Research Article |
Corresponding author: Lisete Chamma Davide ( lisete.ufla@gmail.com ) Academic editor: Viktoria Shneyer
© 2015 Thaís Furtado Nani, Amanda Teixeira Mesquita, Fernanda de Oliveira Bustamante, Sandro Barbosa, João Vítor Calvelli Barbosa, Lisete Chamma Davide.
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:
Nani TF, Mesquita AT, Bustamante FO, Barbosa S, Barbosa JVC, Davide LC (2015) Variation of karyotype and nuclear DNA content among four species of Plectranthus L’ Héritier, 1788 (Lamiaceae) from Brazil. Comparative Cytogenetics 9(4): 549-563. https://doi.org/10.3897/CompCytogen.v9i4.6255
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Plectranthus is a genus which includes species of ornamental and medicinal potential. It faces taxonomic problems due to aggregating species previously belonging to the genus Coleus, a fact that has contributed to the existence of various synonymies. The species Plectranthus amboinicus, Plectranthus barbatus, Plectranthus grandis and Plectranthus neochilus are included in this context. Some authors consider P. barbatus and P. grandis as synonyms. The present work was carried out with the aim of comparing plants of the above-mentioned species, originating from different localities in Brazil, with regards to chromosome number and karyotypic morphology, correlated to the nuclear DNA content. There was no variation in chromosome number among plants of the same species. P. amboinicus was the only species to exhibit 2n=34, whereas the others had 2n=30. No karyotypic differences were found among the plants of each species, except for P. barbatus. The plants of the Plectranthus species revealed little coincidence between chromosome pairs. The nuclear DNA content allowed grouping P. amboinicus and P. neochilus, with the highest mean values, and P. grandis and P. barbatus with the lowest ones. Differences in DNA amount among the plants were identified only for P. barbatus. These results allow the inference that the populations of P. amboinicus and P. neochilus present coincident karyotypes among their plants, and P. grandis is probably a synonym of P. barbatus.
Cytogenetics, Cytotaxonomy, Flow cytometry, Karyotypic evolution
The family Lamiaceae Martinov, 1820 contains approximately 250 genera and 6,500 species (
Plectranthus L’ Héritier, 1788 is one of the most common genera of this family, and comprises about 300 species (
This genus, along with Burnatastrum Briquet, 1897, Coleus Loureiro, 1790 Englerastrum Briquet, 1894, Isodictyophorus Briquet, 1917 and Neomullera unrecorded, has already been placed in the genus Ocimum Linnaeus, 1753. Coleus and Plectranthus have been considered distinct genera only because of morphological differences of the stamen; however, this characteristic has later been considered insufficient for the separation of these taxa. This way, the Coleus species were aggregated to the genus Plectranthus, turning this grouping into a unique genus and independent of Ocimum Linnaeus, 1753 (
In Brazil there are some important species of this genus used as herbal medicines. Plectranthus amboinicus (Loureiro, 1825) Sprengel, 1825 is native to East Asia, later introduced in Cuba and distributed in America (
Cytogenetic differences among plants of the same species may reflect in variation in amount, quality and type of secondary metabolites produced by the plant, as observed by
Knowledge of DNA content, along with cytogenetics and molecular genetics, contributes to the genetic characterization of related species. The correct definition of the taxon, associated to biochemical and pharmacological evaluations, is essential for the correct use of plants for medicinal purposes. This preoccupation becomes even more important considering the recognition by the World Health Organization that about 80% of the population in developing countries makes use of plants or preparations thereof as home and communitarian remedies (
Considering the variety of chromosome number descriptions and the lack of karyotypic information about Plectranthus species, an enhanced investigation of the chromosome complement is necessary with the purpose of supporting taxonomic studies and evolutionary inferences. In this sense, the present work aimed at the characterization and comparison of the karyotype and DNA content of plants, from distinct localities, of the species P. amboinicus, P. barbatus, P. grandis and P. neochilus.
Plants of P. amboinicus, P. barbatus, P. grandis and P. neochilus from Lavras-MG, Campinas-SP and Santa Maria-RS was cytogenetically compared. In each region three cuttings of a plant of each species were collected from plant clumps. Plants from Minas Gerais State were supplied by Medicinal Plant Garden of the University of Lavras (UFLA), the ones from Rio Grande do Sul State was provided by Medicinal Plant Garden of University of Santa Maria (UFSM) and plants from São Paulo State by the Campinas Agronomy Institute (IAC). Voucher specimens were deposited at the Research Center for Chemistry, Biology and Agriculture (CPQBA), State University of Campinas, and the State University of Campinas Herbarium (UEC), São Paulo, Brazil (Table
Species | Voucher specimens |
---|---|
P. amboinicus | CPQBA 364 |
P. barbatus | UEC 121.403 |
P. grandis | CPQBA 1433 |
P. neochilus | CPQBA 1388 |
Slides were prepared by the squash technique, and chromosomes were stained with 1% acetic orcein after enzymatic maceration in pectinase-cellulase solution (100U:200U) for 15 min, at 37 °C.
Metaphases were digitized by means of a bright field microscope (Leica DMLS) equipped with microcamera (Nikon Digital Sight DS-Fil). The chromosomes were measured using the software Image Tool 3.0 from the UTHSCA (University of Texas Health Science Center in San Antonio).
For assembly of the karyograms and idiograms, at least four mitotic metaphases of each plant collected were used. Measurements of short and long arm length (SA/LA) were carried out for each chromosome pair, as well as of total length for each chromosome (TLi = LA + SA), total length of haploid lot (TLHL = STLi), and relative length (RL = TLi/TLHL × 100).
The data on relative length of chromosome pairs of P. grandis as well as of P. barbatus plants were compared by the least significant difference (LSD) at 5% probability, using the statistical program SAS.
Morphological classification of the chromosomes was based on centromere position, as proposed by
Estimation of nuclear DNA content by flow cytometry was obtained from leaf tissue according to the work of
The data on DNA content of plants of each location, as well as the means of each species, were submitted to analysis of variance, and the mean values were compared with help of the statistical program SAS, using Tukey test at 5% probability to compare the plants and species.
No variation was found as to the number of chromosomes among the plants of Plectranthus species. The somatic number of chromosomes was common (2n=30), except for P. amboinicus, which presented 2n=34 chromosomes (Fig.
Mitotic metaphases. P. amboinicus, 2n=34 (A), P. barbatus (from Lavras-MG), 2n=30 (B), P. grandis, 2n=30 (C), P. neochilus, 2n=30 (D). Scale bars: 10 µm.
Differences were observed in chromosome morphology among the karyotypes of the species. P. amboinicus follows the karyotypic formula 13m+4sm, P. grandis 7m+8sm, and P. neochilus 9m+6sm (Table
Karyograms and idiograms. P. amboinicus (A), P. grandis (B), P. neochilus (C). Scale bars: Karyograms (5 µm); idiograms (1 µm).
Karyograms and idiograms for P. barbatus from different localities. Lavras (A), Campinas (B), Santa Maria (C). Scale bars: Karyograms (5 µm); idiograms (1 µm).
Data regarding arm relation and chromosome type in species of Plectranthus genus.
P. a | P. b | P. g | P. n | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
UFLA | IAC | UFSM | |||||||||
1 | 1.43m | 2.35sm | 1.88sm | 1.21m | 1.34m | 1.79sm | |||||
2 | 1.21m | 2.15sm | 1.18m | 1.25m | 1.34m | 1.17m | |||||
3 | 1.37m | 1.42m | 1.98sm | 1.38m | 1.96sm | 2.00sm | |||||
4 | 2.35sm | 1.44m | 1.29m | 1.28m | 1.20m | 2.13sm | |||||
5 | 1.39m | 2.07sm | 1.88sm | 1.79sm | 2.02sm | 1.13m | |||||
6 | 1.36m | 1.32m | 1.17m | 1.37m | 1.18m | 1.19m | |||||
7 | 2.19sm | 1.32m | 1.30m | 2.23sm | 2.07sm | 1.92sm | |||||
8 | 1.27m | 2.22sm | 1.79sm | 1.30m | 1.98sm | 1.11m | |||||
9 | 1.38m | 1.53m | 1.97sm | 2.22sm | 2.27sm | 1.81sm | |||||
10 | 1.99sm | 2.19sm | 1.40m | 1.35m | 2.15sm | 1.25m | |||||
11 | 1.29m | 2.25sm | 1.38m | 1.17m | 1.37m | 1.26m | |||||
12 | 1.36m | 2.01sm | 1.41m | 2.06sm | 2.20sm | 1.39m | |||||
13 | 1.29m | 1.20m | 1.21m | 2.27sm | 2.04sm | 1.43m | |||||
14 | 1.97sm | 1.43m | 2.11sm | 1.47m | 1.21m | 1.84sm | |||||
15 | 1.17m | 1.24m | 1.20m | 1.21m | 1.35m | 1.25m | |||||
16 | 1.18m | ||||||||||
17 | 1.24m |
The position of the centromere was coincident among the four species only for the pairs 6 and 15, with these being classified as metacentric (Table
Also among the plants of P. barbatus little karyotypic similarity could be established. Only the pairs 4, 5, 6 and 15 had chromosomes with coinciding classification. Moreover, these same pairs are also coincident in P. grandis, which presented greater karyotypic similarity with the plants of P. barbatus (Santa Maria), differing only in the pairs 3, 8 and 10 (Table
The contrasts accomplished through the statistical test of least significant difference (LSD) among the plants revealed that P. barbatus (Campinas) differs statistically from P. grandis in relation to the pairs 2 and 12. The pair 2 presents relative length with mean values of 8.78 and 8.10, and the pair 12 shows 5.45 and 5.92 for P. barbatus and P. grandis, respectively. The pair 8 differed among the plants of P. barbatus originated from Lavras and Santa Maria, with respective averages of 6.68 and 6.93 (Tables
Data regarding the total length (µm) and relative length (%) of each chromosome of Plectranthus spp.
Pair | P. amboinicus | P. barbatus | P. grandis | P. neochilus | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
UFLA | IAC | UFSM | ||||||||||
1 | 3.04 | (8.89) | 2.99 | (8.56) | 3.57 | (9.42) | 2.93 | (9.09) | 2.95 | (8.37) | 2.82 | (8.55) |
2 | 2.87 | (8.38) | 2.87 | (8.22) | 3.33 | (8.78) | 2.63 | (8.15) | 2.85 | (8.10) | 2.75 | (8.33) |
3 | 2.49 | (7.26) | 2.75 | (7.87) | 3.09 | (8.15) | 2.52 | (7.82) | 2.72 | (7.72) | 2.55 | (7.75) |
4 | 2.42 | (7.06) | 2.60 | (7.44) | 2.94 | (7.75) | 2.43 | (7.55) | 2.65 | (7.52) | 2.42 | (7.34) |
5 | 2.31 | (6.73) | 2.52 | (7.20) | 2.80 | (7.38) | 2.34 | (7.26) | 2.58 | (7.32) | 2.41 | (7.31) |
6 | 2.13 | (6.23) | 2.51 | (7.17) | 2.72 | (7.17) | 2.27 | (7.03) | 2.50 | (7.09) | 2.36 | (7.15) |
7 | 2.05 | (5.98) | 2.44 | (6.97) | 2.61 | (6.89) | 2.26 | (7.00) | 2.37 | (6.72) | 2.30 | (6.97) |
8 | 2.02 | (5.90) | 2.34 | (6.68) | 2.59 | (6.83) | 2.23 | (6.93) | 2.36 | (6.69) | 2.25 | (6.82) |
9 | 1.94 | (5.68) | 2.23 | (6.37) | 2.35 | (6.20) | 2.10 | (6.50) | 2.31 | (6.57) | 2.14 | (6.48) |
10 | 1.86 | (5.42) | 2.18 | (6.23) | 2.25 | (5.95) | 2.06 | (6.39) | 2.18 | (6.19) | 2.04 | (6.19) |
11 | 1.78 | (5.20) | 2.11 | (6.02) | 2.23 | (5.87) | 2.00 | (6.21) | 2.14 | (6.09) | 1.97 | (5.97) |
12 | 1.72 | (5.02) | 1.95 | (5.58) | 2.07 | (5.45) | 1.84 | (5.71) | 2.09 | (5.92) | 1.89 | (5.72) |
13 | 1.68 | (4.90) | 1.94 | (5.56) | 2.03 | (5.35) | 1.66 | (5.15) | 1.95 | (5.52) | 1.78 | (5.39) |
14 | 1.61 | (4.70) | 1.83 | (5.23) | 1.78 | (4.71) | 1.57 | (4.88) | 1.86 | (5.29) | 1.74 | (5.26) |
15 | 1.57 | (4.59) | 1.71 | (4.89) | 1.56 | (4.10) | 1.40 | (4.34) | 1.72 | (4.89) | 1.57 | (4.76) |
16 | 1.45 | (4.22) | ||||||||||
17 | 1.32 | (3.85) |
Comparison of relative lengths of the chromosome pairs 2, 8 and 12 of Plectranthus plants by LSD test.
P. b (UFLA) | P. b (IAC) | P. b (UFSM) | P. g (IAC) | |
---|---|---|---|---|
P. b (UFLA) | - | ABC | AbC | ABC |
P. b (IAC) | ABC | - | ABC | aBc |
P. b (UFSM) | AbC | ABC | - | ABC |
P. g (IAB) | ABC | aBc | ABC | - |
According to
The studied species of Plectranthus have close karyotypic asymmetry indices (Table
Values of karyotypic asymmetry indices of Plectranthus species, according to criteria proposed by
P. a | P. b | P. g | P. n | |||
---|---|---|---|---|---|---|
UFLA | IAC | UFSM | ||||
A1 | 0.29 | 0.39 | 0.32 | 0.32 | 0.38 | 0.33 |
A2 | 0.23 | 0.17 | 0.22 | 0.19 | 0.15 | 0.16 |
AI | 3.09 | 2.60 | 2.93 | 2.82 | 2.48 | 2.06 |
P. amboinicus has the largest difference in relation to total size of the chromosomes, besides having the greatest asymmetry index as proposed by
In relation to DNA amount in the evaluated Plectranthus plants, two groupings were identified (Table
Flow cytometry histograms. P. amboinicus (A), P. grandis (B), P. neochilus (C), P. barbatus (D–F) from UFLA(D), IAC(E), UFSM(F). The first peak in each histogram refers to the G1 peak of each of the Plectranthus species, and the second G1 peak corresponds to the reference sample (Pisum sativum). The abscissa represents the DNA amount, and the ordinate the number of nuclei.
Mean values of 2C DNA and coefficient of variation obtained by flow cytometry technique for Plectranthus plants.
Species/Plant | DNA (pg) |
CV (%) |
---|---|---|
P. amboinicus (UFLA) | 5.98 a | 0.79 |
P. amboinicus (IAC 465) | 5.79 a | 0.72 |
P. amboinicus (IAC 2193) | 5.81 a | 0.57 |
Mean | 5.86 A | |
P. barbatus (UFLA) | 5.20 a | 0.57 |
P. barbatus (IAC) | 5.17 a | 0.70 |
P. barbatus (UFSM) | 5.69 b | 0.57 |
Mean | 5.35 B | |
P. grandis | 5.23 B | 0.64 |
P. neochilus (UFLA) | 5.99 a | 0.56 |
P. neochilus (IAC) | 5.94 a | 0.54 |
P. neochilus (UFSM) | 6.00 a | 0.55 |
Mean | 5.98 A |
Distinct chromosome numbers for P. amboinicus have already been described in the literature (2n=16, 24, 30, 32, 34, 48 and 56) in works that treated the species with different synonymies (
Karyotypic studies on P. neochilus are rare in the literature.
The occurrence of 30 chromosomes in P. barbatus that was observed for different accessions of this species in the present study corroborates the number reported earlier by different authors (
The statistical differences observed for the pairs 2, 8 and 12 in P. barbatus and P. grandis suggest the occurrence of chromosomal rearrangements, seeing that some of these pairs present variation both in relative length as well as in centromeric position. This way, the chromosomes of pairs 8 and 12 classified as submetacentric may have undergone alterations, namely deficiency in one of the chromosome arms, giving rise to the metacentric form, or duplications in one of the arms of these chromosomes, thus rendering them submetacentric. The differences seen in the pair 2 for P. barbatus (Campinas) and P. grandis did not express variations in centromere position, which suggests events of duplication/deficiency in both chromosome arms.
The remaining chromosome pairs of the evaluated P. barbatus and P. grandis plants did not present significant statistical differences regarding relative length. Nevertheless, the centromere position in some of the pairs of P. barbatus plants and of the P. grandis plant appeared altered. Taking the chromosome pair 1 as example, the plants from Lavras and Campinas had it classified as submetacentric, and that from Santa Maria, together with P. grandis, had the same pair classified as metacentric. These changes in classification of the chromosome pair as to centromere position may be justified by the occurrence of inversions, since the relative lengths are statistically similar. Also, other mechanisms may drastically modify the chromosome structure, among which centromeric repositioning, as reported by
Different pressures exerted by the different environments can be other reason for karyotypic variations mentioned for P. barbatus, since this hypothesis was considered previously by
Passinho et al. (1999) and
The occurrence of differentiated karyotypic formulas for plants of P. barbatus and the fact that P. barbatus presents nuclear DNA content statistically similar to that of P. grandis, are not able to indicate that P. barbatus and P. grandis have enough differences to be considered distinct species. Therefore, more experiments using molecular cytogenetic techniques are needed in order to understand the relationship between both species.
Regarding to asymmetry of karyotype, based on the methods of
Based on asymmetry indexes proposed by
The variation among karyotypes of kindred species and among plants, associated with the differences in nuclear DNA content found in this work, supports the hypothesis that, karyotypically, P. amboinicus and P. neochilus are more stable species and the variation found among plants of P. barbatus, regarding chromosome morphology, express differences among populations.
The populations of P. amboinicus and P. neochilus present coinciding karyotypes among their respective plants.
P. barbatus is a species undergoing active process of karyotypic variation.
The karyotypic intraspecific variation in P. barbatus is an indication that P. grandis is one of the events of variation in the species, since the species exhibit the same morphological characteristics.
The authors thank the Fundação de Amparo à Pesquisa (FAPEMIG) for financial support; to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for granting of the scholarship; to Dr. Glyn Mara Figueira of the Instituto Agronômico de Campinas (IAC), Dr. José Eduardo Brasil Pereira Pinto of the Universidade Federal de Lavras (UFLA), to Dr. Thais Scotti do Canto-Dorow of Universidade Fedral de Santa Maria (UFSM) for supplying plant material; and to Dr. Moacir Pasqual of the Laboratory of Tissue Culture of the UFLA for support in the cytometric analyses.