Re-study of Guangdedendron micrum from the Late Devonian Xinhang forest

Guangdedendron micrum is the Late Devonian tree lycopsid that made up Xinhang fossil forest in Anhui, China, showing the earliest stigmarian rooting system. Based on new specimens of this lycopsid, the roots bearing rootlets, terminal parts of stems, vegetative leaves and monosporangiate strobili containing megaspores are researched in detail. The roots with four robust rhizomorphs are largely expanded and approach the size of those of the Late Carboniferous giant tree lycopsids in swampy forests. The rootlets along rhizomorphic axis leave oval to circular scars after abscission. Narrow-fusiform leaf cushions display a leaf scar, vascular bundle and ligule pit. Cylindrical megasporangiate strobili are borne singly, in pairs, or occasionally once-dichotomized. Of each megasporophyll, the pedicel consists of a keel and possibly undeveloped alations, and the long-triangular lamina presents a heel. Megasporangium is sessile and contains multiple Lagenicula megaspores with distinct spines and a large gula. G. micrum displays large terminal monosporangiate strobili probably adapted to turbulent condition, and its megasporophylls together with multiple Lagenicula-type megaspores hint a possible primitive evolutionary status. These characteristics provide new insights into the evolution of fertile traits of early lycopsids.


Introduction
The evolutionary radiation of vascular plants in the Devonian is one of the major events in the history of life, leading to the establishment of the terrestrial ecosystems [1,2]. During the Devonian, trees originated and evolved independently in three major taxa, including pseudosporochnaleans (a kind of fern-like plants or possible stem ferns), archaeopteridaleans and lycopsids [3][4][5][6]. The Late Devonian is an important period for lycopsids, during which they rapidly evolved or diversified crucial traits such as bipolar growth, heterospory and arborescence [7,8]. Arborescent lycopsids then dominated Carboniferous swamp forests ecosystem [9] and occupied the majority of biomass that turned into coals [10,11]. However, Devonian forests preserved in-situ are rarely known and mostly limited to Euramerica [1,5,6,12,13], and only the Late Devonian (Frasnian) Svalbard forest was reported before 2019 to consist of in-situ fossil lycopsids [1].
South China is regarded as a diversity hotspot of Late Devonian lycopsids as well as a potential research region for arborescent lycopsid evolution and Devonian lycopsid forests [5,14]. Recently, Xinhang forest, a Famennian insitu forest, was reported from Xinhang Town, Guangde City, Anhui Province of China [15], which primarily consists of a species of lycopsid, Guangdedendron micrum, that bears the earliest stigmarian rooting system. With the progress of clay excavation by local company, we keep on collecting plant specimens in Xinhang area. Based on new fossils, this article further studies the vegetative and reproductive characteristics of G. micrum, and provides information about its rhizomorphs, rootlets, vegetative stems bearing leaves, and monosporangiate strobili with megaspores.
Systematics Specific diagnosis (based on descriptions of Wang et al. [15] and this study): As for generic diagnosis. Rhizomorphs over 27.0 cm in depth. Rhizomorphic axes 8.3-31.0 cm long and 0.3-7.8 cm wide, forming angles of 19°-60° to ground surface. Rootlets up to 27.2 cm long and 7.0 mm wide. Rootlet scars circular and 2.5-3.9 mm in diameter. Preserved parts of in-situ stems reaching 88.0 cm in height and up to 18.7 cm in diameter, with rare dichotomy. Vegetative leaves 2.0-9.2 cm long and 1.2-9.0 mm wide, each with a single vein and an entire margin. The length-width ratios of leaf bases or leaf cushions ca. 6:1. Fertile axes with persistent leaves, terminated by strobili. Megasporangiate strobili cylindrical, with maximum length and width (excluding sporophyll laminae) of 23.4 cm and 3.0 cm, respectively. Strobilar axes up to 3.0 mm in width. Sporophyll laminae long-triangular in shape with the maximum length and width of 18.0 mm and 5.8 mm, showing entire margins. Basal portion of each lamina forming a downturned, inverted triangular heel. Sporophyll pedicels 6.0-8.0 mm in length, approximately perpendicular to strobilar axis. Pedicel displaying an abaxial keel 0.5-0.8 mm in height. Megasporangium horizontally elongated, 4.0-7.4 mm long. Megaspore ca. 1.5 mm in polar axis length, consisting of a smooth gula and a spherical body with spiny ornamentations.

Description
The description in this article is based on a new fossil collection of Guangdedendron micrum as well as several specimens introduced in Wang et al. [15]. Plant organs described here include rooting system (Fig. 1, 2), stems and vegetative axes bearing leaves and leaf cushions or leaf bases (Figs. 3,4), and strobili with the 3-D reconstruction (Figs. 5, 6) displaying megaspores (Figs. 7,8).
The stigmarian rhizomorph has four evenly separated axes and then usually dichotomizes once (  Comparison with Guangdedendron micrum described by Wang et al. [15] (See figure on next page.) Fossils in this study are collected from the same sections and morphologically consistent with Guangdedendron micrum, which is considered as a possible monocarpic and dioecious tree lycopsid bearing stigmarian rhizomorphs [15]. This study supports the former conclusions and adds new traits of rootlet scars, stems and axes bearing vegetative leaves, terminal megasporangiate strobili and megaspores. The length and width of rhizomorph axis are expanded, approaching in the size of Stigmaria ficoides from the Middle Pennsylvanian [16]. Stems of 8.0-10.0 cm width (Fig. 1A, C, E, 3A-K) and axes with dichotomy ( Fig. 3L-O) are found again, but they are still unusual, thus supporting G. micrum as a small tree with an advanced rooting system type and a simple crown [15]. The lower portions of stems lack vegetative leaves but show leaf cushions indicating leaf abscission. Vegetative terminal twigs in Fig. 4A-C with leaves gradually shorten upwards may indicate apex of juvenile plants of G. micrum, as reconstructed in Fig. 6A in Wang et al. [15]. The strobili of G. micrum are large and often borne in pairs, and occasionally dichotomize once. tituted different populations and lived far from the fossil locality, or the microsporangiate strobili are difficult to be preserved or identified.

Comparison with Late Devonian heterosporous lycopsids bearing monosporangiate strobili in China
Despite an even larger collection of Guangdedendron micrum, no strobili containing microspores have been found. Previous studies of coeval heterosporous lycopsids often report mega-and microsporangiate strobili [17][18][19]. One possible explanation is that G. micrum has few male individuals and parthenogenesis happened, while other interpretations include that the male individuals consSeveral Late Devonian members of the Isoëtales sensu lato have been reported in China, e.g. Sublepidodendron songziense [17,20,21], Sublepidodendron grabaui [19,22], Minostrobus chaohuensis [18,23], Changxingia longifolia [24] and Changxingia sp. [25]. These taxa, together with Guangdedendron micrum, could be assigned to the Suborder Dichostrobiles, i.e. isoetaleans that produce monosporangiate strobili [26]. Major morphological traits of these plants are compared in Table 1, while a more detailed comparison is given (see Additional file 4: Table S1). Among these plants, most of them are recognized as arborescent, while G. micrum and possibly S. songziense bear stigmarian rooting system. Changxingia longifolia and Changxingia sp. display the smallest sizes of stems and megasporangiate strobili. S. songziense and S. grabaui possess lateral branches, but G. micrum bears a crown with fewer bifurcations. G. micrum shows the leaf bases of similar shape with M. chaohuensis and S. grabaui, but the latter displays only leaf bases with false leaf scars and no typical leaf cushions. Strobili of G. micrum are often in pairs and occasionally dichotomous, and larger than those of other coeval taxa. Alations along sporophyll pedicels are distinct in M. chaohuensis but relatively undeveloped in G. micrum, C. longifolia and S. songziensis. Considering the number of megaspores in each megasporangium, G. micrum and S. songziense display multiple but C. longifolia and Changxingia sp. possibly four, and M. chaohuensis contains four megaspores with some of them aborted. All of these plants exhibit Lagenicula megaspores with distinct gula, which is however larger in G. micrum.

Comparison with Late Devonian heterosporous lycopsids outside of South China
Several Late Devonian heterosporous lycopsids outside South China are known for their bisporangiate strobili. Two such lycopsids were recently reported from Gondwana palaeocontinent. Cymastrobus irvingii is a 3-D preserved bisporangiate strobilus from Famennian in New South Wales, Australia [27]. Its megasporangia contain a  large number of megaspores, up to 500 μm in diameter. Casts of the megaspores show numerous circular pores surrounding the trilete mark and in several rows but no gula, thus differing from the Lagenicula-type megaspores in Guangdedendron micrum. Kowieria alveoformis from the Famennian of South Africa produces up to four Lagenicula megaspores [28] similar with those of G. micrum, while they differ in the number of megaspores per sporangium. In addition, K. alveoformis bears sporophylls homomorphic to vegetative leaves, in contrast with those of G. micrum. Clevelandodendron ohioensis is a Famennian lycopsid from the USA [29]. It displays a straight and totally unbranched stem terminated by a single bisporangiate strobilus, and contains Triletes megaspores and microspores. Unlike species mentioned above, Jurinodendron (= Cyclostigma) kiltorkense bears monosporangiate strobili and was a widespread taxon during the Upper Devonian and Early Mississippian [30]. J. kiltorkense is similar with G. micrum in the shape of sporophylls and Lagenicula-type megaspores [31]. J. kiltorkense differs from G. micrum in its small circular leaf scars (about 1.5 mm in diameter) and lacking of leaf cushions.

Discussion
Most Devonian lycopsid fertile zones or strobili show no bifurcations [8,32]. However, bifurcated strobili or fertile zones occurred in several taxa of the Middle-Late Devonian lycopsids, e.g. the Givetian Yuguangia ordinata [33], Frasnian Kossoviella timanica [34] and an undetermined "type C" [35], and Famennian Hefengstrobus bifucus [36] and Guangdedendron micrum (see details in Table 2). The evolutionary route of this trait is unclear. In most cases, the two resulted parts of strobili show nearly similar width as the strobilus before bifurcation. We suggest that the bifurcated strobili or fertile zones are produced by the dichotomy of an apical meristem after the shoot turns into reproductive growth. Alternatively, the last branching point on the terminal fertile axes may represent an earlier bifurcation than the transition to reproductive growth, i.e., the sequence of reproductive growth differentiation and apical meristem bifurcation controls the growth pattern of lycopsid strobili. In G. micrum, the strobili could be borne in pairs or bifurcated, which together indicate that the apical meristem has a relatively independent potential for bifurcation and sporophyll differentiation. Many tassel-fern species (especially Phlegmariurus) included in living Lycopodiales show similar dichotomized fertile zones indicating ready shift from vegetative branches to fertile branches (strobili) [37]. Large size and multi-dichotomized strobili may correspond to individual's adaptation in this group to the epiphytic habit [37]. Strobili of G. micrum are various in growth patterns and sizes with lengths ranging from 5.0 [15] to 23.0 cm (9.6 cm on average), and individuals bearing large strobili may result in improved production of offsprings and surviving the turbulent condition near coastal area.
The earliest heterosporous lycopsids appeared in the Middle Devonian [33,38,39], and over ten heterosporous lycopsid genera have been reported so far from the Late Devonian [14]. Many of these early heterosporous lycopsids produce the gulate Lagenicula megaspores (see details in Table 3), while Guangdedendron micrum presents the relatively larger gula. Lagenicula appears not only in bisporangiate strobili of Givetian Mixostrobus, Famennian Kowieria as well as Carboniferous Flemingites [28,38,40,41], but also in the megasporangiate strobili of all the members of Suborder Dichostrobiles reported from the Upper Devonian of South China [14,25]. However, the taxa dominating the Carboniferous swamp, e.g. Sigillariostrobus, Lepidocarpon and Achlamydocarpon, bear megaspore types including Tuberculatisporites and Cystosporites [42][43][44]. We consider that there was a significant evolutionary change in the megaspore type between the early and late representatives of heterosporous lycopsids.
During the Middle-Late Devonian, the lycopsids evolved well-differentiated strobili and sporophylls [45]. Each specialized sporophyll is composed of a pedicel and an upturned or curved lamina. Reduction in the number of megaspores per sporangium together with alations widened and upturned to enclose the sporangium are considered as evolutionary trends within the Suborder Dichostrobiles [23,[46][47][48]. In one megasporangium, Minostrobus chaohuensis contains one functional megaspore and three aborted megaspores, which is regarded as the most derived taxon among the Famennian members of Dichostrobiles [23]. As for the other taxa, Changxingia longifolia and Changxingia sp. have probable four megaspores per sporangium, while Guangdedendron micrum and Sublepidodendron songziense [21] show multiple megaspores that are considered primitive. On the other hand, the alations of C. longifolia and S. songziensis resemble those of Achlamydocarpon, which leave the sporangium largely exposed and represent a primary status, while Minostrobus represents the intermediate [23] and Lepidocarpon the most derived [46]. Although carefully serial dégagement has been applied to the G. micrum sporophylls in lateral and face views, the existence of alations still cannot be confirmed, but suggests undeveloped alations probably comparable with C. longifolia and S. songziensis. The relatively primitive characteristic combination of G. micrum hints a basal position in the lineage of Suborder Dichostrobiles. In addition, as suggested by Philips and DiMichele [49], there are many megaspores exposed on the surfaces of compressed strobili, probably indicating the free sporing habit.

Materials and methods
Specimens of fossil plant Guangdedendron micrum in this study were further collected from the same localities and horizons illustrated by Wang et al. [15], i.e., the upper part (Leigutai Member) of the Upper Devonian