The genus of basidiomycetous fungi Cortinarius occurs worldwide, from subtropical to boreal latitudes. Although molecular systematics has triggered the study of these fungi in the Americas and Europe in the last two decades, there is still limited research on its diversity in large portions of the planet, such as the high mountain ranges of Asia. Several collections of Cortinarius were made during mycological field trips conducted between 2014 and 2018 in pure oak forests in the Pakistan Himalayas. An integrative framework combining morphological and phylogenetic data was employed for their study. As a result, the two species C. pakistanicus and C. pseudotorvus are here described as new to science. Detailed macro- and micro-morphological descriptions, including SEM images of spores, and a molecular phylogenetic reconstruction based on nrITS sequence data are provided and used to discriminate the new species from morphologically and phylogenetically close taxa. Whereas our phylogenetic tree inference gave unequivocal support for the inclusion of C. pseudotorvus within C. sect. Telamonia, the assignment of C. pakistanicus to any known sections remained elusive. These species likely establish ectomycorrhizal associations with trees in the genus Quercus, making this type of forest in the Pakistan Himalayas a promising focus for future research on the diversity of Cortinarius.

Biodiversity, ectomycorrhizal fungi, ITS, phylogeny, systematics, taxonomy

Cortinarius (Pers.) Gray (Cortinariaceae) is a relatively well known mushroom-forming genus of basidiomycetous fungi characterized by the fugacious veil forming a fine cobweb (“cortina”) between the stipe and pileus margin and by the production of ornamented, cinnamon brown to fulvous basidiospores (Kirk et al. 2008; Niskanen 2008). It is one of the most species rich, abundant and widespread ectomycorrhizal genera in Agaricales (Geml et al. 2012; Nouhra et al. 2013; Nuske et al. 2019), encompassing ca. 3000 species worldwide (Niskanen et al. 2018). Whereas the distributional ranges for many species are known to be rather restricted (e.g. Ballarà et al. 2017; Soop et al. 2019), other species occur across several regions and continents (Dima et al. 2014; Harrower et al. 2015; Liimatainen et al. 2017). Furthermore, evidence gained in recent years due to the use of high-throughput sequencing techniques has shown that Cortinarius establishes ectomycorrhizal associations with a myriad of plant hosts, including not only members in the important families Fagaceae, Betulaceae, Pinaceae, Salicaceae and Cistaceae, but also some herbaceous plants in the Cyperaceae and Polygonaceae (Geml et al. 2012; Horton et al. 2017; Pérez-Izquierdo et al. 2017; Nuske et al. 2019). However, the vast majority of systematic studies on Cortinarius have been conducted in Europe and the Americas, and more recently in Oceania (Australia, Tasmania and New Zealand), and therefore little is known about its diversity and range of plant hosts in other areas of the planet. In particular, we know virtually nothing about the species of Cortinarius growing in forests in the high mountain ranges north of Pakistan, which belong to the Himalayas.

Taxonomic studies on Cortinarius throughout the whole of Pakistan are in fact scant. Ahmad et al. (1997) reported C. bulliardii (Pers.) Fr., C. cinnamomeus (L.) Gray, C. hinnuleus Fr., and C. phoeniceus (Vent.) Maire from moist temperate forests of Abies, Cedrus, Picea and Pinus in Khanspur, Shogran, Dungagali and adjoining areas of the Pakistan Himalayas. After more than 20 years, Saba et al. (2017) described C. longistipitatus Saba, S. Jabeen, Khalid & Dima as a new species in C. subgenus Telamonia sect. Cinnabarini based on phylogenetic data. This species was collected in mixed Cedrus deodara and Pinus wallichiana forests in the Hazara and Malakand divisions. Recently, C. brunneocarpus Razaq & Khalid has been described from Khanspur growing in a mixed P. wallichiana and Abies pindrow forest, and the phylogenetic analysis found it in C. section Hinnulei (Song et al. 2019). In the same work, an additional species, C. lilacinoarmillatus Semwal & Dima, was described from the Indian Himalayas. All in all, further research on the diversity of this genus in the whole region is required. The aim of the present work is to provide new insights about the diversity of Cortinarius species associated with oaks in the Pakistan Himalayas.

Several collections of Cortinarius were sampled and preliminarily assigned to C. subgenus Telamonia (Fr.) Trog. during a 2014–2018 exploration campaign to oak forests from the Khyber Pakhtunkhwa province (Swat District, Pakistan, Fig. 1). This subgenus initially comprised a very large number of species, but thanks to the advent of molecular techniques, we now know that it is polyphyletic (Høiland and Holst-Jensen 2000; Niskanen et al. 2013a; Soop et al. 2019). Here, we combine data of the nuclear ribosomal internal transcribed spacer (nrITS) region as well as morphology and habitat to propose two novel Cortinarius species for science and study their affinities to any known sections within this genus.

Figure 1. 

A Map of the two surveyed localities in the Swat District (Pakistan) B forest of Quercus oblongata in Shawar Valley C Quercus oblongata trees in the Alpuri forests (Toa). Photo: A. Naseer.

In the present study, Cortinarius pakistanicus and C. pseudotorvus are described as new to science based on an integrative taxonomic approach. The brownish color with occasional lilaceous tinges displayed by basidiomata of these species, which are both of rather small size, suggested their inclusion within the complicated C. subgenus Telamonia s. lat. Although recent phylogenetic surveys have helped to better circumscribe it, telamonioid cortinarii are still poorly known and constitute one of the most taxonomically challenging groups within the genus (Niskanen et al. 2013b; Brandrud et al. 2015, Liimatainen et al. 2015; Garrido-Benavent et al. 2019).

Our phylogenetic inference showed that C. pakistanicus is not in any known sections. The closest taxa to this species were C. bivelus (Fr.) Fr. and C. impennoides Bidaud, Moënne-Locc. & Reumaux in C. sect. Biveli, which produce larger basidiomata that become pale ochraceous when dry, their spores are shorter and they usually grow in subalpine or boreal regions (Brandrud et al. 2015). In any case, phylogenetic relationships among these species were not supported by any of the four different phylogenetic analyses implemented in the present study. Furthermore, other species in C. sect. Biveli usually have abundant whitish veil remnants and lack any traces of lilaceous tinges in basidiomata which are otherwise shown by C. pakistanicus at least on the stipe surface. Young specimens of C. pakistanicus may resemble several species in C. sect. Hydrocybe due to overall morphology, pigmentation and the presence of traces of veil in the surface of pilei (Suárez-Santiago et al. 2009), although our reconstructed phylogenetic tree showed that members of this section are phylogenetically unrelated to the new species. The type species of this section is C. decipiens Fr., which usually shows grayish lamellae and stipe cortex when young, traits that were not noticed on our specimens. Besides, C. decipiens has had various interpretations in the literature and it probably forms a species complex in need of detailed morphological and phylogenetic study (e.g. Esteve-Raventós et al. 2014; Brandrud et al. 2015). Cortinarius gallurae D. Antonini, M. Antonini & Cons. can be larger in size, with flattened pilei ca. 60 mm showing abundant veil traces, and has shorter spores than the new species and associates with thermophilous Quercus spp. in the Mediterranean region (Consiglio et al. 2005). Cortinarius casimiri (Velen.) Huijsman displays a warmer pigmentation than C. pakistanicus and its spores are larger, ca. 12 × 7 µm. Cortinarius subturibulosus Kizlik & Trescol has a distinct smell like orange flower and associates with thermophilous Quercus spp. (Ortega and Mahiques 1995). On the other hand, mature and dry C. pakistanicus specimens with an umbonate pileus may bear a slight resemblance to species in C. sect. Hinnulei, but these normally lack lilaceous tinges, show more evident veil remnants on the stipes and their spores are more coarsely ornamented. Cortinarius saniosus (Fr.) Fr. in C. sect. Saniosi produces basidiomata with a similar size but these display warmer colors and a striking yellowish veil. Members in other sections like C. sect. Brunnei and Uracei commonly associate with coniferous trees and are characterized by producing slender or large basidiomata, with darker pigmentation than C. pakistanicus, sometimes necropigmented, and highly hygrophanous pilei.

On the other hand, the inferred nrITS phylogeny was unequivocal in including C. pseudotorvus within C. sect. Telamonia, together with several representatives of the section’s type species C. torvus. Both species share the habitat under deciduous trees, the fibrillose to felty and matte pileus cuticle, which is slightly more felty in the new species, and the relatively thick and distant lamellae (Brandrud et al. 1992: B13). In C. torvus lamellae are initially grayish, a character that was not noticed in our collections, and then they turn brownish and even darker with age as happens in C. pseudotorvus. Spore dimensions are similar in both taxa, despite verrucae in the new species’ spores seeming to be less prominent. Nevertheless, the main distinguishing character between C. torvus and C. pseudotorvus is that the former produces stouter and larger basidiomata. This is particularly true for C. torvus stipes, whose morphology is also more variable, from cylindrical (as in C. pseudotorvus) to fusiform and especially clavate. A further character that should be tested with new collections of C. pseudotorvus is the smell, which in C. torvus is described as persistently sweet and pleasant. There are other species within C. sect. Telamonia displaying such smell and with abundant veil remnants: C. venustus P. Karst., whose basidiomata are more vividly pigmented, with lilaceous to orangish tinges (Brandrud et al. 1994: C50); and the very hygrophanous C. agathosmus, with dark pilei when moist and with longer stipes (Brandrud et al. 1990: A05). In contrast to C. pseudotorvus, both species fructify in acidophilous, subalpine coniferous forests. Finally, C. tigrinipes Bergeron displays conspicuous bands or girdles on the stipe, which is not thickened at the base (as in C. pseudotorvus), and the spores are smaller (7.5) 8–10.5 × 5–6.5 µm (Bidaud et al. 1999: pl. 250, f. 410).

With the new data provided in the present study, the number of Cortinarius species for all of Pakistan increases to eight. In neighboring countries, such as India, the number of recorded species for this genus seems to be lower than 20 as well (Verma et al. 2019). All in all, these data suggest that our knowledge of the diversity of Cortinarius in high mountain areas in Asia and the Himalayan forests of Pakistan is still in its infancy. By combining morphological and molecular analyses, which has proven to be a straightforward approach to disentangle the diversity of other fungal groups in the region (Saba et al. 2015; Khan et al. 2017; Sarwar et al. 2018; Naseer et al. 2019), we expect to further improve our knowledge of the mycobiota in this area of the planet.

We would like to thank Emma Harrower (US), Jean-Michel Bellanger (FR) and Bálint Dima (HU) for their helpful comments and suggestions.