Genetic diversity of Pisolithus in New Zealand ... - Wiley Online Library

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Genetic diversity of Pisolithus in New Zealand indicates multiple long-distance dispersal from Australia Blackwell Publishing Ltd.

Bernard Moyersoen1, Ross E Beever1 and Francis Martin2 1

Landcare Research, Private Bag 92170, Auckland, New Zealand; 2UMR INRA/UHP 1136 1aM, Centre INRA de Nancy, F-54280, France

Summary Author for correspondence: Bernard Moyersoen Email: [email protected] Received: 11 June 2003 Accepted: 1 August 2003 doi: 10.1046/j.1469-8137.2003.00908.x

• Pisolithus is a common ectomycorrhizal (EcM) associate of prostrate kanuka Kunzea ericoides var. microflora (Myrtaceae) in New Zealand geothermal areas. Here, we report the genetic diversity and phylogeny of Pisolithus and interpret the results in relation to the origin of this fungus in New Zealand. • We determined the genetic variation of Pisolithus on the basis of ITS gene sequences and spore morphology. • We identified three Pisolithus species in New Zealand, each matching Australian species associated with eucalypts and acacias. All three species co-occurred locally in thermal areas, with two species sometimes colonizing root tips in the same soil volume, indicating co-occurrence of species on a smaller scale. • We propose that Pisolithus fungi were introduced to New Zealand from Australia by trans-Tasman airflow during recent geological times. The success of this longdistance dispersal of EcM fungi may be related to the capacity of kanuka to act as a ‘nurse plant’ for wind-blown spores. Key words: Pisolithus, ectomycorrhizas, dispersal, New Zealand, geothermal, Kunzea ericoides var. microflora, Leptospermum scoparium. © New Phytologist (2003) 160: 569–579

Introduction Current knowledge in biogeography suggests typical ectomycorrhizal (EcM) plant and fungus symbionts require overland routes for migration as a consequence of the obligate heterotrophic habit of many EcM associations (Malloch et al., 1980). Because of the presumed necessity of the concomitant spread of seed and fungal inoculum, long-distance dispersal of either the fungus or the host plant is considered unpredictable and unlikely. Initial failure to establish pines outside their natural range as a consequence of absence of suitable EcM fungi in the soil (Mikola, 1980) supports the concept of immobility of obligatorily EcM plants. The importance of host plant biogeographic history in the distribution of EcM fungal species has been repeatedly demonstrated (e.g. Pirozynski, 1983; Halling, 2001). New Zealand (NZ) is an interesting model for biogeographic studies. The NZ landmass has been separated from all other landmasses since the late Cretaceous period, 80 million years ago. Since then it has passed through several major land transformations and climatic changes related to north-east

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movement of the country, marine transgression leading to widespread inundations until the Oligocene, recurrent glaciations during the Pleistocene, and volcanic activity from the Pliocene in the North Island (Stevens, 1980). These events produced major vegetational changes and several massive flora and fauna extinctions (Mildenhall, 1980; Stevens, 1980). Long-distance trans-Tasman dispersal of biota from Australia to NZ contributed a significant proportion of the modern NZ flora (Mildenhall, 1980). The importance of this dispersal process in comparison with migrations via land bridges at earlier geological times, and vicariance and/or evolution in situ is under debate (Mildenhall, 1980; Pole, 1994). The origin of EcM fungus flora associated with the Leptospermoideae (Leptospermum and Kunzea) in NZ is particularly interesting since these EcM host taxa appeared in NZ during recent geological times (Thompson, 1989). Because of their capacity to establish successfully without EcM association (Baylis, 1962; Cooper, 1976), dispersal capacity of these two facultative EcM genera is probably greater than in obligate EcM associations. EcM associations observed in Leptospermum and Kunzea in NZ can be explained by the capacity

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of Leptospermoideae to act as hosts for mycosymbionts of taxonomically different plants such as Nothofagus sp. (Bougher et al., 1994). An alternative long-distance dispersal from Australia to NZ of EcM fungi associating with Leptospermoideae has not yet been reported. Molecular methods have proved particularly powerful in phylogeographic studies to test hypotheses about processes of dispersal, particularly when morphology gives limited information about phylogenetic relationships (e.g. McDaniel & Shaw, 2003). The genus Pisolithus is a good model to test hypotheses about the origin of EcM fungus flora associated with Myrtaceae in NZ. Recent advances in molecular biology, mostly based on ITS, have considerably improved knowledge about the phylogeography of this fungus worldwide (Anderson et al., 1998a, 2001a; Junghans et al., 1998; Martin et al., 1998, 2002; Gomes et al., 1999, 2000; Sims et al., 1999; Díez et al., 2001). However, the genetic diversity of this genus associated with Myrtaceae in NZ (Burns, 1997) has not yet been studied. The cosmopolitan genus Pisolithus was originally considered as monotypic, although morphological and basidiospore variations suggesting the existence of several species were recognized (Bronchart et al., 1975; Calogne & Dring, 1975; Demoulin & Dring, 1975). Recent molecular studies have demonstrated trends in Pisolithus species’ geographical distribution in natural environments as well as in host and probably ecological specificity. At least 11 different clades have been reported, the major ones being recognized as phylogenetic species (Martin et al., 2002). Amongst them, P. arrhizus (Scop. Pers.) Rauschert is distributed widely in the Northern Hemisphere, where it associates in natural ecosystems with pines and oaks (Marx, 1977; Martin et al., 2002). Two additional clades, with a more local distribution and distinctive host and edaphic specificity, are also present in the Northern Hemisphere (Díez et al., 2001; Martin et al., 2002). Several clades, including the genetically well-isolated species P. aurantioscabrosus Watling (Watling et al., 1995), are found in tropical rain forests in SE Asia or Africa, and associate with different tree hosts (Martin et al., 1998, 2002). The natural geographic range of the remaining clades is in Australia, where at least 5 species, including P. microcarpus (Cooke & Massee) G. Cunn., P. albus (Cooke & Massee) M.J. Priest nom. prov. and P. marmoratus (Berkeley) M.J. Priest nom. prov. (provisional names sensu Bougher & Syme, 1998), associate with eucalypts and acacias (Martin et al., 2002). Wide divergence of ITS sequences suggests the centre of diversification of Pisolithus was in Australia (Martin et al., 2002). By contrast with the wide distribution of Pisolithus in Australia, in NZ this genus is restricted to volcanic areas where it is commonly associated with prostrate kanuka (Kunzea ericoides var. microflora (G. Simpson) W. Harris), a dominant plant in NZ geothermal areas (Cunningham, 1944; Burns, 1997). The Pisolithus association with Leptospermum scoparium J.R. Forst & G. Forst, also occurring in geothermal areas, has not yet been reported. There are no records of Pisolithus in association

with exotic hosts (i.e. eucalypts, pines) in NZ and Chu-Chou & Grace (1983) did not observe Pisolithus fruit bodies in a survey of mycorrhizal fungus associates of Pinus radiata D. Don and Eucalyptus spp. NZ Pisolithus was identified as P. tinctorius (Pers. Pers.) Coker and Couch (Cunningham, 1944). Knowledge about NZ Pisolithus genetic diversity and relationships with Pisolithus species elsewhere is necessary to understand how this fungus colonized NZ. Pole (1994) pointed out that NZ is an ideal candidate to study questions of island biogeography and dispersal vs vicariance. NZ Pisolithus natural history might improve knowledge about the dispersal of EcM fungi associated with facultative EcM plant hosts. In this study we define the genetic variation of Pisolithus on the basis of ITS gene sequences, leading to the recognition of three species genetically similar to species recognized in Australia. These three species are shown to co-occur in geothermal areas along the main NZ volcanic zone.

Methods Sampling sites Naturally thermotolerant vegetation in NZ geothermal areas has been described by Given (1980) and Burns (1997). It is distributed as vegetation ‘islands’ totalling 580 ha scattered along the 300 km length of the Taupo volcanic zone. Fruit bodies of Pisolithus were collected along the volcanic zone and a soil core was taken below fruit bodies on several occasions for subsequent EcM identification. Sampling sites, from south-west to north-east of the Taupo volcanic zone (Fig. 1) were Tokaanu thermal park Recreational Reserve (38°58′ S, 175°46′ E, altitude approximately 360 m); Broadlands Road Scenic Reserve, Tauhara (38°40′ S, 176°6′ E, altitude 420– 440 m); Karapiti steam field ‘Craters of the Moon’ (38°39′ S, 176°4′ E, average 435 m altitude); Lake Rotokawa Conservation Area (38°38′ S, 176°11′ E, altitude 320–340 m); Te Kopia Scenic Reserve (38°24′ S, 176°13′ E, altitude 400–420 m); Kuirau Park (38°8′ S, 176°15′ E, altitude approximately 280 m); Parimahana Scenic Reserve, Kawerau (38°4′ S, 176°43′ E, altitude approximately 40 m); and Motuhora Island (Whale Island) Wildlife Management Reserve (37°51′ S, 176°58′ E, altitude 0–100 m). The most intensively surveyed area was Karapiti (36 ha). Specimens were collected in July 2001 (Kuirau, Te Kopia), February 2002 (Tokaanu, Tauhara, Karapiti, Te Kopia, Kawerau), March 2002 (Motuhora Island) and June 2002 (Tauhara, Karapiti, Te Kopia). Vegetation and soil characteristics of Karapiti and Te Kopia sites are described in Given (1980) and Burns (1997), respectively. Morphological descriptions of fruit bodies and isolation of exsiccata EcM and fresh fruit bodies were processed a few hours after harvesting. EcMs were washed from the soil and Pisolithus

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Fig. 1 Distribution map of sampling sites.

EcM tips were identified on the basis of their habit (Weiss, 1992; Watling et al., 1995) and stored at −80°C until molecular analysis. Morphological characteristics of fresh fruit bodies were recorded, and mycelium was excised from the central tissue of the stipe and transition between stipe and immature gleba for future molecular analysis. The excised mycelium was stored at −80°C. Fruit bodies were then dried before making further anatomical descriptions. Spores were mounted in 3% KOH for light microscopy, and measured using an eyepiece graticule. For scanning electron microscopy they were mounted on doublesided tape, gold coated and examined using a Philips 505 SEM (Eindhoven, The Netherlands). Spore colour was assessed using the charts of Kornerup & Wanscher (1963). Dried specimens are deposited in Herbarium PDD (Landcare Research). DNA extraction and ITS PCR Genomic DNA was extracted using the DNAeasy Kit (Qiagen, Düsseldorf, Germany). The ITS of nuclear rDNA were amplified using the primers ITS1f and ITS4b (Gardes & Bruns, 1993). PCR reactions were carried out on a Techne Progene using 50 µl reaction volumes each containing: 1 µl DNA template, 5 µl MgCl2 (25 m), 5 µl dNTP (2 m), 1 µl of each primer (10 µ), 2 units of Amplitaq Gold (Roche, Auckland, New Zealand) polymerase. 2 µl purified BSA (Biolabs, Auckland, New Zealand) were added to 47 µl PCR mix to improve success of amplifications. Cycling parameters were 1 cycle of 95°C for 5 min, 10 cycles including 94°C for 30 s, 55°C


decreasing 1°C at each cycle for 30 s, 72°C for 2 min, 30 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 2 min with a final extension at 72°C for 10 min. Amplification products were electrophoresed in 1% agarose gels stained with ethidium bromide and visualized under UV light. 1 kb + DNA ladder (Life Technologies) was used as marker. Controls with no DNA were included in every set of amplifications. ITS-RFLP Three restriction enzymes were selected using GeneDoc version 2.6 (Nicholas & Nicholas, 1997, GeneDoc: a tool for editing and annotating multiple sequence alignments) software on the basis of their capacity to produce distinctive patterns from Australian Pisolithus collections: Sau3AI (Biolabs), AluI (Roche), TaqI (Life Technologies). Four units of the endonucleases were used for each enzyme to digest 2 µl of the amplified ITS product for 2 h either at room temperature (Sau3AI, AluI) or at 65°C (TaqI). Restriction fragments were separated by electrophoresis in a 4% NuSieve GTG agarose (BMA) gel and stained with ethidium bromide before visualization under UV light. 1 kb DNA ladder (Invitrogen, Auckland, New Zealand) was used as marker. Subcloning and sequencing DNA amplification products belonging to collections representative of each ITS-RFLP pattern were purified with High

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572 Research Table 1 ITS-fragment sizes of 4 Pisolithus ITS-RFLP types following digestion with AluI, Sau3A I and TaqI ITS-RFLP fragment sizes (bp) ITS-RFLP type

ITS size (bp)

AluI

Sau3A I

TaqI

A B C D

850 850 850 850

85,166,406 76,85 140,406 85,166,406 85,140,508

61,112,196,208,275 61,76,196,232,260 61,112,196,208,275 57,176,290

58,68,75,89,99,105,212 61,68,80,110,278 54,68,75,84,170,212,293 61,68,94,123,190,212

Bold figures correspond to intense bands following digestion with TaqI.

Pure PCR Product Purification kit (Invitrogen) and then ethanol precipitated for storage before further analysis. Precipitates were resuspended in purified water. The ITS region was subcloned in the plasmid pCR4-TOPO (Invitrogen) following the manufacturer’s instructions. The subcloned ITS region in the plasmid DNA was amplified using the primers T3 and T7 (Invitrogen). PCR reactions were carried out on a thermocycler GenAmp PCR 9600 (Perkin Elmer, Norwalk, CT, USA) using 27 µl reaction volumes each containing: 2 µl plasmid DNA, 0.5 µl dNTP (2 m), 1 µl of each primer (10 µ), 0.5 units of taq Gold (Roche) polymerase. Cycling parameters were 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 1 min, 60°C for 30 s and 72°C for 3 min, with a final extension at 72°C for 15 min. Amplification products were electrophoresed in 1% agarose gels stained with ethidium bromide and visualized under UV light. Amplification products were purified on a Multiscreen PCR (Millipore, Molsheim, France) 96 well filtration system and sequenced using the Taq Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems Inc., Norwalk, CT, USA), following the manufacturer’s instructions. Sequences were generated for both strands of ITS region using T3 and T7 primers. Reactions were then electrophoresed on an ABI Genotyper 310 automated sequencer (Applied Biosystems Inc.). Gels were tracked using the ABI Prism sequencing programme and raw sequence data were edited using the Sequence Analysis 3.3 (Applied Biosystems Inc.) programme and assembled with Sequencher 3.1.1 (Gene Codes Corporation, Ann Arbor, MI, USA) programme. Sequence analysis 29 ITS 1/5.8S/ITS 2 sequences of Pisolithus spp. belonging to 11 different clades and 3 lineages described in the literature (Martin et al., 2002), were retrieved from GenBank and aligned with sequences generated in this study using the programme Multalin (http://www.toulouse.inra.fr/multalin.html). Alignments were manually optimized using the Sequence Alignment Editor Se-Al V1.Oa1 (Andrew Rambaut, 1996, http:// evolve.zoo.ox.ac.uk/Se-Al/Se-Al.html). To infer phylogenetic relationships between Pisolithus collections in this study and those described elsewhere, aligned sequences were analysed using Maximum Parsimony (MP), Maximum Likelihood

(ML) and Neighbour Joining (NJ) methods in 4.0b10 (Swofford, 2002). Gaps were treated as missing data. Optimal trees were identified using heuristic searches based on 1000 random addition replicates retaining clades compatible with the 50% majority-rule in the bootstrap consensus tree. We used the Nearest Neighbour Interchange (NNI) branchswapping option and saved no more than 100 trees with > 1000 length for each replication. Suillus luteus (GenBank accession no. L54110) and Paxillus involutus (GenBank accession n° AF167700) were used as outgroup taxa. Base differences for pairs of sequences amongst subclones within RFLP types and genetic distances ‘p’ (uncorrected value) between collections were calculated using 4.0b10 (Swofford, 2002). Corrected ML distances (enclosed within parentheses in Table 2) were estimated using the HKY85+Ã model (Hasegawa et al., 1985; Yang, 1994) in 4.0b10 (2002).

Results ITS-RFLP Amplification of the ITS region resulted in a single product of 850 bp for the 24 fruit body and 2 EcM collections tested (listed in Table 4). RFLP patterns grouped these collections in 4 RFLP types (Fig. 2, Table 1). TaqI gave the highest degree of polymorphism, and RFLP types A and C differed only with this restriction enzyme (Table 1). Each RFLP type resulting from the restriction with TaqI could be distinguished on the basis of a limited number of intense reproducible bands ranging from 293 to 170 bp. These bands corresponded to 212 bp (RFLP type A), 278 bp (RFLP type B), 170, 212 and 293 bp (RFLP type C) and 190 and 212 bp (RFLP type D) (Fig. 2, Table 1). TaqI also produced a series of fainter bands corresponding to smaller size restriction products in each RFLP pattern (Fig. 2, Table 1). Phylogenetic analysis of ITS sequences Either one (RFLP types B, C, D) or two (RFLP type A) collections per RFLP type were subcloned. Between three and five subclones were sequenced per collection and a total of 20 sequences was obtained from the five collections. Four groups


Research Table 2 Pair-wise comparison of ITS 1/5.8S/ ITS 2 sequences between Pisolithus collections belonging to four different ITSRFLP types (A, B, C, D). The percentage of divergence was estimated using uncorrected ‘p’ distance and corrected ML distance (within parentheses) following the HKY85+Γ model (Hasegawa et al., 1985; Yang, 1994) in PAUP4.0b10

Genetic distance (%) Collections

REB2047 [C]

REB2078 [B]

REB2047 [C] REB2078 [B] REB2050 [A] REB2111 [D]

–

–

9.4 (14.3) 6.4 (8.5) 17.9 (46.9)

7.4 (10.4) 17.5 (43.1)

Fig. 2 RFLP patterns of Pisolithus collections REB 2047 (type C), REB 2050 (type A), REB 2078 (type B) and REB 2111 (type D) following digestion with TaqI. 1 kb ladder (Invitrogen).

REB2050 [A]

REB2111 [D]

– 17.1 (39.8)

–

the ones identical (RFLP types A, C, D) or closest (RFLP types B) to the consensus sequence in each RFLP type. Sequences from REB 2111 (AY318745), 2078 (AY318746), 2047 (AY318747) and 2050 (AY318748) were selected for further phylogenetic analysis. Uncorrected and corrected genetic distance between these collections is shown in Table 2. Alignment of these sequences displayed several insertions deletions in both ITS1 and 2. Sequences of NZ collections were aligned with sequences available from GenBank belonging to 11 clades (Martin et al., 2002) collected worldwide. Only sequences corresponding to ITS1/5.8S/ITS2 region were used for phylogenetic analysis. The KS781 (Fig. 3) sequence includes a missing region 36 bp long in the 5.8S, and alignment for this collection was optimized manually. A preliminary analysis showed that location of NZ collections in trees was identical, irrespective of subclones selected within RFLP types using the MP method. The same-tree topology was obtained using three different methods (MP, ML, NJ). Tree placement of sequences was also constant in parsimony and distance-based trees. Only results from MP are shown (Fig. 3). REB2111 grouped with WA specimens KS781 and Mu98 105 in the clade ‘species 2’ reported by Martin et al. (2002). This species was in the Lineage A of Martin et al. (2002). The other two NZ species were in Lineage B. REB2078 was genetically similar to the Australian collections Gemas and W15. Martin et al. (2002) included these collections in a taxon referred to as ‘species 7’, a species with high within-species divergence. W15 was referred to as ‘species 1’ by Anderson et al. (1998a, 2001a). REB2047 and REB2050 grouped with two Australian collections CSH4461 and MU986 in the genetically variable Lineage B2, referred to as ‘species 10’ in Martin et al. (2002). Basidiocarp and spore morphology

of sequences matching the four RFLP types were obtained after alignment (data not shown). Within each RFLP type, base differences for pairs of subclone sequences were 0–0.8% (RFLP type A), 0–0.4% (RFLP type B), 0–0.3% (RFLP type C), and 0.4–0.9% (RFLP type D). Base differences were observed both in ITS 1, 2 and 5.8S regions (RFLP types B, D), or only in ITS 1, 2 regions (RFLP types A, C). There were also 1–2 bases insertion/deletion in pairwise subclone comparisons within each RFLP type. Clones selected for phylogenetic analysis are


Basiodiocarps varied widely in gross morphology from globose to oblong to pyriform to more or less turbinate with variable development of sterile base tissue, but with no obvious relationship with RFLP type. Peridium colour in all collections was some shade of brown often bruising black. Spore colour, size and ornamentation have been recognized as useful in discriminating groups within Pisolithus and these are summarized for the collections sequenced in this study. Three groups can be recognized (Table 3).

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Fig. 3 Phylogenetic relationships between New Zealand Pisolithus collections (REB 2050, 2078, 2047, 2111) belonging to four different ITS-RFLP types [A, B, C, D] and Pisolithus collections collected worldwide belonging to at least 11 clades and three lineages (A, B, C) described by Martin et al. (2002), on basis of ITS1/5.8S/ITS2 sequences. Sequences from collections from outside New Zealand were retrieved from GenBank. The 50% majority rule consensus tree is based on parsimony analysis. Bootstrap heuristic search (1000 replicates) was conducted using PAUP4.0b10. Tree length: 1153 changes; consistency index (CI): 0.6826; retention index (RI): 0.8208; rescaled consistency (RC): 0.5602. Tree was rooted using Suillus and Paxillus as an outgroup. Branch lengths are proportional to the number of nucleotides changes. Significant bootstrap frequencies are indicated.

Table 3 Spore characteristics of selected Pisolithus collections

RFLP type D B C A

Collection (PDD n°)

Spore diameter*

Spore ornamentation**

Spore mass colour***

REB 2111 (77289) REB 2078 (77288) REB 2047 (77287) REB 2050 (77286)

10.0–12.5 (11.2 ± 0.7) 8.8–13.2 (11.2 ± 1.2) 7.5–10 (8.6 ± 0.7) 7.6–11.3 (9.4 ± 0.8)

Coarsely echinate (coalescent spines) Coarsely echinulate (non-coalescent spines) Finely echinulate (coalescent spines) Finely echinulate (coalescent spines)

Dark brown (7F6, eye brown) Yellow brown (5E6, mustard brown) Yellowish brown (5D6, honey yellow) Yellowish brown (5D6, honey yellow)

*Range (mean ± SE), based on 30 spores including ornamentation. **Based on light microscopy with SEM observations in brackets. ***Terminology follows Kornerup & Wanscher (1963).


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Table 4 ITS-RFLP type and identification of collections of Pisolithus fruit bodies (REB), ectomycorrhizas (BM) and one culture (REBCC) from 8 different sampling sites along Taupo volcanic zone, in association with Kunzea ericoides var. microflora and Leptospermum scoparium

Locality Tokaanu Tokaanu Tokaanu Tauhara Tauhara Tauhara Tauhara Tauhara Tauhara Tauhara Tauhara Tauhara Karapiti Karapiti Karapiti Karapiti Karapiti Karapiti Lake Rotokawa Te Kopia Te Kopia Te Kopia Kuirau Park Kawerau Kawerau Motuhora Island

N° of visits 1

2

2

1 3

1 1 1

Collection n°

PDD n°

Host

RFLP type

Identification

REB2045 REB2047 REB2046 REB2106 REB2081 REB2116 REB2108 REB2049 REB2105 REB2078 REB2111 BM2082b REBCC910-1 REB2054 REB2024 REB2053 REB2092 REB2099 REB2050 REB2085 REB2056 REB2083 REB2025 BM26 REB2062 REB2064

77420 77287 77421 77435 77428 77437 77436 77422 77434 77288 77289

K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides K. ericoides L. scoparium K. ericoides K. ericoides K. ericoides

A C C A A A C B B B E E A C B B B B A A C B A C E A

P. species 10 P. species 10 P. species 10 P. species 10 P. species 10 P. species 10 P. species 10 P. species 7 P. species 7 P. species 7 P. species 2 P. species 2 P. species 10 P. species 10 P. species 7 P. species 7 P. species 7 P. species 7 P. species 10 P. species 10 P. species 10 P. species 7 P. species 10 P. species 10 P. species 2 P. species 10

72083 77424 74167 77423 77432 77433 77286 77431 77425 77430 74169 77426 77427

Collection REB2111 (RFLP type D) is readily distinguished by its dark brown spore mass, coarsely echinate spore ornamentation with spines ‘coalescent’ (Kope & Fortin, 1990) under SEM, and large spores. Collection REB2078 (RFLP type B) also has large spores, but differs in its pale brown spore mass and coarsely echinulate spore ornamentation, with the spines not coalescent under SEM. Collections REB2047 (RFLP type C) and REB2050 (RFLP type A) form one group, distinguished by its relatively small, finely echinulate spores with spines coalescent under SEM and pale brown spore mass. Distribution of Pisolithus species along the Taupo volcanic zone Between one and three species of Pisolithus were harvested in each site (Table 4). Three RFLP types were observed in the most visited sites (Tauhara, Karapiti, Te Kopia). The most common fruit body species amongst sites belonged to Pisolithus species 10 (RFLP type A, C) and Pisolithus species 7. Observation of Pisolithus species 10 (RFLP type A) on another related plant host taxa (L. scoparium) in Kuirau Park in a geothermally active area (13°C at 2 cm soil depth and 17°C at 8 cm depth) demonstrates the capacity of this Pisolithus species to associate with a broad range of Leptospermoideae.


GenBank accession

AY318747

AY318746 AY318745

AY318748

Co-occurrence of Pisolithus species below ground Ectomycorrhizas were collected beneath six fruit bodies belonging to three different Pisolithus species (Table 5) from 4 sites (Tokaanu, Tauhara, Kuirau Park, Te Kopia). With only one exception, the fruit body RFLP type was found below ground. However, observation in two soil cores of two different EcM RFLP types belonging to either C and D or C and B demonstrates that two different Pisolithus species can coexist in a small scale, in the same soil volume in Tauhara. The presence of Pisolithus species 10 (RFLP type C) below Pisolithus species 10 (RFLP type A) fruit body (REB2106) demonstrates the coexistence of several fungi belonging to different population of the same species within the same soil volume.

Discussion Genetic diversity of NZ Pisolithus and phylogenetic relationships with species elsewhere Three Pisolithus genotypes matching species occurring naturally in Australia were identified along the main volcanic zone of NZ, in geothermal conditions. ITS sequence similarity of

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576 Research Table 5 ITS-RFLP type and identity of Pisolithus ectomycorrhizas collected below single Pisolithus fruit bodies, associated with K. ericoides var. microflora or L. scoparium

Site

Fruit body collection

Fruit body RFLP type

Fruit body identity

EcM RFLP type

EcM identity

Tokaanu Kuirau Park Tauhara

REB2045 REB2025 REB2082

A A

P. species 10 P. species 10 *P. species 2

A A C, D

Tauhara

REB2078

B

P. species 7

C, B

Tauhara Karapiti

REB2106 REB2056

A C

P. species 10 P. species 10

C C

P. species 10 P. species 10 P. species 10, P. species 2 P. species 10, P. species 7 P. species 10 P. species 10

Host K. ericoides L. scoparium K. ericoides K. ericoides K. ericoides K. ericoides

*Identified on basis of spore anatomy.

NZ collections with Australian ones was particularly great for Pisolithus species 2 (REB2111) and Pisolithus species 7 (REB2078) (up to 99.8% and 98.6%, respectively) and lower for Pisolithus species 10 (REB2050, REB2047) (up to 96.9%). The number of Pisolithus species 10 ITS sequences available to date is inadequate to define genetic divergence reliably within this clade. Mapping the predicted restriction sites in the ITS region for REB2050 (RFLP type A) and REB 2047 (RFLP type C) showed that differences in RFLP patterns were due to single base transitions in three different locations (data not shown). Intraspecific variations in RFLP types in Pisolithus have previously been reported by Anderson et al. (2001a) in their Clade I. They mentioned that TaqI produced most within-species RFLP pattern variations. This restriction enzyme was selected in our study to maximize the possibility of observing genetic variation amongst NZ collections. In our case, this enzyme proved efficient to separate out three species, including one with two different RFLP patterns. Recent results using ITS sequence analysis (Anderson et al., 2001a; Martin et al., 2002), ITS RFLP (Anderson et al., 2001a) and intersimple sequence repeat PCR (Hitchcock et al., 2003) support the segregation of the Anderson et al. (2001a) Clade I into two genotypes corresponding to species 7 and 9 in the Martin et al. (2002) phylogenetic tree. Amongst these two species, only one consistent type of RFLP pattern (B), with an ITS sequence similar to Australian collections belonging to species 7, was observed in NZ geothermal areas. Taxonomy of Pisolithus in New Zealand The taxonomy of Pisolithus is confused, and modern treatments that include Australasian material are limited. Cunningham (1944) recognized P. tinctorius and P. microcarpus, the former he considered to be widespread around the world, including both Australia NZ, and the latter restricted to Australia. Grgurinovic (1997) recognized three species in South Australia, P. microcarpus, Pisolithus sp. indet., and P. tinctorius. Bougher & Syme (1998) noted that, according to M. Priest,

there are 6 species in Australia, and provided descriptions of Western Australian material they identified as P. albus and P. marmoratus. The name P. albus is based on Polysaccum album Cooke & Massee, and P. marmoratus is based on Polysaccum marmoratum Berk. Bougher & Syme (1998) concluded that the taxon they identify as P. marmoratus is the Southern Hemisphere counterpart to P. tinctorius, which is a nom. illegit. now accepted as a synonym of P. arrhizus (Rauschert, 1959). Martin et al. (2002), using ITS sequence data alone, demonstrated the existence of five major clades (phylogenetic species) in collections from Australia, excluding some that were recovered only once. Collection REB2111, representing our RFLP type D, clearly falls into Clade II group 4 as recognized by Anderson et al. (2001a), who tentatively recognized this as P. marmoratus sensu Bougher & Syme (1998) on the basis of brown spore mass, spore size, and spore ornamentation of coalesced spines when examined by SEM. In the more comprehensive ITS analysis of Martin et al. (2002), which included many of the sequences used by Anderson et al. (2001a), it falls into Clade species 2 identified as P. marmoratus on the basis of sequence KS781 obtained from the collection used as the basis of the published description of this taxon by Bougher & Syme (1998). Our collection reasonably matches the descriptions of Bougher & Syme (1998) and of Anderson et al. (2001a). We conclude that our RFLP type D represents P. marmoratus sensu Bougher & Syme (1998). The known natural range of this species now extends from Australia (WA, NSW) to NZ. In addition, morphological features of our collection reasonably match the description of P. tinctorius provided by Grgurinovic (1997), suggesting that her South Australian material could represent the same species. Collection REB2078, representing RFLP B, falls into Clade I group 2 as recognized by Anderson et al. (2001a), who tentatively recognized this clade as P. albus sensu Bougher & Syme (1998) characterized by ochraceous spore mass, spore size, and spore ornamentation of non-coalesced spines when examined by SEM. In the analysis of Martin et al. (2002) it


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falls into Clade species 7, considered by these authors to match P. albus morphologically, but there is no sequence available for collections examined by Bougher & Syme (1998). Our collection matches reasonably the description of Clade I group 2 of Anderson et al. (2001a), but appears to lack the ‘persistently white peridium’ reported for P. albus by Bougher & Syme (1998). Our observations extend the known natural range of Pisolithus species 7 from Australia (WA, NSW, Qld, Victoria, ACT) to include NZ. Collections REB2047 and 2050, representing our RFLP types C and A, fall into Clade species 10 of Martin et al. (2002). These authors suggest the possibility that species 10 may comprise separate Western Australian and Eastern Australian species, although our analysis does not support this proposal. No morphological data are presently available for this species. Our data indicate it resembles P. albus in its spore mass colour, but differs in its smaller more finely echinulate spores with coalescent spines under SEM. This conclusion extends the known natural range of this species from Australia (WA, Qld) to include NZ. One taxon of Pisolithus has been described from NZ material, namely P. arenarius var. novozelandicus Henn., 1894, Bot. Jahrb. Syst. 18 (Beibl. 44): 37, as ‘novo-zeelandica’. [Type: ‘Neu-Seeland, Nord-Insel, Wairakei-valley, am Boden (29. August 1889. n.116)’], which was cited incorrectly by Cunningham (1944) as Polysaccum pisocarpium var. novo-zelandica P. Henn. The nature of this specimen, which we note is from the thermal region, would require examination of the type material. However, as it was only described at the varietal level, it is not necessary to recognize it, when considering appropriate names to be applied to NZ taxa. Co-occurrence of Pisolithus species in New Zealand geothermal areas Díez et al. (2001) reported patterns of host and ecological specificity that may account for Pisolithus regional diversity in the Mediterranean. By contrast, we observed rich Pisolithus species communities in association with a single host, in the same habitat, in NZ geothermal areas. The three species were distributed along the Taupo volcanic zone, and our fruit bodies survey showed that all three species can coexist in a single area of approximately 1 ha (Tauhara site) where they associated with the single host K. ericoides var. microflora. Anderson et al. (1998b, 2001b) showed, on the basis of fruit body surveys, a patchy distribution of Pisolithus genets within the same species. Hitchcock et al. (2003) discussed the possibility that the Anderson et al. (2001b) North Wilberforce field site might include a mixture of two species. Our results extend these observations by demonstrating the local cooccurrence in NZ geothermal areas of three Pisolithus species. The two-dimensional, patchy distribution of Pisolithus genets reported by Anderson et al. (2001b), together with the cooccurrence of EcMs from several species in a small soil volume


in this study, suggest a priori that different Pisolithus genotypes may share the same ecological niche. Putative origin of Pisolithus in New Zealand The great genetic similarity of the three Pisolithus species occurring naturally in NZ and Australia raises questions about the processes that led to this disjunct distribution of EcM fungal taxa. The following three processes have been proposed to explain present distribution and host specificity of ectomycorrhizal fungi worldwide (Halling, 2001): ancestral generalist fungi were distributed over large geographic areas that subsequently split during geological times, with possible allopatric speciation after isolation; some fungi switched plant hosts as a consequence of vegetation history; and mycorrhizal communities co-migrated over land or via ‘island hopping’. The only ancient EcM host taxon in NZ with ancestral Gondwanan distribution is Nothofagus (Veblen et al., 1996). Among other EcM plant taxa, Australian genera known today as EcM, such as Eucalyptus, Acacia and Casuarina, were also present in NZ at times, during the Tertiary (Mildenhall, 1980). Leptospermum pollen records (difficult to separate from Kunzea, P. de Lange, pers. com.) are recent. The precise period of appearance of the genus in NZ is under debate (e.g. Harris, 2002), but it is widely accepted that this genus arrived to NZ from Australia by long-distance trans-Tasman dispersal. Establishment in NZ of Leptospermoideae including Leptospermum and Kunzea might have been facilitated by their capacity to associate with local Nothofagus species EcM inoculum. Malloch et al. (1980) proposed the hypothesis that mycosymbionts associated with Eucalyptus and other Leptospermoideae in the Southern Hemisphere spread there with Nothofagus and its ancestors. This hypothesis is favoured by taxonomic and experimental data; and host range broadening by typically Nothofagus associated fungi is the process most often reported in the literature to explain the origin of the mycoflora associated with Myrtaceae (e.g. Bougher et al., 1994; Halling, 2001). However, Pisolithus has not yet been observed with Nothofagus worldwide, therefore the hypothesis of a host change from Nothofagus to Leptospermoideae is unlikely. On the other hand, early NZ separation from Australia would probably have led to allopatric speciation of a Pangean Pisolithus ancestor formerly distributed in NZ and Australia. By contrast, the genetic similarity between Australian and NZ collections suggests recent dispersal of Pisolithus between the two countries. Although the co-introduction of Pisolithus sp. with eucalypts has been documented in several countries (Martin et al., 1998; Gomes et al., 2000; Díez et al., 2001), there is no evidence of introduction of this fungus via eucalypt planting activities in NZ. Various eucalypt species have been in cultivation since the late 1800s, with at least one species present in Taupo volcanic zone (Rotorua) in 1897 (Weston, 1957). Nevertheless, there are no records of Pisolithus associated with eucalypts in the National Fungal Collection

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(Herbarium PDD) and Pisolithus was not recorded in association with Eucalyptus spp. by Chu-Chou & Grace (1983). We have surveyed eucalypts in the vicinity of Kawerau field site and near geothermal areas on the margin of Lake Rotorua and did not observe Pisolithus fruit bodies. An alternative process is long-distance dispersal of Pisolithus between Australia and NZ. Long-distance dispersal has been demonstrated for spores belonging to a wide range of lower plants and microorganisms (Close et al., 1978; McKenzie, 1998; McDaniel & Shaw, 2003). Although eastward migration is favoured by prevailing westerly winds from Australia to NZ, upwind migrations are also responsible for the colonization of NZ flora in Australia (McDaniel & Shaw, 2003). McKenzie (1998) demonstrated that the long-distance dispersal process could also involve biotrophic rust fungi. Taking into account NZ’s biogeographic history and the high genetic similarity between Australian and NZ Pisolithus, we hypothesize that several recent long-distance, trans-oceanic dispersal events of Pisolithus species between Australia and NZ were made possible by a combination of the following circumstances: development of west-wind drift related to separation of Australia from Antarctica in the Paleocene (Fell, 1962); the presence of suitable plant hosts belonging to Leptospermoideae in NZ since the Tertiary and their capacity to cope with particularly harsh edaphic conditions such as are found in geothermal areas (Given, 1980; Burns, 1997) – independent migration of these host plants might have been facilitated by their capacity to associate with AM and/or to share EcM fungi with Nothofagus (Baylis, 1962; Cooper, 1976; Moyersoen & Fitter, 1999); and the development of volcanoes subsequent to the Pliocene in the Central North Island (Stevens, 1980) with associated geothermal areas in which Pisolithus is successful. In conclusion, our study shows that at least three Pisolithus species dispersed from Australia where they are distributed in large areas, successfully established in a new habitat in NZ, characterized by extremely acidic, low nutrient and particularly active geothermal soils. Their co-occurrence in small areas and within the same soil volume suggests the new ecological niche occupied by these species overlaps. Pisolithus in NZ geothermal areas offer an excellent model to understand how three taxonomically closely related EcM fungal species, associated with a single plant host can coexist in similar ecological conditions without mutual competitive exclusion.

Acknowledgements This study was funded by Landcare Research. Nick Singers (Department of Conservation, Turangi) assisted with identifying collection sites and Paul Cashmore (Department of Conservation, Rotorua) provided the collection from Motuhora Island. Molecular analysis at INRA were supported by grants’ programmes ‘Sequencing genomes of symbionts and pathogens’ and LIGNOME. The research utilized in part

the DNA Sequencing Facilities at INRA-Nancy financed by the INRA, Région de Lorraine and the European Commission. We thank B. Burns, V. Demoulin, R. Howitt, C. Delaruelle, D. Park, T. Buckley, B. Rhode for advice and S. Pennycook and P. Johnston for comments on the manuscript.

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