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Botanical Journal of the Linnean Society, 2014, 174, 227–239. With 5 figures
Patterns of hybridization in a multispecies hybrid
zone in the Ranunculus cantoniensis
complex (Ranunculaceae)
TONGJIAN LI†, LINGLING XU†, LIANG LIAO*, HUISHENG DENG and XINGJIE HAN
The College of Life Sciences, Jiujiang University, East Xunyang Road No. 320, 332000 Jiujiang,
Jiangxi Province, China
Received 4 May 2012; revised 12 August 2013; accepted for publication 19 August 2013
Simultaneous hybridizations among three or more parental taxa occur in nature, but these have rarely been
analysed. The present study investigates the natural hybridization of diploid members of the Ranunculus
cantoniensis complex over a microgeographical area in south-western China. Sequence information from maternally inherited plastid DNA (trnQ-rps16 and rpL32-trnL) and biparentally inherited nuclear ribosomal DNA was
used to identify hybrids, and these were further confirmed by a dramatic reduction in pollen viability. The
populations from the contact zone contained individuals that had more than two nuclear ribosomal internal
transcribed spacer (nrITS) types; in addition, a network analysis revealed the presence of hard conflicts between
the nuclear and plastid DNA data. These results prove that R. trigonus and R. silerifolius var. dolicanthus have
relatively little reproductive isolation from each other, leading to a high gene flow rate in contact populations. The
four investigated species can hybridize with each other in this contact zone, forming seven hybrid types. © 2014
The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239.
ADDITIONAL KEYWORDS: introgression – nrITS – pollen viability – rpl32-trnL – trnQ-rps16.
INTRODUCTION
Hybrid zones, which may form after secondary
contact between two partially reproductively isolated
populations, have long been utilized in studies of
speciation and the maintenance of species boundaries
(Barton & Hewitt, 1985; Chatfield et al., 2010). Analyses of hybrid zones have provided a wealth of information on factors that favour hybridization, the
nature of pre- and post-zygotic barriers to gene flow
and the stability of hybrid zones (Rieseberg & Carney,
1998; Buggs, 2007; Currat et al., 2008; Wallace et al.,
2011). Nearly all the hybrid zones that have been
studied so far have involved two parental taxa
(Kaplan & Fehrer, 2007; Peñaloza-Ramírez et al.,
2010). Nevertheless, multispecies hybrid zones also
occur in nature, but these have rarely been analysed.
*Corresponding author. E-mail: [email protected]
†These authors contributed equally to this work.
Multispecies hybrid zones need at least one species
pair to produce some fertile hybrids, which can then
hybridize with a third species (Kaplan & Fehrer, 2007).
A wide range of genotypes, including hybrids between
different species pairs, backcrosses and triple hybrids,
potentially emerge from a multispecies hybrid zone
(Peñaloza-Ramírez et al., 2010). Species abundance
will have an impact on both the hybridization rate
and introgression directionality (Lepais et al., 2009).
Spencer, McArdle & Lambert (1986) modelled the
interaction between two populations that are sympatric, but produce hybrids of zero fitness. They obtained
two possible outcomes: extinction of one of the populations or divergence in mate recognition systems
allowing coexistence (Butlin, 1987). In addition to the
model mentioned above, there are several other theoretical models for hybrid zone dynamics to describe
multispecies hybrid zones (Arnold, 1993; Dodd &
Afzal-Rafii, 2004; Peñaloza-Ramírez et al., 2010). Consequently, the hybrid fitness, dynamics of gene flow
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
227
228
T. LI ET AL.
Table 1. Morphological characters of Ranunculus silerifolius, R. silerifolius var. dolicanthus, R. chinensis and R. trigonus
(Liao et al., 1995; Wang, 1995a, b; Liao & Xu, 1997)
Morphological
character
R. silerifolius var.
silerifolius
R. silerifolius var.
dolicanthus
R. chinensis
R. trigonus
Aggregate fruit
Petals
Stigma
Globose
4–5 mm
Hooked at apex
0.7–1.2 mm
Globose
6–10 mm
Slightly curved at apex
1.0–1.4 mm
Cylindrical
5–6 mm
Unbent apex about
0.2 mm
Globose
3–6 mm
Unbent apex
0.5–0.8 mm
and possible outcomes in multispecies hybrid zones are
poorly known.
The Ranunculus cantoniensis DC. complex was first
defined by Tamura (1978) based on the Japanese flora,
including R. chinensis Bunge. (2x), R. silerifolius
H.Lév. var. silerifolius (2x) and R. cantoniensis DC.
(4x). Liao et al. (1995, 2008) and Liao & Xu (1997)
revised the definition based on morphology, karyotype,
molecular phylogeny and fluorescence in situ hybridization data, including all Tamura’s species plus
R. silerifolius var. dolicanthus L.Liao (2x), and they
further considered R. trigonus Hand.-Mazz. (2x) as an
allied species of the complex. Molecular evidence confirmed the close relationship of the five taxa (Liao
et al., 2008; Emadzade et al., 2011; Hörandl &
Emadzade, 2012). The R. cantoniensis complex
comprises weedy plants, widely spread in tropical and
subtropical Asia. Taxonomically, R. chinensis, R. silerifolius, R. silerifolius var. dolicanthus and R. trigonus
have long been ambiguous and often confused with
each other (Kuo, Yang & Wang, 2005). These species
frequently occur in sympatry throughout southwestern China. Notably in Puer, Yunnan Province,
they have congruent flowering times, allowing natural
hybridization to occur occasionally (Liao et al., 2008).
Observations of individuals intermediate between
these species indicate hybridization. Furthermore,
hybridization between R. chinensis and R. silerifolius
var. silerifolius was demonstrated in experiments
(Okada, 1984; Okada, 1989). Thus, Puer may be a
hybrid zone for the four taxa.
Intra-individual nuclear ribosomal internal transcribed spacer (nrITS) paralogues have been instrumental in detecting patterns of reticulation in a large
number of angiosperms, if concerted evolution fails to
act across the repeat units contributed by different
parent species (Devos et al., 2006; Noyes, 2006;
Guggisberg, Mansion & Conti, 2009). In this study, we
investigated the natural hybridization between the
diploid members of the R. cantoniensis complex, over
a microgeographical area in Puer, using the nrITS
region and two plastid intergenic spacers. We
addressed the following questions: (1) does natural
hybridization occur between the four taxa and, if so,
how many different hybrid types could result?; (2) are
gene flow and hybridization symmetrical?
MATERIAL AND METHODS
STUDY TAXON
Ranunculus chinensis (C), R. silerifolius var. silerifolius (S), R. silerifolius var. dolicanthus (D) and
R. trigonus (T) are all diploids and form a monophyletic group (Liao et al., 2008). Although species limits
were considered to be ambiguous and species could be
confused with each other, they could be distinguished
on the basis of characters of the petals, stigmas and
aggregate fruits (Table 1) (Wang, 1995a, b; Kuo et al.,
2005). Flowers of R. chinensis and R. silerifolius var.
silerifolius are protandrous and selfing (Okada &
Kubo, 1999). The breeding systems of R. silerifolius
var. dolicanthus and R. trigonus are still unknown.
SAMPLING
PROCEDURE
Leaf, flower and fruit samples were collected in five
locations in the contact zone (Fig. 1). As a reference,
three morphologically representative isolated populations situated outside of the contact zone were also
sampled (Fig. 1; Table 2). Representative populations
were chosen on the basis of the non-overlapping part
of their distribution and typical diagnostic characters
of each species. All samples were preliminarily identified on the basis of morphological characters, and
samples with undeveloped fruits were preliminarily
identified as putative hybrids. The samples that produced homologous nrITS and did not show conflict
between nrITS and plastid data were considered as
pure individuals. In contrast, samples that produced
heterozygous nrITS or showed conflict between nrITS
and plastid data were considered as hybrids (hereafter abbreviated as H). At least 20 individuals from
each locality within the contact zone and a maximum
of 15 individuals from each locality outside of the
contact zone were collected haphazardly. Specimens
included in the molecular analyses are summarized in
Table 2 and Supporting Information Table S1.
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
A MULTISPECIES HYBRID ZONE IN RANUNCULUS
229
CM Hybrid Zone
YT
LJ
CS
EH
H
XM
SZ
YK
Figure 1. Sampling localities of the eight populations analysed here. Triangles represent reference populations, and
circles represent populations from the contact zone.
DNA
ISOLATION, AMPLIFICATION AND SEQUENCING
Total genomic DNA was extracted using a cetyltrimethylammonium bromide (CTAB) method (Doyle &
Doyle, 1987). Polymerase chain reactions (PCRs) were
performed in 20-μL volumes containing 1 × buffer
(including 1.5 mM MgCl2), 2 mM additional MgCl2,
200 μM deoxynucleotide triphosphates, 0.2 μM of each
primer and one unit Taq polymerase (Sangon,
Shanhai, China). Amplifications were carried out on a
Mastercycler pro (Eppendorf, Hamburg, Germany)
using the following conditions: an initial hold at 94 °C
for 5 min; 30 cycles of 94 °C for 30 s, 52 °C for 1 min
and 72 °C for 1 min, and final holds of 72 °C for
10 min and 4 °C indefinitely.
Two non-coding regions (trnQ-rps16 and rpL32trnL) were amplified using primers trnQ (UUG) and
rpS16x1, and trnL (UAG) and rpL32-F (Shaw et al.,
2007). The nrITS region (comprising ITS1, 5.8s gene
and ITS2) of nrDNA was amplified with primers 1
and 4 (White et al., 1990). Both strands of the purified
PCR products were sequenced by Shanghai Sangon
Biological Engineering Technology and Service Co.
Ltd (Sangon). Geneious Pro 4.8.5 (Biomatters, Auckland, New Zealand) was used to check the quality of
electropherograms. If direct sequencing of nrITS
amplicons produced ambiguous sites, PCR products
were cloned into the pUCm-T vector (pUCm-T
Cloning Vector Kit, Bio Basic Inc.) according to the
manufacturer’s instructions (Guggisberg, Bretagnolle
& Mansion, 2006). Our aim was to capture the allelic
variation of nrITS gene loci within individuals fully,
especially those of putative hybrids. Thus, ten clones
were sequenced for each putative hybrid. Vector fragments and primer regions were trimmed and
sequences were edited in Geneious Pro 4.8.5 (Biomatters). Because the two plastid intergenic regions are
linked in the haploid genome, sequences were combined and treated as a single marker for analysis.
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
230
T. LI ET AL.
Table 2. Samples included in the study
Population
Species
No. of individuals
No. of hybrids
R. chinensis
15
0
R. trigonus
4
0
R. trigonus
17
0
R. silerifolius var. dolicanthus
R. chinensis
R. silerifolius var. silerifolius
R. trigonus
32
20
17
13
5
0
1
2
R. silerifolius var. dolicanthus
R. silerifolius var. silerifolius
R. trigonus
19
1
17
10
0
5
R. silerifolius var. dolicanthus
R. chinensis
R. silerifolius var. silerifolius
1
7
11
0
0
2
R. silerifolius var. dolicanthus
R. silerifolius var. silerifolius
7
2
0
0
R. silerifolius var. dolicanthus
R. chinensis
R. silerifolius var. silerifolius
R. trigonus
1
8
1
4
0
0
0
0
EH
LJ
CS
CM
XM
YT
SZ
YK
PHYLOGENETIC
ANALYSIS
Directly sequenced nrITS sequences without ambiguous sites were used to construct neighbor-joining (NJ)
phylogenetic trees. Alignments were first produced
automatically with ClustalX (Thompson et al., 1997),
and NJ trees were estimated using MEGA 5 (Tamura
et al., 2011) with the Tamura–Nei model of nucleotide
substitution, and indels were treated as missing data.
To assess the support for individual nodes in the
phylogenetic trees, a bootstrap analysis was conducted with 1000 replicates.
NETWORK
ANALYSIS
Plastid and nrITS sequences, including direct
sequences and cloned sequences, were used in
network analyses, respectively. Alignments were produced automatically with ClustalX, and the nrITS
networks were constructed using the program TCS
version 1.21 (Clement, Posada & Crandall, 2000).
Parsimony probability was set at 98%; therefore, haplotypes related with a probability of parsimony of
> 98% would be connected and those with a probabil-
ity of < 98% would be unlinked. The graph generated
from TCS 1.21 was edited with yEd Graph Editor
(http://www.yworks.com).
TEST
OF RECOMBINATION
Hybrids may contain not only both parental nrITS
repeat regions, but also both recombinant and other
variant nrITS cistrons (Coleman, 2002). To distinguish recombinant and variant nrITS (RV) from all
cloned sequences, the dominating nrITS types (D1/
D2, T56/T131, C191/C192 and S147) were aligned
with all cloned sequences. Potentially parsimony
informative sites in the alignment were used to detect
recombined sequences. Cloned sequences being the
chimaera of two direct sequences were considered to
be recombinant sequences, and cloned sequences
including a site different from any direct sequence
were considered to be variant sequences.
POLLEN
VIABILITY
Pollen grains were stained with iodine + potassium
iodide (IKI) medium (1 g KI and 0.5 g I dissolved in
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
A MULTISPECIES HYBRID ZONE IN RANUNCULUS
100 mL of distilled water) to detect starch (staining it
dark blue or brown; Bolat & Pirlak, 1999). Grains of
pollen from hybrids and their parents were counted to
determine viability after 5 min in IKI medium (Bolat
& Pirlak, 1999). About 300 pollen grains of each
individual were counted under a light microscope.
RESULTS
SEQUENCE
VARIATION
From 307 sequences obtained from 198 individuals,
there were 16 direct sequences of nrITS amplicons
that produced more than one ambiguous site. Nuclear
ITS amplicons with more than one ambiguous site
were cloned into pUCm-T vector and 123 clones were
obtained from these amplicons. However, there were
many cloned sequences different from any direct
sequences. From the total length of 648 bp of the
alignment of nrITS sequences, 87 sites were variable
and 29 of these were potentially parsimony informative. The alignment length of trnL-rpL32 was 783 bp,
with four variable sites. The alignment length of
trnQ-rps16 was 910 bp, with a five-nucleotide indel
and nine variable sites.
PHYLOGENETIC
ANALYSIS BASED ON DIRECTLY
231
H45, H47, H48, H49, H58, H60, H62 and H64, and,
accordingly, four groups of haplotypes, designated C,
D, S and T, were defined. H9, H22 and H34 from the
CM population and H16, H19, H20, H45, H47, H48,
H49, H58, H60, H62 and H64 from the XM population, assigned to R. dolicanthus in the nrITS network,
shared the same haplotypes with R. trigonus. H92,
the cloned sequences of which were assigned to
groups T and C, shared the same plastid haplotype
with R. dolicanthus. Haplotype H96 was the same as
that of R. chinensis, but cloned sequences of H96 were
assigned to groups T and C.
TEST
OF RECOMBINATION
Seven representative examples are given in Figure 5.
Fourteen of the 16 putative hybrids contained more
than two types of cloned sequences that were identical with direct sequences. However, H9 and H39
contained only one type of cloned sequence which was
identical with direct sequences. Eleven putative
hybrids contained variant sequences and eight putative hybrids contained recombinant sequences. Only
one putative hybrid (H96) did not contain any recombinant sequence or variant sequence. No variant or
recombinant sequence was detected more than once
in a putative hybrid, except for H9 and H39.
SEQUENCED NRITS
In the nrITS tree, three clades were fairly well
resolved. All individuals of R. silerifolius var. silerifolius and R. silerifolius var. dolicanthus each formed a
clade. Ranunculus chinensis and R. trigonus together
formed a clade, but all individuals of R. chinensis
were clustered in a subclade with a bootstrap value of
99 (Fig. 2).
NETWORK
ANALYSIS
To uncover the phylogenetic relationships among
putative hybrids and pure individuals, networks were
constructed using cloned and directly sequenced
nrITS sequences and plastid haplotypes, respectively
(Figs 3, 4). The nrITS network resolved four major
groups of nrITS sequence types that correlate with
morphological species delimitation. In the hybrid
zone, there were 14 individuals with more than two
nrITS types. The nrITS clones of H138 and H146 in
the YT population were assigned to two groups (C and
S). Cloned sequences of H10, H39, H92 and H96 from
the CM population were also assigned into two groups
(Table 3). Cloned sequences of H73, H77, H78, H82
and H87 from the XM population were separated into
groups D and T.
The plastid network resolved four phylogenetic lineages. However, haplotypes were specific to species,
with the exception of H9, H16, H19, H20, H22, H34,
POLLEN
VIABILITY
The detection of decreased pollen viability in a plant
is commonly used, in addition to molecular markers,
for the identification of spontaneous hybrids (Bureš
et al., 2010). We examined eight pure individuals and
eight suspected hybrids from the hybrid zone and six
accessions from the reference populations (Table 3
and Supporting Information Table S2). The pollen
viability of pure individuals in the hybrid zone and
reference populations was > 87%. However, pollen of
H10 and H39 showed 7.0% and 36.8% viability,
respectively. No viable pollen was detected in H92,
H96, H138 and H146. Pollen of H82 from the XM
population showed 44.7% viability. H47 from the XM
population, which exhibited hard conflict between the
nuclear and plastid DNA data, showed high pollen
viability (96.4%).
DISCUSSION
EVIDENCE
OF HYBRIDIZATION
Increasing evidence shows that intra-individual variation of nrITS regions should not be considered as an
exceptional occurrence, in spite of the homogenizing
mechanisms known as concerted evolution (Chiang
et al., 2001; Harris, Marshall & Crandall, 2001;
Valbuena-Carabana et al., 2007). nrITS markers have
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
1
1 6
19 4
2
30 6
175
1743
17
1729
16 7
16 5
16 4
16 63
1 62
1 1
16 60
1 58
1
99
87
99
65
2
2 12
21 11
2 0
20 08
20 7
20 6
20 1
19 0
1989
19
1926
2 22
195
194
193
191
97
94
130
88
90
99
R. trigonus
R. chinensis
R. silerifolius var. dolicanthus
R. silerifolius var. silerifolius
69
4 7
4 3
5 4
6 2
5 1
1 2
1
183
2
29
36
44
50
58
1
41
7
5
69
17
21
28_1
35
43
49
55
65
8
89
100
101
103
10
1098
11
11 0
11 1
1 5
1116
1 7
1 18
12 19
1 0
1 21
12 24_
5 1
6
12 9
12 31
1 32
1 3
13 34
1 35
1
56
67
68
69
70
71
72
75
76
80
81
83
112
84
85
1
1 57
15 56
1 5
15 54
14 2
14 9
14 7
14 5
14 4
1423
141
140
139
137
246
93
245
244
242
241
240
239
238
235
234
233
232
229
228
227
2254
22 3
22 1
22 0
22 9
21 8
21 17
2 16
2 15
2 14
2 13
2
6
37
40
54
48
42
33
27_
20 1
25
3
38
45
51
60
4
2 6
T. LI ET AL.
59
232
Figure 2. Neighbor-joining trees based on directly sequenced nuclear ribosomal internal transcribed spacer (nrITS)
sequences. Numbers next to nodes are bootstrap values.
been used to determine the parentage of hybrids
through the detection of additive patterns (e.g.
Valbuena-Carabana et al., 2007). Our results showed
that 14 individuals in the contact zone of four different taxa of the R. cantoniensis complex contain more
than two nrITS types. There are two main possible
explanations for the occurrence of intra-individual
ITS variation. First, it may result from hybridization
between parents containing different ITS sequences
(Baldwin et al., 1995; Sang, Crawford & Stuessy,
1995). Second, divergent intra-individual sequences
might arise by molecular processes unrelated to
hybridization, such as the accumulation of mutations,
exceeding the rate of concerted evolution, nrDNA
array multiplication or pseudogenization (Feliner &
Rosselló, 2007; Kosnar et al., 2012). In this study, the
NJ tree distinguished four taxa, and intra-individual
combination of nrITS types of supposed parents was
found only in the contact zone, suggesting that intraindividual nrITS variation might result from hybridi-
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
A MULTISPECIES HYBRID ZONE IN RANUNCULUS
233
H9_4
H9_6,H9_7,H9_8
D31_1,H34_2
H9_2
H10_11
H82_5
H9_1
H73_8
H77_4
H92_3
H92_1
H16, D17, D18, H19, H20,D21,H22
H6_13
H78_8
H87_8
H77_5
H78_4 H73_5
H78_5
T56,T67,T68,T69,T71,T72
T75,T76,T80,T81,T83,T84
H87_5 H77_6
T85,T90,T94,T97,T100,T101
T103,T108,T109,T110,T111
T115,T116,T117,T118,T119
T120,T121,T125,T129,T70_1
T70_3,T70_8,H73_1,H73_3
H77_3,H78_1,H78_3,H82_4,H87_1
H87_3,H87_6,H87_7,H92_5,H92_6
H92_8,H96_2,H96_5,H96_6,H96_7
H96_8,T124_1,T126_1
H39_6
H73_7
H45, H47, H49, H50, D51
H64
D65,T89,H6_2,H6_3,H6_4,H6_5
H6_8,H6_9,H9_3,H9_5,H10_3
H73_6,H77_8
H78_2,H82_1,H82_3,H82_6,H82_7
H77_7
H6_1
H6_7
H73_4
H10_5
H39_2
H87_7
H10_8
H10_9
H87_2
8
9_
H39_7 H39_5
,3
_3
39
H
H39_4
H87_4
D31_2, H34_1
H10_12
H146_5
H146_1
H146_8
H92_2,92_4
H146_9
H138_8
H138_1
H138_7
C222,H96_1,H96_3,H96_4
H138_2,138_3,138_9,H146_7
H92_7
H138_5
H138_10
H146_4
H146_2
H138_6
H10_1
D10_4, 10_6,D10_7
R. trigonus
R. chinensis
R. silerifolius var. silerifolius
R. silerifolius var. dolicanthus
H138_4
H146_10
H146_3
H146_6,H168_2,168_4,168_5,168_8
H10_2
H168_3
H168_7
H168_6
Figure 3. TCS networks based on nuclear ribosomal internal transcribed spacer (nrITS) sequences. Rectangular areas
in the nrITS network represent nrITS types existing in pure individuals, and circular areas are nrITS sequences that are
detected in hybrids. Blue characters represent sequences of conflicting taxonomic assignment. C, Ranunculus chinensis;
D, R. silerifolius var. dolicanthus; S, R. silerifolius var. silerifolius; T, R. trigonus; H, hybrids.
zation rather than other molecular processes. In
addition to intra-individual nrITS variation, conflict
between nuclear and plastid data was also found in
the contact zone. This could be explained by introgressive hybridization or concerted evolution after
hybridization (Okuyama et al., 2005). Furthermore,
individuals with intra-individual nrITS variations
and plastid–nuclear conflicts showed low pollen
viability, which also proved that hybridization events
occurred in this hybrid zone. Therefore, the combined
results of nrITS, plastid and pollen viability indicate
that a high rate of hybridization occurred among the
four Ranunculus spp.
RECOMBINATION OF NRITS
Concerted evolution can result in recombinant nrITS
types that represent a mixture of two original nrITS
sequences. Alternatively, the recombinant and variant
sequences can arise during PCR (Liu et al., 2007). In
this study, not only sequences from putative parents
but also recombinant and variant sequences were
detected among the cloned sequences of hybrids
(Table 3). PCR-mediated recombination can result in
artefacts, complicating research dealing with hybrid
identification, phylogenetic relationships and evolutionary histories (Cronn et al., 2002; Liu et al., 2007).
Our results show that nrITS types of both maternal
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
234
T. LI ET AL.
H10
D2, D3, D4, D5, H6, D7
C195, C221, H96
H9, H22, H34
D35, D36, D37, D38, H39
D46, H50, D51, D52, D53
D54, D55, D63, D65, H92
D16, H19, H20, H45, H47, H48
H49, D58, H60, D62, H64
H73, T74, T75, T76, H77, H78
T79, T80, T81, H82, T83, T84
T85, T86, H87, T115, T116
T119, T120, T121, T123, T124
T125, T126, T129, T132
S137, H138, S139, S140
S141, S143, S144, S145
H146, S147, S154, S155
S156
Figure 4. TCS haplotype networks based on plastid non-coding regions. Rectangular areas represent plastid haplotypes.
Bold italic characters represent sequences of conflicting taxonomic assignment. C, Ranunculus chinensis; D, R. silerifolius
var. dolicanthus; S, R. silerifolius var. silerifolius; T, R. trigonus; H, hybrids.
and paternal parents could be detected, and there
was no recombinant or variant sequence found more
than once in a hybrid. It seems that PCR-mediated
recombination and Taq polymerase error, rather than
concerted evolution, played an important role in the
formation of recombinant and variant sequences. The
parentage of hybrids can still be inferred through
polymorphic sites, and PCR-mediated recombinations
will not influence the reliability of the methods for the
characterization of hybrids. There was an exception
in H9 and H39, in which only one parental nrITS type
was found, and this might be caused by insufficient or
limited sampling.
PATTERNS
OF HYBRIDIZATION
The nrITS sequences showed an additive pattern
combining the variation of the parental species
(Clement et al., 2000). Eight hybrids and five genotypes were detected in the CM population. Four nrITS
clones of H10 were assigned to R. chinensis (C), three
to R. silerifolius var. dolicanthus (D) and three to
RVs; the plastid haplotypes were grouped in R. chinensis (C). Thus, H10 was a hybrid derived from
R. chinensis and R. silerifolius var. dolicanthus, with
R. chinensis as female parent. This type of hybridization was named CD-C. The results for the remaining
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
A MULTISPECIES HYBRID ZONE IN RANUNCULUS
235
Table 3. The genetic constitution and pollen viability of hybrids in the contact zone
nrITS origin*
Population
Individual
Number
of clones
CM
H6
H9
H10
H22
H34
H39
H92
H96
10
8
11
–
2
7
8
8
H138
H146
10
10
H73
H77
H78
H82
H87
H16
H19
H20
H45
H47
H48
H49
H50
H60
H64
7
7
7
6
8
–
–
–
–
–
–
–
–
–
–
C
4
D
S
9
4
3
1
2
1
1
T
R
V
Haplotype
Pollen
viability (%)
Genotype
3CD
1
4
1
D
T
C
T
T
D
D
C
–
–
7.0
–
–
36.8
0
0
D-T
D-T
CD-C
D-T
D-T
DT-D
CT-D
CT-C
S
S
0
0
CS-S
CS-S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
–
–
–
44.7
–
–
–
–
–
96.4
–
–
–
–
–
DT-T
DT-T
DT-T
DT-T
DT-T
D-T
D-T
D-T
D-T
D-T
D-T
D-T
D-T
D-T
D-T
6
3
4
5
3
YT
5
4
1
2
3CS
3CS
1
1
XM
1
1
1
4
1
1
1
1
1
1
1
1
1
1
1
2
2
3
1
4
4DT
4DT
3DT
1DT
1
2
nrITS, nuclear ribosomal internal transcribed spacer.
*R, chimaera of two direct sequences; V, cloned sequence different from any direct sequence.
hybrids using the same inference process are listed in
Table 3. Three nrITS clones of H92 were assigned to
R. chinensis (C) and five to R. trigonus (T). The
plastid haplotypes were grouped in R. silerifolius var.
dolicanthus (D). Therefore, H92 was a triple hybrid
derived from R. chinensis, R. trigonus and R. silerifolius var. dolicanthus, with R. silerifolius var. dolicanthus as female parent, and named TC-D. Previous
research has documented other triple hybrids, and
they both required the production of fertile hybrid
genotypes between at least two species, and then the
crossing between hybrids and a third species (Kaplan
& Fehrer, 2007; Peñaloza-Ramírez et al., 2010). The
DT-D type (H39) was detected in CM populations, the
pollen grains of which showed 80% viability. Therefore, H92 might be derived from hybridization
between a DT-D-type hybrid and R. chinensis, with
the DT-D-type hybrid as female parent. Kaplan &
Fehrer (2007) reported that a triple hybrid in Potamogeton L. is sterile, and can survive and form local
populations in the parental environment. However,
pollen grains of H92 were nonviable and abnormal,
and only one individual of this hybrid type was
observed in the contact zone; thus, this meant that
the survival of triple hybrids was difficult.
DT-T and D-T types were detected in the XM population. They shared the same plastid haplotype, but
nrITS clones of DT-T types were heterogeneous,
whereas those of D-T types were homogeneous. There
are two possible explanations for this result. One
involves concerted evolution, and the other recurrent
hybridization or introgression (Fuertes Aguilar,
Rossell & Nieto Feliner, 2002; Feliner, Larena &
Aguilar, 2004; Peñaloza-Ramírez et al., 2010; Kosnar
et al., 2012). It was difficult to prove which one plays
a role in this process. Nevertheless, all the D-T-type
hybrids were initially identified as R. silerifolius var.
dolicanthus without molecular data, and it seemed
that recurrent hybridization might contribute to the
homogenization of nrITS. Ranunculus trigonus is a
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
236
T. LI ET AL.
1
10
20
26
1
10
20
26
Consensus
Identity
TRATKCGTGCCGGMGTTGTAGAARGG
Consensus
Identity
TRATTCGNGCCGGCGTTGTRGAARGG
D2
9_1,3,5
9_4,6,7,8
9_2
T56
TAATTCGTGCCGGCGTTGTAGAAGGG
TAATTCGTGCCGGCGTTGTAGAAGGG
TAATGCGTGCCGGAGTTGTAGAAGGG
TAATGCGTGCCGGCGTTGTAGAAGGG
TGATTCGTGCCGGCGTTGTAGAAAGG
D2
77_5,8
77_7
77_6
77_2,3,4
T56
TAATTCGTGCCGGCGTTGTAGAAGGG
TAATTCGTGCCGGCGTTGTAGAAGGG
TGATTCG-GCCGGCGTTGTGGAAGGG
TGATTCGTGCCGGCGTTGTAGAAGGG
TGATTCGTGCCGGCGTTGTAGAAAGG
TGATTCGTGCCGGCGTTGTAGAAAGG
1
10
20
26
Consensus
Identity
TRATTCGTGCYGGCGTTGTAGAARRG
D2
39_6
39_2,3,4,5,7,8
T56
TAATTCGTGCCGGCGTTGTAGAAGGG
TAATTCGTGCCGGCGTTGTAGAAGGG
TGATTCGTGCTGGCGTTGTAGAAAAG
TGATTCGTGCCGGCGTTGTAGAAAGG
1
10
20
26
Consensus
Identity
TGATTYGTGCCGGCRTTRYAGARAGG
T56
96_2,5,6,7,8
96_1,3,4
C191
TGATTCGTGCCGGCGTTGTAGAAAGG
TGATTCGTGCCGGCGTTGTAGAAAGG
TGATTTGTGCCGGCATTACAGAGAGG
TGATTTGTGCCGGCATTACAGAGAGG
1
10
20
26
Consensus
Identity
TGAYTYGTGCCGGCRYTRYAGARAGG
T56
92_3,5,6,8
92_1
92_2,4
92_7
C191
TGATTCGTGCCGGCGTTGTAGAAAGG
TGATTCGTGCCGGCGTTGTAGAAAGG
TGACTCGTGCCGGCGTTGTAGAAAGG
TGATTTGTGCCGGCACTACAGAGAGG
TGATTTGTGCCGGCATTACAGAGAGG
TGATTTGTGCCGGCATTACAGAGAGG
1
10
20
26
Consensus
Identity
TRRTTYGTRCCGGCRTTRYAGRRRGG
D2
10_3,11,12
10_5
10_8
10_9
10_1
10_2,4,6,7
C192
TAATTCGTGCCGGCGTTGTAGAAGGG
TAATTCGTGCCGGCGTTGTAGAAGGG
TAGTTCGTGCCGGCGTTGTAGAAAGG
TAATTCGTGCCGGCGTTACAGGGAGG
TAATTCGTGCCGGCGTTACAGAGAGG
TGATTTGTACCGGCATTGTAGAGAGG
TGATTTGTACCGGCATTACAGAGAGG
TGATTTGTACCGGCATTACAGAGAGG
1
10
20
26
Consensus
Identity
TGATTYRTGYCRRCRTTRYARRRWGG
S147
138_4
138_6
138_8
138_7
138_5
138_1,2,3,9,10
C191
TGATTCATGTCAACGTTGTAAGATGG
TGATTCATGTCAACGTTGTAAGATGG
TGATTCATGTCAACGTTGTAAGAAGG
TGATTCATGTCAACGTTGTAAAGAGG
TGATTCATGTCAACGTTACAGAGAGG
TGATTTGTGTCAGCATTACAGAGAGG
TGATTTGTGCCGGCATTACAGAGAGG
TGATTTGTGCCGGCATTACAGAGAGG
Figure 5. Potentially parsimony informative sites observed in the alignment of cloned and directly sequenced nuclear
ribosomal internal transcribed spacer (nrITS) sequences. C, Ranunculus chinensis; D, R. silerifolius var. dolicanthus; S,
R. silerifolius var. silerifolius; T, R. trigonus.
selfing plant, and R. silerifolius var. dolicanthus has
larger flowers than the other species, and might be
outcrossing according to the theory of sex allocation
(Rademaker & De Jong, 2003). Therefore, this might
be in agreement with the findings of Ruhsam,
Hollingsworth & Ennos (2011), which showed that
introgression is likely to be asymmetric from selfing
to outcrossing lineages. However, D-T types are later
generation DT-T types. Moreover, pollen grains of
some DT-T types (H82) exhibited 44.7% viability, and
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239
A MULTISPECIES HYBRID ZONE IN RANUNCULUS
the proportion of DT-T and D-T types in the XM
population achieved 40.5%. Therefore, it is suggested
that there is a relatively low reproductive isolation
between R. trigonus and R. silerifolius var. dolicanthus, and hybridization between them is asymmetric,
with R. trigonus always serving as maternal parent.
Hybrids were detected in three populations located
in the contact zone. Among them, the CM population
comprised four taxa and formed five genotypes, the XM
population mainly comprised two taxa and formed two
genotypes, and the YT population mainly comprised
two taxa and formed one genotype. The results provide
an opportunity to compare the consequences of
multiple-species and two-species hybridizations. In the
YT population, two CS-S-type hybrids were found and
these had nonviable pollen. Thus, it is difficult for them
to produce a next generation and hybridization will
reinforce the reproductive isolation between R. chinensis and R. silerifolius (Nosil, Crespi & Sandoval,
2003). In the XM population, reproductive isolation
between R. trigonus and R. silerifolius var. dolicanthus was relatively low and many hybrids arose by
asymmetric hybridization. Previous research has
proven that asymmetric hybridization often results in
cytoplasmic introgression (Wu & Campbell, 2005), but
the results cannot provide a clear answer to this
question, and multilocus molecular markers are
needed for further research. Five genotypes arose from
crosses between different species pairs in the CM
population, which contained hybrids between different
species pairs, backcrosses and a triple hybrid.
Multiple-species hybridization did not increase the
hybridization rate, but the number of hybrid genotypes. This finding corroborates the ideas of Lepais
et al. (2009), who suggested that collective evolution
may take place within species belonging to the same
species complex, and the rate of exchange between
these species should not be viewed as a fixed parameter, but as a variable one that depends on several
factors, such as the local composition of the community.
ACKNOWLEDGEMENTS
The authors would like to express their thanks to
Daming Zhang (Centre for Systematic and Evolutionary Botany, Institute of Botany, Beijing, China) for
assistance with finishing the manuscript. This work
was supported by the National Natural Science Foundation of China (NSFC30860027, NSFC31260044)
and the Natural Science Foundation of Jiangxi Province (2009GZN0080).
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Supporting Information Table S1. ID, geographical coordinates, GenBank accession numbers of samples
included in the study.
Supporting Information Table S2. Pollen viability of some samples included in the study.
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 227–239