Introduction
Bark and ambrosia beetles, members of the subfamily Scolytinae (Curculionidae), represent a functionally diverse group of saproxylic insects (Kirkendall et al., Reference Kirkendall, Biedermann, Jordal, Vega and Hofstetter2015). Together with wood-inhabiting fungi, they play an essential role in forest ecosystems as decomposers and nutrient recyclers (Goheen and Hansen, Reference Goheen, Hansen, Schowalter and Filip1993; Siegert et al., Reference Siegert, Clay, Pace, Vissa, Hofstetter, Leverón and Riggins2024; Skelton et al., Reference Skelton, Jusino, Carlson, Smith, Banik, Lindner, Palmer and Hulcr2019). Although commonly referred to collectively as bark beetles, Scolytinae comprises ecologically distinct feeding guilds (Jordal, Reference Jordal2006). Phloeophagous species feed on the inner bark (phloem), xylophagous species bore directly into woody tissues, and xylomycetophagous species, commonly known as ambrosia beetles, cultivate symbiotic fungi within the xylem galleries (Faccoli et al., Reference Faccoli, Blaženec and Schlyter2005; Kirisits, Reference Kirisits, Lieutier, Day, Battisti, Grégoire and Evans2004; Kirkendall et al., Reference Kirkendall, Biedermann, Jordal, Vega and Hofstetter2015). While a limited number of bark beetle species can become major forest pests capable of colonising and killing living trees, this is the exception rather than the norm, and tends to occur only when climatic and host stress conditions are favourable (Fiala et al., Reference Fiala, Holuša, Procházka, Čížek, Dzurenko, Foit, Galko, Kašák, Kulfan, Lakatos, Nakládal, Schlaghamerský, Svatoš, Trombik, Zábranský, Zach and Kula2020; Holuša et al., Reference Holuša, Fiala and Foit2021; Mezei et al., Reference Mezei, Fleischer, Rozkošný, Kurjak and Dzurenko2022; Polizzi et al., Reference Polizzi, Vitale, Garzia, Aiello, Biondi, Gugliuzzo and Criscione2020). In most cases, bark beetles are secondary colonisers that develop in dead or dying wood and do not threaten healthy trees (Gregoire and Evans, Reference Gregoire, Evans, Lieutier, Day, Battisti, Grégoire and Evans2004; Sauvard, Reference Sauvard, Lieutier, Day, Battisti, Grégoire and Evans2004). Ambrosia beetles, in particular, are considered beneficial decomposers that break down woody material and promote nutrient cycling within forest ecosystems (Siegert et al., Reference Siegert, Clay, Pace, Vissa, Hofstetter, Leverón and Riggins2024; Skelton et al., Reference Skelton, Jusino, Carlson, Smith, Banik, Lindner, Palmer and Hulcr2019). Despite their ecological significance, key aspects of the circadian flight activity of Scolytinae remain poorly understood, particularly with respect to diel patterns and dispersal behaviour across different functional feeding guilds (Johnson et al., Reference Johnson, Kendra, Skelton and Hulcr2016; Monterrosa et al., Reference Monterrosa, Joseph, Blaauw, Hudson and Acebes-Doria2022; Reding et al., Reference Reding, Ranger, Oliver and Schultz2013).
Circadian rhythm is a fundamental aspect of insect physiology that produces near-24-h rhythms that are attuned to an exact 24 h by environmental cycles of light and temperature (Saunders, Reference Saunders2009; Reshma et al., Reference Reshma, Prüser, Schulz, Mayer, Ogueta, Stanewsky and Kurtz2024). Biological circadian rhythms are endogenous, have a cycle length close to environmental day, remain accurate, adjust to temperature changes, and sync with external cues like light (Asgari Targhi and Klerman, Reference Asgari Targhi and Klerman2018; Giebultowicz, Reference Giebultowicz2000; Stengl and Schneider, Reference Stengl and Schneider2024). They control a wide variety of behavioural and physiological activities in insects, including daily rhythms of locomotion, feeding, mating, or oviposition (Benita et al., Reference Benita, Menahem, Rath, Scharf and Gottlieb2024; Bloch et al., Reference Bloch, Hazan and Rafaeli2013; Saunders et al., Reference Saunders, Steel, Vafopoulou and Lewis2002). Bark and ambrosia beetles, like other insects, restrict their activity to certain times of the day (Johnson et al., Reference Johnson, Kendra, Skelton and Hulcr2016; Menocal et al., Reference Menocal, Cruz, Kendra, Crane, Ploetz and Carrillo2018; Sittichaya et al., Reference Sittichaya, Permkam and Cognato2012). They may be active during the day (diurnal), at twilight (crepuscular) or at night (nocturnal) (Mendel et al., Reference Mendel, Boneh and Shenhar1991; Menocal et al., Reference Menocal, Cruz, Kendra, Crane, Ploetz and Carrillo2018; Saunders et al., Reference Saunders, Steel, Vafopoulou and Lewis2002). The flight activity may be species specific with unimodal (one peak) or bimodal (two peak) patterns (Johnson et al., Reference Johnson, Kendra, Skelton and Hulcr2016; Lewis and Taylor, Reference Lewis and Taylor1965; Saunders and Knoke, Reference Saunders and Knoke1968; Seo et al., Reference Seo, Martini, Rivera and Stelinski2017). As early as 1965, Lewis and Taylor proposed that insect feeding on decaying matter or fungi, likely locating their food through olfactory cues, tend to fly in dim light. As certain bark and ambrosia beetles have become increasingly important economic species, several papers on their circadian rhythms have been published in recent decades (Saunders and Knoke, Reference Saunders and Knoke1968; Wertman and Bleiker, Reference Wertman and Bleiker2019; Reshma et al., Reference Reshma, Prüser, Schulz, Mayer, Ogueta, Stanewsky and Kurtz2024).
Several studies on the circadian rhythms of Scolytinae have been published from various regions of North America, often focused on specific feeding guilds (Dell and Davis, Reference Dell and Davis2019; Fettig et al., Reference Fettig, Shea and Borys2004; Lobinger, Reference Lobinger1994; Mendel et al., Reference Mendel, Boneh and Shenhar1991; Williams et al., Reference Williams, McMillin, Degomez, Clancy and Miller2008). Phloeophagous species that feed on the inner bark, have been extensively studied. For example, Pityogenes chalcographus (L.) shows a strong temperature-dependent emergence pattern, with diapause induced under specific light conditions at lower temperatures (Führer and Chen, Reference Führer and Chen1979). Similarly, Ips typographus (L.) in Sweden displays a unimodal flight peak, typically occurring in the afternoon, indicating synchronisation with ambient thermal conditions (Byers et al., Reference Byers, Anderbrant and Löqvist1989). In Dendroctonus ponderosae (Hopkins), a clear circadian emergence rhythm has been identified, suggesting control by endogenous clocks beyond environmental cues (Gray et al., Reference Gray, Billings, Gara and Johnsey1972, Reference Gray, Billings, Gara and Johnsey2009; Wertman and Bleiker, Reference Wertman and Bleiker2019).
Ambrosia beetles, which cultivate symbiotic fungi in wood galleries, demonstrate different activity patterns. Xyleborus ferrugineus (F.), responsible for damaging coca trees, exhibits a diurnal rhythm tightly linked to temperature, with flight activity persisting even without light cues (Saunders and Knoke, Reference Saunders and Knoke1968). Xyleborus glabratus (Eichhoff) displays a consistent late afternoon flight peak, reflecting unimodal dispersal dynamics (Brar et al., Reference Brar, Capinera, McLean, Kendra, Ploetz and Peña2012), with subsequent studies reinforcing this pattern and suggesting species-specific flight timing (Seo et al., Reference Seo, Martini, Rivera and Stelinski2017).
Despite these insights, relatively few studies have examined the relationship between circadian rhythms and dispersal strategies in Scolytinae within Central Europe. Available research has mostly focused on abundance and distribution of phloeophagous bark beetles, such as Ips typographus and Pityogenes chalcographus, and has provided limited data on ambrosia beetles (Fiala et al., Reference Fiala, Holuša, Procházka, Čížek, Dzurenko, Foit, Galko, Kašák, Kulfan, Lakatos, Nakládal, Schlaghamerský, Svatoš, Trombik, Zábranský, Zach and Kula2020; Holuša et al., Reference Holuša, Fiala and Foit2021; Mezei et al., Reference Mezei, Fleischer, Rozkošný, Kurjak and Dzurenko2022). This highlights a knowledge gap, particularly concerning the diel activity of ambrosia beetles in temperate European forests.
Understanding how activity patterns differ between phloeophagous and xylomycetophagous trophic guilds remains a key challenge, especially when considering how environmental drivers shape their behaviour over a 24-h cycle. While temperature and light serve as critical regulatory factors (Chen et al., Reference Chen, Aukema and Seybold2020; Fraedrich et al., Reference Fraedrich, Harrington, Rabaglia, Ulyshen, Mayfield, Hanula, Eickwort and Miller2008; Mezei et al., Reference Mezei, Fleischer, Rozkošný, Kurjak and Dzurenko2022), emerging evidence suggests that other variables, such as humidity may be a crucial driver for xylomycetophagous species, which rely on symbiotic fungi for nutrition (Sittichaya et al., Reference Sittichaya, Permkam and Cognato2012). For instance, the abundance of Xyleborini ambrosia beetles decreases as temperature rise, but increases with higher humidity and rainfall (Sittichaya et al., Reference Sittichaya, Permkam and Cognato2012). This pattern probably reflects the fact that dry wood inhibits the growth of symbiotic fungi, which are an essential food source for these members of the tribe Xyleborini (Sittichaya et al., Reference Sittichaya, Permkam and Cognato2012). On the other hand, phloeophagous species specialise in exploiting the inner bark and often show increased flight activity around midday, possibly triggered by chemical signals released from weakened or damaged trees (Byers 1983; Schiebe et al., Reference Schiebe, Unelius, Ganji, Binyameen, Birgersson and Schlyter2019). This diel segregation between ecological and trophic guilds may improve colonization of suitable hosts.
Microhabitat preferences also shape flight periodicity (Williams et al., (Reference Williams, McMillin, Degomez, Clancy and Miller2008); Dell and Davis, Reference Dell and Davis2019). Species adapted to shaded environments tend to have different flight patterns than those preferring sun-exposed habitats (Kautz et al., Reference Kautz, Schopf and Ohser2013; Lobinger, Reference Lobinger1994; Majdák et al., Reference Majdák, Jakuš and Blaženec2021; Vindstad et al., Reference Vindstad, Birkemoe, Ims and Sverdrup Thygeson2020). Research on Xyleborinus saxesenii indicates that light intensity influences flight activity, with individuals preferring low-light conditions (Menocal et al., Reference Menocal, Kendra, Padilla, Chagas, Chagas, Crane and Carrillo2022). Likewise, Pityogenes chalcographus and Ips typographus display temperature-driven emergence patterns, reinforcing the role of both microclimate and phylogenetic lineage in determining diel activity (Lobinger, Reference Lobinger1994; Wermelinger and Seifert, Reference Wermelinger and Seifert1998).
To effectively monitor and manage bark beetle populations, it is essential to understand their daily activity cycles and microhabitat preferences. Evaluating how specific traits might influence circadian rhythms can provide deeper insights into the temporal dynamics of these insects, (Conti et al., Reference Conti, Schmidt Kloiber and Grenouillet2014; Yazdanian et al., Reference Yazdanian, Kankaanpää, Itämies, Leinonen, Merckx, Pöyry, Sihvonen, Suuronen, Välimäki and Kivelä2023). Community analyses based on functional guilds rather than solely on species data allow for a better understanding of differences in temporal activity, as they reflect ecological strategies and responses to environmental conditions (Garnier et al., Reference Garnier, Navas and Grigulis2016). This enables a more precise interpretation of activity patterns across groups and helps explain how different ecological strategies influence their temporal distribution in the environment (Mouchet et al., Reference Mouchet, Villéger, Mason and Mouillot2010).
This study investigates the circadian rhythms of bark beetle species in a Central European forest ecosystem, using flight interception traps sampled every 4 h over a 24-h cycle in the Czech Republic. To guide our investigation, we formulated the following hypotheses: (i) circadian flight patterns will be primarily determined by species identity, with different taxonomic groups exhibiting distinct flight periodicities. (ii) The highest species richness will be observed during evening and nighttime hours. (iii) Scolytini bark beetles (phloeophagous) and Xyleborini ambrosia beetles (xylomycetophagous) are expected to differ in their flight activity patterns across the monitored time intervals. By clarifying these patterns, our study aims to provide new insights into the functional group-specific circadian behaviour of bark beetles. Understanding such temporal dynamics may enhance the timing and placement of monitoring tools (e.g. traps, trap trees), improve the accuracy of population assessments, and optimise the scheduling of control measures, particularly for early detection and targeted interventions during periods of peak activity. This knowledge is therefore valuable for designing more effective and ecologically-informed forest protection strategies.
Methods
Study area
The research site is located in the northern part of the Pálava Protected Landscape Area (CHKO Pálava), within the Děvín National Nature Reserve (NPR Děvín), on a northeastern slope between the villages of Dolní Věstonice and Pavlov, Czechia (fig. 1). The coordinates of the site are 48°52′47.8″ N, 16°39′34.7″ E, and the study area is situated at an altitude of approximately 310 m above sea level. The site is characterised by a thermophilous oak forest, predominantly composed of sessile oak (Qercus petraea L.) (Fagales: Fagaceae) and large-leaved lime (Tilia platyphyllos L.) (Malvales: Malvaceae). Although located within a strictly protected area, parts of the surrounding forest stands have undergone targeted conservation interventions since 2009, aimed at restoring biodiversity. These continuous management actions are part of a broader effort to support biodiversity within historically coppiced forests, which were later converted to high forest. While coppice restoration is ongoing in selected parts of the reserve, it does not apply to the specific plot used in this study.
Study area and design – the geographical context of the research is illustrated by highlighting the Czech Republic (A) and specifying the location of the study site within the NPR děvín (B). The study site boundary (C), corresponding to the area subjected to targeted conservation intervention, is outlined with a dark line. Individual sampling points, where flight interception traps were placed, are represented by 10 black dots.
Experimental design and data sampling
For insect sampling, flight interception traps were utilised (fig. 2). A total of 10 traps were installed at the study site, strategically positioned in varying spatial orientations and at random intervals to avoid disrupting natural flight paths while ensuring a minimum spacing of 5 m between each trap (Lamarre et al., Reference Lamarre, Molto, Fine and Baraloto2012; Russo et al., Reference Russo, Stehouwer, Heberling and Shea2011) (fig. 1). Each trap consisted of a transparent, rigid PVC sheet (0.3 mm thick) with an impact surface measuring 140 cm in width and 100 cm in height. The sheets were positioned perpendicular to the ground and secured using ropes between two nearby trees, ensuring that the impact surface did not make direct contact with the tree trunk. Transparent plastic collection trays filled with water and detergent were placed at ground level on both sides of the impact surface to capture and euthanise intercepted insects. Each side of the PVC sheet was equipped with eight collection trays (fig. 2). A crucial aspect of trap installation was ensuring that the lower edge of the interception sheet was positioned below the level of the collection trays to prevent insects from escaping through potential gaps.
Flight interception trap with plastic containers used to capture the material.
Trap contents were collected every four hours over a 24-h cycle, resulting in six distinct sampling intervals per day: 08:00, 12:00, 16:00, 20:00, 24:00, and 04:00 the following day. For ecological interpretation of patterns in species richness and to assess factors influencing temporal flight activity, these intervals were grouped into three time periods: diurnal (04:00–16:00), crepuscular (16:00–20:00), and nocturnal (20:00–4:00). Sampling was conducted using a fine mesh sieve to filter the water and detergent solution, after which the material was transferred into labelled collection vials and preserved for later identification.
A total of six sampling sessions were conducted on June 2–3, June 6–7, June 15–16, June 20–21, July 14–15, and July 21–22 in 2022; and on June 12–13, June 20–21, and July 4–5 in 2023. Sampling in May, as initially planned, was not feasible due to unforeseen logistical constraints. However, the data collected from early June onward provide a reliable basis for addressing our primary research questions regarding the diurnal rhythms of Scolytinae beetles. The sampling period coincided with increased adult activity, and we recorded a sufficient number of specimens to support robust statistical analyses (Lieutier et al., Reference Lieutier, Day, Battisti, Grégoire and Evans2004). The collected material was subsequently sorted, with insect specimens identified to the most precise taxonomic level possible as per Pfeffer (Reference Pfeffer1955), Szujecki (Reference Szujecki1987), and Burakowski et al. (Reference Burakowski, Mroczkowski and Stefańska1992). Any unidentified material was preserved in 70% ethanol for further identification.
Arthropod classification
Following taxonomic identification, species were classified according to their trophic specialisation (phloeophagous vs. xylomycetophagous), dietary specialisation (oligophagous or polyphagous), and microhabitat preference, inferred from typical gallery locations in trunks, branches, or stumps. These classifications were made using the same literature sources that provided the taxonomic identification. Classification as generalists or specialists was based on the breadth of host and habitat associations: species exhibiting polyphagy across multiple unrelated host plants and diverse habitat types (e.g. open, deciduous, or coniferous forests) were considered generalists, while those consistently associated with a narrow range of host taxa or specific habitats (termophilous Quercus spp.) were categorised as specialists. These classifications are detailed in Supplementary Tables S1 and S2, which list all collected species along with their trophic guilds, habitat preferences, and dominant host trees.
Statistical analysis
We graphically represented activity rhythms over the course of the day using partial canonical correspondence analysis (pCCA). Trap ID and ambient temperature were included as covariates. Given the relatively low abundance of some species in individual years, data were pooled across both years to ensure a more reliable assessment of temporal patterns.
To determine whether the occurrence of individual taxa significantly differed during the 24-h sampling period, we used a constrained permutation test. Temporal correlation between repeated samples from the same trap was addressed by restricting the permutation test within each trap separately. Additionally, insect samples were collected in the same temporal sequence throughout the day, preserving temporal autocorrelation through cyclic shifts.
Species abundances were log10-transformed. The significance of the canonical axis was tested using a restricted Monte Carlo permutation test with 2,000 permutations. Ordination analyses were performed using CANOCO 5 (Ter Braak and Šmilauer, Reference Ter Braak and Šmilauer2012).
To test the relationships between the circadian rhythm intervals of each species and their activity patterns, we performed a fourth-corner analysis with the ‘ade4’ package in R ver.4.2.0 (Thioulouse et al., 2018, R Core Team, 2020). This analysis links three matrices – environmental variables (R), species abundance (L), and species traits (Q) – in order to evaluate the impact of relationships between traits and the environment on community composition. To account for inflated type I error rates, we applied a modified fourth-corner analysis (modeltype = 6; Doledec et al., Reference Doledec, Chessel, Ter Braak and Champely1996), adjusting the significance by taking the maximum p-value from the row- and column-based permutation tests (Ter Braak, Reference Ter Braak2017; Ter Braak et al., Reference Ter Braak, Peres-Neto and Dray2018). We adjusted the p-values for multiple comparisons using the false discovery rate method of Benjamini and Hochberg (Reference Benjamini and Hochberg1995), implemented through the ‘fourthcorner()’ function (Dray, Reference Dray2013), for both the global test (G) and the cell-wise test (D).
Results
Time lapse differentiation with diversity gradient
Between 2022 and 2023, a total of 1,149 bark beetles representing 15 species from the subfamily Scolytinae were captured. pCCA revealed a significant temporal shift in assemblage composition (pCCA, R 2 = 11.91%; Test on All Axes: pseudo-F = 1.4, p = 0.015) (fig. 3). The pCCA biplot (fig. 3) provides a spatial representation of shift in species richness, where the horizontal axis (pCCA1) captures a flight activity gradient, from predominantly nocturnal dispersers (left) to species exhibiting diurnal flight patterns (right), explaining 2.7% of the total variation in standard deviation. The vertical axis (pCCA2) accounts for an additional 2.2% of variability, which may represent unrecognised species-specific temporal variation (fig. 3). Based on the ordination, species richness peaked during the part of nocturnal period (20:00–24:00) and gradually declined towards the diurnal period (08:00–16:00), indicating a clear nocturnal-to-diurnal diversity gradient (fig. 3). Three time intervals (16:00–20:00, 24:00–04:00, and 04:00–08:00) exhibited comparable species richness and occurred between the periods of highest and lowest richness observed (fig. 3). This pattern reflects a dynamic turnover of species with differing temporal flight activity.
Ordination biplot from partial canonical correspondence analysis (pCCA) illustrating temporal shifts in bark and ambrosia beetle assemblage composition between six-time intervals during a 24-h sampling period. The horizontal axis (pCCA1) represents a flight activity gradient, ranging from predominantly nocturnal species (left) to diurnal species (right), explaining 2.7% of the total variation. The vertical axis (pCCA2) accounts for an additional 2.2% of the variation and likely reflects species-specific temporal responses. Trap number was included as a covariate.
Factors affecting the temporal flight activity of bark and ambrosia beetles
Using fourth-corner analysis to test the associations between species traits (including tribal affiliation, trophic specialisation, dietary specialisation, and microhabitat preference) and time intervals, we identified significant relationships between bark and ambrosia beetle traits and their temporal flight activity (table 1). These patterns were further tested with forward selection in a pCCA, which confirmed significant shifts in assemblage composition across time intervals (pCCA, R 2 = 7.57%; test on all axes: pseudo-F = 0.8, p = 0.002) (figs. 4 and 5). The drivers shaping this temporal structuring reflect differences in habitat conditions, resource availability, and dispersal energetics between bark and ambrosia beetles (table 1). The resulting triplots visualise how time intervals align with species- and trait-level variation (figs. 4 and 5).
Results of the fourth-corner analysis testing the relationship between sampling time and species traits
Significant results (adjusted p ≤ 0.05) are highlighted in bold. Abbreviations: neg. – negative association with sampling between 16:00–20:00; na. – no clear association. Xyleborini ambrosia beetles: Anisandrus dispar, Xyleborinus saxesenii, Xyleborus dryographus, Xyleborus monographus, Xylosandrus germanus. Scolytini bark beetles: Scolytus carpini, Scolytus intricatus, Scolytus rugulosus.
Temporal structuring at the tribal level
Trait-specific responses were analysed at the tribal level. In the ordination triplot, the horizontal axis (pCCA1) reflects variation in flight activity across beetle tribes, explaining 2.7% of the total variation (fig. 4). The vertical axis (pCCA2) reflects residual variation, accounting for an additional 2.2% (fig. 4). The fourth-corner test revealed a significant-negative association between the tribe Xyleborini and the crepuscular period (16:00–20:00), suggesting flight suppression potentially driven by internal rhythms or environmental constraints (table 1, fig. 4). In contrast, the tribe Scolytini showed no strong association with specific time intervals (table 1). A boxplot of log-transformed abundances confirmed temporal segregation between Xyleborini ambrosia beetles and Scolytini bark species (fig. S1). Due to the absence of significant patterns, the remaining tribes were excluded from further analysis (fig. 4).
Ordination triplot from partial canonical correspondence analysis (pCCA) illustrating the association between six-time intervals and the occurrence frequency of individual bark and ambrosia beetle species during a 24-h sampling period. The horizontal axis (pCCA1) reflects variation in flight activity across tribal affiliation, explaining 2.7% of the total variation. The vertical axis (pCCA2) reflects residual variation, accounting for an additional 2.2%. Only the tribes that significantly correlated with time intervals were passively projected. Trap number was included as a covariate.
Ordination triplot from partial canonical correspondence analysis (pCCA) showing associations between bark and ambrosia beetle species and six-time intervals during a 24-h sampling period. The horizontal axis (pCCA1) separates stump-associated and xylomycetophagous species from those with other microhabitat and trophic preferences, explaining 2.7% of the total variation. The vertical axis (pCCA2) distinguishes broadleaf specialists from generalists, accounting for an additional 2.2%. Ffunctional traits were passively projected. Trap number was included as a covariate.
Functional traits at the species level
To delineate temporal flight strategies at the species level, we examined functional trait distributions across time intervals (fig. 5). The horizontal axis (pCCA1) segregated bark beetle species based on trophic and microhabitat preferences, with Dryocoetes villosus, which develops beneath the bark of dying oaks or in fresh oak stumps, emerging as the most influential contributor to this pattern (Table S1). The vertical axis (pCCA2) reflects a gradient from dominant broadleaf specialists in the upper part of the diagram (Taphrorychus villifrons, associated with oak and beech) to a heterogeneous group of species in the lower part, including generalists (Xylosandrus germanus, Xyleborinus saxesenii, Anisandrus dispar) and incidental records of species typically developing in fruit trees (Scolytus rugulosus) or conifers (Pityogenes chalcographus), likely representing occasional dispersal from nearby habitats (fig. 5, Table S2). Dietary specialisation based on dominant host plants, whether oligophagous or polyphagous species, did not show significant temporal structuring (table 1). Similarly, no temporal differences were found between generalists and forest specialists (table 1). Due to the lack of significant patterns, these factors were omitted from further analysis.
Trophic specialisation and circadian rhythms
A contrasting pattern emerged between flight activity, represented by full-day time intervals, and the trophic specialisation (table 1). Specifically, xylomycetophagous species exhibited multimodal peak activity from nocturnal to early morning hours (figs. 5 and S1), while a significant negative association was observed in the crepuscular period (16:00–20:00) (table 1). This trend was consistent across taxonomic categories and was primarily driven by Xyleborinus saxesenii, Xyleborus dryographus, Xyleborus monographus, Xylosandrus germanus, and Anisandrus dispar (figs. S1 and 5). These findings likely align with dispersal patterns linked to microhabitat preferences and their trophic specialisation, as all members are xylomycetophages (Table S1, fig. 5).
Microhabitat preference and flight timing
A significant association was found between time intervals and species known to develop in trunks and potentially occur in stumps (Anisandrus dispar, Dryocoetes villosus, Xyleborinus saxesenii, Xyleborus dryographus and Xyleborus monographus), with a negative association detected during the crepuscular period (16:00–20:00) (table 1). The ordination triplot shows that species associated with stumps were found during the 12:00–16:00 interval (fig. 5), a period when overall beetle activity was lowest (fig. 3). This pattern may indicate that bark and ambrosia beetles, which prefer to create galleries in trunks and stumps, take advantage of this time window to disperse when competition for flight space is lower (table 1). These findings indicate that resource availability, combined with species-specific physiological constraints, influences diel flight periodicity within Scolytinae assemblages.
Discussion
Our study provides a comprehensive overview of the circadian rhythms of bark and ambrosia beetles (Scolytinae) based on data collected during the peak activity period in June and July of 2022 and 2023. This seasonal window was selected to capture the most representative patterns of Xyleborini ambrosia and Scolytini bark beetles in Central Europe. Using ordination and fourth-corner analysis, we identified key factors influencing species-specific flight activity, confirming that flight periodicity varies among species and is shaped by phylogenetic affiliation, trophic specialisation, and host substrate preference (Jones et al., Reference Jones, Shegelski, Marculis, Wijerathna and Evenden2019; Menocal et al., Reference Menocal, Cruz, Kendra, Crane, Ploetz and Carrillo2018; Salmane et al., Reference Salmane, Ciematnieks, Ozoliņa Pole, Ralle and Ievinsh2015).
Factors influencing the temporal activity of Scolytinae
Temperature and light
Environmental factors such as temperature and light exposure strongly influence the diel activity patterns of bark and ambrosia beetles. Studies have shown that many Scolytinae species (but not all) exhibit peak flight activity during warm periods, often aligning with late afternoon or evening temperatures (Kendra et al., Reference Kendra, Montgomery, Niogret, Deyrup, Guillén and Epsky2012; Ranger et al., Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016; Seo et al., Reference Seo, Martini, Rivera and Stelinski2017). Temperature-dependent flight behaviour has been well-documented in species such as Ips typographus, which exhibits increased activity under warm conditions (Lobinger, Reference Lobinger1994; Wermelinger and Seifert, Reference Wermelinger and Seifert1998; Zang et al., Reference Zang, Helm, Sparks and Menzel2015). Similarly, Hypothenemus species have been observed to synchronise their flight with optimal thermal conditions, displaying a broad flight window with peak activity around 15:00 (Johnson et al., Reference Johnson, Kendra, Skelton and Hulcr2016; Seo et al., Reference Seo, Martini, Rivera and Stelinski2017).
In our study, species diversity was highest during the evening and night (20:00–24:00), a period characterised by lower ambient temperatures and increased relative humidity in the forest understory, which may influence beetle activity patterns through behavioural or physiological responses to external environmental cues. Notably, xylomycetophagous species exhibited increased nocturnal flight activity, likely driven by their preference for humid microhabitats that support fungal growth (Fraedrich et al., Reference Fraedrich, Harrington, Rabaglia, Ulyshen, Mayfield, Hanula, Eickwort and Miller2008; Kirisits, Reference Kirisits, Lieutier, Day, Battisti, Grégoire and Evans2004).
Dispersal strategies and ecological plasticity
Flight activity in bark and ambrosia beetles is primarily linked to dispersal and colonization rather than routine foraging (Ayres and Lombardero, Reference Ayres and Lombardero2018; Jones et al., Reference Jones, Shegelski, Marculis, Wijerathna and Evenden2019). These insects typically initiate flight to locate new host trees or suitable colonisation sites, with their activity influenced by both environmental conditions and internal physiological cues (Reding et al., Reference Reding, Ranger, Oliver and Schultz2013; Wertman and Bleiker, Reference Wertman and Bleiker2019). In our study, Xyleborini ambrosia beetles exhibited reduced flight activity in the afternoon and evening (16:00–20:00), likely adapting their dispersal to maximise fungal colonisation success (Fraedrich et al., Reference Fraedrich, Harrington, Rabaglia, Ulyshen, Mayfield, Hanula, Eickwort and Miller2008; Menocal et al., Reference Menocal, Kendra, Padilla, Chagas, Chagas, Crane and Carrillo2022; Salmane et al., Reference Salmane, Ciematnieks, Ozoliņa Pole, Ralle and Ievinsh2015). This pattern parallels findings in bark beetles such as Dendroctonus ponderosae, which increase flight activity with temperature but do not strictly synchronise it with daily temperature peaks, suggesting an underlying rhythmic control of dispersal (Fettig et al., Reference Fettig, Shea and Borys2004; Gray et al., Reference Gray, Billings, Gara and Johnsey2009). Although D. brevicomis (LeConte) and D. ponderosae are phloeophagous rather than xylomycetophagous, their seasonal and flight activity patterns suggest that their dispersal timing is finely tuned (Fettig et al., Reference Fettig, Shea and Borys2004; Zang et al., Reference Zang, Helm, Sparks and Menzel2015).
Some species do not demonstrate a clear circadian flight pattern. For example, Hypothenemus brunneus maintains activity throughout the day, likely due to its generalist host strategy and broader ecological tolerance (Johnson et al., Reference Johnson, Kendra, Skelton and Hulcr2016; Seo et al., Reference Seo, Martini, Rivera and Stelinski2017). These differences in circadian flight patterns likely reflect species-specific dispersal strategies and resource utilisation timing, rather than daily feeding needs (Ayres and Lombardero, Reference Ayres and Lombardero2018; Bloch et al., Reference Bloch, Hazan and Rafaeli2013; Jones et al., Reference Jones, Shegelski, Marculis, Wijerathna and Evenden2019). Although our study focused on the months with increased flight, future research should investigate non-peak months to assess the seasonal consistency of circadian patterns and dispersal strategies across species.
Microhabitat and host substrate preference
Temporal differentiation in flight activity is also influenced by host substrate condition. Species colonizing freshly dead trunks-initiated flight activity earlier in the day than those exploiting decomposing wood, likely due to differences in resource availability and host condition. Jones et al. (Reference Jones, Shegelski, Marculis, Wijerathna and Evenden2019) suggest that species preferring decomposing branches delay their activity as part of an energy-optimisation strategy, a pattern also observed in bark beetle flight periodicity studies (Dell and Davis, Reference Dell and Davis2019). Key host characteristics, such as moisture content, decay stage, and volatile emissions, play critical roles in determining the timing of colonisation (Mezei et al., Reference Mezei, Fleischer, Rozkošný, Kurjak and Dzurenko2022).
In our study, species associated with later stages of wood decomposition, such as those colonise stumps or decaying trunks, initiated flight later in the day than those that primarily target recently stressed or dead trees. This supports the idea that colonisation timing is shaped by host material quality and environmental conditions (Beaver, Reference Beaver, Wilding, Collins, Hammond and Webber1989; Mezei et al., Reference Mezei, Fleischer, Rozkošný, Kurjak and Dzurenko2022; Salmane et al., Reference Salmane, Ciematnieks, Ozoliņa Pole, Ralle and Ievinsh2015). Ambrosia beetles (Xylosandrus germanus, Xyleborinus saxesenii) displayed peak activity at night, supporting previous findings that they prefer humid, low-light conditions for dispersal (Fraedrich et al., Reference Fraedrich, Harrington, Rabaglia, Ulyshen, Mayfield, Hanula, Eickwort and Miller2008; Menocal et al., Reference Menocal, Kendra, Padilla, Chagas, Chagas, Crane and Carrillo2022; Ranger et al., Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016). In contrast, phloeophagous species targeting intact or recently killed wood exhibited flight peaks in the early morning and late afternoon, possibly influenced by diurnal variations in host volatile release (Faccoli et al., Reference Faccoli, Blaženec and Schlyter2005; Kelsey et al., Reference Kelsey, Gallego, Sánchez-García and Pajares2014; Schiebe et al., Reference Schiebe, Unelius, Ganji, Binyameen, Birgersson and Schlyter2019). The co-occurrence of both trophic guilds may reflect the presence of freshly cut oak stumps within the vicinity of the study site. These stumps could potentially serve as a marginal yet ecologically significant habitat for the colonisation of bark and ambrosia beetles (Brin et al., Reference Brin, Bouget, Valladares and Brustel2012).
Interspecific interactions
In diverse bark and ambrosia beetle communities, flight activity may be temporally segregated to minimise interspecific competition. Jones et al. (Reference Jones, Shegelski, Marculis, Wijerathna and Evenden2019) reported differences flight strategies at the tribe level, particularly between Ipini, Hylurgini and Tomicini bark beetles, which exhibit distinct dispersal behaviours. Species with limited flight capacity, such as the ambrosia beetle Xyleborus glabratus, exhibit short-range dispersal, typically flying less than 20 m in a 24-h period and showing peak flight activity during late afternoon hours (Seo et al., Reference Seo, Martini, Rivera and Stelinski2017). In contrast, bark beetle species with greater dispersal abilities often rely on host tree volatiles for colonisation and tend to be more active under warm conditions, aligning their flight with peak physiological stress signals from trees (Chu et al., Reference Chu, Yang, Li, Liu, Liang, Wu, Wang, Zhang and Hu2021; Ranger et al., Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016; Roling and Kearby, Reference Roling and Kearby1975).
Implications for microhabitat management
Our findings emphasise the importance of habitat heterogeneity in sustaining functionally diverse bark and ambrosia beetle assemblages. Temperature and humidity are key drivers of functional diversity in saproxylic beetles, highlighting the need for forest management strategies that retain both sun-exposed and shaded deadwood (Balázs et al., Reference Balázs, Bezděk and Šipoš2024; Lieutier et al., Reference Lieutier, Day, Battisti, Grégoire and Evans2004; Menocal et al., Reference Menocal, Cruz, Kendra, Crane, Ploetz and Carrillo2018; Vodka et al., Reference Vodka, Konvička and Čížek2009).
Phloeophagous bark beetles, which primarily colonise fresh phloem tissues, exhibited peak flight activity in the afternoon and early evening. This pattern is likely influenced by host stress signals such as ethanol emissions and volatile organic compounds, which serve as important cues for locating weakened or recently killed trees (Kendra et al., Reference Kendra, Montgomery, Niogret, Deyrup, Guillén and Epsky2012; Ranger et al., Reference Ranger, Reding, Persad and Herms2010; Schiebe et al., Reference Schiebe, Unelius, Ganji, Binyameen, Birgersson and Schlyter2019). Notably, both phloeophagous and xylomycetophagous species respond to these chemical signals despite their differing ecological roles (Kelsey et al., Reference Kelsey, Gallego, Sánchez-García and Pajares2014; Ranger et al., Reference Ranger, Reding, Persad and Herms2010, Reference Ranger, Reding, Schultz and Oliver2012, Reference Ranger, Reding, Schultz, Oliver, Frank, Addesso, Chong, Sampson, Werle, Gill and Krause2016). Xylomycetophagous species, although dependent on symbiotic fungi for nutrition, are also attracted to ethanol and host volatiles that guide them to suitable substrates for fungal cultivation (Kendra et al., Reference Kendra, Montgomery, Niogret, Deyrup, Guillén and Epsky2012; Menocal et al., Reference Menocal, Cruz, Kendra, Crane, Ploetz and Carrillo2018; Ranger et al., Reference Ranger, Reding, Persad and Herms2010). In our study, xylomycetophagous species exhibited peak activity during the night and early morning, reinforcing the importance of high-humidity, shaded microhabitats for their dispersal and colonisation success (Beaver, Reference Beaver, Wilding, Collins, Hammond and Webber1989; Sittichaya et al., Reference Sittichaya, Permkam and Cognato2012; Sterkenburg et al., Reference Sterkenburg, Clemmensen, Lindahl and Dahlberg2019).
From a conservation perspective, deadwood retention is critical for supporting xylomycetophagous species, particularly in humid environments where fungal growth is optimal (Beaver, Reference Beaver, Wilding, Collins, Hammond and Webber1989; Sterkenburg et al., Reference Sterkenburg, Clemmensen, Lindahl and Dahlberg2019). Management practices should prioritise preserving decaying wood while limiting excessive logging, as the removal of moist, shaded deadwood negatively impacts saproxylic beetle populations (Brin et al., Reference Brin, Bouget, Valladares and Brustel2012; Lieutier et al., Reference Lieutier, Day, Battisti, Grégoire and Evans2004; Percel et al., Reference Percel, Laroche and Bouget2019; Sterkenburg et al., Reference Sterkenburg, Clemmensen, Lindahl and Dahlberg2019; Tsikas and Karanikola, Reference Tsikas and Karanikola2022; Vodka et al., Reference Vodka, Konvička and Čížek2009).
For phloeophagous species, understanding the circadian patterns of flight activity can offer valuable insights into their dispersal strategies and host selection behaviour. Although our study did not directly monitor pest outbreaks, the observed patterns of flight periodicity suggest that management interventions, such as selective logging or pheromone-based trapping, could be better timed to coincide with periods of peak seasonal dispersal (Chu et al., Reference Chu, Yang, Li, Liu, Liang, Wu, Wang, Zhang and Hu2021; Jones et al., Reference Jones, Shegelski, Marculis, Wijerathna and Evenden2019). To evaluate the practical relevance of such timing, future research should integrate fine-scale monitoring of flight phenology with pest population dynamics and outbreak risk assessment (Ayres and Lombardero, Reference Ayres and Lombardero2018; Illarionova et al., Reference Illarionova, Tregubova, Shukhratov, Shadrin, Kedrov and Burnaev2024; Luo et al., Reference Luo, Huang and Roques2022).
The recognition of circadian rhythm segregation between Scolytini bark and Xyleborini ambrosia beetles emphasises the importance of aligning ecological monitoring with activity windows of specific species. Ignoring such variation can lead to underestimation of species richness and distorted community assessments (Kendra et al., Reference Kendra, Montgomery, Niogret, Deyrup, Guillén and Epsky2012; Menocal et al., Reference Menocal, Cruz, Kendra, Crane, Ploetz and Carrillo2018; Seo et al., Reference Seo, Martini, Rivera and Stelinski2017). While daily flight patterns are most relevant for optimizing survey design, conservation strategies should prioritise seasonal phenology by timing interventions such as wood removal or thinning to avoid peak dispersal periods, when freshly deadwood is most susceptible to colonisation (Mezei et al., Reference Mezei, Fleischer, Rozkošný, Kurjak and Dzurenko2022; Resnerová et al., Reference Resnerová, Šenfeldová, Horák, Popelková and Holuša2023). Integrating flight activity with phylogenetic and functional data may improve both biodiversity monitoring and the ecological interpretation of bark and ambrosia beetle communities.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485325100357.
Acknowledgements
We wish to express our gratitude to Leonidas Romanos Davranoglou (University of Oxford), a native speaker who improved the language of the manuscript.
Author contributions
Dominik Stočes: writing – original draft, visualization; Jan Šipoš: conceptualization, data curation, formal analysis, visualization, writing – review and editing, supervision; Attila Balázs: investigation; methodology, writing – review and editing; Jiří Procházka: methodology, validation, writing – review and editing, David Kopr: investigation, methodology; Petr Baňař: conceptualization, investigation, methodology, project administration, resources, supervision.
Financial support
The study was supported by the Internal Grant Agency (IGA) of the Faculty of Agronomy, Mendel University in Brno, project AF-IGA2023-IP-062, ‘Circadian activity of saproxylic beetles (Insecta: Coleoptera)’. The research was also supported in part by a grant from the Ministry of Culture of the Czech Republic to the Moravian Museum, Brno (ref. MK000094862), and by Institutional Subsidy (VUKOZ-IP-00027073).
