Research Article |
Corresponding author: María M. Sosa ( mdlmsvg@yahoo.com.ar ) Academic editor: Snejana Grozeva
© 2016 María M. Sosa, Maria B. Angulo, Julian A. Greppi, Verónica Bugallo.
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:
Sosa MM, Angulo MB, Greppi JA, Bugallo V (2016) Chromosome numbers and DNA content in some species of Mecardonia (Gratiolae, Plantaginaceae). Comparative Cytogenetics 10(4): 769-780. https://doi.org/10.3897/CompCytogen.v10i4.10362
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Cytogenetic characterization and determination of DNA content by flow cytometry of five species of Mecardonia Ruiz et Pavon, 1798 (Gratiolae, Plantaginaceae) was performed. This is the first study of nuclear DNA content carried out in the genus. Mitotic analysis revealed a base chromosome number x = 11 for all entities and different ploidy levels, ranging from diploid (2n = 2x = 22) to hexaploid (2n = 6x = 66). The results include the first report of the chromosome numbers for M. flagellaris (Chamisso & Schlechtendal, 1827) (2n = 22), M. grandiflora (Bentham) Pennell, 1946 (2n = 22), M. kamogawae Greppi & Hagiwara, 2011 (2n = 66), and Mecardonia sp. (2n = 44). The three ploidy levels here reported suggest that polyploidy is common in Mecardonia and appear to be an important factor in the evolution of this genus. The 2C- and 1Cx-values were also estimated in all the species. The 2C-values ranged from 1.91 to 5.29 pg. The 1Cx-values ranged from 0.88 to 1.03 pg. The general tendency indicated a decrease in the 1Cx-value with increasing ploidy level. The significance of the results is discussed in relation to taxonomy of the genus.
Gratiolae, chromosome number, DNA content, flow cytometry, polyploidy
Mecardonia Ruiz & Pavon, 1798 belongs to the tribe Gratiolae (Plantaginaceae) and is distributed across the America, reaching its southernmost distribution in Argentina. The species are erect or creeping herbs, annual or perennial, much branched, mostly glabrous, sometimes blackening on drying, gland dotted, and yellow and white flowers (
Mecardonia has ornamental value because some cultivars developed from native species from Northern Argentina were recently introduced in the trade of ornamental plants (
Cytological and cytogenetic studies have proved useful data for taxonomic and evolutionary analyses, which are widely used in processes of conventional or biotechnological genetic improvement (
Nuclear DNA content, understood as genome size, is very variable across angiosperm, and has been revealed as an important character in biodiversity. In Mecardonia species there are no reported measurements of DNA content, but genome size variation has been explored in some genera of Plantaginaceae. DNA C-values are currently available for 204 species belonging to 18 genera of this family, including Callitriche Linnaeus, 1753, Penstemon Schmidel, 1762, Plantago Linnaeus, 1753 and Veronica Linnaeus, 1753 with a range of variation of 0.32–4,.63 pg (
We examined six populations from five species of Mecardonia collected in Argentina. Information about the studied material and the voucher specimens is provided in Table
Mecardonia species analyzed in this study, with their respective chromosome numbers (2n), locations, and voucher specimens.
Species | 2n | Location, voucher specimen | |
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* | M. flagellaris (Cham. & Schldlt.) Rossow | 2n = 2x = 22 | Argentina. Entre Ríos, Dept. Federación, in front of complejo turístico Irupé. Greppi et al. 1411 ( |
2n = 2x = 22 | Argentina. Entre Ríos, Dept. Federación, complejo turístico Irupé Greppi et al. 1190 ( |
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* | M. grandiflora (Benth.) Pennell | 2n = 2x = 22 | Argentina. Misiones, Dept. Guaraní, Ayo. Pepirí Miní o Yabotí. Greppi et al. 1189 ( |
* | M. kamogawae Greppi & Hagiwara | 2n = 6x = 66 | Argentina. Corrientes, Dept. Paso de los Libres, Paso de los Libres to national route 14, Greppi et al. 1081 ( |
M. procumbens (Mill.) Small | 2n = 2x = 22 | Argentina. Córdoba. Dept. Unión, Monte Leña, national route 9, km 491, Greppi 681( |
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* | Mecardonia sp. n. | 2n = 4x = 44 | Argentina. Corrientes. Dept. Empedrado. Greppi and Hagiwara 1410 ( |
Mitotic chromosome preparations were made from root meristems obtained from rooted stems. The roots were pretreated for about 4 h in 0.002 M 8-hydroxyquinoline solution at room temperature, fixed in 5:1 absolute alcohol/lactic acid, and then stained using Feulgen’s technique. Permanent microscope slides were prepared by mounting in Euparal. In all samples at least 20 counts of 7–10 individuals were made to verify the observations.
Permanent microscope slides were examined and photographed using Zeiss Axioplan microscope Carl Zeiss with digital camera Canon Power Shot A 640.
DNA content (in picograms) was estimated by flow cytometry using fresh young leaves. The measurements were calculated from three replicates per individuals. In total we analyzed three individuals per species. The leaves of Zea mays Linnaeus, 1753 cv. ‘CE-777’ (2C = 5.43 pg.,
The absolute value of DNA content (2C) of each sample was calculated by the formula: (X peak of sample × G1 DNA content (2C) of the standard)/X G1 peak of the standard (
The monoploid genome size (1Cx) was calculated dividing the 2C-value by the ploidy level (
The mean, standard deviation and the coefficient of variation of 2C-value were calculated for each species from three different individuals. Differences in 1Cx-value between species were tested by one-way analysis of variance (ANOVA) at a significance level of 5% (a = 0.05). The Tukey 5% post hoc test was used to test differences between each pair of species.
Pearson correlation coefficient was calculated to test whether the 2C–and 1Cx-values were related to chromosome number. Scatter plot was performed to evaluate the relationship between the 1Cx-values and the chromosome numbers (2n) of species. All statistical analyses were performed using the InfoStat software version 2013 (
The chromosome numbers of six populations belonging to five species of Mecardonia were determined. The analyzed species and their chromosome numbers are given in Table
Somatic chromosomes of Mecardonia species. A M. flagellaris: 2n = 2x = 22 B M. grandiflora: 2n = 2x = 22 C Mecardonia sp. n.: 2n = 4x = 44 D M. kamogawae: 2n = 6x = 66. Bar = 5 μm.
The DNA amounts determined for five species of Mecardonia are shown in Table
Chromosome number (2n), ploidy level, 2C-value (pg), CV (coefficient of variation), 1Cx-value (pg) of the Mecardonia species analyzed.
Species | Chromosome number (2n) | Ploidy level | 2C (pg) | CV | 1Cx (pg) |
M. flagellaris | 22 | 2x | 2.06 ± 0.16 | 0.029 | 1.03d |
M. grandiflora | 22 | 2x | 2.05 ± 0.08 | 0.010 | 1.02d |
M. procumbens | 22 | 2x | 1.91 ± 0.06 | 0.012 | 0.95c |
Mecardonia sp. n. | 44 | 4x | 3.71 ± 0.05 | 0.053 | 0.92b |
M. kamogawae | 66 | 6x | 5.29 ± 0.10 | 0.061 | 0.88a |
ANOVA | (F=357.52; P= <0.0001) |
The 2C-values of the species here analyzed varied from 1.91 pg in M. procumbens (2x) to 5.29 pg in M. kamogawae (6x). The 2C-values were strongly and significantly correlated with chromosome number (r= 0.99; P= < 0.0001).
The 1Cx-values, which indicated the DNA content per genome, ranging from 1Cx= 0.88 pg in M. kamogawae to 1Cx= 1.03 pg in M. flagellaris (Table
The chromosome number 2n = 22 found in M. procumbens, is consistent with the chromosome counts recorded in a previous cytological study (
The interest on the study of genome size increased in the last decade. These studies focused on the use of genome size as a taxonomic marker (
The 2C-values of Mecardonia species revealed a positive and significant correlation with chromosome number (r = 0.99, P = < 0.0001). Therefore, in the genus there is a trend for increasing 2C–value with increasing ploidy level. On the other hand, the variation of 1Cx-values is negative and significantly (r = -0.86; P = <0.0001) correlated with chromosome number. Consequently, the values of 1Cx of the species decrease in inverse proportional to the ploidy level. Our data reflect that both polyploids (tetraploid 1Cx = 0.92 pg and hexaploid 1Cx = 0.88 pg) have lesser values of monoploid genome size than diploid species (mean of Cx = 1.00 pg). Many polyploid angiosperms undergo genome downsizing and so have smaller average genome sizes than their diploid relatives (
Recently,
The genus Mecardonia is currently under revision and some closely related species with intermediate morphological characteristics were found. It has been well documented in many plants that chromosome numbers and genome size can be used as extra taxonomic characters for discriminating between closely related taxa, helping to clarify the taxonomy of some species in problematic genus (
Another case is M. kamogawae that is morphologically related to M. procumbens from which it differs in the life-form, root types, leaf texture, and size of bracteoles and pedicels. Regarding chromosome number, M. kamogawae is hexaploid with 2n = 66, while M. procumbens is diploid with 2n = 22. Therefore, both species can be distinguished by morphological features, as well as by the chromosome number.
Mecardonia procumbens and M. flagellaris were diploids with 2n = 22. Although the chromosome number does not distinguish both species, differences in 2C-values were observed. Mecardonia flagellaris had higher value (2C = 2.06 pg.) than Mecardonia procumbens (2C = 1.91 pg.).
The results of this study suggest that chromosome number is useful in distinguishing species of Mecardonia. The different ploidy levels of the taxa showed the importance of polyploidy in the evolution of the genus. The results here obtained combined with those reported previously confirm that the Mecardonia genus has basic number x = 11.
Regarding to the variation of genome size, decreases in DNA content have occurred during the evolution of genome size in the Mecardonia species.
Our results showed that differences in morphological features along with chromosome numbers and DNA content values support Rossow’s criterion (1987).
This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), the Secretaría General de Ciencia y Técnica de la Universidad Nacional del Nordeste (SGCyT-UNNE) and the Instituto de Floricultura belonging to Instituto Nacional de Tecnología Agropecuaria (INTA). Also, the authors thank to Zulma Roa the care of cultivated plants and Laura Mini.