Molecular Phylogeny and Morphology of Amphisphaeria (= Lepteutypa) (Amphisphaeriaceae)

Amphisphaeriaceous taxa (fungi) are saprobes on decaying wood in terrestrial, mangrove, and freshwater habitats. The generic boundaries of the family have traditionally been based on morphology, and the delimitation of genera has always been challenging. Amphisphaeria species have clypeate ascomata and 1-septate ascospores and a coelomycetous asexual morph. Lepteutypa is different from Amphisphaeria in having eutypoid stromata and more than 1-septate ascospores. These main characters have been used for segregation of Lepteutypa from Amphisphaeria for a long time. However, the above characters are overlapping among Amphisphaeria and Lepteutypa species. Therefore, here we synonymized Lepteutypa under Amphisphaeria based on holomorphic morphology and multigene phylogeny. Further, our cluster analysis reveals the relationship between seven morphological traits among Amphisphaeria/Lepteutypa species and suggests those morphologies are not specific to either genus. Three new species (i.e., Amphisphaeria camelliae, A. curvaticonidia, and A. micheliae) are introduced based on morphology and LSU-ITS-RPB2-TUB2 phylogenies. Furthermore, the monotypic genus Trochilispora, which had been accepted in Amphisphaeriaceae, is revisited and synonymized under Hymenopleella and placed in Sporocadaceae.

The 25 µL total volume of PCR mixture contained 9.5 µL of ddH 2 O, 12.5 µL of 2X PCR Master Mix (TIANGEN Co., China), 1 µL of DNA template, and 1 µL of forward and reverse primers (10 µM each) in each reaction. PCR amplified products were checked on 1% agarose electrophoresis gels stained with GoldView I nuclear staining dye (1 µL/10 mL of agarose). Purification and sequencing of PCR products were done by Invitrogen Biotechnology Co. Ltd., Beijing, China. A consensus sequence for each gene region was assembled in SeqMan (DNAStar, Inc., Madison, WI, USA).

Phylogenetic Analyses
Generated ITS, LSU, RPB2, and TUB2 sequences were subjected to BLASTn searches (https://blast. ncbi.nlm.nih.gov) and related sequences were downloaded from GenBank ® ( Table 1). The individual gene matrix was aligned using MAFFT v7 (https://mafft.cbrc.jp/alignment/server/large.html; [28]) with E-INS-i and L-INS-i Iterative refinement methods, respectively, and improved when necessary in BioEdit v. 7.0 [29]. All absent sequences were coded as missing data and characters were assessed to be unordered and equally weighted.
Evolutionary models were estimated by using MrModeltest 2.2 [30] and model parameters were selected independently for different gene regions under the Akaike information criterion (AIC) implemented in PAUP v. 4.0b10. The GTR+G+I model was the best-fit model for all loci.
Single (LSU, ITS, RPB2, TUB2) and combined (LSU-ITS, LSU-ITS-RPB2-TUB2) matrices were used for phylogenetic analyses in order to compare the topology with previous studies which only used LSU-ITS matrices. Maximum parsimony (MP) analyses were carried out using PAUP v.4.0b 10 [31]. The parameters were set up with tree bisection-reconnection (TBR). Trees were inferred using the heuristic search option with 1000 random sequence additions, with maxtrees set at 1000. Tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC), and homoplasy index (HI) were calculated for trees generated under different optimality criteria. The Kishino-Hasegawa tests [32] were performed to determine whether trees were significantly different.
The Bayesian inference (BI) analysis was generated by using Markov Chain Monte Carlo sampling in MrBayes v3.1.2 [35,36] for 3,000,000 generations using four chains with 100 sample frequencies which products 30,000 trees. The first 3000 (10% from total) trees were the burn-in phase and were discarded. The remaining 27,000 trees were used to calculate the posterior probability (PP). The final alignment and tree were registered in TreeBASE under the submission ID: 26768 (http://www.treebase.org).
The resulting trees were viewed in FigTree v. 1.4.0 [37] and the final layout was done with Adobe Illustrator ® CS5 (Adobe Systems, San Jose, CA, USA).
The analysis was conducted using Python 3.8 coupled with SciPy 1.5.1 package. We obtained a dendrogram using the unweighted pair group method with arithmetic mean (UPGMA) algorithm and also obtained the Average distance measure. The dendrogram of obtained results was plotted using Matplotlib 3.3.0 package. The heatmap was obtained in a similar way. Plotting was performed by using Seaborn 0.10.1 (https://docs.scipy.org).
The ITS sequence of "Lepteutypa uniseptata"; CBS 114967 (MH553979) is dubious because it appeared highly similar to Robillarda species in a BLASTn search and clustered in Sporocadaceae in our ITS-based phylogenies, which is why it has been excluded from our final analyses.

Cluster Analysis
The cluster analysis was comprised of 37 species with seven characters ( Figure 2). There are three clusters (A, B and C). The type of species of Amphisphaeria; A. umbrina and the type of "Lepteutypa"; "L. fuckelii" cluster with clusters B and C, respectively. Species in cluster A shared asci l/w greater than 15. Clusters B and C were mainly distinguished by having 1-septate (B) and multiseptate (C) ascospores. All other characteristics were distributed among the species without a unique characteristic for each genus.
J. Fungi 2020, 6, x FOR PEER REVIEW 7 of 27 strain "Trochilispora schefflerae" (COAD 2371) clustered with Hymenopleella austroafricana (CBS 143886) with high statistical support (100%/100%/1.00 PP) in Sporocadaceae. The ITS sequence of "Lepteutypa uniseptata"; CBS 114967 (MH553979) is dubious because it appeared highly similar to Robillarda species in a BLASTn search and clustered in Sporocadaceae in our ITS-based phylogenies, which is why it has been excluded from our final analyses.

Taxonomy
Petrak [10] introduced Lepteutypa to accommodate species with eutypoid stroma and multicellular ascospores different from Amphisphaeria. Lepteutypa species do not possess eutypoid stroma in their morphological descriptions, figures, and illustrations except aggregated ascomata. Jaklitsch et al. [16] proposed a neotype for Lepteutypa fuckelii, the type species of the genus, which has scattered or aggregated ascomata on the host but not eutypoid stroma. Jaklitsch et al. [16] further noted that Petrak [10] interpreted those aggregated perithecial colonies as eutypoid stromata. Among previously known Lepteutypa species, L. alpestris, L. cisticola, L. fusispora, L. hederae and L. tropicalis also possess aggregated perithecia, while L. hexagonalis possesses two perithecia under a single clypeus rarely. The aggregated ascomata also can be observed in A. bertiana and A. seriata.
Amphisphaeria and Lepteutypa were previously separated based on stromatic nature and ascospore septation. However, with the introduction of Amphisphaeria camelliae and A. micheliae, which are typical of Amphisphaeria and not "Lepteutypa" where they cluster, we conclude the Amphisphaeria and "Lepteutypa" are congeneric. There is no distinct characteristic used to separate Amphisphaeria (clade Y) and "Lepteutypa" (clade X) clusters in Figure 1. Both clusters have 2-celled and multicelled ascospores. No single characteristic exists in one clade. The predicament here is whether to follow the molecular data, which indicates there are two distinct genera (Amphisphaeria and Lepteutypa) or the morphological data which indicates the group is one genus (Amphisphaeria). We adopt a single genus, Amphisphaeria, due to the morphological similarity. However, further studies with fresh collections may resolve the taxonomic relationships in Amphisphaeriaceae and sexual asexual connections.
The coelomycetous asexual morph of Amphisphaeria sorbi is the only asexual record in Amphisphaeriaceae. Several morphological records of pestaloid-like asexual morphs for "Lepteutypa" have been suggested, but not confirmed [63]. In our study, we obtained two asexual morphs which are not pestaloid-like, but similar to the coelomycetous asexual morph of A sorbi. Since there is lack of sequence data for most of the "Lepteutypa" species, here we provided five taxonomic combinations of "Lepteutypa" species which have molecular data. However, the remaining "Lepteutypa" species are treated as ambiguous taxa and need to be revisited with molecular and morphology data in further studies. Notes: Amphisphaeria acericola is similar to A. pseudoumbrina in having immersed, oblate ascomata, J+, a discoid apical ring, cylindrical asci with rugose-walled, brown, uniseptate ascospores, but different in having non-clypeate ascomata, peridium with textura angularis cells, and aseptate, cellular paraphyses. The LSU-ITS phylogeny of A. acericola forms a basal clade distinct from other Amphisphaeria species.

Additional Accepted Species in Amphisphaeria
Amphisphaeria     Notes: Our specimens have solitary and aggregated, immersed ascomata with two-layered peridium, unitunicate asci with J+, a discoid apical ring, and brown ascospores similar to amphisphaeriaceous species. Amphisphaeria camelliae possesses 1-septate ascospores similar to A. uniseptata, but differs in having single or aggregated, globose to subglobose (vs. single, subglobose or applanate) ascomata, thin paraphyses (3.1 vs. 5 µm) and large ascospores (l/w 3.1 vs. l/w 2.7). In the phylogenetic analyses, our collection also clusters with A. uniseptata. Based on morphology and phylogeny, our collection is introduced as a novel species A. camelliae.
Amphisphaeria      Notes: Our collection shares immersed, clypeate, globose to subglobose ascomata with inner hyaline and outer brown peridium, sepatate, flexuose paraphyses and ellipsoidal, brown ascospores typical for Amphisphaeria. The J+ discoid apical ring is similar to several other species, including A. flava described from Thailand. However, A. flava differs from our new collection in having a halo on the host surface around the ostiole and 1-septate ascospores. Amphisphaeria mangrovei differs from our strains with J− and an apical ring in asci. In addition, some of the immature ascospores are covered with a thin mucilaginous sheath as in A. flava, A. lusitanica, A. seriata, A. sorbi and A. vibratilis. In addition, our collection has 1 median, slightly constricted at the euseptum, and two distosepta which are characteristic to A. depressa. Wang et al. [2] re-examined the herbarium specimen of A. depressa and noted that this unusual distoseptate ascospores characteristic is not matched with the generic description. However, based on unituniate asci with J+, apical ring, Wang et al. [2] accepted this species in Amphisphaeriaceae. The asexual coelomycetous from the culture of our strain is similar to the only known asexual morph of A. sorbi by having septate, branched, hyaline conidiophores, elongated conical, thin-walled, septate, hyaline, annellidic conidiogenous cells, and elongate-fusiform, 1-celled, hyaline conidia. The asexual morph of our strain is characterized with curved appendage derived from the middle of the conidia cell. Here we introduce our new collection as A. curvaticonidia.
Amphisphaeria Notes: Aptroot [64] transferred Didymosphaeria gaubae to Amphisphaeria and Wang et al. [2] re-examined the holotype and accepted the transfer to Amphisphaeria due to the combination of clypeate perithecia, unitunicate asci with J+ apical ring and 1-septate, slightly curved ascospores.   Notes: Two of our collections have solitary, immersed ascomata with two-layered peridium, unitunicate asci with J+, discoid apical ring, and brown ascospores. This collection also has 1-septate ascospores similar to Amphisphaeria unisepta and A. camelliae. Compared to those two similar species, this collection has subglobose to oblate ascomata and oblong or narrowly fusiform ascospores. The phylogenetic analyses show MFLUCC 20-0121 and HKAS 107012 are closely related to A. sambuci, isolated from partly decorticated branches of Sambucus nigra. Amphisphaeria sambuci is different from our new collection in having large, depressed globose ascomata and oblong-ellipsoid, straight, rarely curved, 2-4(-6)-distoseptate ascospores with a thick mucilaginous sheath. Based on the morphology and phylogeny, here we introduce it as the novel species A. micheliae.
Amphisphaeria Notes: Tsui et al. [67] introduced Amphisphaeria uniseptata on submerged wood from Hong Kong and noted that it is similar to A. pakistanae in having ellipsoidal, brown, 1-sepatate but differs from having thick-walled ascospores. Jaklitsch et al. [16]  Notes: Amphisphaeria yunnanensis has ascomata with narrow and long ostioles, asci with the J-apical ring and ascospores without a gelatinous sheath.

Taxa Needing Further Revisions
Ten species of Lepteutypa lack molecular data and asexual morphs. There are some "Lepteutypa" species that have been considered with pestaloid-like asexual morphs as mentioned in the relevant notes below and it is not possible to place them in Amphisphaeria. Therefore, these "lepteutypa-like" taxa need to be recollected, and sexual-asexual connections, molecular data, and generic affiliations established. We therefore do not treat them in Amphisphaeria sensu stricto.
Lepteutypa alpestris ( Notes: Ellis and Everhart [68] introduced Lepteutypa alpestris with the sexual morph. However, they noted that one perithecium contained stromatic nature. Barr [14] revisited the species and accepted it in "Lepteutypa" which is closely related to L. cupressi by having 3-septate ascospores covered with mucilaginous sheath and their sizes. However, L. cupressi has been accepted as Seiridium cupressi [16,69]. Lepteutypa  Notes: Lepteutypa cisticola has 3-5 gregarious ascomata, asci with J+ apical ring and euseptate, with 3-septate (or occasionally two transverse and one oblique) ascospores [70]. Petrak [71] introduced Adea canariensis while suggesting it was possibly the anamorph of L. cisticola. However, Nag Raj and Kendrick [70] re-described Seiridium canariense (= Adea canariensis). Lepteutypa  Notes: Lepteutypa fusispora has more or less often aggregated (often paired) perithecia and 1-septate, straight or curved ascospores [11]. The author has noted that the specimen was young, and asci and ascospores often shrink, and Diaporthe seposita is often found on the same collection. Nag Raj and Kendrick [70] re-examined the type specimen and remarked that "L. fusispora" is not congeneric with A fuckelii based on the J+ asci and ascospores.
Lepteutypa hederae (Fuckel) Rappaz Notes: Rappaz [72] accommodated the species in Lepteutypa. Jaklitsch et al. [16] re-examined the holotype, which was reported in the protologue to grow on corticated branches of Hedera helix (but as revealed by xylotomy, the host is probably Viburnum sp.) from Switzerland. Lepteutypa hederae possesses consistently 3-septate ascospores, lacking a median euseptum and asci with a J− apical ring. Lepteutypa  Notes: Goh and Hyde [15] introduced Lepteutypa hexagonalis which is characterized by having single or in groups of two, deeply immersed ascomata with a distinct ostiole, cylindrical asci with a J+ apical ring and brown, 3-septate, ascospores with 6-7 longitudinal ridges.
Lepteutypa podocarpi (Butin) Aa, Notes: Barr [14] re-examined the type of the specimen on Serenoa serrulata from Florida. Ascomata are usually gregarious beneath a conspicuous blackened clypeus. Ascospores are verruculose under the narrow hyaline coating. Another collection from Florida on Aralia spinosa is identical in asci and ascospores, but the ascomata form beaks that protrude 330-449 beyond the blackened clypeus. Lepteutypa  Notes: Ascospores of Lepteutypa ulmicola have one median septum and two distosepta with strongly pigmented and irregularly roughened walls. Notes: Hymenopleella is a sexual morph genus and sexual-asexual connection was confirmed through phylogeny by Liu et al. [39] while synonymizing Dyrithiopsis and Neotruncatella under Wijayawardene et al. [80]). Amphisphaeria and Lepteutypa species differ in eutypoid stromata and septation of ascospores in the protologues. However, we consider that these morphologies are not sufficient to distinguish these genera. Here, we synonymize Lepteutypa under Amphisphaeria.

Hymenopleella
Supplementary Materials: The following are available online at http://www.mdpi.com/2309-608X/6/3/174/s1. Figure S1: ML tree revealed by RAxML from an analysis of the LSU-ITS matrix of the Amphisphaeriaceae and selected families of Amphisphaeriales.