A new macrofossil ephedroid plant with unusual bract morphology from the Lower Cretaceous Jiufotang Formation of northeastern China

Background The evolution of the Jehol Biota of western Liaoning in China includes three phases, initiation in the Dabeigou phase, radiation in the Yixian phase, and decline in the Jiufotang phase. Numerous ephedroid macrofossils were reported from the Lower Cretaceous Yixian Formation. However, so far none has been found in the younger Jiufotang Formation (ca. 120.3 Ma) of western Liaoning. Results Here we report a new species Jianchangia verticillata gen. et sp. nov. with unusual morphology from the Lower Cretaceous of the Jiufotang Formation, Lamadong Village, Jianchang County, Liaoning. This species is the first record of gnetophytes from the Jiufotang Formation. It is similar to other ephedroid species from the Yixian Formation in possessing linear leaves with parallel veins, jointed shoots with swollen nodes and longitudinally furrowed internodes, and ovulate cones possessing two whorls of bracts enclosing two chlamydosperms, but differs from all known species by the ovulate cone having multiple fine linear verticillate bracts. Conclusions This study expands our knowledge about the diversity of early gnetophytes in the Lower Cretaceous, and demonstrates the lineage continuity of gnetophytes from the Yixian Formation to the younger Jiufotang Formation.

Here we describe a new macrofossil plant from the Jiufotang Formation, and note that this species possessed certain unusual characters which distinguish it from all other known ephedroid species from the Jianshangou Bed.  1 and 2b). Each ovulate cone containing two whorls of bracts; four or more bracts at each node, linear, ascending. Two chlamydosperms enclosed within the uppermost whorl of bracts; obovoid, ca. 1.8 mm long, 1.5 mm wide, and narrowing towards the apex into a short tube (Fig. 2b).

Gymnosperms
Distribution − This species is only known from Lamadong Village, Jianchang County, Huludao Municipality, Liaoning of northeastern China.
Holotype − PE2018013101 (Fig. 1), deposited in the National Museum of Plant History of China, Institute of Botany, Chinese Academy of Sciences, Beijing.
Etymology − 'Verticillata' refers to the whorled linear bracts of the new species.
Remarks − Gnetophytes are common floristic elements in the Lower Cretaceous. They have been found in Europe, North America, South America, Australia, and northeastern Asia including Mongolia and northeastern China [25,26,30,35,38,39,41,46,47,53]. The richest diversity is in the Yixian Formation [16]. Previous findings of ephedroid plants in northeastern China are restricted to Yixian and Lingyuan Counties, Liaoning Province. The new species described here was discovered in Jianchang County, Liaoning Province, China. This locality belongs to the Jiufotang Formation, which is younger than the Yixian Formation [55,56].
The unique character of our new species lies in the number of foliar organs at the nodes. Though leaves/ bracts are linear and possess two parallel veins as in many other gnetophyte species from the Yixian Formation, four or more of them are whorled at the nodes. The ovulate cones possess two whorls of bracts that are ascending and free from one another. There are two chlamydosperms enclosed in the uppermost whorl of Abbreviations: l, parallel-veined leaves/bracts; mt, micropylar tube; w1, the proximal whorl of bracts; w2, the distal whorl of bracts bracts, which shows similarity to Gurvanella, Callianthus, Beipiaoa, and Ephedra [41,46,47,51,58]. These genera are classified based on their bract morphology (Figs. 4a−c). Our new species shows unusual bract morphology different from these known genera. As a result, we established a new genus here to include the new species based on the current classification of the ephedroid plants.
A one to one relationship between chlamydosperm and its subtending bract is relatively stable in ephedroid plants excepting Siphonospermum, in which chlamydosperms are pedicelled and have no associated bracts [45]. A chlamydosperm is axillary to a subtending bract and a pair of fertile bracts usually enclose two chlamydosperms in an ovulate cone. Sometimes, the paired bracts enclose only a single chlamydosperm because of abortion, e.g. E. monosperma and E. nebrodensis. The unusual feature of our new genus is that the multiple verticellate bracts enclose only two chlamydosperms in an ovulate cone, which fundamentally breaks the rule of chlamydosperm-bract relationship in Ephedraceae. There are two explanations for this pattern: the first is that the multiple bracts in a whorl are derived by division of two bracts, so there are only two chlamydosperms; the second is that the bracts are primary, but the original multiple chlamydosperms are reduced to only two due to loss. We are inclined to  the first explanation, and believe that the multiple bracts of the same whorl may have been divided/ multiplied from two bracts, since opposite phyllotaxis is predominant and presumably ancestral in the gnetophytes.

Discussion
Variation of leaves of early ephedroid plants  [43], the leaves are petiolate and divided and seem to be compound. In Ephedra multinervia Y. Yang & al., the leaves are strap-shaped and elongate and have multiple parallel veins connected by cross-veins, similar to those of Welwitschiaceae [52]. Jianchangia verticillata possesses normal ephedroid leaf shape, i.e. linear and parallel-veined, but is distinct from all known gnetalean fossils in that the foliar organs are verticellate.

Morphological diversification of bracts of early ephedroid plants
Representative types of ephedroid ovulate cones are illustrated in Fig. 5; a comparison between Jianchangia and related fossils from northeastern China is provided in Tables 1 and 2. Jianchangia shows similarities to other known early ephedroid plants from northeastern China in the linear and parallel-veined leaves and the articulate reproductive shoots with swollen nodes and fine longitudinal striations. Siphonospermum simplex differs from all known ephedroid plants [16] in having no typical ovulate cones or spikes, the chlamydosperms being pedicelled and lacking subtending bracts/leaves [45]. Pseudoephedra was reported as incertae sedis [44], but may be related to Siphonospermum in that it shows general ephedroid morphology, and the chlamydosperms are pedicelled and not closely associated with any foliar organs. Jianchangia differs from Siphonospermum and Pseudoephedra in the presence of ovulate cones and the whorled bracts subtending the inner paired chlamydosperms.
Protognetum is a Jurassic macrofossil that also possesses ephedroid vegetative morphology and ovulate spikes; the bracts are linear and leaf-like, but at each node there are only two bracts subtending a whorl of chlamydosperms; these characters indicate its relationship to the Gnetaceae clade [1]. Our new fossil genus Jianchangia can be easily distinguished from Protognetum by the fact that the whorled bracts subtend only two chlamydosperms. Spinobractea has   [43] ovulate spikes with multiple pairs of bracts that appear to be elongate and strap-shaped [43], characters which are different from the whorled linear bracts enclosing only two chlamydosperms in Jianchangia. Gnetum possesses ovulate spikes with multiple whorls of chlamydosperms, with each whorl subtended by a collar of bracts, while Welwitschia has ovulate cones with multiple pairs of fertile bracts each subtending an axillary chlamydosperm. The circular and paired bracts in Gnetum and Welwitschia distinguish them from our new genus. Jianchangia is also similar to a few other macrofossils from the Lower Cretaceous Yixian Formation in its ephedroid vegetative morphology and presence of bracts in ovulate spikes/cones, e.g. Prognetella Krassilov [15], Chengia Y. Yang & al. [16], Liaoningia Y. Yang & L.B. Lin [59], and Liaoxia Cao & Wu [46], but differs from all of them in having only one fertile whorl in each ovulate cone (vs. multiple fertile pairs/whorls in each ovulate cone in the latter five genera) in addition to fine, linear bract morphology. In Prognetella, the bracts are usually elongate and leaf-like, only modified at the basal portion to subtend the partially enclosed chlamydosperms [15], whereas the bracts of Jianchangia are not modified and are organized into whorls and not pairs. In Liaoningia, Chengia, and Liaoxia, the bracts are modified and not leaf-like, and paired at the nodes. Jianchangia differs from all of them in having unspecialized bracts arranged into whorls.
Jianchangia is similar to Gurvanella Krassilov [41], Beipiaoa Dilcher & al. [47], and Ephedra in having ovulate cones with only one fertile whorl in addition to the ephedroid vegetative morphology, but differs from the latter genera mainly in having unspecialized and verticillate bracts [35,40,47,49,51,52,54]. In Gurvanella, the two to three bracts are specialized in shape and possess unusual furcate venation [47], which makes it easily distinguished from Jianchangia. In Beipiaoa and Ephedra, the bracts are also modified but only two or three are inserted at each node [47]. This is also the case in Erenia Krassilov and Callianthus Wang & Zheng, in which the bracts are not leaf-like but specialized and encapsulate a pair of chlamydosperms [58]. Constrobilus and Latibractea probably belong to this complex because both of them have ovulate cones with one pair of chlamydosperms, as in modern Ephedra, but their leaves are unusual, divided in Latibractea and broad and pinnately veined in Constrobilus [43]. The differences between Jianchangia and these two genera are  [43] obvious, the bracts of the latter two genera are specialized and paired, thus differing from the leaf-like and whorled bracts in Jianchangia.
In summary, bracts of these ephedroid macrofossils show intergrading modifications from absence to presence, and from leaf-like to specialized shape. These transformations together with the reduction of the fertile whorls of the ovulate spike may reflect the evolutionary stages and probable relationships among them, but phylogenetic analysis is needed to test the direction of these transformations.

Ecology
Modern Ephedra usually lives in dry areas, in Gobi-type deserts (e.g. E. rhytidosperma Pachom., Fig. 6a), on cliffs (e.g. E. equisetina Bunge, Fig. 6b), or in stony crevices (e.g. E. monosperma Gmel. ex C.A. Mey., Fig. 6c), and they are distributed from Mediterranean regions eastwards to Siberia and northern China, in southwestern U.S.A. and northwestern Mexico, and Andean South America as well [60]. Early ephedroid plants may have occupied more diverse habitats than they do now, probably also in humid swampy places [15], and even in water [49], and these plants evolved a set of characters adapted to the palaeoenvironment. Prognetella is provided with an unusual set of characters, for instance, the fragmented reproductive shoots and frequent cystiform chlamydosperms may have facilitated the dispersal of diaspores in a lacustrine environment [15]. Our new species displays leaf/bract morphology distinct from other species of the Yixian Formation and extant species, which may represent an ecological adaptation. The numerous fine linear leaves of Jianchangia may be functionally analogous to the finely divided leaves of many living species of aquatic flowering plants, e.g. Myriophyllum spicatum L. and Hippuris vulgaris L. [61,62].
Three phases of floristic development were recognized for the Jehol Biota, i.e. the early initiation Dabeigou phase, the middle radiation Yixian phase, and the late declining Jiufotang phase [57]. There are only a few plant fossils of gymnosperms recorded from the Jiufotang Formation, e.g. Ginkgoites truncatus Li, Czekanowskia rigida Heer, and Elatocladus pinnatus Sun & Zheng. All three plants were also recorded from the Jianshangou Bed, Yixian Formation, suggesting a floristic similarity between the Jiufotang Formation and the Yixian Formation. Though ephedroid macrofossils are common in the Yixian Formation, they have not been reported from the younger Jiufotang Formation thus far. Our new species is thus the first ephedroid species from the Jiufotang Formation. This finding confirms the floristic similarity and continuity between the Yixian Formation and the Jiufotang Formation.
The Yixian Formation displays an enormous diversity of ephedroid plants. This early burst of Ephedraceae may have been driven by the turbulent geological environment at the time [15]. It is well known that volcanic activity was frequent in the Yixian Formation [57]. This radiation was not only observed in the gnetophytes, but also in other plant groups, insects, mammals, dinosaurs, and birds [55]. In the younger Jiufotang Formation, the environment was relatively stable [57]. The declining species diversity in the Jiufotang Formation may be attributable to local extinction.

Conclusions
Here we describe the first record of gnetophytes from the Lower Cretaceous Jiufotang Formation, Lamadong Village, Jianchang County, Liaoning Province, northeastern China. This finding demonstrates the lineage continuity of gnetophytes in western Liaoning since the Jiufotang Formation is ca. 5 Ma years younger than the Yixian Formation. This new plant Jianchangia verticillata gen. et sp. nov. also enhances our knowledge of morphological diversity in early gnetophytes. It has multiple foliar bracts, whorled at the nodes, and is distinguished from all other known ephedroid plants.

Methods
Our studied fossil was from Lamadong Village, Jianchang County, Liaoning Province, northeastern China (Fig. 7). The fossil bed belongs to the Jiufotang Formation. The Formation dates back to the Aptian, ca. 120.3 Ma [63], and is well-known for early birds, e.g. Jianchangornis microdonta Zhou & al. [64], Bohaiornis guoi Hu & al. [65], Schirooura lii Zhou & al. [66], Jeholornis palmapenis O'Connor & al. [67], Zhongornis haoae Gao & al. [68,69], and the dinosaur Moganopterus zhuiana Lu & al. [70]. Plant fossils were rarely reported, and only a few gymnosperms have been recorded, e.g. Ginkgoites truncatus Li, Czekanowskia rigida Heer, and Elatocladus pinnatus Sun & Zheng [57]. The new fossil is deposited in the National Museum of Plant History of China, Institute of Botany, Chinese Academy of Sciences, Beijing, China. The plant fossil was preserved as impressions, and is only fragmentary. The fossil was observed with a light microscope (Nikon Eclipse E600) and was photographed with digital cameras (Nikon D700 and Olympus TG-3). The figures were manually edited and created using Adobe Photoshop CS2 ver. 9.0 and CorelDRAW X4. The map of the type locality was generated using ArcGIS 9.3 (ESRI, Redlands, CA, USA; http://www.esri.com).