Studies on Tracheary Element of Several Native Cycad Species in Australia and Two American Plants

Cycas angulata, Cycas armstrongii and Cycas conferta that are native species of Australia were collected in Darwin city of tropical region, Zamia erosa and angiosperm Guaiacum officinale of Zygophyllaceae which are introduced collected in the Darwin Botanic Gardens, and were carried out light microscope and electron microscope observed research to tracheary element and parenchyma tissue cells of leaflet, rachis and stem. The results showed that there are more vessel elements in their xylems; the length of vessel element of Cycadaceae with Zamiaceae are not obvious difference, the length of vessel element of angiosperm Guaiacum officinale is shorter, but their diameter is smaller, we thought that the characteristics which the length of vessel element is longer and the diameter is bigger are more evolutionary, because thus vessel, their number which interconnected points of possessed perforations’ end walls of vessel element is fewer, and the passageway space is larger so that the conduction speed and quantity are faster and more in unit length range and unit time. These vessels are annular vessels, spiral vessels, scalariform vessels, reticular vessels, pitted vessels and scalariform-pitted vessels, etc. In the transverse section, the vessel elements of Cycadaceae and Zamiaceae are circular, polygonous, more similar to vessel characteristics of leaf and stem of some angiosperms; however, the number of circular vessel of Guaiacum officinale is more, about occupy 35%, others are polygonous. The diameter of vessel is that annular vessel, spiral vessel is least, scalariform vessel or scalariform-reticular vessel is medium, reticular vessel and pitted vessel are the bigHow to cite this paper: Huang, Y.Y., Ottley, H., Yep, Y., Wilson, S., Griffiths, D., O’sullivan, N., Han, Y.H. and Hempel, J. (2022) Studies on Tracheary Element of Several Native Cycad Species in Australia and Two American Plants. American Journal of Plant Sciences, 13, 147-174. https://doi.org/10.4236/ajps.2022.131010 Received: December 8, 2021 Accepted: January 25, 2022 Published: January 28, 2022 Copyright © 2022 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

Cycas vessel element is similar to that of Zamia, but the diameter of more vessel elements of Cycas angulata and Cycas conferta is bigger than the latter, and is bigger than that of Guaiacum officinale, although Cycas plants are more primitive than Zamia plants, and Guaiacum officinale is more evolutionary, but the scale of vessel diameter of Cycas plants is similar to Zamia or bigger than the latter, even larger than Guaiacum officinale, these showed that cycads have evolutionary and more developed characteristics in aqueous solution transport system. In Guaiacum officinale, more vessel element has been not seen the ridges in the different directions of side walls which can make vessel has more mechanical supportability, but some possessed the ridges; however, more vessel elements are twist shape and can twist together each other, thus the state can increase their mechanical supportability, and their synergy with fiber elements which is longer with a twist shape, therefore their capability of xylem conduction and the combine mechanical support capability with mechanical tissue are also stronger. In the respect of observed parenchyma tissue, any parenchyma tissue cells are without perforation, all cell walls are smoothed and like a membrane. Aimed at only one or two people pointed the query about Jeffery's method, some scholars have made comparative researches, their research used as following methods: 1) The fresh materials were cut sections 1 -2 mm thick by hand (the query person provided and thought right method), were examined with SEM. 2) The materials were treated by Jeffrey's Fluid and observed with SEM and so on. The results showed that the pit membrane, the remnants in the perforation and the structural characteristics of perforations were not different; and the results of our comparative research in past time and this research all justified that Jeffrey's method is reliable and reasonable. Meanwhile, our research results showed that only vessel element can form perforation, in other all parenchyma tissue cells cannot form perforation are also proved that the perforations of vessel element are naturally inherited character. Analyzed from the structural characteristics, although they distributed so far apart from each other, they are a well and unified taxonomy system; we thought that Australia' most species or all species of Cycadaceae came from Asia, and it is impossible from Africa, because the latter only has one more evolutionary species and lack of fossil of Cycadaceae, these species of Cycadaceae are the descendants of ancestor species; America possessed more genera of Zamiaceae, this family is mutual with Africa and Australia, we thought that these genera of Zamiaceae in Australia and America came from Pangea before continental drift in Jurassic Period, this family is also maybe one of the proofs to Australia drifted from Pangea to present position after Jurassic Period. The characteristics of tracheary element of several different genera of cycads and angiosperm further showed that more primary species or taxon, their more tissue

Introduction
Cycads are very primitive seed plants of ancient; they originated from about the Carboniferous period [1], and still possess primitive characteristics in external morphology and internal structure, such as sperm with flagella. Metasequoia glyptostroboides Hu et Cheng is an ancient, rare "living fossil". Research on more species of cycad structural characteristics of tracheary elements is important to understand their adapted mechanism and evolutional extent. Such research can also promote effective measures to cultivate, breed and create the right conditions for increasing their individual population in the natural community. In 1999, Huang and Zhang published a brief report on the first discovery of vessels in Zamia furfuracea of cycads [2]. Lin & Huang published another brief report on vessel features in the root of Cycas elonga [3], and another brief report on discovered vessel in the plant of Stangeriaceae [4], Huang and Liao [5] published a brief report on discovered vessel in Coniferae and Taxinae, and Huang and Wu [6] discovered vessels in Cycas panzhihuaensis. Huang et al. [7] reported the structural feature of vessels on four genera of cycads and some angiosperms; Huang et al. [8] published the structural characteristics of vessels in three families of Cycadopsida, Huang et al. reported the structural characteristics of vessels of some gymnosperms and angiosperms [9].
The present report is a comparative study on structural features of the xylem of these native cycads of Australia and America a cycad species and angiosperm, in an attempt further research and understanding their characteristics to more species of different regions...

Materials and Methods
The pinna and rachis of Cycas angulata, Cycas armstrongii and Cycas conferta which are Australia native species, pinna and rachis of Zamia erosa and leaf and stem of Guaiacum officinale which are introduced from America were collected from living trees in the George Brown Darwin Botanic Gardens, All materials were random sampling, therefore ensure whether these different plant taxon maybe almost all plants are having the characteristics under the condition of random sampling. After collection, these materials were cut into 1 × 1 cm pieces American Journal of Plant Sciences and fixed immediately in FAA (formalin: acetic acid: glacial ethanol 70%, 5:5:90), then macerated and dissociated by 10% nitric acid + 10% chromate solution, volume proportion 1: 1 (strict control dissociated situation, only then tissue disentwine was stoped at once) [10] [11], and rinsed with water (Jeffrey's method). Some dissociated and section materials were observed and made photography using light microscope; other materials were and were dehydrated in a graded series that 100% ethanol and isogamy acetate were according with the proportion of 1.0:0 → 0.3:0.7 → 0.5:0.5 → 0.3:0.7 → 0: 1.0 and, critical point-dried in CO 2 , then sputter-coated with gold for observation and photography by XL-30 ESEM and JEM-1200Ex/s scanning electron microscope (SEM).

Results
In the leaf, stem tissues of plants of Cycas angulata, Cycas armstrongii, Cycas conferta of Cycadaceae, Zamia erosa of Zamiaceae and Guaiacum officinale of angiosperm, their tracheary elements have perforations in end wall and lateral wall, numerous types of vessel elements were found. The structural constitution of tracheary element in gymnosperms of two families is the same or basic same as this family of angiosperm, but every species has itself feature in detail morphology character. The structural characteristics of these vessels are described next.

Cycas angulata
There are scalariform vessels, reticular vessels, pitted vessels and scalariform-pitted vessels in the pinna, rachis, the mesh of some reticular vessel elements is irregular ( Figure 1(1)), In these vessel elements, perforations are found in the lateral wall, while that of a portion of these elements, near pitted vessels, some reticular vessels, their septa or bars of ornamentation consist of secondary wall shape a lightly sloping arrangement, some have several ridges on the outside of the vessel element ( Figure 1(1), Figure 1(2)), there are more scalariform and reticular perforantions, while there are more near oval or circular perforations in side walls of pitted vessels. In some scalariform vessels, a side is scalariform, and another side is pitted perforations (Figure 1(1)).
In the xylon, there are many fiber elements, they are twisted shape and with a Y. Y. Huang et al.  (2) A pitted vessel element, the end wall was very incline, there was some preforations in end wall and some perforations in the lateral wall and some fibre elements which are twisted shape and are tip in the end. (3) A scalarifum-reticulate vessel element, some sections of the lateral walls have perforations, while others do not, in addition, on the outside of some pits, a pit membrane can still be seen. (4) Some pitted-reticular vessel, many perforations in the side wall, it has a near horizontal end wall with several perforations. In pinna, a reticular vessel, some reticular and near circular perforations in side wall, these perforations are 12 -15 μm length, and 4 -6 μm width. The pitted vessels, the diameter are about 30 -32 μm, in a side wall, some circular perforations arranged a line, in other sides, there still are primary wall or more primary walls in the outside of vessel elements, and can see inside secondary wall ornamentation ( Figure 1(2); Figure 2(3)). In pinna and rechis, the round vessels of Xylem of conduction tissue, there are some accessory transfusion tissue cells, they are near short rectangle shape, the side walls and the horizontal or near horizontal end walls have more pitted or pitted-rectangular perforations ( Figure 1(1), Figure 1(2)); the action of these are bearing the synergy to conduct aqueous solution with vessels of xylem in longitudinal and transverse directions, but the their synergy action main is transverse conduction, they connect other tissues, so that some aqueous solution easier entry these tissues, or opposite direction conduct some nutritive materials entry the vessels.
In the xylem, fibre elements is tip and twist-shaped ( Figure 2(1), Figure   2(2)); in some scalarifum vessel elements, some sections of the lateral walls have perforations, while others do not. In addition, on the outside of some pits, a pit membrane can still be seen; the few pit membranes that remained on the surface of perforations are threadlike, sheet-like, shred-like, and mesh-like in shape ( Figure 2(3)).
In some vessel element of scalariform-pitted vessel, the pits are intercolumn arrangement, some scalariform and pitted perforations in the lateral walls. They showed that a face of side wall is scalariform perforations, and another face of side wall is pitted perforations (Figure 1(4)). In some scalariform-pitted vessels, their end walls of vessel element are near horizontal shape, showed very evolutary characteristics (Figure 2(4)).

Cycas armstrongii
Some spiral vessels, scalariform vessels, pitted vessels and scalariform-reticular vessels are distributed in pinna and rachis. Multiple perforation plates were found in the end walls.
In some scalariform vessels, the septa or bars of ornamentation consist of secondary wall are longer, and more longer perforations in the side walls, the perforations length can reached 25 -28 μm with width 6 -7 μm (Figure 3(1)).
Some pitted vessels possessed a more acuate tip with very inclined perforation plate in the end wall with some perforations, the lateral walls of these pitted vessels have many perforations, in some scalariform vessel elements, their lateral wall possessed many perforations, in end wall, the extent of inclination is large, so that the end wall is rather difficult to distinguish from the lateral wall ( Figure   3(2)).
Y. Y. Huang et al.  Some spiral vessels are smaler, their side walls have more perforations ( Figure   4(1)), pitted vessels possess a more acuate tip with very inclined perforation plate in the end wall with some perforations (Figure 3(3)); in some pitted vessel elements, they are twisted shape, its lateral wall possessed many perforations, the extent of inclination is large so that the end wall is rather difficult to distinguish from the lateral wall, the lateral walls of these pitted vessels have many perforations ( Figure 4(2)).

Cycas conferta
In pinna and rachis, which contained spiral, scalariform, reticular, and reticular-pitted vessels, all vessel elements have more perforations in their end walls and lateral walls.
Some scalariform vessel elements are longer, and their side wall has more large perforations, the diameter is 34 -37 μm, some pitted vessels are bigger, the diameter are 42 -47 μm ( Figure 5(1), Figure 5(3)), in some vessel elements, some sections of the lateral walls have perforations, while others do not, on the outside of some pits, a pit membrane there are some web, silk, filiform, little shred-like membrane remnants in the perforation surface can still be seen ( Figure 5(1)).
In rachis, some spiral vessels near circular shape, in the lateral wall have two ridges which is consist of secondary wall, their side walls have many perforations ( Figure 5(2)). In vessel elements side, some parenchyma tissue cells, they all are without any perforation in their any walls ( Figure 5(2), Figure 5(3)). Some scalariform vessels, in the lateral wall have two ridges which is consist of secondary wall, curved strip partitions connected the redges, this should is the characteristics that from spiral vessel or annular vessel evolved to scalariform vessel ( Figure   5(2)). In rachis, some pitted vessels with twist-shaped, the lateral wall have more perforations ( Figure 5(4)).
Rachis, in reticulate-pitted vessel element, some end wall has a tip, and with two bigger perforations, one of them' perforation is 46 μm length and about 25  (1) Some scalariform and pitted vessel elements, rachis; (P: perforation); (2) Some scalariform vessels near circular shape, in the lateral wall have two ridges which is consist of secondary wall, their lateral wall possessed more perforations; some parenchyma tissue cells, they all are without any perforation in their any walls rachis; (Pa: parenchyma tissue cells); (3) Some pitted vessel elements, their lateral wall have more perforations, some parenchyma tissue cells, they all are also without any perforation in their any walls, rachis; (4) A pitted vessel element, it is a twisted shape, its lateral wall possessed some perforations, its pits are interlaced arrangement, rachis; (5) A reticulate-pitted vessel element, the end wall has a tip, and with two bigger perforations, rachis; (6) Some scalariform vessels possess very inclined perforation plate in the end wall, rachis. μm width ( Figure 5 Pa P Figure 6. (1) Several pitted-reticular vessel and the side wall has some little perforations with some silk, shred-like membrane remnants in the perforation surface, the end wall near horizontal shape, rachis; (2) Some pitted and scalariform-pitted vessel elements, their end walls are near horizontal, and the side walls possessed more perforations, some perforations with some web, silk, little shred-like membrane remnants in the perforation surface, pinna; (3) Several pitted vessel elements, their several side walls have more perforations, some little perforations with threadlike, sheet-like, shred-like, mesh-like or band-like in shape membrane remnants in the surface, their end walls near horizotal shape, pinna; (4) Several pitted vessel elements, some perforation in the incline end wall, rachis. Figure 6(3)). Two adjacent end walls that connect each other are also near horizontal status, in some vessels, the few pit membranes of web, little shred-likere shape mained on the surface of perforations ( Figure 6(2), Figure 6(3)).

Zamia erosa
In the rachis, the scalariform vessel elements have many scalariform perforations in the lateral wall (Figure 7(1)), some pitted vessels is a twist-shape, their lateral walls have more perforations (Figure 7(2)), all parenchyma tissue cells have only a primary wall, wherefore preserve their integrity of wall of thin membrane shape and are without any perforation in their walls (Figure 7  (1) Rachis, several scalariform vessel elements, in the lateral walls have many perforations surface, some perforations with some silk, filiform, shred-like membrane remnants; (P: perforation); (2) Several pitted vessels, their lateral walls have more perforations, rachis; (3) A scalariform vessel, its end wall is inclined, there are more scalariform perforations, rachis; (4) Some parenchyma tissue cells, they all are without any perforation in their any walls, rachis; (Pa: parenchyma tissue cells); (5) Some parenchyma tissue cells and fibre elements, all parenchyma tissue cells are without any perforation in their walls, rachis; (Pa: parenchyma tissue cells); (6) A bigger scalariform vessel, its side walls have more perforations, rachis.
results further justified also that perforations of vessel elements are the result of plant development, and these perforations are only formed in vessel elements, not formed in other parenchyma tissue.
In the pinna, several reticular vessel elements, the diameter are 23 -25 μm, they have an inclined wall, and some perforations in the end wall and lateral walls (Figure 8(1)). In the pinna, though they are together with those vessels which possessed perforations, these parenchyma tissue cells have only a primary wall, and have no perforations (Figure 8(3)), indicating that the primary wall is not dissolved to form perforation by enzymes [12]; perforations can be seen only in vessel elements. In the pinna, some scaraliform vessels's

Guaiacum officinale
In the stem, some spiral vessel elements are little, the diameter is 7.2 -8.2 μm, the lateral walls have some perforations, some perforations are 6.5 μm length, and 5.8 μm width (Figure 9(1), Figure 9(2); Figure 10(1), Figure 10(3)), the end wall is inclined. Some reticular vessels, the diameter are 12 -15 μm, the side walls have more perforations, a part of perforations are bigger, their diameter is 7 μm length with 4.2 μm width (Figure 9(3)). On the vessel element side, there are some accessory transfusion tissue cells, their near short rectangle shape and the side walls and horizontal or near horizontal end walls have more perforations ( Figure 9(1)). (2) Several spiral vessel elements, their side walls have many large perforations, (3) A reticular vessels, it's end wall is horizontal and side wall has more littile peforattions, stem. (4) Several pitted vessels, their end wall are twinst shape, formed a inclined wall and has a tip, in the end wall has a large perforation, stem. (5) Some reticular vessel elements and a spiral vessel, the former' end walls are horizontal or near horizontal, and some perforations in every end wall, formed multiple perforation plate, the lateral walls have also some perforations, stem. (6) The fiber element is thing and longer with a twinst shape, stem.
In the reticular vessels, the diameter is little, only 17 -18 μm, their side wall has more littile perforations; some reticular vessels near circular, the end wall are horizontal, in the side walls have some little perforations (Figure 9(5)). In aspect of pitted vessels, their end wall are twist shape, formed an inclined wall and has a tip, in the end, wall has a large perforation, the diameter of about 7.8 μm, this twist shipe shape is similar to other vessel elements of angiosperms [7] [9], belong to the more primary vessel type (Figure 9(4)).
The fiber element is a thing and longer with a twist shape, the characteristic is same as the fiber of other angiosperms [7] [9] and cycads ( Figure 9(6)). In leaf, (2) Some parenchyma cells, they are without any perforations in their cell walls; (Pa: parenchyma tissue cells); (3) A spiral vessel, its lateral wall formed more perforations, in its around, the cell walls of accessory transfusion tissue have also some perforations. (4) A pitted-reticular vessel element, its side wall and end wall have some perforations, its end wall is horizontal shape, stem; (5) Two pitted-reticular vessel elements with two fibre elements, the lateral wall of the vessel elements have many perforations, their end walls are inclined or tip shape, and there are several perforations formed multiple perforation plate, stem; (6) Two large reticular vessel elements, their side wall have many perforations and every end wall is inclined or near horizontal shape with a bigger perforation, it is whole perforation plate dissolved to form a single perforation plate, stem. the parenchyma cells are without any perforations in their cell walls ( Figure   10(2)).  (5)). The wall of parenchyma cells is like a thin membrane and is not perforated (Figure 10(2)), indicating that the primary wall are not dissolved to form perforation by enzymes [12]; perforations can be seen only in tracheary elements.
Around the spiral vessel elements, the cell walls of accessory transfusion tissue have also some perforations (Figure 10(3)). In the xylem of angiosperm, the accessory transfusion tissue is together with vessel, and the cell walls have some perforations have been not seen report in past time. These perforations are bountiful to aqueous solution of vessels cross transport entered to these tissues, then transport to leaf and other tissues.
In stem, some pitted-reticular vessel element, its side wall and end wall have some perforations, its end wall is horizontal shape, but some end walls of pitted-reticular vessel elements are inclined or tip shape (Figure 10(4), Figure   10 (5)). In some large reticular vessel elements of stem, their side wall have many perforations and every end wall is inclined or near horizontal shape with a bigger perforation, it is whole perforation plate dissolved to form a single perforation plate (Figure 10 (6)).

Analyse the Characteristics of Vessel
Seen these vessel elements of cycad species, their end wall have more inclined, few of them are near acuate, and more state are near horizontal shape, their end walls have several large perforations, some have more perforations, these characteristics are same as vessel of more angiosperms, even apart of characteristics are more evolution than that of angiosperms [7] [9]. In each species, the lateral walls of all types vessels have more perforations, and the diameter is large, these are scalariform, near circular and reticular, etc., these characteristics are the same as leaves of angiosperms [7] and stem of angiosperms [9], the function of perforations main is that making xylem vessel element not only can long transport aqueous solution, but also can cross transport the solution between the vessel elements. Thereby, caused xylems can play the largest overall harmonizes transporting action so that the tissues of root, stem and leaf can obtain the water and nutrients as used or stored.
Compare with these species of Cycadaceae and Zamia erosa of Zamiaceae which are distributed in America in those vessel characteristics of evolution or primary, they are not shown that the latter is more evolution than the former. In the former, the general view is that vessel diameter is bigger, the end wall is more horizontal is more evolutionary. Our research showed that more primary species of Cycas of Cycadaceae possessed more evolutionary vessel characteristics than Zamia of Zamiaceae.
The theory of comparative morphology suggests that vessel elements evolved gradually from tracheids whose end (bottom or top) is pointed in the shape of a cone. Therefore, one or two lateral walls gradually change to form near 90˚ di-   [21], or an elongate, open-ended cell [22]; and vessel element every end possess a or several perforations [13]. The tracheid concept is that tapered cells, with overlapping ends and often spiral thickening of cell wall, rather than being open-ended, perforations [22], tracheid is no perforation cells [13]. In this research, we observed that leaf and stem of Guaiacum officinale possessed accessory transfusion tissue, in the xylem of angiosperm, the accessory transfusion tissue is together with vessel, and the cell walls have some perforations that have been not seen report in past time.
Some vessel element of Michelia alba, Michelia figo and Amygdalus persica of angiosperms and cycads [7] [9], and the vessel elements of Guaiacum officinale possess a twisting shape, that can enable several adjoining vessel elements to connect together. Thus, such a structure improves conduction and confers greater mechanical support at the same time.
In this study, perforations were seen only in vessel elements, the cell wall of parenchyma cells only had primary walls without any perforations, although these cells of parenchyma were distributed in the around or very close to vessel elements, indicating that enzyme action has specificity for tracheary elements and not dissolve parenchyma cell walls, whereby they can preserve the integrity of the thin membrane-like wall.
Same as the vessel elements of angiosperms and gymnosperms [7] [8] [9], in this research, all species in our research, in these vessels, some sites have perfo-

The Measures of Research Vessel Elements
These characters studied here also demonstrate that the experimental methods are stable and scientific. Furthermore, these characters testify that like very few prior workers [23] who surmised that the pit membranes consist of primary wall which may easily be damaged in procedure using Jeffrey's method (10% nitric −10% Chromic acid, 1:1) are wrong. Because this method has been through many replications and shown to be stable and reliable, it does not damage pit membranes, so, since 1940, this important method, [24] is widely utilized to study vessel element structure including angiosperms, ferns, gymnosperms.
Most vessel research uses this method, only a few studies used other methods such as sodium hypochlorite in dissociated solution. These are among the strongest oxidizers, with oxidized intensity higher than Jeffrey's method solution. It should and must be noted, in these solutions of several experimental methods, the solution of Jeffrey' method is most moderate. And that the method procedure requires low than 60˚C, and stops isolation (maceration) then the tissues have to begin separated, this is the safest and stable method [9]. If who make so-called evidence want to testify that Jeffrey' method is a mistake, that who certainly is not in the light of the procedure of Jeffrey method, he certainly is used a such as overpass 100˚C situation, and after tissues have sepa- In the research of Carlquist and Schneider [23], measured which materials were cut sections 1 -2 mm thick by hand, then fixed in aqueous 70% ethanol, and were air-dried on a warming table, exhibited those figures which tracheary elements have many reticulate or porose pit membranes in ferns because they thought that their earlier studies on fern xylem were based on macerations prepared with Jeffrey's fluid. Macerations of dicotyledonous woods and even of xylem of monocotyledonous roots and stems [25] [26] tend to leave pit membranes in lateral walls of vessel elements intact; they, therefore, assumed that these methods would result in similar results with fern xylem. That assumption proved faulty because fern roots and stems are refractory when treated with Jeffrey's fluid. Successful macerations require prolonged treatment, apparently because the xylem is associated with thick fibrous sheaths. Although secondary wall architecture was not damaged by prolonged maceration, they believe that the integrity of primary walls was lost to various degrees by prolonged oxidative treatment. As such, Carlquist and Schneider [23] believed that revisions in their earlier reports are therefore required. Certainly presence of porous pit membranes, reported in their earlier studies, has been confirmed in this study, although with improved preservation and imaging. However, reports of multiple perforation plates, lateral perforation plates, and intermittent perforations in fern tracheids should be regarded as erroneous; reports of perforation plates lacking any pit membrane remnants in such genera as Microgramma and Vandenboschia [27] may be the result of excessive maceration; even if they thought that in their study to several species of cycad, they observed some characteristics which on the surface of pores or perforations were threadlike, sheet-like, shred-like, mesh-like in shape were the result of peroxide [25], but this is error.
But see to their figures of the research which were treated by macerations [27], their figures of experimental results had a very typical character because of artificially excess treated so that all pits in tracheary tissues were shaped perforations in all cell walls, these specimens are without any pit membrane was integrated. Their study had not the situation that all end walls possessed perforations and only a part of sites of lateral walls have perforations, and some sites were without perforation with there were integrated primary wall (without any reticulate or porose pit membranes). Thereby, their research result maybe was an excess treatment by maceration solution. Otherwise, they did not exhibit the pictures of cells of parenchyma tissues and were without any a picture which was that the cells of parenchyma tissues together with vessel elements, if they so excess treat, should have induced to these cells of parenchyma tissues formed perforation at the same time, but they did not notice this content.
They thought that this measure [23] which is cut sections 1 -2 mm thick by hand, using single-edged razor blades. Then fixed in aqueous 70% ethanol and air-dried on a warming table was mild, this is not sure. Because they only exhi- Because the materials with ethanol dried in air are not mild, because ethanol volatilizing quickly it easy make material quickly contract and drag, or transmutation. Thereby, almost all scholars use the new measure that dehydrated in a graded series of ethanol and isoamyl acetate and critical point-dried in CO 2 , because this measure is really mild [9].
Otherwise, their sections from hand used reamer was very easy formed damage to the primary wall that take off the materials of primary wall, those materials of the primary wall shall be stuck in reamer, and be peeled off, then these sections put in 70% ethanol solution, they formed like uniformity porose or reticular primary wall (pit membrane of tracheary element) at once like their figures [23]. These very deserve oppugn and more exploration. They thought also that artifacts, such as tearing or cracking of pit membranes, are readily recognized as stress-induced phenomena. Pit membranes are "striated" (minutely corrugated) occurred both in their earlier studies and in the study [23] ( Figure   3, upper left) and very likely result from kinds of stress, such as heating by the SEM electron beam. Such stress artifacts are easily recognizable as different from the reticulum of pit membranes that results from natural hydrolysis of the cell wall [23].
But this is ungrounded, because if primary wall can be heated by the SEM electron beam so that formed perforation, the process which is hit form perforation shall certainly be seen, because that is impossible occurred that then tracheary element entered the field of vision of SEM, at once all are heated to artifact perforation in split second, they certainly have a changing process can be watched by observer, in our many pieces of research, we were all without seen the phenomena [7] [8] [9]; Moreover, those materials of plants had been sputter-coated a layer gold membrane in their outside, therefore they were firmly fixed and safeguarded, thereby they complete could not be heated formed perforation by SEM.
Otherwise, it is none proof showed why heating by the SEM electron beam stress only locate in tracheary elements, and they were heated formed perfora- They thought also that observed from inside tracheary elements of the several species of cycads there had some spiral secondary thickening, look like pits [28].
But we thought that any secondary wall is formed inside of primary wall of plant tracheary elements, they have spiral, scalariform, pitted and reticular ornamentation, and the primary wall like a membrane, they located in periphery of tracheary elements, then formed perforations, it is the periphery primary walls be dissolved by enzyme, so these perforations are without any relations with those inside ornamentations, and observe the perforation of tracheary element ought to observe the primary wall situation in outside of the tracheary element and are not observe inside of them. In addition, in our former researches, observed from inside of vessel element, many were without seen the spiral thickening phenomenon [7] [8] [9].
Any vessel element is all need to pass through the individual development process that it from the shape which is without any wall perforation, to the shape which wall (pit membrane) have retained thread-like, sheet-like, shred-like or mesh-like membrane remnants of the primary wall in the periphery of the perforations. With some not complete perforations, these remnants are evidence that the primary wall gradually developed small or more than a half perforations, and eventually these wall remnants completely disappeared, leaving large, entire perforations [13] [15] [16]. As these perforations differentiated, the dissolving membrane remnants were reabsorbed by the plant, until the membranes completely disappeared, then developed to the shape that possessed some or many complete perforations. This is a vessel individual development phenomenon; these characteristics exist in this research, these results justified also that this is an individual development process. These are not like the formers [ Thereby, we thought that their hereinbefore parlances have many errors, and are a subjective guesswork.
In our former research [9], we used the method as the literature [23] [28], it is without any dissociated fluid, see the structural characteristics of rachis of Cycas szechuanensis (Figures 4(A)-(D)), all longitudinal section and cross section were the situation that perforations exist in tracheary elements and without perforations in parenchyma cells, especially the cross section (Figure 4(D)), can observed some larger perforations located in end walls and lateral walls inside vessel pipe hole; in these places, where are without go through any dissociated situation, and are without any touch by tool in the process of sectioning by hand, therefore, these perforations are most pure natural situation; these structural characteristics are same as the method that used the isolation process of Jeffrey's.
These results also further adequately proved that Jeffrey's method are scientific and reliable.
In this research, seen the cross section of rachis of Cycas szechuanensis ( On the question, some scholars have already made a specialized comparative experimental study [29], They to the secondary xylem from five woody species used as following four methods made experiment, include the fresh materials were cut sections 1 -2 mm thick by hand [23], in low vacuum and then the same  other places which possessed whole perforations in the wall of vessel, they not observed and not described [9]. So, the first, although their so-call new measure is simple and crude, it was also could basically be used, but the effect is lower than other measures; the second, their reports in several papers which were without any whole vessel element or more proportion of a vessel element can be viewed to the all vessel element' or more places and perforations by reader, only were some very partial sites which have only one or several pits [9]. So these measures and thought [23] [28] are more subjective, on-sidedness and mistake. the latter only has one more evolutionary species and lack of fossil of Cycadaceae [31], of course these species of Cycadaceae are the descendants of ancestor species. America possessed more genera of Zamiaceae, this family is mutual with Africa and Australia, we thought that these genera of Zamiaceae in Australia and America came from Pangea before continental drift in Jurassic Period; this family is also maybe one of proofs to Australia drift from Pangea to present position after Jurassic Period.

Analyse the Evolutionary Extent of Vessel Element of the Two Families
Zamia erosa is the species of Zamia, it was introduced from America, seen these cycads of the research, the species which possess the most primary characteristics is C. armstrongii, its more proportion of end walls of vessel are inclined to shape, and the degree of inclination is bigger. But the characteristics of vessel of C. angulata and C. conferta evolution are more revolutionary, showed the characteristics that the degree of inclination of more end walls of vessels are smaller, and more end walls of vessel are horizontal or near horizontal; Zamia erosa is one of species of the most evolutionary family in Cycadopsida, although it is more evolutionary in the characteristics of vessel than some angiosperms [7] [9], compared with the 3 species of Cyacdaceae, the evolution degree is a slightly higher than C. armstrongii, but lower than that of C. angulata and C. conferta.

Y. Y. Huang et al.
These showed also that more primary species or taxon, their more tissue characteristics are not also more primary, because of the need to adapt the environment, and they are also constantly evolving.

Conclusions
1) In this leaflet and rachis of cycads and leaf and stem of Guaiacum officinale, there are annular vessels, spiral vessels, scalariform vessels, reticular vessels, pitted vessels and scalariform-pitted vessels in their conduction tissues. In the transverse section, the vessel elements of Cycadaceae and Zamiaceae are circular, polygonous, more similar to vessel characteristics of petiole and stem of some angiosperms; however, the number of circular vessel of Guaiacum officinale were more.
2) The length of vessel element of Cycadaceae with Zamiaceae are not obvious difference, the length of vessel element of angiosperm Guaiacum officinale is shorter, but their diameter is smaller, we thought that the characteristics which the length of vessel element is longer and the diameter is bigger are more evolutionary, because thus vessel, their number which interconnected points of possessed perforations' end walls of vessel element is fewer, and the passageway space is larger so that the conduction speed and quantity are faster and more in unit length range and unit time.
3) The diameter of vessel is that annular vessel, spiral vessel is least, scalariform vessel is medium, reticular vessel and pitted vessel are the biggest, the characteristic is the same as angiosperm. The scale of diameter of Cycas vessel element is similar to that of Zamia, but the diameter of more vessel elements of Cycas angulata and Cycas conferta are bigger than the latter, and is bigger than that of Guaiacum officinale, although Cycas plants are more primitive than Zamia plants, and Guaiacum officinale is more evolutionary, but the scale of vessel diameter of Cycas plants is similar to Zamia or bigger than the latter, even larger than Guaiacum officinale, these showed that cycads have evolutionary and more developed characteristics in aqueous solution transport system. 4) Like cycads, in Guaiacum officinale, around the vessel elements, there are accessory transfusion tissues; their cell walls have also some perforations. 5) Different to other cycads or angiosperms species, more vessels of leaf and brach of stem of Guaiacum officinale have not seen the ridges in the different directions of side walls which can make vessel has more mechanical supportability, but some possessed the ridges; however, more vessel elements are twist shape, and can twist together each other, thus the state can increase their mechanical supportability, and their synergy with fiber elements which is longer with a twist shape, therefore their capability of xylem conduction and the combine mechanical support capability with mechanical tissue are also stronger. 6) Combined the comparative studies on research methods that have been made by some scholars made discussed and analysed with our research showed that any parenchyma tissue cells are without perforation, all cell walls are smoothed American Journal of Plant Sciences and like a membrane, justified Jeffrey's method is very reliable and reasonable; only vessel element can form perforation, and in other, all parenchyma tissue cells cannot form perforation are also proved that the perforations of vessel element are natural inherited character. 7) Although they are distributed so far apart from each other, they are a rather well and unified taxonomy system, Australia' most species or all species of Cycadaceae came from Asia, and it is impossible from Africa, because the latter only has one more evolutionary species and lack of fossil of Cycadaceae, these species of Cycadaceae are the descendants of ancestor species; America possessed more genera of Zamiaceae, this family is mutual with Africa and Australia, we thought that these genera of Zamiaceae in Australia and America came from Pangea before continental drift in Jurassic Period, this family is also maybe one of the proofs to Australia drifted from Pangea to present position after Jurassic Period.
8) The characteristics of tracheary element of several different genera of cycads and the angiosperm further showed that more primary species or taxon, their tissue characteristics are not also more primary, because of the need to adapt to the environment, they are also constantly evolving.