Hypomyces pseudolactifluorum sp. nov. (Hypocreales: Hypocreaceae) on Russula sp. from Yunnan, PR China

Abstract Background Hypomyces is a large genus of fungicolous fungi, parasitising the fruiting bodies of Agaricales, Boletales, Helotiales, Pezizales and Polyporales. Hypomyces currently comprises of 147 species widely distributed in Australia, China, France, Germany, Italy, Japan, North America, Sri Lanka, Thailand and UK. Amongst them, 28 species have been recorded in China. New information Hypomyces pseudolactifluorum sp. nov., growing on the fruiting bodies of Russula sp. in subsect. Lactarioideae and collected from Yunnan, China, is described with illustrations and molecular phylogenetic data (combined ITS, LSU, TEF1-α and RPB2 sequence dataset). The new species is characterised by semi-immersed to immersed perithecia and fusiform, apiculate and verrucose ascospores. We also review the species diversity of the genus Hypomyces in China.


Introduction
Fungicolous fungi are a large and diverse ecological group, currently containing more than 1500 taxa distributed in many lineages across the fungal kingdom (Põldmaa 2011, Sun et al. 2019a. Hypomyces (Fr.) Tul. & C. Tul. is an important genus of fungicolous fungi and placed in the family Hypocreaceae (Hypocreales, Sordariomycetes, Ascomycota) (Hyde et al. 2020). Hypomyces was originally introduced as a subgenus of Hypocrea Fr. (Fries 1825) and then Tulasne and Tulasne (1860) revised it to a genus and designated H. lactifluorum (Schwein.) Tul. & C. Tul. from the USA as its type. Hypomyces parasitises the fruiting bodies of Agaricales, Boletales, Helotiales, Pezizales and Polyporales (Rossman et al. 1999, Tamm and Põldmaa 2013, Sun et al. 2019a. Hypomyces is characterised by: superficial or immersed, spherical to ovate, pyriform, papillate and yellow, orange, tawny red or green perithecia in a subiculum; 8-spored, subcylindrical to cylindrical and with a thickened apical asci; and ellipsoid, lanceolate, fusiform to navicular, 0-1-septate or rarely 3-septate, hyaline, spinulose or verrucose and smooth-walled ascospores (Rossman et al. 1999, Zeng andZhuang 2015). Its allied genera include Cladobotryum Nees, Mycogone Link, Sepedonium Link and Stephanoma Wallr (Wijayawardene et al. 2017) and its asexual morphs are Acremonium-, Dactylaria-, Papulaspora-, Trichothecium-or Verticillium-like (Jaklitsch et al. 2006, Hyde et al. 2020. Hypomyces currently comprises of 147 species in Species Fungorum (http://www.speciesfungorum.org/, accessed in April 2020) and is widely distributed in Australia, China, France, Germany, Italy, Japan, North America, Sri Lanka, Thailand and UK , Rossman et al. 2013, Zeng and Zhuang 2016, Zare and Gams 2016, Lechat et al. 2017, Wei and Kirschner 2017, Sun et al. 2019a, Sun et al. 2019b, Zeng and Zhuang 2019. Amongst them, 28 species have been reported in China (Table 1). Fungicolous fungi play important roles in the processes of the growths and degradations of their hosts. With the rapid development of mushroom industries, the fungicolous fungi on mushrooms have received more and more attention (Hyde et al. 2019). In this paper, we introduce a new member of fungicolous fungi, Hypomyces pseudolactifluorum sp. nov., on the fruiting bodies of Russula sp., collected from Yunnan Province, China. At the same time, we review the species diversity of the genus Hypomyces in China.

Collections and Morphology
Hypomyces specimens, including their host mushrooms, were collected in an evergreen broad-leaved forest in Baihualing, Baoshan, Yunnan Province, China. The specimens, as well as collected host mushrooms, were placed on a piece of aluminium foil at first, then rolled the paper into a cylinder, twisted at the ends for sealing and lastly taken back to the laboratory for study (McKnight and McKnight 1997). Colour codes were recorded following those of Kornerup and Wanscher (1978). A Nikon Coolpix P510 camera was used to take photos in the wild. Dried specimens were observed and photographed using an Olympus SZ61 stereomicroscope and a Nikon ECLIPSE Ni compound microscope fitted with a Canon EOS 600D digital camera. Measurements were made using the Tarosoft® Image Frame Work programme v.0.9.7. The colour change of the perithecial wall was tested using 5% potassium hydroxide (KOH). Type specimens are deposited at the Herbarium of Mae Fah Luang University, Thailand (MFLU) and the Herbarium of Cryptogams Kunming Institute of Botany, Chinese Academy of Sciences, PR China (HKAS).
PCR was performed in a 25 μl reaction volume: 12.5 μl Taq PCR Master Mix (Abmgood, Richmond, BC, Canada), 1 μl forward primer, 1 μl reverse primer, 1 μl DNA template and 9.5 μl ddH O. For ITS and LSU, PCR reaction conditions are: 8 min at 94ºC, followed by 30 s at 94ºC, 30 s at 52ºC and 1 min at 72ºC for 35 cycles and a final extension of 10 min at 72ºC. PCR reaction conditions of TEF1-α and RPB2 are: 8 min at 94ºC, followed by 1 min at 95ºC, 45 s at 59ºC for RPB2/55ºC for TEF1-α and 1 min at 72ºC for 35 cycles and a final extension of 10 min at 72ºC. The PCR products were detected using agarose gel electrophoresis and, in the gel documentation system, clear bands were observed. Sequencing was performed by Sangon Biotech (Shanghai) Co. Ltd., PR China; partial impure products were purified using the Cycle-pure-kit (Omega, America) and then cloned into pClone007 Simple vector (TSV-007S from Beijing TsingKe Biotech). Twenty clones of PCR products of each gene were sequenced using the universal primer pairs M13-47/ M13-48.

Sequence alignment and phylogenetic analyses
The parasitic fungus: Hypomyces pseudolactifluorum sp. nov.

Diagnosis
The new species is similar to Hypomyces lactifluorum on Russula and Lactarius spp. from North America (Rogerson and Samuels 1994), but has smaller perithecia and shorter asci. The main differences of the two species are compared in Table 4.

H. pseudolactifluorum
Subiculum Pale yellowish-orange to bright orange (young), in age becoming deep red, reddish-purple to very dark purple (old), occasionally fading to pink, turning purple in 3% KOH.

Etymology
Referring to the most closely-related species, Hypomyces lactifluorum.

Host
On the fruiting bodies of Russula sp. that grew on the humus layer in an evergreen broad-leaved forest of a rainforest. The host mushrooms: basidiocarps medium-sized and infundibuliform, pilei 63−77 mm in diameter. As serious degradation has occurred, the colour and other characters of the host mushrooms cannot be determined. Molecular phylogenetic evidence indicates it is a Russula species.

Notes
Only sexual morph had been discovered on the hosts (Russula sp.) of the new species.  ML tree of Hypomyces pseudolactifluorum sp. nov. and its allies generated from a combined ITS, LSU,TEF1-α and RPB2 gene sequence dataset. Supporting values of MLBP (left, greater than 75%) and BIBP (right, greater than 0.9) are shown at the nodes, respectively. The new species is marked in red.

Analysis Phylogenetic analyses
In the phylogenetic tree, the parasitic fungi MFLU 20-0265 and MFLU 20-0266 are clustered together and formed a distinct lineage with the same branch length and strong supportive values (MLBP = 100%, BIPP = 1), which support them to be conspecific. The parasitic fungi are closely related H. lactifluorum and they form a sister clade also with strong supportive values (MLBP = 100%, BIPP = 1). Comparing the gene sequences of the two species, there are 25 bp (4.3%) differences across 582 bp in ITS, 28 bp (3.2%) differences across 870 bp in LSU, 24 bp (2.6%) differences across 921 bp in TEF1-α and 24 bp (3.2%) differences across 739 bp in RPB2 (Suppl. material 1). Following the recommendations from Jeewon and Hyde (2016), we assign the parasitic fungi as H. pseudolactifluorum sp. nov.
The host mushroom: Russula sp.
According to the ITS phylogenetic tree of the host mushroom and its allies, the host mushroom (MFLU 20-0265) is clustered together with Russula leucocarpa (HGAS-MF 009910 and HGAS-MF 009916) (MLBP = 100%) in subsect. Lactarioideae. However, their ITS sequences have 24 bp (3.5%) differences across 694 bp, which indicated they may be two distinct species. Due to lack of sufficient morphological evidence, the host mushroom was temporarily identified as Russula sp. (Fig. 3).