Phylogeny and biogeography of Rhododendron subsection Pontica, a group with a tertiary relict distribution

MOLECULAR

Molecular Phylogenetics and Evolution 33 (2004) 389–401

PHYLOGENETICSANDEVOLUTION

www.elsevier.com/locate/ympev

Phylogeny and biogeography of Rhododendron subsection Pontica,a group with a tertiary relict distribution

Richard Ian Milne*

Division of Environmental and Evolutionary Biology, School of Biology, University of St. Andrews, St. Andrews, Fife KY16 9TH, UK

Received 28 December 2003; revised 21 May 2004

Abstract

Rhododendron subgenusHymenanthes subsection Pontica is exceptional among Tertiary relict groups in having a high proportion

of species (4 of 11) native to SW Eurasia. A phylogeny based on cpDNA matK and trnL-F indicated that multiple Pontica lineages

colonised each of SW Eurasia, SE North America, and NE Asia, with little or no speciation within regions thereafter. Therefore,

multiple (3–4) Pontica lineages survived the Quaternary in SW Eurasia, in contrast to other Tertiary relict genera. Pontica comprises

two major clades, one of which is wholly Eurasian, and paraphyletic with respect to at least some of the remaining 200 species of

subgenus Hymenanthes, which are all distributed in SE Asia. The other clade has species from W and SE North America, SW Eur-

asia, and NE Asia. According to synonymous matK substitution data, the two clades diverged 9–6 million years ago (mya), whereas

most divergence within them happened 5–3mya. Although the phylogeny indicates probable trans-Atlantic migration for one of two

America-Eurasia disjunctions in Pontica, the timing supports migration via Beringia for both.

� 2004 Published by Elsevier Inc.

Keywords: Divergence times; Tertiary relict floras; Disjunct distributions; Northern hemisphere; Ericaceae; cpDNA sequences; North Atlantic land

bridge

1. Introduction

The modern distribution and abundance of biota

throughout the Northern Hemisphere reflects specia-

tion, migration, and extinction events, which in turn

have been profoundly affected by the climatic fluctua-

tions of the past 65 million years (i.e., the Tertiary per-

iod). For much of this period, the climate in most ofNorth America and Eurasia was warm and humid,

and supported a large circumboreal forest ecosystem

(Tiffney, 1985a,b). As the world cooled from 15mya,

culminating in the Quaternary glaciations, members of

this community became increasingly restricted in distri-

bution and now, for the most part, exist only in refugial

regions in East Asia, western and southeastern North

1055-7903/$ - see front matter � 2004 Published by Elsevier Inc.

doi:10.1016/j.ympev.2004.06.009

* Fax: +44-1334-463366.

E-mail address: [email protected].

America, and southwest Eurasia. Studies of these Ter-

tiary relict floras have been highly informative regarding

at what time and by which routes biota moved between

the continents of the Northern Hemisphere biota

(Donoghue et al., 2001; Milne and Abbott, 2002; Wen,

1999, 2001; Xiang et al., 2000).

Tertiary relict floras are most diverse in eastern Asia,

which probably comprises separate refugial regions inNE and SE Asia (Donoghue et al., 2001; Milne and Ab-

bott, 2002). This results from high niche diversity and

low extinction rates in this region (Milne and Abbott,

2002; Tiffney, 1985a,b; Wen, 1999; Wen et al., 1998).

Phylogeographic examinations of Tertiary relict genera

often reveal multiple lineages within SE and NE Asia,

each exhibiting connections to species in different re-

gions, and therefore each having a separate biogeo-graphic history (e.g., Asarum, Kelly, 1998; Styrax,

Fritsch, 1999; Magnolia, Qiu et al., 1995; and Aralia,

mailto:[email protected].

390 R.I. Milne / Molecular Phylogenetics and Evolution 33 (2004) 389–401

Wen et al., 1998). Additionally, lineages from many Ter-

tiary relict genera have diversified within eastern Asia,

producing monophyletic clades of species endemic to

the region (e.g., Aesculus, Xiang et al., 1998; Asarum,

Kelly, 1998; and Castanea/Castanopsis, Manos and

Stanford, 2001). This indicates a complex history forthe floras of this region, involving separate lineages from

the same genus arriving into or spreading out of this re-

gion by different routes and/or at different times, fol-

lowed by radiation events within the region in some

cases.

By contrast, diversity among Tertiary relict floras in

the western half of Eurasia is very low. Here, Tertiary

relict floras occur only in SW Eurasia, concentratedaround Turkey and the southern Black Sea coast. Most

Tertiary relict groups have either a single species in

southwestern Eurasia (e.g., Aesculus, Xiang et al.,

1998; Asarum, Kelly, 1998; Liquidambar, Li and Donog-

hue, 1999; Styrax, Fritsch, 1999; and Castanea/Castan-

opsis, Manos and Stanford, 2001), or none at all (e.g.,

Liriodendron, Parks and Wendel, 1990; Nyssa, Wen

and Stuessy, 1993; Aralia, Wen et al., 1998; Hamamelis,Wen and Shi, 1999; andMagnolia, Qiu et al., 1995). This

might reflect higher extinction rates in western Eurasia

due to east–west orientated mountain ranges blocking

southward movement of biota (Parks and Wendel,

1990; Tiffney, 1985b), especially as many Tertiary relict

taxa now absent from western Eurasia existed there dur-

Fig. 1. Global distribution of Rhododendron subsection Pontica, and of the r

ing the Tertiary (Milne and Abbott, 2002). However, it

could also be due to lower niche diversity in SW Eur-

asia, relative to eastern Asia, limiting speciation (Milne

and Abbott, 2002; Qian and Ricklefs, 2000; Tiffney,

1985a,b; Wen, 1999). Because of the very small number

of extant species within any Tertiary relict genus in SWEurasia, it is difficult to reconstruct the biogeographic

history of the region in the way that has been possible

for eastern Asia.

Among groups with a Tertiary relict distribution,

Rhododendron subsection Pontica is exceptional in that

it contains four species native to SW Eurasia, out of

only 11 in the group as a whole (Fig. 1). These four spe-

cies (R. caucasicum, R. smirnowii, R. ungernii, and R.

ponticum) all occur in NE Turkey and the adjacent Cau-

casus, in regions of very high rainfall around the south-

east edges of the Black Sea, although R. ponticum alone

extends westwards to SE Bulgaria and has outliers in

SW Europe and Lebanon (Fig. 1). Elsewhere, Pontica

has two species in southeastern N America (R. maximum

and R. catawbiense), one (R. macrophyllum) in western

N America, one (R. hyperythrum) in Taiwan (SE Asia),and three species in NE Asia, among which the R. degro-

nianum species complex is endemic to Japan, R. brachy-

carpum occurs in Japan and S Korea, and R. aureum

occurs in Japan and N Korea, extending across NE Asia

towards Siberia (Chamberlain, 1982; Fig. 1). Pontica is

also very unusual among Tertiary relict groups in that

emaining 23 subsections that with it comprise subgenus Hymenanthes.

R.I. Milne / Molecular Phylogenetics and Evolution 33 (2004) 389–401 391

it has no representatives on mainland SE Asia south of

Korea. However, it is one of 24 subsections that com-

prise subgenus Hymenanthes of Rhododendron, and the

other 23 subsections, which together contain over 200

species, are all distributed entirely in SE Asia (Chamber-

lain, 1982; Fig. 1). The distribution of Pontica thereforeonly overlaps that of the rest of subgenus Hymenanthes

in Taiwan (Fig. 1).

In order to examine the biogeography of Pontica, a

molecular phylogeny based on cpDNA sequences was

generated. In particular, this was used to determine

whether the presence of four species in the Black Sea re-

gion reflects the arrival there of multiple lineages, or

diversification of a single lineage within the region. Also,a sample of four representatives of other subsections of

subgenus Hymenanthes was included in the analysis, to

determine how Pontica is related to these taxa in both

evolutionary and biogeographic terms. The timing of

divergence events in Pontica was also of interest, be-

cause Pontica is evergreen and divergence times are

available for very few evergreen Tertiary relict groups

despite unanswered questions about whether such taxawere able to cross the high-latitude Bering Land Bridge

(Milne and Abbott, 2002; Tiffney, 1985b, 2000; Tiffney

and Manchester, 2001). The matK and trnL-F cpDNA

regions were chosen for this work because both are reg-

ularly used in phylogenetic studies, and exhibit more

variation than does rbcL, making them suitable for use

at the infrageneric level. In addition, matK has previ-

ously been employed for divergence time estimatesamong Tertiary relict taxa (Donoghue et al., 2001) and

phylogenetic work within Rhododendron (Kron, 1997).

Data from matK were therefore used for estimations

of approximate dates of key divergence events within

Pontica.

2. Materials and methods

2.1. Taxon sampling

Both the initial and the final analyses contained the

11 species of subsection Pontica, plus four other mem-

bers ofHymenanthes (each belonging to separate subsec-

tions), whose sequences were obtained from GenBank

(Table 1). Both matK and trnL-F sequences were avail-able for R. fortunei, and R. neriiflorum, whereas only

matK sequences were available for R. falconeri and R.

grande (Table 1). These four species were included to

test the monophyly of subsection Pontica; time and re-

sources did not permit a larger sampling of subgenus

Hymenanthes.

To indicate the direction of mutations within

Hymenanthes, four taxa from subgenus Pentanthera

section Pentanthera, indicated to be the sister group

to Hymenanthes by Kron (1997), were chosen; se-

quences of R. luteum, R. atlanticum, and R. occiden-

tale were generated for the project whereas matK

and trnL-F sequences for R. molle were obtained from

GenBank (Table 1). In the initial analysis every other

Rhododendron species for which both matK and trnL-

F sequence data were available from GenBank (25 intotal, representing each of the other six subgenera of

Rhododendron plus two more sections of subgenus

Pentanthera) was included, plus Empetrum nigrum

and Calluna vulgaris, making 46 species in total. This

was done to confirm the sister relationship between

Pentanthera and Hymenanthes, and hence to confirm

that section Pentanthera was the best outgroup to

Hymenanthes in the analysis.In subsequent analyses all these additional Rhodo-

dendron species were removed except for three: R. cana-

dense (subgenus Pentanthera section Rhodora) which

formed part of the sister group to Hymenanthes, R.

camtschaticum (subgenus Therorhodion) which was sis-

ter to the clade of all other Rhododendron species exam-

ined, and R. ferrugineum, the type species of

Rhododendron whose divergence from the Pontica–Pen-

tanthera clade marked the position of a large polytomy

in the original phylogeny that apparently represented

the first major diversification event within Rhododen-

dron (Kron, 1997). Two species were retained from

the two nearest sister clades to Rhododendron (Kron,

1997), i.e., E. nigrum and C. vulgaris, to serve as out-

groups to Rhododendron. The phylogeny therefore con-

tained 24 species. The sources for all material andsequences are given in Table 1.

2.2. Plant material and sequence generation

Leaf material of the 11 species of subsection Pontica

was obtained from cultivated material at the Royal Bo-

tanic Garden, Edinburgh (Table 1); wherever possible,

material of known wild origin was used. DNA extrac-tion was conducted as in Milne et al. (1999), except that

the extracted DNA was purified using a Wizard DNA

clean-up kit (Promega, 2800 Woods Hollow Road,

Madison, WI 53711-5399, USA).

PCR amplification followed the protocol of Johnson

and Soltis (1994, 1995), for all sequences. Primers used

for matK were those of Johnson and Soltis (1994,

1995) plus some developed by Sang et al. (1997). Inaddition to the matK gene, an intron (of trnK) of

250bp at the 30 end was also sequenced using the same

primer sets. Primers used for trnL-F were those of Tab-

erlet et al. (1991). PCR products were cleaned using a

Wizard PCR prep kit (Promega, address as above),

and also by gel purification in cases where additional

faint bands were detected in addition to the main prod-

uct. Sequencing of purified PCR products was carriedout by PNACL, University of Leicester, UK. Sequences

were aligned manually.

Table 1

Sources, origins, and accession numbers of material and sequences used in the analysis

Species Subgenusa Section or

subsectiona,bOrigin of

accession

Live Accession

No.cGenBank Accession Nos.

matK trnL-F

Material extracted for sequencing

R. aureum Hymenanthes Pontica Cultivated 19450053 AY494177 AY496918

R. brachycarpum Hymenanthes Pontica Honshu, Japan 19660135 AY494176 AY496917

R. catawbiense Hymenanthes Pontica North Carolina, USA 19340114 AY494174 AY496915

R. caucasicum Hymenanthes Pontica Rize, Turkey 19520168 AY494175 AY496916

R. degronianumd Hymenanthes Pontica Cultivated 19341071 AY494179 AY496920

R. hyperythrum Hymenanthes Pontica Cultivated 19410106 AY494181 AY496922

R. macrophyllum Hymenanthes Pontica Cultivated 19734184 AY494173 AY496914

R. maximum Hymenanthes Pontica North Carolina, USA 19800047 AY494171 AY496912

R. ponticum Hymenanthes Pontica Trabzon, Turkey 19773079 AY494172 AY496913

R. smirnowiie Hymenanthes Pontica Cultivated 19698845 AY494180 AY496921

R. ungernii Hymenanthes Pontica Artvin, Turkey 19623836 AY494178 AY496919

R. atlanticum Pentanthera Pentanthera California, USA 19730782 AY494183 AY496924

R. occidentale Pentanthera Pentanthera Cultivated 19582084 AY494182 AY496923

R. luteum Pentanthera Pentanthera Slovenia 19773072 AY494184 AY496925

Species Subgenusa Section or

subsectionbAuthors of matK

sequence

Authors of

trnL-F sequence

GenBank Accession Nos.

matK trnL-F

Sequences withdrawn for GenBank for use in the phylogeny

R. molle Pentanthera Pentanthera Kron (1997) Gao et al.f U61356 AF452211

R. canadense Pentanthera Rhodora Kurashige et al. (1998) Gao et al.f AB012735 AF452212

R. falconerig Hymenanthes Falconera Kron (1997) — U61346 —

R. fortunei Hymenanthes Fortunea Gao et al.f Gao et al.f AF454850 AF394247

R. grandeg Hymenanthes Grandia Kron (1997) — U61336 —

R. neriiflorum Hymenanthes Neriiflora Gao et al.f Gao et al.f AF454851 AF394248

R. ferrugineum Rhododendron Rhododendron Kurashige et al. (1998) Gao et al.f AB012741 AF394254

R. camtschaticum Therorhodion Kurashige et al. (1998) Gao et al.f AB012744 AF394258

Calluna vulgarisg Kron (1997) — U61326 —

Empetrum nigrumh Li et al. (2002) R. Milneh AF519558 AY496911

a Rhododendron species only.b For members of subgenus Hymenanthes, names given are subsections. All subsections in this subgenus belong to a single section, Pontica, under

the current classification (Chamberlain, 1982). For other subfamilies, sections are given where these exist.c All accession numbers are for live material at RBG Edinburgh. Herbarium specimens of each live accession were taken when the material was

extracted and are held at RBG Edinburgh.d Material is R. degronianum spp. degronianum. R. degroninanum comprises a complex of closely related entities, all endemic to Japan, and is here

taken to include taxa that are sometimes given species status, i.e., R. makinoii and R. yakushimanum.e This species� name is sometimes spelt as ‘‘smirnovii.’’f Gao, L.M., Li, D.Z., Yang, Y.B., and Zhang, C.Q. Otherwise unpublished.g trnL-F sequences were not available for these species.h The trnL-F sequence for this species was generated for this project from material wild collected in Skye, UK.


2.3. Phylogenetic analysis

The trnL-F region contained only single-base indels

that either were not phylogenetically informative or could

not be interpreted unequivocally, therefore all indels in

trnL-F were treated as missing data. ThematK gene con-

tained no indels. However, two insertions were present in

the trnK intron, one of which was shared between threespecies while the other was detected in a single species

(see below). These two indels were not included in the

analysis but were mapped on to the trees produced.

Data from matK, the trnK intron, and trnL-F were

combined into a single data matrix for the initial analy-

sis. Sequences from the trnK intron, and in three cases

trnL-F, were not available for taxa whose sequences

were taken from GenBank; these were thus coded as

missing data for these taxa. Data were analysed by heu-

ristic search using PAUP* version 4.0b10 (Swofford,

2002) under the following conditions: optimality crite-

rion=parsimony, maxtrees set to 5000 (10,000 for decay

analysis), collapse and multrees options in effect, TBR

on, steepest descent in operation.The initial analysis was conducted for all 46 taxa, and

then repeated for the subset of 24 taxa in Table 1. Char-

acters and character states were weighted equally (Fitch

parsimony) in all analyses. A heuristic search was con-

ducted with E. nigrum and C. vulgaris designated as out-

groups with the tree rooted from a basal polytomy. A


strict consensus of all most parsimonious trees was gen-

erated. Strength of support for groupings in the strict

consensus tree was assessed using bootstrap analysis of

1000 replicates and a Maxtrees limit of 5000, and also

by decay analysis. The analysis was repeated for the

matK gene and trnL-F regions singly.

2.4. Timing divergence events

Synonymous mutations within a gene region might

offer the best tool to estimate divergence times (Xiang

et al., 2000). To assign approximate dates to divergence

events within Pontica, a phylogram was generated based

on synonymous matK mutations only. This was done bycreating a separate matrix for each of the 19 amino acids

or stop codons that can be coded by more than one

DNA codon, and in each matrix treating each such co-

don as a separate character state, whereas all codons for

another amino acid, or that contain missing data, are

treated as missing data. The 19 resulting matrices were

then combined into one large matrix, and a heuristic

search was run on this data set using PAUP* 4.0, withtopological constraints applied so that the tree topology

could not conflict with that of the strict consensus tree

from the combined data set. Zero length branches were

collapsed, and two most parsimonious trees resulted,

which differed in a single step. Branch lengths within this

phylogram were then used to determine the relative age

of divergence events within the group�s history. Likeli-

hood ratio (LR) tests (Goldmann, 1993) were performedto test whether the hypothesis of a molecular clock

could be applied to this data set, and also to the com-

bined data set and the unaltered matK sequence data

set. Where possible, substitution models were selected

prior to this using Modeltest 3.4 (Posada and Crandall,

1998).

3. Results

3.1. Phylogenetic analysis

Phylogenetic analysis of the full set of 46 taxa con-

firmed that a clade comprising sections Pentanthera

and Rhodora of subsection Pentanthera was the sister

group to subgenus Hymenanthes (tree not shown, butsee Kron, 1997 for a similar tree). However, two spe-

cies of subgenus Pentanthera section Sciarhodion (R.

schlippenbachii and R. albrechtii) did not form part of

this Pentanthera clade, as Pentanthera is Paraphyletic

according to plastid data (Kurashige et al., 1998).

Therefore, for convenience, the clade comprising sec-

tions Pentanthera and Rhodora is referred to as ‘‘subge-

nus Pentanthera’’ for the remainder of this paper. Aswith Kron�s (1997) analysis, members of subgenus

Therorhodion (i.e., R. camtschaticum and R. redowskia-

num) formed a clade sister to all other Rhododendron

species examined, but otherwise the next divergence

event within Rhododendron was an unresolved poly-

tomy. The position of this polytomy was indicated in

subsequent analyses by the inclusion of one species,

R. ferrugineum, that diverged at this point from thelineage comprising subgenera Pontica and Pentanthera.

The analysis was then repeated with only the species

listed in Table 1.

3.1.1. Parsimony analysis of the combined data set

The combined data matrix of matK, the trnK intron

and trnL-F comprised 24 species, and contained 2874

sites. Among these, 2466 were constant, 308 were vari-able but parsimony-uninformative, and 100 were parsi-

mony-informative. The parsimony search swapped to

completion and found 15 most parsimonious trees of

length 498 (Consistency Index [CI]=0.89 or 0.68 exclud-

ing uninformative characters; Retention Index

[RI]=0.79; Rescaled Consistency Index [RC]=0.70).

The strict consensus tree (Fig. 2) was well resolved,

and contained only one polytomy. One of the most par-simonious trees had the same topology as both the strict

consensus tree and the bootstrap consensus tree (Fig. 2).

The data support the monophyly of the subgenus

Hymenanthes (bootstrap support (BS)=98; Decay index

(DI)=6; Fig. 2), but indicate that Pontica is paraphyletic

with respect to all four of the species of Hymenanthes

not in Pontica that were included in the analysis, i.e.,

R. grande, R. fortunei, R. falconeri and R. neriiflorum.Each of these species is the type species of one of the

24 subsections into which Hymenanthes is divided

(Chamberlain, 1982). Therefore Pontica is paraphyletic

with respect to representatives of four other subsections.

These four species form a well-supported clade together

with R. hyperythrum (‘‘Clade H1’’; BS=97, DI=3).

These five, together with R. smirnowii and R. degronia-

num, form one of two clades into which subgenusHyme-

nanthes is divided by the data. This clade, here named

‘‘Clade H,’’ has moderate (BS=72; DI=1) support in

the combined analysis. However, further support for

this clade is provided by an insertion of 3bp in the trnK

intron that is shared between R. smirnowii, R. hypery-

thrum, and R. degronianum. trnK intron sequences were

not available for the other four species in this clade, (i.e.,

R. grande, R. fortunei, R. falconeri, and R. neriiflorum),but their strongly supported grouping with R. hypery-

thrum indicates that they also possess the insertion, un-

less it has subsequently been lost in some lineages. A

second insertion of 6bp in this region was found only

in R. hyperythrum, but might also be shared with some

or all of these four species (Fig. 2). R. degronianum

might be sister to all the other species within clade H,

but the support for the grouping of R. smirnowii withthe other species, but excluding R. degronianum, is not

strong (BS=62, DI=1) (Fig. 2).

Fig. 2. Phylogeny for Rhododendron subsection Pontica (species names in bold), four representatives of other subsections of subgenus Hymenanthes,

five species of subgenus Pentanthera, two other Rhododendron species, and two outgroup taxa. Tree shown is one of 15 most parsimonious trees,

which has identical topology to both the strict consensus and bootstrap consensus trees. Figures above branches are bootstrap support (BS) and

(after slash) decay index (DI); figures below branches are branch lengths in the selected most parsimonious tree. Dotted branches are those not in the

strict consensus tree based on matK data alone. Areas of distribution indicated for species are as follows: ‘‘SE Asia’’ comprises southern China, the

adjacent islands south to Java, the Himalayas and outliers in India and Sri Lanka (Fig. 1). ‘‘NE Asia’’ comprises Japan and Korea with a narrow

band stretching through Manchuria to eastern Siberia for R. aureum only (Fig. 1). ‘‘SW Eurasia’’ comprises the area around the southern Black Sea

coast (mainly N Turkey and Caucasus) with outliers in Lebanon, Spain, and Portugal for R. ponticum only (Fig. 1). ‘‘SE N America’’ indicates

parts of N America east of 86�, from N Georgia to Nova Scotia (Fig. 1). ‘‘W N America’’ indicates areas within 300km of the west coast of the USA

(Fig. 1).


The remaining eight species of subsection Pontica are

resolved in a strongly supported (BS=95; DI=4) mono-

phyletic clade, here called ‘‘Clade P.’’ Within this clade

five species form a reasonably (BS=88, DI=2) well-sup-ported clade which itself comprises two strongly sup-

ported (BS=95, DI=3 for each) subclades (Fig. 2).

The first of these, ‘‘Clade P1,’’ contains R. macrophyllum

and R. catawbiense resolved as sister taxa. Within the

second, ‘‘Clade P2,’’ there is weak support for a group-

ing of R. caucasicum and R. brachycarpum as sister spe-

cies (BS=64, DI=1) with R. aureum sister to this pair.

Relationships involving the remaining three species inClade P are not well resolved. Other relationships in

Clade P were not strongly supported; support for R.

ungernii as the sister group to the clade of Clades P1

and P2 was very weak (BS=55, DI=1), and sister to


these was a weakly supported clade (‘‘Clade P3,’’

BS=59; DI=1) comprising R. maximum and R. ponti-

cum (Fig. 2).

3.1.2. Parsimony analysis of the matK data set

The data matrix of matK alone comprised the same24 species, and contained 1584 sites. Among these,

1350 were constant, 172 were variable but parsimony-

uninformative, and 62 were parsimony-informative.

The parsimony search swapped to completion and

found two most parsimonious trees of length 292 (Con-

sistency Index [CI]=0.87 or 0.65 excluding uninforma-

tive characters; Retention Index [RI]=0.75; Rescaled

Consistency Index [RC]=0.65). The topology of thestrict consensus tree was the same as that of the com-

bined analysis strict consensus and bootstrap consensus

trees, except that two groupings were not present

(Fig. 2). The relative positions of R. degronianum and

R. smirnowii within Clade H were not resolved, and

the position of R. ungernii within Clade P was com-

pletely unresolved. There were no groupings in the strict

or bootstrap consensus trees that contradicted those inthe strict consensus tree of the combined data set. Boot-

strap support for the grouping of R. maximum with

R. ponticum was slightly higher in this (BS=69) than

the combined (BS=59) analysis; otherwise no clade

received higher support in the matK than the combined

analysis.

3.1.3. Parsimony analysis of the trnL-F data set

The data matrix of trnL-F alone comprised 21 spe-

cies (trnL-F data was not available for R. grande and

R. falconeri, or C. vulgaris), and contained 1037 sites.

Among these, 888 were constant, 115 were variable

but parsimony-uninformative, and 34 were parsimo-

ny-informative. The parsimony search swapped to

completion and found 40 most parsimonious trees of

length 178 (Consistency Index [CI]=0.92 or 0.74excluding uninformative characters; Retention Index

[RI]=0.84; Rescaled Consistency Index [RC]=0.77).

The topology of the strict consensus tree was poorly

resolved, though it contained no groupings not present

in the strict consensus tree from the combined analy-

sis. No groupings received higher support in the boot-

strap tree of this analysis than that of the combined

analysis.

3.1.4. Biogeography

There is very little agreement between geographic

location and phylogenetic position within subsection

Pontica. In fact, within Pontica, there is no case of spe-

cies from the same refugial region being sister to one an-

other. However, R. hyperythrum, the only member of

Pontica from the SE Asia refugial region, forms awell-supported clade with four non-Pontica species from

this region (Fig. 2). The other two species that form

Clade H with these five, R. smirnowii and R. degronia-

num, are distributed in the SW Eurasia and NE Asia (Ja-

pan), respectively, giving Clade H a distribution that

stretches across southern Asia from Japan through the

Himalayas to NE Turkey (Figs. 1 and 2).

Clade P contains species from four refugial regions,i.e., SW Eurasia, NE Asia, SE N America, and W N

America; it lacks any species from SE Asia (Fig. 2).

Clade P1 comprises a sister relationship between R.

catawbiense from SE N America and R. macrophyllum

from W N America (Fig. 2). Clade P2 comprises one

species from SW Eurasia and two from NE Asia (Fig.

2), however, the latter are not resolved as sister taxa, in-

stead the SW Eurasian R. caucasicum appears to be sis-ter to R. brachycarpum (NE Asia), with the other NE

Asian species, R. aureum, sister to this pair.

A third, weakly supported clade is Clade P3, compris-

ing R. maximum (SE N America) and R. ponticum (SW

Eurasia). Bootstrap support for this grouping rises to

80% if R. ungernii is removed from the analysis. This

might be because a trnL-F mutation is shared between

R. ponticum and R. ungernii, leading to contradictionin the phylogenetic signal.

3.2. Divergence times

The synonymous matK data set contained 66 substi-

tutions of which 16 were parsimony-informative.

Unconstrained heuristic analysis of this data set pro-

duced a strict consensus tree that failed to resolve anyphylogenetic structure within Pontica, though it did

not conflict with any of the relationships indicated by

the combined data set. Therefore, to generate the phylo-

gram used to date divergence events, the topology was

constrained to that of the strict consensus tree for the

combined data set.

Likelihood ratio (LR) tests (Goldmann, 1993) strongly

rejected the assumption of a molecular clock for the com-bined data set (�lnL=76.64; df=22; p<0.0005) and the

matK sequence data set (�lnL=56.66; df=22;

p<0.0005). When best-fitting substitution models were

first obtained using Modeltest 3.4 (Posada and Crandall,

1998), then LR tests similarly rejected molecular clocks

for the combined (�lnL=79.77; df=22; p<0.0005) and

matK (�lnL=55.61; df=22; p<0.0005) data sets. How-

ever, LR tests accepted the molecular clock assumptionfor the synonymous matK mutation data set

(�lnL=22.47; df=22; p>0.25). Therefore the latter

could be used for dating divergence eventswithout further

transformation. Furthermore, R. camstchaticum ap-

peared to have a slower synonymous substitution rate

than any other lineage (Fig. 3) and if this taxon was ex-

cluded from the analysis then the LR test produced a

much lower value (�lnL=15.43; df=21; p>0.50). Thisindicates that the data set conforms more closely to a

molecular clock if this taxon is excluded.

Fig. 3. Phylogram for Rhododendron subsection Pontica, four representatives of other subsections of subgenusHymenanthes, five species of subgenus

Pentanthera, two other Rhododendron species, and two outgroup taxa. The phylogram is based on synonymous matK mutations only, but

constrained to the topology of Fig. 2. Maximum and minimum age scales are shown for divergence events, based on point A being 89 and 60my old,

respectively. The main figure shows one of two most parsimonious trees generated. The alternative most parsimonious tree differs only in the

arrangement of R. ponticum and R. maximum, as shown in the inset.


Constrained heuristic analysis of the synonymous

matK mutation data set produced two competing phylo-

gram topologies. The first is shown in Fig. 3; the second

differs only in that one mutation links R. ponticum to R.

maximum, following which the branches leading to the

species are 2 and 0 mutations long, respectively (Fig.

3, inset). The following calculations and discussion arebased on the first tree; but all calculations were repeated

for the second, which was one substitution longer.

Fossil records for Rhododendron, in the form of seed

and pollen, date from the upper Palaeocene (60mya;

Zetter and Hesse, 1996, and references therein). This is

the latest time at which the genus could have arisen.

On the phylogram, Point A represents the divergence

of Rhododendron from its sister lineage Empetrum, so

the Rhododendron fossils must represent this or a later

point on the phylogram, therefore Point A is at least

60my old. The family Ericaceae (sensu lato) is up to89my old according to fossil data and a calibrated

Angiosperm phylogeny (Magallon et al., 1999; Wik-

strom et al., 2001) and as Rhododendron is nested within

this family (Kron, 1997), Point A cannot be older than


the family, so Point A cannot be older than 89my old.

The age range of 60–89mya was therefore assigned to

Point A, to allow dating of later events in the phylogram

(Fig. 3).

phylogram represent key divergence events involving

Rhododendron subsection Pontica. Point B is the diver-gence of Pontica and Hymenanthes from the deciduous

sister group subgenus Pentanthera. Point C is the initial

divergence event within Pontica, which in the phylo-

gram is a tetrachotomy. Point D is the initial diver-

gence event involving R. hyperythrum and at least

four species of Hymenanthes excluding Pontica. Point

E is the initial divergence event within clade P, which

in the phylogram is a pentachotomy (Fig. 3).Excluding R. camschaticum, which has a substitution

rate around half the mean rate for all the other Rhodo-

dendron species in the phylogram, the mean number of

substitutions after Point A, between all species, is

21.143, or 21.190 with the alternative phylogram topol-

ogy (Fig. 3). The number of substitutions between Point

A and Points B, C, D, and E are 15, 19, 20, and 20,

respectively. From this, Points B, C, D, and E are0.29, 0.10, 0.054, and 0.054 times as old as Point A.

Therefore, given the age range 60–89mya for Point A,

Points B, C, D, and E are 17.3–25.9, 6.07–9.02, 3.24–

4.81, and 3.24–4.81my old, respectively.

4. Discussion

4.1. The relationships between Pontica and other Rhodo-

dendron subgroups

Rhododendron subsection Pontica is paraphyletic with

respect to representatives of four other subsections of

Hymenanthes, i.e., Grandia, Fortunea, Falconera, and

Neriiflora. These species form a well-supported mono-

phyletic clade together with the only member of Ponticathat occurs in SE Asia, i.e., R. hyperythrum (Clade H1,

Fig. 2). Therefore all five SE Asian species examined

form a well-supported monophyletic clade (Fig. 2) that

does not overlap at all the distribution of Pontica

excluding R. hyperythrum (Fig. 1). More species need

to be examined to determine whether all other sections

of Hymenanthes fall within this clade, making all SE

Asian species of the subgenus a monophyletic group de-rived from within subsection Pontica. R. hyperythrum is

also linked to the species of Hymenanthes excluding

Pontica by the morphological character of red corolla

flecks, whereas the other 10 species of Pontica have

flecks that are orange to greenish (Chamberlain, 1982).

Although R. hyperythrum is linked to subsection Pontica

by inflorescence form and corolla shape (Chamberlain,

1982), these characters might be plesiomorphic withinPontica, and perhaps all of subgenus Hymenanthes, hav-

ing been altered in the species of Clade H1. On this ba-

sis, R. hyperythrum is certainly not a member of

subsection Pontica and is best placed in a subsection

of its own.

Subsection Pontica is not a natural group, and the

morphological characters shared by its species (includ-

ing or excluding R. hyperythrum) are plesiomorphicwithin the subgenus according to the phylogeny. Given

that some or all other subsections of Hymenanthes are

derived from within it, subsection status also seems

inappropriate for Pontica. If Pontica is found to be

paraphyletic with respect to all other subsections, it

would be better to treat Pontica as one of two sections

within Hymenanthes, paraphyletic with respect to a sec-

ond section that comprised R. hyperythrum and theother 23 subsections. Treating Clades P and H as sec-

tions within Hymenanthes would fit better with the phy-

logeny and avoid paraphyly; however, at present, there

are no known morphological characters shared by all

the species of one clade and none of the other, because

of the presence of R. smirnowii and R. degronianum in

Clade H.

4.2. Biogeography of subsection Pontica: diversification

followed by range expansion?

Each of the refugial regions occupied by more than

one species within Pontica comprise at least two lineages

according to the phylogeny. Japan contains R. degronia-

num from Clade H and R. aureum plus R. brachycarpum

from Clade P2, indicating separate links with southwestEurasia (Clade P2) and with SW Eurasia/SE Asia (Clade

H) (Fig. 2). Southeastern N America contains R.

catawbiense from Clade P1 and R. maximum from Clade

P3, indicating separate biogeographic links with western

N America and southwest Eurasia, respectively (Fig. 2).

The presence of two independent lineages of Pontica in

SE N America also supports Donoghue et al.�s (2001)

suggestion that a divide might exist within the SE NAmerica Tertiary relict floras between biotas that ar-

rived at different times. Southwestern Eurasia contains

representatives of three clades, i.e., R. smirnowii (Clade

H), R. caucasicum (Clade P2), and R. ponticum (Clade

P3); the affinities of R. ungernii are not clearly resolved.

Therefore the species in this region exhibit three different

biogeographic links: R. smirnowii is linked to the large

clade of species in SE Asia and to R. degronianum in Ja-pan; R. caucasicum is linked to the NE Asian species R.

brachycarpum and R. aureum; and R. ponticum exhibits a

weakly supported trans-Atlantic link to R. maximum of

southeastern N America (Fig. 2). Notably, there are no

biogeographic links between SE Asia and either south-

eastern or western N America, among the species exam-

ined. Overall, this analysis indicates strongly that

diversification in Pontica occurred before and duringthe spread of the group to the intercontinental range it

now occupies, rather than afterwards.


Pontica comprises two clades that are sister to one

another, and has no basal species that might indicate

an area of origin. Based on the phylogeny, Eurasia ap-

pears a more likely area of origin than North America

as the latter contains only two Pontica lineages, one of

which is in a relatively derived position. The questionof whether Pontica is paraphyletic with respect to all

other sections of Hymenanthes must be answered before

consideration can be given to eastern versus western

Eurasia as the likelier area of origin for Pontica.

Biogeographically, Clades P and H differ in that

Clade P contains American but not SE Asian species,

while Clade H has the reverse (Fig. 2). One possible

explanation for this is that the divergence of these cladesreflects a north-south split within an initial Pontica line-

age that existed within the Tertiary forests of northern

Eurasia. Members of Clade P at some point reached N

America, presumably via the Bering and/or N Atlantic

land bridges, both of which lay at high latitude, which

might indicate a northerly distribution for Clade P dur-

ing the Tertiary. Equally, the fact that no lineage from

Clade H has crossed these high-latitude bridges to reachAmerica, together with the presence of species in SE

Asia, might indicate a more southerly distribution for

this clade. Even in Japan, where Clades P and H over-

lap, the species of Clade H (R. degronianum) has a more

southerly distribution than do R. brachycarpum or R.

aureum (Fig. 1; Chamberlain, 1982). Furthermore,

Clade P contains four highly cold-adapted species i.e.,

R. aureum, R. caucasicum, R. catawbiense, and R. max-

imum (Chamberlain, 1982; Davidian, 1982; Nilsen,

1991).

4.3. Why is Pontica so diverse in SW Eurasia?

Pontica is very unusual among Tertiary relict groups

in having more than one species in the SW Eurasia ref-

ugial region, as Tertiary relict genera tend to have nomore than one species in this region (Milne and Abbott,

2002; Wen, 1999). The presence of four Pontica species

in this region reflects the presence there of three or four

separate lineages within the group (Fig. 2). This indi-

cates that multiple lineages of Pontica survived the Qua-

ternary in western Eurasia and rejects a hypothesis of

later diversification within the region.

Representatives of many other Tertiary relict generawere present in Europe in the middle Tertiary but are

now extinct in the western half of Eurasia (e.g., Lirioden-

dron, Parks and Wendel, 1990; Nyssa, Wen and Stuessy,

1993; Aralia, Wen et al., 1998; and Hamamelis, Tiffney

and Manchester, 2001). The presence of three to four

Pontica lineages in modern SW Eurasia indicates that

at least this many separate lineages of Pontica, each with

different biogeographic affinities, existed in western Eur-asia prior to the Quaternary—something that it has not

been possible to assert for any other Tertiary relict

group. The survival of three to four Pontica lineages in

SW Eurasia might reflect a greater ability to survive

the effects of Quaternary glaciations than was exhibited

by many or most other Tertiary relict genera. This might

be due to higher cold tolerance among species of Pontica

than for most other Tertiary relict genera; many speciesof Pontica are tolerant of very low temperatures (Davi-

dian, 1982; Nilsen, 1991). Ponticamight exhibit a similar

distribution to other Tertiary relict groups not because

of its temperature tolerance range, but because it shares

with them other ecological requirements, such as for re-

gions of high rainfall. Hence the possibility exists that

many Tertiary relict groups comprised multiple lineages

in western Eurasia prior to the Quaternary, but in mostcases these were reduced to one or none in SW Eurasia

during the Quaternary glaciations due to poor cold

tolerance.

4.4. Times of divergence and routes of intercontinental

migration

The term ‘‘migration’’ is here taken to mean themovement of a lineage from one region or landmass to

another, without an implication that this was by dis-

persal or gradual spread of a continuous population.

Clade P comprises four lineages among which relation-

ships are not strongly supported (Fig. 2). These lineages

are R. ponticum (SW Eurasia), R. ungernii (SW Eurasia),

R. maximum (SE N America), and the clade comprising

Clades P1 and P2 (Fig. 2). R. ponticum and R. maximum

are resolved as sister species but with weak support;

enforcing any other arrangement of these four lineages

makes the tree only one step longer than the most parsi-

monious trees. However, a trans-Atlantic link of some

form is implied, however the four lineages at the base

of Clade P are arranged. While migration via east Asia

and Beringia cannot be ruled out, this problem exists

to some extent for all apparent trans-Atlantic disjuncts.Therefore, a trans-Atlantic migration early in the history

of Clade P is indicated by the phylogeny topology,

though the nature of such a migration remains unclear.

The data from synonymous matKmutations conform

to the assumption of a molecular clock, and indicates

that divergence within Pontica is surprisingly recent.

Rhododendron is at least 60my old, given that fossil seed

of the genus existed by �60mya, in the upper Palaeo-cene (Zetter and Hesse, 1996), and recognisable pollen

(Zetter and Hesse, 1996) existed by 50mya. However,

it cannot be older than its family, Ericaceae (sens.

lat.), which is up to 89my old (Magallon et al., 1999;

Wikstrom et al., 2001). Hence the divergence of Rhodo-

dendron from its sister lineage Empetrum (see Kron,

1997 for phylogeny) occurred between 60 and 89mya.

Using this calibration point and the phylogram, Pon-tica/Hymenanthes diverged from its sister group subge-

nus Pentanthera between 17 and 26mya, i.e., in the


late Oligocene/early Miocene (Point B, Fig. 3). This

event might have represented an ecological split, because

Pontica/Hymenanthes comprises only evergreen species

whereas all Pentanthera are deciduous.

The first diversification event within Pontica/Hyme-

nanthes, i.e., the split of Clades P and H (Point C), oc-curred much more recently, i.e., 9.1–6.0mya. This

indicates that the Pontica lineage did not diversify for

some time after splitting from Pentanthera unless other

branching lineages went extinct; however, more com-

plete sampling of subgenus Hymenanthes is required to

confirm this. It appears that Pontica began to diversify

in the late Miocene, a time at which the global climate

had begun the gradual cooling that culminated in theQuaternary glaciations (Milne and Abbott, 2002). Fol-

lowing the split between Clades H and P, R. smirnowii

and R. degronianum diverged within Clade H, respec-

tively becoming disjunct to the west and northeast of

the other species in the clade. Divergence among the

remaining five species in the clade (Point D) occurred

4.9–3.2mya according to the data.

Some form of trans-Atlantic migration is impliedwithin Pontica soon after diversification within Clade

P (Point E), most likely involving R. maximum and R.

ponticum (see above). Point E is 4.9–3.2my old accord-

ing to this analysis. This appears to be far too recent

to be compatible with the lifespan of the N Atlantic

Land Bridge, if this severed >40mya as most geological

data indicates (Milne and Abbott, 2002; Tiffney, 1985b).

The latest time that the bridge might have existed,according to oceanographic studies, is 15mya (Milne

and Abbott, 2002; Poole and Vorren, 1993). However,

divergence estimates for trans-Atlantic disjuncts in Sty-

rax (Fritsch, 1996), Cercis (Donoghue et al., 2001), and

especially Corylus (Whitcher and Wen, 2001) also indi-

cate that trans-Atlantic migration might have continued

even after 15mya. Trans-Atlantic disjuncts in Corylus

diverged <8mya (Whitcher and Wen, 2001), and theseeds (hazelnuts) are certainly too large to have been dis-

persed across substantial bodies of ocean by wind or

birds. Hence the possibility must be considered that a

land connection existed across the N Atlantic, perhaps

only briefly, in the late Miocene 10–5mya. The alterna-

tives are that many apparent trans-Atlantic disjuncts

were actually connected across Beringia and eastern

Asia, but left no extant lineages there despite the lowerextinction rates in this region, or that molecular data

somehow underestimated divergences times in all these

cases including Rhododendron.

The route by which Clade P1 (R. catawbiense and R.

macrophyllum) reached America is equivocal because it

is sister to the Eurasian Clade P2, and both clades in-

clude species on both the E and W sides of their respec-

tive landmasses (Fig. 2). In this case again, divergencebetween the American and Eurasian clades is indicated

to be 4.9–3.2mya old (Point E, Fig. 3). The known life-

span of the Bering Land Bridge (BLB), which existed al-

most throughout the Tertiary up until between 5.5 and

4.8mya (Marinovich and Gladenov, 1999; Tiffney and

Manchester, 2001), is far more suitable to both disjuncts

than that of the N Atlantic land bridge. In addition, the

divergence time of 4.9–3.2mya matches closely those fornine of 11 East Asia–Eastern North America disjuncts

examined by Xiang et al. (2000), all of which were as-

sumed to have probably migrated via Beringia. How-

ever, Pontica is evergreen and the high latitude of the

BLB might have prevented some evergreen taxa from

existing there due to winter darkness (Milne and Ab-

bott, 2002; Tiffney, 2000; Tiffney and Manchester,

2001). However, the BLB moved south throughout theTertiary (McKenna, 1983; Tiffney, 2000; Tiffney and

Manchester, 2001) and this combined with progressive

climatic cooling (Milne and Abbott, 2002) might have

made the bridge progressively less hostile to cold-toler-

ant evergreens throughout its lifespan, as colder winters

might ameliorate the winter darkness effect on ever-

greens (Milne and Abbott, 2002). Therefore, the period

4.9–3.2mya corresponds with what might be the mostlikely period in which members of Pontica could have

existed in Beringia and hence crossed the BLB. Based

on this, the migration of Clade P1 to America is on bal-

ance much more likely to have been via the BLB. The

route by which the other trans-Atlantic migration

(Clade P3) occurred is less certain.

Given that the high latitude of land bridges might

have impeded intercontinental migration by evergreens,it is notable that the America-Eurasia disjunction in

Pentanthera might be older than those in Pontica. R.

molle and R. luteum are the sole E and SW Eurasian spe-

cies of Pentanthera sect. Pentanthera, and divergence be-

tween these and the two American species of the section

examined (R. atlanticum and R. occidentale) was indi-

cated to be 8.9–13.3mya (Fig. 3). This could be because

such deciduous taxa were able to cross the BLB earlierthan were the evergreen members of Pontica.

4.5. The possibility of reticulate evolution and plastid

transfer

A phylogeny based on plastids, however well resolved,

does not necessarily correspond to an evolutionary tree

for the species that contain them, because plastids mayhave different histories from the species that contain

them. This is particularly true of a genus such as Rhodo-

dendron in which species barriers are weak and introgres-

sion is common (Milne et al., 1999), although in stable

habitats there may be mechanisms that greatly restrict

this (Milne et al., 2003). Thus a phylogeny for the same

group based on nuclear genes might provide slightly dif-

ferent results, and if so might indicate a hybrid origin forspecies whose placement differs between the phylogenies.

Candidates for possible reticulate evolution in Pontica


include those species with overlapping distributions

which also share morphological features, but whose

plastids belong to different lineages. Thus R. ungernii

and R. smirnowii, which occur together in Turkey and

certainly hybridise (Chamberlain, 1982; Milne et al.,

1999) share morphological features in common despitebelonging to clades P and H, respectively, indicating pos-

sible transfer of nuclear germplasm between the two.

Likewise, morphological similarity between the three

American species (R. maximum, R. catawbiense, and R.

macrophyllum; Chamberlain, 1982) could reflect germ-

plasm exchange between them, perhaps at a time before

the latter two species diverged from one another.

Though it might not represent accurately the phylog-eny of the species examined, the plastid phylogeny pre-

sented must nonetheless provide a true account of the

biogeography of plastid lineages within Pontica. SW

Eurasia contains three to four separate plastid lineages

with separate biogeographic affinities, regardless of

whether hybridisation complicates the affinities of the

species that contain them. Likewise SE North America

and NE Asia each contain two separate plastid lineages,indicating the original presence of more than one Pon-

tica lineage there, irrespective of whether the species that

now contain these plastid lineages have exchanged

germplasm since.

5. Conclusions

Pontica is, as suggested by its Tertiary relict distribu-

tion, probably the oldest group within subgenus Hyme-

nanthes. It may be separated into two clades, one with a

Tertiary relict distribution excluding SE China/Himala-

yas, and the other concentrated in that region and ab-

sent from America. Similar subdivisions are found in

Aesculus (Xiang et al., 1998) and Liquidambar (Li and

Donoghue, 1999). Such divisions might have reflectedthe division of a Boreotropical forest species into warm-

er-adapted southern and cooler-adapted northern com-

munities. In the case of Pontica, the SE China/

Himalaya lineage is paraphyletic with respect to at least

some other sections of subgenus Hymenanthes. The

hypothesis that it is paraphyletic with respect to all of

them, making all SE Asian species of Hymenanthes a

monophyletic group, can be tested using the insertionsthat define Clade H. Once this has been done, techniques

such as Dispersal-Vicariance analysis (Ronquist, 1997)

could be used to examine further how the distribution

of Pontica/Hymenanthes might have been achieved.

Paraphyly also occurs in SE Asian clades of several

other Tertiary relict genera (Milne and Abbott, 2002),

including Liquidambar (Li and Donoghue, 1999; Shi

et al., 1998) and Aralia (Wen, 2000).Divergence among the elepidote Rhododendrons

(subgenus Hymenanthes) appears to have begun fairly

late in the history of the genus, between 9 and 6mya.

Most diversification among the species examined oc-

curred still later, between 3 and 5mya. This means either

that the North Atlantic land bridge persisted much more

recently than geological or oceanographic data suggest,

or that the two America-Eurasia disjunctions within thegroup arose via the BLB despite its high latitude; other-

wise the molecule-based divergence time estimates have

dramatically underestimated the age of events.

The presence of four species of Pontica in the Black

Sea region reflects the survival of multiple lineages in

the region rather than later diversification within the re-

gion, which might be because Pontica is more cold-toler-

ant than most groups with a Tertiary relict distribution.

Acknowledgments

The author thanks the keeper and staff of the Royal

Botanic Garden, Edinburgh, for access to their live

material, Dr. Richard Abbott for his consistent support

before and during this work, Dr. David Chamberlainfor introducing me to subsection Pontica and its pecu-

liarities, Dr. Amanda Gillies for laboratory assistance,

Dr. Peter Comes for helpful advice, and two reviewers

for constructive comments on the manuscript. This

work was supported by NERC Grant GR9/4767.

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Documents

Phylogeny and biogeography of Rhododendron subsection Pontica, a group with a tertiary relict distribution