Anatomical investigations of the Turkish critically endangered species: Achillea sivasica Çelik et Akpulat (Asteraceae)

Introduction

Asteraceae is the largest flowering plant family, comprising 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 continents 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 represented by 1209 species of which 447 species are endemic with an endemism ratio of 37% (Dogan et al. 2009). According 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 distributed 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 carried out in the past (Metcalfe and Chalk 1950,Milan et al. 2006,Tekin and Meriç 2015). According to a literature survey, it was revealed that, although there are many pharmacognostical 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 species (A. millefolium L., A. stricta Schleich., A. carpatica Bloski 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 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. grandifolia 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, soÇelik and Akpulat (2008) suggested a threat category of “Critically Endangered (CR)” according toIUCN (2001) red list criteria for this species. No anatomical investigations have previously been carried out on A. sivasica, however, the medicinal importance of many Achillea species in Turkey; the chemical composition and phytochemicals, antioxidant, and antityrosinase activities of A. sivasica 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 therapeutic applications.

The aim of this study is to reveal the previously unknown anatomical features and fruit micromorphological features of A. sivasica, pharmacognosy studies of which have been carried out, as mentioned above. For this purpose, root, stem, leaf midrib area and leaf lamina cross sections of plant samples were taken. Photomicroscopic examinations were carried out and achene surface micrographs were examined using a scanning electron microscope.

Materials and methods

Sampling

Species identification was carried out by Dr. M. Tekin according to study ofÇelik 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).

Fig. 1 Geographic distribution area of Achillea sivasica () in Sivas province in Turkey.

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.

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 dying 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. Micrographs were taken using an Olympus BX51 light microscope coupled with an Olympus DP70 digital camera. Anatomical measurements are based on at least 30 measurement results of each tissue or cell group from many sections obtained from plant samples from different locations. As a result of analysis of these measurements in SPSS package program, minimum, maximum, average values and standard deviation were determined. For scanning electron microscopy (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 magnifications. The nomenclature of all taxa followsIPNI (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 multilayered on surface of roots in the early stage of secondary development (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 consisting of 12-16 parenchymatous cell rows. Cortex cells were oval and circular in the younger roots, whereas they were genereally 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 presence of 5-7 secretory ducts formed by 5-12 unilayered secretory 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 occupied 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 corresponding locations. There was distinguishable cambium layer consist of 1-3 cell rows, between the phloem and the xylem in both younger and older root cross sections (Fig. 3,Tab. 1)

Fig. 3 Photomicrographs of root cross sections in the late stage of primary development (A-B) and early stage of secondary development (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 – perisicle, sc – secretory cell, sd – secretory duct, xy – xylem.

Tab. 1 Anatomical measurement results of the root, stem, leaf midrib area and leaf lamina of Achillea sivasica (n = 100, max – maximum, 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	─	─

Stem anatomy

The stem had a circular-pentagonal shape in cross-section 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 lamellar 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 pentagon (Fig. 4A, B). The cortex parenchyma consisted of 4-8 cells rows of oval and circular cells and occasionally contained 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, secretory ducts were observed (Fig. 4D). Beneath the cortex, there was a single cell row of the endodermis layer, consisting of oval cells that separate the cortex layer and vascular bundles from each other. Endodermis cells have indentations and protrusions in the places where they touch adjacent endodermis cells, which strengthens the connection between 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 endodermis 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 bundle 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 many cell rows of phloem. Between phloem and xylem there was cambium consisting of 2-3 cell rows that were dorsiventrally flattened (Fig. 4C). We observed only a fascicular cambium; 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).

Fig. 4 Photomicrographs of stem cross sections of Achillea sivasica (A-E) and detail of non-glandular hairs (F). General view of stem (A); detailed view of stem (B); detailed view of open collateral vascular bundle (C); detailed view of endodermis and secretory duct (green arrows indicated indentations and protrusions between adjacent endodermis cells and yellow arrows indicated intercellular area between cortex parenchyma cells) (D); detailed view of Casparian strips in endodermis (red arrows indicate Casparian strips in endodermis cells). Abbreviations: ca – cambium, cl – collenchyma, co – cortex, eh – epidermal non-glandular hair, en – endodermis, ep – epidermis, ph – phloem, pt – parenchymatous pith, sc – secretory cell, sd – secretory duct, sf – sclerenchymatous fibers, vb – vascular bundle, xy – xylem.

Leaf midrib area anatomy

The midrib area had a triangular shape in 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-photosynthetic ground parenchyma. Between the lower epidermis and the ground parenchyma, there were 2-3 cell layered collenchyma tissue (Fig. 5A, B). There were 5 vascular bundles in the cross section of each leaf midrib area. The main vascular bundle (midrib) was almost circular and was supported by sclerenchymatous fibers from upper and lower side. There were two secretory ducts, one on each side of the sclerenchymatous fibers accompaning xylem. The epithel consisted of 4-5 cells which are unilayered (Fig. 5A, C,Tab. 1).

Fig. 5 Photomicrographs of Achillea sivasica leaf (A-F). Leaf midrib 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 – phloem, 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.

Fruit micromorphology

Achene of A. sivasica were yellowish-brown with lanceolate-oblong shapes. Their surface is ribbed and glabrous. Slime cells were present on achene surface but they are inconspicuous (Fig. 6).

Fig. 6 Achene micrographs of Achillea sivasica. A – general appearance, B – detailed appearance of surface ornamentation.

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 reported 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. sivasica in the early secondary growth phase, in stem and leaves whereas secretory ducts were not reported on any of the vegetative 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 anatomical 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 Tanacetum 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 albipannosum 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 early 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 Menezes 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 ofAkcin and Akcin (2010) A. phrygia and A. gypsicola had endodermis in root and stem, which is the same result as in the present 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 primary 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 unilayered epidermis with abundant non-glandular hairs and lamellar collenchyma at the corners are features similar to those found in A. phrygia and A. gypsicola. In terms of anatomical 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 between cortex and stele as in A. gypsicola, but it is different from A. phrygia which has one- or two-layered endodermises (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 Casparian strip which consists of hydrophobic substance to restrict apoplastic flow of water is clearly obvious in many of the endodermis cells of A. sivasica. There is no report on these endodermis 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 studied (T. albipannosum, T. densum ssp. sivasicum, T. heterotomum, 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 reports on the presence of cambium in A. phrygia and A. gypsicola. The general stem anatomy of A. sivasica, including the presence of unilayered epidermis, epidermal hairs, interrupted collenchyma beneath epidermis, single-layered endodermis is similar to that of the six Tanacetum taxa described 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 toMetcalfe and Chalk (1950), anatomical diversity in taxa of the family Asteraceae is commonly observed in the leaf structure. They reported that there were both anomocytic 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 byAkcin 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 Achillea 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. However, 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 larger 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 Achillea species byGrytsyk 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 epidermis 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 byAkcin and Akcin (2010). Tekin and Meriç (2015) stated that all studied Tanacetum taxa had mesomorphic stomata, located 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 evaluation 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 diagnostic characters in several families, including the Asteraceae (Kreitschitz and Valles 2007,Inceer 2011). However, there are very restricted studies on achene micromorphology 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-lanseolate 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. coarctata 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 Matricaria L. (Inceer 2011), Tripleurospermum Sch. Bip. (Inceer et al. 2012), and Artemisia L. taxa (Yakovleva et al. 2002, Kreitschitz 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 only comprehensive report on the anatomy and achene micromorphology 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 Achillea 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 on stem endodermis. We believe that the results of the present study will contribute to the studies on anatomy and micromorphology 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.