Annals of Botany 104: 1243–1253, 2009 doi:10.1093/aob/mcp232, available online at www.aob.oxfordjournals.org

Early reproductive developmental anatomy in Decaisnea () and its systematic implications

Hua-Feng Wang1, Cynthia Ross Friedman2,*, Zhi-Xin Zhu3 and Hai-Ning Qin3 1Beijing Urban Ecosystem Research Station, State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 100085 Beijing, China, 2Department of Biological Sciences, Thompson Rivers University, Box 3010 900 McGill Road, Kamloops, British Columbia, Canada V2C 5N3 and 3State Key Laboratory of Systematic and Evolutionary Botany, Chinese Academy of Sciences, 100093 Beijing, China Downloaded from Received: 26 May 2009 Returned for revision: 20 July 2009 Accepted: 3 August 2009 Published electronically: 16 September 2009

† Background and Aims Decaisnea insignis, known as ‘dead man’s fingers’ (Lardizabalaceae), is widely distrib- uted in China and the Himalayan foothill countries. This economically important , which is the only species in the , has not been the subject of any embryological studies aside from one brief, older paper that lacks micrographs. Data on Decaisnea are also important because its systematic position has been unstable since the http://aob.oxfordjournals.org/ genus was established in 1855. Therefore, the objectives of this study were: (a) to use modern microscopy to document early reproductive anatomical development in Decaisnea; and (b) to compare qualitatively these early embryological characters with allied taxa in a systematic context. † Methods Decaisnea insignis floral buds and inflorescences were regularly collected from Shaanxi Province, China and prepared for light microscopy. The embryological characters studied were qualitatively compared with those of allied taxa via a thorough examination of the existing literature. † Key Results Early reproductive anatomy in Decaisnea was documented and novel revelations made. It was dis- covered that the pollen is shed when three-celled (not two-celled, as previously reported), and that endosperm

formation is nuclear (not cellular or helobial, as previously reported). These two newly revealed embryological at Library of Chinese Academy Sciences on April 2, 2015 characters are not found in any other members of Lardizabalaceae. Furthermore, neither are persistent antipodal cells, which we confirmed to be present in Decaisnea. † Conclusions Decaisnea and other Lardizabalaceae characteristically have tetrasporangiate anthers, a secretory tapetum, simultaneous microsporocyte cytokinesis, primarily bitegmic, crassinucellate ovules, and a Polygonum type embryo sac. However, in the family, persistent antipodals, nuclear endosperm, and pollen shed at the three- celled stage are only found in Decaisnea. These embryological data prompted the suggestion that Decaisnea needs elevation above the level of genus.

Key words: Decaisnea insignis, embryology, endosperm, Lardizabalaceae, microscopy, pollen, reproductive anatomy, systematics.

INTRODUCTION Parvatia, Sargentodoxa, Sinofranchetia and Stauntonia (Qin, 1997; Chen and Tatemi, 2001). However, Loconte and Estes Decaisnea is a monotypic genus of Lardizabalaceae, with the (1989) state that Decaisea could be treated as a subfamily species, Decaisnea insignis (Griffith) Hook. f. & Thomson named Decaisneoideae within Lardizabalaceae, as indicated (Chen and Tatemi, 2001), widely distributed from central to by an outgroup comparison and parsimony analysis of 34 south-western China, extending into Bhutan, Myanmar, genera within . Further to this, Loconte et al. Nepal, Sikkim and north-eastern India. The plant is nicknamed (1995) conclude that the genus Decaisnea should be a new ‘dead man’s fingers’, as it possesses racemes of striking deep family within a new order Lardizabalales within ranunculids purplish-blue elongated fruits (follicles). The plant is econom- as implied by a morphologically based cladistic analysis on ically important, as it is readily cultivated as an ornamental, 116 ingroup taxa and five outgroups coded for 109 characters and its fruits are deemed to be a delicacy. However, in spite and 192 apomorphic character states. Thorne (2000, 2007) of the value of Decaisnea, it has not been the subject of any acknowledges the elevated status of Decaisnea suggested by dedicated embryological studies aside from a solitary and Loconte and Estes (1989) and Loconte et al. (1995). brief older paper by Swamy (1953), which is limited in that Qin (1989, 1997), however, regards Decaisnea as the only it exclusively presents line drawings. As such, a modern exam- genus within the tribe of Decaisneeae, which, along with three ination of the embryology of Decaisnea is needed. other tribes (Sinofranchetieae, Lardizabaleae and Akebieae) The systematic position of Decaisnea has been unstable since comprise Lardizabalaceae. Many authors support Qin’s taxo- the genus was established in 1855 (Griffith, 1855). Presently, nomic treatment (Qin, 1989). Chen and Tatemi (2001) agree the Lardizabalaceae is generally recognized to have nine that Decaisnea is a genus within Lardizabalaceae; this view is genera: Akebia, Boquila, Decaisnea, Holboellia, Lardizabala, supported by the Angiosperm Phylogeny Group (APG II, 2003) and Mabberly (2008). Using a cladistic analysis of 43 * For correspondence. E-mail [email protected] morphological characters sensu lato, Wang et al. (2002) # The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 1244 Wang et al. — Reproductive anatomy of Decaisnea and systematic implications provide support for Qin’s view (Qin, 1989), but note that the female flowers and bisexual flowers; the strictly monoecious phylogeny of Lardizabalaceae needs further study. Zhang condition is apparently rare. For consistency, only unisexual et al. (2005) also agree with Qin’s taxonomic system (Qin, flowers were examined. In early spring, winter buds are ovoid 1989). Hoot et al. (1995a, b, 1999) constructed a molecular phy- and possess two outer scales (Fig. 1A). The inflorescence is a logeny based on chloroplast and nuclear DNA sequences, which terminal panicle of racemes (Fig. 1B). Each flower is subtended resolved Decaisnea as a member of Lardizabalaceae, but did not by a bract (Fig. 1C). Each flower has six subimbricate sepals and include morphological or developmental characters. lacks petals. Male flowers have six stamens with oblong anthers, Embryological data have been helpful in inferring relation- and three carpellodes that are small and concealed within the ships among genera and families (Bhojwani and Bhatnagar, filament tube (Fig. 1D). Female flowers have staminodes that 1978). Endress and Igersheim (1999) present an excellent are either free, or, less frequently, connate at the base, and review of gynoecium diversity and systematics of the basal have three cyclically arranged straight carpels (not shown). dicots, which includes a useful synopsis of ovular character- The mature fruit consists of three follicles.

istics of the Lardizabalaceae, but does not specifically describe Downloaded from embryology nor focus on the genus Decaisnea. As such, a Development of the anther wall study of embryological ontogeny in Decaisnea would not only contribute new information regarding reproductive devel- The anther is tetrasporangiate (Fig. 2A). The single-celled opment in this important plant, but would also provide a new archesporium is hypodermal and undergoes a periclinal div- suite of characters for systematic studies; Swamy (1953) did ision, resulting in a primary parietal layer and a primary spor- not compare his limited embryological data on Decaisnea ogenous layer. The parietal layer divides periclinally to form http://aob.oxfordjournals.org/ with those of allied taxa nor discuss their systematic impli- two layers: the inner layer contributes to the tapetum cations. Here, anther, pollen, ovule, embryo sac and early (Fig. 2B), while the outer parietal layer undergoes another endosperm development in Decaisnea insignis are studied. periclinal division, resulting in an endothecium toward the These early embryological characters are then compared outside and a middle layer toward the inside (Fig. 2C). The with allied taxa and the systematic implications discussed. mature anther wall is thus comprised of five or six layers: a single-layered epidermis, a single-layered endothecium, two or three middle layers, and a single-layered tapetum MATERIALS AND METHODS (Fig. 2D), and conforms to the dicotyledonous form of at Library of Chinese Academy Sciences on April 2, 2015 Collection and photography of whole floral buds and flowers anther wall development. The tapetum is of dual origin: most of it develops from the About five floral buds or maturing inflorescences of Decaisnea primary parietal layer, but a component also arises from the insignis (Griffith) Hook. f. & Thomson were collected every 5 ground tissue of the connective side (not shown). After for- or 6 d from Taibai Mountain in Shaanxi Province, China (alti- mation of the anther wall but before microsporogenesis, tude 1200–1500 m; voucher: Zhang 20030609, SANU) from 1 about half of the tapetal cells undergo mitosis without cytokin- March 2005 to 1 May 2006 in order to obtain a range of devel- esis, becoming binucleate (Fig. 3A). Upon initiation of micro- opmental stages. Samples were fixed in formalin–acetic acid– sporocyte meiosis, the tapetal cells elongate radially and ethanol (FAA) 2 : 2 : 1 volume/volume (v/v/v). Floral buds and protrude into the anther locule (Fig. 3B). The tapetal cells flowers (about three per sampling date) were dissected and degenerate at their original sites following microsporogenesis. photographed using a Nikon DXM 1200 stereo microscope. Therefore, the tapetum is of the secretory (glandular) type. The epidermis persists at maturity, the endothecium develops Preparation for sectioning and microscopy fibrous thickenings, and the middle layers are ephemeral, degenerating shortly before the microspores develop into Fixed male and female flower buds were dehydrated in an pollen grains. ethanol series (70 %, 85 %, 95 %, 100 % and 100 % ethanol, 2 h each), and embedded in paraffin wax. Approximately ten serial sections of individual flowers (at least two flowers) were Microsporogenesis and microgametogenesis cut at 6–9 mm, stained for 4 h in 4 % Heidenhain’s iron-alum, After formation of the anther wall, each microsporangium con- washed for 40 min with H O, stained for 4 h with 0.05 % hema- 2 tains numerous sporogenous cells (Fig. 2D). Microsporocytes toxylin, washed again with H O (30 min), and mounted on slides 2 originate from the primary sporogenous layer as well as from sec- in a gelatin solution (1 g gelatin, 100 mL H O, 2 g phenol, 2 ondary sporogenous cells (Fig. 3A). Individual microsporocytes 15 mL glycerol; Li, 1978). Photographs were taken with an become enclosed in a thick callose wall when their nuclei Olympus SP-565UZ digital camera mounted on an Olympus enter prophase of meiosis I (Fig. 3B). Meiosis II is followed by BH-2 photomicroscope equipped with Nomarski optics. The simultaneous cytokinesis with centripetally advancing constric- tonal qualities of the images were adjusted, labels were added, tion furrows, and results in tetrahedral microspore tetrads, and plates assembled with Adobe Photoshop CS2 and CS3. which enlarge and acquire thick callose walls (Fig. 3C). Shortly after callose formation, the callose walls promptly RESULTS break down, releasing microspores from the tetrad (Fig. 3D). Then, formation of a large vacuole pushes the single nucleus of Floral bud morphology each freed microspore toward the microspore wall (Fig. 3E). Decaisnea insignis used in this study were found to be Each microspore divides to form a large tube (vegetative) cell polygamo-monoecious, as individuals possessed male flowers, in the vicinity of the large vacuole and a small generative cell Wang et al. — Reproductive anatomy of Decaisnea and systematic implications 1245 Downloaded from http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015

F IG. 1 Inflorescence and floral structure of Decaisnea insignis. (A) Young leaves surrounding an immature inflorescence. One of two outer scales (sc) is visible. (B) Inflorescence branches emanate from the base of the inflorescence axis. The two lowermost branches (lb) are prominent. (C) Flowers: each flower (f) is subtended at its base by a single bract (b). (D) Floral structure: each flower has six subimbricate sepals (se) and lacks petals. Depicted here is a male flower, which possesses six stamens (st) with oblong anthers (two stamens have been removed to reveal inner structure), and the three carpellodes (arrowheads) are small and typically concealed within the filament tube. Scale bars: (A, B) ¼ 5 mm; (C, D) ¼ 1 mm. in the region that housed the original wallward microspore one-celled (Fig. 4B), and transforms into a megasporocyte nucleus (Fig. 3F). The generative cell undergoes a further div- by cutting off a parietal cell (Fig. 4C). The parietal cell under- ision, resulting in two sperm cells (Fig. 3G). Tricolpate pollen goes further divisions to form the nucellar tissue (Fig. 4D), grains are shed at this three-celled stage (Fig 3H). which causes the megasporocyte to become deep-seated within the ovule. Thus, the ovule is crassinucellate. Following parietal cell divisions, the megasporocyte under- Megasporogenesis and megagametogenesis goes meiosis I, resulting in a dyad (Fig. 4D). Meiosis II pro- Numerous ovules are found in two rows on either side of duces a T-shaped tetrad of megaspores (Fig. 4E). The three an adaxial carpel suture (Fig. 4A). The archesporium is micropylar megaspores of the tetrad degenerate, while the 1246 Wang et al. — Reproductive anatomy of Decaisnea and systematic implications Downloaded from http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015

F IG. 2 Development of the anther wall and sporogenous tissue. (A) Cross-section through the six tetrasporangiate anthers (an); arrowheads indicate the four sporangia of a representative anther. c, Carpellode; se, sepal. (B) Periclinal division of the hypodermal archesporium results in a primary sporogenous (s) layer and a primary parietal layer. The latter rapidly undergoes a second periclinal division to produce an inner layer that contributes to the tapetum (t); the outer, secondary parietal (p) layer will soon divide periclinally again. ep, Epidermis. (C) The secondary parietal layer evident in (B) undergoes another periclinal division, resulting in an endothecium (en) toward the outside and a middle layer (md) toward the inside. ep, Epidermis; s, sporogenous layer; t, tapetum. (D) The mature anther wall is comprised of five layers: a single-layered epidermis (ep), a single-layered endothecium (en), two or three middle layers (md) and a single- layered tapetum (t). By the time the anther wall has matured, the sporogenous layer has undergone several divisions to produce numerous sporogenous cells (s). Scale bars: (A) ¼ 100 mm; (B–D) ¼ 20 mm. chalazal megaspore becomes visibly functional, possessing a central cell and remain in this central location until fertiliza- prominent nucleus (Fig. 5A). The functional megaspore devel- tion (Fig. 6A). ops successively into a two-nucleate (Fig. 5B), four-nucleate (Fig. 5C) and, finally, eight-nucleate embryo sac (Fig. 5D, E) by three mitotic divisions. Thus, the mode of embryo sac Double fertilization and development of endosperm formation is of the Polygonum type. Following fertilization of the egg cell, the zygote becomes The three micropylar nuclei become the egg and two densely cytoplasmic (Fig. 6B). Soon after, the primary endo- synergids, collectively comprising the egg apparatus sperm nucleus of the central cell migrates toward the antipo- (Fig. 5D). The unfertilized egg cell is highly vacuolate dals, which persist at the chalazal pole of the embryo sac (Fig. 5D). The two median nuclei become the polar (Fig. 6C). The first division of the primary endosperm nuclei (Fig. 5D, E), and the chalazal nuclei become the nucleus is not accompanied by wall formation (Fig. 6D). three antipodals (Fig. 5D, E). The polar nuclei fuse in Free nuclear endosperm formation ensues within several the centre, forming the fusion (secondary) nucleus of the mitotic divisions (Fig. 6E). As early nuclear endosperm Wang et al. — Reproductive anatomy of Decaisnea and systematic implications 1247 Downloaded from http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015

F IG. 3 Maturation of the tapetum is concurrent with microsporogenesis and microgametogenesis in Decaisnea insignis. (A) Prior to microsporogenesis, about half of the tapetal cells (t) undergo mitosis without cytokinesis, becoming binucleate (arrowheads). Microsporocytes (mi) originate from the primary sporogenous layer as well as from secondary sporogenous cells. (B) Individual microsporocytes (mi) become enclosed in a thick callose wall (arrowheads) when their nuclei enter prophase of meiosis I. Upon initiation of microsporocyte meiosis, the tapetal cells (t) elongate radially and protrude into the anther locule. (C) Simultaneous cytokinesis with centripetally advancing constriction furrows follows microspore meiosis II, resulting in tetrahedral microspore (ms) tetrads, which enlarge and acquire thick callose walls (arrowheads). (D) Microspores (ms) being released from the tetrad, as the callose walls have broken down. (E) Formation of a large vacuole (v) pushes the single nucleus (arrowhead) of each freed microspore toward the microspore wall. (F) Each microspore divides to form a large tube (vege- tative) cell (tc) and a small generative cell (gc). (G) The generative cell undergoes a further division, resulting in two sperm cells (sp). tc, tube cell. (H) Tricolpate pollen grains (arrowheads) will be shed at the three-celled stage. Scale bars: (A, B, H) ¼ 20 mm; (C, D) ¼ 10 mm; (E, F) ¼ 60 mm; (G) ¼ 8 mm. develops, the two synergids become reduced, while the antipo- noticeable when the megasporocyte has completed meiosis II dals persist at the chalazal pole (Fig. 6E). (Fig. 7B), and as the ovule approaches maturity, it gradually becomes anatropous (Fig. 7C), completing curvature when the embryo sac reaches the eight-nucleate stage (Fig. 7D). Ovule development The ovule is bitegmic. The inner integument is initiated simul- Ovule development is concurrent with events of megaspor- taneously with the onset of megasporogenesis (Fig. 7A), while ogenesis and megagametogenesis. The ovule is nearly straight the outer integument is initiated when the embryo sac has early in development (Fig. 7A), but a slight curvature becomes become two-nucleate. The integuments do not complete 1248 Wang et al. — Reproductive anatomy of Decaisnea and systematic implications Downloaded from http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015

F IG. 4 Megasporogenesis in Decaisnea insignis. (A) Numerous ovules (ov) are found in two rows on either side of an adaxial carpel suture. fu, Funiculus; pl, placenta. (B) The archesporium (arrowhead) is one-celled and situated in a hypodermal position. (C) The hypodermal archesporium transforms into a megaspor- ocyte (arrowhead) by cutting off a parietal cell (p). (D) The parietal cell of (C) undergoes further divisions forming parietal cells (p) that will comprise the nucellar tissue of the crassinucellate ovule. Following parietal cell divisions, the megasporocyte undergoes meiosis I, resulting in a dyad; each cell in the dyad is indicated by an arrow. (E) Meiosis II produces a T-shaped tetrad of megaspores. The chalazal megaspore will become the functional megaspore (fm); the other three micropylar megaspores (arrows) will degenerate. ep, Epidermis; p, parietal cells. Scale bars: (A) ¼ 150 mm; (B, E) ¼ 50 mm; (C) ¼ 30 mm; (D) ¼ 40 mm. development until the embryo sac reaches the eight-nucleate There are noteworthy differences in early embryological stage, when the endostomic micropyle becomes evident development of Decaisnea when compared with other genera (Fig. 7D). in the Lardizabalaceae. Development of the anther wall is of the basic type in Sargentodoxa (Liu and Sheng, 2003), and of both the dicotyledonous and basic type in Sinofranchetia DISCUSSION (Zhang et al., 2005). Like Decaisnea, the tapetum is secretory Early embryological features of Decaisnea and other in Holboellia (Bhatnagar, 1965), Sargentodoxa (Liu and Lardizabalaceae Sheng, 2003) and Sinofranchetia (Zhang et al., 2005). In Stauntonia hexaphylla, the tapetal cell walls appear to break Development of the anther wall. It has been found that develop- down (Yoshida and Nakajima, 1978), as is typical of an amoe- ment of the anther wall is of the dicotyledonous type in boid tapetum, but the protoplasts remain in situ, which is Decaisnea, since the endothecium and middle layers originate characteristic of a secretory tapetum. The tapetal cells typi- from a single layer of cells, and the tapetum, which is secretory cally contain no more than two nuclei in Decaisnea and rather than amoeboid, does not originate from connective Holboellia angustifolia (Wang, 2001), while they contain tissue. Most of the present observations on anther development two to four nuclei in Holboellia latifolia (Bhatnagar, 1965). confirm and expand those of Swamy (1953), as wall layer ontogeny is explicitly documented. However, while Swamy Microsporogenesis and microgametogenesis. It has been (1953) suggests that all tapetal cells are binucleate, it is observed that cytokinesis following microsporocyte meiosis noted that some remain uninucleate, even late in development. is simultaneous, and results in tetrahedral tetrads in Wang et al. — Reproductive anatomy of Decaisnea and systematic implications 1249 Downloaded from http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015

F IG. 5 Megagametogenesis in Decaisnea insignis. (A) The three micropylar megaspores of the tetrad have become degenerated megaspores (dm), while the chalazal megaspore is obviously the functional megaspore (fm), as it possesses a prominent nucleus (arrow). (B) The functional megaspore (fm) has developed mitotically into an embryo sac possessing two nuclei (arrows); arrowheads indicate the remnants of the three degenerated megaspores. (C) Embryo sac containing four nuclei (arrows). (D and E) Two consecutive sections of an eight-nucleate embryo sac. Evident in (D) is one synergid (arrowhead), the egg cell (ec) at the micropylar pole, two of three antipodals (a) at the chalazal pole, and one of two polar nuclei (pn1) in the central cell. Note the vacuolate nature of the unfertilized egg cell. Evident in (E) is the other synergid (arrowhead) at the micropylar pole, two of three antipodals (a) at the chalazal pole, including one that was not captured in the previous section, and the second of two polar nuclei (pn2) in the central cell. Scale bars: (A) ¼ 40 mm; (B) ¼ 50 mm; (C–E) ¼ 60 mm.

Decaisnea. The same is true for Holboellia angustifolia megaspore tetrads (Bhatnagar, 1965), as is found in (Wang, 2001), Sargentodoxa (Liu and Sheng, 2003) and Sargentodoxa, although the megaspore tetrads in this genus Sinofranchetia (Zhang et al., 2005). However, tetrads are tetra- are occasionally linear (Sheng et al., 2005). The megaspore hedral or decussate in Holboellia latifolia (Bhatnagar, 1965). It tetrads are linear in Holboellia angustifolia (Wang, 2001) has been found that the mature tricolpate pollen grains of and Sinofranchetia (Zhang et al., 2005). Embryo sac develop- Decaisnea are three-celled at the time of shedding, and not ment in Decaisnea conforms to the Polygonum type; this is two-celled as reported by Swamy (1953). Holboellia latifolia also the case in Holboellia latifolia (Bhatnagar, 1965), (Bhatnagar, 1965), Holboellia angustifolia (Wang, 2001), Akebia spp. (Yoshida and Michikawa, 1973), Stauntonia hex- Sargentodoxa (Liu and Sheng, 2003) and Sinofranchetia aphylla (Yoshida and Nakajima, 1978), Holboellia angustifo- (Zhang et al., 2005) are also reported to have two-celled lia (Wang, 2001), Sargentodoxa (Sheng et al., 2005) and mature pollen grains when shed. Sinofranchetia (Zhang et al., 2005). The present findings show that the antipodals in D. insignis are persistent and Megasporogenesis and megagametogenesis. The present results large; these findings are consistent with Swamy’s report indicate that Decaisnea possesses T-shaped megaspore (Swamy, 1953). In contrast, the antipodals are small and tetrads. Holboellia latifolia also possesses T-shaped ephemeral in Akebia spp. (Yoshida and Michikawa, 1973), 1250 Wang et al. — Reproductive anatomy of Decaisnea and systematic implications Downloaded from http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015

F IG. 6 Events immediately surrounding double fertilization. (A) The two polar nuclei of the central cell fuse to form the fusion nucleus (fn) immediately prior to fertilization. The fusion nucleus resides in a central position until fertilization. (B) Zygote (z) at the micropylar pole. The densely stained cytoplasm of the cell indicates that fertilization has occurred. (C) Soon after fertilization of the fusion nucleus to form the primary endosperm nucleus (pe), it migrates toward the antipodals, which persist at the chalazal pole of the embryo sac (arrowheads). (D) The first division of the primary endosperm nucleus resulting in two endosperm nuclei (e) is not accompanied by wall formation. The antipodals (arrowheads) persist at the chalazal pole. The zygote (z) remains densely cytoplasmic. (E) After several mitotic divisions without cytokinesis, numerous endosperm nuclei (e) are produced. Arrowheads label the persistent antipodals. Scale bars ¼ 20 mm.

Holboellia latifolia (Bhatnagar, 1965) and Stauntonia hexa- into a micropylar and chalazal chamber; while Swamy phylla (Yoshida and Nakajima, 1978). In Decaisnea, the (1953) did not explicitly use the term ‘helobial’, he had effec- polar nuclei fuse before fertilization. While Swamy (1953) tively described such a type of endosperm development similarly notes that the two polar nuclei of Decaisnea fuse, (Raghavan, 1998). However, no evidence of such a division his report suggests that the unfertilized fusion nucleus migrates has been seen, and we maintain that the endosperm in toward the antipodals. We maintain that migration toward the Decaisnea is of the free nuclear type. All members of the antipodals only occurs after double fertilization has taken Lardizabalaceae are generally believed to have ab initio cellu- place, as the large nucleus of the central cell is only found lar endosperm (Johri et al., 1992). near the antipodals following a visible change in the egg cell to a more cytoplasmic state, indicative of fertilization Ovule development. In Decaisnea, ovules are anatropous, biteg- (Raghavan, 1998). mic and crassinucellate (see also Endress and Igersheim, 1999). However, we disagree with Swamy’s (1953) report Double fertilization and development of endosperm. The present regarding the timing of integumentary development. In the findings regarding early endosperm development in Decaisnea present study it was found that the inner integument is initiated do differ somewhat from those of Swamy (1953). Firstly, as simultaneously with the onset of megasporogenesis, the outer mentioned, it is clear that it is the fertilized primary endosperm integument is initiated when the embryo sac has become two- nucleus that migrates toward the antipodals, not the unferti- nucleate, and that the integuments do not complete development lized fusion nucleus. Furthermore, we believe that endosperm until the embryo sac reaches the eight-nucleate stage. Swamy development in Decaisnea is of the free nuclear type, not the (1953), on the other hand, suggests both integuments are initiated helobial type as described by Swamy (1953). Using line draw- simultaneously, and that integumentary development is complete ings, Swamy (1953) documents a first cellular division of the at the four-nucleate stage. Akebia quinata (Endress and primary endosperm nucleus that partitions the endosperm Igersheim, 1999), Holboellia angustifolia (Wang, 2001) and Wang et al. — Reproductive anatomy of Decaisnea and systematic implications 1251 Downloaded from http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015

F IG. 7 Ovule development in Decaisnea insignis. (A) The ovule is nearly straight early in development. The inner integument (i) is initiated simultaneously with the onset of megasporogenesis. fu, Funiculus. The megasporocyte is entering meiosis, and can be better observed in Fig. 4C. (B) A slight ovular curvature becomes noticeable when the megasporocyte has completed meiosis II to form the tetrad (see Fig. 4E for a better view of the megaspore tetrad). fu, Funiculus; i, inner integument. (C) As the ovule approaches the stage where the embryo sac is two-nucleate (two-nucleate embryo sac seen here and more clearly in Fig. 5B), the ovule gradually becomes anatropous, and the outer integument (o) is initiated. fu, Funiculus; i, inner integument. (D) Ovule curvature is complete when the embryo sac reaches the eight-nucleate stage (details of eight-nucleate embryo sac in Fig. 5D, E). At this time, the inner (i) and outer (o) integuments have completed development and the endostomic micropyle has become evident. fu, Funiculus. Scale bars: (A) ¼ 30 mm; (B–D) ¼ 20 mm.

Sinofranchetia (Endress and Igersheim, 1999; Zhang et al., characters: tetrasporangiate anthers (Johri et al., 1992), secretory 2005) also have ovules that are anatropous, bitegmic and crassi- tapetum (Bhatnagar, 1965; Liu and Sheng, 2003; Zhang et al., nucellate. The ovules are hemianatropous in Sargentodoxa 2005), simultaneous cytokinesis in the microsporocytes (Wang, (Sheng et al., 2005). 2001; Liu and Sheng, 2003; Zhang et al., 2005), primarily biteg- mic, crassinucellate ovules (Bhatnagar, 1965; Wang, 2001; Zhang et al.,2005)andaPolygonum type of embryo sac devel- Embryological comparison of Decaisnea with allied groups: opment (Yoshida and Michikawa, 1973; Yoshida and Nakajima, systematic implications 1978; Wang, 2001; Zhang et al., 2005; Sheng et al., 2005). The present study shows that Decaisnea and other genera of However, Decaisnea displays three embryological charac- Lardizabalaceae generally share the following embryological ters that are rarely found in Lardizabalaceae, and are thus of 1252 Wang et al. — Reproductive anatomy of Decaisnea and systematic implications substantial systematic implication. First, the antipodals are per- However, in the family, only Decaisnea has persistent antipo- sistent and relatively large in Decaisnea, while they are dals, nuclear endosperm, and three-celled pollen upon shed- ephemeral and smaller in the other Lardizabalaceae ding. Based on these embryological results, we suggest that (Bhatnagar, 1965; Yoshida and Michikawa, 1973; Yoshida monospecific Decaisnea is in need of taxonomic re-evaluation and Nakajima, 1978; Wang, 2001; Sheng et al., 2005; Zhang and circumscription above the genus level. et al., 2005). Secondly, the endosperm of Decaisnea is free nuclear, while endosperm is ab initio cellular in most Lardizabalaceae (Johri et al., 1992). Thirdly, the pollen ACKNOWLEDGEMENTS grains of Decaisnea are shed when they are three-celled, We are very grateful to Professor Yi Ren at the Shaanxi while those of most Lardizabalaceae are shed at the two-celled Normal University for providing us with material, and we stage (Johri et al., 1992). also thank Ms Jie Wen and Dr Qing Cai for technical assist- With these family-level incongruencies in mind, it is impor- ance. We thank Professor Libing Zhang at the Missouri tant to note that two of the three embryological characters that Botanical Garden, Dr Chunying Xue (Kunming institute of Downloaded from distinguish Decaisnea from other Lardizabalaceae are regu- Botany), Dr Thomas B. Friedman (Thompson Rivers larly observed in Ranunculales, specifically Ranunculaceae. University) and Dr Tomas Rodriguez-Riano (Universidad de Like Decaisnea, members of Ranunculaceae have persistent Extremadura, Spain) for their helpful suggestions on the manu- antipodals (Williams and Friedman, 2004) and free nuclear script. This work was supported by a National Sciences and endosperm (Johri et al., 1992). While pollen grains in most Engineering Research Council of Canada Discovery Grant genera of Ranunculaceae are shed at the two-celled rather (grant number 164375 provided to C.R.F.). The work would http://aob.oxfordjournals.org/ than the three-celled stage (Brewbaker, 1967; Johri et al., not have been possible without laboratory supplies and 1992), Ranunculaceae have a secretory tapetum, simultaneous reagents generously supplied by Professor Yi Ren and the microsporocyte meiosis, primarily bitegmic, crassinucellate Shaanxi Normal University. ovules that are numerous in each carpel, and a Polygonum type embryo sac (Jalan, 1963; Johri et al., 1992), like most Lardizabalaceae. We do not conclude that Decaisnea is more LITERATURE CITED closely related to the Ranunculaceae; however, the embryolo- APG II. 2003. An update of the Angiosperm Phylogeny Group classification gical characteristics of Decaisnea relative to Lardizabalaceae for the orders and families of flowering plants: APG II. Botanical Journal at Library of Chinese Academy Sciences on April 2, 2015 and other Ranunculales may suggest that the systematic pos- of the Linnean Society 141: 399–436. ition of Decaisnea calls for further evaluation and elevation. Bhatnagar SP. 1965. Some observations on the embryology of Holboellia Thus, it would be premature to make formal taxonomic con- latifolia Wall. Current Science 34: 28–29. Bhojwani SS, Bhatnagar SP. 1978. The embryology of angiosperms, 3rd edn. clusions before stronger evidence is obtained and before evol- New Delhi: Vikas Publishing House, 225–226. utionary trends of the gross morphology are finally elucidated; Brewbaker JL. 1967. The distribution and phylogenetic significance of however, we believe our embryological results indicate that binucleate and trinucleate pollen grains in the angiosperms. American Decaisnea is not a simple ‘genus-level fit’ in the Journal of Botany 54: 1069–1083. Lardizabalaceae. The presence of persistent antipodals, free Chen D-Z, Tatemi S. 2001. Lardizabalaceae. In: Wu C-Y. ed. Flora of China, Vol. 6. St Louis, MO, Missouri Botanical Garden Press, 440–454. nuclear endosperm, and three-nucleate pollen upon shedding Endress PK, Igersheim A. 1999. Gynoecium diversity and systematics of the are characters unique to Decaisnea within Lardizabalaceae. basal . Proceedings of the Linnean Society of London 130: Such normally conservative characters can be used to circum- 305–393. scribe taxa above the generic rank (Tobe, 1989). As men- Griffith W. 1855. Lardizabalaceae. Proceedings of the Linnean Society of London 2: 350. tioned, based on morphological data, Loconte and Estes Hoot SB, Culham A, Crane PR. 1995a. Phylogenetic relationships of the (1989) suggest that Decaisnea could be treated as a subfamily Lardizabalaceae and Sargentodoxaceae: chloroplast and nuclear DNA named Decaisneoideae, and Loconte et al. (1995) circum- sequence evidence. Plant Systematics and Evolution [Supplement] 9: scribe Decaisnea as a new family within Lardizabalales. Qin 195–199. (1989, 1997) has treated Decaisnea as a monogeneric tribe Hoot SB, Culham A, Crane PR. 1995b. The utility of atpB gene sequences in resolving relationships in the Lardizabalaceae, including comparisons Decaisneeae. In light of the present embryological study on with rbcL and 18S ribosomal DNA sequences. Annals of the Missouri Decaisnea insignis, we believe such statements deserve Botanical Garden 82: 194–207. serious reconsideration, particularly those of Qin (1989, Hoot SB, Magallon S, Crane PR. 1999. Phylogeny of basal eudicots based on 1997). Our suggestion neither directly confirms nor refutes a three molecular data sets: atpB, rbcL, and 18S nuclear ribosomal DNA sequences. Annals of the Missouri Botanical Garden 86: 1–32. chloroplast and nuclear DNA sequence-based molecular phy- Jalan S. 1963. Studies in the family Ranunculaceae. 4. The embryology of logeny wherein Decaisnea was resolved as a member of Actaea spicata Linn. Phytomorphology 13: 338–347. Lardizabalaceae (Hoot et al., 1995a, b, 1999). However, we Johri BM, Ambegaokar KB, Srivastava PS. 1992. Comparative embryology recommend that our new embryological results be incorporated of angiosperms. Berlin: Springer-Verlag. in future phylogenetic studies. Li ZL. 1978. Technique of making plant slides for microscopy. Beijing: Science Press [in Chinese]. Liu WZ, Sheng XY. 2003. A study on the embryology in Sargentodoxa Conclusions simplicifolia: the formation of microspores and development of male gametes. Journal of Northwestern Polytechnical University 33: 349–352. Decaisnea and other genera of Lardizabalaceae character- Loconte H, Estes JR. 1989. Phylogenetic systematics of Berberidaceae and Ranunculales (Magnoliidae). Systematic Botany 14: 565–579. istically have tetrasporangiate anthers, a secretory tapetum, Loconte H, Campbell LM, Stevenson DW. 1995. Ordinal and familial simultaneous microsporocyte cytokinesis, primarily bitegmic, relationships of Ranunculid genera. Plant Systematics and Evolution crassinucellate ovules and a Polygonum-type embryo sac. [Supplement] 9: 99–118. Wang et al. — Reproductive anatomy of Decaisnea and systematic implications 1253

Mabberley DJ. 2008. Mabberley’s plant-book: a portable dictionary of Tobe H. 1989. The embryology of angiosperms: its broad application to sys- plants, their classifications, and uses, 3rd edn. Cambridge: Cambridge tematic and evolutionary study. Botanical Magazine [Tokyo] 102: University Press. 351–367. Qin HN. 1989. An investigation on carpels of Lardizabalaceae in relation to Williams JH, Friedman WE. 2004. The four-celled female gametophyte of and phylogeny. Cathaya 1: 61–82. Illicium (Illiciaceae; Austrobaileyales): implications for understanding Qin HN. 1997. A taxonomic revision of the Lardizabalaceae. Cathaya 8–9: the origin and early evolution of monocots, eumagnoliids, and eudicots. 1–214. American Journal of Botany 91: 332–351. Raghavan V. 1998. Molecular embryology of flowering plants. Cambridge: Wang F. 2001. Phylogeny and biogeography of the Lardizabalaceae. PhD Cambridge University Press. Thesis, Kunming Institute of Botany, Chinese Academy of Sciences, Sheng XY, Liu WZ, Hu ZH. 2005. A study on the embryology in Kunming, China. Sargentodoxa simplicifolia: megasporogenesis and female gamete devel- Wang F, Li D-Z, Yang J-B. 2002. Molecular phylogeny of the opment. Journal of Northwestern Polytechnical University 35: 63–66. Lardizabalaceae based on trnL-F sequences and combined chloroplast data. Acta Botanica Sinica 44: 971–977. Swamy BGL. 1953. Some observations on the embryology of Decaisnea Yoshida O, Michikawa A. 1973. Embryological studies of genus Akebia insignis Hook et. Thoms. Proceedings of the National Institute of Decaisne. Journal of the College of Arts and Sciences, Chiba Sciences of India – Part B: Biological Sciences 19(B): 307–310.

University B 6: 25–37. Downloaded from Thorne RF. 2000. The classification and geography of the flowering plants: Yoshida O, Nakajima Y. 1978. Embryological studies of Stauntonia hexa- dicotyledons of the class Angiospermae (subclasses Magnoliidae, phylla Decaisne. Journal of the College of Arts and Sciences, Chiba Ranunculidae, Caryophyllidae, Dilleniidae, Rosidae, Asteridae, and University B 11: 45–57. Lamiidae). The Botanical Review 66: 441–647. Zhang XH, Ren Y, Tian XH, Pan LZ. 2005. Anatomical studies on Thorne RF. 2007. An updated classification of the class Magnoliopsida Sinofranchetia chinensis (Lardizabalaceae) and their systematic signifi- (‘Angiospermae’). The Botanical Review 73: 67–182. cance. Botanical Journal of the Linnean Society 149: 271–281. http://aob.oxfordjournals.org/ at Library of Chinese Academy Sciences on April 2, 2015