Research Article |
Corresponding author: Larry R. Noblick ( larryn@montgomerybotanical.org ) Academic editor: Thomas L.P. Couvreur
© 2021 Larry R. Noblick, Bruno F. Sant’anna-Santos.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Noblick LR, Sant’anna-Santos BF (2021) Diversity of leaf anatomy within a single leaflet and between leaflets of four Butia (Arecaceae, Arecoideae) species. PhytoKeys 180: 31-52. https://doi.org/10.3897/phytokeys.180.66018
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This paper is an investigation into how the anatomy changes within a leaflet and between the leaflets of a single leaf blade of Butia. Four species of Butia were studied: Butia paraguayensis, B. eriospatha, B. yatay and B. odorata. Changes in the anatomical characters are important because some have been used in keys to help separate the species. Recently, anatomical mid-rib characters were used in a key to separate species of Butia. We found that characters, such as abaxially projected or rounded mid-rib fibrous ring or number and arrangement of accessory bundles, do change within a single leaflet or between the leaflets of a single leaf blade. Growing conditions and leaf developmental maturity are also important factors that influence leaflet anatomy and may cause one to be misled in an identification key based on anatomical characters. We re-emphasize the importance of always sampling from the same part of the leaf, to have a broader sampling, be attentive to the environmental condition and health of the plant from which you are sampling and to consider population differences.
anatomy, environmental variation, interspecific variation, key
Palms (Arecaceae) are an easy family of plants to identify to family and are divided into five subfamilies: Calamoideae, Nypoideae, Coryphoideae, Ceroxyloideae and Arecoideae (
Leaflet anatomy has been advocated as an alternative method for the identification of palm species. The methodology is simple – choose a middle leaflet and section its centre (or center in American English).
While
After examining leaflet cross-sections of over 100 accessions of Butia from the living collections at Montgomery Botanical Center, the first author began to develop some concerns. What if the exact middle leaflet is not selected for study? How quickly does palm leaflet anatomy change depending on which “middle” leaflet is chosen (closer to the base or closer to the leaf apex)? Does the leaflet anatomy change much depending on where the section is made along the leaflet? Does the anatomy change from one side of the leaf blade to the other? Does the leaflet anatomy change from between a more mature and less mature or lesser developed specimen of the same species? Are there differences in the anatomy of the same species from one population to another? These are questions that have not been adequately addressed by those using leaflet anatomy for the purpose of identification and that includes ourselves.
Fresh material was used to prepare 67 slides from four leaves; one leaf from each of the four Butia species. The specimens, sampled in this study, came from the living collections at Montgomery Botanical Center (MBC), Miami, FL. Butia paraguayensis (Barb.Rodr.) L.H.Bailey (MBC accession 20020856*C) was grown from seed collected from San Estanislão, Paraguay and was sampled more thoroughly than the others. Figure
After much sectioning, it was reasoned that, if one simply “eye-balls” the middle of a leaf, one is not likely to select a middle leaflet more than 20 cm to either side of the true middle of a leaf, nor section that leaflet by more than 5 cm to either side of its centre. Therefore, only three leaflets were sampled from each of other three Butia species, which included B. yatay (Mart.) Becc., B. eriospatha (Mart. ex Drude) Becc. and B. odorata (Barb.Rodr.) Noblick. These three species were chosen, because they all have aboveground stems like B. paraguayensis and live, wild collected specimens were readily available at MBC. Most of these species are also widely distributed geographically, allowing us to test for some degree of variation in morphological and anatomical characters. Butia eriospatha (92271*E) was grown from seed collected from Paraná, Brazil. Butia odorata (20060233*E) was grown from seed collected from Rocha, Uruguay and Butia yatay (20040309*C) was grown from seed collected from Batel, Argentina. The leaflets were collected from the middle and 20 cm to either side of the middle. Each of these leaflets was sampled in three places: the centre and 5 cm to either side of it for a total of nine slides for each species and a total of 27 slides.
Several methods for hand sectioning are covered in
A piece of carrot is cut into a small cube that will fit in the hand microtome as described in
Glass slides with one side frosted for labelling, glass cover slips, artist’s brush, a dropper bottle of 1:1 glycerine and water solution and a dissecting needle are needed for slide preparation. Confirm that the glass slide and glass cover slip are clean before using. Label the frosted portion of the slide and spread a drop or two of the 1:1 glycerine and water solution on to the slide. While looking through the dissecting microscope, select the best sections from the watch glass with the narrow artist’s brush and transfer them into the 1:1 glycerine droplet on the slide. After placing a number of the sections on the slide (ca. 4–6), cover the sections with a glass cover slip. Place one edge of the cover slip at the edge of the glycerine droplet on the slide and gently lower it in 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.
For the collection from Tapes, the samples followed the additional protocol proposed by
The glass slide is now placed under a 40×–2000× Trinocular Biological Compound Microscope available from Amscope (model T490B) and photographed under the 10× objective (100× magnification). Images were taken with a 5 Mb AmScope digital camera. The images were cleaned of background spots, adjusted for brightness and sharpened, if necessary, using Adobe Photoshop. If the entire mid-rib or leaf margin did not fit into the field of view, adjacent images were photo-merged using the automatic photo-merge capabilities of Adobe Photoshop. A stage micrometer was used to apply a scale to each image.
Characters of the leaflet margin (Figure
More characters for the leaflet mid-rib (Figure
In the Butia leaflet margin (Figure
Anatomical characters of leaflet cross-sections A Butia odorata leaflet margin showing isolateral “mirrored” anatomy B B. yatay midrib. ab = accessory bundle; ET = expansion tissue; MFR = mid-rib fibrous ring; mv = miniveins; php = phloem pole; PVB = primary vascular bundle; SVB = secondary vascular bundle; TVB = tertiary vascular bundle; vb = vascular bundle or collateral vascular bundle; vbe = vascular bundle with enlarged sheath. Not all are labelled. Scale bars: 0.3 mm.
In the mid-rib (Figure
The proximal cross-sections are located between the centre and the base of the leaflet and the distal sections are located between the centre and the apex of the leaflet. By the same thinking, the proximal leaflets are located between the middle leaflet and the basal leaflet and the distal leaflets are located between the middle leaflet and the apex or leaf tip.
Table
Anatomical changes of the leaflet margins in a 10 cm region to either side of the leaflet centre: Butia paraguayensis. PVB-SVB = primary vascular bundles transitioning to secondary vascular bundles; SVB-PVB = secondary vascular bundles transitioning to primary vascular bundles. Gone = vascular bundle has disappeared or nearly ended. Scale bar: 0.5 mm.
Anatomical changes of the leaflet margins in a 5 cm region near the leaflet centre A Butia paraguayensis B B. eriospatha. PVB-SVB = primary vascular bundles transitioning to secondary vascular bundles; SVB-PVB = secondary vascular bundles transitioning to primary vascular bundles. Gone = vascular bundle disappeared or nearly so. Scale bars: 0.5 mm.
Changes that occur in the vascular bundles (veins) of the leaflet margins of four different Butia species from the proximal to the distal portion of the leaflet. Number of changes from primary vascular bundles (PVB) to secondary vascular bundles (SVB) and from SVB to PVB in that portion of the margin. Number of vascular bundles (VB) that disappear or nearly disappear (Figures
Name (where sectioned) | PVB-SVB | SVB-PVB | VB nearly gone | Margin bent |
---|---|---|---|---|
B. paraguayensis (proximal 10 cm) | abaxially | |||
B. paraguayensis (proximal 5 cm) | 1 | 2 | 1 | adaxially |
B. paraguayensis (centre) | 1 | adaxially | ||
B. paraguayensis (distal 5 cm) | 2 | 2 | 1 | abaxially |
B. paraguayensis (distal 10 cm) | 1 | 1 | 3 | none |
B. yatay (proximal 5 cm) | none | |||
B. yatay (centre) | none | |||
B. yatay (distal 5 cm) | 1 | none | ||
B. eriospatha (proximal 5 cm) | none | |||
B. eriospatha (centre) | 1 | 2 | none | |
B. eriospatha (distal 5 cm) | none | |||
B. odorata (proximal 5 cm) | none | |||
B. odorata (centre) | none | |||
B. odorata (distal 5 cm) | abaxially |
Table
Anatomical variation of the centre mid-rib cross-section of a middle leaflet, compared to proximal (prox.) and distal sections located 5–10 cm from the centre. ab = accessory vascular bundles, MFR = mid-rib fibrous ring, vb = vascular bundles or collateral vascular bundles, vbe = vascular bundle with enlarged sheath. (Figures
Name (where sectioned) | MFR thickness (# cell layers) | MFR shape | MRF reaches hypodermis | #vbe | #ab | ab completely surround MFR | #vb in MFR |
---|---|---|---|---|---|---|---|
B. paraguayensis (prox. 10 cm) | 9–11 | ab-projected | yes | 2 | 22 | no | 8 |
B. paraguayensis (prox. 5 cm) | 8–9 | ab-projected | yes | 2 | 21 | no | 7 |
B. paraguayensis (centre) | 6–7 | slightly ab-projected | yes | 2 | 17 | no | 5 |
B. paraguayensis (distal 5 cm) | 4–5 | round | yes | 2 | 13 | no | 3–4 |
B. paraguayensis (distal 10 cm) | 3–5 | round | yes | 2 | 9 | no | 3 |
B. yatay (proximal 5 cm) | 6–10 | ab-projected | yes | 2 | 24 | no | 7 |
B. yatay (centre) | 5–10 | ab-projected | yes | 2 | 21 | no | 6 |
B. yatay (distal 5 cm) | 5–8 | slightly ab-projected | yes | 2 | 19 | no | 4 |
B. eriospatha (proximal 5 cm) | 5–8 | ab-projected | yes | 2 | 26 | no | 9–10 |
B. eriospatha (centre) | 4–7 | ab-projected | yes | 1 | 25 | no | 9 |
B. eriospatha (distal 5 cm) | 2–7 | slightly ab-projected | no | 1 | 18 | no | 8 |
B. odorata (proximal 5 cm) | 5–8 | ab-projected | no | 0 | 22 | yes | 8–9 |
B. odorata (centreer) | 4–7 | round | no | 1 | 21 | yes | 5–6 |
B. odorata (distal 5 cm) | 2–7 | round | no | 1 | 16 | yes | 5 |
Table
Within and between leaflet comparisons of Butia paraguayensis cross-sections A centre cross-section and those sections 5 cm and 10 cm to either side of it within the same middle leaflet B centre of middle leaflet and centres of the leaflets 18 cm and 36 cm to either side of it. Scale bars: 0.3 mm.
Between leaflet anatomical changes. Centre cross sections of middle leaflet of A Butia paraguayensis and the leaflets 18 cm to either side of it B B. yatay and the leaflets 20 cm to either side of it C B. eriospatha and the leaflets 20 cm to either side of it D B. odorata and the leaflets 20 cm to either side of it. Scale bars: 0.3 mm.
Anatomical variation of the centre mid-rib cross-section of a middle leaflet, compared to a proximal (prox.) and a distal leaflets located 18–20 cm from the middle and up to 36 cm from the middle in B. paraguayensis. ab = accessory vascular bundles, MFR = mid-rib fibrous ring, vb = vascular bundles or collateral vascular bundles, vbe = vascular bundle with enlarged sheath. (Figures
Name (where sectioned) | MFR thickness (# cell layers) | MFR shape | MRF reaches hypodermis | #vbe | #ab | ab’s completely surround MFR | #vb in MFR |
---|---|---|---|---|---|---|---|
B. paraguayensis (prox. 36 cm) | 8–9 | ab-projected | yes | 2 | 23 | no | 7 |
B. paraguayensis (prox. 18 cm) | 8–9 | ab-projected | yes | 2–3 | 21 | no | 6 |
B. paraguayensis (middle) | 6–7 | ab-projected | yes | 2 | 17 | no | 5 |
B. paraguayensis (distal 18 cm) | 3–4 | round | yes | 2 | 10 | no | 3 |
B. paraguayensis (distal 36 cm) | 2–4 | round | yes | 1 | 8 | no | 3 |
B. yatay (proximal 20 cm) | 7–8 | ab-projected | yes | 2 | 25 | no | 7 |
B. yatay (middle) | 5–9 | ab-projected | yes | 2 | 21 | no | 6 |
B. yatay (distal 20 cm) | 6–8 | slightly ab-projected | yes | 2 | 16 | no | 5 |
B. eriospatha (prox. 20 cm) | 3–7 | ab-projected | yes | 1 | 21 | no | 8–9 |
B. eriospatha (middle) | 4–7 | ab-projected | yes | 1 | 25 | no | 9 |
B. eriospatha (distal 20 cm) | 4–5 | ab-projected | yes | 2 | 20 | no | 6 |
B. odorata (proximal 20 cm) | 6–7 | ab-projected | no | 2 | 22 | yes | 8–9 |
B. odorata (middle) | 5–7 | slightly ab-projected | no | 2 | 21 | yes | 5–6 |
B. odorata (distal 20 cm) | 4–5 | slightly ab-projected | no | 3 | 16 | no | 5 |
Table
Anatomical variation of the centre mid-rib cross-section of a middle leaflet, compared to centres of the two adjacent (adj.) leaflets on the proximal and distal side and the centre of the opposite (opp.) middle leaflet. ab = accessory vascular bundles, MFR = mid-rib fibrous ring, vb = vascular bundles or collateral vascular bundles, vbe = vascular bundle with enlarged sheath. (Figure
Name (where sectioned) | MFR thickness (# cell layers) | MFR shape | MRF reaches hypodermis | #vbe | #ab | ab’s completely surround MFR | #vb in MFR |
---|---|---|---|---|---|---|---|
B. paraguayensis (adj. proximal centre) | 5–7 | ab-projected | yes | 2–3 | 21 | no | 6 |
B. paraguayensis (middle centre) | 6–7 | ab-projected | yes | 2 | 17 | no | 5 |
B. paraguayensis (adj. distal centre) | 5–7 | round | no | 2 | 15 | no | 3 |
B. paraguayensis (opp. middle centre) | 4–6 | ab-projected | yes | 2 | 15 | no | 4 |
To our knowledge, there has never been a study that explored the anatomical changes that occur within a single palm leaf or even within a single palm leaflet. Most studies have been focused on the centre of the middle leaflet, which has been found to be very useful. This is certainly true for
Our study suggests that we need to take a step back and re-examine our previous data to reconfirm and re-evaluate the characters we have been using in our identification keys (
The leaf margins of Butia species contain few characters to distinguish species, but the vasculature within each leaflet changes more frequently in some species than in others over a 10 cm length. Changes were observed to take place in B. paraguayensis and B. eriospatha (Figures
Amongst several characters used by
Population and individual differences in Butia odorata A, B, D–H are from Rocha, Uruguay, C is from Tapes, RS, Brazil A 20060233*E B 20060233*G C contrasting stains make the abs from the Brazil specimen more visible D enlargement of A E enlargement of B F 20060234*M G 20060237*C H 20060240*A. Note that the presence of accessory bundles (ab) surrounding the mid-rib fibrous ring (MFR) is more apparent in the Brazil sample C partially due to the staining, but less apparent or absent from the Uruguay samples. Red arrows pointing out the abs in the Uruguayan samples and abs do not completely surround the MFR in F–H. Scale bars: 0.3 mm (A, B, F–H), 0.1 mm (C), 0.2 mm (D, E).
Montgomery Botanical Center has several wild collected plants that were grown from seed collected from the same mother. The plants, however, have developed differently due to their unique set of growing conditions, like sun/shade exposure, differences in soil type and available moisture. Certainly, the use of fertilisers may be responsible for favouring the growth of vegetative organs and thus modifying the basic histological patterns. It has been reported that Butia pubispatha, under cultivation, showed an accelerated growth, resulting in larger plants than those observed in its natural habitat (
Differences in the centre mid-rib cross-sections within plants of the same species and same accession (seed collected from the same mother plant), but growing under different environmental conditions resulting in a different stage of developmental plant and leaf maturity M, N are Butia paraguayensis plant accession 20060222*M and 20060222*N A, C are Butia yatay. plant accession 20040335*A and 20040335*C. Note the differences in the developmental stage of the plant above and its corresponding anatomy below.
This paper has shown the importance of always collecting leaflets as close to the middle of the leaf blade as possible and sectioning that leaflet as close to its centre as possible for consistent and comparable results. It is also important to expect some population differences, differences in plants of different developmental maturity and differences in those growing under distinctly different conditions than those found in their original habitat. Here, we re-emphasise the importance of a broader sampling exercise when studying leaf anatomy due to possible ecological and developmental variations that may occur in some species. The diversity of leaf anatomy, here observed, also led us to suggest that characters previously used should be re-evaluated in further studies, using wild populations and/or cultivated specimens.
We would like to thank Montgomery Botanical Center for access to their living plant collections.