Genet size and distribution of Amanita muscaria in a suburban park, Dunedin, New Zealand

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1 New Zealand Journal of Botany ISSN: X (Print) (Online) Journal homepage: Genet size and distribution of Amanita muscaria in a suburban park, Dunedin, New Zealand Scott J. Bagley & David A. Orlovich To cite this article: Scott J. Bagley & David A. Orlovich (2004) Genet size and distribution of Amanita muscaria in a suburban park, Dunedin, New Zealand, New Zealand Journal of Botany, 42:5, , DOI: / X To link to this article: Published online: 17 Mar Submit your article to this journal Article views: 246 View related articles Citing articles: 21 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 21 November 2017, At: 06:04

2 New Zealand Journal of Botany, 2004, Vol. 42Amanitamuscariainasuburbanpark: X/04/ The Royal Society of New Zealand Genet size and distribution of Amanita muscaria in a suburban park, Dunedin, New Zealand SCOTT J.BAGLEY DAVID A. ORLOVICH Department of Botany University of Otago P.O. Box 56 Dunedin, New Zealand david.orlovich@botany.otago.ac.nz Abstract Amanita muscaria is a common ectomycorrhizal (ECM) fungus introduced to New Zealand. It most commonly forms associations with introduced host trees, but is also known to form ECM associations with native Nothofagus species. It may act as a "mycorrhizal weed" and could potentially have far-reaching consequences for the diversity of indigenous fungal taxa in New Zealand. Little is known about its population biology. By analysing the banding patterns produced from randomly amplified microsatellite (RAMS) primed polymerase chain reactions, we examined the genetic structure of an established population of A. muscaria in a park in Dunedin, New Zealand. The primer BDB(ACA) 5 was found to have a higher number of polymorphisms than the either of the primers DDB(CCA) 5 or DHB(CGA) 5, and it is possible that within any given area the number of genets detected might be greater, and the size of genets smaller, with the use of more primers. Within a m area the population was found to comprise 28 genotypes, both small (< 6.0 m across) and large (> 6.0 m across). The distribution of genotypes showed no clear pattern of spatial segregation, indicating that single genotypes may comprise multiple ramets rather than contiguous genets. The presence of both small and large genets (or widely separated ramets) indicates that establishment through sexually derived basidiospores and through mycelial spread may play important roles in populations of A. muscaria B04009; Online publication date 9 December 2004 Received 19 March 2004; accepted 23 August 2004 such as the one studied. A. muscaria may employ a strategy combining attributes of C, S, and r-selected ecological strategies. Frequent disturbance at the site may mean that establishment by basidiospore plays a more important role than at other sites with low levels of disturbance. Keywords fungi; Amanita muscaria; random amplified microsatellite analysis INTRODUCTION In New Zealand, Amanita muscaria infects a number of exotic tree species, including Pinus radiata (Stevenson 1962), Betula pendula, Fagus silvatica, Castanea sativa, Quercus robur (Taylor 1981), and Eucalyptusficifolia and E. pauciflora (Ridley 1991). A. muscaria also occurs under cultivated Nothofagus trees (Ridley 1991) and is widespread in the South Island within natural stands of Nothofagus (Stevenson 1958; Johnston & Buchanan 1997; Johnston et al. 1998; Orlovich & Cairney 2004). Invasion of New Zealand's Nothofagus forests is of particular concern as these forests are a vast reservoir of New Zealand fungi (McKenzie et al. 2000). Apart from two records (PDD 64912, PDD 67040), Amanita muscaria generally does not occur in association with the other indigenous ectomycorrhizal (ECM) trees Leptospermum scoparium (manuka) or Kunzea ericoides (kanuka) (Johnston et al. 1998). The role of disturbance in the initial establishment of A. muscaria in Nothofagus forests is unclear. Whether basidiospores or mycelium are the primary means by which A. muscaria colonises and persists in Nothofagus forest is unknown. In Australian Pinus radiata plantations (Sawyer et al. 2001), A. muscaria was found to exist as longlived genets with a complex spatial distribution attributed to the redistribution of soil at the time of tree planting. Here we report a preliminary study aimed at understanding the population biology of A. muscaria in another disturbed site. We assessed the use of random amplified microsatellite (RAMS)

3 940 New Zealand Journal of Botany, 2004, Vol. 42 analysis to determine the size and extent of identical genotypes of A. muscaria in a suburban park containing the introduced tree species Quercus robur and Betula pendula in Dunedin, New Zealand. MATERIALS AND METHODS Site Sporocarps were collected from a small park situated below Stonelaw Terrace on the fringe of the Town Belt in Dunedin, South Island, New Zealand (NZMS 260I44/163803). This park is a small, grassed area sparsely populated with oak (Quercus robur) and silver birch (Betula pendula) trees. A bitumen path runs diagonally across the north-east corner of the study site. This site was chosen for the large number of A. muscaria sporocarps present. A visual estimation of the age of the trees (based on tree size and comparison with trees of known age) was conducted by Mr M. Thompson, Dunedin City Council Horticultural Officer for Trees. Specimen collection All visible sporocarps (excepting those which were deemed to be too degraded and likely to yield poor quality DNA) were collected from a 1600 m 2 (40 40 m) area of the park on 25 May For each specimen the entire sporocarp was carefully dug out of the ground and its location recorded as co-ordinates accurate to the nearest cm. The location and species of trees present within the study area were also noted. Freshly collected specimens were stored, in numbered paper bags, by freezing at -20 C on the same day as collection. Samples remained frozen until DNA extraction. DNA extraction DNA was extracted from a small square of the stipe tissue taken from immediately below the cap of a frozen specimen. Each sample was ground to a fine powder in liquid nitrogen and incubated in 500 μl of CTAB extraction buffer (Carlson et al. 1991) at 65 C for 10 min. Proteins were removed by two successive extractions with chloroform. DNA was precipitated with 2 volumes of 95% ethanol and 1/10 volume of 3 M sodium acetate ph 4.8, and washed twice in 80% ethanol. Pellets were air-dried and resuspended in 50 μl of Tris-EDTA (TE) buffer, ph 8.0. RNA was removed by incubation with 1 μl of RNase at 37 C for 15 min. Amplification and visualisation of RAMS Genomic DNA was amplified with the degenerate-ended primers (Hantula et al. 1996) (5-3 ): BDB(ACA) 5, DDB(CCA) 5, and DHB(CGA) 5 (where B = C, G, or T; D = A, G, or T; and H = A, C, or T) using a Touchdown Thermal Cycling System (Hybaid Limited, Middlesex, UK). Amplification reactions were carried out in a 20 μl reaction volume containing: 1 μl of DNA, 8 pmol of primer, 10 μl 2x Amplitaq Gold PCR Master Mix (Applied Biosystems, California), and water to 20 μl. The reactions were cycled by a method modified from Anderson et al. (2001). The cycling parameters were: initial denaturation at 95 C for 5 min followed by 37 cycles of 95 C for 30 s, annealing at a temperature dependant on the primer (BDB(ACA) 5 = 49 C, DDB(CCA) 5 and DHB(CGA) 5 = 55 C) for 45 s, and 72 C for 2.5 min, and a final extension of 10 min at 72 C. Duplicate PCR reactions were carried out for 50 isolates chosen randomly to ensure reproducibility. A negative control containing no DNA was included in each reaction in order to ensure they were free from contamination. Amplification products were separated by electrophoresis in a 2% agarose gel stained with ethidium bromide. All gels for each primer were run for the same length of time (125 min) and at the same voltage (60 V) to allow easier comparison between gels to be made. The amplification products were visualised under UV light and their sizes estimated by comparison to a 100 base pair (bp) molecular ladder (Invitrogen New Zealand Limited, Auckland) run in the left and right hand lanes of each gel. Two previously run samples were also included on each gel to facilitate comparison between gels. Analysis of RAMS profiles The presence or absence of each amplification product was recorded for each sporocarp. Only distinct, reproducible bands were included. Isolates having identical fingerprints for all three primers were regarded as belonging to a single genotype. Genotypes were subsequently mapped according to the distribution of collected sporocarps at the field site. Using R version (R Development Core Team 2003), with the multivariate analysis (MVA) library, dissimilarity matrices were generated, using Manhattan distance and average clustering, describing the variation within and between primers. These were visualised by performing hierarchical cluster analyses to generate dendrograms for each individual primer and for all three primers combined.

4 Bagley & Orlovich Amanita muscaria in a suburban park 941 This method corresponds to the unweighted pairgroup method with arithmetic means (UPGMA). The size of each genotype was calculated as the distance between the outermost sporocarps yielding identical banding patterns for all three primers. The nearest distance between genotypes was calculated as the nearest distance between sporocarps yielding different banding patterns for at least one primer for each genotype. RESULTS Age of oak trees at the site Although an exhaustive search of the Dunedin City Council archives failed to obtain an exact date for the planting of the oak trees at the study site, a visual assessment by an expert placed the likely age at years old (M. Thompson pers. comm. 2003). Collections and RAMS amplification Of the 169 collections obtained from the study site, 107 were used in the final analysis (Fig. 1). Reproducible fingerprints were obtained for all three primers within the same amplification and between amplifications for the 50 randomly chosen isolates for which duplicate reactions were carried out. Variation observed with each primer The BDB(ACA) 5 was the most variable primer of the three primers used, producing a total of 16 distinct banding patterns. Fingerprints comprised amplification products, ranging in size from 305 to 1300 bp. Several bands (320, 400, 550, 650, 1200, 1300 bp) were common to all isolates. For the primer DDB(CCA) 5, six distinct banding patterns were observed, comprising 9-13 amplification products. Bands ranged in size from 250 to 1400 bp with the bands 250, 360, 680, and 1400 bp common to all isolates. The primer DHB(CGA) 5 was the least polymorphic, producing five distinct fingerprints, comprising 9-10 amplification products ranging from 220 to 930 bp in size. The majority of the markers (220,400,500,600,700,800,930 bp) were common to all isolates. Identification of genotypes In total 28 genotypes were identified at the study site based on the combined analysis of all three primers (Fig. 2,3). Since not all sporocarps were included in the study the total number of genotypes is likely to have been somewhat higher. Each genotype differed by 1 18 markers from one, two, or all three primers. The examined population consisted of both small and larger genets. The largest genet (genet 18; Fig. 3) was approximately 38 m long and comprised five sporocarps. The smallest genets each consisted of a solitary sporocarp (genets 4,7,8,11,13,20,24,and 26; Fig. 3). The smallest genet comprising more than one sporocarp was approximately 2 m long (genet 5; Fig. 3). There were a total of 18 small (< 6.0 m long) genets. The average genet length (for genets comprising multiple sporocarps) was m. At the time of collection the number of sporocarps present with each genotype ranged between 1 and 14. The average number of sporocarps per genet was 3.8. Sporocarps located close to each other were not necessarily of the same genotype, as there appeared to be considerable intermingling of genets (Fig. 3). The closest distance between sporocarps from different genets was approximately 8 cm (genet 10 and genet 9; Fig. 3). DISCUSSION The data presented here, based on the comparison of RAMS fingerprints for the three primers BDB(ACA) 5, DDB(CCA) 5, and DHB(CGA) 5, indicate that at least 28 genets were present at the field site at the time of sampling. Although the presence of sporocarps clearly reflects the presence of subterranean parent mycelia, their absence does not necessarily indicate the absence of mycelia (Gardes & Bruns 1996; Dahlberg et al. 1997). This, coupled with the facts that all sporocarps were collected over a single day and not all sporocarps were included in the final analyses, means that these must be regarded as minimum estimates of genet size and number. Ecological implications of the observed patterns of Amanita muscaria genet distribution The distribution of roots is likely to influence the pattern of mycelial growth, which will have an effect on the size and shape of genets. While the majority of genets in the present study were small, several genets were greater than 20 m long. This indicates that, assuming that bulk soil movement has not occurred, A. muscaria is capable of extending significant distances by mycelial growth in some situations. Sawyer et al. (2001) identified genotypes of A. muscaria in NSW that had persisted for up to 36 yr. Large genets (20-40 m) have been reported for several ECM species including Suillus bovinus

5 942 New Zealand Journal of Botany, 2004, Vol. 42 Stonelaw Terrace B.pendula O p Q. robur.";. o Q. robur ".' Q Q. robur o" O Q. robur o ','_.' Q. robur o metres Fig. 1 Distribution ofamanita muscaria sporocarps included in the final analysis (filled circles) and those not (open circles). The positions of trees at the site are indicated to scale (diameter at breast height (DBH)) by dashed circles and the species (Quercus robur or Betula pendula) labelled. ooo o (Dahlberg & Stenlid 1994), S. variegatus (Dahlberg 1997), S. pungens (Bonello et al. 1998), Pisolithus tinctorius (Anderson et al. 2001), and Cortinarius rotundisporus (Sawyer et al. 1999). The presence of large genets in the population of A. muscaria studied here and the temporal persistence of genotypes for up to 36 yr reported by Sawyer et al. (2001) are characteristics of a C or S strategist (Dahlberg & Stenlid 1990, 1994; Dahlberg 1997; Bonello et al. 1998; Sawyer et al. 1999; Anderson et al. 2001). In natural fungal successions, late-stage fungi should be expected to employ features of C or S strategies (Deacon & Fleming 1992). Species of Amanita are considered to be typical protagonists of late stages of succession in natural situations (Redecker et al. 2001). However, AFLP analysis of A. francheti in

6 Bagley & Orlovich Amanita muscaria in a suburban park 943 CD o S2 LO " o J 1 )OJT OO r-tcor- C\i ~ r7j pt8~ [J [2i ~ 23 [25] [ Fig. 2 Dendrogram combining all three primers and delineating the observed genotypes. Numbers in boxes and arrowed numbers indicate corresponding genets in Fig. 3. late-stage ECM successions (Redecker et al. 2001) indicated that the studied population consisted of single-sporocarp genets and several larger genets up to 4.7 m in length. Similarly, in the same study, a population of the late-stage ECM species Lactarius xanthoglactus was found to consist largely of small genets with the largest detectable distance across any one genet being 7.3 m, and Laccaria amethystina has been found present as numerous <1.5-m-diameter genets in 150-yr-old late-successional beech forest (Gherbietal. 1999). The presence of many smaller genets in fungal populations is thought to indicate the establishment of genets from sexually derived basidiospores (Dahlberg & Stenlid 1995). The majority of A. muscaria genets (c. 65%) in the present study were small (<6.0 m long) indicating that recruitment through spore dispersal also plays an active role in maintain-

7 944 New Zealand Journal of Botany, 2004, Vol. 42 Stonelaw Terrace B.pendula O LO - o - CD -1 ' CD E o CM CM O CO LO CO 0 20 metres Fig. 3 Size and distribution of the 28 genets at the site. Numbers correspond to the clusters in the dendrogram (Fig. 2). The boundaries of the genets were drawn to include all analysed sporocarps with the same genotype and to exclude analysed sporocarps with a different genotype. The positions of trees at the site are indicated to scale (DBH) by dashed circles and the species (Quercus robur or Betula pendula) labelled. 30 ing populations in situations such as that examined here. Small genets are characteristic of an r-selected strategy, considered typical of early colonising species in natural fungal successions (Deacon & Fleming 1992) such as Hebeloma cylindrosporum (Gryta etal. 1997) and L. bicolor(delabastideetal. 1994). However, Pisolithus tinctorius, which could perhaps be considered an early coloniser of disturbed sites, has been demonstrated to form genets of up to 30 m long (Anderson et al. 2001). The presence of both small and large genets in the studied population and the reported temporal persistence of A. muscaria genotypes elsewhere (Sawyer et al. 2001) indicate that A. muscaria may employ a strategy combining C, S, and r-selected attributes. Similar strategies have been suggested for Suillus

8 Bagley & Orlovich Amanita muscaria in a suburban park 945 spp. (Bonello et al. 1998), P. tinctorius (Anderson et al. 2001), and Russula brevipes (Bergemann & Miller 2002). Establishment of S. bovinus by spores was found to be more important at disturbed sites, while mycelial spread increased in importance with decreased disturbance (Dahlberg & Stenlid 1990). Although it is possible that under less disturbed conditions A. muscaria populations of comparable ages may potentially be made up of fewer genets of a larger size, as discussed earlier, it cannot simply be assumed that this will be the case. Individual ECM mycelia may fragment to form a number of genetically identical ramets due to the action of frugivores and/or other forms of disturbance or the edaphic or physical constraints of the soil (Dahlberg & Stenlid 1995). Although in the present situation disturbance by frugivores at the site is unlikely, several other forms of disturbance may affect the population structure of A. muscaria. Aside from frequent disturbance of sporocarps by humans and dogs at the site, the construction of the bitumen path (Fig. 1) involved significant excavation and we have observed the presence of car tyre tracks within the study area. It is impossible to tell from the information presented in this study whether the A. muscaria sporocarps of identical genotype truly represent contiguous genets or multiple fragmented ramets. Variability of RAMS primers A previous study by Sawyer et al. (2001) investigated the genetic variation in A. muscaria from three different sites, separated by as much as 50 km, in Pinus radiata plantations in New South Wales, Australia. Using the RAMS primers (DDB(CCA) 5 and DHB(CGA) 5 ) Sawyer et al. (2001) identified a total of 14 distinct genotypes, 6 of which were common to all three sites. It should be noted, however, that the most variable primer used in our study (BDB(ACA) 5 ) was not included in the NSW study. The maximum number of genotypes found at a single site was 10. These were reported from the largest site at Jenolan Caves (7800 m 2 ). At the other two sites in that study, Forest Lodge (700 m 2 ) and Mount Macquarie State Forest (1500 m 2 ), nine and eight distinct genotypes were detected, respectively. On the basis of these two primers the population at Stonelaw Terrace is separated into 14 distinct genotypes, indicating a higher degree of variation than that found by Sawyer et al. (2001) in NSW, Australia. The greater degree of variation in our study may be due to several factors. The site heterogeneity at Stonelaw Terrace might be greater than at the sites examined in the NSW study (Sawyer et al. 2001). The NSW sites were largely monospecific stands of Pinus radiata whereas the host species at Stonelaw Terrace comprised Betula pendula and Quercus robur. The disturbance histories of the two sites are also likely to differ, with more frequent disturbance at Stonelaw Terrace leading to a higher rate of successful mycelial establishment via sporulation or mycelial translocation. Implications for native ECM fungi Due to the lack of studies investigating the ecology and population genetics of native New Zealand ECM fungi, the specific implications of A. muscaria invading Nothofagus forests remains unclear. The flexible C, S, r-strategy that A. muscaria appears to be employing at the Stonelaw Terrace site may mean that once established in Nothofagus forests A. muscaria may be an effective competitor with the ability to persist and spread through mycelial growth, mycelial transfer, and sporulation. Many of the sites where A. muscaria has been observed in association with Nothofagus are distantly removed from exotic species that would allow them to become established through mycelial extension. SJB has observed A. muscaria growing in Nothofagus forest by the Lakeside Track in Nelson Lakes National Park. Spore dispersal whether natural or mediated by humans or other vectors is likely to have been the method by which at least some of the observed individuals have become established. Although a certain amount of information can be gleaned from the distribution and genetic identity of sporocarps, such studies have significant limitations as previously discussed. Below-ground surveys of A. muscaria will cast further illumination on its ecological attributes and may provide answers to some of the debates raised here. The reproductive ecology of A. muscaria may vary from the picture presented here within the different habitat of Nothofagus forest. It is therefore necessary to investigate the population genetics of A. muscaria within that environment. The development of a species-specific DNA probe for the detection and amplification of A. muscaria on mycorrhizal beech roots will be instrumental and invaluable in this regard. Little is currently known regarding the ecology of native ectomycorrhizal taxa and hence it is difficult to assess the probable impact of A. muscaria on fungal populations within Nothofagus forests. To date 226 ECM species have been recorded from Nothofagus forests (McKenzie et al. 2000). However, this information is largely based on the results

9 946 New Zealand Journal of Botany, 2004, Vol. 42 of sporocarp surveys (Orlovich & Cairney 2004) and, thus, neglects the taxa that do not produce conspicuous fruiting structures and under-represents those that produce few fruit or fruit rarely. In order to accurately describe the effect of invasive fungal species such as A. muscaria on native fungal diversity it is important to answer the fundamental ecological question of "who is there?" (Bruns et al. 2002). Until this question is answered it is difficult to state with any certainty what effect A. muscaria is having on the fungal diversity of New Zealand Nothofagus forests. Studies utilising the abundance and diversity of sporocarps may provide limited information regarding the effects of A. muscaria invasion. However, the widely reported lack of correspondence between species that fruit abundantly and those that are abundant on roots (Gardes & Bruns 1996; Dahlberg et al. 1997; Gehring et al. 1998; Jonsson et al. 1999) highlights the necessity for below-ground surveys within Nothofagus forest. Furthermore, there is need to address the void of knowledge regarding the reproductive strategies and functional ecology of native taxa. ACKNOWLEDGMENTS We thank Martin Thompson, Dunedin City Council Horticultural Officer - Trees for assistance with aging trees; Peter Johnston, Landcare Research, for helpful discussions early in this study; John Cairney, University of Western Sydney, and Peter Buchanan, Landcare Research, for helpful comments on a draft of this manuscript; Vickey Clark for expert technical assistance; Mary Anne Miller, Anne-Maree Oliver, and Lynda McCann for field assistance; and the Department of Botany and the University of Otago Research Grants Committee for financial assistance. REFERENCES Anderson, I. C.; Chambers, S. M.; Cairney, J. W. G. 2001: Distribution and persistence of Australian Pisolithus species genets at native sclerophyll forest field sites. Mycological Research 105: Bergemann, S. E.; Miller, S. L. 2002: Size, distribution and persistence of genets in local populations of the late-stage ectomycorrhizal basidiomycete, Russula brevipes. New Phytologist 156: Bonello, P.; Bruns, T. D.; Gardes, M. 1998: Genetic structure of a natural population of the ectomycorrhizal fungus Suillus pungens. New Phytologist 138: Bruns, T. D.; Bidartondo, M. I.; Taylor, D. L. 2002: Host specificity in ectomycorrhizal communities: What do the exceptions tell us? Integrative and Comparative Biology 42: Carlson, J. E.; Tulsieram, L. K.; Glaubitz, J. C.; Luk, V. M. K.; Kauffelt, C. R. R. 1991: Segregation of random amplified DNA markers in F1 progeny of conifers. Theoretical and Applied Genetics 83: Dahlberg, A. 1997: Population ecology of Suillus variegatus in old Swedish Scots pine forests. Mycological Research 101: Dahlberg, A.; Stenlid, J. 1990: Population structure and dynamics in Suillus bovinus as indicated by spatial distribution of fungal clones. New Phytologist 115: Dahlberg, A.; Stenlid, J. 1994: Size, distribution and biomass of genets in populations of Suillus bovinus (L.: Fr.) Roussel revealed by somatic incompatibility. New Phytologist 128: Dahlberg, A.; Stenlid, J. 1995: Spatiotemporal patterns in ectomycorrhizal populations. Canadian Journal of Botany 73 (Suppl. 1): S1222-S1230. Dahlberg, A.; Jonsson, L.; Nylund, J. E. 1997: Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in south Sweden. Canadian Journal of Botany 74: Deacon, J. W.; Fleming, L. V. 1992: Interactions of ectomycorrhizal fungi. In: Allen, M. F. ed. Mycorrhizal functioning: An integrative plant-fungal process. New York, Chapman and Hall. Pp de la Bastide, P. Y.; Kropp, B. R.; Piche, Y. 1994: Spatial distribution and temporal persistence of discrete genotypes of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton. New Phytologist 127: Gardes, M.; Bruns, T. D. 1996: Community structure of ectomycorrhizal fungi in Pinus muricata forest: above and below ground views. Canadian Journal of Botany 74: Gehring, C. A.; Theimer, T. C.; Whitham, T. G.; Keim, P. 1998: Ectomycorrhizal fungal community structure of piny on pines growing in two environmental extremes. Ecology 79: Gherbi, H.; Delaruelle, M.; Selosse, A.; Martin, F. 1999: High genetic diversity in a population of the ectomycorrhizal basidiomycete Laccaria amethystina in a 150-year-old beech forest. Molecular Ecology 8: Gryta, H.; Debaud, A.; Effosse, A.; Gay, G.; Marmeisse, R. 1997: Fine-scale structure of populations of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune forest ecosystems. Molecular Ecology 6:

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