DNA Barcoding of fishes in South China Sea Junbin Zhang, Christa Maitland and Robert Hanner* Biodiversity Institute of Ontario, University of Guelph * Corresponding author INTRODUCTION RESULTS RESULTS DISCUSSION• Fishes are the main animal protein source for • 321 sequences (all >500 bp) belong to 122 • In case the suspicious congeneric K2P • Except for the introgression for genus Pampus, human beings and they play a major role in the species (one species was identified to the distances in the genera Pampus are excluded, there are no overlaps between genetic variations aquatic ecosystem. Species delimitation and genus level) were ultimately obtained. Specific the pairwise genetic divergences for the other of S (intraspecific genetic distance) and G, and eventual recognition of them is not only of primer combinations and electropherogram individuals among congeneric species are all even so, intraspecific specimens formed interest for taxonomy and systematics, but also trace files for each specimen are available at above 10%. Actually, there are no overlaps monophyletic units and could be definitely is tightly related with authentication of food FSCS project in BOLD (Ratnasingham.&Hebert, between intraspecific K2P distances and those differentiated from other species. products and studies of natural history and 2007). among congeneric species within the same • Complex causes were responsible for deviations ecology such as fishery management, egg • The average K2P distance of intraspecies was family (Fig.2). from taxonomic monophyly, and one potential &larval dispersal and recruitment and spawn 0.319% and it increased sharply to 15.742% • Values of G (interspecific genetic distance cause of polyphyly for species is the occasional area estimates. among individuals of congeneric species. within the congener) fluctuated within a narrow mating between distinct species and resulting in• Recently, DNA barcoding has been proposed Overall, the average of genetic distance among range, but values of F (interspecific genetic gene transfer to hybrid offspring. Mitochondrial as a universal method to identify species and congeneric species is nearly 50 fold than that of distance from different genus but within intra- genes are generally subjected to introgression uncover biological diversity. Hebert (2004) intraspecies. For species within high taxonomic family ) were different between families (Fig.3). more frequently than nuclear ones and demonstrated that sequence divergences for 5 ranks such as families, orders and classes, • In this study, for individuals from different introgression also causes the phylogentic region of the mitochondrial DNA gene pairwise genetic divergences increased gently localities, some intraspecific genetic variations paraphyly, like the hybridization between cytochrome oxidase subunit I (COI) were and reached 20.199%, 24.656% and 25.225%, reduced to zero within families Carangidae Pampus argentenus and Pampus cinereus in generally much greater between species than respectively (Table 1). Sciaenidae and Mullidae. However, some this study. within them for many species and in turn • The interspecific hybridization between Pampus pairwise K2P distances exceeded 1.00% within • Biological mechanisms or the complex water suggests that the approach is widely applicable cinereus and Pampus argentenus was detected the coastal species such as Acentrogobius currents may play the main contribution to the across phylogenetically distant animal groups. in this study. For several specimens, they caninus, Scomber japonicus, Terapon jarbua, fluctuation of intraspecific divergences, in• South China Sea belongs to Indo-west Pacific possessed combinations of morphological Upeneus sulphureus, Elops hawaiensis, particular, for minitypical species lacking the Ocean, which is regarded as the center of the characteristics from Pampus cinereus and Gymnothorax pseudothyrsoideus and gene flow. worlds marine biodiversity. The species-rich Pampus argentenus (Fig.1). Dendrophysa russelii . • An assumed threshold is helpful to expedite biota of this area is particularly attractive to test comparisons taxa number of mean median minimum maximum s.e.# discovering new species and biodiversity, the reliability and efficiency of DNA barcoding. within compariso especially in facing of biotas involving with few ns studies. species 121 453 0.319 0.150 0 2.021* 0.018 METHODS genus 85 397 15.742 16.490 0.154** 25.189 0.292 family 55 848 20.199 19.850 11.532 34.333 0.134 order 15 17881 24.656 — 12.923 39.627 0.024• All specimens were preserved in class 2 29262 25.225 — 15.730 40.800 0.016 70% ethanol. Tissue samples were dissected from the dorsal muscle Table 1 and genomic DNA extracted Above- Genetic divergences (percentage, K2P according to the protocol in Barcode distance) within various taxonomic levels. Data are based on 321 sequences (>500 bp) from 122 of Life (Ivanova et al., 2006). Firstly, species. ** between Pampus cinereus and Pampus fragments of 5 region of argentenus, discussed in the text. mitochondrial COI gene were amplified using C_FishF1t1/ Fig. 3 C_FishR1t1 and C_VF1LFt1/ C_ Right- Boxplot distributions of S, G and F. VR1LRt1 primer combinations Intra-species (S), inter-species among congeneric (Ivanova et al., 2007). species (G), and inter-genera but intra-family (F) K2P• Amplicons were visualized on 2% distances for different families. agarose E-Gel® 96-well system (Invitrogen), and only for plates, which held >70% positive amplicons, were selected for sequencing PCR. . REFERENCES• Final PCR products were directly sequenced using an ABI 3730 capillary sequencer according to 1. Hebert, P.D.N, Penton, E.H., Burns, manufacturer’s instructions. J.M., Janzen, D.H.&Hallwachs W. (2004) PNAS 101,14812–14817• DNA sequences were aligned with 2. Ivanova, N.V., deWaard, J.R.&Hebert SeqScape v.2.1.1 software (Applied ,P.D.N. (2006) An inexpensive, Biosystems, Inc.). Sequence automation-friendly protocol for divergences were calculated using recovering high-quality DNA. Mol Ecol the Kimura two parameter (K2P) Notes, In Press distance model, and unrooted NJ 3. Ivanova, N.V., Zemlak, T.S., Hanner, trees based on K2P distances were Fig. 2 R.H.&Hebert, P.D.N. (2007) Universal created in MEGA software. Above- Box plots of K2P distances. primer cocktails for fish DNA IQR: interval into which the “central” 50% of the data fall. barcoding. Mol Ecol Notes, In Press• Distribution of these categories of Black bar in the box indicates the median. Circle: “Mild K2P distances: intraspecific 4. Ratnasingham, S.&Hebert, P.D.N. outlier” and asterisks: “extreme outliers”. Extreme (2007) BOLD: The Barcode of Life Data distances (S), inter-species within outliers are discussed in the text. Systems (www.barcodinglife.org). Mol the congener (G) and inter-species Eco Notes, In Press from different genus but within intra- family (F) were plotted using the CONCLUSIONS boxplot. Boxplots describe median ACKNOWLEDGEMENTS Effectiveness of species (central bar), interquartile range (IQR: between upper [Q3] and low Identification [Q1] quartile), values lying within • These results suggest DNA barcoding is 1.5× IQR beneath Q1 or 1.5× definitely effective for species above Q3 (“whiskers”) and extreme identification of fishes from South China This research was supported through values (outliers). Mann–Whitney Fig. 1 Sea, and some phylogenetic information funding to the Canadian Barcode of Life tests were performed between S, G Above - Neighbor-joining (NJ) tree of COI sequences. Scale: 5% K2P distance. was also revealed. The Barcode of Life Network from Genome Canada (through the and F distributions to estimate the The front numbers on branches are the process ID and the latter are the sample ID in provided a powerful platform for Ontario Genomics Institute), NSERC and overlap among taxonomic ranks. Project Barcode of Life for specimens. The shadow indicates interspecific hybridization molecular taxonomy. other sponsors listed at www.BOLNET.ca. between Pampus cinereus and Pampus argentenus.