Myosin,microtubules, and microfilaments: co-operation between cytoskeletal components during cambial cell division and secondary vascular differentiation in trees

The immunolocalisation of unconventional myosin VIII ('myosin') in the cells of the secondary vascular tissues of angiosperm (Populus tremula L. x P. tremuloides Michx. and Aesculus hippocastanum L.) and gymnosperm (Pinus pinea L.) trees is described for the first time and related to other cytoskeletal elements, as well as to callose. Both myosin and callose are located at the cell plate in dividing cambial cells, whereas actin microfilaments are found alongside the cell plate; actin and tubulin are both associated with the phragmoplast. Myosin and callose also localise to the plasmodesmata-rich pit fields in the walls of living cells, which are particularly abundant within the common walls between ray cells and between ray cells and axial parenchyma cells in the phloem and xylem. In those xylem ray cells that contact developing vessel elements and tracheids, myosin, tubulin, actin and callose are localised at the periphery of developing contact and cross-field pits; the respective antibodies also highlight the bordered pits between vessels and between tracheids. The aperture of the bordered pits, whose diameter diminishes as the over-arching border of these pits develops, also houses myosin, actin and tubulin. Myosin, actin and callose are also found together around the sieve pores of sieve elements and sieve cells. We suggest that an acto-myosin contractile system (a 'plant muscle') is present at the cell plate, the sieve pores, the plasmodesmata within the walls of long-lived parenchyma cells, and at the apertures of bordered pits during their development.     

A cytoskeletal basis for wood formation in angiosperm trees: the involvement of microfilaments

The cortical microfilament (MF) component of the cytoskeleton within axial elements of the secondary vascular system of the angiosperm tree, Aesculus hippocastanum L. (horse-chestnut) was studied using transmission electron microscopy of ultrathin sections and indirect immunofluorescence microscopy of actin in thick sections. As seen by electron microscopy, MF bundles have a net axial orientation within fusiform cambial cells and their secondary vascular derivatives (i.e. in the axial xylem and phloem parenchyma, xylem fibres, vessel and sieve elements, and companion cells). Immunofluorescence studies, however, reveal that this axial orientation can be more accurately described as a helix of extremely high pitch; it is a persistent feature of all axial secondary vascular elements during their development. Helical MF arrays are the only arrangement seen in secondary phloem cells. However, in addition to helices, other MF arrays are seen in secondary xylem cells. For example, fibres possess ellipses of MFs associated with simple-pit formation, and vessel elements possess circular arrays of MFs that associate with the developing inter-vessel bordered pits, ray–vessel contact pits, and with the perforation plate. Linear MF arrays are seen co-oriented with the developing tertiary wall-thickenings in vessel elements. The possible roles of MFs during the cytodifferentiation of secondary vascular cells is discussed, and compared with that of microtubules. Received: 7 June 1999 / Accepted: 23 December 1999     

Arrangement of cortical microtubules during formation of bordered pit in the tracheids ofTaxus

Summary The arrangement of cortical microtubules during the development of the secondary wall and bordered pits in the tracheids ofTaxus was examined by immunofluorescence and electron microscopy. The cambial region of radial longitudinal sections of developing young shoots (2–3 years old) contains cells at various stages of differentiation from cambial cells to tracheids. At the early stage of formation of bordered pits, circular bands of microtubules were seen to be associated with the inner edge of the border of the developing pit. In other regions than the pit secondary wall of uniform thickness was laid down, and obliquely oriented cortical microtubules ran parallel to one another. These cortical microtubules also covered the surface of the border of the developing pit on the side facing the center of the cell. As the border of the pit developed, a circular band of MTs remained associated with the inner edge of border, suggesting that the MTs were involved in the formation of the rim of the bordered pit, extending the initial border thickening, which consisted of concentrically oriented cellulose microfibrils. After completion of the formation of the bordered pit, helical thickenings became apparent. The obliquely oriented microtubules were organized in bands parallel to one another, being superimposed on the helical thickenings. The involvement of MTs in the formation of bordered pits and helical thickening is discussed.     

Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not

The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however, has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament (MF) components of the cytoskeleton in a secondary tissue  –  active vascular cambium of Aesculus hippocastanum L. (horse-chestnut)  –  and followed the changes in these components during the early stages of differentiation of fusiform cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged. Received: 5 August 1996 /  Accepted: 23 September 1996     

Differences in the timing of cell death,differentiation and function among three different types of ray parenchyma cells in the hardwood <Emphasis Type="Italic">Populus sieboldii</Emphasis> × <Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">grandidentata</Emphasis>

Differences in the timing of cell death, differentiation and function among three different types of ray parenchyma cells in the hardwood Populus sieboldii × P. grandidentata which form uniseriate and homocellular rays were examined and clarified. Ray parenchyma cells died within 5 years, and the disappearance of nuclei from ray parenchyma cells did not occur successively from the pith side, even within individual radial cell lines of a given ray. Cell death occurred earliest in contact cells, which were connected to adjacent vessel elements through pits, in the fourth annual ring from the cambium. Cell death occurred next in intermediate cells, which were located within the same cell lines as contact cells but were not adjacent to vessel elements, in the fourth annual ring from the cambium. Finally, isolation cells, which were located within the other cell lines of a given ray, died in the fifth annual ring from the cambium. Secondary wall thickenings in contact cells and intermediate cells were initiated before those in isolation cells in the current year’s xylem. Most starch grains were localized in intermediate cells, and there were more lipid droplets in contact cells and intermediate cells than in isolation cells. In addition, the largest quantities of protein were found in contact cells. Our results indicate that the position within a ray and neighboring short-lived vessel elements might affect the timing of cell death and differentiation and, thus, the function of long-lived ray parenchyma cells in Populus sieboldii × P. grandidentata.     

Cortical microtubule involvement in bordered pit formation in secondary xylem vessel elements ofAesculus hippocastanum L. (Hippocastanaceae): A correlative study using electron microscopy and indirect immunofluorescence microscopy

Summary A correlative study, using indirect immunofluorescence microscopy (IIF) of anti--tubulin stained sections and transmission electron microscopy (TEM), gave details of the involvement of cortical microtubules (CMTs) in the development of bordered pits in secondary xylem vessel elements ofAesculus hippocastanum L. In addition, aspects of wall cytochemistry were studied during this process using the Thiéry (PATAg) test, immunolocalization with the monoclonal antibodies JIM5 and JIM7, and a range of other cytochemical procedures. IIF showed that the alternately-arranged pits are pre-figured as perforations within a reticulum of randomly-oriented CMTs before any secondary wall thickening is obvious. Each incipient pit border is subsequently delimited by a circle of CMTs whose diameter decreases as deposition of secondary wall takes place around the perforation. These IIF observations are corroborated by a parallel TEM study. During the period of bordered pit formation, the secondary walls of the cell are lignifying. At maturity, however, the pit membrane is unlignified and continues to stain strongly with the monoclonal antibody JIM5, a marker of primary, juvenile wall. The results are discussed in terms of the relationship of the CMT cytoskeleton with development of bordered pits.Abbrevations BSA bovine serum albumin - CMT(s) cortical micro-tubule(s) - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - IIP indirect immunofluorescence - MAP microtubule-associated protein - mf(s) wall microfibril(s) - MTSB microtubule-stabilising buffer - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PBS Phosphate-buffered saline - PIPES piperazine-N,N-bis-[2-ethylsulphonic acid] - SVS secondary vascular system - TEM transmission electron microscopy     

Cambial reactivation in locally heated stems of the evergreen conifer Abies sachalinensis (Schmidt) Masters

A study was made of cambial activity, the localization of storage starch around the cambium, and the localization and occurrence of microtubules in cambial cells from dormancy to reactivation in locally heated (22–26 °C) stems of the evergreen conifer Abies sachalinensis. Heating induced localized reactivation of the cambium in the heated portions of the stem. Erect ray cambial cells resumed cell division 1 d prior to the reactivation of fusiform cambial cells and procumbent ray cambial cells. The re-initiation of the division of fusiform cambial cells occurred first on the phloem side. During the heat treatment, the amount of storage starch decreased in procumbent ray cambial cells and in the phloem parenchyma adjacent to the cambium but increased in fusiform cambial cells. Preprophase bands of microtubules, spindle microtubules and phragmoplast microtubules were observed both in erect ray cambial cells and in procumbent ray cambial cells. By contrast, no evidence of the presence of such preprophase bands of microtubules was detected in fusiform cambial cells. The results suggest that the localized heating of stems of evergreen conifers might provide a useful experimental model system for studies of the dynamics of cambial reactivation in intact trees. Received: 25 May 2000 / Accepted: 12 July 2000     

Variations in the Lengths of Fusiform Cambial Cells and Vessel Elements in Kalopanax pictus

Samples of a mature specimen of Kalopanax pictus, a ring-poroushardwood, were studied to compare the respective lengths offusiform cambial cells and vessel elements in the stem. Thelengths of dormant and reactivated fusiform cambial cells weremeasured with a confocal laser scanning microscope in tissuethat had been macerated by digestion with pectinase and in thicktangential sections. The lengths of early wood and late woodvessel elements were measured in tissues that had been maceratedby Franklin's method. The vessel elements and fusiform cambialcells varied considerably in length within individual samples.The mean length of early wood vessel elements corresponded tothat of fusiform cells in the dormant cambium but not in thereactivated cambium. Significant differences were observed betweenthe mean lengths of dormant and reactivated fusiform cambialcells, between those of reactivated fusiform cambial cells andearly wood vessel elements, between those of reactivated fusiformcambial cells and late wood vessel elements, and between thoseof early wood and late wood vessel elements. The frequency distributionsof lengths of cambial cells were bimodal and differed from thoseof vessel elements, which more closely resembled a normal distribution.The proportion of shorter lengths was higher in the reactivatedcambium than in the dormant cambium, the early wood and thelate wood vessel elements. Our results do not suppot the hypothesisthat the lengths of early wood vessel elements in ring-poroushardwoods change during differentiation. The similar rangesof recorded lengths suggest that short and long vessel elementsmight be derived directly from short and long cambial cells,respectively. Copyright 1999 Annals of Botany Company Kalopanax pictus, cambium, vessel element, confocal laser scanning microscopy, maceration, cell length.     

Bordered pits in ray cells and axial parenchyma: the histology of conduction, storage, and strength in living wood cells

Bordered pits occur in walls of living ray cells of numerous species of woody dicotyledons. The occurrence of this feature has been minimally reported because the pits are relatively small and not easily observed in face view. Bordered pits are illustrated in sectional view with light microscopy and with scanning electron microscopy in face view for dicotyledonous and gnetalean woods. Bordered pits are more numerous and often have prominent borders on tangential walls of procumbent ray cells, but also occur on radial walls; they are approximately equally abundant on tangential and horizontal walls of upright cells, suggesting parallels to cell shape in flow pathway design. Axial parenchyma typically has secondary walls thinner than those of ray cells, but bordered pits or large simple pit areas occur on some cross walls of parenchyma strands. There is no apparent correlation between the phylogenetic position of species and the presence of borders in ray cells or axial parenchyma. Bordered pits represent a compromise between maximal mechanical strength and maximal conductive capability. High rates of flow of sugar solutions may occur if starch in ray cells or axial parenchyma is mobilized for sudden osmotic enhancement of the conductive stream or for rapid development of foliage, flowers, or fruits. Measurement of the secondary wall thickness of ray cells may offer simple inferential information about the role that rays play in the mechanical strength of woods. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 157–168.     

Stem anatomy of Dolichos lablab Linn (Fabaceae): Origin of cambium and reverse orientation of vascular bundles

Secondary growth in the stem of Dolichos lablab is achieved by the formation of eccentric successive rings of vascular bundles. The stem is composed of parenchymatous ground tissue and xylem and phloem confined to portions of small cambial segments. However, development of new cambial segments can be observed from the obliterating ray parenchyma, the outermost phloem parenchyma and the secondary cortical parenchyma. Initially cambium develops as small segments, which latter become joined to form a complete cylinder of vascular cambium. Each cambial ring is functionally divided into two distinct regions. The one segment of cambium produces thick-walled lignified xylem derivatives in centripetal direction and phloem elements centrifugally. The other segment produces only thin-walled parenchyma on both xylem and phloem side. In mature stems, some of the axial parenchyma embedded deep inside the xylem acquires meristematic activity and leads to the formation of thick-walled xylem derivatives centrifugally and phloem elements centripetally. The secondary xylem comprises vessel elements, tracheids, fibres and axial parenchyma. Rays are uni-multiseriate in the region of cambium that produces xylem and phloem derivatives, while in some of the regions of cambium large multiseriate, compound, aggregate and polycentric rays can be noticed.     

INFLUENCE OF PHLOEM BLOCKAGE ON CAMBIAL GROWTH OF SUGAR MAPLE

Circular patches of bark were surgically isolated on the sides of sugar maple (Acer saccharum Marsh.) trees at breast height at various times during the dormant and growing seasons. Subsequently, samples of wood and attached bark were taken from isolated and control sites to determine the effects of isolation of the bark on cambial activity and xylem and phloem development. In control sites cambial activity and xylem and phloem development occurred normally. Isolation of bark during the dormant season (in November, February, or March) prevented initiation of cambial activity and xylem and phloem development in isolated areas of half of the trees. Varying degrees of cambial activity (periclinal divisions) occurred in the remaining isolated areas, but normal cambial activity and xylem and phloem development were prevented. Isolation of bark after initiation of cambial activity and phloem differentiation, but prior to initiation of xylem differentiation, resulted in the formation of very narrow xylem and phloem increments with atypically short vessel members and sieve-tube members, respectively. The xylem increments consisted primarily of parenchyma cells. Isolation of bark after initiation of xylem differentiation resulted in curtailment of secondary wall formation in the last-formed part of many increments. The last-formed vessel members of all these xylem increments were atypically short. Similarly, the last formed sieve-tube members of corresponding phloem increments were atypically short. The atypically short cells in the xylem and phloem of isolated areas reflected the effect of isolation on the cambial region, viz., the subdivision of all fusiform cells into strands of cells. Ultimately, the strands of short fusiform cells lapsed into maturity, leaving only strands of parenchymatous elements between xylem and phloem.     

四合木属四合木(蒺藜科)的木材解剖学研究

在光学显微镜和扫描电子显微镜下观察了蒺藜科四合木属四合木(Tetraena mongolica Maim.)木材结构.其导管分子直径小,管壁厚,分布密度高,分子短,端壁几乎水平,具单穿孔;管间纹孔为对列或互列的具缘纹孔;韧性纤维短、壁厚、壁上有较少的单纹孔;同型单列射线、分布密度高;轴向薄壁组织散生或傍导管生.这些表明四合木的木材解剖特征是与干旱环境条件是相适应的.     

红树族植物次生木质部附物纹孔的电镜观测

应用扫描电子显微镜详细观察了红树族4属、10种、1变种植物次生木质部管状分子附物纹孔的分布和形态, 应用Carnoy 2.0软件和扫描电镜下采集的照片, 测定了管间梯状附物纹孔丰富度指标和管间梯状纹孔数量特征指标。结果显示, 红树族植物次生木质部管状分子侧壁具附物纹孔。所观察的植物附物纹孔的分布和形态变化大。附物纹孔丰富度指标与管间梯状纹孔数量特征指标的逐步回归分析表明, 导管侧壁附物纹孔丰富度随纹孔口面积百分比的增大而增大。据此推测, 红树族植物附物纹孔丰富度与纹孔几何构造及数量特征有关。附物纹孔是红树族植物稳定存在的一个木材解剖性状。综合生态-系统演化的观点, 红树族植物具附物纹孔可能是受系统演化关系控制的生态适应结果。     

水青树特殊管胞的分布位置及其形态特征的研究

针对水青树（Tetracentron sinense）中一类特殊管胞进行较为全面的观察研究,判断细胞种类并分析维管组织输导机理及树木进化过程中的细胞演化规律。通过切片和解离技术,借助光学显微镜和电子显微镜对34年生水青树特殊管胞的分布位置和形态特征进行观察。结果表明：(1)特殊管胞在树木水平方向自内向外径向呈串排列,并贯穿年轮界限,多为一列,少数两列,且较为稀见。每个特殊管胞弦向左右两侧或单侧均与木射线细胞相连通。纵向上,特殊管胞单独或数个上下端接相连。(2)特殊管胞主要有以下3种类型：无端壁的纺锤形,有一个倾斜端壁,以及有两个倾斜端壁。特殊管胞的平均长度为286.44μm;横切面为四边形,平均弦向宽度为55.22μm,其平均壁厚为1.53μm。(3)特殊管胞两端封闭,无穿孔。(4)特殊管胞侧面壁上的纹孔数量较多且纹孔膜明显可见,具体表现为：弦面壁上布满特殊管胞之间的具缘纹孔,呈对列、互列偶见梯状排列;径面壁上存在与射线细胞间的具狭缘单纹孔,呈大圆形至椭圆形,每区域多为2～10个纹孔,呈1～4排横列;径面壁上与正常管胞间几无纹孔。水青树特殊管胞分布有一定规律,其长度远小于水青树正常管胞...     

Formation of successive cambia and stem anatomy of Sesuvium sesuvioides (Aizoaceae)

Mature stems of Sesuvium sesuvioides (Fenzl) Verdc. were found to be composed of successive rings of xylem alternating with phloem. Repeated periclinal divisions in the parenchyma outside the primary phloem gave rise to conjunctive tissue and the lateral meristem that differentiate into the vascular cambium on its inner side. After the formation of the vascular cambium, the lateral meristem external to it became indistinct as long as the cambium was functional. As the cambium ceased to divide, the lateral meristem again became apparent prior to the initiation of the next cambial ring. The cambium was exclusively composed of fusiform cambial cells with no rays. In the young saplings, the number of cambial cylinders in the axis varied from the apex to the base, indicating formation of several rings within the year. In each successive ring of the lateral meristem, small segments differentiated into the vascular cambium and gave rise to vessels, axial parenchyma, fibres and fibriform vessels towards the inside, and secondary phloem on the outer side. In the old stems, non‐functional phloem of the innermost rings was replaced by a new set of sieve tube elements formed by periclinal divisions in the cambial segments associated with the non‐functional phloem. In some places the cambial segments completely differentiate into derivatives leaving no cambial cells between the xylem and phloem. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 548–555.     

Anatomy of the vessel network within and between tree rings of Fraxinus lanuginosa (Oleaceae)

The three-dimensional (3-D) arrangement of vessels and the vessel-to-vessel connections in the secondary xylem of the stem of the ring-porous hardwood tree Fraxinus lanuginosa were studied in series of thick transverse sections with epifluorescence microscope and confocal laser scanning microscope. Vessels were traced in sequential sections, and vessel networks were reconstructed in two segments of wood with dimensions of 2 × 1.4 × 21.2 mm(3) and 2 × 1.4 × 5.8 mm(3) (tangential × radial × axial). The arrangement of vessels and intervessel pits were visualized by scanning electron microscopy in low-density polyethylene microcasts and on exposed tangential faces of growth-ring boundaries. The vessels deviated from the stem axis in the tangential direction and, to a lesser extent, in the radial direction. Some neighboring vessels were twisted around each other. Vessels that appeared solitary in single sections were found to be sequentially contiguous with a number of other vessels, forming networks that extended in the tangential direction and across growth-ring boundaries. In the 21.2-mm wood block, all earlywood vessels at the growth-ring boundary made contact with latewood vessels in the previous tree ring. Within a growth ring however, only a single contact was observed between individual earlywood and latewood vessels. Densely arranged intervessel pits were characteristic in the regions where adjacent vessels made contact with each other. Such bordered pits were abundant in the tangential walls of vessel elements adjacent to growth-ring boundaries. Therefore, bordered pits appear to provide the pathway for the radial transport of water via the vessel network across growth-ring borders. Fiber-tracheids, observed as terminal cells in the tree rings, might also contribute to the apoplastic transfer of water across ring borders.     

Wood and bark anatomy of Gnetutn gnemon L.

Quantitative and qualitative data are presented for seven collections representing two varieties (unlike in habit) of Gnetum gnemon. Tracheids are present, but abundant and intermixed with them are septate fibre-tracheids rich in starch. Axial parenchyma has been reported only once previously for the species. Axial parenchyma is in strands of 4–10 cells, is rich in starch, is primarily vasicentric (paratracheal) in distribution, less commonly diffuse. About equally common are simple and compound perforation plates; the latter are composed of from two to about ten bordered foraminate perforations, the shape of which may be altered by crowding or coalescence, but is clearly still foraminate. Lateral walls of vessels bear pits that are vestured around pit cavities, not facing the pit membrane. Rays are composed mostly of procumbent cells; the tangential walls bear bordered pits. Crystals, present in ray cells and (rarely) axial parenchyma vary widely in size. Crystalliferous sclereids with layered walls, starch-rich parenchyma, and gelatinous secondary phloem fibres are the main components of bark. Early stages in origin of successive vascular cambia in bark are newly described. When representative conditions are derived from study of large numbers of slides, the classical view that Gnetum vessels are unlike those of angiosperms is supported. Features of Gnetum gnemon wood are discussed in the light of ecology and conductive physiology.     

A New Species of Araucarioxylon Kraus from the Early Early Permian,Nei Mongol,China

A kind of silicified fossil wood with mixed pits on the radial tracheid wall is described. The fossil wood was collected from the top of Taiyuan Formation (early Early Permian) in Wuda Mining District, Nei Mongol. Compared with the Paleozoic fossil woods in the world, it is put into Araucarioxylon Kraus and named as A. laoshidanense sp. nov. Based on the character of possessing mixed pittings (alternate and opposite pittings) on the radial tracheid wall, the fossil wood is believed to be one of the unknown primitive conifers.Diagnosis of the new species: Only secondary xylem preserved and consisting of axial tracheids and rays. Growth ring boundary, resin duct and axial parenchyma absent. One to Four (commonly 2or3) seriates of bordered pits mostly alternate but sometimes opposite）on the radial tracheid wall. One to Four (commonly1, rarely2 to 4) Cupressoid pits in each cross-field. Rays usually uniseriate, sometimes partly-biseriate and 2 to 39 (mainly3-5) cells high.     

梣属11种植物次生木质部附物纹孔的电镜观测

应用扫描电镜对梣属11种植物次生木质部导管附物纹孔的分布和形态进行了详细的观察,应用Carnoy2.0软件和扫描电子显微镜采集的照片,测定了附物纹孔丰富度指标和纹孔数量特征指标。电镜观察表明,梣属11种研究植物次生木质部导管分子侧壁附物纹孔的分布和形态变化较大,附物纹孔丰富度指标的统计描述进一步证实附物纹孔的分布变化大;3个附物纹孔丰富度指标分别与管间具缘纹孔数量特征指标的逐步回归分析表明,导管侧壁附物纹孔丰富度2个指标,即导管外纹孔口附物频率与导管纹孔腔附物频率随纹孔口面积百分比的增大而增大,推测梣属植物附物纹孔丰富度与纹孔几何构造及数量特征有关。研究认为,附物纹孔在梣属植物稳定存在,是可界定梣属植物的木材解剖性状。     

Variation of Susceptibility to Aspiration of Bordered Pits in Conifer Wood

The axial gaseous permeability of conifer wood has been measuredbefore and after saturation with a variety of liquids and redrying.It was possible to show that if the surface tension of the saturatingliquid was progressively increased, the permeability of thewood alter redrying decreased as a result. This is in keepingwith existing theory on the mechanism of bordered pit aspirationand known systematic variation of bordered pit structure acrossthe growth ring. The bordered pits were found to fall into threecategories, according to their susceptibility to aspiration.