Pine Processionary Research Articles

Shifted phenology in the pine processionary moth affects the outcome of tree–insect interaction

In the Mediterranean and temperate regions, an increase in the frequency and intensity of drought events has been recorded, probably due to climate change. In consequence, trees will more frequently experience hydric stress, a condition that can be expected to affect insect-tree interactions, while adaptation mechanisms may be further in course. The effect of tree water stress on the performance of two allochronic populations of Thaumetopoea pityocampa was here studied. Namely, we compared a unique population of this insect, in which the larvae develop in the summer (SP), with the typical population having winter larval development (WP), to test the adaptation hypothesis to host plant status. Larvae of each population were fed on needles of young potted Pinus pinaster plants under two water supply regimes: (i) well-watered (control) and (ii) subjected to 3 months of drought stress. Compared to control, stressed plants had higher amounts of soluble sugars, phenols, and higher C/N ratio, whereas water content and chlorophylls concentrations were lower. In general, T. pityocampa larvae had lower performances on water-stressed plants, as shown by lower survival rates, lower needle consumption, and longer development times. Yet, the detrimental effects of tree stress were only significant for the WP larvae, while SP larvae were able to overcome such conditions. Results demonstrate that tree water stress can negatively affect T. pityocampa populations. Furthermore, the evidence is also provided that responses to the physiological condition of the host trees may occur at the population level, as a result of adaptation mechanisms driven by climate change.

Dataset of occurrence and incidence of pine processionary moth in Andalusia, south Spain.

This dataset provides information about infestation caused by the pine processionary moth (Thaumetopoeapityocampa ([Denis & Schiffermüller], 1775)) in pure or mixed pine woodlands and plantations in Andalusia. It represents a long-term series (1993–2015) containing 81,908 records that describe the occurrence and incidence of this species. Data were collected within a monitoring programme known as COPLAS, developed by the Regional Ministry of Environment and Territorial Planning of the Andalusian Regional Government within the frame of the Plan de Lucha Integrada contra la Procesionaria del Pino (Plan for Integrated Control Against the Pine Processionary Moth).In particular, this dataset includes 4,386 monitoring stands which, together with the campaign year, define the dataset events in Darwin Core Archive. Events are related with occurrence data which show if the species is present or absent. In turn, the event data have a measurement associated: degree of infestation.

Forests and allergies

Many research papers have addressed the beneficial qualities of forests in promoting mental and physical health. However, we should also be clear that forest environments could have detrimental effects. Some of them, which are of allergic nature, have hitherto been largely neglected. A comprehensive literature search has allowed us to achieve an initial synthesis on this matter. The emphasis was on temperate forests, especially on French forests. Anemophilous pollen from deciduous trees is the major factor causing respiratory allergies in connection with forests. The risks are focused between mid-winter and late spring. Ash, hornbeam and birch pollen and, more incidentally, oak and beech pollen induce allergic reactions not only in the forests, but also at great distances. As a result of its high humidity and because decaying organic matter is very abundant, the forest environment presents also a breeding ground for mould growth. Accordingly, fungal spores can lead to allergic manifestations recalling pollinosis. Lastly, the "other" allergies mainly include contact dermatitis symptoms, which affect wood workers first and foremost. However, the pine processionary moth, whose zone of infestation is rapidly spreading northwards and whose setae contain several relevant allergens, represents one particular case. Allergic reactions observed in forests or originating in forests are sufficiently frequent to justify to set up a wide-reaching system for aerobiological and epidemiological monitoring.

Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT50 and LT90 of 1 × 106 concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60 days, respectively in leaves application. The LT50 and LT90 of the 1 × 108 concentration of the same isolate were 2.50 and 2.95 days for direct application, and 2.98 and 3.74 days for leaves application. The LT50 of insect and leaves application for 1 × 106 of B. bassiana were 3.75 and 3.49 days, respectively. The LT90 of same concentration for insect application was 4.48 days, while LT90 for leaves application was 4.63 days. Similarly, LT50 of insect and leaves application for 1 × 108 of B. bassiana were 3.03 and 3.31 days, while LT90 were 3.68 and 4.29 days, respectively. Approximate 100% mycosis was observed in all treatments.

Quantifying pine processionary moth defoliation in a pine-oak mixed forest using unmanned aerial systems and multispectral imagery.

Pine processionary moth (PPM) feeds on conifer foliage and periodically result in outbreaks leading to large scale defoliation, causing decreased tree growth, vitality and tree reproduction capacity. Multispectral high-resolution imagery acquired from a UAS platform was successfully used to assess pest tree damage at the tree level in a pine-oak mixed forest. We generated point clouds and multispectral orthomosaics from UAS through photogrammetric processes. These were used to automatically delineate individual tree crowns and calculate vegetation indices such as the normalized difference vegetation index (NDVI) and excess green index (ExG) to objectively quantify defoliation of trees previously identified. Overall, our research suggests that UAS imagery and its derived products enable robust estimation of tree crowns with acceptable accuracy and the assessment of tree defoliation by classifying trees along a gradient from completely defoliated to non-defoliated automatically with 81.8% overall accuracy. The promising results presented in this work should inspire further research and applications involving a combination of methods allowing the scaling up of the results on multispectral imagery by integrating satellite remote sensing information in the assessments over large spatial scales.

Mistletoe generates non‐trophic and trait‐mediated indirect interactions through a shared host of herbivore consumers

AbstractIndirect interactions emerge among a wide range of herbivores sharing the same plant resource. Consumers usually belong to different trophic guilds, from folivores and sapsuckers to parasitic plants. We propose that mistletoes parasitizing pines could play a key role acting as herbivores on host pines and coming indirectly into competition with other herbivores feeding on the same host. Changes caused by mistletoes on its host have been well studied, but its effects running across trophic webs remain unrevealed. In this study, we investigate the effect of European mistletoe (Viscum album subsp. austriacum) on the host‐feeding herbivores via trait‐mediated indirect interactions (TMIIs) across their shared pine host (Pinus nigra subsp. salzmannii). We performed field and laboratory experiments, and analyzed the net effect of different mistletoe parasite loads on three host‐phytophagous species: the sapsucker Cinara pini (Aphididae), the winter folivore Thaumetopoea pityocampa (Thaumetopoeidae), and the summer folivore Brachyderes sp. (Curculionidae), all being members of different functional feeding groups (FFGs). We summarize the mistletoe–host–herbivore interactions by means of a TMII, where mistletoe parasitism causes non‐trophic links and detrimental indirect interactions on pine‐feeding herbivores across its shared host, suggesting a worsening of host quality as food. These indirect interactions vary according to three parameters. First, the intensity has a non‐proportional relation with parasite load, showing an impact threshold on highly parasitized pines. Second, the movement capacity of insect herbivores determines their response, by decreasing the abundance of herbivores with low movement ability (aphids and pine processionary caterpillars) while altering the behavior (plant selection) of more mobile herbivores (pine weevils). Finally, FFG determines the intensity of mistletoe parasitism effects, folivores being more responsive than sapsuckers. Overall, mistletoe generates non‐trophic interaction linkages in the forest able to modify community structure by becoming a nexus of the entire herbivore community of the pine canopy.

Winter temperature predicts prolonged diapause in pine processionary moth species across their geographic range.

Prolonged diapause occurs in a number of insects and is interpreted as a way to evade adverse conditions. The winter pine processionary moths (Thaumetopoea pityocampa and Th. wilkinsoni) are important pests of pines and cedars in the Mediterranean region. They are typically univoltine, with larvae feeding across the winter, pupating in spring in the soil and emerging as adults in summer. Pupae may, however, enter a prolonged diapause with adults emerging one or more years later. We tested the effect of variation in winter temperature on the incidence of prolonged diapause, using a total of 64 individual datasets related to insect cohorts over the period 1964–2015 for 36 sites in seven countries, covering most of the geographic range of both species. We found high variation in prolonged diapause incidence over their ranges. At both lower and upper ends of the thermal range in winter, prolonged diapause tended to be higher than at intermediate temperatures. Prolonged diapause may represent a risk-spreading strategy to mitigate climate uncertainty, although it may increase individual mortality because of a longer exposure to mortality factors such as predation, parasitism, diseases or energy depletion. Climate change, and in particular the increase of winter temperature, may reduce the incidence of prolonged diapause in colder regions whereas it may increase it in warmer ones, with consequences for population dynamics.

Termination of pupal diapause in the pine processionary moth Thaumetopoea pityocampa

AbstractDiapause development is a complex process involving several eco‐physiological phases. Understanding these phases, especially diapause termination, is vital for interpreting the life history of many insect species and for developing suitable predictive models of population dynamics. The pine processionary moth is a major defoliator of pine and a vertebrate health hazard in the Mediterranean region. This species can display either univoltine or semivoltine development, with a pupal diapause extending from a few months to several years, respectively. Although the ecological and applied importance of diapause is acknowledged, its physiological regulation in either case remains obscure. In the present study, we characterize pre‐termination, termination and post‐termination phases of pupae developing as univoltine or remaining in prolonged diapause. Changes in metabolic activity are monitored continuously using thermocouples, comprising a novel method based on direct calorimetry, and periodically by use of O2 respirometry. The two methods clearly detect diapause termination in both types of pupae before any visible morphological or behavioural changes can be observed. Univoltine individuals are characterized by an increase in metabolic activity from pre‐termination through to termination and post‐termination, ultimately resulting in emergence. Remarkably, a synchronous termination is observed in individuals that enter prolonged diapause instead of emerging; however, in these pupae, the increased metabolic activity is only transient. The present study represents a starting point toward understanding the eco‐physiology of diapause development processes in the pupae of the pine processionary moth.

Identification and Quantification of the Most Abondant Hemocytes in the Pine Processionary Caterpillar; ThaumetopoeaPityocampa (Notodontidae)

Thaumetopoeapityocampa is an important pine pest in the Mediterranean basin and central Europe. The insecte is a butterfly, whose larvae are called pine processionary caterpillars. To understand the resistance mechanism of the insect in order to proceed of its control and to diminuate their effect in nature, it was necessary to study of the immune system and reactions of the larva in different stages. The aim of our work is to identify the hemocytes formula of the caterpillar during the larval stages L2, L3, and L4, as well as the quantification of the different cells during each stage. After extraction of the hemolymph by centrifugation, the cells were placed in culture medium and then incubated. Microscopic observation has shown that prohemocytes population appear early in hemolymph, they differentiate into plasmatocytes and granulocytes during the advanced stages. However, the quantification process (THC), carried out in a Malassez counting chamber has shown that granulocytes are the most abundant cell population in the hemolymph of the insect larvae. To investigate the role of hemocytes in immune responses, we have co-incubate T. pityocampa cells with bacteria, entomopathogenic nematodes, and synthetic beads. Both humoral and cellular encapsulation processes have been observed early in larval stages, all hemocytes seem to be involved in the formation of nodules and capsules against bacteria and microbeads. At the opposite, entomopathogenic nematodes (Steinernemafeltiae) were not recognized and encapsulated, but their presence can strongly damage host hemocytes.

We Are What We Eat: A Stoichiometric and Ecometabolomic Study of Caterpillars Feeding on Two Pine Subspecies of Pinus sylvestris.

Many studies have addressed several plant-insect interaction topics at nutritional, molecular, physiological, and evolutionary levels. However, it is still unknown how flexible the metabolism and the nutritional content of specialist insect herbivores feeding on different closely related plants can be. We performed elemental, stoichiometric, and metabolomics analyses on leaves of two coexisting Pinus sylvestris subspecies and on their main insect herbivore; the caterpillar of the processionary moth (Thaumetopoea pityocampa). Caterpillars feeding on different pine subspecies had distinct overall metabolome structure, accounting for over 10% of the total variability. Although plants and insects have very divergent metabolomes, caterpillars showed certain resemblance to their plant-host metabolome. In addition, few plant-related secondary metabolites were found accumulated in caterpillar tissues which could potentially be used for self-defense. Caterpillars feeding on N and P richer needles had lower N and P tissue concentration and higher C:N and C:P ratios, suggesting that nutrient transfer is not necessarily linear through trophic levels and other plant-metabolic factors could be interfering. This exploratory study showed that little chemical differences between plant food sources can impact the overall metabolome of specialist insect herbivores. Significant nutritional shifts in herbivore tissues could lead to larger changes of the trophic web structure.

Réactions allergiques et dermatites à Thaumetopoea pityocampa. Revue critique à propos d’un cas

A case of acute pruritic dermatitis occurring in an adult provided us with an opportunity to review the symptoms associated with pine processionary caterpillars. While out picking mushrooms in a pine forest, the patient developed pruritus (some 30minutes after entering the pine forest in which certain trees were infested with processionary caterpillars) and a papular rash occurred several hours later. The diagnosis was based on patient history, which included previous episodes, the appearance of the lesions, and negative allergy tests for allergens likely to occur in this biotope. Our case report is accompanied by a critical literature review. Allergy to pine processionary caterpillars is not associated with allergy to adult butterflies (imago) of the species. The allergenic protein (thaumetopoein) causes local and regional allergic reactions on exposed areas of skin and in conjunctival and respiratory mucosa (nasal, tracheal, bronchial). The major allergen is Tha p 2; a recombinant allergen, r Tha p 2, exists. Individuals allergic to pine processionary caterpillars may include professionals (forest workers) and non-professionals (walkers, holidaymakers, mushroom gatherers, etc.). Symptoms involve irritation (strophulus-like rash associated with urticating caterpillar hairs) or they may be IgE-dependent (conjunctivitis, rhinitis, asthma, and even anaphylaxis). Diagnosis is made primarily on clinical grounds. Prevention requires both individual and collective measures (nationally coordinated combat against the propagation of processionary caterpillars). It should also be noted that animals (particularly dogs) may also be affected, with potentially extremely serious symptoms in the event of ingestion.

Plant phenotype affects oviposition behaviour of pine processionary moth and egg survival at the southern edge of its range

Morphological traits of Aleppo pine (Pinus halepensis) needles in native and planted stands at the southern edge of its range influence oviposition behaviour of the pine processionary moth (Thaumetopoea pityocampa). Extreme environmental conditions result in a reduction in needle size of the host plant which corresponds to a lower rate of fecundity in the moth. Our results showed that egg batches were laid closer to the needle buds, especially on native trees with short needles, and this resulted in increased egg mortality. Number of eggs laid by the female moths did not vary between native and planted stands, nor did the number of parasitized eggs of the two common Hymenopteran parasitoids, Baryscapus servadeii and Ooencyrtus pityocampae. The observed differences in egg mortality are likely due to abiotic factors associated with the position of the egg batch on the needles. Thaumetopoea pityocampa eggs require a thermal niche for optimal development, and further measurements are required to determine the thermal threshold of these eggs. Understanding the role of climate in T. pityocampa populations will be an important factor for the survival of the Aleppo pine forests and protecting it from desertification.

Mass‐trapping trials for the control of pine processionary moth in a pine woodland recreational area

AbstractThe pine processionary moth, Thaumetopoea pityocampa, causes serious defoliation to Cedrus, Pinus and Pseudotsuga trees, as well as health problems in humans, pets and farm animals due to their urticating hairs. Environmentally friendly strategies for the management of T. pityocampa include: removal of egg batches, removal of nests, trapping of migrant larvae, spraying microbial or Insect Growth Regulator (IGR) insecticides and biocontrol, as well as pheromone‐based adult trapping and mating‐disruption. In the present paper, results on innovative technology for the control of T. pityocampa infestation using pheromone mass‐trapping are reported. Two 1‐ha plots were identified in the study area (central‐south Italy), a pine woodland recreational site growing Pinus halepensis. In the experimental plot (MT‐plot), 10 G‐traps (funnel trap type) baited with (Z)‐13‐hexadecen‐11‐ynyl acetate sex pheromone component were placed for mass‐trapping of adults; the other plot was used as a control‐plot (C‐plot). The T. pityocampa population was monitored using the two central traps in the MT‐plot and two traps positioned in the C‐plot. In addition, the winter nests made by T. pityocampa larvae overwintering on pine trees were counted. After 2 years of mass‐trapping, the number of adults trapped by the monitoring pheromone traps decreased in the MT‐plot, but not in the C‐plot, whereas the number of nests decreased in both plots. Statistical results highlighted significant differences in trap catches between the two plots but not between years. In the case of nests, differences among plots were not significant before the mass‐trapping, but significant after 1‐year treatment. According to our results, the mass‐trapping technique is able to reduce T. pityocampa infestations. This pheromone method can be applied in combination with other control systems in the context of integrated pest management in recreational areas.

Calibrating the Severity of Forest Defoliation by Pine Processionary Moth with Landsat and UAV Imagery.

The pine processionary moth (Thaumetopoea pityocampa Dennis and Schiff.), one of the major defoliating insects in Mediterranean forests, has become an increasing threat to the forest health of the region over the past two decades. After a recent outbreak of T. pityocampa in Catalonia, Spain, we attempted to estimate the damage severity by capturing the maximum defoliation period over winter between pre-outbreak and post-outbreak images. The difference in vegetation index (dVI) derived from Landsat 8 was used as the change detection indicator and was further calibrated with Unmanned Aerial Vehicle (UAV) imagery. Regression models between predicted dVIs and observed defoliation degrees by UAV were compared among five selected dVIs for the coefficient of determination. Our results found the highest R-squared value (0.815) using Moisture Stress Index (MSI), with an overall accuracy of 72%, as a promising approach for estimating the severity of defoliation in affected areas where ground-truth data is limited. We concluded with the high potential of using UAVs as an alternative method to obtain ground-truth data for cost-effectively monitoring forest health. In future studies, combining UAV images with satellite data may be considered to validate model predictions of the forest condition for developing ecosystem service tools.

Variation du taux d’infestation par Thaumetopoea pityocampa du pin d’Alep : effet sur les paramètres dendrométriques dans les forêts de la région de Djelfa (Atlas saharien, Algérie)

The forests of the Saharan Atlas represent the southern edge of the natural range of the Aleppo pine (Pinus halepensis Mill.). These are exposed to climatic stress in relation to drought and attacks of the pine processionary moth (Thaumetopoea pityocampa Schiff., denoted by CP in the French text). The purpose of our work is to study the variation of the infestation rate by the pine processionary moth and its density with climatic factors and dendrometric parameters. The infestation rate (7.82%) by the pine processionary moth and its density (2.74±2.61nests/tree) were lower in our pine forests than in the northern ones. It appeared that the attacks of the moth increase mainly with the number of days of frost and heatwave. The dendrometric parameters were negatively correlated with the moth's density per tree. On the other hand, the latter was positively correlated with the defoliation rate. The results were discussed in a biogeographic framework in the light of the current knowledge of the distribution of the pine processionary moth in the Aleppo pine, in relation with the climatic conditions.

Efficiency of funnel traps in controlling pine processionary moth

Changing climate conditions have allowed the pine processionary moth [Thaumetopoea pityocampa (Den. & Schiff. 1775) (Lepidoptera: Notodontidae: Thaumetopoeidae) and T. wilkinsoni Tams. 1925 (Lepidoptera: Notodontidae: Thaumetopoeidae)] which is one of the most important insects in Mediterranean forest ecosystems, to distribute to larger areas. More effective control of this moth is necessary to prevent its expansion to northern and higher altitudes. Mechanical, biological, chemical, and biotechnical control methods are currently being applied. These methods are either not effective alone, are very expensive, have low impact, or are problematic with respect to the environmental and human health. The purpose of this study was to develop a new approach to control the pine processionary moth and to develop an economical, ecological, and effective trap exploiting its behavioural characteristics. Traps attached to tree trunks caused mature larvae to be trapped as they landed on the soil in convoys to become pupa. In 2013, the average number of nests per tree was 4.7, while it decreased to 2.0 in 2015; furthermore, the average number of larvae decreased from 265 to 57. As a result of this 3-year study, it was found that these traps could catch nearly all of the adult larvae and reduce the moth population; it was also evident that these traps can be used as a new alternative method to control the pine processionary moth, alone or in combination with other methods.

Geographic variation in growth, survival, and susceptibility to the processionary moth (Thaumetopoea pityocampa Dennis & Schiff.) of Pinus halepensis Mill. and P. brutia Ten.: results from common gardens in Morocco

AbstractKey messageAdaptation ofPinus brutiaandPinus halepensisto harsh Moroccan environments varied considerably among populations, with variation following marked geographic clines.ContextMediterranean pines are drought tolerant species that play relevant ecological and economic roles in North Africa. Mediterranean pines harbor huge intraspecific variation in adaptive traits.AimsExploring the relative performance of different seed sources and analyzing the genotype by environment (G × E) interaction becomes, thus, essential for maximizing the efficiency of any reforestation program.MethodsWe present the results of a comprehensive quantitative analysis of growth, survival, and resistance to the processionary moth of 57 populations of P. halepensis Mill. and P. brutia Ten. tested under severe drought conditions in two contrasting Moroccan test sites differing in water regime, soil, and physiographic conditions.ResultsThe results indicated the existence of considerable variation between species and among populations within species in growth, survival, and susceptibility to the processionary moth (Thaumetopoea pityocampa Dennis & Schiff.), but low plasticity across sites, despite the apparently large environmental differences between them. On average, P. brutia performed better than P. halepensis. The G × E interaction was significant for all traits and ages, but an in-depth analysis revealed that the main source of the interaction was due to large differences in residual variances across sites, while the relative population ranks remained highly consistent across sites.ConclusionGrowth and survival of Aleppo pine populations followed well-defined geographical clines, with populations from the Eastern range of the distribution, particularly those of Greece, showing outstanding performance. The two local Aleppo pine populations were, however, also among the best-growing and surviving populations. Superiority of Eastern Aleppo pine populations was not considered high enough to recommend their use in reforestation programs. Aiming to preserve local genetic resources, local seed sources are preferred for reforestation in the dry areas of Morocco. Planting middle to high elevation Turkish populations of the non-native P. brutia is also an alternative, especially in more mesic sites. The reduced G × E suggests that the above recommendations may be valid, even in the context of future climate change.

Prepupal diapause synchronizes adult emergence in the pine processionary moth Thaumetopoea pityocampa (Lepidoptera: Notodontidae)

AbstractInsects with short‐lived adults must synchronize their emergence to maximize fitness. However, pre‐imaginal development time often varies among individuals as a result of exposure to varied abiotic and biotic factors; therefore, mechanisms adjusting pre‐imaginal development time are expected. Larvae of the pine processionary moth feed throughout the winter and conclude their development with characteristic processions in spring, leaving the trees to pupate in soil. The procession period can be as long as 2 months in some regions because of prior desynchronization of larval colonies, whereas the emergence period of the adults in summer remains short (less than 1 month). Through weekly sampling of larvae leaving trees to pupate in soil and subsequent rearing under field and laboratory conditions, it was observed that early prepupae waited longer than late prepupae before moulting into pupa. The differential duration of the prepupal stage was independent of temperature conditions and allowed resynchronization of colonies and overlapping emergences. The prepupal phase therefore appears to be critical for understanding the regulation of adult emergence of this important pest insect.

Determining the edge effect of pine processionary moth (Thaumetopoea pityocampa) in its horizontal distribution in the stand

Pine processionary moth is one of the most important forest pests in the Mediterranean Basin. There is need to explore its distribution behaviour to undertake effective control and determine the edge effect of its horizontal distribution in the stand. Five trial sites damaged by the insect were selected and traps installed in all trees in the sites. The number of nests and larvae was counted and diameter increment cores were taken. The distance to the edge of the stand revealed that there was no statistical difference in the number of nests and larvae up to 25 m while there was a statistically significant difference (0.5%) at > 25 m. There was a 2-fold difference in the density of nests between 0 and 25 m and > 25 meters, and a 3.8-fold difference in the number of larvae. The effect of pine processionary moth on tree diameter was not significant between trees at the edge of the stand and those in the stand.

Revisiting the Distribution of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) and T. pityocampa ENA Clade in Greece

In the present work, we sampled individuals of the processionary pine moth, Thaumetopoea pityocampa (Denis and Schiffermüller; Lepidoptera: Notodontidae) from different areas of Greece between 2014 and 2016. These samples were sequenced for a 760-bp long mtDNA COI locus and the haplotypes retrieved clearly showed that the occurrence of T. pityocampa in Greece is being considerably restricted, with only 8 individuals out of the 221 exhibiting T. pityocampa haplotypes and the rest being identified as T. pityocampa ENA clade haplotypes. To that, one haplotype in particular exhibited the highest abundance and broadest geographic distribution, occurring both in mainland and on islands. Our data suggest a rather recent and rapid population expansion of the ENA clade in Greece and a concomitant recent displacement of T. pityocampa. It thus seems that the relation between T. pityocampa and T. pityocampa ENA clade needs to be further and thorough analyzed before the taxonomic status of T. pityocampa ENA clade can be concluded with confidence.