Genomic relationships among diploid andpolyploid species of the genus Eryngium L. usinggenomic in situ hybridization

Gisele Yvonne Perthuy, Susana Martınez, Eduardo Jose Greizerstein, andLidia Poggio

Abstract: Eryngium L. (Umbelliferae) is a large genus including more than 250 species worldwide. The large morphologi-cal variability in this genus makes it difficult to delimit the species or to establish phylogenetic relationships. The occur-rence of different ploidy levels within the genus might indicate a hybrid origin of the polyploid species. In the presentstudy, the chromosome number and karyotype of E. regnellii are reported for the first time and the ploidy level of a popu-lation of E. paniculatum is confirmed. We compare the genomes of the diploids E. horridum and E. eburneum, the tetra-ploids E. megapotamicum and E. regnellii, and the hexaploids E. pandanifolium (as a representative of the wholepandanifolium complex) and E. paniculatum using genomic in situ hybridization (GISH). Although it was not possible toidentify the parental species of the polyploid taxa analyzed, the GISH technique allowed us to postulate some hypothesesabout their origin. Eryngium horridum and E. eburneum do not seem to be the direct progenitors of the polyploids ana-lyzed. On the other hand, it seems that other diploid species unrelated to E. horridum and E. eburneum are involved intheir origin. Our results are consistent with morphological and phylogenetic studies, indicating a close relationship betweenthe species of the series Latifolia.

Key words: Eryngium, genomic relationships, GISH, polyploidy.

Resume : Le genre Eryngium L. (Umbelliferae) compte plus de 250 especes a l’echelle mondiale. La grande variabilitemorphologique au sein de ce genre rend difficile la delimitation des especes ou l’etablissement de relations phylogeneti-ques. La presence de plusieurs niveaux de ploıdie au sein du genre pourrait indiquer une origine hybride des especes poly-ploıdes. Dans le present travail, les auteurs comparent les genomes des especes diploıdes (E. horridum et E. eburneum),les tetraploıdes (E. megapotamicum et E. regnelli), et les hexaploıdes E. pandanifolium (comme representant de tout lecomplexe pandanifolium) et E. paniculatum par hybridation genomique in situ (GISH). Le nombre de chromosomes et lecaryotype de l’E. regnellii sont rapportes ici pour la premiere fois et la ploıdie d’une population de l’E. paniculatum estconfirmee. Bien qu’il n’ait pas ete possible d’identifier les especes parentales des taxons polyploıdes analyses, la techniqueGISH a permis de mettre de l’avant certaines hypotheses au sujet de leur origine. L’E. horridum et l’E. eburneum ne sem-blent pas les ancetres en droite ligne des polyploıdes analyses. Par ailleurs, il semblerait que d’autres especes diploıdes,sans relation avec l’E. horridum et l’E. eburneum, seraient impliquees. Ces resultats sont conformes avec ceux obtenussuite a des etudes morphologiques et phylogenetiques et indiquent une parente proche parmi les especes de la serie des La-tifolia.

Mots-cles : Eryngium, relations genomiques, GISH, polyploıdie.

Introduction

Eryngium L. is the largest genus of the Umbelliferae fam-ily. It belongs to the Saniculoideae subfamily and includesmore than 250 species grouped in 34 sections (Wolff 1913),which are distributed throughout temperate regions world-wide. Although it is easily distinguished from the other gen-era of the family, the existence of a large morphological

variability at the intragenus level makes it difficult to de-limit the species or to establish phylogenetic relationships(Martınez and Calvino 2007). The basic chromosome num-ber varies from x = 6 to x = 9 (Cerceau-Larrival 1973; Vi-anna and Irgang 1971; Constance 1977; Calvino et al. 2002),the most common being x = 8. The occurrence of differentploidy levels within the genus may indicate a possible hy-brid origin for the polyploid species (Constance 1977). Cal-

Received 21 April 2010. Accepted 26 July 2010.. Published on the NRC Research Press Web site at genome.nrc.ca on 4 October 2010.

Corresponding Editor: M. Puertas.

G.Y. Perthuy,1 E.J. Greizerstein, and L. Poggio. Laboratorio de Citogenetica y Evolucion, Departamento de Ecologıa, Genetica yEvolucion, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Guiraldes 2160, Buenos AiresC1428EGA, Argentina.S. Martınez. Departamento de Biodiversidad y Biologıa Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de BuenosAires, Intendente Guiraldes 2160, Buenos Aires C1428EGA, Argentina.

1Corresponding author (e-mail: gperthuy@ege.fcen.uba.ar).

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vino et al. (2008) carried out a phylogenetic analysis usingnuclear and chloroplast DNA, postulating that the genusevolution has been influenced by the combined effect oflong-distance dispersal, hybridization, and rapid radiation.

In Argentina, the genus is represented by 31 species be-longing to seven sections, mainly distributed in the north-eastern and central regions (Martınez 1999, 2005; Martınezand Calvino 2000; Martınez and Galotti 2001). One third ofthe species are included in the Eryngium sect. Panniculataser. Latifolia, which is characterized by erect plants with‘‘monocotyledonous’’ habit, linear or subulate leaves withstrictly parallel first-order veins, and fruits displaying lateralwings. The species of this group differ by plant size, leafmargin (setose or spiny), shape and size of the capitate inflor-escence, and presence or absence of dorsal vesicles in thefruits. Some of this species are important weeds (E. horri-dum), whereas others are used as forages (E. paniculatum) inarid and semiarid areas of the country (Sabattini et al. 1989).

Different ploidy levels in Eryngium ser. Latifolia are re-ported (Mathias and Constance 1971; Constance 1977), butthere is no information on the ploidy levels of E. balansaeand E. regnellii. In addition, two chromosome numberswere reported for E. paniculatum, i.e., 2n = 4x = 32 fromValparaiso, Chile (Constance et al. 1971) and 2n = 6x = 48from Talca, Chile (Bell and Constance 1960). Studies on theorigin and evolution of the polyploid species in this groupand on their relationship with the diploid taxa have only re-cently been initiated.

Cytogenetic studies to date involve the analysis of chro-mosome number and karyotype (Calvino et al. 2002), mei-otic chromosome behavior (O’Leary et al. 2004), andheterochromatin characterization (Perthuy et al. 2006). Theregular meiotic behavior observed in all the polyploid spe-cies of Eryngium suggests an allopolyploid origin (O’Learyet al. 2004). Based on morphological comparisons, karyo-type morphology, and geographical distribution, O’Leary etal. (2004) proposed a hybrid origin for the tetraploid E.megapotamicum with the diploids E. eburneum and E. horri-dum as putative progenitors. The authors hypothesized thatthese three species could have a role in the origin of the

hexaploid taxa of Eryngium, because the hexaploid levelcan be interpreted as the result of an allopolyploid event be-tween a diploid and a tetraploid species.

Mathias and Constance (1971) proposed that E. pandani-folium and its related entities, the hexaploid species E. mes-opotamicum, E. balansae, and E. chamissonis, represent apolyploid complex. Several processes of hybridization andpolyploidy could have originated the different chromosomenumbers found in this complex (O’Leary et al. 2004).

Genomic in situ hybridization (GISH) is a well recog-nized technique to detect genomic relationships between re-lated species, especially in hybrid plants and allopolyploids,and recently it has been used to complement phylogeneticstudies (for a review see, Markova and Vyskot 2009).

The aim of the present study is to compare the genomesof species of Eryngium ser. Latifolia using GISH and to testthe above-mentioned hypothesis about the origin of thesespecies. We analyze the genomes of the diploids E. horri-dum and E. eburneum, the tetraploids E. megapotamicumand E. regnellii and the hexaploids E. pandanifolium (as arepresentative of the whole pandanifolium complex) and E.paniculatum. The chromosome number and karyotype of E.regnellii are reported for the first time and the ploidy levelof a population of E. paniculatum is confirmed.

Materials and methods

Plant materialAll the material studied was collected by the authors from

different populations in Argentina and Uruguay (Table 1).

Chromosome preparationRoot tips were taken from freshly germinated seeds and

pretreated in a 2 mmol/L 8-hydroxyquinoline solution for3.5 h at room temperature. The tissues were then fixed inabsolute ethanol – acetic acid mixture of 3:1 (v/v) at 4 8Cfor 5 days and then stored in 70% ethanol at 4 8C.

For karyotype analyses, root tips were stained using Feul-gen’s technique for 90 min after hydrolysis in 5 mol/L HClfollowed by digestion with cellulase and pectinase (1.6%

Table 1. Origin of the samples used for cytogenetic analyses.

SpeciesChromosomeno. Origin Collector and date

Eryngium horridum 2n = 16 Santa Ana, Uruguay Greizerstein, January 2006San Jose, Dept. Colon, Entre Rıos Perthuy & Greizerstein, March 2006RN 14 km 25, Dept. Gualeguaychu, Entre Rıos Perthuy & Greizerstein, March 2006RN 14 km 230, Dept. Colon, Entre Rıos Perthuy & Greizerstein, March 2006Park San Carlos, Concordia, Entre Rıos Perthuy & Greizerstein, March 2006Villaguay, Entre Rıos Perthuy & Greizerstein, March 2006

Eryngium eburneum 2n = 16 RN 12, near Gualeguay Martınez, 2005RN 14 km 10, Dept. Gualeguaychu, Entre Rıos Perthuy & Greizerstein, March 2006RN 14 km 170, Dept. Colon, Entre Rıos Perthuy & Greizerstein, March 2006

Eryngium megapotamicum 2n = 32 Park San Carlos, Concordia, Entre Rıos Perthuy & Greizerstein, March 2006Eryngium regnellii 2n = 32 Piriapolis, Uruguay Martınez, January 2005

Sierra de los Padres, Buenos Aires Perthuy, March 2007Eryngium pandanifolium 2n = 48 Stream Los Loros, El Palmar, Dept. Colon, Entre Rıos Perthuy & Greizerstein, March 2006Eryngium paniculatum 2n = 48 Small Circuit, Bariloche, Rıo Negro Molares, 2006

Bariloche, Rıo Negro Perthuy, January & March 2007

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cellulase (Calbiochem), 0.4% cellulose Ozonuka RS(Merck), 20% pectinase (Sigma), in enzyme buffer 6 mmol/Lsodium citrate, 4 mmol/L citric acid, pH 4.6). Chromosomepreparations were made with 0.5% acetic hematoxylin us-ing the squash technique.

For in situ experiments, chromosome squash preparationswere made according to Schwarzacher and Heslop-Harrison(2000) with previous enzymatic treatment of the mitotic tis-sue (1.6% cellulase (Calbiochem), 0.4% cellulase OzonukaRS (Merck), 20% pectinase (Sigma), in enzyme buffer6 mmol/L sodium citrate, 4 mmol/L citric acid, pH 4.6) at37 8C for 1 h. Preparations were stored at –20 8C until use.

Genomic DNA probesTotal genomic DNA from dry collected leaves was iso-

lated using the Wizard Genomic DNA purification kit(Promega) and labeled using DIG High Prime (BoehringerMannheim, Germany) or a Biotin Nick Translation kit(Boehringer Mannheim, Germany) according to manufac-turer’s instructions.

Genomic in situ hybridization (GISH)The preparations were incubated with RNase solution

(100 mg/mL) at 37 8C for 1 h. Then the slides were post-fixed in freshly prepared 4% (w/v) paraformaldehyde, dehy-drated in a graded ethanol series, and air-dried.Chromosome and probe denaturation and in situ hybridiza-tion were carried out as described by Cuadrado and Jouve(1994) with a hybridization stringency of 85%.

Slides were treated with sheep anti-digoxigenin–FITC(Roche) and streptavidine-Cy3 conjugate (Sigma), to detectdigoxigenin- and biotin-labeled probes, respectively.

Slides were counterstained with DAPI (4’,6-diamidino-2-phenylindole) and then mounted in Vectashield (Vector Lab-oratories).

Image analysesThe images were captured with a Leica epifluorescence

microscope equipped with a digital camera (Leica DFC 350FX) using the Leica IM50 version 4.0 program (Leica Mi-crosystems, Cambridge, UK). Images were analyzed usingAdobe Photoshop 7.0 software.

Karyotype analysisChromosome measurements were made using the com-

puter application MicroMeasure version 3.01 (available athttp://www.colostate.edu/Depts/Biology/MicroMeasure) andthe karyotype was made using Adobe Photoshop 7.0 soft-ware.

ResultsEryngium regnellii is a tetraploid species 2n = 4x = 32

(Fig. 1a). Secondary constrictions are observed located atthe short arm of the submetacentric chromosome 11, whichis the largest of the complement, and on the metacentricchromosome 7. The karyotype formula is 10m + 6sm(Fig. 1c).

The E. paniculatum population from Bariloche, Argentina

Fig. 1. Metaphase chromosomes of Eryngium regnellii and Eryngium paniculatum. (a) E. regnellii chromosome number 2n = 4x = 32 withthree satellite chromosomes (arrows); (b) E. paniculatum chromosome number 2n = 6x = 48 with four satellite chromosomes (arrows);(c) karyogram of E. regnellii with a karyotype formula of 10m + 6sm; (d) karyogram of E. paniculatum with a karyotype formula of11m + 6m-sm + 6sm + 1st. Bar indicates 5 mm.

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Fig. 2. GISH on somatic metaphase cells from root tips of diploid and tetraploid taxa of Eryngium. Sites of probe hybridization fluorescered with Cy3-conjugated avidin and green or yellow with antidigoxigenin-FITC, whereas non hybridized sites fluoresce blue with DAPIcounterstaining. (a,b) E. eburneum (2n = 2x = 16). (a) Probed with biotinylated total genomic DNA of E. horridum (2n = 2x = 16), redsignal is weak and dispersed along all chromosomes. (b) DAPI counterstaining overlapped with a. Arrows indicate two hybridized DAPI-positive regions. (c,d) E. horridum. (c) Probed with biotinylated total genomic DNA of E. eburneum, four chromosomes are completelyhybridized (arrows) and one chromosome pair (arrowheads) presents a strong red signal in one chromosome arm. (d) DAPI counterstainingoverlapped with c. (e–g) E. megapotamicum (2n = 4x = 32). (e) Probed with biotinylated total genomic DNA of E. horridum. (f) DAPIcounterstaining overlapped with e. Almost all telomeric DAPI-positive regions remain blue indicating absence of hybridization, arrowheadsshow six of them. (g) E. megapotamicum probed with total genomic DNA of E. eburneum and labeled with digoxigenin-11-dUTP (yellow).(h–k) E. regnellii (2n = 4x = 32). (h) DAPI counterstaining. (i) E. regnellii probed with biotinylated total genomic DNA of E. eburneum.Red signals are dispersed over the entire complement. The telomeric DAPI-positive regions do not show signals of hybridization, arrow-heads indicate two of them. (j) E. regnellii probed with total genomic DNA of E. horridum and labeled with digoxigenin-11-dUTP. All ofthe 32 chromosomes present green signals clustered on certain regions, arrows show three of them. No signal appears on telomeric DAPI-positive regions, arrowheads indicate two of them. (k) DAPI counterstaining overlapped with j. Sites of probe hybridization fluoresce yel-low, whereas non hybridized sites fluoresce blue. Arrows and arrowheads indicate the same as in j. (l–m) E. megapotamicum probed withbiotinylated total genomic DNA of E. regnellii. (l) Red signals appear over the entire complement in an intensity gradient. (m) DAPI coun-terstaining overlapped with l. DAPI-positive regions remain blue, whereas the rest is entirely hybridized. Bar indicates 2.5 mm (a–d) and5 mm (e–m).

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is hexaploid 2n = 6x = 48. It shows secondary constrictionslocated at the metacentric chromosome 9 and the short armof the subtelocentric chromosome 24 (Fig. 1b). The karyo-type formula is 11m + 6m-sm + 6sm + 1st (Fig. 1d).

Genomic relationships using the GISH techniqueEryngium horridum (2x) and E. eburneum (2x) were

studied using GISH without blocking DNA. The total ge-nomic DNA from one species was biotin labeled and usedas a probe on metaphase chromosomes from the other spe-cies.

When a total DNA probe from E. horridum was hybri-dized on chromosome preparations of E. eburneum, the sig-nal was weak and dispersed along all chromosomes. Nohybridization of the DAPI-positive regions was observed ex-cept for one chromosome pair (Figs. 2a and 2b). In the re-ciprocal experiment using a total DNA probe from E.eburneum on chromosome preparations of E. horridum, fourchromosomes were completely hybridized and one chromo-some pair showed a strong signal on one chromosome arm,whereas the remaining chromosomes show weak dispersedsignals. No hybridization of the DAPI-positive regions wasobserved (Figs. 2c and 2d).

To analyze the relationships among diploid and polyploidgenomes, the total genomic DNA from one diploid specieswas labeled with biotin, whereas the total genomic DNAfrom the other diploid species was labeled with digoxigeninfor simultaneous hybridization of the two diploid species onthe polyploid species.

When the E. eburneum (2x) probe was hybridized on E.regnellii (4x) metaphase chromosomes, hybridization signalswere distributed over the entire complement, albeit strongeron certain chromosomal regions. These were moderate inpericentromeric DAPI-positive regions and weak or unde-tectable in terminal ones (Figs. 2h and 2i). When E. horri-dum (2x) was used as a probe, hybridization signals werestrong and clustered on the centromeric and pericentromericregions of all the chromosomes. Similarly to that observedfor E. eburneum, there were hybridization signals in pericen-tromeric DAPI-positive regions but not in the terminal ones(Figs. 2h, 2j, and 2k).

The hybridization of E. megapotamicum (4x) mitoticchromosomes with an E. horridum (2x) biotin-labeled probeand an E. eburneum (2x) digoxigenin-labeled probe showedthe same pattern. For both probes, most chromosomes

showed weak signals, with the exception of certain stronglyfluorescent regions. Hybridization signals were absent inmost terminal DAPI-positive regions (Figs. 2e–2g).

Total genomic DNA from E. regnellii (4x) was biotin-la-beled and hybridized on E. megapotamicum (4x) metaphasechromosomes. Hybridization signals were spread over thegenome showing a variable intensity, except for mostDAPI-positive regions (Figs. 2l and 2m).

A simultaneous hybridization was performed with an E.eburneum (2x) biotin-labeled probe and an E. horridum (2x)digoxigenin-labeled probe on E. paniculatum (6x) metaphasechromosomes. The same hybridization pattern was obtainedwith the two diploid taxa. No hybridization signal was ob-served in DAPI-positive or adjacent regions (Figs. 3a–3c).A rehybridization experiment was carried out on the samemetaphase chromosomes of E. paniculatum using an E.megapotamicum (4x) biotin-labeled probe (in yellow). All48 chromosomes fluoresce yellow showing an intensity gra-dient. The hybridization signal was stronger in approxi-mately 16 chromosomes. Most DAPI-positive regionsshowed hybridization signals (Fig. 3d).

To study the E. pandanifolium (6x) genome, a hybridiza-tion experiment was carried out on the hexaploid chromo-somes using total DNA of E. eburneum (2x) and E.horridum (2x) as probes. Red and green signals were ob-served on the same chromosome regions. Hybridization sig-nals were strong along the length of eight chromosomes,absent in seven chromosomes, and clustered in certain re-gions in the rest of the complement (Figs. 3e–3h). A GISHexperiment was performed on E. pandanifolium chromo-somes using an E. megapotamicum (4x) biotin-labeled probeand an E. horridum (2x) digoxigenin-labeled probe. Whenusing the tetraploid probe, hybridization signals were weakin 16 chromosomes and strong all over or in some regionsof the remaining chromosomes (Figs. 3i and 3j). When usingthe E. horridum probe, eight chromosomes failed to showhybridization signals (Fig. 3k). The combination of these re-sults revealed that E. megapotamicum probe hybridized onthe same chromosome regions as the E. horridum probe.

DiscussionIn the species of the series Latifolia studied in the present

paper, the chromosome number and therefore the ploidylevel of E. regnellii is reported for the first time as being atetraploid species. Only metaphase and submetaphase chro-

Fig. 3. GISH on somatic metaphase cells from root tips of hexaploid taxa of Eryngium. Sites of probe hybridization fluoresce red with Cy3-conjugated avidin and green or yellow with antidigoxigenin-FITC, whereas non hybridized sites fluoresce blue with DAPI counterstaining.(a–d) E. paniculatum (2n = 6x = 48). (a) Probed with biotinylated total genomic DNA of E. eburneum (2n = 2x = 16). (b) DAPI counter-staining overlapped with a. Telomeric DAPI-positive regions do not show signs of hybridization, arrowheads indicate four of them. (c) E.paniculatum probed with total genomic DNA of E. horridum (2n = 2x = 16) and labeled with digoxigenin-11-dUTP. (d) E. paniculatumprobed with total genomic DNA of E. megapotamicum (2n = 4x = 32) and labeled with digoxigenin-11-dUTP (yellow). All the comple-ments show signs of hybridization in an intensity gradient as well as telomeric DAPI-positive regions (arrowheads). (e–k) E. pandanifolium(2n = 6x = 48). (e) Probed with biotinylated total genomic DNA of E. horridum. (f) DAPI counterstaining overlapped with e. Eight chro-mosomes are completely hybridized (arrowheads), whereas seven chromosomes show no hybridization signals (arrows). (g) E. pandanifo-lium probed with total genomic DNA of E. eburneum and labeled with digoxigenin-11-dUTP. (h) Images e and g overlapped show that bothdiploid have the same affinity with the hexaploid. (i) DAPI counterstaining. (j) Same cell as in i, probed with biotinylated total genomicDNA of E. megapotamicum. Hybridization signals are weak on sixteen chromosomes (arrowheads). (k) Same cell as in i, probed with totalgenomic DNA of E. horridum and labeled with digoxigenin-11-dUTP. Eight chromosomes fail to show hybridization signals (arrowheads).Bar indicates 5 mm.

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mosomes are observed in this species and two chromosomepairs with secondary constriction are detected. Eryngiummegapotamicum, the other tetraploid species of Eryngiumser. Latifolia, has a similar karyotype and the same numberof secondary constrictions than E. regnellii but are locatedon different chromosomes (O’Leary et al. 2004).

In E. paniculatum different cytotypes have been found.Basic chromosome numbers of n = 24 and n = 16 havebeen reported in the Chilean populations of E. paniculatumfrom Talca (Bell and Constance 1960) and Valparaiso (Con-stance et al 1971), respectively. The population of E. pani-culatum of the locality of Bariloche studied in this paper

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resulted to be hexaploid similar to the population fromTalca.

To analyze the genomic relationship between the speciesof the series Latifolia, separate GISH experiments were per-formed.

When E. horridum was labeled and hybridized over E.eburneum chromosomes, a weak and disperse signal was ob-served. Therefore, E. eburneum has dispersed repeated DNAsequences in all chromosomes that are also present in E.horridum. On the other hand, the highly repeated DNA se-quences (DAPI-positive) of E. eburneum would not bepresent in E. horridum with the exception of a chromosomepair. A similar homology pattern was observed in the recip-rocal hybridization using biotinylated genomic DNA of E.eburneum, except in four chromosomes where the hybridiza-tion signal was stronger. The absence of hybridization sig-nals in the DAPI-positive regions indicates that the highlyrepeated DNA sequences of E. horridum are not present inthe E. eburneum genome. Importantly, the presence of achromosome pair with only one chromosome arm hybri-dized suggests the presence of intergenomic rearrangements.In summary, the low sequence homology between themmight be a result of a common ancestry, and the lack of se-quence homology in the DAPI-positive bands may suggestdivergent evolution of the heterochromatin regions (Perthuyet al. 2006). The results obtained in the present paper areconsistent with the distinctive morphology of the diploidsand with the results of phylogenetic studies on Eryngium,revealing that E. horridum and E. eburneum belong to dif-ferent monophyletic groups included within a clade of SouthAmerican species (Calvino et al. 2008).

When the tetraploid E. megapotamicum was hybridizedwith E. eburneum and E. horridum probes, a low homologywas observed suggesting that both diploid species were notinvolved in the origin of the allotetraploid. On the otherhand, the lack of hybridization signals in some chromosomeregions of E. megapotamicum might suggest that a diploidspecies unrelated to E. horridum and E. eburneum was im-plicated in its origin. Thus, our data do not support the hy-pothesis of O’Leary et al. (2004) that proposes E.megapotamicum was originated from the studied diploids.Therefore, the observed homology may possibly be a resultof a common ancestry. This is in agreement with the ab-sence of a common ancestor for the studied tetraploid anddiploid species on the phylogenetic tree proposed by Cal-vino et al. (2008).

The GISH experiments on E. regnellii chromosomesshowed strong hybridization signals on discrete centromericand pericentromeric regions with the E. horridum probe, anddisperse ones along the chromosomes with the E. eburneumprobe. Therefore, the tetraploid seems to have a closer rela-tionship with E. horridum than with E. eburneum indicatingthat the former species, or a related one, might have partici-pated in the origin of the tetraploid. This idea is supportedby morphological and ecological data as well as by phyloge-netic studies of the genus in which E. regnellii and E. horri-dum appear as sister species (Calvino et al. 2008).

Our results reveal a close relationship between the tetra-ploids E. regnellii and E. megapotamicum, suggesting thatthey share a common diploid ancestor different from E. hor-

ridum and E. eburneum and that they arose from independ-ent hybridization events.

As for the genomic relationship between diploid and hex-aploid species, E. horridum (2x) and E. eburneum (2x)probes overlap on the same chromosome regions of E. pan-iculatum (6x) chromosomes, suggesting that one of the spe-cies involved in the origin of this hexaploid is closelyrelated to the diploids studied. However, the heterochroma-tin-rich telomeric regions show no homology with those ofthe diploids, indicating loss of these highly repeated sequen-ces or even an independent origin of these sequences in thehexaploid species.

The rehybridization of E. paniculatum (6x) chromosomeswith the E. megapotamicum (4x) probe resulted in a highhomology between both species. Fluorescence was not onlyobserved at sites that show hybridization signals using thediploid species as probes, but also in other regions of thehexaploid genome including heterochromatin-rich telomericregions. Thus, our results support the phylogenetic sistergroup relationship between E. paniculatum and E. megapo-tamicum proposed by Calvino et al. (2008). On this basis,we postulate that E. paniculatum would have derived fromE. megapotamicum or from a diploid species giving rise toboth tetraploids, which would not be closely related to thediploids included in this study.

GISH experiments on E. pandanifolium (6x) chromo-somes using diploids E. horridum and E. eburneum probesshowed a low homology in all the chromosomes. However,the fact that both diploids hybridize overlapping in the samechromosome regions of the hexaploid might indicate a com-mon ancestry. According to Calvino et al. (2008), E. panda-nifolium is phylogenetically less related to E. eburneum thanto E. horridum. However, the latter taxon is grouped into apolytomy with the other species of the pandanifolium com-plex, E. regnellii and a species of Eryngium sect. Areata.This was not reflected with the methodology used in thisstudy, as both techniques demonstrated the divergence andevolution from a different part of the genome.

On the other hand, the hybridization of E. megapotami-cum (4x) probe on the hexaploid E. pandanifolium showedthat the hybridization signals overlapped with some regionshybridized by the diploids. Thus, the observed affinity islikely to be a result of sequences in common with E. panda-nifolium, E. megapotamicum, and the diploids E. horridumand E. eburneum. This proves the occurrence of highly ho-mologous regions within the series Latifolia.

It is interesting to point out the presence of partially hy-bridized chromosomes in the studied polyploid species thatwould indicate the occurrence of chromosome rearrange-ments. This is in agreement with Soltis and Soltis (1999)who postulated that the intra- and intergenomic reorganiza-tion is a frequent event in polyploid species.

In summary, the obtained results in this study are inagreement with Calvino et al. (2008) who proposed that arapid diversification of the group could be both the causeand consequence of hybridization events and subsequent in-trogression. The GISH technique turned out to be a usefultool to analyze genomic relationships, although it was notpossible to identify the parental species of the polyploids an-alyzed. The origin of these polyploid species, which involvehybridization and secondary polyploidy, might be clarified

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with morphologic, cytogenetic, and molecular analysis indiploid species of other series of the genus Eryngium.

AcknowledgementsThis research was supported by grants of the Consejo Na-

cional de Investigaciones Cientıficas y Tecnicas (CONICET,PIP 5927), Agencia Nacional de Promocion Cientıfica yTecnologica (ANPCyT, PICT 14119), and the University ofBuenos Aires (grant No. X317).

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