Taxonomic and ecological significance of seed morphology and ultrastructure in species of Lotus formerly classified in Dorycnium (Leguminosae – Loteae )

Summary . Seed morphology and ultrastructure of Lotus species from sections Dorycnium ( L. dorycnium and L. graecus ), Bonjeanea ( L. rectus , L. hirsutus and L. strictus ) and Lotus (L. corniculatus ) have been studied using light and scanning electron microscopy. The seeds of studied species have a conservative structure typical for most Papilionoideae . Seed size, shape and color only partially allow distinguishing species in the studied group. The most morphologically distinguishable seeds are those of members of the polyphyletic section Bonjeanea , with L. rectus having the smallest and L. strictus the largest seeds. Seed size ranges in the species of the section Dorycnium overlap both between the members of the section and with seeds of L. hirsutus (section Bonjeanea ). Seed surface ultrastructure around the hilum is very stable among studied species, whereas that on the lateral seed side is more variable. The types of surface sculpture on the lateral seed side do not strongly correlate with taxonomic position of species, but some cor-relations with eco-geographical patterns can be traced. The type of seed surface micromorphology with inconspicuous primary sculpture and thick secondary cuticular deposits was revealed in taxa, which distribution is connected with the Mediterranean region ( L. hirsutus , L. dorycnium subsp. gracilis and partially L. rectus ). Foveolate-papillose primary sculpture with thin secondary cuticular deposits is typical for species occurring in milder climate ( L. corniculatus , L. strictus , L. graecus ). Studied species can be subdivided into two groups by endosperm thickness. We believe that thin endosperm revealed in L. strictus and L. corniculatus may be a trait connected with the acceleration of development when spreading from the Mediterranean to the temperate climatic zone.


Introduction
Seeds of Papilionoideae are usually formed from campylotropous ovules. The seed coat (testa) is formed from the outer integument. Tegmen (inner integument) is not specialized and is completely resorbed by the time the seed ripens. Seeds of Papilionoideae are exotestal (Corner, 1976). Typical papilionoid seed coat pattern includes epidermis (Malpighian layer or exotesta), hypodermis (sclereid layer) and parenchyma (Corner, 1976;Gunn, 1981).
The epidermis is formed by one layer of palisade or Malpighian cells, which are radially elongated thick-walled macrosclereids. Malpighian cells have special caps, and below the caps is the light line (Barton, 1965). The light line, according to different points of view, is formed as a result of bending of the outer thickening of epidermal cells (Corner, 1976) or reflecting the high density of cellulose microfibrils (Boesewinkel, Bowman, 1990). Outside, the exotesta is covered with a cuticle, which is thinner in the Papilionoideae subfamily than in the Mimosoideae and Caesalpinioideae and usually sculptured (Ponomarenko, 1985). The variety of cuticle sculpture types in Papilionoideae exceeds that in Caesalpinioideae and Mimosoideae (Manning, Staden, 1987). Features of the cuticle sculpture are used for diagnostic and systematic purposes at different taxonomic levels. Under the cuticle, there is a subcuticular layer developed to varying degrees in different representatives of the subfamily (Barton, 1965;Ponomarenko, 1985Ponomarenko, , 1996. Hypodermis located under the epidermis is represented by single-layer hourglass-shaped osteosclereids (Manning, Staden, 1987). In Papilionoideae, this layer is thicker than in the other Leguminosae subfamilies and is clearly separated from the underlying parenchyma (Ponomarenko, 1985;Manning, Staden, 1987). The cells of the hypodermis are separated by the intercellular spaces and probably participate in carrying water to the asteroid tissue and the tracheid block, which finally evaporates through the fissure in the hilum, when the seed ripens.
The parenchyma consists of a small number of layers of usually flattened cells; in comparison with Mimosoideae and Caesalpinioideae in the Papilionoideae subfamily, its thickness is usually the smallest (Ponomarenko, 1985).
In the area of the hilum, so-called hilum complex is formed. When seed is separated from the end of the funicle, one layer of funicle cells often remains attached to the palisade tissue, forming an outer layer (counter palisades) in the hilum region (Manning, Staden, 1987). The hilum is dissected by a median hilum fissure, which functions as a hygroscopic valve that regulates the evaporation of water by the seed (Hyde, 1954). The hypodermis in the hilum region is usually interrupted, and the parenchyma thickens and is replaced by aerenchyma, or asteroid tissue involved in the dehydration of the seed. Directly under the hilum fissure is the so-called "tracheid island" or tracheid block, which is a characteristic feature of the seed coat of the Papilionoideae subfamily (Hyde, 1954;Barton, 1965). The remains of the funicle can form an epihilum or rim-aril; the latter is noted for representatives of the genus Lotus (Arambarri, 1999), although Ponomarenko (1985) believed that only epihilum was a characteristic of Papilionoideae. Around the hilum, in Papilionoideae seeds a hilar rim is usually present, that is raised and colored differently (Gunn, 1981). It is formed by the asteroid tissue located under the epidermis.
The micropyle is a small fissure or hole; in the Papilionoideae subfamily it is smaller than a hilum (Manning, Staden, 1987). It is located opposite the radicular lobe (Yakovlev, 1991), on the other side of the hilum, than the lens (Gunn, 1981). Three main forms of micropyle are distinguished for Papilionoideae: bifurcated (or Y-shaped), deltoid (or triangular) and punctate (Manning, Staden, 1987;Arambarri, 1999).
Raphe is a continuation of the funicle that runs along the seed from the hilum to the chalaza (Boesewinkel, Bowman, 1990;Yakovlev, 1991). Raphe and chalaza carry a vascular bundle. The opposite section of the seed (from the chalaza to the hilum) is called anti-raphe. Asymmetric seeds of Papilionoideae have a raphe shorter than anti-raphe (Corner, 1976). Often in the area of the raphe the testa has a different color than in other areas.
The lens is a zone in the seed coat, the special structure of which makes it permeable (Yakovlev, 1991). The lens of the Papilionoideae seeds is located below the hilum and is usually a slight projection often colored differently than the rest of the seed coat.
Most representatives of the Papilionoideae subfamily have an endosperm developed to varying degrees (Ponomarenko, 1985).
Seeds of Papilionoideae possess a large embryo, in the cotyledons of which a large amount of reserve nutrients accumulates. Ponomarenko (1985) made the conclusion that the embryo in legumes is a fairly stable structure that does not carry such an important set of representative features. She believed that in the structure of the embryo it is impossible to distinguish the levels of structural organization, unlike the seed coat and the hilum. Arambarri (1999) considered some characteristics of the embryo to be important for the taxonomy of the genus Lotus. Manning and Staden (1987) demonstrated the important taxonomic significance for the differentiation of tribes and genera of the Papilionoideae subfamily of such features of the ultrastructure and anatomy of seeds as the shape of the hilum and micropyle, the nature of cells and thickness of the epidermis and hypodermis, the nature of the hilum aerenchyma, the location and shape of the pits on tracheoids (in the tracheid block), as well as sculpture of the exotesta surface. Arambarri (1999) studied seeds of about 45 species of Lotus (Papilionoideae: Loteae) and 22 species of related genera. She concluded that seed ultrastructure revealed using scanning electron microscope (SEM) (the shape and position of the micropyle, the shape of the rim-aril and testa sculpture), as well as seed vasculature, are most useful for diagnosing species and groups of closely related species of the genus Lotus. Kramina (2014) studied seed micromorphology using SEM in 39 species of Lotus belonging to sections Lotus, Tetragonolobus, Lotea, Pedrosia, Heinekenia, and Chamaelotus and two monospecific genera of the same tribe, Kebirita Kramina et D.D. Sokoloff and Antopetitia A. Rich. Using terminology of Arambarri (1999) to describe seed ultrastructure, Kramina applied the approach of Barthlott (1981) to describe testa sculpture. The study by Kramina (2014) did not reveal strict correspondence between seed ultrastructure and sectional subdivision of Lotus and demonstrated that similar sculpture types can be observed in different sections.
The nature of exotesta sculpture revealed using SEM was often used for taxonomic and diagnostic purposes in various tribes and genera of Papilionoideae (Lersten, 1981), in particular Vigna (Kumar, Rangaswami, 1984;Ojeda et al., 2013), genera of the tribe Genisteae (Pandey, Jha, 1988), Melilotus and Vicia (Voronchikhin, 1992(Voronchikhin, , 1993, Trifolium (Rodriguez, 1995), Onobrychis (Özkan et al., 2015), and others. Manning and Staden (1987) showed that in a number of genera from advanced tribes of Papilionoideae, in particular in Medicago (Trifolieae), Lessertia and Sutherlandia (Galegeae), as a result of changes in the wall chemistry the outer ends of epidermal cells are separated from each other, which leads to the appearance of a foveolate-papillose character of the testa. A similar phenomenon is observed in representatives of the genus Lotus (Arambarri, 1999;Kramina, 2014).
In Papilionoideae, the cuticle on the testa is usually sculptured, only occasionally smooth. The predominant types of sculpture in the subfamily are rugose and reticulate, less often foveolate and papillose. Often different types of sculpture are combined together or transitional types are formed (Manning, Staden, 1987). Manning and Staden (1987) also noted that similar types of cuticle sculpture are presented in different, sometimes unrelated, tribes, which may indicate their independent origin.
The morphology and ultrastructure of seeds in Lotus sections Dorycnium and Bonjeanea formerly classified within the genus Dorycnium are insufficiently studied. Seed morphology in four species from Turkey (L. hirsutus, L. graecus, L. dorycnium, and L. axilliflorus) was investigated by Çelebioğlu (1977), but seed ultrastructure was not studied. Seed morphology and ultrastructure in Lotus The goal of our work was to study seed morphology and ultrastructure in representatives of the genus Lotus belonging to sections Dorycnium and Bonjeanea formerly classified within Dorycnium Mill. (Sokoloff, 2003). We intended to elucidate the relationship between the seed structure and the taxonomic and phylogenetic position of species and their ecological and geographical features.

Material and methods
Morphology and ultrastructure of seeds were studied in six species and four subspecies of the genus Lotus belonging to the sections Dorycnium, Bonjeanea, and Lotus. The seeds were collected in the wild or taken from herbarium specimens stored in Herbaria GAZI, LE, MA, MHA, MW, and P. The seeds were photographed with a stereomicroscope Nikon SMZ1500 equipped with an DS-Fi2 camera. Some stacked micrographs using several optical sections were composed using the software package HeliconFocus 7.7.5.
For scanning electron microscopy (SEM) seeds samples of specimens were collected and drayed on the air. Dry specimens were attached to a SEM stub by carbon conductive tabs, then were coated with gold and palladium using EIKO IB-3 Ion Coater. Gold film deposition in Ar gas at 0.1 Torr pressure. Average film thickness 15-20 nm. The specimens were observed under Camscan-S2. Accelerating voltage 20 kV. SEI mode. MicroCapture software and JEOL, JSM-6380LA. Accelerating voltage 20 kV. SEI mode. SEM Control User Interface Version 7.11 (JEOL) software.
Morphometry was carried out for 105 seeds (from 2 to 22 seeds for each taxon), studies using SEM for two to three samples for each species and subspecies.
When describing the morphology and ultrastructure of seeds, we used the terminology of Arambarri (1999). When describing the types of seed surface sculpture, we used the approach of Barthlott (1981) with the isolation of primary, secondary, and tertiary sculpture. The primary sculpture is determined by the shape of exotesta cells and the degree of curvature of their outer periclinal wall. The secondary sculpture is a fine relief of cell walls defined by cuticular deposits or specific types of thickenings. The tertiary sculpture is defined by epicuticular secretions. The thickness of the endosperm and cotyledons was measured on longitudinal sections of seeds taken in the middle part, and then their thick-ness was calculated as a percentage relative to the total thickness of the seed.
Lotus graecus (Figs. 1B, 2J-L, 4I-K): seeds 1.3-2.4 × 1-1.5 mm, ovate, oblong or irregularly globose; smooth; semiglossy or dull; light-brown or greenish-light-brown; monochrome or purple pointed or mottled. Radicular lobe inconspicuous or slightly discernible. Hilum suborbicular. Rimaril thick. Hilar rim rounded and often discoloured (yellow or brown). Raphe deltoid; testa above raphe darker than the rest of the seed coat. Lens is a small protuberance. Endosperm thick. Epidermis layer rather thick (41-54 µm). Micropyle deltoid-Seed morphology and ultrastructure in Lotus bifurcate. Surface micromorphology on the hilum side: primary sculpture foveolate (a depression in the center of a group of exotesta cells), secondary sculpture rugulate (folds are directed to the center of a group of cells) and reticulate. Surface micromorphology on lateral sides: primary structure foveolate-papillose (papillae correspond to the tops of epidermal cells, and a depression in the center of a group of cells), secondary structure smooth or rugulate.
Lotus rectus (Figs. 1D, 3A-C, 5A-D): seeds 1-1.4 × 0.9-1.3 mm, globose; smooth; semiglossy; dark-brown, rarely light-brown, sometimes mottled. Radicular lobe discernible. Hilum suborbicular. Rimaril thick. Hilar rim rounded and often discoloured. Raphe deltoid. Lens is a small protuberance or inconspicous. Endosperm thick. Epidermis layer moderately thick (32-49 µm). Micropyle deltoid. Surface micromorphology on the hilum side: primary sculpture foveolate (a depression in the center of a group of exotesta cells), secondary sculpture rugulate (folds are directed to the center of a group of cells). Surface micromorphology on lateral sides: primary sculpture foveolate-papillose (papillae correspond to the tops of epidermal cells, and a depression in the center of a group of cells), secondary sculpture smooth or rugulate, sometimes also reticulate.
Lotus strictus (Figs. 1F, 3G-H, 5H-J): seeds 2.3-2.4 × 1.6-1.8 mm, rounded-oblong; smooth; semiglossy; yellowish-brown or brown; mottled. Radicular lobe discernible. Hilum ovate. Rim-aril thin. Hilar rim rounded, discoloured. Raphe deltoid; testa above raphe coloured darker than the rest of the seed coat. Lens is a small protuberance. Endosperm thin. Epidermis layer thick (45-58 µm). Micropyle bifurcate. Surface micromorphology on hilum side: primary sculpture foveolate (a depression in the center of a group of exotesta cells), secondary sculpture rugulate (folds are directed to the center of a group of cells). Surface micromorphology on lateral side: primary sculpture foveolate-papillose (papillae correspond to the tops of epidermal cells, and a depression in the center of a group of cells), secondary sculpture smooth or rugulate.

Discussion
All studied species of Lotus are characterized by a conservative seed structure, which is typical for Papilionoideae seeds (Gunn, 1981).
Seed size, shape and color only partially allow distinguishing species in the studied group. The most pronounced difference was observed among the species of Lotus section Bonjeanea. This correlates with high degree of genetic isolation among species of this section revealed in phylogenetic analyses conducted by both nuclear ribosomal ITS1-2 and a set of plastid DNA markers (Kramina et al., 2021). At the same time, the separation of species within the section Dorycnium is not possible, moreover, seed characters do not allow distinguishing L. hirsutus from species of Lotus section Dorycnium. This is in agreement with recently obtained results of molecular phylogenetic study, which demonstrated the absence of genetic isolation between L. hirsutus and L. dorycnium by plastid DNA markers (Kramina et al., 2022). The analysis of seed ultrastructure revealed that the surface sculpture around the hilum is very conservative among studied species, whereas the surface ultrastructure on the lateral seed side is more variable. Our study demonstrated that the types of surface ultrastructure on the lateral seed side do not correlate with taxonomic position of the species. Similar types of surface ultrastructure are developed in species that are not closely related to each other. For example, foveolate-papillose primary sculpture with smooth or rugulate secondary sculpture is typical for L. graecus (section Dorycnium), L. strictus (section Bonjeanea), and L. corniculatus (section Lotus). The same structure was revealed in the majority of species of Lotus section Lotus and in some other sections (e. g., sections Heinekenia and Lotea) (Kramina, 2014). Lotus dorycnium s. l. (section Dorycnium) and L. hirsutus (section Bonjeanea) were characterized by inconspicuous primary sculpture and roughened or wrinkled secondary sculpture. We consider that the types of surface ultrastructure on the lateral seed size more corresponds to eco-geographical conditions than to taxonomic position. Thus, the type of ultrastructure with inconspicuous primary sculpture and thick cuticular secondary sculpture was revealed in taxa with distribution connected with the Mediterranean region, e. g. L. hirsutus, L. dorycnium subsp. gracilis and partially L. rectus. Thick cuticular deposits hide the primary sculpture of the seed coat and contribute to the adaptation to the dry and hot summer period typical of the Mediterranean type of climate. Species common in milder climates (L. corniculatus, L. strictus, L. graecus) are characterized by less thick cuticle deposits that do not hide the primary sculpture of the seed coat surface. Lersten (1981) pointed out that the testa surface patterns are significant within and between adjacent tribes, e. g. papillose sculpture characteristic for the tribe Vicieae. However, Manning and Staden (1987) revealed the similar types of sculpture in unrelated tribes, e. g. the papillose sculpturing present in Abrus (Abreae), Medicago (Trifolieae), and Erythrina (Phaseoleae). Sometimes, the same type of seed surface ultrastructure may differ among taxa by some quantitative parameters, e. g. cell size or number of epidermis cells in a group (Voronchikhin, 1992). To date, there is still insufficient information to assess the taxonomic significance of seed coat sculpture in many groups of Papilionoideae, including the tribe Loteae.
Our study revealed the difference in the degree of endosperm development between studied species, which were subdivided into groups with thin (L. strictus, L. corniculatus) and rather thick endosperm (remaining species). The degree of endosperm development was studied by Ponomarenko (1985) within the whole Leguminosae family. She identified three levels of seed organization. At the first level, which includes seeds of the genera Brachystegia and Bauhinia (Caesalpinioideae), the endosperm is absent or is represented by remains. At the second level, which includes many representatives of Caesalpinioideae and Mimosoideae, the endosperm is most developed and its surface is ruminated, only some taxa do not have it (Acacia, some Albizzia). At the third level represented in the Papilionoideae subfamily, almost all representatives have an endosperm, but