Melanconis marginalis subsp. marginalis (Peck) Wehm., Pap. Michigan Acad. I. 6: 382 (1926).

Jaklitsch, Walter M. & Voglmayr, Hermann, 2020, The genus Melanconis (Diaporthales), MycoKeys 63, pp. 69-117 : 69

Melanconis marginalis subsp. marginalis (Peck) Wehm., Pap. Michigan Acad. I. 6: 382 (1926). Figures 8 , 9

≡ Diaporthe marginalis Peck, Rep. (Annual) Trustees State Mus. Nat. Hist., New York 39: 52 (1887) [1886] (Basionym).

≡ Melanconis alni var. marginalis (Peck) Wehm., Revision of Melanconis , Pseudovalsa , Prosthecium & Titania : 27 (1941).

= Diaporthe nivosa Ellis & Everh., Proc. Acad. nat. Sci. Philad. 42: 222 (1890).

= Melanconium dimorphum Peck, Ann. Rep. New York State Mus. Nat. Hist. 40: 62 (1887).

Type material.

Holotype of Diaporthe marginalis : USA, New York, Essex County, Elisabethtown, on Alnus alnobetula subsp. crispa (given as Alnus viridis ), May 1885, C.H. Peck (NYSf 1859!; material separated into 2 envelopes NYSf 1859.1 and NYSf 1859.2). Epitype, here designated: Canada, New Brunswick, Charlotte Co., 1.5 km SW of Little Lepreau, on Alnus alnobetula subsp. crispa attached to the tree, soc. Tortilispora aurantiaca , 3 Sep 2019, D. Malloch (WU 37850; ex-epitype cultures CBS 146200 = D321 (from ascospores), D321a (from α-conidia), D321b (from β-conidia); MBT390382).

Description.

Sexual morph: Pseudostromata immersed in bark causing pustules, scattered or aggregated, sometimes fused in pairs, 1.2-3.2 mm diam., pulvinate, circular to elliptic in outline, often elevated beyond bark surface; consisting of an ectostromatic disc and perithecia embedded in an entostroma around a central column, sometimes also acervuli containing α-conidia on the ostiolar level. Ectostromatic discs 0.3-1.5(-2) mm diam. or long, bright white to yellowish or cream, flat, convex or concave, sometimes fissured or with dark stellate stripes around disc on the bark surface, sometimes concealed by ostioles, circular, elliptic or fusoid in outline, typically distinctly projecting up to 1 mm including projecting part of the pseudostroma; central column beneath disc white to yellowish, consisting of hyaline hyphae and colourless crystals. Ostiolar necks cylindrical, laterally attached on perithecia, centrally attached only on centrally arranged perithecia, convergent in the disc margin or crowded at the ends of fusoid discs, sometimes completely filling disc, 1-15(-22) per disc. Visible part of the ostiolar necks (55-)87-153(-230) µm (n = 128) diam., shiny black or brown with black tip, flat discoid to ring-like, papillate to subglobose with pointed tip or conical, sometimes bristle-like and projecting up to 0.6 mm. Entostroma bark coloured, not or only slightly paler than the surrounding bark, consisting of bark cells and some light-coloured hyphae. Perithecia (420-)480-650(-750) µm (n = 34) diam., arranged in valsoid configuration around and below central column, depressed subglobose, collapsing up- or laterally inwards upon drying. Peridium pseudoparenchymatous, consisting of a dark brown small-celled outer and a hyaline to brownish, large-celled inner layer. Hamathecium of broad multiguttulate paraphyses, collapsing, dissolving and usually absent amongst mature asci. Asci floating free at maturity, (46-)56-69(-82) × (10-)11-14.5(-18) µm (n = 116), mostly oblong to fusoid, but also clavate or narrowly ellipsoid, with an apical ring distinct in water and staining in Congo Red but invisible or indistinct in 3% KOH, containing 8 ascospores in bi- or obliquely uniseriate arrangement. Ascospores (13.8-)15.5-18(-20.7) × (3.7-)4.5-5.7(-7.7) µm, l/w (2.4-)2.9-3.7(-4.4) (n = 236), hyaline to yellowish, oblong to ellipsoid, bicellular with equal or slightly unequal cells, slightly to distinctly constricted at the more or less median septum, multiguttulate or with few large and several small guttules when fresh, with a roundish to triangular or broadly oblong to beak-like and truncate appendage (1.1-)1.8-3.5(-6.1) × (2.2-)2.5-3.5(-4.2) µm, l/w (0.4-)0.6-1.2(-2.1) (n = 140) at each end; in 3% KOH, ascospores wider and more ellipsoid; appendages mostly invisible.

Asexual morph acervular, intermingled with pseudostromata of the sexual morph or more frequently developing separately, usually inconspicuous but sometimes becoming conspicuous due to greyish-brown to dark brown conidial deposits to 2.7 mm diam., sometimes confluent from 2 conidiomata and then up to 7 mm long. First white to yellow tissue (central column) forming within the bark, becoming visible by pustulate bark and narrow whitish to yellowish or brownish slit-like discs emerging through bark cracks, usually first followed by the production of β-conidia in olivaceous chambers, followed by fusion of the chambers and production of α-conidia on the same or similar conidiophores, turning the cavity brown and oozing out from ends of the discs or perithecia of the sexual morph formed beneath. Conidiomata ca. 0.9-3 mm long or diam., pulvinate, more or less circular in outline, scattered or aggregated in lines. Covering discs 0.3-0.7 mm long or diam., narrowly fusoid or longish to circular, plane to convex, white-yellowish-brownish, becoming covered and obscured by conidial deposits; discs and pulvinate or conical columns beneath consisting of compact textura intricata of hyaline hyphae and numerous colourless crystals. Conidiophores emerging around the central column from a textura intricata, fasciculate, filiform, branched near the base or sometimes 1-2 fold asymmetrically at higher levels, hyaline, turning brown from their tips; terminal conidiogenous cells (10-)13.5-23(-31) × (1.7-)2-3(-3.5) µm (n = 68), cylindrical and often widened in the middle or towards the base and at the funnel-shaped tips beyond its width, annellidic, producing α- and/or β-conidia. Conidia dimorphic, α-conidia (9-)10.5-13.3(-16.8) × (3.8-)4.5-5.3(- 6) µm, l/w (1.7-)2-2.8(-3.9) (n = 171), first hyaline, soon turning light to medium brown, unicellular, mostly fusoid, but also oblong, oval, citriform or ellipsoid, straight or slightly curved to sigmoid, upper end often subacute, lower end narrowly truncate, containing several guttules or eguttulate, smooth; β-conidia (6-)8-10.5(-12.2) × (1.7-)2.2-2.8(- 3) µm, l/w (2.4-)3-4.6(-6.4) (n = 46), hyaline to dilute brownish, unicellular, oblong to cylindrical, sometimes reniform, straight or curved, thick-walled in water, with few guttules to eguttulate, smooth.

Culture: Colony on CMD at 22 °C circular with slightly uneven margin, hyaline to whitish, forming a broad inner white zone with tooth-like margin and narrow hyaline outer zones; on MEA at room temperature circular, first hyaline to white, margin becoming diffuse, narrow or coarse concentric zones formed, turning brown from the margins, aerial hyphae short, dense, surface sometimes becoming imbricate, sometimes growth limited and ceasing after a few weeks.

Distribution and ecology.

Widespread in North America and also occurring in Japan and eastern Russia on various subspecies of Alnus alnobetula and A. incana ; recorded also from A. rubra ( Sieber et al. 1991; see also material cited below).

Additional material examined.

Canada, British Columbia, Kelowna, June Springs road, June Springs trail, on Alnus incana , 18 Jul 1999, J. Ginns 10834 (DAOM 227767; measurements separately given, see below under Notes); Nelson, on Alnus incana subsp. tenuifolia , soc. Cryptosporella sp., 26 Jun 1930, G.G. Hedgcock (BPI 614844, F.P. 50704); Victoria, Lake Cowichan, Mesachie Lake, 48.7942N 124.1573W, on Alnus rubra , 14 Sep 1988, C. Dorworth (DAVFP 24976, dried culture PFC-051 only); Victoria, Ucluelet, Kennedy Lake, 49.0416N 125.5315W, on Alnus rubra , 16 May 1987, C. Dorworth (DAVFP 24972, dried culture PFC-025 only); Manitoba, W Hawk Lake, on Alnus sp., 5 Jun 1932, G.R. Bisby 4593 (DAOM 202917); Nova Scotia, Kings Co., Glenmont, on Alnus alnobetula subsp. crispa (as Alnus crispa var. mollis ), 25 Jul 1936, I.L. Conners (Ottawa 3798 (DAOM)); Kentville, on Alnus alnobetula subsp. crispa , 11 May 1953, D. Creelman (DAOM 54346); Ontario, District of Nipissing, Temagami Forest Reserve, Lake Temagami, Bear Island, on Alnus alnobetula subsp. crispa (as Alnus viridis var. mollis ), 19 Jun 1933, R.F. Cain 2686 (DAOM 86075); trail at Matagama Point, on Alnus alnobetula subsp. crispa (as Alnus crispa var. mollis ), 23 Jun 1933, R.F. Cain 2687 (DAOM 86074); Sharp Rock Inlet, on Alnus alnobetula subsp. crispa (as Alnus crispa var. mollis ), 29 Jun 1933, R.F. Cain (BPI 614977, F.P. 69748). Japan, Hokkaido, Shirikinai, on Alnus alnobetula subsp. maximowiczii , 1 Sept 1967, T. Oguchi (TFM FPH3290; culture MAFF 410218 = M4-6, ME9). RUSSIA, Sakhalin Island, Lake Dvoynoe, on Alnus alnobetula subsp. maximowiczii , 3 Aug 2000, A. Bogachova, comm. L. Vasilyeva (BPI 748233; culture CBS 109496 = A.R. 3529, ME2). USA, Alaska, Fairbanks, Large Animal Research Station, on Alnus alnobetula , 5 Aug 2011, L. Mejia (BPI 884096; culture A.R. 4864, ME5); same area, on Alnus alnobetula (given as Betula neoalaskana ), 5 Aug 2011, L. Mejia (BPI 884097; culture CBS 133346 = A.R. 4865, ME6); Juneau, on Alnus alnobetula subsp. sinuata , 6 Sep 1936, D.V. Baxter (BPI 615125).

Notes.

The asexual morph of Melanconis marginalis subsp. marginalis is inconspicuous with usually only thin greyish patches of α-conidia. The two types of conidia may be present at the same time or only one is present; acervuli containing α-conidia are sometimes present in pseudostromata of the sexual morph. The specimen DAOM 227767 from Alnus incana differs from all others by very large and conspicuous conidial deposits (Fig. 9c ), slightly larger α-conidia, (13-)14.5-16.5(-17.5) × (5-)5.8-7(-8) µm, l/w (1.8-)2.1-2.8(-3.4) (n = 70) and longer and more slender β-conidia, (7.5-)12.5-16(-17.3) × (1.7-)2.2-3(-3.5) µm, l/w (4-)4.6-6.7(-9) (n = 35) and also by slightly larger asci, (68-)74-88(-95.5) × (10-)12-15.5(-18.2) µm (n = 26), which approach the European subspecies. Although Jensen (1984) gave a range of 9-17 × 3-7 µm for α-conidia and 10-18 × 2-3 µm for β-conidia of M. marginalis , it is unclear, whether all examined specimens, including DAOM 227767, phylogenetically belong to M. marginalis subsp. marginalis or a different subspecies or even species. Jensen (1984) reported exceptionally long ascospores (19-32 µm) for four of his collections from Idaho, which also differed in their colony characters; due to lack of DNA data, the taxonomic status of these collections is unclear. While all our North American and Eastern Asian accessions of M. marginalis subsp. marginalis sequences originated from various subspecies of Alnus alnobetula , the accessions investigated by Jensen (1984) originated from Alnus incana subsp. tenuifolia . Sieber et al. (1991), who investigated M. marginalis from British Columbia, recorded mean conidial sizes of 11.2-11.8 × 4.4-4.7 µm for two isolates from A. rubra , while those from three isolates of Alnus alnobetula were slightly larger (13.6-14 × 5.6-5.9 µm). These data demonstrate the need of additional detailed investigations of the M. marginalis complex in western North America. Kobayashi (1970) determined the following sizes for Japanese collections of M. marginalis : asci 70-93 × 10-15 µm, ascospores 15-23 × 4-6.5 µm, mostly 17-20 × 4.5-5.5 µm, α-conidia 11.5-15 × 4-6.5 µm, β-conidia 7.5-12.5 × 1.5-2.5 µm. He also mentioned that the Japanese collections usually lacked ascospore appendages, which, however, may be due to the use of a mounting medium instead of water in his microscope mounts. This is supported by the fact that he also reported a lack of appendages in his M. pterocaryae , which was disproved by re-investigation of the type ( Voglmayr et al. 2017).

Sizes of asci depend on the age of the material. They shrink with time and in specimens, which are 20 or more years old, they are smaller and do not obtain the original size even in KOH; also, it is very difficult to release ascospores from asci. In fresher specimens, asci are easily separable and ascospores are readily released. Vital asci open readily in mounts. Nonetheless, fresh asci of the epitype of subsp. marginalis were distinctly smaller than fresh asci of subsp. europaea .

Poor representation of the asexual morph in fungarium specimens may be due to the fact that the sexual morph is usually abundant, with numerous white ectostromatic discs; thus, the asexual morph may have been neglected during collecting or even discarded. β-conidia are often absent or scant and old amongst α-conidia in dark conidial deposits, hence they are either not formed or produced before α-conidia.

Figures

Figure 8. Melanconis marginalis subsp. marginalis. Sexual morph a pseudostroma in face view b-d ectostromatic discs (note conical to bristle-like ostiolar necks in c discoid in d; e vertical section showing central column and perithecia f-i, p-u asci j-o, v-y ascospores t, u, y in aqueous Congo Red x in 3 % KOH a, f BPI 614844 b, g, h, t holotype NYSf 1859 c, k, j BPI 748233 d, e, n, o, q-s, v, w, y epitype WU 37850 i, u DAOM 227767 l, m DAOM 202917 p BPI 614977 x DAOM 86074. Scale bars: 500 µm (a-e), 10 µm (f-i, q-u), 7 µm (j-p, v-y).

Figure 9. Melanconis marginalis subsp. marginalis. Asexual morph a early stage of covering disc b, c conidiomata and conidial deposits in face view d, e conidiomata in cross section (d with β-conidia, e with α-conidia) f conidioma with α-conidia in vertical section g-k conidiophores and conidiogenous cells (producing α-conidia in g, h β-conidia in i-k) l-w α-conidia x-e 1 β-conidia g-e 1 in 3 % KOH a, b, d-g, i-k, n-s, x-b 1 epitype WU 37850 = D 321 c, t-w, c 1 - e 1 DAOM 227767 h, l, m BPI 614844. Scale bars: 300 µm (a, e, f), 500 µm (b, d), 1 mm (c), 10 µm (g, h), 7 µm (i, t-v), 5 µm (j-l, n, s, w, y-e 1), 3 µm (m, o-r, x).