Diversity of metaxylem vessel elements in three Syagrus palms ( Arecaceae ) of diff erent habits

(Diversity of metaxylem vessel elements in three Syagrus palms (Arecaceae) of diff erent habits). Vessel elements in Monocotyledons present morphological diff erences according to the organ where they occur. Usually such diff erences have been explained from an evolutionary perspective, with few data on how the vessel elements could be infl uenced by the diff erent functions of organs and the growth habits of the plants. To address this question, three vegetative organs of palms of the same genus, Syagrus, growing in similar environments, but with diff erent habits, were analyzed. Accordingly, we aimed to detect whether the vessel elements would present similar features in all species or whether the vessel elements would change according to their diff erent habits. We found that the width and type of perforation plates varied in the same way among all species, while the lengths varied in an unusual form. First, all species presented very long elements in the roots, either as long, or longer than those of the stems and leaves. Second, the vessel elements of the stems varied considerably among the species. Specifi cally, in Syagrus romanzoffi ana, vessel elements of the stem were equal in size to those of the other organs, while in both Syagrus fl exuosa and Syagrus petraea, shorter vessel elements were found in the stems. We surmise that vessel elements in palm roots may be related to the high pressure-potential required to avoid stem embolism. Th e dimorphism of the vessel elements in the stems most likely refl ects the distinct habits of these species. Large stems, such as of those of the arboreal palms, presented much longer vessels than those of subterranean stems. Based on these anatomical fi ndings, we suggest that the diff erences found among the vessel elements of roots, stem, and leaves may have evolved in response not only to phylogenetic and ecological constraints, but also to specifi cities derived from the diff erent plant habits.


Introduction
Vessel elements in Monocotyledons are known to present marked morphological diff erences among distinct organs of the same plants.Such diff erences include length, width and perforation plates.Th is diversity, in turn, has been directly correlated to the origin and specialization of vessel elements across the diff erent organs of the Monocotyledons.Since vessel elements are thought to have evolved from tracheids, vessels with features similar to them would be regarded as the most primitive, while those with the most dissimilar features would be regarded as the most advanced (Bailey & Tupper 1918;Frost 1930a;b;Bailey 1944).Continuing this logic, these authors further suggested that vessels that are long and narrow with multi-perforated plates would be considered the most primitive and less specialized, while short, wide and simple perforated vessels would be considered both the most advanced and specialized.In a series of studies, Cheadle (1942;1943a;b;1944;1953) described the characteristics of the tracheary elements in a sample of over 300 species belonging to more than 200 genera from most Monocot families and found root vessels to be more specialized than those of either stems or leaves.Accordingly, root vessels would display short, wide and simple perforated plates, while the leaves would present long, narrow and multi-perforated plates.Stems would present intermediate features between roots and leaves.Th ese nearly consistent diff erences seen in the Monocot organs led Cheadle (1943a;b) to infer that the vessel elements in Monocotyledons would have fi rst appeared in the roots, then at the stems and fi nally in the leaves.Th is presumption was supported by the fact that some Monocotyledons would present vessels only in the roots (Cheadle 1943a;b), and in these cases, such vessels would display less specialized features.
Born in a gradist context, the order of specialization fi rst proposed by Bailey & Tupper (1918), and later reinforced by Cheadle (1943a;b), fi nally found support in cladistic studies (Carlquist & Schneider 2002) and in a number of studies with the Monocotyledons (Carlquist 1975;Parthasarathy & Klotz 1976;Klotz 1978a;b;Carlquist & Schneider 1997;1998a;b;2006;2007;Schneider & Carlquist 1997;1998;2005).Th e long series of studies by Carlquist & Schneider (cited above) showed, moreover, that in some families not possessing vessels in stems or leaves, as recorded by Cheadle (1942), vessels would otherwise be present, but with perforations only recognizable through scanning electron microscopy (SEM).Th e reason their perforations were only visible by SEM came from the fact that these perforations possess many pit membrane remnants, a phenomenon interpreted as an indicator of an intermediate stage of perforation plate formation in these plants.Pit membrane remnants were also seen in basal Angiosperms (Schneider & Carlquist 1995;1996) and ferns (Schneider & Carlquist 2000a;b;Carlquist & Schneider 2001;2007).Th ese fi ndings support the idea that the perforation plates would have evolved from the dissolution of the pit membranes in the pit fi elds, as previously proposed by Bailey & Tupper (1918) and Bailey (1944), and that their presence would indicate an intermediate stage of specialization.
Regarding ecological factors, Cheadle and Whitford (1941) and Cheadle (1943a;b) asserted that the diff erences among vessel elements across the plant organs are relatively independent of the environment.Th is is an opinion, however, not entirely shared by Carlquist (1975), who suggested that the Monocotyledons with the most primitive features would actually be growing in aquatic or mesic environments, while those with more advanced features would grow in seasonal environments with a marked dry season.Th is suggestion was further supported both by ecological and experimental studies, which illustrate that the vessel elements tend to show convergences independent of their phylogenetic relationships for plants growing in similar environments (Maherali et al. 2004).For instance, vessel elements tend to reduce in size from mesic to xeric environments (Baas et al. 1983).In addition to convergences, reversions have also been found within many groups, such as Aquifoliaceae in the Eudicots, showing that the so-called "primitive features" may sometimes be selected over "advanced features" (Sperry et al. 2007).In such cases, the morphology of vessel elements is related to both the effi ciency of water conduction and protection in the event of embolism (Carlquist 1975;Baas et al.1983), as well as phylogenetic infl uences.Th at is, while the so-called less specialized vessel elements, with long, narrow and multi-perforated plates, are considered less effi cient for water conduction, they are, at the same time, more protective against embolism since they are more easily refi lled when embolism happens (Canny 2001;Sperry 2003).
Other factors also seem to aff ect the morphology of vessel elements, namely, the function of a given organ and the growth habit of a plant.Storage organs such as rhizomes, for example, were shown either not to possess vessels or to possess vessel elements with more primitive features (Cheadle 1942;Carlquist & Schneider 2006).Th e presence of less specialized vessel elements was related to the apparent slow rate of conduction presented to these organs (Carlquist & Schneider 2006).In terms of growth habit, vessel elements of monocot vines are known to be the widest vessels among the Monocotyledons (Klotz 1978a;b;Tomlinson et al. 2001;Fisher et al. 2002), even though diff erent genera may present diff erent perforation plate types, either simple or multiple (Klotz 1978a;b), thus rebutting a direct correlation between growth habit and level of specialization.Hence, in spite of the clear correlation between the form of vessel elements and the environment, how plant habit might infl uence the morphology of vessel elements remains elusive.One way to address this question would be to examine the morphology of vessel elements in closely related taxa growing in similar environments, but with diff erent habits.
In the present study, we therefore analyzed three palms of the same genus, Syagrus, growing in similar environ-ments, but with fairly diff erent habits.Using this approach, we aimed to detect whether the vessel elements would present similar features in all species, given the taxonomic proximity of these species, or whether the vessel elements would change according to the diff erent growth habits.

Material and methods
Three species of Syagrus (Arecoideae, Cocoseae; Dransfi eld et al. 2005) displaying three diff erent growth habits were sampled in their natural habitats (Table 1).Th e genus Syagrus is neotropical and widely distributed, presenting a considerable range of habits.Here, a singlestemmed solitary palm, a caespitose suckering palm and a palm with underground stem were studied (Table 1).Moreover, at least three individuals of each species were entirely collected (Table 1), and they were all growing in environments marked with seasonal water stress periods.In fact, both the Restingas of São Paulo (forest formations between the dunes and the rainforest) and the Cerrado (Neotropical savannas) possess alternating wet and dry seasons.Our idea was to reduce phylogenetic and environmental variables to the extent possible in order to test exclusively the infl uence of the growth habits in vessel element morphologies.
All specimens were immediately fi xed in FAA 70 (10% formalin, 5% acetic acid, 70% ethanol; Berlyn & Mikshe 1976) and then stored in 70% ethanol.Analyses were made from roots, stems and leaves.Both roots and stems were sectioned in a sliding microtome.Th e roots and stem of Syagrus petraea had their entire circumference crosssectioned, while the remaining stems were too wide and were therefore cut from cubic samples of 4 cm 3 .Finally, for the leaves, we made free-hand sections of the midrib and adjacent leaf blade.Sectioning palm organs can be extremely challenging, since they possess very hard vascular tissues embedded in a matrix of parenchyma.To mitigate this problem, small sections of the roots, stem, and mid portion of the leaves were soft ened in 10% ethylenediamine for up to four days (Carlquist 1982) within a hot chamber at 58 o C. Samples were then gradually embedded in increasingly concentrated solutions of polyethylene glycol 1500 starting at 10% and 20%, until a 100% polyethylene glycol medium was obtained, allowing one day at each concentration (Rupp 1964, modifi ed).Samples were subsequently sectioned with the help of anti-tearing resins made of an expanded polystyrene (foam) solution applied upon the stem before sectioning in a sliding microtome (Barbosa et al. 2010).Sections were double-stained in Astra Blue-Safranine 9:1 in 50% ethanol, a modifi cation of Bukatsch (1972), and mounted in Canada Balsam to make permanent slides.Observation of vessel elements also required the organs to be macerated in a solution of 50% Acetic Acid and 50% Hydrogen Peroxide overnight (Franklin 1945), with subsequent staining in 1% Safranine and 50% ethanol and mounted in semi-permanent slides with glycerin.
At least 50 slides with macerated material were mounted per organ per specimen, and all the quantitative data on length and width were obtained using the Zeiss KS 100 soft ware.Deciding how many vessel elements would be measured per organ per species was made possible by applying the equation for sample size proposed by Eckblad (1991).According to this analysis, 30 vessel elements per organ per species were shown to be a reasonable number to sample.Subsequently, t-tests were performed to investigate whether the means for the length and width of vessel elements were statistically diff erent in the distinct organs.In addition, it should be noted that only metaxylem vessels were analyzed.
To assure that we were analyzing metaxylem vessels, we considered exclusively those vessels that had bordered pits, since protoxylem vessels were either tracheids or vessels with helical and/or annular thickening.
For qualitative analysis, the diversity in vessel element morphology was captured both in the anatomical crosssectioned samples and in the macerations.Such diversity was registered in line drawings produced with the aid of a camera lucida.

Vascular Anatomy
Proper identifi cation of the distributional pattern of vessel elements in the vascular system of diff erent plant organs depends on anatomical analysis; therefore, a brief description is given for each organ.
All roots collected in Syagrus are stem borne (adventitious) (Fig. 1A).Th eir vascular system is formed by protoxylem poles radially followed by metaxylem vessel elements, in alternation with primary phloem (Fig. 1A).Considering the tracheary elements, the increase in width from the protoxylem to the metaxylem is evident, with the late metaxylem vessels being the widest vessels in the roots.Th ere are as many as 20 protoxylem poles.Moreover, when the root is more mature, it can be observed that a great part of the parenchyma becomes sclerifi ed and surrounds all the conducting cells (Fig. 1A).Th e stems are marked by the presence of vascular bundles and fi ber strands scattered in a matrix of parenchyma (Fig. 1B), with two distinct types of vascular bundles detected, some with proto-and metaxylem and others with metaxylem alone (Fig. 1B).In addition to the tracheary elements, the vascular bundles are also composed of primary phloem surrounded by a wide and thick cap of fi bers (Fig. 1E).
Th e leaves of Syagrus possess reduplicate leaf segments with the midrib presenting a central vascular system composed of one large bundle with proto-and metaxylem associated with several primary phloem strands and additional vascular bundles on the sides possessing only metaxylem, the latter juxtapositioned with primary phloem (Fig. 1C).Such vascular system is surrounded by a sheath of fi bers (Fig. 1C).All other bundles in the leaf blade are smaller and present proto-and metaxylem vessels associated with primary phloem, either totally or partially surrounded by a sheath of fi bers (Fig. 1F).In our analyses, we preferentially sampled vessels from the central vascular system of the midrib.

Dimorphism of Vessel Elements
All the numerical values for metaxylem vessel elements can be found in Table 2.Moreover, the diversity of vessel elements, as found in root, stem, and leaves, is presented in Figure 2 in the form of line drawn pictures.It should be noted, however, that Figure 2 shows the diversity of vessel elements found across all organs, not the most abundant vessel element types present.A detailed description of vessel element morphology is provided below.

Perforation plate
Th e perforation plates show an identical pattern in all three Syagrus species.In the roots, there was a predominance of simple and transverse perforation plates (Fig. 1G), with only a few vessels presenting slightly inclined perforation plates with few bars (up to 5; Fig. 2A, D, G).Although some elements presented simple plates in the stems, practically all vessels presented slightly inclined perforation plates with few bars (from 2 to 7) in at least one extremity of the vessel elements (Fig. 1H, 2B, E, H), while on the leaves, all vessels presented inclined perforation plates with many bars (Fig. 1I, 2C, F, I), usually more than twenty.

Width
Th e widths of the vessel elements were consistent in all species studied.Th e vessels in the roots were always the widest (Fig. 1D, 2), with means ranging from 167-272 mm, while the vessels in the leaves presented the narrowest diameters (Fig. 1F, 2), with means ranging from 30-50 mm.Th e vessels in the stems were intermediate (Fig. 1E, 2), ranging from 60-130 mm.Th ese results indicate a decrease in width from the root to the leaves, something corroborated by statistical analysis with P < 0.05.

Length
Contrary to the quantitative consistency in perforation plates and width, much more variability was identifi ed in the lengths of vessel elements in the three Syagrus species studied (Table 2, Fig. 2).For Syagrus romanzoffi ana, all vessels presented similar lengths, from approximately 1300-1700 mm (P > 0.05), while for Syagrus fl exuosa and Syagrus petraea, the stem vessel elements were signifi cantly shorter than those of the root and leaves (P < 0.05).In fact, Syagrus fl exuosa presents roots and leaves with vessel elements of similar means (P > 0.05), ranging from 1100-1600 mm, while the stems possess a considerably diff erent mean (for Syagrus fl exuosa: 940 mm; P < 0.05).For Syagrus petraea, the diff erences were even more pronounced, with stem vessels having less than a third of the length of either root or leaf vessel elements (mean 300 mm).

Vascular parenchyma Paratracheal contact parenchyma tissue
Parenchyma was found closely associated with the vessel elements in all species examined (Fig. 3A-B).Th ese contact parenchyma cells are rectangular in shape (Fig. 3B-C) and present simple to semi-bordered scalariform pits in contact with the vessel elements (Fig. 3B-C).Moreover, they are so intimately associated with the vessel elements that it is possible to see their shape impressed on the vessels even in longitudinal section (Fig. 3B) or in dissociated material (Fig. 3A).

Discussion
Our results confi rm the results of previous investigations in that the vessel elements present marked diff erences between the organs of the Monocotyledons.Importantly, however, it also shows some newly discovered variations in the lengths of vessel elements among the organs.In fact, while the perforation plates and width presented exactly the same results previously reported for other Monocotyledons (Cheadle 1942;1955;Cheadle & Kosakai 1975), i.e., decrease in width and increase in the number of bars in the perforation plates from roots to leaves, we found two unusual patterns for Monocotyledons: i) very long vessel elements in the roots, as long or longer than those of the stems and leaves, and ii) variability in the length of vessel elements in stems., root transverse (TS) section showing vascular cylinder.Several protoxylem poles followed by metaxylem vessels (arrows).Sclerifi ed parenchyma with some portions not sclerifi ed (asterisk).B. Syagrus fl exuosa (Mart.)Becc., stem (TS).Vascular bundles scattered in a matrix of parenchyma cells.Note bundles with proto-and metaxylem (arrows) and bundles with exclusively metaxylem vessels (asterisks).C. Syagrus petraea (Mart.)Becc., leaf-segment midrib (TS), showing a central vascular system with a robust bundle with protoxylem (arrow) and metaxylem associated with several phloem strands (asterisk) and an additional bundle upon it with exclusive metaxylem associated with a phloem strand (asterisk), each surrounded by a sheath of fi bers.D. Syagrus fl exuosa (Mart.)Becc., detail of a root (TS) showing protoxylem poles followed by metaxylem vessels in alternation with primary phloem (asterisks).E. Syagrus romanzoffi ana (Cham.)Glassman, vascular bundles with metaxylem (arrows) juxtapositioned with primary phloem and a large cap of fi bers.When analyzing the lengths of the vessel elements, phylogenetic, environmental and functional aspects must be considered.Phylogenetic aspects were discussed by Cheadle (1942) and Cheadle & Kosakai (1975) who showed that the vessel elements tend to be more specialized in the roots and less specialized in the leaves.Similarly, within the idea that one group could have given rise to other groups, Cheadle (1955) showed that the families considered to be basal would coincidently be those possessing less specialized vessels across all organs, while the contrary would be true for those families thought to be derived.Here, contrary to what is known for other Monocotyledons, we found vessel elements in the roots to be as long, or longer, than the vessel elements found in the stem and leaves, a feature which, as noted previously, is considered to be more primitive, i.e., less specialized.Nonetheless, palms are within Arecales (Commelinids), one of the terminal branches in monocot's phylogeny (APG III 2009); thus, if Syagrus palms present long vessel elements in the roots, it contradicts the trends suggested by Cheadle.
Environmental or ecological features are also known to aff ect the form of vessel elements, with the length of vessel elements decreasing from mesic to xeric environments (Baas et al.1983).Nevertheless, the three Syagrus species analyzed here grow in a similar seasonal environment, i.e., alternating wet and dry seasons, and would be expected to have short rather than long vessels in the roots.
Th e answer for why the vessel elements in the roots are so long may be, then, in their functional role.Specifi cally, long vessels may permit the creation of high root pressure potentials (Davis 1961).High root pressure potentials are known to be eff ective in avoiding and repairing air embolisms.Moreover, since monocotyledonous stems also rely upon the same vessels throughout their lives (Sperry 1985), it can be expected that such a mechanism would be present to avoid embolism and stem tissue damage.High root pres-  sure potential may be one of the most important means of avoiding the formation of gas embolism in palm stems (Davis 1961), with the probable involvement of high pressure potentials in embolism repair during either the wet season, aft er prolonged rain, or during the night.Another system that could aid in refi lling vessels post-embolism is the presence of paratracheal parenchyma closely associated with the vessels (Braun 1984), as found in the three Syagrus species studied.Indeed, it has been suggested that vessel-associated cells could create a positive osmotic gradient between the parenchyma cells and the vessel elements, intermediated by the plasma membrane of the contact parenchyma cells, so that these parenchyma cells could regenerate water fl ux in previously embolized vessels (Czaninski 1968;1977;Sauter 1981;Braun 1984).
Besides the phenomenon of high root pressure potentials, the presence of leaf to stem hydraulic constrictions in palms is thought to be another efficient mechanism to avoid stem vessel embolism (Sperry 1985), showing that palms possess complex mechanisms to avoid stem damage.Hydraulic constrictions are specific regions between the stem and leaves where only protoxylem is present.Under these conditions, the vessels in the leaves would be more susceptible to embolism, but the vessels in the stems more protected (Sperry 1985).The presence of long, narrow and multi-perforated plates in the vessel elements of the leaves is another mechanism that helps in embolism repair (Sperry 2003).
In addition to very long root vessel elements, we identifi ed diff erences in the lengths among the vessel elements of closely related taxa growing in similar environments, a phenomenon which had never been recorded.In the aerial stem of Syagrus romanzoffi ana, the stem vessels were as long as those in the roots and leaves.In Syagrus fl exuosa, on the other hand, stem vessels were found to be shorter than those in the roots and leaves.Finally, in Syagrus petraea, the stem vessels were more than three times shorter than the vessels of roots and leaves.We cannot explain why the vessels in Syagrus romanzoffi ana would all possess the same length.For Syagrus fl exuosa, the presence of vessels in the stem, which are shorter than those in leaves, agrees with what is expected for Monocotyledons as a whole (Cheadle 1942).However, the presence of very short vessels in Syagrus petraea, which is totally dissimilar to the vessels found elsewhere in the plant, may indicate the functional specifi city of this subterranean organ.Studies with other storage and succulent organs, such as rhizomes, obtained similar results, with either the absence of vessels (Carlquist & Schneider 2007) or the presence of minute, less specialized vessels (Carlquist 1975;Carlquist & Schneider 2006;2007).Such storage organs are thought to possess very low water fl ow rates and act primarily as water storage organs (Carlquist & Schneider 2007).
In terms of width and type of perforation plates across distinct plant organs, we can conclude that the vessel elements of Syagrus palms share the same types of variation with all Monocotyledons.However, based on the anatomical results of our study, these palms also present a previously unknown variation in the lengths of vessels elements, most likely related to both growth habit and organ function, as we have demonstrated.Therefore, the present study provides evidence that the differences found among the vessel elements of roots, stem and leaves of the Monocotyledons may have evolved in response not only to environmental and ecological constraints, but also the growth habit and function of the organs within the plant.

Figure 2 .
Figure 2. Morphological diversity of vessel elements across root, stem and leaves.A-C.Syagrus romanzoffi ana (Cham.)Glassman.D-F.Syagrus fl exuosa (Mart.)Becc.G-I.Syagrus petraea (Mart.)Becc.A, D, G. Very long root vessel elements with predominantly simple perforation plates.B, E, H. Stem vessel elements vary greatly in length among the species, but predominantly have multiple perforation plates with few bars.C, F, I. Leaf vessel elements are very long and narrow, with multiple perforation plates.Scale bar: 400 μm.

Table 1 .
List of studied species and collection details.* *All species possess voucher in the SPF herbarium.

Table 2 .
Numerical values for the vessel elements of the three Syagrus palms studied.Numbers represent means with their standard deviations aside.