convergent evolution of sequestrate forms in amanita under

Convergent evolution of sequestrate forms in Amanitaunder Mediterranean climate conditions

Alfredo Justo1

Ingo MorgensternBiology Department, Clark University, 950 Main Street,Worcester, Massachusetts 01610

Heather E. Hallen-AdamsDepartment of Plant Biology, Michigan StateUniversity, 166 Plant Biology Laboratories, EastLansing, Michigan 48824-1312

David S. HibbettBiology Department, Clark University, 950 Main Street,Worcester, Massachusetts 01610

Abstract: The systematic position of secotioid (Tor-rendia) and gasteroid (Amarrendia) forms within theagaricoid Amanita lineage (Agaricales, Basidiomyco-ta) was studied with molecular (nLSU, ITS) data.Secotioid and gasteroid forms occur in four indepen-dent clades nested within agaricoid forms. One cladecorresponds to the secotioid T. pulchella fromsouthern Europe and northern Africa. The otherscorrespond to Torrendia and Amarrendia species fromAustralia. Mediterranean climatic conditions arepostulated as a force driving the convergent evolutionof these secotioid and at least one of the gasteroidforms in geographically distant areas. Species former-ly placed in Torrendia and Amarrendia are transferredto Amanita. A new species of Torrendia from Australiawas discovered during the revision of the collectionsoriginally identified as T. arenaria and is describedhere as Amanita pseudoinculta.

Key words: Amarrendia, ITS, nLSU, phylogeny,sequestrate forms, Torrendia

INTRODUCTION

Secotioid and gasteroid forms have evolved indepen-dently several times from agaricoid/boletoid ances-tors in different groups of fungi (Hibbett 2007), suchas Boletales (Binder and Hibbett 2006), Russulales(Eberhardt and Verbeken 2007), Agaricales (Peintneret al. 2001) and Phallomycetidae (Hosaka et al. 2006).Gasteroid fungi such as false truffles, puffballs andstinkhorns are highly modified for nonballistosporicdispersal, whereas secotioid forms are morphologi-

cally intermediate between gasteroid and their agar-icoid/boletoid ancestors. The advantage of theseforms against loss of moisture under unfavorableconditions (extreme drought or cold) has beenproposed as a main factor favoring their evolution(Thiers 1984). It also has been postulated that naturalselection would act against intermediate (secotioid)forms because they lack the dispersal advantages ofthe agaricoid and gasteroid forms (via air and animalsrespectively) and are only partially adapted toseasonal xeric conditions (Bruns et al. 1989). Peint-ner et al. (2001) showed that it also is possible thatsuccessfully adapted, stable, sequestrate (secotioidand gasteroid) forms tend to radiate but they canradiate only into species with the same basidiome typeor a further reduced type. This study concerns theevolution of secotioid (Torrendia) and gasteroid(Amarrendia) forms within the Amanitaceae (Agar-icales).

The secotioid genus Torrendia was created byBresadola (1902) to accommodate a small, whitish,volvate and stipitate gasteromycete collected inPortugal by C. Torrend. After a careful study of themorphology and development of Torrendia pulchellaBres. a close relationship with the agaricoid genusAmanita Pers. was postulated (Malencon 1955, Bas1975). Miller and Horak (1992) described a secondspecies of Torrendia from Western Australia, T.arenaria O.K. Mill. & E. Horak, very similar in itsexternal morphology to T. pulchella but differing inspore shape and size, presence of sclerobasidia,absence of clamp connections and different mycor-rhizal partners. A variant with yellowing flesh wasgiven the name T. arenaria f. lutescens O.K. Mill & E.Horak. Bougher (1999) described two new speciesalso from Western Australia, T. grandis Bougher,mainly characterized by the relatively big basidiomes,and T. inculta Bougher with a gleba that fragmentsduring stipe elongation. Both species have clampconnections and lack sclerobasidia, which separatesthem from T. arenaria, and possess ellipsoid tooblong spores, which separates them from T.pulchella.

Torrendia has a puzzling distribution pattern; onespecies (T. pulchella) occurs in the MediterraneanBasin, including the Iberian Peninsula (Spain, Portu-gal), northern Africa (Morocco, Algeria), southernFrance, Sardinia (Italy) and Turkey, with putativemycorrhizal partners such as Pinus, Quercus and

Submitted 29 Jun 2009; accepted for publication 17 Oct 2009.1 Corresponding author. E-mail: [email protected]

Mycologia, 102(3), 2010, pp. 675–688. DOI: 10.3852/09-191# 2010 by The Mycological Society of America, Lawrence, KS 66044-8897

675

Cistus (Neville and Poumarat 2004), and the otherthree taxa (T. arenaria, T. grandis, T. inculta) occurin Western Australia with putative mycorrhizal part-ners such as Eucalyptus, Allocasuarina and Leptosper-mum (Miller and Horak 1992, Bougher 1999). Withthis disjunct distribution in both hemispheres amonophyletic Torrendia would imply that the groupis ancient or that there has been long distancedispersal, either by natural or anthropogenic means.

The gasteroid genus Amarrendia Bougher & T.Lebel was created to include species with thesecharacteristics: white to cream peridium and gleba,basidiome flesh fragile and minutely granular; glebaloculate; spores smooth, thin-walled, hyaline, non-amyloid and nondextrinoid, with a large oil droplet,broadly ellipsoid and with a broad apiculus; contexttrama composed of inflated and hyphal elementsintermixed. Bougher and Lebel (2002) describedthree new species of Amarrendia (viz. Amarrendiaoleosa Bougher & T. Lebel, Amarrendia nemoribusBougher & T. Lebel and Amarrendia peridiocrystaliaBougher & T. Lebel) and recombined two speciesformerly placed in Alpova (Amarrendia grandispora[G.W. Beaton, Pegler & T.W.K. Young] Bougher & T.Lebel and Amarrendia lignicolor [G.W. Beaton, Pegler& T.W.K. Young] Bougher & T. Lebel ). The genus isdistributed in Western Australia, Victoria and Tasma-nia with putative mycorrhizal partners such asEucalyptus, Allocasuarina, Acacia and Gastrolobium.Amarrendia was proposed to occupy a systematicposition within a complex of related taxa that alsoincorporates Torrendia and Amanita mainly based inthe presence of inflated elements in the trama andthe characteristics of the spores (Bougher and Lebel2002)

Previous molecular work has shown that T. pul-chella is a secotioid derivative of Amanita with itsclosest agaricoid relatives in sect. Caesareae (Moncalvoet al. 2002). A previous analysis by Hallen et al. (2004)suggests that Torrendia is polyphyletic and also thatamong the five species of Amarrendia only two,Amarrendia oleosa and Amarrendia grandispora, arepart of the Amanita lineage while the other speciesfall outside the Amanitaceae or even the Agaricalesclade. However that analysis was not formally pub-lished and the sequence data were not deposited inGenBank.

The purpose of the present study was to revisit theorigins of Torrendia and Amarrendia with newsequence data obtained from Australian and Europe-an materials to assess the monophyly and systematicposition of Torrendia and Amarrendia among theAmanita lineage, discuss its implications for morpho-logical and ecological evolution and make formaltaxonomic proposals.

MATERIALS AND METHODS

Sequences.—Thirty-six new sequences (nuclear ribosomalRNA large subunit [nLSU] and the internal transcribedspacers [ITS]) were generated from herbarium material ofT. pulchella, T. arenaria, T. inculta, T. grandis andAmarrendia oleosa. Sequences generated from an unidenti-fied herbarium collection (H909), possibly belonging toAmarrendia, also were included in the study. The nLSU datafor Amarrendia grandispora came from Hallen et al. (2004).A total of 167 sequences of Amanita, two of Limacella andone of Torrendia pulchella, the majority coming from theworks of Weiß et al. (1998), Drehmel et al. (1999) andZhang et al. (2004), were retrieved from GenBank(TABLES I, II).

Approximately 0.05 g each herbarium collection wereground in liquid nitrogen, and DNA was extracted with 3%

SDS extraction buffer; DNA then was isolated by thesequential addition of phenol chloroform and chloro-form-isoamyl alcohol; finally, isopropyl alcohol and 3Msodium acetate were added to precipitate the DNA, whichwas washed with 70% EtOH and resuspended in sterilewater. A portion of the nLSU and the complete ITS1 + 5.8 +ITS2 (ITS) regions were amplified by PCR with fungalprimers LR0R and LR5 and ITS1F and ITS4 respectively(Gardes and Bruns 1993, http://www.biology.duke.edu/fungi/mycolab/primers.htm). Amplification products weresequenced with ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction reagents with primers LR0Rand LR5 (with additional LR3R and LR3 for some samples)and ITS1F and ITS4. Sequencing was carried out on an ABI3130 Genetic Analyzer. Raw data were processed withSequencher 4.7 (GeneCodes, Ann Arbor, Michigan). TheITS region of all the Australian taxa of Torrendia andAmarrendia showed a high level of intragenomic variability,so the PCR products were cloned with the TOPO TACloning Kit (Invitrogen, Carlsbad, California) following themanufacturer’s instructions.

Alignment.—Sequences were aligned with MAFFT (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/). Align-ments then were examined and manually corrected inMacClade 4.05 (Maddison and Maddison 2002). They havebeen deposited in TreeBASE under accession numberS2490.

Phylogenetic analysis.—Maximum parsimony (MP), neigh-bor joining (NJ), maximum likelihood (ML) and Bayesiananalyses (BA) were performed with these parameters: (i)MP. Equally weighted parsimony analysis was performedwith PAUP* 4.0.b10 (Swofford 2002). One thousandheuristic search replicates were performed with startingtrees generated by stepwise addition with random additionsequences followed by tree bisection reconnection (TBR)branch swapping. Up to two trees were kept in eachreplicate. Parsimony bootstrap analysis was performed with1000 replicates, each with 10 random taxon additionsequences and branch swapping set to subtree pruningand regrafting (SPR). (ii) NJ. The analysis was run inPAUP* 4.0.b10 (Swofford 2002) with distances estimatedunder a general time reversible (GTR) model. NJ bootstrap

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678 MYCOLOGIA

was performed with 1000 replicates. (iii) ML. The analysiswas run in the RAxML servers (http://8ball.sdsc.edu:8889/cipres-web/Home.do, which implements the search proto-col of Stamatakis et al. 2008) under a GTR model with 1000rapid bootstrap replicates. (iv) BA. The analysis was run withMrBayes 3.1 (Ronquist and Huelsenbeck 2003) for10 000 000 generations under a GTR model with four chainsand trees sampled every 100 generations. After examiningthe graphic representation of the likelihood scores withMicrosoft Excel the burn-in was set to 20 000 generations.All four analyses were performed with the nLSU dataset,while only MP and ML analyses were performed with theITS datasets. MP and ML analyses also were performed on acombined nLSU and ITS dataset although the results areshown only in supplementary material because they yieldedessentially the same results as the analyses of the non-combined datasets.

Morphological descriptions.—Standard methods for describ-ing the basidiocarps were applied with the terminology ofNeville and Poumarat (2004). The notation [120/8/4]indicates that measurements were made in 120 spores in 8samples from 4 collections. The spores of Torrendia lack adistinct lateral view, although a few slightly asymmetricspores with a somewhat lateral apiculus can be observedsometimes in microscopical preparations, therefore thespores were measured in frontal view including the broadcentral apiculus as was done in previous descriptions ofTorrendia species (Miller and Horak 1992, Bougher 1999,Neville and Poumarat 2004).

Abbreviations.—avl 5 average length, avw 5 average width,Q 5 length/width quotient, avQ 5 average quotient,I.C.B.N 5 International Code of Botanical Nomenclature.To avoid confusion Amanita is abbreviated as ‘‘A.’’ andAmarrendia is not abbreviated.

RESULTS

Analyses of the nLSU dataset.—The partial nLSUsequences of Torrendia and Amarrendia obtained inthis study range from 761 base pairs (bp) in Torrendiapulchella to 957 bp in T. grandis. The sequence ofAmarrendia grandispora obtained in Hallen et al.(2004) is 572 bp long. The dataset includes 16Torrendia, three Amarrendia, 81 Amanita and twoLimacella sequences, which were used as outgroup.The final dataset consists of 102 sequences of 1198characters (gaps included), of which 296 are parsi-mony informative. In the MP analysis a total of 50equally most parsimonious trees (MPT) were recov-ered (Length 5 1710, CI 5 0.35, RI 5 0.76).

In all four analyses the general topology withrespect to subgeneric classification of Amanita wasconsistent among all trees and studies on the genus(Weiß et al. 1998, Drehmel et al. 1999, Zhang et al.2004). Two clades corresponding to the traditionalmorphological subgenera Amanita and Lepidella andseven corresponding to sections Amanita, Caesareae,

Amanitopsis (5 Vaginatae), Lepidella, Phalloideae,Validae, including Mappae, and Amidella were recov-ered, although some with low bootstrap support. Twospecies of section Lepidella (A. armillariiformis True-blood & D.T. Jenkins and A. nauseosa [Wakef.] D.A.Reid) were placed outside the Amanita clade; thesespecies are suspected to be non-ectomycorrhizal, andsimilar results about their placement were observed inMoncalvo et al. (2002). The internal topology of somesections and the relations between sections, especiallyin subgenus Amanita, were poorly resolved as shownin the Bayesian phylogram (FIG. 1). Torrendia andAmarrendia species are distributed along the tree infour groups, which we refer to as the Torrendia clade,Amarrendia clade, Pseudoamarrendia clade andArenaria clade.

The Torrendia clade includes the three newsequences of T. pulchella (the type species ofTorrendia) and the existing GenBank sequence,which were placed together with high support in allanalyses. The position of T. pulchella as a member ofsect. Caesareae is highly supported in all four analyses.

The Amarrendia clade includes T. grandis, T.inculta, four collections originally identified as T.arenaria that are here considered to represent adifferent species referred to in the text and figures asAmanita pseudoinculta (see TAXONOMY), Amarrendiaoleosa (the type species of Amarrendia) and Amarren-dia grandispora. The Amarrendia clade is placed as asister group of sect. Caesareae in the MP, ML and NJanalyses, but this relationship receives significantsupport only in the NJ analysis (80%). In the BAanalysis the relationships among the four main cladesof subgenus Amanita are unresolved (FIG. 1).

The Pseudoamarrendia clade includes the uniden-tified Amarrendia sp. that is grouped with two speciesof sect. Amanitopsis (A. verrucosivolva Zhu L. Yangand A. aff. fulva). This clade is highly supported in allanalyses because it is the sister taxa relationshipbetween A. aff. fulva and Amarrendia sp. (FIG. 1). ThePseudoamarrendia clade appears to be the sistergroup of all other species of sect. Amanitopsis in thestudy. The data of this ‘‘Amarrendia sp.’’ come fromthe herbarium collection H909 (CSIRO), but noadditional morphological, ecological or geographicdata are available so no formal description of a newspecies can be made at this moment.

The Arenaria clade includes six collections of T.arenaria including the type collection (VPI 679) andone collection identified as T. arenaria f. lutescens(VPI 364). The Arenaria clade is supported in allanalyses, although its closest relatives cannot beidentified with certainty in BA, ML and NJ analyses,is placed as the sister group of sect. Phalloideae butwith no statistical support.


FIG. 1. Fifty per cent majority rule Bayesian phylogram for the nLSU dataset. Thick branches are supported by posteriorprobabilities of 1 and bootstrap values . 90% in the other analyses (ML/MP/NJ). Other values for branches supported in atleast two of the analyses with posterior probabilities . 0.90 and/or bootstrap values . 70% are shown on the branches (BA/ML/MP/NJ). An asterisk indicates that the branch collapses in the strict consensus tree of the MP analysis.

680 MYCOLOGIA

Analyses of the ITS datasets.—Due to high levels ofsequence divergence, two separate datasets wereconstructed: one only with members of subgenusAmanita with all species of Torrendia and Amarrendiaexcept T. arenaria and a second dataset only withmembers of subgenus Lepidella and T. arenaria. Afteran initial ML analysis of the second dataset (SUPPLE-

MENTARY FIG. 1) species of section Lepidella wereexcluded from the analysis. A similar problem withITS alignment in Amanita has been reported byMoreno et al. (2008).

Only the ITS region of T. pulchella could beobtained through direct sequencing (611 bp).Cloning was necessary to obtain the ITS ofAustralian species of Torrendia and Amarrendia.The final datasets contained two clones of T.inculta (669–675 bp), four clones of Amanitapseudoinculta (683–705 bp), two clones of T.grandis (663–669 bp), three clones of Amarrendiaoleosa (662–664 bp), three clones of Amarrendia sp.(606–613 bp), one clone of T. arenaria from thetype locality (712 bp) and one clone of T. arenariafrom a different locality (715 bp). Considerableintragenomic variability was observed, for examplethe four cloned sequences of A. pseudoincultadiffer in 36 point mutations and four small indels(1 or 2 bases).

Subgenus Amanita. The final dataset consists of 57sequences of 1020 characters (gaps included), ofwhich 482 are parsimony informative. A. phalloides(Vail. ex Fr.: Fr.) Link and A. porphyria (Alb. &Schwein.: Fr.) Alb. & Schwein. were used to root thetree. A total of 180 MPT were recovered (Length 5

2614, CI 5 0.47, RI 5 0.72).The main difference with the nLSU analyses is that

an agaricoid species of Amanita (A. umbrinella E.-J.Gilbert & Cleland) is included in the Amarrendiaclade as the sister taxon of Torrendia inculta andAmanita pseudoinculta. This taxon was not includedin the nLSU dataset.

Subgenus Lepidella. The final dataset consists of 37sequences of 911 characters (gaps included), of which392 are parsimony informative. A. muscaria (L.: Fr.)Lam. and A. vaginata (Bull.: Fr.) Lam. were used toroot the tree. A total of 42 MPT were recovered(Length 5 1783, CI 5 0.55, RI 5 0.68).

Again the ITS analyses yielded results very similar tothe nLSU data. In both MP and ML trees (FIG. 3) fivemain clades are recovered: sections Amidella, Validae,Phalloideae, two species of sect. Lepidella (A. griseo-verrucosa and A. cinereopannosa Bas) and the Arenariaclade. In the ML tree (FIG. 3) the Arenaria clade andthe two species of sect. Lepidella are placed (with nobootstrap support) as a sister group of sect. Phalloi-deae.

Analyses of the combined dataset.—Two datasets (sub-gen. Amanita and subgen. Lepidella) were construct-ed for the combined analysis (nLSU + ITS). No majordifferences with the independent analyses wereobserved (SUPPLEMENTARY FIGS. 3, 4).

TAXONOMY

Torrendia and Amarrendia are polyphyletic entitiesthat are nested within Amanita. To maintain mono-phyletic taxa the species described in Torrendia andAmarrendia must be transferred to Amanita, assuggested by Tulloss and Yang (http://www.njcc.com/,ret/amanita/mainaman.html). The genericnames Torrendia and Amarrendia convey informationabout morphological diversity that unfortunately willbe lost if these names are simply subsumed intoAmanita. To maintain the correspondence betweentaxonomy and phylogeny, while highlighting morpho-logical variation, we suggest that the ‘‘Torrendia’’,‘‘Amarrendia’’, ‘‘Pseudoamarrendia’’ and ‘‘Arenaria’’clades eventually should be classified formally assections or subsections of Amanita. However theseformal recombinations and descriptions of infragener-ic taxa are not made here because more extensivesampling of Amanita species is necessary for a betterresolution of the internal topology of the genus. Thecurrent molecular sampling only accounts for approx-imately 10–15% of the estimated diversity of Amanita(Tulloss 2005). In the meantime the informal cladenames still can be used as such or as ‘‘stirps’’, which aretraditionally used in the taxonomy of Amanita (Bas1969) but are not governed by the rules of the I.C.B.N.Analyses of nLSU sequences suggest that sect. Lepidellais polyphyletic and also requires reclassification, butthat taxonomic problem is not addressed here.

The situation involving Torrendia and Amarrendiais similar to that in other clades of Agaricomycetesthat contain secotioid and gasteroid taxa nestedwithin paraphyletic assemblages of agaricoid orboletoid taxa. Fungal systematists typically haveapproached this recurring problem by combiningsecotioid and gasteroid forms under the genericname of their related agaricoid or boletoid forms, aswe do here. For example Vellinga et al. (2003)recombined the species of the polyphyletic Endopty-chum into Agaricus and Chlorophyllum, which weredescribed based on agaricoid taxa. Similarly Eber-hardt and Verbeken (2004) described a new gasteroidfungus as a species of Lactarius, which is based onagaricoid forms, instead of placing the taxon inArcangeliella or Zelleromyces, which are gasteroid‘‘genera’’ nested within Lactarius. The polyphyleticsecotioid Thaxterogaster also was recombined inCortinarius (Peintner et al. 2002).


Some workers have expressed concern that the lossof generic names of sequestrate taxa reduces theinformation content of classifications (Smith andHealy 2009). We endorse the view that the primary(if not the sole) organizing principle for biologicalclassifications should be phylogeny. At the same time itis an unfortunate paradox of modern taxonomy thatimprovements in understanding of phylogeny cancause the loss of names that highlight unique cladeswith distinguishing morphological features. The rootcause of this situation is the use of taxonomic ranksand the prohibition of having taxa at a given ranknested within other taxa of equal or lower rank. Rank-free classification systems could mitigate such prob-lems and thereby promote stability and continuity inname usage (Hibbett and Donoghue 1998). At presentthere is no generally accepted system of unrankedbiological nomenclature, but the forthcoming Phylo-Code (http://www.ohio.edu/phylocode/) might pro-vide a useful model. In the meantime the solution weadvocate attempts to highlight distinctive clades in aLinnaean taxonomic framework.

Most of the required transfers are straightforward.However a new name is needed for Torrendiapulchella because the combination Amanita pulchellaS. Imai already exists. The name Amanita arenaria K.Syme also exists, but it was not validly published (K.Syme pers com) because there was no Latin diagnosis(I.C.B.N., art. 36) and therefore is available forTorrendia arenaria. The new species discoveredduring revision of the original Torrendia arenariacollections is described here under the name Amanitapseudoinculta.

Amanita arenaria (O.K.Mill. & E. Horak) Justo, comb.nov.

MycoBank 515037Basionym: Torrendia arenaria O.K.Mill & E. Horak,

Mycologia 84(1):65. 1992

Amanita grandis (Bougher) Justo, comb. nov.MycoBank MB 515043Basionym: Torrendia grandis Bougher, Aust. Syst. Bot.

12(1):146. 1999

Amanita grandispora (G.W. Beaton, Pegler & T.W.K.Young) Justo, comb. nov.

MycoBank MB 515038Basionym: Alpova grandisporus G.W. Beaton, Pegler &

T.W.K. Young, Kew Bull. 40(3):580. 1985. (5

Amarrendia grandispora (G.W. Beaton, Pegler &T.W.K. Young) Bougher & T. Lebel, Aust. Syst. Bot.15:518. 2002)

Amanita inculta (Bougher) Justo, comb. nov.MycoBank MB 515039

Basionym: Torrendia inculta Bougher, Aust. Syst. Bot.12(1):149. 1999

Amanita oleosa (Bougher & T.Lebel) Justo, comb.nov.

MycoBank 515040Basionym: Amarrendia oleosa Bougher & T. Lebel,

Aust. Syst. Bot. 15:514. 2002

Amanita torrendii (Bres.) Justo, nom. nov.MycoBank MB 515041

Etymology. Torrendii refers to the Portuguese mycol-ogist Camillo Torrend after whom genus Torrendia wasnamed.

Basionym: Torrendia pulchella Bres., Atti Imp. RegiaAccad. Rovereto, ser III 8:132.1902; non Amanitapulchella S. Imai, Bot. Mag. (Tokyo) 47:427. 1933nec Amanita pulchella (Cooke & Masee) E.-J.Gilbert, Icongr. Mycol. 27, suppl. 1:203. 1941

Amanita pseudoinculta Justo, sp. nov. FIG. 4Amanita inculta similis sed differt in sporis longibus et

pileus non fragilis. Holotypus, hic designatus, OKM25066 (VPI 555).

MycoBank MB 515042Etymology. Pseudoinculta means ‘‘false inculta’’,

referring to the closest relative of the new species, A.inculta.

Published figures. Miller and Horak 1992, FIGS. 17,18.

Pileus 4–10 mm broad, hemispheric, at maturitysometimes slightly depressed at center, not disinte-grating. Peridium up to 0.7 mm broad, smooth orgranular, whitish, in young stages with patches ofuniversal veil attached. Gleba subgelatinous at first,divided in numerous irregularly shaped locules,whitish. Stipe 10–35 3 2–7 mm, cylindrical or slightlynarrower toward the base, whitish, usually coveredwith fibrils and/or scales. Volva membranous, sacci-form, whitish, with upper part free from the stipe atmaturity. Context white, unchanging. Odor and flavorindistinct.

Spores [120/8/4] 11.3–19(19.5) 3 5.2–7.3(8) mm,avl 5 13.1–15.6, avw 5 6.0–6.5, Q 5 1.8–2.9, avQ 5

2.05–2.48 oblong to cylindrical, with a broad centralapiculus, inamyloid. Basidia 20–60 3 10–16 mm,mostly four-spored but two-spored also present, thin-walled. Peridium composed of thin-walled cylindricalhyphae 2–10 mm, nongelatinized, with a subpellis ofglobose, ovoid or irregularly shaped cells up to35(45) mm diam. Stipe context made up of filamentoushyphae (4–20 mm broad) with terminal cylindrical toclavate elements (acrophysalides) 45–150 3 10–40 mm.Volva a mixture of globose, ovoid or irregularlyshaped elements 20–75 3 20–65 mm and denselyinterwoven cylindrical hyphae 2–6 mm broad. Oleifer-

682 MYCOLOGIA

ous hyphae present in the hymenium and stipe trama.Clamp connections present in the hymenium, stipetrama and veil tissue.

Ecology, phenology and distribution: on sandy soilnear Allocasuarina humilis and Eucalyptus. Fruiting inJune–July. Known only from the type locality (Muller-ing Brook, approx. 206 km north or Perth, WesternAustralia).

Collections examined. AUSTRALIA. WESTERN AUS-TRALIA: Mullering Brook, Brand Highway, approx.206 km north of Perth, 3-VII-1991, L. Bailey, M. Bailey,O.K. Miller, H.H. Miller, OKM 25066 (Holotype, VPI555); idem, 3-VII-1991, L. Bailey, M. Bailey, O.K. Miller,H.H. Miller, OKM 25094 (VPI 366); idem, 3-VII-1991, L.Bailey, M. Bailey, O.K. Miller, H.H. Miller, OKM 25070(VPI 558); idem, 6-VI-1989, H.H. Miller, OKM 23825 (VPI411).

Observations. All collections of Amanita pseudoin-culta originally were identified as Torrendia arenaria(5 Amanita arenaria), and except for VPI 555 theywere mentioned in the original description of thatspecies (Miller and Horak 1992). However both themolecular and morphological data indicate that theyrepresent a different species not closely related to A.arenaria (see DISCUSSION). These collections comefrom Mullering Brook (206 km north of Perth),relatively far from the type locality of A. arenaria(Two Peoples Bay, 424 km south of Perth). Themicroscopical analysis revealed several differences withthe morphological concept of A. arenaria; the sporesare cylindrical (avQ 5 2.05–2.48) while A. arenaria hasbroadly ellipsoid to oblong spores (avQ 5 1.23–1.72).Clamp connections and oleiferous hyphae are presentin A. pseudoinculta, while clamps are absent andoleiferous hyphae are absent or scarce in A. arenaria.A. pseudoinculta also differs from A. inculta (its closestrelative in the molecular analyses) by the nondisinte-grating gleba and higher values of avQ (1.84 in T.inculta, Bougher 1999).

Amanita pseudoinculta is unique among the Aus-tralian secotioid species because of its cylindricalspores, a character shared with A. torrendii fromwhich it mainly differs by its mycorrhizal partners andarea of distribution: Allocasuarina and Eucalyptus inWestern Australia (A. pseudoinculta) vs. Pinus,Quercus and Cistus in the Mediterranean Basin (A.torrendii).

DISCUSSION

Phylogenetic position of secotioid species among theAmanita lineage.—The present data clearly rejectthe hypothesis of a single origin for the secotioid taxain Amanita because they are placed in three differentmajor clades, Torrendia (A. torrendii) nested in sect.Caesareae, Amarrendia (A. grandis, A. inculta and A.

pseudoinculta) as an independent group in subgenusAmanita and Arenaria (A. arenaria) in subgenusLepidella, possibly related to sections Phalloideae orValidae. Even the monophyly of the Australiansecotioid species has to be rejected because A.arenaria is placed in a distant position from the otherAustralian taxa. The ITS analysis also suggests twodifferent origins for the secotioid species in theAmarrendia clade (FIG. 2), meaning that the seco-tioid morphology in Amanita has arisen four inde-pendent times.

A. yuaniana consistently appears as the sister taxonof Amanita torrendii or as a part of a more inclusiveclade with other agaricoid species (A. longistriata, A.incarnatifolia), although this relationship gets strongstatistical support only in the combined analysis(SUPPLEMENTARY FIG. 3). A. yuaniana occurs insouthwestern China with Pinus and Quercus (Yang1997) and future research should address thebiogeographical implications and significance of thisrelationship.

A. umbrinella, an Australian species of sectionAmanita, is placed in the ITS analysis as the sistergroup of A. inculta and A. pseudoinculta (FIG. 2).Australian species of Amanita are underrepresentedin the current dataset, so more extensive samplingand sequencing of Australian taxa is necessary toinvestigate the identity of possible agaricoid relatives(or members) of the Amarrendia clade. The positionof the Amarrendia clade among subgen. Amanitaremains unclear although the combined analysis(SUPPLEMENTARY FIG. 3) suggests a relationship withsections Caesareae or Amanitopsis.

A. arenaria is placed in subgen. Lepidella in a moreinclusive clade together with sections Phalloideae andValidae, however the internal topology of this clade isnot well resolved in the analyses. Some speciestraditionally classified in sect. Lepidella also are placedin this more inclusive clade, A. strobiliformis (in thenLSU dataset), A. cinnereopanosa (in the ITS dataset)and A. griseoverrucosa (in both and the combineddataset). Some of these species are placed in theanalyses as the sister taxon of the Arenaria clade butalways with low bootstrap support. Exclusion of thesespecies from the datasets does not result in a betterresolution of the relationships between A. arenariaand sections Phalloideae and Validae (results notshown).

Systematic position of gasteroid species among theAmanita lineage.—The position of Amarrendia oleosa,Amarrendia grandispora (both in the Amarrendiaclade) and Amarrendia sp. (in the Pseudoamarrendiaclade) in the nLSU analysis also rejects a single originfor the gasteroid taxa of Amanita. The ITS dataset,


which did not include Amarrendia grandispora,yielded similar results.

Taxonomy of ‘‘Torrendia arenaria’’.—During therevision of the original collections of T. arenaria theexistence of a different species, here described asAmanita pseudoinculta (see TAXONOMY), was discov-ered. A. pseudoinculta is placed in the Amarrendiaclade, while A. arenaria is placed, in a distantposition, in the Arenaria clade (FIG. 1).

Amarrendia clade. The separation of A. pseudoin-culta and its closest relative, A. inculta, is supported bythe nLSU analysis (FIG. 1) and the ML analysis of theITS dataset (FIG. 2), however in the MP analysis of theITS data A. inculta sequences are paraphyletic(SUPPLEMENTARY FIG. 2). This is probably caused bythe high level of intragenomic variability observed in

the secotioid and gasteroid taxa from Australia thatoverlaps with the interspecific variability between pairsof closely related species. High intragenomic variabilityin the ITS region has been observed in other groups ofectomycorrhizal fungi such as Lactarius (Nuytinck andVerbeken 2007) and Xerocomus (Taylor et al. 2006). InLactarius similar problems with overlapping intrage-nomic and interspecific ITS variation had been detectedin pairs of closely related species, and this situation hasbeen linked to recent speciation events. So far no highlevels of intragenomic ITS variability have beenreported in the agaricoid members of Amanita; theyalso seem to be not present in A. torrendii.

Arenaria clade includes the type collection ofAmanita arenaria (VPI 679), another collection fromthe same locality and date (VPI 412), and collectionsfrom two different localities, Kalamunda (VPI 364,

FIG. 2. Best tree from the ML analysis of the ITS dataset of subgenus Amanita. Thick branches are supported by bootstrapvalues . 90% in the ML and MP analyses. Other bootstrap values . 70% are shown on the branches (ML/MP). An asteriskindicates that the branch collapses in the strict consensus tree of the MP analysis.

684 MYCOLOGIA

365, 551) and Julimar Forest (VPI 363, see TABLE II).The collection VPI 364 was identified as ‘‘Torrendiaarenaria f. lutescens’’. In all analyses of the nLSUdataset there are two subgroups in the Arenaria clade,one with the two collections from the type locality andthe other with the remaining collections (FIG. 1). Inthe ITS dataset one collection from each subgroupwas included and the number of differences betweenthe two ITS sequences was similar to, or even greaterthan, the number of differences between pairs ofclosely related species in Amanita (FIG. 3).

These results suggest either the existence of acryptic species among the collections of A. arenaria orrelatively high genetic divergence among populationsfrom different localities. From a morphological pointof view the collections from Kalamunda and JulimarForest have spores with slightly lower values of avQ(1.28–1.36) than the collections from Two PeoplesBay (1.55–1.72), however they all share similar macro-and micromorphological characteristics, includingthe absence of clamp connections and the absence

or scarcity of oleiferous hyphae. One of the collec-tions from Kalamunda (VPI 364) has yellowing fleshand was identified as ‘‘forma lutescens’’, but othercollections with unchanging flesh have been collectedin the same locality (VPI 365, 551), indicating thatthis character is not sufficiently constant to maketaxonomic distinctions based on it. A more detailedmolecular and morphological study that takes intoaccount collections of A. arenaria from differentlocalities, including those mentioned by Bougher(1999), is necessary to establish whether the divergentnLSU and ITS sequences of A. arenaria fromlocalities other than Two Peoples Bay represent anundescribed cryptic species and whether this geneticvariation is correlated with some morphologicalcharacters such as spore shape.

Evolution of secotioid and gasteroid forms in Amani-ta.—Thiers (1984) suggested that the gasteroid formsin many groups of Basidiomycetes evolved from anagaricoid ancestor via secotioid intermediates and

FIG. 3. Best tree from the ML analysis of the ITS dataset of subgenus Lepidella. Thick branches are supported by bootstrapvalues . 90% in the ML and MP analyses. Other bootstrap values . 70% are shown on the branches (ML/MP). An asteriskindicates that the branch collapses in the strict consensus tree of the MP analysis.


provided examples of this sequential process, bothcomplete series such as Cortinarius (agaricoid),Thaxterogaster (secotioid) and Hymenogaster (gas-teroid) and incomplete series such as Agaricus(agaricoid) and Endoptychum (secotioid). He pos-tulated that incomplete series (the Amanita/Tor-rendia was an example at that time) could becaused by the extinction of the missing stage or simplyreflect the incomplete documentation of fungaldiversity.

Two incomplete series are present in the Amanitalineage, including secotioid forms without additionalgasteroid forms (Torrendia clade) and gasteroidforms without known secotioid relatives (Pseudoa-marrendia clade). It is possible that further explora-tion of fungal diversity in Australia and the Mediter-ranean region of Europe and Africa will reveal themissing stages. Also one example of a complete seriesis present in the Amarrendia clade, with secotioidforms related to agaricoid forms (A. umbrinella) with

additional radiation into other secotioid (A. inculta/A. pseudoinculta) and also gasteroid forms (A.grandis/A. oleosa).

Despite the independent origins of the secotioidmorphology in Amanita all species share a greatsimilarity in their general characteristics both macro-and microscopical. Morphological differences amongtaxa are reduced to basidiome size, structural integrityof the gleba, spore size and shape, and presence orabsence of sclerobasidia, oleiferous hyphae andclamp connections. While the majority of secotioidand gasteroid forms presumably have evolved fromancestors with nonamyloid spores in subgen. Amani-ta, A. arenaria probably has evolved from an amyloid-spored ancestor in subgenus Lepidella but thischaracter was lost during the gasteromycetizationprocess.

Influence of Mediterranean climate in the gasteromyce-tization process in Amanita.—The distribution of the

FIG. 4. Amanita pseudoinculta. a. dried basidiomes (bar 5 1 cm). b. spores. c. basidia. d. volva tissue. Bars 5 10 mm. All fromholotype (VPI 555).

686 MYCOLOGIA

secotioid and gasteroid species of Amanita aroundthe Mediterranean Basin and Western Australia (withthe exception of Amanita grandispora known fromVictoria and Tasmania) suggests a direct link betweenthe gasteromycetization process in Amanita and theMediterranean climate that these areas share. Ama-nita arenaria, A. grandis, A. inculta, A. oleosa and A.pseudoinculta are endemic to Western Australia andin fact are known only from a few localities each.Speciation events under Mediterranean conditionsseem to be the origin for the taxa in the Amarrendiaclade. If the existence of a cryptic species among A.arenaria is confirmed it would be another example.The distribution pattern of these related endemicspecies of fungi in relatively close geographic areasclosely matches the distribution pattern of endemicplants in Mediterranean areas (Cowling et al. 1996).

Amanita torrendii has a larger number of popula-tions that range from northern Africa to southernFrance, spreading eastward to Turkey (Watling andIsiloglu 1991), but the great majority of the records(Calonge 1996) are concentrated in the Mediterra-nean region of the Iberian Peninsula. It appears thatA. torrendii has remained a single, relatively wide-spread, secotioid species, in contrast to the Australiantaxa, which have radiated into multiple species,including secotioid and fully gasteroid forms. Moreresearch on the population level is needed to gainfurther understanding of the speciation and gaster-omycetization events in the Amanita lineage.

ACKNOWLEDGMENTS

The curator of VPI is gratefully thanked for the loan of theoriginal T. arenaria collections. Katrina Syme kindlyprovided complete information about the nomenclatureof A. arenaria. We thankfully acknowledge the technicalsupport of and helpful discussions with Manfred Binder,Dimitris Floudas, Brian Seitzman and Andy Wilson. Thecomments and suggestions of two reviewers helped improv-ing the manuscript. Financial support was received from apostdoctoral grant of the Autonomous Government ofGalicia (Spain) to A. Justo and from the NSF grants IOS-0843278 and DEB-0732968.

LITERATURE CITED

Bas C. 1969. Morphology and subdivision of Amanita and amonograph of its section Lepidella. Persoonia 5:285–579.

———. 1975. A comparison of Torrendia (Gasteromycetes)with Amanita (Agaricales). Beih Nov Hedwig 51:53–61.

Binder M, Hibbett DS. 2006. Molecular systematics andbiological diversification of Boletales. Mycologia 98:971–981.

Bougher N. 1999. New species of Torrendia (Fungi,Agaricales) from remnant woodlands in the wheat-belt

region of Western Australia. Australian Syst Bot 12:145–156.

———, Lebel T. 2002. Australian sequestrate (truffle-like)fungi XII. Amarrendia gen. nov.: an astipitate, seques-trate relative of Torrendia and Amanita (Amanitaceae)from Australia. Australian Syst Bot 15:513–525.

Bresadola G. 1902. Mycetes Lusitanici novi. Atti Imp RegiaAccad Rovereto ser. 3, 8:132.

Calonge FD. 1996. Torrendia pulchella. In: Almaraz T, ed.Bases corologicas de Flora Micologica Iberica. Numeros693–894. Madrid, Espana: Real Jardın Botanico. CuadTrabajo Flora Micol Iberica 9: 211–212.

Cowling RM, Rundel PW, Lamont BB, Arroyo MK,Arianoutsou M. 1996. Plant diversity in Mediterra-nean-climate regions. Trends Ecol Evol 11:362–366.

Drehmel D, Moncalvo JM, Vilgalys R. 1999. Molecularphylogeny of Amanita based on large-subunit ofribosomal DNA sequences: implications for taxonomyand character evolution. Mycologia 91:610–618.

Eberhardt U, Verbeken A. 2004. Sequestrate Lactariusspecies from tropical Africa: L. angiocarpus sp. nov. andL. dolichocaulis comb. nov. Mycol Res 91:1042–1052.

Gardes M, Bruns TD. 1993. ITS primers with enhancedspecificity for basidiomycetes application to the iden-tification of mycorrhizae and rusts. Mol Ecol 2:132–118.

Hallen HE, Bougher N, Lebel T. 2004. Phylogeneticplacement of Amarrendia and Torrendia: sequestrateAmanita or a mixed bag? Inoculum 55:16.

Hibbett DS. 2007. After the gold rush, or before the flood?Evolutionary morphology of mushroom-forming fungi(Agaricomycetes) in the early 21st century. Mycol Res111:1001–1018.

———, Donoghue MJ. 1998. Integrating phylogeneticanalysis and classification in fungi. Mycologia 90:347–356.

Hosaka K, Bates ST, Beever RE, Castellano MA, Colgan IIIW, Domınguez LS, Nouhra ER, Geml J, Giachini AJ,Kenney SR, Simpson NB, Spatafora JW, Trappe JM.2006. Molecular phylogenetics of the gomphoid-phal-loid fungi with an establishment of the new subclassPhallomycetidae and two new orders. Mycologia 98:949–959.

Maddison DR, Maddison WP. 2002. MacClade4: analysis ofphylogeny and character evolution. Sunderland, Mas-sachusetts: Sinauer Associates.

Malencon G. 1955. Le developement de Torrendia pulchellaBres. et son importance morphogenetique. Rev Mycol20:81–130.

Miller OK, Horak E. 1992. Observations on the genusTorrendia and a new species from Australia. Mycologia84:64–71.

Moncalvo JM, Vilgalys R, Redhead SA, Johnson JE, JamesTY, Aime MC, Hofstetter V, Verduin SJW, Larsson E,Baroni TJ, Thorn RG, Jacobsson S, Clemencon H,Miller Jr OK. 2002. One hundred seventeen clades ofeuagarics. Mol Phylogenet Evol 23:357–400.

Moreno G, Platas G, Pelaez F, Bernedo M, Vargas A, Daza A,Santamarıa C, Camacho M, Romero de la Osa L,Manjon JL. 2008. Molecular phylogenetic analysis show


that Amanita ponderosa and A. curtipes are distinctspecies. Mycol Progress 7:41–47.

Neville P, Poumarat S. 2004. Fungi Europaei 9: Amaniteae.Alassio, Italy: Candusso.

Nuytinck J, Verbeken A. 2007. Species delimitation andphylogenetic relationships in Lactarius section Deliciosiin Europe. Mycol Res 111:1285–1297.

Peintner U, Bougher NL, Castellano MA, Moncalvo JM,Moser MM, Trappe JM, Vilgalys R. 2001. Multipleorigins of sequestrate fungi related to Cortinarius(Cortinariaceae). Am J Bot 88:2168–2179.

———, Moser MM, Vilgalys R. 2002. Thaxterogaster is ataxonomic synonym of Cortinarius: new combinationsand new names. Mycotaxon 81:174–184.

Ronquist F, Huelsenbeck JP. 2003. MrBayes3: Bayesianphylogenetic inference under mixed models. Bioinfor-matics 19:1572–1574.

Smith ME, Healy RA. 2009. Otidea subterranea sp. nov.:Otidea goes below ground. Mycol Res 113:858–866.

Stamatakis A, Hoover P, Rougemont J. 2008. A rapidbootstrap algorithm for the RAxML Web servers. SystBiol 75:758–771.

Swofford DL. 2002. PAUP*: phylogenetic analysis usingparsimony (*and other methods) 4.0 Beta. Sunderland,Massachusetts: Sinauer Associates.

Taylor AFS, Hills AE, Simonini G, Both EE, Eberhardt U.2006. Detection of species within the Xerocomussubtomentosus complex in Europe using rDNA-ITSsequences. Mycol Res 110:276–287.

Thiers HD. 1984. The secotioid syndrome. Mycologia 76:1–8.

Tulloss RE. 2005. Amanita distribution in the Americas withcomparison to eastern and southern Asia and notes onspore character variation with latitude and ecology.Mycotaxon 93:189–231.

Vellinga EC, de Kok RPJ, Bruns TD. 2003. Phylogeny andtaxonomy of Macrolepiota (Agaricaceae). Mycologia 95:442–456.

Watling R, Isiloglu M. 1991. Torrendia pulchella Bres. A newand interesting record from Turkiye. Turkish J Bot 15:297–299.

Weiß M, Yang ZL, Oberwinkler F. 1998. Molecularphylogenetic studies in the genus Amanita. Can J Bot76:1170–1179.

Yang ZL. 1997. Die Amanita-Arten von Sudwestchina.Biblioth Mycol 170:1–240.

Zang L, Yang J, Yang ZL. 2004. Molecular phylogeny ofeastern Asian species of Amanita (Agaricales, Basidio-mycota): taxonomic and biogeographic implications.Fungal Divers 17:219–238.

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convergent evolution of sequestrate forms in amanita under

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