Pollen Morphology of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae): Taxonomic Implications

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Flora 206 (2011) 458–467

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Pollen morphology of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae): Taxonomic implications

Juan Chen a,b, Nian-He Xia a,∗ a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, The Chinese Academy of Sciences, No. 723, Xingke Road, Tianhe District, Guangzhou 510650, People’s Republic of China b Graduate University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China article info abstract

Article history: In order to find new non-molecular evidence to support the phylogenetic and taxonomic position, pollen Received 23 March 2010 grains of 20 populations of 16 species of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae) Accepted 19 September 2010 were investigated under SEM and TEM. The pollen grains are spherical and ovoid, nonaperturate. The pollen wall is composed of a very thin exine and a thick intine. The exine is psilate or echinate. The Keywords: intine consists of two layers, i.e., a thick, channeled layer (exintine) and an inner homogenous layer Curcuma (endintine). The results reveal morphological congruence between the pollen grains of species of Curcuma, Boesenbergia which according to DNA sequence data appears to be a polyphyletic genus. However the uniform pollen Zingiberaceae Pollen morphology morphology in Curcuma provides no evidence to divide this genus into separate taxonomic entities. Our results on pollen morphology also do not provide any additional evidence to either unite or segregate Boesenbergia albomaculata and Curcumorpha longiflora in the same genus and demonstrate that more taxonomic data on the genus Boesenbergia and its relatives are needed before a final decision can be made. © 2011 Published by Elsevier GmbH.

Introduction sification and the taxonomic status of various genera have been under investigation. With 57 genera and about 1300 species, the tropical mono- The molecular phylogenetic surveys (Kress et al., 2002) sug- cotyledonous family Zingiberaceae is the largest one of the eight gested the non-monophyly of Curcuma and Boesenbergia. But families in the order Zingiberales (Costaceae, Cannaceae, Heli- Ngamriabsakul and Techaprasan (2006) stated that Boesenbergia coniaceae, Lowiaceae, Marantaceae, Musaceae, Streliziaceae and is monophyletic, based on petA-psbJ spacer. It was also shown that Zingiberaceae). Members of Zingiberaceae are mostly distributed floral gross morphological characters, such as color of bractsand from India to New Guinea (Kress et al., 2002; Larsen et al., 1998). position of inflorescence, which are traditionally used in the tax- The most accepted classification of the Zingiberaceae includes four onomy of Curcuma and Boesenbergia, are highly homoplastic and tribes: Hedychieae, Alpinieae, Zingibereae and Globbeae (Burtt thus often unreliable for the definition of lineages (Ngamriabsakul and Smith, 1972; Holttum, 1950; Larsen et al., 1998; Schumann, and Techaprasan, 2006). Therefore, many relationships identified 1904; Smith, 1981; Takhtajan, 1997) based on vegetative and by morphology have not been found in accordance with molecular flowering features. However, Kress et al. (2002) have brought surveys. new sights into the evolutionary relationships and classification Moreover, chromosome numbers were not useful for delimi- of the Zingiberaceae based on molecular phylogenies. They pro- tation of major lineages, especially in Boesenbergia. Chromosome posed a new classification of the Zingiberaceae and recognized four numbers of Boesenbergia indicate that two different evolutionary subfamilies and four tribes: Siphonochiloideae (Siphonochileae), lines exist in the genus. One has the basic chromosome num- Tamijioideae (Tamijieae), Alpinioideae (Alpinieae, Riedelieae), and ber x = 10, while the other has x = 12. Confirmation of the two Zingiberoideae (Zingibereae, Globbeae). Takhtajan (2009) accepted different evolutionary lines and the monophyly of the genus Boe- Kress’ system in his update system of angiosperm classification. senbergia requires further studies (Eksomtramage et al., 2002; Since then, the congruence of morphological features with this clas- Ngamriabsakul and Techaprasan, 2006). Pollen characters have been used to assess systematic and phylogenetic relationships in many taxonomic groups (Castro et al., 2009; Panajiotidis et al., 2000; Pardo et al., 2000). Pollen morphol- ∗ Corresponding author. Tel.: +86 20 3725 2565. ogy has been of particular significance in taxonomy and phylogeny E-mail address: [email protected] (N.-H. Xia).

Table 1 Accessions of Curcuma and Boesenbergia with vouchers, habitat and source.

Species Vouchers Habitat Source

Curcuma aromatica Salisb.a J. Chen132 (IBSC) Sand soil along the road of Menghai, Yunnan, China FAA-fixed IBSC C. aromatica Salisb. J. Chen54 (IBSC) Sand soil along the road of Mengla, Yunnan, China FAA-fixed IBSC C. aromatica Salisb. J. Chen0812 (IBSC) Dry soil in Nanning, Guangxi, China FAA-fixed IBSC C. amarrisima Roscoe J. Chen144 (IBSC) Sand soil along the road of Menghai, Yunnan, China FAA-fixed IBSC C. elata Roxb J. Chen0813 (IBSC) Wet soil along the road at South China Botanical Living collection IBSC Garden, Guangzhou, China C. aff. elata Roxb.b J. Chen57 (IBSC) Margin of the forest of Xishuangbanna, Yunnan, FAA-fixed IBSC China C. flaviflora S. Q. Tong J. Chen0885 (IBSC) Margin of the forest of Menghai, Yunnan, China FAA-fixed IBSC C. kwangsiensis S. G. Lee & C. F. Liang J. Chen0816 (IBSC) Dry slope at South China Botanical Garden, Living collection IBSC Guangzhou, China C. aff. kwangsiensis S. G. Lee & C. F. Liang J. Chen0841 (IBSC) Sand soil along the road of Menghai, Yunnan, China FAA-fixed IBSC C. longa L. J. Chen0886 (IBSC) Sand soil along the road of Menghai, Yunnan, China FAA-fixed IBSC C. nankunshanensis X. B. Xu, J. Chen & J. Chen0815 (IBSC) In the pot at South China Botanical Garden, Living collection IBSC N. Liu Guangzhou, China C. phaecaulis Valeton J. Chen0828 (IBSC) Beside streams of Gulinqing, Yunnan, China FAA-fixed IBSC C. rubrobracteata Skorniˇ cková,ˇ M. Sabu J. Chen0843 (IBSC) Sand soil along the road of Menghai, Yunnan, China FAA-fixed IBSC & Prasanthkumar. C. sp. ‘pink flower’ J. Chen134 (IBSC) Sand soil along the road of Menghai, Yunnan, China FAA-fixed IBSC C. sichuanensis X. X. Chen J. Chen0850 (IBSC) Beside streams of Menghai, Yunnan, China Living collection IBSC C. wenyujin Y. H. Chen & C. Ling J. Chen0890 (IBSC) Beside the road at South China Botanical Garden, Living collection IBSC Guangzhou, China C. yunnanensis N. Liu & S. J. Chen J. Chen0883 (IBSC) Sand soil along the road of Menghai, Yunnan, China FAA-fixed IBSC C. zanthorrhiza Roxb. J. Chen58 (IBSC) Dry slope at XSBN Tropical Botany Garden FAA-fixed IBSC Boesenbergia albomaculata S. Q. Tong. J. Chen0834-1 (IBSC) Margin of the Forest at XSBN Tropical Botany FAA-fixed IBSC Garden, Yunnan, China B. longiflora (Wallich) Kuntze J. Chen169 (IBSC) Margin of the forest at South China Botanical Living collection IBSC Garden, Guangzhou, China

a If three populations are given for one species, the upper one has been used for statistical analysis of quantitative characters measured under SEM and TEM in order to provide regular procedure in comparison between mean and standard deviations. b The specimens were determined with some doubts. of the family Zingiberaceae (Saensouk et al., 2009; Theilade et al., is provided in Table 1. When available, two or three collections 1993). Liang (1988) studied pollen morphology of Zingiberaceae were sampled for some species in order to be sure about the con- including Costaceae under LM and SEM. The results showed that stancy of pollen characters among different populations of one pollen grains of the Zingiberaceae are spherical, subspherical, ovoid species. Otherwise only one sample was prepared. Pollen obtained and prolate, respectively, 36–225 ␮m in size. The wall is composed mainly from blooming flowers was prepared for SEM, while pollen of a very thin exine and a thick intine. Based on the exine sculp- collected mostly from flower buds at start of anthesis was pre- turing, pollen grains of the Zingiberaceae/Costaceae lineage can pared for TEM observation. Most vouchers and all corresponding be divided into two types: inaperturate in the family Zingiber- microscope slides are deposited in the Herbarium of South China aceae and aperturate in the family Costaceae; and six subtypes: Botanical Garden, Chinese Academy of Sciences (IBSC). Herbarium psilate, spinate, cerebelloid-areolate, striate, verrucate, and fove- acronyms follow Index Herbariorum, 8th edition (Holmgren et al., olate. Liang (1988) emphasized that further palynological studies 1990). Taxonomic names are in accordance with the classifications concerning the phylogenetic placement of Boesenbergia and Cur- of Wu and Larsen (2000), the terminology follows Kress and Stone cumorpha are necessary. Mangaly and Nayar (1990) studied the (1982), Punt et al. (2007), and Hesse et al. (2009). palynology of South Indian Zingiberaceae, and divided the fam- For SEM observation of pollen morphology, pollen grains were ily Zingiberaceae into two groups (sulcate group and inaperturate collected from flowers and fixed in FAA in the field. The pollen group) according to the pollen morphology. Among them, the gen- grains were dispersed on stubs directly without any acid treat- era Curcuma and Boesenbergia belong to sulcate group, which is ments, then gold-coated in a JFC-1600 Auto Fine Coater and different from the inaperturate and nonsulcate type of Liang (1988). observed under a JEOL JSM-6360LV scanning electron microscope They also revealed that the exine is absent only in Kaempferia at 15 kV. and a discontinuous exine layer consisting of circular plates joined For study under TEM, pollen grains were collected from flow- together at margins occurs in Alpinia galanga and Amomum hypoleu- ers resp. opening flower buds fixed in FAA or glutaraldehyde cum. 2% + Alcian Blue 1% in phosphate buffer for 12 h and posted-fixed Following the recent molecular phylogenetic surveys, and the with osmium tetroxide 1% in water + Alcian Blue 1% phosphate extensive morphological and taxonomic studies of these genera buffer, dehydrated in an alcohol series and embedded in Spurr’s (Kress et al., 2002; Larsen et al., 1998; Saensouk and Larsen, 2001; low-viscosity resin (Spurr, 1969). Sections were stained with uranyl Wu and Larsen, 2000; Wu, 1997), it is the time to complement the acetate, followed by lead citrate, and examined with JSM-1010. For pollen data available and to reinterpret pollen morphological varia- each of the measurements about 15–20 pollen grains were used. tion as well as to assess the taxonomic delimitation of these genera based on pollen morphological characters. Results

Materials and methods Description of general pollen morphology

Pollen grains of 20 populations of 16 species of Curcuma and The shape (column 2 in Table 2) of the investigated pollen Boesenbergia (Zingiberaceae) were sampled. The list of voucher grains ranged from ovoid (Figs. 1–36) to spherical (Figs. 37–40). specimens including the ecology of the plants and their sources Their size (column 3 in Table 2) varied from 51.9 ± 7.2 ␮minCur- 460 J. Chen, N.-H. Xia / Flora 206 (2011) 458–467

Table 2 Characteristic features of pollen grains in Chinese representatives of Curcuma and Boesenbergia.

Species Shape Size (␮m) mean ± SD Aperture Exine WT (␮m) Sculpturing

Curcuma aromatica Salisb. Ovoid 60.4 ± 7.2 × 45.3 ± 5.6 N Continuous 5.0–9.5 Psilate C. amarrisima Roscoe Ovoid 63.7 ± 9.1 × 45.7 ± 5.9 N Continuous 2.7–5.4 Psilate C. elata Roxb. Ovoid 59.8 ± 4.2 × 43.9 ± 4.6 N Continuous 4.2–4.6 Psilate C. aff. elata Roxb. Ovoid 65.8 ± 7.7 × 45.7 ± 3.7 N Continuous 2.2–2.7 Psilate C. flaviflora S. Q. Tong Ovoid 55.8 ± 3.7 × 43.6 ± 4.4 N Continuous 2.3–3.0 Psilate C. kwangsiensis S. G. Lee & C. F. Ovoid 49.5 ± 3.8 × 41.3 ± 3.9 N Continuous 2.2–2.5 Psilate Liang C. aff. kwangsiensis Ovoid 62.0 ± 5.4 × 47.4 ± 4.9 N Continuous 2.3–2.6 Psilate C. longa L. Ovoid 61.8 ± 12.1 × 45.1 ± 8.5 N Continuous 4.1–4.6 Psilate C. nankunshanensis X. B. Xu, J. Ovoid 71.6 ± 6.9 × 56.1 ± 4.0 N Continuous 2.4–3.2 Psilate Chen & N. Liu C. rubrobracteata Skorniˇ cková,ˇ Ovoid 62.7 ± 9.4 × 46.7 ± 10.4 N Continuous / Psilate M. Sabu & Prasanthkumar. C. phaecaulis Valeton Ovoid 63.9 ± 3.4 × 39.5 ± 7.9 N Continuous 2.6–3.3 Psilate C. sichuanensis X. X. Chen Ovoid 70.5 ± 5.9 × 51.5 ± 6.9 N Continuous 3.4–4.7 Psilate C.sp, ‘pink flower’ Ovoid 51.9 ± 7.2 × 40.1 ± 3.8 N Continuous 3.1–4.9 Psilate C. wenyujin Y. H. Chen & C. Ling Ovoid 70.1 ± 6.4 × 52.2 ± 4.8 N Continuous / Psilate C. yunnanensis N.Liu&S.J. Ovoid 53.2 ± 7.5 × 43.4 ± 6.7 N Continuous 5.1–5.6 Psilate Chen C. zanthorrhiza Roxb. Ovoid 63.4 ± 7.9 × 44.2 ± 5.0 N Continuous 4.0–5.5 Psilate Boesenbergia albomaculata S. Q. Spherical 83.0 ± 7.8 N Discontinuous / Echinate; spine Tong. uniformly distributed, sharp apex, psilate between spines B. longiflora (Wallich) Kuntze Spherical 109.4 ± 12.5 N Continuous 1.6–4.9 Echinate; spine 4.7–6.5 ␮m long, sharp apex, 0.4–0.6 ␮m thick collared base

Abbreviations: N – nonaperturate; WT – wall thickness. cuma kwangsiensis, to 109.4 ± 12.5 ␮minBoesenbergia longiflora. species it shows an almost uniform thickness. The intine consists of The pollen (column 4 in Table 2) is nonaperturate (Figs. 1–40). Wall two layers, a thick exintine and a thin endintine adjacent to the pro- thickness (column 6 in Table 2) ranges from 2.2 ␮mto9.5␮m. The toplast (Figs. 41–54). The exintine is striated, while the endintine exine is very thin (Figs. 41–55). Most of the exine in the consid- exhibits a distinct minute fine structure. ered species is continuous, except for Boesenbergia albomaculata (column 6 in Table 2). According to the exine sculptures (column 7 Boesenbergia albomaculata S. Q. Tong (Figs. 38 and 40) in Table 2), pollen grains of these genera can be divided into two Pollen grains are spherical, 83.0 ± 7.8 ␮m in diameter (Table 2). types, and two groups of echinate are recognized: The grains appear nonaperturate. Spine uniformly distributed, apex sharp, surface reticulate (Figs. 38 and 40), which is a little different Type 1: Psilate type. Curcuma (Figs. 1–36) belongs to this type. from the collared base spine of Boesenbergia longiflora. Type 2: Echinate type. Group 1: Echinate–psilate. Exine sculpture is echinate but psilate Boesenbergia longiflora (Wallich) Kuntze (Figs. 37, 39 and 55) between spines. Boesenbergia albomaculata (Figs. 38 and 40) falls Pollen grains are spherical, 109.4 ± 12.5 ␮m in diameter under this group. (Table 2). The grains appear nonaperturate. Spines are 4.7–6.5 ␮m Group 2: Echinate-reticulate. Exine sculpture is echinate with long, uniformly distributed, with sharp apex and 0.4–0.6 ␮m thick reticulate surface between spines. Boesenbergia longiflora collared base (Figs. 37, 39 and 55). The exine is 0.4–0.6 ␮m thick. (Figs. 37 and 39) falls under this group. The intine consists of two hyaline layers, a 1.4–2.0 ␮m thick outer lamellate, and an up to 2.0 ␮m thick wavy lamellate one adjacent The thick intine consists of two layers, i.e., a thick, chan- to the protoplast (Fig. 55). The most inner layer is developed only neled layer (exintine) and inner homogenous layers (endintine) in certain regions and appears as excrescences, same as B. tiliiflora (Figs. 41–55). with a circular area observed by Mangaly and Nayar (1990), around The main features of investigated pollen grains are summarized the distal pole where intine is thinner. in Table 2. Selected SEM and TEM micrographs of pollen grains studied are presented in Figs. 1–55. Discussion Curcuma L. (Figs. 1–36, 41–54) Pollen grains are ovoid, varying from 51.9 ± 7.2 × 40.1 ± 3.8 to Because it is difficult to get and store the fresh pollen grains, the 71.6 ± 6.9 × 56.1 ± 4.0 ␮m in size. The grains appear nonaperturate, pollen morphology of the Zingiberaceae is still poorly understood. psilate (Figs. 1–36), but grains of some species have aperture-like However, the main characteristic features of pollen grains in the structures recognized as ridges or furrows (Figs. 13, 15, 21 and 27). genera studied here are similar to those found in earlier research The grain wall consists of a very thin membrane-like exine, a col- on Zingiberaceae (Liang, 1990; Mangaly and Nayar, 1990; Saensouk umellate middle layer (the channeled exintine) and a developed et al., 2009; Theilade et al., 1993). Curcuma and Boesenbergia pollen intine (endintine) (Figs. 41–49, 53, 54). In some species, such as C. grains are spherical or ovoid, nonaperturate. The wall is composed amarissima, the columellate layer is indistinguishable (Figs. 50–52). of a very thin exine and a thick intine. The exine is psilate or echi- Wall thickness ranges from 2.2 to 9.5 ␮m. The thickness of exine is nate. The thick intine consists of two layers: exintine and endintine. consistent, ca. 0.15 ␮m thick. But the thickness of the intine varies The congruence of some important pollen characters with former from 2.1 to 9.3 ␮m among different species. Within one particular classifications of these genera is discussed below. J. Chen, N.-H. Xia / Flora 206 (2011) 458–467 461

Figs. 1–12. SEM micrographs of pollen grains in Chinese Curcuma. Figs. 1 and 4: Curcuma aromatica 1, Figs. 2 and 5: C. aromatica 2, Figs. 3 and 6: C. aromatica 3, Figs. 7 and 10: C. sp., Figs. 8 and 11: C. wenyujin, Figs. 9 and 12: C. sichuanensis. Scale bars: (1–3, 7–9) = 10 ␮m; (12) = 5 ␮m; (4–5, 10, 11) = 2 ␮m;(6)=1␮m. 462 J. Chen, N.-H. Xia / Flora 206 (2011) 458–467

Figs. 13–24. SEM micrographs of pollen grains in Chinese Curcuma. Figs. 13 and 16: Curcuma aff. kwangsiensis, Figs. 14 and 17: C. kwangsiensis, Figs. 15 and 18: C. phaecaulis, Figs. 19 and 22: C. zanthorrhiza, Figs. 20 and 23: C. aff. elata, Figs. 21 and 24: C. yunnanensis. Scale bars: (13–15, 19–21) = 10 ␮m; (17, 18, 22, 23) = 2 ␮m; (16, 24) = 1 ␮m.

Shape this character cannot be used for taxonomic delimitation. Because, quite generally the pollen shape of Boesenbergia species is congru- The basic shape of the pollen grains in most Curcuma species ent and thus insufficient to separate species and natural groups. studied is ovoid, but spherical shapes can also be found in few species (Figs. 1 and 27). The shape of pollen grains may be influ- Aperture enced by the contact of young pollen grains in the tetrad stages. Therefore it can vary among pollen grains from the same flower of The pollen grains of Curcuma and Boesenbergia are usually non- Curcuma. However, the spherical shape is the only characteristic aperturate, but aperture-like structures recognized as ridges or for Boesenbergia albomaculata (Fig. 38) and Boesenbergia longiflora furrows are found in a few species of Curcuma (Figs. 13, 15, 21, 27), (Fig. 37). Although this spherical shape is similar in the two species, e.g. Curcuma kwangsiensis (Fig. 13). In both examined populations J. Chen, N.-H. Xia / Flora 206 (2011) 458–467 463

Figs. 25–36. SEM micrographs of pollen grains in Chinese Curcuma. Figs. 25 and 28: Curcuma longa, Figs. 26 and 29: C. flaviflora, Figs. 27 and 30: C. elata, Figs. 31 and 34: C. amarrisima, Figs. 32 and 35: C. nankunshanensis, Figs. 33 and 36: C. rubrobracteata. Scale bars: (25–27, 31–33) = 10 ␮m; (29, 30, 34–36) = 2 ␮m; (28) = 1 ␮m. of C. kwangsiensis and C. yunnanensis, inaperturate and aperture- Pollen wall structure like structures of grains were found in the same flower. Thus, the presence of aperture-like structures of pollen must not be a char- Kress et al. (1978) reported the pollen grains of the Zingiberales acteristic feature of Curcuma, being eventually formed by tight to be essentially ‘exineless’ with only a thin coat of sporopollenin contact of young pollen in the tetrad stage. Mangaly and Nayar on the surface. Available information about Zingiberaceae indicates (1990) studied the palynology of South Indian Zingiberaceae, and that the pollen wall consists of an extremely reduced thin exine reported that Curcuma and Boesenbergia belong to sulcate type. possessing a channeled medine layer (Saad and Ibrahim, 1965; Rao But Liang (1988) stated that they belong to the inaperturate and and Nijiagunaiah, 1983), and a thin intine. However, the medine nonsulcate type. Our study confirms that these two genera are is treated as a channeled layer of a well-developed intine complex inaperturate. in literature, which consists of an outer, thick, channeled layer I1 464 J. Chen, N.-H. Xia / Flora 206 (2011) 458–467

Figs. 37–40. SEM micrographs of selected pollen grains in Chinese Boesenbergia. Figs. 37 and 39: Boesenbergia longiflora, Figs. 38 and 40: Boesenbergia albomaculata. Scale bars: (37, 38) = 20 ␮m; (39–40) = 10 ␮m. and inner homogeneous layers (e.g. Kress and Stone, 1982; Liang, exine are psilate in Curcuma (Figs. 41–54). In Boesenbergia longiflora, 1990; Mangaly and Nayar, 1990; Saensouk et al., 2009; Theilade the sculpture is echinate with a reticulate surface between spines et al., 1993). (Figs. 37 and 39). However, in Boesenbergia albomaculata, the pollen Pollen grains of most species sampled in our study have grains are also echinate but psilate between spines (Figs. 38 and 40). a thin exine. This is in accordance with the previous results There is also some variation in number, size, shape and arrange- (Erdtman, 1952; Liang, 1990; Mangaly and Nayar, 1990; Zavada, ment of the spines among the studied species. The variation in 1983). The wall thickness of Curcuma varies from 2.2 ␮mto exine sculpturing appears to have particular value in classification 9.5 ␮m among different species (column 6 in Table 2). In Curcuma, at generic rank. Based on this heterogeneous structure the pollen the exine is represented only by a ca. 0.15 ␮m thick outermost of these genera can principally be divided into two types, psilate membrane-like layer (Figs. 41–54). The exine layer may peel off and echinate. as a perforated sheet in Boesenbergia longiflora and B. albomaculata (Figs. 38–40, 55). Pollen morphology in relation to taxonomy and phylogeny The most complex feature of the pollen of Zingiberaceae is the intine. This layer consists of exintine and endintine Curcuma (Figs. 41–49, 53–55). The thickness of the intine varies from 2.1 ␮m Lack of a comprehensive taxonomic revision of the genus Cur- to 9.3 ␮m among different species. It is uniformly thick in Curcuma cuma has hindered a precise estimation of its total number of species (Figs. 41–54). The exintine is thick, bearing crowded bacula- species in the world, with the estimates varying from about 50 like “columellae”, and the endintine is thin (Figs. 41–49, 53–55). But (Smith, 1981)to80(Larsen et al., 1998), 100 (Sirirugsa, 1996) in some species of Curcuma, the “columellae” are not distinguish- or even 120 (Leong-Skorniˇ ckováˇ et al., 2007). Furthermore, high able (Figs. 50–52). The intine of Boesenbergia longiflora looks like intra- and inter-population variation has rendered the identifica- an intine complex consisting of several layers, and the endintine tion of Curcuma species very difficult (Leong-Skorniˇ ckováˇ et al., is developed only in certain regions and markedly thinner at the 2007). Early taxonomists attempted to establish a natural system region without spines (Fig. 55). for the genus Curcuma. Roxburgh (1820) divided the genus into two unnamed sections based on the characters of lateral or central Sculpturing spikes. Baker (1890) separated it into three sections, i.e. Sect. Exan- tha (spikes vernal, always lateral), Sect. Mesantha (spikes autumnal, The patterns on the exine sculpture of Curcuma and Boesen- terminal, bracts not recurved at the tip) and Sect. Hitcheniopsis bergia species studied here are different. The sculptures of the (spikes autumnal, terminal, bracts very obtuse and spreading at the J. Chen, N.-H. Xia / Flora 206 (2011) 458–467 465

Figs. 41–55. TEM micrographs of selected pollen grains in Chinese Curcuma and Boesenbergia. Fig. 41: Curcuma longa, Fig. 42: C. yunnanensis, Fig. 43: C. elata, Fig. 44: C. aff. kwangsiensis, Fig. 45: C. kwangsiensis, Fig. 46: C. phaecaulis, Fig. 47: C. aff. elata, Fig. 48: C. zanthorrhiza, Fig. 49: C. flaviflora, Fig. 50: C. aromatica, Fig. 51: C. sp., Fig. 52: C. amarrisima, Fig. 53: C. nankunshanensis, Fig. 54: C. sichuanensis, Fig. 55: Boesenbergia longiflora. Scale bars: (41–43, 50–52, 54, 55) = 1 ␮m; (44–49, 53) = 500 nm. (E – exine; EI – channeled exintine; I – intine; EN – endintine). tip). Based on the presence or absence of the anther spur, Schumann morphology is still lacking. The present survey indicates that, (1904) divided the genus into two subgenera, i.e. subgen. Eucur- according to intine thickness, the genus can be divided into two cuma K. Schum. (= subgen. Curcuma) and subgen. Hitcheniopsis groups: group 1: pollen < 3 ␮m, most of Curcuma species; group (Bak.) K. Schum., which was accepted by most authors (Burtt, 1972; 2: pollen > 3 ␮m, found in C. aromatica, C. longa, C. yunnanensis. Holttum, 1950; Smith, 1981; Valeton, 1918). Among them, C. elata belongs to the traditional section Exan- For the large and highly diverse genus Curcuma with its com- tha, but C. longa and C. yunnanensis belong to sect. Mesantha. plicated taxonomy a comprehensive information on its pollen Therefore the palynological findings do not coincide with the 466 J. Chen, N.-H. Xia / Flora 206 (2011) 458–467 classification proposed by previous authors, particularly Baker Conclusion (1890) who divided the genus into the three sections Exantha, Sect. Mesantha and Sect. Hitcheniopsis, this classification based The taxonomic complex of Curcuma and Boesenbergia requires on the position of the spikes. At the same time, present results still more detailed phylogenetic analyses to define clearer the show a surprisingly high morphological congruence between the generic boundaries in the family Zingiberaceae and patterns of pollen grains of Curcuma, which, based on molecular systemat- their evolution (Kress et al., 2002; Ngamriabsakul and Techaprasan, ics, appears to be polyphyletic (Kress et al., 2002; Ngamriabsakul 2006). Pollen is one of the useful characters to assist the taxonomic et al., 2004). However, the similarity of the pollen morphol- delimitation and the investigation of relationships between the ogy must not provide unequivocal evidence for phylogenetic family Zingiberaceae and Costaceae (Liang, 1988). Pollen morphol- relationships. ogy confirms the previous taxonomic work on the genus Curcuma (Liang, 1988, 1990). Our study shows both similarities (shape) and differences (sculpturing) between Boesenbergia longiflora and Boe- Boesenbergia senbergia albomaculata, but provides no additional support to place Boesenbergia Kuntze is a genus of 80 species, with a distribu- Curcumorpha longiflora into Boesenbergia. Further pollen studies tion ranging from India to SE Asia (Saensouk and Larsen, 2001), and more extensive molecular analysis of Boesenbergia are needed with three species occurring in China (Wu and Larsen, 2000). to completely understand the evolution within the genus Boesen- Among them, Boesenbergia longiflora (Wallich) Kuntze was trans- bergia s.l. In present paper we have concentrated on SEM and TEM ferred to the monotypic genus Curcumorpha A. S. Rao & D. M. data of two Zingiberaceae genera. Certainly, further detailed pollen Verma as C. longiflora (Wallich) A. S. Rao & D. M. Verma (Rao studies within the whole family Zingiberaceae with its 57 gen- and Verma, 1971). As indicated by Rao and Verma (1971), Cur- era will importantly contribute to a better understanding of the cumorpha differs from Boesenbergia in the inflorescence sprouting evolution of this family. directly from rootstock, spirally arranged bracts and shape of staminodia. After studying material from India, Myanmar and Acknowledgements Thailand, Larsen (1997) recommended to maintain Curcumorpha longiflora in the genus Boesenbergia. He emphasized that the stem- We thank Ms. Xiao-Ying Hu and Ms. Xin-Lan Xu of South China less inflorescence, as an important character, is variable, since, Botanical Garden, the Chinese Academy of Sciences, for their help e.g. B. tenuisicata K. Larsen and B. trangensis K. Larsen bear not in the preparation of SEM and TEM images and in the electron only terminal but also lateral inflorenscences. Furthermore, the microscopy. We are also grateful to Prof. Te-Lin Wu, Dr. W. John spirally arranged bracts, the shape of the labellum, the whole flo- Kress and Prof. Yun-Fei Deng for their comments in the preparation ral morphology and the flower colors of Curcumorpha longiflora of the manuscript. conform with fundamental characters of the genus Boesenbergia. Liang (1988) suggested that the taxonomic position of Curcumorpha References and Boesenbergia requires more discussions based on the differ- ence of pollen exine sculptures between Curcumorpha longiflora Baker, J.G., 1890. Scitamineae. In: Hooker, J.D. (Ed.), Flora of British India, vol. 6. and Boesenbergia rotunda (Linn.) Mansf. According to the molecular Reeve, London, pp. 198–264. surveys, Kress et al. (2002) suggested that Boesenbergia including Burtt, B.L., 1972. Notes on Curcuma. Notes Roy. Bot. Gard. Edinburgh 31, 224–227. Burtt, B.L., Smith, R.M., 1972. Key words to the subfamilies; tribes and genera of two clades might be polyphyletic with several species (such as Zingiberaceae. Notes Roy. Bot. 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