Silvae Genetica 61, 4–5 (2012) 179

Abstract

The description of the karyotype features and idio-gram in Grindelia squarrosa (Pursh) Dunal (Aster-aceae), an invasive plant in Romania, are reported herefor the first time. The diploid chromosome number is2n=2x=12, in agreement with the data published forthe other species of the genus. The karyomorphologicaldata show that the complements of the studied geno-types have small chromosomes (mean chromosomelength is X

–±SE=2.56±0.10 µm, and mean length of

haploid complements is X–±SE=15.33±0.69 µm, with a

range of variability comprised between 12.87–17.51 µm).The karyotypes are made up of six pairs of metacentricand submetacentric chromosomes, with an identical formula of the diploid complement: KF=2n=12=8m+2sm + 2sm-SAT. Satellites are located on the short arms of the chromosomes of pair III. The karyotypesshow a relatively high level of intra-specific uniformityas well as similar symmetry patterns (R=1.29–1.53;TF%=38.78–41.57%; AsI%=54.54–57.61%; A1 = 0.24–0.32; A2=0.08–0.16), belonging to 1A and 2A classes ofsymmetry. The small size of the chromosomes, the pres-ence of only two chromosome morphometric types, andthe preponderance of metacentrics confer a relativelyhigh degree of symmetry to the karyotypes studied.

Key words: Grindelia squarrosa, invasive plant, karyotype,mitotic chromosomes, satellites, asymmetry indexes.

Introduction

Grindelia Willd. is a genus with ca. 45 species inNorth America and 26 species in South America, com-prising annual, biennial, perennial forbs or subshrubswidely distributed in xerophytic and halophytic areas(BAEZA and SCHRADER, 2005; DEBLE and OLIVEIRA-DEBLE,2010). In 1804, Grindelia seeds from Mexico werebrought to Europe (Royal Gardens – Madrid, Spain) andcultivated as Aster spathulatus Hort.; afterwards theywere distributed to other botanical gardens (STEYER-MARK, 1937).

The curlycup gumweed – Grindelia squarrosa (Pursh)Dunal (BRUMMITT and POWELL, 1992), a common weedoriginating in the central prairies of North America isnow largely spread over Eastern, Central and Western

Europe (Russia, the Ukraine, Republic of Moldova, Esto-nia, Lithuania, Czech Republic, Belgium, Sweden,Latvia, Ireland) (SIRBU and OPREA, 2008). Although thespecies is most common in the lower elevations of plainsand foothills, it was met, too, at 3000 m altitude in Col-orado and New Mexico. In Northern Utah, it occurs inTony Grove Canyon (2000 m) in the Cache National For-est, also on disturbed ground throughout the valleys andin the adjacent Wasatch Mountains at elevations of atleast 2200 m (MCDONOUGH, 1975; WALSH, 1993). Report-ed for the first time in the flora of Romania in 1998(SIRBU and OPREA, 1998), G. squarrosa can be consideredan invasive alien plant in this country. Probably, theplant came into Romania by accident from the formerUSSR, carried across by goods or passenger trains. Itwas first identified in the ruderal areas on the side-tracks of the Socola-Iasi railway station. Meanwhile itinvaded anthropic habitats both in the North-East andthe South of the Moldavian Region of Romania (SIRBU

and OPREA, 2011). At present, G. squarrosa is consideredas being fully naturalized in Romania, it having an evi-dent invasive tendency. Although in Romania the inva-sion of this species into agricultural crops or nativeplant communities has not occurred yet, this trend isnot excluded in the future, given its behaviour in theneighbouring countries (SIRBU and OPREA, 2008, 2011).In the Ukraine and the Republic of Moldova, G. squar-rosa is considered as a very aggressive plant, while forSpain it is noted as potentially invasive (SANZ ELORZA etal., 2001).

The invasive alien species, including curlycupgumweed, are seen as a major threat to the native biodi-versity, ecosystem structure and conservation of the pro-tected areas, thus causing damages to agriculture,forestry, fisheries and other human activities, andthreatening human health (STINSON et al., 2006). Exceptthe aspect of its invasiveness – sometimes seen as harm-ful – G. squarrosa is one of the only two officinal plantsin the genus Grindelia (the other is G. robusta) (GHEDIRA et al., 2010). The chemical profile, representedby diterpenes (grindelic acid and its methylesthers),flavonoids (quercetin, kempferol), tannins (5.3%), vita-min P, resin, phenolic acids, and essential oils(0.3–0.5%), confers numerous medical and pharmaceuti-cal valences to plant extracts and bio-preparations.Because of this complex chemical constitution, theextracts are valuable as stimulants, sedatives, astrin-gents, purgatives, emetics, diuretics, antiseptics, anddisinfectants and are used in the treatment of bronchialspasm, whooping cough, asthma, and rashes caused bypoison ivy (Toxicodendron radicans L. Kunze), while thetinctures are useful for bladder and urethra infections(JOHNSON and NICHOLS, 1970; BARE, 1979).

Karyotype traits in Grindelia squarrosa (Pursh) Dunal (Asteraceae), an invasive plant in Romania

By ELENA TRUTA1),*), GABRIELA VOCHITA1), ADRIAN OPREA2) and CULITA SIRBU3)

(Received 3rd February 2012)

1) NIRDBS – Institute of Biological Research, Department of CellBiology and Microbiology, 47 Lascar Catargi Street, 700107Iasi, Romania.

2) Botanical Garden, 7–9 Dumbrava Rosie Street, 700487 Iasi,Romania.

3) University of Agricultural Sciences and Veterinary MedicineIasi, Faculty of Agriculture, 3 Mihail Sadoveanu Street, 700490Iasi, Romania.

*) Corresponding author: ELENA TRUTA. Fax: +40-232218121. E-mail: trutaelena@yahoo.com

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Curlycup gumweed is used as an ornamental plantdue to the intense yellow colour of its flowers as well asof their persistence over a long period of time, even inpoor and dry soils. Because of its deep and extensiveroot system and of its high ability to survive and grow under adverse conditions, curlycup gumweed isutilized for the rehabilitation of disturbed sites(http://www.fs.fed.us/database/feis/plants/forb/grisqu/all.html). The plants are unpalatable to livestock (theunpleasant taste is given by tannins, volatile oils, a bit-ter saponin, and resins) and they can readily absorbselenium from the soil, for which reason their presenceis considered undesirable by many farmers (BARE, 1979).

In Romania, G. squarrosa is a biennial plant, with adeep and palar root (SIRBU and OPREA, 2008, 2011). Inthe first year of vegetation, it forms a rosette, and thefollowing year many branched erect stems develop; thebranches carry numerous yellow flower heads. At theflower base there are shiny, sticky bracts, curved down-ward (hence the name squarrosa). The leaves are alter-nate and oblong, tooth-edged, gland-dotted, and gummy.The fruits are pseudoachenes, without thistledown, andshow a marked heteromorphism (SIRBU and OPREA,2011). The plant shows a high reproductive capacity inRomania, counting up to 76,000 achenes per individualper year. The disc and ray achenes are morphologicallydistinct in this species, and they differ in germinationrate (MCDONOUGH, 1975; SIRBU et al., 2011) (Figure 1).

Since the taxonomic relationships in Grindelia genushave not yet been clarified completely, they are revisedpermanently (TADEY et al., 2009; DEBLE and OLIVEIRA-DEBLE, 2010), this process giving reasons for gettingthoroughly into chromosome analysis, by both classicaland molecular methods. To reach a full impact, it isessential that the new exciting molecular findingsshould be fully integrated with the traditional cytoge-netical data in order to clarify the taxonomy ofGrindelia (STACE, 2000).

There are relatively few cytogenetical works on theGrindelia genus. This is unfortunate since both thechromosome numbers and the karyotype traits areimportant criteria in establishing the evolutionary pat-terns as well as in plant systematics. Nothing has beenpublished on the karyomorphology of G. squarrosa.

Considering the invasive character of G. squarrosa, itspharmacological valences and the quasi-absence of dataon its cytogenetics, the knowledge of detailed karyotypepatterns in this species becomes a necessity, since onlythe diploid chromosome number is available in litera-ture (PINKAVA and KEIL, 1977; LANE and HARTMAN,1996). The deciphering of the genetic constitution is anessential prerequisite in establishing the species struc-ture and polymorphism, its geographical distribution,systematics and evolution within the genus, at inter-and intra-specific levels.

Figure 1. – Grindelia squarrosa (Pursh) Dunal: a, b, c–different phenophases; d. ach-enes; e. flower (photographer: SIRBU, 2011).

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Because G. squarrosa has not been studied yet from akaryomorphological point of view and because it is nec-essary to know whether the invasive character and thenew habitat conditions of Romania have induced somechanges at the level of the genetic material, the purpos-es of this paper are: (1) to establish the chromosomediploid number, (2) to detail the morphological traits ofsomatic chromosomes, (3) to construct the karyotypes,and (4) to construct the idiogram, providing thus valu-able supplementary data on the chromosome constitu-tion of Grindelia genus.

Material and Methods

Plant material

The cytogenetic investigations have been carried outon root tips of seedlings obtained by germinating seedsof G. squarrosa (Pursh) Dunal plants, collected fromnaturally growing populations from the Iasi – Socolaarea (N47°08’23.63“, E27°37’02.14“, Alt. 40 m.s.l.).Voucher specimens have been deposited in the Herbari-um of the University of Agricultural Sciences and Vet-erinary Medicine of Iasi, Romania (Herbar IASI 17915 –17922).

Chromosome preparation

The germination took place at 22°C, in the dark, onwater-moistened filter paper in Petri glass dishes. At10–15 mm in length, the root tips were pre-treated with8-hydroxyquinoline (0.002 mol/L), for 4 h and were fixedin an ethanol-acetic acid mixture (3:l) for 24 h at roomtemperature. The samples were stored in 70% alcohol,at 4°C, until required. For cytogenetic analysis, the roottips were hydrolyzed in 50% hydrochloric acid for 6 min-utes and then stained in a modified carbol fuchsin solu-tion (GAMBORG and WETTER, 1975). The squashed prepa-rations were produced in 45% glacial acetic acid. Themicroscopic investigation was carried out by using aNikon Eclipse 600 microscope and the metaphases withwell-spread chromosomes were photographed with aCool Pix Nikon digital camera, 1600x1200 dpi, 100xobjective. The chromosome metric determinations, theorganization of chromosomes in karyotypes and the dia-grammatic representation of chromosome traits in idio-gram were carried out by using an Adobe Photoshop CS-3 software package.

Biometrics

Our chromosome measurements included the absolutelength of individual chromosomes (CL), long arm length(L), short arm length (S), arm ratio, r (r=L/S), cen-tromeric index, CI (CI=100xS/CL), length of the hap-loid complement (LHC), and the relative length of eachchromosome (CL/LHCx100). For each parameter werecalculated the mean (X

–), the standard error of the mean

(SE), and the standard deviation (SD). X–±SE is repre-

sented graphically. The chromosome designation fol-lowed the terminology recommended by LEVAN et al.(1964), while homology was assigned according to simi-larities in length, morphology, and centromere position,on the basis on CI and r values, respectively. In kary-otypes, the chromosome pairs (I–VI) have been grouped

in the descending order of their length. Average data foreach karyotype – included in tables – are the results ofthe metric determinations realized on fivemetaphases/genotype.

Karyotype asymmetry

To evaluate the karyotype asymmetry, the followingindexes were analyzed: TF%, AsI%, A1 and A2.

AsI% index (synonymous with AsK%) (ARANO andSAITO, 1980; PASZKO, 2006) represents the ratio betweenthe sum of long arm lengths of individual chromosomesand the haploid complement length: AsI%=(�longarms/haploid complement length) x 100.

The total form percent (TF%) (HUZIWARA, 1962) isexpressed by the ratio between the total sum of shortarm lengths of individual chromosomes and the haploidcomplement length: TF%=(�short arms/haploid com-plement length) x 100.

The intra-chromosomal asymmetry index (A1) and theinter-chromosomal asymmetry index (A2) were calculat-ed according to ROMERO ZARCO (1986) and PASZKO (2006):

A1=1– [�(b/B)/n], where b and B are the meanlengths of short and long arms of each pair of homo-logues, and n is the number of homologues. It measuresthe average position of the centromere in karyotype andranges from 0 (completely symmetrical) to 1 (completelyasymmetrical).

A2=SCL/XCL, where SCL is the standard deviation ofchromosome length and XCL is the mean chromosomelength for each genotype. It is a coefficient used toappreciate the heterogeneity of the chromosome length.Both indices (A1, A2) are independent of the chromosomenumber and size.

STEBBINS’s classification (1971), based on the frequen-cy of chromosomes with arm ratio (r) higher than 2 andon the ratio between the lengths of the longest and theshortest chromosomes in the complement (R), wasemployed to establish the karyotype symmetry classes.The asymmetry increases from type 1 to type 4 (as theproportion of chromosomes with r>2 increases) andfrom type A to type C (in relation to the size ratiobetween the largest and smallest chromosomes).

Idiograms of haploid complements were drawn by con-sidering the mean values calculated for the analyzedkaryotypes.

Results

The chromosome complements in the studied geno-types of G. squarrosa displayed a somatic diploid num-ber of 12 small-sized chromosomes (Table 1), grouped insix pairs of homologues (I–VI) after the metric determi-nations of the cytogenetic parameters. The sizes of indi-vidual chromosomes vary between 3.44 µm (the longestchromosome pair of Gsq-1 genotype) and 1.81 µm (theshortest chromosome pair of Gsq-6 genotype), with amean chromosome length/haploid complement rangingfrom 2.91±0.10 µm (Gsq-1) to 2.17±0.09 µm (Gsq-6).The mean value of the length of the haploid comple-ments of the seven karyotypes is 15.33±0.69 µm, with

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variations between 12.87 µm (Gsq-2) and 17.51 µm(Gsq-1). The arm ratio average values calculated pereach genotype show variation from 1.14±0.18 (Gsq-1) to1.60±0.20 (Gsq-4) with limits of variability rangingfrom r=1.04 (Gsq-1) to 2.30 (Gsq-3). Two satellite-bear-ing chromosomes have been identified in all studiedkaryotypes. The secondary constrictions delimitingsatellite bodies are present on the short arms of the sub-metacentric chromosomes of pair III. The mean size ofthe satellites is X

–±SE=0.57±0.02 µm. The small size of

chromosomes and the high level of chromosome conden-sation sometimes hindered the identification of thestructural details concerning the secondary constrictionposition and the presence of satellites.

The karyotype formula of the diploid complements isidentical: KF=2n=12=8m+2sm+2sm-SAT. If we con-sider the values of arm ratios and centromeric indexes,all the karyotypes have exclusively metacentric (m) andsubmetacentric (sm) chromosomes, the metacentricsbeing numerically prevalent (66.66%). Because of thekaryotypes’ resemblance, only metaphase and karyotypeof Gsq-1 genotype are given here as photos (Figures 2, 3).

As to the symmetry/asymmetry degree, consideringthat the numerical predominance of m and sm chromo-somes of approximately the same size defines a symmet-rical karyotype, the detailed analysis of the G. squar-rosa karyotypes shows a relatively high level of intra-specific uniformity for all measured variables. The aver-age centromeric index is X

–±SE=40.07±0.53 (limits of

variability: 38.45–41.95), R ranges from 1.29 to 1.53, thevalues of AsI% are comprised between 54.54 and57.61%, while TF% varies from 38.78 to 41.57%. Theintra- and inter-chromosomal asymmetry indexes arealso similar between genotypes and express the slightkaryotype asymmetry. Thus, A2 index indicates thatthere is little variation among the chromosome sizes inthe studied karyotypes, while the data for A1 index showno sharp inter-karyotype differences of chromosomearms.

Table 1. – Karyotype features in G. squarrosa studied genotypes (all karyotypes show satellite to chromosome pair III).

KF=karyotype formula, LHC=length of haploid complement, CL=chromosome length, CI=centromeric index, R= longest/shortestchromosome pair, AsI%=asymmetry index, TF%=total form percent, A1= intrachromosomal asymmetry index, A2= interchromo-somal asymmetry index, SKC=Stebbins’ karyotype classification; X

– ±SE=mean ± standard error of the mean.

Figure 2. – Metaphase from G. squarrosa (2n=12) – Gsq-1genotype. Scale bar=5 µm.

Figure 3. – Karyotype of G. squarrosa – Gsq-1 genotype (* satellite position). Scale bar = 5 µm.

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If we consider the two parameters used by STEBBINS

(1971) in order to appreciate the karyotype symmetry,we can see that six of the investigated genotypes (Gsq-1– Gsq-5, Gsq-7) have chromosomes with R<2, varyingfrom 1.29 to 1.53, whereas the proportion of chromo-somes with arm ratio (r)<2 ranges in interval 0.99 –0.51 (0.67 for Gsq-4 genotype, and 0.83 for the otherones), so that according to Stebbins’ classification(1971), the G. squarrosa karyotypes are quoted as 2A.Gsq-6 karyotype (classified as 1A) has no chromosomewith r>2, but we should mention that the arm ratio inpair III is very close to 2.0 (1.96). Anyway, both cate-gories (1A and 2A) reflect an increased level of kary-otype symmetry and include primitive karyotypes.Although the differences are small, the comparativeinter-karyotypic analysis could indicate that Gsq-2 andGsq-4 karyotypes are slightly more asymmetrical thanthe other five because they have the greatest values of Rand AsI% index, and the smallest TF% level. Addition-ally, Gsq-4 genotype has the highest proportion of chro-mosomes with an arm ratio greater than 2, which factalso confers it an increased asymmetry.

In order to construct the G. squarrosa idiogram, weused the average values of cytogenetic parametersdetermined for the seven karyotypes (Table 2).

The mean chromosome length calculated for the idio-gram is X

–±SE=2.6±0.1, and the average sizes of the

longest (I) and shortest (VI) chromosome pairs are2.98±0.13 µm and 2.17±0.11 µm, respectively. The rela-tive chromosome length ranges between 14.20±0.59%–19.58±0.78%. The final results stated the presence ofonly one chromosome pair with sub-median placed cen-tromere (sm), namely the satellite-bearing chromosomepair (III). In the idiogram, the average data led to theestablishment of the following karyotype formula for thehaploid complement: n=x=6=5m+1sm-SAT (Figure 4).

Discussion

Taking into account the similarity of some morphologi-cal traits (shape of disc corollas, for example), the samechromosome number, and some molecular data (chloro-plast DNA restriction site), the genus Grindelia is close-ly related to the genera Isocoma Nutt., Olivaea Schultz-Bip. Ex-Benth., Rayjacksonia Hartman & Lane,Stephanodoria Greene, and Xanthocephalum Willd.(LANE and HARTMAN, 1996; NESOM, 2000). However,adjustments of generic boundaries are to be made, andGrindelia is one of the genera from the Asteraceae fami-ly subjected to possible taxonomic revisions becausesome current proposals on systematic affinities and phy-logenetic relationships remain controversial (BARTOLI

and TORTOSA, 2003). A phylogenetic analysis of theGrindelia species based on molecular investigations(internal transcribed spacer nuclear sequence, microsatellite markers, fluorescence in situ hybridizationwith rDNA) is yet unsolved; only some clades have beenrecovered so far (BAEZA and SCHRADER, 2005; MOORE etal., 2009). Therefore, any new scientific data, includingthose provided by classical cytogenetics, can contributeto the clearing of incompletely known aspects of theinter-specific relationships, especially as the availableinformation on karyotype traits in the members of theGrindelia genus is relatively scarce.

Until now, the cytotaxonomic studies in the Grindeliaspecies from South and Central America (Argentina,Chile, Bolivia, Southern Brazil, Paraguay and Uruguay)have indicated only the basic number (x=6) and thesomatic chromosome number (2n=2x=12) (BARTOLI andTORTOSA, 1998; TORTOSA et al., 2000; BAEZA et al., 2004;

Table 2. – Average values of karyotype parameters of the seven G. squarrosa genotypes, calculated for idiogram construction(LHC=15.33±0.69 µm).

X– ±SE=mean ± standard error of the mean; SD=standard deviation; *=satellite location

Figure 4. – Idiogram of haploid complement of G. squarrosa.Bars represent the standard errors of the mean length of longand short arms. Chromosome pairs are marked with Romanfonts.

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BAEZA and SCHRADER, 2005). Few are the details con-cerning karyotype features (BARTOLI and TORTOSA, 2004;BAEZA and SCHRADER, 2005). Both diploids (2n=2x=12)and tetraploids (2n=4x=24) have been reported only forG. chiloensis in South America (BARTOLI et al., 1990,1993; BARTOLI and TORTOSA, 1998), while for NorthAmerica, two levels of chromosome numbers were recog-nized in the Grindelia genus, namely 2n=12 (diploid)and 2n=24 (tetraploid) (G. camporum Greene) (DUN-FORD, 1970, 1983). In G. camporum supernumerarychromosomes (0–3) have been evidenced while inG. humilis and G. stricta var. platyphylla aneuploids aresometimes present (STIEFKENS et al., 2011).

The present study shows that the investigated geno-types of G. squarrosa have exclusively 12 somatic chro-mosomes and our results confirm the basic chromosomenumber x=6 and the diploid number 2n=2x=12, report-ed for other species of the genus (LANE and HARTMAN,1996; TORTOSA et al., 2000; BAEZA et al., 2004; BARTOLI

and TORTOSA, 2004; BAEZA and SCHRADER, 2005).

If the data available in literature for differentGrindelia species have shown constancy of the diploidnumber, some variation is displayed concerning the cen-tromere position and chromosome morphotypes. Forsome Grindelia diploid species, the reported karyotypeformula was KF=2n=12=10m+2sm, with no specifica-tion concerning satellite position (STIEFKENS et al.,2011). A more diversified karyotype formula, comparedto that of G. squarrosa, was established for two SouthAmerican species – G. pygmaea and G. coronensis (TORTOSA et al., 2000; BARTOLI and TORTOSA, 2004) inwhich three pairs of metacentric chromosomes withmedian placed centromere, and three telocentric chro-mosome pairs are recorded: KF=2n=12=6m+4st+2st-SAT. In this case, the satellite is located on the smallestchromosome pair of the complement. Other two SouthAmerican species (G. anethifolia and G. prunelloides),newly analyzed with molecular-cytological methods,have exclusively metacentric chromosomes: 2n=12=10m+2m-SAT, and symmetric karyotypes (AsI%=55.46% and R=1.27, 55.95% and R=1.3, respectively)(BAEZA and SCHRADER, 2005). In these species, the nucle-olar organizing region (NOR) was detected by fluores-cence in situ hybridization (FISH) with 18/25S rDNA inthe satellite-bearing chromosome 2, but some specificsignals were also identified by FISH with 5S rDNA inthe short arms of the chromosomes 3 or 4, which are notsignificantly different in their length.

The results we obtained reveal that the seven kary-otypes of G. squarrosa show low variation concerningchromosome morphometric features and that they dis-play a high degree of symmetry. Consequently, G. squar-rosa karyotypes can be considered as less evolved andless subjected to significant genetic restructurings dur-ing their evolution. The symmetrical karyotypes withsmall chromosomes (<4µm, according to LIMA-DE-FARIA,1980) and predominantly of the metacentric and sub-metacentric type are considered as being more primi-tive, little evolved, because they have not undergone sig-nificant structural changes and rearrangements duringtheir evolution (STEBBINS, 1971; ACOSTA et al., 2005).

Increasing asymmetry can occur either through theshifting of centromere position from median/submedianto terminal/subterminal, or through the accumulation ofdifferences in the relative size between the chromo-somes of the complement, thus making the karyotypemore heterogeneous (PASZKO, 2006).

STEBBINS (1971) stated that the tendency towardskaryotype asymmetrization by the increase of the num-ber of telocentric chromosomes to the detriment of themetacentric and submetacentric ones marks a progres-sive step in karyotype evolution, with repercussions onthe evolution of the species.

The high symmetry and homogeneity of the kary-otypes made difficult the detection of intra-specific differences, as reported for other species as well (MARTINELLO and SCHIFINO-WITTMANN, 2003). Theabsence of basic chromosome numbers other than x=6in the Grindelia species suggests that the diversificationat inter-specific level has occurred through the struc -tural alteration of chromosomes rather than throughnumerical change. These conclusive data on the diploidchromosome number and on the resemblance of karyotype features in G. squarrosa with the otherspecies of the genus additionally prove that the evolu-tion has not been accompanied by large karyotypicchanges, although small chromosomal rearrangementshave certainly occurred (e.g., the number and position of5S rDNA in the karyotype – BAEZA and SCHRADER,2005).

The results of this study contribute to the knowledgeof chromosome constitution in G. squarrosa, an invasiveplant in Romania, ensuring thus the enlargement ofkaryological databases for the Grindelia genus. Obvi-ously, such classical cytogenetic investigations must becompleted and correlated, in the future, with thorough-going advanced approaches such as CMA3/DAPI band-ing, fluorescence in situ hybridization (FISH) or multi-colour fluorescent in situ hybridization (McFISH), foridentifying new molecular markers that could enablethe unequivocal chromosome pairing, or the preciseidentification of the presence of nucleolar organizingregions as well as the formulation of an accurate viewon the karyotype evolutionary trends in the Asteraceaefamily.

Acknowledgements

This work was supported by ANCS Romania, PN IICAPACITATI, project SK-RO 0013-10, contract number474/07.03.2011, and CNCSIS-UEFISCDI Romania, pro-ject number PNII-IDEI_1227. We gratefully thank Prof.Dr. TAMARA LACATUSU from Faculty of Letters, “Al. I.Cuza” University, Iasi, for the quick help in the checkingof English language of the manuscript.

References

ACOSTA, M. G., G. BERNARDELLO, M. GUERRA and E. A.MOSCONE (2005): Karyotype analysis in several SouthAmerican species of Solanum and Lycianthes rantonnei(Solanaceae). Taxon 54: 713–723.

185

ARANO, H. and H. SAITO (1980): Cytological studies infamily Umbelliferae 5. Karyotypes of seven species insubtribe Seselinae. Kromosomo 2: 471–480.

BAEZA, C. M., E. VOSYKA and T. STUESSY (2004): Chromo-some reports from Chilean plants. Darwiniana 42:25–29.

BAEZA, C. and O. SCHRADER (2005): Comparative kary-otype analysis in Haplopappus Cass. and GrindeliaWilld. (Asteraceae) by double FISH with rRNA specificgenes. Plant Systematics and Evolution 251: 161–172.

BARE, J. E. (1979): Wildflowers and weeds of Kansas.Lawrence, KS: The Regents Press of Kansas.

BARTOLI, A., G. G. ROITMAN and N. FRAYSSINET (1990):Estudio comparativo de poblaciones diploides ytetraploides de Grindelia chiloensis (Asteraceae). Dar-winiana 30: 171–177.

BARTOLI, A., G. G. ROITMAN and N. FRAYSSINET (1993):Presencia de hexaploides (2n=36) en Grindelia chiloen-sis (Asteraceae). (Presence of a hexaploid population(2n=36) in Grindelia chiloensis (Asteraceae). Boletín dela Sociedad Argentina de Botánica 29: 139–142.

BARTOLI, A. and R. D. TORTOSA (1998): Intermediacybetween Grindelia chiloensis and Haplopappus pectina-tus (Astereae, Asteraceae). Taxon 47: 337–345.

BARTOLI, A. and R. D. TORTOSA (2003): Architecture of thegenus Grindelia (Asteraceae: Astereae). Flora 198:106–111.

BARTOLI, A. and R. D. TORTOSA (2004): Chromosome num-bers in species of Grindelia (Asteraceae, Astereae) fromthe Meseta del Somuncura (Patagonia, Argentina).Compositae Newsletter 41: 51–53.

BRUMMITT, R. K. and C. E. POWELL (eds.) (1992): Authorsof Plant Names, Royal Botanic Gardens, Kew.

DEBLE, L. P. and A. S. OLIVEIRA-DEBLE (2010): Novelties inGrindelia (Asteraceae: Astereae) from South America.Bonplandia 19: 47–57.

DUNFORD, M. P. (1970): Triploid and tetraploid hybridsfrom diploid x tetraploid crosses in Grindelia (Composi-tae). American Journal of Botany 57: 856–860.

DUNFORD, M. P. (1983): Chromosome analysis of hybridsbetween 4n Grindelia camporum and 2n G. grandiflora(Compositae). American Journal of Botany 70:1320–1324.

GAMBORG, O. L. and L. R. WETTER (eds.) (1975): Plant Tis-sue Culture Methods. National Research Council ofCanada, Prairie Regional Laboratory (Saskatoon, Sask).

GHEDIRA, K., P. GOETZ and R. LE JEUNE (2010): Grindélia:Grindelia squarrosa (Pursh) Dunal (Asteraceae). Phy-tothérapie 8: 314–318.

HUZIWARA, Y. (1962): Karyotype analysis in some genera ofCompositae. VIII. Further studies on the chromosomeof Aster. American Journal of Botany 49: 116–119.

JOHNSON, J. R. and J. T. NICHOLS (1970): Plants of SouthDakota grasslands: A photographic study. Bulletin 566,Agricultural Experiment Station, South Dakota StateUniversity, Brookings.

LANE, M. A. and R. L. HARTMAN (1996): Reclassification ofNorth American Haplopappus (Compositae: Astereae)completed: Rayjacksonia gen. nov. American Journal ofBotany 83: 356–370.

LEVAN, A. K., K. FREDGA and A. A. SANDBERG (1964):Nomenclature for centromeric position on chromosomes.Hereditas 52: 201–220.

LIMA-DE-FARIA, A. (1980): Classification of genes,rearrangements and chromosomes according to thechromosome field. Hereditas 93: 1–46.

MARTINELLO, G. E. and M. T. SCHIFINO-WITTMANN

(2003): Chromosomes of Bromus auleticus Trin. ex Nees (Poaceae), Genetics and Molecular Biology 26:369–371.

MCDONOUGH, W. T. (1975): Germination polymorphism inGrindelia squarrosa (Pursh) Dunal, Northwest Science49: 190–200.

MOORE, A. J., W. L. MOORE, V. R. F. MORRIS and B. G.BALDWIN (2009): Development of microsatellite markersfor Californian Grindelia (Asteraceae, Astereae). Molec-ular Ecology Resources – manuscript [tomato.biol.trini-ty.edu/manuscripts/9-5/mer-09-0038.pdf]

NESOM, G. L. (2000): Generic conspectus of the tribeAstereae (Asteraceae) in North America, Central Ameri-ca, the Antilles, and Hawaii. Sida, BotanicalMiscellany,Botanical Research Institute of Texas, USA: 1–100.

PASZKO, B. (2006): A critical review and a new proposal ofkaryotype asymmetry indexes. Plant Systematics andEvolution 258: 39–48.

PINKAVA, D. J. and D. J. KEIL (1977): Chromosome countsof Compositae from the United States and Mexico.American Journal of Botany 64: 680–686.

ROMERO ZARCO, C. (1986): A new method for estimatingkaryotype asymmetry. Taxon 35: 526–530.

SANZ ELORZA, M, E. DANA and E. SOBRINO (2001): Aproxi-mación al listado de plantas alóctonas invasoras realesy potenciales en España. Lazaroa 22: 121–131.

SIRBU, C. and A. OPREA (1998): Grindelia squarrosa(Pursh) Dunal en Roumanie. Revue Roumaine de Biolo-gie, Série de Biologie végétale 43: 91–93.

SIRBU, C. and A. OPREA (2008): Two alien species in thespreading process in Romania: Reynoutria x bohemicaChrtek & Chrtková and Grindelia squarrosa (Pursh)Dunal. Cercetari Agronomice in Moldova XLI, 2: 41–50.

SIRBU, C. and A. OPREA (2011): Plante adventive in floraRomaniei. Edit. Ion Ionescu de la Brad, Iasi.

SIRBU, C., A. OPREA, E. PAVOL and F. PETER (2011): Newcontribution to the study of alien flora in Romania.Journal of Plant Development 18: 121–134.

SIRBU, C., A. OPREA, E. TRUTA and G. VOCHITA (2011): Astudy on biology and phytosociology of the invasiveplant: Grindelia squarrosa (Pursh) Dunal (Asteraceae)in Romania, pp 54. In: Book of Abstracts of 3rd

International Symposium of Environmental Weeds andInvasive Plants, October 2–7, 2011 (edited by C.BOHREN, M. BERTOSSA, N. SCHÖNENBERGER, M.ROSSINELLI and M. CONEDERA), Monte Verità, Ascona,Switzerland.

STACE, C. A. (2000): Cytology and cytogenetics as a funda-mental resource for 20th and 21st centuries. Taxon 49:451–477.

STEBBINS, G. L. (1971): Chromosomal evolution in higherplants. IV. Chromosomal changes, genetic recombina-tion and speciation, Edward Arnold Ltd, London:101–105.

STEYERMARK, J. A. (1937): Studies in Grindelia. III.Annals of the Missouri Botanical Garden 24: 225–262.

STIEFKENS, L., N. MORENO, M. L. LAS PEÑAS, A. BARTOLI

and G. BERNARDELLO (2011): Estudios citogeneticos en elgrupo “Xanthocephalum” (Machaerantherinae, Aster-eae, Asteraceae): Un aporte a la comprension de susrelaciones intergenericas, Basic&Applied Genetics, vol.XXI (Suppl.) (Resumenes – XL Congreso Argentino deGenetica, 18–21 September 2011, Buenos Aires,Argentina): S121 [http://www.sag.org.ar/Documentos/Actas_XLCongreso.pdf].

186 Silvae Genetica 61, 4–5 (2012)

STINSON, K. A., S. A. CAMPBELL, J. R. POWELL, B. E.WOLFE, R. M. CALLAWAY, G. C. THELEN, S. HALLET, D. PRATI and J. N. KLIRONOMOS (2006): Invasive plantssuppress the growth of native tree seedlings by disrupt-ing below ground mutualisms. Plos Biology 4e140:0727–1731.

TADEY, M., J. C. TADEY and N. TADEY (2009): Reproductivebiology of five native plant species from the MonteDesert of Argentina. Botanical Journal of the LinneanSociety 161: 190–201.

TORTOSA, R. D., A. BARTOLI, N. L. GIL-AD and N. BAR-TOLONI (2000): Analysis of hybridization betweenGrindelia brachystephana and G. chiloensis (Aster-aceae: Astereae) in Patagonia, Argentina. Plant System-atics and Evolution 224: 243–253.

WALSH, R. A. (1993): Grindelia squarrosa. In: Fire EffectsInformation System, [Online]. USDA Forest Service,Rocky Mountain Research Station, Fire Sciences Labo-ratory (Producer) (http://www.fs.fed.us/database/feis/plants/forb/grisqu/all.html) (accessed 2012, February 1).

Abstract

Measurements on growth traits up to 41 years of agefrom 68 progeny sites in eight first-generation breedingzones of coastal Douglas-fir (Pseudotsuga menziesii[MIRB.] FRANCO var. menziesii) in the US Pacific North-west were used to investigate age trends of genetic para-meters and to determine optimum age of selection. Heri-tabilities and age-age genetic correlations were estimat-ed using univariate or bivariate mixed model analyses.Heritability estimates tended to increase with age for

both total growth and growth increment traits. The esti-mates showed different age trends among breedingzones, but the differences were generally small. Age-agegenetic correlations for total growth traits fitted Lam-beth’s model surprisingly well, despite the data beingcollected from multiple breeding zones. Using rotation-age (i.e., 50yr) volume as the selection

criterion, the greatest correlated gains per year wereachieved by making family selection at juvenile ages(i.e., 9 for height, 13 for diameter, and 11 for volume).Similar results were obtained for within-family selectionexcept that the optimum ages of selection were 2~4years later than that from family selection, i.e., 11 forheight, 15 for diameter and volume. Early selection ontotal height was always more efficient and had earlieroptimum ages than on other growth traits. The opti-mum ages of early family selection on total growth were4~11 years earlier than on the corresponding growthincrement traits. It was also evident that the optimumages of selection occurred later for slow-growth trialsthan for fast-growing trials.

Key words: Douglas-fir, growth, growth increment, early selec-tion, selection efficiency, optimum age, heritability, age-agegenetic correlation.

Introduction

Forest trees often have long rotations and produceseed late, and therefore long generation intervals. Selec-tion for growth traits in tree breeding programs usuallytakes place long before rotation age to shorten thebreeding cycle and maximize genetic gain per unit time.The optimum age for selection and the choice of mea-surement traits are vitally important to the long-termefficiency of any tree breeding program (LAMBETH, 1980;ZOBEL and TALBERT, 1984).

The biologically optimum age for selection has beendefined as the selection age when average annual gaintowards the breeding objective is maximized (KANG,1985). It can be determined using theory of quantitativegenetics if the heritabilities of the juvenile and maturetraits and their genetic correlation are known. Whileheritabilities and age-age correlations for growth havebeen reported for many coniferous species such as loblol-ly pine (e.g., MCKEAND, 1988; LAMBETH and DILL, 2001;GWAZE and BRIDGWATER, 2002), radiata pine (e.g., MATH-ESON et al., 1994), lodgepole pine (e.g., XIE and YING,1996), slash pine (e.g., WHITE and HODGE, 1992), Dou-glas-fir (e.g., JOHNSON et al., 1997; DEAN andSTONECYPHER, 2006), Scots pine (e.g., JANSSON et al.,2003) and jack pine (e.g., WENG et al., 2007), these para-meters at mature stages are seldom known, and areusually predicted using attributes measured in juvenileprogeny trials.

Stem volume is usually the most important trait indetermining tree value. Thus, genetic improvement involume growth has been the highest priority in mosttree improvement programs. Early selection for rota-tion-age stem volume is often conducted based on juve-nile height growth (e.g., MCKEAND, 1988; DEAN and

Early Selection for Improving Volume Growth in Coastal Douglas-fir Breeding Programs

By T. Z. YE*) and K. J. S. JAYAWICKRAMA

Department of Forest Ecosystems and Society, Oregon State University, 321 Richardson Hall, Corvallis, OR 97331, USA

(Received 10th February 2012)

*) Corresponding Author: TERRANCE Z. YE. Tel: (541) 737-9881,Fax: (541) 737-1393. E-Mail: Terrance.Ye@oregonstate.edu