An overview on the mycoparasitic Piptocephalis (Zoopagomycota): taxonomic notes and geographic distribution with new occurrences for South America

ABSTRACT Piptocephalis includes mycoparasitic fungi, mainly targeting mucoralean species. Until now, there has been no compilation of data on the taxonomy and geographic distribution of Piptocephalis, which is a barrier to the proper identification of species of this genus by taxonomists. The present study provides an overview of Piptocephalis with taxonomic and occurrence data, in addition to reporting P. graefenhanii and P. xenophila for the first time in South America. Both species were observed parasitizing Mucor spp. The P. graefenhanii was observed growing on paca dung and P. xenophila was observed on guinea-pig dung in Recife, northeastern Brazil. Aspects of their morphology are discussed and a key for the genus is presented.


Introduction
Zoopagomycota comprises fungi parasitic of protozoa (e.g., amoebae), small animals (e.g., rotifers or nematodes) and other fungi, mostly of Mucorales (Mucoromycota) and Mortierellales (Mortierellomycota), rarely infecting species of Ascomycota (Tedersoo et al. 2018;Davis et al. 2019).This phylum includes the Zoopagales, with five families, among which Piptocephalidaceae is the best known (Reynolds et al. 2019).Piptocephalidaceae comprises both Piptocephalis and Syncephalis (Wijayawardene et al. 2022) that are merosporangipherous haustorial fungi that form merosporangia on terminal vesicles, with merospores remaining dry or released in a droplet of fluid at maturity.Zygospores are formed from the union of gametangia produced terminally by apposed or coiled progametangia (Benny et al. 2016).
Piptocephalis species form slender vegetative hyphae, with small appressoria which penetrate the host wall, giving rise to restricted, delicate, and branched haustoria (Benjamin 1959).Merosporangiophores are erect, septate, smooth or longitudinally striate, dichotomously branched several times where the ultimate branch ends in a head cell supporting few to many merosporangia containing one or more merosporangiospores (Ho 2004).Although Piptocephalis has a worldwide distribution (Fig. 1), the majority of species of this genus cannot be confirmed as cosmopolitan due to a lack of inventories of these fungi (Reynolds et al. 2019).For South America, records of Piptocephalis are restricted to Argentina, Brazil and Colombia (GBIF 2022).
Although sequences of Piptocephalis species are available in the GenBank database, identification of this genus has been mainly based on morphological characters due to the obligate parasitic nature of these fungi (Ho 2004;2006).Although we recognize the importance of molecular biology to separate species of Piptocephalis, we believe that morphological identification is also a reliable tool for the identification of these species.Reynolds et al. (2019), after a morphological and phylogenetic (28S and ITS rDNA regions) study of Piptocephalis observed that the Piptocephalis strains formed a monophyletic group with several distinct clades corresponding to morphologically identified species, which indicates that morphological features are sufficient to identify species of Piptocephalis.However, the last morphological key for identification of Piptocephalis was provided by Gräfenhan (1998), not including species described in the last 24 years (until March 2023).
The present study aims to provide an overview of Piptocephalis with taxonomic, ecological and occurrence notes of all species, as well as to illustrate P. xenophila and P. graefenhanii (new records for South America), therefore expanding knowledge on geographic distribution of this still poorly studied genus.In order to fill a gap in time since the last identification key of Piptocephalis was produced, we provide a morphological identification key for the species known until March 2023.

Material and methods
Occurrence and substrates data -GenBank, UNITE and GBIF databases were used to access distribution and substrates information of Piptocephalis spp.
Observation and identification of Piptocephalis spp.-Samples of Cuniculus paca Linnaeus (paca) and Cavia porcellus Linnaeus (guinea-pig) dung were collected in the Parque Estadual de Dois Irmãos, an Ecological Reserve located in the city of Recife, state of Pernambuco, Brazil (Fig. 1).Dung samples were collected with a spatula previously sterilized in 70% alcohol, placed in plastic bags and taken to the laboratory where they were incubated in Petri dishes containing two filter paper sheets moistened with sterile distilled water at room temperature (± 28 ℃) for seven days in alternate periods of light and dark.After four days, merosporangiophores of Piptocephalis were observed growing directly from hyphae of Mucor sp.Microscopic slides were prepared directly from samples using Amann's Cotton Blue containing both Piptocephalis and its host and observed under a light microscope (Leica MD 500).The identification was based on microstructural characters according to Gräfenhan (1998) and Ho (2003Ho ( , 2006)).Since Piptocephalis spp.could not be successfully grown on corn An overview on the mycoparasitic Piptocephalis (Zoopagomycota): taxonomic notes and geographic distribution with new occurrences for South America meal agar (CM), malt extract agar (MEA 2%), malt extractyeast agar (MEYE) and potato dextrose agar (PDA) media, permanent slides prepared directly from the moist chambers using 50% glycerol containing a small amount of Amann's cotton blue (Benny 2008) were deposited in the URM Herbarium of the Universidade Federal de Pernambuco.
Notes: Piptocephalis graefenhanii is reported for the first time in South America from paca dung.Until now, P. graefenhanii had only been reported from soil.It is morphologically similar to P. lepidula.The main feature common to both species is the formation of apical merospores by budding from a basal merospore.However, unlike merosporangiophores of P. graefenhanii, which branch up to six times and are not striated, merosporangiophores of P. lepidula may branch up to eight times and exhibit striation.The primary branching of P. graefenhanii merosporangiophores is longer (from 1800 µm in length) than the primary branch of P. lepidula (up to 700 µm in length).Additionally, there are differences in the number and size of the merosporangia between both species.Piptocephalis graefenhanii head cells sustain 4-14 merosporangia, while head cells of P. lepidula sustain from 15-30 merosporangia.
Habitat and geographic distribution: This species was previously reported on animal dung from the United States of America; leaf litter from the United Kingdom; soil from Afghanistan, Canada, Estonia, Taiwan, the United Kingdom and the United States of America.It has also been reported in Afghanistan, Australia and Japan without substrate indication (Dobbs and English 1954;Gräfenhan 1998;GBIF 2022).This species can parasitize Mucor sp.(Gräfenhan 1998).
Notes: Piptocephalis xenophila is here reported for the first time to South America from guinea-pig dung.Until now, P. xenophila had only been reported from soil.Merosporangiophores of this species form heart-shaped head cells like ones of P. microcephala and P. indica.However, the merosporangiophores of P. xenophila form up to seven dichotomies, different from the ones of P. microcephala which form up to 11 and the ones of P. indica which form up to eight dichotomies.The head cells of P. xenophila sustain up to 12 merosporangia, while those of P. microcephala and P. indica support up to six merosporangia.Furthermore, unlike P. xenophila, merospores of P. microcephala remain in a drop of fluid at maturity.Additionally, P. xenophila forms cylindrical merospores, while merospores of P. microcephala and P. indica are doliform.
Habitat and geographic distribution: This species was previously reported on animal dung from Austria, Germany, Ireland, the United Kingdom and the United States of America.Piptocephalis arrhiza has also been reported in Belgium and Sweden without substrate indication (Gräfenhan 1998;GBIF 2022).This species can parasitize C. recurvatus (Gräfenhan 1998).
Habitat and geographic distribution: This species was previously reported on animal dung from India and Pakistan (Gräfenhan 1998;Genbank 2022).Piptocephalis brijmohanii has also been reported in the Netherlands (from glass jar) and China without substrate indication (GBIF 2022).This species can parasitize C. recurvatus (Gräfenhan 1998).
An overview on the mycoparasitic Piptocephalis (Zoopagomycota): taxonomic notes and geographic distribution with new occurrences for South America Habitat and geographic distribution: This species was previously reported from soil, leaf litter and animal dung from the United Kingdom and Ireland; soil from Japan, Taiwan and the United States of America (Gräfenhan 1998;Ho 2004;GBIF 2022;Genbank 2022).It has also been reported in Argentina, Australia, Austria, Belarus, Belgium, Bulgaria, China, Congo, Colombia, Czechia, Finland, Germany, Greenland, Iceland, Iran, Italy, Kenya, Kyrgyzstan, Mexico, Morocco, South Africa, Thailand, Russia, Sweden and Switzerland without substrate indication (GBIF 2022).Piptocephalis fimbriata can parasitize C. recurvatus (Gräfenhan 1998) and Mucor sp.(Ho 2004).
Habitat and geographic distribution: This species was previously reported from soil in the United States of America and Taiwan (GBIF 2022;Ho & Kirk 2009).It has also been reported in Afghanistan without substrate indication (GBIF 2022).It can parasitize Mucor sp.(Ho & Kirk 2009).
Habitat and geographic distribution: This species was previously reported on animal dung from Brazil, the United Kingdom and the United States of America; soil from the United States of America (Gräfenhan 1998;GBIF 2022;Genbank 2022).It has also been reported in Germany and Morocco without substrate indication (GBIF 2022).Piptocephalis freseniana can parasitize C. recurvatus and Mucor sp.(Gräfenhan 1998).
Habitat and geographic distribution: This species was previously reported on animal dung from India (Mehrotra & Baijal 1964;Gräfenhan 1998); soil from Malaysia, the United States of America and Taiwan (Ho 2003;Genbank 2022).It has also been reported in Afghanistan without substrate indication (GBIF 2022).Piptocephalis indica can parasitize of C. recurvatus (Gräfenhan 1998) and Cunninghamella sp.(Ho 2003).
Habitat and geographic distribution: Piptocephalis lemonnieriana has been reported in France as a parasite of Mucor fragilis Bainier, which was found growing on a birch stump (Vuillemin 1902).It has also been reported on animal dung from Brazil (Santiago et al. 2011).
Habitat and geographic distribution: This species was previously reported on animal dung from Pakistan (Gräfenhan 1998).
Habitat and geographic distribution: This species was previously reported from soil in Japan (Gräfenhan 1998;GBIF 2022).It has also been reported in Australia, Belarus, Finland, Kyrgyzstan, Russia and the United States of America without substrate indication (GBIF 2022).This species can parasitize Mortierella humilis (Gräfenhan 1998).
Habitat and geographic distribution -This species was previously reported from soil in Japan (Gräfenhan 1998;GBIF 2022).This species has also been reported in Panama and Thailand without substrate indication (GBIF 2022).

Piptocephalis sphaerospora
An overview on the mycoparasitic Piptocephalis (Zoopagomycota): taxonomic notes and geographic distribution with new occurrences for South America Notes: According Van Tieghem (1875), this species is characterized by the formation of striate merosporangiophores with 2-3 successive dichotomous branches, globose head cells, merosporangia containing 5-8 globose merospores, which are 2-3 µm in diam.Zygospores were not observed.Spore heads were not indicated in the protolog.

Piptocephalis tieghemiana
Habitat and geographic distribution: This species was previously reported on animal dung from India, Pakistan, Taiwan and the United States of America; beetle larvae from Japan; hog feed and lizard eggs from the United States of America and soy sauce from China (Gräfenhan 1998, GBIF 2022, Genbank 2022).It has also been reported in the Netherlands and South Africa without substrate indication (Gräfenhan 1998).It can parasitize C. recurvatus, Lichtheimia ramosa, Mucor hiemalis, M. racemosus and Rhizopus arrhizus (Gräfenhan 1998;GBIF 2022).

Discussion
In this study we considered 24 Piptocephalis species as valid.Gräfenhan (1998) considered P. dichotomica, P. fusispora, P. lemonnieriana, P. monospora and P. sphaerospora as doubtful.In Benjamin (1959), Gräfenhan (1998) and in this work, P. monospora was not considered as valid because the morphological description by Mangin (1899) was not informative enough to differentiate this species from other Piptocephalis species.In the same way, P. dichotomica described by Krzemieniewska & Badura (1954) was not considered valid in this study.However, morphological features of P. fusispora, P. lemonnieriana and P. sphaerospora are enough to differentiate these species, which is why they were recognized as valid species by Benjamin (1959) and Zycha et al. (1969).In this study we also considered those species as valid despite the fact that Gräfenhan (1998) did not.
According to Reynolds et al. (2019), species of Piptocephalis formed a well-supported monophyletic group with 25 clades including five still unpublished species.Although they concluded that morphological characters are not phylogenetically informative at the generic level, they are sufficient to separate species of Piptocephalis.Among the main morphological characters for identification of Piptocephalis are the following: spore heads (if they are wetor dry-spored), head cell shape, striation and ramification An overview on the mycoparasitic Piptocephalis (Zoopagomycota): taxonomic notes and geographic distribution with new occurrences for South America of merosporangiophores, including pigmentation, diameter, and texture of zygospores (Benjamin 1959;Curtis et al. 1978;Gräfenhan 1998).Among the Piptocephalis species it is known that 10 species form wet spore heads.The spore head are easily observed macroscopically in cultures.This structure was not cited in the descriptions of P. fusispora and P. sphaerospora (Gräfenhan 1998).
Most species of Piptocephalis form striate merosporangiophores, except P. curvata, P. graefenhanii, P. minuta and P. moniliformis.The presence or absence of stolons and rhizoids was used by Van Tieghem (1875) to classify Piptocephalis species.Nevertheless, absence, presence, shape and size of both structures vary according to the substrate, growth temperature and host (Curtis et al. 1978), thus they should not be used to differentiate Piptocephalis species (Benjamin 1959;Gräfenhan 1998).Leadbeater and Mercer (1957) and Benjamin (1959) also indicated zygospore morphology as an important characteristic for the identification of Piptocephalis species.Nonetheless, this structure has not been observed in P. cruciata, P. lemonnieriana, P. macrocephala, P. pseudocephala and P. sphaerospora.In addition, zygospore formation may require specific conditions, thus we decided not to include zygospore characteristics in our taxonomic key.
To the best of our knowledge, species of Piptocephalis have been reported in 43 countries, in temperate (most species) and tropical zones, with the highest number of species reported in the United States of America (10 species), the United Kingdom (eight), Japan (six), and Taiwan (eight).For 12 countries only one species of Piptocephalis has been documented (Fig. 3).Reynolds et al. (2019) considered that some Piptocephalis species have a worldwide distribution, while also highlighting the possible endemism of some species.For example, P. cruciata, P. macrocephala, P. pseudocephala, P. sphaerospora and P. unispora have only been reported once and from just one country (Gräfenhan 1998;GBIF 2022), and the fact that these species have not been found again may suggest that they are endemic in their countries.However, it is difficult to predict whether the majority of the known species belonging to this genus are endemic or not, because inventories of Piptocephalis are rare.This is probably due to the insufficient number of taxonomists specialized in Zoopagales.What we know so far is that all species of Piptocephalis described until March 2023 occur in the temperate zone (Fig. 1), and P. arrhiza, P. benjaminii, P. cruciata, P. fusispora, P. macrocephala, P. microcephala, P. sphaerospora, P. pseudocephala and P. tieghemiana have exclusively been reported in this zone.Fourteen species also occur in the tropical zone but there is no species exclusively observed in the tropics.
Piptocephalis species are mostly reported in soil and dung.However, some species have been reported in leaf litter and other substrates (Gräfenhan 1998).According to Richardson & Leadbeater (1972), the frequent occurrence of Piptocephalis species on dung does not correspond to susceptibility of this substrate to mycoparasites, but to the high prevalence of mucoralean fungi that are common hosts of Piptocephalis on dung, which may also be valid for soil and leaf litter.
The low number of species reported in South America is probably related to the scarcity of studies focused on zoopagalean fungi.Herein we report the first occurrence of two Piptocephalis species in South America, both parasitizing Mucor sp. on herbivore dung, contributing to knowledge of these fungi in the tropical zone.

Mateus Oliveira da Cruz, Leslie Waren Silva de Freitas, Francisca Robervânia Soares dos Santos, Cristina Maria de Souza-Motta and André Luiz Cabral Monteiro de Azevedo Santiago
, P. fusispora has been also reported in China, Italy, the Netherlands and Poland without substrate indication.