TAXONOMY AND GENETIC RELATIONSHIPS OF PANGASIIDAE , ASIAN CATFISHES , BASED ON MORPHOLOGICAL AND MOLECULAR ANALYSES

Pangasiids are economically important riverine catfishes generally residing in freshwater from the Indian subcontinent to the Indonesian Archipelago. The systematics of this family are still poorly known. Consequently, lack of such basic information impedes the understanding of the biology of the Pangasiids and the study of their aquaculture potential as well as improvement of seed production and growth performance. The objectives of the present study are to clarify phylogeny of this family based on a biometric analysis and molecular evidence using 12S ribosomal mtDNA on the total of 1070 specimens. The study revealed that 28 species are recognised as valid in Pangasiidae. Four genera are also recognized as Helicophagus Bleeker 1858, Pangasianodon Chevey 1930, Pteropangasius Fowler 1937, and Pangasius Valenciennes 1840 instead of two as reported by previous workers. The phylogenetic analysis demonstrated the recognised genera, and genetic relationships among taxa. Overall, trees from the different analyses show similar topologies and confirm the hypothesis derived from geological history, palaeontology, and similar models in other taxa of fishes from the same area. The oldest genus may already have existed when the Asian mainland was still connected to the islands in the southern part about 20 million years ago.


INTRODUCTION
Pangasiidae are economically important riverine catfishes generally occurring in freshwater from the Indian subcontinent to the Indonesian Archipelago (Gustiano 2003;Gustiano and Pouyaud 2005;2006).Morphologically, they are recognized by a laterally compressed body, two pairs of barbels, a short dorsal fin with two spines, a well developed adipose fin, a long anal fin, and a strong pectoral spine (Teugels 1996).The systematics of this family are still poorly known (Gustiano 2003;Pouyaud et al. 2005).Consequently, the lack of this basic information is a significant barrier to understanding the biology and hence the study o f the aquaculture po te ntial o f Pangasiids, the improvement of their seed production and growth performance (Legendre 1999).
The objectives of the present study are to clarify phylogeny of this family based on a biometric analysis and molecular evidence using 12S ribosomal mtDNA on the total of 1070 specimens.

Biometrics
Nine hundred and ninety nine specimens were examined including the type of 49 previous described species housed in various museums.For each specimen, 35 point-to-point measurements covering the possible variations of the body were undertaken (Fig. 1).first factor, considered as the size factor, was not taken into account in order to minimise the size differences between samples.An independent PCA was run on the correlation matrix from untransformed data.
Finally, data analysis co nsiste d o f characterising groups from scatter plots between pairs of structuring characters for subsequent use in generic identification keys.

Molecular analysis
Fresh tissue samples were stored in liquid nitrogen for transfer to the laboratory.They were then stored at -20ºC.Mitochondrial analysis consisted of sequencing part of the 12S rDNA gene.One to five individuals were analysed for 28 species of Pangasiidae.One hundred mg of muscle were used to isolate total genomic DNA using a hexadecylmethyl-ammoniumbromide extraction process (Doyle & Doyle, 1987).Primers used for the amplification of the mitochondrial gene included 12S light strand 5'-TTACACATGCAAGTCTCCGC-3' and 12S heavy strand GTTACGACTTGCC TCCCCTT-3' de fine d o n the co mple te mitochondrial sequence of Cyprinus carpio (EMBL X61010, Chang et al. 1994).
DNA amplification and sequencing were performed following the procedures developed in Pouyaud et al. (2000).In order to check sequence accuracy and to correct any ambiguity bases, both strands were sequenced using each one of the two initial PCR primers.
Sequences of both strands were compared with each other and aligned using the sequence editor ESEE (version 3.1 s; Cabot & Beckenbach, 1989).Comparison of absolute numbers of transitions and tranversions were

RESULTS AND DISCUSSION
During the study twenty eight species were recognised as valid in Pangasiidae.Four genera were also recognized (Helicophagus Bleeker, 1858;Pangasianodon Chevey, 1930;Pteropangasius Fowler, 1937;and Pangasius Valenciennes, 1840) instead of two as reported by Vidthayanon (1993).Key below different genera is presented.For each genus, a key to the species is given.

Key to genera
1a. 8-9 pelvic fin rays, long predorsal length (>37% SL), and slender dorsal spine width (3.5-5% HL The phylogenetic analysis demonstrated the recognised genera, and the genetic relationships among the species (Fig. 2).Overall, trees from the osteological (Gustiano 2003), molecular and biometric analyses (Gustiano and Pouyaud 2005) show similar topologies and confirm the hypothesis derived from geological history, palaeontology, and similar models in other taxa of fishes from the same area (Gustiano 2003).The oldest genus may already have existed when the Asian mainland was still connected to the islands in the southern part about 20 million years ago (Gustiano et al. 2003;Pouyaud et al. 2005).

CONCLUSION
The analysis demonstrated the genetic relationships among species of Pangasiids.The present study recognizes four genera i.e.Helicophagus Bleeker, 1858;Pangasianodon Chevey, 1930;Pteropangasius Fowler, 1937;and Pangasius Valenciennes, 1840.At this level, species belonging to the same genus are aggregated using the degree of similarity of morphometric and molecular data.

AKNOWLEDGEMENTS
The authors thank J. Slembrouck, M. Legendre, A. Pariselle, Sudarto, A.H. Kristanto, W. Hadie, D. Sadili, O. Komarudin, and E. Siagian for their support and help in collecting fish samples.Sincere thanks to G. Teugels for his encouragement in the present study.This paper forms a part of INCO.DC Proje ct "Catfish Asia" financed by the European Union (Contract IC 18-CT 96-0043).The Senior Author is also grateful to AARD for the fellowship provided to complete this study through "PAATP" Project at Leuven University, Belgium.