Reinvestigation of the Late Devonian Lycopsid Sublepidodendron grabaui from Anhui Province, South China

Simple Summary The lycopsid Sublepidodendron is cosmopolitan in the Late Devonian and Early Carboniferous. Sublepidodendron grabaui is known from several localities of the Upper Devonian Wutong Formation in South China, with its overall morphology and male reproductive organs (i.e., the microsporangiate strobili and microspores) studied previously. Here we describe new specimens of S. grabaui from Guangde City, Anhui Province, to get further knowledge on this plant, especially its female reproductive organs (i.e., megasporangiate strobili and megaspores). The megasporangiate strobili are borne terminally on fertile axes and occasionally dichotomized, bearing at least eight Lagenicula-type megaspores with a small gula in each sporangium. Based on the previous and present study, this plant is considered as a tree lycopsid exhibiting multiple dichotomized stems, occasionally produced lateral branches and monosporangiate strobili. Abstract South China displays Devonian strata with well-exposed outcrops and is regarded as a diversity hotspot of Late Devonian lycopsids. The heterosporous lycopsid Sublepidodendron grabaui has been studied for over ten years, with its general morphology, aerial stem anatomy, microsporangiate strobili, and growth architecture reported. Based on new specimens from Guangde City, Anhui Province, this study provides further knowledge about the megasporangiate strobili and megaspores of S. grabaui. Its slender megasporangiate strobili occur singly or in pairs and occasionally bifurcate in the middle-upper portion. Each megasporophyll consists of a flattened pedicel and an adaxially curved lamina. The lamina forms a downturned heel at the base. Each sessile megasporangium contains at least eight Lagenicula-type megaspores with a small gula. The other observed characteristics of S. grabaui in this study conform to those previously known and are compared to relative coeval taxa.

Sublepidodendron grabaui is known as the combination of the former morphological species S. wusihense and Lepidostrobus grabaui and is described as an arborescent, heterosporous lycopsid from several localities of the Upper Devonian Wutong Formation in South China [12,23]. The previous studies revealed the general morphology, aerial stem anatomy, microsporangiate strobili, and growth architecture of S. grabaui. In this article, its megaporangiate strobili and megaspores are investigated based on new materials excavated from the Upper Devonian of Guangde City, Anhui Province, China.

Materials and Methods
The specimens were collected from the Yongchuan mine, Xinhang town, Guangde City, Anhui Province, China ( Figure 1). The fossil plant in this study is restricted to the bottom portion of the Leigutai Member, i.e., the upper member of the Upper Devonian (Famennian) Wutong Formation (Figure 2), and is preserved as compressions and impressions in yellow, brown, or grayish mudstones. Steel needles were used to expose the morphology of some dichotomized axes and strobili. The limonitized stele shows the anatomy in the transverse section, which was examined under a stereomicroscope. The specimens were photographed using a Nikon Z6 digital camera and a Leica S9 stereomicroscope. All figures were organized using Adobe Photoshop CC 2018 (ver. 19      Trunk stele with a pith, exarch primary xylem, and secondary xylem; stele of large branches with a small pith, exarch primary xylem; a solid exarch primary xylem strand present in small branches.

Comparison among Specimens of Sublepidodendron grabaui in China
The central strobilar axes are up to 1.5 mm in width, bearing sporophylls in helices. The sporophylls are long-triangular in front view, ca. 2.0 mm at the widest part, showing a single vein in the middle ( Figure 6A,B). Each sporophyll consists of a horizontal pedicel and an adaxially curved lamina ( Figure 6C,E). All strobili were found to contain megaspores and are thus megasporangiate. Each sporophyll bears one sessile sporangium on the adaxial surface of the pedicel. However, only a few specimens show unbroken sporangial walls to recognize the contour of sporangia ( Figure 6C-E). The sporangia are long-ellipsoidal in shape, 2.7-3.3 mm long, and 1.1-1.3 mm high ( Figure 6D,E). One specimen shows two adjacent megaspore tetrads that are probably in one sporangium ( Figure 6D). Isolated megasporophyll-sporangium complexes are ca. 3.2 mm long, showing exposed megaspores ( Figure 6E). The megaspores are Lagenicula-type and 661-943 µm in diameter. The spores are round to pear-shaped in outline ( Figure 6G-K). Each megaspore is composed of a spherical body and a distinct gula, with a gula height-to-body diameter ratio of 0.34 ( Figure 6H,I). Spiny ornamentations, 18-77 µm long, are clear on some megaspores ( Figure 6J,K).

Vegetative axis
Width (

Comparison with Lycopsids Bearing Bisporangiate Strobili (Table 2)
During the Middle and Late Devonian, lycopsids with bisporangiate strobili are widely distributed in the world (e.g., [5,[20][21][22]26]). All these taxa display megasporophylls and microsporophylls in the basal and terminal parts of one strobilus (Table 2), respectively, and thus differ from Sublepidodendron grabaui, which possesses megasporangiate strobili. The Middle Devonian (Givetian) Yuguangia ordinata [5] and the Late Devonian (Frasnian) Kossoviella timanica [22] are bisporangiate lycopsids reported from South China and Northern Russia, respectively. These two plants bear slender and occasionally dichotomized strobili that are morphologically similar to Sublepidodendron grabaui, while they differ in the type of spores and shape of the sporangium.
Kowieria alveoformis [21] from the Famennian of South Africa produces Lagenicula megaspores as in Sublepidodendron grabaui, while the numbers of megaspores per each sporangium in these two plants are different. Furthermore, K. alveoformis bears falcate sporophylls homomorphic to vegetative leaves that are dissimilar to those of S. grabaui. (Table 3) Sublepidodendron songziense occurs in the Upper Devonian Xiejingsi (previously known as Hsiehchingssu) Formation of the Hubei Province and the Wutong Formation of the Anhui Province, China. S. songziense and S. grabaui can be basically distinguished from each other by the different shapes of leaf bases and the ornamentations between them [24]. Detailed studies on these two plants' Lycospora-type microspores show that the distal surface of the S. songziense microspore is granulated [24], while that of S. grabaui is microspinate [12]. Meanwhile, S. songziense is characterized by a complex branching system consisting of many lateral branches along its trunk, whereas S. grabaui probably displays bifurcations in stems but occasional pseudomonopodial branchings in slender axes (Table 3) and thus may exhibit a different architecture with S. songziense.   Longostachys latisporophyllus [6] is a small arborescent lycopsid with megasporangiate strobili from the Middle Devonian (Givetian) of Hunan Province. Its stems are possibly at least four times dichotomized, and its leaves show spiny appendages. Slender strobili consist of spoon-shaped sporophylls and contain Laevigatisporites?-type megaspores. The megasporophyll of Sublepidodendron grabaui shows horizontal pedicel and upturned lamina and produces Lagenicula megaspores.

Comparison with Lycopsids Bearing Monosporangiate Strobili from China
Minostrobus chaohuensis [8,11,28] is another arborescent lycopsid with monosporangiate strobili found in the Wutong formation from South China. Its aerial axes display multiple dichotomies and fusiform leaf bases/cushions. Protoxylem confined to ridges at the periphery of primary xylem. Each megasporangium of M. chaohuensis contains a single tetrad of Lagenicula megaspores. Sublepidodendron grabaui differs from M. chaohuensis in the number of megaspores in each sporangium and the shape of leaf bases/cushions.
Changxingia longifolia is also reported from the Upper Devonian (Famennian) of South China and is interpreted as a small-sized lycopsid with monosporangiate strobili [10,15]. Its rhomboidal leaf cushions and oblanceolate leaf bases are helically arranged on the stems. Both C. longifolia and Sublepidodendron grabaui produce dichotomized stem and terminal megasporangiate strobili in pairs, while the former bears shorter strobili (20-50 mm in length), and each sporangium contains four Lagenicula-type megaspores.
Guangdedendron micrum shares the same fossil locality with Sublepidodendron grabaui and is regarded as the major tree lycopsid that made up the Xinhang fossil forest [14,27]. In the Leigutai Member of Yongchuan Section, G. micrum is widely distributed, while S. grabaui is restricted to the bottom portion. In G. micrum, the strobili are borne singly, in pairs, and occasionally once-dichotomized, resembling some specimens of S. grabaui; however, the S. grabaui stems dichotomize several times, while G. micrum stems rarely branch. Furthermore, S. grabaui shows a much smaller overall architecture than G. micrum: the axes, strobili and leaves of the former are roughly half the size of those of the latter. The strobili of G. micrum are 70-240 mm in length and 13-23 mm in diameter, while those of S. grabaui are 60-150 mm and 6-11 mm (Figure 8). Along the thick stems or the main trunks, G. micrum displays a fusiform leaf cushion (the length/width ratio = 6:1) with an elliptical leaf scar in the middle, while S. grabaui shows slenderer, elongated fusiform leaf bases (the length/width ratio = 12:1) with a fissure-like false leaf scar. In addition, the Lagenicula-type megaspores of the two plants can be clearly distinguished: megaspores of S. grabaui show smaller gula (gula/body = 0.34), whereas those of G. micrum possess larger gula (gula/body = 0.89).  [14], and this study.

Discussion
Heterosporous lycopsids occurred in the Middle Devonian [4][5][6] and underwent their first evolutionary radiation during the Late Devonian [3,5]. The previous phylogenic result shows that heterosporous lycopsids with monosporangiate strobili originated in those with bisporangiate strobili and are proposed to be the most derived clade [29][30][31]. Wang et al. [32] considered that the reproductive diversification of arborescent lycopsids occurred in the Late Devonian according to Lepidostrobus specimens. Paired or bifurcated strobili occurred in Famennian Sublepidodendron grabaui, as well as Guangdedendron micrum and several other taxa, and such a trait was interpreted as a reproductive strategy to produce more sporangia [14].
Lagenicula-type megaspores are characterized by a prominent gula. Till now, almost all Late Devonian lycopsids with monosporangiate strobili found in South China produce Lagenicula-type megaspores (e.g., Minostrobus chaohuensis, Sublepidodendron songziensis, and Guangdedendron micrum). However, diversification also exists among these Lagenicula megaspores, i.e., the gula height-to-body diameter ratio of these Late Devonian lycopsids varies. Such ratio is 0.34-0.52 in S. grabaui, M. chaohuensis, S. songziense and Changxingia longifolia, while 0.89 in G. micrum. The varied dimensions of gula and body among different species could result in a difference in adaptability to the environment, as some evidence suggests that megaspores with a prominent gula could adapt to the dispersal from a highly dense canopy [33]. In this regard, G. micrum is better adapted to the high density of communities than S. grabaui, which is consistent with the quantitative difference we observed between them in the Xinhang Forest.
The reduced number of megaspores per megasporangium is considered as a derived trait in the Suborder Dichostrobiles of Order Isoëtales sensu lato [8]. The Late Devonian Minostrobus and Changxingia show four megaspores in each megasporangium, while the Carboniferous Sigillaria shows numerous ones [34] and may thus hint a mosaic pattern of characteristic evolution of heterosporous lycopsids [30]. Moreover, some other lycopsids,  [14], and this study.

Discussion
Heterosporous lycopsids occurred in the Middle Devonian [4][5][6] and underwent their first evolutionary radiation during the Late Devonian [3,5]. The previous phylogenic result shows that heterosporous lycopsids with monosporangiate strobili originated in those with bisporangiate strobili and are proposed to be the most derived clade [29][30][31]. Wang et al. [32] considered that the reproductive diversification of arborescent lycopsids occurred in the Late Devonian according to Lepidostrobus specimens. Paired or bifurcated strobili occurred in Famennian Sublepidodendron grabaui, as well as Guangdedendron micrum and several other taxa, and such a trait was interpreted as a reproductive strategy to produce more sporangia [14].
Lagenicula-type megaspores are characterized by a prominent gula. Till now, almost all Late Devonian lycopsids with monosporangiate strobili found in South China produce Lagenicula-type megaspores (e.g., Minostrobus chaohuensis, Sublepidodendron songziensis, and Guangdedendron micrum). However, diversification also exists among these Lagenicula megaspores, i.e., the gula height-to-body diameter ratio of these Late Devonian lycopsids varies. Such ratio is 0.34-0.52 in S. grabaui, M. chaohuensis, S. songziense and Changxingia longifolia, while 0.89 in G. micrum. The varied dimensions of gula and body among different species could result in a difference in adaptability to the environment, as some evidence suggests that megaspores with a prominent gula could adapt to the dispersal from a highly dense canopy [33]. In this regard, G. micrum is better adapted to the high density of communities than S. grabaui, which is consistent with the quantitative difference we observed between them in the Xinhang Forest.
The reduced number of megaspores per megasporangium is considered as a derived trait in the Suborder Dichostrobiles of Order Isoëtales sensu lato [8]. The Late Devonian Minostrobus and Changxingia show four megaspores in each megasporangium, while the Carboniferous Sigillaria shows numerous ones [34] and may thus hint a mosaic pattern of characteristic evolution of heterosporous lycopsids [30]. Moreover, some other lycopsids, including Kowieria with bisporangiate strobili, and Wuxia with intercalary megasporangiate fertile zones, also contain four megaspores in each of their megasporangium [16,21]. There-fore, the reduced number of megaspores per megasporangium in lycopsids might be a trait with multiple independent origins beyond the Suborder Dichostrobiles.