Corresponding author: Nikolay A. Vislobokov (
Academic editor: Lorenzo Peruzzi
Micromorphological characters of leaf epidermis were investigated in 69 species of
Vislobokov NA, Fu L-F, Wei Y-G, Nuraliev MS (2021) Leaf epidermal micromorphology in
Despite the usefulness of the vegetative characters of
Micromorphological characters, including those of vegetative organs, sometimes appear sufficiently diverse to serve as a useful instrument for taxonomy and species identification. This approach has already been successfully applied for
Fully developed foliage leaves were collected from living plants of
For SEM, a single fragment ca. 5×5 mm was cut out from the leaf blade by a razor blade for each specimen. The fragment was taken from the central part of the leaf blade (equidistant from petiole/leaf base and leaf apex), equidistantly from midvein and leaf margin, between secondary veins. The dissected material was transferred from 70% ethanol to 100% acetone via 80% and 96% ethanol followed by an 1: 1 mixture of ethanol (96%) and acetone (100%). The material was critical-point dried using an HCP-2 critical point dryer (Hitachi, Japan). Dried samples were divided in two equal parts by a razor blade, which then were mounted onto stubs with different sides exposed, using double-sided sticky tape. The mounted specimens were coated with gold using an Eiko IB-3 ion-coater (Eiko Engineering, Japan) and observed using a CamScan 4DV (CamScan, UK) scanning electron microscope at Moscow State University.
The following traits of leaf epidermis were investigated: (1) size and shape of epidermal cells; (2) fine relief of the outer periclinal cell wall: micro-sculpture of epidermis surface; and (3) curvature of outer periclinal wall: presence and density of papillae on epidermis. Size of epidermal cells and density of stomata were measured once for each specimen. Density of papillae was measured by counting number of papillae within a frame 100×100 µm in at least two repeats in each specimen. Standard terminology of surface sculpturing patterns mainly follows
The identification key was compiled on the basis of original data on leaf micromorphology and data on gross vegetative morphology available from
Leaf epidermis of examined species of
Epidermis surface is smooth or sculptured to various degrees (Figs
SEM images of abaxial leaf epidermis of
Epidermis of about a half of the studied specimens is smooth on both surfaces. In the other specimens, the adaxial and abaxial epidermis is either uniformly or differently micro-ornamented (or one of the surfaces is smooth). Usually the sculpture is pronounced to a greater extent on the abaxial surface than on the adaxial one. Verrucose sculpture, if present, is usually found on both sides, or only abaxially with smooth adaxial epidermis. Rugose epidermis is usually expressed on both leaf sides or rarely only on the abaxial side, with the adaxial side being verrucose. Thus, only the abaxial epidermis is illustrated (Figs
SEM images of abaxial leaf epidermis of
Adaxial epidermis of foliage leaves of all studied specimens is uniformly epapillate. Abaxial epidermis of about a half of the studied specimens bears papillae (e.g. Figs
Additionally, we have found that shape and density of papillae on the secondary veins (not employed in the main part of our study) is different in some specimens from those of the epidermis located between the secondary veins.
SEM images of abaxial leaf epidermis of
The main results of investigation of each species (epidermis sculpture and the presence and density of papillae) are presented in Appendix
Size and shape of epidermal cells of investigated species does not reveal any species-specific pattern: their infraspecific variation is nearly as broad as interspecific variation.
The micro-sculpture of abaxial and adaxial epidermis is, in contrast, generally constant (fixed) at a species level. These traits show stability in at least 16 out of 22 species of
SEM images of abaxial leaf epidermis of
Presence and density of papillae is also stable in 16 out of 22 species of
We built a space of logical possibilities for all the studied specimens with regard to the following characters of vegetative morphology: type of shoot, sculpture of adaxial and abaxial epidermis, and density (and presence) of papillae. The specimens showed 23 combinations of these traits. Considering infraspecific variation of some traits, we combined the studied specimens of
Groups of the examined specimens of
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I | tufted | smooth/verrucose | smooth/verrucose | no papillae |
II | tufted | smooth | smooth/verrucose | low |
III | solitary | smooth/verrucose | smooth | no papillae |
IV | solitary | smooth/verrucose | verrucose | no papillae |
V | solitary | verrucose/rugose | rugose | no papillae |
VI | solitary | smooth/verrucose | smooth | low |
VII | solitary | smooth/verrucose | verrucose | low |
VIII | solitary | rugose | rugose | low |
IX | solitary | smooth | smooth | medium |
X | solitary | smooth/verrucose | verrucose | medium |
XI | solitary | rugose | rugose | medium |
XII | solitary | smooth | smooth | high |
XIII | solitary | verrucose/rugose | verrucose/rugose | high |
Some species possess a unique combination of traits, e.g.
We recognized no correlation between the morphological groups of species outlined above and geographical distribution of the species.
The availability of recognition of the morphological groups indicates that the characters under study show an infraspecific variation that is narrow enough, and the interspecific diversity that is broad enough to be applied to taxonomy for most of the studied species of
Most of the groups of species outlined here on the basis of vegetative characters do not show any correlation with floral traits. We were able to recognize only several cases of such correlation, which are addressed below.
A group of five species,
The general stability of the micromorphological characters at the species level demonstrated here in
Representatives of
Several other taxa with uncertain boundaries form a group here referred to as
1 | Leaves grouped on shoot by 3–5 (3–5 foliage leaves per elementary shoot), sessile; blade narrowly elliptic to linear, 1–5 cm wide, 15–50 times as long as wide |
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– | Leaves solitary (one leaf per elementary shoot), petiolate or sessile; blade of various shape |
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2 | Adaxial epidermis epapillate; abaxial epidermis with sparse papillae (papillae density low: 1–3.5 papillae per 0.01 mm2) |
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– | Adaxial and abaxial epidermis epapillate |
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3 | Epidermis smooth adaxially, verrucose abaxially |
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– | Epidermis smooth on both sides | |
4(2) | Adaxial and abaxial epidermis smooth | |
– | Epidermis verrucose at least on one side |
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5 | Epidermis smooth adaxially, verrucose abaxially |
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– | Epidermis verrucose on both sides |
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6(1) | Adaxial and abaxial epidermis smooth and epapillate; leaf petiolate |
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– | Epidermis sculptured (verrucose or rugose) or papillate at least on one side; leaf petiolate or sessile |
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7 | Plant with aerial erect to ascending stem ca. 50 cm high |
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– | Plant without aerial stem |
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8 | Blade narrowly lanceolate to narrowly elliptic, 1.5–4.5 cm wide | |
– | Blade ovate to elliptic, 5–15 cm wide | |
9(6) | Adaxial and abaxial epidermis epapillate |
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– | Adaxial epidermis epapillate, abaxial epidermis papillate |
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10 | Epidermis verrucose at least on one side (and never rugose) |
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– | Epidermis finely rugose at least on one side |
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11 | Plant with erect stem |
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– | Plant without erect stem |
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12 | Aerial stem up to 50 cm high |
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– | Aerial stem 3–20 cm high |
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13 | Aerial stem 3–5 cm high, blade 8–16 cm long |
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– | Aerial stem ca. 20 cm high, blade 20–25 cm long |
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14(11) | Blade narrowly elliptic to narrowly lanceolate, 8–20 times as long as wide |
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– | Blade elliptic to ovate, 2–7 times as long as wide |
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15 | Petiole 3–5 cm long |
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– | Petiole 15–32 cm long | |
16(14) | Epidermis verrucose on both sides | |
– | Epidermis verrucose on one side, smooth on the other side |
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17 | Petiole longer than blade |
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– | Petiole equal to or shorter than blade | |
18(10) | Epidermis finely rugose on both sides |
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– | Epidermis verrucose adaxially, finely rugose abaxially |
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19 | Blade 1.5–3.7 cm wide | |
– | Blade 4–10 cm wide |
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20(18) | Blade elliptic, 5–11 cm wide | |
– | Blade lanceolate, 1.5–5 cm wide |
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21 | Blade equal to or insignificantly longer than petiole |
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– | Blade 2–10 times as long as petiole |
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22 | Blade 12–20 cm long, 4–6 times as long as wide |
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– | Blade 20–35 cm long, 8–13 times as long as wide |
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23(9) | Density of papillae on abaxial epidermis low (1–3.5 papillae per 0.01 mm2) |
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– | Density of papillae on abaxial epidermis medium to high (4–45.5 papillae per 0.01 mm2) |
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24 | Epidermis finely rugose on both sides |
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– | Epidermis smooth or verrucose on both sides |
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25 | Epidermis smooth on both sides | |
– | Epidermis verrucose at least on one side | |
26(23) | Density of papillae on abaxial epidermis medium (4–7.5 papillae per 0.01 mm2) |
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– | Density of papillae on abaxial epidermis high (8–45.5 papillae per 0.01 mm2) |
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27 | Epidermis finely rugose on both sides |
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– | Epidermis smooth or verrucose on both sides |
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28 | Epidermis verrucose at least on one side | |
– | Epidermis smooth on both sides |
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29 | Petiole 2–5 cm long, blade 3–7 times as long as petiole | |
– | Petiole 9–28 cm long, blade equal to or insignificantly longer than petiole | |
30(26) | Epidermis finely rugose (rarely verrucose) on both sides |
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– | Epidermis smooth on both sides |
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31 | Rhizome with very short internodes (foliage leaves crowded); blade 20–30 × 8.5–13 cm |
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– | Rhizome with long internodes (foliage leaves spaced 8–25 mm apart); blade 10–17 × 5–8 cm |
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32 | Petiole 10–15 cm long, blade 10–12 × 5 cm; epidermis finely rugose on both sides |
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– | Petiole 15–20 cm long, blade 14–17 × 7–8 cm; epidermis finely rugose or verrucose at least on one side |
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33(30) | Blade narrowly lanceolate, 1.5–3 cm wide, 23–30 times as long as wide |
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– | Blade lanceolate, elliptic or ovate, 5–14 cm wide, 6–9 times as long as wide |
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34 | Petiole absent or inconspicuous |
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– | Petiole distinctly present, usually 30 cm long or longer |
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Micromorphological characters of leaf epidermis show sufficiently high diversity in the genus
The authors are grateful to the staff of Botanical Garden Munich-Nymphenburg and personally to Andreas Gröger, staff of the Botanical Institute of the Russian Academy of Sciences and personally to Leonid Averyanov, staff of the Main Botanical Garden of the Russian Academy of Sciences and personally to Mikhail Romanov, staff of Singapore Botanic Gardens and personally to Jana Leong-Škorničková for the opportunity to get material from living collections. The work of N.A. Vislobokov and M.S. Nuraliev was carried out as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No. 121032500082-2. The reported study was funded by RFBR, project number 18-34-20135 and by RFBR and VAST, project number 21-54-54011.
Species and specimens of
Species | Living specimens studied | Herbarium voucher | Country of origin | Type of shoot (leaves solitary / tufted) | Sculpture of adaxial epidermis | Sculpture of abaxial epidermis | Density of papillae distribution on abaxial epidermis** | Group | |
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Greenhouse hosting specimen* | Garden accession number; field collector’s number (if differs from that of herbarium voucher) | ||||||||
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1285 | Vietnam | tufted | smooth | smooth | no papillae | I | |
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s.n. | - | unknown | tufted | smooth | smooth | no papillae | |
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2011/0980; |
- | unknown | tufted | smooth | smooth | no papillae | |
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2013.12433.01 | Vietnam | tufted | smooth | smooth | no papillae | ||
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13394; |
Vietnam | tufted | smooth | smooth | no papillae | ||
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2016.12342.01 | China | tufted | smooth | smooth | no papillae | ||
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2015.11381.01 | Vietnam | tufted | smooth | verrucose | no papillae | ||
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251964 | - | unknown | tufted | verrucose | verrucose | no papillae | |
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96/3122W; |
Vietnam | tufted | smooth | smooth | low | II | |
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2015.11382.01 | Vietnam | tufted | smooth | smooth | low | ||
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11/1394 | unknown | tufted | smooth | smooth | low | ||
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15/1593 | - | unknown | tufted | smooth | smooth | low | |
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2018.14340.01 | - | unknown | tufted | smooth | smooth | low | |
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14386 | Vietnam | tufted | smooth | smooth | low | |||
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s.n. | - | unknown | tufted | smooth | verrucose | low | |
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97/2363 | Vietnam | solitary | smooth | smooth | no papillae | III | |
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2014.12431.01 | Vietnam | solitary | smooth | smooth | no papillae | ||
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2016.12351.01 | China | solitary | smooth | smooth | no papillae | ||
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V/0490 | - | unknown | solitary | verrucose | smooth | no papillae | |
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2016.12354.01 | China | solitary | smooth | smooth | no papillae | ||
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s.n. | Vietnam | solitary | smooth | smooth | no papillae | III | |
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2016.12349.01 | China | solitary | smooth | smooth | no papillae | ||
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2016.12348.01 | China | solitary | smooth | smooth | no papillae | ||
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2225 | unknown | solitary | smooth | smooth | no papillae | ||
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20121925; |
- | Vietnam | solitary | smooth | smooth | no papillae | |
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04/1772 | Vietnam | solitary | smooth | smooth | no papillae | ||
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14448 | China | solitary | smooth | smooth | no papillae | ||
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2011/1397 | China | solitary | smooth | verrucose | no papillae | IV | |
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2019.14670 | Vietnam | solitary | verrucose | verrucose | no papillae | ||
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- | unknown | solitary | verrucose | verrucose | no papillae | |
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from nature | - | Vietnam | solitary | verrucose | verrucose | no papillae | ||
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2011/1395 | China | solitary | verrucose | verrucose | no papillae | ||
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20122018 | Laos | solitary | verrucose | verrucose | no papillae | ||
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1311(1313) | Laos | solitary | verrucose | verrucose | no papillae | |||
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97/2382; |
Vietnam | solitary | smooth | verrucose | no papillae | ||
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- | Laos | solitary | smooth | verrucose | no papillae | |
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Vietnam | solitary | verrucose | verrucose | no papillae | ||
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2015.11347.01 | Vietnam | solitary | verrucose | verrucose | no papillae | ||
from nature | - | Vietnam | solitary | verrucose | verrucose | no papillae | |||
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- | Vietnam | solitary | verrucose | verrucose | no papillae | |
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JLS2962 | Vietnam | solitary | verrucose | verrucose | no papillae | |||
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97/2376 II | Vietnam | solitary | verrucose | verrucose | no papillae | ||
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2015.11350.01 | Vietnam | solitary | verrucose | verrucose | no papillae | IV | ||
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2015.11365.01 | Vietnam | solitary | verrucose | verrucose | no papillae | IV | |
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05/2338 | Thailand | solitary | verrucose | verrucose | no papillae | ||
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2013/2461 | Vietnam | solitary | verrucose | verrucose | no papillae | ||
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2013/2461W | Vietnam | solitary | verrucose | verrucose | no papillae | ||
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- | Vietnam | solitary | verrucose | verrucose | no papillae | ||
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2017.12835.06 | Vietnam | solitary | smooth | verrucose | no papillae | ||
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786 | Vietnam | solitary | verrucose | verrucose | no papillae | ||
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from nature | Vietnam | solitary | rugose | rugose | no papillae | V | ||
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- | Vietnam | solitary | verrucose | rugose | no papillae | |
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97/2360; |
Vietnam | solitary | rugose | rugose | no papillae | ||
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97/2353 | Vietnam | solitary | rugose | rugose | no papillae | |||
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96/3121; |
Vietnam | solitary | verrucose | rugose | no papillae | ||
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86-169 | Vietnam | solitary | verrucose | rugose | no papillae | |||
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CPC1566 | Vietnam | solitary | verrucose | rugose | no papillae | ||
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13266 | Vietnam | solitary | verrucose | rugose | no papillae | ||
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2015.11375.01 | Vietnam | solitary | rugose | rugose | no papillae | ||
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2015.11366.01 | Vietnam | solitary | verrucose | rugose | no papillae | ||
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97/2352; Bogner 2503 | unknown | solitary | verrucose | smooth | low | VI | |
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2016.12350.02 | China | solitary | smooth | smooth | low | ||
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2016.12355.01 | China | solitary | smooth | smooth | low | |||
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97/2375G | Vietnam | solitary | smooth | smooth | low | ||
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2017.13459.01 | China | solitary | smooth | smooth | low | ||
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from nature | Vietnam | solitary | smooth | smooth | low | |||
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13270 | Vietnam | solitary | smooth | smooth | low | VI | |
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from nature | Vietnam | solitary | verrucose | verrucose | low | VII | ||
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97/2358 | Vietnam | solitary | smooth | verrucose | low | ||
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from nature | Vietnam | solitary | verrucose | verrucose | low | |||
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11614 | Vietnam | solitary | smooth | verrucose | low | ||
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from nature | Thailand | solitary | verrucose | verrucose | low | |||
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2015.11355.01 | Thailand | solitary | verrucose | verrucose | low | ||
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2018.14282.01 | Vietnam | solitary | rugose | rugose | low | VIII | |
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95/2832; |
China | solitary | smooth | smooth | medium | IX | |
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260301 | - | Vietnam | solitary | smooth | smooth | medium | |
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96/3126; |
Vietnam | solitary | smooth | smooth | medium | ||
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17304 | Vietnam | solitary | smooth | smooth | medium | |||
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13659 | China | solitary | smooth | smooth | medium | ||
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96/3124; |
Vietnam | solitary | smooth | smooth | medium | ||
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20121895; |
- | Vietnam | solitary | smooth | smooth | medium | |
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97/2369T; |
Vietnam | solitary | smooth | smooth | medium | ||
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18566 | Vietnam | solitary | smooth | verrucose | medium | X | |
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96/3123; |
Vietnam | solitary | smooth | verrucose | medium | |||
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2015.11353.01 | Vietnam | solitary | smooth | verrucose | medium | |||
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96/3125; |
Vietnam | solitary | verrucose | verrucose | medium | ||
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2015.11352.01 | Vietnam | solitary | verrucose | verrucose | medium | |||
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96/3119; |
Vietnam | solitary | smooth | verrucose | medium | ||
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Vietnam | solitary | smooth | verrucose | medium | |||
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13281 | Vietnam | solitary | smooth | verrucose | medium | ||
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2013/2460W; |
- | Vietnam | solitary | verrucose | verrucose | medium | |
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97/2359; |
Vietnam | solitary | verrucose | verrucose | medium | |||
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20111766 | JLS1121 | Vietnam | solitary | verrucose | verrucose | medium | X |
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13/2460; |
- | Vietnam | solitary | verrucose | verrucose | medium | |
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- | Vietnam | solitary | smooth | verrucose | medium | |
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04/1769 | - | Vietnam | solitary | verrucose | verrucose | medium | |
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7242 | Vietnam | solitary | rugose | rugose | medium | XI | |
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9311 | - | Vietnam | solitary | smooth | smooth | high | XII |
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97/2374W | Vietnam | solitary | smooth | smooth | high | |||
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97/2404 | Vietnam | solitary | smooth | smooth | high | |||
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20120086; |
- | Vietnam | solitary | smooth | smooth | high | ||
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11590 | Vietnam | solitary | smooth | smooth | high | ||
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- | Vietnam | solitary | smooth | smooth | high | |
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2016.12423.01; |
Vietnam | solitary | smooth | smooth | high | ||
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20120060; |
- | Vietnam | solitary | verrucose | verrucose | high | XIII |
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1997/2367 | unknown | solitary | verrucose | verrucose | high | |||
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- | Vietnam | solitary | verrucose | verrucose | high | ||
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2015.11376.01 | Vietnam | solitary | verrucose | rugose | high | |||
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s.n. | unknown | solitary | rugose | rugose | high | |||
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Vietnam | solitary | rugose | rugose | high | ||
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20122069 | Vietnam | solitary | rugose | rugose | high | |||
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96/3118; |
Vietnam | solitary | rugose | rugose | high | ||
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2015.11354.01 | Vietnam | solitary | rugose | rugose | high |
* For description of the abbreviations, see Materials and methods. ** Estimated as number of papillae per 0.01 mm2: low = 1–3.5, medium = 4–7.5, high = 8–45.5.