Two new rare species of Candolleomyces with pale spores from China

Abstract Most species of Candolleomyces have brown or dark brown spores. Although pale-spored members are rare in the genus we frequently collected two such species from many Provinces during our investigations in subtropical China from 2016–2020. As revealed by morphological characterisation and multigene phylogenetic analyses (ITS LSU β-tub and tef-1α) these species which we have named C. subcacao and C. subminutisporus are unique and distinct from known taxa. In addition a new combination C. cladii-marisci is proposed on the basis of ITS sequence analysis of the type specimen. Detailed descriptions colour photos illustrations and a key to related species are presented.

According to the research of Wächter and Melzer (2020), Candolleomyces may be more speciose than previously thought and better delimitation of species boundaries is needed. Although controversies still exist regarding some species boundaries, the number of new taxa is steadily increasing (Melzer et al. 2018;Sicoli et al. 2019a;Büttner et al. 2020). This continuous discovery of new taxa with clear boundaries deepens understanding of the species in this genus.
Approximately eight taxa in the genus Candolleomyces have previously been reported from China (Yan 2018). During our investigations in subtropical China from 2016-2020, we frequently collected two unknown Candolleomyces species with pale spores from many Provinces. Spores that are pale or almost colourless in water and 5% potassium hydroxide (KOH) are very rare in this genus. On the basis of our morphological and phylogenetic analyses, the specimens are described as new species in this paper.

Morphological studies
Specimens were deposited in the Herbarium of Mycology, Jilin Agricultural University (HMJAU) and the Herbarium of Fungi, Jiangxi Agricultural University (HFJAU). Macromorphological characters and habitat details were recorded from fresh basidiomata. Colour codes were based on the Methuen Handbook of Colour (Kornerup and Wanscher 1978). More than 30 spores, cystidia and basidia in water and 5% aqueous KOH were measured under a microscope. In subsequent descriptions, measurements are shown as (a)b-c(d), where a is the lowest value, b-c encompasses at least 90% of values and d is the highest value, while Q is the length-width ratio of a spore (Bas 1969;Yu et al. 2020).

Data analyses
Taking into consideration the results of BLAST searching against GenBank and the research of Büttner et al. (2020) and Wächter and Melzer (2020), we analysed ITS, LSU, tef-1α (Tef 1 st , Tef 2 nd and Tef 3 rd ) and β-tub (Tub 1 st and Tub 2 nd ) sequences from 37 taxa. Details are presented in Table 1. Sequences were aligned using the online version of the multiple sequence alignment programme MAFFT v.7 (Katoh and Standley 2013), followed by manual adjustment in BIOEDIT v.7.1.3.0 (Hall 1999 Note: Newly-generated sequences are in bold. for 10 million generations, with sampling every 100 th generation and with the first 25% of trees discarded as burn-in (Ronquist et al. 2012). ML analyses were undertaken by applying the ultrafast bootstrap approximation with 1000 replicates. The sequence alignment has been deposited in TreeBASE (S28074).
Habit and habitat. Scattered on rotten wood or humus in Pinus massoniana and oak forests.  Note. According to the ITS phylogenetic analysis including the type specimen, P. cladii-marisci belongs to Candolleomyces and has a close phylogenetic relationship with C. candolleanus, C. badiophyllus and C. trinitatensis. In addition, the morphological characteristics of this species correspond to Candolleomyces, which lack pleurocystidia. For detailed descriptions and line drawings of this species, see Sicoli et al. (2019a;b).

Discussion
Most species of Candolleomyces have dark brown or brown spores, whereas species with pale spores are rare. Candolleomyces subcacao is very easily confused with C. cacao in the field because of their similar macroscopic characteristics. In addition, these two species have highly similar ITS regions (98%). Nevertheless, some members of Candolleomyces with high ITS similarity are still treated as separate species on the basis of morphological characters (Sicoli et al. 2019a;Büttner et al. 2020;Wächter and Melzer 2020). Candolleomyces subcacao and C. cacao group together, but comprise independent lineages, in the phylogenetic tree (Fig. 1). Moreover, C. cacao has ventricose to broad lageniform cheilocystidia, an indistinct germ pore in 5% KOH and a tropical distribution (Desjardin 2016). On the basis of morphology, C. subcacao has been classified into Psathyrella sect. Spintrigerae using the classification system of Kits van Waveren (1985;1987) and Psathyrella sect. Subatratae, based on the system of Smith (1972). Some species in these sections lack pleurocystidia and may thus actually belong to Candolleomyces, but molecular analyses of type materials are needed prior to their possible reassignment. In this paper, we have, therefore, only compared these species and the new ones with respect to morphology (see the key below). In particular, two species in these sections possess the combined characteristics of small basidiomata, a pale brown and evanescent veil and pale yellow-brown spores with a distinct germ pore: P. lacuum Huijsman, which can be distinguished from C. subcacao by the presence of a veil with dispersed white arachnoid fibrils or flocci, abundant pyriform cells at the marginal of the lamellae and very rare utriform cheilocystidia (Kits van Waveren 1985;Battistin et al. 2014) and P. cordobaensis A.H. Sm., which differs mainly in having a 10 mm wide pileus, an indistinct germ pore and saccate to ellipsoid cheilocystidia (Smith 1972;Desjardin 2016).
Candolleomyces singeri ( (Fig. 1). These species can be separated as follows: C. singeri has larger spores, mostly 6.8-7.8 μm long (Smith 1972, pers. obs. of HMJUA37867 by JQ Yan), whereas C. eurysporus can be separated on the basis of its broader spores, a Q-value of 1.2-1.6(-1.7) and brown lamellae at maturity (Büttner et al. 2020) and C. aberdarensis is distinguished by having larger spores [7.5-8(-8.8) μm long] (Melzer et al. 2018). In addition, two species are morphologically similar to C. subminutisporus in having more-or-less pale spores, germ pores that are indistinct or lacking and no pleurocystidia. These species can be separated from C. subminutisporus as follows: C. halophilus Finally, P. cladii-marisci was described by Sicoli et. al. (2019) and is characterised by the absence of pleurocystidia and the presence of large spores up to 11 μm long (Sicoli et al. 2019a;b). According to our phylogenetic analysis, this species is relatively closely related to C. candolleanus, C. badiophyllus and C. trinitatensis and should be moved to Candolleomyces. A new combination is thus proposed.