ISSN 0373-580 X

Bol. Soc. Argent. Bot. 35 (3-4): 275 - 282. 2000

NATURAL HYBRIDIZATION IN HYPOCHOERIS(ASTERACEAE-CICHORIOIDEAE)*

ALICIA D. BURGHARDT13 and ARTURO F. WULFF23

Summary: Two Hypochoeris L. species: H. chillensis (Kunth) Hieran., H. microcephala var. albiflora (Kuntze)Cabrera and their putative hybrids has been analyzed by numerical methods, using 21 morphometric or qualitativecharacters, in order to study the composition of a hybrid swarm growing in Ciudad Universitaria (BuenosAires). Principal Component Analysis (PCA), Wells’ Distance Diagram, and Wilson's Character Count Procedurewere applied for predicting hybridity. Morphology of pollen and achene surface observed by SEM are reported.The significance of the hybridization is discussed in relation with the geographical distribution and themorphological variation observed inH. chillensis.

Keywords: Hybridization,Hypochoeris, numerical analysis, pollen morphology, achene surface.

Resumen: Hibridación natural en Hypochoeris (Asteraceae-Cichorioideae). Dos especies de Hypochoeris:H. microcephala var. albiflora (Kunth) Hieran., H. chillensis (Kuntze) Cabrera y sus probables híbridos fueroncaracterizados numéricamente usando 21 caracteres. Para diagnosticar esta hibridación se utilizó el Análisisde Componentes Principales (PCA), el diagrama de Wells y el índice de Wilson. El estudio incluyó el análisisde la superficie del aquenio y del polen por medio de microscopio electrónico de barrido (MEB). El significadode esta hibridación es discutido en relación al área de distribución geográfica y variación morfológica observadaen H. chillensis.

Palabras clave: Hibridación, Hypochoeris, análisis numéricos, polen, superficie de aquenio.

regions along the Andes, humid and arid plains inArgentina (including Patagonia) and tropical and

The genus Hypochoeris L. is distributed in subtropical regions of South Brazil.At least, one ofthe most widely distributed South

INTRODUCTION

Western Eurasia and South America. Differentauthors (Stebbins, 1971; Tomb, 1977) reported va- American species, H. chillensis (Kunth) Hieron.

riable number of species for the genus (from 50 to (the” achicoria” or “cerraja”) shows, as H. radicata,

100). The species are classifiedinto five sections and a wide morphological variation, whichhas originatedmore than one confusion at taxonomic level.only one of them -Achyrophorus (Scop) Benth. &

Hook.- is represented in America.Some of the European species (i.e., H.

radicataL.) show considerable variability originatedby natural hybridization. This evolutionary processis responsible of variation in morphologicalcharacters such as leaf shape, root form, and capitu¬la, arising a lot of subspecies, varieties, and forms(Aarssen, 1981).

The South American species, all belongingto the same section, occupy different habitats: high

Hypochoeris chillensis, like H. radicata have aperennial root system.

Hypochoeris chillensis includes two taxapreviously described as species: H. tweedie (Kunth)Cabrera and H. brasiliensis (Less) Benth. & Hook.ex Griseb. Both were considered by Cabrera (1976)as variations without systematic value.

Natural hybridization between H. chillensis andother two species has been previously reported(Wulff, 1992; Wulff & Burghardt, 1994; Burghardt& Wulff, 1996). The present work was carried outin order to analyze, by means of a numerical studybased on 21 qualitative and quantitativemorphological characters, the occurrence of naturalhybridization between H. chillensis and H.

microcephala var. albiflora (Kuntze) Cabrera. Somepalynologic and fruit surface characters, useful forthe analysis, are included as well.

* Dedicated to Prof. Dr. Juan H. Hunzikeron the ocassion ofhis 75th anniversary.

'Depto. de Ciencias Biológicas, Facultad de Ciencias Exactasy Naturales, Universidad de Buenos Aires C1428EHA. CiudadUniversitaria Buenos Aires, ARGENTINA.2Plantas Vasculares, Miembro de la Carrera del InvestigadorCientífico CONICET, e-mail: alibu@bg.fcen.uba.ar

3Genética,Profesional Principal CONICET, e-mail:artulf@bg.fcen.uba.ar

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Bol. Soc. Argent. Bot. 35 (3-4) 2000

Numerical Analysis ofHybridizationFor numerical analysis the following characters

were used (codification of the stages of qualitativecharacters are included):

MATERIALS AND METHODS

Materials

Hypochoeris chillensis

ARGENTINA, Ciudad Autónoma de Buenos Ai¬res. Ciudad Universitaria: 15-11-93 A.F.Wulff 602,603, 605, 606, 608, 796, 797, 798, 799, 800, 801.

1) Shape of the involucre (0=bell-shaped, 1intermediate,2=cylindrical)

2) Apical diameter of the involucre (mm)3) Lateral diameter of the involucre (mm)4) Longof the involucre at the flowering(mm)5) Longof the involucre at the fructification(mm)6) Ligules sticking out of the bracts involucre (0=absent,

l=present)7) Number of flowers per capitulum8) Flower color (0=white, l=pale yellow,2=brillant yellow)9) Palea length without cirrus (mm)

. 10) Cirrus length (mm)11) Corolla-tube length (mm)12) Ligule length (mm)13) Achene color (0=redish brown; 1intermediate;

2=olivaceousbrown)14) Achene ornamentation (H. chillensis like=0,

intermediated,H. microcephala liked)15)Number ofbranchingpoints16) Number ofbasal stems .17)First intemode length(cm)18) Basal diameter of stems (cm)19) Plant height (cm)20) Trichome arrangement oftheachenebeak apex (sparse=0,

fasciculatedanddispersedi, fasciculated=2)21) Achene scales boundary (thin=0, irregular thickeningd,

thick=2)

H. microcephala. var. albifloraARGENTINA, Ciudad Autónoma de Buenos Ai¬

res, Ciudad Universitaria: 15-11-93 A.F. Wulff 802,803, 804, 805, 806, 807, 808, 809, 810, 811, 812.

Putative hybrids

ARGENTINA, Ciudad Autónoma de BuenosAires, Ciudad Universitaria: 15-11-93 A.F. Wulff610, 611, 612, 613, 614, 615, 616, 617, 618.

Vouchers are deposited at the herbarium of theFacultad de Ciencias Exactas y Naturales (BAFC).

Methods

Pollen morphology

Pollen grains fromherbarium specimens for lightmicroscopy (LM) studies were mounted inglycerinejelly and sealed with paraffin wax. Samples forscanning electron microscopy (SEM) were mountedon aluminum stubs using double-sided self-adhesivetape. The SEMpreparations were sputter coatedwitha thin gold layer before viewing and photographing.

Description was made following El-Ghazaly(1980) and Punt et al. (1994). Measurements of theequatorial diameter of pollen grains were made on

Measurements of characters 2, 3, 4, 5, 7, 9,10, 11, and 12 were taken with a stereomicroscopeLeitz and amicrometric ocular. Whenit waspossible,ten measures were made for each character and the

mean was calculated.

Principal Component Analysis

Individuals of the two taxa and the naturallight microscope samples; averages from three sets hybrids were the operative to0nomicalunits (OTU).of 30 pollen grains of each taxon were calculated. A Principal Component Analysis (PCA) was applied

using all the characters above cited and performedusing NT-Sys Program, version 1.8 (Rohlf, 1993).Pollen fertility

Pollen stainability was studied withAlexander's differential staining (1969). For each Wells Distance Diagram (Distance Diagram)individual, 500 pollen grains were observed.

The hybridization hypothesis was analyzedbymeans of the method proposed by Wells (1980). InSEMmorphological traits of achene surface

Achenes from specimens of the three taxa for order to perform the Distance Diagram, each

SEM weremountedin the same way above described, specimen was considered as an operationaltaxonomic unity (OTU) . The same characters used

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A. D. Burghardt & A. F. Wulff, Hybridization in Hypochoeris

for PCA were registered and codified. As reference Solidum tectum (Fig. 1I). Oblongus os.points, individuals of H. chillensis and H.microcephala var. albiflora with morphologicalcharacteristics falling within the described range byCabrera (1963) were utilized.

Pollen fertility was 92-98% for both parentspecies, while the putative hybrids pollen fertilityvaried from 34 to 60%.

SEMmorphological characteristics of acheneCharacter Count Procedure

Hypochoeris chillensis showed achenebeak apexA Character Count Procedure (Wilson, 1992) onthe same individuals was carried out, but only with sparse, adpressed trichotomata of 16.7 pm

considering the quantitative characteristics, as this mean length (13-21pm) (Fig. 2 A). Scales with thin

method indicates. The analysis was as follows: 1) ciliate margin; cilia straight to recúrvate of 19 pm

Each individual was assigned to one of the three (13-24 pm) (Fig. 2 B, C).

groups (H. chillensis,H. microcephala var. albiflora,and the putative hybrids), 2) Characters for dispersed trichotomata, most of them adpressed,

separating parents were selected, 3) For each 18.8 pmlength (8.7-26 pm) (Fig. 2D). Scales with

character, was determined and tabulated whether or irregular boundary, irregular thickening, ciliate,not the values for the hybrids were intermediate, 4) cilia ineurvate, 11.9 pm length (8.7-17.4 pm)

The number of intermediate characters was divided (Fig. 2 E, F).

Putative hybrids beak apex with fasciculate and

Hypochoeris microcephala var. albiflora showedbeak apex with fasciculate trichotomata,not adpressed,31.7 pm length (26.1-39.1 pm ) (Fig. 2 G). Scaleswith thick boundary, ciliate, cilia ineurvate, 7.2 pmlength (4.3-13 pm ) (Fig. 2 H, I).

by the total number of considered characters.

RESULTS

Pollen morphologyNumerical Study ofHybridization

All pollen grains arç equinolophate andtricolporate. Fig. 1shows pollen grains of the threetaxa in polar and equatorial views.

Principal Component AnalysisThe position of the OTUs in a space delimited

Hypochoeris chillensis: Equatorial diameter by the three firstprincipal components revealed three

32 pm-38pm (mean=35 pm). Triangular amb with groups well defined without any continuity among

right supratectal spines (Fig. 1 A). Equatorial ridges them. The hybrid group was in an intermediate

withtwo series ofspines (Fig. 1B). Spinoseparaporal position between individuals of both species that

lacunae (Fig. 1 C). Puhctitegilate tectum (Fig. 1 C). could be its putative parents (Fig. 3).

Circular elliptic os.Distance Diagram

The Distance Diagram is showedinFig. 4. Theindividuals fromboth species were assembledin twodifferent groups and those that show intermediacywere located in an intermediate area between them.These specimens were located into the semicirclethat includes all the samples of H. chillensis and H.

Hypochoeris microcephala var. albiflora: microcephala var. albiflora.Equatorial diameter rather constant: 30 mm - 34

Putativehybrids: Equatorial diameter ranged from30 pmto 38 pm(mean=33.14 pm). Circular amb withslightly curvedsupratectal spines (Fig. 1D).Equatorialridges with a single series of spines (Fig. 1 E).Paraporal lacunae with or without spines (Fig. 1 F).Punctitegilate tectum (Fig. 1 F). Circular-elliptic os.

pm (mean=33.2 pm). Circular amb with slightly ’

curved supratectal spines (Fig. 1 G). Equatorialridges with a single series of spines (Fig. 1 H). putativehybrids showedintermediacy while only oneParaporal lacunae always without spines (Fjg. 1 I). was not intermediate (Table 1).

Character Count ProcedureMeans of nine quantitative characters in the

277

Bol. Soc. Argent. Bot. 35 (3-4) 2000

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Fig.1. Pollenmorphology with SEM. A-C:H. chillensis. D-F: Hybrid. G-I:H. microcephala. A,D and G=polar view; C,D,E,F,Hand1= equatorial view. Bar indicates 10 pm.

278

A. D. Burghardt& A. F. Wulff, Hybridization in Hypochoeris

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Scales. Bar indicates 100 pm.

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Bol. Soc. Argent. Bot. 35 (3-4) 2000

H. chillensisw

H. microcephala2

O#

fHybrids

ft Y COMPONENTmí _1°COMPONENT

Fig.3.FirstPrincipalComponent Analysis (PCA)projectionofthe twoHypochoeris species andtheirputativehybrids.

•*••* N.

Fig. 4. Wells distance diagram ofH. chillensis ( •).H, microcephala var. albiflora ( y) and their putative hybrids (ÿ).

series of spines were traits shared with H:microcephala.

The surface of the achene in the hybrids showedintermediate features between those of the putativeparents. The disposition of trichotomata on the beakapex, thickness of scales margins, and also averageof cilia length were evidently intermediate.

Character CountProcedure was successfullyusedfor inferring hybridity in Astéraceae (Bartoli &Tortosa, 1998a, b). Our results using this procedureindicated that the hypothesis of divergence must berejected. The deviation was' very significant (P0.05%) and confirmed the hypothesis of hybridity.

Principal Component Analysis was used forinferring hybridity or to show swarm structure (Pa¬lacios et al., 1991; Mollard et al., 2000). The resultsherepresented showed three groups ofpoints withoutcontinuity between them and the intermediateindividuals were located in intermediate positionsinto the 3-dimensional space delimited by the threefirst principal components. Accordingly, theintermediate individuals could be considered as F,hybrids.

DISCUSSION AND CONCLUSIONS

Hybridization results in intermediacy betweenparents in many characters. Most of the numericalmethods are based in this feature to demonstratehybridity (Anderson, 1953; Adams, 1982; Wilson,1992). All of the analyses here performed coincidedinshowing thehybridswarmas composedbyhybridsH. microcephala var. albiflora x H. chillensis.

Pollen grain size was rather constant in H.microcephala var. albiflora while H. chillensisexhibits a wider range of size pollen grains. Rangein hybrids includes all pollen grain sizes,of bothputative parents, the mean of the hybrids is smallerthan theH. microcephala one,because most ofhybridgrains were small. This feature may be relatedprobably to deficiencies intheir genomic compositionderivedòfcryptic structuralhybridity (Wulff, 1992).

Some characters of the hybrid pollen grains(punctitegilate tectum, presence of spjnes inparaporal lacunae, circular elliptic os) were sharedwith H. chillensis. Circular amb, curvature ofsüpratectal spines, and equatorial ridges with single

A. D. Burghardt& A. F. Wulff, Hybridization in Hypochoeris

Table1. Character Count Procedure (Willson's Method)

Xx XIntermediateCharacter Ô 8 5

H. microcephalaH.chillensis Hybrid

7.127 0.9983 7.85 0.988 7.844 1.574 +19.635 41.818 6.047170.75 20.356 93.4447 +

2.531 0.413 2.538 0.25810 1.951 0.24 +0.702 4.412 0.295.42 1.077 5.153 +

3.163 0.373 3.481 0.36112 2.208 0.1481.922 3.182 0.7516.917 1.73 4,77815

2.556 1.74 9.091 1.44616 3.25 1.4229.761 2.863 5.451 0.84717 17.792 7.074 +0.233 0.048 0.146 0.02718 0.386 0.12

42.489 8.244 23.845 2.2519 52.625 5.6939:1

and cytogenetics. This work wishes to express animmense gratitude to our teacher in both scientificformation and ethics of life.

In the Diagram Distance, individuals from eachspecies constituted a different group and the putativehybrids were locatedinan intermediate areabetweenthem. The intermediate specimens were located intothe semicircle that includes the specimens of H.chillensis and H. microcephala var. albiflora,another evidence confirming hybridization betweenboth species.

All the numerical methods applied in this paperwere coincident in showingunquestionable evidencesof hybridization between H. chillensis and H.microcephala var. albiflora. Although the structureof thehybrid swarmisnot clear yet, the hybrids seemto be FI, but Distance Diagram evidencedmorphological continuity amçng the individuals ofH. chillensis and the hybrids. This feature would beevidence of introgression.

Karyotypes of these species were first describedby Saez (1949, 1951) and later by Siljak-Yakovlevet al. (1994), Ruas et al. (1995), and Cerbah et al.(1995, 1999). The karyotypes of these species haveshown similarities and these have been reflected ona quasi-regular meiosis in their hybrids (Wulff,1992). However, Siljak-Yakovlev etal. (1994), Ruaset al., (1995), and Cerbah et al. (1995, 1999) haveshown some chromosomal variations onH. chillensiskaryotypes of different provenances that were neverdiscussed.

On the basis of evidences here described, we areable topropose that these differences couldbe arisen,in part due to hybridization or/and introgressionwhich could be promoted, like in H. radicata, forthe presence of a perennial root system.

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We have the honor to dedicate this contributionto Dr. Juan Héctor Hunziker, who has been

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