Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya, IMA Fungus 5(1): 80 (2014) – Figures 5–11

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Figure 11.

Hymenoscyphus fraxineus (Japan). a–g, u. Apothecia emerging from black pseudosclerotia in rachises of Fraxinus mandshurica (u: fresh; a–g: rehydrated); i–k. pseudosclerotium in cross section (i–j: several stromata in one rachis, delimited by black demarcation lines that occur in the xylem along radial rays and through the mark parenchyma); l. median section of stipe base (with black basal surface and internal crystals); m. surface view on ectal excipulum near margin (with yellow exudate); n–t, v–w. ascogenous hyphae with croziers. – Dead state (h–k, m in H2O; l in KOH; n–t KOH+CR; v–w in phloxine B). – Nagano: a, e, k. TNS-F-40074 (Sugadaira); b, g. TNS-F-12761 (ibid.); f, h. TNS-F-17817 (ibid.); v. TNS-F-12503 (Yuzawa); Hokkaido: c, m, s–t. TNS-F-52060 (Tomakomai); d, l, n–r, u. TNS-F-40043 (ibid.); i, j, w. TNS-F-52061 (Sapporo). – Phot. u: YJ Zhao.

• ≡ Chalara fraxinea T. Kowalski, Forest Pathology 36, p. 265 (2006) [anamorph]

• = Hymenoscyphus pseudoalbidus Queloz, Grünig, Berndt, T. Kowalski, T.N. Sieber & Holdenr., Forest Pathology 41, p. 140 (2010) [teleomorph]

Hymenoscyphus honshuanus Baral, nom. nov. – Figure 12 – MycoBank MB 809090

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Figure 12.

Hymenoscyphus honshuanus (holotype of Lambertellinia scutuloides): a. ascospores (partly from inside asci, containing LBs); b. ascogenous hyphae with crozier formation; c. median section of receptacle; d. apothecium; e–f. ascus apices with euamyloid apical ring (Hymenoscyphus-type); g. ectal excipulum at flanks (median section), with gelatinized walls. – All in dead state.

• Basionym: Lambertellinia scutuloides Korf & Lizoň, Mycotaxon 50, p. 168 (1994) [non Hymenoscyphus scutuloides Hengstm., Persoonia 16, p. 199 (1996)]

• ≡ Lambertellinia scutuloides Korf & Lizoň, Inoculum, Newsletter of the Mycological Society of America 44(2), p. 43 (1993); nom. inval., Art. 39.1 (Melbourne)

Type study and circumscription

According to the present redescription of the holotype, which was briefly mentioned also in Galán and Baral (1997), the taxon deviates from H. aesculi and H. albidus in the presence of croziers and some other features (see also under those species). While the ascospores of H. aesculi lose their characteristic central swelling in the dead state, it is unknown whether living spores of H. honshuanus might also show such swelling.

Korf and Lizoň (1994) thought that Lambertellinia scutuloides resembles the genus Lambertella Höhn. in its black substratal stroma and the ascospores getting finally light brown, but the authors placed it in a new genus because of its long-celled excipular structure, the assumed connection to an Idriella anamorph, and probably also because of the deviating scutuloid spore shape, to which the original species epithet refers. Regrettably, no microscopic features were illustrated in the protologue, except for a photograph showing a section of the excipulum.

Korf and Lizoň believed that collections on leaves of Acer rubrum from North America and Berberis vulgaris from Germany are conspecific, though they examined only that on Berberis. The collection on Acer rubrum was treated by Kimbrough and Atkinson (1972) under the name Hymenoscyphus caudatus. Unlike Hy. fraxineus, a strain from this collection formed an Idriella anamorph in pure culture, with slightly falcate conidia measuring ?15 × 2 µm (evaluated from photos), formed in sporodochia in a sympoduloconidial type of development. The original material was not available to Korf and Lizoň, who were sure about its conspecificity with Lambertellinia scutuloides because of the reported brown colouration of the 1–2-septate ascospores when ‘mature’. However, Kimbrough and Atkinson did not observe stromatic tissues around the inhabited midribs and larger veins, although they saw the stipe base to arise from darkened host tissue. Regrettably, Kimbrough and Atkinson did not report the presence or absence of croziers, though they stated that the features of their fungus were essentially the same as in Hy. caudatus as described by White (1943) and others.

Korf and Lizoň (1994) characterized the ectal excipulum of Lambertellinia scutuloides as a hyaline textura porrecta consisting of gelatinized hyphae 3–6.5 µm wide, externally covered by a thin layer of straw- or golden-yellow pigmented hyphae 0.5–1.5 µm wide, and internally delimited by a light yellow layer. The authors considered this construction of the ectal excipulum as different from typical members of Hymenoscyphus. However, the present re-examination of the excipular texture (Figure 12(g)) revealed no substantial differences from, e.g., Hy. albidus, Hy. fraxineus, and Hy. aesculi.

The description given above is based on the present re-examination of the holotype, but it includes also some data of the protologue, mainly in regard to macroscopical features. The petioles from which the apothecia of Hy. honshuanus arise show a dark blackish-grey colour. Korf and Lizoň referred to it as a ‘black substratal stroma’. Similar as in the other species treated in the present paper, a black ring was often seen at the outermost base of the stipe (Figure 12(d)). Contrary to Korf and Lizoň, who stated the asci to be simple-septate, the present examination of two apothecia showed that they arise from croziers (Figure 12(b)). Since the authors did not illustrate the feature, their report of absent croziers remains mysterious. Apart from the abundant hyaline ascospores, only a single spore with a pale ochre-brown wall was seen in the present examination. It must be stated, however, that the asci, spores and paraphyses were distinctly wider than indicated by Korf and Lizoň, who measured them as follows: asci (82–)84.5–100(–116) × (5.6–)6.6–7.5(–8.3) µm, spores (13.8–)15.4–16.9(–18.5) × (2.3–)3.1–3.5(–3.8) µm, paraphyses 1.5 µm wide. The protologue appears to be based exclusively on the Japanese type specimen. Data of the specimen from Germany (on Berberis) were not incorporated, which is obvious from the mentioned larger spore size in the Berberis specimen (Korf & Lizoň 1994, p. 171).

We assume that Hy. honshuanus was incorrectly described by Korf and Lizoň (1994) concerning the ascus base, though it cannot be excluded that the holotype collection represents a mixtum of two species with deviating ascus base characters as well as ascus and spore measurements. In any case, the present data indicate that the here described specimen is not conspecific with either Hy. aesculi or Hy. caudatus. Hy. caudatus was redescribed by White (1943, p. 151, fig. 8), who observed the absence of croziers in most (though not all) of the many examined specimens, including the holotype. Regrettably, White did not specify the aberrant material in which he saw croziers.

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Figure 8.

Hymenoscyphus fraxineus (Europe): a. apothecium in median and rachis in cross section (the black demarcation line covers the outer face of the sclerenchyma), b–f. apothecial stipe in median section (with internal crystals), g. rachis in cross section (with external demarcation line, cells of sclerenchyma containing hyphae), h–k. external view on pseudosclerotial plate (tangential section of rachis surface); cells of cortical parenchyma densely filled with hyaline or ± brown hyphae (textura epidermoidea). – All in living state. – a. H.B. 9596 (DE-BW, Tübingen), b, f–g. H.B. 9589 (DE-BY, Amberg), c–e. H.B. 9593 (DE-SN, Chemnitz), h–k. H.B. 9588 (DE-BY, Hirschau).

Because of the striking morphological similarity with Hy. fraxineus, it would be interesting to determine the ITS sequence of Hy. honshuanus. However, this approach requires careful examination of single apothecia for the presence of croziers, in order to avoid confusion in the case of a mixtum. Actually, a further Japanese record (on petioles of Aesculus sp. kindly sent by T. Hosoya, TNS-F-12758) differs from H. honshuanus in the ascus base, indicating that two different species exist in Japan: the asci arise here from simple septa (Figure 15), and the species is, therefore, included in Hy. aesculi.

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Figure 18.

Hymenoscyphus aesculi: a–b, e, r. fresh apothecia (e: in median section, petioles in cross section); c–d. senescent apothecia (with dark bluish-olive patches on receptacle); f–g. stipe base in median section, showing erumpent growth and absence of crystals; h. cortical parenchyma and sclerenchyma below insertion of apothecial stipe, densely filled with intracellular hyphae; i. sclerenchyma forming a radial ray, loosely filled with hyphae; j–l. ectal excipulum at stipe and (l) at lower flanks in median section, showing VBs in cortical cells; m. ascus apices with euamyloid apical ring; n–q. ascospores (n–o, q: freshly ejected); s–t. surface view on stipe base, showing brown cortical cells and hairs. – Living state, except for m (in KOH+IKI), o (in KOH+CRB), p (in H₂O?). – a. 31.VII.2011 (DE-HS, Darmstadt), b–p. H.B. 9701 (Darmstadt), q–t. C.Y. F/2194 (UK-Yorkshire, Halifax). – Phot. a, p: H. Lotz, q–t: C. Yeates.

With the present data, Korf and Lizoň’s conclusion that Lambertellinia scutuloides has an Idriella anamorph needs to be reinvestigated. Experiments with cultures of the common and plurivorous Hy. caudatus, but also Hy. vacini, a taxon specific to leaves of Acer, kept under varying conditions as proposed by Kimbrough and Atkinson (1972), could clarify whether or not any of these species are connected to an Idriella anamorph.

The German specimen on Berberis (Sydow, Mycotheca Marchica no. 1576, 1887) remains of unclear identity. The apothecia are said to arise from stromatized veins and petioles, but the presence or absence of croziers was not stated by Korf and Lizoň. When dry, the apothecia were ‘dark brown (almost blackish)’. The spores measured 16.2–19.8 × 3.6–4.8 µm and were ‘cylindric-clavate, scutuloid, deep brown to hyaline’. The exsiccatum was issued under the name Helotium berberidis Syd., but without a diagnosis on the label (Korf & Lizoň 1994).

Hy. honshuanus is known with certainty only from the type collection on Aesculus turbinata. Reports under the names He. robergei or Hymenoscyphus albidus on fallen petioles of Aesculus indica in Northern India and Nepal might well concern Hy. honshuanus (or Hy. aesculi), but the ascus base was not studied for the presence of croziers (see discussion under Hy. albidus).

The type locality of Hy. honshuanus is not exactly known, and the collector, Daisuke Shimizu, who was a zoologist specialized in vegetable wasps, passed away in 1998. The small town Oguni(-machi) belongs in the Nishiokitama district and lies 55 km WSW of Yamagata. The village Tsugawa could not be located (it was included in Oguni in 1960), but its altitude was found to be 294 m (T. Hosoya personal communication). Oguni has an altitude of about 140 m; therefore, the type locality must be in one of the adjacent mountain ranges.

Hymenoscyphus aesculi (Velen.) Baral & E. Rubio, comb. nov. – Figures 13–​1919 – MycoBank MB 809091

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Figures 13–15.

Hymenoscyphus aesculi. a. ascospores (partly from inside asci, containing LBs); b. ascus apices with euamyloid apical ring (Hymenoscyphus-type); c. ascus bases arising from simple septa; d. apothecia. – All in dead state.

• Basionym: Helotium aesculi Velen., Opera Bot. Čech. 4, p. 120 (1947)

• ≡ Lanzia aesculi (Velen.) Svrček, Acta Mus. Nat. Pragae 40 B, p. 131 (1985, ‘1984’)

• (?) = Hymenoscyphus albidus var. aesculi W. Phillips, Man. Brit. Discom. p. 138 (1887)

• ≡ Phialea albida var. aesculi (W. Phillips) Saccardo, Syll. Fung. 8, p. 254 (1889)

• ≡ Helotium albidum var. aesculi (W. Phillips) Massee, Brit. Fung.-Fl. 4, p. 260 (1895)

Type studies and circumscription

In the protologue of Helotium aesculi, Velenovský (1947) described the apothecia as white, 2–4 mm diam., later with a black stipe, the spores as 12–17 × 6–7 µm, with an indistinctly acute base and a series of guttules, and the asci as 130–150 × 6–8 µm. In comparison with the here presented description, these measurements fit only concerning spore length and ascus width, whereas apothecial size, spore width and ascus length are extraordinary large and perhaps erroneous, even when taking living elements into consideration.

Svrček (1989) did not describe the type material of He. aesculi, but he stated that it fully concurs with some fresh specimens studied by him. This is astonishing, because he measured the asci in one of his collections with 65–85 × 7–10 µm without discussing the discrepancy to the protologue. He also noted (p. 74) that ‘the original Velenovský diagnosis is not accurate if compared with V. Vacek’s [the finder of the holotype] handwritten notes’.

No such handwritten notes were seen when the holotype was re-examined in the present study. It revealed an ascus size (see Figure 13) compatible with Svrček’s above-mentioned measurement which likewise undoubtedly refers to dead asci. The contents of many paraphyses were bright olivaceous-brown, and in cross section of a petiole a distinct black stroma could be seen which surrounds the sclerenchyma on its outer as well as inner face.

Svrček (1989, pl. 2 fig. 4) illustrated a personal collection from near Praha, which he considered conspecific with the type of Helotium aesculi. Concerning his reasons to transfer the species to Lanzia, Svrček (1985) referred to a paper in press which, however, does not treat this species (see also under Hy. vacini). Probably he proposed the combination because of the blackish stroma, but in 1989 he mentioned merely the dark stipe base, not the substratal stroma. The drawn spores look as if they were in the living state, according to the regular lipid content. Svrček (1989, p. 20) described them as ‘fusiform, clavate, often strongly tapering below and curved, filled with many small and larger guttules’, 15–24.5 × (3.5–)4–5 µm, i.e., rather variable in length. The spores lack the central swelling, however, and the LBs are drawn distinctly smaller than in any of the here studied collections. Svrček examined also a further specimen from Southern Bohemia and one that H. Engel sent to him from Weidhausen near Coburg. The latter collection was also examined in the present study and found to have predominantly strongly fusiform spores with a central bulge and four rather large LBs, unlike the sketch by Engel and Hanff (1989, p. 40) that shows no central bulge and only rather small LBs.

In all collections of Hy. aesculi studied by the first author in the fresh state, the characteristic central swelling of the living spores was noted in a major part of the freshly ejected spores (see Figures 16(i), 16(m); 18(n)). A central swelling is also obvious in some or all spores of fresh collections drawn by B. Grauwinkel (personal communication) from Fallingbostel (Niedersachsen), B. Fellmann (personal communication) from Starnberg (Oberbayern), B. Declercq (personal communication) from Wachtebeke (Eastern Flanders), and Beyer (1998) from Bayreuth (Oberfranken). This swelling is further evident in almost every spore of a British record photographed by C. Yeates (personal communication, Figure 18(p)), and in one from Asturias by E. Rubio (personal communication, Figure 17(c)–(d)).

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Figure 16.

Hymenoscyphus aesculi: a–d, l. fresh apothecia; j–k. dry apothecia (phot. over 3 years after collection); g. cortical ectal excipulum of receptacle in surface view (VBs in living cells, distorted brass-yellow cytoplasm of scattered external hyphae); h. paraphyses in squash mount (containing VBs); e–f. turgescent mature asci; i, m. ascospores (freshly ejected, containing LBs). – All in living state (except for j–k, brass-yellow cells in g). – a–f, h–i. H.B. 8914 (DE-NI, Hamburg); g. H.B. 9843 (DE-BW, Tübingen); l–m. H.B. 9417 (DE-SN, Chemnitz). – Phot. l: B. Mühler.

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Figure 17.

Hymenoscyphus aesculi: a–b. fresh apothecia; j–l, p. rehydrated apothecia (colour was originally white except for base of stipe); f–g, o. asci and paraphyses(o: with olive secondary pigment especially in paraphyses); h. ascus apices with euamyloid apical ring; i: simple-septate ascus base; c–e. ascospores; m–n, q–s. cross section through cortical region of petioles, with black demarcation line surrounding the sclerenchyma on outer and inner face (cortical parenchyma above and phloem beneath being entirely decomposed). – Living state: c–g; dead state: h (in IKI), o (in H₂O). – a–i. E.R.D. 5685 (ES-Asturias, Somiedo); j–o. H.B. 5736 (DE-BW, Gaildorf); p–s. TNS-F-12758 (JP-Honshu, Nagano, Sugadaira). – Phot. a–i: E. Rubio.

However, the peculiar spore shape of Hy. aesculi is state-dependent and well recognizable only in living spores. The loss of the spore bulge under treatment with KOH is illustrated on Figure 18(n)–(o). Apart from the holotype, two specimens of Hy. aesculi were studied in the dead state only (Figure 14–15), and these were first thought to deviate from typical Hy. aesculi in ascospores devoid of a central swelling. From the observed influence of spore turgor on spore shape; however, we assume that they also possessed a central swelling in the living state. In the holotype of Hy. aesculi (Figure 13), a few of the fusoid spores still showed the median swelling characteristic of the living spores. In conclusion, one of the most diagnostic markers to separate Hy. aesculi from Hy. albidus is partially or entirely obscured in herbarium material.

The medullary excipulum in the central part of the receptacle of Hy. aesculi was found to be composed of a rather dense, upwards oriented textura prismatica-porrecta, unlike Hy. albidus and Hy. fraxineus for which we have always observed a loose t. intricata. However, this feature was only tested in one collection (H.B. 9701) and needs further study.

All presently known European records of Hy. aesculi grew on leaves of Aesculus hippocastanum. The species fruits between July and October, predominantly in August. A distribution map under the name Lanzia aesculi for W-Germany is presented in Krieglsteiner (Krieglsteiner 1993, pl. 744), with 13 grid squares distributed in Niedersachsen, Bayern, and Baden-Württemberg (including Northern Switzerland). One of the two examined Japanese specimens on blackened Aesculus petioles (as Lanzia sp.) is here referred to Hy. aesculi (Figure 15, 17(p)–(s)) because of the absence of croziers and the lack of crystals in the stipe base; the other is the type of Hy. honshuanus (≡Lambertellinia scutuloides) and deviates from Hy. aesculi in having croziers.

Size

Apothecial disc diameters were found to vary strongly within a species. In those on leaves, the diameter seems to depend mainly upon whether the apothecia emerge from a thick petiole or from thin veins. Although Hy. vacini grows on thin net veins of skeletonized leaves, this species is able to produce rather large apothecia.

In Hy. fraxineus a tendency towards larger apothecia in comparison with Hy. albidus is confirmed in the present study (see Tables 1 and ​and2).2). A clear trend towards larger apothecia in Hy. fraxineus (maximum 3–7 mm diam.) was reported by Kowalski (2012) in a table, while Queloz et al. (2011) stated that they could not observe any difference in apothecial size between the two species. The disc diameter of Hy. albidus is given in the literature (fresh or rehydrated) as 1–2 mm (Desmazières), up to 2 mm (White, Dennis), 1–2.5 mm (Arendholz), 1–3 mm (Breitenbach & Kränzlin), 1.5–3 mm (Boudier), or 2–4 mm (Srvček). Stipe length is reported as about 1–1.5(–2) mm. This is in concordance with our observations, and gives evidence that this species rarely exceeded 4 mm in diam. Only Patouillard indicated a larger size (3–5 mm), and also Kirisits and Kräutler (2013, fig. 1) observed diameters of up to 5 mm in a collection from the Bretagne. Hy. fraxineus may reach a size of about 8 mm as a maximum, but is often also found with rather small apothecia.

Online photos of Slovenian records under the name Hy. albidus (by N. Ogris, Ljubljana, http://www.zdravgozd.si/prirocnik/zapis.aspx?idso=319 – accessed 23 May 2014) show exceptionally long stipes with red-brown lower half. Possibly, the long stipes result from keeping the collection in a moist chamber in a dark room.

Stroma

A character which has all here treated species in common concerns the dark brown or black, melanized pseudosclerotial plate outside of the sclerenchyma of petioles, rachises, and veins that they inhabit. This stromatized host tissue was reported already in the type of Hy. albidus by Desmazières (1851), who thought that it might be an Asteroma, an anamorph connected mainly to members of the family Valsaceae (Diaporthales). However, there is no doubt that the stroma or pseudosclerotium belongs to the Hymenoscyphus.

Sections through petioles show that the apothecial initials are formed from mycelium inside the pseudosclerotia (Figures 3(a), 3(e), 8(f)). In pure culture on modified Seed agar medium, ‘at first a whitish-grey mycelium was formed, which changed soon to dark brown or black and pseudostromatic areas with a hard surface were developed’ (Pham et al. 2013, p. 447). Contrary to Svrček (1985), who stated that not all apothecia of Hy. albidus emerge from the blackened areas (‘epidermis’), we have never seen apothecia inserted on unblackened parts of the substrate in any of the here treated species.

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Figure 3.

Hymenoscyphus albidus: a–c, e. median section of stipe base (with internal crystals) and cross section of pseudosclerotium in ash rachis (the black demarcation line is restricted to the border between cortical parenchyma and sclerenchyma, hyphae present in all tissues of petiole); d, f–g. external view on pseudosclerotial plate (tangential section of rachis surface), cells of cortical parenchyma and sclerenchyma densely filled with subhyaline hyphae (textura epidermoidea). – All in living state. – a–g. H.B. 9699 (FR-PC, Granzay-Gript).

What appears dark brown to black by macroscopical view is a thin layer (demarcation line or pseudosclerotial plate) in the outer region of the host tissue, which completely surrounds the infected parts beneath, and which separates them laterally from uninfected regions. This dark pigment of the pseudosclerotial plate completely fades when placed in eau de Javelle for about 5 min, as is generally observed with melanized pigments.

In all these foliicolous species, the pseudosclerotial plate develops beneath the epidermis within the large-celled cortical parenchyma, often at the border to the smaller-celled, thick-walled sclerenchyma. During winter the epidermis and cortical parenchyma and also the phloem are degraded and may entirely disappear (Figure 17(m)–(n), 17(s)), but remnants of brown cortical parenchyma or phloem are partly still present when apothecia are formed (Figures 11(k), ​,19).19). Only in rare cases the light-coloured epidermis can still be found loosely attached to the dark stroma beneath by covering large areas of the rachis (Figure 11(c)). The dark stromatic colour originates from black-brown hyphae that form a plectenchyma (textura epidermoidea) on both sides of the host cell walls of the large-celled cortical parenchyma and the outer cells of the sclerenchyma (Figure 3(e)). The lumina of the host cells are densely filled with hyaline or pale brown hyphae (Figures 3(d), 3(f)–(g), 8(i)–(k), 11(h)). The same is true in the host tissue below the apothecial stipe (Figures 3(a), 3(e), 18h). Cells of the sclerenchyma distant from the demarcation line are only sparsely filled with hyphae, lining the inner face of their walls (Figure 8(g), 18(i)).

In Hy. aesculi and Hy. vacini also the inner border of the sclerenchyma is frequently delimited by a black pseudosclerotial plate when viewed in cross section (Figures 17(m), 17(q)–(s), 19(b), ​,22).22). However, also those cells below the inner pseudosclerotial plate contain small amounts of hyphae.

Demarcation lines that delimit the pseudosclerotium from uninfected tissue are infrequently seen in Hy. albidus and Hy. fraxineus when studying cross-cuts of rachises. Especially when several infections took place within a single leaf, the black lines frequently cross outer or inner parts of the mark parenchyma (Figure 11(i)). Also Gross, Zaffarano et al. (2012) observed up to eight different infections and mating type locus (MAT) genotypes in a single rachis colonized by Hy. fraxineus, although it looked homogeneously blackened from outside. A limited (insular) pattern of pseudosclerotia more obviously permits to conclude that a multiple infection of a single rachis took place.

The present study suggests that differences in the extension of the pseudosclerotium occur between Hy. albidus and Hy. fraxineus. The apothecia of Hy. albidus often grow on sharply isolated (insular) black areas on the otherwise pale straw-coloured rachis, though in some specimens, including the type, the stroma covers a main part of the rachises. Hy. fraxineus often stains more or less the entire rachis of a leaf in black, or a major part of it, though also not rarely only insular areas. In Hy. aesculi more or less the entire petiole was found to be occupied by the pseudosclerotium.

Various literature reports of Hy. albidus account for this peculiarity by phrases such as ‘on blackened areas’ or ‘on blackened patches’ (White 1944; Dennis 1956, 1978; Arendholz 1979; Clark 1980; Breitenbach & Kränzlin 1981; Ellis & Ellis 1997). Also Patouillard’s (Patouillard 1885, pl. 382), Boudier’s (1908, pl. 492), and Gams and Arnold (2007, fig. 1A) illustrations show this insular pattern. The difference in the stroma between Hy. albidus and Hy. fraxineus is also recognizable on the two macrophotos of these species in Hietala and Solheim (2011). Likewise, on a photo of rachises from a montane locality without ash dieback in the Austrian Alps (Kirisits 2011, fig. 6), the partial blackening can clearly be seen, but these petioles were not genetically processed and their identity is uncertain. Overview photos of Hy. fraxineus show entirely blackened rachises in virtually all of the many illustrated papers on ash dieback (e.g., Engesser, Forster et al. 2009; Engesser, Meierb et al. 2009; Kirisits 2010; Metzler 2009, 2010; Solheim et al. 2011; Kowalski et al. 2013).

Above the black stipe base a constriction is often seen where it intergrades with the whitish main part of the stipe (Figures 7(n), 8(c), 8(f)). The black basal part of the stipe represents the primordium, from which the development of the stipe and later the receptacle starts. The primordia can be seen as minute blackish spikes on the stromatized rachises. Apparently they appear only in late spring shortly before apothecia are formed.

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Figure 7.

Hymenoscyphus fraxineus (Europe). Apothecia emerging from black pseudosclerotia in rachises and veins of F. excelsior (n, p: in median/longitudinal/cross section). – All in fresh state. – a–b. H.B. 9700 (DE-BW, Tübingen); c, e. 23.VI.2011 (DE-BW, Heidelberg, Ziegelhausen); d. 28.V.2011 (ibid.), f. 17.VI.2011 (Tübingen); g. H.B. 9560 (DE-BW, Reutlingen); h. 30.VI.2012 (BE-LUX, Arlon, phot. G. Marson); i. 23.VI.2012 (Heidelberg, Lobbach); j, m. 18.VII.2011 (DE-TH, Sonneberg, phot. I. Wagner); k. 15.VII.2011 (Ziegelhausen); l, n, p. H.B. 9698 (Tübingen); o. 10.VII.2011 (Heidelberg, Boxberg).

The black pseudosclerotial plate is not yet present when the wilted leaves fall to ground in autumn but is formed prior to the winter season (see Gross & Holdenrieder 2013). Since the fallen rachises lie loosely on the forest floor, they completely dry down during dry weather. The authors found that the mycelium in the pseudosclerotia survives at least 92 days in the dry state, while in nature under repeated drying and rewetting the pseudosclerotia can endure at least 2 years.

Croziers and simple septa

Differences in the ascogenous hyphae among closely related species play an important role in the taxonomy of ascomycetes. A couple of papers have drawn attention to this peculiarity, such as Huhtinen (1990) for Hyaloscypha, Baral (1996, p. 255) and Hengstmengel (1996, p. 192) for Hymenoscyphus. Nevertheless, croziers and simple septa are still being frequently neglected in many groups.

Literature reports on the mode of ascus base development in Hymenoscyphus species with a pseudosclerotium are sparse. According to White’s (1944, p. 608, figs. 19–24) precise illustration of Pl. Crypt. N. France, éd. I. n° 2004 (FH), the asci in the lectotype of Hy. albidus arise from simple septa. White’s statement ‘not originating from croziers’ in the description allows to assume that he saw this feature also in the examined isolectotype collection in FH (éd. II. n° 1604). Besides Arendholz (1979), White’s report is the only known to us from the past century, which mentions the type of ascus development in this species.

V. Ruiz-Badanelli (in Hairaud 2010) and Zhao et al. (2013) presented photographs of croziers of Hy. fraxineus (as Hy. pseudoalbidus). Hairaud gave also a photo of the simple-septate ascus base in Hy. albidus for his collection from July 2012, following personal communication with H.O. Baral. However, his note that he saw the feature also in the 2007 sample must be in error. A statement by Otto and Wagner (2011) about the difference in the ascus base between the two species originates also from such personal communication with the first author of the present paper.

Hy. honshuanus (≡Lambertellinia scutuloides) is here found to possess croziers (Figure 12(b)), although the protologue states the contrary. Korf and Lizoň (1994) observation was not accompanied by an illustration; however, it remains open whether this observation was erroneous, or the type collection represented a mixtum.

Homothallism, monokaryon, relative DNA content of nuclei

The cytological background of the presence vs. absence of croziers is still little understood. In Hy. albidus the absence of croziers is correlated with homothallism and with the absence of an anamorph state (see below under ‘Anamorph and life cycle’ section). Data on the karyological situation in the ascogenous hyphae of the here treated pseudosclerotium-forming species of Hymenoscyphus were presently not available.

Berkson (1966) observed in Chaetomium that species with croziers had dikaryotic ascogenous hyphae whereas others with simple septa were monokaryotic. Also Weber (1992, p. 68) observed monokaryotic ascogenous hyphae in species of Helotiales that lack croziers, although only in a smaller part of them, while in others monokaryotic and dikaryotic cells occurred mixed in the same ascogenous system. Nuclei of monokaryotic cells thereby had the double DNA content compared to those of dikaryotic cells of the same species. In species in which the ascogenous hyphae possess croziers, Weber observed mainly dikaryotic cells. Zickler et al. (1995) reported ‘uninucleate croziers’ in mutant strains of Podospora anserina which normally were dikaryotic. However, their microphotos (Figure 4(a)–(d)) show simple-septate ascogenous hyphae with a partial presence of hook-like protuberances which did not fuse with the cells below. The complete croziers drawn for the uninucleate mutants on their diagrammatic survey (Figure 5(b)–(c)) are not seen on their micrographs.

In her study on the relative DNA content of nuclei in vegetative cells, Weber (1992) revealed for Hy. albidus a value of 4× of the basic value (1×), the lowest found in the Helotiales. Within Hymenoscyphus, this value of 4× was not rarely encountered in her study (e.g., in He. scutula and Hy. vacini), but values of 2×, 3×, 6×, and rarely 1× also occurred. A correlation of these values with the presence or absence of croziers could not be established; however, species with croziers as well as simple septa occurred in quite equal frequency within each value, except for those species with values of 1× (simple-septate) and 2× (croziers), which were too sparsely encountered in order to draw a conclusion. In any case, it would be interesting to measure the relative DNA content of the two so far not investigated species of Hymenoscyphus with a pseudosclerotium, Hy. fraxineus and Hy. aesculi. A survey on the known genome size values in fungi are found in Kullman et al. (2005).

A correlation between croziers and apothecial size similar as in Hy. albidus and Hy. fraxineus was noted in two further species pairs of the Helotiales (Baral in Weber 1992, p. 110, 118): (1) Calycina herbarum (without croziers) and C. aff. herbarum (with croziers and larger apothecia), a very similar species which was currently confused with Ca. herbarum, and which inhabits a comparable spectrum of herbaceous stems; (2) Heyderia pusilla (without croziers, on needles of Pinus) and Hy. cucullata (=Hy. abietis, with croziers and larger fruit bodies, on needles of Picea). Within both species pairs the relative DNA content did not differ (Calycina 2×, Heyderia 6×).

Apical ring

The morphology of the ascus apical thickening in the dead expanded state when stained with iodine is usually very similar among the true members of the large genus Hymenoscyphus. Its characteristic morphology was addressed by Baral (in Baral & Krieglsteiner 1985, p. 119), and later referred to as Hymenoscyphus-type (Baral 1987, p. 126, fig. 10–12, Verkley 1993). The ascus apex in the Sclerotiniaceae and Rutstroemiaceae (Sclerotinia-type) sharply differs hereof and easily permits distinction with the light microscope between sclerotiniaceous/rutstroemiaceous and hymenoscyphoid taxa in most cases (see Baral 1987). White (1944) and Svrček (1985) illustrated the Hymenoscyphus-type of apical ring in Hy. albidus. However, it was perhaps more because of a similar spore shape that White believed in a very close relationship to He. scutula and Hy. caudatus.

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Figure 10.

Hymenoscyphus fraxineus (Europe). a₁, b, c, d₁, e₁, g, h_3. ascospores (freshly ejected, containing LBs); h₄. do. (LBs deteriorated); a₂, j. paraphyses (containing VBs); e₂, i, k. mature asci; f, h₁. overmature ascospores (septate, partly with olive-brown wall); u. ascus apices with euamyloid apical ring; remaining figures: ascogenous hyphae with crozier formation. – Living state: a₁–e₁, a₂, f_1, g (in Waterman blue-black ink), h₁, h₃, i, j (in CRB), k₁, r–s, v; dead state: k₂ (in H₂O), a₃–a₄, d₂–d_4, h₂ (in CR); e₂–e₃, l–n, p–q (in CR_SDS), f₂ (in KOH), o, t (in KOH+CR), h₄ (in ethanol + KOH + phloxine), u₁–u₂ (in IKI). – a. 28.V.2011 (DE-BW, Heidelberg, Ziegelhausen), b. H.B. 9572 (CH-LU, Emmenbrücke), c. H.B. 8220 (DE-ST, Chemnitz), d. 31.VII.2011 (Heidelberg, Schönbrunn), e. 10.VII.2011 (Heidelberg, Boxberg), f. H.B. 9593 (Chemnitz), g. 14.VII.2012 (Heidelberg), h. 23.VII.2011 (DE-BW, Speyer, Böhl-Iggelheim), i. 15.VII.2011 (Ziegelhausen), j. H.B. 9599 (DE-BW, Tübingen), k. H.B. 8509 (DK-Sjaelland, Sorø), l. 18.VII.2011 (DE-TH, Sonneberg), m. 2.VII.2011 (Heidelberg, Handschuhsheim), n. 5.VII.2011 (Ziegelhausen), o. ZT 3298 (CH-BE, Belp), p. 23.VI.2011 (Ziegelhausen), q. 23.VI.2012 (Heidelberg, Lobbach), r. H.B. 9698 (Tübingen), s. H.B. 9700 (Tübingen), t. ZT 3297 (CH-BE, Burgdorf), u. 8.VII.2011 (Sonneberg), v. H.B. 9583 (Tübingen). – Phot. l_1–3, u_1–2: I. Wagner.

Ascospore size and shape

Heteropolar, subapically rounded and with a lateral hook-like protrusion, basally ± pointed ascospores are typical of many of the true members of Hymenoscyphus. This characteristic spore type was called ‘scutuloid’ by Baral (in Baral & Krieglsteiner 1985) according to the situation in the representative species He. scutula, but was rarely also referred to as virguliformis, which means in Latin comma-shaped, e.g., by Patouillard (1885, p. 173), who erroneously wrote in French ‘spores virgultiformes’ instead of ‘virguliformes’. The spore size in the type material of Hy. albidus was given as minimum 15 µm by Desmazières, 11–17 × 3.5–4 µm by Nylander (1868), 15–18 × 3.5–4 µm by Karsten (1871), 15–18 × 3–4 µm by Massee (1895), and 13–17 × 4–5 µm by White (1944).

Hy. fraxineus is said to have slightly longer ascospores, but variation and overlap in this feature forbid recognition of the two species on Fraxinus from spore length alone. Queloz et al. (2011) gave a graphic representation of length values of some selected specimens. Accordingly, spore length ranges were (11–)13–14–16–17(–18) µm in Hy. albidus and (15–)16–17–18–20(–22) µm in Hy. fraxineus, both possibly evaluated from dead spores. The tendency to longer spores in Hy. fraxineus is confirmed from the specimens studied by us. Our spore length values are about 0.5–1 µm over those of Queloz et al. (2011), probably because of a slight shrinking effect between living and dead spores. The longest freshly ejected spores that we measured in Hy. fraxineus were almost 25 µm long.

The characteristic spore shape of Hy. aesculi with a swollen middle part is only present when living spores are studied in a water mount. After releasing the spore turgor by heating or applying killing agents, the spores shrink and more or less completely loose this inflation (see Figure 18(n)–(o)). Also inside the turgescent asci, the spores are distinctly narrower and less swollen in their middle part because of the ascus turgor. When discharged in a water mount, the spores swell to their typical shape within a few seconds.

Ascospore contents

Published illustrations of living spores of the here presented species are rather rare. Particularly freshly ejected living spores, but also those inside fully turgescent asci, show a regular pattern of larger and smaller oil drops (LBs). Hy. albidus, Hy. fraxineus, and Hy. aesculi possess quite the same pattern of rather large, globose LBs (2–3.5 µm diam. in Hy. albidus and Hy. fraxineus, 1.5–2.5 µm in Hy. aesculi), about 1–4 in each half, surrounded by small ones. The spores of Hy. vacini consistently contain only medium-sized LBs (~1–2 µm diam.) among many small ones.

Patouillard’s (1885, pl. 382) drawing, though rather small and inexact, appears to be almost the only published documentation of living mature ascospores within the Hy. albidus aggregate, showing about four rather large globose drops in each spore. Two recent illustrations of living spores are found in Hairaud (2010) and Gross, Holdenrieder et al. (2014, fig. 1h). Even Boudier (1908, pl. 492), who consequently studied ascomycetes in the fresh state, illustrated the living spores of Hy. albidus with only small- to medium-sized drops, the latter about 1.2–1.8 µm diam., though with a rather regular pattern. Perhaps theses spores were not fully mature.

The original lipid pattern can often also be observed in herbarium material, but it requires caution to select those spores which possess enough maturity, and to avoid all those which are overmature or in which the LBs have fused to irregular aggregations. Actually, most reports in the literature concern dead spores that contain irregular rather variably shaped aggregations.

Ascospore reports such as ‘often with a large oil globule’ (Dennis 1956, p. 93) or ‘often with 1–3 oil drops’ (Arendholz 1979, p. 80) are due to fusion of the original oil drops during the influence of lethal mountants or the natural death of spores. Surprisingly, White’s 1944, fig. 24) drawing of the type of Hy. albidus shows a rather regular spore content with a ‘granular’, multiguttulate sporoplasm, the largest LBs exceeding not even 1 µm diam. This is astonishing, since oil drops usually fuse in dead material, but never split into small ones. The explanation for this appears to be that White (1943, p. 137) followed a method described by Martin (1934, p. 264), who placed fungal fragments in alcohol before applying KOH and phloxine. We made a test with a specimen of Hy. fraxineus: the large oil drops in the spores indeed disappeared and only the small ones remained visible. Apparently it is the alcohol that dissolves the large oil drops (see Figure 10(h₃)→(h₄)).

Ascospore sheath and setulae

Distinct setulae at the spore ends were not observed in any of the species treated here, while a delicate sheath around ejected spores was occasionally seen, though not yet in Hy. honshuanus and Hy. vacini. Because of its fragile nature, it is a rarely reported structure. When studying herbarium material, it remains inevitably undetected since it does not detach from the wall when dead spores are rehydrated, due to the absence of cell turgor. The sheath can only be observed when the spores are ejected from living asci in a water mount: after discharge, they rapidly take up water and swell, and the inelastic sheath bursts and eventually floats besides the spores. Gross, Holdenrieder et al. (2014, fig. 1m–n) reported a thick, hyaline to pale brown mucilage secreted from the spores of Hy. fraxineus, by which they are thought to adhere to the leaf surface after wind dispersal. This mucilage was observed on overmature (1-septate or brown) spores during. In fact, the mature spores are hyaline and aseptate during ejection, and soon dehydrated after dispersal, including their mucilage. Possibly the sheath observed by us covers a thin mucilage that gets more abundant during germination and affixes the spore to the leaf when the appressorium is formed.

Overmature ascospores, pigmentation

Apparently all true members of Hymenoscyphus eject hyaline, non-septate ascospores. However, at an overmature stage of development, the spores frequently get 1(–2)-septate and their wall may turn pale to light brown and often also warted. Because brown or septate spores of a Hymenoscyphus are never forcibly ejected, such spores in this genus can only be found inside dead asci or outside asci, but never inside living asci (see also Baral 1992, p. 375).

The presence of pigmented spores has been overestimated as a generic character in the Helotiales. As pointed out by Galán and Baral (1997, p. 61) and Hengstmengel (2009, p. 271), this feature of overmature spores is quite a common character in Hymenoscyphus, though it is seen only inconsistently and sometimes very rarely in a given species. The proportion of brown spores in a preparation depends on the senescence of the apothecia. For some reason, perhaps unfavourable field conditions, some populations produce many such brown spores when senescent, while in others none or only a few can be found.

Spore germination is observed in both hyaline and brown spores. Possibly, the hyaline spores directly infect the living leaves, whereas the brown spores are resting spores being able to survive a longer time. Brown germinating spores were figured by Hosoya et al. (1993) for Hy. fraxineus (as Lam. albida), and by Kowalski and Holdenrieder (2009, fig. 1g). In the present study, brown spores were only two times seen in Hy. fraxineus (Figure 10(f)), once in Hy. honshuanus, and once in Hy. aesculi.

Ectal excipulum

In sections of living specimens of Hy. albidus, the ectal excipulum is a textura prismatica with comparatively thin though slightly gelatinized walls. In dead specimens, or when adding killing agents such as KOH, the cells shrink especially in width, and the gel between the walls becomes more obvious due to imbibition. The shrinking effect was probably the reason why Svrček (1985) found the cells to be only 4–5 µm wide, though he obviously did not see any gelatinization. Likewise, Arendholz (1979), who classified the ectal excipulum of Hy. albidus as a t. intricata, did not see glassy or agglutinated cell walls. White (1944, fig. 22) found the excipular cells in the lectotype 8–10(–15) µm wide, but did not mention or clearly figure a gel between the cell walls.

In Hy. fraxineus strong variation in the width of the excipular cells was noted, which appears to depend mainly on the development stage: in younger apothecia the ectal excipulum forms a t. prismatica-porrecta in both stipe and receptacle, with a cell width of *~6–10 µm, whereas in older and larger apothecia it forms a t. prismatica-angularis with *~10–30 µm wide cells.

Hair-like protrusions

Svrček (1985) mentioned the outside of the receptacle of Hy. albidus to be finely downy, the stipe almost tomentose in the lower part. Also Dennis (1956, p. 94) described and illustrated ‘short, slender, obtuse hairs about 10–15 × 2 µm’ on the excipular surface in this species. We here describe them in Hy. albidus and Hy. fraxineus (Figure 9(l)–(m)), but saw a pubescent stipe also in Hy. aesculi. These protrusions were not present in each of the studied samples of these species, however.

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Figure 9.

Hymenoscyphus fraxineus (Europe): a–b, j–o. median section of stipe, showing golden-yellow external exudate and black basal stroma (ee = ectal excipulum, me = medullary excipulum); c–d. median section of receptacle at flanks (c) and margin (d); e–f. surface view of stipe; g–i, k. crystals in medullary excipulum near stipe base; k–n. hairs at stipe base. – All in living state (i: under polarized light). – a, g, k, l–n. H.B. 9593 (DE-SN, Chemnitz); b. H.B. 9698 (DE-BW, Tübingen); c, h–i. H.B. 9700 (Tübingen); d. H.B. 9571 (CH-LU, Hitzkirch); e–f. H.B. 9583 (Tübingen); j, o. H.B. 9589 (DE-BY, Amberg).

Vacuolar bodies

In all here treated species of Hymenoscyphus, refractive vacuolar bodies (VBs) occupy most of the lumen of the terminal cells of the living paraphyses and partly also of the cortical cells of the marginal ectal excipulum. In young paraphyses, they are globose or somewhat angular, but soon fuse and finally form very elongate vacuoles (see also Baral 1992, p. 363). In the dead state, they are distorted or entirely disappeared. Also KOH applied to living paraphyses irreversibly makes them disappear (Baral 1992), and that without provoking any colour reaction. Staining agents when added to a water mount, provoke a distinct stain to VBs: CRB slowly stains them turqoise, and IKI light yellowish to bright red-brown. In IKI mounts, many minute red-brown granules may extrude into the surrounding medium, but this effect was only inconsistently obtained in Hy. albidus, Hy. fraxineus, and Hy. vacini, and not in Hy. aesculi.

A colour change of the VBs from hyaline towards olivaceous-brown was repeatedly noted in Hy. aesculi, resulting in partially blackish-brown hymenia in herbarium material. In the other species studied, the VBs turned merely yellowish-cream with age. The phenomenon appears to be due to an oxidative chemical process.

Crystals

Arendholz (1979) observed abundant crystals of ‘presumably Calcium oxalate’ in the lower 1/4 of the stipe. We have regularly seen these crystals in both Hy. albidus (Figure 3(c)) and Hy. fraxineus (Figures 8(d)–(e), 9(h)–(i), (k)), and Zheng and Zhuang (2014) report them for Hy. fraxineus and Hy. albidoides. Although crystals occur also inside some of the cells of the infected rachises, those found in the dense tissue of the medullary excipulum of the stipe base are undoubtedly formed by the fungus, not by the host.

The crystals are hyaline, ±rhomboid, and frequently aggregated in druses. They are KOH-inert and do not stain with CRB or IKI. Under polarized light, they are birefringent by changing the direction of the light for about 90° (Figure 9(i)). Crystals occur also in the living rachises of Fraxinus, inside cells of the radial rays between the vascular bundles. However, these are not arranged in druses. Crystals were not observed in Hy. aesculi and Hy. vacini, nor in any other species of Hymenoscyphus examined by us.

Host range

Within Europe, Hy. fraxineus was recorded, apart from the European temperate species F. excelsior, also on the North American F. nigra (Estonia, Drenkhan and Hanso 2010) and on two submediterranean species from Southern and Southeastern Europea, F. angustifolia and F. ornus. Artificial wound inoculation experiments revealed an equally high or slightly higher susceptibility of F. angustifolia to Hy. fraxineus compared to F. excelsior (Matlakova 2009). When inoculated, the fungus also caused symptoms on F. ornus, though less intensely than on the two other species (Matlakova 2009, Gross, Holdenrieder et al. 2014). However, ash dieback symptoms due to natural infections have so far never been observed on flowering ash, neither on woody parts nor on leaves, and this species may be resistant to the fungus (Gross, Holdenrieder et al. 2014; T. Kirisits personal communication). Gross, Holdenrieder et al. (2014) reported the occurrence of apothecia of Hy. fraxineus on petioles of F. ornus, which may indicate that it occurs endophytically in leaves of this ash species.

F. nigra was badly affected with symptoms, F. pennsylvanica only moderately, and F. americana and F. mandshurica were least affected (Drenkhan & Hanso 2010). In its presently known original area of Eastern Asia, the hosts of Hy. fraxineus are F. mandshurica and F. rhynchophylla (partly treated as a subspecies of F. chinensis).

Hy. albidus is known with certainty only from F. excelsior and sometimes F. angustifolia. For two here mentioned records on Acer, voucher specimens have not been preserved. A preserved specimen on a leaf of ‘Pyrus’ from Belgium was found to grow in fact on Fraxinus. Misidentifications of either host or fungus explain in some cases a seemingly wide host spectrum.

Similarly narrow host spectra are noted in the genus Rutstroemia. The common Rutstroemia longipes is said to be confined to rachises of Fraxinus in North America (R.P. Korf personal communication), and the closely related Rutstroemia luteovirescens is frequent on petioles of Acer in Europe. However, the independence of these two fungal species and the extent of their host spectra have been questioned in the past (see above).

Fruiting on twigs

As an exception, both Hy. albidus and Hy. fraxineus were found fruiting on twigs of Fraxinus in the present study, though in both cases also petioles were inhabited. The record of Hy. albidus (Echternach, LUX 047701) concerns a corticated 1 mm thick twig with blackened bark, while in that of Hy. fraxineus (Luzern, H.B. 9571) the apothecia grew on blackened wood of an almost entirely decorticated, 5.5 mm thick twig. The rare occurrence of Hy. fraxineus on twigs was repeatedly reported, e.g., by Gross, Holdenrieder et al. (2014, fig. 1d). The rareness of this behaviour is astonishing, since living twigs are regularly invaded by the fungus. Whether also Hy. albidus is able to invade living woody parts is not known.

Phenology

The slightly earlier production of apothecia of Hy. fraxineus in comparison with Hy. albidus is shown in Table 4, based on the specimens listed in our collection data. Literature data suggest that Hy. albidus occurred also in October (e.g., Krieglsteiner 1999, p. 238, 30.X.1997, near Schweinfurt). A fruiting period of Hy. fraxineus during May–October(–November) was demonstrated by Chandelier et al. (2014, fig. 4) using spore traps and real-time polymerase chain reaction (PCR). This earlier start of fruiting plays a role in the dominance of the pathogen over Hy. albidus.

Japan

Apothecia recorded in Japan on blackened rachises of Fraxinus mandshurica var. japonica were identified as Lam. albida by Hosoya et al. (1993). The very detailed documentation includes a pure culture and an anamorph which fits very well Ch. fraxinea. A sequence (ITS and part of SSU) was deposited at the NBCR (National Biological Resource Center) of the Japanese National Institute of Technology and Evaluation under the number NBRC102368 and was, therefore, overlooked by European researchers. After re-examination of eight specimens collected during 1990–2011, these were found to possess croziers at the ascus base in each of the apothecia tested (see Table 2). Five of these were sequenced and confirmed to belong to Hy. fraxineus (Zhao et al. 2013).

Korea

J.G. Han (personal communication) examined 10 records on rachises of Fraxinus mandshurica and F. rhynchophylla morphologically, four of them also genetically. All were found by him to possess croziers, and his sequences refer them to Hy. fraxineus.

China (northeast)

Zheng and Zhuang (2014) recorded Hy. fraxineus on F. mandshurica at four localities in the northeast of China (Jilin province). The authors observed croziers at the ascus base, and their sequences refer these records to Hy. fraxineus.

Russia (far east)

A collection of Hy. fraxineus from Kedrovaya Pad Reserve (Khasansky District, Primorsky Kray) on F. mandshurica was made in 17.VIII.2005 by E. Popov (personal communication), identified by the presence of croziers (1508-8-Ked). Marčiulynienė et al. (2013) confirmed the presence of Hy. fraxineus from three sites near Primorye by molecular methods.

Russia (westernmost part)

Records of Hy. albidus were made by E. Popov (personal communication) during 2004–2006 from Leningrad Oblast, e.g., from two islands in the Gulf of Finland. Moreover, several finds from Pskov (1998–2009) and one from Kaluga Oblast (2013) were made by him, as well as one from Novgorod Oblast (Valdaysky district, 4.IX.2001, Popov 2005). All these were re-examined by him recently and the absence of croziers was confirmed (documentation not seen). E. Popov observed that Hy. albidus fruited usually very abundantly, and almost everywhere in stands where Fraxinus grows. Only in the Volga-Akhtuba floodplain, where F. pennsylvanica was very commonly introduced, he did not observe Hy. albidus.

In a forest with Alnus incana and F. excelsior in Pskov Oblast visited by E. Popov every year since 1996, with Hy. albidus being common, no symptoms of disease were obvious until 2009 when all ash trees showed strong dieback, all dying after 1 or 2 years, also in several similar communities within a radius of 5 km. Other regions (Kaluga and Orel Oblast) did not show the disease so far. Shabunin et al. (2012) recorded Hy. fraxineus southwest of St. Petersburg (Leningrad Oblast) in 2012 by molecular methods.

Poland

Several records of Hy. albidus can be found in Polish publications. For instance, Lisiewska and Bujakiewicz (1976, p. 57) mentioned a specimen (as He. robergei) for the Dębina reserve in Wielkopolska (Central Poland), and Bujakiewicz (2001, p. 116) reported Hy. albidus from the Ostrów Panieński reserve close to Chełmno (Northern Poland) collected during 1981–1984. Molecular data have not been gained from these specimens, but the occurrence on blackened rachises prior to ca. 1990 leaves little doubt about their identity.

Ash dieback symptoms are known from Northern Poland since 1992 (Kowalski 2006), and since 1998 decline of F. excelsior occurred all over the country (Vasaitis 2013). Nevertheless, apothecia were noticed as part of the disease only since 2008 (Kowalski & Holdenrieder 2009). During surveys in 2005–2006 and 2011, Hy. fraxineus had totally replaced Hy. albidus, according to molecular analyses of 230 apothecia-derived cultures which all represented Hy. fraxineus (Kraj et al. 2012; Kowalski et al. 2013).

Lithuania

Six herbarium specimens under the name Hy. albidus, collected on blackened petioles of F. excelsior from the districts of Kėdainiai and Vilnius in 1999–2002, were recently reviewed by E. Kutorga (personal communication). He could demonstrate the presence of croziers by microphotos in all of them. The distribution of ‘Hy. albidus’ in Lithuania based on specimens (not tested for croziers) and literature data was reported by Treigienė et al. (2010) for the districts of Kėdainiai, Ukmergė and Biržai during field trips in 1997–2005, but these observations most likely also concern Hy. fraxineus.

The ash dieback disease was noticed since about 1996/97 (Juodvalkis & Vasiliauskas 2002), mainly in Biržai and Panevėžys districts (Northern Lithuania), Kėdainiai, Jonava and Ukmergė districts (Central Lithuania, see also Stepanenkova (2010) and Bakys (2013)). In the following years it spread over the whole country. A DNA sequence that matches Hy. fraxineus (AY787704) was for the first time gained in 2001 by Lygis et al. (2005, as Hymenoscyphus sp. 970, later corrected to Ch. fraxinea in GenBank, V. Lygis/E. Kutorga personal communication) from a stem base of F. excelsior in Biržai district (northeast of Lithuania). Mass production of apothecia was noticed by E. Kutorga from about 1999 onwards including 2013.

Estonia and Latvia

Rytkönen et al. (2011) isolated Ch. fraxinea from F. excelsior in 2008 in NW- and SE-Estonia and in 2009 in W-Latvia. Also Drenkhan and Hanso (2010) isolated the fungus in 2009 in SE-Estonia. However, the first indications of ash dieback were as early as 1995 in the northwest of Estonia, where the disease reached most ash stands during 2003, and from where it spread towards the southeast of the country during 2006–2007 (Drenkhan et al. 2014, fig. 4). Also in Latvia massive ash dieback occurred since mid-1995s (Vasaitis 2013).

Finland

Karsten (1871, p. 112) stated that collections from his country were unknown to him. The first record of Ch. fraxinea was in 2007 and 2008 in the SW-part of the country (Åland archipelago and mainland), from where the disease spread towards the southeast. The first symptoms of ash disease were in 2000 in the Åland archipelago (Rytkönen et al. 2011).

Sweden

Hy. albidus is known from a single locality near Helsingborg in Skåne (south of Sweden), where it was abundant in 1994 and 1996 (S.Å. Hanson personal communication). The first observation of the disease was in 2001, and in 2004 the entire distribution area of F. excelsior in the south of Sweden was affected (Timmermann et al. 2011). A frequent occurrence of apothecia was first noted in Skåne in 2002 and particularly from 2004 onwards (S.Å. Hanson personal communication).

Norway

Hy. albidus was recorded in 1985 from the southwest of Norway (Bergen) by S. Olsen (K. Homble personal communication), and is today still present along the west coast, according to molecular data by Hietala and Solheim (2011). Possibly it had occurred also in the southeast of Norway before Hy. fraxineus invaded the country, but K. Homble never found apothecia referable to Hy. albidus in the Oslo area before 2009.

The first symptoms were noted in 2006 in the most southern part of the country. The first isolate of Ch. fraxinea was made in SE-Norway in May 2008 in a nursery in the Østfold region, and in 2009 the disease has spread along the entire coastline there (Talgø et al. 2009; Timmermann et al. 2011). K. Homble (personal communication) noticed mass occurrence of apothecia in VIII.2012 in Kjaglidalen (Bærum, Akershus, 15 km west of Oslo), while H. Solheim (personal communication) observed the first mass occurrence in 2009 near Ås (30 km S of Oslo).

Hietala and Solheim (2011) found by molecular methods that Hy. albidus survived along the west coast of Norway. According to Hietala et al. (2013, p. 5), ‘The low ascospore number of Hy. albidus detected by real-time PCR in our Norwegian ash stand could relate to such a transition. On the other hand, our data suggest that Hy. albidus is still present in the studied forest.’.

Great Britain

Berkeley and Broome (1878, p. 29, No. 1724) list a single record of Hy. albidus located in Southeast England, with the data ‘On ash petioles. [Kent,] East Farleigh, Sept. 13, 1876’. The same record is mentioned by Cooke (1878, p. 127). Also Phillips (1887, p. 138) gave only one though very remote West English record ([Shropshire,] Shrewsbury, Copthorne). Dennis (1956, p. 93, as He. robergei) based his description on two British specimens, the one mentioned by Berkeley, and one in the east of England: Norfolk, Wheatfen [SE of Norwich], 27.VII.1946.

Clark (1980) recorded Hy. albidus rather often on black patches of Fraxinus petioles (but also on Aesculus) in Warwickshire (West Midlands of England), with the first record in 1967. Ellis and Ellis (1997, p. 137, fig. 600) probably relied on Clark’s records when saying that Hy. albidus is common during July–October. A distribution map is shown in Webber and Hendry (2012), which includes also misidentified records, especially on substrates other than Fraxinus.

The British Isles were considered as being free of Hy. fraxineus until January 2012 when ash seedlings were imported from the Netherlands and Germany. At four locations in England they showed the typical signs of the disease (Webber & Hendry 2012). In August 2012 Ch. fraxinea was reported as well from Scotland in a stand of young ashes planted in spring 2009 (Webber & Hendry 2012; Munro 2012). Mainly the eastern part of the country was affected.

An intensive survey carried out since November 2012 confirmed 427 sites with ash trees infected by Ch. fraxinea (status 25.III.2013). Southeastern areas of the British main island are affected the most (http://www.defra.gov.uk/news/2012/11/09/wms-ash/), but confirmed sites are distributed from Wales to Scotland and Ireland.

Denmark

A couple of collections of Hy. albidus have been made between 1989 and 1999, some of which were DNA-checked (McKinney et al. 2012, T. Laessøe personal communication). According to the molecular analysis, other collections made in 2005–2010 turned out to belong always to Hy. fraxineus, including three sites where Hy. albidus apothecia had been collected previously, and it was concluded that Hy. fraxineus had eliminated Hy. albidus in these regions.

The first records of ash disease were in 2002 near Haderslev (a small port on the Baltic side of the Jutland peninsula), and in 2003 in Zealand and Bornholm (Thomsen et al. 2011; Bakys 2013).

The Netherlands

Arnolds et al. (1999) considered Hy. albidus as a comparatively rare species, though rather common in the Ijsselmeerpolders and Zeeland. A Dutch online database (http://www.verspreidingsatlas.nl/622010) shows indeed numerous records throughout the country, many of which dating before 1990. Only Fraxinus petioles are given as substrate. A record made by S. Helleman in Nijmegen (Gelderland) in 2007 is here confirmed to represent Hy. albidus.

Ash dieback was first reported in 2010 at young trees in public parks in Bellingwedde in the far northeast of the country close to the German border (EPPO 2010). L. Deceuninck recorded for the first time apothecia of Hy. fraxineus in 2012 at Schokland (Flevoland, B. Declercq personal communication), while S. Helleman (personal communication) did not observe occurrence of Hy. fraxineus around Boxmeer (Noord-Brabant) until recently (2013).

Belgium and Luxembourg

Mouton (1886, p. 140) reported Hy. albidus from Beaufays near Liège (on petioles of Fraxinus, without any further data). In a preliminary checklist for Flanders, Declercq (personal communication) considered Hy. albidus as rather common, based on numerous records collected and identified by Lieve Deceuninck, Bernard Declercq and Piet Bormans during 1989–2012. Also from Wallonia a lot of records were made during 1993–2008 by B. Declercq. For all of them the absence of croziers was noted (B. Declercq personal communication, documentation not seen). A further record from the southeast of Belgium and three from Luxembourg preserved at LUX (all leg. C. Besch, during 1985–1990) were re-examined in the present study and found to represent Hy. albidus.

According to Roskams and De Haeck (2011), the first record of ash disease was in autumn 2010 in Eastern Flanders (Hainaut), while in Flemish Brabant it was already present as early as 2007. In 2011 the disease was recorded throughout Flanders. For Wallonia (Southern Belgium) the disease was firstly recorded in June 2010 (Chandelier et al. 2011). Apothecia of Hy. fraxineus were detected for the first time in Belgium and Luxembourg in 2012 and 2013 (B. Declercq, G. Marson personal communication, Garnier-Delcourt et al. 2013), and that in great abundance at various sites.

France

Roberge’s specimens of Hy. albidus around Caen (dépt. Calvados) concern the abundant type collection made around 1850, and a scantier later sample. Cooke (1876, p. 132, pl. LXV fig. 297) and Patouillard (1885, p. 173, pl. 382) gave the first illustrations of the species. While Cooke studied the type material (Desm. n° 2004), Patouillard obviously depicted a recent collection (with living spores) but regrettably did not indicate its origin. Boudier (1908, p. 287, pl. 492) provided a detailed coloured illustration based on a record from Fôret de Carnelle north of Paris (dépt. Val-d’Oise), and stated that the species is frequent in summer and autumn.

A further record was made near Paris on 2.IX.1945 by Arnaud (Gams & Arnold 2007). Here the apothecia (on blackened petiole) were parasitized by the hyphomycete Pleurocatena acicularis Arnaud ex Gams & Arnold. From near Besançon a record of Hy. albidus was made in 22.VIII.1996 and illustrated by G. Moyne (personal communication).

From 2007 onwards, M. Hairaud (personal communication) observed Hy. albidus repeatedly in dépt. Deux Sèvres, especially in Marais Poitevin. Based on a molecular identification, Husson et al. (2011) listed 18 recent records of Hy. albidus from Central and West France, one made in 2002 (dept. Calvados), and 17 in 2009 (dépts Côte d’Armor, Haute-Marne, Maine-et-Loire, Saône-et-Loire, Haute-Saône, Moselle, Meurthe-et-Moselle).

According to a map given by Goudet and Piou (2012), the first record of ash disease was in 2008 in dépt. Haute-Saône, though a map in Timmermann et al. (2011) gives 2007 for this region. A photo taken by G. Moyne (personal communication) near Besançon (La Vèze, bois d’Aglans, dépt. Doubs, 13.VIII.2008) showing abundant and rather large apothecia on entirely blackened petioles supports that the disease invaded this part of Eastern France already before 2008. In their molecular study, Husson et al. (2011) identified in the whole northeast of France exclusively Hy. fraxineus (collected in 2009), while in Central and West France only Hy. albidus was recorded (see above). In 2010 the disease was widespread in Northeastern France, but was also recorded in Northern France, and in 2012 in the northwest (Normandie, Jersey).

Germany

When Arendholz (1979, p. 79) performed his study on leaf-inhabiting Helotiales, he had only material of Hy. albidus of the nineteenth century at his disposition: the type collection, the material of Berkeley, and a specimen from Germany (Sachsen, Großer Winterberg [Sächsische Schweiz, Elbsandsteingebirge], X.1898, W. Krieger, Fungi Sax. 1486, HBG). Baral and Krieglsteiner (1985, p. 121) reported collections from the south of Germany made during 1975–1979 along the rivers Rhein and Donau, also from Franken and Eifel, but were unaware of records from Northern Germany. Beyer (1992, p. 67) described a single collection made in August near Burggaillenreuth (Bayreuth), and believed the species to be rare. G.J. Krieglsteiner (1993, pl. 745) presented a distribution map for W-Germany that shows a scattered occurrence of Hy. albidus within Niedersachsen, Rheinland-Pfalz, Baden-Württemberg, and Bayern, but whether it includes also misidentified samples on other substrates or without pseudosclerotium remains unclear.

L.G. Krieglsteiner (1999, p. 238, 2004, p. 601) reported Hy. albidus from 4 localities in the Main area between Schweinfurt and Würzburg (Bayern), and from the Rhön mountains at the edge of Bayern, Hessen and Thüringen with 14 collections. Judging from the phenology data (30.VII.–30.X.) and the occurrence in the years between 1996 and 2004, these records might all concern Hy. albidus. The same is true for nine samples from the Hainich National Park made between 16.VII. and 17.XI. in 2003 made by F. Putzmann, G. Hirsch and A. Gminder (P. Püwert & I. Wagner personal communication). Especially those specimens collected in 2003–2004 should be re-examined for croziers in order to exclude Hy. fraxineus. However, Hy. fraxineus has apparently never been recorded within Thüringen in the years before 2008 (P. Püwert & I. Wagner personal communication); therefore, we strongly suspect that the ash disease was still absent in the Hainich and Rhön region in 2004.

As early as 2002, ash dieback or conspicuous damages on ash were noticed in the northeastern lowlands (Schumacher et al. 2007; Leonhard et al. 2009; Heydeck & Dahms 2012). The first scientific proof of the presence of Ch. fraxinea for Germany was published by Schumacher et al. in 2007 and was detected in material collected from woodlands and nurseries, e.g., from Salzwedel (Altmark, Sachsen-Anhalt). The earliest records of mass fructifications that came to our notice were observed in Sachsen (SN, 2006) and Mecklenburg-Vorpommern (MV, 2007) (see Table 2). In Mecklenburg many ash stands have been cut in order to avoid damage of the timber; therefore, the pathogen was suppressed (T. Richter personal communication).

In Thüringen, the fructifications of Hy. fraxineus became very abundant from 2008 onwards (P. Püwert & I. Wagner personal communication). Although the ascus base was studied by I. Wagner (personal communication) in only a few collections, the absence of abundant fruit bodies in previous years suggests that all these records from the south of Thüringen (around Sonneberg) but also some from the Rhön mountains collected in 2011 belonged to Hy. fraxineus. The presence of Ch. fraxinea was confirmed officially for Thüringen as late as 2009 (Baier et al. 2010; TLWJF 2010).

In Baden-Württemberg ash dieback was first noted in 2006 for a single tree in the eastern part of the country (Gaildorf, Metzler 2010, personal communication). The first detection of Ch. fraxinea dates from 2009 from near Aalen (Schröter et al. 2009, B. Metzler personal communication), and the disease remained almost undetected in SW-Germany before 2009 (Metzler 2009). At four sites in Baden (along the Rhine between Freiburg and Karlsruhe) dieback symptoms could be backdated to 2007 based on repeated yearly infection, followed by proliferation of replacement shoots (Metzler et al. 2012). The first symptoms detected in the region around Stuttgart (Calw, Ludwigsburg, Heilbronn, Schwäbisch Hall, etc.) were all made in May and June 2009 (B. Metzler personal communication). Apothecia were first recorded near Heidelberg in 2009 (M. Bemmann, unpreserved) and near Tübingen in 2011 (H.O. Baral, Table 2).

For Bayern the disease seems to have started in 2008, being recorded at various sites, especially in the north (Main area) and the southeast to east (Leonhard et al. 2009). Apothecia were first recorded in 2009 by P. Karasch in the southeast, and in 2010 in the northeast by H. Ostrow (P. Karasch personal communication).

Austria

Some records of Hy. albidus from Oberösterreich are included in the distribution map of Krieglsteiner (1993, pl. 745). A recent map (Dämon et al. 2009) shows 98 records under the name Hy. albidus, mainly from the north and east of Austria, and merely 7 of Hy. fraxineus. Only a few of the Hy. albidus records date before 2000, whilst the majority was made between 2008 and 2009; therefore, probably most of them concern Hy. fraxineus. For a critical review of these records and a map see Kirisits and Cech (2009). After the invasion of Hy. fraxineus, Hy. albidus has not been observed in Austria anymore (Kirisits & Kräutler 2013).

Ash dieback appears to have invaded Austria from Czechia. The first observations on younger trees date from 2005, while in 2008 symptoms were observed in all Austrian provinces. Ch. fraxinea was first isolated in 2007, and apothecia were first detected in 2009 in great number in various parts of the country (Kirisits & Cech 2011).

Switzerland and Liechtenstein

Breitenbach and Kränzlin (1981) reported Hy. albidus in the cantons Luzern, Obwalden and Nidwalden, and stated the species to be uncommon. Prongué et al. (2004) gave four records for Liechtenstein, and the drawing on our Figure 1 refers to one of them. A recent distribution map of Switzerland (Senn-Irlet 2014) gives four records of Hy. albidus before 1990 and eight between 1991 and 2005, but 26 after 2005. However, the records after 2005 certainly include Hy. fraxineus. Based on molecular identification, Queloz et al. (2011) reported 24 collections of Hy. albidus from the cantons Bern, Glarus, Obwalden, Ticino, Valais, Zug and Zürich, collected mainly in 2009 but also earlier. Most of them originate from montane to subalpine areas in more southern regions of Switzerland (see Figure 23).

The epidemic was not evident before 2007 in Switzerland, and diseased trees were noticed in 2008 only in the north and northwest of the country. During 2009–2011 Hy. fraxineus spread towards the Alps (Engesser & Meier 2012; Pautasso et al. 2013). Apothecia of Hy. fraxineus were recorded by Queloz et al. (2011) during 2009–2011. In the montane southern cantons Ticino, Glarus and Wallis the disease was still absent during that period.

Italy

Rehm (1893, p. 797) mentioned a collection from Südtirol (leg. G. Bresadola), in which apothecia of obviously Hy. albidus arise from blackened patches on the petioles of Fraxinus (other records in Rehm concern Cy. fraxinophila). A record from Vigevano (Pavia) on rotting petioles of Fraxinus (22.IX.1983) is included in the checklist of fungi from the ‘Parco della Valle del Ticino’ (Gaggianese et al. 2002).

In NW-Italy along the Italo-Slovenian border, Ogris, Hauptman, Jurc et al. (2010) recorded in July 2009 ash dieback symptoms and isolated Ch. fraxinea from one canker.

Czechia

Velenovský (1934, p. 205) considered He. albidum as frequent and often abundant in whole Bohemia. However, he confused this species with Cy. fraxinophila (see below). Although two of his four preserved specimens represent Hy. albidus (Central Bohemia: Bilichov 23.VII.1925, PRM 147373; Slané IX.1928, PRM 148933), it can be assumed that his observation of an abundant occurrence was only true for Cy. fraxinophila.Two further Czech specimens were deposited by V. Vacek (Cernošice, 12.VII.1942, PRM 683594; Žarošice, 5.IX.1942, PRM 683595). Revision of this material and also that from Bilichov (PRM 147373) done by M. Tomšovský and O. Koukol based on morphology and especially molecular data (see GenBank HF937562.1, HF937561.1, HF937560.1) confirmed the identification as Hy. albidus. No material of Hy. albidus is present in PRC and BRNM. When searching for Hy. albidus since 2009, no records were made of this species which appears to be extinct.

Ash dieback symptoms were observed already in the mid-1990s, and increasingly in 2004 (Jankovský et al. 2008). In Moravia, the first record of Ch. fraxinea was in 2007 (Jankovský & Holdenrieder 2009). In most parts of Bohemia and Moravia, Hy. fraxineus is now abundant everywhere (O. Koukol personal communication). Collections from Bohemia deposited in PRM are, e.g., from Srbská Kamenice in the north of Czechia (6.VI.2009, PRM 961903, M. Chlebická) and Bílý vrch between Mochov and Celákovice northeast of Praha (8.VII.2011, PRM 899715, M. Kríž). Numerous further collections taken since 2009 from various localities in Czechia belonged exclusively to Hy. fraxineus, with the highest observed altitude at 970 m in Southern Bohemia (Ceské Žleby, 17. IX. 2013, leg. V. Poustka, PRC 1806), whereas Hy. albidus seems to be extinct (O. Koukol personal communication).

Hungary

Bánhegyi (1941, p. 18) reported a collection of Hy. albidus on petioles of Fraxinus (Budapest, Hüvösvölgy, 12.VII.1940), and Svrček (1979, p. 153) one on blackened petioles and veins of Fraxinus angustifolia (PRM 818040, collected by him in 20.IX.1978 during the VII. Congress of European Mycologists in Budapest in the Bugac puszta; a specimen made during this congress is also found in CUP-059500).

Symptoms of ash dieback on F. excelsior and isolation of Ch. fraxinea were first reported in May 2008 in NW-Hungary (Szabó 2009).

Differences between European Hymenoscyphus fraxineus and Hymenoscyphus albidus

Queloz et al. (2011) defined Hy. fraxineus based on 20 nucleotide positions in the CAL gene and 18 positions in the region of the EF1-α gene. At these 38 specifically mentioned, so-called definition positions, Hy. fraxineus consistently differed from Hy. albidus in the available sequences. Therefore, they were selected to constitute the protologue of the new species (see also Queloz et al. (2012) for corrections to these positions). Four further positions represent gaps that were not considered in the protologue definition (V. Queloz, personal communication). These are pos. 235 in the CAL gene, and pos. 232–234 in the EF1-α gene (the alignment at pos. 232–236 is not at an optimum in the table of Zhao et al. 2013)(Table 5).

Table 5.

Part of ITS1 which includes seven critical positions. At two of them (marked in blue: 80, 124) European Hy. fraxineus deviates from NE-Asian Hy. fraxineus. Positions in red (77, 79, 87, 101, 117) concern definition positions against Hy. albidus that apply also for Asian samples (see also Table 6).

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However, also in the ITS rDNA region 11 nucleotide positions can be used to distinguish Hy. fraxineus from Hy. albidus. Based on the presently available data in GenBank, together with the original ITS sequence of Japanese material in NBRC, four sequences published in Shabunin et al. (2012) from Leningrad Oblast, and four gained by V. Queloz (personal communication) from Central European specimens of the first author, European Hy. fraxineus differs from Hy. albidus in the ITS1 region at positions 77 (T/C), 79 (T/C), 80 (gap/G), 87 (C/T), 101 (T/C), 117 (C/T), and 124 (T/C), and in the ITS2 region at positions 355 (G/C), 361 (T/C), 436 (C/A) and 450 (C/A) (Tables 5 and ​and6)6) [position numbers starting with the last five nucleotides of the SSU: CATTA]. One exception must be mentioned: though otherwise perfectly matching Hy. fraxineus, a twice sequenced isolate from Czechia (071026.1, FJ429386, GU586921) shows at position 124 the character C which would be typical of Hy. albidus, as well as Chinese, Korean and Japanese Hy. fraxineus.

Table 6.

Variable nucleotide positions of the ITS1 (77–155), 5.8S (212), and ITS2 (355–453) of Hymenoscyphus albidus and Hy. fraxineus from European and Asian samples [position numbers starting with the last five nucleotides of the SSU: ‘CATTA’]; ***/** = normal case, * = infrequent abnormal case. Definition positions that apply also to Asian samples are marked in red, whereas at the blue markings Asian and European samples of Hy. fraxineus differ, the former matching Hy. albidus. Positions identical to all sequences are omitted, and identical sequences are equally omitted except for a few from Asia.

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Apart from the characteristics in the ITS, Husson et al. (2011, table 4) mentioned a difference of four nucleotides in the partial SSU between the two species: positions 56 (C/T), 90 (C/A), 182 (T/C), and 269 (C/G) [the authors counted from the beginning of their sequences that start with CTTGGTCA]. It must be noted that those critical positions which they listed for the ITS bear a few errors: positions 460, 461, 463 should read 460, 462, 463 (bold = gap, position 460 corresponds to pos. 77 in Figures 23–24), and 607 is an error for 507 (=pos. 124 in Figures 23–24). LSU sequences from Hy. albidus were not available in GenBank for comparison with Hy. fraxineus.

Type sequences of Hymenoscyphus albidus

Husson et al. (2011) succeeded to isolate ITS rDNA from the two ~160 years old duplicates of the type collection of Hy. albidus preserved in the Caen Herbarium. According to their sequences deposited in GenBank, only 67 nucleotides (part of ITS1) were gained from n° 1604 (HM193465), but 510 nucleotides (part of SSU, ITS1, and 5.8S) from n° 2004 (HM193466). Both sequences fully match previously obtained sequences of Hy. albidus in GenBank, thus confirming the identity of the type material. Five critical positions are located in the region of the 67 nucleotides of n° 1604, (77, 79, 80, 87, 101; Tables 5 and ​and6),6), allowing exclusion of Hy. fraxineus, while the sequence from n° 2004 covers all 7 critical positions of the ITS1.

Intraspecific variation in the ITS

Within the many European sequences of Hy. fraxineus variation in the ITS was noted only in on sample (positions 124 and 379, see Table 6). In Hy. albidus three sequences showed variation, which concerns positions 86, 93, and 399. Among the 7 Japanese sequences variation occurred at positions 155, 212, and 443, and among the 6 Chinese sequences only at position 443 (Zheng & Zhuang 2014). The four Korean sequences (KF830850, KF830851, KF830852, KF830853) do not show any variation (J.G. Han personal communication). This infraspecific variation of Hy. albidus and Hy. fraxineus lies in the range of 0–0.5% of the entire ITS1-5.8S-ITS2 region, and the distance between the two species is about 2.2–2.4%.

Does European Hymenoscyphus fraxineus originate from the northeast of Asia?

The phylogenetic analyses by Zhao et al. (2013), J.G. Han (personal communication) and Zheng and Zhuang (2014) have shown that European and Asian Hy. fraxineus cluster together in a clade with rather high conformity and support, sharply separate from Hy. albidus. However, two deviations were noted, which question the asserted invasion of the pathogen from the presently known distribution area in Eastern Asia. According to the results presented by Zhao et al., all seven Japanese ITS sequences fully concur with European Hy. fraxineus, except for two nucleotide positions (80 and 124) at which the Japanese ones show the character of Hy. albidus (80: G instead of a gap, 124: C instead of T; counted when starting with CATTA, see Tables 5–6). The very same peculiarity is shown by all four ITS sequences from South Korea (J.G. Han) and all six from the northeast of China (Zheng & Zhuang 2014). As mentioned above, a sample of Hy. fraxineus from Czechia which was twice sequenced (FJ429386, GU586921) shows at position 124 the character of the Asian samples, while at position 80 it shows the typical gap of European Hy. fraxineus. This sequence further deviates from all sequences of Hy. albidus/Hy. fraxineus at position 379: T vs. C, Table 6).

In the two available sequences of LSU, European Hy. fraxineus (HM145907, Slovenia) deviates from Japanese (NBRC102368) at two positions in the overlapping part. More sequences are needed to clarify whether these two positions are constant markers. In the CAL and EF1-α gene regions, Japanese and European Hy. fraxineus isolates do not consistently deviate from each other, with at best one exception: at position 266 of CAL, Japanese isolates have A and European G, but one Japanese (TNS-F-12503, AB705208) has also G.

Further arguments against an origin from the northeast of Asia were presented by Drenkhan et al. (2014) who showed that the outbreak of the disease in NW-Estonia in 1995 was not associated with the areas where Mandshurian ash had repeatedly been introduced as seeds or seedlings since nearly 150 years.

Variation of Asian Hymenoscyphus fraxineus towards the genome of Hymenoscyphus albidus

Various nucleotide positions are presently known at which Asian Hy. fraxineus shows infraspecific variation. These can be divided into two groups: those positions at which Hy. fraxineus does not differ within Europe from Hy. albidus, and those at which the two differ. In the latter group (the definition positions), the Asian sequences show either the character of Hy. fraxineus or that of Hy. albidus, but never a further possibility. In other words: when deviating from European Hy. fraxineus, variation of Asian Hy. fraxineus was always towards the character of Hy. albidus. This strange phenomenon, which reminds of the two consistent deviations in the ITS between European and Asian Hy. fraxineus, might indicate a lineage from the ancient Asian Hy. fraxineus towards the younger Hy. albidus.

Variation in the ITS region within Asian Hy. fraxineus is noted at three positions (155, 212, 443) which are located outside the definition positions. Variation in the EF1-α gene within Japanese isolates concerns various positions. Among these are 11 definition positions, and all of them show either the character of European Hy. fraxineus or that of Hy. albidus (see Zhao et al. 2013, table 3; gaps not counted, pos. 236 must show a T instead of a gap). Also one position in the CAL gene (244) shows this phenomenon in both Japanese and Chinese specimens, and a further position (49) varies in one Chinese specimen towards Hy. albidus. These variations concern not only transitions but also transversions.

Phylogenetic considerations within the Fraxinus-inhabiting species

According to fossil finds, the genus Fraxinus occurred for the first time in the early Eocene(?) in North America. The land bridge between North America and Asia during this epoch allowed it to spread westwards while forming different species. According to molecular data, Asian species such as F. mandshurica are older than European species such as F. excelsior and F. angustifolia (Wallander 2008; Hinsinger 2010). Parallel to this evolutionary progress, different leaf decaying fungi developed on different Fraxinus species, such as R. longipes in North America (see below), Hymenoscyphus fraxineus in Eastern Asia, and Hymenoscyphus albidus in Europe.

The poor genetic variability of both Hy. albidus and Hy. fraxineus in comparison with a rather high variability of Eastern Asian Hy. fraxineus as detected by Zhao et al. (2013) and Gross, Hosoya et al. (2014) might suggest that not only the invasion of Hy. fraxineus but also that of Hy. albidus to Europe started from a small part of the whole Asian gene pool of Hy. fraxineus. This hypothesis is supported by the striking coincidence in the variable nucleotides of Japanese Hy. fraxineus with the stable nucleotides of European Hy. albidus (Zhao et al. 2013). Possibly, the ancient invasion of Europe derived from a single infection event, which resulted in the homothallic European species Hy. albidus. The loss of an anamorph as well as croziers at the ascogenous hyphae appears to be a consequence of this homothally. If these presumptions were true, the ancient invasion from Asia was perhaps also initially accompanied by ill effects to European ash.