Molecular evidence for hybridization and

introgression across a ploidy barrier between

Spergularia echinosperma and S. rubra
(Caryophyllaceae)

Pavel Kúr, Jiří Košnar, Petr Koutecký & Milan Štech

Department of Botany, Faculty of Science,
University of South Bohemia, České Budějovice

International Conference on Polyploidy, Hybridization, and

Biodiversity
7-10 May 2012, Průhonice near Prague, Czech Republic
 Introduction
Petit et al.
 Interploidal hybridization (1999)

» relatively rare × common homoploid hybridization

» yet, hybrids between species differening in ploidy
levels exist and can facilitate interploidal introgression
» few documented examples

Study group
 Spergularia echinosperma
» annual; rare species of periodically flooded soils,
endemic to Central Europe; 2n = 2x = 18
 S. rubra
» annual(-perennial); ruderal species, widespread
due to human activity; 2n = 4x = 36
 indirect morphologic evidence of interspecific
hybridization
» presumed tetraploid hybrid described as S. ×kurkae
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 Questions
 Does interploidal hybridization between S. echinosperma and
S. rubra take place?
 What mechanisms can the hybridization be attributed to?
 Is the hybrid reproductively isolated from the parental species, or
does introgressive hybridization take place?

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 Methods
Sampling
 88 populations; SW Czech Republic,
N Germany
 plants from both intermixed and pure
populations
 taxa determined by morphology and
flow cytometry

Molecular analyses
 nrDNA – ITS (ITS-1, 5.8S, ITS-2)
» direct sequencing with universal primers
» ribotype-specific amplification
 cpDNA – rpoC1 intron
» direct sequencing
» PCR-RFLP assay
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 Results
ITS (nrDNA)
 direct sequencing
» S. echinosperma (2x) and S. rubra (4x): clear difference
(16 substitutions, 4 indels)

» S. ×kurkae: intragenomic variation; presence of both the

S. echinosperma and S. rubra ribotypes
 ribotype-specific amplification
» two primer pairs for each of the S. echinosperma/S. rubra ribotypes
» confirmation of specificity by sequencing the PCR product
RIf (S. rub-specific)
SIf (universal) EIf (S. ech-specific)

150 bp 500 bp
EIr (S. ech-specific) RIr (S. rub-specific) ITS4i (universal)
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 S. echinosperma (2x): rubra-specific primers
» introgression × ancestral polymorphism

Parezny Parezny
no amplification

Hodemysl Hodemysl
amplification

Vosecky Vosecky
Svihov Svihov
Skopec Skopec
Podhursky Podhursky
rub-specific amplification in

Mlynhor Mlynhor
Malobor Malobor
Kojatin Kojatin
Jensov Jensov
» introgression × (ancestral polymorphism ) S. echinosperma

Chvalovec Chvalovec
Hurka Hurka
Hrachoviste Hrachoviste
(404 samp.)

Driten Driten
(84 samp.)

Cky Cky
RIr
RIf

Babak Babak
Results

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Janus
Chvalovec Chvalovec
ChvalovecRUB ChvalovecRUB
SkopecRUB SkopecRUB
GriebenRUB GriebenRUB
Beranov2011 Beranov2011
Pobocensky Pobocensky
Slavkovicky Slavkovicky
LabeRUB LabeRUB
ech-specific amplification in S. rubra

SvihovRUB
S. rubra (4x): echinosperma-specific primers

SvihovRUB
KliekenRUB KliekenRUB
VoseckyRUB VoseckyRUB
Siglov Siglov
NovyDarkoRU NovyDarkoRU
MlynhorRUB MlynhorRUB
KondracskyRU KondracskyR
HodemyslRUB HodemyslRUB
Heinrichsberg Heinrichsberg
Dvorak Dvorak
BleddinRUB BleddinRUB
BeranovRUB BeranovRUB
Altelbe1 Altelbe1
C.Hora C.Hora
Zavlekov Zavlekov
PolomRUB PolomRUB
DobevRUB DobevRUB
Strmilov Strmilov
Pecihradek Pecihradek
HorMez HorMez
VrbinecRUB VrbinecRUB
TelcStepnice TelcStepnice
PtacovRUB PtacovRUB
MricRUB MricRUB
Vlkov Vlkov
StHlina StHlina
RozmitalRUB RozmitalRUB
PlanavaRUB PlanavaRUB
Pisek Pisek
Pirin Pirin
Nem1 Nem1
Myto Myto
Maj Maj
Luznice Luznice
Kubhut Kubhut
KrLesRUB KrLesRUB
Kosov Kosov

(549 samp.)

(372 samp.)
Klec Klec
Jachymov Jachymov
Havlic Havlic
Cerna Cerna

EIr
EIf
BulhSperg BulhSperg
BrezejcRUB BrezejcRUB
BohdalovRUB BohdalovRUB

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 Results
rpoC1 (cpDNA)
 direct sequencing
» S. echinosperma and S. rubra: clear difference (2 substs., 1 indel)

 PCR-RFLP
» restriction site in the S. rubra haplotype
 S. ×kurkae: exclusively the S. echinosperma
(2x) haplotype

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 Conclusions
 2x S. echinosperma and 4x S. rubra can hybridize across the ploidy
barrier
» allotetraploid S. ×kurkae
 most probably via unreduced egg cells of 2x S. echinosperma
» unilateral hybridization with S. echinosperma as the maternal parent
 hybridization probably accompanied by unidirectional gene
introgression:
S. echinosperma (2x) -> S. ×kurkae (4x) -> S. rubra (4x)
» ancestral polymorphism and incomplete lineage sorting obscures the
detection of introgression
 S. echinosperma/S. rubra represents a suitable model group for the
study of interploidal hybridization and gene introgression

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 E2X haplotype amplifying from S. rubra

S. RUBRA 4x (S5) C G C C G G A A A C G G G G G A C C T T C
S. ECHINOSPERMA 2x (S7) C G T C A A C G G G - - - - G A T T C C T
233_EIf_RC_Beranov_rubra_introgression C G T Y A A C G G G - - - - G A T T C C T
234_EIf_RC_Beranov_rubra_introgression Y G T C A A C G G G - - - - G A T T C C T
236_EIf_RC_Beranov_rubra_introgression C G T C A A C G G G - - - - G A T T C C T
289_EIf_RC_Siglovec_rubra_AP_new ECH haplotype C K T C A A C G G G - - - - G A T T C C T
E92_RC_Janus_rubra_AP_new ECH haplotype C G T C A A C G G G - - - - G G T T C C T
149_EIf_RC_rubra_introgression+AP? n n n y A A C G G G - - - - G A T T C C T
4RIF_210_E2X_Chvalovec_1b_different RUB haplotype C G C C G G A A A C G G G G T A C C T T C

red haplotypes:
introgression echinosperma → rubra, because sequences contain both typical
recent rubra haplotype + in some cases new slightly differing haplotypes
(hidden as paralogs on direct sequencing = IUPAC degenerated bases)
! typical recent rubra haplotype is more frequently amplified than putatively
ancestral haplotypes

blue haplotype:
sequence differs in one base from recent rubra haplotype → probably ancestral
polymorphism (not gene flow rubra → echinosperma); we have never obtained
pure recent rubra haplotype (but could be hidden in paralogous positions!)
 RUB haplotype amplifying from S. echinosperma

S. RUBRA 4x (S5) G T G G G C G C C T C C C C G C C G C C C G G G A A A C G G G G C G C G
S. ECHINOSPERMA 2x (S7) G T G G G C G C C T - T C T G T C G T C C G A A C G G G - - - - C G C G
233_EIr_Beranov_rubra_introgression+AP? G T R G G C G C C T - T C T G T C G T C C G A A C G G G - - - - C G C G
600_EIr_Zavlekov_rubra_introgression_bad R W G G K C R C C T - t C T G t M R T C C g A A C g g g - - - - C G C G
R622_RIr1_E2X_AP_without pure RUB? G T G G G m G C C T y C C y G T C G C C C G G G A A A C G G G G C
R8_RIr1_E2X_AP+pure RUB G T G G G C G C C T C C m y G C C G C C M G G G A A A C G G G G C
R18_RIr1_E2X_AP_KOMPLET G T G G G m G C C T y C C Y G y C G C C C G G G A A A C G G G G C
18_1_RIr1_E2X_AP_clone G T G G G A G C C T T C C T G T C G C C C G G G A A A C G G G G C
18_2_RIr1_E2X_1bp_differing from RUB, not Taq error G T G G G C G C T T C C C C G C C G C C C G G G A A A C G G G G C
18_4_RIr1_E2X_AP_clone G T G G G C A C C T C C C T G T C G C C C G G G A A A C G G G G C
R38_RIr1_E2X_AP+catching ECH G T G G G M G C C T y C C Y G K C G C C C G G G A A A C G G G G C
39_2_RIr1_E2X_pure_R_s_Taq_error? G T G G G C G T C T C C C C G C C G C C C G G G A A A C G G G G C
39_3_RIr1_E2X_pure_R_s_Taq_error? G T G T G C G C C T C C C C G C C G C C C G G G A A A C G G G G C
R41_RIr1_E2X_AP G T G G G M G C y T Y C C Y G Y C G C C C G G G A A A C G G G G C
41_3_RIr1_E2X_1bp_differing from RUB G T G G G C G C C T C C C C G C C G T C C G G G A A A C G G G G C
R42_RIr1_E2X_AP G T G G G M G C y T Y C C Y G Y C G y C C G G G A A A C G G G G C
R119_RIr1_E2X_AP+pure RUB? G T G G G C G C C T n C C C G C M k C C M G G G A A A C G G G G C
119_2_RIr1_E2X_AP G T G G G C G C C T C C C C G C A G C C C G G G A A A C G G G G C
3RIR_119_E2X_35c_prevalent_RUB!!!+AP G T G G G C G C C T C C C C G C m G C C m G G G A A A C G G G G C
4RIR_119_E2X_45c_prevalent_RUB!!!+AP G T G G G C G C s T C C C C k C M G C C m G G G A A A C G G G G C
R293_RIr1_E4X_AP+pure_RUB? G T G G G C G C C T C C C C G Y C r C y m G G G A A A C G G G G C
R297_RIr1_E2X_AP_without pure RUB g T G G G C G C C T C C C C G T C G C C C k G G A A A C G G G G Y
R366_RIr1_E2X_AP_without pure RUB G T G G G C G C C T C C C C G T C G C C m G G G A A A C G G G G C
R424_RIr1_E2X_Apwithout pure RUB, no paralogues G T G G G A G C C C T C C T G T C G C C C G G G A A A C G G G G C
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