Guazia, the earliest ovule without cupule but with unique integumentary lobes

Abstract The earliest ovules in the Late Devonian (Famennian) are surrounded by a cupule that is involved in both protection and pollination, and generally have free integumentary lobes. Here we report a new taxon from the Famennian of China, Guazia dongzhiensis gen. et sp. nov. The terminally borne ovule is apparently acupulate (without cupule) and has four radially arranged wing-like integumentary lobes that are extensively fused, and folded lengthwise and inwards. Guazia provides evidence that not all Devonian seeds possess a cupule and it increases their diversity in integumentary lobes. This genus also suggests that the integuments develop new functions, probably including wind dispersal at the expense of the cupules.


INTRODUCTION
The earliest ovules or seeds (fertilized ovules) in the Late Devonian (Famennian, 372-359 million years ago [Ma]) have been reported from Europe, North America and China [1]. The cupule, derived from fusion of a vegetative branching system, is an accessory structure with free or united segments surrounding one to several ovules [2,3]. Famennian ovules are (usually) borne within a segmented cupule [4] that is thought to have functioned as protection while also aiding in pollen capture [2,5].
Famennian ovules are characterized by an integument (developing into a seed coat covering the megasporangium termed nucellus) that is variously lobed, more or less free from the nucellus and serves to increase protection and pollination [2]. These ovules lack a well-defined micropyle as a small hole in the more complete integument, which was evolved by later seed plants (spermatophytes) and through which the pollen enters [6].
In this study, a new taxon, Guazia dongzhiensis gen. et sp. nov., from China is known to have borne terminal ovules on the branches and represents the oldest acupulate ovule. Hitherto, Warsteinia from Germany was the only Famennian ovule with four wing-like integumentary lobes that may have been adapted for wind dispersal (anemochory), but its attachment and cupule remain unclear [7,8]. Guazia is shown to be another Famennian ovule with four radially symmetrical wing-like integumentary lobes. Its acupulate ovule with unusual integument provides new insights into the early evolution of seed plants.

Locality and stratigraphy
The plant fossils were collected from the Wutong (Wutung) Formation at Xiangkou Section (30 • 03 57 N and 116 • 47 26 E), Xiangyu Town, Dongzhi County, Anhui Province, China (Fig. S1A-C). This formation, widespread in the lower reaches of the Yangtze River including Anhui, consists of the Guanshan Member characterized by quartzose sandstone and conglomerate, and the overlying Leigutai Member with inter-beds of quartzose sandstone and mudstone [9]. Assemblages of plants, fish, conchostracans and especially spores indicate that the Wutong Formation (possibly excluding the uppermost part) is Upper Devonian (Famennian) [9][10][11]. At the Xiangkou Section, the progymnosperm Archaeopteris halliana occurs in the basal part of the Leigutai Member [12]. Guazia dongzhiensis and a pollen organ Placotheca minuta [13] were C The Author(s) 2021. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. found separately ca. 10 m and 11 m above the A. halliana horizon (Fig. S1D). Another pollen organ Telangiopsis sp. [14] sometimes occurs together with G. dongzhiensis. Archaeopteris was distributed worldwide in the Late Devonian [2,15]. These data indicate a Famennian age for G. dongzhiensis. From a layer of muddy siltstone, we obtained ∼300 specimens of ovules preserved as impressions or compressions with coaly material preserved.
Etymology: the generic name is from 'Guazi', the Chinese pinyin for sunflower seeds with a shell, referring to the shape of the organ; the specific epithet is derived from Dongzhi, the name of the county where the fossils were collected.
Holotype: PKUB14701a, b (part and counterpart housed in the Department of Geology, Peking University, Beijing), dichotomous axes with an acupulate ovule borne terminally ( Fig. 1A and B).

Description
Fertile axes are preserved with multiple orders of branching, and the isolated fossils examined show one to three orders of dichotomies. They are twice ( Fig. 1A-E) or once ( Fig. 1F-I), and rarely thrice (Fig. 1J) dichotomously branched at angles of 20-90 • . The longest branching system excluding the ovules is 29.6 mm (Fig. 1A). The internodes are 2.0-18.8 mm long and the fertile axes are 0.2-1.0 mm wide. The branching is probably threedimensional because two axes produced by successive dichotomies depart at different angles ( Fig. 1A, arrows, and C, arrows), with one axis plunging into the rock matrix (Fig. 1A, upper arrow, and C, lower arrow). Some ultimate axes are terminated by a single ovule, whereas others lack ovules or are distally broken (Fig. 1). The boundary between the ultimate axis and ovule is difficult to distinguish, but it may be at the point where the basipetal reduction of width ceases (Fig. S2, arrows). The ultimate axes bearing ovules are 2.8-15.2 mm long and 0.3-0.8 mm wide. In most cases the ovules are detached, and either preserved with the distal part of the ultimate axis ( In transverse sections of the ovules, two opposite integumentary lobes extend along the bedding plane and were originally perpendicular to the other two opposite lobes ( Fig. 4b-n, Figs S8H-Z, a, b, 9D-W, 10a-g, 11A-J, a-f). During diagenesis, however, the other two lobes were usually compressed more or less along the bedding plane. Sections of an ovule show that one integumentary lobe is perpendicular to two opposite lobes ( Fig. 4o-r, Fig. S10D-J) and then compressed ( Fig. 4s and t, Fig. S10K-P). Multiple serial sections demonstrate that the four radially arranged integumentary lobes are narrow in the lower part of an ovule, becoming gradually wider towards the nucellar apex, narrowing again after overtopping the nucellus, and finally converging (Figs S7A-C, 8H-Z, a-d, 9D-W).
In transverse sections of the ovules, the individual integumentary lobes are flattened in the lower parts of an ovule (Figs 4b, j, 5A-A , Figs S10B, C, a-g, 11a), become thick upwards (Figs 4c-e, k, l, or, 5B-B , Fig. S10h), present a large V or U shape when free near the nucellar apex (Figs 4f, g, m, n, s, t, 5C-C , Fig. S11A-E, b, c), form a small V or U shape over the nucellar apex (Figs 4h, i, 5D-D , Figs S11F-I, d-f), and become flattened distally (Figs S8c, d, 11J). Therefore, the integumentary lobes are symmetrically folded along the abaxial side and toward the center. The surface of the integumentary lobes appears uneven with tiny indentations ( Fig. 4D and F, Figs S8e, 9X, 11g), probably representing a parenchymatous outer layer of the integument (sarcotesta). These indentations and taphonomic compression sometimes result in the distortion and interruption of a single V-or U-shaped integumentary lobe in transverse section.
The nucellus may not be easily recognized in some ovules (Figs 1C-G, J, 2C, Figs S3, 7). However, well-preserved ovules and dégagement show that the nucellar apex is dome-shaped in lateral view, 1.0-1.7 mm long and 0.7-1.2 mm wide at the base (Figs 2D-H, 3, 4H, Figs S4-6). Transverse sections occasionally display the nucellar apex (Fig. 4f, arrow, and n, arrow). With the exception of the apex, the nucellus is closely fused to the integument (Figs 3,  arrows, 4H, Figs S4, 5). The nucelli are at least 5.7 mm long (Fig. 2H), reaching ∼11.5 mm in length (Fig. 2D), with a maximum width of 0.8-1.4 mm, and distally surrounded by the free parts of the integumentary lobes. The ovule is reconstructed with the radially arranged integumentary lobes and nucellar apex (Fig. 5).

DISCUSSION
The fertile fronds of the Late Devonian seed plants Elkinsia and Dorinnotheca branch dichotomously [16,17]. The ultimate axes with terminal cupulate ovules of Elkinsia are cruciately arranged in three dimensions, while those of Dorinnotheca are pinnately arranged and the ovules pendulous. The fertile branching of Guazia is, where known, also dichotomous. The ultimate axes are terminated by single ovules and there appears limited evidence that these axes occur in different planes. Of Devonian ovules, Guazia most resembles Pseudosporogonites [18] in its radial symmetry and four broad integumentary lobes. However, the ovule of Pseudosporogonites is surrounded at the base by a collar-shaped cupule; the three or four integumentary lobes are only fused for 1/3 length of the in-tegument and are tangentially arranged around a nucellus; the tips of the integumentary lobes and nucellus are located at approximately the same level. Guazia lacks a cupule, but possesses an integument that surrounds the nucellus (except for its apex) and produces four radially arranged outgrowths. These outgrowths are interpreted as wing-like integumentary lobes. It is sometimes difficult to distinguish the nucellus from the closely adpressed integument.
All Devonian ovules so far described for which attachment is known are surrounded by a cupule [4,18] that was presumably involved in both protection and pollination [2,5,19]. The wing-like ovules of Warsteinia are detached and the cupule unknown [7], but they are assumed to have been cupulate as in other Devonian ovules [18]. The ovules of Guazia are attached to branches and clearly lack a cupule, providing the first convincing evidence for acupulate ovules in the earliest seed plants. The telome theory proposes that the cupule evolved from the coalescence of the surrounding vegetative dichotomous branches [2,20]. In this context, the absence of cupules in Guazia might have resulted from a reduction of these branches.
Cupules disappeared in the Carboniferous (Late Mississippian)-Permian medullosan spermatophytes and later in several other derived lineages; instead, the importance and complexity of the integument increased [2,4]. Like Guazia, some medullosan ovules are borne terminally on dichotomous branches and possess four integumentary wings [21]. Among Devonian ovules, Natl Sci Rev, 2022, Vol. 9, nwab196 Pseudosporogonites and Latisemenia have a very short cupule only surrounding the ovule base [18,22]. The integumentary lobes of these two genera, especially Latisemenia, are so broad and largely fused as to effectively protect the nucellus. The nucellus (except at the tip) of Guazia is adnate or close to the integument and the free portions of the integumentary lobes extend a long distance above the nucellar apex, fold inwards and often curve centripetally. In the absence of a cupule, such an integument highlights its role in protection. In contrast, the free portions of the integumentary lobes of Warsteinia are short, flat, straight and open. The integumentary lobes of early ovules suggest pollen capture by wind, i.e. anemophily as a pollination syndrome [2]. Thus, the integument of Guazia appears to have the same structural function of protection and pollination as the cupule. Guazia may also perform the function of potential wind dispersal by wing-like integumentary lobes, despite losing the protective and pollinating functions of the cupule (see below).
Winged diaspores (e.g. seeds or fruits) of fossil and living seed plants vary greatly in size but can promote (horizontal) wind movement during their abscission from the source plant, especially if given enough height [2,[23][24][25][26]. In addition, having landed, a winged diaspore may be blown along the ground, a movement termed secondary wind dispersal [27]. Late Devonian Warsteinia with four wing-like integumentary lobes suggests its possible adaptation for wind dispersal; the lobes increase floatation and saltation/adherence before and after landing, respectively [7,8]. Guazia is the second Devonian ovule with such radially arranged lobes. The lobes appear thin as in Warsteinia, however, fold lengthwise and usually arch centripetally above the nucellar apex. This ovulate morphology of Guazia may not have been much effective in primary dispersal (airborne movement), but could potentially facilitate the secondary dispersal by wind. Most specimens of Guazia (ca. 90% collections) were found as detached ovules with ultimate axis tips sometimes preserved. They provide clear evidence that the ovules were shed, and the narrow connection of the ultimate axis to the ovule suggests the likely point of disarticulation.

MATERIALS AND METHODS
All specimens in this study were collected in 2006-2008, and are deposited in the Department of Geology, Peking University, Beijing, China. Steel needles were used to expose some fertile axes and ovules. Serial dégagement was employed to reveal the morphology and structure of the ovules. Ovules were embedded in resin, sectioned and ground to display the integumentary lobes. Maceration of the specimens produced only fragments of organic matter, so we have failed to see some details of the ovules (e.g. megaspore). All photographs were taken with a digital camera and Leica microscope. Using Autodesk 3ds Max 2017 software, we generated the reconstruction of the ovule (Figs 4G and H, 5).