Species identification of larval fish in Hawaiian waters using DNA barcoding

Background: Correct identification of fish larvae is notably difficult, given that the morphological features at this stage of development are often poorly defined. However, obtaining such taxonomic information is of considerable importance from the perspectives of fisheries management and conservation. Results: In this study, we collected a total of 95 specimens of larval fish from Hawaiian waters, which we sought to identify using DNA barcoding based on mitochondrial DNA cytochrome oxidase subunit I sequences. Among these, 40 and 52 samples were accordingly identified to the species and genus levels, respectively. The determined average Kimura-2-parameter distances within species, genera, families, and orders were 0.72%, 25.99%, 26.30%, and 27.50%, respectively, and the mean interspecific distance was found to be 36-fold higher than the mean intraspecific distance. Conclusions: The results of this study provide convincing evidence that DNA barcoding can serve as an effective tool for the accurate species identification of larval fish in Hawaiian waters, and could thereby make a valuable contribution to species diversity surveys. The findings of this study can make a valuable contribution to our knowledge of the diversity and dispersal of larval fish in Hawaiian waters.

Molecular and Biological Characterization of Haemaphysalis Leachi (Acari: Ixodidae) in Nigeria West Africa

Ticks constitute a serious threat to the wellbeing of humans and other animals. The accurate identification of ticks is paramount in epidemiological investigations. Genetic markers have been identified and used to overcome the limitations of phenotypic identification of ticks. In this study, the cytochrome c oxidase 1 (Cox1) gene was amplified and sequenced for the identification of Haemaphysalis leachi, the putative vector of Babesia rossi in Nigeria. Amplification was successful in 34 out of 39 (87 %) ticks collected from dogs in three Nigerian states with sequence homology of 99 % to H. leachi in Genbank (GenBank: MN663156.1). Maximum Likelihood phylogenetic analysis showed significant grouping of H. leachi sequences in independent monophyletic subclade with a bootstrap value of 100 %. Genetic distance analysis of H. leachi identified in this study indicated a very low level of intraspecific diversity (0.016 %) compared to 0.150—0.190 % interspecific distance to other Haemaphysalis species. The number of eggs laid by engorged female ticks maintained in the laboratory ranged from 885 to 2190 and was proportional to the ticks’ initial weight. The mean value of other biological parameters; female engorgement weight, pre-oviposition period, oviposition period, total egg mass, egg size, efficiency rates of female ticks in converting their food reservoir to eggs and incubation period are, 147.5 mg, 7.8 days, 13.2 days, 59.5 mg, 485.5 × 348.7 µm, 41.2 % and 26.2 days, respectively. This study reports the first molecular identification of H. leachi in Nigeria.

Molecular diversity of Uzbekistan’s fishes assessed with DNA barcoding

Uzbekistan is one of two doubly landlocked countries in the world, where all rivers are endorheic basins. Although fish diversity is relatively poor in Uzbekistan, the fish fauna of the region has not yet been fully studied. The aim of this study was to establish a reliable barcoding reference database for fish in Uzbekistan. A total of 666 specimens, belonging to 59 species within 39 genera, 17 families, and 9 orders, were subjected to polymerase chain reaction amplification in the barcode region and sequenced. The length of the 666 barcodes was 682 bp. The average K2P distances within species, genera, and families were 0.22%, 6.33%, and 16.46%, respectively. The average interspecific distance was approximately 28.8 times higher than the mean intraspecific distance. The Barcode Index Number (BIN) discordance report showed that 666 specimens represented 55 BINs, of which five were singletons, 45 were taxonomically concordant, and five were taxonomically discordant. The barcode gap analysis demonstrated that 89.3% of the fish species examined could be discriminated by DNA barcoding. These results provide new insights into fish diversity in the inland waters of Uzbekistan and can provide a basis for the development of further studies on fish fauna.

Genotypic identification of Panicum spp. in New South Wales, Australia using DNA barcoding

Australia has over 30 Panicum spp. (panic grass) including several non-native species that cause crop and pasture loss and hepatogenous photosensitisation in livestock. It is critical to correctly identify them at the species level to facilitate the development of appropriate management strategies for efficacious control of Panicum grasses in crops, fallows and pastures. Currently, identification of Panicum spp. relies on morphological examination of the reproductive structures, but this approach is only useful for flowering specimens and requires significant taxonomic expertise. To overcome this limitation, we used multi-locus DNA barcoding for the identification of ten selected Panicum spp. found in Australia. With the exception of P. buncei, other native Australian Panicum were genetically separated at the species level and distinguished from non-native species. One nuclear (ITS) and two chloroplast regions (matK and trnL intron-trnF) were identified with varying facility for DNA barcode separation of the Panicum species. Concatenation of sequences from ITS, matK and trnL intron-trnF regions provided clear separation of eight regionally collected species, with a maximum intraspecific distance of 0.22% and minimum interspecific distance of 0.33%. Two of three non-native Panicum species exhibited a smaller genome size compared to native species evaluated, and we speculate that this may be associated with biological advantages impacting invasion of non-native Panicum species in novel locations. We conclude that multi-locus DNA barcoding, in combination with traditional taxonomic identification, provides an accurate and cost-effective adjunctive tool for further distinguishing Panicum spp. at the species level.

Badis kaladanensis, a new fish species (Teleostei: Badidae) from Mizoram, northeast India

Badis kaladanensis, a new percoid fish is described from the Kaladan basin of Mizoram, northeast India. It belongs to the Badis badis species group but can be easily distinguished from its congeners, except from B. kanabos and B. tuivaiei, in having a dark blotch on the dorsal fin between the base of 3rd to 5th spines. It is further distinguished from B. kanabos in having more scales in lateral row (27–30 vs. 25–26), more circumpeduncular scale rows (18–20 vs. 16–17) and smaller eye (7.5–8.9% SL vs. 9.5–12.7); and from B. tuivaiei in having fewer vertebrae (28–29 vs. 30–31) and more rakers on the first gill arch (9 vs. 6–8). The analysis of the mitochondrial DNA (coi and cytb) revealed the distinctness of B. kaladanensis from all other Badis species with the interspecific distance ranges from 5.4–20.4%. (coi) and 5.1–26.3% (cytb).

Fish diversity in a doubly landlocked country - a description of the fish fauna of Uzbekistan using DNA barcoding

Uzbekistan is one of two doubly landlocked countries in the world, where all rivers are endorheic basins. Although fish diversity is relatively poor in Uzbekistan compared to other regions, the fish fauna of the region has not yet been fully studied. The aim of this study was to establish a reliable barcoding reference database for fish in Uzbekistan. A total of 666 specimens, belonging to 59 species within 39 genera, 16 families, and 9 orders, were subjected to polymerase chain reaction amplification in the barcode region and sequenced. The length of the 666 barcodes was 682 bp. The average K2P distances within species, genera, and families were 0.22%, 6.33%, and 16.46%, respectively. The average interspecific distance was approximately 28.8 times higher than the mean intraspecific distance. The Barcode Index Number (BIN) discordance report showed that 666 specimens represented 55 BINs, of which five were singletons, 45 were taxonomically concordant, and five were taxonomically discordant. The barcode gap analysis demonstrated that 89.3% of the fish species examined could be discriminated by DNA barcoding. These results provide new insights into fish diversity in the inland waters of Uzbekistan and can provide a basis for the development of further studies on fish fauna.

Identification of Vicia Species Native to South Korea Using Molecular and Morphological Characteristics

Recently, within the Fabaceae family, the Vicia genus has been recognized for its vital role in sustainable agriculture. Vicia species are economically important grain and forage crops. However, the presence of complex morphological characteristics makes identification and recognition of native species difficult. In this study, the possibility of using DNA barcoding regions (ITS2, matK, and rbcL) to distinguish among 19 Vicia taxa (59 accessions) found in South Korea was evaluated. The sequence alignment analysis revealed considerable nucleotide diversity (π) between the loci, in which ITS2 showed the highest mean interspecific distance, whereas there was no intraspecific variability among the barcode regions in 12 of the 19 taxa. Phylogenetic analysis of combined barcoding regions revealed well-resolved phylogeny with the highest species level discrimination. Combinations of barcode loci were also used in classification at the subgenera and section levels. The results revealed that the combined barcoding regions can be used effectively to differentiate the following species: Vicia angustifolia var. segetilis, Vicia bungei, Vicia villosa, Vicia cracca, Vicia dasycarpa, Vicia hirsuta, Vicia tetrasperma, Vicia amurensis, Vicia hirticalycina, and Vicia chosenensis. However, it is difficult to differentiate the species of Vicia unijuga, Vicia unijuga var. kaussanensis, Vicia linearifolia, Vicia unijuga f. angustifolia, Vicia nipponica, Vicia amoena, Vicia venosa var. cuspidata, Vicia pseudo-orobus, and Vicia japonica with the tested barcode regions. These species come under sect. Vicilla and are found to be closely related or species that have recently undergone speciation; thus, it has limitation to distinguish with recommended barcodes. Hence, to differentiate the unclassified species, 39 morphological characteristics were investigated, in which 16 useful characteristics were selected for efficient classification. Finally, the 16 selected morphological useful traits efficiently differentiated all the Vicia species. In conclusion, a combination of barcoding loci together with morphological characteristics of this study efficiently discriminated all the Korean Vicia species.

Comparative Analysis of Complete Chloroplast Genome Sequences and Insertion-Deletion (Indel) Polymorphisms to Distinguish Five Vaccinium Species

We report the identification of interspecific barcoding InDel regions in Vaccinium species. We compared five complete Vaccinium chloroplast (cp) genomes (V. bracteatum, V. vitis-idaea, V. uliginosum, V. macrocarpon, and V. oldhamii) to identify regions that can be used to distinguish them. Comparative analysis of nucleotide diversity from five cp genomes revealed 25 hotspot coding and noncoding regions, occurring in 65 of a total of 505 sliding windows, that exhibited nucleotide diversity (Pi) > 0.02. PCR validation of 12 hypervariable InDel regions identified seven candidate barcodes with high discriminatory powers: accD-trnT-GGU, rpoB-rpoA, ycf2-trnL-GAA, rps12-ycf15, trnV-GAC, and ndhE-ndhF. Among them, the rpoB-rpoA(2) and ycf2-trnL-CAA sequences clearly showed the intraspecific and interspecific distance among five Vaccinium species by using a K2P technique. In phylogenetic analysis, included five Vaccinium species (n = 19) in the Bayesian and Neighbor-Joining (NJ) analysis revered all species in two major clades and resolved taxonomic position within species groups. These two locus provide comprehensive information that aids the phylogenetics of this genus and increased discriminatory capacity during species authentication.

Morphometrics and genetics variations of mullets (Pisces: Mugillidae) from Aceh waters. Indonesia

Yulianto D, Batubara IIAS, Nur FM, Rizal S, Siti-Azizah MN, Muchlisin ZA. 2020. Morphometrics and genetics variations of mullets (Pisces: Mugilidae) from Aceh waters, Indonesia. Biodiversitas 21: 3422-3430. Mullets are one of the commercial groups of coastal fish living in Aceh waters, in Indonesia. Presently, there is limited information on the bioecology, especially on the genetics and morphometrics of these fishes. Therefore, the objective of this study is to examine the morphology and genetic variations of Liza macrolepis, Liza tade, and Moolgarda engeli mullets. This study was conducted from January 2018 to July 2019 in four locations along the northern and eastern coasts of Aceh. Data were obtained by measuring the morphometrics of a total of 180 samples of the species in equal ratios. The data were analyzed using the ANOVA and Discriminate function analysis (DFA). The ANOVA test showed that at least 75% of characters are not significantly different among the mullets. Meanwhile, the discriminant function analysis produces the two functions with Eigenvalue of 0.627 and 0.107 with 85.5% and 17.2% total variants, respectively. Function 1 discriminates the mullet samples into two groups; the first was L. tade, and the second belonged to L. macrolepis and M. engeli. In addition, a total of 18 samples of mullets consisting of 8 samples of the L. macrolepis, 2 samples of the L tade, and 8 samples of the M. engeli were successfully amplified from the 5' region of the mitochondrial cytochrome oxidase subunit I (COX1) gene using a pair of primers (Fish F1 and Fish R1). Furthermore, a total of 7 haplotype sequences were produced from the ingroup where L. tade has one haplotype, L. macrolepis and M. engeli had three haplotypes, respectively. The genetic distance analysis showed that the interspecific distance was 0.38% and intraspecific was 8.2%. Therefore, the COI gene successfully discriminated against the mullet into three valid species.

Molecular diversity of freshwater fishes of Bangladesh assessed by DNA barcoding

This study represents the comprehensive molecular identification of freshwater fishes of Bangladesh based on a fragment of the cytochrome c oxidase subunit I (COI) gene in the mitochondrial genome. A total of 315 mitochondrial COI barcode sequences were obtained from 153 species,114 genera, 49 families and 16 orders of fishes. The mean length of the sequences was 652 base pairs. For all the samples, %G was significantly lower compared to the other nucleotides and %GC was lower compared to %AT (p-value ˂ 0.05). Also, a significantly lower %GC content was observed in second and third codon position compared to the first one in all the samples (1st>2nd>3rd, p-value˂ 0.05). The average K2P distances within species, genera, families and orders were 0.38%, 7.02%, 12.75% and 18.68%, respectively. The mean interspecific distance was 18-fold higher than the mean intraspecific distance. The K2P neighbor-joining (NJ) trees based on the sequences generally clustered species according to their taxonomic position. A total of 12 species were newly recorded in Bangladesh. High efficiency in species identification were demonstrated in the present study by DNA barcoding, and concluded that COI sequencing can be used as an authentic identification marker for freshwater fish species. Bangladesh J. Zool. 48(1): 1-19, 2020