Generic relationships and dating of lineages in ... - CNCFlora

Generic relationships and dating of lineages in Winteraceae based on nuclear 

(ITS) and plastid (rpS16 and psbA-trnH) sequence data 

Xavier Marquínez a, *, Lúcia G. Lohmann b , Maria L. Faria Salatino b , Antonio Salatino b , Favio González a 

a Universidad Nacional de Colombia, Apartado Aéreo 7495, Bogotá D.C., Colombia 

b Universidade de São Paulo, Departamento de Botânica, Instituto de Biociências, Rua do Matão, 277, CEP 05508-090, São Paulo, SP, Brazil 

article info 

Article history: 

Received 26 November 2008 

Revised 30 June 2009 

Accepted 1 July 2009 

Available online 4 July 2009 

Keywords: 

Canellales 

Divergence time estimates 

Drimys 

Gondwana biogeography 

Pseudowintera 

Takhtajania 

Tasmannia 

Winteraceae 

Zygogynum 

1. Introduction 

abstract 

Winteraceae was first described as a family by Lindley (1836). 

Its current circumscription comprises five genera ( Drimys, 

Pseudowintera, Takhtajania, Tasmannia, and Zygogynum s.l.) and 

approximately 79 species distributed in Australia, Tasmania, Borneo, 

Celebes, Moluccas, New Caledonia, New Guinea, New Zealand, 

Philippines, Madagascar, and Central- and South America (Vink, 

1993a, 2003). The family belongs to the order Canellales, along with 

Canellaceae (APGII, 2003; Cai et al., 2006). The monophyly of the order 

is supported by molecular and morphological data (e.g., Chase 

et al., 1993; Qiu et al., 1993, 1999; Nandi et al., 1998; Doyle and 

Endress, 2000; Soltis et al., 2000, 2005; Zanis et al., 2002). 

The Winteraceae possess vesselless xylem and plicate carpels, 

two conditions long considered ancestral in the angiosperms 

(van Tieghem, 1900; Bailey and Thompson, 1918; Bailey and 

Swamy, 1951; Takhtajan, 1980; Cronquist, 1981). However, those 

conditions have recently been reinterpreted as secondarily derived 

(Young, 1981; Soltis et al., 2005). The physiological implications of 

vesselless xylem and the presence of stomatal plugs have been 

suggested as a result of the relictual condition of the family by 

Bailey and Nast (1944) and Baranova (1972). A reinterpretion of 

* Corresponding author. Fax: +57 1 3165310. 

E-mail address: xmarquinezc@unal.edu.co (X. Marquínez). 

1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. 

doi:10.1016/j.ympev.2009.07.001 

Molecular Phylogenetics and Evolution 53 (2009) 435–449 

Contents lists available at ScienceDirect 

Molecular Phylogenetics and Evolution 

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

Phylogenetic analyses of representative species from the five genera of Winteraceae (Drimys, Pseudowintera, 

Takhtajania, Tasmannia, and Zygogynum s.l.) were performed using ITS nuclear sequences and a combined 

data-set of ITS + psbA-trnH+rpS16 sequences (sampling of 30 and 15 species, respectively). Indel informativity 

using simple gap coding or gaps as a fifth character was examined in both data-sets. Parsimony 

and Bayesian analyses support the monophyly of Drimys, Tasmannia, and Zygogynum s.l., but do not support 

the monophyly of Belliolum, Zygogynum s.s., and Bubbia. Within Drimys, the combined data-set recovers two 

subclades. Divergence time estimates suggest that the splitting between Drimys and its sister clade (Pseudowintera 

+ Zygogynum s.l.) occurred around the end of the Cretaceous; in contrast, the divergence between 

the two subclades within Drimys is more recent (15.5–18.5 MY) and coincides in time with the Andean 

uplift. Estimates suggest that the earliest divergences within Winteraceae could have predated the first 

events of Gondwana fragmentation. 

Ó 2009 Elsevier Inc. All rights reserved. 

these characters based on a phylogenetic framework indicates that 

they might actually represent adaptations to changes from tropical 

to temperate (stational) habitats since the early Cretaceous (Feild 

et al., 1998, 2000, 2002; Feild and Holbrook, 2000). 

The current available phylogenetic analyses for Winteraceae are 

not conclusive in terms of generic relationships. Vink (1988), based 

on morphological data obtained two alternative topologies: 

(Takhtajania (Drimys + Tasmannia)(Zygogynum s.l. + Pseudowintera)); 

and (Tasmannia (Drimys (Takhtajania (Zygogynum + Pseudowintera)))). 

On the other hand, Endress et al. (2000), primarily based mainly on 

floral morphology, arrived at two topologies depending on the outgroup 

chosen: (Canella (Tahktajania + Pseudowintera + Zygogynum) 

(Drimys + Tasmannia)); and (Degeneria (Zygogynum p.p. 

(Z. thieghemii (Takhtajania + Pseudowintera))) (Drimys winteri 

(Drimys p.p. + Tasmannia piperita) (Tasmannia p.p.)). The analysis 

based on Degeneria as the outgroup casts doubts on the monophyly 

of Drimys, Tasmannia and Zygogynum. Finally, three separate analyses 

based on molecular data (Suh et al., 1993; Karol et al., 2000; 

and Doust and Drinnan, 2004), coincide in the generic relationship 

(Takhtajania (Tasmannia (Drimys (Pseudowintera + Zygogynum 

s.l.)))). The controversial close relationship between Drimys and 

Tasmannia, proposed by Smith (1943b, cf. also Vink, 1970, 1993a) 

is supported by all of the morphological analyses (except by one 

of Vink’s analysis and by the flower developmental data provided 

by Doust and Drinnan, 2004).

436 X. Marquínez et al. / Molecular Phylogenetics and Evolution 53 (2009) 435–449 

The taxonomy of the family has been reviewed by Smith 

(1943a, 1943b) and Vink (1970, 1977, 1983, 1985, 1988, 1993a). 

Within the family, two genera are particularly problematic, Drimys 

and Zygogynum. The current circumscription of Drimys (following 

Smith, 1943b; Ehrendorfer et al., 1979; Rodríguez and Quezada, 

1991, 2001) includes seven species distributed from southern Mexico 

to Tierra del Fuego (Table 1). However, the genus has been 

poorly sampled in phylogenetic analyses (three species by Endress 

et al., 2000, and two species by Doust and Drinnan, 2004), bringing 

to question its monophyly. Zygogynum has traditionally been splitted 

into four independent genera, Belliolum, Bubbia, Exospermum 

and Zygogynum s.s. (van Tieghem, 1900; Smith, 1943a). Alternatively, 

Vink (1985, 1988, 1993a, 1993b) treats all these genera as 

Zygogynum s.l. Molecular analysis (Suh et al., 1993) supports the 

topology (Bubbia comptonii (Exospermum stipitatum, Zygogynum 

acsmithii (Z. pomiferum (Belliolum pancheri, Z. bicolor)))), in which 

Zygogynum s.s. is not monophyletic. 

Three competing hypotheses have been proposed to explain the 

biogeographic history of the Winteraceae: (1) a South American 

origin followed by dispersal to Africa and Madagascar on one side, 

and dispersal to Australasia via Antarctica on the other side (cf. 

Fig. 2A inFeild et al., 2000); (2) an African origin followed by dispersal 

into Madagascar and Antarctica, followed by subsequent 

dispersal into America and Australasia (cf. Fig. 2B inFeild et al., 

2000); and (3) a northern Gondwanic tropical origin followed by 

dispersal into southern temperate Gondwana and subsequent 

migration from America to Antarctica and to Australasia, or from 

Africa to Madagascar, India, and finally Australasia, after the fragmentation 

of Gondwana into Africa and America (cf. Fig. 3 in Doyle, 

2000). 

The aims of the present study are to reconstruct the phylogenetic 

relationships between Drimys and the other genera of Winteraceae, 

to test the monophyly of Drimys, to explore the 

phylogenetic relationships inside Drimys and Zygogynum s.l., and 

Table 1 

Taxonomic history and current geographic distribution of Drimys species. 

Forster and 

Forster, 1776 

Linnaeus f., 

1781 

de Candolle, 

1817, 1824 

Saint-Hilaire, 

1824 

Philippi, 

1856 

to estimate divergence times in order to evaluate alternative biogeographic 

hypotheses for Drimys and the entire Winteraceae. 

2. Materials and methods 

2.1. Sampling 

Table 2 summarizes the voucher specimens and Genbank 

accession numbers for the taxa included in the present study. 

Two data-sets were prepared for the various analyses: (1) A 

data-set including 52 sequences of ITS, representing 30 species of 

Winteraceae and six species of Canellaceae; and (2) a combined 

data-set including 23 sequences of ITS, 21 sequences of psbA-trnH 

and 20 sequences of rpS16 sequences representing 14 species of 

Winteraceae, plus Capsicodendron denissi and Cynnamosma madagascarensis 

(Canellaceae) as the outgroup. In this data-set, sequences 

of Drimys roraimensis and Tasmannia lanceolata were 

missing for rpS16 and psbA-trnH, and sequences of Drimys confertifolia 

were missing for rpS16. 

2.2. DNA extraction, amplification and sequencing 

DNA was extracted from silica-gel dried leaves, air-dried leaves 

and herbarium specimens (Table 2). For DNA extraction, PCR 

amplification and sequencing, we followed the procedures described 

by Ferreira and Grattapaglia (1996). For PCR of ITS1- 

5.8S–ITS2 we used Primers Leu1 (Malcomber, 2002; Doust and 

Drinnan, 2004) and ITS4 (White et al., 1990; Suh et al., 1993). 

Amplification reactions contained 3 ll MgCl2 (1.5 M), 5 ll of 

amplification buffer 10 (160 mM (NH 4)SO 4, 670 mM Tris–HCl 

(ph 8.8), 0.1% Tween-20), 1 ll of bovine serum albumine (BSA; 

5 lg/ll), 2.5 ll of DMSO, 0.5 ll of each primer (10 lM), 3 ll of 

dNTPs (2 mM), 0,25 ll Taq DNA Polimerase, 2 ll of DNA and 

32.75 ll ofH2O. The following PCR conditions were used: 80° for 

Miers, 1858 Reiche, 1898 Smith, 1943a Ehrendorfer et al., 

1979 

D. brasiliensis 

var. roraimensis 

D. angustifolia D. brasiliensis 

var. angustifolia 

D. granadensis D. retorta D. brasiliensis 

D. montana 

vars. retorta 

D. brasiliensis 

& campestris 

D. granadensis D. granadensis D. granadensis D. granadensis D. granadensis a 

D. mexicana 

High mountains 

in Mexico and 

Central America, 

Northern Andes 

of South America 

D. winteri D. winteri D. winteri D. winteri D. winteri vars. 

D. chilensis D. chilensis 

winteri & chilensis 

a Drimys species recognized in this paper. 

D. winteri 

var. andina 

D. winteri 

var. andina 

D. roraimensis a 

(Venezuelan 

tepuyes, 

Roraima) 

D. angustifolia a 

(South of Brazil) 

D. brasiliensis a 

(Brazil) 

Rodríguez and 

Quezada, 

1991, 2001 

D. winteri a 

Southern Andes 

of Chile and 

Argentina, 

Patagonia and 

Tierra del Fuego 

D. andina a 

Nahuelbuta 

mountains and 

Andes of Chile 

and Argentine 

D. confertifolia D. fernandezianus D. confertifolia D. confertifolia a 

Juan Fernandez 

Islands (Chile)

5 min; 35 cycles of 94° for 30 s; 48° for 1 min.; 72° for 90 s; and, 

72° for 7 min. ITS cloning was not performed in Drimys because 

previous studies (Karol et al., 2000; Doust and Drinnan, 2004) 

found only one copy after cloning. Amplifications of psbA-trnH 

and rpS16 used primers and PCR conditions as described by Shaw 

et al. (2005). PCR products were cleaned using the GFX kit 

(Amersham Bioscience, Piscataway, NJ). Purified products were sequenced 

using amplification primers and the ABI Prism Big Dye 

Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, 

Foster City, CA) on an ABI 3700 Ò Automated Sequencer. 

2.3. Phylogenetic analyses 

Forward and reverse sequences were compiled into consensus 

sequences using MacClade 4.06 (Maddison and Maddison, 2003). 

Consensus sequences were aligned with Clustal X (Thompson 

et al., 1997), and subsequently adjusted manually. For psbA-trnH 

and rpS16, manual adjustments followed Borsch et al. (2003) and 

Kelchner (2000); for ITS, adjustments followed Karol et al. 

X. Marquínez et al. / Molecular Phylogenetics and Evolution 53 (2009) 435–449 437 

Fig. 1. Strict consensus tree resulting from parsimony analysis of the ITS data-set, with bootstrap values presented above branches, numbers before scientific names 

corresponds to different accessions of the same species; Zygogynum pomiferum spp. balansae Vink. (A) Considering gaps as missing, the arrowhead and the arrow indicate 

the corresponding clades derived from clones 1 and 2 sequences from Suh et al. (1993). (B) Using the simple gap coding option; numbers in parenthesis below branches 

correspond to gaps that support clades; circle and letters indicate synapomorphies and/or homoplasies (see Table 3). 

(2000). Non-aligned regions between Canellaceae and Winteraceae 

were treated as non-overlapping indels to minimize incorrect 

homology assessments. 

In order to test congruence among data-sets, we implemented 

two tests in PAUP version 4.0b10 (Swofford, 2002): (1) the ILD 

Test (Farris et al., 1994) using combined ITS + rpS16 + psbA-trnH 

partitioned nexus matrix, 1000 replicates of random addition sequences 

and a maximum of 10 trees retained per replicate; starting 

trees were obtained via random stepwise addition with TBR branch 

swapping and (2) the SH test (Shimodaira and Hasegawa, 1999), 

using GTR + I + G model of DNA substitutions to compare the likelihood 

calculated for strict consensus trees of ITS, rpS16 and 

psbA-trnH data-sets under parsimony (cf. Fig. 4); and also for 

majority rule consensus trees of these data-sets obtained under 

Bayesian analyses. 

Two independent analyses of the ITS and combined data-sets 

were performed in PAUP version 4.0b10 (Swofford, 2002), using 

parsimony. These analyses were conducted using a heuristic search 

with 1000 replicates of random addition sequences and a


Fig. 2. (A) Strict consensus tree resulting from a parsimony analysis of the ITS data-set treating gaps as a fifth character, bootstrap values are presented below branches (B) 

Majority rule consensus tree resulting from Bayesian analysis of the ITS data-set using GTR + I + G DNA substitution model; PP values are indicated above branches. Numbers 

in parenthesis below branches indicate nodes used for divergence time estimates, followed by the range of ages in million years according to Table 4. 

maximum of 10 trees retained per replicate, with collapsed 

branches of zero length. Starting trees were obtained via random 

stepwise addition with TBR branch swapping. Branch support 

was calculated using bootstrap and 1000 replicates in a heuristic 

search, 100 replicates for random sequence addition. 

In order to compare the impact of alternative gap coding 

schemes in both analyses, gaps were treated in three ways: (1) 

as missing data (GM); (2) at the end of the matrix as presence-absence 

characters using the simple gap coding (SGC) scheme of Simmons 

and Ochoterena (2000); and (3) using ‘‘gaps as a fifth 

character” (GFC) as implemented in PAUP version 4.0b10 (Swofford, 

2002). Informative gaps, used in the SGC option analyses were 

treated as insertions (ins), deletions (del), duplications of motifs 

(dup) and short (2–4 bp; SHR) or long (7–17 bp; LHR) homonucleotides 

repeats. Table 3 includes information on the gap coding 

treatments used in the various analyses (cf. Figs. 1B and 5A). 

In addition, a Bayesian analysis was performed for each data-set 

using MrBayes 3.1.1. (Huelsenbeck and Ronquist, 2001). 

GTR + I + G was selected as the best model of DNA substitution 

for each of the data-sets using the LRT implemented in MrModel 

Test 3.7 (Nylander, 2004). Analyses using MCMCMC were run for 

3,000,000 generations, initiating with a random starting tree and 

default settings (i.e., four chains, temperature of the heated chain 

set to 0.2). Each chain was sampled every 1000 generations for a 

total of 3000 trees sampled. Sample points collected prior to stationary 

were eliminated (i.e., burn-in;

was processed using r8s which summarizes the distribution of 

divergence times for each node. Canellaceae were excluded from 

these analyses in order to avoid artifactual branching point ages 

due to the high differences of substitution rates with respect to 

the Winteraceae. Three alternative calibration points were explored 

in four scenarios that correspond to combinations of fixed 

or minimum ages for specific nodes in the Bayesian trees obtained 

from the ITS or the combined analyses (Table 4). 

Node 1 in the ITS and combined topologies was fixed using two 

different ages: (1) 120 MY based on the early Cretaceous pollen 

Walkeripollis from Africa (Doyle, 2000) and the age of the split between 

Madagascar and Africa by the Somali basin (121 MY; 

Sanmartin and Ronquist, 2004) and (2) 135 MY, taking into account 

the first Gondwanic fragmentation event by the southern South 

Atlantic ocean that separated Africa from the rest of southern 

Gondwana (Sanmartin and Ronquist, 2004). 

Winteroid pollen fossil from the Campanian of SE Australia (c. 

77 MY; Dettmann and Jarzen, 1990; Doyle, 2000; Feild et al., 

2002), assigned to Tasmannia (node 2; Table 4); finally, pollen fossil 


Fig. 3. Distribution map of Drimys species (Winteraceae), and location of Winteroid fossils. 

from the Late Cretaceous of New Zealand (c. 69 MY; initially reported 

as Zygogynum by Mildenhall, 1980, but later assigned to 

Pseudowintera by Feild et al., 2002) were used to constrict the minimum 

age for nodes 3 or 4 (Table 4). 

Finally, data from scenario 4 (Table 4) were used to calculate 

the following variables: (1) speciation rates for each genus of Winteraceae, 

assuming an equal rate of random speciation Yule model 

(Hughes and Eastwood, 2006); (2) nucleotide substitution rates 

(NSR); and (3) time for speciation (von Hagen and Kadereit, 

2001; Table 5). 

3. Results 

Twenty eight new sequences of ITS, 21 of psbA-trnH and 20 of 

rpS16 were obtained for the present study; two sequences of 

psbA-trnH and rpS16 correspond to Canellaceae and the remaining 

sequences correspond to Winteraceae (Table 2). Sequences of ITS1 

ranged from 235 to 252 bp in Winteraceae and from 244 to 275 bp 

in Canellaceae; the size of 5.8S coding region was 164 bp long for


Table 2 

Taxa included in this study with localities, voucher information and Genbank accession. 

Taxa Country Voucher Sequences 

origin a 

Genbank accession number 

ITS psbAtrnH 

rpS16 

Canellaceae 

Canella winterana (L.) Gaernt. South America LBT 124, NO 1 LO3844 

Capsicodendron dinissii (Schwacke) Occhioni South America Butzkee et al. 12521 (US) 2 AY004132 

Capsicodendron dinissii 

(Schwacke) Occhioni 

Brazil (Parana) Lohmann et al. 727 (COL, SPF) 4 FJ539215 FJ554663 

Cinnamodendron ekmanni 

Sleumer 

Dominican Republic García and Veloz 6866 (SD) 2 AY004143 

Cinnamosma madagascariensis 

Danguy 

Madagascar Lowry 4991 (MO) 2 FJ53 

Cinnamosma madagascariensis 

Danguy 

Madagascar Rabevohitra 4510 (MO) 4 FJ539214 FJ554659 

Pleodendron macranthum (Baill.) Tiegh. Puerto Rico Axelrod 10783 (MO) 2 AY004134 

Warburgia salutaris (G. Bertol.) Chiov. South Africa Goldblatt 11314 (MO) 2 AY004130 

Winteraceae 

Drimys andina (Reiche) Rodr. & Quez. (1) Chile, X region 19961186 RBGE 4 FJ539242 FJ539211 FJ554657 

Drimys andina (2) Argentina, Neuquén Beenken 992 (M) 4 FJ539241 FJ539212 FJ554662 

Drimys andina (Reiche) Rodr. & Quez. (3) Chile, IX region 19961048 RBGE 4 FJ539243 

Drimys angustifolia Miers Brazil, Rio grande do sur Wasum et al. 10779 (US) 4 FJ539232 FJ539203 FJ554651 

Drimys brasiliensis Miers. (1) Brazil, Paraná Lohmann et al. 728, (COL, SPF) 4 FJ539226 FJ539204 FJ554652 

Drimys brasiliensis (2) Brazil, Paraná Lohmann et al. 729 (COL, SPF) 4 FJ539227 FJ539205 FJ554653 

Drimys brasiliensis (3) Brazil, Cunha N.P. Cordeiro 2917 (SP) 4 FJ539228 FJ539206 FJ554654 

Drimys brasiliensis (4) Brazil, Campos do Jordao Lohmann et al. 738 (COL, SPF) 4 FJ539229 FJ539207 FJ554660 

Drimys brasiliensis (St. Hil.) Miers (5) Brazil, Paraná Cordeiro 553 (US) 4 FJ539230 

Drimys brasiliensis (6) Brazil, Paraná Hatschbach 17330 (COL, MBM) 4 FJ539231 

Drimys confertifolia Philippi Chile, Juan Fernandez Bernardello and Anderson 3005 4 FJ539236 FJ539208 

Islands 

(UCA) 

Drimys granadensis L.f. (1) Colombia, Cundinamarca Marquínez 004 (SPF, COL) 4 FJ539233 FJ539201 FJ554649 

Drimys granadensis(2) Colombia, Santander Morales 1765 (COL) 4 FJ539234 FJ539202 FJ554650 

Drimys granadensis (3) Costa Rica Salazar 2630 (BH, COL) 4 FJ539235 

Drimys roraimensis (A.C. Smith) Ehrend. & 

Gottsb. 

Venezuela, Bolivar Holst 3689 (NY) 4 FJ539225 

Drimys winteri Forst. and Forst. (1) Chile, Valparais Simon 142 (UCA) 4 FJ539237 FJ539209 FJ554655 

Drimys winteri (2) Chile, IX Region 19880691 

RBGE 

4 FJ539238 FJ539216 FJ554656 

Drimys winteri (3) Chile XII Region 19670643 

RBGE 

4 FJ539239 FJ539210 FJ554661 

Drimys winteri (4) Argentina, Patagonia Donat 398 (F, M) 4 FJ539240 

Pseudowintera axillaris (Forst. & Forst.) Dandy New Zealand LBT 300 (NO) 1 AY004124 

Pseudowintera colorata (Raoult) Dandy New Zealand LBT 301 (NO) 1 AY004125 

Pseudowintera colorata New Zealand 19981039 RBGE 4 FJ539196 FJ554644 

Takhtajania perrieri (Capuron) Baranova & J.F. 

Leroy 

Madagascar Rakotomalaza et al. 1342 (MO) 2 AY004129 

Takhtajania perrieri Madagascar Rabenantoandro J 219 (MO) 4 FJ539213 FJ554658 

Tasmannia glaucifolia J.B. Williams Australia, NSW (NSW) 198433 (LCR) 863140 3 AY526315 

Tasmannia insipida R.Br. ex DC Australia, Queensland LBT 108 (NO) 1 AY004127 

Tasmannia lanceolata (Poir.) A.C. Smith Australia Berkeley B.G. 60.0052 (NO) 1 AY004128 

Tasmannia piperita (Hook.f.) Miers Irian Jaya (New Guinea) Lowry 5287 (MO) 4 FJ539244 

Tasmannia purpurascens (Vickery) A.C. Smith Australia, NSW (NSW) 209939 

(LCR) 875858 

3 AY526309 

Tasmannia stipitata (Vickery) A.C. Smith Australia, NSW (NSW) 209908 

(LCR) 875857 

3 AY526316 

Tasmannia xerophila (Parment.) M. Gray Australia, NSW (NSW) 215000 

(LCR) 850099 

3 AY526312 

Zygogynum acsmithii Vink. b 

New Caledonia LBT 201 (NO) 1 (clone 2) AY004122 

Zygogynum amplexicaule (Vieill. ex Parment.) 

Vink 

= Bubbia amplexicaulis (Viell. Ex Parment.) 

Dandy 

New Caledonia Mc Pherson 19089 (MO) 4 FJ539222 

Zygogynum bicolor Tiegh. b 


Zygogynum bicolor Tiegh. b 


Zygogynum baillonii Tiegh. b 

New Caledonia Mc Pherson 19206 (MO) 4 FJ539219 FJ539197 FJ554645 

Zygogynum crassifolium (Baill.) Vink 

= Belliolum crassifolium Tiegh. 

= Bubbia crassifolium Burtt 

New Caledonia Lowry 6451 (MO) 4 FJ539221 

Zygogynum comptonii (Baker f.) Vink 

= Bubbia comptonii (Baker f.) Dandy 


b 

Zygogynum comptonii (Baker f.) Vink New Caledonia Mc Pherson 18061 (MO) 4 FJ539199 FJ554647 

Zygogynum fraterculum Vink c 


Zygogynum pauciflorum (Baker f.) Vink c 

= Bubbia pauciflora (Baker f.) Dandy 


Zygogynum pancheri ssp. Arrhantum 

= Belliolum pancheri (Baillon) Tiegh. 

New Caledonia LBT 205 (NO) 1 (clone 2) AY004120


Table 2 (continued) 

Taxa Country Voucher Sequences 

origin a 

Genbank accession number 

ITS psbAtrnH 

rpS16 

Zygogynum pomiferum spp. balansae 

(Tiegh) Vink 

= Zygogynum balansae Tiegh 


b 







Zygogynum stipitatum Baill. 

= Exospermum stipitatum Tiegh. 

New Caledonia Mc Pherson 19231 (MO) 1 (clone 1) AY004113 

b 

Zygogynum stipitatum Baill. 

Exospermum stipitatum Tiegh. 

New Caledonia LBT 202, (NO) 1 (clone 2) AY004121 

b 

Zygogynum tanyostigma Vink c 

New Caledonia Mc Pherson 18986 (MO) 4 FJ539220 FJ539200 FJ554648 

Zygogynum vinkii Sampson New Caledonia Mc Pherson 19141 (MO) 4 FJ539218 FJ539198 FJ554646 

Numbers before scientific names correspond to different accessions of the same species. 

a 

Sequences origin: 1, Suh et al. (1993); 2,Karol et al. (2000); 3,Doust and Drinnan (2004); 4, this research. 

b 

Traditional denomination of species followed by Suh et al. (1993) and Karol et al. (2000). 

c 

This species were described by Vink recently, He indicates that the position of Z. fraterculum and Z. pauciflorum is ‘‘intermediate between the Bubbia and the Zygogynum s.s. 

types” (Vink, 2003). 

Fig. 4. Strict consensus trees resulting from parsimony analyses of the following data-sets: (A) rpS16, (B) psbA-trnH, and (C) ITS. Numbers next to the branches correspond to 

bootstrap values.


Table 3 

Summary of indels in the ITS, psbA-trnH and rpS16 sequences of Canellales. 

ITS (52 OTUS) Combined analyses (24 OTUS) 

ITS psbA-trnH rpS16 

(A) Informative indels (codified using SGC) 

Total # of informative indels 44 38 5 7 

# of informative indels across families a 

19 29 3 5 

# of informative indels in Canellaceae 7 – – – 

Tasmannia (GC)dup (GT)dup – – – 

((Pseudowintera + Zygogynum) Drimys) SHR SHR – – 

Drimys 2 (GC)ins 2 (GC)ins – (CTTT)dup 

2 SHR 2 SHR 

Drimys SW clade – – (GTCAATC) dup – 

Pseudowintera (TG)dup 

(GT)dup 

– – – 

Zygogynum (Clone 1) (CT)dup 

SHR 

– – – 

Zygogynum (Clone 2) (AG)dup (AG)dup – – 

Zygogynum (GTG)dup (GTG)dup – (TC)Del 

SHR SHR 

Indel A SHR – – – 

Indel B (A)del (A)del – – 

Indel C (ATC)dup – – 

Indel D SHR – – – 

Indel E – – (GTAAAT) dup – 

Indel F (GT)dup – – – 

Indel G SHR – – – 

(B) Number of uninformative indels (autapomorphies) 

Duplication of motifs 3 5 2 2 

Insertion 1 1 0 – 

Deletion 5 5 3 2 

SHRs 6 6 1 – 

LHRs 1 1 1 3 

Del, deletion; dup, duplication; Ins, insertion; LHR, long homonucleotide string (>5 bp); SHR, short homonucleotide repeats (2–4 bp). 

a Across families = Indels present in all the species of Winteraceae or Canellaceae but not both. Sequences in 5 0 –3 0 sense. (Indels A–G are plotted in Fig. 2B and/or 5B). 

Fig. 5. Strict consensus tree resulting from analyses of the combined ITS + psbA-trnH + rpS16 data-set parsimony analyses. (A) Considering gaps as missing (MG) or simple 

gap coding options (SGC). (Topologies are identical, see Table 6), first bootstrap values shown above branches correspond to GM analysis; second bootstrap values (shown in 

parenthesis) correspond to the SGC analysis. Numbers in parenthesis below branches indicates numbers of gaps supporting clades in the SGC analysis; circles and letters 

indicate homoplasies (see Table 3); and, (B) Majority rule consensus tree resulting from a Bayesian analysis using GTR + I + G DNA substitution model; PP values are presented 

above branches; numbers in parenthesis below branches represented nodes used for divergence time estimates, followed by the range of ages in million years according to 

Table 4. 

all species of Canellales; ITS2 sequences ranged from 213 to 226 bp 

in Winteraceae and 187–233 bp in Canellaceae. 

3.1. Phylogenetic analyses based on ITS sequences 

The parsimony analysis considering gaps as missing (GM) resulted 

in three trees of 591 steps, with CI = 0.743 and RI = 0.901 

(Table 6). The monospecific Takhtajania is sister to the rest of the 

family. The four non-monospecific genera of Winteraceae appear 

as monophyletic and are supported with bootstrap values > 80% 

(Fig. 1A). Within Tasmannia, T. lanceolata is sister to the rest of 

the genus; in turn, T. insipida is sister to an unresolved clade 

formed by T. piperita (T. xerophila + T. glaucifolia) and (T. stipitata 

+ T. purpurascens). Within Drimys, only one clade (D. andina + D.

Table 4 

Average ages (MY) and confidence intervals (a = 0.05) for the Bayesian consensus tree nodes of ITS (Fig. 3B) and combined analyses (Fig. 5B) estimated using r8s under six 

different scenarios. For exact node locations see Figs. 3 and 5. 

winteri) was recovered; the relationship among the other five species 

remained unresolved. 

The ITS data shows that sequences from Zygogynum s.l. conform 

two main clades: (1) ‘Clone 1’ sequences (Suh et al., 1993) of 

(Zygogynum stipitatum (Z. pomiferum 1+Z. bicolor)); (2) ‘Clone 2’ 

sequences (Suh et al., 1993) of(Z. comptonii (Z. acsmithii, Z. stipitatum 

(Z. pomiferum 1 (Z. pancheri + Z. bicolor)))), as sister to (Z. 

amplexicaule (Z. crassifolium (Z. fraterculum + Z. pauciflorum) and 

(Z. tanyostigma (Z. vinkii (Z. pomiferum 1+Z. baillonii))))). Clade 1 

is supported by two indels (cf. Fig. 1B and Table 3), five substitutions 

in ITS1, one substitution in 5.8S and six substitutions in 

ITS2; Clade 2 is supported by one indel (cf. Fig. 1B and Table 3), 

and two substitutions in each ITS1 and ITS2. 

The parsimony analysis using the SGC option produced three 

trees of 646 steps, CI = 0.751 and RI = 0.912 (Fig. 1B). The strict consensus 

tree resulting from this analysis is similar to that obtained 

from the analysis that considered gaps as missing, but with higher 

bootstrap values in some clades. The main difference in topology 

occurs within Zygogynum, asZ. amplexicaule plus Z. crassifolium 


Scenario 1 Scenario 2 Scenario 3 Scenario 4 

Fixed age (MY) node 1 135 135 120 120 

Minimum age = 77 MY Node 2 Node 2 Node 2 Node 2 

Minimum age = 69 MY Node 3 Node 4 Node 3 Node 4 

(A) ITS analysis 

Node 1 135 ± 0 135 ± 0 120 ± 0 120 ± 0 

Node 2 85.44 ± 2.40 92.38 ± 1.90 77 ± 0 87.01 ± 1.42 

Node 3 76.60 ± 1.93 84.64 ± 1.50 69 ± 0 80.95 ± 1.09 

Node 4 58.03 ± 2.16 69 ± 0 52.24 ± 2.02 69 ± 0 

Node 5 41.97 ± 1.54 48.63 ± 1.47 37.82 ± 1.41 47.81 ± 1.61 

Node 6 11.30 ± 0.84 12.79 ± 0.74 10.29 ± 0.82 12.40 ± 0.76 

Node 7 4.62 ± 0.41 5.18 ± 0.42 4.17 ± 0.41 5.00 ± 0.43 

Node 8 5.93 ± 0.62 6.59 ± 0.54 5.34 ± 0.44 6.29 ± 0.43 

Node 9 10.94 ± 0.88 12.04 ± 1.17 9.85 ± 0.93 11.59 ± 1.04 

Node 10 13.83 ± 0.84 15.70 ± 1.22 12.37 ± 0.71 15.11 ± 1.24 

Node 11 6.91 ± 0.32 7.45 ± 0.29 6.21 ± 0.28 7.01 ± 0.29 

Node 12 4.29 ± 0.31 4.62 ± 0.28 3.86 ± 0.22 4.34 ± 0.22 

Node 13 37.22 ± 1.67 39.94 ± 1.59 33.36 ± 1.48 37.33 ± 1.47 

Node 14 23.71 ± 1.17 25.36 ± 1.16 21.23 ± 0.81 23.66 ± 0.94 

Node 15 17.31 ± 0.92 18.48 ± 0.80 15.49 ± 0.55 17.23 ± 0.69 

Node 16 9.06 ± 0.67 9.66 ± 0.82 6.11 ± 0.64 9.01 ± 0.70 

Node 17 6.91 ± 0.41 7.37 ± 0.57 6.18 ± 0.49 6.87 ± 0.56 

(B) Combined analysis 

Node 1 135 ± 0 135 ± 0 120 ± 0 120 ± 0 

Node 2 83.19 ± 1.22 93.48 ± 1.48 79.88 ± 1.01 87.89 ± 1.14 

Node 3 69 ± 0 82.02 ± 0.86 69 ± 0 79.05 ± 0.69 

Node 4 48.05 ± 1.22 69 ± 0 47.29 ± 1.05 69 ± 0 

Node 5 25.64 ± 0.75 34.12 ± 1.04 24.95 ± 0.75 33.38 ± 1.02 

Node 11 13.95 ± 0.58 16.09 ± 0.63 13.41 ± 0.51 15.19 ± 0.61 

Node 18 9.42 ± 0.37 10.81 ± 0.45 8.99 ± 0.41 10.16 ± 0.41 

Node 19 7.52 ± 0.39 8.61 ± 0.46 7.17 ± 0.35 8.09 ± 0.41 

Node 20 5.34 ± 0.29 6.09 ± 0.37 5.08 ± 0.33 5.72 ± 0.32 

Table 5 

Number of species and estimated nucleotide substitution rate (NSR), speciation rate (SR), and time for speciation (TFS) for genera of Winteraceae, based on ITS and combined 

analyses under scenario 4 (see Table 4). 

Takhtajania Tasmannia Drimys Pseudowintera Zygogynum 

Number of species 1 8 7 4 59 

NSR of ITS (# of substitutions per site/yr 10 10 ) 6.65 7.69 ± 1.84 8.67 ± 4.37 4.96 ± 1.17 10.75 ± 7.73 

NSR of ITS + psbA-trnH+rpS16 

(# of substitutions per site/yr 10 10 3.64 3.26 3.87 ± 1.91 1.45 3.14 ± 0.56 

) 

SR of ITS (sp/MY) 0 0.056 0.278 0.0917 0.085 

SR of ITS + psbA-trnH+rpS16 (sp/MY) 0 – 0.128 – 0.117 

TFSln ITS (MY/sp) – 12.44 2.50 7.55 8.13 

TFSln ITS + psbA+rpS16 (MY/sp) – – 5.41 – 5.88 

resulted sister to Z. pauciflorum plus Z. fraterculum; in turn, these 

four species are sister to Z. pomiferum 2 plus Z. vinkii. The 

clade formed by these six species is sister to Z. tanyostigma plus 

Z. baillonii (Fig. 1A). 

The parsimony analysis using the GFC option resulted in 105 

trees of 922 steps, CI = 0.793 and RI = 0.927; the strict consensus 

tree recovers the same generic relationships that in the analyses 

that used GM and SGS options (Fig. 2A). Resolution within Drimys 

and Zygogynum s.l. is lower than that obtained with the GM and 

SGS options. 

Generic relationships emerging from the Bayesian analysis 

(Fig. 2B) are completely congruent to those obtained using parsimony. 

Within Drimys the topology is similar to that recovered from 

GM or SGC options. Within Zygogynum, ‘clade 1’ sequences recovers 

the same topology obtained in all the analyses; however, in ‘clade 2’, 

Z. comptonni resulted sister to an unresolved clade conformed by Z. 

stipitatum, Z. acsmithii, Z. pomiferum 2, and two subclades: (Z. vinkii 

(Z. tanyostigma, (Z. crassifolium, Z. amplexicaule (Z. pauciflorum + Z. 

fraterculum)))) and (Z. pomiferum 1(Z. pancheri + Z. bicolor)).


Table 6 

Tree statistics for the different gap coding schemes applied for ITS and combined analyses. 

Data-sets (number 

of sequences) 

psbA-trnH (21) rpS16 (20) ITS (23) ITS (52) Combined ITS + psbA-trnH+rpS16 (23) 

Gap coding option Missing Missing Missing Missing Simple gap Fifth Missing Simple gap coding Fifth character 

coding character 

Total number of characters 503 810 752 804 850 804 2065 2115 2065 

Number of non-variable 

characters 

367 695 512 465 465 292 1574 1574 1076 

Number of variable uninformative 

characters 

87 65 93 104 104 114 245 245 509 

Number of informative 

characters 

49 50 147 235 281 398 246 296 480 

Informativity (%) a 

10.3 6.2 19.5 29.1 33.1 49.5 11.9 14 23.2 

L 157 126 326 591 646 922 614 667 1303 

Number of most 

parsimonious trees 

8 4 1 3 3 105 32 32 20 

CI 0.962 0.960 0.850 0.743 0.751 0.793 0.894 0.898 0.911 

RI 0.942 0.946 0.863 0.901 0.912 0.927 0.882 0.895 0.887 

RC 0.906 0.908 0.733 0.670 0.685 0.735 0.789 0.804 0.808 

Total number of 

resolved clades 

5 9 12 32 32 28 12 12 14 

Polytomies in the strict 

consensus tree 

4 5 4 5 5 4 2 2 3 

Figures 4B 4A 4C 1A 1B 2A 5A 5A Not shown 

a Informativity (%) = (number of informative characters/number of total characters) 100. 

3.2. Phylogenetic analyses based on psbA-trnH, rpS16 and ITS 

Three parsimony analyses considering gaps as missing were 

performed individually with the psbA-trnH, rpS16 and ITS datasets. 

The analysis based on psbA-trnH produced eight trees of 

157 steps, CI = 0.962 and RI = 0.942 (Table 6); the strict consensus 

tree recovered Drimys (bootstrap = 79) and a clade within Drimys 

formed by the three Chilean and Argentinian species, D. andina, 

D. confertifolia, and D. winteri (hereafter called the Southwestern 

or SW clade; bootstrap = 88; Fig. 3). Pseudowintera colorata and 

the four species of Zygogynum s.l. were recovered as a well supported 

clade but without internal resolution (bootstrap = 99; 

Fig. 4B). The analysis based on rpS16 produced four trees of 126 

steps, CI = 0.960 and RI = 0.946 (Table 6). The strict consensus tree 

recovered the genera Drimys and Zygogynum s.l. as a well supported 

clades (bootstraps = 87 and 95, respectively; Fig. 4A) conforming 

a trichotomy with Pseudowintera colorata. Drimys 

contains two clades within: the SW clade (bootstrap = 62) and a 

clade formed by the NE South America species D. angustifolia, D. 

brasiliensis, D. granadensis, and D. roraimensis (hereafter called the 

Northeastern or NE clade; bootstrap = 64; Fig. 3). The analysis 

based on ITS produced a single tree of 326 steps (CI = 0.850 and 

RI = 0.863; Table 4; Fig. 4C), essentially similar in topology and 

support to the analysis using 47 taxa (Fig. 1A). 

3.3. Congruence tests 

The ILD test indicates combinability of the psbA-trnH, rpS16 and 

ITS data-sets (P = 0.235). The SH test indicates, on one hand, topological 

congruence between rpS16 and psbA-trnH (P = 0.74 or 0.82, 

depending of the use of consensus trees under parsimony or 

Bayesian analyses); and on the other hand, topological incongruence 

between rpS16 and ITS data-sets (P = 0.00 in both cases). 

The topologies of strict consensus trees (Fig. 4) presented only 

two incongruences: (1) in the rps16 tree Zygogynum vinkii is sister 

to Z. comptonii whereas in the ITS tree Z. vinkii resulted sister to Z. 

baillonii, and Z. comptonii resulted sister to Z. tanyostigma and (2) 

in the rps16 analysis Drimys brasiliensis 1 resulted sister to 

D. angustifolia and D granadensis, whereas in the ITS analysis D. brasiliensis 

1 resulted sister to D. brasiliensis 2 and D. roraimensis. 

Because the SH test has been considered too conservative, as 

hypotheses are too infrequently rejected (cf. Susko, 2003), we 

finally decided to follow the results of the ILD analysis and to combine 

the three data-sets (Fig. 5). 

3.4. Phylogenetic analyses based on the combined molecular data-set 

Parsimony analysis of the ITS + psbA-trnH+rpS16 data-set, considering 

gaps as missing resulted in 32 trees of L = 614 steps, 

CI = 0.894 and RI = 0.882. The four genera of Winteraceae are 

recovered as monophyletic in the strict consensus tree, and are 

supported by bootstrap values of 95% or higher (Fig. 5A; Table 4). 

Within Zygogynum s.l. only a clade (Z. baillonii + Z. vinkii) is recovered. 

The SW and the NE clades of Drimys were obtained with bootstrap 

values of 82 and 58%, respectively. 

The parsimony analysis using the SGC option generated 32 trees 

of L = 667 steps, CI = 0.898 and RI = 0.895; the strict consensus tree 

recovers an identical topology to that obtained considering gaps as 

missing (Fig. 5A); however the bootstrap value for the SW clade is 

higher (90%). 

The analysis using GFC resulted in 20 trees of 1303 steps, 

CI = 0.911 and RI = 0.887. The strict consensus tree (not shown) 

only differs from those obtained in the two previous analyses with 

respect to the relationships within Zygogynum s.l., in which the following 

relationships are encountered (((Z. vinkii + Z. tanyostigma) Z. 

comptonii) Z. baillonii). These relationships are not recovered in any 

other analyses. Within Drimys, both the NE and the SW clades were 

recovered again, the latter being well supported (bootstrap = 92%). 

However, Drimys winteri appears as paraphyletic with respect to D. 

andina. Relationships emerging from the Bayesian analysis are congruent 

to those obtained using parsimony (Fig. 5B). 

3.5. Molecular clock and divergence time estimates 

The likelihood-ratio test (LRT) indicates a significant rate of heterogeneity 

among clades for both data-sets. Estimation of divergence 

times using the Penalized likelihood method and TN 

algorithm implemented in r8s, and exploring four alternative calibration 

scenarios are presented in Table 4. 

3.6. Gap informativity 

In Winteraceae, some informative gaps correspond to short 

duplicate motifs of 2–3 bp in the ITS data-set, 6–7 bp in the

psbA-trnH data-set and 4 bp in the rpS16 data-set. The longest 

duplicate motif (TATTTCATATTGTATA) was identified in the psbAtrnH 

sequence of Cynamosma madagascariensis (Canellaceae) as 

an autopomorphy. An additional proportion of informative gaps 

(33%) corresponds to changes in the length of homonucleotide repeats 

in the ITS data-set, most of them between 1–4 bp; however, 

in the psbA-trnH data-set there is one long complex homonucleotide 

repeat (12–13 bp) that corresponds to a polyA/T region that 

includes a synapomorphic duplication (GTCAATC) for the SW clade 

of Drimys. InrpS16 there are three PolyA/T with lengths of 8–13, 

11–12, and 15–17 bp. ITS has one poliA/T that varies in length between 

5–8 bp. The psbA-trnH data-set presents the longest indels, 

especially across the whole order (up to 23 nucleotide) or, within 

Canellaceae, an indel of 35 nucleotide in Capsicodendron dinissi. 

The simple gap coding (SGC) and gaps as a fifth character (GFC) 

options led to an increase in the number of informative characters 

(%), length, CI and RI of most parsimonious trees (MPT) in both ITS 

and combined analyses (Table 6). ITS analysis using SGC option resulted 

in a consensus tree (Fig. 2A) similar to that obtained from 

the analysis using gaps as missing (GM; Fig. 1A), with differences 

in the Zygogynum topology, and increasing branch support in some 

clades. The analysis of the ITS data-set using GFC option resulted in 

a less resolved topology within Drimys and Zygogynum s.l. with respect 

to that obtained using SGC option (Figs. 1A and 2A, respectively). 

As for Drimys, the specific relationships were less 

resolved in both the ITS and the combined analyses using GFC; in 

addition, the three accessions of D. winteri resulted paraphyletic 

with respect to the two accessions of D. andina in the combined 

analysis using GFC option (data not shown). 

4. Discussion 

All analyses corroborate monophyly of Drimys, Pseudowintera, 

Tasmannia, Zygogynum s.l. and recover the same intergeneric 

relationships (Takhtajania (Tasmannia (Drimys (Pseudowintera 

+ Zygogynum s.l.)))). Furthermore, the use of the combined 

data-set recovered the NW and SW clades within Drimys. Coding 

indels using the SGC option results in produced a similar resolution 

with respect to that obtained with GM options, but increase branch 

support. 

4.1. Orthology vs. paralogy in the ITS sequences 

The ribosomal rDNA that includes the ITS region is known to 

contain multiple copies. Intragenomic rDNA diversity is generally 

thought to be low as a result of concerted evolution of these copies 

(Buckler et al., 1997). In some cases, however, concerted evolution 

is lower than speciation leading to divergent paralogues within a 

single genome (Bradford, 2002). Suh et al. (1993) obtained cloned 

ITS sequences and found two divergent ITS copies in Zygogynum s.l., 

(named here ‘clone 1’ and ‘clone 2’). Additionally, Buckler et al. 

(1997) found that ‘clone 1’ of Zygogynum corresponds to a pseudogene 

and that ‘clone 2’ copies represents the functional gene. 

Buckler et al. (1997) considered that amplifications that use 

denaturing conditions (DMSO or high extension temperatures) 

could favor the amplification of ‘clone 2’ sequences, which are 

structurally more stable and are present in higher numbers. 

Baldwin and Donoghue (unpublished data cited by Baldwin et al., 

1995) performed pooled PCR (without cloning) using samples of 

Zygogynum bicolor and Z. comptonii and obtained only copies that 

correspond to ‘clone 2’. Following Baldwin and Donoghue (unpubl. 

data), we used DMSO and BSA in the PCR reactions, and different 

PCR conditions and primers than those used by Suh et al. (1993) 

in order to rule out that the ITS sequences obtained here could 

correspond to ‘clone 1’ copies. In addition, our analyses support 

a clade conformed by all the sequences newly obtained 


(Z. amplexicaule, Z. baillonii, Z. crassifolium, Z. fraterculum, Z. pauciflorum, 

Z. pomiferum 2 and Z. vinkii) together with the ‘clone 2’ sequences 

of previous studies (Figs. 1 and 2). All these facts strongly 

suggest that, although we did not perform ITS cloning, our newly 

obtained sequences are orthologous functional genes. 

4.2. Systematics 

The generic relationships recovered here are in agreement 

with the topologies obtained by Karol et al. (2000) and Doust 

and Drinnan (2004). That is: (Takhtajania (Tasmannia (Drimys 

(Pseudowintera, Zygogynum s.l.)))). Our increased sampling of Drimys 

(7 spp.), Tasmannia (7 spp.), and Zygogynum s.l. (13 spp.) provided 

additional evidence for the monophyly of these genera. A 

phylogenetic analysis including molecular and morphological data 

is currently underway. However, resolution within genera deserves 

further research due to the low nucleotide substitution rates 

encountered in sequences of ITS, psbA-trnH and rpS16 (Table 5). 

4.2.1. Drimys 

Two subclades were recovered in the Winteraceae combined 

analysis: the NE clade (D. angustifolia, D. brasiliensis, D. granadensis, 

D. roraimensis), and the SW clade (D. andina, D. confertifolia, 

D. winteri). These clades essentially correspond to the two main 

species groups keyed out by Smith (1943b). The Andean species 

of Drimys (D. winteri, D. andina, and D. granadensis) do not conform 

a clade by themselves (Fig. 5). Within the SW clade, the topology 

recovered by the combined analysis ((D. confertifolia) (D. winteri, 

D. andina)) essentially corresponds to the species grouping proposed 

by Smith (1943b). D. confertifolia is closely related to D. winteri, 

although this author considered D. andina as a subspecies of D. 

winteri. On the other hand, Ehrendorfer et al. (1979) grouped species 

of Drimys as follows: (1) the southern group (equivalent to our 

SW clade); (2) D. granadensis; and (3) the eastern group including 

D. angustifolia, D. brasiliensis, and D. roraimensis. Our analyses support 

the monophyly of group 1, but is still inconclusive regarding 

the recognition of group 3. The lack of resolution within the NE 

clade of Drimys further prevents an infrageneric classification; 

the problematic circumscription of this clade has long been recognized 

in previous morphological studies (Saint-Hilaire, 1824; 

Miers, 1858; Smith, 1943a; Ehrendorfer et al., 1979; see Table 1). 

Therefore, the use of additional fast-evolving markers, and a phylogeographic 

approach is urgently needed in order to improve resolution 

inside the genus. 

4.2.2. Tasmannia 

Species circumscription within Tasmannia has long been controversial, 

especially in reference to the circumscription of T. piperita. 

Smith (1943b) recognized six Australian and 30 extra-Australian 

species. Vink (1970) proposed three informal sections: (1) T. 

lanceolata, (2) T. insipida plus T. purpurascens plus T. stipitata, and 

(3) Tasmannia piperita sensu lato, which includes Smith’s 30 extra- 

Australian species plus the Australian T. glaucifolia, 

T. membranea and T. xerophila. Our results suggest that T. glaucifolia 

is sister to T. xerophila. However, the relationship between these 

two species and T. piperita (based on an Irian Jaya specimen) remains 

unresolved (Figs. 1 and 2). According to our results, Vink’s second 

section appears as paraphyletic in the analyses (Figs. 1 and 2). An expanded 

sampling in future phylogenetic analysis (including members 

of Borneo and The Philipines) is urgently needed in order to 

further clarify the phylogenetic relationships within the genus. 

4.2.3. Zygogynum s.l. 

This genus comprises approximately 59 species distributed in 

Australia, New Guinea, Moluccas, and New Caledonia (Vink, 

1993a, 1993b). However, only New Caledonian species have been


included in phylogenetic analyses of the Winteraceae, which casts 

doubts on the monophyly of the genus. The presence of Zygogynum 

s.l. in New Zealand has been discussed by Vink (1988: 697), who 

stated that the New Zealand Harrisipollenites fossil pollen could 

correspond to Zygogynum, and that ‘‘this type was eliminated during 

the Pleistocene, whereas its hardier offspring Pseudowintera 

survived and is now represented by three species”. This assumption 

suggests the possibility of a paraphyletic Zygogynum s.l. with 

respect to Pseudowintera. 

Our results do not support the monophyly of the generic segregates 

Belliolum, Bubbia and the remaining Zygogynum s.s. (Figs. 1 

and 2, Table 2). A phylogenetic analysis based on combined data 

(morphology + molecular) is currently underway to further clarify 

species-level relationships within the Belliolum + Bubbia + 

Exospermum + Zygogynum complex sensu van Tieghem (1900). 

4.3. Nucleotide substitution and speciation rates 

Our nucleotide substitution rates (NSR) for ITS in Pseudowintera 

and Zygogynum fluctuated around 4.96 10 10 and 10.75 10 10 

substitutions/year, respectively (Table 5). Previously, Suh et al. 

(1993) estimated that NSR for Pseudowintera and Zygogynum fall 

into the 3.2–5.2 10 10 substitutions/year and 3.6–5.7 10 10 

substitutions/year intervals, respectively. Differences between 

our higher estimates of NSR for Zygogynum in relation with the values 

reported by Suh et al. (1993), could be related with the model 

of DNA substitutions, sequences sampling, and/or estimation of 

ages of the splitting between Pseudowintera and Zygogynum. Suh 

et al. (1993) used the Kimura model of DNA substitutions and five 

sequences of Zygogynum s.l., whereas we used GTR + I + G model of 

DNA substitutions and 17 sequences of Zygogynum s.l.; additionally, 

Suh et al. (1993) assumed that the splitting between 

Pseudowintera and Zygogynum s.l. occurred at least 50–80 MY by 

the separation of New Zealand from Australia according to Raven 

and Axelrod (1974), the splitting between these two areas is based 

on the presence of Zygogynum s.l. in Australia (species not sampled 

by Suh et al., 1993); whereas our estimated ages of Pseudowintera – 

Zygogynum s.l. splitting ranged from 52.24 to 69 MY (Table 4, node 

4; cf. Fig. 2B); these estimates are based on the NSRs and in the fossil 

record, rather than the geographical events considered by Suh 

et al. (1993). 

As expected, speciation rates in Winteraceae (between 0.056 

and 0.278 sp/MY; Table 5) and Drimys (0.128–0.278 sp/MY) are extremely 

low when compared to other high Andean taxa, such as 

Gentianella (1.71–3.2 sp/MY; von Hagen and Kadereit, 2001), 

Lupinus (1.73–2.78 sp/MY; Hughes and Eastwood, 2006), or Valeriana 

(0.8–1.34 sp/MY; Bell and Donoghue, 2005). In addition, the 

time for speciation (TFS) in Drimys (2.50–5.41 MY/sp; Table 5) is 

high when compared to those in Gentianella (0.22–0.40 MY/sp; 

von Hagen and Kadereit, 2001) orDendrosenecio (0.24–0.29 MY/ 

sp; Knox and Palmer, 1995). 

Suh et al. (1993) suggested that the long generation time, the 

frequent vegetative propagation by runners in Pseudowintera and 

Tasmannia, and a low numbers of pollinators of Pseudowintera 

(associated with self-incompatibility) could be related to the extremely 

low rates of molecular evolution and speciation in these genera. 

Furthermore, Suh et al. (1993: 1054) stated that ‘‘possibly the 

slow rate of molecular evolution may be correlated with a similar 

slow rate of morphological changes in the family [...] even though it 

is often claimed that molecules do not march to the same rhythm 

as morphological traits”. 

In Zygogynum s.l. nucleotide substitution rates are similar or 

slightly higher than those found in Drimys (Table 5). However, 

diversification rates are higher in Drimys, which contrasts to the 

time for speciation, which is higher in Zygogynum s.l. The relatively 

higher morphological diversification of Zygogynum s.l. (at least in 

New Caledonia; cf. Vink, 1993b) with respect to that of Drimys 

(cf. Smith, 1943b) is consistent to the differences in the inferred 

time from the branching point of the extant species in both genera 

(37.8–48.6 MY vs. 6.9–7.5 MY based on the ITS data-set; 48–69 MY 

vs. 13.4–16.1 based on the combined data-set Figs. 2B and 5B; 

Table 4). The capacity of the species of Zygogynum s.l. to live in 

tropical lowlands could also be related to the higher diversification 

of this genus (Feild et al., 2002). 

4.4. Biogeography 

The current distribution of the Winteraceae has been used in order 

to arrive at area cladograms. In the study by Crisci et al. (1991), 

based on 17 species-level area cladograms, the Winteraceae (represented 

by species of Drimys, Tasmannia and possibly Pseudowintera), 

contributed to the area cladogram (SSA(AUS(NG(NC)))); (for acronyms 

see Fig. 6). As a result, two general area cladograms were obtained 

by ‘biogeographic parsimony’ (Fig. 6A and B) and two by a 

quantification of a component analysis (Fig. 6C and D). 

On the other hand, Linder and Crisp (1995) used 13 species-level 

area cladograms. The Winteraceae, contributed to the area 

cladogram (MAD(AUS,SSA)(NZ(AUS,NC,NG)))), to finally obtain 

the general area cladogram shown in Fig. 6E. 

More recently, Sanmartin and Ronquist (2004) used 73 area 

cladograms. Two area cladograms were derived from the Winteraceae 

(), as follows: (MAD(AUS(SSA(NZ(NC,NG))))) and (NG(MA- 

D(AUS(SSA(NZ,NC))))). However, although the authors cited Karol 

et al. (2000) as the phylogenetic framework for these two area 

cladograms, the latter does not correspond to any of the phylogenies 

obtained by Karol et al. (2000). The two optimal areas 

cladograms obtained by Sanmartin and Ronquist (2004) are summarized 

in Fig. 6F. 

The area cladogram that summarizes the topology of Winteraceae 

distribution obtained from our research can be summarized 

as follows: (((((NZ)(NC,AUS,NG))(SWSA,NESA))(AUS,TAS,SEA,NG)) 

(MAD)). In this area cladogram, South America has been splitted 

into two different areas, Southwest South America (SWSA) and 

North-east South America (NESA; using the 30° south latitude proposed 

as a limit by Humphries and Parenti, 1999; Fig. 3), in order to 

show the geographic distribution of our SW and NE clades of 

Drimys. Thus, our phylogenetic analysis do not exactly reflect the 

geological reconstructions of Gondwanic fragmentation events 

presented by Linder and Crisp (1995), or by Sanmartin and 

Ronquist (2004; see Fig. 6G). 

In our area cladogram two ‘‘biogeographic” politomies remain 

unresolved: The first (SEA, NG, AUS, TAS) is due to the distribution 

of Tasmannia, a genus primarily distributed in Tasmania and Australia 

(except T. piperita, widespread in Australia, New Guinea 

and SE Asia), including the Philippines and Borneo. The second 

polytomy (NC, AUS, NG) is due to the presence of Zygogynum s.l. 

in Australia, New Caledonia and New Guinea (cf. Vink, 1983, 

1985, 1988, 1993b, 2003). The inclusion of species of Zygogynum 

from Australia and New Guinea in further phylogenetic analysis 

will help to clarify such polytomy. 

4.5. Divergence time estimates 

The assumption of a more widespread geographical distribution 

of Winteraceae during the Cretaceous in Gondwana is consistent 

with the presence of fossils of Walkeripollis in Israel and Gabon 

(c. 120 MY; Doyle, 2000) and in Patagonia (Argentina, c. 100 MY; 

Barreda and Archangelsky, 2006; Fig. 3), and the vesselless fossil 

wood of Winteroxylon jamesrossi in West Antarctica at approximately 

85 MY (Fig. 3; Doyle, 2000). 

The estimates of the branching point between Tasmannia and 

the rest of the family (Drimys + Pseudowintera + Zygogynum s.l) is

77–92.38 MY (node 2 in Figs. 2B and 5B; Table 4); if so, it could 

have occurred before the separation of South America plus Australia 

from New Caledonia plus New Zealand by the Tasman Sea, 

which has been dated around 80 MY (Fig. 6; cf. Sanmartin and Ronquist, 

2004). 

The age of the branching point between the New Zealand species 

of Pseudowintera and the species of the New Caledonian Zygogynum 

s.l. included in our study (52.24–79 MY; node 4 in Fig. 2B and Table 

4) corresponds to estimate of c. 70–60 MY (Fig. 6G) given by Sanmartin 

and Ronquist (2004) and by Swenson et al. (2001). The biogeographic 

implications of the Pseudowintera + Zygogynum s.l. clade 

require further investigation and an increase in the samples especially 

of the members of the complex Zygogynum. 

Our results (Figs. 1 and 2) show that the early divergent species 

of Tasmannia are in Australia plus Tasmania. The splitting between 

T. piperita from New Guinea and its sister Australian relatives is between 

15.5 and 18.5 MY (node 15 in Fig. 2B and Table 4). These 

ages are more recent than the New Guinea–Australia splitting by 

the Coral Sea Basin, which occurred at approximately 30 MY 


Fig. 6. (A–D) General area cladogram from Crisci et al. (1991); (E) General area cladogram from Linder and Crisp (1995); (F) One of the two possible optimal area cladograms 

for the plant data-set from Sanmartin and Ronquist (2004); (G) Geological area cladogram representing the relationships between the southern hemisphere land masses, 

based on paleogeographic evidence. Time of vicariance is assumed as the primary fragmentation event. ( ) dated as 70–60 MY in alternative reconstructions (Sanmartin and 

Ronquist, 2004). AFR, Africa; AUS, Australia; ESA, East South America; HOL, Holartic; IND, India; NA, North America; NC, New Caledonia; NG, New Guinea; NSA, Northern 

South America; MAD, Madagascar; SEA, South East Pacific; SSA, Southern South America; SWP, South West Pacific; TAS, Tasmania, and WANT, West Antarctica. 

(Fig. 6G; Sanmartin and Ronquist, 2004). However, according to 

Swenson et al. (2001), two alternative estimates exists for New 

Guinea–Australia splitting: (1) 25 MY, based on the beginning of 

the uplift of New Guinea; (2) 15–10 MY, based on the expansion 

of arid conditions in Australia, and the fragmentation of mesic 

environments; these estimates predates and postdates, respectively 

the age of the splitting between T. piperita and its sister relatives 

(18.5–15.5 MY). 

Speciation of Pseudowintera is estimated to have started by mid- 

Miocene (11.3–17.3 MY; node 10 in Fig 2B and Table 4), after the 

‘‘Oligocene bottleneck” in the New Zealand biota (Winkworth 

et al., 2002). At that time, New Zealand was reduced to a scattered 

archipelago, formed by islands of low elevation. The presence of 

pollen likely assigned to Zygogynum s.l. from the Oligocene to the 

Pleistocene of New Zealand indicates that the genus was more 

widespread in the past (Vink, 1988). 

The branching point between Drimys and its sister group (69– 

84.6 MY; node 3, Fig. 2B and Table 4) could have predated the 

formation of the Tasman Sea (80 MY, Fig 6G) and the separation


of South America and Australia (52–35 MY, Fig. 6G). The oldest fossil 

assignable to Drimys is pollen of approximately 58.5 MY (Fig. 3; 

Doyle, 2000). However, the speciation of Drimys appears to have 

occurred much later (around 13.4–16.1 MY (node 11, Fig. 5B and 

Table 4; using the combined data-set), which coincides in time 

with the beginning of the Central Andean uplift (30 and 25% of 

their modern elevation between 14 and 20 MY, respectively; 

Wodzicki, 2000). Ehrendorfer et al. (1979:78) stated that the precursors 

of Drimys ‘‘reached the ancient fragments of Gondwana 

land masses which are now part of S. America (Guyana, Brazilian 

shield, etc.) after their separation from African fragment but before 

their fusion by the Andean Cordilleras emerging during the Tertiary. 

D. roraimensis and D. brasiliensis could represent relicts from 

that time”. This assumption is not supported by the estimates of 

age of splitting between SW and NE clades of Drimys (13.4– 

16.1 MY; node 11, Fig. 5B).Further age estimations within Drimys 

is prevented by the lack of resolution in our phylogenetic analyses, 

except for the splitting between the Juan Fernandez endemic 

D. confertifolia and its sister clade (D. andina + D. winteri), dated 

in 9–10.8 MY (node 18, Fig. 5B and Table 4) predates the formation 

of the Juan Fernandez Archipelago, dated on 4 MY (Anderson et al., 

2001). 

5. Conclusions 

Our increased sampling of Drimys, Tasmannia and Zygogynum s.l. 

supports the monophyly of these genera, but does not support the 

monophyly of Bubbia, Belliolum, and Zygogynum s.s. Within Drimys, 

we propose two clades, the SW and the NE clades. Divergence time 

estimates indicate that the earliest cladogenetic events that gave 

rise to the genera of Winteraceae could have predate the 

Gondwanic fragmentation. The splitting between Drimys and its 

sister group (Pseudowintera + Zygogynum s.l.) could have occurred 

by late Cretaceous, but speciation of Drimys seems to have started 

much later, during late Miocene. The area cladogram derived from 

our phylogenetic analyses does not correspond to any of the previous 

area cladograms based on the Winteraceae. 

The use of gaps as characters, following alignment criteria proposed 

by Kelchner (2000) and Borsch et al. (2003) and the SGC option 

proposed by Simmons and Ochoterena (2000) increases 

resolution and branch support values. In contrast, the treatment 

of gaps as a fifth character decreases resolution and could cause 

artifactual topologies. 

Acknowledgments 

X.M. is indebted to the Instituto para el Desarrollo de la Ciencias 

y la Tecnología ‘‘Francisco José de Caldas”. (COLCIENCIAS) for a fellowship 

received from the ‘‘Programa de apoyo a doctorados nacionales 

2004”, and to the Botany Department of the University of 

São Paulo (Brazil) for academic support during lab work. A.S. and 

M.L.F.S. are fellow researchers of CNPq (Conselho Nacional do 

Desenvolvimento Científico e Tecnológico, Brazil). We thank G.J. 

Anderson (University of Connecticut, USA), I. Cordeiro (Instituto 

de Botânica do Estado de São Paulo, Brazil), J. Salazar (Cornell University, 

USA), A. Townsmith (DNA Bank Manager, Missouri 

Botanical Garden, USA), and the Director of Horticulture of the Royal 

Botanic Garden Edinburgh (UK), for provide silica-gel dried samples. 

We also thank Dr. Frederico Arzolla (Campos de Jordão) for 

supporting field work, L. F. Garcia (Universidad Nacional de Colombia) 

for helping with Bayesian analysis. We acknowledge IBAMA 

for collecting permits, and the curators of the herbaria M, NY, UC, 

and US, for the loans received at COL. Reviews of an early manuscript 

by J. Salazar, J. Lynch (Universidad Nacional de Colombia), 

and R. Callejas (Universidad de Antioquia, Colombia) improved 

the manuscript. We thank two anonymous reviewers for critical 

reading of the manuscript. 

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