Key to Syagrus identification using leaflet margin anatomy: Supplement to “A revision of Syagrus (Arecaceae)”

Abstract Presented here is an alternative method of identification for species of the Neotropical palm genus Syagrus. It makes use of anatomical characters found in the leaflet margins and can be used for identification when few other vegetative or reproductive morphological characters are available. This anatomical study demonstrates the vast diversity found in a single palm genus and may also help to gain understanding of some possible relationships within the genus.


Introduction
The following is meant to be a supplement to a revision of Syagrus (Noblick 2017). Palm leaflet anatomy has been useful in identification and has been used to suggest systematic relationships in the family. Tomlinson (1961) examined and described the leaflet anatomy of some 250 species of palms in 137 genera and suggested systematic relationships among genera. Horn et al. (2009) took it a step further and mapped lamina anatomy on the phylogenetic tree for the palm family based on plastid sequence data (Asmussen et al. 2006) in order to understand the evolution of lamina anatomy. Tomlinson's brief survey (Tomlinson 1961) inspired Glassman to examine anatomy of the genus and its closely related genera in greater detail (Glassman 1972(Glassman , 1987. Glassman (1972) emphasized that his survey of the genus was "based on mostly one collection for each taxon." However by the time Glassman (1987) completed his revision, slides of two or more specimens were made for most taxa. His key was written as a convenient tool for identification and was not intended to show relationships. I produced a key based on leaflet anatomy to aid in the identification of 25 difficult to taxonomically distinguish Syagrus species with short, subterranean stems and was able to infer some relationships (Noblick 2013) that had been previously resolved by a molecular analysis (Meerow 2009). The key included herein has been completely rewritten and is meant to aid in the identification of all known 65 species and two subspecies of Syagrus. Meerow et al. (2009) showed how leaflet anatomy supported the molecular relationships between Allagoptera, Parajubaea and Polyandrococos (now a synonym of Allagoptera). Tomlinson et al. (2011) expanded his original 1961 work and presented information on 183 palm genera (out of 185 now recognized) and suggested relationships based on anatomy and the use of modern phylogenetic approaches. Glassman (1972) emphasized various anatomical characters, several of which this paper makes use of, such as: (1) location of larger veins, (2) frequency and location (adaxial, abaxial, or in the middle) of intermediate and minor veins, (3) relative abundance, shape and location (adaxial and abaxial, or adaxial only) of clusters of nonvascular fibers, called fiber bundles (Tomlinson et al. 2011, Noblick 2013, (4) relative size of fiber bundles and veins at extremities of leaflets. Tomlinson et al. (2011) only showed two Syagrus cross-sections in their publication: S. orinocensis and S. weddelliana (formerly Lytocaryum). However he suggested many anatomical features that vary in Syagrus, but the only ones used in this current study are: (1) the presence of trichomes, (2) the abundance and location of adaxial non-vascular fibers, varying from an almost continuous layer within the hypodermis to few fibers, (3) the extent and location of abaxial non-vascular fibers, (4) the extent to which the minor abaxial veins are in contact with the abaxial hypodermis, (5) the degree to which the inner sheath of major veins develop fibrous extensions to the upper surface layers.

Plants examined
Both fresh and preserved (herbarium) material were used in this project. Dried material can be sectioned after rehydrating in a 5% solution of Contrad 70® (Decon Labs, King of Prussia, PA, U.S.A.) for a period of 24 hours (Tomlinson et al. 2011), though better results are obtained with fresh material.
The living material used in this study came mainly from the collections at Montgomery Botanical Center (MBC, Miami, FL) and the Jardim Botânico Plantarum (Nova Odessa, São Paulo, Brazil). A few were collected from Fairchild Tropical Botanic Garden (Coral Gables, FL). The dried material was often from air-dried specimens made while doing fieldwork, from the herbarium at Jardim Botânico Plantarum (HPL, Novo Odessa, São Paulo, Brazil), and from the Fairchild Tropical Botanic Garden herbarium (FTG, Miami, FL). A few specimens were from the following herbaria: G, IBGE, IPA, K, MO, NY and US.

Anatomical preparation
There are a number of ways to hand section leaflet margins and most of these are covered in Noblick (2013). This type of research neither requires expensive hardware or use of chemicals and dyes. To start with, the leaflet sampled should always be collected from the same place on the plant. In this study, leaflets were sampled from the middle of a central leaflet. The following equipment was used: a hand microtome, a sharp knife, a straight razor, a double sided razor blade, a small artists brush (one of the smallest ones), a dropper bottle of water, a watch glass, a stone or plate to sharpen the straight razor blade, and a carrot ( Figure 1A). The hand microtome was purchased from a home schooling site (Homesciencetools.com, Billings, MT, U.S.A.) for about $45. The traditional straight razor I use can be purchased from an online shaving store, though the one I purchased from no longer exists. One of the cheapest pre-sharpened stainless steel straight razors for beginners will probably do fine, and they can be purchased for as little as $14 from Jet.com (Hoboken, NJ, U.S.A.). I use a Dovo hollow ground stainless steel straight razor, which is available online for about $80. I prefer the traditional straight razor blade over those with replacement blades. Finally, to keep a razor-sharp edge, one needs a sharpening stone. The Dia-sharp 3 micron 8000 mesh (DMT D8EE 8" Extra Extra Fine Diamond Stone) (DMTsharp.com or Diamond Machining Technology, Marlbourough, MA, U.S.A) was found to be an excellent choice. Using it frequently between sectionings will maintain the sharpness required for making clean, thin sections.

Preparing the section
Using a small sharp knife, a piece of carrot is cut into a small cube that will fit in the hand microtome ( Figure 1B, C). A perpendicular, vertical slit is cut in the top of the cube with the double-sided razor blade, but not all the way through. A piece of the palm leaf margin is placed into the vertical carrot slit and the carrot cube is clamped into the hand microtome. The carrot cube is adjusted down until it is barely showing above the microtome plate. The first cut is made across the carrot to cut off the excess leaf material and carrot. The blade is re-sharpened using the Diamond Stone and water ( Figure 1D). Lubricate the surface of the carrot with a drop of water and slide the straight razor across the microtome in a horizontal slicing movement, while pressing the side of the blade firmly against the plate ( Figure 1E). If at first, it does not cut anything, adjust the hand Figure 1. Materials and methods. A Materials used in making leaflet sections B Cutting carrot into a cube, slitting the cube and inserting the leaflet margin C Leaflet margin inserted in carrot cube ready for sectioning D Sharpening the straight razor on the wet stone E Clamping the carrot cube with leaflet margin into the hand microtome and preparing to section with the straight razor F Leaflet sections teased from the carrot into a watch glass with water. microtome up about a ¼ of a turn and try again. Always keep the specimen lubricated with water using the eye dropper. After obtaining a section, tease the carrot away from the section or drop the section with attached carrot into a watch glass with water and tease the carrot from the section using the narrow artist brush, leaving the section in the watch glass ( Figure 1F). After several sections have been successfully made, examine the sections in the watch glass under a dissecting microscope.

Preparing the slide
To prepare the slide, some glass slides, glass cover slips, artist brush, a bottle of 1:1 glycerin and water solution, and a dissecting needle are needed. Glass slides with frosted glass along one side to write on are preferred. The glass slide and glass cover slip should always be cleaned with distilled water or 70 % alcohol before using. Label the frosted portion of the slide and spread a drop or two of the 1:1 glycerin and water solution on the slide. While looking through the dissecting microscope, select the thinnest sections from the watch glass with the narrow artist brush and place them into the 1:1 glycerin droplet on the slide. After placing a number of the sections on the slide (ca. 6), then cover the sections with the glass cover slip. The best way to insure that no bubbles are left behind under the cover slip is to use a dissecting needle. Place one edge of the cover slip at the edge of the glycerin droplet with the sections and begin gently lowering it into place over the sections. By placing the dissecting needle tip on its side under the other edge of the cover slip, while slowly pulling out the needle as the cover slip lowers into place, most of the air bubbles should exit from under the cover slip on the side of the exiting needle.

Photography
The glass slide is now placed under a compound light microscope and photographed under the 10× objective (100× magnification). Images were taken with a Nikon Coolpix 4500 digital camera with a Leitz periplan 10 x/18 eye piece adapter. The camera was powered by a Powerline Universal AC adapter so there was no need to rely on battery power. The camera was set to P mode, white balance was adjusted to the light source of the microscope, and the lens was set to Fisheye 2. The images were cleaned of background spots, adjusted for brightness and contrast, and sharpened if necessary using Adobe Photoshop. Two adjacent images near the tip were photo merged using Adobe Photoshop. A stage micrometer was used to apply a scale to each image and replaced by a scale bar to aid in assembling these into a plate, while maintaining the scale.

Characters defined
This paper is focused mainly on characters of the more easily sectioned leaflet margin and not on the more difficult to section midrib. Epidermis and dark staining idioblasts (tanniniferous cells) were also not examined. Characters assessed during this study follow some of Glassman's and Tomlinson's characters as listed above in the introduction. Figure 2 clarifies much of the terminology for characters used in this paper. To be clear, in each leaflet cross-section the upper or superior side of the lamina is called the adaxial surface. The lower or inferior side is called the abaxial (Dransfield et al. 2008, Esau 1977. A Syagrus vagans with large adaxial fiber bundles reaching nearly ½ across the mesophyll, fiber bundles scattered throughout the mesophyll, mesophyll minor veins, abaxial fibers and a primary vein that is nearly connected to the hypodermis B S. lilliputiana showing a large marginal vein with an exaggerated fibrous sheath, adaxial minor veins, and an air bubble artifact C S. coronata with a large marginal fiber bundle, large adaxial fiber bundles, a small vein with an exaggerated fibrous sheath, and unattached primary vein D S. harleyi with a sclerenchymous hypodermis on the margin and along the adaxial surface, along with adaxial fiber bundles and abaxial minor vein symmetry (mirrored anatomy) and fibrous extensions from minor veins forming a girder E S. orinocensis with a mesophyll differentiated into palisade and spongy layers, marginal vein with fibrous sheath noticeably thickened adaxially, many fiber bundles along the adaxial surface, many of which are only one-cell thick and others reaching less than 1/5 across the mesophyll, also note very small abaxial fiber bundles often alternating with abaxial minor veins F S. vermicularis with an adaxial fiber bundle forming a continuous single layer of fiber cells in the hypodermis. abf = abaxial fiber bundle adf = adaxial fiber bundle abv = abaxial minor vein adv = adaxial minor vein b = trapped air bubble artifact c = cuticle e = epidermis fb = larger fiber bundle g = girder vein h = hypodermis hf = fibrous hypodermis m = mesophyll mf = marginal fiber bundle mp = palisade mesophyll ms = spongy mesophyll mv = mesophyll minor vein pv = primary vein sv = secondary vein vex = vein with exaggerated fibrous sheath w = wax. Bar scale = 0.25 mm.
The outer most layer of the leaf is the cuticle (Figure 2, c), a non-cellular waxy layer produced by the epidermis (Dransfield et al. 2008). The cuticle is followed by the epidermis, "outer skin" (Figure 2, e), followed by the hypodermis, "under skin" (Figure 2, h), which is finally followed by the mesophyll or "middle leaf " region ( Figure 2, m). The mesophyll can be undifferentiated (m) or differentiated into a distinct palisade mesophyll (mp) and spongy mesophyll (ms). The palisade mesophyll near the adaxial surface is composed of vertically linear cells; the spongy mesophyll near the abaxial surface is composed of round cells and intercellular spaces of various sizes. Within the mesophyll are veins of various sizes, which are also known as vascular bundles or fibro vascular bundles (Tomlinson et al. 2011). In this paper they will be referred to as primary veins ( Figure  . Some primary and secondary veins are often attached to the adaxial hypodermis and sometimes to both adaxial and abaxial surfaces by fibrous sheath extensions. If the attachment extends to both surfaces via a narrow fibrous sheath extension, the vein appears girder-like and is indeed referred to as a girder (Tomlinson et al. 2011) ( Figure 2D, g). In some veins the fibrous sheath becomes so enlarged with fibers that they are referred to as veins with exaggerated fibrous sheaths (Tomlinson et al. 2011) ( Figure 2B, C, D, vex). Some veins have exaggerated sheaths both adaxially and abaxially as in S. guimaraesensis ( Figure 5) or S. pimentae and S. procumbens (Figure 7). In addition to the veins, the laminal tissues are supported by nonvascular fibers or fiber bundles of various sizes. Some have large fiber bundles adjacent to or near their margins ( Figure  2C, fb). Many fiber bundles are adaxial and may reach close to 1/3 to ½ or more across the mesophyll (

Terminology defined
Mirrored anatomy-anatomy in which the abaxial surface is identical or similar to the abaxial surface. One surface appears to be reflected in the other. In this study, this term is not used exactly in the classical sense, where adaxial veins are exactly opposite other abaxial veins or adaxial fiber bundles are opposite abaxial fiber bundles (again, one surface reflected in the opposite surface). Here the term is used to describe the situation where structures are lined up perfectly opposite each other as if in a reflection. For example, adaxial fiber bundles may lie exactly opposite from abaxial veins or adaxial veins are opposite abaxial fiber bundles as in S. mendanhensis ( Figure 6) or S. pleioclada (Figure 7). Again it is a reflection, but not necessarily of the same structures. This arrangement is not common in Syagrus.
Dorsiventral anatomy -anatomy in which the adaxial surface is very different from the abaxial surface. This is the anatomy most commonly seen in most Syagrus species.

Characters utilized
The following characters were examined and used in this key:

Fibers
• Presence/absence of adaxial fiber bundles • Size of the adaxial fiber bundles (reaching 1/3 to ½ across mesophyll, reaching less than 1/3, reaching less than 1/5 across mesophyll) • Shape of adaxial fiber bundles (long and fat, long and skinny, elliptical, oblong, wedge-shaped, fat or skinny icicle-shaped, short and fat, irregular, rounded) • First adaxial fiber bundle (the largest, ca. same size as others) • Quantity of adaxial fiber bundles (many, occasional to none or few) • Alignment of adaxial fiber bundles across from abaxial minor veins (aligned and opposite or not aligned) • Mirrored versus dorsiventral anatomy (as defined above) • Small adaxial fiber bundles alternating with larger adaxial fiber bundles • Adaxial fiber bundles opposite abaxial veins (opposite each other, no such arrangement) • Presence/absence of large to extra-large fiber bundle at or near the margin This key was designed for use in the field using simple tools and simple methods, which means using minimal equipment, no staining, low magnification (no higher than 100×), and the use of simple characters. Refer to the characters in the methods and Figure 2 for clarification. 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 of equipment.

Results
This key contains headings, which will aid the reader to move through the key more rapidly. The headings refer back to the couplet that was responsible for bringing the reader to this section of the key. The number of the couplet that brought the reader to this section of the key is enclosed in parentheses following the heading. Besides aiding the reader to move more quickly through the key, the headings show the major subsections of the key. It is a way for the reader to understand where the major branches are in the key and which character separates species in that section of the key.

Discussion
Variation I have examined several specimens for each species. However, I make no claim that this has been an exhaustive study. In making use of this key the user must allow for slight variation due to environmental and population differences from the published images. As more specimens were sampled, I expected to see more variation, but was surprised to see how many species stayed true to their basic arrangement of veins and fiber bundles, as well as agreed with previous work done by Glassman (1972Glassman ( , 1987. Nonetheless, a few do vary and for this reason more than one image is used to represent some species. Examples of this are seen in Syagrus cerqueirana (Figure 3) where the marginal vein with the exaggerated fibrous sheath varies in size along with the number of adaxial and abaxial minor veins and fiber bundles. Another example is S. cocoides (Figure 3), where the marginal tip shape varies, along with the quantity and frequency of adaxial fiber bundles. Syagrus glazioviana (Figure 4, 5) is an extreme example of variation. The variation seen in this species either represents true variation within a single species or it is revealing a complex of several closely related species. Environment definitely plays a part in this variation as seen in S. harleyi ( Figure 5) where the low elevation form has a sclerenchymous hypodermis on the margins and along the adaxial surface making it more drought resistant, while the high elevation form has a sclerenchymous margin, but has less sclerenchymous tissue along the adaxial surface. The variation seen in S. hoehnei ( Figure 5) represents two different populations (Harri Lorenzi, pers. comm.). Syagrus minor also represents two specimens separated by over 100 km with one having adaxial veins, which are nearly absent in the other. The variation seen in Syagrus vagans ( Figure 8) clearly demonstrates the futility of trying to use marginal shape for species determination, at least in Syagrus.

Molecular clades versus phenetic (key) branch similarities
Although I was not attempting to show close relationships in his key, nevertheless, certain relationships are suggested based on how these species resolved in the key. Such relationships can only be confirmed further by an in depth phylogenetic molecular analysis of the genus. An outline of the key is presented (Figure 9, 10) to allow one to more clearly visualize the branches of the key and is not meant to depict a phylogenetic analysis. This is not a cladogram. An interesting, but unintentional consequence of producing this key has been to discover how species might be related based on their anatomical phenetics, i.e. possible relationships based on their overall similarity in anatomy or the organization of their anatomical characters. It must be emphasized that this is not an actual phenetic analysis using some kind of distance coefficient. In some cases, branches of the key contain species that had been previously shown to be related by molecular analyses (Meerow et al. 2009,  Table 1, species of the former genus Lytocaryum all resolve in the same branch of the key (Figure 10, branch 53) as they did in the molecular analysis of those analyzed. Species of the strongly supported Rain Forest clade (Meerow 2009(Meerow , 2014 all emerge together in a branch of the key containing mostly Amazonian, Caribbean, An-     dean and few Atlantic forest species (Figure 10, branch 60). The Eastern Brazilian species of the molecular analyses are not as neatly grouped, but of the few species analyzed, they emerge in two portions of the key, but usually among other Eastern or Central-Western Brazilian species (Table 1, Figure 9, branches 18 and 43'). Cluster stemmed species mostly emerge in the same branch of the key (Table 1, Figure 9, branch 3').  also resolved in the molecular analyses (Meerow 2009(Meerow , 2014. A similar Amazonian/ Atlantic Forest connection pops up in branch 67' between the pre-Amazonian species of S. vermicularis and the Atlantic Forest species, S. pseudococos. This Amazonian connection shown both here and in the molecular analyses offers further evidence that the two forests were connected at one time. Finally, several Andean species emerge in branch 60' with S. smithii (Colombia, Ecuador, and Peru), S. sancona (Venezuela to Bolivia), and S. cardenasii (Bolivia).

Conclusion
As demonstrated in the figures, there is a great deal of diversity in the leaflet anatomy of the genus Syagrus. This key has made use of this diversity to create a valuable and easy tool for the identification of Syagrus species using anatomical characters found in leaflet margin cross-sections. It will especially be found to be useful when the palms have not yet produced any reproductive material and little else is known about the palm under study. It can also be used to help confirm identifications.
The key can be seen as a crude means to infer certain relationships within the genus. The anatomical key based on phenetics, not only repeated some of the same groupings or clades that were seen in the molecular analyses, but also grouped the species geographically as well. Only further, more inclusive, molecular analyses will determine if the relations found here are substantiated by the DNA sequences.