Contribution to the biosystematics of the wild Vaccinium species distributed in Turkey Текст научной статьи по специальности «Биологические науки»

Turczaninowia 27, 1: 151-170 (2024) K9ISSN 1560-7259 (print edition)

DOI: 10.14258/turczaninowia.27.1.18 ШЩ TURCZANINOWIA

http://turczanin0wia.asu.ru fciieiSSN 1560-7267 (online edition)

УДК 582.912.46(560)

Contribution to the biosystematics of the wild Vaccinium species distributed in Turkey

G. Yilmaz1' 3*, K. Coskun^elebi1, 4, M. E. Guzel1 5, S. Makbul2' 6

1 Karadeniz Technical University, Department of Biology, Kanuni Campus, Ortahisar, Trabzon, 61080, Turkey 2 Recep Tayyip Erdogan University, Zihni Derin Campus - Fener Mahallesi, Rize, 53100, Turkey 3E-mail:gulsahyilmaz@ktu.edu.tr; ORCiD iD: https://orcid.org/0000-0002-1806-2344

4E-mail:kamil@ktu.edu.tr; ORCiD iD https://orcid.org/0000-0001-5713-6628 5E-mail: muraterdemguzel@hotmail.com; ORCiD iD http://orcid.org/0000-0002-7923-7443 6E-mail: serdar.makbul@erdogan.edu.tr; ORCiD iD http://orcid.org/0000-0001-8798-0926

* Corresponding author

Keywords: Ericaceae, fruit, leaf, pollen grain, seed.

Summary. The genus Vaccinium L. (Ericaceae) consists of shrubs or dwarf shrubs species widely distributed in Southeast Asia, America, and Europe. In the present paper, members of the genus (V. arctostaphylos, V. myrtillus, V. uliginosum, V. vitis-idaea) naturally grown in Northeast Anatolia were compared in terms of leaf venation, seed micromorphology, and palynological properties based on 16 populations in detail. Present findings revealed that number of areoles (the small area bounded by secondary veinlet) is 8-9 for V. arctostaphylos, 5-7 for К myrtillus, 3-4 for V. uliginosum and V. vitis-idaea. Scanning electron microscope observation shows that the leaf adaxial surface is fiber-fluted in К arctostaphylos, ruminate in V. myrtillus, V. uliginosum, and К vitis-idaea due to epicuticular waxes. In addition, the seed shape of V. arctostaphylos in outline varies from triangular, tetragonal to oval, whereas they are lunate to reniform or falcate in the rest of the examined taxa. Epidermal cells of the seed are quadrilateral with straight anticlinal walls in V. arctostaphylos, however, they are most elongate rectangle with weakly undulate anticlinal walls in the rest species. The mean tetrad diameter (D) of pollen belonging to all examined species is 29.89 (21.47-43.22) ^m while the lowest mean D was found in V. uliginosum (24.59 ^m) and the highest mean D was found in V. arctostaphylos (36.74 ^m). Principal component analysis based on 47 traits (flower: 9, leaf: 18, seed and fruit: 9 and pollen: 11) showed that the leaf length and width, teeth numbers, tetrad diameter, equatorial axis, and polar axis were the most valuable characters to distinguish the examined taxa in the present paper.

К биосистематике диких видов Vaccinium, распространенных в Турции

Г. Илмаз1*, К. Джошкунчелеби1, М. Э. Гюзель1, С. Макбул2

1 Черноморский технический университет, г. Трабзон, 61080, Турция 2 Университет им. Реджепа Тайипа Эрдогана, Кампус Зихни Дерин - Фенер Махаллеси, г. Ризе, 53100, Турция

Ключевые слова: вересковые, лист, плод, пыльцевое зерно, семя.

Аннотация. Род Vaccinium L. (Ericaceae) состоит из кустарниковых и кустарничковых видов, широко распространенных в Азии (гл. обр. Юго-Восточной), Америке и Европе. В настоящем исследовании было прове-

Поступило в редакцию 12.10.2022 Принято к публикации 06.05.2024

Submitted 12.10.2022 Accepted 06.05.2024

дено сравнительное изучение жилкования листьев, микроморфологии семян и палинологических особенностей 16 популяций четырёх представителей Уасстшш (V. arctostaphylos, V. тутЫНт, V. ы^тозыт, V. уШ$-14аеа), естественно произрастающих в Северо-Восточной Анатолии. Полученные результаты показали, что для V. arctostaphylos характерно наличие 8-9 ареол, для V. тугШ1ш - 5-7, для V. ы^то$ыт и V. vitis-idaea - 3-4 ареолы, образованные вторичной жилкой. Данные СЭМ показали, что адаксиальная поверхность листа у V. arctostaphylos покрыта волокнистыми бороздками, у V. тугЫНш, К и^^зит и V. vitis-idaea она рифленая из-за эпикутикулярных восков. Кроме того, форма семян V. arctostaphylos варьирует от треугольной и четырехугольной до овальной, в то время как у остальных исследованных таксонов она лунообразная, почковидная или серповидная. Эпидермальные клетки семени четырехугольные, с прямыми антиклинальными стенками у V. arctostaphylos, однако у остальных видов представляют собой вытянутый прямоугольник со слабоволнистыми антиклинальными стенками. Средний диаметр тетрады (Б) пыльцы всех исследованных видов составляет 29,89 (21,47-43,22) мкм, наименьший у V. uliginosum (24,59 мкм), а наибольший - у V. arctostaphylos (36,74 мкм). Анализ основных компонент, основанный на 47 признаках (9 признаков цветка, 18 - листа, 9 - семени и плода и 11 - пыльцы), показал, что длина, ширина и число зубцов листа, диаметр тетрады, экваториальная ось и полярная длина пыльцевых зерен являются наиболее ценными признаками для разграничения исследованных видов.

Introduction

The genus Vaccinium L., the third largest genus in Ericaceae, contains approximately 450 species worldwide (Cronquist, 1981). The genus is mostly distributed in Southeast Asia, America and Europe including subtropical or tropical montane regions on most continents (Vander Kloet, 1988). Members of Vaccinium have been subjected to several taxonomical studies on both regional and global scale up to now. Sleumer (1941) reported that the inflorescence type, calyx tube shape, corolla shape, and type of stamen appendages are valuable taxonomical traits for the members of Vaccinium. Likewise, Palser (1961) confirmed the sectional classification for Vaccinium taxa distributed in the United States mainly based on the floral structure. However, according to Stevens (1971), many floral characters like calyx shape, corolla shape, and stamen supply limited information to distinguish taxa within the genus Vaccinium. On the other hand, Nekrasova (1952) emphasized that leaf indumentum is valuable to distinguish Vaccinium members in the "Flora of the USSR". However, it is reported that the vegetative characters are much less important than floral characters in circumscribing sections in the genus (Odell, Vander Kloet, 1991). Schultz (1944) reported that the only difference between V. scoparium Leiberg ex Coville and V. myrtillus L. is berry color at a lower elevation. Both puberulent and glabrous twigs may occur in both taxa (often on the same plant) and puberulent twigs are not restricted to V. myrtillus. Fang and Stevens (2005) used leaf venation and fruit size to distinguish V. myrtillus, V. uliginosum L., and V. vitis-idaea L. as key characters in the "Flora of China". It is well known that the carpological investigation provides large

number of diagnostic features for the classification of taxa at several level (Szkudlarz, 1999b) however there are limited carpological studies related to members of Vaccinium at a specific level (Vander Kloet, 1988; Szkudlarz, 1999a, 2001).

The genus Vaccinium is known to have a basic chromosome number of x = 12 (Darrow et al., 1944). Although diploid chromosome numbers are common in the genus, several polyploidy cytotypes were also recorded in V. uliginosum, V. arctostaphylos L., and V. myrtillus (Hagerup, 1928; Darrow et al., 1944). However, there seems to be no correlation between morphological characters and ploidy level (Young, 1970; Vander Kloet, 1988).

Sarwar et al. (2006) evaluated palynological features of 37 Vaccinium species in detail and reported that palynological characters are not in accordance with the current infrageneric classification, but they provide useful information to figure out the taxonomic problem within the genus. Goldy et al. (1984) also reported that the exine sculpture contributes to the delimitation of Vaccinium taxa. According to Cockerham and Gal-letta (1976), tetrad diameter was calculated 11 % larger in the tetraploid pollen than diploid pollen in the genus Vaccinium. Similarly, slight differences in tetrad size and exine sculpture were recorded between the New World blueberry species and Old World blueberry species by Sarwar et al. (2006). The smallest pollen grains were also found in V. uliginosum by Sarwar and Takahashi (2007).

The most recent and widely used molecular phylogenetic data also improved our knowledge of the taxonomy of Vaccinium. Firstly, Sleumer (1941) revealed the polyphyletic origin of the genus. However, the most comprehensive phylogenetic studies on the genus at sectional level (sect.

Macropelma Klotzsch and sect. Myrtillus Dumortier and sect. Hemimyrtillus Sleumer) and on the tribe Vaccinieae Rchb. were performed by Powell and Kron (2002) and Kron et al. (2002), respectively. The latter two molecular studies also revealed that Vaccinium is a polyphyletic genus as previously reported by Sleumer (1941). Vaccinium arctostaphylos is placed within a clade sister to V. uliginosum (Powell, Kron, 2002). Correspondingly, Sultana et al. (2020) reported that V myrtillus and V macrocarpon Ait. are closely related species and formed a sister clade. Kron et al. (2002) suggested that more morphological and molecular data are necessary to recognize members of Vaccinium within the tribe Vaccinieae. Despite many advantages of phylogenetic works, the representation of the Vaccinium in phylogenetic studies is still too limited to draw taxonomic conclusions, so Pedraza-Penalosa and Luteyn (2011) described seven new species based on traditional circumscription from Andean South America.

The first comprehensive taxonomic study on the Turkish native species of Vaccinium (V. arctostaphylos, V. myrtillus, V. uliginosum, and V vitis-idaea) was presented by Stevens (1978). Afterward, the systematics of the Turkish Vaccinium was updated by Terzioglu (2012) in the checklist of the "Flora of Turkey" without any further taxonomic contributions. Subsequently, many researchers mostly focused on chemical properties of the Turkish

Vaccinium. Ayaz et al. (2001) firstly examined the levels of fructose and glucose in V. arctostaphylos and V. myrtillus at three different maturity stages in NE Turkey. Similarly, anthocyanin and antioxidant contents in the berries of two species were reported from Turkey (Qolak et al., 2016). Furthermore, altitude variation in the volatile composition of blueberry species (V arctostaphlyos, V. uliginosum, V. vitis-idaea, and V. myrtillus) distributed in the NE Anatolia was examined by Erik et al. (2020). Some phenological characteristics and distribution patterns of the Turkish Vaccinium were compiled from accessible sources by Qelik and Ser^e (2015).

In the present paper, we aimed to explore surface and venation patterns of leaf, seed macro-micromorphology, and pollen features of the Vaccinium species distributed in NE Anatolia, contribute filling the gaps in the biosystematics features of the examined taxa and determine valuable characters to distinguish the examined Vaccinium species.

Materials and methods

Specimens

Samples for biosystematic studies were collected from sixteen populations of V. arctostaphlyos, V. uli-ginosum, V. vitis-idaea, and V. myrtillus in Turkey within the scope of the MSc thesis of the first author

Fig. 1. Leaf areolar (vein islets) pattern: a - Vaccinium arctostaphylos; b - V. myrtillus; c - V. uliginosum; d -V. vitis-idaea. Arrow shows the primary (1), secondary (2), tertiary (3), and quaternary veins (4).

(Table 1). The specimens were labeled, numbered, annotated with the date of collections, the locality and the name of the collector during the field studies. After identification of the samples according to Stevens (1978), all vouchers were stored in the herbarium of the Department of Biology at Karadeniz Technical University (KTUB). Additionally, healthy leaves and fruits including seeds for morphological studies and flowers for palynological studies were also collected from the same population during the field trips. Taxonomic names followed Terzioglu (2012), and distribution details were given according to the Turkey Grid System (Davis, 1965).

Protocols for leaf venation studies

At least 3-4 entire mature leaves from each investigated population of the Turkish Vaccinium were immersed in 2.5 % Sodium Hydroxide for 1520 days (Panda, Kirtania, 2016). After that, all leaves were rinsed with distilled water and then stained with safranin for 15 minutes (Vardar, 1962). After staining, all leaves were again rinsed with distilled water before taking photos with a Leica DFC 490 digital camera attached to a stereomicroscope. Areoles number (the area bounded by veins) and divergence angle of secondary, tertiary, and quaternary veins were calculated from at least 3-4 different part

Fig. 2. Adaxial leaf surface, scanning electron microscope: a - Vaccinium arctostaphylos; b - V myrtillus; c - V uliginosum; d - V vitis-idaea.

of each stained leaves belonging to each investigated population according to Saibaba and Rao (1990) and Panda and Kirtania (2016).

Protocols for leaf micromorphology studies

Firstly at least three pieces of dried leaves about 1 cm2 belong to each investigated population were cut out from the middle part by a razor blade and then all pieces were transferred onto aluminum stubs, coated with gold in a sputter-coater and examined under the JEOL-JSM 6610 scanning electron microscope (SEM) at the Central Research Laboratory of Recep Tayyip Erdogan University at a voltage 10 kV. All micrographs were taken 200x and 1000x magnification. At least ten leaf micro-

graphs taken 200x and 1000x magnification per investigated population was observed for micromor-phological description. The terminology of Simpson (2012) and Stearn (1985) was adopted for the leaf micromorphological descriptions.

Protocols for seed macro and micromorphology studies

A total of 12 dried fruits from each investigated population were boiled in water and then all seeds were extracted from fruits. Then, the seeds were stored in anhydride-sulfuric acid (9 : 1) for 24 hour at room temperature. After that, all seeds were rinsed with distilled water, dried in oven at 50 °C for 24 hour and photographed by a Leica DFC 490

Fig. 3. Seed morphology, light microscope: a - Vaccinium arctostaphylos; b - V myrtillus; c - V uliginosum; d - V vitis-

stereomicroscope attached to a digital camera. Dried seeds also were transferred on aluminum stubs with double-sided adhesive carbon tape and coated with gold for 4 min in a sputter-coater for SEM studies. Seed micrographs were observed by a JEOL-JSM 6610 microscope at the Central Research Laboratory of Recep Tayyip Erdogan University at 50x, 300x, 1000x, and 2000x magnification. The terminology of Barthlott (1981), Stearn (1985), Co§kun^elebi et al. (2000, 2016) and Khorasani et al. (2020) was mainly followed for the seed macro-micromorphological descriptions. Qualitative characters of seed were described according to terminology provided by Khorasani et al. (2020).

Protocols for palynological studies

Several randomly selected flowers were collected from each population belonging to investigated species. Pollen grains were extracted from the flower according to the acetolysed method proposed by Erdtman (1952) and then one half was immersed in an alcohol solution of glycerine (for LM) to obtain

permanent slides and the other in ethyl alcohol 96 % (for SEM). Permanent slides were examined using Leica S6D light microscope to measure or determine the following parameters: Tetrad axis (D), equatorial axis (E or d), polar axis (P), P/E ratio, D/d ratio, colpus length (2f), colpus width (W), septum (total exine) thickness (Se), exine (Ex), 2f/W ratio, 2f/D ratio. For each analyzed population, thirty mature and correctly formed pollen grains were measured by light microscopy. For SEM studies, air-dried acetolysed pollen grains were transferred onto aluminum stubs and coated with gold for 4 min in a sputter-coater and examined by a JEOL-JSM 6610 microscope at the Central Research Laboratory of Recep Tayyip Erdogan University at a voltage 10 kV. Pollen photographs and micrographs were taken using Leica S6D light microscope and JEOL-JSM 6610 electron microscope, respectively. For the pollen grains, the description terminology of Sarwar (2007), Punt et al. (2007) and Park and Song (2010) were mainly followed.

Fig. 4. Seed morphology, scanning electron microscope: a1-4 - Vaccinium arctostaphylos; b - V. myrtillus; c - V. uliginosum; d - V. vitis-idaea.

Numerical analysis

Forty-seven traits (Table 2) related to leaf, flower, fruit, seed and pollen grains were measured or scored using a light microscope. All scores or measurements were checked at least three or four times for each population. Average values and standard deviations (SD) of the all traits were given in Table 4 for each population. A data matrix including scores of 47 traits was subjected to cluster analysis (CA) and principal component analysis (PCA). For CA, a pair-wise matrix of resemblance values was calculated from the standardized data matrix, using Gower's coefficient of resemblance designed for mixed data sets (Sneath, Sokal, 1973). For PCA, the standardized data were used to create a covariance matrix. Five eigenvectors were extracted, providing axis onto which the standardized data were projected to give a two-dimensional plot of the examined taxa. All analyses were performed using MVSP computer programs for multivariate data analysis in ecology and systematics (Kovach, 2007).

Results

Traits of leaf, seed and pollen grains

Findings related to leaf (shape, margins, and venation), seed, and pollen grains of each investigated taxon were given alphabetically in the following.

Vaccinium arctostaphylos L. 1753, Sp. Pl. 351

Leaf: Elliptic-broadly lanceolate with serrate or serrulate margin in shape (Fig. 1a:). The secondary veins are opposite at the base of a primary vein and then alternatively arranged with a divergence angle of 25°-45° towards the primary vein tip and free-ending. The numbers of areoles formed by the secondary, tertiary, and quaternary veins are 8-9, 5-6 and 3-6, respectively (Fig. 1a2). The leaf adaxial surface under SEM is weakly striate; the epidermal cell borders are hardly indistinct (Fig. 2a1-3).

Seed: Seeds are 1.3-1.5 mm length, brown, triangular, tetragonal or oval (Fig. 3a). Epidermal cells of seed coat are mostly polygonal in outline,

Fig. 5. Pollen morphology, scanning electron microscope: a1-4 - Vaccinium arctostaphylos; b1-4 - V myrtillus; c1-4 - V uliginosum; d - V vitis-idaea.

anticlinal walls straight, raised, smooth, strongly thickened, periclinal cell walls smooth and concave (Fig- 4a1_4).

Pollen grains: Pollen grains are in tetrahedral tetrads, tetrad axis (D) 28.55-43.22 (36.7) |m, oblate-spheroidal, equatorial diameter (E or d) 22.34-31.02 (29.86) ^m, polar axis (P) 15.15-23.99 (20.58) |m, P/E ratio 0.63-0.94 (0.81) |m, D/d ratio 1.27-1.38 (1.34) |m. Colpus is narrowing, colpus margin distinct. Ora is in the middle of the colpus, length (2f) 17.45-24.23 (21.95) |m, width (W) 1.98-3.65 (2.69) |m. Septum thickness (Se) 1.22-1.89 (1.46) |m thick, exine (Ex) 1.52-1.9 (1.72) |m thick (Fig. 5a4). 2f/W ratio 9.04-9.45 (9.16) |m, 2f/D ratio 0.33-0.62 (0.47). Pollen grains are verrucate, microgranulate in

the population of VA1, regulate in the rest examined populations. Representative micrographs of pollen under the SEM are given in Fig. 5a .

Vaccinium myrtillus L. 1753, Sp. Pl. 349

Leaf: Broadly elliptic or ovate, sharply serrate margin in shape (Fig. 1Ц). The secondary veins are alternatively arranged with a divergence angle of 30-55° toward a primary vein tip and free-ending. The numbers of areoles formed by the secondary, tertiary, and quaternary veins are 5-7, 5-9, and 3-6, respectively (Fig. 1b2). The leaf adaxial surface under SEM is without striate; epidermal cell borders are slightly visible (Fig. 2b ).

Fig. 6. UPGMA dendrogram of Vaccinium taxa based on morphological data (Details of the population numbers are given in Table 1).

Seed: Seeds are 1.2-1.3 mm length, light or dark brown, lunate to reniform in outline (Fig. 3b). Epidermal cells of seed coat are elongated, mostly rectangle in shape, anticlinal walls straight, raised, slightly striated, thickened, the surface of outer periclinal cell wall is smooth and concave (Fig. 4bi-4).

Pollen grains: Pollen grains are in tetrahedral tetrads, tetrad axis (D) 25.68-35.4 (31.08) |m, oblate, equatorial axis (E or d) 20.58-29.14 (24.13) |m, polar axis (P) 13.7-26.17 (18.32) |m, P/E ratio 0.65-0.97 (0.82), D/d ratio 1.13-1.36 (1.27). Colpus is narrowing, colpus margin distinct. Ora is in the middle of the colpus, length (2f) 9.45-19.78 (17.04) |m, width (W) 1.43-3.08 (2.35) |m. Septum thickness (Se) 1.32-2.09 (1.69) |m thick, exine (Ex) 1.44-1.68 (1.59) |m thick, regulate. 2f/W ratio 6.08-8.35 (7.09) |m, 2f/D ratio 0.37-0.62 (0.51) |m. Representative micrographs of pollen under the SEM are given in Fig. 5b .

Vaccinium uliginosum L. 1753, Sp. Pl. 350

Leaf: Ovate-obovate with entire margin in shape (Fig. 1c:). The secondary veins are opposite at the base of a primary vein and then alternatively arranged with a divergence angle of 25-45° towards the primary vein tip and anastomose. The numbers of areoles formed by the secondary, tertiary, and quaternary veins are 3-4, 1-3, and 1-2, respectively (Fig. 1c2). However the quaternary veins are generally freely ending without forming any areoles. The leaf adaxial surface under SEM is covered by a distinctly rare, more or less dispersed epicuticular wax; the epidermal cell borders are slightly visible (Fig. 2c1-3).

Seed: Seeds are 1-1.1 mm length, light or dark brown, lunate to elliptic in outline (Fig. 3c). Epidermal cells of seed coat are elongated, irregular in shape, anticlinal walls obvious, smooth, thickened, slightly undulate, periclinal surfaces smooth and flat (Fig. 4SJ.

PC 1

Fig. 7. Ordination resulting from principal components analysis (PCA) of Vaccinium based on 47 characters (Details of the population numbers and traits are given in Table 1, 2 respectively).

Voucher data of the studied taxa Vaccinium

Table 1

Taxa Pop. Code Grid of Davis (1970) Locality Altitude (m) Voucher number

V arctostaphylos VA1 A7 Gumu^hane: Kurtun above Alan Plateau 1845 Co§kungelebi & Yilmaz 1

VA23 A7 Trabzon: Tonya above Kalin^am Village 1483 Co§kungelebi & Yilmaz 23

VA47 A8 Rize: Arde^en, Siprona Plateau 1032 Co§kungelebi & Yilmaz 47

VA91 A8 Artvin: Murgul, Tiryal Mountain 2234 Co§kungelebi & Yilmaz 91

V myrtillus VM6 A7 Gumu^hane: Kurtun, Sume Village 2081 Co§kungelebi & Yilmaz 6

VM94 A7 Trabzon: Multat Plateau 2811 Co§kungelebi & Yilmaz 94

VM97 A8 Artvin: §av§at, Kirazli Village 2533 Co§kungelebi & Yilmaz 97

VM102 A9 Ardahan: Hanak, Alabalik Plateau 2613 Co§kungelebi & Yilmaz 102

V uliginosum VU66 A8 Artvin: §av§at, Karagol National Park 2537 Co§kungelebi & Yilmaz 66

VU76 A9 Ardahan: Posof, U^li^kin Hill 2405 Co§kungelebi & Yilmaz 76

VU78 A9 Ardahan: Hanak, Alabalik Plateau 2588 Co§kungelebi & Yilmaz 78

VU93 A7 Trabzon: Multat Plateau 2811 Co§kungelebi & Yilmaz 93

V vitis-idaea VV63 A8 Artvin: §av§at, Karagol National Park 2533 Co§kungelebi & Yilmaz 63

VV69 A9 Ardahan: Posof, Sesodile Hill 2376 Co§kungelebi & Yilmaz 69

VV80 A9 Ardahan: Alabalik Plateau 2579 Co§kungelebi & Yilmaz 80

VV100 A8 Artvin: §av§at, Kocabey Plateau 2491 Co§kungelebi & Yilmaz 100

Table 2 Characters

Acronym Examined characters and their states

X1 Leaf (evergreen: 0; deciduous: 1)

X2 Leaf (papery: 0; coriaceous: 1)

X3 Leaf width (cm)

X4 Leaf length (cm)

X5 Leaf shapes (elliptic-lanceolate: 1; elliptic-ovoid: 2; ovate-oblong: 3; ovate-oblong: 4)

X6 Leaf margin (entire: 1; serrate 2; serrulate: 3)

X7 Leaf teeth numbers in 1 cm long (digit)

X8 Leaf apex (acuminate: 1; acute: 2; obtuse: 3)

X9 Leaf base (oblique: 1; attenuate: 2; rounded: 3)

X10 Leaf abaxial surface (pubescent: 0; glabrous: 1)

X11 Leaf adaxial surface (pubescent: 0; glabrous: 1)

X12 Petiole length (mm)

X13 Petiole (glabrous: 0; hairy: 1)

X14 Leaf venation (reticulate: 0; anastomose: 1)

X15 Inflorescence (terminal: 0; axilary rasemes: 1)

X16 Flower number (digit)

X17 Corolla shape (campanulate: 1; urceolate: 2; spherical: 3)

X18 Corolla color (white-mock: 1; white - dark red: 2; dark red-yellowish green: 3)

X19 Corolla width (mm)

X20 Corolla length (mm)

X21 Calyx length (mm)

X22 Calyx margin (narrow-wide triangular: 1; wavy: 2; rounded-undulate: 3)

X23 Calyx (pubescent: 0; glabrous: 1)

X24 Pedicel length (mm)

X25 Fruit color (coal black: 1; black: 2; indigo: 3; sour cherry: 4)

X26 Average weight of 10 air dried fruit/mg

X27 Pedicel length in fruiting (cm)

X28 Dried fruit diameter (mm)

Table 2 (continuation)

Acronym Examined characters and their states

X29 Seed length (mm)

X30 Leaf surface under SEM (fibros-striat: 1; ruminate: 2; concave-postulate :3)

X31 Areoles shape (ovoid: 0; diagonal: 1)

X32 Number of secondary veins (10-16: 0; 8-10: 1)

X33 Areoles number of secondary vein (16-18: 1; 10-14: 2; 6-8: 3)

X34 Seed surface under SEM (wrinkled: 0; wavy: 1)

X35 Seed epidermal cells shape (isolateral or quadrilateral: 0; elengote: 1)

X36 Anticlinal wall of the seed epidermal cells (smooth: 0; ruminate: 1)

X37 Tetrad axis (D) (|m)

X38 Equatorial axis (E or d) (|m)

X39 Polar length (P) (|m)

X40 P/E (|m/|m)

X41 D/E (|m/|m)

X42 Colpus length (2f) (|m)

X43 Colpus width (W) (|m)

X44 Septum thickness (Se) (|m)

X45 Exine (Ex) (|m)

X46 2f/W (|m/|m)

X47 2f/D (|m/|m)

Pollen grains: Pollen grains are in tetrahedral tetrads, tetrad axis (D) 21.47-28.95 (24.59) |m, oblate, equatorial axis (E or d) 17.83-25.43 (20.77) |m, polar axis (P) 11.96-17.94 (14.51) |m, P/E ratio 0.63-0.77 (0.64) |m, D/d ratio 1.14-1.39 (1.27) |m. Colpus is narrowing, colpus margin distinct. Ora is in the middle of the colpus, length (2f) 10.3-17.45 (15.47) |m, width (W) 1.83-2.88 (2.48) |m. Septum thickness (Se) 0.97-1.44 (1.25) |m thick, exine (Ex) 1.32-1.53 (1.42) |m thick, rugulose. 2f/W ratio 5.63-6.99 (6.26) | m, 2f/D ratio 0.49-0.88 (0.65) |m. Representative micrographs of pollen under the SEM are given in Fig. 5c1-4.

Vaccinium vitis-idaea L. 1753, Sp. Pl. 351

Leaf: Ovate-obovate with entire margin in shape (Fig. 1d1). The secondary veins are opposite at the base of a primary vein and then alternatively arranged with a divergence angle of 25°-45° towards the primary vein tip and free-ending. The numbers of areoles formed by the secondary, tertiary, and quaternary veins are 3-4, 1-3, and 1-3, respectively (Fig. 1d2). The leaf adaxial surface is covered by a distinctly dense dispersed epicuticular waxes; the epidermal cell borders are slightly visible (Fig. 2d1-3).

Seed: Seeds are 1.3-1.5 (1.4) mm length, light or dark brown, lunate to falcate (Fig. 3d). Epidermal cells of seed coat are elongated, irregular in shape, anticlinal walls obvious, smooth, thickened, slightly undulate, periclinal surfaces smooth and flat (Fig. 4d).

Pollen grains: Pollen grains are in tetrahedral tetrads, tetrad axis (D) 24.38-34.74 (27.14) |m, oblate-spheroidal, equatorial axis (E or d) 12.9622.48 (18.7) |m, polar axis (P) 12.03-18.32 (14.86) |m, P/E ratio 0.71-0.92 (0.84) |m, D/d ratio 1.321.39 (1.36) |m. Colpus is narrowing, colpus margin distinct. Ora is in the middle of the colpus, length (2f) 14.37-23.85 (17.68) |m, width (W) 1.52-3.15 (2.67) |m. Septum thickness (Se) 0.89-1.52 (1.18) |m thick, exine (Ex) 1.18-1.26 (1.2) |m thick, regulate. 2f/W ratio 5.45-9.12 (7.94) |m, 2f/D ratio 0.61-0.83 (0.81) |m. Representative micrographs of pollen under the SEM are given in Fig. 5d1-4.

Numerical results

Scores related to leaf, flower, fruit and seed and pollen traits of 16 populations belonging to four wild Vaccinium were given in Table 4. The dendrogram resulting from the cluster analysis (UPGMA) based on the combined dataset (36 morphological: flower: 9, leaf: 18, seed and fruit: 9) and 11 palynological characters) is given in Fig. 6. The dendrogram revealed that the examined V. arctostaphylos specimens (label b) are distinctly separated from the rest of the examined Vaccinium specimens (label a) at a distance value of 67.5. Moreover, specimens of V. uliginosum and V. vitis-idaea are aggregated at a distance value of 7.5, which shows that they are the most closely related taxa based on both morphological and palynological characters (Fig. 6).

Scores of forty seven traits of examined population belong to Vaccinium (Explanation of the population number and traits are given in Table 1,2 respectively

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XI 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0

X2 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1

X3 Mean 34.70 33.00 41.50 46.60 9.00 12.75 14.25 20.33 6.43 5.60 8.58 6.72 8.62 7.75 7.92 6.50

SD ±0.16 ±0.12 ±0.14 ±0.20 ±0.26 ±0.18 ±0.16 ±0.25 ±0.55 ±0.39 ±0.47 ±0.35 ±0.15 ±0.22 ±0.18 ±0.21

X4 Mean 65.85 75.99 67.3 77.87 13.25 24.00 27.75 32.30 12.01 9.49 13.65 10.09 16.36 14.89 14.61 13.68

SD ±1.25 ±1.28 ±1.17 ±1.21 ±0.95 ±1.04 ±0.93 ±0.97 ±0.24 ±0.32 ±0.28 ±0.17 ±0.22 ±0.28 ±0.19 ±0.21

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X6 2 3 2 3 3 3 2 2 1 1 1 1 1 1 1

X7 Mean 23.44 30.53 29.11 25.16 19.5 19.25 13.5 17.00 0 0 0 0 0 0 0

SD ±1.12 ±0.85 ±0.96 ±1.02 ±0.65 ±0.74 ±1.17 ±1.11 0 0 0 0 0 0 0 0

X8 1 1 1 1 2 2 2 2 1 3 3 3 3 3 3 3

X9 1 1 1 1 3 3 3 3 1 1 1 1 2 2 2 2

X10 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1

Xll 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1

X12 Mean 0.1 0.1 0.1 0.1 0.98 1.06 0.8 0.95 0.94 1.11 0.95 1.06 1.7 1.72 1.61 2.01

SD ±0.24 ±0.19 ±0.25 ±0.29 ±0.17 ±0.22 ±0.14 ±0.12 ±0.01 ±0.02 ±0.04 ±0.03 ±0.02 ±0.01 ±0.03 ±0.04

X13 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1

X14 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0

X15 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 1

X16 Mean 5.30 4.70 5.90 6.20 0 0 0 0 1.50 2.00 1.50 1.50 4.00 4.00 5.50 4.00

SD ±0.46 ±0.75 ±0.63 ±0.92 0 0 0 0 ±0.21 ±0.49 ±0.73 ±0.54 ±0.25 ±0.15 ±0.42 ±0.50

X17 1 1 1 1 3 3 3 3 2 2 2 2 1 1 1 1

X18 2 2 2 2 3 3 3 3 1 1 1 1 2 2 2 2

X19 Mean 5.7 4.02 5.89 4.06 4.5 3.87 4.25 5.48 3.02 2.72 2.87 2.95 3.35 3.4 4.07 3.43

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Eigen values of the characters on the first five principal components (Explanation of traits are given in Table 2)

Acronym PC 1 PC 2 PC 3 PC 4 PC 5

X1 0.005 0.029 -0.062 -0.132 -0.027

X2 -0.005 -0.029 0.062 0.132 0.027

X3 0.430 -0.160 -0.781 0.312 0.029

X4 0.795 -0.323 0.332 -0.229 -0.123

X5 -0.025 -0.033 0.003 0.000 0.001

X6 0.018 0.078 0.025 0.001 -0.036

X7 0.344 0.769 0.089 0.028 0.104

X8 -0.022 -0.025 0.025 0.090 0.033

X9 -0.007 0.089 0.027 0.170 0.057

X10 -0.012 -0.046 0.010 -0.008 -0.006

X11 -0.012 -0.046 0.010 -0.008 -0.006

X12 -0.016 -0.012 0.043 0.107 0.039

X13 -0.005 -0.029 0.062 0.132 0.027

X14 -0.006 -0.017 -0.051 -0.141 -0.034

X15 0.000 -0.059 0.017 -0.018 -0.015

X16 0.038 -0.234 0.135 0.358 0.021

X17 -0.008 0.101 -0.087 -0.104 -0.004

X18 0.005 0.076 0.034 0.160 0.049

X19 0.021 0.051 -0.053 0.166 0.079

X20 0.043 -0.023 0.077 0.124 0.108

X21 0.021 -0.001 0.020 0.009 -0.050

X22 -0.014 0.024 -0.121 -0.265 -0.054

X23 -0.007 0.042 -0.069 -0.123 -0.019

X24 0.031 0.252 -0.014 0.279 -0.848

X25 -0.029 -0.002 0.018 0.167 0.086

X26 0.005 0.032 -0.003 -0.011 -0.004

X27 0.021 0.026 -0.011 0.085 0.174

X28 0.056 0.157 -0.195 0.067 0.273

X29 0.002 -0.001 0.019 0.042 0.022

X30 -0.019 -0.016 0.055 0.142 0.035

X31 -0.000 0.059 -0.017 0.018 0.015

X32 -0.012 -0.046 0.010 -0.008 -0.006

X33 -0.025 -0.033 0.003 0.000 0.001

X34 -0.013 0.012 -0.007 0.009 0.008

X35 -0.013 0.012 -0.007 0.009 0.008

X36 -0.005 -0.029 0.062 0.132 0.027

X37 0.143 0.143 0.247 0.219 0.098

X38 0.130 0.157 -0.093 -0.364 0.088

X39 0.075 0.155 0.018 0.046 0.228

X40 0.001 0.004 0.013 0.028 0.001

X41 7,70E-01 -0.005 0.010 0.018 -0.000

X42 0.073 -0.047 0.246 0.202 0.154

X43 0.001 -0.014 0.022 0.023 -0.019

X44 0.002 0.023 -0.010 -0.001 0.020

X45 0.005 0.016 -0.009 -0.026 -0.005

X46 0.028 -0.046 0.130 0.216 0.065

X47 -0.003 -0.011 -0.003 0.021 0.004

Eigenvalues 1013,18 45.638 7.787 5.054 4.212

Percentage 93.613 4.264 0.719 0.466 0.389

Cumulative Percentage 93.566 97.877 98.596 99.062 99.451

The positions of all investigated populations belonging to the Turkish Vaccinium and variable that explained most of the total variation using the first two components of the PCA are given in Fig. 7. Although the first extracted components explain 93.61 % of the total variation among the examined taxa, the first three components were considered due to height Eigenvalues and those account for 93.61 %, 4.21 %, and 0.71 % of the total variation, respectively (Table 3). As seen in Table 3, the first component emphasizes that the characters contributing most to the separation of the examined taxa are leaf length (0.79), leaf width (0.43), leaf teeth numbers in 1 cm long (0.34), tetrad axis (0.14), equatorial axis (0.13) and polar axis (0.07). The second component emphasizes that both leaf teeth numbers in 1 cm long (0.76) and leaf length (0.32) and also pedicel length (0.25) contributing most to the separation of the examined taxa. Besides the third component emphasized that leaf width (0.78) and leaf length (0.33) and both tetrad axis and colpus length (0.24) contribute most to the separation of the examined taxa (Table 3).

Discussion

The genus Vaccinium was investigated worldwide by several researchers (Sleumer, 1941; Camp, 1945; Stevens 1969, 1971) based on limited morphological characters (Vander Kloet, 1988). Although Odell and Vander Kloet (1991) reported that vegetative and generative characters are valuable to distinguish members of Vaccinium, Stevens (1971) emphasized that the many floral characters like calyx shape, corolla shape, and stamen supply limited information to distinguish taxa within the genus Vaccinium. Present study showed that leaf and pollen traits supply more informative data at the species level than flower and fruit traits to separate the examined Vaccinium taxa in Turkey (Table 2, 3).

The current study also supplies several new data for the surface sculpturing and venation type of leaf and seed length which are not specified before by Stevens (1978) in the "Flora of Turkey" and any other studies regarding Vaccinium in Turkey (Tables 2, 3). Although veining type was not investigated by Stevens (1978), its taxonomic importance was emphasized in the "Flora of China" (Fang, Stevens, 2005). Moreover, Pedraza-Penalosa et al. (2013) reported that leaf venation type is not affected by environmental conditions. In the present study, it was found that the vein islets of secondary veins are 8-9 in V. arctostaphylos, 5-7 in V. myrtillus, 3-4 in

V. uliginosum, and V. vitis-idaea (Fig. 1), which are compatible with the "Flora of China" (Fang, Stevens, 2005) and Pedraza-Penalosa et al. (2013). However, Powell and Vander Kloet (1997) reported that the foliar venation does not always supply valuable data at the species level as seen in V. uliginosum and V. vitis-idaea in the present study.

Odell and Vander Kloet (1991) used vegetative characters such as surface features and leaf blade size measurements to distinguish the Vaccinium taxa. Similarly, Pedraza-Penalosa and Luteyn (2011) described seven new species based on the leaves (shape, margins, indumentum) and floral traits (merosity, calyx, corolla, pedicel) from Andean South America. The numerical analyses indicated that leaf length (X4), leaf width (X3), and leaf teeth numbers in 1 cm long (X7) are the most valuable characters for separating the investigated Vaccinium species. Semerdjieva et al. (2003) indicated that the secondary sculpture (according to Barthlott, 1981) of leaf adaxial surface and adaxial cuticle thickness varied among V. uliginosum, V. vitis-idaea, and V. myrtillus however epicuticular structures were recorded only in V. vitis-idaea and V. myrtillus. Our observations also revealed that the leaf adaxial of V. arctostaphylos (Fig. 2a) is like fiber-fluted while it is ruminate in V. myrtillus (Fig. 2b) and V. uliginosum (Fig. 2c) and pustulate in V. vitis-idaea (Fig. 2d) due to epicuticular waxes. However epidermal cell borders are hardly indistinct in V. arctostaphylos (Fig. 2a), they are slightly visible in other species (Fig. 2b-d).

Szkudlarz (1999b) reported a relationship between the genus Oxycoccus Hill (O. microcarpus Turcz. ex Rupr. and O. quadripetalus Gilib.) and V. vitis-idaea based on number of locule in fruits as previously reported by Gray (1868 cited in Szkudlarz, 1999b) and frequently placing V. vitis-idaea in a separate genus or section. Additionally, chemotaxonomic studies also indicated differences of V. vitis-idaea from the remaining Vaccinium taxa (Gugnacka-Fiedor, 1994). However, the updated taxonomic position of O. microcarpus and O. quadripetalus Gilib. (The Plant List, 2010), the variation in the chemical composition of Turkish blueberries (Erik et al., 2020) and present findings did not support the view of Gray (1868 according to Szkudlarz, 1999b) and Gugnacka-Fiedor (1994).

The seed of V. myrtillus, V. uliginosum, and V. vitis-idaea varies from lunate to reniform or falcate in outline (Fig. 3b-d) and it is triangular, tetragonal or oval in V. arctostaphylos (Fig. 3a). Contrary Szkudlarz (2001) indicated that the seed shape varies

from oval to ovate in V. myrtillus, V. uliginosum, and V. vitis-idaea. However, the seeds length is very similar with finding of Szkudlarz (2001). The seed micromorphology of V. arctostaphylos was observed for the first time, however, the seed micromorphology of the rest species was investigated by Vander Kloet (1988) and Szkudlarz (2001). According to Vander Kloet (1988), Szkudlarz (2001) and Anisimova et al. (2005), the cell walls of exotesta cells are strongly lignified, thickened and obvious in V. myrtillus, V. uliginosum, and V. vitis-idaea. Present findings mainly support the researcher's observations.

It is well known that the dispersal unit of pollen (monads or tetrads) is an important feature in several genera of Ericaceae (e. g. Erica L.) (Wronska-Pilarek et al., 2018). On this basis, there are no differences in the examined Vaccinium taxa, however, there are significant differences among the tetrads axis. The mean measurement of tetrads is in general consistent with those reported by palynologists (Sarwar et al., 2006; Sarwar, Takahashi, 2007), although higher value ranges are generally obtained for the examined features.

Palynological features of V. myrtillus, V. uli-ginosum, and V. vitis-idaea were examined in many studies (Cockerham, Galle tta, 1976; Kocon et al., 1981; Goldy et al., 1984, Sarwar et al., 2006; Sarwar, Takahashi, 2007). However, pollen features of V. arctostaphylos were documented for the first time and also detailed palynological observations were performed in detail based on multiple wild populations belonging to the rest of the examined Vaccinium taxa. The surface ornamentation of pollen grain varies from rugulate to psilate in Vaccinium (Sarwar et al., 2006). On the other hand, pollen surface is broadly fine verrucate in the Old World Vaccinium species and they have a wider variety of exine sculptures (Sarwar, Takahashi, 2007). Although pollen grains in V. myrtillus were recorded as verrucate by Kocon et al. (1981), the current study based on three populations showed that they are regulate (Fig. 5b). On the other hand, the pollen surface is verrucate in V. arctostaphylos (Fig. 5a), therefore it easily distinguishes itself from the other taxa based on the pollen surface ornamentation. Among the examined taxa, pollen grains of V. arctostaphylos have the longest tetrad axis (D: 28.5543.22 ^m) and that coincides with the palynological features of V. sect. Hemimyrtillus Sleumer (D: 30.145.0 ^m) (Sarwar, Takahashi, 2007). Tetrad axis (X37) is among the most important palynological traits to delimitate the examined taxa (Table 2, 3). Correspondingly, all studied populations belonging

to V. arctostaphylos (label b) are exclusively grouped based on the combined dataset (Fig. 6). Sarwar and Takahashi (2007) noted that the lowest value of tetrad axis presents in V. uliginosum and also observed similarities in exine. In the present study, the smallest pollen grain was found in V. uliginosum (D: 21.47-28.95 цт), nevertheless, minimum values of exine were measured in V. vitis-idaea (Ex: 1.1-1.37 ^m). Our results corroborate Sarwar et al. (2006) which consider slight differences in tetrad size and exine surface sculpture to be corresponding to the geographical distribution. Goldy et al. (1984) noted that exine sculpture may provide beneficial data on taxonomic relationships in Vaccinium. However, exine does not seem to be effective to separate Vaccinium taxa. Powell and Kron (2002), reported a relationship between V. cylindraceum Smith endemic to Azores and V. arctostaphylos based on both nuclear and chloroplast markers. However, present pollen findings of V. arctostaphylos (P: 20.58 ^m) and V. cylindraceum (P: 44.92 ^m) reported by Morgado et al. (2018) are not supporting a relationship between these species.

Stevens (1978) did not use any subgeneric classification in the treatment of Turkish Vaccinium. However, Nekrasova (1952) accepted sectional treating for the genus Vaccinium in the "Flora of the USSR". According to Nekrasova (1952), V. myrtillus and V. uliginosum belong to V. sect. Euvaccinium A. Gray, V. vitis-idaea, and V. arctostaphylos belong to V. sect. Vitis-idaea (Moench) Koch. However, present findings do not support treating V. arc-tostaphylos under the V. sect. Vitis-idaea contrary to Nekrasova (1952). The results clearly showed that V. arctostaphylos distinguishes from the rest of the examined species with several characters (e. g. leaf adaxial surface, the seed shape and epidermal cells of the seed) and it should be treated under the V. sect. Hemimyrtillus as stated by Powell and Kron (2002). Gailite et al. (2020) reported a close relationship between V. myrtillus and V. uliginosum based on genetic and geographic distribution. A similar relationship was reported for V. uliginosum and V. vitis-idaea by Vander Kloet and Avery (2010) based on phenological characters such as timing of flowering and fruiting, the number of plump seeds per berry. The present findings (Figs. 6-7) indicated that V. uliginosum and V. vitis-idaea are closely related taxa as stated by Vander Kloet and Avery (2010) and Bjedov et al. (2021). According to Powell and Kron (2002), molecular phylogeny, however, showed that V. myrtillus was neither closely related to V. vitis-idaea in Circumboreal Region.

Acknowledgements Center, Recep Tayyip Erdogan University, Rize,

We kindly thank Dr. Murat §irin (Central Turkey) for his technical assistance during the SEM Research Laboratory Application and Research studies.

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