Description of the Fifth New Species of Russula subsect. Maculatinae from Pakistan Indicates Local Diversity Hotspot of Ectomycorrhizal Fungi in Southwestern Himalayas

Russula subsect. Maculatinae is morphologically and phylogenetically well-defined lineage of ectomycorrhizal fungi associated with arctic, boreal, temperate and Mediterranean habitats of Northern Hemisphere. Based on phylogenetic distance among species, it seems that this group diversified relatively recently. Russula ayubiana sp. nov., described in this study, is the fifth in the group known from relatively small area of northern Pakistan situated in southwestern Himalayas. This is the highest known number of agaric lineage members from a single area in the world. This study uses available data about phylogeny, ecology, and climate to trace phylogenetic origin and ecological preferences of Maculatinae in southwestern Himalayas. Our results suggest that the area has been recently colonised by Maculatinae members migrating from various geographical areas and adapting to local conditions. We also discuss the perspectives and obstacles in research of biogeography and ecology, and we propose improvements that would facilitate the integration of ecological and biogeographical metadata from the future taxonomic studies of fungi in the region.


Introduction
Russula Pers. (Agaricomycotina, fungi) is an important genus of ectomycorrhizal (EcM) fungi with a high species diversity of which many are used as edible mushrooms in different parts of the world [1]. The research on Russula toxins and enzymes also shows a perspective in discoveries of compounds with potential medical and food industry uses [2]. It was hypothesized that previously described species from Europe and North America occur also in Asia [3][4][5]. Implementation of molecular techniques and use of ITS rDNA sequence as a barcoding tool proved that Asia has EcM taxa endemic to the continent [6]. Systematic research on some Russula lineages suggests existence of multiple diversity pools in the area of southern Asia [7], but diversity of northern boreal part of Asia probably in major parts overlaps with Europe [8]. Evidence regarding the endemic EcM species to southeastern and southern Asia boosted efforts to describe the local diversity. Therefore, multiple studies have recently emerged with new species from the area [9]. The current stage of Asian Russula research is exploratory, with a focus on species description and low attention to non-taxonomic reader.
This study began with the detection of new genetic variability within Russula subsection Maculatinae (Konrad and Joss.) Romagn in Pakistan. The known diversity of Russula in Pakistan is overrepresented within this group and four species have already been described as new to science from relatively small area of southwestern Himalayas located in northern Pakistan. There are only seven other novel species of Russula described from Pakistan Life 2021, 11, 662 2 of 14 and supported by molecular phylogenetic analysis [9][10][11][12][13][14][15]. New collections from Pakistan with 97.75% similarity to closest published sequence in GenBank (MK966687 labelled as R. dryadicola originated from China) suggest that they may represent fifth Maculatinae species from this area. Recent studies have detected the highest number of known Maculatinae members in Europe with six species confirmed molecularly, from which four are agaric and two sequestrate species [16,17]. Southwest Himalayas in Pakistan seem to be the diversity hotspot of the group. Occurrence of high number of species indicates that there is a strong niche differentiation that acted as evolutionary driver and it also suggests that this is a possible ancestral area of the lineage [18]. However, low number of total Russula species published so far from western Himalaya (including Indian and Chinese territories) and locations of Pakistani Maculatinae records in a relatively small area suggest that the current knowledge of Maculatinae distribution in the Himalayas is incomplete and largely dependent on human factor. Publications describing Maculatinae members from Pakistan were facilitated by efficient morphological delimitation of the group and available European phylogenies [8].
In addition to standard taxonomic ambition to describe and circumscribe potentially new species of Russula subsection Maculatinae from Pakistan, the authors wanted to understand why so many species of the lineage occur on a relatively small area. To trace phylogenetic relationships and evolutionary history of the Pakistani Maculatinae we performed the multi-loci phylogenetic reconstruction. We analysed available ecological, climate and geographical data to understand niche specialization.

Sampling
The morphological description of new Russula member is based on our two collections from Khyber Pakhtunkhwa province. Morphologically they were assigned to R. globispora lineage based on the red pileus colour, brownish yellow spots on the stipe and yellow lamellae. They originate from moist temperate forests at altitudes above 2400 m dominated by Abies pindrow (Royle ex D.Don) Royle, Cedrus deodora (Roxb.) G.Don, Pinus wallichiana A.B. Jackson and with minor presence of evergreen broad leaf Quercus species. To understand niche specialization of Pakistani members of Russula subsection Maculatinae, we retrieved data about ecology and geographical position of already published R. abbottabadensis Saba and Adamčík [8], R. mansehraensis Saba, Caboň and Adamčík [19], R. quercus-floribundae Kiran and Adamčík [20] and R. rubricolor Jabeen, Naseer and Khalid [21].

Sequence Alignment and Phylogenetic Analyses
Sequences were edited in the BioEdit Sequence Alignment Editor version 7.2.5 [28] or Geneious version R10 [29]. Intra-individual polymorphic sites having more than one signal were marked with IUPAC ambiguity codes (https://www.bioinformatics.org/sms/ iupac.html, accessed on 31 May 2021). For phylogenetic analyses, our sequence data were supplemented by available sequences of published species from Pakistan (for sources see references in Sampling), other Asian regions [9,30], Europe [8] and North America [31]. Members of Cuprea lineage were selected as an outgroup following Caboň et al. [8]. We searched GenBank (https://www.ncbi.nlm.nih.gov/genbank, accessed on 31 May 2021) and UNITE (https://unite.ut.ee, accessed on 31 May 2021) databases for all available ITS sequences with Asian origin and with 97% similarity to closest Maculatinae member. Sequences used in this multi-loci phylogenetic analysis are listed in the Supplementary Table S1. Partial alignments of individual loci were aligned by MAFFT version 7 using the strategy E-INS-i [32], further manually improved and concatenated into single multi-loci dataset using SeaView version 4.5.1 [33].
Phylogenetic analyses were computed using the CIPRES Science Gateway [34]. The topology of ITS tree was not congruent with partial trees of other genes. As ITS, as a fungal barcode, is the most represented in public databases, the analysis of this region was run separately. The final multi-loci dataset of LSU, mtSSU, rpb2 and tef1α was analysed with two methods: Bayesian Inference (BI) and Maximum Likelihood (ML). For the ML analysis, the concatenated alignment was uploaded as FASTA files and analysed using RAXML-HPC2 on XSEDE (8.2.12) [35] as a partitioned dataset under the GTR + GAMMA model with 1000 bootstrap iterations. For the BI analysis, the dataset was divided into seven partitions: nrLSU, mtSSU, intronic positions of tef1α, and the 1st + 2nd and 3rd codon positions of rpb2 and tef1α. The best substitution model for each partition was computed jointly in PartitionFinder 1.1.1 [36]. The aligned FASTA dataset was converted to nexus format using Mesquite 3.61 [37] and further analysed using MrBayes 3.2.6 [38] on XSEDE (8.2.12) [35]. Bayesian runs were computed independently twice with four MCMC chains for 10 million generations until the standard deviation of split frequencies fell below the 0.01 threshold. The convergence of runs was visually assessed using the trace function in Tracer 1.6 [39]. The ITS dataset was analysed as a single partition by ML under the same settings as the multi-loci analysis. The resulting trees were visualized and annotated by TreeGraph 2 [40] and graphically improved in CorelDRAW X5 (Ottawa, ON, Canada).

Morphological Observations
Macromorphological characters were observed on fresh basidiomata. Microscopic characters were studied on dried specimens. Most of the microscopic objects (except of spores) were measured in Congo red solution with ammonia after a short treatment in aqueous 10% KOH under Olympus BX-41 microscope (Olympus, Japan) using an eyepiece micrometer and at magnification of 1000×. Spores were scanned with an Artray Artcam 300MI camera and measured by Quick Micro Photo (version 2.1) software. Enlarged scanned pictures of spores were used for measuring with an accuracy of 0.1 µm and for making line drawings. All other elements are measured with an accuracy of 0.5 µm. All drawings of microscopic structures, with the exception of basidiospores, were made with the aid of a camera lucida (Olympus U-DA, Olympus, Japan) at a projection scale of 2000×. Morphological description, terminology and used chemical reactions follow morphological Russula standards proposed by Adamčík et al. [9]. Statistics of microscopic dimensions are based on 30 or 20 measurements per sampled collection and given as a mean value (in italics) plus/minus standard deviation; values in parentheses give measured minimum or maximum values.

Analyses of Ecological, Geographical and Climate Data
Data regarding the geographical position of known Maculatinae samples from Pakistan, presence of associated tree species (as potential symbiotic partners) and other collecting data are summarised in the Table 1. Based on geographical position, we assigned global environmental zone and biome to each collection following high resolution bioclimate map of the world [41]. This map is built on 42 bioclimatic variables and has 30 arcsec resolutions (equivalent to 0.86 km 2 at the equator). The map segment of northern Pakistan was digitalised and further edited in GRASS GIS 7.8.5 Geographic Resources Analysis Support System (GRASS) of the Geographic Information System (GIS) environment version 7.8.5, which was released under the GNU/GPL license. Global environments are classified into 7 biomes, 18 global environmental zones and 125 global environmental strata (GEnS) following Metzer et al. [41].

Phylogeny
ITS tree (Figure 1) showed that the studied Pakistani collections formed a wellsupported clade within R. globispora lineage. However, the topology of this lineage was in a large part polytomy showing only good support at some terminal nodes, from which at least some apparently correspond to the already described species. All Pakistani species received support: R. abbottabadensis, R. rubricolor and R. quercus-floribundae together with the two our Pakistani collections are all member of R. globispora lineage, while R. mansehraensis belonged to R. maculata lineage. Other Asian sequences from public databases did not form a supported cluster with any clade of Pakistani species. Collections of R. globispora lineage from southeastern Asia included R. heilongjiangensis G.J. Li and R.L. Zhao, R. tengii G.J. Li and H.A. Wen, two other undescribed species clades and a few singleton collections from Korea, Japan and China. The only Middle East sequence retrieved from GenBank was from Turkey and was placed close to European R. globispora (ML = 90). Within R. maculata lineage, Asian collections formed a monophyletic clade (ML = 92) sister to European members (ML = 95) with support for additional species from China and other singleton sequences from both China and Papua New Guinea. Multi-loci analysis of LSU, mtSSU, rpb2 and tef1α confirmed that studied Pakistani collections form a strongly supported clade within R. globispora lineage and are described in this study as R. ayubiana sp. nov. (Figure 2). The tree strongly supported relationship of the new species with alpine-arctic species R. dryadicola R. Fellner and Landa and R. tengii. This clade was further supported as sister to sequestrate R. mattiroloana (Cavara) T. Lebel. Other two Pakistani species, R. abbottabadensis and R. quercus-floribundae formed a wellsuported clade. R. mansehraensis was confirmed to be a member of R. maculata lineage. The multi-loci phylogeny demonstrated existence of at least three lineages with independent origin in Pakistan.  (Table 1). Their altitude range from 1109 to 2472 m, but apparently altitude did not limit distribution of at least two species, i.e., R. rubricolor and R. mansehraensis were both collected below 1500 m and also above 2000 m. Maculatinae collections were located in six geographical environmental strata (GEnS) that are further classified to four geographical environmental zones and two biomes (Figure 3). Three species; R. abbottabadensis, R. ayubiana and R. quercus-floribundae; are only present in single GEnS but this is due to low dispersal of collections that originate from single or a couple of collecting areas as different mycelia or different time duplicates (same mycelium origin collected at different dates). Russula mansehraensis and R. rubricolor have more dispersed collections that were each located in two to three GEnS, respectively, both were collected in two zones and the first also in two biomes. To our knowledge, R. abottabadensis and R. mansehraensis were clearly associated with pines (Pinus roxburghii Sarg.) and R. quercusfloribundae with oak (Quercus floribunda Lindl. ex Camus). It was impossible to decide about host tree of R. ayubiana because of mixture of different trees present at collecting site and authors of R. rubricolor also reported both conifer and deciduous tree as associated trees.   Figure 4) small to medium-sized, 60-90 mm diam., plano-convex with a slightly depressed centre, becoming applanate when old; margin deflexed, smooth, tuberculate-striate; cuticle smooth, wet-viscous and shiny, often with attached particles of debris, pruinose towards the centre, near the margin brownish pink to red, in the centre dark red and sometimes discolouring to pale olive. LAMELLAE moderately distant, equal, adnate-emarginate, pale yellow; lamellulae absent, furcations rare near the stipe; edge even, concoloured. STIPE 50-75 × 12-15 mm, obclavate to almost cylindrical, longitudinally striate, pruinose, white, with brownish yellow spots especially near the base. CONTEXT white, yellowish brown upon bruising, compact. SPORE PRINT not observed.  Note: Russula ayubiana has field appearance typical for Maculatinae: red pileus cuticle, pale yellow lamellae (suggesting that the spore print is yellow) and the surface is covered by brownish yellow spots. The spores are clearly smaller (in average shorter than 9 µm) compared with all known members of R. globispora lineage and in size they match rather R. maculata lineage [42]. The new species differs from R. maculata Quél. & Roze and its relatives in spores with relatively dispersed line connections and more prominent elements of the ornamentation. Pileocystidia have frequently three and more cells that is another character distinguishing it from the majority of known species of R. globispora lineage and shared with R. tengii and R. quercus-floribundae [8,20].

Discussion
Our study clearly demonstrated that all five Pakistani Maculatinae species are only known to be located in the southwestern Himalayas and belong to at least three independent lineages. Sequences of Pakistani Maculatinae from other foreign regions are not represented in public databases and none Maculatinae members are reported in the Russulaceae list from adjacent Indian region of Uttarakhand located in southwestern Himalayas [43]. No evidence of long dispersal of Pakistani Maculatinae members was detected that is similar to broad distribution of boreal-arctic species of Northern Hemisphere [44,45]. The results suggest that the area is a unique biodiversity region for the genus Russula; different from southeastern Asia, northern Eurasia and Europe.
According to analyses of global metadata associated with publicly available ITS sequences, the genus Russula evolved ca. 55 million years ago (Mya) in temperate areas and Maculatinae belongs to the crown clade that splitted at ca. 44 Mya [44]. The individual species origins are estimated for an average of 3.3 Mya for extratropical taxa. When looking into details, the species relations and their age might be closer, e.g., age of Russula subsection Roseinae shows approximately similar branch length and phylogenetic divergence to Maculatinae that was estimated to 12.2 Mya, and the age of crown Roseinae clade that may correspond to R. globispora lineage was estimated to 6.96 Mya, with the majority of species younger than 2.6 and evolved in Pliocene [46].
All known Maculatinae collections from Pakistan are located in Himalaya biodiversity hotspot (https://www.conservation.org/priorities/biodiversity-hotspots, accessed on 3 May 2021). Biological diversity of the south Himalayas corresponding to this hotspot was formed by five major geophysical upheavals from Indian subcontinent collision at 45 Mya until the beginning of the quaternary climate fluctuations at 2.6 Mya [47]. Southwestern part of Himalayas was specifically influenced by climate drying and cooling in central Asia at 8-7 Mya associated with change of woodland to savannah and this resulted to decreased plant species diversity compare to southeastern Himalayas [48,49]. Sufficient data for molecular dating are not available, but if taking Russula subsection Roseinae Sarnari as an equivalent, it is most likely that Maculatinae migrated to southwestern Himalayas in several lineages after aridification of central Asia and before quaternary climate fluctuations between 8 to 2.6 Mya. Contemporary events in this period may support the hypothesis of host tree switch and expansion as driving Russula diversification [44].
Available data about Pakistani species are very limited in current form and do allow very few conclusions about ecological characteristics or niche specification. Russula ayubiana is the only species showing clear link between phylogenetic history and its current niche. The species is nested in a clade with two boreal-arctic-alpine species R. dryadicola and R. tengii (Figure 2). The new Pakistani species was collected at higher altitudes without apparent preference for host tree that roughly correspond to ecological characterisation of other two Asian species [8]. Russula abbottabadensis and R. quercus-floribundae are placed on sister positions, but they sit on long branches, which suggests that the resulting support on higher note may be result of a long-branch attraction. Including more Asian samples repre-sented now only in ITS data may eliminate the heterotachy and reveal further relationships within this clade [50]. Russula abbottabadensis and R. mansehraensis both form apparently mycorrhiza with Pinus roxburghii and they are vicariant together with R. rubricolor. Two collections of R. quercus-floribundae associated with Quercus floribunda also do not serve as sufficient sampling to decide about niche differences from other Pakistani Maculatinae members. Our analysis of bioclimatic zones revealed vicariance and broad ecological amplitude of at least a part of Maculatinae members in western Himalayas. This can be explained as a result of adaptation to specific ecological conditions at a small, local scale [51]. Ecological function of ectomycorrhizal fungi resulting from their enzymatic activities involved in symbiotic interaction with trees also play an important role in shaping fungal communities [52]. However, to understand speciation processes, we need to focus on local adaptation and gene flow rather than distribution pattern and coexistence of species [53]. Important factor that shapes speciation processes in Russula is a fundamental function of symbiosis [54]. Above we discussed how much we could understand from the available data about evolutionary processes, ecological adaptations, niche specialisations, speciation drivers, etc. Our data are not sufficient to make conclusions. The structure of recent publications describing new Russula species from Asia is often simple and straightforward: they describe a single or a pair of species based on ITS sequence data, usually defined by unique and distinct morphological characters. Species are picked opportunistically from different unrelated groups. There are only a few very recent publications using more DNA loci (including single-copy genes) describing new Asian Russula species within a lineage of closely related species; for example Wang et al. [55] described two new species of Russula subsect. Substriatinae X.H. Wang and Buyck from China, Wisitrassameewong et al. [7] described three species in the Russula sect. Amoeninae Buyck and Caboň et al. [8] described three species of R. globispora lineage from Asia. The latter study includes R. abbottabadensis from Pakistan and the data become a solid base of other species descriptions and are well-implemented in our study. Such publications facilitate further research of the diversity of Russula lineages globally.
More collections data about wild fungi are required to understand their commercial use as addition to human diet and their conservation status. Little is known about edible fungi in Pakistan [56]. According to our communication with local people, few Russula species are collected for consumption but there is not a single published report about any edible Russula sp. in the country [57,58]. Russula is apparently among the most valuable traded edible fungi in China [59] and also in Himalayan regions of Northern India and Nepal, but they are usually members of subgenera Heterophyllidiae Romagn. and Brevipedum Buyck and V. Hofst. [60,61]. We did not find any evidence about Maculatinae used as edible fungi globally [58,62]. In Europe, many Russulas are collected as edible based on mild taste of lamellae context (https://www.wildfooduk.com/articles/identifying-russulas/, accessed on 4 May 2021) and if applying this taste test on Maculatinae of Pakistan, probably all species of R. globispora lineage will qualify as edible. However, this taste testing should be considered with a caution because of a high number of edible Russula species with look-alike poisonous counterparts that often co-occur alongside [63]. Sufficient knowledge about current ecological variation and distribution of Russula members can reveal their conservation concern and contribute to protection of endangered habitats where fungal role is still underestimated [64].

Conclusions
All five species of Russula subsect. Maculatinae occurring in Pakistani region of western Himalayas are members of at least three independently evolved lineages, but current data do not allow explaining their vicariance, ecological adaptations and evolutionary history. Authors of taxonomic literature frequently do not realise that data about differences in morphology and DNA sequences are often useful only for taxonomists [65]. Ecological data represented by a statistically significant number of observations are an important part of fungal functional traits [66]. The message of this paper is "let's pay more attention to collection sampling and ecological data to understand the biological identity of species and nature itself".
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/life11070662/s1, Table S1: Specimens and GenBank accession numbers of DNA sequences used in the multi-loci phylogenetic analysis.