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<strong>Generic</strong> <strong>relationships</strong> <strong>and</strong> <strong>dat<strong>in</strong>g</strong> <strong>of</strong> <strong>l<strong>in</strong>eages</strong> <strong>in</strong> W<strong>in</strong>teraceae based on nuclear<br />
(ITS) <strong>and</strong> plastid (rpS16 <strong>and</strong> psbA-trnH) sequence data<br />
Xavier Marquínez a, *, Lúcia G. Lohmann b , Maria L. Faria Salat<strong>in</strong>o b , Antonio Salat<strong>in</strong>o b , Favio González a<br />
a Universidad Nacional de Colombia, Apartado Aéreo 7495, Bogotá D.C., Colombia<br />
b Universidade de São Paulo, Departamento de Botânica, Instituto de Biociências, Rua do Matão, 277, CEP 05508-090, São Paulo, SP, Brazil<br />
article <strong>in</strong>fo<br />
Article history:<br />
Received 26 November 2008<br />
Revised 30 June 2009<br />
Accepted 1 July 2009<br />
Available onl<strong>in</strong>e 4 July 2009<br />
Keywords:<br />
Canellales<br />
Divergence time estimates<br />
Drimys<br />
Gondwana biogeography<br />
Pseudow<strong>in</strong>tera<br />
Takhtajania<br />
Tasmannia<br />
W<strong>in</strong>teraceae<br />
Zygogynum<br />
1. Introduction<br />
abstract<br />
W<strong>in</strong>teraceae was first described as a family by L<strong>in</strong>dley (1836).<br />
Its current circumscription comprises five genera ( Drimys,<br />
Pseudow<strong>in</strong>tera, Takhtajania, Tasmannia, <strong>and</strong> Zygogynum s.l.) <strong>and</strong><br />
approximately 79 species distributed <strong>in</strong> Australia, Tasmania, Borneo,<br />
Celebes, Moluccas, New Caledonia, New Gu<strong>in</strong>ea, New Zeal<strong>and</strong>,<br />
Philipp<strong>in</strong>es, Madagascar, <strong>and</strong> Central- <strong>and</strong> South America (V<strong>in</strong>k,<br />
1993a, 2003). The family belongs to the order Canellales, along with<br />
Canellaceae (APGII, 2003; Cai et al., 2006). The monophyly <strong>of</strong> the order<br />
is supported by molecular <strong>and</strong> morphological data (e.g., Chase<br />
et al., 1993; Qiu et al., 1993, 1999; N<strong>and</strong>i et al., 1998; Doyle <strong>and</strong><br />
Endress, 2000; Soltis et al., 2000, 2005; Zanis et al., 2002).<br />
The W<strong>in</strong>teraceae possess vesselless xylem <strong>and</strong> plicate carpels,<br />
two conditions long considered ancestral <strong>in</strong> the angiosperms<br />
(van Tieghem, 1900; Bailey <strong>and</strong> Thompson, 1918; Bailey <strong>and</strong><br />
Swamy, 1951; Takhtajan, 1980; Cronquist, 1981). However, those<br />
conditions have recently been re<strong>in</strong>terpreted as secondarily derived<br />
(Young, 1981; Soltis et al., 2005). The physiological implications <strong>of</strong><br />
vesselless xylem <strong>and</strong> the presence <strong>of</strong> stomatal plugs have been<br />
suggested as a result <strong>of</strong> the relictual condition <strong>of</strong> the family by<br />
Bailey <strong>and</strong> Nast (1944) <strong>and</strong> Baranova (1972). A re<strong>in</strong>terpretion <strong>of</strong><br />
* Correspond<strong>in</strong>g author. Fax: +57 1 3165310.<br />
E-mail address: xmarqu<strong>in</strong>ezc@unal.edu.co (X. Marquínez).<br />
1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.<br />
doi:10.1016/j.ympev.2009.07.001<br />
Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
Contents lists available at ScienceDirect<br />
Molecular Phylogenetics <strong>and</strong> Evolution<br />
journal homepage: www.elsevier.com/locate/ympev<br />
Phylogenetic analyses <strong>of</strong> representative species from the five genera <strong>of</strong> W<strong>in</strong>teraceae (Drimys, Pseudow<strong>in</strong>tera,<br />
Takhtajania, Tasmannia, <strong>and</strong> Zygogynum s.l.) were performed us<strong>in</strong>g ITS nuclear sequences <strong>and</strong> a comb<strong>in</strong>ed<br />
data-set <strong>of</strong> ITS + psbA-trnH+rpS16 sequences (sampl<strong>in</strong>g <strong>of</strong> 30 <strong>and</strong> 15 species, respectively). Indel <strong>in</strong>formativity<br />
us<strong>in</strong>g simple gap cod<strong>in</strong>g or gaps as a fifth character was exam<strong>in</strong>ed <strong>in</strong> both data-sets. Parsimony<br />
<strong>and</strong> Bayesian analyses support the monophyly <strong>of</strong> Drimys, Tasmannia, <strong>and</strong> Zygogynum s.l., but do not support<br />
the monophyly <strong>of</strong> Belliolum, Zygogynum s.s., <strong>and</strong> Bubbia. With<strong>in</strong> Drimys, the comb<strong>in</strong>ed data-set recovers two<br />
subclades. Divergence time estimates suggest that the splitt<strong>in</strong>g between Drimys <strong>and</strong> its sister clade (Pseudow<strong>in</strong>tera<br />
+ Zygogynum s.l.) occurred around the end <strong>of</strong> the Cretaceous; <strong>in</strong> contrast, the divergence between<br />
the two subclades with<strong>in</strong> Drimys is more recent (15.5–18.5 MY) <strong>and</strong> co<strong>in</strong>cides <strong>in</strong> time with the Andean<br />
uplift. Estimates suggest that the earliest divergences with<strong>in</strong> W<strong>in</strong>teraceae could have predated the first<br />
events <strong>of</strong> Gondwana fragmentation.<br />
Ó 2009 Elsevier Inc. All rights reserved.<br />
these characters based on a phylogenetic framework <strong>in</strong>dicates that<br />
they might actually represent adaptations to changes from tropical<br />
to temperate (stational) habitats s<strong>in</strong>ce the early Cretaceous (Feild<br />
et al., 1998, 2000, 2002; Feild <strong>and</strong> Holbrook, 2000).<br />
The current available phylogenetic analyses for W<strong>in</strong>teraceae are<br />
not conclusive <strong>in</strong> terms <strong>of</strong> generic <strong>relationships</strong>. V<strong>in</strong>k (1988), based<br />
on morphological data obta<strong>in</strong>ed two alternative topologies:<br />
(Takhtajania (Drimys + Tasmannia)(Zygogynum s.l. + Pseudow<strong>in</strong>tera));<br />
<strong>and</strong> (Tasmannia (Drimys (Takhtajania (Zygogynum + Pseudow<strong>in</strong>tera)))).<br />
On the other h<strong>and</strong>, Endress et al. (2000), primarily based ma<strong>in</strong>ly on<br />
floral morphology, arrived at two topologies depend<strong>in</strong>g on the outgroup<br />
chosen: (Canella (Tahktajania + Pseudow<strong>in</strong>tera + Zygogynum)<br />
(Drimys + Tasmannia)); <strong>and</strong> (Degeneria (Zygogynum p.p.<br />
(Z. thieghemii (Takhtajania + Pseudow<strong>in</strong>tera))) (Drimys w<strong>in</strong>teri<br />
(Drimys p.p. + Tasmannia piperita) (Tasmannia p.p.)). The analysis<br />
based on Degeneria as the outgroup casts doubts on the monophyly<br />
<strong>of</strong> Drimys, Tasmannia <strong>and</strong> Zygogynum. F<strong>in</strong>ally, three separate analyses<br />
based on molecular data (Suh et al., 1993; Karol et al., 2000;<br />
<strong>and</strong> Doust <strong>and</strong> Dr<strong>in</strong>nan, 2004), co<strong>in</strong>cide <strong>in</strong> the generic relationship<br />
(Takhtajania (Tasmannia (Drimys (Pseudow<strong>in</strong>tera + Zygogynum<br />
s.l.)))). The controversial close relationship between Drimys <strong>and</strong><br />
Tasmannia, proposed by Smith (1943b, cf. also V<strong>in</strong>k, 1970, 1993a)<br />
is supported by all <strong>of</strong> the morphological analyses (except by one<br />
<strong>of</strong> V<strong>in</strong>k’s analysis <strong>and</strong> by the flower developmental data provided<br />
by Doust <strong>and</strong> Dr<strong>in</strong>nan, 2004).
436 X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
The taxonomy <strong>of</strong> the family has been reviewed by Smith<br />
(1943a, 1943b) <strong>and</strong> V<strong>in</strong>k (1970, 1977, 1983, 1985, 1988, 1993a).<br />
With<strong>in</strong> the family, two genera are particularly problematic, Drimys<br />
<strong>and</strong> Zygogynum. The current circumscription <strong>of</strong> Drimys (follow<strong>in</strong>g<br />
Smith, 1943b; Ehrendorfer et al., 1979; Rodríguez <strong>and</strong> Quezada,<br />
1991, 2001) <strong>in</strong>cludes seven species distributed from southern Mexico<br />
to Tierra del Fuego (Table 1). However, the genus has been<br />
poorly sampled <strong>in</strong> phylogenetic analyses (three species by Endress<br />
et al., 2000, <strong>and</strong> two species by Doust <strong>and</strong> Dr<strong>in</strong>nan, 2004), br<strong>in</strong>g<strong>in</strong>g<br />
to question its monophyly. Zygogynum has traditionally been splitted<br />
<strong>in</strong>to four <strong>in</strong>dependent genera, Belliolum, Bubbia, Exospermum<br />
<strong>and</strong> Zygogynum s.s. (van Tieghem, 1900; Smith, 1943a). Alternatively,<br />
V<strong>in</strong>k (1985, 1988, 1993a, 1993b) treats all these genera as<br />
Zygogynum s.l. Molecular analysis (Suh et al., 1993) supports the<br />
topology (Bubbia comptonii (Exospermum stipitatum, Zygogynum<br />
acsmithii (Z. pomiferum (Belliolum pancheri, Z. bicolor)))), <strong>in</strong> which<br />
Zygogynum s.s. is not monophyletic.<br />
Three compet<strong>in</strong>g hypotheses have been proposed to expla<strong>in</strong> the<br />
biogeographic history <strong>of</strong> the W<strong>in</strong>teraceae: (1) a South American<br />
orig<strong>in</strong> followed by dispersal to Africa <strong>and</strong> Madagascar on one side,<br />
<strong>and</strong> dispersal to Australasia via Antarctica on the other side (cf.<br />
Fig. 2A <strong>in</strong>Feild et al., 2000); (2) an African orig<strong>in</strong> followed by dispersal<br />
<strong>in</strong>to Madagascar <strong>and</strong> Antarctica, followed by subsequent<br />
dispersal <strong>in</strong>to America <strong>and</strong> Australasia (cf. Fig. 2B <strong>in</strong>Feild et al.,<br />
2000); <strong>and</strong> (3) a northern Gondwanic tropical orig<strong>in</strong> followed by<br />
dispersal <strong>in</strong>to southern temperate Gondwana <strong>and</strong> subsequent<br />
migration from America to Antarctica <strong>and</strong> to Australasia, or from<br />
Africa to Madagascar, India, <strong>and</strong> f<strong>in</strong>ally Australasia, after the fragmentation<br />
<strong>of</strong> Gondwana <strong>in</strong>to Africa <strong>and</strong> America (cf. Fig. 3 <strong>in</strong> Doyle,<br />
2000).<br />
The aims <strong>of</strong> the present study are to reconstruct the phylogenetic<br />
<strong>relationships</strong> between Drimys <strong>and</strong> the other genera <strong>of</strong> W<strong>in</strong>teraceae,<br />
to test the monophyly <strong>of</strong> Drimys, to explore the<br />
phylogenetic <strong>relationships</strong> <strong>in</strong>side Drimys <strong>and</strong> Zygogynum s.l., <strong>and</strong><br />
Table 1<br />
Taxonomic history <strong>and</strong> current geographic distribution <strong>of</strong> Drimys species.<br />
Forster <strong>and</strong><br />
Forster, 1776<br />
L<strong>in</strong>naeus f.,<br />
1781<br />
de C<strong>and</strong>olle,<br />
1817, 1824<br />
Sa<strong>in</strong>t-Hilaire,<br />
1824<br />
Philippi,<br />
1856<br />
to estimate divergence times <strong>in</strong> order to evaluate alternative biogeographic<br />
hypotheses for Drimys <strong>and</strong> the entire W<strong>in</strong>teraceae.<br />
2. Materials <strong>and</strong> methods<br />
2.1. Sampl<strong>in</strong>g<br />
Table 2 summarizes the voucher specimens <strong>and</strong> Genbank<br />
accession numbers for the taxa <strong>in</strong>cluded <strong>in</strong> the present study.<br />
Two data-sets were prepared for the various analyses: (1) A<br />
data-set <strong>in</strong>clud<strong>in</strong>g 52 sequences <strong>of</strong> ITS, represent<strong>in</strong>g 30 species <strong>of</strong><br />
W<strong>in</strong>teraceae <strong>and</strong> six species <strong>of</strong> Canellaceae; <strong>and</strong> (2) a comb<strong>in</strong>ed<br />
data-set <strong>in</strong>clud<strong>in</strong>g 23 sequences <strong>of</strong> ITS, 21 sequences <strong>of</strong> psbA-trnH<br />
<strong>and</strong> 20 sequences <strong>of</strong> rpS16 sequences represent<strong>in</strong>g 14 species <strong>of</strong><br />
W<strong>in</strong>teraceae, plus Capsicodendron denissi <strong>and</strong> Cynnamosma madagascarensis<br />
(Canellaceae) as the outgroup. In this data-set, sequences<br />
<strong>of</strong> Drimys roraimensis <strong>and</strong> Tasmannia lanceolata were<br />
miss<strong>in</strong>g for rpS16 <strong>and</strong> psbA-trnH, <strong>and</strong> sequences <strong>of</strong> Drimys confertifolia<br />
were miss<strong>in</strong>g for rpS16.<br />
2.2. DNA extraction, amplification <strong>and</strong> sequenc<strong>in</strong>g<br />
DNA was extracted from silica-gel dried leaves, air-dried leaves<br />
<strong>and</strong> herbarium specimens (Table 2). For DNA extraction, PCR<br />
amplification <strong>and</strong> sequenc<strong>in</strong>g, we followed the procedures described<br />
by Ferreira <strong>and</strong> Grattapaglia (1996). For PCR <strong>of</strong> ITS1-<br />
5.8S–ITS2 we used Primers Leu1 (Malcomber, 2002; Doust <strong>and</strong><br />
Dr<strong>in</strong>nan, 2004) <strong>and</strong> ITS4 (White et al., 1990; Suh et al., 1993).<br />
Amplification reactions conta<strong>in</strong>ed 3 ll MgCl2 (1.5 M), 5 ll <strong>of</strong><br />
amplification buffer 10 (160 mM (NH 4)SO 4, 670 mM Tris–HCl<br />
(ph 8.8), 0.1% Tween-20), 1 ll <strong>of</strong> bov<strong>in</strong>e serum album<strong>in</strong>e (BSA;<br />
5 lg/ll), 2.5 ll <strong>of</strong> DMSO, 0.5 ll <strong>of</strong> each primer (10 lM), 3 ll <strong>of</strong><br />
dNTPs (2 mM), 0,25 ll Taq DNA Polimerase, 2 ll <strong>of</strong> DNA <strong>and</strong><br />
32.75 ll <strong>of</strong>H2O. The follow<strong>in</strong>g PCR conditions were used: 80° for<br />
Miers, 1858 Reiche, 1898 Smith, 1943a Ehrendorfer et al.,<br />
1979<br />
D. brasiliensis<br />
var. roraimensis<br />
D. angustifolia D. brasiliensis<br />
var. angustifolia<br />
D. granadensis D. retorta D. brasiliensis<br />
D. montana<br />
vars. retorta<br />
D. brasiliensis<br />
& campestris<br />
D. granadensis D. granadensis D. granadensis D. granadensis D. granadensis a<br />
D. mexicana<br />
High mounta<strong>in</strong>s<br />
<strong>in</strong> Mexico <strong>and</strong><br />
Central America,<br />
Northern Andes<br />
<strong>of</strong> South America<br />
D. w<strong>in</strong>teri D. w<strong>in</strong>teri D. w<strong>in</strong>teri D. w<strong>in</strong>teri D. w<strong>in</strong>teri vars.<br />
D. chilensis D. chilensis<br />
w<strong>in</strong>teri & chilensis<br />
a Drimys species recognized <strong>in</strong> this paper.<br />
D. w<strong>in</strong>teri<br />
var. <strong>and</strong><strong>in</strong>a<br />
D. w<strong>in</strong>teri<br />
var. <strong>and</strong><strong>in</strong>a<br />
D. roraimensis a<br />
(Venezuelan<br />
tepuyes,<br />
Roraima)<br />
D. angustifolia a<br />
(South <strong>of</strong> Brazil)<br />
D. brasiliensis a<br />
(Brazil)<br />
Rodríguez <strong>and</strong><br />
Quezada,<br />
1991, 2001<br />
D. w<strong>in</strong>teri a<br />
Southern Andes<br />
<strong>of</strong> Chile <strong>and</strong><br />
Argent<strong>in</strong>a,<br />
Patagonia <strong>and</strong><br />
Tierra del Fuego<br />
D. <strong>and</strong><strong>in</strong>a a<br />
Nahuelbuta<br />
mounta<strong>in</strong>s <strong>and</strong><br />
Andes <strong>of</strong> Chile<br />
<strong>and</strong> Argent<strong>in</strong>e<br />
D. confertifolia D. fern<strong>and</strong>ezianus D. confertifolia D. confertifolia a<br />
Juan Fern<strong>and</strong>ez<br />
Isl<strong>and</strong>s (Chile)
5 m<strong>in</strong>; 35 cycles <strong>of</strong> 94° for 30 s; 48° for 1 m<strong>in</strong>.; 72° for 90 s; <strong>and</strong>,<br />
72° for 7 m<strong>in</strong>. ITS clon<strong>in</strong>g was not performed <strong>in</strong> Drimys because<br />
previous studies (Karol et al., 2000; Doust <strong>and</strong> Dr<strong>in</strong>nan, 2004)<br />
found only one copy after clon<strong>in</strong>g. Amplifications <strong>of</strong> psbA-trnH<br />
<strong>and</strong> rpS16 used primers <strong>and</strong> PCR conditions as described by Shaw<br />
et al. (2005). PCR products were cleaned us<strong>in</strong>g the GFX kit<br />
(Amersham Bioscience, Piscataway, NJ). Purified products were sequenced<br />
us<strong>in</strong>g amplification primers <strong>and</strong> the ABI Prism Big Dye<br />
Term<strong>in</strong>ator Cycle Sequenc<strong>in</strong>g Ready Reaction Kit (Applied Biosystems,<br />
Foster City, CA) on an ABI 3700 Ò Automated Sequencer.<br />
2.3. Phylogenetic analyses<br />
Forward <strong>and</strong> reverse sequences were compiled <strong>in</strong>to consensus<br />
sequences us<strong>in</strong>g MacClade 4.06 (Maddison <strong>and</strong> Maddison, 2003).<br />
Consensus sequences were aligned with Clustal X (Thompson<br />
et al., 1997), <strong>and</strong> subsequently adjusted manually. For psbA-trnH<br />
<strong>and</strong> rpS16, manual adjustments followed Borsch et al. (2003) <strong>and</strong><br />
Kelchner (2000); for ITS, adjustments followed Karol et al.<br />
X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449 437<br />
Fig. 1. Strict consensus tree result<strong>in</strong>g from parsimony analysis <strong>of</strong> the ITS data-set, with bootstrap values presented above branches, numbers before scientific names<br />
corresponds to different accessions <strong>of</strong> the same species; Zygogynum pomiferum spp. balansae V<strong>in</strong>k. (A) Consider<strong>in</strong>g gaps as miss<strong>in</strong>g, the arrowhead <strong>and</strong> the arrow <strong>in</strong>dicate<br />
the correspond<strong>in</strong>g clades derived from clones 1 <strong>and</strong> 2 sequences from Suh et al. (1993). (B) Us<strong>in</strong>g the simple gap cod<strong>in</strong>g option; numbers <strong>in</strong> parenthesis below branches<br />
correspond to gaps that support clades; circle <strong>and</strong> letters <strong>in</strong>dicate synapomorphies <strong>and</strong>/or homoplasies (see Table 3).<br />
(2000). Non-aligned regions between Canellaceae <strong>and</strong> W<strong>in</strong>teraceae<br />
were treated as non-overlapp<strong>in</strong>g <strong>in</strong>dels to m<strong>in</strong>imize <strong>in</strong>correct<br />
homology assessments.<br />
In order to test congruence among data-sets, we implemented<br />
two tests <strong>in</strong> PAUP version 4.0b10 (Sw<strong>of</strong>ford, 2002): (1) the ILD<br />
Test (Farris et al., 1994) us<strong>in</strong>g comb<strong>in</strong>ed ITS + rpS16 + psbA-trnH<br />
partitioned nexus matrix, 1000 replicates <strong>of</strong> r<strong>and</strong>om addition sequences<br />
<strong>and</strong> a maximum <strong>of</strong> 10 trees reta<strong>in</strong>ed per replicate; start<strong>in</strong>g<br />
trees were obta<strong>in</strong>ed via r<strong>and</strong>om stepwise addition with TBR branch<br />
swapp<strong>in</strong>g <strong>and</strong> (2) the SH test (Shimodaira <strong>and</strong> Hasegawa, 1999),<br />
us<strong>in</strong>g GTR + I + G model <strong>of</strong> DNA substitutions to compare the likelihood<br />
calculated for strict consensus trees <strong>of</strong> ITS, rpS16 <strong>and</strong><br />
psbA-trnH data-sets under parsimony (cf. Fig. 4); <strong>and</strong> also for<br />
majority rule consensus trees <strong>of</strong> these data-sets obta<strong>in</strong>ed under<br />
Bayesian analyses.<br />
Two <strong>in</strong>dependent analyses <strong>of</strong> the ITS <strong>and</strong> comb<strong>in</strong>ed data-sets<br />
were performed <strong>in</strong> PAUP version 4.0b10 (Sw<strong>of</strong>ford, 2002), us<strong>in</strong>g<br />
parsimony. These analyses were conducted us<strong>in</strong>g a heuristic search<br />
with 1000 replicates <strong>of</strong> r<strong>and</strong>om addition sequences <strong>and</strong> a
438 X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
Fig. 2. (A) Strict consensus tree result<strong>in</strong>g from a parsimony analysis <strong>of</strong> the ITS data-set treat<strong>in</strong>g gaps as a fifth character, bootstrap values are presented below branches (B)<br />
Majority rule consensus tree result<strong>in</strong>g from Bayesian analysis <strong>of</strong> the ITS data-set us<strong>in</strong>g GTR + I + G DNA substitution model; PP values are <strong>in</strong>dicated above branches. Numbers<br />
<strong>in</strong> parenthesis below branches <strong>in</strong>dicate nodes used for divergence time estimates, followed by the range <strong>of</strong> ages <strong>in</strong> million years accord<strong>in</strong>g to Table 4.<br />
maximum <strong>of</strong> 10 trees reta<strong>in</strong>ed per replicate, with collapsed<br />
branches <strong>of</strong> zero length. Start<strong>in</strong>g trees were obta<strong>in</strong>ed via r<strong>and</strong>om<br />
stepwise addition with TBR branch swapp<strong>in</strong>g. Branch support<br />
was calculated us<strong>in</strong>g bootstrap <strong>and</strong> 1000 replicates <strong>in</strong> a heuristic<br />
search, 100 replicates for r<strong>and</strong>om sequence addition.<br />
In order to compare the impact <strong>of</strong> alternative gap cod<strong>in</strong>g<br />
schemes <strong>in</strong> both analyses, gaps were treated <strong>in</strong> three ways: (1)<br />
as miss<strong>in</strong>g data (GM); (2) at the end <strong>of</strong> the matrix as presence-absence<br />
characters us<strong>in</strong>g the simple gap cod<strong>in</strong>g (SGC) scheme <strong>of</strong> Simmons<br />
<strong>and</strong> Ochoterena (2000); <strong>and</strong> (3) us<strong>in</strong>g ‘‘gaps as a fifth<br />
character” (GFC) as implemented <strong>in</strong> PAUP version 4.0b10 (Sw<strong>of</strong>ford,<br />
2002). Informative gaps, used <strong>in</strong> the SGC option analyses were<br />
treated as <strong>in</strong>sertions (<strong>in</strong>s), deletions (del), duplications <strong>of</strong> motifs<br />
(dup) <strong>and</strong> short (2–4 bp; SHR) or long (7–17 bp; LHR) homonucleotides<br />
repeats. Table 3 <strong>in</strong>cludes <strong>in</strong>formation on the gap cod<strong>in</strong>g<br />
treatments used <strong>in</strong> the various analyses (cf. Figs. 1B <strong>and</strong> 5A).<br />
In addition, a Bayesian analysis was performed for each data-set<br />
us<strong>in</strong>g MrBayes 3.1.1. (Huelsenbeck <strong>and</strong> Ronquist, 2001).<br />
GTR + I + G was selected as the best model <strong>of</strong> DNA substitution<br />
for each <strong>of</strong> the data-sets us<strong>in</strong>g the LRT implemented <strong>in</strong> MrModel<br />
Test 3.7 (Nyl<strong>and</strong>er, 2004). Analyses us<strong>in</strong>g MCMCMC were run for<br />
3,000,000 generations, <strong>in</strong>itiat<strong>in</strong>g with a r<strong>and</strong>om start<strong>in</strong>g tree <strong>and</strong><br />
default sett<strong>in</strong>gs (i.e., four cha<strong>in</strong>s, temperature <strong>of</strong> the heated cha<strong>in</strong><br />
set to 0.2). Each cha<strong>in</strong> was sampled every 1000 generations for a<br />
total <strong>of</strong> 3000 trees sampled. Sample po<strong>in</strong>ts collected prior to stationary<br />
were elim<strong>in</strong>ated (i.e., burn-<strong>in</strong>;
was processed us<strong>in</strong>g r8s which summarizes the distribution <strong>of</strong><br />
divergence times for each node. Canellaceae were excluded from<br />
these analyses <strong>in</strong> order to avoid artifactual branch<strong>in</strong>g po<strong>in</strong>t ages<br />
due to the high differences <strong>of</strong> substitution rates with respect to<br />
the W<strong>in</strong>teraceae. Three alternative calibration po<strong>in</strong>ts were explored<br />
<strong>in</strong> four scenarios that correspond to comb<strong>in</strong>ations <strong>of</strong> fixed<br />
or m<strong>in</strong>imum ages for specific nodes <strong>in</strong> the Bayesian trees obta<strong>in</strong>ed<br />
from the ITS or the comb<strong>in</strong>ed analyses (Table 4).<br />
Node 1 <strong>in</strong> the ITS <strong>and</strong> comb<strong>in</strong>ed topologies was fixed us<strong>in</strong>g two<br />
different ages: (1) 120 MY based on the early Cretaceous pollen<br />
Walkeripollis from Africa (Doyle, 2000) <strong>and</strong> the age <strong>of</strong> the split between<br />
Madagascar <strong>and</strong> Africa by the Somali bas<strong>in</strong> (121 MY;<br />
Sanmart<strong>in</strong> <strong>and</strong> Ronquist, 2004) <strong>and</strong> (2) 135 MY, tak<strong>in</strong>g <strong>in</strong>to account<br />
the first Gondwanic fragmentation event by the southern South<br />
Atlantic ocean that separated Africa from the rest <strong>of</strong> southern<br />
Gondwana (Sanmart<strong>in</strong> <strong>and</strong> Ronquist, 2004).<br />
W<strong>in</strong>teroid pollen fossil from the Campanian <strong>of</strong> SE Australia (c.<br />
77 MY; Dettmann <strong>and</strong> Jarzen, 1990; Doyle, 2000; Feild et al.,<br />
2002), assigned to Tasmannia (node 2; Table 4); f<strong>in</strong>ally, pollen fossil<br />
X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449 439<br />
Fig. 3. Distribution map <strong>of</strong> Drimys species (W<strong>in</strong>teraceae), <strong>and</strong> location <strong>of</strong> W<strong>in</strong>teroid fossils.<br />
from the Late Cretaceous <strong>of</strong> New Zeal<strong>and</strong> (c. 69 MY; <strong>in</strong>itially reported<br />
as Zygogynum by Mildenhall, 1980, but later assigned to<br />
Pseudow<strong>in</strong>tera by Feild et al., 2002) were used to constrict the m<strong>in</strong>imum<br />
age for nodes 3 or 4 (Table 4).<br />
F<strong>in</strong>ally, data from scenario 4 (Table 4) were used to calculate<br />
the follow<strong>in</strong>g variables: (1) speciation rates for each genus <strong>of</strong> W<strong>in</strong>teraceae,<br />
assum<strong>in</strong>g an equal rate <strong>of</strong> r<strong>and</strong>om speciation Yule model<br />
(Hughes <strong>and</strong> Eastwood, 2006); (2) nucleotide substitution rates<br />
(NSR); <strong>and</strong> (3) time for speciation (von Hagen <strong>and</strong> Kadereit,<br />
2001; Table 5).<br />
3. Results<br />
Twenty eight new sequences <strong>of</strong> ITS, 21 <strong>of</strong> psbA-trnH <strong>and</strong> 20 <strong>of</strong><br />
rpS16 were obta<strong>in</strong>ed for the present study; two sequences <strong>of</strong><br />
psbA-trnH <strong>and</strong> rpS16 correspond to Canellaceae <strong>and</strong> the rema<strong>in</strong><strong>in</strong>g<br />
sequences correspond to W<strong>in</strong>teraceae (Table 2). Sequences <strong>of</strong> ITS1<br />
ranged from 235 to 252 bp <strong>in</strong> W<strong>in</strong>teraceae <strong>and</strong> from 244 to 275 bp<br />
<strong>in</strong> Canellaceae; the size <strong>of</strong> 5.8S cod<strong>in</strong>g region was 164 bp long for
440 X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
Table 2<br />
Taxa <strong>in</strong>cluded <strong>in</strong> this study with localities, voucher <strong>in</strong>formation <strong>and</strong> Genbank accession.<br />
Taxa Country Voucher Sequences<br />
orig<strong>in</strong> a<br />
Genbank accession number<br />
ITS psbAtrnH<br />
rpS16<br />
Canellaceae<br />
Canella w<strong>in</strong>terana (L.) Gaernt. South America LBT 124, NO 1 LO3844<br />
Capsicodendron d<strong>in</strong>issii (Schwacke) Occhioni South America Butzkee et al. 12521 (US) 2 AY004132<br />
Capsicodendron d<strong>in</strong>issii<br />
(Schwacke) Occhioni<br />
Brazil (Parana) Lohmann et al. 727 (COL, SPF) 4 FJ539215 FJ554663<br />
C<strong>in</strong>namodendron ekmanni<br />
Sleumer<br />
Dom<strong>in</strong>ican Republic García <strong>and</strong> Veloz 6866 (SD) 2 AY004143<br />
C<strong>in</strong>namosma madagascariensis<br />
Danguy<br />
Madagascar Lowry 4991 (MO) 2 FJ53<br />
C<strong>in</strong>namosma madagascariensis<br />
Danguy<br />
Madagascar Rabevohitra 4510 (MO) 4 FJ539214 FJ554659<br />
Pleodendron macranthum (Baill.) Tiegh. Puerto Rico Axelrod 10783 (MO) 2 AY004134<br />
Warburgia salutaris (G. Bertol.) Chiov. South Africa Goldblatt 11314 (MO) 2 AY004130<br />
W<strong>in</strong>teraceae<br />
Drimys <strong>and</strong><strong>in</strong>a (Reiche) Rodr. & Quez. (1) Chile, X region 19961186 RBGE 4 FJ539242 FJ539211 FJ554657<br />
Drimys <strong>and</strong><strong>in</strong>a (2) Argent<strong>in</strong>a, Neuquén Beenken 992 (M) 4 FJ539241 FJ539212 FJ554662<br />
Drimys <strong>and</strong><strong>in</strong>a (Reiche) Rodr. & Quez. (3) Chile, IX region 19961048 RBGE 4 FJ539243<br />
Drimys angustifolia Miers Brazil, Rio gr<strong>and</strong>e do sur Wasum et al. 10779 (US) 4 FJ539232 FJ539203 FJ554651<br />
Drimys brasiliensis Miers. (1) Brazil, Paraná Lohmann et al. 728, (COL, SPF) 4 FJ539226 FJ539204 FJ554652<br />
Drimys brasiliensis (2) Brazil, Paraná Lohmann et al. 729 (COL, SPF) 4 FJ539227 FJ539205 FJ554653<br />
Drimys brasiliensis (3) Brazil, Cunha N.P. Cordeiro 2917 (SP) 4 FJ539228 FJ539206 FJ554654<br />
Drimys brasiliensis (4) Brazil, Campos do Jordao Lohmann et al. 738 (COL, SPF) 4 FJ539229 FJ539207 FJ554660<br />
Drimys brasiliensis (St. Hil.) Miers (5) Brazil, Paraná Cordeiro 553 (US) 4 FJ539230<br />
Drimys brasiliensis (6) Brazil, Paraná Hatschbach 17330 (COL, MBM) 4 FJ539231<br />
Drimys confertifolia Philippi Chile, Juan Fern<strong>and</strong>ez Bernardello <strong>and</strong> Anderson 3005 4 FJ539236 FJ539208<br />
Isl<strong>and</strong>s<br />
(UCA)<br />
Drimys granadensis L.f. (1) Colombia, Cund<strong>in</strong>amarca Marquínez 004 (SPF, COL) 4 FJ539233 FJ539201 FJ554649<br />
Drimys granadensis(2) Colombia, Sant<strong>and</strong>er Morales 1765 (COL) 4 FJ539234 FJ539202 FJ554650<br />
Drimys granadensis (3) Costa Rica Salazar 2630 (BH, COL) 4 FJ539235<br />
Drimys roraimensis (A.C. Smith) Ehrend. &<br />
Gottsb.<br />
Venezuela, Bolivar Holst 3689 (NY) 4 FJ539225<br />
Drimys w<strong>in</strong>teri Forst. <strong>and</strong> Forst. (1) Chile, Valparais Simon 142 (UCA) 4 FJ539237 FJ539209 FJ554655<br />
Drimys w<strong>in</strong>teri (2) Chile, IX Region 19880691<br />
RBGE<br />
4 FJ539238 FJ539216 FJ554656<br />
Drimys w<strong>in</strong>teri (3) Chile XII Region 19670643<br />
RBGE<br />
4 FJ539239 FJ539210 FJ554661<br />
Drimys w<strong>in</strong>teri (4) Argent<strong>in</strong>a, Patagonia Donat 398 (F, M) 4 FJ539240<br />
Pseudow<strong>in</strong>tera axillaris (Forst. & Forst.) D<strong>and</strong>y New Zeal<strong>and</strong> LBT 300 (NO) 1 AY004124<br />
Pseudow<strong>in</strong>tera colorata (Raoult) D<strong>and</strong>y New Zeal<strong>and</strong> LBT 301 (NO) 1 AY004125<br />
Pseudow<strong>in</strong>tera colorata New Zeal<strong>and</strong> 19981039 RBGE 4 FJ539196 FJ554644<br />
Takhtajania perrieri (Capuron) Baranova & J.F.<br />
Leroy<br />
Madagascar Rakotomalaza et al. 1342 (MO) 2 AY004129<br />
Takhtajania perrieri Madagascar Rabenanto<strong>and</strong>ro J 219 (MO) 4 FJ539213 FJ554658<br />
Tasmannia glaucifolia J.B. Williams Australia, NSW (NSW) 198433 (LCR) 863140 3 AY526315<br />
Tasmannia <strong>in</strong>sipida R.Br. ex DC Australia, Queensl<strong>and</strong> LBT 108 (NO) 1 AY004127<br />
Tasmannia lanceolata (Poir.) A.C. Smith Australia Berkeley B.G. 60.0052 (NO) 1 AY004128<br />
Tasmannia piperita (Hook.f.) Miers Irian Jaya (New Gu<strong>in</strong>ea) Lowry 5287 (MO) 4 FJ539244<br />
Tasmannia purpurascens (Vickery) A.C. Smith Australia, NSW (NSW) 209939<br />
(LCR) 875858<br />
3 AY526309<br />
Tasmannia stipitata (Vickery) A.C. Smith Australia, NSW (NSW) 209908<br />
(LCR) 875857<br />
3 AY526316<br />
Tasmannia xerophila (Parment.) M. Gray Australia, NSW (NSW) 215000<br />
(LCR) 850099<br />
3 AY526312<br />
Zygogynum acsmithii V<strong>in</strong>k. b<br />
New Caledonia LBT 201 (NO) 1 (clone 2) AY004122<br />
Zygogynum amplexicaule (Vieill. ex Parment.)<br />
V<strong>in</strong>k<br />
= Bubbia amplexicaulis (Viell. Ex Parment.)<br />
D<strong>and</strong>y<br />
New Caledonia Mc Pherson 19089 (MO) 4 FJ539222<br />
Zygogynum bicolor Tiegh. b<br />
New Caledonia LBT 200 (NO) 1 (clone 1) AY004111<br />
Zygogynum bicolor Tiegh. b<br />
New Caledonia LBT 200 (NO) 1 (clone 2) AY004118<br />
Zygogynum baillonii Tiegh. b<br />
New Caledonia Mc Pherson 19206 (MO) 4 FJ539219 FJ539197 FJ554645<br />
Zygogynum crassifolium (Baill.) V<strong>in</strong>k<br />
= Belliolum crassifolium Tiegh.<br />
= Bubbia crassifolium Burtt<br />
New Caledonia Lowry 6451 (MO) 4 FJ539221<br />
Zygogynum comptonii (Baker f.) V<strong>in</strong>k<br />
= Bubbia comptonii (Baker f.) D<strong>and</strong>y<br />
New Caledonia LBT 204 (NO) 1 (clone 2) AY004123<br />
b<br />
Zygogynum comptonii (Baker f.) V<strong>in</strong>k New Caledonia Mc Pherson 18061 (MO) 4 FJ539199 FJ554647<br />
Zygogynum fraterculum V<strong>in</strong>k c<br />
New Caledonia Mc Pherson 19092 (MO) 4 FJ539224<br />
Zygogynum pauciflorum (Baker f.) V<strong>in</strong>k c<br />
= Bubbia pauciflora (Baker f.) D<strong>and</strong>y<br />
New Caledonia Mc Pherson 19119 (MO) 4 FJ539223<br />
Zygogynum pancheri ssp. Arrhantum<br />
= Belliolum pancheri (Baillon) Tiegh.<br />
New Caledonia LBT 205 (NO) 1 (clone 2) AY004120
X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449 441<br />
Table 2 (cont<strong>in</strong>ued)<br />
Taxa Country Voucher Sequences<br />
orig<strong>in</strong> a<br />
Genbank accession number<br />
ITS psbAtrnH<br />
rpS16<br />
Zygogynum pomiferum spp. balansae<br />
(Tiegh) V<strong>in</strong>k<br />
= Zygogynum balansae Tiegh<br />
New Caledonia LBT 203 (NO) 1 (clone 1) AY004112<br />
b<br />
Zygogynum pomiferum spp. balansae<br />
(Tiegh) V<strong>in</strong>k<br />
New Caledonia LBT 203 (NO) 1 (clone 2) AY004120<br />
Zygogynum pomiferum spp. balansae<br />
(Tiegh) V<strong>in</strong>k<br />
New Caledonia Mc Pherson 19231 (MO) 4 FJ539217<br />
Zygogynum stipitatum Baill.<br />
= Exospermum stipitatum Tiegh.<br />
New Caledonia Mc Pherson 19231 (MO) 1 (clone 1) AY004113<br />
b<br />
Zygogynum stipitatum Baill.<br />
Exospermum stipitatum Tiegh.<br />
New Caledonia LBT 202, (NO) 1 (clone 2) AY004121<br />
b<br />
Zygogynum tanyostigma V<strong>in</strong>k c<br />
New Caledonia Mc Pherson 18986 (MO) 4 FJ539220 FJ539200 FJ554648<br />
Zygogynum v<strong>in</strong>kii Sampson New Caledonia Mc Pherson 19141 (MO) 4 FJ539218 FJ539198 FJ554646<br />
Numbers before scientific names correspond to different accessions <strong>of</strong> the same species.<br />
a<br />
Sequences orig<strong>in</strong>: 1, Suh et al. (1993); 2,Karol et al. (2000); 3,Doust <strong>and</strong> Dr<strong>in</strong>nan (2004); 4, this research.<br />
b<br />
Traditional denom<strong>in</strong>ation <strong>of</strong> species followed by Suh et al. (1993) <strong>and</strong> Karol et al. (2000).<br />
c<br />
This species were described by V<strong>in</strong>k recently, He <strong>in</strong>dicates that the position <strong>of</strong> Z. fraterculum <strong>and</strong> Z. pauciflorum is ‘‘<strong>in</strong>termediate between the Bubbia <strong>and</strong> the Zygogynum s.s.<br />
types” (V<strong>in</strong>k, 2003).<br />
Fig. 4. Strict consensus trees result<strong>in</strong>g from parsimony analyses <strong>of</strong> the follow<strong>in</strong>g data-sets: (A) rpS16, (B) psbA-trnH, <strong>and</strong> (C) ITS. Numbers next to the branches correspond to<br />
bootstrap values.
442 X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
Table 3<br />
Summary <strong>of</strong> <strong>in</strong>dels <strong>in</strong> the ITS, psbA-trnH <strong>and</strong> rpS16 sequences <strong>of</strong> Canellales.<br />
ITS (52 OTUS) Comb<strong>in</strong>ed analyses (24 OTUS)<br />
ITS psbA-trnH rpS16<br />
(A) Informative <strong>in</strong>dels (codified us<strong>in</strong>g SGC)<br />
Total # <strong>of</strong> <strong>in</strong>formative <strong>in</strong>dels 44 38 5 7<br />
# <strong>of</strong> <strong>in</strong>formative <strong>in</strong>dels across families a<br />
19 29 3 5<br />
# <strong>of</strong> <strong>in</strong>formative <strong>in</strong>dels <strong>in</strong> Canellaceae 7 – – –<br />
Tasmannia (GC)dup (GT)dup – – –<br />
((Pseudow<strong>in</strong>tera + Zygogynum) Drimys) SHR SHR – –<br />
Drimys 2 (GC)<strong>in</strong>s 2 (GC)<strong>in</strong>s – (CTTT)dup<br />
2 SHR 2 SHR<br />
Drimys SW clade – – (GTCAATC) dup –<br />
Pseudow<strong>in</strong>tera (TG)dup<br />
(GT)dup<br />
– – –<br />
Zygogynum (Clone 1) (CT)dup<br />
SHR<br />
– – –<br />
Zygogynum (Clone 2) (AG)dup (AG)dup – –<br />
Zygogynum (GTG)dup (GTG)dup – (TC)Del<br />
SHR SHR<br />
Indel A SHR – – –<br />
Indel B (A)del (A)del – –<br />
Indel C (ATC)dup – –<br />
Indel D SHR – – –<br />
Indel E – – (GTAAAT) dup –<br />
Indel F (GT)dup – – –<br />
Indel G SHR – – –<br />
(B) Number <strong>of</strong> un<strong>in</strong>formative <strong>in</strong>dels (autapomorphies)<br />
Duplication <strong>of</strong> motifs 3 5 2 2<br />
Insertion 1 1 0 –<br />
Deletion 5 5 3 2<br />
SHRs 6 6 1 –<br />
LHRs 1 1 1 3<br />
Del, deletion; dup, duplication; Ins, <strong>in</strong>sertion; LHR, long homonucleotide str<strong>in</strong>g (>5 bp); SHR, short homonucleotide repeats (2–4 bp).<br />
a Across families = Indels present <strong>in</strong> all the species <strong>of</strong> W<strong>in</strong>teraceae or Canellaceae but not both. Sequences <strong>in</strong> 5 0 –3 0 sense. (Indels A–G are plotted <strong>in</strong> Fig. 2B <strong>and</strong>/or 5B).<br />
Fig. 5. Strict consensus tree result<strong>in</strong>g from analyses <strong>of</strong> the comb<strong>in</strong>ed ITS + psbA-trnH + rpS16 data-set parsimony analyses. (A) Consider<strong>in</strong>g gaps as miss<strong>in</strong>g (MG) or simple<br />
gap cod<strong>in</strong>g options (SGC). (Topologies are identical, see Table 6), first bootstrap values shown above branches correspond to GM analysis; second bootstrap values (shown <strong>in</strong><br />
parenthesis) correspond to the SGC analysis. Numbers <strong>in</strong> parenthesis below branches <strong>in</strong>dicates numbers <strong>of</strong> gaps support<strong>in</strong>g clades <strong>in</strong> the SGC analysis; circles <strong>and</strong> letters<br />
<strong>in</strong>dicate homoplasies (see Table 3); <strong>and</strong>, (B) Majority rule consensus tree result<strong>in</strong>g from a Bayesian analysis us<strong>in</strong>g GTR + I + G DNA substitution model; PP values are presented<br />
above branches; numbers <strong>in</strong> parenthesis below branches represented nodes used for divergence time estimates, followed by the range <strong>of</strong> ages <strong>in</strong> million years accord<strong>in</strong>g to<br />
Table 4.<br />
all species <strong>of</strong> Canellales; ITS2 sequences ranged from 213 to 226 bp<br />
<strong>in</strong> W<strong>in</strong>teraceae <strong>and</strong> 187–233 bp <strong>in</strong> Canellaceae.<br />
3.1. Phylogenetic analyses based on ITS sequences<br />
The parsimony analysis consider<strong>in</strong>g gaps as miss<strong>in</strong>g (GM) resulted<br />
<strong>in</strong> three trees <strong>of</strong> 591 steps, with CI = 0.743 <strong>and</strong> RI = 0.901<br />
(Table 6). The monospecific Takhtajania is sister to the rest <strong>of</strong> the<br />
family. The four non-monospecific genera <strong>of</strong> W<strong>in</strong>teraceae appear<br />
as monophyletic <strong>and</strong> are supported with bootstrap values > 80%<br />
(Fig. 1A). With<strong>in</strong> Tasmannia, T. lanceolata is sister to the rest <strong>of</strong><br />
the genus; <strong>in</strong> turn, T. <strong>in</strong>sipida is sister to an unresolved clade<br />
formed by T. piperita (T. xerophila + T. glaucifolia) <strong>and</strong> (T. stipitata<br />
+ T. purpurascens). With<strong>in</strong> Drimys, only one clade (D. <strong>and</strong><strong>in</strong>a + D.
Table 4<br />
Average ages (MY) <strong>and</strong> confidence <strong>in</strong>tervals (a = 0.05) for the Bayesian consensus tree nodes <strong>of</strong> ITS (Fig. 3B) <strong>and</strong> comb<strong>in</strong>ed analyses (Fig. 5B) estimated us<strong>in</strong>g r8s under six<br />
different scenarios. For exact node locations see Figs. 3 <strong>and</strong> 5.<br />
w<strong>in</strong>teri) was recovered; the relationship among the other five species<br />
rema<strong>in</strong>ed unresolved.<br />
The ITS data shows that sequences from Zygogynum s.l. conform<br />
two ma<strong>in</strong> clades: (1) ‘Clone 1’ sequences (Suh et al., 1993) <strong>of</strong><br />
(Zygogynum stipitatum (Z. pomiferum 1+Z. bicolor)); (2) ‘Clone 2’<br />
sequences (Suh et al., 1993) <strong>of</strong>(Z. comptonii (Z. acsmithii, Z. stipitatum<br />
(Z. pomiferum 1 (Z. pancheri + Z. bicolor)))), as sister to (Z.<br />
amplexicaule (Z. crassifolium (Z. fraterculum + Z. pauciflorum) <strong>and</strong><br />
(Z. tanyostigma (Z. v<strong>in</strong>kii (Z. pomiferum 1+Z. baillonii))))). Clade 1<br />
is supported by two <strong>in</strong>dels (cf. Fig. 1B <strong>and</strong> Table 3), five substitutions<br />
<strong>in</strong> ITS1, one substitution <strong>in</strong> 5.8S <strong>and</strong> six substitutions <strong>in</strong><br />
ITS2; Clade 2 is supported by one <strong>in</strong>del (cf. Fig. 1B <strong>and</strong> Table 3),<br />
<strong>and</strong> two substitutions <strong>in</strong> each ITS1 <strong>and</strong> ITS2.<br />
The parsimony analysis us<strong>in</strong>g the SGC option produced three<br />
trees <strong>of</strong> 646 steps, CI = 0.751 <strong>and</strong> RI = 0.912 (Fig. 1B). The strict consensus<br />
tree result<strong>in</strong>g from this analysis is similar to that obta<strong>in</strong>ed<br />
from the analysis that considered gaps as miss<strong>in</strong>g, but with higher<br />
bootstrap values <strong>in</strong> some clades. The ma<strong>in</strong> difference <strong>in</strong> topology<br />
occurs with<strong>in</strong> Zygogynum, asZ. amplexicaule plus Z. crassifolium<br />
X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449 443<br />
Scenario 1 Scenario 2 Scenario 3 Scenario 4<br />
Fixed age (MY) node 1 135 135 120 120<br />
M<strong>in</strong>imum age = 77 MY Node 2 Node 2 Node 2 Node 2<br />
M<strong>in</strong>imum age = 69 MY Node 3 Node 4 Node 3 Node 4<br />
(A) ITS analysis<br />
Node 1 135 ± 0 135 ± 0 120 ± 0 120 ± 0<br />
Node 2 85.44 ± 2.40 92.38 ± 1.90 77 ± 0 87.01 ± 1.42<br />
Node 3 76.60 ± 1.93 84.64 ± 1.50 69 ± 0 80.95 ± 1.09<br />
Node 4 58.03 ± 2.16 69 ± 0 52.24 ± 2.02 69 ± 0<br />
Node 5 41.97 ± 1.54 48.63 ± 1.47 37.82 ± 1.41 47.81 ± 1.61<br />
Node 6 11.30 ± 0.84 12.79 ± 0.74 10.29 ± 0.82 12.40 ± 0.76<br />
Node 7 4.62 ± 0.41 5.18 ± 0.42 4.17 ± 0.41 5.00 ± 0.43<br />
Node 8 5.93 ± 0.62 6.59 ± 0.54 5.34 ± 0.44 6.29 ± 0.43<br />
Node 9 10.94 ± 0.88 12.04 ± 1.17 9.85 ± 0.93 11.59 ± 1.04<br />
Node 10 13.83 ± 0.84 15.70 ± 1.22 12.37 ± 0.71 15.11 ± 1.24<br />
Node 11 6.91 ± 0.32 7.45 ± 0.29 6.21 ± 0.28 7.01 ± 0.29<br />
Node 12 4.29 ± 0.31 4.62 ± 0.28 3.86 ± 0.22 4.34 ± 0.22<br />
Node 13 37.22 ± 1.67 39.94 ± 1.59 33.36 ± 1.48 37.33 ± 1.47<br />
Node 14 23.71 ± 1.17 25.36 ± 1.16 21.23 ± 0.81 23.66 ± 0.94<br />
Node 15 17.31 ± 0.92 18.48 ± 0.80 15.49 ± 0.55 17.23 ± 0.69<br />
Node 16 9.06 ± 0.67 9.66 ± 0.82 6.11 ± 0.64 9.01 ± 0.70<br />
Node 17 6.91 ± 0.41 7.37 ± 0.57 6.18 ± 0.49 6.87 ± 0.56<br />
(B) Comb<strong>in</strong>ed analysis<br />
Node 1 135 ± 0 135 ± 0 120 ± 0 120 ± 0<br />
Node 2 83.19 ± 1.22 93.48 ± 1.48 79.88 ± 1.01 87.89 ± 1.14<br />
Node 3 69 ± 0 82.02 ± 0.86 69 ± 0 79.05 ± 0.69<br />
Node 4 48.05 ± 1.22 69 ± 0 47.29 ± 1.05 69 ± 0<br />
Node 5 25.64 ± 0.75 34.12 ± 1.04 24.95 ± 0.75 33.38 ± 1.02<br />
Node 11 13.95 ± 0.58 16.09 ± 0.63 13.41 ± 0.51 15.19 ± 0.61<br />
Node 18 9.42 ± 0.37 10.81 ± 0.45 8.99 ± 0.41 10.16 ± 0.41<br />
Node 19 7.52 ± 0.39 8.61 ± 0.46 7.17 ± 0.35 8.09 ± 0.41<br />
Node 20 5.34 ± 0.29 6.09 ± 0.37 5.08 ± 0.33 5.72 ± 0.32<br />
Table 5<br />
Number <strong>of</strong> species <strong>and</strong> estimated nucleotide substitution rate (NSR), speciation rate (SR), <strong>and</strong> time for speciation (TFS) for genera <strong>of</strong> W<strong>in</strong>teraceae, based on ITS <strong>and</strong> comb<strong>in</strong>ed<br />
analyses under scenario 4 (see Table 4).<br />
Takhtajania Tasmannia Drimys Pseudow<strong>in</strong>tera Zygogynum<br />
Number <strong>of</strong> species 1 8 7 4 59<br />
NSR <strong>of</strong> ITS (# <strong>of</strong> substitutions per site/yr 10 10 ) 6.65 7.69 ± 1.84 8.67 ± 4.37 4.96 ± 1.17 10.75 ± 7.73<br />
NSR <strong>of</strong> ITS + psbA-trnH+rpS16<br />
(# <strong>of</strong> substitutions per site/yr 10 10 3.64 3.26 3.87 ± 1.91 1.45 3.14 ± 0.56<br />
)<br />
SR <strong>of</strong> ITS (sp/MY) 0 0.056 0.278 0.0917 0.085<br />
SR <strong>of</strong> ITS + psbA-trnH+rpS16 (sp/MY) 0 – 0.128 – 0.117<br />
TFSln ITS (MY/sp) – 12.44 2.50 7.55 8.13<br />
TFSln ITS + psbA+rpS16 (MY/sp) – – 5.41 – 5.88<br />
resulted sister to Z. pauciflorum plus Z. fraterculum; <strong>in</strong> turn, these<br />
four species are sister to Z. pomiferum 2 plus Z. v<strong>in</strong>kii. The<br />
clade formed by these six species is sister to Z. tanyostigma plus<br />
Z. baillonii (Fig. 1A).<br />
The parsimony analysis us<strong>in</strong>g the GFC option resulted <strong>in</strong> 105<br />
trees <strong>of</strong> 922 steps, CI = 0.793 <strong>and</strong> RI = 0.927; the strict consensus<br />
tree recovers the same generic <strong>relationships</strong> that <strong>in</strong> the analyses<br />
that used GM <strong>and</strong> SGS options (Fig. 2A). Resolution with<strong>in</strong> Drimys<br />
<strong>and</strong> Zygogynum s.l. is lower than that obta<strong>in</strong>ed with the GM <strong>and</strong><br />
SGS options.<br />
<strong>Generic</strong> <strong>relationships</strong> emerg<strong>in</strong>g from the Bayesian analysis<br />
(Fig. 2B) are completely congruent to those obta<strong>in</strong>ed us<strong>in</strong>g parsimony.<br />
With<strong>in</strong> Drimys the topology is similar to that recovered from<br />
GM or SGC options. With<strong>in</strong> Zygogynum, ‘clade 1’ sequences recovers<br />
the same topology obta<strong>in</strong>ed <strong>in</strong> all the analyses; however, <strong>in</strong> ‘clade 2’,<br />
Z. comptonni resulted sister to an unresolved clade conformed by Z.<br />
stipitatum, Z. acsmithii, Z. pomiferum 2, <strong>and</strong> two subclades: (Z. v<strong>in</strong>kii<br />
(Z. tanyostigma, (Z. crassifolium, Z. amplexicaule (Z. pauciflorum + Z.<br />
fraterculum)))) <strong>and</strong> (Z. pomiferum 1(Z. pancheri + Z. bicolor)).
444 X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
Table 6<br />
Tree statistics for the different gap cod<strong>in</strong>g schemes applied for ITS <strong>and</strong> comb<strong>in</strong>ed analyses.<br />
Data-sets (number<br />
<strong>of</strong> sequences)<br />
psbA-trnH (21) rpS16 (20) ITS (23) ITS (52) Comb<strong>in</strong>ed ITS + psbA-trnH+rpS16 (23)<br />
Gap cod<strong>in</strong>g option Miss<strong>in</strong>g Miss<strong>in</strong>g Miss<strong>in</strong>g Miss<strong>in</strong>g Simple gap Fifth Miss<strong>in</strong>g Simple gap cod<strong>in</strong>g Fifth character<br />
cod<strong>in</strong>g character<br />
Total number <strong>of</strong> characters 503 810 752 804 850 804 2065 2115 2065<br />
Number <strong>of</strong> non-variable<br />
characters<br />
367 695 512 465 465 292 1574 1574 1076<br />
Number <strong>of</strong> variable un<strong>in</strong>formative<br />
characters<br />
87 65 93 104 104 114 245 245 509<br />
Number <strong>of</strong> <strong>in</strong>formative<br />
characters<br />
49 50 147 235 281 398 246 296 480<br />
Informativity (%) a<br />
10.3 6.2 19.5 29.1 33.1 49.5 11.9 14 23.2<br />
L 157 126 326 591 646 922 614 667 1303<br />
Number <strong>of</strong> most<br />
parsimonious trees<br />
8 4 1 3 3 105 32 32 20<br />
CI 0.962 0.960 0.850 0.743 0.751 0.793 0.894 0.898 0.911<br />
RI 0.942 0.946 0.863 0.901 0.912 0.927 0.882 0.895 0.887<br />
RC 0.906 0.908 0.733 0.670 0.685 0.735 0.789 0.804 0.808<br />
Total number <strong>of</strong><br />
resolved clades<br />
5 9 12 32 32 28 12 12 14<br />
Polytomies <strong>in</strong> the strict<br />
consensus tree<br />
4 5 4 5 5 4 2 2 3<br />
Figures 4B 4A 4C 1A 1B 2A 5A 5A Not shown<br />
a Informativity (%) = (number <strong>of</strong> <strong>in</strong>formative characters/number <strong>of</strong> total characters) 100.<br />
3.2. Phylogenetic analyses based on psbA-trnH, rpS16 <strong>and</strong> ITS<br />
Three parsimony analyses consider<strong>in</strong>g gaps as miss<strong>in</strong>g were<br />
performed <strong>in</strong>dividually with the psbA-trnH, rpS16 <strong>and</strong> ITS datasets.<br />
The analysis based on psbA-trnH produced eight trees <strong>of</strong><br />
157 steps, CI = 0.962 <strong>and</strong> RI = 0.942 (Table 6); the strict consensus<br />
tree recovered Drimys (bootstrap = 79) <strong>and</strong> a clade with<strong>in</strong> Drimys<br />
formed by the three Chilean <strong>and</strong> Argent<strong>in</strong>ian species, D. <strong>and</strong><strong>in</strong>a,<br />
D. confertifolia, <strong>and</strong> D. w<strong>in</strong>teri (hereafter called the Southwestern<br />
or SW clade; bootstrap = 88; Fig. 3). Pseudow<strong>in</strong>tera colorata <strong>and</strong><br />
the four species <strong>of</strong> Zygogynum s.l. were recovered as a well supported<br />
clade but without <strong>in</strong>ternal resolution (bootstrap = 99;<br />
Fig. 4B). The analysis based on rpS16 produced four trees <strong>of</strong> 126<br />
steps, CI = 0.960 <strong>and</strong> RI = 0.946 (Table 6). The strict consensus tree<br />
recovered the genera Drimys <strong>and</strong> Zygogynum s.l. as a well supported<br />
clades (bootstraps = 87 <strong>and</strong> 95, respectively; Fig. 4A) conform<strong>in</strong>g<br />
a trichotomy with Pseudow<strong>in</strong>tera colorata. Drimys<br />
conta<strong>in</strong>s two clades with<strong>in</strong>: the SW clade (bootstrap = 62) <strong>and</strong> a<br />
clade formed by the NE South America species D. angustifolia, D.<br />
brasiliensis, D. granadensis, <strong>and</strong> D. roraimensis (hereafter called the<br />
Northeastern or NE clade; bootstrap = 64; Fig. 3). The analysis<br />
based on ITS produced a s<strong>in</strong>gle tree <strong>of</strong> 326 steps (CI = 0.850 <strong>and</strong><br />
RI = 0.863; Table 4; Fig. 4C), essentially similar <strong>in</strong> topology <strong>and</strong><br />
support to the analysis us<strong>in</strong>g 47 taxa (Fig. 1A).<br />
3.3. Congruence tests<br />
The ILD test <strong>in</strong>dicates comb<strong>in</strong>ability <strong>of</strong> the psbA-trnH, rpS16 <strong>and</strong><br />
ITS data-sets (P = 0.235). The SH test <strong>in</strong>dicates, on one h<strong>and</strong>, topological<br />
congruence between rpS16 <strong>and</strong> psbA-trnH (P = 0.74 or 0.82,<br />
depend<strong>in</strong>g <strong>of</strong> the use <strong>of</strong> consensus trees under parsimony or<br />
Bayesian analyses); <strong>and</strong> on the other h<strong>and</strong>, topological <strong>in</strong>congruence<br />
between rpS16 <strong>and</strong> ITS data-sets (P = 0.00 <strong>in</strong> both cases).<br />
The topologies <strong>of</strong> strict consensus trees (Fig. 4) presented only<br />
two <strong>in</strong>congruences: (1) <strong>in</strong> the rps16 tree Zygogynum v<strong>in</strong>kii is sister<br />
to Z. comptonii whereas <strong>in</strong> the ITS tree Z. v<strong>in</strong>kii resulted sister to Z.<br />
baillonii, <strong>and</strong> Z. comptonii resulted sister to Z. tanyostigma <strong>and</strong> (2)<br />
<strong>in</strong> the rps16 analysis Drimys brasiliensis 1 resulted sister to<br />
D. angustifolia <strong>and</strong> D granadensis, whereas <strong>in</strong> the ITS analysis D. brasiliensis<br />
1 resulted sister to D. brasiliensis 2 <strong>and</strong> D. roraimensis.<br />
Because the SH test has been considered too conservative, as<br />
hypotheses are too <strong>in</strong>frequently rejected (cf. Susko, 2003), we<br />
f<strong>in</strong>ally decided to follow the results <strong>of</strong> the ILD analysis <strong>and</strong> to comb<strong>in</strong>e<br />
the three data-sets (Fig. 5).<br />
3.4. Phylogenetic analyses based on the comb<strong>in</strong>ed molecular data-set<br />
Parsimony analysis <strong>of</strong> the ITS + psbA-trnH+rpS16 data-set, consider<strong>in</strong>g<br />
gaps as miss<strong>in</strong>g resulted <strong>in</strong> 32 trees <strong>of</strong> L = 614 steps,<br />
CI = 0.894 <strong>and</strong> RI = 0.882. The four genera <strong>of</strong> W<strong>in</strong>teraceae are<br />
recovered as monophyletic <strong>in</strong> the strict consensus tree, <strong>and</strong> are<br />
supported by bootstrap values <strong>of</strong> 95% or higher (Fig. 5A; Table 4).<br />
With<strong>in</strong> Zygogynum s.l. only a clade (Z. baillonii + Z. v<strong>in</strong>kii) is recovered.<br />
The SW <strong>and</strong> the NE clades <strong>of</strong> Drimys were obta<strong>in</strong>ed with bootstrap<br />
values <strong>of</strong> 82 <strong>and</strong> 58%, respectively.<br />
The parsimony analysis us<strong>in</strong>g the SGC option generated 32 trees<br />
<strong>of</strong> L = 667 steps, CI = 0.898 <strong>and</strong> RI = 0.895; the strict consensus tree<br />
recovers an identical topology to that obta<strong>in</strong>ed consider<strong>in</strong>g gaps as<br />
miss<strong>in</strong>g (Fig. 5A); however the bootstrap value for the SW clade is<br />
higher (90%).<br />
The analysis us<strong>in</strong>g GFC resulted <strong>in</strong> 20 trees <strong>of</strong> 1303 steps,<br />
CI = 0.911 <strong>and</strong> RI = 0.887. The strict consensus tree (not shown)<br />
only differs from those obta<strong>in</strong>ed <strong>in</strong> the two previous analyses with<br />
respect to the <strong>relationships</strong> with<strong>in</strong> Zygogynum s.l., <strong>in</strong> which the follow<strong>in</strong>g<br />
<strong>relationships</strong> are encountered (((Z. v<strong>in</strong>kii + Z. tanyostigma) Z.<br />
comptonii) Z. baillonii). These <strong>relationships</strong> are not recovered <strong>in</strong> any<br />
other analyses. With<strong>in</strong> Drimys, both the NE <strong>and</strong> the SW clades were<br />
recovered aga<strong>in</strong>, the latter be<strong>in</strong>g well supported (bootstrap = 92%).<br />
However, Drimys w<strong>in</strong>teri appears as paraphyletic with respect to D.<br />
<strong>and</strong><strong>in</strong>a. Relationships emerg<strong>in</strong>g from the Bayesian analysis are congruent<br />
to those obta<strong>in</strong>ed us<strong>in</strong>g parsimony (Fig. 5B).<br />
3.5. Molecular clock <strong>and</strong> divergence time estimates<br />
The likelihood-ratio test (LRT) <strong>in</strong>dicates a significant rate <strong>of</strong> heterogeneity<br />
among clades for both data-sets. Estimation <strong>of</strong> divergence<br />
times us<strong>in</strong>g the Penalized likelihood method <strong>and</strong> TN<br />
algorithm implemented <strong>in</strong> r8s, <strong>and</strong> explor<strong>in</strong>g four alternative calibration<br />
scenarios are presented <strong>in</strong> Table 4.<br />
3.6. Gap <strong>in</strong>formativity<br />
In W<strong>in</strong>teraceae, some <strong>in</strong>formative gaps correspond to short<br />
duplicate motifs <strong>of</strong> 2–3 bp <strong>in</strong> the ITS data-set, 6–7 bp <strong>in</strong> the
psbA-trnH data-set <strong>and</strong> 4 bp <strong>in</strong> the rpS16 data-set. The longest<br />
duplicate motif (TATTTCATATTGTATA) was identified <strong>in</strong> the psbAtrnH<br />
sequence <strong>of</strong> Cynamosma madagascariensis (Canellaceae) as<br />
an autopomorphy. An additional proportion <strong>of</strong> <strong>in</strong>formative gaps<br />
(33%) corresponds to changes <strong>in</strong> the length <strong>of</strong> homonucleotide repeats<br />
<strong>in</strong> the ITS data-set, most <strong>of</strong> them between 1–4 bp; however,<br />
<strong>in</strong> the psbA-trnH data-set there is one long complex homonucleotide<br />
repeat (12–13 bp) that corresponds to a polyA/T region that<br />
<strong>in</strong>cludes a synapomorphic duplication (GTCAATC) for the SW clade<br />
<strong>of</strong> Drimys. InrpS16 there are three PolyA/T with lengths <strong>of</strong> 8–13,<br />
11–12, <strong>and</strong> 15–17 bp. ITS has one poliA/T that varies <strong>in</strong> length between<br />
5–8 bp. The psbA-trnH data-set presents the longest <strong>in</strong>dels,<br />
especially across the whole order (up to 23 nucleotide) or, with<strong>in</strong><br />
Canellaceae, an <strong>in</strong>del <strong>of</strong> 35 nucleotide <strong>in</strong> Capsicodendron d<strong>in</strong>issi.<br />
The simple gap cod<strong>in</strong>g (SGC) <strong>and</strong> gaps as a fifth character (GFC)<br />
options led to an <strong>in</strong>crease <strong>in</strong> the number <strong>of</strong> <strong>in</strong>formative characters<br />
(%), length, CI <strong>and</strong> RI <strong>of</strong> most parsimonious trees (MPT) <strong>in</strong> both ITS<br />
<strong>and</strong> comb<strong>in</strong>ed analyses (Table 6). ITS analysis us<strong>in</strong>g SGC option resulted<br />
<strong>in</strong> a consensus tree (Fig. 2A) similar to that obta<strong>in</strong>ed from<br />
the analysis us<strong>in</strong>g gaps as miss<strong>in</strong>g (GM; Fig. 1A), with differences<br />
<strong>in</strong> the Zygogynum topology, <strong>and</strong> <strong>in</strong>creas<strong>in</strong>g branch support <strong>in</strong> some<br />
clades. The analysis <strong>of</strong> the ITS data-set us<strong>in</strong>g GFC option resulted <strong>in</strong><br />
a less resolved topology with<strong>in</strong> Drimys <strong>and</strong> Zygogynum s.l. with respect<br />
to that obta<strong>in</strong>ed us<strong>in</strong>g SGC option (Figs. 1A <strong>and</strong> 2A, respectively).<br />
As for Drimys, the specific <strong>relationships</strong> were less<br />
resolved <strong>in</strong> both the ITS <strong>and</strong> the comb<strong>in</strong>ed analyses us<strong>in</strong>g GFC; <strong>in</strong><br />
addition, the three accessions <strong>of</strong> D. w<strong>in</strong>teri resulted paraphyletic<br />
with respect to the two accessions <strong>of</strong> D. <strong>and</strong><strong>in</strong>a <strong>in</strong> the comb<strong>in</strong>ed<br />
analysis us<strong>in</strong>g GFC option (data not shown).<br />
4. Discussion<br />
All analyses corroborate monophyly <strong>of</strong> Drimys, Pseudow<strong>in</strong>tera,<br />
Tasmannia, Zygogynum s.l. <strong>and</strong> recover the same <strong>in</strong>tergeneric<br />
<strong>relationships</strong> (Takhtajania (Tasmannia (Drimys (Pseudow<strong>in</strong>tera<br />
+ Zygogynum s.l.)))). Furthermore, the use <strong>of</strong> the comb<strong>in</strong>ed<br />
data-set recovered the NW <strong>and</strong> SW clades with<strong>in</strong> Drimys. Cod<strong>in</strong>g<br />
<strong>in</strong>dels us<strong>in</strong>g the SGC option results <strong>in</strong> produced a similar resolution<br />
with respect to that obta<strong>in</strong>ed with GM options, but <strong>in</strong>crease branch<br />
support.<br />
4.1. Orthology vs. paralogy <strong>in</strong> the ITS sequences<br />
The ribosomal rDNA that <strong>in</strong>cludes the ITS region is known to<br />
conta<strong>in</strong> multiple copies. Intragenomic rDNA diversity is generally<br />
thought to be low as a result <strong>of</strong> concerted evolution <strong>of</strong> these copies<br />
(Buckler et al., 1997). In some cases, however, concerted evolution<br />
is lower than speciation lead<strong>in</strong>g to divergent paralogues with<strong>in</strong> a<br />
s<strong>in</strong>gle genome (Bradford, 2002). Suh et al. (1993) obta<strong>in</strong>ed cloned<br />
ITS sequences <strong>and</strong> found two divergent ITS copies <strong>in</strong> Zygogynum s.l.,<br />
(named here ‘clone 1’ <strong>and</strong> ‘clone 2’). Additionally, Buckler et al.<br />
(1997) found that ‘clone 1’ <strong>of</strong> Zygogynum corresponds to a pseudogene<br />
<strong>and</strong> that ‘clone 2’ copies represents the functional gene.<br />
Buckler et al. (1997) considered that amplifications that use<br />
denatur<strong>in</strong>g conditions (DMSO or high extension temperatures)<br />
could favor the amplification <strong>of</strong> ‘clone 2’ sequences, which are<br />
structurally more stable <strong>and</strong> are present <strong>in</strong> higher numbers.<br />
Baldw<strong>in</strong> <strong>and</strong> Donoghue (unpublished data cited by Baldw<strong>in</strong> et al.,<br />
1995) performed pooled PCR (without clon<strong>in</strong>g) us<strong>in</strong>g samples <strong>of</strong><br />
Zygogynum bicolor <strong>and</strong> Z. comptonii <strong>and</strong> obta<strong>in</strong>ed only copies that<br />
correspond to ‘clone 2’. Follow<strong>in</strong>g Baldw<strong>in</strong> <strong>and</strong> Donoghue (unpubl.<br />
data), we used DMSO <strong>and</strong> BSA <strong>in</strong> the PCR reactions, <strong>and</strong> different<br />
PCR conditions <strong>and</strong> primers than those used by Suh et al. (1993)<br />
<strong>in</strong> order to rule out that the ITS sequences obta<strong>in</strong>ed here could<br />
correspond to ‘clone 1’ copies. In addition, our analyses support<br />
a clade conformed by all the sequences newly obta<strong>in</strong>ed<br />
X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449 445<br />
(Z. amplexicaule, Z. baillonii, Z. crassifolium, Z. fraterculum, Z. pauciflorum,<br />
Z. pomiferum 2 <strong>and</strong> Z. v<strong>in</strong>kii) together with the ‘clone 2’ sequences<br />
<strong>of</strong> previous studies (Figs. 1 <strong>and</strong> 2). All these facts strongly<br />
suggest that, although we did not perform ITS clon<strong>in</strong>g, our newly<br />
obta<strong>in</strong>ed sequences are orthologous functional genes.<br />
4.2. Systematics<br />
The generic <strong>relationships</strong> recovered here are <strong>in</strong> agreement<br />
with the topologies obta<strong>in</strong>ed by Karol et al. (2000) <strong>and</strong> Doust<br />
<strong>and</strong> Dr<strong>in</strong>nan (2004). That is: (Takhtajania (Tasmannia (Drimys<br />
(Pseudow<strong>in</strong>tera, Zygogynum s.l.)))). Our <strong>in</strong>creased sampl<strong>in</strong>g <strong>of</strong> Drimys<br />
(7 spp.), Tasmannia (7 spp.), <strong>and</strong> Zygogynum s.l. (13 spp.) provided<br />
additional evidence for the monophyly <strong>of</strong> these genera. A<br />
phylogenetic analysis <strong>in</strong>clud<strong>in</strong>g molecular <strong>and</strong> morphological data<br />
is currently underway. However, resolution with<strong>in</strong> genera deserves<br />
further research due to the low nucleotide substitution rates<br />
encountered <strong>in</strong> sequences <strong>of</strong> ITS, psbA-trnH <strong>and</strong> rpS16 (Table 5).<br />
4.2.1. Drimys<br />
Two subclades were recovered <strong>in</strong> the W<strong>in</strong>teraceae comb<strong>in</strong>ed<br />
analysis: the NE clade (D. angustifolia, D. brasiliensis, D. granadensis,<br />
D. roraimensis), <strong>and</strong> the SW clade (D. <strong>and</strong><strong>in</strong>a, D. confertifolia,<br />
D. w<strong>in</strong>teri). These clades essentially correspond to the two ma<strong>in</strong><br />
species groups keyed out by Smith (1943b). The Andean species<br />
<strong>of</strong> Drimys (D. w<strong>in</strong>teri, D. <strong>and</strong><strong>in</strong>a, <strong>and</strong> D. granadensis) do not conform<br />
a clade by themselves (Fig. 5). With<strong>in</strong> the SW clade, the topology<br />
recovered by the comb<strong>in</strong>ed analysis ((D. confertifolia) (D. w<strong>in</strong>teri,<br />
D. <strong>and</strong><strong>in</strong>a)) essentially corresponds to the species group<strong>in</strong>g proposed<br />
by Smith (1943b). D. confertifolia is closely related to D. w<strong>in</strong>teri,<br />
although this author considered D. <strong>and</strong><strong>in</strong>a as a subspecies <strong>of</strong> D.<br />
w<strong>in</strong>teri. On the other h<strong>and</strong>, Ehrendorfer et al. (1979) grouped species<br />
<strong>of</strong> Drimys as follows: (1) the southern group (equivalent to our<br />
SW clade); (2) D. granadensis; <strong>and</strong> (3) the eastern group <strong>in</strong>clud<strong>in</strong>g<br />
D. angustifolia, D. brasiliensis, <strong>and</strong> D. roraimensis. Our analyses support<br />
the monophyly <strong>of</strong> group 1, but is still <strong>in</strong>conclusive regard<strong>in</strong>g<br />
the recognition <strong>of</strong> group 3. The lack <strong>of</strong> resolution with<strong>in</strong> the NE<br />
clade <strong>of</strong> Drimys further prevents an <strong>in</strong>frageneric classification;<br />
the problematic circumscription <strong>of</strong> this clade has long been recognized<br />
<strong>in</strong> previous morphological studies (Sa<strong>in</strong>t-Hilaire, 1824;<br />
Miers, 1858; Smith, 1943a; Ehrendorfer et al., 1979; see Table 1).<br />
Therefore, the use <strong>of</strong> additional fast-evolv<strong>in</strong>g markers, <strong>and</strong> a phylogeographic<br />
approach is urgently needed <strong>in</strong> order to improve resolution<br />
<strong>in</strong>side the genus.<br />
4.2.2. Tasmannia<br />
Species circumscription with<strong>in</strong> Tasmannia has long been controversial,<br />
especially <strong>in</strong> reference to the circumscription <strong>of</strong> T. piperita.<br />
Smith (1943b) recognized six Australian <strong>and</strong> 30 extra-Australian<br />
species. V<strong>in</strong>k (1970) proposed three <strong>in</strong>formal sections: (1) T.<br />
lanceolata, (2) T. <strong>in</strong>sipida plus T. purpurascens plus T. stipitata, <strong>and</strong><br />
(3) Tasmannia piperita sensu lato, which <strong>in</strong>cludes Smith’s 30 extra-<br />
Australian species plus the Australian T. glaucifolia,<br />
T. membranea <strong>and</strong> T. xerophila. Our results suggest that T. glaucifolia<br />
is sister to T. xerophila. However, the relationship between these<br />
two species <strong>and</strong> T. piperita (based on an Irian Jaya specimen) rema<strong>in</strong>s<br />
unresolved (Figs. 1 <strong>and</strong> 2). Accord<strong>in</strong>g to our results, V<strong>in</strong>k’s second<br />
section appears as paraphyletic <strong>in</strong> the analyses (Figs. 1 <strong>and</strong> 2). An exp<strong>and</strong>ed<br />
sampl<strong>in</strong>g <strong>in</strong> future phylogenetic analysis (<strong>in</strong>clud<strong>in</strong>g members<br />
<strong>of</strong> Borneo <strong>and</strong> The Philip<strong>in</strong>es) is urgently needed <strong>in</strong> order to<br />
further clarify the phylogenetic <strong>relationships</strong> with<strong>in</strong> the genus.<br />
4.2.3. Zygogynum s.l.<br />
This genus comprises approximately 59 species distributed <strong>in</strong><br />
Australia, New Gu<strong>in</strong>ea, Moluccas, <strong>and</strong> New Caledonia (V<strong>in</strong>k,<br />
1993a, 1993b). However, only New Caledonian species have been
446 X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
<strong>in</strong>cluded <strong>in</strong> phylogenetic analyses <strong>of</strong> the W<strong>in</strong>teraceae, which casts<br />
doubts on the monophyly <strong>of</strong> the genus. The presence <strong>of</strong> Zygogynum<br />
s.l. <strong>in</strong> New Zeal<strong>and</strong> has been discussed by V<strong>in</strong>k (1988: 697), who<br />
stated that the New Zeal<strong>and</strong> Harrisipollenites fossil pollen could<br />
correspond to Zygogynum, <strong>and</strong> that ‘‘this type was elim<strong>in</strong>ated dur<strong>in</strong>g<br />
the Pleistocene, whereas its hardier <strong>of</strong>fspr<strong>in</strong>g Pseudow<strong>in</strong>tera<br />
survived <strong>and</strong> is now represented by three species”. This assumption<br />
suggests the possibility <strong>of</strong> a paraphyletic Zygogynum s.l. with<br />
respect to Pseudow<strong>in</strong>tera.<br />
Our results do not support the monophyly <strong>of</strong> the generic segregates<br />
Belliolum, Bubbia <strong>and</strong> the rema<strong>in</strong><strong>in</strong>g Zygogynum s.s. (Figs. 1<br />
<strong>and</strong> 2, Table 2). A phylogenetic analysis based on comb<strong>in</strong>ed data<br />
(morphology + molecular) is currently underway to further clarify<br />
species-level <strong>relationships</strong> with<strong>in</strong> the Belliolum + Bubbia +<br />
Exospermum + Zygogynum complex sensu van Tieghem (1900).<br />
4.3. Nucleotide substitution <strong>and</strong> speciation rates<br />
Our nucleotide substitution rates (NSR) for ITS <strong>in</strong> Pseudow<strong>in</strong>tera<br />
<strong>and</strong> Zygogynum fluctuated around 4.96 10 10 <strong>and</strong> 10.75 10 10<br />
substitutions/year, respectively (Table 5). Previously, Suh et al.<br />
(1993) estimated that NSR for Pseudow<strong>in</strong>tera <strong>and</strong> Zygogynum fall<br />
<strong>in</strong>to the 3.2–5.2 10 10 substitutions/year <strong>and</strong> 3.6–5.7 10 10<br />
substitutions/year <strong>in</strong>tervals, respectively. Differences between<br />
our higher estimates <strong>of</strong> NSR for Zygogynum <strong>in</strong> relation with the values<br />
reported by Suh et al. (1993), could be related with the model<br />
<strong>of</strong> DNA substitutions, sequences sampl<strong>in</strong>g, <strong>and</strong>/or estimation <strong>of</strong><br />
ages <strong>of</strong> the splitt<strong>in</strong>g between Pseudow<strong>in</strong>tera <strong>and</strong> Zygogynum. Suh<br />
et al. (1993) used the Kimura model <strong>of</strong> DNA substitutions <strong>and</strong> five<br />
sequences <strong>of</strong> Zygogynum s.l., whereas we used GTR + I + G model <strong>of</strong><br />
DNA substitutions <strong>and</strong> 17 sequences <strong>of</strong> Zygogynum s.l.; additionally,<br />
Suh et al. (1993) assumed that the splitt<strong>in</strong>g between<br />
Pseudow<strong>in</strong>tera <strong>and</strong> Zygogynum s.l. occurred at least 50–80 MY by<br />
the separation <strong>of</strong> New Zeal<strong>and</strong> from Australia accord<strong>in</strong>g to Raven<br />
<strong>and</strong> Axelrod (1974), the splitt<strong>in</strong>g between these two areas is based<br />
on the presence <strong>of</strong> Zygogynum s.l. <strong>in</strong> Australia (species not sampled<br />
by Suh et al., 1993); whereas our estimated ages <strong>of</strong> Pseudow<strong>in</strong>tera –<br />
Zygogynum s.l. splitt<strong>in</strong>g ranged from 52.24 to 69 MY (Table 4, node<br />
4; cf. Fig. 2B); these estimates are based on the NSRs <strong>and</strong> <strong>in</strong> the fossil<br />
record, rather than the geographical events considered by Suh<br />
et al. (1993).<br />
As expected, speciation rates <strong>in</strong> W<strong>in</strong>teraceae (between 0.056<br />
<strong>and</strong> 0.278 sp/MY; Table 5) <strong>and</strong> Drimys (0.128–0.278 sp/MY) are extremely<br />
low when compared to other high Andean taxa, such as<br />
Gentianella (1.71–3.2 sp/MY; von Hagen <strong>and</strong> Kadereit, 2001),<br />
Lup<strong>in</strong>us (1.73–2.78 sp/MY; Hughes <strong>and</strong> Eastwood, 2006), or Valeriana<br />
(0.8–1.34 sp/MY; Bell <strong>and</strong> Donoghue, 2005). In addition, the<br />
time for speciation (TFS) <strong>in</strong> Drimys (2.50–5.41 MY/sp; Table 5) is<br />
high when compared to those <strong>in</strong> Gentianella (0.22–0.40 MY/sp;<br />
von Hagen <strong>and</strong> Kadereit, 2001) orDendrosenecio (0.24–0.29 MY/<br />
sp; Knox <strong>and</strong> Palmer, 1995).<br />
Suh et al. (1993) suggested that the long generation time, the<br />
frequent vegetative propagation by runners <strong>in</strong> Pseudow<strong>in</strong>tera <strong>and</strong><br />
Tasmannia, <strong>and</strong> a low numbers <strong>of</strong> poll<strong>in</strong>ators <strong>of</strong> Pseudow<strong>in</strong>tera<br />
(associated with self-<strong>in</strong>compatibility) could be related to the extremely<br />
low rates <strong>of</strong> molecular evolution <strong>and</strong> speciation <strong>in</strong> these genera.<br />
Furthermore, Suh et al. (1993: 1054) stated that ‘‘possibly the<br />
slow rate <strong>of</strong> molecular evolution may be correlated with a similar<br />
slow rate <strong>of</strong> morphological changes <strong>in</strong> the family [...] even though it<br />
is <strong>of</strong>ten claimed that molecules do not march to the same rhythm<br />
as morphological traits”.<br />
In Zygogynum s.l. nucleotide substitution rates are similar or<br />
slightly higher than those found <strong>in</strong> Drimys (Table 5). However,<br />
diversification rates are higher <strong>in</strong> Drimys, which contrasts to the<br />
time for speciation, which is higher <strong>in</strong> Zygogynum s.l. The relatively<br />
higher morphological diversification <strong>of</strong> Zygogynum s.l. (at least <strong>in</strong><br />
New Caledonia; cf. V<strong>in</strong>k, 1993b) with respect to that <strong>of</strong> Drimys<br />
(cf. Smith, 1943b) is consistent to the differences <strong>in</strong> the <strong>in</strong>ferred<br />
time from the branch<strong>in</strong>g po<strong>in</strong>t <strong>of</strong> the extant species <strong>in</strong> both genera<br />
(37.8–48.6 MY vs. 6.9–7.5 MY based on the ITS data-set; 48–69 MY<br />
vs. 13.4–16.1 based on the comb<strong>in</strong>ed data-set Figs. 2B <strong>and</strong> 5B;<br />
Table 4). The capacity <strong>of</strong> the species <strong>of</strong> Zygogynum s.l. to live <strong>in</strong><br />
tropical lowl<strong>and</strong>s could also be related to the higher diversification<br />
<strong>of</strong> this genus (Feild et al., 2002).<br />
4.4. Biogeography<br />
The current distribution <strong>of</strong> the W<strong>in</strong>teraceae has been used <strong>in</strong> order<br />
to arrive at area cladograms. In the study by Crisci et al. (1991),<br />
based on 17 species-level area cladograms, the W<strong>in</strong>teraceae (represented<br />
by species <strong>of</strong> Drimys, Tasmannia <strong>and</strong> possibly Pseudow<strong>in</strong>tera),<br />
contributed to the area cladogram (SSA(AUS(NG(NC)))); (for acronyms<br />
see Fig. 6). As a result, two general area cladograms were obta<strong>in</strong>ed<br />
by ‘biogeographic parsimony’ (Fig. 6A <strong>and</strong> B) <strong>and</strong> two by a<br />
quantification <strong>of</strong> a component analysis (Fig. 6C <strong>and</strong> D).<br />
On the other h<strong>and</strong>, L<strong>in</strong>der <strong>and</strong> Crisp (1995) used 13 species-level<br />
area cladograms. The W<strong>in</strong>teraceae, contributed to the area<br />
cladogram (MAD(AUS,SSA)(NZ(AUS,NC,NG)))), to f<strong>in</strong>ally obta<strong>in</strong><br />
the general area cladogram shown <strong>in</strong> Fig. 6E.<br />
More recently, Sanmart<strong>in</strong> <strong>and</strong> Ronquist (2004) used 73 area<br />
cladograms. Two area cladograms were derived from the W<strong>in</strong>teraceae<br />
(), as follows: (MAD(AUS(SSA(NZ(NC,NG))))) <strong>and</strong> (NG(MA-<br />
D(AUS(SSA(NZ,NC))))). However, although the authors cited Karol<br />
et al. (2000) as the phylogenetic framework for these two area<br />
cladograms, the latter does not correspond to any <strong>of</strong> the phylogenies<br />
obta<strong>in</strong>ed by Karol et al. (2000). The two optimal areas<br />
cladograms obta<strong>in</strong>ed by Sanmart<strong>in</strong> <strong>and</strong> Ronquist (2004) are summarized<br />
<strong>in</strong> Fig. 6F.<br />
The area cladogram that summarizes the topology <strong>of</strong> W<strong>in</strong>teraceae<br />
distribution obta<strong>in</strong>ed from our research can be summarized<br />
as follows: (((((NZ)(NC,AUS,NG))(SWSA,NESA))(AUS,TAS,SEA,NG))<br />
(MAD)). In this area cladogram, South America has been splitted<br />
<strong>in</strong>to two different areas, Southwest South America (SWSA) <strong>and</strong><br />
North-east South America (NESA; us<strong>in</strong>g the 30° south latitude proposed<br />
as a limit by Humphries <strong>and</strong> Parenti, 1999; Fig. 3), <strong>in</strong> order to<br />
show the geographic distribution <strong>of</strong> our SW <strong>and</strong> NE clades <strong>of</strong><br />
Drimys. Thus, our phylogenetic analysis do not exactly reflect the<br />
geological reconstructions <strong>of</strong> Gondwanic fragmentation events<br />
presented by L<strong>in</strong>der <strong>and</strong> Crisp (1995), or by Sanmart<strong>in</strong> <strong>and</strong><br />
Ronquist (2004; see Fig. 6G).<br />
In our area cladogram two ‘‘biogeographic” politomies rema<strong>in</strong><br />
unresolved: The first (SEA, NG, AUS, TAS) is due to the distribution<br />
<strong>of</strong> Tasmannia, a genus primarily distributed <strong>in</strong> Tasmania <strong>and</strong> Australia<br />
(except T. piperita, widespread <strong>in</strong> Australia, New Gu<strong>in</strong>ea<br />
<strong>and</strong> SE Asia), <strong>in</strong>clud<strong>in</strong>g the Philipp<strong>in</strong>es <strong>and</strong> Borneo. The second<br />
polytomy (NC, AUS, NG) is due to the presence <strong>of</strong> Zygogynum s.l.<br />
<strong>in</strong> Australia, New Caledonia <strong>and</strong> New Gu<strong>in</strong>ea (cf. V<strong>in</strong>k, 1983,<br />
1985, 1988, 1993b, 2003). The <strong>in</strong>clusion <strong>of</strong> species <strong>of</strong> Zygogynum<br />
from Australia <strong>and</strong> New Gu<strong>in</strong>ea <strong>in</strong> further phylogenetic analysis<br />
will help to clarify such polytomy.<br />
4.5. Divergence time estimates<br />
The assumption <strong>of</strong> a more widespread geographical distribution<br />
<strong>of</strong> W<strong>in</strong>teraceae dur<strong>in</strong>g the Cretaceous <strong>in</strong> Gondwana is consistent<br />
with the presence <strong>of</strong> fossils <strong>of</strong> Walkeripollis <strong>in</strong> Israel <strong>and</strong> Gabon<br />
(c. 120 MY; Doyle, 2000) <strong>and</strong> <strong>in</strong> Patagonia (Argent<strong>in</strong>a, c. 100 MY;<br />
Barreda <strong>and</strong> Archangelsky, 2006; Fig. 3), <strong>and</strong> the vesselless fossil<br />
wood <strong>of</strong> W<strong>in</strong>teroxylon jamesrossi <strong>in</strong> West Antarctica at approximately<br />
85 MY (Fig. 3; Doyle, 2000).<br />
The estimates <strong>of</strong> the branch<strong>in</strong>g po<strong>in</strong>t between Tasmannia <strong>and</strong><br />
the rest <strong>of</strong> the family (Drimys + Pseudow<strong>in</strong>tera + Zygogynum s.l) is
77–92.38 MY (node 2 <strong>in</strong> Figs. 2B <strong>and</strong> 5B; Table 4); if so, it could<br />
have occurred before the separation <strong>of</strong> South America plus Australia<br />
from New Caledonia plus New Zeal<strong>and</strong> by the Tasman Sea,<br />
which has been dated around 80 MY (Fig. 6; cf. Sanmart<strong>in</strong> <strong>and</strong> Ronquist,<br />
2004).<br />
The age <strong>of</strong> the branch<strong>in</strong>g po<strong>in</strong>t between the New Zeal<strong>and</strong> species<br />
<strong>of</strong> Pseudow<strong>in</strong>tera <strong>and</strong> the species <strong>of</strong> the New Caledonian Zygogynum<br />
s.l. <strong>in</strong>cluded <strong>in</strong> our study (52.24–79 MY; node 4 <strong>in</strong> Fig. 2B <strong>and</strong> Table<br />
4) corresponds to estimate <strong>of</strong> c. 70–60 MY (Fig. 6G) given by Sanmart<strong>in</strong><br />
<strong>and</strong> Ronquist (2004) <strong>and</strong> by Swenson et al. (2001). The biogeographic<br />
implications <strong>of</strong> the Pseudow<strong>in</strong>tera + Zygogynum s.l. clade<br />
require further <strong>in</strong>vestigation <strong>and</strong> an <strong>in</strong>crease <strong>in</strong> the samples especially<br />
<strong>of</strong> the members <strong>of</strong> the complex Zygogynum.<br />
Our results (Figs. 1 <strong>and</strong> 2) show that the early divergent species<br />
<strong>of</strong> Tasmannia are <strong>in</strong> Australia plus Tasmania. The splitt<strong>in</strong>g between<br />
T. piperita from New Gu<strong>in</strong>ea <strong>and</strong> its sister Australian relatives is between<br />
15.5 <strong>and</strong> 18.5 MY (node 15 <strong>in</strong> Fig. 2B <strong>and</strong> Table 4). These<br />
ages are more recent than the New Gu<strong>in</strong>ea–Australia splitt<strong>in</strong>g by<br />
the Coral Sea Bas<strong>in</strong>, which occurred at approximately 30 MY<br />
X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449 447<br />
Fig. 6. (A–D) General area cladogram from Crisci et al. (1991); (E) General area cladogram from L<strong>in</strong>der <strong>and</strong> Crisp (1995); (F) One <strong>of</strong> the two possible optimal area cladograms<br />
for the plant data-set from Sanmart<strong>in</strong> <strong>and</strong> Ronquist (2004); (G) Geological area cladogram represent<strong>in</strong>g the <strong>relationships</strong> between the southern hemisphere l<strong>and</strong> masses,<br />
based on paleogeographic evidence. Time <strong>of</strong> vicariance is assumed as the primary fragmentation event. ( ) dated as 70–60 MY <strong>in</strong> alternative reconstructions (Sanmart<strong>in</strong> <strong>and</strong><br />
Ronquist, 2004). AFR, Africa; AUS, Australia; ESA, East South America; HOL, Holartic; IND, India; NA, North America; NC, New Caledonia; NG, New Gu<strong>in</strong>ea; NSA, Northern<br />
South America; MAD, Madagascar; SEA, South East Pacific; SSA, Southern South America; SWP, South West Pacific; TAS, Tasmania, <strong>and</strong> WANT, West Antarctica.<br />
(Fig. 6G; Sanmart<strong>in</strong> <strong>and</strong> Ronquist, 2004). However, accord<strong>in</strong>g to<br />
Swenson et al. (2001), two alternative estimates exists for New<br />
Gu<strong>in</strong>ea–Australia splitt<strong>in</strong>g: (1) 25 MY, based on the beg<strong>in</strong>n<strong>in</strong>g <strong>of</strong><br />
the uplift <strong>of</strong> New Gu<strong>in</strong>ea; (2) 15–10 MY, based on the expansion<br />
<strong>of</strong> arid conditions <strong>in</strong> Australia, <strong>and</strong> the fragmentation <strong>of</strong> mesic<br />
environments; these estimates predates <strong>and</strong> postdates, respectively<br />
the age <strong>of</strong> the splitt<strong>in</strong>g between T. piperita <strong>and</strong> its sister relatives<br />
(18.5–15.5 MY).<br />
Speciation <strong>of</strong> Pseudow<strong>in</strong>tera is estimated to have started by mid-<br />
Miocene (11.3–17.3 MY; node 10 <strong>in</strong> Fig 2B <strong>and</strong> Table 4), after the<br />
‘‘Oligocene bottleneck” <strong>in</strong> the New Zeal<strong>and</strong> biota (W<strong>in</strong>kworth<br />
et al., 2002). At that time, New Zeal<strong>and</strong> was reduced to a scattered<br />
archipelago, formed by isl<strong>and</strong>s <strong>of</strong> low elevation. The presence <strong>of</strong><br />
pollen likely assigned to Zygogynum s.l. from the Oligocene to the<br />
Pleistocene <strong>of</strong> New Zeal<strong>and</strong> <strong>in</strong>dicates that the genus was more<br />
widespread <strong>in</strong> the past (V<strong>in</strong>k, 1988).<br />
The branch<strong>in</strong>g po<strong>in</strong>t between Drimys <strong>and</strong> its sister group (69–<br />
84.6 MY; node 3, Fig. 2B <strong>and</strong> Table 4) could have predated the<br />
formation <strong>of</strong> the Tasman Sea (80 MY, Fig 6G) <strong>and</strong> the separation
448 X. Marquínez et al. / Molecular Phylogenetics <strong>and</strong> Evolution 53 (2009) 435–449<br />
<strong>of</strong> South America <strong>and</strong> Australia (52–35 MY, Fig. 6G). The oldest fossil<br />
assignable to Drimys is pollen <strong>of</strong> approximately 58.5 MY (Fig. 3;<br />
Doyle, 2000). However, the speciation <strong>of</strong> Drimys appears to have<br />
occurred much later (around 13.4–16.1 MY (node 11, Fig. 5B <strong>and</strong><br />
Table 4; us<strong>in</strong>g the comb<strong>in</strong>ed data-set), which co<strong>in</strong>cides <strong>in</strong> time<br />
with the beg<strong>in</strong>n<strong>in</strong>g <strong>of</strong> the Central Andean uplift (30 <strong>and</strong> 25% <strong>of</strong><br />
their modern elevation between 14 <strong>and</strong> 20 MY, respectively;<br />
Wodzicki, 2000). Ehrendorfer et al. (1979:78) stated that the precursors<br />
<strong>of</strong> Drimys ‘‘reached the ancient fragments <strong>of</strong> Gondwana<br />
l<strong>and</strong> masses which are now part <strong>of</strong> S. America (Guyana, Brazilian<br />
shield, etc.) after their separation from African fragment but before<br />
their fusion by the Andean Cordilleras emerg<strong>in</strong>g dur<strong>in</strong>g the Tertiary.<br />
D. roraimensis <strong>and</strong> D. brasiliensis could represent relicts from<br />
that time”. This assumption is not supported by the estimates <strong>of</strong><br />
age <strong>of</strong> splitt<strong>in</strong>g between SW <strong>and</strong> NE clades <strong>of</strong> Drimys (13.4–<br />
16.1 MY; node 11, Fig. 5B).Further age estimations with<strong>in</strong> Drimys<br />
is prevented by the lack <strong>of</strong> resolution <strong>in</strong> our phylogenetic analyses,<br />
except for the splitt<strong>in</strong>g between the Juan Fern<strong>and</strong>ez endemic<br />
D. confertifolia <strong>and</strong> its sister clade (D. <strong>and</strong><strong>in</strong>a + D. w<strong>in</strong>teri), dated<br />
<strong>in</strong> 9–10.8 MY (node 18, Fig. 5B <strong>and</strong> Table 4) predates the formation<br />
<strong>of</strong> the Juan Fern<strong>and</strong>ez Archipelago, dated on 4 MY (Anderson et al.,<br />
2001).<br />
5. Conclusions<br />
Our <strong>in</strong>creased sampl<strong>in</strong>g <strong>of</strong> Drimys, Tasmannia <strong>and</strong> Zygogynum s.l.<br />
supports the monophyly <strong>of</strong> these genera, but does not support the<br />
monophyly <strong>of</strong> Bubbia, Belliolum, <strong>and</strong> Zygogynum s.s. With<strong>in</strong> Drimys,<br />
we propose two clades, the SW <strong>and</strong> the NE clades. Divergence time<br />
estimates <strong>in</strong>dicate that the earliest cladogenetic events that gave<br />
rise to the genera <strong>of</strong> W<strong>in</strong>teraceae could have predate the<br />
Gondwanic fragmentation. The splitt<strong>in</strong>g between Drimys <strong>and</strong> its<br />
sister group (Pseudow<strong>in</strong>tera + Zygogynum s.l.) could have occurred<br />
by late Cretaceous, but speciation <strong>of</strong> Drimys seems to have started<br />
much later, dur<strong>in</strong>g late Miocene. The area cladogram derived from<br />
our phylogenetic analyses does not correspond to any <strong>of</strong> the previous<br />
area cladograms based on the W<strong>in</strong>teraceae.<br />
The use <strong>of</strong> gaps as characters, follow<strong>in</strong>g alignment criteria proposed<br />
by Kelchner (2000) <strong>and</strong> Borsch et al. (2003) <strong>and</strong> the SGC option<br />
proposed by Simmons <strong>and</strong> Ochoterena (2000) <strong>in</strong>creases<br />
resolution <strong>and</strong> branch support values. In contrast, the treatment<br />
<strong>of</strong> gaps as a fifth character decreases resolution <strong>and</strong> could cause<br />
artifactual topologies.<br />
Acknowledgments<br />
X.M. is <strong>in</strong>debted to the Instituto para el Desarrollo de la Ciencias<br />
y la Tecnología ‘‘Francisco José de Caldas”. (COLCIENCIAS) for a fellowship<br />
received from the ‘‘Programa de apoyo a doctorados nacionales<br />
2004”, <strong>and</strong> to the Botany Department <strong>of</strong> the University <strong>of</strong><br />
São Paulo (Brazil) for academic support dur<strong>in</strong>g lab work. A.S. <strong>and</strong><br />
M.L.F.S. are fellow researchers <strong>of</strong> CNPq (Conselho Nacional do<br />
Desenvolvimento Científico e Tecnológico, Brazil). We thank G.J.<br />
Anderson (University <strong>of</strong> Connecticut, USA), I. Cordeiro (Instituto<br />
de Botânica do Estado de São Paulo, Brazil), J. Salazar (Cornell University,<br />
USA), A. Townsmith (DNA Bank Manager, Missouri<br />
Botanical Garden, USA), <strong>and</strong> the Director <strong>of</strong> Horticulture <strong>of</strong> the Royal<br />
Botanic Garden Ed<strong>in</strong>burgh (UK), for provide silica-gel dried samples.<br />
We also thank Dr. Frederico Arzolla (Campos de Jordão) for<br />
support<strong>in</strong>g field work, L. F. Garcia (Universidad Nacional de Colombia)<br />
for help<strong>in</strong>g with Bayesian analysis. We acknowledge IBAMA<br />
for collect<strong>in</strong>g permits, <strong>and</strong> the curators <strong>of</strong> the herbaria M, NY, UC,<br />
<strong>and</strong> US, for the loans received at COL. Reviews <strong>of</strong> an early manuscript<br />
by J. Salazar, J. Lynch (Universidad Nacional de Colombia),<br />
<strong>and</strong> R. Callejas (Universidad de Antioquia, Colombia) improved<br />
the manuscript. We thank two anonymous reviewers for critical<br />
read<strong>in</strong>g <strong>of</strong> the manuscript.<br />
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