Acta Botanica 1-2017 - za web.indd 32 ACTA BOT. CROAT. 76 (1), 2017 Acta Bot. Croat. 76 (1), 32–40, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0045 ISSN 0365-0588 eISSN 1847-8476 Morphology, anatomy and karyology of endangered Turkish endemic Physoptychis haussknechtii Bornm. (Brassicaceae) from Central Anatolia Mehmet Tekin1*,#, Esra Martin2 1 Cumhuriyet University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 58140, Sivas, Turkey 2 Necmettin Erbakan University, Faculty of Science, Department of Biotechnology, 42090, Meram, Konya, Turkey # present address: Trakya University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 22030 Edirne, Turkey Abstract – In this study, the morphology, anatomy, pollen morphology, leaf and seed micromorphology and karyology of the Turkish endangered endemic Physoptychis haussknechtii were investigated in detail for the fi rst time. The species was studied in terms of detailed morphology. Basal leaves are narrowly spatulate and cauline leaves are linear to oblanceolate; both leaves are covered with fl attened stalked fi ve-armed trichomes. Raceme length was found to be up to 6 cm and 9 cm in the fl owering and fruiting stages, respectively. Sepals are linear-oblong, rarely elliptic with obtuse to subacute apex. Petals are spatulate with emarginate apex. Some morphological characteristics such as dimensions of cauline leaves and ovary and length of pistil were measured for the fi rst time. In an anatomical study, root, stem, basal leaf and cauline leaf were examined. The anatomical results showed that the plants have secondary growth in roots, primary growth in stems, amphis- tomatic and equifacial basal and cauline leaves with anisocytic stomata. Pollen grains are radial symmetrical, isopolar and tricolpate. The ratio of polar axis to equatorial diameter is 1.16, and pollen shape is subprolate. Exine sculpturing is reticulate under light microscope and scanning electron microscope. Seeds are obovate- suborbicular, compressed and pale brown. The chromosome number of P. haussknechtii was defi ned to be 2n=16. The karyotype formula of P. haussknechtii consists of fi ve metacentric chromosome pairs and three submetacentric chromosome pairs. Keywords: anatomy, Cruciferae, endemic, karyology, morphology, Physoptychis haussknechtii, pollen, Turkey * Corresponding author, e-mail: mtekin2280@gmail.com Introduction The Brassicaceae family is represented by 321 genera and about 3660 species in the world, and with these num- bers it is known as a huge dicot family (Al-Shehbaz 2012). The major distribution centers of the family are the Medi- terranean, Irano-Turanian and Saharo-Sindian regions (Hedge 1976). Turkey is one of the richest countries in this respect, with 653 native species belonging to 61 genera of the Bras- sicaceae family (Al-Shehbaz et al. 2007). Physoptychis Boiss. is one of the genera of Alysseae tribe, which comprises in all 24 genera with about 277 spe- cies (Španiel et al. 2015) distributed mainly in Eurasia (Re- šetnik et al. 2013). Morphologically, the tribe Alysseae is characterized by a herbaceous or sub-shrubby growth form, a dense pubescence consisting usually of stellate trichomes, yellow or white (rarely pale pink) corollas, fi laments usual- ly possessing wings, teeth or other appendages, latiseptate or terete, mostly few-seeded siliques, often winged seeds and a base chromosome number of x=8 (Warwick et al. 2008, Rešetnik et al. 2013). In addition, a common physio- logical characteristic of the taxa of the tribe Alysseae is their ability to hyperaccumulate some heavy metals (Teren- teva et al. 2014). According to Davis (1965) and Cullen (1965), Physop- tychis is morphologically related to the genera Alyssoides Adans. and Fibigia Medik. and it can be separated from Fibigia by having infl ated fruits and narrower seed wings. On the other hand, possessing fruits in diameter ca. 20 mm, the genus Physoptychis is different from the genus Alyssoi- des, which has the fruits ca. 10 mm in diameter (Davis 1965). Recently, some phylogenetic investigations were carried out using molecular methods based on nuclear ITS regions (Warwick et al. 2008, 2010, German et al. 2009, Cecchi et al. 2010, Cecchi 2011) or nuclear and chloroplast DNA se- quence data on the tribe Alysseae (Rešetnik et al. 2013). According to Rešetnik et al. (2013), Physoptychis is phylo- genetically related to the genera Alyssoides, Clastopus Fair- maire, Fibigia and Degenia Hayek. STRUCTURAL AND KARYOLOGICAL STUDIES ON PHYSOPTYCHIS HAUSSKNECHTII ACTA BOT. CROAT. 76 (1), 2017 33 The genus Physoptychis is represented by two species, P. haussknechtii Bornm. and P. gnaphalodes (DC.) Boiss. in Turkey (Davis 1965). While P. haussknechtii is an en- demic species to Turkey known from only two localities in Sivas province, P. gnaphalodes is non-endemic, grows in the Caucasus, Iran, Northern Iraq and Turkey (Boissier 1867, Cullen 1965, Appel and Al-Shehbaz 2003). Recently, some other localities of P. haussknechtii were found in Sivas and Erzincan provinces. According to the Flora of Turkey (Da- vis 1965), P. gnaphalodes is known from one locality, in Hakkari province of Turkey, and grows in Armenia, Iraq and Iran. Botschantzeva (1976) treated P. gnaphalodes as synonym of P. caspica (Hablitz) V.V. Botscantz. In 2007, a new species for the genus, P. purpurascens Çelik & Akpu- lat very similar to P. haussknechtii was found and pub- lished. It has pink-purple petals, unlike the yellow petals known in P. haussknechtii from Sivas province of Turkey (Çelik et al. 2007). After this study, German et al. (2016) published a revision of the genus Physoptychis and found that Bornmüller’s mention of yellow petals in P. hauss- knechtii was erroneous. Thus, P. purpurascens was reduced to being a synonym of P. haussknechtii. Currently, the ge- nus Physoptychis is represented by two species (P. hauss- knechtii and P. caspica) in the world. According to IUCN criteria P. haussknechtii is treated as an Endangered (EN) species in the Red data book of Turkish plants (Ekim et al. 2000). There is great defi ciency of morphological knowledge on P. haussknechtii in the Flo- ra of Turkey (Cullen 1965). The present study is the fi rst comprehensive study of the morphology, and the fi rst study of the anatomy, palynology, seed micromorphology and karyology of P. haussknechtii. Furthermore, the present study is the fi rst comprehensive structural study not only of P. haussknechtii, but also of the genus Physoptychis. There are only a few cytogenetic studies that have been carried out on P. caspica (syn. P. gnaphalodes) and the so- matic chromosome number of this species is 2n=16 (Küp- fer 1980, Maassoumi 1980, Carrique and Martínez 1984, Warwick and Al-Shehbaz 2006). There is no report on the karyology or ideogram of this species, so our study, con- ducted to determine the karyology and ideogram of P. haussknechtii, is the fi rst study for the genus. The aim of this study is to investigate the morphologi- cal, anatomical, palynological, micromorphological and ka- ryological characteristics of the endangered Turkish ende- mic P. haussknechtii, and to reveal their contribution to the taxonomy of the genus. Materials and methods P. haussknechtii samples were collected during the fl ow- ering and fruiting period from different natural habitats in Sivas province. Localities of P. haussknechtii samples are listed in Tab. 1. Specimens for morphological studies were dried according to standard herbarium techniques and were stored in the Cumhuriyet University, Science Faculty Her- barium (CUFH). Identifi cation was made according to Flo- ra of Turkey (Davis et al. 1965) by the fi rst author. Fresh samples were used for morphological measurements. The materials necessary for anatomical studies were collected from natural habitats and preserved in 70% alcohol. For anatomical analysis, transverse sections of root, stem, basal and cauline leaves were used. From these organs, hand sec- tions were taken with a razor blade and sections were stained with Alcian blue (Sigma) for pectic substances and Safranine O (Sigma) for lignin substances in the ratio of 3:2. The sections were put in prepared dye about 5 minutes for staining. The well-stained sections were mounted in glycerin-gelatin to obtain permanent preparations (Jensen 1962). Sections were examined and measured by using an Olympus light microscope BX21. Microphotographs were captured using a digital camera Olympus DP70 connected with the Olympus BX51light microscope. For palynologi- cal studies, pollen grains were prepared for light microsco- py (LM) by using the Wodehouse method (Wodehouse 1959). In micromorphological studies using scanning elec- tron microscopy (SEM), the pollen grains, a part of the dried and scraped basal leaf, a part of the dried cauline leaf and mature seed were transferred to stubs prepared with double-sided adhesive tape. After that, the stubs were coat- ed with the gold for 5 minutes. The surface ornamentation detail and the aperture characteristics of the pollen grains and surface characteristics of the leaves and seeds were in- vestigated under the LEO 440 scanning electron micro- scope, and the micrographs were taken at different magnifi - Tab. 1. Collection data of the investigated specimens of Physoptychis haussknechtii. Locality Collector and collector number Date Herbarium B6 Sivas: Ulaş, Kurtlukaya village to Deliilyas village, 1474 m, 39°23’02.8″N, 36°55’48.9″E M. Tekin, 1244 M. Tekin, 1408 M. Tekin, 1565 M. Tekin, 1542 M. Tekin, 1654 12.06.2012 11.05.2013 05.06.2014 16.05.2014 30.04.2015 CUFH B6 Sivas: Hafi k, Gürlevik mountain, serpentine slopes, 1932 m, 39°34’31.8″N, 37°30’14.9″E M. Tekin, 1591 M. Tekin, 1662 11.06.2014 11.05.2015 CUFH B6 Sivas: Divriği-Kangal road, near Pınargözü village, serpentine slopes, 1549 m, 39°15’06.6″N, 37°41’06.1″E M. Tekin, 1669 22.05.2015 CUFH B7 Sivas: Divriği, Dumlucadağ mountain (Type locality), fer mine road, serpentine slopes, 1862 m, 39°23’32.0″N, 38°02’35.0″E M. Tekin, 1670 22.05.2015 CUFH TEKİN M., MARTİN E. 34 ACTA BOT. CROAT. 76 (1), 2017 cations. For palynological study, the terminology of Punt et al. (1994) was followed. The statistical analyses of anatom- ical measurements were made by the SPSS package pro- gram (ver. 15). For karyological analyses, root meristems from germinating seeds collected in the wild were used. The root tips were pretreated with α-monobromonaphthalene at 4 °C for 16 h. The root tips were fi xed with Carnoy for 24 h at 4 °C. Before staining, the material was hydrolyzed with 1 N HCl for 12 minutes at room temperature. The chromo- somes were stained with 2% acetic orcein and mounted in 45% acetic acid. Permanent slides were made by using the standard liquid nitrogen method. Microphotographs were taken through an Olympus BX51 light microscope. Chro- mosomes were classifi ed according to the nomenclature of Levan et al. (1964). The ideogram was drawn based on cen- tromeric index, and was arranged in decreasing order of size. Results Morphological characteristics Perennial herb, plant height 5–22 cm. Caespitose, tap- root thick with a few secondary branches. Stem much branched, covered densely with whitish grey trichomes and with numerous lateral sterile rosettes at the densely foliose base. Basal leaves 18–42×3–6.5 mm in dia., sessile, nar- rowly spatulate with subacute to obtuse apex and covered in whitish grey fl attened stalked fi ve-armed trichomes. Cauline leaves 20–55×2–4 mm in dia. sessile, linear to ob- lanceolate with subacute to obtuse apex, usually narrower than basal leaves and covered with whitish grey fl attened stalked fi ve-armed trichomes. Racemes densely fl owered, up to 6 cm and 9 cm long at the fl owering and fruiting stage, respectively. Pedicels up to 8 mm and 12 mm at the fl owering and fruiting stage, respectively, and covered with whitish grey fl attened stalked fi ve-armed trichomes. Sepals 4–8×1.8–2.4 mm in dia., linear-oblong, rarely elliptic, outer side green-purplish with densely whitish grey fl attened stalked fi ve-armed trichomes, inner side green and smooth, margin membranous, apex obtuse to subacute. Petals 9–13× 2–3 mm in dia., spatulate with emarginate apex, pink-pur- ple colored. Stamens dorsifi xed, tetradynamous, long fi la- ments 3.5–4.4 mm, short fi laments 2.5–3.3 mm. Anthers 1–1.5 mm, longitudinal dehiscence. Pistils 3–4 mm, covered with whitish grey trichomes. Ovaries obovate or elliptic, 1.7– 2.5×0.8–1.3 mm in dia., styles slender and distinct, stigma capitate. Siliculae conical in outline, 10–26×9–16 mm in dia., acuminate at apex, straw-colored at maturity and cov- ered densely with whitish grey fl attened stalked fi ve-armed trichomes. Seeds 3–6×2–4 mm in dia., obovate-suborbicu- lar, compressed, and pale brown. Seed margins membra- nous, wing-like and seed surface ornamentation coarsely reticulate (Figs. 1, 2J–K and Tab. 2). Palynological characteristics The pollen grains of P. haussknechtii are radial sym- metrical, isopolar and tricolpate. Pollen grain shape is el- liptical in equatorial view (Figs. 2A, E) and orbicular in Fig. 1. General view of Physoptychis haussknechtii in fl owering stage (A) and fruiting stage (B) in natural habitat (Photos by M. Tekin). Fig. 2. Pollen grain microphotographs under light microscope (A–D) and scanning electron microscope (E–H), and seed micro- graphs under scanning electron microscope (J–K) of Physoptychis haussknechtii: A) equatorial view (high focus), B) aperture and exine ornamentation in equatorial view (low focus), C) polar view (high focus), D) apertures and exine ornamentation in polar view (low focus); E) equatorial view, F) aperture and exine ornamenta- tion in equatorial area, G) polar view, H) apertures and exine orna- mentation in polar area; J) general view of seed, K) ornamentation of seed coat. STRUCTURAL AND KARYOLOGICAL STUDIES ON PHYSOPTYCHIS HAUSSKNECHTII ACTA BOT. CROAT. 76 (1), 2017 35 polar view (Figs. 2C, G). The length of the polar axis (P) and equatorial diameter (E) are 24.11–29.34 μm and 21.02– 25.38 μm, respectively. The ratio of P/E is 1.16 and pollen shape is subprolate. Exine thickness is 1.03–1.46 μm. Exine ornamentation is reticulate in both the equatorial and the polar sides (Figs. 2B, D, F, H). Anatomical characteristics Root: In the cross-section, the root exhibits a secondary structure. There is a periderm which makes up the outer- most layer of the root and consists of multilayered dark col- ored cells, crushed, broken up and sometimes fallen out. Beneath the periderm, there is collenchyma tissue which consists of 2–5 cell layers, and cells are usually depressed elliptical, rarely rectangular or square shaped. Cortex is composed of 3–10 parenchymatous cell layers under the collenchyma. Cortex cells are usually elliptical, occasional- ly irregularly shaped and are bigger on the outer cross-sec- tion of the root than on the inner cross-section. Sometimes, there are wide gaps between the cortex cells. The phloem is located under the cortex, and is composed of 4–14 layered overlapping sequence of cells. Cambium cells are usually compressed, rectangular or irregular shaped, and are 2–4 layered between phloem and xylem. The region from the cambium layer to the root center consists of parenchymatic and tracheal cells belonging to xylem (Figs. 3A–C and Tab. 3). Tab. 2. Morphological results comparison of Physoptychis hauss knechtii and its synonym, Physoptychis purpurascens. Characteristic P. haussknechtii (Present study) P. purpurascens (Çelik et al. 2007) Plant height (cm) 5–22 15–18 Basal leaves dimension (mm) 18–42×3–6.5 25–40×3–5 Basal leaves shape narrowly spatulate narrowly spatulate to linear Basal leaves apex subacute to obtuse obtuse to subacute Cauline leaves dimension (mm) 20–55×2–4 ─ Cauline leaves shape linear to oblanceolate lanceolate to linear Cauline leaves apex subacute to obtuse ─ Raceme length up to 6 cm in fl owering and 9 cm in fruiting time up to 8 cm Pedicel length up to 8 mm in fl owering and 12 mm in fruiting time 3–5 mm Sepal dimension (mm) 4–8×1.8–2.4 3–5 Sepal shape linear–oblong linear–oblong Petal dimensions (mm) 9–13×2–3 6–7 Petal shape spatulate obovate Petal apex emarginate emarginate Petal color pink–purple pink–purple Long fi lament length (mm) 3.5–4.4 ca. 4 Short fi lament length (mm) 2.5–3.3 ca. 3 Anther length (mm) 1–1.5 2.5–3.5 Pistil length (mm) 3–4 ─ Ovary dimension (mm) 1.7–2.5×0.8–1.3 ─ Silicula dimension (mm) 10–26×9–16 20–25×10–12 Silicula shape conical conical Seed dimension (mm) 3–6×2–4 3–4 Seed shape obovate–suborbicular suborbicular Seed surface ornamentation reticulate smooth Fig. 3. Microphotographs of root (A–C) and stem (D–F) cross sec- tion of Physoptychis haussknechtii; ca – cambium, cl – collenchy- ma, co – cortex, eh – non-glandular trichome, ep – epidermis, pd – periderm, ph – phloem, pr – pith ray, pt – pith, sc – sclerenchy- ma, st – stoma, tr – trachea, xy – xylem, xyp – xylem parenchyma. TEKİN M., MARTİN E. 36 ACTA BOT. CROAT. 76 (1), 2017 Stem: In the cross section of the middle part, the stem is ovoid shaped in outline and shows the primary structure. There is a thin cuticle layer on the uniseriate epidermis. The epidermis is composed of compactly arranged square, rect- angular, elliptical or circular cells. Epidermis has stomata. Whitish grey trichomes are located intensely on the epider- mis. Below epidermis, there is cortex tissue which consists of 7–18 layered elliptical or circular shaped parenchyma- tous cells. Outermost layer of cortex is composed of small collenchymatous cells (Fig. 3F). Under the cortex, there are phloem elements and sclerenchymatous tissue which forms continuous ring in the stem. The cambium is clearly distin- guishable and is located between phloem and xylem elements. The cells of cambium tissue are typically rectangular shaped and are arranged as 2–5 layers. Under the cambium, xylem tissue occurs. The pith of the stem is formed from elliptical or circular parenchymatous cells. In some parts of the pith region, there are gaps which are formed by the fragmenta- tion of the some pith parenchymatous cells (Figs. 3D–F and Tab. 3). Basal leaf: There is a single-layered epidermis on the up- per and lower surfaces of the leaf. There are unicellular fl at- tened stalked fi ve-armed trichomes and thin cuticle on both surfaces of the epidermises. Epidermis cells of both epider- mis layers are oval or occasionally rectangular and square shaped. According to the mesophyll layer, leaf is equifacial. Upper palisade parenchyma consists of 2–4 cell layers, lower palisade parenchyma consists of 2–3 cell layers, and the shape of palisade parenchyma cells is cylindrical, rectangular or occasionally oval. Between the two palisade parenchyma layers, there is spongy parenchyma which consists of 2–5 cell layers. Spongy parenchyma cells are usually oval or al- most circular shaped. Midrib is ovoid shaped, and is located close to the upper epidermis. Type of vascular bundle is col- lateral. All vascular bundles are surrounded by parenchyma- tous bundle sheath cells. Leaf is amphistomatic and stomata are anisocytic (Figs. 4A–B and Tab. 3). Cauline leaf: In the cross section of the cauline leaf, there is single layered epidermis covered by unicellular fl at- tened stalked fi ve-armed trichomes and a thin cuticle layer Tab. 3. The anatomical measurements of Physoptychis haussknechtii. Width (μm) Length (μm) Min–Max Mean±SD Min–Max mean±SD Root Collenchyma cells 7.95–32.33 17.78±6.96 12.86–74.60 32.34±18.46 Cortex cells 13.75–46.30 25.54±7.58 18.21–59.88 39.81±9.37 Cambium cells 2.59–8.96 4.79±1.58 8.12–18.92 14.38±2.53 Diameter of trachea 14.09–69.82 33.93±17.78 ─ ─ Stem Cuticle 0.31–0.87 0.53±0.18 ─ ─ Epidermis cells 4.65–18.24 11.95±3.85 11.50–23.41 17.25±3.30 Cortex cells 6.58–25.02 15.27±4.78 8.41–33.99 18.38±6.64 Phloem elements 1.60–5.86 3.21±1.11 2.04–8.31 4.29±1.54 Cambium cells 1.42–5.01 3.40±0.86 5.04–8.08 6.40±0.84 Pith cells 8.22–37.12 21.05±7.37 9.24–40.84 23.67±7.96 Diameter of trachea 10.58–25.47 15.30±3.88 ─ ─ Basal leaf Upper epidermis cells 10.69–26.81 19.00±3.81 11.25–43.82 25.09±8.04 Lower epidermis cells 9.33–22.99 15.37±3.92 11.23–26.85 17.83±4.36 Upper palisade parenchyma cells 14.85–32.21 21.61±4.07 26.54–53.70 39.59±6.24 Lower palisade parenchyma cells 12.14–28.58 20.17±3.91 26.63–76.16 45.83±11.00 Spongy parenchyma cells 18.68–31.80 25.48±3.50 21.95–45.03 33.17±5.62 Mesophyll tissue 231.96–329.66 278.94±32.58 ─ ─ Leaf 272.05–361.65 316.97±33.90 ─ ─ Cauline leaf Upper epidermis cells 10.12–31.90 20.38±5.21 16.15–42.29 24.67±6.12 Lower epidermis cells 8.91–21.77 15.60±3.59 11.39–33.06 19.48±4.28 Upper palisade parenchyma cells 20.34–46.81 29.18±5.43 35.23–82.44 48.65±9.68 Lower palisade parenchyma cells 12.72–37.18 21.10±5.79 24.94–61.88 40.89±9.11 Spongy parenchyma cells 14.56–43.38 27.52±6.70 16.61–53.20 34.09±8.24 Mesophyll tissue 324.34–381.06 348.41±12.75 ─ ─ Leaf 361.14–417.31 386.36±12.75 ─ ─ STRUCTURAL AND KARYOLOGICAL STUDIES ON PHYSOPTYCHIS HAUSSKNECHTII ACTA BOT. CROAT. 76 (1), 2017 37 on the upper and lower surfaces. Upper and lower epider- mis cells are oval, rectangular or squarish. According to the mesophyll layer, the leaf is equifacial. Upper palisade pa- renchyma consists of 2–4 layered rectangular shaped cells, while the lower palisade parenchyma is composed of 2–3 layered cylindrical and rectangular shaped cells. Between the two palisade parenchyma layers, there is spongy paren- chyma that consists of 2–4 layered oval or circular shaped cells. Midrib is triangular shaped. Type of vascular bundle is collateral and vascular bundles are surrounded by paren- chymatous bundle sheath cells. Leaf is amphistomatic and stomata are anisocytic (Figs. 4D–E and Tab. 3). Karyological characteristics Our study has shown that the chromosome number of P. haussknechtii is 2n=16 (Fig. 5A). The shortest chromosome length is 2.14 μm, the longest is 6.01 μm, and haploid chro- mosome length is 31.90 μm. The chromosome arm ratios are measured as 1.15–2.58. The centromeric index varies between 2.74 and 7.81, relative lengths vary from 6.71 to 18.83. The karyotype formula of P. haussknechtii consists of fi ve metacentric chromosome pairs and three submetacen- tric chromosome pairs. The ideogram is shown in Fig. 5B. Discussion Physoptychis is one of the small genus of the Brassica- ceae family, and is described with two species, one of which, P. haussknechtii, is a Turkish endemic. Morphologi- cal knowledge on the genus Physoptychis in the Flora of Turkey (Cullen 1965) is defi cient. The type species of the Physoptychis genus is P. caspica, a species that has yellow petals, so this characteristic has been used to specify the ge- nus Physoptychis (Boissier 1867). According to German et al. (2016), a servant of Bornmüller collected plant samples from the mountain Dumlucadağ, in the Divriği district of Sivas province, Turkey in 1893, and Bornmüller published it as a new species with the name P. haussknechtii (Born- müller 1899). Type samples of P. haussknechtii were all at the fruiting stage so P. haussknechtii was described solely on fruiting material. Because of its morphological similari- ty to the yellow fl owered P. caspica, Bornmüller (1899) suggested this color was characteristic of the whole genus (German et al. 2016). As a result of German et al. (2016), this suggestion was found to be erroneous and the petal col- or of P. haussknechtii was determined to be pink-purple. Morphological data on P. haussknechtii have been poor- ly described in Flora of Turkey (Cullen 1965), providing only information on general appearance of the species with- out measuring quantitative morphological characters. In the present study, all quantitative measurements such as height of stem, raceme, pedicel, long stamen, short stamen, anther, pistil and dimension of basal leaf, cauline leaf, sepal, petal, ovary, siliculae and seed have been carried out. In addition, in the study of Cullen (1965) the plant leaves were not dis- tinguished as basal and cauline and all the leaves were de- scribed as being in shape narrowly spatulate to linear, but here, the leaves have been studied separately. In the present study, the shape of basal leaves is described as narrowly spatulate, but cauline leaves have been found to be narrow- er than basal leaves, linear to oblanceolate, unlike in the study of Cullen (1965). Only a few morphological charac- teristics such as the dense infl orescence and the conical si- liculae with acuminate apices are results shared with the study of Cullen (1965). Recent morphological studies on P. haussknechtii were made by Çelik et al. (2007) who described a new species P. purpurascens which was later reviewed to be a synonym of P. haussknechtii. They studied the specimens of only one population. In the present study, morphological characteris- tics of P. haussknechtii were studied from the specimens growing in four separate localities, one of which is the type locality, thus the distribution area of P. haussknechtii has been expanded (Tab. 1). When we compare our morpho- logical results with the results of Çelik et al. (2007), there Fig. 4. Microphotographs of the leaf cross section under light mi- croscopy (A, B, D, E) and micrographs of the leaf surface under scanning electron microscopy (C, F) of Physoptychis haussknechtii: (A–B) Cross section of the basal leaf, (C) View of basal leaf scraped upper surface, (D–E) Cross section of stem leaf, (F) View of stem leaf unscraped lower surface; bs – bundle sheath, eh – epidermal hair, le – lower epidermis, ph – phloem, pp – pali- sade parenchyma, sp – spongy parenchyma, st – stoma, ue – upper epidermis, xy – xylem. Fig. 5. The microphotograph of somatic chromosomes (A), and ideogram (B) of Physoptychis haussknechtii. TEKİN M., MARTİN E. 38 ACTA BOT. CROAT. 76 (1), 2017 are some considerable differences, which are shown in Ta- ble 2. In addition, some morphological characteristics such as cauline leaf dimension, pistil length and ovary dimen- sion of P. haussknechtii have been measured for the fi rst time in the present study, and the morphological description of the species has been expanded. Trichome morphology of eleven genera of tribe Alys- seae was studied in Ančev and Goranova (2006). They rec- ognized four trichome types: (1) simple, (2) stalked 2 to 5 armed, (3) stellate and (4) dendritic trichomes. In the pres- ent study, type (2) leaf trichomes were found on P. hauss- knechtii: they are 5 armed with a fl attened stalk and smooth or minutely warty (Fig. 4F). According to the study of An- čev and Goranova (2006), 2–5 armed trichomes are present on the leaves of Draba korabensis Kümmerle & Degen, Draba muralis L., Draba siliquosa M. Bieb. and Erophila verna (L.) DC. subsp. spathulata (Láng) Walters which have leaf indumentum similar to P. haussknechtii, while stellate trichomes cover the leaves of some taxa belonging to the genera Aurinia Desv. and Clypeola L. and Alyssoides of the tribe Alysseae. P. haussknechtii stem has the general anatomical char- acteristics of the Cruciferae family (Figs. 3D–F). They are in accordance with results of Metcalfe and Chalk (1950), who pointed out that the pith region is parenchymatous, and occupies a large proportion of the total diameter of the stem in the Cruciferae family. In addition, they stated that the stomata of Cruciferae family are of the cruciferous (aniso- cytic) type and those of P. haussknechtii are identical. As mentioned above, Fibigia is a genus related to Physopty- chis, and morpho-anatomical adaptations of Fibigia trique- tra (DC.) Boiss., which is endemic to Mid-Dalmatia area of Me diterranean region, were studied by Damjanović and Ste vanović (1993). In a comparison of their results on F. triquetra and our results on P. haussknechtii, in the leaf anatomy of both species, mesophyll is equifacial, spongy parenchyma is weakly developed and stomata are tiny. While in the leaves of F. triquetra, upper and lower epider- mis cells are equal in size and leaf surfaces are covered by stellate trichomes, in P. haussknechtii leaves, upper epider- mis cells are usually larger than lower epidermis cells, and leaf surfaces are covered with fl attened stalked 5 armed tri- chomes (Fig. 4F). The genus Alyssum L. belongs to the Alysseae tribe and it is related to Physoptychis. Anatomical characteristics of Alyssum obtusifolium Steven ex DC. were studied by Orcan and Binzet (2003). The results of our study show that P. haussknechtii has similar anatomical proper- ties as A. obtusifolium, such as having roots in secondary structure, equifacial and amphistomatic leaves with aniso- cytic stomata. While the stem exhibits the primary structure and palisade parenchyma of the leaves have 2–4 layers in P. haussknechtii, the stem of A. obtusifolium exhibits the sec- ondary structure and leaves have 2–3 cell layered palisade parenchyma. One of the Turkish endangered endemics is Matthiola anchoniifolia Hub.-Mor. which is a species of the Anchonieae tribe in Brassicaceae. The anatomy of M. ancho- niifolia was studied by Tekin et al. (2013). While general root and stem anatomical characteristics of P. hauss knechtii are similar to M. anchoniifolia, there are considerable dif- ferences in terms of leaf anatomy. The leaves of both spe- cies are equifacial, but palisade and spongy parenchyma in cauline leaves of P. haussknechtii consist of 2–4 cell layers, while the leaves of M. anchoniifolia have 1–2 cell layered palisade parenchyma, and 5–12 cell layered spongy paren- chyma. These results have shown that the spongy parenchy- ma is weakly developed in P. haussknechtii, and well devel- oped in M. anchoniifolia. Brassicaceae is a stenopalynous family and polen grains are usually tricolpate and reticulate and pollen shape is var- ied more or less among genera within tribes but very rarely among species within the same genus (Erdtman 1972, Reile 1992, Abdel Khalik et al. 2002). The pollen grains of P. haussknechtii are tricolpate types, and exine ornamentation is reticulate in LM (Figs. 2A) and SEM (Figs. 2E), like the general pollen structure of the Brassicaceae family (Erdt- man 1972). In addition, the pollen shape of P. hauss knechtii was observed as subprolate, and pollen grains were radial symmetrical and isopolar. The genera Degenia and Fibigia are phylogenetically related to the Physoptychis. Pollen morphology of one of the Croatian critically endangered endemics Degenia velebitica (Degen) Hayek was studied by Mitić and Halbritter (2008). According to their study, D. velebitica has tricolpate pollen grains with reticulate exine ornamentation, and pollen shape is prolate. In present study, pollen grains of P. haussknechtii were found to be tricolpate with reticulate exine ornamentation, which is identical to D. velebitica. The pollen shape was found as a difference be- tween the two species. While, pollen grains of D. velebitica are prolate, they are subprolate with 1.16 P/E ratio in P. haussknechtii. Palynological characteristics of some Turk- ish Fibigia taxa [F. clypeata (L.) Medik subsp. clypeata var. clypeata, F. clypeata subsp. clypeata var. eriocarpa (D.C.) Post, F. clypeata subsp. anatolica A. Duran & Tuştaş, F. macrocarpa (Boiss.) Boiss., F. suffruticosa (Vent.) Sweet] were studied by Çetin et al. (2012) and they found all pol- lens have tricolpate aperture type and reticulate exine orna- mentation. These fi ndings are identical for P. hauss knechtii. Furthermore seed surface ornamentation of mentioned Fibigia taxa were reported as reticulate (Çetin et al. 2012) which is the same ornamentation for the seeds of P. haussknechtii (Fig. 2K). In micromorphological study under SEM, basal leaf, cauline leaf and seed surface properties of P. haussknechtii were determined. All leaf surfaces were covered by dense unicellular fl attened stalked 5 armed trichomes and had an- isocytic stomata (Figs. 4C, F). The seed margins were mem- branous and wing-like and seed surfaces were observed as coarsely reticulate (Fig. 2J–K). There are some karyological studies on the Physopty- chis genus. The chromosome number of P. caspica and its synonym P. gnaphalodes were determined as 2n=16 (Küp- fer 1980; Maassoumi 1980; Carrique and Martínez 1984; Warwick and Al-Shehbaz 2006). P. haussknechtii is related to P. caspica and we showed that the somatic chromosome number is the same, 2n=16. The base chromosome number of tribe Alysseae is reported as x=8 (Warwick et al. 2008, Rešetnik et al. 2013) There is no report on karyotype study of Physoptychis species. As the genus Physoptychis, Alys- STRUCTURAL AND KARYOLOGICAL STUDIES ON PHYSOPTYCHIS HAUSSKNECHTII ACTA BOT. CROAT. 76 (1), 2017 39 soides is one of the tribe Alysseae genera and there are some cytological studies on Alyssoides species such as A. cretica (L.) Medik., A. graeca Javorka, A. sinuate Medik. and A. utriculata (L.) Medik. and the chromosome numbers of all these species are reported as 2n=16 (Chichiricco and Tammaro 1980, Montmollin 1986, Nikolov 1991, Baltis- berger 2006). The results of our karyological study show that the chromosome number of P. haussknechtii is identi- cal to that of the mentioned related Alyssoides species. Also, one of the other genera of the Alysseae tribe is Clasto- pus Bunge ex Boiss. and the chromosome numbers of two species of this genus, C. erubescens Hausskn. and C. vesti- tus (Desv.) Boiss., were counted as 2n=16 (Maassoumi 1980; Khosravi and Maassoumi 1998). Degenia is one of the other genera of Alysseae tribe and chromosome number of taxa of this genus is reported as 2n=16 (Warwick and Al- Shehbaz 2006). In a study conducted by Španiel et al. (2015), the tribe Alysseae includes 24 genera and 277 taxa, and they report- ed that the chromosome numbers and polyploid levels of 171 of them are known. Of these, 95 (55.6 %) taxa are dip- loids, 43 (25.1 %) are polyploids, and 33 (19.3 %) involve both diploids and polyploids. The most common base chro- mosome number in the tribe is x=8 and less frequently it is x=7. The highest variation in base chromosome numbers (x=7, 8, 11, 15) is found in the genus Hormathophylla (Španiel et al. 2015). In the present study, base and diploid chromosome number of P. haussknechtii was found as x=8 and 2n=16, respectively. These results are consistent with the mentioned literature. Španiel et al. (2015) determined that there are defi cient karyotype analyses on the taxa of tribe Alysseae and suggested carrying out karyotype analy- ses on the taxa of this tribe. So, the karyotype analysis on P. haussknechtii is a signifi cant event to eliminate this defi - ciency for the tribe Alysseae. One of the ot her genera of Alysseae tribe is Fibigia and chromosome number and ka- ryotype analysis of some taxa of this genus such as F. cly- peata subsp. clypeata var. clypeata, F. clypeata subsp. cly pe- ata var. eriocarpa, F. macrocarpa and F. suffruticosa were studied by Çetin et al. (2012). While the chromosome num- ber of these Fibigia taxa are identical with P. haussknechtii, karyotype analyses are quite different and karyotype for- mula of P. haussknechtii is not congruous with any Fibigia taxa of the study Çetin et al. (2012). In addition, results of chromosome measurement obtained for P. haussknechtii were quite different. As a conclusion, in this study, morphological, anatomi- cal, palynological, micromorphological and karyological characteristics of P. haussknechtii, an endangered endemic species for Turkey, were reported in detail for the fi rst time. At the same time, this paper is the fi rst comprehensive study conducted on the morphology, anatomy, pollen mor- phology, leaf and seed micromorphology and karyology of Physoptychis genus. In addition, all our results revealed by this study will contribute to the taxonomy of P. hauss- knechtii and Physoptychis genus. 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