INTRODUCTION

Lasianthus Jack is a large pantropical genus in

Rubiaceae comprising more than 180 species. Of these,

c. 160 species occur in tropical Asia, with one extending

to Australia, c. 20 in tropical Africa and three in tropical

America. The members of Lasianthus are exclusively

confined to primary rainforests throughout their

geographic ranges. The distribution pattern of Lasianthus

appears to be important for understanding biogeography

and speciation in tropical rainforests (Zhu, 2002).

Some regional taxonomic revisions have been made

for Lasianthus [e.g., Verdcourt (1976); Denys (1981)

for Africa, Wong (1989) for the Malay Peninsula; Deb

and Gangopadpyay (1991) for India, Zhu (2001) for

Thailand, and Zhu (1994, 1998, 2002) for Eastern Asia].

However, the delimitation of Lasianthus has always

been controversial and remains unsettled. Jack (1823)

originally described Lasianthus as a 4-locular ovary

bearing a single basally erected ovule per locule, and a

drupe with four pyrenes. Blume (1826) enlarged Jack¡¦

s original circumscription to include species with 4-9

locular ovaries and drupes with 4-9 pyrenes. Wight (1846)

and Korthals (1851) added some species with 2-locular

ovaries, developing into 2-pyrene drupes. Later these

species were transferred to Saprosma (Schumann, 1891;

Boerlage, 1899). In addition, the Madagascar genus

Saldinia, with 2-locular ovaries and drupes with 1-pyrene,

was once placed under Lasianthus as a subgenus, (Baillon,

1880). Furthermore, Bremekamp (1957) proposed a new

classification of Lasianthus as species with two or more

locules per ovary and two or more pyrenes per drupe with

Botanical Studies (2007) 48: 227-232.

*

Corresponding author: E-mail: zhuh@xtbg.ac.cn;

Telephone: +86-871-5110721; Fax: +86-871-5160916.

a thick wall. He also restored Saldinia as a separate genus,

and merged part of the species with

2-locular ovaries

developing into 2-pyrene drupes in Lasianthus.

The tribal position of Lasianthus has also been

controversial. Traditionally, Lasianthus was placed in

the tribe Psychotrieae based on aestivation of the corolla

lobes and the position, attachment, and types of its ovules

(Hooker, 1880; Schumann, 1891). Petit (1964) proposed

new circumscriptions for Psychotrieae and Morindeae

and transferred Lasianthus to Morindeae based on its

seeds, which have soft oily endosperm and large embryos.

However, molecular data based on a few samples (Bremer,

1996; Andersson and Rova, 1999; Piesschaert et al., 1999;

Bremer and Manen, 2000) indicated that Lasianthus

appeared to be related to Pauridiantha, Perama,

Trichostachys, and Saldinia. Bremer and Manen (2000)

placed Lasianthus, along with Saldinia and Trichostachys,

in the tribe Lasiantheae.

In addition, the only available

comprehensive infrageneric classification of Lasianthus

was Hooker¡¦ classification (1880) based mainly on

quantitative characters, such as the size of stipules, the

occurrence of bracts, and peduncles. Hooker divided

Lasianthus into four sections: Bracteatae, Nudiflorae,

Stipulares, and Pedunculatae.

The identity of the Asian monotypic genus Litosanthes,

L. biflorus, has also been controversial. Litosanthes is

characterised by its imbricate corolla, forked stipules, and

pedunculate inflorescences. Some Asian Lasianthus with

pedunculate inflorescences were transferred to Litosanthes

(Deb and Ganopadhyay, 1989; 1991) and recently

returned to a section of Lasianthus (Gangopadhyay and

Chakrabarty, 1992). In the Flora of China, however,

Litosanthes biflorus is treated as a monotypic genus (Lo,

1999).

plaNT BIOSySTemaTICS

paraphyly and phylogenetic relationships in Lasianthus

(Rubiaceae) inferred from chloroplast rps16 data

Long-Qian XIAO and Hua ZHU*

Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, 650223, Yunnan, P.R. China

(Received November 21, 2005; Accepted August 25, 2006)

aBSTRaCT.

A phylogenetic analysis of Lasianthus and some representatives of tribes in subfamily

Rubioideae based on the chloroplast rps16 data indicates that Lasianthus as currently circumscribed is

paraphyletic, because Saprosma crassipes, representative of the species with two locules per ovary developing

into two pyrene s per drupe with a thin wall, and Litosanthes biflora, the species in the monotypic genus

Litosanthes, are nested within the highly supported Lasianthus clade. The present delimitation of the tribe

Lasiantheae, which includes Saldinia and Trichostachys, is supported by our results. Finally, our results are

inconclusive for evaluating the monophyly of the infrageneric classification of Lasianthus.

Keyword: Lasianthus; Lasiantheae; Litosanthes; Paraphyly; rps16 intron.

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Botanical Studies, Vol. 48, 2007

Here we present a ribosomal protein S16 (rps16) intron

phylogenetic analysis with 11 Lasianthus from the tropical

Africa, America, and Asia, and 28

representatives from the

recognized tribes in the subfamily Rubioideae based on

the classification proposed by Bremer and Manen (2000).

The rps16 intron was chosen because the marker has

proven useful for inferring phylogenetic relationships at

generic or higher levels (e.g. Andersson and Rova, 1999;

Bremer and Manen, 2000; Nie et al., 2005). Additionally,

many published Rubioideae rps16 sequences are available

for our studies. Pairwise comparisons of the 17 chloroplast

introns shared between tobacco (Nicotiana tabacum L.)

and rice (Oryza sativa L.) indicate that the rps16 intron is

one of the most divergent, with 67% sequence similarity

(Downie et al., 1996). The following questions are to be

addressed in particular: (1) Is the current circumscription

of the genus Lasianthus monophyletic. (2) What are the

relationships of Lasianthus with other Lasiantheae genera.

(3) What are the infrageneric relationships of Lasianthus

from tropical America, Africa and Asia. (4) And, finally, is

this phylogeny consistent with Hooker¡¦s classification.

maTeRIalS aND meTHODS

Eleven species representing Lasianthus from tropical

Africa, tropical America, and tropical Asia, and 28

species representing all recognized tribes in the subfamily

Rubioideae (except Spermacoceae and Theligoneae)

(Bremer and Manen, 2000) were sampled as ingroups.

The outgroups were designated as Ixora amplexicaulis

(Ixoroideae) and Cinchona pubescens (Cinchonoideae)

based on the identification of the monophyly of subfamily

Rubioideae (Bremer et al., 1995). The materials collected

in this study were identified by the second author, Dr

Zhu, H., a specialist on Lasianthus. All sequences have

been deposited in GenBank. (For accession numbers

for the rps16 intron sequences and vouchers/references

information see Table 1.)

Total genomic DNA was extracted from silica-dried or

fresh leaves using a modified CTAB procedure (Doyle and

Doyle, 1987). The primers rpsF and rpsR

2

described by

Oxelman et al. (1997) were used for amplifying the rps16

intron from the genomic DNA. PCR reaction volumes

(30 ul) contained 1.5 U of Ampli Ta q DNA polymerase

(Perkin-Elmer 9600). Reactions were incubated at 95¢XC

for 3 min, then cycled 35 times (95¢XC for 1 min, 55¢XC for

1 min, 72¢XC for 1.5 min), followed by a final extension for

10 min at 72¢XC. Double-stranded products were purified

using the E.Z.N.A. Cycle-Pure Kit (Omegabio-tek, USA).

Sequencing reactions were performed using PRISM Dye

Terminator Cycle Sequencing Ready Reaction kit

(Applied

Biosystems, Foster City, Calif.). The products of the

sequencing reaction were electrophoresed on an ABI 3700

automated sequencer.

Contiguous DNA sequences were edited using

SeqMan (DNASTAR package) and subsequently adjusted

manually. All sequences were aligned using MEGALIGN

(DNASTAR package) and then adjusted manually.

Deletions were coded as missing data.

Maximum parsimony (MP) analysis was performed

using PAUP 4.0b10 (Swofford, 2001) treating gaps as

missing data using heuristic search options with 1,000

random replications of stepwise data addition and TBR

swapping and Multrees on no tree limit with all characters

weighted equally and unordered. Bootstrap analysis

(Felsenstein, 1985) was performed with 1,000 replicates to

evaluate internal support.

ReSUlTS

All the newly acquired sequences were submitted to

GenBank (Table 1). The total length of 1,191 nucleotides

of the rps16 intron sequences in the data matrix, including

41 species, was determined, and 327 were parsimony-

informative (27.5%). A parsimony analysis of the

rps16 intron data matrix resulted in 970 equally most

parsimonious trees, each with 819 steps, CI = 0.6119,

and RI = 0.807. The strict consensus tree is shown in

Figure 1. Saprosma crassipes, Litosanthes biflorus, and all

sequenced Lasianthus species formed a strongly supported

(BP = 92) monophyletic group. This mostly Lasianthus

clade was resolved as sister to a highly supported (BP =

98) clade containing Saldinia and Trichostachys. However,

the support for this sister-group relationships was weak

(BP = 58). The Lasiantheae clade was in turn resolved

as sister to the tribe Perameae, represented by Perama

hirsuta. The highly supported (BP = 100) Lasiantheae-

Perameae clade was resolved with strong support (BP

= 100) as sister to a highly supported (BP = 98) large

clade containing Saprosma ternatum and the remaining

sequenced Rubioideae taxa, formally classified into

eleven tribes (Morindeae, Gaertnereae, Schradereae,

Psychotrieae, Craterispermeae, Anthospermeae,

Paederieae, Argostemmeae, Danaideae, Coussarieae,

Urophylleae, and Ophiorhizeae). The two studied species

of the genus Saprosma, S. crassipes and S. ternatum,

did not form a clade. Finally, the African Lasianthus

batangensis and the Neotropical L. lanceolatus formed a

monophyletic group while the sequenced Asian Lasianthus

did not group together as a clade.

DISCUSSION

Delimitation of Lasianthus

Lasianthus as presently delimited is not monophyletic,

unless Litosanthus and Saprosma crassipes are included.

In other words, the species with two locules per ovary

developing into two pyrenes per drupe with a thin wall

should be transferred to Lasianthus, an d Litosanthes

biflorus should not be separated from Lasianthus.

Our results further support the placement of Lasianthus

in Lasiantheae as proposed by Bremer and Manen (2000).

However, we find no support for the position of the genus

in Psychotrieae (e.g., Schumann, 1891) or Morindeae

XIAO and ZHU ¡X Paraphyly and relationships of

Lasianthus

229

Table 1. GenBank accession, Vouchers or references information and the species sampled in this study.

Species

Tribe

Vouchers /References

Origin GenBank aceession

number

Lasianthus hirsutus (Roxb.) Merr.

Lasiantheae Gong, 04298

Vietnam *DQ282637

Lasianthus attenuatus Jack

Lasiantheae Zhu, 03122

Malaysia *DQ282638

Lasianthus sikkimensis Hook. f.

Lasiantheae Zhu, 03155

China

*DQ282644

Lasianthus rhinocerotis Bl.

Lasiantheae Zhu,03123

Malaysia *DQ282639

Lasianthus chinesis (Champ.) Benth.

Lasiantheae Xiao, 04010

China

*DQ282641

Lasianthus verticillatus (Lour.) Merr.

Lasiantheae Zhu, 03156

China

*DQ282640

Lasianthus hookeri Clarke ex Hook. f.

Lasiantheae Zhu, 03157

China

*DQ282643

Lasianthus chrysoneurus (Korth) Miq.

Lasiantheae Zhu, 03159

China

*DQ282642

Lasianthus batangensis Schum.

Lasiantheae Andersson & Antonelli, 2005 Congo AY538439

Lasianthus lanceolatus (Griseb.) Urb.

Lasiantheae Andersson & Rova, 1999 Puerto Rico AF004062

Lasianthus coffeoides Fyson

Lasiantheae Andersson & Rova, 1999 India

AF004061

Litosanthes biflorus Bl.

Lasiantheae Zhou, 2655

China

*DQ282649

Saldinia sp.

Lasiantheae Piesschaert et al., 1999

Madagascar AF129275

Trichostachys microcarpa Schum.

Lasiantheae Piesschaert et al., 1999

Congo AF191491

Perama hirsuta Aubl.

Lasiantheae Andersson & Rova, 1999 Guiana AF004070

Coussarea sp.

Coussareeae Andersson & Rova, 1999 Guiana AF004041

Declieuxia dusenii Standl.

Coussareeae Andersson & Rova, 1999 Brazil AF004045

Craterispermum laurinum (Poiret) Benth. Craterispermeae ¡X

.

AF331645

Gaertnera paniculata Benth.

Gaertnereae Andersson & Rova, 1999 Congo AF002736

Morinda angustifolia Roxb.

Morindeae Zhu, 03160

China

*DQ282648

Gynochthodes epiphytica AC Sm. & S.Darwin Morindeae Andersson & Rova, 1999 Fiji

AF001440

Psychotria peduncularis (Salisb.) Steyerm. Psychotrieae Andersson, 2002

.

AF410742

Amaracarpus kochii Valeton

Psychotrieae Andersson, 2002

.

AF410679

Saprosma crassipes Lo.

.

Xiao, 04009

China

DQ282645

Saprosma ternatum Hook. f.

.

Zhu, 03161

China

DQ282646

Schradera sp.

Schradereae Andersson & Rova, 1999 Colombia AF003617

Danais sp.

Danaideae Andersson, 2000

.

AF331648

Anthospermum tricostatum Sond

Anthospermeae ¡X

.

AF257898

Galopina crocylloides Schinz

Anthospermeae Andersson & Rova, 1999 South Africa AF002764

Argostemma rupestre Ridl

Argostemmateae Andersson & Rova, 1999 Malaysia

AF002756

Mycetia malayana Craib

Argostemmateae Andersson & Rova, 1999 .

AF002771

Paederia scandens (Lour.) Merr.

Paederieae Zhu, 03162

China

*DQ282647

Plocama pendula Aiton

Paederieae Andersson & Rova, 1999 .

AF004071

Rubia fruticosa Aiton

Rubieae

Andersson & Rova, 1999 .

AF004078

Didymaea mexicana Hook. f.

Rubieae

Andersson & Rova, 1999 Mexico AF004047

Pauridiantha lyallii (Baker) Bremek.

Urophylleae Andersson & Rova, 1999 Madagascar AF004067

Urophyllum glabrum Jack

Urophylleae Andersson & Rova, 1999 Singapore AF004089

Amphidasya colombiana (Standl.) Steyerm. Urophylleae Andersson & Rova, 1999 Angola AF242906

Ophiorrhiza mungos L.

Ophiorrhizeae Andersson & Rova, 1999 .

AF004064

Cinchona pubescens Vahl.

Andersson & Rova, 1999 .

AF004035

Ixora amplexicaulis Gillespie

¡X

.

AF242969

Accession numbers marked with* represent the samples which were sequenced in this study.

230

Botanical Studies, Vol. 48, 2007

(Petit, 1964). Our analysis also supports the monophyly

of Lasiantheae sensu Bremer and Manen (2000) and the

exclusion of Saldinia from Lasianthus (Figure 1).

The relationships of Lasianthus

Lasianthus has been postulated to be closely related to

the genera Psychotria, Morinda, Saprosma, Pauridiantha,

Perama, Saldinia and Trichostachys (Schumann, 1891;

Petit, 1964; Verdcourt, 1976; Robbrecht, 1988; Bremer,

1996; Andresson and Rova, 1999; Bremer and Manen,

2000). Our present data confirm that Saldinia and

Trichostachys are the closest relatives of Lasianthus, and

they constitute the tribe Lasiantheae together, as sister to

Perameae. This is largely congruent with most previous

molecular phylogenetic analyses with few and different

samples (Andersson and Rova, 1999; Piesschaert et al.,

1999; Bremer and Manen, 2000). However, a previous

rbcL phylogenetic analysis suggested that Lasianthus

and Pauridiantha, possessing completely different fruit

types respectively, come into a single clade (Bremer,

1996). This may result from long branch attraction for

the too sparse samples in that analysis. Morphologically,

Figure 1. T h e s t r i c t

consensus tree of 970 equally

parsim onious trees ba sed on

rps16 intron sequences. Length

=819, CI=0.6119, RI=0.8070.

Numbers above braches

indicate bootstrap percentage

(BP), and branch lengths are

below branches. Names of

t he m ajor c lade s a re s hown

on the right. A-A-C = tropical

America-Africa Clade.

XIAO and ZHU ¡X Paraphyly and relationships of

Lasianthus

231

Lasianthus, Saldinia, and Trichostachys share the same

seed morphology and wood structure, with features such

as fibre tracheids and solitary vessels (Piesschaert et al.,

1999).

The infrageneric relationships of

Lasianthus

In traditional taxonomic treatment (Hooker, 1880),

Lasianthus was divided into four sections, i.e., Bracteatae,

Nudiflorae, Stipulares and Pedunculatae, determined by

the size of stipules and by the occurrence of bracts and

peduncles. Our chloroplast DNA phylogenetic tree does

not resolve the infrageneric classification well, but the

species from tropical America and tropical Africa form

a clade with a full bootstrap percentage. They were,

however, placed into Section Nudiflorae and Section

Lasianthus, respectively (Hooker, 1880). Our results are

thus inconclusive for testing the monophyly of Hooker¡¦s

section of Lasianthus. Morphologically, the species

from tropical America and tropical Africa share common

characters, having eight or more locules per ovary and

pyrenes per drupe while the others possesses fewer than

eight locules per ovary and pyrenes per drupe (with only

one exception not sampled in this analysis). Further insight

into the infrageneric classification of Lasianthus will

require more extensive taxa sampling for comprehensive

analyses through molecular data combined with

morphological characters.

The rps16 sequences have shown much higher

divergence (1.722-1.825%) than some other chloroplast

markers (atpB-rbcL: 0.551-0.735%; rbcL: 0.376-0.601%)

between the species in Kelloggia, which is a rather small

genus in Rubiaceae with only two species (Nei et al.,

2005). It is thus interesting to mention that all members

of the Lasianthus clade have short branch lengths (Figure

1). This indicates that their pan-tropical distribution may

result from a relatively recent inter-continent dispersal

and that these species may have undergone a recent

rapid radiation in tropical Asia, perhaps related to the

tropical rain forest fragmentation and secondary sympatry.

Lasianthus has limited potential for developmental and

physiological acclimation to intense light. Consequently,

the individuals of Lasianthus are absent in forest gaps and

exclusive found in the understory of primary forests (Cai,

2005). Therefore, the lack of fierce species competition

for lots of vacant ecological niches in the understory,

coupled with the infrequent migration between isolative

forest patches, has contributed to the rapid speciation.

This is also implied by the sympatric occurrence of some

tropical Asian Lasianthus species in relative narrow

habitats (Personal observation by Zhu), and by the very

asymmetric species richness between continents (twenty

species in tropical Africa, three in tropical America, and

160 in tropical Asia, respectively), which shows the

marked differences in their species diversification rates

between continents. However, a decrease of nucleotide

substitution in the rps16 intron sequence, remains difficult

to exclude as an alternative explanation for the observed

diversification pattern in Lasianthus.

Acknowledgements. This project was funded by the

National Natural Science Foundation of China (30570128).

The project was completed in the laboratory of

phylogenetics and conservation biology, Xishuangbanna

Tropical Botanical Garden, the Chinese Academy of

Sciences. We express our thanks to Dr. Li Jie, Mrs. Xia

Yongmei and Mr. Li Zhiming in this laboratory for their

considerable help in the experiment and data analysis,

to Dr. Wen Jun, Dr. Gong Xun and Mr. Shi Ji-Pu for

collecting some materials for this study, and to Dr. Sylvain

Razafimandimbison and another anonymous reviewer for

their constructive comments and grammar improvements.

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