DOI: 10.5061/dryad.f4qrfj6tq
title: Home is where the heart rot is: Violet click beetle, Limoniscus violaceus (Müller, 1821), habitat attributes and volatiles

description: The decreasing number of veteran trees in Europe threatens old-growth
 habitats and the fauna they support. This includes rare taxa, such as the
 violet click beetle, Limoniscus violaceus (Müller, 1821). Samples of wood
 mould were taken from all beech trees in Windsor Forest previously
 confirmed to have contained L. violaceus larvae, and from trees where L.
 violaceus had not previously been detected, the latter categorised as
 having high, medium or low likelihood of containing the beetle during
 recent surveys. Habitat characteristics were measured, and volatile
 profiles determined using gas-chromatography mass-spectrometry. Water
 content significantly differed between tree hollows of different violet
 click beetle status, high-potential habitats having higher and relatively
 stable water content compared with habitats with medium or low potential
 of beetle occupancy. Several volatile organic compounds (VOCs) were
 significantly associated with L. violaceus habitats. No differences in
 other characteristics were detected. The distinction in water regime
 between habitats highlights that recording this quantitatively could
 improve habitat surveys. Several potential L. violaceus attractant VOCs
 were identified. These could potentially be integrated into existing
 monitoring strategies, such as through volatile-baited emergence traps or
 volatile-based surveying of habitats, for more efficient population
 monitoring of the beetle.
description: <strong>Study sites and sample
 collection</strong> All samples and data were
 collected from Windsor Forest (51°26’02.5”N, 0°38’37.2”W). Windsor Forest
 contains many veteran trees with large hollows that afford habitat for
 many saproxylic invertebrates (JNCC 2019; Fowles 2020).
 <em>Limoniscus violaceus</em> was first recorded in Windsor
 Forest in 1937 and this location was thought to be the home of the largest
 UK population of the beetle at the time of this study. Windsor Forest is
 designated a Special Area of Conservation (SAC) under Annex II of the EC
 Habitats Directive. The tree hollow microhabitats at
 Windsor Forest have all been assessed and each rated according to
 <em>L. violaceus</em> occupancy potential (i.e. habitat
 status): confirmed, and low, medium or high potential of beetle presence.
 Habitat statuses were determined in 2014-15 by entomological experts from
 Natural England and Buglife contracted to survey the site. Veteran beech
 trees were categorised as high potential if they had basal hollows, blown
 tops and evidence of heart rot comprising black wood mould within the
 hollow. Medium potential trees lacked obvious basal hollows but had their
 top blown off, trunk damage or early-stage basal hollows. Low potential
 trees were intact beech with no obvious or accessible hollows, or
 substrate too dry and exposed to be <em>L. violaceus</em>
 habitat. Breeding populations of <em>L. violaceus</em> were
 confirmed in five trees by presence of larvae, the surveys taking place 1
 to 2 years before this study, thus very likely that beetles remain given
 their 1 to 2 year larval development. The number of
 trees sampled in the present study was based around the number of
 confirmed <em>L. violaceus</em> habitats, all of which were
 sampled alongside a similar number from each other survey category.
 Non-confirmed trees were selected based upon proximity to confirmed trees,
 the volume and accessibility of wood mould, and to satisfy approximate
 equivalence to the number of confirmed trees. Wood mould was collected
 from each sampled Windsor Forest tree in April, May and June 2016, the
 period during which adult <em>L. violaceus</em> is active
 (pers. comm. Sarah Henshall, Buglife). Samples were taken from 16 trees at
 Windsor Forest, comprising five trees with confirmed activity of
 <em>L. violaceus</em> (all of the confirmed <em>L.
 violaceus</em> habitat trees present at the time of collection), and
 three, four and four trees considered to have, respectively, high, medium
 and low likelihood of violet click beetle presence. All sampling was
 carried out with a Natural England habitat disturbance license. Samples
 were taken from standing trees and a single fallen tree. Substrate samples
 of approximately 100 cm3 in volume were removed by gloved hand from basal
 hollows. One substrate sample (~15 cm3) was taken from each tree hollow in
 each month by taking wood mould from a few centimetres below the surface
 in a gloved hand and sealing it in a universal tube for analysis of
 volatile organic compounds (VOCs).  The tree diameter at 1.3 m from the
 ground, and hollow entrance dimensions halfway up and along each hole were
 measured. The substrate samples were transported to the laboratory at
 Cardiff University on the same day and stored at 4 °C.
 <strong>Wood mould
 characterisation</strong> Density (oven dry mass
 / fresh volume; g cm-3), water content (% oven dry mass) and water
 potential (MPa) were determined for each sample. Each of these is
 intimately associated with the decay process: density determines the
 physical resistance and affects moisture relations; water content is
 relevant both in terms of whether there is enough for physiological
 processes to occur or too much, which restricts aeration, but suffers from
 the drawback that its value varies not only depending on the amount of
 water present but also on the density of the material; and water potential
 indicates the availability of water, the latter known to be particularly
 important in facilitating fungal activity. Density and water content were
 determined by measuring fresh volume and mass, oven-drying for 5 days at
 60 °C, and reweighing. Water potential of fresh wood mould samples was
 determined in a Decagon Devices WP4C Dew Point PotentiaMeter (METER Group
 Inc., Pullman, WA, USA) at 25 °C. <strong>VOC
 analysis</strong> The VOCs present in each wood
 mould sample were determined using thermal desorption gas-chromatography
 time-of-flight mass-spectrometry (TD-GC-TOF-MS) by tipping the wood mould
 into plastic food-grade roasting bags and sealing them for 30 min at room
 temperature (~25 °C), allowing the VOCs to diffuse into the bag’s
 headspace. Using an Easy-VOC manual hand pump (Markes International Ltd.,
 Llantrisant, UK), 500 ml of the air from the headspace was extracted over
 a SafeLokTM thermal desorption tube (TenaxTA/Sulficarb, Markes
 International Ltd.). A control sample was prepared by pumping 500 ml of
 air from the laboratory atmosphere over a thermal desorption tube for each
 round of sampling. A retention standard was prepared by directly loading 1
 µl of C8-C20 alkane standard solution (Sigma-Aldrich, St. Louis, MO, USA)
 into a thermal desorption tube. The thermal desorption
 tubes were placed in a Markes International TD-100 Thermal Desorber
 (Markes International Ltd.) which desorbed the tubes at 100 °C for 5 min,
 followed by 280 °C for 5 min with a 40 ml min-1 trap flow. Trap desorption
 and transfer were carried out with a temperature increase of 20 °C sec-1,
 a maximum temperature of 300 °C for 3 min, with a split flow of 5 ml
 min-1. The VOCs were then separated in an Agilent 7890A GC system (Agilent
 Technologies, Santa Clara, CA, USA) with helium used as a carrier gas at 2
 ml min-1 under constant flow conditions for 2 min at 40 °C. This process
 was followed by a temperature increase of 5 °C min-1 up to a maximum
 temperature of 240 °C, at which point the temperature remained constant
 for 5 min. The mass spectra of the separated VOCs were then recorded from
 m/z 30-350 in a time-of-flight ALMSCO BenchTOF-dx (Markes International
 Ltd.). Data from GC-MS were processed using MSD
 ChemStation (Agilent Technologies Inc. 2005) and AMDIS, with a custom
 retention-indexed mass spectral library of compounds, a compiled list of
 compounds identified across all of these samples and others taken from
 wood mould that have been checked and verified against the NIST 2011
 library. All VOCs scoring more than 80 % in forward and backward fit in
 the NIST 2011 library were included in the custom library of mass spectra.
 Data were normalised as proportions of each volatile profile and were
 square root transformed to prevent large values from biasing the
 results.