Morphological characterization and DNA barcoding of freshwater fishes collected from riverine ecosystem

A study on the fish fauna of River Jindi at Charsadda, Khyber Pakhtunkhwa, Pakistan was conducted, with an aim to determine fresh records of fish fauna from March to July, 2017. For fish distribution a 20 km belt of the river was explored for five months and every effort was made to collect the fish specimens by any legal mean. In this study, a total of 14 fish species belonging to 5 orders, 6 families and 9 genera was recorded. Cyprinidae was the richest family and was represented by 6 species, Sisoridae by 3 species, Channidae by 2 species, Mastacembelidae, Nemacheidae and Belonidae by single species. The fish species collected are Barilius vagra, Barilius pakistanicus, Barilius modestus, Carassius auratus, Puntius ticto, Puntius conchonius, Schistura alepidota, Glyptothorax punjabensis, Gagata pakistanica, Gagata cenia, Mastacembelus armatus, Channa punctatus, Channa gachua and Xenentodon cancila. The present studies was very helpful to evaluate the present fish fauna of River Jindi and also the need of conservation of different fish species in this river in future.

Fish are diverse aquatic organisms, economically significant as a primary source of food and nutrients. Accurate species identification is crucial for conservation, taxonomy and management. This study combined morphological and COI gene-based approaches to identify and characterize fish diversity from three locations in Malakand Division, Khyber Pakhtunkhwa Pakistan. All species were identified morphologically. DNA was extracted using phenol-chloroform method followed by PCR and sequencing. The consensus sequences were then utilized as search queries in BLASTn. BioEdit was used for trimming and MEGA X for phylogenetic study. Fourteen specimens representing eight species (Tariqilabeo latius, Mastacembelus armatus, Tor putitora, Carassius auratus, Cyprinus carpio, Garra gotyla, Channa punctata and Channa gachua) from three orders were analyzed. Results showed AT (54.23%) and GC (45.77%) contents, and a high Simpson’s diversity index (0.925), indicating rich species presence and evenness. Density, relative density, frequency, relative frequency and morphometric measurements were also calculated. This study validates the effectiveness of integrating morphological and molecular identification using COI gene for fish species in the region, highlighting its potential for broader application. Biodiversity metrics provide insights into its distribution in the area. Our findings contribute to the understanding of fish diversity in Pakistan and inform conservation and management efforts.

The present study explores the ichthyodiversity of a Cyprinid fish. A total of 622 fish samples were collected from five different sites of the Kurram River and identified using standard keys for identification. Out of 622 collected fish samples, 188 were related to the family Cyprinidae comprised of 19 species such as Barilius vagra, Barilius modestus, Barilius pakistanicus, Cyprinus carpio, Labeo rohita, Carassius auratus, Salmophasia punjabensis, Amblypharyngodon mola, Gara gotyla, Puntius conchonius, Puntius chola, Puntius sophore, Puntius ticto, Tor putitora, Schizothorax esocinus, Schizothorax plagiostomus, Schizothorax labiatus, Labeo diplocheilus, and Crossocheilus diplocheilus. The current study explores 5 new species, i.e., Schizothorax labiatus, Schizothorax esocinus, Amblypharyngodon mola, Puntius chola, and Salmophasia punjabensis for the first time, which are not reported in the earlier work. Principal component analysis (PCA) and diversity indices were analyzed using XLSTAT in conjunction with Microsoft Excel 2019 to assess the correlation and richness of fish diversity. To check the water quality of the River Kurram, the following parameters were examined: temperature, copper, pH, nitrate, alkalinity, chlorine, total hardness, iron, nitrite, and lead. All the recorded physicochemical parameters remained within the safe limits throughout the study period.

The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.

ABSTRACTThis paper addresses the relationship between fish and environmental variables from the Seti Gandaki River Pokhara, Nepal. The water bodies previously had higher abundance and distribution of fishes, which are declining probably due to deterioration of abiotic characteristics and degradation of habitat. Five study sites were: three along the main channel and two along the tributaries. Water sampling was conducted fortnightly and fish sampling was done monthly. Altogether 30 species of fishes belonging to 5 orders, 9 families and 24 genera were recorded. The redundancy analysis (RDA) revealed significant correlations between fish assemblage and environmental variables. The first two axes accounted for 44.15% of the variation of which RDA1 explained 30.32% and correlated with conductivity, pH, dissolved oxygen, free carbon dioxide, ortho-phosphate, nitrite and silicates; while RDA2 explained 13.83% and correlated with depth, width, discharge and nitrates. Likewise, RDA1 revealed a gradient from species that require more oxygenated waters of upstream sites (Schizothorax richardsonii, Pseudecheneis eddsi, Naziritor chelynoides, Garra annandalei, Schistura rupecula and Lepidocephalichthys guntea) to less oxygenated urban and downstream sites (Puntius sophore, Pethia conchonius, Barilius bendelisis, Barilius vagra, Garra gotyla, Mastacembelus armatus and Channa orientalis). RDA2 revealed the gradient from species inhabiting urban and downstream sites to upstream sites.

The use of DNA barcodes is a molecular method commonly used for the identification of specimens that are difficult to pinpoint accurately by traditional morphological methods. DNA barcoding uses standard short genomic regions that are universally present in target lineages and has sufficient sequence variation to identify species in the genus. The hawkmoth genus Cechetra (Zolotuhin & Ryabov, 2012) was proposed for a group of morphologically similar, stripe-patterned species. Based on the seven specimens of the three species of Cechetra genus collected from the Central of Vietnam, we used morphological analysis to initially identified their scientific name of Cechetra lineosa (Walker, 1856) for 04 specimens (LB_F01, LB_G01, LB_H01 and LK_G05); 02 specimens (MK_H04 and MK_B07) were identified with the scientific name of Cechetra minor (Butler, 1875) and 01 specimen (SG_D7) with scientific name Cechetra subangustata (Rothschild, 1920). Then, the COI (Cytochrome C oxidase I) sequence was used for molecular analysis. Total DNA was extracted from the legs of specimens, COI with over 600 bp in length from each specimen was amplified by PCR reactions. The PCR products are hence purified and sequenced. COI gene cloning results showed a very high level of genetic similarity (over 99%) and 7 specimens are of the genus Cechetra, Sphingidae family. For three homomorphic species belonging to the genus Cechetra Zolotuhin & Ryabov, 2012 including Cechetra lineosa (Walker, 1856); Cechetra minor (Butler, 1875) and Cechetra subangustata (Rothschild, 1920). The studied sequences have been added to the current database at GenBank (NCBI) with accession numbers MT994230, MT994231, MT994232, MT994233, MT994234, MT994235, and MT994236.

Assessment of snake DNA barcodes based on mitochondrial COI and Cytb genes revealed multiple putative cryptic species in Thailand

Distinguishing Black Bullhead (Ameiurus melas) from Brown Bullhead (A. nebulosus) can be difficult, in part, due to conflicting evidence regarding reliable meristic and morphological characters with which to base identifications. The possibility of hybrids with intermediate traits further complicates diagnosis. Identification guides have typically focused on adults, leaving guidance for juveniles largely undescribed. We compared morphological identifications of Black Bullhead and Brown Bullhead with a cytochrome c oxidase I (COI) barcode and microsatellite assignments. Specifically, we examined: (1) gill-raker count, (2) anal-fin ray count, (3) prominence of pectoral-spine serrae, (4) DNA barcoding using the COI gene, and (5) cluster analysis of nine microsatellite loci. With the exception of anal-fin ray counts, we found a high degree of agreement among all methods. Microsatellite analysis identified two clearly distinct genetic groups, and these groups corresponded to individuals identified as Black Bullhead and Brown Bullhead based on morphology. Although Black Bullhead and Brown Bullhead did not share COI haplotypes (except for one individual), we found greater intraspecific genetic distances among individuals identified as Brown Bullhead than between Brown Bullhead and Black Bullhead, demonstrating that caution is warranted against using COI haplotypes as a barcode to solely distinguish the two species. Pectoral-spine serrae prominence was a useful character to distinguish the two species and worked especially well on individuals <125 mm total length. Gill-raker counts of euthanized fish in the laboratory were appropriate for the identification of all sizes of Black Bullhead and Brown Bullhead (using the lowest number of gill rakers on the first gill arch if asymmetry was observed). Finally, microsatellites did not show evidence of recent hybridization at our sampling sites. We recommend using pectoral-spine serrae prominence to distinguish between Black Bullhead and Brown Bullhead in the field and using gill-raker counts for laboratory based identification.

Unveiling cryptic diversity of the anemonefish genera Amphiprion and Premnas (Perciformes: Pomacentridae) in Thailand with mitochondrial DNA barcodes

We adopted DNA barcoding technique using a 658-bp fragment of the mitochondrial cytochrome c oxidase I (COI) gene to identify shrimp species collected from the different areas of Bangladesh. A total of 24 sequences were generated belonging to 14 species including four new records- Macrobrachium nipponense, Macrobrachium kistnense, Exopalaemon carinicauda and Alpheus malleator. Genetic distance measured with Kimura 2 parameter showed that genetic divergence increased with higher taxonomic rank. The mean genetic divergence was evaluated and found to be 0.935%, 22.67% and 30.92% within species, genus and family, respectively. In addition to the barcode-based species identification system, phylogenetic relationships were established where individuals belonging to the same species were grouped under the same clade. Maximum likelihood (ML) was preferred as the statistical method and as expected, the phylogenetic tree complemented and ensured the conventional taxonomy. The present study evidently showed that DNA barcoding can be served as an effective tool to discriminate the shrimp species and this will enhance the understanding on evolution and conservation biology.&#x0D; Bioresearch Commu. 7(1): 941-946, 2021 (January)

This study was undertaken to investigate the diversity, abundance ratio, and distribution of the fish species and to record the water quality of Konhaye Stream, district Dir Lower, Pakistan. A total of 16 fish species were recorded, belonging to 4 orders (Cypriniformes, Channiformes, Siluriformes and Mastacebilformes), and 5 families including Cyprinidae (Schizopyge esocinus, Racoma labiata, Cyprinion watsoni, Cyprinus carpio, Barilius pakistanicus, B. vagra, B. modestus, Crossocheilus diplocheilus, Garra gotyla, Puntius ticto, and P. sophore), Channidae (Channa punctatus and Channa gachua), Nemacheilidae (Schistura macrolepis), Sisoidae (Glyptothorax punjabensis), and Mastacembelidae (Mastacembelus armatus). Different ichthyo-diversity indices [Simpson's biodiversity index (D=0.918), Simpson's reciprocal index (1/D=1.088), Simpson's evenness index (E-1/D=0.068), species richness (S), Shannon-Weiner's index (H'=3.775), Menhinick's index (D-mn=0.804), and Margalef's index (D-mg=2.510)] were calculated for the stream. The physicochemical parameters [temperature (23.125 +/- 3.514 degrees C), dissolved Oxygen (9.003 +/- 0.627 mg L-1), pH (7.333 +/- 0.201), turbidity (76.5 +/- 6.403 NTU), electric conductivity (201.68 +/- 11.31 mu s cm(-1)), free CO2 (124.75 +/- 9.912 ppm), total dissolved solids (126.1 +/- 9.477 ppm), total alkalinity (4.325 +/- 0.171 mg L-1), total suspended solids (127.1 +/- 6.864 ppm), total hardness (5.225 +/- 0.341 mg L-1), salinity (42.25 +/- 6.292 ppt), calcium hardness (2.975 +/- 0.670 mg L-1), magnesium hardness (105 +/- 9.954 mg L-1), potassium (0.0145 +/- 0.001 mg L-1), sodium (16.55 +/- 3.861 mg L-1), chloride (1.825 +/- 0.727 mg L-1), and nitrate (0.053 +/- 0.006 mg L-1) level] were monitored and found to be in varying but permissible ranges. The stream was found to be harboring a number of economically valuable fish species. An exotic fish species, C. carpio, was found well flourished, indicating the potential of the stream to be used for mitigating the declining ichthyofaunal diversity in the main nearby rivers. Two species, P. sophore and P. ticto, were recorded for the first time from the district. Proper management, appropriate fish stocking, and implementing environmental/fishing laws are suggested for the maintenance of the diversity and alleviating anthropogenic stress/threats.

The study evaluates the morphometric parameters of freshwater fish from the Cyprinidae family in Khyber Pakhtunkhwa, Pakistan at the Panjkora River. The ten species of the family Cyprinidae (Barilius pakistanicus, Barilius vagra, Carassius auratus, Cyprinus carpio, Tor putitora, Labeo rohita, Labeo diplocheilus, Schizothorax plagiostomus, Schizothorax esocinus, and Schizothorax labiatus) were identified from a total of 50 fish specimens collected from the study area. Seven morphometric parameters (Total length, fork length, standard length, head length, body depth, eye diameter, and snout length) were statistically analyzed using Excel and SPSS software. Ten species were categorized into four subfamilies within the family Cyprinidae. The two species belonging to the subfamily Cyprininae, three species to the subfamily Danioninae, two species to the subfamily Labeoninae, and three species to the subfamily Barbinae. This study serves as a baseline for future taxonomic studies on ichthyofauna.

Tipulidae, commonly known as true crane flies, represent one of the most species-rich dipteran families, boasting approximately 4,500 known species globally. Their larvae serve as vital decomposers across diverse ecosystems, prompting their frequent and close observation in biomonitoring programs. However, traditional morphological identification methods are laborious and time-consuming, underscoring the need for a comprehensive DNA barcode reference library to speed up species determination.In this study, we present the outcomes of the German Barcode of Life initiative focused on Tipulidae. Our DNA barcode library comprises 824 high-quality cytochrome c oxidase I (COI) barcodes encompassing 76 crane fly species, counting for ca. 54% of the German tipulid fauna. Our results significantly increased the number of European tipulid species available in the Barcode of Life Data System (BOLD) by 14%. Additionally, the number of barcodes from European tipulid specimens more than doubled, with an increase of 118%, bolstering the DNA resource for future identification inquiries.Employing diverse species delimitation algorithms — including the multi-rate Poisson tree processes model (mPTP), Barcode Index Number assignments (BIN), Assemble Species by Automatic Partitioning (ASAP), and the TaxCI R-script — we successfully match 76-86% of the morphologically identified species. Further validation through neighbor-joining tree topology analysis and comparison with 712 additional European tipulid barcodes yield a remarkable 89% success rate for the species identification of German tipulids based on COI barcodes.This comprehensive DNA barcode dataset not only enhances species identification accuracy but also serves as a pivotal resource for ecological and biomonitoring studies, fostering a deeper understanding of crane fly diversity and distribution across terrestrial landscapes.

Event Abstract Back to Event Complementing morphological classification of Anguilliform leptocephali with DNA barcoding Alessandra Anibaldi1, Claudia Benassi Franciosi1, Fausto Tinti1, 2, Corrado Piccinetti1 and Giulia Riccioni1* 1 University of Bologna, Department of Biological, Geological and Environmental Sciences, Italy 2 University of Bologna, Department of Biological, Geological and Environmental Sciences, Italy DNA barcoding is a molecular tool that enables rapid and accurate identification of biological species by sequencing a short, standardized region of the mitochondrial gene cytochrome c oxidase I ( COI) as internal species tag. The aim of the present study was to use DNA barcoding in addition to classical taxonomy in order to obtain a more reliable species-identification of leptocephali, the characteristic larval forms of the superorder Elopomorpha, often difficult to identify and to match with their adult stage. Based on the examination of external morphology, meristics, and pigmentation, 2785 leptocephalus larvae collected in the Adriatic Sea between 2010 and 2012, were ascribed to 7 morphotypes, belonging to Anguilliform order (Ariosoma balearicum, Conger conger, Gnathophis mystax, Nettastoma melanurum, Dalophis imberbis, Chlopsis bicolor, Facciolella sp.) and 69 specimens were sequenced for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (COI) to confirm the previous morphological analysis. The highly consistent results obtained revealed a good performance of COI barcoding as a diagnostic method for the identification of these larvae, but the limited number of leptocephali species annotated in the reference databases for barcode (Barcode of Life Data Systems and GenBank) allowed to validate only partially the morphological analysis. Moreover two species, Gnathophis mystax and Facciolella sp., showed unexpected outcomes. The data obtained in this work represent the first results of a wider project aimed at the creation of a new barcode database for the assessment of leptocephali diversity in the Mediterranean Sea (Barcoding of the Adriatic Leptocephali [BAL]), contributing to the knowledge of these unusual larvae and of their adult forms. Keywords: barcoding, Leptocephali, Adriatic sea, COI, Identification of larvae Conference: XV European Congress of Ichthyology, Porto, Portugal, 7 Sep - 11 Sep, 2015. Presentation Type: Oral Presentation Topic: Phylogeny, Systematics and Genetics Citation: Anibaldi A, Benassi Franciosi C, Tinti F, Piccinetti C and Riccioni G (2015). Complementing morphological classification of Anguilliform leptocephali with DNA barcoding. Front. Mar. Sci. Conference Abstract: XV European Congress of Ichthyology. doi: 10.3389/conf.fmars.2015.03.00021 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 24 Oct 2015; Published Online: 04 Nov 2015. * Correspondence: PhD. Giulia Riccioni, University of Bologna, Department of Biological, Geological and Environmental Sciences, Fano, 61032, Italy, giulia.riccioni@unibo.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni Google Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni Google Scholar Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni PubMed Alessandra Anibaldi Claudia Benassi Franciosi Fausto Tinti Corrado Piccinetti Giulia Riccioni Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Seven hundred and twenty four fish specimens were captured from March to September 2016. The materials used in the current study were cast nets, hand nets. Eight cyprinid fish species were studied for their length-weight relationships. Parameter b in the LWR was 3.03, 3.06, 3.02, 2.29, 2.82, 3.43, 2.73 and 2.47 for Schizothorax plagiostomus, Schizothorax esocinus, Racoma labiata, Tor putitora, Barilius vagra, Garra gotyla, Puntius ticto and Arassius auratus respectively. Current study is the first attempt on the LWRs of cyprinid species, provide a baseline approach for conservation and /management of local fish fauna of economic importance.