OPCE-STR.vp Acta Bot. Croat. 69 (1), 47–64, 2010 CODEN: ABCRA 25 ISSN 0365–0588 Comparative study of Salvia limbata C.A. and S. palaestina Bentham (sect. Aethiopis Bentham, Labiatae) from East Anatolia, Turkey AHMET KAHRAMAN*, MUSA DOGH AN Middle East Technical University, Department of Biological Sciences, 06531 Ankara, Turkey Morphological characteristics of stems, leaves, bracts, calyces and corollas are taxonomi- cally discriminating characters in Salvia. In this paper we present morphological, anatomi- cal and ecological features of S. limbata C.A. Meyer and S. palaestina Bentham as well as micromorphological characteristics of their pollen grains and nutlets using scanning electron microscopy. Anatomical characters such as size of cortex and vascular tissue, number of palisade parenchyma rows and vascular bundles are found to be important spe- cies specific characters. Pollen grains in the species are different in shape and size, but they look similar in their exine sculpturing; shape, size and ornamentation of nutlets are found to be different. The two investigated Salvia species grow on clayey-loamy and loamy soils, with pH 7.6–7.9, with 0.4–2.1% of organic matter, 0.006–0.026% of total salt content, 4.2–21.0 mg kg–1 phosphorus and 87.0–445.8 mg kg–1 potassium. Key words: Morphology, anatomy, ecology, palynology, nutlet, Salvia limbata, Salvia palaestina Introduction The family Labiatae includes 250 genera and about 7000 species distributed all over the world (THORNE 1992). It is the third largest family in Turkey with 45 genera and 574 spe- cies, 256 of which are endemic. The rate of endemism is 44.5% in this family (DAVIS 1965–1985, GÜNER et al. 2000). The genus Salvia L. represents a cosmopolitan assemblage of nearly 1000 species, distri- buted in three regions of the world: Central and South America (500 species), western Asia (200 species) and eastern Asia (100 species) (WALKER and SYTSMA 2007). The lever mechanism regulates pollination, selection and evolution in the group (CLAßEN-BOCKHOFF et al. 2004). The genus has been the subject of a number of studies on morphology (HEDGE 1982), anatomy (METCALFE and CHALK 1972, KAHRAMAN et al. 2009a, 2010a, b), palynology (ERDTMAN 1945, CANTINO et al. 1992) and nutlet micromorphology (MARIN et al. 1996). ACTA BOT. CROAT. 69 (1), 2010 47 * Corresponding author, e-mail: ahmetk@metu.edu.tr U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:20 Color profile: Disabled Composite 150 lpi at 45 degrees As many as 107 species of Salvia has been recognized in the Flora Orientalis, 75 of which were recorded from Turkey (BOISSIER 1875). These species have been placed under seven sections using BENTHAM’s (1833) sectional delimitation. These sections are as fol- lows: Eusphace Benth., Hymenosphace Benth., Aethiopis Benth., Plethiosphace Benth., Horminum Benth., Drymosphace Benth. and Hemisphace Benth. After that sect. Eusphace was changed to sect. Salvia (HEDGE 1972). Salvia limbata and S. palaestina were placed in the section Aethiopis (BENTHAM 1833, BOISSIER 1875). Section Aethiopis comprises bien- nial or perennial herbs or chamaephytes. The characteristic features of the section are tubu- lar or campanulate calyx, more or less falcate upper lip of corolla and not annulate corolla tube. Staminal connectives are longer than filaments, arms are unequal, the sterile shorter and more or less flattened distally. The first revision of Salvia in Turkey was made by HEDGE (1982), who recognized 87 species, one of them however being doubtful. Between 1982 and 2005, four more species, S. nydeggeri (HUBER-MORATH 1982), S. aytachii (VURAL and ADIGÜZEL 1996), S. hedgeana (DÖNMEZ 2001), S. anatolica (HAMZAOGH LU et al. 2005), were published from Turkey. Since 2005, as a part of a revision of the genus Salvia in Turkey, the authors have carried out ex- tensive field studies and collected a large number of specimens. Population size, habitat, phenological and ecological properties of the species were observed and their GPS coordi- nates and photographs were taken in the field. The studies have revealed two new species, S. marashica (I · LÇI · M et al. 2009) and S. ekimiana (CELEP and DOGH AN 2010) and new records of S. macrosiphon Boiss. (KAHRAMAN et al. 2009b) and S. viscosa Jacq. (CELEP et al. 2009). Salvia limbata and S. palaestina grow in eastern Anatolia and have similar morphologi- cal features and confused taxonomy. In this paper for the first time we give descriptions of S. limbata and S. palaestina, their comparative root, stem, leaf and petiole anatomy, palynology and nutlet micromorphology. Materials and methods Specimens of S. limbata and S. palaestina were collected from six localities in Turkey (Tab. 1, Fig. 1). The specimens have been stored in the Middle East Technical University (METU) Department of Biological Sciences and Ankara University Herbarium (ANK). Morphological studies were carried out on living as well as herbarium materials. 25 in- dividuals of each species and their 25 characters were studied and measured so as to deter- mine their morphological characteristics using a Leica S8AP0 stereomicroscope. Mini- mum and maximum ranges of measured characters are presented. Anatomical studies were carried out on specimens kept in 70 % alcohol. The paraffin method was used for the transverse sections of the root, the stem, the leaf and the petiole, and surface sections of leaves. The specimens were embedded in paraffin and then sec- tioned with a Leica RM2125RT rotary microtome. All sections were stained with Safranin and Fast Green and then mounted in Canada Balsam or Entellan (JOHANSEN 1944). Mea- surements and photographs were taken using a Leica DM1000 binocular light microscope and a Leica DFC280 camera. For palynological investigations, pollen material was obtained from herbarium sam- ples. The pollen slides were prepared according to WODEHOUSE (1935) technique. Mea- surements and observations were made using the Leica DM1000 binocular light micro- 48 ACTA BOT. CROAT. 69 (1), 2010 KAHRAMAN A., DOGH AN M. U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:20 Color profile: Disabled Composite 150 lpi at 45 degrees scope and Leica DFC280 camera. Polar length, equatorial length, colpus length, exine and intine thickness for 30 pollen grains were measured under the light microscope (magnifica- tion of 1000´). Pollen grain exine sculpturing was observed using a JEOL-6060 scanning electron microscope. The pollen terminology of FAEGRI and IVERSON (1975) has been used. The nutlets were examined using the Leica S8AP0 stereomicroscope to ensure their size and maturity. In order to determine the average seed sizes, 30 mature nutlets were mea- sured. For SEM, the mature nutlets were placed on stubs directly and covered with gold (DOGH AN 1988). After that, they were observed and photographed with a JEOL JSM-6400 SEM. For ecological studies, soil samples were taken from suitable habitats of S. limbata and S. palaestina. All soil samples were analyzed at the Soil, Fertilizer and Water Resources Central Research Institute, Ankara. Soil texture, organic matter, total salt, pH, CaCO3, P and K analysis were made using standard techniques (BAYRAKLI 1987) and the results have been evaluated according to KAÇAR (1972). ACTA BOT. CROAT. 69 (1), 2010 49 A COMPARATIVE STUDY OF SALVIA SECT. AETHIOPIS (LABIATAE) Tab. 1. Collection data of Salvia limbata and S. palaestina from Turkey. Collection data and collector number S. limbata B8 Erzurum: Ilica to Erzurum, 5 km to Erzurum, 38° 55’ 00’’ N 41° 12’ 42’’ E, 1817 m, 10.7.2006, AKahraman 1293. B9 Van: Bahçesaray to Van, near Naren village, 38° 09’ 568’’ N 43° 01’ 452’’ E, 2281 m, 9.7.2007, AKahraman 1437. C10 Hakkari: Hakkari to Van, 1 km to Van city border, 37° 48’ 015’’ N 44° 05’ 274’’ E, 1839 m, 7.6.2008, Akahraman 1571. S. palaestina B6 Malatya: Kangal to Hekimhan road, 39° 02’ 132’’ N 37° 44’ 240’’ E, 1460 m, 2.6.2008, AKahraman 1528B. B8 Diyarbakir: Lice to Diyarbakir, 65 km to Diyarbakir, 38° 18’ 675’’ N 40° 31’ 895’’ E, 789 m, 4.6.2008, AKahraman 1533A. C9 Şirnak: 5 km from Şirnak to Cizre, 37° 30’ 891’’ N 42° 25’ 613’’ E, 1090 m, 5.6.2008, AKahraman 1544. Fig. 1. Distribution map of Salvia limbata (�) and S. palaestina (�) in Turkey. U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:20 Color profile: Disabled Composite 150 lpi at 45 degrees Results Morphological characteristics Stems of Salvia limbata C.A. Meyer (synonym: S. chrysadenia Freyn) are 30–120 cm. The stem indumentum is retrorsely scabridulous below and with sessile glands above. Leaves are (3.5–) 5–16 ´ (2–) 4–10 cm, ovate-oblong and erose-dentate. The leaf indu- mentum is glabrous and glandular-punctate below, and sparsely pilose above. Petiole is 2–12 cm. Inflorescence is 15–80 cm. Verticillasters are 2–6 (–8)-flowered. Bracts are 2–7 mm, pale green. Pedicels are 2–6 mm. Calyces are ovate-campanulate, 6–10 mm in flower and 11–13 mm in fruit. The calyx indumentum is eglandular scabrid or densely pilose with hairs with many sessile glands. Its upper lip is shortly tridentate and recurved. Corolla is 15–25 mm and white with pale yellow lip. Corolla tube is 5–9 mm and squamulate. Fila- ments are 3–4 mm. Fertile anthers are 3–4 mm. Upper thecae are 15–20 mm and lower thecae are 1–2 mm. Style is 35–40 mm (Fig. 4). Stems of Salvia palaestina Bentham (synonym: S. lorentii Hochst.) are 20–65 cm. The stem indumentum is hirsute with long flattened eglandular hairs below and densely glandu- lar pilose (sometimes with long flattened eglandular hairs) above. Leaves are 4.5–15 (–20) ´ 1.5–7 (–9) cm, oblong to ovate and erose. The leaf indumentum is tomentose. Petiole 2.5–15 cm. Inflorescence is 10–50 cm. Verticillasters are (2–) 3–6-flowered. Bracts are 15–25 ´ 10–20 mm, often tinged pink or purple. Pedicels are 2–5 mm. Calyces are almost tubular, often pink or purple (rarely green), 12–16 mm in flower and 17–25 mm in fruit. The calyx indumentum is papillose-glandular with some longer hairs. Its upper lip is equally tridentate and spinulose. Calyx teeth are 2–4 mm, corolla 20–35 mm long, lilac or whitish-lilac. Corolla tube is 10–20 mm long and not squamulate. Filaments are 2–3 mm long. Fertile anthers are 3–4 mm. Upper thecae are 12–15 mm and lower thecae are 1–2 mm. Style is 25–45 mm (Fig. 5). 50 ACTA BOT. CROAT. 69 (1), 2010 KAHRAMAN A., DOGH AN M. Fig. 2. Cross-sections of root. A–B. S. limbata, C–D. S. palaestina. p – periderm, c – cortex, sc – sclerenchyma, ph – phloem, x – xylem, pr – pith ray, pi – pith. U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:23 Color profile: Disabled Composite 150 lpi at 45 degrees Anatomical characteristics Root anatomy: Cross-sections taken from the root of Salvia limbata have revealed that the periderm layer on the outermost surface of the root is thin and its cells are irregular. A multilayered parenchymatic cortex is present under the periderm. There are several sclerenchyma groups above the phloem. The xylem (800–1300 mm) is composed of vessels and tracheids. Those cells underlying the xylem are thick-walled and narrower. Pith rays comprise 2–6-rowed rectangular cells. The pith consists of polygonal or orbicular paren- chymatous cells (Fig. 2A–B, Tab. 2). Cross-sections taken from the root of Salvia palaestina have showed that the multilay- ered periderm layer on the outermost surface of the root is thick and its cells are squashed or breaking up. Under the periderm, there is a parenchymatic cortex with a few layers. Phloem and xylem are distinguished from one another by sclerenchyma and ground tissue. Phloem is embedded in sclerenchyma tissue as several groups. The xylem (1500–2000 mm) con- sists of regular vessels and tracheids. Pith rays are composed of 1–8(–10)-rowed rectangu- lar cells. The pith comprises polygonal, oval or orbicular parenchymatous cells (Fig. 2C–D, Tab. 2). Stem anatomy: Cross-sections taken from the stem of Salvia. limbata have exhibited a monolayer epidermis covered by an undulate cuticle. The epidermis is composed of oval, ACTA BOT. CROAT. 69 (1), 2010 51 A COMPARATIVE STUDY OF SALVIA SECT. AETHIOPIS (LABIATAE) Tab. 2. Comparative anatomy of root, stem, leaf and petiole of S. limbata and S. palaestina. S. limbata S. palaestina Width (mm) Length (mm) Width (mm) Length (mm) Min. – Max. Min. – Max. Min. – Max. Min. – Max. Root Periderm cell 15–50 10–45 30–100 30–70 Cortex cell 30–50 10–20 25–50 15–20 Pith ray 15–20 15–40 10–35 15–60 Stem Cuticle 4–7 5–7 Epidermis cell 15–55 10–20 20–45 10–20 Cortex cell 30–120 25–80 30–105 20–65 Trachea cell 20–40 30–50 25–85 25–110 Pith cell 50–180 50–150 45–150 50–150 Leaf Cuticle 3–7 1.5–4 Upper Epidermis Cell 25–65 15–35 15–55 10–25 Lower Epidermis Cell 10–30 7–25 15–50 10–25 Palisade Parenchyma 11–16 25–60 10–15 20–50 Spongy Parenchyma 10–16 15–20 8–20 10–20 Petiole Abaxial Epidermis 15–25 10–20 15–25 15–20 Adaxial Epidermis 10–20 10–20 15–30 15–20 Cortex Cell 30–105 30–115 30–125 30–130 U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:23 Color profile: Disabled Composite 150 lpi at 45 degrees ovate or rectangular cells. Underneath the epidermis, multilayered collenchyma cells (60–90 mm) are located at the corners and there are 1–2 rows of chlorenchyma cells be- tween them. The cortex tissue (250–400 mm) consists of 7–10 layers of oval, ovate or orbic- ular parenchymatous cells. The phloem (80–120 mm) is surrounded by more or less sclerenchymatous fibers. Cambium is distinguishable. The xylem (500–1150 mm) consid- erably bulges at ridges. The pith comprises hexagonal or orbicular parenchymatous cells with intercellular spaces (Fig. 3A, Tab. 2). 52 ACTA BOT. CROAT. 69 (1), 2010 KAHRAMAN A., DOGH AN M. Fig. 3. Cross-sections of stem. A. S. limbata, B. S. palaestina. e – epidermis, gh – glandular hair, eg – eglandular hair, co – collenchyma, c – cortex, sc – sclerenchyma, ph – phloem, x – xylem, pi – pith. Fig. 4. General appearance of S. limbata. Scale bar: 2 cm. Fig. 5. General appearance of S. palaestina. Scale bar: 2 cm U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:28 Color profile: Disabled Composite 150 lpi at 45 degrees Cross-sections taken from the stem of S. palaestina display a monolayer epidermis cov- ered by an undulate cuticle. The epidermis consists of oval or rectangular cells. Multilay- ered collenchyma cells (70–130 mm) are located at the corners. There are 1–3 rows of chlorenchyma (20–30 mm) with a large number of chloroplasts between the corners. The cortex (150–200 mm) is composed of 4–7 layers of oval or orbicular parenchymatous cells. The phloem with less or more sclerenchymatic fibers measures 25–55 mm. Cambium is not clearly distinguishable. Size of the xylem is 90–300 mm and it immediately bulges at the ridges. The pith region comprises large hexagonal, polygonal or circular parenchymatous cells (Fig. 3B, Tab. 2). Leaf anatomy: Cross-sections of the lamina and surface sections of upper and lower epidermises of S. limbata have showed that both epidermises are covered with eglandular hairs and sessile glands and they consist of uniseriate rectangular or oval cells with undu- late cuticles. Cells of the upper epidermis are clearly larger than the lower. The leaf is equifacial and amphistomatic, with diacytic stoma type. Stomata on the lower surface show higher frequencies than those on the upper surface. On the upper surface, the length of stomata varies from 26–29 mm while the width of stomata ranges from 15–20 mm. On the lower surface, the length of stomata varies from 22–30 mm whereas the width of stomata ranges from 16–20 mm. Mesophyll (135–220 mm) comprises elongated palisade and isodiametric spongy parenchyma cells. Palisade parenchyma is 2–3-rowed under the upper and lower epidermis. Spongy parenchyma is 1–2-rowed in the middle. The midrib region, which forms a projecting part, comprises 1–3 layers of collenchyma adjacent the epidermal cells. A single large vascular bundle is located in the center (Fig. 6A–D, Tab. 2). Cross-sections of the lamina and surface preparations of both epidermises of S. palaestina have revealed that the upper and lower epidermises are covered with glandular and eglandular hairs and they are composed of uniseriate rectangular or oval cells with un- dulate cuticles. Cells of the upper epidermis cells are nearly equal to the lower. The equifacial leaf is amphistomatic, with diacytic stoma type. Number of stomata on the lower surface is nearly equal to that of stomata on the upper surface. On the upper surface, the length of stomata varies from 38–45 mm while the width of stomata ranges from 30–36 mm. On the lower surface, the length of stomata varies from 34–43 mm whereas the width of stomata ranges from 23–30 mm. Mesophyll region (60–180 mm) consists of 1–2 layers of elongated palisade parenchyma cells found on both sides of 1–2 layers of isodiametric spongy parenchyma cells, occupying a small part in the middle. The midrib region, which forms a projecting part, comprises 2–3 collenchyma layers below the epidermal cells. There is a single large vascular bundle in the center (Fig. 6E–H, Tab. 2). Petiole anatomy: In cross-sections taken from the petiole of S. limbata it has been ob- served that the epidermal cells of both surfaces are oval, rectangular or squarish epidermal cells. The adaxial epidermis cells are slightly larger than the abaxial epidermis cells. There are several layers of collenchyma cells under the epidermis. Four broad vascular bundles are located in the middle. Also, there are four small vascular bundles in each of the petiolar wings. Vascular bundles are of collateral type. The sclerenchyma tissue is well developed outside of the phloem and the xylem (Fig. 7A–B, Tab. 2). Cross-sections taken from the petiole of S. palaestina showed that the epidermal cells of both surfaces are oval, rectangular or squarish epidermal cells. Adaxial and abaxial epi- dermis cells are equal in size. 2–5-layered collenchyma are located under the epidermis. ACTA BOT. CROAT. 69 (1), 2010 53 A COMPARATIVE STUDY OF SALVIA SECT. AETHIOPIS (LABIATAE) U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:28 Color profile: Disabled Composite 150 lpi at 45 degrees Two or three broad vascular bundles are located in the middle. Moreover, there are two small vascular bundles in each of the petiolar wings. The vascular bundle tissue surrounded by parenchymatic cells seems as a shallow crescent and collateral. There are several sclerenchymatous cells on the phloem (Fig. 7C–D, Tab. 2). 54 ACTA BOT. CROAT. 69 (1), 2010 KAHRAMAN A., DOGH AN M. Fig. 6. Cross and surface sections of leaf. A–D. S. limbata, E–H. S. palaestina. cu – cuticle, eg – eglandular hair, ue – upper epidermis, le – lower epidermis, pc – parenchyma cell, x – xylem, ph – phloem, pp – palisade parenchyma, sp – spongy parenchyma, st – stomata. U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:29 Color profile: Disabled Composite 150 lpi at 45 degrees Ecological characteristics Both Salvia limbata and S. palaestina prefer clayey-loamy and loamy soils that are slightly alkaline. The organic matter and CaCO3 content of the soil of S. limbata are 2.07–2.10% (a middle level) and 0.94–16.90% (a very small to a high level), whereas the organic matter and CaCO3 content of the soil of S. palaestina are 0.44–2.04% (a very low to middle level of organic matter) and 3.89–72.84% (a small to a very high level) (Tab. 3). ACTA BOT. CROAT. 69 (1), 2010 55 A COMPARATIVE STUDY OF SALVIA SECT. AETHIOPIS (LABIATAE) Fig. 7. Cross-sections of petiole. A–B. S. limbata, C–D. S. palaestina. ad – adaxial epidermis, ab – abaxial epidermis, co – collenchyma, pc – parenchyma cell, vb – vascular bundle, x – xylem, ph – phloem, sc – sclerenchyma. Tab. 3. Soil features of S. limbata and S. palaestina. Parameter S. limbata S. palaestina Sand (%) 43.9–46.6 23.0–42.8 Silt (%) 25.9–30.5 31.3–38.2 Clay (%) 22.9–30.2 25.9–38.8 Texture Clayey-loamy and loamy Clay loam and loamy pH 7.56–7.93 7.60–7.82 Organic matter (%) 2.07–2.10 0.44–2.04 Total salt (%) 0.014–0.016 0.006–0.026 Total CaCO3 (%) 0.94–16.90 3.89–72.84 P (ppm) 9.36–16.00 4.21–21.01 K (ppm) 113.55–296.31 87.00–445.84 U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:31 Color profile: Disabled Composite 150 lpi at 45 degrees Salvia limbata shares its habitat with mainly herbaceous plants including S. nemorosa L., S. poculata Náb, S. xanthocheila Boiss. ex Bentham, S. macrochlamys Montbret et Aucher ex Bentham, S. atropatana Bunge, S. syriaca L., S. multicaulis Vahl, S. aethiopis L., S. virgata Jacq., S. verticillata L. subsp. verticillata and amasiaca (Freyn et Born.) Bornm., Senecio sp., Euphorbia sp., Vicia sp., Dactylis sp., Verbascum sp., Hordeum sp. and Centaurea sp. Other species growing in association with S. palaestina are S. cryptan- tha Montbret et Aucher ex Bentham, S. euphratica Montbret et Aucher ex Bentham var. euphratica and var. leiocalycina (Rech. fil.) Hedge, S. candidissima Vahl subsp. candi- dissima, S. ceratophylla L., S. syriaca L., S. multicaulis Vahl, S. aethiopis L., S. virgata Jacq., S. verticillata L. subsp. amasiaca (Freyn et Born.) Bornm., Tripleouspermum sp., Crupina sp., Psorolea sp., Phlomis sp., Crepis sp., Tragopogon sp., Avena sp., Trigonella sp., Torillis sp., Aegilops sp., Mellilotus sp., Alopecurus sp., Scabiosa sp. and Alkanna sp. Pollen characteristics Shape of pollen grains of S. limbata is oblate-spheroidal (Fig. 6A–B). Polar axis is 52.41 ± 4.69 mm and its equatorial axis is 59.82 ± 4.89 mm. The ratio of P/E is 0.88. Colpus length is 49.25 ± 4.78 mm and colpus width is 7.57 ± 1.61 mm. The exine thickness is 1.33 ± 0.20 mm and the intine thickness is 0.55 ± 0.09 mm (Tab. 4). The exine sculpturing is bireticulate-perforate (Fig. 8C–D). Shape of pollen grains of S. palaestina is prolate-spheroidal (Fig. 9A–B). Polar axis is 47.72 ± 3.85 mm and equatorial axis is 45.40 ± 3.73 mm. The ratio of P/E is 1.05. Colpus length is 42.07 ± 3.99 mm and colpus width is 3.79 ± 0.72 mm. The exine thickness is 1.37 ± 0.21 mm and the intine thickness is 0.75 ± 0.03 mm (Tab. 4). The exine sculpturing is bireticulate-perforate (Fig. 9C–D). 56 ACTA BOT. CROAT. 69 (1), 2010 KAHRAMAN A., DOGH AN M. Tab. 4. Pollen morphological data of S. limbata and S. palaestina. Parameter S. limbata S. palaestina Polar axis* 41.52–65.03 52.41 ± 4.69 40.00–51.96 47.72±3.85 Equatorial axis* 50.16–74.10 59.82 ± 4.89 38.35–48.99 45.40±3.73 P/E 0.88 1.05 Colpus length* 39.61–59.15 49.25 ± 4.78 33.08–46.24 42.07±3.99 Colpus width* 5.25–11.78 7.57 ± 1.61 3.00–5.21 3.79±0.72 Exine thickness* 1.03–1.67 1.33 ± 0.20 1.07–1.70 1.37±0.21 Intine thickness* 0.42–0.70 0.55 ± 0.09 0.71–0.81 0.75±0.03 Shape oblate-spheroidal prolate-spheroidal Exine surface bireticulate-perforate bireticulate-perforate * range, mean and standard deviation U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:31 Color profile: Disabled Composite 150 lpi at 45 degrees ACTA BOT. CROAT. 69 (1), 2010 57 A COMPARATIVE STUDY OF SALVIA SECT. AETHIOPIS (LABIATAE) Fig. 8. LM and SEM micrographs of the pollen grains of S. limbata. A–B Equatorial and polar view in LM micrographs, C–D Equatorial view and exine sculpturing SEM. Fig. 9. LM and SEM micrographs of the pollen grains of S. palaestina. A–B Equatorial and polar view in LM micrographs, C–D Equatorial view and exine sculpturing in SEM. U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:34 Color profile: Disabled Composite 150 lpi at 45 degrees Nutlet micromorphology The nutlets of Salvia limbata are ovate to oblong in their outline. Mature nutlets of S. lim- bata are 2.5–3.0 mm long and 1.8–2.1 mm wide. Their hilum diameter is 0.40–0.55 mm. The nutlet surface is glabrous and has irregular protuberances and undulating ridges (Fig. 10 A–B). The mature nutlet is light brown. The nutlets of Salvia palaestina are spherical in their outline. Mature nutlets of S. pala- estina are 2.4–2.8 mm long and 2.2–2.6 mm wide. Their hilum diameter is 0.23–0.38 mm. The nutlet surface is glabrous and smooth to slightly papillaes (Fig. 10 C–D). The mature nutlet is light brown. Habitat and phenology Salvia limbata grows on stony slopes, roadsides, steppe and cornfields between 1060– 2400 m. It flowers in June to August. Salvia palaestina grows on limestone and igneous rocky slopes, in Quercus scrub, vineyards, fallow fields and roadsides between 300 and 1460 m. It flowers in April to July. Discussion The main morphological characters useful for Salvia species determination are stem, leaf and calyx indumentum, bract and calyx length, bract and corolla colour. The stem tex- ture of S. limbata is retrorsely scabridulous below and with sessile glands above, whereas 58 ACTA BOT. CROAT. 69 (1), 2010 KAHRAMAN A., DOGH AN M. Fig. 10. SEM photos of the nutlets of Salvia limbata and S. palaestina. A–B General appearance and surface of the nutlets of S. limbata, C–D General appearance and surface of the nutlets of S. palaestina. U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:34 Color profile: Disabled Composite 150 lpi at 45 degrees that of S. palaestina is hirsute with long flattened eglandular hairs below and often glandu- lar pilose above. In S. limbata the leaf texture is glabrous and glandular-punctate below and sparsely pilose above, but in S. palaestina the leaf indumentum is tomentose. The calyx texture is eglandular scabrid or densely pilose in S. limbata but papillose-glandular in S. palaestina. The length of bracts and calyces in S. limbata are shorter than that of bracts and calyces in S. palaestina. In S. limbata bract is pale green and corolla is white with pale yel- low lip whereas in S. palaestina bract colour is pink or purple and corolla colour is lilac or whitish-lilac. S. limbata has taller corolla tube, upper thecae and filaments than S. pala- estina. Morphological characters of S. limbata and S. palaestina are compared in Tab. 5. Our findings usually agree with the description of the Flora of Turkey (HEDGE 1982), but some differences were found here and so their descriptions were emended and ex- panded. It is reported that the stem length of S. limbata was 30–100 cm, its leaf size was 10–16 cm in length and 6–10 cm in width, its bract size was 2–6 mm, its corolla length was c. 25 mm, its calyx length was 8–10 mm in flower and to c. 12 mm in fruit while the stem length of S. palaestina was 30–60 cm, its leaf size was 5–13 (–20) cm in length and 1.5–7 (–8.5) cm in width, its bract size was c. 15 ´ 18 mm, its corolla length was c. 25 mm, its ca- lyx length was c. 15 mm in flower and to c. 17 mm in fruit (HEDGE 1982). According to our study, the stem length of S. limbata was 30–120 cm, its leaf size was (3.5–) 5–16 cm in length and (2–) 4–10 cm in width, its bract size was 2–7 mm, its corolla length was 15–25 mm, its calyx length was 6–10 mm in flower and 11–13 mm in fruit, but the stem length of S. palaestina was 20–65 cm, its leaf size was 4.5–15 (–20) cm in length and 1.5–7 (–9) cm in width, its bract size was c. 15–25 ´ 10–20 mm, its corolla length was 20–35 mm, its ca- lyx length was 12–16 mm in flower and 17–25 mm in fruit. Additionally, inflorescence, co- rolla tube, filament, fertile anther, style, upper thecae and lower thecae features are pre- sented the first time in this study. ACTA BOT. CROAT. 69 (1), 2010 59 A COMPARATIVE STUDY OF SALVIA SECT. AETHIOPIS (LABIATAE) Tab. 5. Diagnostic morphological features of Salvia limbata and S. palaestina. Character S. limbata S. palaestina Stem indumentum retrorsely scabridulous below and with sessile glands above hirsute with long flattened eglandular hairs below and densely glandular pilose above Leaf indumentum glabrous and glandular-punctate below and sparsely pilose above tomentose Bract length (mm) 2–7 15–25 Bract colour pale green pink or purple Calyx length (mm) 6–10 in flower, 11–13 mm in fruit 12–16 in flower, 17–25 in fruit Calyx indumentum eglandular scabrid or densely pilose hairs papillose-glandular Corolla colour white with pale yellow lip lilac or whitish-lilac Corolla length (mm) 15–25 20–35 Corolla tube length (mm) 5–9 10–20 Corolla tube shape Squamulate Not squamulate Upper theca length (mm) 15–20 12–15 Filament length (mm) 3–4 2–3 U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:34 Color profile: Disabled Composite 150 lpi at 45 degrees Root anatomy of the family Labiatae (METCALFE and CHALK 1972) is characterized by pith rays of roots composed of 2–12 or more rowed cells. Our study shows that pith rays of S. limbata consist of 2–6 rowed cells while those of S. palaestina comprise 1–8 (–10) rowed cells. It was reported that S. macrochlamys Boiss. et Kotschy (sect. Salvia) has mainly 1–4 rays of cells (KAHRAMAN et al. 2010a) and S. ballsiana (Rech. fil.) Hedge has 1–3 (–4) rays of cells (KAHRAMAN 2010b). The number of pith ray rows may be used to dis- tinguish Salvia species at the sectional level. In addition, xylem size is a diagnostic charac- ter in the two species. In S. limbata xylem size in is 800–1300 mm while in S. palaestina it is 1500–2000 mm. The stems of Labiatae are rectangular, have well-developed collenchyma covering a broad area at the corners and have a sclerenchymatic tissue surrounding the vascular bun- dle tissue (METCALFE and CHALK 1950). We found the same anatomical features in the stems of S. limbata and S. palaestina and also detected a few layers of chlorenchyma cells below the epidermis between the corners. However, vascular and cortex size are different in the two species. In S. limbata phloem is 80–120 mm in width while in S. palaestina phloem is 25–55 mm in width. Xylem is 500–1150 mm in S. limbata, however, xylem is 90–300 mm in S. palaestina. The cortex tissue of S. limbata (250–400 mm) is larger than that of S. palaestina (150–200 mm). In addition, S. limbata has a 7–10-layered cortex while S. palaestina has a 4–7-layered cortex. Mesophyll is completely parenchymatic and midrib is surrounded by collenchyma cells in Salvia species (METCALFE and CHALK 1972). We observed the same characteristics in the leaves of S. limbata and S. palaestina. However, the number of palisade parenchyma rows in the two species is different. The mesophyll of S. limbata is composed of 2–3 layers of palisade parenchyma cells on both sides while that of S. palaestina consists of 1–2 layers of palisade parenchyma cells. Cells of the upper epidermis of S. limbata are clearly larger than the lower epidermis, but those of the upper epidermis of S. palaestina are almost equal to the lower epidermis. Additionally, stomata of S. limbata (22–30 mm in length and 15–20 mm in width) are smaller than stomata of S. palaestina (34–45 mm in length and 23–36 mm in width) on both surfaces of leaves. The structure of the vascular bundles in the petiole structure of the family Labiatae is a taxonomically significant character (METCALFE and CHALK 1950). In the petiole of Salvia limbata, there are four broad vascular bundles in the middle and also there are four small vascular bundles in each of the petiolar wings. In the petiole of S. palaestina, there are two or three large vascular bundles in the middle and also there are two small vascular bundles in each of the petiolar wings. S. ballsiana (KAHRAMAN 2010b) has a broad vascular bundle in its middle part of the petiole and four or six small bundles on its wings, and S. macrochlamys (KAHRAMAN et al. 2010a) has a single large vascular bundle in the center of the petiole and two small bundles its wings. To sum up, number of vascular bundles in the petiole can be used to distinguish the species. Salvia limbata and S. palaestina grow on clayey loamy and loamy soils with slightly al- kaline (pH 7.56–7.93) and a low or medium level of organic matter (0.44–2.10%). The amounts of P, K and total salt present vary between 4.21–21.01 ppm (enough to very much), 87.00–445.84 ppm (very much) and 0.006–0.026% (saltless to slightly salty), re- spectively. However, the soils of S. limbata (0.94–16.90%) are less limy than that of S. palaestina (3.89–72.84%). Both the species generally shares their habitats with herbaceous plants. 60 ACTA BOT. CROAT. 69 (1), 2010 KAHRAMAN A., DOGH AN M. U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:34 Color profile: Disabled Composite 150 lpi at 45 degrees ACTA BOT. CROAT. 69 (1), 2010 61 A COMPARATIVE STUDY OF SALVIA SECT. AETHIOPIS (LABIATAE) Fig. 11. Boxplots of the nutlet length, width and hilum diameter of Salvia limbata and S. palaestina. Nutlet length (a), width (b) and hilum diameter (c) of S. limbata. Nutlet length (d), width (e) and hilum diameter (f) of S. palaestina. Tab. 6. Comparison of anatomical, palynological and nutlet characters of Salvia limbata and S. palaestina. Character S. limbata S. palaestina Root Xylem size in (mm) 800–1300 1500–2000 Stem Phloem size (mm) 80–120 25–55 Xylem size (mm) 500–1150 90–300 Cortex size (mm) 250–400 150–200 Cortex layer 7–10-layered 4–7-layered Leaf Number of palisade parenchyma rows 2–3 1–2 Size of stomata (mm) 22–30 ´ 15–20 34–45 ´ 23–36 Upper epidermis versus lower epidermis Large Equal Petiole Number of vascular bundles 4 large in the center and 4 small in each wing 2–3 large in the center and 2 small in each wing Pollen Size (mm) 52.41± 4.69 ´ 59.82 ± 4.89 47.72 ± 3.85 ´ 45.40 ± 3.73 Shape oblate-spheroidal prolate-spheroidal Nutlet Size (mm) 2.5–3.0 ´ 1.8–2.1 2.4–2.8 ´ 2.2–2.6 Shape ovate to oblong spherical Hilum diameter (mm) 0.40–0.55 0.23–0.38 Ornamentation irregular protuberances and undulating ridges smooth to slightly papillaes U:\ACTA BOTANICA\Acta-Botan 1-10\Kahraman.vp 9. travanj 2010 12:26:35 Color profile: Disabled Composite 150 lpi at 45 degrees Pollen characteristics of the family Labiatae have considerable taxonomic importance (ERDTMAN (1945). The classification of genera in Labiatae has been revised (CANTINO et al. (1992), with Salvia placed within the subfamily Nepetoideae because it had hexacolpate pollen. Light microscope observations in this study show pollen size and shape in different species is different. However, scanning electron micrographs reveal that their exine sculp- turing is similar to each other. Salvia limbata has a pollen size of 52.41 ± 4.69 ´ 59.82 ± 4.89 mm while S. palaestina has a pollen size of 47.72 ± 3.85 ´ 45.40 ± 3.73 mm (Tab. 4). In addition, pollen shape is oblate-spheroidal in S. limbata but it is prolate-spheroidal in S. palaestina. The exine sculpturing of the two species are bireticulate-perforate. Pollen size, shape and sculpturing of S. indica L. (section Aethiopis) are 44.98 ± 4.22 ´ 52.24 ± 4.41 mm, suboblate and bireticulate-perforate, respectively (KAHRAMAN 2009a). S. anatolica Hamzaoglu et A. Duran and S. bracteata Banks et Sol. were observed to be oblate- -spheriodal shaped pollen grains. The sculpturing in S. anatolica is euryreticulate, however the sculpturing in S. bracteata is suprareticulate (HAMZAOGH LU et al. 2005). Seed surface micromorphology was found to have systematic value at generic and spe- cific levels (HEDGE 1970, MARIN et al. 1996). The nutlets of S. sclarea were observed to be rounded-trigonous, c. 3 ´ 2 mm (HEDGE 1982) and its surface had protuberances (MARIN et al. 1996). The nutlets of S. verticillata L. (sect. Hemisphace) were analyzed to be c. 2.2 ´ 1.3 mm (HEDGE 1982) and its surface had reticulo-papillosae (MARIN et al. 1996). Accord- ing to our study, the nutlets of S. limbata are ovate to oblong, 2.5–3.0 mm long and 1.8–2.1 mm wide while the nutlets of S. palaestina are spherical, 2.4–2.8 mm long and 2.2–2.6 mm wide. Additionally, hilum diameter in the former species is 0.40–0.55 mm, however in the latter species it is 0.23–0.38 mm. The nutlet surface of S. limbata has irregular protuber- ances and undulating ridges in S. palaestina it is smooth to slightly papillaes. Their seed length, width and hilum diameter are illustrated in figure 11. Consequently, seed size, shape and ornamentation characters appear to have significant taxonomic value in distin- guishing S. limbata and S. palaestina. Diagnostic anatomical, palynological and nutlet characteristics of the two species are summarized (Tab. 6). Acknowledgements We wish to thank The Scientific and Technological Research Council (TÜBI · TAK -TBAG-104 T 450) for their financial assistance, professor Sevil Pehlivan and Dr. Birol Baser for taking SEM pictures and Mr. Ferhat Celep making valuable suggestions. References BAYRAKLI, F., 1987: Analysis of soil and plant. Ondokuz Mayis University Agricultural Faculty Publications, 17. University of Samsun, Samsun. BENTHAM, G., 1833: Labiatarum genera et species. Ridgway, London. BOISSEIR, E. P., 1875: Flora Orientalis. 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