Botanical Studies (2009) 50: 89-100.

*

Corresponding author: E-mail: crsheue@mail.ncyu.edu.tw;

Tel: +886-05-2717827; Fax: +886-05-2760164.

**Co-Corresponding author. E-mail: tsaicc9017@yahoo.

com.tw; Tel: +886-08-7746735; Fax: +886-08-7229466.

INTRODUCTION

Ceriops Arn. is one of the mangrove genera in the

family Rhizophoraceae, with a widespread geographical

range from eastern Africa throughout tropical Asia, and

northern Australia to Melanesia, and through Micronesia

north to southern China (Tomlinson, 1986). It typically

grows in the inner mangroves, often forming pure stands

on better drained sites or becoming stunted in exposed and

highly saline sites, within the reach of occasional tides

(Hou, 1958).

The last revision of the genus Ceriops was done by

Hou (1958), with two species recognized: C. tagal (Perr.)

C. B. Rob. and C. decandra (Griff.) Ding Hou. Some 20

additional names, including several infraspecific names

were synonymized for them, but a variety name C. tagal

(Perr.) C. B. Rob. var. australis C. T. White named by

White (1926) was not listed.

White (1926) noticed a form of C. tagal in which the

propagules had smooth, terete hypocotyls rather than

the angled or ribbed hypocotyls typical of C. tagal from

Australia and Papua New Guinea. He initially intended to

describe this form as a new species distinct from C. tagal,

based on the "less distinctly veined, and more inclined

to recurved" leaves (White, 1926). After examining

additional specimens, however, he found those differences

between the new form and C. tagal were not constant

Reevaluating the taxonomic status of Ceriops australis

(Rhizophoraceae) based on morphological and

molecular evidence

Chiou-Rong SHEUE

1,

*, Yuen-Po YANG

2

, Ho-Yi LIU

2

, Fu-Shan CHOU

3

, Hsiu-Chin CHANG

1

,

Peter

SAENGER

4

, Christopher P. MANGION

5

, Glenn WIGHTMAN

5

, Jean W. H. YONG

6

, and Chi-Chu

TSAI

7,

**

1

Department of Biological Resources, National Chiayi University, 300 Syuefu Rd., Chiayi 600, Taiwan

2

Department of Biological Sciences, National Sun Yat-sen University, 70 Lien-hai Rd., Kaohsiung 804, Taiwan

3

Liouguei Research Center, Taiwan Forestry Research Institute, 198 Chunghsing Village, Liouguei 844, Kaoshiung

County, Taiwan

4

School of Environmental Science and Management, Southern Cross University, Lismore NSW 2480, Australia

5

Department of Natural Resources, Environment and the Arts, PO Box 496, Palmerston NT 0831, Australia

6

Natural Sciences, National Institute of Education, 1 Nanyang Walk, Nanyang Technological University, 637616

Singapore

7

Kaohsiung District Agricultural Improvement Station, 2-6 Dehe Rd., Changihih Township, Pingtung County 908, Taiwan

(Received December 24, 2007; Accepted August 19, 2008)

ABSTRACT.

Ceriops australis (White) Ballment, Smith & Stoddart, a member of the mangrove family

Rhizophoraceae, was originally recognized as C. tagal var. australis White but was raised to species rank

based solely on isozyme features and the only distinctive morphological feature of the hypocotyl. Therefore,

it was considered a sibling species of C. tagal (Perr.) C. B. Rob. The goal of this study was to test the

previous assessment that C. australis and C. tagal differ consistently only in hypocotyl morphology, in order

to reevaluate the taxonomic status and to establish its geographic range. Principal components analysis was

employed to analyze 29 morphological characters of herbarium specimens from Australia, Madagascar, and

Sumatra tentatively identified as C. australis and C. tagal, and two well differentiated distinct taxa were

recognized. In addition, both of the detailed morphological features based on fresh and herbarium materials

and the intron sequences of trnL gene from plastid DNA support this conclusion. This finding disagrees

with previous assessment and supports the current taxonomic status of C. australis. Here, a key to these two

species is provided, and a revised distribution range of C. australis is established. This is the initial report of C.

australis¡¦ occurrence in a part of Indonesia, in addition to areas of Australia and Papua New Guinea.

Keywords: Australia; Ceriops tagal; Ceriops; Distribution; Indonesia; Mangroves; Papua New Guinea; Plastid

DNA; Principal components analysis.

TAxONOmy

90

Botanical Studies, Vol. 50, 2009

except for the hypocotyl morphology. Thus, White

described the form as a variety, C. tagal var. australis.

Based on the analysis of starch gel electrophoresis of

isozymes from C. tagal var. tagal, C. tagal var. australis

and C. decandra in northern Australia, Ballment et al.

(1988) found a uniform genetic structure within each

taxon and a high level of genetic divergence among taxa.

For each taxon having a distinct isozyme profile and the

evidence of reproductive isolation, the authors proposed

three distinctive species and hence raised White¡¦s variety

to specific rank as C. australis, despite the fact that the

extent of divergence in morphological characters other

than propagule morphology remained unclear (Ballment et

al., 1988). Ceriops australis was then claimed as a sibling

species of C. tagal (Ballment et al., 1988).

Due to the confusion regarding diagnostic characters,

it is still unknown how far north of Australia C. australis

extends (Duke, 2006). Misidentification of these two

morphologically similar taxa has been quite common in

herbaria (Sheue, personal observation). Making a field

identification of C. australis is very difficult at any time

other than the fruiting stage with a hypocotyl. Thus,

a detailed study of these two morphologically similar

species is vital.

The goals of this study, therefore, are to detect the

differences between C. australis and C. tagal, based on

a broad and detailed morphological assessment aided by

molecular data, and to establish the geographic distribution

range of C. australis. Here we apply principal components

analysis to morphometric data obtained from herbarium

specimens and investigate the DNA features of the trnL

intron of cpDNA. We also use detailed characters from

fresh and herbarium materials to reevaluate the taxonomic

status of these species. The results will be useful for

field work identification and herbarium examination,

conservation, and for clarification of the relationship

between these species.

mATERIALS AND mETHODS

Herbarium specimens and morphometric

analysis

As it has been reported that only viviparous seedlings

could be used to differentiate Ceriops australis from

C. tagal, the former having terete (smooth) hypocotyls

and the latter having ridged hypocotyls (White, 1926;

Ballment et al., 1988), specimens of branches with both

vegetative and reproductive features, including viviparous

seedlings, were examined for this morphometric study.

Fifteen specimens from the Northern Territory (OTUs

1-8) and Queensland (OTUs 9-15) of Australia, tentatively

identified as C. australis, and 15 herbarium specimens

tentatively identified as C. tagal representing populations

of Northern Territory from Australia (OTUs 16-22),

Madagascar (OTUs 23-26), and Sumatra (OTUs 27-30)

were used in the morphometric study (Appendix).

Principal components analysis (PCA) was conducted to

analyze 29 morphological characters (25 quantitative and

4 binary characters, Table 1), and the PC-ORD package

(McCune and Mefford, 1999) was used to analyze

character variable matrices. The possible differentiated

characters identified in this analysis will be used to detect

diagnostic features in the following analysis.

Fresh plant materials for morphological

characterization

Fresh plant materials of C. australis and C. tagal

were sampled from Cape York, Cairns, and Cardwell of

northeastern Queensland and from the Darwin area of

the Northern Territory of Australia during 2005 to 2007

for morphological characters investigation and molecular

study. Three branches from each of five individuals in a

population were collected. Characters of fresh materials

were investigated by a Leica MZ75 stereoscope and

photographed with an Olympus C7070 digital camera.

Voucher specimens were deposited at the Herbarium of

the Department of Biological Resources, National Chiayi

University (CHIA).

Herbarium specimens for determining

distribution range

The loaned specimens (Appendix) from herbaria BM,

DNA, GH, K, L and MO were identified as C. australis

or C. tagal through the following two steps. In the first

step, specimens with viviparous seedlings attached on the

shoots were determined and used for getting diagnostic

features for identification. In the second step, specimens

lacking hypocotyls were identified according to the

diagnostic features obtained from the previous first step.

Each specimen was carefully examined at least thrice. In

addition, a few specimens of C. australis examined from

Herbaria BO and CAL were incorporated in the results.

molecular evidence

Materials. Populations of C. australis and C. tagal

were mainly sampled at five sites on the northeast

Queensland coast and north Northern Territory coast

in Australia during the period from 2003 to 2007. In

addition, C. tagal collected from Singapore and India

and C. decandra collected from India were analyzed

together (Table 2). Three to five leaves were taken from

each individual and stored with silica gel in zip-lock

plastic bags until DNA isolation. Voucher specimens were

deposited at the Herbarium of National Chiayi University

(CHIA).

DNA extraction. Using the cetyltrimethylammonium

bromide (CTAB) method described previously (Doyle

and Doyle, 1987), total DNA was extracted from fresh

etiolated leaves. Ethanol-precipitated DNA was dissolved

in TE (Tris-EDTA) buffer and stored at -20oC. Qiagen

(Valencia, CA, USA) columns were used to clean the

DNA samples, which were difficult to amplify by PCR.

The approximate DNA yields were then determined using

a spectrophotometer (model U-2001, Hitachi).

SHEUE et al. ¡X Reevaluating the taxonomic status of

Ceriops australis

91

PCR amplification and electrophoresis. The protocols

for PCR were as follows. A 50-£gl mixture contained 40

mM Tricine-KOH (pH 8.7), 15 mM KOAc, 3.5 mM Mg

(OAc)

2

, 3.75 £gg/ml BSA, 0.005% Tween 20, 0.005%

Nonidet-P40, four dNTPs (0.2 mM each), primers (0.5

£gM each), 2.5 units of Advantage 2 DNA polymerase

(Clontech), 10 ng genomic DNA, plus a 50-£gl of mineral

oil. Amplification reactions were carried out in a dry-block

with two-step thermal cycles (Biometra). The universal

primers for amplifying the trnL intron of chloroplast

DNA were the same as described by Taberlet et al. (1991).

The first step of PCR reaction conditions for the trnL

intron were: incubation at 94oC for 3 min, 10 cycles of

denaturation at 94oC for 30 s, annealing at 68oC for 10

s, and extension at 72oC for 45 s. The second step was

carried out with 30 cycles of denaturation at 94oC for 30

s, annealing at 66oC for 10 s, extension at 72oC for 45 s,

and a final extension for 5 min at 72oC. The PCR products

were analyzed by agarose gel electrophoresis (1.0%, w/v

in TBE), stained with 0.5 £gg/ml ethidium bromide, and

photographed under UV light exposure.

DNA recovery and sequencing. The PCR products

in this study were recovered using glassmilk (BIO 101,

California) and directly sequenced following the method

of dideoxy chain-termination using an ABI377 automated

sequencer with the Ready Reaction Kit (PE Biosystems,

California) of the BigDye. Terminator Cycle Sequencing.

Primers for sequencing were the same as those used for

PCR. Each sample was sequenced two or three times

to ensure the accuracy of the sequences. The reactions

were performed following the recommendation of the

manufacturers. These reactions were performed based on

the recommendations of the manufacturer.

Data analyses. DNA sequence alignment was

conducted using the program Clustal W multiple alignment

in BioEdit (Hall, 1999). Genetic relationships were then

determined using the program MEGA version 2.1 (Kumar

et al., 2001). The genetic distance matrix was calculated by

the two-parameter method of Kimura (1980) and then used

to construct the phylogenetic trees using the Neighbor-

joining (NJ) method (Saitou and Nei, 1987). Maximum

parsimony (MP) analyses (Fitch, 1971) were done using

code modified from the Close-Neighbor-Interchange (CNI)

algorithm (Rzhetsky and Nei, 1992) in MEGA version 2.1

(Kumar et al., 2001). Bootstrapping (1000 replicates) was

carried out to estimate the support for both NJ and MP

topologies (Felsenstein, 1985; Hillis and Bull, 1993). The

strict consensus parsimonious tree was then constructed

using the program MEGA version 2.1 (Kumar et al.,

2001).

RESULTS

morphometric analysis

We performed a principal coordinate analysis, and the

result is shown in Figure 1. These two species are well

separated, and 51.8% of the variation can be explained by

the first two principal coordinates. Only the first ordination

axis was considered (Table 1). For components, the ten

highest eigenvector values belonged to reproductive

characters, except leaf length; accordingly, these include

surface of hypocotyl (HS), length of fruit (FL), length of

hypocotyl (HL), length of calyx lobe/ width of calyx lobe

(CLL/CLW), width of calyx lobe (CLW), thickness of

the middle part of calyx lobe (CLT), width of hypocotyl

(HW), width of fruit (FW), length of style (STL) and

leaf length (LL). The highest three eigenvector values of

vegetative characters were leaf length (LL), stipule length

at the naturally expanded stage (SL) and leaf width (LW).

These characteristic variables represented the relative

contribution of the first component in explaining the total

variation within the dataset. Each two selected diagnostic

characters of organs belonging to leaf (LL, SL), flower

(CLW, STL), and fruit (HS, FL) are suggested for use in

identification and are shown in Figure 1.

morphological features

Ceriops australis and C. tagal have very similar

morphological characteristics, including a grey-white

trunk and stem with buttressed base, elliptic-obovate

leaves with reflexed margins, and small flowers with white

petals (Figure 2). The most distinctive basis upon which

to differentiate the two species is the viviparous seedling

(hypocotyl), as reported before. Based on field experience,

C. tagal usually has dark green and elliptic to obovate

leaves and longer stipules (the expanded stipules usually

longer than 1.5 cm) than C. australis, which has more

yellow-green and obovate leaves and shorter stipules (the

expanded stipules usually less than 1.2 cm) (Figure 3;

Table 1).

Figure 1. P CA ordination diagram of OTUs and prominent

variables. OUTs 1-15: Ceriops australis; OUTs 16-30: C. tagal.

Each two selected diagnostic characters belonged to organs

of leaf (LL, SL), flower (CLW, S TL) and fruit (FL, HS) for

differentiating the two species are s uggested. Abbreviations:

CLW: Width of the base of calyx lobe; FL: length of fruit; HS:

surface of hypocotyl; LL: leaf length; SL: stipule length at the

naturally expanded stage; STL: length of style.

92

Botanical Studies, Vol. 50, 2009

Table 1. A List of the selected 29 morphological characters, examined from each 15 herbarium specimens of Ceriops australis and

C. tagal from Madagascar, Sumatra and Australia for principal components analysis. Characters 1-11 are leaf characters, 12-22 are

flower characters, 23-29 are fruit and hypocotyl characters.

No. Character

(unit) or (character state)

Character

abbreviation

C. australis

Mean

¡Ó

std or

character state

C. tagal

Mean

¡Ó

std or

character state

Eigenvector value

of axis 1

1 Leaf blade length (mm)

LL

55.7

¡Ó

5.3

68.5

¡Ó

8.2

-0.2169

2 Leaf width (mm)

LW

27.5

¡Ó

3.4

32.3

¡Ó

5.1

-0.1792

3 The length between the maximum width of leaf

to leaf apex (mm)

LWmax

22.9

¡Ó

3.6

27.7

¡Ó

5.1

-0.1680

4 Petiole length (mm)

PL

18.4

¡Ó

4.4

19.1

¡Ó

4.5

-0.0326

5 Leaf length/ leaf width

LL/LW

2.05

¡Ó

0.20

2.13

¡Ó

0.23

-0.0278

6 The ratio of leaf length to the length between the

maximum width of leaf to leaf apex

LL/LWmax 2.5

¡Ó

0.3

2.5

¡Ó

0.3

-0.0058

7 Leaf length/ petiole length

LL/PL

3.2

¡Ó

0.8

3.7

¡Ó

0.6

-0.1105

8 Leaf apex

LAE

0 = 6

0 = 12

-0.1260

with (0) or without emarginated (1)

1= 9

1 = 3

9 The degree of acute of leaf apex

LAA

61.7

¡Ó

5.0

63.1

¡Ó

7.8

-0.0471

10 Number of lateral vein

V

7.3

¡Ó

0.8

8.0

¡Ó

0.7

-0.1334

11 Stipule length of naturally expanded (mm)

SL

11.8

¡Ó

1.4

16.1

¡Ó

2.7

-0.2060

12 Length of calyx lobe (mm)

CLL

4.3

¡Ó

0.3

4.1

¡Ó

0.4

0.0784

13 Thickness of the middle part of calyx lobe (mm) CLT

0.24

¡Ó

0.04

0.35

¡Ó

0.05

-0.2402

14 Width of the base of calyx lobe (mm)

CLW

1.4

¡Ó

0.2

2.0

¡Ó

0.1

-0.2530

15 Length of calyx lobe/width of calyx lobe

CLL/CLW 3.0

¡Ó

0.4

2.1

¡Ó

0.2

0.2538

16 Length of petal (not included bristle) (mm)

PL

2.8

¡Ó

0.3

3.04

¡Ó

0.19

-0.1519

17 Number of bristles of each petal apex

B

3.5

¡Ó

0.7

3.0

¡Ó

0.0

0.1228

18 Length of bristle (mm)

BL

0.77

¡Ó

0.15

0.61

¡Ó

0.11

0.1554

19 Length of enlarged part of bristle (mm)

BHL

0.23

¡Ó

0.06

0.31

¡Ó

0.05

-0.1902

20 Width of enlarged part of bristle (mm)

BHW

0.08

¡Ó

0.01

0.13

¡Ó

0.04

-0.2180

21 Trichome on abaxial surface of petal

PT

0 = 15

0 = 6

0.1991

with (0) or without (1)

1 = 9

22 Length of style (mm)

STL

3.07

¡Ó

0.39

2.08

¡Ó

0.49

0.2259

23 Length of fruit (mm)

FL

11.6

¡Ó

1.39

19.3

¡Ó

1.62

-0.2645

24 Width of fruit (mm)

FW

6.2

¡Ó

0.7

9.2

¡Ó

1.28

-0.2258

25 Length of fruit/ width of fruit

FL/FW

1.9

¡Ó

0.3

2.1

¡Ó

0.2

-0.1377

26 Persistent calyx lobe

CLR

0 = 7

0 = 15

-0.1651

reflex (0) or patent (1)

1 = 8

27 Length of hypocotyl (mm)

HL

106.6

¡Ó

23.0

241.3

¡Ó

38.6

-0.2596

28 Width of hypocotyl (mm)

HW

3.2

¡Ó

0.6

5.9

¡Ó

1.11

-0.2393

29 Surface of hypocotyl

HS

-0.2818

smooth (0) or ridged (1)

0 = 15

1 = 15

SHEUE et al. ¡X Reevaluating the taxonomic status of

Ceriops australis

93

It was evident that features like calyx lobe, petal

morphology, style (Figure 4), fruit length, and hypocotyl

surface (Figure 3C) could aid the differentiation of these

sibling species. Ceriops australis has longer flowers

(Figure 4A), narrower and longer calyx lobes (Figure 4C;

Table 1), longer petals (Figure 4D-E) and longer styles

(Figure 4F) than C. tagal (Figure 4B, C, E-F). Three to

five more slender clavate appendages were commonly

found on the petal apex of C. australis, but only three

such appendages (more short) were observed on C. tagal

(Figure 4D-E; Table 1).

DNA evidence

Sequence alignment and characteristics. PCR products

from each sample studied were directly sequenced. The

accession numbers of those plastid DNA sequences

Table 2. A list of molecular study for 14 accessions of the Ceriops australis and 15 accessions of C. tagal, as well as three outgroup

accessions of C. decandra, and their different geographical distributions.

Abb.

Taxon

Collection location

Accessions No.

Rh-13

C. australis

Moreton Bay, QLD, Australia (AU)

EF118948

Rh-70

C. australis

Cairns, QLD, Australia (AU)

EF118971

Rh-71

C. australis

Darwin, NT, Australia (AU)

EF118949

Rh-72

C. australis

Darwin, NT, Australia (AU)

EF118950

Rh-73

C. australis

Darwin, NT, Australia (AU)

EF118951

Rh-113

C. australis

Cardwell, QLD, Australia (AU)

EF673713

Rh-114

C. australis

Cardwell, QLD, Australia (AU)

EF673714

Rh-115

C. australis

Cardwell, QLD, Australia (AU)

EF673715

Rh-120

C. australis

Cardwell, QLD, Australia (AU)

EF673717

Rh-132

C. australis

Cardwell, QLD, Australia (AU)

EF673721

Rh-133

C. australis

Cardwell, QLD, Australia (AU)

EF673722

Rh-134

C. australis

Cardwell, QLD, Australia (AU)

EF673723

Rh-135

C. australis

Cardwell, QLD, Australia (AU)

EF673724

Rh-136

C. australis

Cardwell, QLD, Australia (AU)

EF673725

Rh-31

C. tagal

West Sundarbans, India (IN)

EF118987

Rh-32

C. tagal

West Sundarbans, India (IN)

EF118964

Rh-33

C. tagal

West Sundarbans, India (IN)

EF118965

Rh-65

C. tagal

Cairns, QLD, Australia (AU)

EF118966

Rh-85

C. tagal

Cairns, QLD, Australia (AU)

EF118986

Rh-86

C. tagal

Cairns, QLD, Australia (AU)

EF118988

Rh-66

C. tagal

Darwin, NT, Australia (AU)

EF118967

Rh-67

C. tagal

Darwin, NT, Australia (AU)

EF118968

Rh-68

C. tagal

Cape York, QLD, Australia (AU)

EF118969

Rh-69

C. tagal

Cape York, QLD, Australia (AU)

EF118970

Rh-82

C. tagal

Pulau Ubin, Singapore (SING)

EF118972

Rh-116

C. tagal

Cardwell, QLD, Australia (AU)

EF673716

Rh-121

C. tagal

Cardwell, QLD, Australia (AU)

EF673718

Rh-127

C. tagal

Cardwell, QLD, Australia (AU)

EF673719

Rh-131

C. tagal

Cardwell, QLD, Australia (AU)

EF673720

Rh-26

C. decandra

Pichavarum, India (IN)

EF118952

Rh-28

C. decandra

West Sundarbans, India (IN)

EF118953

Rh-29

C. decandra

West Sundarbans, India (IN)

EF118954

Abbreviations: AU: Australia, IN: India; QLD: Queensland; NT: Northern Territory; SING: Singapore.

94

Botanical Studies, Vol. 50, 2009

from the 14 accessions of C. australis and 15 accessions

of C. tagal plus three outgroup accessions are shown

in Table 2. Those sequences were aligned and resulted

in 606 characters, from which 13 were variable sites.

The sequence alignment was submitted to TreeBase

(Submission ID: SN4033). Each variable site was a

potentially informative parsimony site. Neither C. australis

nor C. tagal showed any sequence variation at the species

level. The genetic distance between C. australis and C.

tagal was 0.003 using the 2-parameter method of Kimura

(1980). Two stable transitions were found within this DNA

region between C. tagal and C. australis (data not shown).

Phylogeny reconstruction. The phylogenetic tree

for the intron of trnL used characters that were equally

weighted. Based on the MP method, the analysis yielded

270 equally parsimonious trees with a length of 13 steps, a

consistency index (CI) of 1.0, and a retention index (RI) of

1.0. The strict consensus tree is shown in Figure 5. More

than 50% of the bootstrap values are shown below/above

the supported branches for MP tree. The NJ tree and the

MP strict consensus tree constructed from plastid DNA

data were highly congruent (Figure 5, MP tree presented

only). Based on the phylogenetic tree, accessions of C.

australis formed a clade supported by a 69% bootstrap

value, and accessions of C. tagal formed a clade supported

by a 72% bootstrap value. Molecular data also supported

the distinctness of C. australis and C. tagal, even in

the sympatric populations of Queensland and Darwin,

Australia.

Distribution range of C. australis

The whole distributional range of C. australis includes

eastern (Moreton Bay) and northern Queensland (Cape

York, Nassau River), the coast of the Northern Territory,

through northern and northwestern Western Australia (to

the Ashburton River), the southern part of Papua New

Guinea (Port Moresby, Daru Island), through Timor,

Flores, Sumbawa, Java and Pulau Bilinton, close to

Sumatra, Indonesia (Figure 6). This is the first report that

C. australis occurs in parts of Indonesia.

According to the examination of herbarium specimens,

C. australis has a much wider distribution range than

C. tagal in Australia, although C. tagal is widely

distributed from East Africa through India and Asia to

New Caledonia. However, C. tagal is only found in

northeastern and northern Queensland, through Cape York,

Arnhem Land, and Melville Island in Australia. There are

only about five colonies with a few individuals of C. tagal

growing closely with C. australis found in the Darwin area

(Sandy Creek) in the Northern Territory according to our

field survey.

DISCUSSION

In this study, both of the morphological features

revealed by PCA and molecular evidence demonstrate that

C. australis should be recognized as distinct from C. tagal,

rather than as a sibling species only slightly different

Figure 2. Habitats of Ceriops australis (A-C) and C. tagal (D). A, Close-up of C. australis with flowers and viviparous seedlings with

smooth surface; B, The grey-white bark with buttress base of C. australis at Cairns, Queensland; C, Flowers of C. australis. Note the

evident long style; D, C. tagal with viviparous seedlings with ridges.

SHEUE et al. ¡X Reevaluating the taxonomic status of

Ceriops australis

95

in hypocotyl feature and genetic structure as proposed

by White (1926) and Ballment et al. (1988). However,

for a practical application of the concept of species,

it is necessary to provide some diagnostic characters.

According to the morphometric results obtained in this

study, the most distinctive characters differentiating the

two species are reproductive features. The diagnostic

characters of style length (STL) and width of calyx lobe

(CLW) are recommended for the plants with flowers;

those of hypocotyl surface (HS) and fruit length (FL) are

recommended for plants with fruits. Nevertheless, we

suggest that the features of leaf length (LL) and stipule

length of the naturally expanded stage (SL) could also aid

the identification of plants in the field without flower or

fruit.

Based on the results of morphological features and

PCA, a key to differentiating the populations of C.

australis and C. tagal is here provided:

Key to C. australis and C. tagal

1a.

Leaf blade usually shorter than 6.5 cm in length;

stipule less than 1.2 cm long at the naturally expanded

stage; base of calyx lobe 12-15 mm in width; style

2.7-4 mm in length; fruit 9-14 mm long; hypocotyl

terete (without longitudinal ridges), 5-12 cm in length

..................................................................... C. australis

1b.

Leaf blade usually longer than 6.5 cm in length; stipule

longer than 1.4 cm long at naturally expanded stage;

base of calyx lobe 18-25 mm in width; style 1.5 mm in

length (-3.5 of populations from Darwin area, Northern

Territory of Australia); fruit 18-25 mm long; hypocotyl

angular (with longitudinal ridges), 15-35 cm in length

...........................................................................C. tagal

According to Wightman (2006), populations of C. tagal

in Northern Territory generally have elliptic leaves and

relative shorter petiole length (usually less than 1/4 of the

blade length) than the mostly obovate leaves and relative

longer petiole length (generally reaching 1/3 or more of

the blade length) of C. australis. Based on the observation

of this study, we agreed with Wightman¡¦s statement and

found that the populations of C. australis in Western

Australia have the most typical obovate and smaller leaves

than other populations. It is likely that C. australis is

the only one species of this genus occurring in Western

Australia, which results in much less opportunity to have

gene flow with other species of Ceriops, if compared to

the other sympatric populations of Ceriops.

In addition, we noted some of the detailed differences

between these two taxa, including the number of colleters

inside the adaxial base of the stipule (Sheue, 2003), the

thickness of the middle of the calyx lobe, and the number

and shape of the clavate appendages on the petal apex.

Figure 3. Characters of leaves, fruits and hypocotyls of Ceriops australis and C. tagal. A, Leaves of C. australis tend to be more

obovate in shape and stipules at naturally expanded stage are usually less than 1.2 cm; B, Leaves of C. tagal are more oblong in shape,

and stipules at naturally expanded stage are usually longer than 1.4 cm; C, The fruit is smaller and the hypocotyl is shorter and ridge-

free of C. australis (A & a); while the fruit is larger and the hypocotyl is longer and ridged of C. tagal (B & b). Abbreviations: NT:

Northern Territory, QLD: Queensland, WA: Western Australia.

96

Botanical Studies, Vol. 50, 2009

However, to observe these delicate features a hand lens

(10X) or a stereoscope may be needed.

It is notable that the observation of herbarium

specimens in this study revealed no evident morphological

variations of C. tagal between the populations of

Madagascar and Sumatra and those from northern

Australia. The low levels of morphological variation

across a big geographic range of C. tagal noted by this

study were consistent with the inter simple sequence

repeat (ISSR) markers of C. tagal studied in Asia (Ge and

Sun, 2001) and the trnL intron sequences of plastid DNA

from different locations of C. tagal in this study.

Correct information for identification is essential to

getting an accurate biogeographic description. Since

the confusion in diagnostic characters applies to these

two taxa in Australia, obtaining accurate information on

their distribution ranges is not easy. This is perhaps why

Australia¡¦s Virtual Herbarium (AVH) could not supply

the correct information for these two taxa (http://www.

anbg.gov.au/cgi-bin/avhxml.cgi). In terms of AVH

Mapper, C. australis only occurs in the Nothern Territory

and northeastern Western Australia while C. tagal has a

much wider distribution range from Queensland, through

the Northern Territory to Western Australia. Based on a

detailed examination of herbarium specimens in this study,

we have reconstructed the geographic range of C. australis

and C. tagal in Australia. The dominant species of Ceriops

in Australia is C. australis. It ranges from Western

Australia and the Northern Territory to Queensland. This

result is consistent with the report of Duke (2006). In

Papua New Guinea, C. australis has only been observed

from Port Moresby and Idlers Bay. This was consistent

with the observations of White (1926), McCusker (1984),

and Wells (1983).

It is quite interesting that C. australis occurs in Timor,

Flores, Sumbawa, Java and Pulau Bilinton of Indonesia.

The first collector of C. australis from Indonesia may

have been Teijsmann in 1875 (specimens found at BO

herbarium, Sheue, personal observation). Due to the

limited herbarium specimens available from Indonesia,

an extensive field survey for C. australis from the nearby

islands of Indonesia would be useful. This information

would be valuable for mangrove conservation and the

study of phytogeography and dispersal ecology.

Figure 4. Flower morphology of of Ceriops australis and C. tagal. A, Lateral view of a flower of C. australis; B, Lateral view of a

flower of C. tagal; C, Calyx lobes with abaxial and adaxial sides of C. australis (left) and C. tagal (right); D, Petals of C. australis,

with 3-5 more slender clavate appendages; E, Petal of C. tagal, usually with 3 short clavate appendages; F, Lateral view of detached

flowers of C. australis (left) and C. tagal (right) showing calyx lobe, anther and style. Scale bars: A-B = 5 mm, C-F = 1 mm.

SHEUE et al. ¡X Reevaluating the taxonomic status of

Ceriops australis

97

Figure 5. The strict consensus parsimonious tree of 14 accessions of Ceriops australis and 15 accessions of C. tagal plus three

outgroup accessions of C. decandra derived from the trnL intron sequence. Bootstrap values > 50% are shown on each branch.

Figure 6. The distribution range of Ceriops australis and the sympatric localities of C. tagal in Australia. The arrows indicate the new

localities of C. australis in Indonesia first reported in this study.

98

Botanical Studies, Vol. 50, 2009

These two species are sympatric in Papua New Guinea

and northern Queensland (White, 1926; McMillan,

1986), and both occur on the northern coast of Northern

Territory (Wells, 1983). No intermediate forms between

them has been recorded, as reported by McCusker (1984).

After the examination of numerous herbarium specimens

and limited fresh materials collected from Darwin area,

we found that several characters of flowers of C. tagal

collected from Northern Territory and Papua New Guinea

are closer to those of C. australis. Namely, the populations

of C. tagal from the Northern Territory and Papua New

Guinea have narrower and oblong calyx lobes, longer

clavate appendages on the petal apex and longer styles than

C. tagal from other populations in the world. However,

the characters of fruit and propagule of C. tagal from

this area resemble those of other global populations of C.

tagal. We assume that a possible hybridization between

these two taxa may have occurred. According to Duke et

al. (1984), the major flowering season of the populations

from northeastern Australia are November and January to

March for C. australis and C. tagal, respectively. Based on

the observation of herbarium specimens, a broader period

of flowering season for both species could be inferred.

The possible overlap of flowering season may increase the

opportunity of hybridization between these two species.

An anecdotal report notes that hybridization has occurred,

and some trees with both types of propagules from the

Murray River, Admiralty Island, and Pigeon Island in

northeastern Queensland have been observed (Ballment

et al., 1988). However, except for the flower variation in

the Northern Territory and Papua New Guinea previously

mentioned, we did not find such intermediate forms in this

study.

A further study to compare the morphological and

genetic variations of populations of C. tagal in the

Northern Territory, Australia, and Papua New Guinea and

the other populations from the world should be useful and

interesting. Moreover, the factors influencing the sympatric

populations of C. australis and C. tagal in the Northern

Territory and Queensland may be worth exploring, in

order to reveal why a possible hybridization only occurs in

the Northern Territory, but not in northern Queensland.

Acknowledgements. The authors thank T. Lammers and

M. S. B Ku for improving the manuscript, D. Foster and V.

Sarafis for helping to collect some materials in Australia,

K. N. Ganeshaiah for helping with the distribution map,

and the following herbaria for permission to study and/or

borrow specimens: BM, BO, CAL, DNA, GH, K, L, and

MO. This study was supported by the National Science

Council (NSC94-2311-B-020-001-) and by National

Chiayi University (NCYU 96T001-02-06-018) of Taiwan.

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Appendix. Specimens list (herbarium acronyms follow Index Herbariorum, available at http://sweetgum.nybg.org/ih/).

Specimens lis t for m orphometric analys is for principal com ponents analys is. Ceriops australis: AUSTRALIA: Northern

Territory: Bowman & Wilson 263 (DNA), Dunlop 3629 (DNA), Dunlop & Munns 7512 (L), Egan 2821 (DNA), Forster &

Russell-Smith PIF5920 (DNA), Henshall 857 (DNA), Latz 3192 (DNA, L, MO), Must 1649 (DNA), Russell-Smith & Lucas

5884 (DNA). Queensland: Smith 4825 (L), 12441 (GH, L) Stoddart 4536, 4699, 4761, 4903 (L, MO), 4992 (L). Ceriops tagal:

AUSTRALIA: Northern Territory: Brock 116 (DNA), Dunlop 3899 (DNA), Dunlop & Wightman 9709 (DNA), J. & Eurell

78/20 (DNA, MO), Scarlett 164 (DNA), Wightman 458, 786 (DNA). MADAGASCAR: Birkinshaw & Jules 13 (MO), Darcy

& Rakotozafy 15470 (MO), Dorr & Koenders 3063 (GH, MO), Rahajasoa 356 (MO). SUMATRA: Iwatsuki et al. S1319 (MO),

S1321 (L), Schmad 146 (L), Teijsmamn & Miquee s. n. [no date] (K).

Specimens examined for revising the distribution range of C. australis and the sympatric localities of C. tagal in Australia.

Ceriops australis: AUSTRALIA: New Holland: Banks & Solander s. n. [1770] (BM), Queensland: Blake 14127 (MO),

Clarkson 2016 (MO), 3875 (DNA, MO), Cribb & Newton s. n. [1950] (BM), Dietrich s. n. [1863-65], 657 (MO), Durrington (L),

Everist 7881A (L), Fosberg 61833 (MO), Macnae s. n. [1962], Mrs. Stephenson 569 (BM), Neldner & Clarkson 2993 (DNA),

Smith 4825, 11435 (L), 12441 (GH, L), Stoddart AQ14784 (K), 4510 (MO), 4527, 4536, 4699, 4761, 4786, 4903 (L, MO), 4992

(L), Webster & Hildreth 15005 (GH), White s. n. [1915] (BM), 3372A (K, type), 3373A (GH); Northern Territory: Bardsley s

.n. [1985] (DNA), Barlow 506 (DNA), Blake 17050 (K, GH), Bowman & Wilson 263 (DNA), Brennan 2619 (DNA), Brooker

3258 (DNA), Byrnes NB275 (DNA), Byrnes & Maconochie 1077 (DNA), Calliss 63 (DNA), Chippendale s. n. [1961], 8180

(DNA), Clark 948 (DNA), Cowie 5183 (DNA), Cowie & Dunlop 4131, 7926 (DNA), Dunlop 1869 (DNA), 2782 (DNA, MO),

3984 (DNA), Dunlop & Leach 8062 (DNA), Dunlop & Munns 7512 (L), Dunlop & Wightman 9203 (DNA), Egan 2391, 2821

(DNA), Forster & Russell-Smith PIF5920 (DNA), Gill s. n. [1970] (GH), Henry 88 (DNA), Henshall 857 (DNA), Hodder s. n.

[1971] (K), D4044 (DNA), Latz 3192 (DNA, L, MO), 3390 (DNA), Leach 3993, 4231 (DNA), Leach & Cowie 3641 (DNA),

Martensz & Schodde AE737 (DNA), McKean B142, 974 (DNA), Michell & Ingraham 27 (DNA), Must 884, 1310 (DNA), 1348,

1649 (DNA, MO), Nelson 1078 (DNA), Rankin 1171, 1248, 1380, 2220 (DNA), Ridpath Mck B7 (DNA), Russell-Smith 8918

(DNA), Russell-Smith & Lucas 4375, 5642, 5884, 8368 (DNA), Scarlett 163 (DNA), Shaw & Dunlop 3629 (DNA, MO), Smith

1030, Specht 591 (GH), Story 8337 (DNA), Thomson 661, 1878, 2621, 2661 (DNA), van Kerckhof 29, 33, 39 (DNA), Waddy

560 (DNA), Wells s. n. [1975, 1978] (DNA), Wheelwright DW8 (DNA), 24 (DNA), Wightman 475, 488, 504, 506, 520, 543, 619,

673, 701, 814, 1070, 1544, 1663, 2290, 2389, 2453, 2472 (DNA), 4603 (DNA, MO), 6162, 6652 (DNA), Wightman & Dunlop

551, 563 (DNA), Wightman & Giulian 2926 (DNA), Wightman & Smith 3531, 4523 (DNA), Williams 350 (DNA), Williams

& Wightman 135 (DNA), Wilson 790 (DNA); Western Australia: Croat 52316A (MO), Cunningham 235 (K), Fstyguold

s. n. [1906] (BM), George 12724 (DNA), 14829 (K), Hartley 14587 (DNA), Mitchell 5949 (DNA), Morrison s. n. [1950],

Paijmans 2469 (DNA), Perry 2548 (DNA), Wightman 7111 (DNA); PAPUA NEW GUINEA: Central District: near Barune:

Frodin UPNG4444 (K); Fairfax Harbour: Gillison NGF22159 (GH); Kairuku subdistriction: Darbyshire 773 (K); near Lae Lae:

Schodde 2681 (GH); Kappa Kappa Papua: Brass 786 (BM); Port Moresby: Frodin & Millar UPNG562 (L). INDONESIA:

Timor: Anonymous s. n. [1923] (BO), L. v. d. Pijl 820 (BO); Flores: M. Kew s. n. [1905] (BO); Subawa: Kostermans & Wirawan

348 (BO); Java: Teijsmann s. n. [no date] (CAL); Bilinton Island: Teijsmann s. n. [1875] (BO).

Ceriops tagal: AUSTRALIA: no data: Leschenault s. n. [1802] (BM), Queensland: Stoddart 4086, 4113, 4154 (MO), 4317 (MO,

L), 4385 (MO), 4634 (MO, L), 4637 (MO), Smith 11409 (GH), 11618 (K), 11619 (L), 12445 (GH), 12521 (L), Smith & Webb

3243 (L), Mrs. Stephenson 486, 545, 570, 606 (BM), Thom 4168, 4170, 4171, 4172 (MO), Clarkson 3387 (MO). Northern

Territory: Bardsley 15 (DNA), Brennan 4563, 2627, 2877 (DNA), Brock 116 (DNA), Byrnes & Maconochie NB1078 (DNA),

Cowie 3397, 5140, 6924 (DNA), Dunlop 3899 (DNA), Dunlop & Wightman 6541, 9709, 9739 (DNA), Egan 2713 (DNA), Eurell

s.n. (MO), s. n. [1978] (GH), J. & Eurell 78/ 20 (DNA, MO), Kerrigan & Risler 57 (DNA), Scarlett 164 (DNA), Stocker GS79

(DNA), Wells s. n. [1975] (DNA), Wightman 458, 786, 1970, 4113, 4185, 4457, 6506 (DNA), Wightman & Giuliana 2993 (DNA),

Wightman & Smith 3531 (DNA).