Molecular Phylogenetics and Evolution 55 (2010) 1008–1017

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Phylogeny and biogeography of the eastern Asian–North American disjunctwild-rice genus (Zizania L., Poaceae)

Xinwei Xu a, Christina Walters b, Michael F. Antolin c, Mara L. Alexander d, Sue Lutz e, Song Ge f, Jun Wen e,f,*

a Freshwater Ecological Field Station of Liangzi Lake, Wuhan University, Wuhan 430072, Chinab USDA/ARS National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USAc Department of Biology, Colorado State University, Fort Collins, CO 80523-1878, USAd San Marcos National Fish Hatchery and Technology Center, 500 East McCarty Lane, San Marcos, TX 78666, USAe Department of Botany, National Museum of Natural History, MRC 166, Smithsonian Institution, Washington, DC 20013-7012, USAf State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

a r t i c l e i n f o

Article history:Received 3 September 2009Revised 17 November 2009Accepted 18 November 2009Available online 26 November 2009

Keywords:Adh1a geneBiogeographyIntercontinental disjunctionWild-riceZizania

1055-7903/$ - see front matter Published by Elsevierdoi:10.1016/j.ympev.2009.11.018

* Corresponding author. Address: Department of BoInstitution, Washington, DC 20013-7012, USA. Fax: +

E-mail address: wenj@si.edu (J. Wen).

a b s t r a c t

The wild-rice genus Zizania includes four species disjunctly distributed in eastern Asia and NorthAmerica, with three species (Z. aquatica, Z. palustris, and Z. texana) in North America and one (Z. latifolia)in eastern Asia. The phylogeny of Zizania was constructed using sequences of seven DNA fragments(atpB-rbcL, matK, rps16, trnL-F, trnH-psbA, nad1, and Adh1a) from chloroplast, mitochondrial, and nucleargenomes. Zizania is shown to be monophyletic with the North American species forming a clade and theeastern Asian Z. latifolia sister to the North American clade. The divergence between the eastern Asian Z.latifolia and the North American clade was dated to be 3.74 (95% HPD: 1.04–7.23) million years ago (mya)using the Bayesian dating method with the combined atpB-rbcL, matK, rps16, trnL-F, and nad1 data.Biogeographic analyses using a likelihood method suggest the North American origin of Zizania and itsmigration into eastern Asia via the Bering land bridge. Among the three North American species, theorganellar data and the haplotype network of the nuclear Adh1a gene show a close relationship betweenZ. palustris and the narrowly distributed endangered species Z. texana. Bayesian dating estimated thedivergence of North American Zizania to be 0.71 (95% HPD: 0.12–1.54) mya in the Pleistocene. Thenon-monophyly of Z. palustris and Z. aquatica in the organellar and nuclear data is most likely causedby incomplete lineage sorting, yet low-frequency unidirectional introgression of Z. palustris intoZ. aquatica is present in the nuclear data as well.

Published by Elsevier Inc.

1. Introduction

The intercontinental disjunction between eastern Asia andNorth America is a well-known biogeographic pattern in the north-ern hemisphere and has attracted considerable attention fromplant biologists (Xiang et al., 1998, 2000; Wen, 1999, 2001; Manosand Donoghue, 2001). The phylogenetic relationships of disjunctlineages, the timing of the disjunctions, and migration pathwaysof many taxa have been investigated based on molecular data(e.g., Xiang et al., 1998; Wen, 2000; Nie et al., 2005, 2006a,b; Pengand Wang, 2008). Most of these studies have focused on woodyplants or terrestrial herbs, but few studies have examined aqua-tic/wetland plants. At present, investigators have mainly discussedthe questions on the processes of the formation of the interconti-nental disjunct pattern, such as estimating divergence times andinferring ancestral areas. Questions on subsequent diversification

Inc.

tany, MRC 166, Smithsonian1 202 786 2563.

and evolution of the disjunct taxa in both continents have rarelybeen examined (Wen, 1999; Xiang et al., 2004; Wen et al., 2009).

The wild-rice genus Zizania L. belongs to the rice tribe (Oryzeae,Poaceae) and is an aquatic/wetland genus with four species dis-junctly distributed between eastern Asia and North America (Ter-rell et al., 1997). In North America, two annual species Z. palustrisL. (with var. palustris and var. interior (Fassett) Dore) and Z. aquaticaL. (with var. aquatica and var. brevis Fassett), are widespread in theGreat Lake region and along the Atlantic coastal plains, respec-tively, and have some overlapping ranges (Aiken et al., 1988; Ter-rell et al., 1997). The perennial Z. texana Hitchcock is restricted to a2.4 km area of the upper San Marcos River in southcentral Texas. Itis an endangered species geographically isolated from all otherZizania taxa by at least 640 km (Terrell et al., 1978). Zizania latifolia(Griseb.) Turcz. ex Stapf is a perennial widely distributed in easternAsia (Wu et al., 2006). Of the four species, two are economicallyimportant as field crops. Zizania palustris has served as a traditionalstaple for native Americans for centuries (Johnson, 1969) and as aspecialty commercial crop more recently (Hayes et al., 1989; Oelke,1993). Zizania latifolia was once used as an important grain in

X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017 1009

ancient China and has been cultivated as an aquatic vegetable be-cause the young shoots become swollen, soft and edible after beinginfected by the fungus Ustilago esculenta P. Henn. (Thrower andChan, 1980; Zhai et al., 2001; Guo et al., 2007).

Previous phylogenetic studies of the rice tribe have supportedthe placement of Zizania in Oryzeae (Zhang and Second, 1989; Duv-all et al., 1993; Ge et al., 2002; Guo and Ge, 2005; Tang et al., 2010).Zizania is most closely related to the South American genusRhynchoryza Baill. At the infrageneric level, the Asian Z. latifolia iswell differentiated from the North American species by its chromo-some number and morphology (Duvall, 1987; Terrell et al., 1997),whereas the relationships among three North American species arenot fully resolved. Because of the overlap in distributional rangesand the existence of morphological intermediates, the two annualspecies, Z. palustris and Z. aquatica, were once treated as a singlespecies (Fassett, 1924; Hitchcock and Chase, 1951). Later studiesbased on spikelet anatomy (Duvall and Biesboer, 1988a), artificialhybridization (Duvall and Biesboer, 1988b), and isozyme patterns(Warwick and Aiken, 1986) supported the recognition of two an-nual species. A close relationship between Z. texana and Z. aquaticais supported by the isoelectric focusing (IEF) profiles of seed pro-tein (Duvall and Biesboer, 1989), whereas crossing behaviors sug-gest that Z. texana is more closely related to Z. palustris (Duvall,1987). The latter relationship is supported by Horne and Kahn(1997) based on isozyme and nuclear ribosomal ITS sequence anal-ysis. Nevertheless the hypothesis needs to be further tested be-cause Horne and Kahn (1997) sampled only 3–4 individuals froma single population of each species and they used the distantly re-lated Oryza sativa L. as the outgroup. The studies by Horne andKahn (1997) and Xu et al. (2008) only examined the North Ameri-can or the eastern Asian species of Zizania, respectively.

The objectives of this study are to (1) construct the phylogenyto resolve the interspecific relationships in Zizania, (2) estimatethe divergence times between intercontinental species and amongintracontinental species, and (3) reconstruct the biogeographic his-tory of Zizania between eastern Asia and North America.

2. Materials and methods

2.1. Plant materials

Nineteen populations of North American Zizania were collectedin the USA and Canada (Table 1). All species and varieties were rep-resented in our sampling: six populations of Z. palustris var. palus-tris (PWIA, PWIB, PWIC, PWID, PVTA, and PVTB), one Z. palustrisvar. interior (PMN), eight populations of Z. aquatica var. aquatica(AWIA, AWIB, ASC, AMD, ADL, ANJ, AVTA, and AVTB), three Z. aqu-atica var. brevis (AQCA, AQCB, and AQCC), and one Z. texana (TTX)from the only known locality of the taxon. Young and healthyleaves were collected in the field and dried with silica gel for sub-sequent DNA extraction. Voucher specimens from each populationwere deposited in the United States National Herbarium (US). Pop-ulations of Z. latifolia were described in Xu et al. (2008). Four clo-sely related species Rhynchoryza subulata (Nees) Baill., Zizaniopsismiliacea (Michx.) Doell & Aschers, Luziola peruviana Juss. ex J.F.Gmel. and Hygroryza aristata (Retz.) Nees were included as out-groups based on the recent phylogenetic study of the rice tribe(Tang et al., 2010).

2.2. DNA extractions, amplification, and sequencing

Total genomic DNA was extracted from silica-dried leaves usingthe DNeasy Plant Mini Kits (Qiagen). Polymerase chain reaction(PCR) amplifications were performed using 10–30 ng of genomicDNA, 5 pmol of each primer, 0.2 mM of each dNTP, 2 mM MgCl2,

and 0.6 U GoTaq DNA polymerase (Promega) in a volume of25 lL under the following conditions: 3 min at 95 �C, followed by35 cycles of 30 s at 94 �C, 30 s at 50–55 �C, and 90 s at 72 �C, andthen a final 5 min extension at 72 �C. Amplifications were carriedout in a PTC-225 Peltier Thermal Cycler.

The amplification and sequencing primers of the organellar re-gions were from the following sources: (1) the atpB-rbcL spacer(Manen et al., 1994); (2) the trnH-psbA spacer (Hamilton, 1999);(3) the trnL-F spacer (primers ‘‘c” and ‘‘f” of Taberlet et al., 1991);(4) the rps16 intron (Oxelman et al., 1997); (5) the matK gene(Ge et al., 1999); and (6) the nad1 intron 2 (Demesure et al.,1995; Guo and Ge, 2005). Two individuals from each populationwere sequenced for the organellar regions and additional individ-uals from sympatric populations and the Texas population wereused. The nuclear Adh1a gene was amplified and sequenced withthe Zizania-specific primers (Adh1aF1: 50-CTGACAGAGGTG-TAATGCTTA-30, Adh1aF2: 50-TCGGGACTTCGACCTTCAGT-30, andAdh1aR, Xu et al., 2008) from six individuals of each populationof the annual species and 30 individuals of Zizania texana. The re-gion of the outgroups was amplified and sequenced with primersAdh1F6 and Adh1R7 (Zhang and Ge, 2007).

All PCR products were purified using the polyethylene glycol(PEG)/NaCl method of Kusukawa et al. (1990). Purified PCR prod-ucts were sequenced using the BigDye Terminator Cycle Sequenc-ing Ready Reaction kit (Applied Biosystems). Sequencing reactionswere purified by gel filtration chromatography using Sephadex col-umns (Amersham Pharmacia Biotech) and run on an ABI 3730xlDNA analyzer (Applied Biosystems). The program Sequencher 4.5(GeneCodes Corporation) was used to evaluate chromatogramsfor base confirmation and to edit contiguous sequences. Individu-als in Zizania can be either homozygous or heterozygous at theAdh1a locus. For a heterozygote, sequences of two alleles can notbe separated in the chromatogram when multi-point mutationsor length differences caused by insertions or deletions exist be-tween the two alleles. In such cases, purified PCR products werecloned using the TOPO TA cloning kit (Invitrogen), and then thetwo alleles were determined separately by sequencing multipleclones. All sequences of different haplotypes were deposited intothe GenBank (Accession Nos. GU177208–GU177454).

2.3. Data analyses

All sequences were aligned using the program Mafft 6.7 (Katohet al., 2005) using the L-INS-i algorithm with ‘‘maxiterate” set to1000. We combined atpB-rbcL, matK, rps16, trnL-F, trnH-psbA, andnad1 data for phylogenetic analyses, collapsing all individuals withthe same haplotype to a single representative. Congruence be-tween the chloroplast and the mitochondrial data was examinedusing the incongruence length difference (ILD) test (Farris et al.,1994) implemented in PAUP� 4.0b10 (Swofford, 2003). This testemployed 100 replicates, each with 10 random sequence additions,and the resulting P value was used to determine whether the twodatasets had significant incongruence (0.05).

Maximum parsimony (MP) searches were performed with 1000random taxon addition replicates followed by tree bisection-recon-nection branch swapping in PAUP� 4.0b10. Gaps were treated asmissing data. Parsimony bootstrap (PB) for the clades was exam-ined with 1000 bootstrap replicates using the same options asabove. Maximum likelihood (ML) analysis was implemented inGARLI 0.951 (Zwickl, 2006) starting from random trees and using10,000,000 generations per search. The ML bootstrap (LB) supportwas estimated from 100 bootstrap replicates in GARLI. For the MLanalysis the K81uf + I substitution model was identified underAkaike information criterion (AIC) implemented in Modeltest 3.7(Posada and Crandall, 1998). Bayesian inference (BI) was imple-mented in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). A

Table 1Voucher information of 19 Zizania populations from North America. Frequencies of the nuclear Adh1a haplotypes (1–63) are also indicated. All voucher specimens are deposited atthe US National Herbarium (US) of the Smithsonian Institution.

Population Species Collection voucher n Location Coordinate (N/W) Haplotype of Adh1a

PWIA Zizania palustris var. palustris J. Wen 9918 6 Taylor Co., WI 45�190510 0/90�260440 0

44, 54(3), 55(4), 56(2), 57, 58

PWIB Zizania palustris var. palustris J. Wen 9936 6 Polk Co., WI 45�230440 0/92�120490 0

45, 49(2), 59(6), 60, 61, 62

PWIC Zizania palustris var. palustris J. Wen 9937 6 Polk Co., WI 45�290500 0/92�290220 0

59(12)

PWID Zizania palustris var. palustris J. Wen 9938 6 Douglas Co., WI 46�260470 0/92�090540 0

49(9), 59(3)

PVTA Zizania palustris var. palustris Xu et al. 106 6 Addison Co., VT 43�480420 0/73�190100 0

51(12)

PVTB Zizania palustris var. palustris Xu et al. 108A 6 Addison Co., VT 44�130350 0/73�160390 0

36(10), 52, 53

PMN Zizania palustris var. interior J. Wen 9963 6 Houston Co., MN 43�490130 0/91�160470 0

43(3), 45(3), 46, 47(2), 48, 49, 50

AWIA Zizania aquatica var. aquatica J. Wen 9912 6 Portage Co., WI 44�310290 0/89�350500 0

1(6), 7(3), 22, 41, 42

AWIB Zizania aquatica var. aquatica J. Wen 9914 and 9915 6 Wood Co., WI 44�200510 0/89�580050 0 7(12)ASC Zizania aquatica var. aquatica J. Wen 10018 6 Berkeley Co., SC 33�110460 0/

79�570120 032(7), 33(2), 34(3)

AMD Zizania aquatica var. aquatica J. Wen 10401 6 Prince George Co., MD 38�470050 0/76�420120 0

1(2), 5, 6, 7(2), 8, 9(2), 10, 11, 12

ADL Zizania aquatica var. aquatica J. Wen 10408 6 Sussex Co., DL 38�330500 0/75�400160 0 1(6), 2(4), 3, 4ANJ Zizania aquatica var. aquatica Xu et al. 101 6 Atlantic Co., NJ 39�360270 0/

74�350300 01, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23

AVTA Zizania aquatica var. aquatica Xu et al. 108B 6 Addison Co., VT 44�130350 0/73�160390 0

7(2), 27(2), 35, 36(2), 37, 38, 39, 40(2)

AVTB Zizania aquatica var. aquatica Xu et al. 109 6 Chittenden Co., VT 44�370330 0/73�140220 0

7(8), 40(4)

AQCA Zizania aquatica var. brevis J. Wen 10435 6 Montmagny Co., QC 46�560090 0/70�440030 0 24, 25(8), 26, 27(2)AQCB Zizania aquatica var. brevis J. Wen 10436 6 Montmagny Co., QC 47�010520 0/70�280110 0 25(2), 27(7), 28, 29, 30AQCC Zizania aquatica var. brevis J. Wen 10437 6 L’Islet Co., QC 47�100480 0/70�180140 0 25(2), 27(7), 29(2), 31TTX Zizania texana Texas WR 30 Hays Co., TX 63(60)

1010 X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017

mixed model Bayesian analysis assigned model parameters foreach gene partitions identified by AIC in Modeltest 3.7 (Table 2).Two independent runs of Metropolis-coupled Markov chain(MCMC) analysis were conducted simultaneously, with each runhaving one cold chain and three incrementally heated chains andall started randomly in the parameters space. Four million genera-tions were run and every 100 generations were sampled with thefirst 25% of samples discarded as burn-in. Tracer 1.4 (Rambautand Drummond, 2004) was used to check whether the chains wereconverged. The remaining trees were sampled from the posteriordistribution to calculate the posterior probability (PP).

For the nuclear Adh1a gene, we also performed MP, ML, and BIanalyses using non-redundant haplotypes only. The same optionsas described above were used except that the best-fit model forAdh1a gene was TrN + I + G and 10 million generations were runin the BI analysis. Furthermore, a haplotype network of Adh1a se-quences was constructed using the Median-Joining model imple-

Table 2Characteristics of six chloroplast and mitochondrial regions and nuclear Adh1a gene(including outgroups).

Alignedlength(bp)

Number ofvariable sites

Number ofinformativesites

Modelselected byAIC

atpB-rbcL 757 52 19 HKY + GmatK 1536 118 58 TVM + GtrnH-psbA 568 23 14 HKY + Irps16 820 53 15 HKYtrnL-F 975 53 18 K81uf + Inad1 1510 31 15 TVMCombined

organellar data6166 330 139 K81uf + I

Adh1a 1508 290 195 TrN + I + G

mented in NETWORK 4.5 (Bandelt et al., 1999). In this analysis,indels were treated as single mutation events and coded as substi-tutions (A or T). Genetic distances among species were calculatedemploying Tamura and Nei’s (1993) distance using MEGA 4 (Tam-ura et al., 2007).

We used the combined atpB-rbcL, matK, rps16, trnL-F, and nad1data to estimate the divergence time of Zizania between easternAsia and North America with 29 taxa sampled from the Oryzeae.Sequences of 21 species were obtained from GenBank. Some taxawere coded with missing data in the trnL-F region because only se-quences of the trnL region were available. A likelihood ratio testwas carried out to determine if there was a significant differencein evolutionary rates among lineages (Felsenstein, 1988). Thehypothesis of a molecular clock was rejected (P < 0.05). Diver-gence-time estimation was performed using a Bayesian methodas implemented in BEAST 1.4.7 (Drummond and Rambaut, 2007).We used the GTR model of nucleotide substitution with a gammadistribution with four rate categories, under an uncorrelated log-normal relaxed clock model (Drummond et al., 2006). Posteriordistributions of parameters were approximated using two inde-pendent MCMC analyses of 20,000,000 generations with 10%burn-in. Results were checked using Tracer 1.4 (Rambaut andDrummond, 2004) to ensure plots of two analyses were convergingon the same area and then combined. Two calibration points wereused to estimate the divergence time within Zizania: 34.5 ± 6.8million years ago (mya) for the stem node of Oryzeae, and 7 myaas the minimum age for the stem node of Leersia. The first calibra-tion point was obtained from Vicentini et al. (2008). They esti-mated the divergence times of major grass lineages using sixfossils as calibration points and several calibration schemes. Thereare two reported macrofossil records for Oryzeae. One is the fertilelemmas and paleas (anthoecia) of Archaeoleersia nebraskensisThomasson preserved as silicifications from the late Miocene in

X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017 1011

the Ash Hollow Formation of the Ogallala Group in Nebraska. Thisfossil compares most closely to the anthoecia of living Leersia ligu-laris Trin. (Thomasson, 1980). The other fossil of the tribe was thespikelets found in a Miocene excavation in Germany and was iden-tified as Oryza exasperate (A. Braun) Heer (Heer, 1855). Marshallet al. (1979) indicated that the top of the Ash Hollow Formationin Nebraska is slightly younger than 7 mya. Tang et al. (2010) used5 mya as minimum age constraint for the Oryza fossil. We thusconstrained the split of Leersia and Oryza with the minimum ageof 7 mya based on the Archaeoleersia record.

5 changes

9

10

6

--

6

Z. latifoliaZ. latifol

Z

Hygroryza aristata

Rhynchoryza subulata

100/100/1.00

81/74/0.89

94/100/1.00

100/100/1.00

Fig. 1. The ML tree of Zizania inferred from combined atpB-rbcL, matK, trnL-F, trnH-psbA,ML analysis and Bayesian posterior probabilities. The population codes are indicated in

The biogeographic range evolution of Zizania and its close rela-tives was inferred using dispersal-vicariance analysis (DIVA; Ron-quist, 1997) and a maximum likelihood based method,LAGRANGE (Ree et al., 2005; Ree and Smith, 2008). The analyseswere conducted on a fully resolved topology for Zizania and itsclose relatives with nodal ages, obtained from Fig. 2 in Tang et al.(2010). Three areas of endemism were defined according to theirdistribution: (A) Asia, (B) North America, and (C) South America.The maximum number of areas in ancestral ranges was set to betwo in both analyses. The A–C area was disallowed in the

Z. texana_TTX2,13,23,27

Zizania palustris var. palustris_PVTB2

Z. palustris var. palustris_PWIA1,3

Z. palustris var. palustris_PWIA2

Z. palustris var. palustris_PWIA6; C1

Z. palustris var. palustris_PWIB1,6

Z. palustris var. interior_PMN3

Z. palustris var. palustris_PVTA1,4Z. palustris var. palustris_PVTB1Z. palustris var. palustris_PVTB6

Z. aquatica var. aquatica_AVTA1,3,5,6; B3,4Z. aquatica var. aquatica_AVTB1

Z. palustris var. interior_PMN1

Z. palustris var. palustris_PWID1,6

Z. aquatica var. aquatica_AVTA2,B6Z. aquatica var. aquatica_AVTA4Z. aquatica var. brevis_AQCC6

Z. aquatica var. aquatica_AWIA1,3; B1,4 &Z. aquatica var. brevis_AQCA1,2; B1,2; C2

Z. aquatica var. aquatica_AMD5

Z. aquatica var. aquatica_ADL1,6; NJ5Z. aquatica var. brevis_AQCC1

Z. aquatica var. aquatica_AMD2Z. aquatica var. aquatica_ASC3,5

Z. aquatica var. aquatica_ANJ1

81/84/1.00

64/70/0.89

3/96/1.00

0/100/1.00

5/60/1.00

/58/1.00

1/73/1.00

_YNia_HLJ101,BJ10,SD210,ZJ01

izaniopsis miliaceaLuziola peruviana

rps16, and nad1 data. Numbers at nodes are bootstrap values obtained from MP andTable 1.

Hap 36Hap 52Hap 43Hap 56Hap 54Hap 58Hap 57

Hap 46Hap 47

Hap 50Hap 55Hap 10Hap 53

Hap 61Hap 49Hap 51Hap 48Hap 62

Hap 60Hap 59

Hap 63Hap 45Hap 27Hap 44

Hap 35Hap 40

Hap 24Hap 29

Hap 26Hap 30

Hap 5Hap 33Hap 4

Hap 28Hap 39

Hap 18Hap 20Hap 1Hap 8

Hap 23Hap 32

Hap 9Hap 6

Hap 3Hap 34

Hap 31Hap 25

Hap 19Hap 17

Hap 14Hap 15Hap 13

Hap 21Hap 22

Hap 12Hap 16

Hap 2Hap 7Hap 41Hap 42Hap 11Hap 37Hap 38

Hap BHap AHap I

Hap DHap HHap CHap Ehap JHap FHap G

5 changes

Rhynchoryza subulataHygroryza aristata

Luziola peruvianaZizaniposis miliacea

Zizania latifolia

Zizania aquatica

Zizania palustris

Zizania texana

100/100/1.00

100/100/1.00

100/100/1.00

98/94/0.99100/100/1.00

99/99/1.00100/100/1.00

86/92/0.99

--/53/0.97

60/60/0.99

54/54/0.95

77/87/1.00

61/84/1.0051/66/--

85/89/1.00

65/61/0.98

--/--/0.99--/--/0.97

--/--/0.99

--/--/0.99

--/--/0.97

Fig. 2. The ML tree of Zizania inferred from the nuclear Adh1a data. Numbers at nodes are bootstrap values obtained from MP and ML analyses and Bayesian posteriorprobabilities.

1012 X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017

LAGRANGE analysis because it required prior extinction in theirintervening area.

3. Results

3.1. Phylogenetic and haplotype network analysis

The statistics of the chloroplast atpB-rbcL, matK, trnL-F, trnH-psbA, and rps16 and mitochondrial nad1 regions are shown in Ta-ble 2. A partition homogeneity test suggested that the chloroplastand the mitochondrial data sets were congruent (P = 0.354). Thus

phylogenetic analyses were performed on the combined organellardata. A single most parsimonious tree was generated with a lengthof 365 steps, a consistency index (CI) of 0.94, a CI excluding unin-formative characters of 0.87, and a retention index (RI) of 0.95. Thetrees in ML and Bayesian analyses were the same in topology as thesingle MP tree. The ML tree is presented in Fig. 1 along with boot-strap support values from MP and ML analyses and posterior prob-abilities from Bayesian analysis. The monophyly of Zizania wasstrongly supported by all analyses (PB = 100%, LB = 100%,PP = 1.00). Among the multiple populations of Zizania, two distinctclades were resolved with robust support, corresponding to the

Table 3Pairwise comparisons of genetic distances with the Tamura–Nei model among threeNorth American Zizania species based on the nuclear Adh1a data.

Z. aquatica Z. palustris Z. texana

Z. aquatica 0.0116 ± 0.0023a

Z. palustris 0.0116 ± 0.0023 0.0038 ± 0.0011b

Z. texana 0.0127 ± 0.0028 0.0084 ± 0.0025

a Distance between the two varieties of Z. aquatica (var. aquatica and var. brevis).b Distance between the two varieties of Z. palustris (var. palustris and var. interior).

X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017 1013

Asian clade of Z. latifolia (PB = 94%, LB = 100%, PP = 1.00) and theNorth American clade of Z. aquatica, Z. palustris, and Z. texana(PB = 100%, LB = 100%, PP = 1.00). In the North American clade,populations of the three species grouped into two separate clades.One clade included only populations of Z. aquatica with high sup-port values (PB = 81%, LB = 84%, PP = 1.00); and the other clade in-cluded all samples of Z. palustris and Z. texana, and seven samplesfrom two populations of Z. aquatica with low support values(PB = 64%, LB = 70%, PP = 1.00). Populations of Z. aquatica appar-ently did not form a monophyletic group (Fig. 1). Moreover, Zizaniapalustris and Z. texana were supported to be very closely related(Fig. 1). The Z. texana population only differed from all populationsof Z. palustris by no more than 7 mutations in the 5987 bp ofaligned nucleotide positions of the combined atpB-rbcL, matK,trnL-F, trnH-psbA, rps16, and nad1 data, whereas it differed fromtwo populations of Z. aquatica in the same clade by a minimumof eight mutations (indels treated as single mutation events).

The ML tree from the nuclear Adh1a gene is shown in Fig. 2. TheML tree, the MP consensus tree and the Bayesian tree only hadminor differences, and the support values of major clades in bothML and MP analyses were congruent with posterior probabilitiesfrom Bayesian analysis. The monophyly of Zizania and the two dis-tinct clades corresponding to eastern Asia and North America withstrong support were recovered, as in the combined organellar data(Figs. 1 and 2). The North American clade was divided into twoclades with weak support: one consisting of haplotypes exclusivelyfrom Z. aquatica, and the other including all haplotypes from Z.palustris and Z. texana and 14 haplotypes from Z. aquatica (Fig. 2).To clarify the relationship of North American wild-rice, a haplotypenetwork was constructed using the Median-Joining method. The63 haplotypes of North American wild-rice were split into threegroups (A, P, and T in Fig. 3) corresponding to the three species,Z. aquatica, Z. palustris, and Z. texana (Fig. 3). Group A consistedof 40 haplotypes from Z. aquatica; group T included the single hap-lotype (63) from Z. texana; and group P included 22 haplotypes, ofwhich 20 were unique to Z. palustris, one (10) was unique to Z. aqu-atica, and one haplotype (36) was shared between Z. palustris and Z.aquatica (Fig. 3). Haplotype 45 from Z. palustris was closest to thesingle haplotype 63 from Z. texana. Within group A, haplotypesof the two varieties of Z. aquatica (var. aquatica and var. brevis) eachdid not form a monophyletic group, and one haplotype (27) wasshared between them. Within group P, haplotypes of the two vari-eties of Z. palustris (var. palustris and var. interior) were each notmonophyletic, and two haplotypes (45 and 49) were shared be-tween them (Fig. 3). The genetic distance between Z. texana andZ. aquatica (0.0127 ± 0.0028) was higher than that between Z. tex-

Fig. 3. The Median-Joining haplotype network of the nuclear Adh1a fragment in North Aproportional to their relative frequency. Black dots and crossed bars represent putativegroups are defined corresponding to the three species with A referring to Z. aquatica, P

ana and Z. palustris (0.0084 ± 0.0025), and a higher infraspecific ge-netic distance was present in Z. aquatica (Table 3).

3.2. Biogeographic analysis

The chronogram and results of divergence-time estimationbased on the combined data of atpB-rbcL, matK, rps16, trnL-F, andnad1 from the Bayesian approach are shown in Fig. 4. The disjunc-tion of Zizania between eastern Asia and North America was esti-mated at 3.74 mya (with a 95% highest posterior density [HPD]interval of 1.04–7.23 mya), which yields time estimates in the lateTertiary. The age of the crown North American Zizania was esti-mated at 0.71 mya (95% HPD: 0.12–1.54 mya).

Both DIVA and LAGRANGE analyses suggested that the ancestralarea of the Zizania node was Asia and North America (Fig. 5). How-ever, the DIVA results conflicted with those of LAGRANGE at somenodes. For the ancestral range of the Zizania-Rhynchoryza node,North America was supported by LAGRANGE, whereas both Asiaand South America were inferred by DIVA (Fig. 5).

4. Discussion

4.1. Phylogenetic relationships

Within Zizania, the eastern Asian Z. latifolia is sister to the cladeof North American species consisting of Z. aquatica, Z. palustris, andZ. texana. This relationship is strongly supported by both organellarand nuclear data (Figs. 1 and 2). The early divergence of Z. latifoliawithin the genus was supported by a suite of morphological andcytological characters. First, the Asian species has chromosomenumber n = 17, whereas all the North American Zizania taxa haven = 15 (Brown, 1950; Dore, 1969; Chen et al., 1990). Second, thepanicle of Z. latifolia possesses mixed branches bearing both stami-nate and pistillate spikelets, whereas the North American specieshave a panicle with the lower branches bearing male spikelets

merican Zizania. Circles and squares represent different haplotypes (1–63) with sizehaplotypes. Each line connecting haplotypes represents one mutational step. Threelargely to Z. palustris (with exceptions of haplotypes 10 and 36), and T to Z. texana.

MIOCENE

QU

ATER-

NAR

Y

PLIO-CENEOLIGOCENEEOCENE

E M L E LE LM L

5101520 25303540

Zizania palustris

Potamophila parviflora

Leersia oryzoides

Leersia perrieri

Hygroryza aristata

Oryza rhizomatis

Oryza officinalis

Oryza punctata

Zizaniopsis villanensis

Phyllostachys aurea

Oryza granulata

Oryza sativa

Leersia hexandra

Leersia tisserantti

Oryza glaberrima

Chikusichloa aquatica

Zizania latifolia

Luziola leiocarpa

Oryza brachyantha

Zizania texana

Oryza meridionalis

Oryza coarctata

Luziola peruviana

Oryza australiensis

Zizaniopsis miliacea

Ehrharta erecta

Rhynchoryza subulata

Luziola fluitans

Zizania aquatica12

3.74mya (95%HPD:1.04-7.23)

0.71mya (95%HPD:0.12-1.54)

0 mya

Fig. 4. Chronogram of Oryzeae inferred from combined atpB-rbcL, matK, trnL-F, rps16, and nad1 data using BEAST. Clade constraints are indicated with blank asterisks. Grayboxes indicate 95% highest posterior density intervals. Node indicated by number 1 is the disjunction between eastern Asian Zizania and North American Zizania. Nodeindicated by number 2 is the divergence among North American Zizania.

Zizania palustris

Zizania aquatica

Zizania latifolia

Rhynchoryza

Hygroryza

Luziola

Zizaniopsis

B

B

A/B

B

B

C

A

BC

BC

A

BAB

ACB

B CB

BA

AB AC

Fig. 5. Ancestral area reconstruction for Zizania and its close relatives using DIVAand LAGRANGE. The tree was obtained from Tang et al. (2010) with branch lengthsproportionate to time. Three areas of endemism were defined according to theirdistribution: (A) Asia, (B) North America and (C) South America. The letters abovebranches and below branches are referred from DIVA and LAGRANGE, respectively.The ancestral areas inferred from LAGRANGE are the ones with the highestlikelihood scores and the highest probabilities among the alternatives.

1014 X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017

and the upper branches bearing female spikelets (Fassett, 1924;Terrell et al., 1997; Wu et al., 2006). Third, the pistillate lemmasof Z. latifolia have numerous hairs and siliceous papillae and pits,whereas hairs and siliceous papillae and pits are absent or nearlyso in the pistillate lemmas of all North American Zizania species(Terrell and Wergin, 1981).

Among the three North American wild-rice species, the geo-graphically isolated Z. texana is supported to be most closely relatedto Z. palustris based on our organellar and nuclear Adh1a data (Figs. 1and 3). The analysis by Horne and Kahn (1997) using ITS sequencesalso suggested a close relationship between the perennial Z. texanaand the annual Z. palustris. This relationship is consistent to the factthat Z. texana can hybridize only with Z. palustris var. interior underartificial conditions (Duvall, 1987). The interspecific genetic dis-tance with the Tamura–Nei model between these two species issmall, ranging from 0.006 to 0.01 (Table 3). Zizania texana has beenrecognized as a highly distinct species based on its habitat in deeprunning water, perennial life history, distinct morphology withstoniferous rhizomes and geniculate culms, and geographicallyisolated distribution (Terrell et al., 1978).

X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017 1015

Zizania aquatica and Z. palustris were each not monophyletic inboth the organellar and the nuclear phylogenies (Figs. 1 and 2).This pattern of non-monophyly was probably due to incompletelineage sorting, not introgression, based on several lines of evi-dence. First, introgression (i.e., chloroplast capture) often resultsin the discordance of nuclear and chloroplast phylogenies (Riese-berg and Soltis, 1991; Soltis and Kuzoff, 1995), whereas our organ-ellar and nuclear trees had similar topologies (see Figs. 1 and 2).Both the organellar and the nuclear trees had two major clades,with one including samples from Z. aquatica only and the otherconsisting of samples from both species (Figs. 1 and 2). Second, re-cently introgressions are expected to be presented in both speciesmore commonly in the areas near contact zones or sympatric local-ities (Barbujani et al., 1994), whereas this pattern did not appear inour organellar and nuclear data (except haplotype 36). The haplo-types of Z. aquatica grouped into the mixed clades were not closelyrelated to haplotypes of Z. palustris from the sympatric localities.Therefore, we prefer incomplete lineage sorting to explain thenon-monophyly of the two annual wild-rice species.

Introgression, however, may have been supported in the sym-patric populations of Z. aquatica and Z. palustris in northwest Ver-mont, USA. It is noted that Adh1a haplotype 36 was shared by Z.aquatica and Z. palustris and nested within group P (Fig. 3). More-over, it was only present in two sympatric populations (AVTAand PVTB from northwest Vermont, Table 1). This phylogeographicpattern is seemingly in favor of introgression rather than incom-plete lineage sorting. Furthermore, only two haplotypes (10 and36) of Z. aquatica were nested within group P of Z. palustris, butnone of the haplotypes of Z. palustris was nested within group Aof Z. aquatica (Fig. 3), suggesting possible unidirectional introgres-sion from Z. palustris to Z. aquatica. This is consistent with the factthat a unilateral interspecific crossability barrier exists between Z.aquatica and Z. palustris. Artificial hybridization demonstrated thatinterspecific hybrids were produced only when Z. aquatica was theovulate parent (Duvall and Biesboer, 1988b). Unidirectional intro-gression was also suggested by the seed protein IEF data (Duvalland Biesboer, 1989). We plan to expand our study to sample acrossthe distribution ranges of both annual species with special empha-sis on the zones of sympatry to test the introgression hypothesisbetween Z. aquatica and Z. palustris.

At the infraspecific level, the two varieties of Z. aquatica (var.aquatica and var. brevis) are geographically separated by a distanceof ca. 130 km, and are morphologically easily distinguishable bytheir plant height and the length of their lemma awns (see Darby-shire and Aiken, 1986; Aiken et al., 1988). The two varieties of Z.palustris, on the other hand, overlap in their distribution and some-times it is difficult to separate var. palustris from var. interior mor-phologically (Terrell et al., 1997). The genetic distance betweenvar. aquatica and var. brevis is three times higher than that be-tween var. palustris and var. interior based on the nuclear Adh1adata (Table 3). Only one of 42 haplotypes in Z. aquatica was sharedbetween var. aquatica and var. brevis, whereas two of 21 haplo-types in Z. palustris were shared between var. palustris and var.interior (Fig. 3). The shared haplotypes and non-monophyly of eachvariety in both species (Figs. 1 and 3) indicated recent divergenceand/or recurrent gene flow at the infraspecific level in the NorthAmerican wild-rice, supporting their recognition at the variety le-vel taxonomically.

4.2. Biogeographic diversification of Zizania

The Bayesian dating using the combined atpB-rbcL, matK, rps16,trnL-F, and nad1 sequence data suggests the divergence time ofZizania between eastern Asia and North America to be 3.74 (95%HPD: 1.04–7.23) mya in the late Tertiary. Palynological evidenceindicated that temperate taxa became important elements in Alas-

ka and northwestern Canada area after 7 mya associated with theglobal climatic cooling in the late Tertiary (White et al., 1997; Gra-ham, 1999). The Bering land bridge was suitable for exchanges oftemperate deciduous plants and remained available for floristic ex-changes until about 3.5 mya (Wen, 1999). Thus dispersal via theBering land bridge seems to be the most plausible explanationfor Zizania. Similar divergence times have also been reported inother temperate aquatic and wetland intercontinental disjunctplants with an eastern Asian–North American distribution, suchas Acorus L. (A. calamus L. and A. americanus (Raf.) Raf., Tian et al.,in press), Lysichiton Schott (L. camtschatcensis (L.) Schott and L.americanus Hulten & H. St. John, Nie et al., 2006b), and Symplocar-pus R.A. Salisb. ex Nutt. (S. foetidus (L.) Salisb. ex W. Barton and S.renifolius Schott ex Tzvelev, Nie et al., 2006b). These results suggestthat dispersal via the Bering land bridge in the late Tertiary may beimportant for the migration of wetland plants between easternAsia and North America.

Our DIVA and LAGRANGE results suggested Asia and NorthAmerica as the ancestral area of Zizania. A widespread Zizaniaancestor between Asia and North America is thus preferred bythe analyses. Using the phylogenetic framework of the rice tribeby Tang et al. (2010), the ancestral area of the Zizania–Rhynchoryzaclade was detected to be Asia and South America in DIVA, andNorth America in LAGRANGE (Fig. 5). This conflict may be attrib-uted to the parsimony criterion not considering branch lengthsor time, and only vicariance being assigned for subdivision of wide-spread ancestors in DIVA (Clark et al., 2008). The LAGRANGE anal-yses incorporate geological information and allow priorhypotheses for range and dispersal constraints, and infer the rangeevolution in a likelihood framework (Ree and Smith, 2008). TheDIVA results of the ancestor of Zizania and Rhynchoryza in both Asiaand South America seem to be unreasonable because no fossil spe-cies occurred in their intervening area North America, and long-distance dispersal between Asian and South America seems muchless likely. We prefer the hypothesis that Zizania originated fromits ancestor in North America and then dispersed from NorthAmerica to Asia based on the LAGRANGE results. A recent phylog-eographic analysis of the eastern Asian Z. latifolia showed that theall three clades of Adh1a haplotypes occurred in northeastern Chi-na while two clades and one clade occurred in eastern and central/southern China, respectively (Xu et al., 2008). The haplotype occur-rence in Z. latifolia suggested that this species colonized in thesouthward direction from the northeast part of eastern Asia andachieved its wide distribution across the entire eastern Asia. Thephylogeographic structure of Z. latifolia thus supports the hypoth-esis that Zizania migrated from the New World to the Old Worldvia the Bering land bridge and then migrated/dispersed south-wardly in eastern Asia.

Bayesian dating estimated the divergence of Zizania aquaticafrom other North American Zizania to be 0.71 (95% HPD: 0.12–1.54) mya, suggesting that the North American Zizania taxa beganto diverge in the middle Pleistocene. The intracontinental diversi-fication in North America was thus relatively recent, consistentwith the electrophoretic seed protein profiles data (Duvall andBiesboer, 1989). These workers detected no taxon-specific bandingpatterns in North American Zizania. Furthermore, in a survey for 11enzyme systems in 33 populations of the two annual species inNorth America, only four enzymes are species specific (Warwickand Aiken, 1986), showing a low level of genetic divergence be-tween the two species. Future phylogeographic study with abroader sampling scheme should be useful and are planned to fur-ther analyze the population history and diversification of NorthAmerican Zizania.

The perennial Zizania texana differs from the two annual NorthAmerican species in several morphological characters including itsprostrate, submerged habit and geniculate culms that root at the

1016 X. Xu et al. / Molecular Phylogenetics and Evolution 55 (2010) 1008–1017

basal nodes (Duvall, 1987; Terrell et al., 1997). Yet our results sug-gested that it diverged recently from the widespread North Amer-ican Z. palustris (Fig. 4). The unusual morphology was noted to benot maintained in the common garden (Duvall, 1987, p. 77), andmay be predominantly determined by the unique habitat in theSan Marcos River of Texas where the only population of Z. texanainhabits. It is also notable that no polymorphism was detected inZ. texana in the nuclear Adh1a gene (Table 1 and Fig. 3), eventhough our samples of the Adh1a haplotype network covered allthe stands of Z. texana in the San Marcos River (from river sectionsA to K, see figure in Richards et al., 2007). The lack of Adh1a genediversity may be due to the small population size within a narrowgeographic range of Z. texana. This species was presumed to be pre-dominantly asexual because monitoring of the stands in the riversince the 1960s revealed little or no flowering (Emery, 1977;Power, 1996). However, high heterozygosity and few duplicategenotypes were detected in Z. texana using microsatellite makers,suggesting that sexual reproduction occurs more often (Richardset al., 2007). The difference in the level of genetic diversity revealedby the Adh1a gene in our study and microsatellite markers (Rich-ards et al., 2007) is most likely due to the much higher mutationrates in microsatellite DNA.

5. Conclusions

Our molecular data support the monophyly of the wild-ricegenus Zizania. Biogeographic analyses suggest that the genus mostlikely originated in North America and then dispersed into easternAsia via the Bering land bridge. The North American Zizania speciesformed a clade, which is sister to the eastern Asian Z. latifolia. Thedivergence of the intercontinental disjunction in Zizania is esti-mated to be in the late Tertiary. The divergence of modern NorthAmerican Zizania species occurred recently in the Pleistocene.Incomplete lineage sorting is responsible for the non-monophylyof the two widespread species Z. palustris and Z. aquatica, whereaslow-frequency unidirectional introgression of Z. palustris into Z.aquatica has occurred as well. A close relationship between thenarrowly distributed endangered species Z. texana and Z. palustrisis supported in this study.

Acknowledgments

This study was supported by grants from the John D. and Cath-erine T. MacArthur Foundation (to J. Wen), the Chinese Academy ofSciences (to J. Wen and S. Ge) and the National Natural ScienceFoundation of China (to J. Wen and T.-S. Yi), and by the Laboratoryof Analytical Biology of the Smithsonian Institution. We thank Pat-rick Reeves, two anonymous reviewers, and Associate Editor LenaHileman for constructive comments, and Guy Jolicoeur, GregKearns, Ken and Bess Weston, Dan Redondo, Dail Laughinghouseand Jinmei Lu for advice and field assistance. The views presentedhere do not necessarily reflect those of the U.S. Fish and WildlifeService.

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