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. 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 (M. coxii Cochayne, 1902, M. divaricata Cunningham, 1839, M. kermadecensis Cheeseman, 1887, M. nummularia (Hooker f.) Hooker f., 1867, M. salicina (Hooker f.) Hooker f., 1864, M. argentea Heenan et de Lange, 1998, M. oliveri Allan, 1961, M. chathamica Mueller, 1864; M. africana Linnaeus, 1753; M. sandwicensis Candolle, 1841, M. seguinii Léveille, 1914, M. semiserrata Wallich, 1824, M. australis (A. Richard, 1832) Allan, 1947, M. capitellata Wallich, 1824), and just three occurs in America continent (M. matensis (Mez, 1902) Otegui, 1998; M. guianensis (Aublet, 1775) Kuntze, 1891, M. 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.

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 M. umbellata and P. sativum with CV below 5%. Owing to the linearity parameter, P. sativum was a more adequate standard relative to S. 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 M. 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 V. 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 M. africana, can be related to the C value of V. radiata used as reference.

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 (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. 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 M. coriacea. Although clustered (Fig. 3b), these chromosomes are cytogenetically distinct, with 22 being acrocentric and 23 submetacentric (Fig. 2b, Table 2). Likewise, distinct chromosomes clustered in M. parvifolia (Fig. 3a, Table 2) and M. umbellata (Fig. 3c, Table 2). Chromosome 1 of M. coriacea and 1 and 2 of M. parvifolia and M. umbellata presented the highest contrast, considering the morphology and Euclidean distances (Fig. 2, Fig. 3). Similarities and differences regarding relative size (% size in relation to sum of the mean values of total length, Table 1) were shown between M. coriacea and M. 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 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 (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.

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.