Introduction
The Poaceae Barnhart is one of the most diverse plant families, represented by about 12,000 species and 780 genera 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 foxtail genus) is a genus of the subtribe Alopecurinae with about 50 species all around the world, including many alpine species (Doğan 1988), the centre of diversity of the genus 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 endemic 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 essential 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 taxafound 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 recently described A. goekyigitiana Cabi & Soreng is counted (Cabi et al. 2017). All of the species in this section are mountainous 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 palea 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 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 anatomical characters for Alopecurus species in the taxonomy of this genus.
In recent years, studies evaluating the phylogenetic relationships 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 analyses made by Boudko (2014), it was shown that the infrageneric 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 infrageneric 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.
Material 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 habitats including high mountainous regions. Most of the plant specimens examined were collected from natural populations during field trips between the years 2018 and 2020. We used herbarium materials deposited in AKDU and the collection data of the taxa used for our analyses are presented inTab. 1. A total of 45 individuals, three individuals from 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 materials 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 microscope at a magnification of 10–40 × (general anatomical pattern) or 100 × (detailed shape of the epidermal and bundle sheath cells).
The leaf preparations were analyzed in detail and anatomical 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 principal coordinate analysis (PCA) with the use of PAST Version 4.03 computer software. The anatomical characters determined for this study and evaluation of these characters for each taxon are presented inTab. 2. In addition, the scatter biplot diagram of the studied Alopecurus species as operational taxonomic units (OTUs) based on the leaf anatomical characters determined was created. Ellis (1976,1979) was used as the main source for choice of leaf anatomical 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 adaxial ribs, and vascular bundles. The cross-sections taken from the tiller leaf blades of all species studied are shown inFig. 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 tiller leaf cross-section, except for A. vaginatus, which has a U- to O-shaped cross-section. In contrast, leaf cross-sections of A. aequalis (sect. Alopecurium) and A. arundinaceus (sect. Alopecurus) are flat. Characteristics determined in relation 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 studied except A. aequalis.
In the species studied, the arrangement of adaxial sclerenchyma strands (C4) can be scattered as few groups, regular 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 bundles 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 abaxial 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 eigen vector value of the components are given in the Appendices, 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 sclerenchyma 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 species 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 separating these two species groups are C9 (the number of adaxial ribs) and C11 (the number of vascular bundles). The number of adaxial ribs is between 6–9 in the first group, whereas they are between 12–20 in the secondgroup. The adaxial ribs are between 17–20 in Alopecurus aucheri and are between 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 evaluations and measurements of the species studied are also given inTab. 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 bundles was evaluated under two categories, and this character is very useful to separate the species groups in the sect. Colobachne.
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 easily 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 reliable characters are C6 (Depth of adaxial furrows in comparison 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 sclerenchyma cells; depth of adaxial furrows quarter to one half the leaf thickness; outer walls of epidermal cells arched but not papillose……………………… A. arundinaceus
2. Abaxial and adaxial surfaces not connected with sclerenchyma 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 present study. All Alopecurus species adapted to subalpine and alpine mountainous areas in Turkey are caespitose perennials classified in the Sect. Colobachne (Doğan 1985,1997,1999,Cabi et. al. 2017). The tiller leaves of these morphologically 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 characters 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 specified by Ellis (1976,1979) could not be used, such as vascular bundle, vascular bundle sheath, and leaf mesophyll (chlorenchyma and colourless parenchyma cells) in the present study. The results obtained showed that especially the number of both adaxial and abaxial ribs, and features of sclerenchyma strands such as number, arrangement and girders are very important and useful anatomical characters. Metcalfe (1960) stated that the ribs are generally characteristic 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 furrows. 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 according 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 evaluated 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 dendrogram obtained, the relationships among the taxa are similar to the results obtained previously by different researchers, from both morphological and phylogenetic studies (e.g., Doğan1997,1999,Cabi et al. 2017). This is an indication 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 annual and grows in damp places and marshy habitats, has distinct 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 different branch. It differs markedly from the other species studied in that the midrib is connected to both the abaxial and adaxial surfaces via sclerenchyma cells, and its leaves are flat 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 includes 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 species 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 related 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 characters 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 anatomical 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. With this study, it has been shown that tiller leaf anatomical characters will contribute greatly to the systematics of the genus.