Annotated Checklist of the Lichenicolous Fungi of Hungary

: Knowledge of lichenicolous fungi is limited at a worldwide level and needs further basic information, as in the case of Central and Southern Europe. The literature sources for “Revised checklist of the Hungarian lichen-forming and lichenicolous fungi” by L˝okös and Farkas in 2009 contained 54 lichenicolous and other microfungi species of 38 genera. Due to recent ﬁeld studies and microscopic work, the number of known species has increased to 104 lichenicolous species in 64 genera during the last decade, including 53 new species for the country. Old records of ﬁve species were conﬁrmed by new collections. Key characteristics of some of the most interesting species are illustrated by microscopic views and two distribution maps are provided. Recent biodiversity estimates suggest that the number of currently known species could be 1.5 (–2) times higher with more detailed work on ﬁeld collections. Although lichenicolous fungi have been less well studied in Hungary in the past, the relative diversity of lichenicolous fungi there, as indicated by Zhurbenko’s lichenicolous index, was found to be slightly higher than the mean value calculated for the world.


Introduction
The symbiosis of lichenized fungi in the strict sense consists of one fungal (mycobiont) and one photosynthetic (photobiont) partner [1]. However, a series of close associations are known, where further lichen associated symbionts accompany these two compulsory components [2][3][4]. One or two additional photosynthetic partners occur typically in cephalodia, which are morphologically more or less well-defined special region on the thallus surface or inside the thallus; the number of partners may therefore reach even four in this way. However, if the number of fungi is increased by one or more additional species, the number of partners can similarly be three (a tripartite symbiotic relationship) or more. These additional fungi are the lichen-inhabiting or lichenicolous fungi. There is also the special case of the four-membered symbiotic relations, where a lichenized fungus lives together with another lichenized fungus, forming a miniature ecosystem. Today, due to the wide range of developed systems in microscopy, microbiology, and molecular genetics, the nature of this ecosystem can be extended further when coexisting bacteria [3] and basidiomycete yeasts [4] are considered. This interpretation of mini-ecosystems was proposed by [5] and extended by others [6][7][8][9], focusing on lichen-associated animals such as mites, snails, and slugs.
Lichenicolous fungi are generally very small and inconspicuous, since their major part is often hidden inside the lichen thallus and only the appearance of their fruiting bodies indicates their presence; therefore, they often remain unnoticed by collectors [10]. Lichenicolous fungi are usually studied by lichen specialists and not by mycologists working on free-living fungi, since the former, when visiting the field, notice the unusual morphological characters or fruit-bodies produced by the additional fungus and frequently collect a specimen. In recent decades, numerous lichenologists-mycologists have mostly

Materials and Methods
New collections were identified with the help of various literature sources [10,[32][33][34]. The morphology and anatomy were studied by means of a NIKON Eclipse/NiU (DIC, epifluorescence) compound microscope (Nikon Corporation, Tokyo, Japan), Nikon SMZ18 stereo microscope (Nikon Corporation, Tokyo, Japan), and Olympus SZX9 and Olympus BX50 (DIC) microscopes (Olympus Corporation, Tokyo, Japan). Micrographs were prepared by Olympus E450 camera (with Quick Photo Camera 2.3 software; Olympus Corporation, Tokyo, Japan) and Nikon DS-Fi1c and Fi3 camera (with NIS-Elements BR ML software; Nikon Corporation, Tokyo, Japan) with the indicated microscopes. Microscopic examinations were carried out in water, where it was necessary in 10% KOH (K) (Reanal, Budapest, Hungary), Lugol's iodine (Merck KGaA, Darmstadt, Germany), directly (I) or after 10% KOH pre-treatment (K/I).
The nomenclature mainly follows the IndexFungorum [35], similarly to the previous checklist [28], but the work by Lawrey and Diederich 2018 [36], several chapters by Paul Diederich in [33], and other literature sources by Hawksworth, Kocourková, and Santesson et al. [18,32,37,38] were also employed. The nomenclatures of old literature data were checked repeatedly and revised. Information on the host is generally based on specimens collected in Hungary. In the case of literature records, we could only rely on the original text available.
Distribution maps were constructed by the computer program for geographical information system QGIS 3.18.2 'Zürich', released in 2020, applying an adaptation of the Central European grid system [40,41]; the symbols (dots) represent units of c. 5 km × 6 km areas.

Results
The following checklist contains 104 species; a nomenclatural revision of the taxa treated by Lőkös and Farkas in 2009 [28] and identification of specimens recently collected mostly by N. Varga throughout Hungary. In total, 53 lichenicolous fungi have been confirmed as new to the country, and old records of five species have been confirmed by new collections. Host lichen species are named wherever possible, and geographical distribution is provided according to the main regions within the country. Anatomical and morphological annotations refer to the specimens collected in Hungary in some cases, especially if they differ in some respects from the known description.

List of Lichenicolous Fungi
New species for Hungary are indicated by an asterisk (*).

1.
Abrothallus Distribution maps were constructed by the computer program for geographical information system QGIS 3.18.2 'Zürich', released in 2020, applying an adaptation of the Central European grid system [40,41]; the symbols (dots) represent units of c. 5 km × 6 km areas.

Results
The following checklist contains 104 species; a nomenclatural revision of the taxa treated by Lőkös and Farkas in 2009 [28] and identification of specimens recently collected mostly by N. Varga throughout Hungary. In total, 53 lichenicolous fungi have been confirmed as new to the country, and old records of five species have been confirmed by new collections. Host lichen species are named wherever possible, and geographical distribution is provided according to the main regions within the country. Anatomical and morphological annotations refer to the specimens collected in Hungary in some cases, especially if they differ in some respects from the known description.

List of Lichenicolous Fungi
New species for Hungary are indicated by an asterisk (*).  [44], and in the Mecsek Mts (BP 97037; BP 97038).
Note: Former specimens identified as A. tulasnei have also been included here.     The old literature source [30] mentions this species with possible hosts but without exact locality and voucher specimens; therefore, it must be regarded as a dubious record.

Bryostigma apotheciorum
Note: Tremelloid galls have been found on many host specimens in recent collections from Hungary. According to recent studies [11,68,69], tremelloid species can be distinguished mostly by molecular genetic methods. In the lack of basidia and/or basidiospores, the presence of these taxa could not be confirmed [70].   48. Lichenopeltella cetrariicola (Nyl.) R. Sant., Nordic J. Bot. 9(1): 99 (1989) Host: Cetraria islandica Recorded without exact locality and voucher specimens [30]. Its host species is rare in Hungary. An old herbarium specimen infected by Lichenopeltella cetrariicola has been found recently (BP 83751); however, it also lacks an exact locality.    Hosts: Peltigera spp. Old, dubious literature record, as Nectriella robergei, without exact locality and voucher specimens but with possible hosts [29].

Discussion
The 104 listed species represent a relatively good level of diversity compared to data of various European countries and the mean value of the world calculated according to the lichenicolous index (LI) [85], previously adopted for the Bulgarian lichenicolous fungi [20]; updated LI values for several other countries are provided in Table 1, the value for lichenicolous fungi in Hungary being just above the mean value for the world. There are countries or regions that reach more than twice the average value for the world (e.g., Bavaria (Germany), Great Britain, Belgium), but countries such as Fennoscandia, Ukraine, and France, which have more humid oceanic and/or Mediterranean climates or a more diverse landscape and habitats or a larger altitudinal range, and thereby would be expected to be important for lichens and lichenicolous fungi, have values not much higher than that of Hungary. Since lichenicolous species belong to a large number of higher taxa (64 genera, 43 families, and 33 orders), they represent a rather high systematic diversity. The most species-rich genera are Stigmidium with nine, Abrothallus with seven, and Didymocyrtis with four, while 49 genera contain only one species.
Lichenicolous fungi are found on c. 80 host species in Hungary, which is a relatively low number compared to the total number of lichens (926). This suggests that lichenicolous fungi might be discovered on further host species in the future, even if not all lichen species are known as hosts of lichenicolous fungi. It is not known why some lichens are found more frequently with lichenicolous fungi, while others are usually without; the effect of lichen secondary metabolites might be a possible explanation [7]. We can also assume that the knowledge of lichenicolous fungi is far from complete globally, and a great number of new taxa are to be described. According to a recent estimate, only c. 3-8% of the world's fungi is known [103].
Additional fieldwork may result in a more detailed knowledge of the distribution of taxa with the possibility for preparing distribution maps for the known species similarly to the current examples, Abrothallus caerulescens ( Figure 1) and Stigmidium eucline ( Figure 5). Further floristical novelties for Hungary are expected by detailed investigations of the frequent host species in the genera Xanthoria, Phaeophyscia, Physcia, and Lecanora, as well as the understudied genera such as Anaptychia, Pseudevernia, Ramalina and Usnea.