Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 55-63, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-646 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: M. Rojas-Gómez, A. García- Piñeres, P. Bolaños-Villegas, G. Arri- eta-Espinoza, E.J. Fuchs (2020) Genome size and chromosome number of Psidium friedrichsthalianum (O. Berg) Nied (“Cas”) in six populations of Cos- ta Rica. Caryologia 73(3): 55-63. doi: 10.13128/caryologia-646 Received: October 03, 2019 Accepted: May 31, 2020 Published: December 31, 2020 Copyright: © 2020 M. Rojas-Gómez, A. García-Piñeres, P. Bolaños-Villegas, G. Arrieta-Espinoza, E.J. Fuchs. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Genome size and chromosome number of Psidium friedrichsthalianum (O. Berg) Nied (“Cas”) in six populations of Costa Rica Mónica Rojas-Gómez1,*, Alfonso García-Piñeres2, Pablo Bolaños-Vil- legas3,4, Griselda Arrieta-Espinoza5, Eric J. Fuchs6 1 Centro Nacional de Innovaciones Biotecnológicas (CENIBiot), CeNAT-CON- ARE,1174-1200 San José, Costa Rica 2 Centro de Investigación en Biología Celular y Molecular and Escuela de Química, Uni- versidad de Costa Rica, 11501-2060 San José (Costa Rica) 3 Fabio Baudrit Agricultural Research Station, Universidad de Costa Rica, La Garita, Alajuela 20101, Costa Rica 4 Lankester Botanical Garden, University of Costa Rica, P.O. Box 302-7050, Cartago, Costa Rica 5 Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Cos- ta Rica 11501-2060 San José, Costa Rica 6 Escuela de Biología, Universidad de Costa Rica, 11501-2060 San José, Costa Rica *Corresponding author. E-mail: morogo27@gmail.com; mrojas@cenat.ac.cr. Abstract. Psidium friedrichsthalianum (O. Berg) Nied is a species found from south- ern Mexico, Central America; and there are reports that it is also found in Venezuela and Ecuador. It is a common fruit component of the Costa Rican diet, and it is val- ued industrially for its high content of polyphenols, mainly proanthocyanidins (PACs). This crop is not completely domesticated and there are no improved varieties produced through plant breeding. Genome size or ploidy levels have not been investigated in Costa Rican populations of Psidium friedrichsthalianum. Information about chromo- some number and genome size is paramount for plant breeding strategies. Therefore, the main objective of our study was to determine chromosome number using pollen meiocytes and genome size by flow cytometry in six populations of P. friedrichsthali- anum in Costa Rica. We found x = 11 bivalent chromosomes in all meiocytes analysed, classifying these populations as diploid. All populations had an average nuclear DNA content of 2C = 1.960 ± 0.005 pg. No statistically significant differences in nuclear DNA content were found among populations. We conclude that the consistency in chromosome number and genome size among populations suggests a common ori- gin among them. Our estimates of the number of chromosomes and genome size of P. friedrichsthalianum determined in this study will be essential for future breeding pro- grams, hybridization practices and development of QTL (Quantitative Trait Loci). Keywords: ploidy, fluorescent microscopy, 2C nuclear DNA, flow cytometry, Costa Rican Guava, plant breeding. 56 Mónica Rojas-Gómez et al. INTRODUCTION Psidium friedrichsthalianum (O. Berg) Nied is a tropical species in the family Myrtaceae, subfam- ily Myrtoideae, tribe Myrteae (Lucas et al. 2019); com- monly known as “Cas”, “Sour Guava” or “Costa Rican Guava”. It is a medium-sized tree with reddish branch- es and abundant foliage of intense green color. Flow- ers are perfect, possibly allogamous and pollination is performed by bees and occasionally by hummingbirds (Barahona and Rivera 1995). Fruits are fleshy globose berries, between 5 and 10 cm in diameter with a green- ish to yellow exocarp and a very distinct soft and acidic pulp. In addition, it is presumed that its center of ori- gin is in Costa Rica (Barahona and Rivera 1995; Rojas- Rodríguez and Torres-Córdoba 2013). “Cas” fruits are characterised by abundant polyphenol content, main- ly proanthocyanidins (PACs); these metabolites have important antioxidant, anti-inflammatory, antimicro- bial and vasodilatory properties (Cuadrado-Silva et al. 2017; Flores et al. 2013; Rojas-Garbanzo et al. 2019; Granados-Chinchilla et al. 2016; González et al. 2012). Vasconcelos et al. (2019) described the chemical com- position and allelopathic properties of essential oils extracted from P. friedrichsthalianum, suggesting that this oil may be used as a natural weed control compara- ble in efficacy, to synthetic herbicides. This fruit is con- sidered an important resource due to its photochemical properties; however, few studies have been conducted on this tropical fruit. The germplasm of P. friedrichsthalianum in Costa Rica has not yet been genetically characterized, however, Srivastava (1977), reported this species as diploid (2n = 2x = 11), while Hirano (1967) reported tetraploid and hexaploid individuals in Central America samples. The diversity in chromosomal number previously reported for P. friedrichsthalianum may be a consequence of its ongoing domestication process. Information on chro- mosome numbers in the Myrtaceae is generally scarce, the fairly small chromosomes found in this taxonomic group, which rarely exceed 2 mm (Costa 2004), may curb chromosome determination. Presently, genome sizes have been reported for Psidium acutangulum, Psid- ium cattleianum, Psidium guajava L. (white cultivar), Psidium guajava L. (red cultivar), Psidium guineense and Psidium grandifolium (Costa and Forni-Martins 2006b, Costa et al. 2008; Machado-Marques et al. 2016; Coser et al. 2012; Souza et al.2015) (Table 1). However, the genome size or the 2C value of P. friedrichsthalianum have not been analyzed yet. Estimates of the number of chromosomes and genome size for P. friedrichsthalianum are essential for the design of effective improvement strategies, such as hybridization practices, the development of QTL (Quan- titative Trait Loci), as well as to better understand the effects of inbreeding and heterosis (Birchler 2013; Wash- Table 1. Chromosomes number and genome size from different species of Psidium and Eucalyptus (Myrtaceae) determined in previous studies. The content of holoploid nuclear DNA (2C, pg DNA) and the content of monoploid DNA (1C, pg DNA) are also provided. Species 2n Ploidy level Nuclear DNA content Reference 2C (pg) 1C (pg) 1C (Mbp)* Genus Eucalyptus Eucalyptus microcorys 22 2x 1.040 0.520 508.56 Almeida-Carvalho et al.(2017) Eucalyptus botryoides 22 2x 1.350 0.675 660.15 Almeida-Carvalho et al.(2017) Genus Psidium Psidium guajava 22 2x 0.950 0.475 464.55 Machado-Marques et al.(2016); Coser et al. (2012) Psidium guajava (purple) 18 2x 0.990 0.495 484.11 Souza et al.(2015) Psidium guajava (“Paluma”) 22 2x 1.020 0.510 498.78 Souza et al.(2015) Psidium guajava (white cultivar) 22 2x 0.507 0.253 247.43 Coser et al. (2012) Psidium guajava (red cultivar) 22 2x 0.551 0.275 268.95 Coser et al. (2012) Psidium grandifolium var. cinereum 44 4x 1.280 0.640 625.92 Costa and Forni-Martins (2009) Psidium grandifolium var. argenteum 44 4x 0.820 0.410 400.98 Costa and Forni-Martins (2009) Psidium cattleianum 44 4x 1.053 0.526 514.42 Costa and Forni-Martins (2006b) Psidium cattleianum 44 4x 1.990 0.995 973.11 Souza et al.(2015) Psidium guineense 44 4x 2.020 1.010 987.78 Souza et al.(2015) Psidium guineense 44 4x 1.850 0.925 904.65 Machado-Marques et al.(2016) Psidium acutangulum 44 4x 1.167 0.583 570.17 Costa and Forni-Martins (2009) *1pg DNA = 978 Mbp (Dolezel et al. 2003; Bennett et al. 2000). 57Genome size and chromosome number of Psidium friedrichsthalianum (O. Berg) Nied (“Cas”) in six populations of Costa Rica burn and Birchler 2014). Additionally, with the current development of second and third generation sequenc- ing techniques (NGS), information on genome size or C values are essential to establish appropriate experimen- tal conditions, to effectively prepare genomic libraries and sequencing of complete genomes (Leitch and Leitch 2008). The C-values reported here may be used as a tool for genomic analysis in this species, which should ben- efit genetic improvement practices in this species. Therefore, given the absence of information on ploidy level and nuclear DNA content in populations of Psidium friedrichsthalianum; we aimed to determine the chromosome number via fluorescent DAPI stain and flow cytometry to determine the nuclear DNA content of this tropical fruit in six populations of Costa Rica, its likely centre of origin. MATERIALS AND METHODS Sample collection We analysed individuals from six populations of P. friedrichsthalianum in Costa Rica. Samples were col- lected from local small-scale plantations from different regions in the country (Table 2). Plantations were locat- ed at different elevations ranging from sea level to over 1500 m asl (metres above sea level). Samples were always taken from reproductive trees and care was taken to col- lect samples from individuals that were separated by at least 10 meters to avoid collecting possible genets. Chromosomal count using DAPI stain Chromosome counts were performed on pollen mother cells in meiotic metaphase. At least seven flower buds were collected from each of six populations; flower buds ranged between 0.7 cm and 0.8 cm in length. Flow- er buds were fixed in FAA solution (96% ethanol, 5% glacial acetic acid and 40% formaldehyde) for 24 hours. As suggested by Dyer (1979), flower buds were dissect- ed to 3/4 of their final bud size. Anthers were placed on slides and subjected to mechanical disaggregation (macerating anthers with a thin spatula) adding occa- sional drops of acetic acid to prevent desiccation. Mac- erated anthers were stained with DAPI fluorochrome (Sigma-Aldrich, Ilinois, USA) and were incubated in the dark for 5 min. We used an epifluorescence microscope (Olympus BX50, Olympus Corporation, Tokyo, Japan) to visualize and photograph stained cells. Only cells that were in a state of meiotic metaphase were photographed. For each population at least five slides were evaluated and at least one cell was recorded in a state of meiotic metaphase. Finally, to facilitate chromosome counts, image color adjustments were performed with Adobe Photoshop CS82 (Adobe Systems, San Jose, CA) and the ImageJ software (US National Institutes of Health, 2007) was used to count chromosomes. Flow cytometry estimates of DNA content We used flow cytometry to estimate genome size on P. friedrichsthalianum. We collected fruits from at least 10 individuals per population. Seeds were manu- ally extracted, washed with tap water and dried in open air for a week to remove moisture. After one week, seeds were sown in pots with vermiculite soil and placed in a greenhouse for 12 weeks until seedling emergence. Ten seedlings per population were analyzed in a BD FACS- Calibur TM (Becton Dickinson, San Jose, CA, USA) flow cytometer. After initial parameter adjustment in the flow cytometry equipment, samples were prepared following the protocol by Dolezel et al. (2007) with some modifica- tions. We used 1 mg of leaf sample from Glycine max as a reference (2C = 2.50 pg) (Dolezel et al. 2007) and 5 mg of leaf tissue from P. friedrichsthalianum. Leaves were Table 2. Collection sites of “Costa Rican Guava” used to determine genome size and chromosomal number. PPT: mean annual precipitation (mm); Samples CMF: number of seedlings used for flow cytometry; Samples NM: number of samples used to determine chromosomes count. Population name Geographical coordinates Altitude (m a.s.l.) PPT (mm) Temp (°C) Samples CMF Samples NM Cervantes 09°53´28.3˝N 83°47´24.0˝W 1465 2500 24 10 5 Guápiles 10°13´42.1˝N 83°46´06.3˝W 262 4535 27 10 5 Tacacorí 10°03´07.3˝N 84°12´52.3˝W 952 2100 23 10 5 Ciruelas 09°59´05.7˝N 84°15´26.3˝W 910 1900 23 10 5 Batán 10°04´34.4˝N 83°22´37.2˝W 114 3567 28 10 5 Escazú 09°55´08.3˝N 84°07´42.6˝W 2428 1929 24 10 5 58 Mónica Rojas-Gómez et al. placed in a petri dish on ice, then 1 ml of OTTO-I lysis buffer (0.1 M citric acid, 0.5% (vol / vol) Tween 20) sup- plemented with 2 mM dithiothreitol (DTT) was added to the leaf cutouts (Otto 1990). Subsequently, leaves were cut with a razor blade until homogenization. The extract was filtered through a 41µm Nylon mesh, onto a 2.0 ml microcentrifuge tube. The filtrate was centrifuged at 10,000 rpm for 5 minutes. The supernatant was discard- ed and the pellet was resuspended in 100 µl of OTTO- I lysis buffer and incubated for 15 min at 4 ° C. After incubation and prior to analysis in a flow cytometer, 300 µl of OTTO-II buffer (0.4 M Na 2 HPO 4 · 12 H 2 O) (Otto 1990), 20 µl of propidium iodide (50 µg / ml) and 2 µl of RNase (50 µg / ml) were added to the mixture. All measurements were based on the fluorescence of at least 5000 total events (total nuclei). We analyzed two independent replicas of each sample on different days and estimated an average nuclear DNA content. Mean fluorescence intensity (MFI), number of events per peak and variation coefficient were all calculated using the FCS Express 4 Flow Cytometry software (De Novo Soft- ware, Los Angeles, CA). Finally, the nuclear DNA con- tent was calculated according to Dolezel et al. (2007) as follows: A = (B × C) D Where A = 2C (pg) nuclear DNA content concen- tration of P. friedrichsthalianum; B = Mean fluorescence intensity (MFI) of the G0 / G1 peak of P. friedrichsthali- anum; C = 2C (pg) nuclear DNA content of the internal standard; D = MFI of the G0 / G1 peak of the internal standard. Genome size was estimated from DNA con- tent as 1 picogram (pg) of DNA being equivalent to 978 megabase pairs (Mbp) (Bennett et al.2000; Dolezel et al. 2003). The 2C nuclear DNA content data of all individu- als was compared among populations with a one-way ANOVA, followed by Tukey’s test to determine individu- al differences (p <0.05). Statistics were done using R 3.5.0 software (R Core Team 2018). RESULTS AND DISCUSSION We consistently found 11 bivalent chromosomes in all meiocytes from Psidium friedrichsthalianum (Figure 2) across all populations (Table 2). Taking into account that the basic chromosome number of the Myrtaceae Figure 1. (a) Seedlings of P. friedrichsthalianum used to measure genome size by flow cytometry. (b and c) Flower buds of P. friedrichsthali- anum used for cytogenetic observations. 59Genome size and chromosome number of Psidium friedrichsthalianum (O. Berg) Nied (“Cas”) in six populations of Costa Rica family is x = 11 (Atchison 1947; Raven 1975) we classi- fied all Costa Rican samples of P. friedrichsthalianum as diploid (2n=2x=22). The diploid nature of the Costa Rican guava mirrors the results from Srivastava (1977), who similarly found a 2n = 2x = 22 diploid chromo- some count in different genotypes of Psidium friedrich- sthalianum. Costa and Forni-Martins (2006a, b, 2007) also described chromosome numbers for 50 species in the Myrtaceae, and found a predominance of 2n = 2x = 22 diploid species. Naitani and Srivastava (1965), Coser et al. (2012), Éder-Silva et al. (2007), and Souza et al. (2015), all found predominantly diploid species in the Psidium genus, such as in Psidium chinense and Psidium guajava. Previous results reported P. friedrichsthalianum individuals with 2n=4x=44 and even 2n=6x=66 (Hirano 1967), suggesting that this species may have tetraploid and hexaploid members. These results clearly indi- cate that there may be variation in ploidy levels among populations of P. freidrichsthalianum in different areas. In contrast, our results show that at least in Costa Rica, cultivated populations are consistently diploid. This chromosomal uniformity may be the result of a com- mon historical origin among populations. Alternatively, our results may also be a consequence of artificial selec- tion by farmers who selected cytotypes with specific homogenous traits of interests such as fruit size of pulp content. Multiple cytotypes have also been found in other Psidium congeners, for example in Psidium catt- leyanum the cytotypes 2n = 44, 66, 77 and 88 have been described (Costa and Forni-Martins 2006a; Costa 2009). Multiple cytotypes have been also found in populations of Psidium guineense and Psidium guajava (Srivastava 1977; Costa and Forni-Martins 2006a; Éder-Silva et al. 2007; Souza et al. 2015). Polyploidy is recognized as one of the main evolutionary forces in angiosperms (Soltis et al. 2015); and it is frequently associated with interspecif- ic hybridization followed by chromosomal duplication to restore hybrid fertility (Soltis et al. 2009). Results from congeners suggests that P. friedrichsthalianum may also have the potential to create other cytotypes may repre- sent important prospects for future breeding programs. Our study found bivalent and univalent chromo- somes in meiocytes of P. freidrichsthalianum (Figure 2a-2c). Chromosomes were also observed in a trivalent state (Figure 2d) and this is consistent with previous observations by Srivastava (1977) in this species. Uni- valent chromosomes are frequently observed in plants; these can arise through three different ways: (i) when a chromosome is not matched completely in zygotene stage; (ii) when paired bivalents separate in diplotene because robust chiasmata have not yet formed between them; (iii) due to premature disjunction of the bivalents during anaphase (Pires-Bione et al. 2000). The premature migration of univalent chromosomes to the poles during cell division is common in plants, giving rise to micro- nuclei (Pagliarini 1990; Pagliarini and Pereira 1992; Con- solaro et al. 1996). Alternatively, univalent chromosomes may occasionally occur in plants due to environmental factors such as temperature fluctuations (Heilborn 1934; Katayama 1935). Some of our sites differ drastically in climatic conditions, however, further studies are needed to better understand the cytology of this species. Our flow cytometry estimates were very consistent across all plant samples. Our coefficients of variation were all less than 5% (Table 3), which confirms that our suspensions had a sufficient number of stoichiometri- cally stained and intact nuclei. Additionally, DTT used in nuclei suspensions proved to be effective inhibiting cytosolic interfering compounds which resulted in clear histograms. DTT is commonly used in flow cytometry studies because of its broad antioxidant activity (Dolezel et al. 2007). In our study, DTT was very efficient because many woody species in the Myrtaceae, as is the case of P. friedrichsthalianum, contain abundant secondary metabolites that may interfere with DNA content stain- ing (Loureiro et al.2006) (Ohri and Kumar 1986). We determined a mean nuclear value of 2C = 1.960 ± 0.005 pg for P. friedrichsthalianum, equivalent to Figure 2. Bivalent chromosomes of P. friedrichsthalianum in mei- otic metaphase, stained with DAPI, scale bar 10um. (a, b and c) Samples from the populations of Cervantes, Tacacorí and Escazú respectively, showing 11 bivalent chromosomes. (d) Image showing chromosomes in trivalent, bivalent and univalent states. 60 Mónica Rojas-Gómez et al. 1916.88 Mbp (Bennett et al. 2000) (Figure 3, Table 3). Nuclear DNA content did not statistically vary among all six populations (F=0.29; df=5; p = 0.917). Leitch et al. (1998) and Soltis et al. (2003) classified species with 1C ≤ 1.4 pg content as species with a very small genomes compared to other angiosperms. Therefore, given our 1C estimates (1C = 0.98 ± 0.005 pg) (Table 3) the Costa Rican guava should also be classified as a small genome species. Consistently, Machado-Marques et al. (2016) found that Psidium guajava and Psidium guineense, also have very small genomes as 1C = 0.475 pg and 1C = 0.925 pg, respectively (Table 1). Almeida- Carvalho et al. (2017) determined that 25 species of the genus Euca- lyptus (Myrtaceae), all had 1C values between 1C=0.40 pg and 1C=0.75 pg which may indicate that the Myrta- ceae family may typically contain species with smaller genomes. On the other hand, our 2C estimates (2C =1.960 ± 0.005 pg) are within the range described by Souza et al. (2015), who also used flow cytometry on different species of Psidium and found 2C values that ranged between 2C=0.99 pg and 2C=5.48 pg. However our estimates are significantly higher than those found for different varieties of Psidium guajava; for example, Coser et al. (2012) found 2C = 0.507 pg for the white varieties, and 2C = 0.551 pg or 2C = 0.950 pg for the red varieties; while Souza et al. (2015) found 2C = 0.990 pg and 2C = 1.020 pg in purple and “Paluma” varieties respectively (Table 1). These differences in genome size may be due to (i) natural or bred adaptations of these species to different environmental conditions (Cavallini and Natali 1990), for example, to new cultivation envi- ronments; (ii) hybridization events, or (iii) changes in repetitive DNA sequences (Martel et al.1997). Several authors have suggested that transposable elements (TE) may be important in the evolution of genome sizes in plants (Wang et al. 2016; Wendel et al. 2016; Zhao et al. 2016). For example, Almeida-Carvalho et al. (2017) com- pared the genome size of two Eucalyptus species, E. bot- ryoides (2C=1.350 pg) and E. microcorys (2C=1.040 pg); and found big differences in genome size between them, although both had the same chromosome number (2n = 2x = 22) (Table 1). They argued that variations in 2C values in these Eucalyptus were caused by chromosome rearrangement and possibly TE elements. Therefore, our results on ploidy level and genome size of P. friedrichsthalianum, contribute to the cytoge- netic characterization of this economically impor- tant fruit species. This information may be used to design regional conservation strategies that preserve local genetic resources. Flow cytometry may be used to assess ploidy level in in vitro propagated plants (Ochatt et al. 2011), to screen for plants with higher ploidy lev- els, which may have new features of economic interest such as increased fruit size, or better juicing capabilities. Additionally, results from our study could aid the taxo- nomic definition of P. friedrichsthalianum species and the understanding of phylogenetic relationships among other members in the genus Psidium. CONCLUSIONS Populations of Psidium friedrichsthalianum from six different regions of Costa Rica, the likely centre of origin Table 3. Parameters obtained by flow cytometry to determine the genome size of Psidium friedrichsthalianum. NE: Number of events obtained; CV: Coefficient of variation obtained; 2C (pg): holoploid nuclear DNA content obtained; 1pg DNA = 978 Mbp (Dolezel et al. 2003; Bennett et al. 2000). Species NE CV 1C (pg) 2C (pg) Mbp Psidium friedrichsthalianum 2452 ± 0.001 2.95 ± 0.007 0.980 ± 0.005 1.960 ± 0.005 1916.88 Glycine max (standard) 2756 ± 0.005 3.01 ± 0.005 Figure 3. Relative fluorescence intensity (propidium iodide (PI)) histogram obtained after a simultaneous cytometric analysis of nuclei of reference standard (Glycine max, 2C=2.50 pg of DNA) and Psidium friedrichsthalianum (2C= 1.960 ± 0.005 pg). 61Genome size and chromosome number of Psidium friedrichsthalianum (O. Berg) Nied (“Cas”) in six populations of Costa Rica of this species, have a chromosome number equal to 2n = 2x = 22, indicating that cultivated populations in Cos- ta Rica, are all consistently diploid. Furthermore, these populations have an average 2C nuclear DNA content of 1.960 ± 0.005 pg. The uniformity found across pop- ulations in terms of chromosomal number and nuclear DNA content, suggests a common origin among them. ACKNOWLEDGEMENTS We acknowledge the help of Estación Experimental Agrícola Fabio Baudrit Moreno (Universidad de Costa Rica); Centro Nacional de Innovaciones Biotecnológicas (CENIBiot) at CeNAT-CONARE; Finca Los Diamantes at Instituto Nacional de Innovación y Transferencia en Tecnología Agropecuaria (INTA). This work would not have been possible without local “Cas” farmers who pro- vided free samples from their farms: David Badilla, Gre- gorio Menocal, Juan Pablo Orozco, Alfonso Ruíz, José Fuentes. This work was conducted under permit # 133- 2018 from the Comisión de Biodiversidad-UCR at Uni- versidad de Costa Rica. GEOLOCATION DATA Geolocation data is found on Table 2. FUNDING DETAILS This work was supported by the Vicerrectoría de Investigación de la Universidad de Costa Rica (UCR) under grant 111-B7-261; Consejo Nacional de Rectores (CONARE) under grant FEES-15-2019. REFERENCES Almeida-Carvalho GM, Carvalho CR, Ferrari-Soares FA. 2017. Flow cytometry and cytogenetic tools in euca- lypts: genome size variation × karyotype stability. Tree Genetics & Genomes. 13: 106 Atchison E. 1947. Chromosome numbers in the Myrta- ceae. American Journal of Botany. 34: 159-164. Barahona M, Rivera G. 1995. Development of jocote (Spondias purpurea L.) and cas (Psidium friedrich- sthalianum Niedz) in the premontano humid forest of Costa Rica. Mesoamerican Agronomy. 6: 23-31. Bennett MD, Bhandol P, Leitch IJ. 2000. Nuclear DNA amounts in angiosperms and their modern uses - 807 new estimates. 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