Design of mini-barcode for catfishes for assessment of archival biodiversity.

Recovery of DNA barcode sequences is often challenging from the archived specimens. However, short fragments of DNA may be recovered, which would significantly improve many unresolved taxonomic conflicts. Here, we designed a mini-barcode for catfishes comprising several species and many cryptic taxa. We analysed a data set of 3048 publicly available COI barcode sequences representing 547 worldwide catfish species and performed 152 628 interspecies comparisons. A significantly more positively correlated interspecies distance was detected with transversion (0.78, P < 0.001) than with transition (0.70, P < 0.001). This suggested that transversions were better diagnostics for species identification. In the aligned data set, two transversion-rich fragments (53 bp and 119 bp) were identified. Transition/transversion bias value was 1.04 in 53-bp fragment, 1.23 in 119-bp fragment and 1.50 in full-length barcode. The interspecies distance with full-length barcode was 0.212 ± 0.037, while that with 53-bp and 119-bp fragments was 0.325 ± 0.039 and 0.218 ± 0.045, respectively. Survey of 53-bp fragment showed a possibility of only 1144 barcodes, while that of 119-bp fragment showed >4 million barcodes. Thus, the 119-bp fragment is a viable mini-barcode for catfishes comprising >3000 extant species. Experiment with 82 archived catfishes showed successful recovery of this mini-barcode using the designed primer. The mini-barcode sequences showed species-specific similarity in the range of 98-100% with the global database. Therefore, survey of a transversion-rich fragment within the full-length barcode would be an ideal approach of mini-barcode design for biodiversity assessment.

The species dilemma of Northeast Indian Mahseer (Actinopterygii: Cyprinidae): DNA barcoding in clarifying the riddle.

BACKGROUND: The taxonomic validity of Northeast Indian endemic Mahseer species, Tor progeneius and Neolissochilus hexastichus, has been argued repeatedly. This is mainly due to disagreements in recognizing the species based on morphological characters. Consequently, both the species have been concealed for many decades. DNA barcoding has become a promising and an independent technique for accurate species level identification. Therefore, utilization of such technique in association with the traditional morphotaxonomic description can resolve the species dilemma of this important group of sport fishes. METHODOLOGY/PRINCIPAL FINDINGS: Altogether, 28 mahseer specimens including paratypes were studied from different locations in Northeast India, and 24 morphometric characters were measured invariably. The Principal Component Analysis with morphometric data revealed five distinct groups of sample that were taxonomically categorized into 4 species, viz., Tor putitora, T. progeneius, Neolissochilus hexagonolepis and N. hexastichus. Analysis with a dataset of 76 DNA barcode sequences of different mahseer species exhibited that the queries of T. putitora and N. hexagonolepis clustered cohesively with the respective conspecific database sequences maintaining 0.8% maximum K2P divergence. The closest congeneric divergence was 3 times higher than the mean conspecific divergence and was considered as barcode gap. The maximum divergence among the samples of T. progeneius and T. putitora was 0.8% that was much below the barcode gap, indicating them being synonymous. The query sequences of N. hexastichus invariably formed a discrete and a congeneric clade with the database sequences and maintained the interspecific divergence that supported its distinct species status. Notably, N. hexastichus was encountered in a single site and seemed to be under threat. CONCLUSION: This study substantiated the identification of N. hexastichus to be a true species, and tentatively regarded T. progeneius to be a synonym of T. putitora. It would guide the conservationists to initiate priority conservation of N. hexastichus and T. putitora.

Identification and re-evaluation of freshwater catfishes through DNA barcoding.

BACKGROUND: Catfishes are globally demanded as human food, angling sport and aquariums keeping thus are highly exploited all over the world. North-East India possess high abundance of catfishes and are equally exploited through decades. The strategies for conservation necessitate understanding the actual species composition, which is hampered due to sporadic descriptions of the species through traditional taxonomy. Therefore, actual catfish diversity in this region is important to be studied through the combined approach of morphological and molecular technique of DNA barcoding. METHODOLOGY/PRINCIPAL FINDINGS: Altogether 75 native catfish specimens were collected from across the North-East India and their morphological features were compared with the taxonomic keys. The detailed taxonomic study identified 25 species belonging to 17 genera and 9 families. The cytochrome oxidase c subunit-I gene fragment were then sequenced from the samples in accordance with the standard DNA barcoding protocols. The sequences were compared with public databases, viz., GenBank and BOLD. Sequences developed in the current study and from databases of the same and related taxa were analyzed to calculate the congeneric and conspecific genetic divergences using Kimura 2-parameter distance model, and a Neighbor Joining tree was created using software MEGA5.1. The DNA barcoding approach delineated 21 distinct species showing 4.33 folds of difference between the nearest congeners. Four species, viz., Amblyceps apangi, Glyptothorax telchitta, G. trilineatus and Erethistes pusillus, showed high conspecific divergence; hence their identification through molecular approach remained inconclusive. On the other hand, the database sequences for three species, viz., Mystus horai, Bagarius yarrelli and Clarias batrachus, appeared mislabeled. CONCLUSION: The efficiency of DNA barcoding was reaffirmed from its success by easily identifying the major share (84%) of the studied catfish into 21 distinct species. The study contributed 27 new barcodes for 7 species and confirmed the range expansion of 2 important species in NE India.