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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.

The

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.

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.

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 oceans are harsh environments where insects are not expected to thrive, yet a few skaters of the genus Halobates Eschscholtz, 1822 (Hemiptera: Heteroptera: Gerridae) have completely adapted to life on the open seas. There are five oceanic Halobates species that have well-established and distinct distributions, but little is known about their population dynamics and intraspecific genetic variation. Moreover, existing knowledge on most species has been largely based on limited sample sizes. We examined the phylogeographic patterns and inferred past population dynamics of three Halobates species (H. micans Eschscholtz, 1822, H. sobrinus White, 1883 and H. splendens Witlaczil, 1886) based on an unprecedented large number of specimens (73–199 individuals each) collected from the Eastern Tropical Pacific (ETP) Ocean. These species have distinct biogeographies, with H. sobrinus occurring mostly along coastal Mexico, H. micans in the ETP north of the equator, and H. splendens largely south of the equator in the cold tongue of water derived from the Peru (Humboldt) current. We did not find evidence for sub-population structure within each species over distances as far as 6000–7000 km. Populations of all three species were found to deviate from neutrality, with evidence of past population growth. Genetic diversity and haplotype genealogies varied between species, implying distinct evolutionary trajectories. Coalescent analyses using Bayesian skyline plots suggested that H. splendens underwent a population expansion ~ 1 Ma, whereas H. sobrinus and H. micans experienced demographic growth ~ 120 Ka to 100 Ka, respectively. The period of population expansion of H. splendens roughly corresponds to the establishment of cool, productive waters in the cold tongue starting ~ 1 Ma and reaching modern temperatures ~ 800 Ka. Population expansions of both H. micans and H. sobrinus north of the equator occurred mostly during the last interglacial period, characterized by increased frequency and dominance of El Nino conditions, and a relatively southerly position of the Intertropical Convergence Zone of high rainfall. Intensification of El Nino conditions between ~ 75 and 125 Ka may have produced a warmer, low wind and nutrient-depleted habitat favoring population growth for both H. micans and H. sobrinus. Key insights drawn from the results of this study, alongside future resolution of evolutionary relationships among Halobates species, will complete our understanding of how these remarkable insects conquered the high seas where no other insect could.

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.

Wang, Wendy Y., Yong, Gordon W.J., Jaitrong, Weeyawat (2021): Revision of the elusive ant genus Rhopalomastix (Hymenoptera, Formicidae, Myrmicinae) in Thailand based on morphology and DNA barcodes, with descriptions of three new species - Corrigendum. European Journal of Taxonomy 746 (1): 186-187, DOI: https://doi.org/10.5852/ejt.2021.746.1329, URL: http://zoobank.org/66f27603-7c26-4492-aad6-64a41e249d7e

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’ 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=3162) 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 amongst 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 *1 allele of one sample. Our findings highlights 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. We were unable, however, to verify selected haplotypes predicted by Ray et al (PMID 30520769). 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.

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 litoralissp. 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.

South-east Asian apple snails, Pila spp., have been declining since the introduction of globally invasive, confamilial South American Pomacea spp., yet Pila ecology remains poorly studied, with most occurrence records unconfirmed. Pila scutata, a previously widespread species, presumed native to the Malay peninsula and assessed as Least Concern in the IUCN Red List, was formerly harvested for food, and may have experienced anthropogenic translocations. We surveyed the Malay peninsula (specifically Peninsular Malaysia and Singapore) to investigate the current distribution and genetic diversity of P. scutata. Six populations were found in Singapore, but only one in Peninsular Malaysia. Mitochondrial COI and 16S sequencing revealed that the Malaysian population shared a single haplotype of both genes with the Singapore populations (500 km distant). This low genetic diversity could stem from a recent anthropogenic introduction, which brings into question the true native range of P. scutata and, coupled with poorly resolved taxonomy of the genus, necessitates a reassessment of its IUCN Red List status. Introduced populations pose a dilemma, and the lack of genetic diversity is of concern in light of Pila decline throughout South-east Asia. Our results highlight that conservation management of P. scutata and its congeners must therefore be better informed by greater taxonomic resolution and more comprehensive investigations of their ecology, both in native and introduced ranges.

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory, is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrate that one flowcell can generate barcodes for ∼500 specimens despite high base-call error rates of MinION™. The pipeline overcomes the errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. These barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. We thus complement the barcode caller with an optional error correction pipeline that uses conserved amino-acid motifs from publicly available barcodes to correct the indel errors. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (1) 511 specimens of a mixed Diptera sample, (2) 575 specimens of ants, and (3) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION barcodes for 490 specimens which were assessed against reference Sanger barcodes (N=471). Overall, the MinION barcodes have an accuracy of 99.3%-100% and the number of ambiguities ranges from 90%). We estimate that up to 1000 barcodes can be generated in one flowcell and that the cost of a MinION barcode can be

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

Carnivorous plants avoid below-ground competition for nitrogen by utilizing an alternative nitrogen resource—invertebrate prey, but it remains unclear if sympatric carnivorous plants compete for prey resources. The aim of this study was to investigate if exploitative prey-resource competition occurs between the two sympatric pitcher plant species, Nepenthes rafflesiana and N. gracilis in Singapore. We first investigated if prey-resource partitioning occurs between these two species, and then investigated niche shift in N. gracilis by examining its pitcher contents along an in situ gradient of N. rafflesiana interspecific competition. Our results showed clear evidence of resource partitioning between the two species, but contrary to the expectation of competition, proximity to N. rafflesiana pitchers correlated with higher total prey numbers in N. gracilis pitchers. Our multivariate model of prey assemblages further suggested that N. rafflesiana facilitates N. gracilis prey capture, especially in several ant taxa that are trapped by both species. Concurrently, we found strong evidence for intraspecific competition between N. gracilis pitchers, suggesting that prey resources are exhaustible by pitcher-predation. Our results show that resource partitioning can be associated with facilitative interactions, instead of competition as is usually assumed. Facilitation is more typically expected between phylogenetically distant species, but divergences in resource acquisition strategies can permit facilitation between congeners.

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 litoralissp. 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.

Various crab taxa are known to use their external colouration as visual courtship signals. Early research on the mangrove crab Parasesarma peninsulare Shahdadi et al., 2018 indicated a potential role in sexual signalling for its bright blue-green facial bands, which are highly conspicuous against mangrove mud. To advance this work, we conducted experiments investigating both male responses to female stimuli and female responses to male stimuli. Two sets of binary choice experiments were performed—one with live crabs and one using androgyne images where the only sex-specific cue was facial band colour—to test whether the crabs could distinguish between male and female conspecifics based on the colour of their facial band alone, i.e. independent of auditory, chemical and other visual cues. In both cases, the crabs showed a significantly greater preference for conspecifics of the opposite sex (i.e. males preferred females and vice versa). These results provide evidence for colour vision in Parasesarma and suggest that the facial bands are used by the crabs for sexual discrimination and mate recognition.

The conversion of natural habitats to croplands is a major cause of the global loss of species diversity and ecosystem services. One potentially effective method for enhancing the biodiversity of important groups such as insects, and their associated ecosystem services within croplands, is to increase the environmental complexity of the agricultural matrix. This can be achieved in perennial tree crops by planting mixed-age stands. There is, however, very little evidence for the effects of stand age on biodiversity in tropical tree crops. In this study, we assess how ground-foraging ant communities differ in relation to oil palm stand age, currently the dominant tree crop across tropical Southeast Asia. Oil palm age did not affect overall ant species richness. However, the numbers of species that were either common or numerically-dominant decreased with increases in canopy cover, which positively correlated with oil palm age, suggesting greater species evenness in older oil palm. In mature, but not young, oil palm, ant species co-occurred non-randomly, suggesting competitive exclusion and species segregation. Species responses to variables associated with oil palm age depended on their functional group. Generally, opportunists and generalized myrmicines were less abundant and less diverse, whereas specialist predators were more abundant and diverse, in older oil palm. Our results show that ground-foraging ant communities in oil palm change significantly in composition, but not in species richness, with crop age. These results suggest that mixed-age stands could support compositionally variable communities and help boost insect diversity in otherwise homogeneous perennial monocultures.

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.

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.

Bright colours are a key visual signal in many animal communication systems. Here we examine the function of the blue-green facial bands found in two species of mangrove crabs, Perisesarma eumolpe (De Man) and P. indiarum (Tweedie). Food (mangrove leaves) deprivation and re-feeding experiments demonstrated that facial band brightness and saturation changed significantly with nutritional status. These results suggest that the colours are diet-derived and could be carotenoid-based. Manipulation of the facial bands had significant effects on inter-crab behaviour. When P. indiarum and P. eumolpe males were given a choice between a female with its facial band blacked out and a similar-sized control female with natural colour, they consistently chose the latter. In a resource holding potential (RHP) experiment, male crabs defending burrows responded differently to male con-specific intruders with blacked out facial bands compared to non-blacked out controls. Together, the results show that brightly coloured facial bands in P. eumolpe and P. indiarum are important for communication; either as a way to identify conspecifics, or to convey nutritional status and hence physical quality.