Bark and Cambial Variation in the Genus Clematis (Ranunculaceae) in Taiwan

Background Studies on the anatomical characteristics of stems of Taiwanese species from the Clematis genus (Ranunculaceae) are scarce. The aim of this study was to investigate and compare cambial variation in stems of 22 Clematis species. The rhytidome (outer bark) was either cogwheel-like or continuous, except for in the species Clematis tashiroi. Key features of the genus were eccentric to elliptical or polygonous-lobed stems, wedge-like phloem, wedge-like rays, indentations in the axial parenchyma, and ray dilatation. The cortical sclerenchyma bers were embedded in the phloem rays with approximately 23% of the Clematis species. Both C. psilandra and C. tsugetorum had restricted vessels. There were three vascular bundle patterns, with approximately 27% of the Clematis species in Taiwan having 12 vascular bundles. The vessels dispersed throughout the stem were semi-ring-porous in most species, but were ring-porous in others. No species had diffuse-porous vessels. Only two species had a primary xylem ring located around the pith. Secondary xylem rays split the secondary xylem into parts, increasing stem diameter. The developmental stage of each sample was determined, with the initial ring-like periderm being produced in the primary phloem during the second stage. The cambial variations described in this study provide a foundation for further morphological studies of the Clematis genus.


Abstract
Background Studies on the anatomical characteristics of stems of Taiwanese species from the Clematis genus (Ranunculaceae) are scarce. The aim of this study was to investigate and compare cambial variation in stems of 22 Clematis species.

Results
The rhytidome (outer bark) was either cogwheel-like or continuous, except for in the species Clematis tashiroi. Key features of the genus were eccentric to elliptical or polygonous-lobed stems, wedge-like phloem, wedge-like rays, indentations in the axial parenchyma, and ray dilatation. The cortical sclerenchyma bers were embedded in the phloem rays with approximately 23% of the Clematis species. Both C. psilandra and C. tsugetorum had restricted vessels. There were three vascular bundle patterns, with approximately 27% of the Clematis species in Taiwan having 12 vascular bundles. The vessels dispersed throughout the stem were semi-ring-porous in most species, but were ring-porous in others. No species had diffuse-porous vessels. Only two species had a primary xylem ring located around the pith. Secondary xylem rays split the secondary xylem into parts, increasing stem diameter. The developmental stage of each sample was determined, with the initial ring-like periderm being produced in the primary phloem during the second stage.

Conclusions
The cambial variations described in this study provide a foundation for further morphological studies of the Clematis genus.

Background
The vascular cambium of climbing plants produces xylem and phloem under normal conditions, and both types of tissue have large amounts of parenchyma cells. All parenchyma cells in climbing plants can be redifferentiated into meristematic cells, which may give rise to vascular bundles, cork cambia, dilatation tissue, or adventitious buds (Mauseth 1988). Due to the uneven deposition of secondary xylem, stems are generally irregularly shaped after secondary growth (Carlquist 1991a;Rajput et al. 2014) as diverse cambial variants are formed. One cambial variant consists of xylem parts that are separated by wider rays (the 'xylem in plate' variant). Many families develop this variant, including Ranunculaceae (Carlquist 2001).
The Ranunculaceae family comprises approximately 60 genera and 2500 species, including approximately 300 Clematis species worldwide (Wang and Bartholomew 2007), 22 of which are found in Taiwan (Yang and Huang 1996). The life forms of Clematis species include shrubs, herbs, and perennial climbers (lianas). Within the Ranunculaceae, several cambial variants have been reported (Angyalossy et al. , 2015Rajput and Gondaliya 2017;.
Previous anatomical studies of Ranunculaceae have mainly concentrated on the genus Clematis (Gregory 1994;Smith 1928;Carlquist 1995;Sieber and Kucera 1980). The axial parenchyma of C. alpina, C. columbiana, C. hirsutissima, and C. recta are paratracheal with semi-ring-porous wood and distinct annual rings, while C. ammula, C. vitalba, and C. viticella have ring-porous wood with annual rings (Schweingruber et al. 2011). Secondary xylem rays are initiated in C. ammula, C. hirsutissima, and C. viticella (Isnard et al. 2003b;Schweingruber et al. 2011). Interfascicular cambia ray width increases with increasing stem diameter in C. alpina and C. viticella (Isnard et al. 2003b), and rays are wedge-like in shape (Schweingruber et al. 2011). In C. alpina and C. vitalba, the phellogen produces cork cells and the phellem (cork) layers outside the phloem form rhytidome (outer bark), which is composed of cork and dead phloem (Sieber and Kucera 1980). In C. vitalba, 12 vascular bundles are divided into two types in the primary state, the pith cavity forms, secondary phloem are composed of parenchyma cells, an arc of sclerenchyma phloem bers develop, and rhytidome appears in a cogwheel-like form (Sieber and Kucera 1980). In C.vitalba, the cambium is dented towards the pith in the region of the broad rays (Sieber and Kucera 1980), and in C. alpina and C. pickerringii the fascicular areas always have indentations (Carlquist 1995). The indentations are strands of thin-walled axial parenchyma near the broader rays. Isnard et al. (2003aIsnard et al. ( , 2003b de ned four developmental stages of C. ammula var. maritima, C. recta, and C. vitalba by the appearance of cambial characteristics, such as the initial periderms and phloem bers. The xylem in plate variant is one of the cambial variants found in the Ranunculaceae (Yang and Chen 2015), but other stem characteristics of the Clematis genus in Taiwan have not been described. As cambial variations constitute an extremely diverse morphology, the present study aimed to 1) provide detailed photographs of the features discussed and 2) provide a bracketed key based on the anatomical characteristics of the stems to facilitate the identi cation of irregular cambial activity in the Ranunculaceae family in Taiwan.

Research Materials
Multiple samples of 22 Clematis species of the family Ranunculaceae recorded in the Flora of Taiwan (Yang and Huang 1996) were collected. The habits of these species included annual and perennial herbs, shrubs, and lianas growing in different forests. The dataset included species scienti c name, collector, herbarium and voucher number, and collection locality. The voucher species information of all Clematis species is presented in Table 1. Approximately 48% of the species were endemic to Taiwan. Among them, C. psilandra and C. tsugetorum are shrubs growing at high elevations of approximately 2,000 m and 3,500 m, respectively. The remaining 20 species are climbing vines in which the species C. montana grows at the highest elevation, approximately 3,600 m. Clematis pseudootophora is a herb and rarity in the eld. This species consists of only a few populations located at an elevation of 1,500-1,900 m, in eastern Taiwan.
Multiple samples of each species were collected, and stems with thick bark and visible secondary growth characteristics were selected in the eld. To keep the material fresh and retain humidity, the stems were stored in a collecting bag. Different diameters of each plant were collected to compare various developmental stages and to accurately assess the position of various vascular bundle tissues. One or two samples with obvious and easy-to-observe cambial variations were selected per species for photographs and the scoring of morphological characteristics. Cambial variations in the investigated species were used to construct a comparison table.
In the laboratory, the fresh materials were cut into pieces approximately 5 cm long, and a freehand cross-section of each stem was made with a razor blade.
The stem cross-section was immediately photographed using a Nikon D7100 SLR digital camera with a 1:1 lens (Lens AF Micro Nikon 60 mm 1:2.8D; Nikon Corporation, Tokyo, Japan). Cambial characteristics were measured and described. Quantitative anatomical traits, such as stem diameter, bark thickness, mean xylem width, and mean primary ray width were determined using Image-J software (Ferreira and Rasband 2011). All specimens were oven-dried at 60 °C for 4-5 days and then stored at −20 °C for 3-4 days. They were then deposited in the Provincial Pingtung Institute (PPI) herbarium at the National Pingtung University of Science and Technology, Pingtung, Taiwan, for subsequent identi cation. The nomenclature follows the Flora of Taiwan volume II (Yang and Huang 1996). The materials of C. pseudootophora were permanently preserved in 75% aqueous alcohol because of its rarity.
The ontogenetic stage of all 22 Clematis species was determined based on Isnard et al. (2003a, b). During the rst ontogenetic stage, stems have an epidermis, a cortex, a vascular cylinder ring, and dense or strand bundles of primary phloem bers. During the second stage, stems have an initial periderm that is linked with dead and collapsed cortical parenchyma, and secondary phloem bers formed from the vascular cambium. In the third stage, sequent periderms develop, which initiate within the secondary phloem; the cortex and primary phloem are detached into a continuous bark segment or into cogwheellike bark; and a wider ray and many larger vessels are formed. In the last stage, sequent periderm is produced in progressively deeper layers in the secondary phloem, periderms are detached after forming decorticated tissue (rhytidome), and secondary xylem rays are formed.

Bark Morphologies
The anatomical and morphological details of stems for the 22 species investigated in this study are listed in Tables 2 and 3 Table 2). The bark of C. tashiroi was deep green in color and glabrous without any rhytidome in spite of large stem sizes (4.0-17.8 mm), and its stem cross-section was hexagonous. Rhytidome is comprised of successive cork and dead phloem to form dead outer bark. According to the arrangement and detachment degree of rhytidome, it can be divided into two forms: cogwheel-like rhytidome (ring bark) and continuous segment rhytidome (scale bark) (Esau 1958;Sieber and Kucera 1980;Evert 2006). The rhytidome did not last long on the bark in the 22 Clematis species. Nineteen species had wrinkled rhytidome; among them, 11 species had continuous segment rhytidome and eight species had cogwheel-like rhytidome, which peeled and teared easily. The rhytidome of C. crassifolia ( Fig. 2B) was continuous segment and was the thickest (1. 8-3.4 mm). That of C. lasiandra ( Fig. 3E) was cogwheel-like and 0.5-2.7 mm in thickness. The remaining species were thinner than the above two species.

Cambial Variant Types
The stems of the 22 species investigated were shallowly grooved or angulated, and the stems were round (Fig. 1A, B) or hexagon and deeply grooved in shape (Figs. 2F, 6A, 6D). Only C. henryi var. morii (Fig. 3F) had an irregular conformation, forming a deeply polygonous lobe. The stem of C. formosana (Fig. 2D) was eccentric to oval or elliptical at the last stage. The Clematis stems examined generally developed axial vascular elements in segments, and the xylem were separated by wider rays, forming the xylem in plate type. This type is derived from a single cambium according to Angyalossy et al. (2012). Except for the xylem in plate type, C. gouriana subsp. lishanensis (Figs. 2D) formed discontinuous wedge-like phloem.

Variation in Vascular Elements
The secondary rays were always linear, but that of C. akoensis (Fig. 1B), C. grata (Fig. 3C), C. henryi var. henryi (Fig. 3D), C. henryi var. morii, C. pseudootophora (Fig. 6A), and C. tsugetorum (Fig. 8B) were wedge-like ( Table 2, RWL column). The interfascicular cambia made the rays of C. alpina wider and wedge-like (Schweingruber et al. 2011); this character was apparent in six of the Clematis species studied. The wedge-like ray form could be referred from the average width of the primary rays. For example, C. henryi var. henryi and C. grata had the widest primary rays, with a maximum width of 668 µm and 642 µm, respectively.
Owing to the presence of thin-walled axial parenchyma in latewood adjacent to the thin-walled ray cells, wedge-shaped indentations can be seen in the interfascicular region (Carlquist 1995). The fascicular areas of stem cross-sections of Clematis species were investigated. There was an obvious indentation in the region of the wider rays (Fig. 5B), except in C. akoensis (Fig. 1B), C. chinensis var. tatushanensis (Fig. 1E), C. montana (Fig. 5C), C. psilandra (Fig. 6C), and C. tsugetorum (Fig. 8B). This is because the stem diameters of these species were too small to develop wider rays. The cortical sclerenchyma bers of ve species, C. chinensis var. chinensis (Fig. 1C, D), C. grata (Fig. 3C), C. lasiandra (Fig. 4B), C. uncinata var. okinawensis (Fig. 8C, D), and C. uncinata var. uncinata (Fig. 8E, F), were connected with the plate of sclerenchyma bers that were embedded in the phloem rays and formed an arc outside the fascicular regions.
The axial parenchyma of C. psilandra (Fig. 5E) and C. tsugetorum (Fig. 8B) were very scarce, and vessel distribution was limited to the central portions of the fascicular xylem area with growth rings. The vessels of the remaining 20 species were distributed along the edge of the fascicular areas.
Species were grouped based on the number of vascular bundles they contained, with the 'central type' having 12 bundles, the 'many type' having >12 bundles, and the 'few type' having <12 bundles (Smith 1928). In this study, the number of vascular bundles observed in Clematis species ranged from 6-21. Among them, three species were classi ed as few type (14%), 13 species were classi ed as many type (59%), six species were classi ed as central type (27%) ( Table  2, VB column). Only one species, C. pseudootophora (Fig. 6A), had six vascular bundles ( Table 2).

Developmental Stages
Based on the characteristics observed in stem cross-sections of 50 samples from 22 species, samples were divided into four ontogenetic stages ( Table 2). If multiple samples were collected of the same species (which was the case for 16 species), each sample was investigated separately. Therefore, these species were assigned 2-3 developmental stages.
A key to the 22 Clematis species in Taiwan, based on the characteristics of bark and vascular bundles, is provided below. in the phloem rays formed an arc, which is characteristic of Lardizabalaceae and Sabiaceae (Carlquist 1984(Carlquist , 1991bYang et al. 2019). This feature has not been previously reported in the Ranunculaceae family.
Clematis psilandra (Fig. 6C) and C. tsugetorum (Fig. 8B) had few axial parenchyma and had vessel restriction. They also had growth in a different location than the other Clematis species studied, but similar to that of Xanthorhiza apiifolia (Carlquist 1995), which grows in temperate regions. Xanthorhiza is a primitive genus according to its vessel restriction pattern (Carlquist 1995). This pattern was also found in C. psilandra and C. tsugetorum, suggesting that they might be more primitive than the other Taiwanese Clematis species.
Clematis species often have 12 vascular bundles, with Smith (1928) nding this feature in 67% of 138 species studied. Therefore, this is generally considered the central type for this genus. Conversely, in Taiwan, only 27% of the 22 Clematis species had 12 vascular bundles. Five of the species in our study had inconsistent numbers of vascular bundles compared with the ndings of Smith (1928). Clematis henryi var. henryi (Fig. 3D) had ten vascular bundles in our study, but only six were recorded by Smith (1928). Moreover, C. chinensis var. chinensis (Fig. 1C, D) had 20 bundles in our study, C. lasiandra (Fig. 4B) had 14, C. meyeniana (Fig. 5B) had 21, and both C. uncinata var. okinawensis (Fig. 8C, D) and C. uncinata var. uncinata (Fig. 8E, F) had 12-14. Conversely, Smith (1928) recorded only 12 vascular bundles in these species. The inconsistent results might be due to different sample sizes or environmental factors; further investigation is required.
Vascular bundle numbers are usually constant for a given species, but in some species the numbers can vary. We examined multiple samples from C. leschenaultiana (Fig. 4C, 4D-F, 5A), C. terni ora var. garanbiensis (Fig. 7E, F, 8A), C. uncinata var. uncinata (Fig. 8E, F), and C. uncinata var. okinawensis (Fig.  8C, D) and found that the number of vascular bundles varied between growth stages. This might be related to the differentiation of meristematic cells or the action of interfascicular cambium. To con rm this, future work should focus on collecting and observing more specimens from different stem positions of the same species.
Clematis henryi is taxonomically treated as C. henryi var. henryi (Fig. 3D) and C. henryi var. morii (Fig. 3F) (Yang and Huang 1996), but C. henryi var. morii is sometime treated as a species, C. morii (Wang and Bartholomew 2007). In this study, these two taxa had wedge-like rays and continuous segment of rhytidome, but the stem of C. henryi var. henryi was round while that of C. henryi var. morii was a deeply polygonous lobe. These stem shape characteristics provide a way to distinguish these two taxa. The vessel arrangement of the Clematis species in this study was semi-ring-porous in almost all species, with no species having diffuse-porous vessels, which is consistent with previous reports (Schweingruber et al. 2011).
In the present study, one to three secondary xylem rays were observed in some Clematis species. This has been recorded in the species Aristolochia macrophylla (Aristolochiaceae) (Carlquist 1993;Schweingruber et al. 2011), in the Cucurbitaceae (Carlquist 1992), and in the genus Cyclea (Menispermaceae) (Yang and Chen 2016). According to Carlquist (1995), secondary xylem rays are new rays that originate abruptly and are relative to the vine habit. Secondary xylem rays split the secondary xylem into two or more parts and the amount of bark increases as some of the splits are near the secondary phloem. Secondary xylem rays increased stem diameter and signi cantly increased the amount of bark. However, secondary xylem rays were not found in all of the species investigated in our study. The presence of secondary xylem rays might be related to stem diameter size, but this needs further investigation.
The pith cavity in C. vitalba results from non-ligni ed walls in the inner-most pith parenchyma cells (Sieber and Kucera 1980). However, we only observed this characteristic in C. uncinata var. uncinata (Fig. 8E, F); the pith cavity formed a hexagon shape around the primary xylem ring. Isnard et al. (2003a, b) used stem size to de ne four ontogenetic stages of three Clematis species. However, in this study we failed to observe the different developmental stages of each species due to low abundance, limited localities, and small stem diameters in some species. Further work collecting specimens and examining the characteristics of different developmental stages of these species is needed, especially species that only contain inner bark. However, the diagnostic features, such as the rhytidome, wedge-like phloem, xylem in plate type, restricted vessel pattern, indentations, and secondary phloem bers, could be used to identify the species in the Clematis genus. The characteristics of the 22 Clematis species described here and provide evidence for systematic problems within this genus.

Conclusions
The bark and cambial variations in the Ranunculaceae family are diverse. The habits of Taiwanese Clematis species include two shrubs and twenty vines, in which three species grow at high altitudes ranging from 2,000 m to 3,600 m. Our results showed that the rhytidome (outer bark) is a key diagnostic characteristic for Clematis species identi cation, and can either take the form of continuous segments bark or cogwheel-like bark. Among the 22 Taiwanese Clematis species, C. tashiroi was the only species that did not form rhytidome. Three cambial variants of the Clematis genus were found-stems with polygonous lobes, wedge-like phloem, and the xylem in plate type. Most Clematis species had ray dilatation and indentation of the axial parenchyma near the wider rays. The cortical sclerenchyma bers embedded in the phloem rays and numbers and sizes of vascular bundles varied among the Clematis species. The vessels of C. psilandra and C. tsugetorum were restricted to the central portions of the fascicular xylem, which was different from the other Clematis species. The xylem vessels dispersed throughout the stem were mostly semi-ring-porous, but a few were ring-porous with annual rings. No diffuse-porous stems were observed. The secondary xylem rays split the vascular elements into different segments, increasing stem diameters. Further collection of fresh materials and observations of different developmental stages are still needed. Interestingly, we found unusual wood features, such as indentation near the wider rays, vessel restriction, and the presence of secondary xylem rays. In conclusion, bark and cambial variations could facilitate future studies addressing Clematis taxonomy.