Occurrence of the Invasive Bark Beetle Phloeosinus aubei on Common Juniper Trees in the Czech Republic

The small cypress bark beetle Phloeosinus aubei is considered an invasive pest in several central European countries, and we have determined its current distribution on common juniper trees (Juniperus communis) in the Czech Republic. The results indicated that P. aubei is widely distributed in the country but is more common in the east than in the west. The beetle was mainly detected on older, damaged trees and on stems with diameters > 3 cm. The apparently widespread and abundant populations of P. aubei could explain infestations of the beetle on Thuja spp. and Juniperus spp. in gardens (three confirmed cases during the last 10 years). We consider P. aubei to be a potential pest on older, naturally occurring J. communis in protected areas where its population density could increase on weakened and damaged trees. We suggest that P. aubei can be monitored via simple inspection of dying and dead J. communis trees in the field.


Introduction
Bark beetles (Coleoptera: Curculionidae, Scolytinae) are among the most damaging pests of trees, because the adults and larvae tunnel beneath the bark; such tunnelling can eventually girdle and kill hosts.Most bark beetles breed in recently felled trees and especially in trees weakened by drought, defoliation by other insects, or diseases [1][2][3].Many bark beetles are invasive and are easily transported with wood products, wooden packaging materials, nursery stock, and bonsai trees [4,5].

Materials and Methods
Field data were collected throughout the Czech Republic from January to October 2018 in 27 localities in protected areas and forests with >10% of one or more of the following J. communis habitats (Figure 1): T3.4A (broad-leaved dry grasslands with orchids and J. communis), T3.4B (broad-leaved dry grasslands with few or no orchids and with J. communis), T8.1A (dry lowland and colline heaths with J. communis), and T8.2A (secondary submontane and montane heaths with J. communis).The areas and forests with these habitats were selected based on the updated mapping of Natura 2000 [44] from the years 2007-2018.Each plot (one plot per locality) was surveyed only one time.
In each of the 27 plots, which ranged in area from 0.1 to 103.5 ha (habitat boundaries), we checked all J. communis trees with dry twigs (Figure 2).We debarked the trunk of such trees and checked the exposed wood and bark for galleries and beetles typical of Phloeosinus spp.The removed bark was 50 cm long on the vertical plane and included the entire circumference (Figure 2).Any bark beetles present were removed with tweezers, stored in alcohol, and identified to species by examination with a dissecting microscope (Bresser Advanced ICD Microscope 10x -160x; Bresser GmbH, Rhede, Germany).The detection of a specimen of P. aubei was considered to indicate that P. aubei was present in the locality; the detection of galleries alone was not considered evidence of P. aubei.We also recorded the time required to detect the first P. aubei specimen (the 'finding time') in a plot; the finding time was considered an indicator of P. aubei abundance.
GLM analyses (Poisson distribution, LN link function) were used to determine the relationships between the 'finding time' and the locality characteristics indicated in the previous sentence plus locality management (managed vs. unmanaged).As noted earlier, finding time served as a surrogate measure of P. aubei population density.All tests were performed with Statistica 12.0 sofware (StatSoft CR, s.r.o.; Prague, Czech Republic).

Results
P. aubei was found in 16 of the 27 localities studied in 2018; the localities with P. aubei in 2018 were scattered throughout the Czech Republic and over a wide range of altitudes (160 to 720 m) (Figure 1).All localities with P. aubei in 2018 also contained Phloeosinus thujae (Perris, 1855).Phloeosinus aubei was found in all localities in the eastern part of the Czech Republic but in only about half of the localities in the western part of the country (Figure 3); this trend was statistically significant (χ 2 =14.89; p < 0.001).The habitats were also different in eastern vs. western parts of the country: protected areas were regularly maintained by grazing or cutting in the eastern localities but were overgrown with Frangula alnus Mill., 1768, Prunus spinosa L., 1753, Rosa spp., and other species in the western localities (Figure 4, Appendix A).The probability of P. aubei detection was not related to locality altitude (z = 0.87; p > 0.05), locality area (z = 1.66; p > 0.10), distance of the locality to the nearest juniper (z = −0.30;p > 0.05), or juniper population density in the locality (z = 1.73; p > 0.10) (Figure 5).Across all localities, the mean (± standard deviation) finding time was 14 (± 16.6) min.Finding time was unrelated to locality altitude, locality area, distance of the locality to the nearest juniper, juniper population density in the locality, or locality management (F = 0.76; p > 0.10).
In addition to being found in 16 localities in 2018, P. aubei was previously found in 8 other localities (Figure 1).In all 24 localities where P. aubei has been detected, the host tree was usually J. communis, although the host tree was not indicated in some earlier reports and was Thuja spp. in one earlier report (Appendix A).Across all 24 localities, P. aubei was mainly found on J. communis stems thicker than 3 cm (Appendix A).In the one case in which P. aubei was found on P. thujae, the beetle was also found on branches thinner than 1 cm.In all localities where P. aubei was detected on J. communis, the trees had been damaged by cattle or game (Figure 2).Data from private collections and museum collections were also summarized.Data from "Finding Database of Agency of Protection Nature and Landscape ČR" [45] are included as well as data from unpublished entomological reports.
The relationship between the presence/absence of P. aubei and longitude was analysed using a logistic model (Quassi-Newton method of estimation).Mann-Whitney U-tests were used to compare the following variables between localities with and without P. aubei: altitude, locality area, distance of the locality from the nearest J. communis locality, and population density of junipers.GLM analyses (Poisson distribution, LN link function) were used to determine the relationships between the 'finding time' and the locality characteristics indicated in the previous sentence plus locality management Forests 2019, 10, 12 (managed vs. unmanaged).As noted earlier, finding time served as a surrogate measure of P. aubei population density.All tests were performed with Statistica 12.0 sofware (StatSoft CR, s.r.o.; Prague, Czech Republic).

Results
P. aubei was found in 16 of the 27 localities studied in 2018; the localities with P. aubei in 2018 were scattered throughout the Czech Republic and over a wide range of altitudes (160 to 720 m) (Figure 1).All localities with P. aubei in 2018 also contained Phloeosinus thujae (Perris, 1855).Phloeosinus aubei was found in all localities in the eastern part of the Czech Republic but in only about half of the localities in the western part of the country (Figure 3); this trend was statistically significant (χ 2 = 14.89; p < 0.001).The habitats were also different in eastern vs. western parts of the country: protected areas were regularly maintained by grazing or cutting in the eastern localities but were overgrown with Frangula alnus Mill., 1768, Prunus spinosa L., 1753, Rosa spp., and other species in the western localities (Figure 4, Appendix A).The probability of P. aubei detection was not related to locality altitude (z = 0.87; p > 0.05), locality area (z = 1.66; p > 0.10), distance of the locality to the nearest juniper (z = −0.30;p > 0.05), or juniper population density in the locality (z = 1.73; p > 0.10) (Figure 5).Across all localities, the mean (±standard deviation) finding time was 14 (±16.6)min.Finding time was unrelated to locality altitude, locality area, distance of the locality to the nearest juniper, juniper population density in the locality, or locality management (F = 0.76; p > 0.10).
Forests 2018, 9, x FOR PEER REVIEW 4 of 13 Finding time was unrelated to locality altitude, locality area, distance of the locality to the nearest juniper, juniper population density in the locality, or locality management (F = 0.76; p > 0.10).
In addition to being found in 16 localities in 2018, P. aubei was previously found in 8 other localities (Figure 1).In all 24 localities where P. aubei has been detected, the host tree was usually J. communis, although the host tree was not indicated in some earlier reports and was Thuja spp. in one earlier report (Appendix A).Across all 24 localities, P. aubei was mainly found on J. communis stems thicker than 3 cm (Appendix A).In the one case in which P. aubei was found on P. thujae, the beetle was also found on branches thinner than 1 cm.In all localities where P. aubei was detected on J. communis, the trees had been damaged by cattle or game (Figure 2).

Discussion
The small cypress bark beetle P. aubei is native to the Caucasus, Asia Minor, and the Mediterranean area [6] from Israel [27] to southwest France [46].Over the last 100 years or so, P. aubei has been spreading north from the Mediterranean area into Central Europe, where it has been reported as an alien pest [24,25,32,[47][48][49][50][51][52].In addition to being found in 16 localities in 2018, P. aubei was previously found in 8 other localities (Figure 1).In all 24 localities where P. aubei has been detected, the host tree was usually J. communis, although the host tree was not indicated in some earlier reports and was Thuja spp. in one earlier report (Appendix A).Across all 24 localities, P. aubei was mainly found on J. communis stems thicker than 3 cm (Appendix A).In the one case in which P. aubei was found on P. thujae, the beetle was also found on branches thinner than 1 cm.In all localities where P. aubei was detected on J. communis, the trees had been damaged by cattle or game (Figure 2).
The presence of P. aubei in the Czech Republic was first mentioned in the literature by Pfeffer [17], who referred to a single specimen captured by J. Picka.Although the details regarding that specimens are few, we do know that it was collected in the southeastern part of the country (i.e., in the historical country of Moravia) and probably in the village of Čejč.Its year of collection is unknown, but it was probably collected in the 1960s.The first dated finding of P. aubei in the Czech Republic was in 1947, and that specimen was found in the east-central part of the country (i.e., in the historical country of Bohemia) (current study).Previous studies had reported the detection of P. thujae [53,54] in the Czech Republic but not P. aubei.In the 1990s, P. aubei was reported from southern Moravia [55] and from Bohemia [56]; the latter report concerned outbreaks in several places in Prague and central Bohemia on Juniperus spp., Chamaecyparis spp., Thuja spp., and Cupressus x leylandii A.B. Jacks & Dallim., 1926.
In the Czech Republic, P. aubei has perhaps been spreading west (Figure 1), because P. aubei detection decreased from east to west (Figure 3).All studied variables (altitude, locality area, distance of the locality to the nearest juniper, and juniper population density) had large ranges in values in localities with and without P. aubei occurrence (Figure 5).Accordingly, these variables cannot be used to predict where the beetle will spread to, and only the presence of host trees appears to be important.A strong influence of host is typical for bark beetles [57].That P. aubei is able to detect its host is confirmed by the fact that the 'finding time' was unrelated to host variables (area, distance of the nearest juniper, and density).Phloeosinus bark beetles have strong dispersal capabilities and can fly over 24 km [58,59].The abilities to disperse and to detect hosts promote the spread of invading populations in a landscape matrix [60].
The severe drought of 2003 possibly increased the susceptibility of juniper trees in Central Europe to bark beetles.For Western Europe, climate change models predict increasing summer drought and heat waves, which will increase the susceptibility of trees to secondary insect pests such as Phloeosinus spp.[25,72].The finding of P. aubei in 1996 in the United Kingdom can be attributed to global trade, because P. aubei was found on Thuja spp.imported from Italy [52].
The main host of P. aubei in the Czech Republic is the common juniper, J. communis, which is listed in the ICUN Red List as near-threatened in the country [36].Given the fragmented and small populations of J. communis (see Figure 1), gene flow is probably limited, with potential implications for the tree's long-term fitness and survival even where viable seed production occurs [73][74][75].In such places, P. aubei can be considered a threat to the tree.This beetle kills J. communis trees that are weakened by drought or damaged by cattle grazing or antler rubbing by game [76,77].Older stands of J. communis in the Czech Republic are in poor condition, and in many places they are overgrown (Figure 4).Factors that weaken the trees increase the spread of P. aubei and P. thujae, but precise quantification of both beetles is difficult because their galleries are very similar.
The frequent occurrence of P. aubei in many regions of the Czech Republic could lead to attack on thujas and junipers in ornamental gardens and cities, as has already occurred in the towns of Písek and Mariánské Lázně (Appendix A) and in Prague and other places in central Bohemia [56].In contrast to P. aubei, P. thujae has not been reported on thujas and junipers in ornamental gardens and cities.In addition, P. aubei is a vector of pathogens and especially of the causal agent of cypress canker, S. cardinale ( [18,51,78].
The monitoring of invasive bark beetle is necessary [79].The use of pheromone trapping for monitoring P. aubei is not currently possible, because P. aubei pheromones have yet to be identified [23].At present, P. aubei populations can be monitored by the debarking of symptomatic juniper and thuja trees in the field.Such field monitoring can be conducted throughout the year, because P. aubei adults and larvae are present under the bark throughout the year [19,34].
Expansion of either the true range or the outbreak range is observed in several model species/groups of major insect guilds in boreal and temperate biomes.Effects of climate change on forest insects are demonstrated for a number of species and guilds, although generalizations of results available so far are difficult because of species-specific responses to climate change.There is evidence that recent warmer temperatures have permitted the expansion of bark beetle outbreaks to higher latitudes and elevations than in the past [80].
Climate and weather can have direct effects on trees, as drought and storms can weaken trees and predispose them to attack by bark beetles and pathogens.Climate can also have direct effects on insects as they are small poikilotherms with limited thermoregulation ability [81].P. aubei is a typical case of this pattern and may be followed by other species from Mediterranean countries.Climate change could also result in the spread of insect species from the opposite direction.Ips duplicatus (C.R. Sahlberg, 1836), a native bark beetle on Norway spruce in Scandinavia, currently is spreading to Central, Eastern, and Southern Europe.The outbreak is supported by the planting of spruce out of its original distribution, physiological weakness of the tree, and the consequent occurrence of fungal pathogens on host trees [82].
On the contrary, the spread direction of alien bark beetles in Europe could be different.Bark beetles mainly travel in wood and in wooden packing materials such as crating, dunnage, and pallets [83], so the places of introduction can be different.A North American species, Gnathotrichus materiarius (Fitch, 1858), and an East Asian species, Xylosandrus germanus (Blandford, 1894), currently occur in the Czech Republic, but they have been spreading from west to east from Germany [84].

Conclusions
The small cypress bark beetle has probably been present in the Czech Republic since the 1950s, and we have determined its current distribution on common juniper trees.The results indicate that P. aubei is widely distributed in the country but is more common in the east than in the west.The apparently widespread and abundant populations of P. aubei could explain infestations of the beetle on Thuja spp.and Juniperus spp. in gardens.We consider P. aubei to be a potential pest on older, naturally occurring J. communis in protected areas where its population density could increase on weakened and damaged trees.

Figure 1 .
Figure 1.Occurrence of Phloeosinus aubei in the Czech Republic.Positive detections as documented in records before 2017 are indicated by small black circles (with detection year), and positive detections in 2018 are indicated by large black circles.Negative detections in 2018 are indicated by empty circles.Small green circles indicate juniper habitats (T3.4A,T3.4B, T8.1A, and T8.2A) according to Natura 2000 mapping in the Czech Republic (see Materials and Methods for details).

Figure 1 .
Figure 1.Occurrence of Phloeosinus aubei in the Czech Republic.Positive detections as documented in records before 2017 are indicated by small black circles (with detection year), and positive detections in 2018 are indicated by large black circles.Negative detections in 2018 are indicated by empty circles.Small green circles indicate juniper habitats (T3.4A,T3.4B, T8.1A, and T8.2A) according to Natura 2000 mapping in the Czech Republic (see Materials and Methods for details).

Figure 2 .
Figure 2. Dying junipers (Juniperus communis) in the Rajchéřov locality (a, the dying trees are red), galleries of Phloeosinus aubei on J. communis in the Mariánské Lázně locality (b), and a juniper tree damaged by antler rubbing in the Vrbička locality (c).

Figure 2 .
Figure 2. Dying junipers (Juniperus communis) in the Rajchéřov locality (a, the dying trees are red), galleries of Phloeosinus aubei on J. communis in the Mariánské Lázně locality (b), and a juniper tree damaged by antler rubbing in the Vrbička locality (c).

Figure 2 .
Figure 2. Dying junipers (Juniperus communis) in the Rajchéřov locality (a, the dying trees are red), galleries of Phloeosinus aubei on J. communis in the Mariánské Lázně locality (b), and a juniper tree damaged by antler rubbing in the Vrbička locality (c).

Figure 3 .
Figure 3. Logistic model for the probability of detecting Phloeosinus aubei in the Czech Republic as related to longitude.On the y-axis, 0 and 1 indicate that P. aubei was not detected or was detected, respectively.

Figure 3 .
Figure 3. Logistic model for the probability of detecting Phloeosinus aubei in the Czech Republic as related to longitude.On the y-axis, 0 and 1 indicate that P. aubei was not detected or was detected, respectively.

Figure 4 .
Figure 4. Junipers overgrown by Prunus spinosa and Rosa spp.(a), and a juniper habitat regularly maintained by grazing at the Vrbička locality (b).

Figure 5 .
Figure 5. Relationships between the presence and absence of Phloeosinus aubei and the following characteristics of the localities: altitude, area, distance from the nearest juniper, and juniper population density.Small squares indicate medians, boxplots indicate the 25% and 75% quartiles, and lines indicate minimum and maximum values.

Figure 4 .
Figure 4. Junipers overgrown by Prunus spinosa and Rosa spp.(a), and a juniper habitat regularly maintained by grazing at the Vrbička locality (b).

Figure 4 .
Figure 4. Junipers overgrown by Prunus spinosa and Rosa spp.(a), and a juniper habitat regularly maintained by grazing at the Vrbička locality (b).

Figure 5 .
Figure 5. Relationships between the presence and absence of Phloeosinus aubei and the following characteristics of the localities: altitude, area, distance from the nearest juniper, and juniper population density.Small squares indicate medians, boxplots indicate the 25% and 75% quartiles, and lines indicate minimum and maximum values.

Figure 5 .
Figure 5. Relationships between the presence and absence of Phloeosinus aubei and the following characteristics of the localities: altitude, area, distance from the nearest juniper, and juniper population density.Small squares indicate medians, boxplots indicate the 25% and 75% quartiles, and lines indicate minimum and maximum values.