Corresponding author: Wellington Ronildo Clarindo ( firstname.lastname@example.org )
Academic editor: Marcelo Guerra
© 2017 Renata Flávia de Carvalho, Paulo Marcos Amaral-Silva, Micheli Sossai Spadeto, Andrei Caíque Pires Nunes, Tatiana Tavares Carrijo, Carlos Roberto Carvalho, Wellington Ronildo Clarindo.
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: Carvalho R, Amaral-Silva P, Spadeto M, Nunes A, Carrijo T, Carvalho C, Clarindo W (2017) First karyotype description and nuclear 2C value for Myrsine (Primulaceae): comparing three species. Comparative Cytogenetics 11(1): 163-177. https://doi.org/10.3897/CompCytogen.v11i1.11601
Cytogenetic studies in Primulaceae are mostly available for herbaceous species, and are focused on the chromosome number determination. An accurate karyotype characterization represents a starting point to know the morphometry and class of the chromosomes. Comparison among species within Myrsine, associating these data with the nuclear 2C value, can show changes that led the karyotype evolution. Here, we studied three Myrsine species [Myrsine coriacea (Swartz, 1788) Brown ex Roemer et Schultes, 1819, Myrsine umbellata Martius, 1841 and Myrsine parvifolia Candolle, 1841] that show different abilities to occupy the varied types of vegetation within the Brazilian Atlantic Forest. Cytogenetic characterization showed some individuals with 2n = 45 chromosomes for M. parvifolia and M. coriacea, with most individuals of the three species having 2n = 46. The first karyograms for Myrsine were assembled and presented morphologically identical and distinct chromosome pairs. In addition, differences in the mean 2C nuclear value and chromosome morphometry were found. Therefore, the first description of the Myrsine karyotype has been presented, as well as the nuclear 2C value. The procedures can be applied to other Myrsine species for future investigations in order to better understand its effects on the differential spatial occupation abilities shown by the species in Brazilian Atlantic Forest.
Atlantic Forest, cytogenetics, flow cytometry, karyogram, Myrsinaceae, Rapanea
Previous studies regarding the chromosome number in Primulaceae (s.
The cosmopolitan Myrsine Linnaeus is one of the main genera of Primulaceae, considering species richness, represented by tree and shrub species (
One interesting ecological aspect observed in Neotropical species of Myrsine that occur in Brazil is that some of them occur in more than one biome, as Cerrado, Atlantic Forest, and Amazonian Forest, while others are restricted of one of these biomes, as Atlantic Forest (BFG 2015). Among species that occur in Atlantic Forest, for example, some are able to occupy different types of vegetation within this biome, including Restinga Vegetation, High Altitude Campos, Rocky Outcrops, Ombrophyllous and Mixed Ombrophyllous Forests, while others are able to occupy just one type of vegetation (
Studies combining cytogenetics and nuclear DNA content have offered data for understanding evolutionary processes in different species (
Three species were selected for this study: 1. Myrsine coriacea (Voucher – T.T. Carrijo 1458, VIES herbarium), which is a widespread species in Atlantic Forest found in all types of vegetation, including open areas within Ombrophyllous and Mixed Ombrophyllous Forests, Rock Outcrops, High Altitude Campos, and Restinga Vegetation; 2. Myrsine umbellata (Voucher – T.T. Carrijo 1467, VIES herbarium), which is found in mostly all types of vegetation of M. coriacea, except High Altitude Campos; and 3. Myrsine parvifolia (Voucher – T.T. Carrijo 2232, VIES herbarium), a species restricted to Restinga vegetation (BFG 2015).
Fruits and leaves of all species were collected. Myrsine coriacea and M. umbellata were sampled from October 2012 to July 2015 in a forest remnant located in Iúna municipality, Espírito Santo (ES) State, Brazil (20°21'6"S – 41°31'58"W), characterized as Rocky Outcrops, at 600 (M. coriacea) and 1,100 m.s.m (M. umbellata). M. parvifolia was collected in a forest remnant located in Guarapari municipality, ES, Brazil (20°36'15"S – 40°25'27"W), characterized as coastal sandy plains vegetation (Restinga) at sea level. Leaves and fruits of Solanum lycopersicum L. and Pisum sativum L. (internal standards for flow cytometry – FCM, 2C = 2.00 pg and 2C = 9.16 pg, respectively,
Fruit pericarp was manually removed and the seeds were desinfested according to
In order to adapt the FCM for Myrsine, the following procedures were done: (a) initially, from leaves collected in the field of male and female individuals (samples) and of the two standards; (b) afterward, replacing the dithiothreitol antioxidant by polyethylene glycol (PEG) in nuclei isolation buffer; and (c) from leaves of the samples and P. sativum plantlets in vitro cultivated.
Nuclei suspensions were obtained by co-chopping (
Roots were cut from the in vitro plantlets, treated with 5.0 μM amiprofos-methyl (APM) (Agrochem KK Nihon Bayer) for 12, 15, 18 or 24 h at 4°C, rinsed in distilled water (dH2O) for 20 min and fixed in methanol:acetic acid (3:1) for 24 h. The fixative solution was changed three times and the material was stored at -20°C (
Slides were prepared and stained according to
Using chromosome morphometric data (total, short and long arm length), the standardized Euclidean Distance and Unweighted Pair-Group Method Average (UPGMA) was applied to each species. In addition, the value of the relative size (% size in relation to sum of the mean values of total length, Table
Morphometry and chromosome class performed at least 10 prometaphases/metaphases. In all species were found chromosomes morphologically indentical, similar and distinct.
|M. parvifolia||M. coriacea||M. umbellata|
|Chrom.||Total ± SD||Short||Long||r||Class||Relative size (%)||Total ± SD||Short||Long||r||Class||Relative size (%)||Total ± SD||Short||Long||r||Class||Relative size (%)|
|1||2.64 ± 0.29||1.01||1.63||1.61||SM||5.60||2.79 ± 0.09||1.24||1.55||1.25||M||6.14||2.72 ± 0.06||1.14||1.59||1.39||M||6.60|
|2||2.47 ± 0.23||1.09||1.37||1.25||M||5.24||2.45 ± 0.11||1.02||1.42||1.38||M||5.38||2.67 ± 0.06||1.14||1.54||1.35||M||6.48|
|3||2.45 ± 0.22||0.86||1.59||1.85||SM||5.19||2.35 ± 0.10||1.09||1.26||1.15||M||5.17||2.13 ± 0.16||0.94||1.19||1.26||M||5.16|
|4||2.44 ± 0.27||0.68||1.75||2.55||SM||5.17||2.30 ± 0.05||1.02||1.27||1.24||M||5.06||2.13 ± 0.08||0.84||1.29||1.53||SM||5.16|
|5||2.24 ± 0.18||0.71||1.53||2.13||SM||4.76||2.29 ± 0.08||0.99||1.30||1.30||M||5.04||2.08 ± 0.14||0.74||1.34||1.80||SM||5.04|
|6||2.21 ± 0.17||0.73||1.48||2.00||SM||4.69||2.22 ± 0.17||0.86||1.36||1.57||SM||4.88||1.88 ± 0.11||0.64||1.24||1.92||SM||4.56|
|7||2.18 ± 0.25||0.81||1.37||1.68||SM||4.62||2.17 ± 0.12||0.78||1.39||1.77||SM||4.77||1.83 ± 0.11||0.79||1.04||1.31||M||4.44|
|8||2.16 ± 0.27||0.61||1.55||2.51||SM||4.59||2.12 ± 0.11||0.78||1.34||1.71||SM||4.67||1.83 ± 0.09||0.59||1.24||2.08||SM||4.44|
|9||2.15 ± 0.29||0.86||1.29||1.49||M||4.55||2.04 ± 0.15||0.81||1.23||1.50||SM||4.49||1.83 ± 0.09||0.59||1.24||2.08||SM||4.44|
|10||2.13 ± 0.25||0.61||1.51||2.45||SM||4.51||2.00 ± 0.10||0.78||1.23||1.56||SM||4.41||1.78 ± 0.12||0.59||1.19||2.00||SM||4.32|
|11||2.09 ± 0.22||0.79||1.31||1.65||SM||4.44||2.00 ± 0.17||0.75||1.26||1.67||SM||4.41||1.68 ± 0.13||0.59||1.09||1.83||SM||4.08|
|12||1.99 ± 0.16||0.75||1.23||1.63||SM||4.22||1.89 ± 0.10||0.71||1.18||1.64||SM||4.16||1.68 ± 0.08||0.59||1.09||1.83||SM||4.08|
|13||1.97 ± 0.23||0.66||1.31||1.96||SM||4.19||1.89 ± 0.07||0.57||1.32||2.31||SM||4.16||1.68 ± 0.10||0.49||1.19||2.40||SM||4.08|
|14||1.95 ± 0.14||0.65||1.30||2.00||SM||4.14||1.84 ± 0.06||0.55||1.29||2.32||SM||4.06||1.68 ± 0.14||0.66||1.02||1.52||SM||4.08|
|15||1.93 ± 0.16||0.72||1.21||1.67||SM||4.11||1.81 ± 0.11||0.65||1.16||1.78||SM||3.98||1.58 ± 0.06||0.64||0.94||1.46||M||3.84|
|16||1.85 ± 0.13||0.65||1.20||1.82||SM||3.93||1.81 ± 0.08||0.57||1.24||2.17||SM||3.98||1.58 ± 0.06||0.69||0.89||1.29||M||3.84|
|17||1.85 ± 0.23||0.72||1.13||1.56||SM||3.93||1.78 ± 0.04||0.66||1.11||1.66||SM||3.91||1.58 ± 0.09||0.69||0.89||1.29||M||3.84|
|18||1.84 ± 0.19||0.70||1.15||1.63||SM||3.92||1.71 ± 0.13||0.65||1.06||1.63||SM||3.77||1.58 ± 0.11||0.59||0.99||1.67||SM||3.84|
|19||1.82 ± 0.22||0.63||1.20||1.89||SM||3.87||1.68 ± 0.11||0.55||1.13||2.03||SM||3.70||1.58 ± 0.08||0.59||0.99||1.67||SM||3.84|
|20||1.75 ± 0.18||0.68||1.06||1.55||SM||3.71||1.67 ± 0.09||0.58||1.09||1.85||SM||3.69||1.58 ± 0.13||0.49||1.09||2.20||SM||3.84|
|21||1.68 ± 0.14||0.79||0.89||1.13||M||3.56||1.55 ± 0.16||0.49||1.06||2.17||SM||3.42||1.43 ± 0.14||0.59||0.84||1.42||M||3.48|
|22||1.66 ± 0.16||0.58||1.08||1.85||SM||3.53||1.55 ± 0.04||0.35||1.20||3.33||A||3.42||1.38 ± 0.11||0.49||0.89||1.80||SM||3.36|
|23||1.66 ± 0.30||0.58||1.08||1.86||SM||3.53||1.52 ± 0.07||0.39||1.13||2.88||SM||3.34||1.28 ± 0.10||0.59||0.69||1.17||M||3.13|
Approximately 60 days after in vitro inoculation, plantlets were obtained for the three Myrsine species. All plantlets exhibited sufficient and morphologically normal leaves and roots for FCM and cytogenetic analyses, respectively.
FCM analysis performed on leaves collected in the field did not result in histograms showing profile G0/G1 peaks. So, dithiothreitol antioxidant was replaced by PEG in the nuclei isolation buffer OTTO I. This change provided G0/G1 peaks, exhibiting a coefficient of variation (CV) less than 5% for M. umbellata and the two internal standards. The channel of the P. sativum G0/G1 peak however was closer to M. umbellata than S. lycopersicum. Thus, based on linearity international criteria for FCM, P. sativum was the standard chosen for the next measurements. The mean 2C value of the male (2C = 6.65 pg ± 0.02) and female (2C = 6.67 pg ± 0.11) M. umbellata individuals were statistically identical by the F test. Considering these previous results, the 2C value was measured from leaves of in vitro plantlets. The mean values were 2C = 4.81 pg ± 0.05 for M. parvifolia, 2C = 6.60 pg ± 0.14 for M. coriacea and 2C = 6.63 pg ± 0.13 for M. umbellata. The mean value of the M. umbellata in vitro plantlets was statistically identical to the males and females in the field. Therefore, the mean value adopted for this species was 2C = 6.65 pg, which was statistically equal to the M. coriacea.
Roots exposed to a 12 h APM provided prometaphases, exhibiting chromosomes at a distinct chromatin compact level, and metaphases. Enzymatic maceration in 1:5 pectinase solution ensured the chromosomes remained inside the cell, allowing an accurate determination of 2n = 45 or 2n = 46. Chromosome number of 2n = 45 was found for 12.60% individuals of M. parvifolia and 8.45% of M. coriacea, with 2n = 46 for the three species. Based on these results, the next slides were made from roots of particular seedlings with 2n = 45 or 2n = 46. Root maceration with 1:20 enzymatic pool for 1h 30 min supplied chromosomes no damage to the chromatin structure, without overlapping, with well-defined centromeres and free of cytoplasm debris (Fig.
First images of the Myrsine chromosomes. Karyotype of a M. parvifolia individual with 2n = 45 (a) and another with 2n = 46 (b) chromosomes. Note the different levels of chromatin compaction between the chromosomes of the two karyotypes. The distinct chromatin compact level was highlighted in (c), where the same submetacentric chromosome of M. parvifolia (above) and the same acrocentric chromosome of M. coriacea (below) were taken from two different prometaphases (I and II) and one metaphase (III). Bar = 5 µm.
Karyotype characterization was possible only after carefully testing the time and concentration of the APM antitubulin and cell wall enzymes. Myrsine parvifolia presented a greater total sum of the length of the chromosomes despite having less nuclear DNA content. For this species only, we found prometaphase chromosomes showing low level of chromatin compaction (Fig.
Myrsine karyograms displaying 2n = 45 (a M. parvifolia and b M. coriacea) or 2n = 46 chromosomes (a–c the three species). In all M. parvifolia (a) and M. coriacea (b) individuals with 2n = 45, the odd chromosome number was well-marked by absence of the homologue pair of the chromosome 23. Metacentric and submetacentric chromosomes prevailing in the karyograms of the three species, with only one acrocentric chromosome was identified in M. coriacea (b chromosome 22). Although showing approximately 2C = 1.50 pg less DNA, M. parvifolia (a) displayed the same chromosome number in relation to the other species (b M. coriacea c M. umbellata). For all species, morphometric analyses showed identical, similar and distinct chromosome pairs with regard to morphometry and class. The similarity of some chromosomes was highlighted from the metacentric chromosome pairs 4 and 5 (d above) and submetacentric 15 and 16 (d below) of M. coriacea. In contrast, other chromosomes showed singular morphology, as the chromosome 1 and 2 of all species, the 22 of M. coriacea, which is the single acrocentric chromosome, and the chromosome 23. Bar = 5 µm.
Morphometric analysis was used to classify the chromosomes and evidence similarities and differences among species karyotypes. Myrsine parvifolia presented three metacentric (2, 9 and 21) and 20 submetacentric (1, 3–8, 10–20, 22 and 23) chromosome pairs, M. coriacea showed five metacentric (1–5), 17 submetacentric (6–21 and 23) and one acrocentric (22) chromosome pairs, and M. umbellata displayed nine metacentric (1–3, 7, 15–17, 21 and 23) and 14 submetacentric (4–6, 8–14, 17, 18, 20 and 22) chromosome pairs (Fig.
Morphologically similar and identical chromosomes groups were found in all species. Myrsine parvifolia presented sets of two chromosome pairs (5–6, 13–14, 16–17 and 22–23), as did M. coriacea (4–5, 10–11, 13–14, 15–16 and 19–20), and M. umbellata presented three sets of two (11–12, 16–17 and 18–19) and one set of three chromosome pairs (8–10). The other chromosome pairs in each species were considered morphologically distinct (Fig.
a–c Multivariate clustering generated from chromosome morphometric data (total, long and short arms length). Mojena’s criteria showed three clusters for M. parvifolia (a), M. coriacea (b) and M. umbellata (c) with cut point between 1.5 to 1.8. This analysis confirmed the morphological discrepancy of the chromosome 1, and the similarity of other chromosomes (d) Graphic provided by comparison between mean relative size (% size in relation to sum of the mean values of total length, Table
As the mean 2C values of M. coriacea (6.60 pg) and M. umbellata (6.65 pg) were statistically identical, the Scott-Knott test was used to compare the relative size (Table
Chromosome groups of the Myrsine karyotype suggested from karyogram evaluation (Fig.
|Species||*Karyogram evaluation||**UPGMA clustering||***Confirmed chromosome groups|
|M. parvifolia||5–6; 13–14; 16–17; and 22–23||1 and 2; 3–11; and 12–23||5–6; 13–14; 16–17; and 22–23|
|M. coriacea||4–5; 9–10; 13–14; 15–16; and 19–20||1; 2–11; and 12–23||4–5; 9–10; 13–14; 15–16; and 19–20|
|M. umbellata||8–10; 11–12; 16–17; and 18–19||1 and 2; 3–5, 7; and 6, 8–23||8–10; 11–12; 16–17; and 18–19|
The first step in FCM was to define the best antioxidant and internal standard. The presence of secondary metabolites in the Myrsine leaves, such as tannins, saponins, flavonoids and steroids (
Secondary metabolite interference was completely resolved for other Myrsine species using in vitro plantlets propagated in a controlled environment. FCM measurements from leaves collected in the field may have been influenced by environmental conditions. Secondary metabolite production is influenced by humidity, temperature, light intensity and the availability of water and nutrients (
Genome size in Myrsine had only been reported for M. africana as 2C = 2.46 pg (
Values close to M. umbellata and M. coriacea species were reported for Cyclamen purpurascens Mill. (2C = 6.60 pg) and Dodecatheon meadia L. (2C = 5.58 pg). Higher DNA contents were described for Cyclamen coum Mill. (2C = 13.56 pg), Soldanella pusilla Baumg. (2C = 12.36 pg), and lower values for Soldanella hungarica Simonk (2C = 3.16 pg) and Primula vulgaris Huds (2C = 0.47 pg) (Bennett and Leitch 2012). The interspecific variation for the 2C DNA value found in this study, as for other species of Primulaceae (Bennett and Leitch 2012), suggests the occurrence of karyotype changes.
As well as for FCM, karyotype data about Myrsine species in the literature are very limited, with only the chromosome number reported (
Chromosome number 2n = 46 (
Some chromosome groups determined by statistical analysis are morphologically distinct, such as chromosomes 22 and 23 of M. coriacea. Although clustered (Fig.
Based on the constant chromosome number displayed by Myrsine species, interspecific variation of the nuclear 2C value between M. parvifolia compared to M. coriacea and M. umbellata was also caused by karyotype alterations. The changes to the nuclear DNA content have also been attributed to structural rearrangements and/or heterochromatin polymorphisms (
In conclusion, the first karyotype description and data about nuclear 2C value were shown for three Myrsine species. Besides of the comparison between them, these data represent the basis to understand karyotype evolution in Myrsine.
The authors Carvalho RF, Amaral-Silva PM, Spadeto MS and Clarindo WR conceived, designed and conducted the tissue culture, flow cytometry and cytogenetic approaches. Carvalho CR contributed the flow cytometry analysis. Amaral-Silva PP and Carrijo TT collected and identified the Myrsine species. Nunes ACP did the statistical analysis. All authors contributed equally to manuscript editing and revision and approved the final manuscript for submission.
The authors declare they have no conflict of interest.
We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília – DF, Brazil), the Fundação de Amparo à Pesquisa do Espírito Santo (FAPES, Vitória – ES, Brazil, grant: 61860808/2013) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília – DF, Brazil) for financial support.