Ecological and Landscape Factors Affecting the Spread of European Mistletoe (Viscum album L.) in Urban Areas (A Case Study of the Kaliningrad City, Russia)

Green spaces are very important for an urban environment. Trees in cities develop under more stressful conditions and are, therefore, more susceptible to parasite including mistletoe infestation. The aim of this study was to investigate the ecological, microclimatic, and landscape factors causing the spread of European mistletoe (Viscum album L.) in urban conditions. The most numerous hosts of mistletoe were Tilia cordata (24.4%), Acer platanoides (22.7%), and Populus nigra (16.7%). On average, there were more than 10 mistletoe bushes per tree. The mass mistletoe infestations (more than 50 bushes per the tree) were detected for Populus × berolinensis, Populus nigra, and Acer saccharinum. The largest number of infected trees was detected in the green zone (city parks), historical housing estates, and green zone along water bodies. Based on the results of principal component analysis (PCA), the main factors causing the spread of mistletoe on the urban territories are trees’ age and relative air humidity. The factors reflecting environmental pollution (the content of heavy metals in the soil and the concentration of nitrogen dioxide in the air) did not statistically affect the mistletoe distribution in the study area. However, this result may be due to the heterogeneity of other parameters in the studied areas. Therefore, additional research is required to more accurately interpret the data on the relationship between environmental pollutions and distribution of mistletoe infestation of trees in urban areas.


Introduction
Urban green spaces have been increasingly recognized as key components of urban planning [1]. The trees as urban keystone flora have a multitude of functions, such as to reduce air pollution, support biodiversity, mitigate heat island effects, increase land value, improve aesthetics, and even improve human health [2,3]. However, the urban environments present enormous challenges for tree growth, including elevated temperatures, water and nutrient constraints, and lack of adequate root and crown volumes [4]. Under the influence of adverse environmental conditions, urban trees become more sensitive to biotic stressors (e.g., hemiparasitic plants, herbivorous insects, or pathogens). The hemiparasitic plants that infest trees and cause particular concern are mistletoes, which have been determined as one of the increasing agents of forest decline worldwide [5].
Most mistletoes are an evergreen plant that develops stable haustorium in the host tree. The plants assimilate their own carbon due to photosynthesis, which determines its green color, while completely Plants 2020, 9,394 3 of 13

Hosts' Preference of European Mistletoe in the Reserch Area
In the investigated area of Kaliningrad, over 43,000 mistletoe specimens were recorded on 3661 host trees. Table 1 presents the species of trees on which mistletoe bushes were found. V. album was identified on 50 taxa belonging to 17 genera and nine families. As a result of studies, no infected conifers were identified, which indicates the distribution of the subspecies Viscum album L. subsp. album only in the city.

Intensity of Mistletoe Infestation Depending on Tree Species
Assessment of the mistletoe infection degree of trees showed that on average there were more than 10 mistletoe bushes per tree (Table 1). Depending on the number of bushes on one tree, low (less than 10 bushes), medium (10 to 30 bushes), high (30 to 50 bushes), and very high (more than 50 bushes) infection rates were distinguished. The first group included 62% of infected plants, the second group 29%, the third group 6%, and the last one about 2%.
The results of the studies of the degree of the hosts' crown infestation in relation to the host species are presented in Table 2. Despite the fact that the largest number of infected trees was detected among the species Tilia cordata and Acer platanoides, most of these trees were characterized by a low degree of mistletoe infection. The species of host trees on which the foci of mass mistletoe were detected (more than 50 bushes on the tree) included, first of all, Populus × berolinensis, Populus nigra, and Acer saccharinum. More than 5% of the trees of these species belonged to the fourth group, a "very high" degree of infection.

Distribution of Mistletoe Infestation under the Urban Conditions
At the first stage of the study, a general assessment of the geographical distribution of mistletoe in the city of Kaliningrad was performed. Geographical distribution analysis of mistletoe-infected trees in the city showed that the main distribution areas are mosaic and located inconsistently ( Figure 1A,B). A comparison of the results presented in Figure 1A,B shows that the areas with the maximum number of mistletoe-infected trees almost completely coincided with the areas in which the number of mistletoe bushes was also high. This result indicates that the average infection rate (the number of mistletoe bushes per tree) was independent of the mistletoe distribution areas.  Table 3.
In the next phase of research to identify the main ecological and landscape factors affecting the mistletoe distribution in the city, within the research area, seven zones differing in the degree of anthropogenic load were selected. For these zones, some landscape, microclimatic, and environmental parameters were investigated. The results are shown in Table 3.  In the next phase of research to identify the main ecological and landscape factors affecting the mistletoe distribution in the city, within the research area, seven zones differing in the degree of anthropogenic load were selected. For these zones, some landscape, microclimatic, and environmental parameters were investigated. The results are shown in Table 3.
The studied zones differed significantly in the presence of mistletoe-infected trees in their territory. The largest number of infected trees was detected in the green zone (city parks), historical housing estates, and green zone along the water bodies. The zone of modern multistory apartments was characterized by the lowest degree of infestation. The city center, zone of medium-rise apartments, and zone of private residential buildings were characterized by an average degree of infestation. The diameter of mistletoe bushes in the studied zones varied widely from 20 to 180 cm, but averaged from 45 to 75 cm.
The presence of a higher density of roads and buildings in the city center and in the zone of modern multistory apartment block estates was accompanied by a higher average air temperature and lower relative humidity. The city center was also characterized by a higher concentration of nitrogen dioxide in the air. However, the content of lead and zinc in the soil was higher in zone Z3 (medium-rise apartment block estates). The results of a multivariate statistical analysis of the obtained experimental data are presented in Figure 2 in the form of a principal component analysis (PCA) biplot. The studied zones differed significantly in the presence of mistletoe-infected trees in their territory. The largest number of infected trees was detected in the green zone (city parks), historical housing estates, and green zone along the water bodies. The zone of modern multistory apartments was characterized by the lowest degree of infestation. The city center, zone of medium-rise apartments, and zone of private residential buildings were characterized by an average degree of infestation. The diameter of mistletoe bushes in the studied zones varied widely from 20 to 180 cm, but averaged from 45 to 75 cm.
The presence of a higher density of roads and buildings in the city center and in the zone of modern multistory apartment block estates was accompanied by a higher average air temperature and lower relative humidity. The city center was also characterized by a higher concentration of nitrogen dioxide in the air. However, the content of lead and zinc in the soil was higher in zone Z3 (medium-rise apartment block estates).
The results of a multivariate statistical analysis of the obtained experimental data are presented in Figure 2 in the form of a principal component analysis (PCA) biplot. humidity, and parameters of mistletoe infestation. The closest connection was established between the age of the trees and the diameter of the mistletoe bushes. The amount of mistletoe increased with increasing relative humidity in the area studied. However, between the parameters of mistletoe distribution and landscape factors (the density of the road network and the density of buildings), an inverse correlation was revealed. The areas studied that were characterized by a higher local temperature, had a lower level of mistletoe infestation. No correlation was found between the factors reflecting environmental pollution (the content of heavy metals in the soil and the concentration of nitrogen dioxide in the air) and the parameters of mistletoe distribution.

Discussion
Despite the accumulated experimental material on species susceptible to mistletoe infestation, the strategy for choosing a host tree by mistletoe is still unclear. For example, the in the paper by Barney et al. [28] a list of 452 taxa, on which European mistletoe was identified in Europe, Asia, and California, was presented. Moreover, it is possible that the preferences of mistletoe also depend on the geographical region of its growth and the characteristics of local populations. Linden, maple, and poplar trees predominated among the recorded infected tree species in this study. These species are often used in urban woodland, especially along roads. The strong susceptibility of linden trees to mistletoe infestation was also established earlier for some European cities. However, this species was not recorded among the most infected in a number of studies on the distribution of European mistletoe in cities in Poland bordering the Kaliningrad region [22,27]. In addition, in the same studies, mistletoe was rarely found on Acer platanoides, which was severely affected by mistletoe in Kaliningrad. It is believed that European mistletoe prefers trees with soft wood [14]. However, the significant frequency of its occurrence on maple, belonging to solid species of trees, suggests that it is not possible to single out one factor that determines the choice of host tree by mistletoe. In addition, a study of the distribution of mistletoe in the territory of the Botanical Garden showed that introduced tree species were more susceptible to mistletoe infestation. Among the infected tree species in the territory of the Botanical Garden were species such as Styphnolobium japonicum, Juglans mandshurica, and Aesculus glabra. The number of infected Acer saccharinum trees was 5 times lower compared to the number of infected. However, the result could be explained with a generally low percentage of this species in the wooded area of the observed city, as almost 100% of Acer saccharinum was proven to be infected. A strong susceptibility of Acer saccharinum to mistletoe infestation has also been reported in previous studies [14,22,27]. In this regard, one of the measures to reduce the distribution of mistletoe is to plant the trees that are less likely to be attacked by mistletoe (for example, oaks, hornbeams, and beech trees).
The largest number of infected trees was detected in the green areas (city parks), historical housing estates, and green zone along the natural or artificial lakes, ponds, and waterways. Similar results were obtained in a study of the mistletoe distribution in Warsaw [27]. At the same time, as well as for Warsaw, Kaliningrad was not characterized by the presence of mistletoe in urban forests, in which trees infected with mistletoe were found only at the outer borders. As noted by several authors, an adequate amount of sunlight is an important condition for the mistletoe distribution [29,30]. For the same reason, some authors believe that thinning trees will contribute to the mistletoe distribution [31,32]. The city center, featuring a higher road and construction density, was observed to have a relatively low level of mistletoe distribution. This could be associated not only with its generally low concentration of "green" areas, but also with a better and more regular maintenance of old and damaged trees.
A higher level of mistletoe-infected trees in the historical housing estates was due to the prevalence of older trees there. The age of the tree is also a factor in the mistletoe distribution. Despite the fact that mistletoe can also affect young trees, in most studies there was reported a direct correlation between the mean age of trees in green spaces and the level of mistletoe infestation [14,27,33].
The presence of a direct correlation between the degree of mistletoe infestation and air humidity, as well as the predominance of mistletoe-affected trees in green areas along water bodies, can be due to several reasons. First, tree species that have an increased water balance and hygrophytes are most often susceptible to infection. The moisture content is a prerequisite for both the germination of mistletoe seeds, and for its successful development. At the first stages of ontogenesis, when the mistletoe is just beginning to damage the tree, the moisture in the air is an additional source of water for it. Secondly, natural and artificial reservoirs and streams in cities represent a rich forage base for various species of birds, which are the main carriers of mistletoe seeds and contribute to its distribution. The most important birds for mistletoe dispersal are the mistle thrush (Turdus viscivorus), fieldfare (Turdus pilaris), waxwing (Bombycilla garrula), and blackcap (Sylvia atricapilla) [6]. According to the Atlas of Breeding Birds of Kaliningrad [34], Turdus pilaris and Sylvia atricapilla nest in the city of Kaliningrad. The highest nesting density of Sylvia atricapilla was recorded in the western part of the city, including in the historical residential area, where a high level of mistletoe infestation in trees was revealed. Bombycilla garrula do not nest in the research area, but are represented by a rather large population in the city and, according to bird watchers, are the main distributors of mistletoe.
According to available literature data, one of the most important factors determining the mistletoe distribution is temperature. It is believed that increasing average annual temperatures and climate change contribute to the mistletoe distribution in new areas [35]. However, in our study, an inverse correlation was found between temperature and mistletoe distribution parameters. Such a contradictory result is probably due to the fact that higher temperatures in the area studied were set in the center of the city, which, as mentioned above, was characterized by a smaller share of tree plantings and more careful care for them. Another possible reason for the lower mistletoe infestation in areas with higher temperature is lower relative humidity detected in these zones. For example, previous studies in the city of Kaliningrad showed the negative effect of reducing air humidity in the city center, as a result of heat island formation, on the urban flora especially on biodiversity of epiphytic lichens [36]. The result obtained in the presented study indicates that insufficient moisture is the main factor limiting the mistletoe distribution in an urban environment. In the study by Molnár and Végvári [37], it was shown that dryness in July was the main factor limiting the mistletoe spread, followed only by average July temperature and amount of air temperature during the growing period.
Despite the fact that some authors point to the relationship between the level of nitrogen dioxide in the air and the degree of mistletoe infestation of trees [22], no correlation between these parameters was established in this study. The result obtained may be due to the heterogeneity of other parameters in the studied areas (density of the road network, building density, humidity, presence of birds, accessibility of host trees, etc.). Therefore, additional research is required to more accurately interpret the data obtained. This conclusion also refers to the results indicating the absence of a correlation between the content of heavy metals in the soil and the parameters of the mistletoe infestation of trees. The study by Sharm et al. indicated that in areas with a higher content of heavy metals in the soil, mistletoe infestation of trees was also higher [38]. However, as the authors themselves noted, the effect of heavy metals on the mistletoe distribution was indirect and was primarily associated with the deterioration of the physiological state of trees under their influence, which complicated the unambiguous interpretation of the data.

Research Area
A study of the distribution of mistletoe and identification of trees affected by it was carried out in the city of Kaliningrad (54 • 43 N, 20 • 30 E) from April to December 2019. Kaliningrad is the administrative center of the Kaliningrad region, located in the western part of Russia on the Baltic coast. From the south, the Kaliningrad region borders Poland, and in the north and east, Lithuania. Kaliningrad is located in the Primorsk Lowland, on the banks of the Pregolya River at its confluence with the Kaliningrad (Vistula) Bay. The climate of Kaliningrad is transitional from temperate to temperate continental. Air temperature in winter is -5 to 5 • C and in summer is 18 to 23 • C. The warmest month of the year is July, with an average daily temperature of 18.1 • C. The coldest is January, with an average Plants 2020, 9, 394 9 of 13 temperature of -1.5 • C. The average annual temperature is 7.9 • C. The average annual rainfall is 804 mm (1981-2010). The total area of the city is 223 km 2 . The population is 482,400 people (as of 1 January 1 2019). The total area of the territory studied was 25 km 2 and included the central part of the city (Figure 3). Plants 2020, 9, x FOR PEER REVIEW 10 of 14 with the Kaliningrad (Vistula) Bay. The climate of Kaliningrad is transitional from temperate to temperate continental. Air temperature in winter is -5 to 5 °C and in summer is 18 to 23 °C. The warmest month of the year is July, with an average daily temperature of 18.1 °C. The coldest is January, with an average temperature of -1.5 °C. The average annual temperature is 7.9 °C. The average annual rainfall is 804 mm (1981-2010). The total area of the city is 223 km 2 . The population is 482,400 people (as of 1 January 1 2019). The total area of the territory studied was 25 km 2 and included the central part of the city (Figure 3).

Distribution of Mistletoe Infestation in the City
Due to the fact that the spread of mistletoe was not previously studied in the city of Kaliningrad, at the first stage of the presented study, the general distribution of mistletoe was explored. For this purpose, in the area shown in Figure 3 all mistletoe-infected trees were identified. For each infected tree the following information was recorded: Coordinates, species, tree diameter (at a height at 1.3 m), number of mistletoe bushes on tree, and average diameter of the mistletoe bushes (the minimum and maximum diameter was also recorded in the case of heterogeneity of sizes). Scientific species' names and species' authorities for plants were given according to the World Flora Online (http://www.worldfloraonline.org/) [39].
Four categories were distinguished according to the degree of damage to the crown of mistletoe trees: Low (less than 10 bushes per tree), medium (10 to 30 bushes), high (30 to 50 bushes on a tree), and very high (more than 50 bushes per tree). The classification proposed by the authors was based on the Barbu [40] rating system of mistletoe infestation intensity, which takes into account the site of mistletoe damage (lateral branches, crown, stem) and the percentage of the affected crown, as well as the presence and amount of dried branches infested with mistletoe.
To calculate the spatial patterns of mistletoe distribution and identify relationships, QGIS 3.6 with the Spreadsheet Layers module was used. The SasPlanet software package (stable version v. 190707) was used to obtain a raster substrate for the studied areas. In order to increase the accuracy of estimating the European mistletoe distribution, the method of zonal statistics with elements of focal statistics was chosen. The size of the computational cell was set to 250 m*250 m (6.25 ha), which is most optimal for cities of such a size as Kaliningrad. Using GIS, the total number of affected trees and total number of mistletoe bushes, as well as road and building density for each studied square, was determined.

Distribution of Mistletoe Infestation in Zones Differed by Anthropogenic Load
In order to assess the features of the spread of mistletoe depending on the anthropogenic load in the surveyed area, seven zones were selected. These zones differed in a certain type of habitat,

Distribution of Mistletoe Infestation in the City
Due to the fact that the spread of mistletoe was not previously studied in the city of Kaliningrad, at the first stage of the presented study, the general distribution of mistletoe was explored. For this purpose, in the area shown in Figure 3 all mistletoe-infected trees were identified. For each infected tree the following information was recorded: Coordinates, species, tree diameter (at a height at 1.3 m), number of mistletoe bushes on tree, and average diameter of the mistletoe bushes (the minimum and maximum diameter was also recorded in the case of heterogeneity of sizes). Scientific species' names and species' authorities for plants were given according to the World Flora Online (http://www.worldfloraonline.org/) [39].
Four categories were distinguished according to the degree of damage to the crown of mistletoe trees: Low (less than 10 bushes per tree), medium (10 to 30 bushes), high (30 to 50 bushes on a tree), and very high (more than 50 bushes per tree). The classification proposed by the authors was based on the Barbu [40] rating system of mistletoe infestation intensity, which takes into account the site of mistletoe damage (lateral branches, crown, stem) and the percentage of the affected crown, as well as the presence and amount of dried branches infested with mistletoe.
To calculate the spatial patterns of mistletoe distribution and identify relationships, QGIS 3.6 with the Spreadsheet Layers module was used. The SasPlanet software package (stable version v. 190707) was used to obtain a raster substrate for the studied areas. In order to increase the accuracy of estimating the European mistletoe distribution, the method of zonal statistics with elements of focal statistics was chosen. The size of the computational cell was set to 250 m*250 m (6.25 ha), which is most optimal for cities of such a size as Kaliningrad. Using GIS, the total number of affected trees and total number of mistletoe bushes, as well as road and building density for each studied square, was determined.

Distribution of Mistletoe Infestation in Zones Differed by Anthropogenic Load
In order to assess the features of the spread of mistletoe depending on the anthropogenic load in the surveyed area, seven zones were selected. These zones differed in a certain type of habitat, from seminatural to the most altered as a result of human activity. The allocation of zones was carried out on the basis of the master plan of the city of Kaliningrad. The size of each zone was 1 km 2 (100 ha). A brief description of each zone is presented in Table 4.
In addition to the assessment of the level of mistletoe distribution, the following parameters were determined for each zone: Microclimatic features (temperature, humidity), soil contamination with heavy metals (average content of lead, zinc, and copper in soil), and atmospheric air pollution with nitrogen dioxide.

Climatic Parameters and Enviromental Pollutants Measuring
Temperature and humidity in the areas studied were determined using portable sensors (UNI-T UT330C USB). For this purpose, three trees were selected in each of the seven zones, on which sensors were mounted on the south side at a height of 3 meters from the ground. Temperature and humidity were recorded around the clock during the first week of April, July, and December 2019. Then the data on temperature and humidity were averaged for each zone and used for statistical analysis.
The metal content in soil samples taken from the territories of the studied zones was determined by X-ray fluorescence analysis on a Spectroscan Max-G instrument (NPO Spectron LLC, Russia). Then, 5-6 samples were taken from each zone, which were then combined into a mixed sample and used for analysis. Sample preparation and analysis were carried out according to [41].
Measurements of nitrogen dioxide in the air of the research zones were carried out by using a passive sampling method with spectrophotometric determination as described in [42].

Statistical Analysis
The statistical analysis of the obtained experimental data was performed using OriginPro 2019b (OriginLab Corporation, Northampton, MA, USA). To evaluate the relationship between the parameters of mistletoe infestation (number of infected trees, number of mistletoe specimens, diameter of mistletoe shrub) and ecological and landscape factors, the principal component analysis (PCA) was used. The assessment of loadings' significance in PCA was performed using the bootstrap (N = 999) according to Peres-Neto et al. [43]. The 95% bootstrapped confidence intervals for the eigenvalues were evaluated using Past v. 4.01. One-way analysis of variance (ANOVA) and Kruskal-Wallis tests were used for comparing the research zones. Difference among means was determined by Tukey's test at a significance level of p ≤ 0.05.

Conclusions
Performed for the first time, a study of the spread of mistletoe in the city of Kaliningrad revealed that the main tree species susceptible to mistletoe infestation were Tilia cordata (24.4 %), Acer platanoides (22.7 %), and Populus nigra (16.7%). In addition, it was found that most of Acer saccharinum trees in Kaliningrad are infested by mistletoe, which would allow us to recommend excluding it from urban plantings and favoring more resistant species such as oaks, hornbeams, and beech trees. An analysis of the environmental and landscape factors causing mistletoe spreading under urban conditions (with reference to the city of Kaliningrad) showed that the main factors that positively affected the spread of mistletoe were the age of the trees and the relative humidity. The factors reflecting environmental pollution (the content of heavy metals in the soil and the concentration of nitrogen dioxide in the air) did not statistically affect the mistletoe distribution in the study area. However, additional research is required to more accurately interpret the data on the relationship between environmental pollutions and distribution of mistletoe infestation of trees in urban areas.