cytotyPe variation and clonal diverSity in PolyPloid

aPomictic PoPulationS of PiloSella (comPoSiteae,

cichorieae) introduced to Southern Patagonia

variación citotíPica y diverSidad clonal en PoblacioneS PoliPloideS

y aPomícticaS de PiloSella (comPoSitae, cichorieae) introducidaS en

el Sur de la Patagonia

Anna Krahulcová and František Krahulec*

Summary

Introduction and objectives: The genus Pilosella is native to Europe and Asia, but its

Institute of Botany of the Czech

Academy of Sciences, CZ-25243

Průhonice, Czech Republic

species are successful invaders on most continents. These species form an agamic

complex with common apomixis. Apomictic species hybridize and have diﬀerent degree

of residual sexuality. Main aim of this paper was to determine if interspeciﬁc hybridization

already occurred in Patagonia.

M&M: We analysed seed progeny collected at thirteen populations of Pilosella in southern

Argentina and Chile. The taxonomic identity of plants, DNA ploidy level (using ﬂow

cytometry), chromosome number, reproduction, formation of parthenogenetic seeds

and clonal identity (using isozyme phenotypes) were examined.

*frantisek.krahulec@ibot.cas.cz

Citar este artículo

KRAHULCOVÁ, A. & F. KRAHULEC.

021. Cytotype variation and

2

Results: No mixed-species population was recorded. Two apomictic clones of P.

oﬃcinarum (one pentaploid and the other hexaploid) were found in populations:

eight were hexaploid and one was mixed in cytotype composition. A new species for

Patagonia, the apomictic pentaploid P. caespitosa, was represented by plants from two

Argentinean populations. Some of the progeny plants, cultivated from seeds sampled

at three localities, represented seed-fertile aneuploids the morphology of which suggest

a hybrid origin with P. oﬃcinarum as one of the parental species.

Conclusions: The presence of seed-fertile, aneuploid and parthenogenetic hybrids

among the cultivated plants signiﬁes an increased risk of the formation of new

hybridogeneous genotypes of Pilosella in southern Patagonia.

clonal diversity in polyploid

apomictic populations of Pilosella

(

Compositeae, Cichorieae)

introduced to southern Patagonia.

Bol. Soc. Argent. Bot. 56: 307-326.

DOI: h ttps://doi.

org/10.31055/1851.2372.v56.

n3.32767

Key wordS

Alien plants, aneuploidy, clonal diversity, cytotypes, facultative apomixis, hybridization,

Patagonia, Pilosella, plant invasion, polyploidy, South America.

reSumen

Introducción y objetivos: El género Pilosella es nativo de Europa y Asia, pero sus

especies son invasoras exitosas en la mayoría de continentes. Estas especies forman

un complejo agámico en el que la apomixis es común. Las especies apomícticas

hibridan y presentan diferentes grados de sexualidad residual. El objetivo de este

trabajo fue determinar la existencia de hibridación interespecíﬁca en poblaciones del

sur de la Patagonia.

M&M: Se analizó la descendencia de semillas de Pilosella recolectadas en trece

poblaciones del Sur de Argentina y Chile. Se determinó la identidad taxonómica de las

plantas, el nivel de ploidía (empleando citometría de ﬂujo), el número cromosómico, el

tipo de reproducción, la formación de semillas partenogenéticas y su identidad clonal

(caracterizando fenotipos isoenzimáticos).

Resultados: No se registró ninguna población mixta entre especies. Se encontraron

dos clones apomícticos de P. oﬃcinarum (uno pentaploide y otro hexaploide) en varias

poblaciones: ocho de ellas fueron hexaploides; mientras que una presentó ambos

citotipos. La presencia de la especie apomíctica y pentaploide P. caespitosa en dos

poblaciones de Argentina supone el primer registro de esta especie en la Patagonia.

Algunas semillas muestreadas en tres localidades mostraron una descendencia

aneuploide fértil, cuya morfología indicó un origen híbrido con P. oﬃcinarum como una

de las especies parentales.

Conclusiones: La presencia de híbridos partenogenéticos, aneuploides y con semillas

fértiles entre las plantas cultivadas, implica un aumento del riesgo de formación de

nuevos genotipos híbridos de Pilosella en el sur de la Patagonia.

Recibido: 19 Abr 2021

Aceptado: 18 Ago 2021

Publicado impreso: 30 Set 2021

Editora: Viviana Solis Neffa

PalabraS clave

ISSN versión impresa 0373-580X

ISSN versión on-line 1851-2372

AméricadelSur, aneuploidía, apomixisfacultativa, citotipos, diversidadclonal, hibridación,

invasión de plantas, Patagonia, Pilosella, plantas invasoras, poliploidía.

307

Bol. Soc. Argent. Bot. 56 (3) 2021

introduction

name of Hieracium pilosella L., ﬁrst published by

Moore, 1983 and many authors later), P. aurantiaca

The agamic complex Pilosella Hill., formerly (L.) F. W. Schultz et Sch. Bip. (H. aurantiacum

placed in the genus Hieracium L. in its broad sense, L.: Zuloaga & Morrone, 1999; Ugarte et al., 2011;

includes strictly sexual diploids and polyploids Ariza Espinar & Cerana, 2015; Rodriguez et al.,

(up to the octoploid level, the basic chromosome 2018), P. praealta (Gochnat) F. W. Schultz et Sch.

number x = 9) that are predominantly facultatively Bip. (H. praealtum Gochnat: Zuloaga & Morrone,

apomictic and less commonly sexual (Krahulcová 1999; Ugarte et al., 2011; Ariza Espinar & Cerana

et al., 2000). All Pilosella species are native to 2015 – the ﬁgure indicates P. piloselloides (Vill.)

Eurasia and most of them occur in Europe (Fehrer Soják; Rodriguez et al., 2018), P. ﬂagellaris (Willd.)

et al., 2007). Multiple species or cytotypes often Arv.-Touv. (H. ﬂagellare Willd.: Moore, 1983) and

coexist within a population, and interspeciﬁc and P. ﬂoribunda (Wimm. et Grab.) Fr. (H. ﬂoribundum

intercytotype hybridization is rather common (Fehrer Wimm. et Grab.: Krahulec & Krahulcová, 2011).

et al., 2007; Krahulcová et al., 2009, 2014). The Surprisingly, P. oﬃcinarum F. W. Schultz et Sch. Bip.

still commonly used taxonomic concept is primarily (H. pilosella) has been reported to not occur in Flora

based on morphological characters, distinguishing of Argentina (Ariza Espinar & Cerana, 2015, but it is

between ‘basic‘ species and ‘intermediate‘ species often reported in ecological papers, e.g. Rauber et al.,

(Zahn, 1922–1930; Bräutigam, 2017). Basic species 2013, Braun et al., 2019).

possess unique morphological characters whereas Both native and alien populations of Pilosella

intermediate species combine morphological have primarily been studied with respect to

characters of two or more basic species (Zahn, ecological traits supporting invasiveness, such as

1922–1930; Fehrer et al., 2007; Bräutigam, 2017). seed germination and adaptation to environmental

Supposed hybrid origins of intermediate species conditions (Beckmann et al., 2011), dynamics of

have in many cases been inferred from early crossing vegetative and generative reproduction (Makepeace,

experiments (Peter, 1884; Nägeli & Peter, 1885). The 1985; Beckmann et al., 2009), and the relationship

morphological characters of intermediate species are between environmental and ecological factors

expressed using hybrid formulas that reﬂect either a influencing population dynamics and spread

balanced contribution of both parents (sign ‘–’) or (Cipriotti et al., 2010, 2012; Day & Buckley,

a prevalence/less distinct inﬂuence of either parent 2011; Rauber et al., 2014; Cook et al., 2021).

(

2

signs ‘>’ or ‘<’; Zahn, 1922–1930; Bräutigam, Follow-up research aimed to devise appropriate

017). management practices to restrict the spread of

Preferring unproductive habitats, Pilosella species invasive P. oﬃcinarum in natural grasslands, for

are capable colonizers of disturbed habitats (Fehrer example in New Zealand (e.g. Scott, 1993; Walker

et al., 2007). Several polyploid species have become et al., 2016) or in Argentinian Patagonia (Cipriotti et

a weed problem in the secondary distribution area of al., 2012, 2014). The geographic origin of invasive

the genus (e.g. Makepeace, 1985; Jenkins & Jong, populations of Pilosella was traced by comparing

1996; Wilson et al., 2006; Loomis & Fishman, 2009; the DNA molecular markers in natural populations

Cipriotti et al., 2010). Their success as invaders in in Europe and in alien populations in New Zealand

North and South America, New Zealand and North- (Trewick et al., 2004) and in North America (Wilson

eastern Asia is mainly enabled by their reproductive et al., 2006; Loomis & Fishman, 2009). Evidence

versatility (for an overview of the secondary of recent interspeciﬁc/intraspeciﬁc hybridization

distribution area, see Fehrer et al., 2007). Whereas in facultatively apomictic alien populations of

facultatively apomictic reproduction, together with Pilosella in New Zealand is based on chloroplast

clonal growth, facilitates the establishment and DNA markers (Trewick et al., 2004), inter-simple

rapid spread of new populations, residual sexuality sequence repeats and allozyme markers (Chapman

maintains a potential for hybridization allowing the et al., 2003), as well as species-speciﬁc parental

generation of new genotypes (Fehrer et al., 2007; genome sizes reﬂected in hybrids (Morgan-Richards

Krahulcová et al., 2014). To date, ﬁve Pilosella et al., 2004; Suda et al., 2007).

species have been reported from Patagonia: P.

The reproductive system largely determines the

oﬃcinarum F. W. Schultz et Sch. Bip. (under the ease of progeny formation and progeny diversity.

308

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

These aspects are important for invasion success, material and methodS

but they have until recently been overlooked in

alien populations of Pilosella in Patagonia. Our Seed sampling and recovery of progeny plants

previous study, focused on karyological, reproductive

In the late summer of 2013, Petra Šarhanová

and clonal diversity in the seed progeny of P. (Leibnitz-Institut für Pflanzengenetik und

officinarum originating from three localities in Kulturpflanzenforschung, Gatersleben, Germany)

Chilean Patagonia, was the ﬁrst to take account of and Erwin Domínguez (INIA Kampenaike, Punta

reproductive traits of Pilosella in South America Arenas, Chile) collected, independently of each

(

Krahulec & Krahulcová, 2011). The cultivated other, seeds in thirteen populations of Pilosella in

plants belonged to two clones of P. oﬃcinarum, Chilean Patagonia (both collectors) and Argentinian

one pentaploid and the other hexaploid. Both clones Patagonia (exclusively P. Šarhanová; Table 1).

exhibited a high degree of apomictic reproduction The two collectors used diﬀerent seed-sampling

facilitating easy seed formation independent of strategies. Because of the limited time spent at each

a compatible pollinating parent (Krahulec & locality, Petra Šarhanová sampled seeds together from

Krahulcová, 2011). Later on, in 2013, our colleagues all available fruiting Pilosella plants at each locality.

took samples of seeds in populations of Pilosella at This sampling strategy is likely to have increased

an additional thirteen localities in southern Argentina the heterogeneity of these seed samples (accessions

and Chile (Table 1). The study presented here is 2102–2111 in Table 1 and Table 2). The reason for

based on the evaluation of individual mature plants this simpliﬁed seed sampling was that was carried

raised from these seeds, especially with respect to out during an expedition to Patagonia conducted as

their (a) taxonomic identity, (b) DNA ploidy level/ part of a diﬀerent project. Erwin Domínguez sampled

chromosome number, (c) reproductive behaviour (i.e. separately the fruiting inflorescences (capitula)

the potential for parthenogenetic seed formation), and of individual maternal plants of P. officinarum

(d) clonal identity illustrating genotype diversity. If (accessions 2113–2117 in Table 1 and Table 2).

the cultivated progeny proved to be heterogeneous A map of all collection sites is provided in Fig. 1.

in either morphological/karyological characters or The maximum distance between localities in the

in clonal structure, the remaining available seeds northwest to southeast direction was ca 500 km and

were used for the determination of seed origin. the maximum distance in the southwest to northeast

The methodical approach followed the one used direction was ca 170 km (Fig. 1). All the collection

previously for analysing seeds of Pilosella from the sites were situated in the close vicinity of roads.

ﬁeld and cultivated progeny plants (e.g. Krahulec & Herbarium specimens of fruiting maternal plants

Krahulcová 2011; 2018).

were not collected.

Because of the known occurrence of at least

The material used is based on progeny plants that

ﬁve morphologically distinct species of Pilosella were grown from seeds, alternatively on remaining

in Patagonia (Moore, 1983; Zuloaga & Morrone, seeds that were not used for cultivation of the

1999; Cipriotti et al., 2010; Krahulec & Krahulcová, progeny. Examining the cultivated progeny plants,

2011, Ariza Espinar & Cerana, 2015), we tried to the following methodological approach was used: 1)

ascertain whether each of the thirteen populations assessment of morphological appearance that resulted

sampled is composed of a single species or whether to determination of particular species (Bräutigam &

at least some of them were composed of more than Greuter, 2007-2009; Bräutigam, 2017); 2) search for

one species. Among the progeny grown from seeds apomictic reproduction, i.e., for an ability to produce

we especially looked for traits that may be indicative the seed progeny that replicates the maternal parent

of sexual reproduction potentially taking place (both the cultivated progeny plants and remaining

in the ﬁeld. Such traits are, for example, speciﬁc seeds were used for this purpose); 3) determination

combinations of certain parental morphological of ploidy level/chromosome number reﬂecting the

characters reflecting interspecific hybridization, cytotypic diversity; 4) search for the clonal diversity

aneuploidy and intraspeciﬁc clonal diversity within within particular species/morphotypes and cytotypes

populations. Better knowledge of reproductive traits using the isoenzyme analysis; 5) evaluation of the

and population structure may help to understand the pollen quality (reﬂected in pollen stainability) in two

possibly advancing invasion of Pilosella in Patagonia. selected representatives of an euploid basic species

309

Bol. Soc. Argent. Bot. 56 (3) 2021

Table 1. Collection data of Pilosella plants from southern Patagonia. Plants were grown from seeds

sampled in the ﬁeld by Petra Šarhanová (PŠ) and Erwin Domínguez (ED). Taxonomic determination of

cultivated plants was done by S. Bräutigam and F. Krahulec.

Collector

Locality Accession

No. label

Elevation

(msnm)

of

seeds,

date

Species

Country, location

Argentina, Santa

Cruz, S of Estancia Nothofagus forest

Cancha Carrera, 30 in upper Rio

Habitat

Coordinates

in open, patchy

PŠ, 2

Feb

013

51°24’41”S,

72°13’42”W

1

.

2102

P. caespitosa

428

15

2

km N of Rio Turbio

Turbio valley

overgrazed steppe

with all plants small 52°31’17”S,

no bushes), lot of

Empetrum cushions

Chile, Region XII,

PŠ, 4

Feb

2013

2

2103

P. oﬃcinarum Tierra del Fuego,

(

69°29’40”W

W of Manantiales

Chile, Region XII,

P. oﬃcinarum Tierra del Fuego,

W of Manantiales

PŠ, 4

Feb

2013

grass dominated

steppe

52°43’60”S,

69°03’33”W

3

.

2104

2105

67

5

Chile, Region XII,

P. oﬃcinarum Tierra del Fuego,

W of Manantiales

PŠ, 7

Feb

2013

grass dominated

steppe

52°32’05”S,

69°23’51”W

4

bog vegetation with

Berberis empetrifolia 54°27’45”S,

within Nothofagus

forest area

Argentina, Tierra

P. brachiata /

PŠ, 5

Feb

2013

5.

6.

7.

2106

2108

2109

del Fuego, N of

99

P. piloselliﬂora

67°12’22”W

Tolhuin along R3

Argentina, Tierra del

Fuego, at coast of

PŠ, 6

Feb

2013

P. brachiata /

meadow on top

of the hill

54°49’28”S,

67°30’03”W

Beagle Channel, 10

P. piloselliﬂora

110

11

km W of Estancia

Harberton

Chile, Region XII,

Tierra del Fuego,

coast E of Pt.

Nuevo along Y71

PŠ, 7

Feb

53°21’15”S,

69°22’11”W

P. oﬃcinarum

not speciﬁed

2013

Chile, Region XII,

Tierra del Fuego,

along R257 S of

Cerro Sombrero

PŠ, 7

Feb

grass dominated

steppe

53°11’23”S,

69°16’35”W

8

.

2110

2111

P. oﬃcinarum

73

2013

PŠ, 7

Feb

Argentina, Santa

P. caespitosa

lava rocks and

outcrops

52°04’30”S,

69°34’53”W

9

131

Cruz, Laguna Azul

2013

ED,

Chile, XII Reg.,

Prov. Mag., Sector

Barranco Amarillo,

Com. Punta Arenas

53°05’00” –

53°05’01”S,

70°52’25” –

70°52’26”W

5 Jan

2013

and 23

Feb

2

113 +

114

10.

P. oﬃcinarum

not speciﬁed

44 – 45

2013

Chile, XII Reg.,

Prov. Mag., Sector

ED,

18 Jan

53°17’31”S,

70°56’12”W

11.

2115

P. oﬃcinarum

0

805, Punta Arenas,

2013

Ruta No 9

Chile, XII Reg.,

P. oﬃcinarum Prov. Mag., Sector

Los Cineces

ED,

18 Jan

2013

5

7

3°20’13”S,

0°57’03”W

1

2.

3.

2116

2117

not speciﬁed

0

Chile, XII Reg.,

P. oﬃcinarum Prov. Mag., Sector

Agua Fresca

ED,

18 Jan

2013

5

7

3°23’39”S,

0°59’25”W

1

310

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

Table 2. Cytotype, reproductive and clonal-identity attributes of Pilosella plants that were grown from

seeds sampled in the ﬁeld in southern Patagonia.

Nr of

plants

grown

DNA

ploidy

level

Production of

parthenogenetic

seeds

Accession

label

Sampling

design

Chromosome

number (2n)

Isozyme

Species

phenotype

2

102

103

P. caespitosa

P. oﬃcinarum

CP

20

22

5x (20)

6x (22)

6x (20)

45M (1)

n.d.

yes (3)

yes (4)

yes (2)

b (3)

a (2)

2

54 (1)

2

104

P. oﬃcinarum ×

CP

21

<

6x (1)

51 (1)

54 (1)

46 (4)

n.d.

a (1)

a (2)

unknown parent

2

105

106

P. oﬃcinarum

CP

20

18

6x (20)

5x (18)

yes(2)

yes (2)

P. brachiata /

P. piloselliﬂora

2

c (5), d (12), h (1)

5

x (20)

44 (13)

39 (1)

54 (1)

45M (2)

45 (1)

n.d.

c (2), d (16), e (2)

P. brachiata /

P. piloselliﬂora

2

108

109

CP

21

<5x (1)

6x (9)

f (1)

a (2)

b (3)

g (7)

a (3)

a (1)

2

P. oﬃcinarum

P. caespitosa

CP

9

yes (3)

yes (2)

yes (3)

n.d.

2

110

CP

22

16

14

28

30

28

6x (22)

5x (16)

5x (14)

6x (28)

6x (30)

6x (28)

2

111

CP

2

113

114

115

116

117

IFC (6)

IFC (10)

P. oﬃcinarum

n.d.

P. oﬃcinarum

n.d.

54 (1)

n.d.

References: Accession labels refer to the respective localities listed in Table 1. Design of seed sampling:

accessions 2102 – 2111 represent the composite progenies (CP) of all seeding maternal plants that were

available at locality; accessions 2113 – 2117 comprise the progeny arrays sourced from individual fruiting

capitula (IFC) sampled separately at each locality. The number of fruiting capitula sampled and the number

of analysed plants are given in parentheses. The DNA ploidy level was determined using ﬂow cytometry,

the basic chromosome number in Pilosella is x = 9. The diﬀerent isozyme phenotypes (clones), designated

by speciﬁc letters, were distinguished using a combined pattern of four enzyme systems analysed (see

M & M section for details). The isozyme phenotype ‘fʼ within a multiclonal accession 2108 refers to plant

with a somatic chromosome number of 2n = 39 presented in the same row. Some of the diverse cytotypes

that undoubtedly represent diﬀerent clones, still shared identical isozyme phenotypes: speciﬁcally, the

phenotype ‘aʼ was shared by two cytotypes within accession 2104 and phenotypes ‘cʼ and ‘dʼ were shared

by two cytotypes comprising the accessions 2106 and 2108. The isozyme phenotype ‘aʼ has previously been

detected in hexaploid apomictic P. oﬃcinarum originating from two other localities in Patagonia whereas the

isozyme phenotype ‘gʼ has previously been detected in pentaploid apomicic P. oﬃcinarum originating from

another locality in this region (Krahulec & Krahulcová, 2011). Abbreviations and symbols used: M – a long

hemizygous (marker) chromosome is present in the karyotype; n.d. – not determined.

and of an aneuploid putative hybrid, respectively: this the experimental garden of the Institute of Botany,

indicative test allowed to estimate if the transfer of Průhonice, Czech Republic. Individual seedlings

the paternal genome to progeny is possible by means were replanted into seedling trays and the DNA

of pollen.

ploidy level of adequately grown young plants was

Seeds were sown into pots with sterilized garden determined (see below). Later on, the plants were

soil and were left to germinate in a greenhouse in repotted and transferred to outdoor garden beds for

311

Bol. Soc. Argent. Bot. 56 (3) 2021

Fig. 1. Map of localities (black circles) in southern Patagonia of Pilosella species under study. The accession

labels, corresponding to those presented in Table 1, refer to the following species: P. oﬃcinarum (accessions

2

103, 2104, 2105, 2109, 2110, 2113 + 2114, 2115, 2116 and 2117), P. caespitosa (accessions 2102 and

111) and P. brachiata/P. piloselliﬂora (accession 2106 and 2108).

continued cultivation. A total of 299 progeny plants (FCM). The DAPI staining method (Otto, 1990)

were raised from the seeds (Table 2). Their accession was applied using the ice-cold nuclei-extracting

labels correspond to the respective localities where buﬀer (Otto I) and the staining buﬀer (Otto II)

−1

the seeds were sampled (Tables 1 and 2). Accessions supplemented with 2 µl·ml mercaptoethanol as an

113 and 2014 share one locality because they antioxidant. Fluorescence intensity was determined

2

were sampled from six and ten nearby occurring using a CyFlow Ploidy Analyser PA II (Partec

maternal plants, respectively (Table 2). Herbarium GmbH, Münster, Germany) equipped with an HBO

specimens of cultivated and analysed progeny high-pressure mercury lamp for UV excitation.

plants are deposited in the Herbarium of the Institute For details of the procedure, see Krahulcová et al.

of Botany, Průhonice (PRA). Determination of (2004). The diploid Pilosella lactucella (Wallr.) P.

species was done by Siegfried Bräutigam (Dresden, D. Sell & C. West (DNA content 4.07 pg/2C, Suda

Germany) using both live plants grown in the et al., 2007) was used as the internal standard. When

experimental garden and their herbarium specimens. evaluating the FCM analyses, only histograms in

The nomenclature follows Bräutigam & Greuter which the coeﬃcient of variance of peaks (hereafter

(

2007–2009) and Bräutigam (2017).

referred to as CV) did not exceed 3% were accepted.

A total of 3000 nuclei were scored for each sample.

The reproduction mode was principally

determined using the emasculation/open pollination

Ploidy level and reproduction mode in recovered

progeny

DNA ploidy levels (Suda et al., 2006) were experiment, which allows us to distinguish

determined in seedlings using flow cytometry parthenogenetic (assumed to be apomictic), sexual

312

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

and seed-sterile plants (Gadella, 1984; Krahulcová et enzymatic systems provided suﬃcient resolution

al., 2004). Emasculation was carried out by cutting within individual cytotypes of numerous polyploid

oﬀ the whole upper part of an unopened inﬂorescence taxa of Pilosella (Krahulec et al., 2004; Krahulcová

(capitulum) before anthesis; the anthers and stigmas et al., 2009, 2014; Krahulcová & Krahulec, 2020).

were removed, but the ovaries remained untouched.

Given that fertilization-independent seed formation Chromosome numbers and pollen stainability

is predominantly coupled with apomeiosis in wild-

Chromosome numbers were determined

type apomicts within Pilosella, this test is an especially for selected plants which represented

approximation for apomictically reproducing plants individual accessions and individual clones as

(

Bicknell & Koltunow, 2004; Hand et al., 2015).

implied from speciﬁc isozyme phenotypes. In those

sporadic aberrant plants that were distinguishable

by morphological traits from other plants within

Clonal structure

Clonal (genotypic) identity was examined each of the two accessions 2104 and 2108, the

in each accession and plants representing an chromosomes were counted as well (Table 2). Plants

identical taxon/cytotype were compared among that were uniform in morphology and belonged

accessions (Table 2). An extent of morphological to a common hexaploid clone of P. oﬃcinarum

and karyological uniformity within an accession (i.e. bearing the isozyme phenotype labelled ‘a’ in

was preliminarily assessed, based on a general Table 2) were cytotyped in only ﬁve out of the ten

appearance of plant phenotypes (morphotypes) and accessions in which this clone was recorded (Table

on their corresponding cytotypes, respectively. If the 2). Chromosomes were counted in metaphases

cultivated plants showed high morphological and in root-tip meristems. Root-tips were pre-treated

karyological uniformity within an accession, one to with a saturated solution of α-bromonaphtalene

three individuals per accession were chosen for the for 3 hours at room temperature, rinsed in water

determination of clonal identity. Individuals that and ﬁxed in cold acetic-ethanol (1: 3) overnight.

showed some diversity in morphological features The ﬁxed material was stored in 70% ethanol at 4

within an accession were included in addition; °C until required. The maceration was carried out

6

9 plants were analysed for clonal structure in in 1N HCl at 60 °C for 7 min. The root-tips were

total (Table 2). Clonal identity was inferred from then rinsed in water and the cut meristems were

unique patterns of isozyme phenotypes (Table squashed in a drop of lacto-propionic orcein (Dyer,

2

) derived from a combination of four tested 1963). Only temporary slides were made.

enzymes (AAT, LAP, 6-PGDH/PGM and EST). An indicative evaluation of pollen fertility

Electrophoresis was performed on crude protein was carried out for one plant representing the

extracts of leaf material. Tissue was ground in common eu-hexaploid P. oﬃcinarum (2n = 54)

ice-cold Tris-HCl extraction buﬀer: 0.1 M Tris- and for one aneuploid plant (2n = 46) of a putative

HCl pH 8.0; 70 mM 2-mercaptoethanol, 26 mM hybrid origin, representing the morphotype of

sodium metabisulﬁte, 11 mM ascorbic acid, 4% the hybridogeneous species P. brachiata/ P.

polyvinyl-pyrrolidone. Roughly 60 mg of fresh leaf piloselliflora (for explanation see Discussion,

material was homogenized with Dowex.Cl (1-X8) Apomictic nature…). Pollen fertility was estimated

on ice in 0.7 ml of extraction buﬀer. Extracts were as pollen stainability using Alexanderʼs stain

centrifuged for 10 min at 13,000 rpm and clear (Alexander, 1969). The stain was applied to fresh

supernatants were stored at ̶ 75 °C. All enzyme pollen, following the procedure described by

systems were investigated on polyacrylamide gels Rotreklová & Krahulcová (2016). A total of 300

8% acrylamide, discontinuous tris-glycine buﬀer pollen grains were evaluated per plant.

system, pH 8.3). The staining procedures followed

Vallejos (1983) LAP (Fast Black K), AAT (+ Fast Reproductive origins in seeds and embryo ploidy

Violet B) and Wendel & Weeden (1989) 6-PGDH, levels

PGM (NADP), EST-colorometric (b-naphtyl Reproductive origins and embryo ploidy levels

(

̶

phosphate, Fast Blue BB) with modifications. were screened in accessions 2104, 2105 and 2108,

The isoenzyme patterns were visually compared using the remaining seeds that were sampled in

among individual plants. In previous studies, the ﬁeld but not used to obtain progeny. Flow

313

Bol. Soc. Argent. Bot. 56 (3) 2021

cytometric seed screening (FCSS) was used (Matzk room temperature, stained and analysed (Krahulcová

et al., 2000) with the following modiﬁcations for & Suda, 2006; Krahulcová et al., 2011). The

Pilosella: The seeds were kept in a fridge in order maternal ploidy peak was used as the internal ploidy

to retain endosperm tissue, allowing us to get standard and histograms with CVs below 5% were

interpretable endosperm peaks in ﬂow-cytometric considered. This threshold of accuracy of analyses

histograms for up to several months. Before the was still acceptable because two simultaneous

analysis, ﬁlled (well developed) seeds were screened peaks in a histogram are indicative of a diﬀerence

under a stereomicroscope by putting appropriate in DNA content between two components within a

pressure with tweezers on each seed. Filled seeds sample, corresponding to double the CV (Doležel &

were pooled either two at a time (Table 3) or ten at Göhde, 1995). The quantiﬁcation of embryo ploidy

a time (Table 4), so either seed doublets or groups classes was based on the proportions of nuclei in

comprising ten seeds each were prepared for FCM their respective embryo ploidy peaks (Krahulcová

analysis. If possible, 5000 nuclei were scored for & Suda, 2006).

each sample analysed. Basically, the procedure was

the same as the one used for the detection of ploidy ploidy level of progeny embryos (when analysing

level in seedlings (see above). both the seed doublets and pooled samples of

The FCSS analysis allows us to detect both the

When analysing seed doublets, two cypselae ten seeds) and their reproductive origins (when

(

achenes) were chopped in the nuclei-isolating analysing the seed doublets). FCSS analyses of

2

buﬀer with a razor blade together with 0.05 cm

pooled seed samples sourced from facultatively

of leaf tissue of Bellis perennis L. (DNA content apomictic mothers commonly reveal the most

.96 pg/2C, Leong-Škorničková et al., 2007) as frequent embryos of apomictic origin that maintain

3

the reference standard. The suspension was ﬁltered the maternal ploidy level; in addition, this procedure

and incubated for 10 min at room temperature, can distinguish and quantify the number of potential

stained and analysed (Krahulcová et al., 2009, less frequent embryos diﬀering in ploidy level

2

011). Histograms with CVs below 3.5% were from the maternal parent, namely, the products

considered. When analysing pooled samples of (a) heteroploid crosses, (b) sexual mating via

comprising ten seeds, ten cypselae were chopped in unreduced gametes and (c) haploid parthenogenesis

the nuclei-isolating buﬀer with a razor blade, and the (Krahulcová & Suda, 2006). This way, the potential

suspension was ﬁltered and incubated for 10 min at diversity in embryo ploidy level can be traced.

Table 3. Flow cytometric seed screening (FCSS) of remaining seeds (seed doublets) of Pilosella from the

ﬁeld that were not used to obtain progeny. The accession symbols and chromosome numbers that were

detected paralelly among cultivated plants, follow Table 2. The basic chromosome number in Pilosella is

x = 9. Symbols of species: OFF – P. oﬃcinarum; BRA – P. brachiata; PF – P. piloselliﬂora. The symbol of

seed origin 2n + 0 (Harlan and de Wet 1975) refers to apomictic origin of embryo. For description of both

FCSS methods used and for their detection capacity, see M & M section.

Plants grown from the same

FCSS analysis of seed doublets

source of seeds

Seed origin

Chromosome

number

2n + 0

Embryo

Other

Embryo

Total Nr of seeds

Accession Species

Nr of

ploidy

seeds

ploidy

seeds

2

n = 6x = 54,

2104

2105

2108

OFF

6x

20

12

–

0

20

12

2n = 51

2n = 6x = 54

n = ca 5x =

6x

–

2

BRA/PF

ca 5x

4

4, 2n = 39

52

314

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

Table 4. Flow cytometric seed screening (FCSS) of remaining seeds (bulked samples of ten seeds) of

a

Pilosella from the ﬁeld that were not used to obtain progeny. For explanation see Table 3. The embryo

ploidy level corresponds to polyhaploid origin (n + 0, Fig. 3C).

FCSS analysis of bulked samples comprising 10 seeds

Chromosome

Accession

Species

Maternal

Other embryo

ploidy

Total Nr

of seeds

number

Nr of seeds

embryo ploidy

2

n = 6x =

a

2

104

108

OFF

6x

47

3x

3

50

54, 2n = 51

2

n = ca 5x =

2

BRA/PF

ca 5x

50

–

0

44, 2n = 39

100

reSultS

In addition, 39 plants cultivated from seeds

obtained from two other localities in Argentina

Taxonomic identity, cytotypes and pollen (accessions 2106 and 2108 in Table 1 and

stainability Table 2) were inconclusively determined as P.

The plants cultivated from seeds originating brachiata (P. officinarum × P. piloselloides/

from nine populations in Chile represented the bauhini, hybrid formula P. officinarum > P.

basic species Pilosella officinarum (Table 1, piloselloides/bauhini) or P. piloselliflora (P.

Table 2, Fig. 1). Hexaploids (2n = 54) prevailed officinarum × P. floribunda, hybrid formula P.

whereas pentaploids (2n = 45) were detected only officinarum ≥ P. floribunda). These plants were

among plants of P. officinarum which made up a highly homogeneous in morphology within

mixed pentaploid/hexaploid population situated each of the two recovered accessions, with the

close to Punta Arenas (accessions 2113 and 2114 exception of a single plant within accession

in Table 2, Fig. 1). While both hexaploids and 2108, which later turned out to be of a deviating

pentaploids morphologically matched typical cytotype (see below). Although the overall

P. officinarum, the rather deviating appearance morphological appearance was slightly different

of a single plant within hexaploid accession between accessions 2106 and 2108, the variation

2

104 suggested that this plant is a hybrid of of all cultivated plants corresponded to the

P. officinarum and another, unknown species species P. brachiata/P. piloselliflora. These

Table 2). This atypical plant turned out to be plants belonged to three aneuploid cytotypes:

(

aneuploid (2n = 51, Fig. 2A) lacking the three hyperpentaploid (2n = 46, accession 2106

chromosomes when compared with the eu- in Table 2, Fig. 2B), hypopentaploid (2n =

hexaploid cytotype (2n = 54).

44, accession 2108 in Table 2, Fig. 2C), and

Another basic species that was recorded hypertetraploid (a sporadic cytotype with 2n

among the progeny plants was Pilosella = 39, accession 2108 in Table 2, Fig. 3A). The

caespitosa (Dumort.) P.D. Sell & C. West from overall morphological appearance of the last

two localities in Argentina (accessions 2102 aberrant individual (2n = 39) still allowed to

and 2111 in Table 1 and Table 2). All 36 raised distinguish it from the other aneuploid (2n =

plants of P. caespitosa were pentaploid (Table 44) plants constituting accession 2108. Pollen

2

). Chromosome counting of three selected stainability reached 80% and 88% in an eu-

plants of P. caespitosa revealed the chromosome hexaploid (2n = 54) plant of P. officinarum

number of 2n = 45, which included a long (accession 2115) and in the aneuploid (2n = 46)

hemizygous chromosome (symbol ‘M’ following plant of P. brachiata/P. piloselliflora (accession

the chromosome number in Table 2).

2106), respectively.

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Bol. Soc. Argent. Bot. 56 (3) 2021

Fig. 2. Chromosomes in aneuploid cytotypes of Pilosella. Aneuploid plants were raised from seeds

originating from altogether three populations in southern Patagonia (accession numbers follow Table 1 and

Table 2). The mitotic metaphases were obtained from root-tip meristems. A: accession 2104, a putative

hybrid of P. oﬃcinarum with another (unknown) species, 2n = 51. B: accession 2106, a hybridogeneous

taxon of P. brachiata/P. piloselliﬂora, 2n = 46. C: accession 2108, a hybridogeneous taxon of P. brachiata/P.

piloselliﬂora, 2n = 44.

Clonal structure

from two localities in Chilean Patagonia that had

All obtained hexaploids assigned to P. been examined in a previous study (Krahulec

oﬃcinarum shared an identical isozyme phenotype & Krahulcová, 2011) revealed a concordance

(

labelled ‘a’ in Table 2). A simultaneous analysis in all analysed isozyme phenotypes, suggesting

of these plants with hexaploid P. oﬃcinarum isoclonality. One hypo-hexaploid plant,

316

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

Fig. 3. Flow-cytometric ﬂuorescence histograms. A: Pilosella brachiata/P. piloselliﬂora, accession 2108:

simultaneous analysis of DAPI-stained nuclei of four hypo-pentaploid seedlings (peak 2, chromosome number

determined as 2n = 44) and a single hyper-tetraploid seedling (peak 1, chromosome number determined as 2n =

39). B: P. oﬃcinarum, accession 2105: simultaneous FCSS analysis of two seeds sampled from plants in the ﬁeld;

diploid P. lactucella used as the internal standard (peak 1). The histogram represents the ﬂuorescence intensity

of DAPI-stained nuclei of apomictically derived hexaploid embryos (6x, peak 2) and of dodecaploid autonomously

formed endosperm (12x, minor peak 3). C: P. oﬃcinarum, accession 2104: FCSS analysis of a pooled sample of

ten seeds sampled from plants in the ﬁeld. The histogram represents the ﬂuorescence intensity of DAPI-stained

nuclei of a trihaploid embryo (3x, minor peak 1), of other hexaploid embryos (6x, peak 2) and of dodecaploid

endosperm (12x, minor peak 3) attributed to hexaploid embryos. The peak of hexaploid endosperm, which is

attributed to a trihaploid embryo, is overlapped by peak 2. All accession numbers follow Table 1 and Table 2.

317

Bol. Soc. Argent. Bot. 56 (3) 2021

corresponding in morphology to a hybrid of P. therefore highly probably apomictic reproduction.

oﬃcinarum and an unknown parent (2n = 51, Emasculation experiments were not done in

accession 2104 in Table 2), also exhibited isozyme time in the case of aneuploid accession 2108,

phenotype ‘a’, which was assigned to typical and plants produced normal seeds following

hexaploid P. oﬃcinarum. Seven pentaploid plants open-pollination. The seed set was not evaluated

of P. oﬃcinarum that constituted accession 2113 in one aneuploid individual (2n = 51) from

from Chile had a concordant isozyme phenotype accession 2104 (Table 2) because of damage to

(

labelled ‘g’ in Table 2) with an analogous fruiting capitula caused by insects. Emasculation

pentaploid P. oﬃcinarum that had previously experiments were not carried out for accessions

been recorded at another locality in the country 2103, 2113 + 2114, 2115, 2116 and 2117, which

(

Fig. 1, Krahulec & Krahulcová, 2011). Six were found to be isoclonal within the hexaploid

analysed plants that represented P. caespitosa and cytotype of P. oﬃcinarum (see above) and shared

were sourced from two localities in Argentina isozyme phenotypes with two apomictic clones of

(

accessions 2102 and 2111) shared a common P. oﬃcinarum previously identiﬁed in the region

isozyme phenotype (labelled ‘b’ in Table 2).

Clonal diversity within an individual cytotype

under study (Table 2).

was only recorded in two accessions, 2106 and Reproductive origins of seeds from the ﬁeld

108, from populations in Argentina, assigned A total of 52 seeds from three localities were

2

to P. brachiata/P. piloselliﬂora (Table 2). These screened for seed origin. All the FCSS analyses

were eighteen hyper-pentaploid plants (2n = 46) of seed doublets revealed an apomictic (2n +

analysed within accession 2106, which possessed 0) origin of these seeds (accessions 2104, 2105

three isozyme phenotypes, as well as twenty and 2108 in the Table 3, Fig. 3B). The embryos

analysed hypopentaploid plants (2n = 44) within ascribed to P. oﬃcinarum (accessions 2104 and

accession 2108 (Table 2). Comparison between 2105) were hexaploid. The ploidy of embryos

accessions 2106 and 2108 found that plants in accession 2108 (assigned to P. brachiata/P.

from these accessions shared the most common piloselliﬂora) corresponded to an approximately

isozyme phenotype ‘d’ and also the less common pentaploid ploidy level, which is in agreement

isozyme phenotype ‘c’ (Table 2). In general, with a hypopentaploid chromosome number of

all ﬁve isozyme phenotypes (labelled ‘c’, ‘d’, 2n = 44 that was detected in parallelly cultivated

‘

e’, ‘f’ and ‘h’ in Table 2) that were detected plants (Table 3). FCSS analyses of pooled seed

in accessions 2106 and 2108 were similar to samples comprising ten seeds (100 seeds were

each other in their isozyme patterns, but certain analysed in total) revealed a prevalence of

differences were still recorded among them. hexaploid embryos in seeds of P. oﬃcinarum.

Speciﬁcally, the isozyme phenotypes ‘c’, ‘d’, In addition, three embryos (i.e. 6% out of all

‘

e’ and ‘h’ had a rather distinct isozyme pattern analysed embryos attributed to P. oﬃcinarum)

of the enzyme EST (for a demonstration of were triploid, indicating a polyhaploid origin

isozyme phenotypes in selected plants, see Fig. (accession 2104 in the Table 4, Fig. 3C). All

4

) whereas the enzymes AAT, LAP and 6-PGDH/ analysed embryos in pooled seed samples of P.

PGM showed a uniform pattern. The isozyme brachiata/P. piloselliﬂora were approximately

phenotype ‘f’ was characterized by a distinct pentaploid (accession 2108 in the Table 4), as

pattern of the enzyme 6-PGDH whereas the other were the embryos in the seed doublets (see above).

three enzymes had a uniform pattern.

Analyses of pooled seed samples yielded a minor

endosperm peak besides the major embryo peak

Potential for parthenogenesis in plants grown in most histograms (Fig. 3C). This endosperm

from seeds peak was always positioned at approximately

A total of 21 raised plants selected to cover double the distance of the major embryo peak,

all recorded taxa were tested for parthenogenetic implying that an overwhelming majority of

seed formation (Table 2). All these plants set a analysed embryos were of apomictic origin (2n

normal amount of seeds in emasculated capitula + 0). Each of these embryos had the same ploidy

(

not quantiﬁed), implying parthenogenetic and level as the respective apomictic mother.

318

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

2

011). The hexaploid clone of P. officinarum

probably has a broader distribution in southern

Patagonia because we recorded it at nine additional

localities in the present study (Fig. 1, Table 1, Table

2

). Furthermore, we identiﬁed both previously

reported clones/cytotypes of P. oﬃcinarum in one

mixed pentaploid/hexaploid population (accessions

2

113 and 2114 in Table 2, Fig. 1), which constitutes

the ﬁrst record of a mixed-cytotype population of

Pilosella in Patagonia. Each individual leaf rosette

of P. officinarum forms a single inflorescence

(capitulum; Bräutigam, 2017) and both cytotypes

recorded in southern Patagonia produce clonal

apomictic seeds (Krahulec & Krahulcová, 2011).

From the aforesaid mixed population, fruiting

capitula were sampled separately and progeny

originating from a single capitulum was always

homogeneous as to its ploidy level; that is, it was

either hexaploid or pentaploid (Table 2). Therefore,

Fig. 4. Illustration of isozyme patterns in Pilosella each of the either hexaploid or pentaploid capitula

acquired from analysis of enzyme system esterases

must correspond to an individual hexaploid/

(EST). The numbers at the top (codes of laboratory

pentaploid maternal rosette of P. oﬃcinarum in

the source mixed-cytotype population and no inter-

cytotype was recorded.

The pentaploid isoclonal apomictic plants raised

from seeds originating from two localities in

southern Argentina (accessions 2102 and 2111 in

samples) correspond to individual cultivated plants;

the letters at the bottom correspond to speciﬁc

isozyme phenotypes as presented in Table 2. The

numbers 66, 68 and 80–91 represent cultivated

accession 2106 (Pilosella brachiata/P. piloselliﬂora)

whereas the number 60 represents cultivated

accession 2111 (Pilosella caespitosa). Only some Table 1 and Table 2) were classiﬁed as Pilosella

of the plants constituting accessions 2106 and 2111 caespitosa, which is a new species for Patagonia.

(

Table 2) are included here.

Similarly, the discovery of aneuploid apomictic

and polyclonal plants classiﬁed as P. brachiata/P.

piloselliﬂora implies the occurrence of another

species of Pilosella in Argentinian Patagonia

(accessions 2106 and 2108 in Table 1 and Table 2).

Based on morphology, however, the plants in these

two accessions were determined unequivocally

diScuSSion

Taxonomic, clonal and cytotype diversity

Here we report P. caespitosa as a new species for as either P. brachiata or P. piloselliﬂora. These

the ﬂora of Argentina. With all probability it will be two hybridogeneous species of Pilosella share

found also in the Chilean part of Patagonia in due P. oﬃcinarum as one of the parents (Bräutigam

time.

& Greuter, 2007–2009; Bräutigam, 2017). The

The history of the invasion of Pilosella common predominance of morphological characters

oﬃcinarum in Patagonia is quite recent. Soon of P. oﬃcinarum in the species P. brachiata and

after it was recorded in Patagonia for the ﬁrst time P. piloselliflora, respectively, can hinder their

(Moore, 1983), this species was reported to invade identiﬁcation. Interestingly, both these accessions

the northern Tierra del Fuego rangelands (Livraghi of P. brachiata/P. piloselliﬂora shared two most

et al., 1998). Later, two apomictic cytotypes/clones common isozyme phenotypes (labelled ‘c’and ‘d’in

of P. oﬃcinarum were identiﬁed in southernmost Table 2) that were speciﬁc to this taxon whereas the

Chile, the Magallanes province, namely a hexaploid other three distinct isozyme phenotypes (labelled

clone occurring at two localities and a pentaploid ‘e’, ‘f’and ‘h’in Table 2) were similar to each other.

clone occurring at another (Krahulec & Krahulcová, Such similarity in the pattern of isozyme phenotypes

319

Bol. Soc. Argent. Bot. 56 (3) 2021

may imply a close genetic relation between two populations. Both the eu-hexaploid P. oﬃcinarum

populations of P. brachiata/P. piloselliﬂora that (2n = 54) and the hyper-pentaploid P. brachiata/P.

are situated approximately 45 km apart (accessions piloselliﬂora (2n = 46) exhibited fairly high pollen

2

106 and 2108 in Fig. 1). Nevertheless, each of stainability implying a capacity for sexual mating

these two separate populations represents a speciﬁc as the pollen parent. This agrees with the fact

genotype(s), as implies the cytotype diversity of that, despite autonomous apomixis independent

raised apomictic and aneuploid progeny (Table 2). of pollen, most polyploid apomictic biotypes of

Evidently, aneuploidy is compatible with viability Pilosella produce sufficient amounts of viable

in polyploid Pilosella biotypes, as has already been reduced pollen allowing them to serve as the pollen

shown in mixed-cytotype populations in Europe parent (Rotreklová & Krahulcová, 2016).

(e.g. Krahulcová et al., 2009, 2014). The unavailability of maternal plants from the

Summarizing all previously published data (see ﬁeld and the design of the seed sampling did not

the Introduction for references) and those presented allow us to explain with certainty the origin of

in this study, the alien ﬂora of Patagonia currently the aneuploids grown from the seeds. Each of the

includes seven Pilosella species, both basic (P. respective accessions 2104, 2106 and 2108 (Table

aurantiaca, P. caespitosa, P. oﬃcinarum and P. 2) was composed of pooled progeny of an unknown

piloselloides) and hybridogeneous (P. ﬂagellaris, P. number of maternal individuals (see the Material

ﬂoribunda and tentatively classiﬁed P. brachiata/P. and methods). Because living plants were not

piloselliﬂora). No mixed-species population was collected, neither morphological nor karyological

detected among those examined because each of traits could be compared between any maternal

the thirteen populations sampled were composed of plant from the ﬁeld and the respective progeny array

a single taxon.

raised from the seeds sampled. Therefore, each

of the aneuploid progeny individuals originated

Apomictic nature of populations and origins of either de novo within an euploid population or as

variation a copy of the maternal aneuploid genome inherited

The studied polyploid populations of Pilosella through apomixis. The single aneuploid individual

in southern Patagonia are apomictic, as can be in accession 2104 was most likely generated de

inferred from (a) the formation of parthenogenetic novo within a hexaploid population of P. oﬃcinarum

seeds in raised progeny plants (Table 2), (b) the because it was raised among eu-hexaploid plants

apomictic origins of analysed seeds that were grown from seeds sampled from the same

produced in the ﬁeld (Tables 3 and 4), and (c) the population (Table 2). Moreover, one morphological

maintenance of maternal ploidy level in progeny peculiarity distinguished this single aneuploid plant

embryos (Tables 3 and 4). The low frequency of from the other raised eu-hexaploid plants of P.

trihaploid embryos recorded among hexaploid oﬃcinarum, suggesting occasional hybridization

embryos of P. oﬃcinarum (Table 4) suggests a of P. oﬃcinarum with another (unknown) species.

low degree of residual sexuality in this apomictic The putative hybridizing parents were most likely

cytotype. This ﬁnding corresponds with the well- a hexaploid of P. oﬃcinarum and a pentaploid of an

known facultativeness of apomixis in Pilosella unknown species of Pilosella because the aneuploid

(

2

e.g. Bicknell & Koltunow, 2004; Fehrer et al., hybrid progeny (2n = 51) had a chromosome

007). The apomictic character of P. oﬃcinarum number between the pentaploid (2n = 45) and the

and P. caespitosa in Patagonia is also reﬂected in hexaploid (2n = 54) ploidy level. However, potential

the clonal homogeneity of the raised plants within heterogeneity among fruiting plants in the ﬁeld

individual species and cytotypes (Table 2). At least allows for an alternative scenario. Speciﬁcally,

ﬁve diﬀerent but probably closely related aneuploid the parent of the hybrid apomictic plant with an

genotypes were found in two apomictic accessions aneuploid the chromosome number of 2n = 51 could

of P. brachiata/P. piloselliflora sampled from theoretically be already established at the locality

two separate populations (Table 2, Fig. 1). The and its single aneuploid progeny individual could

minor genotype and cytotype diversity recorded have happened to be raised from an apomictic seed.

between and within these two accessions may

Progeny accessions 2106 and 2108 most

imply occasional sexual mating within the source likely originated from two aneuploid apomictic

320

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

populations of P. brachiata/P. piloselliflora is both sexual and apomictic in Europe (Rotreklová

that were already established in the ﬁeld. This et al., 2005). In spite of the expected apomictic

assumption is based on (a) the reproduction mode reproduction, the putative parental biotypes might

and morphological appearance, (b) variation in occasionally hybridize as either parent owing to

chromosome numbers, and (c) clonal structure. residual sexuality operating in apomictic mothers

Speciﬁcally, (a) the remaining seeds from which and to the production of viable pollen (e.g. Fehrer

plants of accession 2108 were raised were of et al., 2007; Rotreklová & Krahulcová, 2016).

apomictic origin (Tables 3 and 4). Although The two apomictic populations of P. brachiata/P.

no remaining source seeds of accession 2106 piloselliﬂora in Patagonia exhibited slight diversity

were available, the obtained progeny produced in their aneuploid chromosome numbers and

parthenogenetic seeds, implying inherited apomictic also in their isozyme phenotypes (Table 2). Such

reproduction as well (Table 2). A great uniformity variation could be derived from occasional sexual

in overall morphological appearance was observed mating that potentially shaped the progeny of

among the plants cultivated from seeds of both the primary interspecific hybrids. The diverse

accessions, which also supports an apomictic origin aneuploid cytotypes could then have arisen by

of the seeds sampled in the ﬁeld. (b) All cytotyped irregular male/female meiosis during sporogenesis.

progeny cultivated from seeds in accessions 2106

and 2108 was exclusively aneuploid; the aneuploid Increasing risk of invasive hybrids in Patagonia

chromosome numbers were clustered into three

The plants raised from seeds appear to be

cytotypes diﬀering by two chromosomes in most apomictic and species-uniform within populations

of the plants analysed (Table 2). (c) As inferred in southern Patagonia examined to date. Residual

from isozyme phenotypes, the clonal structure of sexuality and viable pollen are two crucial attributes

both these accessions indicated the sharing of two allowing sexual mating of a facultative apomict as

most common clones (genotypes), the other three either parent. Both attributes needed for occasional

clones being considerably rarer (Table 2). All ﬁve hybridization were found in the hexaploid cytotype

detected clones are probably closely related to each of P. oﬃcinarum, which is not rare in southern

other, as implied by the similarity of their respective Patagonia. Indeed, the properties of plants obtained

isozyme phenotypes.

by the cultivation of seeds sampled in the ﬁeld

It is probable that these two aneuploid imply sporadic hybridization of P. oﬃcinarum

populations of P. brachiata/P. piloselliﬂora arose with another species. However, the way seeds

recently in Argentinian Patagonia by occasional were sampled for this study does not allow us to

interspeciﬁc hybridization. Pilosella oﬃcinarum ascertain whether the putatively hybrid plants had

as well as P. piloselloides and P. ﬂoribunda occur already been established in the ﬁeld, or whether

as non-indigeneous taxa in Patagonia (Zuloaga the hybrid seeds originated de novo. Regardless of

&

Morrone, 1999; Krahulec & Krahulcová, this missing information, the following traits that

2

011), and the presence of these putative parental may be indicative of ongoing hybridization in the

species is suﬃcient for the formation of both ﬁeld were observed in the seed-cultivated plants:

P. brachiata (DC.) F. W. Schultz & Sch. Bip. clonal and cytotype diversity among progeny

(

P. officinarum × P. piloselloides/P. bauhini) originating from certain populations, aneuploidy

and P. piloselliﬂora (Nägeli & Peter) Soják (P. and a morphological aﬃliation of putative hybrids

oﬃcinarum × P. ﬂoribunda; Bräutigam, 2017). with P. oﬃcinarum, which was most likely one of

The shared putative parent Pilosella oﬃcinarum is their parents. One of the aneuploid hybrids that

apomictic in Patagonia, but the rarity of polyhaploid were tentatively classiﬁed as the hybridogeneous

progeny (Table 4) suggests some degree of residual species P. brachiata/P. piloselliflora produced

sexuality operating in the common hexaploid clone. fairly viable pollen, similarly to that of eu-hexaploid

Facultatively apomictic reproduction might also be P. oﬃcinarum. Consequently, both the biotypes,

expected in P. ﬂoribunda in Patagonia because this the putatively parental one (P. oﬃcinarum) and

is the reproduction mode of most populations of the derived one (P. brachiata/P. piloselliflora)

this species in Europe (Krahulec et al., 2004, 2008). can take part in sexual mating as the male parent.

The third putative parental species, P. piloselloides, Furthermore, the existence of a mixed-cytotype

321

Bol. Soc. Argent. Bot. 56 (3) 2021

population of P. oﬃcinarum, which was recorded reported from Chilean Patagonia, were found

in Patagonia for the ﬁrst time, may increase the in additional populations, of which eight were

likelihood of inter-cytotype hybridization within hexaploid and one was mixed in its cytotype

this species.

composition. A new species for Patagonia, the

Hybrids of many angiosperm taxa have apomictic pentaploid P. caespitosa, was represented

become invasive in their secondary distribution by isoclonal plants grown from seeds originating

areas regardless of whether a native species had from two populations in Argentina. Some of the

hybridized with an introduced one, or whether progeny plants cultivated from seeds sampled at

both the parents had been introduced (Ellstrand three localities represented seed-fertile aneuploids

&

Schierenbeck, 2000). In general, polyploidy the morphology of which implied a hybrid origin

and clonal growth are rather common among and indicated P. oﬃcinarum as one of the parents.

successful plant invaders, especially those capable Speciﬁcally, one aneuploid (2n = 51) hybrid plant

of uniparental reproduction, including apomixis of P. oﬃcinarum and an undetermined species

(e.g. Rambuda & Johnson, 2004; te Beest et al., was found among other eu-hexaploid (2n = 54)

2

012). However, invasive or potentially invasive plants of P. oﬃcinarum constituting a progeny

apomictic hybrids that themselves had originated accession sourced from a population in Chile.

from a facultatively apomictic parent or parents are Other accessions, originating from two populations

not common. Such hybrids have been detected, for in Argentina, were exclusively aneuploid. Their

example, in the genera Amelanchier (Ellstrand & morphology resembled either P. brachiata (P.

Schierenbeck, 2000) and Rubus (Clark & Jasieniuk, officinarum > P. piloselloides/bauhini) or P.

2

012) of the Rosaceae family. Another example are piloselliﬂora (P. oﬃcinarum ≥ P. ﬂoribunda), which

non-indigenous species of Pilosella (Asteraceae) are two mutually related hybridogeneous species

hybridizing in New Zealand (Morgan-Richards native in Europe. These two aneuploid accessions

et al., 2004; Trewick et al., 2004). Similarly, the comprised a single hypertetraploid plant (2n = 39)

present study recorded some signs of interspeciﬁc and seventeen plants that were polyclonal within

hybridization of non-native Pilosella in southern each of two approximately pentaploid cytotypes

Patagonia. Because of polyploidy, clonal growth (2n = 44, 2n = 46). The presence of seed-fertile,

and facultative apomixis allowing occasional aneuploid and parthenogenetic hybrids among

hybridization, non-indigenous biotypes of Pilosella the cultivated plants signiﬁes an increased risk of

pose a potential risk that invasive hybrids will arise the formation of new hybridogeneous genotypes

in this part of the species’ secondary distribution of Pilosella in southern Patagonia. If such new

area.

genotypes are formed, they may have greater

evolutionary and invasive potential.

concluSionS

author contributionS

The following species of Pilosella have so far

been mentioned as alien in Patagonia: P. aurantiaca,

AK did all experimental work and prepared

P. ﬂagellaris, P. ﬂoribunda, P. oﬃcinarum and ﬁrst draft of the paper; FK participated on plant

P. piloselloides. The present study inquires into determination and prepared ﬁnal version of the

the diversity of progeny plants grown from seeds paper.

sampled at thirteen populations of Pilosella in

southern Argentina and Chile. The plants were

examined for their taxonomic identity, DNA ploidy acKnowledgementS

level (using ﬂow cytometry), chromosome number,

formation of parthenogenetic seeds and clonal

We thank Petra Šarhanová (University of

identity (using isozyme phenotypes). No mixed- Vienna, Austria) and Erwin Domínguez (INIA

species population was recorded. Two apomictic Kampenaike, Punta Arenas, Chile) who collected

clones of P. oﬃcinarum (one pentaploid and the seeds in Patagonia, Ivana Plačková (Průhonice) for

other hexaploid), which had previously been isozyme studies, Siegfried Bräutigam (Germany,

322

A. Krahulcová and F. Krahulec - Pilosella in Patagonia

Dresden) for help with plant determination, Juan BRÄUTIGAM, S. 2017. Pilosella Vaill. In: Jäger, E. J.

Manuel Gorospe for the translation of Summary

into Spanish, and Fred Rooks (Průhonice) for

language correction of the manuscript. Jan Wild

(ed.), Rothmaler Exkursionsﬂora von Deutschland,

Gefäβpﬂanzen: Grundband, Ed. 21. pp. 817−829.

Spektrum Akademische Verlag, Heidelberg, Berlin.

is acknowledged for the preparation of the map. CHAPMAN, H.M., G. J. HOULISTON, B. ROBSON &

Two reviewers are acknowledged for their critical

remarks and suggestions. This work was supported

by the Czech Academy of Sciences (long-term

research & development project No. RVO

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