ACTA BOT. CROAT. 80 (1), 2021 91 Acta Bot. Croat. 80 (1), 91–98, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-006 ISSN 0365-0588 eISSN 1847-8476 Anatomical investigations of the Turkish critically endangered species: Achillea sivasica Çelik et Akpulat (Asteraceae) Mehmet Tekin*, Şeyda Akdere Trakya University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 22030 Edirne, Turkey Abstract – In this study, the root, stem, leaf midrib and leaf lamina anatomy and achene micromorphology of the Turkish critically endangered endemic Achillea sivasica were investigated for the first time. In this study, the root was found in late primary growth and in early secondary growth stage. It has a large cortex layer consisting of 12-16 cell rows beneath the periderm. Secretory ducts formed by 5-12 secretory cells embedded in the cortex and located near the vascular bundle were found at the root, which was in the early stage of secondary development. The stem was cir- cular-pentagonal in cross-section. There was lamellar collenchyma beneath epidermis of pentagon corners, and cortex parenchyma between corners. Secretory ducts located near the phloem, between the cortex and endodermis on the interfascicular region, were also observed. An endodermis layer was evident and its cells have indentations and pro- trusions where they touch adjacent endodermis cells, which strengthens the connection between them. In addition, casparian strips were conspicuous in many endodermis cells. The leaf midrib area had a triangular cross section. There were secretory ducts, consisting of 4-5 secretory cells observed on both sides of the sclerenchymatous fibers that accompany the xylem. The leaf lamina was amphistomatic and stomata type was anomocytic. Mesophyll layer was equifacial. There was a large secretory duct and its diameter is bigger than the nearest main lamina vascular bundle. Achene shape of A. sivasica was lanceolate-oblong and its surface was ribbed and glabrous. Keywords: Achillea sivasica, anatomy, Asteraceae, endemic, Turkey Introduction Asteraceae is the largest flowering plant family, compris- ing 23,000 species distributed in 13 subfamilies, 44 tribes and over 1600 genera worldwide (Funk et al. 2009, Panero et al. 2014). This family is distributed naturally on all con- tinents outside of Antarctica, and its phylogenetic origin is thought to be in South America (Heywood 1978, Bremer 1994). In the flora of Turkey, the Asteraceae family is repre- sented by 1209 species of which 447 species are endemic with an endemism ratio of 37% (Dogan et al. 2009). Accord- ing to these numbers, Asteraceae is the largest plant family in Turkey, with the most endemic species. Achillea L. is one of the youngest evolutionary genera of the Asteraceae family, with more than 100 species widely distributed throughout the world (Arabacı and Yıldız 2006, Goli et al. 2008). Turkey is one of the main diversity centers of the genus Achillea. In all, 47 species of Achillea are dis- tributed and grow naturally in Turkey, and 24 of them are endemic for Turkey with a rate of endemism of 51% (Agar et al. 2015). Anatomical studies on Asteraceae family have been car- ried out in the past (Metcalfe and Chalk 1950, Milan et al. 2006, Tekin and Meriç 2015). According to a literature sur- vey, it was revealed that, although there are many pharma- cognostical and phytochemical studies of taxa of the genus Achillea, there are very few anatomical studies. Grytsyk et al. (2016) studied the morpho-anatomy of four Achillea spe- cies (A. millefolium L., A. stricta Schleich., A. carpatica Blos- ki ex Dubovik and A. distans Wald. et Kit.) in the western region of Ukraine. Sulborska and Weryszko-Chmielewska (2006) studied the morphology, anatomy and ultrastructure of floral nectaries of A. millefolium. Akcin and Akcin (2010) studied the morphological and anatomical characteristics * Corresponding author e-mail: mtekin2280@gmail.com TEKIN M., AKDERE Ş. 92 ACTA BOT. CROAT. 80 (1), 2021 Anatolia and is in the Iranian-Touranian phytogoeraphical area and has a continental climate. Localities where the plant samples were collected: Locality 1: B6 Sivas: Sivas to Ulaş district, Ziyarettepe, 39°33'08.7'' N, 37°01'12.2'' E, al- titude 1441 m a.s.l., M. Tekin 1430, 11.05.2013; ibid. M. Tekin 1812, 06.06.2018 (This locality is also the locus clas- sicus). Locality 2: B6 Sivas: Kangal to Gürün district, 8th km, calcareous area, 39°07'56.3'' N, 37°13'48.0'' E, altitude 1570 m a.s.l., M. Tekin 1252, 21.06.2012; ibid. M. Tekin 1484, 19.07.2013; ibid. M. Tekin 1836, 04.07.2019. Sampling Species identification was carried out by Dr. M. Tekin according to study of Çelik et Akpulat (2008) and dried plant samples were kept with collector name and number in Cumhuriyet University Faculty of Science Herbarium (CUFH) (Figs. 1, 2). Ten plant samples which best represent the population were collected from each locality for anatomical studies. These samples were stored at +4 °C in 70% ethyl alcohol. Transverse sections were taken manually from the root, stem and leaf of A. sivasica using a razor. Root sections were taken from secondary roots. Stem sections were taken 15-20 cm above the ground and leaf sections were taken from the middle part. Also, superficial hand-made sections of the lower surface of the leaf lamina were taken with a razor. In order to determine pectin- or lignin-containing tissues, and thus to separate the tissues from each other, all sections were double stained with 1% Safranin (Sigma) and 1% Alcian Blue (Sigma) dyes in the ratio of 3/2 (Davis and Barnett 1997), and sections were kept about 5 min in the dye. Washing was performed in adistilled water/glycerin 1:1 mixture after dy- ing to remove excess paint from the sections. The stained sections were prepared with a glycerin-gelatin mixture (Brown 1960). Anatomical investigations on these sections were made using an Olympus BX22 light microscope. Mi- crographs were taken using an Olympus BX51 light micro- scope coupled with an Olympus DP70 digital camera. Ana- tomical measurements are based on at least 30 measurement of two Turkish endemic Achillea; A. phrygia Boiss. et Bal. and A. gypsicola Hub.-Mor. Also, Akcin and Akcin (2014) studied the achene micromorphology of seven Achillea taxa including A. biebersteinii Afan., A. coarctata Poir., A. gran- difolia Friv., A. millefolium ssp. millefolium, A. millefolium ssp. pannonica (Scheele) Hayek, A. teretifolia Willd. and A. biserrata Bieb. from Turkey. Achillea sivasica Çelik et Akpulat was found as a new species in very limited area in Sivas province, Turkey (Çelik and Akpulat 2008). It is a local endemic, so Çelik and Akpulat (2008) suggested a threat category of “Critically Endangered (CR)” according to IUCN (2001) red list criteria for this species. No anatomical investigations have pre- viously been carried out on A. sivasica, however, consider- ing the medicinal importance of many Achillea species in Turkey; the chemical composition and phytochemicals, an- tioxidant, and antityrosinase activities of A. sivasica are re- cently being studied (Haliloglu et al. 2017, Özek et al. 2018). According to these studies A. sivasica is characterized by an extremely high percentage of the C18:3 fatty acid, and is a rich source of valuable volatile constituents such as β-pinene, 1,8-cineole camphor, and β-bisabolol. Haliloglu et al. (2017) reported that A. sivasica could be considered a natural source of active constituents for food supplements and ther- apeutic applications. The aim of this study is to reveal the previously un- known anatomical features and fruit micromorphological features of A. sivasica, pharmacognosy studies of which have been carried out, as mentioned above. For this pur- pose, root, stem, leaf midrib area and leaf lamina cross sec- tions of plant samples were taken. Photomicroscopic exam- inations were carried out and achene surface micrographs were examined using a scanning electron microscope. Materials and methods Study area Achillea sivasica samples were collected from two dis- tricts of Sivas (Turkey) province. Sivas is located in Inner Fig. 1. Geographic distribution area of Achillea sivasica ( ) in Sivas province in Turkey. ANATOMICAL STUDIES ON ACHILLEA SIVASICA ACTA BOT. CROAT. 80 (1), 2021 93 results of each tissue or cell group from many sections ob- tained from plant samples from different locations. As a re- sult of analysis of these measurements in SPSS package pro- gram, minimum, maximum, average values and standard deviation were determined. For scanning electron micros- copy (SEM) analysis, fruit samples were transferred onto lead staples using double layer tape and then coated with gold. Micromorphological observations were made using the LEO 440 model SEM on the surface of the gold-coated staples and micrographs were taken at different magnifica- tions. The nomenclature of all taxa follows IPNI (2020). Results Root anatomy The root was of late primary and early secondary growth stage and had a circular cross-section (Fig. 3). The outermost layer, the periderm, consisted of phellem, phellogen and phelloderma, and all these layers were conspicuous. Phellem was only 1-3 layered on the surface of roots in the late stage of primary development (younger root), and was multilay- ered on surface of roots in the early stage of secondary de- velopment (older root). Phellem and phellogen were single layered in both developmental types of root (Fig. 3). Under the periderma there was a very large layered cortex consist- ing of 12-16 parenchymatous cell rows. Cortex cells were oval and circular in the younger roots, whereas they were genere- ally rectangular-oval and dorsiventrally pressed in the older roots. With the development, while young roots only have periclinal and anticlinal cell divisions along 6-7 cells rows close to the vascular bundle, in older roots, these divisions were seen in all cortex layer cell rows. In older roots, the pres- ence of 5-7 secretory ducts formed by 5-12 unilayered secre- tory cells embedded in the cortex near the vascular bundle was found as an important difference compared to the younger roots (Fig. 3C, D). Most of the root volume was oc- cupied by the cortex. The endodermis and pericycle consist of single cell row and they were prominent in older root but inconspicuous in younger root (Fig. 3B, D). In the center of the root, there was a tetrarch xylem and between its arms there was phloem which covering a small area at the corre- sponding locations. There was distinguishable cambium lay- er consist of 1-3 cell rows, between the phloem and the xylem in both younger and older root cross sections (Fig. 3, Tab. 1) Stem anatomy The stem had a circular-pentagonal shape in cross-sec- tion and was covered by a single-layered epidermis. Its cells generally were oval, circular, rarely squarish or rectangular with abundant non-glandular, elongated hairs (Fig. 4A, B, F). Beneath the epidermis, there were 7-10 cell rows of lamel- lar collenchyma at locations corresponding to the corners of the pentagonal cross-section. The cortex parenchyma was located under the epidermis between the corners of the pen- tagon (Fig. 4A, B). The cortex parenchyma consisted of 4-8 cells rows of oval and circular cells and occasionally con- tained intercellular spaces. Cortex parenchyma cells located close to the epidermis were usually small and had abundant chloroplasts, while those adjacent to the endodermis were larger and had fewer chloroplasts. Near the phloem, between the cortex and endodermis on the interfascicular region, se- Fig. 2. Calcareous rocks are the natural habitat of Achillea sivasica (A) and the general appearance of the species in its habitat (B) Photos by M. Tekin. Fig. 3. Photomicrographs of root cross sections in the late stage of primary development (A-B) and early stage of secondary devel- opment (C-D) of Achillea sivasica. Abbreviations: ca – cambium, co – cortex, en – endodermis, lu – lumen, pd – phelloderma, pg – phellogen, ph – phloem, pl – phellem, pm – periderm, pr – peris- icle, sc – secretory cell, sd – secretory duct, xy – xylem. TEKIN M., AKDERE Ş. 94 ACTA BOT. CROAT. 80 (1), 2021 cretory ducts were observed (Fig. 4D). Beneath the cortex, there was a single cell row of the endodermis layer, consist- ing of oval cells that separate the cortex layer and vascular bundles from each other. Endodermis cells have indenta- tions and protrusions in the places where they touch adjacent endodermis cells, which strengthens the connection be- tween them (Fig. 4D). In addition, a Casparian strip was clearly obvious in many endodermis cells (Fig. 4E). Many open collateral vascular bundles located below the endoder- mis were arranged more or less in a regular ring, which is typical for dicotyledons. Sclerenchymatic fibers are found in vascular bundles densely on the phloem. There were 3-7 cell rows of sclerenchymatous fibers layer on each vascular bun- dle and also these fibers occupied the area around the xylem, so that it fills the interfascicular area and forms a continuous internal ring throughout the stem. Underneath it, there were Tab. 1. Anatomical measurement results of the root, stem, leaf midrib area and leaf lamina of Achillea sivasica (n = 100, max – maxi- mum, min – minimum, SD – standard deviation). Width/Diameter (µm) Length (µm) Min–Max Mean ± SD Min–Max Mean ± SD Root Cortex cells 9.45 – 38.65 25.6 ± 8.26 11.59 – 52.50 33.05 ± 11.70 Cambium cells 2.04 – 8.05 4.36 ± 1.29 3.73 – 11.31 8.35 ± 1.62 Trachea diameter 12.13 – 35.27 21.89 ± 5.14 – – Stem Cuticle thickness 0.61 – 1.23 0.85 ± 0.19 – – Cortex cells 13.50 – 40.45 24.04 ± 7.81 14.02 – 44.09 26.91 ± 8.62 Epidermis cells 8.51 – 32.81 20.31 ± 5.82 16.52 – 39.75 25.66 ± 5.46 Endodermis cell 14.23 – 36.18 24.89 ± 5.48 19.44 – 43.84 32.28 ± 6.96 Cambium cells 4.48 – 8.08 5.93 ± 0.83 9.80 – 16.36 12.19 ± 1.69 Pith cells 20.54 – 83.65 46.34 ± 20.58 21.76 – 91.41 51.03 ± 22.11 Trachea diameter 11.64 – 23.96 19.39 ± 2.89 – – Leaf midrib area Cuticle thickness 0.65 – 1.07 0.86 ± 0.09 – – Epidermis cells 9.27 – 41.36 21.69 ± 8.49 19.96 – 51.66 31.43 ± 7.54 Chlorenchyma cells 15.09 – 42.52 21.70 ± 5.72 18.10 – 51.77 31.98 ± 9.03 Non-photosynthetic parenchymatous cells 7.62 – 59.52 30.99 ± 15.35 15.77 – 69.13 39.42 ± 16.79 Trachea diameter 6.10 – 18.08 12.23 ± 3.79 – – Leaf lamina Cuticle thickness of upper epidermis 0.52 – 1.23 0.76 ± 0.20 – – Cuticle thickness of lower epidermis 0.60 – 1.33 0.97 ± 0.19 – – Upper epidermis cell 12.16 – 37.72 25.59 ± 6.83 21.76 – 41.04 32.87 ± 4.77 Lower epidermis cell 19.28 – 47.33 30.14 ± 7.74 25.52 – 55.98 37.22 ± 6.54 Palisade parenchyma cell 13.12 – 24.06 19.73 ± 3.29 32.21 – 80.33 54.73 ± 12.52 Spongy parenchyma cell 11.66 – 28.97 20.51 ± 3.73 16.43 – 39.84 27.34 ± 5.29 Mesophyll thickness 170.26 – 302.60 248.12 ± 38.81 – – Leaf lamina thickness 222.66 – 378.82 314.68 ± 37.83 – – Fig. 4. Photomicrographs of stem cross sections of Achillea siv- asica (A-E) and detail of non-glandular hairs (F). General view of stem (A); detailed view of stem (B); detailed view of open collat- eral vascular bundle (C); detailed view of endodermis and secretory duct (green arrows indicated indentations and protrusions be- tween adjacent endodermis cells and yellow arrows indicated intercellular area between cortex parenchyma cells) (D); detailed view of Casparian strips in endodermis (red arrows indicate Cas- parian strips in endodermis cells). Abbreviations: ca – cambium, cl – collenchyma, co – cortex, eh – epidermal non-glandular hair, en – endodermis, ep – epidermis, ph – phloem, pt – parenchyma- tous pith, sc – secretory cell, sd – secretory duct, sf – sclerenchy- matous fibers, vb – vascular bundle, xy – xylem. ANATOMICAL STUDIES ON ACHILLEA SIVASICA ACTA BOT. CROAT. 80 (1), 2021 95 many cell rows of phloem. Between phloem and xylem there was cambium consisting of 2-3 cell rows that were dorsiven- trally flattened (Fig. 4C). We observed only a fascicular cam- bium; no interfascicular cambium had been yet formed. The center of the stem is filled by pith parenchyma. Pith cells were mostly circular, hexagonal, sometimes oval, and there were wide intercellular spaces between them. There was no pith cavity in the center of the stem (Fig. 4, Tab. 1). Leaf midrib area anatomy The midrib area had a triangular cross section (Fig. 5A). It was covered by a single layered upper and lower epidermis composed of cells of a similar size, oval and squarish in shape. On the surface of both epidermises were found non- glandular trichoms. Beneath the epidermis, there were 2-6 cell layered chlorenchyma which are loosely aligned and have wide intercellular spaces. Below it, and especially around the midrib, there was a tightly aligned, non-photo- synthetic ground parenchyma. Between the lower epidermis and the ground parenchyma, there were 2-3 cell layered col- lenchyma tissue (Fig. 5A, B). There were 5 vascular bundles in the cross section of each leaf midrib area. The main vas- cular bundle (midrib) was almost circular and was support- ed by sclerenchymatous fibers from upper and lower side. There were two secretory ducts, one on each side of the scler- enchymatous fibers accompaning xylem. The epithel con- sisted of 4-5 cells which are unilayered (Fig. 5A, C, Tab. 1). Leaf lamina anatomy The leaf lamina was surrounded by a single layered epi- dermis on the upper and lower sides in cross-section. Both epidermis cells were oval and rectangular, occasionally cir- cular or squarish. There was a thick cuticle layer on the sur- face of both epidermises (Tab. 1). Stomata were observed on both sides of the lamina so the leaves belong to the amphis- tomatic type. The stomata were anomocytic and hydrophyt- ic (Fig. 5D, E). Beneath upper and lower epidermis, there were 1-3 cell rows of palisade parenchyma, consisting of cy- lindrical shaped cells and large intercellular spaces. Between palisade parenchymas, there were 2-4 cell rows of spongy parenchyma the cells of which are generally oval and some- times circular. According to the mesophyll layer arrange- ment, lamina was equifacial. The vascular bundles were em- bedded in the spongy parenchyma and surrounded by the single cell row parenchymatous bundle sheath. Above the main vascular bundle of lamina, there was a secretory duct consisting of 4-5 secretory cells. Secretory duct was sur- rounded by the ground parenchyma cells and its diameter was bigger than the nearest main vascular bundle of lamina (Fig. 5D, F, Tab. 1). Fruit micromorphology Achene of A. sivasica were yellowish-brown with lanceo- late-oblong shapes. Their surface is ribbed and glabrous. Slime cells were present on achene surface but they are in- conspicuous (Fig. 6). Fig. 5. Photomicrographs of Achillea sivasica leaf (A-F). Leaf mid- rib area in cross sections, general view (A), detailed view (B) and detailed view of midrib (C). Leaf lamina superficial section of lower epidermis (D). Cross sections of leaf lamina, general view (E) and detailed view (F). Abbreviations: cl – collenchyma, ch – chlorenchyma, eh – non-glandular hair, ep – epidermis, le – lower epidermis, lu – duct lumen, pc – parenchymatous cells, ph – phlo- em, pp – palisade parenchyma, sc – secretory cell, sd – secretory duct, sf – seclerenchymatous fibers, sp – spongy parenchyma, st – stoma, ue – upper epidermis, vb – vascular bundle, xy – xylem. Fig. 6. Achene micrographs of Achillea sivasica. A – general appearance, B – detailed appearance of surface ornamentation. TEKIN M., AKDERE Ş. 96 ACTA BOT. CROAT. 80 (1), 2021 Discussion This study is the first report focused on investigations of anatomical and fruit micromorphological characters of the Turkish critically endangered A. sivasica. Metcalfe and Chalk (1950) determined the general anatomical characters of the family Asteraceae. Anatomical features of A. sivasica obtained as a result of the present study in general overlap with those given by the mentioned authors. These authors described the existence of secretory system elements in the roots of taxa belonging to the Asteraceae family. It was re- ported that presence of secretory elements is an important taxonomical character, and their limited distribution has a great diagnostic value (Metcalfe and Chalk 1950, Fahn 1979). We observed secretory ducts in the roots of A. sivasi- ca in the early secondary growth phase, in stem and leaves whereas secretory ducts were not reported on any of the veg- etative organs of A. phrygia and A. gypsicola (Akcin and Akcin 2010). Achillea sivasica has both primary and secondary root structure according to growth stage, whereas the anatomi- cal description of A. phrygia and A. gypsicola shows only a secondary root structure (Akcin and Akcin 2010). Tekin and Meriç (2015) studied the anatomical characters of six Turkish endemic Tanacetum L. (Asteraceae) taxa which are very similar taxonomically to Achillea. The roots of Tanac- etum taxa studied by Tekin and Meriç (2015), were found only in the primary growth stage. However, in roots studied by us, primary xylem ridges were determined as tetrarch whereas they were found as triarch in Tanacetum albipan- nosum Hub. Mor. et Grierson, T. densum (Lab.) Schultz Bip. ssp. sivasicum Hub. Mor. et Grierson, T. haussknechtii (Bornm.) Grierson and T. heterotomum (Bornm.) Grierson, pentarch in T. cappadocicum (DC.) Schultz Bip. and hexarch in T. argenteum (Lam.) Willd. ssp. argenteum according to mentioned study. Secretory ducts were observed in the ear- ly secondary growth roots of A. sivasica while no secretory ducts were found in the roots of any of the samples studied by Tekin and Meriç (2015) Tanacetum taxa. The existence of an endodermis on the vegetative organs was reported by some studies on Asteraceae taxa (Melo-De Pinna and Mene- zes 2002, Topsakal et al. 2019). Tekin and Meriç (2015) was found the presence of endodermis layer in root and stem of all studied Tanacetum taxa. According to study of Akcin and Akcin (2010) A. phrygia and A. gypsicola had endoder- mis in root and stem, which is the same result as in the pres- ent study. In A. sivasica there is typical endodermis in early secondary growth stage of root and primary growth stage of stem. In addition, the pith area was occupied by a prima- ry xylem element in A. sivasica, which is the same as in A. phrygia and A. gypsicola. While most of the root volume in A. sivasica is filled by the cortex parenchyma, in A. phrygia and A. gypsicola it is filled by the secondary xylem (Akcin and Akcin 2010). In the stem anatomy of A. sivasica, the presence of unilay- ered epidermis with abundant non-glandular hairs and la- mellar collenchyma at the corners are features similar to those found in A. phrygia and A. gypsicola. In terms of ana- tomical features of the stem, a significant difference between A. phrygia, A. gypsicola and A. sivasica is epidermis cell size, the largest being found in A. sivasica (Tab. 2). A. sivasica has a single layered endodermis which is the boundary layer be- tween cortex and stele as in A. gypsicola, but it is different from A. phrygia which has one- or two-layered endodermis- es (Akcin and Akcin 2010). Endodermis cells of A. sivasica have indentations and protrusions where they touch adjacent endodermis cells. We suggest it is important to increase the durability of the endodermis layer. In addition, the Caspar- Tab. 2. Comparison of the anatomical measurement results of root, stem and leaf lamina obtained A. sivasica with present study and the results of A. phrygia and A. gypsicola studied by Akcin and Akcin (2010). Mean ± standard deviation is presented. Width (µm) Length (µm) A. sivasica n = 100 A. phrygia – A. gypsicola – A. sivasica n=100 A. phrygia – A. gypsicola – Root Cortex cells 25.6 ± 8.26 15.00 ± 2.51 13.20 ± 1.62 33.05 ± 11.70 30.95 ± 6.18 37.20 ± 5.07 Trachea diameter 21.89 ± 5.14 26.30 ± 11.79 20.45 ± 2.38 – – – Stem Epidermis cells 20.31 ± 5.82 9.00 ± 1.24 14.05 ± 2.36 25.66 ± 5.46 11.90 ± 1.66 17.90 ± 2.84 Cortex cells 24.04 ± 7.81 11.05 ± 2.39 22.50 ± 4.74 26.91 ± 8.62 18.15 ± 3.97 38.35 ± 5.16 Endodermis cells 24.89 ± 5.48 15.90 ± 3.21 16.90 ± 3.21 32.28 ± 6.96 22.25 ± 4.72 30.60 ± 10.79 Pith cells 46.34 ± 20.58 38.30 ± 12.33 45.70 ± 7.38 51.03 ± 22.11 – – Trachea diameter 19.39 ± 2.89 15.50 ± 3.47 25.30 ± 3.30 – – – Leaf lamina Upper epidermis cells 25.59 ± 6.83 9.35 ± 1.18 10.25 ± 2.20 32.87 ± 4.77 15.05 ± 2.56 22.30 ± 5.58 Palisade parenchyma cells 19.73 ± 3.29 11.20 ± 3.06 11.45 ± 2.27 54.73 ± 12.52 31.50 ± 6.14 25.50 ± 4.53 Spongy parenchyma cells 20.51 ± 3.73 10.20 ± 2.25 12.75 ± 3.60 27.34 ± 5.29 11.45 ± 2.91 17.45 ± 2.06 Lower epidermis cells 30.14 ± 7.74 21.15 ± 6.84 12.90 ± 3.73 37.22 ± 6.54 22.80 ± 7.08 22.45 ± 5.82 ANATOMICAL STUDIES ON ACHILLEA SIVASICA ACTA BOT. CROAT. 80 (1), 2021 97 ian strip which consists of hydrophobic substance to restrict apoplastic flow of water is clearly obvious in many of the en- dodermis cells of A. sivasica. There is no report on these en- dodermis features in A. phrygia and A. gypsicola. The stem of A. sivasica is in the primary growth stage and has no pith cavity in the center, whereas the stems of A. phrygia and A. gypsicola are in the secondary growth stage and contain a pith cavity in the center of the stem. This feature of the stem was reported by Tekin and Meriç (2015) only for T. haussknechtii, whereas the other five Tanacetum taxa stud- ied (T. albipannosum, T. densum ssp. sivasicum, T. heteroto- mum, T. cappadocicum T. argenteum ssp. argenteum) had no pith cavity in the center of the stem. The fascicular cambium is very distinguishable in A. sivasica while there were no re- ports on the presence of cambium in A. phrygia and A. gyp- sicola. The general stem anatomy of A. sivasica, including the presence of unilayered epidermis, epidermal hairs, inter- rupted collenchyma beneath epidermis, single-layered en- dodermis is similar to that of the six Tanacetum taxa de- scribed by Tekin and Meriç (2015). The only difference is that some of these taxa have no distinguishable cambium, as was observed very clearly in A. sivasica. According to Metcalfe and Chalk (1950), anatomical di- versity in taxa of the family Asteraceae is commonly observed in the leaf structure. They reported that there were both ano- mocytic and anisocytic stomata types for representatives of Asteraceae. Stomata of A. sivasica were found as anomocytic type which is the same as with mentioned study. In the cross section of the A. sivasica leaf we found single layered upper and lower epidermis, parenchymatous bundle sheath and anomocytic stomata, which is similar to the general anatomy of A. phrygia and A. gypsicola leaves described by Akcin and Akcin (2010). But there is important difference between the epidermis cell sizes of the mentioned species. Both epidermis cell sizes of A. sivasica are significantly larger than those in A. phrygia and A. gypsicola (Tab. 2). Grytsyk et al. (2016) were compared anatomically and morphologically, different Ach- illea species. They reported A. stricta Schleich. ex. W.D.J. Koch, A. distans Waldst. et Kit. ex. Willd. and A. carpatica Blocki ex Dubovik have anomocytic type stomata which is the same result as in the present study. The most important difference in terms of leaf lamina anatomy is the presence of equifacial mesophyll in A. sivasica, while mesophyll was found as dorsiventral in A. phrygia and A. gypsicola. How- ever, there are no glandular and non-glandular hairs on A. sivasica leaf lamina, as was described for A. phrygia and A. gypsicola leaves. In A. sivasica leaves the mesophyll consists of 1-3 cell rows palisade parenchyma and 2-4 cell rows spongy parenchyma, while in the leaf of A. phrygia and A. gypsicola palisade parenchyma was composed of 1-2 and single cell rows and spongy parenchyma composed of 6-8 and 4-5 cell rows, respectively. According to these results palisade and spongy parenchyma cell sizes of A. sivasica are distinctly larg- er than those in A. phrygia and A. gypsicola. In our study we found big secretory ducts near main vascular bundle of leaf lamina, which is similar with the results obtained for Achil- lea species by Grytsyk et al. (2016). In contrast, Akcin and Akcin (2010) did not report the presence of secretory ducts in the leaf lamina of A. phrygia and A. gypsicola. In leaves of A. sivasica stomata were observed on both sides of lamina in almost equal number, whereas in A. phrygia and A. gypsicola, stomata were reported as densely present on the lower epider- mis as compared to the upper epidermis (Akcin and Akcin 2010). In A. sivasica we found hydrophtic type of stomata that were located higher than adjacent epiderma cells. There were no reports of this character of A. phrygia and A. gypsicola by Akcin and Akcin (2010). Tekin and Meriç (2015) stated that all studied Tanacetum taxa had mesomorphic stomata, lo- cated at the same level as the adjacent epidermal cells. The anatomical and micromorphological features of the achene surface and its sculpturing have been studied using SEM in some Asteraceae genera, and these characters have been reported to provide additional information for evalua- tion regarding family classification (Zhu et al. 2006). Also, in flowering plants, the presence of slime cells on the surface of the fruit and slime envelope formation are known as di- agnostic characters in several families, including the Astera- ceae (Kreitschitz and Valles 2007, Inceer 2011). However, there are very restricted studies on achene micromorphol- ogy of the genus Achillea (Akcin and Akcin 2010, 2014). In those mentioned two studies, the authors described achene shape as oblong (A. phrygia, A. biebersteinii, A. biserrata, A. coarctata, A. millefolium ssp. millefolium, A. millefolium ssp. pannonica) oblong lanceolate (A. grandifolia, A. teretifolia) and ovoid or obovoid (A. gypsicola). In the present study, achene shape of A. sivasica were found to be oblong-lanseo- late which is the same shape as A. grandiflora and one of the other Turkish endemics, A. teretifolia. Akcin and Akcin (2014) reported that achene colour of A. biserrata and A. co- arctata is yellowish-brown. This feature is the same for A. sivasica which has yellowish-brown achene. There are some studies on fruit surface and slime cell features on Matricar- ia L. (Inceer 2011), Tripleurospermum Sch. Bip. (Inceer et al. 2012), and Artemisia L. taxa (Yakovleva et al. 2002, Kreitsch- itz and Valles 2007) of the Asteraceae family. In addition, Akcin and Akcin (2010, 2014) studied slime cell features on achene of some taxa of the genus Achillea in Turkey. Akcin and Akcin (2010) found slime (myxogenic) cells on achene surface of A. phrygia and A. gypsicola, but they did not give these cells’ shapes because they were not obvious enough. Accordingly, in present study, the slime cells found on achene surface are not conspicuous. Akcin and Akcin (2014) reported slime cells of ladder-like columns in parallel to the long axis of the fruit found in A. teretifolia and A. coacrata. As a conclusion, the present study is the first and the on- ly comprehensive report on the anatomy and achene mic- romorphology of the recently found critically endangered Turkish endemic A. sivasica. This study is important for the taxonomy of the genus, in terms of identifying the secretory system elements as well as determination of the significant anatomical differences between A. sivasica and related Ac- hillea taxa; they were found to have equifacial mesophyll layer, hydrophylic type stomata although it is a plant living in arid environment and having significant Casparian strip TEKIN M., AKDERE Ş. 98 ACTA BOT. CROAT. 80 (1), 2021 on stem endodermis. We believe that the results of the pres- ent study will contribute to the studies on anatomy and mi- cromorphology that will be carried out in the future aimed at determining the structural features of other Achillea taxa and investigating their place in the systematics. 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