FOLIAR TRICHOMES OF CROTON L . ( EUPHORBIACEAE : CROTONOIDEAE ) FROM CHINA AND ITS TAXONOMIC IMPLICATIONS

Foliar trichomes of 21 species of the genus Croton L. from China have been examined using stereomicroscopy and scanning electron microscopy. Five trichome types characterized by their morphology are identified, viz., stellate, lepidote, simple, dendritic and appressed-rosulate. Only stellate trichome is observed in most species, with only six species that are found to maintain two or three trichome types. Trichome types and density are useful for species identification and sectional classification for Chinese species. Based on the trichome types and other morphological characters, 21 Chinese species are proposed to be placed in five sections. Croton crassifolius belongs to sect. Andrichnia; C. cascarilloides belongs to sect. Monguia; C. mangelong, C. kongensis, C. laevigatus and C. laniflorus belong to sect. Argyrocroton; C. lauioides, C. howii and C. damayeshu belong to sect. Adenophylli. The remaining Chinese Croton species might be placed into sect. Croton. A key for Chinese Croton species based on trichome morphology is provided. Introduction Croton L. (Euphorbiaceae s.s.) is one of the largest genera of flowering plants, with about 1300 species of herbs, shrubs, trees and occasionally lianas that are ecologically prominent and important elements of secondary vegetation in the tropical and subtropical regions worldwide (Webster, 1993; Radcliffe-Smith, 2001). Croton belongs to subfamily Crotonoideae (APG, 2009; Wurdack and Davis, 2009), is characterized by mostly lactiferous taxa having pollen with an unusual (crotonoid) exine pattern of triangular supratectal elements attached to a network of muri with short columellae (Nowicke, 1994). The synapomorphy that characterizes Croton is the inflexed conformation of the tips of the staminal filaments in bud, which causes the anthers to be introrsely inverted until anthesis (Berry et al., 2005). Because of the large number of species and extensive morphological variation, it has been proved difficult to define and delimit sections and subsections within the genus Croton (Webster et al., 1996) despite the efforts of many taxonomists (Pax and Hoffmann, 1931; Webster, 1993). Webster (1993) established the most recent infrageneric classification, recognizing 40 sections in the genus mainly based on the New World taxa. Among them, three were reassigned generic status by Radcliffe-Smith (2001). Webster (1993) pointed out that his treatment of Old World taxa was much more cursory than that of New World taxa because of lack of familiarity with the living plants in Africa, Madagascar and Asia. Berry et al. (2005) presented a molecular systematic analysis of the genus Croton and tribe Crotoneae using nrITS and trnL-trnF DNA sequences data to test the validity of Webster’s classification. van Ee et al. (2011) revised the infrageneric classification and proposed a new system for New World Croton dividing into four subgenera and 31 sections including some species described as new ones. Corresponding author. Email: hfliu@scbg.ac.cn


Introduction
Croton L. (Euphorbiaceae s.s.) is one of the largest genera of flowering plants, with about 1300 species of herbs, shrubs, trees and occasionally lianas that are ecologically prominent and important elements of secondary vegetation in the tropical and subtropical regions worldwide (Webster, 1993;Radcliffe-Smith, 2001).Croton belongs to subfamily Crotonoideae (APG, 2009;Wurdack and Davis, 2009), is characterized by mostly lactiferous taxa having pollen with an unusual (crotonoid) exine pattern of triangular supratectal elements attached to a network of muri with short columellae (Nowicke, 1994).The synapomorphy that characterizes Croton is the inflexed conformation of the tips of the staminal filaments in bud, which causes the anthers to be introrsely inverted until anthesis (Berry et al., 2005).
Because of the large number of species and extensive morphological variation, it has been proved difficult to define and delimit sections and subsections within the genus Croton (Webster et al., 1996) despite the efforts of many taxonomists (Pax and Hoffmann, 1931;Webster, 1993).Webster (1993) established the most recent infrageneric classification, recognizing 40 sections in the genus mainly based on the New World taxa.Among them, three were reassigned generic status by Radcliffe-Smith (2001).Webster (1993) pointed out that his treatment of Old World taxa was much more cursory than that of New World taxa because of lack of familiarity with the living plants in Africa, Madagascar and Asia.Berry et al. (2005) presented a molecular systematic analysis of the genus Croton and tribe Crotoneae using nrITS and trnL-trnF DNA sequences data to test the validity of Webster's classification.van Ee et al. (2011) revised the infrageneric classification and proposed a new system for New World Croton dividing into four subgenera and 31 sections including some species described as new ones.
One of the most significant characters for infrageneric classification of Croton is the trichome morphology.Previous studies showed that trichome types have great variation within Croton (Webster et al. 1996;de Sá-Haiad et al., 2009;Senakun and Chantaranothai, 2010).Müller (1866) characterized the taxa as having stellate and lepidote hairs.Solereder (1908) and Metcalfe and Chalk (1950) noted that stellate and lepidote hairs also occur in other genera of subfamily Crotonoideae.Webster et al. (1996) identified foliar trichome characteristics for 120 species from 36 sections in Croton and established the possible evolutionary relationships among the different sections based on trichome characters.Senakun and Chantaranothai (2010) observed 23 Thai species and recognized seven trichome types.
In China, 23 species of Croton are recorded, including 15 endemic species (Li and Esser, 2008).Among them, only five species are placed in the 40 sections of Webster (1993).Moreover, foliar trichomes in Croton from China are not well-studied.Chang (1996) and Li and Esser (2008) described two main trichome types in Chinese Croton species, peltate scales [same as lepidote of Webster et al. (1996)] and stellate.Previously, foliar trichomes of only seven Chinese Croton species have been observed (Webster, 1993;Senakun and Chantaranothai, 2010).In the present work, we characterize the foliar trichomes of young to mature leaves of 21 species from China, including 15 endemic species.The objectives of this study are to provide descriptions, illustrations, and a survey of the trichomes in these 21 species using stereomicroscopy and scanning electron microscopy (SEM) and to propose the infrageneric classification for Chinese species.

Materials and Methods
Leaf samples of 21 species of Croton were obtained from dry specimens deposited at the herbarium of South China Botanical Garden, Chinese Academy of Sciences (IBSC).Both young and mature leaves were observed for each species.Two to four samples were examined for each species.A list of investigated materials is given in Table 1.Density of foliar trichomes was observed under Zeiss Stemi SV 11 stereomicroscopy, and photographed with an AxioCam MRC digital camera.
Both young and mature leaves were washed in 95% ethanol.Whole sections of young leaves and 0.5 × 0.5 cm of mature leaf fragments were bisected and mounted on copper stubs so that both adaxial and abaxial surfaces faced upwards.The mounts were air-dried, and coated with gold in a JFC-1600 sputter coater (JEOL Ltd, Tokyo, Japan).Observations and digital images were collected with a JEOL JSM-6360LV SEM (JEOL Ltd, Tokyo, Japan).The terminology follows Webster et al. (1996) and Senakun and Chantaranothai (2010).

Results
The main types of the trichomes and their density among the Croton species studied are summarized in Table 2. Selected SEM micrographs of trichome types are presented in Figure 1.Foliar trichomes of 21 Chinese Croton can be separated into five types; stellate, lepidote, simple, dendritic and appressed-rosulate.Glandular trichomes are not observed.
Density of trichome distribution is variable on different surfaces even within the same species.In general, trichomes are much denser on the abaxial surface than on the adaxial surface.Among 21 observed species, six species are glabrous on the adaxial surface even when they are at very In some species, trichomes are observed on both surfaces when young, but fall off completely on either both surfaces or on a single surface when mature.Variation in trichome type can be used to differentiate the Croton species examined in this study.In most species, only stellate trichome is observed.Only six species are found to have two or three trichome types.In C. chunianus, a few appressed-rosulate and few appressed-stellate with porrect radius trichomes are observed on the abaxial surface (Fig. 1B) and its adaxial surface is glabrous even very young.In C. cnidophyllus, a few dendritic and few appressed-stellate trichomes occur on the adaxial surface (Fig. 1C), and only dendritic trichome occurs on the abaxial surface.In C. crassifolius, three types of trichomes (appressed-stellate with porrect radius, simple and dendritic) are observed on the adaxial leaf surface (Fig. 1E), and only dendritic trichome occurs on the abaxial surface (Fig. 1L).This species can also be easily distinguished from other Chinese Croton species by possessing simple trichome which is not found in any other species.In C. dinghuensis, appressed-stellate with porrect radius trichome is found on the adaxial surface and appressed-rosulate trichome is observed on the abaxial surface (Fig. 1N).In C. lachnocarpus, it is observed that appressed-stellate with porrect radius trichome occurs on the adaxial surface (Fig. 1A) and dendritic trichome occurs on the abaxial surface (Fig. 1M).The dendritic with porrect radius trichome type and few appressed-stellate with porrect radius trichomes are found in C. yanhuii (Fig. 1D).In addition, two subtypes of trichomes are found in C. cascarilloides; dentate-lepidote trichome sometimes with porrect radius occurs on the adaxial surface (Fig. 1J), and leptidote-subentire trichome occurs on the abaxial surface (Fig. 1K).

Discussion
Among the 21 species we observed, seven species were also observed earlier by Webster et al. (1996) and Senakun and Chantaranothai (2010).Compared to their works, trichomes of C. lachnocarpus, C. laevigatus and C. tiglium are characterized as identical to their observation.Webster (1993) reported that C. kongensis had stellate trichome and was accordingly placed into Sect.Cascarilla, but we observed the lepidote-subentire trichome type as observed by Senakun and Chantaranothai (2010) based on Thai material.We cannot discuss more about the differences of the observation between our studies and Webster (1993), because we did not see the material observed by them.Only lepidote-subentire trichome was observed in C. cascarilloides by Senakun and Chantaranothai (2010), but we find that dentate-lepidote trichome sometimes with porrect radius occurs on the adaxial surface and lepidote-subentire trichome occurs on the abaxial surface.Senakun and Chantaranothai (2010) observed three types of trichomes (fasciculate, dendritic and glandular) in C. crassifolius, but we find that appressed-stellate with porrect radius, simple, and dendritic trichomes occur on the adaxial surface, and dendritic trichome occurs on the abaxial surface.Our observation accords with the previous studies (Webster et al., 1996;Chayamarit and van Welzen, 2005;Li and Esser, 2008).We could not check their voucher specimen of C. crassifolius observed by Senakun and Chantaranothai (2010) and therefore presume that their material was misidentified.Webster et al. (1996) indicated that the number and length of radii of trichomes could vary considerably on different leaves of a single specimen.It is supported by our observation.It also showed that the number and length of radii vary considerably even on same leaf of a single specimen.For example, it has 6-17 radii, 0.18-1.1 mm in diam. in C. yanhuii (Fig. 1D).
The foliar trichome is one of the most important characters to define sections in the genus Croton (Webster, 1993).According to the trichome types and other morphological characters, Chinese Croton species can be divided into five sections.Two species, C. cascarilloides and C. crassifolius, have multifid styles.They can be easily distinguished from each other by foliar trichome type.Webster (1993) placed C. crassifolius into sect.Croton, which had lepidote trichome.However, C. crassifolius has stellate trichome and it might be a member of sect.Andrichnia.Webster (1993) uncertainly listed C. cascarilloides under both sect.Anisophyllum, having appressed-stellate trichome, and sect.Monguia, having lepidote trichome.This is the same as Senakun and Chantaranothai (2010) observed, C. cascarilloides has lepidote trichome and it is suggested to be placed in sect.Monguia.Among species with bifid styles, C. mangelong, C. kongensis, C. laevigatus and C. laniflorus are different from other Chinese species in having lepidote trichome and belong to sect.Argyrocroton which is characterized by the bifid style and lepidote trichomes.Croton laevigatus and C. laniflorus were placed by Müller (1866) in sect.Decapetalon, however, they are not related to the species of sect.Decapetalon because they have glands on the leaf blades while eglandular in sect.Decapetalon.Croton lauioides, C. howii and C. damayeshu have stellate trichome and belong to sect.Adenophylli.Croton hancei and C. purpurascens with appressed-rosulate trichomes and the remaining Chinese Croton species having appressed-stellate trichomes might be placed into sect.Tiglium according to the classification of Webster (1993).However, the correct name for sect.Tiglium is sect.Croton because the section including C. tiglium, the type of the genus.Although Webster et al. (1996) superseded Small's (1913) choice of C. tiglium as the lectotype of Croton and designated C. aromaticus L., the valid lectotype of the genus is C. tiglium (Britton, 1918;van Ee and Berry, 2010).
A key to species of Croton from China is provided as follows.
For example, trichomes fall off completely on both surfaces in C. damayeshu, C. dinghuensis, C. euryphyllus and C. purpurascens, or on the adaxial surface in C. cascarilloides, C. cnidophyllus, C. hancei, C. kongensis and C. laui, or on the adaxial surface in C. chunianus and C. howii.

Table 1 . List of Croton species used in the present study.
*Species endemic to China.