The stem anatomy of the Clematis species (Ranunculaceae) in Taiwan

Background Studies on the stem anatomical characteristics of Taiwanese species from the Clematis genus (Ranunculaceae) are scarce. This study aimed to investigate and compare the patterns of secondary growth in stems of 22 Clematis species. The rhytidome is composed of periderm and non-conducting phloem and formed either cogwheel-like or continuous segment bark. Key features of the genus were stem with an irregular conformation, wedge-like phloem and rays, indentations in the axial parenchyma, ray dilatation, and narrow rays. Approximately eight Clematis species formed bark arc shape, which developed the cogwheel- like rhytidome. There were 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. The vessel restriction pattern was only found in the two shrubs, C. psilandra and C. tsugetorum. The primary xylem ring was located around the pith in C. uncinata var. uncinata, making its pith cavity hexagon in shape. Four species had the pith cavity feature. Narrow rays that occurred in the secondary xylem increased with increasing stem diameter. The objectives of this study were to understand the stem anatomy of the 22 Taiwanese Clematis species in the Ranunculaceae family. Our results showed that due to successive layers of periderm combined with non-conducting phloem, the cogwheel-like and continuous segments rhytidome were formed in most Clematis species. Three cambial variants of the Clematis genus were found—stems with an irregular conformation, wedge-like phloem, and xylem in plate. Most Clematis species had the indentation of the axial parenchyma near the wider rays, ray dilatation, and varied in the numbers and sizes of vascular bundles. The vessel restriction pattern was found in two shrubs, C. psilandra and C. tsugetorum, which were different from the other vine Clematis species. The narrow rays occurred in some secondary xylem, and no diffuse-porous vessels dispersed throughout the stems. Further fresh materials collection and observations of different developmental stages are still needed. In conclusion, the data presented here provide important basic information on lianas addressing Clematis taxonomy to understand their morphology and ensure the conservation of their diversity.


Abstract
Background Studies on the stem anatomical characteristics of Taiwanese species from the Clematis genus (Ranunculaceae) are scarce. This study aimed to investigate and compare the patterns of secondary growth in stems of 22 Clematis species.

Results
The rhytidome is composed of periderm and non-conducting phloem and formed either cogwheel-like or continuous segment bark. Key features of the genus were stem with an irregular conformation, wedge-like phloem and rays, indentations in the axial parenchyma, ray dilatation, and narrow rays. Approximately eight Clematis species formed bark arc shape, which developed the cogwheel-like rhytidome. There were 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. The vessel restriction pattern was only found in the two shrubs, C. psilandra and C. tsugetorum. The primary xylem ring was located around the pith in C. uncinata var. uncinata, making its pith cavity hexagon in shape. Four species had the pith cavity feature. Narrow rays that occurred in the secondary xylem increased with increasing stem diameter.

Conclusions
The cambial variants 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. These cells can be re-differentiated into meristematic cells, which may give rise to secondary vascular tissue, 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 1991;Rajput et al. 2014) as diverse cambial variants, or secondary growth patterns are formed. The stems examined generally developed axial vascular elements in segments separating by the wider xylem and phloem rays, forming xylem in plate type. This type is derived from a single cambium, according to Angyalossy et al. (2012Angyalossy et al. ( , 2015. 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), of which 22 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. 2012(Angyalossy et al. , 2015. Previous anatomical studies of Ranunculaceae have mainly concentrated on the genus Clematis (Smith 1928;Sieber and Kucera 1980;Gregory 1994;Carlquist 1995). Few secondary rays begin abruptly and occur in C. cirrbosa, C. ammula, C. montevidensis, C. vitalba, and C. viticella (Schweingruber et al. 2011) with increasing the stem diameter. Carlquist (1995) indicated that new rays are wide multiseriate from their origin, and are initiated abruptly rather than as narrow rays that gradually increase in width in Clematis genus. Beck (2011) described that closely spaced, narrow rays occurred in groups will simulate large rays which terms as aggregate rays. The secondary or new rays also occur in woody climbers in two families, Cucurbitaceae (Carlquist 1992) and Aristolochiaceae (Carlquist 1993). The cambium in the secondary vascular tissue forms increasing ray-like parenchyma in Clematis ammula var. maritima (Isnard et al. 2003a). Interfascicular cambia ray width increases with increasing stem diameter in C. alpina subsp. alpina, that the rays are wedge-like in shape (Schweingruber et al. 2011).
The phellogen of C. alpina and C. vitalba produces cork cells and forms the phellem (cork) layers is located outside the secondary phloem (Schweingruber et al. 2011). The complex tissue region of the periderm and enclosed non-conducting phloem is called the rhytidome (Beck, 2011), which is generally comprised of successive cork and non-conducting phloem. According to the arrangement and detachment degree of rhytidome, two types were formed: cogwheel-like rhytidome (ring bark) and continuous segment rhytidome (scale bark) (Esau 1958;Sieber and Kucera 1980;Evert 2006). C. vitalba is a vine with big and small vascular bundles (six each) in its primary state. It forms the pith cavity, its bark tissue develops an arc, and the stem appears in a cogwheel-like form due to the different activities in the fascicular and interfascicular regions (Sieber and Kucera 1980). In C. vitalba, the cambium is dented towards the pith in the broad rays region (Sieber and Kucera 1980), and in C. alpina and C. pickerringii, the interfascicular areas always have indentations (Carlquist 1995). 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 (Sieber and Kucera 1980;Carlquist 1995). However, the indentations are strands of thinwalled axial parenchyma near the broader rays. Isnard et al. (2003a, b) de ned four developmental stages of C. ammula var. maritima, C. recta, and C. vitalba by the appearance of the periderms, phloem bers, rhytidome, and wide rays. 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).
The xylem in plate variant was found in the genus Clematis (Carlquist 1995, Isnard et al. 2003b, Yang and Chen 2015, but the information about the patterns of secondary growth or cambial variants in Taiwan 22 Clematis species were scarce. As cambial variants 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 Clematis genus in Taiwan.

Research Materials
Multiple samples of 22 Clematis species of the family Ranunculaceae 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. The voucher species information of all Clematis species is presented in Table 1. Approximately 48% of the species were endemic to Taiwan. Among them, Clematis psilandra and C. tsugetorum are shrubs growing at high elevations of approximately 2,300 m and 3,200 m, respectively. The species C. pseudootophora is an herb rarely found in the eld. The remaining 19 species are climbing vines in which C. montana grows at the highest elevation of approximately 3,200 m. This species consists of only a few populations located at an elevation of 1,500-1,900 m in eastern Taiwan.

Research Methods
Multiple samples of each species were collected, stems with thick bark and secondary growth characteristics were selected in the eld. To keep the materials 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 accurately assess the position of various vascular bundle tissues. One or three samples with obvious and easy-to-observe cambial variants were selected per species for photographs and the scoring of morphological characteristics. The morphological features of stem cross-sections 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 The ontogenetic stage of all 22 Clematis species was determined based on two lianas and one herb reported in Isnard et al. (2003a, b). During the rst ontogenetic stage, stems have a cortex and a vascular cylinder, collenchyma and primary sclerenchyma bers (primary phloem bers) as a continuous ring or dense bundles in liana, but as separate bundle sheaths in herb. The primary phloem bers are located between the collenchyma and secondary phloem.
During the second stage, the stem has secondary tissue and an initial periderm (Esau 1958) formed beneath the primary phloem in liana, but cortex suberisation and parenchyma ligni ed in herb. Secondary phloem bers are produced along the secondary phloem. In the third stage, sequent periderms develop within the secondary phloem; the cortex and primary phloem are detached into a ringbark or a continuous segment (Esau 1958); possess a wide ray and forms many larger vessels; secondary phloem bers increases. In the last stage, sequent periderm is produced in progressively deeper layers in the secondary phloem; periderms are detached after forming decorticated tissues.

Variation in Vascular Elements
There was an obvious indentation in the region of the wider rays, except in C. chinensis var. tatushanensis (Fig. 1E), C. montana (Fig. 4E), C. psilandra (Fig. 5D), and C. tsugetorum (Fig. 7B). Perhaps, this is because the stem diameters of C. chinensis var. tatushanensis, C. montana were too small to develop wider rays. The wedge-shaped indentations developed in the interfascicular region were only found in C. henryi var. morii due to its thin-walled axial parenchyma adjacent to the thin-walled ray cells (Sieber and Kucera 1980). The axial parenchyma of C. psilandra and C. tsugetorum were very scarce, and vessel distribution was limited to the central portions of the fascicular area with growth rings. Almost all Clematis species in this study had semi-ring-porous vessels, except for C. crassifolia ( Fig. 2A) and C. lasiandra (Fig. 3F), which had ring-porous vessels with distinct annual rings. None of the species had diffuseporous vessels, which is consistent with previous reports (Schweingruber et al. 2011).
In this study, the number of vascular bundles observed in Clematis species ranged from 6-21. Of which, three were classi ed as few type (14%), 13 as many type (59%), and six as central type (27%) ( Table 2, VB column). Only one species, C. pseudootophora (Fig. 5B), had six vascular bundles.

Developmental Stages
Based on the characteristics observed in stem cross-sections of 42 samples from the 22 species, stem developments were divided into four ontogenetic stages ( Table 2,  Rhytidomes were not observed in three samples of C. tashiroi, and its epidermis was deep green in color and glabrous; the stem cross-section was hexagon in shape. However, according to the stem features, three developmental stages of C. tashiroi can be divided. In the rst stage, the stem had primary phloem bers as strands along the secondary phloem (Figs. 6B). In the second stage, secondary phloem became triangular with secondary phloem bers, and a wider ray and many larger vessels were formed (Figs. 6C). In the third stage, rays became unequal in width, and narrow rays were formed (Figs. 6D).
Bracket key to 22 clematis species A key to the 22 Clematis species in Taiwan, based on the characteristics of bark and vascular bundles, is provided below.
The stem cross-section of C. henryi var. morii and C. formosana were a lobe with asymmetrical conformation and eccentric to oval or elliptical, respectively. This generates as an irregular conformation, found in some Apocynaceae and Malpighiaceae (Angyalossy et al. 2015). Clematis gouriana subsp. lishanensis forms discontinuous wedge-like phloem, a common characteristic of the family Bignoniaceae (Pace et al. 2011). In summary, except for xylem in plate variant, two cambial variants are found in the Ranunculaceae family-stems with an irregular conformation, and phloem arcs/wedges.
Indentation toward the pith in the wider rays region is obvious in Clematis species (Sieber and Kucera 1980;Carlquist 1995). This feature was found in most Clematis species investigated in this study, except for those with a small stem diameter ( Table 2, ID column). The bark texture formed an arc shape outside the fascicular region in C. vitalba (Sieber and Kucera 1980); this feature was also found in eight species apparently ( Table 2, AR column). The arc feature comprised several layers of periderm combined with the non-conducting phloem (e.g., Fig. 1D. The characteristic of arc-like bark indeed is diagnostic evidence for Clematis species comparison. The vessel restriction pattern was found in Xanthorhiza apiifolia of Ranunculaceae (Carlquist 1995), Valeriana of Valerianaceae (Carlquist 1983), Launea of Astraceae (Carlquist 1988), and Dicentra and Hunnemannia of Papaveraceae. This pattern appeared in Clematis psilandra (Fig. 5C-D) and C. tsugetorum (Fig. 7B) studied, but few vessels were in contact with the rays; this phenomenon also appeared in the Clematis genus (Carlquist and Zona 1988). Carlquist and Zona (1988) suggested that the species clearly showed vessel restriction patterns; stems had limited diameter and duration, and they were either woody herbs or short-lived shrubs. This unusual wood feature of Clematis psilandra and C. tsugetorum was useful as a potential indicator of relationship (Carlquist 1995).
Clematis species often have 12 vascular bundles, with Smith (1928) nding this feature in 67% of 138 species studied. This is generally considered the central type for this genus. 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). In our study, Clematis henryi var. henryi (Fig. 3B) had 10 vascular bundles, but only six were recorded by Smith (1928). Moreover, C. chinensis var. chinensis ( Fig. 1C-D) had 20 − 23 bundles, C. lasiandra (Fig. 3F) had 12 − 14, C. meyeniana (Fig. 4D) had 21, and both C. uncinata var. okinawensis (Fig. 7C, D) and C. uncinata var. uncinata (Fig. 7E, F) had 12-14, but Smith (1928) recorded only 12 vascular bundles in these species. The inconsistent results might be due to different sample sizes, or developmental stages; further investigation is required to elucidate this discrepancy.
Vascular bundle numbers are usually constant for a given species, but the numbers can vary in some species. We examined three samples of C. leschenaultiana ( Fig. 4A-C . We checked that within 21 vascular bundles in the second stage, 10 might aggregate closely into ve groups, each group was composed of a big and a small vascular bundle, and the vascular bundle seems to be 16. In the fourth stage, some groups gradually separated, the number of vascular bundles seems to change to be 19. These processes could indicate that the bigger vascular bundle and fascicular zone were more active than the smaller vascular bundle and interfascicular zone. 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 and C. henryi var. morii (Yang and Huang 1996), but C. henryi var. morii is sometimes treated as a species, C. morii (Wang and Bartholomew 2007). In this study, these two taxa had wedge-like rays and a continuous rhytidome segment, but the stem of C. henryi var. henryi was round while that of C. henryi var. morii was an irregular conformation variant. These stem shape characteristics provide a way to distinguish these two taxa.
The site of phellogen initiation is often in an outer layer of cortical parenchyma cells, one or two layers beneath the epidermis (Beck, 2011). However, the phellogen location was highly variable and formed deep within the cortex, in the outer secondary phloem, or even within the secondary phloem (Isnard et al. 2003a), such as the Clematis genus. Compared with the characteristics of the developmental stage described in Isnard et al. (2003a, b), some features were different or could not be found in the present study. In the rst stage, the stem had the primary phloem bers in dense bundles or strands, not in a continuous ring; and in the fourth stage, narrow rays were found.
The one to two samples of two shrub species, C. psilandra, and C. tsugetorum, were not enough to assess their developmental stages. Assessing their development stage by the features of lianas and herb (Isnard et al. 2003a, b), we suggested they should belong to the fourth stage due to develop the narrow rays. However, further study collecting specimens of these species and examining their characteristics of different developmental stages is needed, especially species without the rhytidome.
In the present study, different narrow rays were observed in nine Clematis species (Table 2, NR column). These rays had been recorded in the species Aristolochia macrophylla (Aristolochiaceae) (Carlquist 1993;Schweingruber et al. 2011), in the Cucurbitaceae family (Carlquist 1992), and the Cyclea genus (Menispermaceae) (Yang and Chen 2016). The characteristics of narrow rays and activity of the bigger vascular bundles and the fascicular areas of C. grata are similar to those of C. vitalba, showing that they could be related to stem bark thickness and the formation of arc-like or cogwheel-like bark. However, the narrow rays were not found in all of the species investigated in our study, and its presence might be related to stem diameter size, but this needs further investigation.
The pith cavity resulted from non-ligni ed walls in the inner-most pith parenchyma cells; this characteristic was found in four species, Clematis parviloba subsp. bartlettii, C. psilandra, C. uncinata var. okinawensis, and C. uncinata var. uncinata.
Moreover, the stem features of the Clematis genus, such as the arc-like bark, rhytidome, wedge-like phloem, ray, and indentations, restricted vessel pattern, secondary phloem bers, and narrow rays, are very important diagnostic characteristics used to identify the species in the Clematis genus. The stem anatomy of the 22 Clematis species described here provided evidence for systematic problems within this genus.  formosana; secondary phloem ber strands (open arrowhead). C. C. formosana; secondary phloem bers strands, black, continuous segment rhytidome with 2 layers, 32 narrow rays, eccentric pith (circle). D. C. gouriana subsp. lishanensis; ring-like sequent periderm, discontinuous wedge-like phloem (star), 12 wide rays, 14 narrow rays. E. C. grata; stem with 6 shallow grooves, phloem ber in dense bundles beneath the angular extension of stem, 6 big and 6 small vascular bundles. F. C. grata; ring-like initial periderm, yellow, cogwheel-like rhytidome with 1 layer.