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Leaf epidermal micromorphology defining the clades in Cinnamomum (Lauraceae)
expand article infoZeng Gang§, Bing Liu, Jens G. Rohwer|, David Kay Ferguson, Yong Yang#
‡ Institute of Botany, the Chinese Academy of Sciences, Beijing, China
§ Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, China
| Universität Hamburg, Hamburg, Germany
¶ University of Vienna, Vienna, Austria
# Nanjing Forestry University, Vienna, Austria
Open Access

Abstract

In this study, we sampled 48 species of Asian Cinnamomum covering the species groups that were identified in recent phylogenetic studies and conducted leaf micromorphological observations using both light microscopy (LM) and scanning electron microscopy (SEM). Synapomorphies were determined by means of mapping micromorphological characters on a phylogenetic tree. The results indicate that Cinnamomum exhibits two different types of leaf upper epidermis: Type I has smooth/non-reticulate periclinal walls whereas Type II has reticulate periclinal walls and is unusual in the family Lauraceae. We found that the two types of micromorphological characters are clade-specific, sect. Camphora s.s. possesses Type I leaf upper epidermis, and sect. Cinnamomum s.l. has Type II leaf upper epidermis. Our study also reveals that C. saxatile, a member of sect. Camphora s.l. in the traditional classification, actually has Type II leaf upper epidermis, thus reinforcing the result of a recent molecular phylogeny that has this species in a clade consisting mainly of species of sect. Cinnamomum.

Keywords

Anatomy, Cinnamomum, Lauraceae, scanning electron microscope (SEM), systematics

Introduction

In the family Lauraceae, there are some named generic complexes according to molecular systematic studies, e.g. the Beilschmiedia group, the Persea group, the Litsea group, the Alseodaphne group, and the Cinnamomum group (e.g. Chanderbali et al. 2001; Rohwer et al. 2009, 2014; Huang et al. 2016; Trofimov et al. 2016, 2019; Mo et al. 2017; Rohde et al. 2017; Trofimov and Rohwer 2020; Xiao et al. 2020; Liu et al. 2021). A number of macromorphological characters have been used in the past to define the genera in each complex, but it now seems that these macromorphological characters were either plesiomorphic (e.g. in Ocotea Aubl. s.l.) or originated through parallelism, i.e. evolved several times. For instance, in the Litsea group, Lindera Thunb. differs from Litsea Lam. in the number of anther locules (2-locular in Lindera vs. 4-locular in Litsea). Phylogenetic studies based on DNA sequences suggest that Lindera is polyphyletic, and comprises many different clades (Li et al. 2004; Fijridiyanto and Murakami 2009). How these clades can be recognized using morphological characters has become an important question in the taxonomy of the group.

The Cinnamomum group is amphi-Pacific and distributed in tropical America and tropical to subtropical Asia with relatively few species found in Africa and Australia (Rohwer 1993; van der Werff 2001). The group belongs to the Laureae-Cinnamomeae clade of the core Lauraceae (Chanderbali et al. 2001; Rohwer and Rudolph 2005; Song et al. 2017, 2020), and consists of several closely related genera, i.e. Cinnamomum Schaeff., Aiouea Aubl. and the Ocotea complex (Chanderbali et al. 2001; Huang et al. 2016; Rohde et al. 2017). The group is thought to have originated ca. 55 mya and was once widely distributed in the palaeotropical Arcto-Tertiary flora of Laurasia during the Eocene, then migrated southwards and, with cooling temperatures, split, resulting in the modern amphi-Pacific disjunct distribution (Huang et al. 2016).

Cinnamomum is generally considered to consist of ca. 300 species, with the highest diversity in tropical Asia (Rohwer 1993; van der Werff 2001) and only a few species in Australia (Hyland 1989) and some Pacific Islands. Species of the genus are characterized by inflorescence Type II of van der Werff and Richter (1996), i.e. paniculate inflorescences with strictly opposite ultimate cymes, flowers with nine fertile stamens plus three staminodia with a conspicuous cordate to sagittate glandular head, and a more or less developed cupule with or without persistent (remnants of) tepals (van der Rohwer 1993; van der Werff 2001). Traditionally, the Asian species have been classified into two sections: Camphora Meisn. (1864: 24) and Cinnamomum. Species in sect. Camphora have alternate leaves, usually with domatia in the axils of lateral veins, pinnate to subtriplinerved venation, and often perulate buds. Species in sect. Cinnamomum have (sub)opposite and tripliveined leaves lacking domatia in the axils of lateral veins, and no perulate buds (Fig. 1, e.g. Li et al. 1982). Recent phylogenetic studies have consistently suggested that Asian Cinnamomum is not monophyletic and contains two robust clades (Huang et al. 2016; Trofimov and Rohwer 2020). Cinnamomum sect. Camphora s.s. (Clade 1) excluding three species of the traditional sect. Camphora, and sect. Cinnamomum s.l. (Clade 2) containing three species previously attributed to sect. Camphora s.l. (C. saxatile H.W. Li (1975: 44), C. longipetiolatum (Gamble) N. Chao ex H.W. Li (1975: 47), and an unidentified species labelled C. sp. C684; Huang et al. 2016). The species of the Neotropical clade (Clade 3) recognized by Huang et al. (2016) have recently been transferred to Aiouea (Rohde et al. 2017), as the result of a study of nrITS sequences and two chloroplast spacers (psbA-trnH and trnG-trnS).

Figure 1. 

Morphology of the two sections of the Asian Cinnamomum A–C Cinnamomum camphora of sect. Camphora A perulate terminal buds B branch portion displaying the alternate leaf arrangement C a leaf showing the pinnate venation and the domatia in axils of lateral veins D, E Cinnamomum japonicum of sect. Cinnamomum D terminal buds lacking helically arranged scales E branch portion exhibiting the subopposite leaf arrangement F a leaf displaying the tripliveined venation and the absence of domatia in axils of lateral veins.

Various studies have suggested different topologies, e.g. sect. Cinnamomum s.l. as sister to the Neotropical clade in Huang et al. (2016), but sect. Camphora s.s. appears to be sister to the Neotropical clade in the ITS result of Rohde et al. (2017). According to Rohde et al. (2017), sect. Camphora s.s. and the Neotropical clade share alternate, penninerved to moderately triplinerved leaves. Huang et al. (2016) suggested that alternate and penninerved leaves, perulate buds and domatia are potential synapomorphies for sect. Camphora s.s., but these characters do not seem to be very reliable (Huang et al. 2016). Though Trofimov and Rohwer (2020) confirmed that Asian Cinnamomum is diphyletic, they gave different relationships of the two sections of the Asian Cinnamomum: sect. Camphora appears to be sister to Sassafras J. Presl, and together they constitute a clade which appears to be sister to the Neotropical Ocotea complex, whereas sect. Cinnamomum is sister to Kuloa Trofimov & Rohwer, an African genus recently described. Liu et al. (2021) found pervasive conflicts between plastid data and nrITS, and suggested that Asian Cinnamomum was paraphyletic with respect to Sassafras. They included only a single Neotropical sample from the Cinnamomeae, Nectandra angustifolia (Schrad.) Nees et Mart. (1833: 48). In any case, Asian Cinnamomum contains two robust clades. However, it remains unclear how the clades of Cinnamomum should be defined morphologically.

Leaf epidermal micromorphology has been considered to be of taxonomic importance within the Lauraceae (Christophel et al. 1996; Nishida and Christophel 1999; Nishida and van der Werff 2007, 2011, 2014; Yang et al. 2012; Zeng et al. 2014; Nishida et al. 2016; Trofimov and Rohwer 2018), but its systematic significance has rarely been discussed within a phylogenetic context. In this study, we report micromorphological observations in Asian Cinnamomum, and discuss their systematic significance.

Material and methods

Mature leaf materials were taken from herbarium specimens. Our sampling covered the two clades that were identified in recent molecular phylogenetic studies (Huang et al. 2016; Rohde et al. 2017), and contained 48 species of Asian Cinnamomum. Three Sassafras species were also sampled for comparison because the result based on psbA-trnH and trnG-trnS sequences in Rohde et al. (2017) suggested that Sassafras was closely related to at least some species of Cinnamomum.

Leaf samples were obtained from herbarium specimens deposited in the Herbarium of the State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany (PE), the Chinese Academy of Sciences (Table 1). Earlier studies have suggested that leaf epidermal characters are stable within species (e.g. Nishida and Christophel 1999; Nishida and van der Werff 2007, 2011; Yang et al. 2012). Therefore, in most cases, one specimen was sampled to represent the species in this study. For the purpose of comparison and to eliminate variation that might be caused by sampling from different leaf areas, we took samples close to the basal portion on the left hand side of the midvein of a leaf. The leaf materials were then cut into small rectangular pieces (ca. 3 mm×10 mm).

Table 1.

Voucher information of leaf samples for this study.

Latin Name Collection Locality
Cinnamomum appelianum Schewe (1925: 20) S.H. Chun 10175 China, Guangxi
C. aromaticum Nees (1831b: 74) Anshun Exped. 049 China, Guizhou
C. austrosinense H.T. Chang (1959: 20) S.Y. Chang 3133 China, prov. unknown
C. bejolghota (Buch.-Ham.) Sweet (1826: 344) S.Z. Cheng & B.S. Li 03985 China, Xizang
C. bodinieri H. Lév. (Léveillé 1912: 369) P.C. Tsoong 34 China, Guizhou
C. burmannii (Nees & T. Nees) Blume (1826: 569) G.Z. Li 15650 China, Guangxi
C. camphora (L.) J. Presl (1825: 47) S.Y. Chang 4819 China, Zhengjiang
C. camphora L.D. Duan 2601 China, Hunan
C. camphora C.F. Liang 33262 China, Guangxi
C. chartophyllum H.W. Li (1975: 49) B. Liu 1366 China, Yunnan
C. chekiangense Nakai (1939: 23) H. Zou 01435 China, Anhui
C. daphnoides Siebold & Zucc. (Siebold and Zuccarini 1846: 402) T. Yahara 6641 Japan, Kyushu
C. doederleinii Engl. (Engler 1884: 57) M. Furuse 43512 Japan, Kyushu
C. glanduliferum (Wall.) Meisn. (Meisner 1864: 25) Y.M. Shui 2217 China, Yunnan
C. ilicioides A. Chev. (Chevalier 1918: 855) F.C. How 72957 China, Hainan
C. iners (Reinw. ex Nees et T. Nees) Blume (1826: 570) Y. Tsiang 12772 China, Yunnan
C. insularimontanum Hayata (1913: 158) T.Y.A. Yang et al. 08378 China, Taiwan
C. japonicum Siebold (1830: 23) Zhejiang Bot. Exped. 27696 China, Zhejiang
C. jensenianum Hand.-Mazz. (Handel-Mazzetti 1921: 63) T.T. Yü 3125 China, Sichuan
C. liangii C.K. Allen (1939: 58) S.K. Lau 26252 China, Hainan
C. litseifolium Thwaites (1861: 253) M. Poilane 14784 Cambodia
C. longipaniculatum (Gamble) N. Chao ex H.W. Li (1975: 48) Z.W. Yao 3567 China, Sichuan
C. macrostemon Hayata (1913: 160) Y.H. Lai 27 China, Taiwan
C. mairei H. Lév. (Léveillé 1914: 174) Z.W. Yao 4980 China, Yunnan
C. micranthum (Hayata) Hayata (1913: 160) M. Poilane 10707 Cambodia
C. migao H.W. Li (1978: 90) Beijing Team 891144 China, Guangxi
C. osmophloeum Kaneh. (Kanehira 1917: 428) C.M. Wang 05395 China, Taiwan
C. ovalifolium Gardner ex Meisn. (Meisner 1864: 11) T. Koyama 13513 Sri Lanka
C. parthenoxylon (Jack) Meisn. (Meisner 1864: 26) Sichuan Bot. Exped. 2355 China, Sichuan
C. parthenoxylon IBCAS Team. 814 China, Jiangxi
C. pauciflorum Nees (1831b: 75) Mt. Ziyun Exped. 411 China, Hunan
C. pedunculatum (Thunb.) J. Presl (1825: 37) Jiangxi Exped. 947 China, Jiangxi
C. pingbienense H.W. Li (1978: 91) B. Liu 1363 China, Yunnan
C. pittosporoides Hand.-Mazz. (Handel-Mazzetti 1925: 19) Yunnan Exped. of CAS 311 China, Yunnan
C. pseudopedunculatum Hayata (1913: 161) M. Furuse 52925 Japan, Kyushu
C. randaiense Hayata (1911: 238) T.Y. Liu et al. 201 China, Taiwan
C. reticulatum Hayata (1911: 239) G.F. Zhong et al. 1374 China, Taiwan
C. rigidissimum H.T. Chang (1959: 19) C.F. Wei 122561 China, Hainan
C. saxatile H.W. Li (1975: 44) B. Liu 1327 China, Yunnan
C. scortechinii Gamble (1910: 219) M. Poilane 11143 Unknown locality
C. septentrionale Hand.-Mazz. (Handel-Mazzetti 1936: 213) H. Yu 177 China, Sichuan
C. subavenium Miq. (Miqeul 1858: 902) W.C. Cheng 3649 China, Zhejiang
C. subavenium M.J. Wang 3768 China, Anhui
C. tamala (Buch.-Ham.) T. Nees & Eberm. (Nees 1831a: 2) Qinghai-Xizang Veg. Exped. 4584 China, Xizang
C. tenuifolium (Makino) Sugim. (Sugimoto 1928: 57) M. Furuse 8173 Japan
C. tetragonum A. Chev. (Chevalier 1918: 855) M. Poilane 378 Unknown locality
C. tonkinense (Lecomte) A. Chev. (Chevalier 1918: 856) Liu Bing 1326 China, Yunnan
C. tsangii Merr. (Merrill 1934: 26) Jiangxi Exped. 124 China, Jiangxi
C. validinerve Hance (1882: 80) X.G. Li 200631 China, Guangdong
C. verum J. Presl (1825: 37) N. Wallich 2573B Unknown locality
C. wilsonii Gamble (1914: 66) Z.C. Luo 191 China, Hunan
C. zollingeri Lukman. (Lukmanoff 1889: 4) S. Saito 1388 Japan, Nagato
Sassafras albidum (Nutt.) Nees (1836: 490) S.C. Chen et al. 715 China, Taiwan
S. randaiense (Hayata) Rehder (1920: 244) W.M. Wang 93 USA, Georgia
S. tzumu (Hemsl.) Hemsl. (Hemsley 1907: 55) B. Liu 1372 China, Yunnan

For light microscopic observation, the samples were dipped in 40% NaClO at 60 °C until the samples began to bleach. The samples were then washed in distilled water. The epidermis of both surfaces of the leaves was peeled off under a light microscope (Zeiss Stemi 2000), then stained in 1% safranin-50% ethanol for 30 minutes, dehydrated with gradations of ethanol, and treated with gradations from ethanol to xylene, and finally the epidermis pieces were mounted in Canada balsam (Yang et al. 2012; Zeng et al. 2014). The preparations were dried at 40 °C in an incubator. Photographs were taken using a Zeiss Axio Imager A1 light microscope with a 10× eyepiece and 40× objective.

For SEM observations, leaf samples were cut into small pieces of ca. 3 × 3 mm. Leaf samples were soaked in 100% ethanol for 15 minutes, followed by ultrasonic cleaning for 10 minutes at 100 hz, after which the ethanol was replaced by isoamyl acetate, and critical-point dried using carbon dioxide for five hours (equipment: HCP-2; Yang et al. 2012; Zeng et al. 2014). The treated leaf pieces were then fixed on stubs with the inner surface of leaf epidermis exposed, coated with palladium under 15 mA for 110 s, observed and photographed under a HITACHI s-4800 scanning electron microscope (10.0KV; State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences). To clarify the ornamentation of the upper leaf epidermis, the peeled upper leaf epidermis was also observed.

Observed features of leaf epidermis included the following: (1) epidermal cell shape, (2) anticlinal walls of normal epidermal cells, i.e. non-stomatal cells, (3) periclinal walls of normal epidermal cells, and (4) the stomatal complex (including subsidiary cells). Line-drawings were made with Adobe Photoshop CS2 ver. 9.0 using the Pen Tool.

Because our aim was to understand the evolution of micromorphological characters in the context of phylogeny, we selected published sequences according to our sampling for the micromorphological studies to reconstruct a phylogeny. Phoebe zhennan, P. hungmoensis, Alseodaphnopsis rugosa, and A. hainanensis were selected as the outgroup for phylogenetic analysis of Cinnamomum. Micromorphology of these outgroup species was observed but has not been published yet (Zeng 2018). All sequences were downloaded from NCBI (Table 2). We used three nuclear genes to reconstruct the phylogeny of the Asian Cinnamomum, i.e. nrITS, LEAFY intron II, and RPB2. Sequences were aligned with MAFFT v7.304b (Katoh and Standley 2013). MrModeltest 2.3 (Nylander 2008) and Paup* v.4.0b10 (Swofford 2003) were used to select the best-fit evolutionary model using Akaike Information Criterion (AIC). Bayesian inference (BI) analysis was performed using MrBayes 3.2.6 on XSEDE (Huelsenbeck and Ronquist 2001). The Markov Chain Monte Carlo (MCMC) algorithm was run for 3,000,000 generations, sampling one out of every 1000 generations. The first 25% trees were discarded as burn-in. The remaining trees were used to calculate the posterior probabilities (PP) and construct the consensus tree. Maximum Likelihood (ML) analyses were performed using RAxML-HPC2 on XSEDE with the GTRCAT model to search the best-scoring ML tree and generate a tree block at the same time. 1000 bootstrap replicates were performed in each analysis to obtain the confidence support. The ML tree block was read in FigTree v1.4.0 and saved as a nexus file which was then opened in Mesquite v3.04. Micromorphological characters were collected based on leaf anatomy in this study, and were manually input into the “Character Matrix” in Mesquite. All characters were treated as unordered and equally weighted. To reconstruct character evolution, a maximum likelihood approach using Markov k-state 1 parameter model (Mk1; Lewis 2001) was used. We selected the “Trace-Character-Over-Trees” command to calculate ancestral states at each node including probabilities in the context of likelihood reconstructions. To carry out these analyses, characters were plotted onto trees that were sampled in ML analyses. The results were finally summarized as percentage of changes of character states on a given branch among the stored trees utilizing the option of “Average-frequencies-across-trees”. Trees with reconstructed ancestral character states were then exported as pdf files which were then manually adjusted in Adobe Illustrator CS6.

Table 2.

Sequences obtained from the GenBank for phylogeny of Asian Cinnamomum.

Taxon ITS RPB2 LEAFY
Alseodaphnopsis hainanensis (Merr.) H.W. Li & J. Li (2016: e0186545 (9)) FJ755440 KU140409 HQ697006
A. rugosa (Merr. & Chun) H.W. Li & J. Li (2016: e0186545 (9)) HQ697183 KU140410 HQ697012
Cinnamomum appelianum KU139817 KU140330 KU140244
C. austrosinense KU139818 KU140331 KU140245
C. bejolghota KU139822 KU140335 KU140249
C. bodinieri KU139824 KU140336 KU140251
C. burmannii KU139825 KU140337 KU140252
C. camphora KU139826 KU140338 KU140253
C. chartophyllum KU139832 KU140344 KU140259
C. chavarrianum (Hammel) Kosterm. (Kostermans 1988: 442) AF272261 KU140345
C. chekiangense MF110041 KU140346 KU140260
C. daphnoides FM957803 KU140352 KU140266
C. doederleinii KU139842 KU174408
C. glanduliferum KU139843 KU140354 KU140269
C. iners KU139849 KU140360 KU140275
C. insularimontanum KY271510 KU140361 KU174418
C. japonicum KU139851 KU140361 KU140277
C. jensenianum KU139853 KU140363 KU140279
C. liangii KU139856 KU140366 KU174422
C. longipaniculatum KX546754 KT248715 KU140283
C. macrostemon GU598521
C. mairei KU139859 KU140368 KU174423
C. micranthum KY271519 KU140369 GQ260581
C. osmophloeum KY271528 KU140375
C. parthenoxylon KU139871 KU140377 KU140295
C. pauciflorum KU139872 KU140378 KU140296
C. pingbienense KU139873 KU140379 KU140297
C. pittosporoides KU139874 KU140380 KU140298
C. reticulatum KU139879 KU174432
C. rigidissimum KU139881 KU140386 KU140305
C. saxatile KU139882 KU140387 KU140306
C. septentrionale KU139883 KU140388 KU140307
C. subavenium KU139888 KU140393 KU140312
C. tamala KX822090 KU140396 KU174439
C. tenuifolium KU139892 KU140397 KU140316
C. tenuifolium KU140316
C. tonkinense KU139895 KU140400 KU140319
C. tsangii KU139900 KU140405 KU140324
C. verum MF110061 KU140407 KU140326
C. wilsonii KU139904 KU140408 KU140328
Phoebe hungmoensis S.K. Lee (1963: 190) HQ697206 KU140413 HQ697138
P. zhennan S.K. Lee & F.N. Wei (1979: 61) HQ697212 KT248761 HQ697161

Results

Leaf epidermal micromorphology is presented in Figs 2, 3. Illustrations in Fig. 4 display main characters and their variation. The main results are presented in Table 3.

Table 3.

Micromorphology of the leaf epidermis of Asian Cinnamomum under light microscopy (LM) and scanning electron microscope (SEM).

Clade Latin name Cell shape Periclinal wall Anticlinal wall Lower stomatal ledge (LM) Stomatal surface (SEM) Section
Clade 1 C. bodinieri polygonal non-reticulate straight/rounded wide lip-shaped eyelid-shaped Sect. Camphora
C. camphora polygonal non-reticulate straight/rounded wide lip-shaped or bat-shaped globose
C. chartophyllum polygonal non-reticulate straight/rounded narrow lip-shaped circular
C. glanduliferum polygonal non-reticulate straight/rounded wide lip-shaped or bat-shaped globose
C. longepaniculatum polygonal non-reticulate straight/rounded bat-shaped lip-shaped
C. micranthum polygonal non-reticulate straight/rounded wide lip-shaped circular
C. parthenoxylon polygonal non-reticulate straight/rounded wide lip-shaped globose
C. septentrionale polygonal non-reticulate straight/rounded wide lip-shaped globose
Note 1 C. ilicioides polygonal non-reticulate straight/rounded wide lip-shaped eyelid-shaped
C. migao polygonal non-reticulate straight/rounded narrow lip-shaped lip-shaped
Clade 2 C. appelianum irregular reticulate Sinuous wide lip-shaped or bat-shaped eyelid-shaped Sect. Cinnamomum
C. austrosinense irregular reticulate Sinuous wide lip-shaped or bat-shaped eyelid-shaped
C. bejolghota irregular reticulate Sinuous - globose
C. burmannii irregular reticulate Sinuous butterfly-shaped eyelid-shaped
C. cassia irregular reticulate Sinuous butterfly-shaped eyelid-shaped
C. chekiangense irregular reticulate Sinuous butterfly-shaped eyelid-shaped
C. iners irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. insularimontanum irregular reticulate Sinuous wide lip-shaped or butterfly-shaped eyelid-shaped
C. japonicum irregular reticulate Sinuous wide lip-shaped or butterfly-shaped eyelid-shaped
C. jensenianum irregular reticulate Sinuous butterfly-shaped eyelid-shaped
C. mairei irregular reticulate Sinuous wide lip-shaped invisible
C. osmophloem irregular reticulate Sinuous wide lip-shaped or butterfly-shaped eyelid-shaped
C. pauciflorum irregular reticulate Sinuous butterfly-shaped eyelid-shaped
C. pedunculatum irregular reticulate Sinuous wide lip-shaped or butterfly-shaped eyelid-shaped
C. pingbienense irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. randaiense irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. rigidissimum irregular reticulate Sinuous wide lip-shaped eyelid-shaped
#C. saxatile irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. subavenium irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. tamala irregular reticulate Sinuous wide lip-shaped or butterfly-shaped lip-shaped
C. tenuifolium irregular reticulate Sinuous wide lip-shaped or butterfly-shaped eyelid-shaped
C. tonkinense irregular reticulate Sinuous wide lip-shaped or bat-shaped eyelid-shaped
C. tsangii irregular reticulate Sinuous wide lip-shaped invisible
C. verum irregular reticulate Sinuous wide lip-shaped or narrow lip-shaped eyelid-shaped
C. wilsonii irregular reticulate Sinuous wide lip-shaped or butterfly-shaped eyelid-shaped
C. zollingeri irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. daphnoides polygonal reticulate straight/rounded butterfly-shaped invisible
C. doederleinii polygonal reticulate straight/rounded wide lip-shaped eyelid-shaped
C. pittosporoides polygonal reticulate straight/rounded wide lip-shaped globose
C. reticulatum polygonal reticulate straight/rounded bat-shaped eyelid-shaped
C. scortechinii polygonal reticulate straight/rounded butterfly-shaped globose
*Note 2 C. liangii irregular reticulate Sinuous wide lip-shaped eyelid-shaped Sect. Cinnamomum
C. litseifolium irregular reticulate Sinuous narrow lip-shaped eyelid-shaped
C. macrostemon irregular reticulate Sinuous butterfly-shaped eyelid-shaped
C. pseudopedunculatum irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. tetragonum irregular reticulate Sinuous wide lip-shaped eyelid-shaped
C. validinerve polygonal/irregular reticulate undulate/rounded wide lip-shaped eyelid-shaped
C. ovalifolium polygonal/irregular reticulate undulate/rounded wide lip-shaped eyelid-shaped
Figure 2. 

Leaf epidermal micromorphology of the Asian Cinnamomum, displaying the Type I upper leaf epidermis with polygonal cell shape, periclinal walls lacking reticulate ornamentations, and variable thickness of anticlinal walls A C. parthenoxylon B C. inunctum C C. bodinieri. Bars: 20 μm.

Micromorphology

The upper leaf epidermal micromorphology of Asian Cinnamomum species falls clearly into two types according to the reticulation of the periclinal walls of leaf upper epidermis, cell shape and straightness of anticlinal walls. Type I is characterized by polygonal epidermal cells, the anticlinal walls being straight or nearly so, the periclinal walls smooth and not reticulate (Figs 2, 4a). This type of epidermal cell is rather homogeneous. Variation occurs in the thickness of the anticlinal walls, e.g. not thickened, somewhat beaded (Fig. 2A), prominently thickened (Fig. 2B), or more or less thickened (Fig. 2C). Thickening of the anticlinal walls was based on visual perception and no measurements were made. Type II possesses epidermal cells with irregular outlines, with the anticlinal walls undulate to sinuous, and the periclinal walls reticulate (Figs 3, 4b, d). The cell shape and anticlinal walls are variable in straightness. In most species, the epidermal cells have an irregular shape, the anticlinal walls being either sinuous (Fig. 4b) or extremely sinuous, almost stellate in appearance (Fig. 4c). In a few species, the epidermal cells are polygonal and the anticlinal walls are straight or curved, e.g. C. daphnoides, C. doederleinii, C. pittosporoides, C. reticulatum, C. scortechinii, C. validinerve, and C. ovalifolium. The reticulation of the periclinal walls of the Type II results from the uneven thickening of the periclinal walls (Fig. 5A–D). In Type I, the periclinal walls are evenly thickened (Fig. 5E, F). The leaf epidermal characters such as the anticlinal wall straightness, cell shape and periclinal wall ornamentation are stable within species and not influenced by external stimuli.

Figure 3 

. Leaf epidermal micromorphology of Cinnamomum, displaying the Type II upper leaf epidermis with irregular or polygonal cell shape, reticulate periclinal walls, and straight, sinuous to extremely sinuous anticlinal walls A C. iners B C. appelianum C C. pittosporoides. Scale bars: 20 μm.

Figure 4. 

Line-drawing displaying variation of the leaf upper epidermis a type I, displaying the non-reticulate periclinal wall, the polygonal cells, and the round to polygonal cell shape b type II, displaying the sinuous anticlinal wall, the irregular cell shape, and the reticulate periclinal wall c type II, displaying an extremely sinuous anticlinal wall, the irregular cell shape, and the reticulate periclinal wall d type II, displaying the straight or nearly so anticlinal wall, the polygonal cell shape, and the reticulate periclinal wall.

Figure 5. 

A comparison between the upper leaf epidermis under scanning electron microscope and light microscope, SEM images of internal surface of the upper leaf epidermis displaying the possible origin of the reticulations of the periclinal walls A, B C. aromaticum C, D C. daphnoides E, F C. ilicioides. Scale bars: 10 μm; (A, C); 20 μm; (E); 50 μm (B, D, F).

The leaf micromorphology of Sassafras is presented in Fig. 6. The upper leaf surface is very similar to Type I of Cinnamomum, the epidermal cells being rectangular or polygonal, the anticlinal walls straight or nearly so, and the periclinal walls not reticulate (Fig. 6A–C). The stomata are elliptic in outline, with reniform subsidiary cells, or lip-shaped with narrower subsidiary cells, the subsidiary cells usually raised. The periclinal walls of epidermal cells are slightly wrinkled and immersed in S. albidum, but raised in S. tzumu and S. randaiense.

Figure 6. 

Leaf epidermal micromorphology of Sassafras displaying the non-reticulate periclinal wall in the genus A S. albidum B S. randaiense C S. tzumu. Scale bars: 20 μm (A–C).

Lower leaf epidermis comprises epidermal cells and stomata. Epidermal cells are polygonal (e.g. C. camphora, C. daphnoides, and C. glanduliferum), round (e.g. C. bodinieri, C. glanduliferum, C. migao, and C. porrectum) or irregular/amoeboid in shape (e.g. C. burmannii, C. randaiense, C. saxatile and C. subavenium). Anticlinal walls are straight and angular (e.g. C. camphora and C. glanduliferum), or round (e.g. C. bodinieri, C. glanduliferum and C. porrectum), or sinuous (e.g. C. iners and C. saxatile), thickened (e.g. C. daphnoides, C. randaiense, and C. subavenium) or not (e.g. C. camphora, C. longepaniculatum, C. migao, and C. porrectum). Periclinal walls of epidermal cells are either smooth (e.g. C. camphora, C. glanduliferum, C. longepaniculatum, C. migao, and C. porrectum) or reticulate (e.g. C. burmannii, C. iners, and C. randaiense). The lower stomatal ledges of Cinnamomum under LM include different types, e.g. wide lip-shaped (e.g. C. randaiense, Fig. 7E), narrow lip-shaped (e.g. C. migao, Fig. 7C), bat-shaped (e.g. C. longepaniculatum, Fig. 7D), butterfly-shaped (e.g. C. burmannii and C. daphnoides, Fig. 7A, B, Table 3). The wide lip-shaped and narrow li-shaped sometimes concur in a certain species (e.g. C. verum, Fig. 7F). Stomatal surfaces under SEM possess at least five different types, i.e. circular (e.g. C. chartophyllum, Fig. 8A), eyelid-shaped (e.g. C. tonkinense and C. jensenianum, Fig. 8G and 8H), globose (e.g. C. septentrionale and C. camphora, Fig. 8E and 8F), lip-shaped (e.g. C. longepaniculatum and C. migao, Fig. 8C and 8D), and invisible when the stomata are densely covered with wax/appendages (Table 3).

Figure 7. 

Lower leaf epidermis of Cinnamomum under light microscope (LM) A C. daphnoides displaying butterfly-shaped stomata B C. burmannii displaying butterfly-shaped stomata C C. migao displaying narrow lip-shaped stomata D C. longepaniculatum displaying bat-shaped stomata E C. randaiense displaying narrow lip-shaped stomata F C. verum displaying wide lip-shaped/wide lip-shaped stomata. Scale bars: 50 μm.

Figure 8. 

Lower leaf epidermis of Cinnamomum under scanning electron microscope displaying stomatal features A, B circular stomata A C. chartophyllum B C. micranthum C, D lip-shaped stomatal C C. migao D C. longepaniculatum E, F globose stomata E C. septentrionale F C. camphora G, H eyelid-shaped stomata G C. tonkinense H C. jensenianum. Scale bars: 10 μm.

Phylogeny and character evolution

Asian Cinnamomum diverged into two robust clades (BS: 100; PP: 1.00, Fig. 9), one containing the species of sect. Camphora s.s. except C. saxatile, the other including the species of sect. Cinnamomum plus C. saxatile, which was previously ascribed to sect. Camphora. This latter clade is considered as sect. Cinnamomum s.l. here. However, relationships within the two clades were not completely resolved. A number of nodes were only poorly supported, bootstrap values were less than 50 and posterior probabilities were less than 0.70.

Figure 9. 

Phylogeny of the Asian Cinnamomum incorporating ML and BI trees. Upper number of the slash refers to the bootstrap value of the ML tree and the lower number of the slash refers to the posterior probabilities of the BI tree.

When simply mapping on the phylogenetic tree, the two character states of the periclinal wall reticulation allowed clear separation into two clades: non-reticulate for sect. Camphora s.s. and reticulate for sect. Cinnamomum s.l.; there is no overlap. However, neither epidermal cell shape nor anticlinal wall straightness are clear-cut. Sect. Cinnamomum s.l. usually possess sinuous anticlinal walls and irregular cell shapes, but a few species with straight or curved anticlinal walls and polygonal cell shapes were found to belong to this clade, e.g. C. reticulatum, C. doederleinii, C. daphnoides, and C. pittosporoides. Straight or curved anticlinal walls and polygonal cell shapes were common in sect. Camphora s.s., and we found no exception. For evolutionary history of periclinal wall reticulation (Fig. 10), the ancestral node A of sect. Cinnamomum s.l. was reticulate with high probability (95.18%), and the ancestral node B of sect. Camphora s.s. was non-reticulate with very high likelihood (99.99%). Anticlinal wall straightness and epidermal cell shape resulted in the same reconstruction (Fig. 11), the ancestral node of sect. Camphora s.s. possessed straight or curved anticlinal walls (node B), and polygonal cell shapes (99.17%), while it was uncertain whether the ancestral node of sect. Cinnamomum s.l. had sinuous anticlinal walls and irregular cell shapes or not, the probability being only 56.54% (node A).

Figure 10. 

Ancestral character reconstruction of the periclinal wall reticulation by applying a ML tree block in Mesquite with a maximum likelihood approach and MK1 model. The common ancestor of Node A possesses reticulate periclinal wall with high likelihood (95.18%), and the ancestral Node B is reticulate with high likelihood (99.99%).

Discussion

In this study, leaf epidermal micromorphology of 48 species representing the two macromorphological sections of Asian Cinnamomum was studied. Our sampled species largely overlapped with the species sampling of the two recent molecular phylogenetic studies (Huang et al. 2016; Rohde et al. 2017), permitting an assessment of the systematic significance of leaf epidermal micromorphology within a phylogenetic context.

The polygonal to irregular epidermal cell shape and the straight to sinuous anticlinal walls have been described in previous reports (e.g. Christophel et al. 1996; Nishida and Christophel 1999; Nishida and van der Werff 2007, 2011; Yang et al. 2012; Trofimov and Rohwer 2018), but our study suggests that sect. Cinnamomum s.l. possesses an unusual reticulate periclinal wall which has not been studied carefully before in Lauraceae. We studied the reticulate periclinal wall under SEM, and hypothesize that uneven thickening of the periclinal wall gives rise to the reticulation under LM (Fig. 5). The reticulations of the periclinal wall are usually coarse in sect. Cinnamomum s.l., the spaces dividing the periclinal wall into reticulations are narrow. The reticulations in sect. Cinnamomum s.l. are rarely fine and appear to be ‘punctate’, e.g. in C. iners and C. japonicum, which is similar to that of a few species of Beilschmiedia Nees (Nishida and van der Werff 2007), where the spaces are wide. We prefer to describe the unusual periclinal wall in sect. Cinnamomum as reticulate but not punctate because they appear to be coarse but not dot-like.

Asian Cinnamomum species are classified into two sections according to the persistence of tepals, presence of perulate buds, leaf arrangement either alternate or subopposite, and leaf venation, i.e. sect. Camphora s.l. and sect. Cinnamomum s.s. (syn.: sect. Malabathrum Meisn. (1864: 10)). This classification was proposed by Meisner (1864) and followed by subsequent authors (e.g. Li et al. 1982). A recent phylogeny based on three nuclear sequences (ITS, RPB2 and LEAFY) suggests that a few taxa placed in sect. Camphora based on macromorphological characters actually belong to the clade consisting mainly of sect. Cinnamomum s.l., namely C. saxatile, C. longipetiolatum and an unidentified sample (Huang et al. 2016). Sect. Camphora s.s. is characterized by alternate, pinnately veined or weakly tripliveined leaves, mostly perulate buds and presence of domatia in the axils of lateral veins. However, these features occur also in sect. Cinnamomum s.l. when the clade includes C. saxatile and C. longipetiolatum. As a result, the current definition of both sect. Cinnamomum s.l. and Camphora s.s. using presence or absence of these morphological characters is problematic.

Our study suggests that the leaf epidermal micromorphology can be divided into two different types and the two types of leaf epidermal micromorphology are surprisingly congruent with the clades retrieved in the analysis of Huang et al. (2016): the taxa of sect. Camphora s.s. possess the smooth upper epidermis, while those of sect. Cinnamomum have the reticulate upper epidermis. The reticulate type of periclinal walls is derived, because this type has not been found in any other groups of the family (see Christophel et al. 1996; Nishida and Christophel 1999; Nishida and van der Werff 2007, 2011, 2014; Yang et al. 2012; Zeng et al. 2014; Nishida et al. 2016). In the character reconstruction, the ancestral node of the Asian Cinnamomum possesses a non-reticulate type of periclinal walls with high probability (95.34%). The two types of periclinal walls are clade-specific (Fig. 10), and the reticulate type appears to have originated in the ancestor of sect. Cinnamomum s.l. The reticulate type is shared by sect. Cinnamomum s.l. and its ancestor with a probability of 95.18%, and the non-reticulate type is shared by sect. Camphora s.s. and its ancestor with a probability of 100%. We consider that the reticulate type of periclinal walls is a synapomorphy of sect. Cinnamomum s.l., and is useful in classification of the two clades.

Both leaf epidermal cell shape and the straightness of anticlinal walls are not clade specific and transitional between the two groups/clades of Asian Cinnamomum (Fig. 11). In sect. Cinnamomum s.l., a few species possess polygonal epidermal cell shape and straight/curved anticlinal walls, which are common in sect. Camphora s.s., e.g. C. daphnoides, C. doederleinii, C. pittosporoides, C. reticulatum, and C. scortechinii. These species possess opposite triveined/tripliveined leaves. A few other species were not examined in phylogenetic studies, but they too possess reticulate periclinal walls, and opposite/subopposite, triveined/tripliveined leaves lacking domatia, viz. C. liangii, C. litseifolium, C. macrostemon, C. pseudopedunculatum, C. tetragonum, C. validinerve, and C. ovalifolium. We thus expect them to belong to sect. Cinnamomum s.s. Another two species examined here, C. ilicioides and C. migao possess non-reticulate periclinal walls, polygonal cell shape, straight/rounded anticlinal walls, perulate buds, and alternate and penninerved leaves which bear inconspicuous domatia in axils of lateral veins, so they clearly belong to sect. Camphora s.s.

Figure 11. 

Ancestral character reconstruction of the epidermal cell shape and the straightness of anticlinal wall by applying a ML tree block in Mesquite with a maximum likelihood approach and MK1 model. Node A: the ancestral node of sect. Cinnamomum s.l. had sinuous anticlinal walls and irregular cell shapes or not, the probability being only 56.54%; Node B: the ancestral node of sect. Camphora s.s. possessed straight or curved anticlinal walls and polygonal cell shapes, the probability being 99.17%.

Phylogenetic relationships of Sassafras have not been resolved. Rohde et al. (2017) gave conflicting phylogenetic results on Sassafras based on nrDNA and cpDNA sequences. The phylogeny based on nrITS indicates that Sassafras is sister to the Cinnamomum+Aiouea+Ocotea complex, nevertheless, the phylogeny based on cpDNA suggests that Sassafras forms a clade together with two species of sect. Camphora, i.e. C. bodinieri and C. glanduliferum, making the sect. Camphora polyphyletic. Trofimov and Rohwer (2020) indicated that Sassafras is sister to sect. Camphora based on nrITS and psbA-trnH. Liu et al. (2021) reported conflicts between nuclear and plastid phylogenetic results. In their analysis, Sassafras is either sister to sect. Camphora (nrDNA phylogeny) or to a clade consisting of C. caudiferum and C. porrectum (plastome phylogeny), but their result on Sassafras is not conclusive due to poor sampling of Cinnamomum. Our leaf anatomy indicates that Sassafras does possess Type I upper leaf epidermis as in sect. Camphora (and most other Lauraceae), i.e. polygonal epidermal cells, straight anticlinal walls, and non-reticulate periclinal walls.

The genus Cinnamomum was formerly considered to be amphi-Pacific (Rohwer 1993; Lorea-Hernandez 1996; van der Werff 2001), but a recent phylogenetic study (Rohde et al. 2017) suggested that the American species are closer to the likewise predominantly American Ocotea complex than to Asian Cinnamomum; they have now been accommodated in Aiouea (Rohde et al. 2017). The Old World Cinnamomum is thus a diphyletic group, and includes two clades (Huang et al. 2016; Rohde et al. 2017, and this study). Sect. Cinnamomum appears to be sister to the Neotropical clade in Huang et al. (2016) but it is the sect. Camphora that appears to be sister to the Neotropical clade in nrITS analysis of Rohde et al. (2017). Whichever is correct, the Asian Cinnamomum is not a monophyletic group and should be further subdivided into two genera. Our new study clearly suggests that use of leaf epidermal micromorphological characters leads to the recognition of two distinct groups that are clade-specific and highly predictive. We thus provide micromorphological support to classify the Asian Cinnamomum into two genera.

Acknowledgements

The authors thank D.J. Mabberley and H. van der Werff for their instructive suggestions on the manuscript. Thanks are also due to F. Alves, S. Nishida and an anonymous reviewer for their valuable suggestions. This work was supported by the National Natural Science Foundation of China [31770211, 31470301, 31270238] and the Metasequoia funding of Nanjing Forestry University to YY.

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