Genetics and Molecular Biology, 40, 3, 610-619 (2017) Copyright © 2017, Sociedade Brasileira de Genética. Printed in Brazil DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2016-0264 Research Article Intrachromosomal karyotype asymmetry in Orchidaceae Enoque Medeiros-Neto1, Felipe Nollet1, Ana Paula Moraes2,3 and Leonardo P. Felix1 1Departamento de Ciências Biológicas, Laboratório de Citogenética Vegetal, Centro de Ciências Agrárias, Universidade Federal da Paraíba, Campus II, Areia, PB, Brazil. 2Departamento de Genética, Instituto de Biociências, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Brazil. 3Instituto de Ciências e Tecnologia, Universidade Federal de São Paulo, São José dos Campos, SP, Brazil. Abstract The asymmetry indexes have helped cytotaxonomists to interpret and classify plant karyotypes for species delimita- tion efforts. However, there is no consensus about the best method to calculate the intrachromosomal asymmetry. The present study aimed to compare different intrachromosomal asymmetry indexes in order to indicate which are more efficient for the estimation of asymmetry in different groups of orchids. Besides, we aimed to compare our re- sults with the Orchidaceae phylogenetic proposal to test the hypothesis of Stebbins (1971). Through a literature re- view, karyotypes were selected and analyzed comparatively with ideal karyotypes in a cluster analysis. All karyotypes showed some level of interchromosomal asymmetry, ranging from slightly asymmetric to moderately asymmetric. The five tested intrachromosomal asymmetry indexes indicated Sarcoglottis grandiflora as the species with the most symmetrical karyotype and Christensonella pachyphylla with the most asymmetrical karyotype. In the cluster analysis, the largest number of species were grouped with the intermediary ideal karyotypes B or C. Con- sidering our results, we recommend the combined use of at least two indexes, especially Ask% or A1 with Syi, for cytotaxonomic analysis in groups of orchids. In an evolutionary perspective, our results support Stebbins’ hypothesis that asymmetric karyotypes derive from a symmetric karyotypes. Keywords: Asymmetry index, karyotype symmetry, chromosome, cytogenetic, evolution. Received: October 03, 2016; Accepted: December 19, 2016. Introduction many other authors (for a detailed discussion see Peruzzi The karyotype is the first phenotypic expression of and Ero lu, 2013). For a long time, these indexes were em- the genotype and provides an overview of the organization ployed by many cytogeneticists and cytotaxonomists to of the genetic material in the chromosome (Guerra, 2008). discuss the taxonomic relationships among related species Among the information that can be extracted from karyo- (Dematteis, 1998; D’Emerico et al., 1999; Selvi et al., types, i.e. number and morphology of the chromosomes, di- 2006; Felix et al., 2007; Peruzzi et al., 2009; Souza et al., versity of heterochromatic bands, gene location, etc., a very 2010). Stebbins (1971) suggested that asymmetric karyo- peculiar characteristic stands out: the karyotype asymme- types were originated from symmetrical ones, which has try, which is the subject of long debates. Changes in karyo- not been properly tested until now. type symmetry often involve modifications in chromosome The existing indexes are separated into two groups: size and morphology usually caused by DNA sequence interchromosomal asymmetry indexes, which quantify the expansions or deletions or by centric fusion/fissions (ac- heterogeneity in chromosome size, and intrachromosomal companied by disploidy) (Weiss-Schneeweiss and Schne- asymmetry indexes, which quantify the relative differences eweiss, 2013). in the centromere position among chromosomes of a com- The search for an index that reflected the karyotype plement (Stebbins, 1971; Peruzzi and Erolu, 2013). asymmetry started with Lewitsky (1931) and was followed Among the interchromosomal asymmetry indexes, A2 (Ro- by Huziwara (1962), Arano (1963), Stebbins (1971), and mero-Zarco, 1986) and CVCL (Paszko, 2006) are the most used due to their accuracy in the evaluation of chromosome dissimilarities (Chiarini and Barboza, 2008; Souza et al., Send correspondence to Felipe Nollet. Departamento de Ciências 2010; Pierozzi, 2011; Alves et al., 2011; Assis et al., 2013). Biológicas, Laboratório de Citogenética Vegetal, Centro de Ciên- cias Agrárias, Universidade Federal da Paraíba, Campus II, However, there is no consensus about the best method for 58.397-000 Areia, PB, Brazil. E-mail: nolletmedeiros@ya- calculating the intrachromosomal asymmetry (Romero- hoo.com.br Zarco, 1986; Peruzzi and Erolu, 2013). gmb-40-3.pdf 60 8/28/2017 3:37:29 PM Medeiros-Neto et al. 611 Among the proposed intrachromosomal asymmetry ån (/BbBb-+ ) i=1 iiii indexes, the following stand out: A = n The four categories of Stebbins (1971): from A to D according to the proportion of acrocentric and/or telo- The coefficient of variation of the centromeric index centric chromosomes in a karyotype, i.e. proportion of CVCI (Paszko, 2006): based on the index of interchro- chromosomes with a ratio between chromosome arms < mosomal asymmetry A2 (standard deviation/total average 2:1. The four categories have subtypes 1 to 3 according to of the chromosome length): the ratio between the large/small chromosome arms, giving a total of 12 categories (Table 1). CVCI =A2 ´ 100 The total form percentage (TF%; Huziwara, 1962): ratio between the sum of the short arms (p) length and the With so many ways to calculate the intrachromo- sum of the total chromosomes length: somal asymmetry, Zuo and Yuan (2011) developed six models of ideal karyotypes to test the accuracy of these S S TF% = p/ total karyotype length methods. These authors observed that the CVCI does not re- flect the intrachromosomal asymmetry in karyotypes when The karyotype asymmetry index percentage (Ask%; it is composed by telocentric and/or acrocentric chromo- Arano, 1963): ratio between the length of the long arms somes. Since the standard deviation decreases in acrocen- (q) of the chromosome set and the total length of the chro- tric or telocentric chromosomes, the CV is more suitable mosome set: CI to indicate the heterogeneity of the centromeric index, i.e. Ask% = Sq/Stotal karyotype length how different is the position of the centromeres among the chromosomes of the complement (see Zuo and Yuan, The symmetric index (Syi; Greilhuber and Speta, 2011). 1976): ratio between the average length of the short arms Peruzzi and Erolu (2013) discouraged the use of any (p) and the average length of the long arms, multiplied by intrachromosomal asymmetry index for karyotypes with 100: small chromosomes (£ 1 mm), due to the inaccuracy in the Syi =(Smean of p length/Smean of q length) ´ 100 arms’ measurement. However, the mean chromosome size in plants is 1.5–2.0 mm. Moreover, if the suggestion of The intrachromosomal asymmetry index A1 (Rome- Peruzzi and Erolu (2013) is followed, the chromosome ro-Zarco 1986): sum of the ratio between the average symmetry analysis will be prohibitive in a large number of length of the short arms in each homologous pairs (bi) and plant species, including a great part of Bromeliaceae, the average length of the long arms in each homologous Fabaceae and Orchidaceae species. pair (Bi) divided by the number of homologous chromo- some pairs (n): Considered one of the most diverse and taxonomi- cally complex plant families among the angiosperms, ån ()b/B Orchidaceae comprises 25,971 species with global distri- A =-1 i=1 ii 1 n bution (Pridgeon et al., 1999; Joppa et al., 2011). The Orchidaceae present a large karyotype variation, with all This proposal was later modified by Watanabe et al. types of chromosome morphology distributed in species (1999), who created the asymmetry index A, and followed with chromosome numbers varying from 2n =12in by Peruzzi and Erolu (2013), who called the same index Erycina pusilla (L.) N.H.Williams & M.W.Chase (Felix MCA. This index results from the sum of the ratio between and Guerra, 1999) to 2n = 240 in Epidendrum the differences in the long arm length (Bi) and the short arm cinnabarinum Salzm. ex Lindl. (Guerra, 2000; Felix and length (bi) of each chromosome and the sum of the lengths Guerra, 2010; Assis et al., 2013). Except for the subfamily of the long and short arms of each chromosome (Bi +bi). Cypripedioideae, Orchidaceae species are characterized by The sum is divided by the haploid chromosome number (n): small chromosomes (Larsen, 1968; Okada, 1988). Table 1 - Intrachromosomal asymmetry indexes. The total of 12 indexes is composted of the four categories of Stebbins (1971)-AtoDaccording to the proportion of acrocentric and/or telocentric chromosomes in a karyotype - and subtypes 1 to 3 according to the ratio between the large/small chromosome arms in each of these. Ratio: largest/smallest chromosomes Proportion of chromosomes with arm ratio < 2:1 0.0 0.01 – 0.5 0.51 – 0.99 1.0 <2:1 1A 1B 1C 1D 2:1–4:1 2A 2B 2C 2D >4:1 3A 3B 3C 3D gmb-40-3.pdf 61 8/28/2017 3:37:29 PM 612 Intrachromosomal asymmetry The wide karyotype diversity observed in consensus. Subsequently, only one consensus tree was Orchidaceae makes this plant family an excellent group for stored. The software Dendroscope® (Huson and Scorna- evaluating the applicability of karyotype asymmetry in- vacca, 2012) was used to root the tree with the most ideal dexes, especially the intrachromosomal index. Thus, the symmetrical karyotype (karyotype A) as an outgroup, fol- present study aimed to compare different intrachro- lowing the hypothesis of Stebbins (1971). mosomal asymmetry indexes, in order to indicate the most efficient for the estimation of karyotype asymmetry in or- Statistical analysis chids, including species with small chromosomes. Besides, we aimed to compare our results with the Orchidaceae To test the hypothesis of Stebbins (1971), the mean phylogenetic proposal to test the hypothesis of Stebbins values of the interchromosomal index A2 and the intra- (1971) that asymmetric karyotypes derived from symmet- chromosomal asymmetry indexes were separately used, in ric ones. order to compare the karyotype asymmetry levels for each subfamily.