Kimura 2-parameter Distances Research Articles

Identification of Edible Fish Species of Pakistan Through DNA Barcoding

Fish is a fundamentally healthy food, loaded with essential nutrients, high protein content, vitamin D, and omega-three fatty acid. Mislabeling is a common problem in the fish industry that causes an imbalance in prices and fluctuation in the market. DNA barcoding is a potential technique for authentication of mislabeled and misidentified fish species. In this study, 11 freshwater and 6 marine fish species were used for DNA barcoding and further authentication using the mitochondrial cytochrome b (Cyto b) gene. Cyto b was amplified using PCR, producing an average read length of 1,141 bp. The obtained sequences were compared to the National Center for Biotechnology Information database using the Basic Local Alignment Search Tool. The average AT content (55.20%) was higher than the average GC content (44.78%) in marine and freshwater fish species. The mean genetic Kimura 2-parameter distances for species, genus, families, and orders were 0.311, 0.308, 0.023, and 0.337, respectively. Phylogenetic tree analysis revealed that most of the freshwater fish species clustered together due to the fact that they were in the same order or family, while the marine fish species clustered distantly. Single nucleotide polymorphism (SNP) analysis of all species in the study revealed distinct features regarding unique sites. All fish species could be identified based on their unique SNP profiles. Based on SNP data, DNA sequence based QR codes were developed for accurate identification of fish species. This is the first study to develop DNA-based QR barcodes for proper authentication of species during the chain of custody using simple technology.

Ontogenetic comparison of larvae and juveniles of Diaphus garmani and Benthosema pterotum (Myctophidae, Pisces) collected from Korea

During June 2017, we collected two postflexion larvae (6.01 and 7.56 mm in standard length [SL]) and two juveniles (7.72 and 9.62 mm SL) belonging to Myctophidae in the waters of Jejudo Island. Those four individuals were identified as Diaphus garmani, which had not been reported in Korea. They were distinguished from Benthosema pterotum by melanophores in the abdominal cavity (absent in D. garmani vs. present in B. pterotum) and the development of photophores (developed in D. garmani vs. rudimentary in B. pterotum) when shorter than 10.0 mm SL. Analysis of 16S rRNA sequences showed that the sequences of four individuals matched those of adult D. garmani (Kimura 2-parameter distance: 0.6–0.8%). This is the first record of larvae and juveniles of D. garmani in Korean waters, and we propose a new Korean name, Gar-ma-ni-sat-bi-neul-chi.

DNA barcoding of coral reef fishes from Chuuk State, Micronesia

The fish diversity of Chuuk Micronesia is currently under threat due to rapid changes in the coral reef ecosystem. Thus, accurate fish identification using DNA barcodes is fundamental for exploring species biodiversity and resource protection. In this study, we analyzed 162 fish mitochondrial DNA cytochrome c oxidase I (COI) barcodes from Chuuk Micronesia. Consequently, we identified 95 species from 53 genera in 26 families and seven orders. The average Kimura 2-parameter genetic distances within species, genera, families, and orders were calculated as 0.17%, 11.78%, 15.63%, and 21.90%, respectively. Also, we have utilized DNA barcodes to perform genetic divergence and phylogenetic analysis of families recognized as dominant groups in Chuuk State. Our findings confirm that DNA barcodes using COI are an effective approach in identifying coral reef fish species. We anticipate that the results of this study will provide baseline data for the protection of coral reef fish biodiversity at Chuuk Micronesia.

Cryptic diversity and mtDNA phylogeography of the invasive demon shrimp, Dikerogammarus haemobaphes (Eichwald, 1841), in Europe

The regions of the Black, Caspian, and Azov seas are known for being both (i) the place of extensive crustacean radiation dated to the times of Paratethys and Sarmatian basins, and (ii) present donors of alien and invasive taxa to many areas worldwide. One amphipod morphospecies,Dikerogammarus haemobaphes, is known both as native to rivers draining to the Black and Caspian seas as well as a successful invader (nicknamed demon shrimp) in Central and Western European rivers. Based on mitochondrial (COI and 16S) and nuclear (28S) datasets and 41 sampling sites, representing both the native (19) and the invaded (22) range, we assessed cryptic diversity, phylogeography and population genetics of this taxon. First, we revealed the presence of two divergent lineages supported by all markers and all species delimitation methods. The divergence between the lineages was high (18.3% Kimura 2-parameter distance for COI) and old (ca. 5.1 Ma), suggesting the presence of two cryptic species withinD. haemobaphes. Lineage A was found only in a few localities in the native range, while lineage B was widespread both in the native and in the invaded range. Although genetic divergence within lineage B was shallow, geographic distribution of 16S and COI haplotypes was highly heterogeneous, leading us to the definition of four Geo-Demographic Units (GDUs). Two GDUs were restricted to the native range: GDU-B1 was endemic for the Durugöl (aka Duruşu) Liman in Turkey, whereas GDU-B2 occurred only in the Dniester River. GDU-B3 was both present in several localities in the native range in the Black Sea drainage area and widespread in Central and Western Europe. The GDU-B4 was found exclusively in the Moskva River in Russia. Extended Bayesian Skyline Plot indicated steady growth of GDU-B3 population size since 30 ka, pointing to the rather old history of its expansion, first in the late Pleistocene in the native range and nowadays in Central and Western Europe. The analysis of haplotype distribution across the present distribution range clearly showed two invasion routes to Central and Western Europe. The first one, originating from the lower Dnieper, allowed the demon shrimp to colonize Polish rivers and the Mittellandkanal in Germany. The second one, originating from the Danube delta, allowed to colonize the water bodies in the upper Danube basin. The UK population has originated from the Central Corridor, as only a haplotype found exclusively along this route was recorded in the UK. Population genetics analysis showed that the invasion of the demon shrimp along the Central Corridor was not associated with the loss of genetic diversity, which might contribute to the success of this invader in the newly colonized areas.

First record of Euphlyctis kalasgramensis (Anura: Dicroglossidae) from Punjab, Pakistan

The present study documented the first record of Euphlyctis kalasgramensis from Punjab, Pakistan. The specimens were collected during field visits from June through August 2018. Various morphometric measurements of E. kalasgramensis were taken and compared with Euphlyctis cyanophlyctis. Snout-vent length (SVL) was 38.11 ± 0.87 mm (n = 5), snout length was 3% of SVL, foot length was 55% of SVL, head length was 32% of SVL and weight was 8.01 ± 0.12 g (n = 5). A few specimens (n = 2) were euthanized and preserved for molecular analysis through mitochondrial 16S rRNA gene sequences. The newly obtained DNA sequences of E. kalasgramensis were submitted to GenBank and accession numbers were obtained (MK881165.1 and MK920114.1). The Maximum likelihood and Neighbor-joining trees based on Kimura 2-parameter distance resulted in similar phylogenetic trees. Euphlyctis kalasgramensis was out group in both phylogenetic trees. The interspecific divergence of E. kalasgramensis and E. cyanophlyctis was high ranging from 4% to 6% as compared to low intraspecific divergence 0% and 1%. The diversity and distribution ranges of many amphibians species are not well known in Pakistan due to lack of taxonomic information. In our recommendation, a large scale DNA barcoding is required to report more cryptic or new species from Pakistan.

Hidden biodiversity revealed by DNA barcoding in black fly genus Simulium

The black fly genus Simulium Latreille is one of the most important medical insect group of the family Simuliidae (Diptera) and many species of this genus are important pests of human and animals, while some of them also represent vectors of pathogens. Correct species identification is essential to the implementation of control measures for species of medical or agricultural importance. In this study, the usefulness of DNA barcoding was discussed in distinguishing species of Simulium. Analysis showed hidden biodiversity, usually referred to in Simuliidae as cryptic species, which was detected in 15 species. Firstly, intraspecific divergences of eleven species was unexpectedly high and the maximum distances of them ranged from 5.1-16.8%. Based on the differential of K2P (Kimura 2-Parameter) distances, sequences were subdivided into two or three groups, respectively. Secondly, extremely low interspecific divergences were detected in eight groups of species, and shared haplotypes were also found among them. Furthermore, the subdivision within species and shared haplotypes among some species were all supported by the NJ (Neighbour-Joining) analysis. Our results confirmed that DNA barcoding was a powerful tool for revealing hidden species diversity of black flies. Further work is needed to reveal ambiguous species delimitation in some problematic species groups.

Reassessment of species distribution and occurrence of mud crab (Scylla spp., Portunidae) in Malaysia through morphological and molecular identification.

This study utilized genetic and morphometric approaches to assess the molecular and morphometric differentiation among commercially important species of mud crab. Molecular investigations were derived from 542 bp mitochondrial DNA COI on 249 individuals within genus Scylla from nine states in Malaysia represents four marine regions; South China Sea, Sulu Sea, Straits of Singapore and Straits of Malacca. Four specimens were obtained from Indonesia to give a robust analysis in this study. For species delimitation, Automatic Barcode Gap Discovery (ABGD) method on a web interface was employed. Analysis on phylogenetics was implemented utilizing Neighbour joining (NJ) and Maximum Parsimony (MP) methods. The inter- and intraspecies genetic distances (Ds) was computed using Kimura 2-parameter distance and executed in MEGA version 5.05. All samples were genetically and morphologically identified and clustered into four distinct species. Among the species, S. olivacea was the most abundant (n = 111), on the other hand the occurrence of S. paramamosain in Malaysia was very low (n = 29). No single individual of S. serrata from Malaysia was recorded in this study. Both genetic distance and phylogenetic approaches exhibited a correlative monophyletic association among all specimens analysed. This present study is crucial as it reports the reassessment of all species within genus Scylla in Malaysia, eventually could be employed as a reference source for subsequent research mainly on mariculture and other conservation efforts for the species.

The combination of ITS2 and psbA-trnH region is powerful DNA barcode markers for authentication of medicinal Terminalia plants from Thailand.

The dried fruits of Terminalia plant (Combretaceae) called "Samo" have been used as herbal medicine in Thai traditional medicine. Four "Samo" crude drugs, namely, Samo thai, Samo thed, Samo dee-ngu, and Samo phiphek, are used as the main ingredients in Triphala and Trisamo recipes. Their commercial products are available in processed and powdered form, but are difficult to authenticate by conventional methods. In this study, we aimed to discriminate species of genus Terminalia for the identification of their crude drugs by a DNA barcoding technique. A total of 208 closely related nucleotide sequences were obtained from nine Terminalia species collected from Thailand and the DDBJ/EMBL/GenBank database. An effective DNA barcode marker was selected from six DNA loci (matK, rbcL, psbA-trnH, ITS, ITS1, and ITS2) and their two-locus combination. All sequences were analyzed by three major methods: (1) BLAST search; (2) the genetic divergence method using Kimura 2-parameter (K2P) distance matrices; and (3) tree topology analysis based on the neighbor-joining method. Comparison of the six candidate DNA loci indicated that ITS identified Terminalia with 100% accuracy at the species and genus levels in the BLAST1 method. ITS2 showed the highest K2P variability. The data from the single markers and the two-locus combinations revealed that only the two-locus combinations, namely, the combinations of rbcL, ITS, ITS1, and ITS2 with psbA-trnH, clearly discriminated all the species. From the results of DNA sequence analysis and the three methods, ITS2 is recommended for the identification of Terminalia species to supplement psbA-trnH.

Morphological description and DNA barcoding of Hydrobaenus laticaudus Sæther, 1976 (Diptera: Chironomidae: Orthocladiinae) from Amur River basin (Russian Far East).

Illustrated redescription of adult male and description for the first time of pupa and fourth instar larva, as well as DNA barcoding, of Hydrobaenus laticaudus Sæther in comparison with close related known species of Hydrobaenus Fries, and an updated part of a key to species of Hydrobaenus from the Russian Far East are provided. A reference 658 bp barcode sequence from a fragment of the mitochondrial gene cytochrome oxidase I (COI) was used as a tool for species delimitation. For the estimation of interspecific distances and constructing detailed Bayesian tree within Hydrobaenus, we have obtained DNA barcoding for the two other species, H. distinctus and Hydrobaenus sp.1. The intraspecific sequence divergence of H. laticaudus based on the Kimura-2-parameter (K2P) distance ranged from 0.0-0.031, whereas the interspecific sequence divergence based on the K2P distance ranged from 0.106-0.197% between H. laticaudus and other species of genus Hydrobaenus. The well-supported monophyly as well as results of an ABGD analysis confirms the validity of H. laticaudus.

A19 The origin of acute HCV in HIV-infected men who have sex with men in the Netherlands

In recent years, several outbreaks of hepatitis C virus (HCV) infections have been observed among HIV-infected men who have sex with men (MSM) in Europe. In the Netherlands, high incidence rates of 10/1,000 person-years are reported. In this analysis, we describe whether acute HCV is linked to specific transmission networks. A total of 50 Dutch HIV-infected MSM, diagnosed with acute HCV genotype 1a between 2013 and 2014, were included. Target enrichment for viral nucleic acid separation and deep sequencing were used to recover whole HCV genomes. Phylogenetic trees were constructed by use of the maximum likelihood method. Pairwise distance matrices were generated by use of the Kimura-2 parameter distance estimation method. The consistency of the phylogenetic clustering was tested by bootstrap analysis with 1,000 replicates. A cluster was defined as having a genetic distance of at most 1.5 per cent and bootstrap values of 100 per cent. The most recent common ancestor was estimated with a coalescent-based model with a Bayesian statistical framework. Four transmission clusters were identified that included a total of 38 patients (76% of the total). The clusters were indicative of recent outbreaks, as highlighted by small genetic distances and a most recent common ancestor after the year 2000, when the first cases of HCV infection in HIV-infected MSM were reported. The HCV epidemic among HIV-infected MSM is a young epidemic, with most of the acute infections linked within the four major transmission networks. Prevention strategies identifying and targeting these transmission networks can potentially curb the epidemic.

Two Hexapleomera species from Japan, with a new species descriptionand discussion of phylogenetic relationships within Hexapleomera(Crustacea: Tanaidacea).

We describe Hexapleomera sasuke sp. nov. and provide a supplemental redescription of Hexapleomera urashima Tanabe et al., both of which are based on specimens collected in Japan. Hexapleomera sasuke closely resembles H. urashima, sharing a uropod with four articles, maxillipedal endites with two tiny dorso-subdistal and two distal spiniform setae, a labium with the palp fused to the outer lobe, and the pereopod-1 propodus with an inner subdistal plumose seta. The new species differs from H. urashima in having the basal article of pleopod 3 with three outer plumose setae, the chelipedal carpus with three or four dorsodistal simple setae, the region between the bases of the chelipedal dactylus and fixed finger with three or four simple setae, and deeply pigmented pleopodal rami. We determined partial sequences of the cytochrome c oxidase subunit I (COI) gene (655 nt) from H. sasuke, which proved to be 15.0-15.3% divergent from H. urashima in Kimura 2-parameter (K2P) distance; also in partial sequences of 18S rRNA gene (1888 nt after alignment), 0.8% divergent (K2P distance) was detected between two species. Based on morphology and molecular data, we discuss phylogenetic relationships within Hexapleomera, and propose two morphologically distinct groups, the robusta group and the wombat group.

DNA barcode and minibarcode identification of freshwater fishes from Cerrado headwater streams in Central Brazil.

The extraordinary species diversity of the Neotropical freshwater fish fauna is world renown. Yet, despite rich species diversity, taxonomic and genetic resources for its Cerrado ichthyofauna remain poorly developed. We provide a reference library of 149 DNA barcodes for 39 species/lineages of Cerrado headwater stream fishes from the Brazilian Distrito Federal and nearby areas and test the utility of distance-based criteria, tree-based criteria and minibarcodes for specimen identification. Mean Kimura 2-parameter genetic distances within species to orders ranged 1·8-12·1%. However, mean intraspecific v. congeneric-interspecific distances (0·9-1·3%) overlapped extensively and distance-based barcoding failed to achieve correct identifications due to c. 4-12·1% error rates and 19·5% ambiguous identifications related to the presence of singletons. Overlap was reduced and best-match success rates improved drastically to 83·5% when Characidium barcodes representing potential misidentifications or undescribed species were removed. Tree-based monophyly criteria generally performed similarly to distance methods, correctly differentiating up to c. 85% of species/lineages despite neighbour-joining and Bayesian tree errors (random lineage-branching events, long-branch attraction). Five clusters (Ancistrus aguaboensis, Characidium spp., Eigenmannia trilineata, Hasemania hanseni and Hypostomus sp. 2) exhibited deep intraspecific divergences or para-/polyphyly and multiple Barcode Index Number assignments indicative of putative candidate species needing taxonomic re-examination. Sliding-window analyses also indicated that a 200 bp minibarcode region performed just as well at specimen identification as the entire barcode gene. Future DNA barcoding studies of Distrito Federal-Cerrado freshwater fishes will benefit from increased sampling coverage, as well as consideration of minibarcode targets for degraded samples and next-generation sequencing.

Insight into the species diversity of the quill mite genus Betasyringophiloidus Skoracki, 2011 (Prostigmata: Syringophilidae) on the basis of the DNA barcodes

Betasyringophiloidus Skoracki, 2011 is a genus of quill mites (Prostigmata: Syringophilidae) that is believed to contain mono-, steno- and polyxenous parasites associated with a wide range of passerine birds (Passeriformes) across the world. In this work we applied the DNA-barcode marker (mitochondrial cytochrome c oxidase subunit I gene fragment, COI) to verify whether Betasyringophiloidus schoeniclus (Skoracki, 2002) and Betasyringophiloidus seiuri (Clark, 1964) are actual steno- and polyxenous species associated with the currently recognised host ranges, or their populations are highly host-specific, cryptic species. Our results revealed that a population living on the Tristram's bunting Emberiza tristrami Swinhoe (Emberizidae) in Russia, so far classified as B. schoeniclus, is a new cryptic species Betasyringophiloidus emberizae sp. nov. Both topologies of the neighbor-joining and maximum likelihood phylogenetic trees as well as genetic distance (11.9% Kimura 2-parameter distance) (K2P) support species status of the mite population from E. tristrami. The same data support previously established conspecific status of B. seiuri found on the ovenbird Seiurus aurocapilla (Linnaeus) (Parulidae) (type host) and the northern waterthrush Parkesia noveboracensis (Gmelin) (Parulidae) and expand its range with a population found on a new host species Icterus pustulatus (Wagler) (Icteridae) with intraspecific K2P distance up to 1.9% and interpopulation distances ranging from 1.3 to 3.1%.

Insights into cryptic diversity and adaptive evolution of the clam Coelomactra antiquata (Spengler, 1802) from comparative transcriptomics

The clam Coelomactra antiquata (Spengler, 1802) is one of the most economically important marine bivalves in China. Previous genetic analysis shows that C. antiquata possibly consists of two cryptic species, a northern and a southern lineage. Some differences between the two lineages are observed in their morphology and fitness in aquaculture. However, little is known about the genetic basis of these differences. In this study, a comparative transcriptomics study was performed to provide additional information for delineating the cryptic species of C. antiquata, as well as insights into the adaptive evolution in the southern lineage of C. antiquata. A total of 4377 orthologous genes were identified using a phylogenetic tree-based approach. The Kimura 2-parameter genetic distances were 0.0385 between the two lineages of C. antiquata, and 0.006 within the northern lineage. The sequence divergences supports that the lineages represent two cryptic species. We identified 28 positively selected genes (PSGs) from the southern lineage of C. antiquata using a modified branch-site model. The PSGs were implicated in innate immune responses, metabolism, development, cell division, transmembrane transport, and signaling processes, which could be related to the higher disease resistance, growth rate, and adaptability of the southern lineage than its northern counterpart. The comparative transcriptome analysis provides a foundation for future investigations on the biological differences between the two lineages of C. antiquata.

Molecular identification of the Danzhou chicken breed in China using DNA barcoding

Mitochondrial cytochrome C oxidase subunit I (COI) has been used as a DNA barcode to identify population genetic diversity and distinguish animal species as it is variable enough to distinguish between species, yet suitably conserved. A new native chicken breed, named Danzhou chicken was discovered in Hainan, China in 2014, although identification is difficult by morphological examination alone. The mitochondrial COI genes of six chicken breeds, including four local and two imported breeds (Danzhou, Wenchang, Bawang, Beijing-You, Hy-Line Brown, and Ross) were compared and assessed in terms of their efficacy for DNA barcoding. The results showed that the number of COI gene variants in Danzhou chickens was less than those of other breeds, except Bawang and the genetic structure was relatively stable. The Kimura 2-parameter genetic distance between Danzhou chickens and the five other breeds was from ∼0.001 to 0.734. The genetic distance of the six breeds was ∼0.001–0.339, with that of Danzhou being the highest (0.339). Danzhou chickens clustered with Bawang and Wenchang chickens in the phylogenetic tree due to geographic closeness. Danzhou chickens could be identified more accurately using COI barcoding. Multiple molecular markers combined with morphological differences were more persuasive for identifying species.

DNA barcoding reveals underestimated species diversity of mantis shrimp larvae (stomatopods) in Banten Bay, Indonesia

Abstract. Abinawanto, Intan MDB, Wardhana W, Bowolaksono A. 2019. DNA barcoding reveals species diversity of mantis shrimp larvae (stomatopods) from Banten Bay, Indonesia. Biodiversitas 20: 1758-1763. Larvae of stomatopods can be morphologically identified by linking between larval and adult form, but it's difficult applied in the laboratory because of requirement of condition to rearing larvae collected either from plankton, or spawning adults in the laboratory. Molecular methods are the alternative approach to gather accurate taxonomic identification. One of molecular methods is DNA barcoding based upon sequence diversity in cytochrome c oxidase subunit 1 (COI). Research on stomatopod larvae diversity and distribution at six stations in Banten Bay has been studied from October 2013 to March 2014. This study was conducted to identify the diversity of stomatopod larvae through DNA barcoding and also to examine the distribution of stomatopod larvae in Banten Bay. DNA barcoding was used to identify mantis shrimp larvae in Banten Bay, Indonesia. Partial mitochondrial COI were obtained for 37 larvae, revealing five distinct molecular operational taxonomic units (MOTUs). All MOTUs distinctively fell into Squillidea clade. Two Molecular Operational Taxonomic Units, MOTUs were successfully identified to species level (i.e. Anchisquilla fasciata), while three others remained identified to genus level (i.e. Stomatopoda spp). The intraspecific variation of COI ranged from 0% to 3.1% (Kimura 2-parameter distance), whereas interspecific divergence ranged from 5.9% to 20.4%. These results imply that COI genes are suitable for species identification of stomatopod larvae in this area, though the number of unidentified species was higher than the identified ones. This is likely because of the limited database in GenBank. In addition, the two identified species represents new records for this area, suggesting that the mantis shrimp in the area have been underestimated based on traditional biodiversity measures. We recommend to develop a taxonomically-comprehensive DNA barcoding database to improve the accuracy and feasibility of using DNA barcoding for species identification of Indonesia mantis shrimps.

Genetic status of indigenous poultry (red jungle fowl) from India

The global biodiversity of domesticated red jungle fowl (Gallus gallus) is gradually eroding by replacement with commercial poultry breeds and results loss of valuable genetic and physical traits like resistance to disease, extreme environment, etc. posing a threat to the poultry genetic resources. Very fewer reports exist on Indian poultry diversity, especially native chicken of India. Therefore, species identification and inventorying of the poultry genetic resource is indispensable. Thus, the present study aimed to characterize indigenous chicken from bio-diversity hotspot of Sunderban and Northeast India using DNA sequence based barcoding approach. A total of 15 CO1 (Cytochrome c Oxidase-I) DNA barcode of different indigenous chicken were newly sequenced along with 6 previously published sequences from our laboratory and compared with the available data of distinctive genera of Phasianidae as per the standard protocol and are identified as Gallus gallus. About 98.96% of the Phasianid birds were successfully delimitated into the respective species except for 12 congeneric pairs whose minimum interspecific K2P (Kimura 2-parameter) distance overlaps with the maximum intraspecific distance (3.9%). The least genetic divergence is observed between G. gallus and G. varius (0.013%) and highest between G. gallus and G. lafayettei (0.059%). The NJ tree showed a cohesive clustering of indigenous chicken with G. gallus and distinct with respect to all the different species under study, thereby revealing their taxonomic position except for few G. sonneratti that showed mixed clustering with G. gallus. This may be due to the genetic introgression between the species. Nevertheless, the study for the first time provided the molecular identification tag of indigenous poultry from biodiversity hotspot of East and Northeast India and will remain as a potential guide to recognize inimitable and valuable poultry genetic resources for future needs.

DNA barcoding of fishes from Lake Lanao, Philippines

Lake Lanao, the second largest lake in the Philippines and one of the 15 ancient lakes in the world, used to contain 20 endemic cyprinid species, which had attracted the attention of evolutionary biologists in the past. Over the years, there has been a steady decline in the abundance and diversity of endemic fishes in the lake because of overfishing and introduction of non-native species. This study represents the first molecular survey of the ichthyofauna of Lake Lanao. A total of 75 specimens of 12 different species belonging to nine genera, eight families, and five orders were DNA barcoded using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Average Kimura 2-parameter genetic distances were 0.24% (within species), 8.31% (between species), 9.69% (within family), and 24.86% (between families). Possible hybrids between Oreochromis species were detected. Glossogobius spp. was highlighted for further taxonomic investigation because barcoding indicated unidentified species of this genus. The partial sequence of mitochondrial COI gene was found to be a good DNA barcode for fast and accurate species identification of fishes in Lake Lanao and for tagging species that warrant further taxonomic investigation.

Mermithid parasitism of shoot borer (Conogethes punctiferalis) infesting ginger and turmeric and its biocontrol potential

A mermithid nematode parasitic to larvae ofConogethes punctiferalis, a serious pest of ginger and turmeric was recorded under field conditions during 2015 at Peruvannamuzhi (Kerala, India). The infection reached epizootic levels during July to September 2015 exceeding 50% mortality in host insect populations and the parasitism ranged from 18.2 to 80.6% and 17.9 to 66.7% inC. punctiferaliscollected from ginger and turmeric, respectively. The level of host parasitism by the mermithid was positively correlated with rainfall and negatively influenced by maximum temperature. Molecular analysis of the partial 18S small ribosomal subunit gene region and phylogenetic analysis with other mermithid sequences available in the GenBank indicated that the present nematode clustered with other nematodes belonging to different genera and hence could not be attributed to a specific genus. The pairwise Kimura 2‐parameter (K2P) distance revealed that the closest taxon to the study nematode was an undescribed mermithid species reported to infect slugs with a K2P distance of 0.009. The epizootics caused by this mermithid nematode in the natural populations ofC. punctiferalissuggest its potential as a biocontrol agent against a polyphagous pest of international importance. The results of our studies provide a basis for using this nematode as a biocontrol agent for developing integrated pest management strategies againstC. punctiferalis.

DNA barcoding of marine fish species from Rongcheng Bay, China.

Rongcheng Bay is a coastal bay of the Northern Yellow Sea, China. To investigate and monitor the fish resources in Rongcheng Bay, 187 specimens from 41 different species belonging to 28 families in nine orders were DNA-barcoded using the mitochondrial cytochrome c oxidase subunit I gene (COI). Most of the fish species could be discriminated using this COI sequence with the exception of Cynoglossus joyneri and Cynoglossus lighti. The average GC% content of the 41 fish species was 47.3%. The average Kimura 2-parameter genetic distances within the species, genera, families, and orders were 0.21%, 5.28%, 21.30%, and 23.63%, respectively. Our results confirmed that the use of combined morphological and DNA barcoding identification methods facilitated fish species identification in Rongcheng Bay, and also established a reliable DNA barcode reference library for these fish. DNA barcodes will contribute to future efforts to achieve better monitoring, conservation, and management of fisheries in this area.