Comparative morphological, palynological and anatomical ...

Plant Syst Evol (2015) 301:125–137 DOI 10.1007/s00606-014-1059-z

ORIGINAL ARTICLE

Comparative morphological, palynological and anatomical characteristics of Turkish rare endemics Helianthemum germanicopolitanum and Helianthemum antitauricum (Cistaceae) Emine Burcu Yes¸ ilyurt • Sadık Erik ¨ zmen • Galip Akaydın Edibe O

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Received: 9 January 2014 / Accepted: 1 April 2014 / Published online: 24 April 2014 Ó Springer-Verlag Wien 2014

Abstract The genus Helianthemum Mill. includes 12 annual or perennial species in the Flora of Turkey, four of which are endemic. In this study, morphological, micromorphological, anatomical and palynological characteristics of the rare endemic H. germanicopolitanum Bornm. and H. antitauricum P.H. Davis & Coode are studied for the first time. H. germanicopolitanum is grown on chalky and limestone slopes in Çankırı province. H. antitauricum is grown on rocky limestone slopes in Adana and Kayseri provinces. In morphological studies, detailed descriptions of these species and characteristic features are given. Besides, pollen and seed morphology are studied by SEM. The pollen grains of both species are tricolporate, medium sized and prolate-spheroidal. Exine ornamentation is identified as perforate under LM and striate-perforate under SEM. The seed characteristics clearly appear to have taxonomic significance. The seed coat of H. germanicopolitanum characterized by the rugulate–verrucate ornamentation clearly differs from that of H. antitauricum by striate. Finally, anatomical research characters are studied on H. germanicopolitanum and H. antitauricum samples. Keywords Cistaceae Helianthemum germanicopolitanum Helianthemum antitauricum Morphology Anatomy Pollen Seed

¨ zmen E. B. Yes¸ ilyurt (&) S. Erik E. O Botany Section, Department of Biology, Faculty of Science, University of Hacettepe, Beytepe, 06800 Ankara, Turkey e-mail: [email protected] G. Akaydın Department of Biology Education, Faculty of Education, University of Hacettepe, Beytepe, 06800 Ankara, Turkey

Introduction The genus Helianthemum includes approximately 110 species in the world (Mabberly 1997) and shows a wide spread in Europe, America and from North Africa to Central Asia. Although the names Helianthemum and Cistus were in use earlier, Tournefourt (1700) recognized two genera on the basis of their capsule differences. Linnaeus (1753, 1754) did not accept Tournefort’s Helianthemum. Instead he combined it with Cistus, which he placed in the first order (Monogynia) of his thirteenth class (Polyandria). Nomenclaturally, the genus is attributed to Miller (1754) in his Gardeners Dictionary, whose Fourth Edition is still an acceptable source of generic names. In his ‘‘Flora Orientalis’’, Boissier (1867) recognized 13 taxa of Helianthemum from Turkey and placed them under five sections, all of which had been recognized previously by Dunal (1824) and Willkomm (1856). These sections are as follows: Brachypetalum Dunal, Eriocarpum Dunal, Pseudocistus Dunal, Polystachyum Willk. and Euhelianthemum Dunal. Later, Grosser (1903) informed 64 species of Helianthemum under two subgenera and seven sections: Subgen. I. Ortholobum Willk., Polystachyum Willk., Euhelianthemum Dunal, Pseudomacularia Gross., Eriocarpum Dunal, Brachypetalum Dunal, Subgen. II. Plectolobum Willk., Chamaecistus Willk., Macularia Dunal. In ‘Flora of Turkey’ (Coode 1965), the species were not referred to sections. According to published works, habit, pedicel, calyx, stamen, style and capsule are the most important diagnostic characters for distinguishing sections and species within Helianthemum (Dunal 1824; Willkomm 1856; Boissier 1867; Grosser 1903; Coode 1965). Because the species were discovered later than Boissier (1867) and Grosser (1903) (Bornmu¨ller 1930; Davis and Coode 1965), H. germanicopolitanum and H. antitauricum were not

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included in the sectional delimitation of Turkish Helianthemum. Most species of Helianthemum have been subject to numerous studies based on various data: pollen, seed, anatomy, phylogeography and ecology (Proctor 1958; Rivas 1979; Georghiou et al. 1992; Ukraintseva 1993; Nandi 1998a, b; Gonzales-Benito and Perz-Garcia 2006; Guzman and Vargas 2009; Soubani 2010; Sanderson et al. 2011). The palynological, anatomical and micromorphological features of H. germanicopolitanum and H. antitauricum have not been studied previously. Thus, the aim of this study was to execute the possible similarities and the differences between H. germanicopolitanum and H. antitauricum. These species were mentioned as closest relative species in Flora of Turkey. However, these two species are exactly different in morphology, habitat and habitus. For this reason morphology, anatomy, palynology and micromorphology of H. germanicopolitanum and H. antitauricum under LM and SEM were studied.

E. B. Yes¸ ilyurt et al.

Seeds were observed by the Tronic-Kameram 122CU stereomicroscope for checking their size and maturity. Twenty mature seeds for each species were measured to determine the average seed size. For SEM studies, mature seeds were placed on stubs directly, covered with gold and then their surface sculptures were analyzed by Zeiss EVO50 EP. For the identification of seed surface ornamentation Stearn (1992) and Koch et al. (2008) were followed. For anatomical studies, living material was kept in 70 % alcohol. The paraffin method was used for cross section of roots, stems, leaves, petioles and stipules. The specimens were embedded in paraffin wax and then sectioned at 10–20 lm thickness with a Leica RM2125RT rotary microtome. All sections were stained with safranin-astra blue and mounted with Canada balsam (Johansen 1944). Also cross section of roots, stems and leaves were performed by hand from material preserved in Sartur reagent. The Sartur reagent was composed of KI-I, aniline, sudanIII, lactic acid, alcohol and water (Çelebiog˘lu and Baytop 1949). Measurements and photos were taken by Leica DM4000 B binocular light microscope with Leica DFC320 camera.

Materials and methods Between years 2011 and 2012, field studies were realized by first author. During the field trips, populations were observed on account of density and dispersal area and the conservation status of species were decided according to IUCN (2001) criteria. Besides, H. germanicopolitanum and H. antitauricum species were collected from natural populations and materials were prepared as herbarium samples. These herbarium samples have been conserving in the Hacettepe University Herbarium (HUB), Department of Biology, Ankara, Turkey. Morphological measurements were carried out on the herbarium samples and observations were made on the living materials during the field studies. The detailed descriptions of species were created by measuring about 80 morphological characters for each sample. Palynological features were investigated on pollen material obtained from the herbarium samples. The pollen slides were prepared by asetolysis method (Erdtman 1969). Polar axes (P), equatorial axes (E), length of colpi (Clg), wide of colpi (Clt), length of pori (Plt), wide of pori (Plg), pollen diameters in polar view (Amb), apocolpia, apoporia and exine thicknesses were measured from at least 50 pollen grains under LM until the gauss curve is reached. The LM images were taken by the imaging system Olympus CX41-E330. For SEM studies, the pollen grains were directly placed on double-sided adhesive tape covered aluminum stubs and sputter coated with gold. Then, SEM images were taken by Zeiss EVO-50 EP. Punt et al. (2007) was followed for the terminology of palynology.

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Results Distribution and conservation status According to Coode (1965), the type location of H. germanicopolitanum has been given as [Turkey A4 Çankırı] In Paphlagoniae australis, in valle Tschakmaklidere, Tschangiri (=Çankırı, Germanicopolitanum, nom. vet.), 750–800 m, 16 vi 1929, Bornmu¨ller 13319 (E!, BM!) and distributed A4 Çankırı: S of Çankırı, 750 m, Hub.-Mor. 14855. Between years 2011 and 2012, we collected the species from a natural population in the Çankırı province of Turkey (Fig. 1). Collection data are as follows: A4 C ¸ ankırı, Karakıs¸ la way, the upper side of C ¸ akmaklıdere, gypsiferous slopes, 659 m, 16.6.2011, 40°290 3000 N–033°390 3600 E, EBY 1067; A4, ÇANKIRI, Kalecik-C ¸ ankırı highway,

Fig. 1 Distribution map of Helianthemum germanicopolitanum (filled circle) and H. antitauricum (filled diamond)

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Fig. 2 a–c Helianthemum germanicopolitanum: a habitat, b habitus, c flowers; d–f H. antitauricum: d habitat, e habitus, f flowers

Fig. 3 a, b Helianthemum germanicopolitanum: a without articulation of pedicel, b doubly curved style; c, d H. antitauricum: c with articulation of pedicel, d doubly curved style

division of ˙Inandık way, gypsiferous and marly slopes, 831 m, 16.6.2011, 40°230 1500 N–033°350 0100 E, EBY 1057 (Fig. 2).

The type location of H. antitauricum has been given as [Turkey B6 Adana] d. Saimbeyli, Bozog˘lan Dag˘, above Obruk Yayla, 2,000–2,100 m, 7 vii 1952, Davis, Dodds &

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Table 1 Comparison of morphological characters and habitats of Helianthemum germanicopolitanum and H. antitauricum

Stem (cm) Leaf (mm)

Indumentum (above)

H. germanicopolitanum

H. antitauricum

Ascending, 15.0–25.0 ±woolly

Procumbent, up to 15.0

adpressed-stellate and spreading-bifurcate hairy

adpressed-stellate-canescent hairy

Elliptic-lanceolate

Obovate-elliptic, in axillary fascicles

Upper leaves longer than, lower leaves shorter than the internodes

–

11.0–30.0 9 2.0–6.0

Stem leaves 11.0–20.0 9 3.5–6.5; fascicular leaves 5.5–10.0 9 2.0–4.0

±Strongly revolute

Stem leaves ± revolute; fascicular leaves strongly revolute

±Woolly, adpressed-stellate-tomentose

Stem leaves adpressed-stellate-tomentose; fascicular leaves spreading-stellate-canescent

Petiole (mm)

1.0–2.5

Stem leaves 1.5–3.5; fascicular leaves 1.0–1.5

Stipula (mm)

Acuminate

Obtuse to acuminate

2.0–7.0 9 0.5–1.75

Stem leaves 1.5–5.5 9 0.5–1.0; fascicular leaves 1.0–2.0 9 0.5

Inflorescence

Panicle in a panicle with 3–30-flowered

Raceme or rarely simple 1 or 2–4 flowered

Pedicel (mm)

4.5–8.0 in flower, 6.5–8.0 in fruit

Articulate, 7.5 in flower, 8.5–9.0 in fruit

Inner sepal (mm)

Broadly ovate, mucronate

Orbicular, obtuse to acuminate

Tip in flower

Erect, 3.0–5.5 9 2.5–3.5

Erect, 6.0 9 5.5 Erect, 8.5–9.5 9 6.5–7.5

Tip in fruit

Recurved, 5.0–6.5 9 3.5–5.2

Inner surface

Adpressed-stellate, bifurcate and simple hairy

Adpressed-bifurcate, stellate, fascicular hairy

Outer surface

±Woolly, spreading-stellate hairy, on nerves simple, bifurcate, stellate setose

Spreading-stellate, bifurcate, simple, fascicular hairy

Outer sepal (mm)

Acute

Obtuse

2.5–3.5 9 0.5 in flower, 3.0–4.0 9 0.5–0.75 in fruit

3.0–3.5 9 0.75 in flower, 4.5–5.5 9 0.75 in fruit

Petal (mm)

6.5–10.0 9 6.5–9.0

10.5–11.5 9 7.5–9.0

Style (mm)

4.5–5.0 in flower, 4.5–6.0 in fruit

4.0 in flower, 5.5 in fruit

Capsule

5.0–6.0 9 4.0–4.5

5.0 9 4.5

Seed (mm)

6–17-seed in per capsule

10–12-seed in per capsule

Ampuliform-ovate

Ampuliform

1.6–2.3 9 1.3–2.0

1.5–2.0 9 1.5–1.7

Flowering time

June

July

Habitat

Gypsiferous and marly slopes, 650–850 m

Rocky slopes, 1,800–2,100 m

C ¸ etik, D. 19719 (holo. E!) and distributed B5 Kayseri: Bakır Dag˘, 1,800–1,900 m, D. 19378! Between 2011 and 2012, we was collected the species from a natural population in the Adana province of Turkey (Fig. 1). Collection data are as follows: B6 Adana, between Arslantas¸ and Ayvat village, rocky slopes, 38°220 2900 N–036°110 1000 E, *2,000 m, 11.08.2011, EBY 1126; 13.07.2012, EBY 1320 (Fig. 2). According to IUCN (2001) criteria H. germanicopolitanum is evaluated as EN (Endangered). This species was recorded from two localities on roadside. This species produces populations with low density and covers restricted area. It is in danger of erosion and roadwork. Thus, we approved to place in EN (Endangered) category.

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H. antitauricum is utilized as LC (Least Concern). There are densely grazing in the habitat of H. antitauricum. This population is damaged and decreased. In our opinion, threat category of H. antitauricum would be more appropriate as EN (Endangered). Distribution of H. germanicopolitanum is in Irano-Turanian area; on the other hand, H. antitauricum is an East Mediterranean element. Morphological characteristics H. germanicopolitanum Suffruticose perennial herb, ascending flowering stems 15.0–25.0 cm tall (Fig. 2). Indumentum ± woolly,


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Fig. 4 LM images of Helianthemum germanicopolitanum pollen grains: a polar view (high focus), b equatorial view (high focus), c polar view (low focus) and d equatorial view (low focus). LM

images of H. antitauricum pollen grains: e equatorial view (high focus), f equatorial view (low focus), g polar view (high focus), h polar view (low focus)

adpressed-stellate and spreading-bifurcate hairy. Leaves opposite, elliptic-lanceolate, 11.0–30.0 9 2.0–6.0 mm, ±strongly revolute, ±woolly, adpressed-stellate-tomentose above, spreading-stellate canescent below, denser beneath, upper leaves longer than the internodes, but lower leaves shorter than the internodes. Petiole 1.0–2.5 mm. Stipules subulate, sessile, acuminate, 2.0–7.0 9 0.5–1.75 mm, consistent. Flowers pendulous in a panicle with 3–30 flowered. Pedicels without articulation, 4.5–8.0 mm in flower, 6.5–8.0 mm in fruit (Fig. 3). Inner sepals broadly ovate, mucronate, 3.0–5.5 9 2.5–3.5 mm in flower, 5.0–6.5 9 3.5–5.2 mm in fruit, elongating in fruit, tips of inner sepals erect in flower, but recurved in fruit, the inner surface adpressed-stellate, bifurcate and simple hairy, the outer surface ± woolly, spreading-stellate hairy, on the nerves simple, bifurcate and stellate setose, 3–4 nerves. Outer sepals subulate, acute, 2.5–3.5 9 0.5 mm in flower, 3.0–4.0 9 0.5–0.75 mm in fruit, the inner surface adpressed-stellate, bifurcate, simple hairy, the Outer surface spreading-stellate hairy, simple, bifurcate, stellate setose. Petals yellow, obovate, undulate, longer than inner sepals, 6.5–10.0 9 6.5–9.0 mm. Stamens numerous, multiseriate,

filament 2.0–4.0 mm long, anthers 0.5 9 0.5 mm. Style doubly curved (Fig. 3), 4.5–5.0 mm in flower, but 4.5–6.0 mm in fruit. Stigma capitate. Capsules with spreading-stellate hairy, the tips longer and denser simple bifurcate hairy, 5.0–6.0 9 4.0–4.5 mm.

H. antitauricum Suffruticose perennial herb, procumbent flowering stems up to 15.0 cm tall (Fig. 2). Indumentum adpressed-stellatecanescent hairy. Leaves in axillary fascicles, obovateelliptic, stem leaves 11.0–20.0 9 3.5–6.5 mm broad, ±revolute, adpressed-stellate-tomentose above, spreadingstellate-canescent below; fascicular leaves 5.5–10.0 9 2.0–4.0 mm, spreading-stellate-canescent above, spreading-stellate-canescent below, strongly revolute. Petiole stem leaves 1.5–3.5 mm, fascicular leaves 1.0–1.5 mm. Stipules subulate, sessile, obtuse to acuminate, stem leaves 1.5–5.5 9 0.5–1.0 mm; fascicular leaves 1.0–2.0 9 0.5 mm; consistent. Flowers pendulous in a raceme or rarely simple, 1 or 2–4 flowered. Pedicels with articulation,

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Fig. 5 SEM images of Helianthemum germanicopolitanum pollen grains: a general view, b equatorial view, c polar view. SEM images of H. antitauricum pollen grains: d general view, e equatorial view, f polar view

7.5 mm in flower, 8.5–9.0 mm in fruit (Fig. 3). Inner sepals orbicular, obtuse to acuminate, 6.0 9 5.5 mm broad in flower, 8.5–9.5 9 6.5–7.5 mm in fruit, elongating in fruit, tips of inner sepals erect in flower and fruit, the inner surface adpressed-bifurcate, stellate, fascicular hairy, the Outer surface spreading-stellate, bifurcate, simple, fascicular hairy, 4–5 nerves. Outer sepals subulate, obtuse, 3.0–3.5 9 0.75 mm in flower, 4.5–5.5 9 0.75 mm in fruit, the inner surface adpressed-stellate,

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bifurcate, simple, fascicular hairy, the Outer surface adpressed-stellate, bifurcate, fascicular hairy. Petals yellow, obovate, entire, longer than inner sepals, 10.5–11.5 9 7.5–9.0 mm. Stamens numerous, multiseriate, filament 2.0–3.0 mm long, anthers 0.5 9 0.25 mm. Style doubly curved, 4.0 mm long in flower, but 5.5 mm in fruit (Fig. 3). Stigma capitate. Capsules with spreadingstellate hairy, the tips longer and denser simple bifurcate hairy, 5.0 9 4.5 mm.

Comparative morphological, palynological and anatomical characteristics of Turkish rare endemics Table 2 Comparison of palynological measurements of Helianthemum germanicopolitanum and H. antitauricum H. germanicopolitanum

H. antitauricum

E

29.0–36.0 (32.18 ± 1.49) l

33.0–42.0 (37.62 ± 2.32) l

P

33.0–39.0 (36.04 ± 1.31) l

37.0–48.0 (42.26 ± 2.79) l

P/E

1.12

1.12

Amb

27.0–32.0 (29.52 ± 1.18) l

33.0–44.0 (37.98 ± 2.42) l

Exine

2.0–2.5 (2.06 ± 0.17) l

2.0–3.0 (2.36 ± 0.35) l

Clg

28.0–34.0 (30.66 ± 1.52) l 1.0–3.0 (2.01 ± 0.45) l

32.0–43.0 (37.78 ± 2.80) l 1.0–2.0 (1.15 ± 0.29) l

Clt Plg

4.0–6.0 (4.94 ± 0.65) l

4.0–9.0 (6.30 ± 1.05) l

Plt

4.0–7.0 (5.20 ± 0.73) l

4.0–7.0 (5.46 ± 0.95) l

Apocolpium

3.0–7.0 (5.10 ± 1.08) l

4.0–7.0 (5.86 ± 0.83) l

Apoporium

18.0–24.0 (21.10 ± 1.50) l

24.0–35.0 (28.78 ± 2.74) l

E equatorial axis, P polar axis, Amb diameter in polar view, Clg colpus longitude, Clt colpus latitude, Plg porus longitude, Plt porus latitude

Summary of morphological measurements is given in Table 1. Pollen characteristics Pollen grains of H. germanicopolitanum are isopolar and tricolporate. Shape of pollen grains is prolate-spheroidal, P 36.04 (±1.31) l, E 32.18 (±1.49) l, P/E 1.12. Pollen grains are circular in polar view, Amb 29.52 (±1.18) l. Apocolpial areas are rather narrow, apocolpia 5.10 (±1.08) l, apoporia 21.10 (±1.50) l. Colpi are long and narrow, Clg 30.66 (±1.52) l, Clt 2.01 (±0.45) l. Pori are circular and wider than colpi, Plg 4.94 (±0.65) l, Plt 5.20 (±0.73) l. Exine is very thin, 2.06 (±0.17) l. Exine ornamentation is identified as perforate under LM (Fig. 4). Despite that, the SEM analysis showed that the exine ornamentation was striate-perforate at the equatorial area and rugulate-perforate at the polar area (Fig. 5). Pollen grains of H. antitauricum are isopolar, tricolporate. Shape of pollen grains is prolate-spheroidal, P 42.26 (±2.79) l, E 37.62 (±2.32) l, P/E 1.12. Pollen grains are circular in polar view, Amb 37.98 (±2.42) l. Apocolpial areas are rather narrow, apocolpia 5.86 ± 0.83 l, apoporia 28.78 ± 2.74 l. Colpi are long and narrow, Clg 37.78 (±2.80) l, Clt 1.15 (±0.29) l. Pori are circular and wider than colpi, Plg 6.30 (±1.05) l, Plt 5.46 (±0.95) l. Exine is very thin, 2.36 ± 0.35 l. Exine ornamentation is identified as perforate under LM (Fig. 4). Nevertheless, the SEM analysis showed that the exine ornamentation was striate-

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perforate at the equatorial area and rugulate-perforate at the polar area (Fig. 5). Summary of palynological measurements is given in Table 2. Seed morphology H. germanicopolitanum There are 6–17 seeds per capsule. Seeds are ampuliformovate and dark brown or fawn. Size of seeds is 1.6- to 2.3-mm long and 1.3- to 2.0-mm broad. The surface of seeds are densely to sparsely pruinose-punctate under LM. Because of the unclear anticlines, cells of seed coat are not distinguished one by one under SEM. In the general term, the seed coat ornamentation is rugulate–verrucate and warts are in irregular groups. Besides, anticlinal lines are clearly seen on hilum (Fig. 6). H. antitauricum There are 10–12 seeds per capsule. Seeds are ampuliform and dark brown. Size of seeds is 1.5- to 2.0-mm long and 1.5- to 1.7-mm broad. The surface of seeds is pruinosepunctate under LM. Under SEM, the anticlines are distinct, anticlinal fields are striate and central fields have convex hunches with striate ornamentation. In addition, anticlinal lines are clearly seen on hilum (Fig. 6). Anatomy Root H. antitauricum and H. germanicopolitanum resemble each other in transverse section. The periderm layer on the outermost surface is thick and its cells are squashed or breaking up as it is two perennial dicotyledon plants. Under the periderm, there is a thick cortex layer. Next to the cortex there are well-developed conduction bundles. Although the number of phloem cells is few, xylem cells occupy a wider area. Tracheas and tracheides can be distinguished clearly in the xylem tissue (Fig. 7). Stem The stem of H. germanicopolitanum and H. antitauricum is more or less circular in cross section. The partially stellate hairy epidermis of H. germanicopolitanum is covered by a thin and undulate layer of cuticle and consists of 1–2 layers of oval, squarish or rectangular cells. In spite of this, H. antitauricum stem shows a multilayer periderm structure in the outermost. Also, young stems of H. antitauricum have epidermis with stellate hairs. In the stem, cross

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Fig. 6 SEM images of seeds: a–c Helianthemum germanicopolitanum: a general appearance, b hilum ornamentation, c surface ornamentation; d–f H. antitauricum: d general appearance, e hilum ornamentation, f surface ornamentation

section of H. germanicopolitanum, 3–4 layered chlorenchyma and 5–7 layered of irregularly oval, squarish or rectangular collenchyma are composed under the epidermis. In H. antitauricum, only 1–2 layered collenchyma appears under the periderm. Vascular bundles are next to each other for both species. The phloem is surrounded by 1–5 layered sclerenchyma in H. germanicopolitanum; however, it is 1–3 layered in H. antitauricum. The cambium is located between the phloem and xylem. The xylem

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is enclosed by 8–15 layered sclerenchyma in H. germanicopolitanum and the sclerenchyma is 8–11 layered in H. antitauricum. The vascular bundles are arranged circular in stem of either species (Fig. 8). Leaf The cross section of leaf indicates that the epidermis is covered by a thin cuticula layer on the outside. In addition,


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Fig. 7 a Transverse section of roots: a Helianthemum germanicopolitanum and b. H. antitauricum. Pe periderm, C cortex, Ph phloem, Xy xylem)

Fig. 8 Transverse section of the stem. a, b Helianthemum germanicopolitanum: a general view, b detailed view; c, d. H. antitauricum: c general view, d detailed view. Pi pith, Xy xylem, Ca cambium, Ph

phloem, Sc sclerenchymatic cell groups, Ch chlorenchyma, Co collenchyma, E epidermis, Pe periderm)

both surface of the leaf are coated by stellate hairs as seen on the stem. The indumentum of H. antitauricum is denser than H. germanicopolitanum. Either of upper and lower epidermis consist of rectangular or squarish cells. Upper epidermis cells are larger than lower ones. The leaf type is equifacial (isolateral). The surface anatomy of the leaf shows that the anomocytic stomata are framed by the epidermis cells (Fig. 9). Furthermore, there are mesophytic stomata on the both surfaces (amhistomatic). The

mesophyll is composed of elongated rectangular palisade parenchyma cells and irregularly arranged spongy parenchyma cells. Palisade parenchyma is 2–3 layered above and 1 layered below. The spongy parenchyma is 2–4 layered in H. antitauricum and 4–5 layered in H. germanicopolitanum with large intercellular spaces. There is well-developed collenchyma upper the lower epidermis. The type of the vascular bundles is open-collateral. There is a sclerenchyma tissue below the phloem (Fig. 10).

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Fig. 9 Leaf cross sections: a, b Helianthemum germanicopolitanum, a transverse section, b surface section; c, d H. antitauricum, c transverse section, d surface section

Discussion H. germanicopolitanum and H. antitauricum are geographically isolated from each other. While H. germanicopolitanum grows in Çankırı, gypsiferous and marly slopes, 650–850 m, H. antitauricum grows in Adana/ Kayseri, rocky slopes, 1,800–2,100 m. At the same time, these species are also seasonally isolated. H. germanicopolitanum blooms in June and fruits in June to July. Against this, H. antitauricum blooms in July and fruits in August. Morphological differences between two species are as follows: H. germanicopolitanum is ascending and 15.0–25.0 cm tall, but also H. antitauricum procumbent and up to 15 cm. Leaves of H. germanicopolitanum are opposite, but H. antitauricum’s leaves in axillary fascicles. Inflorescence of H. germanicopolitanum is panicle up to 30-flowered, as H. antitauricum has simple or rasem up to 4-flowered. Pedisel of H. antitauricum is articulated, but H. germanicopolitanum pedisel is not (Table 1). Pollen grains of both species are isopolar, tricolporate and prolate-spheroidal. Pollen diameters (E, P, Amb) of H. germanicopolitanum are longer than H. antitauricum pollen diameters (Table 2). Exine ornamentation of both

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species is perforate under LM (Fig. 4), under SEM striateperforate at the equatorial area and rugulate-perforate at the polar area (Fig. 5). Rivas (1979), Ukraintseva (1993), Perveen and Qaiser (1998), Hassan (2011) and Yes¸ ilyurt et al. (2012) studied on the pollen morphology of Family Cistaceae. Rivas (1979) researched 10 species of Helianthemum as part of this study. According to the study, pollen grains of Helianthemum are 3-colporate, prolate to subprolate and striate or rugulate (only H. sanguineum pollen). In addition, he indicated that all pink-flowered Cistus species have rugulate pollen grains and H. sanguineum is also pink flowered. Examined samples of our study, H. antitauricum and H. germanicopolitanum, are yellow flowered and our SEM analysis showed that the pollen grains of these two species are striate-perforate at equatorial area and rugulate-perforate at polar area. Pollen SEM images show that Rivas (1979) analyzed the equatorial area of pollen grains for deciding the sculpture and also he ignored the perforations between stria which seem clearly in the images. Ukraintseva (1993) studied the pollen morphologies of 65 species from eight genus of Cistaceae under LM. According to this study 6 types of pollen are characterized


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Fig. 10 Transverse section of the leaf: a the lamina of Helianthemum germanicopolitanum, b the midrib of Helianthemum germanicopolitanum, c the lamina of H. antitauricum, d the midrib of H. antitauricum

such as Cistus-type, Helianthemum-type, Crocanthemumtype, Fumana-type, Hudsonia-type and Lechea-type. The types of pollen grains, recognized in the family Cisteceae, from 3 palynological groups: 1. Cisteae, 2. Hudsonia, 3. Lechea. The Cisteae group include 2 pollen subtypes: 1.1. Cistinae (Cistus-type and Helianthemum-type) and 1.2. Fumaninae (Fumana-type and Crocanthemum-type). According to Ukraintseva (1993), the pollen types differ clearly from one another and almost always correspond to genera. On the other hand the pollen morphology is insufficient separation of the Helianthemum species. Ukraintseva’s Helianthemum-type pollen grains are 3-colporate, prolate-spheroidal to prolate and finely reticulate. The researcher reported that Helianthemum pollen type is characteristic only for the genus Helianthemum. Our study brought out the sculpture of pollen grains as perforate under LM. Reticulum is a network-like pattern but tectum of Helianthemum pollen consists irregular holes less than 1 lm. Perveen and Qaiser (1998) investigated the pollen morphology of Helianthemum lippii. Researchers described the pollen of H. lippii as 3-colporate, prolate-spheroidal, triangular in polar view and striate-rugulate. They also ignored the perforations in tectum.

Hassan (2011) indicated that pollen size, shape, aperture, and exine sculpturing are valuable characters for Cistaceae family. The author recognized three pollen types among the studied taxa of Cistaceae (Hassan, 2011). According to Hassan (2011) the taxa within genus Helianthemum, except H. salicifolium, have been grouped into pollen type III. This pollen type has been containing three subtypes; striate, striate-reticulate, and striate-rugulate. Pollen grains of H. germanicopolitanum and H. antitauricum look like Hassan’s striate-reticulate subtype. Besides, the images from the study of Hassan (2011) show that the pollen grains were containing holes \1 lm between striae instead of a network pattern. For this reason, the subtype should be described as striate-perforate. Furthermore, in the present study it emerged that the pollen grains showed different ornamentation in polar and equatorial area. Yes¸ ilyurt et al. (2012) studied the pollen morphology of H. germanicopolitanum. According to this study, pollen grains were tricolporate, medium sized and prolate-spheroidal. Exine ornamentation has been identified as perforate under LM, striate at equatorial area and rugulate-perforate at polar area under SEM. The researchers disregarded the perforations in equatorial area. The results of the present

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study are consistent with the study of Yes¸ ilyurt et al. (2012). The consequence of our anatomical research was accordant with Metcalfe and Chalk (1957). According to Metcalfe and Chalk (1957), in Cistaceae, stomata present on both leaf surface, mesophyll always including welldeveloped palisade tissue. Druse crystals were occured in the phloem of leaf and stem of these species. Besides, it is reported that there are rhomboidal calcium oxalate crystals in axial and ray parenchyma in the wood of Cistus (Fahn et al. 1986; Carlquist 2001). Endodermis clearly defined in young stems of certain Helianthemum species. Xylem and phloem present in the form of a closed cylinder surrounding the pith. In the genus Lechea, either raylessness that persists for the duration of a stem or instances in which wood is rayless at first and then develops rays (Boureau 1957; Carlquist 2001). In any case, in the present study the both species have ray cells in xylem tissue. In our study, the cross section of H. germanicopolitanum stem was cut in early-phase. So the epidermis layer was seen on the outside. Otherwise, the cross section of H. antitauricum stem was cut in late-phase. Therefore, the outside was periderm. As a conclusion, there was no detailed study on Turkish rare endemics H. germanicopolitanum and H. antitauricum before this research. Our study is accordingly the first extensive research of morphology, pollen, anatomy and seed on both species. The results demonstrated that the studied taxa are generally similar to each other in terms of palynology and anatomy. However, morphological characters and nutlet micromorphology of samples indicated some differences. At the same time, there is an geographical isolation between the species. Acknowledgments We wish to thank the Scientific Research Projects Coordination Unit of Hacettepe University (Project No. 01001601006) for financial support. We are grateful to Dr. Hatice Nurhan Buÿu¨kkartal from Ankara University and Dr. Cahit Dog˘an from Hacettepe University for their valuable comments and suggestions to improve this paper.

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