Fungal communities associated with forests in the Afromontane region of Ethiopia

ESCUELA TÉCNICA SUPERIOR DE INGENIERÍAS AGRARIAS SUSTAINABLE FOREST MANAGEMENT RESEARCH INSTITUTE DOCTORAL DISSERTATION / TESIS DOCTORAL Fungal communities associated with forests in the Afromontane region of Ethiopia Comunidades de hongos asociadas a los bosques en la región de Afromontana de Etiopía Presentada por Demelash Alem Ayana para optar al grado de doctor por la Universidad de Valladolid Dirigida por: Dr. Pablo Martín-Pinto Dr. Tatek Dejene Bekele Acknowledgement i Acknowledgment First of all, I would like to thank the Glorious God for enabling me to successfully accomplish my study. I forward great gratitude to my Advisors, Prof. Dr. Pablo Martin Pinto and Dr. Tatek Dejene for their unreserved technical support throughout my study period. I greatly thank Professor Pablo Martin-Pinto for his positive attitude and continuous motivation throughout my study period. I thank Dr. Tatek Dejene for his dedicated support, mentoring, frequent follow-up and provision of relevant reference materials and data. I would like to thank Dr. Wubalem Tadesse for his support and encouragement throughout my study period. Without him, my PhD study would not have happened. I would like to thank the University of Valladolid for acceptance as a PhD student and EEFRI for allowing me to pursue my study. I thank Dr. Jozsef Geml for mentoring and helping me develop data analysis skills especially in R software while I was in the Netherlands for such training. I would also forward great gratitude to Mr. Sewale Wondimneh and Mr. Anteneh Yenesew (Bahir Dar Environment and forest Research Center) for their support in field data collection. I thank Banja, Farta and Libokemkem district offices experts and religious leader of Taragedam monastery for allowing me to conduct the studies in these forests. I also thank forest guards for their support during field data collection. This research was supported by the projects SUSTIFUNGI_ET (Sustfungi_Eth: 2017/ACDE/002094) and MYCOPROED_ET (Mycoproed_Eth: 2019/ACDE/000921) funded by the Spanish Agency for International Development and Cooperation. This study was also co-funded by the Spanish Ministry of Education and Culture under a Salvador de Madariaga grant agreement, n° PRX17/00315. I would like to thank all members of the Sustfungi_Eth project who, in way or another, contributed to the success of my study. Acknowledgement ii I extend my grateful thanks to the staff members of Ethiopian Environment and Forest Research Institute (EEFRI) and Bahir Dar Environment and Forest Research Center for supporting and facilitating my study. I would like to thank my wife Zenebech Chekol, for her continuous motivation and shouldering family cases while I was busy of my study. The smiley faces and laughter of my kids (Natnael, Yordanos and Mikias) were excellent energizers for my study especially during the period of COVID-19 pandemic. I thank my colleagues Dr. Mulugeta Atnaf, Dr. Demeke Mewa, Chalachew Abebe, Tsehaynesh Gurmu and Dr. Mehari Alebachew for their moral support. My brothers, sisters, my brothers-in-law and my sisters-in-law are greatly thanked for their encouragement and moral support. I would like to thank my father, Kes Alem Ayana, and my mother, Mrs. Hibist Yeshu, my father-in-law, Chekol Gelaw, and my mother-inlaw, Emawayish Weldelul, for their pray and encouragement. Thanks God for keeping all of us safe. Dedication iii Dedication Those who lost their lives due to Corona Virus (COVID-19) all over the world and conflicts in Ethiopia Table of contents v CONTENTS Acknowledgment............................................................................................................ I Abstract ........................................................................................................................ VII Resumen ....................................................................................................................... XI List of original articles ............................................................................................... XV Outline of the thesis ................................................................................................. XVII 1. Introduction ............................................................................................................... 1 1.1. CLIMATE, GEOLOGY AND ECOLOGY OF ETHIOPIA ...................................................................................... 1 1.2. NATURAL FORESTS IN ETHIOPIA ................................................................................................................. 1 1.3. PLANTATION FOREST IN ETHIOPIA .............................................................................................................. 4 1.4. WHAT ARE FUNGAL RESOURCES? .............................................................................................................. 6 1.5. IMPORTANCE OF FUNGI ............................................................................................................................... 6 1.6. FACTORS GOVERNING FUNGAL DISTRIBUTION ............................................................................................ 8 1.7. RESEARCH AND CONSERVATION STATUS OF FUNGI ................................................................................... 9 1.8. OVERVIEW OF MYCOLOGICAL STUDIES IN ETHIOPIA ................................................................................. 11 1.9. WHY THE CURRENT STUDY? ..................................................................................................................... 14 1.10. HYPOTHESES AND RESEARCH QUESTIONS............................................................................................. 16 1.11. SCOPE OF THE STUDY ............................................................................................................................. 17 2. Objectives of the thesis .......................................................................................... 21 3. Material and methods ............................................................................................. 25 3.1. DATA SOURCES ......................................................................................................................................... 25 3.2. THE STUDY AREAS ..................................................................................................................................... 25 3.3. ESTABLISHMENT OF FIELD PLOTS ............................................................................................................. 28 3.3.1. Plot establishment in Dry Afromontane forests to study soil fungal community composition and diversity in relation to forest fire .................................................. 28 3.3.2. Plot establishment in P. patula plantation forest of different age to study the soil fungal communities and succession ....................................................................... 28 3.3.3. Plot establishment to study sporocarps diversity and production and soil fungal community composition and diversity in fragmented church forests in Dry Afromontane forest systems in Northern Ethiopia .................................................. 29 3.4. SAMPLING .................................................................................................................................................. 29 3.4.1. Sporocarp sampling ................................................................................................ 29 3.4.2. Soil sampling for DNA extraction ............................................................................ 29 3.4.3. Soil sampling for physico-chemical analysis ........................................................... 30 3.4.4. Vegetation and climate data collection ................................................................... 30 3.5. LABORATORY ANALYSIS AND TAXA IDENTIFICATION ................................................................................. 31 3.5.1 Sporocarp taxa identification and classification ....................................................... 31 3.5.2. Molecular analysis .................................................................................................. 32 Table of contents vi 3.6. DATA ANALYSIS ......................................................................................................................................... 32 3.6.1 Bioinformatics analysis ............................................................................................ 32 3.6.2. Statistical analysis ................................................................................................... 33 4. Results ..................................................................................................................... 37 4.1. TAXA COMPOSITION OF SOIL FUNGI ........................................................................................................... 37 4.2. MACROFUNGAL TAXA COMPOSITION ......................................................................................................... 39 4.3. THE EFFECT OF FIRE, STAND AGE AND ABOVEGROUND PLANT DIVERSITY ON FUNGAL RICHNESS, DIVERSITY AND PRODUCTION..................................................................................................................... 40 5. Discussion ............................................................................................................... 43 5.1. SOIL FUNGAL TAXA COMPOSITION ........................................................................................................... 43 5.2. MACROFUNGAL TAXA COMPOSITION ......................................................................................................... 47 5.3. EFFECT OF FIRE, STAND AGE, AND ABOVEGROUND PLANT DIVERSITY ON FUNGAL RICHNESS, DIVERSITY AND PRODUCTION ...................................................................................................................................... 50 7. Conclusiones ........................................................................................................... 59 8. References ............................................................................................................... 61 Original articles ........................................................................................................... 83 Abstract vii Abstract The Afromontane region of Ethiopia has natural and plantation forest systems that provide high socioeconomic and ecological value, including the biodiversity conservation. However, the natural forests in this region are facing challenges in which their degradation is framed for decades. In response to this, exotic tree species have been introduced to decrease the pressure on the natural forests. Thus, the plantations are managed to maximize the value of the wood and reduce the gap between wood demand and supply in the country. Accordingly, some natural forests have been conserved as priority forests, but without generating tangible benefits for the local community. In this context, some non-timber forest products, notably mushrooms, are neglected and not included in Ethiopia's forest management plans and strategies of the country. Consequently, studies on the effects of forest management on the diversity and composition of fungal communities are very limited. Therefore, our objective was to generate information on the composition and diversity of the fungal community in the forest systems of the Afromontane region, including natural and plantation forests. In the natural forests, the spatial distribution of fragmented church forests and the time after disturbance in the forest were taken into consideration for the study. In the plantation forests, the stand age was taken into account to analysis the existing fungal communities and succession in Pinus patula plantations. We studied the soil fungal communities in all the forests systems. Specifically, we also conducted the fruit body collection in the fragmented natural forests of the Northwest of the country, since this particular information was already available for the rest of the systems included in this study. In the fragmented Dry Afromontane forests in Northwestern Ethiopia, three forest types were selected. A total of 27 plots (2 m × 50 m), nine in each forest type, were established for the collection of the sporocarps and soil fungi. To assess the effect of fire on soil fungi, three similar plots were sampled in each of the three forests that differed in their fire history (unburned, 10-years old burned and 36-years old burned stands). Likewise, a total of nine plots were established in the plantation forests in three age categories (5-, 11or 36year old stands). List of original articles xv List of original articles This thesis is based on four original works, which are referred in the text with Roman numerals (I – IV). All except the forth one are already published. The fourth is a manuscript. Authors, coauthors, and the stage of the publication are presented below: I. Demelash Alem, Tatek Dejene, Juan Andrés Oria-de-Rueda, József Geml, Carles Castaño, Jane E. Smith, Pablo Martín-Pinto. 2020. Soil fungal communities and succession following wildfire in Ethiopian Dry Afromontane forests, a highly diverse underexplored ecosystem. For. Ecol. Manage. 474, xx– xx.(118328) https://doi.org/10.1016/j.foreco.2020.118328 II. Demelash Alem, Tatek Dejene, Juan Andrés Oria-de-Rueda, József Geml, Pablo Martín-Pinto. 2020. Soil Fungal Communities under Pinus patula Schiede ex Schltdl. & Cham. Plantation Forests of Different Ages in Ethiopia. Forests 11, 1109. https://doi.org/10.3390/f11101109 III. Demelash Alem, Tatek Dejene, Juan Andrés Oria-de-Rueda, Pablo MartínPinto. 2021. Survey of macrofungal diversity and analysis of edaphic factors influencing the fungal community of church forests in Dry Afromontane areas of Northern Ethiopia. For. Ecol. Manage. 496, xx–xx.(119391). https://doi.org/10.1016/j.foreco.2021.119391 IV. Demelash Alem, Tatek Dejene, József Geml, Juan Andrés Oria-de-Rueda and Pablo Martín-Pinto. 2021. Soil fungal communities in fragmented Dry Afromontane Church forests in Northern Ethiopia (Manuscript) Outline of the thesis xvii Outline of the thesis This thesis consisted of four studies important to describe the status of fungal communities from two forest systems in the Dry Afromontane region of Ethiopia. The first study (Study I) focused on the community composition, diversity and richness of soil fungi under different successional stages after fire in the Dry Afromontane forest systems of Southern Ethiopia where the recurrent forest fire is common. The second (Study II) is on fungal succession in relation to stand development of Pinus patula where the effects of stand age of P. patula on the community composition, diversity and richness of soil fungi are discussed. The third and the fourth (Study III and IV) are focused on macrofungi and soil fungi composition of the Dry Afromontane church forest systems of Northwestern Ethiopia respectively. In the third paper, the effects of aboveground vegetation, climatic, spatial and edaphic variables on the community composition, diversity and richness of total macrofungi as well on functional groups are discussed. In paper IV, the impacts of the variables indicated in paper III on the community composition, diversity and richness of soil fungi and functional guild are studied. The findings have implications for the sustainable conservation and use of both the natural and plantation forests in Ethiopia through mycosilvicultural management approaches. Also, the conservation of biological diversity of the forest system through the provision of complementary income for the local communities through sporocarps production was emphasized. The taxa composition is also explained in terms of edaphic variables, stand age, vegetation types and climatic variables. Conceptual map of the study including the four studies is shown below (Fig. 1). Outline of the thesis xviii Figure 1: Conceptual map of the thesis including the 4 studies Introduction Introduction 1 1. Introduction 1.1. Climate, Geology and Ecology of Ethiopia Ethiopia is situated in central part of the horn of Africa and it is a land locked country which spans 30 24' to 140 53'N and 330 00' to 48° 00'E, encompassing approximately 1270 kms in North-south and 1650 kms in East-west directions. The country has varied topography ranging from 123 m below sea level to 4533 m above sea level and majority of area have > 2000 m elevation unlike other countries in Africa (Friis et al., 2010). The mean annual rainfall ranges from 500 to 2800 mm and temperature 10oC to 30oC (Demissew and Nordal, 2010). The highland of the country is dissected by the East African rift valley. The oldest rocks in Ethiopia are part of the crystalline basement, which is pre-Cambrian in origin. The original igneous and sedimentary rocks are interblended with schists and gneisses and subsequent igneous intrusions. The whole system is referred to as the basement complex (Friis et al., 2010). Furthermore, the country is an ecologically diverse country owing to the varied topographic features and altitudinal variations (Gebretsadik, 2016). 1.2. Natural forests in Ethiopia According to Friis et al. (2010), the vegetation of Ethiopia is classified into 12 types based on the elevation zones in which they occurred: (1) Desert and semi-desert shrub land, (2) Acacia-Commiphora woodland and bush land, (3) Wooded grassland of the Western Gambela Region, (4) Combretum-Terminalia woodland and wooded grasslands, (5) Dry Afromontane forest and grasslands complex, (6) Moist Afromontane forest, (7) Transition rainforest, (8) Ericaceous belt, (9) Afroalpine vegetation, (10) Riverine vegetation, (11) Fresh water, lakes, lakes shores, marshes, swamps and flood plain vegetation and (12) Salt-water, lakes, lakes shores, salt marshes and plain vegetation. The natural high-elevation forests (Fig 2), that include the Afromontane vegetation, are exclusively found in the highland regions of Ethiopia between 1500 to Introduction 2 3400 m above sea level (Lemenih and Bekele, 2008) that occupy more than 44% of the country’s land area (Kidanu, 2004; McCann, 1995). Dry Afromontane forests is a complex ecosystem characterized by high humidity, a variable rainfall pattern, and a prolonged dry season (Friis et al., 2010). These forests provide important ecosystem services such as watershed protection and carbon sequestration (Wassie et al., 2005). The dominant tree species in these forests are Juniperus procera, Podocarpus falcatus, Hagenia abyssinica and Olea africana, which are the main source of timber in the country. These forests also harbour various types of non-timber forest products (Fig 2B), including wild edible mushrooms (Dejene et al., 2017b). Figure 2: The natural forests (A) and the collected non timber forests products (B) in Ethiopia High levels of historical human landscape alteration and land-use pressure have resulted in widespread deforestation and the degradation of Ethiopian forests (Aerts et al., 2016; Aynekulu et al., 2016; Darbyshire et al., 2003; Nyssen et al., 2014). A recent review of forestry in Ethiopia revealed that deforestation is a continuous process (Gebru, 2016). When all forest types were included, a deforestation rate of 0.93% per year was calculated in 2010 (FAO, 2020; Zewdie et al., 2010). The ever-increasing demand for wood products as well as crop and grazing land expansion, stimulated by rapid population and livestock growth are the factors aggravating the degradation of the Dry Afromontane forests in the country (Bekele and Lemenih, 2008). Human-induced fire is also one of the most important reasons for the depletion and degradation of natural resources in Ethiopia (Lemenih and Bekele, 2008; Wassie et al., 2005). Fire is more common in the highland areas, where the dry Afromontane forest is found, and Introduction 3 has a direct impact on the biodiversity in the forest ecosystem (Lemenih and Bekele, 2008). As a result, many physical and biological changes have occurred in these forest systems in Northern Ethiopia. These forest currently are the most fragmented ecosystems (Dessie, 2007; Lemenih and Bekele, 2008; Miles et al., 2006; Wassie et al., 2010). Loss of biodiversity could also occur in the forest soil, which harbours a great diversity of microbial organisms (Fierer and Jackson, 2006), including fungi. Depending on the severity and frequency, fire could directly or indirectly affect edaphic variables in the forest ecosystem (Reazin et al., 2016), which in turn could have an impact on fungal communities dwelling in the soil (Cairney and Bastias, 2007; Dahlberg et al., 2001; Rincón and Pueyo, 2010). Figure 3: The fragmented Dry Afromontane church forests in Northern part of Ethiopia Studies have evaluated the conservation value of fragmented forests in the Northern landscapes of Ethiopia (Aerts et al., 2016; Aynekulu et al., 2016; Wassie et al., 2010, 2005; Nyssen et al., 2014). Most of these fragment forests survive in the landscape because of the cultural or religious values held by local communities that are found as forest islands (Aynekulu et al., 2016). These forests belong to the church or are located around church forest territories (Aerts et al., 2016; Aynekulu et al., 2016; Wassie et al., 2009) (Fig 3). Forest fragmentation affects biodiversity (Lemenih and Bongers, 2011; Wassie et al., 2005) and the population viability in the long-term (Fernández et al., 2020). The condition probably reflected on the fungal communities constituting the forests systems. Additionally, the forest fragmentation impacts soil Introduction 10 fungal species (56) had been evaluated for the International Union for conservation of Nature (IUCN) Red List compared to plants (25,452 species) and animals (68,054) (Kew, 2018). Despite recent advances in determining the diversity and composition of forest fungi in various biomes, fundamental questions regarding their distribution and function, and the factors that influence them remain unanswered, particularly in undersampled biomes (Guo et al., 2013; Krashevska et al., 2015). The majority of soil fungi are unexplored and, the functional relationship between fungi, soil, and plants remains understudied (Bridge and Spooner, 2011; van der Heijden et al., 2008). Previous investigations have estimated that there are about 5.1 million fungal species worldwide (Taylor et al., 2014). Of these, 2–6% have been described (O’Brien et al., 2005) and ~1200 new species are described each year (Hibbett and Thorn, 2001), indicating that there are many more fungal species to be explored, named, and identified. Furthermore, to date, most studies of soil fungal communities have focussed on temperate and Mediterranean forest ecosystems; less consideration has been given to soil fungal communities in tropical forest ecosystems (Taudière et al., 2017). Therefore, further studies are required to increase our understanding of the dynamics of soil fungi and their community structure (Dhruba et al., 2015) and the impact of various environmental and anthropogenic factors. Also, most studies of soil fungal communities currently have focussed on temperate and Mediterranean forest ecosystems; less consideration has been given to soil fungal communities in tropical forest ecosystems (Taudière et al., 2017). Therefore, further studies are required to increase our understanding of the dynamics of soil fungi and their community structure (Dhruba et al., 2015) and the impact of various environmental and anthropogenic factors. On the other hand, fungi are facing threats related with climate change, pollution, over-exploitation, and habitat destruction and fragmentation (Dahlberg et al., 2010; Kew, 2018). Despite these, the fungi are often neglected in conservation due to knowledge gap between mycologists and conservationists in fungal distributional and ecological data. This problem is more complicated mainly due to the invisible, indeterminate form and their tendency to switch between forms (Dahlberg and Mueller, 2011). Although the noticeable and abundant spectacles of fruiting structures of fungi Introduction 11 (e.g. mushrooms) produced by some fungal species, in general fungi are difficult to identify and count since, when not fruiting, most are composed of nothing more substantial than a wispy network of mycelium. Fungi therefore contain a great, and yet largely obscured, presence within soil and inside other living things (Kew, 2018). This makes them difficult subjects to characterize, survey, and monitor them. Fortunately, a general shift towards conserving whole ecosystems is now underway due to their ecological importance in nutrient cycling (Heilmann-Clausen et al., 2015). In general, long-term and large-scale data, data from tropical, experimental data from fungi associated with trees and data from multiple simultaneous drivers of change for fungal community change are the major information gaps on global fungal resources (Kew, 2018). 1.8. Overview of mycological studies in Ethiopia Despite fragmentation and deforestation, studies have evaluated the conservation value of different forests in the Dry Afromontane regions in Ethiopia (Aerts et al., 2016; Aynekulu et al., 2016; Nyssen et al., 2014; Wassie et al., 2010). Most of these studies focused on the above story components of these forest systems and very limited numbers of studies have investigated fungal communities from the Dry Afromontane region of Ethiopia. These studies were focused on above-ground fungal communities (Dejene et al., 2017a) while the soil fungal communities associated with the dry Afromontane forests in Ethiopia are undescribed. Furthermore, the potential effect of fire, edaphic, aboveground vegetation, climate and spatial variables on soil fungal communities in these ecosystems has not yet been evaluated. Limited focuses have been given to the ecology and conservation status of the soil fungal and macrofungal diversity of the Dry Afromontane forests system despite their wider distribution in the country. Consequently, the fungal taxonomy and ecology are very poorly described and, hence, fungi are neglected when decisions need to be made regarding forest management and conservation actions in this region. Previous studies conducted on plantation forest resources in the Dry Afromontane regions of the country focused on ways to assist the management and Introduction 12 development of these plantations for wood products. The study of soil fungal communities in these forest systems in the Dry Afromontane region is scarce. Recently, Castaño et al. (2019) investigated the soil fungal community and ecological guilds associated with Eucalyptus grandis plantations in Ethiopia. However, currently there has been an interest in surveying fungi in particular habitats (Alem et al., 2020b), to describe and predict the extent of their diversity on a larger scale (Danielsen et al., 2005; Peay, 2014). However, studies on the soil microbial community, including fungi associated with Pinus plantations, are very limited. Sporocarps associated with a P. patula plantation during a single rainy season have previously been reported (Averill et al., 2014). Although sporocarps represent a unique step in the complex life of fungi (Averill et al., 2014), they do not reflect the entire soil biota (Ortega-Martínez and MartínezPeña, 2008). Aboveground vegetation affects the community composition and diversity of fungi. The practice of using plant communities as surrogates to predict fungal diversity has been reported by previous studies (McMullan-Fisher et al., 2010; Rudolf et al., 2013). Despite fragmentation, the forests in the study areas are suggested to be relatively rich in plant species (Aerts et al., 2016; Wassie et al., 2010). The tree species composition of the fragmented forests also varied with their status, topography and altitude (Bongers and Tenngkeit, 2010), with a wide distribution over the landscapes (Aerts et al., 2016). However, there is no evidence that the high level of plant diversity in the church Dry Afromontane forests system anticipates correspondingly high macrofungal diversity. Moreover, there is no information on how habitat fragmentation may affect fungal communities or limit fungal processes is relatively limited (Edman et al., 2004; Grilli et al., 2012; Mangan et al., 2004). In addition, as yet, the environmental variables that govern fungal communities in these fragmented forest systems have not been identified given that these forests vary in their status (size, density, species composition etc.), topography and altitude (Bongers and Tenngkeit, 2010). Previous studies in Ethiopia have been focused on ethnomycological knowledge and mushroom consumption habit of the local people, diversity and community composition of macrofungi and soil fungi. More attention has been given to studies on Introduction 13 AMF associations and edible fungi. Less emphasis was given to total fungal studies, both above ground and below ground. In terms of study locations, more studies were conducted in southern Ethiopia (Alem et al., 2020a; Beenhouwer et al., 2015; Castaño et al., 2019; Chauhan et al., 2019; Dejene et al., 2017b, 2017a; Dobo et al., 2018b, 2018a, 2016; Hailemariam et al., 2013; Megersa et al., 2017; Michelsen, 1993; Muleta et al., 2013, 2008; Sewnet and Tuju, 2013; Tuno, 2001; Wubet et al., 2009) followed by north Ethiopia (Birhane et al., 2018b, 2018a, 2017a, 2012, 2010; Delelegn et al., 2018; Weldekiros et al., 2017; Welemariam et al., 2018). Northwestern and central Ethiopia had been given less attention in mycological studies. Since Ethiopia has diversified climatic, vegetation, topographic and edaphic features (Friis et al., 2010; Gebretsadik, 2016) which in turn affect the type of fungi that exist in the area (Semwal et al., 2014) additional studies in understudied areas of the country are required. Location specific studies carried out in Ethiopia showed the existence of huge potential of mushrooms (Abate, 1999; Weldekiros et al., 2017) and high species numebr per unit area (Dejene et al., 2017a). These studies also indicated the potential of the resources for the sustainable management of forest resources and contribution to food security of the local community living around these forests in general. It was also indicated that more number of macrofungal species and new ones can be explored if different forest systems are included (Dejene et al., 2017b, 2017d; Megersa et al., 2017). In many countries, edible wild mushrooms have been identified, cultivated and incorporated as staple foods (Boa, 2004) and extensive collections and herbarium data have also been documented (Beluhan and Ranogajec, 2011). However, in Ethiopia the mushroom cultivation practices and consumption is very low compared to other countries in Europe and Asia (Gebrelibanos et al., 2016) despite the country possesses numerous species of wild mushrooms (Abate, 1999; Weldekiros et al., 2017). The forest management in the country neglected such important resources. The overall review of the available published data indicated that, still there is a gap in quantifying the fungal diversity of the country (Dejene et al., 2017d). Introduction 14 1.9. Why the current study? Sustainable development though the conservation of natural resources is a major challenge for developing countries like Ethiopia. Ethiopia is mentioned as one of the countries rich in diverse plant and animal species (Gebretsadik, 2016). However, the forest resource of the country are vanishing at an alarming rate because of various reasons such as high human and animal pressure and unsustainable use of forest resources (Gebru, 2016). To avert the problem, different forest development approaches have been tried and symbolic achievements have been recorded in some localities (Winberg, 2011). However, many remnant natural forests in Ethiopia have been closed as “reserve forests” to promote the conservation of the natural forests. Plantation forests especially fast growing tree species have been introduced in the country and planted mainly for fuelwood and timber. However, in the management of Ethiopian forests, the value of forest resources other than timber have been overlooked (Desalegn and Tadesse, 2004) and the NTFPs such as mushroom are not included in forest management plan of both natural and plantation forests resource of the country despite their potential contribution to the sustainable management of forest ecosystems (Amma et al., 2018) and ensuring food security of the nation. Because of this problem sustained deforestation and degradation has been recorded in Ethiopia (Asfaw and Etefa, 2017). Devising cheap and ethically widely accepted inocula production methods and better ways of management for effective restoration of degraded lands will also remain to be important research areas (Asmelash et al., 2016). Therefore, alterative forest management approaches that bring economic benefits to the local community and provides intermediate income from forest resources (Melesse and Abtew, 2015) are important for the sustainable management of Ethiopian forest resources (Yadav and Mekonnen, 2013). One of these alternative forest management interventions is the use of NTFPs such as gums and resin, wild honey, medicinal plants and fungi/mushrooms. However, the fungal resources and the tradition of mushroom consumption are being disappearing parallel with the forest resource of the country (Weldekiros et al., 2017). These resources are also under dynamic change due to sever habitat degradation in the country (Dejene et al., 2017d). Introduction 15 Review of existing literature on fungal studies of the country indicated that the fungal resource of the country is poorly studied, documented and not properly utilized. This implies the need for conducting more detailed and wider scale fungal studies to document the fungal resource of the country including fungal community compositions, mushroom production and driving factors for their distribution (Dejene et al., 2017d). Such information will help to devise strategies for the sustainable management and utilization (Hailemariam et al., 1990), formulate effective and integrated policies, provide prioritized management recommendations (Dahlberg et al., 2010) of these resources. Conservation of biodiversity depends on reliable information about the kinds of organism present, total number of species in each of these group, their genetic diversity, their habitats, distribution pattern, ecology, population size, evolutionary history, and their trends both in time and space (Bhandari and Jha, 2018). The biodiversity data of fungi are useful indicators to assess the current status of an ecosystem and important for maintaining and managing the ecosystem of a forest. It was in the light of this background information that the present study was conducted in different agro-ecologies and different forest systems of Ethiopia so as to generate valuable information on total species richness, abundance, diversity, evenness and composition of soil and macrofungi community and factors affecting these. Due to the key ecological role that fungi play in ecosystem functioning, the information about how ecological factors affect the fungal communities in the church fragmented forests can be crucial to enable the integration of these forests into global biodiversity conservation strategies and to understand what actions must be undertaken to conserve these forests, and their biological components. Information generated from this research is believed to help design conservation and sustainable use of fungi in particular and of forest resource of Ethiopia in general. The output of the research will also add valuable information to the local, national and global fungal community diversity. It will also contribute to the achievement of national environmental and forest related goals as well as to the national GDP, food security of the nation and the country’s vision to become middle income country by 2030. This research in line with the international and national development strategies of the country such as Climate Introduction 16 Resilient Green Economy of Ethiopia, Millennium Development Goals, and Forest Sector development of Ethiopia, Growth and transformation plan of Ethiopia and other forest related national and international conventions. Thus, study the effect of various environmental variables soil on the fungal community composition and sporocarps production in different ecosystems is of paramount importance to have a general understanding of processes in the forests ecosystems (Hanson et al., 2012; Hazard et al., 2013) to set up their management and conservation strategies. Investigating the fungal community composition and how this community changes across sites in fragmented forests should help us to understand different aspects of fungal interaction within these systems and their function in the ecosystem (Genevieve et al., 2019). This information would also enable the integration of fragmented forests into global biodiversity conservation strategies (Hundera et al., 2013; Aerts et al., 2016; Aynekulu et al., 2016) and to understand what actions are required to conserve these forest systems their biological components, including fungi (Burgess et al., 2006). Furthermore, the macrofungal study in fragmented Dry Afromontane forests is also a means to understand how to improve natural fungal richness and sporocarp production and help us to facilitate the conservation of economically and ecologically important macrofungal species in these fragmented high priority forest systems. Such information could help to guide management and conservation strategies for these priority forests and supplement our knowledge of macrofungal species in Ethiopia. Similarly, the knowledge of fungi and their community structure in relation to different age categories of plantation forests also helps to determine proper management strategies of plantation forest in Ethiopia. Furthermore, knowledge of the edible mushrooms produced in these forests could provide an opportunity for harvesting edible mushrooms for either subsistence or commercial use. 1.10. Hypotheses and research questions In our study regarding the effect of fire on soil fungal community composition and diversity, we hypothesized that the richness and composition of the entire and functional soil fungal communities would change substantially during a post-fire forest succession Introduction 17 and would differ from those in unburned forest and the community turnover would partially be explained by edaphic variables. As a consequence, we expected ruderal, generalist saprotrophic fungi be more abundant and species-rich shortly after fire than in unburned forests. By contrast, root-associated symbiotic fungi were expected to be more diverse in older burned forests and unburned forests. In our soil fungal study under P. patula plantation, we hypothesized that substantial change in the composition of soil fungal communities would be detected along the chronosequence of P. patula plantations. Specifically, we hypothesized that there would be changes in the total and functional fungal diversity and community composition along the chronosequence of the plantations. In our macrofungal and soil fungal studies in the three fragmented church forests of the Dry Afromontane areas in North Ethiopia, we hypothesized that the fungal diversity of the church forests would be high in terms of total fungal species and functional status given that fungal diversity is related positively to plant richness (Tedersoo et al., 2014b). We also hypothesized that the composition of macrofungal communities would differ among the studied forests, resulting in an overall higher richness value for the study sites and their community composition is driven by vegetation and site conditions such as soil fertility (Castaño et al., 2018; Vašutová et al., 2017), climatic and spatial variability (Glassman et al., 2017; Li et al., 2020; Tedersoo et al., 2014a; Tedersoo et al., 2014b). 1.11. Scope of the study To our knowledge, this research is the first systematic attempts focused to describe the fungal communities’ structures and sporocarp production in fragmented forests in Dry Afromontane region of Ethiopia in relation to multiple environmental factors such as forest fire, vascular plant diversity, edaphic characteristics, climatic and other spatial variables. This research is also the first attempt to describe the soil fungal community composition in relation to stand development stage of P.patula plantation under Ethiopian condition. The field studies in the Dry Afromontane forests were based Introduction 18 both on soil fungal sampling and sporocarp collections. Sporocarp collections were done during the pick rainy season of the year in the studied forests. However, the study was conducted during the main rainy season of a single year in the Dry Afromontane forests and P.patula plantation in Ethiopia. Therefore, its wider application in other forest systems of the country shall be taken with caution. Long term sampling period and wider scale studies are required to know more about the fungal resource of the country. However, the results provide relevant information for developing sustainable management strategies of Dry Afromontane forests in Ethiopia by integrating fungi/mushrooms which in turn help to conserve these forest systems and their biological components, including fungi. The study on sporocarps including edible mushrooms in fragmented church forests could also provide an opportunity for harvesting edible mushrooms for either subsistence or commercial use and help for sustainable management of these forest systems. Objectives Material and Methods 26 Wondo Genet natural forest area, where the study on soil fungal community in relation to fire conducted, covers about 797 ha of natural forests land (Ango and Bewket, 2007; Belaynesh, 2002; Fenta, 2014). The forest is characterized by remnant Dry Afromontane forest patches (Ango and Bewket, 2007; Belaynesh, 2002; Fenta, 2014) and harbours important fauna and flora (Belaynesh, 2002; Fenta, 2014). The climate is characterized by the Weyna-Dega agro-climatic zone, with a bimodal rainfall pattern: the main rainy season is in the summer and a lesser rainy season is in spring (Belay, 2016; Fenta, 2014). Part of the native vegetation in Wondo Genet study sites, where the study on effect of P.patula stand age on soil fungal community and diversity conducted (Fig. 6), was destroyed for cultivation (Teshome, 2011). In recent decades, a mass planting scheme of exotic tree species has been undertaken on those areas and resulted in approximately 100 ha of non-native plantations of Cupressus lusitanica, Grevillea robusta and P. patula (Bekele et al., 2013; Teshome, 2011). Figure 6. Plantation of Pinus patula where soil fungal data was collected (photo credit, Dejene et al., 2017) Figure 7. Dry Afromontane forests (A,Wondo Genet; B, Taragedam; C,Alemsaga; D, Banja) selected for our study (photo credit:7A, Dejene et al., 2017; 7B-7D, Demelash Alem) Material and Methods 27 The Taragedam (Fig. 7C) and Banja (Fig. 7D) forests were designated as reserves in 1979 (Zegeye et al., 2011) and 1994 (Abere et al., 2017), respectively, to prevent any kind of encroachments. The Alemsaga forest (Fig. 7B) was designated as a priority forest in 1978 to serve as a seed source, to conserve the remnant natural forest, and to rehabilitate the degraded area in the Northern part of the country (Masresha et al., 2015). Descriptions of the study forests are provided in Table 1. Table 1. Characteristics of the study sites Descriptions Forests Taragedam Alemsaga Banja Wondo Genet Geographical location 12°06'–12°07' N 37°46'– 37°47' E 11°54'–11°56'N 37°55'–37°57'E 10°57'–11° 03'N 36°39'– 36°48'E 7°06' –7°07' N 38°37' –38°42' E Altitude range (m asl) 2142–2484 2180–2470 1870–2570 1600-2580 Mean annual precipitation (mm) 1098 1926 1884.3 1210 Mean annual temperature (°C) 19.5 15.8 18.7 20 Forest area (ha) 875 814 897 797 Sand (%) 58.89(2.93) 51.78(2.99) 68.67(2.21) 56.55(2.14) Silt (%) 28.44(2.38) 32.44(2.13) 20.00(1.76) 20.83(1.87) Clay (%) 12.67(1.37) 15.78(1.93) 11.33(1.33) 23.23(1.81) pH H2O 1:2.5 7.04(7.03) 5.85(6.59) 5.60(6.24) 6.46(0.12) EC (dS/m) 0.43(0.05) 0.28(0.03) 0.81(0.14) 0.17(0.03) Ex.Ca (cmol(+)/kg) 13.95(0.60) 9.19(0.52) 13.55(0.87) 22.03(2.48) Ex.Mg (cmol(+)/kg) 6.16(0.10) 4.58(0.15) 5.54(0.20) 7.49(0.89) Ex.Na (cmol(+)/kg) 1.95(0.05) 2.05(0.10) 1.82(0.12) 0.97(0.09) Ex.K (cmol(+)/kg) 0.73(0.06) 0.61(0.04) 0.77(0.06) 0.60(0.07) CEC (cmol(+)/kg) 47.21(1.36) 34.89(0.92) 44.51(1.96) 42.8(3.25) Organic matter (%) 4.46(0.60) 3.35(1.34) 4.87(0.10) 9.14(1.03) Nitrogen (%) 0.23(0.01) 0.17(0.02) 0.26(0.01) 0.49(0.05) P (ppm) 17.18(5.72) 7.8(0.73) 17.64(6.05) 32.38(2.85) Dominant species in each plots Maytenus obscura, Carissa edulis, Olea sp. Acacia abyssinica, Buddleja polystachya, Acacia nilotica Albizia gummifera, Prunus africana, Brucea antidysenterica Juniperus procera, Podocarpus falcatus, Hagenia abyssinica Olea africana References Gedefaw and Soromessa (2014) Zegeye et al. (2011) Zerihun et al. (2013) Birhane et al. (2017) Masresha et al. (2015) Wubet et al. (2004) Abere et al. (2017) (Alem et al., 2020a; Belaynesh, 2002; Costa et al., 2014) Note: Numbers in parentheses are standard error of the mean Material and Methods 28 3.3. Establishment of field plots Plots in Dry Afromontane natural forests (study I) and plantations of P. patula (study II) of Wondo Genet study area were established by considering the similarity of the areas in climate, altitude, soil and other ecological conditions. We used the information from the Department of Forest Management in Wondo Genet College of Forestry (WGCF) to find areas with similar fire history in natural forests and stand ages classes in plantations. The plots established in Taragedam, Alemsaga and Banja forests (study III and IV) were based on their history and the composition of vascular plants. Field observations and published data sources were used to describe the sites. Within each of the selected areas in all studies, plots were placed systematically (Luoma et al., 1991) far enough from each other in order to provide relatively independent estimates as possible. 3.3.1. Plot establishment in Dry Afromontane forests to study soil fungal community composition and diversity in relation to forest fire (study I) Sample plots were established in the forest in 2015. The control stand of unburned natural forest (UB) was representative of the original natural forest and had not been affected by fire for at least 40 years. Burned stands selected for the study were similar in terms of fire severity, i.e., the canopy and understory had burned and the soil organic layer had been consumed (Rincón and Pueyo, 2010). In these burned areas, two forest stands were selected based on fire history: (1) one-year-old burned forest (B1); and (2) ten-year-old burned forest (B10). Within each of these forest stands, three transects (a total of 9 plots) were established about 250 m apart from each other. Each transect covered an area of 100 m2, with a rectangular shape (2 m × 50 m). 3.3.2. Plot establishment in P. patula plantation forest of different age to study the soil fungal communities and succession (study II) In P. patula plantations, stands of three different age groups (5-, 11and 36years-old stands) were selected and three 2 m × 50 m plots were established in each age category (Gassibe et al., 2011) for soil sampling both for molecular work and for Material and Methods 29 soil physico-chemical analysis. A minimum distance of about 120 m was used between plots within a stand (Luoma et al., 1991). 3.3.3. Plot establishment to study sporocarps diversity and production (study III) and soil fungal community composition and diversity (study IV) in fragmented church forests in Dry Afromontane forest systems in Northern Ethiopia In total, 27 sample plots were established, nine in each of the three church forests, as described in Gassibe et al. (2011) and (Hernández-Rodríguez et al., 2013). Each plot was rectangular in shape (2 m × 50 m). Within each of the selected church forests, we studied three different sites including three plots per site. The plots were established about a minimum distance of 500 m apart. 3.4. Sampling 3.4.1. Sporocarp sampling (study III) All fungal fruit bodies found in each plot were harvested weekly. Fresh weight measurements were taken in situ to determine fruit body production in kilograms per hectare per year. The number of individuals of each species in each plot was also recorded. Specimens were photographed in the field and their morphological features and ecological characteristics were noted to facilitate taxonomic identification processes in the laboratory (Adeniyi et al., 2018). Specimens of each macrofungus were taken to the laboratory and dried to preserve as herbaria specimens, and then used for morphological taxa identification. 3.4.2. Soil sampling for DNA extraction (Study I, II & IV) Five cores were extracted in each plot using a cylindrical (2 cm radius, 20 cm deep, 250 cm3) soil borer (De la Varga et al., 2012; Taylor, 2002) along the centerline of each transect and 5 m apart to collect spatial variability and minimize the probability of sampling the same genet repeatedly. Soil sample cores from each plot were pooled Material and Methods 30 to form a composite sample for DNA extraction. Soil cores were dried, sieved through a 1 mm mesh and grounded to a fine powder using a mortar and pestle. A subsample was stored at -20ºC until submitted for molecular analysis. 3.4.3. Soil sampling for physico-chemical analysis (Studies I, II, III & IV) To relate soil fungal composition to edaphic variables, additional soil samples were collected from each transects. Soil samples, from the center and from the four corners of each plot in each study, were extracted to a depth of 20 cm with the aid of an auger and spade after clearing plant matter and debris. A composite soil sample of approximately 500 g from each plot was placed in a plastic bag and transported to the laboratory for the determination of edaphic variables. After air drying the soil in shade, important chemical and physical properties of the soil were determined using DTPA extraction, KH2PO4 extraction, Olsen, Kjeldahl digestion, Walkley–Black, ammonium acetate and instrumental methods respectively. The analysis was conducted by Water Works Design and Supervision Enterprises, laboratory service sub process, soil fertility section at Addis Ababa, Ethiopia (Studies I and II) and Amhara Design and Supervision Works Enterprise at Bahir Dar, Ethiopia (studies III & IV). Main edaphic variables for the natural forest in Wondo Genet (Table 1 in study I), P. patula stands (Table 1 in Study II) and church forests of fragmented natural forests of in Northern Ethiopia (Table 1 in study III & Table 1 in study IV) are summarized. 3.4.4. Vegetation and climate data collection (Studies III & IV) To relate the vegetation characteristics to macrofungal (study III) and soil fungal (study IV) richness and diversity, vegetation inventories were conducted in the plots established for fungal sampling. Vascular plant identified in each plots were recorded using their vernacular names. For those species difficult to identify their scientific name in the field, specimens were collected and their taxonomic identification was conducted using published volume of the flora of Ethiopia and Eritrea (Hedberg and Sue, 1989). Large trees growing outside the plots were included in the survey if their crowns Material and Methods 31 overhung the plots because tree crown projection areas can affect fungal occurrence (Collins et al., 2018). Furthermore, large trees create their own microhabitat and develop a large root system, providing more space for fungal associations (Schön et al., 2018). Vascular plant species richness and diversity parameters were determined (Table 3 in study III). Plant parameters and their correlations were also used for further interpretation of fungal pattern from each study areas. The mycorrhizal status of the vascular tree species found in each of the studied plots were checked using freely accessible databases (Soudzilovskaia et al., 2020). Since rainfall and temperature affect fungal community composition and diversity (Bahram et al., 2012; Djelloul and Samraoui, 2011), rainfall and temperature data of the nearby meteorological stations were procured from the Ethiopian National Meteorological Agency (NMA), Bahir Dar meteorological service center. This was done to relate climatic variables with soil fungal species composition and diversity. 3.5. Laboratory analysis and taxa identification 3.5.1 Sporocarp taxa identification and classification (Study III) In the laboratory, the morphological features of the fruit bodies were examined using appropriate monographs, including Antonin (2007), Hama et al. (2010), Heinemann (1956), Hjortstam and Ryvarden (1996), Morris (1990), Pegler (1968, 1969, 1977), Rammeloo and Walleyn (1993), and Singer (1965), to determine the genus and species of the macrofungal specimens. Up-to-date fungal taxa names and authors’ names were obtained from the Mycobank database (http://mycobank.org). Ecological functions at the genus level were identified using a FUNGuild (www.funguild.org) search and provided (Table 2, study III). In addition, the edibility of the fruiting bodies collected from the study sites was assessed following the criteria used by Bonet et al. (2004). Taxa described in the literature as both non-edible and edible in the literature were classified as non-edible. Taxa described in the literature as having doubtful edibility were classified as non-edible. Only species classified as edible by a large majority of the literature consulted were classified as edible fungi (E). Material and Methods 32 3.5.2. Molecular analysis (Studies I, II & IV) DNA was extracted from 0.25 g of soil per sample using a PowerSoil™ DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, USA). PCR reactions were performed in triplicate for each sample to minimize PCR biases. PCR reactions were performed in 20 μl reaction volumes containing 11.22 μl of MQ water, 1.60 μl of DNA template, 2.00 μl of 10× buffer, 1.40 μl of MgCl2 (50 mM), 1.60 μl dNTPs (10 mM), 0.50 μl BSA (2%), 0.80 μl of reverse and forward primers (10 μM) and 0.08 μl Platinum Taq polymerase (Invitrogen, Carlsbad, CA, USA). We used the following PCR conditions: an initial denaturation step at 94°C for 3 min; then 35 cycles of 94°C for 45 s, 50°C for 1 min and 72ºC for 1.5 min; and a final cycle of 72°C for 10 min. The ITS2 rDNA region was amplified using the forward primer fITS7 (Ihrmark et al., 2012) and the barcoded reverse primer ITS4 (White et al., 1990). The ITS4 primer was labelled with samplespecific Multiplex Identification DNA-tags. A negative control consisting of MQ water instead of DNA was included in each PCR run. The absence of bands on gels indicated that negative controls were amplicon free. Ion Torrent sequencing was carried out at the Naturalis Biodiversity Center. The sequencing Ion 318TMChip was used to allow for the highest possible sequencing coverage. 3.6. Data analysis 3.6.1 Bioinformatics analysis (Studies I, II & IV) Raw sequence reads comprising demultiplexed sample reads were obtained from the Ion Torrent output. Primers and poor-quality ends were removed based on a 0.02 error probability limit in Geneious Pro 8.1.8 (BioMatters, New Zealand). Next, all sequences were truncated to 200 bp and then filtered with USEARCH v.8.0 (Edgar, 2010) to discard sequences with an expected error of >1. The remaining sequences were collapsed into unique sequence types on a per-sample basis using USEARCH v.8.0 (Edgar, 2010) while preserving read counts. First, we discarded singleton sequence types before grouping the remaining high-quality sequences into operational taxonomic units (OTUs) with USEARCH at a 97% sequence similarity level while simultaneously excluding OTUs with <70% similarity or <150 bp pairwise alignment Material and Methods 33 length to a fungal sequence. Sequences were assigned to taxonomic groups based on pairwise similarity searches against the curated UNITE+INSD fungal ITS sequence database, which contains identified fungal sequences with assignments to species hypothesis groups (Kõljalg et al., 2013). Up-to-date fungal taxa names and authors’ names were obtained from Mycobank database (http://www. mycobank.org). The FUNGuild database (http://www.funguild.org) was initially used to perform functional classification of OTUs at the genus level and it was manually checked afterwards. OTUs with >90% similarities to a fungal SH with known ecological function were assigned to functional groups. For genera that are known to comprise species from multiple functional guilds, their ecological function was assigned individually based on available ecological information for the matching SH in the UNITE database. 3.6.2. Statistical analysis (Studies I-IV) All explanatory environmental variables were subjected to appropriate data transformation when needed to achieve the parametric criteria of normality and homoscedasticity. Shannon’s H’ diversity indices, H = –Σpi(lnpi) (Shannon and Weaver, 1949), were estimated, where p indicates the relative abundance of fungal OTUs (Kent and Coker, 1993). The Simpson’s diversity indices, D = 1 – Σ (pi2), where pi is the importance probability in element i; and the Evenness, J = H′/H′max, where H′ is the number derived from the Shannon diversity index and the H′ max is the maximum possible value of H′ were also calculated (Magurran, 1988). In addition, the richness values of all fungal OTUs (S) based on treatment type were estimated. All diversity measures were calculated using the BiodiversityR package (Kindt and Coe, 2005) in R (R Core Team, 2020). For studies which ANOVA assumptions were met, diversity indices and richness were compared across treatments using one-way ANOVA using R (R Core Team, 2020) and Tukey HSD was used to determine significant differences between means (P ≤ 0.05) among treatments. Linear Mixed Effects models (LME, Pinheiro et al., 2016) was used to prevent the false positive associations due relatedness structure in the sampling (study III). The most significant variables used for interpretation of fungal diversity and community composition were selected using the forward.sel function of adegraphics package (Siberchicot et al., 2017) in R. Separate Material and Methods 34 analysis was conducted on functional groups of fungi to determine whether their abundance and diversity were affected by explanatory environmental variables. Ordination techniques based on Hellinger-transformed fungal abundance data were used to discern changes in fungal community composition among treatment and to identify significant explanatory variables related to taxa composition. The abundance data matrices against edaphic variables were subjected to a canonical correspondence analysis (CCA) using PC-ORD v. 6.0 software (McCune and Mefford., 2011) (study II). Non-parametric data analysis methods were used when transformations did not provide the appropriate results or a clear interpretation (Ágreda et al., 2014). Non-metric multidimensional scaling (NMDS) was conducted using metaMDS function of the vegan package in R on Hellinger-transformed abundance of fungal data matrices against explanatory variables (Study III and IV). Correlation of ordinations axes scores with edaphic, vegetation and climatic variables were assessed using linear regression. A multiple-response permutation procedure (MRPP) and a permutation-based nonparametric MANOVA (PerMANOVA) (Anderson, 2001) were run using Bray–Curtis distance and adonis function of the vegan package (Oksanen et al., 2019) in R (R Core Team, 2020) to analyze differences in fungal communities across forests or among treatments. An analysis of similarity percentages (SIMPER; Clarke, 1993) was also performed using the simper function of the vegan package (Oksanen et al., 2019) in R (R Core Team, 2020) to identify fungal species that were most responsible for the observed patterns and determine the percentage contribution of fungal taxa to significant dissimilarities between the three forests (Parravicini et al., 2010). Indicator species analysis was determined using the multipatt function of the indicspecies package (Caceres and Legendre, 2009) in R. Fungal species accumulation curves and the Rényi diversity profile were also generated using a sample-based estimator of EstimateS Version 9 (Colwell, 2013) to compare fungal richness and diversity among different treatments. A Rényi diversity profile (Tóthmérész, 1995) was also used to depict the diversity curves among treatment. Unless stated, all data analysis was conducted using R Software version 4.0.3 (R Core Team, 2020). Results Discussion 43 5. Discussion 5.1. Soil Fungal Taxa Composition (Studies-I,-II, and-IV) Fragmentation poses major threats to Dry Afromontane forest ecosystems of Ethiopia. However, these forests are considered to be major reservoirs of biodiversity (Aerts et al., 2016; Aynekulu et al., 2016; Darbyshire et al., 2003; Nyssen et al., 2014). Similarly, we found a huge soil fungal diversity in these forest systems with clear differences in community composition among the three study areas located in Northern Ethiopia (Study IV). The diverse fungal species in such forest systems could be mainly explained by higher tree species diversity (Chen et al., 2017) and due to the improved soil fertility from added nutrients in the decomposition process of wood materials that provide the required nutrients for diverse groups of fungal species (Siciliano et al., 2014). In all studies, the fungal taxa were dominated by Ascomycota, which is congruent with other studies in different forest ecosystems (Geml et al., 2014; Reazin et al., 2016; Smith et al., 2017; Tedersoo et al., 2014). The dominancy of Ascomycota could be due to their higher genomic potential for resource utilization, competition, and stress tolerance (Egidi et al., 2019). In all our studies it was observed that significant numbers of fungal taxa were not identified down to genus and species level indicating lack of data from understudied tropical and subtropical forest ecosystems (Tedersoo et al., 2014) such as the forest systems in Ethiopia. The largest proportions of identified fungal species in our studies were saprophytic which play a role in decomposition of organic matter, as a source of human food (Kirk et al., 2008) and as biocontrol agent in agriculture (Rossman et al., 1999; Samuels, 1996). Our study on soil fungal community under different age groups of P. patula plantation showed that the functional groups of fungi from the whole study plots were saprotrophs (41%) followed by plant pathogenic (7%) and ECM (2%) fungi. The low proportion of ECM fungi detected under P.patula plantations might be due to the Discussion 44 conversion of the native vegetation of the area to crop cultivation many years ago (Teshome, 2011). Studies indicated that the proportion of mycorrhizal fungi in tropical regions is low and the majority of plant species in the region do not form mycorrhizal association with fungi (Brundrett, 2009). Absence of ECM fungi in the dry Afromontane forests of Ethiopia was also reported previously (Dejene et al., 2017a). In contrast, we encountered higher relative proportion of ECM fungi in such forest systems (Fig 2B in study-I; Fig 1B in study-IV). This association may be due to the diverse vegetation (Friis et al., 2010) and the presence of more trees that host mycorrhizal fungi, or may be due to the dispersion of mycorrhizal inocula from nearby plantation forests dominated by Eucalyptus and Pinus species (Castaño et al., 2019; Dejene et al., 2017a, 2017b; Urcelay et al., 2017). High number of ECM species in such forest systems could also be justified by the older and denser forests that maintain temperature and adequate moisture (Fernández-Toirán et al., 2006; Pinna et al., 2010; Toivanen et al., 2012). The root systems of the old trees also facilitate the occurrence of ECM fungi (Mölder et al., 2014). In study-II, some fungal species under the genera Tomentella, Ramaria and Inocybe were found associated with P. patula trees at all age stages of tree development. Tomentella and Inocybe are cosmopolitan species that inhabit Eucalyptus plantations in Ethiopia (Castaño et al., 2019). Species of Rhizopogon were also associated more with younger stands (5and 11-year-old stands) in this study, which supports previous findings, that they are early colonizer fungal species (Tedersoo et al., 2016b). The Rhizopogon species are known as spore bank species that facilitate the establishment of trees in formerly non-forest habitats. Amanita, which was also found in our study site of P. patula plantation are well-known for their association with conifer forests as the genera is characteristic of late-stage pine stands that are 30–40 years old (Chu-Chou and Grace, 1982; Visser, 1995). Previous studies indicated that the composition of ECM fungi in the soil is correlated with soil fertility and the growth status of the host trees (Cozzolino et al., Discussion 45 2016; Wang and Wang, 2008). Similarly, we found that the 36-year-old stand and the 5year old stand of P. patula had distinctive soil fungal communities. The distinct composition of ECM fungi in young and old stands under P. patula plantations (study II) might be related to site quality factors, such as soil fertility and stand age factors. Given that the amount of OM, available P and the C/N ratio of 5and 11-year-old stands were not significantly different, this may have enabled 11-year-old stands to develop an association with only a limited number of ECM fungi, but a higher relative abundance of these ECM fungi, which could indicate increased dependence of P. patula trees on a limited number of dominant symbionts species. We observed differences in fungal community composition and diversity due to forest fire. This could be due to a change in vegetation (Hart et al., 2005) and loss of host plants after fire (Pattinson et al., 2006; Smith et al., 2005). More fungal species were detected in the burned forest areas where the soil fertility was relatively low than in unburned areas, which could be related to depositions of ash after the fire (Hul et al., 2015). Ash depositions could create empty niches for rapid colonization of the area by early stages colonizer fungi (Fritze et al., 1993). Agaricus campestroides was highly abundant in one-year-old burned stands but much less abundant in ten-year-old burned and unburned stands. The species might be partially responsible for the differences between stands, suggesting that time after fire is also probably responsible for the variation in the dominance of some species and their exclusive occurrence in certain stands. This is supported by previous findings that, for a given stand, certain fungal species tend to be abundant and characterize its composition (Zhu et al., 2010). Distinct fungal community composition pattern was also observed in our soil fungal study in the studied Dry Afromontane forest systems in North Ethiopia (study-IV). Such differences indicate the site specific nature of fungal assembly such as ecological gradients (Egidi et al., 2019) and vegetation (Egidi et al., 2019; Tedersoo et al., 2016).Vegetation is known to affect fungi composition along with the processes that influence the nature and quantity of resources entering into the soil (Wardle et al., 2004) which further implies that fungal community is structured through the environmental Discussion 46 factors (Hanson et al., 2012; Hazard et al., 2013) that regulate the assemblage of vegetation. Studies demonstrated that fungal community composition can be governed by various environmental variables and landscape heterogeneity (Bahram et al., 2015; Ferrari et al., 2016; Peay et al., 2010; L. Tedersoo et al., 2014). Thus, evaluating the fungal communities in different ecosystems is essential to filter out the relative contributions of environmental factors to fungal diversity and composition in an ecosystem (Tian et al., 2018). Soil characteristics strongly affect fungal community structures (Straatsma et al., 2001; Zakaria and Boddy, 2002; Lauber et al., 2008; Reazin et al., 2016; Yang et al., 2017), community composition (Castaño et al., 2019; Delelegn et al., 2018; Lauber et al., 2008) and distribution (Claridge et al., 1993). Specific fungal species are likely to respond to environmental variables, mainly edaphic parameters, in different ways (Cozzolino et al., 2016; Koide et al., 2014), and, thus, in turn, the composition of the fungal community is directly correlated with edaphic variables (Cozzolino et al., 2016). Nitrogen and Phosphorous significantly affected the community composition of entire soil fungal in our fire experiment in Dry Afromontane forest systems (study I). Previous reports also indicated that high availability of N and P could negatively affect the structure of fungi in the soil, particularly of the mycorrhizal fungi (Zhao et al., 2018) by decreasing plant dependency on fungi and reducing carbon allocation to fungi (Liu et al., 2019) which eventually could cause competition among the fungal species and lead to the formation of distinct fungal composition (Wang and Wang, 2008; Zhao et al., 2018). Available P also influenced the composition of soil ECM fungi in P. patula plantation (Table 4 in study-II), which was similar to the findings reported by Rosenstock et al. (2016). However, we noted positive correlation between N and entire soil fungal community composition in Dry Afromontane forests in Ethiopia (study-IV). This most probably indicates that the majority of plant species in the studied forest are independent of mycorrhizal fungi. Discussion 47 Soil pH is reported as important factors governing the fungal composition (Ullah et al., 2019). Our study on soil fungi in the Dry Afromontane forests in North Ethiopia (study-IV) indicated that fungal composition is maintained at a lower pH level. However, some fungal taxa are directed towards the higher pH level which could be due to the ability and adaptability of these taxa to grow in a comparatively alkaline soil condition (Nevarez et al., 2009; Tian et al., 2018). Cations play an important role in many physicochemical processes, such as photosynthesis (He et al., 2017) and, hence, the amount of carbon that is available to soil fungi (Shi et al., 2014) in forests soils. Our study showed significant effect of Ca, Mg, and K on soil fungal community composition in the Dry Afromontane forests (studyIV). The climatic and vegetation characteristics are reported to influence the spatial variation and thus, the composition of fungal communities (Newsham et al., 2016). In our soil fungal study in the Dry Afromontane forest systems in North Ethiopia (study IV), we observed a strong association of soil fungi with daily temperature and annual rainfall. In line with our results, other studies indicated that temperature shapes the composition of soil fungal communities in forest ecosystems (Newsham et al., 2016) since air temperature together with the moisture in the soil increases the metabolic activity of fungi, extends the period for which fungi are active each year, and enables a switch from survival to growth strategies. Thus, the distinct soil fungal communities found in each of the church forests (Study-IV) may follow the variation of the rainfall of the three areas, the plant community in each of the forests, or both (Hawkes et al., 2011). 5.2. Macrofungal Taxa composition (Study-III) Despite habitat fragmentation is reported as negatively affecting the fungi community in forest systems (Sapsford et al., 2017), we found a high number of macrofungal species in fragmented church forests in Dry Afromontane region of Ethiopia. The diverse fungal species in such forest systems could be mainly explained by higher tree species diversity (Chen et al., 2017) and due to the improved soil fertility Discussion 48 from added nutrients in the decomposition process of wood materials that provide the required nutrients for diverse groups of fungal species (Siciliano et al., 2014). Significant numbers of fungal taxa were not identified down to genus and species level indicating lack of data from understudied tropical and subtropical forest ecosystems (Tedersoo et al., 2014) such as the forest systems in Ethiopia. The largest proportions of identified macrofungal species in our study were saprophytic. Such functional groups are important for decomposition of organic matter and are valuable food sources for humans (Kirk et al., 2008). Some of the species are also important in agriculture as biological control agents (Rossman et al., 1999; Samuels, 1996). The sporocarp productions obtained in this study were not high. Although further research is needed, this could partially be explained by the single tone species. Some of the species were collected in a single time during the collection period. Moreover, majority of the species were saprophytic fungi which are characterized by low biomass productions (Gassibe et al., 2011; Mediavilla et al., 2014). However, valuable edible macrofungal species belonging to the Calvatia, Laetiporus, Pleurotus, Termitomyces sp., and Macrolepiota genera were also collected in this study. Among these edible species, Termitomyces sp. is highly regarded by local people in southwest Ethiopia because of its good taste and aroma (Abate, 2014). This also provides a starting point in terms of broadening the management and conservation of fragmented forests for the production of non-timber forests products in Ethiopia. Although, previous studies indicated the low proportion of ECM species in the tropics (Dejene et al., 2017a; Tedersoo et al., 2014), we found more ECM taxa (14% of the total). This could be attributed to higher vascular plant diversity (Friis et al., 2010), availability of more trees that host mycorrhizal fungi (Hailemariam et al., 2013; Wubet et al., 2003) or the dispersion of mycorrhizal inocula from the nearby plantation forests. This finding indicates the important implication of the indigenous forest system for the maintenance of functional fungal diversity in Ethiopia (Dejene et al., 2017a). Discussion 49 Soil pH is known to be the most critical edaphic variable affecting the composition and structure of fungal communities (Docherty et al., 2015; Fierer and Jackson, 2006b; Zhang et al., 2016). Similarly, in this study, soil pH correlated with fungal species composition and the presence of greater numbers of macrofungal species was associated with lower pH values. This was in line with the findings of Puangsombat et al. (2010) and Zhang et al. (2016) who reported negative influence of higher pH levels on fungal community structure, probably because a higher pH restrains the expansion of fungi and the production of sporocarps. Organic matter influences mycelial outgrowth and network formation (Zakaria and Boddy, 2002) and fungal community through its impact on the water-holding capacity and soil and nutrient availability (Harrington, 2003). Thus, a high level of organic matter accumulation implies a high level of macrofungal assembly, particularly of saprophytic species. Similarly, we found OM associated with the composition of macrofungi in the studied fragmented church forests of Dry Afromontane forests systems (Figure 6 in study III). We also found that CEC associated with macrofungal composition. Crabtree et al. (2010) observed that fungal species richness was low, particularly when the CEC was high. This is probably because the CEC influence nutrient availability, soil pH, and soil reactions to other ameliorants in the soil (Ogeleka et al., 2017). CEC is vital in many physicochemical processes, such as photosynthesis (He et al., 2017) and thereby influence the amount of carbon available to fungi in the soil (Shi et al., 2014). Maximum and minimum temperatures also affected macrofungal composition. This may be due to the fact that the mycelium of the fungal species is more readily affected by atmospheric changes (Salerni et al., 2002), being more superficial specifically for those saprotrophs species that constitute the majority of macrofungal taxa in our studied forests. Temperature can also play important role in nutrient cycling process (Geng et al., 2017) which in turn results in the formation of distinct fungal communities, particularly of the fungi that are soil dependent as a substrate (Nicolás et al., 2019). Discussion 50 5.3. Effect of fire (study-I), stand age (study-II), and aboveground plant diversity (study-III and study-IV) on fungal richness, diversity and production Our study on the effect of fire on soil fungal community (study-I) revealed that higher total richness values were found in forest stands recently affected by fire than unburned stands. This could be attributed to the new ecological conditions created by differences in fire severity, which may incite or support spore germination of several fungal species in the soil (Heino, 2012) following the fire in the investigated forests. In addition, the mycelium of fungal species in the rhizosphere may persist (Cowan et al., 2016; Shen et al., 2016) or the fungal community may be resilient to the effects of fire to some extent (Cowan et al., 2016; Jennings et al., 2012). The study also revealed absence of significant difference in fungal diversity between fire affected areas. This could be due to the less fuel consumed and heat produced during fire (Reazin et al., 2016; Semenova-Nelsen et al., 2019). Rather the change might have been driven by indirect effect of fire in soil properties or by the change in the plant communities (Oliver et al., 2015; Ponder et al., 2009; Trappe et al., 2009). Also, fungi in a recurrent forest ecosystem may be adapted to frequent fires (Semenova-Nelsen et al., 2019) (Dean et al., 2015; Hart et al., 2005) (SemenovaNelsen et al., 2019). Furthermore, the intensity of the fire might not have been high enough to affect the below-ground fungal communities (Bárcenas-Moreno et al., 2009; Egidi et al., 2016). Thus, the responses of soil fungi to recurring low-intensity fire also appear to be minimal (Johnson et al., 2013; Oliver et al., 2015) and ephemeral (Hart et al., 2005). Time since the fire occurrence did not affect fungal guild diversity. In agreement with our finding, Egidi et al. (2016) noted absence of a significant change in fungal diversity following fire. This might be due to low fuel loads that resulted in little heat transferred to the soil (Lunt and Morgan, 2002).. Discussion 51 Our study also revealed that both the richness and diversity of ECM fungi were higher in in the recently burned stands, which could indicated an immediate post-fire mycorrhizal colonization in fire-affected forest stands (Dahlberg, 2002; Rincón et al., 2015). The ECM taxa may also have established dominance immediately after burning owing to their tolerance of fire effects (Dahlberg, 2002; Kipfer et al., 2010) or they may have survived in a mycelial state during the fire event (Hewitt et al., 2013). In contrast, , other studies reported that older stands support greater diversity of ECM fungi (Fernández-Toirán et al., 2006; Pinna et al., 2010; Toivanen et al., 2012) and generally have higher abundance of ECM (Mölder et al., 2014). The relatively higher diversity indices of ECM fungi in the 5-year-old stand than in the 11-year-old stand under P. patula may possibly be attributed to the less developed tree canopy at the earlier stage of stand development, which may have allowed diverse ECM fungi to interact extensively with the root systems of understory plants (Dang et al., 2017) and/or the previous land use of the plantation area (agricultural crop production). Deacon and Fleming, (1992) demonstrated that when afforestation takes place on land initially used for other purposes, the ECM fungal spores are the fundamental inoculum during the early stage of ECM succession. The spore banks might have also derived from other nearby mycorrhizal-associated plantations such as Eucalyptus plantations (Castaño et al., 2019; Dejene et al., 2017d). The higher diversity indices of ECM species detected in the 36-year-old stand than in the 11-year-old stand may be due to the thinning carried out in this stand unlike the other two age groups of P. patula (Dejene et al., 2017b). Chen et al. (2015) because thinning could increase the relative abundance of mycorrhizal fungi as it opens up the forest canopy, which in turn, increases soil temperature and moisture (Wang et al., 2019). Thinning may therefore have a positive effect on microbial activity (Pang et al., 2013) because soil temperature and moisture influence the reaction of microbial enzymes and, thus, shift the microbial community composition by altering substrates and extracellular enzyme activity (Hassett and Zak, 2005). Dove and Keeton (2015) and Tomao et al. (2020) suggested that fungal diversity can be conserved or even increased using forest management practices that enhance the structural complexity of stands and Conclusions 58 of valuable wild mushrooms should be incorporated into management and conservation strategies in these fragmented forest systems. 4. Our soil fungal and macrofungal studies in fragmented church forests of the dry Afromontane forest systems indicated that the promotion of vascular tree diversity in these forest systems through enrichment plantings or assisted natural regeneration management systems would offer suitable habitats with variable microclimates that should assist fungal species. In addition, the effects of the aforementioned management practices on soil fertility should be taken into consideration owing to the important relationship between edaphic variables and fungal composition in these forests. 5. Data obtained in all the studies in this thesis will significantly contribute to the body of knowledge regarding soil fungal communities in Ethiopia and provided relevant information required for the management and conservation of these forest systems and the central roles played by fungi in the management and conservation of such forests systems including provision of food resources for poor populations during times of food scarcity. 6. In all our studies it was observed that significant number of fungal taxa was not identified down to genus and species level. This indicates that the fungal diversity in Ethiopian forest systems is as yet largely undescribed and likely includes many taxa unknown to science. Thus, we advise that additional long term scientific investigations are needed to consolidate the Ethiopian fungal biodiversity database. Conclusions 59 7. Conclusiones 1. El estudio reveló que los bosques eclesiásticos fragmentados de los sistemas forestales secos afromontanos en Etiopía albergan muchos hongos diversificados importantes para el manejo sostenible de estos sistemas forestales. Por lo tanto, el resultado presenta una visión de la conservación y el manejo de gremios funcionales valiosos en los suelos de los sistemas forestales fragmentados de la iglesia Dry Afromontane de Etiopía. La diversidad de plantas vasculares se encontró como un factor para la diversidad fúngica del suelo. Se encontraron hongos ECM importantes en estos sistemas forestales. Sistemas forestales etíopes que podrían respaldar la importancia de la conservación de los hongos en los sistemas forestales afromontanos secos de Etiopía. Es inevitable que los bosques eclesiásticos fragmentados de los sistemas forestales secos afromontanos necesiten una conexión, ya sea mediante el establecimiento de un nuevo bosque de protección adyacente a los bosques remanentes o el establecimiento de plantaciones forestales asociadas con micorrizas. Esto también proporcionaría diversos nichos para el crecimiento y desarrollo de diferentes especies de hongos y permitiría un fácil movimiento de esporas entre los tipos de bosques. 2. Nuestros estudios destacaron que la composición de los hongos difería en una cronosecuencia después de un incendio en los bosques secos fragmentados de Afromontano, variaba con la edad del rodal en las plantaciones de P. patula, difería debido a la composición de especies de árboles de los bosques secos de Afromontano en Etiopía y su composición se explica por factores vasculares. diversidad arbórea, variables edáficas, espaciales y climáticas. Por lo tanto, el efecto de las prácticas de manejo forestal como el aclareo y la cosecha debe tenerse en cuenta debido a la importante relación entre estos parámetros ecológicos y la composición fúngica del suelo en los sistemas forestales de Etiopía. 3. Nuestro estudio de macrofungos reveló la presencia de valiosas especies de macrofungos comestibles pertenecientes a los géneros Tricholoma, Suillus y Conclusions 60 Termitomyces, que potencialmente podrían comercializarse y, por lo tanto, podrían proporcionar ingresos suplementarios a la población local y los administradores forestales que dependen de los bosques. Por lo tanto, la producción de valiosos hongos silvestres debe incorporarse a las estrategias de manejo y conservación en estos sistemas forestales fragmentados. 4. Nuestros estudios sobre hongos y macrofúngicos del suelo en bosques eclesiásticos fragmentados de los sistemas forestales secos afromontanos indicaron que la promoción de la diversidad de árboles vasculares en estos sistemas forestales mediante plantaciones de enriquecimiento o sistemas de gestión de regeneración natural asistida ofrecería hábitats adecuados con microclimas variables que deberían ayudar especies. Además, deben tenerse en cuenta los efectos de las prácticas de manejo antes mencionadas sobre la fertilidad del suelo debido a la importante relación entre las variables edáficas y la composición fúngica en estos bosques. 5. Los datos obtenidos en todos los estudios de esta tesis contribuirán significativamente al cuerpo de conocimiento sobre las comunidades de hongos en el suelo en Etiopía y proporcionaron información relevante requerida para el manejo y conservación de estos sistemas forestales y los roles centrales que juegan los hongos en el manejo y conservación de dichos sistemas forestales, incluida la provisión de recursos alimentarios para las poblaciones pobres en épocas de escasez de alimentos. 6. En todos nuestros estudios se observó que no se identificó un número significativo de taxones de hongos hasta el nivel de género y especie. Esto indica que la diversidad de hongos en los sistemas forestales de Etiopía aún no se ha descrito en gran medida y probablemente incluye muchos taxones desconocidos para la ciencia. Por lo tanto, advertimos que se necesitan investigaciones científicas adicionales a largo plazo para consolidar la base de datos de biodiversidad fúngica de Etiopía. References 61 8. 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Madrid 44, 34071 Palencia, Spain bEthiopian Environment and Forest Research Institute, Addis Ababa, Ethiopia c MTA-EKE Lendület Environmental Microbiome Research Group, Eszterházy Károly University, Leányka u. 6, H-3300 Eger, Hungary d USDA Forest Service Pacific Northwest Research Station, Forestry Sciences Laboratory, 3200 SW Jeferson Way, Corvallis, OR 97331, USA For. Ecol. Manage. 474(118328) https://doi.org/10.1016/j.foreco.2020.118328 Resumen Los bosques secos afromontanos de Etiopía son ecosistemas complejos que tienen importantes funciones económicas y ecológicas. Sin embargo, los incendios recurrentes han sido una fuente de perturbación para estos bosques. Evaluamos el efecto del fuego en las comunidades de hongos del suelo en un bosque afromontano seco remanente en Wondo Genet, en el sur de Etiopía, mediante el análisis de muestras de suelo recolectadas de rodales no quemados y de rodales uno y diez años después del incendio utilizando metabarcoding de ADN del ADNr ITS2. El análisis indicó que la comunidad de hongos del suelo era más diversa poco después de una perturbación por incendio y disminuyó con el tiempo. La composición de la comunidad fúngica también difirió entre los rodales. Nuestros resultados también indicaron que las diferencias en la diversidad de hongos dependían del rodal y no de la cronología de la historia de los incendios en este sistema forestal. Encontramos un mayor número de especies de micorrizas en rodales quemados, lo que sugiere que estos simbiontes de hongos podrían compensar los efectos del estrés nutricional causado por el fuego en estas áreas. La composición de la comunidad fúngica también se correlacionó significativamente con el contenido de materia orgánica, potasio y magnesio en el suelo. Este trabajo podría considerarse como un estudio de caso ya que las parcelas se establecieron en un solo rodal para cada tratamiento en los bosques secos afromontanos de Etiopía. Por lo tanto, recomendamos estudios adicionales y las conclusiones con respecto a otros rodales deben tomarse con precaución. Palabras clave: Variable edáfica, Etiopía, Incendio forestal, grupos funcionales de hongos, secuenciación de torrente de iones, trópicos. fungi in this order can also be saprotrophic, entomopathogenic, and mycoparasitic (Rossman et al., 1999). In addition to their ecological and economic importance, the Hypocreales are also considered to be the most important regulators of insect and fungal populations and, therefore, are used in agriculture as biocontrol agents (Carruthers and Hural, 1990; Esser and El-Gholl, 1993; Rossman et al., 1999; Samuels, 1996). The second largest order of Ascomycota detected in this study was the Pleosporales. This order comprises saprotrophs or fungi that are parasites of vascular plants (Kruys et al., 2006). Some species from this order are also found on animal dung (Kruys et al., 2006), a small number occur as lichens (Semenova-Nelsen et al., 2019) and as rockinhabiting fungi (Ruibal et al., 2009). The epiphytic or endophytic fungi of the Pleosporales are mainly saprotrophic but also play a key role in causing plant diseases such as stem canker (Zhang et al., 2009). A considerable number of fungi belonging to the order Chaetothyriales were also detected in this study. This order includes fungi that are known to be epiphytes, colonizing the leaves and the bark of trees in tropical forest ecosystems (Arnold et al., 2000; Batista and Ciferri, 1962). The order Agaricales was the largest order of Basidiomycota detected in this study: members of this order produce the familiar gilled mushroom (Binder et al., 2005; Hibbett and Thorn, 2001; Stajich, 2015). Agaricales are widespread in diverse ecosystems (Kirk et al., 2008) and many form ectomycorrhizae by engaging in mutualistic symbioses with vascular plants (Alexopoulos et al., 1996). Some Agaricales are known to be termite symbionts, some are valuable as a Fig. 3. Numbers and proportional distribution of fungal operational taxonomic units (OTUs) representing all taxonomic phyla and orders found in soil samples collected from the dry Afromontane forest of Wondo Genet, Ethiopia. D. Alem, et al. Forest Ecology and Management 474 (2020) 118328 6 source of food for animals, including humans (Kirk et al., 2008), whereas others have hallucinogenic properties or produce toxins lethal to humans (Nichols, 2003). Most of the species of Agaricales detected in this study are well known soil saprotrophs, such as those belonging to the genera Agaricus,Calvatia,Coprinellus,Gymnopilus,Leucoagaricus, Lycoperdon,Marasmius,Psathyrella and Psilocybe, have been reported previously as fruit bodies from our study area (Dejene et al., 2017b) providing validation of our molecular techniques. 4.2. Fungal richness and diversity changes after fire Our results from the post-fire successional chronosequence showed that soil fungal richness was related to fire. In this study, we found higher total richness and diversity values in the forest stands recently affected by fire than unburned stands. This may be attributed to the new ecological conditions created owing to differences in fire severity, which may incite or support spore germination of several fungal species in the soil (Heino, 2012) following the fire in the investigated forests. In addition, the mycelium of fungal species in the rhizosphere may persist (Cowan et al., 2016; Shen et al., 2016) or the fungal community may be resilient to the effects of fire to some extent (Cowan et al., 2016; Jennings et al., 2012). Furthermore, the intensity of the fire might not have been high enough to affect the below-ground fungal communities given that low-intensity fires may have little effect on below-ground fungal communities (Bárcenas-Moreno et al., 2009; Egidi et al., 2016). Thus, the responses of soil fungi to reoccurring low-intensity fire also appear to be minimal (Johnson et al., 2013; Oliver et al., 2015) and ephemeral (Hart et al., 2005). Contrary to our expectations, we found that the amount of time since the fire did not seem to affect fungal guild Fig. 4. Mean total fungal community richness values in a dry Afromontane forest of Wondo Genet, Ethiopia, following fire. Abbreviations: UB, unburned stand; B1, one-year-old burned stand; B10, ten-year-old burned stand. Bars denote standard deviation. Different letters above the bars indicate a significant difference in richness between stand types (P < 0.01, n = 3 transects per stand). Fig. 5. Mean soil fungal community diversity and evenness estimated for functional guilds detected in three types of forest stand with different fire histories. Abbreviations: UB, unburned stand; B1, one-year-old burned stand; B10, ten-year-old burned stand; Key: Shannon = Shannon diversity values; Simpson = Simpson diversity values; Evenness = evenness values. Bars denote standard deviation, n = 3 transects per stand. Fig. 6. Relative proportions of fungal operational taxonomic units (OTUs) in different ecological guilds in unburned stands (UB), one-year-old burned stands (B1) and ten-year-old burned stands (B10). Bars denote standard deviation. D. Alem, et al. Forest Ecology and Management 474 (2020) 118328 7 diversity. These results agree with the findings of a meta-analysis of fire effects on soil fungi (Egidi et al., 2016), which highlighted the absence of a significant change in fungal diversity following fire. This might be because little heat is transferred to the soil because fuel loads are low (Lunt and Morgan, 2002) or might indicate that the fungal guild communities in burned and unburned forest stands shared similar gene profiles, which may promote functional similarities among fungal communities with differing compositions (Mundra, 2015). On the other hand, the absence of significant difference in fungal richness and diversity in fire affected areas might be due to the fact that recurrent fires consume less fuel and produce less heat, which does not penetrate into soil as deeply as during high-intensity fires (Reazin et al., 2016; Semenova-Nelsen et al., 2019). Accordingly, fungal community shifts in such recurrent fire ecosystems, like that of the dry Afromontane forest, may be relatively modest (Choromanska and DeLuca, 2001; Korb et al., 2004) and the change may be driven by indirect fire-induced changes in soil properties or by the change in the plant communities (Hart et al., 2005; Oliver et al., 2015; Ponder et al., 2009; Trappe et al., 2009). Also, fungi in a recurrent forest ecosystem may be adapted to frequent fires. Some fungi produce heatand smoke-activated spores (SemenovaNelsen et al., 2019) and some may benefit from post fire ash deposits (Dean et al., 2015; Hart et al., 2005) or reduced competition from other species (Semenova-Nelsen et al., 2019). However, factors other than fire might have a greater effect on the richness and diversity of soil fungal communities. Therefore, further research is needed to better understand the dynamics and characteristics of soil fungal communities. A previous study reported the absence of ectomycorrhizal fungi in the dry Afromontane forests of Ethiopia (Dejene et al., 2017a). This finding was not exceptional as the majority of tropical woody tree species are unable to form associations with ectomycorrhizal fungi (Brundrett, 2009). However, in this study, we observed different groups of mycorrhizal fungi and they were identified and classified as ectomycorrhizal and arbuscular mycorrhizal (Fig. 2). This association may be due to the diverse vegetation (Friis et al., 2010) and, hence, there may be more trees present that can act as hosts for mycorrhizal fungi, or may be due to the dispersion of mycorrhizal inocula from nearby plantation forests, which are dominated by Eucalyptus and Pinus species (Castaño et al., 2019; Dejene et al., 2017a; Urcelay et al., 2017). Thus, the findings presented here may have important implifications for the indigenous forest system for the maintenance of functional guild diversity in Ethiopia given that mycorrhizal fungi have previously only been reported from exotic tree plantations (Dejene et al., 2017a). However, the importance of ectomycorrhizae and arbuscular mycorrhizae in indigenous forest systems in Ethiopia needs empirical data to confirm. In addition, the coexistence of these fungi has many practical advantages, such as the exchange of water and nutrients through mycorrhizal hyphal networks (Brundrett, 2002, 2004). Thus, our analysis of the fungal communities in these forest soils presents an insight into the conservation of functional guilds in the forest system in the study area. The vegetation changes after a fire may affect the soil microbial community (Hart et al., 2005). Previous studies have reported that the loss of host plants after fire decreases mycorrhizal fungal diversity (Pattinson et al., 2006; Smith et al., 2005). In our study, both the richness and diversity of ectomycorrhizal fungi increased in the recently burned stands, which could indicated an immediate post-fire mycorrhizal colonization in fire-affected forest stands (Dahlberg, 2002; Rincón et al., 2015), while the saprotrophic fungi mineralize nutrients and stabilize the soil moisture after the fire (Dighton et al., 1986). The ectomycorrhizal taxa may also have established dominance immediately after burning owing to their tolerance of fire effects (Dahlberg, 2002; Kipfer et al., 2010) or they may have survived in a mycelial state during the fire event (Hewitt et al., 2013). However, the Fig. 7. . Detrended Correspondence Analysis ordination plot for soil fungal communities detected in the three treatment groups: B1, plots in one-year-old burned stands; B10, ten-year-old burned stands; UB, unburned stands. Plots with the same symbol are in the same treatment group. Table 2 Canonical correspondence analysis showing the significance (P < 0.05) of edaphic variables based on simple term effects on the mycorrhizal fungal species. Variable Simple term effects Explains % pseudo-F P Mg 22.6 1.71 0.032 K 19.7 1.63 0.036 OM 17.0 1.40 0.047 Fig. 8. . Canonical Correspondence Analysis (CCA) of the species level community composition of ectomycorrhizal fungi in a dry Afromontane forest in Ethiopia. Abbreviations: OM, organic matter; Mg, magnesium; K, potassium. Species names are abbreviated (the full names of the ectomycorrhizal species used in the ordination are provided in supplementary Table S2). D. Alem, et al. Forest Ecology and Management 474 (2020) 118328 8 colonization of mycorrhizal fungi could also be governed by burn severity and by the depth of burning in the soil profile (Hewitt et al., 2013). Thus, the effect of fire on mycorrhizae could be reduced when the fire only occurs at the soil surface, and the effect of the fire reduces with soil depth (Danielson, 1984; Pattinson et al., 2006; Visser, 1995). Thus, the fire that occurred in our forest study area might not have been strong enough to affect the mycorrhizal fungi or may have only affected fungi on the soil surface. It may also be influenced by the host plant's response to fire. However, in Ethiopia the mycorrhizal-associations for most plants are not yet well known. Thus, this should be investigated in future studies, including ectomycorrhizal root-tip samples, to learn more about the diversity of ectomycorrhizal host tree species and their associated fungi in dry Afromontane forests. 4.3. Soil fungal communities and environmental variables The DCA indicated that the fungal communities detected in the three stand types were different. The SIMPER analysis also distinguished the total dissimilarity between stands and the relative contribution of each fungal species to the observed dissimilarity. The species making the highest contributions to the dissimilarity between the oneand ten-year-old burned stands (10.95%) and the one-year-old burned stands and the unburned stands (10.92%) was Agaricus campestroides. This species was highly abundant (N ~ 14916) in one-yearold burned stands but much less abundant in ten-year-old burned and unburned stands (N = 2 and N = 386, respectively). The contribution of the species might be partially responsible for the differences between stands, suggesting that time after fire is also probably responsible for the variation in the dominance of some species and their exclusive occurrence in certain stands. This is supported by previous findings that, for a given stand, certain fungal species tend to be abundant and characterize its composition (Zhu et al., 2010). Thus, a species with a consistently high contribution to the dissimilarity is a good discriminating species (Clarke, 1993). Soil microorganisms, including fungi, are influenced by edaphic parameters (Drenovsky et al., 2004; Lauber et al., 2009, 2008). Our edaphic data from the dry Afromontane forest showed that more fungal species were detected in the burned forest areas (B1 and B10) where the soil fertility was relatively low than in unburned areas, which could be related to depositions of ash after the fire (Hul et al., 2015). Ash depositions could create empty niches that provide opportunities for the area to be rapidly colonized by fungal species at the early stages of succession (Fritze et al., 1993). However, the dry Afromontane forest area has suffered erosion caused by heavy rainfall soon after the fire events. As a result, there is a potential for sediment transportation from fire-affected areas and, thus, changes in soil fertility levels among stands. For instance, pH was assumed to be increased in newly burned areas, owing to the production of oxides and hydroxides (Hul et al., 2015). However, in our study forests, we recorded slightly high soil pH values in unburned forest stands. We found also a significant influence of the N, C/N ration and P on the entire fungal community in this study. For example, N and P reported could affect the structure of fungi in the soil, particularly of the mycorrhizal fungi (Zhao et al., 2018). The higher availability of these elements could decrease plant dependency on mycorrhizal fungi. This condition could also reduce the carbon allocation to fungi (Liu et al., 2019), which could increase competition and affect community composition (Wang and Wang, 2008; Zhao et al., 2018). Our result also confirmed that the fungal richness is low in stands where the soil C/N ratio is higher. Previous studies have reported that after fire, the abundance of ectomycorrhizal fungi is reduced owing to the loss of host plants (Hart et al., 2005). However, in our study, the total fungal OTU richness in fire-affected stands, which had poor soil fertility, was high compared with that of unburned stands (Fig. 4), although such conditions remain to be interpreted. However, Castaño et al. (2019), reported high levels of ectomycorrhizal fungi in stands with poor soil quality. The occurrence of mycorrhizal species in poor quality soils suggests that the nutrient stress created in the fire-affected area could be compensated for by the increased dependency of trees on fungal symbionts (Read and Perez-Moreno, 2003). In this regard, the mycorrhizal ruderal guild in the spore bank would play an important role by quickly colonizing roots of plants, and will likely aid the survival of trees after the fire (Glassman et al., 2016). Species of Wilcoxina,Tomentella,Tricholoma and Laccaria were among the ectomycorrhizal species represented in the fire-affected stands, where soil fertility was low. Some of these genera such as Laccaria are considered ruderal species (Ishida et al., 2007) and are known to form an ectomycorrhizal association with several host tree species (Glassman et al., 2016; Hul et al., 2015). 4.4. Conclusions This pioneer study is the first attempt to describe the soil fungal community in a dry Afromontane forest system of Ethiopia using nextgeneration sequencing and to investigate the effect of fire disturbance on these fungal communities. Data obtained in this study will significantly contribute to the body of knowledge regarding soil fungal communities in Ethiopia; however, the taxonomy of these fungi remains challenging and about 20% of the fungal species detected have not been described even at the phylum level. We conclude that, in general, the fungal diversity in Ethiopian forest systems is as yet largely undescribed and likely includes many taxa unknown to science. Thus, we advise that additional scientific investigations of this highly diverse but unexplored forest ecosystem are needed to consolidate the Ethiopian fungal biodiversity database. Also in this study, the soil fungal communities expected to be changed substantially along a post-fire forest succession and in comparison to those in unburned forest. However, we found that fire did not have a significant negative effect on fungal richness and diversity in the burned stands. Our study also highlighted that soil fungal composition differed across a chronosequence after fire and was correlated with soil fertility conditions and the changes would be explained partially by the edaphic conditions. Contrary to our expectation, root-associated symbiotic fungi like that of the mycorrhizal fungi were not lacking in fire-affected stands. We assume that mycorrhizal fungi present in the spore bank were able to colonize the roots of plants that survived the fire. In the fire-affected forests, we also found fungi that are known to form ectomycorrhizal associations with several host tree species. This key ecological role could provide support for the importance of fungal conservation in the dry Afromontane forest systems in Ethiopia. Similarly, vital edaphic variables such as OM and K also appear to be important in shaping the composition of mycorrhizal soil fungi in different ways. Thus, the effect of forest management practices such as thinning and harvesting on soil fertility should be taken into consideration owing to the important relationship between these ecological parameters and the soil fungal composition in the dry Afromontane forests of Ethiopia. CRediT authorship contribution statement Demelash Alem: Formal analysis, Writing - review & editing. Tatek Dejene: Methodology, Writing - review & editing. Juan Andrés Oriade-Rueda: Supervision. József Geml: Formal analysis, Writing - review & editing. Carles Castaño: Writing - review & editing. Jane E. Smith: Writing - review & editing. Pablo Martín-Pinto: Conceptualization, Methodology, Supervision, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. D. Alem, et al. 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Bosques de plantaciones de diferentes edades en Etiopía Demelash Alem1,2, Tatek Dejene2, Juan Andrés Oria-de-Rueda1, József Geml3 and Pablo Martín-Pinto1,* 1 Sustainable Forest Management Research Institute, University of Valladolid (Palencia), Avda. Madrid 44, 34071, Palencia, Spain; [email protected] (D.A.); [email protected] (J.A.O.-d.-R.) 2 Ethiopian Environment and Forest Research Institute, Forest Products Innovation Research Directorate, P.O. Box 24536, 1000 Addis Ababa, Ethiopia; [email protected] 3 MTA-EKE Lendület Environmental Microbiome Research Group, Eszterházy Károly University, Leányka u. 6, H-3300 Eger, Hungary Forests 11, 1109. https://doi.org/10.3390/f11101109 Resumen Es probable que el cultivo de plantaciones forestales cambie la diversidad y composición de las comunidades de hongos del suelo. En la actualidad, hay poca información sobre estas comunidades en los sistemas forestales de plantaciones de Etiopía. Evaluamos las comunidades de hongos del suelo en Pinus patula Schiede ex Schltdl. & Cham. Stands de 5, 11 o 36 años de edad utilizando metabarcoding de ADN de amplicones ITS2. Las condiciones ecológicas de cada parcela, como clima, altitud y suelo, fueron similares. La edad del rodal y la fertilidad del suelo influyeron en la diversidad de especies de hongos del suelo y los gremios ecológicos. En total, se identificaron 2262 unidades taxonómicas operativas de hongos, de las cuales el 2% eran ectomicorrízicas (ECM). La diversidad de hongos ECM fue mayor en los rodales de 5 y 36 años que en los rodales de P. patula de 11 años. Contrariamente a nuestras expectativas, se observó un alto nivel de diversidad de especies de ECM en rodales jóvenes, lo que sugiere que estas especies de ECM podrían compensar los efectos del estrés nutricional en estos rodales. Nuestros resultados también sugirieron que la abundancia de patógenos vegetales y saprótrofos no se vio afectada por la edad del rodal. Este estudio proporciona información de referencia sobre los cambios en las comunidades de hongos en los rodales de árboles de diferentes edades en las plantaciones de P. patula en Etiopía que probablemente estén relacionados con los hongos ECM en rodales jóvenes donde prevalece una fertilidad del suelo relativamente baja. Sin embargo, dado que las parcelas se establecieron en un solo rodal para cada clase de edad para cada tratamiento, este estudio debe considerarse como un caso de estudio y, por tanto, se debe tener precaución al aplicar las conclusiones a otros rodales. Palabras clave: hongos ectomicorrízicos; Secuenciación ion torrent; metabarcoding; Pinus patula; diversidad de hongos en el suelo; soportar la edad Article Soil Fungal Communities under Pinus patula Schiede ex Schltdl. & Cham. Plantation Forests of Different Ages in Ethiopia Demelash Alem 1,2, Tatek Dejene 2, Juan Andrés Oria-de-Rueda 1, József Geml 3,4 and Pablo Martín-Pinto 1,* 1Sustainable Forest Management Research Institute, University of Valladolid (Palencia), Avda. Madrid 44, 34071 Palencia, Spain; [email protected] (D.A.); [email protected] (J.A.O.-d.-R.) 2Ethiopian Environment and Forest Research Institute, Forest Products Innovation Research Directorate, P.O. Box 24536, 1000 Addis Ababa, Ethiopia; [email protected] 3Biodiversity Dynamics Research Group, Naturalis Biodiversity Center, Darwinweg 2, P.O. Box 9517, 2300 RA Leiden, The Netherlands; [email protected] 4MTA-EKE Lendület Environmental Microbiome Research Group, Eszterházy Károly University, Leányka u. 6, H-3300 Eger, Hungary *Correspondence: [email protected]; Tel.: +34-979-108-340; Fax: +34-979-108-440 Received: 3 September 2020; Accepted: 14 October 2020; Published: 19 October 2020   Abstract: The cultivation of plantation forests is likely to change the diversity and composition of soil fungal communities. At present, there is scant information about these communities in Ethiopian plantation forest systems. We assessed the soil fungal communities in Pinus patula Schiede ex Schltdl. & Cham. stands aged 5, 11, or 36-years-old using DNA metabarcoding of ITS2 amplicons. The ecological conditions of each plot, such as climate, altitude, and soil, were similar. Stand age and soil fertility influenced soil fungal species diversity and ecological guilds. In total, 2262 fungal operational taxonomic units were identified, of which 2% were ectomycorrhizal (ECM). The diversity of ECM fungi was higher in the 5 and 36-year-old stands than in the 11-year-old P. patula stands. Contrary to our expectations, a high level of ECM species diversity was observed in young stands, suggesting that these ECM species could compensate for the effects of nutrient stress in these stands. Our results also suggested that the abundance of plant pathogens and saprotrophs was not affected by stand age. This study provides baseline information about fungal community changes across tree stands of different ages in P. patula plantations in Ethiopia that are likely related to ECM fungi in young stands where relatively low soil fertility prevails. However, given that the plots were established in a single stand for each age class for each treatment, this study should be considered as a case study and, therefore, caution should be exercised when applying the conclusions to other stands. Keywords: ectomycorrhizal fungi; Ion torrent sequencing; metabarcoding; Pinus patula; soil fungal diversity; stand age 1. Introduction A recent review of forestry in Ethiopia revealed that deforestation is a continuous process [ 1 ]. When all forest use was included, a deforestation rate of 0.93% per year was calculated in 2010 [ 2 , 3 ]. Despite this, establishing plantations of fast-growing exotic tree species is becoming a major part of forestry practice in Ethiopia [ 4 , 5 ]. Exotic tree species plantations are now estimated to cover 1,000,000 ha of land [ 5 , 6 ]. One of these introduced tree species is Pinus, which is mainly being grown to meet the increasing demand for woody raw materials [ 6 – 8 ]. As a consequence, Pinus patula Schiede ex Schltdl. Forests 2020,11, 1109; doi:10.3390/f11101109 www.mdpi.com/journal/forests Forests 2020,11, 1109 8 of 18 variables and plantation age. The PerMANOVA analysis also indicated that there were species compositional differences between stands in terms of ECM fungi (F =3.21, p=0.003). 2 (a) (b) Figure 3. ( a ) Detrended correspondence analyses and ( b ) canonical correspondence analysis (CCA) ordination plots based on Hellinger-transformed abundance data of mycorrhizal fungal species at the order level of fungal communities associated with three age groups of Pinus patula stands in the study area in Ethiopia. Plots shown in the same color are in the same stand (yellow, plots in the 5-year-old stand; black, plots in the 11-year-old stand; red, plots in the 36-year-old stand). Edaphic variables are shown in green. The percentages of cumulative explained variation by each axis are shown in (b). Table 4. Canonical correspondence analysis based on simple term effects showing the significance (p<0.05) of edaphic variables when considering the Hellinger transformed data of ectomycorrhizal species at the order level of fungal community in the study area. Variable Simple Term Effects Explains % Pseudo-F p Age 26.8 2.60 0.010 Phosphorus 19.0 2.10 0.052 Several species belonging to the orders Gomphales, Sebacinales, and Thelephorales were associated with plots in the oldest stand, where the soil is characterized by relatively high fertility (based on the organic matter content and the C/N ratio). In 5 and 11-year-old stands, which are characterized by low soil fertility (based on organic matter content and the C/N ratio), the species belonging to the order Cantharellales formed an association with other fungal species (Figure 3). Species in the orders Boletales, Pezizales and Agaricales were associated with all age groups of P. patula plantations (Figure 3). Forests 2020,11, 1109 9 of 18 4. Discussion 4.1. Diversity of Fungal OTUs Fungi have been described as the most interesting, enigmatic and species-rich organisms on Earth [ 69 ]. The use of molecular methods in recent years has dramatically increased the number of fungal OTUs detected worldwide [ 70 ]. In this study, we detected a total of 2262 high-quality OTUs, of which 1303 OTUs (58%) belonged to the Ascomycota, the largest phylum of fungi [ 71 ], indicating the dominance of Ascomycota in the P. patula forests investigated in this study. Fungi often interact with other organisms, forming beneficial or mutualistic associations. Conifers in the Pinaceae usually form symbiotic relationships with ECM fungi. Thus, ECM fungi were likely to play an important role in the P. patula plantations in our study area. However, of the total OTUs identified at genus level and classified by ecological function, 41% were saprotrophs, 7% were plant pathogens, and only 2% were ECM fungi. An explanation for the small proportion of ECM fungi detected might be that the plantation area was located in a non-ECM biome area. The native vegetation that originally grew in the study area was destroyed many years ago by logging and clearance for crop cultivation [ 10 ]. Therefore, fungal symbionts compatible with P. patula may be absent in the native fungal community in the soil, enabling ECM fungi introduced to the plantation area along with P. patula to co-invade the soil habitat [ 72 ]. However, the ecological function of 48% of the fungi identified in this study is unknown, indicating that we have hardly scratched the surface in terms of understanding the role played by fungi in these plantation forest systems. It might also an indication for the lack of scientific studies on the local fungal flora in the country. Thus, they are highlighting the need for further studies in the study area. 4.2. Diversity of Functional OTUs along the Chronosequence of Stands The Shannon and Simpson diversity indices were determined to explain variations in the soil fungal functional groups at different stages of P. patula stand development. Only ECM fungi showed differences in their diversity among the stand age groups. Initially, we expected that as stands developed, soil fertility would decrease over time and that these conditions would lead to higher ECM fungal diversity along the chronosequence. By contrast, the soil fertility of young P. patula stands was expected to be higher than that of old stands and, therefore, the trees were expected to exert less influence on the fungal microbiome. However, our investigations revealed that the 5 and 36-year-old stands had more diverse ECM fungi in their soils than the 11-year-old stand and were more fertile. Despite this, the relative proportion of ECM abundance was higher in the 11-year-old stand than in the 5 and 36-year-old stands due to the dominance of some ECM species in the 11-year-old stand. Soil microorganisms, including fungi, are influenced by stand development in several ways [ 73 , 74 ]. As the stand develops, the amount of tree cover directly modifies the amount of light available, which affects the composition of the understory, which regulates carbon allocation, nutrient cycling and soil water content [ 75 ]. The relatively higher diversity indices of ECM fungi in the 5-year-old stand than in the 11-year-old stand may possibly be explained by the less developed tree canopy at the earlier stage of stand development, which may have allowed diverse ECM fungi to interact extensively with the root systems of understory plants [ 75 ]. Another explanation for the relatively higher diversity indices of ECM fungi in the 5-year-old stand may be the previous land use of the plantation area. Deacon and Fleming [ 76 ] demonstrated that when afforestation takes place on land initially used for other purposes, the ECM fungal spores are the fundamental inoculum during the early stage of ECM succession. In this case, prior to the establishment of P. patula plantations, the study area was previously used for agricultural purposes, which might have contributed to the diversity of ECM fungi in young stands of P. patula trees due to the primary succession of ECM fungi through inocula in the spore bank derived from other nearby mycorrhizal-associated plantations such as Eucalyptus sp. L’H é r. plantations [24,36]. Forests 2020,11, 1109 10 of 18 The relatively higher diversity indices of ECM species in the 36-year-old stand than in the 11-year-old stand may reflect a difference in the management of the 36-year-old stand. Chen et al. [77] indicated that thinning could increase the relative abundance of mycorrhizal fungi because thinning opens up the forest canopy, which increases soil temperature and moisture [ 78 ]. Thinning may therefore have a positive effect on microbial activity [ 79 ] because soil temperature and moisture influence the reaction of microbial enzymes and, thus, shift the microbial community composition by altering substrates and extracellular enzyme activity [ 80 ]. Dove and Keeton [ 81 ] and Tomao et al. [ 82 ] have suggested that fungal diversity can be conserved or even increased using forest management practices that enhance the structural complexity of stands and the late-successional characteristics of the forest and by carrying out low-impact logging operations. In this study, the 36-year-old stand had undergone thinning as part of a management operation in the study area [ 8 ], which could have enhanced the root growth of the remaining trees [ 83 , 84 ], providing new environments for soil microbes, which could lead to an improvement in fungal diversity through root attachments. Castaño et al. [ 12 ] observed that the species diversity of soil fungi remained stable after thinning, regardless of its intensity, when sufficient host trees and functional roots from thinned trees were retained. This finding has been supported by a number of different reports. For example, according to Mölder et al. [ 85 ], thinning could maintain a high level of ECM fungal diversity in mature stands. Chen et al. [ 86 ] and Dang et al. [ 75 ] also hypothesized that when thinning operations are performed, light availability, water, and nutrients increase, which improves the forest microclimatic conditions and, hence, the diversity of ECM fungi could be improved by this type of management practice. Furthermore, Goldmann et al. [ 87 ] reported that ECM fungi were less diverse in unmanaged forests than in highly managed stands. Thus, we suggest that forest management practices such as thinning could be one of the factors that impact ECM fungal diversity along the chronosequence of P. patula stands, although this should be further studied. 4.3. Fungal Composition and Edaphic Variables Forest soils contain a diverse range of fungal species. The composition of soil fungal communities is influenced by different factors, such as dispersal, plant diversity, soil properties, land use, and climate, which are key components of forest systems [ 88 , 89 ]. Specifically, different fungal species are likely to respond to environmental drivers in different ways, depending on their characteristic traits [ 90 , 91 ], and, thus, in turn, the composition of soil fungal communities is directly correlated with soil fertility and plant growth status [ 92 ]. Mycorrhizal species are a particularly important part of the soil fungal community because they form a beneficial symbiotic association with plants, providing them with nutrients in return for photosynthetically fixed carbon [ 93 ], which is especially relevant under nutrient-limited conditions [ 94 ]. ECM fungi also play a key role in alleviating the drought stress of the host tree [ 95 ]. The composition of ECM fungi in the soil is also correlated with soil fertility and the growth status of the host trees [ 92 , 96 ]. In this study, we found that both stand age and soil fertility were factors that affected the fungal community composition in our study area. Our ordination analysis indicated that the 36-year-old stand and the 5-year-old stand had distinctive soil fungal communities, characterized by a relatively high number of ECM species. Previous studies have related similar findings regarding fungal community composition to several factors, such as changes in soil fertility [ 24 ], changes in root density [ 97 ], specific life-history events that have occurred since the stand was established or changes in microclimate conditions [ 12 ]. For example, less litter accumulation in young P. patula stands resulted in less organic matter and a lower C/N ratio in the 5 and 11-year-old stands in this study compared with the 36-year-old stand. This situation leads to trees having a greater dependence on mycorrhizal fungal associations for enhanced nutrient and water uptake and availability [ 24 ]. Similarly, the greater diversity of ECM species in young stands may indicate that suitable symbionts are present. However, older stands have a greater capacity to reduce fluctuations in temperature and to maintain adequate moisture levels [ 98 – 100 ], which is particularly important for the occurrence of ECM fungi. The abundance of ECM fungi in older stands is generally greater than in younger stands, which could be facilitated by the root systems of old trees, which could increase the Forests 2020,11, 1109 11 of 18 chances of ECM associations forming [ 85 ], thereby facilitating the easy uptake of nutrients by trees [ 94 ]. Thus, the distinct composition of ECM fungi in young and old stands might not only be related to site quality factors, such as soil fertility, but also to stand age factors (e.g., the increasing area of tree root exploration in the soil with stand age); however, this needs to be investigated further. Given that the amount of organic matter, available P, and the C/N ratio of 5 and 11-year-old stands showed no greater difference in their values, this may have enabled 11-year-old stands to develop an association with only a limited number of ECM fungi, but a higher relative abundance of these ECM fungi, which could indicate increased dependence of P. patula trees on a limited number of dominant symbiont species. In any case, the survival of P. patula in soils in which ECM species comprise only a small proportion of the microbial community, together with other factors, supports the view that P. patula is well adapted to the conditions in this study area; however, this also needs further study. Fertile soil contains nutrients that enable the growth and development of a soil fungal community [ 101 ]. Thus, in turn, the fungi are directly influenced by edaphic parameters [ 102 – 104 ]. In this study, edaphic cation elements, such as Mg and K, were also correlated with the overall fungal community from the whole data set, which indicates that soil cation concentrations could influence the composition of the fungal community [ 105 ]. Cations in general play an important part in a number of physicochemical processes, such as photosynthesis [ 106 ] and, thus, can affect plant photosynthesis and, hence, the amount of carbon that is available to soil fungi and bacteria [ 107 ]. Of the various cations, Ca is one of the main edaphic factors that influence the structure of soil fungal communities worldwide [ 108 ]. Other edaphic elements have also been reported to influence the composition of fungal communities in forest systems. For example, in this study, available P and tree age influenced the composition of mycorrhizal fungi (Table 4), which was similar to the findings reported by Rosenstock et al. [ 109 ]. The composition of fungi in the soil particularly that of mycorrhizal fungi, can also be influenced by N availability [ 110 ]. High levels of available N could decrease the dependency of the host plant on mycorrhizal fungi, which could reduce the amount of carbon allocated to fungi [ 111 ], which eventually could cause competition among the fungal species and could lead to changes in their composition [ 96 , 110 ]. In this study, the C/N ratio of soil in 5 and 11-year-old stands was relatively low compared with that of 36-year-old P. patula stands (Table 1). This result is inconsistent with Wang and Wang [ 96 ] who reported that a high C/N ratio negatively influenced fungal community structure, probably because a high concentration of N restrains the expansion of fungi. Our results also confirmed this, in the sense that the fungal diversity was low in stands where the soil C/N ratio was high, indicating that a high C/N ratio might not favor the fungal community in the forests in the study area. Soil organic matter could also impact the composition of soil fungal communities because fungi generally extend their mycelia at the soil–litter interface [ 112 ]. The amount of organic matter affects the water holding capacity of soil and nutrient availability, which could affect mycelial outgrowth and network formation [ 113 , 114 ]. However, soil acidity can also influence the composition of soil fungal communities [115,116] through its influence on spore germination and mycelial development [117]. Fungi have different life-history strategies and, in plantations, early colonizer fungal species that relish disturbance colonize first, followed by superior competitors that can outcompete the early colonizer species in older stands where resources are getting scarcer. In this study, we found that some fungal species associated with P. patula trees were detected at all age stages of tree development, such as those belonging to the genera Tomentella,Ramaria, and Inocybe. These genera have several hundreds of species, many still undescribed, and their strategies do not seem to be conserved at the genus level. Of these, Tomentella and Inocybe are cosmopolitan species that inhabit Eucalyptus plantations in Ethiopia [ 24 ]. Species of Rhizopogon were also associated more with younger stands (5and 11-year-old stands) in this study, which supports previous findings that they are early colonizer fungal species [ 118 ]. The Rhizopogon species are known as spore bank species that facilitate the establishment of trees in formerly non-forest habitats. Other taxa belonging to the genera Amanita have also been reported in our P. patula plantation forests. These fungi species are well-known for their association with conifer forests as the genera is characteristic of late-stage pine stands that are 30–40 years old [119,120]. Forests 2020,11, 1109 12 of 18 5. Conclusions Our study explained the soil fungal community composition associated with a P. patula plantation in Ethiopia. The diversity value of vital fungi such as ECM was relatively higher in the youngest stand than in the two older stands. Stand age and soil fertility were also found to affect fungal community composition. Some ECM fungi were found as early colonizer species in the young stand and were replaced by superior competitor species as the P. patula stands developed. The overall low level of ECM species richness detected in the P. patula stands is probably because this is a plantation of an ECM tree in a non-ECM biome. Due to the importance of fungi in plantation forest system, the results of this study could be relevant for the promotion and conservation of forests in Ethiopia through the promotion of non-wood forest products such as fungi, which could also provide food resources for poor populations during times of food scarcity. Thus, it is imperative to investigate how soil fungal communities respond to management regimes such as thinning and clear-cutting. The high diversity and relative abundance of plant pathogenic fungi detected in this study also highlights the need to protect Ethiopian plantations from plant diseases and pests. Although the ecological conditions of all studied plots were similar in terms of climate, altitude, and soil, the results of this study should be considered as a case study, given that the plots were established in a single stand for each age class for each treatment and, therefore, the applicability of any conclusions to other stands should be treated with caution. Furthermore, additional scientific investigations of the plantation forest ecosystem are needed in order to consolidate the Ethiopian fungal biodiversity database. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4907/11/10/1109/s1, Table S1: Summary of the Similarity Percentage (SIMPER) analysis showing contrasts between the cumulative total contribution (50% cut-off) and the contribution (%) of the most influential fungal operational taxonomic units to the dissimilarity of the soil fungi detected in three Pinus patula stands of different age groups in a plantation in Wondo Genet, Ethiopia. Author Contributions: Conceptualization, P.M.-P., J.A.O.-d.-R. and T.D.; methodology, J.G., P.M.-P. and T.D.; software, D.A., P.M.-P. and T.D.; validation, P.M.-P.; formal analysis, P.M.-P., J.G. and T.D.; investigation, P.M.-P., T.D. and D.A.; data curation, D.A.; writing—original draft preparation, T.D. and D.A.; writing—review and editing, P.M.-P., T.D. and J.A.O.-d.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research work was partially supported by the Erasmus Mundus-Dream project grant and by the project SUSTIFUNGI_ET (Sustfungi_Eth:2017/ACDE/002094) funded by the Spanish Agency for International Development and Cooperation. This work was also co-funded by the Spanish Ministry of Education and Culture under a Salvador de Madariaga grant agreement, n◦PRX17/00315. Acknowledgments: We would like to express our gratitude to the people involved in the field work of this study. Conflicts of Interest: The authors declare no conflict of interest. References 1. Gebru, T. Deforestation in Ethiopia: Causes, Impacts and Remedy. Int. J. Eng. Dev. Res. 2016,4, 204–209. 2. FAO. Global Forest Resources Assessment. Rome, Italy. Available online: http://www.fao.org/3/i1757e/ i1757e00.htm (accessed on 1 September 2020). 3. Zewdie, M.; Tesfaye, A.; Girma, Y. Management Plan for Forest Plantations of WGCF-NR; Final Document; WGCF-NR: Wondo Genet, Ethiopia, 2010. 4. Moges, Y. 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[CrossRef] Forest Ecology and Management 496 (2021) 119391 4 2.5. Vegetation sampling To relate the vegetation characteristics to the macrofungal richness and diversity, vegetation inventories were conducted in the plots established for macrofungal species sampling as described above. Vascular plants identified in each plots were recorded using their vernacular names. For those species difficult to identify their scientific name in the field, specimens were collected and their taxonomic identification was conducted using published volume of the flora of Ethiopia and Eritrea (Hedberg and Sue, 1989). Large trees growing outside the plots were included in the survey if their crowns overhung the plots because tree crown projection areas can affect macrofungal occurrence (Collins et al., 2018). Furthermore, large trees create their own microhabitat and develop a large root system, providing more space for fungal associations (Sch¨ on et al., 2018). Then, the vascular plant species richness and diversity parameters were determined (Table 3). Plant parameters and their correlations were also used for further interpretation of macrofungal pattern from each study areas. The mycorrhizal status of the vascular tree species found in each of the studied plots were checked using freely accessible databases (Soudzilovskaia et al., 2020) and the data is provided (Table S1). 2.6. Statistical analysis Data were transformed when needed to achieve the parametric criteria of normality and homoscedasticity. The macrofungi data were normalized by rarefying the abundance data to the smallest number of macrofungi per plot. Also, the data from soil variables were scaled using base R and used for subsequent statistical analyses. Shannon’s H′diversity index, H′=–Σpi (lnpi) (Shannon and Weaver, 1949), was estimated for each forest, where pi indicates the relative abundance of the species (Kent and Coker, 1993). Simpson’s diversity, D =1 −Σ(pi2), where pi is the importance probability in element i; and the evenness, J =H′/H′max, where H′is the number derived from the Shannon diversity index and the H′max is the maximum possible value of H′were also calculated (Magurran, 1988). In addition to species richness values, macrofungi biomass production levels in each forest were estimated and converted in to Kg bases. All diversity measures for macrofungi and vascular plants were analyzed using the Biodiversity R package (Kindt and Coe, 2005) in R version 4.0.3 (R Core Team, 2020). The difference in the soil, vegetation and sporocarps variables across forests were assessed by Linear Mixed Effects models (LME, Pinheiro et al., 2016), where block (a set of plots in a same site in each forest) was defined as random and forest was defined as fixed factor. The LME used to prevent the false positive associations due relatedness structure in the sampling. Tukey Test was later used to check significant differences (p ≤0.05) between forests when needed. Species accumulation curves were constructed to compare the rate at which new fungal species were found in the three forests and to provide an estimate of macrofungal species richness. Curves were generated using a sample-based estimator of EstimateS Version 9 (Colwell, 2013). The number of fungal species collected during each weekly visit to a plot within a forest constituted the sample. Curves were generated based on the total of the weekly sampling datasets. A R´ enyi diversity profile (T´ othm´ er´ esz, 1995) was also used to depict the diversity curves of the three church forests. When parameter alpha =0, this function gives the total species number and when alpha =1, this gives an index proportional to the Shannon index. The relationship of macrofungal composition with the edaphic, climate and location parameters was visualized using non-metric multidimensional scaling (NMDS), based on absence and presence species data matrix and environmental scaled data. A permutation-based nonparametric MANOVA (PerMANOVA) (Anderson, 2001) using Bray–Curtis distance was conducted to analyze differences in macrofungal communities across forests. The isolines of the elevation also plotted on the NMDS ordinations using the ordisurf function. The correlation of NMDS axes scores with explanatory variables was assessed using envfit function in R. To test the influence of categories of the edaphic, climate and location variables on the fungal community, we used Mantel Test (Bray-Curtis distance) on total species matrix and scaled environmental parameters. Also, an analysis of similarity percentages (SIMPER; Clarke, 1993) was performed to identify macrofungal species that were most responsible for the observed patterns and was also used to determine the percentage contribution of macrofungal species to significant dissimilarities between the three forests (Parravicini et al., 2010). The SIMPER analysis was performed using the sim function of the Vegan package in R (R Core Team, 2020). 3. Results 3.1. Macrofungal richness and diversity In total, 13,736 sporocarps were collected from the three church Table 1 Characteristics of the study sites and selected edaphic properties. Descriptions Forests Taragedam Alemsaga Banja Geographical location 12◦06′–12◦07′N 37◦46′– 37◦47′E 11◦54′–11◦56′N 37◦55′–37◦57′E 10◦57′–11◦03′N 36◦39′– 36◦48′E Altitude range (m asl) 2142–2484 2180–2470 1870–2570 Mean annual precipitation (mm) 1098 1926 1884.3 Mean annual temperature (◦C) 19.5 15.8 18.7 Forest area (ha) 875 814 897 Density of trees ha −1 48.11 17.19 43.13 Sand (%) 58.89 ±2.93b 51.78 ±2.99b 68.67 ±2.21a Silt (%) 28.44 ±2.38a 32.44 ±2.13a 20.00 ±1.76b Clay (%) 12.67 ±1.37a 15.78 ±1.93a 11.33 ±1.33a pH H2O 1:2.5 7.04 ±7.03a 5.85 ±6.59b 5.60 ±6.24c EC (dS/m) 0.43 ±0.05b 0.28 ±0.03b 0.81 ±0.14a Ex.Ca (cmol (+)/kg) 13.95 ±0.60a 9.19 ±0.52b 13.55 ±0.87a Ex.Mg (cmol (+)/kg) 6.16 ±0.10a 4.58 ±0.15c 5.54 ±0.20b Ex.Na (cmol (+)/kg) 1.95 ±0.05a 2.05 ±0.10a 1.82 ±0.12a Ex.K (cmol (+)/kg) 0.73 ±0.06a 0.61 ±0.04a 0.77 ±0.06a CEC (cmol (+)/kg) 47.21 ±1.36a 34.89 ±0.92b 44.51 ±1.96a Organic matter (%) 4.46(0.60)a 3.35(1.34)b 4.87(0.10)a Nitrogen (%) 0.23 ±0.01a 0.17 ±0.02b 0.26 ±0.01a P (ppm) 17.18 ±5.72a 7.8 ±0.73b 17.64 ±6.05a Dominant species in each plots Maytenus obscura, Carissa edulis, Olea sp. Acacia abyssinica, Buddleja polystachya, Acacia nilotica Albizia gummifera, Prunus africana, Brucea antidysenterica References Gedefaw and Soromessa (2014), Zegeye et al. (2011), Zerihun et al. (2013) Birhane et al. (2017), Masresha et al. (2015), Wubet et al. (2004) Abere et al. (2017) Note: Values shown are means; standard errors of the means are indicated in parentheses. Values with different lowercase letters are significantly different (p <0.05). The mean annual precipitation and mean annual temperature are given based on nearby stations data of each study area by the year 2019. Abbreviations: EC, electrical conductivity; CEC, cation exchange capacity; m, meter; mm, millimeter; asl, above sea level. The references listed are related to the climatic and geographical descriptions of the study areas. D. Alem et al. Forest Ecology and Management 496 (2021) 119391 5 Table 2 Fungal sporocarps collected in July and August in three church forests in Northern Ethiopia. Taxa Order Family T A B E LM Agaricus augustus Fr. Agaricales Agaricaceae x E SS Agaricus bitorquis (Qu´ el.) Sacc. Agaricales Agaricaceae x E SS Agaricus campestris L. Agaricales Agaricaceae x x x E SS Agaricus cupreobrunneus (Jul.Sch¨ affer & Steer ex F.H.Møller) Pil´ at Agaricales Agaricaceae x x E SS Agaricus megalosporus J. Chen, R.L. Zhao, Karun. & K.D. Hyde Agaricales Agaricaceae x x x E SS Agaricus moelleri Wasser Agaricales Agaricaceae x x E SS Agaricus murinaceus Bull. Agaricales Agaricaceae x E SS Amanita vaginata (Bull.) Lam. Agaricales Amanitaceae x x EM Amanita sp. Pers. Agaricales Amanitaceae x x E EM Amanita verna (Bull.) Lam. Agaricales Amanitaceae x x x E EM Ampulloclitocybe clavipes (Pers.) Redhead, Lutzoni, Moncalvo & Vilgalys Agaricales Tricholomataceae x x E LS Artomyces pyxidatus (Pers.) Jülich Russulales Amylostereaceae x x x WS Auricularia auricula-judae (Bull.) Qu´ el. Auriculariales Auriculariaceae x E WS Bisporella citrina (Batsch) Korf & S.E.Carp. Helotiales Helotiaceae x WS Bjerkandera adusta (Willd.) P.Karst. Polyporales Meruliaceae x x WS Bolbitius sp. Fr. Agaricales Bolbitiaceae x x DS Bovista aestivalis (Bonord.) Demoulin Agaricales Agaricaceae x x SS Bovista plumbea Pers. Agaricales Agaricaceae x SS Calvatia cyathiformis (Bosc) Morgan. Agaricales Agaricaceae x x E SS Calvatia gigantea (Batsch) Lloyd Agaricales Agaricaceae x E SS Calvatia sp. Fr. Agaricales Agaricaceae x E SS Cantharellula umbonata (J.F.Gmel.) Singer Agaricales Tricholomataceae x x LS Cantharellus cinnabarinus (Schwein.) Schwein. Cantharellales Hydnaceae x E EM Chlorophyllum molybdites (G. Mey.) Massee Agaricales Agaricaceae x x x E LS Chlorophyllum rhacodes (Vittad.) Vellinga Agaricales Agaricaceae x x x E LS Clavaria falcata Pers. Agaricales Clavariaceae x SS Climacodon septentrionalis (Fr.) P. Karst. Polyporales Phanerochaetaceae x WS Clitocybe carolinensis H.E. Bigelow & Hesler Agaricales Tricholomataceae x x E LS Clitocybe cistophila Bon & Contu Agaricales Tricholomataceae x E LS Clitocybe foetens Melot. Agaricales Tricholomataceae x x x E LS Clitocybe fragrans (With.) P.Kumm. Agaricales Tricholomataceae x x x E LS Clitocybe geotropa (Bull.ex DC.) Qu´ el Agaricales Tricholomataceae x E LS Clitopilus hobsonii (Berk. & Broome) P.D. Orton Agaricales Entolomataceae x x x LS Conocybe apala (Fr.) Arnolds Agaricales Bolbitiaceae x SS Conocybe aurea (Jul.Sch¨ aff.) Hongo Agaricales Bolbitiaceae x x SS Conocybe dumetorum (Velen.) Svrcek Agaricales Bolbitiaceae x x SS Conocybe tenera (Schaeff.) Fayod Agaricales Bolbitiaceae x x x SS Conocybe velutipes (Velen.) Hauskn. & Svrcek Agaricales Bolbitiaceae x x x SS Coprinellus disseminatus (Pers.) J.E.Lange Agaricales Psathyrellaceae x x SS Coprinellus micaceus (Bull.) Vilgalys, Hopple & Jacq. Johnson Agaricales Psathyrellaceae x x x SS Coprinopsis sp. P. Karst. Agaricales Coprinaceae x x SS Coprinus comatus (O.F.Müll.) Pers. Agaricales Coprinaceae x x x E DS Coprinus lagopus (Fr.) Fr. Agaricales Coprinaceae x x DS Coprinus micaceus (Bull.) Fr. Agaricales Coprinaceae x E DS Coprinus niveus (Pers.) Fr. Agaricales Coprinaceae x x x E DS Cortinarius rubellus Cooke Agaricales Cortinariaceae x x EM Craterellus ignicolor (R.H. Petersen) Dahlman, Danell & Spatafora Cantharellales Hydnaceae x E EM Crepidotus applanatus (Pers.) P. Kumm. Agaricales Inocybaceae x x x E WS Crepidotus mollis (Schaeff.) Staude Agaricales Inocybaceae x x x E WS Crucibulum laeve (Huds.) Kambly Agaricales Agaricaceae x LS Cystodermella granulosa (Batsch) Harmaja Agaricales Agaricaceae x x LS Dacrymyces palmatus (Schwein.) Burt Dacrymycetales Dacrymycetaceae x WS Daedaleopsis confragosa (Bolton) J.Schr¨ ot. Polyporales Polyporaceae x x WS Daldinia concentrica (Bolton) Ces. & De Not. Xylariales Hypoxylaceae x WS Deconica montana (Pers.) P.D. Orton Agaricales Strophariaceae x x x LS Entoloma asprellum (Fr.) Fayod. Agaricales Entolomataceae x x x SS Entoloma olivaceohebes Noordel. & Hauskn. Agaricales Entolomataceae x x SS Entoloma poliopus (Romagn.) Noordel. Agaricales Entolomataceae x x SS Entoloma sp. Fr. ex P. Kumm. Agaricales Entolomataceae x x SS Entoloma undatum (Gillet) M.M. Moser Agaricales Entolomataceae x x SS Favolaschia calocera R. Heim Agaricales Marasmiaceae x WS Galerina badipes (Pers.) Kühner. Agaricales Strophariaceae x x x WS Geastrum triplex Jungh. Geastrales Geastraceae x x x LS Geoglossum sp. Pers. Geoglossales Geoglossaceae x x SS Gymnopilus sp1. P.Karst. Agaricales Omphalotaceae x x WS Gymnopilus sp2. P.Karst. Agaricales Omphalotaceae x x WS Gymnopilus sp3. P.Karst. Agaricales Omphalotaceae x WS Gymnopus dryophilus (Bull.) Murrill Agaricales Omphalotaceae x x x LS Gymnopus luxurians (Peck) Murrill Agaricales Omphalotaceae x LS Gymnopus putillus (Fr.) Antonín, Halling & Noordel. Agaricales Omphalotaceae x x LS Hebeloma eburneum Malençon Agaricales Strophariaceae x EM Hemimycena delectabilis (Peck) Singer. Agaricales Tricholomataceae x x x LS Hexagonia tenuis (Hook.) Fr. Polyporales Polyporaceae x x x WS Hohenbuehelia petalodes (Bull.) Schulzer. Agaricales Pleurotaceae x WS (continued on next page) D. Alem et al. Forest Ecology and Management 496 (2021) 119391 6 Table 2 (continued) Taxa Order Family T A B E LM Hygrocybe chlorophana (Fr.) Wünsche Agaricales Hygrophoraceae x x x E SS Hygrocybe chlorophana var. aurantiaca Bon. Agaricales Hygrophoraceae x E SS Hygrophoropsis aurantiaca (Wulfen) Maire Boletales Hygrophoropsidaceae x x x LS Hygrophorus hypothejus Fr. (Fr.) Agaricales Hygrophoraceae x x x E EM Hymenagaricus sp1. Heinem. Agaricales Agaricaceae x E SS Hymenagaricus sp2. Heinem. Agaricales Agaricaceae x SS Inocybe viridiumbonata Pegler Agaricales Inocybaceae x EM Laccaria glabripes McNabb. Agaricales Hydnangiaceae x EM Laccaria laccata (Scop.) Cooke Agaricales Hydnangiaceae x EM Laetiporus sulphureus (Bull.) Murrill Polyporales Fomitopsidaceae x x x E PP Lentinellus cochleatus (Pers.) P. Karst. Russulales Auriscalpiaceae x x E WS Lepiota cristata (Bolton) P.Kumm. Agaricales Agaricaceae x LS Lepiota ermine (Fr.) P.Kumm. Agaricales Agaricaceae x x LS Lepiota himalayensis Khalid & Razaq Agaricales Agaricaceae x x LS Lepiota sp1. (Pers.) Gray Agaricales Agaricaceae x LS Lepiota sp2. (Pers.) Gray Agaricales Agaricaceae x LS Lepiota sp3. (Pers.) Gray Agaricales Agaricaceae x x LS Leptonia lampropus (Fr.) Qu´ el. Agaricales Entolomataceae x x x SS Leucoagaricus americanus (Peck) Vellinga. Agaricales Agaricaceae x x x E SS Leucoagaricus purpureolilacinus Huijsman Agaricales Agaricaceae x x x E SS Leucoagaricus sp1. Locq. ex Singer Agaricales Agaricaceae x x E SS Leucoagaricus sp2. Locq. ex Singer Agaricales Agaricaceae x SS Leucocoprinus cepaestipes (Sowerby) Pat. Agaricales Agaricaceae x x SS Leucocoprinus fragilissimus (Berk. &M.A.Curtis) Pat. Agaricales Agaricaceae x SS Lyophyllum infumatum (Bres.) Kühner Agaricales Lyophyllaceae x EM Macrolepiota procera (Scop.) Singer Agaricales Agaricaceae x E LS Macrolepiota sp. Singer Agaricales Agaricaceae x E LS Marasimus sp1. Fr. Agaricales Marasmiaceae x E LS Marasmiellus chamaecyparidis (Hongo) Hongo Agaricales Omphalotaceae x LS Marasmius arborescens (Henn.) Beeli Agaricales Marasmiaceae x x LS Marasmius candidus Fr. Agaricales Marasmiaceae x E LS Marasmius guyanensis Mont. Agaricales Marasmiaceae x x x E LS Marasmius oreades (Bolton) Fr. Agaricales Marasmiaceae x x E LS Marasmius purpureostriatus Hongo Agaricales Marasmiaceae x x x E LS Marasmius scorodonius (Fr.) Fr. Agaricales Marasmiaceae x E LS Marasmius siccus Schwein. ex Fr. Agaricales Marasmiaceae x x E LS Marasmius sp2. Fr. Agaricales Marasmiaceae x x x E LS Marasmius sp3. Fr. Agaricales Marasmiaceae x x E LS Marasmius undatus (Berk.) Fr. Agaricales Marasmiaceae x x x E LS Micropsalliota sp. H¨ ohn. Agaricales Agaricaceae x SS Mycena griseoviridis A.H. Sm. Agaricales Mycenaceae x x LS Mycena interrupta (Berk.) Sacc. Agaricales Mycenaceae x LS Mycena rhenana Maas Geest. & Winterh. Agaricales Mycenaceae x x x LS Mycena rosea Gramberg Agaricales Mycenaceae x LS Mycena sp1. (Pers.) Roussel Agaricales Mycenaceae x LS Mycena sp2. (Pers.) Roussel Agaricales Mycenaceae x LS Mycena stipata Maas Geest. & Schw¨ obel Agaricales Mycenaceae x x x LS Mycena tenerrima (Berk.) Qu´ el. Agaricales Mycenaceae x LS Neopaxillus plumbeus Singer & Lodge. Boletales Serpulaceae x x SS Onnia tomentosa (Fr.) P.Karst. Hymenochaetales Hymenochaetaceae x x WS Panaeolina foenisecii (Pers.) Maire Agaricales Psathyrellaceae x x x SS Panaeolus fimicola (Fr.) Qu´ el. Agaricales Psathyrellaceae x DS Panaeolus papilionaceus (Bull.) Qu´ el Agaricales Psathyrellaceae x x DS Panellus mitis (Pers.) Singer Agaricales Mycenaceae x x WS Phaeolus schweinitzii (Fr.) Pat. Polyporales Fomitopsidaceae x x WS Phellinus noxius (Corner) G. Cunn. Hymenochaetales Hymenochaetaceae x x PP Phellinus populicola Niemel¨ a Hymenochaetales Hymenochaetaceae x PP Pholiota aurivella (Batsch) P. Kumm. Agaricales Strophariaceae x x E WS Pleurotus luteoalbus Beeli Agaricales Pleurotaceae x x x E WS Pleurotus populinus O.Hilber &O.K.Mill. Agaricales Pleurotaceae x x E WS Pleurotus pulmonarius (Fr.) Qu´ el. Agaricales Pleurotaceae x x E WS Pluteus longistriatus (Peck) Peck Agaricales Pluteaceae x LS Pluteus mammillatus (Longyear) Minnis, Sundb. & Methven. Agaricales Pluteaceae x LS Pluteus umbrosus (Pers.) P. Kumm. Agaricales Pluteaceae x x x LS Polyporus brumalis (Pers) Fr. Polyporales Polyporaceae x x x WS Polyporus tenuiculus (P. Beauv.) Fr. Polyporales Polyporaceae x x WS Polyporus varius (Pers.) Fr. Polyporales Polyporaceae x x x WS Psathyrella candolleana (Fr.) Maire Agaricales Psathyrellaceae x x x WS Psathyrella corrugis (Pers.) Konrad & Maubl. Agaricales Psathyrellaceae x x WS Psathyrella multipedata (Peck) A.H. Sm. Agaricales Psathyrellaceae x x x WS Psathyrella gracilis (Fr.) Qu´ el. Agaricales Psathyrellaceae x x x WS Psathyrella ammophila (Durieu &L´ ev.) P.D. Orton Agaricales Psathyrellaceae x x x WS Psathyrella piluliformis (Bull.) P.D.Orton Agaricales Psathyrellaceae x x x WS Psathyrella sp1. Fr. ex Qu´ el. Agaricales Psathyrellaceae x WS Psathyrella sp2. Fr. ex Qu´ el. Agaricales Psathyrellaceae x WS Psathyrella sp3. Fr. ex Qu´ el. Agaricales Psathyrellaceae x x WS (continued on next page) D. Alem et al. Forest Ecology and Management 496 (2021) 119391 7 forests and classified into 258 fungal taxa (Table 2). Although identification of sporocarps down to species level was not possible, out of the total taxa collected, 155 (60%) were identified to species level, 33 (13%) to genus level and further 69 (27%) were completely unidentified. The unidentified sporocarps were excluded from further analysis. The Basidiomycota was the dominant phylum and was represented by 10 orders, 62 families, 90 genera, and 180 species. Ascomycota was represented by three orders, seven families, seven genera, and eight species (Table 2). Among the taxa identified, the Agaricaceae was the most diverse family with 58 different taxa, followed by Psathyrellaceae (26), Tricholomataceae (22), and Mycenaceae (20), which together accounted for 33.6% of the total collected taxa (Table 2). The most abundant genera were Termitomyces, Psathyrella, Leucoagaricus, Marasmius, and Mycena. The proportions of macrofungal taxa at the genus level are provided (Fig. 2A). The Agaricales was the most prevalent order in the three forests (77.66%). Since many Agaricales are conspicuous macrofungi, it is not surprising to find a higher abundance during sampling. The family to genus and genus to species ratios were 0.70 and 0.50, respectively. Total numbers of fungal taxa per family encountered in the three studied forests are provided (Fig. 2B). In terms of the trophic groups, the majority of species were saprophytic (81%) followed by ectomycorrhizal (14%) and parasitic taxa (4%). The accumulation curves (Fig. 3A) generated for the taxa identified in the three forests show that the saturation of macrofungal richness was not reached during the survey given that the curves showed a steady increase with additional samplings. Although there was no significant difference in species richness between the three forests (p >0.05), the taxa accumulation curve for Banja forest showed a relatively steeper rising slope and yielded higher macrofungal richness values than the other forests. The highest macrofungal diversity values were obtained for Taragedam forest; however, diversity was not significantly different to that of the other two forests (Fig. 3B). The occurrence of macrofungi was more uneven in Banja forest than in the other forests (Table 3), with no fungal species found at all sampling events and certain macrofungal Table 2 (continued) Taxa Order Family T A B E LM Psathyrella sp4. Fr. ex Qu´ el. Agaricales Psathyrellaceae x WS Psathyrella sp5. Fr. ex Qu´ el. Agaricales Psathyrellaceae x x x WS Psathyrella sp6. Fr. ex Qu´ el. Agaricales Psathyrellaceae x WS Pseudoclitocybe cyathiformis (Bull.) Singer Agaricales Tricholomataceae x LS Pseudohydnum gelatinosum (Scop.) P.Karst. Auriculariales Exidiaceae x x x WS Pseudoomphalina pachyphylla (Fr.) Knudsen. Agaricales Tricholomataceae x LS Psilocybe ovoideocystidiata Guzm´ an & Gaines Agaricales Strophariaceae x x LS Psilocybe samuiensis Guzm´ an, Bandala & J.W.Allen Agaricales Strophariaceae x LS Ramaria stricta (Pers.) Qu´ el. Gomphales Gomphaceae x x x E EM Rhizopogon luteolus Krombh. Boletales Rhizopogonaceae x x x E EM Rhizopogon pseudoroseolus A.H. Sm. Boletales Rhizopogonaceae x E EM Russula gracillima Jul. Sch¨ aff. Russulales Russulaceae x EM Russula ochroleuca Pers. Russulales Russulaceae x x x EM Sarcoscypha occidentalis (Schwein.) Sacc. Pezizales Sarcoscyphaceae x x x WS Scleroderma areolatum Ehrenb. Boletales Sclerodermataceae x EM Scleroderma aurantium (L.) Pers. Boletales Sclerodermataceae x EM Sebacina concrescens (Schwein.) P. Roberts Auriculariales Exidiaceae x EM Skeletocutis carneogrisea A.David Polyporales Polyporaceae x x WS Suillus luteus (L.) Roussel Boletales Suillaceae x E EM Suillus sp. Gray Boletales Suillaceae x EM Terfezia leonis (Tul. & C.Tul.) Tul. Pezizales Terfeziaceae x x E EM Termitomyces clypeatus R.Heim Agaricales Lyophyllaceae x x x E LS Termitomyces microcarpus (Berk. & Broome) R. Heim Agaricales Lyophyllaceae x x E LS Termitomyces robustus (Beeli) R. Heim Agaricales Lyophyllaceae x x E LS Termitomyces sp. R. Heim Agaricales Lyophyllaceae x x x E LS Termitomyces schimperi (Pat.) R.Heim Agaricales Lyophyllaceae x x x E LS Trichaptum biforme (Fr.) Ryvarden Polyporales Polyporaceae x WS Tricholoma portentosum (Fr.) Qu´ el. Agaricales Tricholomataceae x E EM Tricholoma saponaceum (Fr.) P.Kumm. Agaricales Tricholomataceae x E EM Tricholoma sp. (Fr.) Staude Agaricales Tricholomataceae x E EM Tricholomopsis rutilans (Schaeff.: Fr.) Sing. Agaricales Tricholomataceae x x x WS Volvariella speciosa (Fr.) P.Kumm. Agaricales Pluteaceae x x LS Wilcoxina mikolae (Chin S. Yang & H.E. Wilcox) Chin S. Yang & Korf Pezizales Pyronemataceae x x E EM Xeromphalina caulicinalis (Bull.) Kühner & Maire Agaricales Mycenaceae x x x WS Xeromphalina tenuipes (Schwein.) A.H.Sm. Agaricales Mycenaceae x x x WS Xerula radicata (Relhan) D¨ orfelt Agaricales Physalacriaceae x x PP Xylaria hypoxylon (L.) Grev. Xylariales Xylariaceae x WS Xylaria scruposa (Fr.) Fr. Xylariales Xylariaceae x x WS Note: Abbreviations: T =the Taragedam forest group; A =the Alemsaga forest group; B =the Banja forest group; x =sporocarp production; E =edible; LM =mode of life; PP =Plant pathogen; EM =ectomycorrhizal, SS =Soil saprotroph, WS =Wood saprotroph, LS =Litter saprotroph, DS =Dung saprotroph. Table 3 Macrofungal and vascular plant richness and diversity indices in three church forests in Northern Ethiopia. Forest status Banja forest Taragedam forest Alemsaga forest All macrofungi Richness 22.56 ±3.02a 18.44 ±2.34a 22.67 ±1.84a Shannon 2.03 ±0.23a 2.57 ±0.13a 2.06 ±0.20a Simpson 0.73 ±0.05b 0.88 ±0.02a 0.77 ±0.05ab Evenness 0.38 ±0.03c 0.60 ±0.03a 0.47 ±0.02b Vascular plants Richness 5.78 ±0.55c 16.89 ±1.25a 12.67 ±1.04b Shannon 1.38 ±0.12b 2.18 ±0.08a 2.04 ±0.07a Simpson 0.67 ±0.05b 0.83 ±0.02a 0.82 ±0.02a Evenness 0.73 ±0.04a 0.55 ±0.03b 0.63 ±0.03ab Ectomycorrhizal fungi Richness 3.88 ±0.64a 2.57 ±0.3a 2.67 ±0.21a Shannon 1.09 ±0.1a 0.80 ±0.06a 0.90 ±0.07a Simpson 0.61 ±0.03a 0.53 ±0.02a 0.57 ±0.02a Evenness 0.85 ±0.05a 0.91 ±0.05a 0.94 ±0.03a Note: Values shown are means ±the SE of the mean. Different lowercase letters indicate a significant difference (p <0.05) in richness or diversity between forests. D. Alem et al. Forest Ecology and Management 496 (2021) 119391 8 species were more dominant in Banja forest than in the other two forests. The Shannon index and richness for vascular plants were significantly correlated with Shannon and Simpson diversity indices for the fungal communities (Fig. 4). Interestingly, for all these variables, the highest values were found in Taragedam forests and the lowest values were observed in Banja forests (Table 3). Although the three forests were not significantly different (p >0.05; Table 3) in terms of measure of diversity of their ectomycorrhizal fungal species and richness, more ectomycorrhizal species were collected from Banja forest (20) than from Taragedam (15) or Alemsaga (7) forests (Table 2). 3.2. Sporocarp production Taragedam forest produced the greatest quantity of sporocarps (25.4 kg ha −1 ), although production levels were not significantly different (p =0.63) to those of Alemsaga forest (21.6 kg ha −1 ; Fig. 5). However, both of these forests produced significantly greater quantities of sporocarps than Banja forest (p <0.05). Sixty eight (36%) of the total macrofungi collected were deemed to be edible (Table 2). Banja forest produced the greatest quantity of edible fungi (mean fresh weight, 1.8 kg ha −1 ) and Alemsaga forest produced the least (0.4 kg ha −1 ); however, the production of edible species did not differ significantly among the three forests (Fig. 5; p =0.01). Fig. 2. (A) The proportions of macrofungal taxa at the genus level (name of genus; the number of species; percentage); and (B) total numbers of fungal taxa per family encountered in the three studied forests. Fig. 3. Taxa accumulation curves generated for the fungal community found in the three studied forests using a rarefaction sample-based estimator (A) and R´ enyi diversity profiles (B). D. Alem et al. Forest Ecology and Management 496 (2021) 119391 9 3.3. Macrofungal communities and edaphic variables The perMANOVA analyses indicated the three church forests differed significantly in their macrofungal composition (F =2.05, R 2 =0.14, p = 0.001; Fig. 6). With respect to the explanatory variables, categorized edaphic, climate and location parameters were correlated to the macrofungal community composition (p <0.05; Table 4). Of these, Mantel test confirmed that location variables aggregately had a strongly significant effect on macrofungal community structure (p =0.000) than that of the climate (p =0.009) and the edaphic variables (p =0.112). The significance of each explanatory variable and their aggregated contribution to the difference of the macrofungal community compositions is provided (Table 4). The SIMPER analysis also identified macrofungal species that distinguished between the three forests (Table 5). The overall betweengroup dissimilarity (Sørensen) was 88.73% for Taragedam and Alemsaga forests, 94.44% for Alemsaga and Banja forests, and 93.76% for Taragedam and Banja forests. In this regard, the Coprinellus species are found the most important in distinguishing all forest locations along with the others (Table 5). The cumulative contribution of the most influential macrofungal species for the dissimilarity between these forests is shown in Table 5. 4. Discussion Although fragmentation poses major threats to forest ecosystems, the Dry Afromontane forests in the highland region of Ethiopia, including forest fragments owned by the church or located around church forest territories, are considered to be major reservoirs of biodiversity (Aerts et al., 2016; Aynekulu et al., 2016; Darbyshire et al., 2003; Nyssen et al., 2014). This study provides a comprehensive analysis of macrofungal communities and showed the differences in fungal community compositions of the fragmented forest systems in Northern Ethiopia. The difference in macrofungal species among the three forests might be due to the difference in vegetation composition or the variation in ecological factors such as soils, which are among the most important factors that could affect macrofungal species (Oria-de-Rueda et al., 2010). The availability of suitable substrates due to the difference in plant inputs on Fig. 4. Scatter plot matrices showing correlation coefficients between the entire tree and macrofungal variables and their significance levels. Abbreviations: T = tree, F =fungi. On the bottom of the diagonal, bi-variate scatter plots with a fitted line are displayed. On the top of the diagonal, the value of the correlation is shown, plus the significance level of the p-values, which are indicated by red asterisks. p-values: ***, <0.001; **, <0.01; and *, <0.05; *<0.1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) D. Alem et al. Forest Ecology and Management 496 (2021) 119391 10 the forest floor could be also a factor explaining the variation in fungal species composition among the three forests. The retention of plant residues is thought to enhance fungal activity by promoting moisture retention and providing a source of organic carbon, which is important for fungal survival and growth (Blumfield and Xu, 2003). Thus, the differences in substrate richness among these three forests can influence the diversity and richness of macrofungal species (Reverchon et al., 2010). Besides, the variation in macrofungal species among the three forests probably reflect the heterogeneity of these habitats, resulting in Fig. 5. Fresh weight of sporocarps collected from three forests in Northern Ethiopia. Dark-gray bars indicate the total fungal species collected; light-gray bars indicate edible fungal species. The data shown are means ±the SE of the mean. Values with different lowercase letters are significantly different (p <0.05). Fig. 6. Non-metric Multidimensional Scaling (NMDS) ordination graph with fitted explanatory variables based on dissimilarities calculated using the Bray–Curtis index of macrofungal communities compositions from plots in the three forests in Northern Ethiopia with altitude displayed as isolines. Arrows represent environmental variables that were most significantly (p <0.005) related to ordination. Ellipses indicate forest groups with the names indicated. The explanatory variables are shown in blue color: CEC, cation exchange capacity; OM, organic matter; Tmax, maximum daily temperature; and Tmin, minimum daily temperature. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 4 Significance of the explanatory variables for macrofungal community compositions. Numbers in bold indicate a highly significant effects (p <0.001). Sources Contribution% Variables pseudo-F p Edaphic variables 7.13% pH 0.4358 0.004 CEC 0.3191 0.010 OM 0.2767 0.017 Climate 14.11% Tmax 0.3441 0.004 Tmin 0.6150 0.001 Spatial factors 33.92% Latitude 0.6162 0.001 Note: the variables are: CEC, cation exchange capacity; OM, organic matter; Tmax, maximum daily temperature and Tmin, minimum daily temperature. Table 5 Summary of similarity percentage (SIMPER) results showing the cumulative total contribution (50% cut-off) and the contribution (%) of the most influential species to the dissimilarity between stands in the three forests in Northern Ethiopia. Species Individual contribution to the dissimilarity Cumulative contribution to the dissimilarity Edibility status Alemsaga and Banja forests Coprinellus disseminatus 13.07 13.07 Coprinellus micaceus 10.37 23.44 Coprinellus micaceus 6.49 29.93 Geastrum triplex 5.35 35.27 Marasmius guyanensis 4.19 39.46 edible Psathyrella sp. 3.33 46.31 Agaricus megalosporus 3.06 49.37 edible Taragedam and Alemsaga forests Coprinellus micaceus 7.42 7.42 Sarcoscypha occidentalis 6.02 13.44 Geastrum triplex 5.36 18.80 Psathyrella sp3. 3.83 30.81 Psathyrella candollena 3.60 34.41 Gymnopus dryophilus 3.14 37.55 Marasmius guyanensis 2.92 40.47 edible Phellinus noxius 2.74 43.20 Termitomyces robustus 2.24 45.44 edible Psathyrella sp. 2.04 47.49 Marasmius guyanensis 1.71 49.19 edible Polyporus varius 1.69 50.88 Taragedam and Banja forests Coprinellus disseminatus 13.21 13.21 Coprinellus micaceus 10.45 23.67 Sarcoscypha occidentalis 5.68 29.34 Marasmius guyanensis 3.90 33.24 edible Geastrum triplex 3.50 40.63 Agaricus megalosporus 3.08 43.71 edible Crepidotus mollis 2.37 46.08 edible Xylaria scruposa 1.96 48.04 Crepidotus applanatus 1.85 49.89 edible Psathyrella corrugis 0.93 67.45 Psathyrella candollena 0.89 68.35 Psathyrella candolleana 0.87 69.22 Psathyrella sp. 0.84 70.05 D. Alem et al. Forest Ecology and Management 496 (2021) 119391 11 variations in microclimate and, hence, variations in moisture, temperature, and other factors among these different forest systems (Suggitt et al., 2011) that influence the richness and productivity of fungi (G´ omez-Hern´ andez and Williams-Linera, 2011). However, the characteristics of the macrofungi themselves could also explain the variation in fungal species among the three forests. Many macrofungal species are believed to fruit spontaneously, with no consistent pattern of occurrence at any time given favorable environmental conditions and suitable substrates (Piepenbring et al., 2012; Tibuhwa et al., 2011). Furthermore, fungal sporocarps are short-lived and may last only a few days before decomposing or being eaten and, therefore, may not have been observed during our weekly surveys (Maurice et al., 2021). Habitat fragmentation can influence the fungal communities in forests (Sapsford et al., 2017). Lack of symbiotic fungal colonization in these systems may be a limiting factor for seedling establishment, which is the main regeneration ecological process in the studied forests. Thus, trees species more dependent on mycorrhizal fungi could potentially have a substantial decrease in recruitment, particularly in the rehabilitation or conservation scheme of the forests (Tonn and Ib´ a˜ nez, 2017). Although, recently studies reported the availability of Ectomycorrhizal (ECM) hosts plant from the tropic regions (Tedersoo et al., 2010), the ECM associations has long been considered rare or absent from tropical forest ecosystems (Corrales et al., 2018), particularly of the African forests like that of Ethiopian. A previous study also reported the absence of ECM fungi in the Dry Afromontane forests of Ethiopia (Dejene et al., 2017a). Though the majority of the species collected in this study were saprophytic, about 14% were characterized as ectomycorrhizal. Species from the general of Amanita, Entoloma, Geastrum, Laccaria, Russula and Rhizopogon were reported from these studied forests. Although the mycorrhizal status of each tree species in the study area are unknown (Table S1), the existence of ECM species may be due to the diverse vegetation (Friis et al., 2010a,b) and, hence, there may be more trees present that can act as hosts for mycorrhizal fungi (Hailemariam et al., 2013; Wubet et al., 2003). Also, the existence of mycorrhizal species in the studied forests can be explained by the dispersion of mycorrhizal inocula from nearby plantation forests that are supposed to host trees. The plantations are constituted by Eucalyptus camaldulensses, Eucalyptus globulus, Pinus patula and other highland Aacacia species. Thus, the findings presented here may have important implication for the indigenous forest system for the maintenance of functional fungal diversity in Ethiopia (Dejene et al., 2017a). Besides, the coexistence of mycorrhizal fungi with natural forests has many practical advantages, such as the exchange of water and nutrients through hyphal networks (Brundrett, 2002; Brundrett, 2004). They are also commonly the key determinants of plant population and community dynamics in the forests systems (Tedersoo et al., 2020). This result presents an insight into the conservation of fungal functional groups in the forest system in the study areas as these functional groups are important for the rehabilitation and conservation of these fragmented forests as the fungi, particularly of the ECM, species could potentially have a substantial role in recruitments seedlings (Tonn and Ib´ a˜ nez, 2017). Thus, further studies on tropical ectomycorrhizae are deeply needed, particularly in Africa where the vegetation resource is immense with significant livelihood and environmental benefits. In Ethiopia, wild mushrooms have been used for their nutritional and medicinal properties (Abate, 2014; Dejene et al., 2017b; Tuno, 2001). Equally to other wild edibles, they have also been used as a coping food during food shortage periods (Alemu et al., 2012; Sitotaw et al., 2020). In some local markets mushrooms are also available where they are sold by the local people to earn some income to supplement the household economy (Abate, 2014). The sporocarp productions obtained in this study were not high. Although further research is needed to verify the claim, the lower biomass yield reported here could be explained by the single tone species and the species composition. Some of the species were collected in a single time during the collection period. Majority of the species were saprophytic fungi and are characterized by low biomass productions (Gassibe et al., 2011; Mediavilla et al., 2014). However, valuable edible macrofungal species belonging to the Calvatia, Laetiporus, Pleurotus, Termitomyces sp., and Macrolepiota genera were also collected in this study. Among these edible species, Termitomyces sp. is highly regarded by local people in southwest Ethiopia because of its good taste and aroma (Abate, 2014). Although the overall quantity of sporocarp biomass produced in the studied forests was low, the most productive species had biomass values of approximately 0.46 kg ha –1 yr −1 , which provides an insight into the potential production levels of valuable sporocarp species. This also provides a starting point in terms of broadening the management and conservation of fragmented forests for the production of non-timber forests products in Ethiopia. In addition, important ectomycorrhizal species such as Tricholoma, Rhizopogon, and Suillus were also found in this study. The presence of these species in the study areas may be due to the high level of plant diversity in church forests, which may provide ectomycorrhizal fungi with a very broad host range (Roy et al., 2008; Smith and Read, 2008) and, hence, there may be a number of trees that can act as hosts for mycorrhizal fungi (Hailemariam et al., 2013; Wubet et al., 2003). Interestingly, some of the fungi in the genera of Trichoderma could also function as biocontrol activity (Vinale et al., 2008) in the forests. In addition, the overall landscape connectivity of exotic tree plantations to nearby fragmented natural forests could also contribute to the presence of ectomycorrhizal fungi in the fungal community assembly (Boeraeve et al., 2018; Peay and Bruns, 2014; Vannette et al., 2016). In these kind of plantations, the local communities in Ethiopia are collecting edible mushrooms, particularly in the Southwest part of the country for their subsistence use or to generate income in some cases (Dejene et al., 2017c). However, this finding may have important implications for indigenous forest systems in terms of the maintenance of valuable macrofungal species for commercial production in Ethiopia (Dejene et al., 2017a). Thus, our survey of macrofungi provides an insight into the valuable fungal functional groups present in the fragmented Dry Afromontane forest system of Ethiopia, which may aid their conservation and management through increasing their economic outputs through NTFPs production in addition to other forests products. Vascular plants are often used as a surrogate for total biodiversity (Schmit et al., 2005; Sætersdal et al., 2004). Thus, the vascular plants have also been considered a useful indicator of fungal diversity in management programs based on the fact that a species-rich plant community assumed to have more ecological niches or microhabitats available for fungi than a species-poor community (Chiarucci et al., 2005). However, in this study reported a lack of congruence between the species richness of vascular plants and macrofungi in line with Rudolf et al. (2013) who indicted the negative correlation between the two communities regarding species richness. Such correlation might be due to the fact that higher species richness of vascular plants could cause variation of light availability for the ground species, including macrofungi, due to canopy (H¨ ardtle et al., 2003). Thus, the fungal community and their species richness could be influenced by the amount and variation of light availability on the forest floor (Rudolph et al., 2018). The low correlation of species richness of vascular plant and fungi might be due to the fact that the pooled plant species richness not always maximize species richness of other organisms, including all macrofungi (Chiarucci et al., 2005). This is probably because of the special ecological requirements of the fungal that constitute the composition of the community, which are linked to substrate or other factors related to habitats such as edaphic variables (Liang et al., 2015; Rillig et al., 2015). In contrary to this, however, we found an indication of the positive correlation in the Shannon diversity index values of the two communities. Such association could suggests that the tree species identity can be used as a factor for macrofungal diversity (Otsing et al., 2021). Gabel and Gabel (2007) and McMullan-Fisher et al. (2010) also reported positive correlations between plant identities and fungal diversity based on abundance as a measure of diversity. This association is particularly evident for saprotrophic fungi because saprotrophic fungi increase their D. Alem et al. Forest Ecology and Management 496 (2021) 119391 12 community diversity through the provision of wider variety of substrates from the diverse vegetation to establish facilitative interactions in the systems (Gessner et al., 2010; Wu et al., 2019; Zhang et al., 2018), which in turn promote their higher levels of diversity (Ye et al., 2019). The observed correlation of plants and macrofungi diversity indices may suggest the influence of habitat microheterogeneity, causing a positive correlation between both plant diversity and macrofungal diversity (Rudolf et al., 2013). Thus, the promotion of vascular tree plantations in these fragmented forest systems, such as enrichment plantings or assisted natural regeneration systems, should offer suitable habitats with variable microclimates that would influence and/or assist the diversity and productivity of fungal species in the fragmented Dry Afromontane forests of Ethiopia. Studies demonstrated that fungal community composition can be governed by various environmental variables and landscape heterogeneity (Bahram et al., 2015; Ferrari et al., 2016; Peay et al., 2010; Tedersoo et al., 2014b). Thus, evaluating the fungal communities in different ecosystems is essential to filter out the relative contributions of environmental factors to fungal diversity and composition in an ecosystem (Tian et al., 2018). In this study, the NMDS ordination against the environmental variables is shown distinct macrofungal pattern of the three studied forests. Of the categorized variables, the spatial factors contributed highly for driving the macrofungi assembly together with climate and edaphic variables. This may an indication that the climate, and soil characteristics together are vital in setting spatial variation (Chen et al., 2015), reflecting the combined effects of these variables on the vegetation and thus on macrofungal community (Li et al., 2020). Although the relative degree to which organisms can move is determined by multiple factors, Golan and Anne (2017) indicated that distances as a spatial factor could affect the dispersal of fungal propagules. This could affect the large scale connectively of the different fungal species to form similarity in community structure or morphology (Calhim et al., 2018). However, this needs further investigation to provide an ecological meaningful explanation from our study areas. Conversely, specific fungal species are likely to respond to environmental variables, mainly edaphic parameters, in different ways (Cozzolino et al., 2016; Koide et al., 2014), and, thus, in turn, the composition of the fungal community is directly correlated with edaphic variables (Cozzolino et al., 2016). In particular, pH is known to be the most critical soil characteristic affecting the composition and structure of fungal communities across different continents (Docherty et al., 2015; Fierer and Jackson, 2006; Zhang et al., 2016). Similarly in this study also, soil pH appeared to be correlated with fungal species composition. We found that the presence of greater numbers of macrofungal species was associated with lower pH values. A relatively lower pH values were found in the Alemsaga and Banja forests. This supports the findings of Puangsombat et al. (2010) and Zhang et al. (2016) who reported that higher pH levels negatively influenced fungal community structure, probably because a higher pH restrains the expansion of fungi and the production of sporocarps. However, the species from the Taragedam forests showed exceptional ordination towards a relatively higher end point of the pH gradient. This might be associated with their adaptability of the species to higher pH values in the soil. We also found that CEC and EC are explanatory factors for macrofungal composition. Although the exact role that the CEC and EC play in macrofungal composition and sporocarp production is not fully understood, Crabtree et al. (2010) observed that fungal species richness was low, particularly when the CEC was high. This is probably because the CEC and EC influence nutrient availability, soil pH, and soil reactions to other ameliorants in the soil (Ogeleka et al., 2017). The majority of species in our ordination were directed towards plots with low CEC and EC values. This is probably also because soils with a high CEC are less susceptible to the discharge of base saturation as base saturation is an important factor in the distribution of macrofungal species. Base saturation indicates the proportion of sites occupied by basic cations such as Ca 2+ , Mg 2+ , Na + , and K + (Zheng et al., 2019). These elements are vital in many physicochemical processes, such as photosynthesis (He et al., 2017) and, thus, can affect plant photosynthesis and, hence, the amount of carbon that is available to fungi in the soil (Shi et al., 2014). Organic matter also appeared to be an important factor associated with the composition of macrofungi in the studied forests. This is likely because fungi typically extend their mycelia at the soil–litter interface (Boddy et al., 2009) and, thereby, organic matter influences mycelial outgrowth and network formation (Zakaria and Boddy, 2002). Organic matter also influences the fungal community through its impact on the water-holding capacity of soil and nutrient availability (Harrington, 2003). Thus, a high level of organic matter accumulation implies a high level of macrofungal assembly, particularly of saprophytic species. However, the accumulation of organic matter in some cases may also attract the ectomycorrhizal fungi as some of the ECM species can be benefit from organic matter decomposition in a similar manner to freeliving saprotrophs; that is, as a source of reduced C compounds to support metabolism (Lindahl and Tunlid, 2015). Nitrogen was also correlated with the composition of fungal species. This finding is in line with those of Kranabetter et al. (2009) and Reverchon et al. (2010), who reported that fungi assembly increased along soil N gradients. This is because nitrogen can influence the formation of mycelium in the soil and play a role in sporocarp formation (Trudell and Edmonds, 2004). Furthermore, many fungal species can adapt to more nitrogen-rich sites (Kranabetter et al., 2009; Toljander et al., 2006). In addition to the edaphic variables, the analysis also showed a significant role of max and minimum temperature on the composition of macrofungal composition. This may be due to the fact that the mycelium of the fungal species is more readily affected by atmospheric changes (Salerni et al., 2002), being more superficial specifically for those saprotrophs species that constitute mainly the community composition of our studied forests. Furthermore, the temperature can play role in nutrient cycling process (Geng et al., 2017). An increase in temperature generally facilitates the decomposition organic matter in the soil and accelerates the availability of nutrients. Thus, the fungal species likely are responding to this condition and form distinct communities, particularly of the fungi that are soil dependet as a substrate (Nicol´ as et al., 2019). 5. Conclusions We investigated the diversity and composition pattern of macrofungi in three church forests in Northern Ethiopia to help us to understand the strategies required for the management and conservation of these remnant Dry Afromontane forests and the crucial roles played by fungi in the management and protection of these forest systems. The diversity indexes and community composition of macrofungi in the study areas were influenced by site conditions, including vascular plant diversity and soil fertility gradients. From the analysis on fungi and plant diversity indices, we can see that the species richness of macrofungi is independent of the diversity and richness of vascular plant communities. In our analysis, no correlations were observed for richness, suggesting that richness of vascular plants cannot be used as a proxy for macrofungi richness. However, a positive correlation was found between the two communities for their diversity Shannon index, indicating the tree identity might be used as a factor for macrofungal diversity as there was a highest fungal diversity value in forests with the highest level of tree diversity values. Unsurprisingly, macrofungal communities as a whole were influenced by edaphic variables given that edaphic variables are the main factors affecting mycelial development and, hence, the production of sporocarps by different macrofungal species. Thus, the promotion of vascular tree diversity in fragmented forest systems by enrichment plantings or assisted natural regeneration management systems would offer suitable habitats with variable microclimates that should assist macrofungal species diversity and productivity in the fragmented Dry Afromontane forests of Ethiopia. In addition, the effects of the aforementioned management practices on soil fertility should be taken into consideration owing to the important relationship between D. Alem et al. Forest Ecology and Management 496 (2021) 119391 13 edaphic variables and macrofungal composition in these forests. Forests and sites showed a significant influence in the composition of the fungal communities associated. Therefore, conservation of a higher number of these fragmented forests, can lead to the conservation of a higher fugal richness in an overall landscape scale. Our survey also revealed the presence of valuable edible macrofungal species belonging to the Tricholoma, Suillus, and Termitomyces genera, which could potentially be marketed and, hence, could provide supplementary incomes to forestdependent local people and forest managers. Thus, we suggest that the production of valuable non-timber forest products such as wild mushrooms should be incorporated into management and conservation strategies for these fragmented forest systems. Moreover, the application of the baseline information provided in this study could assist other countries that are facing similar forest conservation issues due to deforestation and forest fragmentation. CRediT authorship contribution statement Demelash Alem: Investigation, Data curation, Writing - original draft. Tatek Dejene: Supervision, Investigation, Writing - review & editing. Juan Andr´ es Oria-de-Rueda: Conceptualization, Methodology. Pablo Martín-Pinto: Supervision, Conceptualization, Methodology, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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