﻿A compendium of macrofungi of Pakistan by ecoregions

﻿Abstract Macrofungi form fruiting bodies that can be detected with the naked eye in the field and handled by hand. They mostly consist of basidiomycetes, but also include some ascomycetes. Mycology in Pakistan is still in its infancy, but there have been many historical reports and checklists of macrofungi occurrence from its 15 ecoregions, which range from Himalayan alpine grasslands and subtropical pine forests to deserts and xeric shrublands. In this work, we searched and reviewed the historical literature and the GenBank database for compiling a comprehensive list of macrofungi reported from Pakistan to date. We recorded 1,293 species belonging to 411 genera, 115 families and 24 orders. These occurrences were updated taxonomically following the classification system currently proposed in the Index Fungorum website. The highest represented order by taxon number is Agaricales (47%) with 31 families, 146 genera and 602 species, followed by Polyporales (11%), Russulales (9%) and Pezizales (8%). Genera occurrence reported therein are presented for each ecoregion to the best of our ability given the data. We also discussed the currently known macrofungi diversity between different ecoregions in Pakistan. Overall, this work should serve as a solid foundation for the inclusion of Pakistan macrofungi in global biodiversity and conservation studies.


Introduction
Fungi are amongst the most diverse groups of organisms on earth. There have been numerous estimates regarding the total number of fungi worldwide. Bisby and Ainsworth (1943) recorded the total number to be about 100,000 and later, Hawksworth (1991) hypothesised the total number of fungal species to be 1.5 million. Later, Blackwell (2011) estimated the total number of fungi to be around 3.5 -5.1 million. More recently, Hawksworth and Lucking (2017) predicted the total number to be in the range of 2.8 to 3.8 million. To date, 149,974 species have been recognised (Index Fungorum 2021). The current rate of fungal species discovery per year averages at 2,000 as compared to 1,000 to 2,000 a decade ago (Cheek et al. 2020).
Macrofungi form fruiting bodies that can be detected with the naked eye in the field and handled by hand. They mostly consist of basidiomycetes, but also include some ascomycetes. They play many essential roles in ecosystems as mutualists, pathogens, decomposers or saprotrophs (Volk 2013). Some are edible, medicinal or toxic to humans. About 20,000 macrofungal species have been recognised worldwide (Hawskworth 2001), but many belong to cryptic species complexes and many more await discovery, particularly from poorly explored regions of the world.
A major hindrance of traditional systematics in fungal discovery and identification is the presence of limited taxonomic features (Wu et al. 2019). The traditional identification techniques utilised morphological features, ecological characters, physiology and biochemistry of tissues (Wang et al. 2016). The boom in molecular methods in the 1980s and a remarkable paper by White et al. (1990) describing rRNA primers in fungi spurred the beginning of molecular data utilisation in fungal classification and species identification. Phylogenetic studies have shown that morphologically similar taxa might belong to different lineages (e.g. Hibbett et al. 1997;Moncalvo et al. 2002). DNA sequences can also be helpful for detecting and distinguishing amongst cryptic taxa sharing similar morphological traits (e.g. Moncalvo and Buchanan 2008;Schoch et al. 2012;Wu et al. 2019).
Before the partition of British India, mycoflora of the region (presently India and Pakistan) was listed by Butler and Bisby (1931) and Mundkur (1938). These checklists recorded only 198 species of this region expanding on 30,000 square miles (77700 km 2 ). Later, Ahmad et al. (1997) recorded about 4,500 fungal taxa in a list that included all groups of fungi, i.e. macrofungi as well as microfungi and lichens. In the last two decades, many new records and description of new species have been added, based on morphological characters alone or in combination with molecular data (e.g. Sarwar et al. 2011;Saba et al. 2019a;Bashir et al. 2020a;Khalid in press), but none of these was comprehensively addressing macrofungal diversity in Pakistan and the ecoregions of their occurrence.
From a biodiversity conservation perspective, ecologists have been concerned about the factors that affect the delimitation of ecological units and how it affects our knowledge of ecological processes (Weins et al. 1985;Gosz 1991). Numerous efforts have been made to categorise geographical zones with analogous features. In a remarkable paper, Olson et al. (2001) defined ecoregions as broad areas of land or water that consist of geographically distinct assemblages of taxa, natural communities and environmental conditions. They presented an ecoregion map for its utilisation at global as well as regional scales. They based their map on biogeographic information and this was built with the collaboration of more than 1,000 experts in biogeography, taxonomy, conservation biology and ecology from all over the world. Ecoregions were classified by taking into account biogeographic features like endemism, species richness and special evolutionary perspectives. The unique feature of this global biodiversity map is that it focuses on species allocation and communities more precisely than the earlier models, based on biophysical characters, for instance, rainfall and temperature (Holdridge 1967;Walter and Box 1976;Schultz 1995;Bailey 1998) or vegetation structure (UNESCO 1969;de Laubenfels 1975;Schmidthüsen 1976). In Olson et al. (2001), the terrestrial world is divided into 14 biomes, eight biogeographic realms and 867 ecoregions; out of which, nine biomes, two realms and 15 ecoregions are found in Pakistan. This country covers a wide altitudinal range from sea level (Arabian Sea) to the second highest peak of the world, K2. The variety of ecoregions from Himalayan alpine grasslands and subtropical pine forests to deserts and xeric shrublands promotes a great deal of fungal diversity that still remains largely unaccounted for.
In this study we compiled a compendium of macrofungi reported from Pakistan to date from searches in the historical literature as well as in the GenBank database. We have included fungi with prominent fruiting bodies visible to the naked eye in this taxonomic list. We have excluded taxa in Ascomycota which are immersed or half immersed structures, galls or non-prominent fruiting structures on animal dung. We also categorised the reported macromycetes into ecoregions, based on available data.

Compendium of Macromycetes of Pakistan
For compiling a comprehensive compendium of macromycetes of Pakistan, data were gathered from extensive literature searches of checklists and published papers, as well as in the GenBank sequence database. Sequence data in GenBank (2020, 2021) was retrieved using a Python script written by Santiago Sanchez-Ramirez (available upon request) on 09-10-2020 for Basidiomycota and on 22-06-2021 for Ascomycota. The list was arranged following the current classification system in Index Fungorum (2021) with great care about eliminating synonymy.

Division of Macromycetes of Pakistan into Ecoregions
In order to attribute ecoregion occurrence of the taxa we retrieved, we used their locality-based information to consult various repositories, such as Ecoregion 2017 (Dinerstein et al. 2017), DOPA explorer (Dubois et al. 2018) and the ArcGis search tool (2021). Ecoregion allocation of genera was graphically represented on an MS excel spreadsheet for analyses. Genera were listed in rows and ecoregions in columns. The presence or absence of a genus in an ecoregion was scored "1" or "0", respectively. The sum and percentage of each genus in each ecoregion were then calculated.

Results
Table 1 provides a comprehensive record of the macrofungal biota of Pakistan known to date, to the best of our knowledge. It lists 1,293 species belonging to 411 genera, 115 families and 24 orders. Out of which, 1,117 species, 338 genera, 83 families and 16 orders belong to Basidiomycota and 176 species, 73 genera, 32 families and eight orders are from Ascomycota. The source reference in Table 1 indicates that most entries are from the extensive checklist by Ahmad et al. (1997;874 entries). The highest order recorded is Agaricales (27%) with 31 families, 146 genera and 602 species, followed by Polyporales (11%), Russulales (9%) and Pezizales (8%). The orders of least occurrences are Atheliales, Leotiales and Trechisporales representing one taxon in a single genus and family. The proportion of respective families, genera as well as species are shown in Fig. 1. Table 2 indicates the ecoregions from which each genus was recorded. Fig. 2 shows that the highest macrofungal diversity is found in the western Himalayan     (Ahmad et al. 1997

Discussion
The compendium presented in Table 1 gives an overview of the macrofungal diversity of Pakistan known to date. It largely reposes on the checklist by Ahmad et al. (1997;866 entries) published over two decades ago and its recent update (Khalid, in press), as well as taxa recently described with the use of molecular data, for example, Russula foetenoides (Razaq et al. 2014), Leucoagaricus lahorensis (Qasim et al. 2015a), Tulsotoma ahmadii (Hussain et al. 2015b), Phaeocollybia pakistanica (Khan et al. 2016a), Descolea quercina (Khan et al. 2017a), Amanita griseofusca   (2020) worked on the cultivation potential of two wild indigenous species of Agaricus, i.e. A. bisporus and A. subrufescens and obtained promising results for spawn production locally. In the future, more edible mushrooms can be worked on for their possible cultivation and commercialisation prospects. We recorded 1,293 species belonging to 411 genera, 115 families and 24 orders. For comparison, Vaco-Palacios and Franco-Molano (2013) listed 1,239 macrofungal species from Colombia. Flores et al. (2012) reported 315 taxa, 163 genera and 20 orders from Guatemala. Kinge et al. (2020) recently presented an elaborate checklist of macrofungi in South Africa listing 1,008 species, 251 genera and 72 families. For comparison with a well-studied area, 3,173 species have been reported from Quebec (mycoquébec.org). Approximately 20,000 species of macrofungi are known worldwide (Hawksworth 2001). Unsurprisingly, since Agaricales is the largest order of macrofungi (Money 2016), it is by far the most commonly represented order with 47% species in the present taxonomic list followed by Polyporales (11%), Russulales (9%) and Pezizales (8%).
The highest number of taxa was recorded in the western Himalayan broadleaf forests ecoregion, which belongs to the temperate broadleaf and mixed forest biome and has been reported to be the richest in central China and eastern North America (Zhao et al. 1990;Martin et al. 1993). The second highest diversity was found in the north-western thorn scrub forests, which are categorised under deserts and xeric shrublands. This thorn scrub is considered as a degraded form of tropical dry forests (e.g. Champion and Seth 1968;Puri et al. 1989). This ecoregion includes semi-arid to arid climatic zones and a mean annual rainfall of less than 750 mm and a temperature range of 45 degrees or more in summers to temperatures dropping below freezing point in winters. Furthermore, ecoregions in tropical and subtropical coniferous forests and temperate coniferous forest biomes also show good macrofungal taxa representation. The ecoregion of western Himalayan subalpine conifer forests plays an important ecological role to vanguard the alpine meadows to the north. For instance, many Himalayan birds and mammals migrate seasonally between the steep mountain slopes, relying on adjacent habitats when the original ones are disturbed. Likewise, large-scale collection of morel mushrooms (Morchella spp.) from this ecoregion by local people for export overlaps with the breeding season of many pheasants and some mammals. Therefore, maintaining the biodiversity composition and ecological processes within this geologically young, highest mountain range on Earth requires particular conservation policies for this unique ecoregion (Wikramanayake et al. 2002). Finally, the ecoregions of flooded grasslands and savannahs, mangroves and montane grasslands and shrublands have either less than five percent or no representation. So, clearly there are missing data due to very limited exploration in several regions.
Although the data presented here will be useful to taxonomists, ecologists and conservation biologists, conclusive trends cannot be drawn as there are gaps in data due to extensive sampling in a few ecoregions, whereas other areas have been either neglected or unexplored. Therefore, the unexplored ecoregions of Pakistan need to be sampled extensively to give a full picture of the fungal diversity and endemism therein. Many countries and regions around the world have identified and listed endemic species, including the United States (Stein 2002), Russia (Griffin 1999), the Tuscan Region in Italy (Foggi et al. 2014) and New Caledonia (Wulff et al. 2013). The International Union for Conservation of Nature (IUCN) recently published a report on endemic threatened species on the Red List for each country (IUCN 2019). Redhead (1997) listed rare macrofungi of British Columbia, Canada, for each ecoregion. More recently, Enns et al. (2020) generated a list of endemic species, including a few fungal species as well, highlighting the status of target species for conservation.
In conclusion, this study provides a comprehensive list of macrofungi recorded in Pakistan as of the year 2020 and their known distribution by ecoregions. The otherwise scattered data have now been arranged and are available to be utilised by mycologists and other scientists as well as by amateur citizens. Most importantly, it can serve as a baseline information for further conservation studies and policy-making. Furthermore, these data also highlight the need for more sampling from less sampled areas like Sindh and Baluchistan Provinces. Our next step is to develop an online portal for fungi of Pakistan, where revisions of the current compendium can be done and new reports can be continuously added.