Molecular phylogeny

Phylogenetic relationships of chalara-like fungi in Leotiomycetes were partly resolved based on the analyses of three different datasets (2 for LSU + SSU, 1 for LSU + ITS). The phylogenetic trees were generated by BI and ML analyses and were largely congruent. All phylogenetic analyses with inclusion of a broad diversity of chalara-like fungi in this study clearly demonstrated that the chalara-like fungi in Leotiomycetes were paraphyletic and scattered in more than 4 different families of Leotiomycetes.

LSU and SSU dataset (I)

For the first dataset with only a few representative species under each family of Leotiomycetes, the combined nuclear LSU and SSU sequences were analyzed to determine the phylogenetic relationship of the chalara-like fungi within the Leotiomycetes. The final alignment included 95 strains representing 82 species in 44 genera and 11 families of the Leotiomycetes. which consisted of 1897 characters including gaps (deposited in TreeBASE 29633). The ML tree is shown in Fig. 1. In ML and BI analyses, 52 species of chalara-like fungi scattered on 16 distinct lineages that represented genera or natural groups of species; furthermore, these species clustered in 7 distinct clades. The first clade (Pezizellaceae) includes 13 species of Chalara s. lat. with septate conidia and +/− setae (named as Nagrajchalara in this study), Bloxamia cyatheicola, Zymochalara cyatheae, Z. lygodii and one unnamed chalara-like fungus, all with aseptate conidia and no setae. The second clade included two strains identified as Chalara fusidioides (the type species of Chalara s. lat.) and Xenochalara juniperi, characterized by aseptate conidia and no setae. The third clade (Hamatocanthoscyphaceae) included Chalara austriaca, C. constricta, Infundichalara microchona, and an undescribed chalara-like species, all characterized by aseptate conidia and absence of setae. The fourth clade (Pezizellaceae) consisted of two strongly supported subgroups, one with Bloxamia elongata, B. truncata, and several Chalara s. lat. species (C. africana, Chalara crassipes, C. eucalypticola, C. fungorum, C. parvispora and C. riisgaardii), all characterized by aseptate conidia and +/− setae; the other one consisted of Chalara aurea, C. hughesii, C. kendrickii and an unnamed chalara-like fungus, all characterized by septate conidia and +/− setae. The fifth clade included all known genera of Neolauriomycetaceae (Exochalara, Lareunionomyces, Neolauriomyces, and an undescribed chalara-like species). The sixth clade (Helotiaceae) consisted of four specie of Hymenoscyphus and three of them, H. albidus, H. albidoides and H. globus were with chalara-like anamorphs. The seventh clade consisted of two Chalara s. lat. species, C. hyalina and C. schoenoplecti, characterized by absence of setae, hyaline to subhyaline conidiophores and aseptate conidia without basal frills. These two species did not belong to Leotiomycetes and should be correctly placed in Pyxidiophoraceae under Pyxidiophorales.

Fig. 1

Maximum likelihood (ML) tree based on 18S rDNA and 28S rDNA sequence data for the chalara-like anamorphic fungi in Leotiomycetes. Bootstrap support values ≥ 60%, Bayesian posterior probability values ≥ 0.80 are shown at the nodes. Fuscocatenula variegata Wu12520 was chosen as the outgroup. Highlighted generic names are members of chalara-like fungi

LSU and SSU dataset (II)

Analyses of the second dataset of combined nuclear SSU and LSU sequences including more species and genera in Leotiomycetes obtained similar tree topologies as the first dataset. The final alignment included 159 strains representing 118 species in 47 genera and 11 families of Leotiomycetes, which consisted of 1901 characters including gaps (deposited in TreeBASE 29633). The ML tree is shown in Fig. 2. The overall results were very similar to those from the first dataset (Fig. 1). The 86 species of chalara-like fungi of Leotiomycetes were grouped on 16 distinct lineages that represent genera or natural groups of species in seven distinct clades with strong support. The first clade (92 bs/0.95 pp) (Pezizellaceae) consisted of 40 species of Chalara s. lat. with septate conidia and +/− setae, Bloxamia cyatheicola, Zymochalara cyatheae, Z. lygodii and an unnamed chalara-like fungus, all with aseptate conidia and absence of setae. The second clade (84 bs/– pp) (Hamatocanthoscyphaceae) included Chalara austriaca, C. constricta, C. ellisii, C. holubovae, C. hyalocuspica, C. recta, C. longipes, Infundichalara microchona, and an undescribed chalara-like species, all characterized by absence of setae and aseptate conidia without basal frill. The third clade (96 bs/– pp) (Pezizellaceae) consisted of Bloxamia elongata, B. truncata, Calycina populina, C. vulgaris, eleven Chalara s. lat. species (Chalara affinis, C. africana, C. brevipes, C. clidemiae, C. crassipes, C. eucalypticola, C. fungorum, C. parvispora, C. pseudoaffinis, C. qiandaohuensis, C. riisgaardii), and 5 undescribed chalara-like species, all characterized by aseptate conidia and +/− setae; the fourth clade (96 bs/0.98 pp) was with Chalara aurea, C. hughesii, and C. kendrickii and one undescribed species, characterized by uniseptate conidia and +/− setae. The fifth clade (89 bs/0.98 pp) (Helotiaceae) consisted of six specie of Hymenoscyphus and four of them, H. albidus, H. albidoides, H. globus, H. koreanus were with chalara-like anamorphs, characterized by reduced conidiophores, and aseptate, short-cylindrical or globose conidia. The sixth clade (90 bs/– pp) was with a strain of Chalara fusidioides among other genera. The seventh clade (97 bs/– pp) included all known genera of Neolauriomycetaceae (Exochalara, Lareunionomyces, Minichalara, Neolauriomyces). Finally, C. vaccinii formed an independent lineage, which was not related to Leotiomycetes and should be correctly placed into Lasiosphaeriaceae (Sordariales). The two Chalara s. lat. species C. hyalina and H. schoenoplecti, with hyaline conidiophores and conidiogenous cells and members of Pyxidiophoraceae, were not included in the analysis due to their far distance from other species.

Fig. 2

Maximum likelihood (ML) tree based on 18S rDNA and 28S rDNA sequence data for the chalara-like anamorphic fungi in Leotiomycetes. Bootstrap support values ≥ 60%, Bayesian posterior probability values ≥ 0.80 are shown at the nodes. Fuscocatenula variegata Wu12520 was chosen as the outgroup. Species names given in bold are the new sequences generated in this study. Species names given in bold and marked in blue color are the new sequences generated from the ex-type material in this study. Ex-type strains are indicated with “T” in the end of the taxa labels

LSU and ITS dataset

For the third dataset with inclusion of broad diversity of species and genera in Leotiomycetes, the combined nuclear 28S and ITS sequences were analyzed, and the ML tree was shown in Fig. 3. The final alignment included the sequences of 265 strains of 169 species in Leotiomycetes, which consisted of 1591 characters including gaps (deposited in TreeBASE 29633). The result was very similar to those from the first two datasets (Figs. 1, 2). This further confirmed that the chalara-like fungi within Leotiomycetes affinity were paraphyletic. The 101 species of chalara-like fungi in Leotiomycetes were grouped on 19 distinct lineages that represented genera or natural groups of species, scattered in 4 distinct clades that represented different families of Leotiomycetes.

Fig. 3

Maximum likelihood (ML) tree based on 28S rDNA and ITS sequence data for the chalara-like anamorphic fungi in Leotiomycetes. Bootstrap support values ≥ 60%, Bayesian posterior probability values ≥ 0.80 are shown at the nodes. Neotainosphaeria microsperma 44779 and Parabahusutrabeeja minima 55337 were chosen as the outgroup. Species names given in bold are the new sequences generated in this study. Species names given in bold and marked in blue color are the new sequences generated from the ex-type material in this study. Ex-type strains are indicated with “T” in the end of the taxa labels

Clade I (87 bs/0.91 pp): consisted of members of Pezizellaceae, and within this clade six strongly supported subclades formed. The first subclade (Subclade I, 97 bs/– pp) consisted of 49 species of Chalara s. lat., all (except for C. strobilina) with septate conidia and +/– setae, named as Nagrajchalara in this work, and Calycellina leucella; the second subclade (Subclade II, 99 bs/1 pp) consisted of Calycellina fagina, Mollisina uncinata, Phialina lachnobrachyoides and P. ulmariae, and among them only M. uncinata was known to produce chalara-like anamorph with solitary conidiophores and aseptate conidia; the third subclade (Subclade III, 94 bs/0.99 pp) consisted of Bloxamia cyatheicola, Zymochalara cyatheae, Z. lygodii and one new chalara-like fungus named as Parachalara olekirkii in this study, characterized by solitary conidiophores or sporodochial conidiomata, and aseptate conidia; the fourth subclade (Subclade IV, 99 bs/1 pp) consisted of 3 species of Bloxamia, 11 species of Calycina (including the type species C. herbarum, and C. alstrupii, C. claroflava, C. cortegadensis, C. discreta, C. lactea, C. languida, C. marina, C. populina, C. sulfurina, C. vulgaris), 9 species of Chalara s. str. with aseptate species (except for C. eucalypticola with 0–1-septate conidia), and Mollisina rubi; The fifth subclade (Subclade V, 93 bs/1 pp) consisted of 8 species of Chalara s. str., all with aseptate conidia and +/– setae; the sixth subclade (Subclade VI, 100 bs/1 pp) consisting of two species of Calycina, C. citrina and C. shangrillana, no chalara-like anamorph were reported for them; the seventh subclade (Subclade VII, 97 bs/0.98 pp) consisted of 6 species of Chalara s. lat., all with septate conidia and +/– setae, named as Cylindrocephalum in this study. In addition, Brachyalara straminea (the type species), Neochalara spiraeae (the type species) and Remieria rhododendricola scattered among these subclades.

Clade II (100 bs/0.87 pp): included all known genera of Neolauriomycetaceae (Exochalara, Lareunionomyces, Neolauriomyces) and the new genus Minichalara. Roseodiscus sinicus (only ITS sequence was available) also clustered into this clade, but this needs to be further validated in future study.

Clade III (99 bs/1 pp): consisted of 7 species of Hymenoscyphus in Helotiaceae and five of them, H. albidus, H. albidoides, H. fraxineus, H. globus, H. koreanus were reported to produce chalara-like anamorphs.

Clade IV (100 bs/– pp): included members of 8 species of Chalara s. lat. (C. constricta, C. ellisii, C. holubovae, C. hyalocuspica, C. longipes, C. recta, C. piceae-abietis, and one new species), all with aseptate conidia and absence of setae, and named under Constrictochalara, Cylindrochalara in this study, 3 species Hamatocanthoscypha (the type species H. laricionis, H. podocarpi and H. straminella), Infundichalara microchona (the type species) 2 species of Microscypha (M. ellisii and one unnamed species), and Xenochalara juniperi (the type species) in Hamatocanthoscyphaceae. The chalara-like anamorphs in this family are characterized by absence of setae, and aseptate and narrow conidia without basal frills.

Phylogenetic placement of chalara-like fungi in Leotiomycetes

Based on the above phylogenetic analyses, it can be concluded that the chalara-like fungi within Leotiomycetes are paraphyletic, and the analyzed species belong to 20 genera in five families of Leotiomycetes. The other six chalara-like genera (Ascoconidium, Chalarodendron, Bioscypha, Didonia, Phaeoscypha and Tapesina) were not included in the analysis due to lacking living strain or DNA sequence, and they were listed under Pezizellaceae or Leotiomycetes genera incertae sedis (Baral 2002; Ekanayaka et al. 2019; Johnston et al. 2019). In addition, three existing species, Chalara hyalina, C. schoenoplecti and C. vaccinii should be excluded from Leotiomycetes and reclassified.

Arachnopezizaceae: includes Leochalara, a newly created genus for a Chalara-like fungus with hyaline conidiophores and aseptate conidia. In a different phylogenetic analysis with another dataset of LSU and ITS sequence, the new genus Leochalara, was assigned to Arachnopezizaceae. Morphologically it is similar to Pyxidiophora but phylogenetically distinct. Morphologically it is similar to Pyxidiophora but phylogenetically distinct.

Hamatocanthoscyphaceae: includes Constrictochalara, Cylindrochalara, Infundichalara and Stipitochalara. The new genus Constrictochalara is introduced for Chalara constricta, C. ellisii, C. holubovae and one undescribed species. They were characterized by short conidiophores consisting of 1–2-celled basal stalk and a terminal phialide with sharp transition from venter to collarette, obvious constriction and darker between venters and collarettes, and hyaline, aseptate, clavate, subcylindrical or cylindrical conidia (Nag Raj and Kendrick 1975; Koukol 2011). Cylindrochalara is introduced for C. hyalocuspica which clustered together with Infundichalara microchona (the type species of the genus), but the two species differs in shape of collarette and conidia. In I. microchona, collarettes were typical funnel-shaped, and conidia were aseptate and clavate; while in C. hyalocuspica, collarettes and conidia were cylindrical. The new genus Stipitochalara is established for Chalara longipes, C. picea-abietis and C. recta, all characterized by multi-septate conidiophores, terminal phialides with abrupt transition from venter to collarette, and hyaline, aseptate, cylindrical conidia without basal frill. Other included genera in this family were Xenochalara, Microscypha, and two species of Hamatocanthoscypha.

Helotiaceae: includes Hymenoscyphus. Five species of Hymenoscyphus (H. albidus, A. albidoides, H. fraxineus, H. globus and H. koreanus) were known to produce Chalara-like anamorphs, such as. The Chalara-like anamorphs in this clade are characterized by brown and reduced conidiophores consisting of 1–2 basal cells and a terminal phialide, gradual or disrupt transition from venter to collarette, and hyaline, aseptate, subglobose, short cylindrical to cylindrical conidia. In H. globus, the phialides have obvious constriction between venter and collarette and the conidia are slightly clavate-cylindrical. All these species are well-connected with their Hymenoscyphus teleomorphs.

Neolauriomycetaceae: includes Exochalara, Lareunionomyces, Minichalara and Neolauriomyces. These four genera form a strongly supported clade as a distinct family in all phylogenetic analyses. Exochalara, Lareunionomyces and Neolauriomyces and are well-defined monophyletic genera. The three strains of Chalara microspora and one unidentified species from China clustered together as a subclade, and morphologically the two species are very similar in producing pale colored conidiophores, short conidiophores consisting of a 1–3-septate basal stalk and s terminal phialides with gradual transition from venter to collarette, and hyaline, aseptate, cylindrical conidia with rounded ends and no basal frill. The new genus Minichalara is established for the two species. None of these fungi in this family was known with teleomorph (Crous et al. 2016a, b, 2018a, b, 2019).

Pezizellaceae: includes Bloxamia, Bloxamiella, Calycina, Calycellina, Chalara s. str., Cylindrocephalum, Mollisina, Nagrajchalara, Parachalara, Phaeoscypha, Tapesina, Xenochalara and Zymochalara. In the phylogenetic trees, these genera scattered as a few strongly supported lineages under Pezizellaceae clade.

Leotiomycetes genera incertae sedis: include Ascoconidium, Chalarodendron, Bioscypha, Didonia, Phaeoscypha and Tapesina. All these genera are known with Chalara-like anamorphs, but their phylogenetic relationship within Leotiomycetes can’t be determined due to lacking living strain and DNA sequences for molecular phylogenetic analysis.

Preliminary analyses of ITS as barcode for chalara-like anamorphs

Lengths of ITS sequences of the analyzed chalara-like anamorphs (114 species) of Leotiomycetes were between 462 and 468 base pairs. The ITS barcode separated intraspecific from interspecific variability for almost all genera, although occasionally a few species in a given genus were closely related and the genetic distance between them was between intraspecific and interspecific variability observed in other species. The intraspecific variability of the analyzed species was usually below 0.86% in sequence identity, although it was higher (up to 2.16%) in three species of Nagrajchalara. The interspecific sequence divergences were usually > 0.86%, except for the four pathogenic species of Hymenoscyphus with chalara-like anamorphs, which were very similar in morphology and also identity of the ITS sequences (Supplementary Table 2–8).

For the genus Chalara s. str., the lengths of ITS sequences were between 464 and 467 base pairs of the 21 analyzed species. The ITS barcode gap separated intraspecific from interspecific variability. The intraspecific variability among the analyzed species was 0–0.86% in sequence identity. The interspecific sequence divergences were 1.72–22.76%: the minimum is 1.48% between C. fungorum and C. sessilis, while the maximum one was 22.76% between C. longiphora and C. oxenbolliae.

For the genus Nagrajchalara, the lengths of ITS sequences were between 464 and 473 base pairs among the 129 strains representing 39 species. The ITS barcode gap separated intraspecific from interspecific variability, except for the closely related species N. acuaria and N. acuariella; the genetic distance between these two closely species (0.86–1.08%) was between intraspecific and interspecific variability observed in other species. The intraspecific variability of the 39 analyzed species was 0–2.16% in sequence identity. The greatest intraspecific variability was found in N. cannonii (up to 1.28%). N. tubakii (up to 2.16%) and N. yongnianii (up to 1.51%). The interspecific sequence divergences were of the 39 species are 0.86–30.7%: the minimum was 0.86–1.08% between N. acuaria and N. acuariella, while the maximum one was 30.7% between N. jonesii and N. paraunicolor.

Morphology

Morphology of chalara-like fungi in Leotiomycetes was documented by Nag Raj and Kendrick (1975). The common diagnostic characters are pale to dark brown, obclavate, lageniform or subcylindrical phialides consisting of a variously shaped venter and a cylindrical collarette with a deeply seated sporulating locus, and hyaline, aseptate or septate, cylindrical, clavate or other shaped conidia usually with obtuse apex and truncate base, and extruded in short or long chains (Figs. 4, 5, 6, 7).

Fig. 4

Conidiomata, conidiophores and conidiogenous cells of chalara-like fungi in Leotiomycetes. a, b Sporodochial conidiomata (a) and cylindrical phialides (b) of Bloxamia elegans. c Apically branched conidiophores in Neolauriomyces crousii. d, f Apically branched conidiophores of N. beijingensis. e Conidiophores bearing branches and terminal phialides in Lareunionomyces minimus. g Upper part of conidiophores bearing branches and terminal phialides L. syzgii. h Solitary conidiophores bearing phialides with significant constriction between collarette and venter in Constrictochalara clavatospora. i, j Sessile phialides with significant constriction between venter and collarette in Hymenoscyphus globus. k Well-developed and multiseptate conidiophores in Chalara longiphora. l Subhyaline conidiophores bearing terminal phialides with gradual transition from venter to collarette and significant constriction in Minichalara aseptata. m Setae of Nagrajchalara mutabilis. Scale bar: 20 μm for a, 10 μm for d, e, k, m; 5 μm for b, c, f–j, l

Fig. 5

Conidiophores and conidiogenous cells of chalara-like fungi in Leotiomycetes. a Nagrajchalara inflatipes. b N. unicolor. c N. pulchra. d N. knudsonii. e N. puerensis. f N. yongnianii. g Cylindrocephalum clavatisetosum. Scale bar: 5 μm

Fig. 6

Conidiophores and conidiogenous cells of chalara-like fungi in Leotiomycetes. a Nagrajchalara sivanesanii. b N. aspera. c N. yinglaniae. d N. septata. e N. tubakii. f Cylindrocephalum clavatisetosum. g N. mutabilis. h Chalara parilis. i N. cannonii. j C. pengii. k Pyxidiophora schoenoplecti. l C. affinis. m N. strobilina. Scale bar: 5 μm

Fig. 7

Conidia of chalara-like fungi in Leotiomycetes. a Leochalara danxiashanensis. b Hymenoscyphus globus. c Lareunionomyces loeiensis. d Chalara crassipes. e Constrictochalara aseptata. f Pyxidiophora schoenoplecti. g Bloxamia elongata. h C. sporendocladioides. i, j Nagrajchalara angionacea. k N. acuaria. l N. yongnianii. m, n N. pulchra. o N. unicolor. p N. strobilina. Scale bar: 5 μm

Sporodochial or synnematous conidiomata are only found in Bloxamia, Bloxamiella, Chalarodendron and Chalara longipes, while the conidiophores in all other genera are solitary or aggregated in small groups (Nag Raj and Kendrick 1975; Koukol 2011; Guatimosim et al. 2016).

Sterile or fertile setae present in several species which were traditionally classified under Chaetochalara. The setae are usually associated with conidiophores at base, or scattered among conidiophores, dark brown, septate, longer than conidiophores, with sterile or fertile apex (Sutton and Pirozynski 1965; Nag Raj and Kendrick 1975; Kendrick 1980; Seifert et al. 2011). Kendrick (1980) was in doubt of difference between the two closely related genera Chalara and Chaetochalara, differentiated by a single character of absence or presence of sterile setae. Kirk and Spooner (1984) merged the two genera and transferred all Chaetochalara species known by that time into Chalara. Cai et al. (2009) in their phylogenetic analyses further confirmed the congeneric of the two genera, and concluded presence or absence of sterile setae was not a reliable character in delimiting the two genera. This study with inclusion of more species with setae further confirmed that presence of setae among the conidiophores can’t serve as the diagnostic character for Chaetochalara, and the two genera are congeneric.

Conidiophores in the chalara-like fungi arise as lateral outgrowths of individual or aggregated vegetative hyphae or from short, broad, thick-walled cells of pseudoparenchymatous layers of synnemata or sporodochia (Nag Raj and Kendrick 1975). When borne directly on the vegetative hyphae, they are usually solitary and scattered or sometimes gregarious, but when they originate from the aggregated hyphae or cells of prosenchymatous layers, they are arranged in loose or compact fascicles as seen in C. connari. The conidiophores of chalara-like fungi are usually morphologically distinct from the vegetative hyphae. In Lareunionomyces and Neolauriomyces the conidiophores are apically branched and with multiple phialides bearing chained conidia; while other genera are usually with simple and unbranched conidiophore, consisting of a basal stalk and a terminal phialide. Percurrent or sympodial proliferation are seen only in a few species such as Chalara constricta, C. nigricollis and C. prolifera. Morphology of the basal stalks varies a lot among species and can be used as one of the characters to separate species. In some species such as C. ampullula. C. emodensis, C. fusidioides, C. curvata, C. microspora and C. sessilis, the basal stalk is absent and the phialides directly arise from superficial vegetable hyphae. However, in most other species the basal stalks are present and consist of one basal cell, and this is found in C. brevipes, C. dictyoseptata, C. hughesii and C. pulchra) to multiseptate and cylindrical (such as C. bicolor, C. cylindrosperma, C. inaequalis, C. insignis, C. longipes, C. nothofagi, C. stipitata, C. tubifera and C. urceolata. Morphology of conidiophores seems to have limited value indicating the phylogeny.

The characteristic conidiogenous cell of chalara-like fungi is a terminal phialide of peculiar morphology. The basic features of this cell are a more or less expanded lower portion, or venter, and a narrower, more or less tubular, open ended collarette bearing a deeply seated sporulating locus (Nag Raj and Kendrick 1975). Based on morphology of venter, Nag Raj and Kendrick (1975) defined four typical morphotypes of the conidiogenous cells in chalara-like fungi, namely ampulliform or lageniform with globose, subglobose or ellipsoidal venter, obclavate with ellipsoidal venter, subcylindrical with cylindrical venter and urceolate with obconical. However, shapes of venters are much more variable than those typical morphotypes among different species or even within the same species. Collarettes in shape of cylindrical, conical or obconical are morphologically distinct from venter in most species of chalara-like fungi, but the venters and collarettes are indistinguishable in Bloxamia and Lareunionomyces, and some species of Chalara s. lat. such as C. microspora (Nag Raj and Kendrick 1975). Transition from venter to collarette may be abrupt (often marked by a perceptible constriction as in C. ampullula, C. insignis, C. inaequalis, C. tubifera, etc.), gradual (C. agathidis, and many other species) or barely perceptible (C. microspora). Cell wall of venter and collarettes are usually smooth in most species, but verrucose appearances are also found in several species such as C. aspera, C. brunnipes, C. curvata, C. cylindrica, C. emodensis, C. bohemica, C. panamensis, and C. scabrida. The phialidic conidiogenous cells among the chalara-like fungi are concolorous or versicolorous, and from hyaline (Leochalara danxiashanensis), pale brown to dark brown. Within the cylindrical collarettes, the monosporulating loci are seated deeply at the basal part of collarette or in the transition region of venter and collarette. All these characters of the conidiogenous cells can be used to separate species, however, they have limited value indicating the phylogeny (Cai et al. 2009).

Conidia in chalara-like fungi with Leotiomycetes affinity are usually endogenous, hyaline, aseptate or septate, cylindrical or subcylindrical, and extruded in short or long chains. However, other conidial shapes such as clavate, ellipsoidal, short cylindrical, subglobose are also seen in some species. The ends of the conidia are usually blunt or truncate, but they are rounded at one end, or both ends in some species. The conidia with truncate base usually bear a minute but distinct marginal frills. The conidia in Chalara rubi are unique in having a fringe of wall material extending for some microns from both ends (Nag Raj and Kendrick 1975; Baral 2002). The shape, size and septation of conidia are the most important characters to delimit species. Aligned with the research result from Cai et al. (2009), the phylogenetic analyses in this study also show that the conidial septation is phylogenetically informative. In the phylogenetic trees majority of the chalara-like species with aseptate and septate conidia phylogenetically clustered into different clades with strong support. Based on this, the polyphyletic genera such as Bloxamia, Chaetochalara and Chalara are revised, and several new genera are established.

Most of the chalara-like fungi of Leotiomycetes can be cultivated in laboratory. The conidia usually germinate easily on PDA, but most of these fungi grow slowly in artificial media. Morphology of colony, including growth rate, color, pigmentation, aerial mycelium etc. is useful for distinguishing genera and species in some cases. For example, the living culture of Hymenoscyphus globus produces characteristic green colony on PDA plate; the living cultures of all Cylindrocephalum species produce orange-colored pigment diffused into agar on PDA plate, which is used as one of the characters to distinguish it from Nagrajchalara.

Taxonomy

Based on the systematic study with an integrated approach of literature study, morphological observation and phylogenetic analyses, 152 species of chalara-like fungi with diversified morphology and phylogeny (115 species were with living strains and DNA sequence data) were classified into 26 genera in more than 5 families, i.e., Leochalara in Arachnopezizaceae; Constrictochalara, Cylindrochalara, Stipitochalara and Xenochalara in Hamatocanthoscyphaceae; Hymenoscyphus in Helotiaceae; Exochalara, Lareunionomyces, Minichalara and Neolauriomyces in Neolauriomycetaceae; Bloxamia, Bloxamiella, Calycellina, Calycina, Chalara, Chalarodendron, Cylindrocephalum, Mollisina, Neochalara, Nagrajchalara, Parachalara, Phaeoscypha and Zymochalara in Pezizelaceae; and Ascoconidium, Bioscypha, Didonia and Tapesina as Leotiomycetes genera incertae sedis, in which the chalara-like anamorphs were reported in literatures, but no living strain or DNA sequence was available for phylogenetic analysis (Table 2; Ekanayaka et al. 2019; Johnston et al. 2019). The species of each genus formed a strongly supported monophyletic clade distinct from others in the phylogenetic trees. Most of these genera were also morphologically well-delimitated. However, some of these genera could hardly be distinguished from each other in morphology. For example, Neochalara was similar to Nagrajchalara, and Calycina, Cylindrochalara and Stipitochalara were similar to Chalara s. str. An identification key for all these accepted genera is provided below.

Table 2 A list of families and genera with chalara-like anamorphs in Leotiomycetes (New genera are in bold; *genera not confirmed by molecular phylogeny)

Among these 26 accepted genera, fifteen genera, Chalara, Chalarodendron, Constrictochalara, Cylindrochalara, Cylindrocephalum, Leochalara, Lareunionomyces, Minichalara, Neochalara, Neolauriomyces, Nagrajchalara, Parachalara, Stipitochalara, Xenochalara and Zymochalara, were asexually typified and without known teleomorphs; while the other eleven genera, Ascoconidium, Bioscypha, Bloxamia, Bloxamiella, Calycellina, Calycina, Didonia, Hymenoscyphus, Mollisina, Phaeoscypha and Tapesina were known with both anamorphs and teleomorphs.

Based on morphological and phylogenetic study of more than 90 species representing a high diversity of morphotypes, the polyphyletic genera Chaetochalara and Chalara with solitary conidiophores, cylindrical collarettes and deeply seated conidiogenous loci, and hyaline aseptate or septate conidia, were revised with monophyletic generic concepts and reclassified into 14 different genera by: (a). redelimitation of Chalara s. str. in narrow concept, (b). adaption of the emended Calycina to accommodate asexually typified chalara-like fungi, (c). reinstatement of Cylindrocephalum, (d). acceptance of four known genera (Ascoconidium, Hymenoscyphus, Neochalara, Zymochalara), and (e). introduction of seven new genera: Constrictochalara W.P. Wu & Y.Z. Diao, Cylindrochalara W.P. Wu & Y.Z. Diao, Leochalara W.P. Wu & Y.Z. Diao, Minichalara W.P. Wu & Y.Z. Diao, Nagrajchalara W.P. Wu & Y.Z. Diao, Parachalara W.P. Wu & Y.Z. Diao and Stipitochalara W.P. Wu & Y.Z. Diao. Chaetochalara became a synonym of Chalara s. str., and the known species were transferred into Chalara s. str. and Nagrajchalara. The polyphyletic genus Bloxamia was also redefined by introducing the new genus Bloxamiella W.P. Wu & Y.Z. Diao for Bloxamia cyatheicola. Chalara breviclavata and C. vaccinii were excluded from Leotiomycetes, and the new genera Chalarosphaeria W.P. Wu & Y.Z. Diao (Chaetosphaeriaceae) and Sordariochalara W.P. Wu & Y.Z. Diao (Lasiosphaeriaceae) in Sordariomycetes were established for them respectively. Two hyaline Chalara species, C. hyalina and C. schoenoplecti were also excluded from Leotiomycetes and reclassified as new combinations of Pyxidiophora (Pyxidiophoraceae) in Laboulbeniomycetes.

For biodiversity assessment a total of 147 species in 26 genera, including 63 new species and 1 new name, were documented in this paper. Among them, 80 species in 12 genera, including 60 new species, 17 new records and 1 new name, were discovered and documented from China (Table 3). In addition, five species including 3 new species were also reported from Japan. In connection to this revision, a total of 44 new combinations were made. All these species are described and illustrated, and identification keys are provided for most of these genera and species. The DNA barcodes (ITS and LSU) were generated for all studied genera and species with pure cultures preserved.

Table 3 A list of species documented in this work

Future research area for these fungi should be the phylogenetic relationship of the several sexually typified genera such as Bioscypha, Calycellina, Calycina, Didonia, Phaeoscypha, Rodwayella and Tapesina, and systematic revision of existing species under the generic names Bloxamia, Chaetochalara and Chalara.

Key to genera with the chalara-like anamorphs in Leotiomycetes

(*difficult to be distinguished in anamorphs, but differs in teleomorph and/or phylogeny)

1.

Conidiomata sporodochial or synnematous………2

1.

Conidiomata absent; conidiophores solitary or loosely aggregated………4

2.

Conidiomata sporodochial/synnematous, composed of aggregated conidiophores………3