Morphotaxonomy and seasonal distribution of ...

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Arabian Sea*. Sonia MUNIR 1, **, Zaib-un-nisa BURHAN 1, Tahira NAZ 1, P. J. A. SIDDIQUI 1, ... Sonia Munir. ** Corresponding ...... Nakaike T, Malik S, eds.
Chinese Journal of Oceanology and Limnology Vol. 31 No. 2, P. 267-281, 2013 http://dx.doi.org/10.1007/s00343-013-2150-y

Morphotaxonomy and seasonal distribution of planktonic and benthic Prorocentrales in Karachi waters, Pakistan Northern Arabian Sea* Sonia MUNIR1, **, Zaib-un-nisa BURHAN1, Tahira NAZ1, P. J. A. SIDDIQUI1, Steve L. MORTON2 1

Centre of Excellence in Marine Biology, University of Karachi-75270, Pakistan

2

National Oceanic and Atmospheric Administrations, 219 Fort Johnson Road, Charleston SC29412, USA

Received Jun. 7, 2012; accepted in principle Aug. 14, 2012; accepted for publication Nov. 19, 2012 © Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2013

Abstract Morphotaxonomy and seasonal abundance of dinoflagellates of the genera Prorocentrum and Mesoporos (Prorocentrales) were studied from nutrient-rich waters, Karachi Harbor and the mouth of the Manora Channel, Pakistan during May 2002–July 2003. Using both light and scanning electron microscopy, 13 species of Prorocentrales were identified according to cell shape, size, ornamentation of thecal plates, and architecture of apical platelets, apical pore area, marginal pores, and intercalary bands. P. sigmoides, P. arcuatum, P. scutellum, P. donghaiense, P. balticum, P. minimum, P. emarginatum, P. lima, P. faustiae, and Mesoporos perforatus constitute new records for sindh coast of Pakistan. The most abundant species were P. minimum/P. balticum (4.5×103 cells/L), P. micans (1.1×103 cells/L), P. gracile/P. sigmoides (2.5×102 cells/L) and P. donghaiense (6.6×103 cells/L) at temperatures of 29–31°C and salinities of 35–40. Maximum abundance was observed in winter and lower abundance in summer. There was no significant change in the distribution of species between stations except for the benthic species which occurred close to Karachi Harbor waters. Significant positive correlations were observed between Prorocentrum spp. and temperature (R2=0.27) and negative correlations with salinity (R2=-0.32) except for P. minimum and P. emarginatum which has negative correlation with temperature (R2=-0.24) and positive with salinity (R2=0.08, 0.19). The finding of potential okadaic-acid producing species of benthic Prorocentrum call for monitoring for possible human health problems in this region. Keyword: ecology; morphology; planktonic/benthic Prorocentrales; harmful algae; scanning electron microscopy; northern Arabian Sea

1 INTRODUCTION Harmful algae and phycotoxin production by dinoflagellates is a global and economic problem throughout coastal areas of the world, generating adverse effects on marine fish resources and human health (Nakajima et al., 1981; Anderson et al., 1989; Hallegraeff, 1995). Planktonic dinoflagellate species such as Prorocentrum micans Ehrenberg, P. gracile Schutt, P. sigmoides and P. triestinum Schiller, P. arcuatum Issle, P. compressum (Bailey) Abé ex Dodge, P. balticum (Lohmann) Loeblich III, P. dentatum Stein have been reported to cause blooms and occasionally fish kills (Steidinger, 1983), and are distributed worldwide (Dodge, 1975). To date, none

of these planktonic species have been found to produce toxic compounds. During the past two decades, many new epiphytic/benthic species of Prorocentrum have been described using both morphological and molecular characters. Many of these species produce the toxin, okadaic acid (Murakami et al., 1982; Faust, 1990, 1993a, 1993b, 1994; Morton, 1998; Morton et al., 1998; Holmes et al., 2001; Hoppenrath and Leander, 2008; Aligizaki et al., 2009, Nagahama et al., 2011, Chomerat et al.,

* Supported by Scholarship (IRSIP) of Higher Education Commission, Islamabad, Pakistan for Doctoral student Miss. Sonia Munir ** Corresponding author: [email protected]

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2011). These okadaic-acid producing species include P. concavum (Fukuyo, 1981), the P. lima complex (Ehrenberg) Dodge (Faust, 1991; Aligizaki et al., 2009), P. maculosum Faust (Faust et al., 1993b), P. belizeanum Faust (Faust, 1994), P. hoffmannianum Faust (Morton et al., 1994; Faust et al., 1999), P. faustiae Morton (Morton, 1998), P. tsawwassenense (Hoppenerath and Leander, 2008), P. glenanicum sp. nov., and P. pseudopanamense (Chomerat et al., 2011). These benthic species favor variable substrate conditions (macrophytes, rocks, sand, and corals) (Bomber et al., 1988; Fraga et al., 2012) compared to planktonic species, that form blooms in the water column (Fraga et al., 2012). Currently, the order Prorocentrales comprises the two genera, Prorocentrum and Mesoporos (Hoppenerath and Leander, 2008). A taxonomic revision by Dodge (1975) merged Prorocentrum and Exuviella due to the presence and absence of an apical spine and the lack of a sulcus and a cingulum. To date more than 60 species have been recognized in the genus Prorocentrum and 6 in the genus Mesoporos (Steidinger and Tangen, 1996). Morphologically, the Prorocentrales are a group of armored dinoflagellates with 2 lateral plates (concave or convex in lateral view). The anterior margin has 5–16 periflagellar platelets of which an apical spine may arise at the anterior end of the valve. Surface marking varies from pores to areolate to spines (Dodge, 1975; Steidinger and Tangen, 1996). Taxonomic characters used in the identification of this dinoflagellate group include cell shape, size, arrangement of the valves, valve surface, intercalary bands, marginal pores, periflagellar platelets, presence or absence of an apical spine (Dodge, 1975; Faust, 1990, 1993b, 1999; Steidinger and Tangen, 1996). Previous ecological studies in Pakistan have reported only 6 species: P. micans (Hassan and Saifullah, 1971), P. compressum, P. dentatum (Ghazala et al., 2006), P. gracile, and P. rhathymum (Gul and Saifullah, 2011), plus an unconfirmed report of P. lima (Baig, 2004). This preliminary research explores the biodiversity of the Prorocentrales species in relation to seasonality, distribution and identification at taxonomical level, from the northern Arabian Sea and the Manora coast of Karachi, Pakistan.

2 MATERIAL AND METHOD Manora Channel is located at the northern boundary of the Arabian Sea and the northeast section of the Indian Ocean. A total of 180 samples were collected

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bimonthly from two stations near the shore, Karachi Harbor (Stn. A) 24°49'46''N/66°57'55''E and the mouth of the Manora Channel (Stn. B) 24°47'51''N/66°58'52''E from May 2002 to July 2003, along the coast of Karachi, Pakistan (Fig.1). Triplicate samples were collected using a 1-L Niskin bottle (1 m depth) from both stations. Water temperature and salinity were measured by thermometer and refractometer. For qualitative study, all Lugol’s fixed samples were analyzed using the Utermöhl (1958) technique under inverted light microscope at 20× magnification. Cell abundance was expressed as cells per liter and these values also log-transformed. Samples were examined using an Olympus BX-51 (Tokyo, Japan) fitted with fluorescence microscopy and with phase and using a DIC fitted with an Olympus DP71 digital camera. Interesting samples were then examined using scanning electron microscopy (SEM). For SEM, Lugol’s fixed samples were desalted using a 10% step gradient from seawater to freshwater on polycarbonate filter paper and dehydrated by using a step gradient of acetone (10%–100%), coated with 1.5 nm of gold using a Denton sputter-edge coater (Moorestown, USA) and examined under a SEM JEOL 5600LV (Tokyo, Japan). The measurement of the cells is based on SEM imaging using MicroSuite 5 (Olympus, Tokyo, Japan). The data has been tested for significance by paired t-tests and Pearson correlation coefficients between the abundance of Prorocentrum species and both temperature and salinity.

3 RESULT 3.1 Morphotaxonomy of Prorocentrales found in Karachi waters 3.1.1 Heart or leaf shaped planktonic Prorocentrum species The body is heart shaped, widest at anterior end and pointed at posterior end with an apical spine at the right valve. The theca is bounded by an intercalary band in horizontal position. The thecal surface is rugose with numerous trichocyst pores which have minute areolated depressions. The apical plate area is small and shallow with two apical pores; 1) a flagellar pore and 2) an auxiliary pore. A well developed apical spine arises at the flagellar pore, the other pore being covered by curved periflagellar plate. The cells are dorsoventrally flattened, tear drop- or heart-shaped e.g. P. micans and P. scutellum and P. arcuatum

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26° N

25° Karachi coast Karachi Harbor ● Stn. A Mangr

24°

oves

▲ Stn.

Manora

B

km 0 23° 65°

50

66°

100

67°

E 68°

Fig.1 Map of Karachi coast, Pakistan. Locations indicated by circle (red dot) for Stn. A and triangle (red triangle) for Stn. B

a

b

(Fig.2a–d, e, f–g), slender, long and sigmoid e.g P. gracile and P. sigmoides (Fig.3 a–d) a) Prorocentrum micans: Cells are small to medium in size (L (length): 35.49–41.6 μm and W (width): 20.64–23.47 μm). Cells are slightly indented around the apical area anteriorly but pointed posteriorly. The valves or thecal surface are areolated and marked with surface depression and trichocyst pores (Fig.2b). The trichocyst pores are about 40–85 per valve. The space between each valve pore is 0.69– 0.96 μm on the marginal edge. The apical plate area is shallow (3.1 μm) with a wing spine (L: 6.4–7.5 μm) at the flagellar pore (Fig.2c and Table 1). Both valves are bounded with a horizontal intercalary band (Fig.2d). b) Prorocentrum scutellum: Cells are medium in size (L: 38.32 μm and W: 23.48 μm) and the thecal surface was areolated with trichocyst pores (60 per

d

c

×2 300

10 μm

269

×4 000

×2 200 10 μm

5 μm

×1 600 10 μm e

f

g

×1 500 10 μm

Fig.2 Scanning electron microscopy of Prorocentrum micans (a–d), Prorocentrum scutellum (e), light and scanning electron microscopy of Prorocentrum arcuatum (f–g) a–e. Heart shaped and flattened in right valve; b. Cell are areolated with trichocysts pores (black arrows) on periphery edge and marginal pores (white arrow); c. Small apical spine inserted at the anterior end of apical plate (black arrow); d. Intercalary band is horizontal striated (black arrow); e. Cell surface has depression and trichocyst pores and prominent with small apical spine present at the median position (white arrow); f–g. Cells are oblong and lanceolate, broadest above the apical end and pointed at the posterior end; g. The apical spine is long and sharp, and it arises at the apical pore of periflagellar area (white arrow).

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b

a

×1 600 ×1 600

10 μm

10 μm

d

c

×1 900

10 μm

×9 000

2 μm

Fig.3 Scanning electron microscopy of Prorocentrum gracile (a–b), Prorocentrum sigmoides (c–d) a–b. Cells are slender, rounded at anterior end and pointed at posterior end; a. Valve is prominent with scattered trichocyst pores on the valve surface (black arrow). Apical spine is adjacent to apical plate (white arrow); b. Intercalary band is horizontal striated (black arrow); c–d. Cells are S shaped with twisted apical spine; d. Deep depression is present at the apical area (black arrow).

valve). The pores are spaced between 1.4 and 2.5 μm from each other. This species can be differentiated by the wing spine (3 μm) present at the median position of the apical region (Fig.2e and Table 1). c) Prorocentrum arcuatum: Cells are lanceolate and medium to large (L: 45.2–50.8 μm, W: 19.3– 22.2 μm) (Table 1). This species can be differentiated by the anterior end, broadest at the middle of the cell and attenuated at the posterior end (Fig.2f, 2g). The valve pores are 60–70 per valve and scattered irregularly. The distance between the valve pores is 2–4 μm. The apical spine is long, sharp (L: 10.2– 11.3 μm) and broadest at the base (Fig.2g and Table 1). d) Prorocentrum gracile: Cells are pyriform or

slender in shape and much longer than broad (L: 45.5–52.8 μm, W: 15.9–20.4 μm) (Table 1). It has a broadly rounded apical end and is pointed posteriorly (Fig.3a, 3b). The valve pores are 65–70 per valve, arranged in radial rows. Each pore is spaced between 1 and 2 μm. The long flagellar spine (L: 7.4–9.2 μm) is pointed in shape (Fig.3a and Table 1). The valves are bounded by a horizontal intercalary band (Fig.3b) e) Prorocentrum sigmoides: Cells are sigmoid, S-shaped and large (L: 50.48–55.01 μm and W: 20.26–22.07 μm) (Fig.3c, 3d and Table 1) which can be different in character from P. gracile. It has 65–70 pores per valve that also present radially. It has a long flagellar spine (L: 10.8–14.2 μm) and it is flanged

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Table 1 Measurements of cell length, width, apical spine, valve pores, periflagellar area and collar are characteristic features of Prorocentrum species and Mesoporos perforatus in Pakistan waters Species

Length (Mean, μm)

Width (Mean, μm)

Apical spine (Mean, μm)

Valve pores (Mean, μm)

Periflagellar area (Mean, μm)

Collar (Mean, μm)

Figures

P. arcuatum

45.23–50.87 (n=10)

19.28–22.22

10.23–11.34

0.63–0.75

NA

NA

Fig.2, f–g

P. balticum

13.12–14.16 (n=4)

11.29–13.38

0.58–0.8

0.49–0.76

0.66

NA

Fig.5, a–c

P. compressum

34.99–40.55 (n=5)

28.43–32.92

1.55–2.18

0.26–0.39

NA

4.96

Fig.4, a–c

P. donghaiense

18.38–20.62 (n=3)

8.42–8.60

NA

0.49–0.52

2.16

1.03

Fig.5, g–i

P. emarginatum

30.45–41.18 (n=5)

24.60–34.26

NA

0.15–0.22

8.78

NA

Fig.6, c–e

P. faustiae

46.58 (n=1)

38.31

NA

0.25–0.59

13.2

NA

Fig.6, b

P. gracile

45.54–52.78 (n=11)

15.92–20.39

7.38–9.21

0.57–0.83

3.12

NA

Fig.3, a–b

P. lima

35.60–40.05 (n=2)

20.2–25.76

NA

0.22–0.33

6.17

NA

Fig.6, a

P. micans

35.49–41.6 (n=21)

20.64–23.74

6.44–7.54

0.69–0.96

3.12

NA

Fig.2, a–d

P. minimum

14.51–18.04 (n=2)

13.43–17.46

NA

NA

NA

NA

Fig.5, d–e

P. sigmoides

50.48–55.01 (n=5)

20.26–22.07

10.76–14.21

0.59–0.83

2.89

NA

Fig.3, c–d

P. scutellum

38.32 (n=1)

23.48

3.00

0.49–0.77

NA

NA

Fig.2, e

12.51–35.81 (n=2)

11.00–46.42

NA

0.80–3.25

NA

NA

Fig.5, f

Mesoporos perforatus

n: number of observation; NA: no observed features or lack of features.

a

b

×2 000

10 μm

c

×2 500

10 μm

×2 500

10 μm

Fig.4 Scanning electron micrograph of Prorocentrum compressum (a–c) a–c. Round, compressed in lateral view; a. Smooth thecal surface and trichocyst pores. Apical area is short has two apical projections or small apical spines (black arrow); b. Intercalary band is smooth, wide and transversely striated; c. Valve pores large to small scattered on the valve surface (black arrows). Scale bar=10 μm.

with a deep depression at the apical end (Fig.3d and Table 3). The valves are bounded by a horizontal intercalary band. 3.1.2 Smooth, round, planktonic Prorocentrum species The body shape is round, compressed, with a shallow depression and trichocyst pores. A small apical spine or projection arises at the anterior end of

the apical plate. Both valves are bound together by a horizontal striated intercalary band. a) Prorocentrum compressum: Cells are round, but ovate in valve view and compressed in lateral view (Fig.4a–c), and small (L: 35–40.6 μm, W: 28.4– 32.9 μm) (Table 1). The valve surface is smooth, marked with trichocyst pores (approximately 100 per valve). Large valve pores and small pores were 0.2–

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Table 2 Significance, correlation coefficient and t-test values between Prorocentrum species and temperature and salinity at Station A, Karachi harbour Temperature (°C)

Species

Correlation coefficient

Salinity

t-test significant

Correlation coefficient

t-test significant

P. minimum

-0.27

3.18 (P=0.007)*

0.08

3.09 (P=0.008)*

P. micans

0.31

2.06 (P=0.058)*

-0.22

1.79 (P=0.096)*

P. gracile

0.36

1.07 (P=0.112)

-0.31

0.75 (P=0.465)

P. arcuatum

0.47

1.56 (P=0.170)

-0.26

1.25 (P=0.246)

P. donghaiense

0.28

0.93 (P=0.379)*

-0.36

0.91 (P=0.391)*

P. compressum

0.33

1.38 (P=0.302)

-0.12

1.03 (P= 0.412)

P. scutellum

0.27

-7.65(P=0.000)

-0.02

-11.76 (P=0.000)

P. lima

0.47

0.52 (P=0.642)*

-0.37

-0.08 (P=0.941)*

P. emarginatum

-0.27

0.99 (P=0.427)*

0.19

0.94 (P=0.447)*

*: Significance level P