Evaluating the seasonal efficacy of commonly used chemical treatments on Varroa destructor (Mesostigmata: Varroidae) population resurgence in honey bee colonies.

The purpose of this research was to determine how common chemical treatments influence Varroa destructor (Anderson and Trueman) population resurgence rates (defined as time posttreatment for mite populations to reach 3 mites/100 adult bees) in managed honey bee (Apis mellifera L.) colonies seasonally. We conducted 2 experiments that followed the same basic protocol to address this purpose. We established 6 treatment groups in Experiment 1 in the fall of 2014: untreated control, Apivar, Apistan, CheckMite+, ApiLifeVar, and Mite Away II applied to 10 colonies per treatment. In Experiment 2, we applied 8 chemical treatments to each of 4 seasonal (spring, summer, fall, and winter) cohorts of honey bee colonies to determine how mite populations are influenced by the treatments. The treatments/formulations tested were Apivar, Apistan, Apiguard, MAQS, CheckMite+, oxalic acid (dribble), oxalic acid (shop towels), and amitraz (shop towels soaked in Bovitraz). In Experiment 1, Apivar and Mite Away II were able to delay V. destructor resurgence for 2 and 6 months, respectively. In Experiment 2, Apiguard, MAQS, oxalic acid (dribble), and Bovitraz treatments were effective at delaying V. destructor resurgence for at least 2 months during winter and spring. Only the Bovitraz and MAQS treatments were effective at controlling V. destructor in the summer and fall. Of the 2 amitraz-based treatments, the off-label Bovitraz treatment was the only treatment to reduce V. destructor populations in every season. The data gathered through this study allow for the refinement of treatment recommendations for V. destructor, especially regarding the seasonal efficacy of each miticide and the temporal efficacy posttreatment.

Developing a method to rear Varroa destructor in vitro.

Varroa destructor is a significant mite pest of western honey bees (Apis mellifera). Developing a method to rear and maintain populations of V. destructor in vitro would provide year-round access to the mites, allowing scientists to study their biology, behavior, and control more rapidly. In this study, we determined the impact of various rearing parameters on V. destructor survival and reproduction in vitro. This was done by collecting V. destructor from colonies, placing them in gelatin capsules containing honey bee larvae, and manipulating the following conditions experimentally: rearing temperature, colony source of honey bee larva, behavioral/developmental stages of V. destructor and honey bee larva, and mite:bee larva ratio. Varroa destructor survival was significantly impacted by temperature, colony source of larvae and mite behavioral stage. In addition, V. destructor reproduction was significantly impacted by mite: larva ratio, larval developmental stage, colony source of larva, and temperature. The following conditions optimized mite survival and reproduction in vitro: using a 4:1 mite:larva ratio, beginning the study with late stage uncapped larvae, using mites collected from adult bees, maintaining the rearing temperature at 34.5° C, and screening larval colony source. Ultimately, this research can be used to improve V. destructor in vitro rearing programs.

Oxalic acid application method and treatment intervals for reduction of Varroa destructor (Mesostigmata: Varroidae) populations in Apis mellifera (Hymenoptera: Apidae) colonies.

Oxalic acid (OA) is a popular miticide used to control Varroa destructor (Mesostigmata: Varroidae) in western honey bee (Apis mellifera L.) (Hymenoptera: Apidae) colonies. Our aim was to investigate which method of OA application (dribbling, fogging, or vaporizing) was the most effective at reducing V. destructor infestations (Experiment 1) and to improve upon this method by determining the treatment interval that resulted in the greatest V. destructor control (Experiment 2). We used the product Api-Bioxal (97% OA) and maintained 40 honey bee colonies (10/treatment) in both experiments. In Experiment 1, the treatments included (i) dribbling 50 ml of 3% OA solution, (ii) vaporizing 4 g of solid OA, (iii) using an insect fogger supplied with 2.5% OA dissolved in ethyl alcohol, and (iv) an untreated control. After 3 weeks, only the vaporization method reduced V. destructor infestations (from 9.24 mites/100 bees pretreatment to 3.25 mites/100 bees posttreatment) and resulted in significantly increased brood amounts and numbers of adult bees over those of the controls. In Experiment 2, all colonies were treated with 4 applications of OA via vaporization at a constant concentration of 4 g OA/colony. In this experiment, the groups were separated by treatment intervals at either 3-, 5-, or 7-day intervals. We observed that 5- and 7-day treatment intervals significantly reduced V. destructor populations from pretreatment levels over that of the controls and 3-day intervals. Our data demonstrate the efficacy of OA in reducing V. destructor infestation, particularly vaporizing 4 g every 5-7 days as the most effective method of application.

Toxic evaluation of Proclaim Fit® on adult and larval worker honey bees.

Impacts to honey bees due to exposure to agricultural pesticides is one of the most serious threats to the beekeeping industry. Our research evaluated toxicity of the formulated insecticides Lufenuron+Emamectin benzoate (Proclaim Fit®) on the European honey bee Apis mellifera L. at field-realistic concentration (worst-case scenario). Newly emerged (≤24-h old) and forager (unknown age) worker bees were treated with the field recommended concentration of Proclaim Fit® using three routes of exposure including residual contact, oral, and spray within the laboratory. We also assessed the effects of Proclaim Fit® on the specific activity of some well-known detoxifying enzymes including α-esterase, ß-esterase, and Glutathione S-transferase (GST) in the honey bees. In addition, toxicity of the formulation was tested on 4th instar larvae within the hive. Based on estimated median survival times (MSTs), Proclaim Fit® was highly toxic to the bees, especially when applied as spray. According to our estimated relative median potency (RMP) values, newly emerged bees were 1.72× more susceptible than foragers to Proclaim Fit® applied orally. Enzyme assays revealed the considerable involvement of the enzymes, especially GST and α-esterase, in detoxification of the Proclaim Fit®, but their activities were significantly influenced by route of exposure and age of bee. Notably, Proclaim Fit® was highly toxic to 4th instar honey bee larvae. Our results generally indicate a potent toxicity of Proclaim Fit® toward honey bees. Therefore, its application requires serious consideration and adherence to strict guidelines, especially during the flowering time of crops.

Integrated Pest Management Control of Varroa destructor (Acari: Varroidae), the Most Damaging Pest of (Apis mellifera L. (Hymenoptera: Apidae)) Colonies.

Varroa destructor is among the greatest biological threats to western honey bee (Apis mellifera L.) health worldwide. Beekeepers routinely use chemical treatments to control this parasite, though overuse and mismanagement of these treatments have led to widespread resistance in Varroa populations. Integrated Pest Management (IPM) is an ecologically based, sustainable approach to pest management that relies on a combination of control tactics that minimize environmental impacts. Herein, we provide an in-depth review of the components of IPM in a Varroa control context. These include determining economic thresholds for the mite, identification of and monitoring for Varroa, prevention strategies, and risk conscious treatments. Furthermore, we provide a detailed review of cultural, mechanical, biological, and chemical control strategies, both longstanding and emerging, used against Varroa globally. For each control type, we describe all available treatments, their efficacies against Varroa as described in the primary scientific literature, and the obstacles to their adoption. Unfortunately, reliable IPM protocols do not exist for Varroa due to the complex biology of the mite and strong reliance on chemical control by beekeepers. To encourage beekeeper adoption, a successful IPM approach to Varroa control in managed colonies must be an improvement over conventional control methods and include cost-effective treatments that can be employed readily by beekeepers. It is our intention to provide the most thorough review of Varroa control options available, ultimately framing our discussion within the context of IPM. We hope this article is a call-to-arms against the most damaging pest managed honey bee colonies face worldwide.

Comparing four methods of rearing Varroa destructor in vitro.

The parasitic mite Varroa destructor Anderson and Trueman continues to devastate western honey bee (Apis mellifera L.) colonies throughout most of the world where they are managed. The development of a method to rear Varroa in vitro would allow for year-round Varroa research, rapidly advancing our progress towards controlling the mite. We created two separate experiments to address this objective. First, we determined which of four in vitro rearing methods yields the greatest number of Varroa offspring. Second, we attempted to improve the rearing rates achieved with that method. The four methods tested included (1) rearing Varroa on honey bee pupae in gelatin capsules, (2) rearing Varroa on in vitro-reared honey bees, (3) group rearing Varroa on honey bee pupae in Petri dishes, and (4) providing Varroa a bee-derived diet. The number of reproducing females and the number of fully mature offspring were significantly higher in the gelatin capsules maintained at 75% RH than in any other method. A 2 × 3 full factorial design was used to test combinations of gelatin capsule size (6 and 7 mm diameter) and relative humidity (65, 75, or 85%) on Varroa rearing success. Varroa reproduction and survival were significantly higher in 7-mm-diameter gelatin capsules maintained at 75% RH than in those maintained in 6-mm capsules and at the other humidities. By identifying factors that influence Varroa reproductive success in vitro, this work provides an important foundation for the development of future rearing protocols.

Effects of Tropilaelaps mercedesae on midgut bacterial diversity of Apis mellifera.

Tropilaelaps mercedesae is an ectoparasite of Apis mellifera in Asia and is considered a major threat to honey bee health. Herein, we used the Illumina MiSeq platform 16S rDNA Amplicon Sequencing targeting the V3-V4 regions and analysed the effects on the midgut bacterial communities of honey bees infested with T. mercedesae. The overall bacterial community in honey bees infested with T. mercedesae were observed at different developmental stages. Honey bee core intestinal bacterial genera such as Gilliamella, Lactobacillus and Frischella were detected. Tropilaelapsmercedesae infestation changed the bacterial communities in the midgut of A. mellifera. Tropilaelapsmercedesae-infested pupae had greatly increased relative abundances of Micrococcus and Sphingomonas, whereas T. mercedesae-infested 15-day-old workers had significantly reduced relative abundance of non-core microbes: Corynebacterium, Sphingomonas, Acinetobacter and Enhydrobacter compared to T. mercedesae-infested newly emerged bees. The bacterial community was significantly changed at the various T. mercedesae-infested developmental stages of A. mellifera. Tropilaelapsmercedesae infestation also changed the non-core bacterial community from larvae to newly emerged honey bees. Bacterial communities were significantly different between T. mercedesa-infested and non-mite-infested 15-day-old workers. Lactobacillus was dominant in T. mercedesae-infested 15-day-old workers compared to non-mite-infested 15-day-old workers.

The discovery of Varroa destructor on drone honey bees, Apis mellifera, at drone congregation areas.

Varroa is an external parasitic mite of honey bees and is a vector of multiple viruses that can severely weaken or cause the failure of western honey bee colonies if untreated. Effective Varroa control is dependent upon a thorough understanding of Varroa biology, including how Varroa move between host colonies. Here, we highlight that drone (male) honey bees may also play a role in Varroa dispersal. Drones were collected and the number of Varroa per 100 drones was calculated for each of five drone congregation areas (mating sites). This study is the first to confirm that drones present at drone congregation areas do carry Varroa. Further experimentation is needed to determine the extent to which drone-mediated movement may play a role in Varroa life history and/or to develop practical management strategies to limit drone-mediated movement of Varroa between honey bee hives.

The impacts of chlorothalonil and diflubenzuron on Apis mellifera L. larvae reared in vitro.

Chlorothalonil is a broad-spectrum fungicide and diflubenzuron is an insect growth regulator used to control many insect larvae feeding on agricultural, forest and ornamental plants. Honey bee larvae may be exposed to both via contaminated pollen, in the form of beebread, added to their diet by their adult nurse sisters. In this study, we determined how single (acute: 72 h mortality) and repeated (chronic: mortality until emergence as adults) exposure to chlorothalonil and diflubenzuron in their diet affected honey bee larvae reared in vitro. The tested doses of chlorothalonil (20, 100, or 200 mg/L) did not impact 72 h larval mortality acutely relative to that of the solvent control. The 72 h mortality of larvae exposed to 1.6 mg/L and higher doses of diflubenzuron acutely in their diet (47.2-63.9% mortality) was significantly higher than that of larvae fed the solvent control, with no predictable dose dependent pattern observed. In the chronic toxicity tests, consuming an artificial diet with 30 or 100 mg/L chlorothalonil and 0.8, 1.3 or 2 mg/L diflubenzuron significantly lowered the survival of honey bee larvae over that of larvae feeding on the solvent control diet. We calculated risk quotients (RQs) for both compounds using the data we generated in our experiments. Collectively, the RQs suggest that neither compound is likely to affect larval mortality directly at field relevant doses given that pollen composes only a fraction of the total larval diet. Nevertheless, our data do not preclude any sublethal effects that chronic exposure to either compound may cause.

Genotyping of whole genome amplified reduced representation libraries reveals a cryptic population of Culicoides brevitarsis in the Northern Territory, Australia.

BACKGROUND: The advent of genotyping by Next Generation Sequencing has enabled rapid discovery of thousands of single nucleotide polymorphism (SNP) markers and high throughput genotyping of large populations at an affordable cost. Genotyping by sequencing (GBS), a reduced representation library sequencing method, allows highly multiplexed sequencing of genomic subsets. This method has limitations for small organisms with low amounts of genomic DNA, such as the bluetongue virus (BTV) vectors, Culicoides midges. RESULTS: This study employed the GBS method to isolate SNP markers de novo from whole genome amplified Culicoides brevitarsis genomic DNA. The individuals were collected from regions representing two different Australian patterns of BTV strain distribution: the Northern Territory (NT) and the east coast. We isolated 8145 SNPs using GBS. Phylogenetic analysis conducted using the filtered 3263 SNPs revealed the presence of a distinct C. brevitarsis sub-population in the NT and this was confirmed by analysis of mitochondrial DNA. Two loci showed a very strong signal for selection and were unique to the NT population. Bayesian analysis with STRUCTURE indicated a possible two-population cluster. CONCLUSIONS: The results suggest that genotyping vectors with high density markers in combination with biological and environmental data is useful. However, more extensive sampling over a wider spatial and temporal range is needed. The presence of sub-structure in populations and loci under natural selection indicates the need for further investigation of the role of vectors in shaping the two Australian systems of BTV transmission. The described workflow is transferable to genotyping of small, non-model organisms, including arthropod vectors of pathogens of economic and medical importance.

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.

Evaluating the Efficacy of Active Ingredients Used in Roach Baits against Small Hive Beetle (Aethina tumida) and Their Safety to Honey Bees (Apis mellifera).

Beekeepers need new treatment options for controlling small hive beetles (Aethina tumida), a devastating honey bee (Apis mellifera) pest. For many years, commercial beekeepers in the U.S. have used gel roach baits off-label as a method for treating SHBs. Herein, we evaluated the acute toxicity of active ingredients commonly found in gel roach baits, including abamectin, clothianidin, hydramethylnon, fipronil, and indoxacarb through topical and oral routes of exposure against SHBs and honey bees. Additionally, coumaphos, the active ingredient of the only registered in-hive control treatment for SHBs, was evaluated to provide a comparison to the gel roach bait active ingredients. Fipronil was the most toxic compound to SHBs topically (LD50 = 0.23 ng/SHB) and through pollen (LC50 = 0.06 µg/g pollen). Fipronil (LD50 = 0.31 ng/honey bee) had a selectivity ratio of 1.3, suggesting that it is more toxic to SHBs than it is to honey bees, but only to a small degree. Abamectin, clothianidin, hydramethylnon, and indoxacarb had a higher toxicity to honey bees than to SHBs through topical exposure. Our results suggest that gel roach baits and their active ingredients are toxic to honey bees and pose a serious risk to colony safety if used as in-hive treatments.

Profiling age and body fluid DNA methylation markers using nanopore adaptive sampling.

DNA methylation plays essential roles in regulating physiological processes, from tissue and organ development to gene expression and aging processes and has emerged as a widely used biomarker for the identification of body fluids and age prediction. Currently, methylation markers are targeted independently at specific CpG sites as part of a multiplexed assay rather than through a unified assay. Methylation detection is also dependent on divergent methodologies, ranging from enzyme digestion and affinity enrichment to bisulfite treatment, alongside various technologies for high-throughput profiling, including microarray and sequencing. In this pilot study, we test the simultaneous identification of age-associated and body fluid-specific methylation markers using a single technology, nanopore adaptive sampling. This innovative approach enables the profiling of multiple CpG marker sites across entire gene regions from a single sample without the need for specialized DNA preparation or additional biochemical treatments. Our study demonstrates that adaptive sampling achieves sufficient coverage in regions of interest to accurately determine the methylation status, shows a robust consistency with whole-genome bisulfite sequencing data, and corroborates known CpG markers of age and body fluids. Our work also resulted in the identification of new sites strongly correlated with age, suggesting new possible age methylation markers. This study lays the groundwork for the systematic development of nanopore-based methodologies in both age prediction and body fluid identification, highlighting the feasibility and potential of nanopore adaptive sampling while acknowledging the need for further validation and expansion in future research.

Testing new compounds for efficacy against Varroa destructor and safety to honey bees (Apis mellifera).

BACKGROUND: Varroa destructor is among the greatest threats to honey bee health worldwide. Acaricides used to control Varroa are becoming increasingly ineffective due to resistance issues, prompting the need for new compounds that can be used for control purposes. Ideally, such compounds would exhibit high toxicity to Varroa while maintaining relatively low toxicity to bees and beekeepers. We characterized the lethal concentrations (LC50 ) of amitraz, matrine, FlyNap®, the experimental carbamates 2-((2-ethylbutyl)thio)phenyl methylcarbamate (1) and 2-(2-ethylbutoxy)phenyl methylcarbamate (2), and dimethoate (positive control) for Varroa using a glass vial assay. The test compounds also were applied to honey bees using an acute contact toxicity assay to determine the adult bee LD50 for each compound. RESULTS: Amitraz was the most toxic compound to Varroa, but carbamate 2 was nearly as active (within 2-fold) and the most selective due to its lower bee toxicity, demonstrating its promise as a Varroa control. While carbamate 1 was less toxic to honey bees than was amitraz, it was also 4.7-fold less toxic to the mites. Both matrine and FlyNap® were relatively ineffective at killing Varroa and were moderately toxic to honey bees. CONCLUSION: Additional testing is required to determine if carbamate 2 can be used as an effective Varroa control. As new chemical treatments are identified, it will be necessary to determine how they can be utilized best alongside other control techniques as part of an integrated pest management program. © 2021 Society of Chemical Industry.

Helicoverpa genus on the edge of the continental U.S.: Flight phenology, analysis of hybrid presence, and insecticide performance in high-input field crops in Puerto Rico.

The genus Helicoverpa includes several agricultural pests globally. Helicoverpa armigera was reported in several countries in South America in 2013, and in Puerto Rico, in 2014. This territory is considered an agricultural hub, with a high-input system of seed production in the southern region of the island, and also at the edge of the continental U.S. Possible natural dispersion of populations of H. armigera from the Caribbean or other Central American regions poses a continuing risk to the U.S. This study was performed during the post-detection scenario of H. armigera in Puerto Rico, from 2018 to 2021. A year-round pheromone trapping program of adult males indicated an increase in the population from October to March and differences in the occurrence of Helicoverpa spp. between the municipalities Juan Diaz and Salinas. The proportion of H. armigera/H. zea and detection of congeneric hybrids between these species were assessed based on genital morphology and DNA analysis. Interestingly, neither H. armigera nor expected hybrids were detected in the present study. The susceptibility of H. zea populations to the insecticides Spinetoram, Emamectin benzoate, Chlorantraniliprole, and Esfenvalerate was assessed, and an overall significant effect of insecticide susceptibility was detected. Chlorantraniliprole and Emamectin benzoate had the highest efficacy. These results contribute to the Integrated Pest Management and Insect resistance management programs to Helicoverpa spp. in Puerto Rico. In addition, provide validated information to be considered in mitigation plans, in the scenario of an invasion of H. armigera in the continental U.S.

Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing.

DNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools have a tradeoff between false positives and false negatives and present a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools, including a consensus approach, METEORE ( https://github.com/comprna/METEORE ), based on the combination of the predictions from two or more tools that shows improved accuracy over individual tools. Snakemake pipelines are also provided for reproducibility and to enable the systematic application of our analyses to other datasets.

Evaluating the Efficacy of Oxalic Acid Vaporization and Brood Interruption in Controlling the Honey Bee Pest Varroa destructor (Acari: Varroidae).

A successful Integrated Pest Management approach to Varroa destructor Anderson and Trueman control in managed colonies of western honey bees Apis mellifera Linnaeus (Hymenoptera: Apidae) must be an improvement over conventional control methods and include cost-effective treatments that can be readily employed by beekeepers. Herein, we tested the efficacy of oxalic acid (OA) vaporization and brood interruption as Varroa controls. Sixty experimental colonies were randomly assigned to one of six treatment groups with 10 colonies per group. The six treatments were: 1) OA applied once, 2) OA applied three times, 3) brood interruption, 4) OA applied once + brood interruption, 5) OA applied three times + brood interruption, and 6) no OA or brood interruption. The OA was applied via vaporization, with each application being 1 g OA applied through the hive entrance (label rate), on the bottom board. Brood interruption was accomplished by caging a colony's queen in a queen cage for a period of 24 d. An additional 10 colonies were treated with amitraz (Apivar - positive control). Varroa levels were estimated before, during, and after treatment applications using sticky boards left in colonies for 3 d. Our data suggest that queen caging to achieve brood interruption during the fall season can negatively impact colony strength and survival. We observed high colony mortality in some treatments, despite diligent colony management to alleviate the side effects of the treatments. Colonies treated with amitraz were healthier and had better survival than those treated with OA vaporization. In conclusion, OA and/or brood interruption did not provide sufficient Varroa control.

Modifying culture conditions in chemical library screening identifies alternative inhibitors of mycobacteria.

In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection.

Ontogenetic colour changes in an insular tree species: signalling to extinct browsing birds?

* Animals often use colours to hide from predators (crypsis) or advertise defences (aposematism), but there is little evidence for colour-based defence in plants. * Here, we test whether ontogenetic changes in leaf colour of lancewood (Pseudopanax crassifolius) may have been part of a defensive strategy against flightless browsing birds called moa, which were once the only large herbivores in New Zealand. We tested this hypothesis by conducting spectrographic measurements on different-sized plants grown in a common garden. We also compared these results with observations on a closely related, derived species that evolved in the absence of moa on the Chatham Islands. * Spectrographic analyses showed that birds would have difficulty distinguishing seedling leaves against a background of leaf litter. Conversely, brightly coloured tissues flanking spines on sapling leaves are highly conspicuous to birds. Once above the reach of the tallest known moa, adults produce leaves that are typical in appearance to adult leaves. The Chatham Island species lacks ontogenetic colour changes entirely. * Overall, the results indicate that P. crassifolius goes through a remarkable series of colour changes during development, from cryptically coloured seedlings to aposematically coloured saplings, which may have formed a defensive strategy to protect against giant browsing birds.

Pervasive admixture between eucalypt species has consequences for conservation and assisted migration.

Conservation management often uses information on genetic population structure to assess the importance of local provenancing for ecological restoration and reintroduction programs. For species that do not exhibit complete reproductive isolation, the estimation of population genetic parameters may be influenced by the extent of admixture. Therefore, to avoid perverse outcomes for conservation, genetically informed management strategies must determine whether hybridization between species is relevant, and the extent to which observed population genetic patterns are shaped by interspecific versus intraspecific gene flow. We used genotyping by sequencing to identify over 2,400 informative single nucleotide polymorphisms across 18 populations of Eucalyptus regnans F. Muell., a foundation tree species of montane forests in south-eastern Australia. We used these data to determine the extent of hybridization with another species, Eucalyptus obliqua L'Hér., and investigate how admixture influences genetic diversity parameters, by estimating metrics of genetic diversity and examining population genetic structure in datasets with and without admixed individuals. We found hybrid individuals at all sites and two highly introgressed populations. Hybrid individuals were not distributed evenly across environmental gradients, with logistic regression identifying hybrids as being associated with temperature. Removal of hybrids resulted in increases in genetic differentiation (F ST), expected heterozygosity, observed heterozygosity and the inbreeding coefficient, and different patterns of isolation by distance. After removal of hybrids and introgressed populations, mountain ash showed very little population genetic structure, with a small effect of isolation by distance, and very low global F ST(0.03). Our study shows that, in plants, decisions around provenancing of individuals for restoration depend on knowledge of whether hybridization is influencing population genetic structure. For species in which most genetic variation is held within populations, there may be little benefit in planning conservation strategies around environmental adaptation of seed sources. The possibility for adaptive introgression may also be relevant when species regularly hybridize.