Seed micromorphology and anatomy of 36 Muscari (Asparagaceae) taxa from Turkey with notes on their systematic importance

Introduction

The genus Muscari Mill. is found across the European continent, Mediterranean region and northwest Asia (Jafari and Maassoumi 2011). According to the latest checklist study, the genus is represented by 51 species worldwide (Govaerts 2019). According to other recent studies, Muscari includes 40 species belonging to four subgenera as Muscari, Leopoldia and Botryanthus and Pseudomuscari with controversial status in Turkey, 26 of which are endemic (Dizkırıcı et al. 2019,Eker 2019a,b,Demirci-Kayıran et al. 2019). The genus is characterized by its bulbs, basal leaves, inflorescences, pedicels, flower form and colour, filament placement relative to the tube, and capsule shape (Davis and Stuart 1984). Major taxonomic problems of the genus include the many synonyms among taxa, the fact that type specimens are often cultivated material of unknown origin, that widespread taxa show a lot of variation and the color difference between fresh and dried flowers (Davis and Stuart 1984). Moreover, reliable classification is impossible in the genus because morphological characteristics and karyological information are not complete or consistent enough to make uncontroversial taxonomic judgements (Dizkırıcı et al. 2019). The genus was placed in Hyacinthaceae until the Angiosperm Phylogeny Group (APG) re-evaluated its taxonomic position as a result of subsequent studies, and placed it within the family Asparagaceae (Reveal and Chase 2011,Guner et al. 2012,Demirci and Özhatay 2017).

Several morphological, anatomical, cytological, palynological and ecological studies on taxa belonging to various genera of Asparagaceae have been performed previously (Shoub and Halevy 1971,Bentzer et al. 1974,Küçüker 1990,Uysal 1999,Herrmann et al. 2006,Lynch et al. 2006,Uysal et al. 2007,Gürsoy and Şık 2010,Kahraman et al. 2010,Doğu and Bağcı 2009,Doğu et al. 2011,Sezer et al. 2013). However, the morphological and anatomical features of seeds have been largely ignored in the systematics of taxa in the family, except in a few new species descriptions (Yıldırım 2015,2016,Doğu and Uysal 2019).

The purpose of this study is to: (i) examine the morphological and anatomical characteristics of seeds of 36 taxa in subgenera Muscari, Leopoldia, Pseudomuscari and Botryanthus of the genus Muscari in Turkey, and (ii) debate the taxonomic use of these characters. The study will also serve as a guide to further related studies on various genera in the family.

Material and methods

The plant specimens were collected from various phytogeographical regions of Turkey during the fruiting season and were deposited at VANF (Van Yüzüncü Yıl University Herbarium). Details are provided inTab. 1.

Macromorphological features of the seeds including colour, shape and size were documented for 100 seeds of 10 individuals per species utilizing a Leica EZ4 binocular microscope with a HD camera (On-line Suppl. Fig. 1,Tab. 2). For the micromorphological features of surface ornamentation, anticlinal and periclinal cell walls, and the form of epidermal cells, the samples were studied with a Scanning Electron Microscope (On-line Suppl. Fig. 2,Tab. 3). Seeds were first placed on the stub with silver epoxy and coated with gold, then examined with a Zeiss LEO 440 SEM.

A survey of seed anatomical characters was done with dry herbarium materials. Cross-sections were taken from the middle of the seed with a fully automatic microtome (Thermo Shonda Met Finesse, Thermo). They were brought through a series of alcohol and xylene, dyed with hematoxylin and eosin-Y in a staining device (ASC 720 Medite) and mounted using Entellan (On-line Suppl. Fig. 3,Tab. 4) (Karaismailoğlu 2015,Karaismailoğlu and Erol 2018,Karaismailoğlu and Güner 2019). Anatomical characteristics were examined with an Olympus CX31 light microscope and Kameram Imaging Software (KAMERAM12 CCD, Argenit Micro System Ltd., Turkey).

The terminology used for seed morphological and anatomical characteristics is compatible withStearn (1985).

Grouping of taxa was performed using the clustering analysis method (UPGMA) in MultiVariate Statistical Package (MVSP) in accordance with the 44 characters inTables 2,3 and4 (Fig. 1). Characters used in statistical analysis were: seed colour (1); shape: orbicular (2), ovate (3), oblong (4), elliptic (5), lanceolate (6); sizes: length (7), width (8), L/W (9); surface ornamentation: reticulate (10), alveolate (11), areolate (12), verrucate (13), ruminate (14), foveate (15), rugose (16), scalariform (17); anticlinal cell walls: sunken (18), raised (19), unclear (20); periclinal cell walls: convex (21), concave (22), unclear (23); epidermal cell structure: polygonal (24), alveolar (25), rectangular (26), flat (27), unclear (28); anatomical structure of the epidermis: flat (29), rectangular (30), crushed (31), polygonal (32), scleranchymatous cells (33), parenchymatous cells (34); anatomical structure of the subepidermis: crushed (35), flat (36), orbicular (37), square (38), polygonal (39), rectangular (40), scleranchymatous cells (41), parenchymatous cells (42); testa thickness (43); presence of crystals (44). The dissimilarity matrix of the studied taxa was created with MVSP (Kovach 2007) (On-line Suppl. Tab. 1). A dendrogram was created. Also, the cophenetic correlation coefficient is designed to explain the relation between the dendrogram and similarity matrix (On-line Suppl. Tab. 1,Fig. 1).

Fig. 1 Cluster analysis of the studied taxa.

Results

This work assesses macromorphologically the seed features of the studied taxa, including colour, shape and dimensions. All of the taxa examined have the same seed color (black) but the shape and size of seeds vary considerably. Seeds examined can be divided into 7 shapes; orbicular, ovate-orbicular, ovate, oblong-ovate, oblong-elliptic, elliptic and elliptic-lanceolate. Orbicular is the most common type (found in 20 taxa). However, oblong-ovate, oblong-elliptic and elliptic-lanceolate are characteristic types for Muscari mirum, M. longipes and M. macbeathianum, respectively. Seed dimensions range from 1.66 mm to 3.21 mm in length, and from 1.12 mm to 2.63 mm in width. While M. erdalii and M. racemosum have the largest seeds, M. macbeathianum has the smallest seeds (Tab. 2, On-line Suppl. Fig. 1).

The surface ornamentation, anticlinal and periclinal cell walls, and epidermal cell structures of the seeds have been micromorphologically evaluated in this study. Seed surface ornamentation is grouped into nine types: ruminate, reticulate, reticulate-areolate, reticulate-foveate, alveolate, scalariform, rugose, verrucate and areolate. The most common form is ruminate, while areolate, reticulate-foveate and scalariform ornamentation forms were found to be taxon-specific (Tab. 3, On-line Suppl. Fig. 2). The reticulate-foveate (in M. elmasii), areolate (M. neglectum), and scalariform (M. azureum) ornamentation types are each displayed by only one taxon. The anticlinal cell walls in the studied taxa are raised, sunken or unclear. While sunken cell walls are widely seen in the alveolate, verrucate, areolate, reticulate-areolate and scalariform ornamentation types, the reticulate and reticulate-foveate ornamentation types are found where epidermal cells are enclosed by raised walls. Rugose and ruminate types are associated with unclear form (Tab. 3). No clear relationship exists between convex or concave periclinal cell walls and surface ornamentation types; however, ruminate and rugose types are found only with unclear periclinal cells. The shape of epidermal cells on the seed surface has also showed diversity and may be grouped into polygonal, alveolar, rectangular and unclear categories. The most common cell type is unclear, while rectangular and alveolar are fairly rare (Tab. 3).

The results of the examination of the anatomical structures of the seeds are indicated in On-line Suppl. Fig. 3 andTab. 4. Testa structures of the seeds of the examined taxa generally consisted of 2 main layers, the epidermis and the subepidermis, formed in either the scleranchymatous or parenchymatous tissue. The epidermis layer displays important variations in cell form, consisting of flat, rectangular, crushed, or polygonal cells, in 1-3 layers, and has undulated or straight wall structure. The most frequent form is flat, while the rarest ones are the rectangular and polygonal types (Tab. 4, On-line Suppl. Fig. 3). The subepidermis layer consists of crushed, orbicular, rectangular, square, flat or polygonal cells in 1-10 layers. The most commonly seen types are crushed and polygonal, whereas the rarest ones are the orbicular and square types. The subepidermis layer is not found in some of the examined taxa (M. discolor, M. inconstrictum, M. parviflorum, M. botryoides and M. turcicum) (Tab. 4). The thickness of the epidermis layers varies between 16.64 μm (in M. turcicum) and 128.46 μm (in M. longipes). Raphide crystals are seen in the epidermis or subepidermis layers of seeds in M. comosum, M. tenuiflorum, M. babachii, M. discolor and M. vuralii (Tab. 4, On-line Suppl. Fig. 3).

A dendrogram indicating differences and similarities among the studied taxa was created by numerical analyses of the seed morphological and anatomical characters, based on the variation of 44 characteristics in 36 taxa. The cophenetic correlation between the similarity matrix and dendrogram has been computed as 0.59, representing a good match. Cluster A2 includes the highest number of taxa when compared to other clusters. Muscari sandrasicum forms a clade separate from these clusters in the dendrogram (Fig. 1). M. discolor and M. parviflorum are the most closely related taxa (with a dissimilarity coefficient of 1.01), the most distantly related taxa recorded are M. sandrasicum and M. turcicum (with a dissimilarity coefficient of 136.31) (On-line Suppl. Tab. 1).

Discussion

The morphological features of seeds offer valuable information about evolutionary relationships among flowering plants (Corner 1976,Karaismailoğlu and Erol 2018). However, seed morphological and anatomical features have so far not been extensively used to elucidate inter-species relationships within genera of the family Asparagaceae. This is the first study to reveal the morphological and anatomical features of the seeds of a genus in the family, and it will be a model for subsequent studies on various genera.

The macromorphological characters of seeds display variation among the examined Muscari taxa, with the exception of seed colour, which is consistently black. The general appearance among populations, including floristic characters and capsule structures, of M. macrocarpum and M. racemosum in subgenus Muscari, M. caucasicum and M. weissii in subgenus Leopoldia, M. aucheri and M. armeniacum in subgenus Botryanthus are very similar, but they can be easily distinguished using seed shape and size.

Comparison of the surface micromorphological structure of seeds is of taxonomical importance (Karaismailoğlu and Erol 2018).Heywood (1971) discusses the significance and efficiency of scanning electron microscopy in elucidating taxonomic problems and distinguishing taxa. However, there are few studies on the importance of seed micromorphology in the family Asparagaceae (Yıldırım 2015,2016). This study on 36 Muscari taxa shows that seed microstructures are useful characteristics in separating the taxa within the family. Almost all of the studied taxa have been examined in this way for the first time, with the exceptions of M. elmasii (smooth) and M. atillae (smooth) (Yıldırım 2015,2016). We recorded nine seed surface ornamentation types in this study. In the genus, the most common seed ornamentation types are ruminate and reticulate. In contrast to this study, reticulate and reticulate-areolate types have been commonly seen among taxa from various angiosperm families (Tantawy et al. 2004,Karaismailoğlu 2015,Karaismailoğlu and Erol 2018). Two closely related taxa in the subgenus Muscari, M. macrocarpum and M. racemosum, have the same reticulate surface ornamentation type; however, M. macrocarpum has different secondary cuticular protrusions. Seed surface ornamentation is a useful character in distinguishing the taxa of the subgenus Leopoldia, which exhibits five ornamentation types in 11 taxa. In the subgenus Pseudomuscari, M. coeleste and M. azureum taxa are very similar in terms of population appearance, flowers and fruit capsule characteristics; however, they are distinctly different in terms of seed ornamentation types: ruminate and scalariform, respectively. In the subgenus Botryanthus, ornamentation types are diverse (seven types), and the distinct surface ornamentation in nearly identical taxa, such as M. armeniacum-M. aucheri, M. armeniacum-M. bourgaei, M. armeniacum-M. microstomum is proof of the taxonomical significance of this characteristic in the subgenus.

Earlier seed surface studies have indicated that the views and structures of anticlinal and periclinal cell walls are good diagnostic characters in the establishment of inter-species relationships (Barthlott 1981,Karaismailoğlu 2015,2016). The types of anticlinal and periclinal cell walls, and epidermal cell structures of the examined taxa vary among the taxa, except for those of the subgenus Muscari.

Revisions of the anatomy of the testa of the various angiosperm families are influential in solving systematic problems (Vaughan et al. 1976,Karaismailoğlu and Erol 2018).Koul et al. (2000) have shown that testa structures may be utilized as a valuable characteristic in the separation of the taxa and the clarification of their phylogenetic relationships.

The seed anatomical characters are frequently as useful as morphological characters for plant taxonomy, and they are valuable in the discrimination of closely correlated taxa in various families and genera (Karamian et al. 2012,Karaismailoğlu and Erol 2018,Karaismailoğlu et al. 2018). A detailed review of the literature has not found a previous study aiming at the exploration of phylogenetic relationships among the taxa with a comparative investigation of anatomical structures of the testa in members of the family Asparagaceae. This work is the first such study for the family and is the precursor to subsequent investigations. In this study, we found that the testae mostly consist of two layers, the epidermis and the subepidermis, in the sclerotic or parenchymatous structure. The epidermis type differs among the taxa. This 1-3 layered epidermis may consist of flat, rectangular, crushed, or polygonal cells. The most frequent form is flat, while the rarest are the rectangular and polygonal types. The structure of the subepidermis layer, which is mostly a compressed tissue under the epidermis layers, also displays significant differences among the taxa. The subepidermis layer consists of crushed, orbicular, rectangular, square, flat or polygonal cells in 1-10 layers, except for M. discolor, M. inconstrictum, M. parviflorum, M. botryoides and M. turcicum, which do not have a subepidermis layer. Testa characters such as the structures of the epidermis and subepidermis, thickness of the testa, and the presence or absence of crystals are fairly effective and beneficial in discriminating almost all of the studied taxa, especially in the pairs of closely correlated taxa M. macrocarpum-M. racemosum, M. caucasicum-M. weissii, M. coeleste-M. azureum, and M. aucheri-M. armeniacum. This can be interpreted as follows: the anatomy of the testa is a useful additional character in the Muscari, and it can aid in the classification of this huge genus. The results obtained are also in agreement with similar previous studies performed on seed structure of some taxa of the genera Crocus L. and Romulea Maratti in the closely related family Iridaceae, in terms of the differences observed at interspecific level in testa anatomical structures such as epidermis cell types and thickness of the testa (Grilli Caiola et al. 2010,Karaismailoğlu 2015,Karaismailoğlu et al. 2018).

The dendrogram showing two main clusters largely agree with the results ofDavis and Stuart (1984). The seed morphological and anatomical variations have been observed at the species level and subgenus level, especially in shapes, ornamentation types, dimensions, and thicknesses and structures of epidermis and subepidermis layers. The proximity between taxa belonging to subgenera Muscari and Pseudomuscari has been preserved; however, there are taxon transitions between Leopoldia and Botryanthus subgenera. While M. atillae, M. latifolium, M. microstomum and M. armeniacum taxa are among the taxa belonging to Botryanthus subgenus, M. mirum and M. caucasicum taxa are located between Leopoldia taxa.

In conclusion, the study of morphological and anatomical seed characteristics of the studied Muscari taxa offers important insights into the systematics of taxa within the genus.