SHORT COMMUNICATION

Proof of ectomycorrhizal status of Sistotrema confluens Pers.,the type species of the polyphyletic genus Sistotrema

Ben Bubner & Christine Morgner & Wolfgang Stark &

Babette Münzenberger

Received: 28 November 2013 /Revised: 18 February 2014 /Accepted: 20 February 2014# German Mycological Society and Springer-Verlag Berlin Heidelberg 2014

Abstract The genus Sistotrema is supposed to be polyphy-letic. This idea is supported by the existence of saprotrophicand ectomycorrhizal Sistotrema species. We show for the firsttime that the type species Sistotrema confluens Pers. isectomycorrhizal by ITS rDNA sequence identity of fruitingbodies and underneath growing ectomycorrhizas. The accom-panying anatomical description of the ectomycorrhiza is incorrespondence with previously published descriptions ofSistotrema ectomycorrhizas, especially the presence ofampullate hyphae. The ectomycorrhizal status and the deviat-ing fruiting body morphology (partially stipitate versus resu-pinate in all other Sistotrema species) indicate a relationship ofS. confluens to the ectomycorrhizal genus Hydnum. Aphylogram of ITS rDNA sequences cannot resolve the basalpolytomies in the genus Sistotrema but confirms the mono-phyly of the genus Hydnum. Thus, S. confluens is not moreclosely related to Hydnum than to ectomycorrhizal Sistotremaspecies. The proof of the ectomycorrhizal status of S. confluenscan be of value in a future revision of phylogenetic relationships

within the group of Hydnum and ectomycorrhizal Sistotremaspecies.

Keywords Ampullate hyphae . Anatomotype .

Ectomycorrhiza . ITS rDNA . Sistotrema confluens

Question

A series of publications demonstrated the ectomycorrhizalstatus of several species within the genus Sistotrema(Nilsson et al. 2006; DiMarino et al. 2008; Münzenbergeret al. 2012). However, it was also shown that Sistotrema is apolyphyletic genus that contains both ectomycorrhizal andsaprophytic species (Nilsson et al. 2006). Two large-scalephylogenetic studies in the euagarics indicated that severalclades can be better supported by trophic status than bymorphological similarities (Hibbett et al. 2000; Moncalvoet al. 2002). An example at the genus level is Tapinellaatrotomentosa (Batsch) Šutara that was for a long time includ-ed in the genus Paxillus. Molecular studies confirmed that thewood rotting genus Tapinella is clearly separated from theectomycorrhizal genus Paxillus s. str. (Bresinsky et al. 1999).With respect to the polyphyletic genus Sistotrema, Larsson(2007) proposed that the ectomycorrhizal Sistotrema speciesare closely related to the ectomycorrhizal genus Hydnum,while the remaining saprophytic Sistotrema species have tobe placed in other genera.

In a future revision of the genus Sistotrema it is of interestwhether the type species of the genus Sistotrema confluensPers. is ectomycorrhizal or saprophytic. Based on aphylogram of 18S rDNA sequences in which S. confluensgrouped with ectomycorrhizal Sistotrema species, Nilssonet al. (2006) proposed that S. confluens might beectomycorrhizal. Here we present the direct proof by molec-ular methods that S. confluens forms ectomycorrhizae. We

B. Bubner (*)Thünen Institute of Forest Genetics, Eberswalder Chaussee 3A,15377 Waldsieversdorf, Germanye-mail: ben.bubner@ti.bund.de

C. Morgner :W. StarkAm Brandteich 1, 08239 Bergen, Germany

C. Morgnere-mail: cmorgner@freenet.de

W. Starke-mail: cmorgner@freenet.de

B. MünzenbergerLeibniz-Centre for Agricultural Landscape Research (ZALF),Institute of Landscape Biogeochemistry, Eberswalder Straße 84,15374 Müncheberg, Germanye-mail: bmuenzenberger@zalf.de

Mycol ProgressDOI 10.1007/s11557-014-0973-3

also include a short morphological and anatomical descriptionof the S. confluens ectomycorrhiza and a phylogram ofSistotrema and Hydnum ITS rDNA sequences.

Collecting the fungus

Fruiting bodies of Sistotrema confluens were collected nearBergen, Vogtland, Germany on September 15, 2007 (MTB5539/231, road from Bergen to Theumar, right-hand side,508–513 m above sea level). The species was identified onthe basis of the fruiting body morphology (Fig. 1a). Fruitingbodies grew next to a road under birch (Betula pendula Roth)and aspen (Populus tremula L.). Seedlings of spruce (Piceaabies (L.) Karst) were found at a 10 m distance. The fruitingbodies were collected together with the first 10 cm of top soil.Morphology and anatomy of ectomycorrhizae found belowthe fruiting bodies were analyzed two days later in the labo-ratory and microphotographs of morphology and mantle anat-omy were prepared. On October 2, 2013 fruiting bodies couldbe observed at the same location. Again, ectomycorrhizal roottips were collected beneath the fruiting bodies to preparemicro drawings of the mantle anatomy as a complement tothe description and microphotographs of 2007.

Molecular proof

Mycorrhized root tips and fruiting bodies were ITS sequencedaccording to the methods described by Münzenberger et al.(2009) using the primers ITS1F and ITS4, both for PCR andsequencing. The forward and reverse sequences of each samplewere assembled to a 644 bp double-stranded sequence. Onehundred percent identity (644/644) of fruiting body sequence(laboratory code 971, GenBank accession no. KC86586) andmycorrhized root tip sequence (laboratory code 972, GenBankaccession no. KC86587) is the proof that the analyzedectomycorrhiza is formed by Sistotrema confluens (Fig. 1f).The strength of evidence that S. confluens is ectomycorrhizal iscomparable to the evidence published for S. muscicola (Pers.)S. Lundell, S. alboluteum (Bourdot & Galzin) Bondartsev &Singer, (Nilsson et al. 2006), and the unknown Sistotremaspecies RA 14583 (DiMarino et al. 2008). In these three speciesproof of ectomycorrhizal status is also based on ITS sequenceidentity of fruiting bodies and ectomycorrhizae.

Ectomycorrhizal description

The short description of the ectomycorrhiza of Sistotremaconfluens follows the principles published in Agerer et al.(1998–2006) and the online key DEEMY (Agerer andRambold 2004–2013).

Morphology: Ectomycorrhizae monopodial-pyramidallyramified or as solitary tips (Fig. 1b). System up to 2.7 mmlong, unramified ends 0.4 to 0.6 mm long and 0.3 mmdiam. Colour light yellowish brown, hydrophobic surfacewith a silvery shine. Abundant emanating hyphae (cot-tony to woolly surface).Mantle anatomy: plectenchymatic in all mantlelayers. Hyphae of the outer mantle layer without aspecial pattern (mantle type B) but streaks of par-allel hyphae (Fig. 1c). Emanating hyphae extendfrom the outer mantle (arrow in Fig. 1c). Hyphaeof the outer mantle mostly cylindrical (ø 3–6.5 μm,length 15–60 μm, wall thickness 0.5 μm). Somecells irregularly shaped or with slightly inflatedends or constrictions at septa. Hyphae ramify atangles of 120°. Surface of hyphae smooth, hyphaecolourless to slightly membranaceously brownish.The arrangements of the middle and the inner man-tle layer are similar to each other. Basicallyplectenchymatic but shorter hyphae as in outer man-tle (ø 4–6 μm, length 12–25 μm, wall thickness0.5 μm in middle mantle; ø 4–6 μm, length 10–30 μm, wall thickness 0.5 μm in inner mantle).Hyphae o f inne r man t l e accompan ied bypseudoparenchymatic cells (Fig. 1d). Surface of hy-phae smooth, hyphae colourless to slightlymembranaceously brownish.Emanating elements: Emanating hyphae (ø 3–4 μm,cells up to 100 μm long, wall thickness 0.5 μm)clamped, ramification at angles of 90°, one or twohyphal diameters below septum. At normal septaclamps in lateral view semicircular with a radiusequal to the hyphal width, in dorsal view cylindri-cal and with the same width as the hypha.Ampullate inflations up to 6 μm wide at one sideof the septum (Fig. 1e). Cell surface smooth, cellscolourless to slightly membranaceously brownish,rhizomorphs absent.

Anatomical comparison with other Sistotremaectomycorrhizae

Because of the lack of a detailed ectomycorrhizal de-scription of S. muscicola and S. alboluteum (Nilssonet al. 2006), the S. confluens ectomycorrhiza can onlybe compared with the two descriptions in DiMarinoet al. (2008) and Münzenberger et al. (2012). All threeectomycorrhizae are similar in having lightly colouredmantle surfaces with a silvery appearance and manyemanating hyphae (woolly to cottony surface). At theanatomical level they have the plectenchymatic mantle

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in common (between type B/C, when ring-like struc-tures present type A), and in all three species clampedsepta are present.

The most striking difference is the presence of nu-merous rhizomorphs in the ectomycorrhizae of RA

14583 (DiMarino et al. 2008), while they are absentor not conspicuous in S. confluens and in the unknownSistotrema species EW/BM63 (Münzenberger et al.2012). The presence or absence of rhizomorphs inSistotrema deserves further attention because it can be

Fig. 1 Sistotrema confluens: a fruiting body, identified by morphology bectomycorrhizal root tip below fruiting body, 40x, depicted root wassequenced c upper mantle with an emanating hypha (arrow), 1000x dlower mantle, 1000x e emanating hyphae with inflated ends, 1000x fMaximum Likelihood (ML) phylogram based on a 653 bp alignment ofthe rDNA ITS region. Reference sequences are from UNITE database(http://unite.ut.ee) and from references (1), (2), and (3). The ML tree was

generated with the RAxML algorithm and 100 bootstrap replicates(Stamatakis et al. 2008). A second tree was generated with theNeighbour-Joining (NJ) algorithm and 1000 bootstrap replicates(PAUP* 4.0b10, Sinauer, USA). At nodes with support values above 50they are given in the order ML/NJ. Bold sequences belong to publishedproofs of the ectomycorrhizal status of other Sistotrema species.

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characteristic for a genus as e.g. in Xerocomus andLeccinum (Agerer 2001). It might be that therhizomorph differences between the Sistotremaectomycorrhizas are due to ontogenetic stage. DiMarinoet al. (2008) observed the rhizomorphs at olderectomycorrhizas of field collections while Münzenbergeret al. (2012) described the morphology of EW63 from21 weeks old axenically cultured mycorrhizas.

One anatomical detail is common for S. confluens, the twounknown Sistotrema species (DiMarino et al. 2008;Münzenberger et al. 2012), and also for S. muscicola andS. alboluteum (Nilsson et al. 2006): the presence of hyphaewith ampullately inflated ends. This feature is most promi-nently found in the emanating hyphae but also in rhizomorphswhen they are present. Future investigations should evaluatewhether ampullate inflations are of taxonomic value as aunifying character of the ectomycorrhizal species in the genusSistotrema.

Comparison of ITS sequences

A Maximum-Likelihood phylogram was generated usingITS rDNA sequences from the GenBank and UNITEdatabases as reference sequences (Fig. 1f). Note that inaddition to Sistotrema confluens ectomycorrhiza, it isthe first time that the sequence of the unknownSistotrema species RA 14583 (DiMarino et al. 2008) isincluded into a phylogram. The phylogram confirms thatall four published ectomycorrhizas (S. muscicola,S. alboluteum, EW/BM63, and RA14583), and the new-ly described ectomycorrhiza of S. confluens belong eachto a separate species. Due to the large ITS sequencedivergence between the species (less than 90 % se-quence homology in most cases), many subterminalnodes of the tree cannot be supported by Maximum-Likelihood (ML) or Neighbour-Joining (NJ) bootstrapvalues. Therefore, although species can be easily delin-eated by ITS rDNA sequences, their relationships toeach other remain unclear. It is to note that S. confluensis not included in any monophyletic group (Fig. 1e).Thus once more, the phylogram confirms the taxonomicdifficulties and the polyphyletic status of the genusSistotrema.

The large genetic distance of S. confluens to the otherectomycorrhizal Sistotrema species is also reflected in mor-phological characteristics. Despite being the type species ofthe genus Sistotrema, S. confluens deviates from all otherSistotrema species in the habit of the fruiting body. Whilethe remaining Sistotrema species have resupinate fruitingbodies (Larsson 2007), those of S. confluens are partiallystipitate (Fig. 1 a). In that respect S. confluens resemblesHydnum species that are ectomycorrhizal and have stipitate

fruiting bodies (Larsson 2007). This leads to the hypothesisthat S. confluens might be more closely related to Hydnumspecies than to ectomycorrhizal but resupinate Sistotremaspecies. Therefore, Hydnum species have been included inthe ITS phylogram (Fig. 1f). In contrast to Sistotrema se-quences, Hydnum sequences form a well supported clade(node support 65/75, Fig. 1f). Since S. confluens is not includ-ed in thisHydnum cluster, it is possible to reject the hypothesisthat S. confluens is more closely related to Hydnum speciesthan to ectomycorrhizal Sistotrema species. The exact positionand taxonomic status of S. confluens within the group ofHydnum and ectomycorrhizal Sistotrema species can only bedetermined in future investigations that are based on a broadersampling of confirmed ectomycorrhizal Sistotrema species.

Acknowledgments We thankKatja Kühdorf for assistance in preparingthe micro drawings of mantle anatomy and Monika Roth and PetraKnauer for assistance in the laboratory. Comments of Reinhard Agererimproved the short description of the ectomycorrhiza.

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