Female Cone Development in Fokienia, Cupressus, Chamaecyparis and Juniperus (Cupressaceae)

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1 Acta Botanica Sinica 2004, 46 (9): Female Cone Development in Fokienia, Cupressus, Chamaecyparis and Juniperus (Cupressaceae) ZHANG Quan 1, 2, Sodmergen 1, HU Yu-Shi 2, LIN Jin-Xing 2* (1. College of Life Sciences, Peking University, Beijing , China; 2. Institute of Botany, The Chinese Academy of Sciences, Beijing , China) Abstract: The ontogeny and vascular systems of female cones of the Fokienia, Cupressus, Chamaecyparis and Juniperus were investigated in detail using scanning electron microscopy (SEM) and conventional light microscopy. In the species examined, in the axils of the bracts, the first recognizable structure was a broad meristematic swelling, from which ovules developed. No ovuliferous scales developed during the ontogeny of the female cones. The number of ovules and ovule developing sequence displayed considerable variation in different species. However, development of the bracts was similar in all of the investigated species. Following pollination, the foliage-like bracts became peltate bract scales due to intercalary expansion, and global cones formed. In addition, the vascular system in the bract scales became intricate, and inverted vascular bundles emerged in the adaxial of the mature bracts. Based on these observations, a morphological interpretation and possible evolutionary trend of the Cupressaceae female reproductive structures was discussed. Key words: Cupressaceae; female cone development The female cone of conifers has received considerable attention, not only for its systematic significance (Li, 1953; Hart and Price, 1990; Fu et al., 1999; Gadek et al., 2000), but also for its intriguing morphology (Florin, 1954; Farjon and Garcia, 2003; Schulz et al., 2003). In a series of detailed studies of fossil and living conifers, Florin (1954) came to the widely accepted conclusion that the female cones of most conifers are compound structures in which the ovuliferous scale represents a modified branch borne in the axil of a bract scale. Many scientists initially believed that, in the Cupressaceae, the ovuliferous scale and bract scale were completely fused (Sporne, 1971; Owens and Molder, 1980; Gifford and Foster, 1989; Page, 1990). However, subsequent studies have shown that, in female cones of certain genera, no ovuliferous scale develops (Takaso and Tomlinson, 1989; Zhang et al., 2000; 2001; Tomlinson and Takaso, 2002). These results imply that there must be an alternative explanation of the morphological nature of the female reproductive structures in the Cupressaceae. In the present study, we examined female cone development in the Chamaecyparis, Cupressus, Fokienia and Juniperus genera, as part of a series of studies on conifer female reproductive structures (Hu et al., 1989; Napp-Zinn and Hu, 1989; Shi and Wang, 1989; Wang et al., 1995; Lin and Hu, 2000). The overall aim is to determine the morphological nature and evolutionary trends of conifer female reproductive structures, with reference to other studies on the ontogeny of female cones. 1 Materials and Methods Female cones of Juniperus chinensis Linnaeus and Chamaecyparis pisifera (Siebold and Zuccarini) Endlicher were collected in the Beijing Botanical Garden, Institute of Botany, The Chinese Academy of Sciences. Cones of other species, such as Cupressus duclouxiana Hickel, Cupressus funebris Endlichler, Cupressus torulosa D. Don, Chamaecyparis lawsoniana (A. Murray bis) Parlatore, Fokienia hodginsii (Dunn) A. Henry and H. H. Thomas, Juniperus recurva var. coxii (A. B. Jackson) Melville, J. gaussenii W. C. Cheng, were collected in the Kunming Botanical Garden, Kunming, Yunnan. All collections were made throughout the annual cycle from mature plants. Materials were fixed in FAA (formalin-acetic acid-alcohol). For scanning electron microscopy (SEM) observation, the fixed young seed cones were dehydrated and dissected in a graded alcohol series, critical-point dried and coated with gold palladium. Specimens were observed under a Hitachi S-800 scanning electron microscope operated at 30 kv. For histology and vascular analysis, fixed cone samples Received 9 Mar Accepted 21 Jun Supported by the Knowlege Innovation Program of The Chinese Academy of Sciences (KSCXZ-SW-108) and the National Science Fund of China for Distinguished Young Scholars ( ). * Author for correspondence. Tel: +86 (0) ext. 6211; Fax: +86 (0) ; <linjx@ns.ibcas.ac.cn>.

2 collected one month before pollination, at the pollination stage, and near maturity, were prepared for paraffin sectioning. Serial longitudinal and transverse sections at 8 12 µm thickness were cut and stained in hematoxylin and safranin. 2 Results 2.1 Female cone development in Fokienia There is only one species in the genus Fokienia, F. hodginsii. The female cone of this species consists of six to nine pairs of decussate bracts, initiated on the terminal branchlet in early September. In the axils of each fertile bract in the middle of the cone, two ovule primordia were observed to initiate (Fig.1a). The ovule developed in a distinct acropetal manner (Fig.1b). When all structures of the cone had been established, there were, at most, 28 ovules formed along the long axis of the cone (Fig.1c). However, only four to eight ovules and subtending bracts in the middle of the cone were fully developed after pollination. Two or three pairs of the bracts in the middle of the cone developed into a peltate scale, and the cone assumed a globular shape. The other ovules were aborted, with the subtending bracts remaining poorly developed (Fig.2). The development of the vascular system of the F. hodginsii young cone up to the time of pollination was Fig.1. Scanning electron microscopy (SEM) photographs of dissected female cones of Fokienia hodginsii at successive stages of development. a. Ovule primordia initiated. b. Ovules developed in an acropetal manner. c. A cone with all ovules fully developed. Bar = 200 µm. Abbreviations: B, removed bract; O, ovule; OP, ovule primordia. Fig.2. Diagrammatic longitudinal sections of Fokienia hodginsii cones at different developmental stages. a. Ovules initiated in an acropetal manner. b. Bracts opened at the time of pollination. c. Bracts developed into peltate scales and vascular system become intricate in mature cone. B, bract; O, ovule; S, seed; V, vascular. Dotted lines indicate the vascular bundles that had developed after pollination.

3 ZHANG Quan et al.: Female Cone Development in Fokienia, Cupressus, Chamaecyparis and Juniperus (Cupressaceae) very similar to that of the vegetative shoot. Each bract contained a single centrally situated vascular bundle. During the intercalary growth of the bract following pollination, the vascular system in the female cone developed into an intricate pattern. At the base of the bract scale, apart from the central trace, two lateral bundles diverged from the cone axis, and each bundle immediately branched into several bundles, forming two series that branched into the abaxial and adaxial sides of the bract scale. The adaxial series of bundles were inverted, with the xylem on the abaxial side and the phloem on the adaxial side (Fig. 2c; Fig. 6a). All of the species investigated in this study displayed similar vascular development pattern in the cone. 2.2 Female cone development in Cupressus The genus Cupressus contains about 17 species, of which three were examined: C. funebris, C. duclouxiana and C. torulosa. Female cones of these species were found Fig.3. Scanning electron microscopy (SEM) photographs of dissected female cones from Cupressus funebris (a c), Cupressus duclouxiana (d f) and Cupressus torulosa (g i). a, d, g. The broad meristematic swelling (arrows) formed in the axil of the bract. b, e, h. Ovule primordia initiated. Numbers indicate the order of ovule initiation. c, f, i. Cones with all ovules developed. Bar = 200 µm. Abbreviations: B, removed bract; I, integument; N, nucellus; O, ovule; OP, ovule primordia.

4 to have numerous ovules and a similar developmental pattern. The C. funebris female cone initiated in early July and had three to five pairs of bracts by the end of the month. In the axil of each bract, a broad swelling was observed (Fig. 3a), on which two series of ovules were produced in basipetal order. The first series contained three ovule primordia, and the second series with two ovule primordia appeared later and alternated with the first series (Fig.3b). Multiple ovules clustered between the bracts, obscuring the relationships between the ovules and the subtending bracts (Fig.3c). No ovule developed in the axils of the uppermost pairs of bracts (Fig.3b, 3c). Ovules development in C. duclouxiana (Fig.3d f) and C. torulosa (Fig.3g i) cones were similar to that of C. funebris except that several ovules initiated at the axils of the uppermost bracts. 2.3 Female cone development in Chamaecyparis Six species of the Chamaecyparis genus occur in East Asia and North America, and we examined two of these species, Ch. lawsoniana and Ch. pisifera. In mid-july, a subset of the vegetative buds of Ch. lawsoniana developed into female reproductive buds. The newly formed female cone contained four to six pairs of bracts. Ovule primordia initiated simultaneously in the axils of two or three pairs of bracts in the middle of the cone (Fig.4a c). Generally, two or four ovules developed in the axil of each fertile bract. In the bracts containing four ovules, the lateral two ovules initiated later than the two middle ovules. Abortion of the middle two ovule primordia was observed (Fig.4c). The ontogeny of the Ch. pisifera female cone was similar to that of Ch. lawsoniana, except that the axil of each fertile bract always generated two ovules (Fig.4d f). 2.4 Female cone development in Juniperus Of the approximately 60 species in the Juniperus genus, we investigated three species: J. chinensis, J. gaussenii, and J. recurva var. coxii. The female cone of J. chinensis consisted of three pairs of bracts whose arrangement followed that of the leaves in a decussate manner. In July, the first distinguishable structure in the axil of each bract in the middle of the cone was a broad meristematic swelling Fig.4. Scanning electron microscopy (SEM) photographs of dissected female cones of Chamaecyparis lawsoniana (a c) and Chamaecyparis pisifera (d f). a. Ovule primordia initiated. b. All ovules developed in a cone. c. Two ovule primordial (fat arrows) that failed to develop into ovules. d. The broad meristematic swelling (arrow) formed in the axil of the bract. e. Nucellus and integument developed. f. A cone with all ovules developed. Bar = 200 µm. Abbreviations: B, removed bract; I, integument; N, nucellus; O, ovule; OP, ovule primordia.

5 ZHANG Quan et al.: Female Cone Development in Fokienia, Cupressus, Chamaecyparis and Juniperus (Cupressaceae) (Fig.5a), on which two ovule primordia initiated (Fig.5b). A typical female cone of J. chinensis consisted of two pairs of decussate bracts with two ovules in the axil of each of the lower bracts (Fig.5c). J. gaussenii female cones had two or three pairs of decussate bracts at the ends of very short branchlets. In general, a typical female cone of J. gaussenii consisted of two pairs of decussate bracts and one ovule developed at the axil of each of the lower fertile bracts (Fig.5d). Generally, the apices of the cones were flat, but in a few cones the apices continued to develop to form a central columella (Fig.5e). In one cone, the apices developed into an ovule (Fig.5f). In addition, an abnormal cone with a bract initiated at the position corresponding to an ovule (Fig.5g). The J. recurva var. coxii female cone had only one ovule on the top of the cone axis, it was the simplest cone structure observed in all of the species studied. The bracts were either decussate (Fig.5h) or in a whorl of three (Fig. 5i). Fig.5. Scanning electron microscopy (SEM) photographs of dissected female cones of Juniperus chinensis (a c), Juniperus gaussenii (d g) and Juniperus recurva var. coxii (h and i). a. The broad meristematic band (arrow) formed in the axil of the bract. b. Ovule primordia initiated. c. A typical female cone of J. chinensis. d. A typical female cone of J. gaussenii. e. A cone apex developed into a column. f. An ovule developed at the cone apex. g. An abnormal cone with a bract (star) initiated at the position corresponding to an ovule. h, i. The female cone of J. recurva var. coxii has only one ovule terminal on the cone axis. Bar = 100 µm. Abbreviations: B, removed bract; O, ovule; OP, ovule primordia.

6 Fig.6. Diagrammatic transverse section through the middle of bract scales, indicating three types of vascular system in the female cones of the Cupressaceae. Arrows point to adaxial side. Circles indicate the vascular bundles. The black half is xylem and the white half is phloem. Dotted lines divide the vascular bundles into three series that are derived from three traces (a central trace and two lateral traces) at the base of the bract scale. 3 Discussion In the systematics of the Cupressaceae, Cupressus and Chamaecyparis have often been placed in the Cupresseae, and Fokienia has also been placed in this tribe (Li, 1953; Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita, 1978; Gadek et al., 2000). Our demonstration of a strong similarity in the female cone development of the Cupressus and Chamaecyparis genera is consistent with previous studies. Because the ovules of Fokienia cones initiate in a distinct acropetal manner, it may be appropriate to remove Fokienia from the Cupresseae tribe. One evolutionary trend in the female cones of the Cupressaceae was constituted by a reduction in the number of ovules and bracts (Li, 1953). In the report of a study on seed cones of fossil and extant Cupressaceae and Taxodiaceae, Ohsawa (1994) proposed that the ancestral seed cone of the Cupressaceae had an elongated form with many bract-scale complexes. Later, reduction of the cone axis probably took place to form a small spherical cone, consisting of a small number of bract-scale complexes. We previously found that ovule primordia of Thuja occidentalis abort regularly (Zhang et al., 2001). Abortion of ovules was also observed in Ch. lawsoniana, indicating that cones of its ancestor may have contained more ovules. Cupressus cones contain numerous ovules, whereas Juniperus has the simplest cones in the Cupressaceae, with just one ovule on each cone in J. recurva var. coxii. Most strikingly, although many ovules (up to 28) initiated in young Fokienia cones, only four to eight ovules succeeded in developing into mature seeds. Taking the evolutionary trends of female cones into account, Fokienia is probably a relatively primitive genus in the Cupressaceae, Juniperus is advanced, and Cupressus and Chamaecyparis fall between Fokienia and Juniperus. It is now generally accepted that conifer seed cones are compound structures in which the seed scale represents a modified branch borne in the axil of a bract scale. In female cones of the Cupressaceae, the ovuliferous scale and bract scale are completely fused (Owens and Molder, 1980; Gifford and Foster, 1989). The present study of the ontogeny of female cones of the Cupressaceae implied that conifer seed cones are compound structures is acceptable in principle. In the axil of the fertile bract, ovules initiate on a broad meristematic swelling that might represent the secondary branch system. However, the idea about a congenital fusion of a bract with an ovuliferous scale was misleading. Because many researchers have claimed completely fusion of the bract scale and ovuliferous scale mainly on the basis of the inverted vascular bundles in the scale of mature Cupressaceae cones (Lemoine-Sebastian, 1972; Gifford and Foster, 1989). In our studies, we identified three types of vascular systems in the female cones of mature Cupressaceae cones: (A) The Juniperus, Cupressus, Chamaecyparis, Fokienia, and Callitris (Takaso and Tomlinson, 1989) had globular cones with peltate bract-scales, as well as radial branching of the strands forming two series of vascular bundles, including an adaxial series of strands with an inverted xylem orientation and an abaxial system consisting of normally oriented bundles (Figs.2, 6a). (B) In Calocedrus (unpublished data), Platycladus (Zhang et al., 2000), and Libocedrus (Tomlinson and Takaso, 2002), a single series of inverted vascular bundles was generated in the adaxial side of the thick, flat bract scale (Fig.6b). (C) The flat bract scale of Thuja occidentalis contains no inverted vascular bundle (Zhang et al., 2001) (Fig.6c). The results presented here indicate that the inverted vascular bundles are the result of bract scale development after pollination in conjunction with the intercalary growth of the bract. It appears that the arrangement of the vascular system in mature cones is related to the morphologies of the cone and the bract scale. As the seed cone maturing, the bract functions as a seed-protecting structure, similar to the seed scale in the Pinaceae.

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