Additions to the genus Chroogomphus (Boletales, Gomphidiaceae) from Pakistan

Abstract With only three published reports, the genus Chroogomphus (Boletales, Gomphidiaceae) is poorly studied in Pakistan. During recent sampling events in Khyber Pakhtunkhawa province, Pakistan, several collections of Chroogomphus were made, representing undescribed taxa. Based on morphological and molecular data, two new species are described: Chroogomphus pakistanicus and C. pruinosus. We present a description and illustrations for both taxa. A molecular phylogenetic reconstruction, based on the internal transcribed spacer (ITS1–5.8S–ITS2) barcode region, shows that C. pakistanicus and C. pruinosus are placed in two different subgenera of Chroogomphus (subg. Chroogomphus and subg. Siccigomphus, respectively).


Introduction
Chroogomphus (Singer) Mill. was initially recognised as a sub-genus of Gomphidius Fr. (Singer 1948). It was Miller (1964) who elevated it to genus level. More than 33 taxa are currently recognised worldwide, including species, subspecies and varieties, but the number of accepted species in the genus is ambiguous (Miller and Aime 2001;Miller 2003;Watling 2004;Li et al. 2009;Martín et al. 2016;Razaq et al. 2016;Scambler et al. 2018). Members of the genus are characterised by ochraceous basidiomata; orange to somewhat ochraceous, decurrent lamellae; a fibrous veil; and grey to black spore deposit. Other useful characters are the pileipellis hyphae (moist to glutinous or viscid) and the stipe base (with hyphae that are amyloid in Melzer's reagent) (Miller 1964;Miller and Aime 2001;Li et al. 2009;Martín et al. 2016).
Chroogomphus species are economically very important because of their ectomycorrhizal association with pines and applications as drugs and food (Agerer 1990(Agerer , 1991Miller 1964;Xie et al. 1986;Yu and Liu 2005;Dai and Tolgor 2007). They are found in Europe, America and Asia (Miller 1964;Miller and Aime 2001;Legon and Henrici 2005;Li et al. 2009;Knudsen and Taylor 2012;Scambler et al. 2018). In Pakistan, the genus is underexplored with only three published reports. These are C. helveticus (Singer) M.M. Moser, C. roseolus Yan C. Li & Zhu L. Yang and C. rutilus (Schaeff.) O.K. Mill. (Ahmad et al. 1997;Razaq et al. 2016). Here, we describe two new species of Chroogomphus belonging to two different subgenera, based on their morphoanatomical features and molecular phylogenetic analysis.

Morphological observations
Macro-morphological characters of fresh basidiomata were recorded and colour codes were assigned using Munsell Soil Color Charts (1975). Macro-morphological characters included the size, shape and colour of pileus; colour of gills and mode of attachment to the stipe; colour of stipe and attachment to the pileus; presence or absence of annular ring and volva. Micro-morphological features were observed using a compound light microscope (MX4300H, Meiji Techno, Japan). For detailed microscopic examination, sections of lamellae, pileipellis and stipitipellis from dried specimens were observed in 5% potassium hydroxide (KOH), Congo red stain and Melzer's reagent. Anatomical features were measured using ScopeImage software version 1.0.0 (BioImager, Maple, Canada). Measurements of basidiospores were made under oil immersion. A minimum of 60 basidiospores, 20 basidia and 20 cystidia were measured. The abbreviations 'n/m/p' indicates number of basidiospores 'n', measured from 'm' basidiomata from 'p' collections. Basidiospores dimensions are given as length × width with extreme values given in parentheses; avQ = average Q of all spores ± standard deviation. Voucher specimens are deposited in LAH (Department of Botany, University of the Punjab, Pakistan).

DNA extraction, PCR amplifications and sequencing
Genomic DNA was extracted from dried tissue employing a modified CTAB protocol (Gardes and Bruns 1993). Amplification of the internal transcribed spacer (ITS, including ITS1, 5.8S and ITS2) barcode region of the nuclear ribosomal DNA was done using the primer pair ITS1F and ITS4 (Gardes and Bruns 1993;White et al. 1990). Polymerase chain reaction (PCR) was performed in a reaction volume of 20 µl containing 10 µl of 2× PCR buffer (Sigma-Aldrich, St. Louis, Missouri), 0.1 µl of each 0.6 nM primer, 8.8 µl of ddH 2 O and 1 µl of template DNA under the following cycling parameters: initial denaturation at 94 °C for 1 min; followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min and extension at 72 °C for 1 min; and a final extension at 72 °C for 8 min. Amplified PCR products were directly sequenced in both directions by Sanger sequencing, using the same primers (Macrogen Inc., South Korea). Consensus sequences were generated using BioEdit software version 7.2.5.0 (Hall 1999) and then blasted against the NCBI GenBank database (https://blast.ncbi.nlm.nih.gov/).

Sequence alignment and phylogenetic analysis
We constructed an ITS dataset of our newly generated sequences along with closely related sequences that were downloaded from GenBank (Li et al. 2009;Martín et al. 2016;Scambler et al. 2018). We included species of Gomphidius Fr. as outgroup taxa (Scambler et al. 2018). Multiple sequence alignment was done using MUSCLE (Edgar 2004) available from EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/muscle/). The final alignment was submitted to TreeBASE under study ID: S24298.
A Bayesian Inference (BI) phylogeny was estimated using BEAST version 1.8.4 (Drummond et al. 2012) with an uncorrelated lognormal relaxed clock, allowing for evolutionary rates to vary across branches. We selected a Birth-Death Incomplete Sampling speciation model (Stadler 2009) tree prior and appropriate substitution models as determined by jModelTest2 (Darriba et al. 2012) under AICc. Models were TrNef+G (ITS1, -lnL = 2028.8929), JC (5.8S, -lnL = 320.6928) and TPM3+G (ITS2, -lnL = 1905.6932). Four independent runs were performed from a random starting tree for 40 million generations with a sampling frequency of 4000. The analyses were run from the BEAST on XSEDE tool on the Cipres Science Gateway (Miller et al. 2010). Resulting log files were entered in Tracer (Rambaut et al. 2014) to check trace plots and burn-in values. Effective sample sizes were well over 200 for all sampled parameters for each run and so we selected a standard burn-in of 10%. After the removal of 10% of each run as burn-in, log files and trees files were combined in LogCombiner. TreeAnnotator was used to generate consensus trees (with 0% burn-in) and to infer the Maximum Clade Credibility tree.

Phylogenetic analyses
Amplification of the ITS from three basidiomata of C. pruinosus resulted in 670 bp sequences (GenBank accession numbers MK509768, MK509769 and MK509770). All of these sequences showed 97% similarity to C. roseolus (LT576117, Pakistan) with 100% query coverage. The ITS sequences obtained from two basidiomata of C. pakistanicus (MK509771, MK509772) were 650 bp in length and showed 98% similarity to C. filiformis Yan C. Li & Zhu L. Yang (EU706324, China) with 95% query coverage.
Habitat. On forest floor under mixed conifers.
Chroogomphus britannicus was included in sect. Filiformes by Scambler et al. (2018). In our phylogenetic tree, its position is unresolved within subg. Chroogomphus. Morphologically, it can be easily distinguished from the new species. Chroogomphus britannicus has larger basidiospores (20.3 × 7.1 µm), amyloid lamellar trama and inamyloid pileal trama (Scambler et al. 2018). The morphology of Chroogomphus pakistanicus is similar to C. mediterraneus, which can be distinguished by a subconical to convex pileus ranging in colour from grey to olivaceous to brown to red to pink to purplish, in combination with differently shaped cystidia, ranging from cylindrical, subfusiform, subutriform to sometimes subcapitate (Scambler et al. 2018). Chroogomphus vinicolor is another species related to C. pakistanicus, but the cystidia of C. vinicolor are thick-walled (5-7.5 µm) and it has a differently coloured pileus (Miller 1964;Singer and Kuthan 1976). Furthermore, geographically, members of the section Vinicolores have thus far only been reported from North America (Scambler at al. 2018). Chroogomphus jamaicensis may also be confused with C. pakistanicus, but it can be separated from the latter in having different micromorphological characters including thick-walled (4-5 µm) fusiform caulocystidia, which are occasionally amyloid towards the base (Miller 1964). Etymology. Referring to the pruinose surface of pileus and stipe.
Notes. Chroogomphus pruinosus differs from all other members of the genus in having pileocystidia. This new species is phylogenetically most closely related to C. roseolus, a species that has been reported from China and Pakistan (Li et al. 2009;Razaq et al. 2016). The macro-and micro-morphology of C. pruinosus is different from C. roseolus in the following characters: C. pruinosus possesses an obtusely conic to broadly convex, yellowish-orange, pruinose, larger pileus; presence of pileocystidia and caulocystidia in C. pruinosus; and the pileal and lamellar trama and stipitipellis of C. pruinosus are inamyloid, whereas those of C. roseolus are amyloid or partially amyloid (Li et al. 2009;Razaq et al. 2016). Chroogomphus helveticus is another close relative of C. pruinosus and has also been reported from China and Pakistan (Li et al. 2009;Razaq et al. 2016). However, no herbarium specimens are available for the Pakistani reports of C. helveticus (Ahmad et al. 1997) and it is likely that these collections represent C. roseolus, as discussed by Razaq et al. (2016). Chroogomphus roseolus is an Asian native species, whereas reports of C. heleveticus have so far only been confirmed in Europe, generally in association with 5-needle pines -mostly Pinus cembra (Li et al. 2009), which does not occur in Pakistan. A striking feature of C. helveticus is the presence of a pinkish mycelium at the base of the stipe (Li et al. 2009;Razaq et al. 2016;Scambler et al. 2018), which is not observed in C. pruinosus. Chroogomphus rutilus and C. purpurascens are morphologically very similar to C. pruinosus. However, C. rutilus has larger basidiomata (20-90 mm) with vinaceous brown or ochraceous-buff to vinaceous red, reddish-brown to purplish, umbonate pileus, buff to yellowish mycelium on the base of the stipe, slightly larger basidiospores (18.0 × 6.2 µm), cylindrical to subfusiform thick walled cystidia and lamellar trama composed of amyloid hyphae (Singer 1949;Miller 1964;Singer and Kuthan 1976;Gerhardt 1984;Breitenbach and Kränzlin 1991;Villarreal and Heykoop 1996;Horak 2005;Li et al. 2009;Scambler et al. 2018). Chroogomphus purpurascens is distinguished by a grey to brown then purple pileus that is slightly depressed, an ochraceous stipe, salmon to purple pink mycelium on the base of the stipe, thin-walled cystidia and deeply amyloid pileal trama. Moreover, the species is only known to be in association with Pinus cembra, P. koraiensis and P. tabuliformis, three pine species that are not found in Pakistan (Vassiljeva 1950(Vassiljeva , 1973Azbukina 1990;Li et al. 2009). Chroogomphus tomentosus, a species that has been reported from Asia (Li et al. 2009), can be distinguished by its larger basidiospores [15-25 × 6-8(9) µm], thick-walled cystidia (2-4 µm) and strongly amyloid lamellar and pileal trama (Miller 1964).  Sochorová et al. 2019). This was also shown to be a useful approach in the delimitation of species within Chroogomphus (Scambler et al. 2018). The genus can be found throughout the Northern Hemisphere with the exception of only one species, C. papillatus, which was reported from the Southern Hemisphere by Raithelhuber (1974). There is morphological and molecular evidence of intercontinental distribution for C. purpurascens and C. rutilus, which both occur in Europe and Asia (Miller and Aime 2001;Li et al. 2009;Martín et al. 2016;Scambler et al. 2018). Our phylogenetic tree, obtained from ML and BI analyses (Figure 1), is in accordance with Scambler et al. (2018), with the division of the genus into the subgenera Chroogomphus, Floccigomphus and Siccigomphus. Subg. Chroogomphus was further subdivided by Scambler et al. (2018) into four sections -sect. Chroogomphus, sect. Confusi, sect. Filiformes and sect. Fulminei -and one informal clade, Vinicolores. Two identical sequences of C. pakistanicus are nested within subg. Chroogomphus sect. Filiformes and three identical sequences of C. pruinosus cluster within subg. Siccigomphus. In our phylogeny, sect. Filiformes is not monophyletic; the position of C. britannicus within subg. Chroogomphus is unresolved. The other sections are retrieved as monophyletic in our phylogeny with high support: sect. Chroogomphus (with C. orientirutilus, C. purpurascens and C. rutilus), sect. Confusi (C. confusus and C. mediterraneus), sect. Fulminei (C. fulmineus and C. subfulmineus) and the informal Vinicolores clade (C. jamaicensis and C. vinicolor).

Key to species of Chroogomphus reported from Pakistan
The subgenera in our phylogenetic analyses are also supported morphologically. Members of clade II fall in subg. Siccigomphus found all over the Northern Hemisphere and are similar in having comparatively smaller basidiospores and inamyloid lamellar trama. They can be distinguished from the members of clade III, which belong to subg. Chroogomphus and have a narrow pileipellis and shiny pileus surface and distributed throughout Eurasia, but not in North America. Clade I represents subg. Floccigomphus, with members that are found in North America and Asia, but not in Europe and recognised by non-gelatinised pileipellis hyphae and amyloid lamellar trama.
Based on the distinct and well-supported molecular phylogenetic placement of our Pakistani collections in combination with morphological differences with their closest described relatives, we confirm that they represent two new species in the genus Chroogomphus.