Lilapila – Facesoffungi number: FoF14388

Lilapila Baral & G. Marson

Index Fungorum number: IF 813441; Facesoffungi number: FoF14388

Type species: Lilapila oculispora Baral & G. Marson

Etymology: named according to the deep purple-lilac colour of the apothecial tissue and hairs when seen under transmitted light.

Latin diagnosis: Apothecia in statu rehydratato subsessilia, superficialia, hymenio obscure purpureo-lilaceo vel atro, pilis longis lilaceo-nigris dense confertis. Asci cylindrici, apice in statu emortuo rotundati, tunica tenui, non amyloidei (IKI), octospori, ad basim uncinati. Ascosporae monostichae in ascis vivis, globosae vel subglobosae, intus corpusculo refringente lentiforme ad tunicam appresso. Paraphyses deorsum ramosae, ad apicem non inflatae, non cohaerentes. Pili cylindrici, plerumque 50–150 µm longi, crassitunicati, 3–7 septati, in toto intense lilaceo-purpurei, tunica dense minute granulosa, apice rotundato. Excipulum ectale e textura prismatica-angulari, cellulis hyalinis, extus crassitunicatis, exsudato brunneo-lilaceo tectis, excipulum medullare in parte inferiore textura oblita leniter vel valde gelatinosa. Inter cellulis hymenii, subhymenii et excipuli medullaris exsudatum abundans e multis granulis lilaceo- rubris compositum. Habitat in ramis vulneratis siccis Piceae et Pini, vivis vel leniter putridis, in corona arborum, ad corticem vel plerumque resinam nigram, in Alpibus meridio-occidentalibus.

Description: — TELEOMORPH: Apothecia rehydrated dark purple-lilac-blackish, round, somewhat elliptical or irregular if large, slightly to medium gelatinous, scattered to gregarious in small groups; disc medium concave to flat, margin and exterior densely covered by fascicles of blackish-purple hairs; sessile or mostly with a ± abrupt stipe, stipe sometimes also with scattered purple-brown hairs and completely immersed in blackish layer of associated hyphomycetes; dry black, contracted, sometimes ± hysterioid, disc completely covered by the converging hairs. Asci *(43–)50–80(–84) × (4.5–)5–10.3(–11.5) µm, (44–)49–65(–70) × (4–)4.3–5.8(–6.7) µm, 8-spored (rarely 1–2 spores aborted), spores (*) uniseriate, sometimes sub biseriate, orientation irregular but SBs preferably pointing sideways; apex (†) hemispherical to very slightly truncate or conical (irrespective of the viewing direction), apical wall only slightly thickened [†0.3–0.4(–0.5) µm {3}], without internal wall thickening, wall CR–; base with a medium long, thick, slightly flexuous stalk, arising from croziers of variable shape, mostly with a 1–3 µm long, ± slit-like perforation (up to 6 µm in ascogenous hyphae). Ascospores *(3–)3.3–4.5(–5.5) × (2.5–)2.8–4.5(– 5.5) µm, †2.8–4.2 × 2.3–4 µm, smooth, globose to subglobose, slightly flattened (e. g., 4 × 3.7 × 2.8 µm, distinctly subglobose only in profile view); SBs (0.3–)0.4–0.8(–1) × 1.4–3.3 µm, strongly refractive, lens- to calotte-shaped, circular in front view, very broadly attached to the wall where flattened spore is broadest (simulating terminal attachment, Pls 31: 1a, 2a, 3a; 32: 4b); with 1–7 minute LBs; germination by germ tubes once seen in an old apothecium. Paraphyses apically uninflated or only slightly inflated or attenuated, *(1.7–)2–3(–3.7) µm wide, ± equalling the living asci (protruding 0–5 µm over dead asci), ± equidistantly septate, branched only near base, here sometimes with anastomoses, paraphyses and asci ± easily separable, hymenium bright purple-lilac by numerous exudate granules. Medullary excipulum 50–200 µm thick, hyaline but young with dense, later scattered patches of purple-lilac granules, especially in the perihymenial region and towards the ectal excipulum, granules embedded in an invisible gel matrix; 2-layered: upper part non- or medium gelatinized, ~ 40–150 µm thick, of medium dense to dense, horizontally or ± upwards oriented textura intricata, hyphae *1.8–2.5 µm wide, intermingled with many inflated, globose to ellipsoid, catenate cells *5–15(–20) × 5–10(–15) µm; lower part of slightly to strongly gelatinized t. intricata-porrecta-oblita, 10–100 µm thick, irregularly oriented or at 0–50° towards the very sharply delimited ectal excipulum, individual cells *17–30 × 1.7–3(–5) µm, 1.2–3 µm wide, if strongly gelatinized then with slightly refractive gelatinous sheath ~ 1 µm thick. Ectal excipulum hyaline to pale yellowish-ochraceous, near base with bright to dark ochre-brown to purple-lilac intercellular exudate, of vertically oriented t. angularis- prismatica from base to mid flanks, cells *(7–)9–19(–22) × (5–)7– 10(–14) µm, (†) strongly gelatinized (wall swelling in dead state to 1.5–3 µm), outer wall of cortical cells *1.5–2 µm thick, lateral wall *0.5–1.5 µm, glassy; marginal cortical cells more elongated, oriented at a 20–30° angle to the surface. Hairs covering the whole exterior but sometimes lacking at base and flanks, inserted on cortical cells, free or in converging, agglutinated fascicles, at margin and flanks (30–)80–150(–200) × 5–9(–11) µm, cylindrical, straight to slightly flexuous, with (1–)3–8(–11) refractive septa (0.3–1.3 µm thick), apex rounded, whole surface densely covered by low but distinct warts, wall surface and warts dark purple-lilac in H₂O, sometimes ochre-brown when dead, marginal hairs ± paler towards apex (upper 5–20 µm or more), sometimes almost smooth; lateral wall */†1–2.5 µm thick {3}, 2-layered, warts 0.1–0.3 µm high, 0.2–0.8 µm wide, sometimes almost confluent, glassy processes absent. Anchoring hyphae sparse to very abundant, restricted to base, subhyaline to pale yellowish-cream, (3.5–)4–5.5(–6.5) µm wide, walls *(0.2–)0.5–1.3 µm thick [†(0.2–)1– 1.5(–2) µm], surface finely or mostly roughly and densely warted, rarely some hyphae smooth. SCBs in terminal cells of paraphyses absent or globose to ellipsoid, sparse, very slightly or medium refractive; in cells of medullary excipulum regularly present, 1.5–4 µm diam., strongly refractive, ± globose or often ring-shaped, unstained in CRBA; VBs sometimes present inside vacuoles of asci, paraphyses and medullary excipulum: one or a few strongly refractive, globose, hyaline, trembling bodies 0.3–1 µm diam., staining tardily blue in CRBA, disappearing in KOH; LBs never containing carotenoids. Exudate: bright to deep purple-lilac granules or clods 0.3–1.5 µm diam., abundant in hymenium and medullary excipulum, between paraphyses from base to apex or subapex, not covering the tips, in ectal excipulum abundant between the cells, also covering the entire hair wall as a thin film; exterior covered by a ± continuous reddish-brown layer, in stipe pale yellowish- ochraceous; anchoring hyphae warted by a hyaline exudate.

Chemical reactions: purple-lilac exudate in 5 % KOH, turning light to dark dirty grey- to olive-brown, warts on hairs not dissolved; adding strong acids to the KOH-mount fully recovers the purple-lilac colour. CRB stains the warted exudate on the anchoring hyphae (turquoise-)blue and a thin gel layer beneath lilac and the medullary excipular gel faintly lilac.

ANAMORPH: Colonies whitish to pale yellowish, slow-growing (see below). Conidiophores and conidia unknown (but see under L. oculispora).

Habitat: on resinous, usually blackened wounds of corticated, living or recently dead, still-attached branches or standing trunks of conifers, never directly on wood, wounds either with small to large, resinous decorticated areas or completely overgrown by bark, apothecia often only on bark that borders the decorticated wound, preferably growing in the niche between bark and wood; often also on the decorticated resinous area but then on a ± thick layer of resin covered by a black layer of dark brown hyphomycetes, green algae sparse or lacking. Growing in association with various resinicolous ascomycetes.

Recognized species: L. jurana, L. oculispora, L. oculisporella, L. gallica nom. prov.

Notes: The genus Lilapila is quite extraordinary by it purplish-black apothecia covered with large, deep purple, septate, thick-walled, finely warted hairs and by (sub)globose ascospores with a single, broad and thin, lens- shaped spore body. Further characteristics are: asci arising from croziers, with rounded, thin-walled apex, paraphyses simple, non-inflated at apex, with purple-lilac exudate between, ectal excipulum of thick-walled, angular to prismatic, vertically oriented cells, medullary excipulum sharply delimited, of gelatinized textura intricata.

Some variation was noted in the rate of gelatinization of the outer medullary excipulum, but this seems to depend on the development stage: in younger apothecia the gel was more refractive and therefore more obvious. The purple-lilac pigment of Lilapila belongs to the most spectacular characteristics of the genus. It is best seen under transmitted light in a water mount, but also in acidic solutions. Unlike Liladisca, the pigment turns greyish-olivaceous in alcali, moreover, it is granular in the inner regions of the apothecium, and only on the surface of the hairs it forms a continuous layer. In aged but still-living apothecia the lilac pigment on the hairs may change to red-brown in water without application of alkali. Thus, the hairs at the flanks may appear brownish while the younger marginal hairs are still bright lilac.

Species delimitation: Although the four taxa within Lilapila are morphologically very similar, we have separated them based on strong molecular evidence. However, a correlation of molecular data with differences in ascus and ascospore measurements was noted between some of them. This variation was observed when sequences were not yet available, and was considered as quite unusual within the species level.

Cultural characteristics and anamorph. Ascospores of L. oculispora and L. oculisporella tardily germinated when shot on agar (CMA:5), and the whitish to very pale yellowish mycelium was very slow-growing. No conidiophores and conidia could be obtained in culture under different CMA concentrations.

Phylogeny: Sequences from 28 samples of Lilapila spp. were obtained, comprising ITS and LSU (D1–D2, often also D3–D4 and D5–D6; 2 sequences only ITS), 10 also SSU (V7–V9). Two sequences (L. oculispora H.B. 10122, L. jurana H.B. 10123) include SSU V1–V9, LSU D1–D12, L. oculispora also IGS. Sequence similarity and phylogenetic analysis support affiliation of the genus in Orbiliomycetes. BLAST search for SSU (V7– V9) yields Lecophagus spp. as closest hit (distance 2 %). When analysing 5.8S+LSU or SSU+ITS+LSU, Lilapila clustered in a clade with Amphosoma, Bryorbilia, Lecophagus, and Retiarius with medium or low support, respectively (Phyls 1, 2). However, a high distance to these and other genera is observed, ranging in the LSU (D1–D2) at 6.2–7.5 % (Amphosoma), 8.3–10 % (Bryorbilia), 8.7–10.5 % (Lecophagus), 9–10.7 % (Retiarius),10–12 % (Mycoceros), and 13–19 % (Hyalorbilia).

Nucleotide positions specific for the genus. A few motifs occur in the more frequently sequenced rDNA regions that characterize Lilapila. In the SSU V9 at pos. 287 Lilapila has AGTAAGT (L. oculispora, L. oculisporella) or AGTGAGT (L. jurana, L. gallica), whereas other Orbiliomycetes have T (mostly AGTTGGT). In the LSU D1 domain pos. 226 is unique (GTTGACGGC or in L. oculisporella GTCGTCGGC; in all other Orbiliomycetes mainly C, sometimes T or A but never G), and also pos. 300 and 311 are unique (AGGTAAATTTCT), except for O. pilifera which has likewise A + T. In a few more variable regions of the LSU D2 domain the following motifs are almost unique for Lilapila: TCGGTGCAATG (pos. 561–571) also occurs in Retiarius bovicornutus, and CGCGATCGAG (pos. 580–589) in section Lentiformes p.p. (O. foliicola, O. cucumispora, O.pluristomachia). In the D3 domain the motifs AGGTGGGAGCCGC (pos. 717–729) and adjacent AAGGTGCACCATCG (pos. 730–743) each were otherwise only observed in O. crenatofalcata.

In the region of the three ‘Orbiliales-specific’ primers (Smith & Jaffee 2009, see Tabs 18–19), Lilapila concurs with different other genera of Orbiliomycetes. For primer Orb5.8s1F this is Amphosoma, Bryorbilia, Lecophagus, Retiarius, and the Vermispora clade of Hyalorbilia, whereas for Orb5.8s3F it is Mycoceros and Orbilia p.p.maj. Lilapila is variable in the primer region Orb28s2R: L. oculispora concurs here mainly with Mycoceros, L. oculisporella with Retiarius, Amphosoma, and Lecophagus, and L. jurana/L. gallica show a further variant which is so far unique within Orbiliomycetes. However, especially in Orb28s2R various non-orbiliaceous ascomycetes concur with Lilapila. Congruence of Lilapila with Mycoceros and Hyalorbilia Phylogeny. Sequences from 28 samples of Lilapila spp. were obtained, comprising ITS and LSU (D1–D2, often also D3–D4 and D5–D6; 2 sequences only ITS), 10 also SSU (V7–V9). Two sequences (L. oculispora H.B. 10122, L. jurana H.B. 10123) include SSU V1–V9, LSU D1–D12, L. oculispora also IGS. Sequence similarity and phylogenetic analysis support affiliation of the genus in Orbiliomycetes. BLAST search for SSU (V7– V9) yields Lecophagus spp. as closest hit (distance 2 %). When analysing 5.8S+LSU or SSU+ITS+LSU, Lilapila clustered in a clade with Amphosoma, Bryorbilia, Lecophagus, and Retiarius with medium or low support, respectively (Phyls 1, 2). However, a high distance to these and other genera is observed, ranging in the LSU (D1–D2) at 6.2–7.5 % (Amphosoma), 8.3–10 % (Bryorbilia), 8.7–10.5 % (Lecophagus), 9–10.7 % (Retiarius),10–12 % (Mycoceros), and 13–19 % (Hyalorbilia).

The S1506 intron is absent in all sequences of the four recognized species. The S943 intron is present in L. oculispora (380 nt) but absent in L. oculisporella and L. jurana. This enables recognition of L. oculispora already from the PCR product on gel without sequencing, using the primer SR5 which binds upstream of the S943 intron. Also L2449 and L2563 introns are present in L. oculispora but not in L. jurana (this region so far is not covered in L. oculisporella). Deviations in the SSU region occur at 2 positions in the V9 region: at pos. 238 and 241 L. oculispora has CTCTCGG, L. jurana and L. gallica CTTTCGG, and L. oculisporella CTCTCAG (other Orbiliomycetes have T+G); in addition, L. jurana and L. gallica deviate from the other two at pos. 287 by A vs. G (see above). All this supports the existence of three four different species.

Ecology. Members of Lilapila were so far only detected in montane to subalpine, orotemperate to orosub- or rarely suprasubmediterranean, humid to slightly semihumid, often pure conifer forests of Southern French Alps (Rhône-Alpes and Provence-Alpes-Côte d’Azur) and Massif central (Auvergne) in southern Europe, from orotemperate humid Northern and Central Alps and High Tatras, and from (alti)montane, orotemperate humid French and Swiss Jura. The forests are often on steep north-, east-, or south-exposed slopes, but also on the top of high plateaus. The geology was almost always calcareous, but in the samples from Central Alps and High Tatras it was acidic bedrock. Apothecia were exclusively found on black resinous wounds of still-attached, xeric branches or trunks of Picea and Pinus, which were often still viable by carrying green needles at their ends. The wounds have been injured some years ago, perhaps as a result of avalanches and snowslides or other influences of snow and frost during winter, or by rockfall, also by passing forestry machines.

Apothecia of all species usually grow in a black mould of dark brown hyphae of some dematiaceous hyphomycetes that abundantly cover the resin. The dry contracted apothecia appear blackish and can hardly be detected when growing in black mould, but even after rehydration the lilac pigment appears blackish in external view (similar as in Liladisca). Although the apothecia are comparatively large and occur in numbers of dozens on a given branch, the genus appears to have escaped notice on account of its camouflage, xeric habitat, and ecological restriction. The currently known phenology of the genus Lilapila (mainly summer and autumn) is certainly only accidental. It results from the difficulty during winter and spring to travel and collect in mountainous areas rich in snow. The detection by E. Stöckli (pers. comm.) of mature apothecia of L. jurana in late January and early March proved our assumption. A striking correlation is observed in the distribution area of samples on Picea and Pinus in the Southern French Alps. Those on Picea are more in the north (L. oculispora, L. oculisporella) and those on Pinus more in the south (L. oculisporella). Together with an altitude of sometimes below 1000 m in the Pinus samples, it appears that L. oculisporella is adapted to a warmer, strictly orosub- or rarely suprasubmediterranean climate. Because L. oculisporella occurred at about equal frequency on Picea and Pinus and was recorded even at the type locality of L. oculispora (Col du Labouret) on Picea, a sharp geographical or host specificity limit cannot be recognized. Therefore, a number of older collections remained undetermined due to the lack of ascus or spore measurements, voucher specimens, or DNA data.

Species