Phylogenetic placement of the enigmatic and critically endangered genus Saniculiphyllum (Saxifragaceae) inferred from combined analysis of plastid and nuclear DNA sequences

Molecular Phylogenetics and Evolution 64 (2012) 357–367

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Phylogenetic placement of the enigmatic and critically endangered genus Saniculiphyllum (Saxifragaceae) inferred from combined analysis of plastid and nuclear DNA sequences Chun-Lei Xiang a, Matthew A. Gitzendanner b, Douglas E. Soltis b, Hua Peng a, Li-Gong Lei a,⇑ a b

Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, No. 132, Lanhei Road, Kunming, Yunnan 650201, PR China Department of Biology, University of Florida, Gainesville, Florida 32611, USA

a r t i c l e

i n f o

Article history: Received 12 July 2011 Revised 17 March 2012 Accepted 11 April 2012 Available online 24 April 2012 Keywords: China Endemic Heucheroid clade Saniculiphyllum guangxiense Saxifragales Phylogenetic analysis

a b s t r a c t Saniculiphyllum, a monotypic genus distributed in Southwest China, was thought to be extinct before our recent rediscovery. The taxonomic position of this genus has been enigmatic ever since its publication. It was originally treated as the only member of a distinct tribe Saniculiphylleae in the family Saxifragaceae. Some proposed a new family, Saniculophyllaceae, to accommodate this genus, although its affinities are clearly with members of Saxifragaceae. Here we analyzed six DNA regions, the nuclear ribosomal ITS and 26S rDNA and the plastid rbcL, matK, trnL-trnF, psbA-trnH genes, spacers, and intron to explore the phylogenetic position of Saniculiphyllum within Saxifragaceae. The combined nuclear and chloroplast dataset includes 63 ingroup species, representing all genera but Hieronymusia in the family. Results from likelihood, parsimony and Bayesian phylogenetic methods corroborate earlier results. Two clades of Saxifragaceae, the Heucheroid and Saxifragoid clades, were recovered. The topologies obtained from different analyses confirm the placement of Saniculiphyllum in Saxifragaceae, but our analyses reveal that Saniculiphyllum is embedded within the large Heucheroid clade. However, the closest relatives of Saniculiphyllum within the Heucheroid clade remain unclear. Combined with morphological data, our results suggest that Saniculiphyllum should best be regarded as a highly distinctive lineage within the Heucheroid clade of Saxifragaceae. Morphological novelties and conservation status of Saniculiphyllum are also presented. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Over the past two decades the traditionally recognized and broadly defined Saxifragaceae s.l. (e.g., Cronquist, 1981) have been shown to be polyphyletic (Chase et al., 1993; Morgan and Soltis, 1993; Soltis and Soltis, 1997; Soltis et al., 2000). As a result, the family has experienced major revision (Johnson and Soltis, 1994, 1995; Soltis et al., 2001a; Soltis, 2007) and now it is generally treated as a number of smaller families. Recently, the APG classification (APG III, 2009) clarified the circumscription of the family. Saxifragaceae are now recognized as a modest sized family of 33 genera and approximately 600 species, with a nearly worldwide distribution, but mainly found in temperate regions. Approximately one half of the genera are monotypic (Soltis et al., 2001a; Soltis, 2007). Genera of Saxifragaceae from China include Astilbe Buch.Ham., Astilboides Engler, Bergenia Moench, Chrysosplenium L., Mitella L., Mukdenia Koidzumi, Oresitrophe Bunge, Rodgersia A. Gray,

⇑ Corresponding author. Fax: +86 871 5213916. E-mail addresses: [email protected] (C.-L. Xiang), [email protected]fl.edu (M.A. Gitzendanner), [email protected]fl.edu (D.E. Soltis), [email protected] (H. Peng), [email protected] (L.-G. Lei). 1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.04.010

Saxifraga L., Tanakaea Franchet and Savat., Tiarella L., and the recently described Saniculiphyllum C.Y. Wu and T.C. Ku (Wu and Ku, 1992; Pan et al., 2001), which has been distinguished from other genera of Saxifragaceae because of its unusual morphology, including palmately lobed leaves, 3-5-carpellate and 3-5-loculed ovary (Fig. 1). The monotypic Saniculiphyllum, comprising the single species S. guangxiense C.Y. Wu and T.C. Ku, is a clonal aquatic herb clinging to the wet rocks or stone in brooks in southeast Yunnan Province and Northwest of the Guangxi Zhuang Autonomous Region (Wu et al., 2007). In the protologue to the description (Wu and Ku, 1992), two specimens were cited. One was collected in 1989 (Guangxi Zhuang Autonomous Region, Tianlin County), this specimen was selected as HOLOTYPE (PE). Another specimen was collected in 1968 (Yunnan, Funing County, Lida), this specimen was a paratype (KUN, PE). The species is considered to be endangered and was actually thought to be extinct before the recent rediscovery of several populations in Yunnan. The populations in Tianlin County of Guangxi Zhuang Autonomous Region of southern China, from where the type specimens of the species were collected in 1989, had disappeared due to seasonal drying of the streams and plants never been found in this area again.

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Fig. 1. Photographs of Saniculiphyllum guangxiense in its natural habitat. (a and b) habitat of S. guangxiense in Funing County. (a) Group of individuals growing on cliff near water; (b) the plants of S. guangxiense cling to stones in stream; (c) details of S. guangxiense showing the leaves palmately deeply lobed; (d and e) floral form of the species; (d) cymes with 7–10 flowers; (e) flower, showing five red petals, five stamens, and three carpels; (f, g, and i) rhizomes and fibrous roots; (f) long and horizontal rhizomes cling to stone; (g) densely fibrous roots; (i) details of fibrous roots under stereo microscope; (h) ovules; (j) details of seeds showing that these seeds generally hypogenetic. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The systematic position of Saniculiphyllum has been enigmatic ever since its publication. When establishing the genus, Wu and Ku (1992) placed it within its own tribe, Saniculiphylleae within Saxifragaceae, but the circumscription of the family Saxifragaceae at that time was much broader compared to our current understanding. In the protologue (Wu and Ku, 1992), Saniculiphyllum was considered to be related to Saxifraga and Mukdenia because all have axile placentation. However, the number of petals, sepals, stamens and habit of Saniculiphyllum is clearly different from the latter two genera. Saniculiphyllum was also deemed to have a close relationship to Chrysosplenium, but the latter genus has parietal placentation. Compared with other genera such as Bolandra Gray, Boykinia Nutt., Jepsonia Small, Peltoboykinia (Engler) H. Hara and Sullivantia Torrey and Gray ex Gray, which all have axile placentation, Saniculiphyllum is easily distinguished by having a 10-lobed floral disk, completely inferior ovary, short anther filament, and creeping flat rhizomes (Fig. 1f, g, and i; Wu and Ku, 1992). Mabberley (1997) elevated the genus to the rank of family, but as Saniculophyllum and Saniculophyllaceae. Wu et al. (2003) suggested that the genus may be a highly specialized taxon within Saxifragaceae.

Based on its thick rhizomes and palmately lobed leaves, Soltis (2007) thought that this genus might belong to the Darmera group in Saxifragaceae. However, at the same time, he also stressed that it was more appropriate at that point to consider the exact placement of the genus within Saxifragaceae as unknown. During the past two decades, tremendous progress has been made in understanding the phylogeny of Saxifragaceae (Morgan and Soltis, 1993; Johnson and Soltis, 1994; Soltis and Soltis, 1997; Soltis et al., 2001a,b). Saniculiphyllum, however, has never been included in these comprehensive molecular phylogenetic studies, because of its extremely rare occurrence and the unavailability of material from which to obtain DNA. This Chinese endemic remains one of the most taxonomically enigmatic members of Saxifragaceae (Soltis, 2007). Furthermore, a better understanding of the relationships of Saniculiphyllum may elucidate evolutionary processes across Saxifragaceae. The systematic position and possible allies of Saniculiphyllum therefore require more rigorous evaluation in a phylogenetic context. Between 2008 and 2011, we found five new populations of Saniculiphyllum guangxiense in Funing County of Yunnan in Southwest

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Fig. 2. Geographical distribution of Saniculiphyllum guangxiense in China. The solid circle is sample collecting site in Yunnan, and the filled box is the extinct distribution area in Guangxi Zhuang Autonomous Region.

China (Fig. 2). Unfortunately, we have not been able to find the species again at the type collection site in Tianlin County of the Guangxi Zhuang Autonomous Region in South China, despite our repeated efforts. However, our discovery of new populations in Yunnan Province enabled us to obtain DNA data to investigate the systematic position of Saniculiphyllum. In this study, we use nuclear [internal transcribed spacer (ITS), and 26S] and chloroplast data [matK, rbcL, trnL-trnF (consisting of the trnL intron and the trnL-trnF intergenic spacer), psbA-trnH] in an attempt to elucidate the phylogenetic position of Saniculiphyllum and provide new insights into the phylogeny of family Saxifragaceae These sequences are widely used to assess the affinities of taxonomically enigmatic angiosperm taxa (e.g., Herbert et al., 2006; Chandler and Bayer, 2000; Bayer and Cross, 2002), as well as phylogenetic relationships within Saxifragaceae (Hibsch-Jetter et al., 1997; Soltis and Soltis, 1997; Morgan and Soltis, 1993; Johnson and Soltis, 1994, 1995; Soltis et al., 1996a,b, 2001a). Consequently, a large data base of these sequences is now available representing Saxifragaceae, permitting analyses across the phylogenetic diversity of the family. Given the longstanding confusion regarding the relationship of Saniculiphyllum to other genera within Saxifragaceae, the objectives of this study were: (1) investigate the phylogenetic placement of Saniculiphyllum in Saxifragaceae, (2) identify the major lineage(s) that are related to Saniculiphyllum, (3) further contribute to a comprehensive phylogenetic framework for the Saxifragaceae. 2. Materials and methods 2.1. Field work Materials of Saniculiphyllum were collected from Ligong Village, Lida Town, Funing County, Yunnan Province, Southwest China (Fig. 2). Fresh leaves of two populations were collected for the purpose of DNA extraction. Materials of the other ingroup and

outgroup taxa were either collected in the wild or obtained from cultivated plants in Kunming Botanic Garden (Table 1). Voucher specimens are deposited in the Herbarium of Kunming Institute of Botany (KUN), Chinese Academy of Sciences. 2.2. Taxon sampling Familial circumscriptions and nomenclature are based on the treatment of the Saxifragaceae by Soltis et al. (2001a) and Soltis (2007). Outgroup taxa were selected based on the well supported placement of Iteaceae, Grossulariaceae, and Pterostemonaceae as closest relatives to Saxifragaceae (Morgan and Soltis, 1993; Soltis et al., 1993, 2001a; Johnson and Soltis, 1995). Therefore, one species from Pterostemonaceae (Pterostemon rotundifolius Ramírez), two species of Iteaceae (Itea virginica L., I. yunnanensis Franch.), and five species of Grossulariaceae (Ribes glaciale Wall., R. maximowiczianum Kom., R. moupinense Franch. var. tripartitum (Batalin) Jancz., R. soulieanum Jancz., R. tenue Jancz.) were selected as outgroup taxa. The ingroup included 63 species, and included all genera of Saxifragaceae except one, Hieronymusia Engler, a monotypic genus restricted to remote areas of Argentina and Bolivia, for which we have been unable to obtain suitable material. However, Hieronymusia was actually included in Suksdorfia A. Gray by Gornall and Bohm (1985) and is clearly closely related to that genus based on morphology and chemistry. Therefore, following the Gornall and Bohm (1985) treatment, all genera of Saxifragaceae were included in our study. In total, the study material consisted of 74 accessions representing 71 species, 25 of which were newly sequenced as part of this study. Other sequences came from previous studies (Soltis et al., 1991a, 1993, 1996a,b, 2001a; Soltis and Kuzoff, 1995; Johnson and Soltis, 1995, 1998; Kuzoff et al., 1998, 1999; Conti et al., 1999; Fishbein et al., 2001; Senters and Soltis, 2003; Okuyama et al., 2005, 2008; Okuyama and Kato, 2009).

Taxon

Bergenia purpurascens⁄ B. cordifolia Darmera peltata Mukdenia rosii Oresitrophe rupifraga O. rupifraga⁄ Rodgersia aesculifolia⁄ Bergenia cordifolia R. pinnata Leptarrhena pyrolifolia Tanakaea radicans Saniculiphyllum guangxiense⁄ Saniculiphyllum guangxiense⁄ Cascadia nuttallii Saxifragodes albowiana Saxifraga aizoides S. balfourii⁄

Grable 11668 (WS) Gornall 0101 (UBC) Rieseberg 1110 (WS) Soltis & Soltis 2309 (WA) Gornall 11214 (WS) Wolf 151 (WS) Botanical Gardens, Univ. Tokyo, Cult. (WS) Yin et al. 1862 (KUN) Xiang 443 (KUN) UCBG 82-1325 (UC) Lang, Soltis & Soltis, s.n. (WS) Soltis & Soltis 1608 (WS) Soltis & Soltis 2179 (WS) Soltis & Soltis 1949 (WS) Kuzoff 95-03 (WS) Johnson & Brunsfeld 1908 (WS) Soltis & Soltis 2113 (WS) Soltis 2555 (WS) Soltis & Soltis 1903 (WS) WS 32167 (WS) Soltis & Soltis 2253 (WS) Soltis & Soltis 2217 (WS) Ying et al. 1342 (KUN) Horandl 2703 (WS) Wendel s.n. (WS) Lei 20090402 (KUN) Gan 1912 (KUN) Gan 1875 (KUN) Gan 2063 (KUN) Nikko Botanical Garden, cult. Palmengarten Botanical Gardens, Germany, Cult. (WS) Lei KBG01. (KUN) Komarov Botanical Institute, Russia, Cult. (WS) University of California, Berkley (WS) University British Columbia, Botanical Gardens (WS) Beijing Botanical Gardens, China, Cult. (WS) Lei KBG02 (KUN) Yin et al. 1668 (KUN) Komarov Botanical Institute, Russia, Cult. (WS) Palmengarten Botanical Gardens, Germany, Cult. (WS) Soltis & Soltis 2237 (WS) Nikko Botanical Garden, Japan (WS) Lei 20090403-1 (KUN) Lei 20090403-5 (KUN) University of Washington 3446 (WTU) Arroyo et al. 941179 (WS) Brochmann 92-78-1 (OS) Yin et al. 2265 (KUN)

Locality

GenBank No./reference matK

rbcL

psbA-trnH

trnL-trnF

ITS

26S

Washington, United States California, United States California, United States Washington, United States Oregon, United States Wyoming, United States Japan, Cult. Yunnan, China Yunnan, China California, United States United States United States Washington, United States United States California, United States Precise locality unknown Alaska, United States Japan Oregon, United States Precise locality unknown Oregon, United States Washington, United States Yunnan, China Precise locality unknown Iowa, United States Yunnan, China Hubei, China Hubei, China Hubei, China Japan Palmengarten Botanical Gardens, Germany, Cult.

L34117 L34118 L34128 L34146 L34113 L34148 CQ386964 JN102180 JN102181 AF37429 L34112 L34122 L34124 C – L34134 L34149 AB116692 L34152 AF115484 L20131 E JN102182 AF115493 L34120 JN102183 JN102184 JN102185 – L34138 L34115

U06209 L11175 U06211 – U06219 U06221 – JN102248 JN102249 A – – U06210 L01925 – – U06222 – U06223 E L01953 A JN102250 AF374732 L19935 JN102251 JN102252 JN102253 – U06213 U06207

AF374766 AF374767 AF374765 – AF374768 AF374764 AF374771 JN102204 JN102205 AF374772 – – AF374761 AF374763 – AB492501 AF374762 – AF374760 AF374756 AF374758 AF374757 JN102206 AF374759 AF374747 JN102207 JN102208 JN102209 JN102210 AF374746 AF374750

AF374809 AF374810 AF374808 – AF374811 AF374807 AF374814 JN102272 JN102273 AF374815 – – AF374804 AF374806 – AB116715 AF374805 AB116717 AF374803 AF374799 AF374801 AF374800 JN102274 AF374802 AF374790 JN102275 JN102276 JN102277 JN102278 AF374789 AF374793

U51255 U51248 U51262 U51257 U51258 U51261 B – – B AF158953 AB292020 D D AF158951 AB163495 D AF006834 D B B B – AF374827 B JN102226 JN102227 JN102228 JN102229 AB248847 B

AF374857 AF274638 AF374856 – AF274668 AF374855 AF374860 JN102223 JN102224 AF374861 – – AF374853 AF374854 AF036501 – AF036500 – AF374852 AF374849 AF274666 AF374850 JN102225 AF374851 AF274641 – – – – AF036499 AF374843

Yunnan, China Komarov Botanical Institute, Russia, Cult. University of California, Berkley, UC Cult. University British Columbia, Botanical Gardens, Cult. Beijing Botancal Gardens, China, Cult. Yunnan, China Yunnan, China

JN102186

JN102254

JN102211

JN102279

JN102230

–

L34123 L34137

L11180 U06212

AF374752 AF374751

AF374795 AF374794

D B

AF374845 AF374844

Palmengarten Botanical Gardens, Germany, Cult.

E JN102187 JN102188 L34116 L34139

E JN102255 JN102256 U06208 U06214

AF374749 JN102212 JN102213 AF374753 AF374748

AF374792 JN102280 JN102281 AF374796 AF374791

B JN102231 JN102232 D U51264

AF374842 – – AF374846 AF374841

Vancouver Island, Canada Japan Yunnan, China Yunnan, China Oregon, United States Chile Precise locality unknown Yunnan, China

L34129 L34147 JN102189 JN102190 AF115483 AF374729 E JN102191

L11191 U06220 JN102257 JN102258 A AF374731 E JN102259

AF374769 AF374770 JN102214 JN102215 AF374755 AF374754 AF374744 JN102216

AF374812 AF374813 JN102282 JN102283 AF374798 AF374797 AF374787 JN102284

F U51263 JN102233 JN102234 B AF374825 AF087594 JN102235

AF374858 AF374859 – – AF374848 AF374847 AF374839 –

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Ingroups Bolandra oregana Boykinia rotundifolia Jepsonia parryi Suksdorfia violacea Sullivantia oregana Telesonix hercheriformis Astilbe microphylla A. chinensisi⁄ A. rivularis⁄ Saxifragopsis fragarioides Bensoniella oregona Conimitella williamsii Elmera racemosa Heuchera micrantha Lithophragma trifoliatum Mitella nuda Tellima grandiflora Tiarella polyphylla Tolmiea menziesii Micranthes tolmiei M. integrifolia M. punctata M. pallida⁄ M. stellaris Chrysosplenium iowense C. hydrocotylifolium⁄ C. lanuginosum var. gracile⁄ C. nepalense⁄ C. microspermum⁄ Peltoboykinia tellimoides Astilboides tabularis

Voucher/herbarium

360

Table 1 Voucher information and GenBank accession numbers for taxa used in this study. Vouch specimens are deposited in the following herbaria: K = Royalo Botanic Gardens, Kew, England; KUN = Kunming Institute of Botany, China; OS = Ohio State University, United States; UC = University of California, United States; WA = University of Warsaw, Poland; WS = Washington State University, United States; UBC = University of British Columbia, Canada; WTU = University of Washington, United States. If accession numbers of some sequences are not available from GenBank, then the source publications were listed. Regions not sampled are indicated by an en dash (–). A: Soltis et al. (1993); B: Johnson and Soltis (1998); C: Johnson and Soltis (1995); D: Soltis and Kuzoff (1995); E: Soltis et al. (1996a); F: Soltis et al. (1996b). Asterisk: newly sequenced taxon in present study.

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361

AF274650 – AF274663 AF274665 – – – – – AY231368 JN102242 AY231369 AF426382 JN102243 JN102244 JN102245 JN102246 JN102247 AF374818 JN102291 AF374817 AF374816 JN102292 JN102293 JN102294 JN102295 JN102296 AF374775 JN102217 AF374774 AF374773 JN102218 JN102219 JN102220 JN10221 JN102222 L11188 JN102266 L11203 L11204 JN102267 JN102268 JN102269 JN102270 JN102271 United States Yunnan, China Mexico Washington, United States Yunnan, China Yunnan, China Yunnan, China Yunnan, China Yunnan, China D. M. E. Ware 94 (WILI) Xiang 444 (KUN) Jordan s.n. (WS) Soltis & Soltis 2220 (WS) Yin et al. 1675 (KUN) Yin et al. 1601 (KUN) Yin & Dong 0438 (KUN) Yin & Dong 0434 (KUN) Yin et al. 1935 (KUN)

AF274618 JN102198 AF274630 L34153 JN102199 JN102200 JN102201 JN102202 JN102203

AF374833 AF374829 AF374823 AF374819 AF375781 AF374776 AF374738 AF374733 E AF374730 Precise locality unknown Chile Brochmann 93-219 (OS) Arroyo et al. 950914 (?)

E AF374728

Yunnan, China Alaska, United States Precise locality unknown Precise locality unknown Yunnan, China Oregon, United States Royal Botanic Gardens, Kew Precise locality unknown Precise locality unknown Precise locality unknown Precise locality unknown Kunming Botanic Garden, Cult. Yunnan, China Yunnan, China Precise locality unknown Yunnan, China Yin et al. 2169 (KUN) Brochmann 92-31-22 (OS) Ferguson 1994-04 (WS) Soltis 2519 (WS) Yin et al. 1951 (KUN) Saxifraga mertensiana Bong (WS) Kew 1975-4135, Cult. (K) Brochmann s.n. (WS) Gornall 0101 (UBC) Kew 361-84-03742, Cult. (K) BBG 001-91-75-10, Cult. (K) Lei s. n. (KUN) Yin et al. 1276 (KUN) Yin et al. 2109 (KUN) Parker s.n. (WS) Yin et al. 1923 (KUN)

JN102192 L34140 E E JN102193 L34142 E E L34118 E E JN102194 JN102195 JN102196 E JN102197

JN102260 U06215 E E JN102261 U06216 E E L11175 E E JN102262 JN102263 JN102264 E JN102265

– AF374736 AF374734 AF374737 – AF374735 AF374739 AF374745 AF374767 AF374741 AF374742 – JN102216 – AF374743 –

JN102285 AF374779 AF374777 AF375780 JN102286 AF374778 AF374782 AF374788 AF374810 AF374784 AF374785 JN102287 JN102288 JN102289 AF374786 JN102290

JN102236 B AF087599 AF374821 JN102237 AY231367 B AF087608 U51248 B AF087596 JN102238 JN102239 JN102240 AF087601 JN102241

– AF374831 AF374830 AF374832 – AF036498 AF374834 AF374840 AF374835 AF374836 AF374837 – – – AF374838 –

2.3. DNA extraction, PCR amplification and sequencing Total DNA was obtained from freshly-collected and silica-geldried leaf fragments. All accessions were identified using published keys and compared to herbarium specimens. Total genomic DNA was isolated using CTAB procedure of Doyle and Doyle (1987) as modified by Soltis et al. (1991b) for fresh or frozen samples, or the Bioteke’s plant mini Kit (Bioteke Corporation, Beijing, China) following the manufacturer’s protocol. After extraction, the DNA was resuspended in TE buffer and kept at 40 °C for further use. PCR amplifications were performed using a Biometra T1 thermocycler (Biometra, Göttingen, Germany). The 50 ll volume polymerase chain reaction (PCR) contained 2 ll DNA solution (adjusted to approximately 20 ng), 5 ll PCR reaction buffer, 5 ll dNPT mix (0.2 mM), 2 ll of each primer, and 1.5 U Taq DNA polymerase (Chenlü, Kunming, China). Amplification and sequencing of the ITS region (ITS1, 5.8S rDNA, ITS2) were performed with primers ITS4 and ITS5 (White et al., 1990) or N-nc18S10 and C26A (Wen and Zimmer, 1996). PCR and sequencing of the trnL intron and trnL-trnF intergenic spacer was performed using the universal primers of Taberlet et al. (1991), either as one fragment using primers ‘‘c’’ and ‘‘f’’ or as two separate fragments using primers ‘‘c’’ and ‘‘d’’, and ‘‘e’’ and ‘‘f’’, respectively. The primers used for amplifying and sequencing the psbA-trnH region were ‘‘psbA’’ and ‘‘trnH’’ as described in Hamilton (1999). Amplifications were performed using a program consisting of 3 min at 94 °C followed by 35 cycles of 45 s denaturation (94 °C), 1 min annealing (53 °C) and 3 min extension (72 °C), ending with a final 7 min extension at 72 °C. The PCR amplification protocols were identical for all above three fragments. The matK region was divided into three overlapping fragments using the following PCR primer combinations for most taxa: trnK-3914F and matK-1470R; trnK-710F and matK-2200R; matK1412F and trnK-2R, as sequencing primers. The base composition of these primers is given in Johnson and Soltis (1994, 1995). For some taxa, these primers failed to produce usable double-stranded products, thus the primers matK-3268F, and matK-2200R of Okuyama et al. (2005) were used. The amplification conditions were set as follows: denaturation at 94 °C for 4 min, 30 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min, and a final extension of 7 min at 72 °C. Primers Z1 and Z-1351R were used for amplifying and sequencing the rbcL region (Chandler and Bayer, 2000). The PCR conditions used were: 2 min at 94 °C, then 30 cycles with 45 s at 94 °C, 90 s at 45 °C and 90 s at 72 °C, and finally 5 min at 72 °C. Amplified products were purified using a QIAquick PCR purification Kit (BioTeke Corporation, Beijing, China) following the manufacturer’s instructions. Sequencing reactions were performed with the dideoxy chain termination method running on an ABI PRISM 3730 automated sequencer. The same primers described above for PCR were used for the sequencing reactions. All regions were sequenced for both strands where there was an overlap of at least 70%. All sequences used in this study together with their GenBank accession numbers or source publications are listed in Table 1.

Outgroups Itea virginica I. yunnanensis⁄ Pterostemon rotundifolius Ribes aureum R. glaciale⁄ R. maximowiczianum⁄ R. moupinense var. tripartitum⁄ R. soulieanum⁄ R. tenue⁄

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

brunonis⁄ cernua cymbalaria fortunei hispidula⁄ mertensiana oppositifolia osloensis rotundifolia scardica spathularis stolonifera 1⁄ stolonifera 2⁄ strigosa var. ramosa⁄ tricuspidata diversifolia var. haematophylla⁄ S. hirculus Saxifragella bicuspidata

2.4. Sequence analysis and phylogenetic reconstruction Datasets for 26S rDNA and rbcL were easily aligned by eye. The remaining datasets, which were much more difficult to align, were aligned using an iterative strategy. The simultaneous alignment and tree estimation program SATé (Liu et al., 2009; Yu and Holder, 2010) was used to obtain alignments and single gene phylogenetic estimates for ITS, matK, psbA-trnH and trnL-trnF. SATé is not able to perform alignments of multi-gene datasets, so each gene region was analyzed individually. These alignments were then concatenated and analyzed with RAxML 7.2.7 (Stamatakis, 2006). As the

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SATé alignments had a lot of gaps, we used Phyutility (Smith and Dunn, 2008) to eliminate positions where more than 60% of the taxa had gap characters for that position. This dataset was then analyzed with RAxML with 1000 bootstrap replicates. SATé frequently made little or no improvement over the initial MAFFT alignment on single gene datasets even after 24 h of iteration over alignment and tree building. Thus, we redid the alignments with SATé using the topology of the tree obtained from the analysis of the reduced dataset above as a starting topology in SATé. Effectively this is similar to the iterative process SATé employs, but making use of the information in the multi-gene phylogeny, rather than being limited by each single gene topology. Alignments generated from the initial unguided alignment were different than those obtained using the starting tree, though no formal statistics were calculated. After this realignment in SATé, datasets were once again concatenated and reanalyzed with RAxML, again with 1000 bootstrap replicates and six independent gene-based partitions (estimating a single set of branch lengths). Data alignment and trees were deposited in Treebase (Study number S12254). The final SATé alignment was taken as the best available alignment of the genes and used for additional analyses of the dataset with maximum parsimony (MP) (Swofford et al., 1996) and Bayesian inference (BI) (Ronquist and Huelsenbeck, 2003). Maximum parsimony analyses were performed with the heuristic search option of the computer package: Phylogenetic Analysis Using Parsimony (PAUP) version 4.0b10 (Swofford, 2003). Gaps were treated as missing data. All unambiguous characters and character-transformations were unordered and weighted equally. Support for clades was calculated via bootstrap analyses (Felsenstein, 1985) from 1000 replicates as described earlier (Li et al., 2009). Bayesian inference (BI) based on Markov Chain Monte Carlo methods (Yang and Rannala, 1997) was carried out with MrBayes version 3.1.2 parallel version (Ronquist and Huelsenbeck, 2003). Prior to analysis, the appropriate nucleotide sequence evolution model for each marker was selected using the Akaike Information Criterion (AIC) as implemented in jModeltest version 0.1.1 (Guidon and Gascuel, 2003; Posada, 2008). The TVM + G model was shown to best fit our data of matK, trnL-trnF, psbA-trnH, the GTR + I + G model best fit rbcL, and GTR + G was best for ITS and the combined datasets. Model parameters were estimated directly during the runs. For each analysis, two simultaneous runs were performed, starting from random trees for 2  106 generations, having three heated and one cold chain. Markov chains were sampled every 100th generation. At the end of the run we considered the sampling of the posterior distribution to be adequate if the average standard deviation of split frequencies was <0.01 (Ronquist et al., 2005). To assess whether the MCMC chain reached stationarity we examined the InL plots using Tracer V. 1.5.0 (Rambaut and Drummond, 2009). Also, to visually check for convergence we used the program AWTY online (Wilgenbusch et al., 2004). The states of the chain that were sampled before stationarity (i.e., the ‘‘burn in’’ of the chain) were

discarded, and the posterior probability (PP) values were determined from the remaining trees. The Shimodaira–Hasegawa (SH) test (Shimodaira and Hasegawa, 1999) was employed in the CONSEL package (Shimodaira and Hasegawa, 2001) to compare the likelihood scores of trees derived from two different data partitions (nuclear vs. plastid). 3. Results 3.1. Sequence and alignment Sequence lengths were as follows in Saniculiphyllum guangxiense: 841 nucleotides (nt) for the trnL-trnF spacer, 274 nt for the psbA-trnH spacer, 1402 nt for matK, 1296 nt for rbcL, and 666 nt for the ITS1-5.8S-ITS2 region. The resulting combined and aligned sequence matrix contained 7137 positions (including gaps) of which 1350 positions belong to the trnL-trnF partition, 804 positions to the psbA-trnH partition, 1512 positions to the matK partition, 1461 positions to the rbcL partition, 819 positions to the ITS partition, and 26S gene contributed 1191 bp. Of the 7137 nucleotides 41% were variable (29% parsimony-informative) in the dataset. Table 2 summarizes the properties contributed by each aligned data partition. 3.2. Incongruence among data partitions We divided the combined dataset into two process partitions: chloroplast dataset (trnL-trnF, psbA-trnH, matK, and rbcL); and nuclear dataset (ITS and 26S). While the results of the SH test were highly significant (P = 0.000), visual inspection indicates that there are very few ‘‘hard’’ conflicts between the nuclear vs. plastid trees (e.g., Bull et al., 1993; Mason-Gamer and Kellogg, 1996; Oh and Potter, 2005; Quicke et al., 2007). In this study, two large clades, Saxifragoid clade and Heucheroid clade, were well supported in both plastid dataset (ML BS: 100, 75, Supplementary Fig. 1) and nuclear dataset (ML BS: 100, 100, Supplementary Fig. 2). Therefore, we combined the datasets for simultaneous analyses. 3.3. Phylogenetic analyses Phylogenetic relationships are very similar between the total evidence trees obtained in the ML, MP and BI analyses (Supplementary Figs. 3 and 4), although somewhat lower resolution was obtained with MP and BI. The ML topology from the combined dataset will therefore be the primary tree for discussion of phylogenetic relationships. Analyses of the combined dataset yielded a single ML tree (Fig. 3), and the topology obtained is basically congruent with a previous study based on a smaller number of taxa (e.g. Soltis et al., 2001a). The ingroup (Saxifragaceae) is well supported as monophyletic (ML BS: 100, MP BS: 100, PP: 1.00; all values reported in this order below). The ingroup consists of two large

Table 2 The statistics from analyses of the chloroplast and nuclear data sets for parsimony analysis. Data matrix

Aligned positions

No. informative sites

No. MPTs

Tree Length

CI (excluding uninformative characters)

RI

RC

matK rbcL trnL-trnF psbA-trnH Combined chloroplast data matrix ITS ITS + 26S All combined data matrix

1512 1461 1350 804 5127 819 2010 7137

494 174 363 365 1396 481 657 2053

36 313 110 26 24 24 45 2

1683 653 1249 1645 5582 3695 4560 10,198

0.633 0.538 0.692 0.535 0.566 0.329 0.317 0.465

0.803 0.781 0.820 0.687 0.734 0.706 0.695 0.711

0.508 0.420 0.567 0.367 0.416 0.232 0.241 0.330

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clades in the combined molecular dataset. One of these is the Saxifragoid clade (100, 100, 1.00) comprising the genera Saxifraga s.s. and Saxifragella Engler. Many relationships within Saxifraga s.s. are well supported. Saxifraga mertensiana, S. stolonifera, and S. fortunei form a well-supported clade (100, 100, 1.00) that is sister to the remaining members of the clade (100, 100, 1.00). The next branching member is Saxifragella, which is sister to the remainder

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of the Saxifragoid clade, which is again well supported (100, 100, 1.00). Saxifraga tricuspidata then follows as sister to a well-supported (100, 100, 1.00) core group of taxa, as evidenced by Soltis et al. (2001a). The second major subclade of Saxifragaceae is the Heucheroid clade which receives low support in the present study (73, 68, 1.00). Saniculiphyllum is embedded well within the Heucheroids.

Fig. 3. Maximum likelihood analysis based on a combined data set of trnL-trnF, psbA-trnH, matK, rbcL, ITS, and 26S sequences. All branches are drawn to scale. The clade names for the family Saxifragaceae sensu stricto are those suggested by Soltis (2007); the geographical distribution of each taxon is shown on the tree. RAxML and MP BS support values (>50%) are indicated above or below the branches as RAxML BS/MP BS. In Bayesian analysis, PP > 0.95 are indicated with thick branch. GenBank accession numbers for the six genes of each species were presented in Table 1.

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In our study a well supported (100, 100, 1.00) Saxifragodes + Cascadia clade is sister to the remaining Heucheroids which form a weakly supported clade (70, –, 0.96). Following Saxifragodes + Cascadia a well supported (100, 100, 1.0) Boykinia group is then sister to the remainder of the Heucheroids which also form a weakly supported clade (61, 91, 1.00). Following the Boykinia group, a wellsupported (100, 100, 1.00) Astilbe + Saxifragopsis clade is sister to the remaining Heucheroids (59, –, –). At this point in the Heucheroid topology, the two samples of Saniculiphyllum formed a well-supported clade (100, 100, 1.00) and are sister to the remaining Heucheroids, which form a clade that is only weakly supported (64, –, 1.00). These remaining Heucheroids comprise: Leptarrhena + Tanakaea (100, 100, 1.00), Darmera group (99, 95, 1.00; Astilboides, Bergenia, Darmera Voss, Mukdenia, Oresitrophe Bunge, and Rodgersia), Peltoboykinia group (89, 98, 1.00; Chrysosplenium and Peltoboykinia), Micranthes group [100, 100, 1.00; formerly Saxifraga section Micranthes (Haw.) D. Don], and Heuchera group (100, 100, 1.00; Bensoniella C.V. Morton, Conimitella Rydb., Elmera Rydb., Heuchera L., Lithophragma (Nutt.) Torr. and A. Gray, Mitella, Tellima R. Br., Tiarella, as well as Tolmiea Torr. and A. Gray) (Fig. 3). 4. Discussion 4.1. Overview of Saxifragaceae and placement of Saniculiphyllum The present study is based on a combined analysis of four molecular markers matK, psbA-trnH, rbcL, trnL-trnF from the chloroplast genome, and two molecular markers ITS, 26S rDNA from the nuclear genome; these have been used previously to infer relationships within family Saxifragaceae and among Saxifragales (Fishbein et al., 2001; Soltis et al., 2001a). While our current study represents increased taxonomic sampling (25 additional samples were employed) from previous work (Soltis et al., 1991a,b, 1993, 2001a; Morgan and Soltis, 1993; Johnson and Soltis, 1995; Soltis and Soltis, 1997), the phylogenetic tree obtained here is in close agreement with previously published results based on molecular data (Soltis et al., 2001a). Support values are also comparable, with all groups that receive strong support in agreement with previous studies relying on the same molecular markers (e.g., Soltis et al., 2001a). This major split determined here for Saxifragaceae based on DNA sequence analyses (Saxifragoids vs. Heucheroids) agrees with previous studies and is accompanied by some general morphological differences. The Saxifragoid clade receives strong support (100, 100, 1.0), and Saxifraga is the core member of this clade (Soltis, 2007). As reviewed elsewhere (Webb and Gornall, 1989; Soltis, 2007), Saxifraga s.s. has a relatively uniform floral morphology. The genus usually has actinomorphic flowers, 5 sepals, 5 petals, 10 stamens, and 2 carpels. Saniculiphyllum is embedded within the second large subclade of Saxifragaceae, the Heucheroid clade, which here receives low support (73, 68, 1.00). The Heucheroid clade encompasses more morphological variation than seen in the Saxifragoid clade (Soltis, 2007). For example, the Heucheroid clade includes actinomorphic and zygomorphic forms, as well as variation in the number of sepals, petals, stamens and carpels. 4.2. Saniculiphyllum as a distinct lineage within Saxifragaceae The distinctive morphology and phylogenetic placement of Saniculiphyllum together suggest that the genus is not a member of any of the well-supported subclades (groups) recognized previously within Saxifragaceae (e.g., Heuchera, Boykinia, Darmera groups, etc.; see Soltis, 2007). As noted, our analyses place Saniculiphyllum

as embedded well within the Heucheroids, a clade that receives low support (73, 68, 1.00). Within the Heucheroid clade, Saxifragodes + Cascadia, followed by the Boykinia group and then Astilbe + Saxifragopsis, are subsequent sisters to the remainder of the Heucheroids, which in turn form a weakly supported subclade (59, –, –). Saniculiphyllum appears as sister to the remaining genera of Heucheroids, a clade which also receives low support (64, –, 1.00). These remaining members of the Heucheroid clade comprise, however, a number of well-supported subclades: Heuchera group (100, 100, 1.00), Micranthes (100, 100, 1.00), Peltoboykinia/Chrysosplenium (100, 98, 1.00); Darmera group (100, 94, 1.00) (reviewed in Soltis, 2007). Hence, relationships among the well-supported subclades (groups) in the Heucheroid clade are poorly supported, as was the case in Soltis et al. (2001a). This lack of resolution is probably the result of a relatively rapid radiation (Soltis, 2007). Thus, while we can conclude that Saniculiphyllum appears to be a member of the Heucheroid clade, the relationship of the genus to other Heucheroids remains uncertain. The systematic position of Saniculiphyllum has been heavily debated. When establishing the genus, Wu and Ku (1992) placed Saniculiphyllum in subfamily Saxifragoideae (sensu Engler, 1930) as a tribe (Saniculiphylleae). In the original description, they thought the tribe Saniculiphylleae was closely related to tribe Saxifrageae because both have axile placentation, but differ in the number of petals, sepals, and stamens. Saniculiphyllum was also considered to be related to Chrysosplenium in that some species of the latter genus share some characters with Saniculiphyllum (e.g., short styles exserted from a thick disk). But, Chrysosplenium is characterized by parietal placentation which differs from Saniculiphyllum. When all morphological data are considered, Saniculiphyllum is a highly distinctive and unusual taxon, and it is quite different from other members of Saxifragaceae in having a 10-lobed floral disk, inferior ovary, short filament and creeping flat rhizomes, as well as 3–5 carpels as reported in the present study. Because of its distinctive characters, Mabberley (1997) elevated the genus to the rank of a family as Saniculophyllaceae. Although the present study clearly places the genus within Saxifragaceae (and within the Heucheroids), the uncertain position of Saniculiphyllum within the Heucheroids agrees with the highly distinctive morphology of the genus. Results from our analyses suggest that Saniculiphyllum is embedded within the Heucheroid clade and is sister to a clade comprising five groups including Leptarrheana group, Darmera group, Peltoboykinia group, Micranthes group, and Heuchera group, but this position receives low support. Thus, Saniculiphyllum can be regarded as a highly distinctive lineage within the Heucheroid clade of Saxifragaceae based on a combination of evidence from molecular phylogeny and morphological characters. 4.3. Comparison of topologies with earlier analyses Despite strong overall similarities there are also some noteworthy differences from the trees we report here and the topology recovered in Soltis et al. (2001a). There are two likely contributing factors to differences between the topologies of Soltis et al. (2001a) and those provided here. First, the present study has included 25 additional samples, including a distinctive genus, Saniculiphyllum. The addition of Saniculiphyllum could impact relationships among members of the Heucheroid clade, particularly given the poor internal support in this part of the tree. Perhaps a more important factor is sequence alignment, particularly alignment of the nuclear ITS region, and the trnL-trnF, psbA-trnH plastid regions, as noted below. The total evidence topology of Soltis et al. (2001a) is highly similar to that obtained here, but the relationships within the Heucheroid clade differ between the two studies. The Heucheroids form two major subclades in Soltis et al. (2001a). The Boykinia group

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(BS = 100%) + Leptarrhena + Tanakaea group (BS = 100%) and Astilbe + Saxifragopsis (BS = 100%) form one weakly supported major subclade (BS = 58%). A clade of the Heuchera group (BS = 100%), Darmera group (BS = 93%), Micranthes group (BS = 100%), Cascadia + Saxifragodes (BS = 100%), and Peltoboykinia + Chrysosplenium (BS = 100%) form a second weakly supported major lineage within Saxifragaceae (BS = 75%). In contrast, in the present study, the Heucheroids do not form two major subclades. Differences between the total evidence topology presented here and in Soltis et al. (2001a) are not well-supported. In both analyses there are well-supported smaller clades (e.g., Heuchera, Darmera, Micranthes, Peltoboykinia + Chrysosplenium groups, etc.), but relationships among these groups are generally poorly supported in both studies. The plastid tree of Soltis et al. (2001a) is very similar to the plastid tree reported here. Heucheroid and Saxifragoid clades are recovered in both analyses and both are well-supported. Relationships within the Heucheroid clade differ in the placement of some of the groups (e.g., Astilbe + Saxifragopsis), but few relationships among these groups have support >50%. In the present study, Saniculiphyllum is sister to Leptarrhena + Tanakaea, but with low support (BS = 68%); Saniculiphyllum was not included in Soltis et al. (2001a). The matK and rbcL regions are straightforward in terms of alignment and the major split of Heucheroids and Saxifragoids was apparent even with matK alone and the two genes combined revealed the two subclades with strong support (Soltis et al., 1996b). However, the trnL-trnF, psbA-trnH plastid regions are very difficult to align across the entire Saxifragaceae and whereas these regions were aligned by eye in Soltis et al. (2001a), alignment in the present study was done computationally. The close agreement of the Soltis et al. (2001a) plastid tree and the plastid tree recovered here is not surprising and likely reflects the strong matK + rbcL framework. The biggest difference between the present study and Soltis et al. (2001a) is in the ITS + 26S tree. In the present study the Heucheroids are not monophyletic. Instead, the Saxifragoid clade is sister to a subset of the Heucheroids: Darmera, Micranthes, and Chrysosplenium + Peltoboykinia. This clade of Saxifragoids plus some Heucheroids receives moderate support (BS = 84%). However, ITS + 26S provided minimal resolution in Soltis (2007). Saxifragoids were recovered with strong support (BS = 100); but Heucheroids formed a clade but without BS support >50% and relationships throughout the tree were poorly supported. This difference in nuclear trees (ITS + 26S) is probably best attributed to alignment differences with ITS. The 26S rDNA region was straightforward to align, but is highly conserved and provides little information at this level. The highly variable ITS region was easy to align by eye within subgroups (e.g., within the Boykinia group, or Darmera group, etc.), but very difficult to align among these well-supported groups. In Soltis et al. (2001a) the alignment was done by eye; in the present study, alignment was accomplished, as noted, with SATé. We may be pushing the limits of the utility of the entire ITS region at this taxonomic depth in Saxifragaceae. 4.4. The search for synapomorphies Incongruence between morphology and molecular data is common in angiosperms, for example in Saxifraga (Vargas, 2000), as well as in bryophytes, such as Leptodon D. Mohr (Sotiaux et al., 2009) and Neckera Hedw. (Olsson et al., 2011). The Saxifragaceae are a morphologically highly diverse group, variability of characters such as carpels, sepals, petals, stamens, is generally considered to be of taxonomic importance. Morphological characters supporting the clades found in the molecular analyses are as yet unknown or limited, although some morphological trends, which corroborate

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the relationships based on DNA sequence date, are becoming apparent (Nakazawa et al., 1997; Soltis et al., 1993, 2001a). While there are some broad morphological trends within clades it remains difficult to identify morphological synapomorphies. The search for morphological synapomorphies, especially in the Heucheroids clade, remains a challenge given the enormous morphological variation encompassed by members of this clade. Earlier results on the morphological evolution in Saxifragaceae (Soltis et al., 1991b, 2001a) showed that certain morphological states have multiple origins. For example, at least three separate instances of floral reduction (from 5 to fewer petals) occurred in Saxifragaceae. In the present study our phylogenetic inferences imply that several morphological character states, especially carpel number that were held as unique and characteristic of Saniculiphyllum, actually evolved independently. Most species of Saxifragaceae standardly have 2 carpels, as do closely related genera (e.g., Itea, Ribes). Five genera, Astilbe of Astilbe group, Conimitella, Micranthes, Bergenia and Rodgersia of Heuchera group occasionally have 3 carpels. Only the genus Lithophragma of the Heuchera group, is characterized by 3 carpels. However, based on our investigation of 491 flowers, Saniculiphyllum has three different carpel numbers. The percentages of 3, 4 and 5 carpels are 28.72%, 45.62% and 25.66% respectively, of which 4 and 5 carpels are unique to Saniculiphyllum within Saxifragaceae (Fig. 4), while 2 carpels, which was reported in the original description (Wu and Ku, 1992), could not be found in our samples. 4.5. Distribution, ecology and conservation status of Saniculiphyllum Saniculiphyllum grows only in wet habitats in Southwest China, and water is a decisive environment factor. The water of the streams have a pH of about 5.4 and an average temperature of 10–15 °C. Herbarium records indicate that the genus is confined to Yunnan and Guangxi. The four known populations are confined to an area about 10 square kilometers near a village in Funing County of southwestern Yunnan of southwestern China. The type and other early collections were from adjacent Guangxi Zhuang Autonomous Region. But as noted plants had disappeared from the type location due to continuing seasonal drought in southwestern China in recent years. Plants of Saniculiphyllum generally cling to stones in streams or wet, drippy rocks in water falls with dense and fibrous adventitious roots. The vegetation type of the habitat of S. guangxiense is broad-leaved evergreen forests dominated by Cyclobalanopsis glaucoides Schottky (Fagaceae). Additional associated plants include Acorus calamus L. (Araceae), Elatostema sp. (Urticaceae) and widespread cultivated species, Musa nana Lour. (Musaceae). Saniculiphyllum is highly threatened, and currently only known from a single small area in Funing County from Yunnan Province (Fig. 2). We found only five populations. Actually, five populations were initially rediscovered, but one which was seen in 2009 at a site nearest to the village disappeared in 2010 due to the polluted water draining out from the village; now only four populations remained. Hence, human activity has to be considered the biggest threat to the survival of the species. The vegetation in this region is severely fragmented by roads and agricultural land. During our field surveys we also visited another locality, Tianlin County from Guangxi Zhuang Autonomous Region, where the type plants were collected. However, we were unable to find any plants at this site. It is hard to estimate the exact number of plants that remain. Plants of the species propagate through rhizomes and form clones on stones in the streams or on cliffs near waterfalls. Two of the populations comprise perhaps 20–50 clones along streams. The two other populations occur near waterfalls and consist of a greater number of clones. The recently published red-list of vascular plants from China considers Saniculiphyllum to be in the Endangered IUCN category

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Fig. 4. Three types of carpels of Saniculiphyllum guangxiense. (a) Three carpels; (b) four carpels; (c) five carpels.

(EN; Wang and Xie, 2004). However, from our field experience we believe that until additional extant populations are found, S. guangxiense falls within the Critically Endangered category (CR, based on criteria B2a) of the red list guidelines of the World Conservation Union (IUCN, 2001). Acknowledgments The authors are grateful to Xi Lu, Zhi-Jian Yin, Hong-Jing Dong, Qi-Liang Gan, and Yong-Zuo Shi for their assistance in sample collection. We also thank Dr. Amy Litt and two anonymous reviewers for their constructive suggestions that greatly improved the paper. The research was supported by the National Natural Science Foundation of China (No. 30770152) granted to LGL and partially financed by the Angiosperm Tree of Life Project (NSF EF-0431266) to DES. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2012.04. 010. References APG III, 2009. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG III. Bot. J. Linn. Soc. 161, 105–121. Bayer, R.J., Cross, E.W., 2002. A reassessment of tribal affinities of the enigmatic genera Printzia and Isoetopsis (Asteraceae), based on three chloroplast sequences. Aust. J. Bot. 50, 677–686. Bull, J.J., Huelsenbeck, J.P., Cunningham, C.W., Swofford, D.L., Waddell, P.J., 1993. Partitioning and combining data in phylogenetic analyses. Syst. Biol. 42, 384– 397. Chandler, G.T., Bayer, R.J., 2000. Phylogenetic placement of the enigmatic western Australian genus Emblingia based on rbcL sequences. Plant Spec. Biol. 15, 67–72. Chase, M.W., Soltis, D.E., Olmstead, R.G., Morgan, D., Les, D.H., Mishler, B.D., Duvall, M.R., Price, R.A., Hills, H.G., Qiu, Y.L., Kron, K.A., Rettig, J.H., Palmer, J.D., Manhart, J.R., Sytsma, K.J., Michaels, H.J., Kress, W.J., Karol, K.G., Clark, W.D., Hedren, M., Gaut, B.S., Jansen, R.K., Kim, K.J., Wimpee, C.F., Smith, J.F., Furnier, G.R., Strauss, S.H., Xiang, Q.Y., Plunkett, G.M., Soltis, P.S., Swensen, S., Williams, S.E., Gadek, P.A., Quinn, C.J., Eguiarte, L.E., Golenberg, E., Learn Jr., G.H., Graham, S.W., Barrett, S.C.H., Dayanandan, S., Albert, V.A., 1993. Phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Ann. Mo. Bot. Gard. 80, 528–580. Conti, E., Soltis, D.E., Harding, T.M., Schneider, J., 1999. Phylogenetic relationships of the Silver Saxifrages (Saxifraga, Sect. Ligulatae Haworth): implications for the evolution of substrate specificity, life histories, and biogeography. Mol. Phylogenet. Evol. 13, 536–555. Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants. Columbia University Press, New York, USA. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochem. Bull. 19, 11–15.

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