DOI: 10.5061/dryad.xd2547dt2
title: Data from: Oviposition and larval mycelia preference of the saproxylic European stag beetle

description: In the light of evidence-based conservation, we analysed the relation
 between the European stag beetle (Lucanus cervus) and a selection of ten
 common wood-decomposing fungi. We studied log preference and
 number of offspring for logs inoculated with different fungi during
 oviposition in a cafeteria experiment. A second experiment assessed
 survival and relative growth rates of larvae placed in substrates
 colonized by the different fungal species. The selected fungi used in both
 experiments are phylogenetically diverse and represent different rot
 types. Females laid no eggs in logs with brown-rot fungi and all
 larvae died in brown-rot mycelia within 33 days due to black spot. All
 white-rot fungi were accepted for oviposition, with no species-specific
 preferences. The larval survival rate in white-rot species was at least
 57% and the relative growth rate of larvae was positive (except for the
 toxic Omphalotus illudens). At rot type level, we can
 confirm the optimal oviposition hypothesis, a match between oviposition
 and larval habitat. Our findings contradict the association of brown-rot
 fungal species with stag beetles, as reported in some literature, which is
 probably based on the presence of fruiting bodies. We conclude that the
 European stag beetle is a white-rot dependent species and accepts numerous
 species of white-rot fungi without a clear preference. This is relevant
 for evidence-based conservation when restoring its habitat or building
 artificial habitats (such as log piles) as well as for ex-situ breeding
 campaigns.
description: Ten fungal species were selected for the larval as
 well as the oviposition experiments (Table 1). All selected species have a
 wide distribution in Europe and are capable of colonising subterranean
 deciduous dead wood. Some of the selected species have been mentioned in
 the literature to be associated with the European stag beetle (Table 1).
 Furthermore, species were selected to represent different rot types (based
 on Funguild, Nguyen <em>et al.,</em> 2016) and taxonomic
 groups (Basidiomycota and Ascomycota). Finally, <em>Omphalotus
 illudens (French and Garrettson)</em> (Schwein.) Bresinsky &
 Besl was included as it contains the toxin illudin, which is at least
 toxic to humans and rats (French & Garrettson, 1988; Tanaka
 <em>et al.,</em> 1996). 
 <strong>Table 1.</strong> Selected fungus species in
 alphabetical order within rot type used in the two experiments with
 abbreviated name (Abbr.), rot type (Nguyen <em>et al.,</em>
 2016), division, reference for its association with <em>L.
 cervus,</em> and number of successful inoculated logs.
 
 Abbr. 
 Scientific name English
 name Rot type
 Division 
 Association with <em>L. cervus</em>
 Successful log inoculations
 Am
 <em>Armillaria mellea</em>
 (Vahl) P. Kumm. Honey
 fungus White rot
 Basidiomycota 
   
 6/10 
 Fv <em>Flammulina
 velutipes</em> (Curtis) Singer 
 Velvet Shank White
 rot Basidiomycota
   
 10/10 
 Gl <em>Ganoderma
 lucidum </em>(Fr.) P. Karst. 
   White rot
 Basidiomycota 
 <em>Ganoderma applanatum </em>(Pers.)
 Pat.; Rink, 2006; Rink & Sinsch, 2008 
 10/10 
 Gf 
 <em>Grifola frondosa</em> (Dicks.) Gray
 Hen of the Woods 
 White rot 
 Basidiomycota 
   10/10
 Km
 <em>Kuehneromyces
 mutabilis</em> (Schaeff.) Singer & A.H.Sm.
 Sheathed woodtuft 
 White rot 
 Basidiomycota 
   2/10
 Oi
 <em>Omphalotus illudens (French and
 Garrettson)</em> (Schwein.) Bresinsky & Besl
 Jack o' Lantern 
 White rot 
 Basidiomycota 
   0/10
 Tv
 <em>Trametes versicolor</em>
 (L.) Lloyd Turkeytail
 White rot 
 Basidiomycota 
   10/10
 Fp
 <em>Fomitopsis pinicola</em>
 (Sw.) P. Karst. Redbelted
 Bracket Brown rot
 Basidiomycota 
 Tochtermann, 1992 
 10/10 
 Ls <em>Laetiporus
 sulphureus</em> (Bull.) Murrill 
 Chicken-of-the-woods 
 Brown rot 
 Basidiomycota Scherf,
 1985 8/10 
 Cg 
 <em>Chaetomium globosum</em>
 Kunze   
 Soft rot 
 Ascomycota  
 0/10 
 <em>Oviposition
 experiment</em> A cafeteria experiment was set up
 to study the oviposition preference of females for the different selected
 fungi. For this, fresh beech logs (<em>Fagus sylvatica
 </em>Linnaeus, 1753) of 40 cm length and diameters between 10-15 cm
 were collected at Begijnenbos in Buizingen (Belgium, 50°45'N,
 4°18'E) from a stand thinning at the end of January 2023. Hundred
 logs (with similar volume) were inoculated by placing 100 spawn dowels in
 each log, ten logs for each fungal species. The inoculation was performed
 by the company Mycelia (Deinze, Belgium) in a laminar flow chamber and
 subsequently logs were sealed in mushroom bags with aeration strips to
 avoid contamination. Logs were stored at 25 °C and 90% RH for four months
 to allow the mycelia to colonise the logs. Colonization
 of some species was unsuccessful, resulting in only seven species
 remaining with a varying number of logs (2-10, Table 1). Secondly,
 species-specific decomposition rates resulted in a varying degree of log
 density (hardness). The only two logs of <em>Kuehneromyces
 mutabilis</em> (Schaeff.) Singer & A.H.Sm. were cut in half
 to gain a total of 68 logs used in the oviposition experiment. The length
 and diameter at both ends and weight of each log was measured before the
 start of the experiment in order to calculate its volume and
 density. Ten plastic boxes measuring (l x w x h) 80 x
 60 x 42 cm with 16 6 mm aeration holes at the side were subdivided into
 ten uniform compartments of 16 x 30 x 30 cm using plastic biplex sheets.
 Each compartment was filled randomly with either a vertically positioned
 log and covered with wood compost (gabco Kompostierung GmbH, Aachen,
 Germany), or solely with wood compost (n=32) so that each fungus species
 was used only once in each box (Table S1). The substrate height was 30 cm
 presenting a flat surface with the biplex sheets and only the logs
 protruding on top allowing accessibility for the adult females to all
 compartments by the 12 cm of free space up to the lid. The boxes were
 filled on June 13th, 2023, and placed in a climate chamber with a constant
 temperature of 17 °C. Ten adult male and
 female stag beetles were collected from three nearby populations
 (Huizingen, Overijse, and Sint-Genesius-Rode) in Belgium between the 21st
 and 23rd of June 2023. These different populations belong to a once
 continuous population but are currently quite small, isolated, and
 genetically impoverished (Cox <em>et al.,</em> 2020). When
 placing one couple in each of the designated boxes on June 30th, the male
 and female were selected from a different population as the final
 offspring will be used for restocking with genetic enriched individuals
 (Table S2). For this experiment, we will not consider the population
 genetics and fitness in different environments. The
 beetles were fed with beetle jelly (beetlejelly.eu) and subjected to
 weekly monitoring. Dead adult beetles (parents) were removed and if
 required about 1 L of water was added to dry compartments.
 On October 4th, 2023, the number of offspring was counted a first
 time by carefully emptying each compartment. The number of eggs and three
 larval instars (L1, L2, and L3) found in the substrate or on the surface
 of the wood was noted and removed from the oviposition boxes. Furthermore,
 the number of galleries in the logs containing most likely one or more
 larvae were also noted. The logs were placed back and the number of
 remaining larvae was counted again on May 3rd 2024 by breaking open all
 the logs with galleries. The total number of offspring as well as the log
 preference (logs with at least one egg or larva) was calculated by
 compiling both assessments. Both were analysed, log preference represents
 females acceptance of certain fungi while number of larvae might also
 account for the females' preference to lay more eggs or for higher
 survival rates of eggs and larvae in certain fungi. Furthermore, female
 stag beetles lay eggs in groups and groups become smaller with female age
 (Tochtermann, 1992) which might interfere with offspring size.
 The mycelia of several species grew outside the log and colonised
 the surrounding wood compost. Especially the mycelia of the brown-rot
 fungi <em>F. pinicola</em> and <em>L.
 sulphureus</em> were found to grow strongly in the compost, even in
 adjacent compartments. These mycelia can be easily recognised by their
 wet, slimy texture and a strong sulphuric odour. The presence of brown-rot
 in other compartments was therefore noted on October 4th and the logs of
 these two species were removed from the boxes and further reared
 separately. <em>Larval
 experiment</em> To gain larvae, three stag beetle
 pairs were collected in Glons, Belgium (50°45'N, 5°32'E) in June
 2022 and placed in breeding containers with white-rotten oak
 (<em>Quercus</em> spp.) wood buried in fermented beech
 sawdust. The population in Glons is part of the well-connected population
 along the Sambre-Meuse valley which is isolated (86 km) from the Brabant
 population where the adults for the oviposition experiment came from. The
 populations were selected for restocking and reintroduction goals and not
 specifically for the experiments. The adults were fed with beetle jelly
 and remained in the containers. For the rearing
 substrate, two mushroom bags with 5 l of beech sawdust were inoculated for
 each of the ten selected fungal species (Table 1). The sawdust was
 intermixed with whole wheat grains, wheat bran, gypsum, and limestone
 powder as additives to optimise initial fungal growth. The substrates were
 produced by Mycelia (Deinze, Belgium) with their normal rearing standards.
 These mature mycelia substrates were used to rear the stag beetle
 larvae. On October 5th, 2022, 69 larvae (58 L2, 11 L1)
 were collected from the rearing containers. Consequently, 69 1-litre
 transparent plastic containers (l x w x h) 15.5 x 10.5 x 7 cm with seven 3
 mm-aeration holes just under the lid were filled with mycelia substrate,
 seven for each fungal species except for <em>Chaetomium
 globosum</em> Kunze with six replicates. The larvae were weighed,
 the larval instar determined, and randomly assigned to one of the
 containers. The larvae were placed in a climate chamber
 at 16 °C and checked about weekly. On November 7th, 2022, i.e. on day 33,
 surviving larvae were weighed and the experiment ceased as some substrates
 were starting to get covered with green moulds while other mycelia were
 growing very vigorously resulting in encapsulation of the larvae (see
 results). Relative growth rate (RGR) was calculated from the initial (i)
 and final (f) weight as: RGR = (Weight-f – Weight-i) /
 Weight-i.
description: # Data from: Oviposition and larval mycelia preference of the saproxylic
 European stag beetle
 [https://doi.org/10.5061/dryad.xd2547dt2](https://doi.org/10.5061/dryad.xd2547dt2) ## Description of the data and file structure ### Files and variables #### File: LarvalExperiment.csv **Description:** Data from the larval experiment ##### Variables * Fungi: Abbreviated name of the fungi inocculated in the substrate, see table 1 of the associated article, factor * RotType: Rot type to which the fungi belongs to, factor * Survival: Survival of the stag beetle (L. crvus) larvae, binomial * RGR: Relative growth rate of the stag beetle (L. crvus) larvae, numerical. NA for larvae that didn't survive the experiment. #### File: OvipositionExperiment.csv **Description:** Data from the oviposition experiment ##### Variables * Box: Box in which the compartement was present, numerical * Compartment: Code of individual compartment, factor * Fungus: Abbreviated name of the fungi inocculated in the log, see table 1 of the associated article, factor * RotType: Rot type to which the fungi belongs to, factor * EggsOct: Number of eggs found in October, numerical * L1Oct: Number of L1 larvae found in October, numerical * L2Oct: Number of L2 larvae found in October, numerical * L3Oct: Number of L3 larvae found in October, numerical * GalleriesOct: Number of additional galleries found in October indicating additional larvae deeper in the wood, numerical * L2May: Number of L2 larvae found in May, numerical * L3May: Number of L3 larvae found in May, numerical * OffspringTotal: Sum of EggsOct, L1Oct, L2Oct, L3Oct, L2May and L3May, numerical * ContaminationBrownrot: Whether contamination by brown rot mycelia of neighbouring compartments was present, binomial * LogVolume: Volume of the log present in this compartment, numerical. NA for compartments without a log.  * LogDensity: Density of the log present in this compartment, numerical. NA for compartments without a log.  ## Code/software The two files are .csv files which can be opened in the statistcal program r, in Microsoft Excel and many more.  ## Access information Other publicly accessible locations of the data: * Non Data was derived from the following sources: * Non