Anther and ovule development of Clematis serratifolia (Ranunculaceae)–with new formation types in megaspore and nucellus

Morphological indices of vegetative organs or reproductive organs, which are often used to analyze the evolution and classify Clematis, indicate that Clematis serratifolia and C. glauca could be related members at similar evolutionary levels. However, this assumption differs with phylogenetic studies based on genetics. Embryonic characteristics, which are more stable, are commonly used to estimate the phylogeny and evolution of angiosperms. We studied the microsporogenesis, microgametogenesis, megasporogenesis and macrogametogenesis development of C. serratifolia, and compared the early embryological characteristics among C. serratifolia, C. serratifolia and other Clematis species reported to provide a reference for the taxonomy of the genus Clematis. Our results showed that C. serratifolia and C. glauca differ in megaspore formation and nucellus types suggesting that they have originated from different ancestors. The differences among Clematis were mainly found in the type of the anther wall development, tapetum, pollen grains, megaspore formation and nucellus types.


Introduction
The genus Clematis belongs to the Ranunculaceae family, which is one of the ancestral groups of angiosperms, particularly, the eudicots [1]. It is a diverse genus of about 355 species with highly variable morphology and a wide distribution. The morphological indices of vegetative organs or reproductive organs of Clematis [2][3][4][5] have long been used as the basis for classifying Clematis. However, these indices have proved unstable in different environments. For instance, C. pinnata, C. brevicaudata and C. heracleifolia exhibit variable leaf morphology in different regions [6]. The classification of Clematis, therefore, has been controversial. In recent years, karyotype [7][8][9], pollen grain morphology [10] and molecular techniques [11][12][13] have been used to classify Clematis. However, the results are easily affected by the equipment and methods employed [7][8][9]14], and can be contradictory [11][12][13]. It is, therefore, necessary to find relatively stable morphological characteristics that can reflect the phylogeny and evolution to assist with the taxonomic study of Clematis.
Embryonic characteristics, which are commonly used to estimate the phylogeny and evolution of angiosperms [15,16], are relatively stable traits even in complex and changeable environments. Extensive embryological studies on Ranunculaceae have been carried out in recent years [17][18][19][20][21][22]. In Clematis, the anther development of C. 'Ernest Markham' [23] and C. hexapetala [24] have been studied, and the anther and ovule development of C. fusca [25], C. terniflora var. mandshurica [26], C. heracleifolia [27] and C. glauca [28] also have been observed in detail. The morphological indices of Clematis serratifolia and C. glauca are similar. Therefore, they were considered to be related members at similar evolutionary levels [4]. However, this assumption differs from the findings of phylogenetic studies, which were based on ITS and chloroplasts DNA sequence [13]. Here, we studied the microsporogenesis, microgametogenesis, megasporogenesis and macrogametogenesis development of C. serratifolia to elucidate its early embryological characteristics. We analyzed the characteristics between C. serratifolia and C. glauca, and identified the similarities and differences in the early embryological characteristics of Clematis, to provide a reference for the taxonomy of Clematis.

Materials and methods
C. serratifolia plants were grown from seed (collected from the natural site of Chimney Hill, Panshi city, Jiling, China) at Qingdao Agricultural University (36˚20 0 N, 120˚12 0 E, Qingdao city, Shandong, China) in April 2015. The mean annual temperature in this region was about 12.6˚C.
Flower buds were collected every day from June to early September in 2018 and fixed in FAA (formalin: acetic acid: 50% ethanol, 1:1:18, v/v/v) immediately upon collection. Samples were embedded in paraffin by conventional methods and sliced by rotary microtome (Leica RM-2145, Shanghai Leica Instrument Co. Ltd., Shanghai, China). Ovary blocks were sectioned longitudinally with a thickness of 10 μm and anther blocks were sectioned transversely with a thickness of 8 μm. Sections were stained with Ehrlich's hematoxylin and eosin [29], and examined using a light microscope (Leica DM 500, Shanghai Leica Instrument Co. Ltd., Shanghai, China).
After freeze-dring with tert-Butyl alcohol, pollen grains of C. serratifolia were coated with gold-palladium in a spatter coater (JFC-1600, JEOL, Japan) and observed under a scanning electron microscopy (2 kV, JSM-7500 F, JEOL, Japan). Characteristics of pollen grains, including the length of the polar axis (P), and the length of the equatorial axis (E), and spinule height and density were measured using Image J (National Institutes of Health, Bethesda, Maryland, United States of America).

Microspore and microgametophyte development
Early in June, the flower of C. serratifolia began to differentiate. The initiation and development patterns of floral organs were both centripetal (Fig 1A and 1B). After the development of perianth primordia, stamen primordia initiated and developed, and then matured gradually.
Stamen primordia were initially located under the epidermal cells and composed of actively dividing cells, from which the archesporial cells differentiated (Fig 1C). After the periclinal division of the archesporial cells, the outer primary parietal cells and inner primary sporogenous cells were formed ( Fig 1D). The outer primary parietal cells then divided periclinally and anticlinally to form two layers of secondary parietal cells (Fig 1E), the outer layer of which divided again to form the endothecium and the middle layers, while the inter layer of secondary parietal cells developed directly into the tapetum. A mass of microspore mother cells were formed by several mitotic divisions of the primary sporogenous cells (Fig 1F).
The microspore mother cells then separated from each other and entered meiosis to form the tetrads (Fig 1G-1L). The tetrads were separated by the callose wall (Fig 1L and 1M), and the middle layers and tapetum cells gradually degenerated (Fig 1G-1M).
After the disintegration of the callose wall, the microspores were released from the tetrads (Fig 1N and 1O). With the expansion of its vacuoles, the microspore's nucleus was displaced to one side of the endothecium ( Fig 1P). Next, mitotic division of the microspore's nucleus occured, resulting in a larger vegetative cell and a smaller generative cell (Fig 1P-1R). At this point, the middle layers and tapetum cells degenerated entirely, or left traces near the endothecium cells (Fig 1P-1R). Then, the radial and tangential walls of the endothecium were thickened, leading to anther dehiscence ( Fig 1S). Until the shedding of the pollen grains, grains were two-celled monads.

Pollen morphology
The pollen of C. serratifolia was symmetrical monads, with three evenly distributed germ furrows (Fig 2A-2C). The outlines were trifid-round with an axis 20.66 μm in length in the polar view and an axis 30.57 μm in length in the equatorial view (Fig 2B and 2C; Table 1). On the pollen surface, perforation and microechinate ornamentation were clearly evident (Fig 2C and  2D).

Megaspore and megagametophyte development
The ovary of C. serratifolia had one chamber with several ovules, of which only one ovule developed normally.
Initially, a dome-shaped ovule primordium differentiated on the placenta epidermis, then formed a finger-like structure and developed into the nucellus. Subsequently, a single archesporial cell with large volume, dense cytoplasm and conspicuous nuclei appeared below the nucellus ( Fig 3A). Two cells then formed after the periclinal division of the archesporial cell ( Fig 3B and 3C). The outer (parietal) cell developed into the parietal tissue layer, and the inner (primary sporogenous) cell then directly differentiated into a megaspore mother cell (Fig 3D). At this point, the megaspore mother cell was surrounded by 2-3 layers of nucellus cells ( Fig  3D). The ovules gradually curved (Fig 3D-3P), and a unitegmic integument formed ( Fig 3D). Thus, the ovule of C. serratifolia was anatropous, unitegmic and crassinucellate.
A linear tetrad of megaspores was formed after the megasporocyte meiosis (Fig 3D-3G), with the chalazal megaspore developing normally and differentiating into the functional megaspore while the remaining megaspores becoming reduced (Fig 3H). In subsequent stages, the functional megaspore increased in volume ( Fig 3I) and entered mitosis (Fig 3J-3O). It divided successively to form an embryo sac with 2 nuclei (Fig 3J), 4 nuclei (Fig 3K), and then 8 free nuclei (Fig 3L). The 8 free nuclei embryo sac entered cellularization and matured. The mature embryo sac (Fig 3M-3O) included three larger binucleated antipodal cells in the chalazal

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region, a diploid central cell, two synergids and an egg cell in the micropylar region. The embryo sac was similar to that found in Polygonum.

Development relationship between gametophytes and flower bud morphology
In the same flower of C. serratifolia, the anther primordium developed before the pistil primordium. The female gametophytes developed slower than the male gametophytes, with a delay of about three to four stages at the beginning. However, the male and female gametophytes matured at approximately the same time before blooming. The developmental relationship between the gametophytes and the flower bud morphology were presented in Table 2.

Microspore and microgametophyte development
We observed that the microspore and microgametophyte development of C. serratifolia and C. glauca were with only minor differences in the structure of the mature anther wall (Table 3). Features such as centripetal development of floral organs, tetrahedral microspores tetrads, simultaneous microsporocyte cytokinesis, and two-celled mature pollen grains may be the common anther development characteristics of Clematis. There were different in the type of anther wall development, tapetum, pollen grains and the structure of mature anther wall (Table 3). It has previously been reported that the evolutionary trend in Ranunculaceae was from centripetal floral organs to centrifugal flower organs [21], from the amoeboid tapetum to glandular tapetum [31], from the basic type of anther wall development to monocotyledonous type [32], and from the tricolpate to the pantocolpate and the pantoporate [10]. Thus, C. serratifolia, along with the six species of Clematis (Table 3), showed a mix of ancestral and derived features.

Megaspore and megagametophyte development
We observed that the megaspore and megagametophyte development of C. serratifolia and C. glauca were different in the megaspore formation and nucellus types (Table 3). Although the

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reported nucellus characteristics of C. fusca [25] were the same as C. serratifolia, evidence for this came from some fuzzy pictures and there was no conclusive information regarding the division of the archesporial cell and the nucellus type. It was, therefore, difficult to confirm the relevant characteristics of C. fusca, and we did not compare the ovule development characteristics of C. fusca with C. serratifolia here. Studies have shown that the characteristics of megagametophyte development in Ranunculaceae were highly variable, including species with unitegmic integument (Tribe Helleboreae,  [33], species with tenuinucellate, crassinucellate or both types of nucellus [18,34], species with linear, T-shaped tetrad of megaspores, or both [35,36], and species with uninucleate, dikaryotic or multinucleate antipodal cells [18,22]. There were also variations in the number of archesporial cells. Tamura considered that species in Ranunculaceae usually had only one archesporial cell, which can be observed in Anemone, Aquilegia, Actaea, Adonis, Helleborus and Pulsatilla [20]. However, species in Caltha, Ranunculus, Delphinium and Cimicifuga had 1-3 archesporial cells, and varieties of Ranunculus septentrionalis had 2-13 archesporial cells [20]. It can be seen that species of Clematis are anatropous and unitegmic, with a Polygonum-type embryo sac, a linear tetrad of megaspores, three large dikaryotic antipodal cells, and only one archesporial cell (Table 3). These may be common characteristics during the ovule development of Clematis. There were numerous convergences and differences in the early embryological characteristics between Clematis and other genera in Ranunculaceae. Tamura considered that the megasporocyte of species in Ranunculaceae was usually formed by further division of the archesporial cell [37], as observed in Trollius buddae [20]. However, the single archesporial cell of Helleborus thibetanus [19] and Caltha palustris [20] developed directly into a megasporocyte, and the crassinucellate nucellus of the two species was derived from the division of the epidermis. It can be seen that the megasporocyte of C. heracleifolia, C. glauca and C. terniflora var. mandshurica all with tenuinucellate nucellus was formed directly by the archesporial cell, while that of C. serratifolia with crassinucellate nucellus was formed by further division of the single archesporial cell (Table 3). Thus, there were two megaspore formation types and two nucellus types in Clematis, and which of these types is more common needs to be studied further.
The characteristics of ovule development, especially integumentum, nucellus and ovule types, were of great reference value for species evolution and taxonomic studies. In dicotyledon, anatropous ovules, which were widely existed in the ancient plant groups such as Nymphaeaceae and Ranunculales, were generally considered as primitive characteristics [33,[38][39][40][41]. Crassinucellate ovules commonly existed in the ancient plant groups, and bitegmic integument usually existed in the polypetalous, were both regarded as other primitive characteristics [41,42]. It can be seen that species of Clematis were commonly with ancestral features (anatropous ovules) and derived features (unitegmic integument). Studies have shown that species with crassinucellate ovules were usually with bitegmic integument [33,41]. However, there were two nucellus types in Clematis. The genus Clematis was overall not very original and its speciation process may be complex.

Conclusion
Although C. serratifolia and C. glauca had similar morphological indices, there were differences in their early embryological characteristics, primarily in megaspore formation and nucellus types. Thus, C. serratifolia and C. glauca may have originated from different ancestors.
Common features of Clematis include centripetal development of floral organs, tetrahedral microspores tetrads, simultaneous microsporocyte cytokinesis, two-celled mature pollen grains, anatropous and unitegmic characteristics, Polygonum-type embryo-sac, a linear tetrad of megaspores, three larger dikaryotic antipodal cells and only one archesporial cell. The differences in early embryological characteristics among Clematis species were mainly found in the type of anther wall development, tapetum, pollen grains, megaspore formation and nucellus types.
There were numerous convergences in the early embryological characteristics within the species of the genus Clematis, or between Clematis and other genera in Ranunculaceae. The speciation process of Clematis was relatively complex, and interspecific hybridization could have occurred in its early differentiation.
Supporting information S1