The Earliest Fleshy Cone of Ephedra from the Early Cretaceous Yixian Formation of Northeast China

Bracts of female cones of extant gymnosperm Ephedra (Joint fir) are either colorful and fleshy (section Ephedra), or dry-winged and membranous (section Alatae), or dry and coriaceous (section Asarca), which have played a crucial role in long-distance seed dispersal that is responsible for a wide distribution of the genus in semiarid and arid areas of Eurasia, North Africa, North America, and South America. Recent molecular systematic studies on Ephedra have suggested that the fleshy bracts in character evolution may be plesiomorphic relative to the dry, membranous and coriaceous bracts. However, little is known about when the fleshy bracts of Ephedra have made their debut in the geological past. Herein, we describe a novel, fleshy bract-bearing female cone macrofossil from the Early Cretaceous (ca. 120—125 Ma) Yixian Formation in Liaoning, northeastern China. This cone bears three ellipsoid seeds subtended by only one whorl of fleshy bracts. Each seed has a thin outer envelope and an inner integument that extends upward and passes through the opening of the outer envelope, forming a thin and straight micropylar tube. Such a syndrome shows the closest similarity to an extant triovulate species Ephedra intermedia in the section Ephedra, but the latter bears a whorl of terminal fertile bracts and more than one whorl of inferior sterile bracts, and a thick outer envelope. Hence, we establish a new fossil species Ephedra carnosa. Our discovery provides the first direct macrofossil evidence for the previous molecular systematics of Ephedra, implying that the origin of fleshy bracts in Ephedra should not have been later than that of the membranous and coriaceous bracts by at least the Early Cretaceous.


Introduction
The gymnospermous genus Ephedra L. (Joint fir) contains about 50 living species, native to semiarid and arid areas of Asia, Europe, North Africa, North America, and South America ( [1][2][3][4][5][6][7]; Fig. 1). This genus has three types of female cones upon which a once widely accepted sectional classification is based [8], i.e., Sect. Alatae Stapf bears free, dry, winged, and membranous bracts ( Fig. 2A), Sect. Asarca Stapf has free, dry, but coriaceous bracts (Fig. 2B), while Sect. Ephedra possesses thickened, colorful, and fleshy bracts (Fig. 2C). Recent molecular phylogenetic studies have suggested that the three morphological sections are not natural and none of them is monophyletic, but the earliest diverged branch within the genus comprises species with fleshy cones from the Mediterranean region [9][10][11][12]. Meanwhile, Sect. Alatae and Sect. Asarca are nested within Sect. Ephedra, implying that the fleshy cone is plesiomorphic in Ephedra. However, when the first fleshy cone occurred in the fossil record remains unknown. Ephedra macrofossils (especially female cones) will provide an historical perspective for the early evolution, taxonomy, and biogeography of the genus.
Early Ephedra might have transformed bracts of female cones into fleshiness and vivid color to assist seed dispersal [13][14][15], resulting in a wide intercontinental distribution of the genus. However, little is known about when such characteristic fleshy cones occurred in Ephedra. According to molecular clock data, modern Ephedra was estimated to have a minimum age 8-32 Ma (i.e., an Oligocene origin) [11,16]. However, it lags behind the first occurrence of Early Cretaceous meso-and macrofossils bearing clear morphology that can be reliably circumscribed within Ephedra, e.g., E. archaeorhytidosperma Yang et al. [17], E. portugallica Rydin et al., E. drewriensis Rydin et al. [18], and E. hongtaoi Wang et Zheng [19]. Therefore, pruning of early stem groups and extinctions of ancient lineages may have played an important role in the early evolution of Ephedra [11,12,20]. It is expected that the putatively plesiomorphic character bearing fleshy cones in Ephedra would have made their debut earlier than Oligocene.
In recent decades, numerous Ephedra and Ephedra-like meso-and macrofossils have been reported from the Early Cretaceous of South Europe, Northeast China, Mongolia, North America, and South America [17][18][19][21][22][23]. Seed mesofossils with in situ pollen were reported from the Early Cretaceous of South Europe (Portugal) and North America [18,24]. Macrofossils of reproductive shoots or female cones were found in the Early Cretaceous of South America [25,26], and Mongolia [27,28] and adjacent Northeast China [17,19,[21][22][23]29,30]. Early Cretaceous strata of Northeast China contain a number of well-preserved ephedroid macrofossils that may shed light on the early evolution of Ephedra. They show high reproductive diversity but similar vegetative morphology, e.g., dichasial branching pattern (sometimes branches being clustered due to highly shortened internodes), long and linear leaves usually with two parallel veins, and internodes with longitudinal fine striations, which can be divided into three groups. The first group bears female cones with multiple whorls of fertile bracts, e.g. Liaoxia Cao et Wu ( = Ephedrites Göppert et Berendt) [21,31]; the second group bears female cones with only one whorl of fertile bracts, e.g., Ephedra hongtaoi and E. archaeorhytidosperma [17,19] and several species ascribed to Gurvenella Krassilov ( = Chaoyangia Duan) bearing trichotomous complex surrounding female cones [27,32,33]; the third group is Siphonospermum Rydin et Friis which has female cones without supporting bracts [22]. So far, all the previous studies have not provided any fossil evidence for the origin of fleshy female cones of Ephedra.
In this paper, we aim to describe a new, freshy cone-bearing fossil species, Ephedra carnosa Yang et Wang sp. nov., from the Early Cretaceous of Liaoning Province, Northeast China. Our discovery provides the first direct macrofossil evidence for the previous molecular systematics of Ephedra, implying that the origin of fleshy bracts in Sect. Ephedra should not have been later than that of the membranous and coriaceous bracts in Sect. Alatae and Sect. Asarca by at least the Early Cretaceous.
The macrofossils are preserved as impressions lacking cuticle and include a part and a counterpart from a gathering slab of finely laminated light grey to yellowish siltstone. Figures 1, 3 presented here were redrawn from the base maps, respectively [5,40]. Voucher specimens (WH Lipsky 4181 and L Benson 15280) of Ephedra californica Watson and E. strobilacea Bunge were examined at the China National Herbarium (PE), including two female cones used in Figure 2A-B. The female cone of Ephedra intermedia Schrenk et Mey. presented in Figure 2C was photographed by the senior author at Mt Renshoushan of Gansu Province, Northwest China. Macrofossils were photographed with digital cameras (Nikon D700 and Panasonic DMC-FZ30) and under a microscope (Nikon Eclipse E600). Comparisons were made in Table 1 with some coeval, similar Ephedra and ephedroid meso-and macrofossils. All figures were arranged by Adobe Photoshop 6.0 and CorelDraw 10.0 programs. Terminology on the female cones of Ephedra [6,[42][43][44] used in the specimen descriptions was adopted. The specimens (including two gatherings PE 20120319A and 20120319B, 2012071006) are deposited at the China National Herbarium (PE), Institute of Botany, Chinese Academy of Sciences, Beijing, P. R. China.

Nomenclature
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Results
The three extant gymnospermous genera, Ephedra L., Gnetum L., and Welwitschia Hooker, have been widely treated as a natural taxon, either a class Gnetopsida or an order Gnetales, with their own monogeneric families [3,6,7,45,46]. Therefore, our new fossil species of Ephedra is classified as follows: Gnetopsida

Diagnosis
Triovulate cones have but only one whorl of bracts. Each bract is apparently thickened and spreading and subtends a seed. The bract bears two veins sub-parallel in the middle-upper part and divergent toward the basal part. Seeds are ellipsoid. The outer envelope is thin. The inner integument is fused with the nucellus, but only its apical part extends upward and passes through the outer envelope opening, forming a slim and straight micropylar tube.

Description
The fossils presented here are triovulate cones, having but only one whorl of bracts [ Fig. 4A Fig. 4F-G]. The interval between the two veins is wider at the base (up to 620 mm) and becomes narrower (ca. 207 mm) in the middle part, and then the two veins are parallel (ca. 138 mm) to each other towards the apex. Each bract subtends a seed. Seeds are ellipsoid, flat at the ventral side and arched at the dorsal side, about 1.5 mm wide and 2.6-3 mm long. The outer envelope is thin. The inner integument appears to be fused with the nucellus, but only its apical part extends upward and passes through the opening of the outer envelope to form a micropylar tube [Fig. 4E]. The micropylar tube is slim and straight, the exposed part is approximately1.3 mm long.

Etymology
The specific epithet is derived from Latin ''carnosus'', denoting the apparently thickened (thereby fleshy) bracts of female cones.

Stratigraphic horizon and age
Yixian Formation, the early Aptian-earliest late Aptian of the Early Cretaceous.

Comparisons
The new fossil species Ephedra carnosa Yang et Wang is noticeably different from any other known extant and fossil species in Ephedra and other ephedroids [ Table 1].

Remarks
Bracts of female cones of Ephedra are modified foliar organs in nature. In living Ephedra, cone bracts have three states according to their mature morphology, e.g., fleshy bracts, dry and coriaceous bracts, and dry and membranous bracts (see Introduction, Fig. 2A-C). The thickness of median portion of cone bracts between the two parallel vascular bundles varies in the three sections of Ephedra, Sect. Ephedra (1.2-1.8 mm in E. intermedia), Sect. Asarca (ca. 80 mm in E. californica), and Sect. Alatae (ca. 40 mm in E. torreyana) (unpublished personal observations). This demonstrates that the fleshy cone bracts are markedly thicker than dry (either coriaceous or membranous) bracts in Ephedra. Similarly, the triovulate cones of our new macrofossil species Ephedra carnosa bear apparently thickened bracts without clearly defined margins, which are far more likely to be compared with those fleshy bracts in living Ephedra. In addition, two veins in each bract are sub-parallel in the middle-upper part (ca. 138-207 mm apart) but apparently divergent toward the basal part (up to 620 mm apart), implying that the bract would be most possibly swollen in life. Hence, the cone bracts of macrofossils Ephedra carnosa presented here are ripe and fleshy.
The triovulate cones appear to have abscised from the reproductive shoots as a mature disseminule (or diaspore). Almost intact micropylar tubes in fossils imply that the cones have not been transported far from the parent plants before fossilization.
On the basis of its trimerous nature, three seeds of each cone must be subtended by three verticillate bracts, which are 120 degrees distant from each other. Two bracts are visible, so the third bract might have run into the embedding rock after the cone fell into soft sediments, fossilized and then opened along the cutting plane. Building on the above morphological description and taphonomic inference, we present here a schematic reconstruction of the triovulate cone and its seed [ Fig. 5].
There is an associated axis in the specimens besides the female cone described above. The axis is straight, 1.3 cm long and 0.4 mm wide, with fine longitudinal striations (Fig. 4A-B). These features imply that the axis is likely to be the vegetative twigs of a same parent plant (or population) as the female cone. Due to lacking organic connection and other useful epidermal evidence between them, we do not include the axis into the description of this new species.

Characteristic thin outer envelope
The outer envelope of the new macrofossil species Ephedra carnosa seems very thin and vulnerable, so it is easy to be transformed after compression. Also, the integument extended into a micropylar tube enclosed in the outer envelope is visible. In contrast, modern Ephedra with fleshy bracts usually bears a thicker and harder outer envelope and a very thin integument with only 1-2-cell thick [44,45]. In addition, Sect. Alatae usually bears thin outer envelope and integument while Sect. Asarca and Sect. Ephedra usually bear a thickened outer envelope. As a result, our new fossil species is quite unique in that it bears fleshy bracts and a very thin outer envelope.
The outer envelope of modern Ephedra has three types of ornamentations, including smooth surface (e.g., E. intermedia, E. distachya L., E. aphylla Forssk., and E. tweediana Fisch. et Mey.), papillate type (e.g., E. major Host, and E. funerea Coville et Morton), and transverse lamellar sculpture (e.g., E. rhytidosperma Pachomova, E. trifurca Torr. [6,47,48]. The smooth and/or striated surfaces are very common in both extant and fossil species while the other two types are only restricted to a few species. The transverse lamellar Table 1. Key to extant and fossil species in Ephedra and other ephedroids. sculpture can also be found in fossil species from the Early Cretaceous [17,[49][50][51] and might have multiple origins [52]. Our new fossil species Ephedra carnosa bears the outer envelope with the smooth type of seed surface. Surface sculpture of the outer envelope may be variable in the developmental sequence. In both E. equisetina Bunge and E. rhytidosperma, the outer envelope is generally smooth at the early stages of development while specialized surface ornamentations only occur in the late stages of development [6]. In modern Ephedra, fleshiness is correlated with maturity of female cones. As a result, we infer that our new fossil species has ripe reproductive units with smooth outer envelope. Such a thin outer envelope may have some physiological functions, e.g., regulating water loss. Two alternative hypotheses may be used to explain the thin outer envelope of this new fossil Ephedra. One is that this new species represents the stem lineage of Ephedra and it is the beginning of fleshy cones in response to animal (probably reptiles and birds) dispersal, and subsequent thickened outer envelope would have been evolved into the modern form; the other hypothesis is that the fossil species could not adapt to animal dispersal and become extinct because of its thin envelope. Hence it is an evolutionary blind alley of Ephedra.

Ecological implications for the fleshy bracts
Three kinds of agents are known for the dispersal of Ephedra. The dry-winged, membranous bracts type of mature female cones is dispersed by wind while the coriaceous bract type is distributed by seed-catching rodents, and the fleshy bract type is dispersed by frugivorous birds [14,15]. During Jurassic and Cretaceous, vertebrate-mediated (e.g., early mammals and early birds) seed dispersal interactions may have been important drivers of seed cone evolution in such conifers as Podocarpaceae Endl. and Taxaceae S. F. Grey, resulting in shifts of female cones from the lax open cones typical of Paleozoic and Triassic conifers to the more compact and reduced seed cones that are associated with fleshiness [53]. Remarkably, a seed-eating bird fossil has been discovered from the Yixian Formation of western Liaoning [39,41]. Probably this is also the case in Ephedra, so fleshiness as an effective vertebrate-mediated seed dispersal mechanism may have accounted for the wide distribution of ephedroid plants in southern Europe, northeastern Asia, eastern North America, and South America during the Early Cretaceous.