Fungal Ecology xxx (2018) 1e9 Contents lists available at ScienceDirect Fungal Ecology journal homepage: www.elsevier.com/locate/funeco Molecular identification and enzymatic profiling of Trypodendron (Curculionidae: Xyloterini) ambrosia beetle-associated fungi of the genus Phialophoropsis (Microascales: Ceratocystidaceae) ** * Maximilian Lehenberger a, , 2, Peter H.W. Biedermann b, 1, J. Philipp Benz a, , 1 a Wood Bioprocesses, Holzforschung Munich, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Bavaria, Germany b Research Group Insect-Fungus Symbiosis, Department of Animal Ecology and Tropical Biology, University of Wuerzburg, Germany article info abstract Article history: Ambrosia fungi are a polyphyletic group from currently seven ascomycete and basidiomycete lineages Received 15 February 2018 that independently evolved an obligate farming mutualism with wood-boring weevils. One long known, Received in revised form but understudied, association is the mutualism between the scolytine beetle genus Trypodendron (Cur- 3 July 2018 culionidae: Xyloterini) and the Microascales fungal genus Phialophoropsis (Ascomycota: Ceratocystida- Accepted 25 July 2018 ceae) for which a species-specific association has not been safely established yet. Moreover, the fungal Available online xxx wood degrading capabilities are completely unknown. Corresponding Editor: Henrik Hjarvard de Here, the ambrosia fungi of three Xyloterini species, Trypodendron domesticum, Trypodendron lineatum Fine Licht and Trypodendron signatum, were isolated and identified using culture-dependent methods. T. lineatum was confirmed to be exclusively associated with Phialophoropsis ferruginea, whereas T. domesticum and Keywords: T. signatum are associated with a closely related but putatively novel Phialophoropsis species. In- Ambrosia vestigations of their wood decomposing potential revealed that both fungi mainly depolymerize xylan Phialophoropsis but are weak mannan decomposers. In addition, robust cellulolytic activity was observed, indicating Scolytinae cellulose as another main carbon source. Fungal morphology © 2018 Elsevier Ltd and British Mycological Society. All rights reserved. Enzymatic activity Hemicelluloses Cellulose 1. Introduction Analogous to the polyphyletic ambrosia beetles, fungal mutu- alists e the so-called ambrosia fungi e have repeatedly evolved Fungus-farming evolved in three groups of insects: attine ants, from at least five ascomycete (Ophiostomataceae, Ceratocystida- macrotermites and wood-boring weevils in the subfamilies Scoly- ceae, Nectriaceae, Bionectriaceae, Saccharomycetaceae) and two tinae and Platypodinae (Farrell et al., 2001; Muller et al., 2005). basidiomycete families (Peniophoraceae, Meruliaceae) (Hulcr and Farming weevils, the so-called ambrosia beetles, currently Stelinski, 2017). While an earlier hypothesis suspected tight co- encompass more than 3400 species in at least eleven lineages that evolution and species-specificity between beetles and fungi independently evolved a nutritional mutualism with fungi (Hulcr (Francke-Grosmann, 1967; Muller et al., 2005), current data sug- et al., 2007). All ambrosia beetles are dependent on the presence gests that fungal lineages are switched between beetle lineages of woody plants, which are typically recently dead or in an un- (e.g. Vanderpool et al., 2017) and that the tightness of the associ- healthy state, in which they drill their tunnel systems (“galleries”). ation depends on the beetle taxon (Kostovcik et al., 2014; Mayers Therein, they actively farm one or several fungal mutualists, which et al., 2015). The best studied Xyleborini ambrosia beetle lineage, serve as their essential source of food (Francke-Grosmann, 1967; for example, encompasses apparently tightly evolved associations Kirkendall et al., 2015). in the genera Anisandrus, Xylosandrus and Xyleborinus, but also * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M. Lehenberger), [email protected] (J.P. Benz). 1 Shared senior authorship. 2 Present address: Research Group Insect-Fungus Symbiosis, Department of Animal Ecology and Tropical Biology, University of Wuerzburg, Germany. https://doi.org/10.1016/j.funeco.2018.07.010 1754-5048/© 2018 Elsevier Ltd and British Mycological Society. All rights reserved. Please cite this article in press as: Lehenberger, M., et al., Molecular identification and enzymatic profiling of Trypodendron (Curculionidae: Xyloterini) ambrosia beetle-associated fungi of the genus Phialophoropsis (Microascales: Ceratocystidaceae), Fungal Ecology (2018), https:// doi.org/10.1016/j.funeco.2018.07.010 2 M. Lehenberger et al. / Fungal Ecology xxx (2018) 1e9 more flexible beetle-fungus associations in the genus Xyleborus. 2. Materials and methods However, the causes for these potential switches of the symbionts are unknown. Possible reasons are unspecific vertical transmission 2.1. Beetle collections of fungi between natal and newly founded nests and/or horizontal fungal exchange between neighboring nests when different beetle Beetles were caught in March of 2017 with pheromone-baited species colonize the same host tree (Bateman et al., 2015). traps (“Lineatin Kombi” by Witasek, Germany; Suppl. Fig. 1 A & B) Typically, partner fidelity between beetles and fungi is main- during 5 d sampling at the Bavarian Forest National Park (Neu- tained over generations by vertical transmission of fungal symbi- schonau,€ Bohmerweg,€ between 850 and 966 m NHN, Germany) and onts in mycetangia, which are gland-rich pouches that are 11 d sampling in the “Plantage” in Freising (between 497 and 513 m specialized for fungal spore transmission and thought to be more or NHN, Germany). Subsequently, the beetles were individually stored less selective (Francke Grosmann, 1956; Batra, 1963; Six, 2003). The in 1.5 ml Eppendorf tubes filled with a piece of damp tissue and morphology and location differs between species, and mycetangia transported to the lab for morphological identification (Grüne, can be located internally or externally (Hulcr and Stelinski, 2017). 1979; Bussler and Schmidt, 2008). Individual beetles were kept in Within the galleries, ambrosia fungi line the tunnel walls with the cooling room (3e6 C) and fungi were isolated immediately asexual fruiting structures (conidia on conidiophores), which serve after beetle identification. as the main food source for the beetles and their larvae (Norris, Active Trypodendron galleries in beech and fir(Fagus sylvatica, 1979; Biedermann et al., 2013). To date, only about 5% of the Picea abies) were identified and excised with a chainsaw in June fungal symbionts of known ambrosia beetle species have been 2017 in the Bavarian Forest National Park (Suppl. Fig. 1C & D) at the described (Hulcr and Stelinski, 2017). Even less studied is the same locations where the pheromone-baited traps were placed. identity and role of other mycelial fungi, yeasts and bacteria that The wood pieces containing the beetle's galleries were subse- are frequently found to co-inhabit the ambrosia beetle galleries quently cooled and transported to the lab. (Haanstad and Norris, 1985). Hitherto, little is known about the metabolic characteristics of 2.2. Artificial rearing of beetles ambrosia fungi in general. A few studies investigated the utilization of different carbon sources by some ambrosia fungi. For instance, Some of the caught beetles were used for lab-rearing on a the ambrosia fungi Raffaelea brunnea and Raffaelea tritirachium are modified sawdust-agar substrate (Biedermann et al., 2009; known to assimilate various carbon compounds like xylose or Suppl. Fig. 1 J). Briefly, the following ingredients were used for cellobiose (Batra, 1967). Using a minimal nutrient assay, the fungal 12e14 50 ml conical Falcon tubes: 100 g sawdust (spruce, beech or symbiont of Xyleborus ferrugineus was found to metabolize oak), 0.63 g Wesson salt mixture (W1374 Sigma-Aldrich), 2.5 g dextrose but not cellulose (Norris and Baker, 1968). The first yeast-extract, 15 g agar, 280 ml water, 5 ml wheat-germ oil and 4 ml extensive enzymatic profiling of an ambrosia association investi- of ethanol (absolute). In contrast to the reported medium, addition gated the whole gallery of Xyleborinus saxesenii and showed the of any simple sugars to stimulate fungal growth was omitted. After ability to depolymerize hemicellulose, such as xylan, glucomannan autoclaving of the half-liquid media and some minutes of cooling, and callose (De Fine Licht and Biedermann, 2012). Besides these the ethanol was added, mixed again and finally distributed into findings, we are unaware of any study examining the enzymatic sterile tubes. The media inside the tubes was compacted with a activity of individual ambrosia fungi on a wood-based medium, sterile spatula and dried for 2 d. The surface of collected Trypo- elucidating the fungal ability to utilize the wood polysaccharides. dendron females and males were briefly sterilized with 70% ethanol The main purpose of this study was to examine the specificity to reduce the potentially vectored contaminants (e.g. molds), fol- and fungal physiology of the Phialophoropsis-Trypodendron mutu- lowed by a short rinse in sterile, deionized water. Finally, the fe- alism. In particular, we focused on the fungal mutualists of males were individually placed into the tubes, which subsequently Trypodendron lineatum, Trypodendron domesticum and were loosely closed, horizontally stored and incubated in ambient Trypodendron signatum, which were not