Molecular phylogenetics and biogeography of the eastern .../menu/standa… · Molecular...

Molecular phylogenetics and biogeography of theeastern Asian–eastern North American disjunctMitchella and its close relative Damnacanthus(Rubiaceae, Mitchelleae)

WEI-PING HUANG1,2, HANG SUN1, TAO DENG1, SYLVAIN G. RAZAFIMANDIMBISON4,ZE-LONG NIE1* and JUN WEN3*

1Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy ofSciences, Kunming, Yunnan 650204, China2University of Chinese Academy of Sciences, Beijing 100049, China3Department of Botany, National Museum of Natural History, MRC 166, Smithsonian Institution,Washington DC, 20013-7012, USA4Bergius Foundation, The Royal Swedish Academy of Sciences and Department of Botany, StockholmUniversity, SE-10691, Stockholm, Sweden

Received 25 April 2012; revised 23 July 2012; accepted for publication 12 September 2012

Mitchella is a small genus of the Rubiaceae with only two species. It is the only herbaceous semishrub of the familyshowing a disjunct distribution in eastern Asia and eastern North America, extending to Central America. Itsphylogeny and biogeographical diversification remain poorly understood. In this study, we conducted phylogeneticand biogeographical analyses for Mitchella and its close relative Damnacanthus based on sequences of the nuclearinternal transcribed spacer (ITS) and four plastid markers (rbcL, atpB-rbcL, rps16 and trnL-F). Mitchella ismonophyletic, consisting of an eastern Asian M. undulata clade and a New World M. repens clade. Our results alsosupport Michella as the closest relative to the eastern Asian Damnacanthus. The divergence time between the twointercontinental disjunct Mitchella species was dated to 7.73 Mya, with a 95% highest posterior density (HPD) of3.14-12.53 Mya, using the Bayesian relaxed clock estimation. Ancestral area reconstructions suggest that thegenus originated in eastern Asia. The semishrub Mitchella appears to have arisen from its woody ancestor ineastern Asia and then migrated to North America via the Bering land bridge in the late Miocene. © 2013 TheLinnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 395–412.

ADDITIONAL KEYWORDS: Bering land bridge – intercontinental disjunction.

INTRODUCTION

The well-known biogeographical disjunction betweeneastern Asia and eastern North America has attractedmuch attention from plant biologists not only becauseit exhibits a unique distribution pattern, but alsobecause it offers an excellent opportunity to exploreplant differentiation and evolution in allopatry (Bouf-ford & Spongberg, 1983; Hong, 1993; Wen, 1999,2001; Wen et al., 2010). Fossil, molecular phylogenetic

and geological data all indicate that this disjunctpattern originated multiple times in multiple areasthroughout the Tertiary (Tiffney, 1985a, b; Wen,1999). Much progress has been made concerning theevolution of this pattern (Wen, 1998, 2001; vonDohlen, Kurosu & Aoki, 2002; Dane et al., 2003; Fuet al., 2005; Wen et al., 2010). Molecular data havebeen employed extensively to estimate divergencetimes (Xiang et al., 2000; Nie et al., 2006a, 2010;Meng et al., 2008; Bremer & Eriksson, 2009) and toinfer ancestral areas of the disjunct groups (Wen,2000; Xiang & Soltis, 2001). However, few studieshave examined the evolution of the disjunct pattern

*Corresponding authors. E-mail: [email protected];[email protected]

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Botanical Journal of the Linnean Society, 2013, 171, 395–412. With 4 figures

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 395–412 395

in herbaceous taxa (Tiffney, 1985a, b; Wen, 1999; Nieet al., 2005).

Mitchella L. is a herbaceous semishrub genus in themostly tropical and woody family Rubiaceae thatexhibits the classical intercontinental disjunctionbetween eastern Asia and eastern North America (Li,1952). Mitchella is composed of only two species: theeastern Asian M. undulata Siebold & Zucc. and theeastern North American M. repens L., which extends toCentral America (Li, 1952; Rogers, 2005; Chen et al.,2011). This genus can be distinguished from theherbaceous genera of Rubiaceae by a combinationof several characters, including its rather longunbranched primary shoots, paired flowers on a shortpeduncle with the base of the calyx fused and two reddrupaceous fused fruits with campylotropous ovulesinserted in the upper part of the septum (Robbrecht,Puff & Igersheim, 1991; Yamazaki, 1993; Rogers, 2005;Chen et al., 2011). Both species are evergreen. TheAsian M. undulata grows mostly on forest floors inTaiwan, Korea, Japan and southeastern China (Yama-zaki, 1993; Liu & Yang, 1998; Chen et al., 2011), andthe eastern North American M. repens occurs in moistor dry woods, along stream banks and on sandy slopesthroughout eastern North America southwards toMartin County, Florida, with disjunct extensions toCentral America (Li, 1952; Rogers, 2005) (Fig. 1).

Although the generic status of Mitchella hasnever been questioned, its phylogenetic position in

Rubiaceae has been controversial. Mitchella has beenplaced in various tribes, such as ‘Guettardidae’(Lindley, 1846), Anthospermeae (Hooker, 1873), Chio-cocceae (Baillon, 1880) and Paederieae (Puff, 1982).Based on a detailed morphological study, Robbrechtet al. (1991) pointed out that Mitchella is close toDamnacanthus Gaertn.f., a shrubby genus compris-ing about 13 species with evergreen leaves and awide distribution in the understorey of natural laurelforests of south China, Taiwan, Japan, Korea,Vietnam, Laos, Myanmar and Assam, India (Yama-zaki, 1993; Liu & Yang, 1998; Chen et al., 2011). Aunique characteristic of Damnacanthus is hetero-phylly associated with sympodial growth and pairedthorns (Robbrecht et al., 1991; Naiki & Nagamasu,2003, 2004). Recent molecular phylogenetic studiesalso support the close relationships between Mitchellaand Damnacanthus (Andersson & Rova, 1999;Bremer & Manen, 2000; Razafimandimbison, Rydin &Bremer, 2008). A new tribe Mitchelleae Razafim. &B.Bremer, including only these two genera, wasestablished by Razafimandimbison et al. (2008),which belongs to the subfamily Rubioideae.

The New World M. repens and the eastern AsianM. undulata are morphologically similar (Robbrechtet al., 1991). No molecular studies have focusedparticularly on this genus and almost all previousmolecular analyses have included only one speciesof Mitchella. Xiang et al. (2000) suggested the

Mitchella undulata Mitchella repens

Figure 1. Distribution map of Mitchella showing disjunction between eastern Asia and eastern North America (includingCentral America).

396 W-P. HUANG ET AL.

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 395–412

divergence between eastern Asian and easternNorth American species of Mitchella at about5.89 ± 2.38 Mya based on rbcL sequences. Becausetaxa once suspected as sister disjunct species basedon morphological similarities may not represent realsister species (Wen, 1999), whether the two species ofMitchella are phylogenetically closest to each other orgenetically distant from each other needs to be exam-ined in a broader phylogenetic framework, especiallywith a comprehensive sampling scheme including itsclose relative Damnacanthus.

Here, we used four plastid fragments (atpB-rbcL,rbcL, trnL-F and rps16) and the nuclear ribosomalinternal transcribed spacer (ITS) region to addressthe following questions. (1) Is Mitchella a mono-phyletic genus? (2) What is the phylogenetic relation-ship between Mitchella and Damnacanthus? (3) Whatare the most likely hypotheses to explain the biogeo-graphical disjunction of Mitchella between easternAsia and the New World? The molecular markersselected and most sequences in the dating analysishave been used widely in previous studies inRubiaceae (e.g. Bremer, Andreasen & Olsson, 1995;Andersson & Rova, 1999; Rova et al., 2002; Church,2003; Razafimandimbison, Kellogg & Bremer, 2004;Nie et al., 2005; Razafimandimbison et al., 2008,2009; Bremer & Eriksson, 2009).

MATERIAL AND METHODSTAXON SAMPLING

The voucher information for all the materials andGenBank accessions are presented in Table 1. Oursampling included both species of Mitchella:M. repens from North and Central America (sevenaccessions) and M. undulata from eastern Asia (threeaccessions). Damnacanthus is supported to be theclosest relative of Mitchella (Robbrecht et al., 1991;Razafimandimbison et al., 2008) and eight of the 13species were sampled in this study. To test the mono-phyly of Mitchella with all available data, sequencesof two Mitchella accessions from GenBank were com-bined with our dataset (Table 1). Based on Razafi-mandimbison et al. (2008), five species of Morindeaeand three species from Gaertnereae (sequences fromGenBank) were chosen as outgroup taxa in the phy-logenetic analysis (Table 1).

DNA EXTRACTION, AMPLIFICATION AND SEQUENCING

Total DNA of field-collected material was extractedfrom silica gel-dried leaf tissue using the Plant TotalDNA Extraction Kit (BioTeKe, Beijing, China). Iso-lated DNA was amplified and sequenced followingRydin et al. (2008) for the atpB-rbcL spacer, Bremeret al. (1995) for rbcL, Oxelman et al. (1997) for rps16,

Razafimandimbison & Bremer (2002) and Razafi-mandimbison, Kellogg & Bremer (2004) for ITS andRazafimandimbison & Bremer (2002) for trnL-F. Allpolymerase chain reactions (PCRs) were run in aPTC-100 thermocycler (MJ Research, Ramsey, MN,USA). PCR products were purified using an agarosegel DNA purification kit (Takara, Shiga, Japan), fol-lowing the manufacturer’s instructions. Sequencingwas performed with BigDye Terminator 3.1 (AppliedBiosystems, Foster City, CA, USA) on an ABI PRISM3730 Sequencer using the same primers as employedfor the PCR amplifications. All sequences were ana-lysed and assembled with Sequencher ver.4.14 (GeneCode, Ann Arbor, MI, USA).

PHYLOGENETIC ANALYSES

The computer program CLUSTALX (Thompson et al.,1997) was used for an initial alignment of all thesequences, followed by manual alignment usingBioEdit (Hall, 1999). All datasets were analysedunder maximum parsimony (MP) with PAUP 4.0b10(Swofford, 2003) and Bayesian inference (BI) withMrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). Forthe MP analyses, we used heuristic searches withtree bisection–reconnection (TBR) branch swapping,MULTREES option on and 1000 replicates of randomtaxon addition. All characters were unordered andequally weighted, and gaps were treated as missingdata in the analyses. Bootstrap tests of the data used1000 pseudoreplicates to evaluate clade support.

For the Bayesian analyses, the best-fitting modelsof sequence evolution for the plastid and ITS datasetswere chosen by MrModeltest v. 3.7 (Nylander, 2004)under the Akaike information criterion (Akaike,1973). Bayesian analyses were conducted underfour independent Markov chain runs for 10 millionMetropolis-coupled generations, sampling trees every1000 generations. The first 10% of trees were dis-carded as burn-in (average split deviations betweenparallel runs < 0.01). In the combined plastid and ITSanalyses, we set the matrices into two unlinked par-titions (the plastid data and the ITS data). All Baye-sian analyses were run twice with random startingtrees, and a consensus tree was constructed using thesaved trees by the two independent runs. Clades withposterior probabilities (PPs) over 95% were regardedas strongly supported.

DIVERGENCE TIME ESTIMATION

After assessing the sequences generated, and thoseavailable from GenBank, we chose to use the com-bined ITS, atpB-rbcL, rbcL, rps16 and trnL-F data toestimate the divergence time of Mitchella between theNew World and eastern Asia. A likelihood ratio test

PHYLOGENY AND BIOGEOGRAPHY OF MITCHELLA 397


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rejected the molecular clock hypothesis (P < 0.05). Wethus used a Bayesian relaxed method implementedin BEAST 1.7.1 (Drummond & Rambaut, 2007) toestimate the divergence times. With our focus onthe divergence time of genus Mitchella, and with theconsideration of minimizing the influence from topo-logical uncertainties in our analyses on dating ofthe phylogeny, we excluded some Damnacanthustaxa. To allow multiple fossil calibrations in a broadphylogenetic framework of Rubiaceae, sequences of63 additional taxa were obtained from GenBank(see Appendix). Gelsemium sempervirens (L.) J.St.-Hil. (Gelsemiaceae) was selected as the outgroup inour dating analysis.

We used the GTR model of nuclear substitution,gamma distribution for four rate categories, uncorre-lated log-normal relaxed clock model and Yule processtree prior in the Bayesian dating analyses. Two sepa-rate BEAST runs were set to perform 50 milliongenerations with 10% burn-in, and each run waschecked for convergence with Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/).

Fossils of Rubiaceae have been widely used to esti-mate the divergence time of the family or certainclades in the family (Nie et al., 2005; Antonelli et al.,2009; Bremer & Eriksson, 2009; Smedmark, Eriksson& Bremer, 2010). Although there have been manydescribed leaves and pollen fossils of Rubiaceae sincethe Cretaceous and Palaeocene (Graham, 2009), themost convincing fossil of Rubiaceae is from Cephalan-thus L., which was reported from the late Eocene tothe Pliocene in almost 20 fossil sites (Dorofeev, 1960,1963; Friis, 1985; Mai & Walther, 1985; Antonelliet al., 2009). We followed Antonelli et al. (2009) inusing the oldest fossil of this genus to place aminimum age constraint of 33.9 Ma, which was fixedby using the ending point of the geological epoch towhich the fossil belongs as the stem age of Cephalan-thus. The pollen fossils of Faramea Aubl. have beenreported from the late Eocene (~34–40 Ma) inPanama to the Pliocene in Veracruz, Mexico (Graham,2009); we thus used 37Ma, the mean age of the lateEocene, to set a minimum age of the Faramea stemnode. Saenger (1998) reported two pollen fossil ages ofScyphiphora C.F.Gaertn.: 16 Ma from Japan and23 Ma from the Marshall Islands. Scyphiphora is theonly extant genus of Rubiaceae that belongs to man-grove vegetation (Bremer & Eriksson, 2009), and itspollen characters are well defined and unique inRubiaceae. We thus used 23 Ma as a minimum ageprior for the Scyphiphora stem node.

Morinda chinensis Shi, Liu & Jin was recentlydescribed as a well-preserved fossil dated back to thelate early to the early late Eocene (Shi et al., 2012).This fossil has a head-shaped infructescence (multiplefruits or syncarps), which is developed from a capitu-

lum composed of about 20–30 flowers, the fruits ofwhich are fused into one unit (Shi et al., 2012), andthese characters fit well with Morinda. Shi et al.(2012) argued that, based on its shape and number ofsimple fruits of the infructescence, the fossil fruitshould be placed in Morinda section Roioc DC.However, the authors also point out that its infruct-escence contains fewer simple fruits than otherspecies of this section. Molecular phylogenetic analy-sis also suggests that Morinda is paraphyletic(Razafimandimbison et al., 2009). As the phylogeneticposition of this species is unclear, we used this fossilto calibrate the stem age of Morinda with the priorset to 44.5 + 3 Ma, between 40.6 and 48.4 Ma (lateearly Eocene–early late Eocene).

Four fossils were selected as calibration points inour analyses, three of which (Cephalanthus, Farameaand Scyphiphora) were the same as in Bremer &Eriksson (2009). We used the new fossil to calibratethe stem age of Morinda to enhance the accuracy forthe dating of Mitchella, because these two generabelong to the sister tribes Morindeae and Mitchelleae,respectively (Razafimandimbison et al., 2009). To rootthe tree, 78 Ma was enforced as the split timebetween Rubiaceae and other Gentianales, based onBremer, Friis & Bremer (2004), who used a broadsampling of asterids and multiple fossils.

BIOGEOGRAPHICAL ANALYSES

We defined two areas of endemism to assess thehistorical biogeography of the Mitchella clade, easternAsia (A) and North America to Central America (B),based on the extant distributions of the species in theBayesian tree and geological history. Although manyanalyses on the disjunct taxa (Baird et al., 2010; Nieet al., 2010; Xu et al., 2010) have used dispersal–vicariance analysis (DIVA; Ronquist, 1996) to inferancestral distributions, DIVA requires fully bifurcatedtrees. Because our Bayesian trees were not fullyresolved, we used RASP 1.1 (Yu, Harris & He, 2011),which implements the S-DIVA (statistical dispersal–vicariance analysis) method (Yu, Harris & He, 2010)and allows uncertainties in the phylogenetic trees. Weused Bayesian trees from the phylogenetic analyses(10 000 trees, excluding the remote outgroup of Gaer-tnereae) as input for S-DIVA. The condensed tree wascomputed using these 9000 trees (excluding theburn-in 1000 trees); the ‘maxarea’ was set to two andstate frequencies were estimated.

RESULTS

The statistics of the sequences are shown in Table 2.The ITS dataset had the highest percentage of poten-tially parsimony informative (PI) sites (21.16%),



although ITS was the shortest of the fragments. Asthe individual plastid markers had limited PI (5.31%–6.05%, Table 2) and generated unresolved trees in ourseparate analyses, we combined these four regions toreconstruct a combined plastid phylogeny. As therewere no statistically supported conflicts of the ITSand plastid trees, our discussion is based on resultsfrom concatenated plastid and ITS data. The MP andBI analyses produced similar results, and only theBayesian tree, with parsimony bootstrap (PB) andBayesian PPs, is presented in Fig 2.

Mitchella was strongly supported as monophyletic(PP = 1.00, PB = 87%; Fig. 2) with two well-supportedgroups: the New World M. repens (PP = 1.00, PB =98%) and the eastern Asian M. undulata (PP = 1.00,PB = 92%). In all analyses, tribe Mitchelleae (Mitch-ella and Damnacanthus) was supported as mono-phyletic (PP = 1.00, PB = 100%). In Damnacanthus,four lineages were recognizable with strong support(Fig. 2): (1) D. giganteus Nakai, D. labordei (H.Lév.)H.S.Lo, D. officinarum C.C.Huang and D. macrophyl-lus Siebold ex Miq. (accessions of Nie2241 andNie2240); (2) D. henryi (H.Lév.) H.S.Lo and D. hain-anensis (H.S.Lo) Y.Z.Ruan (PP = 1.00, PB = 100%); (3)D. macrophyllus Deng109 and D. major Siebold &Zucc. (PP = 1.00, PB = 100%); and (4) the two acces-sions of D. indicus C.F.Gaertn.

The chronogram and estimated divergence timesfrom the dating analyses at the family level areshown in Fig. 3. The combined tree resolvedRubiaceae into three major lineages, formally recog-nized as subfamilies Cinchonoideae, Ixoroideae andRubioideae. The divergence between the easternNorth American and the eastern Asian species wasestimated at 7.33 Ma in the late Miocene, with a 95%highest posterior density (HPD) of 3.14-12.53 Ma,covering a period from the late–middle Miocene tothe Pliocene. The S-DIVA analyses clearly inferred

eastern Asia as the ancestral area of Mitchella(Fig. 4).

DISCUSSIONPHYLOGENETIC RELATIONSHIPS

Our results based on the combined analysis of plastidand nuclear data support the monophyly of Mitchella(Fig 2), which comprises two widely disjunct species:M. undulata from eastern Asia and M. repens fromeastern North America extending to Central America.These two species share many features, such as acreeping habit with dark evergreen leaves and rootingat the nodes (Rogers, 2005; Chen et al., 2011), andusually heterostylous flowers (i.e. some individualshave exserted stamens and an included style,whereas others possess included stamens and anexserted style) (Blaser, 1954; Ganders, 1975; Hicks,Wyatt & Meagher, 1985; Yamazaki, 1993; Chen et al.,2011). These two species are morphologically sosimilar that M. undulata has sometimes been reducedto infraspecific rank as M. repens var. undulata (Sieb.& Zucc.) Makino (Makino, 1909; Robbrecht et al.,1991). In spite of the low level of morphological vari-ation, molecular results support a clear separation ofthe eastern Asian and the New World clades (Fig. 2).Nevertheless, minor morphological and phenologicaldifferences can be observed between them. Mitchellarepens usually has leaves obtuse at the apex andentire margins, and flowers in April to June (Miller &Miller, 2005; Rogers, 2005), whereas M. undulata hasleaves acuminate to rounded at the apex and some-times undulate at the margins, and usually flowers inJune to August (Makino, 1909; Yamazaki, 1993; Liu &Yang, 1998; Chen et al., 2011).

Our results also strongly support Mitchella asthe closest relative of Damnacanthus (PP = 1.00,

Table 2. Sequence characteristics of Mitchella and its relatives used in this study

ITS rbcL rps16 atpB-rbcL trnL-F Plastid Plastid + ITS

Length of aligned matrices(bp)

534 1318 985 694 1103 4100 4634

Number of potentiallyparsimony-informativecharacters (PICs)

113 70 59 42 59 230 343

Percentage of potentiallyparsimony-informativesites

21.16 5.31 5.99 6.05 5.35 5.61 7.40

Retention index 0.7789 0.8079 0.9160 0.8968 0.9167 0.8466 0.8098Consistency index 0.7500 0.7706 0.9346 0.8807 0.9353 0.8500 0.8100Best tree length 352 170 153 109 170 618 979

ITS, internal transcribed spacer.



PB = 100%, Fig. 2) and confirm the monophyly of tribeMitchelleae as proposed by Razafimandimbison et al.(2008). Baillon (1880) first suggested the close affini-ties between Damnacanthus and Mitchella. Robbre-cht et al. (1991) further pointed out that Mitchellaand Damnacanthus were closely related based on adetailed morphological study. Recently, moleculardata supported the close relationship of Mitchella andDamnacanthus (Bremer, 1996; Andersson & Rova,1999; Bremer & Manen, 2000; Razafimandimbisonet al., 2008, 2009). The two genera share a numberof morphological characters: campylotropous (rarelyseen in other genera of Rubiaceae), pitted endocarp,

red syncarpous fruits, fused two flowers with eachsole flower having four carpels, placenta inserted inthe upper part of the septum and chromosomenumber of 2n = 22 (Robbrecht et al., 1991).

Robbrecht et al. (1991) also stressed differences invarious important character states which support theseparation of Damnacanthus and Mitchella as twodistinct genera. Damnacanthus is a shrubby genuswith conspicuously heterophyllous evergreen leaves,whereas Mitchella spp. are small creeping semi-shrubs (herbaceous-like) with green uniform leaves.In Mitchella, lateral branches originate on older partsof rather long unbranched primary shoots, whereas a

Gynochthodes coriacea

Damnacanthus henryi Nie3941


Appunia guatemalensis

Mitchella undulata

Morinda citrifolia

Mitchella repens Wen10102

Damnacanthus indicus Huang042

Mitchella undulata Deng081

Gaertnera sp.

Gynochthodes sp.

Caelospermum monticola


Damnacanthus giganteus Nie2093

Damnacanthus macrophyllus Nie2241


Mitchella repens Patterson and Mayfield 7369

Damnacanthus officinarum Xie426

Pagamea guianensis

Damnacanthus macrophyllus Deng109

Damnacanthus major Deng108

Mitchella repens

Damnncanthus hananensis Nie3967



Mitchella repens Breedlove et al. 32460


Damnacanthus labordei Zhang484

Damnacanthus giganteus Xie428

Gaertnera phyllosepala


1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.81

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

100

60

75

100

100

100

88

97

100

99

89 98

100

57

5392

100

10073

100

100

59

98

87

92

outgroup

easternNorth America

and Mexico

easternAsia

Figure 2. Bayesian majority-rule consensus tree of Mitchella, inferred from combined sequence data of four plastidmarkers (rbcL, atpB-rbcL, rps16 and trnL-F) and internal transcribed spacer (ITS). Bayesian posterior probabilities areshown above the branches and maximum parsimony (MP) bootstrap values are shown below.



0 Ma1020304050607080

Manostachya ternifolia

Exostema lineatum

Lasianthus lanceolatus

Damnacanthus indicus Huang054Damnacanthus giganteus Nie2093

Nauclea orientalis

Arcytophyllum aristatum


Cinchona calisaya


Paederia bojeriana

Triainolepis_mandrarensis

Parapentas silvatica

Saprosma foetens

Joosia umbellifera

Scyphiphora hydrophyllacea

Ophiorrhiza elmeri

Theligonum cynocrambe


Coussarea macrophylla

Faramea multiflora

Mitchella repens


Neurocalyx championii

Normandia neocaledonica

Jaubertia aucheri

Crossopteryx febrifuga

Anthospermum herbaceum


Gelsemium sempervirvens

Coccocypselum condalia

Kraussia floribunda

Cremaspora triflora

Sabicea diversifolia


Sipanea biflora

Pavetta abyssinica

Cinchonopsis amazonica

Mitchella undulata

Psychotria amboniana

Cruckshanksia hymencodon

Colletoecema dewevrei

Hamelia papillosa


Morinda citrifolia



Prismatomeris beccariana

Cephalanthus salicifolius

Rondeletia odorata

Isertia occinea

Serissa foetida

Posoqueria latifolia


Condaminea corymbosa


Alberta magna

Emmenopterys henryi

Coussarea hydrangeifolia

Stilpnophyllum grandifolium

Mitchella repens Patterson & Mayfield 7369

Cosmibuena grandiflora

Knoxia platycarpa

Spermacoce hispida

Guettarda speciosa



Hymenodictyon floribundum

Kerianthera praeclara

Chiococca alba

Xanthophytum borneense

Placopoda virgata

Geophila obvallata

Ladenbergia amazonensis

Vangueria madagascariensis

Aulacocalyx asminiflora

Spiradiclis bifida

Cubanola domingensis

Dunnia sinensis

Declieuxia cordigera

Mitchelleae

Cinchonoideae

Ixoroideae

Rubioideae

Figure 3. Chronogram of Mitchella and its relatives from Rubiaceae based on the combined internal transcribed spacer(ITS), atpB-rbcL, rbcL, rps16 and trnL-F data estimated from BEAST. Calibration points are indicated with black stars.Node bars represent 95% highest posterior distribution of node age estimates.



regular sympodial branching pattern prevails inDamnacanthus. The two neighbouring flowers arefused by their ovaries in Mitchella, but separate inDamnacanthus. Mitchella has ‘compound’ drupes,whereas fruits of Damnacanthus are mostly paired,but have separate pedicels. The morphological dif-ferences mentioned above are consistent with ourmolecular results of the clear separation of Damna-canthus and Mitchella (Fig. 2). However, Razafimand-imbison et al. (2008) suggested that Damnacanthus isparaphyletic with Mitchella nested in it. Furtherstudies with complete sampling of Damnacanthusand further molecular data are needed to test therelationships between these two genera and tocircumscribe species of Damnacanthus.

BIOGEOGRAPHICAL DIVERSIFICATION OF MITCHELLA

Our dating results, calibrated with four fossils, aresimilar to those of Antonelli et al. (2009), but muchyounger than those of Bremer & Eriksson (2009). The

difference in the root age set may explain the differ-ence, as we set the root of the family at 78 Ma,whereas Bremer & Eriksson (2009) set 45 Ma as theminimum age prior for the family. The divergencetime between the New World M. repens and theeastern Asian M. undulata was estimated to be about7.33 Ma (95% HPD, 3.14-12.53 Ma) in the lateMiocene (Fig. 3). This estimate is similar to that ofXiang et al. (2000), which was 5.89 ± 2.38 Ma basedon the rbcL gene using an average synonymous sub-stitution rate of 22 species belonging to 11 plantgroups [Rs = (1.23 ± 0.128) ¥ 10-9 substitutions persite per year].

Wen et al. (2010) reported that the divergence timeof most Asian–North American temperate disjunctlineages is between 3 and 25 Ma. The divergence timeof Mitchella in our study is consistent with results ofother studies on eastern Asian and eastern NorthAmerican disjunct taxa dating back to the late Terti-ary to early Pleistocene (Wen, 2000; Dane et al., 2003;Nie et al., 2005; Baird et al., 2010).

AB

AB




Appunia guatemalensis

Mitchella undulata

Morinda citrifolia




Gynochthodes sp.

Caelospermum monticola


Damnacanthus giganteus Nie2093



Mitchella repens Patterson and Mayfield 7369


Damnacanthus macrophyllus Deng109

Damnacanthus major Deng108

Mitchella repens







Damnacanthus giganteus Xie428


Figure 4. Ancestral area reconstruction of Mitchella using statistical dispersal–vicariance (S-DIVA) in RASP: A, easternAsia; B, eastern North America and Central America.



The ancestors of modern eastern Asian–easternNorth American disjunct genera have been hypoth-esized to have originated in various areas andattained their present distribution via multiple path-ways. At least three hypotheses have been proposed:migration through the North Atlantic land bridges(Tiffney, 1985a); migration across the Bering landbridge (Tiffney, 1985b); and long-distance dispersal(Tiffney, 1985b; Wen, 1999; Wen et al., 2010). Ourbiogeographical analyses inferred the ancestral areaof Mitchella as eastern Asia (Fig. 2) with a divergencetime of the disjunction in the late Miocene (Fig. 3).The intercontinental disjunction of Mitchella is mostlikely to be explained as a migration from Asia toNorth America via the Bering land bridge. During thelate Miocene and early Pliocene, the Bering landbridge was available for floristic exchanges of temper-ate plants until about 3.5 Ma (Hopkins, 1967; Wen,1999).

The North Atlantic land bridge is a less likely routefor the Mitchella disjunction, because this routemostly contributed to the dispersal of more tropicalelements, and this floristic connection was no longerviable by the middle Miocene (Parks & Wendel, 1990;Tiffney & Manchester, 2001). Mitchella is a smallsemishrub with red drupes, which are often dispersedby small mammals over only short distances (Eriks-son & Bremer, 1991; Willson, 1993; Bremer & Eriks-son, 1992). Long-distance dispersal is thus consideredquite unlikely to explain the intercontinental disjunc-tion in Mitchella. We favour a hypothesis based on amigration scenario across the Bering land bridge,which has been proposed in a number of other tem-perate groups in the late Miocene and the Pliocene.For instance, Phryma L. (Phrymaceae) shows a clas-sical intercontinental disjunction between easternAsia and eastern North America, and was explainedby the Beringian migration in the late Miocenewith the divergence time estimated as 3.68 ± 2.25–5.23 ± 1.37 Ma (Nie et al., 2006a). Similar cases canalso be found in Penthorum L. (Xiang et al., 2000),Circaea L. (Xie et al., 2009), Saxifraga rivularis L.(Westergaard et al., 2010), Symplocarpus Salisb. (Nieet al., 2006b) and Astilbe Buch.-Ham. ex D.Don (Kimet al., 2009; Zhu et al., in press).

Mitchella mostly occupies the subtropical to tem-perate region, whereas most genera of Rubiaceae aredistributed in tropical regions (Ehrendorfer, Manen &Natali, 1994; Manen, Natali & Ehrendorfer, 1994;Bremer & Eriksson, 2009). However, some popula-tions of M. repens are found in Central America,which can be explained by a southward expansionfrom eastern North America, as accessions fromMexico are nested in the eastern North Americanclade (Fig. 2), although they are sister to the easternNorth American samples in the BEAST phylogenetic

tree, as shown in Fig. 4. Except for some M. repenspopulations from Central America, Mitchella has amore northern distribution than most Damnacanthusspp. (Chen et al., 2011). Our phylogenetic resultssuggest that Mitchella may have adapted to a coldclimate and evolved to the herbaceous life form fromits woody Damnacanthus-like ancestor (Fig. 2), asindicated by the woody basal stem of Mitchella. Pal-aeontological evidence suggests that, in the earlyTertiary, the Boreotropical flora was continuously dis-tributed across the north temperate zone (Leopold &MacGinitie, 1972; Wolfe, 1972; Hong, 1993; Graham,1972). With the global cooling in the late Tertiary,thermophilic plants, including Rubiaceae, movedsouthwards, except a few taxa, such as Mitchella,which most probably survived as relict herbaceouselements in the temperate regions of the NorthernHemisphere. This type of adaptation has also beenreported in other taxa, such as ParthenocissusPlanch., one of the few temperate genera of Vitaceae,which was most likely a derivative of the EoceneBoreotropical element (Nie et al., 2010).

ACKNOWLEDGEMENTS

This study was supported by grants from the NaturalSciences Foundation of China (NSFC 30970193 toZ.-L. Nie, 30828001 and 30625004 to J. Wen and T. Yi,40930209 and 31061160184 to H. Sun), the OneHundred Talents Program of the Chinese Academy ofSciences (2011312D11022 to H. Sun), the UnitedFund of the NSFC and Yunnan Natural Science Foun-dation (U1136601 to H. Sun) and the John D. andCatherine T. MacArthur Foundation to J. Wen, R. Reeand G. Mueller. Laboratory work was performed atand partially supported by the Laboratory of Analyti-cal Biology of the National Museum of NaturalHistory, Smithsonian Institution. Fieldwork in NorthAmerica was supported by the Small Grants Programof the National Museum of Natural History, theSmithsonian Institution. We thank the MEXU her-barium for loaning the sampled Mexican Mitchellarepens used in this study.

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APPENDIXSEQUENCES OBTAINED FROM GENBANK AND USED IN THE DIVERGENCE TIME ANALYSES

Species ITS atpB-rbcL rbcL rps16 trnL-F Reference

Alberta magnaE.mey.

AJ224842 – Y18708 EU145491 AJ620118 Andersson & Rova, 1999;Rydin et al., 2008;Kainulainen et al., 2009

Anthospermumherbaceum L.f.

FM204677 AJ234028 X83623 EU145496 EU145544 Bremer et al., 1995; Bremer& Manen, 2000; Rydinet al., 2008; Kainulainenet al., 2009

Arcytophyllumaristatum Standl.

AM182061 FJ695343 AJ288595 AF333348 AF333349 Bremer & Manen, 2000;Andersson et al., 2002;Wolff & Liede-Schumann,2007; Rydin et al., 2009b



APPENDIX Continued


AulacocalyxjasminifloraHook.f.

FM204688 DQ131704* EU817413 EF205639 EU817455 Mouly et al., 2007, 2009;Kainulainen et al., 2009

Cephalanthussalicifolius Humb.& Bonpl.

AJ346886 GQ851993 AJ346975 GQ852381 AJ346920 Razafimandimbison &Bremer, 2002; Manns &Bremer, 2010

Chiococca alba (L.)Hitchc.

AY763882 – L14394 AF004034 AY763813 Olmstead et al., 1993;Motley et al., 2005;Manns & Bremer, 2010

Cinchona calisayaWeed.

AY538352 GQ852003 AY538478 AF242927 GQ852482 Rova et al., 2002;Andersson & Antonelli,2005; Manns & Bremer,2010

Cinchonopsisamazonica(Stand.)L.Andersson

AY538357 GQ852002 AY538482 AY538428 AY538452 Andersson & Antonelli,2005; Manns & Bremer,2010

Coccocypselumcondalia Pers.

EU145358 EU145420 AM117217 EU145499 EU145547 Rydin et al., 2008, 2009a;Bremer & Eriksson, 2009

Colletoecemadewevrei (DeWild.) E.M.A.Petit

EU145353 DQ131713* FJ209067 AF191491 EU145532 Piesschaert et al., 2000;Rydin et al., 2008, 2009a;Sonké et al., 2008

Condamineacorymbosa (Ruiz& Pav.) DC.

FJ984973 – Y18713 FJ884645 AF102406 Andersson & Rova, 1999;Motley et al., 2005

Cosmibuenagrandiflora (Ruiz& Pav.) Rusby

GQ852120 GQ852007 AY538483 AF242929 AF152686 Rova et al., 2002;Andersson & Antonelli,2005; Manns & Bremer,2010

Coussareahydrangeifolia(Benth.) Benth. &Hook.f. exMüll.Arg.

EU145360 EU145326 EU145460 EU145501 EU145549 Rydin et al., 2008, 2009a

Coussareamacrophylla(Mart.) Müll.Arg.

– – Y11847 AF004040 – Bremer & Thulin, 1998;Andersson & Rova, 1999

Cremaspora triflora(Thonn.) K.Schum.

AJ224824 DQ131718* Z68856 AF200990 AF201040 Andreasen & Bremer, 1996;Andreasen et al., 1999;Persson, 2000

Crossopteryxfebrifuga (Afzel.ex G.Don) Benth.

FM204689 DQ131719* JF265372 FM204717 FM207123 Kainulainen et al., 2009

Cruckshanksiahymenodon Hook.& Arn.

– AJ234004 AJ288599 EU145502 EU145550 Bremer & Manen, 2000;Rydin et al., 2008

Cubanoladomingensis(Britton) Aiello

AY763891 DQ131720* X83632 AF004044 AF152701 Rova et al., 2002; Motleyet al., 2005

Declieuxia cordigeraMart. & Zucc. exSchult. & Schult.f.

EU145361 EU145327 AM117224 AM117298 EU145551 Rydin et al., 2008, 2009a;Bremer & Eriksson, 2009

Dunnia sinensisTutcher




APPENDIX Continued


Emmenopteryshenryi Oliv.

FJ984985 DQ131728* |Y18715 AF242941 AF152637 Bremer et al., 1999; Rovaet al., 2002

Exostema lineatum(Vahl) Schult.

AY763901 DQ131732* AY538484 AF242944 AY763833 Andersson & Antonelli,2005; Motley et al., 2005

Faramea multifloraA.Rich.

EU145363 EU145328 Z68796 AF004048 AF102422 Andreasen & Bremer, 1996;Struwe et al., 1998;Andersson & Rova, 1999;Rydin et al., 2008, 2009a

Gelsemiumsempervirens (L.)J.St.-Hil.

AB454364 AJ233985 L14397 DQ660581 AF159696 Olmstead et al., 1993;Bremer & Manen, 2000;Rova et al., 2002; Simõeset al., 2007; Motohashiet al., 2009

Geophila obvallataDidr.

AM945196 – AM117228 JN643111* JN643390 Bremer & Eriksson, 2009

Guettarda speciosaL.

AY763904 GQ852025 JF738600* AF242964* AY763835 Motley et al., 2005; Manns& Bremer, 2010

Gynochthodescoriacea Blume

AM945192 AM945219 AJ288603 AM117311 AJ847407 Bremer & Manen, 2000;Alejandro et al., 2005;Razafimandimbison et al.,2008; Bremer &Eriksson, 2009

Hamelia papillosaUrb.

GQ852134 AJ233992 AY538487 AF004053 AF102439* Andersson & Rova, 1999;Bremer & Manen, 2000;Andersson & Antonelli,2005; Manns & Bremer,2010

Hymenodictyonfloribundum(Hochst. & Steud.)B.L.Rob.

AJ346905 DQ131742* AY538488 AF004058 AY538454 Razafimandimbison &Bremer, 2002; Andersson& Antonelli, 2005

Isertia coccinea(Aubl.) J.F.Gmel.

GQ852140 – GQ852337 GQ852405 AF152689 Rova et al., 2002; Manns &Bremer, 2010

Jaubertia aucheriGuill.

FJ695456 FJ695383 DQ662178 DQ662202 DQ662145 Backlund et al., 2007;Rydin et al., 2009b

Joosia umbelliferaH.Karst.

AY538361 – AY538492 AY538433 GQ852521 Andersson & Antonelli,2005; Manns & Bremer,2010

KeriantherapraeclaraJ.H.Kirkbr.

AY538362 – AY538493 AF242970* AY538459 Andersson & Antonelli,2005

Knoxia platycarpaArn.

AM267002 FJ695363 AJ288631 AM266826 AM266915 Bremer & Manen, 2000;Kårehed & Bremer, 2007;Rydin et al., 2009b

Kraussia floribundaHarv.

– DQ131746* JF265494* AM117325 AM117368 Bremer & Eriksson, 2009

LadenbergiaamazonensisDucke

AY538363 – AY538494 AY538434 AY538460 Andersson & Antonelli,2005

Lasianthuslanceolatus(Griseb.) Urb.

EU145367 EU145331 AM117238 AF004062 EU145554 Andersson & Rova, 1999;Rydin et al., 2008, 2009a;Bremer & Eriksson, 2009



APPENDIX Continued


ManostachyaternifoliaE.S.Martins

FJ695446 EU542973 AM117246 EU543042 EU543127 Bremer & Eriksson, 2009;Groeninckx et al., 2009;Rydin et al., 2009b

Mitchella repens L. AF072019* AM945223 Z68805 AF001441 FJ906973 Bremer, 1996; Andersson &Rova, 1999;Razafimandimbison et al.,2008; Razafimandimbisonet al., 2009

Morinda citrifolia L. GU222395* AJ234003 X83651 AJ320078 AF152616 Bremer et al., 1995; Bremer& Manen, 2000; Novotnyet al., 2002; Rova et al.,2002

Nauclea orientalis(L.) L.

AJ346897 EU145320 X83653 AY538440 AJ346958 Bremer et al., 1995;Razafimandimbison &Bremer, 2002; Andersson& Antonelli, 2005; Rydinet al., 2008

Neurocalyxchampionii Benth.ex Thwaites

EU145376 – EU145463 EU145509 EU145563 Rydin et al., 2008, 2009a

NormandianeocaledonicaHook.f.

AF257930* FJ695375 FJ695375 AF257931* AM409177 Khan et al., 2008; Rydinet al., 2009b

Ophiorrhiza elmeriMerr.


Paederia bojeriana(A.Rich. ex DC.)Drake

FJ695454 DQ131757* DQ662181 DQ662206 DQ662152 Backlund et al., 2007;Rydin et al., 2009b

Parapentas silvatica(K.Schum.)Bremek.

AM267023 AJ234021 X83657 AM266849 AM266937 Bremer et al., 1995; Bremer& Manen, 2000; Kårehed& Bremer, 2007

Pavetta abyssinicaFresen.

FM204696 – Z68863 FM204726 FM207133 Andreasen & Bremer, 1996;Kainulainen et al., 2009

Placopoda virgataBalf.f.

AM267064 FJ695382 Z68815 AM266894 AM266980 Bremer, 1996; Kårehed &Bremer, 2007; Rydinet al., 2009b

Posoqueria latifolia(Rudge) Schult.

DQ787409* – Z68850 AF242998* AF152680 Andreasen & Bremer, 1996;Rova et al., 2002

Prismatomerisbeccariana (Baill.ex K.Schum.)J.T.Johanss.

AM945206 AM945238 AF331651* AF331652* – Razafimandimbison et al.,2008

PsychotriaambonianaK.Schum.

AM945215 AM945248 AM945302 AM945328 AJ847409 Alejandro et al., 2005;Razafimandimbison et al.,2008

Rondeletia odorataJacq.

AY730307* EU145321 Y11857 EU145490 AF152741 Bremer & Thulin, 1998;Rova et al., 2002, Rydinet al., 2008

Sabicea diversifoliaPers.

AJ846883 DQ131781* AM117268 EU145494 AJ847396 Alejandro et al., 2005;Bremer & Eriksson, 2009

Saprosma foetens(Wight) K.Schum.

FJ695460 FJ695386 DQ662193 DQ662218 DQ662168 Backlund et al., 2007;Rydin et al., 2009b



APPENDIX Continued


ScyphiphorahydrophyllaceaC.F.Gaertn.

– – Y18717 DQ923045 FM207140 Bremer et al., 1999;Kainulainen et al., 2009

Serissa foetida (L.f.)Lam.

FJ980385* AJ234034 Z68822 AF004081 AF152618 Bremer, 1996; Andersson &Rova, 1999; Bremer &Manen, 2000

Sipanea biflora (L.f.)Cham. & Schltdl.

AY555116 DQ131788* AY538509 AF004085 AF152675 Andersson & Rova, 1999;Delprete & Cortes, 2004;Andersson & Antonelli,2005

Spermacoce hispidaL.

AM939540 EU543011 AJ288623 EU543073 EU543162 Rova et al., 2002; Kårehedet al., 2008; Groeninckxet al., 2009; Rydin et al.,2009b

Spiradiclis bifidaKurz


StilpnophyllumgrandifoliumL.Andersson

AY538375 GQ852090 AY538510 AY538446 AY538476 Andersson & Antonelli,2005; Manns & Bremer,2010

Theligonumcynocrambe L.

FJ695470 FJ695393 X83668 AF004087 FJ695427 Bremer et al., 1995;Andersson & Rova, 1999;Rydin et al., 2009b

TriainolepismandrarensisHomolle exBremek.

AM267068 FJ695394 FJ695250 AM266899 AM266985 Kårehed & Bremer, 2007;Rydin et al., 2009b

VangueriamadagascariensisJ.F.Gmel.

AJ224839 – X83670 EU821636 FM207146 Bremer et al., 1995;Andreasen et al., 1999;Cortés-B et al., 2009

Xanthophytumborneense(Valeton) Axelius


*Sequence unpublished.



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