Comparative morphology on leaves of Daphniphyllum ( Daphniphyllaceae )

A comparative anatomical study on the leaves of nine out of 29 species of the genus Daphniphyllum was performed to seek support for the present infrageneric classification. Daphniphyllum is composed of two sections, Lunata (with one subsection Lunata) and Daphniphyllum (with two subsections, Daphniphyllum and Staminodia). The glabrous leaves with brachyparacytic stomata appear in all species in this study; hemiparacytic, laterocytic or anomocytic stomata occur in some taxa. Two (out of three) species of section Lunata show irregular epidermal cells with undulate anticlinal walls on both surfaces and hemiparacytic stomata. Of section Daphniphyllum seven (out of 26) species were sampled; they have curved, straight or undulate anticlinal cell walls and laterocytic or anomocytic stomata. The anatomical features found in this study are useful for identification on the specific level, but there is no support for the current infrasectional classification of section Daphniphyllum; further study on section Daphniphyllum is necessary.

Textures and shapes of the leaves are variable in Daphniphyllum (Croizat & Metcalf 1941, Huang 1965).The staminate flowers and drupes only provided limited characters for species identification, also in combination with leaf morphology (Huang 1965).Leaf anatomical features proved to be of great value in many taxa not only in identifying species, but also in understanding relationships between taxa (Baas 1981, Pan et al. 1990, Mentink & Baas 1992, Kong 2001, Sheue et al. 2003, Gonzalez et al. 2004, Kocsis et al. 2004, Carpenter 2005, 2006).The leaf anatomical study of Daphniphyllum was extremely meager up to now.Metcalfe & Chalk (1988, 1989) described paracytic stomata, clusters of crystals and papillae on the abaxial surface for Daphniphyllum.The stomatal complex of four species of Daphniphyllum, D. chartaceum, D. macropodum, D. triangulatum and D. yunnanense, was studied by Zhang & Lu (1989).They showed that the paracytic type occurs in all four species.Huang (1965) described the leaf anatomy for five taxa, D. calycinum, D. gracile, D. glaucescens subsp.oldhamii, D. himalaense subsp.angustifolium and D. himalaense subsp.macropodum, and mentioned that "the upper, lower epidermis, sponge and palisade cells are different within species level".
The purpose of this comparative study is to provide morphological and anatomical characters, which may be helpful in species identification and can be used to support the present classification of the genus.

MATERIALS AND METHODS
Of the 29 Daphniphyllum species, two (out of three) species, D. calycinum and D. majus, were sampled to represent sect.Lunata; the other seven species (out of 26) were selected to represent sect.Daphniphyllum (Table 1).For two species dried leaf fragments of herbarium specimens were used.Fresh and desiccated materials of the other species were collected in the field in China, Japan, the Philippines and Taiwan.Vouchers are deposited in the herbarium of SYSU.
were cleared with 3 % sodium hydroxide at 40 °C for 5-14 days and stained with 1 % Safranin O. Paraffin sections and cleared leaves were observed and photographed with a Leica DME light microscope and a Nikon D70s digital camera.The mean length of stomatal guard cells was measured from each specimen with 40 replications.
Terminology follows Dilcher (1974) except for the term 'laterocytic stomata' (Carpenter 2005) that was used to describe the unequal parts of asymmetric stomata.

RESULTS
Leaf characteristics of the epidermis and transverse sections of Daphniphyllum are summarized in Table 2. Palisade tissue, stomatal and epidermal features of all studied taxa appear to be constant on the species level.Typical leaf morphological characters for sections, subsections and series are summarized in Table 3.

Palisade and spongy tissues
The leaf structure of Daphniphyllum is dorsiventral.The nine species can be divided into two groups on the basis of cell form and arrangement of palisade tissues (Table 2).One group shows columnar and compactly arranged palisade cells (Fig. 2c, f

Epidermal cells
The adaxial epidermal cells show in transverse section two types of cellular shapes, square or oblong (Table 2).Square adaxial epidermal cells occur in D. buchananiifolium (Fig. 2c), D. borneense (Fig. 2g) and D. gracile of subsect.Daphniphyllum, and oblong epidermal cells appear in all the other species of both sect.Daphniphyllum and sect.Lunata.
The anticlinal walls on both surfaces of the epidermal cells of Daphniphyllum are straight, curved or undulate (Fig. 3).The type of anticlinal cell walls is constant on the species level, but varies between the species (Table 2).The two species of sect.
Lunata show undulate anticlinal walls on both leaf surfaces (Fig. 3a -c).The anticlinal cell walls on both surfaces are straight, straight to curved or curved in all studied species of sect.Daphniphyllum except somewhat undulate in D. oldhamii (Fig. 3l).
The epidermal cells are polygonal, irregular, elongate and isodiametric in shape on both surfaces (Fig. 3, Table 2).Three species, D. calycinum, D. majus (both sect.Lunata) (Fig. 3c) and D. oldhamii (sect.Daphniphyllum) (Fig. 3l), have irregular epidermal cells on the adaxial side only, while the other species of sect.Daphniphyllum have polygonal adaxial epidermis (Fig. 3e, f, i, Table 2).Four epidermal shapes could be distinguished abaxially; seven species always show two shapes and only two species have a single type.The isodiametric shape is found only in leaves of D. teijsmannii (Fig. 3j) and restricted to the abaxial surface.

Stomata types
The stomata are restricted to the abaxial leaf surface only and randomly distributed.Four types of stomata are found, i.e. anomocytic, brachyparacytic, laterocytic and hemiparacytic (Table 2).The most common type is brachyparacytic, which is present in all nine taxa (Fig. 3a, b, d, g, h, j, k).The present study revealed three additional types of stomata in five species.
The mean length of the guard cells varies between 20.2-26.9µm (Table 2).Daphniphyllum macropodum has the longest guard cells and D. calycinum has the shortest ones.
We summarized the typical leaf morphological characters for different taxa in Table 3. First, hemiparacytic stomata, irregular epidermal cells and undulate anticlinal cells on two sides  3 The typical leaf morphological characters for sections, subsections and series of Daphniphyllum observed in this study.are characteristic for sect.Lunata.Secondly, the outstanding character typical for subsect.Staminodia and Daphniphyllum of sect.Daphniphyllum is the shape of the palisade cells (conical to round vs columnar).Finally, we also can distinguish ser.Longicalycifera and Unicalycifera from subsect.Daphniphyllum based on laterocytic stomata and a 1-layered hypodermis, but there is no other available character to further differentiate both series.The characters mentioned above may serve as remarkable taxonomic characteristics for the genus Daphniphyllum.Huang (1965) reported that the genus Daphniphyllum shows two distinct types of mesophyll tissue, i.e. well differentiated palisade and spongy layers and poorly differentiated palisade and spongy layers.This is confirmed by our study.Huang (1965) also indicated that D. angustifolium (sect.Daphniphyllum subsect.Staminodia) possesses a well-differentiated mesophyll and an adaxial hypodermis.However, the specimens of the two investigated species (D. macropodum and D. longeracemosum) of the same subsection did not reveal Huang's characters at all.Moreover, these features "well differentiated mesophyll tissue and adaxial hypodermis" as Huang found for D. angustifolium (subsect.Staminodia) were only found in two of the five species of subsect.Daphniphyllum in this study.Based on these results, we assume that a relatively close relationship may occur between D. angustifolium and subsect.Daphniphyllum.

DISCUSSION
Four types of stomata are found, i.e. brachyparacytic, hemiparacytic, laterocytic and anomocytic; the latter two types are first reported here.These distinctive stomatal types can be important characters for an infrageneric classification.Paracytic stomata were the only type reported for Daphniphyllum so far (Huang 1965, Metcalfe & Chalk 1988, Zhang & Lu 1989).However, the brachyparacytic type may just be a more specific term than the one used in any of the earlier articles (Huang 1965in Fig. 3, Zhang & Lu 1989 in Plate 1).Furthermore, laterocytic stomata are first reported here, but we notice that they were already visible for D. gracile in Huang (1965: f. 3).Anomocytic stomata  Huang (1996Huang ( , 1997) ) treated D. gracile (ser.Longicalycifera) and D. buchananiifolium (ser.Unicalycifera) in different series.However, the anatomical characters like hypodermis, straight anticlinal adaxial cell walls and laterocytic stomata may point to a closer relationship between these species.The various ranges of stomatal types may be an aid to recognize the different infrageneric taxa within the Daphniphyllaceae.
Within the nine studied species, D. oldhamii and D. teijsmannii are similar in morphological character.Huang (1966) distinguished them by papillose/epapillose on the abaxial surface of leaves.However, Huang (1965) and Wang (1981) mentioned that the character of papillae is unstable in D. oldhamii, so that identification of the two species is difficult.Our results show distinguishing differences in the epidermis.Daphniphyllum oldhamii has undulate and irregular adaxial epidermal cells, while D. teijsmannii not only shows straight and curved anticlinal cells on the adaxial surface but it also has isodiametric abaxial epidermal cells.Other studied species of the series can also be distinguished by two characters, shape of epidermal and size of guard cells (Table 2).For example, D. calycinum and D. majus of sect.Lunata can be distinguished by the characters oblong abaxial epidermis/smaller (20.2 µm) guard cells vs dome-shape abaxial epidermis/bigger (24.0 µm) guard cells.Due to the overlap of anatomical characters among the species of subsect.Staminodia, and between the species of ser.Longicalycifera and Unicalycifera, the size of guard cells is the only one character to support the classification.
The undulate anticlinal cell walls of both epidermal layers, the irregular epidermal cells of upper and lower surfaces, and the hemiparacytic stomata are typical characters of sect.Lunata.Anatomically, these three characters may well support the theory that sect.Lunata is likely to be a natural group.However, the leaf anatomical characters within sect.Daphniphyllum are highly divergent, which may imply that the current infrasectional classification is artificial.Furthermore, Huang (1965) pointed out that the two sections Staminodia (currently subsect.Staminodia of sect.Daphniphyllum) and Calycifera (currently subsect.Daphniphyllum) are artificial sections.The limited samples of sect.Daphniphyllum in this study may be inadequate to draw firm conclusions for classification of sect.Daphniphyllum.Therefore, more comprehensive studies on sect.Daphniphyllum are still warranted.
Comparison of leaf anatomical characters of nine species of Daphniphyllum observed from paraffin transverse sections (T.S.) and leaf clearings.Data between parentheses occur occasionally.* Values are means ± standard error.