J. Jpn. Bot. 91 Suppl.: 201–216 (2016)

Anatomy and Development of Leaves in Chamaecrista mimosoides and C. nomame (Leguminosae-)

a, b c Tomoyuki Nemoto *, Hiroyoshi Ohashi and Te-Lin Wu

aDepartment of Biological Sciences, Faculty of Science and Engineering, Ishinomaki Senshu University, 1 Shinmito, Minamisakai, Ishinomaki, 986-8580 JAPAN; bBotanical Garden, Tohoku University, Sendai, 980-0862 JAPAN; cHerbarium, South China Institute of Botany, Chinese Academy of Sciences, Wushan, Guangzhou, Guangdong 510650, P. R. CHINA *Corresponding author: [email protected]

(Accepted on August 31, 2016)

Chamaecrista mimosoides (L.) Greene has a unique even-pinnate leaf with crenate-crested protuberances on the adaxial side of the rachis between leaflet pairs, which is one of the important characteristics for distinguishing it from other with leaf rachises canaliculate. The present study clarified anatomical and developmental features of the leaves with the crenate-crested rachis in C. mimosoides in comparison with those with a canaliculate rachis in C. nomame (Makino) H. Ohashi. Transverse sections showed that the crenate-crested rachis has a protuberance constructed by continuous parenchymatous tissues composed of cells rich in chloroplasts, whereas the canaliculate one has two ridges constructed by two independent parenchymatous tissues. Developmental observations showed that the crenate-crested rachis is initiated as a single swelling of tissues, whereas the canaliculate one initiated as two parallel swelling of tissues. Both rachises have commonly two ridge bundles divided from the main vascular bundles in the petiole, but the two ridge bundles are fused into one bundle in the proximal half of the crenate-crested protuberance between leaflet pairs, and then divided into two again in the distal half. In contrast, the two ridge bundles remain separated throughout the length in the canaliculate rachis. The fused ridge bundles in the crenate-crested protuberance, moreover, branch a small bundle into the protuberance. At the distal part of the petiole there is an extrafloral nectary (EFN) in both species. The vascular bundles are supplied to the EFN from each of two ridge bundles in the petiole in C. mimosoides, whereas from the main vascular bundles in C. nomame. The repetition of fusion and separation of two ridge bundles in the crenate-crested leaf rachis seems to support the idea that the rachis is produced by fusion of two ridges of the canaliculate rachis. The chloroplast-rich cells observed in the crenate-crested protuberance as well as many stomata in the epidermis suggest that the protuberance may improve the ability of photosynthesis.

Key words: Anatomy, Caesalpinioideae, Cassieae, Chamaecrista mimosoides, Chamaecrista nomame, development, extra floral nectary, Leguminosae, leaf, leaf rachis.

The genus Chamaecrista Moench is a South America and tropical Africa (Lewis 2005). pantropical and subtropical genus of about 330 Species of this genus were formerly placed as species, with greatest diversity in Central and parts of the genus L. (Candolle 1825,

—201— 202 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Bentham 1865, 1871, Baker 1878, De Wit 1955, characters in distinguishing between the species Ohwi 1953, 1965, Ohashi 1982, Chen 1988), in Madagascar. but the genus Cassia is commonly recognized Lock (2007) considered that the solid crest of as segregated into three genera, Cassia sensu the crenate-crested rachis is produced by fusion stricto, and Chamaecrista according to of two ridges of the canaliculate rachis. Leaf Irwin and Barneby (1981, 1982) in recent floras structure including rachises has been examined in (e.g., Pedley 1998, Chen et al. 2010, Ohashi Chamaecrista (or as Cassia) by several authors 2016). Chamaecrista is divided into six sections such as Watari (1934, as Cassia mimosoides (Irwin and Barneby 1981, 1982). In Asia, 16 var. nomame), Saheed and Illoh (2010, species have been recognized in total (Lock and Cassiinae including Chamaecrista mimosoides), Simpson 1991, Kumar and Sane 2003, Lock Coutinho et al. (2013, Chamaecrista sect. Absus and Heald 1994, Lock and Ford 2004, Zhu et al. subsect. Baseophyllum), Francino et al. (2015, 2007, Choi 2007, Ohashi 1999), and almost all Chamaecrista sect. Absus). But Chamaecrista Asian species except C. absus (sect. Absus) are mimosoides（sect. Chamaecrista）itself and the attributed to sect. Chamaecrista according to unique crenate-crested leaf rachis have not been Irwin and Barneby’s key (1982). examined so far. Although the unique rachis Among these Asian species, Chamaecrista feature has been recognized, our understandings mimosoides (L.) Greene is the most widely of the structural relationships between the distributed species, extending into Africa (Lock crenate-crested leaf rachises and canaliculate 1989) and Australia (Pedley 1998). The leaf ones have been poor. rachis of C. mimosoides was described as “of the The present study aims to clarify the young leaves furnished on its upper side with a structural relationships between the crenate- crenated margin” by Wight and Walker-Arnott crested rachises and the canaliculate ones in (1834, as Cassia angustissima Lam.). The leaf Chamaecrista. For this objective we performed rachis, distinctly crenated or crenate-crested on anatomical and developmental analyses on the the adaxial side between leaflet pairs, has been crenate-crested rachises of C. mimosoides in regarded as unique to C. mimosoides (or Cassia comparison with the canaliculate ones of C. mimosoides) and adopted as a useful character nomame (Makino) H. Ohashi. distinguishing it from other species with distinct leaf rachis grooved, canaliculate or channeled Materials and Methods along the adaxial side in floras of various regions Morphology and structure of leaf (Ghesquière 1932, Steyaert 1950, Brenan 1967, rachises were examined anatomically and Larsen et al. 1980, Rudd 1991, Larsen and Ding organogenetically in Chamaecrista mimosoides 1996, Pedley 1998, Singh 2001, Du Puy 2002). (L.) Greene and C. nomame (Makino) H. Brenan (1967) noted the leaf rachis as one of Ohashi. Materials of C. mimosoides were the most significant features for separating the collected from Hushan hill, Jianfeng, Ledong species under the genus Cassia sensu lato in the Co, Hainan, China on 21 Oct. in 1993 (Voucher: tropical East Africa and that there is a channel T. Nemoto et al. 1021001) and those of C. running along the upper side of the leaf rachis nomame collected from Narusegawa River, in most species, while some species including Junai, Nakaniida-machi, Kami-gun, Miyagi Cassia mimosoides have a raised wing-like Prefecture, Japan on 18 Aug. in 1983 (Voucher: projection running along the upper side of the T. Nemoto 2277). Collected materials were rachis. Du Puy (2002) also listed the presence preserved in FAA. Both voucher specimens are or absence of a crenate crest along the upper kept in TUS. side of the leaf rachis as one of the most useful Leaves were examined according to the December 2016 Nemoto et al.: Anatomy and development of leaves in Chamaecrista 203

Fig. 1. Morphology of leaf rachis in Chamaecrista mimosoides (A, C, E) and C. nomame (B, D, F). A. Crenate- crested leaf rachis, showing crenate protuberances (arrows) on the adaxial side of the rachis between leaflet pairs. B. Canaliculated leaf rachis, showing two ridges (arrows) on the adaxial side of the rachis between leaflet pairs. C. Transverse section of the crenate-crested leaf rachis between leaflet pairs, showing a protuberance (arrow). D. Transverse section of the canaliculated leaf rachis between leaflet pairs, showing two ridges (arrows) and groove formed between these ridges. E. Transverse section of the crenate-crested leaf rachis between leaflet pairs, showing two but joined ridge bundle (arrowhead) supplied to a protuberance. F. Transverse section of the canaliculated leaf rachis between leaflet pairs, showing two ridge bundles (arrowheads) supplied to two ridges. lt, leaflet; mb, main .(scar of leaflet removed. Scale bars: 200 μm (A, B); 50 μm (C, E); 100 μm (D, F ,٭ ;vascular bundles of rachis developmental stages along some shoots from were made using a Leica RM2165 rotary proximal to distal orders. Within a leaf the distal microtome at a thickness of 2 or 3 µm, stained half was used for SEM observations and the with 0.05% Toluidine Blue O in benzoate remaining proximal half used for anatomical buffer, pH 4.4 (Feder and O’Brien 1968) and ones. For morphological observations using then mounted with Entellan. Observations were SEM, leaves were dissected and half of the performed with a Leica DMRX light microscope leaflets were removed for enabling observation and photographs were taken with a Leica DC of the rachis above between leaflet pairs, then 200 digital camera. dehydrated through an ethyl alcohol series, transferred to t-butyl alcohol, freeze-dried in a Results Freeze Dryer VFD-21S, mounted on brass stubs, Morphology and anatomy of the leaf rachis coated with gold, and observed by a JEOL JSM- Leaves are even-pinnate both in 6380LV scanning electron microscope. For Chamaecrista mimosoides and C. nomame. anatomical observations, leaves were dehydrated The leaf is composed of pulvinus, petiole and through an ethanol series and embedded in a rachis with many regions of leaflet pair insertion. methacrylate resin (Technovit 7100). Sections Mature leaves of C. mimosoides have crenate- 204 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue crested ridges between leaflet pairs on the unicellular trichomes are already borne on the adaxial side of their rachises and the ridges are abaxial side of the rachis, they are not on the ca. 200 μm height from the upper margin of the adaxial side. leaflet base (Fig. 1A). The surface of the ridges In the leaf of ca. 3 mm long (Fig. 2C, D), is almost glabrous. Mature leaves of C. nomame the length of the rachis between leaflet pairs have rachises grooved or canaliculate on their is ca. 94 μm. A protuberance is observed on adaxial side between leaflet pairs (Fig. 1B). Two the adaxial side of the rachis between leaflet ridges forming the groove are covered with pairs. The height of the protuberance from the trichomes. upper margin of the leaflet base is ca. 25 μm. Transverse sections of leaf rachises showed Unicellular trichomes have been borne between that the crenate-crested ridge of Chamaecrista the protuberances on the adaxial side of the mimosoides is composed of just a single rachis. swelling of tissues (Fig. 1C, E), whereas the In the leaf of ca. 6.5 mm long (Fig. 2E, F), canaliculate rachis of C. nomame is composed the length of the rachis between leaflet pairs is of two swellings of tissues forming the groove ca. 135 μm. The protuberances between leaflet (Fig. 1D, F). The vascular tissues form a pairs become conspicuous, more than 50 μm in cylinder surrounded by sclerenchyma cells or height and crenate in form. Although there are fibers. There is one additional vascular bundle in few colleters and trichomes between the crenate C. mimosoides and two bundles in C. nomame. protuberances, the protuberance itself is still The number of these vascular bundles is in glabrous. accordance with the number of ridges between In the leaflet of almost mature leaf of ca. 28 leaflet pairs, and they are those called ridge mm long (Fig. 2G), the protuberance becomes bundles (Watari 1934), rib traces (Howard more than 400 μm long in length and more 1979) or accessory bundles (Coutinho et al. than 130 μm in height. The surface is almost 2013, Francino et al. 2015). These bundles are glabrous, but many stomata are observed also abaxially or externally associated with differentiating on it (Fig. 2H). sclerenchyma cells. In C. mimosoides, moreover, Chamaecrista nomame ― In the leaf of a small seemingly vascular tissue is found in the ca. 3.4 mm long (Fig. 3A, B), leaflet pairs are crenate-crested swelling (Fig. 5, arrowhead). borne close to each other and short bottle-shaped Chloroplasts contained in the parenchyma cells colleters are borne on the adaxial side of the between the epidermis and the vascular tissues rachis. Each colleter is located just between are more conspicuous in C. mimosoides than C. leaflet pairs. The adaxial surface of the rachis nomame (Fig. 1E, F). between colleters is ca. 40 μm long and flat, on which there are no protuberances observed. The Development of leaf rachis between leaflet pairs main vascular bundles of the rachis have begun Chamaecrista mimosoides ― In the leaf of differentiating. Although unicellular trichomes ca. 1.8 mm long (Fig. 2A, B), leaflet pairs are are already borne on the abaxial side of rachis, borne close to each other and club-shaped or they are not on the adaxial side. short bottle-shaped colleters are borne on the In the leaf of ca. 4.2 mm long (Fig. 3C, D), adaxial side of the rachis. Each colleter is located the length of the rachis between leaflet pairs just between leaflet pairs. The adaxial surface of is ca. 80 μm. Two protuberances are borne in the rachis between colleters is ca. 20 μm long parallel on the adaxial side of the rachis between and flat, on which there are no protuberances leaflet pairs or between colleters at this stage. observed. The main vascular bundles of the The height of the protuberances from the upper rachis have begun differentiating. Although margin of the leaflet base is less than 25μm. December 2016 Nemoto et al.: Anatomy and development of leaves in Chamaecrista 205

Fig. 2. Leaf rachis development in Chamaecrista mimosoides. A. Leaf of ca. 1.8 mm long, showing the adaxial side of rachis with no protuberances initiated (arrows) between leaflet paris. B. Transverse section of A at the rachis between leaflet pairs, showing the adaxial surface of rachis still being flat (arrow). C. Leaf of ca. 3 mm long, showing initiation of protuberances (arrows) along the adaxial side of rachis between leaflet pairs. D. Transverse section of C at the rachis between leaflet pairs, showing a protuberance (arrow). E. Leaf of ca. 6.5 mm long, showing protuberances (arrows) growing along the adaxial side of rachis between leaflet pairs. F. Transverse section of E at the rachis between leaflet pairs, showing a protuberance (arrow). G. Almost mature leaf of ca. 28 mm long, showing a protuberance (arrow) along the adaxial side of rachis between leaflet pairs. H. The protuberance of G enlarged, showing stomata (arrowheads) on the ;(scar of leaflet removed. Scale bars: 20 μm (A, B ,٭ ;surface. c, colleter; lt, leaflet; mb, main vascular bundles of rachis 50 μm (C–F); 100 μm (G, H). 206 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Fig. 3. Leaf rachis development in Chamaecrista nomame. A. Leaf of ca. 3.4 mm long, showing the adaxial side of rachis with no protuberances initiated (arrows) between leaflet pairs. B. Transverse section of A at the rachis between leaflet pairs, showing the adaxial surface of rachis still being flat (arrow). C. Leaf of ca. 4.2 mm long, showing initiation of protuberances (arrows) along the adaxial side of rachis between leaflet pairs. D. Transverse section of C at the rachis between leaflet pairs, showing two protuberances (arrows). E. Leaf of ca. 6 mm long, showing protuberances (arrows) growing and covered by trichomes. F. Transverse section of E at the rachis between leaflet pairs, showing two ,٭ ;protuberances growing and forming two ridges (arrows). c, colleter; lt, leaflet; mb, main vascular bundles of rachis scar of leaflet removed. Scale bars: 20 μm (A, B); 50 μm (C–F).

There are no unicellular trichomes borne on the in depth between them on the adaxial side of the surface of the protuberances. rachis. In the leaf of ca. 6 mm long (Fig. 3E, F), the length of the rachis between leaflet pairs is ca. Anatomical features from node to petiole 125 μm. The number of colleters between leaflet The leaf is supplied with three foliar traces, pairs increases to two and unicellular trichomes or trilacunar type, in both species (Fig. 4A, E). are borne on and between the protuberances. After the two lateral traces divide into stipule Two protuberances form a groove of ca. 40 μm traces, the three traces fuse with each other and December 2016 Nemoto et al.: Anatomy and development of leaves in Chamaecrista 207

Fig. 4. Vascular system from the node of stem to petiole (A–H) and extrafloral nectary (EFN) (I–L) in Chamaecrista mimosoides and C. nomame. A–D, I and J. C. mimosoides. E–H, K and L. C. nomame. A, E. Node of stem, showing the departure of three foliar traces (arrows) from the vascular cylinder of stem. B, F. Middle part of pulvinus, showing the main bundles formed in arc by three leaf traces fused. C, G. Near distal end of pulvinus, showing a pair of ridge bundles (arrows) separated from the both margins of the arc of petiolar bundle. D, H. Slender part of petiole between pulvinus and extrafloral nectary, showing slight two ridges (D) vs. obvious ones (H), a pair of ridge bundles (arrows) running beneath the two ridges (D, H), main vascular bundles in arc (D) vs. those divided into three (H), and fibers externally collateral with vascular bundles (D, H). I, K. EFN borne on the distal end of petiole, showing the position of EFN closer to the first leaflet pair in I than in K and the differences in size and shape. J, L. Transverse section of petiole at EFN, showing traces (arrowhead) supplied to EFN from ridge bundle in J and from both margins of the arc of main vascular bundles in L. lt, .scar of first leaflet removed , ٭;first leaflet; mb, main vascular bundles of rachis; n, EFN; p, crenate-crested protuberance Scale bars: 100 μm. 208 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue December 2016 Nemoto et al.: Anatomy and development of leaves in Chamaecrista 209 form a central arc in the pulvinus (Fig. 4B, F). in C. nomame (Fig. 4L). The vascular tissues At the distal part of the pulvinus two vascular enter within the proximal area, and they appear bundles, or ridge bundles, are separated from to be composed mainly of phloem cells. The the central arc toward the adaxial side (Fig. 4C, epidermis of the EFN is slightly elevated at the G). The adaxial ridges of the petiole are slight in central area of the top in both species (Fig. 4J, Chamaecrista mimosoides (Fig. 4D), whereas L). they are obvious in C. nomame, but a pair of ridge bundles obviously runs along the ridges Vascular system in leaf rachis through the region in both species (Fig. 4D, H). The central arc of leaflet pair insertion running in the pulvinus is still as an arc in the Chamaecrista mimosoides ― The main petiole in C. mimosoides, whereas it divides into vascular bundles are arranged in a circle or three bundles in C. nomame. These three bundles cylinder surrounded by a complete ring of of C. nomame become an arc connecting to each fibers throughout the rachis (Fig. 5A–L). other in the distal part of the petiole (Fig. 4L). The ridge bundles running along the crenate- At the distal end of the petiole, immediately crested protuberance form a single bundle at the below the first leaflet pair, there is one extrafloral proximal half of the region between leaflet pairs nectary (EFN) in both species (Fig. 4I, K). (Fig. 5A, B, I–L). Around the middle part of the The EFN appears slightly sunken in a shallow region between leaflet pairs, the height of the hollow surrounded by the ridges on the adaxial crenate-crested protuberance becomes the tallest side of the petiole. The EFN of C. mimosoides and the ridge bundle is divided into two bundles is flat and dish-shaped, while those of C. (Fig. 5C). These two ridge bundles run toward nomame is larger, rounded and thicker than C. the next region of leaflet pair insertion (Fig. 5D– mimosoides. The EFN tissues are composed of F). Near the region of leaflet pair insertion the a layer of epidermis and parenchyma below the leaflet trace begins to depart from both sides of layer. The parenchyma appears to be composed the main vascular cylinder of the rachis (Fig. of two areas: the distal area composed of cells 5F). Each of two ridge bundles branches off a with dark-stained cytoplasm, and the proximal bundle laterally and outward, which connects area composed of cells without such stained with each leaflet trace on both sides of the cytoplasm. The vascular tissues are supplied to region of leaflet pair insertion (Fig. 5G, H), then the EFNs from each of two ridge bundles in C. two ridge bundles fuse to each other forming mimosoides (Fig. 4J), whereas they are supplied a single ridge bundle when the crenate-crested from both margins of the central vascular arc protuberance becomes increasing the height,

Fig. 5. Vascular system in leaf rachis through the region of leaflet pair insertion in Chamaecrista mimosoides. Serial transverse sections are arranged from proximal to distal parts across the region. A–C. Leaf rachis between leaflets pairs and proximal half, increasing the height of crenate-crested protuberance toward the tallest middle part, showing single ridge bundle (arrow in A, B), an additional small bundle (arrowhead in A–C) and division of the single ridge bundle into two (arrows in C) around the middle part. D–F. Leaf rachis between leaflet pairs and distal half, decreasing the height of crenate-crested protuberance toward the region of leaflet pair insertion, showing two ridge bundles remaining separated to each other (arrows), leaflet bundles starting departure from the ring of main vascular bundles (F), and additional small bundle becoming invisible across these region (E, F). G–I. Leaf rachis at the region of leaflet pair insertion, showing the connection between ridge bundle and leaflet bundle when the height of ridge almost lost (G), and two ridge bundles fusing into a single bundle again as the height of crenate-crested protuberance increasing (H, I). J–L. Leaf rachis above the region of leaflet pair insertion, showing the increasing height of crenate-crested protuberance, single ridge bundle (arrow), and an additional small bundle (arrowhead) divided from the ridge bundle into the protuberance. lb, leaflet bundle; lt, leaflet; mb, main vascular bundles of rachis. Scale bar: 100 μm. ◀ 210 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Fig. 6. Vascular system in leaf rachis through the region of leaflet pair insertion inChamaecrista nomame. Serial transverse sections are arranged from proximal to distal parts across the region. A. Leaf rachis below the region of leaflet insertion, showing two ridge bundles (arrows) running along two ridges and leaflet bundles starting departure from the ring of main vascular bundles. B–G. Leaf rachis at the region of leaflet pair insertion, showing the division of each ridge bundle into two external and internal ones (B), the external bundles connecting with leaflet bundles on both sides (C, D), the internal bundles becoming next two ridge bundles (arrows) (D–F), and small bundle (arrowhead) apparently connecting these two ridge bundles (C–E). H, I. Leaf rachis above the region of leaflet pair insertion, showing the increasing height of ridge, the increasing depth of groove between two ridges, and two ridge bundles running under two ridges. lb, leaflet bundle; lt, leaflet; mb, main vascular bundles of rachis. Scale bar: 100 μm. December 2016 Nemoto et al.: Anatomy and development of leaves in Chamaecrista 211 even before separating leaflets at the region pairs, whereas the latter is made up of two (Fig. 5I). The ridge bundle continues fusing protuberances of tissues forming two ridges until the middle of the region between leaflet parallel to each other between leaflet pairs. pairs (Fig. 5J–L), and then again divides into The vascular system within the rachis has the two bundles (Fig. 5A–C). Within the crenate- following similarities in both: (1) there are a crested protuberance an additional small bundle vascular ring or cylinder, called siphonostele, at is also observed departing from the fused single the center, and (2) there are basically a pair of ridge bundle at the proximal part of the region ridge bundles that are detached from the adaxial between leaflet pairs (Fig. 5K, L). The small margins of the vascular arc near the distal end bundle runs until the middle part of the region of the pulvinus and run along the adaxial side of between leaflet pairs and simultaneously moves the petiole and the rachis of the leaf. However, toward the distal part, or the adaxial part, of the a pair of ridge bundles fuses into one at the protuberance (Fig. 5A–D), and then the bundle proximal half of the region between leaflet can’t be confirmed (Fig. 5E). pairs in the crenate-crested rachis and they are Chamaecrista nomame ―The main separated into two again in the distal half of the vascular bundles are arranged in a circle or region and in the region of leaflet pair insertion cylinder surrounded by a complete ring of fibers in C. mimosoides. Moreover, an additional small throughout the rachis (Fig. 6A–I). The two ridge vascular bundle is observed in the proximal half bundles run along the adaxial two ridges of the of the crenate-crested protuberance, which is region between leaflet pairs toward the next departed from the fused ridge bundle and runs region of leaflet pair insertion (Fig. 6A, I). Two upward and in the center of the protuberance. In leaflet traces begin departing from the main contrast, a pair of ridge bundles is never fused vascular cylinder before the region of leaflet pair into one throughout the canaliculate rachis and insertion (Fig. 6A). At the region of leaflet pair there are no additional bundles supplied in the insertion each of two ridge bundles branches off two ridges in C. nomame. a bundle laterally and outward, which connects with each leaflet trace on both sides of the region Vascular system from node to petiole (Fig. 6B–D). The two ridge bundles appear Leaves of Chamaecrista mimosoides and connected to each other by another bundle C. nomame are both supplied with three foliar branched laterally (Fig. 6C–E, arrowheads). traces, the nodal structure of which is that called Although the adaxial two protuberances forming the trilacunar type (Sinnott 1914) and this type the ridges are absent throughout this region, two of nodal structure has been known as common in ridge bundles remain separating (Fig. 6B–G). Leguminosae (Sinnott 1914, Sinnott and Bailey After two leaflets are separated from both sides 1914, Watari 1934). The vascular bundles of the of the rachis at the region, two ridges become pulvinus form the central vascular arc and divide obvious (Fig. 6H, I). two ridge bundles from the adaxial margins of the arc at the distal part of the pulvinus in both Discussion species. A pair of ridge bundles in the petiole Comparison in external and internal is commonly observed in Cassia, Senna and morphology between two leaf rachis types Chamaecrista (Watari 1934, as Cassia sensu The crenate-crested leaf rachis of lato). Chamaecrista mimosoides and the canaliculate After dividing two ridge bundles in the one of C. nomame are obviously different in pulvinus, the main vascular bundles present their external features: the former is made up an arc shape in the petiole in C. mimosoides, of one protuberance of tissues between leaflet whereas they are divided into three bundles in 212 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

C. nomame. Variation of vascular bundles in the clarified structural diversity of the EFNs as petiole was shown by Watari (1934) in Cassia well as their features of secretion. Although no sensu lato, and the feature of his sole material histochemical tests were applied in the present of Chamaecrista, C. nomame (as Cassia study, two parenchyma areas similar to the mimosoides var. nomame), is consistent with EFNs of sect. Absus were found in the EFNs the present result. In Chamaecrista sect. Absus, of C. mimosoides and C. nomame: the distal variations in the number of ridge bundles (as non-vascularized area, which is composed of accessory bundles), two or four to six, and in parenchyma with dark-stained cells, appears disposition of the vascular bundles within the to correspond to the “nectary parenchyma” of petiole were reported (Coutinho et al. 2013). sect. Absus (Couthinho et al. 2012, 2015), and Phylogenetic significance of such variations has the proximal vascularized area without dark- not yet confirmed critically in Chamaecrista stained cells corresponds to the “subnectary sect. Absus. With respect to sect. Chamaecrista, parenchyma”. The central area of the top of the anatomical data of leaf rachises have not EFN, where the epidermis was more or less yet accumulated enough for confirming its elevated, is assumed the site of nectar exudation phylogenetic significance. also in C. mimosoides and C. nomame. Two types of vascularization toward Vascular system related with extrafloral nectary the EFNs were reported in the sect. Absus The presence of an extrafloral nectary (Couthinho et al. 2012, 2015): vascularization (EFN) is one of important characteristics for supplied either from accessory vascular bundles, distinguishing Chamaecrista from Senna which correspond to the ridge bundles in the and Cassia: the EFN is present in some of present study, and that from the median vascular Chamaecrista and Senna, but absent in Cassia; cylinder of the rachis. The former vascularization the EFN of Chamaecrista is dish- or cup- was observed in Chamaecrista mimosoides and shaped, rarely flat, while that of Senna is ovoid, the latter in C. nomame in the present study. The globose, mounded, claviform or phalloid (Irwin vascular tissues of these two species are also and Barneby 1982). In Chamaecrista the EFNs mainly composed of phloem cells like the EFNs are known from four sections (Apoucouita, in the sect. Absus. Caliciopsis, Chamaecrista and Xerocalyx) and a part of sect. Absus (Irwin and Barneby 1982, Structural relationships between crenate-crested Coutinho et al. 2012, Coutinho and Meira 2015), and canaliculate leaf rachises and the EFNs are present on the petiole/rachis, The crenate-crested leaf rachis of Chamae- therefore also called “petiolar glands” (Irwin crista mimosoides is initiated as a single and Barneby 1982). The molecular phylogenetic protuberance on the adaxial surface, whereas the analyses supported the presence of EFNs as canaliculate one of C. nomame is initiated as two synapomorphic in Chamaecrista (Conceição et parallel ridgelines. Both types of leaf rachises are al. 2009). different from their initiation of development. Chamaecrista mimosoides and C. nomame The vascular system between leaflet pairs is also have similar EFNs on the petiole immediately different in both species, there is a single ridge below the first leaflet pair, but the EFNs are bundle in C. mimosoides and two separated different in the vascular tissues supplied as well ridge bundles in C. nomame. In C. mimosoides, as the size and shape between both species. however, the single ridge bundle partly divides Couthinho et al. (2012, 2015) investigated into two ridge bundles in the region between morphology, anatomy and histochemistry of leaflet pair when approaching to the region of the EFNs in Chamaecrista sect. Absus and they leaflet insertion. Then, the two bundles are fused December 2016 Nemoto et al.: Anatomy and development of leaves in Chamaecrista 213 into a single bundle again after connecting with The unique crenate-crested leaf rachis leaflet traces in the region of leaflet insertion. was recognized as an important taxonomic Although the developmental process does not characteristic for Chamaecrista mimosoides imply the structural relationship between two by Ghesquière (1932, as Cassia), and then types of leaf rachis, the vascular system appears Steyaert (1950, as Cassia) mentioned the utility to imply that the rachis with a pair of ridge of the rachis character for distinguishing species bundles is original state and that the crenate- in Asian and African Chamaecrista. Brenan crested rachis is formed by the fusion of two (1967) described the crenate-crested leaf rachis ridges as considered by Lock (2007). According in four species of African Chamaecrista (as to Lock (2007) the top of the crest becomes Cassia exilis Vatke, Cassia gracilior (Ghesq.) flattened, sometimes with two rows of hairs in Steyaert, Cassia mimosoides L. and unknown some regions of Africa, although the margins are Cassia sp. B). He, moreover, recognized huge firmly fused so that there is no groove between variation within Chamaecrista mimosoides (as them. For understanding structural relationships Cassia mimosoides) in Africa and distinguished of two rachis types further anatomical studies on seven groups, called Groups A–G without these variations will provide useful evidence. giving their names, within the species. In It is noteworthy that the parenchyma cells Madagascar Du Puy (2002) recognized in the crenate-crested protuberance are rich in 10 species, five of which have the crenate- chloroplasts and there are many stomata present crested leaf rachises. Although Ghesquière in the epidermis in Chamaecrista mimosoides. (1935) and Viguier (1949) attributed some of This implies that the unique crenate-crested them to Chamaecrista mimosoides (as Cassia protuberance in the genus is related with mimosoides), Du Puy (2002) distinguished improvement of the function of photosynthesis. them from C. mimosoides at species level with Because leaves and leaflets of C. mimosoides recognition their closer relationships with the are obviously smaller than C. nomame and other ‘Chamaecrista mimosoides’ complex in Africa. related species of the genus, the crenate-crested The ‘Chamaecrista mimosoides’ complex in protuberances appear to increase whole leaf area Africa and Madagascar appears to be important of one leaf for photosynthesis. materials for understanding structural variation and systematic implication of the crenate-crested Systematic implication of crenate-crested leaf leaf rachis. rachis With respect to New World Chamaecrista, The present study clarified the anatomical and Steyaert (1950) mentioned the similar crenate- developmental features of the unique crenate- crested leaf rachis present in Chamaecrista crested leaf including the parts of pulvinus, flexuosa (L.) Greene (as Cassia flexuosa L.). petiole, EFN and rachis in Chamaecrista Irwin and Barneby (1982) established the mimosoides and revealed differences in these new ser. Flexuosae under Chamaecrsita sect. features from the common canaliculate leaf of Chamaecrista in having the leaf rachis as C. nomame. Although there was the opinion that narrowly winged between leaflet pairs as one of these two species are conspecific (e.g., Baker the important characteristics. However, except 1878, Matsumura 1902, Makino 1917, Ohashi for this characteristic there are no characteristics 1966, 1982), the present evidence supports the supporting closer relationships between treatment of them as two different species (e.g., C. mimosoides of Asia and Africa and ser. Honda1938, Ohashi 1989, Huang and Ohashi Flexuosae of New World. The crenate-crested 1993, Singh 2001, Ohashi et al. 2013, Ohashi rachis may be evolved independently in both 2016). regions. 214 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Because the crenate-crested leaf rachis is rare Press, Beijing and Missouri Botanical Garden Press, St. in the genus Chamaecrista and the species with Louis. Chen P.-Y. 1988. Cassia L. In: Chen T.-C. (ed.), Flora the canaliculate leaf rachis is located at the base Republicae Popularis Sinicae 39: 123–140. Science in a recent molecular phylogenetic tree including Press, Beijing (in Chinese). Indian Cassia, Senna and Chamaecrista Choi B.-H. 2007. Lindl. In: Flora of Korea (Seethapathy et al. 2015), the crenate-crested Editorial Committee (ed.), The Genera of Vascular leaf rachis is considered to be apomorphic in of Korea, pp. 585–622. Academy Publishing Co., Seoul. Chamaecrista. For clarifying the systematic Conceição A. S., Queiroz L. P., Lewis G. P., Andrade M. J. implication of these characteristics in details, G., Almeida P. R. M., Schnadelbach A. S. and van den morph-anatomical investigations and molecular Berg C. 2009. Phylogeny of Chamaecrista Moench phylogenetic analyses are needed on the whole (Leguminosae-Caesalpinioideae) based on nuclear and chloroplast DNA regions. Taxon 58: 1168–1180. of Chamaecrista sect. Chamaecrista as well as Coutinho Í. A. C., Francino D. M. T., Azevedo A. A. and the C. mimosoides complex, especially in Old Meira R. M. S. A. 2012. Anatomy of the extrafloral World. nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427–435. We are grateful to Yoichi Tateishi (University Coutinho Í. A. C., Francino D. M. T. and Meira R. M. S. A. of the Ryukyus), Yasuhiko Endo (Ibaraki 2013. Leaf anatomical studies of Chamaecrista subsect. University), Tadashi Kajita (University of the Baseophyllum (Leguminosae, Caesalpinioideae): Ryukyus), Tao Chen (Fairy Lake Botanical new evidence for the up-ranking of the varieties to the Garden, Shenzhen & CAS), Binhui Chen, specices level. Syst. Evol. 299: 1709–1720. Coutinho Í. A. C., Francino D. M. T. and Meira R. M. S. Fuwu Xing, Huagu Ye (South China Botanical A. 2015. New records of colleters in Chamaecrista Garden, CAS) and Chiajui Chen and Xiangyun (Leguminosae, Caesalpinioideae s.l.): structural Zhu (State Key Laboratory of Systematic diversity, secretion, functional role, and taxonomic and Evolutionary Botany, IB, CAS) for their importance. Int. J. Plant Sci. 176: 72–85. Coutinho Í. A. C. and Meira R. M. S. A. 2015. Structural cooperation during the field trip in South China diversity of extrafloral nectaries in Chamaecrista sect. performed in 1993. This study was supported Apoucouita. Botany 93(6): 379–388. in part by Grant-in-Aid for Scientific Research De Wit H. C. D. 1955. A revision of the genus Cassia as from the Japan Society for the Promotion of occurring in Malaysia. Webbia 11: 197–292. Science (No. 04041019 to H. Ohashi). Du Puy D. J. 2002. Tribe Cassieae: 7. Chamarcrista. In: Du Puy D. J., Labat J.-N., Rabevohitra R., Villiers J.- F., Bosser J. and Moat J. (eds.), The Leguminosae of References Madagascar. pp. 94–103. Royal Botanic Gardens, Kew. Baker J. G. 1878. Cassia. In: Hooker J. D., The Flora of Feder N. and O’Brien T. P. 1968. Plant microtechnique: British India 2: 261–267. Reeve & Co., London. some principles and new methods. Amer. J. Bot. 55: Bentham G. 1865. Leguminosae. In: Bentham G. and 123–142. Hooker J. D., Genera Plantarum 1: 434–600 (Cassia: Francino D. M. T., Coutinho Í. A. C., Dalvi V. C., Azevedo 571–573). Reeve & Co., London. A. A., Conceição A. de S. and Meira R. M. S. A. Bentham G. 1871. Revision of the genus Cassia. Trans. 2015. Anatomical interpretations of the of Linn. Soc. 27: 503–591. Chamaecrista (L.) Moench sect. Absus (Leguminosae- Brenan J. P. M. 1967. Leguminosae (Part 2): Subfamily Caesalpinioideae). Plant Syst. Evol. 301: 2087–2103. Caesalpinioideae. In: Milne-Redhead E. and Polhill Ghesquière J. 1932. Contribution a l'étude des R. M. (eds.), Flora of Tropical East Africa. (Cassia: Légumineuses principalement du Congo Belge. I. Les 47–103). Crown Agents, London. Cassia africains de la section Chamaecrista Benth. Candolle A. P. de. 1825. Leguminosae. In: Prodromus Bull. Jard. Bot. État Brux. 9: 139–169. Systematis Naturalis Regni Vegetabilis 2: 93–524 Ghesquière J. 1935. Matériaux pour l’étude des Cassia de (Cassia: 489–507). Treuttel & Würtz, Paris. la Province malgache. Rev. Zool. Bot. Afr. 26: 125– Chen D.-Z., Zhang D.-X. and Larsen K. 2010. Tribe 154. Cassieae. In: Wu Z.-Y., Raven P. H. and Hong D.-Y. Honda M. 1938. Plantae Sieboldianae In: Japanisch- (eds.), Flora of China 10 (Fabaceae): 27–34. Science Deutsche Kulturinstitut Geselschaft (ed.), Siebold December 2016 Nemoto et al.: Anatomy and development of leaves in Chamaecrista 215

Kenkyu. pp. 517–595. Iwanami, Tokyo. (Roxb.) Lock (Leguminosae-Caesalpinioideae). J. Jpn. Howard R. A. 1979. The Petiole. In: Metcalfe C. R. and Bot. 67: 215–216 (in Japanese). Chalk L. (eds.), Anatomy of the Dicotyledons, 2nd ed. Ohashi H. 1999. The genera, tribes and subfamilies of 1: 88–96. Clarendon Press, Oxford. Japanease Leguminosae. Sci. Rep. Tohoku Univ. 4th Huang T. C. and Ohashi H. 1993. Leguminosae. In: Huang ser. (Biol.) 40: 187–268. T. C. (ed.), Flora of Taiwan, 2nd ed. 3: 160–396. Ohashi H. 2016. Leguminosae. In: Ohashi H., Kadota Y., Editorial Committee of the Flora of Taiwan, Second Murata J., Yonekura K. and Kihara H. (eds.), Wild Editon, Taipei. Flower of Japan. 2: 240–306 (in Japanese). Irwin H. S. and Barneby R. C. 1981. Tribe Cassieae Bronn. Ohashi H., Nemoto T. and Ohashi K. 2013. Taxonomic In: Polhill R. M. and Raven P. H. (eds.), Advances position and nomenclature of Chamaecrista nomame in Legume Systematics 1: 97–106. Royal Botanic (Leguminosae). J. Jpn. Bot. 88: 67–76. Gardens, Kew. Ohwi J. 1953. Flora of Japan. Shibundo, Tokyo (in Irwin H. S. and Barneby R. C. 1982. The American Japanese). Cassiinae, synoptical revision of Leguminosae tribe Ohwi J. 1965. Flora of Japan (in English). Smithsonian Cassieae subtribe Cassiinae in the New World. Mem. Institution, Washington D.C. New York Bot. Gard. 35(2): 455–918. Pedley L. 1998. Chamaecrista. In: McCarthy P. M. (ed.), Kumar S. and Sane P. V. 2003. Legumes of South Asia: a 12, Mimosaceae (excl. Acacia), Check-list. Royal Botanic Gardens, Kew. Caesalpiniaceae. pp. 138–145. CSIRO Australia, Larsen K. and Ding H. 1996. Chamaecrista. In: Ding H., Melbourne. Larsen K. and Larsen S. S. (eds.), Flora Malesiana, Ser. Rudd V. E. 1991. Fabaceae (Leguminosae) Subfamily 1, 12(2), Caesalpiniaceae. pp. 565–570. Cip-Gegevens Caesalpinioideae. In: Dassanayake M. D. and Fosberg Koninklijke Bibliotheek, Den Haag. F. R. (eds.), A Revised Handbook of the Flora of Larsen K., Larsen S. S. and Vidal J. E. 1980. Légumineuses- Ceylon 7: 34–107. A. A. Balkema, Rotterdam. Césalpinioïdées. In: Aubréville A. and Leroy J.-F. Saheed S. A. and Illoh H. C. 2010. A taxonomic study of (eds.), Flore du Cambodge du Laos et du Viêt-nam 18: some species in Cassiinae (Leguminosae) using leaf 1–227. Muséum National d’Histoire Naturelle, Paris. epidermal characters. Not. Bot. Hoprt. Agrobot. Cluj Lewis G. P. 2005. Tribe Cassieae. In: Lewis G., Schrire 38: 21–27. B., Mackinder B. and Lock M. (eds.), Legumes of the Seethapathy G. S., Ganesh D., Santhosh Kumar J. U., World. pp. 110–125. Royal Botanic Gardens, Kew. Senthilkumar U., Newmaster S. G., Ragupathy S., Uma Lock J. M. 1989. Legumes of Africa: A Check-list. Royal Shaanker R. and Ravikanth G. 2015. Assessing product Botanic Gardens, Kew. adulteration in natural health products for laxative Lock J. M. 2007. Chmaecrista Moench. In: Timberlake J. yielding plants, Cassia, Senna, and Chamaecrista, in R., Pope G. V., Polhill R. M. and Martins E. S. (eds.), Southern India using DNA barcoding. Int. J. Legal Flora Zambesiaca 3(2): 121–144. Royal Botanic Med. 129: 696–700. Gardens, Kew. Singh V. 2001. Monograph on Indian Subtribe Cassiinae Lock J. M. and Ford C. S. 2004. Legumes of Malesia: a (Caesalpiniaceae). Scientific Publishers (India), Check-list. Royal Botanic Gardens, Kew. Jodhpur. Lock J. M. and Heald J. 1994. Legumes of Indo-China: a Sinnott E. W. 1914. Investigations on the phylogeny of the Check-list. Royal Botanic Gardens, Kew. Angiosperms. 1. The anatomy of the node as an aid Lock J. M. and Simpson K. 1991. Legumes of West Asia: a in the classification of Angiosperms. Amer. J. Bot. 1: Check-list. Royal Botanic Gardens, Kew. 303–322. Makino T. 1917. A contribution to the knowledge of the Sinnott E. W. and Bailey I. W. 1914. Investigations on the flora of Japan. J. Jap. Bot.1 : 15–18. phylogeny of the Angiosperms. 3. Nodal anatomy and Matsumura J. 1902. A conspectus of the Leguminosae the morphology of stipules. Amer. J. Bot. 1: 441–453. found growing wild, or cultivated in Japan, Loochoo Steyaert R. L. 1950. Note sur des Cassia africains et and Formosa. Bot. Mag. (Tokyo) 16: 91–105. asiatiques de la section Chamaecrista avec description Ohashi H. 1966. Leguminosae. In: Hara H. (ed.), The Flora de nouvelles especes. Bull. Jard. Bot. État. Brux. 20: of Eastern Himalaya. pp. 135–166 (Cassia: 142–145). 233–268. University of Tokyo Press, Tokyo. Viguier R. 1949. Leguminosae Madagascarienses Novae. Ohashi H. 1982. Leguminosae. In: Satake Y., Ohwi J., Notul. Syst. (Paris) 13: 333–369. Kitamura S., Watari S. and Tominari T. (eds.), Wild Watari S. 1934. Anatomical studies of some leguminous Flower of Japan, Herbaceous Plants (including Dwarf leaves with special reference to the vascular system in Subshurubs). 2: 186–212 (in Japanese). petioles and rachises. J. Fac. Sci. Univ. Tokyo (Sect. III, Ohashi H. 1989. The new scientific name instead ofCassia Botany) 4: 225–365. nomame (Sieb.) Honda or Chamaecrista dimidiata Wight R. and Walker-Arnott G. A. 1834. Prodromus Florae 216 The Journal of Japanese Botany Vol. 91 Centennial Memorial Issue

Peninsulae Indiae Orientalis 1. Parbury, Allen, & Co, of China: a Check-list. The ILDIS at the School London. of Biological Sciences, the University of Reading, Zhu X. Y., Du Y. F., Wen J. and Bao B. J. 2007. Legumes Reading.

a b c 根本智行 ，大橋広好 ，吴 德邻 ：マメ科ジャケツイ バラ亜科カワラケツメイ属 Chamaecrista mimosoides お よび C. nomame における葉の解剖および発生 カ ワ ラ ケ ツ メ イ 属 Chamaecrista mimosoides (L.) 束 (ridge bundle) が供給される．しかし，円鋸歯型葉軸 Greene は東アジアからアフリカまで分布し，葉は比較 では小葉の付着点の直後に 1 本に融合し，次の小葉付着 的小型の小葉をつける偶数羽状複葉で，約 80 対にまで 点までの中央付近で，再度 2 本に分岐し，次の小葉付着 達する多数の小葉対からなる．さらに，葉軸の小葉対と 点を過ぎると再度一本に融合する．溝型葉軸では，葉軸 小葉対の間の向軸側に特異な半円形の円鋸歯型隆起を 中で稜維管束は 2 本のままである．また，円鋸歯型葉軸 もつことで，近縁種から識別されてきた．カワラケツ では，融合した稜維管束から隆起に向かって分枝する 1 メイ属植物の葉軸は，一般に，小葉対と小葉対の間の 本の小さな維管束が確認された．葉柄の先端部にはどち 向軸側に 2 列の畝状の隆起があり，中央には溝ができ らも花外蜜腺を 1 個もつが，C. mimosoides では稜維管 る．C. mimosoides にみられる円鋸歯型葉軸はたいへん 束から花外蜜腺に，C. nomame では中央維管束の両端 希な特徴であるが，溝型葉軸との構造的な関連について から，いずれも主に篩部からなる維管束が供給される． 詳細は知られていない．本研究では，C. mimosoides と 本研究の結果は，これまで同一種とする見解もあった 同じ Sect. Chamaecrista に属し，一般的な溝型葉軸をも C. mimosoides と C. nomame を区別する新規の形質を明 ち，かつては同一種として扱われることもあったカワラ らかにした．また，円鋸歯型葉軸の 2 本の稜維管束が ケツメイ C. nomame (Makino) H. Ohashi と解剖学的お 小葉付着点間で融合と分岐を繰り返す走向パターンは， よび発生学的に比較することで，両者の構造的な関連を 円鋸歯型葉軸が溝型葉軸の 2 列の畝が合着してできた 明らかにすることを目的とした． とする Lock (2007) の考えを支持した．さらに，円鋸歯 Chamaecriata mimosoides の円鋸歯型隆起は葉緑体を 型隆起を構成する柔組織に多数の葉緑体が含まれ，表皮 多く含む単一の柔組織からできており，C. nomame の に多数の気孔が観察されることから，円鋸歯型葉軸は葉 溝型葉軸の 2 列の隆起はそれぞれ独立した柔組織から の光合成能力の向上に係わっていることが示唆された． できている．両者は発生過程初期から異なっており，前 （a石巻専修大学理工学部生物科学科， 者では単一の隆起が，一方後者では 2 つの隆起が生じた． b東北大学植物園津田記念館， どちらも葉柄の中央脈から向軸側の隆起に 2 本の稜維管 c中国科学院華南植物研究所）