CYP2D6 copy number determination using digital PCR.

Background: CYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the "One-pot" single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5'UTR, intron 6, and exon 9 regions. Methods: Genomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0-6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5'UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA. Results: Overall, results between the two platforms and digestions methods were consistent. The "One-pot" digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference. Conclusion: The Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.

Characterization of Reference Materials for DPYD: A GeT-RM Collaborative Project.

The DPYD gene encodes dihydropyrimidine dehydrogenase (DPD), which is involved in the catalysis of uracil and thymine, as well as 5-fluorouracil (5-FU), which is used to treat solid tumors. Patients with decreased DPD activity are at risk of serious, sometimes fatal, adverse drug reactions to this important cancer drug. Pharmacogenetic testing for DPYD is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for clinical DPYD testing. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 33 DNA samples derived from Coriell cell lines for DPYD. Samples were distributed to four volunteer laboratories for genetic testing using a variety of commercially available and laboratory-developed tests. Sanger sequencing was used by one laboratory and publicly available whole-genome sequence data from the 1000 Genomes Project were used by another to inform genotype. Thirty-three distinct DPYD variants were identified among the 33 samples characterized. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

New Zealand nurses perceptions of caring for patients with influenza A (H1N1).

AIM: This study aimed to explore the perceptions of the highly specialized nurses who provided extracorporeal membrane oxygenation therapy for the mostly young and critically ill patients during the 2009 H1N1 pandemic. BACKGROUND: The 2009 influenza A (H1N1) virus caused a global pandemic and also affected New Zealand during that winter. Nine H1N1-infected adult patients with severe acute respiratory distress syndrome were admitted into an intensive care unit of a large urban hospital for rescue extracorporeal membrane oxygenation therapy. DESIGN: The study used a two-phase mix methods study design. METHODS: Phase 1 of the study involved five nurses attending a focus group interview to collect their views of the challenges and issues of caring for these patients. The results of the focus group were used to formulate the phase 2 survey. In total, 25 eligible nurses were invited to complete an anonymous survey; 18 completed and returned surveys giving a 72% response rate. RESULTS: The survey identified issues including the acuity and high mortality rate of those affected, nurses working in an isolated environment because of infection control requirements, limited support and being asked to work extra shifts. CONCLUSION: Despite these challenges, the nurses felt positive about their experience of caring for the H1N1 patients, and felt the experience advanced their skills and improved job satisfaction. RELEVANCE TO CLINICAL PRACTICE: For future pandemics, this study identified the need for all staff to have a basic understanding of extracorporeal membrane oxygenation; strengthen inter-professional collaboration and communication; provision for more support and recognition of these highly specialized nurses, along with providing regular pandemic updates and offering counselling services.

Scrutinising an inscrutable bark-nesting ant: Exploring cryptic diversity in the Rhopalomastix javana (Hymenoptera: Formicidae) complex using DNA barcodes, genome-wide MIG-seq and geometric morphometrics.

Overlooking cryptic species diversity has grave implications on assessments of climate change impacts on biodiversity, ecosystems and organismal populations. Discriminating between cryptic species has long been challenging even for seasoned taxonomists, as interspecies morphological differences are often indiscernible by visual observation. Multi-disciplinary methods involving genetic analyses in conjunction with quantitative morphological data, should therefore be used to investigate boundaries between cryptic species. We adopted an integrated approach combining analyses of mitochondrial COI barcodes, a genome-wide dataset obtained via multiplexed inter-simple sequence repeats (ISSRs) genotyping by sequencing (MIG-seq), and geometric morphometrics to investigate species divergences in the inscrutable Rhopalomastix javana species complex. Objective clustering of COI suggested five putative molecular species units divergent from each other by thresholds within 4.2-10.6% uncorrected pairwise distance. Phylogenetic analyses based on concatenated MIG-seq data also recovered and strongly supported the monophyly of five major lineages in agreement with COI clusters. Co-ancestry analyses based on MIG-seq data using fineRADstructure resolved variable patterns of admixture linked to geography, and potential genetic drift within some putative species. Geometric morphometric analyses of specimen images further detected statistically significant differences in at least one of three anatomical aspects (Head, Meso, Profile) between all pairs of putative species. Head shape (full-face view) was determined to be the most informative character for species diagnosis, with relatively high classification accuracy. Thin-plate spline deformation grids highlighted areas of high variation between species in each shape for deeper taxonomic scrutiny. The presence of species from multiple distinct lineages existing in near-sympatry firmly demonstrates that R. javana comprises more than one closely-related species, but exact species boundaries are difficult to ascertain. Differences in elevation and its associated abiotic effects on ant adaptations and reproductive phenology may contribute to restricting gene flow and maintaining species boundaries between sympatric populations of the R. javana complex. We further assess the advantages and limitations of geometric morphometrics as a taxonomic tool. Despite its drawbacks, our combined approach has helped draw important insights on cryptic diversity in R. javana, and also identified gaps of knowledge that await address. Results from this study will inform and prime future in-depth taxonomic investigation on the R. javana complex, including formal descriptions and establishment of the five putative species.

Impact of CYP2C:TG Haplotype on CYP2C19 Substrates Clearance In Vivo, Protein Content, and In Vitro Activity.

A novel haplotype composed of two non-coding variants, CYP2C18 NM_000772.3:c.*31T (rs2860840) and NM_000772.2:c.819+2182G (rs11188059), referred to as "CYP2C:TG," was recently associated with ultrarapid metabolism of various CYP2C19 substrates. As the underlying mechanism and clinical relevance of this effect remain uncertain, we analyzed existing in vivo and in vitro data to determine the magnitude of the CYP2C:TG haplotype effect. We assessed variability in pharmacokinetics of CYP2C19 substrates, including citalopram, sertraline, voriconazole, omeprazole, pantoprazole, and rabeprazole in 222 healthy volunteers receiving one of these six drugs. We also determined its impact on CYP2C8, CYP2C9, CYP2C18, and CYP2C19 protein abundance in 135 human liver tissue samples, and on CYP2C18/CYP2C19 activity in vitro using N-desmethyl atomoxetine formation. No effects were observed according to CYP2C:TG haplotype or to CYP2C19*1+TG alleles (i.e., CYP2C19 alleles containing the CYP2C:TG haplotype). In contrast, CYP2C19 intermediate (e.g., CYP2C19*1/*2) and poor metabolizers (e.g., CYP2C19*2/*2) showed significantly higher exposure in vivo, lower CYP2C19 protein abundance in human liver microsomes, and lower activity in vitro compared with normal, rapid (i.e., CYP2C19*1/*17), and ultrarapid metabolizers (i.e., CYP2C19*17/*17). Moreover, a tendency toward lower exposure was observed in ultrarapid metabolizers compared with rapid metabolizers and normal metabolizers. Furthermore, when the CYP2C19*17 allele was present, CYP2C18 protein abundance was increased suggesting that genetic variation in CYP2C19 may be relevant to the overall metabolism of certain drugs by regulating not only its expression levels, but also those of CYP2C18. Considering all available data, we conclude that there is insufficient evidence supporting clinical CYP2C:TG testing to inform drug therapy.

Characterization of Reference Materials for CYP3A4 and CYP3A5: A (GeT-RM) Collaborative Project.

Pharmacogenetic testing for CYP3A4 is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the CYP3A4 variants included in clinical tests. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 30 DNA samples derived from Coriell cell lines for CYP3A4. Samples were distributed to five volunteer laboratories for genotyping using a variety of commercially available and laboratory-developed tests. Sanger and next-generation sequencing were also utilized by some of the laboratories. Whole-genome sequencing data from the 1000 Genomes Projects were utilized to inform genotype. Twenty CYP3A4 alleles were identified in the 30 samples characterized for CYP3A4: CYP3A4∗4, ∗5, ∗6, ∗7, ∗8, ∗9, ∗10, ∗11, ∗12, ∗15, ∗16, ∗18, ∗19, ∗20, ∗21, ∗22, ∗23, ∗24, ∗35, and a novel allele, CYP3A4∗38. Nineteen additional samples with preexisting data for CYP3A4 or CYP3A5 were re-analyzed to generate comprehensive reference material panels for these genes. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

The Identification of Novel CYP2D6 Variants in US Hmong: Results From Genome Sequencing and Clinical Genotyping.

Objective: Hmong individuals represent a unique East Asian subpopulation in whom limited information concerning pharmacogenetic variation exists. The objectives of this study were to comprehensively characterize the highly polymorphic CYP2D6 gene in Hmong, estimate allele and phenotype frequencies and to compare results between two testing platforms. Methods: DNA from 48 self-identified Hmong participants were sequenced using a targeted next-generation sequencing (NGS) panel. Star allele calls were made using Astrolabe, manual inspection of NGS variant calls and confirmatory Sanger sequencing. Structural variation was determined by long-range (XL)-PCR and digital droplet PCR (ddPCR). The consensus diplotypes were subsequently translated into phenotype utilizing the activity score system. Clinical grade pharmacogenetic testing was obtained for 12 of the 48 samples enabling an assessment of concordance between the consensus calls and those determined by clinical testing platforms. Results: A total of 13 CYP2D6 alleles were identified. The most common alleles were CYP2D6*10 and its structural arrangements (37.5%, 36/96) and the *5 gene deletion (13.5%, 13/96). Three novel suballeles (*10.007, *36.004, and *75.002) were also identified. Phenotype frequencies were as follows: ultrarapid metabolizers (4.2%, 2/48), normal metabolizers (41.7%, 20/48) and intermediate metabolizers (52.1%, 25/48); none of the 48 participants were predicted to be poor metabolizers. Concordance of diplotype and phenotype calls between the consensus and clinical testing were 66.7 and 50%, respectively. Conclusion: Our study to explore CYP2D6 genotypes in the Hmong population suggests that this subpopulation is unique regarding CYP2D6 allelic variants; also, a higher portion of Hmong participants (50%) are predicted to have an intermediate metabolizer phenotype for CYP2D6 compared to other East Asians which range between 27 and 44%. Results from different testing methods varied considerably. These preliminary findings underscore the importance of thoroughly interrogating unique subpopulations to accurately predict a patient's CYP2D6 metabolizer status.

The Impact of the CYP2D6 "Enhancer" Single Nucleotide Polymorphism on CYP2D6 Activity.

rs5758550 has been associated with enhanced transcription and suggested to be a useful marker of CYP2D6 activity. As there are limited and inconsistent data regarding the utility of this distant "enhancer" single nucleotide polymorphism (SNP), our goal was to further assess the impact of rs5758550 on CYP2D6 activity toward two probe substrates, atomoxetine (ATX) and dextromethorphan (DM), using in vivo urinary metabolite (DM; n = 188) and pharmacokinetic (ATX; n = 70) and in vitro metabolite formation (ATX and DM; n = 166) data. All subjects and tissues were extensively genotyped, the "enhancer" SNP phased with established CYP2D6 haplotypes either computationally or experimentally, and the impact on CYP2D6 activity investigated using several linear models of varying complexity to determine the proportion of variability in CYP2D6 activity captured by each model. For all datasets and models, the "enhancer" SNP had no or only a modest impact on CYP2D6 activity prediction. An increased effect, when present, was more pronounced for ATX than DM suggesting potential substate-dependency. In addition, CYP2D6*2 alleles with the "enhancer" SNP were associated with modestly higher metabolite formation rates in vitro, but not in vivo; no effect was detected for CYP2D6*1 alleles with "enhancer" SNP. In summary, it remains inconclusive whether the small effects detected in this investigation are indeed caused by the "enhancer" SNP or are rather due to its incomplete linkage with other variants within the gene. Taken together, there does not appear to be sufficient evidence to warrant the "enhancer" SNP be included in clinical CYP2D6 pharmacogenetic testing.

Characterization of Novel CYP2D6 Alleles across Sub-Saharan African Populations.

The CYP2D6 gene has been widely studied to characterize variants and/or star alleles, which account for a significant portion of variability in drug responses observed within and between populations. However, African populations remain under-represented in these studies. The increasing availability of high coverage genomes from African populations has provided the opportunity to fill this knowledge gap. In this study, we characterized computationally predicted novel CYP2D6 star alleles in 30 African subjects for whom DNA samples were available from the Coriell Institute. CYP2D6 genotyping and resequencing was performed using a variety of commercially available and laboratory-developed tests in a collaborative effort involving three laboratories. Fourteen novel CYP2D6 alleles and multiple novel suballeles were identified. This work adds to the growing catalogue of validated African ancestry CYP2D6 allelic variation in pharmacogenomic databases, thus laying the foundation for future functional studies and improving the accuracy of CYP2D6 genotyping, phenotype prediction, and the refinement of clinical pharmacogenomic implementation guidelines in African and global settings.

Characterization of Reference Materials for TPMT and NUDT15: A GeT-RM Collaborative Project.

Pharmacogenetic testing is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the TPMT and NUDT15 variants included in clinical tests. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) coordination program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 19 DNA samples derived from Coriell cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using a variety of commercially available and laboratory developed tests and/or Sanger sequencing. Of the 12 samples characterized for TPMT, newly identified variants include TPMT∗2, ∗6, ∗12, ∗16, ∗21, ∗24, ∗32, ∗33, and ∗40; for the 7 NUDT15 reference material samples, newly identified variants are NUDT15∗2, ∗3, ∗4, ∗5, ∗6, and ∗9. In addition, a novel haplotype, TPMT∗46, was identified in this study. Preexisting data on an additional 11 Coriell samples, as well as some supplemental testing, were used to create comprehensive reference material panels for TPMT and NUDT15. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

CYP2C8, CYP2C9, and CYP2C19 Characterization Using Next-Generation Sequencing and Haplotype Analysis: A GeT-RM Collaborative Project.

Pharmacogenetic tests typically target selected sequence variants to identify haplotypes that are often defined by star (∗) allele nomenclature. Due to their design, these targeted genotyping assays are unable to detect novel variants that may change the function of the gene product and thereby affect phenotype prediction and patient care. In the current study, 137 DNA samples that were previously characterized by the Genetic Testing Reference Material (GeT-RM) program using a variety of targeted genotyping methods were recharacterized using targeted and whole genome sequencing analysis. Sequence data were analyzed using three genotype calling tools to identify star allele diplotypes for CYP2C8, CYP2C9, and CYP2C19. The genotype calls from next-generation sequencing (NGS) correlated well to those previously reported, except when novel alleles were present in a sample. Six novel alleles and 38 novel suballeles were identified in the three genes due to identification of variants not covered by targeted genotyping assays. In addition, several ambiguous genotype calls from a previous study were resolved using the NGS and/or long-read NGS data. Diplotype calls were mostly consistent between the calling algorithms, although several discrepancies were noted. This study highlights the utility of NGS for pharmacogenetic testing and demonstrates that there are many novel alleles that are yet to be discovered, even in highly characterized genes such as CYP2C9 and CYP2C19.

Resolving discordant CYP2D6 genotyping results in Thai subjects: platform limitations and novel haplotypes.

Aim: Several CYP2D6 Luminex xTAG genotype calls were identified as inconsistent or suspicious among Thai subjects and further characterized to identify the root causes. Material & methods: Forty-eight subjects were followed-up with long-range-PCR, quantitative copy number assays and/or Sanger sequencing. Results: Most of the Luminex-duplication calls were either negative or had hybrid structures involving CYP2D6*36 in various configurations. Ten samples were inaccurately called as CYP2D6*2, *29 or *35 alleles. Sequencing revealed three novel haplotypes, CYP2D6*142, *143 and *144 of which two are nonfunctional. Conclusion: The Luminex platform produced a relatively high number of false genotype calls for Thai subjects. Our findings underscore the need for the systematic characterization of the CYP2D6 locus in diverse populations and rigorous platform validation.

Maritime trap-jaw ants (Hymenoptera, Formicidae, Ponerinae) of the Indo-Australian region - redescription of Odontomachus malignus Smith and description of a related new species from Singapore, including first descriptions of males.

The maritime trap-jaw ant Odontomachus malignus Smith, 1859 is thought to be widespread throughout islands in the Indo-Pacific and parts of the Oriental realm. Because of its unique nesting preference for harsh littoral habitat and distinct morphology, O. malignus has usually been assumed to consist of only one species. We, however, describe a new species similar to O. malignus found in the mangroves of Singapore, Southeast Asia - Odontomachus litoralis sp. nov. We find strong evidence of both species existing in (near) sympatry, and also distinct morphological differences between O. malignus and the new species. Additional complementary DNA evidence in the form of COI barcodes (313 bp) supporting putative species identification and delimitation is provided. Defining morphological characteristics for the O. malignus species group (nested within the larger O. infandus clade) are given in detail for the first time. The worker and queen castes of the new species are described; a redescription of the worker caste of O. malignus, based on specimens from Singapore and the Philippines in addition to the holotype, is also given. The males of both species are also described for the first time, including male genitalia. A preliminary key to most known species of the O. infandus group based on the worker caste is provided.

Long-Distance Phasing of a Tentative "Enhancer" Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions.

BACKGROUND: The CYP2D6 gene locus has been extensively studied over decades, yet a portion of variability in CYP2D6 activity cannot be explained by known sequence variations within the gene, copy number variation, or structural rearrangements. It was proposed that rs5758550, located 116 kb downstream of the CYP2D6 gene locus, increases gene expression and thus contributes to variability in CYP2D6 activity. This finding has, however, not been validated. The purpose of the study was to address a major technological barrier, i.e., experimentally linking rs5758550, also referred to as the "enhancer" single-nucleotide polymorphism (SNP), to CYP2D6 haplotypes >100 kb away. To overcome this challenge is essential to ultimately determine the contribution of the "enhancer" SNP to interindividual variability in CYP2D6 activity. METHODS: A large ethnically mixed population sample (n=3,162) was computationally phased to determine linkage between the "enhancer" SNP and CYP2D6 haplotypes (or star alleles). To experimentally validate predicted linkages, DropPhase2D6, a digital droplet PCR (ddPCR)-based method was developed. 10X Genomics Linked-Reads were utilized as a proof of concept. RESULTS: Phasing predicted that the "enhancer" SNP can occur on numerous CYP2D6 haplotypes including CYP2D6*1, *2, *5, and *41 and suggested that linkage is incomplete, i.e., a portion of these alleles do not have the "enhancer" SNP. Phasing also revealed differences among the European and African ancestry data sets regarding the proportion of alleles with and without the "enhancer" SNP. DropPhase2D6 was utilized to confirm or refute the predicted "enhancer" SNP location for individual samples, e.g., of n=3 samples genotyped as *1/*41, rs5758550 was on the *41 allele of two samples and on the *1 allele of one sample. Our findings highlight that the location of the "enhancer" SNP must not be assigned by "default." Furthermore, linkage between the "enhancer" SNP and CYP2D6 star allele haplotypes was confirmed with 10X Genomics technology. CONCLUSIONS: Since the "enhancer" SNP can be present on a portion of normal, decreased, or no function alleles, the phase of the "enhancer" SNP must be considered when investigating the impact of the "enhancer" SNP on CYP2D6 activity.

Discovery of a new ant species of the elusive termitophilous genus Metapone in Singapore (Hymenoptera, Formicidae, Myrmicinae), with the first detailed description of male genitalia of the genus.

A new species of the rare ant genus Metapone, Metapone murphyi sp. nov., is described based on museum material consisting of a single nest series (workers, queens, and males) collected from a decayed coconut palm stump on Pulau Sakra, previously an offshore island south of mainland Singapore. Workers can be distinguished from other named congeners mainly by the following characters: 1) subpetiolar lamella subrectangular; 2) short median longitudinal ventral subpetiolar edge and roundly obtuse posteroventral subpetiolar angle; 3) outer margin of posterior subpetiolar face in posteroventral view forming a continuous, U-shaped, translucent, laminate carina; and 4) petiole subtrapezoidal in dorsal view with extended blunt tooth-like posterolateral corners. Detailed description and illustrations of male genitalia of the genus are given for the first time. The key to Asian species of Metapone is updated to include the new species.

The ant genus Rhopalomastix (Hymenoptera: Formicidae: Myrmicinae) in Southeast Asia, with descriptions of four new species from Singapore based on morphology and DNA barcoding.

The true diversity of the Asian ant genus Rhopalomastix Forel is poorly understood. We use an integrated approach to review the known species and subspecies of Rhopalomastix in Southeast Asia. Based on morphology and supporting DNA evidence, we recognize six species. We raise two subspecies of R. rothneyi Forel to species rank (R. johorensis Wheeler stat. n, R. javana Wheeler stat. n.), synonymize R. janeti Donisthorpe (syn. nov.) with R. johorensis, and describe four new species from Singapore: R. glabricephala sp. n., R. murphyi sp. n., R. striata sp. n., and R. tenebra sp. n. All six species found in Southeast Asia are distinct from each other based on morphology; morphological delimitation of these species is further supported by and congruent with mOTUs generated from objective clustering of short fragment COI barcodes using the best close match criteria. Different castes and sexes of most species are described, including redescriptions of the queen of R. javana and male of R. johorensis. A key to the Southeast Asian species based on the worker caste is also provided. Variation among sympatric and also geographically distant populations, and the possibilities of cryptic species, are discussed.

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.

The effects of forest conversion to oil palm on ground-foraging ant communities depend on beta diversity and sampling grain.

Beta diversity - the variation in species composition among spatially discrete communities - and sampling grain - the size of samples being compared - may alter our perspectives of diversity within and between landscapes before and after agricultural conversion. Such assumptions are usually based on point comparisons, which do not accurately capture actual differences in total diversity. Beta diversity is often not rigorously examined. We investigated the beta diversity of ground-foraging ant communities in fragmented oil palm and forest landscapes in Sabah, Malaysia, using diversity metrics transformed from Hill number equivalents to remove dependences on alpha diversity. We compared the beta diversities of oil palm and forest, across three hierarchically nested sampling grains. We found that oil palm and forest communities had a greater percentage of total shared species when larger samples were compared. Across all grains and disregarding relative abundances, there was higher beta diversity of all species among forest communities. However, there were higher beta diversities of common and very abundant (dominant) species in oil palm as compared to forests. Differences in beta diversities between oil palm and forest were greatest at the largest sampling grain. Larger sampling grains in oil palm may generate bigger species pools, increasing the probability of shared species with forest samples. Greater beta diversity of all species in forest may be attributed to rare species. Oil palm communities may be more heterogeneous in common and dominant species because of variable community assembly events. Rare and also common species are better captured at larger grains, boosting differences in beta diversity between larger samples of forest and oil palm communities. Although agricultural landscapes support a lower total diversity than natural forests, diversity especially of abundant species is still important for maintaining ecosystem stability. Diversity in agricultural landscapes may be greater than expected when beta diversity is accounted for at large spatial scales.