Cambial Variants Combine Successive Cambia and Intraxylary Phloem in Convolvulaceae in Taiwan

Secondary stem growth develops different cambial variants in Convolvulaceae. Here, the cambial variant features of 17 species in seven genera (Con-volvulaceae) in Taiwan are presented. The xylem rings produce various successive cambial types and the primary xylem and intraxylary phloem are produced in the outer and inner pith, respectively. The two Argyreia species have round or elliptical stems with continuous secondary xylem and phloem rings. In the eight Ipomoea species, secondary growth has two to four layers of successive cambia and xylem, dispersed with parenchyma tissue, except for I. nil and I. violacea with one layer of successive cambia. The first secondary xylem segments are separated by few or many xylem rays: I. cairica and I. obscura have three to five xylem rays and I. triloba has numerous xylem rays. The first secondary xylem of Erycibe henryi is divided by numerous rays in small stems, but develops more layers, as adjacent segments separated by xylem rays, in larger stems. Owing to continuous vascular bundle division, it has a dissected xylem. The secondary growth of Distimate tuberosa and Operculina turpethum produces two layers of successive cambia; the secondary successive cambium is divided evenly by rays into many arcs/segments. The stem


Introduction
The vascular cambium of climbing plants is usually located around the circumference of the stem, and if the activity of the cambium is not continuous, some of the circumferential regions produce tissue that is very different from the adjacent areas. Such irregular growth may be associated with the cambium or with morphogenetic factors derived from the cambium that affect cell activity [1].
Under normal conditions, each cambium produces xylem centripetally and phloem centrifugally, but 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 [1]. The stem cross-section generally changes from a regular circular to an irregular shape after secondary growth, owing to the uneven deposition of secondary xylem and the eccentricity of successive cambia [2]. As the stem twines around a support, cambial activity increases the amount of xylem and phloem on the opposite side of the stem [3].
Cambial variants are divided into two types: those that originate from a single cambium and those that originate from multiple cambia [4]. Within Convolvulaceae, several cambial variants have been reported, such as successive cambia, interxylary phloem, xylem parts dispersed by parenchyma, and intraxylary phloem [4] [5] [6] [7] [8]. The successive cambia are divided into concentric bands and non-concentric bands [9]. The activity of concentric bands produces vascular bundles of the same width, and parenchyma cells with alternating, well-defined xylem and phloem rings; the pith is usually located in the center.
The activity of non-concentric bands produces an asymmetric band in different directional successive cambia, leading to intensified development of one to three sides of the stem and an off-center pith [10].
The genus Ipomoea has successive cambia, interxylary phloem, and fibrous woody parts dispersed by parenchyma proliferation [11]. In an early stage of primary growth, some Ipomoea species stems form primary xylem [14]. Under normal developmental conditions, the vascular bundles in the genus Ipomoea usually develop precisely in their positions. Initially, the secondary growth of Ipomoea is normal, producing both xylem and phloem. This is followed by the activity of additional cambia, each forming around a single vascular bundle or Lowell and Lucansky [15] reported that the primary growth of Ipomoea hederifolia L. comprises a bicollateral bundle stage and a cambium-like meristem stage, whereas the secondary growth consists of a normal cambial stage, an anomalous stage, and a supernumerary cambial stage. Rajput et al. [16] indicated that the discontinuous segments of the secondary xylem in the second ring in I. hederifolia showed xylem with reverse orientation, producing secondary xylem centrifugally and secondary phloem centripetally. Patil et al. [17] also showed that one of the successive cambial layers develops anomalous cambial arcs/segments with reverse orientation in I. hederifolia. The stem thickness of Ipomoea obscura (L.) Ker Gawl. and Ipomoea pes-caprae (L.) R. Br. in cross-section consist of three to four layers of secondary xylem and alternating phloem rings, giving rise to multiple/successive cambia [2] [18]. The stem of I. quamoclit L. subsequently develops lobed, concentric, and diffuse (foraminate) anomalous secondary growth [19]; and I. turbianata Lag. forms internal cambium and intraxylary phloem [7].
Multiple types of cambial variants are found within Convolvulaceae, but the growth patterns of the stem cross-sections of many members of Convolvulaceae in Taiwan have not been described. Various stem structures and identifications have been reported in the Menispermaceae and Fabaceae families in Taiwan [35] [36]. In the present study, we examined the stem cross-sections of Convolvulaceae vines in Taiwan, and how the cambial variants combined with successive cambia and intraxylary phloem. As cambial variations constitute an extremely diverse morphology, the present study attempts 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 identification of irregular cambial activity in the Convolvulaceae family in Taiwan.

Research Materials
The species datasets included species scientific name, collector, herbarium and To keep the material fresh and retain humidity, the stems were stored in the collecting bag. One or two samples with obvious and easy-to-observe cambial variations were selected per species for photographs and the scoring of morphological characters.

Research Methods
In the laboratory, the fresh stems 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 1:1 lens (Lens AF Micro Nikon 60 mm 1:2.8D; Nikon Corporation, Tokyo, Japan). All specimens were oven-dried at 60˚C for 4 -5 days and then stored at −20˚C for 3 -4 days, and deposited in the Provincial Pingtung Institute (PPI) herbarium at the National Pingtung University of Science and Technology, Pingtung, Taiwan, for subsequent identification. The nomenclature follows Flora of Taiwan Volume 4 [21] with the dissolution of the Convolvulaceae tribe Merremieae, and the new classification of the constituent genera [37].
The morphological characteristics of the irregular stem structures of each species were investigated as followed: stem diameter (mm), shape of the fresh stem cross-section, number of successive cambium layers, segment number of the first secondary xylem, minimum and maximum diameter of vessels (μm), mean diameter of vessels (μm) (mean ± standard deviation, SD, n = 25), directional successive cambia (+/−), cambial arcs/segment (+/−), and cork (+/−). Stem diameter was assessed using the Radford [38] definition, according to the plan length/width proportion. Vessels with two distinct diameter classes, and wood that was not ring-porous, were measured using the larger size class, and 25 measured vessels (mean ± SD) are required by the definition of Wheeler et al. [39]. Quantitative anatomical traits such as the diameter of the stems (length and width, mm × mm) or minimum and maximum sizes of the xylem vessels were determined using Image-J software [40]. The diameter of nearly circular stems was measured from the average of four diagonal lines; whereas non-circular stems were measured using the minimum and maximum diameter as length and width.

Cambial Variants Photographs and Explanation
Cambium variations in stems, combined with successive cambia and intraxylary phloem, were assessed in approximately 17 species in seven genera in the Convolvulaceae family in Taiwan. The dataset of these species is provided in Table 1.   The epidermis is one of the diagnostic features of these species. Thus, a noticeable cork (phellem) was observed on the larger stems of A. formosana ( Figure  1(c)), but absent in A. akoensis. Among the 10 Ipomoea species, five species also developed cork ( Table 2). The primary growth of the stem was similar among all the species-a cambium-like meristem was formed, and intraxylary phloem and primary xylem were produced in the inner and outer pith respectively. The secondary growth developed one to four successive cambial rings among the studied species. The successive rings of the secondary xylem alternate with phloem rings (Figure 1(b), Figure 2(d), Figure 2(e), Figures 3(c)-(f)). The first layer of the successive cambium developed normal secondary tissue, which produced secondary xylem centripetally and secondary phloem centrifugally in all species (Figures 1-4). The second or third layers of secondary tissue developed from the anomalous cambial stage to the supernumerary cambial stage, forming a ring of small cambial arcs/segments (Figure 1 Figure 4(d)). These small cambial arcs/segments were fused to a continuous cambial ring and the adjacent segments separated by rays. The supernumerary cambial stage in the first secondary growth of the stem formed a discontinuous xylem ring generally separated by a few wide rays (Figure 1  henryi (Figure 3(a)), Ipomoea nil (Figure 3(b)), I. pes-caprae subsp. brasiliensis (Figure 3(d)), and I. triloba (Figure 3(e), Figure 3(f)) were separated by several thin rays. Parenchyma proliferation is often present in some species, which results in thick stems with mucilaginous canals (Figure 2 Table 2).

Species Characterization
Based on the characteristics of the stem cross-sections, we created a bracketed key for identification of the 17 climbing species. This key allows species identification when leaves are not available.

Discussion
In the present study, the stem of the genus Argyreia was usually round or elliptical in cross-section and the secondary xylem and secondary phloem usually formed concentric rings without rays. Parenchyma proliferation was not obvious ( Figure 1). In contrast, the stem of the genus Ipomoea is irregular in cross-section, the secondary cambium rings are arcs/segments-like, and parenchyma proliferation is obvious (Figure 2(b), Figure 2(c), Figure 2(f), Figure   3(a), Figure 3(c), Figure 4(a)). The texture of the stems of Ipomoea is quite soft with irregularly distributed patches of thin-walled parenchyma, but there are almost no irregular patches of parenchyma in Argyreia. The vegetative morphologies of Argyreia and Ipomoea are quite similar, resulting in some species being erroneously described within the genus Ipomoea or Argyreia [41]. The cambial variations in their stems could be used in the debate around the species delimitation of these two genera. Vasicentric or aliform-confluent paratracheal parenchyma cells around the vessels of Argyreia and Ipomoea have been previously recorded [42], and the presence of vasicentric paratracheal parenchyma was confirmed around the vessels of the 17 species in this study. Metaxylem elements (also called the primary xylem) differentiated from the cambium-like meristem on the primary growth were observed in these 17 species from Taiwan, and the results confirmed the metaxylem characteristics described in I. hederifolia by Lowell and Lucansky [15]. During secondary growth, the anomalous cambial stage produces the secondary xylem and secondary phloem. If the formation of secondary xylem is greater in one region than in another, it resulted in a grooved stem [16]. In this study, the stem cross-sections of I. hederifolia had two deep lobes of successive cambia. The characteristics of these lobed stems have not been described in previous reports. They might be related to stem size and the supernumerary cambial stage, and warrant further studies.
The asymmetry in the cross-sections of vines is often related to the supports used by the twining vines and results from increased tissue production in the vascular bundles. The site of the increased cambial activity is often located on the side of the stem that is not in contact with the support, e.g., in I. quamoclit [19]. In the present study, significant secondary xylem and phloem growth was The characteristics of the small arcs/segments of internal cambium in I. hederifolia were developed during the supernumerary cambial stage of secondary growth, as described by Lowell and Lucansky [15], Patil et al. [17], and Rajput et al. [18]. In this study, the diagnostic characteristics of the cambium arc/segment rings were present in D. tuberosa, E. henryi, I. cairica, I. obscura, I. pes-caprae subsp. brasiliensis, I. triloba, and Operculina turpethum. The species E. henryi developed more layers as adjacent segments separated by xylem rays, which would form a dissected xylem [6] in larger stems.
The supernumerary cambial stage is a common developmental stage in Ipomoea [19]. The supernumerary cambial refers to the presence of many successive rings. The stems of I. nil (Figure 3(b)) and I. violacea (Figure 4 Cambial variants have been used as diagnostic characteristics for the identification of climbing plants in different families and genera, such as Aristolochiaceae, Menispermaceae, and Vitaceae. A variety of cambial variances developed in the Convolvulaceae family (Figures 1-4), as well as some climbing plants with regular secondary growth (Figures 1(a) Figure 4(b)) that may be asymmetrical or symmetrical, intraxylary phloem, cambial arc/segment rings, and dissected xylems, all caused by the parenchyma proliferation as well as succession cambia. These variants may also be found in different combinations. Whether or not these variances have developmental relationships warrants further study.

Conclusion
Studies on the cambial variants of climbing plants in the family Convolvulaceae in Taiwan are scarce. In this study, we examined species in which cambial variants in the stem were combined with successive cambia and intraxylary phloem in Convolvulaceae from Taiwan. The experimental results showed that the stems of the two Argyreia species are round or elliptical and the secondary xylem and phloem rings are continuous. The cambial variants of Ipomoea species, except I. nil and I. violacea, include irregular conformations, successive cambia, and xylem parts dispersed by rays into segments. The first secondary xylem in E. henryi, I. nil, I. pes-caprae subsp. brasiliensis, and I. triloba is di-