Pollen morphology of the Central European broomrapes (Orobanchaceae: Orobanche, Phelipanche and Orobanchella) and its taxonomical implications

Pollen grains of 450 samples of 25 species of the genus Orobanche and Phelipanche occurring in Central Europe were investigated using light and scanning electron microscopy. Palynological data on 18 species are reported here for the first time. The usefulness of micromorphological studies on pollen of Orobanche and Phelipanche is demonstrated. Previous research showed the separation of Phelipanche from Orobanche, a finding which is also supported by pollen aperture type and exine ornamentation. The pollen of Phelipanche is tricolpate, while that of Orobanche is inaperturate. Our research showed that one of the species included so far within Orobanche, namely O. coerulescens, has tricolpate pollen with microreticulate sculpture. As a consequence of the finding of tricolpate pollen and intermediate macromorphological characteristics of this species between Orobanche and Phelipanche, it is proposed to transfer it to a new genus, Orobanchella, described in the paper. A comparison of the main characteristics distinguishing Orobanche, Phelipanche, Boulardia and Orobanchella is also presented and discussed.


Introduction
The family Orobanchaceae contains 15 obligate parasitic genera with ca 250 species (Pusch and Günther 2009). The family has a worldwide distribution, but the main centres of distribution are the Mediterranean, western and central Asia, northern Africa and North America (Kreutz 1995). Orobanche s.l. is the largest genus and comprises more than 200 species, which lack chlorophyll and are holoparasites of the roots of other vascular plants (Uhlich et al. 1995;Pusch and Günther 2009). In Europe, these species usually grow in the warmest regions, with the highest species diversity in the Mediterranean countries. In central and northern parts of Europe the genus comprises about 30 species, which are mostly rare, endangered or declining (e.g. Zázvorka 1997Zázvorka , 2000Pusch and Günther 2009;Halamski and Piwowarczyk 2008;Przemyski 2009, 2010;Piwowarczyk et al. , 2011Piwowarczyk 2011Piwowarczyk , 2012aPiwowarczyk , b, c, d, e, f, g, h, 2013Nobis et al. 2014).
The flowers in Orobanche are always bisexual, with superior ovaries, more or less zygomorphic, with four didynamous stamens and two or three lobed stigma, characterised by cross-pollination or, rarely, by the absence of insects, i.e. self-pollination (Beck von Mannagetta 1930;Teryokhin 1997;Jones 1991). There are many adaptations of flowers for pollination: large or small flowers gathered in dense inflorescences; contrasting colouration and shine of the corolla and stigma; a wide range of often bright colours; the smell of the flowers, either pleasant (scent of cloves, e.g. O. alba, O. caryophyllacea, O. crenata, O. gracilis) or unpleasant (the smell of carrion, e.g. O. foetida, O. rigens, O. rapum-genistae, O. variegata) (the majority of species have a weak scent or none); the lower lip of the corolla serving as an alighting place for pollinators (two bright folds, coated with hairs); the production of nectar from coloured spots. Most species are pollinated by bumblebees and bees (Apidae), as well as, albeit less importantly, by species of the families Syrphidae, Vespidae, or Formicidae or carrion flies (Diptera) (Piwowarczyk, unpublished). Preliminary studies in relation to pollination and other shared interactions between parasitic plants and their hosts are presented by Ollerton et al. (2007).
Orobanchaceae is a eurypalynous family, with different types of pollen. Nevertheless, in many cases the biometric and morphological features of pollen grains possess a high diagnostic significance and are used in taxonomy. Pollen taxonomy can also explain the relationship and phylogenetic connections between particular genera of the family. Exine sculpture is one of the most important features and is a good tool in the recognition of genera or sections within a genus. Unfortunately, there are still not many papers dedicated to the pollen morphology of the family Orobanchaceae (e.g. Tiagi 1951;Rao 1963;Erdtman 1966;Polo 1987;Minkin and Eshbaugh 1989;Teryokhin 1997;Shahi Shavvon and Saeidi Mehrvarz 2010;Zare and Dönmez 2013;Zare et al. 2013;) and further studies within the whole family are required.
The aim of the study was to present (1) the macro-and micromorphological variability of pollen in the Central European Orobanche and Phelipanche taxa; (2) the taxonomic implications of pollen morphology in the examined taxa.

Plant material
This study is based on plant material deposited mainly in the KTC herbarium. In total, 25 taxa of three genera, Orobanche, Phelipanche and Orobanchella, were analysed. A list of voucher specimens used in the study is given in Table 1. Each sample corresponds to a single plant, but pollen grains originating from different flowers have not been distinguished.

Light microscopy observations
The pollen grains were prepared with the standard method, Erdtman's acetolysis (Erdtman 1969;Faegri and Iversen 1989). Following acetolysis, the pollen grains were mounted in glycerine jelly and prepared for LM observation. Some characteristics, such as polar and equatorial axis and exine thickness, were examined by LM (Axio Imager 2, Zeiss) for 30 pollen grains under 1,0009 magnifications with the help of the AxioVision computer program (version 4.8.1.0, Carl Zeiss Imaging Solutions). Measurements were taken under the light microscope only in accordance with changes in pollen shape and dimensions occurring during its preparation for SEM.

Scanning electron microscopy (SEM) observation
Using SEM observation, we analysed 450 samples of 25 taxa. For SEM observation, samples were coated with gold using a JFC-1100E Ion Sputter manufactured by JEOL. Micromorphological structures of pollen grains were observed and photographs taken by means of Hitachi S-4700 and Philips XL 20 scanning electron microscopes at various magnifications. The terminology was adopted from Punt et al. (2007) and Hesse et al. (2009).

Morphometric analysis
The statistical analyses are based on 1,170 fully-developed pollen grains from 25 taxa. Characteristics were measured in 30 grains of each specimen studied (Table 1). Finally, a total of the seven most informative quantitative and qualitative morphological characteristics was chosen for the analyses: width of exine (mean lm); polar axis (mean lm); equatorial diameter (mean lm); polar/equatorial ratio; number of furrows on grain; sculpture of pollen grains (1-granules look like a ball of wool, 2-granules consisted of a few smaller round elements knitted together into a smooth surface, 3-microreticulate); arrangement of granules on the pollen surface: 1-no areas without granules are present, 2-areas without granules are present, 3-no granules are present (different type of pollen sculpture). The characteristics used are listed in Table 2. The mean value of the characteristics measured or scored from all samples of the taxon was analysed using cluster analysis to illustrate the general relationships and similarities between them. The analysis was performed in order to obtain results providing a general overview and to determine whether different morphological groups exist and are separated into distinct clusters. Similarity between samples was calculated on the basis of Gower's general similarity coefficient. The dendrogram was prepared using the UP-GMA method. Subsequently, principal coordinate analysis (PCoA) was performed on the basis of all quantitative and qualitative features. The goal of PCoA was the positioning of objects (individuals) in a space of reduced dimensionality while preserving their distance relationships. Data analyses were performed using MVSP 3.1 (Kovach 1999).

LM and SEM study
Both LM and SEM observations enabled the team to distinguish, among Orobanche and Phelipanche pollen, two main groups with distinctly different features regarding pollen shape and exine micromorphology. Group 1 contains pollen grains in monads, suboblate (P. arenaria), oblate spheroidal (P. purpurea, P. bohemica, P. ramosa and Orobanche coerulescens) (according to P/E ratio) and prolate spheroidal (P. caesia), and tricolpate (but some single grains with 2 or 4 colpi) forms. Some differences in colpi shape and distribution are visible (colpi were sometimes fused, wide at the equator and narrow near the poles) but these features appear in all members of group 1 (Tables 2, 3 According to differences in the size and distribution of granules, two main patterns of pollen surface ornamentation can be found: 1-granules distributed over the whole pollen grain, but small areas without granules are present; the granules are of different sizes but always smaller than 1 lm; most granules are joined to others, but isolated granules are also present; 2-the surfaces are similar to those described above, but there are no areas without granules (Figs. 1, 2).

Statistical analysis
Cluster analysis (UPGMA) performed on the complete set of data (Table 2) resulted in the delimitation of two main subclusters. The first comprises a group of taxa with distinctly tricolpate pollen grains with reticulate surfaces, whereas the second comprises uncolpate grains with granulate surfaces (Figs. 3, 4, 5, 6). A similar pattern of results is shown by the ordination diagrams from the principal coordinate analysis (PCoA, not shown), also based on the complete quantitative and qualitative set of data. The first subcluster in UPGMA effectively separates the species P. ramosa, P. bohemica, P. arenaria, P. purpurea, P. caesia and Orobanchella coerulescens from all the rest of the specimens belonging to Orobanche placed in the second cluster. In the Phelipanche subcluster, all taxa are very similar in general habit, with tricolpate pollen grains. In cluster analysis, P. caesia differs slightly from the other members, with a higher polar/equatorial ratio and a thinner exine. However, pollen characteristics in this group of species are characterised by generally higher variance (Fig. 7). Although all the species located in the  picridis, the mean polar axis of the pollen grains is \23.9, the equatorial mean is \20.8 and the exine is generally narrower (0.8-0.9 lm). In all the remaining taxa from the subclade, the mean polar axis of pollen grains varies from 23.3 to 28.8, the equatorial mean is 21.3-24.2 and the exine width is (0.8-) 0.9-1.1 lm (Table 2; Fig. 8).
In the Phelipanche group, a thinner exine has been noted in P. caesia, but at the same time, it has the highest value for equatorial and polar axis. The largest pollen grains in the Orobanche group were noted e.g.

Discussion
Analysis of the pollen grains of Central European representatives of Orobanche s.l. showed that they are characterised by high variability, both inter-and intra-specific, as well as in relation to oligo-and polyphagous species parasitic on various hosts. In addition, some taxa may produce heteromorphic pollen grains (Zare et al. 2013), but this issue requires further study. Surely pollen characteristics, i.e. the number of apertures and the ornamentation of exines, cannot be overlooked, because they bear valuable information to help analyse evolutionary relationships and classifications within the Orobanchaceae family. The characteristics of pollen are important criteria for distinguishing genera or sections, but are less important in distinguishing species. However, it is worth mentioning the subtle yet noticeable differences in the size and sculpture of pollen of species treated in different taxonomic units, i.e. species of the problematic complex O. alsatica s.l., namely O. alsatica, O. bartlingii and O. mayeri, all three of which are parasites of Apiaceae (Piwowarczyk 2011(Piwowarczyk , 2012c. In another example, O. pallidiflora, the species is treated differently in various studies: as a separate taxon, or in the rank of subspecies or variety within Orobanche reticulata (Beck von Mannagetta 1890, 1930Zázvorka 2000;Pusch and Günther 2009). O. reticulata and O. pallidiflora differ in morphology, habitat and altitudinal ranges as well as host preferences (e.g. Kreutz 1995; . Our study showed that O. pallidiflora has thinner exines and smaller equatorial and polar axes than O. reticulata, along with differences in sculpture. Orobanche bohemica as a separate species was first described by Č elakovský (1879). It has been assigned to different taxonomic ranks, e.g., that of a variety, by Beck von Mannagetta (1930) and Hayek (1914); as a subspecies of Phelipanche purpurea, by e.g. Zázvorka (2000) and Carlón et al. (2008); or as a separate species, by Holub and Zázvorka (1999), Pusch (2006), Pusch and Günther (2009), and Pollen morphology of the Central European broomrapes 803 Piwowarczyk (2012f, g). Palynological analysis also revealed auxiliary micromorphological differences: P. bohemica has a thicker exine and larger equatorial and polar axes than P. purpurea, along with differences in sculpture. A Central European species, parasitic mainly on Centaurea, considered until recently as O. elatior s.l. and now recognised as two distinct species, O. elatior and O. kochii (Zázvorka 2010), also demonstrates subtle differences in the size and level of sculpture of pollen grains (Tables 2, 3, 4). Our study of pollen morphology showed various characteristics of Orobanche coerulescens. O. coerulescens has a Eurasian distribution. It occurs in the Far East, Japan, China, the Caucasus, Latvia, Ukraine and Romania. Very rare in Central Europe, it is known from Slovakia, the Czech Republic, Austria, Germany and Poland. As a typical steppe plant, it has relict features of post-glacial Artemisia steppes in the western part of its range. The species is recognised as extinct in the majority of its localities at the western limit of its distribution and its populations are very scarce in Central Europe. It is mainly parasitic on Artemisia campestris (Zázvorka 2000;Pusch and Günther 2009;Piwowarczyk 2012d). Traditionally, the species was included in the section Orobanche, grex Coerulescentes Beck (Beck von Mannagetta 1890), or, according to the new classification, in the section Inflatae (Beck) Tzvel., subsection Coerulescentes (Beck) Teryokhin (Teryokhin et al. 1993). This subsection contains about 10 species, poorly known and often lacking a very clear taxonomic position, of mainly steppe parasites involving the genus Artemisia. They are mostly endemic, occurring mainly in eastern Asia and Russia (Teryokhin et al. 1993;Piwowarczyk 2012d). We have shown that the pattern of pollen grains of O. coerulescens clearly belongs to a Phelipanche tricolpate type of pollen with microreticulate  (Figs. 3, 5). Previously, microreticulate sculpture of the exine was known only in Cistanche and Phelipanche (Zare et al. 2013 Schneeweiss et al. (2004b), and Weiss- Schneeweiss et al. (2006), it is shown for the first time to be a member of the Eurasian lineage O. coerulescens. Phylogenetic relationships also show a degree of separation of O. coerulescens from the rest of the species of the section Orobanche and place it in an intermediate position between Orobanche, Boulardia and Phelipanche (Schneeweiss et al. 2004a;Piednoël et al. 2012).