A critical review and a new proposal of karyotype asymmetry indices

B. Paszko

W. Szafer Institute of Botany, Polish Academy of Sciences, Krakow, Poland

Received March 22, 2005; accepted September 19, 2005Published online: March 8, 2006 Springer-Verlag 2006

Abstract. In literature seven dierent methods ofevaluating karyotype asymmetry the TF%, theAsK%, Stebbins classication, the Rec and the Syi,theA1 and theA2, theDI, and theA are used for theelucidation of phylogenetic relationships and taxo-nomic treatmentswithin a particular groupor taxon.The investigation of these sevenmethods reveals thatthe intervals used by Stebbins to separate thedierent types of karyotype asymmetry are verybroad and only one quantitative parameter, the A2index, correctly describes the variation in chromo-some length in a complement. A new asymmetryindex (AI) is proposed to measure karyotype asym-metry and a newparameter, theCVCI, is oered, thatprecisely assesses the relative variation in centromereposition in a complement. The AI index, the CVCIand the CVCL (=A2 100) have the potential todisplay even minor karyotypic variations. Thus,these three indices together increase the precision ofresults in comparison with other existing methods.All this has important consequences as regardsthe interpretation of the results of karyologicalstudies.

Key words: Asymmetry index, chromosomes,karyotype morphology.

Introduction

Stebbins (1971), writing about karyotypeasymmetry, referred to the Russian school of

comparative karyotype morphology, led byG. Levitsky (1931), who developed the conceptof its symmetry vs. asymmetry. A symmetricalkaryotype is characterised by the predomi-nance of m and sm chromosomes of approx-imately the same size. Increasing asymmetrycan occur either through the shift of centro-mere position from median/submedian toterminal or subterminal, or through the accu-mulation of dierences in the relative sizebetween the chromosomes of the complement,thus making the karyotype more heteroge-neous. These two tendencies are not correlatedwith one other, though they may be in somegroups (Stebbins 1971).

Stebbins (1971) distinguished twelve cate-gories with respect to karyotype asymmetry,only ten of which were known to occur inhigher plants. He established these by recog-nising three degrees of dierence (AC)between the largest and smallest chromosomeof the complement, and four degrees (14) withrespect to the proportion of chromosomeswhich are metacentric with an arm ratio of lessthan 2:1 (Table 1).

The classication of Stebbins (1971) is themost frequently used qualitative method forassessing karyotype-symmetry conditions anddescribing the karyotypic relationshipsbetween dierent taxa (e.g. Lathyrus, Seijo

Pl. Syst. Evol. 258: 3948 (2006)DOI 10.1007/s00606-005-0389-2

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and Fernandez 2003; Ophrys, Bernardos et al.2003; between Davidia involucrata and Camp-totheca acuminata, He et al. 2004).

The total form percent (TF%), was de-scribed by Huziwara (1962) to analyse thekaryotypes in the genus Aster. The TF% indexis expressed by the ratio of the sum of thelengths of the short arms of individual chro-mosomes to the total haploid length of thecomplement:

TF%

Length of short arms in chromosome setTotal chromosome length in set

100:

The TF% index has frequently been used todescribe karyotype asymmetry and to deter-mine the karyotypic relationships betweenspecies of genera (e.g. Phaseolus, Mercado-Ruaro and Delgado-Salinas 1998; Mikania,Ruas et al. 2000; in the Alismataceae family,Costa and Forni-Martins 2003).

Arano (1963) introduced another karyo-type asymmetry index, the As K%, which wasused to determine the phylogenetic relationsbetween and within the genera Pertya andAinsliaea. The As K% index is expressed by theratio of the sum of the lengths of the long armsof individual chromosomes to the total haploidlength of the chromosome complement:

As K%

Length of long arms in chromosome setTotal chromosome length in set

100:

The As K% index was used subsequently toanalyse chromosome evolution between spe-cies in the Umbelliferae family (Arano andSaito 1980), as well as between species ofHypochaeris (Weiss-Schneeweiss et al. 2003),

Armeria (Coulaud et al. 1999), Metagentiana(Ho et al. 2002), Solms-laubachia, and tworelated genera (Yue et al. 2004).

Greilhuber and Speta (1976) developed twoindices, the index of karyotype symmetry andthe index of chromosomal size resemblance, toevaluate karyotype asymmetry. These two indi-ces were later called by Venora et al. (2002) theSyi index and the Rec index, respectively. TheSyi value indicates the ratio of the mean lengthof the short arms against the mean length of thelong arms in a chromosome set. The Rec indexexpresses the mean of the ratios of the length ofeach chromosome (CLi) to that of the longestone (LC):

Syi Mean length of the short armsMean length of the long arms

100;

Rec Pn

i1CLiLC

n 100;

where n is the number of analysed chromo-somes. Both indices have been used to estimatekaryotype asymmetry and to discuss therelationships between species of Scilla andPuschkinia (Greilhuber and Speta 1976), andTriticum (Venora et al. 2002) only.

Romero Zarco (1986) provided an alterna-tive method for measuring karyotype asym-metry by using quantication and graphicrepresentation. He proposed two numericalparameters to estimate karyotype asymmetry.The rst was named the intrachromosomalasymmetry index (A1) and the second theinterchromosomal asymmetry index (A2).

The intrachromosomal asymmetry index(A1), ranging from 0 to 1, can be calculated forevery sample using the following equation:

Table 1. The classication of karyotypes in relation to their degree of asymmetry according to Stebbins(1971)

Ratio Proportion of chromosomes with arm ratio

A1 1Pn

i1qipi

n;

where qi is the mean length for short, and pi forlong arms in every homologous chromosomepair or group; n is the number of homologouschromosome pairs or groups. The interchro-mosomal asymmetry index (A2) is the ratiobetween the standard deviation (sCL) and themean chromosome length (xCL):

A2 sCLxCL :

Both Romero Zarco indices tend to be themost frequently used estimates of karyotypeasymmetry between dierent taxa (e.g. Lathy-rus, Seijo and Fernandez 2003; Ligularia, Liu2004; Mediterranean orchids, Cozzolino et al.2004).

Mugnier and Siljak-Yakovlev (1987) usedthe asymmetry index (AsI) which is probably asynonym of the As K% (Arano 1963), as bothare expressed by the ratio between the sum ofthe lengths of the long arms of individualchromosomes and the sum of chromosomelength in its set. Two years later Barghi et al.(1989), using the AsI index, referred to Aranoand Saito (1980). The AsI index was used toanalyse chromosome evolution among speciesof Mikania (Ruas and Aguiar-Perecin 1997).

Lavania and Srivastava (1992) introducedanother chromosomal parameter they calledthe dispersion index (DI), which is the pro-portionate measure of the centromeric gradi-ent (CG) with respect to the coecient ofvariation of chromosome length (CV), calcu-lated from the following equations:

CG Median length of short armMedian length of chromosome

100;

CV sCLxCL

100;

DI CG CV100

;

where sCL is the standard deviation of chro-mosome length, and xCL is the mean chromo-some length. The DI index was used to discussphylogenetic dierentiation and origin in the

genus Papaver (Lavania and Srivastava 1992,1999).

Watanabe et al. (1999) dened degree ofasymmetry of karyotype (A) as:

A Pn

i1piqipiqi

n;

where p and q are the lengths of a long armand a short arm of chromosome i, respectively,in a cell, and n is the haploid chromosomenumber of an individual or taxon.

The karyotype asymmetry index is a goodexpression of the general morphology of plantchromosomes. It would therefore be advanta-geous to have a uniform system whereby thekaryotypes of related genotypes and speciescould be compared. As shown above, scientistshave developed to date, a variety of methodsfor assessing and analysing karyotype asym-metry in a chromosome set. The primary aimof this study was to discuss the validity andsensitivity of these methods, which are used toassess karyotype asymmetry between andwithin dierent taxa.

Materials and methods

Eight accessions of ve Calamagrostis Adanson(Poaceae) taxa were collected (Table 2). Thevoucher specimens are deposited in the herbariumof the W. Szafer Institute of Botany PAS inKrakow (KRAM). The root tips were stainedfollowing the Feulgen method. No less than vecells per individual and ve plants per species wereexamined. Idiograms were drawn using a computerprogram Mr. Karyo (A. Joachimiak, Institute ofBotany, Jagiellonian University, Krakow).

For the numerical characterisation of thekaryotypes the following parameters were calcu-lated: (1) shortest (SC) and longest (LC) chromo-some length; (2) ratio of longest to shortestchromosome (LC/SC); (3) mean long arm length(p); (4) mean and median of short (q) and of totalchromosome length (CL); (5) percentage of chro-mosomes with an arm ratio of less than 2:1; (6)mean centromeric index (CI 100 length of shortarm/total chromosome length); and (7) karyotypeformula (Table 3).

Seven dierent methods were used to assess thedegree of karyotype asymmetry (Table 4). The

B. Paszko: A critical review and a new proposal of karyotype asymmetry indices 41

strength of the association between karyotypeasymmetry indices (excluding the degrees of Steb-bins (1971)) was tested using Pearson correlationanalysis (Table 5). Coecients of variation (CVCL,CVCI) were calculated by dividing the standarddeviations by the means (Table 6) (Sokal and Rolf1981). Cluster analysis using the Euclidean distancewas employed to compare dierent data, andaverage linkage methods of hierarchical clusteranalysis were used for clustering on standardiseddata. Statistical evaluation was carried out usingthe Statistica 6.1 software package (StatSoft 1995).

Results and discussion

This study is based on the eight accessions ofCalamagrostis (Table 2). Karyotype formulaeobtained and the parameters analysed aresummarized in Table 3. The respective idio-grams (Fig. 1) are based on mean valuespresented in Table 3. Accessions of Calama-grostis arundinacea (A-55) and C. villosa (vi-58) exhibited the most variation in chromo-some length, but only C. villosa (vi-58) ischaracterised by the highest level of variationof the centromeric index (Table 3; Fig. 1).

The karyotype asymmetry was assessed,based on seven dierent methods (Table 4).Among these methods, one qualitative classi-cation and eight dierent quantitative indicescan be marked out. The analysis of indexformulae and the association between quanti-tative indices and three karyotype characteris-

tics (LC/SC, CL, CI) reveals three groupsamong them (Table 5). Five indices theTF%, the Syi, the As K%, the A1, and the A have been formulated so far by dierentresearchers to evaluate the variation in centro-mere position in a chromosome complement.The As K% index has a perfect negativecorrelation with two indices: the TF% andthe Syi. None of these ve indices is either arelative standard deviation (RSD) or a coe-cient of variation (CV), and for that reason allof them inadequately demonstrate relation-ships between studied accessions.

Two indices, the Rec and the A2, werecreated to assess the variation in chromosomelength in a complement. The A2 index is arelative standard deviation of chromosomelength, and from the statistical point of viewit is a sensible parameter, which adequatelyassesses the relative variation in chromosomelength in a complement. The Rec index is awrong parameter and does not reect relation-ships between karyotypes too closely or at all.

The DI index was developed by Lavaniaand Srivastava (1992) in order to give a singlevalue that evaluated the karyotype asymmetry.It was the rst attempt to create one karyotypeasymmetry index, but one of two parametersused, the centromeric gradient (CG), cannotcorrectly assess the variation in centromericposition. As a result, the DI index is unable toevaluate karyotype asymmetry.

Table 2. Taxa, accessions, chromosome numbers, ploidy level, and collection number of the examinedindividuals from the genus Calamagrostis

Taxon Accession Chromosomenumber (2n)

Ploidylevel

Vouchernumber (in KRAM)

C. arundinacea (L.) Roth A-55 28 4 525974-977C. canescens (Weber) Roth C-57 28 4 525964-968C. epigejos (L.) Roth E-00 28 4 525946-947

E-01 28 4 525948-952C. villosa (Chaix ex Villars)J. F. Gmelin

vi-58 70 10 525935-940

C. hartmaniana ha-40 28 4 525953-957[C. arundinacea (L.) Roth ha-41 28 4 525958-963C. canescens (Weber) Roth] ha-56 28 4 525969-973

42 B. Paszko: A critical review and a new proposal of karyotype asymmetry indices

According to Levitsky (1931) and Stebbins(1971), the karyotype asymmetry of a comple-ment is determined by the variation inchromosome length and the variation in cen-tromere position. Only four methods Steb-bins classication (1971), Rec and Syi indices(Greilhuber and Speta 1976), Romero Zarco(1986) indices (A1 and A2), and the dispersionindex (DI) (Lavania and Srivastava 1992) usea combination of both types of variation thataect karyotype asymmetry. Relationshipsbetween Calamagrostis accessions based onthese four methods are shown in Figs 2, 3, and5. The remaining methods, the TF% (Huziw-ara 1962), the As K% (Arano 1963), and the Aindex (Watanabe et al. 1999), try to describeonly the variation in centromere position in achromosome complement and they have aperfect or almost perfect positive or negativecorrelation with the Syi index (Table 5).

The relative variation in centromere posi-tion in a chromosome set can be assesseddirectly on the basis of the coecient ofvariation for the centromeric index (CVCI).The Calamagrostis villosa (vi-58) is character-ised by the highest value of CVCI, thenfollowed by C. hartmaniana (ha-56) andC. arundinacea (A-55), which have lower val-ues of CVCI. Remaining samples are charac-terised by much lower values of CVCI (Table 6,Fig. 4). All these do not correspond withclusters in the dendrogram based on veindices which try to describe the variation incentromere position (the TF%, the Syi, theAs K%, the A1, and A) (Fig. 6). These veindices are parameters incapable of describingthe variation in centromere position and theydo not reect the real variation in centromereposition among the Calamagrostis accessionsstudied.

The CVCI parameter is evaluated as theratio between the standard deviation (sCI) andthe mean centromeric index (xCI):

CVCI sCIxCI 100:

The relative variation in chromosome length(CVCI A2 100) in a complement is evalu-Ta

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