Extreme habitats that emerged in the Pleistocene triggered divergence of weedy Youngia (Asteraceae) in Taiwan

Molecular Phylogenetics and Evolution 63 (2012) 486–499

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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: [email protected] (K. Nakamura), [email protected]. 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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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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13

12

14

Japan

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.

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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  109 to 7.83  109 substitutions site1 year1 (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  108 to 3  108 substitutions site1 year1 (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  108 substitutions site1 year1) 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  109 and 7.83  109), 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  108) and standard deviation (s.d.) of 0.59  108, which covered a range from 1.00  108 to 3.00  108 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  109) and s.d. of 2.29  109, which covered a range from 4.87  1010 to 8.24  109 (the lowest and highest rates for cpDNA noncoding

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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

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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).

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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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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

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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. References Babcock, E.B., Stebbins, G.L., 1937. The genus Youngia. Carnegie Inst. Wash. Publ. 484, 1–106. Baker-Brosh, K.F., Peet, R.K., 1997. The ecological significance of lobed and toothed leaves in temperate forest trees. Ecology 78, 1250–1255. Barker, W.R., Barker, R.M., Jessop, J.P., Vonow, H.P., 2005. Census of South Australian Vascular Plants, fifth ed. Botanic Gardens of Adelaide and State Herbarium, Adelaide, Australia (J. Adelaide Bot. Gardens (Suppl. 1)). Barrett, S.C.H., 1982. Genetic variation in weeds. In: Charudattan, R., Walker, H.L. (Eds.), Biological Control of Weeds with Plant Pathogens. John Wiley and Sons, New York, USA, pp. 73–98.

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