ACTA BOT. CROAT. 82 (1), 2023 1 Acta Bot. Croat. 82 (1), 1–9, 2023 CODEN: ABCRA 25 DOI: 10.37427/botcro-2022-025 ISSN 0365-0588 eISSN 1847-8476 Taxonomic importance of leaf anatomical characters for the genus Alopecurus L. (Poaceae) Sinem Günaydın1, Candan Aykurt2* 1 Akdeniz University, Graduate School of Applied and Natural Sciences, Department of Biology, 07058, Antalya, Turkey 2 Akdeniz University, Faculty of Science, Department of Biology, 07058, Antalya, Turkey Abstract – Tiller leaf anatomical features of nine Alopecurus species collected from high mountainous areas in Turkey were evaluated in the present study. Detailed leaf anatomical descriptions of the studied taxa and an identification key generated using the anatomical features are given. Clustering and principal coordinate analysis (PCA) methods based on a total of 14 leaf anatomical characters were applied. Anatomical differences among species were detected and ana- tomical characters of taxonomic interest were identified in this genus. With clustering analyses, a unweighted pair group method with arithmetic mean (UPGMA) tree was obtained to show the relationship between the species stud- ied. Based on the results of PCA, the arrangement of adaxial sclerenchyma cells, the number of adaxial ribs, the num- ber of abaxial sclerenchyma strands, the size of epidermal cells and the number of vascular bundles are designated as the most reliable characters to separate the species. Keywords: Alopecurus, leaf anatomy, numerical taxonomy, systematics Introduction The Poaceae Barnhart is one of the most diverse plant families, represented by about 12,000 species and 780 gen- era worldwide (Clayton and Renvoize 1986, Kellogg 2015, Christenhusz and Byng 2016, Soreng et al. 2017). In Turkey, Poaceae consists 658 species and infraspecific taxa within 146 genera (Cabi and Doğan 2012). Alopecurus L. (the fox- tail genus) is a genus of the subtribe Alopecurinae with about 50 species all around the world, including many al- pine species (Doğan 1988), the centre of diversity of the ge- nus being in southwest Asia (Boudko 2014). According to Cabi and Doğan (2012) and Cabi et al. (2017), the genus Alopecurus is represented by 27 taxa, seven of which are en- demic to Turkey. Turkish Alopecurus species occupy a wide variety of habitats ranging in elevation from sea level to high mountain steppes (Doğan 1985). The grass family has highly specialized and reduced flowers and fine morphological distinctions are often essen- tial to define differences among taxa (Ellis 1976, 1986). Therefore, Ellis (1976) indicated that anatomical data are regarded as being of undoubted importance in the jigsaw of complete systematic evidence. Leaf anatomical characters in cross-section have been the main supplementary tools to add to the morphological features for characterizing some genera of difficult taxonomy within the grass family (e.g., Ellis 1976, 1986, López and Devesa 1991, Martínez-Sagarra et al. 2017, Aykurt et al. 2022) such as Festuca L. (e.g., Martínez-Sagarra et al. 2017). According to Doğan (1985), the Alopecurus taxa found in Turkey were classified under four sections: Sect. Alopecurus, Sect. Colobachne P.Beauv., Sect. Pseudophalaris Tzvelev., and Sect. Tozzettia (Savi) Endl. Then, as a result of the numerical taxonomy of the genus, Alopecurus was divided into three different sections: Alopecurus, Alopecurium Dumort. and Colobachne (Doğan 1997). According to Doğan (1985, 1997) the most diverse section in Turkey is Colobachne, which contains a total of 10 species if the re- cently described A. goekyigitiana Cabi & Soreng is counted (Cabi et al. 2017). All of the species in this section are moun- tainous caespitose perennials that grow in the subalpine or alpine zone (Doğan 1985, 1999, Cabi et al. 2017) with ovoid panicles and branches bearing 1–6 spikelets (Doğan 1985, Boudko 2014). The glumes gradually attenuate into long points (Doğan 1985) and are longer than the lemma. The pa- lea of taxa in the Sect. Colobachne is generally present and rarely absent (Boudko 2014). The Turkish species included in this section, except for three species, which are A. anatolicus Doğan, A. glacialis K.Koch and A. laguroides Balansa, were * Corresponding author e-mail: candan@akdeniz.edu.tr GÜNAYDIN S., AYKURT C. 2 ACTA BOT. CROAT. 82 (1), 2023 evaluated within the scope of our study. In addition, A. aequalis Sobol. and A. arundinaceus Poir. were included in the study, because they also occur in high mountain areas. They are included in the sections Alopecurium and Alopecurus, respectively. The sections Alopecurium and Alopecurus consist of both annual and perennial species and the palea of the species is absent (Doğan 1985, Boudko 2014). The panicles are cylindrical to oblong and with 1–5 spikelets in the section Alopecurium. The glumes are acute to obtuse and are longer than or equal to the lemma. The section Alopecurus has ovoid to cylindrical panicles with each branch bearing 1–10 spikelets. Glumes are acute, equal to or longer than the lemma and connate in the lower half (Boudko 2014). Although numerous studies conducted on the morphology of the genus Alopecurus can be found (e.g., Doğan 1997, 1999, Soreng et al. 2007, Boudko 2014), there are limited studies conducted on the importance of leaf an- atomical characters for Alopecurus species in the taxonomy of this genus. In recent years, studies evaluating the phylogenetic re- lationships between the members of this genus have been conducted, and infrageneric and interspecific relationships have been interpreted with the use of both nuclear (ITS and ETS) and plastid markers (trnTLF, rpoB-trnC and matK) (e.g., Soreng et al. 2007, Boudko 2014, Cabi et al. 2017). As a result of the plastid (TF+rpoB+matK) and combined (ETS+ITS+TF+rpoB+matK) maximum parsimony analy- ses made by Boudko (2014), it was shown that the infrage- neric classification of the genus accepted by Doğan (1999) (sect. Alopecurus incl. A. myosuroides, sect. Alopecurium and sect. Colobachne) is not monophyletic. Phylogenetic studies involving more species are very important in order to resolve the phylogenetic relationships in both infrage- neric and interspecific classification of the genus Alopecurus (Boudko 2014). A large number of specimens belonging to nine Alopecurus species were collected in the high mountainous zone of the Western Taurus (Antalya, Turkey) during our project on the diversity of the Poaceae, and their leaf anatomy (in cross-section view) was evaluated. There are some difficulties during the identification and separation of some Alopecurus species with the use of morphological characters. This study aimed to (i) determine the leaf anatomical features in the Alopecurus species studied; (ii) determine the anatomical characters useful in identification of the species of the genus; (iii) evaluate the relationship between the taxa using the anatomical data. Materials and methods The tiller leaf anatomical features of mountain Alopecurus species that occur in subalpine and alpine areas in Turkey were evaluated in the present study. We investigated a total of nine Alopecurus species, seven of which are included in the Sect. Colobachne, which are A. aucheri Boiss., A. davisii Bor., A. gerardii (All.) Vill., A. goekyigitiana, A. lanatus Sm., A. textilis Boiss. and A. vaginatus (Willd.) Pall. ex Kunth.  Alopecurus goekyigitiana and A. lanatus, endemic to Turkey. The other two species investigated, A. aequalis and A. arundinaceus, are common foxtails in different hab- itats including high mountainous regions. Most of the plant specimens examined were collected from natural popula- tions during field trips between the years 2018 and 2020. We used herbarium materials deposited in AKDU and the col- lection data of the taxa used for our analyses are presented in Tab. 1. A total of 45 individuals, three individuals from Tab. 1. Studied Alopecurus taxa and collection information of the specimens. OTU – operational taxonomic unit OTU Species Locality data of specimens studied OTU1 A. aequalis Isparta, Gölcük Nature Park, 1390 m, 10.07.2013, C. Aykurt 3397. OTU2 A. arundinaceus Antalya, Kaş, Gömbe, Ak Mountain, around Yeşilgöl, 1825 m, 29.07.2018, C. Aykurt 4779. Antalya, Gündoğmuş, between Eğrigöl and Söbüçimen Plateau, 2087 m, 20.06.2019, C. Aykurt 5345. OTU3 A. vaginatus Antalya, Serik, Bozburun Mountain, 1855 m, 24.05.2019, C. Aykurt 4940a. Antalya, Alanya, Ak Mountain, 1985 m, 18.06.2019, C. Aykurt 5245. OTU4 A. textilis subsp. textilis Antalya, Akseki, Gidengelmez Mountains, 1950 m, 13.06.2012, C. Aykurt 3224. Antalya, Kumluca, Sarıkaya Wildlife Development Area, Bey Mountains, 2754 m, 28.06.2021, SWDA 97-1-12. OTU5 A. gerardii var. gerardii Antalya, Alanya, Başyayla environs, 1579 m, 17.06.2019, C. Aykurt 5153. Antalya, Alanya, Ak Mountain, 2044 m, 16.07.2019, C. Aykurt 5514. OTU6 A. goekyigitiana Antalya, Gündoğmuş, Eğrigöl to Hadim, 2205 m, 20.06.2019, C. Aykurt 5327. Antalya, Gündoğmuş, between Eğrigöl and Söbüçimen Plateau, 2087 m, 20.06.2019, C. Aykurt 5344. OTU7 A. aucheri Bitlis, Tatvan, Nemrut Mountain, 2480 m, 28.05.2019, L.Y. Konuralp. OTU8 A. davisii Izmir: Kemalpaşa, Mahmut Mountain, 1250-1368 m, 28.04.1992, A. Aksoy 744. OTU9 A. lanatus Antalya, Kumluca, Sarıkaya Wildlife Development Area, Bey Mountains, 2754 m, 28.06.2021, SWDA 97-1-11. Antalya, İbradı, Toka Plateau, 1514 m, 24.06.2020, C. Aykurt 5624. LEAF ANATOMY OF ALOPECURUS SPECIES IN TURKEY ACTA BOT. CROAT. 82 (1), 2023 3 each location, belonging to nine species were used for the anatomical measurements carried out within the scope of the study. At least three tiller leaves from each individual were analyzed. Short pieces of tiller leaves taken from herbarium mate- rials were kept in distilled water for approx. five minutes. Then, cross-sections ± 0.05 mm thick were cut by free hand from pieces of leaf fixed in styrofoam. This was done under a stereomicroscope using reflected light and the sections were stained in a drop of water with toluidine blue. After 1–3 minutes, depending on stainability, the sections were washed in distilled water and studied under a light micro- scope at a magnification of 10–40 × (general anatomical pattern) or 100 × (detailed shape of the epidermal and bun- dle sheath cells). The leaf preparations were analyzed in detail and ana- tomical features were determined for each taxon. As a result, 14 anatomical characters that can be used for the genus Alopecurus were scored for each taxon and included in the numerical analysis. In this study, two different numerical analysis methods were used: clustering (UPGMA) and prin- cipal coordinate analysis (PCA) with the use of PAST Ver- sion 4.03 computer software. The anatomical characters de- termined for this study and evaluation of these characters for each taxon are presented in Tab. 2. In addition, the scat- ter biplot diagram of the studied Alopecurus species as op- erational taxonomic units (OTUs) based on the leaf ana- tomical characters determined was created. Ellis (1976, 1979) was used as the main source for choice of leaf ana- tomical characters. Results The results of our study are presented in two parts, in which the general leaf anatomical features and anatomical characters of Alopecurus taxa are evaluated taxonomically with numerical analyses. The tiller leaf anatomical characters determined within the scope of the study are generally related to the epidermis, sclerenchyma strands and girders, both abaxial and adaxi- al ribs, and vascular bundles. The cross-sections taken from the tiller leaf blades of all species studied are shown in Fig. 1. The first anatomical character was determined as the shape of the leaves in cross-section. Almost all species of Sect. Colobachne included in the study have a U-shaped til- ler leaf cross-section, except for A. vaginatus, which has a U- to O-shaped cross-section. In contrast, leaf cross-sec- tions of A. aequalis (sect. Alopecurium) and A. arundinaceus (sect. Alopecurus) are flat. Characteristics determined in re- lation to the epidermis are the size of epidermal cells (C13), the size of bulliform cells (C14), papillae on the epidermal cells (C3) and density of macro-hairs (C7). The epidermal cells of all Alopecurus species studied are single-layered and generally polygonal in shape. The size of epidermal cells was categorized under three different groups according to their cell length. Accordingly, the epidermal cells of the species included in sect. Colobachne are quite different in size. In addition, bulliform cells are prominent in all species stud- ied except A. aequalis. Tab. 2. Tiller leaf anatomical characters for the genus Alopecurus and their scorings determined within the scope of the study and used in the numerical analyses. Character Description C1 The shape of the tiller leaves in cross-section: flat (0) / U- to O-shaped (1) C2 The midrib larger than the laterals (1) / not larger (0) C3 Papillae on the epidermal cells: Outer walls of epidermal cells arched but not papillose (0) / Entire or major part of epidermis composed of thin-walled wide papillae (1) / Thin-walled wide papillae scattered throughout the epidermis (2) C4 Arrangement of adaxial sclerenchyma strands as: scattered few groups (0) / regular groups at the level of the vascular bundles (1) / extended along the lobes (2) / T-shaped (3) C5 The number of abaxial sclerenchyma strands: 5–14 (0) / 17–21 (1) / 24–28 (2) C6 Depth of adaxial furrows in comparison to the leaf thickness: Medium furrows; quarter to one half the leaf thickness (0) / Deep furrows; more than one half the leaf thickness (1) C7 Density of macro hairs: few and scattered (0) / dense (1) C8 Abaxial ribs: absent (0) / slightly lobed (1) / distinct 14–21 (2) C9 The number of adaxial ribs: 6–9 (0) / 12–16 (1) / 17–20 (2) C10 Abaxial sclerenchyma strands as: very small or small strands (0) / big strands like a cap (1) / with well-developed girder (2) C11 The number of vascular bundles: 6–10 (0) / 11–18(1) C12 The connection of midrib with epidermal layer: midrib not connected (0) / midrib connected with abaxial side (1) / midrib connected to both sides (2) C13 The size of epidermal cells: up to 12.5 µm long (0) / up to 17.99 µm long (1) / up to 24 µm long (2) C14 The size of bulliform cells: not distinct (0) / 16–26 µm (1) / 35–45 µm (2) GÜNAYDIN S., AYKURT C. 4 ACTA BOT. CROAT. 82 (1), 2023 Fig. 1. Tiller leaf cross-sections of Alopecurus taxa studied. a – A. aequalis (from C. Aykurt 3397), b – A. arundinaceus (from C. Aykurt 5345), c – A. vaginatus (from C. Aykurt 4940a); d – A. textilis subsp. textilis (from C. Aykurt 3224), e – A. gerardii var. gerardii (from C. Aykurt 5514), f – A. goekyigitiana (from C. Aykurt 5327), g – A. aucheri (from L.Y. Konuralp); h – A. davisii (from A. Aksoy 744), i – A. lanatus (from C. Aykurt 5624). Scale bars: 50 µm. In the species studied, the arrangement of adaxial scle- renchyma strands (C4) can be scattered as few groups, reg- ular groups at the level of the vascular bundles, extended along the lobes or T-shaped. The adaxial sclerenchyma strands are: regular groups at the level of the vascular bun- dles in A. aequalis and A. arundinaceus; a few scattered groups in A. vaginatus, A. gerardii and A. goekyigitana; and extended along the lobes in A. aucheri and A. textilis. Only in A. davisii are the adaxial sclerenchyma strands T-shaped. The abaxial sclerenchyma strands (C 10) are: very small or small strands; big strands like a cap; or with well-developed girder. Abaxial sclerenchyma strands with well-developed girders appear only in A. arundinaceous. In A. textilis and A. aucheri, the abaxial strands are big and resemble a cap. The rest of the species studied have very small or small ab- axial sclerenchyma strands. Based on the PCA results, the variance value of the first two components is 59.58%, and the variance value of the first four components is 91.06% (Fig. 2.). The eigenvalue and the percentage of eigenvalue of the components, and the ei- gen vector value of the components are given in the Appen- dices, respectively. According to the results of the numerical analysis PC1, the first five most reliable characters are C4 (arrangement of adaxial sclerenchyma strands), C9 (the number of adaxial ribs), C5 (The number of abaxial scleren- chyma strands), C13 (the size of epidermal cells) and C11 (the number of vascular bundles), respectively; based on PC2, it is seen that they are C12 (the connection of midrib with epidermal layer), C10 (the number adaxial ribs), C14 (the size of bulliform cells), C3 (Papillae on the epidermal cells) and C2 (the ratio of midrib size to the laterals). The obtained UPGMA dendrogram shows that A. aequalis (Sect. Alopecurium) is separated from all other spe- cies studied (Fig. 3). Alopecurus arundinaceus is located close to the cluster of the Sect. Colobachne species. Alopecurus Sect. Colobachne species are grouped together into two main branches. Alopecurus vaginatus, A. gerardii and A. goekyigitiana are grouped together and close to A. textilis (first group), whereas A. lanatus and A. davisii are grouped in a different branch close to A. aucheri (second group). The anatomical characters most useful for separat- ing these two species groups are C9 (the number of adaxial ribs) and C11 (the number of vascular bundles). The num- ber of adaxial ribs is between 6–9 in the first group, where- LEAF ANATOMY OF ALOPECURUS SPECIES IN TURKEY ACTA BOT. CROAT. 82 (1), 2023 5 as they are between 12–20 in the second group. The adaxial ribs are between 17–20 in Alopecurus aucheri and are be- tween 12–16 in A. davisii and A. lanatus. While the number of vascular bundles varies between 6–10 in the first group; in the second group, it varies between 11–18. Detailed eval- uations and measurements of the species studied are also given in Tab. 3. The abaxial ribs (C8) are very prominent in only two of the species studied, which are A. textilis and A. aucheri. The abaxial ribs are slightly lobed in A. aequalis and are absent in the other species studied. The number of vascular bun- dles was evaluated under two categories, and this character is very useful to separate the species groups in the sect. Colobachne. Fig. 2. Principal component analysis (PCA) biplot scatter diagram of studied Alopecurus species as operational taxonomic unit (OTUs) along PC1 and PC2 axes based on 14 leaf anatomical characters described in Tab 2. Fig. 3. Unweighted pair group method with arithmetic mean (UPGMA) dendrogram created according to the Gower similarity index by using anatomical character matrix of Alopecurus species studied (see Tab. 2). GÜNAYDIN S., AYKURT C. 6 ACTA BOT. CROAT. 82 (1), 2023 Ta b. 3 . E va lu at io n, m ea su re m en ts a nd sc or in g of th e c ha ra ct er s u se d in n um er ic al a na ly se s a cc or di ng to th e t ill er le af a na to m ic al fe at ur es o f A lo pe cu ru s s pe ci es st ud ie d as d es cr ib ed in T ab . 2 . Le af a na to m ic al ch ar ac te rs A . a eq ua lis A . a ru nd in ac eu s A . v ag in at us A . t ex til is A . g er ar di i A . g oe ky ig iti an a A . a uc he ri A . d av isi i A . l an at us Th e s ha pe o f t he ti lle r l ea ve s in cr os s- se ct io n (C 1) Fl at (0 ) Fl at (0 ) U - t o O -s ha pe d (1 ) U -s ha pe d (1 ) U -s ha pe d (1 ) U -s ha pe d (1 ) U -s ha pe d (1 ) U -s ha pe d (1 ) U -s ha pe d (1 ) Th e m id rib la rg er th an th e la te ra ls or n ot (C 2) N ot la rg er th an th e la te ra ls (0 ) La rg er th an th e la te ra ls (1 ) La rg er th an th e la te ra ls (1 ) La rg er th an th e la te ra ls (1 ) La rg er th an th e la te ra ls (1 ) La rg er th an th e la te ra ls (1 ) N ot la rg er th an th e l at er al s (0 ) N ot la rg er th an th e l at er al s (0 ) N ot la rg er th an th e l at er al s (0 ) Pa pi lla e o n th e e pi de rm al ce lls (C 3) Th in -w al le d w id e pa pi lla e s ca tte re d th ro ug ho ut th e ep id er m is (2 ) O ut er w al ls of ep id er m al ce lls ar ch ed b ut n ot pa pi llo se (0 ) En tir e o r m aj or pa rt o f e pi de rm is co m po se d of th in -w al le d w id e pa pi lla e ( 1) O ut er w al ls of ep id er m al ce lls ar ch ed b ut n ot pa pi llo se (0 ) En tir e o r m aj or pa rt o f e pi de rm is co m po se d of th in -w al le d w id e pa pi lla e ( 1) O ut er w al ls of ep id er m al ce lls ar ch ed b ut n ot pa pi llo se (0 ) O ut er w al ls of ep id er m al ce lls ar ch ed b ut n ot pa pi llo se (0 ) O ut er w al ls of ep id er m al ce lls ar ch ed b ut n ot pa pi llo se (0 ) O ut er w al ls of ep id er m al ce lls ar ch ed b ut n ot pa pi llo se (0 ) A rr an ge m en t o f a da xi al sc le re nc hy m a st ra nd s (C 4) Re gu la r g ro up s a t th e l ev el o f t he va sc ul ar b un dl es (1 ) Re gu la r g ro up s a t th e l ev el o f t he va sc ul ar b un dl es (1 ) Sc at te re d fe w gr ou ps (0 ) Ex te nd ed a lo ng th e l ob es (2 ) Sc at te re d fe w gr ou ps (0 ) Sc at te re d fe w gr ou ps (0 ) Ex te nd ed a lo ng th e l ob es (2 ) T- sh ap ed (3 ) Ex te nd ed a lo ng th e l ob es (2 ) Th e n um be r o f a ba xi al sc le re nc hy m a st ra nd s ( C 5) 24 -2 6 (2 ) 24 -2 8 (2 ) 5– 7 (0 ) 17 –1 8 (1 ) 9– 11 (0 ) 12 –1 4 (0 ) 17 –2 1 (1 ) 17 –1 8 (1 ) 17 –1 8 (1 ) D ep th o f a da xi al fu rr ow s i n co m pa ris on to th e l ea f th ic kn es s ( C 6) D ee p fu rr ow s (1 ) M ed iu m fu rr ow s (0 ) M ed iu m fu rr ow s (0 ) M ed iu m fu rr ow s (0 ) M ed iu m fu rr ow s (0 ) M ed iu m fu rr ow s (0 ) M ed iu m fu rr ow s (0 ) M ed iu m fu rr ow s (0 ) M ed iu m fu rr ow s (0 ) D en sit y of m ac ro -h ai rs (C 7) Fe w a nd sc at te re d (0 ) Fe w a nd sc at te re d (0 ) Fe w a nd sc at te re d (0 ) Fe w a nd sc at te re d (0 ) Fe w a nd sc at te re d (0 ) Fe w a nd sc at te re d (0 ) Fe w a nd sc at te re d (0 ) D en se (1 ) D en se (1 ) Th e n um be r o f a ba xi al ri bs (C 8) Sl ig ht ly lo be d (1 ) A bs en t (0 ) A bs en t (0 ) 16 –1 9 (2 ) A bs en t (0 ) A bs en t (0 ) 14 -2 1 (2 ) A bs en t (0 ) A bs en t (0 ) Th e n um be r o f a da xi al ri bs (C 9) 15 –2 0 (2 ) 15 –1 6 (1 ) 6– 8 (0 ) 7– 9 (0 ) 6– 7 (0 ) 6– 7 (0 ) 11 –2 0 (2 ) 13 –1 5 (1 ) 12 –1 3 (1 ) A ba xi al sc le re nc hy m a st ra nd s ( C 10 ) Ve ry sm al l o r s m al l st ra nd s ( 0) W ith w el l-d ev el - op ed g ird er (2 ) Ve ry sm al l o r sm al l s tr an ds (0 ) Bi g st ra nd s l ik e a ca p (1 ) Ve ry sm al l o r sm al l s tr an ds (0 ) Ve ry sm al l o r sm al l s tr an ds (0 ) Bi g st ra nd s l ik e a ca p (1 ) Ve ry sm al l o r sm al l s tr an ds (0 ) Ve ry sm al l o r sm al l s tr an ds (0 ) Th e n um be r o f v as cu la r bu nd le s ( C 11 ) 12 –2 0 (1 ) 17 –2 0( 1) 7– 8 (0 ) 7– 10 (0 ) 6– 7 (0 ) 7– 8 (0 ) 12 –1 4 (1 ) 15 –1 7 (1 ) 14 –1 8 (1 ) Th e c on ne ct io n of m id rib w ith ep id er m al la ye rs (C 12 ) M id rib n ot co nn ec te d (0 ) M id rib co n - ne ct ed to b ot h sid es (2 ) M id rib n ot co nn ec te d (0 ) M id rib co n - ne ct ed w ith a ba x- ia l s id e (1 ) M id rib n ot co nn ec te d (0 ) M id rib n ot co nn ec te d (0 ) M id rib n ot co nn ec te d (0 ) M id rib n ot co nn ec te d (0 ) M id rib n ot co nn ec te d (0 ) Th e s iz e o f e pi de rm al ce lls (µ m × µ m ) ( C 13 ) 11 .2 4– 14 .7 9 × 8. 57 –8 .8 7 (1 ) 15 .3 1– 20 .3 3 × 8. 67 –1 0. 11 (2 ) 9. 36 –1 7. 57 × 7. 57 –1 2. 42 (1 ) 10 .1 1– 11 .9 0× 5. 65 –8 .3 3 (0 ) 11 .5 1– 17 .8 7 × 7. 87 –9 .3 9 (0 ) 10 .6 2– 16 .3 2 × 3. 88 –6 .9 9 (0 ) 17 .5 1– 24 .2 0 × 8. 91 –1 0. 50 (2 ) 13 .2 2– 17 .3 5 × 6. 33 –9 .0 9 (1 ) 10 .0 8– 12 .3 5 × 7. 05 –8 .2 3 (0 ) Th e s iz e o f b ul lif or m ce lls (µ m × µ m ) ( C 14 ) N ot d ist in ct (0 ) 40 .4 6– 44 .7 9 × 15 .0 2– 26 .5 8 (2 ) 19 .0 9– 20 .9 0 × 8. 48 –1 0. 28 (1 ) 16 .0 7– 22 .6 × 9. 31 –1 6. 45 (1 ) 16 .3 6– 19 .3 9 × 12 .1 2– 15 .7 5 (1 ) 16 .4 8– 23 .6 7 × 12 .3 9– 17 .2 8 (1 ) 20 .2 6– 26 .3 6 × 11 .5 1– 13 .9 3 (1 ) 32 .5 0– 35 .2 6 × 17 .9 0– 2 4. 51 (2 ) 22 .9 4– 25 .8 8 × 9. 41 –1 7. 64 (1 ) LEAF ANATOMY OF ALOPECURUS SPECIES IN TURKEY ACTA BOT. CROAT. 82 (1), 2023 7 The depth of adaxial furrows is more than one half of the leaf thickness (C6), and thin-walled wide papillae are easily visible as scattered throughout the epidermis (C3) in A. aequalis. With these characters, A. aequalis can be eas- ily distinguished from other species. Alopecurus arundinaceus is the only species having a midrib connected to both sides (C12). The sclerenchyma bands around the mature vascular bundles of this species are elongated to both abaxial and adaxial surfaces. Results from the PCA analysis showed that the least re- liable characters are C6 (Depth of adaxial furrows in com- parison to the leaf thickness) and C7 (Density of macro- hairs) according to PC1. Identification key for Alopecurus taxa studied 1. Leaves flat in cross-section . . . . . . . . . . . . . . . . . . . . . . . . 2 1. Leaves U- or O-shaped in cross-section . . . . . . . . . . . . 3 2. Abaxial and adaxial surfaces connected with sclerenchy- ma cells; depth of adaxial furrows quarter to one half the leaf thickness; outer walls of epidermal cells arched but ot papillose . . . . . . . . . . . . . . . . . . . . . . . . A. arundinaceus 2. Abaxial and adaxial surfaces not connected with scleren- chyma cells; depth of adaxial furrows more than one half the leaf thickness; thin-walled wide papillae scattered throughout the epidermis . . . . . . . . . . . . . . . A. aequalis 3. The number of vascular bundles 6–10 . . . . . . . . . . . . . . 4 3. The number of vascular bundles 11–18 . . . . . . . . . . . . . 7 4. Adaxial sclerenchyma strands scattered in several groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4. Adaxial sclerenchyma strands extended along the lobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. textilis 5. Outer walls of epidermal cells arched but not papillose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. goekyigitiana 5. Entire or major part of epidermis composed of thin- walled wide papillae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6. Epidermal cells up to 12.5 µm long . . . . . . . . A. gerardii 6. Epidermal cells up to 17.99 µm long . . . . . . A. vaginatus 7. Abaxial ribs distinct . . . . . . . . . . . . . . . . . . . . . A. aucheri 7. Abaxial ribs absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8. Adaxial sclerenchyma bands T-shaped; epidermal cells up to 17.99 µm long . . . . . . . . . . . . . . . . . . . . . . . A. davisii 8. Adaxial sclerenchyma bands not T-shaped; epidermal cells up to 12.5 µm long . . . . . . . . . . . . . . . . . . A. lanatus Discussion We focused on the tiller leaf anatomical characters of the high mountain Alopecurus species in Turkey in the pres- ent study. All Alopecurus species adapted to subalpine and alpine mountainous areas in Turkey are caespitose peren- nials classified in the Sect. Colobachne (Doğan 1985, 1997, 1999, Cabi et. al. 2017). The tiller leaves of these morpho- logically similar species are filiform, narrowly lanceolate and generally convolute. The mountain Alopecurus species studied exhibit leaf anatomical characters similar to those of high mountain Festuca species. Abaxial sclerenchyma patterns, number of vascular bundles and number of ribs are among the most distinctive morphological characters for Festuca (Martínez-Sagarra et al. 2017). Considering the results obtained from our study, it is seen that these char- acters are similar to the most important characters in PC1 in the high mountain species of the genus Alopecurus (C4, C9, C5 and C8). The anatomical characters analyzed within the scope of our study can be easily observed in leaf cross-sections that can be taken from herbarium materials. Since dry samples were used, some distinguishing anatomical characters spec- ified by Ellis (1976, 1979) could not be used, such as vascular bundle, vascular bundle sheath, and leaf mesophyll (chlor- enchyma and colourless parenchyma cells) in the present study. The results obtained showed that especially the num- ber of both adaxial and abaxial ribs, and features of scle- renchyma strands such as number, arrangement and gird- ers are very important and useful anatomical characters. Metcalfe (1960) stated that the ribs are generally character- istic of and more fully developed on the adaxial than the abaxial surface. Adaxial ribs are distinct in all of the species evaluated within the scope of the study, while abaxial ribs are distinct only in A. textilis included in the first group and A. aucheri included in the second group. According to Ellis (1976), ribs and furrows may vary in depth, transverse shape, spacing and location in different species. In A. aequalis the adaxial furrows are distinct and much deeper than in the other species studied, which have medium fur- rows. In the PCA results obtained, the length of epidermal cells is one of the most significant anatomical characters. Alopecurus textilis, A. gerardii and A. goekyigitiana have the smallest epidermal cells, in contrast to A. arundinaceus and A. aucheri, which have the biggest epidermal cells. The least significant leaf anatomical characters accord- ing to the PC1 results are depth of adaxial furrows (C6), density of macro hairs (C7), size of bulliform cells (C14) and papillae on the epidermal cells (C3), respectively. We evalu- ated C7 as scattered and dense; accordingly, A. davisii and A. lanatus, which have densely hairy leaves, were scored differently from the rest of the species studied. In the den- drogram obtained, the relationships among the taxa are similar to the results obtained previously by different re- searchers, from both morphological and phylogenetic stud- ies (e.g., Doğan 1997, 1999, Cabi et al. 2017). This is an in- dication that anatomical characters are useful for the taxonomy of the genus Alopecurus. Alopecurus aequalis was separated into a different branch from all other species. Among the studied species, A. aequalis, which is an annu- al and grows in damp places and marshy habitats, has dis- tinct anatomical differences from the others in terms of the characters C3, C6, C8 and C14. Although A. arundinaceus is close to other species studied, it is separated into a differ- ent branch. It differs markedly from the other species stud- ied in that the midrib is connected to both the abaxial and adaxial surfaces via sclerenchyma cells, and its leaves are flat GÜNAYDIN S., AYKURT C. 8 ACTA BOT. CROAT. 82 (1), 2023 in cross-section. Doğan (1985) stated that A. arundinaceus is rare in South Anatolia. However, we observed during the field studies that this species was widespread in the high mountainous areas of southern Anatolia. The section Colobachne is divided into two groups in the dendrogram according to the number of adaxial ribs (C9), the number of vascular bundles (C11) and the size of midrib in comparison to the laterals (C2). In the first group composed of A. vaginatus, A. gerardii, A. goekyigitiana and A. textilis, the number of vascular bundles is between 6–10, the number of adaxial ribs is between 6–9 and the midvein is larger than the laterals. In the second group, which in- cludes A. aucheri, A. davisii and A. lanatus, the number of vascular bundles is between 11–18, the number of adaxial ribs is between 12–20 and the midvein is not larger than the laterals. Alopecurus goekyigitiana as described by Cabi et al. (2017) was evaluated as closely related to A. gerardii and A. vaginatus. According to our results, it is similar to these spe- cies in terms of its leaf anatomical features. Unlike these species, however, the outer walls of the epidermal cells in A. goekyigitiana are arched but not papillose (C3). The entire or major part of the epidermis in A. gerardii and A. vaginatus is composed of thin-walled wide papillae. It was also noted by Codignola et al. (1987) that A. gerardii has a bulbous epidermis. When evaluated in terms of this character (C3), A. goekyigitiana resembles A. lanatus and A. davisii. Alopecurus davisii and A. lanatus, indicated as closely re- lated species by Doğan (1985), have some differences in terms of their leaf anatomical characters such as C4, C13 and C14 (Tab. 3). The most distinctive anatomical feature that can be used to distinguish between these two species is the arrangement of the adaxial sclerenchyma strands (C4); the adaxial sclerenchyma bands are T-shaped in A. davisii, whereas the sclerenchyma strands are extended along the lobes in A. lanatus. Among the studied species, only A. aucheri and A. textilis have prominent abaxial ribs. Although these two species are similar in many anatomical characters, they differ in the most reliable characters, C9 and C13. This has caused these species to be included in separate groups. The results obtained show that the leaf anatomical char- acters we determined within the scope of the study are very useful in grouping close species and separating species from each other for the genus Alopecurus. The tiller leaf anatom- ical features of the species groups and of the species should be clarified by further analysis of the anatomical features by using fresh materials for this genus. 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