sing utng teng , chui pin leaw , hong chang lim and po ...repository.um.edu.my/40137/1/teng et al....

DOI 10.1515/bot-2012-0194 Botanica Marina 2013; aop

Sing Tung Teng , Chui Pin Leaw , Hong Chang Lim and Po Teen Lim *

The genus Pseudo - nitzschia (Bacillariophyceae) in Malaysia, including new records and a key to species inferred from morphology-based phylogeny Abstract: Species of the diatom Pseudo-nitzschia are known

to produce domoic acid that is responsible for amnesic

shellfish poisoning (ASP). To investigate the potential risk

of ASP and species occurrence of toxic or potentially toxic

Pseudo-nitzschia in Malaysian waters, plankton samples

were collected from 17 locations. Samples were examined

through transmission electron microscopy. Species of

Pseudo-nitzschia were identified on the basis of the frustule

morphology and morphometric measurements. Twenty-

two well-described species were recorded, of which 14

are new records for Malaysia. A new morphotype, closely

resembling species in the pseudodelicatissima complex,

was also discovered. The morphotype differs from other

species in the complex by its lower densities of fibulae

and striae. Nine of the species have previously been asso-

ciated with ASP events worldwide. Our study recorded for

the first time high species richness of Pseudo-nitzschia in

the confined coasts of Malaysia. In addition, we performed

a morphology-based phylogeny and proposed a key to

Pseudo-nitzschia species, with a special emphasis on the

poroid structure of the striae, to aid in species identification.

Keywords: diatom; Malaysia; morphology; new morpho-

type; Pseudo-nitzschia .

*Corresponding author: Po Teen Lim, Faculty of Resource Science

and Technology, Universiti Malaysia Sarawak, Kota Samarahan,

94300 Sarawak, Malaysia,

e-mail: [email protected]; [email protected]

Sing Tung Teng and Chui Pin Leaw: Institute of Biodiversity and

Environmental Conservation, Universiti Malaysia Sarawak, Kota

Samarahan, 94300 Sarawak, Malaysia

Hong Chang Lim: Faculty of Resource Science and Technology, Universiti

Malaysia Sarawak, Kota Samarahan, 94300 Sarawak, Malaysia

Introduction Pseudo-nitzschia H. Peragallo, a genus of pennate chain-

forming diatoms, has drawn significant attention and

scientific interest after an incidence of amnesic shellfish

poisoning (ASP) in Prince Edward Island, Canada, in

1987 ( Bates et al. 1989 ). Identification of Pseudo-nitzschia

species based on morphology requires detailed examina-

tion of the frustule ultrastructure, which is only feasible

through electron microscopy. Morphological characteris-

tics such as shape; length and width of the valve; pres-

ence or absence of a central nodule; and number of

fibulae, striae, and poroids are among the detailed fea-

tures used for species delineation. Even though morpho-

metric characterizations of Pseudo-nitzschia have been

well documented (e.g., Hasle et al. 1996 ), it is challenging

to distinguish some closely related pseudo-cryptic species

such as P. cuspidata - P. pseudodelicatissima ( Lundholm

et al. 2003 ) and cryptic species such as P. delicatissima -

P. arenysensis ( Quijano-Scheggia et al. 2009 ). This taxo-

nomic complexity could be a significant barrier to the

early detection and mitigation of harmful algal blooms

(HABs) particularly in the genus Pseudo-nitzschia .

The recent detection of the ASP toxin, domoic acid

(DA), in shellfish from Southeast Asian countries ( Bajarias

et al. 2006 , Dao et al. 2006, 2009a , Takata et al. 2009 )

indicates the potential risk for ASP events in the region.

In Malaysia, harmful algal research and monitoring are

confined to locations with known incidences of paralytic

shellfish poisoning toxins produced by the dinoflagellates

Pyrodinium bahamense Plate ( Usup et al. 2012 ) and Alex-andrium spp. ( Lim et al. 2004, 2005 ). Very little attention

has been given to other groups of HAB species, especially

species of Pseudo-nitzschia .

The present study was conducted to remedy the insuf-

ficient number of background studies on the occurrence

of Pseudo-nitzschia species in Malaysian waters, in parti-

cular on Peninsular Malaysia, which is virtually lacking

such information. Several studies were previously under-

taken but mainly to focus on selected sites along the coasts

of Malaysian Borneo ( Lim et al. 2010, 2012a ). Detailed

morphology and genetics of P. brasiliana Lundholm,

Hasle et Fryxell, P. micropora Priisholm, Moestrup et Lundholm, P. dolorosa Lundholm et Moestrup, P. pungens

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2 S.T. Teng et al.: Species of Pseudo-nitzschia in Malaysia

(Grunow ex Cleve) Hasle, P. cuspidata (Hasle) Hasle, and a

new species, P. circumpora Lim, Leaw et Lim were recently

described from the Sabah and Sarawakian waters ( Lim

et al. 2012b ), while P . calliantha Lundholm, Moestrup et Hasle, P. delicatissima (Cleve) Heiden, and P. multistriata

Takano were previously discovered from Sabah ( Skov

et al. 2004 ).

The aims of this survey were to compile a list of

Pseudo-nitzschia species along the coasts of Malaysia and

provide in-depth descriptive information of each species

based on morphology. Here, we document 22 species of

Pseudo-nitzschia recorded in Malaysian coastal waters,

including a new morphotype. Fourteen of them consti-

tute new records for Malaysia. In addition, we attempt

to group Pseudo-nitzschia species based on a morpho-

logical phylo genetic analysis to assist in obtaining a more

accurate species recognition of Pseudo-nitzschia in field

studies, and to generate a key to species of the genus, with

a special emphasis on the poroid structure of the striae.

Materials and methods

Sampling

Sampling took place along the coasts of Malaysia from

June 2009 to October 2011. Seventeen sites were selected,

including 11 sites from the coasts of Borneo and six from

Peninsular Malaysia ( Figure 1 , Supplement 1).

Plankton samples were collected by vertical plank-

ton haul using a 20- μ m-mesh plankton net. Concentrated

samples were either preserved directly in 1% acidic Lugol ’ s

solution or filtered through a 0.2- μ m nylon membrane

filter (Whatman, Piscataway, NJ, USA) and preserved with

95% modified saline ethanol ( Miller and Scholin 2000 ) in

a 50-ml centrifuge tube. Samples were kept at 4 ° C until

analysis. Cells were counted with an Olympus BX51 light

microscope (Olympus, Tokyo, Japan), using a Sedgewick-

Rafter coun ting chamber ( McAlice 1971 ).

Species identification

Samples were shaken to resuspend cells and a 2-ml

aliquot was centrifuged at 4000× g for 10 min (Mikro 120;

Hettich Zentrifugen, Tuttlingen, Germany). Cell pellets

were rinsed three to four times with distilled water to

remove excess Lugol ’ s or saline ethanol, followed by

acid cleaning of the cells using saturated KMnO 4 , 37%

HCl ( Bargu et al. 2002 ), and 10% oxalic acid to remove

the organic material. Cleaned samples were mounted

on Formvar-coated copper grid and air-dried over-

night. Dried samples were examined using a JEM-1230

transmission electron microscope (TEM) (JEOL, Tokyo,

Japan). TEM micrographs were taken with an Erlangshen

ES500W camera and analyzed using Digital Micro-

graph software (Gatan, Pleasanton, CA, USA). Pseudo-nitzschia terminology mainly follows Hasle et al. (1996)

and Hasle and Syvertsen (1997) , and the morphological

characters of each Malaysian Pseudo-nitzschia observed

were referred to related literature. Morphological char-

acters and morphometric measurements were obtained

through TEM.

99ºE 102ºE 105ºE 108ºE 112ºE 115ºE 118ºE 121ºE

Strait of Malacca

South China Sea

Kota Kinabalu

Sarawak

Sabah

MuarJohore Bharu

Teluk Batik

Queen Bay Kuala Terengganu

Santubong

Semariang

GerigatKabung

Bintulu

Kuala Penyu

Kota BeludKudat Pulau Banggi

Sempurna

6ºN

2ºN

N

Penisular Malaysia

Malaysian Borneo

200km

Figure 1 Map showing the 17 stations sampled along the coasts of Malaysia between June 2009 and October 2011.



S.T. Teng et al.: Species of Pseudo-nitzschia in Malaysia 3

Morphological character coding and phylogenetic analysis

Twenty-two morphological characters were scored for

the morphologically described Pseudo-nitzschia taxa and

two outgroup taxa, Nitzschia navis-varingica Lundholm et Moestrup and Bacillaria paxillifer (M ü ller) Marsson (Sup-

plement 2). Character information of the 24 taxa recorded

in this study and 43 published taxa obtained from the lit-

erature were used in the character scoring (Supplement 3).

A character matrix was constructed using the program

Nexus Data Editor v. 0.5.0 ( Page 2001 ) with equal weight,

and treated as unordered (Supplement 4).

The characters are based on morphological features

normally used in diatom identification. Two characters

(A, B) are commonly used to distinguish the genus Pseudo-nitzschia , while 20 characters were used to delineate to

species level. The central interspace (D) and transapical

axis (T) are the two common characters that were used to

delineate species of Pseudo-nitzschia in the seriata and del-icatissima groups ( Hasle et al. 1996 ). The valve morphology

was assigned with six characters (M – O and S – U), while the

poroid morphology of the striae was assigned characters C

and E – I ( Figures 2 – 10 ). Characters P – R were assigned to the

features for the valvocopula bands (Supplement 2).

Maximum parsimony (MP) analysis was undertaken

for the morphological data, using PAUP* ver. 4.0 ( Swofford

2000 ). Weighted and unweighted analyses were performed.

Retention index (RI) values were used to reweight the char-

acters. The reweighted characters were then used to obtain

Figure 2 – 10 Poroid morphology of striae in Pseudo-nitzschia species, TEM.

(2 – 7) Striation with one row of poroids. (2) Poroids in four to five hymen sectors with a large space at the center. (3) Rounded edge poroids

in two to three hymen sectors with a central dot sometimes present. (4) Poroids forming two hymen sectors. (5) Poroids in three to four

hymen sectors with a narrow central space. (6) Poroids in more than seven hymen sectors, with a central sector rarely present. (7) Poroids

in 6 – 10 hymen sectors, with a central sector rarely present. (8 – 10) Striation with two rows of poroids. (8) Poroids in four to six hymen

sectors. (9) Hexagonal poroids without hymen sector. (10) Rounded poroids without hymen sector. Scale bars: 0.5 μ m (Figures 3 and 7), 0.2

μ m (Figures 2, 6, 8, and 9), and 0.1 μ m (Figures 4, 5, and 10).




the optimized MP tree under DELTRAN and ACCTRAN

(delay and accelerated character transformations).

An identification key to species of Pseudo-nitzschia

was illustrated based on the cladistics analysis in con-

junction with documented morphological data.

Results

Species diversity

Species of Pseudo-nitzschia were found at all sampling

locations ( Figure 11 ). Twenty-two well-described species,

including a new morphotype, were identified. Bintulu

had the highest species richness with 17 species observed,

followed by Port Dickson and Pulau Banggi with 15 and

13 species, respectively. The lowest species richness was

recorded at five locations, where only two species were

found, P. brasiliana and P. pungens . Both of these species

were present at all locations investigated.

The number of known toxic species throughout

all locations ranged from two to nine and included

P. brasiliana, P. caciantha Lundholm, Moestrup et Hasle,

P. calliantha, P. cuspidata , P. delicatissima , P. multistri-ata , P. pseudodelicatissima , P. pungens , and P. turgidula

(Hustedt) Hasle. Among the 17 locations, only Bintulu,

Pulau Banggi, Semporna, and Port Dickson had more than

five toxic or potentially toxic species of Pseudo-nitzschia .

The species descriptions as well as their distribution

in Malaysian waters are described below, and their mor-

phometric data are summarized in Table 1 .

6ºN

2ºN




P. pseudodelicatissimalP. cuspidataP. callianthaP. caciantha

P. manniiP. hasleanaP. circumpora

A

B

C

P. sp. Port DicksonP. sinica

6ºN

2ºN

P. brasiliana

P. americanaP. linea

P. multistriata

P. delicatissimalP. arenysensis

P. decipiens

P. dolorosa

P. microporaP. cf. lineola

6ºN

2ºN

P. pungensP. turgidula

P. subfraudulenta

P. inflatula

Figure 11 Spatial distribution of Pseudo-nitzschia species within the (A) pseudodelicatissima group; (B) delicatissima group; and

(C) P . pungens , P . subfraudulenta , and P . turgidula in Malaysian coastal waters.




Tabl

e 1

Va

lve

sh

ap

e a

nd

mo

rph

om

etr

ic d

ata

fo

r s

pe

cie

s o

f Ps

eudo

-nitz

schi

a re

cord

ed

in

Ma

lays

ian

co

as

tal

wa

ters

.

Taxo

nVa

lve

shap

eTr

ansa

pica

l ax

is ( μ

m)

Apic

al

axis

( μ m

)CI

SFi

bula

e in

10

μ m

Stria

e in

10

μ m

Row

of

poro

ids

Poro

id

stru

ctur

e a Po

roid

s in

1 μ

m n

P. p

seud

odel

icat

issi

ma*

/ P. c

uspi

data

* Li

ne

ar/

lan

ceo

late

1.8

– 2

.17

6 –

91

+ (

3 –

6)

17

– 2

03

1 –

42

1 +

(2

– 4

)5

– 7

12

P. h

asle

ana

Lin

ea

r to

la

nce

ola

te1

.2 –

2.1

36

– 1

00

+ (

5 –

7)

14

– 1

73

8 –

44

1 +

(2

– 4

)5

– 7

23

P. ca

cian

tha*

La

nce

ola

te2

.5 –

3.0

58

– 8

5 +

(4

)1

5 –

18

28

– 3

11

+ (

2 –

7)

4 –

58

P. ca

llian

tha*

Li

ne

ar

1.9

– 2

.65

6 –

88

+ (

4 –

6)

18

– 2

03

3 –

36

1 +

(3

– 1

2)

4 –

61

2

P. m

anni

i Li

ne

ar

1.8

– 2

.65

4 –

67

+ (

3 –

5)

18

– 2

12

8 –

36

1 +

(2

– 7

)4

– 6

6

P. ci

rcum

pora

La

nce

ola

te1

.9 –

2.9

73

+ (

4)

18

– 2

23

5 –

36

1 +

( > 7

)1

– 3

5

P. si

nica

Li

ne

ar

3.5

11

5 +

(2

)1

11

71

+ (

2)

1 –

21

P. in

flatu

la

Lan

ceo

late

, in

fla

ted

at

ap

ex

1.0

– 2

.14

8 –

10

8 +

(3

– 5

)1

5 –

23

30

– 3

91

+ (

2 –

4)

5 –

74

2

P. d

elic

atis

sim

a*/P

. are

nyse

nsis

Li

ne

ar

to l

an

ceo

late

1.2

– 2

.23

2 –

61

+ (

2 –

3)

20

– 2

63

2 –

40

2-

10

– 1

21

1

P. d

ecip

iens

La

nce

ola

te1

.1 –

2.4

27

– 4

6 +

(3

– 5

)2

0 –

26

42

– 4

62

-1

1 –

13

12

P. d

olor

osa

Lan

ceo

late

1.6

– 2

.53

5 –

72

+ (

3 –

5)

18

– 2

13

2 –

38

1 –

2-

6 –

74

P. c

f. li

neol

a La

nce

ola

te3

.38

7 +

(4

)1

42

41

– 2

-3

– 4

1

P. a

mer

ican

a La

nce

ola

te a

nd

re

cta

ng

ula

r2

.4 –

3.4

14

– 2

9-

18

– 2

22

6 –

35

2(3

)-

9 –

11

12

P. li

nea

Lin

ea

r a

nd

re

cta

ng

ula

r2

.1 –

2.7

12

– 1

7-

21

– 2

24

0 –

42

2-

9 –

12

3

P. b

rasi

liana

* La

nce

ola

te2

.0 –

3.4

21

– 4

6-

21

– 2

82

2 –

29

2(3

)-

8 –

10

33

P. m

icro

pora

La

nce

ola

te1

.3 –

1.8

31

– 4

7-

25

– 2

64

2 –

45

2-

11

– 1

23

P. m

ultis

triat

a*

Lan

ceo

late

1.4

– 2

.54

3 –

91

-2

3 –

25

38

– 4

02

– 3

-1

1 –

12

9

P. tu

rgid

ula*

La

nce

ola

te2

.4 –

2.8

72

– 1

05

+ (

3 –

4)

14

– 1

72

2 –

27

2-

7 –

85

P. p

unge

ns*

Lin

ea

r to

la

nce

ola

te1

.9 –

4.0

70

– 1

48

-9

– 1

39

– 1

32

-2

– 4

16

P. su

bfra

udul

enta

Li

ne

ar

4.0

– 6

.05

6 –

12

2 +

(4

– 5

)1

3 –

15

23

– 2

81

– 2

+ (

3 –

6)

5 –

62

8

P. s

p.

Po

rt D

ick

so

nLi

ne

ar

2.0

– 2

.66

4 –

11

4 +

(3

– 4

)1

1 –

15

21

– 2

91

+ (

2 –

4)

3 –

52

6

*, T

oxi

c s

pe

cie

s;

CIS

, ce

ntr

al

inte

rsp

ace

pre

se

nt

( + )

or

ab

se

nt

(-),

wit

h l

en

gth

of

CIS

in

nu

mb

ers

of

str

iae

; a p

oro

id s

tru

ctu

re w

ith

( +

) a

nd

wit

ho

ut

hym

en

se

cto

r (-

), n

um

be

rs i

n p

are

nth

es

es

de

no

te

nu

mb

er

of

hym

en

se

cto

rs;

n , n

um

be

r o

f s

pe

cim

en

s e

xam

ine

d.




Pseudo-nitzschia pseudodelicatissima (Hasle) Hasle/P. cuspidata (Hasle) Hasle (Figures 12 – 17)

References Lundholm et al. (2003) , p. 801, 804, figs. 1A – G,

3A – G; Cusack et al. (2004) , p. 62 figs. 6A – F; Kaczmarska

et al. (2005) , p. 12, figs. 35 – 39; Fehling et al. (2006) , p. 102,

fig. 6C; Churro et al. (2009) , p. 47, figs. 19 – 20; Trainer et al.

(2007) , p. 455, figs. 8A – B; Trainer et al. (2009) , p. 1469, figs.

8A – J; Leandro et al. (2010) , p. 293, fig. 3B; Moschandreou

and Nikolaidis (2010) , p. 164, figs. 33 – 37.

Morphology In valve view, the cells are linear and

symmetrical, with short tapering and pointed apices

(Figures 12 and 13). Both proximal and distal mantles are

one poroid high (Figures 14 and 16). The stria consists

of one row of round poroids, with five to seven poroids

in 1 μ m (Figures 14 and 16). Poroid hymenes are mainly

divided into two to four sectors (Figures 15 and 17). Some

cells differ by having rarely three to four sectors in the

poroids (Figure 17).

Malaysian distribution New records for Bintulu in

Sarawak, Borneo, and Kota Belud, Kota Kinabalu, and

Pulau Banggi in Sabah, Borneo (Figure 11).

Pseudo-nitzschia hasleana Lundholm (Figures 18 – 21)

Reference Lundholm et al. (2012) , p. 439, figs. 1A – M.

Morphology The cells are linear to lanceolate and asym-

metrical in valve view (Figure 18). The apices are pointed

(Figure 19). Each stria contains one row of poroids, with

five to seven poroids in 1 μ m. The proximal mantle is three

poroids high, while the distal mantle is two poroids high

Figures 12 – 21 Valve shape and ultrastructure of Pseudo-nitzschia species, TEM.

(12 – 17) P. pseudodelicatissima/P. cuspidata . (12) Linear valve in valve view. (13) Valve apex. (14) Striation showing one row of poroids. (15)

Poroids with two hymen sectors. (16) Mid-valve showing a central interspace (arrowhead). (17) Poroids with two to four hymen sectors.

(18 – 21) P. hasleana . (18) Lanceolate valve in valve view. (19) Valve apex. (20) Mid-valve showing a central interspace (arrowhead). (21)

Poroids with hymen sectors. Scale bars: 20 μ m (Figure 12), 10 μ m (Figure 18), 2 μ m (Figure 19), 1 μ m (Figure 13), 0.5 μ m (Figures 16, 20, and

21), 0.2 μ m (Figures 14, 17, and 21), and 0.1 μ m (Figure 15).




(Figure 20). The poroids are round and consist of two to

four hymen sectors (Figure 21).

Malaysian distribution New records for Bintulu and

Port Dickson (Figure 11).

Pseudo-nitzschia caciantha Lundholm, Moestrup et Hasle (Figures 22 – 25)

References Lundholm et al. (2003) , p. 806, figs. 5A – F;

Congestri et al. (2008) , p. 205, figs. 4A – B; Quijano-Scheg-

gia et al. (2010) , p. 401, figs. 2C – D.

Morphology The cells are lanceolate and slightly asym-

metrical, with rounded apices (Figures 22 and 23). Each

stria consists of one row of poroids, with four to five

poroids in 1 μ m (Figure 24). The poroids are round and the

hymenes are divided into two to seven sectors, but mainly

four to five (Figure 25). Some poroids (7 – 14%) have central

sectors.

Malaysian distribution New records for Johore Bharu,

Santubong, Bintulu, Kudat, and Pulau Banggi (Figure 11).

Pseudo-nitzschia calliantha Lundholm, Moestrup et Hasle (Figures 26 – 28)

References Lundholm et al. (2003) , p. 801, figs. 2A – G;

Larsen and Nguyen (2004) , p. 32, pl. 4, fig. 6; Caroppo

et al. (2005) , p. 765, figs. 3A – C; Spatharis et al. (2007) , p.

813, figs. 2A – D; Quijano-Scheggia et al. (2008) , p. 350, figs.

2B, E and G; Quijano-Scheggia et al. (2010) , p. 402, figs.

2E, F, and P; Churro et al. (2009) , p. 44, figs. 17 – 18.

Morphology The cells are linear with rounded apices.

Each stria contains one row of poroids, with four to six


(22 – 25) P. caciantha . (22) Lanceolate valve in valve view. (23) Valve apex. (24) Mid-valve showing a central interspace (arrowhead), striae,

and poroids. (25) Poroids with hymenate structure. (26 – 28) P. calliantha . (26) Details of striae and poroids. (27) Mid-valve showing a central

interspace (arrowhead). (28) Poroids with four to ten hymen sectors. (29 – 33) P. mannii. (29) Linear valve in valve view. (30) Valve apex. (31)

Mid-valve showing central part of valve, with a central interspace (arrowhead). (32) Poroids with mainly four to five hymen sectors. (33)

Valve center showing a central interspace (arrowhead). (34 – 38) P. circumpora. (34) Lanceolate valve in valve view. (35) Valve apex. (36)

Mid-valve showing a central interspace (arrowhead). (37) Poroid structure. (38) Close up of poroid. Scale bars: 10 μ m (Figures 22, 29, and

34), 2 μ m (Figures 26, 30, and 31), 1.0 μ m (Figure 36), 0.5 μ m (Figures 23, 24, 27, 33, 35, and 37), 0.2 μ m (Figures 25, 28, 32), and 0.1 μ m

(Figure 38).




poroids in 1 μ m (Figure 26). The flower-like poroid pattern

consists of 5 – 12 hymen sectors, and some poroids (20 – 30%)

possess perforated central sectors (Figures 26 and 28).

Malaysian distribution Bintulu, Pulau Banggi, and

Semporna in the Malaysian Borneo, and Port Dickson in

the Strait of Malacca (Figure 11).

Pseudo-nitzschia mannii Amato et Montresor (Figures 29 – 33)

References Amato and Montresor (2008) , p. 490, figs. 1 – 5;

Moschandreou and Nikolaidis (2010) , p. 164, figs. 23 – 26.

Morphology The cells are linear and symmetrical

(Figure 29), and the apices are rounded (Figure 30).

Each stria contains one row of poroids, with four to six

poroids in 1 μ m (Figure 31). The poroids are rounded to

square-rounded and have two to seven perforated sectors

(Figures 32 and 33).


Pulau Banggi in Borneo, Port Dickson, and Teluk Batik in

the Strait of Malacca (Figure 11).

Pseudo-nitzschia circumpora Lim, Leaw et Lim (Figures 34 – 38)

Reference Lim et al. (2012b) , p. 1239, figs. 7A – H.

Morphology In valve view, the cells are lanceolate

and asymmetrical with rounded apices (Figures 34 and

35). Each stria consists of one row of poroids, with one

to three poroids in 1 μ m (Figure 36). The poroids are

rounded or rectangular, and the hymenes have more than

seven sectors (Figure 37). The small sectors are regularly

arranged around the poroid margin (Figure 38).

Malaysian distribution Bintulu and Semporna in Borneo,

and Port Dickson in the Strait of Malacca (Figure 11).

Pseudo-nitzschia sinica Qi, Ju et Lei (Figures 39 – 42)

References Priisholm et al. (2002) , p. 167, figs. 52 – 57; Li

et al. (2010b) , p. 305, figs. 30 – 32.

Morphology The cells are highly silicified, linear, and

symmetrical (Figure 39), with short, tapered, and rounded


(39 – 42) P. sinica . (39) Linear valve in valve view. (40) Valve apex. (41) Mid-valve showing a central interspace (arrowhead). (42) Poroid struc-

ture. (43 – 47). P. inflatula . (43) Lanceolate valves in valve view. (44) Valve apex. (45) Mid-valve showing a central interspace (arrowhead).

(46 and 47) Striation showing one to two rows of poroids. Scale bar: 10 μ m (Figures 39 and 43), 2 μ m (Figures 40 and 45), 1 μ m (Figure 41),

0.5 μ m (Figures 44 and 46), and 0.2 μ m (Figures 41 and 47).




apices (Figure 40). Each stria contains one to two rows of

rounded poroids (Figure 42), with one to two poroids in

1 μ m (Figure 41). The hymen is most often divided into two

sectors (two to four sectors) (Figures 41 and 42).

Malaysian distribution New record and found rarely in

Pulau Banggi (Figure 11).

Pseudo-nitzschia inflatula (Hasle) Hasle (Figures 43 – 47)

References Priisholm et al. (2002) , p. 167, figs. 48 – 51;

Larsen and Nguyen (2004) , p. 38, pl. 6, figs. 1 – 3; Congestri

et al. (2008) , p. 202, figs. 2G – I.

Morphology In valve view, the cells are clearly lan-

ceolate, inflated in the middle part and at both ends of

valve, asymmetrical, and have pointed apices (Figures 43

and 44). Each stria consists of one row of small poroids

(Figures 45 and 46), with five to seven poroids in 1 μ m

(Figures 45 and 46). The hymen is divided into two sectors

(Figure 47).

Malaysian distribution New records for Bintulu, Sem-

porna, Johore Bharu, and Port Dickson (Figure 11).

Pseudo-nitzschia delicatissima (Cleve) Heiden/ P. arenysensis Quijano-Scheggia, Garc é s et Lundholm (Figures 48 – 51)

References Lundholm et al. (2006) , p. 467, figs. 1A – G;

Congestri et al. (2008) , p. 203, figs. 3A – E; Kaczmarska

et al. (2008) , p. 767, figs. 2 – 7; Quijano-Scheggia et al.

(2008) , p. 350, figs. 2C, F and H; Quijano-Scheggia et al.

(2009) , p. 498, figs. 8 – 14; Quijano-Scheggia et al. (2010) ,


(48 – 51) P. delicatissima/P. arenysensis . (48) Lanceolate valve in valve view. (49) Valve apex. (50) Mid-valve showing a central interspace

(arrowhead). (51) Striation showing two rows of poroids. (52 – 55) P. decipiens . (52) Lanceolate valve in valve view. (53) Valve apex. (54)

Mid-valve showing a central interspace (arrowhead). (55) Striation showing two rows of poroids. (56 – 58) P. dolorosa . (56) Lanceolate valve

in valve view. (57) Mid-valve showing a central interspace (arrowhead) and striation with one to two rows of poroids. (58) Poroid structure.

(59 – 62) P. cf. lineola . (59) Lanceolate valve in valve view. (60) Valve apex. (61) Part of valve showing stria structure. (62) Detail of striae and

poroid structure. Scale bar: 10 μ m (Figures 52, 56, and 59), 5 μ m (Figures 48 and 49), 2 μ m (Figures 53, 54, and 61), 1 μ m (Figure 57), 0.5 μ m

(Figures 50, 60, and 62), and 0.2 μ m (Figures 51, 55, and 58).




p. 402, figs. 2G – H; Churro et al. (2009) , p. 44, figs. 21 – 22;

Klein et al. (2010) , p. 221, figs. 4C and D; Leandro et al.

(2010) , p. 293, fig. 3C; Yap-Dejeto et al. (2010) , p. 8, figs.

16A – C; Stonik et al. (2011) , p. 126, figs. 18 – 21.

Morphology The cells are linear to lanceolate and sym-

metrical in valve view (Figure 48), with rounded apices

(Figure 49). The striae consistently contain two rows of

poroids, with 10 – 12 poroids in 1 μ m (Figure 50). Each

small poroid has an irregular hexagonal shape. The perfo-

rations of the hymen are arranged in a hexagonal pattern

(Figure 51).

Malaysian distribution Bintulu, Pulau Banggi, Sem-

porna, and Port Dickson (Figure 11).

Pseudo-nitzschia decipiens Lundholm et Moestrup (Figures 52 – 55)

Reference Lundholm et al. (2006) , p. 472, figs. 3A – G.

Morphology In valve view, the cells are lanceolate and

asymmetrical, with rounded apices (Figures 52 and 53).

The striae contain two rows of poroids, with a high density

of poroids (11 – 13) in 1 μ m (Figure 54). The poroids have a

trapezoid or hexagonal shape (Figure 55).

Malaysian distribution New records for Bintulu, Kota

Kinabalu, Pulau Banggi, and Semporna in Malaysian

Borneo waters, and Johore Bharu in the Tebrau Strait

(Figure 11).

Pseudo-nitzschia dolorosa Lundholm et Moestrup (Figures 56 – 58)

References Lundholm et al. (2006) , p. 470, figs. 2A – I;

Marchetti et al. (2008) , p. 653, figs. 1A – D; Churro et al. (2009) ,

p. 46, figs. 23 – 24; Lim et al. (2012b) , p. 1238, figs. 4A – C.

Morphology The valves are lanceolate and asymmetri-

cal, with rounded apices (Figure 56). Each stria contains

one to two rows of poroids, with six to seven poroids in

1 μ m (Figure 57). The poroids are unevenly shaped, from

kidney-shaped to hexagonal (Figure 58).

Malaysian distribution Kuala Penyu, Pulau Banggi,

Semporna, and Port Dickson (Figure 11).

Pseudo-nitzschia cf. lineola (Cleve) Hasle (Figures 59 – 62)

References Hern á ndez-Becerril (1998) , p. 80, figs. 12 – 16;

Almandoz et al. (2008) , p. 439, figs. 7E – H; Lundholm et al.

(2012) , p. 446, figs. 3A – K.

Morphology The valves are lanceolate and asymmetri-

cal, with rounded apices (Figures 59 and 60). Each stria

contains one to two rows of poroids, with one to two

poroids in 1 μ m (Figures 61 and 62). The poroids are irreg-

ularly rounded to kidney shaped (Figure 62).

Malaysian distribution New record for Bintulu

(Figure 11).

Pseudo-nitzschia americana (Hasle) Fryxell (Figures 63 – 65)

References Hern á ndez-Becerril (1998) , p. 79, figs. 2 – 4;

Lundholm et al. (2002) , p. 483, figs. 1 – 20; Orlova and

Shevchenko (2002) , p. 336, figs. 1A – F; Churro et al. (2009) ,

p. 42, figs. 5 – 11; Klein et al. (2010) , p. 221, fig. 4A; Li et al.

(2010a) , p. 852, figs. 2A – G.

Morphology In valve view, the cells are slightly linear,

rectangular, and symmetrical (Figure 63), with broadly

rounded apices (Figure 64). Each stria contains two rows

of poroids, with 9 – 11 poroids in 1 μ m (Figure 63). The

shape of poroids is irregular, from round to hexagonal

(Figure 63). The valvocopula contains 45 – 48 striae in 10

μ m, each being two poroids wide and three to four poroids

high (Figure 65).

Malaysian distribution New record for Bintulu, Johore

Bharu, Port Dickson, and Teluk Batik (Figure 11).

Pseudo-nitzschia linea Lundholm, Hasle et Fryxell (Figures 66 – 69)

References Lundholm et al. (2002) , p. 487, figs. 46 – 58;

Quijano-Scheggia et al. (2010) , p. 402, figs. 3A – B;

Yap-Dejeto et al. (2010) , p. 3, figs. 12A – C.

Morphology In valve view, the cells are linear, rectan-

gular, and symmetrical (Figure 66), with broadly rounded

apices (Figure 67). Each stria contains two rows of poroids,

with 9 – 12 poroids in 1 μ m (Figure 68). The valvocopula

contains 45 – 48 striae in 10 μ m; each being two poroids

wide and four to five poroids high (Figure 69).


Kuala Penyu (Figure 11).

Pseudo-nitzschia brasiliana Lundholm, Hasle et Fryxell (Figures 70 – 73)

References Lundholm et al. (2002) , p. 484, figs. 21 –

45; Larsen and Nguyen (2004) , p. 30, pl. 3, figs. 3 – 4;

Quijano-Scheggia et al. (2008) , p. 350, figs. 2A and D;




Quijano-Scheggia et al. (2011) , p.193, figs. 5A and D; Li

et al. (2010a) , p. 852, figs. 2H – N; Yap-Dejeto et al. (2010) ,

p. 5, figs. 13A – C.

Morphology In valve view, the cells are lanceolate, with

broadly rounded apices (Figures 70 and 71). Each stria

consists of two rows, rarely three, with 8 – 10 poroids in 1

μ m (Figures 72 and 73).

Malaysian distribution Reported from all sampling

locations (Figure 11).

Pseudo-nitzschia micropora Priisholm, Moestrup et Lundholm (Figures 74 – 77)

References Priisholm et al. (2002) , p. 155, figs. 1 – 14, 20 –

25; Quijano-Scheggia et al. (2011) , p. 193, figs. 5A and D;

Lim et al. (2012b) , p. 1238, figs. 5A – E.

Morphology The cells are lanceolate in valve view,

tapering from the middle to the apices (Figures 74 and 75).

Each stria contains two rows of poroids, with 11 – 12 poroids


(63 – 65) P. americana . (63) Linear to lanceolate valve in valve view. (64) Valve apex broadly rounded. (65) Valvocopula structure. (66 – 69)

P. linea . (66) Linear valve in valve view. (67) Valve apex. (68) Poroid and stria structure. (69) Valvocopula with pattern of 2 × 4 – 5. (70 – 73)

P. brasiliana . (70) Lanceolate valve in valve view. (71) Valve apex. (72) Striation showing two rows of poroids. (73) Striation with incomplete

third row of poroids. Scale bar: 5 μ m (Figure 70), 2 μ m (Figures 63 and 66), 0.5 μ m (Figures 64, 67, and 71), and 0.2 μ m (Figures 65, 68, 69,

72, and 73).





(74 – 77) P. micropora . (74) Lanceolate valve in valve view. (75) Valve apex. (76) Striation showing two rows of poroids. (77) Stria and poroid

structure. (78 – 81) P. multistriata . (78) Lanceolate valve in valve view. (79) Valve apex. (80) Central part of valve; note the absence of a

central interspace. (81) Striae with uneven row arrangement of poroids. (82 – 85) P. turgidula. (82) Lanceolate valve in valve view. (83) Valve

apex. (84) Mid-valve showing a central interspace (arrowhead). (85) Striation showing two rows of poroids. Scale bar: 10 μ m (Figures 78 and

82), 5 μ m (Figure 74), 2 μ m (Figures 79, 80, and 83), 0.5 μ m (Figures 75, 76, and 84), and 0.2 μ m (Figures 77, 81, and 85).

in 1 μ m (Figure 76). The poroids have perforations in a

trapezoid to hexagonal pattern (Figure 77).

Malaysian distribution Port Dickson (Figure 11) and

Kota Kinabalu ( Lim et al. 2012b ).

Pseudo-nitzschia multistriata (Takano) Takano (Figures 78 – 81)

References Rhodes et al. (2000) , p. 464, figs. 1A – F;

Orsini et al. (2002) , p. 250, figs. 1 – 13; Larsen and Nguyen

(2004) , p. 41, pl. 7, figs. 1 – 4; Congestri et al. (2008) , p. 200,

figs. 1A – B; Quijano-Scheggia et al. (2008) , p. 350, figs.

3B and E – F; Quijano-Scheggia et al. (2010) , p. 402, figs.

3C and D; Churro et al. (2009) , p. 46, figs. 29 – 33; D ’ Alelio

et al. (2009) , p. 10, fig. 1; Moschandreou and Nikolaidis

(2010) , p. 164, figs. 29 – 32; Yap-Dejeto et al. (2010) , p. 9,

figs. 19A – C; Stonik et al. (2011) , p. 128, figs. 36 – 39.


(Figure 78). Each stria consists of two to three rows of

poroids, with 11 – 12 poroids in 1 μ m. The striae are distri-

buted unevenly (Figures 80 and 81).




Malaysian distribution Bintulu in Sarawak Borneo and

Port Dickson in the Strait of Malacca (Figure 11).

Pseudo-nitzschia turgidula (Hustedt) Hasle (Figures 82 – 85)

References Rhodes (1998) , p. 540, fig. 1; Almandoz et al.

(2008) , p. 439, figs. 7I – L.


(Figure 82). Each stria contains two rows of extremely

small round poroids, with seven to eight poroids in 1 μ m

(Figures 84 and 85).


Semporna (Figure 11).

Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle (Figures 86 – 88)

References Hern á ndez-Becerril (1998) , p. 82, figs. 17 –

20; Cho et al. (2001) , p. 209, fig. 1A; Li et al. (2005) , p. 419,

figs. 1A – H; Fehling et al. (2006) , p. 98, fig. 7D; Quijano-

Scheggia et al. (2008) , p. 350, figs. 3C and G; Churro et al.

(2009) , p. 48, figs. 47 – 51; Moschandreou and Nikolaidis

(2010) , p. 165, figs. 38 – 41; Stonik et al. (2011) , p. 130, figs.

45 – 48.

Morphology In valve view, the cells are linear to lanceo-

late (Figure 86), with pointed apices (Figure 87). Each stria

has two rows of round poroids, with three to four poroids

in 1 μ m (Figure 88). Some striae contain an incomplete

third row of poroids (Figure 88).

Malaysian distribution Reported from all sampling

locations (Figure 11).

Pseudo-nitzschia subfraudulenta (Hasle) Hasle (Figures 89 – 94)

References Hasle et al. (1996) , p. 145, figs. 62 – 64; Cho

et al. (2001) , p. 209, fig. 1F; Larsen and Nguyen (2004) , p.

46, pl. 9, figs. 6 – 8; Moschandreou and Nikolaidis (2010) ,

p. 165, figs. 42 – 45.

Morphology In valve view, the cells are linear and sym-

metrical (Figure 89), with pointed apices (Figure 90). Each

stria contains two rows of poroids, with five poroids in

1 μ m (Figure 91). Sometimes the striae near the apex show

only one row of poroids. The poroid hymenes consist of

four to five sectors (Figures 92 and 93). Each stria of the

valvocopula is two poroids wide and five to seven poroids

high (Figure 94).

Malaysian distribution New records for Bintulu,

Gerigat, Port Dickson, Teluk Batik, and Pulau Banggi

(Figure 11).

Pseudo-nitzschia sp. Port Dickson (Figures 95 – 100)

Morphology The cells are linear and symmetrical, with

an apical axis of 64 – 114 μ m and a transapical axis of

2.0 – 2.6 μ m (Figure 95). The valve margins are gradually

tapered toward both rounded apices (Figure 96). A central

interspace is present and occupies three to four striae in

length (Figure 98). There are 11 – 15 fibulae in 10 μ m and

21 – 29 striae in 10 μ m (Figure 97). Each stria contains one

row of poroids divided by two to four sectors, with three

to five poroids in 1 μ m (Figures 98 and 99). The mantle

is two poroids high (Figure 97). The valvocopula consists

of one to two rows of perforated sectors, with 35 striae in

10 μ m. Each band stria is one to two poroids wide and

three poroids high (Figure 100).

Malaysian distribution New records for Bintulu, Pulau

Banggi, Semporna, Johore Bharu, Port Dickson, and Teluk

Batik (Figure 11).

Morphological phylogenetic inference

The maximum parsimony analysis on the morphomet-

ric data of Pseudo-nitzschia resulted in 26,200 most par-

simonious trees with a length of 98 steps (consistency

index = 0.541, RI = 0.792, rescaled consistency = 0.428), with

the majority-rule tree shown in Figure 101 . The analysis of

RI reweighted characters did not result in a resolved clad-

ogram, and was thus not included in the study. Outgroups

were consistently branched off from the Pseudo-nitzschia

clades and they were pruned for simplification.

Two monophyletic clades were formed, i.e., clade

I and clade II. The grouping was supported by the char-

acter state distribution of poroid hymenes divided into

sectors (character C), with all taxa in clade I possessing

poroids whose hymenes were divided into sectors, while

clade II taxa had poroids without sectors. Within clade I, a

subclade (S1) was formed with 14 taxa mostly recognized

in the P. pseudodelicatissima complex, which was desig-

nated as the pseudodelicatissima group. The subclade S1

was not well resolved, however, because the polytomic

position of eight taxa ( P. caciantha , P . sp. Port Dickson,

P. roundii Hern á ndez-Becerril, P. hasleana , P. fryxelliana ,

P . circumpora , P. cuspidata , and P. calliantha ) was formed

in the subclade. The other two taxa [ P. subfraudulenta and





(86 – 88) P. pungens . (86) Lanceolate valve in valve view. (87) Valve apex. (88) Striation showing two rows of poroids with an incomplete

insertion of the third row of poroids. (89 – 94) P. subfraudulenta . (89) Lanceolate valve in valve view. (90) Valve apex. (91) Mid-valve showing

a central interspace (arrowhead). (92) Striation showing two rows of poroids with hymen sectors. (93) Teratological specimen showing one

row of poroids per stria. (94) Valvocopula. Scale bar: 10 μ m (Figures 86 and 89), 5 μ m (Figure 90), 2 μ m (Figures 87, 88, 91, and 94), and 0.5

μ m (Figures 92 and 93).

P. fraudulenta (Cleve) Hasle] remained as sisters on outer

branches. There was no clear grouping in clade II. The

clade comprised 25 taxa from seriata group sensu Hasle

and delicatissima complex sensu latto . Within clade II,

subclade S2 formed a group containing species in deli-catissima and americana complexes.

Discussion

Species richness and geographical distribution

This survey of the genus Pseudo-nitzschia in Malaysian

coastal waters revealed high species richness, with 22

taxa reported thus far. Ten potentially toxic Pseudo-nitzschia species ( P. brasiliana , P. caciantha , P. calliantha ,

P. cuspidata , P. delicatissima , P. micropora , P. multistriata ,

P. pseudodelicatissima , P. pungens , and P. turgidula ) were

distributed throughout the various sampling sites along

the Malaysian coasts. Unfortunately, toxin analyses on

field samples were not conducted due to the low cell

abundances of Pseudo-nitzschia species. Toxin analyses

based on culture samples, however, showed undetectable

DA concentrations among strains of P. pungens , P. brasili-ana ( Lim et al. 2010 ), and P. circumpora ( Lim et al. 2012b ).

Among the sampling sites, four were identified as

potential ASP hotspots (Port Dickson, Bintulu, Pulau

Banggi, and Semporna), with at least five potentially toxic

Pseudo-nitzschia species reported. Nonetheless, species

blooms have hardly been recorded in Malaysian waters

(Su 2011). In the previous 3-year survey, with > 50 sampling

days, from 2007 to 2010 at Santubong and Samariang,

Sarawak Borneo, only a small amount of Pseudo-nitzschia

cells was observed (0 – 250 cells l -1 ), except on one occasion




in October 2007, where a cell density of 30 × 10 3 cells l -1 was

recorded (Su 2011). The relative abundance of Pseudo-nitzschia species was very low in the phytoplankton

assemblages, with 1.3 – 14% at Port Dickson, 0.5 – 20% at

Bintulu, 8.6% at Pulau Banggi, and 8.1% at Semporna.

Pseudo-nitzschia brasiliana and P. pungens were

present at all locations in Malaysian waters, which is not a

surprise knowing that they are reported as cosmopolitan

species ( Hasle 2002 , Lundholm et al. 2002 , Lundholm and

Moestrup 2006 ). Pseudo-nitzschia brasiliana was recently

reported as a toxic species by Sahraoui et al. (2011) and it

has been frequently reported in coastal waters of South-

east Asia ( Lundholm et al. 2002 , Skov et al. 2004 , Li et al.

2010b ), and more recently from China ( Wang et al. 2012 ).

Isolates of P. brasiliana from Sarawak, however, were

found to be non-toxic ( Lim et al. 2010 ).

Pseudo-nitzschia caciantha was one of the species

with a wide distribution, particularly in the coastal

region of Malaysian Borneo. The species was first disco-

vered from Mexican waters ( Lundholm et al. 2003 ), and

subsequently, it was reported from wide geographical

regions ( Lelong et al. 2012 ). It was known as a non-toxic

species ( Lundholm et al. 2003 ); surprisingly, however,

strains from Vietnam were reported as toxic and could

be responsible for DA in the bivalve Spondylus versicolor

Schreibers in Nha Phu Bay ( Dao et al. 2009b , Fukuyo et al.

2011 ). Another species, P. pseudodelicatissima , which was

found only in northern Borneo, was recently reported to

cause red tide incidents in Hong Kong. The species was

proven to be toxic (Hong Kong Agriculture, Fisheries

and Conservation Department 2008 ). Recently, strains of

P. pseudodelicatissima from the Mediterranean Sea were

also reported to be toxic ( Moschandreou et al. 2010 ).

Several species of Pseudo-nitzschia are confined to

specific latitudinal ranges and are thus classified as non-

cosmopolitan ( Lelong et al. 2012 ). Surprisingly, our data

showed that some species, previously reported as being

cold-water or temperate species, were present in the tropi-

cal Malaysian coastal waters, such as P. turgidula , P. decip-iens , and P. lineola ( Almandoz et al. 2008 , Marchetti et al.

Figures 95 – 100 Pseudo-nitzschia sp. Port Dickson, TEM.

(95) Lanceolate valve in valve view. (96) Valve apex. (97) Mid-valve showing a central interspace (arrowhead). (98) Mid-valve showing a

central interspace (arrowhead) and one row of poroids per stria. (99) Poroid structure showing hymen sectors. (100) Valvocopula. Scale bar:

20 μ m (Figure 95), 2 μ m (Figures 96 and 97), 0.5 μ m (Figures 98 and 100), and 0.2 μ m (Figure 99).




2008 , Lundholm et al. 2012 ). The history of occurrence for

these species in Malaysian waters is unknown. Although

the species were found mainly in major port areas (e.g.,

Bintulu, Kota Kinabalu, and Johore Bharu), it is premature

to speculate that they were dispersed or introduced via

ships ’ ballast waters. Attempts to obtain cultures of these

species are ongoing to elucidate the origin and dispersal

mechanisms of these species.

Pseudo-nitzschia morphology

The taxonomy of Pseudo-nitzschia , like most other diatoms,

is inferred from the frustule morphology ( Hasle 1994 ).

However, the discovery of increasing numbers of cryptic/

pseudo-cryptic species has made species identification of

Pseudo-nitzschia ambiguous and difficult. In the absence

of genetic information, the identification of species can

be achieved by making subgroups based on valve width

( Hasle et al. 1996 , Hasle and Syvertsen 1997 ) and species-

complex characteristics (e.g., Lundholm et al. 2002, 2003 ,

2012, Amato and Montresor 2008 , Quijano-Scheggia et al.

2009 ). However, several features used in the grouping

are sometimes dubious, uninformative, or too subtle to

be detected, leading to the inability to recognize the true

pseudo-cryptic diversity in the species; this can only be

resolved by further corroboration using genetic informa-

tion ( Amato and Montresor 2008 , Lundholm et al. 2012 ).

In this study, we propose a new taxonomic grouping

of Pseudo-nitzschia species based on a morphological

phylogenetic analysis to assist in obtaining more accurate

species recognition of Pseudo-nitzschia in field studies.

Grouping of species before their detailed morphological

comparisons (often this requires TEM) would enhance

precision in species identification, result in fewer errors,

and require less time, especially when genetic informa-

tion is not available. Although species identification by

molecular tools will undoubtedly become feasible in the

future, particularly with the development of quantita-

tive polymerase chain reaction assay, automated riboso-

mal intergenic spacer analysis (ARISA), and microarrays

(reviews in Kudela et al. 2010 , Medlin and Kooistra 2010 ,

Trainer et al. 2012 ).

In this analysis, the genus was divided into two clades,

I and II (Figure 101). This classification is generally con-

gruent with several groupings of complexes, for instance

the P . pseudodelicatissima , americana , and delicatissima

complexes, notwithstanding the slight differences from

the previous classification ( seriata and delicatissima

group sensu Hasle; Hasle et al. 1996 ), although characters

used here were based mainly on those previously defined.

It is noteworthy that poroid morphology may potentially

be of significant taxonomic diagnostic value in classifica-

tion, as it is no doubt related to species circumscription.

The split of clade I and II was supported by the poroid

structure, wherein all taxa in clade I possess poroids with

hymenes divided into sectors, in contrast to those in clade

II. In this study, two groups were designated in clade I,

the pseudodelicatissima group, including an inflatula sub-

group, and the fraudulenta group. In comparison, there is

only the delicatissima group in clade II, which includes

the americana subgroup.

Clade I: pseudodelicatissima group The group com-

prises species that possess poroid hymenes divided into

sectors and striae with one row of poroids (Figure 101).

These main characters supported the monophyly of this

group and distinguished it from its basal sister taxa (the

fraudulenta group). Taxa in this group mainly belong to

the pseudodelicatissima complex, which comprises the

originally defined species, Pseudo-nitzschia pseudodeli-catissima , P. cuspidata , P. caciantha and P. calliantha , and

P. mannii (after Lundholm et al. 2012 ), and three recently

described species, P. hasleana , P. fryxelliana ( Lundholm

et al. 2012 ), and P. circumpora ( Lim et al. 2012b ). All

species in this complex were clustered in the group owing

to their similarity in the poroid morphology (character C,

poroid structure), as the poroids have hymen sectors. The

number of hymen sectors in the group was used to distin-

guish among the closely related species.

Taxonomic confusion between P. pseudodelicatissima

and P. cuspidata has been demonstrated in many studies

(e.g., Lundholm et al. 2012 ). One of the familiar features

used to differentiate the two species is the valve shape

( Lundholm et al. 2003 ), which can sometimes be dubious

and misleading. As demonstrated in our phylogenetic

analysis, valve width and shape were homoplastic, thus

uninformative (Figure 101).

The morphology of P. caciantha reported in this study

generally agrees with the type specimen of Lundholm

et al. (2003) , with the exception of a smaller range of

valve width (2.5 – 3.0 μ m; Table 1). Narrow valves were also

reported by Congestri et al. (2008) and Quijano-Scheggia

et al. (2010) . It differs from other species in the group

by having hymenes divided by 2 – 7 sectors (mainly 4 – 5

sectors) as opposed to P. pseudodelicatissma (2 sectors),

P. cuspidata (2 – 4 sectors), P . fryxelliana (1 – 3 sectors),

P. hasleana and P. sinica (2 – 6 sectors), P. calliantha (3 – 12

sectors), and P. circumpora ( > 7 sectors). Pseudo-nitzschia caciantha shares a similar range of sectors with P. mannii ; however, they differ from each other by a lanceolate valve

shape in P . caciantha and higher densities of fibulae and

band striae in P. mannii .




Cells of P. calliantha reported from the Malaysian

coastal waters have a slightly greater valve width than the

type species ( Lundholm et al. 2003 ), but they are closer

to those described from southern Brazil ( Fernandes and

Brandini 2010 ), the Mediterranean Sea ( Caroppo et al.

2005 , Quijano-Scheggia et al. 2008 , 2010), and Vietnam-

ese waters ( Larsen and Nguyen 2004 ). It is very similar

to P. circumpora ( Lim et al. 2012b ). The only difference

is that the poroids in P. calliantha have a central sector

and 3 – 12 hymen sectors, whereas in P. circumpora the

hymenes are divided into more than seven (some-

times > 20) sectors (Figures 34 – 37). Pseudo-nitzschia cal-liantha differs by its number of sectors from P. cuspidata

(two to four sectors), and P. mannii and P. caciantha (two

to seven sectors).

Pseudo-nitzschia mannii has been reported from the

western North Pacific, Greek coastal waters, and Daya

Bay, China ( Amato and Montresor 2008 , Li et al. 2010a ,

Moschandreou and Nikolaidis 2010 , Stonik et al. 2011 ). The

Malaysian P. mannii is generally similar to the type species

and those described previously, except that it has a lower

density of striae with 28 – 36 in 10 μ m (Table 1). The species

can easily be distinguished from P. pseudodelicatissima / P. cuspidata by the number of hymen sectors, whereas it

differs from P. calliantha by having poroids divided into

two to seven (mainly four) sectors. The linear valve shape

is used to distinguish it from P. caciantha , which has lan-

ceolate valves.

Pseudo-nitzschia sinica was only reported from Pulau

Banggi, northern Borneo. The distribution history of

this species is scanty, with only two limited distribution

records known thus far in Vietnam ( Larsen and Nguyen

2004 ) and China ( Qi et al. 1996 , Li et al. 2010a ). Cells of

P. sinica have lower densities of fibulae and striae com-

pared with P. pseudodelicatissima / P. cuspidata and

P. inflatula (Table 1).

Pseudo-nitzschia hasleana and P. circumpora are

new species that were recently delineated from the

P. subfraudulentaP. fraudulentaP. cacianthaP. sp. Port DicksonP. roundiiP. hasleanaP. fryxellianaP. circumporaP. cuspidataP. callianthaP. manniiP. sinicaP. pseudodelicatissimaP. inflatulaP. graniiP. subcurvataP. turgidulaP. galaxiaeP. turgiduloidesP. subpacificaP. prolongatoidesP. heimiiP. seriataP. obtusaP. antarcticaP. lineolaP. pungiformisP. pungens var. pungensP. pungens var. cingulataP. pungens var. aveirensisP. australisP. multiseriesP. dolorosaP. decipiensP. arenysensisP. delicatissimaP. microporaP. multistriataP. americanaP. brasilianaP. linea

Fraudulenta-groupClade I

Pse

udod

elic

atis

sim

a-gr

oup

inflatula -subgroup

S1

Del

icat

issi

ma-

grou

p

Americana-subgroup

D T I E C

C. Poroid structureWith hymen sectorsWithout hymen sectors

D. Central interspacePresentAbsent

T. Transapical axis<3>3

I. Poroids in 1 μm1–23–67–1011–13Scatter

E. Rows of poroids1

3–45–6Scatter

2

Clade II

S2

Figure 101 Unweighted cladogram of majority-rule tree based on Pseudo-nitzschia morphological characters and biometric data.

Columns to the right of tree show character mapping, with the numerical code converted into a color code. Code key for the character states

is shown in Supplement 2. Species data are compiled from the literature as in Supplement 3.




pseudodelicatissima complex ( Lim et al. 2012b , Lundholm

et al. 2012 ). Cells of P. hasleana reported in this study

have a slightly smaller transapical axis and a higher

stria density, while other morphological features are in

agreement with the original diagnosis ( Lundholm et al.

2012 ). Specimens of P. circumpora reported here have a

higher density of fibulae than that described by Lim et al.

(2012b) . Pseudo-nitzschia circumpora is morphologically

closer to P. calliantha . It has one row of poroids per stria,

which is similar to P. calliantha . The main difference is the

numbers of hymen sectors and the absence of a central

sector in P. circumpora (Table 1).

The new morphotype, Pseudo-nitzschia sp. Port

Dickson, was found in the coastal waters of Malaysia.

This morphotype is common, as it was found at most

of the sampling sites. The morphotype is morpho-

logically close to species in the pseudodelicatissima

complex. The distinctive features of this morphotype are

its much lower densities of fibulae and striae (Table 1,

Figure 102 B,C). Among the species in the pseudodelicatis-sima complex, the morphotype resembles P. hasleana and

P . pseudodelicatissima / P. cuspidata in sharing a similar

number of poroid sectors (Table 1). However, it can be

distinguished from both species by its mantle structure of

two poroids high (Figures 97 – 98), and lower densities of

striae and band striae (Table 1, Figure 102). The identity of

this new morphotype can be confirmed only after cells are

isolated and maintained in culture, and molecular infor-

mation is gathered; this work is ongoing.

The inflatula subgroup This subgroup consists of

three taxa, Pseudo-nitzschia inflatula , P. granii , and P. sub-curvata , supported by the characters for the valve shape

and poroid structure. The valves of P . inflatula are inflated

in the middle and at the apex, which clearly differentiates

it from the other species. The presence of a central inter-

space delineates it from P. granii and P. subcurvata . Very

small poroids make P. inflatula unique and distinct from

P. calliantha , P. caciantha , P. mannii , P. cuspidata , and P. pseudodelicatissima ( Priisholm et al. 2002 , Larsen and

Nguyen 2004 , Congestri et al. 2008 ). The morphology of

P. inflatula from the Malaysian material agrees with that of

cells from Vietnam ( Larsen and Nguyen 2004 ), but differs

from cells from the Andaman Sea ( Priisholm et al. 2002 )

by having a slightly lower density of fibulae and striae

(Table 1).

Clade I: fraudulenta group In our cladistic analy-

sis, Pseudo-nitzschia fraudulenta and P. subfraudulenta

that branched off from the subclade S1 were assigned to

the fraudulenta group. These basal taxa of S1 possessed

more than one row of poroids in their striae (character L).

4.0A B C

D E F

3.5

3.0

2.5

Valu

e w

ith (μ

m)

2.0

1.0

1.5

10

8

6

Por

oids

(1μm

)

4

0cac cus cir pde man has fry PDcal cac cus

Pseudo-nitzschia species

cir pde man has fry PDcal cac cus cir pde man has fry PDcal

2

25

20

15

Sec

tors

in p

orio

ds

10

0

5

30

35

20

Fibu

lae

in10

μm

15

10

50

40

30

20

Stri

ae in

10 μ

m

60

50

40

30

10

20

0

Ban

d st

riae

in10

μm

Figure 102 Box plots of the variation in (A) valve width, and the numbers of (B) fibulae, (C) striae, (D) poroids, (E) sectors in poroids, and (F)

striae on valvocopula for nine species of Pseudo-nitzschia .

Data are presented as minimum and maximum values (gray box), mean ( + ), median and standard error. cac, P . caciantha ; cal, P . calliantha ;

cus, P. cuspidata ; cir, P . circumpora ; pde, P. pseudodelicatissma ; man, P . mannii ; has, P . hasleana ; fry, P. fryxelliana (data from Lundholm

et al. 2012); PD, P. sp. Port Dickson.




Pseudo-nitzschia subfraudulenta resembles P. fraudulenta ,

but differs by the densities of fibulae, striae, and band

striae (Table 1). The Malaysian P. subfraudulenta agreed

morphologically with previous reports ( Hasle et al. 1996 ,

Cho et al. 2001 , Larsen and Nguyen 2004 , Moschandreou

and Nikolaidis 2010 ).

Clade II: delicatissima group This group com-

prises nine species (Figure 101). The grouping is gener-

ally in agreement with previously described species in

the delicatissima complex ( Lundholm et al. 2006 , Qui-

jano-Scheggia et al. 2009 ) and in the americana complex

( Pseudo-nitzschia americana , P. brasiliana , and P. linea ;

Lundholm et al. 2002 ). Generally, the poroid structure is

without dividing sectors for the delicatissima group.

We failed to differentiate P. delicatissima from P. are-nysensis because of a lack of molecular data. The two

species are morphologically indistinguishable (thus term

cryptic), so that they can only be distinguished by genetic

differences ( Quijano-Scheggia et al. 2009 ). The Malaysian

specimens were assigned P. delicatissima/P . arenysensis .

This agrees morphologically with the original description

( Lundholm et al. 2006 ) and several subsequent studies

( Kaczmarska et al. 2008 , Churro et al. 2009 , Quijano-

Scheggia et al. 2010 , Stonik et al. 2011 ). They resembled

cells from the northeast Spanish coast ( Quijano-Scheggia

et al. 2008 ), by having a smaller transapical axis. Mor-

phologically, P. delicatissima / P . arenysensis resembles

P. micropora and P. decipiens . The presence of a central

interspace in P. delicatissima / P . arenysensis differentiates

it from P. micropora . When compared with P. decipiens , it

has a lower stria density (Table 1).

The morphology of the Malaysian specimens of

P. micropora agreed with the original diagnosis from

Thailand ( Priisholm et al. 2002 ). Pseudo-nitzschia micro-pora was placed in the delicatissima group based on the

poroid structure, which showed no sectors. It shares

morphological features, however, with P. delicatissima /

P. arenysensis and P. decipiens . The distinctive feature dis-

tinguishing it from the other species in the group is the

absence of a central interspace.

The morphology of P. decipiens in this study agrees

totally with that of the original description ( Lundholm

et al. 2006 ). It differs from its closest allied species,

P. delicatissima , by having a wider transapical axis, a

higher density of striae, and more striae on the cingu-

lar bands (Table 1). The fine perforations in the poroids

resemble those in P. micropora , but it can be distinguished

from the latter by the presence of a central interspace.

The morphology of the Malaysian P. dolorosa is in

agreement with the type species ( Lundholm et al. 2006 ),

except for its slightly shorter valve width (Table 1). The

y

x

y

x

y

xA

P. brasiliana P. americana P. linea0

0.5

1.0

1.5

x:y

B

Figure 103 (A) Drawing of cells of P. brasiliana , P. americana , and

P. linea in valve view showing the transapical axes in the apices ( x )

and the maximum widths ( y ). Scale bar: 5 μ m. (B) Comparison of x

and y ratio in the three species.

Malaysian specimens of P. multistriata generally agreed

morphologically with previous descriptions ( Rhodes

et al. 2000 , Orsini et al. 2002 , Hasle and Lundholm 2005 ,

Congestri et al. 2008 , Quijano-Scheggia et al. 2008 , 2010,

Churro et al. 2009 , D ’ Alelio et al. 2009 , Moschandreou

and Nikolaidis 2010 , Stonik et al. 2011 ), except for a larger

transapical axis (2.2 – 4.5 μ m). The distinctive feature of

this species is the uneven arrangement of the striation.

Its frustule morphology closely resembles P. delicatissima

and P. decipiens in the delicatissima complex, but can be

easily distinguished by the absence of a central interspace.

The americana subgroup The americana complex

comprises Pseudo-nitzschia americana , P. brasiliana , and

P. linea , as originally described by Lundholm et al. (2002) .

Their unique rectangular valves are distinctive among the

species of Pseudo-nitzschia . The three species, however,

are sometimes confused because of their broad valve




>3 μm width

>3 μm width

P. pungiformis

P. subpacifica

P. heimii

CIS+

fibulae< striae in 10 μm

Fibulae=striae in 10 μm

19–28 striae in 10 μm

4–8 poroids in 1 μm

7–10 poroids in 1 μm27–32 striae in 10 μm

1–2 rows

0 non consistent tiny poroids, no stria pattern in valvocopulaP. galaxiae

P. turgiduloides

18–22 fibulae and 30–37 striae in 10 μm,40–47 striae in the valvocopula

10–18 fibulae and 22031 striae in 10 μm,22–34 striae in the valvocopula

P. dolorosaP. lineola

P. delicatissima/ P. arenysensisP. decipiens

2 rows

P. prolongatoides

P. turgidula 29–33 striae in 10 μm with 11–13 poroids in 1 μm

19–28 striae in 10 μm with 7–9 poroids in 1 μm

2–3 rows

Small poroids with 7–10 poroids in 1 μm

Large poroids with 4–8 poroids in 1 μm

P. pungens var. pungensP. pungens var. aveirensisP. pungens var. cingulata

1/31/4 cell overlapping, valvocopula=3–4x3–5; 11–19 striae in 10 μm

1/71/9 cell overlapping, valvocopula=2x2–4; 28–32striae in 10 μmP. obtusa

P. australis2 rows

2(3) rows

Linear to lanceolate, 3–5 rows of poroids/stria

Lanceolate, 2–4 rows of poroids/stria

P. multiseries

P. seriata>3 rows

<3 μm width

<3 μm width

CIS-

2 rows

2–3 rows Lanceolate and sigmoid

Linear

>40 striae in 10 μmLinear tolanceolate

Striae >fibulae, valvocopula=2x3

Striae ≈ fibulae, valvocopula=1x1

P. multistriata

P. lineaP. americana

P. brasiliana< 40 striae in 10 μm

P. micropora

>40 striae in 10 μm

<40 striae in 10 μm

Key to Pseudo-nitzschia species in Clade II

>7hymen sectors

4–12hymen sectors

2–7hymen sectors

>18 Fibulae in 10 μm P. cuspidata

P. circumpora

P. calliantha

P. caciantha

P. mannii

1 row of poroids

Gradually taper from middle to the end

with poroids D < 0.15 μm

CIS+

CIS-

P. inflatula

P. sp. Port Dickson

1–3hymen sectors

2–4 hymen sectors

2hymen sectors

<18 fibulae in 10 μm

P. hasleana

P. fryxelliana

P. pseudodelicatissima

Valvocopula = 2x1–3

Valvocopula =1x2–3

31–40 striae in 10 μm, mantle with 2–3 poroids high,Valvocopula = 2x3–6

21–29 striae in 10 μmValvocopula = 1x5

Lanceolatewith poroids D > 0.15 μm

Inflated at both apices

P. granii

P. subcurvata

Lanceolate

Slightly concave

Pseudodelicatissima-group

Fibulae < striae in 10 μmValvocopula = 2–3x5–9 P. subfraudulenta

1–3 rows of poroids

Fibulae = striae in 10 μmValvocopula = 3x6–9 P. fraudulenta Fraudulenta-group

Key to Pseudo-nitzschia species in Clade I

P. roundiiP. sinica >3 μm width

<3 μm width

CIS with 2 poroids/stria

CIS with3–5 poroids/stria

CIS with 5–7 poroids/stria

44–48 band striaein 10 μm

33–40 band striaein 10 μm

(valvocopula=1x1)

(valvocopula=1x2–3)

(valvocopula=1–2x2–4)

Figure 104 Identification key to species of Pseudo-nitzschia based on the current groupings in Figure 101 and in conjunction with the mor-

phological differences of documented frustules as in Supplements 2 and 3.

Valvocopula metrics represent width and height of striae in poroid numbers. CIS, central interspace; + , presence; -, absence; D, diameter.




shapes. The diagnostic features used to differentiate them

are the fibula and stria density.

These three species coexist in Malaysian waters,

which may lead to their misidentification. The morphology

of the Malaysian P. linea and P. brasiliana closely agrees

with that of the type species ( Lundholm et al. 2002 ) and

specimens reported subsequently ( Hasle and Lundholm

2005 , Quijano-Scheggia et al. 2008 , 2010, Li et al. 2010b ).

The Malaysian P. americana has a slightly higher density

of striae (Table 1) than that reported by Lundholm et al.

(2002) , Hasle and Lundholm (2005) , and Li et al. (2010b) ,

but agrees with that reported for the Bay of Fundy, Canada

( Kaczmarska et al. 2005 ).

The three species can be distinguished by their valve

shape, which is more lanceolate in P. brasiliana , slightly

lanceolate in P. americana , and linear in P. linea . The ratio

of the transapical axis at the apex ( x ) to the one at the mid-

valve maximum width ( y ) can be used as one of the mor-

phometrics for delineating these species ( Figure 103A ).

In this case, P. brasiliana has the smallest ratio (0.5), P. americana has 0.6, and P. linea has the largest (1.0) (Figure

103B). This ratio can be used to distinguish P. americana

from P. linea (t-test, p < 0.0001). The other feature used

to clearly distinguish P. americana from P. linea is the

number of striae, which is much greater in P. americana

than in P. linea (Table 1).

As a taxonomic remark, the frustule morphology of

the Malaysian P. turgidula agreed with that reported previ-

ously ( Rhodes 1998 , Hasle and Lundholm 2005 , Alman-

doz et al. 2008 , Leandro et al. 2010 ), with the exception

of the shape, which is rhomboid to sigmoid, instead of

the reported rhomboid shape. The Malaysian specimens

showed two rows of well-distributed poroids per stria,

compared with the report of uneven rows of poroids.

Furthermore, an identification key to Malaysian

species of Pseudo-nitzschia based on cladistic groups

in conjunction with their morphological differences is

provided to aid species identification ( Figure 104 ). This

key, together with the morphological data collected, will

serve as the framework for building an online interactive

identification key for Pseudo-nitzschia ; this project is cur-

rently in progress.

Conclusion This study revealed a considerably high species rich-

ness of Pseudo-nitzschia in the Malaysian coastal

waters, with more than half of the species thus far being

reported for the first time in these waters. Nine species

are currently known to be toxic or potentially toxic. It is

notable that these potentially toxic species coexist with

non-toxic species at most of the sampling sites. This

emphasizes the importance and urgency to detect these

species rapidly and accurately in Malaysian waters. This

morphology-based grouping approach, as well as the

key to species of Pseudo-nitzschia , could be useful as

an identification tool for field studies. Molecular tech-

niques such as whole-cell fluorescence in situ hybridi-

zation and ARISA using species-specific oligonucleotide

probes/primers to detect these species in Malaysian

waters are currently being applied. The species inven-

tory gathered from this study provides essential taxo-

nomic information for developing molecular probes and

for allowing government authorities to monitor HABs

better in Malaysia.

Acknowledgments: The authors are grateful to the anony-

mous reviewers for their constructive comments. Special

thanks to Stephen S. Bates for valuable suggestions and

English revision of the manuscript. S.T. Teng was partially

supported by UNIMAS postgraduate fellowship. This

study was funded by the Malaysian Government through

MOSTI ScienceFund (02-01-09-SF0054) to C.P. Leaw, Sci-

enceFund (04-01-02-SF0092), and MoHE JSPS-Asian Core

Program to P.T. Lim.

Received August 13, 2012; accepted June 29, 2013

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