Abstract
The asymmetry indexes have helped cytotaxonomists to interpret and classify plant karyotypes for species delimitation 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 results with the Orchidaceae phylogenetic proposal to test the hypothesis of Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p.. Through a literature review, 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. Considering 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
Introduction
The karyotype is the first phenotypic expression of the genotype and provides an overview of the organization of the genetic material in the chromosome (Guerra, 2008Guerra M (2008) Chromosome numbers in plant cytotaxonomy: Concepts and implications. Cytogenet Genome Res 120:339-350.). Among the information that can be extracted from karyotypes, i.e. number and morphology of the chromosomes, diversity of heterochromatic bands, gene location, etc., a very peculiar characteristic stands out: the karyotype asymmetry, which is the subject of long debates. Changes in karyotype symmetry often involve modifications in chromosome size and morphology usually caused by DNA sequence expansions or deletions or by centric fusion/fissions (accompanied by disploidy) (Weiss-Schneeweiss and Schneeweiss, 2013Weiss-Schneeweiss H and Schneeweiss G (2013) Karyotype diversity and evolutionary trends in Angiosperms. In: Greilhuber J, Dolezel J and Wendel JF (eds) Plant Genome Diversity vol. 2. Springer, Vienna, pp 209-230.).
The search for an index that reflected the karyotype asymmetry started with Lewitsky (1931)Lewitsky GA (1931) An essay on cytological analysis of the fixing action of the chromacetic formalin and the chromic formalin. Bull Appl Bot Genet Plant Breed 27:187-240. and was followed by Huziwara (1962)Huziwara Y (1962) Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromosome of Aster. Amer J Bot 49:116-119., Arano (1963)Arano H (1963) Cytological studies in subfamily Carduoideae (Compositae) of Japan. IX. The karyotype analysis and phylogenic consideration of Pertya and Ainsliaea. Bot Mag 76:32-39., Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p., and many other authors (for a detailed discussion see Peruzzi and Eroğlu, 2013Peruzzi L and Eroğlu HE (2013) Karyotype asymmetry: Again, how to measure and what to measure? Comp Cytogenet 7:1-9.). For a long time, these indexes were employed by many cytogeneticists and cytotaxonomists to discuss the taxonomic relationships among related species (Dematteis, 1998Dematteis M (1998) Karyotype analysis in some Vernonia species (Asteraceae) from South America. Caryologia 51:279-288.; D’Emerico et al., 1999D’Emerico S, Grünanger P, Scrugli A and Pignone D (1999) Karyomorphological parameters and C-bands distribution suggest phyletic relationship within the subtribe Limodorinae Bentham (Orchidaceae). Plant Syst Evol 217:147-161.; Selvi et al., 2006Selvi F, Coppi A and Bigazzi M (2006) Karyotype variation, evolution and phylogeny in Borago (Boraginaceae), with emphasis on subgenus Buglossites in the Corso-Sardinian system. Ann Bot 98:857-868.; Felix et al., 2007Felix WJP, Almeida A, de Melo NF and Felix LP (2007) Citogenética de duas espécies de Zephyranthes herb (Amaryllidaceae-Hipeastreae) cultivadas. Rev Bras Biocienc 5:294-296.; Peruzzi et al., 2009Peruzzi L, Leitch IJ and Caparelli KF (2009) Chromosome diversity and evolution in Liliaceae. Ann Bot 103:459-475.; Souza et al., 2010Souza LGR, Crosa O and Guerra M (2010) Karyological circumscription of Ipheion Rafinesque (Gilliesioideae, Alliaceae). Plant Syst Evol 287:119-127.). Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p. suggested that asymmetric karyotypes were originated from symmetrical ones, which has not been properly tested until now.
The existing indexes are separated into two groups: interchromosomal asymmetry indexes, which quantify the heterogeneity in chromosome size, and intrachromosomal asymmetry indexes, which quantify the relative differences in the centromere position among chromosomes of a complement (Stebbins, 1971Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p.; Peruzzi and Eroğlu, 2013Peruzzi L and Eroğlu HE (2013) Karyotype asymmetry: Again, how to measure and what to measure? Comp Cytogenet 7:1-9.). Among the interchromosomal asymmetry indexes, A2 (Romero-Zarco, 1986Romero-Zarco C (1986) A new method for estimating karyotype asymmetry. Taxon 35:526.) and CVCL (Paszko, 2006Paszko B (2006) A critical review and a new proposal of karyotype asymmetry indices. Plant Syst Evol 258:39-48.) are the most used due to their accuracy in the evaluation of chromosome dissimilarities (Chiarini and Barboza, 2008Chiarini FE and Barboza GE (2008) Karyological studies in Jaborosa (Solanaceae). Bot J Lin Soc 156:467-478.; Souza et al., 2010Souza LGR, Crosa O and Guerra M (2010) Karyological circumscription of Ipheion Rafinesque (Gilliesioideae, Alliaceae). Plant Syst Evol 287:119-127.; Pierozzi, 2011Pierozzi NI (2011) Karyotype and NOR-banding of mitotic chromosomes of some Vitis L. species. Rev Bras Frutic 33:564-570.; Alves et al., 2011Alves LIF, Lima SAA and Felix LP (2011) Chromosome characterization and variability in some Iridaceae from Northeastern Brazil. Genet Mol Biol 34:259-267.; Assis et al., 2013Assis FNM, Souza BCQ, Medeiros-Neto E, Pinheiro F, Silva AEB and Felix LP (2013) Karyology of the genus Epidendrum (Orchidaceae: Laeliinae) with emphasis on subgenus Amphiglottium and chromosome number variability in Epidendrum secundum. Bot J Linn Soc 172:329-344.). However, there is no consensus about the best method for calculating the intrachromosomal asymmetry (Romero-Zarco, 1986Romero-Zarco C (1986) A new method for estimating karyotype asymmetry. Taxon 35:526.; Peruzzi and Eroğlu, 2013Peruzzi L and Eroğlu HE (2013) Karyotype asymmetry: Again, how to measure and what to measure? Comp Cytogenet 7:1-9.).
Among the proposed intrachromosomal asymmetry indexes, the following stand out:
The four categories ofStebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p.: from A to D according to the proportion of acrocentric and/or telocentric chromosomes in a karyotype, i.e. proportion of chromosomes with a ratio between chromosome arms < 2:1. The four categories have subtypes 1 to 3 according to the ratio between the large/small chromosome arms, giving a total of 12 categories (Table 1).
Intrachromosomal asymmetry indexes. The total of 12 indexes is composted of the four categories of Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p. - A to D according 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.
The total form percentage (TF%; Huziwara, 1962Huziwara Y (1962) Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromosome of Aster. Amer J Bot 49:116-119.): ratio between the sum of the short arms (p) length and the sum of the total chromosomes length:
The karyotype asymmetry index percentage (Ask%; Arano, 1963Arano H (1963) Cytological studies in subfamily Carduoideae (Compositae) of Japan. IX. The karyotype analysis and phylogenic consideration of Pertya and Ainsliaea. Bot Mag 76:32-39.): ratio between the length of the long arms (q) of the chromosome set and the total length of the chromosome set:
The symmetric index (Syi; Greilhuber and Speta, 1976Greilhuber J and Speta F (1976) C-banded karyotypes in the Scilla hohenackeri group, S. persica, and Puschkinia (Liliaceae). Plant Syst Evol 126:149-188.): ratio between the average length of the short arms (p) and the average length of the long arms, multiplied by 100:
The intrachromosomal asymmetry index A1 (Romero-Zarco 1986Romero-Zarco C (1986) A new method for estimating karyotype asymmetry. Taxon 35:526.): sum of the ratio between the average length of the short arms in each homologous pairs (bi) and the average length of the long arms in each homologous pair (Bi) divided by the number of homologous chromosome pairs (n):
This proposal was later modified by Watanabe et al. (1999)Watanabe K, Yahara T, Denda T and Kosuge K (1999) Chromosomal evolution in the genus Brachyscome (Asteraceae, Astereae): Statistical tests regarding correlation between changes in karyotype and habit using phylogenetic information. J Plant Res 112:145-161., who created the asymmetry index A, and followed by Peruzzi and Eroğlu (2013)Peruzzi L and Eroğlu HE (2013) Karyotype asymmetry: Again, how to measure and what to measure? Comp Cytogenet 7:1-9., who called the same index MCA. This index results from the sum of the ratio between the differences in the long arm length (Bi) and the short arm length (bi) of each chromosome and the sum of the lengths of the long and short arms of each chromosome (Bi + bi). The sum is divided by the haploid chromosome number (n):
The coefficient of variation of the centromeric index CVCI (Paszko, 2006Paszko B (2006) A critical review and a new proposal of karyotype asymmetry indices. Plant Syst Evol 258:39-48.): based on the index of interchromosomal asymmetry A2 (standard deviation/total average of the chromosome length):
With so many ways to calculate the intrachromosomal asymmetry, Zuo and Yuan (2011)Zuo L and Yuan Q (2011) The difference between the heterogeneity of the centromeric index and intrachromosomal asymmetry. Plant Syst Evol 297:141-145. developed six models of ideal karyotypes to test the accuracy of these methods. These authors observed that the CVCI does not reflect the intrachromosomal asymmetry in karyotypes when it is composed by telocentric and/or acrocentric chromosomes. Since the standard deviation decreases in acrocentric or telocentric chromosomes, the CVCI is more suitable to indicate the heterogeneity of the centromeric index, i.e. how different is the position of the centromeres among the chromosomes of the complement (see Zuo and Yuan, 2011Zuo L and Yuan Q (2011) The difference between the heterogeneity of the centromeric index and intrachromosomal asymmetry. Plant Syst Evol 297:141-145.).
Peruzzi and Eroğlu (2013)Peruzzi L and Eroğlu HE (2013) Karyotype asymmetry: Again, how to measure and what to measure? Comp Cytogenet 7:1-9. discouraged the use of any intrachromosomal asymmetry index for karyotypes with small chromosomes (≤ 1 μm), due to the inaccuracy in the arms’ measurement. However, the mean chromosome size in plants is 1.5–2.0 μm. Moreover, if the suggestion of Peruzzi and Eroğlu (2013)Peruzzi L and Eroğlu HE (2013) Karyotype asymmetry: Again, how to measure and what to measure? Comp Cytogenet 7:1-9. is followed, the chromosome symmetry analysis will be prohibitive in a large number of plant species, including a great part of Bromeliaceae, Fabaceae and Orchidaceae species.
Considered one of the most diverse and taxonomically complex plant families among the angiosperms, Orchidaceae comprises 25,971 species with global distribution (Pridgeon et al., 1999Pridgeon AM, Cribb PJ, Chase MW and Rasmussen F (1999) Genera Orchidacearum, vol.1. Oxford University Press, Oxford, 240 p.; Joppa et al., 2011Joppa LN, Roberts DL and Pimm SL (2011) How many species of flowering plants are there? Proc R Soc Lond B Biol Sci 278:554-559.). The Orchidaceae present a large karyotype variation, with all types of chromosome morphology distributed in species with chromosome numbers varying from 2n = 12 in Erycina pusilla (L.) N.H.Williams & M.W.Chase (Felix and Guerra, 1999Felix LP and Guerra M (1999) Chromosome analysis in Psygmorchis pusilla (L.) Dodson et Dressler: The smallest chromosome number known in Orchidaceae. Caryologia 52:165-168.) to 2n = 240 in Epidendrum cinnabarinum Salzm. ex Lindl. (Guerra, 2000Guerra M (2000) Patterns of heterochromatin distribution in plant chromosomes. Genet Mol Biol 23:1029-1041.; Felix and Guerra, 2010Felix LP and Guerra M (2010) Variation in chromosome number and the basic number of subfamily Epidendroideae (Orchidaceae). Bot J Linn Soc 163:234-278.; Assis et al., 2013Assis FNM, Souza BCQ, Medeiros-Neto E, Pinheiro F, Silva AEB and Felix LP (2013) Karyology of the genus Epidendrum (Orchidaceae: Laeliinae) with emphasis on subgenus Amphiglottium and chromosome number variability in Epidendrum secundum. Bot J Linn Soc 172:329-344.). Except for the subfamily Cypripedioideae, Orchidaceae species are characterized by small chromosomes (Larsen, 1968Larsen K (1968) Brief notes on Neuwiedia singapureana in Thailand. Nat Hist Bull Siam Soc 22:330-331.; Okada, 1988Okada H (1988) Karyomorphological observations of Apostasia nuda and Neuwiedia veratrifolia (Apostasiaceae, Orchidaceae). Jpn J Bot 63:16-22.).
The wide karyotype diversity observed in Orchidaceae makes this plant family an excellent group for evaluating the applicability of karyotype asymmetry indexes, especially the intrachromosomal index. Thus, the present study aimed to compare different intrachromosomal asymmetry indexes, in order to indicate the most efficient for the estimation of karyotype asymmetry in orchids, including species with small chromosomes. Besides, we aimed to compare our results with the Orchidaceae phylogenetic proposal to test the hypothesis of Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p. that asymmetric karyotypes derived from symmetric ones.
Material and Methods
Chromosome measurements
A literature search was performed to select informative photographic records of metaphases quality – clear identification of centromere and secondary constrictions – and the available voucher (Table 2).
Karyomorphometric data for the representatives of the family Orchidaceae and respective references, with diploid chromosome number (2n), karyotype formula, chromosome size variation (μm), total sum of the haploid chromosome length (hcl), the interchromosomal index (Inter Index) by Romero-Zarco (A2), and the intrachromosomal indexes: Huziwara index (TF%), Arano index (Ask%), Greilhuber and Speta index (Syi), Romero-Zarco (A1) and Watanabe index (A). The bold and underlined numbers indicate the most symmetrical and asymmetric karyotypes for each index, respectively.
The arm ratio (r = length of the long arm/length of the short arm) was used to classify the chromosomes as metacentric (M: r = 1.00 to 1.49), submetacentric (S: r = 1.50 to 2.99), acrocentric (A: r ≥ 3.00) and telocentric (T: r = ∞), according to Guerra (1986)Guerra M (1986) Reviewing chromosome nomenclature of Levan et al. Rev Bras Gen 4:741-743.. We did not consider differences between acrocentric and telocentric chromosomes. For chromosome measurements we used Imagetool® software version 3.0 (available at http://compdent.uthscsa.edu/dig/itdesc.html) calibrated with scales available on the selected images.
The interchromosomal asymmetry was calculated using A2 by Romero-Zarco (1986)Romero-Zarco C (1986) A new method for estimating karyotype asymmetry. Taxon 35:526.. Five different indexes were estimated for the intrachromosomal asymmetry: TF% (Huziwara, 1962Huziwara Y (1962) Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromosome of Aster. Amer J Bot 49:116-119.); Ask% (Arano, 1963Arano H (1963) Cytological studies in subfamily Carduoideae (Compositae) of Japan. IX. The karyotype analysis and phylogenic consideration of Pertya and Ainsliaea. Bot Mag 76:32-39.); Syi (Greihuber and Speta, 1976); A1 (Romero-Zarco, 1986Romero-Zarco C (1986) A new method for estimating karyotype asymmetry. Taxon 35:526.); and A (Watanabe et al., 1999Watanabe K, Yahara T, Denda T and Kosuge K (1999) Chromosomal evolution in the genus Brachyscome (Asteraceae, Astereae): Statistical tests regarding correlation between changes in karyotype and habit using phylogenetic information. J Plant Res 112:145-161.). In addition, the ideal karyotypes (Zuo and Yuan, 2011Zuo L and Yuan Q (2011) The difference between the heterogeneity of the centromeric index and intrachromosomal asymmetry. Plant Syst Evol 297:141-145.) were used to establish a comparative standard for the analyzed karyotypes. The ideal karyotypes, ranked from A to F, present chromosome morphology based on Levan et al. (1964)Levan A, Fredgra K and Sandberg AA (1964) Nomenclature for centromeric position on chromosomes. Hereditas 52:201-220.. However, here we followed the nomenclature proposed by Guerra (1986)Guerra M (1986) Reviewing chromosome nomenclature of Levan et al. Rev Bras Gen 4:741-743. based on the percentage of acrocentric/telocentric chromosomes: (1) up to 30% (slightly asymmetric - SA); (2) from 31 to 50% (moderately asymmetric - MA) and (3) more than 50% (strongly asymmetric - StA).
Cluster analysis
The intrachromosomal index values were separately used for cluster analysis. The obtained values were categorized and used to define the limits of each category (Table S1). For the cluster analysis we used the UPGMA algorithm (unweighted pair-group method with arithmetic means) implemented in the software Mesquite® (Maddison and Maddison, 2015Maddison WP and Maddison DR (2015) Mesquite: A modular system for evolutionary analysis. Version 3.02, http://mesquiteproject.org (April 10, 2016).
http://mesquiteproject.org...
). Ten trees were generated for each index using the distances from the data matrix by majority consensus. Subsequently, only one consensus tree was stored. The software Dendroscope® (Huson and Scornavacca, 2012Huson DH and Scornavacca C (2012) Dendroscope 3: An interactive tool for rooted phylogenetic trees and networks. Syst Biol 61:1061-1067.) was used to root the tree with the most ideal symmetrical karyotype (karyotype A) as an outgroup, following the hypothesis of Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p..
Statistical analysis
To test the hypothesis of Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p., the mean values of the interchromosomal index A2 and the intrachromosomal asymmetry indexes were separately used, in order to compare the karyotype asymmetry levels for each subfamily. The variation between the mean values of asymmetry indexes for the subfamilies was compared statistically by ANOVA followed by Tukey's test using BioEstat v.5.3 (Ayres et al., 2007Ayres M, Ayres Jr M, Ayres DL and Santos AA (2007) BioEstat - Aplicações estatísticas nas áreas das ciências bio-médicas, version 5.3. Instituto de Desenvolvimento Sustentável Mamirauá, Tefé, Brazil.).
Results
Karyomorphometric analysis
The metaphases of 64 species, distributed throughout four subfamilies (Cypripedoideae, Epidendroideae, Orchidoideae and Vanilloideae) were selected across 16 references (Table 2). No karyotype analysis was found for Apostasioideae. Chromosome numbers ranged from 2n = 12 in Erycina pusilla (Epidendroideae) to 2n = 56 in Eurystyles actinosophila (Barb. Rodr.) Schltr (Orchidoideae). The Campylocentrum neglectum (Rchb.f. & Warm.) Cogn. (Epidendroideae) presented the smallest chromosomes, ranging from 0.62 μm to 1.20 μm, while Paphiopedilum randsii Fowlie (Cypripedioideae) presented the largest chromosomes, ranging from 10.74 μm to 22.48 μm (Table 2). The total sum of haploid chromosome length (hcl) ranged from 27.78 μm in Pabstiella fusca (Lindl.) Chiron & Xim.Bols. (Epidendroideae) to 395.88 μm in Paphiopedilum dianthum Tang & F.T.Wang (Cypripedioideae) (Table 2).
Regarding interchromosomal asymmetry (A2), the most symmetric karyotype was found in Christensonella pumila (Hook.) Szlach. (A2 = 0.12), while the most asymmetrical were found in Cephalanthera damasonium (Mill.) Druce and Pteroglossa lurida (M.N.Correa) Garay (A2 = 0.60; see Table 2). However, when comparing the subfamilies, the statistical test failed to show any difference (F = 1.6526, p = 0.1988; Figure 1).
Interchromosomal index A2 values for Orchidaceae subfamily. For each subfamily the mean value (dot), the amplitude of variation (bar), the number of species analyzed and mode value (last two data in the parenthesis, respectively) are presented.
The five tested intrachromosomal asymmetry indexes provided the same result: the most symmetric karyotype was found in Sarcoglottis grandiflora (Hook.) Klotzsch (44M + 2S; see bold numbers in Table 2) and the most asymmetric karyotype was found in Christensonella pachyphylla (Schltr. ex Hoehne) Szlach., (20S + 18A; see underlined numbers in Table 2).
Cluster analysis for intrachromosomal indexes
Cluster analysis using the values found for TF% grouped most species with the ideal karyotype C, the Sarcoglottis grandiflora with the ideal karyotype B and Christensonella pachyphylla with the ideal karyotype D (Figure S1). Ask% and A1 indexes presented identical trees (Figure S2) in the cluster analysis, with most species grouped with ideal karyotype B, plus a clade, separated into two groups: (1) a polytomy with the ideal karyotypes D, E and F and (2) a group with Christensonella pachyphylla, C. subulata (Lindl.) Szlach. and the ideal karyotype C. The cluster analysis for A and Syi also formed identical trees (Figure S3), similar to trees obtained with Ask% and A1 (Figure S2). A difference was found with Christensonella subulata, which was grouped with most species and the ideal karyotype B (instead of C). The indexes Ask% and A1 (Figure S2), and Syi and A (Figure S3) grouped Sarcoglottis grandiflora as a sister group of karyotype A. The indexes Ask%, A1, Syi and A provided more consistent groups (Figure 2), reflecting the species karyotype composition in the ideal karyotypes, as proposed by Zuo and Yuan (2011)Zuo L and Yuan Q (2011) The difference between the heterogeneity of the centromeric index and intrachromosomal asymmetry. Plant Syst Evol 297:141-145..
Ideograms of the ideal karyotypes A, B and C, as well as the most similar species, grouped by UPGMA, equally obtained by the indexes Ask%, A1, Syi and A. The numeric scale at the right side of the ideogram is given in micrometers (μm).
Comparing the four most congruent intrachromosomal indexes, Ask%, A1, Syi and A, with the current proposed Orchidaceae phylogeny (Chase et al., 2015Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, van den Berg C and Schuiteman A (2015) An updated classification of Orchidaceae. Bot J Linn Soc 177:151-174.), all indexes presented similar mean and mode values for Orchidoideae and Cypripedioideae (Figure 3). Index A did not detected a difference among subfamilies, after Tuckey's test (despite the F = 4.1420, p = 0.0203). The indexes Ask% and Syi indicated that karyotypes from Epidendroideae and Orchidoideae are more asymmetrical than Cypripedioideae - the most basal subfamily among the three (F = 4.4915 and 4.7008, respectively; p = 0.01 for both indexes). The A1 index suggested Epidendroideae as the most asymmetric karyotype among subfamilies (F = 5.77, p = 0.0054).
Intrachromosomal asymmetry values obtained by Ask% (blue), A1 (red), A (green) and Syi (orange) indexes for the Orchidaceae subfamily. The numeric scale at the right side indicates the mean value for the four intrachromosomal indexes. The Syi value was divided by 100. Subfamilies indicated by the same letters are not significantly different (Tukey test, p < 0.05).
Discussion
The inter and intrachromosomal asymmetry values observed here corroborate previous studies, with a slight variation for some species, such as Epidendrum paniculatum Ruiz & Pav., E. fulgens Brongn. (Assis et al., 2013Assis FNM, Souza BCQ, Medeiros-Neto E, Pinheiro F, Silva AEB and Felix LP (2013) Karyology of the genus Epidendrum (Orchidaceae: Laeliinae) with emphasis on subgenus Amphiglottium and chromosome number variability in Epidendrum secundum. Bot J Linn Soc 172:329-344.), Cyclopogon calophyllus (Barb.Rodr.) Barb.Rodr. and C. elatus (Sw.) Schltr. (Grabiele et al., 2013Grabiele M, Cerutti JC, Hojsgaard DH, Almada RD, Daviña JR and Honfi AI (2013) Comparative cytogenetics in Cyclopogon (Orchidaceae). Biologia 68:48-54.). Therefore, we can observe that the relationship between the two kinds of asymmetry (intra and interchromosomal) is not always unidirectional, but it is a result of complex rearrangements that modify both the centromere position and the chromosome size in a karyotype.
The interchromosomal index
The A2 index employed here yielded values close to zero for some species, mainly in the subfamilies Epidendroideae and Orchidoideae. In such cases, the index reflects a conservation among chromosome size in the karyotype; other species, however, presented high A2 values.
The highly asymmetric karyotypes could be the result of chromosome rearrangements, what could also cause bimodality, as observed in Cephalanthera damasonium (Epidendroideae; Moscone et al., 2007Moscone EA, Samuel R, Schwarzacher T, Schweizer D and Pedrosa-Harand A (2007) Complex rearrangements are involved in Cephalanthera (Orchidaceae) chromosome evolution. Chromosome Res 15:931-943.) and Pteroglossa lurida (Orchidoideae; Martinez 1984Martinez A (1984) The chromosomes of orchids, vol. 3, Spiranthinae and Cranichidinae. Kew Bulletin 40:139-147.), both with A2 = 0.60. The origin of bimodal karyotypes could be due to the loss of chromosome segments after polyploidy, resulting in the formation of smaller chromosomes (Weiss-Schneeweiss and Schneeweiss, 2013Weiss-Schneeweiss H and Schneeweiss G (2013) Karyotype diversity and evolutionary trends in Angiosperms. In: Greilhuber J, Dolezel J and Wendel JF (eds) Plant Genome Diversity vol. 2. Springer, Vienna, pp 209-230.), or due to unequal translocations (Stebbins, 1971Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p.), differential amplification of heterochromatic regions (de la Herrán et al., 2001de la Herrán R, Robles F, Cuñado N, Santos JL, Ruiz-Rejón M, Garrido-Ramos MA and Réjon CR (2001) A heterochromatic satellite DNA is highly amplified in a single chromosome of Muscari (Hyacinthaceae). Chromosoma 110:197-202.), or even in the hybridization between species with different chromosome sizes. All these events increase the interchromosomal asymmetry by increasing the morphological discontinuities between chromosomes in a karyotype.
The intrachromosomal indexes
Regarding the intrachromosomal asymmetry, we showed that Orchidaceae karyotypes ranged from slightly asymmetric to moderately asymmetric. The intrachromosomal asymmetry is defined by the presence of a greater number of acrocentric/telocentric chromosomes in relation to the metacentric and submetacentric ones, a consequence of changes in centromere position (Stebbins, 1971Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p.) – in which case the chromosome rearrangement could affect all chromosomes in the same way and even increase the karyotype asymmetry. Therefore, the efficacy of the intrachromosomal asymmetry indexes is dependent on the precise identification of the centromere and a well-defined chromosome morphology, and not on chromosome size. The indexes Ask% and A1 proved to be more useful in determining the intrachromosomal asymmetry, even in species with small chromosomes, like Campylocentrum neglectum.
The extreme symmetry (ideal karyotype A) or the extreme asymmetry (ideal karyotype F) karyotypes are hardly found in nature. However, in the present analysis, an extreme of symmetric karyotype was found in Sarcoglottis grandiflora, grouped with the ideal karyotype A. Christensonella pachyphylla showed the most asymmetric karyotype, but this species was grouped with ideal karyotypes C and D and not with the extreme ideal karyotype F.
The occurrence of asymmetric karyotypes is probably a consequence of chromosomal structural changes, especially centric fusion/fission, a very common rearrangement in Orchidaceae (Pinheiro et al., 2009Pinheiro F, Koehler S, Corrêa AM, Salatino MLF, Salatino A and Barros F (2009) Phylogenetic relationships and infrageneric classification of Epidendrum subgenus Amphiglottium (Laeliinae, Orchidaceae). Plant Syst Evol 283:165-177.; Yamagishi-Costa and Forni-Martins, 2009Yamagishi-Costa J and Forni-Martins E (2009) Hybridization and polyploidy: Cytogenetic indications for Hoffmannseggella (Orchidaceae) species evolution. Int J Bot 5:93-99.; Assis et al., 2013Assis FNM, Souza BCQ, Medeiros-Neto E, Pinheiro F, Silva AEB and Felix LP (2013) Karyology of the genus Epidendrum (Orchidaceae: Laeliinae) with emphasis on subgenus Amphiglottium and chromosome number variability in Epidendrum secundum. Bot J Linn Soc 172:329-344.; Moraes et al., 2012Moraes AP, Leitch IJ and Leitch AR (2012) Chromosome studies in Orchidaceae: Karyotype divergence in Neotropical genera in subtribe Maxillariinae. Bot J Linn Soc 170:29-39., 2013Moraes AP, Chinaglia M, Palma-Silva C and Pinheiro F (2013) Interploidy hybridization in sympatric zones: The formation of Epidendrum fulgens × E. puniceoluteum hybrids (Epidendroideae, Orchidaceae). Ecol Evol 3:3824-3837.; 2016Moraes AP, Simões AO, Alayon DIO, Barros F and Forni-Martins ER (2016) Detecting mechanisms of karyotype evolution in Heterotaxis (Orchidaceae). PLoS One 11:e0165960.). Christensonella Szlach. presents a dysploidy variation (2n = 36 and 38) and the occurrence of centric fusion/fission is suggested as the main cause. This is illustrated by the double DAPI+ band holding the centromere in C. fernandiana, as the remainder species present just one DAPI+ band (Koehler et al., 2008Koehler S, Cabral JS, Whitten WM, Williams NH, Singer RB, Neubig KM, Guerra M, Souza AP and Amaral MCE (2008) Molecular phylogeny of the Neotropical genus Christensonella (Orchidaceae, Maxillariinae): Species delimitation and insights into chromosome evolution. Ann Bot 102:491-507.; Moraes et al., 2012Moraes AP, Leitch IJ and Leitch AR (2012) Chromosome studies in Orchidaceae: Karyotype divergence in Neotropical genera in subtribe Maxillariinae. Bot J Linn Soc 170:29-39.). The centric fusion/fission is suggested to be the cause of the frequent dysploidy in other genera in subtribe Maxillariinae (Moraes et al., 2012Moraes AP, Leitch IJ and Leitch AR (2012) Chromosome studies in Orchidaceae: Karyotype divergence in Neotropical genera in subtribe Maxillariinae. Bot J Linn Soc 170:29-39.; 2016Moraes AP, Simões AO, Alayon DIO, Barros F and Forni-Martins ER (2016) Detecting mechanisms of karyotype evolution in Heterotaxis (Orchidaceae). PLoS One 11:e0165960.) and Orchidaceae (Pinheiro et al., 2009Pinheiro F, Koehler S, Corrêa AM, Salatino MLF, Salatino A and Barros F (2009) Phylogenetic relationships and infrageneric classification of Epidendrum subgenus Amphiglottium (Laeliinae, Orchidaceae). Plant Syst Evol 283:165-177.; Felix and Guerra, 2010Felix LP and Guerra M (2010) Variation in chromosome number and the basic number of subfamily Epidendroideae (Orchidaceae). Bot J Linn Soc 163:234-278.; Assis et al., 2013Assis FNM, Souza BCQ, Medeiros-Neto E, Pinheiro F, Silva AEB and Felix LP (2013) Karyology of the genus Epidendrum (Orchidaceae: Laeliinae) with emphasis on subgenus Amphiglottium and chromosome number variability in Epidendrum secundum. Bot J Linn Soc 172:329-344.). The same can be detected in other plant groups as the genus Tristagma Poepp. (as Ipheion Raf., Souza et al., 2010Souza LGR, Crosa O and Guerra M (2010) Karyological circumscription of Ipheion Rafinesque (Gilliesioideae, Alliaceae). Plant Syst Evol 287:119-127.): Tristagma tweedieanum (Baker) Traub (2n = 14, karyotypic formula 14A, A1 = 0.86) and T. uniflorum (Lindl.) Traub (2n = 12, karyotypic formula = 2SM + 10A, A1 = 0.78), family Iridaceae (Goldblatt, 1990Goldblatt P (1990) Phylogeny and classification of Iridaceae. Ann Missouri Bot Gard 77:607-627.; Alves et al., 2011Alves LIF, Lima SAA and Felix LP (2011) Chromosome characterization and variability in some Iridaceae from Northeastern Brazil. Genet Mol Biol 34:259-267.; Moraes et al., 2015Moraes AP, Souza-Chies TT, Stiehl-Alves EM, Piccolli P, Eggers L, Yakolev SS, Brown SC, Chaveau O, Nador S, Bourge M, et al. (2015) Evolutionary trends in Iridaceae: New cytogenetic findings from the New World. Bot J Linn Soc 177:27-49.), Asteraceae (Brachyscome Cass.; Watanabe et al., 1999Watanabe K, Yahara T, Denda T and Kosuge K (1999) Chromosomal evolution in the genus Brachyscome (Asteraceae, Astereae): Statistical tests regarding correlation between changes in karyotype and habit using phylogenetic information. J Plant Res 112:145-161.) and Sapindaceae (Serjania Mill.; Coulleri et al., 2012Coulleri JP, Dematteis M and Ferrucci MS (2012) A new insight into Serjania Mill. (Sapindaceae, Paullinieae) infrageneric classification: A cytogenetic approach. Plant Syst Evol 298:1743-1753.).
The Stebbins’ hypothesis
The relationship between karyotype asymmetry and species evolution could be discussed based on intrachromosomal indexes, since the interchromosomal index does not differ among subfamilies. The intrachromosomal asymmetry indexes indicated the karyotypes of some representatives of the subfamily Epidendroideae as the most asymmetric – in agreement with the hypothesis of Stebbins (1971)Stebbins GL (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London, 216 p. that asymmetric karyotypes had been originated from symmetrical ones. Based on the statistical results and cluster analysis the congruent indexes Ask%, A1 and Syi indicated Epidendroideae as the most derivate subfamily, presenting the most asymmetrical karyotype, while the representatives of the subfamily Cypripedioideae have more symmetrical karyotypes.
Conclusions
Considering our results, the indexes Ask% (Arano, 1963Arano H (1963) Cytological studies in subfamily Carduoideae (Compositae) of Japan. IX. The karyotype analysis and phylogenic consideration of Pertya and Ainsliaea. Bot Mag 76:32-39.), A1 (Romero-Zarco, 1986Romero-Zarco C (1986) A new method for estimating karyotype asymmetry. Taxon 35:526.) and Syi are recommended for the estimation of intrachromosomal asymmetry in cytotaxonomic studies, especially in a combined fashion. We showed that the critical point for the efficacy of an asymmetric index is the well-preserved chromosome morphology and precise definition of the centromere position – and not the size of chromosomes. Moreover, the higher karyotype asymmetry associated with the derivative subfamily Epidendroideae supports Stebbins’ hypothesis that asymmetric karyotypes tend to derive from symmetric karyotypes.
Acknowledgments
The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Proc. 400209/2014-4 and Proc. 460990/2014-4), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) and Fundação de Apoio à Pesquisa de São Paulo (FAPESP 2011/22215-3) for financial support and research grants, and Instituto Nacional do Semiárido (INSA) for technical support.
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Supplementary Material
The following online material is available for this article:
Table S1 - Asymmetry values for ideal karyotypes.
Figure S1 - UPGMA analysis using the intrachromosomal asymmetry values from TF% index.
Figure S2 - UPGMA analysis using the intrachromosomal asymmetry values from Ask% and A1 indexes.
Figure S3 - UPGMA analysis using the intrachromosomal asymmetry values from Syi and A indexes.
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Associate Editor: Marcelo Guerra
Publication Dates
-
Publication in this collection
22 June 2017 -
Date of issue
Jul-Sep 2017
History
-
Received
03 Oct 2016 -
Accepted
19 Dec 2016