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ORIGINAL PAPER Karyological studies in Deschampsia antarctica Desv. (Poaceae) Susana Cardone Patricia Sawatani Pablo Rush Ana Marı´a Garcı´a Lidia Poggio Gustavo Schrauf Received: 27 May 2008/Revised: 10 October 2008/Accepted: 14 October 2008 Springer-Verlag 2008 Abstract Deschampsia antarctica Desv. (Poaceae),known as Antarctic hairgrass, is the only grass species andone of the two vascular plant species native to Antarcticaand it is a valuable source for gene discovery associatedwith freezing-tolerance. In this work the karyotype of D. antarctica collected near Jubany Antarctic Base wasreported for the first time. This species presents a chro-mosome number of 2 n = 2 x = 26, with karyotypicformula 10 m ? 6sm ? 8st ? 2 t . The nucleolar organizerregion is located in the short arm in one of the submeta-centric pairs forming a terminal satellite. Aneusomaty,a phenomenon already reported in the genus, was detectedduring this study. The cytogenetic information, togetherwith the recent phylogenetic data will be useful forbreeding strategies in agronomically valuable crops. Keywords Deschampsia antarctica Karyotype Chromosomes Aneusomaty Introduction Deschampsia P. Beauv. is a cosmopolitan genus that con-sists of around 40 species (Parodi 1949; Garcı´a Sua´rezet al. 1997; Chiapella 2000; Chiapella and Probatova 2003), including annual and perennial grasses that arewidespread in temperate and cold regions of the world andrestricted to high altitudes in tropical areas. Many of thespecies belonging to this genus, such as D. alba , D. nubi-gena , and D. alpina , inhabit extreme polar environments(Index Kewensis 1997; Chiapella 2000). The high mor- phological diversification makes the taxonomy of the groupvery difficult. Deschampsia was divided into two sections that weresegregated into two genera, Deschampsia and Avenella ,based on morphological differences (Frey 1999). Later, thephylogenetic study by Chiapella (2007), based on nuclearribosomal internal transcribed spacer (ITS) and plastid trnL intron sequences, showed independent srcins for these twogenera. This was corroborated by Quintanar et al. (2007), who analyzed the phylogeny of the tribe Aveneae using thesame nuclear and plastid sequences.Most species of the genus Deschampsia possess a 2 n chromosome number of 26, being 13 the basic value.Polyploid genotypes, with 52 chromosomes and triploidhybrids with 39 chromosomes have also been reported(Table 1).Twospeciesthatpresentabasicnumberof x = 7, Deschampsia flexuosa (2 n = 28) and D. atropurpurea (2 n = 14), deserve a special consideration because molec-ular and morphological evidences suggest that they shouldbe included in separate genera (Chiapella 2007). S. Cardone P. Rush G. Schrauf Facultad de Agronomı´a de la Universidad de Buenos Aires(FAUBA), Av. San Martı´n, 4453-1417 Buenos Aires, Argentinae-mail:
[email protected]. Rushe-mail:
[email protected]. Schrauf e-mail:
[email protected]. Sawatani A. M. Garcı´a L. Poggio ( & )Facultad de Ciencias Exactas y Naturales de la Universidad deBuenos Aires (FCEyN), Intendente Gu¨iraldes 2160, CiudadUniversitaria, C1428EGA Buenos Aires, Argentinae-mail:
[email protected]. M. Garcı´ae-mail:
[email protected] 1 3 Polar BiolDOI 10.1007/s00300-008-0535-8 The D. caespitosa complex is the most studied taxon inthe genus. This is a tussock-forming perennial grass widelydispersed around the globe, particularly in the northernhemisphere (Kawano 1963; Bush and Barret 1993). Kawano (1963), Garcı´a Sua´rez et al. (1997) and Winter-feld (2004) have described different karyotypes of D. caespitosa , all with 2 n = 26. Intraindividual variationin chromosome number (aneusomaty) varying between 2 n = 15–26 and different ploidy levels (2 n = 26 to 4 n = 52)have also been reported (Lo¨ve and Lo¨ve 1961; Kawano 1963; Alberts 1978; Nkongolo et al. 2001). Deschampsia antarctica , known as ‘‘Antarctic hair-grass’’, is the only perennial gramineae that colonizesAntarctica. This species shows no photoinhibition on clearand cold days in Antarctica or growth inhibition in thelaboratory at low temperatures (Xiong et al. 1999). Its freezing-tolerance (Bravo et al. 2001; Bravo and Griffith 2005; Sawatani 2006), together with its ability to become established rapidly by seed and to tolerate prolonged snowcover and exposure to wind (Greene and Holtom 1971) explains its success in colonizing Antarctica (Day et al.1999; Alberdi and Corcuera 1991). Otherwise, D. antarc-tica increase its population size in response to the warmingmore rapidly than that of the dominant cryptogamic group,therefore D. antarctica may be useful as bioindicators of climate change in Antarctica (Lewis Smith 1994). The native Antarctic vegetation certainly must have one orvarious mechanisms that allow the maintenance of metabolism at low temperature during the Antarctic sum-mer (growing season) and survival during the winter(Bravo et al. 2001). Along with several plants of the Northern Hemisphere, this species is a valuable source forgene discovery of vascular plants associated with freezing-tolerance, allowing the development of breeding strategiesin agronomically valuable crops (John and Spangenberg2005). D. antarctica is also found in South America (Tierra delFuego and Andean sites at ca. 34 S) and has a wide Table 1 Chromosome numbers in Deschampsia speciesSpecies 2 n References Species 2 n References D. alpina (ssp) a, b 52 Lo¨ve and Lo¨ve (1975) D. koelerioides 26 Tzvelev (1976)50 Engelskjon (1979) D. komarovi 52 Petrovsky and Zhukova(1981)39, 49, 52 Alberts (1980) 26 Zhukova (1980) D. antarctica b 26 Present work D. liebmanniana 26, 52 Taken from Fedorov (1969) D. argentea—maderensis 26 Dalgaard (1991) D. mackenzieana a 52 Purdy and Bayer (1995) D. atropurpurea 14 Alberts (1972) D. macrothyrsa a 26 Probatova (1984) D. beringensis a 26, 42 Sokolovoskaya and Probatova (1975) D. media 26 Alberts (1980) D. borealis a 26, 28 Tzvelev (1976) 28 Kerguelen (1975) D. bottnica a, b 26 Alberts (1980) D. mildbraedii 52 Morton (1993) D. brevifolia a 52 Krogulevich (1976) D. obensis 52 Zhukova and Petrovsky(1980)52 Petrovsky and Zhukova (1981) D. orientalis a 52 Petrovsky and Zhukova(1981) D. caespitosa b 26 (0-2B) Pashuk (1980) 26 Lo¨ve and Lo¨ve (1981)26, 52 Stoeva (1982) D. parviflora a 26, 28 Kerguele´n (1993)26 Beuzenberg and Hair (1983) D. pamirica a 2626 Strid and Franze´n (1983) D. pumila 26 Dalgaard (1989)26, 52 Rhotera and Davy 1986 D. refracta 26 Alberts (1980) D. chapmanii b 26 Edgar (1993) D. danthonioides b 26 Taken from Fedorov (1969) D. rhenana a 49–52 Alberts (1980) D. elongata b 26 Taken from Fedorov (1969) D. setaceae 14 Hubbard (1984) D. festucaelifolia a 27, 28 Osada (1993) 14 Lo¨vkvist and Hultgard (1999) D. flexuosa 28 Stoeva; Arohonka (1982) D. sukatschewii 52 Zhukova (1980)28 Strid and Franzen 1983 D. tenella b 26 Edgar and Connor (2000)26 Druskovic and Lovka (1995) D. wibeliana 26 Alberts (1980)28 Petrova and Stoyanova (1998) 26 (05B) Mesı´cˇek (1992) D. glauca a 26, 52 Zhukova and Petrovsky (1975), (1976) a Also reported as subspecies of D. caespitosa b Species grouped by Chiapella (2007) in the core of the genus Deschampsia Polar Biol 1 3 distribution on all the subantarctic islands, includingKerguele´n island in the Indian Ocean (Parodi 1949; Greene and Holtom 1971; Chiapella 2000). In Antarctica, the growing season of D. antarctica begins in November and takes place by seed germination orby regrowth of ramets of the previous year (Corte 1961).This species is self-compatible and its flowers often remainclosed, such that self-pollination is enforced throughcleistogamy (Moore 1983). On the other hand, both cleis- togamic and chasmogamic flowers were found in samplesof the South American continent (Corte 1961).The phylogenetic position of D. antarctica has beeninvestigated in relation to other species of this genus(Ferna´ndez Souto et al. 2006). Chiapella (2007) has poin- ted out the need for obtaining data of different geographicalsrcins and ploidy levels in order to clarify the phyloge-netic relationships of the species within the genus. Thechromosome number is often an effective indicator of taxonomic relationships, also providing information onpossible karyotypic rearrangements and direction of thechromosome changes in related groups (Soltis et al. 2005).The karyotype, which is a basic tool for plant breedingand biotechnology has not yet been described for thisspecies. In this paper, we report the first cytogeneticinformation on D. antarctica in terms of chromosomenumber, ploidy level and chromosome morphology. Thecytogenetic features are compared with the availablephylogenetic data. Materials and methods The plants studied grew from seeds obtained from theAntarctic soil (Base Jubany-Argentinean Antarctica). Thepopulation established from these seeds consisted of 14plants about 2 years old that were kept in a greenhouse(located in Buenos Aires Latitude: 34 28 0 S, Longitude:58 28 0 ) where the temperature available was between 20and 25 C (day) and between 10 and 14 C (night).The mitotic chromosomes were prepared from root tips,pre-treated with colchicine (0.25%) and fixed in Carnoysolution. Feulgen staining (Darlington and La Cour 1962) followed the hydrolysis with HCl 1 N for 10 min at 60 C.For the mitotic cycle analysis, fixation and staining wasperformed in the same way, but without the pre-treatment.Chromosome morphology was assigned followingLevan et al. (1964), which classifies chromosomes according to the centromere index (CI: length of the shortarm 9 100/total length of the chromosome) as metacentric(m), submetacentric (sm), subtelocentric (st), and telocen-tric (t). We studied ten metaphases from each individual of the population. For the idiograms and the chromosomemeasurements, we analyzed representative metaphases.Indexes A 1 and A 2 , intra and inter-chromosome asymmetryindexes respectively, were also calculated according toRomero Zarco (1986), as follows: A 1 ¼ 1 P n i ¼ 1 b i B i n where ‘‘ n ’’ is the number of homologous chromosome pairs,‘‘ b i ’’ is the average length for short arms in everyhomologous chromosome pair and ‘‘ B i ’’ is the averagelengthforlongarmsineveryhomologouschromosomepair; A 2 ¼ S X which is the ratio between the standard deviation ( S ) andthe mean ( X ) of chromosome length for each sample. Results All the individual of D. antarctica analyzed had a chro-mosome complement based on five metacentric, threesubmetacentric, four subtelocentric, and one telocentric inthe haploid set (Fig. 1a, b). The nuclear organizer region(NOR) is located at terminal position in one of the sub-metacentric chromosome, pair 7 (Figs. 1 b, c, 2a). This pair of chromosome appeared as heteromorphic in severalstudied cells suggesting the existence of small rearrange-ment or differences in heterochromatic blocks.Total length of the karyotype was 78.67 ± 6.56 l m.The analysis of the karyotype asymmetry showed a valueof 0.52 for A 1 and 0.24 for A 2 .All the individuals showed a diploid number of 26(2 n = 26). The analysis of the mitotic stages allowed thedetection of some irregularities such as failures in theformation of the spindle and delayed chromosomes inanaphase (Fig. 2b, c) leading to cells with variable chro-mosome number (2 n = 28 and 2 n = 13), in low frequency(Fig. 2a, d). The analysis of cells showing 2 n = 28allowed the detection of two ‘‘extra’’ chromosomes possi-bly corresponding to chromosomes 1 and 11 of thekaryotype, thus constituting a double trisomy (Fig. 2a).The aneusomaty was recorded in 5 out of the 14 individualsof the analyzed population. Discussion In the genus Deschampsia , so far the chromosome numberhas been reported in 33 out of the 40 known species(Table 1). The only species of the genus with a describedkaryotype is D. caespitosa . Kawano (1963) and Garcı´aSua´rez et al. (1997) reported 2 n = 26 for several Polar Biol 1 3 Fig. 1 a Mitotic metaphase of Deschampsia antarctica with2 n = 26. b Haploidiogram. c Karyogram with(10 m ? 6sm ? 8st ? 2 t ). Bars correspond to 10 l m Fig. 2 a Mitotic prometaphasewith 2 n = 28. Single arrows show chromosome 1, doublearrows show chromosome 11. Oval heat arrows showchromosome 7. b The arrow shows a delayed chromosomesin anaphase. c Tripolar spindle. d Cell with 2 n = 13. Bars correspond to 10 l mPolar Biol 1 3 populations of the Northern Hemisphere and north of Spain. Kawano (1963) showed an asymmetrical karyotype with two pairs of rather long metacentric chromosomes,whereas Garcı´a Sua´rez et al. (1997) described a chromo- some morphology similar to that of the D. antarctica described in the present work, where the karyotypes of D. caespitosa and D. antarctica have all chromosomessimilar in size, even for chromosome 1, which is twice aslarge as the others. In fact, when chromosome 1 wasexcluded of the A 2 index calculation in D. antarctica, thisindex diminished from 0.24 to 0.17, indicating that thischromosome is responsible for the asymmetry (Fig. 1). Onthe other hand, Winterfeld (2004) described, for a popu-lation of D. caespitosa from Germany, a karyotype of 2 n = 2 x = 26, which has morphological characteristicsvery different both from those of Deschampsia antarctica showed in this work, and from those described for D.caespitosa by Kawano (1963) and Garcı ´a Sua´rez (1997). Infact Winterfeld (2004) showed heteromorphisms thatwould indicate the existence of several chromosomerearrangements.Inter and intra individual variability in chromosomenumber, aneuploidy and aneusomaty were noticed in D. caespitosa by Kawano (1963), who recorded countings with 2 n = 26–28, and by Nkongolo et al. (2001), who found a great intraindividual variation with several cells of the same plant, containing fewer than 26 chromosomes(2 n = 15–26). Lo¨ve and Lo¨ve (1961) and Kawano (1963) have reported various chromosome number for this species(2 n = 27, 28, 49, 39, 52). This result suggests that chro-mosomic variation found in this species is mainly due tohybridization and polyploidy. Moreover, the aneusomatyphenomenon could contribute to the reported variation. Themitotic studies described in the present work lead us toconsider that aneusomaty could be due to a constitutionaltendency to non-disjunction during mitosis.In this paper, intrapopulation karyotypic variability wasnot detected in D. antarctica ; however, aneusomaty wasrecorded in 5 out of the 14 individuals (Fig. 2a). The an-eusomatyisrareamongangiospermsandonlyfewexampleshave been reported such as the variation in the chromosomenumber between individuals as well as within individuals inseveral populations of Orobranche gracilis , due to abnor-malities in the mitotic cycle (Greilhuber and Weber 1975). The genetic cause of aneusomaty may be either the presenceof certain alleles that affect the spindle mechanisms or agenedosageeffectofapolysomiccondition(GreilhuberandWeber 1975). In sunflower ( Helianthus annuus L.) chro-mosome number variation resulted from anaphases andmetaphases with lagging chromosomes and from anaphaseswith bridges (Cavallini and Cremonini 1985); Cremonini and Cavallini 1986). These anomalies decrease progres-sively during the development of the plant and disappearbefore the onset of meiosis in the anthers, and thus are nottransmitted to the progeny. It is not known whether thesomatic aneusomaty found in D. antarctica is transmitted tothe progeny.Humpreys (1978) observed that the increase in temper-ature from 25 to 30 C caused a significant increase in therate of elimination of chromosomes in interspecific hybridsof Hordeum vulgare 9 H. bulbosum . Moreover, changesin environmental conditions can also alter DNA content(Cullis 1990). These examples suggest that the aneusomaty found in D. antarctica in the present work may be due togrowing conditions (e.g. temperature) much different fromtheir natural environment. However, several reports(Kawano 1963; Nkongolo et al. 2001) suggest that chro- mosome instability would be an intrinsic characteristic of the genus.Based on the analysis of nuclear sequences and plastids,Chiapella (2007) established groups with separate genericstatus where D. flexuosa and D. atropurpurea present an x = 7, whereas the group corresponding to D. caespitosa (core of the genus) presents an x = 13.The chromosome number described in the present work for D. antarctica (2 n = 26) is in agreement with the abovephylogenetical hypothesis where this species is grouped inthe Deschampsia genus core.The chromosome data reported previously in the genusand the present karyological study are in accordance withthe hypothesis of Kawano (1963) and Alberts (1978). These authors have proposed that the polyploids wouldhave arisen from duplication ( x = 7–14), whereas thespecies with the chromosome number 2 n = 26 would havearisen by dysploidy (28–26) from polyploidy species. D. antarctica seems to be one of the species that experi-mented the process of dysploidy during its evolution.Theresultsofpresentwork,togetherwithfurtherresearchon the polyploidy and dysploidy processes, will improve theunderstanding of the organization, structure, and evolutionof the genome of Deschampsia . Besides their evolutionaryinterest the present data also provide a basic cytogeneticframework for genetic and biotechnologic applications,considering the fact that Deschampsia antarctica is animportant source of genes related to cold resistance. Acknowledgments The authors wish to thank Ulrik John andGerman Spangenberg for the encouragement to obtain the informa-tion provided in the present work. Financial support by CONICET(PIP5927), FONCYT (PICT 14170) and UBA (G097). L.P. andA.M.G. belong to CONICET. References Alberdi M, Corcuera LJ (1991) Cold acclimation in plants. Phyto-chemistry 30:3177–3184Polar Biol 1 3
