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(American Journal of Botany. 2004;91:361-368.)
© 2004 Botanical Society of America, Inc.

Structure and Development

Ontogeny of triovulate cones of Ephedra intermedia and origin of the outer envelope ofovules of Ephedraceae1

Yong Yang2

Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing 100093, People's Republic of China

Received for publication July 22, 2003. Accepted for publication October 21, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The origin of the two envelopes of ovules of extant species of Ephedraceae has been discussed previously in different ways based only on investigation of bimerous female cones. To generalize the characteristics of the two envelopes, ontogenetic and metamorphic patterns of the two envelopes of ovules of trimerous female cones of Ephedra intermedia Schrenk et C. A. Meyer were studied using scanning electron microscopy. The outer envelope of the ovules of triovulate female cones of Ephedra intermedia is initiated as two adaxial lateral protuberances. Thus, the outer envelope of extant Ephedraceae may have been derived from three foliar components of the second proximal whorl of the secondary reproductive shoot of the ancestral compound female cone. Two adaxial lateral foliar components likely gave rise to the outer envelope by fusion, while the third abaxial foliar component was reduced. These new findings suggest that the Gnetales derived their female cones from trimerous multiaxial female reproductive organs of their progenitors. Some evolutionary aspects of female cones of Ephedra are also discussed.

Key Words: Ephedra intermedia • Ephedraceae • ontogeny • origin • outer envelope of ovule • trimerous female cone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The Gnetales have female reproductive organs bearing ovules with more than one envelope that are unique within the four extant groups of gymnosperms—cycads, Ginkgo, conifers, and Gnetales. The characteristic female reproductive organs of the Gnetales have aroused much discussion, and many diverse opinions have been proposed to explain the origin and evolution of reproductive organs of gnetalean plants, especially the additional envelopes (Van Tieghem, 1869 ; Strasburger, 1879 ; Von Wettstein, 1907 ; Arber and Parkin, 1908 ; Thoday and Berridge, 1912 ; Chamberlain, 1935 ; Eames, 1952 ; Martens, 1971 ; Crane, 1985 ; Takaso, 1985 ; Doyle and Donoghue, 1986 ; Takaso and Bouman, 1986 ; Doyle, 1994 ; Nixon et al., 1994 ; Endress, 1996 ; Hasebe, 1999 ; Shindo et al., 1999 , 2001 ; Bowe et al., 2000 ; Chaw et al., 2000 ; Gugerli et al., 2001 ; Yang, 2001 ; Magallón and Sanderson, 2002 ). However, that question has by far remained unclear.

The extant Gnetales consist of three monotypic families, Ephedraceae (Ephedra L.), Gnetaceae (Gnetum L.), and Welwitschiaceae (Welwitschia Hooker f.). Many features of Ephedraceae account for a basal position of the family within the Gnetales (e.g., presence of archegonia, developmental patterns of male and female gametophytes, embryo without a feeder, and molecular characters) (Chamberlain, 1935 ; Bierhorst, 1971 ; Crane, 1985 ; Friedman, 1990 ; Chaw et al., 2000 ; Gugerli et al., 2001 ).

The ovule of extant Ephedra consistently bears two envelopes. The nature of the two envelopes has been in dispute since Van Tieghem (1869) and was reviewed by Thoday and Berridge (1912) , Pearson (1929) , Martens (1971) , and Takaso (1985) . The outer envelope has been regarded variously as a second integument (Strasburger, 1879 ; Thoday and Berridge, 1912 ; Meeuse, 1978 ), two median foliar components (Van Tieghem, 1869 ; Eames, 1952 ), two lateral foliar components (Strasburger, 1872 ; Jaccard, 1894 ; Pearson, 1929 ; Mehra, 1950 ), three foliar components (Lignier and Tison, 1911 ), or four foliar components (Land, 1904 , 1907 ; Coulter and Chamberlain, 1917 ; Chamberlain, 1935 ), while the inner envelope has been regarded either as an integument (Van Tieghem, 1869 ; Strasburger, 1872 , 1879 ; Yang, 2001 ), two foliar components (Land, 1904 , 1907 ; Coulter and Chamberlain, 1917 ; Chamberlain, 1935 ), or three foliar components (Lignier and Tison, 1911 ). However, detailed data of the two envelopes have been based only on bimerous female cones. Because extant Ephedra has both bimerous and trimerous cones, previous arguments of the two envelopes are certainly incomplete. Takaso (1985) emphasized that trimerous cones should be investigated to generalize characteristics of the two envelopes. As a result, this study focused on trimerous female cones of Ephedraceae to elucidate the origin and evolution of the two envelopes, which will definitely lend itself to understanding the evolution of female cones in the family, and accordingly, to understanding the Gnetales.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Terminology
In Ephedra, ovulate units of female cones are addressed in different ways, e.g., ovules, flowers, and female reproductive units (Pearson, 1929 ; Eames, 1952 ; Yang, 2001 ). In this paper, I follow Yang (2001) and call the ovulate units of female cones "female reproductive units (FRUs)."

Plant material
None of the extant species of Ephedra had strictly trimerous female cones, and trimerous female cones often coexisted with bimerous female cones within a single species. Because the focus of this study is only on trimerous female cones and because triovulate cones must be trimerous, a species such as E. intermedia Schrenk et C. A. Meyer with frequent triovulate cones is deemed suitable for this study.

Ephedra intermedia is distributed in the north and northwest of China as well as the vast arid region of Central Asia. This species is characteristically an herb-like subshrub or shrub. Woody stems are absent, short, and prostrate, or sometimes strong and erect, and usually densely ramified. Three leaves, or sometimes two, are connate at the nodes, and the free parts of the leaves are obtuse or sometimes subulate. The female cone usually bears five whorls or pairs of bracts, among which only the uppermost whorl is fertile. The bracts become red and fleshy at maturity. Each female cone bears three or sometimes two FRUs, and each FRU has a leathery outer envelope and a membranous inner envelope. The inner envelope develops into a micropylar tube that is up to 5 mm long and is tortuous. Mature cones bear three, sometimes two, seeds (Fu et al., 1999 ).

Samples of E. intermedia were collected from Ürümqi Shi of Xinjiang Uygur Zishiqu, People's Republic of China. The voucher specimens (XJ 01005) are preserved in the Herbarium (PE), Institute of Botany, Chinese Academy of Sciences.

Sample preparation
Female cones at various developmental stages were fixed in formalin-acetic acid-alcohol (FAA) from the beginning of April to the beginning of May 2001. The fixed ovulate cones were dissected and dehydrated in an alcohol series, critical-point dried, and coated with gold palladium. Observations and photographs were made using a Hitachi S-800 scanning electron microscope operating at 30 kV.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Development of triovulate cones
Female cones of E. intermedia are sessile and axillary to the leaves of the previous year's shoots, which may be trimerous or bimerous depending on whether they arise on branches with three leaves or two leaves at a node. Each female cone possesses usually five whorls of verticillate or opposite bracts. Normally, only the uppermost whorl of bracts is fertile. The bimerous cone bears two FRUs, one in the axil of each fertile bract. In a trimerous cone, there are commonly three FRUs, one in each axil of the uppermost whorl of fertile bracts. Each FRU possesses two envelopes.

Reproductive shoots and FRU primordia
Male cones of E. intermedia appear earlier than female cones. In Ürümqi Shi, axillary young buds of the previous year's shoots shift from vegetative growth to reproductive growth in late April. Female cones occur in the form of axillary buds (Fig. 1A) that are protected by five alternate whorls of verticillate bracts. Soon after the appearance of the fifth whorl of bracts, the apex of the young bud differentiates and gives rise to a globular FRU primordium in each axil of the uppermost whorl of bracts. The FRU primordia are oriented outward and in an oblique direction from the reproductive axis (Fig. 1B). The apical bud of the female reproductive axis is surrounded by the three FRUs and is discernible at this time.



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  		Fig. 1. Scanning electron micrographs of development of trimerous cones of Ephedra intermedia. (A) Terminal bud of young cone before differentiation (bar: 40 µm). (B) Initiation of FRU (bar: 100 µm). (C) Initiation of outer envelope (bar: 50 µm). (D) Hippocrepiform initial complex of outer envelope (bar: 100 µm). (E) Initiation of inner envelope (bar: 100 µm). (F) Circular swelling of initials of inner envelope (bar: 100 µm). (G) Further development of two envelopes (bar: 50 µm). (H) Inner envelope protruding out of outer envelope to form micropylar tube (bar: 125 µm). (I) Further development of micropylar tube and three Female reproductive units (FRUs) (bar: 250 µm). Abbreviations: tb, terminal bud; b1, b2, b3, b4, b5, bract whorl numbered acropetally; fp, female reproductive unit primordium; ioe, initiation of outer envelope; iie, initiation of inner envelope; oe, outer envelope; ie, inner envelope; mt, micropylar tube; fru, female reproductive unit.

 
Initiation and development of the outer envelope
The outer envelope of an FRU makes its first appearance as two lateral adaxial protuberances at the base of the FRU primordium (Fig. 1C). This initiation complex of the outer envelope undergoes a rapid anisomerous growth and produces a hippocrepiform structure (Fig. 1D, E); at this time, the abaxial side of the outer envelope begins to grow. The apical bud of the female cone is prominent during this period (Fig. 1D). The inner envelope is initiated at this time.

Initiation and development of inner envelope
The inner envelope of the FRU comes out as an annular swelling between the hippocrepiform outer envelope initiation complex and the inner ovular primordium (Fig. 1F). The annular swelling together with the outer envelope undergo a rapid zonal growth until the two developing envelopes surround most of the central globular primordium (Fig. 1G), that is the early nucellus of the ovule in nature. In this phase, the abaxial side of the outer envelope grows much quicker than the adaxial side, while the annular swelling of the inner envelope develops simultaneously. Soon after the developing inner envelope surrounds the ovular primordium, the outer envelope grows slower than the inner envelope, and the inner envelope protrudes out of the outer envelope and yields a micropylar tube. The adaxial side of the inner envelope grows much more rapidly than the abaxial side, which leads to the outward orientation of the opening of the micropylar tube (Fig. 1H, I).

Asymmetrical and abortive development of FRUs
Abortion of FRUs or FRU primordia occurs frequently in the developing female cones of Ephedra intermedia. In the first case, one of the three FRUs in a triovulate cone aborted when the micropylar tubes of the other two FRUs were protruding out of the outer envelopes (Fig. 2A). In the second case, a residual protuberance was in place of an FRU in a trimeous female cone that had five whorls of verticillate bracts (Fig. 2B). The inner envelope of the other two FRUs of this cone stayed at the early stage of annular protuberance between the outer envelope and the inner ovular primordium. The residual apical bud of this cone was discernible. In the third case, one FRU of a biovulate cone only gave rise to its outer envelope while the inner envelope of the other FRU of the same cone was at the beginning of protruding out of the outer envelope (Fig. 2C). In the fourth case, a female cone bore a single FRU (Fig. 2D). This uniovulate cone had five pairs of bracts, and the inner envelope stayed inside of the outer envelope.



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  		Fig. 2. Scanning electron micrographs of metamorphic patterns during the development of female cones of Ephedra intermedia. (A) Abortive female reproductive unit in triovulate cone (bar: 125 µm). (B) Residue of FRU primordium in trimerous cone (bar: 40 µm). (C) Abortive FRU and asymmetrical development of biovulate cone (bar: 40 µm). (D) Uniovulate cone (bar: 50 µm). (E) Proliferated bracts in biovulate cone (bar: 100 µm). (F) Asymmetrical development of triovulate cone with three proliferated bract-like structures (bar: 125 µm). (G) Two foliar organs in position of one FRU in triovulate cone (bar: 125 µm). (H) Proliferated terminal bud of female cone (bar: 125 µm). (I) Cleft of outer envelope and four protuberances in trimerous cone (bar: 100 µm). Abbreviations: af, abortive FRU; apf, abortive primordium of FRU; bl, bract like structures; fc, foliar component; pb, proliferated bud; p1, p2, p3, p4, four protuberances

 
Metamorphic patterns in the development of female cones of Ephedra intermedia
A pair of bract-like structures, decussate to the fertile pair of subtending bracts of the female cone, develops between the two FRUs (Fig. 2E). Micropylar tubes of the twin FRUs form, and their openings are obliquely outward. The bract-like structures are strip-like and incurved. This kind of strip like bract is also observable in a triovulate cone (Fig. 2F). However, only two bracts are alternately arranged with two of the three FRUs, while the third bract-like structure is missing. The developmental stage of the FRUs in this triovulate cone is slightly earlier than that of the FRUs in the former biovulate cone (Fig. 2E).

In a female cone with verticillate bracts, a pair of bracteoles appears in place of the third FRU in the axil of one of bracts of the uppermost whorl (Fig. 2G). The two bracteoles are nearly opposite and laterally arranged to the false fertile subtending bract. No structure is observed between the twin bract-like structures. The micropylar tubes of the other two FRUs just protrude out of the outer envelope and the openings of the micropylar tube are oblique outwards.

In a triovulate cone, the apical bud is proliferated (Fig. 2H) and gives rise to a column like structure. It is worth pointing out that one of the three FRUs in this cone was removed for clear observation of the column like structure, leaving a scar. The developmental stages of the other two FRUs in this cone are almost the same as those of Fig. 2F.

In a peculiar female cone (Fig. 2I) with verticillate bracts, some strange structures are remarkable. A normal FRU is at a developmental stage identical to that of Fig. 2G. The adaxial side of the outer envelope of this FRU is slightly split, while the envelope of the other FRU is cleft. Both of the inner structures of the two mentioned FRUs are similar to that of a normal FRU. Moreover, there are three small, round protuberances (Fig. 2I, p1, p2, p4) and one conical or column-like structure (Fig. 2I, p3). The round protuberance (Fig. 2I, p1) is in the position of the apical bud of a normal female cone. The column-like protuberance (Fig. 2I, p3) is opposite another protuberance (Fig. 2I, p2); these two structures together with the abaxial protuberance (Fig. 2I, p4) occupy the position of the third FRU in a triovulate cone.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Different opinions have been put forth as to whether the two envelopes of FRUs of Ephedra are homologous or not. Strasburger (1879) and Land (1904 , 1907 ) assumed that the two envelopes are homologous in nature, but Van Tieghem (1869) , Strasburger (1872) , Eames (1952) , Crane (1985) , Takaso (1985) , and Yang (2001) believed that they are different. The two envelopes will be discussed separately because in this study they are different in nature. By the way, to better understand the origin of the two envelopes, it is important to know if the female cones of Ephedraceae are compound, which has been suggested in most previous studies because the ovulate structures are in the axils of bracts (Coulter and Chamberlain, 1917 ; Chamberlain, 1935 ; Doyle, 1994 ; Hickey and Taylor, 1996 ; Yang, 2001 ).

Origin of the outer envelope of FRUs
All gymnosperms except Gnetales have an ovule bearing one single envelope. In Gnetales, the ovule of Ephedra has two envelopes and that of Gnetum and Welwitschia has three (Martens, 1971 ; Endress, 1996 ). The origin and evolution of these additional envelopes have been considered of great evolutionary significance to the phylogeny of Gnetales and discussed in different ways (Thoday and Berridge, 1912 ; Eames, 1952 ; Martens, 1971 ; Crane, 1985 ; Takaso, 1985 ; Hasebe, 1999 ; Shindo et al., 1999 ; Yang, 2001 ). For Ephedra, different opinions also have been expressed. (1) Strasburger (1872) first proposed that the outer envelope of Ephedra corresponds to the first pair of leaves of an axillary shoot, and Jaccard (1894) , Pearson (1929) , Hagerup (1934) , Takaso (1985) , Kubitzki (1990) , and Yang (2001) held the same view. (2) Strasburger (1879) changed his opinion and regarded the outer envelope as a second integument; Thoday and Berridge (1912) and Meeuse (1978) agreed. (3) Van Tieghem (1869) believed that the outer envelope is comparable to the second pair of leaves of an axillary shoot; Mehra (1950) , Eames (1952) , Bierhorst (1971) , and Foster and Gifford (1974) agreed. (4) Lignier and Tison (1911) assumed that the outer envelope is made up of three leaves that are represented by the three angles of seeds. (5) Coulter and Chamberlain (1917) and Chamberlain (1935) agreed with Land (1904 , 1907 ) that the outer envelope of Ephedra trifurca Torr. ex Wats. consists of four bracts. (6) Herzefeld (1922) argued that the outer envelope is an organ comparable to the "Fruchtschuppe" of Taxaceae. (7) Finally, Hagerup (1934) assumed that the outer envelope consists of prophylls. In short, three fundamental questions are involved in the discussion of the origin and evolution of the two envelopes of Ephedra. What is the nature of the outer envelope: integumental or foliar? How many components are involved in formation of the outer envelope if it is foliar? How was the outer envelope derived?

The outer envelope of Ephedra was considered as an integument or leaf in nature. Some authors assumed that the outer envelope is comparable to the outer integument of angiosperms (Strasburger, 1879 ; Thoday and Berridge, 1912 ; Meeuse, 1978 ). This hypothesis is, however, disproved by ontogenetic evidence in both this study and previous studies. The developmental pattern of the two envelopes of Ephedra is totally different from that of the two integuments of angiosperms. In angiosperms, the initiation of the two integuments is basipetal or almost simultaneous (Foster and Gifford, 1974 ; Endress, 1996 ), whereas it is distinctly acropetal in both E. intermedia of this study and E. sinica (Yang, 2001 ). Accordingly, the present author believes that the outer envelope of Ephedra is different from the outer integument of angiosperms. In addition, if the outer envelope is another integument, it should have a similar initiation pattern to the inner integument; however, this is not the case. The outer envelope initiates as two adaxial lateral protuberances, while the inner integument initiates as an annular swelling. This corroborates the idea that the outer envelope is not a second integument. As a result, the second integument hypothesis of the outer envelope (Strasburger, 1879 ) is not favored by this study.

On the contrary, most other previous researchers believed that the outer envelope of Ephedra was derived from foliar structures of the ancestral secondary reproductive shoot (Van Tieghem, 1869 ; Strasburger, 1872 ; Land, 1904 , 1907 ; Lignier and Tison, 1911 ; Coulter and Chamberlain, 1917 ; Eames, 1952 ; Takaso, 1985 ; Endress, 1996 ; Yang, 2001 ). However, no unequivocal evidence has been obtained supporting this theory until this study.

In this study, the outer envelope does always initiate as two adaxial lateral protuberances in triovulate cones of E. intermedia (Fig. 1C, D). This initiating pattern is identical to that of bimerous female cones of E. distachya and E. sinica. It indicates that the outer envelope probably includes two foliar components. Two other metamorphic patterns of female cones provide strong evidence for foliar origin of the outer envelope. In a trimerous female cone of E. intermedia, the outer envelope of an FRU is replaced by a pair of basally fused bracteoles that encloses an ovule bearing a very short micropylar tube (Fig. 2I), suggesting that the outer envelope is composed of two foliar organs.

This inference is further corroborated by another trimerous female cone under a reversed condition. In this trimerous female cone (Fig. 2G), a pair of free bracteoles occurs in place of an FRU, and the arrangement of that pair of bracteoles is similar to that of the two components of Fig. 2I. It may be caused by an asymmetrical development of the outer envelope and the ovule of the female cone in which the outer envelope is reversed into foliar bracts while the ovule was suppressed. Both types of metamorphic female cones support the foliar theory of the outer envelope of Ephedra. By the way, transfusion tissue is defined as the tracheids and associated parenchyma cells. It exists at the periphery of the leaf veins in all extant gymnosperms (Foster and Gifford, 1974 ). Thoday and Berridge (1912) found that vascular bundles of the outer envelope of E. distachya are accompanied by transfusion tissue. This implies that the outer envelope is foliar in nature because the transfusion tissue is present only in foliar organs of gymnosperms, which further corroborates the foliar theory of the outer envelope. Moreover, it is suggested by the two reversed trimerous cones that two foliar organs are involved in the formation of the outer envelope. In addition, the outer envelope is always initiated by two protuberances (Takaso, 1985 ; Yang, 2001 ; and this study), which indicates that the outer envelope is made up of two foliar organs as well. As a result, it is clear now that the outer envelope of Ephedra is modified from two foliar organs by fusion, and the hypotheses of Lignier and Tison (1911) and Land (1904 , 1907 ) are not supported in this study.

The origin and evolution of the outer envelope has been controversial within those foliar theories, and four different views have emerged (Van Tieghem, 1869 ; Strasburger, 1872 ; Land, 1904 , 1907 ; Lignier and Tison, 1911 ). (1) Strasburger (1872) first proposed that the outer envelope of Ephedra corresponds to the first pair of leaves of an axillary shoot; Jaccard (1894) , Pearson (1929) , Hagerup (1934) , Takaso (1985) , Kubitzki (1990) , Endress (1996) , and Yang (2001) held the same point. (2) Van Tieghem (1869) believed that the outer envelope is comparable to the second pair of leaves of an axillary shoot; Mehra (1950) , Eames (1952) , Bierhorst (1971) , and Foster and Gifford (1974) , agreed with him. (3) Lignier and Tison (1911) assumed that the outer envelope is made up of three leaves, one dorsal and two lateral. (4) Coulter and Chamberlain (1917) and Chamberlain (1935) agreed with Land (1904 , 1907 ) that the outer envelope of Ephedra trifurca consists of four bracts. It is possible to generalize about the origin and evolution of the outer envelope based on the early development of both trimerous cones of this study and bimerous ones of previous studies (Takaso, 1985 ; Yang, 2001 ).

In both bimerous and trimerous female cones (Martens, 1971 ; Takaso, 1985 ; Yang, 2001 ; this study), the outer envelope of Ephedra without exception is initiated as two protuberances that are positioned laterally and adaxially but not strictly oppositely. Meanwhile, the outer envelope has nothing abaxial at the beginning of initiation but develops later by means of zonal growth led by the two initiation protuberances. This strongly indicates that originally three foliar organs, two adaxial and one abaxial (Fig. 3A, B), may have been at the node in the place of the outer envelope. The two initiating protuberances might represent the two original adaxial lateral foliar organs while the abaxial one was reduced in the evolution of the outer envelope. The arrangement of the three foliar organs implies that they belonged to the second proximal whorl of the ancestral secondary reproductive shoot because the verticillate foliar organs are alternately arranged along an axis. Vasculature of the outer envelope has similar implication. According to Strasburger (1879) , Thoday and Berridge (1912) , and Eames (1952) , three large vascular bundles enter the base of the outer envelope, and two adaxial bundles traverse the outer envelope while the abaxial one dies out very early and does not contribute anything towards the supply of the outer envelope. Each of the three vascular bundles may represent a foliar organ. The two adaxial bundles traverse the outer envelope suggesting that the outer envelope was fused from two foliar organs. The third abaxial bundle dies out quite early, indicating that the abaxial foliar organ was reduced before the formation of the outer envelope. Interestingly, four protuberances occur in a trimerous cone (Fig. 2I). This reversed condition can be easily explained with the new viewpoint described on the origin of the outer envelope. According to that, p1 is the terminal bud of the cone, while p2, p3, and p4 may represent the three foliar organs of the second proximal whorl of the secondary reproductive shoot. Among p2, p3, and p4, p2 and p3 are lateral and adaxial, while p4 is abaxial; meanwhile, p4 is less conspicuous than p2 and p3. This condition thus may have occurred slightly earlier than the loss of the abaxial foliar organ.



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  		Fig. 3. Diagram of evolution of the outer envelope of Ephedra. (A), (B), (C), and (D), front view; (E), (F), (G), and (H), view from top. (A, E) Early evolutionary stage 1. (B, F) Early evolutionary stage 2. (C, G) Evolutionary stage 3. (D, H) Extant Ephedra. Abbreviations: ab2, abaxial bracts of second proximal whorl of secondary reproductive shoot; ad2, adaxial bracts of second proximal whorl of secondary reproductive shoot; b, bract of primary axis of female cones; b1, bract of first proximal whorl of secondary reproductive shoot; fru, female reproductive unit; o, ovule; oe, outer envelope; pa, primary axis of female cone; sa, axis of secondary reproductive shoot

 
This new scenario of origin and evolution of the outer envelope is illustrated in Fig. 3. The female cone of progenitor of Ephedra is a shortened multiaxial reproductive shoot system that bears secondary reproductive axes in axils of bracts (Yang, 2001 ). The phyllotaxes of both the primary and secondary axes of the ancestral female cone are trimerous but not opposite and decussate, and there are at least two whorls of bracteoles along the secondary axes of the progenitor of Ephedra (Fig. 3A, E). Evolutionary changes of the female cone have gone with the shortening of the internodes of both primary and secondary axes that leads to a structural reduction and loss of secondary axes of the female cone and produces compact female cones. With the shortening of the secondary axes, the proximal whorl of bracteoles is reduced and lost (Fig. 3B, F). With further shortening of the secondary axes, the abaxial bracteole of the second whorl is lost (Fig. 3C, G). Subsequent combined growth of the two adaxial bracteoles gave rise to the outer envelope of ovules of Ephedra (Fig. 3D, H). The two constituent adaxial bracteoles of the outer envelope have preserved their original position in this evolutionary process.

From this new theory, a new architecture for the ancestor of Ephedraceae as well as the Gnetales is proposed. The extant Gnetales might have derived their female cones from trimerous multiaxial female reproductive organs of their progenitors. The prominent relict ancestral structures of the extant Gnetales are the additional envelopes of ovules. Evolution of the outer envelope of Ephedraceae might have paralleled that of Gnetaceae and Welwitschiaceae because Ephedraceae and the ancestor of the other two gnetalean families diverged from their common ancestor. The Ephedraceae preserve not only the trimerous phyllotaxis but also the position of the two initiation protuberances of the outer envelope while the other two gnetalean families have entirely lost their ancestral phyllotaxis and their outer envelopes have two opposite initiation components (Takaso and Bouman, 1986 ).

This new scenario partially revives the opinion of Lignier and Tison (1911) but has no similarity to those of Van Tieghem (1869) , Stasburger (1872) , and Land (1904 , 1907 ). I partially agree with Lignier and Tison (1911) in the arrangement pattern of the three foliar organs, but disagree with them in that the abaxial foliar organ is reduced before the formation of the outer envelope according to my new theory. In addition, Lignier and Tison (1911) believed the inner envelope includes three foliar organs, which is different from my opinion as well.

Homology of the inner envelope of FRUs
The inner envelope is regarded by most researchers as (1) comparable to the integument of other gymnosperms (Van Tieghem, 1869 ; Strasburger, 1879 ; Jaccard, 1894 ; Arber and Parkin, 1908 ; Thoday and Berridge, 1912 ; Herzefeld, 1922 ; Pearson, 1929 ; Mehra, 1950 ; Eames, 1952 ; Lehmann-Baerts, 1967 ; Fagerlind, 1971 ; Foster and Gifford, 1974 ; Meeuse, 1978 ; Kubitzki, 1990 ; Endress, 1996 ). However, (2) Lignier and Tison (1911) proposed that it consists of three bracts, (3) Coulter and Chamberlain (1917) and Chamberlain (1935) accepted Land's (1904 , 1907 ) idea that it derives from a fusion of two bracts, and (4) Hagerup (1934) looked upon it as a sporophyll.

The initiation complex of the inner envelope as an annular swelling in Ephedra intermedia is identical to that of E. sinica (Yang, 2001 ) and E. distachya (Takaso, 1985 ). In addition, the inner envelope is always a companion to its inner ovular structure together with which it disappeared (Fig. 2G). Such an ontogenetic pattern does not support the leaf nature of the inner envelope (Land, 1904 , 1907 ; Lignier and Tison, 1911 ; Coulter and Chamberlain, 1917 ; Hagerup, 1934 ; Chamberlain, 1935 ), and no other evidence validates this hypothesis (Takaso, 1985 ). On the contrary, the present new evidence supports the inner envelope to be a true integument that may be comparable to the single integument of cycads, Ginkgo, and conifers. Moreover, recent studies have shown that the Gnetales have a close relationship with conifers (Bowe et al., 2000 ; Chaw et al., 2000 ; Gugerli et al., 2001 ; Magallón and Sanderson, 2002 ), which, coupled with my explanation of the female cone of Ephedra (Yang, 2001 ), could account for the homological nature of the inner envelope of Ephedra to the integument of cycads, Ginkgo, and conifers.

Evolution of female cones within Ephedra
Female cones of Ephedra have two basic patterns including bimerous female cones and trimerous female cones. Cladistic analyses have suggested that bimerous phyllotaxy (manifested in leaves and bracts) is the synapomorphic state of gnetalean plants (Crane, 1996). Hufford (1996) assumed that the trimerous state is derived in Gnetales. However, Lignier and Tison (1911) believed that the trimerous state is primitive. This study provides certain data that allow a discussion of evolution of female cones of Ephedra.

Previous studies have demonstrated that female cones of the extant Ephedraceae are compound in nature (Von Wettstein, 1907 ; Pearson, 1929 ; Chamberlain, 1935 ; Hagerup, 1938 ; Eames, 1952 ; Martens, 1971; Endress, 1996 ; Hickey and Taylor, 1996 ; Yang, 2001 ). Further data supporting this theory were obtained in this study (Fig. 2E, F), in which the proliferated bracts made the FRUs in the axils of bracts of the lower whorl. This theory suggests that the extant female cones of Ephedraceae are derived from ancestral mutiaxial structures (Fig. 3A) that have indeterminate terminal buds of the primary cone axes. In extant species, the terminal buds of female cones have been transformed into three states that bear evolutionary significance. The first is that the terminal bud is preserved in the early development of female cones as shown in the trimerous triovulate cones of this study (Fig. 1B–D). The second occurs in species with bimerous biovulate cones such as E. distachya (Takaso, 1985 ) and E. sinica (Yang, 2001 ), in which the terminal buds have disappeared, but where there is a furrow between the two FRUs. This state still preserves the space of the terminal bud. The third state is that an FRU directly develops from the apex of the primary axis of the bimerous uniovulate cone and the terminal bud has entirely disappeared, e.g., E. altissima (Thoday and Berridge, 1912 ; Pearson, 1929 ). Comparing the three extant states of terminal buds of female cones of Ephedraceae with that of ancestral multiaxial structures, the first state is closer to the ancestral state, the second is less so, and the third state deviates most distantly from the ancestral state. Thus, the trimerous triovulate cone may be most primitive in the genus, the biovulate cone may be transitional, and the uniovulate cone may be most derived. In this study, I have concluded that the outer envelope is derived from an ancestral three-leaved state. It implies that the trimerous female cone is primitive within Ephedraceae as well. In addition, this hypothesis is corroborated by data obtained in this study. Figure 2A–D shows a reductive series of female cones from triovulate to uniovulate, which occurs on account of asymmetrical development of FRUs within the female cones. The asymmetrical development of FRUs can happen in varied stages, e.g., developing FRU in Fig. 2A, FRU primordium in Fig. 2B, and entirely disappearing in Fig. 2C–D. It is worth pointing out that the asymmetrical development also works in the derivation of uniovulate cones from biovulate cones within Ephedra rhytidosperma Pachomova (personal observation). As a result, this mechanism of asymmetrical development seems an important mode for the reduction of female cones of Ephedra. Some other studies yielded results in agreement with my viewpoint. Thoday and Berridge (1912) suggested that the uniovulate cone is a modification from the biovulate condition. Mussayev (1978) believed those species with uniovulate cones are most derived within Ephedra. In conclusion, female cones of extant Ephedra have an evolutionary tendency from trimerous to bimerous and from three ovules to one ovule via two ovules.


   FOOTNOTES
 
1 This paper is extracted from the author's Ph.D. thesis. The author is grateful to Prof. De-zhi Fu, Dr. Guang-hua Zhu, and Dr. Jin-xing Lin for their direction. Thanks are also given to Nicholas J. Turland of the Missouri Botanical Garden for his nice suggestions on the English of the manuscript. This work was supported by the Natural Science Foundation of China (30370105) and Project Ksxc2-sw-108 (Chinese Academy of Sciences). Back

2 ephedra{at}ns.ibcas.ac.cn Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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C. RYDIN, K. R. PEDERSEN, P. R. CRANE, and E. M. FRIIS
Former Diversity of Ephedra (Gnetales): Evidence from Early Cretaceous Seeds from Portugal and North America
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