Phylogenetic analysis

Sequences acquired in this study were supplemented with those retrieved from GenBank (Barnes et al. 2001, Cunnington 2007, Tsopelas et al. 2007, Maharachchikumbura et al. 2015) and aligned using MAFFT v. 7 (Katoh et al. 2002, Katoh & Standley 2013). Alignments were checked and concatenated in MEGA v. 6.06 (Tamura et al. 2013). Maximum-likelihood (ML) analyses for both single-locus and concatenated alignments were performed with RAxML-HPC2 on XSEDE v. 8.2.10 (Stamatakis 2014) using a GTR+GAMMA substitution model with 1 000 bootstrap iterations. For the Bayesian inference (BI) analyses, the optimal substitution model for ITS, TEF and TUB was determined to be HKY+I+G and for RPB2, GTR+I+G, using MrModeltest software v. 2.2. (Nylander 2004). The BI analyses were computed with MrBayes v. 3.2.6 (Ronquist et al. 2012) with four simultaneous Markov Chain Monte Carlo chains from random trees over 5 M generations, ending the run automatically when standard deviation of split frequencies dropped below 0.01. Both RAxML and Bayesian analyses were run on the CIPRES Science Gateway portal (Miller et al. 2012). Maximum Parsimony (MP) analyses were conducted with PAUP v. 4.0b10 (Swofford 2002), inferring trees with the heuristic search option with TBR branch swapping and 1 000 random sequence additions. The robustness of equally parsimonious trees was evaluated by 1 000 bootstrap replications. Alignments and trees were deposited in TreeBASE (www.treebase.org; study 21661).

Taxonomy

Based on the results of the combined multi-gene phylogenies (Fig. 1), morphological observations, measurements of fungarium specimens, cultures (Fig. 3) and ecological data, five novel species of Seiridium are described, a lectotype is designated for S. cupressi, and epitypes are selected for S. cupressi and S. eucalypti. Overall, from four clades (3, 5, 7 and 8) all available cultures were sterile. However, DNA was extracted and sequenced from mycelia of these cultures and for three of the clades (3, 5 and 7) morphological characters were studied from the associated herbarium specimens.

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Fig. 3

Boxplots reflecting the distribution of variance in conidial measurements in micrometres as acquired from living culture material on SNA (in white) or from herbarium material (in grey). The boxes show the first and third quartiles. Lower and upper whiskers extend from the boxes to the extreme values or 1.5 times the inter quartile range when the extreme values are outside this range, in which case outlying values are indicated by black dots. Strains are ordered by clade as in Fig. 1 and in the background coloured accordingly to the clade colours in Fig. 1: a. Conidial length; b. conidial width; c. length of basal appendage; d. length of apical appendage.

Seiridium Nees, Syst. Pilze (Würzburg): 22. 1817

Synonyms. Hyaloceras Durieu & Mont., Expl. Sci. Algerie 1: 587. 1849.

Adea Petr., Bot. Jahrb. Syst. 62: 144. 1928.

Type species. Seiridium marginatum Nees.

Ascomata perithecial, immersed to semi-erumpent, depressed, globose to pyriform, scattered or confluent; peridium dark brown, pseudoparenchymatous. Ostioles central, slightly papillate, black, periphysate. Paraphyses hyaline, smooth, filiform. Asci cylindrical, 8-spored, unitunicate, thin-walled, stipitate, with an apical amyloid ring. Ascospores cylindrical-oblong, euseptate, septa often thicker than the wall, yellow- to dark brown, guttulate. Conidiomata acervuloid to pycnidioid, semi-immersed to erumpent, uni- to plurilocular, brown or black, glabrous, dehiscing by irregular splits in the upper wall. Conidiophores lining the cavity of the conidioma, septate and sparsely branched at the base, or reduced to conidiogenous cells, hyaline, smooth. Conidiogenous cells discrete, integrated, ampulliform to lageniform or subcylindrical, hyaline, smooth, proliferating percurrently at the apex. Conidia fusiform, distoseptate (septal pores present or not), end cells hyaline, median cells dark brown to brown, wall thick, smooth or with striations, constricted at septa or not; apical cell with a single, cellular, unbranched or branched, appendage; basal cell with or without a centric, unbranched or sometimes branched appendage.

Notes — Seiridium includes coelomycetes producing versicolorous, 5-septate conidia with appendages and typically forming acervuli on the plant host. The original description of the generic type, S. marginatum, dates back over 200 years ago and was re-described by Shoemaker et al. (1966), Sutton (1980) and Nag Raj (1993). Recently, the generic type S. marginatum was epitypified (Jaklitsch et al. 2016) from which additional sequence data was generated in this study and included in the present phylogeny (Fig. 1).

Seiridium cancrinum Bonthond, Sandoval-Denis & Crous, sp. nov. — MycoBank MB823296; Fig. 4

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Fig. 4

Seiridium cancrinum sp. nov. (IMI 52256, holotype). a. Herbarium specimen; b. conidiogenous cells and conidia; c. conidia. — Scale bars: b–c = 10 μm.

Etymology. From the three strains that Jones (1953, 1954a, b) conducted pathogenic experiments on, this fungus (strain A) was the most aggressive. Therefore, the fungus is named after the canker formation it induces on Cupressus.

Conidia lunate to falcate, curved, 5-septate, occasionally 4-septate, not striate, bearing two appendages, euseptate with no visible pores, (20–)22–26(–30.5) × (7–)7.5–9(–10) μm, mean ± SD = 24.0 ± 2.1 × 8.2 ± 0.7 μm (n = 35); basal cell obconic with truncate base, hyaline, walls smooth, bearing marginal frills, 1.2–5.8 μm; four median cells, varying in colour ranging from pale to dark brown, smooth, cylindrical to doliiform (second cell from base 3–5.5 μm long; third cell 3–5 μm long; fourth cell 3.5–5.5 μm long; fifth cell 3.5–6.5 μm long); apical cell conical, hyaline, smooth, 2–4.5 μm long; apical appendage single, centric, 1–13 μm; basal appendage, single, cylindrical, centric, 0.5–14.5 μm.

Known distribution — Kenya, South Africa.

Materials examined. Kenya, from cankers in branches of Cupressus macrocarpa, 1950, D.R. Jones (holotype IMI 52256, isotype BRL 1119, culture ex-type CBS 226.55). – South Africa, from Cupressus lusitanica, unknown collection date, M.J. Wingfield (CBS 907.85 = CMW 320).

Notes — The pathogenicity of the strain IMI 52256 was studied by Jones (1953, 1954a, b), who classified the fungus as Monochaetia unicornis strain A. Jones identified strain A as the most aggressive of the three strains that would eventually be described as S. cupressi (Guba 1961). However, here we show that the three strains (A: IMI 52256 = CBS 226.55, B: IMI 52254 = CBS 224.55, IMI 52255 = CBS 225.55 and IMI 52258 = CBS 227.55 and D: IMI 52257 = CBS 228.55) are three different species, which in turn correlates with the observations originally made by Jones (1953, 1954a, b). Although both cultures (CBS 226.55 = IMI 52256 and CBS 907.85) remained sterile on culture media, conidia could be studied from herbarium material (IMI 52256). Based on conidial morphology, S. cancrinum is highly similar to S. cupressi, but on average has longer basal appendages. Conidia with basal appendages up to almost 15 μm can be observed, whereas conidia of S. cupressi bear basal appendages that do not exceed 10 μm in length.

Seiridium cardinale (W.W. Wagener) B. Sutton & I.A.S. Gibson, CMI Descriptions of Pathogenic Fungi and Bacteria: 36. 1972 — Fig. 5, ​,66

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Fig. 5

Seiridium cardinale (CBS 909.85, reference strain). a–d. Colony morphology in 90 mm Petri dishes after 2 wk at 22 °C on PDA, CMA, MEA, SNA, respectively; e. conidioma on PDA partially immersed in agar; f. sporodochia on SNA immersed in agar; g. conidia; h–i. conidiophores; j–k. conidiogenous cells. — Scale bars: e–f = 100 μm; g–k = 10 μm.

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Fig. 6

Seiridium cardinale (IMI 75045 isotype of Coryneum cardinale). a. Herbarium specimen; b–c. conidiomata and conidia in vivo; d. conidia in vitro. — Scale bars: b–c = 20 μm; d = 10 μm.

Basionym. Coryneum cardinale W.W. Wagener, J. Agric. Res. 58: 8. 1939.

Conidiomata on PDA sporodochial, pseudostromatic, globose or clavate, at the edge of the colony, dark brown to black; on SNA sporodochial, pseudostromatic, globose, scattered, irregular in outline, mostly immersed in agar. Conidiophores septate, cylindrical, irregularly branched, branch lengths variable (20–85 μm long), hyaline, thin- and smooth-walled, invested in mucus. Conidiogenous cells discrete, hyaline, subcylindrical to lageniform, thin- and smooth-walled, 4–15 × 1.5–2.5 μm, proliferating percurrently multiple times, with collarettes and minute periclinal thickenings. Conidia fusiform, straight to slightly curved, 5-septate, not striate, bearing two short appendages, euseptate with pores clearly visible, (20–)20.5–24(–27.5) × (7.5–)8–9(–9.5) μm, mean ± SD = 22.3 ± 1.8 × 8.4 ± 0.4 μm (n = 30); basal cell obconic with a truncate base, hyaline, walls smooth, bearing minute marginal frills, 2–3 μm; four median cells, smooth, cylindrical to doliiform, young conidia concave cylindrical to subcylindrical, pale brown to brown, septa darker than the rest of the cells (second cell from base 3.9–4.8 μm long; third cell 3.8–4.8 μm long; fourth cell 4–4.9 μm long; fifth cell 3.9–4.8 μm long); apical cell conical, hyaline, smooth, 2–3 μm long; apical appendage single, centric, short, < 1 μm; basal appendage, single when present, cylindrical, centric, < 1 μm.

Culture characteristics — Colonies on PDA circular, reaching 60–61 mm diam after 14 d at 22 °C, flat at centre and margin, white- to pale luteous-coloured, with aerial mycelium abundant on the surface, sporulating poorly at the margin of the colony and not within 2 wk with pycnidioid conidiomata. On CMA circular, reaching 79–81 mm diam after 14 d at 22 °C, flat at the centre and margin, white-, buff- to hazel-coloured in the centre, hazel-coloured at the inner margin to white at the outer margin, with aerial mycelium formed abundantly on the surface, sporulating poorly, after at least 4 wk with black spore masses produced in sporodochia. On MEA circular, reaching 45–49 mm diam after 14 d at 22 °C, hazel-coloured at the centre surrounded by a grey olivaceous to hazel ring and a white margin, flat at centre and margin, with aerial mycelium abundantly on the surface, sporulating poorly with few sporodochia producing black spore masses. On SNA circular to irregular, reaching 37–39 mm diam after 14 d at 22 °C, flat at centre and margins, with moderate aerial mycelium mostly at the margin, sporulation after approximately 2 wk, sporodochia scattered and immersed in agar.

Known distribution — Africa, Asia, Australia, Europe, New Zealand, North and South America.

Materials examined. New Zealand, Christchurch, 1981, from Cupressocyparis sp., H.J. Boesewinkel (CBS H-18011, culture CBS 523.82 = CPC 23793). – South Africa, East Transvaal, Mac Mac State Forest, 1985, M.J. Wingfield (CBS H-18015, culture CBS 908.85 = CMW 616); Algeria State forest, from Cupressus lusitanica, 1985, M.J. Wingfield (CBS H-18012 reference strain designated here, culture CBS 909.85 = CMW 635). – USA, California, Atherton, from Cupressus macrocarpa, 16 Mar. 1934, W.W. Wagener (isotype of Coryneum cardinale IMI 75045).

Notes — Seiridium cardinale (clade 1) forms a separate cluster in the combined phylogeny with four sister clades including specimens collected from Cupressaceae: S. neocupressi (clade 2), S. cupressi (clade 3), S. unicorne (clade 4) and S. cancrinum (clade 5). Morphologically, S. cardinale is clearly distinct from members of these sister clades by its reduced basal and apical appendages. Since a culture from the correct locality is currently not available, we designated CBS 909.85 as a reference strain, which matches the characteristics of the isotype (IMI 75045). The isotype consists of two slides. One of the slides includes conidia but lacks conidiophores and conidiogenous cells. The second slide is better preserved and contains sections of the fungus in the host, forming acervuli (Fig. 6). By designating a reference strain, we aim to provide a specimen of S. cardinale that contains both a detailed morphological description and is represented by multiple loci in GenBank to promote consistent use in future studies until an appropriate culture is collected from Cupressus macrocarpa in California that can be designated as epitype to supplement the current materials.

Seiridium cupressi (Guba) Boesew., Trans. Brit. Mycol. Soc. 80: 545. 1983 — Fig. 7, ​,88

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Fig. 7

Seiridium cupressi (IMI 52254, lectotype of Cryptostictis cupressi). a. Herbarium specimen; b–c. conidiogenous cells and conidia; d. conidia. — Scale bars: b–d = 10 μm.

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Fig. 8

Rhynchosphaeria cupressi (IMI 40096). a–b. Herbarium specimen; c. ascomata; d. ascoma with asci; e–j. paraphyses and asci. — Scale bars: d–j = 20 μm.

Basionym. Cryptostictis cupressi Guba, Monograph of Monochaetia and Pestalotia: 47. 1961.

Synonyms. Rhynchosphaeria cupressi Nattrass et al., Trans. Brit. Mycol. Soc. 46: 103. 1963.

Lepteutypa cupressi (Nattrass et al.) H.J. Swart, Trans. Brit. Mycol. Soc. 61: 79. 1973.

Conidia on dried PDA cultures from Jones (1953) lunate to falcate, curved, 5-septate, not striate, bearing two appendages, euseptate, (18–)22–29.5(–36) × (5–)6–8.5(–11.5) μm, mean ± SD = 25.8 ± 3.6 × 7.4 ± 1.2 μm (n = 61); basal cell obconic with a truncate base, hyaline, 1.5–6 μm; four median cells, smooth, cylindrical to doliiform, pale to dark brown (second cell from base 3–6.5 μm long; third cell 2.5–5.5 μm long; fourth cell 2.5–6 μm long; fifth cell 3–7.5 μm long); apical cell conical, hyaline, 1.5–7 μm long; appendages cylindrical, attenuated; apical appendage single, centric, 0.5–9.5 μm long, occasionally branched near the tip; basal appendage consistently present, single, cylindrical, excentric, 0.5–15 μm long.

Known distribution — Greece, Kenya, Uganda.

Materials examined. Greece, from Cupressus sp., A. Graniti (CBS 122616 = CMW 1646). – Kenya, non pathogenic isolate from Cupressus sp., collection data unknown (CBS 320.51); on Cupressus macrocarpa, July 1948, R.M. Nattrass (IMI 37158 holotype of Rhynchosphaeria cupressi); on Cupressus macrocarpa, Dec. 1949, R.M. Nattrass (IMI 40096); from cankers in branches of Cupressus macrocarpa, 1953, D.R. Jones (IMI 52254 lectotype of Cryptostictis cupressi designated here (MBT379187), BRL 1117 isolectotype, CBS 224.55 epitype designated here (MBT379208), metabolically inactive culture, culture ex-epitype CBS 224.55); from cankers in branches of Cupressus forbesii, 1949, D.R. Jones (IMI 52255 dried culture = BRL 1118, living culture CBS 225.55).

Notes — Clade 3 of the multi-gene phylogeny (Fig. 1) is in the present study identified as S. cupressi based on morphological comparison of the authentic material listed in the protologue (Guba 1961). These materials include one specimen with the sexual morph on Cupressus macrocarpa, collected in Dec. 1949 in the ‘Uplands’, Kenya. Within the IMI fungarium only IMI 40096, Lepteutypa cupressi, carries exactly this label. The other herbarium specimen Guba (1961) cited is from the same host, collector and date, but from Nairobi instead of the Uplands, Kenya and is without mention of a sexual morph. Therefore, we conclude that IMI 40096 is authentic and the specimen from Nairobi was either not preserved, or represents the same strain (Nairobi and the Uplands are only ~ 30 km apart). In addition, Guba (1961) used dried cultures from cankers on Cupressus and Juniperus, collected by Jones in Kenya and Uganda, including the strains A, B, C and D. The IMI fungarium contains five dried cultures that were deposited by Jones (IMI 52254 from Cupressus macrocarpa, IMI 52255 from C. forbesii, IMI 52256 from C. macrocarpa, IMI 52257 from Juniperus procera and IMI 52258 from C. macrocarpa). Together with IMI 40096, these specimens represent the authentic material of S. cupressi. Cultures of the same strains were also deposited in the CBS collection and the dried materials are thus linked to the living cultures CBS 224.55, CBS 225.55, CBS 226.55, CBS 228.55 and CBS 227.55, respectively. Jones (1953, 1954a, b) studied the pathogenic characteristics of the fungus prior to the formal description of S. cupressi by Guba (1961), and classified the fungi in four different strain categories, based on their aggressiveness. Whereas strain A was the most aggressive pathogen to different Cupressus hosts and strain B a mild pathogen, strain D was only pathogenic on Juniperus and appeared to be saprophytic on Cupressus. Strain C comprised mutated clones that had arisen in culture and was never deposited in the IMI or the CBS (see Jones 1954a). To re-evaluate the species and select a lectotype, we examined the herbarium specimen collected by Nattrass (IMI 40096) as well as the dried cultures from Jones (1953, 1954a), with the exception of the specimen from Uganda (IMI 52258 = CBS 227.55). In addition, all loci used in this study were sequenced from the living cultures, which are linked to the authentic dried cultures in the IMI collection. None of the living cultures were fertile, nor were any other cultures examined in this clade (CBS 320.51 and CBS 122616). Despite thorough examination of the material from IMI 40096 only the sexual-morph was observed (Fig. 8), making it impossible to compare this strain to the other authentic material or the original description itself. Therefore, IMI 40096 could not be used for the re-evaluation of S. cupressi. The same applied to the holotype of Rhynchosphaeria cupressi (Nattrass 1963; IMI 37158), in which also no asexual-morph was found, impeding us to link the sexual-morph to the other examined specimens. Furthermore, phylogenetic analysis revealed that the remaining five specimens (the dried cultures from Jones 1953, 1954a) were distributed over three distinct clades (clades 3, 5 and 7; Fig. 1), thus constituting three different species. Morphological comparison (see Fig. 3) showed the specimens in clade 3 (CBS 224.55 = IMI 52254 and CBS 225.55 = IMI 52255) to be similar to the original description. This clade also includes the Ugandan specimen (CBS 227.55 = IMI 52258) and thus the majority of the authentic material. We therefore designate IMI 52254 as lectotype of Cryptostictis cupressi (basionym of S. cupressi), while CBS 224.55, a strain derived from the same collection is designated as epitype in order to provide a stable platform for DNA data comparisons. Because the work of Jones (1953, 1954a, b) preceded the formal description of Seiridium cupressi, each of the dried cultures carries the label ‘Monochaetia unicornis’, including the here designated lectotype IMI 52254. The lectotype material consists of four parts; two dried slant cultures on CMA and two dried plate cultures on PDA. Similar to IMI 52255 the material is in relatively poor condition, but the features of the conidia are clearly recognizable as S. cupressi (Fig. 7). Cultures derived from the other two specimens, CBS 226.55 (= IMI 52256) and CBS 228.55 (= IMI 52257), clustered in clades 5 (S. cancrinum) and 9 (S. kenyanum), respectively. Furthermore, the pathogenic differences that Jones (1953, 1954a, b) identified between the strains (A, B and D) are congruent with the topology of the multi-gene phylogeny (Fig. 1), indicating the three species (S. cancrinum, S. cupressi and S. kenyanum) have different pathology. Morphologically, IMI 52257 deviates most from the other specimens and the original description by bearing considerably larger conidia. This strain was also the only isolate retrieved from Juniperus sp. Instead of Cupressus sp. Specimen IMI 52256 (S. cancrinum; clade 5) is morphologically more similar to the fungarium specimens of S. cupressi (IMI 52254 and IMI 52255; clade 3) and the original description. However, conidia generally have a longer basal appendage (mean ± SD = 5.8 ± 2.4) compared to IMI 52254 and IMI 52255 (mean ± SD = 2.0 ± 2.0 and 3.9 ± 2.6, respectively). Since the living cultures presently available are sterile, future collections from Kenya are required to add more morphological details of this fungus in culture.

Seiridium eucalypti Nag Raj, Coelomycetous anamorphs with appendage-bearing conidia (Ontario): 862. 1993 — Fig. 9

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Fig. 9

Seiridium eucalypti (CBS 343.97, culture ex-epitype). a–d. Colony morphology in 90 mm dishes after 2 wk at 22 °C on PDA, CMA, MEA, SNA, respectively; e–g. sporodochia on SNA; h. conidia; i–j. conidiophores; k. conidiophore; l. conidiogenous cell. — Scale bars: e–g = 100 μm; h–l = 10 μm.

Conidiomata on PDA sterile. On SNA erumpent sporodochia, mostly globose, solitary and compact, producing large black spore masses, sporodochia immersed in agar more aggregated and amorphous. Conidiophores relatively long, varying in length, scattered, septate, cylindrical, irregularly branched, hyaline, smooth- and thin-walled, occasionally reduced to conidiogenous cells, 13–85 μm long. Conidiogenous cells discrete, hyaline, subcylindrical to cylindrical, smooth- and thin-walled, 5–18 × 1.5–3.5 μm, percurrent proliferation rarely observed, with minute periclinal thickenings. Conidia lunate to falcate, straight to curved, 5-septate, not striate, bearing two appendages, euseptate with visible pores, (23.5–)27.5–33(–36) × (8–)9–10.5(–11) μm, mean ± SD = 30.3 ± 2.7 × 9.6 ± 0.7 μm (n = 32); basal cell obconic with a truncate base, hyaline, walls smooth, 4–6 μm; four median cells, smooth, cylindrical to doliiform, brown to dark brown, and septa darker than the rest of the cells (second cell from base 4.5–8 μm long; third cell 4–6.5 μm long; fourth cell 3.5–6 μm long; fifth cell 4.5–7 μm long); apical cell conical, hyaline, smooth, 2.5–6 μm long; apical appendage single, centric, 8.5–15.5 μm long; basal appendage, single, cylindrical, centric and excentric, 6.5–10 μm long.

Culture characteristics — Colonies on PDA irregular, reaching 15–20 mm diam after 14 d at 22 °C, flat at centre, white to pale luteous-coloured at the centre, slightly raised at the margin, white-coloured, with abundant aerial mycelium, not sporulating. On CMA almost circular, reaching 37–42 mm diam after 14 d at 22 °C, flat at centre and margin, white-coloured, with abundant aerial mycelium, sporulating poorly and not within 4 wk, pycnidioid sporodochia producing black spore masses. On MEA irregular, reaching 21–25 mm diam after 14 d at 22 °C, flat at centre and margin, white-coloured, with abundant aerial mycelium, no sporulation. On SNA circular, reaching 33–35 mm diam after 14 d at 22 °C, flat at centre and margin, white-coloured, abundant aerial mycelium, sporulation after 2 wk with scattered compact sporodochia.

Known distribution — Continental Australia, Tasmania.

Materials examined. Australia, South Australia, Adelaide, Mt Lofty Summit, on leaves of Eucalyptus sp., 16 Oct. 1979, B. Kendrick (holotype DAOM 215255); Tasmania, on Eucalyptus delegatensis, 13 Sept. 1996, Z.Q. Yuan (epitype designated here CBS H-23145 (MBT379188), ex-epitype culture CBS 343.97).

Notes — Seiridium eucalypti (clade 10 in Fig. 1) forms a separate clade in the combined phylogeny, sister to S. pseudocardinale and S. kenyanum (clades 8 and 9, respectively). Morphologically, S. eucalypti is characterised by conidia with distinctively long appendages, in particular the apical appendage (Fig. 3). Other Seiridium spp. isolated from Eucalyptus spp. (including S. kartense sp. nov. and S. papillatum) are not monophyletic in the combined phylogeny, nor in any of the single-locus phylogenies (Fig. 2). Seiridium eucalypti is known to inflict lesions on a wide range of Eucalyptus spp. Its pathogenicity to members of Eucalyptus was studied in detail by Yuan & Old (1995) and Yuan & Mohammed (1997, 1999, 2001). The strain examined in this work (CBS 343.97) matches the protologue of S. eucalypti (Nag Raj 1993), and is therefore designated as epitype.

Seiridium kartense Bonthond, Sandoval-Denis & Crous, sp. nov. — MycoBank MB823297; Fig. 10

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Fig. 10

Seiridium kartense sp. nov. (CBS 142629, culture ex-holotype). a–d. Colony morphology in 90 mm dishes after 2 wk at 22 °C on PDA, CMA, MEA, SNA, respectively; e. sporodochia on SNA immersed in agar; f. sporodochia on SNA erumpent from agar; g. conidia; h–i. conidiophores; j. polyblastic conidiogenesis; k. conidiogenous cell proliferating percurrently showing multiple collarettes. — Scale bars: e–f = 100 μm; g–k = 10 μm.

Etymology. Named after the location where the fungus was collected which is known as ‘Kangaroo Island’ and by aboriginals from the mainland of Australia as Karta, meaning ‘Island of the dead’.

Conidiomata on SNA sporodochial, globose to amorphous, solitary, immersed, 160–300 μm diam. Conidiophores short and compact, septate, cylindrical, irregularly branched, hyaline, smooth- and thin-walled, 27–44 μm long. Conidiogenous cells discrete, hyaline, cylindrical to globose, smooth- and thin-walled, 5.4–11.5 × 2.1–3.5 μm, proliferating multiple times percurrently, with minute periclinal thickenings and collarets visible, occasionally polyblastic. Conidia lunate to falcate, curved, 5-septate, not striate, bearing two appendages, euseptate with visible pores, (23.5–)27.5–30.5(–32) × (6.5–)7.5–8.5(–9) μm, mean ± SD = 28.5 ± 1.8 × 8.0 ± 0.4 μm (n = 38); basal cell obconic with a truncate base, hyaline, walls smooth, 4–5.5 μm; four median cells, smooth, cylindrical to doliiform, dark brown (second cell from base 5.5–6.5 μm long; third cell 5–5.5 μm long; fourth cell 4.5–5 μm long; fifth cell 4.5–5.5 μm long); apical cell conical, hyaline, smooth, 3–4 μm long; appendages cylindrical, attenuated, unbranched, slightly spathulate; apical appendage single, centric, 6.5–8.5 μm long; basal appendage consistently present, single, cylindrical, excentric, 6–8.5 μm long.

Culture characteristics — Colonies on PDA circular, reaching 56–59 mm diam after 14 d at 22 °C, flat, from centre to margin; salmon- to umber- to white-coloured, with moderate aerial mycelium, no sporulation. On CMA circular, reaching 65–68 mm diam after 14 d at 22 °C, flat to slightly crateriform, at the centre salmon-coloured, at the margin white-coloured, with abundant aerial mycelium, no sporulation. On MEA circular, reaching 42–44 mm diam after 14 d at 22 °C, raised, ochreous- and honey-coloured at the centre and white-coloured at the margin, with dense aerial mycelium, no sporulation. On SNA circular, reaching 51–54 mm diam after 14 d at 22 °C, flat, white-coloured, with moderate areal mycelium more densely produced at the margin, sporulation after 2 wk with scattered compact erumpent sporodochia in the centre.

Known distribution — Kangaroo Island, Australia.

Material examined. Australia, Kangaroo Island, on leaves of Eucalyptus cladocalyx, 15 Dec. 2012, W. Quaedvlieg (holotype CBS H-23146, ex-type culture CBS 142629 = CPC 20183).

Notes — Seiridium kartense forms a monotypic clade (clade 11 in Fig. 1), and was collected from Kangaroo Island (Australia) on Eucalyptus cladocalyx. Seiridium eucalypti (Nag Raj 1993; clade 10 in Fig. 1) and S. papillatum (Yuan & Mohammed 1997; clade 17 in Fig. 1) have both also been collected from Australia on Eucalyptus spp. Morphologically, S. kartense differs from S. eucalypti based on its conidial length and length of its apical appendage (Fig. 2), which are in both cases shorter than those of S. eucalypti. Seiridium papillatum (see Yuan & Mohammed 1997) is different from S. kartense by its shorter appendages.

Seiridium kenyanum Bonthond, Sandoval-Denis & Crous, sp. nov. — MycoBank MB823301; Fig. 11

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Fig. 11

Seiridium kenyanum (IMI 52257, holotype). a. Herbarium specimen; b–c. conidia. — Scale bars: b–c = 10 μm.

Etymology. Named after the country where it was isolated, Kenya.

Conidia lunate to falcate, curved, 5-septate, not striate, bearing two appendages, euseptate with no visible pores, (20–)22–26(–30.5) × (7–)7.5–9(–10) μm, mean ± SD = 24.0 ± 2.1 × 8.2 ± 0.7 μm (n = 33); basal cell obconic with truncate base, hyaline, walls smooth, bearing marginal frills, 1–6 μm; four median cells, varying in colour ranging from pale to dark brown, smooth, cylindrical to doliiform (second cell from base 3–5.5 μm long; third cell 3–5 μm long; fourth cell 3.5–5.5 μm long; fifth cell 3.5–6.5 μm long); apical cell conical, hyaline, smooth, 2–4.5 μm long; apical appendage single, centric, 0.5–13 μm; basal appendage, single, cylindrical, centric and excentric, 0.5–14.5 μm.

Materials examined. Kenya, from cankers in branches of Juniperus procera, 1951, D.R. Jones (holotype IMI 52257, culture ex-type CBS 228.55); BRL 1121 isotype.

Notes — Phylogenetic analysis and morphological examination of IMI 52257 (= CBS 228.55) revealed this strain to be different from S. cupressi (see notes on S. cupressi). Jones (1953, 1954a, b) classified this fungus as Monochaetia unicornis strain D and already determined that it was ecologically different from Seiridium cupressi and S. cancrinum (Monochaetia unicornis strain B and M. unicornis strain A, respectively) by being non-pathogenic to Cupressus spp. The source of this fungus was Juniperus procera, on which it was a pathogen and induced cankers. Furthermore, its conidia deviate from S. cupressi and S. cancrinum by being considerably larger.

The fungus appears to be genetically highly similar to the sterile cultures CBS 122613 and CBS 122614, as well as to the type of S. pseudocardinale. However, the conidia bear long appendages, and are morphologically clearly different from the latter taxon, as this fungus is characterised by lacking or having reduced appendages (Wijayawardene et al. 2016). Since only an ITS sequence is available for S. pseudocardinale, it is likely that the short distance in the phylogenetic tree between the two species is a result of the limited ability of the ITS rDNA region to delineate species within Seiridium (Fig. 2a but see also Viljoen et al. 1993, Barnes et al. 2001, Maharachchikumbura et al. 2015).

Seiridium neocupressi Bonthond, Sandoval-Denis & Crous, sp. nov. — MycoBank MB823299; Fig. 12

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Fig. 12

Seiridium neocupressi sp. nov. (CBS 142625, culture ex-holotype). a–d. Colony morphology in 90 mm dishes after 2 wk at 22 °C on PDA, CMA, MEA, SNA, respectively; e. conidioma on PDA; f–g. sporodochia on SNA immersed in agar; h. conidia; i. conidiophores and paraphyses-like structures; j. microconidia (spermatia) on PDA; k–l. conidiophores. — Scale bars: e–g = 100 μm; h–l = 10 μm.

Etymology. neo- cupressi denotes the fact that this phylogenetic clade has been repeatedly incorrectly assumed to represent S. cupressi.

Conidiomata on PDA pycnidioid sporodochial, globose to amorphous, in some strains exuding hyaline mucus, solitary, immersed. On SNA sporodochial, amorphous, immersed in agar. Conidiophores compact, septate, cylindrical, irregularly branched, hyaline, thin-walled, smooth, 13–160 μm long, in some strains conidiophores are intermingled with unbranched, hyaline, paraphyses-like hyphae, up to 115 μm long. Conidiogenous cells discrete, hyaline, cylindrical to globose, sometimes elongated, smooth- and thin-walled, (4–)3.5–24(–65) × (1.5–)2–3(–4) μm, with numerous percurrent proliferations. Conidia lunate to falcate, curved, 5-septate, not striate, bearing two appendages, euseptate without visible pores, (21–)26.5–31(–35.5) × (7–)8.5–10(–10.5) μm, mean ± SD = 28.7 ± 2.3 × 9.2 ± 0.6 μm (n = 94); basal cell obconic with a truncate base, hyaline, 2.5–5 μm; four median cells, smooth, cylindrical to doliiform, pale to brown (second cell from base 2.5–7 μm long; third cell 4–6.5 μm long; fourth cell 4–6.5 μm long; fifth cell 3.5–6.5 μm long); apical cell conical, hyaline, 2.5–5 μm long; appendages cylindrical, attenuated; apical appendage single, centric, 6.5–12 μm long, occasionally branched near the tip; basal appendage consistently present, single, cylindrical, centric and excentric, 3.5–10.5 μm long. Microconidia or spermatia, when produced only on CMA and PDA, cylindrical, hyaline, on PDA (14.5–)18.5–24(–26) μm long.

Culture characteristics — Colonies on PDA circular to irregular, reaching 43–55 mm diam after 14 d at 22 °C, flat at the centre and flat to elevated at the margin, white to pale luteous, sometimes with a white outer ring, with abundant aerial mycelium, sporulating poorly and not within 2 wk with black, scattered pycnidioid conidiomata. On CMA circular, reaching 51–68 mm diam after 14 d at 22 °C, flat at centre and margin to slightly crateriform, buff to saffron with hazel coloured patches in the centre, buff to white coloured at the margin, with moderate aerial mycelium, sporulating after at least 4 wk with black, pycnidioid conidiomata. On MEA irregular, reaching 19–30 mm diam after 14 d at 22 °C, elevated at the centre, flat at the margin, buff to pale green, with aerial mycelium on the surface, no sporulation. On SNA circular to rhizoid, reaching 19–49 mm diam after 14 d at 22 °C, flat, with moderate aerial mycelium, sporulation after a few days with black sporodochial conidiomata.

Known distribution — Australia, Italy.

Materials examined. Australia, Victoria, Torquay, from Cupressus leylandi, 18 Dec. 2006, A. Hoffert (CBS H-23149, culture CBS 142627 = CPC 28351 = VPRI 40665). – Italy, Bari, from Cupressus sempervirens, 23 Nov. 2007, unknown collector (CBS H-23148, culture CBS 142626 = CPC 23789); Bari, from Cupressus sempervirens, 23 Nov. 2007, unknown collector (holotype CBS H-23147, ex-type culture CBS 142625 = CPC 23786).

Notes — The morphology of S. neocupressi bears similarity to the original description of S. cupressi and to the authentic strains that Jones (1953, 1954a) designated as ‘strain B’. Strains CMW 420, CMW 5282, VPRI 15696, VPRI 16083, VPRI 32740, VPRI 40658 and VPRI 40665 were for this reason originally identified as S. cupressi (Barnes et al. 2001, Cunnington 2007, Tsopelas et al. 2007). However, although in the combined phylogenetic analysis the strains A, B and D from which S. cupressi was described clustered in genetically different clades (3, 5 and 9 in Fig. 1), none clustered within clade 2, and thus this clade is distinct from isolates representing S. cupressi. Morphologically, conidia are slightly longer and wider and bear longer basal appendages in comparison to S. cancrinum and S. cupressi.

Seiridium phylicae Crous & M.J. Wingf., Persoonia 29: 187. 2012

Description and illustration — Crous et al. (2012).

Known distribution — UK, Overseas territory of Saint Helena, Ascension, Tristan da Cunha islands.

Material examined. uk, Saint Helena, Ascension and Tristan da Cunha, Inaccessible Island, Blenden Hall, from stems of Phylica arborea, Sept. 2011, P.G. Ryan (holotype CBS H-21089, ex-type cultures CBS 133587 = CPC 19964, CPC 19962, CPC 19965, CPC 19970).

Notes — Seiridium phylicae was described and treated in detail in Crous et al. (2012). Similar to S. spyridicola, this fungus was isolated from a plant host in the Rhamnaceae. Given the sole isolation of this fungus from the Tristan da Cunha archipelago, it seems to be geographically isolated. The species is a problematic pathogen to the Tristan da Cunha endemic Phyilica arborea (Ryan et al. 2014).

Seiridium pseudocardinale Wijayaw. et al., Fung. Diversity 77: 248. 2016

Description and illustration — Wijayawardene et al. (2016).

Known distribution — Italy, Portugal.

Materials examined. Portugal, from Cupressus sp, collection date unknown, A. Graniti (CBS 122613 = CMW 1648); from Cupressus sp., collection date unknown, A. Graniti (CBS 122614 = CMW 1649).

Notes — Seiridium pseudocardinale was recently described by Wijayawardene et al. (2016). In GenBank, only an ITS sequence is available for the type specimen (MFLUCC 13-0525) and thus in the combined phylogeny (Fig. 1) the other loci are missing data for this species. Given the limited resolution on the ITS region alone (Fig. 2a), its phylogenetic placement should thus be interpreted with care. This fungus was isolated from Cupressus glabra in Italy and based on ITS sequence seems to be closely related to cultures CBS 122612 and CBS 122613, which were isolated from Cupressus species in Portugal, and CBS 228.55 (= IMI 52257; S. kenyanum sp. nov.). However, morphologically, the fungus is different from IMI 52257 by the lack of conidial appendages and the poor delineation from CBS 228.55 is likely a result of the absence of informative loci. Since none of the two cultures from Portugal sporulated, we were unable to investigate this clade morphologically and compare it with the description in the protologue. To resolve the phylogenetic relation of S. pseudocardinale to S. kenyanum and the sterile cultures CBS 122613 and CBS 122614, sequence data of at least TUB, but preferably RPB2 and TEF are required from the type (MFLUCC 13-0525).

Seiridium spyridicola Bonthond, Sandoval-Denis & Crous, sp. nov. — MycoBank MB823300; Fig. 13

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Fig. 13

Seiridium spyridicola sp. nov. (CBS 142628, culture ex-holotype). a–d. Colony morphology in 90 mm dishes after 2 wk at 22 °C on PDA, CMA, MEA, SNA, respectively; e–f. sporodochia on SNA immersed in agar; g. conidia; h–j. conidiophores; k. conidiogenous cell. — Scale bars: e–f = 100 μm; g–k= 10 μm.

Etymology. Named after the host genus, Spyridium, from which it was isolated.

Conidiomata on PDA sporodochial, solitary or aggregated, immersed in agar, dark brown to black. Conidiophores septate, cylindrical, irregularly branched, hyaline, smooth- and thin-walled, 9–52 μm long. Conidiogenous cells discrete, hyaline, subcylindrical to globose, smooth- and thin-walled, 5.5–12 × 2–4 μm, proliferating multiple times percurrently, with minute periclinal thickenings. Conidia straight to falcate, straight to slightly curved, 5-septate, sporadically 6-septate, not striate, bearing two appendages, euseptate, pores visible, (22–)24–28.5(–33.5) × (4.5–)8–10.5(–11) μm, mean ± SD = 26.4 ± 2.3 × 9.1 ± 1.2 μm (n = 31); basal cell obconic with a truncate base, hyaline, walls smooth, bearing minute marginal frills, 3–4.5 μm long; four median cells, smooth, doliiform, brown to dark brown, septa darker than the rest of the cells (second cell from base 5–6 μm long; third cell 4.5–5.5 μm long; fourth cell 4.5–5.5 μm long; fifth cell 4.5–5.5 μm long); apical cell conical, hyaline, smooth, 2.5–4 μm long; apical appendage single, excentric and typically oriented perpendicular to conidium, cylindrical, often spatulate, 4–6.5 μm; basal appendage single, cylindrical, often spatulate, mostly centric, 3.5–5.5 μm.

Culture characteristics — Colonies on PDA irregular, reaching 12–16 mm diam after 14 d at 22 °C, flat, from centre to margin, white-coloured, with dense aerial mycelium, sterile. On CMA circular, reaching 46–50 mm diam after 14 d at 22 °C, flat to slightly umbonate, white-coloured, with abundant aerial mycelium on the surface in the centre, mycelium at the margin immersed in agar, sterile. On MEA circular to irregular, reaching 27–29 mm diam after 14 d at 22 °C, flat, white-coloured, with dense aerial mycelium, sterile. On SNA circular, reaching 22–27 mm diam after 14 d at 22 °C, slightly umbonate, cinnamon-coloured at the centre, cinnamon- to white-coloured at the margin, with moderate areal mycelium, sporulation within 2 wk with scattered sporodochia around the centre.

Known distribution — Australia.

Material examined. Australia, Western Australia, from Spyridium globosum, 19 Sept. 2015, P.W. Crous (holotype CBS H-23150, ex-type culture CBS 142628 = CPC 29108).

Notes — This species was isolated from Spyridium globosum (Rhamnaceae) and in the combined phylogeny forms a basal lineage to clades 1 to 6, including most of the Cupressaceae pathogens as well as S. phylicae (Crous et al. 2012), which was isolated from Phylica arborea, a host also belonging to the Rhamnaceae. Conidia are, in comparison to the other studied Seiridium spp. of average length, but the appendages are notably shorter and the apical appendage is typically oriented perpendicular to the long axis of the conidium.

Seiridium unicorne (Cooke & Ellis) B. Sutton, Mycol. Pap. 138: 74. 1975 — Fig. 14, ​,1515

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Fig. 14

Seiridium unicorne (CBS 538.82, reference strain). a–d. Colony morphology in 90 mm dishes after 2 wk at 22 °C on PDA, CMA, MEA, SNA, respectively; e. conidioma on PDA erumpent from agar partially covered with mycelium; f–g. sporodochia on SNA erumpent from agar; h. conidia; i–j. conidiophores produced from sporodochia; k. conidiophore; l. conidiogenous cells — Scale bars: e–g = 100 μm; h–l = 10 μm.

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Fig. 15

Seiridium unicorne (IMI 5816, holotype of Pestalozzia unicornis). a. Herbarium specimen; b. conidiomata and conidia in vivo; c–f. conidiogenous cells and conidia in vitro. — Scale bars: b = 20 μm; c–f = 10 μm.

Basionym. Pestalotia unicornis Cooke & Ellis, Grevillea 7: 6. 1878.

Synonym. Monochaetia unicornis (Cooke & Ellis) Sacc. & D. Sacc., Syll. Fung. 18: 485. 1906.

Conidiomata on PDA pycnidial to sporodochial, globose or clavate, mostly solitary, erumpent from agar, partially immersed in mycelium, producing large black spore masses. On SNA, sporodochial, mostly aggregated, erumpent from agar, producing large black spore masses. Conidiophores septate, cylindrical, relatively long and irregularly branched, brown or colourless, thin-walled, 29–68 μm long. Conidiogenous cells discrete, hyaline, cylindrical, smooth- and thin-walled, 3.5–29.5 × 1.5–3 μm, proliferating percurrently, with visible collarettes and minute periclinal thickenings. Conidia lunate to falcate, curved, 5-septate, rarely 4- or 6-septate, not striate, bearing two appendages, euseptate with no visible pores, (21.5–)23.5–26.5(–27.5) × (7–)8–9(–9.5) μm, mean ± SD = 24.9 ± 1.6 × 8.5 ± 0.6 μm (n = 31); basal cell obconic with a truncate base, hyaline, walls smooth, bearing minute marginal frills, 3–5.5 μm; four median cells, varying in colour from pale to dark brown, smooth, cylindrical to doliiform (second cell from base 3.5–5.5 μm long; third cell 3.5–5 μm long; fourth cell 4–5.5 μm long; fifth cell 4–5.5 μm long); apical cell conical, hyaline, smooth, 2–5 μm long; apical appendage single, mostly centric, 5–10 μm; basal appendage, single, cylindrical, mostly excentric, 4–6 μm long.

Culture characteristics — Colonies on PDA circular, reaching 65–68 mm diam after 14 d at 22 °C, slightly umbonate, citrine to olivaceous coloured, with aerial mycelium abundant on the surface, sporulating in between centre and margin of the colony within 2 wk. On CMA circular, reaching 58–59 mm diam after 14 d at 22 °C, flat at the centre and margin, citrine to olivaceous coloured, with moderate aerial mycelium on the surface, sporulating in the centre within 2 wk. On MEA irregular, reaching 35–40 mm diam after 14 d at 22 °C, flat to crateriform, slightly sunk into the agar, malachite green coloured at the centre becoming white at the margin with saffron spots or areas in between centre and margin, with dense mycelium on the surface, not sporulating. On SNA circular, reaching 20–21 mm diam after 14 d at 22 °C, umbonate, with aerial mycelium at the surface, sporulation within 2 wk.

Known distribution — New Zealand, South Africa, USA.

Materials examined. New Zealand, from Cryptomeria japonica, 1981, H.J. Boesewinkel (CBS H-23151 reference strain designated here, culture CBS 538.82 = CPC 23783 = IFO 32684). – South Africa, from Cupressus sempervirens, 1999, I. Barnes (culture CBS 120306 = CMW 5596). – USA, New Jersey, associated with Chamaecyparis thyoides, 1878, J.B. Ellis (IMI 5816 holotype of Pestalotia unicornis).

Notes — Culture CBS 120306 remained sterile on all media tested. The holotype material of S. unicorne (IMI 5816) consists of two slides. One of the slides includes conidia, but conidiophores and conidiogenous cells are not discernible, while the second slide contains sections of the fungus in host tissue (Fig. 15). Conidia were produced by CBS 538.82 which were morphologically, apart from slightly longer basal appendages (mean ± SD = 5.35 ± 1.14 opposed to 2.75 ± 2.10), highly similar to the examined holotype material from P. unicornis (see Fig. 3). Despite the highly similar morphological characters, both host and geographic origin of CBS 538.82 (Cryptomeria japonica and South Africa) are different from the holotype which was collected from Cupressus sempervirens in New Jersey. We therefore designate CBS 538.82 as reference strain of S. unicorne, providing a new description of the species and linking DNA sequence data to the morphological description. Pestalozzia unicornis (Cooke & Ellis 1878) was placed in Seiridium by Sutton (1980) and the typically broad host range has traditionally been regarded as an important character of the species. As described by Guba (1961), S. unicorne was found on the Cupressaceae genera Chamaecyparis, Cupressus and Juniperus, but also on Anacardiaceae (Rhus), Caprifoliaceae (Lonicera), Hamamelidaceae (Hamamelis), Rosaceae (Crataegus, Malus, Rosa) and Vitaceae (Vitis). Given that these observations preceded the use of DNA sequence data, it cannot be ruled out that these observations included additional cryptic species.