Molecular Phylogenetics and Evolution 57 (2010) 961–977

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Molecular Phylogenetics and Evolution

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The history of extant Ilex species (Aquifoliaceae): Evidence of hybridizationwithin a Miocene radiation

Jean-François Manen a,b, Gabrielle Barriera b, Pierre-André Loizeau a,b, Yamama Naciri a,b,⇑a Université de Genève, Laboratoire de Systématique Végétale et Biodiversité, Chemin de l’Impératrice 1, CH-1292 Chambésy, Switzerlandb Conservatoire et Jardin Botaniques, Laboratoire de Systématique Végétale et Biodiversité, Unité de Phylogénie et Génétique Moléculaires, Chemin de l’Impératrice 1,CH-1292 Chambésy, Switzerland

a r t i c l e i n f o

Article history:Received 6 March 2009Revised 3 September 2010Accepted 9 September 2010Available online 24 September 2010

Keywords:MCMC Bayesian analysesMolecular datingMost common recent ancestor (MRCA)Ancestral areasIntrogressionHybridizationHorizontal gene transferITSnepGSPlastid sequences

1055-7903/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ympev.2010.09.006

⇑ Corresponding author at: Laboratoire de SystématUnité de Phylogénie et Génétique Moléculaires, ChemiChambésy, Switzerland. Fax: +41 22 418 5101.

E-mail address: Yamama.Naciri@ville-ge.ch (Y. Na

a b s t r a c t

The history and diversification of the genus Ilex (Aquifoliaceae), based on 108 different species (116 spec-imens), are inferred from the analysis of two nuclear (ITS and nepGS) and three plastid (rbcL, trnL-F andatpB-rbcL) sequences. Nuclear and plastid trees are highly incongruent and the nuclear tree is more com-patible with current taxonomic classifications than the plastid one. The most recent common ancestor(MRCA) of extant species is dated from the Miocene, although the Ilex stem lineage can be traced backto the late Cretaceous, according to fossil records. This suggests extensive lineage extinctions betweenthe Cretaceous and Miocene and may also explain the difficulties encountered in defining the relation-ships between Ilex and its closest relatives. The MRCA ancestral area was identified as being in the NorthHemisphere (North America and/or East Asia). Several bidirectional North America/East Asia and NorthAmerica/South America dispersal events are proposed to explain observed geographic and phylogeneticpatterns. Hybridization and introgression events between distantly related lineages are also inferred,indicating weak reproductive barriers between species in Ilex.

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1. Introduction Southeast Asia, Central America, the Caribbean and North America.

The Aquifoliaceae is a large lineage of the earliest divergingorder Aquifoliales in Euasterid II, or Campanulids (http://tol-web.org/Aquifoliales/20704/2002.01.01). The precise relationshipsof the Aquifoliaceae within the Aquifoliales remain unclear. Withinthe Aquifoliales two clades are found, the first containing theCardiopteridaceae and Stemonuraceae and the second the Helwin-giaceae, Phyllonomaceae and Aquifoliaceae. Within the latterclade, it is unclear whether the Helwingiaceae and Aquifoliaceae(Olmstead et al., 2000; Soltis et al., 2000) or the Phyllonomaceaeand Helwingiaceae are most closely related (Bremer et al., 2002).

The present Aquifoliaceae consists of the single genus Ilex(Powell et al., 2000). This sub-cosmopolitan genus comprises al-most 600 tropical and temperate species of dioecious trees orshrubs (Loizeau et al., 2005, in press) that are evergreen ordeciduous. The main regions of extant Ilex diversification are EastAsia and South America. The genus is also well-represented in

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ique Végétale et Biodiversité,n de l’Impératrice 1, CH-1292

ciri).

One species is known from tropical Africa, two species are found innorthern tropical Australia (see below) and four closely relatedspecies are present in Europe. Endemic species also occur in theCanary Islands, Madeira and the Azores (Ilex canariensis), Hawaiiand Tahiti (Ilex anomala), New Caledonia (Ilex sebertii) and on Fiji(Ilex vitiensis).

The history of the genus is controversial. The Tertiary macrofos-sil record is mainly distributed in the northern hemisphere, begin-ning at the Maastrichtian (Knobloch and Mai, 1986). Macrofossilsof Ilex are absent from the southern hemisphere pre-Pliocenesediments (see a review in Manen et al. (2002)). As defined byBoufford and Spongberg (1983) and Lavin and Luckow (1993),the genus Ilex would be a typical Arcto-Tertiary lineage, with arecent colonization of South America from North America havingoccurred late in the Tertiary (Graham, 1999). On the other hand,because of the occurrence of Cretaceous pollen of Ilex in southernAustralia and Antarctica (Martin, 1977; Dettmann and Jarzen,1990; Askin, 1992; Specht et al., 1992), as well as in other partsof the world (see Cuénoud et al., 2000; Manen et al., 2002 for areview), the lineage was thought to be cosmopolitan in the lateCretaceous (Cronquist, 1988). These pollen fossils may indicate aGondwanan origin of the genus (Raven and Axelrod, 1974), butthey need a re-evaluation (Manen et al., 2002).

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The phylogeny of Ilex is still not completely resolved and theseminal monograph of Loesener (1901) is not fully supported byrecent biosystematic studies (Baas, 1973, 1975; Lobreau-Callen,1975, 1977; Loizeau and Spichiger, 1992), or by chloroplast or nu-clear DNA sequence comparisons (Cuénoud et al., 2000; Setoguchiand Watanabe, 2000; Manen et al., 2002; Gottlieb et al., 2005). Asstated by Baas (1978), ‘‘the pattern of anatomical variation withinthe genus Ilex, which is reticulate rather than revealing distinctinfrageneric taxa, would not be incompatible with an importantrole of hybridization in its evolution”. Recent DNA analyses haveaccordingly highlighted cases of inter-specific introgression in Ilex(Manen et al., 2002; Setoguchi and Watanabe, 2000; Manen, 2004).Suspected poor reproductive barriers leading to inter-specificintrogression makes the Ilex lineage very complex, as observed inother tree genera such as Quercus (Petit et al., 1997; Zoldos et al.,1999; Belahbib et al., 2001; Muir and Schlötterer, 2006), Pinus(Matos and Schall, 2000), Fraxinus (Gérard et al., 2006; Heuertzet al., 2006) and Betula (Palme et al., 2004). The present study re-examines the worldwide phylogeography and inter-lineagehybridization of Ilex by integrating plastid and nuclear DNA se-quences for 116 Ilex specimens, representing 108 species, aug-menting the earlier sampling of Manen et al. (2002). The taxonsampling, on which this work is based, does encompass all the sub-genera and series of the genus Ilex (Loesener, 1901; Galle, 1997),together with the former genus Nemopanthus, now included in Ilex.Consequently, all the basal lineages proposed by taxonomic assess-ments are included. The discovery that some Ilex ITS sequences(Internal Transcribed Spacer of the ribosomal operon, a multi-copygene) were pseudogenes (Manen, 2004; Gottlieb et al., 2005) hadimplications for the interpretation of earlier phylogenies. Herein,detected ITS pseudogenes were eliminated and replaced by ITSfunctional sequences in I. anomala, I. brasiliensis, I. brevicuspis,I. integerrima, I. purpurea and I. rugosa. A new phylogeny, includingnewly sampled taxa, is proposed using Bayesian MCMC1 analysesand taking into account past introgression. This allows for the deter-mination of the ancestral geographic distribution and dating of themost recent common ancestor of extant Ilex species.

2. Materials and methods

2.1. Specimens used and DNA sequencing

The sampling incorporates 116 individuals representing 105 outof the 119 species described in Table 1 of Cuénoud et al. (2000), i.e.those for which sequences for all five loci could be obtained. Ilexmacrophylla of Cuénoud et al. (2000) is an illegitimate name for I.wallichii thus I. macrophylla is treated as synonymous with I. walli-chii here. Three additional taxa were recently obtained: I. sebertii(New Caledonia), I. arnhemensis subsp ferdinandii and Ilex sp. nov.(Australia). Ilex argentina (specimen ‘‘Gilberti 507” G) was re-ex-tracted and all loci sequenced de novo because mislabelling wassuspected in Manen et al. (2002), as suggested by Gottlieb et al.(2005). Eight species (I. cornuta, I. fargesii, I. integra, I. purpurea,I. rotunda, I. theezans, I. wallichii and I. yunnanensis) were repre-sented by two specimens each, in order to examine within-speciesvariation and/or possible species misidentification.

The amplification and sequencing methods for the plastidatpB-rbcL, trnL-trnF and rbcL sequences are described in Cuénoudet al. (2000) and Manen et al. (2002). Those for the nuclear ITS,and nepGS (nuclear encoded plastid Glutamine Synthetase, a sin-gle-copy gene) are given in Manen et al. (2002) and Manen

1 Abbreviations used: MCMC, Markov chain Monte Carlo method; MRCA, mostrecent common ancestor; dF, degree of freedom; MY, million years; HGT, horizontalgene transfer; nDNA, nuclear DNA; cpDNA, chloroplast or plastid DNA.

(2004), respectively. Because of frequent heterogeneity in ITS,due either to pseudogene amplification, incomplete gene conver-sion or heterozygosity, it was necessary to sequence cloned PCRproducts. ITS pseudogenes, characterized by low GC content andhighly divergent sequences are frequently found in Ilex (Manen,2004; Gottlieb et al., 2005). It was shown that among parameterssuch as RNA folding, conserved 5.8S nucleotides or low GC content,the latter was a good criterion for identifying such pseudogenes(Manen, 2004). Only Ilex ITS sequences with GC contents higherthan 57% were retained in this study. Heterozygosity was alsofound in some nepGS sequences due to single point mutations.Only one nepGS copy per species was used, since the divergentnepGS sequences found within species were always monophyletic.Genbank accession numbers of all analyzed sequences, 335 ofthem new (FJ394587–FJ394914 and FJ418155–FJ418161), arelisted in Table 1.

Four genera could be used as outgroups to reconstruct the phy-logeny of Ilex and to date its MRCA: Helwingia. Phyllonoma, Gono-caryum and Irvingbaileya. Together with Ilex, these five generarepresent the Aquifoliales lineage. In this study, five different se-quences were used: rbcL, atpB-rbcL, trnL-F, ITS, nepGS. The onlyalignable sequence for these five genera is rbcL. Large fragmentsof atpB-rbcL, trnL-F, ITS, nepGS are unalignable between Ilex anddistantly related outgroup taxa, such as Phyllonoma, Gonocaryumand Irvingbaileya. At the end, only Helwingia is alignable with Ilexfor the five studied sequences. In order to have a nuclear datasetfor all Aquifoliales we used a few 18S rDNA sequences that areunambiguously alignable. Thus, age calculation relied on two dif-ferent datasets. The first one was based on rbcL and the few avail-able 18S rDNA for five Ilex species (representing 4 continents) andall available outgroups (namely Helwingia, Phyllonoma, Gonocar-yum and Irvingbaileya). The second one included 108 Ilex speciesand Helwingia for five sequences (rbcL, atpB-rbcL, trnL-F, ITS andnepGS).

2.2. Phylogenetic analysis, dating and ancestral geographicdistribution

2.2.1. Bayesian MCMC analysisAlignment of ITS and nepGS sequences (as well as some regions

of the atpB-rbcL spacer and of trnL-F) of Phyllonoma are problem-atic. In this study Phyllonoma was excluded and analyses werebased on Helwingia and Ilex. All analyses were conducted usingthe BEAST program v1.4.8 (Drummond and Rambaut, 2007). Thisprogram builds time-measured phylogenies and calculates themost probable ancestral geographic distribution (BEAST v1.5.2 –released in October 2009). Inferences are calculated using strictor relaxed molecular clocks, using different nucleotide substitutionmodels. BEAST input files were created with BEAUti v1.4.8 fromDNA sequence matrices in a NEXUS format. Nucleotide substitu-tion models were tested on all sequence alignments separately(atpB-rbcL, trnL-trnF, rbcL, ITS and nepGS) and selected accordingto the Akaike information criterion (AIC) score using ModelTestv3.8 (Posada and Crandall, 1998). BEAUti v1.4.8 only uses HKYand GTR substitution models as priors, which means that any othermodels to be tested should be manually edited in BEAUti files, asdescribed in the Tutorials of BEAST (http://beast.bio.ed.ac.uk/Nucleotide_Substitution_Models). In all analyses a ‘‘Yule process”(selected as tree prior in BEAUti v1.4.8) was used. This is a simplemodel of speciation that is appropriate when considering se-quences from different species. Burn-in was chosen at 10% and ver-ified from statistic traces. Analyses of the BEAST output were donewith Tracer v1.4.1 (Rambaut and Drummond, 2007), LogCombinorv1.4.8 and TreeAnnotator v1.4.8 (all distributed with the BEASTpackage) as well as with FigTree v1.2.2 (http://tree.bio.ed.ac.uk/software/figtree). The comparison and selection of a strict or

Table 1GenBank accession numbers of DNA sequences used in this work.

Species Nuclear Plastid

18S rDNA ITS nepGS atpB-rbcL rbcL trnL-trnF

Gonocaryum litorale AF206919 AJ235779Irvingbaileya sp. AJ235999 AF156733Phyllonoma laticuspis U42546 AF471727 L11201 AJ492565Helwingia japonica U42524 AJ275343 FJ394798 X94941 L11226 AJ275344Ilex aculeolata 140 FJ394656 FJ394799 AF471590 FJ394587 FJ394729Ilex amara H301 FJ394657 FJ394800 AF471597 FJ394588 FJ394730Ilex amelanchier 100 AJ275340 FJ394801 AF471593 X98722 AJ275349Ilex anomala 101 AJ786505 FJ394802 AF471594 X98723 AJ492567Ilex aquifolium 136 FJ418158 FJ394658 FJ394803 AF471596 FJ394589 FJ394731Ilex argentina 139 FJ394659 FJ394804 AF471592 AJ492699 AJ492570Ilex arnhemensis 502 FJ394660 FJ394805 FJ418155 FJ394590 FJ394732Ilex asperula H303 FJ394661 FJ394806 AF471598 FJ394591 FJ394733Ilex x attenuata 74 FJ394723 FJ394908 AF471719 FJ394650 FJ394792Ilex bioritsensis 142 FJ394662 FJ394807 AF471602 FJ394592 FJ394734Ilex brasiliensis 102 AJ786506 FJ394808 AF471600 X98735 AJ492575Ilex brevicuspis 105 AJ786507 FJ394809 AF471603 X98719 AJ492576Ilex buergeri 11 FJ394663 FJ394810 AF471601 FJ394593 FJ394735Ilex canariensis 90 AJ275339 FJ394811 AF471608 X98727 AJ275348Ilex cassine 06 FJ394664 FJ394812 AF471620 AJ492704 AJ492580Ilex chamaedryfolia 117 FJ394665 FJ394813 AF471612 FJ394594 FJ394736Ilex ciliospinosa 168 FJ394666 FJ394814 AF471613 FJ394595 FJ394737Ilex cissoidea 163 FJ394667 FJ394815 AF471610 FJ394596 FJ394738Ilex colchica 67 FJ394668 FJ394816 AF471617 FJ394597 FJ394739Ilex collina 81 AJ492679 FJ394817 AF471615 AJ492716 AJ492592Ilex corallina 88 FJ394669 FJ394818 AF471619 FJ394598 FJ394740Ilex coriacea 147 FJ394670 FJ394819 AF471609 FJ394599 FJ394741Ilex cornuta 57 FJ394671 FJ394820 AF471624 FJ394600 FJ394742Ilex cornuta 61 FJ418159 FJ394672 FJ394821 AF471625 FJ394601 FJ394743Ilex crenata 14 AJ492677 FJ394822 AF471618 L01928 AJ492590Ilex cumulicola 160 FJ394673 FJ394823 AF471623 FJ394602 FJ394744Ilex curtissii 122 FJ394674 FJ394824 AF471622 FJ394603 FJ394745Ilex cymosa 172 FJ394675 FJ394825 AF471621 FJ394604 FJ394746Ilex cyrtura 60 FJ394676 FJ394826 AF471614 FJ394605 FJ394747Ilex decidua 73 AJ492680 FJ394827 AF471626 X98724 AJ492593Ilex dimorphophylla 08 FJ394677 FJ394828 AF471628 FJ394606 FJ394748Ilex discolor 108 FJ394678 FJ394829 AF471629 FJ394607 FJ394749Ilex dugesii 98 FJ394679 FJ394830 AF471627 FJ394608 FJ394750Ilex dumosa 103 AJ492657 FJ394831 AF471630 X98725 AJ492571Ilex fargesii 68 FJ394680 FJ394832 AF471633 FJ394609 FJ394751Ilex fargesii 124 FJ394681 FJ394833 AF471635 FJ394610 FJ394752Ilex ficoidea 85 FJ394682 FJ394834 AF471634 FJ394611 FJ394753Ilex fragilis 152 FJ394683 FJ394835 AF471636 FJ394612 FJ394754Ilex geniculata 125 FJ394684 FJ394836 AF471638 FJ394613 FJ394755Ilex georgei 65 FJ394685 FJ394837 AF471641 FJ394614 FJ394756Ilex glabra 143 AJ275342 FJ394838 AF471639 AJ492720 AJ275351Ilex goshiensis 10 AJ492687 FJ394839 AF471640 X98734 AJ492600Ilex guianensis H7 AJ492668 FJ394840 AF471642 AJ492706 AJ492582Ilex havilandii 131 FJ394686 FJ394841 AF471643 FJ394615 FJ394757Ilex hookeri 72 FJ394687 FJ394842 AF471645 FJ394616 FJ394758Ilex hylonoma 138 FJ394688 FJ394843 AF471646 FJ394617 FJ394759Ilex integerrima 106 AJ786508 FJ394844 AF471649 X98726 AJ492577Ilex integra 05 FJ394689 FJ394845 AF471651 FJ394618 FJ394760Ilex integra 09 FJ394690 FJ394846 AF471647 FJ394619 FJ394761Ilex kinabaluensis 166 FJ394691 FJ394847 AF471653 FJ394620 FJ394762Ilex kingiana 144 FJ394692 FJ394848 AF471652 FJ394621 FJ394763Ilex x kiusiana 170 FJ394724 FJ394909 AF471720 FJ394651 FJ394793Ilex kusanoi 158 FJ394693 FJ394849 AF471654 FJ394622 FJ394764Ilex laevigata 114 FJ394694 FJ394850 AF471657 FJ394623 FJ394765Ilex latifolia 62 AJ492691 FJ394851 AF471655 X98731 AJ492604Ilex laurina H1 AJ492651 FJ394852 AF471660 AJ492730 AJ492566Ilex leucoclada 159 AJ492690 FJ394853 AF471656 AJ492728 AJ492603Ilex liebmannii 71 AJ492659 FJ394854 AF471659 AJ492700 AJ492573Ilex liukiuensis 17 FJ394695 FJ394855 AF471661 FJ394624 FJ394766Ilex longipes 77 FJ394696 FJ394856 AF471658 FJ394625 FJ394767Ilex macrocarpa 76 AJ492689 FJ394857 AF471663 AJ492727 AJ492602Ilex macropoda 91 AJ492688 FJ394858 AF471662 AJ492726 AJ492601Ilex maingayi 148 FJ394697 FJ394859 AF471666 FJ394626 FJ394768Ilex x makinoi 169 FJ394725 FJ394910 AF471721 FJ394652 FJ394794Ilex matanoana 12 FJ394698 FJ394860 AF471669 FJ394627 FJ394769Ilex maximowicziana 02 AJ492678 FJ394861 AF471674 AJ492715 AJ492591Ilex mertensii 03 FJ394699 FJ394862 AF471671 FJ394628 FJ394770Ilex micrococca 79 AJ492684 FJ394863 AF471670 X98721 AJ492597Ilex microdonta 118 AJ492665 FJ394864 AF471665 AJ492702 AJ492578

(continued on next page)

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Table 1 (continued)

Species Nuclear Plastid

18S rDNA ITS nepGS atpB-rbcL rbcL trnL-trnF

Ilex mitis 63 FJ418160 AJ275338 FJ394865 AF471667 X98730 AJ275347Ilex montana 121 FJ394700 FJ394866 AF471676 FJ394629 FJ394771Ilex mucronata 153 FJ418161 AJ275336 FJ394867 AF471672 X69747 AJ275345Ilex mutchagara 95 AJ492677 FJ394868 AF471673 AJ492712 AJ492587Ilex nervulosa 165 FJ394701 FJ394869 AF471678 FJ394630 FJ394772Ilex nitida 80 FJ394702 FJ394870 AF471677 FJ394631 FJ394773Ilex nothofagifolia 82 FJ394703 FJ394871 AF471708 FJ394632 FJ394774Ilex opaca 92 AF206938 FJ394704 FJ394872 AF471679 FJ394633 FJ394775Ilex oppositifolia 134 AJ492685 FJ394873 AF471680 AJ492719 AJ492598Ilex paraguariensis 146 FJ394705 FJ394874 AF471684 FJ394634 FJ394776Ilex pedunculosa 75 AJ275341 FJ394875 AF471682 X98728 AJ275350Ilex perado 56 AJ492693 FJ394876 AF471687 X98729 AJ492606Ilex pernyi 53 FJ394706 FJ394877 AF471685 FJ394635 FJ394777Ilex pseudobuxus 104 FJ394707 FJ394878 AF471688 X98736 AJ492574Ilex pubescens 69 AJ492686 FJ394879 AF471689 AJ492722 AJ492599Ilex purpurea 123 AJ786509 FJ394880 AF471690 AJ492711 AJ492586Ilex purpurea 97 FJ394708 FJ394881 AF471681 AJ492710 AJ492585Ilex quercetorum 126 FJ394709 FJ394882 AF471691 FJ394636 FJ394778Ilex repanda 119 AJ492654 FJ394883 AF471694 AJ492697 AJ492569Ilex revoluta 132 AJ492676 FJ394884 AF471699 AJ492714 AJ492589Ilex rotunda 167 FJ394710 FJ394885 AF471692 FJ394637 FJ394779Ilex rotunda 04 AJ786510 FJ394886 AF471695 X98720 AJ492596Ilex rubra 83 FJ394711 FJ394887 AF471693 FJ394638 FJ394780Ilex rugosa 16 AJ786511 FJ394888 AF471698 X98733 AJ492605Ilex sebertii 401 FJ394712 FJ394889 FJ418156 FJ394639 FJ394781Ilex serrata 78 AJ492682 FJ394890 AF471604 AJ492718 AJ492595Ilex shennongjiaensis 156 AJ492670 FJ394891 AF471601 AJ492707 AJ492583Ilex sp. nov. 501 FJ394713 FJ394892 FJ418157 FJ394640 FJ394782Ilex spicata 133 FJ394714 FJ394893 AF471703 FJ394641 FJ394783Ilex spinigera 93 FJ394715 FJ394894 AF471700 FJ394642 FJ394784Ilex sugerokii 149 AJ492671 FJ394895 AF471702 AJ492709 AJ492584Ilex teratopis H10 AJ492649 FJ394896 AF471707 AJ492695 AJ492564Ilex theezans 115 FJ394716 FJ394897 AF471705 FJ394643 FJ394785Ilex theezans 116 AJ492666 FJ394898 AF471706 AJ492703 AJ492579Ilex tolucana 89 FJ394717 FJ394899 AF471710 FJ394644 FJ394786Ilex triflora 129 AJ492675 FJ394900 AF471711 AJ492713 AJ492588Ilex tsoi 151 FJ394718 FJ394901 AF471712 FJ394645 FJ394787Ilex verticillata 59 AJ492681 FJ394902 AF471713 AJ492717 AJ492594Ilex vomitoria 66 AJ492653 FJ394903 AF471715 M88583 AJ492568Ilex wallichii 154 FJ394719 FJ394904 AF471675 FJ394646 FJ394788Ilex wallichii 173 FJ394720 FJ394905 AF471716 FJ394647 FJ394789Ilex warburgii 01 FJ394721 FJ394906 AF471718 FJ394648 FJ394790Ilex wilsonii 86 FJ394722 FJ394907 AF471717 FJ394649 FJ394791Ilex yunnanensis 157 FJ394726 FJ394911 AF471724 FJ394653 FJ394795Ilex yunnanensis 54 AJ275337 FJ394912 AF471722 AJ275346 AJ275337Ilex zhejiangensis 99 FJ394727 FJ394913 AF471725 FJ394654 FJ394796Ilex zygophylla 130 FJ394728 FJ394914 AF471726 FJ394655 FJ394797

Voucher information can be found in Cuénoud et al. (2000).

964 J.-F. Manen et al. / Molecular Phylogenetics and Evolution 57 (2010) 961–977

relaxed molecular clock (uncorrelated lognormal) was done withTracer v1.4.1 on the BEAST output, for both clock models, by calcu-lating the marginal likelihood and the Bayes factor using a likeli-hood ratio test (LRT) which corresponds to a goodness-of-fitbetween the two clock models. Critical values were obtained fromthe chi-square distribution with n � 2 degree of freedom, n beingthe number of samples.

Both the nuclear and chloroplast phylogenies were obtainedusing BEAST as follows: five independent MCMC runs were set toperform 10,000,000 generations, sampling once every 1000 gener-ations. The combined effective sample size (ESS) statistics valueswere checked. ESS lower than 100 indicated a low number of effec-tively independent draws, and a global poor mixing (http://beast.-bio.ed.ac.uk). The 45,000 trees resulting from the five runs werecombined using LogCombinor v1.4.8. A maximum clade credibility(MCC) chronogram was then selected by TreeAnnotator v1.4.8.

2.2.2. DatingFor Ilex, two dates are available to calibrate trees. One is 69 mil-

lion years ago (69 MY), representing the most ancient recognized

macrofossil (seed) record of Ilex from Central Europe and set asthe date of Ilex emergence (Knobloch and Mai, 1986, see Manenet al. (2002) for a discussion on the validity of this record). The sec-ond, 80 MY, is set as the most recent common ancestor (the crownage) of the clade comprising Phyllonoma, Helwingia and Ilex. Thiswas inferred from estimates given by Davies et al. (2004) on anangiosperm tree [approximately 83 MY for (Phyllonoma (Helwingia,Ilex))], and by Bremer et al. (2004) on an Asterid tree [approxi-mately 78 MY for ((Phyllonoma, Helwingia) Ilex)]. Using BEAUtiv1.4.8., a log-normal distribution was assumed as prior for the 69MY calibration date, with a lower bound fixed at 69 MY and a95% interval confidence (IC) ranging from 69.3 to 72.1 MY, a periodof time corresponding to the age of the upper Maastrichtiangeological stratum from which the fossil was extracted (the Maas-trichtian era spanned 65.6–70.6 MY). In this case, a constrainedlog-normal distribution was chosen because the node cannot beyounger than the fossil, by definition (Ho, 2007). Again, followingHo (2007), a normal time distribution was assumed as prior forthe 80 MY date since this calibration point comes from previouscalibrated trees. The normal distribution was centred on 80 MY,

J.-F. Manen et al. / Molecular Phylogenetics and Evolution 57 (2010) 961–977 965

with a standard deviation of 6 MY (95% IC ranging from 70.1 to89.9 MY) which allowed to cover almost all the late Cretaceousera spanning 65–90 MY.

The calculation of the age of the most recent common ancestorof extant species of Ilex, was performed in two ways:

First, it was computed using the 80 MY calibration age on fiveIlex species. Four of them were chosen as to be representative ofthe four chloroplast clades/phylogroups found by Manen et al.(2002), in addition to Ilex mucronata, the former monospecificgenus Nemopanthus, previously considered as sister of the genusIlex. This selection therefore represents a large phylogenetic rangeand four different continents. The five species were sequenced for18S RNA and rbcL for this purpose and the corresponding Genbankaccessions are listed in Table 1: I. opaca (North America, 18S rRNAfrom Genbank: AF206938, rbcL: this study), I. mucronata (NorthAmerica, 18S rRNA and rbcL: this study), I. cornuta (Asia, 18S rRNAand rbcL: this study), I. mitis (Africa, 18S rRNA and rbcL: this study)and I. aquifolium (Europe, 18S rRNA and rbcL: this study). In addi-tion, four Aquifoliales species (sensu Karehed, 2001) collected fromGenbank or sequenced for this study were included: Gonocaryumlitorale (18S rRNA: AF206919, rbcL: AJ235779, Cardiopteridaceae),Irvingbaileya australis (18S rRNA: AJ235999, rbcL: AF156733,Stemonuraceae), Phyllonoma laticuspis (18S rRNA: U42546, rbcL:L11201, Phyllonomaceae) and Helwingia japonica (18S rRNA:U42524, rbcL: L11226, Helwingiaceae). For this dataset, ModelTestwas used to select the most appropriate substitution model. Bothstrict and relaxed clocks were used and for both genes (9 se-quences), one run was set to perform 10,000,000 generations, sam-pling once every 1000 generations.

To test the results obtained on a restricted sample of Aquifoli-ales taxa, two other calculations were run separately using the nu-clear and plastid datasets of 116 Ilex specimens in addition toHelwingia. A 69 MY time calibration (first occurrence of Ilex inthe fossil record) was used for the divergence of Ilex from Helwin-gia. This calculation was performed using either a relaxed molecu-lar clock or a strict molecular clock to test for their effects on thedating.

2.2.3. Ancestral geographic distributionThe most recent versions of BEAST (v1.5.2) and FigTree (v1.2.3)

(http://beast.bio.ed.ac.uk/Main_Page) allowed the calculation ofancestral geographic distributions based on a discrete phylogeo-graphic analysis using a standard continuous-time Markov chain(CTMC, Lemey et al., 2009). Each taxon can be allocated to a givenstate, here a geographic location, corresponding to the distributioncentre of each species. The ancestral reconstruction based on dis-crete states (or areas as we referred to) assumes that the ancestralorganisms at the internal nodes were distributed in locations fromwhich samples have been taken. For each node BEAST is able toreconstruct the probability distribution for the different states.Moreover, if the hypothesis is that an ancestral area is A + B foran internal node, it is possible to see that A and B share most ofthe posterior probability mass, which would suggest a vicarianceevent. Twelve areas were selected: East Asia, Southeast Asia, NorthAmerica, South America, Central America, Caribbean Islands, Can-ary Islands, Hawaii, New Caledonia, Australia, Africa and Europe.The beast.xml file was edited according to the BEAST Tutorial ‘‘Dis-crete Phylogenetic Analysis (http://beast.bio.ed.ac.uk/Dis-crete_Phylogeographic_Analysis). From nuclear and plastid data,BEAST v1.5.2 calculates the most probable ancestral geographicdistribution of each tree node using a 10,000,000 MCMC chainlength, saving trees every 1000 steps. Within the 9000 trees pro-duced for each data set, one was found identical to the chronogramdisplaying the maximum clade credibility, using TreeAnnotatorv1.5.2, (Figs. 1 and 2). These two trees were therefore selected

and printed with FigTree v 1.2.3. Each geographic location was gi-ven a different color.

2.3. Evaluation of nuclear introgression or plastid capture

Different algorithms are available that identify horizontal genetransfer events (HGT) by comparing a species tree to a gene tree.They find the most parsimonious scenario to explain disparities be-tween the two trees by means of HGT events (Makarenkov et al.,2006). In order to examine whether a similar mechanism can ex-plain the differences observed between the nuclear and the plastidphylogeny of Ilex, the nuclear phylogeny was here considered asthe species phylogeny (see below), and the plastid phylogeny asthe gene phylogeny. The two phylogenies are assumed to be differ-ent because of HGT, in our case due to horizontal plastid capture.The online program ‘‘HGT-detection” from the T-REX package(www.trex.uqam.ca, Makarenkov, 2001) was used to infer suchHGT. Instead of the trees based on 116 specimens that would haveled to too many HGT, nuclear and plastid ‘‘summary trees” wereused. For this purpose, twenty-five taxa were chosen, as to repre-sent all lineages defined by the nuclear dataset, using at leastone species per lineage (outlined in blue in Figs. 3 and 4). Severaldistances were used for comparative purposes: the Robinson andFoulds distance, the bipartition distance and the last-squaresoptimization.

3. Results

The nuclear matrix (1575 sites, of which 806 are variable) con-tains ITS and nepGS for 116 Ilex specimens (108 species) with onlyHelwingia japonica as an outgroup. The sequences of Phyllonomaruscifolia were indeed unalignable with both Ilex and Helwingia(see below). The plastid matrix (2995 sites, of which 399 are var-iable) contains atpB-rbcL, trnL-trnF and rbcL for 116 Ilex speci-mens, again with Helwingia japonica as outgroup. Alignmentswere performed manually as no alignment problem in bothcpDNA and nDNA datasets were found for Ilex, except for somenucleotide repeats whose length seems random and/or due topossible amplification errors. These regions were therefore dis-carded from the alignments. A few regions of Helwingia sequenceswere difficult to align with Ilex and were also excluded from theanalyses.

3.1. Phylogeny based on nuclear data

According to the AIC score, ModelTest selected the GTR+I+Gsubstitution model for ITS and the TVM+I+G model (a model closeto GTR+I+G, except that both transitions have equal frequencies)for nepGS. When working on both sequences, ModelTest favouredthe GTR+I+G and analyses were run using this model on the com-bined data. According to the very high Bayes factor (BF), a relaxedclock (uncorrelated Lognormal) was preferred to the strict molec-ular one (BF = 220, dF = 115, P < 0.001). Fig. 1 shows the MCC chro-nogram. ESS values were all higher than 150. The branchsubstitution rate was set proportional to branch width, and except-ing deep branches for which posterior credibility values (CV) weregiven, CV were indicated by different colors, red to blue indicatinghigh to low credibility, and listed in Appendix A. In order to illus-trate the differences in branch lengths between Helwingia and Ilex,a phylogram is presented as an insert in Fig. 1.

The Ilex tree reveals a clear cleavage, outlined by an arrowhead.The first clade is well-supported (CV = 0.96), whereas the secondphylogroup is not (CV = 0.51). The first clade includes New Worldspecies, depicted in green, with a small Asian clade nested within(colored in red, I. mutchagara, I. crenata, I. maximowicziana,

Fig. 1. Maximum clade credibility (MCC) chronogram based on nuclear data (ITS and nepGS). The small insert on left represents the corresponding phylogram. In order toincrease legibility, branch thickness was set proportional to substitution rates, and colors were used for correspondence with high to low posterior credibility values (red toblue). The exact credibility values are indicated for deep branches. The red taxa are from the Old World, the green taxa are from the New World. Species from the Atlantic (I.canariensis) and Pacific Islands (I. sebertii and I. anomala) were depicted using the color of the clade/phylogroup to which they belong. The small arrowheads outline clades/phylogroups also found in the plastid tree with the same composition. The big arrow points to a cleavage discussed in the text. Informal names were given to the differentclades and are documented in Appendix A.

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Fig. 2. Maximum clade credibility (MCC) chronogram based on plastid data (atpB-rbcL, trnL-F and rbcL). The small insert on left represents the corresponding phylogram. Inorder to increase legibility, branch thickness was set proportional to substitution rates, and colors were used for correspondence with high to low posterior credibility values(red to blue). The exact credibility values are indicated for deep branches. Red taxa are from the Old World, green taxa are from the New World. Species from the Atlantic (I.canariensis) and Pacific Islands (I. sebertii and I. anomala) were depicted using the color of the clade/phylogroup to which they belong. The small arrowheads outline clades/phylogroups also found in the nuclear tree with the same composition. The big arrow points to an indel, described by Selbach-Schnadelbach et al. (2009), and found in theplastid psbA-trnH spacer.

J.-F. Manen et al. / Molecular Phylogenetics and Evolution 57 (2010) 961–977 967

Fig. 3. CTMC spatial reconstruction of the MCC chronogram based on nuclear data (ITS and nepGS). Lineages are colored according to the highest posterior probability foundfor location. Probabilities higher than 0.97 were removed. Blue taxa were selected for the T-REX analysis described in Fig. 5.

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I. revoluta, and I. triflora). The large second phylogroup includes Ilexspecies that are mainly distributed in the Old World. This largephylogroup comprises one phylogroup and a well-supportedsub-clade. The geographic distribution of the phylogroup (I. canari-ensis–I. wallichii, CV = 0.88) is broad. It comprises three smallAmerican clades (in green): (1) I. coriacea, I. glabra and I. liebmannii;(2) I. argentina, I. anomala (from Hawaii), I. microdonta, I.

pseudobuxus, I. quercetorum, I. guianensis, I. asperula, I. brevicuspis,I. brasiliensis, I. theezans and I. integerrima; and (3) I. laevigata andI. verticillata. This whole subgroup is found in temperate and sub-tropical East Asia, tropical southeast Asia (including Australia andNew Caledonia), America, the Atlantic and Pacific Islands and Afri-ca. The sub-clade (CV = 1.00) is mainly distributed in temperateand subtropical Eurasia (I. rugosa–I. cyrtura).

Fig. 4. CTMC spatial reconstruction of the MCC chronogram based on plastid data (atpB-rbcL, trnL-F and rbcL). Lineages are colored according to the highest posteriorprobability found for location. Probabilities higher than 0.97 were removed. Blue taxa were selected for the T-REX analysis described in Fig. 5.

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3.2. Phylogeny based on plastid data

ModelTest selected the GTR+G model for the atpB-rbcL spacer,the GTR+I+G model for rbcL and the TVM+G model for trnL-trnFat first with the model GTR+G model being selected in a second in-stance. On the combined dataset, ModelTest selected the TVM+I+G

model first and then the GTR+I+G model, among the 24 tested.Analyses were subsequently run using the GTR+I+G model,although it is not the one favoured for all genes, because it is theleast constrained and is directly available in BEAUti v1.4.8. Accord-ing to the high Bayes factor (BF = 78), a relaxed molecular clock(uncorrelated Lognormal) was chosen, although the difference

Table 2Age in MY of the most recent common ancestor of extant species of Ilex calculatedwith the program BEAST V1.4.8.

DNA sets Sequences Mean 95% HPD 95% HPDLowerbound

Upperbound

Aquifoliales 18S (relaxed clock) 14.6 4.0 28.018S (strict clock) 12.8 4.6 22.6rbcL (relaxed clock) 31.2 15.3 49.7rbcL (strict clock) 28.2 17.6 39.4rbcL 1–2 (relaxedclock)

48.9 28.9 70.5

rbcL 1–2 (strict clock) 49.9 31.8 59.5rbcL 3 (relaxed clock) 22.1 6.1 45.6rbcL 3 (strict clock) 13.9 7.8 28.2

Helwingia/Ilex

Nuclear (relaxed clock) 33.6 21.4 47.3

Nuclear (strict clock) 13.4 11.3 15.6Plastid (relaxed clock) 46.5 29.6 69.8Plastid (strict clock) 15.6 12.3 18.9

Aquifoliales: calculations were made on 18S rDNA and rbcL DNA sets comprisingnine taxa of Aquifoliales including five representative Ilex species. The calibrationpoint was 80 MY using a normal distribution (with 6 MY as standard deviation) forthe node Phyllonoma/Helwingia/Ilex (strict or relaxed molecular clock with theTrNef+I substitution model for 18S rDNA and the TVM+I substitution model forrbcL). Helwingia/Ilex: calculations made on the nuclear and the plastid DNA setscomprising Helwingia and 116 Ilex specimens. The calibration point was 69 MYusing a constrained log-normal distribution (with 2 MY as standard deviation) forthe divergence of Helwingia/Ilex (strict or relaxed molecular clock with the GTR+I+Gsubstitution model).

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between the two clocks was not found to be significant (dF = 115,P > 0.05). Fig. 2 presents the results for the chloroplast data, withthe same color scheme as in Fig. 1. ESS values were all higher than435. The resolution of the plastid tree was low, especially in thedeep branches. However, as already observed in Cuénoud et al.(2000) and in Manen et al. (2002), it is highly geographically struc-tured and contains four clades or phylogroups:

(1) Taxa from the North American/East-Asian area. Ilex canarien-sis has a low probability to belong to this clade and wasfound in an isolated (or indefinite) position as in Cuénoudet al. (2000) and in Manen et al. (2002). When I. canariensisis not taken into account, the clade is well sustained(CV = 1.00).

(2) Taxa from North America, East Asia, Southeast Asia (includ-ing Australia), and the single African species I. mitis. Thisphylogroup is not well-supported (CV = 0.43).

(3) North American taxa and all the South American speciesincluded in this study, the Hawaiian and the New Caledo-nian species (I. anomala and I. sebertii, respectively). Thisphylogroup is moderately supported (CV = 0.84).

(4) Eurasian taxa, including species of the Canary Islands andNorth Africa (I. perado) on the one side and Borneo (I. kina-baluensis) on the other. This clade is well-supported(CV = 0.93) and is also inferred from nuclear data.

The relationship between the American phylogroup 3 and theEurasian clade 4 is poorly supported. These clades/phylogroupshowever share a specific indel that has recently been discoveredin the plastid psbA-trnH spacer of Ilex (Selbach-Schnadelbachet al., 2009). Mapping this indel on the plastid tree described here(see arrow in Fig. 2) indicates that Helwingia, phylogroups 1(I. canariensis–I. triflora) and 2 (I. cissoidea–I. aculeolata) share a31–74 pb insertion, and that the corresponding deletion linksphylogroups 3 (I. teratopis–I. microdonta) with clade 4 (I. kinabal-uensis–I. kingiana).

3.3. Comparison of the nuclear and plastid trees

In order to facilitate interpretation, informal names were givento the different clades/phylogroups inferred from Fig. 1. When amolecular clade/phylogroup was found to be identical or close toa section, subsection or series as defined by Loesener (1901, up-dated 1942) and/or by Galle (1997), the name of the clade/phylo-group was given according to these authors. Otherwise the term‘‘alliance” has been added to a representative section/series or spe-cies name (e.g. Megalae alliance or Yunnanensis alliance). For thetime being, these names have no taxonomic value. Appendix Acontains a description of clade/phylogroup names according toLoesener’s and Galle’s classifications, in addition to the credibilityvalue, geographic distribution and habitat zone of all inferredclades/phylogroups.

The trees inferred by nuclear data (Fig. 1) and by plastid data(Fig. 2) are very different. Few clades, displaying identical compo-sition, are inferred by both datasets and are outlined by smallarrowheads in both figures. This is the case for the well-supportednuclear clade Aquifolium (CV = 1) that comprises exactly the samespecies as the plastid clade 4 (CV = 0.93). As first published inManen et al. (2002), the plastid sequence of I. argentina, resultingfrom re-analyzed DNA data (see Appendix A), was found to be sis-ter to Polyphyllae, as defined by the nuclear data.

3.4. Dating

Age calculations were first performed using 18S rDNA and rbcLfrom four outgroups and five representative Ilex species with

80 MY as calibration age for the clade comprising Phyllonoma,Helwingia and Ilex. ModelTest selected the TrNef+I substitutionmodel for 18S rDNA sequences and the model TVM+I for rbcLsequences, according to the AIC score. The ESS values were allhigher than 5000. According to the very low BF factors, neither ofthe two clocks was preferred (BF = 4.4 and 0.02 for 18S rDNA andrbcL, respectively; dF = 7, P > 0.05). Using a strict molecular clockhypothesis the MRCA age for Ilex was 12.8 MY (95% range4.6–22.6 MY) for 18S rDNA, and 28.2 MY (95% range 17.6–39.4 MY)for rbcL (Table 2). First and second codon positions were mainlyresponsible for the older age given by rbcL (rbcL 1–2: 49.9 (95% range31.8–59.5 MY; rbcL 3: 13.9 MY (95% range 7.8–28.2 MY). Agescalculated using a relaxed clock hypothesis were similar (see Table2), as expected from BF factors.

These results are in contradiction with those obtained using nu-clear and plastid data for 116 Ilex specimens. In this case, the age ofthe most recent common ancestor (MRCA) of extent species of Ilexwas calculated by calibrating trees with the divergence of Ilex andHelwingia, estimated at 69 MY. Using the relaxed clock hypothesis,suggested by the BF factor, the age of the Ilex MRCA was higher:33.6 MY (95% range 21.4–47.3 MY) for nuclear data and 46.5 MY(95% range 29.6–69.8 MY) for plastid data (Table 2). It should benoticed that the substitution rate for Helwingia was 2–10 timeshigher that the substitution rate for Ilex, including its stem branch(see Figs. 1 and 2). When a strict molecular clock was used insteadof the relaxed molecular clock (all other parameters being identi-cal), the age of the most recent common ancestor of Ilex was13.4 MY (95% range 11.3–15.6 MY) for nuclear data and 15.6 MY(95% range 12.3–18.9 MY) for plastid data (Table 2).

3.5. Ancestral distribution

Using the same BEAST input files with attached geographiclocations, the probabilities of ancestral geographic distribution ofIlex lineages were calculated and are summarized in Figs. 3 and 4(nuclear and plastid data, respectively). For better legibility, distri-bution credibility values higher than 0.97 were removed.

J.-F. Manen et al. / Molecular Phylogenetics and Evolution 57 (2010) 961–977 971

Nuclear data clearly suggest that the genus Ilex is divided intotwo stems, a North American lineage and an East Asian lineage(Fig. 3). The location of the most recent common ancestor of Ilexis likely to be East Asia as the geographic distribution of the out-group species Helwingia japonica is similarly Asia. The North Amer-ican lineage dispersed twice to South America (Polyphyllae andRepandae) and then from South America to Asia (Stigmatophorae).The East Asian lineage divided into two different lineages: the firstone (Aquifolium) remained in East Asia with dispersal to Europeand a minor dispersal to Southeast Asia. The second dispersed toNorth America twice (Glabra and Verticillata alliance, respec-tively), to South America (Megalae alliance) and to Southeast Asia(Indico-malaicae alliance).

Chloroplast data unambiguously suggests that the origin of themost recent common ancestor of the plastid of Ilex is East Asia(Fig. 4). Focusing on the most important geographic regions, therewere three plastid dispersal events from East Asia to North Amer-ica; one dispersal from East Asia to South America; one dispersalfrom South America to North America; two dispersal events fromEast Asia to Southeast Asia and one dispersal from East Asia toEurope.

3.6. Intraspecific polymorphism and hybridizations

Eight species were represented by two specimens each (I. corn-uta, I. fargesii, I. integra, I. purpurea, I. rotunda, I. theizans, I. wallichiiand I. yunnanensis). Only 2 of them (I. purpurea and I. rotunda) donot show polymorphism at the nuclear and chloroplast levelswhereas I. theezans is polymorphic for the nuclear data only.

Three natural hybrids of Ilex have been analyzed in this study.Ilex � attenuata (I. opaca � I. cassine, Galle, 1997) is found to beclose to I. cassine for the nuclear genes, and either to I. cassine orI. cumulicola for the chloroplast sequences (both relationships areequally well-supported). The hybrid I. �makinoi (I. leucoclada � I.

Fig. 5. The most parsimonious scenario of introgression (plastid capture). The program T-nuclear (species) topology and the corresponding plastid topology based on 25 selectedbeing discussed in the text. Green arrows refer to introgression events between ancestorand red arrows to those between ancestors of species that presently inhabit different georeferences to color in this figure legend, the reader is referred to the web version of thi

rugosa, Galle, 1997) is close to I. leucoclada in the nuclear treeand I. fargesii in the chloroplast tree (but this latter relationshipis not supported). Ilex � kiusiana (I. buergeri � I. integra, Galle,1997) is close to I. integra and I. liukiuensis in the nuclear treeand I. ficoidea and I. georgei in plastid tree (but this latter relation-ship is again not supported).

Comparing the topology of the nuclear tree (considered as a spe-cies tree) and the plastid tree (considered as a gene tree) T-REXfound one most parsimonious scenario comprising 15 horizontalgene transfers events (in this case nuclear introgressions orconversely plastid captures) to reconcile both trees. The resultingscenario was identical whether calculated with the Robinson andFoulds distance, the bipartition distance or the last-squares optimi-zation. Fig. 5 shows that seven horizontal transfers must haveoccurred between species that are now found in the same geo-graphic area (green arrows). This is the case for introgressions 1,3, 7 (East Asia), 4, 5, 11 (South America) and 6 (North America).However, 8 transfers might have occurred between species thatnow inhabit different areas (red arrows). This is the case for intro-gressions 2 (East Asia and Africa), 8 (East Asia/North America/Africaand Australia), 9 (southeast Asia and East Asia), 10 (South Americaand North America), 12 (South America and New Caledonia), 13(Asia/East Asia/Australia/New Caledonia and North America), 14(South America and East Asia/North America) and 15 (SouthAmerica and Eurasia).

4. Discussion

One of the major points of interest in this study is the great dis-crepancy between nuclear and plastid phylogenies. This is ex-pected, to some extent, since chloroplasts and nuclei displaydifferent inheritance patterns. Plastids are indeed generally unipa-rentally transmitted through seeds in most angiosperms (Birky,2008). Plastid or nuclear capture via introgression is known to lead

REX computes a unique scenario of horizontal transfers inferred from a summarizedIlex species. Numbered arrows refer to potential introgression events, some of thems of species presently found in the same area (introgressions 1, 3, 4, 5, 6, 7 and 11),graphic areas (introgressions 2, 8, 9, 10, 12, 13, 14 and 15). (For interpretation of thes article.)

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to incongruence between nuclear and chloroplast phylogenies(Soltis and Kuzoff, 1995; Rieseberg et al., 1996). Such introgressionevents are now well-documented in the genus Ilex (Manen et al.,2002; Setoguchi and Watanabe, 2000; Manen, 2004). Past or cur-rent gene flow between Ilex species therefore explains the greatdiscrepancy between the plastid phylogeny and the nuclear phy-logeny (Setoguchi and Watanabe, 2000; Manen et al., 2002; Gott-lieb et al., 2005) as well as that found between the plastidphylogeny and classical morphological and biosystematic classifi-cations (Baas, 1973; Loesener, 1901, 1942; Galle, 1997, 1975;Lobreau-Callen, 1975, 1977; Loizeau and Spichiger, 1992).

4.1. Taxonomy

The nuclear tree in Fig. 1 is similar to the trees obtained, onmuch less taxa, by Gottlieb et al. (2005) using ITS and AFLP. Thepresent study shows, for the first time, that the nuclear phylogenyis closer to the morphological classification than the plastid phy-logeny. Two traditional classifications (Loesener, 1901, 1942 andGalle, 1997) were compared with the Ilex phylogeny inferred fromnuclear data (Appendix A). Because of the process of introgressionleading to disjunction or homogenization of morphological andanatomical characters, molecular and morphological data are notexpected to be totally congruent. However, in several cases thedata are more or less in accordance, namely Polyphyllae (Galle),Stimatophorae (Galle), and Aquifolium (Galle). The more recentclassification of Galle (1997) better fits the molecular nuclear data.On the other hand, newly defined lineages (based on nuclear DNA)fit relatively well with geographic distribution patterns (amongothers: Aquifolium: Eurasia; Megalae alliance: South America(excluding the Andes); Indico-malaicae alliance: Southeast Asiaincluding Australia and New Caledonia).

Attempts to map morphological characters onto the nucleartree were disappointing, except for one character: abaxially punc-tate leaf lamina. All the nine species of the clade comprising Poly-phyllae and Stimatophorae display such a character. However, fourother species from other clades/phylogroup (I. laurina, I. coriacea,I. glabra and I. spicata) also have punctate leaves (cork warts, Baas,1975) that could be interpreted either as convergence or as inter-lineage introgression (see below). The function of these featuresis poorly known and is supposedly a wound response to the dete-rioration of large stomata (Korn and Fredrick, 1973). An anatomicalcomparison of this character in the Polyphyllae and Stimatophoraeclades, and elsewhere in the tree, is therefore needed.

4.2. Dating

Ilex dating is complicated due to several factors. First, rootingthe genus Ilex is difficult to assess because extensive extinctionsmust have occurred that blur the relationships of this genus withits putative close relatives, Helwingia and Phyllonoma. This situa-tion introduces bias in substitution rates and node dating. Second,full alignments of Phyllonoma with Ilex were impossible for ITS,nepGS and for some regions of trnL-F and atpB-rbcL. This supportsthe hypothesis of Olmstead et al. (2000) and Soltis et al. (2000) thatthe relationship should be (Phyllonoma (Ilex, Helwingia)). This prob-lem meant that only a single outgroup exists which does not per-mit the definition of the exact position of the root of the Ilex-Helwingia pair and makes dating questionable using calibrationof this root node at 69 MY (the assumed date of the emergenceof Ilex according the fossil record). Low age values were obtainedfrom 18S rDNA and rbcL sequences using four outgroups (Gonocar-yum, Irvingbaileya, Phyllonoma and Helwingia) and five representa-tive Ilex species (Table 2). The age of Ilex MRCA was found to bearound 13 and 30 MY for 18S rDNA and rbcL, respectively, which-ever type of molecular clock was used for analyses.

The first insight into Ilex dating is the discrepancy found be-tween 18S rDNA and rbcL dating. Table 2 shows a discrepancy be-tween ages derived from first and second versus third codonpositions in rbcL. A similar codon effect has been observed withrbcL, in calculating the age of angiosperms (Sanderson and Doyle,2001), with first and second rbcL codons giving a higher age thaneither the third codon or 18S rDNA. It can be argued that firstand second codon positions may be less saturated than the thirdone, thus the age given by the former might be more accurate.However, the substitution model used to calculate branch lengthsexplicitly permits multiple hits and should partly correct for this(Sanderson and Doyle, 2001). Magallon and Sanderson (2005) re-ported a similar codon effect for rbcL but not for other plastid genessuch as atpB, psaA and psbB. Sequence comparison of rbcL in the116 Ilex specimens analyzed here shows particularly low transi-tion/transversion (0.70) and synonymous/replacement substitu-tion ratios (0.81), as well as a low number of substitutions at thethird codon position (41% of the total number of substitutions, datanot shown). Similar results were previously seen in Ilex by Manenet al. (1998) again with rbcL. One explanation might be the influ-ence of neighboring base composition that has been shown toinfluence the transition/transversion ratio (Morton, 1995). What-ever the explanation, the singular discrepancy associated with co-don positions of rbcL should be investigated further before thisgene can be used for age calculation, as already mentioned bySanderson and Doyle (2001).

The second insight into Ilex dating is the discrepancy found be-tween age values obtained from Ilex nuclear and plastid phyloge-nies using a relaxed or a strict clock (33.6 versus 13.4 MY and46.5 versus 15.6 MY, respectively, see Table 2). Fig. 1 (nuclear data)shows that, with the suggested relaxed clock model, the branchsubstitution rate of Helwingia is much higher (5.6 � 10�3 substitu-tions per site and per MY) than the one of the stem branch of Ilex(2.7 � 10�3 substitution per site and per MY). The difference iseven higher for plastid data (Fig. 2): 7.8 � 10�4 and 1.9 � 10�4 sub-stitution per sites and per MY, respectively (data not shown). In-serts in Figs. 1 and 2 represent the nuclear and plastidphylograms, respectively, where branch lengths are proportionalto the number of substitutions. These phylograms contradict thefact that none of the two clock models was preferred for the nine18S rDNA and rbcL sequences studied in Aquifoliales using theBayesian factor criteria. This illustrates the difficulty encounteredin rooting the trees shown in Fig. 1 and 2. Without an additionaloutgroup, any DNA substitution model will artificially determinethe relative substitution rate of both root branches, especially ifone branch is represented by a single species (Helwingia japonica)whereas the other corresponds to the stem branch of 108 species(Ilex). An overestimation of the age of the most recent commonancestor of Ilex is therefore suspected in the relaxed clock model.

The situation is different with a strict molecular clock (Table 2).Using the same model of substitution as before (GTR+I+G), the ageof Ilex MRCA is found to be 13.1 MY and 13.6 MY based on nuclearand plastid data, respectively. Omitting the first and second codonpositions of rbcL, these age estimates better fit the age values ob-tained previously with four outgroups and five representative Ilexspecies using 18S rDNA and, as explained previously (Table 2),remaining cautious about the use of rbcL sequences.

Compared with the oldest fossils of Ilex (69 MY), the commonancestor of extant species is then relatively recent: around 15 MYwhich corresponds to Mid-Miocene. This discrepancy between ex-tent DNA phylogeny and fossil record can be due to major extinc-tions of sister lineages of Ilex, leading to the confusion between thestem age and the age of the most recent ancestor of extant Ilex, andto a huge phylogenetic gap between Ilex and its now widely ac-cepted sister lineages, Helwingia and Phyllonoma. The history ofthe phylogenetic position of Aquifoliaceae supports this hypothe-

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sis: in the past, Ilex was placed in Cornales, Theales, Celastrale orothers orders. The difficulty in placing the Ilex lineage withinangiosperms comes from the lack of any apparent transition be-tween the genus and all other angiosperm lineages. Many sisterlineages of extant Ilex must therefore have disappeared, leavingthis lineage ‘‘without a root”. This indirect evidence points to theexistence of a very large gap between the time of the divergenceof the most ancient ancestor of Ilex and the age of the most recentcommon ancestor of extant species. A similar situation has beenreported for Pinus, for which the phylogenetic position is howeverknown since a long time: Willyard et al. (2006) indeed reportedthat a recent divergence of the most recent ancestor of extant Pinusspecies may have been obscured by the antiquity of the stem line-age indicated by fossil records.

Because the time of diversification of extant Ilex species israther imprecise and covers a significant span of geologic time,when including standard deviations, we do not want to speculatetoo deeply on the date of the evolutionary events that shaped Ilex.It can be however noted that North American/East Asian disjunc-tions are found frequently in Ilex. In a study of eleven diverseangiosperms lineages it has been observed that North American/East Asian disjunctions are relatively recent, and date back to12.5–0.3 MY ago (Xiang et al., 2000). This further supports a ratherrecent origin of Ilex MRCA in the Miocene.

It is interesting to determine until when the North-AtlanticBridge and/or the Bering Strait may have favoured Ilex dispersions.Fossil records indicate that these dispersions are rather recent. Fos-sils of Ilex have been found in Iceland as recently as in the late-Mio-cene/Pliocene (Rundgren and Ingolfsson, 1999) and Holocene(Levac et al., 2001), indicating the possibility of North-Atlanticcommunications at that time. Interestingly, Rundgren and Ingolfs-son (1999), working in Iceland, have reported a transition from aflora dominated by American elements during the Miocene toone with more European affinities later on. This indicates Europetowards North America flux for later migrations as opposed tothe reverse direction for earlier ones. The Bering Strait may haveplayed a role in dispersion during approximately the same period:late-Miocene/Pliocene (Tiffney and Manchester, 2001). Pollen ofIlex from the late Miocene has been found in Alaska (Reinink-Smithand Leopold, 2005). Thus, all North American/East Asian disjunc-tions observed in Ilex may have occurred relatively recently, some-thing that does not contradict the recent age of the most recentcommon ancestor of the extant Ilex species.

Another frequent disjunction in Ilex is observed between NorthAmerica and South America. Studying fossil data from 14 paleoflo-ras located from southern North America to Central America andnorthern South America, Graham (1999) reported a gradient of Ilexfossils from Eocene to Pleistocene in North America, no fossil olderthan Pliocene in Costa Rica, and again no fossil older than recenttime in Panama. This is in agreement with a very recent migrationof Ilex species from North America to South America, probably fol-lowing the rise of the Isthmus of Panama about 3–3.5 MY ago. Sucha recent colonization time for South America is also compatiblewith the time of divergence of all extant species estimated here.

4.3. Phylogeography

Based on fossil records, the genus Ilex was claimed to be cosmo-politan at the end of the Cretaceous period (Cronquist, 1988), and afurther worldwide diversification seems to have occurred duringthe Eocene (see Cuénoud et al., 2000). However, the most recentcommon ancestor of extant Ilex species is estimated here to be rel-atively recent (Miocene), which suggests that extinctions of earlierdiverging lineages probably took place between the late Creta-ceous period and the Oligocene or even later. Kvacek et al. (2009)reported that as late as at the Pliocene in continental Europe

several Ilex species existed where only three remain today(I. aquifolium, I. spinigera and I. colchica). If the most recent ancestorof the Ilex plastome originated in Miocene in East Asia, according toour estimates (Fig. 4), the geographical origin of the genus itself isuncertain. An East Asian ancestry is suggested (Fig. 3), as being themost likely because the Helwingia outgroup is Asian. This locationassumes however that no large extinctions and or geographicalshifts have occurred historically in Helwingia or Ilex, which seemsrather unlikely. From extant geographic locations, Fig. 3 suggeststhat the most ancient species of the genus Ilex were distributedin the Northern Hemisphere (East Asia and North America) in theMiocene.

Most of the dispersal events suggested in Fig. 3 involve classicaldisjunctions (East Asia/North America (in both directions), NorthAmerica/South America (in both directions), East Asia/southeastAsia (including North Australia), East Asia/Africa, North America/Caribbean Islands, and North America/central America. Someexceptions exist, among them Asian/South American disjunctions.For instance, the Stigmatophorae clade, sister of I. teratopis distrib-uted in the Andes, dispersed to East and Southeast Asia directlyfrom South America. We hypothesize that this apparent long dis-tance dispersal could have occurred via North America, as sug-gested on a large scale by the analysis of plastid clades/phylogroups 3 and 4. Indeed, the American phylogroup 3 and theEurasian clade 4 are linked by a deletion in the plastid psbA-trnHspacer (Selbach-Schnadelbach et al., 2009), although this link isnot statistically supported in our analysis. The Eurasian clade 4 isalso found in the nuclear tree, but the American phylogroup isnot (Fig. 3). This phylogroup comprises all South American speciesstudied in this work (including central American and Caribbeanspecies) and it is split into four well-supported clades in the nucle-ar tree, Cassinoides, Repandae and Polphyllae on the one hand, andMegalae alliance on the other. As it contains some North Americanspecies (in Cassinoides and Repandae), one can postulate that thehub of communication between clades 3 (Eurasian) and 4 (mostlySouth American) was North America, as suggested for Stimatopho-rae. This example also suggests, since all South American plasto-mes are genetically related although their nucleus belong tounrelated nuclear backgrounds, that hybridization, chloroplastcapture and loss might explain the geographic homogenization ofthe plastid genome in South America, as already suggested byManen et al. (2002).

Inference on the origin of some island species could be donesuccessfully. Both nuclear and plastid data point to a South Amer-ican origin for the Hawaiian species I. anomala, to a North Ameri-can origin for the Carribean species I. repanda. Based on nucleardata, the New Caledonian species I. sebertii is suspected to havedispersed from Southeast Asia (Fig. 3), but its plastome surpris-ingly seems to originate from South America. Finally, nothing addi-tional can be said about I. canariensis, a species found exclusively inthe Canary Islands, because of its undetermined positions both inthe nuclear and the plastid trees, as already observed by Manenet al. (2002).

4.4. Hybridization and introgressions

As foreseen for Ilex species that are known to potentially expe-rience inter-specific hybridization (Baas, 1978; Setoguchi andWatanabe, 2000; Manen et al., 2002; Manen, 2004), the nuclearand chloroplast trees are not phylogenetically congruent, probablybecause of chloroplast capture, recombination and gene conversionin the nuclear genome. Nuclear introgression from a species (thedonor) to another species (the recipient) can result from inter-specific hybridization when large amounts of pollen of the donorare loaded on the recipient. The recipient species conserves itsmaternally inherited plastid but, after some generations, its

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nuclear genetic background can be replaced by the one of the do-nor species, through successive backcrosses. This situation simu-lates the capture of the plastome of the recipient species by thedonor species (Rieseberg, 1995; Rieseberg et al., 1996). A similarresult could also be observed following gene conversion or con-certed evolution when nuclear sequences of hybrids are convertedtowards the paternal type, leading to a maternal plastome inte-grated into a paternal nuclear background, although conversionto the maternal type seems more frequent (Chiang et al., 2001;Rønsted et al., 2007).

In this study, the influence of hybridization has been assessed inthree ways: (1) analysis of three recognized natural hybrids, (2)analysis of sequence variability within eight species representedby two specimens and (3) modeling of hybridization events usingT-REX.

(1) Natural hybridsFor I. x kiusiana, known as a hybrid of I. buergeri and I. integra,

only the male parent was confirmed to be I. integra. No inferencescould be done on the maternal donor (I. buergeri) because of thepoorly supported position of I. x kiusiana in the plastid phylogeny.Either recurrent backcrosses with the paternal I. integra or geneconversion toward the integra nuclear genome may have happenedin this case. The same is true for I. x makinoi, assumed to be a nat-ural hybrid between I. leucoclada and I. rugosa. The paternal donoris probably I. leucoclada, but because of a poorly supported positionof I. x makinoi in the plastid phylogeny, nothing can be said aboutthe mother. Finally, for I. x attenuata, reported to be a hybridbetween I. opaca and I cassine, the maternal donor is probablyI. cassine which was found close to the former species in the chlo-roplast tree, but nothing conclusive was found on the paternal par-ent since the hybrid was also close to I. cassine for the nucleardataset. This could be due to gene conversion toward the maternalparent. The first two examples would correspond to classical chlo-roplast capture due to pollen loading disequilibrium, leading torecurrent backcrosses, or to paternal gene conversion whereasthe last example would be better explained by maternal geneconversion.

(2) Sequence variabilityIn only two out of the eight species represented by two spec-

imens (I. purpurea and I. rotunda), similar sequences were foundfor both the nuclear and the chloroplast sequences. For all otherspecies, divergent and often paraphyletic sequences were found.In the case of I. fargesii, and I. yunnanensis, the two analyzed spec-imens were assigned to different subspecies/varieties: I. fargesiivar. brevifolia versus I. fargesii subsp. melanotricha and I. yunnan-ensis versus I. yunnanensis var. gentilis, respectively (Cuénoudet al., 2000). This classification has been confirmed using morpho-logical characters by Chen et al. (2008) who also recognizedI. fargesii subsp. melanotricha as I. melanotricha. For the remainingfour species (I. theezans, I. integra, I. cornuta and I wallichii), noindication of subspecies variability exists. Explanations for suchdifferences within specimens of the same species could be misi-dentification or, again, partial inter-specific hybridization. Sincespecimens have been carefully examined, the existence of poly-morphism within species for the analyzed sequences could reflecthybridization with either past or ongoing introgression, or chloro-plast captures.

(3) Modeling of hybridization events using T-REXIn agreement with suspected incidences of hybridization, T-REX

gave an estimate of 15 hybridization events needed to reconcilethe nuclear tree with the plastid tree for only 25 selected species.Interestingly, hybridization (and chloroplast capture) was ex-pected to have occurred both between closely related species (asobserved above in natural hybrids) and between more geneticallydistant species (as inferred by T-REX). This indicates that reproduc-tive barriers are weak among Ilex species and that species that have

been isolated for a while can hybridize when they come into sec-ondary contact. Two examples have been chosen to illustrate suchmechanisms.

(1) Seven introgressions happened between unrelated speciesfound in the same geographic area. In these instances, the con-cerned lineages probably arose at different times and were origi-nally not in contact with each other, becoming sympatric laterdue to dispersal. This could be the case of I. argentina which be-longs to the Megalae alliance, a lineage that dispersed to SouthAmerica from East Asia (Fig. 3). Looking at the plastid tree, its plas-tome is not found to be related to Megalae alliance, but is rathersister to I. laurina, a member of Repandae and close to Polyphyllae,both lineages that are assumed to have dispersed to South Americafrom North America (Fig. 4). T-REX (arrows 4 and 11, Fig. 5) indi-cates that I. argentina is the donor species in an horizontal transferwith I. laurina (Repandae) and I. amara (Polyphyllae) as recipients,respectively. The explanation could be that, when they dispersedto South America, Polyphyllae or Repandae (or their ancestor)came into contact with the ancestor of I. argentina, representativeof the Megalae alliance. Introgression of the ancestor of I. argentinainto ancestors of Repandae or Polyphyllae may have lead to thecurrent species I. argentina, having the genetic background of theMegalae alliance and the plastid of Repandae/Polyphyllae. The factthat I. argentina is tetraploid (Barral et al., 1995) gives some sup-port to this hybridization scenario. Similar examples can be foundin other tree genera, Quercus, for instance, contains several exam-ples of chloroplast replacements, one between Q. suber and Q. ilexin Morocco (Belahbib et al., 2001), the other between Q. roburand Q. petraea in Europe (Petit et al., 1997) or in Betula with exten-sive chloroplast sharing between B. nana, B. pendula and B. pubes-cens (Palme et al., 2004).

(2) In 8 other cases, species involved in introgression events de-tected by T-REX are geographically distant today. Either the ances-tors of these species were in contact in the past, or introgressionevents resulted from long distance dispersals. We dismiss the lat-ter explanation for the South American–Asian disjunction shownto have occurred through North America. We suggest that this isthe case for the South American Megalae alliance that dispersedfrom East Asia (Fig. 3). In the plastid tree (Fig. 4) most membersof the Megalae alliance form a clade sister to a clade comprisingall members of the North American clade Cassinoides. T-REX (ar-row 10, Fig. 5) indicates that I. brevicuspis (Megalae alliance) isthe donor in a horizontal transfer with I. cassine (Cassinoides) as re-cipient. This would indicate that the dispersal of Megalae alliancefrom East Asia to South America occurred via North America,which would not require a long distance dispersal event. Megalaealliance would thus be the result of hybridization with an ancestorof Cassinoides in North America before dispersing in SouthAmerica.

4.5. Perspectives

Data is not sufficient to obtain fully resolved trees. However,our results suggest the presence of weak reproductive barriers be-tween species, resulting in frequent natural hybridization events. Itis questionable whether the biological species concept, based oncomplete reproductive isolation, is applicable to this genus. If retic-ulate evolution is confirmed, particularly in deep branches of theIlex phylogenetic tree, the methods used here to reconstruct thehistory of this genus (phylogenetics, dating and phylogeography)would need refining as they are based on simple branch dichot-omy. Hybridization and reticulation would explain the poor resolu-tion of phylogenetic trees and the possible underestimation of theMRCA age because of genome homogenization driven by lowreproductive barriers and DNA sequence conversion. Moreover, ifone considers that hybridization has played an important role in

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plant speciation events (Stebbins, 1959; Mallet, 2005, 2007) thecomplexity seen in the genus Ilex might be the rule, rather thanthe exception, in most tree lineages (Grimm et al., 2007).

According to our results, further explorations into Ilex historycould be done in two ways. The first approach would be to analysemultiple specimens per species, sampled in different localities. Thiswould permit the screening of species that show signs of hybrid-ization from the others as observed for I. canariensis (Manen,2004). In addition, phylogeographic analysis could be conductedfocusing on lineages of species found in the same geographic area,as suggested by Barraclough and Nee (2001). This means that agroup of species could be selected to investigate hybridization be-tween close or distant species, chloroplast capture and gene con-version. Going deeper into the phylogeographic pattern of somesympatric species, using larger sampling sizes per taxon, couldfacilitate the exploration of the different mechanisms suggestedto explain the discrepancy between the two phylogenies. Popula-tion studies on five Ilex species, I. paraguariensis (Gauer and Caval-li-Molina, 2000), I. aquifolium (Rendell and Ennos, 2003),I. leucoclada (Torimaru et al., 2003, 2004), I. canariensis (Manen,2004) and I. cornuta (Son et al., 2007) confirm a rather high popu-lation diversity and the polymorphism observed in the presentstudy for six out of eight species indicates that more investigationsare needed at population or species level to ensure a better charac-terization of Ilex species. The existence of different sequences with-in the same species, as found in several examples in this study, is afirst indication of some phylogeographic structure for the con-cerned taxa (Schaal et al., 1998).

A second approach would be to sequence more species in an at-tempt to obtain better resolved trees. A complementary approachwould be to add other single copy nuclear genes, like nepGS, toavoid the concerted evolution or gene conversion observed in mul-ticopy ribosomal sequences (Rønsted et al., 2007). Better resolvedtrees would lead to better estimates of node ages and ancestralareas, and facilitate the examination of whether ancestors of extantlineages that are now geographically isolated were once in contact,and whether introgression could have occurred. It is foreseen that,adding more species or genes to the dataset, based on the frequenthybridization events seen in the Ilex genus, would lead to morehybridization/recombination events being outlined. This wouldthen result in even more divergent phylogenies revealing an evenmore complex history then the one depicted here. The rooting ofIlex will remain a problem as it will preclude the use of some se-quences that cannot be aligned with divergent outgroups, as itwas the case here for Phyllonoma.

The phylogenies and the inferred lineages presented here arethus a starting-point from which to revisit the classification of Ilex.At this point it would be interesting to investigate whether thewell-supported clades actually contain morphological features thathave so far been overlooked.

Acknowledgments

We thank Lorène Aeschbach and Gisèle Vuille-dit-Bille fortechnical assistance in the lab. We thank Jérôme Munzinger (IRD,New-Caledonia), Lyn Craven (CSIRO, Australia) and Darren Crayn(National Herbarium of New South Wales, Australia) for providingkey specimens for this study as well as the two anonymous refer-ees for their constructive comments.

Appendix A

Below is a description of all clades/phylogroups observed inFig. 1 (nuclear phylogeny) in parallel to the Loesener’s and Galle’sclassifications. In Loesener (1901, updated in 1942), species are ar-ranged in subgenera, series (Reihe), sections and subsections. In

Galle (1997) they are arranged in subgenera, sections, subsections,and series. The clades credibility values, their geographic distribu-tions and habitat zones are given.

Clade I. amara–I. dumosa (Polyphyllae) comprises, in the subge-nus Euilex, members of sections Polyphyllae and Brachythyrsae ofLoesener. However, the three species belong to serie Polyphyllaein the subgenus Byronia of Galle (well sustained: CV = 1.00; distri-bution: South America, excluding Andes; habitat zone: tropical totropical montane).

The sister clade I. teratopis–I. triflora (Stigmatophorae) com-prises the monospecific subgenus Yrbonia, and members of sec-tions Polyphyllae, Microdontae and Rugosae (subgenus Euilex) ofLoesener. It comprises all studied members of serie Stigmatopho-rae, one member of section Byronia (subgenus Byronia) and onemember of serie Hookerianae (subgenus Aquifolium) of Galle(weakly sustained: CV = 0.66, well sustained when I. teratopis is ex-cluded: CV = 1.00; distribution: Andes, East Asia and South-East-Asia; habitat zone: temperate, tropical to tropical montane).

Clade I. tolucana–I. nitida (Repandae) comprises members ofsubsection/serie Repandae, one member of subsection/serieVomitoriae and one member of section/serie Daphnophyllae forboth authors (well sustained: CV = 1.00; distribution: NorthAmerica, Central-America, Caribbean Islands and South Americaincluding Andes; habitat zone: subtropical, tropical to tropicalmontane).

Clade I. collina–I. montana (Prinoides) comprises members ofsubgenus Prinus/Prinos, section Prinoides for both authors, exceptI. curtissi and I. collina described after the Loesener’s monograph,thus unclassified by him (well sustained: CV = 1.00; distribution:North America; habitat zone: temperate).

Clade I. amelanchier–I. mucronata (Mucronata alliance) I. amel-anchier belongs to subgenus Prinus/Prinos and section Prinoidesfor both authors who do not classify I. mucronata (see Powellet al., 2000) considering it as belonging to genus Nemopanthus(well sustained: CV = 1.00; distribution: North America; habitatzone: temperate).

Clade I. cassine–I. rubra (Cassinoides) comprises members ofsection/serie Cassinoides for both authors, except I. x attenuataand I. cumulicola not classified by Loesener (well sustained:CV = 1.00; distribution: North America and Caribbean Islands; hab-itat zone: temperate, subtropical to tropical montane).

Clade I.canariensis–I. liebmanni (Glabra) belongs to section/serieCassinoides for both authors, except I. liebmannii, species unknownby Loesener (not sustained: CV = 0.59, well sustained when exclud-ing I. canariensis: CV = 1.00; distribution: North and Central-Amer-ica, Canary Islands; habitat zone: temperate, subtropical to tropicalmontane).

Clade I. argentina–I integerrima (Megalae alliance) comprisestaxa of different series and sections of Loesener (serie Lioprinus,section Excelsae, serie Paltoria, section Vacciniifoliae and serieAquifolium, sections Micranthae, Megalae and Microdontae andserie Eubyronia). The clade is not better recognized by Galle. Bothauthors recognize the sub-clade I. brasiliensis–I. theizans compris-ing all the studied species of section/serie Megalae. It is to be notedthat I. argentina belongs to this clade as suggested by Gottlieb et al.(2005) and in disagreement with Manen et al. (2002) in which anerror must have occurred (well sustained: CV = 1.00; distribution:South America, excluding Andes, Central-America, Caribbean Is-lands and Hawai; habitat zone: tropical, subtropical to tropicalmontane).

Clade I. pubescens–I. purpurea (Rotunda alliance) belongs to ser-ie/section Lioprinus and Aquifolium for both authors (well sus-tained: CV = 0.98; distribution: East-Asia; habitat zone:subtropical to tropical).

Clade I. geniculata–I. verticillata (Verticillata alliance) belongs tosection Euprinus (Loesener) or Prinos (Galle) (well sustained:

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CV = 1.00; distribution East Asia and North America; habitat zone:temperate).

Clade I. pedunculosa–I. shennongjiaensis (Yunnanensis alliance)comprises species of section/serie Cassinoides of Loesener and Galle,except I. shennongjiaensis unclassified by Loesener and I. pedunculosathat belongs to subsection/serie Umbelliformes (well sustained:CV = 0.98; distribution East-Asia; habitat zone: temperate).

Phylogroup I. mitis–I. aculeolata (Micrococca alliance) mainlybelongs to subgenus Prinos, sections Micrococca, Prinos, Prinoidesand Pseudoprinos with the exception of I. mitis (subgenusAquifolium, serie Lemurenses) for Galle. The clade is not better rec-ognized by Loesener. The sub-clade I. macrocarpa–I. aculeolata be-longs to sections Prinoides and Pseudoprinos of Galle and tosection Prinoides of Loesener except for I. kusanoi, I. aculeolataand I. tsoi described after 1901 (relatively well sustained:CV = 0.96; distribution: Africa, East-Asia; habitat zone: temperateto subtropical).

Clade I. wilsonii–I. goshiensis (Longecaudatae alliance) belongs tosection Pseudoaquifolium serie Longecaudatae of Galle. Loesenerput I. wilsonii in serie Aquifolium, subsection Sideroxyloides, anddid not classify I. goshiensis (well sustained: CV = 1.00; distribu-tion: East-Asia; habitat zone: subtropical).

Phylogroup I. sebertii–I. wallichii (Indico-malaicae alliance) com-prises, in Loesener and in Galle, taxa of different series/sections,belonging to different subgenera. Both authors agree with serie/section Eubyronia/Byronia (I. nervulosa, I. cymosa, I. wallichii, I. arn-hemensis and I. macrophylla), serie/section Lioprinus (I. sebertii) andserie/section Thyrsoprinus (I. spicata and I. cissoidea), in sectionIndico-Malaicae for Loesener and in serie Spicatae for Galle. Theydo not match with the classification of the three species left (I. hav-ilandii, I. oppositifolia and I. maingayi). I. zygophylla is not classifiedby Loesener. This clade contains all the studied members of sectionIndico-malaicae. Galle recognizes the sub-clade I. oppositifolia–I.zygophylla as ‘‘Opposite-leaved” (not sustained: CV = 0.71, wellsustained when excluding I. sebertii, I. spicata and I. havilandii:CV = 1.00; distribution: South-East-Asia, Australia and New Cale-donia; habitat zone: tropical to tropical montane).

Clade I. rugosa–I. cyrtura (Aquifolium). This large Eurasian cladecomprises all members of serie Aquifolium of Loesener, excludingthe Asian species I. revoluta, I. maximowicziana, I. maingayi, I. goshi-ensis, I. triflora, I. pubescens, the African species I. mitis and Ameri-can species I. argentina, I. discolor, I. tolucana, I. nitida, I.paraguariensis, I. repanda I. brevicuspis, I. microdonta, I. vomitoria,I. laurina, I. brasiliensis, I. theezans, I. integerrima, I. guianensis. Thisclade is closer to section Aquifolium of Galle, only excluding I. rev-oluta, I. maingayi, I. mitis, I. argentina, I. discolor, I. tolucana, I. nitida,I. paraguariensis, I. repanda. One species of this clade, I. matanoana,was not classified in serie/section Aquifolium but in serie/sectionPaltoria by both authors. This position of I. matanoana is confirmedby cpDNA data (see also Fig. 3). I. kinabaluensis recently describedin Borneo (Andrews, 1998) belongs to this clade, extending thearea of secondary distribution of this clade to South-East-Asia.The phylogenetic position of hybrids I. x kiusiana (natural hybridof I. integra and I. buergeri) and I. x makinoi (natural hybrid of I. rug-osa and I. leucoclada) fits well with the phylogenetic position of oneof their parents (well sustained: CV = 1.00; distribution: East Asiato southeast Asia and Europe to North Africa and the Canary Is-lands; habitat zone: temperate, subtropical, tropical montane).

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