Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(3): 105-115, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-772 Citation: A. Teixeira Mesquita, M.V. Romero-da Cruz, A.L. Sousa Azevedo, E.R. Forni-Martins (2019) Chromo- some number and genome size diver- sity in five Solanaceae genera. Caryo- logia 72(3): 105-115. doi: 10.13128/ caryologia-772 Published: December 13, 2019 Copyright: © 2019 A. Teixeira Mesqui- ta, M.V. Romero da Cruz, A.L. Sousa Azevedo, E.R. Forni Martins. 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. Chromosome number and genome size diversity in five Solanaceae genera Amanda Teixeira Mesquita1,*, Marìa Victoria Romero-da Cruz1, Ana Luisa Sousa Azevedo2, Eliana Regina Forni-Martins1 1 Departamento de Biologia Vegetal, Universidade Estadual de Campinas, Rua Monteiro Lobato 255, CEP: 13.083-970 Campinas (SP), Brasil 2 Embrapa Gado de Leite, Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Rua Eugênio do Nascimento 610, CEP: 36.038-330 Juiz de Fora (MG), Brasil *Corresponding author: mesquita.at@gmail.com Abstract. Sixteen species of Solanaceae, belonging to five genera, were studied karyo- logically through chromosome counting, chromosomal measurement, and karyotype symmetry. Genome size (GS) estimation was performed on fifteen species using flow cytometry. The chromosome number 2n=24 was found in all Solanum species and Acnistus arborescens, 2n=22 was found in Brunfelsia uniflora, and 2n=16 in Cestrum representatives. Physalis pubescens was the only specie with evidence of polyploidy, showing 2n=4x=48 chromosomes. The chromosome numbers of S. adspersum, S. inodo- rum, S. flaccidum, S. sanctae-catharinae, and B. uniflora were reported for the first time. Haploid karyotype length (HKL) was statistically different between the studied species. The polyploid P. pubescens showed the largest HKL value, 93.10 µm. In general, kary- otypes were symmetrical with predominance of metacentric chromosomes. Chromo- some size was small in most species (<4 µm), while S. diploconos, C. laevigatum, and C. mariquitense, species with high HKL values, exhibited larger chromosomes. Genome size estimation were unpublished for ten studied species and were the first estimation for the genera Acnistus, Brunfelsia and Physalis. Were observed about eight-fold differ- ences between species with averages varying from 2C=2.57 pg to 2C=20.27 pg. As both HKL and GS showed a continuous variation. We observed partial similarity in the spe- cies ordered according to HKL and GS. The Solanaceae genera showed a constant chro- mosome number and a tendency to posse symmetrical karyotypes. The genome size also showed differences, which suggests that chromosome evolution in the group could be driven by alterations in the repetitive fractions of the genome. Keywords. Acnistus, Brunfelsia, Cestrum, Physalis, Solanum, karyotype evolution. INTRODUCTION The Solanaceae family comprises about 2,500 species and 100 genera and have cosmopolitan distribution. The greatest diversity of the family is found in Neotropical regions (D’Arcy 1991; Hunziker 2001). Members of Solanace- ae have great ecological and morphological diversity, characteristics which favoured the occupation of diverse habitats, such as desert regions, tropi- 106 Amanda Teixeira Mesquita et al. cal rainforests, and even disturbed areas (D’Arcy 1991; Knapp 2002). The family includes several species of important global food crops with high economic value, such as tomatoes (Solanum lycopersicum), potatoes (Solanum tuberosum), eggplants (Solanum melongena), and chilli peppers (Capsicum spp.), widely used drug plants, such as tobacco (Nicotiana tabacum), “datura” (Datura stra- monium), and “angel’s tears” Brugmansia suaveolens, as well as many ornamental plants, such as species of the genus Brunfelsia, Cestrum and Petunia. Many Solan- aceae species, including tomatoes, potatoes, and tobacco, are model organisms for various biological studies, and their genomes are some of the most well studied among angiosperms (Knapp et al. 2004). Karyotype information about species and groups are important for taxonomic and evolutionary stud- ies, whereas karyological changes accompany specia- tion and, consequently, the diversification of the groups (Guerra et al. 2008, 2012, Chiarini et al. 2018). The chro- mosome number, nuclear DNA content, total length of the chromosome complement, asymmetry indices, and number and location of the rDNA sites and heterochro- matic bands are the main data used in cytotaxonomic studies. Chromosome number data is the most available information and is not influenced by external agents, such as age of individuals, environmental conditions, and gene expression, providing accurate data about spe- cies evolution (Dobginy et al. 2004, Guerra et al. 2008, 2012). Cytogenetic characterization, accompanied by a genome size (GS) study, can offer important informa- tion about genome organization, phylogenetic relation- ships, and evolutionary trends. This approach has been successful used in some Solanaceae (Mishiba et al. 2000, Moscone 2003, Chiarini et al. 2014). Chromosome data is available for some genera of Solanaceae, while for other genera there is not enough data or information about their chromosomes. Lycium and Solanum present constant chromosome number (2n=24 and polyploids) (Bernadello and Anderson 1990; Bernadello et al. 1994; Chiarini and Bernadello 2006; Rego et al. 2009; Stiefkens et al. 2010; Melo et al. 2011; Chiarini et al. 2014), while Capsicum shows 2n=24 and 2n=26 (Moscone 1993; Moscone et al. 2007; Aguilera et al. 2014; Grabiele et al. 2014; Romero-da Cruz and For- ni-Martins 2015; Romero-da Cruz et al. 2017). For the Cestreae tribe, composed of Cestrum, Sessea, and Vestia, the only chromosome number reported to date is 2n=16 (Fregonezi et al. 2006; Las Peñas et al. 2006; Fernandes et al. 2009; Urdampilleta et al. 2015). The greatest range in chromosome number is found in Nicotiana (n=12 to n=32, and polyploids, Chase et al. 2003). Only about 8% of Solanaceae taxa have available GS data. This character has more variability than chromo- some number (Soltis et al. 2003). In Solanum, the GS ranges of from forty-fold in species with 2n=24 chromo- somes. The smallest reported C-value is in S. chacoense, 1C=0.63 pg (Bennett and Smith 1976), while the largest value is 1C= 24.80 pg, found in S. hartwegii (Pringle and Murray 1991). Nevertheless, there are still many gaps in karyotypic knowledge for the Solanaceae family and such informa- tion (i.e. genome size, chromosome number, and kar- yotype variables) is important to complete current data and to better understand the systematic relationships and chromosome evolution of the family. Therefore, the objectives of this study were: (1) to report original chro- mosome numbers and describe the karyotype variables in distinct genera of the Solanaceae family, (2) to deter- mine the genome size (GS) using flow cytometry for the first time for many species. MATERIAL AND METHODS Plant material Sixteen species from the genera Acnistus, Brunfelsia, Cestrum, Solanum, and Physalis were collected in South- eastern Brazil. Voucher specimens were deposited into the Herbarium at the University of Campinas (UEC). Data collection is detailed in Table 1. Chromosome preparations Seeds of at least three individuals per species were germinated in Petri dishes. In some cases, 1 ml gibberel- lic acid (GA3) was applied to break seed dormancy (Ellis et al. 1985). According to previous tests, root meristems were pre-treated with different solutions to block the cell cycle to obtain good chromosome spread and condensa- tion (Table 2). The root apices were fixed in 3:1 ethanol: acetic acid (v:v) mixture that was stirred for a minimum of 12 h at room temperature (RT) and stored at -6ºC until slide preparation. Slides were made using root mer- istems that were previously digested in a solution of 1% macerozima, 2% cellulase, and 20% pectinase for 10-15 minutes at 37ºC and squashed in a drop of 45% acetic acid. Coverslips were removed after freezing in liquid nitrogen for 15 minutes. The cells were photographed under a microscope Olympus BX51 with a DP72 camera attached and images were captured using Olympus DP2 BSW program (Olympus Corporation). 107Chromosome number and genome size diversity in five Solanaceae genera Karyotype analysis Five metaphases of each species, with the same degree of chromosome condensation, were used to deter- mine the chromosome number. The measurements were taken using the MicroMeasure© software (3.3). Ideo- grams were made using measurements of the following means for each chromosome pair: S (short arm length), L (long arm length) and C (total chromosome length) using the formula C= S+L. In addition, haploid karyo- type length (HKL) was calculated by the sum of the hap- loid chromosome lengths. The arm ratio (r) was calcu- lated using the formula r= L/S and was used to classify chromosomes according to Levan et al. (1964). For ideo- grams, chromosomes were first grouped by morphology (r=1.00-1.69 metacentric-m; r=1.70-2.99 submetacentric- sm; r=3.00-6.99 subtelocentric-sm) and then by decreas- ing size order within each group. The karyotype symmetry was described using the indices A1= 1-[(Σbi/Bi)/n] (bi = mean of the short arm Table 1. Cytogenetics data of Solanaceae species: Species and voucher specimen; provenance of materials; chromosome number, haploid karyotype formula (HKF), median haploid karyotype length (HKL), variation in chromosome length (VCL); symmetry indices A1 and A2; median DNA content (2C values). Species (Voucher specimen) Provenance 2n HKF HKL – µm (CI) VCL – µm A1 A2 2C values – pg (CI) Acnistus arborescens Schltdl. (Monge 2787) Brazil: Rio Grande do Sul; Aratinga 24 12m 45.93 (2.78) 3.17-4.38 0.17 0.10 6.56 (0.06) Brunfelsia. uniflora D. Don (Mesquita 15) Brazil; São Paulo; Campinas 22 7m+4sm 50.51 (0.50) 3.89-5.37 0.32 0.10 6.58 (0.13) Cestrum laevigatum Schltdl. (Mesquita 12) Brazil; São Paulo; Campinas 16 6m+2sm 78.72 (2.96) 7.92-10.88 0.23 0.10 20.27 (0.43) C. mariquitense Kunth (Mesquita 14) Brazil; São Paulo; Campinas 16 7m+1sm 73.91 (6.38) 7.35-11.39 0.21 0.12 - Physalis pubescens L. (Vasconcellos Neto 00-068) Brazil; São Paulo; Jundiaí 48 19m+5sm 93.10 (2.87) 1.43-2.80 0.32 0.18 12.98 (0.09) Solanum Cyphomandra clade Solanum diploconos (Mart.) Bohs (Mesquita 23) Brazil; São Paulo; Jundiaí 24 8m+4sm 74.72 (1.86) 4.63-7.49 0.32 0.14 19.22 (0.43) Dulcamaroid clade S. flaccidum Vell. (Mesquita 07) Brazil; São Paulo; Campinas 24 9m+2sm+1st 26.73 (1.14) 1.83-2.50 0.27 0.10 2.57 (0.25) S. inodorum Vell. (Vasconcellos Neto 20401) Brazil; São Paulo; Jundiaí 24 5m+7sm 38.33 (3.90) 2.83-3.86 0.39 0.09 4.63 (0.06) Geminata clade S. pseudocapsicum L. (Mesquita 24) Brazil; São Paulo; Jundiaí 24 9m+3sm 28.61 (7.70) 1.76-2.72 0.28 0.13 2.94 (0.11) Leptostemonum clade Acanthophora section S. acerifolium Sendt. (Mesquita 02) Brazil; São Paulo; Campinas 24 10m+2sm 36.17 (1.09) 1.71-3.87 0.26 0.23 5.69 (0.15) S. palinacanthum Dunal (Mesquita 20) Brazil; São Paulo; Ubatuba 24 5m+7sm 37.86 (0.72) 2.51-3.86 0.41 0.13 5.00 (0.10) Torva section S. adspersum Witasek (Monge 2748 c 240) Brazil; Rio de Janeiro; Arraial do Cabo 24 9m+3sm 25.09 (1.62) 1.77-2.44 0.25 0.09 3.19 (0.04) S. scuticum M. Nee (Vasconcellos Neto 8503) Brazil; São Paulo; Jundiaí 24 9m+3sm 27.66 (2.10) 1.95-2.79 0.31 0.04 3.42 (0.06) S. variabile Mart (Monge 2324) Brazil; São Paulo; Itacaré 24 9m+3sm 33.45 (0.51) 2.15-3.26 0.24 0.11 3.54 (0.09) Uncertain position S. concinnum Schott ex Sendtn. (Mesquita 08) Brazil; São Paulo; Campinas 24 6m+6sm 31.82 (0.37) 2.26-2.86 0.39 0.09 3.65 (0.25) S. sanctae-catharinae Dunal (Vasconcellos Neto 20873) Brazil; São Paulo; Jundiaí 24 10m+2sm 23.15(2.56) 1.68-2.35 0.26 0.09 3.79 (0.07) CI – Confidence interval at 95% of semi range. 108 Amanda Teixeira Mesquita et al. of each chromosome pair, Bi = average of the long arm of each chromosome pair, n = number of chromosome pairs) and A2=x/s (s = standard deviation; x = average chromosome complement length) (Zarco 1986). A1 index measures intrachromosomal asymmetry which indicates differences in the size of chromosome arms. A2 index measures the interchromosomal asymmetry and indi- cates the variation in chromosome lengths. In terms of length, chromosomes were classified according to Lima de Faria (1980) as very small (≤1 µm), small (>1 µm and ≤4 µm), intermediate (>4 and ≤12) and big (>12 and ≤60). Flow cytometry The same species that were cytogenetically analysed (except for Cestrum mariquitense) were cultivated in a greenhouse and used for GS measurements. For each species, three individuals were measured in three repeti- tions, for a total of nine samples. Approximately 1 cm2 of young leaf tissue was used to prepare the nuclear suspen- sions, according to Dolezel et al. (2007). The material of each species of interest and a piece of internal leaf stand- ard (Pisum sativum “Ctirad” 2C=9.09 pg ( Dolezel et al. 1998) were sliced with a razor blade and placed into a Petri dish on ice. About 1 ml of LB01 buffer (Dolezel et al. 1989) was used to extract the nuclei. A nylon mesh with 40 microns was used to filter the sample (CellTrics, PARTEC), then, 25 µL 1 mg/mL propidium iodide and 25 µL 1 mg/mL RNAse were added to the nuclear suspen- sion. The measurement was performed on a BD FACS Calibur flow cytometer, for each sample an average of 10,000 nuclei were analysed. The 2C value was calculated using the linear relationship between fluorescence signals from stained nuclei of the unknown sample and the ref- erence standard. The nomenclature for genome size clas- sification followed Leitch et al. (1998) with modification by Soltis et al. (2003): values <1.4 pg and between 1.4 to 3.5 pg correspond to “very small” and “small” genomes, respectively. On the other hand, values between 3.51- 13.99 pg, >14 pg and >35 pg are considered “intermedi- ate,” “large”, and “very large” genomes, respectively. Statistical analyses The HKL values, as well of GS values of each species, were compared using Past 3.18 ® (Øyvind Hammer, Nat- ural History Museum, University of Oslo). The Kruskal- Wallis nonparametric test was performed to compare the averages among the species and Dunn’s post-hoc test (Dunn 1954) was carried out after significant Kruskal- Wallis test. RESULTS Karyotype analysis The somatic chromosome numbers were 2n=2x=24 (Acnistus and Solanum), 2n=2x=22 (Brunfelsia), 2n=2x=16 (Cestrum) and 2n=4x=48 (Physalis) (Table 1; Fig. 1). Although the differences in HKL between some species were significant (p<0.05) according to statisti- cal analysis (Table 1; Fig. 2), this variation was gradual, and no groups were formed. Solanum sanctae-catharinae showed the lowest median value (23.15 µm) with a vari- ation of 1.68-2.35 µm from the smallest to largest chro- mosome pair. Other Solanum species also presented low HKL (except for S. diploconos), with values reach- ing 38.33 µm in S. inodorum (2.83 to 3.86 µm). Species with intermediate HKL values were A. arborescens (45.93 µm, 3.17 to 4.38 µm) and B. uniflora (50.51 µm, 3.89 to 5.38 µm). High HKL values were found in C. mariq- uitense with 73.91 µm (7.35 to 11.39 µm), S. diploconos with 74.72 µm (4.63 to 7.49 µm), and C. laevigatum with 78.72 µm (7.92 to 10.88 µm). Physalis pubescens showed Fig. 1. Somatic metaphases of five genera of Solanaceae. a Solanum flaccidum. b S. adspersum. c S. sanctae-catharinae. d S. inodorum. e Physalis pubescens. f Acnistus arborescens. g Brunfelsia uniflora. h S. diploconos. i Cestrum laevigatum. Bar=10 µm. 109Chromosome number and genome size diversity in five Solanaceae genera the highest HKL value (93.10 µm), even though it is a polyploid species with chromosomes ranging from 1.43 to 2.8 µm. Karyotypes are symmetrical with A1 and A2 values for each species ranging from 0.17 to 0.41 and from 0.04 to 0.23, respectively. Most species presented a predomi- nance of metacentric chromosomes (Table 1, Fig. 3) that characterized most intrachromosomal symmetry shown in the A1 index. Acnistus arborescens had the most sym- metrical karyotype, composed of only metacentric chro- mosomes and A1=0.17. Three species had less symmet- rical karyotypes: Solanum inodorum and S. palinacan- thum showed predominance of submetacentric chromo- somes (5m+7sm) and A1 value of 0.39 and 0.41, respec- tively. Solanum concinnum also presented A1=0.39, but karyotype formulae 6m+6sm. Interchromosomal index A2 showed that all spe- cies have few variations in chromosome size of the kar- yotypes. Solanum scuticum showed the small A2 value (0.04) and Solanum acerifolium presented the highest A2 value (0.23), characterizing the most interchromosomal asymmetry among studied species (Table 1). C-value Genome size estimates of all the studied species are shown in Table 1 and histograms for selected spe- cies are shown in Fig. 4. According to statistical analy- sis, GS showed significant differences among some of the studied species (Fig. 5). A variation of about eight-fold was observed, ranging from 2C=2.57 pg (S. flaccidum, Fig. 4a) to 2C=20.27 pg (C. laevigatum, Fig. 4d). The GS presented continuous variation, so distinct groups were not characterized (Fig. 5). Most species had small (2C=2.57 pg in S. flacidum to 2n=3.79 pg in S. sanctae- catharinae) and intermediate genomes (2C=4.63 pg in S. inodorum to 6.56 pg in A. arborescens and 6.58 pg in B. uniflora). The species with larger genomes were P. pube- scens (2n=12.98 pg), S. diploconos (2C=19.22 pg), and C. laevigatum (2C=20.27 pg). DISCUSSION Chromosome number The chromosome number data found here are new for S. adspersum, S. inodorum, S. flaccidum, and S. sanc- tae-catharinae, with 2n=24 chromosomes, as well as for B. uniflora, with 2n=22. For the remaining species, the chromosome number obtained corroborated with data found in the literature for Acnistus (2n=24), Cestrum (2n=16), Solanum (2n=24), and Physalis (2n=48) (Heiser 1963; Pedrosa et al. 1999; Fernandes et al. 2009; Rego et al. 2009; Urdampilleta et al. 2015). All the species in this study, except for P. pubescens, which is a tetraploid, are diploid. Although diploid is the most frequent ploidy level (including other species of Physalis), polyploidization has played an important role in the evolution of some Solanaceae genera (e.g., Nico- tiana, Chase et al. 2003; S. elaeagnifolium, Scaldaferro et al. 2012). The chromosome number most frequent in the family is 2n=24, found in more than 85% of the previ- ously studied Solanaceae species (Olmstead et al. 2008) though a diploid series from 2n=14 to 2n=26 is present in some genera (eg. Petunia and Calibrachoa, Mishiba et al. 2000, Cestrum, Sessea and Vestia, Las Peñas et al. 2006, Capsicum, Moscone et al. 2007). Many authors have postulated hypotheses for the ancestral chromosome base number in the family. Raven (1975) proposed x=7 and 12 for the order Sola- nales and Solanaceae family, respectively, while Badr et al. (1997) suggested the hypothesis of x=7 or x=8. Moscone (1992) corroborate with the proposition of Badr et al. (1997), suggested x=7 as the basic chromo- some number for Solanaceae. Olmstead and Palmer (1992) and Olmstead et al (2008) based in phylogenetic studies, suggests an ancestral position of subfam. Ces- troideae (x=8), and x=12 as a derivate basic chromo- some number the family. Fig. 2. Boxplots illustrating the continuous variability of HKL (Haploid Karyotype Length), as inferred from de Kruskal Wallis analysis. The numbers on the x axis represent the species ordered by crescent HKL values (in µm): S. sanctae-catharinae (1), S. adspersum (2), S. flaccidum (3), S. scuticum (4), S. pseudocapsicum (5), S. concinnum (6), S. variabile (7) S. acerifoium (8) S. palinacan- thum (9) S. inodorum (10), A. arborescens (11) B. uniflora (12) C. mariquitense (13) S. diploconos (14) C. laevigatum (15) P. pubescens (16). The central box represents 50% of the data from de upper to lower quartile. The horizontal bar expresses the median position. The extremity of the vertical lines indicates minimum and maxi- mum values of HKL, if they are no outliners. When outliners are present, they are represented by circles. 110 Amanda Teixeira Mesquita et al. Fig. 3. Ideograms of the investigated Solanaceae species based on median chromosome values. Bar=5 µm. 111Chromosome number and genome size diversity in five Solanaceae genera The lack of chromosomal data for several genera and for the Solanaceae sister group, the family Convolvu- laceae, as well as the presence of distinct basic numbers in other Solanales families, as in Hydroleaceae, x=8 and 10 (Constance 1963) and Montiniaceae, x=12 (Goldblatt 1979), has hampered to establish a consensus about a basic chromosome number and understand the direction of chromosome number evolution for the family. Karyotype structure Differences in chromosome size were seen between the Solanaceae species here investigated. The relatively small chromosome size and HKL observed in the species here of Solanum, except for S. diploconos (statistically distinct, and previously consid- ered a species of the distinct genus Cyphomandra), and P. pubescens, have been reported in some studies for Solanum (Bernardello and Anderson 1990; Acosta et al. 2005; Chiarini et al. 2006; Rego et al. 2009; Melo et al. 2011; Moyetta et al. 2013), and another Solanaceae gen- era, as Lycianthes and Vassobia (Rego et al. 2009) and Lycium, (Stiefkens and Bernadello 2002,) Acnistus arborescens and B. uniflora shows chromo- somes and consequently, HKL values, with intermedi- ate size, when compared to Solanum and Physalis. These karyotype characteristics are also present in Capsicum (Moscone 1996), Sclerophylax and Nolana (Lujea and Chiarini 2017), genera also belonging to Solanaceae. Although the intermediate size of the chromosomes, a constant chromosome number, karyotype symmetry and chromosomes majority metacentric appear to maintain in these groups. The tribe Cestreae (subfam. Cestroideae) embrac- es the genera Cestrum, Sessea and Vestia, presents the largest chromosomal sizes of the family (Fregonezi et al. 2006, Peñas et al. 2006). Cestrum laevigatum and C. mariquitense, investigated here, showed the largest chro- mosome size high HKL values, confirming the trend for the tr. Cestreae (Fregonezi et al. 2006). Such increase in chromosome size for the tribe can be due to the absence of Arabidopsis-type telomeres (TTTAGGG)n for short interstitial telomeric sequences (SITS), leading to the lack of control of the telomerase-dependent replication. These sequences may associate with other DNA sequenc- Fig. 4. Flow cytometry histograms (iodide propidium fluorescence intensity of nuclei) showing DNA amounts from leaf tissues of some Solanaceae species. 1 Pisum sativum “Ctirad” (standard). 2 S. flaccidum. 3 P. pubescens. 4 A. arborescens. 5 C. laevigatum. Fig. 5. Boxplots illustrating the continuous variability of GS (Genome Size), as inferred from de Kruskal Wallis analysis. The numbers on the x axis represent the species ordered by crescent C-values (in pg): S. flaccidum (1), S. pseudocapsicum (2), S. adsper- sum (3), S. scuticum (4), S. variabile (5), S. concinnum (6), S. sanctae- catharinae (7) S. inodorum (8) S. palinacanthum (9) S. acerifolium (10), A. arborescens (11) B. uniflora (12) P. pubescens (13) S. diplo- conos (14) C. laevigatum (15). The central box represents 50% of the data from de upper to lower quartile. The horizontal bar expresses the median position. The extremity of the vertical lines indicates minimum and maximum values of HKL, if they are no outliners. When outliners are present, they are represented by circles. Table 2. Pretreatments used for each genus of Solanaceae studied. Genus Pretreatments Acnistus and Physalis 8-hydroxyquinoline 0.002M + cycloheximide 25mg/L (1:1), 8 h, 4ºC Brunfelsia 8-hydroxyquinoline 0.002M, 6 h, 14ºC Cestrum Colchicine 0.1% 6 h, RT* Solanum Saturated solution of r-dichlorobenzene 2 h, RT* Solanum (S. diploconos) Saturated solution of r-dichlorobenzene 5 h, RT* *RT=room temperature. 112 Amanda Teixeira Mesquita et al. es and assist in their dispersion leading to an increase in genome size (Sykorova et al. 2003a, b). Besides chromosome number, other widely con- served karyotype characters in Solanaceae genera, are chromosome morphology and karyotype symmetry. Symmetrical karyotypes with a predominance of meta- centric chromosome pairs are found in the five genera studied here. Acnistus arborescens was the unique spe- cie that had only metacentric chromosomes, thus, had the most symmetrical karyotype and only S. flaccidum showed a subtelocentric chromosome pair. Other gen- era of the family Solanaceae in which it is possible to observe these characteristics areCapsicum (Pozzobon et al. 2006; Moscone et al. 1993, 2007), Lycium (Stiefkens and Bernadello 2012), Lycianthes, and Vassobia (Rego et al. 2009). Among the angiosperms, karyotype asymmetry can be associated with derivate taxa (Stebbins 1971) In some groups of the Solanaceae family, intermediate asymme- try values, can be observed, such as the tr. Cestreae (Las Peñas et al. 2006), Solanum sect. Acanthophora (Chiarini et al. 2014) However, for species, the karytotype asym- metry was not associate with basal or derived position of the taxa in the phylogeny. Regarding karyotype asym- metry, no evolutive trend was found for the analysed genera or among the representatives of Solanum. Over- all, karyotype asymmetry seems to occur randomly within some groups of the family. Our study analysed five species from other sections (Acantophora and Torva) of the Leptostemonum clade. In sect. Acantophora (S. acerifolium and S. palinacan- thum), we observed greater HKL and karyotype asym- metry than in sect. Torva (S. adspersum, S. scuticum and S. variabile). Karyotype asymmetry was previously reported for the Lepstotemomun clade (Chiarini et al. 2014). In other cases, asymmetry was random within a group, as in Solanum Morelloid and Dulcamaroid clades (Moyetta et al. 2013). Both species belonging to clade Dulcamaroid that were studied corroborated this data, while S. inodorum (HKL=38.33 µm) presented a more asymmetrical karyotype (5m + 7sm and A1=0.39, A2=0.09) than S. flaccidum (HKL=26.73 µm, 9m + 2sm + 1st and A1=0.27, A2=0.10). The karyotype characteristics described for the stud- ied species and genera as well as in other Solanaceae groups, a constancy in the chromosome number, karyo- type symmetry and chromosome morphology, indicates karyotype orthoselection, which preserves similar chro- mosomal complements, regardless of chromosome size (Acosta et al. 2005; Moscone et al. 2003). According to Wu and Tanksley (2010), inversions have occurred at a much higher rate than translocations throughout the evolutionary history of Solanaceae, thereby preserving chromosome morphology favouring chromosomal uni- formity C-value/DNA content Despite the great number of representatives in Sola- naceae, the GS estimation is available for a small propor- tion of species and genera. Only 12 Solanaceae genera have data about GS, representing approximately 10% and 186 species that corresponding to 7% of Solanaceae representatives. Genome size data for A. arborescens, B. uniflora and P. pubescens are the first estimation for the relative genera. Some species of Cestrum and Solanum have their GS measured but the data here obtained are unpublished for C. laevigatum, S. flaccidum, S. inodor- um, S, adspersum, S, scuticum, S. variabile, S. concinnum and S. sanctae-catharinae. The GS variation observed in the species studied partially coincides with the variation observed in HKL. In general, species with small, intermediary, or high HKL presented the same GS classification. Among the five species with small HKL, four presented small values of DNA content (S. adspersum, S. flaccidum, S. pseudo- capsicum and S. scuticum). Similarly, of the six species with high HKL, five showed high values of DNA content (A. arborescens, B. uniflora, S. diploconos, C. laeviga- tum and P. pubescens). The estimation of nuclear DNA using flow cytometry is more accurate than measuring chromosomes. This accuracy is supported by statistical tests and by species boxplots, where the dispersion of HKL data (Figure 2) was greater than GS data (Figure 5). Calculating HKL is more subject to external effects (Stace 2000). Methodological standardization, especially the degree of chromosomal condensation in the mitotic metaphase, is important for obtaining chromosomal siz- es and comparing the results obtained between species (Stace 2000). Although angiosperms have high diversity in their DNA content, the predominance of a small genome size causes a tendency with modal values equal to 1C = 0.7 pg (Leitch et al. 1998). This distribution, strongly skewed towards small genomes, is associated with the ancestral condition of the group and large genomes could have arose more than once during angiosperm evolution (Leitch et al. 1998; Soltis et al. 2003). Among the species studied, and in the Solanaceae family in general, we observed a predominance of small genomes (see Bennett and Leitch 2012). The cosmopoli- tan distribution of the family and occurrence in a wide variety of habitats (D’Arcy 1991; Hunziker 2001; Knapp 2002) are related to some phenotypic characteristics that 113Chromosome number and genome size diversity in five Solanaceae genera are correlated to the low DNA content. Species with low DNA content tend to be found in varying habitats and those with very large genomes appear to be excluded from extreme habitats (Knight and Ackerly 2002). Despite the predominance of small genomes in Sola- naceae, there are some groups with intermediary or large genome sizes, such as species of the genera Nico- tiana, Cestrum, Capsicum, and the Cyphomandra clade of Solanum (see Bennett and Leitch 2012). There are two main factors associated with increased genome size in plants, polyploidy events or whole genome duplication (Soltis et al. 2003; Leitch and Leitch 2013; Wendel et al. 2015) and an increase in the repetitive elements of DNA (mainly transposable elements) (Leitch and Leitch 2013; Bennetzen and Wang 2014). In Solanaceae, it is likely that the changes in genome size of these groups is relat- ed to repetitive elements, since there are few groups with polyploidy relatives causing an increase in DNA content. CONCLUSIONS We conclude that some karyotype characters are well conserved in the Solanaceae family at the generic level. Chromosome numbers are very constant, with few reports of polyploidy and aneuploidy and the predomi- nance of chromosome morphology and karyotype sym- metry. The family represents a model for karyotypic orthoselection and the karyotype evolution in Solan- aceae may have been driven by repetitive DNA reorgani- zation that led to GS diversification, but did not affect chromosome number and morphology. Acknowledgments: The authors thanks the Comis- são de Aperfeiçoamento de Pessoal do Nível Supe- rior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant number 2016/17096-9) for the financial support. We thank the Empresa Brasileira de Pesquisas Agro- pecuária (EMBRAPA), unidade Gado de Leite for the infrastructure granted to execute part of the study. We thank Espaço da Escrita – Coordenadoria Geral da Uni- versidade – Universidade Estadual de Campinas (UNI- CAMP) - for the language services provided. FUNDING DETAILS This work was supported by the Fundação de Amp- aro à Pesquisa do Estado de São Paulo (FAPESP) under the Grant 2016/17096-9. 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