Genetic Species Identification Research Articles

Accurate population genetic measurements require cryptic species identification in corals

Correct identification of closely related species is important for reliable measures of gene flow. Incorrectly lumping individuals of different species together has been shown to over- or underestimate population differentiation, but examples highlighting when these different results are observed in empirical datasets are rare. Using 199 single nucleotide polymorphisms, we assigned 768 individuals in the Acropora hyacinthus and A. cytherea morphospecies complexes to each of eight previously identified cryptic genetic species and measured intraspecific genetic differentiation across three geographic scales (within reefs, among reefs within an archipelago, and among Pacific archipelagos). We then compared these calculations to estimated genetic differentiation at each scale with all cryptic genetic species mixed as if we could not tell them apart. At the reef scale, correct genetic species identification yielded lower FST estimates and fewer significant comparisons than when species were mixed, raising estimates of short-scale gene flow. In contrast, correct genetic species identification at large spatial scales yielded higher FST measurements than mixed-species comparisons, lowering estimates of long-term gene flow among archipelagos. A meta-analysis of published population genetic studies in corals found similar results: FST estimates at small spatial scales were lower and significance was found less often in studies that controlled for cryptic species. Our results and these prior datasets controlling for cryptic species suggest that genetic differentiation among local reefs may be lower than what has generally been reported in the literature. Not properly controlling for cryptic species structure can bias population genetic analyses in different directions across spatial scales, and this has important implications for conservation strategies that rely on these estimates.

Fresh is best: Accurate SNP genotyping from koala scats.

Maintaining genetic diversity is a crucial component in conserving threatened species. For the iconic Australian koala, there is little genetic information on wild populations that is not either skewed by biased sampling methods (e.g., sampling effort skewed toward urban areas) or of limited usefulness due to low numbers of microsatellites used. The ability to genotype DNA extracted from koala scats using next‐generation sequencing technology will not only help resolve location sample bias but also improve the accuracy and scope of genetic analyses (e.g., neutral vs. adaptive genetic diversity, inbreeding, and effective population size). Here, we present the successful SNP genotyping (1272 SNP loci) of koala DNA extracted from scat, using a proprietary DArTseq™ protocol. We compare genotype results from two‐day‐old scat DNA and 14‐day‐old scat DNA to a blood DNA template, to test accuracy of scat genotyping. We find that DNA from fresher scat results in fewer loci with missing information than DNA from older scat; however, 14‐day‐old scat can still provide useful genetic information, depending on the research question. We also find that a subset of 209 conserved loci can accurately identify individual koalas, even from older scat samples. In addition, we find that DNA sequences identified from scat samples through the DArTseq™ process can provide genetic identification of koala diet species, bacterial and viral pathogens, and parasitic organisms.

Research Article New insights into the genetic diversity and species identification of the native apricots in Southern Xinjiang of China

Apricot is a staple stone fruit crop cultivated in Southern Xinjiang of China. This crop is important for the rural communities, as they generate significant employment and income. Here, seventy-eight apricot genotypes, including seventy-six common apricots (Prunus armeniaca L.) and two purple apricots (Prunus dasycarpa Ehrh.), were mainly collected from Aksu, Kashgar, Hetian and Bayingolin. Start Codon Targeted (SCoT) markers and ITS (internal transcribed spacers) sequences were used to investigate the genetic diversity and species identification respectively. Based on POPGENE showed that apricot cultivars from Aksu group exhibited the highest genetically diverse as compared with other groups, cluster analysis of SCoT markers (basede on UPGMA and PCoA method) showed that these apricot cultivars could be divided into three major clusters, which was in agreement with their geographic distribution and pedigrees. It was indicated that there were possible three primary diversity centers in the area: Aksu, Kashgar and Hetian, and also possible introgression among these populations. Furthermore, based on the complete ITS sequences, the phylogenetic analysis showed that P. dasycarpa clustered separated from other the section armeniaca of species. Therefore, it was proposed that P. dasycarpa would be a hybrid species. Our results indicated that SCoT markers are informative and could be used to evaluate genetic diversity of apricots, and ITS could be used effectively to identify P. dasycarpa. These results will provide much more useful information for the native apricots protection and utilization strategies.

Молекулярные маркеры, применяемые для определения генетического разнообразия и видоидентификации дикорастущих растений.

Молекулярные маркеры, применяемые для определения генетического разнообразия и видоидентификации дикорастущих растений.

The complete mitochondrial genome of the short snouted seahorse Hippocampus hippocampus Linnaeus 1758 (Syngnathiformes: Syngnathidae) and its phylogenetic implications

The short-snouted seahorse Hippocampus hippocampus Linnaeus 1758 is a native European seahorse within the Family Syngnathidae. To facilitate its conservation genetic studies, the complete mitochondrial genome of H. hippocampus was determined and analyzed. The circular genome of the H. hippocampus is 16,529 bp in length, with a overall nucleotide composition of 32.03, 24.06, 15.05 and 28.86% for A, C, G and T, respectively, indicating an obvious AT rich feature (60.89%). Similar to other Hippocampus species, it contains 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, one origin of replication on the light-strand and one control region. The 13 PCGs encode 3798 amino acids in total, most of which use the initiation codon ATG except COI gene starts with GTG. For the stop codon, six genes use an an incomplete stop codon T, while the other seven genes apply TAA or TAG as the stop codon. The lengths of 12S and 16S rRNA are 937 and 1695 bp, respectively. Phylogenetic tree analysis based on the complete mitochondrial genome showed that H. hippocampus is closely related to the lined seahorse H. erectus. This work provides basic genetic data of H. hippocampus which will be helpful for further researches on genetic diversity and species identification.

A survey of zoonotic nematodes of commercial key fish species from major European fishing grounds—Introducing the FP7 PARASITE exposure assessment study

Harvesting and exploiting limited fisheries resources in a sustainable manner also implies achieving maximum added value from the raw material. However, the presence of parasites in the products may adversely affect consumer perception and/or pose a direct health hazard. As a major stepping-stone of the PARASITE project, an epidemiological survey was carried out to provide the basis for analysis and prediction of consumer exposure risk due to the presence of anisakid nematodes in fish from European wild-catch fisheries. The project consisted of nine separate workpackages (WP) where the exposure risk assessment survey was organized within WP2. The surveillance task also provided the data or samples needed for data management and sample storage (WP3, Biobank), molecular and genetic parasite species identification (WP4), and statistical modelling and inference (WP8). In total 17,760 fish belonging to 16 teleost species were examined for anisakids, with special emphasis on economically and ecologically important species such as Atlantic mackerel, herring, European hake, Atlantic cod and anchovy. The target fish species were sampled at four major European fishing areas including the Barents Sea, North Sea, Baltic Sea, Grand Sole Bank, waters off NW Spain and Portugal, central and western parts of the Mediterranean Sea, and the Adriatic Sea. Thus, the survey represents the largest and most comprehensive epidemiological data compilation of anisakids ever generated in terms of geographical range as well as number of fish host species and sample size. An important requirement of the survey was the use of commonly accepted nematode detection methods, i.e. the UV-press method or artificial digestion, to quantify infection level and spatial distribution of anisakid larvae in the target fish species. The basic layout, set-up and principles of the method, along with a discussion of possible source of errors are described. Additionally, the molecular and genetic markers which were used to identify and characterize different species and populations of anisakids from the targeted fish host species and geographical areas, are reviewed as well. Some basic parasite infection characteristics of each fish host species, and any relationships with the presumably most important infection predictors, i.e. fish host body size and fishing locality, are presented and discussed. Emphasis is put on anisakid occurrence in the flesh of the fish. Based on the findings, a graphical exposure risk profile is introduced, including fish species or products thereof, which due to common processing or preparation practices, are at highest risk to act as source of anisakiasis in Europe.

Genetic identification and antimicrobial susceptibility of clinically isolated anaerobic bacteria: A prospective multicenter surveillance study in Japan

This prospective multicenter surveillance study was designed to provide antimicrobial susceptibility profiles of clinical anaerobic bacteria with genetic species identification in Japan. In 2014, a total of 526 non-duplicate clinical anaerobic isolates were collected from 11 acute-care hospitals in the Kyoto and Shiga regions of Japan. Genetic identification was performed using 16S rRNA sequencing. Minimum inhibitory concentrations were determined in the central laboratory and were interpreted using the CLSI criteria. Genetic analysis provided species-level identification for 496 isolates (83 species in 40 genera) and genus-level identification for 21 isolates (13 genera). Among these 517 isolates, the most frequent anaerobes were Bacteroides spp. (n = 207), Prevotella spp. (n = 43), Clostridium spp. (n = 40), and Peptoniphilus spp. (n = 40). B. fragilis was the most common species (n = 107) and showed 91.6%–97.2% susceptibility to β-lactam/β-lactamase inhibitor combinations (BLBLIs; ampicillin-sulbactam, amoxicillin-clavulanate, and piperacillin-tazobactam) and carbapenems (imipenem and meropenem) as well as 100% susceptibility to metronidazole. Gram-negative anaerobes were highly susceptible to metronidazole (99.0%) followed by BLBLIs and carbapenems (>90% each). BLBLIs or carbapenems also retained activity against Gram-positive anaerobes (99.5%–100%) except Clostridioides difficile. All isolates were susceptible to combinations of metronidazole with BLBLIs or carbapenems. Thus, BLBLIs or carbapenems are first choices for empirical therapy of anaerobic infections in Japan, and these antimicrobials in combination with metronidazole should be reserved for very severe infections and targeted therapy.

Anisakis species composition and infection characteristics in Atlantic mackerel, Scomber scombrus, from major European fishing grounds — reflecting changing fish host distribution and migration pattern

Atlantic mackerel (Scomber scombrus) ranks among the most valuable fish species in Europe. The NE Atlantic mackerel population is considered to comprise three main stocks (southern, western and North Sea), with variable proportions of these three intermixing at the northerly feeding grounds. The southern and western mackerel stocks have moved over the past 4–5 years further north- and westward. Consequently, large-scale mackerel fishing and processing have become thriving industries in Iceland and the Faroe Islands in just a few years. The mackerel population structure in the Mediterranean Sea is less well known but seems to comprise at least one, more or less isolated, spawning component. Although mackerel is an important food resource, systematic and concerted epidemiological surveys of Anisakis species in Atlantic mackerel from European fishing grounds have been lacking. As part of the EU FP7 PARASITE project (GA no. 312068), occurrence and specific identity of Anisakis spp. from 1801 mackerel from Northeast Atlantic and Mediterranean waters was investigated. In general, mackerel caught at the Atlantic fishing grounds exhibited markedly higher Anisakis spp. infection levels than fish from the Mediterranean localities. Mackerel caught off NW Spain and Portugal (ICES VIIIc, IXa) showed highest overall and muscular prevalence, reaching 87% and 52%, respectively, which differed significantly from all other Atlantic samples. Lowest overall Anisakis spp. prevalence and abundance was recorded in mackerel from Faroe Isles waters, while lowest muscular infection levels were found in the samples from the North Sea. Genetic nematode species identification showed that A. simplex (sensu stricto) is the dominating species in mackerel from the Atlantic areas, while A. pegreffii dominated in the samples from the Mediterranean Sea. The latter species showed generally low prevalence and intensity in the flesh, not exceeding 6% and one larva, respectively. While A. simplex (s. s.) and A. pegreffii seem to co-occur in mackerel from off NW Spain and Portugal, several A. pegreffii were also recorded in mackerel from the North- and Norwegian Seas. These findings imply that the actual mackerel started their feeding migration in waters south to the British Isles, which include parts of the sympatric area of the two sibling species. Thus, A. pegreffii could prove a useful supplementary marker to track migration routes of the different mackerel stock components in NE Atlantic waters.

Discovering the dominance of the non-native European eel in the upper reaches of the Tone River system, Japan

To investigate the presence of non-native anguillid eels in Japanese waters, 141 eels were collected from seven sampling sites throughout the Tone River system. Genetic species identification showed an extraordinary dominance of the non-native European eel Anguilla anguilla in the uppermost site of the study area. Estimation of age from otoliths suggested that the European eels were introduced into the river in the 1990s, corresponding to previous reports from other Japanese water systems. Comparison of the von Bertalanffy growth curve parameters indicated that the European eels caught in the Tone River system appeared to have a similar or even higher growth rate than the same species in the original habitats in Europe. The long-term inhabitation and the normal development of European eels in Japanese waters suggest that regulations prohibiting the release of non-native eels and safeguards against accidental escape from culture ponds must be strictly maintained in order to ensure the conservation of the native Japanese eel.

Genetic identification of macroalgal species on Japanese tsunami marine debris and genetic comparisons with their wild populations

Since 2012 a huge amount of marine debris caused by the 2011 Great East Japan Earthquake and Tsunami has been arriving on Northeastern Pacific shores. Often healthy macroalgae were attached to them, which may become introduced to the Northwestern Pacific coasts and disturb their ecosystems. In order to elucidate the diversity of those macroalgae, and to establish a basis for detecting their new introduction to Northwestern Pacific coasts, we have examined their species diversity by morphology and genetic identifications. We have obtained gene sequences for 205 specimens, and identified 49 species as Japanese Tsunami Marine Debris (JTMD) macroalgae. Most of them are known to be distributed in Japan and showed identical or very closely related genetic types to those of Tohoku, and confirmed to be originated from the area. Several species such as Ceramium sungminbooi, Ectocarpus crouaniorum, Polysiphonia koreana, etc. have not been reported from Japan, but this is explained by the shortage of taxonomic information.

Saccharomyces jurei sp. nov., isolation and genetic identification of a novel yeast species from Quercus robur.

Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain.

Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions.

The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment length polymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.

Genetic identification of prey species from teeth in faeces from the Endangered leopard cat Prionailurus bengalensis using mitochondrial cytochrome b gene sequence

To understand the dietary ecology of the leopard cat (Prionailurus bengalensis), DNA analysis was performed to identify prey species using DNA isolated from teeth harvested from the faeces of this feline species. From 70 DNA samples, a total of 52 mitochondrial DNA (mtDNA) cytochrome b (cytb) gene sequences of mammals were identified. The results of a sequence identity test indicated that those sequences were derived from four rodent species (Apodemus agrarius, Apodemus peninsulae, Eothenomys regulus and Tamias sibiricus) and two shrew species (Crocidura lasiura and Crocidura shantungensis). The sequences contained nine unique cytb sequences from site 1 and 13 from site 2. These results indicate that the leopard cat hunts rodents and shrews, and at least nine animals at site 1 and 13 animals at site 2 were eaten. These findings suggest that the animal molecular signatures that remain undigested in the faeces may provide useful ecological information about food items and may contribute to a better understanding of the leopard cat’s feeding ecology.

Genetic species identification in weatherfish and first molecular confirmation of Oriental Weatherfish Misgurnus anguillicaudatus (Cantor, 1842) in Central Europe

The Oriental Weatherfish is considered a globally invasive fish species. In Europe, several reported feral populations of Oriental Weatherfish display an overlapping distribution range with native weatherfish Misgurnus fossilis , a declining species of international conservation and aquatic management concern. Morphologically distinguishing the different weatherfish species can be difficult, as their coloration is highly variable, many species reveal high phenotypic plasticity, and morphological traits like coloration might be not obvious or might be degraded during field sampling and after preservation. Herein, we analysed suspicious weatherfish specimens from southern Germany, demonstrating the usefulness of molecular genetic species identifications in this genus. We present the first molecular genetic species record of Misgurnus anguillicaudatus in Central Europe, and confirm the range expansion of Oriental Weatherfish into the river Inn catchment in southern Germany. As accurate species identification is crucial both in the context of monitoring and conserving native endangered species, and in early detection and prevention of biological invasion, we suggest the standard use of genetic species identification if morphological traits are not obvious.

Genetic identification of Aeromonas species using a housekeeping gene, rpoD, in cultured salmonid fishes in Gangwon-Do

Genetic identification of Aeromonas species using a housekeeping gene, rpoD, in cultured salmonid fishes in Gangwon-Do

Isolation and characterization of polymorphic microsatellite markers for the chub mackerel (Scomber japonicus) and cross-species amplification in the blue mackerel (S. australasicus).

In this study, 10 polymorphic microsatellite markers were developed in Scomber japonicus and were examined on 30 individuals collected from the North Pacific. The number of alleles per locus ranged from 4 to 17. The observed and expected heterozygosities per locus ranged from 0.2759 to 0.8621 and from 0.43071 to 0.9177, respectively. The polymorphism information content (PIC) was from 0.3931 to 0.8939. One locus showed moderate polymorphism (0.25 < PIC < 0.5), while the rest were highly polymorphic (PIC > 0.5). Two loci showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni corrections (P < 0.005). No linkage disequilibrium was detected among the loci. Results of cross-species amplification showed that 10 microsatellite markers were successfully amplified in 29 individuals of S. australasicus and 9 indicated polymorphisms. These markers will be useful for investigating the genetic structure, gene flow, and species identification of S. japonicus and S. australasicus, its closely related species.

Genetic identification of new microalgal species from Epe Lagoon of West Africa accumulating high lipids

The green microalgal strains are favorable alternatives for producing biodiesel. In spite of the worldwide awareness on microalgae-derived oil to potentially replace the conventional fossil fuel, information on potential microalgal strains from Nigerian lagoons is scarce. In view of this, environmental samples were collected from the Epe Lagoon, Nigeria to assess the biodiversity of microalgae and identify robust strains for biodiesel production. The field samples were collected and genomic DNA was isolated from the monoclonal cultures. Microalgal species were identified based on the PCR amplified DNA sequences of 18S ribosomal DNA. The sequences of PCR products from the unknown samples were compared with the DNA database. Using the phylogenetic maximum-likelihood trees, the green algal strains Desmodesmus sp., Desmodesmus costato-granulatus, Tetradesmus sp. and Chlorella sp. were identified, while two strains belonging to the Chlorococcaceae and Family Selenastraceae need further validation. Microalgal species morphology was validated using electron microscopy. Lipid analysis was carried out using Fatty Acid Methyl Ester (FAMEs) analysis, Gas Chromatography and Mass Spectrometry (GC–MS). The strain of the Family Selenastraceae produced the maximum amount of poly-unsaturated fatty acids (56%), which has potential for food processing and other bio-based products; while Tetradesmus sp. with 35.16mg (FAMEs per gram of dry cell weight) had a balanced lipid composition (44% saturated fatty acid, 39% mono-unsaturated fatty acid, 15% poly-unsaturated fatty acids) making it a good candidate for exploitation as a biodiesel feedstock.

Early life history of oxudercine gobyPseudapocryptes elongatusin the Mekong Delta, Vietnam

Pseudapocryptes elongatusis one of the oxudercine gobies, which show varying degrees of amphibious behaviour and capacities to breathe air. There is little information on the early life history ofP. elongatus, particularly of their morphology and larval habitat and duration. This study focused on the life history of larval and juvenile stages ofP. elongatusinvestigated by genetic species identification, morphological observation and otolith analyses using specimens collected in June and October 2012 from estuaries in Bac Liêu Province, southern Vietnam (09°14′N 105°43′E). Genetically identified juvenileP. elongatuswere characterized by (1) a slender body form, (2) the anterior edges of both the anal and second dorsal fins located at the midpoint along the body axis, (3) scarce chromatophores over the body surface, (4) melanophores in the parietal region between the eyes, and (5) a single row of melanophores along the base of the anal fin. Mean age at recruitment to estuaries was 38.0 ± 4.1 days, and otolith Sr/Ca ratios ranged from 8.9 to 9.9 mmol mol−1, suggesting that larval migration from their spawning sites requires more than 1 month in saline environments.

Species identification of echinoderms from the North Sea by combining morphology and molecular data

BackgroundTaxonomic uncertainties in the morphological species identification and taxonomic revisions in individual groups are known for all echinoderm classes. These uncertainties in morphological species identification and discrimination have spawned the application of molecular genetic identification techniques. However, as the fundamental step to allow and ensure future molecular species identification, valid and comprehensive reference library entries comprising morphological and molecular species information together with various metadata are essentially needed. In our study we compare morphological and molecular genetic species identification techniques for representatives of North Sea echinoderm classes, i.e. the Asteroidea, Ophiuroidea, Echinoidea and Holothuroidea.MethodsIndividuals were sampled during different surveys in different regions of the North Sea, identified to species level based on morphological diagnostic features, and were genetically analysed using a fragment of the mitochondrial cytochrome c oxidase subunit I (COI).Results and DiscussionThe morphological determination revealed 32 species including one taxon determined only to genus level. In contrast to this, the COI analysis supported 34 monophyletic clades with pronounced differences between the intra- and the inter-specific genetic variability (a barcoding gap of 4.93 %) with highest intra-specific variabilities found in the ophiuroid species Amphiura filiformis, A. chiajei and Ophiura sarsii. In 94 % of the investigated species, morphological identification and COI sequence clusters were congruent whereas for two asteroid species we found an underestimated diversity. For Astropecten irregularis, one of the most common starfish species of the North Sea, we found two distinct and possibly depth-related clades, probably sibling species, differing by 11.1–11.9 % sequence divergences (p-distances). For two starfish individuals, morphologically identified as Henricia sanguinolenta, the COI analysis revealed two monophyletic clades, of which one was classified as H. cf. oculata by comparison to published sequences.ConclusionsThis newly established sequence reference library for the North Sea Echinodermata allows and ensures future molecular species identification for various life-cycle stages including juveniles and meroplanktonic larvae and provides sequences for phylogeographic studies and the detection of sibling as well as cryptic species.

Threading: A novel insilico indagation method for genetic characterization of some diplostomoid metacercariae (Digenea:Diplostomidae Poirier, 1886)

The protein encoding zone of Mitochondrial DNA region (inherited from single lineage) seems most suitable and effective for taxonomic, systematic, ecological, evolutionary, DNA barcoding, cryptic species and population studies, exploiting nucleotide/amino acid datasets (1D/2D/3D conformational level). Nowadays, expeditious computerized methods are in trend for analyzing genetic material to demonstrate variations at various levels of protein structures. Structural proteomics have implemented here for genetic identification, differentiation and relationship of species from information rich data of mt COI gene of the family Diplostomidae with inclusion of molecular tools. Various aspects have been utilized herein for re-validation and infallible discrimination of Trematode diplostomoid metacercariae (Tetracotyle lucknowensis Pandey, 1971; T. xenentodoni Chakrabarti, 1970; T. fausti Rai and Pande, 1969; T. muscularius Chakrabarti, 1970 and Diplostomulum minutum Pandey, 1968), the infective stage in the life cycle, causing severe damage to fish host, whose adults are found mainly in fish eating birds and mammals.