Orobanche elatior and O. kochii (Orobanchaceae) in Poland: distribution, taxonomy, plant communities and seed micromorphology

Species of the genus Orobanche (Orobanchaceae), parasitic on Centaurea in Central Europe, were previously considered to belong to the O. elatior group. At present, the taxon is differentiated into two species, O. elatior Sutton and O. kochii F.W. Schultz. The paper presents for the first time the distribution of O. elatior and O. kochii in Poland based on a critical revision of herbarium and the literature data, as well as the results of field studies conducted between 1999 and 2014. The majority of the species’ localities are in south Poland: Silesia-Cracow, Małopolska and the Lublin Uplands. The distribution of both species in Poland is mapped and chronologically organized, and is thus the most recent in Europe. The taxonomy, host preferences, and ecology are also discussed. Seeds of both species were also investigated using light and scanning electron microscopy, which resulted in the designation of diagnostic features. The new color form of O. kochii f. citrina is described and illustrated. An account of all revised herbarium specimens collected from Poland, deposited in Poland and neighboring countries, is presented.


Introduction
Orobanche and Phelipanche (syn. O. sect. Trionychon), are the largest genera in the Orobanchaceae family, and comprise more than 200 species, that lack chlorophyll and are root holoparasites of other vascular plants. Broomrapes are subcosmopolitan, with largest number of species being found in the warmer parts of the Mediterranean, Northern Africa, North America and Western and Central Asia. Several representatives, like P. ramosa, P. aegyptiaca, and O. crenata, parasitize important crops, e.g. tomatoes, tobacco, carrots [1][2][3]. In Europe they usually grow in the warmest regions, mostly in the Mediterranean countries. On the contrary, in central and northern parts of Europe the genera comprise about 30 species (17 established and 2 ephemerophytes from Poland), which are mostly rare, endangered or declining [e.g. 2,[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. These parasitic genera consist of plants, which possess very reduced vegetative organs and are highly variable. They are widely known for notorious difficulties in identifying, especially in herbarium material. For these reasons, there are a lot of erroneous determinations of species.
Research into the species of the genus Orobanche parasitic on Centaurea scabiosa, previously considered as Orobanche elatior group, in Central Europe revealed the existence of two distinct and unrelated species. Their proper names proved to be O. kochii F.W. Schultz and O. elatior Sutton [21]. Centaurea scabiosa is the main host of O. kochii although single records have been reported on C. jacea, C. tenuifolia, C. triumfettii subsp. axillaris, C. stoebe, C. sadleriana, C. montana, and C. ruthenica; O. elatior is mainly parasitic on C. scabiosa and sporadically on C. jacea and C. tenuifolia [9,13,[21][22][23]. It is interesting that two separate species parasitize the same host and some unrelated broomrapes have been found to parasitize the same host, e.g. Phelipanche bohemica (syn. Prior to studies conducted by Zázvorka [21], the name Orobanche elatior had been commonly used for over 100 years for an undifferentiated taxon of Eurasian distribution extending from Britain to China [1, 2,24,25]. The distribution area of O. elatior (excluding O. kochii) is restricted to Western and Central Europe, also Nordic and Baltic countries. Orobanche elatior occurs mainly in England, the Netherlands, southern Sweden, southern Norway, Denmark, Switzerland, France, Italy and Germany, while eastwards its distribution Poland. We performed more than 100 phytosociological relevés, but in this paper we presented the 39 (20 for O. elatior, 19 for O. kochii) most representative or significant. The nomenclature of vascular plants follows Mirek et al. [34]. The nomenclature of syntaxa is based on Matuszkiewicz [35].

Data processing
The localities of the both species are listed alphabetically as ATPOL cartogram units (10 × 10 km) based on Zając [36]. Only localities recorded in our observations and identified or confirmed as well as verified herbarium data are listed. Published data not confirmed by us in the field or undocumented by herbarium materials are not reliable due to frequent determination errors and these incorrect/ doubtful localities are listed separately ("List of localities of Orobanche elatior and O. kochii in Poland" section below) and marked with a special symbol on the map.
Localities are described as follows: ATPOL grid unit, geographic location, habitat description, abundance in brackets. The following information is also given for most localities: geographic coordinates and altitude (above sea level), and for revised exsiccata the collector and collection date, exsiccatum number and the herbarium acronym.

Host analysis
Host plants were observed by delicately exposing the soil with a gardening shovel. A total of 80 soil pits were observed in the field and in the analysis of herbarium materials containing an attached host.

SEM analysis of seeds
Seeds were extracted from 16 samples from 8 localities coming from Poland. The investigated material of O. elatior (4 localities: Baldram, Gipsowa Góra, Leszczany, Pińczów) and O. kochii (4 localities: Pęczelice, Wesołówka, Toporowice, Boria), has been collected in the field by the first author (see "List of localities of Orobanche elatior and O. kochii in Poland" section below); these specimens are deposited in KTC. Seeds were extracted from dried plants, mounted on stubs, coated with gold, and examined using a Joel JSM-7400F SEM, at various magnifications. Microscopic observations (LM) were carried out with a stereo microscope NIKON SMZ800 and a biological microscope NIKON Eclipse 50i.
Twelve characters (length, width, and outline of the seed and of the cells on their surface; height, width, and ornamentation of cell walls; presence of a median trough; form and diameter of seed wall perforation) were recorded. For each investigated taxon at least 30 seeds were examined. The terminology of seed surface is given after Barthlott [37,38], Stearn [39], Plaza et al. [40], Bojnansky and Fargasová [41], and Black et al. [42]. The collected data were subject to elementary statistical procedures [43], the mean, the standard deviation, and the lower and upper quartiles were calculated for each character of each of the three studied taxa. Statistical calculations were performed with Statistica 7.1.  [66,67].

Orobanche elatior Sutton
Zázvorka [21] rightly renewed the division into O. elatior and O. kochii after nearly 100 years. During investigations into the genus Orobanche in Poland, the first author also noticed a lack of homogeneity of specimens considered as O. elatior. Lightly colored specimens with a dense inflorescence were initially treated as hypochromatic plants [68]. The above examples clearly show that many different taxa were placed in O. elatior and that this group of species requires further studies. A historical survey of Orobanche parasitizing Centaurea and the history and taxonomic contents of the related names are briefly discussed in a study by Zázvorka [21].

The most important characters distinguishing Orobanche elatior from O. kochii
Both species are quite easily distinguishable when fresh. The corolla and stem in O. elatior are yellowish to ochre to light-brown or dirty-pale-pink (very rarely to dark-purple, rusty black, e.g. locality in Podskale) while O. kochii has a characteristic carrot-red to whitish-rosaceous or raspberry coloration (Fig. 1). Other characters are helpful in determining herbarium materials. Orobanche elatior is usually a large plant, with dense flowers; the corolla is regularly curved throughout, leaves are long and markedly narrow, linear-lanceolate. Orobanche kochii is mainly medium-sized; the corolla back is straighter in the middle, lower leaves on the stem are ovate to triangular, the spike subcylindrical and below lax. The two species also differ by the flowering period. Orobanche elatior flowers briefly and early, usually in late June, while O. kochii has a long flowering period, from late June until late August. A list of distinguishing characters is given in Tab. 1.
Lemon-yellow forms of O. kochii are found in Boria and Jaworzno-Długoszyn. However, they are sometimes an extremely small part of a normally-colored population (Boria), or comprise 50% of the population (Jaworzno-Długoszyn). The yellow color of the stems in typically pigmented populations was observed during two summer seasons. This new taxon is very similar to typical O. kochii, and is compliant with morphometric characters in the description of the plant after Zázvorka [21], parasitizing C. scabiosa, but differs significantly by color; in typical O. kochii they are red or rosaceous, sometimes pale rosaceous-creamy, whereas in the form citrina whole plant are lemon-yellow with brownish leaves and bracts, drying out much earlier than the rest of the stem (Fig. 1). In addition, the citrina form individuals are slightly smaller -plants are shorter and sometimes have a shorter corolla. Interestingly, the creamy color corolla and stems are found in O. ritro, parasitizing Echinops, a taxon considered being a synonym of O. kochii [21] or a separate species by some authors [62] (see also "Taxonomic notes"). DESCRIPTION. Plants medium-sized, flowering stem simple, (5-)15-25(-40) cm high, lemon-yellow, glandularpubescent, glands whitish to pale yellow; dried specimens light-brown, cream-brown or cinnamon (do not always retain the lighter color after drying). Leaves broad at the base, ovate-triangular. Inflorescence (5-)12-15(-20) cm long, subcylindrical, tapering to the apex, usually 15-30-flowered. Bracts as long as the corolla or shorter. During florescence the bracts dry out quickly and are light-brown, particularly on their ends, which is very clearly seen in contrast to the lemon-yellow corolla and stem. Calyx segments are free or occasionally connate at base, 12-16 mm long, asymmetrically ovate and unequally bidentate. Corolla lemon-yellow (rarely whitish-yellow, whitish-lemon), (15-)20-28 mm long, dorsal line curved at the base, than nearly straight, glandular-pubescent, glands pale lemon-yellow or whitish. Stamens inserted 4-6 mm above the corolla base. Style sparsely glandular, stigma lobes deep yellow and usually darker than the corolla. Flowering period (late June) July to August. Parasitizing C. scabiosa (in Poland).
DISTRIBUTION AND ECOLOGY. Orobanche kochii f. citrina is currently known only from S Poland -Małopolska and the Silesia-Cracow Uplands (Boria and Jaworzno-Długoszyn). It is found in xerothermic grasslands of the Cirsio-Brachypodion pinnati alliance, on gentle slopes of hills, on soils rich in calcium carbonate, southern inclination, altitude 160-280 m.
ETYMOLOGY. The form name originated from the lemon-yellow coloring of plants.

Seeds micromorphology
Orobanche elatior seeds are oblongoid, rare ovoid or subrectiangular, (300-)386(-450) × (190-)242(-320) μm. Epidermal seed coat cells are isodiametric to elongated; rare irregular and elongated, (40-)105(-160) × (30-)59(-100) μm. Perforation diameter ranged (4-)7.9(-13) μm, and its shape is elliptic to subcircular. The narrow trough is always present on the upper side of cell walls and is continuous. Preliminary results of micromophological examinations of seeds of O. kochii and O. elatior (then treated as two forms of O. elatior s.l.) have revealed differences in seed ornamentation, especially of the periclinal walls [68]. Our detailed studies demonstrated significant differences of diagnostic importance between O. elatior and O. kochii. The most important factor is the diameter and shape of the wall perforation. In O. elatior the perforation diameter is much larger, 4-13 μm, and is usually elliptical, while in O. kochii the diameter is smaller, 2-4 μm, and subcircular. Other characters of seed size and their cells are less important, but the analysis of a larger sample clearly demonstrated that seeds of O. elatior are larger than those of O. kochii (Tab. 2, Fig. 2).

Determination problems and discriminating from other species
Orobanche elatior belongs to a diversified section Orobanche (syn.: sect. /grex/ Curvatae Beck) [27], containing about 15 species. However, O. kochii probably has a relationship to another section Minores, this is supported by morphological characteristics, and preliminary molecular evidence (Piwowarczyk et al. unpublished); it was also initially indicated in the work of Carlón et al. [63].
Typical Monstrous shoots of O. elatior, branched into 2-4 inflorescences, with a tape-like stem, multiflorous inflorescence, with incorrectly placed flowers, are encountered occasionally (rarely), e.g. Leszczany in the Lublin region and DG-Błędów in the Dąbrowa Basin. This is probably related to herbicide on farmland near localities.

Distribution in Poland
The revision of and detailed field investigations into O. elatior and O. kochii in Poland have revealed considerable differences in the distribution, especially in relation to the previous distribution map of O. elatior s.l. [70]. The study also significantly changed and updated the previous distribution map for both species in central Europe prepared by Zázvorka [21].
At present O. elatior in Poland mainly occurs in the Silesia-Cracow, Małopolska and Lublin Uplands, rarely in the Lower Vistula Valley, Central Sudetes Mts and the Carpathian Foothills. Orobanche kochii, which is more common than O. elatior, has numerous localities but almost exclusively in the Polish uplands in a very compact range (Fig. 3, Fig. 4).
Only data verified in the field or confirmed by the herbarium material or photographic documentation is listed below. Due to frequent incorrect determinations, localities reported in the literature and unpublished localities that are not documented by herbarium material and not verified by us in the field are listed separately and marked with a special symbol on the map. Localities in the maps have been differentiated into three time periods, in order to illustrate the most current and the actual state distribution of both species (Fig. 3, Fig. 4).

Prefered habitats, plant communities and host
Orobanche elatior and O. kochii are clearly dinstinguished by their phytoceonoses. Orobanche elatior prefers sunny hillsides and slopes of hillocks, hills and valleys but also flat areas, infrequently also the colder horizon sector, at altitudes (40)200-400(700) m above sea level. Its highest sites are in the Góry Orlickie Mts (Central Sudetes Mts) and the Silesian Beskid Mts (Western Carpathians). It prefers alkaline soils, usually shallow rendzinas formed on chalk marl and limestone, dolomite, loess, gypsum, and chernozem. The abundance of specimens in populations is usually small and is 5-20 shoots but sometimes only a single inflorescence is noted. The most abundant populations occur in the Sudetes Mts (one site of a few hundreds shoots) [86] and in the central part of the Silesia-Cracow Upland (a few sites with over 100 shoots). Fluctuations in the population size are noticeable at localities observed over a few vegetative seasons.
Orobanche elatior prefers a broad phytocoenotic range. It comprises intermediate communities and is often difficult to classify. This reflects the transitional and mosaic type of habitats colonized by the species and their dynamic-succession condition. Orobanche elatior occurs in xerothermic grasslands of the alliance Cirsio-Brachypodion pinnati (Festuco-Brometea class) and communities of juniper scrub, e.g. in the Lublin Land. The species also often occurs in abandoned fields, fallows or wastelands, roadsides, along railways, in intermediate communities between xerothermic grasslands (Festuco-Brometea, Festucetalia valesiacae), thermophilous meadows (Molinio-Arrhenetheretea, Arrhenatheretalia), pioneer semiruderal xerothermic communities (Agropyretea intermedio-repentis) and ruderal communities (Artemisietea vulgaris, Onopordetalia, suballiance Dauco-Melilotenion, especially Dauco-Picridetum hieracioidis, and Echio-Meliloletum associations). It is fairly frequently recorded in fringe communities of the alliance Geranion sanguinei (Trifolio-Geranietea class). Orobanche elatior is also encountered within or on the margins of cultivations of, e.g. rape, cereal crops, or on the margins and baulks of calcareous fields, with a contribution of species of the alliance Centauretalia cyani, e.g. Caucalidion lappulae (Stellarietea mediae class) (Appendix S1). In the Sudetes Mts, the species was noted in thermophilous grasslands on forest margins of the alliance Bromion erectii [86].
Orobanche kochii prefers sunny sites, usually gentle S or SW-facing slopes but it is also found at N facing or flat sites; altitude about (40)200-400(430) m. These include xerothermic grasslands, thermophilous shrublands; located on slopes of hills, river valleys, gullies, margins of forests and fields, in former quarries, baulks, fallows and ruderal sites. It prefers alkaline soils, usually shallow rendzinas formed on chalk marl and limestone, dolomite, loess, and chernozem (black-earth soil). It is recorded most frequently in communities of the alliance Cirsio-Brachypodion pinnati (class Festuco-Brometea), especially on flowery or initial dry grasslands, i.e. Adonido-Brachypodietum pinnati, Inuletum ensifoliae, Thalictro-Salvietum pratensis, and on hills, in former gypsum excavation pits in the Nida Basin, also in Seslerio-Scorzoneretum purpureae, and in the community Carex glauca-Tetragonolobus maritimus. In the Silesia-Cracow Upland it occurs mostly on strongly xerothermic fallow rendzinas and on secondary calcareous grasslands formed close to old limestone quarries, usually on steep slopes, less frequently in sligtly thermophilous north-facing associations. In the Cracow-Częstochowa Upland, the species also sometimes grows in the ecotone of rocky grasslands at foothills of limestone mogotes (Festucetum pallentis) and thermophilous scrub (Geranion sanguinei), usually in the association Origano-Brachypodietum pinnati. As a result of natural plant succession in grasslands, the communities have a high contribution or a mosaic of species of thermophilous herbaceous forest edge communities of the alliance Geranion sanguinei (class Trifolio-Geranietea sanguinei) and shrub communities, particularly of the alliance Berberidion (class Rhamno-Prunetea), as well as xeric meadow species of the alliance Arrhenatherion elatioris (class Molinio-Arrhenatheretea). The species is also often encountered on former fallows and wastelands, especially in rendzina soil conditions, in communities of the class Artemisietea vulgaris with a high contribution of xerothermic and meadow species (Appendix S2). The abundance of populations varies greatly and ranges from year to year. Its populations are more abundant than those of O. elatior and comprise 15 shoots on average, rarely app. 100-250, e.g. 100 Gartatowice, 150 (Wojkowice) and 250 (between Wojsławice and Cynków). The abundance behaviour of O. kochii is similar to that of O. picridis [14]. It rapidly and densely colonizes abandoned areas after which its number drops suddenly as a result of succession changes. However, in the central part of the Silesia-Cracow Upland, the majority of its localities are less numerous (also less numerous than O. elatior), and sometimes only single specimens could be found. At these sites the species is difficult to find (in dry forms), especially because specimens are often very sparse in other Orobanche, mostly in the much more numerous O. lutea. All the communities mentioned above include a high contribution of host species of C. scabiosa, with the coverage being about (5)25(50)% (Appendix S1, Appendix S2).

Discussion
Orobanche kochii is more common and widely distributed in Poland, the Czech and Slovak Republics, and in much of Central Europe, with the distribution area shifting from central Europe towards Eastern Europe and Asia. Orobanche elatior is noticeably scarcer in Central Europe and its distribution is confined to Western and Central Europe. Their ranges overlap in Central Europe.
A revision of herbarium materials and field investigations into O. elatior and O. kochii in Poland was conducted between 1999 and 2014. Considerable differences in the distribution were found, especially in relation to the previous distribution map of O. elatior s.l. in Poland [70] and in Central Europe [21]. At present O. elatior occurs in Poland mainly in the Silesia-Cracow, Małopolska and Lublin Uplands, rarely in the Lower Vistula Valley, Central Sudetes Mts, Western Carpathian Mts and Carpathian Foothills. Orobanche kochii, which is definitely more common than O. elatior, has numerous localities especially in the Polish Uplands: Silesia-Cracow and Małopolska Uplands, and Lublin-Lviev, Volhynia Uplands, Roztocze and Polesie (Fig. 3, Fig. 4 Zázvorka [21] stated that O. kochii and O. elatior had never been found growing together at the same site, but they sometimes occur together in Poland (e.g. DG-Błędów, DG-Łosy, DG-Gołonóg, Podskale, Jaworzno-Ciężkowice, Lędziny, Imielin/Mysłowice).
The new form described as O. kochii f. citrina, and found in the populations of typical individuals, within the same habitat conditions, was previously known from Poland only, but may possibly be identified in a wider geographical range. We still need to confirm the cream color forms described as O. kochii and presented in photographs taken in the Crimea (Tepe-Oba Mts near Teodosia and Mys Fiolent), without any certain information on a host plant (http://www.plantarium. ru). However, herbarium materials at LE from the Crimea (Mys Fiolent) include individuals of O. ritro, parasitizing Echinops ritro [20], with a characteristic creamy or less common yellow color of corolla [27,62]. Many species of the genus Orobanche, e.g. O. alba, O. alsatica s.l., O. caryophyllacea, O. hederae, O. laxissima group, are often lighter in color than the typical, commonly occur form, but these forms almost never constitute the majority of a population. Such pigmentation is not typical and sometimes may also result from low insolation, soil pH, hosts, or incomplete plant pigmentation. Yellow and albinotic forms usually occur in shaded sites [10][11][12]68,[131][132][133], but sometimes also in places exposed to strong sunlight.
The role of seed features in the identification of species Orobanche to relate such characters to the systematics of the group, have been presented in several studies (e.g. [11,40,68,132,134,135]). The use of micromorphological characters, i.e. seed and pollen ornamentation (SEM) or a cross-section of the exine of dust seeds (TEM), have revealed additional differences between related species of O. alsatica agg. (O. alsatica, O. bartlingii, O. mayeri) [132]. Concerning the seeds of the investigated species, the best taxonomic character is the diameter and the shape of wall perforations. In O. elatior the perforation diameter is much larger, 4-13 μm, and is usually elliptical, while in O. kochii the diameter is smaller, 2-4 μm, and subcircular. The size and shape of seeds is less important for species identification. The position of a very large number of seeds in the ovary may also determine their shape as well as that of their cells [19,136]. Palynological analysis also revealed auxiliary micromorphological subtle differences in the size and level of sculpture of pollen grains between both species [137]. Differences in morphology, preliminary molecular studies, seed and pollen sculpture, and some ecological preferences confirm the separation of the examined taxa at species level.
Orobanche elatior s.l. is a partially protected species in Poland (regulation 2014). It is not a common species and is included on the "Red list of vascular plants in Poland" as rare -R [138] and vulnerable (VU) in the Carpathian Mts [79]. It is also included on regional red lists and books: Gdańsk Pomerania, CR, Western Pomerania, E [139,140]; Western Pomerania and Wielkopolska, E [139]; Wielkopolska, CR [141]; Sudetes Mts, CR [142]; Opole province, CR [56], endangered, EN, in the Lower Silesia province [143], Kujawy-Pomerania region, I (indeterminate) [144], Małopolska Upland, VU [145]. The species is also vulnerable in neighboring countries, i.e. Germany [146,147], the Czech Republic [148] and Slovakia [149]. These threat categories require revision as they concern O. elatior s.l., which contained the rare O. elatior and considerably more common O. kochii. Both species are included together only in the vascular plant red list of the Silesia province (O. elatior -category VU, vulnerable and O. kochii -NT, near threatened) [150], coauthored by us.
The progressing process of secondary succession of treeand shrub vegetation, the density of the herb layer and the cessation of former cultivation methods, especially the loss of grazing, are the main threats to both species. Their sites are often found near arable fields where they are exposed to chemical agents. Orobanche elatior is especially vulnerable as it often prefers habitats in the ecotone of arable fields, and tetralogic and deformed forms affected by weed-killing agents are often observed. Similarly due to the impact of EU subsidies on cultivation profitability, O. kochii localities on old fallows are reploughed. Land development is a direct threat in many cases as the species' habitats are often in scenic landscapes that are demanded for housing estates, especially in the central part of the Silesia-Cracow Upland, where numerous localities of both O. kochii and O. elatior are now on the margin of one of the largest European conurbations. Some localities are on the edge of operating, expanding quarries. A part of the population of O. kochii on the dolomite quarry in the DG-Ząbkowice has disappeared in this way. The arrival of invasive or expansive species also poses a considerable threat. Heracleum sosnowskyi has been moving worryingly close to its population. Mass self-sowing of Solidago canadensis, S. gigantea and Calamagrostis epigejos is observed at many sites. Only a few localities are protected: O. elatior -Bielinek res. (not confirmed here for many years), Gipsowa Góra hill, Machnowska Góra hill, Polana Polichno; O. kochii -Zimne Wody, Żmudź, Murawy Dobromierskie, Biała Góra hill, Skarpa Dobużańska, Rogów, Opalonki, Przęślin, Skorocice, Krzyżanowice, Skotniki Górne; also protected as ecological sites or within Natura 2000 sites. This does not ensure the preservation of the species as active protection is needed. Many localities are at disturbed sites or at operating excavation sites (limestone, gypsum). At least some of the localities should be protected as ecological sites and active protection measures should be implemented: mowing or grazing of excessively developing field-and herbaceous vegetation, while preserving the unique mosaic systems and fringe communities, and nature monitoring. The abundance of the host species C. scabiosa should also be monitored as this is usually neglected or overlooked in the protection of parasitic plants. Special