# Mass Spectrometry: A Rosetta Stone to Learn How Fungi Interact and Talk
## 1 Introduction  
Fungi comprise a monophyletic yet unresolved kingdom. At the moment, it is still not possible to diagnose fungi based on specific characters and they should be regarded as a group without synapomorphies [1]. Nevertheless, it is clear that the evolution of both osmotrophic and rigid cells, which are widespread among fungi, are among the evolutionary developments that allow their diversification and ecological success [1]. It is estimated that 1.5 to 3.8 million fungal species occur on Earth, despite only around 100,000 species being known to science [2; 3; 4; 5]. This indicates that fungi are far more diverse than other kingdoms, and a perspective research area. Fungi have been very successful in inhabiting the world, and they can be found in terrestrial and aquatic environments, as symbiotic associations as well as saprophytic or parasitic organisms. Fungal ecological success is also a consequence of their huge metabolic machinery that allows them to produce diverse chemical compounds, which are important for their ecological interactions and survival [6; 7; 8; 9].  
Fungi produce a large diversity of secondary metabolites (hereafter referred as specialized metabolites), from which more than 15,600 are unique to them [10]. The production of specialized metabolites by fungi is an evolutionary advantage, as they use metabolites to defend themselves, takedown defenses, as well as to takedown defenses of their potential food sources, and for intra-/interspecific chemical communication [9; 11; 12; 13]. For example, fungi communicate and interact within and with other species either via volatile organic compounds (VOCs) or non-volatile secreted compounds [14; 15]. Most of these compounds have been identified using mass spectrometry (hereafter MS). Therefore, MS is a powerful tool in understanding fungi metabolism. Fungal metabolites are being continuously discovered and the rate of these discoveries has been accelerated significantly during the last two decades. This is thanks to the development of MS, which has been used in mycological studies such as biotyping, ecology and monitoring the production of predominantly specialized metabolites. Indeed, in a search within the Web of Science portal, we found that the number and pattern of scientific publications per year involving fungal metabolites are almost identical to the number and pattern of mycological studies involving MS techniques (Figure 1). The development of MS instruments and techniques during the last three decades has also stimulated the increasing number of publications related to fungal metabolites, which heavily increased from around 750 released publications in 1985 up to around 16,000 in 2019 (Figure 1).  
The inherent sensitivity and rapid data acquisition capabilities of MS instruments, in addition to new MS-based analytical techniques, have thus prompted the further development of metabolomic studies on fungi. However, the use of cutting-edge MS instruments and techniques alone is not enough, if there are not clear analytical protocols that allow the researches to correctly address and achieve their study goals. Thereby, we believe that it is necessary and of current interest for a combination of sampling and analytical techniques used in MS-based fungal metabolomic studies. Our goal in this review is to show the versatility and potential use of MS-based techniques in mycological studies by providing information on the diverse metabolites known from fungi, their ecological role and the MS-based techniques used.  
Figure 1: Development of fungal studies involving fungal metabolites and mass spectrometry. Publications from 1985 to 2020 involving metabolites (blue bars) and mass spectrometry (orange bars) in mycological studies. The search was carried out in the portal of the Web of Science using the following search terms: (i) “fungal OR fungi AND metabolite”; and (ii) “fungal OR fungi AND mass spectrometer*”.