Both live material and preserved herbarium material (Table 3) were used in this project. The living material used in this study came from the collections at Montgomery Botanical Center (MBC, Miami, FL). The dried material was from collections made while doing fieldwork in Brazil, from the garden and herbarium at Jardim Instituto Plantarum (HPL, Novo Odessa, São Paulo, Brazil) and from dried collections at the Fairchild Tropical Botanic Garden herbarium (FTG, Miami, FL) and a few specimens from the following herbaria: G, IBGE, IPA, K, MO, NY and US.

Two methods were employed for expedient identification. First, one side of the middle section of a middle leaflet was folded back and forth on itself in accordion fashion; the folded leaflet was then held down on a cutting board, while using a double-edged razor blade to cut thin cross-sections. The sections were rinsed into a watch glass with water and a thin brush was used to select the thinnest sections under a dissecting scope and then placed on a microscope slide in a droplet of 1:1 glycerin/water solution. A cover glass was placed over the specimen and the slide was placed under a compound light microscope and photographed under the 10× objective (100× magnification). Most of the sections were unstained, but in rare cases toluline blue (0.01%) was tested to see if it made it easier to view certain characters, which it did not (Fig. 3C, 6D).

In the second method, better suited when material is limited, a small square of carrot of the appropriate size is cut to fit in an inexpensive hand-held student microtome. I purchase my hometrainingtools hand-held microtome online. A vertical slit is cut in the carrot and a small piece of leaflet is inserted in the appropriate orientation. The carrot is clamped into the hand-held microtome. The microtome is screwed to the appropriate level and an ordinary folding straight edge razor, the kind used for shaving, is utilized to cut the cross-sections and honed occasionally to keep it sharp. Sections are handled the same way as above. Scale was later added using a stage micrometer. Dried material can also be sectioned and photographed after rehydrating in a 5% solution of Contrad 70® (Decon Labs, King of Prussia, Pennsylvania) for a period of 24 hours (Tomlinson et al. 2011).

This paper is focused mainly on characters of the more easily sectioned marginal and laminal portions of the leaflet and not so much on the harder to section midrib. Trichomes, epidermis and dark staining bodies were also not looked at.

Characters examined during this study follow some of Glassman’s 4, 5 and 10 characters listed above and Tomlinson’s characters 4, 5, 6, and 7 listed above. Figure 2 will clarify much of the terminology and characters used in this paper. In each leaf cross-section the upper or superior side of the lamina is called the adaxial surface, meaning “towards the axis”, since this side of the leaf faces towards the axis or center of the plant as it grows out. The lower or inferior side is called the abaxial, meaning “away from the axis”, since this side faces away from the center of the plant (Dransfield et al. 2008, Esau 1977). The outer most layer of the leaf is the cuticle (Fig. 2D, 2E green arrow), a non cellular waxy layer produced by epidermis (Dransfield et al. 2008). The cuticle is followed by the epidermis, “outer skin” (Fig. 2D, 2E orange arrows), followed by the hypodermis, “under skin” (Fig. 2D, 2E purple arrows), which is finally followed by the mesophyll, “middle leaf”, region (Fig. 2 white stars). Within the mesophyll are vascular bundles, or fibrovascular bundles or veins of various sizes (Tomlinson et al. 2011) that will be referred to as major veins (Fig. 2 white arrows), intermediate veins (Fig. 2 blue arrows), and minor veins (Fig. 2 pink arrows). Some major and intermediate veins are often attached to the adaxial hypodermis and sometimes to both the surfaces by fibrous sheath extensions. If the attachment extends to both surfaces via a fibrous sheath extension, the vein becomes girder-like and is indeed referred to as a girder (Tomlinson et al. 2011) (Fig. 5A). In some veins the fibrous sheath becomes so enlarged with fibers that such veins are referred to as veins with exaggerated fiber sheaths (Tomlinson et al. 2011) (Fig. 2 black arrows). In addition to the veins, the laminal tissues are supported by nonvascular fibers or fiber bundles of various sizes. Some have major fiber bundles adjacent to or near their margins (Fig. 2 yellow arrows). Many fiber bundles are adaxial and may reach close to 1/2 the distance across the mesophyll (Fig. 2B, 2D). Minor, intermediate and major fiber bundles can be found adaxially (Fig. 2A red arrows; 2B, 2C yellow arrows). Most minor fiber bundles are mainly abundant abaxially (Fig. 2B, 2C red arrows) and occasionally scattered throughout the mesophyll (Fig. 2C red arrows).

To keep things simple for field examination, the following qualitative characters were examined: (1) location, attachment or lack of attachment of the major veins to one or both surfaces and method of attachment (fiber sheath extension or not); (2) location, attachment or unattachment of intermediate veins to one or both surfaces and method of attachment (e.g. sheath extensions, formation of girders); (3) location of the minor veins (e.g. adaxial, abaxial, abaxial and adaxial, middle, marginal); (4) presence, size and location of veins with an exaggerated fibrous sheath (large ones often located on the leaflet margin); (5) presence, location, size and sometimes cross-sectional shape of fiber bundles and the extent they reach across the mesophyll. These characters can also be further summarized as follows:

1. Major vein location {adjacent to the margin; near the margin but not adjacent to it (this means that along a horizontal plane there is a maximum of one minor vein or one fiber bundle separating it from the actual margin); not adjacent to nor near the margin}
2. Major vein attached where {unattached; attached to adaxial hypodermal surface only; attached to both adaxial and abaxial hypodermal surfaces}
3. Major vein attachment how{attached by a short or long fibrous sheath extension; attachment not by a fibrous sheath extension}
4. Intermediate veins attached {unattached; attached to adaxial surface only; attached to both surfaces}
5. Intermediate vein attachment {to both surfaces by fibrous sheath extension (girders); attached to adaxial surface only by fibrous sheath extension; attached but without fibrous sheath extension}
6. Minor vein location {adjacent to both the adaxial and abaxial surface; a few adjacent to the adaxial but most on the abaxial surface; near the middle of the mesophyll; adjacent to the abaxial surface or at least in the lower third of the mesophyll; only adjacent to the abaxial surface}
7. Presence of major marginal vein with large exaggerated fibrous sheath {absent; present}
8. Presence of minor marginal vein with exaggerated fibrous sheath {absent; one present; two or more present}
9. One major rounded fiber bundle adjacent to the margin {absent; present}
10. First fiber bundle on the adaxial surface the largest {absent; present}
11. Adaxial fiber bundles size if present {reach 1/3 to 1/2 across the mesophyll; reach 1/5 to 1/4 across the mesophyll}
12. Fiber bundles shape {mostly long and skinny: mostly long and thick: mostly short and thick}
13. Fibers or minor fiber bundle locations {adaxial only; adaxial and abaxial only; adaxial, abaxial and scattered in the mesophyll}
14. Minor fiber bundles adjacent to the margin {absent; present}.
15. Minor fiber bundles abundance {none; few along the adaxial and abaxial surface; only a few along the abaxial surface alternating with the minor veins; many along the abaxial surface}
16. Thick walled hypodermis protecting the margin {absent; present}

The key was designed for field use, which means minimal equipment, no staining, and low magnification and the use of simple characters. Refer to the characters in the methods for clarification of terminology. By using the methods listed above and following many of the simple techniques mentioned by Tomlinson et al. (2011), rapid results can be achieved in a laboratory provided with only the simplest equipment. This simple approach was also successfully used in a significant study of palm leaf development by Nowak et al. (2007).

Species of the Rain Forest clade (Fig. 1), which includes many Amazonian species, are distinguished anatomically by an almost continuous adaxial fibrous layer, one or a few cells thick just under the epidermis (the hypodermal layer) (Fig. 3A, 3B, 3C, 3D). I speculate that perhaps this nearly continuous fibrous layer strengthens the lamina while maintaining its flexibility (Vincent 1982), helps the leaf shed water and discourages fungus infection. Species of the Eastern Brazilian clade (Fig. 1) are distinguished by thicker and stiffer leaflets reinforced by many adaxial, thick, multicellular fiber bundles along the adaxial side of the leaf and these fiber bundles may extend as far as 1/2 the distance across the mesophyll (Fig. 4A, 4B, 4C, 4D). Fibers assume much of the load-bearing capacity of the lamina (Horn et al. 2009, Vincent 1982). These fibers and fiber bundles help the leaflet to retain its shape, flexibility and form when leaf turgidity wanes during the periodic dry spells that frequent the seasonally dry forests, cerrados and caatingas of Eastern Brazil. The Cluster-stem clade (perhaps a misnomer, since not all are cluster-stemmed) is not as clear. Syagrus campylospatha (Figs 5A, 6A) appears anatomically different from the rest of the group having girder type intermediate veins that are attached to both sides of the leaf by fibrous sheath extensions. Syagrus flexuosa and Syagrus macrocarpa have nearly identical anatomies with minor, sparsely spaced fiber bundles running along the adaxial surface (Fig. 5B, 5C) and Syagrus cerqueirana (formerly identified as Syagrus petraea) has a similar aspect but replaces these adaxial fiber bundles with minor veins, which are found on both sides of the leaf (Fig. 5D).

Most acaulescent Syagrus exhibit the Eastern Brazilian pattern (e.g. Syagrus gouveiana; Fig. 7A) with the large, multicellular fiber bundles running along the adaxial side of the leaflet and the Cluster-stem pattern, similar to that of Syagrus cerqueirana (Fig. 5D, 6D, 6H), with minor veins on both surfaces (e.g. Syagrus lilliputiana, Fig. 6G), each attached to either the adaxial or abaxial surface by short, fibrous extensions. Since most acaulescent palms grow in seasonally dry areas (cerrados) that require stiffer leaflets, it is perhaps understandable why the Rain Forest pattern is not seen among them.

Some of the problems of identifying acaulescent Syagrus species were covered previously in the introduction concerning the lack of good label information in relation to how leaves and leaflets are displayed or arranged on the plant before pressing and drying. Having observed most of these variations personally in the field has led me to the challenging process of trying to straighten out this much neglected complex of species. For me, it started in Bahia, Brazil with the misidentification of the acaulescent cerrado palm, Syagrus glazioviana. Many palm taxonomists, including Glassman and myself (Noblick 1991), have erroneously identified it as Syagrus petraea. Initially, Glassman (1965) placed it in synonomy with Syagrus petraea, based on the shape and size of their female flowers. A few years later, he considered them distinct species (Glassman 1968) after he had seen the lectotype for Syagrus glazioviana due to differences in the width of the leaflets and shape of the leaflet tips (symmetrical verses asymmetrical). Finally in his revision (Glassman 1987), he synonomized it once again with Syagrus petraea, concluding that the differences seen must have been due to favorable versus unfavorable growing conditions. In western Bahia it is often used to make brooms. It has meter-long leaves with long, regular to loosely clustered pinnae and a spike inflorescence. In the same cerrados, one will encounter another acaulescent, spicate palm with shorter leaves and tightly clustered pinnae that are unsuitable for broom making, which I had previously identified as Syagrus petraea as well. I currently believe that the smaller western Bahian one is Syagrus loefgrenii, which has also been proposed as a synonym of Syagrus petraea (Henderson et al. 1995), but the leaflet anatomy of Syagrus petraea (Fig. 6M) is very different from the anatomy of both Syagrus loefgrenii (Fig. 6I) and Syagrus glazioviana (Fig. 7E). It has been an unfortunate fact that most acaulescent, spicate Syagrus have gotten automatically classified as Syagrus petraea. In truth, acaulescent palms with a sparsely branched inflorescence have also been classified as Syagrus petraea (i.e. Syagrus cerqueirana from Paraguay). However Syagrus petraea is not the only problematic acaulescent Syagrus. In his anatomy paper (Glassman 1972), Glassman misidentified Syagrus cerqueirana (Swallen 9520 from Mato Grosso) as Syagrus graminifolia. He then proceeded to use the anatomy of that misidentified specimen to represent Syagrus graminifolia in his publications (Glassman 1972, 1987). When I examined the anatomy of collections of Syagrus graminifolia from Goiás I discovered that they had a very different anatomy from what Glassman had published, but I resolved the issue by comparing the anatomy of my specimens with that of a leaflet fragment borrowed from the original 1827 holotype (Burchell 5956) and found them to be a match. In addition, Glassman mistakenly reported Burchell’s collection from the state of Piauí, but Burchell’s field notes and itinerary (Smith and Smith 1967) clearly place him in southern Goiás at the time.

Many of the Syagrus petraea-types have very different leaflet anatomies. Their visible field characters (Table 1) and their distinctive anatomy has justified splitting up the complex (Noblick and Lorenzi 2010; Lorenzi et al. 2010) by resurrecting formerly synonymized names (e.g. Syagrus glazioviana, Syagrus loefgrenii) and by describing several new species (e.g. Syagrus allagopteroides, Syagrus angustifolia, Syagrus caerulescens, Syagrus cerqueirana, Syagrus evansiana, Syagrus gouveiana, Syagrus itacambirana, Syagrus minor, Syagrus pleiocladoides, Syagrus procumbens, Syagrus rupicola). Currently, there are about 26 taxa of Syagrus without visible above ground stems or with very short stems and it is strongly suspected that there will be several more based on the anatomy that has so far been observed. As a disclaimer, I feel that this key is still not the final word and will need further revision as new species are discovered and others get reworked. The same species grown under different growing conditions or adult and juvenile forms may look slightly different, but the anatomy maybe an important tool in helping us to resolve these issues. I suspect that some species which appear to be morphologically different but anatomically similar may turn out to be the same species (e.g. Syagrus allagopteroides and Syagrus minor are suspicious).

In conclusion, leaflet anatomy has been found to be useful in helping to confirm or verify relationships discovered through the molecular analysis and in identifying some of the difficult acaulescent Syagrus species.