malcolm North 4 and Thomas D. Bruns 1 Introduction Hypogeous fungi are important components of the food web in western coniferous forests (e.g. Maser et al. 1978). They are also very species-rich yielding as many as 65-80 species across a single hectare of mixed-conifer forest (North 2002). Their importance is further supported by studies in the Pacific Northwest which show that hypogeous sporocarp production can be much greater and is more temporally stable (North et al. 1997, Smith et al. 2002) than epigeous sporocarp production. While they are abundant and important based on the sporocarp record, hypogeous fruiting taxa are noticeably absent in studies of ectomycorrhizal (ECM) root communities making up no more than 9% of the ECM species composition (Kennedy et al. in press) and 15% of the ECM root biomass (Stendell et al. 1999). In this study we attempt to resolve this discrepancy to gain better insight into the ECM carbon- capturing potential (root biomass vs sporocarp record) and the potential biases inherent in our approaches to ECM root community analysis. 1 Dept of Plant and Microbial Biology, University of California, Berkeley, 2 Dept of Wildlife, Fish, and Conservation Biology, University of California, Davis, 3 Dept of Forest Science, Oregon State University, 4 Sierra Nevada Research Center, Dept of Environmental Horticulture, University of California, Davis hypogeous/secotioid epigeous epigeous low visibility nonfruiting resupinate unknown/unclear 0.20 0.18 0.37 0.13 0.27 0.09 0.27 0.20 0.13 0.08 0.05 0.03 relative species composition 1) Species which fruit in hypogeous/secotioid manner are more prevalent on ECM roots than previously seen • Methodological bias is certainly one reason why this important group has been missed. Directed effort to sample hypogeous fruiting for ECM community studies and addition of more sequences which allow species-level designations will allow us more insight into the contribution of this important group of fungi across more forest types. • Teakettle forest conditions may be important factors in prominence of hypogeous abundance but a more direct comparative study is needed. The relative proportions of hypogeous:epigeous root biomass is similar to sporocarp biomass ratios in the Pacific Northwest (North et al. 1997). Ratios of hypogeous:epigeous species on roots however are much higher than were seen in an Oregon old-growth forest sporocarp study (Smith et al. 2002). 2) Direct molecular analysis of fungi in small mammal scat is feasible and allows new views into fungal fruiting and dispersal ecology. While our molecular approach did not allow a full view of the small mammal diet, molecular analysis of small mammal diet was demonstrated. Rhizopogon species sequences in particular were readily identified and appear to represent most Rhizopogon species in the forest based on species accumulation curves (not shown). Combining this analysis with other Sierra National Forest studies allows us to find more widespread trends across numerous life stages in this important genus. Root match bp Best match (accession number){frequency in scat} % similarity/ bp overlap no match 234 Endoptychum sp. SNF190 {1} 97%/234 no match 163 unidentified ascomycota sp. RH 10-1 (AJ301722){26} 98%/163 no match 197 Ramaria apiculata (AJ408385){1} 92%/69 no match 189 Clavariadelphus ligula (AF347099){1} 98%/156 no match 247 Alpova trappei (AF074920){1} 97%/275 no match 173 Melanogaster tuber {2} 90%/159 no match 273 Rhizopogon arctostaphyli holotype (AF377167){2} 100%/223 Rhizopogon1 224 Rhizopogon subcaerulescens 99%/224 no match 223 Rhizopogon vulgaris (AF062931), Rhizopogon sp TK1616 {2} 100%/223 no match 223 Rhizopogon rubescens (AF158018){6} 99%/223 no match 195 Rhizopogon subsalmonius BCC-MPM 1653 (AJ515424){3} 1 97%/128 Rhizopogon8 294 Rhizopogon sp. (rubescens?) TK1626 {1} 99%/294 no match 240 Pholiota spumosa (AF345654){4} 89%/226 no match 220 Pholiota spumosa (AF345654){1} 87%/79, 89%/57 Pezizales3 220 Geopora cooperi SNF96 {1} 100%/184 1 partial sequence of ITS1 (99 bp) and full ITS2 was used for identification due to difficulties in sequencing through ITS1 Results of scat-root matching • successful PCR amplification of 55/58 scat samples • only 3 matches to root species • full characterization limited by direct sequence approach and primer bias ECM sequence type bp Best match (accession number if not from this study) % similarity/bp overlap Ascomycota29 207 Geopora cooperi SNF96 94%/207 Basidiomycota51 168 Leucogaster rubescens SNF171 91%/135 Basidiomycota8 174 Leucogaster rubescens 96%/143 Choiromyces2 212 Choiromyces alveolatus (AF501258.1) 91%/212 Gautieria2 650 Gautieria sp. Dinkey2230CA (AF377085) 99%/652 Gautieria3 656 Gautieria monticola isolate SNF346CA (AF377101) 99%/656 Gautieria4 196 Gautieria monticola isolate ORHF20 (AF377094.1) 98%/189 Gymnomyces1 190 Gymnomyces abietis SNF168 95%/168 Lactarius7 230 Arcangeliella crassa SNF203 98%/230 Pezizales1 284 Hydnotrya variformis 91%/284 Pezizales3 207 Geopora cooperi SNF96 100%/207 Rhizopogon1 224 Rhizopogon subcaerulescens (salebrosus) 100%/224 Rhizopogon7 293 Rhizopogon parksii (AF062930) 90%/99 Rhizopogon8 296 Rhizopogon rubescens 99%/296 Russula22 217 Martellia sp 99%/217 Russula4 216 Gymnomyces fallax JMT19916 99%/216 Russuloid2 194 Leucophleps spinispora SNF91 98%/183 Russuloid3 171 Leucogaster rubescens SNF171 91%/135 Russuloid4 201 Leucophleps sp 96%/178 Russuloid5 177 Leucogaster rubescens 97%/150 Russuloid6 206 Leucogaster sp 92%/190 Tuber2 220 Tuber maculatum SNF54 83%/179 ECM sequence types which are potentially hypogeous but not included in the analysis Lactarius1 215 Lactarius mitissimus (AF157412) Arcangeliella borziana (AF373599)] [ 97%/213 [94%/215] Pezizales2 184 motifs similar to Hydnotryopsis spp., no close Genbank match n/a Russula26 216 Macowanites ammophilus (AF230890) Russula pectinata (AY061706) 98%/216 Results of sporocarp-root matching • 21 root species identified as hypogeous species Methods The ECM root community centered around Abies spp. was characterized across 3 years (Izzo, unpublished). 104 species groups were identified by sequence analysis 78 sporocarps across 28 genera (17 new to the databases) were chosen from the North (2002) study and lab herbarium samples directed at genera which were underrepresented in Genbank by ITS sequence Scat samples from Glaucomys sabrinus and Tamais speciosus were collected fromTeakettle Forest across the same 3 years as the root study and the mammal mycophagy characterized by spore typing (Meyer 2003). Fifty-eight samples were chosen across mammal species, year, location, and season to maximize potential to detect unique species All samples were compared by sequence analysis (direct sequencing of PCR product) of the internally transcribed spacer region (ITS) Study site - Teakettle Experimental Forest • located in the southern Sierra Nevada (California, USA) • old growth mixed-conifer • Abies spp. dominated Conditions in this forest should increase the importance of hypogeous fungi a) summers are hot and dry b) a heavy litter layer has resulted from fire suppression and forms barriers to sporocarp emergence (image on left) c) mammal mycophagy activity is high Rhizopogon species in the Sierra National Forest Detection type small mammal scat sporocarp collection ectomycorrhizal root spore bank bioassay 0.005 substitutions/site NJ tree based on ITS1 spacer region.. Numbers represent NJ bootstrap support (1000 replicates) R. evadens 86 96 79 97 65 98 100 98 100 100 94 83 100 R. occidentalis R. olivaceotinctus R. sp 9 R. vulgaris R. roseolus R. sp 8 R. sp 7 R. salebrosus R. arctostaphyli R. ellenae } } R. arctostaphyli +R. salebrosus • strong dispersal potential from mammals • fruits abundantly • widespread on roots • frequent and responsive from spore R.olivaceotinctus + R. occidentalis • rare fruiters • rare on roots • frequent and responsive from spore R. vinicolor R.villosulus Conclusions relative root biomass [email protected]
