The European Mistletoe (Viscum Album L.): Distribution, Host Range, Biotic Interactions and Management Worldwide with Special Emphasis on Ukraine

Botany

The European mistletoe (Viscum album L.): distribution, host range, biotic interactions and management worldwide with special emphasis on Ukraine

Journal: Botany

Manuscript ID cjb-2020-0037.R1

Manuscript Type: Review

Date Submitted by the 25-Mar-2020 Author:

Complete List of Authors: Krasylenko, Yuliya; Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, Molecular Biotechnology; Centre of the Region Haná for Biotechnological and Agricultural Research, Draft Sosnovsky, Yevhen; Ivan Franko National University of Lviv, Botanical Garden Atamas, Natalia; I.I. Schmalhausen Institute of Zoology, National Academy of Science of Ukraine, Department of Animal Monitoring and Conservation, Laboratory of Population Ecology Popov, Grigory; I.I. Schmalhausen Institute of Zoology, National Academy of Sciences of Ukraine, Department of Entomology and Collection Management Leonenko, Volodymyr ; Green Clinic Life Science Ukraine Janošíková, Kateřina ; Сenter for Science Communication, Palacký University Olomouc Sytschak, Nadiya; Institute of Ecology of the Carpathians NAS of Ukraine Rydlo, Karol; Brno University of Technology, Faculty of Information Technology Sytnyk, Dmytro; Institute of Mathematics, National Academy of Sciences, Numerical Mathematics Department

Keyword: hemiparasite, hosts, distribution range, seed dispersal, associates

Is the invited manuscript for consideration in a Special IUFRO 2019 Dwarf Mistletoes Symposium Issue? :

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The European mistletoe (Viscum album L.): distribution, host range, biotic interactions and

management worldwide with special emphasis on Ukraine

Yuliya Krasylenko1,2*, Yevhen Sosnovsky3, Atamas Natalia4, Grigory Popov5, Volodymyr

Leonenko6, Kateřina Janošíková7, Nadiya Sytschak8, Karol Rydlo9, and Dmytro Sytnyk10

1Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of

Science, Palacký University Olomouc, Šlechtitelů, 27, 78371, Olomouc, Czech Republic,

[email protected]

2Department of Cell Biology and Biotechnology, Institute of Food Biotechnology and Genomics, National Academy

of Sciences of Ukraine, Osipovskogo Str., 2a, 04 123, Kyiv, Ukraine, [email protected]

3Botanical Garden, Ivan Franko National UniversityDraft of Lviv, Cheremshyny Str., 44, 79014, Lviv, Ukraine,

[email protected]

4Department of Animal Monitoring and Conservation, Laboratory of Population Ecology, I.I. Schmalhausen Institute

of Zoology, National Academy of Science of Ukraine, B. Khmelnytskoho Str., 15, 01601, Kyiv, Ukraine,

[email protected]

5Department of Entomology and Collection Management, I.I. Schmalhausen Institute of Zoology, National Academy

of Sciences of Ukraine, B. Khmelnytskoho Str., 15, 01 601, Kyiv, Ukraine, [email protected]

6Green Clinic Life Science Ukraine, LLC Druzhby Narodiv 10, 03083, Kyiv, Ukraine,

[email protected]

7Сenter for Science Communication, Palacký University Olomouc, 17. listopadu 7, 78371, Olomouc, Czech

Republic, [email protected]

8Department of Nature Ecosystems Protection, Institute of Ecology of the Carpathians, National Academy of

Sciences of Ukraine, Kozelnytska Str., 4, Lviv, 79026, Ukraine, [email protected]

9Faculty of Information Technology, Brno University of Technology, Božetěchova, 2, 61266, Brno, Czech Republic,

[email protected]

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10Department of Numerical Mathematics, Institute of Mathematics, National Academy of Sciences of Ukraine,

Tereshchenkivska Str., 3, 01601, Kyiv, Ukraine, [email protected]

*Corresponding author: Yuliya [email protected]

Abstract: The hemiparasitic European mistletoe, Viscum album L. (Viscaceae), displays a rapid and remarkable expansion into natural and urban ecosystems in Ukraine. The monitoring and management of this rapidly spreading species is increasingly difficult as new plant species become hosts. Unlike other local mistletoe species, the European mistletoe has a broad distribution and thus requires a countrywide pest status assessment for control. This review outlines the major taxonomic and evolutionary issues pertinent to V. album with an emphasis on the characters used to distinguish its Draft five currently recognized subspecies. The review also provides an updated distribution map and host range for the three V. album subspecies in Ukraine

(V. album subsp. album, V. album subsp. abietis (Wiesb.) Janch. and V. album subsp. austriacum

(Wiesb.) Vollm.), addressing the current knowledge of their biology and ecology. A significant portion of the paper is devoted to the diversity of V. album associated organisms including herbivores, endophytes, and parasites, drawing particular attention to major pollination and dispersal vectors as well as potential biocontrol agents of this mistletoe species.

Key words: hemiparasite, hosts, distribution range, seed dispersal, associates, control.

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Introduction

The common name “mistletoe” refers to a polyphyletic group of organisms with a similar

life history, e.g., life form, type of plant-to-plant biotic interaction, host-dependence, cryptic

mimicry to the hosts, and the presence of a parasite-specific organ called the haustorium (Kuijt

and Hansen 2015; Okubamichael et al. 2016). The haustorium anchors the parasite to the host

and creates a xylem-xylem/xylem-phloem vascular bridge between both plants. This connection

allows the parasite to acquire water and nutrients, which can cause water stress and minerals

deficiencies in the host. The haustorium also allows the transfer of materials such as mRNA,

proteins and viruses (Liu et al. 2019a). Mistletoes are represented by several families in the order

Santalales, namely Loranthaceae (ca. 900 species), Viscaceae (ca. 500 species), and less

frequently Santalaceae, Amphorogynaceae,Draft and Misodendraceae (Nickrent et al. 2010).

Mistletoes play an ambiguous role in ecosystems that creates uncertainty for foresters and

conservationists. They are considered a major biotic stress on host plants, along with herbivores,

nematodes, and pathogenic microorganisms, which can cause host mortality and large losses in

wood production (Kuijt and Hansen 2015). Nevertheless, most mistletoes are not detrimental

pests (except some Arceuthobium spp.) and play a crucial ecological role as biodiversity hotspots

and keystone resources in forests and woodlands as they contribute both trophically and

structurally (e.g., by providing a specific micro-habitat) to diverse interactions within ecosystems

(Watson and Herring 2012).

The European or common mistletoe (Viscum album L.) is well-known due to its cytostatic

and hypotensive medicinal properties as well as ritual uses since the Neolithic Age (Kuijt and

Hansen 2015; Kope et al. 2020). The oldest fossil records of mistletoe found in central Europe

are V. morlotii (Unger) Erw.Knobloch & Kvaček from the Early Miocene and V. miquelii (Geyl.

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& Kink.) Czeczott from the Pliocene (Holý et al. 2012). Five subspecies of V. album occur mainly in Europe with some successful cases of introduction in the USA (Sonoma County,

California), Canada (Victoria, British Columbia), Ireland and the northern part of Great Britain

(Zuber and Widmer 2009; Maul et al. 2019). The patchy distribution of this insect- and wind- pollinated hemiparasite depends on the available hosts, presence of dispersal vectors such as birds and human-imposed control agents. The distribution of V. album does not overlap exactly with the range of host tree species, since the latter is typically much wider (Wangerin 1937). The range of V. album fluctuates significantly, spreading both northward (“horizontally”) and uphill

(“vertically”), likely following the ranges of susceptible hosts that are expanding due to climate change (Dobbertin et al. 2005).

The need to review the currentDraft state of V. album in Eastern Europe and in particular

Ukraine, is underscored by the rapid and remarkable dissemination of this hemiparasite into natural and urban ecosystems, its ability to switch hosts, and the challenges related to its monitoring and control. There are three mistletoe species from Santalales in Ukraine: V. album

L., which has broad geographical and host ranges, yellow mistletoe (Loranthus europaeus Jacq.), found predominantly in Southwestern Ukraine on oaks and is recommended for protection at the regional level as a rare species (Krasylenko et al. 2019), and juniper dwarf mistletoe

(Arceuthobium oxycedri (DC.) M.Bieb.), which resides on the Crimean Peninsula with native and adventive representatives of Cupressaceae and seems not to be seriously harming host trees

(Krasylenko et al. 2017). Only one subspecies – V. album subsp. album – requires special phytoquarantine treatment and detailed studies of associated birds, insects, fungi, etc., as it causes host tree decline, harvest reduction and timber destruction.

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Taxonomic overview

Reconstructing the taxonomic history of mistletoes has proven to be quite challenging, as

several taxa have evolved this growth habit independently in Santalales (Der and Nickrent 2008;

Nickrent et al. 2010). The Old World’s genus Viscum L. contains up to 150 species prevailing in

Africa and Southern Asia with V. album as the type species. Viscum album was long affiliated

with Loranthus Jacq. and close allies, first within Viscaceae and then in Loranthaceae (Nickrent

et al. 2010). Later, as more Santalalean groups were discovered, molecular studies regarded

Viscum-related taxa as a monophyletic clade phylogenetically closer to Santalaceae rather than

Loranthaceae, which was also supported by morphological, cytological, embryological, and

biogeographic data (Der and Nickrent 2008; Mathiasen et al. 2008; Nickrent et al. 2010).

Consequently, Angiosperm Phylogeny GroupDraft classifications (APG, 2016) merged Viscaceae into

the heterogenous Santalaceae s.l., while Nickrent et al. (2010) reestablished this family by

splitting Santalaceae s.l. up into six monophyletic groups. Recent studies combining genomics

and inflorescence morphology show Viscaceae to be the most derived Santalalean family, which

stems from its remarkably increased substitution rates in the nuclear and chloroplast genomes

(Chen et al. 2019 ; Nickrent et al. 2019) and in the mitochondrial genome as compared not only

to the rest of Santalales, but also to other hemi-/holoparasitic angiosperm taxa (Zervas et al.

2019). These genetic changes have resulted in the accumulation of phenotypic evolutionary

changes in Viscaceae evident in the multiple reductions of morphological traits and blurred

distinction between reproductive and vegetative structures (Nickrent et al. 2019). Despite the

molecular evidence and adoption of Viscaceae by some of the major taxonomies (Kuijt and

Hansen 2015), many recent floristic and taxonomic treatments retain Viscaceae as part of

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Santalaceae s.l. (e.g., Jäger 2017; Lauber et al. 2018; WCVP 2019) or even Loranthaceae (Uotila

2011).

Among the Viscaceae, generally having clustered sessile flowers or very short racemose or spike-like inflorescences, Viscum is distinguished by a 1-3(-5)-flowered determinate (cymose) inflorescence and usually non-connate cushion-like anthers (Kuijt and Hansen 2015; Nickrent et al. 2019). Infrageneric classification of Viscum is controversial due to high morphological variability, convergent reduction in vegetative organs, overlapping host preferences, as well as puzzling evolutionary switches from dioecy to monoecy and complex dispersal patterns

(Mathiasen et al. 2008; Maul et al. 2019). The most comprehensive phylogenetic study of

Viscum, encompassing 59 taxa (Maul et al. 2019), favors geography over morphology as a predictor of phylogenetic relatedness withinDraft the genus. However, this is likely insufficient for taxonomy if used alone as the distribution of extant taxa is blurred by unresolved dispersal histories.

Viscum album, described by Linnaeus (1753) as the sole European mistletoe species, exemplifies major taxonomic issues within the genus and may serve as a model for microevolutionary processes. It has the broadest range of all Viscum species both in terms of geographic distribution and host association (Maul et al. 2019), resulting in the lack of consensus regarding the taxonomic delineation of its geographic and host-specific races (Zuber and Widmer

2009; Okubamichael et al. 2016; Jäger 2017). The European mistletoe is currently well- distinguished by genetics, fruit morphology and phyllotaxis from the red-berry mistletoe

(V. cruciatum Sieber ex Boiss.) which is also found in Europe and overlaps geographically with

V. album (Zuber 2004; Zuber and Widmer 2009). Vegetative morphology and fruit color allow differentiation of V. album from the closely related and rather polymorphic Korean mistletoe (V.

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coloratum (Kom.) Nakai) residing in Eastern Asia (Qiu and Gilbert 2003; Zuber 2004). The two

species also differ in terms of composition of volatile compounds (Hayashi et al. 1996). Current

taxonomies (Uotila 2011; Lauber et al. 2018; WCVP 2019) recognize five V. album subspecies

with distinct host preferences, and, to some extent, geographic distribution and morphology.

Viscum album subsp. album L. (Fig. 1, A) parasitizes 452 host species of deciduous

dicotyledonous trees in 96 genera and 44 families (Barney et al. 1998) within its range across

Europe and eastward to the Himalayas (Nepal) (Qiu and Gilbert 2003), often occurring as a

synanthropic species.

Viscum album subsp. abietis (Wiesb.) Janch. (Fig. 1, B) prefers Abies spp. as a primary

host and occurs from the Pyrenees to Germany and through Eastern and Southern Europe to the

Caucasus and Asia Minor (Catalán and DraftAparicio 1997; Jäger 2017). In turn, Viscum album subsp.

austriacum (Wiesb.) Vollm. (Fig. 1, C) is found on several conifer taxa, mostly on Pinus and

more rarely on Larix and Picea (Dobbertin et al. 2005). It extends from North-West Africa

(Morocco) and the central Iberian Peninsula through Central and Southern Europe to the

Caucasus and Asia Minor (Catalán and Aparicio 1997; Uotila 2011). Morphologically, this

subspecies is distinguished from V. abietis by the shape of the hypocotyl tip. In subsp. abietis the

hypocotyl tip is swollen due to a constriction below it, while in subsp. austriacum such a

constriction is absent (reviewed by Kahle-Zuber 2008). The two subspecies are also differentiated

based on leaf length and shape, while both differ from the typical V. album with convex versus

straight seed edges and a smaller number of embryos (Jäger 2017).

Viscum album subsp. creticum N.Böhling, Greuter, Raus, B.Snogerup, Snogerup & Zuber

was described from Pinus halepensis subsp. brutia (Ten.) Holmboe (synonym of P. brutia Ten.)

at two locations in eastern Crete. Fruit and seed characters link this subspecies with the other two

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conifer-associated taxa, from which it is differentiated by morphology (e.g., hypocotyl shape), genetics, and a highly localized distribution (Böhling et al. 2002).

Viscum album subsp. meridianum (Danser) D.G.Long is the easternmost and currently the only non-European subdivision of V. album, occurring in South-Eastern Asia including Bhutan,

China, India, Myanmar, and Vietnam (Qiu and Gilbert 2003). It differs from the typical V. album, largely in fruit color and shape, with the host range encompassing some deciduous taxa of Acer,

Carpinus, Juglans, Prunus, and Sorbus (Qiu and Gilbert 2003).

Full synonymy of the aforementioned names can be found in Qiu and Gilbert (2003), and

Uotila (2011). The systematic significance of the morphological characters (such as fruit shape and color, leaf shape and size, hypocotyl anatomy, etc.) used to distinguish these entities has been debated suggesting these taxa should notDraft be treated at the species rank (Zuber 2004; Zuber and

Widmer 2009). Nevertheless, these subspecies are well differentiated genetically based on chloroplast DNA, with haplotype variation suggesting the existence of geographically distinct cryptic races (Zuber and Widmer 2009). The first three European subspecies are also distinguished by variation in isozyme loci and viscotoxins (Schaller et al. 1998). Following Maul et al. (2019), these host-specialized derivatives of the generalist parasite likely present recently evolved lineages, implying that both host shifts and geographic patterns are important speciation drivers in this genus.

Morphology, anatomy, life cycle and phenology

Viscum album is a mostly solitary globose dioecious perennial evergreen shrub with dichasial branching, sometimes reaching 1.5 m in diameter (exophyte) (Fig. 2, A; Fig. 3, A, B), and is anchored into host wood by a persistent endophytic system (endophyte) (Fig. 3, C)

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(Wangerin 1937; Zuber 2004; Kahle-Zuber 2008). Near the attachment site, V. album causes

multiple malformations including radial expansion, hyper- or atrophy and even the die-back of

host branches (Fig. 3, C). Dendrochronology estimates the lifespan of the European mistletoe to

be ca. 10-20 years (Kuijt and Hansen 2015) or up to 30 years based on the number of annual

rings (Wangerin 1937). Viscum album is also able to resprout through adventitious shoot

formation often at times of mechanical or temperature stress (Walldén 1961).

The leaves of V. album subsp. album are leathery (due to ca. 10 μm-thick waxy cuticle),

sessile, obovate-oblong, equifacial, oppositely arranged, highly variable in shape, and contain

three to five clear veins which are nearly parallel and interconnected by diffuse reticulate venules

(Fig. 2, F; Fig. 3, C, D) (Wangerin 1937). Starting at 4–5 years after germination and intrusive

endogenous growth, V. album becomesDraft mature and annually develops one dichasial shoot with

two internodes (short and long) bearing two pairs of leaves (scale and foliar types) per leaf axil

(Fig. 3, F−J) (Zuber 2004; Kahle-Zuber 2008; Kuijt and Hansen 2015).

Viscum album flowers annually beginning at 5–6 years of age. The staminate and

pistillate flowers are produced on separate plants which promotes outcrossing (Fig. 2, C−E; Fig.

3, D, E) (Kahle-Zuber 2008). The dichasial inflorescence of V. album subsp. album contains a

pair of usually fused bracts (also called a bracteal cup) and inconspicuous sessile yellowish-green

flowers arranged in triads by 3(–5) with one terminal and two lateral flowers (Fig. 3, K–P)

(Kahle-Zuber 2008). Staminate flowers have four tepals arranged in two whorls, with each tepal

containing 6–20(–50) adnate pollen chambers (Fig. 2, B, C; Fig. 3, E) (Wangerin 1937). Four-

tepalled pistillate flowers are smaller with a rotund stigma, while lacking a style, ovule, funicle,

nucellus, and integument (Fig. 2, D, E; Fig. 3, D) (Kuijt and Hansen 2015). The flowering time of

V. album is independent of the host species and phenology and predominantly stable, with

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infrequent climate-driven fluctuations (Wangerin 1937; Walldén 1961). In Ukraine, V. album subsp. album flowers in March–April with rare cases of shifted flowering in the Crimean

Mountains, where it was seen flowering in early December 2019 during the unusually warm winter (O. Kukushkin, pers. comm.).

The globose or rarely pyriform fruits of V. album subsp. album are viscous berries (a pseudoberries) up to 10 mm in diameter, containing 1 to 4 “seeds” (Fig. 2, G; Fig. 3, Q). The

“seed” is a green embryo enclosed in a thin endocarp, thick mesocarp consisting of double- layered mucilaginous viscin (so called “birds’ glue”) (Fig. 2, H, I; Fig. 3, S–U), and white or slightly yellowish epicarp having a terminal ring of four lateral (tepalar) and one central (stigmal) scars (Fig. 3, R) (Wangerin 1937; Zuber 2004; Kuijt and Hansen 2015). The “seeds” of V. album can be polyembryonal, with all embryosDraft surrounded with a chlorophyll-containing endosperm being functional and able to germinate (Kuijt and Hansen 2015). The ripening and dispersal of V. album subsp. album fruits start in November−December and last through winter, with some fruits remaining attached until spring and thus co-existing with newly emerged flowers (Wangerin

1937). In Ukraine, fruit dispersal by birds starts in late November and lasts through April.

Germination and self-sustaining growth of V. album occurs in late March−April under the optimal temperature, light and humidity conditions and ends when the holdfast forms from the hypocotyl (Fig. 2, I), followed by intrusive growth, and the switch to a hemiparasitic mixotrophic lifestyle starts after the successful establishment of haustorial xylem-xylem connections (Kuijt and Hansen 2015; Kahle-Zuber 2008).

The endophytic, or “hidden” part of the mistletoe is highly-branched (especially in the case of systemic invasion) and includes the parasite-specific organs called haustoria and cortical strands, which grow through the host wood with the help of cell wall-lytic enzymes such as

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xyloglucan endotransglycosylases, glucanases, and expansins (Zuber 2004; Ko et al. 2014; Kuijt

and Hansen 2015). The holdfast (adhesive disk) produces a single achlorophyllous primary

haustorium, which penetrates the host cambium and connects with xylem to acquire water and

nutrients. The chlorophyll-containing cortical strands running through host parenchyma or

phloem give rise to secondary haustoria (sinkers) which spread laterally and/or longitudinally

(Zuber 2004). The haustoria of V. album display host-dependent morphology, with considerable

variation in their length and branching pattern. A diverse array of haustoria were found in V.

album subsp. album during tree trimming aimed at rescuing host trees, which included closed

(terminal), semi-closed (transitional), strongly branched and other types of haustoria (V.

Leonenko, unpublished data). Draft

Distribution patterns of V. album in Ukraine

Similarly to other European counties (Zuber and Widmer 2009; Maul et al. 2019), the

distribution of V. album in Ukraine is highly fragmented and irregular, with the western and

central parts of Ukraine encompassing the bulk of known locations (Fig. 4). Our compiled

countrywide distribution data set (Table S1) includes 1338 records, of which 17 are for V. album

subsp. abietis, 28 – for V. album subsp. austriacum, and the rest (1293) – for V. album subsp.

album. Among the administrative regions (oblasts) of Ukraine, the most numerous records came

from Lviv Region (325 records), Kyiv Region (219), and Crimea (129), which altogether account

for more than half of all the country’s records of V. album. In general, the mistletoe appears most

widespread in some of the western, central, and eastern regions, with a large exclave in the

Crimean Mountains and adjacent foothills (Fig. 4). The gap in the forest-steppe and steppe

regions of the Southern (Odesa, Mykolaiv, Kherson, and northern and central parts of the

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Crimean Peninslula) and Eastern Ukraine (Luhansk, Donetsk, Zaporizhzhia regions) corroborates previous data of Rybalka et al. (2012) and Taran et al. (2008).

Unlike the conifer-associated subspecies found only sporadically with no discernible spatial trends, V. album subsp. album shows an appreciable predisposition towards urban areas such as cities, villages, industrialized sites, as well as tree stands between fields and along intercity roads, which may stem from a similar distributional patterns of suitable hosts and birds, which are the main dispersers. A much lower density of the mistletoe is observed in wild areas and natural forests, as well as orchards and gardens around private houses, although the latter is likely a consequence of mistletoe being regularly removed by landowners. The elevational distribution of V. album subsp. album in Ukraine ranges between 100 m a.s.l. (Zakarpattya

Region, Chop city northern outskirts, alongDraft Latorytsia River banks) and ca. 950 m a.s.l. (Karabi-

Yalta at the Crimean Mountains, near the source of Baysu River). Thus, the species seems to avoid high altitudes but can move slightly higher in warmer climates, which is consistent with its distribution throughout Europe (Zuber 2004).

The lack of host specificity in V. album subsp. album has enabled a much greater geographic expansion into new areas compared to the other mistletoes occurring in Ukraine, namely A. oxycedri and L. europaeus. Arceuthobium oxycedri is confined to the distribution of its principal host Juniperus deltoides R.P.Adams along the southern and south-eastern coasts and the south-western foothills of Crimea (Krasylenko et al. 2017), while L. europaeus forms small patches mostly in south-western Ukraine where it targets oaks and chestnut (Krasylenko et al.

2019). When comparing our data with older records for the eastern range limit of V. album

(Kharkiv, Izyum, Slovyansk, Sumy regions, etc.), we observe an eastward expansion with the boundary containing both highly infested host stands and solitary trees with male or female

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shrubs temporarily isolated from the rest of population. However, it remains to be verified

whether this eastward expansion has purely anthropogenic origins or follows climate change as it

was predicted by Jeffree and Jeffree (1996).

Host range of V. album in Ukraine

Mistletoes are known to display a variety of host specificity patterns, from very narrow to

as broad as including hundreds of species (Hawskworth and Wiens 1996; Barney 1998; Kuijt and

Hansen 2015; Maul et al. 2019). Host specificity is also the main distinguishing character for the

European subspecies of V. album. Viscum album subsp. album is a generalist targeting deciduous

dicotyledonous woody plants, while the other two subspecies − V. album subsp. abietis and subsp. austriacum − show pronouncedDraft host preferences towards some coniferous taxa. In Ukraine, century-long studies (Larionov, 1912; Kochno 1960; Bulgakova 1977; Doroshenko

2005; Rumyankov 2010) and data from herbaria, on-line databases, personal communications,

and our own observations indicate that V. album subsp. album occurs on at least 123 host species

(including 83 allochthonous species and subspecies) from 37 genera and 17 families (Fig. 5, A;

Table S1). Of all 1293 records compiled in our data set for this subspecies (Table S1), 559

records have identified hosts at least at the generic level. The majority of hosts belong to

Rosaceae (45 species), Betulaceae and Salicaceae (15 species each), and Sapindaceae (13

species). Families such as Fagaceae, Juglandaceae, Malvaceae, and Oleaceae are represented by

5−6 host species, while the rest include one or two species. It is noteworthy that in Ukraine cases

of hyper-parasitism have been observed, i.e., mistletoe parasitizing individuals of its own species.

Comparison of the host families based on the number of records (Fig. 5, B) shows Rosaceae and

Salicaceae to be the most frequent host taxa, accounting for over half of all the records with

identified hosts. Less frequent hosts include members of Betulaceae, Sapindaceae, and 13

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Malvaceae. Interestingly, some of the families with low host species number appear to include important hosts of V. album, such as the family Fabaceae of which the majority of records came from a single species – Robinia pseudoacacia L. At the generic level, several host categories can be recognized based on the number of records: primary hosts with 37−91 records (including

Acer, Betula, Crataegus, Malus, Populus, Robinia, Salix, and Tilia), secondary hosts with 12−18 records (Fraxinus, Prunus, Pyrus, Quercus, and Sorbus), and accidental (or minor) hosts with

1−6 records (the remaining 24 genera).

The presence of several climatic zones with boreal, alpine, steppe, asiatic and mediterranean elements in Ukraine may explain the high variety of host species, representing almost one third of the all-European host taxa reviewed by Barney (1998). Host records of V. album subsp. album from other EasternDraft European countries list 35 taxa from 17 genera in

Slovakia, 53 taxa (including five hybrids) in the Czech Republic, and 194 taxa in Poland

(reviewed by Kahle-Zuber 2008). Earlier reports from Central and Western Europe indicated V. album subsp. album parasitizes species of 36 genera (Kahle-Zuber 2008). Among southern

European countries, 52 hosts (33 autochthonous and 15 allochthonous species, two cultivars and two hybrids) have been recorded in Croatia and 25 hosts (of which 4 allochthonous species) in

Slovenia (Kahle-Zuber 2008). In the Mediterranean, there are 12 host records from the Pyrenees and 53 from the Iberian Peninsula (Kahle-Zuber 2008). However, to be comparable, all this data requires taxonomical update and unification.

The other two V. album subspecies have been spotted in Ukraine on only two coniferous hosts: Abies alba Mill. (parasitized by V. album subsp. abietis) and Pinus sylvestris L. (host for

V. album subsp. austriacum) (Table S1). Although the latter subspecies is known to associate

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with two more coniferous host genera (Larix and Picea), no records of them hosting V. album are

known from Ukraine so far.

Besides the taxonomically recognized V. album subspecies, other definitions

characterizing mistletoe host preference have been applied, such as forms, varieties, ecomorphs,

and host races. Formation of genetically distinct host-specific races possessing selective

advantages contributes to microevolutionary processes in many groups of parasitic organisms,

e.g. mistletoes, fungi, insects (lice, herbivores, and parasitoids), trematodes, and viruses

(Okubamichael et al. 2017; Maul et al. 2018). Among the factors shaping mistletoe host

specificity are host abundance and compatibility (genetic, morphological, physiological, and

chemical), life history, environmental conditions, pollination (e.g., important for isolation of the

sympatric Tristerix species) (Medel et al.Draft 2002), and seed dispersal vectors (e.g., bright exocarp

coloration to target specific frugivorous birds) (Okubamichael et al. 2017).

Biotic interactions of V. album

Birds as primary vectors of V. album seed dispersal

Bird-mediated seed dispersal, or ornithochory, is predominant among mistletoes (Wenny

2001). The paradigmatic mistletoe-host-vector mutualistic relationships are mainly represented

by two co-existing systems: highly specialized frugivorous dispersers, which co-evolve with the

mistletoe, and mixed dispersal systems with the input of both specialist and generalist bird

species (Watson and Rawsthorne 2013). The generalist frugivores are less efficient vectors

compared to specialists; however, they can establish new infection sites and facilitate long-

distance seed dispersal (Montaño-Centellas 2012).

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According to Reid (1991), Pleistocene glaciation periods lead to a disruption in the coevolution of mutualistic mistletoe-host-vector systems in the northern hemisphere. Accordingly, mistletoes of Holarctic ecosystems as well as V. album are only dispersed by generalist vectors.

Among the generalists, mistle thrush (Turdus viscivorus Linnaeus, 1758) is the main V. album seed vector in Europe (Marisova 1963; Cupedo 1985; Snow and Snow 1988; Szmidla et al.

2019). Other European thrushes – song thrush (T. philomelos C.L.Brehm, 1831), redwing

(T. iliacus Linnaeus, 1758), fieldfare (T. pilaris Linnaeus, 1758), blackbird (T. merula Linnaeus,

1758), and ring ouzel (T. torquatus Linnaeus, 1758) – also disperse mistletoe seeds (Walldén

1961; Mellado and Zamora 2014).

Viscum album is an important nutrient resource for T. viscivorus in winter (Skórka and

Wójcik 2005). Within the last decade, Draft the number of T. viscivorus has been increasing in the

Khmelnytskyi region and Rivne Nature Reserve in Western Ukraine as well as the Rostov and

Stavropol regions in Russia (Belik 2003; Novak 2010; M. Franchuk, pers. comm.). It is noteworthy that T. viscivorus defends its wintering territory with abundant food resources against conspecifics and other frugivores such as T. merula, T. iliacus, T. philomelos, waxwing

(Bombycilla garrulus Linnaeus, 1758) and bullfinch (Pyrrhula pyrrhula Linnaeus, 1758) during the non-breeding period (Snow and Snow 1988; Skórka and Wójcik 2005). The discrete distribution and cluster character of V. album facilitate T. viscivorus defense of its feeding territory. Moreover, mistle thrush abundance in Poland and Hungary positively correlates with V. album fruiting (Figarski 2009; Varga et al. 2014).

Bombycilla garrulus is a highly specialized frugivore and an important V. album vector in some northern regions of Europe (e.g., Scandinavia, Northern Germany) and Asia (e.g., Russia)

(Walldén 1961; Cupedo 1985). In Ukraine, B. garrulus is a wintering and migratory species from

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Fennoscandia and Eastern Russia (Poluda 2017). While the number of birds may vary during the

winter and among different years, B. garrulus is considered a key vector of V. album in some

urban landscapes of Eastern Ukraine (Vergeles 2009). The driving factor of such food preference

is likely a competition between thrushes (Turdus ssp.) and waxwings for their primary food

source – rowan (Sorbus aucuparia) fruit, causing waxwings to switch their winter food sources to

V. album (Horban et al. 2005).

The main dispersers of the related V. coloratum are B. garrulus and B. japonica in China,

Japan, Korea and Russian Far East, while the Asian thrushes – the Naumann’s thrush

(T. naumanni Temminck, 1820) and dusky thrush (T. eunomus Temminck, 1831) – seem to be a

secondary vector (Nechaev 2008; Atroshenko et al. 2011). The waxwings disperse V. album

seeds a short distance due to their extremelyDraft fast passing through the bird’s digestive system

(Holthuijzen and Adkisson 1984; Zubar 2004).

Blackcap (Sylvia atricapilla (Linnaeus, 1758)) is a crucial V. album disperser in the

Mediterranean region (e.g., France and Spain) (Snow and Snow 1988; Vallauri 1998; Mellado

and Zamora 2014), though this insectivorous bird is a sedentary species consuming V. album

fruits only in winter due to the lack of its usual food. However, in northern France, the blackcap

is a migratory species consuming V. album during the last cold days of spring when it returns to

its breeding sites before insects appear (Frochot and Sallé 1980). In Western Ukraine, such

foraging was observed during early spring in the first week of April (O. Dubovyk, pers. comm.).

Sylvia atricapilla demonstrates a different type of feeding compared to waxwings and thrushes. It

does not swallow and defecate the large V. album fruits, but consumes only the pulp, squeezing

out the sticky seeds and allowing them to adhere to the host tree stems and branches.

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Mellado and Zamora (2014) found that S. atricapilla is quite an efficient epizoochorous mistletoe vector for the Austrian pine (Pinus nigra Arm.) as a host, since it tends to deposit the seeds on a suitable site for germination. On the other hand, T. viscivorus provides a low- efficiency service due to the non-targeted “seed rain” strategy and an abundant fruit loss, though playing an important role because of the large quantity of consumed fruits. Surprisingly, the most efficient mistletoe seed dispersers on P. niger are dietary opportunists – blue tit (Cyanistes caeruleus Linnaeus, 1758), great tit (Parus major Linnaeus, 1758), and coal tit (Periparus ater

Linnaeus, 1758). Meanwhile, for deciduous tree hosts, blue tit, great tit and marsh tit (Poecile palustris Linnaeus, 1758) are mentioned strictly as mistletoe seed predators, which forage V. album seeds from the branches, thus preventing mistletoe dispersal (Frochot and Sallé 1980;

Vallauri 1998). Draft

Thus, V. album is dispersed in Europe by two groups of generalists with distinct feeding approaches: 1) low-efficient thrushes and waxwings due to non-targeted dispersal and massive seed loss via the defecation at some distance from the parental host tree; 2) comparatively efficient targeted blackcaps that consume only the mistletoe fruit pulp, leaving the viscin-coated mistletoe seeds at a suitable site for germination on the parent or neighboring host trees. Hence, the dual dispersal mechanism – endo- (thrushes) and ectozoochorous (blackcaps) – promotes an effective dispersal system for V. album in Europe by generalist bird species, although Mellado and Zamora (2014) deals with a single region and a specific coniferous host.

Overall, the dispersal efficiency analysis disregarding the spatial arrangement of the dispersed seeds appears insufficient for understanding the colonization process and defining the role of particular bird species in the long-distance mistletoe dispersal and establishment of novel colonization patches (Overtone 1994). Due to its feeding strategy, S. atricapilla seems to be an

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efficient V. album vector among the already infected patches in Southern Europe, but not for the

generation of new ones, although this viewpoint needs further research. Thrushes are sometimes

considered as vectors for long-distance mistletoe dispersal along their migratory routes,

generating the new sites of colonization (Cupedo 1985; Zuber 2004). However, experimental data

on spatio-temporal and metapopulation structure of V. album is still lacking.

The Corvidae – magpie (Pica pica Linnaeus, 1758), jay (Garrulus glandarius Linnaeus,

1758), nutcracker (Nucifraga caryocatactes Linnaeus, 1758) and rook (Corvus frugilegus

Linnaeus, 1758) – were mentioned in some sources as potential vectors for V. album, but data on

the efficiency of seed germination after passing through their digestive system are absent

(Frochot and Sallé 1980; Horban et al. 2005; Nechaev 2008). Generally, the mistle thrush,

waxwing, blackcap and blackbird are assumedDraft as main alleged V. album vectors in Ukraine (see

Fig. 6.).

Mistletoes support a biodiversity of bird communities and promote species richness

providing nesting and roosting sites, foraging substrate and nutritional resource in many world’s

biotas (Cooney et al. 2006; Watson and Herring 2012). Some mistletoe species produce fruits

during times when food is lacking, e.g., in winter (Zuria et al. 2014). In Europe, V. album is an

important source for birds foraging during non-breeding and winter periods (Snow and Snow

1988; Horban et al. 2005; Wojtatowicz and Pietrzykowska 2018). Turček (1961) reported 28 bird

species to be trophically associated with V. album fruits and seeds in Europe, while Nechaev

(2008) listed 18 species feeding on the related mistletoe species V. coloratum in the Russian Far

East.

However, the role of V. album and related Viscum species as a keystone resource for bird

nesting and roosting still remains unknown. Our recent study of mistletoes used as a nesting site

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or nest substrate in Ukraine revealed 21 associated bird species, including black kite (Milvus migrans Boddaert, 1783), long-legged buzzard (Buteo rufinus Cretzschmar, 1829), common buzzard (Buteo buteo Linnaeus, 1758), magpie (Pica pica), jay (Garrulus glandarius), hooded crow (Corvus cornix Linnaeus, 1758), raven (Corvus corax Linnaeus, 1758), wood pigeon

(Columba palumbus Linnaeus, 1758), collared dove (Streptopelia decaocto Frivaldszky, 1838), song thrush (Turdus philomelos), fieldfare (Turdus pilaris), blackbird (Turdus merula), long- tailed tit (Aegithalos caudatus Linnaeus, 1758), golden oriole (Oriolus oriolus Linnaeus, 1758), house sparrow (Passer domesticus Linnaeus, 1758), tree sparrow (Passer montanus Linnaeus,

1758), chaffinch (Fringilla coelebs Linnaeus, 1758), linnet (Linaria cannabina Linnaeus, 1758), goldfinch (Carduelis carduelis Linnaeus, 1758), greenfinch (Chloris chloris Linnaeus, 1758), and hawfinch (Coccothraustes coccothraustesDraft Linnaeus, 1758) (Srebrodolskaya 1998; Atamas et al., unpublished data).

Many aspects of the mistletoe-host-vector interaction remain to be addressed. These include the role and efficiency of particular bird species in dispersing V. album and shaping its spatio-temporal population structure, the impact of various factors on host colonization success, as well as the importance of V. album as the nesting site and food source for birds and its effect on local patterns of bird diversity and abundance.

Insects

The diversity of insects found to interact with V. album in some way can be divided into three groups: flower pollinators/visitors, primary herbivores, and secondary feeders or “transient” species. Despite the combination of entomo- and anemophilous features in Viscum flowers and presumed domination of wind pollination in most species, at least when male and female plants

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are in close proximity (Wangerin 1937; Walldén 1961; Hatton 1965), the input of wind

pollination in V. album is believed to be minor (Kujit 1969; Hawksworth and Wiens 1996; Kahle-

Zuber 2008). Insect pollination is crucial for V. album, as evidenced by studies reporting that the

pollen chambers remain intact and do not release the barbed pollen covered with pollen kitt

forming clumps until an insect’s visit to the flower, and the fact that male flowers have an intense

scent (Wangerin 1937; Walldén 1961; Kahle-Zuber 2008).

The main pollinators of V. album are believed to be flies (order Diptera) (Walldén 1961;

Hatton 1965; Kahle-Zuber 2008). These include members of the Calliphoridae (Calliphora vicina

Robineau-Desvoidy, 1830, Pollenia rudis (Fabricius, 1794), P. vespillo (Fabricius, 1794)),

Heleomyzidae (Heteromyza rotundicornis (Zetterstedt, 1846)), Milichiidae (Madiza glabra

Fallén, 1820), Muscidae (Dasyphora cyanellaDraft (Meigen, 1826), Helina reversio (Harris, 1780),

Musca autumnalis De Geer, 1776, Opsolasia spp.), Scatopsidae (Reichertella pulicaria (Loew,

1846)), Sepsidae (Sepsis cynipsea (Linnaeus, 1758), S. flavimana (Meigen, 1826)) and others

(Walldén 1961; Hatton 1965; Kahle-Zuber 2008).

While both staminate and pistillate flowers produce nectar, the latter are more

nectariferous (Walldén 1961). Male flowers have more intense odour, which attracts bees (Apis

mellifera (Linnaeus, 1758)) and bumble bees (Bombus terrestris (Linnaeus, 1758))

(Hymenoptera, Apidae) (Walldén 1961; Kahle-Zuber 2008). It is noteworthy that under the same

environmental conditions female flowers open earlier than male flowers (Tubeuf 1923).

Herbivorous insects that frequently (and often exclusively) live on fresh green and dead

mistletoe parts and thus regarded to be primary mistletoe feeders include members of three insect

orders: Coleoptera (Agrilus spp. (Buprestidae), Ixapion variegatum (Wencker, 1864), Liparthrum

bartschi Mühl, 1891 (Curculionidae)), Hemiptera (Pinalitus viscicola (Puton, 1888), Hypseloecus

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visci (Puton, 1888) (Heteroptera, Miridae), Anthocoris visci Douglas, 1889 (Heteroptera,

Anthocoridae), Carulaspis juniperi (Bouché, 1851), C. visci (Schrank, 1781) (Sternorrhyncha,

Diaspididae), Cacopsylla visci (Curtis, 1835) (Sternorrhyncha, Psyllidae)), and Lepidoptera

(Synanthedon loranthi (Králíček, 1966) (Sesiidae)) (Schumacher 1918; Tubeuf 1923; Varga et al.

2012b; García Morales et al. 2016; Szmidla et al. 2019). The most common beetle species colonizing and consuming mistletoe shoots, which may potentially contribute to mistletoe mortality belong to the genus Agrilus (Curtis, 1825), e.g., A. graecus Obenberger, 1916

(= viscivorus Bílý, 1991), A. jacetanus Sánches Sobrino & Tolosa Sánchez, 2004, A. kutahyanus

Królik, 2002, and A. roscidus Kiesenwetter, 1857 (Jendek and Poláková 2014; Jendek 2016).

Although Agrilus species seem prospective biocontrol agents against V. album, this can only be achieved when there is a mass occurrenceDraft of the species on the plants (Varga et al. 2012b).

Nevertheless, a relatively long lifespan of V. album leaves (up to 3 years; Wangerin 1937) potentially allows the insects to produce several generations until leaf senescence, gradually increasing their numbers.

Secondary feeders and transient insect species, switching from mistletoe to other hosts and being rarely found on V. album, include a number of beetle species, namely from the

Anthribidae, Laemophloeidae, Cerambycidae, Corylophidae, Curculionidae, and Ptinidae

(Schumacher 1918; Tubeuf 1923; Varga et al. 2012b; Szmidla et al. 2019). Other rare finds reported from V. album include some hemipterans, namely Campyloneura virgule (Herrich-

Schaeffer, 1835) (Heteroptera, Miridae), Pentatoma rufipes (Linnaeus, 1758) (Heteroptera,

Pentatomidae), Aphis fabae group spp., Tuberaphis coreana Takahashi, 1933, T. viscisucta

(Zhang, 1985) (Sternorrhyncha, Aphididae), a number of polyphagous scale insects from the

Coccidae, Diaspididae, Lecanodiaspididae and Pseudococcidae (Sternorrhyncha, Coccoidea), as

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well as an ant species Lasius brunneus (Latreille, 1798) (Hymenoptera, Formicidae) (Varga et al.

2012b; García Morales et al. 2016; Blackman and Eastop 2020). Finally, two rare moths, Celypha

woodiana (Barrett, 1882) and Ditula angustiorana (Haworth, 1811) (Tortricidae) have also been

noted to develop on V. album (Schumacher 1918; Tubeuf 1923; Szmidla et al. 2019).

Fungi, bacteria and lichens

Despite the presence of viscotoxins in V. album believed to have a strong inhibitory effect

on some plant pathogens (Giudici et al. 2004), the species can support quite diverse

microorganismal communities. Endophytic micromycetes spontaneously associating with V.

album have been studied in Austria and Germany (Peršoh 2013), Hungary (Varga et al. 2012d),

India (Sadananda et al. 2014), Serbia Draft(Karadžić and Lazarov 2005), and Turkey (Kotan et al.

2013). These studies combined with miscellaneous reports (e.g., Ruszkiewicz-Michalska et al.

2015; Rashmi et al. 2019) have yielded ca. 90 fungal taxa from 50 families, 32 orders, and 13

classes associated with V. album (Table S2). These exclude a large number of OTUs (operational

taxonomic units) unclassifiable below the order or phylum ranks detected by molecular methods

(Peršoh 2013), as well as taxa found to be associated with V. album only experimentally

(Czeczuga et al. 2010). Ascomycota appear prevalent both in terms of diversity and frequency

(ca. 65 taxa from 34 families), dominated by the Dothideomycetes (especially Capnodiales and

Pleosporales) and Sordariomycetes (Hypocreales, Xylariales, etc.).

The majority of the above fungi are saprobic hyphomycetes (or mould-forming fungi)

with broad ecological potential frequently found as plant endophytes (Sridhar 2019). Common

species include some Capnodiales (Cladosporium), Eurotiales (Aspergillus, Penicillium),

Glomerellales (Verticillium), Hypocreales (Acremonium, Fusarium), Pleosporales (Alternaria),

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and Zygomycota (Mortierella, Rhizopus). Other fungal groups include ascomycetous

(Debaryomyces, Dipodascus) and basidiomycetous yeasts (Cryptococcus, Erythrobasidium,

Malassezia, Trichosporon), as well as several wood-decaying and corticioid taxa

(Peniophoraceae, Xylariales). Some of the sequences obtained by Peršoh (2013) from Pinus- associated V. album leaves and stems matched basidiomycetous fungi from Boletaceae,

Cantharellaceae, Russulaceae, and Thelephoraceae which are known to be ectomycorrhizal associates of woody plants (Table S2). Other infrequent taxa include the resinicolous discomycete Sarea difformis (Fr.) Fr. (Trapeliales) and the mycophile Trichoderma viride Pers.

(Hypocreales) (Karadžić and Lazarov 2005; Peršoh 2013). Endophytic occurrence of S. difformis in the Pinus-associated V. album subsp. austriacum (Peršoh 2013) is likely related to its narrow substrate specificity to conifer resins (BeimfordeDraft et al. 2020). Unless misidentified, all these fungi may compose a “latent” part of Viscum-associated mycobiome comprising a diversity of functional guilds of which the litter-decomposing fungi are presumably the dominant group

(Peršoh 2013). A number of identified fungi are known to be potential plant pathogens, particularly those from the genera Alternaria, Aureobasidium, Botryosphaeria, Colletotrichum,

Cylindrodendrum, Leptosphaeria, Nectria, etc. (Table S2). This group also includes several specific associates of V. album, i.e. Botryosphaeria (= Gibberidea, Sphaeropsis) visci (Kalchbr.)

Arx & E.Müll., Phyllosticta visci (Sacc.) Allesch., and Rhabdospora (= Septoria) visci (Bres.)

Died., which have been referred to as potential biocontrol agents against mistletoes (Kotan et al.

2013). Community-level studies (Peršoh, 2013) indicate that diversity and distribution patterns of fungal endophytes in V. album subsp. austriacum are largely determined by plant organ selectivity by fungi (i.e., young vs. old leaves vs. stems) and composition of fungal communities in the surrounding environment. The host-plant preference of fungi towards V. album and its host

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Pinus sylvestris may vary depending on spatiotemporal factors, leading to similarities or

substantial differences in terms of fungal communities between the two plant associates.

By contrast, reports on epiphytic symbiont communities of V. album appear to be

lacking. Our own observations indicate that green vegetative parts of V. album, especially the

more lignified sites such as stem nodes, frequently become colonized by various lichen species

also growing on the bark of host plants (Fig. 7). In addition, the fruits of V. album have been

experimentally shown to be a suitable substrate for some aquatic fungi and fungus-like

organisms, such as Gonapodya prolifera (Cornu) A.Fisch. (Chytridiomycota) and various

Oomycota (Achlya, Leptomitus, Pythium, Saprolegnia, etc.; Table S2) often collectively termed

“water moulds” (Czeczuga et al. 2010). These organisms are known to commonly occur in water-

submerged decaying plant materials Draft and capillary networks of soils, and sporadically as

endophytes of terrestrial plants (Jones et al. 2014). Hence, they may constitute an important part

of microorganismal communities involved in the decomposition of fallen fruits of V. album

(Czeczuga et al. 2010).

Endophytic bacteria of V. album have only been studied by Kotan et al. (2013), who

identified 30 genera belonging to 7 classes of Gram-positive (Actinobacteria and Firmicutes) and

Gram-negative (Bacteroidetes and Proteobacteria) bacteria isolated from V. album growing at

several locations in Turkey (Table S2). With respect to taxonomic diversity, dominant groups

were Actinobacteria (9 genera from 5 families) and Proteobacteria (14 genera from 12 families).

Most abundant genera (based on the number of isolated strains) included Acinetobacter,

Pseudomonas, Stenotrophomonas (Proteobacteria), and Bacillus (Firmicutes). Ecologically, these

bacteria include many ubiquitous saprobes (particularly in Proteobacteria and Firmicutes), though

a large number of plant endophytes is known among Actinobacteria (Govindasamy et al. 2014).

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No published data on V. album microbiome is currently known from Ukraine and the study has just been initiated by co-authors of the present paper.

Considering the specific ecological niche of V. album and its inclusion in multiple trophic chains (as indicated by its diverse symbiont communities), more studies are needed to reveal the contribution of the associates and complex interaction patterns to V. album distribution and fitness. For instance, recent findings indicate that the composition of epiphytic microorganismal communities may influence, through volatile emissions, the functioning of other trophic levels, such as insect parasitoid-hyperparasitoid systems (Goelen et al. 2020), which is likely mirrored by similar effects on other insect groups including herbivores and inquilines. Furthermore, nectar-dwelling microbiota affecting foraging insects (Álvarez-Pérez and Herrera 2013; Pozo et al. 2020) may contribute to pollination patternsDraft and, ultimately, genetic diversity and dispersal of

V. album.

Plant hyperparasites

Viscum album subsp. album was found to parasitize L. europaeus in Austria, Switzerland, as well as Italy and was even recommended as a biocontrol agent (Grazi and Urech 1985).

However, in the New World, Viscaceae have rarely been reported on Loranthaceae with the exception of Phoradendron iltisiorum Kuijt on Cladocolea pringlei Kuijt (Nickrent 2010).

Monitoring and management of V. album

A balanced strategy for the sustainable management of mistletoe needs to combine the benefits of accurate detection and monitoring with specifically tailored reduction strategies. The presence of mistletoe can be revealed through high resolution imagery of tree canopies using

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modern image recognition tools based on machine learning (Liu et al. 2019b). For forests and

rural areas, such imagery is commonly acquired using unmanned aerial vehicles (UAV) (Zhang

2016; Nevalainen 2017) that are becoming ubiquitous and increasingly affordable. In such areas,

UAVs allow the gathering of images in a repeatable manner by automatically following a pre-

programed path, which is only limited by the maximum flight time of the vehicle (typically

30−120 min, depending on UAV type). This method of mistletoe monitoring is limited to areas of

20−30 km around existing road infrastructure. Data for more remote locations can be

extrapolated from the measurements in available localities, assuming the information on bird

populations and average forest canopy composition is available. A combination of fieldwork and

airborne imaging spectroscopy helped to map both host trees (Quercus lobata, Q. douglasii, and

Q. kellogii) and hemiparasitic PhoradendronDraft leucarpum with high accuracy, and to evaluate the

mistletoe prevalence over oak trees located in different landscape contexts (Barbosa et al. 2016).

Due to the rapidly changing landscape and restriction of flying in populated areas, the

automated operations of UAV are not permitted in urban regions. For such places street view

image data such as “Street View” on Google Maps 2019, “Baidu Total View” on Baidu or

“Panorama” on Mapy.cz containing the panoramic photos of locations in the vicinity of existing

roads and postal addresses can be used. These image data with global coverage are being updated

automatically and are freely available for academic use. The achievements of several existing

projects (Branson 2018; Chen 2019b; Stubbings 2019) using street view image data to estimate

the distribution as well as composition of vegetation in urban ecosystems can be adapted for

mistletoe observation and the evaluation of the infection rate in Ukraine. This could also be tested

using a special UAV called “Druid Drone” with a functional manipulator adapted to mistletoe

observation and collection (Krasylenko, unpublished data).

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Several approaches for mistletoe control, namely mechanical, chemical, biological, and hands-on, exist. Since their presence on hosts contributes to increased loading from rain, frost and snow, mistletoes have to be regularly pruned, sometimes with parts of the affected host branches, or bandaged for the prevention of re-sprouting (Shaw and Mathiasen 2013). In Ukraine, the radical removal of affected branches or even whole trees is a common practice for mistletoe management. A less aggressive method based on cutting the exophyte on the host branch, excising the haustoria within the host, and filling the wound with a special paste containing wound regeneration stimulants, fungicides and antibiotics has been recently introduced into the arboristic practice (V. Leonenko, unpublished data). Chemical approaches include spraying the infected trees with growth regulators such as monocompounds or composite mixtures, e.g., ethylene-releasing defoliants and ripeningDraft enhancers (Florel®, ethephon ((2-chloroethyl)- phosphonic acid), systemic herbicides (synthetic auxins 2,4-dichlorophenoxyacetic, 4-(2,4- dichlorophenoxy)butyric or 2,4,5-trichlorophenoxyacetic acids, glyphosate), arboricides and organic solvents on the waxy leaf surfaces of mistletoes (reviewed by Kahle-Zuber 2008).

However, the use of herbicides, especially in urban areas, is ecologically questionable, inefficient

(often targeting only the exophyte with the endophyte still anchored inside the host wood), challenging for hosts, and expensive (Kahle-Zuber 2008).

Biological methods include the avoidance of tree monocultures and common host species such as poplars, maples, lindens, and especially rosaceous fruit trees whose long-term cultivation in orchards, often in close proximity to other suitable hosts is believed to account for the increase of V. album subsp. album in urban areas (Wangerin 1937). This should be combined with the planting of the deciduous trees along with conifers as natural or artificial mixed lines of buffer strips in areas adjacent to the zone of mistletoe mass occurrence, as well as the introduction of the

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rarely infected and relatively resistant hosts (e.g., Quercus, Cercis, Paulownia, Koelreuteria,

Larix, Ulmus, Catalpa, Platanus, Ailanthus, Ginkgo, Cotynus, etc.). The application of

phytopathogenic organisms also shows promise in V. album biocontrol. The European mistletoe

tends to be affected by several pathogens (see Table S2), many of which can cause disease

symptoms but have either broad host-plant range (e.g., Botryosphaeria dothidea (Moug.) Ces. &

De Not., Nectria cinnabarina (Tode) Fr., etc.) or limited pathogenic capacity, which renders them

unlikely biocontrol agents (Karadžić and Lazarov 2005; Kotan et al. 2013). Pathogenicity tests

conducted by Kotan et al. (2013) for a range of bacteria and fungi suggested the host-specific

strains of Alternaria alternata (Fr.) Keissl. and Sarocladium (= Acremonium) kiliense (Grütz)

Summerb. as prospective candidates for mistletoe biocontrol, which can be applied both by

injection and spray methods. However,Draft no further studies of the biology and effectiveness of

these strains are known. The fungus Botryosphaeria visci is increasingly researched for its high

efficiency against V. album and ability to cause severe infection of all mistletoe parts regardless

of the application method (Varga et al. 2012d), although certain systemic herbicides have been

noted to inhibit the fungus (Varga et al. 2012c). Recently, B. visci has been studied both

genetically (Varga et al. 2012d; Poczai et al. 2015) and with regard to cultivation and production

techniques to facilitate its use in biocontrol (Varga et al. 2012a); however, many aspects of its

ecology, population dynamics, and co-evolution with V. album remain unknown.

Finally, hands-on approach involves V. album targeted harvesting and collection for

pharmacological, forage and medicinal needs as well as for the decorative purposes (Fig. S1).

Conclusions and future perspectives

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In Ukraine, the range of the promiscuous (polyphagous) V. album subsp. album is characterized by a patchy distribution of populations with gaps in the south and east of the country, while the highly specialized V. album subsp. abietis (Wiesb.) Janch. and V. album subsp. austriacum (Wiesb.) Vollm. are confined mostly to the western and central regions and the mountainous part of the Crimean Peninsula. It was found that the multicomponent flora of

Ukraine has a plethora of V. album hosts comprising 123 auto- and allochthonous representatives of 17 families, among which Rosaceae, Salicaceae, Betulaceae, and Sapindaceae are most common. Future research of V. album in Ukraine should focus on the climatic and edaphic factors limiting its distribution revealed by GIS-modelling, trophic interactions and contribution to organismal communities, as well as the efficiency of different approaches for its monitoring and management, including those involving DraftUAVs.

Acknowledgements

We apologize to all colleagues whose original works could not be cited due to space constraints.

The authors are grateful to the curators of the consulted Ukrainian herbaria namely CBR

(Carpathian Biosphere Reserve, Rakhiv), CHER (Yu. Fedkovych Chernivtsi National University,

Chernivtsi), KW (M.G. Kholodny Institute of Botany, National Academy of Sciences (NAS) of

Ukraine, Kyiv), KWHA (M.M. Gryshko National Botanical Garden, NAS of Ukraine, Kyiv),

KWHU (O.V. Fomin Botanical Garden of National Taras Schevchenko University of Kyiv,

Kyiv), LW (Ivan Franko National University of Lviv, Lviv), LWS (State Museum of Natural

History, NAS of Ukraine, Lviv) and LWKS (Institute of Ecology of the Carpathians, NAS of

Ukraine, Lviv). Nadya Kapets (M.G. Kholodny Institute of Botany, NAS of Ukraine, Kyiv) identified lichen species on Viscum. The respondents of the Ukrainian Botanical Group

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(https://www.facebook.com/groups/flora.ukraine/) and UkrBIN (http://www.ukrbin.com/), whose

names are listed in Table S1, and especially Yulya Yurchenko, are appreciated for personal

observations of new localities of V. album in Ukraine. We cordially thank Oleg Kukushkin

(Crimea) for organizing the botanical field trips at the Crimean Peninsula during 2001−2020.

Microscopy studies of mistletoe morphology using V16 Stereo Zoom system (Carl Zeiss,

Germany) were partially supported by the European Regional Developmental Fund (ERDF)

project “Plants as a tool for sustainable global development”

(No. CZ.02.1.01/0.0/0.0/16_019/0000827). The authors also greatly appreciate the invitation

from D.C. Shaw (Oregon State University, Corvallis, Oregon) and S.F. Shamoun (Natural

Resources Canada, Ottawa, Ontario) to contribute to the mistletoe special issue. Private joint

stock company (PJS) “Carlsberg Ukraine”Draft (Kyiv, Ukraine) supported the development of UAV

called Druid Drone for mistletoe observation and samples collection. We thank Daniel Steele

(University of California, Davis) for improving the language as a native speaker.

Authors’ contributions

YK designed the project and worked on mistletoe biology, host preferences, and management.

YS reviewed mistletoe taxonomy. YK, YS and NS summarized distribution data, DS generated

the distribution map. YK, YS and NS collected and evaluated the host data, AN treated mistletoe-

bird interactions, and YS and GP described associations with insects and microorganisms. KJ

made line drawings and analyzed microscopy images. YK, VL, DS, KR, and YS prepared the

methodological recommendations on mistletoe control in Ukraine. All co-authors contributed to

the manuscript writing and approved the final version.

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Conflict of interest

Authors declare that they have no conflict of interest associated with this study.

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Figure legends

Fig. 1. Subspecies of V. album occurring in Ukraine: (A) V. album subsp. аlbum, host Malus

pumila Mill.; (B) V. album subsp. аbietis, host Picea abies (L.) H.Karst.; (C) V. album subsp.

аustriacum, host Pinus sylvestris L. Photos: (A) Yevhen Sosnovsky; (B, C); Yuliya Krasylenko.

Fig. 2. Viscum album subsp. album graphical drawing: (A) habit; (B) part of branch with male

(staminate) flower; (C) male (staminate) flower and corolla lobe with anther; (D, E) female

(pistillate) flower; (F) primordium; (G) fruit on the pedicle; (H) seed without viscin coat; (I)

germinating seed. Scale bar: (B, C, D) = 1 cm. Illustrations: Kateřina Janošíková. Draft

Fig. 3. Morphology of Viscum album subsp. album: (A) infestation of host trees by V. album

associated with branch failure of host tree, Pyrus eleagrifolia Pall. (Kapshor village

surroundings, South Coast of the Crimean Peninsula, March 23, 2019); (B) close-up of an

exophyte; (C) hypertrophy of a host branch; (D) female (pistillate) flower; (E) male (staminate)

flower; (F−P) primordia; (Q) fruit; (R) terminal ring of four lateral (tepalar) and one central

(stigmal) scars; (S) viscin-coated seed; (T) seed with dried viscin; (U) seed with mechanically

destroyed viscin. Scale bars: (C, D, E, L, M, P, Q, T, U) 2 cm; (F−P, K, O, S) 1 cm; (R) 500 μm.

Photos: (A, B) Oleg Kukushkin; (C) Yevhen Sosnovsky; (D−U) Yuliya Krasylenko.

Fig. 4. Distribution of three Viscum album subspecies in Ukraine. Circles designate V. album

subsp. аlbum, squares – V. album subsp. аbietis, and triangles – V. album subsp. аustriacum. The

intensity of the color reflects the number of observations. The map was constructed using QGIS

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software (A Free and Open Source Geographic Information System, www.qgis.org) based on own observations during 2017 – 2020 field trips, data derived from consulted herbaria and public databases (such as UkrBIN, iNaturalist, and Plantarium), and personal communications (see

Table S1 for localities and name references).

Fig. 5. Host-plant families of Viscum album subsp. album in Ukraine and their frequency based on the number of species (A) and observations (B).

Fig. 6. Birds as Viscum album vectors in Ukraine: (A) Bombycilla garrulus (Kharkiv); (B)

Turdus viscivorus (Kharkiv); (C) Turdus merula (Kyiv); (D) Sylvia atricapilla (Lviv). Photos:

(A, B) Igor Kozitskiy; (C) Oleg Snitzar;Draft (D) Olexiy Dubovyk.

Fig. 7. Lichens growing on Viscum album: Xanthoria c.f. parietina (L.) Th.Fr. (Teloschistaceae)

(yellow) and Physcia c.f. tenella (Scop.) DC. (Physciaceae) (grey or greenish): (A−D) host

Crataegus sp., photos by Yuliya Krasylenko and Oleg Kukushkin (Kok-Asan-Bogaz Pass,

Crimean Peninsula, January 17, 2020); (E, F) host Crataegus monogyna Jacq., photos by Yevhen

Sosnovsky (Lviv city, March 19, 2020). Lichens were identified by Nadya Kapets. Scale bars:

(C, D) – 1 cm; (E, F) − 2 mm.

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Fig. 1. Subspecies of V. album occurring in Ukraine: (A) V. album subsp. аlbum, host Malus pumila Mill.; (B) V. album subsp. аbietis, host Picea abies (L.) H. Karst.; (C) V. album subsp. аustriacum, host Pinus sylvestris L. Photos: (A) Yevhen Sosnovsky; (B, C); Yuliya Krasylenko.

208x53mm (300 x 300 DPI)

Draft

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Draft

Fig. 2. Viscum album subsp. album graphical drawing: (A) habit; (B) part of branch with male (staminate) flower; (C) male (staminate) flower and corolla lobe with anther; (D, E) female (pistillate) flower; (F) primordium; (G) fruit on the pedicle; (H) seed without viscin coat; (I) germinating seed. Scale bar: (B,C,D) = 1 cm. Illustrations: Kateřina Janošíková.

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Fig. 3. Morphology of Viscum album subsp. album: (A) infestation of host trees by V. album associated with branch failure of host tree, Pyrus eleagrifoliaDraft Pall. (Kapshor village surroundings, South Coast of the Crimean Peninsula, March 23, 2019); (B) close-up of an exophyte; (C) hypertrophy of a host branch; (D) female (pistillate) flower; (E) male (staminate) flower; (F−P) primordia; (Q) fruit; (R) terminal ring of four lateral (tepalar) and one central (stigmal) scars; (S) viscin-coated seed; (T) seed with dried viscin; (U) seed with mechanically destroyed viscin. Scale bars: (C, D, E, L, M, P, Q, T, U) 2 cm; (F−P, K, O, S) 1 cm; (R) 500 μm. Photos: (A, B) Oleg Kukushkin; (C) Yevhen Sosnovsky; (D−U) Yuliya Krasylenko.

213x122mm (300 x 300 DPI)

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Draft

Fig. 4. Distribution of three Viscum album subspecies in Ukraine. Circles designate V. album subsp. аlbum, squares – V. album subsp. аbietis, and triangles – V. album subsp. аustriacum. The intensity of the color reflects the number of observations. The map was constructed using QGIS software (A Free and Open Source Geographic Information System, www.qgis.org) based on own observations during 2017 – 2020 field trips, data derived from consulted herbaria and public databases (such as UkrBIN, iNaturalist, and Plantarium), and personal communications (see Table S1 for localities and name references).

1237x874mm (72 x 72 DPI)

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Fig. 5. Host-plant families of Viscum album subsp. album in Ukraine and their frequency based on the number of species (A) and observations (B). Draft

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Draft

Fig. 6. Birds as Viscum album vectors in Ukraine: (A) Bombycilla garrulus (Kharkiv); (B) Turdus viscivorus (Kharkiv); (C) Turdus merula (Kyiv); (D) Sylvia atricapilla (Lviv). Photos: (A, B) Igor Kozitskiy; (C) Oleg Snitzar; (D) Olexiy Dubovyk.

161x119mm (300 x 300 DPI)

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Draft

Fig. 7. Lichens growing on Viscum album: Xanthoria c.f. parietina (L.) Th.Fr. (Teloschistaceae) (yellow) and Physcia c.f. tenella (Scop.) DC. (Physciaceae) (grey or greenish): (A−D) host Crataegus sp.; photos by Yuliya Krasylenko and Oleg Kukushkin (Kok-Asan-Bogaz Pass, Crimean Peninsula, January 17, 2020; (E, F) host Crataegus monogyna Jacq., photos by Yevhen Sosnovsky (Lviv city, March 19, 2020). Lichens were identified by Nadya Kapets. Scale bars: (C, D) – 1 cm; (E, F) − 2 mm.

135x113mm (300 x 300 DPI)

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