﻿New studies on Apiospora (Amphisphaeriales, Apiosporaceae): epitypification of Sphaeriaapiospora, proposal of Ap.marianiae sp. nov. and description of the asexual morph of Ap.sichuanensis

﻿Abstract In the present work, an epitype for Sphaeriaapiospora, the basionym of the type species of the genus Apiospora, Apiosporamontagnei, is selected among collections growing in the host plant species reported in the original protologue, Arundomicrantha. 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.


Introduction
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, 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 & (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.

Isolates
Methods employed to isolate the sexual and asexual states are described in Pintos and Alvarado (2021). The samples were deposited in the fungarium of the Muséum National d´Histoire Naturelle (PC; Paris, France) and the Fungarium of the University of Vienna (WU; Vienna, Austria). Living cultures were deposited in Fungal collection at the Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands).

Morphology
Samples were studied with a Zeiss Axioscope compound microscope operating with differential interference contrast (DIC). Images were obtained with a FLIR camera using open source software Microscopia Oberta (A. Coloma). Measurements were taken with FIJI win64 ImajeJ software, and reported as follows: maximum value in parentheses, range between the mean plus and minus the standard deviation, minimum value in parentheses, and the number of elements measured in parentheses. For some images of conidiophores, the image stacking software Zerene Stacker v. 1.04 (Zerene Systems LLC, Richland, WA, USA) was employed. Morphological descriptions were based on fertile cultures growing on 2% MEA (20 g/L malt extract, 20 g/L soy peptone, 15 g/L agar, pH 7) at room temperature.

Results
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 ( , 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.
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). 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.

Apiospora sichuanensis
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 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). 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.
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.
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 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 andAlvarado 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) 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.