Leaf architecture in Rhododendron subsection Rhododendron (Ericaceae) from the Alps and Carpathian Mountains: Taxonomic and evolutionary implications

Accepted Manuscript Title: Leaf architecture in Rhododendron subsection Rhododendron (Ericaceae) from the Alps and Carpathian Mountains: taxonomic and evolutionary implications Authors: Yevhen Sosnovsky, Viktor Nachychko, Andriy Prokopiv, Vitaliy Honcharenko PII: DOI: Reference:

S0367-2530(17)33159-6 http://dx.doi.org/doi:10.1016/j.flora.2017.03.003 FLORA 51100

To appear in: Received date: Revised date: Accepted date:

21-10-2016 17-1-2017 4-3-2017

Please cite this article as: Sosnovsky, Yevhen, Nachychko, Viktor, Prokopiv, Andriy, Honcharenko, Vitaliy, Leaf architecture in Rhododendron subsection Rhododendron (Ericaceae) from the Alps and Carpathian Mountains: taxonomic and evolutionary implications.Flora http://dx.doi.org/10.1016/j.flora.2017.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Leaf architecture in Rhododendron subsection Rhododendron (Ericaceae) from the Alps and Carpathian Mountains: taxonomic and evolutionary implications

Yevhen Sosnovskya,*, Viktor Nachychkoa, Andriy Prokopiva, b, Vitaliy Honcharenkob

a

Botanical Garden, Ivan Franko National University of Lviv, Cheremshyna str. 44, 79014

Lviv, Ukraine; b

Department of Botany, Faculty of Biology, Ivan Franko National University of Lviv,

Hrushevsky str. 4, 79005 Lviv, Ukraine

* Corresponding author. E-mail address: [email protected] (Y. Sosnovsky)

Highlights 

The European alpine rhododendrons are understudied anatomically.

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Leaf structure proves taxonomically valuable in Rhododendron subsect. Rhododendron.

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Leaf traits new for the subsection, genus, and family are described.

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Interspecific hybrids show greater leaf trait variation than the parental species.

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Leaf anatomy suggests a closer affinity between R. ferrugineum and R. myrtifolium.

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ABSTRACT The European Rhododendron subsect. Rhododendron is noteworthy due to its unresolved geographical isolation from Asian relatives, yet it is understudied anatomically. This study examines qualitative leaf architecture of the alpine rhododendrons to reveal its taxonomic significance and improve understanding of the evolutionary relationships in these species. Leaf samples of R. ferrugineum, R. hirsutum, and R. × intermedium were obtained from localities in the Alps and those of R. myrtifolium were from the Carpathians. Herbarium material was also used. Leaf architecture was examined under LM and SEM and the traits were clustered to estimate relationships between the species and hybrid entities. Characters such as leaf shape, epicuticular wax morphology, position of stomata, diversity and anatomy of trichomes, occurrence of hypodermis-like cells and mesophyll anatomy varied depending on the species and were unrelated to altitude and slope exposition of plant growth. Epicuticular wax types new for the genus and the family and a hair type new for the subsection were described. Clustering leaf traits separated well the species and hybrid entities, showing a higher level of similarity between R. ferrugineum and R. myrtifolium. Leaf architecture in Rhododendron subsect. Rhododendron proves taxonomically valuable and suggests closer affinity between R. ferrugineum and R. myrtifolium, while a comparatively higher trait variation in hybrids may be regarded contributive to their reported superior fitness under favourable conditions.

Keywords: alpine rhododendrons, evolutionary relationships, hybrids, leaf anatomy, qualitative traits, taxonomic value.

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1. Introduction The angiosperm genus Rhododendron L. is the most species-rich one within the family Ericaceae, comprising more than 1000 species distributed mainly over the northern hemisphere. Rhododendrons inhabit a wide range of environments (e.g., from humid tropical and subtropical forests to the alpine belt of mountains) and display striking morphological diversity (Cox and Cox, 1997; Cullen, 2005). The complicated classification of Rhododendron has long been a challenge for botanists applying floral and vegetative features as well as molecular data for delimiting and identification of its infrageneric taxa, although the genus still appears understudied with regard to leaf architecture (e.g., Sinclair, 1937; Copeland, 1943; Sleumer, 1949, 1980; Philipson, 1985; Kurashige et al., 2001; Gao et al., 2002; Goetsch et al., 2005; Wang et al., 2007a, b; Sarwar and Takahashi, 2013). While the diversity and concentration of Rhododendron species reach their maxima in southeastern Asia, the European part of the genus range is highly fragmented as a hypothetical result of relatively recent glacial extensions and climatic changes, causing the raised scientirfic interest to speciation and evolutionary patterning in this area (Irving and Hebda, 1993). Among such isolated pockets of species, the morphologically and biogeographically distinct Rhododendron subgen. Rhododendron subsect. Rhododendron remains enigmatic with respect to dispersal and evolution, although being distributed in one of the world’s most comprehensively studied regions. The subsection comprises three small shrubby evergreen clump-forming species restricted to high-mountain habitats: the central European R. ferrugineum L. and R. hirsutum L., as well as the eastern European R. myrtifolium Schott & Kotschy (Cox and Cox, 1997; Cullen, 1980, 2005; Boratyński et al., 2006). The first two species often intercross, producing hybrids known as R. × intermedium Tausch (Milne and Abbott, 2008; Bruni et al., 2016). Despite traditional consideration of the subsection as a natural entity well-distinguished from other members of the section (e.g.,

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Cullen, 1980, 2005; Cox and Cox, 1997), there is still no molecular evidence on its monophyly, and hence the evolutionary relationships of the species are not known yet. Morphological and poor molecular data (the latter based solely on R. ferrugineum) suggest close affinity of this subsection to the eastern Asian Rhododendron subsect. Lapponica (Balf.f.) Sleumer and subsect. Rhodorastra (Maxim.) Cullen (Cullen, 1980, 2005; Cox and Cox, 1997; Kurashige et al., 2001; Goetsch et al., 2005), thus indicating a large disjunction between these related groups. The central European members of the subsection have extensively been under research focus with regard to population structure and genetics unlike R. myrtifolium poorly investigated in many respects (e.g., Pornon and Doche, 1996; Pornon et al., 1997; Bruni et al., 2012, 2016; Voloshchuk, 2012; Charrier et al., 2013, 2014). Few studies touching upon leaf architecture in this plant group have been published so far, and those were predominantly devoted to macromorphology and functional anatomy of certain species (Filella and Peñuelas, 1999; Mircea, 2005; Mayr et al., 2010; Voloschuk and Prokopiv, 2011; Voloschuk, 2012) or provided very brief general leaf descriptions in taxonomic treatments and identification books (Hayes et al., 1951; Popova, 1972; Cullen, 1980, 2005; Cox and Cox, 1997). The present leaf qualitative structural examination of Rhododendron subsect. Rhododendron aimed: (1) to enhance anatomical differentiation of the species and provide their additional diagnostic criteria of potential wider application in the genus; (2) to reveal whether leaf traits may help diagnosing interspecific hybrids in rhododendrons; and (3) to suggest probable evolutionary trends within the subsection reflected in leaf architecture.

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2. Material and methods 2.1. Plant material collection Leafy shoots of Rhododendron ferrugineum, R. hirsutum, and R. × intermedium were collected at 17 locations in the Austrian and Italian Alps and those of R. myrtifolium in 5 locations in the Carpathian Mountains during 2015-2016. The locations were chosen to represent variation in growth conditions such as elevation and slope exposition. Additionally, leaf samples of the herbarium vouchers from Natural History Museum of Vienna (W) and Department of Botany and Biodiversity Research, University of Vienna (WU) were examined to expand the sampling geography (Table 1). For this, most recently collected and chemically untreated vouchers available were chosen to ensure the retention of fine microstructures in the leaves. The morphologically recognizable hybrids R. × intermedium were sampled in the areas known to represent the transition zones between acidic and basic soils (C. Pachschwöll and L. Ehrendorfer-Schratt, University of Vienna, Austria, ‘pers. comm.’). Two to three individuals of each species were randomly sampled from each location, thereupon dried and herbarized. Plants growing at least 5 m from each other were selected to avoid the collection of material from the same genet (Voloshchuk, 2012). From each sample, twelve young leaves (from the unlignified shoots) and twelve mature leaves (from upper part of lignified shoots) without evident infections were selected for anatomical study. To ensure that the shoot phenology did not influence leaf architecture, equal number of leaves was taken from the vegetative and flower-bearing shoots of the same sample. Plant specimens used in this study were deposited at the Herbarium of M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine (KW).

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2.2. Leaf microscopic studies and statistical data analysis For light microscopic study, half of the herbarium material was softened in the equal solution of distilled water, glycerin, and alcohol. Leaf anatomy was examined on the glycerinembedded temporary preparations of the lamina freehand paradermal and transverse sections. Up to three sections were taken from the base, middle, and tip of each leaf blade, and transverse sections of the veins of particular orders were also obtained. Lignified tissue was identified by placing vein sections in 0.5% phloroglucinol and then in concentrated hydrochloric acid (for 30 sec each) and embedding in glycerin (Metcalfe and Chalk, 1979). The rest of the dry material was used for scanning electron microscopy, for which two samples of 6×6 mm per leaf (for adaxial and abaxial surface, respectively) were sputtercoated with gold and observed under microscopes Jeol JSM-T220A (at Ivan Franko National University of Lviv) and Jeol JSM-IT300 (at the University of Vienna). For statistical analysis, qualitative leaf traits revealed were converted into numerical codes by assigning numbers to particular character states (Table 2). The traits showing no evident variation among the species and hybrid entities were not analysed. To estimate the level of similarity between species based on leaf architecture, the data were clustered, using the percent disagreement calculated by the complete linkage method as the distance metric (StatSoft, 2013). Leaf architecture terminology follows Hickey (1973) and Leaf Architecture Working Group (1999) for leaf shape and venation characters, Barthlott et al. (1998) for epicuticular waxes, Seithe (1980) and Cullen (2005) for trichomes, as well as Metcalfe and Chalk (1979), Baranova (1985), and Fahn (1990) for other leaf traits.

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3. Results 3.1. Main leaf architecture patterns of the species Leaf examination of Rhododendron subsect. Rhododendron showed that lamina is coriaceous in R. ferrugineum and R. myrtifolium and subcoriaceous in R. hirsutum, oblong to elliptic or to rounded, with convex or retuse to rounded apex, decurrent to cuneate to (concavo-)convex base, and entire to crenate or revolute margin (Table 2; Fig. 2). Lamina venation is five-ordered, festooned semicraspedodromous, with mostly irregular secondary vein spacing and weak intersecondaries (Fig. 2). The single-layered leaf epidermis consists of irregularly polygonal cells with straight or arched to slightly undulate walls, often elongated on the veins. In R. myrtifolium, one layer of hipodermis-like cells irregularly occurs on either leaf side, adaxially connecting bundle sheaths of two neighbouring bundles and abaxially underlaying scale trichomes near leaf veins (Figs. 3 and 4). Cuticle is smooth with epicuticular wax forming crust-like layer (or smooth layer in R. hirsutum abaxially) and crystalloids of specific morphology. Wax rodlets triangular in crosssection occur adaxially in R. ferrugineum, being more numerous on mature leaves (Fig. 5). The rodlets are variable in length and width, mostly lying bunched on the leaf surface or being suberect, and distributed more or less evenly throughout the leaf. On some leaves, the rodlets are intermixed with loosely arranged irregular wax platelets. In R. hirsutum, entire and irregular wax platelets and threads co-occur on either leaf side, being usually more prominent on mature leaves. The platelets vary in size, the irregular ones being larger, and are arranged into variously shaped groups often with parallel within-group orientation. The two types of platelets are either intermixed or form separate clusters. The loosely arranged waxy threads are mostly curved and have a thickened base (Fig. 5). Waxes of R. myrtifolium produce crystalloids abaxially varying in morphology among the young and old leaves. The younger

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leaves (those on the unlignified shoots) bear membraneous waxy platelets, which are usually destroyed on the older leaves and substituted by wax granules (Fig. 6). Trichomes are of four types: 1. Papillae on the abaxial leaf side in R. ferrugineum and R. myrtifolium. In the former they are longer and their lateral sides are usually (nearly) parallel to each other (Figs 4 and 5). 2. Bristle-like hairs – long multicellular hairs of R. hirsutum with one or two apical cells and individual cells projecting near the tip, fringing the lamina and sometimes occurring adaxially (Figs. 2, 7, and 8). 3. Needle-shaped hairs – tiny unicellular stiff hairs with irregular surface sculpturing (probably of wax nature) covering the midrib adaxially. In R. ferrugineum and R. myrtifolium, they are typically present only on the youngest few leaves along the whole midrib (in R. myrtifolim) or mostly at its base. In R. hirsutum, they remain on mature leaves and sometimes also occur on secondary veins (Figs. 7 and 8). 4. Peltate scales of entire (in R. hirsutum) or undulate type. The scale stalk consists of three to five columns of horizontally elongate cells (three to six cells in each column) with basal cells evident (in R. ferrugineum) or not. The head contains large cells radiating from the stalk and overlaid by smaller upper-central cells, as well as the marginal cells forming a rim. The scale head anatomy is different among the examined species (Table 2; Figs. 4 and 7). The scales of R. ferrugineum occur mostly abaxially and overlap, causing rusty colour of the leaf surface. In R. myrtifolium, abaxial scales are contiguous, while adaxial ones are sparse (several scale diameters apart) or absent. In R. hirsutum, the scales are one to several scale diameters apart, usually denser abaxially or equal on both leaf sides. Leaves are hypostomatic. Stomata are of anomocytic type, evenly distributed throughout between veins, and either protruded from the epidermis (in R. ferrugineum and R. myrtifolium) or almost on the level (in R. hirsutum) (Figs. 3 and 4). Mesophyll is clearly

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differentiated into palisade and spongy parenchyma, the latter consisting of irregularly shaped to isodiametric cells in R. ferrugineum and R. hirsutum. In R. myrtifolium, the palisade often shows unclear or gradual transition to the sponge consisting of rounded to vertically elongate cells. Leaf vascular bundles are collateral and the bundle sheaths extend to both upper and lower epidermis in all veins, the adaxial extensions being collenchymatous and the abaxial ones parenchymatous.

3.2. Leaf architecture in Rhododendron × intermedium Tausch The specimens identified as hybrids were represented by several morphological types (morphotypes) described below. 1. Type 1 – with intermediate leaf traits (characteristic of specimens HY1, HY3, HY6, HY7, and HY9 in Table 1). Lamina is (sub-)coriaceous, oblong to wide elliptic, with convex and shortly acuminate apex, mostly cuneate base, and entire to slightly crenate margin. Wax crystalloids form parallel entire and irregular platelets adaxially and are mostly absent abaxially. Abaxial epidermal cells are prominent but do not form papillae. Bristle-like hairs are very few, only on leaf margins and mostly at the laminar base, or absent. Midrib hairs are persistent, sparse or absent. Scales are polymorphous, some being of R. ferrugineumtype, others of R. hirsutum-type or rather of intermediate structure; the basal cells are usually not evident. Abaxial scales are up to two, their diameters apart or contiguous and adaxial ones are less dense (one to several scale diameters apart or almost absent). Stomata slightly protrude from the epidermis. 2. Type 2 – morphologically resempling R. ferrugineum (characteristic of specimens HY2 and HY8). Lamina is (sub-)coriaceous and glossy, elliptic to oblong, with cuneate to decurrent base and entire to slightly crenate and/or revolute margin. Waxes form loosely arranged triangular rodlets adaxially, sometimes intermixed with irregular platelets.

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Abaxial epidermal cells produce comparatively short papillae with their lateral sides being not parallel to each other. Bristles are mostly absent. Midrib hairs are mainly at the young leaf base and absent on the older leaves; twinned hairs sometimes occur among the separate ones. Scales are mostly of R. ferrugineum-type with evident basal cells or intermediate, sometimes resembling those in R. hirsutum. Abaxial scales do not overlap, being usually one scale diameter apart or contiguous, and the leaf surface is not rustycoloured. Adaxial scales are very sparse or absent. Stomata slightly protrude from the epidermis. 3. Type 3 – morphologically resembling R. hirsutum (characteristic of specimens HY4 and HY5). Lamina is more or less papery, elliptic to rounded, with cuneate to convex base and crenate unrevolute margin. Waxes form entire and irregular platelets on either leaf side. Abaxial papillae are absent. Bristles are very few, along the whole margin, mostly at the laminar base, or absent. Midrib hairs are persistent, sparse or dense. Scales are of R. hirsutum-type, one to several scale diameters apart, and adaxially few or absent. Stomata are (almost) on the same level with epidermis.

3.3. Cluster analysis Clustering leaf structural traits listed in Table 2 showed a comparatively high level of resemblance between R. ferrugineum and R. myrtifolium, while R. hirsutum appeared quite apart (Fig. 9). The traits contributing to similarity between the former included leaf outline, presence of papillae, caducous midrib hairs, absence of bristle-like hairs, type and anatomy of scales, and position of stomata relative to the ordinary epidermal cells. Rhododendron ferrugineum and R. hirsutum shared the same leaf apex, absence of hypodermis, and anatomy of mesophyll, while R. hirsutum and R. myrtifolium were only similar in the absence of scale basal cells. The first two hybrid types occupied rather intermediate position, showing quite

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distant relation to the other entities (Fig. 9A). Analysis excluding leaf shape characters resulted in similar graph (not shown) with the entities being even more closely linked within their clusters and the distance between the latter being more significant. However, based on the leaf shape characters only, R. ferrugineum and R. hirsutum with respectively resembling hybrid entities and the hybrid type 1 fell into three subclusters separate from R. myrtifolium cluster (Fig 9B).

4. Discussion 4.1. Taxonomic value of the leaf architecture in Rhododendron subsect. Rhododendron The features of foliar structure in Rhododendron subsect. Rhododendron described are in accordance with the general patterns observed in Rhododendron (Copeland, 1943; Hayes et al., 1951; Cox and Cox, 1997; Cullen, 2005; Wang et al., 2007b). Particular traits may display a level of variation within the sampled localities as described above, although they appear unrelated to the higher-scale environmental conditions such as altitude and slope exposition of plant growth. The three examined species and the hybrids share many similarities in leaf architecture, exhibiting the uniform shape of epidermal cells, type of cuticle and stomata, venation pattern, and the structure of vascular system and bundle sheath, the latter showing constancy even among different vein orders. However, a number of the leaf traits appear notably diagnostic and each of them will be further discussed. Leaf shape. Diagnostic value of the leaf shape in plants has aquired extensive coverage both in classical taxonomic works and modern studies utilizing a diversity of statistical and digital approaches (e.g., Hickey, 1973; Fuller and Hickey, 2005; Cardozo et al., 2014). Primary literature on Rhododendron identification and classification (Copeland, 1943; Cullen, 1980, 2005; Cox and Cox 1997) provides general leaf outline description of the species, while none of the available taxonomic/phylogenetic studies of these plants uses such features in

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their analyses. We demonstrate that different species and hybrid types of Rhododendron subsect. Rhododendron show distinct variation trends of the number of leaf shape characters despite their partial overlap. For instance, an elliptic leaf outline occurring in all species examined tends to elongate in R. ferrugineum and R. myrtifolium and to become rounded in R. hirsutum, with simultaneous occurrence of the both trends only in hybrids. Such a variation is usually observed at the scale of separate individuals or among individuals at a particular locality, enabling to distinguish between the species and hybrid entities. This highlights diagnostic value of the leaf macroscopic traits in Rhododendron and proves them promising for taxonomic, statistical, and phylogenetic investigations. Epicuticular wax depositions. Leaf epicuticular waxes of Rhododendron have poorly been studied. Ditsch and Barthlott (1997), in their SEM study on numerous plant species, observed no crystalline wax forms in Rhododendron subsect. Rhododendron, although they found wax irregular platelets, tubules, and coiled rodlets in some members of hypothetically allied Rhododendron subsect. Lapponica and subsect. Rhodorastra as well as other subsections of the section. In contrast, our results show remarkably polymorphous wax crystalloids (such as granules, triangular rodlets, threads, as well as parallel grouped entire, irregular and membraneous platelets) in the former. Five of these types and orientation patterns (except triangular rodlets) are recorded for the first time in the genus and two of them (i.e., parallel grouped entire and irregular platelets) also in the family. The triangular rodlets and entire platelets (the latter without mention on specific arrangement patters) have also been found in more distantly related groups of Rhododendron subgen. Pentanthera G.Don and subgen. Hymenanthes (Blume) K.Koch (Ditsch and Barthlott, 1997), while the parallelgrouped platelets have only been known from Hypericaceae, Myrtaceae, Proteaceae, and Haloragaceae (Barthlott et al., 1998). Wax morphology is found species-specific and independent of plant habitat conditions such as altitude and slope exposition, which concurs

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with previous studies also reporting wax chemistry to be independent of altitude (Barthlott and Wollenweber, 1981; Lütz and Gülz, 1985; Salasoo, 1987; Weiller et al., 1994; Barthlott et al., 1998; Meusel et al., 1999). In some species, however, the patterns of epicuticular waxes might change with leaf age (Salasoo, 1983; Bringe et al., 2006), as also shown for R. myrtifolium in the present study. Soil conditions, presence of air pollutants, and other microclimatic factors may also affect chemical and quantitative features of epicuticular waxes (Barthlott and Wollenweber, 1981; Percy et al., 1994; Kravkina, 2000). Trichomes. Cowan (1950) and Seithe (1960, 1980) described trichome types and introduced their terminology in Rhododendron, while Hardin and Gensel (1982) questioned some of their viewpoints by revealing new trichome features in the North American species and thus substantiated the need for reconsidering trichome classification. The present study describes a number of previously unreported leaf indumentum features in Rhododendron subsect. Rhododendron, among which is the presence of needle-shaped midrib-covering hairs and scales adaxially in all three species. The species-differentiating hair characters are their life span (persistent during leaf maturation or not) and location (along the whole midrib or only at its base). The encountered hair type may correspond to the “acicular” one described by Cowan (1950) and later classified within the “filiform” type by Seithe (1960, 1980) who considered them not diagnostic at supraspecific level in Rhododendron. Previous studies reported upper leaf surface glabrous and described only one type of leaf hairs (i.e., the bristles of R. hirsutum) in this subsection (Cullen, 1980, 2005). The latter anatomically resemble “loriform” hairs named so by Cowan (1950) and Seithe (1960, 1980) and found in species of Rhododendron subsect. Edgeworthia (Hutch.) Sleumer and subsect. Trichoclada (Balf.f.) Cullen. Thus the present study is the first report of this hair type in Rhododendron subsect. Rhododendron. The presence/absence and relative size of papillae also appear species-specific within the subsection (Table 2, Fig. 4), which conflicts with Hayes et al. (1951) who reported

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equal-sized papillae in all the three species. The entire scales found in R. hirsutum were previously reported characteristic of the whole subsection together with many other lepidote rhododendron taxa, while the undulate scales were suggested specific of only Rhododendron subsect. Lapponica (Seithe, 1980), thus disagreeing with our findings. Our study also indicates several scale trichome features of diagnostic value: head margin shape, shape/size difference between the marginal and surrounded cells of the head, and distinctness of basal cells, of which only the first one was highlighted in the former studies (e.g., Cowan, 1950; Seithe, 1960, 1980; Cullen, 1980, 2005). Detailed trichome examination in many Rhododendron species would clarify importance of the above features in species recognition and delimitation of higher taxa in the genus. Stomata. Stomatal type, distribution pattern, and position relative to the ordinary epidermal cells are often considered species-diagnostic (Metcalfe and Chalk, 1979). Anomocytic stomata of solely abaxial distribution featuring the examined species are commonplace in the genus, although the occurrence of diacytic and pericytic types have also been noted (Wang et al., 2006; Wang et al., 2007b). Similarly to the encountered species here, which represent the characteristic position of stomata in Rhododendron subsect. Rhododendron, other species of the section may also bear stomata protruded to a different extent (e.g., as can be inferred from the photographs in Wang et al., 2007b). This suggests protruded stomata to be related to the occurrence of abaxial papillae in Rhododendron, although additional evidence is needed. Hypodermis. The presence of hypodermis-like cells appears specific of R. myrtifolium, although this layer is not continuous throughout a leaf (Figs. 3 and 4). The amount of these cells seems unrelated to altitude and slope exposition, suggesting their strong genetic determination. Development intensity of (sub-)epidermal cell layers in leaves is generally considered light-dependent, although it can be a good taxonomic criterion as well (Metcalfe

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and Chalk, 1979; Arens, 1997; Fahn, 1990). The origin of the encountered cells needs additional investigation to reveal whether they represent a hypodermis, an inner epidermal layer, or an atypically extended bundle sheath. The multilayered epidermis has previously been reported in the members of Rhododendron subgen. Hymenanthes (Blume) K.Koch sect. Pontica G.Don, subgen. Azaleastrum Planch. sect. Azaleastrum (Planch.) Maxim., and more rarely in Rhododendron subgen. Rhododendron sect. Rhododendron except subsect. Rhododendron (Hayes et al., 1951; Cullen, 2005). Mesophyll. Hayes et al. (1951) reported several leaf mesophyll characters of taxonomic value in Rhododendron, namely the palisade mesophyll thickness and the number of layers, the size of intercellular spaces in the sponge, and the presence of water tissue. Our study indicates the number of mesophyll layers and the sharpness of transition between palisade and sponge species-discriminative in Rhododendron subsect. Rhododendron. However, as soon as the former is partly overlapping (Table 2) and the latter shows a degree of variability in R. myrtifolium (Fig. 7), the mesophyll features appear generally of low diagnostic value and should be used together with other traits to distinguish the species.

4.2. Identification of Rhododendron × intermedium hybrid classes Morphological differentiation of R. × intermedium often appears problematic in view of its highly superficial description provided in present taxonomic treatments and identification guides (e.g., Cox and Cox, 1997). We demonstrate comparatively high variation of qualitative structural traits in this hybrid most probaly resulted from the increased genetic diversity, although combinations of such traits may serve good diagnostic criteria. The morphotypes of R. × intermedium described herein are presumed, although without molecular evidence, to represent different hybrid classes commonly produced in this hybridization system. In geologically transitional areas, the intercrossing R. ferrugineum and R. hirsutum

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are known to form populations dominated by F1 hybrid fertile individuals of intermediate morphology, which tend to further produce F2s and backcrosses in either direction, usually leading to the occurrence of introgressive races, the phenomenon also typical of other sympatric Rhododendron species (Milne et al., 1999, 2003; Milne and Abbott, 2008; Bruni et al., 2016). Hence, the second and the third hybrid types described here theoretically correspond to such “impure” species because of their close morphological similarity to respective parents, while the first hybrid type can be assigned to putative F1 generation (Table 2; Fig. 9). Clustering leaf shape characters precisely fits the expected position of the hybrid entities (Fig. 9B). The analysis of both macro- and microstructural traits (also with the exclusion of the former) shows a different graph probably resulting from slightly asymmetrical trait distribution among the entities and a level of similarity between R. ferrugineum and R. myrtifolium (Fig. 9A). Relatively small number of metrics used for the analysis may have also affected the result. Hybridization among highly interfertile species has been shown to have a number of evolutionary outcomes, among which is fostering species diversity and fitness of populations by fixation of novel adaptations, reinforcement of species barriers, or, to the other extreme, promoting biodiversity reduction due to species assimilation by their congeners (Buerkle et al., 2003; Milne et al., 2003; Milne and Abbott, 2008). The higher trait variation in the putative hybrids compared to their parents revealed (Table 2) may contribute to their superior fitness under favourable growth conditions reported by Milne and Abbott (2008) and Bruni et al. (2016) for this hybridization system.

4.3. Intrasectional relationships suggested by leaf architecture Relatively high morphological similarity among the species of Rhododendron subsect. Rhododendron is opposed by their dual vicariancy: while R. ferrugineum and R. hirsutum occur in sympatry on acidic and basic soils respectively, the acid-loving R. myrtifolium is well

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separated geographically (Cullen, 1980; Czekalski et al., 2000; Boratyński et al., 2006; Milne and Abbott, 2008; Frey and Lösch, 2010). The present study suggests a closer affinity between the latter and R. ferrugineum based on leaf architecture, which corresponds to their resemblance in ecology (i.e., soil preference) as well as other vegetative and floral characters formerly serving as criteria for their conspecific treatment (Cullen, 1980, 2005). Assuming monophyly of the subsection, we speculate on allopatric origin of R. ferrugineum and R. myrtifolium and early sympatric segregation of R. hirsutum as likely speciation trends that were promoted by ancient geological events and climate fluctuations resulted in fragmentation and isolation of their habitats (Billings, 1974; Irving and Hebda, 1993; Coyne and Orr, 2004; Stöcklin et al., 2009). However, the lack of molecular, phylogeographic, and fossil data (e.g., Irving and Hebda, 1993) impedes any precise conclusions regarding dispersal and evolutionary pathways in this plant group.

Funding This work was supported by Ivan Franko National University of Lviv [grant number PNS-010515 to YS and VN]; and Austrian Agency for International Cooperation in Education & Research, OeAD-GmbH (granted a scholarship to YS under the Stipendien Lemberg Programme financed by the Austrian Federal Ministry of Science, Research and Ecomony).

Acknowledgements The authors are thankful to Brigitta Erschbamer (University of Innsbruck) for collecting plant material for this study in the Tyrolean Alps. Thanks to Josef Greimler, Clemens Pachschwöll, Christian Gilli, and Luise Ehrendorfer-Schratt (University of Vienna) for their support and assistance in collecting material in the Eastern Alps. Thanks to Walter Till (University of Vienna) and Bruno Wallnöfer (Natural History Museum of Vienna) for the

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permissions to extract leaf samples from herbarium vouchers for anatomical study. Heidemarie Halbritter (University of Vienna) and Yuriy Datsyuk (Ivan Franko National University of Lviv) provided technical assistance in scanning electron microscopy. Constructive comments of the two anonymous reviewers are gratefuly acknowledged.

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Figure legends Fig. 1. Location of Rhododendron specimens sampled in the Alps (A) and Carpathians (B). HY, R. × intermedium; RF, R. ferrugineum; RH, R. hirsutum; RM, R. myrtifolium. Localities for HY7 and HY8 specimens are indicated based on their descriptions on the herbarium vouchers with no geospatial data provided. Studied populations are abbreviated according to Table 1.

26

Fig. 2. Leaf shape and venation in Rhododendron ferrugineum (A), R. hirsutum (B), and R. myrtifolium (C). Scale bar = 10 mm. Drawing credit: Y. Sosnovsky.

27

Fig. 3. Leaf cross-section of R. myrtifolium RM1 (A) and leaf adaxial (B) and abaxial (C) epidermis of R. hirsutum RH1 (camera lucida). Scale bars: 100 μm (A) and 50 μm (B and C). 1, cuticle; 2, hypodermis-like cells; 3, mesophyll cells with chloroplasts; 4, bundle sheath cells; 5, stomata; 6, vascular bundle; 7, cells overlying vascular bundle. Drawing credit: Y. Sosnovsky. Studied populations are abbreviated according to Table 1.

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Fig. 4. Scale trichome cross-section and surface view of R. ferrugineum RF1 (A) and R. hirsutum RH1 (B) and cross-sections of R. myrtifolium RM1 scale trichome and trichome stalk (on the left) with three and five cell columns respectively (C) (camera lucida). Scale bars = 80 μm. 1, cuticle; 2, stomata; 3, marginal cells; 4, upper-central cells; 5, abaxial epidermis; 6, mesophyll; 7, trichome basal cells; 8, vascular bundle; 9, bundle sheath cells; 10, hypodermis-like cells; 11, cell columns in trichome stalk. Drawing credit: Y. Sosnovsky. Studied populations are abbreviated according to Table 1.

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Fig. 5. SEM photographs of triangular wax rodlets in R. ferrugineum (A-B), papillae and stomata in R. ferrugineum (C), waxy crust abaxially in R. ferrugineum (D), waxy threads and platelets in R. hirsutum (E), different types of wax platelets co-occurring in R. hirsutum (F), and entire (G) and irregular (H) wax platelets in R. hirsutum. Scale bars: 20 μm (A, B, C, and E), 10 μm (D and G), 40 μm (F), and 5 μm (H).

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Fig. 6. SEM photographs of abaxial leaf surface in R. × intermedium morphotype 1 (A) and morphotype 2 (B), orientation patterns of the wax platelets in R. × intermedium morphotype 3 (C-D), stoma and wax platelets in R. × intermedium morphotype 3 (E), stoma and wax cover of R. myrtifolium young leaf (F), membraneous wax platelets in R. myrtifolium (G), and stoma and wax granules on the old leaf of R. myrtifolium (H). Scale bars: 50 μm (A), 10 μm (B, C, D, F, and H), and 5 μm (E and G). The proposed morphotypes are explained under “Results”.

31

Fig. 7. SEM photographs of scale trichomes in R. ferrugineum (A), R. hirsutum (B), R. × intermedium morphotype 2 (C) and R. myrtifolium (D), top of bristle-like hair of R. hirsutum with projecting individual cells (arrow) (E), needle-shaped hairs along the leaf midrib of R. hirsutum (F), separate and twinned (arrow) midrib hairs of R. × intermedium morphotype 2 (G), and midrib hair surface of R. × intermedium morphotype 1 (H). Scale bars: 100 μm (A, B, C, and D), 20 μm (E and G), 200 μm (F), and 3 μm (H). The proposed morphotypes are explained under “Results”.

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Fig. 8. Cross-sections of bristle-like marginal hair base and top (A) and needle-shaped hair (B) of R. hirsutum RH1 (camera lucida). Scale bars: 100 μm (A) and 50 μm (B). 1, adaxial epidermis; 2, mesophyll. Drawing credit: Y. Sosnovsky. Studied populations are abbreviated according to Table 1.

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Fig. 9. Dendrograms gained from cluster analysis of both macro- and microstructural qualitative leaf traits (A) and leaf shape characters only (B) in Rhododendron subsect. Rhododendron. Numbers indicate linkage distance. The leaf traits analysed are presented in Table 2.

34

Table 1. Collection data of Rhododendron specimens examined No.a

Location

Rhododendron ferrugineum RF1 Austria, Tyrol, Axamer Lizum RF2

Austria, Tyrol, Ötztal, Obergurgl, Schönwieshütte RF3 Austria, Tyrol, Ötztal, Obergurgl, Bruggenboden RF4 Austria, Brenner, Venntal (Ochsenboden) RF5 Austria, Styria, Schladminger Tauern, Liezen county RF6 Austria, Styria, Fischbacher Alpen, Bruck-Mürzzuschlag county, Stuhleck Rhododendron hirsutum RH1 Austria, Tyrol, Birgitzköpflbahn, Axamer Lizum RH2 Italy, South Tyrol, Passo Costalunga, Latemar group RH3 Italy, South Tyrol, Passo Costalunga, Latemar group RH4 Austria, Styria, Schladminger Tauern, Liezen county RH5 Austria, Styria, Schladminger Tauern, Liezen county RH6 Austria, Styria, Mürzsteger Alpen, Bruck-Mürzzuschlag county, Schneealpe Rhododendron × intermedium HY1 Austria, Styria, Schladminger Tauern, Liezen county HY2 Austria, Styria, Schladminger Tauern, Liezen county HY3 Austria, Styria, Schladminger Tauern, Liezen county HY4 Austria, Styria, Schladminger Tauern, Liezen county HY5 Austria, Styria, Mürzsteger Alpen, Bruck-Mürzzuschlag county, Schneealpe HY6 Austria, Styria, Kirchkogel HY7

HY8

Austria, Lower Austria, between Mt. Kuhschneeberg and Mt. Kaiserstein Austria, Styria, Hochschwab

HY9

Austria, Tyrol, Hintertux

Geospatial datab Lat. / Long. Alt.

Slope expositionc

Specimen origind

47.19043° / 11.31030° 46.84959° / 11.01912° 46.85912° / 11.01676° 47.00903° / 11.56083° 47.301806° / 13.672194° 47.573250° / 15.790500°

1771

W

Erschbamer, 2015

2250

SSE

Erschbamer, 2015

2029

N

Erschbamer, 2015

2164

W

Erschbamer, 2015

1635

SE

1768

SE

Sosnovsky, Pachschwöll & Gilli, 2016 Sosnovsky & Pachschwöll, 2016

47.19339° / 11.316730

2045

W

Erschbamer, 2015

46.38815° / 11.60478° 46.39081° / 11.59434° 47.279889° / 13.638750° 47.273861° / 13.631278° 47.697611° / 15.610361°

2413

E

Erschbamer, 2015

2056

N

Erschbamer, 2015

1995

S

2327

S

1750

SW

Sosnovsky, Pachschwöll & Gilli, 2016 Sosnovsky, Pachschwöll & Gilli, 2016 Sosnovsky & Pachschwöll, 2016

47.280778° / 13.644889° 47.280833° / 13.645028° 47.279639° / 13.640056° 47.279697° / 13.639208° 47.697472° / 15.610806

1985

S

1982

S

1982

S

1989

S

1738

SW

47.350000° / n/i 15.316667° n/i 1600

(N)

W 2006-16474

W

W 2005-19614

n/i

WU ICQ.-J.-Nr. 004245 WU 0061147

n/i

1820

47.083333° / 210011.650000° 2300

(NE)

Sosnovsky, Pachschwöll & Gilli, 2016 Sosnovsky, Pachschwöll & Gilli, 2016 Sosnovsky, Pachschwöll & Gilli, 2016 Sosnovsky, Pachschwöll & Gilli, 2016 Sosnovsky & Pachschwöll, 2016

35

Rhododendron myrtifolium RM1 Ukraine, Zakarpattya region, Rakhiv district, Marmaros Mts. RM2 Ukraine, Zakarpattya region, Rakhiv district, Marmaros Mts. RM3 Ukraine, Ivano-Frankivsk region, Verkhovyna district, Chornohora Mts. RM4 Ukraine, Zakarpattya region, Rakhiv district, Chornohora Mts. RM5 Ukraine, Zakarpattya region, Rakhiv district, Chornohora Mts. RM6 Romania, Prahova county, Munții Bucegi, surroundings of Cabana Caraiman RM7 Romania, Sibiu county, Munții Făgăraș, between Lacul Bâlea and Șaua Caprei

47.923917° / 1935 24.328194° 47.970222° / 1710 24.435833° 48.046661° / 2026 24.627474°

E On top SW

Sosnovsky & Nachychko, 2015 Sosnovsky & Nachychko, 2015 Sosnovsky & Nachychko, 2015

48.159027° / 2019 24.501500°

SE

Sosnovsky & Nachychko, 2015

48.158778° / 1248 24.353889°

N

Honcharenko, 2015

45.408528° / 2132 25.485889°

E

WU 0068666

45.603028° / 2152 24.621222°

NW

WU 0068734

a

Specimen numbers refer to their locations indicated on the map (Fig. 1). n/i = not identified. c Information presented parenthetically was obtained based on the geospatial data provided. d Specimen origin indicates the name of collector and year of collection for particular specimens. Herbarium vouchers examined are indicated by their accession numbers. b

36

Table 2. Matrix of qualitative leaf traits of Rhododendron subsect. Rhododendron used for cluster analysis (Fig. 9) Leaf traits 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Plant taxa R. R. R. × intermedium R. × intermedium R. × intermedium R. ferrugineum hirsutum morphotype 1 morphotype 2 morphotype 3 myrtifolium

0 1 0 0 1 0 1 0 0 1 1 1 1 1 0 0.5 0

1 1 1.5 1.5 0.5 0.5 0 1.5 1 0 0 0 0 0 0 0 0

0.5 1 1 1 0.5 0 0 1 0.5 0.5 0.5 0.5 0 0.5 0 0.5 0

0 1 0.5 0.5 1 0 0.5 0.5 0 0.5 0.5 0.5 0.5 0.5 0 0.5 0

0.5 1 1.5 1.5 0.5 0.5 0 1 1 0 0 0 0 0 0 0.5 0

0 0 1 0.5 0 1 0.5 0 0 1 1 1 0 0.5 1 1 1

1. Laminar shape: 0 = elliptic to oblong; 0.5 = elliptic to oblong or rounded; 1 = elliptic to rounded. 2. Apex shape: 0 = retuse to rounded; 1 = convex and shortly accuminate. 3. Base shape: 0 = decurrent; 0.5 = decurrent to cuneate; 1 = cuneate; 1.5 = cuneate to (concavo-)convex. 4. Margin shape: 0 = entire and revolute; 0.5 = slightly crenate and revolute; 1 = slightly crenate and straight; 1.5 = crenate and straight. 5. Epicuticular wax crystalloid type adaxially: 0 = absent; 0.5 = entire and irregular platelets; 1 = triangular rodlets and irregular platelets. 6. Epicuticular wax crystalloid type abaxially: 0 = absent; 0.5 = entire and irregular platelets; 1 = membraneous platelets and granules. 7. Papilla-like trichomes: 0 = absent; 0.5 = short; 1 = long. 8. Bristle-like hairs: 0 = absent; 0.5 = rarely present; 1 = few on leaf margins; 1.5 = on leaf margins and adaxially. 9. Midrib-covering hairs on mature leaves: 0 = absent; 0.5 = present or absent; 1 = always present. 10. Peltate scale type: 0 = entire; 0.5 = intermediate; 1 = undulate. 11. Shape/size difference between marginal and upper-central cells of scales: 0 = gradual; 0.5 = gradual to sharp; 1 = sharp. 12. Scale margin: 0 = straight; 0.5 = variable; 1 = slightly dentate. 13. Scale basal cells: 0 = not evident; 0.5 = sometimes evident; 1 = evident. 14. Position of stomata relative to the ordinary epidermal cells: 0 = on the level; 0.5 = slightly protruded; 1 = protruded. 15. Hypodermis-like cells: 0 = absent; 1 = present. 16. Number of palisade mesophyll layers: 0 = one-two layers; 0.5 = two-three layers; 1 = two-four layers. 17. Transition between palisade and spongy mesophyll: 0 = abrupt; 1 = often unclear or gradual.

37