INTRODUCTION
The genus Pentactina Nakai comprises two species (Yakubov, 2014; Chung et al., 2017; Jeon et al., 2025). Pentactina rupicola Nakai is distributed in Mt. Kumgang on the Korean Peninsula (Nakai, 1917; Lee et al., 2007). The other species, P. schlothauerae (Ignatov et Vorosch.) Jakubov is distributed in the Khabarovsk region of the Far East Russia (Yakubov, 2014). This genus is a penta-apocarpous perennial shrub, which is restricted to 600 m high mountainous woodland habitats, typically Pinus pumila Nakai dominated forests (Nakai, 1917; Yakubov, 2014). Due to recent ongoing habitat and population decline, P. rupicola has been placed on the critically endangered (CR) species listed by the Korea National Arboretum (Chang et al., 2016). According to the IUCN Red List categorization, the current global status of P. rupicola is listed up as CR under criteria B1ab(iii) (Kim et al., 2016). Also, P. schlothauerae is an endemic species with limited distribution in the Khabarovsk state of Russia (Yakubov, 2014). Recently, the molecular phylogenetic studies on Pentactina have been performed, and the taxonomic revision was well represented (Jeon et al., 2025).
On the other hand, several extensive studies of the floral anatomy (Song et al., 2020b), palynology (Lee et al., 1993; Song et al., 2017), seed morphology (Song et al., 2020a; Ahn et al., 2023), chromosome study (Sax, 1931), and molecular characteristics (Lee and Hong, 2011; Choi et al., 2023; Won et al., 2024; Jeon et al., 2025) of P. rupicola have been conducted to elucidate the evolutionary relationships among genera in the tribe Spiraeeae of the Rosaceae. However, P. schlothauerae has been relatively poorly studied. Biochemical study reported differences in the qualitative composition of phenolic compounds, supporting its difference from Spiraea (Kostikova, 2018; Kostikova and Petrova, 2021). A chromosomal study of P. schlothauerae was reported it to be tetraploid but lacked photographic evidence (Probatova et al., 2017).
Concerning the reproductive morphological features of the tribe Spiraeeae, Petrophytum (Torr. et A. Gray) Rydb., Sibiraea Maxim., and Spiraea L. remain poorly documented, showing limited available information. Only a few reproductive morphological characteristics of these genera have been reported (Webb, 1902; Davis, 1966; Johri et al., 1992), and information is insufficient for a comparative study within the tribe Spiraeeae. In particular, Kalkman (2004) and Potter et al. (2007a) published several studies concerning floral anatomy and reproductive morphology. However, they did not provide sufficient information regarding reproductive morphological features. Therefore, more information on the reproductive morphological features at the genus level is needed to provide useful taxonomic characteristics.
Although the genus Pentactina was first described as a new genus by Nakai in 1917, its taxonomic treatment has been subject to varying interpretations. Schulze-Menz (1964) did not mention Pentactina in his treatment of the tribe Spiraeeae, and both Hutchinson (1964) and Kalkman (2004) treated Pentactina as a synonym of Spiraea. In contrast, Takhtajan (1997) recognized Pentactina as a distinct genus within the tribe Spiraeeae (Potter et al., 2007a).
Until the early 21st century, phylogenetic analyses of the tribe Spiraeeae did not include Pentactina, and only eight genera were recognized within the tribe (Potter et al., 2007b). Molecular phylogenetic studies of P. rupicola were conducted by Lee and Hong (2011), Choi et al. (2023), and Won et al. (2024) further elucidating the genus’s taxonomic placement.
Subsequently, Spiraea schlothauerae, which had been collected from Khabarovsk in the Far East of Russia (Voroshilov and Ignatov, 1987), was reclassified as Pentactina schlothauerae by Yakubov (2014), and recognized as an oligotypic genus consisting of two species (Chung et al., 2017). As a result, P. rupicola and P. schlothauerae came to be recognized as endemic species of Mt. Kumgang on the Korean Peninsula and the Khabarovsk region of the Far East of Russia, respectively. More recently, molecular phylogenetic analyses that included two species of Pentactina were reported by Jeon et al. (2025). Based on their results, Pentactina was confirmed as a distinct genus within the tribe Spiraeeae. Consequently, the tribe Spiraeeae is now recognized as comprising nine genera: Aruncus L., Holodiscus (K. Koch) Maxim., Kelseya (S. Watson) Rydb., Luetkea Bong., Pentactina, Petrophytum, Sibiraea, Spiraea, and Xerospiraea Henr. (Kalkman, 2004; Potter et al., 2007b; Jeon et al., 2025).
Reproductive morphological characteristics have been provided still good evidence of taxonomic relationships in flowering plants (Tobe, 1989). However, no reproductive morphological study of the genus Pentactina has been conducted. In this study, we present the reproductive morphological characteristics of the genus Pentactina in an effort to identify useful characteristics for understanding the generic relationships within the tribe Spiraeeae of Rosaceae.
MATERIALS AND METHODS
Pentactina rupicola is a rare plant species endemic to the Korean Peninsula. The only source of this plant outside North Korea is in the Royal Botanical Garden in Edinburgh, Scotland. Samples of P. schlothauerae were collected from the Far East of Russia in 2024 (Table 1). For this study, young flower buds and open flowers were fixed in F.A.A. (5 parts formalin, 5 parts glacial acetic acid, and 90 parts 50% ethanol) and dehydrated in an ethanol series. Dehydrated samples were then replaced with a resin 7100 solution to make a resin block. Then, the samples were embedded in Technovit 7100 resin solution. After hardening, the resin blocks were sectioned at a thickness of ca. 5 μm using a rotary microtome (Leica RM 2235, Heidelberg, Germany). Slides containing sections were stained with Toluidine Blue O and dried on a slide warmer. After drying, the slides were mounted with Entellan (Merck, Germany). The number of cells in mature pollen was counted by staining with 1% acetocarmine (Tobe and Raven, 1984). Permanent slides of different stages were observed under a BX-50 microscope (Olympus Co., Tokyo, Japan). Photos were obtained with a camera attached to the microscope (Olympus DP 70, Olympus Co.). For scanning electron microscope (SEM) observation, mature seeds were dehydrated using critical point drying. After drying, the mature seeds were coated with platinum using an ion sputter (Hitachi E1010, Tokyo, Japan) and observed by SEM (Supra 55VP, Carl Zeiss, Jena, Germany).
Terminology to describe the anther wall formation followed that proposed by Davis (1966) and seed coat terminology followed that proposed by Corner (1976) and Schmid (1986). The seed characteristics of P. rupicola were previously reported by Ahn et al. (2023). Therefore, it was not described in this study. Also, due to the lack of flower samples, the fertilization and developmental embryo characteristics could not be examined. If samples of fertilized flowers and young seed stages become available, these characteristics will be examined in the future.
RESULTS
The reproductive morphological characteristics of P. rupicola and P. schlothauerae were found to be highly similar, and were summarized as follows (also see Table 2).
Anthers and microspores
Both species of the genus Pentactina had a tetrasporangiate extrorse anther (Figs. 1A, B, 2A, C). The anther wall typically comprised four cell layers: the epidermis, endothecium, middle layer, and a tapetum (Figs. 1C, 2B). The anther wall showed only one middle layer (Figs. 1C, 2D). Therefore, the anther wall formation conformed to a dicotyledonous type (Fig. 2B) (Davis, 1966), although it could not be confirmed in P. rupicola due to the lack of the youngest flowers. The tapetum was glandular, and its cells had two nuclei (Figs. 1B, C, 2C, D). The endothecium developed fibrous thickenings during maturation (Figs. 1E, F, 2G, H). The epidermal cells were flattened but persistent (Figs. 1F, 2H). The middle layers were all crushed (Figs. 1F, 2H). Simultaneous cytokinesis was observed in the microspore mother cell during meiosis (Figs. 1D, 2E, F). Resultantly, tetrahedral microspores were produced (Figs. 1D, 2F). The anther dehisced through a longitudinal slit, and it was dithecal (Figs. 1E, 2G). Pollen grains were two-celled at the time of shedding (Figs. 1G, 2I).
Megagametophyte and nucellus
The ovule was anatropous (Figs. 3A, 4A) and crassinucellate (Figs. 3B, 4D). Both single and multiple archesporial cells were differentiated at the hypodermis of the nucellar apex in P. schlothauerae (Fig. 4B, C). Archesporial cells underwent periclinal division to give rise to primary parietal and sporogenous cells. The sporogenous cell developed into a megaspore mother cell, and the main parietal cell divided into two or three additional parietal cells (Figs. 3B, 4D). Megaspore mother cells underwent meiosis to produce a dyad (Figs. 3C, 4E), a linear tetrad of megaspores (Figs. 3D, 4F). The megaspore at the chalazal end acted as a functional megaspore while the three megaspores at the micropyle end degenerated (Figs. 3D, 4F). The functional megaspore underwent successive nuclear divisions and formed two-nucleate (Figs. 3E, F, 4G), four-nucleate (Figs. 3G, 4H), and finally eight-nucleate embryo sac (Figs. 3H, 4I). The embryo sac developed from a single megaspore after meiosis. Hence, the embryo sac development was of the Polygonum type. Rarely, twin mature embryo sacs have been observed (Fig. 3I). The mature embryo sac was ellipsoidal (Figs. 3H, 4I). It comprised an egg cell, two synergids, two polar nuclei, and three antipodal cells. During megasporogenesis, apical epidermal cells of the nucellus divided periclinally to form the nucellar cap (Figs. 3D, E, 4D). Neither the hypostase nor the obturator was differentiated. Starch grains were not observed in embryo sacs.
Integuments
The flowers of P. rupicola and P. schlothauerae had five carpels (Figs. 1A, 2A). Each of them had two ovules (Figs. 3A, 4A). The ovule was anatropous and unitegmic (Figs. 3A, 4A). The ovule position was was epitropous-ventral (Figs. 3A, 4A). The outer integument comprised four to five cell layers (Figs. 3A, 4A, 5A). The micropyle was formed by only an outer integument as it was unitegmic (Figs. 3A, 4A, 5A).
Seed and seed coat
The seed and seed coat characteristics of P. rupicola were identified previously by Ahn et al. (2023). Therefore, the present study described only the seed and seed coat characteristics of P. schlothauerae. It had dark brown fusiform-shaped seeds without seed appendages (Fig. 5B). The seed coat was comprised of an outer integument, i.e., it was unitegmic (Fig. 5A). This species had reticulated seed surface sculptures creating rectangular-reticulate cells in mature seeds (Fig. 5C). The mature seed had copious cotyledons with thin endosperm (Figs. 5D–F). The mature seed coat of P. schlothauerae showed one layer of oblong exotesta in a transverse section (Fig. 5E), narrowly round exotesta in a longitudinal section (Fig. 5F), crushed mesotesta, and one layer of sclerenchyma long cuboidal endotesta (Figs. 5E, F). Therefore, the seed coat of P. schlothauerae was an endotestal type (Figs. 5D–F) (see Corner, 1976; Schmid, 1986). The endotesta cells accumulated as a tannin substance and functioned to protect the embryo and thin endosperm (Figs. 5E, F).
DISCUSSION
Comparison with the tribe Spiraeeae and Rosaceae
According to molecular data, Pentactina was closely related to Petrophytum, Sibiraea, and Spiraea (Lee and Hong, 2011; Jeon et al., 2025). However, the reproductive morphological characteristics of these genera have not been described until now, and only a few reproductive morphological features of Sibiraea and Spiraea have been reported (Webb, 1902; Davis, 1966; Johri et al., 1992). Therefore, we aimed to compare the intergeneric relationships based on reproductive morphological characteristics at the family level. All members of the tribe Spiraeeae were woody shrubs, except for the polymorphic species Aruncus dioicus (Walter) Fernald (Table 3) (Kalkman, 2004).
When comparing the reproductive morphological characteristics of P. rupicola and P. schlothauerae (Table 2), it was not possible to observe the anther wall developmental type of P. rupicola due to limited flower materials. However, during microsporogenesis, both species shared similar features, including the number of microsporangia, persistent epidermis, fibrous endothecium, glandular tapetum, simultaneous cytokinesis of microspores, and the presence of two-celled mature pollen at the time of shedding. Megasporogenesis also exhibited common characteristics such as anatropous, crassinucellate, unitegmic ovules, multiple archesporial cells, nucellar cap formation, Polygonum-type embryo sac development, two ovules per carpel, and the absence of the hypostase and obturator structures. Additionally, both species exhibited similar features, including the absence of seed appendages, a thin endosperm in the mature seed, and an endotestal seed coat type. Thus, the reproductive morphological characteristics of these two species were highly similar. When comparing P. rupicola and P. schlothauerae, they were found to be closely related to each other in many of the reproductive morphological features mentioned above (Table 2).
Pentactina shared many reproductive morphological characteristics with at least Aruncus, Holodiscus, Sibiraea, and Spiraea in the tribe Spiraeeae (Table 3). Among these were persistent anther epidermis, fibrous endothecium, tetrahedral tetrads, two-celled pollen at shedding time, multi-celled ovule archesporium, crassinucellate nucellus, a formed nucellar cap, undeveloped endothelium, and the absence of a vascular bundle in the integuments. Ovules of Rosaceae were consistently crassinucellate and more primitive than the tenuinucellate (Webb, 1902; Davis, 1966; Sporne, 1969; Takhtajan, 1997).
When the genus Pentactina was compared with other genera within the tribe Spiraeeae (Table 3), Holodiscus, Pentactina, Sibiraea, and Spiraea were found to be deciduous shrubs excluding the herbaceous Aruncus (Henrickson, 1985; Kalkman, 2004). In contrast, genera such as Kelseya, Luetkea, Petrophytum, and Xerospiraea were evergreen and xerophytic plants (Henrickson, 1985; Kalkman, 2004). Also, Aruncus and Sibiraea were unisexual and dioecious, whereas Pentactina and the other genera were bisexual and monoecious, as a symplesiomorphy (Figs. 1A, 2A) (Kalkman, 2004). Furthermore, there was some variation in the number of ovules per carpel. Holodiscus, Pentactina, and Xerospiraea consistently had two ovules per carpel (Figs. 3A, 4A, Table 3) (Henrickson, 1985; Kalkman, 2004), while the remaining genera produced two to eight ovules (Henrickson, 1985; Kalkman, 2004). In terms of endosperm features, the Rosaceae exhibited both albuminous and exalbuminous seeds (Cronquist, 1981; Johri et al., 1992). Within the tribe Spiraeeae, both traits coexist in Aruncus and Spiraea, while Kelseya, Luetkea, Petrophytum, and Xerospiraea produced exalbuminous seeds (Table 3) (Kalkman, 2004). However, Holodiscus, Pentactina, and Sibiraea maintained a thin endosperm as a symplesiomorphy when the seed reached maturity (Table 3) (Kalkman, 2004).
Broadening the scope of the comparison to the family level revealed that Rosaceae typically possessed leaves with stipules, whereas genera within the tribe Spiraeeae shared the evolutionary derived character of stipule loss (Schulze-Menz, 1964). The absence of stipules is regarded as a synapomorphic character found in the tribe Spiraeeae within the Rosaceae. At the family level, the characteristics of the ovule integuments included both bitegmic and unitegmic types (Davis, 1966; Sterling, 1966). However, the unitegmic condition was consistently observed, excluding genera with no available data within the tribe Spiraeeae, and was considered a synapomorphy (Davis, 1966; Potter et al., 2007b).
In terms of seed characteristics, all genera of the tribe Spiraeeae shared the plesiomorphic character of lacking seed appendages (Table 3) (Cronquist, 1981). Within the Rosaceae, the mesotestal seed coat type was widely reported (Corner, 1976). However, both Pentactina and Spiraea exhibited an endotestal seed coat type in the tribe Spiraeeae, which is considered a synapomorphic character within the Rosaceae. Notably, the seed surface sculpture of Pentactina exhibited a rectangular-reticulated pattern (Fig. 5C) (Ahn et al., 2023), which differed from the polygonal-reticulated pattern found in Spiraea (Song et al., 2020b). This distinctive feature, together with the linear petal shape, was considered an autapomorphic characteristics of Pentactina. With regard to fruit characteristics, all eight genera including Pentactina, produced follicles as a plesiomorphic character within the tribe, whereas Holodiscus bore an achene (Table 3) (Spjut, 1994; Kalkman, 2004). However, when various characteristics including reproductive morphology and floral morphology were compared, Pentactina appeared to possess both primitive and derived characteristics within the Rosaceae. Among the genera of the tribe Spiraeeae, nevertheless, it was considered most closely related to Spiraea, as exemplified by the fact that P. schlothauerae was originally described as a member of the genus Spiraea (Table 3) (Voroshilov and Ignatov, 1987).
However, in this study, the only reproductive morphological characteristics that clearly distinguished Pentactina from Spiraea were the number of ovules per carpel and the seed surface sculpture pattern. Pentactina consistently possessed two ovules per carpel, whereas Spiraea exhibited variations ranging from two to eight ovules (Henrickson, 1985). Additionally, the petal shape was distinctly obovate in Spiraea and other genera of the tribe Spiraeeae, while both species of Pentactina shared a linear petal shape in floral characteristics (Table 3). This character was considered an autapomorphy in the tribe Spiraeeae.
In conclusion, the two species of Pentactina exhibited highly consistent reproductive morphological characteristics, with the only reproductive morphological differences from the closely related genus Spiraea being the number of ovules per carpel and the seed surface sculpture pattern. A comparative analysis incorporating reproductive morphological characteristics, as well as other vegetative morphology, biochemical compounds (Kostikova, 2018; Kostikova and Petrova, 2021) and floral morphological characteristics (Evans and Dickinson, 1999), supported the interpretation of Pentactina as a distinct genus separate from Spiraea, in agreement with the molecular phylogenetic results of Lee and Hong (2011) and Jeon et al. (2025).
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