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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 Myrsine parvifolia and Myrsine 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.
Previous studies regarding the chromosome number in Primulaceae (s. APG 2016) are available for some genera, as: Cyclamen Linnaeus, 1753 (Bennett and Grimshaw 1991, Ishizaka 2003), Anagallis Linnaeus, 1753 (Aguilera et al. 2011, Bennett and Leitch 2012), Lysimachia Linnaeus, 1753 (Baltisberger and Kocyan 2010, Bennett and Leitch 2012, Chalup and Seijo 2013), Androsace Linnaeus, 1753 (Chepinoga et al. 2009), Elingamita Baylis, 1951 (Dawson 1995), Trientalis Linnaeus, 1753 (Vickery and Miller 2008), Ardisia Swartz, 1788 (Koyama and Kokubugata 1998), Primula Linnaeus, 1753 (Abou-El-Enain 2006, Casazza et al. 2012, Theodoridis et al. 2013), and Dodecatheon Linnaeus, 1753 (Oberle et al. 2012), and Myrsine Linnaeus, 1753 (Beuzenberg and Hair 1983, Dawson 1995, 2000, Hanson et al. 2003, Rice et al. 2015). Except the genus Cyclamen and Myrsine, these taxa comprise annual and biennial herbaceous species.
The cosmopolitan Myrsine Linnaeus is one of the main genera of Primulaceae, considering species richness, represented by tree and shrub species (Heenan and Lange 1998). Its members are generally dioecious plants, characterized by ramiflorus and congested inflorescences, and flowers with oppositipetalous stamens. Despite Myrsine being one of the largest and most important genera of Primulaceae, only eighteen species, among the 300 estimated from this genus, have been studied regarding cytogenetic aspects. Fifteen of these species occur in the African, Asian and Oceania continents (Myrsine coxii Cochayne, 1902, Myrsine divaricata Cunningham, 1839, Myrsine kermadecensis Cheeseman, 1887, Myrsine nummularia (Hooker f.) Hooker f., 1867, Myrsine salicina (Hooker f.) Hooker f., 1864, Myrsine argentea Heenan et de Lange, 1998, Myrsine oliveri Allan, 1961, Myrsine chathamica Mueller, 1864; Myrsine africana Linnaeus, 1753; Myrsine sandwicensis Candolle, 1841, Myrsine seguinii Léveille, 1914, Myrsine semiserrata Wallich, 1824, Myrsine australis (A. Richard, 1832) Allan, 1947, Myrsine capitellata Wallich, 1824), and just three occurs in America continent (Myrsine matensis (Mez, 1902) Otegui, 1998; Myrsine guianensis (Aublet, 1775) Kuntze, 1891, Myrsine coriacea (Swartz, 1788) Brown ex Roemer et Schultes, 1819. The chromosome number (2n = 46 or 2n = 48) was the only karyotype data reported, without any images of the chromosomes. In addition, the evolutionary aspects that culminated in the karyotype diversification within the genus are poorly understood.
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 (Freitas and Kinoshita 2015). Considering the distinct ecological aspects, cytogenetic studies are relevant to show other differences between these species.
Studies combining cytogenetics and nuclear DNA content have offered data for understanding evolutionary processes in different species (Clarindo and Carvalho 2008, Kolář et al. 2013). Measurement of the nuclear DNA content is complementary to cytogenetic information and is useful for detecting genome size variations between related species (Marhold et al. 2010, Kolář et al. 2013). Fine adjustments in cytogenetic procedures, combining advances in microscopy and image analysis systems, can provide accurate karyotype characterization for Myrsine species. Here, we study three species of Myrsine that occur in contrasting types of vegetation of the Brazilian Atlantic Forest, aiming to determine the chromosome number, describe the karyotype and measure the nuclear DNA.
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 Myrsine 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 Myrsine 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 (Myrsine coriacea) and 1,100 m.s.m (Myrsine umbellata). Myrsine 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, Praça-Fontes et al. 2011) were supplied by Dr. Jaroslav Doležel (Experimental Institute of Botany, Czech Republic).
Fruit pericarp was manually removed and the seeds were desinfested according to Oliveira et al. (2013) and germinated in a medium composed of MS salts (Sigma) and vitamins (Murashige and Skoog 1962), 30 g L-1 sucrose (Sigma), 7 g L-1 agar and 2.685 µM naphthaleneacetic acid (NAA, Sigma). Solanum lycopersicum and Pisum sativum seeds were subjected to the same disinfestation procedure and inoculated in medium without NAA. Germination was done at 25 °C under a 16/8 hours (light/dark) regime.
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 Pisum sativum plantlets in vitro cultivated.
Nuclei suspensions were obtained by co-chopping (Galbraith et al. 1983) leaf fragments (1 cm2) cut from each sample (Myrsine species) and standard (Solanum lycopersicum or Pisum sativum). The suspensions were processed and stained following Otto (1990) and Praça-Fontes et al. (2011) and analyzed with the flow cytometer Partec PAS II/III (Partec GmbH). Myrsine genome size was measured by multiplying the 2C value of the internal standard using the fluorescence intensity corresponding to G0/G1 nuclei peak. Mean 2C values were compared by the F test at 5% probability.
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 (Carvalho et al. 2007). The roots were washed, macerated in 1:5 pectinase solution (enzyme:dH2O) for 3 h at 34°C, or 1:20 enzymatic pool (4% cellulase – Kinki Yakult MFG, 1% macerozyme – Kinki Yakult MFG, and 0.4% hemicellulase – Sigma) for 1 h 30 min or 1 h 45 min at 34°C, washed in dH2O, fixed, and stored at -20°C.
Slides were prepared and stained according to Carvalho et al. (2007) and analyzed on a Nikon eclipse Ci-S microscope (Nikon). Prometaphases and metaphases were captured using the 100× objective and a CCD camera (Nikon EvolutionTM) coupled to a Nikon microscope 80i (Nikon). About 100 slides were analyzed for each Myrsine species. Chromosome morphometry was characterized and the class was determined as proposed by Levan et al. (1964) and reviewed by Guerra (1986).
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 Table1) of1) of each chromosome was compared among species by the Scott-Knott test at 5% probability. Analyses were made using the software R 3.2.4 (R Core Team 2016).
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 Myrsine umbellata and the two internal standards. The channel of the Pisum sativum G0/G1 peak however was closer to Myrsine umbellata than Solanum lycopersicum Thus, based on linearity international criteria for FCM, Pisum 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) Myrsine 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 Myrsine parvifolia, 2C = 6.60 pg ± 0.14 for Myrsine coriacea and 2C = 6.63 pg ± 0.13 for Myrsine umbellata. The mean value of the Myrsine 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 Myrsine 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 Myrsine parvifolia and 8.45% of Myrsine 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. (Fig.11).
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. (Fig.2a),2a), resulting in a higher sum of the total length (Table (Table11). Myrsine coriacea and Myrsine umbellata did not show pronounced variation in chromatin compaction, but the quality of the chromosomes allowed us to characterize the karyotype and to assemble the karyogram (Fig. 2b–c, Table Table11).
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, Myrsine coriacea showed five metacentric (1–5), 17 submetacentric (6–21 and 23) and one acrocentric (22) chromosome pairs, and Myrsine 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. (Fig.2,2, Table Table11).
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 Myrsine coriacea (4–5, 10–11, 13–14, 15–16 and 19–20), and Myrsine 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. (Fig.2,2, Table Table1,1, ,2).2). Using morphometric data and applying the UPGMA statistical analysis, the chromosomes of each Myrsine species were grouped in three clusters in all species (Fig. 3a–c, Table Table2).2). Chromosome groups formed by qualitative analysis of all species were clustered by UPGMA, supporting previous findings.
As the mean 2C values of Myrsine coriacea (6.60 pg) and Myrsine umbellata (6.65 pg) were statistically identical, the Scott-Knott test was used to compare the relative size (Table (Table1)1) of each chromosome of these species. Chromosomes 1, 2, 6, 7, 11, 14, 19 and 23 differed between the species, while the others were statistically identical (Fig. (Fig.3d,3d, Table Table22).
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 (Abbi et al. 2011) made this challenging. These compounds probably prevented us from measuring the 2C value in individuals from the field when the OTTO I buffer (Otto 1990) was supplemented with dithiothreitol. Cytosolic compounds can reduce or inhibit the interaction of the fluorochromes and DNA during the nuclei staining step (Noirot et al. 2003). Antioxidants inhibit this interference, preserving the chromatin structure (Shapiro 2003). Nevertheless, the dithiothreitol was not efficient at providing nuclei suspensions suitable for FCM. Thus, this compound, which is more specific for molecules that possess free sulfhydryl groups, was replaced by PEG because of its wide spectrum for antioxidant activities, an effect called PEGylation (Term Fisher Scientific 2016). Due to this effect, PEG was more efficient at inhibiting the action of cytosolic compounds, resulting in G0/G1 peaks for Myrsine umbellata and Pisum sativum with CV below 5%. Owing to the linearity parameter, Pisum sativum was a more adequate standard relative to Solanum lycopersicum, which reduced measurement errors.
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 (Akula and Ravishankar 2011). Thus, the conditions at each elevation gradient can be associated with the FCM result, suggesting a differentiated production of secondary metabolic compounds for Myrsine at distinct altitudes.
Genome size in Myrsine had only been reported for Myrsine africana as 2C = 2.46 pg (Hanson et al. 2003), which was measured by Feulgen microdensitometry using Vigna sp. as standard. Levels of endoreduplication in cells of Vigna radiata, varying from 2C to 64C, were reported by Pal et al. (2004). Thus, the differences, which were about 200% between the values found for Myrsine species in this study and the value observed for Myrsine africana, can be related to the C value of Vigna radiata used as reference.
Values close to Myrsine umbellata and Myrsine 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 (Beuzenberg and Hair 1983, Dawson 1995, Dawson 2000, Molero et al. 2002, Molero et al. 2006, Rice et al. 2015). In vitro Myrsine plantlets were fundamental for providing sufficient quantities of roots for the cytogenetic study independent of the reproductive period. Meticulous standardization of the antimitotic agent and enzymatic maceration were also essential for accurate chromosomal characterization.
Chromosome number 2n = 46 (Beuzenberg and Hair 1983, Dawson 1995, Dawson 2000, Molero et al. 2002, Rice et al. 2015, present study) and 2n = 48 (Molero et al. 2006) had been reported, but this was the first record of 2n = 45. The odd chromosome number 2n = 45 was well-marked by absence of the homologue pair of the chromosome 23 (Fig. (Fig.2).2). So, other cytogenetic approaches should be performed from Myrsine individuals separately to know the cause of this aneuploidy.
Some chromosome groups determined by statistical analysis are morphologically distinct, such as chromosomes 22 and 23 of Myrsine coriacea. Although clustered (Fig. (Fig.3b),3b), these chromosomes are cytogenetically distinct, with 22 being acrocentric and 23 submetacentric (Fig. (Fig.2b,2b, Table Table2).2). Likewise, distinct chromosomes clustered in Myrsine parvifolia (Fig. (Fig.3a,3a, Table Table2)2) and Myrsine umbellata (Fig. (Fig.3c,3c, Table Table2).2). Chromosome 1 of Myrsine coriacea and 1 and 2 of Myrsine parvifolia and Myrsine umbellata presented the highest contrast, considering the morphology and Euclidean distances (Fig. (Fig.2,2, Fig. Fig.3).3). Similarities and differences regarding relative size (% size in relation to sum of the mean values of total length, Table Table1)1) were shown between Myrsine coriacea and Myrsine umbellata through the Scott-Knott test. The similarities, which were shown for some chromosomes, imply that these species could have originated from a common ancestor. The distinct chromosomes are likely to be attributed to karyotype changes that happened throughout their evolution, altering the chromosome relative size and contributing to taxa diversification. Comparative investigations of the karyotypes of related species have usually been applied to infer the evolutionary role of karyotypic modifications in different taxa and to describe the pattern and directions of chromosomal evolution within a group (Stebbins 1971, Soltis and Soltis 2012, Amaral-Silva et al. 2016).
Based on the constant chromosome number displayed by Myrsine species, interspecific variation of the nuclear 2C value between Myrsine parvifolia compared to Myrsine coriacea and Myrsine 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 (Pellicer et al. 2014, Amaral-Silva et al. 2016).
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.
Carvalho RF, Amaral-Silva PM, Spadeto MS, Nunes ACP, Carrijo TT, Carvalho CR, Clarindo WR (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