DNA barcodes enable higher taxonomic assignments in the Acari

Although mites (Acari) are abundant in many terrestrial and freshwater ecosystems, their diversity is poorly understood. Since most mite species can be distinguished by variation in the DNA barcode region of cytochrome c oxidase I, the Barcode Index Number (BIN) system provides a reliable species proxy that facilitates large-scale surveys. Such analysis reveals many new BINs that can only be identified as Acari until they are examined by a taxonomic specialist. This study demonstrates that the Barcode of Life Datasystem’s identification engine (BOLD ID) generally delivers correct ordinal and family assignments from both full-length DNA barcodes and their truncated versions gathered in metabarcoding studies. This result was demonstrated by examining BOLD ID’s capacity to assign 7021 mite BINs to their correct order (4) and family (189). Identification success improved with sequence length and taxon coverage but varied among orders indicating the need for lineage-specific thresholds. A strict sequence similarity threshold (86.6%) prevented all ordinal misassignments and allowed the identification of 78.6% of the 7021 BINs. However, higher thresholds were required to eliminate family misassignments for Sarcoptiformes (89.9%), and Trombidiformes (91.4%), consequently reducing the proportion of BINs identified to 68.6%. Lineages with low barcode coverage in the reference library should be prioritized for barcode library expansion to improve assignment success.

DNA barcoding of native Caucasus herbal plants: potentials and limitations in complex groups and implications for phylogeographic patterns

DNA barcoding has rapidly become a useful complementary tool in floristic investigations particularly for identifying specimens that lack diagnostic characters. Here, we assess the capability of three DNA barcode markers (chloroplast rpoB, accD and nuclear ITS) for correct species assignment in a floristic survey on the Caucasus. We focused on two herbal groups with potential for ornamental applications, namely orchids and asterids. On these two plant groups, we tested whether our selection of barcode markers allows identification of the “barcoding gap” in sequence identity and to distinguish between monophyletic species when employing distance-based methods. All markers successfully amplified most specimens, but we found that the rate of species-level resolution amongst selected markers largely varied in the two plant groups. Overall, for both lineages, plastid markers had a species-level assignment success rate lower than the nuclear ITS marker. The latter confirmed, in orchids, both the existence of a barcoding gap and that all accessions of the same species clustered together in monophyletic groups. Further, it also allowed the detection of a phylogeographic signal.The ITS marker resulted in its being the best performing barcode for asterids; however, none of the three tested markers showed high discriminatory ability. Even if ITS were revealed as the most promising plant barcode marker, we argue that the ability of this barcode for species assignment is strongly dependent on the evolutionary history of the investigated plant lineage.

Keep Garfagnina alive. An integrated study on patterns of homozygosity, genomic inbreeding, admixture and breed traceability of the Italian Garfagnina goat breed

The objective of this study was to investigate the genetic diversity of the Garfagnina (GRF) goat, a breed that currently risks extinction. For this purpose, 48 goats were genotyped with the Illumina CaprineSNP50 BeadChip and analyzed together with 214 goats belonging to 9 other Italian breeds (~25 goats/breed), whose genotypes were available from the AdaptMap project [Argentata (ARG), Bionda dell’Adamello (BIO), Ciociara Grigia (CCG), Di Teramo (DIT), Garganica (GAR), Girgentana (GGT), Orobica (ORO), Valdostana (VAL) and Valpassiria (VSS)]. Comparative analyses were conducted on i) runs of homozygosity (ROH), ii) admixture ancestries and iii) the accuracy of breed traceability via discriminant analysis on principal components (DAPC) based on cross-validation. ROH analyses was used to assess the genetic diversity of GRF, while admixture and DAPC to evaluate its relationship to the other breeds. For GRF, common ROH (more than 45% in GRF samples) was detected on CHR 12 at, roughly 50.25–50.94Mbp (ARS1 assembly), which spans the CENPJ (centromere protein) and IL17D (interleukin 17D) genes. The same area of common ROH was also present in DIT, while a broader region (~49.25–51.94Mbp) was shared among the ARG, CCG, and GGT. Admixture analysis revealed a small region of common ancestry from GRF shared by BIO, VSS, ARG and CCG breeds. The DAPC model yielded 100% assignment success for GRF. Overall, our results support the identification of GRF as a distinct native Italian goat breed. This work can contribute to planning conservation programmes to save GRF from extinction and will improve the understanding of the socio-agro-economic factors related with the farming of GRF.

SNP Markers as a Successful Molecular Tool for Assessing Species Identity and Geographic Origin of Trees in the Economically Important South American Legume Genus Dipteryx

Dipteryx timber has been heavily exploited in South America since 2000s due to the increasing international demand for hardwood. Developing tools for the genetic identification of Dipteryx species and their geographical origin can help to promote legal trading of timber. A collection of 800 individual trees, belonging to 6 different Dipteryx species, was genotyped based on 171 molecular markers. After the exclusion of markers out of Hardy–Weinberg equilibrium or with no polymorphism or low amplification, 83 nuclear, 29 chloroplast, 13 mitochondrial single nucleotide polymorphisms (SNPs), and 2 chloroplast and 5 mitochondrial INDELS remained. Six genetic groups were identified using Bayesian Structure analyses of the nuclear SNPs, which corresponded to the different Dipteryx species collected in the field. Seventeen highly informative markers were identified as suitable for species identification and obtained self-assignment success rates to species level of 78–96%. An additional set of 15 molecular markers was selected to determine the different genetic clusters found in Dipteryx odorata and Dipteryx ferrea, obtaining self-assignment success rates of 91–100%. The success to assign samples to the correct country of origin using all or only the informative markers improved when using the nearest neighbor approach (69–92%) compared to the Bayesian approach (33–80%). While nuclear and chloroplast SNPs were more suitable for differentiating the different Dipteryx species, mitochondrial SNPs were ideal for determining the genetic clusters of D. odorata and D. ferrea. These 32 selected SNPs will be invaluable genetic tools for the accurate identification of species and country of origin of Dipteryx timber.

Keep Garfagnina alive. An integrated study on patterns of homozygosity, genomic inbreeding, admixture and breed traceability of the Italian Garfagnina goat breed

The objective of this study was to investigate the genomic background of the Garfagnina (GRF) goat breed that faces the risk of extinction. In total, 48 goats genotyped with the Illumina CaprineSNP50 BeadChip were analyzed together with 214 goats belonging to 9 Italian breeds (~25 goats/breed) from the AdaptMap project [Argentata (ARG), Bionda dell’Adamello (BIO), Ciociara Grigia (CCG), Di Teramo (DIT), Garganica (GAR), Girgentana (GGT), Orobica (ORO), Valdostana (VAL) and Valpassiria (VSS)]. We estimated i) runs of homozygosity (ROH), ii) admixture ancestries and iii) traceability success via discriminant analysis on principal components (DAPC) based on cross-validation. For GRF, an excess of frequent ROH (more than 45% in the GRF samples analyzed) was detected on CHR 12 at, roughly 50.25-50.94Mbp (ARS1 assembly), spanned between the CENPJ (centromere protein) and IL17D (interleukin 17D) genes. The same area was also present in DIT, while the broader region (~49.25-51.94Mbp) was shared among the ARG, CCG, and GGT. Admixture analysis depicted the uniqueness of the GRF breed, with a small part of common ancestry shared with BIO, VSS, ARG and CCG breeds. The DAPC model resulted in a 100% assignment success. We hope this work will contribute to the efforts of preventing the GRF from extinction and to add value to all the socio-agro-economic factors related with the farming of the GRF breed.

Using nuclear microsatellite data to trace the gene flow and population structure in Czech horses

Based on a data set comprising 2879 animals and 17 nuclear microsatellite DNA markers, we propose the most comprehensive in-depth study mapping the genetic structure and specifying the assignment success rates in horse breeds at the Czech population scale. The STRUCTURE program was used to perform systematic Bayesian clustering via the Markov chain Monte Carlo estimation, enabling us to explain the population stratification and to identify genetic structure patterns within breeds worldwide. In total, 182 different alleles were found over all the populations and markers, with the mean number of 10.7 alleles per locus. The expected heterozygosity ranged from 0.459 (Friesian) to 0.775 (Welsh Part Bred), and the average level reached 0.721. The average observed heterozygosity corresponded to 0.709, with the highest value detected in the Czech Sport Pony (0.775). The largest number of private alleles was found in Equus przewalskii. The population inbreeding coefficient F_IS ranged from –0.08 in the Merens to 0.14 in the Belgian Warmblood. The total within-population inbreeding coefficient was estimated to be moderate. As expected, very large genetic differentiation and small gene flow were established between the Friesian and Equus przewalskii (F_ST= 0.37, Nm = 0.43). Zero F_ST values indicated no differences between the Czech Warmblood–Slovak Warmblood and the Czech Warmblood–Bavarian Warmblood. A high level of breeding and connectivity was revealed between the Slovak Warmblood–Bavarian Warmblood, Dutch Warmblood–Oldenburg Horse, Bavarian Warmblood–Dutch Warmblood, and Bavarian Warmblood–Oldenburg Horse. The breeds’ contribution equalled about 6% of the total genetic variability. The overall proportion of individuals correctly assigned to a population corresponded to 82.4%. The posterior Bayesian approach revealed a hierarchical dynamic genetic structure in four clusters (hot-blooded, warm-blooded, cold-blooded, and pony). While most of the populations were genetically distinct from each other and well-arranged with solid breed structures, some of the entire sets showed signs of admixture and/or fragmentation.

Parentage determination of cuttlefish (Sepiella japonica) based on microsatellite DNA markers

Microsatellite markers have been used for more than ten years to elucidate parentage relationships in aquaculture species. This study aimed to assess the feasibility of utilizing microsatellite markers for parentage determination in cuttlefish (Sepiella japonica) using simulations and real data analysis. We developed a panel of eight microsatellite markers in our lab. These markers were highly polymorphic with a mean of 10.1 alleles and an average expected heterozygosity value of 0.809. Using five simple sequence repeat markers, an allele frequency data-based simulation indicated that the combined exclusion probability values would be over 99%, whereas the rate of assignment success for the real data set was 91.8%. Mismatches caused by null alleles and scoring errors at microsatellite loci were the major reasons for the discrepancies between the simulations and real data analysis. We concluded that microsatellite markers can be used as a powerful tool to evaluate the effectiveness of enhancement and release programs for S. japonica.

Regional variation in otolith geochemistry of juvenile Atlantic cod (Gadus morhua) in coastal Newfoundland

We examined spatial variation in otolith geochemistry as a natural tag in juvenile Atlantic cod (Gadus morhua) to resolve geographic patterns during early life history. Individuals from 54 inshore sites spanned five embayments in eastern Newfoundland. Otolith composition differed at all spatial scales and related inversely to spatial scale. Classification analysis revealed increasing discrimination at coarser spatial scales: site (26%–58%), bay (49%), and coast (76%). Assignment success declined by ∼10% per added site with increasing sampling sites per bay, demonstrating fine-scale (<100 km) variation. When we partitioned environmental variability from observed otolith chemistry using predictive models, assignment success improved by 56%, 14%, and 5% for site, bay, and coast, respectively. Our results demonstrate environmental influence on spatial structure of otolith chemistry and illustrate the importance of resolving baseline variability in otolith chemistry when conducting assignment tests. Collectively, our results describe the potential utility of juvenile otolith composition in evaluating contributions of subpopulations to the Northwest Atlantic cod stock and highlight important limitations imposed by environmental variation at scales less than 100 km.