Lectures 3 and 4 Mycorrhizal networks: Evidence and ecological significance

1. Lectures 3 and 4Mycorrhizal networks: Evidence and ecological significance

2. Common Mycorrhizal Networks • Evidence supporting their existence • Identification of transfer pathways • Factors limiting or disrupting CMNs • Carbon and mineral nutrient transfer between plants • Factors regulating carbon transfer • The influence of CMNs on plant communities

3. What is a Common Mycorrhizal Network? two or more plant root systems connected by mycorrhizal fungal hyphae Read et al. (1985)

4. 1 B A 3a 3b 3b Z Y X 4 Possible pathways for nutrient transfer between plants • Soil-only pathway (1) • Root grafts (2) • CMN pathway (3a) • Disrupted CMN pathway (3b) • Unlinked mycelial pathway (4) 2

5. Anastomoses-Potential for CMNs • Anastomoses (=fusion to form a dikaryon) between genetic variants of fungal species are thought to be prevelant (Sen et al. 1999 and Giovannetti et al. 2001) • Supports the idea that hyphal linkages between plants are probable Giovannetti et al. 1999

6. Network Diversity -Networks in nature likely much larger • Cliquish • Nearest neighbors highly connected by one or more fungal genets • Not cliquish • Easily traversed because few steps between neighbors • Cliquish • Easily traversed • A few highly connected nodes

7. Host Generalists -Potential for CMNs? Many studies have found ECM fungal species, and more recently individuals, are shared amongst two or more hosts Morphotyping: PCR/RFLP: DNA Sequencing: Microsatellites:

8. Microsatellites • For distinguishing individuals • Microsatellites are loci (regions within DNA sequences) where short sequences of DNA (nucleotides; A, T, G, C) are repeated in tandem arrays.  This means that the sequences are repeated one right after the other.  • The lengths of sequences used most often are di-, tri-, or tetra-nucleotides. • What makes microsatellites useful is the fact that at the same location within the genomic DNA, the number of times the sequence (ex. AC) is repeated often varies between individuals.  • So one individual may commonly have 13 AC's repeated in a row while another population has 18 AC's repeated at the same location within the genomic DNA.

9. Genet Distribution-Potential for CMNs • Different genetic variants of a fungal species may show specificity towards a single host species (Vrålstald et al. 2002) • Host selectivity can also be present in AMF (Helgason et al. 2002) • But most genet studies show that populations of ECM fungi extend over an area encompassing multiple plants of the same or different species • In a study following fruiting bodies, genets of Amanita muscaria ranged in size from1 m up to 40 m (Sawyer et al. 2001)

10. Genet Distribution-Potential for CMNs • Followed the distribution of Suillus grevillei genets based on sporocarps, mycorrhizas and mycelium using microsatellite technique • Found that most mycorrhizas and mycelium of a single genet covered an area of < 100 cm Zhou et al. 2001

11. Connected by Individuals Recent studies in Japan have used microsatellite markers to show that several trees (Pinus densiflora) were interconnected by a single fungal individual (Tricholoma matsutake) in a fairy ring (Lian et al. 2006).

12. Genet structure of Tricholoma matsutake sporcarps in Pinus densiflora forests. Dotted line around sporocarps in same genet. Different colours are different genets. Pale blue is fairy ring. Lian et al. 2006

14. Multi-cohort stand, 15-94 years old 30x30m DNA sequencing of ITS region to determine taxa Microsatellite markers of fungal and tree DNA to distinguish individuals

15. R. vesiculosus genets (blue) R. vinicolor genets (pink) Up to 19 trees per genet All trees interconnected by < 3 degrees of separation Largest hub tree connected to 47 other trees (65 total) Trees, sampling locations, root lengths

16. Heterogenous (scale-free) network: Highly interconnected, easily traversed, living and growing Resilient to random removal of links but vulnerable to targeted removal of key node Size of nodes ∞ tree diameter Color shade ∞ tree cohort

17. What is the functional evidence for CMNs and transfer of nutrients between plants belowground?

18. 14CO2 Laboratory Evidence for CMNs • Laboratory studies have provided best evidence for the existence and specific functioning of CMNs • Pinus densiflora inoculated with an unknown ECM fungus • Shoots of one seedling labeled with 14CO2 • Time-lapse autoradiography over 7 days Wu et al. 2001

19. Wu et al. 2001

20. Laboratory Evidence for CMNs • Showed label stayed with the fungus and was not transferred to the receiver tissue • Demonstrated bi-directional movement of 14C between two pine seedlings Wu et al. 2001

21. Functional Evidence for CMNs • Transfer shown using 14C autoradiography • Corallorhiza trifida gained 6-14% in weight when linked to an autotrophic conifer with a CMN, but lost 13% of its weight when not linked McKendrick et al. 2002

22. How do networks function to affect survival and growth? • Mycorrhization • Carbon transfer • Water transfer • Other mechanisms?

23. 0.5μm=soil transfer only 35μm=soil + hyphae 250μm=soil + hyphae + rhizomorphs No=soil + hypahe + rhizomorphs + short distance explorers

24. Survival of 2-yr-old germinants (%) Carbon transferred to –yr germinants (excess 13C (mg)) 0.5μm=soil transfer only 35μm=soil + hyphae 250μm=soil + hyphae + rhizomorphs No=soil + hypahe + rhizomorphs + short distance explorers

25. 0.5 m 0.5 m 1 m 2.5 m 5 m Proximity to mature trees affects network response Francois Teste et al. 0.5 μm: soil solution 35 μm: fine hyphae 250 μm: rhizomorphs No mesh: roots

26. 0.5 m a a a a b b b b Proximity to mature trees affects network response Francois Teste et al. 1. Network with rhizomorphs facilitated germinant survival. 2. Survival and growth declined outside tree crown (>2.5 m), perhaps due to declining network effect. 3. Survival worst at 5 m where network potential eliminated (data not shown).

29. % ECM shared Jones et al. 1997 Twieg et al. p<0.01

30. Carbon transfer study • ECM birch, ECM Douglas-fir, AM western redcedar • Douglas-fir in 100%, 50% and 5% of full sunlight • Dual isotope labelling with 13CO2 and 14CO2 • Experiments repeated 2 years in a row Simard et al. 1997

31. There was net transfer from paper birch to Douglas-fir in 2nd year a b b b c c p<0.01 Simard et al. 1997

32. 9.5% a 4.3% 2.7% b c Net C transfer from birch to Douglas-fir increased with Douglas-fir shading Simard et al. 1997 p<0.01

33. Source-sink gradient for C & N between birch and fir * * * * * p<0.05 Simard et al. 1997

34. Leanne Philip, PhD

35. Labeling Times Summer Fall Birch Fir Philip

36. Effect of tree phenology on carbon transfer b ab AB a B A Bi-directional Net • Bi-directional and net transfer were affected by phenology. • Bi-directional transfer was greater in fall than spring. • Net transfer was to birch in spring and fall, and to fir in • summer. Philip

38. Carbon transfer between AM-linked C3 and C4 plants (Watkins et al. 1996) • deviation from shoot δ13C used as measure of transfer to roots • 0-41% (<10%) C transferred to Cynodon, mostly via links • Transferred C remained in roots of Cynodon but moved into roots and shoots of Plantago C3 Plantago lanceolata δ13C~-28‰ C4 Cynodon dactylon δ13C~-13.5‰ 1) 0.45 μm mesh prevented links 2) 20 μm mesh allowed links AMF grassland inoculum

39. Carbon transferred between AM plants remains in fungal tissues (Fitter et al. 1998) C3 Plantago lanceolata • harvested first shoot, clipped, harvested second shoot • Large C transfer, moreso into Plantago • no mesh effect • No C moved from roots into shoots (in fungal structures) C4 Cynodon dactylon 1) 0.45 μm mesh prevented links 2) 20 μm mesh allowed links Glomus mosseae

40. Carbon transfer between AM plants (Lerat et al. 2002) Erythronium americanum Acer saccharum

41. One-way transfer from trout lily to maple in spring birch seedling maple seedling dpm g-1 dwt maple 3884 a birch 290 b lily (extra unlabelled) 224 b 14CO2 trout lily P < 0.01 Lerat et al. 2002 Spring

42. Transfer was reversed in fall, from maple to trout lily corms dpm g-1 dwt birch 0 lily (7 of 22) 4244 14CO2 birch seedling maple seedling Trout lily corms Fall Lerat et al. 2002

43. Where did transferred carbon go?-ECM versus AM-field versus lab

44. Factors regulating transfer • Source-sink in plants • Physiology (Ps, nutrients, C allocation) • Phenology (seasonal, diurnal) • Growth form or growth stage • Altering sink strength (shade, clipping) • Altering source strength (CO2, fertilizer, N2 fixer) • Fungal ecology • Mycorrhizal dependency • Species and genet variation • Degree of mycorrhization, frequency of links • Phenological or seasonal variation

45. Root Compartment Hyphae Earthworm Hyphal Compartment Soil food web can affect transfer through CMNs • Transfer of 32P from one plant to another was higher when earthworms fed on and disrupted the CMN than when it was left intact without earthworms Tuffen et al. 2002

46. Influence of CMNs on communities • Seedling establishment • Plant competition: plants, fungi • Plant diversity

47. Increased ECM diversity and Douglas-fir photosynthesis where seedlings in contact with trees versus isolated • In contact with trees: • Doubled ECM diversity and richness • 20X more Rhizopogon • 6X less Thelephora • 8X more strand-forming fungi • 2X higher Douglas-fir net photosynthetic rate • No difference in light, soil water, or soil nutrients Simard et al. (1997)

48. Ectomycorrhizal CMN facilitated survival of ECM tree species but inhibited AM species * * Booth (2004)

49. CMN facilitated growth of ECM pine * Booth (2004)

50. Seedlinginfection Resource availability Seedling growth Competition/facilitation trade-offs (decreasing neighbour distance )