1. 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.
world's 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.