In the present work, an epitype for Sphaeria apiospora, the basionym of the type species of the genus Apiospora, Apiospora montagnei, is selected among collections growing in the host plant species reported in the original protologue, Arundo micrantha. Most samples obtained from localities near that of the lectotype (Perpignan, France) belong to the same species, which is not significantly different from the clade previously named Ap. phragmitis, suggesting that this name is a later synonym of Ap. montagnei. In addition, the name Ap. marianiae is here proposed to accommodate a newly discovered species found in the Balearic Islands (Spain), and the asexual state of Ap. sichuanensis is described for the first time from samples growing in the same islands.

Apiosporaceae, Ascomycota, Sordariomycetes

Apiospora Sacc. is the type genus of family Apiosporaceae K.D. Hyde, J. Fröhl., Joanne E. Taylor & M.E. Barr. It occurs worldwide, and includes important pathogens and saprophytes of animals, plants and seaweeds (Heo et al. 2018, Park et al. 2018, Wang et al. 2018, Kwon et al. 2021). Genus Apiospora was built around Apiospora montagnei Sacc. (Saccardo 1875), a replacement name for Sphaeria apiospora Durieu & Mont. (Bory de Saint-Vincent and Durieu de Maisonneuve 1849). For a long time, Apiospora was considered a sexual state of genus Arthrinium Kunze, and both were even formally synonymyzed by Crous and Groenewald (2013), but recently shown to represent independent clades and separated again by Pintos and Alvarado (2021). These authors concluded that although the morphology of the original collections of S. apiospora (≡ Ap. montagnei) does not allow to link them with a unique phylogenetic clade, they should nest inside the clade containing most other species with basauxically-generated rounded/lenticular conidia that occur mainly on Poaceae (but also many other plant families, seaweeds and animals) worldwide (including tropical and subtropical regions), and differ from species in the clade of Arthrinium, which have variously shaped conidia, a narrower host range (mainly, but not exclusively, Cyperaceae and Juncaceae), and occur in temperate, cold or alpine (but not tropical or subtropical) regions. This way, Pintos and Alvarado (2021) selected a lectotype for S. apiospora (≡ Ap. montagnei), and fixed the phylogenetic limits of Apiospora, proposing the necessary combinations at species rank. Later authors followed this approach (Crous et al. 2021; Tian et al. 2021), and genomic analyses seem to confirm their taxonomic decision (Sørensen et al. 2022). A third group of species formerly placed within Arthrinium, including Ar. urticae M.B. Ellis (Ellis 1965) and Ar. trachycarpi C.M. Tian & H. Yan (Yan et al. 2019), are probably unrelated to Arthrinium or Apiospora (Tang et al. 2021), and therefore deserve to be classified in a different genus.

Despite these important taxonomic changes, the exact identity of the type species of Apiospora, Ap. montagnei, still remains uncertain. Pintos and Alvarado (2021) discussed the host plants mentioned by Bory de Saint-Vincent and Durieu de Maisonneuve (1849), concluding that the lectotype (collected near Perpignan, France) was found on Arundo micrantha or Aru. donaciformis. Only four species of Apiospora, Ap. iberica (Pintos & P. Alvarado) Pintos & P. Alvarado, Ap. italica (Pintos & P. Alvarado) Pintos & P. Alvarado, Ap. marii (Larrondo & Calvo) Pintos & P. Alvarado and Ap. phragmitis (Crous) Pintos & P. Alvarado had been recorded in Arundo spp. (Pintos et al. 2019, Pintos and Alvarado 2021), but since Ap. iberica and Ap. italica are relatively rare, Ap. marii and Ap. phragmitis were considered the most probable synonyms of Ap. montagnei (Pintos and Alvarado 2021).

In the present work, several collections of Apiospora growing on Arundo aff. micrantha in northeastern Spain and the Balearic Islands were analyzed, and an epitype of Ap. montagnei selected among them to fix the identity of this species. In addition, a newly discovered species found in the same region is described and given a new name, and the asexual state of Ap. sichuanensis Samarak., Jian K. Liu & K.D. Hyde is described for the first time.

The phylogenetic analysis of sequenced species of Apiospora including ITS1-5.8S-ITS2 and LSU rDNA, as well as exon and intron regions from tef1 and tub2 genes (Fig. 1) resulted in six significantly supported major clades: 1) /sorghi (apparently containing a single species, Ap. sorghi (J.D.P. Bezerra, C.M. Gonçalves & C.M. Souza-Motta) X.G. Tian & Tibpromma = Ar. taeanense S.L. Kwon, S. Jang & J.J. Kim), 2) /jatrophae, 3) /hysterina, 4) /arundinis, 5) /montagnei, and 6) /phaeospermum. These clades, identified in the present work for the first time, maybe represent monophyletic lineages that could be interpreted as sections or subgenera inside Apiospora. However, this hypothesis should be further tested with additional data from less variable DNA regions, since ITS1 rDNA and introns can be easily misaligned.

Figure 1. 

Majority rule consensus (50%) ITS rDNA- 28S rDNA- tef1- tub2 phylogram of the Apiosporaceae obtained in MrBayes from 4 875 sampled trees. Nodes were annotated if supported by > 0.95 Bayesian PP (left) or > 70% ML BP (right).

Among the samples analyzed in the present study (Suppl. material 2), five collections of Apiospora were found growing on Arundo aff. micrantha (the supposed host plant of the lectotype of Sphaeria apiospora) in Girona (northeastern Spain) and Mallorca (Balearic Islands, Spain). Four of them were not genetically different from Ap. phragmitis, and one matched Ap. sichuanensis. Another seven samples found growing on Arundo donax in Mallorca matched Ap. phragmitis, and one Ap. sichuanensis. Due to these results and the data available from Mediterranean species of Apiospora, Ap. phragmitis is here considered the most probable synonym of Ap. montagnei, and an epitype for this species selected among the samples analyzed in the present work. Finally, two samples found on Phleum pratense in Mallorca turned out to represent a previously unknown phylogenetic lineage, which is given a new name below.

Taxonomy

 Apiospora montagnei Sacc., Atti Soc. Veneto-Trent. Sci. Nat. 4: 85. 1875.

Fig. 2C–2N

Sphaeria apiospora Durieu & Mont., Expl. Sci. Alg., Fl. Algér. 1, livr. 13: 492. 1849. [replaced name]

Hypopteris apiospora (Durieu & Mont.) Berk., Hooker’s J. Bot. Kew Gard. Misc. 6: 227. 1854.

Arthrinium phragmitis Crous, IMA Fungus 4: 147. 2013.

Apiospora phragmitis (Crous) Pintos & P. Alvarado, Fungal Systematics and Evolution 7: 206. 2021.

Sexual morph

Stromata solitary to gregarious, immersed to erumpent, fusiform, with the long axis broken at the top by one or two cracks, (0.5–)2.1–2.9(–4) × (0.2–)0.25–0.35(–0.5) mm (n = 20). Ascomata uniseriate or irregularly arranged beneath stromata, pseudothecial, black, globose to subglobose with a flattened base, (150–)159–183(–200) µm high × (200–)247–278(–300) µm wide (n = 35), with a conspicuous periphysate ostiole. Peridium composed of 5 or 6 layers of brown to hyaline cells arranged in textura angularis. Hamathecium paraphyses hyphae-like, up to 4 µm wide. Asci broadly cylindrical, clavate, with an indistinct pedicel, rounded at the apex, lacking apical apparatus, (72–)99–111(–115) × (14–)15.5–16.5(–18) µm (n = 25). Ascospores uniseriate or biseriate, clavate to fusiform, straight or slightly curved, with narrowly rounded ends, composed of a large upper cell and a small lower cell, hyaline, smooth-walled, measuring (21–)23–24.5(–25) × (6–)6.3–7.1(–8) µm (n = 30).

Figure 2. 

A, B Gymnosporium arundinis (original material from PRM) A substrate of the type B label of the original collection of Gymnosporium arundinis C–N Apiospora montagnei (AP301120) C stromata on host D, E asci and periphyses F–I ascospores J–L conidiogenous cell with conidia M–N conidia in face and side view. Scale bars: 100 µm (C); 5 µm (D–I); 10 µm (J–N).

Asexual morph

Mycelium consisting of hyaline, smooth, branched, septate hyphae 1–4 μm in diam. (n = 20). Conidiophore mother cell from hyaline to brown, solitary or aggregated in groups on hyphae, subsphaerical to lageniform or ampuliform, measuring (4–)6.6–8(–10) × (3–)4.5–5.1(–6) µm (n = 10). Conidiophores cylindrical, straight to flexuous, some of them branched, hyaline, measuring (10–)18–34(–45) × (1.5–)1.6–1.8(–2) µm (n = 20). Conidiogenous cells doliiform to lageniform or ampuliform, hyaline, measuring (10–)11.5–13.1(–15) × (2–)4.3–5.1(–6) µm (n = 20). Conidia ellipsoidal to ovoid, smooth to finely roughened, with an equatorial germ slit of paler pigment, measuring (9–)10.3–11.3(–12) µm in surface view, (5–)6.2–7.2(–8) µm in side view (n = 25). Sterile cells ellipsoidal to clavate, measuring 13–16 µm (n = 25).

Culture characteristics

Colonies flat, spreading, with moderate aerial mycelium. On MEA, surface dirty white with pale rose patches, reverse luteous. Occupying an entire 90 mm Petri dish in 14 days at room temperature, sporulating four weeks after culture.

Epitype

Spain: Catalonia, Girona, L´Escala, on Arundo micrantha, 30 November 2020, leg. Marc Grañem, AP301120 (epitype selected here PC:0125164, ex-type culture CBS 148707; iso-epitype WU-MYC0044524, ex-type culture CBS 148708).

Other specimens examined

Spain: Balearic Islands, Mallorca, Esporlas, on Arundo donax, 14 December 2020, leg. Ángel Pintos, AP141220 (WU-MYC0044527). Balearic Islands, Mallorca, Palma de Mallorca, Torrente de Establiments, on Arundo micrantha, 19 April 2021, leg Ángel Pintos, AP19421 (WU-MYC0044523). Balearic Islands, Mallorca, Puerto de Alcudia, on Arundo donax, 2 April 2021, leg. Ángel Pintos, AP2421 (WU-MYC0044522). Balearic Islands, Mallorca, Puerto de Andratx, on Arundo donax, 5 April 2021, leg. Ángel Pintos, AP5421 (WU-MYC0044529). Balearic Islands, Mallorca, Puerto de Soller, on Arundo donax, 3 April 2021, leg. Ángel Pintos, AP3421 (WU-MYC0044528). Balearic Islands, Mallorca, Puigpunyent, on Arundo micrantha, 11 December 2020, leg. Ángel Pintos, AP111220A. Balearic Islands, Mallorca, Soller, on Arundo donax, 4 April 2021, leg. Ángel Pintos, AP4421 (WU-MYC0044530). Balearic Islands, Mallorca, Universitat de les Illes Balears (UIB), on Arundo donax, 28 December 2020, leg. Ángel Pintos, AP281220 (WU-MYC0044521). Catalonia, Barcelona, Premia de Dalt, on Arundo donax, 10 October 2020, leg Miguel Mir, AP101020. Catalonia, Girona, Bescanó, on Arundo micrantha, 29 November 2020, leg. Marc Grañem, AP291120 (WU-MYC0044526).

Notes

The phylogenetic boundaries of Apiospora were recently discussed by Pintos and Alvarado (2021), who selected a lectotype (PC:0125160) for S. apiospora, the basionym of the type species Ap. montagnei. In the present study, an epitype (PC:0125164) of Ap. montagnei is selected among modern collections growing on the same host in Girona, Spain (about 100 km south of the type locality, Perpignan, France). All samples growing on the same host collected in Girona or the Balearic Islands (Spain) are genetically identical to the epitype, and match the phylogenetic clade formerly known as Ap. phragmitis, excepting one that matches Ap. sichuanensis, but the ascospores of this species (29–48 × 7–10.5 μm, Samarakoon et al. 2022) clearly exceed those of Ap. montagnei (21–25 µm, Pintos and Alvarado 2021). Therefore, on the basis of these results, it is here hypothesized that Ap. montagnei is a prioritary synonym of Ap. phragmitis.

 Apiospora marianiae sp. nov. Pintos & P. Alvarado

MycoBank No: 843732

Fig. 3

Etymology

The epithet refers to Marian Mateu, the person who found the holotype collection and beloved wife of the first author.

Figure 3. 

Apiospora marianiae (AP18219) A colony on culture B–E conidiophore mother cell with septate conidiophore giving rise to conidia, in C irregularly lobate sterile cell F conidia in face and side view G conidiophore mother cell with irregular conidia from agar. Scale bars: 100 µm (A); 5 µm (B–G).

Holotype

Spain: Balearic Islands, Palma de Mallorca, on Phleum pratense, 18 February 2019, leg. Marian Mateu AP18219 (holotype CBS 148710).

Asexual morph

Mycelium branched, septate, brown to dark brown. Conidiomata sporodochial, punctiform, scattered or confluent, black, (150–)169–203(–220) µm long × (70–)76–88(–100) µm wide (n = 30). Conidiophore mother cells on the surface of the stroma lageniform to ellipsoidal or doliiform, hyaline to brown, measuring (12–)13.4–14.2(–15) × (4–)6–7.2(–8) µm (n = 10). Conidiophores arising from conidiogenous mother cells, basauxic, cylindrical, straight to flexuous, hyaline except the thin transverse septa, smooth, measuring (19–)28–44(–55) × 3–3.6(–4) µm (n = 25). Conidiogenous cells monoblastic, integrated, terminal and intercalary, cylindrical. Conidia brown, solitary; face view: globose to ovate or ellipsoidal, with pale germ slit, (11–)12.1–13.5(–18) µm in diam. (n = 70); side view: lenticular, (8–)8.4–9.2(–10) µm in diam. (n = 30). Sterile cells only seen in culture, brown, granulate, irregularly lobed, (19–)25–31(–35) × (6–)8.15–8.45(–12) µm diam. (n = 40).

Culture characteristics

colonies in MEA white and cottony, with gray patches, reverse gray. Reaching 80–90 mm in diam, in 14 days at room temperature, sporulating after 5 weeks.

Other specimens examined

Spain: Balearic Islands, Palma de Mallorca, Establiments, on Phleum pratense, 30 November 2019, leg. Angel Pintos, AP301119.

Notes

According to phylogenetic inference, Ap. ovata is the species most closely related to Ap. marianiae, but their ITS rDNA sequences are only 94% similar (including gaps). Their conidia are both oval to broadly ellipsoid, but those of Ap. marianiae measure 11–15 µm in diam., while those of Ap. ovata are longer, measuring about 18–20 µm in diam. in surface view.

 Apiospora sichuanensis Samarak., Jian K. Liu & K.D. Hyde, in Samarakoon, Hyde, Maharachchikumbura, Stadler, Gareth Jones, Promputtha, Suwannarach, Camporesi, Bulgakov & Liu, Fungal Diversity 112: 21. 2022.

Fig. 4

Asexual morph

Mycelium branched, septate, brown. Conidiomata on host parallel to the longitudinal axis of the stem, subepidermal, opening after the dehiscence of the host epidermis, containing a black conidial mass, measuring (400–)600–950(–1000) × (275–)300–550(–600) µm (n = 40). Conidiophore mother cells arising from the stroma, lageniform to ampuliform, pale brown, with superficial granular depositions, (5–)6–10(–16) × (3–)5–7(–8) µm (n = 30). Conidiophores basauxic, cylindrical, straight or flexuous, sometimes with a thin septum, hyaline to brown, smooth, with granular pigments, (20–)43–67(–80) µm in length × (2–)2.2–3.4(–4) µm wide (n = 50). Conidia globose, subcylindrical to ovate, polygonal or obpyriform, with a lateral germ slit over the entire length, brown, smooth, irregularly lobed, measuring (10–)23–31(–35) × (5–)9–13(–14) µm (n = 30).

Figure 4. 

Apiospora sichuanensis (AP121220) A conidiomata on host B germinated conidia C–E conidiophore mother cell giving rise to septate conidiophore with conidia F–H conidia. Scale bars 100 µm (A); 10 µm (B–H).

Culture characteristics

colonies on MEA 70–90 mm in diam. after 14 days at room temperature, flat, spreading, first white and cottony, later becoming gray, reverse dark gray. On PDA (200 g/L potato, 20 g/L dextrose, 20 g/L agar, pH 7.0), 80–90 mm in diam, after 14 days at room temperature, sporulating after 4–5 weeks, white cottony at first, then becoming gray with luteous patches, reverse dark gray.

Specimens examined

Spain: Balearic Islands, Mallorca, Palma de Mallorca, Torrente de Soller, on Arundo donax, 12 December 2020, leg. Ángel Pintos, AP121220 (WU-MYC0044525). Catalonia, Girona, Bescanó, on Arundo micrantha, 15 November 2020, leg. Marc Grañem. AP151120.

Notes

Ap. sichuanensis is genetically close to Ap. pseudoparenchymatica (M. Wang & L. Cai) Pintos & P. Alvarado, but the fruiting body of the former is an acervulus and that of the latter a sporodochium. In addition, the conidia of Ap. sichuanensis are 10–35 × 5–14 µm, longer and narrower than those of Ap. pseudoparenchymatica, which measure 13.5–27.0 × 12.0–23.5.

In the present work, several samples of Apiospora growing on Arundo aff. micrantha (the most probable host plant of the lectotype collection of S. apiospora, PC:0125160, Pintos and Alvarado 2021) were analyzed in order to clarify the identity of the type species of Apiospora, Ap. montagnei (≡ Sphaeria apiospora). In previous works (Crous and Groenewald 2013, Pintos et al. 2019), four species of Apiospora were found on Arundo spp.: Ap. iberica, Ap. italica, Ap. marii, and Ap. phragmitis. In the present work, the first record of Ap. sichuanensis in Europe was found growing also on Arundo spp. However, the ascospore dimensions reported in the protologues of Ap. iberica (29–34 × 6–8 μm, Pintos et al. 2019) and Ap. sichuanensis (29–48 × 7–10.5 μm, Samarakoon et al. 2022), clearly exceed those measured on the lectotype of S. apiospora (PC 0125160, 21–25 µm, Pintos and Alvarado 2021), and other original material of this species (23–28 µm, Hyde et al. 1998). On the contrary, ascospores of Ap. italica, Ap. marii and Ap. phragmitis are not significantly different from those of the lectotype of S. apiospora.

All samples of Apiospora found on Arundo aff. micrantha with an ascospore size matching that of Ap. montagnei are genetically identical to Ap. phragmitis. A single collection of Ap. italica (MA-Fungi 91733, Pintos et al. 2019), and another one of Ap. marii (MA-Fungi 91735, Pintos et al. 2019) were previously found on Arundo donax, a host plant were Ap. phragmitis and Ap. sichuanensis can occur too. Despite the lack of collections confirming it, it is certainly possible that Ap. italica and Ap. marii grow also on Arundo aff. micrantha, as these species have been found also on other host plants, especially Phragmites, but also Ampelodesmos and many others (Arundinaria, Beta, oats, seaweeds). Apiospora marii has been found in southern, central and northern Europe (Spain, Italy, Austria, The Netherlands, Sweden) and Asia (China, Korea), being most probably a widespread species. By way of contrast, Ap. italica and Ap. phragmitis have been found only in the Mediterranean region.

Given the wide host plant range observed in Apiospora, other species which have not been found yet on Arundo could be collected on this host plant genus in the future, reducing the reliability of this character for diagnosis. Of those species occurring in the Mediterranean region, some present ascosopores differing in size from Ap. montagnei (i.e., Ap. balearica (Pintos & P. Alvarado) Pintos & P. Alvarado, Ap. hysterina (Sacc.) Pintos & P. Alvarado). Others, such as Ap. descalsii (Pintos & P. Alvarado) Pintos & P. Alvarado, are apparently rare, and the probability of a synonymy with Ap. montagnei is therefore low. The sexual state of Ap. rasikravindrae (Shiv M. Singh, L.S. Yadav, P.N. Singh, Rah. Sharma & S.K. Singh) Pintos & P. Alvarado produces ascospores measuring 21.5–24.5 × 7–9.5 µm (Dai et al. 2017), therefore fitting the size range observed in S. apiospora lectotype, but the synonymy is here rejected because this species has never been found yet on Arundo sp. (only known to grow on ornamental Phyllostachys and bamboo plants in Mallorca). Unfortunately, the sexual state of multiple species occurring in the Mediterranean region (i.e., Ap. aurea (Calvo & Guarro) Pintos & P. Alvarado, Ap. esporlensis (Pintos & P. Alvarado) Pintos & P. Alvarado, Ap. hispanica (Larrondo & Calvo) Pintos & P. Alvarado, Ap. mediterranea (Larrondo & Calvo) Pintos & P. Alvarado, Ap. piptatheri (Pintos & P. Alvarado) Pintos & P. Alvarado, Ap. serenensis (Larrondo & Calvo) Pintos & P. Alvarado, as well as the new species introduced in the present work, Ap. marianiae) is still unknown, and therefore they cannot be compared yet with the lectotype of S. apiospora (≡ Ap. montagnei). However, these seem to be rare species, and they have never been found on Arundo yet, so the synonymy is here considered much less probable.

A classical candidate synonym of Ap. montagnei, Ap. arundinis (Corda) Pintos & P. Alvarado (Crous and Groenewald 2013), has not been found yet in the western Mediterranean region (Pintos et al. 2019, Pintos and Alvarado 2021), but it seems to be widespread elsewhere, occurring in temperate, cold and also subtropical countries (Crous and Groenewald 2013). In Spain, it has been found in ornamental Bambusa plants in Galicia (north-western Spain), but not in the Mediterranean border with France (closer and ecologically more similar to Perpignan, the type locality of Ap. montagnei). Sequenced samples of Ap. arundinis found growing in Arundo are currently lacking, but the type collection of its basionym, Gymnosporium arundinis Corda was reported to grow on reeds and grasses near Prague by Corda (1838). An original sample kindly loaned by the Prague herbarium (PRM 155522) was found to present globose conidia 5–7 µm in diam., a size compatible with that observed in the clade identified as Ar. arundinis by Crous and Groenewald (2013), but also others, such as Ap. descalsii, Ap. italicum, Ap. jiangxiensis (M. Wang & L. Cai) Pintos & P. Alvarado, Ap. malaysiana (Crous) Pintos & P. Alvarado, Ap. pseudospegazzinii (Crous) Pintos & Alvarado or Ap. sacchari (Speg.) Pintos & P. Alvarado. Interestingly, the host plant of this original collection of G. arundinis loaned by PRM was not Arundo, but very probably Phalaris arundinacea. The identity of Ap. arundinis needs to be further investigated, and an epitype from Prague selected, to ascertain if the name is being correctly applied.

Therefore, on the basis of the data currently available (host plants, ascospore sizes, abundances, distributions), it is here hypothesized that the lectotype of S. apiospora (≡ Ap. montagnei) is not genetically different from the clade of Ap. phragmitis. An epitype of S. apiospora (≡ Ap. montagnei) from Girona (Spain, about 100 km south of Perpignan, the locality where the lectotype was found) is here chosen, and a synonymy between Ap. montagnei and Ap. phragmitis is suggested.

The authors wish to thank Juan Planas for assembling the image plates, as well as Jan Holec, curator of PRM herbarium for kindly allowing the authors to study original collections of Gymnosporium arundinis. Finally, Marc Grañem, Marian Mateu and Miguel Mir are thanked also for collaborating in finding samples of Apiospora.