Forensic DNA Analysis of Pacific Salmonid Samples for Species and Stock Identification

Identification of salmonid tissue samples to species or population of origin has been conducted for over 20 forensic cases in British Columbia. Species identification is based on published sequence variation in exon and intron regions of coding genes. Identification of source populations or regions is carried out using microsatellite and major histocompatibility complex allele frequency data collected from populations throughout the species range and with standard genetic stock identification (GSI) methods. Regional contributions to mixture samples are estimated using maximum likelihood mixture analysis and classification of individual genotypes is carried out with Bayesian methods. DNA has been obtained successfully from salmon scale samples, fresh, frozen and canned tissue samples and bloodstains in clothing. Results from DNA analyses have been instrumental in a number of convictions. A major benefit has been cost savings resulting from the number of guilty pleas entered after disclosure to the defendant of results from genetic testing. In two cases, GSI analysis resulted in exoneration of suspects under investigation for possible illegal sales of Fraser River sockeye salmon by substantiating their claim that the fish originated from the Skeena River watershed. DNA analysis has generally corroborated the species and stock identification carried out by fishery officers, but has revealed that species identification of samples from sources such as restaurants and fish plants can be erroneous. Forensic DNA analysis has facilitated the conviction of those who purchase fish not caught under the authority of licence, thus bringing those who buy fish illegally as well as those involved in illegal harvest and sales within the scope of law enforcement.     

Species identification of small fish in Xixuan Island coastal waters of Zhoushan using DNA barcoding

This study aimed to assess the fish species composition in Xixuan Island coastal waters and confirm the applicability of DNA barcoding in fish identification. Fish samples were caught with a gill net in December 2016 and in January, February, April, May and June 2017, and the fish species in these samples were analyzed and identified via both traditional morphological methods and DNA barcoding (mitochondrial 12S rRNA). The sample contained 1,357 individuals, the majority of which were Tridentiger barbatus (829 individuals). A total of 154 individuals were selected for DNA barcode analysis, and 147 sequences were obtained. A total of 35 species, which belonged to six orders and 16 families, were accurately identified. One species was identified to the genus level (Repomucenus), and only one was identified to the family level (Moridae). Most of the species belonged to Gobiidae (12 species). With the exception of those in Moridae, the identified species were commonly detected on the Zhejiang coast. The average Kimura two-parameter (K2P) distances within species, genera, families, and order and between orders were 0.15%, 4.44%, 14.96%, 20.37%, and 26.20%, respectively. The intraspecific K2P distance was markedly greater than the interspecific distance. The A + T content (55.41%) was higher than the G + C content (44.58%). Phylogenetic tree analysis showed that individuals belonging to the same species were clustered together and could be clearly distinguished. Mitochondrial 12S rRNA can be effectively used for juvenile and adult fish identification and has good application prospects in fish species identification. The investigation and species identification of small fish collected monthly from Xixuan Island coastal waters are conducive to understanding the distribution characteristics and species composition of fish in the Zhoushan coastal area.     

物种与物种多样性

本文首先讨论生物物种的科学概念和生物学本质，分析物种客观存在的自然属性和物种概念的局限性，认为物种的生物学属性和物种多样性的科学属性之间有着本质联系。物种多样性研究的实质是研究生物物种的生物学多样性。度量物种多样性程度有多种方法，但物种数目是物种多样性程度最直接、也是最基本的表达，估计物种多样性数目是当前国际上物种多样性研究的核心与热点内容。物种多样性产生的根源是物种形成，物种绝灭速率是维持物种多样性的关键因素。本文简要总结了物种形成与绝灭的基本模式和机制，通过分析生物地理区系与物种多样性研究的密切关系，说明物种的区系成份分析是物种多样性大尺度格局研究的重要内容。     

Species Identification of Dermatophytes by MALDI-TOF MS

MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) is a new tool for the identification of microorganisms, inclusive of dermatophytes. The technique is faster, more straightforward, and powerful when compared to conventional dermatophyte identification methods (culture and microscopy). Accurate species identification in dermatophytes is not only essential to survey epidemiologic situations, but also for an appropriate medical treatment and to locate the source of infection (zoophilic or anthropophilic). Multiple platforms from a number of well-established commercial manufacturers (Andromas SAS, Bruker Daltonics, bioMérieux) have been used for dermatophyte identification with different success. Independent from the platform used, all of the studies reviewed here report on problems with the identification of phylogenetically closely related anthropophilic and zoophilic dermatophyte species. Thus, supplementation of the databases/libraries as well as standardized extraction protocols and cultivation methods are the precondition required for optimal species identification.     

Species identification of birds through genetic analysis of naturally shed feathers

Genetic analysis of noninvasively collected bird feathers is of growing importance to avian ecology; however, most genetic studies that utilize feathers make no mention of the need to verify their species of origin. While plumage patterns and collection location often are indicative of species identity, broad‐scale feather collections may require definitive species identification prior to analysis. Genetic species identification has been applied to noninvasively collected samples from a wide range of taxa but, to date, these techniques have not been widely used on bird feathers. Here, we develop and test a polymerase chain reaction (PCR)‐based technique for identifying eastern imperial eagle (Aquila heliaca) samples among a vast number of noninvasively collected feathers. Species identification is accomplished by amplifying a fragment of the mitochondrial cytochrome c oxidase I gene, then digesting that fragment with a restriction enzyme. The resulting species‐specific restriction fragment length polymorphisms (RFLPs) are easily visualized by gel electrophoresis. We tested this PCR‐RFLP assay on over 300 individuals that had been genetically identified from noninvasively collected feathers and demonstrated that the assay is both reliable and robust for DNA of low quality and quantity. The genetic methods of species identification used to develop this assay can readily be applied to other bird assemblages, making them particularly relevant to a broad range of future avian research.     

A frequency distribution approach to hotspot identification

We present a new global method for the identification of hotspots in conservation and ecology. The method is based on the identification of spatial structure properties through cumulative relative frequency distributions curves, and is tested with two case studies, the identification of fish density hotspots and terrestrial vertebrate species diversity hotspots. Results from the frequency distribution method are compared with those from standard techniques among local, partially local and global methods. Our approach offers the main advantage to be independent from the selection of any threshold, neighborhood, or other parameter that affect most of the currently available methods for hotspot analysis. The two case studies show how such elements of arbitrariness of the traditional methods influence both size and location of the identified hotspots, and how this new global method can be used for a more objective selection of hotspots.     

Can members of the south‐western Gila robusta species complex be distinguished by morphological features?

The goal for this project was to re‐examine key morphological characters hypothesized to differentiate Gila intermedia, Gila robusta and Gila nigra and outline methods better suited for making species designations based on morphology. Using a combination of meristic counts, morphological measurements and geometric morphometrics, morphological dissimilarities were quantified among these three putative species. Traditional meristic counts and morphological measurements (i.e. distances between landmarks) were not useful for species identification. Geometric morphometrics, however, identified differences among species, while also suggesting an effect of geographic location on morphological variation. Using canonical variate analysis for the 441 fish sampled in this study, geometric morphometrics accurately predicted true group membership 100% of the time for G. nigra, 97% of the time for G. intermedia and 91% of the time for G. robusta. These results suggest that geometric morphometric analysis is necessary to identify morphological differences among the three species. Geometric morphometric analysis used in this study can be adopted by management officials as a tool to classify unidentified individuals.     

Rapid Identification of Mutans Streptococcal Species

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.     

Diet analysis in piscivorous birds: What can the addition of molecular tools offer?

In trophic studies on piscivorous birds, it is vital to know which kind of dietary sample provides the information of interest and how the prey can be identified reliably and efficiently. Often, noninvasively obtained dietary samples such as regurgitated pellets, feces, and regurgitated fish samples are the preferred source of information. Fish prey has usually been identified via morphological analysis of undigested hard parts, but molecular approaches are being increasingly used for this purpose. What remains unknown, however, is which dietary sample type is best suited for molecular diet analysis and how the molecular results compare to those obtained by morphological analysis. Pellets, feces, and regurgitated fish samples of Great Cormorants (Phalacrocorax carbo sinensis) were examined for prey using both morphological hard part analysis and molecular prey identification. The sample types and methods were compared regarding number of species detected (overall and per sample) as well as the prey species composition and its variability among individual samples. Via molecular analysis, significantly higher numbers of prey species were detected in pellets, feces, and fish samples. Of the three sample types, pellets contained the most comprehensive trophic information and could be obtained with the lowest sampling effort. Contrastingly, dietary information obtained from feces was least informative and most variable. For all sample types, the molecular approach outperformed morphological hard part identification regarding the detectable prey spectrum and prey species composition. We recommend the use of pellets in combination with molecular prey identification to study the diet of piscivorous birds.     

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 μl. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.Download video file.^{(106M, mp4)}     

Validity Analysis of the Morphological Identification of Three Highly Morphologically Similar Species Belonging to the Genus Niviventer (Rodentia: Muridae)

Cryptic species are prevalent among mammals, and identifying morphological methods or measurements that can effectively distinguish cryptic species has high significance in taxonomy. Three of the white-bellied rats, Niviventer confucianus, N. fulvescens and N. huang (Rodentia: Muridae), have such similar morphological characteristics that taxonomists have not been able to effectively identify them according to their morphologies. Recent studies have determined that the N. fulvescens species complex contains multiple species, indicating that previous morphological comparisons were potentially based on inaccurate species identifications, leading to false conclusions. To clarify whether the morphological differences among these three species can be used for identification, a variety of morphological methods and measurements were combined with molecular species identification to ensure accurate identifications prior to comparing morphological characteristics. The results showed that: (1) only N. confucianus has a white tail tip, distinguishing this species from the other two species but not N. fulvescens from N. huang; and (2) the tail length of N. fulvescens and ear length of N. confucianus are greater than those of the other species. Traditional morphological methods cannot differentiate these species well, with the discrimination rate reaching more than 90%; therefore, they can only be used as auxiliary methods for morphological identification. Additionally: (3) the geometric morphological results of the four skull views can be combined to distinguish the three species, among which the discrimination rate of lateral view of mandible reaches 98%, making it effective for differentiating these species; and (4) the phallic morphologies of the three species differed significantly in the presence of a dorsal groove, thickness of the distal baculum, and the positional relationship between the distal and proximal baccula; thus, this approach can completely distinguish these species. This study examines the applicability of various morphological measurements for the identification of highly morphologically similar species, provides a reference for distinguishing cryptic species produced by convergent evolution or incomplete differentiation by morphology, and suggests that phallic morphology should be the primary characteristic for differentiating such species in the future.

     

Species Delimitation and Analysis of Cryptic Species Diversity in the XXI Century

The potentials and limitations of different approaches to revealing species boundaries and describing cryptic species are discussed. Both the traditional methods of species delimitation, mostly based on morphological analysis, and the approaches using molecular markers are considered. Besides, the prospects of species identification using digital image recognition and machine learning are briefly considered. It is concluded that molecular markers provide very important material for species delimitation; the value of these data increases manifold if they can be compared with information on morphology, geographic distribution, and ecological preferences of the studied taxa. In many cases, only a practicing taxonomist who knows his or her group thoroughly can correctly interpret the molecular data and incorporate them into the existing knowledge system in order to make a taxonomic decision.

     

Molecular identification of three Ompok species using mitochondrial COI gene

A DNA-based barcode identification system that is applicable to all animal species will provide a simple, universal tool for the identification of fish species. The barcode system is based on sequence diversity in subunit 1 cytochrome c oxidase (COI) gene. Identification and characterization of fish species based on morphological characters are sometimes found to be erroneous and environmentally affected. There are no studies on the genus Ompok in India at molecular level and species identification of the Ompok is usually carried out through morphological features. A total of 106 samples from three species Ompok pabda, O. pabo and O. bimaculatus were collected from eight sampling sites of seven Indian rivers. One hundred and six sequences were generated from COI region of three Ompok species and 21 haplotypes were observed. The sequence analysis of COI gene revealed three genetically distinct Ompok species and exhibited identical phylogenetic resolution among them. The partial COI gene sequence can be used as a diagnostic molecular marker for identification and resolution of taxonomic ambiguity of Ompok species.     

青海省6种裂腹鱼COI基因作为DNA条形码的适用性初探

青海省裂腹鱼鱼类至少有20种,占土著鱼类的40%以上,具有重要的生态价值。由于生态环境的恶化和人为因素的干扰,很多物种群已濒临灭绝。快速和准确的物种鉴定对于这些物种的保护至关重要,而基于形态学的传统分类法很难满足这一需求。因此,本研究通过DNA条形码技术初步探讨了在青海省裂腹鱼物种鉴定中的适用性。本研究中,测序获得了青海26尾裂腹鱼(6个物种)的细胞色素c氧化酶亚基I (COI)基因,并经GenBank数据库进行比对;基于K2P模型分析COI序列变异;运用贝叶斯法(BI)和最大似然法(ML)构建系统发育树。结果显示,6种裂腹鱼COI序列检测得到15个单倍型,且各物种间无共享的单倍型。基于K2P模型,最大种内遗传距离和最小种间遗传距离分别为(0.621±0.297)%和(2.792±0.644)%。种间平均遗传距离为(12.205±1.307)%,约为种内平均遗传距离((0.327±0.162)%)的37倍,表明各物种的COI序列间已经形成明显"条形码间隙"。AMOVA分析显示,遗传变异主要来自种间,约占97.05%;FST=0.970 54,p<0.01,说明各物种间分化程度极高。此外,基于BI和ML方法构建的系统树具有一致的拓扑结构,分辨率较高;各物种均单独聚为一个发育枝,拓扑结构合理。以上结果表明,COI基因作为DNA条形码在青海裂腹鱼物种鉴定中具有较高的适用性。     

Taking a Comparative Approach: Analysing Personality as a Multivariate Behavioural Response across Species

Animal personality, repeatable behaviour through time and across contexts, is ecologically and evolutionarily important as it can account for the exhibition of sub-optimal behaviours. Interspecific comparisons have been suggested as important for understanding the evolution of animal personality; however, these are seldom accomplished due, in part, to the lack of statistical tools for quantifying differences and similarities in behaviour between groups of individuals. We used nine species of closely-related coral reef fishes to investigate the usefulness of ecological community analyses for the analysis of between-species behavioural differences and behavioural heterogeneity. We first documented behavioural carryover across species by observing the fishes' behaviour and measuring their response to a threatening stimulus to quantify boldness. Bold fish spent more time away from the reef and fed more than shy fish. We then used ecological community analysis tools (canonical variate analysis, multi-response permutation procedure, and permutational analysis of multivariate dispersion) and identified four 'clusters' of behaviourally similar fishes, and found that the species differ in the behavioural variation expressed; some species are more behaviourally heterogeneous than others. We found that ecological community analysis tools are easily and fruitfully applied to comparative studies of personality and encourage their use by future studies.     

Hydroacoustic characteristics of mass fishes of the Ob-Irtysh Basin

Hydroacoustic characteristics of mass fish species of the Ob-Irtysh Basin are investigated for elaboration of instrumental methods of fish identification by the results of hydroacoustic surveys. Linear-logarithmic regression equations of average values of the acoustic “target strength” are obtained, depending on the body length and weight of located objects. Instant values of the form of the echo signal envelope of amplitudes of echo signals from fish are analyzed. The numerical values of their statistical parameters are obtained. The characteristics of the backscattering from different species may be used for the solution of practical tasks of identification of fish and estimation of bioresources on inland water bodies.     

Species delimitation through DNA barcoding of freshwater leeches of the Glossiphonia genus (Hirudinea: Glossiphoniidae) from Eastern Siberia,Russia

The study of biodiversity is a priority task of biological science. The structural unit of biodiversity is a species that has a clear identification in a taxonomic system. Morphological features are traditionally the main criteria for species discrimination in zoological studies. However, the presence of inter- and intraspecific polymorphism and phenotypic plasticity makes it difficult to identify species in many groups of invertebrates. To solve this problem, in this research, we analyzed morphological and genetic data in combination to delimit species among the Eastern Siberia Glossiphonia leeches using different approaches. Morphology analysis revealed phenetically distinct groups, suggesting the existence of at least two species in the region, G. verrucata, a rare Palaearctic species, and a potentially new species Glossiphonia sp. Moreover, sequence-based species delimitation methods congruently supported eight distinct species groups (including two Siberian species) within the available molecular dataset of the Glossiphonia world fauna, using phylogenetic (ML and BI), coalescent (ABGD and GMYC) methods, and pairwise analysis of sequences. The detected p-distances (modal value of 0.11) between these 8 groups and the level of genetic polymorphism (max. 0.0041) within groups indicate that the groups are 8 independent species according to the DNA barcoding. Our results once again proved the usefulness of molecular systematics. At the same time, we detected several inaccuracies in the leech species identification, as well as many ambiguous sites in sequences uploaded on GenBank, which affects the analysis and impedes progress of DNA barcoding technology.     

罗霄山脉地区鱼类物种多样性

罗霄山脉是赣江和修水流域与湘江流域的分水岭, 是中国生物多样性保护的关键地区之一。然而, 罗霄山脉地区的鱼类缺乏系统性的研究, 其鱼类物种组成、分布以及受威胁因素尚不清楚。为此, 我们于2014-2018年对罗霄山脉地区11条河流的鱼类进行了系统的调查。结果表明, 该地区共有鱼类5目17科64属113种, 山脉东坡鱼类108种, 高于西坡的72种。从生态类型看, 罗霄山脉鱼类以肉食性、底栖性、定居性类群为主。区系组成上以东亚江河平原类群为主。从物种多样性看, 遂川江、袁水、蜀水和修河的鱼类物种多样性较高, 锦江和富水的鱼类物种多样性较低; β多样性指数揭示遂川江与锦江、禾水、富水间鱼类物种出现一定的分化现象。     

Use of shape to distinguish Chamelea gallina and Chamelea striatula (Bivalvia: Veneridae): Linear and geometric morphometric methods

The commercially fished striped venus clams Chamelea gallina and C. striatula (Bivalvia: Veneridae) are difficult to distinguish by inexperienced observers and the taxonomy of these species is still an issue of discussion. The differences in shape between C. gallina and C. striatula from Portuguese coastal waters were studied through conventional linear and geometric morphometric analysis, using both contour (elliptic Fourier analysis) and landmark-based methods. The relationships shell length vs. height, width, and total weight were significantly different between species. However, because there was a considerable overlap in the data sets, the species could not be distinguished using any combination of those linear measurements. Geometric morphometric methods provided shape variables that led to 0-6% misclassification rates between species; linear morphometric measures led to 16.8% error. Contour analysis revealed differences primarily in the shell umbo and lunular area. The umbo was more "sharp" and the lunula less pronounced in C. striatula than in C. gallina. Generalized procrustes superimposition (landmark analysis) showed that the main differences between species reside in the length of the pallial sinus. Thus, an index was developed (PI: Pallial Index = pallial sinus length/shell length), which was successfully used to separate the species (with 100% correct classification), i.e., specimens with PI lower than 0.119 belonged to C. gallina, whereas greater PI values were attributed to C. striatula. The use of these geometric morphometric methods allowed the detection of differences in shape between these two species and to develop an easy-to-use identification index. We encourage the development of analogous indices that apply the methods of geometric morphometrics to distinguish between other species whose identification is complicated.