210 AvailableNew on-lineZealand at: Journal http://www.newzealandecology.org/nzje/ of Ecology, Vol. 36, No. 2, 2012 Quantification of mycorrhizal limitation in beech spread Ian A. Dickie1*, Murray Davis2 and Fiona E. Carswell1 1Landcare Research, PO Box 40, Lincoln 7640, New Zealand 2Scion, PO Box 29237, Fendalton, Christchurch 8540, New Zealand *Author for correspondence (Email: [email protected]) Published on-line: 13 May 2012 Abstract: Establishment of Nothofagus spp. into grasslands can be limited by a lack of ectomycorrhizal inoculum, but the degree of mycorrhizal inoculum limitation and how far mycorrhizal inoculum spreads from forest edges has not been quantified. Further, it has been hypothesised, but not confirmed, that establishedKunzea ericoides (a native Myrtaceae tree with both ectomycorrhizal and arbuscular mycorrhizal associations) could serve as an alternative host for ectomycorrhizal fungi and thus facilitate mycorrhizal infection of Nothofagus. To confirm and quantify these hypotheses, first we used anex situ, intact-soil-core bioassay of soils collected near Nothofagus solandri var. cliffortioides forest, near established Kunzea, and in grassland distant from trees of either species. Second, we collected soils along transects of increasing distance from Nothofagus forest into adjacent grasslands. Mycorrhizal infection of Nothofagus solandri var. cliffortioides seedlings was high in soils from Near-Nothofagus and Near-Kunzea (74% and 67% of root tips, respectively) and lower in soils Distant from trees (28% of root tips). Seedlings in soils from Near-Nothofagus also had 3.4× greater biomass than those in soils Distant from trees. In the transects, mycorrhizal infection declined in a stepwise fashion at approximately 16 m distance from the forest edge, and seedling biomass was positively correlated with mycorrhizal infection. These data confirm that a lack of mycorrhizal inoculum can limit seedling establishment and show thatKunzea can provide an alternative host for Nothofagus-compatible mycorrhizal inoculum. Further, they provide quantitative data for spatially explicit models of woody establishment. Forty percent of seedlings in soils collected distant from trees had greater than 20% infection, suggesting that a lack of mycorrhizal inoculum is not a complete barrier to woody establishment, but instead may act as one of multiple environmental filters slowing beech spread. Keywords: alternative hosts; facilitation; mycorrhizal inoculum; symbiosis; woody succession Introduction in both studies. Any seedlings without mycorrhizal infection would be unlikely to reach that height. Nonetheless, these Nothofagus is a dominant or co-dominant tree genus in around findings suggest thatNothofagus spp. seedlings can sometimes 70% of New Zealand’s indigenous forest area, yet shows limited establish in areas with limited inoculum. Once established, regeneration into grasslands (Wardle 1984; Wiser et al. 2011). seedlings may then accumulate inoculum over time (Dickie A lack of ectomycorrhizal inoculum more than ‘one to two et al. 2002). Similarly, 2-month-old Nothofagus menziesii tree heights’ from established trees was invoked by Baylis seedlings in riverbeds lacked ectomycorrhizas yet were able (1980) to explain the lack of Nothofagus spp. establishment to persist, while older individuals had mycorrhizas (Wardle into grasslands. This appears to have been the first report of 1980). A lack of mycorrhizal infection may therefore slow mycorrhizal inoculum limitation of seedling establishment at a tree establishment, particularly in combination with other local scale. Since that seminal paper, a similar ectomycorrhizal- environmental filters such as competition, but cannot be taken inoculum limitation has been reported for a wide range of as an absolute barrier to tree expansion (Dickie et al. 2007). other species, including various Pinaceae (Borchers & Perry Further, Leptospermum or Kunzea, widespread native 1990; Teste & Simard 2008; Collier & Bidartondo 2009; genera of Myrtaceae trees, could serve as alternative hosts Nuñez et al. 2009), Quercus (Dickie et al. 2002), Salix and for ectomycorrhizal fungi, and thereby facilitate Nothofagus Betula (Nara & Hogetsu 2004). Further, seedlings establishing establishment (Wardle 1980; Burrows & Russell 1990; Burrows distant from trees have been found to be infected by a lower & Lord 1993). Leptospermum and Kunzea are able to form diversity of mycorrhizal fungi and distinct community of symbioses with arbuscular-mycorrhizal fungi in addition to fungi when compared with seedlings establishing near trees ectomycorrhizal fungi (Moyersoen & Fitter 1999; Weijtmans (Deacon et al. 1983; Cline et al. 2005; Dickie & Reich 2005). et al. 2007), which may permit them to establish into arbuscular- Limitation by a lack of mycorrhizal inoculum has also been mycorrhizal grasslands and accumulate ectomycorrhizal- invoked as a possible explanation for the slow post-glacial fungal inoculum over time. Facilitation of seedling mycorrhizal spread of Nothofagus (Burrows & Russell 1990). infection across plant species has been demonstrated in some In contrast to the mycorrhizal limitation hypothesis, two other systems (e.g. Arctostaphylos–Pseudotsuga (Horton et al. studies have found that all Nothofagus menziesii seedlings 1999), Helianthemum–Quercus (Dickie et al. 2004), Salix– greater than 10 cm in height had mycorrhizal infection, Betula and Larix (Nara 2006), Arbutus–Quercus (Richard despite being distant from established trees (Allen 1987; et al. 2009)), but quantitative evidence is lacking for Wardle’s Rogers 1989). In part these results may reflect the fact that proposed Leptospermum–Nothofagus interaction or any other only seedlings greater than 10 cm in height were measured Myrtaceae–Fagales interaction. Direct evidence is needed, New Zealand Journal of Ecology (2012) 36(2): 210-215 © New Zealand Ecological Society. Dickie et al.: Mycorrhizal limitation of beech spread 211 as many fungi have high levels of plant symbiont-preference Experiment 2 quantified the decline in mycorrhizal (Tedersoo et al. 2008, 2010; Dickie et al. 2010), which might inoculum with distance from Nothofagus. We established be expected to limit sharing of ectomycorrhizal fungi across five replicate transects with samples taken at 0, 4, 8, 12, 16, lineages of ectomycorrhizal plants. 20 and 30 m from the edge of Nothofagus canopy (n = 35). Thus, a number of key details remain unresolved more than The 0-m and 30-m samples were included in Experiment 1 as 30 years after Baylis’s first report of mycorrhizal limitation. Near-Nothofagus and Distant replicates, respectively (n = 10). Notably, (1) at what distance from trees is mycorrhizal For both experiments, we obtained potted soil samples inoculum limiting, (2) does mycorrhizal inoculum decline on 9 November 2010 by driving a metal coring device, lined gradually or abruptly, and (3) do ectomycorrhizal Kunzea with a 65-mm-diameter, 100-mm-long PVC pipe, into the or Leptospermum provide an alternative host for fungi, and soil, removing the top 1 cm with a sharp knife, and closing thus facilitate mycorrhizal infection of Nothofagus seedlings the bottom of the pipe with 1.5 mm nylon mesh, effectively establishing in their vicinity? Understanding these details is potting the soil without disturbance. The coring device was critical to any attempt to integrate mycorrhizal limitation into scrubbed with a 50% bleach solution between samples. We more general models of forest establishment and spread, and bagged individual potted soil samples and transported these carbon sequestration. within 8 h to a 4°C storage room overnight before placing them on individual aluminium trays (to prevent water flow between pots) in a glasshouse and watering for 5 days. Methods Into each pot of soil (n = 55) we then planted a single Nothofagus solandri var. cliffortioides seedling that had been We used ex situ intact-soil-core bioassays to measure aseptically germinated in an autoclaved 50:50 vermiculite and mycorrhizal inoculum in soils, as we did not believe it would peat moss substrate 60 days prior to potting. A 62% cover be feasible to maintain first-yearNothofagus seedlings in the shade-cloth was placed over potted seedlings, and temperatures field. Intact soil cores introduce an unnatural element to the were maintained between 10°C and ambient. Seedlings were experimental design and may disrupt mycelial networks in soil. harvested after 190 days, on 23 May 2011. Prior experience had On the other hand, they have the advantage of isolating edaphic shown that shorter duration experiments resulted in difficulty factors from above-ground effects of vegetation (Dickie et al. in assessing mycorrhizal status (Dickie et al. 2002). 2005) and correspond well with fungal communities observed At harvest, we soaked seedling roots to loosen soil before in the field, provided disruption of soil is minimised (Avis & washing them in flowing tap water. We examined all roots Charvat 2005). for ectomycorrhizal infection, counting all fine root tips, and We conducted two parallel experiments, with some recording whether each tip was ectomycorrhizal. For most replicates shared between the two experiments. Both seedlings, 50% of the root system was examined using a experiments consisted of taking intact soil samples at different clear plastic grid and examining alternate rows under a stereo locations relative to a forest–grassland ecotone, or in proximity microscope (4× to 50×). On seedlings with fewer than