Parallel evolution of leaf morphology in gnetophytes

Parallel evolution of leaf morphology in gnetophytes

Yong Yang, Longbiao Lin & David K. Ferguson

Organisms Diversity & Evolution ISSN 1439-6092 Volume 15 Number 4 Org Divers Evol (2015) 15:651-662 DOI 10.1007/s13127-015-0226-6

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Author's personal copy Org Divers Evol (2015) 15:651–662 DOI 10.1007/s13127-015-0226-6

ORIGINAL ARTICLE

Parallel evolution of leaf morphology in gnetophytes Yong Yang 1 & Longbiao Lin 2 & David K. Ferguson 3

Received: 15 January 2015 / Accepted: 9 July 2015 / Published online: 26 July 2015 # Gesellschaft für Biologische Systematik 2015

Abstract In the present paper, an ephedroid macrofossil species from the Early Cretaceous Yixian Formation of western Liaoning of China is described as new to science: Ephedra multinervia Yang et Lin, sp. nov. This species has typical ephedroid morphology, e.g. the dichasial branching shoot system, swollen nodes, internodes having many fine longitudinal striations and opposite phyllotaxy. Ephedra multinervia has strap-shaped leaves with multiple dichotomizing veins and reduced female cones with a single pair of fertile bracts forming a cupule enclosing two inner seeds. Ephedra multinervia is similar to Ephedra archaeorhytidosperma Yang et al. and Ephedra hongtaoi Wang et Zheng in its reduced bi-ovulate female cone, but differs from the latter two species by the lengthy strap-like leaves bearing multiple parallel veins and its sessile female cones. A new evolutionary hypothesis of the gnetophytes is proposed based on a synthesis of reproductive morphology of macrofossils from the Early Cretaceous and modern representatives. A Chengia-like precursor might have given rise to the Gnetum-Welwitschia clade by diversification of leaf morphology and female reproductive

Electronic supplementary material The online version of this article (doi:10.1007/s13127-015-0226-6) contains supplementary material, which is available to authorized users. * Yong Yang [email protected] 1

State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, The Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing 100093, China

2

China Railway Group Limited, 69 Fuxing Road, Beijing 100039, China

3

Department of Paleontology, University of Vienna, 1090 Vienna, Austria

organs. According to this new explanation, the Welwitschialike strap-like leaves with multiple parallel veins in E. multinervia result from convergence. Keywords China . Early Cretaceous . Ephedra . Evolution . Gnetophytes . Gnetum . Liaoning . Morphology . Palaeobotany . Welwitschia . Yixian Formation

Introduction The living gnetophytes encompass only three living genera, viz. Ephedra L., Gnetum L. and Welwitschia Hook. f. (Pearson 1929; Martens 1971; Gifford and Foster 1989; Price 1996). The genus Ephedra is represented by over 50 species widely distributed in the north temperate zone and high mountains of the Andes in South America (Stapf 1889; Cutler 1939; Florin 1933; Cheng and Fu 1978; Kubitzki 1990; Stevenson 1993; Price 1996; Fu et al. 1999). Gnetum contains 30 or more species ranging across the pantropical region (Kubitzki 1990; Price 1996; Hou et al. 2015). Welwitschia includes only one species endemic to southwestern Africa (Markgraf 1926; Pearson 1929; Martens 1971; Gifford and Foster 1989). The phylogeny of the three genera is well resolved; Ephedra is basal and sister to a clade encompassing Gnetum and Welwitschia (Fig. 1, e.g. Crane 1985; Doyle and Donoghue 1986; Chaw et al. 2000; Zhong et al. 2010). It is well known that female reproductive organs play an important role in understanding the evolution of gymnosperms (e.g. Chamberlain 1935; Eames 1952; Sporne 1971; Bierhorst 1971; Gifford and Foster 1989; Yang 2002, 2004, 2014; Mundry and Stützel 2004). Female cones of the three living genera of gnetophytes are markedly different, each genus is provided with a definitive pattern (Fig. 1). In Ephedra, female cones have one to 13 pairs/whorls of bracts but only

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Fig. 1 Phylogeny of the modern gnetophytes indicating the diversified morphology of leaves and female cones

the uppermost pair/whorl of bracts is fertile, each subtending a female reproductive unit (an enveloped ovule/seed), or these bracts are adnate/fused to form a cupule enclosing one to three seeds (depending on abortion of one or two ovules). The female cone of Gnetum is remarkable in that the female spike has prominent internodes and the female reproductive units are organized in whorls; thus, the cone is a loose reproductive shoot or spike. The cone is lax in the longitudinal direction but becomes complicated in the horizontal direction. Welwitschia possesses a typical female cone; it bears opposite and decussate bracts on the cone axis and each bract usually subtends a female reproductive unit. Though these cones were all considered to be compound, it remains an unanswered question how the reduced female cone of Ephedra gave rise to the two different female cones in Gnetum and Welwitschia. Leaves of Ephedra, Gnetum and Welwitschia are markedly different in form, size and venation and provide diagnostic characters to define each of them (Fig. 1). Ephedra possesses extremely reduced, small, linear leaves connate at the base forming a sheath surrounding nodes and basal portion of internodes; each leaf is provided with two (rarely three) parallel veins (Fig. 2a, b, Foster 1971). Gnetum bears dicot-like, medium-sized, broad leaves with pinnately (or reticulate) venation (Fig. 2c, Rodin 1967; Gifford and Foster 1989). Welwitschia has giant straplike leaves, and each leaf is traversed by numerous parallel longitudinal veins that are interconnected by minor veins (Fig. 2d, Sykes 1911; Pearson 1929; Martens 1971; Butler et al. 1973a, b; Gifford and Foster 1989). It is extremely important to discover how these leaves evolved, since the three genera share a common ancestor, and Gnetum and Welwitschia have diverged from an ancestor close to Ephedra.

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Palaeobotanical evidence is important to answer the abovementioned questions. Fortunately, many gnetalean macrofossils have been described from the Early Cretaceous strata, in western Liaoning of northeastern China in particular (e.g. Miki 1964; Guo and Wu 2000; Sun et al. 2001; Liu 2005; Liu et al. 2008; Guo et al. 2009; Rydin et al. 2006b; Krassilov 2009; Yang and Wang 2013; Yang et al. 2005, 2013; Wang and Zheng 2010; Rydin and Friis 2010). Diversified morphology was observed in the Early Cretaceous gnetalean plants. A synthetic study of morphology, anatomy and palaeobotany may offer some clues regarding the evolutionary transitions leading to the disjunct morphology encountered in modern gnetophytes (Fig. 1). Macrofossils bearing a combination of morphological characters of two or three genera are particularly interesting. This study reports a new ephedroid macrofossil from the Early Cretaceous strata of western Liaoning, NE China. A new working hypothesis is given to explain the morphological transitions from Ephedra to Gnetum and Welwitschia based on a synthesis of palaeobotanical evidence.

Materials and methods The fossil specimens were collected from Dawangzhangzi Village, Lingyuan City, Liaoning Province, northeastern China (Fig. 3). The plant fossils are preserved as impressions. Three specimens of the freshwater fish, Lycoptera davidi Sauvage co-occur on the single slab. The stratum containing the present specimens belongs to the Daxinfangzi Bed in the lower part of the Yixian Formation, where a few famous angiosperm macrofossils have been found, e.g. Leefructus mirus Sun et al., Hyrcantha decussata (Leng et Friis) Dilcher et al., and Archaefructus sinensis Sun et al. (Dilcher et al. 2007; Sun et al. 2002, 2011). The Daxinfangzi Bed mainly consists of yellowish grey and grey sandstone intercalated with grey siltstone, tuffaceous silt and fine-grained sandstone (Sun et al. 2011). The Daxinfangzi Bed of the Yixian Formation has an age of about 122.6–125.8 million years old according to radiometric dating (e.g. Swisher et al. 1999; Peng et al. 2003; Zhang et al. 2006; Meng et al. 2008), which corresponds to the Early Aptian–earliest Late Aptian of the Early Cretaceous in the International Stratigraphic Chart (Gradstein et al. 2012; Walker et al. 2012). The fossil was photographed with digital cameras (Nikon D700, Olympus STYLUS TG3 and Panasonic DMC-FZ30) and under a microscope (Nikon Eclipse E600). Previously published ephedroid macrofossils from the lower part of the Yixian Formation of Huangbanjigou Village, Beipiao City, Liaoning Province, Northeast China were examined at the Institute of Botany, the Chinese Academy of Sciences, Beijing, and Nanjing Institute of Geology and Palaeontology, the Chinese Academy of

Author's personal copy Parallel evolution of leaf morphology in gnetophytes

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Fig. 2 Leaf venation of the modern gnetophytes. a–b Ephedra (from Foster 1971). c Gnetum (from Rodin 1967). d Welwitschia (from Martens 1971)

Sciences, Nanjing. The reconstructions were drawn using a pointed pen and black ink.

Results Systematics Gnetidae Pax

Diagnosis Reproductive shoots noded, internodes finely striated, usually one to two lateral branches axillary to a leaf. Leaves opposite at nodes, long and strap-shaped, with multiple parallel veins. Female cones sessile, bi-ovulate, with one pair of bracts; each bract subtends a female reproductive unit. Bracts adnate to each other or fused for ca. 2/3 of their length. Integumentary tube short and straight. This new species differs from other known species of Ephedra by its long and strap-shaped leaves bearing up to eight parallel veins.

Ephedrales Dumort. Ephedraceae Dumort. Ephedra multinervia Y. Yang et L.B. Lin, sp. nov. (Figs. 4–8).

Fig. 3 Type locality of Ephedra multinervia Y. Yang et L.B. Lin.

Fig. 4 A reproductive shoot of Ephedra multinervia showing the ephedroid morphology. c female cone, l leaf, n node


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Description The fossil represents a reproductive shoot, ca. 14 cm long, 9.5 cm wide (Fig. 4). The shoot is dichasially branched and has nodes and internodes. Nodes are usually swollen and bear a pair of long and strap-like leaves (Fig. 5a). Internodes are up to 5.5 cm long, 2 mm in diameter in the proximal portion, shortened and attenuate distally. These internodes possess many fine longitudinal striations. The proximal node bears a pair of leaves (Fig. 5a), the left leaf is preserved in profile, while the right leaf is preserved with venation pattern (Fig. 5a, b). The left leaf is lanceolate, ca. 4.9 cm long, ca. 4 mm wide in the middle part (Fig. 5a). The right leaf is parallel veined. At least two veins are present at the base, which dichotomize to produce at least eight veins distally (Fig. 5b). The leaf subtends one to two branches. Female cones are usually opposite at nodes or rarely solitary at nodes or terminally, ovoid, obovoid, ellipsoid or globose (Fig. 6a–c). These cones possess a pair of bracts enclosing two seeds (Fig. 7a–d). The pair of bracts is adnate or probably fused for ca. 2/3 of their length (Fig. 7c–d). Seeds are probably ovoid or ellipsoid, acute or acuminate at the apex, each with a straight and short (ca. 0.2 mm) micropylar tube (Fig. 7a–b). Etymology The specific epithet ‘multinervia’ is derived from the venation pattern of the strap-shaped leaves. Fig. 6 Reproductive shoots of Ephedra multinervia magnified showing details. a A reproductive branch displaying increasingly shortened internodes towards the distal ends, and the dichasial ramification with irregular branching. b–c Reproductive shoots magnified showing cone clusters with shortened internodes

Holotype PE 2014031401 (Fig. 3) (here designated). Repository Chinese National Herbarium (PE), Institute of Botany, Chinese Academy of Sciences, Beijing, China. Type locality Dawangzhangzi Village, Songzhangzi Town, Lingyuan City, Chaoyang District, Liaoning Province, China (Fig. 3). Stratigraphic horizon and age Early Cretaceous, Daxinfangzi Bed, Yixian Formation.

Fig. 5 Nodal portion of a shoot of Ephedra multinervia. a The swollen node and the lengthy strap leaf. b The multiple dichotomizing parallel veins. c Connections of veins. l leaf, v vein, n node

Remarks The preserved reproductive shoot bears a number of characters typical of Ephedra, e.g. the dichasial branching pattern, branches jointed, nodes swollen, internodes bearing fine longitudinal striations, female cones bearing one pair of bracts forming a cupule enclosing two seeds, and each seed possessing an apical micropylar tube. The strap-shaped leaves bear multiple parallel veins, which distinguish this fossil from all other known species of Ephedraceae. The plant is reconstructed in Fig. 8.


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Fig. 7 Reproductive shoots of Ephedra multinervia magnified displaying details of cone characters. a Opposite and subsessile female cones showing paired FRUs, enlarged obdeltoid receptacle and the apical straight micropylar tube. b A female cone showing micropylar tube. c Opposite and sessile female cones magnified showing bracteal fusion up to more than half of their length. d Bracts adnate but not connate. mt micropylar tube, r receptacle, fb fused bracts, ab adnate bracts

Discussion Comparison with other Early Cretaceous ephedroid fossils

Fig. 8 Reconstruction of Ephedra multinervia displaying dichasial branching pattern, the lengthy and strap-shaped leaves and the sessile two-seeded female cones having a receptacle. b bract, fru female reproductive unit, l leaf, r receptacle

The geological history of the gnetophytes was poorly known before this century. A few macrofossils were described as having affinities with the group, but these were demonstrated to be unlike their modern counterparts (Crane 1996). After 1996, a number of macrofossil taxa were reported having affinities to the Gnetales, in northeastern China in particular (Cao et al. 1998; Duan 1998; Sun et al. 2001; Akhmetiev and Krassilov 2002; Yang et al. 2005, 2013, 2013; Tao and Yang 2003; Wang 2004; Rydin et al. 2003, 2004, 2006a, b; Cladera et al. 2007; Liu et al. 2008; Krassilov 2009; Wang and Zheng 2009, 2010; Rydin and Friis 2010; Yang 2010; Han et al. 2013; Yang and Wang 2013). Some of them were described as ‘angiosperms’, but actually belong to the gnetophytes (e.g. Cao et al. 1998; Duan 1998; Wang and Zheng 2009; Wang 2010; Friis et al. 2011; Han et al. 2013). All these fossils have characteristic ephedroid morphology, including jointed shoots, opposite phyllotaxy, linear leaves if present and female reproductive units having an integumentary tube. Ephedra multinervia is preserved as a reproductive shoot having a dichasially branched shoot system, leaves and female cones. This species shows similar ephedroid morphology to Siphonospermum simplex Rydin et Friis (Rydin and Friis 2010), Chengia laxispicata Yang et al. (Yang et al. 2013), Liaoxia spp. (Rydin et al. 2006b; Liu et al. 2008), Ephedra


hongtaoi Wang et Zheng (Wang and Zheng 2010), Ephedra c a r n o s a Ya n g e t Wa n g ( Ya n g a n d Wa n g 2 0 1 3 ) , E. archaeorhytidosperma Yang et al. (Yang et al. 2005), Beipiaoa Dilcher et al. (Sun et al. 2001) and Gurvanella dictyoptera Krassilov (syn.: Gurvanella exquisita Sun et al., Krassilov 1982; Duan 1998; Wu 1999; Sun et al. 2001), e.g. dichasial shoot system, swollen nodes and internodes bearing many fine longitudinal striations, opposite leaves possessing parallel veins, the paired seeds enclosed within a pair of bracts and each seed having an apical micropylar tube. However, our new species has a unique combination of characters and differs from other known macrofossil species. It differs from S. simplex by the female reproductive units assembled into female cones (vs. FRUs terminal to twigs and not assembled into a compact cone) and strap-shaped leaves having multiple parallel veins (vs. linear leaves having three veins). This new species differs from Prognetella Krassilov by the compact female cone (vs. female reproductive units sessile and axillary to leaf-like linear bracts, corresponding to a laxly arranged female spike in Prognetella) and the strap-shaped leaves bearing multiple veins (vs. linear leaves bearing two to four parallel veins). The new species differs from C. laxispicata and Liaoxia spp. by the female cone having only one pair of bracts (vs. the female cone consisting of several pairs of fertile bracts in Liaoxia and Chengia) and the strap-shaped leaves with multiple veins (vs. linear leaves with usually two veins in Liaoxia and Chengia). Ephedra multinervia has a compact female cone bearing only one pair of bracts adnate to one another or fused into a cupule enclosing the two seeds and shows similarity to E. archaeorhytidosperma, E. hongtaoi, E. carnosa, G. dictyoptera and Beipiaoa, but it differs from E. hongtaoi by the sessile female cones (vs. usually terminal on twigs in E. hongtaoi) and the less branched shoot system (vs. profusely branched system in E. hongtaoi), from G. dictyoptera by the female cone bearing no furcated appendages (vs. furcated appendages surrounding the seeds in Gurvanella). The new species differs from E. carnosa by the less specialized bracts (vs. fleshy bracts in the latter species), from Beipiaoa by the obtuse bracts (vs. distal portion of the bracts usually modified into spines in Beipiaoa). In addition, E. multinervia usually has bracts equal to the inner seeds in length while Beipiaoa and E. carnosa have bracts longer than the enclosed seeds. The new species differs from modern species of Ephedra by its strap-shaped leaves with multiple veins (vs. linear leaves usually fused into a sheath at nodes and each leaf having usually two but rarely three veins), and the female cone possessing only one pair of bracts (vs. two or more pairs of bracts). We cannot compare vegetative morphology of E. multinervia with Beipiaoa and E. carnosa because the latter two species are only known as female cones. This new species differs from E. archaeorhytidosperma by the sessile female cones (vs. female cones terminal to twigs) and the strap-shaped leaves (vs. associated leaves triangular and much shorter).

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Protoephedrites eamesii Rothwell et Stockey of the Protoephedraceae is a recently described macrofossil species from the Early Cretaceous (Valanginian-Barremian) of Vancouver Island of Canada, with anatomical structures of female cones (Rothwell and Stockey 2013). The female cone is compound and consists of a primary axis on which bracts are opposite and decussate, with each bract usually subtending an axillary fertile shoot. The secondary fertile shoot bears one to two pairs of decussately arranged bracteoles and a pair of sporophylls with erect seeds at the apex. Seeds/ovules are not enveloped by bracteoles. This species might be a direct descendent of a hypothetical ancestor of the Ephedraceae (and perhaps the modern gnetophytes). The ancestor is supposed to bear compound female cones comparable to Cordaitales, which consist of a primary axis and bracts and their subtending secondary reproductive shoots. This plant differs from E. multinervia in reproductive details; the secondary reproductive shoot possesses megasporophylls and does not form the enveloped female reproductive unit (vs. a wellestablished female reproductive unit in E. multinervia). Evolution and phylogeny of the gnetophytes Female reproductive organs play an important role in understanding evolutionary relationships among seed plants. Modern gnetophytes share the enveloped female reproductive unit and the inner integument elongated into a micropylar tube, but they are markedly different from each other by the diversified patterns of female reproductive organs in organization: Ephedra possesses an extremely reduced compact female cone with only the uppermost pair/whorl of bracts fertile and the lower bracts abortive, Gnetum has a very lax spike with prominent internodes but extreme complicated FRUs at each whorl, Welwitschia possesses a compact female cone with multiple pairs of fertile bracts (Fig. 1, Pearson 1929; Maheshwari and Vasil 1961; Martens 1971; Gifford and Foster 1989). This disjunct morphology of female cones of gnetophytes can be explained with macrofossil evidence. Evolution of the female reproductive organs of the gnetophytes consists of the shortening of pedicles of FRUs and internodes of the cone primary axes, modification of foliar organs into bracts, reduction of lower whorls of bracts and fusion of bracts into additional cupules of FRUs. Yang et al. (2013) proposed a reduction and sterilization evolutionary hypothesis of the female reproductive organs of ephedroid macrofossils and modern Ephedra. We follow their major thoughts. Female reproductive organs of the gnetophytes have experienced an evolutionary aggregation, reduction and sterilization. Protoephedrites eamesii is considered as the outgroup because the characteristic female reproductive unit of modern gnetophytes was not well established in that species. Siphonospermum seems to represent the beginning of the evolutionary process towards the modern gnetophytes. It has


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loosely arranged spikes with pedicled female reproductive unit terminal or axillary to unspecialized leaf-like bracts that are linear and free from each other. With losses of pedicles of the FRUs and shortening of the internodes of the cone axis, the female spike/cone became increasingly compact in a morphological series displayed by Prognetella, Chengia, Liaoxia and then to Beipiaoa, Ephedra and Gurvanella. The subtending foliar organs/bracts in Siphonospermum and Prognetella were gradually modified by shortening and broadening into more and more specialized bracts which are finally adnate/fused to form a cupule enclosing the inner FRUs in Beipiaoa, Gurvanella and Ephedra. Both Gnetum and Welwitschia have sessile female reproductive units and modified bracts of female cones, their common ancestor might have diverged from a sister lineage of Chengia in the Ephedraceae. From the female spike of Chengia, Gnetum retained the lax cone axis with conspicuous internodes but acquired a horizontal multiplication and synorganization of the FRUs. Welwitschia might have experienced a parallel evolutionary process such as that from Chengia to Liaoxia by further shortening of internodes of the reproductive shoot. It is relatively easy to place other macrofossils related to the Gnetum-Welwitschia clade within this evolutionary hypothesis (Fig. 9). Khitania columnispicata Guo et al. is the only known macrofossil having Gnetaceae affinity (Guo et al. 2009). It is a compact spike having numerous circular bract rings similar to Gnetum and better placed as the sister group of

the modern Gnetum on account of resemblance between their male spikes. Welwitschiostrobus murili Dilcher et al. shows extreme similarities to male cones of modern Welwitschia mirabilis (Dilcher et al. 2005) and may belong to the crown group of the Welwitschiaceae lineage. Drewria potomacensis Crane et Upchurch is a reproductive shoot from the Early Cretaceous Potomac Group of the USA. It possesses opposite and decussate phyllotaxy, oblong leaves bearing parallel venation and loose female spikes, thus shows a morphology intermediate between Ephedra and Welwitschia. Its habit, leaf form and venation are close to E. multinervia, but its female spike bears multiple pairs of fertile bracts similar to Chengia and Welwitschia. Drewria potomacensis probably represents an earlier evolutionary branch of Welwitschiaceae before the divergence between W. murili and Welwitschia mirabilis. It is highly improbable that the complicated female cone type of Welwitschia mirabilis was derived from the extremely reduced female cone type of E. multinervia or vice versa. The vegetative similarity of leaf morphology between E. multinervia and Welwitschia mirabilis must be attributed to convergence. Based on our studies of reproductive organs of the modern and Early Cretaceous gnetophytes, a new phylogenetic scheme of these macrofossils and extant gnetalean plants is outlined here (Fig. 10). A census of macrofossil species from the Early Cretaceous indicates that most morphological patterns of the gnetophytes are to be found in the Early Cretaceous

Fig. 9 Evolution of female cones of the gnetophytes. a Hypothesized ancestor of modern gnetophytes, b Transitional form as in Siphonospermum showing the lax reproductive shoot, the leaf-like bracts and the pedicled female reproductive unit. c Transitional form as in Prognetella showing the lax reproductive shoot, the leaf-like bracts and the sessile female reproductive unit. d The female cone type of Chengia-

Liaoxia becoming more and more compact and bearing multiple pair/ whorls of modified fertile bracts. e Female cone types of fossil and modern Ephedra-Gurvanella-Beipiaoa complex having only one pair/whorl of fertile bracts. f The lax reproductive shoot but specialized and verticillate reproductive units at nodes in Gnetum. g The compact cone of Welwitschia bearing opposite and decussate fertile bract


Fig. 10 Supposed evolutionary relationships among Cretaceous and modern gnetophytes

Jehol Biota of northeastern China plus Mongolia while fossils from other regions are either not comparable with modern gnetophytes or provide little evolutionary information; Drewria is an exception (Supplementary material 1). The Jehol Biota includes a few species bearing a set of characters transitional between the different gnetalean genera. This area was probably one of the centres of origin of modern gnetophytes (Yang et al. 2013).

Fossil calibration in dating divergence of modern gnetophytes The origin and evolution of the gnetophytes remain poorly resolved. Macro- and meso-fossils from the Early Cretaceous of Asia, Europe, N America and S America clearly indicate that the genus Ephedra originated in the Early Cretaceous (118–125 mya, Guo and Wu 2000; Sun et al. 2001; Yang et al. 2005; Rydin et al. 2006a, b; Cladera et al. 2007; Liu et al. 2008; Wang and Zheng 2010; Rydin and Friis 2010; Yang et al. 2013; Yang and Wang 2013). Fossils related to Gnetum and Welwitschia were reported from the Early Cretaceous of China, e.g. Khitania Guo et al. (125 mya, Guo et al. 2009), N America, e.g. Drewria Crane et Upchurch (120 mya, Crane and Upchurch 1987) and Brazil, e.g. Cratonia Rydin et al. (110 mya, Rydin et al. 2003) and Welwitschiostrobus Dilcher et al. (110 mya, Dilcher et al. 2005). A few other fossils from the Early Cretaceous of Mongolia are related to the gnetophytes (see review in Krassilov 2009).

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A number of recent studies have used molecular clocks constrained with fossils to estimate the divergence of modern gnetophytes (Huang and Price 2003; Wang et al. 2005; Won and Renner 2003, 2006; Ickert-Bond et al. 2009; Lu et al. 2014). Huang and Price (2003), Wang et al. (2005), and Won and Renner (2003) estimated the age of Ephedra and Gnetum with a strict clock, while Ickert-Bond et al. (2009) and Lu et al. (2014) dated the divergence of modern gnetophytes using a Bayesian clock. Won and Renner (2006) used both strict and Bayesian clock hypotheses in their estimates. In general, dating using a strict clock gave much younger ages than with a Bayesian relaxed clock (Won and Renner 2006). Huang and Price (2003) and Wang et al. (2005) used landmark events to estimate the age of modern Ephedra as 8– 32 mya (Oligocene to Late Miocene). Won and Renner (2003, 2006) constrained the node between Ephedra and Gnetum + Welwitschia with Gurvanella Krassilov/ephedroid fossils (125 mya), and the node between Gnetum + Welwitschia with Cratonia Rydin et al. (110–115 mya), to date the origin of Gnetum to around 26.7 mya. However, they obtained different dating results when they constrained the node of origin of modern Ephedra with ephedroid fossil seeds (ca. 125 mya). In this case, the age of Gnetum is ca. 44 mya (Eocene), which is much earlier than 26 mya. This suggests that constraints with fossils having different ages can give different dating results. Recently, Hou et al. (2015) calibrated the node between Gnetum + Welwitschia with the same fossil Cratonia that Won and Renner (2006) used and concluded that the crown group of Gnetum originated in the Late Cretaceous (ca. 81 mya), which is markedly different from the result of Won and Renner (2006). Using the macrofossil Cratonia cotyledon Rydin et al. (Santana Formation, 110–125 mya) to constrain the node of Gnetum and Welwitschia, Ickert-Bond et al. (2009) estimated the divergence of Ephedra from Gnetum + Welwitschia at around 167 mya. Lu et al. (2014) used Eoantha (ca. 125 mya) to calibrate the crown node of modern gnetophytes and arrived at an age of ca. 146 mya for the node between Ephedra and Gnetum + Welwitschia. Renner (2009) suggested that the Ephedraceae diverged from the GnetaceaeWelwitschiaceae clade around 159 mya and the Gnetaceae split from the Welwitschiaceae at 138 mya. Macrofossil taxa of Ephedra, e.g. E. archaeorhytidosperma (Yang et al. 2005), E. hongtaoi (Wang and Zheng 2010), Ephedra-type seeds (Rydin et al. 2006a) and our new species E. multinervia in this study are all stem groups of the genus. It is problematic to use these fossils to constrain the molecular clock, because the divergence time of the gnetophytes would then be much earlier than it should be if these fossils are used to calibrate the divergence node of modern Ephedra. On the other hand, the divergence of modern Ephedra would be much more recent than it should be if we use these fossils to constrain the


divergence node between Ephedra and Gnetum + Welwitschia. Using our new fossil species to constrain the molecular clock will not change our current knowledge of evolution in the gnetophytes, because macrofossils with the same age have already been used to constrain the node between Ephedra and Gnetum + Welwitschia (Won and Renner 2006) and the node for diversification of modern Ephedra (Won and Renner 2006). Parallel evolution of leaf morphology in the gnetophytes Phylogenetic relationships of the three extant lineages of the gnetophytes are well resolved; the genus Ephedra being basal and more primitive, while Gnetum and Welwitschia are closer to one another than to Ephedra (Fig. 1). However, how the morphological transition took place in evolutionary process remains poorly known. Abundant macrofossils bearing female reproductive organs make a discussion possible. Leaf morphology of E. multinervia markedly differs from both modern and extinct species of Ephedraceae. Ephedra multinervia possesses strap-shaped leaves ca. 4.9 cm long and 4 mm wide, and each leaf has multiple veins due to dichotomization. Modern Ephedra has linear leaves that are more or less connate into a thin sheath with two or three triangular teeth, rarely free from each other, each leaf usually possesses two (rarely three) brown parallel veins (Foster 1971; Gifford and Foster 1989). Fossil species from Europe and North America are only represented by seeds (Rydin et al. 2006a), while macrofossils from Australia and South America bear no leaves (Krassilov et al. 1998; Cladera et al. 2007; Mendes et al. 2011), so it is impossible to compare leaf characters of these species with the new species. Those fossil species from the Early Cretaceous of western Liaoning in China and Mongolia are frequently preserved with leaves (Guo and Wu 2000; Rydin et al. 2006b; Liu et al. 2008; Fig. 11 Leaf venation patterns of Cretaceous gnetophytes. a Siphonospermum simplex Rydin et Friis. b–d Drewria potomacensis Crane et Upchurch (redrawn from Crane and Upchurch 1987)

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Rydin and Friis 2010; Krassilov and Bugdaeva 1999; Yang et al. 2013). These leaves are free and linear and bear two (e.g. Liaoxia and Prognetella) or three parallel veins (e.g. in S. simplex). The new species thus provides a set of new and unique apomorphic characters in Ephedraceae. The living gnetophytes have extremely diversified leaves and venation patterns (Fig. 2a, d). Ephedra possesses linear leaves that are free or fused at the base forming a sheath, each leaf bears two (rarely three) parallel veins (Fig. 2a, b, Foster 1971; Gifford and Foster 1989). Gnetum has very specialized broad and pinnately veined leaves similar to dicotyledons; four to 13 vascular bundles pass through the petiole and enter the leaf blade and these bundles dichotomize and the dichotomies are anastomosed to form the pinnate or reticulate venation (Fig. 2c, Rodin 1967). In Welwitschia, two veins enter the base of the leaf blade and then dichotomize gradually to establish the parallel venation (Fig. 2d, Martens 1971). Leaf morphology of E. multinervia reminds us of Gnetum and Welwitschia. Two veins enter the strap-shaped leaf and then dichotomize to increase the vein number and establish the parallel venation as in Welwitschia (Takeda 1913). In Gnetum, the reticulate/pinnate venation is also established by the dichotomy of parallel veins and fusion of these minor veinlets at the margin (Rodin 1967). The similarity of leaf form and venation between the new species and Welwitschia is interesting. It can be explained in two different ways. The most straightforward hypothesis is that Welwitschia is derived from this lineage if emphasis is laid on the morphological similarity in both leaf form and venation pattern. However, the female cone of E. multinervia is quite specialized and reduced, with only one pair of adnate/fused bracts enclosing two seeds. It is highly improbable that this could have given rise to the much more complicated female cone of Welwitschia. Modern Welwitschia morphology might have


been modified from that of Drewria (Crane and Upchurch 1987). The second scenario is that this similarity is superficial and resulted from parallel evolution. The strap-shaped leaves might have adaptive significance. The Cretaceous opens a new era of land vegetation because of the origin and radiation of angiosperms, with broad-leaved species becoming increasingly dominant. A few gnetalean lineages made a similar attempt to evolve new morphology to adapt to the new environment. Siphonospermum simplex Rydin et Friis possesses parallel veins showing clearly dichotomous branching in distal portion (Fig. 11a). Drewria potomacensis Crane et Upchurch has broad strap-shaped leaves having parallel veins and cross veinlets (Fig. 11b, d). Gnetum’s broad leaves might have originated at this stage, and E. multinervia and Welwitshia both evolved lengthy strap-shaped leaves. Parallel evolution of characters is quite common in the gnetophytes, many of which copied angiosperms, e.g. the dicot-like broad leaves in Gnetum, reduced female gametophytes lacking archegonia in Gnetum and Welwitschia, flower-like reproductive organs, double fertilization, style-like micropylar tube, spinulose pollen grains in Gnetum and vessels in wood anatomy, etc. (Martens 1971; Muhammad and Sattler 1982; Doyle 1996; Yao et al. 2004). However, parallel evolution of characters within the gnetophytes remains unknown. The strap-shaped leaves with dichotomizing venation in E. multinervia and W. mirabilis is such a case, though the leaf size is different in the two species. Welwitschia mirabilis has much larger leaves than E. multinervia. The lineage Gnetum evolved its broad leaf and pinnate venation as an adaptation to the tropical and subtropical environment; Welwitschia developed its strap-shaped leaf as an adaptation to the coastal environment in southwestern Africa. Early Ephedraceae have a highly diversified leaf morphology (e.g. leaf shape, fusion and venation), which is at least partially attributable to diverse niches created by the volcanic activities during the Cretaceous (Yang et al. 2013). Molecular clock dating suggested that modern Ephedra diversified since Oligocene (Ickert-Bond et al. 2009). Oligocene glaciations might have had a great impact on morphological evolution of Ephedraceae; those ancient lineages with lengthy and strap-shaped leaves went extinct in the late Cretaceous and Cenozoic, and the modern reduced linear and connate leaf with two (to three) parallel veins was derived as an adaptation to the dry and cold habitat.

Conclusions A huge diversity of ephedroid macrofossils is to be found in the Early Cretaceous worldwide. Ephedra multinervia Yang et Lin from the Early Cretaceous of western Liaoning in China is described as new to science. The abundant macrofossils from

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this epoch provide us with an opportunity to discuss their evolutionary and phylogenetic significance, particularly those macrofossils from western Liaoning. While character convergence between the gnetophytes and angiosperms is well known, it was so far unknown within the gnetophytes. The new species shows general ephedroid morphology, e.g. dichasial branching pattern, opposite and decussate phyllotaxy, swollen nodes, internodes having many fine longitudinal striations and a female reproductive unit possessing a straight micropylar tube. Ephedra multinervia possesses sessile and reduced female cones with only one pair of bracts forming a cupule enclosing the two seeds, while the Welwitschia-like leaf shape markedly distinguishes this species from all other known Ephedraceae. The similarity of leaves between E. multinervia and Welwitschia resulted from parallel evolution and presumably had adaptive significance in the Early Cretaceous. This is the first report on parallel evolution within the gnetophytes.

Acknowledgments We thank Mr. Y. B. Sun for his help with the line drawing. This work was supported by the National Natural Science Foundation of China (J1310002, 31270238, 31470301) and Fairy Lake Botanical Garden. Conflict of interest The authors declare that they have no competing interests.

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