Molecular Phylogenetics and Evolution 63 (2012) 486–499
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Extreme habitats that emerged in the Pleistocene triggered divergence of weedy
Youngia (Asteraceae) in Taiwan
Koh Nakamura a,⇑, Kuo-Fang Chung b, Chiun-Jr Huang a, Yoshiko Kono a, Goro Kokubugata c,
Ching-I Peng a,⇑
a
b
c
Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan
School of Forestry and Resource Conservation, National Taiwan University, Daan, Taipei 106, Taiwan
Department of Botany, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba, Ibaraki 305-0005, Japan
a r t i c l e
i n f o
Article history:
Received 17 February 2011
Revised 29 January 2012
Accepted 30 January 2012
Available online 12 February 2012
Keywords:
Coalescent theory
Divergence time
Glacial periods
Incomplete lineage sorting
Pleistocene
Weed
a b s t r a c t
Weeds with broad distributions and large morphological variation are challenging for systematists and
evolutionarily intriguing because their intensive dispersal would likely prevent local morphological differentiation. Study on weeds will help to understand divergence in plants unlikely to be affected by geographical and ecological barriers. We studied Youngia japonica based on nrDNA and cpDNA sequences.
This is a widespread native in Asia and invasive worldwide; nevertheless, three subspecies (japonica, longiflora, and formosana) and an undescribed variant occur in Taiwan.
Bayesian and the most parsimonious phylogenies revealed that subspecies longiflora is a different linage and independently arrived in Taiwan during the Pleistocene via land connection to the Asian Continent. Bayesian time estimation suggested that Youngia in Taiwan diverged in the lower Pleistocene or
more recently. Extreme habitats that emerged in the Pleistocene, i.e., cold mountain ranges for subspecies
formosana and xeric, raised coral reefs for the undescribed Youngia variant probably had triggered the
divergence.
Components of Youngia in Taiwan are not monophyletic; a coalescent-based test suggested incomplete
lineage sorting. Nevertheless, the samples within each taxon share unique morphological features suggesting a common gene pool and each taxon has different dominant ITS and/or cpDNA types; these conditions suggest ongoing process toward monophyly via coalescent processes and support the delimitation
of intraspecific taxa.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
In weedy plants with broad distribution ranges, the origin of local
morphological variants is evolutionarily intriguing because intensive dispersals over their native ranges would likely prevent genetic
and morphological differentiations among populations (Kawecki
and Ebert, 2004). In fact, weedy plants with broad native ranges
sometimes indicate a lack of genetic structure (e.g., Schönswetter
et al., 2008). Thus revealing processes shaping morphological variant lineages in weedy plants is interesting in terms of understanding
causes and timing of divergence in plants that are unlikely to be affected by most geographical and ecological barriers. However, previous molecular analyses of weeds have focused on invasive
populations to elucidate the origins, introduction pathways, and/
or genetic variability in conservation contexts (e.g., Meekins et al.,
2001; Novak and Mack, 1993; Okada et al., 2009; Pairon et al.,
2010). Weedy plants with broad distribution ranges are notorious
⇑ Corresponding authors. Fax: +886 2 2789 1623.
E-mail addresses: kohnakamur@gmail.com (K. Nakamura), bopeng@sinica.edu.
tw (C.-I. Peng).
1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2012.01.023
for taxonomic difficulties (Brown, 1939; Davis, 1983; Le Roux and
Wieczorek, 2009) because they generally exhibit large morphological variations in diverse environmental conditions (Barrett, 1982;
Geng et al., 2007; Lodge, 1993). This variation may be either a consequence of phenotypic plasticity or a result of genetic differentiation
among populations (Molina-Montenegro et al., 2010; Müller-Schärer and Fischer, 2001; Kim and Donoghue, 2008b). Either of these
cases can lead to different taxonomic opinions on the recognition
of intraspecific taxa and the delimitation from allied species (Le
Roux et al., 2006; Schönswetter et al., 2008). Furthermore, different
taxonomic treatments can lead to different hypotheses for the origin, evolution, and genetic connectivity of morphological variant
populations. Knowledge of genealogical relationship is especially
useful for the proper taxonomy of morphological variants in weedy
plants (Le Roux et al., 2006; Wetzel et al., 1999). In this study, we explore these issues, i.e., phylogenetic systematics and evolution of
morphological variants of a weedy plant genus Youngia Cass. (Asteraceae) in Taiwan.
Youngia (tribe Cichorieae. Note that the name Cichorieae has
priority over the name Lactuceae) is a taxonomically difficult group
because of the weedy nature of some species that are highly
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K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
polymorphic (Babcock and Stebbins, 1937; Peng et al., 1998). The
genus comprises about 30 species (Mabberley, 2008) that are
widely distributed in Asia. They occur throughout China, southern
Siberia to the north, Taiwan and Japan to the east, Afghanistan to
the west, Malay Archipelago to the south, and disjunctly in Ceylon,
with the center of species diversity in the Himalaya region (Babcock and Stebbins, 1937; Ling and Shih, 1983; Peng et al., 1998).
Despite the high species diversity of the genus in the Asian Continent, there is only one species, namely Y. japonica (L.) DC. in
Taiwan (Peng and Chung, 1999; Peng et al., 1998). Youngia japonica,
occurring in Taiwan, Japan, China, Korea, Philippines, the Malay
Peninsula, and India (Ling and Shih, 1983), is the most widely
ranged species in the genus, and a polymorphic annual or biennial
herb with a basal rosette, growing up to 20–100 cm tall (Babcock
and Stebbins, 1937; Peng et al., 1998). Youngia japonica is
commonly found over a broad range of environmental conditions
from seaside to mountain ranges, from forest margin to disturbed
sites such as wastelands, cultivated fields, and roadsides (Peng
et al., 1998); but mostly in moist and semi-shaded habitats. As is
suggested by its broad range of habitats, Y. japonica is an invasive
weed worldwide (Barker et al., 2005; Botha, 2001; Botond and
Zoltán, 2004; PIER, 2010). Its wide distribution range and invasive
nature, together with its achenes (small dry indehiscent fruits)
having feathery pappi, suggest intensive dispersal in the native
distribution range. Thus, it is intriguing to note that three intraspecific taxa, namely Y. japonica subsp. japonica, Y. japonica subsp. longiflora Babc. and Stebbins, and Y. japonica subsp. formosana
(Hayata) Kitam. are recognized in Taiwan, a small island of ca.
36,000 km2 (Babcock and Stebbins, 1937; Hu, 1969; Kitamura,
1937; Peng and Chung, 1999; Peng et al., 1998; Table 1, Fig. 1).
Youngia japonica subsp. longiflora is a rare taxon restricted to a
littoral area in northern Taiwan, southeastern China, and recently
reported from Korea (Pak et al., 2000; Peng et al., 1998). Youngia
japonica subsp. formosana is endemic to Taiwan in mountain
ranges (1500–2500 m) (Babcock and Stebbins, 1937; Peng and
Chung, 1999; Peng et al., 1998). However, there are different taxonomic opinions; Y. japonica subsp. longiflora was recognized as a
distinct species while Y. japonica subsp. formosana was synonymized with Y. japonica subsp. japonica (Ling and Shih, 1983),
although no explanation was given for the taxonomic treatments
in the study. These different taxonomic treatments suggest different scenarios of the origin, evolution, and genetic connectivity
among the morphological variants in Taiwan. To elucidate processes of differentiation and proper taxonomy, molecular analysis
are needed, but no attempt has hitherto been made to resolve
the species phylogeny of the genus.
In addition to the above mentioned three intraspecific taxa, several populations of an atypical Youngia likely representing an undescribed taxon were recently discovered on Hsiao Liuchiu Islet and in
coastal regions of Kaohsiung City, southwestern Taiwan (Fig. 1). In
these areas, this unusual plant was restricted to littoral areas of
raised coral reefs that are exposed to direct sunlight. It shows
resemblance to Y. japonica but differs in several reproductive and
vegetative traits, especially by its velvety leaves (Table 1). In Taiwan, distribution of littoral raised coral reefs is restricted to Hsiao
Liuchiu Islet, small areas of Kaohsiung, and a part of Hengchun Peninsula of the southern tip of Taiwan Island (Hsieh and Shen, 1994).
Given the unique habitat preference of this undescribed Youngia,
we were interested in its origin and phylogenetic relationship to
the other congeners in Taiwan.
Despite its relatively small size, Taiwan warrants attention in
evolutionary studies because of its rugged terrain and geohistory.
Taiwan is a continental, mountainous island, with more than 100
peaks over 3000 m. In the neighboring Ryukyu Archipelago, all islands except for Yakushima Island are lower than 1000 m. Indeed,
Taiwan Island has exceptionally well-developed mountain systems
amongst the East Asian islands. Taiwan Island underwent extensive changes in land configuration during the Neogene (Chiang
and Schaal, 2006; Teng, 2007). This island emerged as the Luzon
arc collided with the Eurasian margins about 5 million years ago
(Ma). Taiwan Island quickly assumed the present shape at about
2 Ma through mountain building (Ho, 1982; Shaw, 1996). During
the Plio-Pleistocene, the island was repeatedly connected to the
Asian Continent as a result of eustatic sea level changes driven
by glacial advances and retreats. On the other hand, islets scattered
offshore Taiwan Island are either raised coral reefs (e.g., Hsiao Liuchiu) or volcanic islets (e.g., Lanyu and Lutao) that have more recent origin during the Pleistocene (Hsieh and Shen, 1994). In this
study, we endeavored to examine causal connections between
the geohistory and differentiation of Taiwanese Youngia.
We conducted molecular analyses based on nrDNA and cpDNA
sequences, aiming to test if morphological taxa of weedy and polymorphic Youngia are phylogenetically delimited, and to elucidate
the causes and timing of their differentiation in Taiwan.
2. Materials and methods
2.1. Plant materials
Four taxa of Youngia, namely Y. japonica subsp. japonica, Y. japonica subsp. longiflora, Y. japonica subsp. formosana, and the undescribed Youngia were collected (Table 2; Fig. 1). A comparison of
salient morphological features among them is shown in Table 1.
We cultivated collected plants in the experimental greenhouse of
Academia Sinica and ascertained that these morphological features
were stable in organs newly developed under cultivation (data not
shown). We collected 31 plants of Y. japonica subsp. japonica samples from 15 localities in Taiwan, Japan and China. Their habitats
ranged from coastal to hilly ranges (ca. 1000 m alt.), and from undisturbed forest margins to urban areas. Youngia japonica subsp. longiflora in Taiwan is restricted to a very narrow area of northern tip of
Taiwan Island and grows in littoral environment; five plants were
collected from a locality. Five plants of Y. japonica subsp. formosana
were collected from three localities in mountain ranges in northern
and central Taiwan. Twelve plants of the undescribed Youngia were
Table 1
Comparison of salient morphological features among the subspecies of Youngia japonica and the undescribed Youngia primarily based on Peng et al. (1998), Peng and Chung
(1999), and authors’ observation.
Taxon
Youngia japonica
subsp. japonica
subsp. longiflora
subsp. formosana
The undescribed
Youngia
Head width
(mm)
Achene color and length
Leaf shape
Inner involucre
length (mm)
Hair on leaf and inflorescence
axes
5–8
>15
12–15
Brown, ca. 1.8 mm
Dark purplish brown, 2–2.6 mm
Reddish brown to dark purple, 2–
2.5 mm
Reddish brown to dark purplish
brown, ca. 2.5 mm
Entire to lyrate-pinnatifid
Lyrate-pinnatifid
Highly-dissected and
runcinate-pinnatifid
Thicker, lyrate-pinnatifid
4–6
6–8
ca. 5
Glabrous or slightly puberulous
Glabrous or slightly puberulous
Glabrous or slightly puberulous
ca. 5
Leaves velvety; inflorescence
axes puberulous
ca. 13
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K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
13
12
Japan
14
26
China
25
11
15
6 8
23 24
10
9
7
Taiwan
subsp. japonica
subsp. formosana
subsp. longiflora
Undescribed Youngia
N
22
1
16
17
18
21
1 km
19
N
2
20
5
Altitude
3
4
50 km
Hsiao Liuchiu
Fig. 1. Collection localities of Youngia in Taiwan, Japan, and China. The numbers correspond to those in Table 2: 1–15, Y. japonica subsp. japonica; 16, Y. japonica subsp.
longiflora; 17–19, Y. japonica subsp. formosana; 20–22, the undescribed Youngia; 23, Y. heterophylla; 24, Y. cf. pseudosenecio; 25, Y. pseudosenecio; 26, Y. erythrocarpa. For details
of the localities, refer to the table. In the lower right panel, areas in different colors indicate approximate distribution ranges of the three subspecies of Youngia japonica and
the undescribed Youngia in Taiwan. Lower left panel shows topography of Taiwan.
collected from all of the three known localities: two in Hsiao Liuchiu
Islet, and one in Kaohsiung of Taiwan Island. When different taxa
were found in proximity, we collected both taxa there because this
was a suitable setting to test their genetic distinctiveness with no
geographical barrier. The locality of Y. japonica subsp. longiflora
was very proximate to a locality of Y. japonica subsp. japonica (no.
1, Longtou). Also, the population of the undescribed Youngia in
Kaohsiung was very proximate to a population of Y. japonica subsp.
japonica (no. 2, Chaishan) although their habitats were different. In
addition, Y. heterophylla (Hemsl.) Babc. and Stebbins, Y. pseudosenecio (Vaniot) Shih, and Y. erythrocarpa (Vaniot) Babc. and Stebbins, all
endemic to mainland China (Ling and Shih, 1983), were included in
the study. These species are considered to be closely related to Y.
japonica, sharing characteristics such as developed main stem, lack
of residue of old petioles, and phyllaries without abaxial apical
appendages (Ling and Shih, 1983). An ITS phylogeny of Cichorieae
representing the entire tribe (including 83 out of ca. 93 genera in
all) indicated the sister relationship between Youngia and Crepidiastrum (Kilian et al., 2009). We used C. keiskeanum (Maxim.) Nakai,
C. lanceolatum (Houtt.) Nakai, and C. platyphyllum (Franch. and
Sav.) Kitam. as outgroup taxa. Based on anatomical and karyological
data, Crepidiastrum and Paraixeris are the most closely related with
each other (Pak and Kawano, 1990). Thus we also used P. denticulata
(Houtt.) Nakai as an outgroup. Paraixeris denticulata was once
treated as Youngia denticulata (Houtt.) Kitam., but anatomical,
karyological and molecular studies indicated that P. denticulata is a
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K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
Table 2
Collection locality, and ITS and cpDNA sequence types of Youngia from Taiwan, Japan, and China.
Taxon no.
Locality and voucher no.
Y. japonica subsp. japonica
1
Longtou, Taipei, Taiwan
Nakamura 20100144
Nakamura 20100145
Nakamura 20100141
2
Chaishan, Kaohsiung, Taiwan
Nakamura 20100327 g
Nakamura 20100327i
3
Sizugang1, Kaohsiung, Taiwan
Nakamura 20100328e
Nakamura 20100328f
Nakamura 20100328 g
Nakamura 20100328 h
Nakamura 20100328i
4
Sizugang2, Kaohsiung, Taiwan
Nakamura 20100329f
Nakamura 20100329 g
Nakamura 20100329i
5
Gushan, Kaohsiung, Taiwan
Huang 3186-1
6
Tabarugawa, Yonaguni Is., Japan
Nakamura 20100247
Nakamura 20100248
7
Ohara, Iriomote Is., Japan
Nakamura 20100245
8
Maesato, Ishigaki Is., Japan
Nakamura 20100238
Nakamura 20100239
Nakamura 20100240
9
Ginowan, Okinawa Is., Japan
Nakamura 20100268
10
Ogimi, Okinawa Is., Japan
Nakamura 20100446
11
Kumejima, Kumejima Is., Japan
Nakamura 20100476
12
Kochi, Shikoku, Japan
Nakamura 20100228
13
Ibaraki, Honshu, Japan
Nakamura 20101504
14
Mozi, Chengdu, Sichuan, China
Peng 22575-1
Peng 22575-3
Peng 22575-5
15
Chengdu, Sichuan, China
Peng 22607-1
Peng 22607-2
Peng 22607-3
Y. japonica subsp. longiflora
16
Nanya, Taipei, Taiwan
Nakamura 20100147
Nakamura 20100149
Nakamura 20100151
Huang 3187A
Huang 3187B
Y. japonica subsp. formosana
17
Jianshi, Hsinchu, Taiwan
Peng 22472a
Peng 22472b
18
Hsinchu, Taiwan
Peng s.n.
19
Hehuanshan, Nantou, Taiwan
Nakamura 20100557a
Nakamura 20100557c
The undescribed Youngia
20
Chaishan, Kaohsiung, Taiwan
Huang 3183-1
Huang 3183-2
Huang 3183-3
Huang 3183-4
Huang 3180
nrDNA
cpDNA
Type
ITS
Type
atpB–rbcL
trnT–trnL
trnL–trnF
rpl16
rps16
A
A
Aa
AB598556
AB598556
AB598556
A
A
B
AB598567
AB598567
AB598567
AB598614
AB598614
AB598614
AB598603
AB598603
AB598603
AB598577
AB598577
AB598577
AB598590
AB598590
AB598591
B
B
AB598557
AB598557
A
A
AB598567
AB598567
AB598614
AB598614
AB598603
AB598603
AB598577
AB598577
AB598590
AB598590
B
B
B
B
B
AB598557
AB598557
AB598557
AB598557
AB598557
A
A
A
A
A
AB598567
AB598567
AB598567
AB598567
AB598567
AB598614
AB598614
AB598614
AB598614
AB598614
AB598603
AB598603
AB598603
AB598603
AB598603
AB598577
AB598577
AB598577
AB598577
AB598577
AB598590
AB598590
AB598590
AB598590
AB598590
B
B
B
AB598557
AB598557
AB598557
A
A
A
AB598567
AB598567
AB598567
AB598614
AB598614
AB598614
AB598603
AB598603
AB598603
AB598577
AB598577
AB598577
AB598590
AB598590
AB598590
B
AB598557
A
AB598567
AB598614
AB598603
AB598577
AB598590
B
B
AB598557
AB598557
A
A
AB598567
AB598567
AB598614
AB598614
AB598603
AB598603
AB598577
AB598577
AB598590
AB598590
B
AB598557
A
AB598567
AB598614
AB598603
AB598577
AB598590
B
B
B
AB598557
AB598557
AB598557
A
A
A
AB598567
AB598567
AB598567
AB598614
AB598614
AB598614
AB598603
AB598603
AB598603
AB598577
AB598577
AB598577
AB598590
AB598590
AB598590
B
AB598557
A
AB598567
AB598614
AB598603
AB598577
AB598590
A
AB598556
A
AB598567
AB598614
AB598603
AB598577
AB598590
A
AB598556
A
AB598567
AB598614
AB598603
AB598577
AB598590
A
AB598556
A
AB598567
AB598614
AB598603
AB598577
AB598590
A
AB598556
A
AB598567
AB598614
AB598603
AB598577
AB598590
B
B
B
AB598557
AB598557
AB598557
C
C
C
AB598567
AB598567
AB598567
AB598614
AB598614
AB598614
AB598603
AB598603
AB598603
AB598580
AB598580
AB598580
AB598590
AB598590
AB598590
B
B
B
AB598557
AB598557
AB598557
A
A
A
AB598567
AB598567
AB598567
AB598614
AB598614
AB598614
AB598603
AB598603
AB598603
AB598577
AB598577
AB598577
AB598590
AB598590
AB598590
C
C
Ca/Aa
Ca/Aa
C
AB598558
AB598558
AB598558/AB598556
AB598558/AB598556
AB598558
D
D
D
D
D
AB598568
AB598568
AB598568
AB598568
AB598568
AB598615
AB598615
AB598615
AB598615
AB598615
AB598604
AB598604
AB598604
AB598604
AB598604
AB598578
AB598578
AB598578
AB598578
AB598578
AB598592
AB598592
AB598592
AB598592
AB598592
D
D
AB598559
AB598559
B
B
AB598567
AB598567
AB598614
AB598614
AB598603
AB598603
AB598577
AB598577
AB598591
AB598591
D
AB598559
B
AB598567
AB598614
AB598603
AB598577
AB598591
D
D
AB598559
AB598559
B
B
AB598567
AB598567
AB598614
AB598614
AB598603
AB598603
AB598577
AB598577
AB598591
AB598591
A
A
A
A
A
AB598556
AB598556
AB598556
AB598556
AB598556
E
E
E
E
E
AB598567
AB598567
AB598567
AB598567
AB598567
AB598616
AB598616
AB598616
AB598616
AB598616
AB598605
AB598605
AB598605
AB598605
AB598605
AB598579
AB598579
AB598579
AB598579
AB598579
AB598593
AB598593
AB598593
AB598593
AB598593
(continued on next page)
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K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
Table 2 (continued)
Taxon no.
nrDNA
cpDNA
Type
ITS
Type
atpB–rbcL
trnT–trnL
trnL–trnF
rpl16
rps16
A
A
A
A
AB598556
AB598556
AB598556
AB598556
E
E
E
E
AB598567
AB598567
AB598567
AB598567
AB598616
AB598616
AB598616
AB598616
AB598605
AB598605
AB598605
AB598605
AB598579
AB598579
AB598579
AB598579
AB598593
AB598593
AB598593
AB598593
A
Aa
Aa
AB598556
AB598556
AB598556
E
F
B
AB598567
AB598567
AB598567
AB598616
AB598617
AB598614
AB598605
AB598603
AB598603
AB598579
AB598577
AB598577
AB598593
AB598591
AB598591
Y. heterophylla
23
Guangxi, China
Peng 21125
E
AB598561
G
AB598569
AB598619
AB598608
AB598582
AB598594
Youngia cf. pseudosenecio
24
Guilin, Guangxi, China
Chung s.n.
F
AB598562
H
AB598571
AB598618
AB598606
AB598581
AB598596
Y. pseudosenecio
25
Mozi, Chengdu, Sichuan, China
Peng 22575-2
Peng 22575-4
Peng 22575-6
Peng 22575-7
G
G
H
G
AB598560
AB598560
AB598565
AB598560
I
J
K
I
AB598572
AB598572
AB598572
AB598572
AB598620
AB598621
AB598622
AB598620
AB598607
AB598607
AB598607
AB598607
AB598583
AB598583
AB598584
AB598583
AB598595
AB598595
AB598595
AB598595
Y. erythrocarpa
26
Dujiangyan, Sichuan, China
Peng 22579
Peng 22583
Peng 22584
Peng 22592
I
I
I
I
AB598566
AB598566
AB598566
AB598566
L
L
L
M
AB598570
AB598570
AB598570
AB598570
AB598623
AB598623
AB598623
AB598624
AB598609
AB598609
AB598609
AB598609
AB598585
AB598585
AB598585
AB598585
AB598597
AB598597
AB598597
AB598598
Crepidiastrum keiskeanum
–
Shizuoka, Honshu, Japan
Sadamu Matsumoto 991111-1
J
AB598564
N
AB598576
AB598628
AB598613
AB598589
AB598602
C. lanceolatum
–
Yomitan, Okinawa Is., Japan
Kokubugata 2492
K
AB598563
O
AB598575
AB598627
AB598612
AB598588
AB598601
C. platyphyllum
–
Kanagawa, Honshu, Japan
Kokubugata 1193
L
AY876259
P
AB598573
AB598625
AB598610
AB598586
AB598599
Paraixeris denticulata
–
Kanagawa, Honshu, Japan
Kokubugata 1212
M
AY876265
Q
AB598574
AB598626
AB598611
AB598587
AB598600
21
22
a
Locality and voucher no.
Wuguidong, Hsiao Liuchiu, Taiwan
Huang 3176-1
Huang 3176-2
Huang 3176-3
Huang 3176-4
Meirendong, Hsiao Liuchiu, Taiwan
Huang 3168-1
Huang 3168-2
Huang 3168-3
Sequences obtained by cloning are marked with an asterisk in ‘‘Type’’ column.
member of Paraixeris (Pak and Kawano, 1990; Park et al., 2003).
Voucher specimens were deposited in the Herbarium of Biodiversity
Research Center, Academia Sinica (HAST).
2.2. DNA extraction, amplification, and sequencing
Total DNA was isolated from leaf tissue using the cetyl trimethyl
ammonium bromide (CTAB) method of Doyle and Doyle (1987). The
following six molecular markers were amplified using PCR: ITS region (including ITS1 and ITS2 spacer regions and the 5.8S rRNA
gene) of nrDNA, and the intergenic spacers between atpB and rbcL
(atpB–rbcL), trnT (UGU) and trnL (UAA) 50 exon (trnT–trnL), trnL
(UAA) 30 exon and trnF (GAA) (including the trnL intron; trnL–trnF),
and introns of rpl16 and rps16 genes of cpDNA. PCR reactions were
performed in 25 lL total volume with the following reagents: about
10 ng of genomic DNA, 1 unit of Taq DNA polymerase master mix
(Ampliqon, Rødovre, Denmark), 0.4 lM of each primer, and 2%
DMSO. The primers used for PCR amplification and the PCR cycle
conditions were as follows: ITS, primers ITS1 and ITS4 (White
et al., 1990), 95 °C for 5 min, 1 cycle of 97 °C for 2 min, 50 °C for
1 min, 72 °C for 1 min, 25 cycles of 95 °C for 1 min, 50 °C for
2 min, 72 °C for 3 min, and 72 °C for 10 min: atpB–rbcL, primers
atpB2F and rbcL2R (Nakamura et al., 2006), 95 °C for 5 min, 35
cycles of 95 °C for 1 min, 57 °C for 1 min, 72 °C for 2 min, and
72 °C for 10 min (Nakamura et al., 2007). For the other four regions,
the same PCR condition (94 °C for 2 min, 35 cycles of 94 °C for 30 s,
52 °C for 60 s, 72 °C for 105 s, and 72 °C for 10 min) was used with
the following primers: trnT–trnL, primers trnT(UGU)-a and
trnL(UAA)-b (Taberlet et al., 1991): trnL–trnF, primers trnL(UAA)-c
and trnF(GAA)-f (Taberlet et al., 1991): rpl16 intron, primers
rpL16F71 and rpL16R1516 (Small et al., 1998): rps16 intron, primers
rpS16F and rpS16R2 (Oxelman et al., 1997).
For samples which shared the same (or very similar) cpDNA
haplotype with other taxa, cloning of the ITS region was conducted
to test hybridity using the pGEM-T vector system kit (Promega,
Madison, WI, USA) and ECOS 101 competent cell (Yeastern Biotech,
Taipei, Taiwan). Colonies were amplified using PCR in 20 lL volumes containing 11.8 lL of sterile water, 2 lL of 10 Prime reaction
buffer, 2 lL of a 10 lM solution of each SP6 primer (GATTT AGGTG
ACACT ATAG) and T7 primer (TAATA CGACT CACTA TA), 0.2 lL of
Prime Taq polymerase, and 1 colony. PCR condition was as follows:
initial template denaturation at 94 °C for 5 min, followed by 25
cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s and a final extension at 72 °C for 7 min. Cloning of the ITS region using the same protocol was also conducted for putative hybrids which showed
double-peak nucleotide signals in the ITS by direct sequencing.
K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
The PCR fragments were purified with shrimp alkaline phosphatase and exonuclease I (Promega). Purified PCR fragments were
used as templates for cycle sequencing reactions with the same
primers used in the PCR, and direct sequencing was performed
on an ABI Prism 3730 DNA analyzer (Applied Biosystems, Foster
City, CA, USA). The sequence data were deposited in the DDBJ/
EMBL/GenBank databases (AB598556–AB598628; Table 2).
2.3. Phylogenetic analyses
The DNA sequences were aligned using the program ClustalX
ver. 1.8 (Thompson et al., 1997) and then adjusted manually using
BioEdit ver. 7.0.9.0 (Hall, 1999). The combinability of the five
cpDNA regions was assessed with the incongruence length difference (ILD) test (Farris et al., 1994) using the partition homogeneity
test implemented in PAUP ver. 4.0b10 (Swofford, 2002). For
cpDNA sequences, indices of genetic diversity, i.e. the haplotype
diversity (h) and nucleotide diversity (p) were calculated for
samples from Taiwan using DnaSP ver. 5 (Librado and Rozas,
2009). Statistics of Tajima’s D (Tajima, 1989) and Fu’s Fs (Fu,
1997) were calculated for Taiwanese samples with DnaSP. Expectations of these statistics are nearly zero in genetic admixture
while positive value can indicate non-admixture (i.e. subdivision
of a data set). Significance of deviation from zero was examined
based on 10,000 coalescent simulations.
Phylogenetic analyses were conducted based on Bayesian and
parsimony criteria. In Bayesian analysis, the Hierarchical Likelihood Ratio Tests (hLRT) implemented in MrModeltest ver. 2.2
(Nylander, 2004) was used to estimate the appropriate evolutionary model of nucleotide substitutions. The software MrBayes ver.
3.1.2 (Ronquist and Huelsenbeck, 2003) was used and two separate
runs of Metropolis-coupled Markov chain Monte Carlo (MCMCMC)
analyses were performed, with a random starting tree and four
chains for each run (one cold and three heated). The MCMCMC
length was five million generations, and the chain was sampled
every 100th generation from the cold chain. The default setting
was used for swapping frequency (swapfreq = 1) and chain heating
(temp = 0.2). We ascertained that the cold chain successfully
swapped states with the heated chains and the Metropolis coupling worked well, by checking the resultant log files. The mixing
and convergence of the MCMC chains of the two runs was assessed
by inspection of the trace plots of parameters using the program
Tracer ver. 1.5.0 (Drummond and Rambaut, 2007). Burn-in was
determined by plotting tree log likelihoods against generations;
trees from generations before the plot reached plateau were discarded. In the ITS and cpDNA analyses, the first 12,500 sample
trees (25% of the total 50,000 sample trees) were discarded. After
the burn-in, the effective sample sizes (ESS) of all parameters were
more than 1000, indicating that the analyses sampled the posterior
distributions of each parameter satisfactorily, and the values of
Average Standard Deviation of Split Frequency (ASDSF) were below
0.01. The 50% majority rule consensus tree of all the post-burn-in
trees was generated using TreeView ver. 1.6.6 (Page, 1996).
The program PAUP was used for parsimony analyses. Indels
were treated as missing data. Characters were treated as unordered, and character transformations were weighted equally. The
branch collapse option was set to collapse at a minimum length
of zero. A heuristic parsimony search was performed with 200
replicates of random additions of sequences, with the ACCTRAN
character optimization, tree-bisection–reconnection (TBR) branch
swapping, MULTREES, and STEEPEST DESCENT options on. Statistical support for each clade was assessed by bootstrap analysis
(Felsenstein, 1985). One thousand replicates of heuristic searches,
with the TBR branch swapping option on and MULTREES option
off, were performed to calculate bootstrap values.
491
2.4. Divergence time estimation
To estimate approximate times of lineage divergences based on
each of the ITS and cpDNA sequence variations, the molecular clock
hypothesis of the data was tested using the likelihood ratio (LR) test
(Felsenstein, 1988) implemented in PAUP. The hypothesis of rate
constancy was evaluated by comparing the log likelihood (L) of
Maximum likelihood trees with and without assuming a molecular
clock, based on the substitution model GTR + I + G determined by
Modeltest ver. 3.7 (Posada and Crandall, 1998). The LR was calculated as 2 (ln Lnoclock ln Lclock) and assumed to follow a chi-squared
distribution with the number of degrees of freedom (d.f.) equal to
the number of samples minus two (Muse and Weir, 1992). The LR
tests rejected the molecular clock hypothesis for the ITS and cpDNA
data at the significance level of P < 0.0001 (ITS, ln Lnoclock = 163
8.52, ln Lclock = 1923.27, LR = 569.50, d.f. = 65, P < 0.0001; cpDNA,
ln Lnoclock = 5849.03,
ln Lclock = 6058.23, LR = 418.39, d.f. = 65,
P < 0.0001). On the basis of these results, Bayesian estimation of
divergence times was performed using the program BEAST ver.
1.5.4 (Drummond and Rambaut, 2007). The analyses were conducted under a relaxed-clock model; this was because simulation
studies indicated that it performed well when the data were nonclocklike and when clocklike (Drummond et al., 2006). We used
uncorrelated lognormal distribution model for rate variation among
lineages, which is robust even when data are generated based on
different types of models (Drummond et al., 2006). We employed
the Yule tree prior for the branching rates and applied the
GTR + I + G nucleotide substitution model. An unweighted pairgroup method of arithmetic averages (UPGMA) was used to construct a starting tree.
Because there is no fossil of Youngia nor of its phylogenetically
allied taxa for molecular clock calibration, divergence times were
calculated using reported substitution rates. There are multiple
estimates of ITS substitution rates in Asteraceae with a minimum
generation time of 2–3 years, ranging from 2.51 10 9 to
7.83 10 9 substitutions site 1 year 1 (Kay et al., 2006; Richardson et al., 2001). These values are similar to values reported in
other plant families (Kay et al., 2006; Richardson et al., 2001).
Some lineages of temperate herbaceous Asteraceae, however, were
reported to have higher ITS substitution rates, ranging from ca.
1 10 8 to 3 10 8 substitutions site 1 year 1 (Schmidt and
Schilling, 2000). These higher rates were calculated based on fossil
evidence (Graham, 1996) and broad phylogeny of Asteraceae (Kim
and Jansen, 1995; Schmidt and Schilling, 2000), and were
supported by another study using vicariance-based calibration
(Bremer and Gustafsson, 1997). Recently application of the higher
ITS substitution rate (1.95 10 8 substitutions site 1 year 1) also
generated reasonable divergence time estimates in other Asteraceae genus (Ainsliaea, Mitsui et al., 2008). Using the general ITS
substitution rates (the mean of 2.51 10 9 and 7.83 10 9), the
age of the most recent common ancestor (MRCA) of Youngia in
our data was estimated as 68 million years, about 1.7 times older
than the earliest Asteraceae fossil record (Graham, 1996). However, because the subfamily Cichorioideae is not a basal lineage
of Asteraceae and Youngia belongs to the recently diverged subtribe Crepidinae within the subfamily (Funk et al., 2009; Kilian
et al., 2009), the estimated old age of the MRCA of Youngia is highly
unlikely, suggesting that Youngia might have a higher molecular
evolution rate. Consequently the higher ITS substitution rates were
employed. We used a normal distribution prior with the mean
(2.00 10 8) and standard deviation (s.d.) of 0.59 10 8, which
covered a range from 1.00 10 8 to 3.00 10 8 in the 95% range
(mean ± 1.69 s.d.) of the distribution. In the analysis of cpDNA data,
we used a normal distribution prior with the mean (4.36 10 9)
and s.d. of 2.29 10 9, which covered a range from 4.87 10 10
to 8.24 10 9 (the lowest and highest rates for cpDNA noncoding
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K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
region of plants with a minimum generation time of 2–3 years,
Richardson et al., 2001) in the 95% range of the distribution.
For the remaining parameters, default priors were used. MCMC
chains were run for 40 million generations and sampled every
1000 generations. We ran two separate analyses and the first
4000 of the 40,000 sampled generations in each run were discarded as ‘‘burn-in’’. We combined log files from the two runs
using LogCombiner ver. 1.5.4 of the BEAST package and used Tracer
ver. 1.5 program of the package to ensure convergence, to measure
the effective sample size, and to calculate the mean of divergence
time estimates.
2.5. Distinguishing incomplete lineage sorting and chloroplast capture
Because cpDNA phylogeny was incongruent with morphological
delimitation of some samples of the undescribed Youngia and Y.
japonica (see result, Section 3.2.), we conducted an analysis to distinguish two possible causes that can lead to the incongruence: incomplete lineage sorting (ILS) and chloroplast capture (or introgression)
via hybridization. ILS is the failure of ancestral polymorphisms to
track speciation (or a divergence event) and generates a phylogenetic pattern very similar to that observed for chloroplast capture
events (Buckley et al., 2006; Holder et al., 2001; Holland et al.,
2008; Joly et al., 2009; Kim and Donoghue, 2008a). We used a coalescent-based method to test the plausibility of ILS (Pelser et al., 2010).
Coalescent theory predicts that ancestral polymorphisms coalesce
within approximately 5Ne generations (Ne: effective population
size) with high probability, and thereafter monophyly of lineages
is probable (Degnan and Rosenberg, 2009; Hudson and Turelii,
2003; Pelser et al., 2010; Rosenberg, 2003). Ne that must be assumed
to explain the incongruence by ILS was calculated by dividing an
estimate of the duration of ILS event by the generation time of Youngia (about 2 years) and the constant of 5. If estimated Ne are much
larger than population sizes observed in the wild, ILS hypothesis
can be excluded and chloroplast capture is favored as the likely
explanation for the observed incongruence (Pelser et al., 2010). In
a scenario of ILS, a speciation event is assumed to be the approximate timing of the onset of an ILS event (Joly et al., 2009; Pelser
et al., 2010). The MRCA of a morphologically incongruent lineage(s)
and its sister lineage species predates the speciation event, and thus
the age of the MRCA represents the oldest limit of the onset of the ILS
event (Joly et al., 2009). The age of the MRCA of morphologically
incongruent lineages of the undescribed Youngia and its sister
lineage species was estimated (Comes and Abbott, 2001).
Each population of the undescribed Youngia in Hsiao Liuchiu
Islet comprised several hundred plants in a very rough estimate,
summing up to less than 2000 plants in the islet. The population
in Kaohsiung was much smaller. However, the habitats in the islet
have been exploited to construct walking trails. Therefore, it is
reasonably assumed that there were more plants in the past. As a
Youngia population in Taiwan generally ranges between several
hundreds to several thousands of individuals, we considered that
10,000 is the maximum of long-term effective population size for
the undescribed Youngia in Hsiao Liuchiu Islet.
3. Results
3.1. NrDNA analyses
In Youngia, the aligned sequence length of ITS was 649 bp. 79
nucleotide substitutions were found in 74 variable sites, and
among them, 20 sites were parsimony informative. In total, nine
types of ITS sequences were recognized (A–I, Table 2). Sequences
obtained by cloning are indicated in Table 2. In Y. japonica, four
types of ITS sequences were obtained (A–D). Youngia japonica
subsp. japonica had types A and B. Three plants of Y. japonica subsp.
longiflora had type C while the other two were heterozygotes of
types C and A. All samples of Y. japonica subsp. formosana had type
D. All of the undescribed Youngia samples had type A.
In the Bayesian analysis, the hLRT selected the SYM + G substitution model. The 50% majority rule consensus tree with mean
branch length of all the post-burn-in trees is depicted in Fig. 2 with
Bayesian posterior probabilities (PP). MP analysis yielded 77
equally parsimonious trees of 144 steps with a consistency index
(CI) = 0.931, a retention index (RI) = 0.985, and a rescaled consistency index (RC) = 0.916. The topology of the MP strict consensus
tree (not shown) was exactly the same as that of the Bayesian tree.
The bootstrap percentages (BP) are plotted on the Bayesian tree.
Concerning the indels (except the ones related to mononucleotide
repeats), two insertions and one deletion of 1–2 bp were found in
the ingroups, and a 1 bp-deletion was found in the ingroups except
Y. erythrocarpa.
Youngia japonica was not monophyletic. Youngia japonica subsp.
longiflora with ITS type C formed a clade (PP = 0.99/BP = 86.9), and
this clade was not sister to a clade comprising the other Y. japonica
samples (including subspecies japonica and formosana, and
heterozygotes of subspecies longiflora) plus the undescribed
Youngia (PP = 1.00/BP = 99.8) because a clade of Y. pseudosenecio
(PP = 1.00/BP = 87.3) was located between the two clades. In the
clade of Y. japonica plus the undescribed Youngia, Y. japonica subsp.
formosana formed a clade (PP = 1.00/BP = 86.1) and clustered in a
polytomy with the other samples.
3.2. CpDNA analyses
In Youngia, the aligned lengths of the atpB–rbcL, trnT–trnL,
trnL–trnF, rpl16, and rps16 were 681, 990, 747, 778, and 593 bp,
respectively. The ILD test did not show significant incongruence
among the five regions (P = 0.687). Therefore, all further analyses
were conducted using the combined data of 3789 bp. In Youngia,
60 nucleotide substitutions were found in 60 variable sites, and
among them, 39 sites were parsimony informative. In total, 13
haplotypes were recognized (A–M, Table 2). In Y. japonica, four
haplotypes were obtained (A–D). Youngia japonica subsp. japonica
had types A–C; Y. japonica subsp. longiflora had type D; Y. japonica
subsp. formosana had type B. The undescribed Youngia had three
haplotypes B, E, and F.
The haplotype (h) and nucleotide (p 103) diversities in
Taiwanese samples are indicated in Table 3. Youngia japonica
(including the three subspecies) showed smaller h compared to
the value of a data set of Y. japonica subsp. japonica and subsp. formosana plus the undescribed Youngia. On the other hand, the former showed larger p than the latter. All the estimates of Tajima’s
D and Fu’s Fs values indicated positive values (Table 3); among
them, the estimate of Fu’s Fs value for the data set of Y. japonica
(including the three subspecies) was significantly deviated from
zero (P < 0.05).
In the Bayesian analysis, the hLRT selected the GTR + I + G substitution model. The 50% majority rule consensus tree of all the
post-burn-in trees is depicted in Fig. 3. MP analysis yielded seven
equally parsimonious trees of 138 steps with a consistency index
(CI) = 0.913, a retention index (RI) = 0.975, and a rescaled consistency index (RC) = 0.890. The topology of the MP strict consensus
tree was the same as that of the Bayesian tree, except for the clade
of Y. pseudosenecio (PP = 0.66/BP = 55.5) and the clade of haplotypes A and C of Y. japonica subsp. japonica (PP = 0.80/BP < 50) that
both collapsed in the MP strict consensus tree. The bootstrap percentages are plotted on the Bayesian tree.
Youngia japonica (including the three subspecies) and the undescribed Youngia clustered in a polytomy with Y. pseudosenecio
(PP = 1.00/BP = 100). Within this clade, Y. japonica subsp. japonica
K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
493
Paraixeris denticulata 1212 (M)
Crepidiastrum platyphyllum 1193 (L)
Crepidiastrum lanceolatum 2492 (K)
1.00 / 98.5
Crepidiastrum keiskeanum 991111-1 (J)
1.00 / 100
Y. erythrocarpa 22579 (I)
Y. erythrocarpa 22583 (I)
Y. erythrocarpa 22584 (I)
Y. erythrocarpa 22592 (I)
1.00 / 100
Y. heterophylla21125 (E)
subsp. longiflora 147 (C)
subsp. longiflora 149 (C)
0.99 / 86.9
subsp. longiflora 151 (C)
subsp. longiflora 3187A (C)
subsp. longiflora 3187B (C)
Y. cf. pseudosenecio s.n. (F)
1.00 / 87.3
Y. pseudosenecio 22575-6 (H)
Y. pseudosenecio 22575-2 (G)
0.99 / 64.6
Y. pseudosenecio 22575-4 (G)
0.98 / 63.2
Y. pseudosenecio 22575-7 (G)
subsp. japonica 141 (A)
1.00 / 96.1
subsp. japonica 144 (A)
subsp. japonica 145 (A)
subsp. japonica 446 (A)
subsp. japonica 476 (A)
subsp. japonica 228 (A)
subsp. japonica 1504 (A)
Undescribed Youngia 3183-1 (A)
Undescribed Youngia 3183-2 (A)
Undescribed Youngia 3183-3 (A)
Undescribed Youngia 3183-4 (A)
1.00 / 92.7
Undescribed Youngia 3180 (A)
Undescribed Youngia 3176-1 (A)
Undescribed Youngia 3176-2 (A)
Undescribed Youngia 3176-3 (A)
Undescribed Youngia 3176-4 (A)
Undescribed Youngia 3168-1 (A)
Undescribed Youngia 3168-2 (A)
Undescribed Youngia 3168-3 (A)
subsp. longiflora 151 (A)
subsp. longiflora 3187A (A)
subsp. japonica 327g (B)
subsp. japonica 327i (B)
1.00 / 99.8
subsp. japonica 328e (B)
subsp. japonica 328f (B)
subsp. japonica 328g (B)
subsp. japonica 328h (B)
subsp. japonica 328i (B)
subsp. japonica 329f (B)
subsp. japonica 329g (B)
subsp. japonica 329i (B)
subsp. japonica 3186-1 (B)
subsp. japonica 247 (B)
subsp. japonica 248 (B)
subsp. japonica 245 (B)
subsp. japonica 238 (B)
subsp. japonica 239 (B)
subsp. japonica 240 (B)
subsp. japonica 268 (B)
subsp. japonica 22575-1 (B)
subsp. japonica 22575-3 (B)
subsp. japonica 22575-5 (B)
subsp. japonica 22607-1 (B)
subsp. japonica 22607-2 (B)
subsp. japonica 22607-3 (B)
subsp. formosana 22472a (D)
subsp. formosana 22472b (D)
subsp. formosana s.n. (D)
1.00 / 86.1
subsp. formosana 557a (D)
0.2
subsp. formosana 557c (D)
1.00 / 100
Clade 1
Y. japonica subsp. longiflora
Y. japonica subsp. japonica
Undescribed Youngia
Y. japonica subsp. longiflora
Clade 2
Y. japonica subsp. japonica
Y. japonica subsp. formosana
Fig. 2. Bayesian majority-rule consensus tree with mean branch length based on ITS for three subspecies of Youngia japonica and the undescribed Youngia. The topology of the
MP strict consensus tree was totally the same. Numbers in sample names correspond to voucher numbers in Table 2. Sequence types are indicated in parentheses. The
numerals beside branches are Bayesian posterior probabilities (left) and bootstrap percentages (right). Scale bar indicates 0.2% sequence divergence. For clades 1 and 2, age
estimate was conducted.
Table 3
Estimates of cpDNA haplotype diversity (h), nucleotide diversity (p), and Tajima’s D
and Fu’s Fs statistics of Youngia japonica and the undescribed Youngia in Taiwan.
Sequence set
No. of
sequences
h
p 103
Tajima’s
D (95%
upper
limit)
Fu’s Fs
(95%
upper
limit)
Y. japonica (including
three subspecies)
and the undescribed
Youngia
Y. japonica (including
three subspecies)
Y. japonica subsp.
japonica, Y. japonica
subsp. formosana
and the undescribed
Youngia
36
0.756
1.13
1.200
(1.880)
4.736
(5.916)
1.176
(1.820)
1.796
(1.892)
5.232a
(5.147)
3.946
(5.334)
24
0.627
0.82
31
0.690
0.79
a
Significantly deviated from 0 at the 5% level, as estimated based on 10,000
coalescent simulations.
and subsp. formosana plus two samples of the undescribed Youngia
formed a clade, although the statistical support was weak
(PP = 0.89/BP = 67.5). The rest of the undescribed Youngia samples
formed a well-supported clade (PP = 1.00/BP = 94.1); thus, the
cpDNA phylogeny was incongruent with the morphological
delimitation of the undescribed Youngia. Youngia japonica subsp.
longiflora also formed a robust clade (PP = 1.00/BP = 99.0).
3.3. Divergence time estimation
Estimates of divergence time are summarized in Table 4. We
used geometric means because the time distributions based on
the ITS were skewed with long but very discrete upper tails (95%
HPD upper values more than 20 Ma in Y. japonica). It is reported
that a distribution of divergence time estimates is never strictly
symmetrical (e.g., Morrison, 2008) and the use of geometric means
reduces the problem of overestimation of divergence times with
arithmetic means (Battistuzzi et al., 2010). In our estimates, the
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K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
Crepidiastrum keiskeanum 991111-1 (N)
Crepidiastrum lanceolatum 2492 (O)
Crepidiastrum platyphyllum 1193 (P)
Paraixeris denticulata 1212 (Q)
1.00 / 100
1.00 / 82.6
1.00 / 96.8
Y. erythrocarpa 22592 (M)
Y. erythrocarpa 22579 (L)
Y. erythrocarpa 22583 (L)
0.90 / 64.2 Y. erythrocarpa 22584 (L)
Y. heterophylla 21125 (G)
Y. pseudosenecio 22575-6 (K)
Y. cf. pseudosenecio s.n. (H)
Y. pseudosenecio 22575-2 (I)
0.66 / 55.5
Y. pseudosenecio 22575-7 (I)
Y. pseudosenecio 22575-4 (J)
subsp. longiflora 147 (D)
subsp. longiflora 149 (D)
1.00 / 99.0
1.00 / 96.6
subsp. longiflora 151 (D)
subsp. longiflora 3187A (D)
subsp. longiflora 3187B (D)
1.00 / 99.9
Undescribed Youngia 3183-1 (E)
Undescribed Youngia 3183-2 (E)
Undescribed Youngia 3183-3 (E)
Undescribed Youngia 3183-4 (E)
1.00 / 94.1 Undescribed Youngia 3180 (E)
Undescribed Youngia 3176-1 (E)
1.00 / 100
Undescribed Youngia 3176-2 (E)
Undescribed Youngia 3176-3 (E)
Undescribed Youngia 3176-4 (E)
Undescribed Youngia 3168-1 (E)
subsp. japonica 141 (B)
subsp. formosana 22472a (B)
subsp. formosana 22472b (B)
subsp. formosana s.n. (B)
subsp. formosana 557a (B)
subsp. formosana 557c (B)
Undescribed Youngia 3168-3 (B)
Undescribed Youngia 3168-2 (F)
subsp. japonica 144 (A)
subsp. japonica 145 (A)
subsp. japonica 327g (A)
subsp. japonica 327i (A)
0.89 / 67.5
subsp. japonica 328e (A)
subsp. japonica 328f (A)
subsp. japonica 328g (A)
subsp. japonica 328h (A)
subsp. japonica 328i (A)
subsp. japonica 329f (A)
subsp. japonica 329g (A)
subsp. japonica 329i (A)
subsp. japonica 3186-1 (A)
subsp. japonica 247 (A)
subsp. japonica 248 (A)
subsp. japonica 245 (A)
0.80 / < 50
subsp. japonica 238 (A)
subsp. japonica 239 (A)
subsp. japonica 240 (A)
subsp. japonica 268 (A)
subsp. japonica 446 (A)
subsp. japonica 476 (A)
subsp. japonica 228 (A)
subsp. japonica 1504 (A)
subsp. japonica 22607-1 (A)
subsp. japonica 22607-2 (A)
subsp. japonica 22607-3 (A)
subsp. japonica 22575-1 (C)
subsp. japonica 22575-3 (C)
subsp. japonica 22575-5 (C)
Clade 1
Y. japonica subsp. longiflora
Undescribed Youngia
Y. japonica subsp. japonica
Y. japonica subsp. formosana
Undescribed Youngia
Clade 2
0.2
Y. japonica subsp. japonica
Fig. 3. Bayesian majority-rule consensus tree with mean branch length based on cpDNA sequences for three subspecies of Youngia japonica and the undescribed Youngia. The
topology of the MP strict consensus tree was the same as that of the Bayesian tree, except that the clade of Y. pseudosenecio (PP = 0.66/BP = 55.5) and the clade of haplotypes A
and C of Y. japonica subsp. japonica (PP = 0.80/BP < 50) collapsed. Numbers in sample names correspond to voucher numbers in Table 2. Haplotypes are indicated in
parentheses. The numerals beside branches are Bayesian posterior probabilities (left) and bootstrap percentages (right). Scale bar indicates 0.2% sequence divergence. For
clades 1 and 2, age estimate was conducted.
Table 4
Age estimates for the most recent common ancestors (MRCAs) based on ITS and
cpDNA data.
Geometric mean
(Median)
The MRCA of Youngia japonica, the undescribed Youngia, and Y. pseudosenecio
(clade 1)
ITS
2.52 Ma
(2.20 Ma)
CpDNA
0.70 Ma
(0.59 Ma)
The MRCA of Y. japonica subsp. japonica and formosana, and the undescribed
Youngiaa (clade 2)
ITS
1.78 Ma
(1.56 Ma)
CpDNA
0.50 Ma
(0.42 Ma)
a
In the cpDNA estimate, two samples of the undescribed Youngia with haplotypes B and F were analyzed.
geometric means indicated very similar values to the medians in
the ITS and cpDNA analyses (Table 4), and thus the geometric
means seemed suitable as representative values. The geometric
mean ages of the MRCA of Y. japonica, the undescribed Youngia,
and Y. pseudosenecio (clade 1 in Figs. 2 and 3) were 2.52 Ma and
0.70 Ma based on the ITS and cpDNA, respectively. The age of the
MRCA of Y. japonica subsp. japonica, Y. japonica subsp. formosana,
and the undescribed Youngia (clade 2 in Fig. 2) was 1.78 Ma based
on the ITS. Based on the cpDNA, the age of the MRCA of Y. japonica
subsp. japonica and subsp. formosana plus the two samples of the
undescribed Youngia with haplotypes B and F (clade 2 in Fig. 3)
was 0.50 Ma.
3.4. Distinguishing incomplete lineage sorting and chloroplast capture
We estimated the age of the MRCA of the morphologically
incongruent lineages of the undescribed Youngia (haplotypes B
and F) and its sister lineages Y. japonica subsp. japonica and subsp.
K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
formosana (haplotypes A, B, and C). When we took the geometric
mean age (0.5 million years) of the MRCA, Ne was calculated as
50,000, which did not support the ILS scenario. Note, however,
the age of the MRCA represents the oldest limit of the onset of
the ILS event. When we took the minimum value of 95% HPD interval (0.029 million years), the minimum Ne was calculated as 2900,
and the explanation based on ILS was retained.
4. Discussion
4.1. Origin of Youngia taxa in Taiwan triggered by new Pleistocene
habitats
The molecular analyses revealed the generally shallow divergence of Taiwanese Youngia; the subspecies of Y. japonica and the
undescribed Youngia clustered in a polytomy in the cpDNA phylogeny (and in the ITS phylogeny, except Y. japonica subsp. longiflora),
and they shared the same or closely related ITS and/or cpDNA sequences with each other (Figs. 2 and 3). However, they were at
the same time more or less differentiated from each other because
they had ITS and/or cpDNA sequences unique to or nearly exclusively found in each of them. In our analyses, samples of Y. japonica
subsp. japonica were collected broadly from Taiwan, Japan, and
China; however, very few sequence divergences were observed
(Figs. 2 and 3, Table 2). This is congruous with the expectation that
Y. japonica subsp. japonica intensively disperses across its distribution range. As such, processes that have triggered the differentiation of Youngia in Taiwan are intriguing. Age estimates for the
MRCA of Y. japonica plus the undescribed Youngia and Y. pseudosenecio were 2.52 and 0.70 million years based on the ITS and
cpDNA respectively (Table 4). Thus, the divergence events in the
lower clades are considered to have occurred in the lower or middle Pleistocene (2.58–0.78 Ma and 0.78–0.13 Ma, respectively;
Gibbard et al., 2010) or more recently. Because the estimate times
have some difference between the ITS and the cpDNA, the chronological order, rather than exact times, of evolutionary events are
discussed below.
4.1.1. Pleistocene land connection and Y. japonica subsp. longiflora
The haplotype and nucleotide diversities in Taiwanese Youngia
samples (h = 0.627–0.756 and p 103 = 0.79–1.13; Table 3) are
comparable to the higher half of reported values for other plants
from Taiwan and its vicinities (h = 0.104–0.761 and p 103 =
0.16–1.90; Cheng et al., 2005; Chung et al., 2007; Huang et al.,
2002, 2004; Hwang et al., 2003; Kuo et al., 2010; Nakamura
et al., 2010; Wu et al., 2006). The relatively high genetic diversity
of Taiwanese Youngia is attributed to the genetic differentiation
among taxa because genetic diversity within each taxon is low.
The estimate of Fu’s Fs value in Y. japonica (including the three subspecies) was significantly deviated from zero (Table 3), suggesting
that these samples were drawn from historically non-admixed
populations (i.e. subdivision of this data set). This is presumably
attributed to the distant phylogenetic relationship between
Y. japonica subsp. longiflora and the other two subspecies.
Possessing unique sequence types in ITS and cpDNA (C and D,
respectively), Y. japonica subsp. longiflora was clearly separated
from the other subspecies. The ITS phylogeny indicated that
Y. japonica is not monophyletic because Y. pseudosenecio intervened Y. japonica subsp. longiflora and the other two subspecies
(Fig. 2). The cpDNA phylogeny collapsed into polytomy (Fig. 3);
however, the smaller h and larger p in Y. japonica (including all
three subspecies) compared to those in the data set of Y. japonica
subsp. japonica and subsp. formosana plus the undescribed Youngia
indicates that Y. japonica subsp. longiflora is more distantly related
to the other subspecies than the undescribed Youngia is (Table 3).
495
Concerning the geographical origin of Y. japonica subsp. longiflora, this taxon is also distributed in southeastern China (Ling and
Shih, 1983) and Korea (Pak et al., 2000). Considering that Y. heterophylla, Y. erythrocarpa, and Y. pseudosenecio are all endemic to
mainland China (Ling and Shih, 1983), the Asian Continent origin
of Y. longiflora and its dispersal to Taiwan independent of Y. japonica subsp. japonica is the parsimonious scenario based on the ITS
tree (Fig. 2), because this scenario does not need to assume reverse
dispersals of Y. japonica subsp. longiflora and Y. pseudosenecio into
the Asian Continent. Taiwan was paleogeographically repeatedly
connected with the Asian Continent by a land bridge due to the
lowering of sea level during the Pleistocene glaciation (Liu, 1988;
Nakamura et al., 2009). This land connection has been considered
to explain the phylogeographical connectivity between Taiwan
and the Asian Continent (Chiang and Schaal, 2006; Wang et al.,
2004; Wei et al., 2010). The achenes of Youngia with feathery pappi
suggest anemochory (wind dispersal). To our knowledge, there is
no report of exo- or endo-zoochory of Youngia, but the achenes
are small enough to be accidentally dispersed by nonstandard
means (Higgins et al., 2003). Empirical and simulation studies suggested that fruits adapted for anemochory are expected to travel
rather short distances (few tens meters) under common wind conditions and long-distance dispersals (more than several kilometers) occur only under extremely strong and turbulent winds
(Higgins et al., 2003). On the other hand, some studies indicated
that haphazard long-distance dispersal events are more influential
than repeated short-distance dispersals in range expansion
(Nathan, 2008). Thus we considered that the both scenarios of a
single long-distance dispersal and repeated short-distance dispersals should be retained. In the latter scenario, Pleistocene land connections between Taiwan and the Asian Continent likely enabled
the migration. Note that Taiwan Strait between Taiwan and the
Asian Continent is shallower (only 40 m deep) in the north and
central part but deeper (up to 400 m deep) in the south (Zeng,
1994). The land connection with the Asian Continent is considered
to have lasted for the longest duration in northern Taiwan (Ota,
1998). This land connection might explain the distribution of Y.
japonica subsp. longiflora in the northern tip of Taiwan Island.
Migration of Y. japonica subsp. japonica is considered to have occurred earlier, because of its broad distribution in Taiwan and
much broader range in the Ryukyu Archipelago and the main islands of Japan. The restricted distribution of Y. japonica subsp. longiflora in Taiwan may be attributable to the limited niche available.
The dominating Y. japonica subsp. japonica, as early-arrived lineages, expanded into suitable habitats and likely reduced chances
for establishment of subsequently arrived Y. japonica subsp.
longiflora.
The ITS data revealed that two samples of Y. japonica subsp.
longiflora were actually heterozygotes of two phylogenetically
distant haplotypes C and A, suggesting that these samples are
hybrids between Y. japonica subsp. longiflora and Y. japonica subsp.
japonica or the undescribed Youngia that carries haplotype A. However, considering that only Y. japonica subsp. japonica grows sympatrically with Y. japonica subsp. longiflora in northern Taiwan (Peng
and Chung, 1999; Peng et al., 1998) and that the undescribed Youngia is known only from southwestern Taiwan, it is highly likely that
Y. japonica subsp. japonica represents one of the parental lineages.
We have carefully reexamined the morphological features (Table
1) of the Y. japonica subsp. longiflora samples. The putative hybrids
were not distinguished from the other samples in head width, leaf
shape, inner involucre length, and hair on leaf and inflorescence
axes; however, the achenes of the putative hybrid NK20100151
(Huang3187A had no achene) were dark brown, not dark purplish
brown, and this might indicate the hybridity with Youngia japonica
subsp. japonica, which has brown achenes. This hypothesis is further supported by the fact that both subspecies have the same
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K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
chromosome number and similar chromosome morphologies
(Kono Y., Academia Sinica, Taiwan, unpublished data), suggesting
their hybrid compatibility. Based on the above-shown migratory
scenario, the hybridization is considered to be a result of secondary
contact of the two subspecies in northern Taiwan.
4.1.2. Cold mountain ranges in Pleistocene glaciations and Y. japonica
subsp. formosana
Youngia japonica subsp. formosana was monophyletic in the ITS
phylogeny but shared a single cpDNA haplotype (haplotype B) with
Y. japonica subsp. japonica and the undescribed Youngia. It is likely
that Y. japonica subsp. formosana was derived from Y. japonica
subsp. japonica, considering the broader distribution of subspecies
japonica than the undescribed Youngia and occasional proximity of
the two subspecies. Youngia japonica subsp. formosana grows in
mountain ranges (1500–2500 m), and thus the origin is likely
explained in relation to altitudinal migration. During the Pleistocene, strong climatic oscillations occurred at regular intervals of
ca. 100,000 years of cold and dry glacial periods and ca.
10,000 years of warm and moist interglacial periods (Webb and
Bartlein, 1992). These alternating climatic conditions prompted
allopatric speciation in the Northern Hemisphere, associated with
altitudinal migration of species (Comes and Kadereit, 1998; Hewitt,
1996, 2000). In Taiwan, the temperature during glacial periods was
8.0–11.0 °C cooler compared to present-day temperatures
(Tsukada, 1966) and these climatic changes are considered to have
influenced plant phylogeographical patterns (Cheng et al., 2005;
Chung et al., 2007; Lee et al., 2006; Lin, 2001). Altitudinal migrations caused by glacial–interglacial temperature fluctuations were
suggested for multiple plant lineages (Hwang et al., 2003; Shih
et al., 2006). Youngia japonica subsp. formosana is also considered
to have experienced altitudinal migration. Broad environmental
tolerance and/or rapid adaptation, as is generally observed in weedy plants (Sahli et al., 2008; Whitney and Gabler, 2008), presumably enabled Y. japonica to survive in extreme habitats of cold
mountain ranges in Pleistocene glacial periods. Among the characters that separate Y. japonica subsp. formosana from the other
Youngia in Taiwan, the highly-dissected leaves might be adaptively
favored in cold environments. It was reported that lobed leaves of
plants in cold environments have higher rates of photosynthesis
and transpiration early in the spring growing season, and hydathodes in lobes prevent the flooding of intercellular airspaces under
conditions where freeze–thaw embolisms are prevalent (BakerBrosh and Peet, 1997; Royer et al., 2008). Note that, unlike regions
in high latitudes, Taiwan did not experience extensive glaciations
over most of its landscape (Chung et al., 2007). Given this, cold-tolerant lineages of Y. japonica could likely retain populations in
mountain ranges even during glacial periods, leading to differentiation from cold-intolerant lineages that retreated to the lower
altitude.
4.1.3. Raised coral reefs with recent Pleistocene origin and the
undescribed Youngia
The ITS phylogeny and the distribution ranges of the subspecies
of Y. japonica strongly suggest that the undescribed Youngia was
derived from Y. japonica subsp. japonica (Figs. 1 and 2). In cpDNA
phylogeny, most samples of the undescribed Youngia formed a
clade except two from Hsiao Liuchiu Islet (Fig. 3). One of the two
exceptional samples shared haplotype B with Y. japonica subsp.
japonica and subsp. formosana, and the other sample had haplotype
F closely related to the haplotypes A, B, and C of the two
subspecies.
Incongruence between morphological and phylogenetic delimitations can be explained by ILS or chloroplast capture via hybridization (Joly et al., 2009; Pelser et al., 2010). The coalescent-based test
of the plausibility of ILS indicated the equivocal result. The duration
of the ILS event based on the geometric mean age (0.5 million years)
requires the minimum effective population size of Ne = 50,000,
which does not support the ILS scenario; on the other hand, the
duration of the ILS event based on the minimum value of 95% HPD
interval (0.029 million years) requires the Ne = 2900, and the explanation based on ILS is retained. There are, however, additional
evidences in favor of ILS as the explanatory hypothesis. The incongruent lineages of the undescribed Youngia were morphologically
clearly different from the subspecies of Y. japonica and did not show
an intermediate morph. Also, the subspecies of Y. japonica were not
found in our thorough field survey on the Hsiao Liuchiu Islet, the
small islet of 6.8 km2. Therefore, the coalescent based test may
suggest that the actual age of the divergence event was younger
than the geometric mean age.
Based on the phylogenetic trees, it remained unclear whether the
undescribed Youngia originated in Hsiao Liuchiu Islet or Kaohsiung.
However, because island populations were more likely to be isolated from Y. japonica populations in Taiwan Island, Hsiao Liuchiu
Islet origin seems more likely. Raised coral reefs of Hsiao Liuchiu
Islet were formed in the middle Pleistocene or more recently due
to the lowering of sea level (Shih et al., 1991). Once the habitats
were formed, the weedy Youngia, with its broad environmental
tolerance, likely migrated into and rapidly adapted to the new and
extreme habitat, which is xeric, saline, alkali, and exposed to direct
sunlight. It is known that pubescent plants, compared with glabrous
or less pubescent plants, have smaller risk of overheating and
subsequent excess water loss in xeric environments, and can protect
photosynthetic tissues against high solar irradiance (e.g., Manetas,
2003). Rapid morphological differentiation of the undescribed
Youngia in a short evolutionary time likely resulted in ILS. In Taiwan,
ILS is also present in other plant species among populations, intraspecific taxa, or allied species (Chiang et al., 2006; Chung et al.,
2007; Huang et al., 2002; Hwang et al., 2003; Shih et al., 2006).
Geotectonic evolution which formed the present shape of Taiwan
at ca. 2 Ma (Shaw, 1996; Ho, 1982) and subsequent Pleistocene
glacial–interglacial fluctuations provided various new habitats
and presumably triggered very recent divergences in plant lineages.
Probably because of this reason, ILS is commonly observed in multiple plant lineages in Taiwan.
4.2. Taxonomic delimitation of recently diverged weeds
It is known that recently diverged taxa have low probability to
achieve reciprocal monophyly, and this is especially true if the
effective population sizes are large relative to divergence times
(Knowles and Carstens, 2007; Maddison and Knowles, 2006). In
the case of ILS, other sources of information, such as shared morphology and distribution patterns, can suggest a common gene
pool within a taxon and help taxonomic delimitation (de Queiroz,
2005a,b; Mayden, 1997).
The undescribed Youngia shared the same ITS sequence type A
with Y. japonica subsp. japonica, while it had the unique and dominant cpDNA haplotype E. This pattern is unexpected, considering
that ITS sequences generally have higher substitution rates than
cpDNA sequences (Kay et al., 2006), but was also reported for other
species (e.g., Streptocarpus of Gesneriaceae, Hughes et al., 2005).
This may be attributed to the larger effective population size in
nrDNA than in cpDNA, and/or higher levels of gene flow in nuclear
markers via pollen dispersal, if pollinators travel well, than in
cpDNA markers dispersed only via seeds, and/or the shorter
sequence length of the ITS than the cpDNA examined and stochastic error. The undescribed Youngia was not monophyletic in the ITS
and cpDNA phylogenies. However, considering the multiple
morphological features shared within the taxon (Table 1), which
suggest a common gene pool, and the dominance of the unique
cpDNA haplotype, the undescribed Youngia is expected to achieve
K. Nakamura et al. / Molecular Phylogenetics and Evolution 63 (2012) 486–499
monophyly in the cpDNA via coalescent process. The undescribed
Youngia deserves to be treated as a subspecies of Y. japonica.
Description of the new subspecies with data on the morphological
details will be dealt with in a companion paper.
Youngia japonica subsp. formosana was monophyletic in the ITS
phylogeny, although it shared the same cpDNA haplotype (B) with
Y. japonica subsp. japonica and the undescribed Youngia. The lower
substitution rates of cpDNA may explain this result. Youngia japonica subsp. formosana has unique reproductive (reddish-brown to
dark purple achenes) and vegetative (highly-dissected and runcinate-pinnatifid leaves) traits (Table 1) as well as has a habitat preference (mountain ranges). On these bases, we consider that it is
appropriate to retain the subspecific status of Y. japonica subsp. formosana (Babcock and Stebbins, 1937; Peng and Chung, 1999; Peng
et al., 1998), and we disagree with the treatment of synonymizing
this subspecies with Y. japonica subsp. japonica (Ling and Shih,
1983).
In this study, Youngia japonica subsp. longiflora was the most
clearly separated from the other subspecies, because Y. pseudosenecio intervened them in the ITS phylogeny. Although the secondary
contact and hybridization with Y. japonica subsp. japonica was observed, the dominance of the non-hybrids in the Y. japonica subsp.
longiflora population suggests that these two subspecies are not
merging. Therefore, we agree to treat this taxon as a distinct species, Y. longiflora (Babc. and Stebbins) C. Shih (Ling and Shih,
1983). Morphological characters supporting this treatment include
both the reproductive traits (wider heads, longer and dark purplish
brown achenes) and vegetative features (much larger involucres)
(Table 1). The limited range in Taiwan Island (Fig. 1) also separates
this taxon from the other subspecies.
5. Conclusions
Our study suggests that extreme habitats that arose in the Pleistocene likely triggered divergence of the weedy Youngia in Taiwan.
This result would enhance our understanding of the causes and timing of differentiation in plants unlikely to be affected by most geographical and ecological barriers. Because of the very recent
divergences, most Youngia taxa in Taiwan have not achieved reciprocal monophyly in the nrDNA and cpDNA phylogenies. However,
the samples within each taxon share multiple unique morphological features suggesting a common gene pool and each taxon has different dominant ITS and/or cpDNA types. These conditions suggest
ongoing process toward monophyly via coalescent processes and
support to delimit intraspecific taxa in the recently diverged weed.
Acknowledgments
We thank our lab members for assistance in molecular experiments, filed collection, and cultivation of plant materials in the
experimental greenhouse. T.G.Lammers and D.E. Boufford kindly
read and improved the manuscript. This study was supported in
part by a postdoctoral fellowship from Academia Sinica to K.N.
and a research grant from Academia Sinica to C.I.P.
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