Diversity of hyperparasitic fungi on Meliolales (Sordariomycetes, Ascomycota): new species, records, and molecular data from Benin and Panama

Meliolales (black mildews) is an order of plant parasitic ascomycetous fungi in the tropics and subtropics. They are frequently overgrown and parasitized by other fungi, known as hyperparasites. During the last few years, species of hyperparasitic fungi on Meliolales have been collected in Benin and Panama. A new species of Paranectria and seven new reports of hyperparasites of different systematic groups are presented here with detailed descriptions and illustrations, together with new data concerning fungal hosts and host plants. The new species is called Paranectria longiappendiculata, characterized by exceptionally long appendages carried by the ascospores. New records for Benin and Panama are Calloriopsis herpotricha, Dimerosporiella cephalosporii, Isthmospora glabra, Isthmospora trichophila, Malacaria meliolicola, Paranectriella hemileiae, and Paranectriella minuta. Calloriopsis herpotricha is recorded for Africa and D. cephalosporii and P. hemileiae for America for the first time, suggesting an apparently pantropical distribution. Findings show a blatant lack of investigation on hyperparasitic fungi in the tropics. The phylogenetic positions of three of these newly reported species, C. herpotricha, D. cephalosporii, and P. minuta, are shown based on the analysis of internal transcribed spacer (ITS), large subunit (LSU), and small subunit (SSU) rDNA sequences. These sequences were generated in the context of the present study for the first time.


Introduction
Meliolales (Sordariomycetes, Ascomycota), commonly known as "black mildews", form a large order of biotrophic, obligate plant parasitic fungi in the tropics and subtropics. Species of this order develop on leaves, petioles, twigs, and sometimes fruits of vascular plants (Piepenbring et al. 2011;Hongsanan et al. 2015;Zeng et al. 2017). Black mildews cause a reduction of chlorophyll, starch, sugar, proteins, and amino acids in the plant tissues they infect, as well as alterations in the photosynthetic and respiratory rates (Old et al. 2003).
Meliolales are frequently infected by hyperparasites (Hawksworth 1981;Gams et al. 2004). There are approximately 200 species of fungi reported to be hyperparasitic on colonies of Meliolales (Bermúdez-Cova et al. 2023), but we expect a much greater number of species to exist in the tropics. Fungal hyperparasites belong to diverse taxonomic groups, and therefore comprise species producing a high diversity of reproductive structures, such as apothecia, catathecia, perithecia, pycnidia, and synnemata, among others. They are generalists concerning genera of Meliolales, but many of these hyperparasites seem to be restricted only to meliolalean hosts (Bermúdez-Cova et al. 2022).
Knowledge of species diversity of black mildews in the tropics is still limited. Only three species are known for Benin and 105 for Panama (Piepenbring et al. 2011;Piepenbring et al. 2020;Hofmann and Piepenbring 2021). Information on hyperparasitic fungi of Meliolales in Benin and Panama is inexistent (Bermúdez-Cova et al. 2022). For a better understanding of the diversity, evolution, and biology of hyperparasitic fungi, it is necessary to increase sampling efforts and to undertake further morphological, molecular, and ecological studies.

Sample collection and morphological characterization
Samples of leaves infected with black mildews were opportunistically collected in Western Panama from January to March 2020 and in Benin in February as well as September 2022. For the present study, colonies of Meliolales parasitized by hyperparasites were considered. Infected leaves were dried in a plant press and deposited in the herbarium at the Universidad Autónoma de Chiriquí (UCH, specimens from Panama) and in the mycological herbarium of the University of Parakou (UNI-PAR) in Benin. If a given sample was large enough, a duplicate was deposited in the Botanische Staatssammlung München (M).
Dried specimens were observed by stereomicroscopy and by light microscopy. Measurements of at least 20 ascospores, conidia, and other structures have been made for each specimen at a magnification of × 600 and × 1000. Measurements are presented as mean value ± standard deviation with extreme values in parentheses. Line drawings were made freehand on scaled paper. Images and drawings were edited with Photoshop (Adobe, San Jose, California).

Host plant identification
Host plants were identified by morphological characteristics and in some cases by molecular sequence data. Morphological identifications were made by comparison with herbarium specimens, literature (e.g., Akoègninou et al. 2006;Condit et al. 2011), and with the help of local botanists. Molecular sequence data for species identifications were obtained by polymerase chain reaction (PCR) for the amplification of the partial region of chloroplast rbcL with the primer pairs rbcLa-F (Levin et al. 2003) and rbcLa-R (Kress et al. 2009). DNA was extracted from approx. 0.05 g of leaf tissue dried with silica gel using the innuPREP plant DNA kit (Analytik Jena, Germany) and following the manufacturer's instructions. Protocols for PCR were carried out as described by Fazekas et al. (2012).

DNA extraction, PCR amplification, and sequencing of fungal DNA
DNA was isolated from the ascomata of dry specimens using the EZNA forensic DNA extraction kit, following the manufacturer's instructions. To extract total genomic DNA, a small amount of clean ascomata were transferred into a sterile Eppendorf tube with approx. 200 μL of distilled water using sterilized tweezers, and trying to avoid picking cells of any other organism associated with the leaves and the colonies of black mildews. The samples were frozen for 24 h at − 20 °C and later homogenized for 10-12 min. using a Retsch mixer mill MM301 with TL buffer and 2.5-mm zirconia beads. Isolated DNA was re-suspended in elution buffer and stored at − 20 °C.
Two partial nuclear gene regions (ribosomal loci) were amplified and sequenced: For the large subunit nuclear ribosomal DNA (nrLSU, 28S rDNA), the primers LSU1Fd and LSU3Rd (Crous et al. 2009), NL1 andNL4 (O'Donnell 1993), LR0R (Wagner and Ryvarden 2002), and LR5 (Vilgalys and Hester 1990) were used. For small subunit nuclear ribosomal DNA (nrSSU, 18S rDNA), the primers SSU1Fd and SSU3Rd (Crous et al. 2009) were used. For the internal transcribed spacer region of ribosomal DNA (ITS), the primers ITS5 and ITS4 (White et al. 1990) were used. The PCR mixtures consisted of 1 μL genomic DNA, 15 × MgCl 2 reaction buffer (Bioline, Luckenwalde, Germany), 25 mM MgCl 2 , 25 μM of each dNTP, 10 μM of each primer, and 5 U Taq DNA polymerase (VWR) in a total volume of 30 μL. Cycling parameters of the PCR for ITS, LSU, and SSU were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of amplification [denaturation at 94 °C for 30 s, primer annealing at 52 °C for 30 s and primer extension at 72 °C for 45 s], and a final extension at 72 °C for 5 min, followed by storage at 8 °C. PCR products were checked on 1.5% agarose electrophoresis gels containing HDGreenPlus DNA stain. Amplified PCR products were purified with the Cycle Pure Kit (VWR-Omega, USA). Sequencing was performed at Seqlab GmbH, Germany.
Numerous attempts were made to obtain DNA sequence data of ITS, LSU, and SSU regions from all the specimens collected in the context of this study. Except for four specimens, these attempts failed.

Apothecioid hyperparasites
Anamorph -Not known.   (Pfister 1976); on Meliola sp. on living leaves of herbs in Puerto Rico (Pfister 1976); without host data in Guadeloupe, France (Pfister 1976); on Meliola sp. on living leaves of Nyssa sp. (Nyssaceae) in the USA (Pfister 1976); on Meliola sp. on living leaves of Coffea arabica (Rubiaceae) in Benin (this study). C. arabica is a new host of C. herpotricha, and the hyperparasite and the species of Meliolales are new records for Benin. This is also the first record of C. herpotricha in Africa.
Illustrations -This species was illustrated by Pfister (1976). Notes -Two species of apothecioid hyperparasitic fungi have been reported to parasitize Meliolales, namely, Calloriopsis herpotricha and Unguiculella meliolicola, both belonging to the Leotiomycetes (Phacidiales and Helotiales, respectively; Bermúdez-Cova et al. 2022). The monotypic genus Calloriopsis was proposed by Sydow and Sydow (1917) and is based on a parasitic discomycete which occurs on Meliola and related dark parasites. Calloriopsis herpotricha differs from other fungi of the Leotiomycetes by the parasitic habit and the gelatinous material that is present in all parts of the apothecium, including the hymenium (Pfister 1976;Baral and Marson 2001).
Sequence data -The SSU rDNA sequence obtained from fresh material of C. herpotricha (specimen AK4H) is 948 bp long. In the tree inferred from the analysis of SSU sequences of 14 specimens of Leotiomycetes and Lecanoromycetes ( Fig. 1), the sequence of C. herpotricha is located within a strongly supported clade that comprises sequences of species of Lecanoromycetes which were obtained from lichenized fungi. Some lineages of non-lichenized Ascomycota are known to be derived from lichenized ancestors by the loss of the lichen symbiosis in favor of a saprotrophic, lichenicolous, or parasitic mode of nutrition (Lutzoni et al. 2001;Hawksworth 2015;Honegger 2022). Examples of these include non-lichenized members of Arthoniales (Arthoniomycetes) and Ostropales (Lecanoromycetes; Kendrick 2017). The foregoing and the fact that the sequences of the species of Calloriopsis clustered together with other sequences of species of Ostropales suggest that the genus Calloriopsis may belong to the Lecanoromycetes and not to the Leotiomycetes as previously assumed. A sequence for C. herpotricha is provided here for the first time.
Four sequences of an unidentified species of Calloriopsis are available in GenBank (accession numbers: MF322776, MF322774, OM103051, and OQ800930). The specimens that yielded these sequences were found on decayed twigs and branches of Cornus sanguinea L. (Cornaceae) and Fraxinus excelsior L. (Oleaceae) in Luxembourg (unpublished data provided by the herbarium LUX). These sequences also fall within the Lecanoromycetes. However, these sequences lack the SSU region; thus, it was not possible to compare them with the sequence of Calloriopsis herpotricha. We also obtained a DNA sequence of the ITS region of C. herpotricha (GenBank accession number: OR243608). This sequence presents 94% identity with the aforementioned sequences of Calloriopsis and other members of the Lecanoromycetes, confirming the systematic placement of C. herpotricha in the Lecanoromycetes.
Teleomorph -Trichothyrium-like (according to Hughes 1953 (Stevens 1918); on Appendiculella sororcula on living leaves of Calea pittieri (Asteraceae) in Panama (this study). A. sororcula and C. pittieri are new hosts of I. glabra, and the hyperparasite is a new record for Panama.
Illustrations -This species was illustrated by Hughes (1953). Notes -The genus Isthmospora (Microthyriaceae, Microthyriales) was proposed by Stevens (1918) and comprises two species of dematiaceous hyphomycetes with dark conidia consisting of two approximately equal halves connected by an isthmus. Both species of the genus, I. glabra and I. spinosa, are associated with colonies of black mildews (Damon 1953).
Isthmospora glabra is characterized by the presence of dark smooth central cells with large hyaline appendages (Stevens 1918;Hughes 1953). However, according to Damon (1953), this species is congeneric with Spegazzinia chandleri.
Isthmospora glabra has always been found on the hyphal mat of Trichothyrium reptans, a catathecioid hyperparasite of Meliolales. Therefore, I. glabra is considered to be the anamorphic stage of T. reptans (Hughes 1953). The specimen examined (MP5326) was also found together with a species of Trichothyrium (Fig. 3a, b); however, the size of the ascospores (10-12 × 4-6 μm) does not match with the size of ascospores of T. reptans (15-20 × 5-6.5 μm; Hughes 1953). There is no molecular evidence that supports the anamorph-teleomorph connection between these fungi.
Notes -Isthmospora trichophila (Microthyriaceae, Microthyriales) is morphologically similar to species of Spegazzinia (Apiosporaceae, Sordariomycetes), and several known species are easily confused (Damon 1953). However, the complex morphology of the isthmospores and the association with species of Meliolales are strong features to distinguish I. trichophila from other species of dematiaceous hyphomycetes (Damon 1953;Hughes 1953).
Isthmospora trichophila has always been recorded growing on the hyphal mat of Trichothyrium asterophorum (Microthyriaceae, Microthyriales), a catathecioid hyperparasite of Meliolales; thus, it is considered to be the anamorphic stage of T. asterophorum (Hughes 1953). The specimens examined were also found together with a species of Trichothyrium, which could not be identified because it was not fertile. There is no molecular evidence that supports the anamorph-teleomorph connection between these two species of fungi. Illustrations -This species was illustrated by Gams (1971, anamorph only), Pirozynski (1977, and Samuels (1988).
Notes -Approximately 70 species of perithecioid fungi are reported as hyperparasites of Meliolales (Bermúdez-Cova et al. 2022). Among these species, Dimerosporiella cephalosporii (Bionectriaceae, Hypocreales) is one of the most common parasites in Uganda (Hansford 1946;Gams 1971;Gams et al. 2004;Bermúdez-Cova et al. 2022). The genus Dimerosporiella was proposed by Spegazzini (1908) and now comprises species that were previously placed in the Nectria leucorrhodina group or treated within Nectriopsis (Samuels 1976(Samuels , 1988Rossman 1983). Species of the genus are fungicolous (i.e., growing on other fungi) and grow on colonies of species of Asterina, Meliolales, or Schiffnerula (Rossman et al. 1999 primarily by features of the surface of ascomatal walls and characteristics of the ascospores. For a detailed key to species of the genus, see Rossman et al. (1999). Dimerosporiella cephalosporii is similar to D. sensitiva, from which it differs by a simple ascal apex and perithecial hairs (Samuels 1988). Both species are commonly associated with an Acremonium-like anamorph with thick-walled conidiophores and phialides (Pirozynski 1977). The conidial form is always found together with the perithecia, but there is no molecular evidence that supports this anamorph-teleomorph connection.
Sequence data -The LSU rDNA sequence obtained from fresh material of D. cephalosporii (specimen MB139) is 498 bp long and presented 32 ambiguous bases. In the tree  MB139). a A leaf of Olyra latifolia parasitized by Meliola sp. Note that some of the black colonies are whitish/greyish (arrows) due to the presence of the hyperparasite; b orange perithecia between the setae of Meliola sp.; c a leaf of Paullinia pinnata infected by Meliola pinnatae (MB139). Note that some of the black colonies are whitish/ greyish due to the presence of D. cephalosporii; d perithecium on a hypha of Meliola sp.; e perithecial hairs; f ascus and ascospores. Scale bars: 1 cm (a, c); 1 mm (b); 13 μm (d); 5 μm; 4.5 μm (f) inferred from the analysis of LSU sequences of 24 specimens (Fig. 4), D. cephalosporii is located within a strongly supported clade that comprises sequences of Acremonium spp. and other species within the Bionectriaceae. It does not cluster with any sequence of Dimerosporiella, because no sequences are available for this genus up to now. Colonies white, hyphae growing closely appressed to the dark hyphae of Meliolales, 1-2 μm wide, hyaline, thin-walled. Pseudothecia superficial, growing between the synnemata of Atractilina parasitica, ovate to elongate ovate with rounded apex, 150-200 × 100-140 μm, dark vinaceous when seen macroscopically, dark cinnamon or brick when seen by light microscopy, not changing color in KOH, smooth. Pseudothecial wall 12-17 μm thick, composed of angular cells with 6-15 μm diam. (surface view). Asci bitunicate, narrowly clavate to cylindric, apex rounded, (40-)52-56(-64) × (9.5-)10-12(-16) μm, 8-spored. Pseudoparaphyses unbranched, abundant, up to 120 μm long, 1-2 μm wide, septate, hyaline, rounded at the ends, with Known hosts and distribution -On Irenina glabra on leaves of Coffea robusta (Rubiaceae) in Uganda (Hansford 1941). On Meliola sp. on leaves of Hamelia erecta (Rubiaceae) in Venezuela (Rossman 1987). On Meliola sp. on leaves of Coffea arabica (Rubiaceae) in Benin (this study). C. arabica is a new host of M. meliolicola, and the hyperparasite is recorded here for Benin for the first time.
Etymology -Named for the long appendages of the ascospores.
Known distribution -On colonies of Meliola sp. on living leaves of Angylocalyx oligophyllus (Fabaceae) in Benin.
Notes -The genus Paranectria was proposed by Saccardo (1878), with P. affinis as type species, a wood-inhabiting species of the Hypocreales (Sordariomycetes). The genus initially comprised species with hyaline, 3-septate ascospores that carry appendages at both tips (Rossman 1987). Based on this description, Stevens (1918), Hansford (1941Hansford ( , 1946 and other authors proposed new species in this genus, all with fungicolous lifestyle. However, none of these authors seemed to notice that many of these fungi have bitunicate asci, a feature that is present in the Dothideomycetes and not in the Sordariomycetes. Therefore, Pirozynski (1977) transferred many of these species to the genus Paranectriella (P. Henn.) Piroz., a genus that comprises eight species of tropical hyperparasites of plant parasitic fungi that resemble Paranectria, but differ fundamentally in possessing bitunicate asci. In addition to this, cells of perithecial walls of species of Bionectriaceae and Nectriaceae (Hypocreales) typically are thin-walled and elongated parallel to the surface of the perithecia as seen in longitudinal sections (Rossman et al. 1999), while corresponding cells of species of Paranectriella are isodiametric (see the examples of Paranectriella hemileiae and Paranectriella minuta below).
Paranectria longiappendiculata (specimens MB169, MB175) resembles species of the genera Paranectria and Paranectriella by the fungicolous lifestyle and partly 3-septate ascospores with appendages at the tips. In comparison to Paranectria affinis (spores 24-34 μm long; Saccardo 1878), the ascospores of P. longiappendiculata are shorter (up to 21 μm long). Paranectriella hemileiae and Paranectriella minuta produce hairs on the surface of the ascomata like P. elongata, but P. elongata differs by ascospores with long terminal appendages that can reach a length of up to 40 μm. Appendages of all the other known species of Paranectria and Paranectriella only reach up to 20 μm (Saccardo 1878;Rossman 1987). Asci were not found in the examined specimens, so it is not possible to assign them to Sordariomycetes or Dothideomycetes based on details of the walls of asci. The cells of the asci of P. longiappendiculata, however, resemble those of species of hypocrealean fungi within the Bionectriaceae and Nectriaceae (Rossman et al. 1999).
Sequence data -The LSU rDNA sequence obtained from fresh material of P. longiappendiculata (specimen MB175) is 811 bp long. Based on a MegaBLAST search in the NCBI GenBank nucleotide database using the LSU sequence data of P. longiappendiculata, the closest match was Acremonium acutatum (GenBank MH872055; identities 726/799, i.e., 90.86%), as well as other species of hypocrealean fungi. The morphological features discussed above, together with the results of the MegaBLAST search, confirm the placement of P. longiappendiculata in the Hypocreales and in the genus Paranectria. In the tree inferred from the analysis of LSU sequences of 24 specimens (Fig. 4), P. longiappendiculata is located within a strongly supported clade that comprises sequences of Acremonium spp. and other species within the Bionectriaceae.
Known hosts and distribution -On sori of Hemileia vastatrix (Pucciniales) on leaves of Coffea robusta (Rubiaceae) in Uganda (Rossman 1987); on Meliola sp. on leaves of Xylopia frutescens (Annonaceae) in Panama (this study). Meliola sp. and X. frutescens are new hosts of P. hemileiae, and the hyperparasite is recorded here for mainland America (Panama) for the first time.
Specimens Known hosts and distribution -On Meliola paulliniae on leaves of Paullinia pinnata (Sapindaceae) in Uganda (Hansford 1941); on Meliola sp. on leaves of Coffea arabica (Rubiaceae) in Benin (this study); on Meliola sp. on leaves of Opilia celtidifolia (Opiliaceae) in Benin (this study). C. arabica and O. celtidifolia are new hosts of P. minuta, and the hyperparasite is recorded here for Benin for the first time.
Notes -Paranectriella minuta is similar to P. hemileiae, but the ascomatal hairs of P. minuta are located mostly close to the apex of the pseudothecium. The presence of appendages on the ascospores and small, translucent ascomata can also occur in some species of the genus Hyalocrea, but Hyalocrea spp. are characterized by the absence of pseudoparaphyses (Rossman 1987). Pseudoparaphyses, however, were not found in the specimen examined (AK38H). Nevertheless, we identify the specimens from Benin as P. minuta, because the ascospores of these specimens are smaller than those of hyperparasitic species of Hyalocrea (e.g., H. meliolicola, 26-35 × 7-9 μm; Rossman 1987).
Sequence data -The LSU rDNA sequence obtained from fresh material of P. minuta (specimen AK38H) is 494 bp long. Based on a MegaBLAST search in the NCBI GenBank nucleotide database using the LSU sequence data of P. minuta, the closest match was Quixadomyces hongheensis (GenBank MW264194; identities 460/491, i.e., 93.69%), as well as other species of Pleosporales. Hyde et al. (2013) designated the family Paranectriellaceae to accommodate hyperparasitic species of Dothideomycetes with bright colored ascomata, ascospores with transverse septa and prominent appendages. However, there is no molecular DNA sequence data that supports this designation. Ours represent the first DNA sequence of a fungus of the genus Paranectriella, and more sequences are necessary to evaluate this hypothesis.

Discussion
Hyperparasitic fungi on Meliolales have been collected in the past mainly in Brazil, Dominican Republic, and Puerto Rico in America, as well as in Ghana, Sierra Leone, and Uganda in Africa (Bermúdez-Cova et al. 2022). In the context of the present study, we analyzed 16 specimens of Meliolales associated with hyperparasites, corresponding to eight species of hyperparasitic fungi. Seven species represent new records: five for Benin and four for Panama. One species is new to science. Calloriopsis herpotricha is recorded for the first time for Africa and Dimerosporiella cephalosporii and Paranectriella hemileiae for mainland America. These findings are based on only three months of fieldwork and show a blatant lack of investigation on hyperparasitic fungi in the tropics.
Patterns of distribution of hyperparasitic fungi have been studied mainly for hyperparasites of rusts and powdery mildews (Zewdie et al. 2021), but never for those infecting black mildews. The distribution of hyperparasitic fungi is restricted to that of their host (Sun et al. 2019). As Meliolales are restricted to tropical and subtropical areas (Piepenbring 2015), hyperparasites are expected to be found in these regions as well. We also expect wide distribution areas of hyperparasitic fungi on Meliolales because of their broad spectra of host species (Bermúdez-Cova et al. 2022). In fact, the data presented in this study suggest that at least part of the species of hyperparasitic fungi of Meliolales have a pantropical distribution, as they have been recorded both in paleotropical and neotropical regions. This is consistent with the assumptions made by Samuels et al. (2002) regarding the pantropical distribution  (AK4H, AK38H). a Pseudothecia on black hyphae of Meliola sp. on a living leaf of Coffea arabica; b pseudothecium on hyphae of Meliola sp.; c ascus with ascospores; d ascospores. Scale bars: 500 μm (a); 15 μm (b); 10 μm (c); 3 μm (d) of tropical perithecioid fungi. Extensive additional fieldwork is needed in order to unravel distribution patterns of hyperparasitic fungi on meliolalean hosts.
It is difficult to obtain molecular sequence data from hyperparasites especially because of their incapability of growing in artificial media and the fact that they develop intermingled with the primary parasite and many other organisms (Bermúdez-Cova et al. 2022). As a consequence, isolating and sequencing hyperparasitic fungi is a challenging task. There is also a lack of sequences of hyperparasitic fungi in public databases. Therefore, the sequences obtained in the context of the present work can be related to existing species concepts only based on morphology, and issues such as anamorph-teleomorph connections cannot be confirmed. Nevertheless, in this study for the first time ever, DNA sequences of hyperparasitic fungi on Meliolales are published. This example emphasizes that field work paired with molecular analysis still plays a crucial role for modern mycology, especially for challenging fungal groups, such as hyperparasites.