These authors contributed equally to this work.
Phylogeography of the ectomycorrhizal Pisolithus species as inferred from nuclear ribosomal DNA ITS sequences
Article first published online: 1 FEB 2002
Volume 153, Issue 2, pages 345–357, February 2002
How to Cite
Martin, F., Díez, J., Dell, B. and Delaruelle, C. (2002), Phylogeography of the ectomycorrhizal Pisolithus species as inferred from nuclear ribosomal DNA ITS sequences. New Phytologist, 153: 345–357. doi: 10.1046/j.0028-646X.2001.00313.x
- Issue published online: 1 FEB 2002
- Article first published online: 1 FEB 2002
- Received: 22 June 2001 Accepted: 3 October 2001
- ectomycorrhizal fungus;
- internal transcribed spacers;
- phylogenetic species;
- ribosomal DNA
- Top of page
- Materials and Methods
- •The fungal genus Pisolithus is cosmopolitan in warm temperate regions and forms ectomycorrhizal associations with a wide range of woody plants. To delimit phylogenetic Pisolithus species and identify their geographical distribution, 102 collections were made worldwide and their rDNA internal transcribed spacer (ITS) was sequenced.
- •Phylogenetic analyses of these sequences, together with 46 additional GenBank accessions, identified 11 species.
- •A strong phylogeographical pattern was observed related to the native geographical origin of the host plants. In the Holarctic, P. tinctorius was widely distributed, associated with Pinus and Quercus. It has been co-introduced with pines to other biogeographical regions. Several Pisolithus lineages, including P. aurantioscabrosus, occurred in restricted biogeographical regions associated with endemic plants, such as Afzelia in eastern Africa. Pisolithus albus, P. marmoratus and P. microcarpus were associated with Australasian hosts (Eucalyptus, Acacia) and were distributed with their hosts worldwide. By contrast, two additional unnamed species were restricted to Australia.
- •The present study shows that evolutionary lineages within Pisolithus are related to the biogeographical origin of the hosts. In addition, regional floras and endemic plants could act as hosts of endemic species of Pisolithus.
- Top of page
- Materials and Methods
The fungal genus Pisolithus is cosmopolitan in warm temperate regions of the world and forms ectomycorrhizal associations with a wide range of woody plants (Marx, 1977; Chambers & Cairney, 1999) including members of the Pinaceae, Myrtaceae, Fagaceae, Mimosaceae, Dipterocarpaceae and Cistaceae. Although considerable heterogeneity exists in terms of sporocarp, spore and isolate culture morphology, taxa within the genus Pisolithus have been widely regarded as conspecific and grouped as Pisolithus tinctorius (Chambers & Cairney, 1999). Pisolithus collections from throughout the world differ in their ecology, morphological and molecular characteristics, symbiotic efficiency, physiology and geographical distribution (Lamhamedi et al., 1990; Burgess et al., 1994, 1995; Anderson et al., 1998, 2001; Martin et al., 1998; Gomes et al., 2000; Díez et al., 2001). Based on mating tests, Kope & Fortin (1990) proposed that the genus Pisolithus comprises several biological species. DNA-based analysis further supported the occurrence of several phylogenetic species of Pisolithus. So far, four major clades were identified within two main phylogenetic lineages (Anderson et al., 1998, 2001; Martin et al., 1998; Gomes et al., 2000; Díez et al., 2001). Several morphological species of Pisolithus are common in forests and woodlands in Australia (P. microcarpus (Cke & Mass.) Cunn.) (Cunningham, 1942; Bougher & Syme, 1998), in tropical rainforests of southeast Asia, [P. kisslingi Fisch. and P. aurantioscabrosus Wat. (Watling et al., 1995, 1999)], and in New Caledonia (P. pusillum Pat.). Two additional Australian species have recently been provisionally described: P. albus (Cooke & Mass.) M. J. Priest, nom. prov., and P. marmoratus (Berk.) Priest, nom. prov. (Bougher & Syme, 1998). The total number of identified Pisolithus species may be as many as 10 (Sims et al., 1999; Anderson et al., 2001). There is thus growing consensus that the genus Pisolithus is more genetically diverse than was previously assumed and that taxonomic revision on a global scale is required (Bougher & Syme, 1998; Martin et al., 1998; Chambers & Cairney, 1999). The accurate delimitation and naming of those species is crucial because a wealth of biological information is linked with the current names.
The dispersal of ectomycorrhizal trees, such as eucalypts and pines, outside their natural range, has resulted in the introduction of a narrow range of exotic ectomycorrhizal fungi into forest plantations in many countries of Europe, South America and Africa (Marx, 1977; Garbaye et al., 1988; Martin et al., 1998; Gomes et al., 2000; Díez et al., 2001). Whether the distribution of these fungi extend beyond these areas depends on the host range of the fungi and their ability to compete with indigenous species.
Studies that are based on DNA sequences serve to re-evaluate earlier classifications and provide more accurate species delimitation (Taylor et al., 2000). Studies of phylogeny and its implications for understanding the biogeography of ectomycorrhizal fungi showing cryptic speciation have been progressing significantly in recent years as a result of DNA sequencing (Kretzer et al., 1996; Jarosh & Bresinsky, 1999; Wu et al., 2000). To date, the phylogeny of Pisolithus has been assessed using a single locus, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), on a limited number of isolates from a restricted number of geographical regions: New South Wales (NSW) (eastern Australia) (Anderson et al., 1998), Kenya (Martin et al., 1998), Brazil (Gomes et al., 2000) and the western Mediterranean region (Díez et al., 2001).
The objectives of this study were first to determine the phylogenetic relationships among Pisolithus species and second to extend the understanding of their host specificity and biogeographical patterns.
Materials and Methods
- Top of page
- Materials and Methods
The 102 isolates of Pisolithus analysed in this study were collected from a variety of geographical localities (i.e. ecosystems) throughout the world (Table 1) and deposited at the Collections of Ectomycorrhizal Fungi of the Trees–Microorganisms Interactions Unit (INRA-Nancy, France), School of Biological Sciences and Biotechnology (Murdoch University, WA, Australia), and CSIRO Division of Forestry and Forest Products (Wembley, WA, Australia). Morphological descriptions of representative specimens were reported in Burgess et al. (1995), Watling et al. (1995, 1999), Anderson et al. (1998, 2001), Martin et al. (1998), Cairney et al. (1999), and Díez et al. (2001). Samples collected by the authors were excised from the central tissues of the basidiome stipe to avoid contamination by other fungi. Cultures obtained from basidiome were maintained on modified Melin Norkrans’s (MMN) medium agar (Marx, 1981) or Pachlewski medium (Martin et al., 1998) at 25°C with subculturing every 2–3 months.
|Isolate no.||Host1||Locality||Code||GenBank accession|
|(1) 5105||Afzelia quanzensis||Arabuko-Sokoke Forest Reserve, Kenya||5105_Afz_Ara_Ken||AF0039153|
|(2) K915||A. quanzensis||Arabuko-Sokoke Forest Reserve, Kenya||K915_Afz_Ara_Ken||AF2286535|
|(3) COI 24||Acacia holosericea||Sinthian Malène, Senegal||COI24_Aca_Sin_Sen||AF374622|
|(4) COI 07||A. mangium||Casamance, Senegal||COI07_Aca_Cas_Sen||AF374623|
|(5) CSH4461||Acacia sp.||Townsville, Queensland||CSH4461_Aca_Two_Qld||AF374624|
|(6) Gemas||A. mangium||Gemas, Malaysia||Gemas_Aca_Gem_Mal||AF374638|
|(7) Pasoh01||Shorea macroprera||Forest Service, Pasoh, Malaysia||Pasoh01_Sho_Pas_Mal||AF415226 & AF415227|
|Pinus, Quercus or Cistus|
|(8) Pt301||Pinus sp.||Georgia, USA||Pt301_Pin_Geo_USA||AF1432334|
|(9) MH208||P. pinaster||unknown, Portugal||MH208_Pin_unk_Por||AF374626|
|(10) MP9812 (CSH557)||Pinus sp.||Voeltjiesdord, South Africa||MP9812_Pin_Voe_SAf||AF374627|
|(11) MP9813 (CSH558)||P. radiata||Grabouw, South Africa||MH9813_Pin_Grb_SAf||AF374628|
|(12) PtJap (MH175)||P. pumila/Betula ermanii||Mt Iou, Shiretoko Peninsula,||PtJapan_Pin_Iou_Jap||AF374629|
|(13) Hel1 (MH177)||P. pinaster/Quercus suber||unknown, Portugal||Hel1_Pin/Que_unk_Por||AF374630|
|(14) MSN (MH181)||P. kesiya||Mae Sa Nam, Chiangmai||MSN_Pin_Mae_Tha||AF374625|
|(15) THAI 1 (MH140)||P. kesiya||Chiangmai Province,||THAI1_Pin_Chi_Tha||AF374631|
|(16) MARX270||P. elliotii||Georgia, USA||MARX270_Pin_Geo_USA||AF374632|
|(17) 5111||P. caribaea||Arabuko, Kenya||5111_Pin_Ara_Ken||AF0039163|
|(18) esp07||P. caribaea||Arabuko, Kenya||esp07_Pin_Ara_Ken||AF228647|
|(19) NIC (S382)||P. caribaea||Puerto Cabezas, Nicaragua||NIC(S382)_Pin_Pue_Nic||AF374633|
|(20) OAK||Pinus sp.||Georgia, USA||OAK_Que_Geo_USA||AF374634|
|(21) ATH||Pinus sp.||Georgia, USA||ATH_Que_Geo_USA||AF374635|
|(22) Pt92JG (MH171)||P. pinaster/Q. suber||Alcácer du Sal, Portugal||Pt92JG_Pin/Que_Alc_Por||AF374636|
|cab01||Cistus ladanifer||Cabañeros National Park, Ciudad Real, Spain||cab01_Cis_Cab_Spa||AF2286445|
|(23) ch01||Q. ilex/C. ladanifer||Chapiñeria, Madrid, Spain||ch01_Que_Cha_Spa||AF2286455|
|(25) ch02||Q. ilex/C. ladanifer||Chapiñeria, Madrid, Spain||ch02_Que_Cha_Spa||AF2286465|
|(26) cr01||C. ladanifer||Abenojar, Ciudad Real, Spain||cr01_Cis_Abe_Spa||AF2286415|
|(27) cr02||C. ladanifer||Abenojar,Ciudad Real, Spain||cr02_Cis_Abe_Spa||AF2286425|
|(28) cr04||C. ladanifer||Abenojar, Ciudad Real, Spain||cr04_Cis_Abe_Spa||AF2286435|
|(29) gr13||P. halepensis/Q. coccifera||Granada, Spain||gr13_Pin/Que_Gra_Spa||AF2286505|
|(30) m14||P. halepensis||Moratalla, Murcia, Spain||m14_Pin_Mor_Spa||AF2286525|
|(31) pt03||Q. ilex/Q. coccifera||Valencia, Spain||pt03_Que_Val_Spa||AF2286485|
|(32) pt04||Q. ilex||Fuentidueña, Spain||pt04_Que_Fue_Spa||AF2286495|
|(33) pt05||Q. ilex||Fuentidueña, Spain||pt05_Que_Fue_Spa||AF2286515|
|(34) MH143||P. yunnanensis||Kunming, China||MH143_Pin_Kun_Chi||AF374717|
|(35) H529||Pinus sp.||unknown, USA||H529_Pin_unk_USA||AF374709|
|(36) F22||P. pinaster||Lac d’Aureilhan, Mimizan, France||F22_Pin_Mim_Fra||AF374707|
|(37) H614||P. elliotii||Kaiping, China||H614_Pin_Kai_Chi||AF374710|
|(38) H613||Pinus/Eucalyptus||Kaiping, China||H613_Pin/Euc_Kai_Chi||AF374711|
|(39) H237||Pinus sp.||unknown, France||H237_Pin_unk_Fra||AF374712|
|Eucalyptus plantations and native forests in Australia|
|(40) MU98/20||E. calophylla||Karridale, Western Australia||MU98/20_Euc_Kar_WA||AF374637|
|(41) MU98/15||E. calophylla||North Augusta, Western Australia||MU98/15_Euc_Aug_WA||AF374639|
|(42) WM01||Eucalyptus sp.||Westmead, New South Wales||WM01_Euc_Wes_NSW||AF0047342|
|(43) CS01||Eucalyptus sp.||unknown, New South Wales||CS01_Euc_unk_NSW||AF0047322|
|(44) LJ07||Eucalyptus sp.||N. Turramurra, New South Wales||LJ07_Euc_Tur_NSW||AF0047332|
|(45) R01||Eucalyptus sp.||unknown, New South Wales||R01_Euc_unk_NSW||AF0047352|
|(46) MU98/1||E. marginata||Augusta, Western Australia||MU98/1_Euc_Aug_WA||AF374640|
|(47) MU98/2||E. marginata||Augusta, Western Australia||MU98/2_Euc_Aug_WA||AF374641|
|(48) MU98/3||E. marginata||Augusta, Western Australia||MU98/3_Euc_Aug_WA||AF374642|
|(49) MU98/4||E. marginata||N. Augusta, Western Australia||MU98/4_Euc_Aug_WA||AF374643|
|(50) MU98/5A||E. globulus||Kudardup, Western Australia||MU98/5A_Euc_Kud_WA||AF374644|
|(51) MU98/5B||E. globulus||Kudardup, Western Australia||MU98/5B_Euc_Kud_WA||AF374645|
|(52) MU98/6||E. globulus||Scott R, Western Australia||MU98/6_Euc_Sco_WA||AF374646|
|(53) MU98/7||E. globulus||Scott R, Western Australia||MU98/7_Euc_Sco_WA||AF374647|
|(54) MU98/8||E. globulus||Scott R, Western Australia||MU98/8_Euc_Sco_WA||AF374648|
|(55) MU98/9||E. globulus||Scott R, Western Australia||MU98/9_Euc_Sco_WA||AF374649|
|(56) MU98/10||E. marginata||W. Manjimup, Western Australia||MU98/10_Euc_Man_WA||AF374650|
|(57) MU98/11||E. marginata||E. Manjimup, Western Australia||MU98/11_Euc_Man_WA||AF374651|
|(58) MU98/12||Eucalyptus spp.||Manjimup, Western Australia||MU98/12_Euc_Man_WA||AF374652|
|(59) MU98/13||Eucalyptus spp.||Manjimup, Western Australia||MU98/13_Euc_Man_WA||AF374653|
|(60) MU98/14||Eucalyptus spp.||South-west, Western Australia||MU98/14_Euc_Sou_WA||AF374654|
|(61) MU98/16||E. globulus||Kudardup, Western Australia||MU98/16_Euc_Kud_WA||AF374655|
|(62) MU98/17||E. globulus||Kudardup, Western Australia||MU98/17_Euc_Kud_WA||AF374656|
|(63) MU98/18||E. globulus||Kudardup, Western Australia||MU98/18_Euc_Kud_WA||AF374657|
|(64) MU98/19||E. globulus||Kudardup, Western Australia||MU98/19_Euc_Kud_WA||AF374658|
|(65) MU98/21||E. globulus||Albany, Western Australia||MU98/21_Euc_Alb_WA||AF374659|
|(66) MU98/22||E. globulus||Albany, Western Australia||MU98/22_Euc_Alb_WA||AF374660|
|(67) MU98/101||E. camaldulensis||S. Eneabba, Western Australia||MU98/101_Euc_Ene_WA||AF374661|
|(68) MU98/102||Eucalyptus/Acacia||Eneabba, Western Australia||MU98/102_Euc/Aca_Ene_WA||AF374662|
|(69) MU98/103||Eucalyptus/Acacia||Eneabba, Western Australia||MU98/103_Euc/Aca_Ene_WA||AF374663|
|(70) MU98/104||E. globulus||N. Albany, Western Australia||MU98/104_Euc_Alb_WA||AF374664|
|(71) MU98/105||E. patens||Kalamunda, Western Australia||MU98/105_Euc_Kal_WA||AF374665|
|(72) CSH4338||Eucalyptus sp., Melaleuca sp.||Atherton, Queensland||CSH4338_Euc_Ath_Qld||AF374666|
|(73) CSH4742||E. grandis||Mt Spec, Queensland||CSH4742_Euc_MtS_Qld||AF374667|
|(74) CSH5822||Eucalyptus sp.||New England National Park, New South Wales||CSH5822_Euc_New_NSW||AF374668|
|(75) MH19 (CSH445)||E. marginata||Jarrahdale, Western Australia||MH19_Euc_Jar_WA||AF374669|
|(76) H4937 (MH132)||E. tereticornis, E. tesselaris||Fanning River, Queensland||H4937_Euc_FR_Qld||AF374670|
|(77) MH155 (CSH1101)||E. globulus||Northcliffe, Western Australia||MH155_Euc_Nor_WA||AF374671|
|(78) MH156 (CSH4334)||Eucalyptus sp.||Atherton Tableland, Quensland||MH156_Euc_Ath_WA||AF374672|
|(79) MH158 (CSH6386)||E. urophylla||Guangdong, China||MH158_Euc_Gua_Chi||AF374673|
|(80) MH730 (CSH4111)||Eucalyptus sp.||Beerwah, Queensland||MH730_Euc_Bee_Qld||AF374674|
|(81) CSH4339 (MH731)||Eucalyptus sp.||Atherton Tableland, Queensland||MH731_Euc_Ath_Qld||AF374675|
|(82) MH732 (CSH6291)||Eucalyptus sp.||Cardwell, Queensland||MH732_Euc_Car_Qld||AF374676|
|(83) MH97||Eucalyptus sp., Melaleuca sp.||N. Morawa, Western Australia||MH97_Euc_Mow_WA||AF374713|
|(84) MH115||Eucalyptus/Acacia||Wittenoom, Western Australia||MH115_Euc/Aca_Wit_WA||AF374714|
|(85) MH22||Eucalyptus/Acacia||Wubin, Western Australia||MH22_Euc/Aca _Wub_WA||AF374715|
|(86) H4111||Eucalyptus sp.||unknown, Queensland||H4111_Euc_unk_Qld||AF374718|
|(87) MH56||Eucalyptus/Acacia||Kalbarri, Western Australia||MH56_Euc/Aca _Klb_WA||AF374708|
|(88) SA01||Eucalyptus sp.||Uraidla, Adelaide Hills, South Australia||SA01_Euc_Ura_SA||AF2707876|
|(89) QLD05||Eucalyptus sp.||Mareeba, North Queensland||QLD05_Euc_Mar_Qld||AF2707866|
|(90) QLD01||Eucalyptus sp.||Wangetti, North Queensland||QLD01_Euc_Wan_Qld||AF2707856|
|(91) NSW5||Eucalyptus sp.||Ballina, New South Wales||NSW5_Euc_Bal_NSW||AF2707846|
|(92) QLD02||Eucalyptus sp.||Wangetti, North Queensland||QLD02_Euc_Wan_Qld||AF2707836|
|(93) CA02||Eucalyptus sp.||Canberra, Australia Capital Territory||CA02_Euc_Can_ACT||AF2707826|
|(94) VIC01||Eucalyptus sp.||Warrandyte, Victoria||VIC01_Euc_War_Vic||AF2707816|
|(95) WM02||Eucalyptus sp.||Westmead, New South Wales||WM02_Euc_Wes_NSW||AF2707806|
|(96) W34||Eucalyptus sp.||N. Wilberforce, New South Wales||W34_Euc_Wil_NSW||AF2707796|
|(97) R08||Eucalyptus sp.||Wattamolla, New South Wales||R08_Euc_Wat_NSW||AF2707786|
|(98) LJ30||Eucalyptus sp.||N. Turramurra, New South Wales||LJ30_Euc_Tur_NSW||AF2707746|
|(99) BW02||Eucalyptus sp.||Patonga, New South Wales||BW02_Euc_Pat_NSW||AF2707736|
|(100) BW01||Eucalyptus sp.||Patonga, New South Wales||BW01_Euc_Pat_NSW||AF2707726|
|(101) BTH1||Eucalyptus sp.||Bathurst, New south Wales||BTH1_Euc_Bat_NSW||AF2707716|
|(102) MH2||E. saligna||Perth, Western Australia||MH2_Euc_Per_WA||AF374716|
|(103) KS781||E. calophylla||Denmark, Western Australia||KS781_Euc_Den_WA||AF374719|
|Eucalyptus plantations outside Australia|
|(104) mam17||Eucalyptus sp.||Mammora Forest, Morocco||mam17_Euc_Mam_Mor||AF2286575|
|(105) mar01||Eucalyptus sp.||Mammora Forest, Morocco||mar01_Euc_Mam_Mor||AF2286545|
|(106) mar02||Eucalyptus sp.||Mammora Forest, Morocco||mar02_Euc_Mam_Mor||AF2286555|
|(107) ast05||Eucalyptus sp.||The Miravete Pass, Cáceres, Spain||ast05_Euc_Mir_Spa||AF2286565|
|(108) 5110||E. camaldulensis||Gede Forest Station, Kenya||5110_Euc_Ged_Ken||AF0039143|
|(109) 441||E. citriodora||Sao Paulo, Brazil||441_Euc_Sao_Bra||U626661|
|(110) Pt90A||Eucalyptus sp.||Vizcosa, Brazil||Pt90A_Euc_Viz_Bra||AF1405474|
|(111) MH210||E. globulus||unknown, Portugal||MH210_Euc_unk_Por||AF374677|
|(112) MH727 (CSH705)||E. urophylla||Zenhai, China||MH727_Euc_Zen_Chi||AF374678|
|(113) MH728 (CSH708)||Eucalyptus sp.||Yanxi, China||MH728_Euc_Yan_Chin||AF374679|
|(114) MH729 (CSH709)||E. grandis||Yanxi, China||H729_Euc_Yan_Chi||AF374680|
|(115) MH745 (CSH710)||E. urophylla||Guangdong, China||MH745_Euc_Gua_Chi||AF374681|
|(116) MP9811 (CSH556)||Eucalyptus sp.||Wiesonhof, South Africa||MP9811_Euc_Wie_SAf||AF374682|
|(117) MP9814 (CSH753)||E. tereticornus||Tamil Nadiu, India||MP9814_Euc_Tam_Chi||AF374683|
|(118) HCX1||E. maidenii||Chuxiong, China||HCX1_Euc_Chu_Chi||AF374684|
|(119) COI03||E. camaldulensis||Bandia, Senegal||COI03_Euc_Ban_Sen||AF374685|
|(120) COI05||E. camaldulensis||Djibelor, Senegal||COI05_Euc_Dji_Sen||AF374686|
|(121) COI12||E. camaldulensis||Kabrousse, Senegal||COI12_Euc_Kab_Sen||AF374687|
|(122) COI15||E. camaldulensis||Casamance, Senegal||COI15_Euc_Cas_Sen||AF374688|
|(123) COI16||E. camaldulensis||Casamance, Senegal||COI16_Euc_Cas_Sen||AF374689|
|(124) COI22||E. camaldulensis||Casamance, Senegal||COI22_Euc_Cas_Sen||AF374690|
|(125) COI25||E. camaldulensis||Casamance, Senegal||COI25_Euc_Cas_Sen||AF374691|
|(126) COI26||E. camaldulensis||Casamance, Senegal||COI26_Euc_Cas_Sen||AF374692|
|(127) COI27||E. camaldulensis||Casamance, Senegal||COI27_Euc_Cas_Sen||AF374693|
|(128) COI30||E. camaldulensis||Ngan, Senegal||COI30_Euc_Nga_Sen||AF374694|
|(129) R15||E. globulus||Castelo Branco, Portugal||R15_Euc_Cst_Por||AF374695|
|(130) UFSC-Pt22||Eucalyptus sp.||Três Barras, SC, Brazil||UFSC22_Euc_Trê_Bra||AF374696|
|(131) UFSC-Pt23||Eucalyptus sp.||UFSC, Florianópolis, SC, Brazil||UFSC23_Euc_Flo_Bra||AF374697|
|(132) UFSC-Pt25||Eucalyptus sp.||UFSC, Florianópolis, SC, Brazil||UFSC25_Euc_Flo_Bra||AF374698|
|(133) UFSC-Pt26||Eucalyptus sp.||UFSC, Florianópolis, SC, Brazil||UFSC26_Euc_Flo_Bra||AF374699|
|(134) UFSC-Pt27||Eucalyptus sp.||UFSC, Florianópolis, SC, Brazil||UFSC27_Euc_Flo_Bra||AF374700|
|(135) UFSC-Pt44||Eucalyptus sp.||UFSC, Florianópolis, SC, Brazil||UFSC44_Euc_Flo_Bra||AF374701|
|(136) UFSC-Pt49||Eucalyptus sp.||UFSC, Florianópolis, SC, Brazil||UFSC49_Euc_Flo_Bra||AF374702|
|(137) UFSC-116||E. dunni||Três Barras, SC, Brazil||UFSC116_Euc_Trê_Bra||AF374703|
|(138) UFSC-132||E. dunni||Três Barras, SC, Brazil||UFSC132_Euc_Trê_Bra||AF374704|
|(139) UFSC-145||Eucalyptus sp.||UFSC, Florianópolis, SC, Brazil||UFSC145_Euc_Flo_Bra||AF374705|
|(140) ITA6||Eucalyptus sp.||Itamarandiba, MG, Brazil||ITA6_Euc_Ita_Bra||AF374706|
|(141) RS26||Eucalyptus sp.||Vizcosa, Brazil||RS26_Euc_Viz_Bra||AF1429914|
|(142) PF||Unknown||unknown, France||PF_unk_unk_Fra||AF1432344|
|(144) W15||Unknown||N. Wilberforce, New South Wales||W15_unk_Wil_NSW||AF0047362|
|(145) W16||Unknown||N. Wilberforce, New South Wales||W16_unk_Wil_NSW||AF0047372|
|(146) SU1||Unknown||North Sumatra||SU1_unk_unk_NSu||AF2707776|
|(149) H714||Unknown||unknown, Western Australia||H714_unk_unk_WA||AF374720|
Samples for DNA extraction were excised from fresh basidiomata, basidiomata preserved in glycerol/ethanol/water (30/30/40) or the outer part of mycelial agar cultures. DNA was isolated from each isolate with the DNAeasy Kit (Qiagen, Dusseldorf, Germany). The ITS of nuclear rDNA were amplified as described by Martin et al. (1998) with the ITS1 and ITS4 primers (White et al., 1990). Amplifications were performed on a GeneAmp 9600 PCR system (Perkin Elmer, Norwalk, CT, USA). The two ITS alleles amplified from the fruiting body of isolate Pasoh01 from P. aurantioscabrosus were subcloned in the plasmid pCR4-TOPO (Invitrogen, Groningen, The Netherlands) before sequencing. DNA amplification products were purified with BIO 101 GeneClean III Kit (BIO 101, Inc., Carlsbad, CA, USA) 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 using the ITS1 and ITS4 primers. Reactions were then electrophoresed on either an ABI 377 (at Murdoch University, WA, Australia) or ABI Genotyper 310 (at INRA-Nancy) 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. About 20% of the amplified ITS were sequenced on both machines to check for base calling reproducibility. Sequences were deposited in the National Center for Biotechnology Information (NCBI) GenBank (accession n° AF374622 to AF374720, AF415226 & AF415227) (Table 1).
Sequence and data analyses
Search for sequence identity in the GenBank DNA database was conducted by Gapped BlastN (NCBI) (Altschul et al., 1997). The 102 sequences from the present survey and 46 additional Pisolithus sequences retrieved from GenBank were aligned using the programme MultAlin (http://www.toulouse.inra.fr/multalin.html) (Corpet, 1988). The resulting multiple alignments were optimized visually. Only unambiguous alignments were used in the phylogenetic and distance analyses. The aligned sequences were exported to a NEXUS file and analysed using PAUP*4.08b (PPC/Altivec) (Swofford, 1999). The final alignment of the ITS1/5.8S/ITS2 region had a total of 706 nonambiguously aligned sites: 133 characters were constant, 92 variable characters were parsimony-uninformative and 481 characters were parsimony-informative. The final alignment of Pisolithus sequences is available on request.
Maximum parsimony (MP) was the primary method to infer phylogenetic relationships between the Pisolithus isolates, and all final results were compared with neighbour joining (NJ) and maximum likelihood (ML) (Swofford, 1999) to detect any algorithm-specific results. MP analysis was carried out with data sets that included and excluded gaps, to detect any character type-specific results. MP trees were identified using heuristic searches based on 1000 random sequence additions, retaining clades compatible with the 50% majority-rule in the bootstrap consensus tree, using the Nearest-Neighbour Interchange (NNI) branch-swapping option and saving no more than 100 trees with > 1000 length for each replication and MULPART in effect (Swofford, 1999). All characters were treated as unordered and with equal weight. Trees were drawn with TreeView (Page, 1996). ITS sequences of Paxillus involutus (GenBank accession n° AF167700) and Suillus luteus (GenBank accession n° L54110) were used as outgroup taxa. Pairwise distance between isolate sequences were calculated according to the Jukes–Cantor model (Jukes & Cantor, 1969). The criteria used to define a phylogenetic species were two-fold: the existence of a monophyletic terminal clade and a ITS sequence divergence among isolates within the clade ≤ 12%.
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The phylogeny of Pisolithus
We obtained the same overall topology for well-resolved phylogenetic trees regardless of the methods MP, NJ or ML (data not shown). The placement of single sequences was also constant in parsimony and distance-based trees (data not shown). The 50% majority-rule consensus tree, generated by bootstrap heuristic search (Swofford, 1999), resulted in three distinct Pisolithus lineages, A, B and C with strong bootstrap support (Fig. 1). These lineages comprised 11 major terminal clades (referred to as phylogenetic species). Lineage C only comprised P. aurantioscabrosus, which formed a basal clade. P. tinctorius and P. marmoratus grouped in lineage A, whereas P. albus and P. microcarpus belonged to lineage B.
Lineage A branched into two well-supported sublineages: AI and AII (Fig. 2a). The sublineage AI terminated in three well supported clades (species 1, 3 and P. marmoratus) and an additional isolate of New South Wales (NSW), LJ30. Isolates of species 1 were collected under an ectomycorrhizal Caesalpinioideae of the coastal Miombo-like woodlands of Kenya, Afzelia quanzensis (Martin et al., 1998). Isolates of species 2 were found in eucalypt plantations in the coastal lowlands of Kenya (Martin et al., 1998), in native sclerophyllous forests of NSW (Anderson et al., 1998, 2001) and in natural forests and plantations in WA. Species 2 is P. marmoratus as the sequence of the voucher specimen KS781 (WA Herbarium), which was used for the published description in Bougher & Syme (1998), falls within this clade. Species 3 included isolates that were found in association with Cistus in Spain (Díez et al., 2001).
Sublineage AII branched into three terminal clades with strong bootstrap support (species 4, 5 and P. tinctorius) (Fig. 2a). Species 4 included isolates found in association with pines and oaks on basic soils in Spain (Díez et al., 2001). It also comprised two isolates collected under pines in Japan and South Africa. Isolates of species 5 were collected under various pine and eucalypt species in Thailand and China. They do not correspond with the south-east Asian taxon, P. aurantioscabrosus (Watling et al., 1995, 1999) (Fig. 1), but to a species closely related to P. tinctorius. Isolates of P. tinctorius were found in association with pine and oak species in North America and Europe, and in pine plantations in Nicaragua, Kenya and South Africa.
Lineage B included isolates associated with eucalypts and acacias. The sublineage BI (Fig. 2b) ramified into several moderately-to-well supported branches and terminal clades. The topology of this sublineage was similar regardless of the methods (MP, NJ and ML). It identified three main groups (species 7–9). Isolates of species 7 showed the morphological features of P. albus sensuBougher & Syme (1998). The sequences of isolates H4339 and H4937 were 100% identical to the voucher specimen T25070 (Oregon State University Herbarium) deposited as P. albus. They were found in plantations of eucalypts and acacias and in forests in various regions of Australia (ACT, NSW, Qld, Southern Australia, Victoria and WA) and in plantations of eucalypts and acacias in China, India, Malaysia, Morocco, Spain and Senegal. The ITS sequence variation within this group was significant (= 10.2%). Species 8 only included isolates collected under acacias and eucalypts in WA. Isolates of species 9 exhibited the morphological features of P. microcarpus (Cunningham, 1942) and the sequenced isolates UFSCPt132 and UFSCPt145 (Fig. 2b) correspond to two voucher specimens (Herbarium of the Universidad Federal de Santa Catarina, Brazil) deposited as P. microcarpus (Giachini et al., 2000). P. microcarpus isolates were found in plantations of eucalypts and acacias and forests in Australia (WA, NSW and Qld) and in plantations throughout the world (e.g. Brazil, China, Morocco, Portugal, Senegal, South Africa).
Isolates of species 10 (lineage BII) were collected under eucalypts in WA and are probably a new species. It showed 12% genetic distance with the acacia-associated isolate CSH4461 from Queensland, which might represent a further species.
Evolutionary distance between and within species
Percentage pairwise sequence divergence across the entire ITS sequence ranged from 7% (between species 5 and P. tinctorius) to (36%) (species 1 and 10). Within-species divergence ranged from 0.1% (P. marmoratus) to 9.7% (P. tinctorius). Maximum ITS divergence between species associated with eucalypts and acacias was 32% (P. marmoratus vs species 10), whereas the highest divergence within Pisolithus associated with Holartic hosts was 23% (species 3 vs species 5) (Table 2).
|1 Species 1||–|
|2 P. marmoratus (sp. 2)||22||–|
|3 Species 3||25||16||–|
|4 Species 4||25||17||19||–|
|5 Species 5||27||20||23||14||–|
|6 P. tinctorius(sp. 6)||26||20||21||14||7||–|
|7 P. albus (sp. 7)||33||28||31||32||9||29||–|
|8 Species 8||32||29||32||31||29||29||7||–|
|9 P. microcarpus||34||29||32||33||31||30||8||10||–|
|10 Species 10||36||32||33||36||33||33||23||5||21||–|
|*11 P. aurantioscabrosus (sp. 11)||37||38||40||41||40||39||42||41||41||42||–|
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Within Pisolithus, biological species that are difficult or even impossible to distinguish based on morphological features were revealed using mating tests (Kope & Fortin, 1990). DNA-based phylogenetic analyses of a restricted set of isolates suggested the occurrence of several phylogenetic species within Pisolithus (Anderson et al., 1998, 2001; Martin et al., 1998; Cairney et al., 1999; Díez et al., 2001). In the present survey, we have extended these previous phylogenetic studies to delimit Pisolithus taxa among 148 collections from natural vegetation and forest plantations throughout the world. There are significant similarities among tree topologies generated with the MP, NJ and ML methods in this study, strengthening the evolutionary relationships inferred. The bootstrap value of clades used to define phylogenetic species was moderate (78% for species 5) to high (100%) (Fig. 1). The evolutionary distance between terminal clades indicated the likelihood that they represent at least 11 distinct phylogenetic species. Whether additional terminal clades (e.g. isolates LJ30, CS01, and CSH4461) correspond to new phylogenetic species will await additional sampling and sequencing.
Pisolithus tinctorius as a lineage associated with Holartic hosts
It has been suggested (Bougher & Syme, 1998; Díez et al., 2001) that P. tinctorius is confined to Holartic hosts. In the present analysis, P. tinctorius associated with pines and oaks (e.g. MH208) in the northern hemisphere. P. tinctorius also occurred in pine plantations in Nicaragua (NIC(S382)), South Africa (MH9813) and Kenya (5111, Martin et al., 1998, esp07, Díez et al., 2001), in northern Sumatra (SU1) and the Philippines (PH1). Thus, the present study supports the hypothesis that this Pisolithus species was introduced to the southern hemisphere with pines (Dunstan et al., 1998; Díez et al., 2001). Two isolates from pine plantations in South Africa (MP9812) and Japan (PtJap) did not belong to P. tinctorius, but they are closely related to species 4 occurring in basophilous woodlands in Spain (Díez et al., 2001), suggesting that they have been introduced from the Mediterranean region into South Africa and Asia along with pines.
In this survey, P. tinctorius isolates were not found in Australia, even though extensive basidiocarp surveys have been undertaken in eucalypt forests and plantations. By contrast to America and Africa, there is no reliable record of P. tinctorius from pine plantations in Australia and New Zealand (Dunstan et al., 1998). P. tinctorius does not occur in Australia, at least not in natural ecosystems (Bougher & Syme, 1998; Cairney et al., 1999). P. tinctorius probably was not among the Holartic ectomycorrhizal fungi brought by early settlers into Australia with soil or pine seedlings. Later, stringent quarantine regulations on the importation of soils and plants probably precluded the introduction of P. tinctorius.
Pisolithus lineages and regional floras
Several species identified in the present analysis consisted of isolates limited to specific geographical regions (species 1, 3, 4, 5), most of them confined to endemic plants (species 1, 3 and 4). Species 1 is composed of isolates associated with an ectomycorrhizal Caesalpinioideae of the coastal Miombo-like woodlands of Kenya, Afzelia quanzensis Welw. (Martin et al., 1998). Species 3 consisted of isolates associated with Cistus ladanifer L. (Cistaceae) in closed shrublands on clayey slate-derived soils in the western Mediterranean region (e.g. cr01; Díez et al., 2001). Isolates of species 4 associated with basophilous pines and sclerophyllous oaks in the Mediterranean region (e.g. m14 and pt03; Díez et al., 2001).
Isolates collected in pine plantations in China and Thailand and in eucalypt plantations in China did not belong to P. tinctorius, but to a related species. The success of plantations of pine and eucalyptus in southeast Asia was probably facilitated by the occurrence of such compatible indigenous strains of Pisolithus, rather than facilitated by the co-introduction of exotic ectomycorrhizal fungi on seedlings. The isolates of the so-called ‘southeast Asia’-type (species 5) were collected in P. kesiya plantations in Thailand (e.g. MSN, THAI1), in P. yunnanensis plantations in China (e.g. MH143), and in eucalypt plantations in China (e.g. MH727). Specimens with similar sporocarp and spore morphologies have been observed in natural stands of P. kesiya and P. merkusii in Thailand, Indonesia and the Philippines, and P. yunnanensis in China (B. Dell, unpublished). We hypothesized that this species could correspond to P. aurantioscabrosus, a taxon found in native forests of Malaysia (Watling et al., 1995, 1999). We thus analysed a sample collected under the dipterocarp Shorea macroprera in the locality where the holotype was collected and morphologically identified as P. aurantioscabrosus (LS See, Forest Service, Pasoh, Malaysia). The phylogenetic analysis (Fig. 1) showed that species 5 does not correspond to P. aurantioscabrosus.
Pisolithus lineages in Australia and their recent dispersal worldwide
Australian material from native forests accounts for all of lineage B and part of lineage A. The wide divergence of their ITS sequences (≤ 42%) (Table 2) suggests that the centre of diversification of the genus Pisolithus may well be Australasia. The ancestral Pisolithus was probably a generalist mycorrhizal symbiont. Allopatric speciation probably led to the radiation of this genus after the break up of Pangaea into the northern and southern landmasses of Laurasia and Gondwana in the Triasic, as suggested for other mycorrhizal genera (e.g. Tylopillus cf. in Halling, 2001).
Because eucalypts have evolved in isolation from the ectomycorrhizal fungal flora associated with Pinus and Quercus in the northern hemisphere, Pisolithus tinctorius is probably not able to associate with eucalypts in silva (Bougher & Syme, 1998; Cairney et al., 1999). P. marmoratus is considered the southern hemisphere counterpart of P. tinctorius. They are morphologically very similar (Bougher & Syme, 1998), but P. marmoratus is associated with eucalypts, while P. tinctorius is not. The species P. marmoratus and P. albus are very common in WA (Bougher & Syme, 1998). The isolates found in sclerophyllous NSW forests (LJ07 and WW01; Anderson et al., 1998, 2001) correspond to P. marmoratus (Bougher & Syme, 1998), together with 13 isolates collected in native woodlands and open forests of E. marginata/E. calophylla, and in plantations of E. globulus in WA.
The lineage B is composed of four major terminal clades, which represent species specifically associated with Australasian acacias (e.g. Acacia mangium) and eucalypts. The sublineage BI includes three species (P. albus, 8 and P. microcarpus), whereas the sublineage BII comprises species 10. The evolutionary relationships within the sublineage BI were not well resolved (Fig. 2b). The ITS region did not have enough parsimony informative characters to fully resolve the topology of this part of the tree, in terms of both branch length and bootstrap support. However, good support in terminal clades allowed us to delineate species boundaries. We considered species 7 to be P. albus based on morphological features. Isolates of these species were collected throughout the world (e.g. India, Senegal, Spain) in eucalypt plantations. Species 8 and 10 were only found in WA and might represent endemic species in concordance with other Pisolithus taxa, such as species 3, 4 and 5. Species 9 corresponds to the morphological species P. microcarpus, illustrated in Bougher and Syme (1998). Although native to Australia, isolates of this species were collected in exotic plantations worldwide (e.g. Brazil, China, Portugal, Senegal, South Africa).
The lack of significant ITS divergence among isolates from different continents within P. albus or P. microcarpus (Fig. 2b), and the significant divergence among isolates belonging to these different species from the same geographical region (e.g. WA) (Fig. 2b; Table 2) are consistent with recent cosmopolitan dissemination of these species with eucalypts through human activity. Australian eucalypts were introduced to southeast Asia, India, Africa, South America, South Africa and southern Europe in the last 200 yr as seeds and saplings and this facilitated the dispersal of Australian Pisolithus. Similarly, it has been suggested that dissemination of the basidiomycetous yeast Cryptococcus neoformans, a human pathogen, associated with Eucalyptus leaves in southern California (Casadevall & Perfect, 1998) and India (Chakrabarti et al., 1997), resulted from the transplantation of Eucalyptus from Australia. The evidence here does not indicate the precise geographical origin of Eucalyptus disseminated through the world, although their likely place of origin within Australia is the east coast (Victoria, NSW, Queensland) where many commercial species of plantation eucalypts emanated. To trace the geographical origin of Pisolithus found in plantations worldwide, more isolates of each species from numerous geographical regions in Australia must be analysed using multilocus genetic approaches (e.g. microsatellites) or mitochondrial DNA. The mechanisms of dispersion facilitated by eucalypt plantations worldwide could place certain isolates of divergent lineages (e.g. eucalypt-associated- and Afzelia-associated Pisolithus) in close proximity, and subsequent mating among them could generate hybrids. Recent hybridization between fungal species has been reported between the poplar rusts Melampsora medusae and M. occidentalis (Brasier, 2000), and between major lineages of Cryptococcus neoformans (Xu et al., 2000). However, no genetic exchange has yet been found in silva between pine-, eucalypt- and Afzelia-associated Pisolithus in woodlands of Kenya (Martin et al., 1998) or in the Mediterranean region where natural and introduced Pisolithus coexist (Díez et al., 2001).
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The present study clearly shows that the evolutionary lineages within Pisolithus are related to biogeographical origin of the hosts. Each phylogenetic species of Pisolithus was confined to hosts from one single biogeographical realm. Living in symbiotic association for a long evolutionary time in the same biogeographical area could have lead to the acquisition of a significant degree of host-fungus specificity. This specificity might be strengthened by ecological factors (Molina et al., 1992), which probably accounts for the strict host–fungus association patterns observed in silva. The present phylogeographical survey also showed that regional floras and endemic plants (e.g. Afzelia and Cistus) could act as hosts of endemic species of Pisolithus. We included in the present study specimens deposited in herbaria and identified by classical taxonomy in order to assign morphological species to the obtained phylogenetic species. Mating tests are needed to delimit biological species, in particular which of the phylogenetic species are reproductively isolated.
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We wish to thank the many field collectors and colleagues who provided isolates of Pisolithus: M. Abourouh, A. Ba, T. Burgess, A. Casares, C. Chamberlain, M. Duccousso, W. Dunstan, R. Duponnois, J. Garbaye, A. Hernandes, M. Honrubia, M. Ivory, L. Khabar, F. Lapeyrie, L. S. See, H. Machado, N. Malajczuk, D. Marx, V. Lopes de Oliveira, U. Sangwanit, D. Sylvia and J. Trappe. We are grateful to I. Alexander, I. Anderson, B. Anta, N. Bougher, J. Cairney, M. Honrubia, M. Ivory, F. Lapeyrie, F. Le Tacon, J. L. Manjón, N. Malajczuk, G. Moreno, Romero de la Osa, and J. Trappe for their helpful discussion over the years. Additional technical support at MU was provided by R. Tothill and M. Farbey. This work was supported by an EU postdoctoral fellowship (Contract HPMF-CT-1999–00174) and a ‘Ramon y Cajal’ contract from the MCYT (Spain) to JD, research grants from the INRA and Bureau des Ressources Génétiques to FM, and MU Special Research Grants to BD. The present study is a joint project between the School of Biological Sciences and Biotechnology (Murdoch University, Perth, WA, Australia) and the UMR IaM (INRA-Nancy, France).
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