Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples

Many studies in molecular ecology rely upon the genotyping of large numbers of low‐quantity DNA extracts derived from noninvasive or museum specimens. To overcome low amplification success rates and avoid genotyping errors such as allelic dropout and false alleles, multiple polymerase chain reaction (PCR) replicates for each sample are typically used. Recently, two‐step multiplex procedures have been introduced which drastically increase the success rate and efficiency of genotyping. However, controversy still exists concerning the amount of replication needed for suitable control of error. Here we describe the use of a two‐step multiplex PCR procedure that allows rapid genotyping using at least 19 different microsatellite loci. We applied this approach to quantified amounts of noninvasive DNAs from western chimpanzee, western gorilla, mountain gorilla and black and white colobus faecal samples, as well as to DNA from ~100‐year‐old gorilla teeth from museums. Analysis of over 45 000 PCRs revealed average success rates of > 90% using faecal DNAs and 74% using museum specimen DNAs. Average allelic dropout rates were substantially reduced compared to those obtained using conventional singleplex PCR protocols, and reliable genotyping using low (< 25 pg) amounts of template DNA was possible. However, four to five replicates of apparently homozygous results are needed to avoid allelic dropout when using the lowest concentration DNAs (< 50 pg/reaction), suggesting that use of protocols allowing routine acceptance of homozygous genotypes after as few as three replicates may lead to unanticipated errors when applied to low‐concentration DNAs.     

A new cost‐effective and fast direct PCR protocol for insects based on PBS buffer

Insect DNA barcoding is a species identification technique used in biodiversity assessment and ecological studies. However, DNA extraction can result in the loss of up to 70% of DNA. Recent research has reported that direct PCR can overcome this issue. However, the success rates could still be improved, and tissues used for direct PCR could not be reused for further genetic studies. Here, we developed a direct PCR workflow that incorporates a 2‐min sample preparation in PBS‐buffer step for fast and effective universal insect species identification. The developed protocol achieved 100% success rates for amplification in six orders: Mantodea, Phasmatodea, Neuroptera, Odonata, Blattodea and Orthoptera. High and moderate success rates were obtained for five other species: Lepidoptera (97.3%), Coleoptera (93.8%), Diptera (90.5%), Hemiptera (81.8%) and Hymenoptera (75.0%). High‐quality sequencing data were also obtained from these amplifiable products, allowing confidence in species identification. The method was sensitive down to 1/4th of a 1‐mm fragment of leg or body and its success rates with oven‐dried, ethanol‐preserved, food, bat guano and museum specimens were 100%, 98.6%, 90.0%, 84.0% and 30.0%, respectively. In addition, the pre‐PCR solution (PBS with insect tissues) could be used for further DNA extraction if needed. The workflow will be beneficial in the fields of insect taxonomy and ecological studies due to its low cost, simplicity and applicability to highly degraded specimens.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

Old and new DNA unweave the phylogenetic position of the eastern Atlantic gall crab Detocarcinus balssi (Monod, 1956) (Decapoda: Cryptochiridae)

Phylogeny reconstructions based on mtDNA and nDNA have become the standard in studies on relationships between taxa. Difficulties in obtaining material, for example because of small endemic distributions, often lead to gaps in datasets. Collections in natural history museums can provide us with material to fill these gaps, but extracting DNA from historical specimens can be challenging. We used a PCR protocol for small amounts of sample material and high PCR yield on eggs of specimens of the coral‐dwelling gall crab family Cryptochiridae collected in 1984, including material from the eastern Atlantic species Detocarcinus balssi. We obtained DNA sequences from seven older museum specimens using newly developed primers, which we combined with COI sequences from recently collected material. Results show that D. balssi is closest to the Indo‐Pacific species Utinomiella dimorpha and not closely related to one of the other three Atlantic Cryptochiridae species. The remaining newly acquired DNA sequences from museum material cluster with the respective sequences from recently collected specimens.     

A molecular tool to identify Anastatus parasitoids of the brown marmorated stink bug

Globally, Anastatus species (Hymenoptera: Eupelmidae) are associated with the invasive agricultural pest Halyomorpha halys (Stål) (Hemiptera: Pentatomidae). In Europe, the polyphagous Anastatus bifasciatus (Geoffroy) is the most prevalent native egg parasitoid on H. halys eggs and is currently being tested as a candidate for augmentative biological control. Anastatus bifasciatus frequently displays behavior without oviposition, and induces additional host mortality through oviposition damage and host feeding that is not measured with offspring emergence. This exacerbates accurate assessment of parasitism and host impact, which is crucial for efficacy evaluation as well as for pre‐ and post‐release risk assessment. To address this, a general Anastatus primer set amplifying a 318‐bp fragment within the barcoding region of the cytochrome oxidase I (COI) gene was developed. When challenged with DNA of three Anastatus species —A. bifasciatus, Anastatus japonicus Ashmead, and Anastatus sp.—, five scelionid parasitoid species that might be encountered in the same host environments and 11 pentatomid host species, only Anastatus DNA was successfully amplified. When applied to eggs of the target host, H. halys, and an exemplary non‐target host, Dendrolimus pini L. (Lepidoptera: Lasiocampidae), subjected to host feeding, no Anastatus amplicons were produced. Eggs of the two host species containing A. bifasciatus parasitoid stages, from 1‐h‐old eggs to pupae, and emerged eggs yielded Anastatus fragments. Confirmation of parasitoid presence with dissections and subsequent PCRs with the developed primer pair resulted in 95% success for 1‐h‐old parasitoid eggs. For both host species, field‐exposed sentinel emerged eggs stored dry for 6 months, 100% of the specimens produced Anastatus amplicons. This DNA‐based screening method can be used in combination with conventional methods to better interpret host‐parasitoid and parasitoid‐parasitoid interactions. It will help address ecological questions related to an environmentally friendly approach for the control of H. halys in invaded areas.     

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome‐wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red‐tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site‐associated DNA marker approach (DArTseq^?). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq‐based method to produce reliable genome‐wide SNP data from traditional museum specimens.     

DNA barcodes of the native ray‐finned fishes in Taiwan

Species identification based on the DNA sequence of a fragment of the cytochrome c oxidase subunit I gene in the mitochondrial genome, DNA barcoding, is widely applied to assist in sustainable exploitation of fish resources and the protection of fish biodiversity. The aim of this study was to establish a reliable barcoding reference database of the native ray‐finned fishes in Taiwan. A total of 2993 individuals, belonging to 1245 species within 637 genera, 184 families and 29 orders of ray‐finned fishes and representing approximately 40% of the recorded ray‐finned fishes in Taiwan, were PCR amplified at the barcode region and bidirectionally sequenced. The mean length of the 2993 barcodes is 549 bp. Mean congeneric K2P distance (15.24%) is approximately 10‐fold higher than the mean conspecific one (1.51%), but approximately 1.4‐fold less than the mean genetic distance between families (20.80%). The Barcode Index Number (BIN) discordance report shows that 2993 specimens represent 1275 BINs and, among them, 86 BINs are singletons, 570 BINs are taxonomically concordant, and the other 619 BINs are taxonomically discordant. Barcode gap analysis also revealed that more than 90% of the collected fishes in this study can be discriminated by DNA barcoding. Overall, the barcoding reference database established by this study reveals the need for taxonomic revisions and voucher specimen rechecks, in addition to assisting in the management of Taiwan's fish resources and diversity.     

Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR‐free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here, we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (three different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome skimming), respectively. We found that, even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read–biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for eight of 13 (amplicons B1FR‐450 bp, FF130R‐130 bp) or four of 13 (amplicon FFFR, 658 bp) species. Combining the results of all three COI amplicons (multiamplicon approach) improved the read–biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large‐scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.     

Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA

Despite advances that allow DNA sequencing of old museum specimens, sequencing small‐bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small‐bodied (3–6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58–159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1–10 ng). We also explored low‐cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low‐input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens.     

Which specimens from a museum collection will yield DNA barcodes? A time series study of spiders in alcohol

We report initial results from an ongoing effort to build a library of DNA barcode sequences for Dutch spiders and investigate the utility of museum collections as a source of specimens for barcoding spiders. Source material for the library comes from a combination of specimens freshly collected in the field specifically for this project and museum specimens collected in the past. For the museum specimens, we focus on 31 species that have been frequently collected over the past several decades. A series of progressively older specimens representing these 31 species were selected for DNA barcoding. Based on the pattern of sequencing successes and failures, we find that smaller-bodied species expire before larger-bodied species as tissue sources for single-PCR standard DNA barcoding. Body size and age of oldest successful DNA barcode are significantly correlated after factoring out phylogenetic effects using independent contrasts analysis. We found some evidence that extracted DNA concentration is correlated with body size and inversely correlated with time since collection, but these relationships are neither strong nor consistent. DNA was extracted from all specimens using standard destructive techniques involving the removal and grinding of tissue. A subset of specimens was selected to evaluate nondestructive extraction. Nondestructive extractions significantly extended the DNA barcoding shelf life of museum specimens, especially small-bodied species, and yielded higher DNA concentrations compared to destructive extractions. All primary data are publically available through a Dryad archive and the Barcode of Life database.     

Sorting specimen‐rich invertebrate samples with cost‐effective NGS barcodes: Validating a reverse workflow for specimen processing

Biologists frequently sort specimen‐rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a “reverse workflow” is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next‐generation sequencing (NGS) barcoding pipeline that allows for cost‐effective high‐throughput generation of short specimen‐specific barcodes (313 bp of COI; laboratory cost <$0.50 per specimen) through next‐generation sequencing of tagged amplicons. We applied our approach to a large sample of tropical ants, obtaining barcodes for 3,290 of 4,032 specimens (82%). NGS barcodes and their corresponding specimens were then sorted into molecular operational taxonomic units (mOTUs) based on objective clustering and Automated Barcode Gap Discovery (ABGD). High diversity of 88–90 mOTUs (4% clustering) was found and morphologically validated based on preserved vouchers. The mOTUs were overwhelmingly in agreement with morphospecies (match ratio 0.95 at 4% clustering). Because of lack of coverage in existing barcode databases, only 18 could be accurately identified to named species, but our study yielded new barcodes for 48 species, including 28 that are potentially new to science. With its low cost and technical simplicity, the NGS barcoding pipeline can be implemented by a large range of laboratories. It accelerates invertebrate species discovery, facilitates downstream taxonomic work, helps with building comprehensive barcode databases and yields precise abundance information.     

‘Direct PCR’ optimization yields a rapid,cost‐effective,nondestructive and efficient method for obtaining DNA barcodes without DNA extraction

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.     

Recovery of DNA barcodes from blackfly museum specimens (Diptera: Simuliidae) using primer sets that target a variety of sequence lengths

In this study, we evaluated the efficacy of various primers for the purpose of DNA barcoding old, pinned museum specimens of blackflies (Diptera: Simuliidae). We analysed 271 pinned specimens representing two genera and at least 36 species. Due to the age of our material, we targeted overlapping DNA fragments ranging in size from 94 to 407 bp. We were able to recover valid sequences from 215 specimens, of which 18% had 500‐ to 658‐bp barcodes, 36% had 201‐ to 499‐bp barcodes and 46% had 65‐ to 200‐bp barcodes. Our study demonstrates the importance of choosing suitable primers when dealing with older specimens and shows that even very short sequences can be diagnostically informative provided that an appropriate gene region is used. Our study also highlights the lack of knowledge surrounding blackfly taxonomy, and we briefly discuss the need for further phylogenetic studies in this socioeconomically important family of insects.     

Noninvasive recovery and detection of possum Trichosurus vulpecula DNA from bitten bait interference devices (WaxTags)

The brushtail possum is a major agricultural and ecological pest in New Zealand. A novel noninvasive DNA sampling tool for detecting its presence (WaxTags, or WT) was tested. DNA was recovered from saliva left on WT, and two lengths (407 bp and 648 bp) of the cytochrome c oxidase I (COI) barcoding region were amplified by polymerase chain reaction (PCR). PCR products were considered (+) when a DNA band was clearly visible by electrophoresis. Different factors that might affect PCR (+) were investigated with captive possums: (i) both extraction protocols of the QIAGEN DNeasy Blood and Tissue Kit, (ii) effect of an overnight or longer delay of up to 3 weeks before DNA extraction on both COI amplicons, and (iii) effect of the individual, order and magnitude of the bite. Extraction protocols were not significantly different. The effect of the overnight delay was not significant, and amplification of the short amplicon was significantly higher (100%) than for the long fragment (48%). After a two or 3‐week delay, the short amplicon had 94% and 56% PCR (+), success rates, respectively. Individual, order and magnitude of a bite had no significant effect. The delay trial was repeated with WT from the wild, for which PCR (+) rate of the short amplicon was 63%, regardless of freshness. Four microsatellites were amplified from captive WT samples. We conclude that DNA from saliva traces can be recovered from WT, a potential new tool for noninvasive monitoring of possums and other wildlife.     

Detecting invertebrate species in archived collections using next‐generation sequencing

Invertebrate biodiversity measured at mostly family level is widely used in biological monitoring programmes to assess anthropogenic impacts on ecosystems. However, next‐generation sequencing (NGS) could allow development of new more sensitive biomonitoring tools by allowing rapid species identification. This could be accelerated if archived invertebrate collections and environmental information from past programmes are used to understand species distributions and their environmental responses. In this study, we take archived macroinvertebrate samples from two sites collected on multiple occasions and test whether NGS can successfully detect species. Samples had been stored in 70% ethanol at room temperature for up to 12 years. Three amplicons ranging from 197 to 274 bps within the DNA barcode region were amplified from samples and compared to DNA barcoding libraries to identify species. We were able to amplify partial DNA barcodes from most samples, and species were often detected with multiple amplicons. However, some singletons and taxa poorly covered by DNA barcoding were missed. This suggests additional DNA barcodes will be required to fill ‘gaps’ in current DNA barcode libraries for aquatic macroinvertebrates and/or that it may not be possible to detect all taxa in a sample. Furthermore, older samples often detected fewer taxa and were less reliable for amplification, suggesting NGS is best used on samples within 8 years of collection. Nevertheless, many common taxa with existing DNA barcodes were reliably identified with NGS and were often present at sites across multiple years, showing the potential of NGS for detecting common and abundant species in archived material.     

Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques

A well‐covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self‐primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four‐sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400–500 bp without bringing or inducing any sequence errors. About one‐third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well‐covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA‐labelled next‐generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality.     

Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish

The effectiveness and accuracy of detection using environmental DNA (eDNA) is dependent on understanding the influence laboratory methods such as DNA extraction and PCR strategies have on detection probability. Ideally choice of sampling and extraction method will maximize eDNA yield and detection probability. Determining the survey effort required to reach a satisfactory detection probability (via increased PCR replicates or more sampling) could compensate for a lower eDNA yield if the sampling and extraction method has other advantages for a study, species or system. I analysed the effect of three different sampling and extraction methods on eDNA yield, detection probability and PCR replication for detecting the endangered freshwater fish Macquaria australasica from water samples. The impact of eDNA concentration, PCR strategy, target amplicon size and two marker regions: 12S (a mitochondrial gene) and 18S (a nuclear gene) was also assessed. The choice of sampling and extraction method and PCR strategy, rather than amplicon size and marker region, had the biggest effect on detection probability and PCR replication. The PCR replication effort required to achieve a detection probability of 0.95, ranged from 2 to 6 PCR replicates depending on the laboratory method used. As all methods yielded eDNA from which M. australasica was detected using the three target amplicons, differences in eDNA yield and detection probability between the three methods could be mitigated by determining the appropriate PCR replication effort. Evaluating the effect sampling and extraction methods will have on the detection probability and determining the laboratory protocols and PCR replication required to maximize detection and minimize false positives and negatives is a useful first step for eDNA occupancy studies.     

The case of an arctic wild ass highlights the utility of ancient DNA for validating problematic identifications in museum collections

Museum collections are essential for reconstructing and understanding past biodiversity. Many museum specimens are, however, challenging to identify. Museum samples may be incomplete, have an unusual morphology, or represent juvenile individuals, all of which complicate accurate identification. In some cases, inaccurate identification can lead to false biogeographic reconstructions with cascading impacts on paleontological and paleoecological research. Here, we analyzed an unusual Equid mandible found in the Far North of the Taymyr peninsula that was identified morphologically as Equus hemionus, an ancestor of present‐day Asiatic wild asses. If correct, this identification represents the only finding of a putative Late Pleistocene hemione in the Arctic region, and is therefore critical to understanding wild ass evolution and paleoecology. To confirm the accuracy of this specimen's taxonomic assignment, we used ancient DNA and mitochondrial hybridization capture to identify and place this specimen in the larger equid phylogeny. We find that the specimen is actually a member of E. caballus, the ancestor of domestic horses. Our study demonstrates the utility of ancient DNA to validate morphological identification, in particular of incomplete, otherwise problematic, or taxonomically unusual museum specimens.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

A minimalist barcode can identify a specimen whose DNA is degraded

A DNA barcode based on 650 bp of mitochondrial gene cytochrome c oxidase I is proving to be highly functional in species identification for various animal groups. However, DNA degradation complicates the recovery of a full‐length barcode from many museum specimens. Here we explore the use of shorter barcode sequences for identification of such specimens. We recovered short sequences — i.e. ～100 bp — with a single PCR pass from more than 90% of the specimens in assemblages of moth and wasp museum specimens from which full barcode recovery was only 50%, and the latter were usually less than 8 years old. Short barcodes were effective in identifying specimens, confirming their utility in circumstances where full barcodes are too expensive to obtain and the identification comparisons are within a confined taxonomic group.