Fishing profile and commercial landings of shark and batoids in a global elasmobranchs conservation hotspot.

This paper describes the fishing profile and the temporal variation in the commercial landings of elasmobranchs in a global hotspot for their conservation and investigates the variables that influenced the landings. Census data on commercial catches were obtained between April 2008 and October 2010 from nine landing sites in Bragança (Pará, northern Brazil). Five vessel types, four fishing gears, and eight fishing techniques engaged with elasmobranch capture were identified. A total of 2,357 landings were recorded, with a total production of 354 t. The highest yields were recorded in 2009, with sharks being harvested mostly by small and medium-sized vessels, and batoids, by small vessels and canoes. Drifting nets and longlines played a prominent role in elasmobranch fisheries. The results show that the landings were influenced by days at sea, which is common in tropical fisheries. The elasmobranch data series is discontinuous as statistics are absent for most fishing sites albeit imperative for proper management, as well as relevant for decision-makers focusing on their conservation.

SynGenes: a Python class for standardizing nomenclatures of mitochondrial and chloroplast genes and a web form for enhancing searches for evolutionary analyses.

BACKGROUND: The reconstruction of the evolutionary history of organisms has been greatly influenced by the advent of molecular techniques, leading to a significant increase in studies utilizing genomic data from different species. However, the lack of standardization in gene nomenclature poses a challenge in database searches and evolutionary analyses, impacting the accuracy of results obtained. RESULTS: To address this issue, a Python class for standardizing gene nomenclatures, SynGenes, has been developed. It automatically recognizes and converts different nomenclature variations into a standardized form, facilitating comprehensive and accurate searches. Additionally, SynGenes offers a web form for individual searches using different names associated with the same gene. The SynGenes database contains a total of 545 gene name variations for mitochondrial and 2485 for chloroplasts genes, providing a valuable resource for researchers. CONCLUSIONS: The SynGenes platform offers a solution for standardizing gene nomenclatures of mitochondrial and chloroplast genes and providing a standardized search solution for specific markers in GenBank. Evaluation of SynGenes effectiveness through research conducted on GenBank and PubMedCentral demonstrated its ability to yield a greater number of outcomes compared to conventional searches, ensuring more comprehensive and accurate results. This tool is crucial for accurate database searches, and consequently, evolutionary analyses, addressing the challenges posed by non-standardized gene nomenclature.

Comparative mitochondrial genome brings insights to slight variation in gene proportion and large intergenic spacer and phylogenetic relationship of mudskipper species.

Fish mitochondrial genome have been largely studied worldwide for evolutionary and other genetic purposes and the structure and gene organization are commonly conservative. However, several studies have demonstrated that this scenario may present variations in some taxa, showing differentiation on the gene rearrangement. In this study, the complete mitogenome of terrestrial fish Boleophthalmus dussumieri was generated and compared with other species of the Exudercidae fishes. The newly complete mitogenome generated is circular and 16,685 bp of length, and it contained 13 protein-coding genes (PCGs), two ribosomal RNA (rRNAs), 22 transfer RNA genes (tRNAs), and one control region (CR), with high conservative structure, like other Mudskippers. Most of the PCG showed similar codon usage bias. The gene length was found to be different specially for the CR, 12S rRNA gene and ND5 gene in some taxon. All the Boleophthalmus species showed a gene duplication in the CR, except for B. dussumieri, and they presented a long intergenic spacer specially on the tRNA-Pro/ OH Tandem duplication/random loss (TDRL) and dimer-mitogenome and nonrandom loss (DMNL) are suitable to explain the mitogenome rearrangement observed in this study. The phylogenetic analysis well supported the monophyly of all mudskipper species and the analysis positioned the Periophthalmus clade as the most basal of the terrestrial fishes. This finding provides basis and brings insights for gene variation, gene rearrangements and replications showing evidence for variety of mitochondrial structure diversity within mudskippers.

First neurotranscriptome of adults Tambaquis (Colossoma macropomum) with characterization and differential expression between males and females.

The Tambaqui is one of the most representative Amazon fish species, being highly exploited in fisheries, aquaculture and as a research model. Nonetheless, data about functional genome are still required to evaluate reproductive and nutrition parameters as well as resistance to pathogens. The of next-generation sequencing has allows assessing the transcriptional processes in non-model species by providing comprehensive gene collections to be used as a database in further genomic applications and increased performance of captive populations. In this study, we relied on RNAseq approach to generate the first transcriptome of the telencephalon from adult males and females of Colossoma macropomum, resulting in a reference dataset for future functional studies. We retrieved 896,238 transcripts, including the identification of 267,785 contigs and 203,790 genes. From this total, 91 transcripts were differentially expressed, being 63 and 28 of them positively regulated for females and males, respectively. The functional annotation resulted in a library of 40 candidate genes for females and 20 for males. The functional enrichment classes comprised reproductive processes (GO:0,048,609; GO:0,003,006; GO:0,044,703; GO:0,032,504; GO:0,019,953) being related to sex differentiation (e.g., SAFB) and immune response (e.g., SLC2A6, AHNAK, NLRC3, NLRP3 and IgC MHC I alpha3), thus indicating that the genes in the neurotranscriptome of Tambaqui participate in sex differentiation and homeostasis of captive specimens. These data are useful to design the selection of genes related to sex determination and animal welfare in raising systems of Tambaqui.

Molecular delimitation methods validate morphologically similar species of red snappers (Perciformes: Lutjanidae).

In this study, we tested the taxonomic validation of red snappers species (Southern red snapper Lutjanus purpureus; Silk snapper L. vivanus; Blackfin snapper L. buccanella; and Pacific red snapper L. peru) based on comparative analysis, using four methods for species delimitation. These methods were based on either genetic similarity or phylogenetic trees inferred from two mitochondrial (Cytochrome b and D-loop) and two nuclear (Myostatin and S7 introns) markers. On one hand, the genetic results corroborated the presence of four red snapper species, confirming their taxonomic validation despite their remarkable morphological similarity. On the other hand, few incongruencies in the species delimitation methods were observed according to the phylogenetic reconstruction method (maximum likelihood or Bayesian inference) when using. Based on the phylogenetic results, L. buccanella should represent a more ancient lineage in relation to the clade that encompasses L. purpureus, L. peru and L. vivanus. The single-locus phylogenetic analysis based on Cytb recovered each the red snapper species as a well-supported clade. Overall, this study provided a DNA-based validation of the traditional morphological taxonomy of red snappers.

DNA barcode reveals occurrence of threatened species and hidden diversity on Teleost fish trade in the Coastal Amazon.

This study aimed to identify the teleost fish species sold in Bragança, a major fishing hub on the north coast of Brazil. The COI gene analysis was performed for the identification of fish species. The local market uses common names that are not accurate and do not reflect the diversity of the species. 204 sequences were obtained, with 119 haplotypes. 83 species were identified by comparing with public databases and constructing phylogenetic trees, with Carangidae being the most prevalent family. The study also found Haemulon atlanticus, Menticirrhus cuiaranensis and Hoplias misioneira, a newly described species from the Amazon basin, among the samples. Additionally, 73 commercial names were recorded, including 10 categories, and the illegal trade of Epinephelus itajara was detected. The DNA Barcode method proved to be effective for discriminating the species. The study highlights that common and commercial names are vague and underestimate the fish diversity, and that Brazil needs to revise its regulations for commercial and scientific names.

Genetic connectivity and population expansion inferred from multilocus analysis in Lutjanus alexandrei (Lutjanidae-Perciformes), an endemic snapper from Northeastern Brazilian coast.

Previous studies about the genetic diversity, connectivity and demographic history in Lutjanidae fishes have reported a common pattern of genetic homogeneity and expansion in populations from Western South Atlantic. In the present work, we inferred the population structure, the levels of genetic diversity and the demographic history of the Brazilian snapper Lutjanus alexandrei, a recently described and endemic species from Northeastern coast of Brazil. Five different fragments, including mitochondrial DNA (Control Region, Cyt b and ND4) and nuclear DNA (Myostatin and S7) regions were analyzed in 120 specimens of L. alexandrei from four localities in Northeastern Brazil, representing the first study of population genetics in this species. High levels of genetic diversity were observed following a panmictic pattern, probably related to the larval dispersal by the current tides along the Brazilian coast. In addition, both demographic history and neutrality tests indicated that L. alexandrei has undergone population expansion during Pleistocene. In this sense, the sea level variation from this period could have increased the available resources and suitable habitats for the Brazilian snapper.

Molecular characterization of juvenile fish from the Amazon estuary using DNA barcoding approach.

The efficiency of the DNA barcoding relies on sequencing fragment of the Cytochrome C Subunit I (COI) gene, which has been claimed as a tool to biodiversity identification from distinct groups. Accordingly, the goal of this study was to identify juvenile fish species along an estuary of Caeté River in the Brazilian Blue Amazon based on. For this purpose, we applied the DNA barcoding and discuss this approach as a tool for discrimination of species in early ontogenetic stages. A 500-bp fragment was obtained from 74 individuals, belonging to 23 species, 20 genera, 13 families and seven orders. About 70% of the 46 haplotypes revealed congruence between morphological and molecular species identification, while 8% of them failed in identification of taxa and 22% demonstrated morphological misidentification. These results proved that COI fragments were effective to diagnose fish species at early life stages, allowing identifying all samples to a species-specific status, except for some taxa whose COI sequences remain unavailable in public databases. Therefore, we recommend the incorporation of DNA barcoding to provide additional support to traditional identification, especially in morphologically controversial groups. In addition, periodic updates and comparative analyses in public COI datasets are encouraged.

First record of rainbow shrimp, exotic species Mierspenaeopsis sculptilis (Heller, 1862), in the Brazilian coastal amazon, validated by DNA barcode.

BACKGROUND: This is the first record of the alien shrimp Mierspenaeopsis sculptilis in Brazil. The invasion was detected within Marine Extractive Reserves based on eight specimens accidentally caught by local fishermen using trawlnets focused on fisheries of native species. These specimens were transported to the Laboratory of Applied Genetics and morphologically identified as Mierspenaeopsis sculptilis (rainbow shrimp). The taxonomic status of analyzed samples was confirmed by DNA barcoding using a 627-bp fragment of the Cytochrome C Oxidase Subunit I (COI) gene. RESULTS: A single haplotype was recovered from the eight specimens, being identical to a haplotype reported in India, where this species naturally occurs, and in Mozambique, where the rainbow shrimp is considered an invasive species. The present analyses indicated a putative invasive route (i.e., India-Mozambique-Brazil) mediated by shipping trade. CONCLUSIONS: This study presents the first record of Mierspenaeopsis sculptilis in Brazil, in areas of extractive reserves on the Amazon coast. Notably exotic species can cause imbalance in the ecosystem, harming native species. In view of this, the registration of new invasions is essential as they contribute to the implementation of control plans.

Quality analysis of genomic DNA and authentication of fisheries products based on distinct methods of DNA extraction.

Molecular genetic techniques are an effective monitoring tool, but high-quality DNA samples are usually required. In this study, we compared three different protocols of DNA extraction: NaCl (saline); phenol-chloroform and commercial kit (Promega)-from three biological tissues of five individuals of Lutjanus purpureus under two methods of storage. The evaluated items included DNA concentration and purity, processing time and cost, as well as the obtaining of functional sequences. The highest average values of DNA concentration were obtained using the saline procedure and the commercial kit. Pure DNA was only obtained using the saline protocol, evaluated by the ratio of 260/280. The saline and phenol-chloroform protocols were the least expensive methods. The commercial kit costs are counterbalanced by the short time required. The procedure based on phenol-chloroform presented the worst results regarding DNA yield and the time required to perform all steps. The saline and commercial kit protocols showed similar results concerning the amount and quality of extracted DNA. Therefore, the final choice should be based on the available financial resources and the available time for carrying out each procedure of DNA extraction.

Implant digital impression accuracy using extraoral scanners: a three-dimensional analysis

Aim: To analyze the accuracy of extraoral systems (Ceramill Map400+, AutoScan-DS200+, and E2) in full implantprosthetic rehabilitation three-dimensionally. Methods: A metallic edentulous maxilla with four implants was digitalized by a contact scanner (MDX-40 - Roland, control) and used as a control image to compare with other images generated by three laboratory scanners (10 samples per group). Letters identified all the four components: A and D angled 45º, and B and C parallel. The BioCAD software exported the images (.STL) to compare and verify deviations of the analogs on the X, Y, and Z axes. The nonparametric Kruskal-Wallis test and the two-way ANOVA on ranks with a post hoc Tukey test analyzed the data with 5% significance. Results: No statistical differences were observed in the accuracy between the extraoral scanners (p=0.0806). However, when analyzing only the components, component D was more accurate when scanned with Ceramill Map400+ compared with AutoScan DS200+ (p<0.001) and with E2 (p=0.002). Conclusions: All extraoral systems assessed showed digitalization accuracy but with more deviations in angled implants. The Ceramill Map400+ scanner showed the best results for the digital impression of a complete arch

Phylogeography of Baryancistrus xanthellus (Siluriformes: Loricariidae), a rheophilic catfish endemic to the Xingu River basin in eastern Amazonia.

Baryancistrus xanthellus (Loricariidae) is an endemic fish species from the Xingu River basin with its life history in the shallow rapid waters flowing over bedrock substrates. In order to investigate the genetic diversity and demographic history of B. xanthellus we analyzed sequence data for one mitochondrial gene (Cyt b) and introns 1 and 5 of nuclear genes Prolactin (Prl) and Ribosomal Protein L3 (RPL3). The analyses contain 358 specimens of B. xanthellus from 39 localities distributed throughout its range. The number of genetically diverged groups was estimated using Bayesian inference on Cyt b haplotypes. Haplotype networks, AMOVA and pairwise fixation index was used to evaluate population structure and gene flow. Historical demography was inferred through neutrality tests and the Extended Bayesian Skyline Plot (EBSP) method. Five longitudinally distributed Cyt b haplogroups for B. xanthellus were identified in the Xingu River and its major tributaries, the Bacajá and Iriri. The demographic analysis suggests that rapids habitats have expanded in the Iriri and Lower Xingu rivers since 200 ka (thousand years) ago. This expansion is possibly related to an increase in water discharge as a consequence of higher rainfall across eastern Amazonia. Conversely, this climate shift also would have promoted zones of sediment trapping and reduction of rocky habitats in the Xingu River channel upstream of the Iriri River mouth. Populations of B. xanthellus showed strong genetic structure along the free-flowing river channels of the Xingu and its major tributaries, the Bacajá and Iriri. The recent impoundment of the Middle Xingu channel for the Belo Monte hydroelectric dam may isolate populations at the downstream limit of the species distribution. Therefore, future conservation plans must consider the genetic diversity of B. xanthellus throughout its range.

New insights about species delimitation in red snappers (Lutjanus purpureus and L. campechanus) using multilocus data.

Lutjanus campechanus and Lutjanus purpureus are two commercially important lutjanid fishes (snappers) with non-sympatric distribution throughout Western Atlantic. Even though both taxa have traditionally been regarded as valid species, their taxonomic status remains under debate. In the present study, we used phylogeographic approaches and molecular methods of species delimitation to elucidate the taxonomic issues between both species, based on 1478 base pairs from four genomic regions. We found haplotypes shared between the two species, particularly in relation to nuclear DNA (nuDNA) sequences. The molecular delimitation of species supported the discrimination of L. purpureus and L. campechanus as distinct evolutionary units. Nonetheless, a unidirectional gene flow was found from L. campechanus towards L. purpureus. Therefore, it seems plausible to infer that L. campechanus and L. purpureus are two evolutionary units in which the apparent sharing of haplotypes should be driven by introgression.

Forensic analysis reveals fraud in fillets from the "Gurijuba" Sciades parkeri (Ariidae - Siluriformes): a vulnerable fish in Brazilian Coastal Amazon.

The utilization of molecular tools for the certification of fishery products has been increasing over the last years. In general, economically important species are replaced by less valuable species, characterizing a commercial fraud. We evaluated the authenticity of 107 frozen fillets tagged as Gurijuba (Sciades parkeri) and Uritinga (Sciades proops) from local markets in northern amazon coast by sequencing two mitochondrial genes: Cytochrome oxidase subunit I and cytochrome b (Cyt b). About 16% of fillets putatively related to S. parkeri were replaced by S. proops. The Gurijuba faces high fishing pressure, being currently listed by the International Union for Conservation of Nature as vulnerable. Forensic analysis with DNA markers, proved to be highly efficient in the discrimination of the processed seafood products, providing unequivocal identification of species, revealing commercial fraud in the fillets of the Gurijuba, and revealing the utility of Cytb sequences as barcode in fishes.

Genomics overrules mitochondrial DNA, siding with morphology on a controversial case of species delimitation.

Species delimitation is a major quest in biology and is essential for adequate management of the organismal diversity. A challenging example comprises the fish species of red snappers in the Western Atlantic. Red snappers have been traditionally recognized as two separate species based on morphology: Lutjanus campechanus (northern red snapper) and L. purpureus (southern red snapper). Recent genetic studies using mitochondrial markers, however, failed to delineate these nominal species, leading to the current lumping of the northern and southern populations into a single species ( L. campechanus). This decision carries broad implications for conservation and management as red snappers have been commercially over-exploited across the Western Atlantic and are currently listed as vulnerable. To address this conflict, we examine genome-wide data collected throughout the range of the two species. Population genomics, phylogenetic and coalescent analyses favour the existence of two independent evolutionary lineages, a result that confirms the morphology-based delimitation scenario in agreement with conventional taxonomy. Despite finding evidence of introgression in geographically neighbouring populations in northern South America, our genomic analyses strongly support isolation and differentiation of these species, suggesting that the northern and southern red snappers should be treated as distinct taxonomic entities.

Multiloci analyses suggest synonymy among Rhomboplites, Ocyurus and Lutjanus and reveal the phylogenetic position of Lutjanus alexandrei (Lutjanidae: Perciformes)

Lutjanidae comprises 21 genera and 135 species widespread throughout Atlantic, Indian and Pacific oceans. Nonetheless, the phylogenetic relationships of Lutjaninae remain uncertain. Furthermore, phylogenetic hypotheses for Lutjanus alexandrei, an endemic species from northeastern Brazilian coast, in Lutjanidae are absent so far. Therefore, we carried out multiloci analyses, combining both mitochondrial and nuclear DNA sequences in Lutjaninae species from Western Atlantic focusing on the controversial relationships among Lutjanus, Rhomboplites, and Ocyurus. Besides, we determined the phylogenetic position and dated the origin of L. alexandrei. The phylogenetics trees based on the 4.4 kb for 11 species corroborated the synonym among Lutjanus and the putative monotypic genera. For the dating of L. alexandrei, another nucleotide dataset (3.0 kb; 40 species) validated the genetic identity of this species that diverged from the sister taxon L. apodus between 2.5 - 6.5 Mya, probably as a result of the barrier caused by the muddy outflow from Orinoco and Amazon rivers along the coastal zone. This report is the most robust multiloci analysis to confirm the synonymy of the three genera of Lutjaninae from Western Atlantic and the first reliable inference about the phylogenetic relationships and origin of L. alexandrei.(AU)

A Família Lutjanidae compreende 21 gêneros e 135 espécies, distribuídas ao longo dos oceanos Atlântico, Índico e Pacífico. As relações filogenéticas dos Lutjaninae são incertas. Além disso, a espécie Lutjanus alexandrei, endêmica da costa nordeste do Brasil, não foi inclusa em nenhuma hipótese filogenética até o presente. Assim, realizamos uma análise integrando DNA mitocondrial e nuclear para espécies de Lutjaninae do Atlântico Ocidental, direcionada para a controversa relação entre Lutjanus, Rhomboplites e Ocyurus. Além disso, alocamos filogeneticamente L. alexandrei e datamos sua origem. As árvores filogenéticas baseadas em 4.4 kb de 11 espécies corroboraram a sinonímia entre os monotípicos e Lutjanus. Para a datação de L. alexandrei, outro banco de nuclueotídeos foi analisado (3.0 kb; 40 espécies), validando geneticamente a espécie e a colocando como irmã de L. apodus, da qual se separou entre 2.5 - 6.5 Mya, o que provavelmente foi provocado pela faixa enlameada na região costeira, influenciada pelas descargas dos rios Amazonas e Orinoco, que funciona como barreira. Este trabalho representa a mais robusta análise multiloci direcionada para a sinonimização dos três gêneros de Lutjaninae e a primeira hipótese filogenética a propor um posicionamento e origem para L. alexandrei.(AU)

Comparative analyses of species delimitation methods with molecular data in snappers (Perciformes: Lutjaninae).

The integration of approaches that allow the incorporation of stochasticity of gene histories with phylogenetic methods resulted in new approaches for the old issue of species delimitation. Nevertheless, coalescent methods seem problematic for taxa with large effective population size and shallow temporal diversification (like marine fishes). Here, we investigate the performance of single-locus (cytochrome oxidase 1, commonly used in DNA barcoding initiatives) methods for molecular species delimitation in snappers of Lutjaninae from the Western Atlantic and Pacific Eastern. Our results show incongruences among methods. ABGD, PTP and mPTP trend towards a lower number of estimated species. Phylogenetic-coalescent methods with single threshold were majority congruent for a same number of lineages. On the other hand, algorithms with multiple thresholds tend to estimate a higher number of potential species. We do not endorse the use of single-locus for species delimitation, but we do reinforce that single-locus data is sufficient to flag many problems.

Molecular authentication of Pargo fillets Lutjanus purpureus (Perciformes: Lutjanidae) by DNA barcoding reveals commercial fraud

The Caribbean Red Snapper (Pargo) Lutjanus purpureus is the most economically important snapper in Brazil, which is sold, among other forms, as frozen fillets. During the process of transformation into fillets there is the removal of the distinctive morphological traits, being able to favor the substitution by less valued species. In addition, there is no national legislation requiring the insertion of the specific name on the product label. However, according to a Normative Instruction (IN N ° 29/2015 MAPA) that correlates the common and specific names of the products destined to the national trade, in Brazil only L. purpureus and L. campechanus can be denominated "Pargo". Thus, the DNA barcode tool was used to identify the fillets sold in north of Brazil, labeled "Pargo", with the aid of sequences from the public and control databases. The results showed that among 142 fillets examined, 78% was identified as L. purpureus and 22% as Rhomboplites aurorubens, a snapper with low commercial value in the country, revealing commercial fraud. The molecular identification method successfully used in this study to authenticate fillets snappers may also be used by surveillance authorities in the quality control of processed fish products, towards ensuring consumer rights.(AU)

O Pargo Lutjanus purpureus, lutjanídeo mais importante economicamente no Brasil, é vendido, entre outras formas, como filés congelados. Durante a transformação em filés, há a remoção das características morfológicas distintivas, podendo favorecer a substituição por espécies menos valorizadas. Além disso, não há legislação nacional que exija a inserção do nome específico no rótulo. Porém, de acordo com uma Instrução Normativa (IN N° 29 /2015 MAPA) que correlaciona os nomes comuns e específicos dos produtos destinados ao comércio nacional, no Brasil somente L. purpureus e L. campechanus podem ser denominados "Pargo". Assim, a ferramenta DNA barcode foi usada para identificar os filés vendidos no norte do Brasil, rotulados como "Pargo", com o auxílio de sequências dos bancos de dados públicos e banco controle. Os resultados mostraram que entre os 142 filés examinados, 78% foi identificado como L. purpureus e 22% como Rhomboplites aurorubens, um lutjanídeo com baixo valor comercial no país, revelando fraude comercial. O método de identificação molecular, utilizado com êxito neste estudo para autenticar filés de lutjanídeos, pode também ser utilizado pelas autoridades de vigilância no controle de qualidade de produtos processados derivados de peixes em geral, para garantir os direitos dos consumidores.(AU)

Development of EPIC-PCR Markers for Lutjanus purpureus (Lutjanidae-Perciformes) and their Potential Applicability in Population Analyses.

In the present study, a novel set of eight EPIC primers were developed for Lutjanus purpureus and assayed in five other marine teleosts including three lutjanids, one scianid and one anablepid. Most of the genomic regions used in this study presented genetic diversity indexes equal or greater than the intragenic regions commonly used in population genetics studies. Moreover, six out of eight markers showed cross-amplification with other taxa. Thus, the primers described here may be used to elucidate questions at the intraspecific level for a large number of taxa.

Molecular protocol for authentication of snappers (Lutjanidae-Perciformes) based on multiplex PCR.

Fraud involving fish products is regularly reported and investigated using genetic methods. However, no such approach has been applied to snappers, despite the commercial relevance of these fish. This study proposes an authentication protocol with multiplex PCR for three species of snappers (Lutjanus purpureus, L. synagris, and Ocyurus chrysurus). The protocol yielded a distinct triple-banding pattern for L. purpureus, whereas L. synagris and O. chrysurus showed a double banding pattern of different sizes, thereby allowing differentiation of the three species. The protocol was validated using fillets labeled as snapper or "Pargo" in Brazil, previously identified using DNA sequencing, amongst which substitution with Rhomboplites aurorubens was detected. When subjected to the new protocol, banding characteristic of L. purpureus were detected whilst R. aurorubens generated only the control band. Our study provides a practical tool for investigating substitutions and might assist in quality control and increase food safety for consumers.