Molecular barcode and morphological
analysis of Smilax purhampuy Ruiz,
Ecuador
Pilar Soledispa1 , Efrén Santos-Ordóñez2 ,3 , Migdalia Miranda4 ,
Ricardo Pacheco3 , Yamilet Irene Gutiérrez Gaiten5 and Ramón Scull5
1
Facultad de Ciencias Químicas. Ciudadela Universitaria ‘‘Salvador Allende’’, Universidad de Guayaquil,
Guayaquil, Ecuador
2
Facultad de Ciencias de la Vida, Campus Gustavo Galindo, ESPOL Polytechnic University, Escuela Superior
Politécnica del Litoral, ESPOL, Guayaquil, Ecuador
3
Centro de Investigaciones Biotecnológicas del Ecuador, Campus Gustavo Galindo, ESPOL Polytechnic
University, Escuela Superior Politécnica del Litoral, ESPOL, Guayaquil, Ecuador
4
Facultad de Ciencias Naturales y Matemáticas, ESPOL Polytechnic University, Escuela Superior Politécnica
del Litoral, ESPOL, Guayaquil, Ecuador
5
Instituto de Farmacia y Alimentos, Universidad de La Habana, Ciudad Habana, Cuba
ABSTRACT
Submitted 5 August 2020
Accepted 8 February 2021
Published 18 March 2021
Corresponding author
Efrén Santos-Ordóñez,
gsantos@espol.edu.ec
Academic editor
Rogerio Sotelo-Mundo
Additional Information and
Declarations can be found on
page 12
DOI 10.7717/peerj.11028
Copyright
2021 Soledispa et al.
Distributed under
Creative Commons CC-BY 4.0
Smilax plants are distributed in tropical, subtropical, and temperate regions in both
hemispheres of the world. They are used extensively in traditional medicines in a
number of countries. However, morphological and molecular barcodes analysis,
which may assist in the taxonomic identification of species, are lacking in Ecuador.
In order to evaluate the micromorphological characteristics of these plants, cross
sections of Smilax purhampuy leaves were obtained manually. The rhizome powder,
which is typically used in traditional medicines, was analyzed for micromorphological
characteristics. All samples were clarified with 1% sodium hypochlorite. Tissues were
colored with 1% safranin in water and were fixed with glycerinated gelatin. DNA
was extracted from the leaves using a modified CTAB method for molecular barcode
characterization and PCR was performed using primers to amplify the different loci
including the plastid genome regions atpF-atpH spacer, matK gene, rbcL gene, rpoB
gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer; and the nuclear DNA
sequence ITS2. A DNA sequence similarity search was performed using BLAST in the
GenBank nr database and phylogenetic analysis was performed using the maximum
likelihood method according to the best model identified by MEGAX using a
bootstrap test with 1,000 replicates. Results showed that the micromorphological
evaluation of a leaf cross section depicted a concave arrangement of the central vein,
which was more pronounced in the lower section and had a slight protuberance. The
micromorphological analysis of the rhizome powder allowed the visualization of a
group of cells with variable sizes in the parenchyma and revealed thickened xylematic
vessels associated with other elements of the vascular system. Specific amplicons
were detected in DNA barcoding for all the barcodes tested except for the trnH–psbA
spacer. BLAST analysis revealed that the Smilax species was predominant in all the
samples for each barcode; therefore, the genus Smilax was confirmed through DNA
barcode analysis. The barcode sequences psbK-psbI, atpF-atpH, and ITS2 had a better
resolution at the species level in phylogenetic analysis than the other barcodes we
tested.
OPEN ACCESS
How to cite this article Soledispa P, Santos-Ordóñez E, Miranda M, Pacheco R, Gutiérrez Gaiten YI, Scull R. 2021. Molecular barcode
and morphological analysis of Smilax purhampuy Ruiz, Ecuador. PeerJ 9:e11028 http://doi.org/10.7717/peerj.11028
Subjects Agricultural Science, Biochemistry, Genetics, Molecular Biology, Plant Science
Keywords atpF-atpH spacer, matK , rbcL, rpoB, rpoC1, psbK –psbI spacer, ITS2, Medicinal
INTRODUCTION
The genus Smilax (in the family Smilacaceae) consists of 310 species that are distributed
in tropical, subtropical and temperate regions in both hemispheres of the world (Qi et al.,
2013). According to Cameron & Fu (2006), Smilacaceae are taxonomically confused and
belong to the cosmopolitan family of Liliales. Due to morphological analysis, the division
of Smilacaceae includes at least seven genera and five sections within the large genus
Smilax. Plants are dioecious, vine, herbaceous, or rarely, sub-shrubs or shrubs. Leaves
are simple, and alternating with petioles that have tendrils; the primary venation are
acrodomous. Thicker stems are rippled while aerial stems are generally aculeate (Martins
et al., 2013b). In Ecuador, the genus is not well-recorded, although approximately ten
species of this genus have been reported in the country according to Gaskin (1999).
Smilax is used a variety of ways in traditional medicine. For instance, in Brazil, Smilax
longifolia Rich and Smilax syphilitica Humb & Bonpl. ex Wild are used as diuretics and
in the treatment of venereal diseases (Breitbach et al., 2013) and Smilax quinquenervia
Vell is used as a tonic for rheumatism and as an anti-syphilitic (Andreata, 1997). In
Central America, several species of Smilax are used as diuretics, and for dermatological
infections, gastrointestinal disorders, rheumatism, vaginitis, contraception, menstrual
regulation, anemia, snake bites, and arthritis. In Ecuador, Smilax species are used for the
elimination of cholesterol and triglycerides, the treatment of arthritis, intestinal, stomach
and prostate inflammations, chronic gastritis, and cysts (Ferrufino & Gómez, 2004).
Several pharmacological properties have been demonstrated, including glucose-lowering
(Romo-Pérez, Escandón-Rivera & Andrade-Cetto, 2019), anti-hyperuricemic (Huang et
al., 2019), anti-inflammatory and analgesic (Khana et al., 2019), diuretic (Pérez-Ramírez
et al., 2016), and antioxidant (Fonseca et al., 2017) effects. Several chemical compounds
have been identified in the genus, including polysaccharides (Zhang, Pan & Ran, 2019),
steroidal saponins (Luo et al., 2018), and flavonoids (Wang et al., 2019), among others.
Smilax purhampuy is native to the Amazon and is distributed throughout Ecuador,
Peru, Nicaragua, Colombia, Bolivia, Costa Rica, Venezuela, Honduras, and Brazil
(Rivas et al., 2017). S. purhampuy is traditionally known for its healing and therapeutic
properties. It has been used to treat cholesterol and triglycerides, chronic gastritis, cysts,
arthritis, and intestinal, stomach, and prostate inflammations (JR Global del Perú, 2011).
However, information on S. purhampuy is limited.
Morphological and molecular barcode analysis are lacking for S. purhampuy in
Ecuador, despite its medicinal use. Phylogenetic analysis of the Smilax genus includes
microsatellites (Martins et al., 2013a; Ru et al., 2017; Qi et al., 2017) which are also
informative tools for genetic diversity and gene flow studies. Other methods for the
phylogenetic analysis of plants includes DNA barcodes, which may be used as a complementary tool in the taxonomic identification of the species; for instance, the plastid
Soledispa et al. (2021), PeerJ, DOI 10.7717/peerj.11028
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genome regions atpF-atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–
psbI spacer, and trnH–psbA spacer have been tested as universal plant barcodes (CBOL
Plant Working Group, 2009). The chloroplast genes rbcL and matK, are recommended to
characterize land plants as a 2-locus combination (CBOL Plant Working Group, 2009).
The first reported use of the DNA barcode in Smilax species included the rDNA ITS
sequence (Cameron & Fu, 2006) inferring phylogenetic relationships that elucidated the
evolutionary and biogeographic history of the genera from the Smilacaceae family.
Sulistyaningsih et al. (2018) used the DNA barcode rbcL for phylogenetic analysis of
Smilax spp. in Java, Indonesia. Qi et al. (2013) used the DNA barcodes ITS, matK and
the rpl16 intron in Smilacaceae indicating that the phylogenetic relationships largely
contradicted the traditional morphological classification of the family. Wang et al. (2014)
used the DNA barcode psbA-trnH to distinguish Smilax glabra from its related species,
and Kritpetcharat et al. (2011) used the trnH-psbA spacer barcode for Smilax china and
S. glabra indicating that measuring the genetic distance may be used to discriminate
between the two species. In a broader study which includes four species of Smilax and
other trees, Liu et al. (2015) used the DNA barcodes rbcL, matK, ITS, ITS2, and trnH-psbA
to analyze the diversity and species resolution, concluding that the combination of the
loci rbcl + ITS2 is an effective tool for documenting plant diversity in the Dinghushan
National Nature Reserve in China. Other loci in medicinal plants have been proposed
for their characterization, including the nuclear sequence ITS2 (Zhang et al., 2016).
Furthermore, DNA barcodes could be used to distinguish adulterated drugs (Kumari
& Kotecha, 2016). Therefore, DNA barcode analysis should be performed for Smilax
species to verify the results of taxonomic and morphological studies. We investigated
the micromorphological and molecular barcode characterization of S. purhampuy Ruiz
collected in Ecuador and found that the barcodes psbK-psbI, atpF-atpH, and ITS2 could
be used in Smilax plants for a better resolution at the species level.
MATERIALS AND METHODS
Study area
The climate of the study area is rainy megathermal with an average monthly temperature
between 22 ◦ C and 26 ◦ C and average rainfall between 2,000 to 3,000 mm per year. The
study area is a tropical humid forest.
Collection of plant material
Plant material was collected from three specimens of S. purhampuy Ruiz in the Francisco
de Orellana Province in Ecuador (coordinates 1◦ 10′ 03.7′′ S 76◦ 56′ 30.9′′ W) in March
and April of 2019. The samples were collected from shaded-exposed plants. Branches
containing leaves, fruits, and the rhizome were transferred to the GUAY herbarium of the
Faculty of Natural Sciences of the University of Guayaquil for taxonomic characterization.
Samples were identified as S. purhampuy Ruiz (voucher number 13,117).
Plant material preparation
Leaves and rhizomes were washed with water. The leaves used in the micromorphological
study were analyzed and stored at −80 ◦ C for DNA extraction. The rhizomes were dried
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in a Mettler Toledo stove at 40 ◦ C and then samples of the rhizomes were crushed with a
manual knife mill and stored in amber glass jars for analysis.
Micromorphological analysis
Leaf samples were taken from the middle of the lamina for the evaluation of their
micromorphological characteristics. The mid-rib was cut transversely according to the
manual method (Miranda & Cuéllar, 2000). The maximum sample width was 1 cm
including the mid-rib. Transversal cuts of fresh leaves were hydrated and clarified with
1% sodium hypochlorite. Tissues were colored with 1% safranin in water and fixed with
glycerinated gelatin according to the method by Gattuso & Gattuso (1999). The powder
obtained from the rhizomes was hydrated, clarified with 1% sodium hypochlorite and
colored with 1% safranin in water and fixed with glycerinated gelatin (Gattuso & Gattuso,
1999; Miranda & Cuéllar, 2000). We performed a histochemical reaction with Lugol
reagent to detect starch in the powdered drug obtained from the rhizomes (Gattuso &
Gattuso, 1999). Morphological analysis was performed using a NOVEL light microscope
at 10x magnification, attached to an HDCE-50B digital camera, model 146 HDCE-50B.
DNA extraction and PCR
Leaf samples from the three S. purhampuy Ruiz plants (with codes CIBE-010, CIBE-011,
CIBE-012) were ground with MM400 (Retsch, Haan, Germany) and liquid nitrogen
and were stored at −80 ◦ C. DNA extraction was performed for each Smilax plant
independently. A modified CTAB protocol was used for total DNA extraction according
to Pacheco Coello et al. (2017). The master mix GoTaq R 2x (Cat# M7123, Promega)
was used for PCR analysis according to the manufacturer’s instructions using 0.5 µM
for each primer according to the barcode used (Table S1) in a 50 µL PCR reaction. The
conditions for the PCR were: 95 ◦ C for 3 min for initial denaturation; 35 cycles of 95 ◦ C
for 30 s, 50 ◦ C/56 ◦ C/60 ◦ C (depending of the barcode, Table S1) for 60 s, 72 ◦ C for 60
s; and a final extension of 72 ◦ C for 10 min. Amplicons were detected by sampling 5 µL
in agarose gel (1.5%) electrophoresis. The remaining 45 µL was purified and sequenced
commercially (Macrogen, Rockville, MD, USA). At least two technical replicates were
sequenced and a consensus was generated for each biological replicate.
Bioinformatic analysis
Sequences were processed using MEGAX (Stecher, Tamura & Kumar, 2020). Technical
replicates were aligned with MUSCLE and a consensus sequence was generated for
each barcode. Consensus sequences were analyzed by BLAST (Zhang et al., 2000) in
the GenBank non-redundant nucleotide database (nr). The nr database included the
accessions of the complete plastid genomes of Smilax spp. representing three species
and also accessions containing sequences of single locus, indicating that the results were
dependent on the sequence availability in the database at the time of the analysis (28th
June 2020). Accessions were selected for phylogenetic analysis based on BLAST analysis.
For each barcode, the accessions and samples sequences were aligned using MUSCLE and
the recommended model from MEGAX was used. The aligned sequences were trimmed at
the ends to allow for all sequences to maintain the same range. The maximum likelihood
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Figure 1 Microscopic characteristics of the leaf from Smilax purhampuy Ruiz. Transversal section
of the central vein of the leaf (A, B). AbE, abaxial epidermis; AdE, adaxial epidermis; FP, fundamental
parenchyma; Me, mesophyll; PP, palisade parenchyma; SP, spongy parenchyma; ST, sclerenchyma tissue;
Vs, vascular system.
Full-size DOI: 10.7717/peerj.11028/fig-1
method was performed according to the best model found by MEGAX using bootstrap
test (1,000 replicates).
RESULTS
Morphological analysis
The micromorphological evaluation of a cross section of the leaf sample (Fig. 1A) showed
a concave arrangement of the central midrib, which was more pronounced in the lower
part with a slight protuberance. The mesophyll showed a well-defined adaxial and abaxial
uniseriate epidermis with a fine cuticle on the lateral sides of the central vein. Below the
adaxial epidermis, we observed a palisade parenchyma forming two or three continuous
layers of elongated cells. The spongy parenchyma exhibited cells of variable size that
bordered the abaxial epidermis. An enlargement of the central vein (Fig. 1B) showed the
fundamental parenchyma, which was formed by many isometric cells. The sclerenchyma
tissue was characterized by lignified thickened walled cells surrounding a well-defined
vascular system (xylem and phloem) near the middle of the central vein, which harbored
six vascular bundles of variable size.
Micromorphological analysis of the drug obtained from the rhizome (Fig. 2) allowed
the visualization of a group of cells of the parenchyma of variable size (Fig. 2A) and
revealed thickened xylematic vessels associated with other elements of the vascular
system (Fig. 2B). Elongated, fusiform, and pointed structures were also visualized, which
corresponded to fibers and may suggest a type of filiform sclerides (Fig. 2C). We observed
xylematic thickening vessels with holes in another sample of the powder drug (Fig. 2D).
Numerous starch granules of variable size were observed showing a blackish coloration
with the Lugol reagent (Fig. 2E).
Molecular barcodes for Smilax purhampuy Ruiz plants
Specific PCR amplification was detected for all barcodes except for trnH-psbA (data not
shown). BLAST analysis was performed for each barcode sequence (Data S1 and S2)
and the best hit for all samples for each barcode indicated the Smilax species (Table 1).
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Figure 2 Microscopic characteristics of the powder drug from Smilax purhampuy Ruiz rhizome. (A)
Parenchyma cells. (B) Xylematic veseels and other elements of the vascular system. (C) Fibers (filiform
sclerides). (D) Xylematic veseels, (E) starch granules.
Full-size DOI: 10.7717/peerj.11028/fig-2
BLAST analysis indicated the presence of Smilax spp. using the sequences available at
the nr database, including plastid genomes and single locus sequence. The best hits in
BLASTn for the different species included: S. nipponica for psbK-psbI (96.57%, 96.56%,
and 93.83% of identity for the three biological replicates, respectively); S. nipponica and
S. china for rpoB (99.73% for the three biological replicates); S. nipponica, S. china, and
S. aspera for rpoC1 (100%); S. sieboldii f. inermis for atpF-atpH (90.66%, 90.69%, and
96.47%), S. fluminensis (99.65%, 100%), S. bona-nox (99.88%) and S. laurifolia (99.88%)
for matK ; S. aspera (99.82%, 99.82%) and S. laurifolia (99.82%) for rbcL; and S. excelsa
(80.49%, 80.05%) for ITS2 (only two biological replicates were sequenced successfully for
ITS2).
We determined the best models for phylogenetic analysis were: T92+G+I (rbcL),
T92+G (matK, ITS2, psbK-psbI, atpF-atpH ), T92 (rpoB), and JC (rpoC1) after alignment
of the barcode sequences between the S. purhampuy from this study and different
accessions including other genera. Phylogenetic analysis revealed that for psbK-psbI, the
S. purhampuy sequences shared a clade (99 bootstrap) with different Smilax species,
including S. china, S. nipponica, and S. glycophylla; and with two accessions from another
genus, including Hemidesmus indicus (Fig. 3). However, the S. purhampuy sequences
CIBE-010 and CIBE-011 are grouped in a subclade (bootstrap 96), while S. purhampuy
CIBE-012 shared a clade with both S. purhampuy (bootstrap 30). The other major clades
in the phylogenetic tree corresponded to other genera. For the rpoB the phylogenetic tree
revealed that the S. purhampuy samples were in a clade (98 bootstrap) with other Smilax
species; while other genera including Brahea spp., Philesia magellanica, Tricyrtis macropoda, Fritillaria spp., and Lilium spp. were in other clades (Fig. S1). The S. purhampuy
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Soledispa et al. (2021), PeerJ, DOI 10.7717/peerj.11028
Table 1 Blastn analysis for seven different barcodes of Smilax purhampuy Ruiz plants (CIBE-010, CIBE-011, CIBE-012). Results were ranked for the first three with
the highest percentage of identity. Species with the best results are bold for each barcode.
Barcode
Code
Blastn rank
1
psbK-psbI
rpoB
rpoC1
atpF-atpH
matK
rbcL
ITS2
2
3
Organism
Accesion
% identity
Organism
Accesion
% identity
Organism
Accesion
% identity
CIBE-010
Smilax nipponica
MT261170.1a
96.57%
Smilax china
MT261168.1a
95.91%
Smilax glycophylla
MT261169.1a
92.69%
CIBE-011
Smilax nipponica
MT261170.1a
96.56%
Smilax china
MT261168.1a
95.91%
Smilax glycophylla
MT261169.1a
92.67%
CIBE-012
Smilax nipponica
a
MT261170.1
93.83%
Smilax china
a
MT261168.1
93.20%
Smilax glycophylla
MT261169.1a
89.32%
CIBE-010
Smilax nipponica
MT261170.1a
99.73%
Smilax glycophylla
MT261169.1a
99.73%
Smilax china
MT261168.1a
99.47%
CIBE-011
Smilax nipponica
a
MT261170.1
99.73%
Smilax glycophylla
a
MT261169.1
99.73%
Smilax china
MT261168.1
a
99.46%
CIBE-012
Smilax nipponica
MT261170.1a
99.73%
Smilax glycophylla
MT261169.1a
99.73%
Smilax china
MT261168.1a
99.46%
CIBE-010
Smilax nipponica
a
MT261170.1
100.00%
Smilax china
a
MT261168.1
100.00%
Smilax aspera
EU531650.1
100.00%
CIBE-011
Smilax nipponica
MT261170.1a
100.00%
Smilax china
MT261168.1a
100.00%
Smilax aspera
EU531650.1
100.00%
CIBE-012
Smilax nipponica
MT261170.1a
100.00%
Smilax china
MT261168.1a
100.00%
Smilax aspera
EU531650.1
100.00%
CIBE-010
Smilax sieboldii f. inermis
JN417282.1
90.66%
Smilax sieboldii
JN417281.1
90.64%
Hemidesmus indicus
NC_047471.1
89.84%
CIBE-011
Smilax sieboldii f. inermis
JN417282.1
90.69%
Smilax sieboldii
JN417281.1
90.68%
Smilax glycophylla
MT261169.1a
89.03%
CIBE-012
Smilax sieboldii f. inermis
JN417282.1
96.47%
Smilax sieboldii
JN417281.1
96.31%
Smilax china
MT261168.1a
94.69%
CIBE-010
Smilax fluminensis
JF461414.1
99.65%
Smilax coriacea
KJ719950.1
98.72%
Smilax havanensis
KF782873.1
98.72%
CIBE-011
Smilax fluminensis
JF461414.1
100.00%
Smilax coriacea
KJ719950.1
99.05%
Smilax havanensis
KF782873.1
99.05%
CIBE-012
Smilax bona-nox
KC511353.1
99.88%
Smilax laurifolia
JF461393.1
99.88%
Smilax ligneoriparia
KX432989.1
99.76%
CIBE-010
Smilax aspera
KX394660.1
99.82%
Smilax aspera
KX394659.1
99.82%
Smilax aspera
KM609079.1
99.82%
CIBE-011
Smilax aspera
KX394660.1
99.82%
Smilax aspera
KX394659.1
99.82%
Smilax aspera
KM609079.1
99.82%
a
CIBE-012
Smilax laurifolia
JF944386.1
99.82%
Smilax china
MT261168.1
99.45%
Smilax gaudichaudiana
KX394669.1
99.45%
CIBE-010
Smilax excelsa
JF461354.1
80.49%
Smilax aspera
KJ719926.1
80.24%
Smilax aspera
KJ719924.1
80.24%
CIBE-011
Smilax excelsa
JF461354.1
80.05%
Smilax aspera
KJ719926.1
79.81%
Smilax aspera
KJ719924.1
79.81
Notes.
a
Accession numbers refer to plastid complete genome.
7/16
Figure 3 Phylogenetic tree of the psbK-psbI spacer with accessions from the genus Smilax and different genera selected from the blastn results. Three species from the genus Paris was used as outgroup.
Maximum Likelihood method based on Tamura 3-parameter model. Boostrap test with 1,000 replicates
was performed. The tree with the highest log likelihood (−1728.93) is shown. The percentage of trees in
which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of
pairwise distances estimated using the Tamura 3 parameter model, and then selecting the topology with
superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter = 1.6020)). The tree is drawn to scale, with branch
lengths measured in the number of substitutions per site. This analysis involved 26 nucleotide sequences.
There were a total of 368 positions in the final dataset. Evolutionary analyses were conducted in MEGA X
(Kumar et al., 2018; Stecher, Tamura & Kumar, 2020). Blue arrows indicate Smilax purhampuy Ruiz from
Ecuador.
Full-size DOI: 10.7717/peerj.11028/fig-3
samples for the rpoC1 were in a clade with different species of Smilax, including S. aspera
(accession number EU531650), S. nipponica (MT261170), S. china (MT261168), and S.
herbacea (HQ594138, HQ594139, Fig. S1). The atpF-atpH phylogenetic analysis revealed
that several Smilax species were in the same clade. However, the S. purhampuy samples
from this study are in a subclade (bootstrap 99). For the matK and rbcL barcodes, the
same pattern was observed where a clade sharing different Smilax species was encountered
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(Fig. S1), although a subclade was formed with the two S. purhampuy samples (CIBE-010,
CIBE-011) and S. fluminensis; while for the S. purhampuy CIBE-012 sample, a branch was
shared with S. aspera for matK. Furthermore, in the rbcL phylogenetic tree, a subclade was
formed for S. purhampuy samples CIBE-010 and CIBE-011 with other species including
S. aspera, while for the S. purhampuy CIBE-012 a clade was shared with S. domingensis
and S. lauriflora. The ITS2 phylogenetic tree (Fig. 4) revealed that the two S. purhampuy
samples (CIBE-010 and CIBE-011) were in a different clade (bootstrap 99) apart from
the other Smilax species, including S. aspera, S. stans, S. menispermoidea, S. trachypoda,
S. aberrans, S. retroflexa, S. excelsa, S. lunglingensis, S. hispida, S. japonica, S. china, and S.
pumila.
DISCUSSION
Microscopic analysis
We made a detailed assessment of the herbal drugs and used microscopy to identify them
based on their known histological characteristics (Shailesh et al., 2015). Micromorphological studies are essential for the quality control of plant-derived drugs, since significant
details are used to correctly identify the plant and possible adulterants.
The cross section of the S. purhampuy leaf indicated that the abaxial and adaxial epidermis are uniseriate, with the existence of an easily perceptible cuticle. Morphoanatomic
studies performed on the leaves of various Smilax species (S. brasiliensis, S. campestris,
S. cisoides, S. fluminensis, S. goyazana, S. oblongifolia, and S. rufescens) revealed a nonstratified epidermis with thick cuticle (Martins et al., 2013b). These results correspond to
those of previous studies and may be a distinctive anatomical characteristic of the genus.
In most plants, the leaf mesophyll harbors palisade and spongy tissue, which differ
in location, cell morphology, and function. In plants with a dorsiventral mesophyll, the
palisade tissue is located on the adaxial side and the spongy tissue on the abaxial side;
this distribution makes a greater contribution to the photosynthesis process (Yahia et
al., 2019). Studies by various researchers have shown that the mesophyll could vary
from one leaf to another of the same individual or of different individuals, depending
on light intensity and salt concentration (Gapińska & Glińska, 2014). In S. purhampuy a
dorsiventral mesophyll was observed where the palisade parenchyma is toward the adaxial
side and the spongy parenchyma was located toward the abaxial side, which supports the
results of Martins & Appezzato-da Glória (2006) for S. polyantha and Martins et al. (2013b)
for S. brasiliensis, S. campestris, S. cissoids, S. goyazana, S. oblongifolia and S. rufescens.
The presence of six vascular bundles of variable size at the level of the central vein of the
leaf was unique and differed from other Smilax species (S. brasiliensis, S. cissoides, and S.
fluminensis), which present three vascular bundles (Martins et al., 2013b).
The presence of parenchyma tissue, xylematic vessels, fibers and starch granules are
reported in S. domingensis from Cuba and Guatemala (Cáceres et al., 2012; González
et al., 2017), which was supported by the present study. The difference lies within the
morphology and arrangement of these structures. For example, thickened scalariformly
xilematic vessels and fibrotraqueids (fibers) were detected in S. domingensis, while
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Figure 4 Phylogenetic tree of the ITS2 with accessions from the genus Smilax and different genera
selected from the blastn results. Three species from the genus Tulipa was used as outgroup. Maximum
Likelihood method based on Tamura 3-parameter model. The tree with the highest log likelihood
(−1405.71) is shown. The percentage of trees in which the associated taxa clustered together is shown
next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying
Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura 3
parameter model, and then selecting the topology with superior log likelihood value. A discrete Gamma
distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter
= 0.8022)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per
site. This analysis involved 30 nucleotide sequences. There were a total of 436 positions in the final dataset.
Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018; Stecher, Tamura & Kumar, 2020).
Blue arrows indicate Smilax purhampuy Ruiz from Ecuador.
Full-size DOI: 10.7717/peerj.11028/fig-4
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thickened xilematic vessel with holes and filiform sclerides (fibers) were observed in the
studied species.
Molecular barcode sequences
Genetic analysis has proven to be an important tool in the standardization of medicinal
plants. The genotypic characterization of plant species is important since most plants
may show considerable variation in morphology, although they belong to the same genus
and species. DNA analysis is useful for the identification of cells, individuals, or species
and could help distinguish genuine from adulterated drugs (Kumari & Kotecha, 2016).
Different methods may be applied for genotyping in plants. Microsatellite (Martins et
al., 2013a; Ru et al., 2017; Qi et al., 2017) and DNA barcodes (Cameron & Fu, 2006; Qi
et al., 2013) have been used for genotyping Smilax species and have shown phylogenetic
relationships between different Smilax species. Additionally, Sulistyaningsih et al. (2018)
used the DNA barcode rbcL for phylogenetic analysis of Smilax spp. in Java, Indonesia,
concluding that rbcL could be used to identify at the genus but not at species level.
The analysis of DNA barcodes could be used as a complementary analysis for the identification of plants species, especially when Smilax species show considerable phenotypic
variations within populations (Cameron & Fu, 2006). The recommended barcodes for
species identification are 2-locus rbcL and matK (CBOL Plant Working Group, 2009).
Generally, the BLASTn analysis relies in the presence of those species in the GenBank
for species identification; consequently, the results presented in the BLAST analysis and
in the phylogenetic trees depended on the sequences available in the nr database in the
GenBank. Therefore, a complete analysis using different species of Smilax should be
performed in the future for all the DNA barcodes tested.
We analyzed the three samples taxonomically identified as S. purhampuy Ruiz (CIBE010, CIBE-011, CIBE-012) which were more similar than other Smilax species, including
S. nipponica, S. glycophylla, S. herbacea, S. china, S. sieboldin, S. aspera, S. stans, S. menispermoidea, S. trachypoda, S. aberrans, and S. pumila. These results were also observed in
the phylogenetic trees for psbK-psbI spacer, atpF-atpH spacer, and ITS2. However, few
accessions were encountered in the GenBank for the DNA barcodes psbK-psbI spacer and
atpF-atpH spacer. There should be additional study of the different Smilax species for
these barcodes; however, other DNA barcodes may accurately identify the genus level. The
ITS2 revealed a low percentage of identification (80.49%) with BLASTn, suggesting that
species differentiation could be detected using the ITS2. The results from other studies
have indicated a better resolution for species identification using the ITS2 in medicinal
plants (Techen et al., 2014; Zhang et al., 2016; Bustamante et al., 2019; Sarmiento-Tomalá et
al., 2020).
Our results determined that the rpoC1 sequence was not accurate at the species
level, and that matK could not be used to discriminate between S. purhampuy and S.
fluminensis. Furthermore, the rbcL barcode could not be used for species differentiation
in the Smilax genus, as a low bootstrap value was observed in the different clades formed.
The psbK-psbI, atpF-atpH, and ITS2 had a better resolution at the species level for S.
purhampuy. Future research should include the sequencing of selected barcodes (rbcL,
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matK, psbK-psbI spacer, atpF-atpH spacer, and ITS2) for different species of the Smilax
genus found in Ecuador with biological replicates. Further studies should establish a
reliable DNA barcode analysis and test different 2-locus combinations to determine which
barcode should be used for species identification in the Smilax genus.
CONCLUSIONS
We determined the morphological characteristics and conducted molecular barcode
analysis on S. purhampuy Ruiz plants collected in Ecuador. The micromorphological
characteristics of the leaves and rhizomes were described for the first time, which
constitutes a novel contribution to the botanical characterization of the species. The
taxonomic classification of Smilax was confirmed by the molecular barcodes used,
including psbK-psbI, rpoB, rpoC, atpF-atpH, mat K, rbcL, and ITS2. Furthermore, the
barcodes sequences psbK-psbI, atpF-atpH, and ITS2 indicated a better resolution at the
species level than the other barcodes tested in this study. These barcodes (psbK-psbI, atpFatpH, and ITS2) could be used to identify other species in the genus Smilax. However,
further molecular barcode analysis should be performed on Smilax spp. from Ecuador
to determine its diversity and to complete its taxonomic classification. Furthermore, the
medicinal properties of the Smilax plants used in this study should be studied in greater
detail.
ACKNOWLEDGEMENTS
Identification of samples by the GUAY herbarium of the Faculty of Natural Sciences of
the Guayaquil University is acknowledged.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
The authors received no funding for this work.
Competing Interests
The authors declare there are no competing interests.
Author Contributions
• Pilar Soledispa conceived and designed the experiments, performed the experiments,
analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the
paper, and approved the final draft.
• Efrén Santos-Ordóñez conceived and designed the experiments, analyzed the data,
prepared figures and/or tables, authored or reviewed drafts of the paper, and approved
the final draft.
• Migdalia Miranda conceived and designed the experiments, analyzed the data, authored
or reviewed drafts of the paper, and approved the final draft.
• Ricardo Pacheco performed the experiments, analyzed the data, authored or reviewed
drafts of the paper, and approved the final draft.
Soledispa et al. (2021), PeerJ, DOI 10.7717/peerj.11028
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• Yamilet Irene Gutiérrez Gaiten performed the experiments, analyzed the data, prepared
figures and/or tables, authored or reviewed drafts of the paper, and approved the final
draft.
• Ramón Scull performed the experiments, authored or reviewed drafts of the paper, and
approved the final draft.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving
body and any reference numbers):
Samples were collected for identification in the framework of the GUAY Herbarium,
Facultad de Ciencias Naturales, Universidad de Guayaquil.
Data Availability
The following information was supplied regarding data availability:
Sequences are available in the Supplemental Files and at GenBank: MT740231,
MT740232, MT740233, MT740234, MT740235, MT740236, MT740237, MT740238,
MT740239, MT740240, MT740241, MT740242, MT734663, MT734664, MW300280,
MW300281, MW300282, MW300277, MW300278, MW300279.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.
7717/peerj.11028#supplemental-information.
REFERENCES
Andreata RHP. 1997. Revisão das espécies brasileiras do gênero Smilax Linnaeus
(Smilacaceae). Pesqui Bot 47:7–244.
Breitbach B, Niehues M, Lopes NP, Faria JEQ, Brandão MGL. 2013. Amazonian
Brazilian medicinal plants described by C.F.P. von Martius in the 19th century.
Journal of Ethnopharmacology 147:180–189.
Bustamante K, Santos-Ordóñez E, Miranda M, Pacheco R, Gutiérrez Y, Scull R. 2019.
Morphological and molecular barcode analysis of the medicinal tree Mimusops
coriacea (A.DC.) Miq. collected in Ecuador. PeerJ 7:e7789
DOI 10.7717/peerj.7789.
Cáceres A, Cruz SM, Martínez V, Gaitán I, Santizo A, Gattuso S, Gattuso M. 2012.
Ethnobotanical, pharmacognostical, pharmacological and phytochemical studies on Smilax domingensis in Guatemala. Brazilian Journal of Pharmacognosy
22(2):239–248 DOI 10.1590/S0102-695X2011005000211.
Cameron KM, Fu C. 2006. A nuclear rDNA phylogeny of Smilax (Smilacaceae). Aliso
22:598–605 DOI 10.5642/aliso.20062201.47.
CBOL Plant Working Group. 2009. A DNA barcode for land plants. Proceedings of the
National Academy of Sciences of the United States of America 106(31):12794–12797
DOI 10.1073/pnas.0905845106.
Soledispa et al. (2021), PeerJ, DOI 10.7717/peerj.11028
13/16
Ferrufino L, Gómez J. 2004. Estudio morfológico de Smilax L. (Smilacaceae) en Costa
Rica, con implicaciones sistemáticas. Lankesteriana 4(1):5–36.
Fonseca JC, Barbosa MA, Silva ICA, Duarte-Almeida JM, Castro AHF, Dos Santos
Lima LAR. 2017. Antioxidant and allelopathic activities of Smilax brasiliensis Sprengel (Smilacaceae). South African Journal of Botany 111:336–340
DOI 10.1016/j.sajb.2017.04.003.
Gapińska M, Glińska S. 2014. Salt-mediated changes in leaf mesophyll cells of Lycopersicon esculentum Mill. plants. Journal of Central European Agriculture 15(3):219–235
DOI 10.5513/JCEA01/15.3.1478.
Gaskin J. 1999. Smilacaceae. In: Jørgensen PM, León-Yánez S, eds. Catalogue of the
vascular plants of Ecuador.Monographs in Systematic Botany from the Missouri
Botanical Garden. St. Louis: Missouri Botanical Garden Press, 75.
Gattuso MA, Gattuso SJ. 1999. Manual de procedimientos para el análisis de drogas en
polvo. Rosario: Editorial de la Universidad Nacional de Rosario Urquiza.
González YJ, Monan M, Cuéllar A, De Armas T, Gómez E, Dopico E. 2017. Pharmacognostic and phytochemical studies of Smilax domingensis Willd. in Cuba. American
Journal of Plant Sciences 8:1462–1470 DOI 10.4236/ajps.2017.86100.
Huang L, Deng J, Yuan C, Zhou M, Liang J, Yan B, Shu J, Liang Y, Huang H.
2019. The anti-hyperuricemic effect of four astilbin stereoisomers in Smilax
glabra on hyperuricemic mice. Journal of Ethnopharmacology 238:111777
DOI 10.1016/j.jep.2019.03.004.
JR Global del Perú S.A.C. 2011. Ficha Técnica-Zarzaparrilla.. Available at http:// www.
inkaplus.com/ media/ web/ pdf/ Zarzaparrilla.pdf (accessed on 08 July 2019).
Khana AK, Singha PD, Reeseb PB, Howdena J, Thomasa TT. 2019. Investigation of
the anti-inflammatory and the analgesic effects of the extracts from Smilax ornata
Lem. (Jamaican sarsaparilla) plant. Journal of Ethnopharmacology 240:111830
DOI 10.1016/j.jep.2019.111830.
Kritpetcharat O, Khemtonglang N, Kritpetcharat P, Daduang J, Daduang S, Suwanrungruang K, Bletter N, Sudmoon R, Chaveerach A. 2011. Using DNA markers and
barcoding to solve the common problem of identifying dried medicinal plants with
the examples of Smilax and Cissus in Thailand. Journal of Medicinal Plants Research
5(15):3480–3487.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary
genetics analysis across computing platforms. Molecular Biology and Evolution
35:1547–1549 DOI 10.1093/molbev/msy096.
Kumari R, Kotecha M. 2016. A review on the Standardization of herbal medicines.
International Journal of Pharma Sciences and Research 7(2):97–106.
Liu J, Yan H-F, Newmaster SG, Pei N, Ragupathy S, Ge X-J. 2015. The use of DNA
barcoding as a tool for the conservation biogeography of subtropical forests in
China. Diversity and Distributions 21(2):188–199 DOI 10.1111/ddi.12276.
Luo R, Gao XL, Cha Shi, Wei YX, Hou PY, Li HG, Wang SQ, Anderson S, Zhang
YW, Wu XH. 2018. Phytochemical and chemotaxonomic study on the rhizomes
Soledispa et al. (2021), PeerJ, DOI 10.7717/peerj.11028
14/16
of Smilax riparia (Liliaceae). Biochemical Systematics and Ecology 76:58–60
DOI 10.1016/j.bse.2017.12.005.
Martins AR, Abreu AG, Bajay MM, Villela PM, Batista CE, Monteiro M, AlvesPereira A, Figueira GM, Pinheiro JB, Appezzato-da Glória B, Zucchi MI. 2013a.
Development and characterization of microsatellite markers for the medicinal plant
Smilax brasiliensis (Smilacaceae) and related species. Applications in Plant Science
1(6):apps.1200507 DOI 10.3732/apps.1200507.
Martins AR, Appezzato-da Glória B. 2006. Morfoanatomia dos órgãos vegetativos de
Smilax polyantha Griseb. (Smilacaceae). Brazilian Journal of Botany 29:555–567
DOI 10.1590/S0100-84042006000400005.
Martins AR, Bombo AB, Soares AN, Appezzato-da Glória B. 2013b. Aerial stem and
leaf morphoanatomy of some species of Smilax. Brazilian Journal of Pharmacognosy
23(4):576–584 DOI 10.1590/S0102-695X2013005000043.
Miranda MM, Cuéllar AC. 2000. Manual de prácticas de laboratorio. Ciudad Habana:
Farmacognosia y productos naturales 25–49, 74–79.
Pacheco Coello R, Pestana Justo J, Factos Mendoza A, Santos Ordoñez E. 2017.
Comparison of three DNA extraction methods for the detection and quantification
of GMO in Ecuadorian manufactured food. BMC Research Notes 10(1):758
DOI 10.1186/s13104-017-3083-x.
Pérez-Ramírez IF, Enciso-Moreno JA, Guevara-González RG, Gallegos-Corona MA,
Loarca-Piña G, Reynoso-Camacho R. 2016. Modulation of renal dysfunction by
Smilax cordifolia and Eryngium carlinae, and their effect on kidney proteome in obese
rats. Journal of Functional Foods 20:545–555 DOI 10.1016/j.jff.2015.11.024.
Qi Z, Cameron KM, Li P, Zhao Y, Chen S, Chen G, Fu C. 2013. Phylogenetics, character
evolution, and distribution patterns of the greenbriers, Smilacaceae (Liliales), a
near-cosmopolitan family of monocots. Botanical Journal of the Linnean Society
173:535–548 DOI 10.1111/boj.12096.
Qi ZC, Shen C, Han YW, Shen W, Yang M, Liu J, Liang ZS, Li P, Fu CX. 2017. Development of microsatellite loci in Mediterranean sarsaparilla (Smilax aspera; Smilacaceae) using transcriptome data. Applications in Plant Sciences 5(4):apps.1700005
DOI 10.3732/apps.1700005.
Rivas MDPP, Muñoz DGL, Ruiz MAC, Fernández LFT, Muñoz FAC, Pérez NM. 2017.
Global Biodiversity Information Facility (GBIF). Available at https:// www.gbif.org/
species/ 5295631 (accessed on 05 August 2019).
Romo-Pérez A, Escandón-Rivera SM, Andrade-Cetto A. 2019. Chronic hypoglycemic
effect and phytochemical composition of Smilax moranensis roots. Revista Brasileira
de Farmacognosia 29(2):246–253 DOI 10.1016/j.bjp.2019.02.007.
Ru Y, Cheng R, Shang J, Zhao Y, Li P, Fu C. 2017. Isolation and characterization of
microsatellite loci for Smilax sieboldii (Smilacaceae). Applications in Plant Sciences
5(3):apps.1700001 DOI 10.3732/apps.1700001.
Soledispa et al. (2021), PeerJ, DOI 10.7717/peerj.11028
15/16
Sarmiento-Tomalá G, Santos-Ordóñez E, Miranda-Martínez M, Pacheco-Coello R,
Scull-Lizama R, Gutiérrez-Gaitén Y, Delgado-Hernández R. 2020. Short communication: molecular barcode and morphology analysis of Malva pseudolavatera Webb
& Berthel and Malva sylvestris L from Ecuador. Biodiversitas 21(8):3554–3561.
Shailesh LP, Suryawanshi AB, Gaikwad MS, Pedewad SR, Potulwar AP. 2015. Standardization of herbal drugs: an overview. The Pharma Innovation Journal 4(9):100–104
DOI 10.7897/2277-4572.04224.
Stecher G, Tamura K, Kumar S. 2020. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Molecular Biology and Evolution 37(4):1237–1239
DOI 10.1093/molbev/msz312.
Sulistyaningsih LD, Abinawanto A, Ardiyani M, Salamah A. 2018. Short communication: phylogenetic analysis and molecular identification of Canar (Smilax spp.)
in Java, Indonesia based on DNA barcoding analysis. Biodiversitas 19(2):364–368
DOI 10.13057/biodiv/d190202.
Techen N, Parveen I, Pan Z, Khan IA. 2014. DNA barcoding of medicinal plant
material for identification. Current Opinion in Biotechnology 25:103–110
DOI 10.1016/j.copbio.2013.09.010.
Wang S, Fanga Y, Yud X, Guoa L, Zhanga X, Xiaa D. 2019. The flavonoid-rich fraction
from rhizomes of Smilax glabra Roxb. ameliorates renal oxidative stress and
inflammation in uric acid nephropathy rats through promoting uric acid excretion.
Biomedicine & Pharmacotherapy 111:162–168 DOI 10.1016/j.biopha.2018.12.050.
Wang ZT, Hao H, Lin LZ, Yu Y, Li SY. 2014. Identification of Smilax glabra and its
related species based on psbA-trnH sequence. Zhong Yao Cai 37(8):1368–1371.
Yahia EM, López AC, Malda BG, Suzán AH, Queijeiro BM. 2019. Chapter 3—
photosynthesis. In: Postharvest physiology and biochemistry of fruits and vegetables.
47–72.
Zhang D, Jiang B, Duan L, Zhou N. 2016. Internal transcribed spacer (ITS), an ideal
DNA barcode for species discrimination in Crawfurdia Wall. (Gentianaceae). African
Journal of Traditional, Complementary and Alternative Medicines 13(6):101–106.
Zhang Y, Pan X, Ran S. 2019. Purification, structural elucidation and anti-inflammatory
activity in vitro of polysaccharides from Smilax china L. International Journal of
Biological Macromolecules 139:233–243 DOI 10.1016/j.ijbiomac.2019.07.209.
Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm for aligning DNA
sequences. Journal of Computational Biology 7(1–2):203–214
DOI 10.1089/10665270050081478.
Soledispa et al. (2021), PeerJ, DOI 10.7717/peerj.11028
16/16