Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

DNA barcoding echinoderms from the East Coast of South Africa. The challenge to maintain DNA data connected with taxonomy.

Echinoderms are marine water invertebrates that are represented by more than 7000 extant species, grouped in five classes and showing diverse morphologies (starfish, sea lilies, feather stars, sea urchins, sea cucumbers, brittle and basket stars). In an effort to further study their diversity, DNA barcodes (DNA fragments of the 5' end of the cytochrome c oxidase subunit I gene, COI) have been used to complement morphological examination in identifying evolutionary lineages. Although divergent clusters of COI sequences were reported to generally match morphological species delineations, they also revealed some discrepancies, suggesting overlooked species, ecophenotypic variation or multiple COI lineages within one species. Here, we sequenced COI fragments of 312 shallow-water echinoderms of the East Coast of South Africa (KwaZulu-Natal Province) and compared morphological identifications with species delimitations obtained with four methods that are exclusively based on COI sequences. We identified a total of 103 morphospecies including 18 that did not exactly match described species. We also report 46 COI sequences that showed large divergences (>5% p-distances) with those available to date and publish the first COI sequences for 30 species. Our analyses also identified discordances between morphological identifications and COI-based species delimitations for a considerable proportion of the morphospecies studied here (49/103). For most of them, further investigation is necessary to keep a sound connection between taxonomy and the growing importance of DNA-based research.

Large-scale and small-scale population genetic structure of the medically important gastropod species Bulinus truncatus (Gastropoda, Heterobranchia).

BACKGROUND: Gastropod snails remain strongly understudied, despite their important role in transmitting parasitic diseases. Knowledge of their distribution and population dynamics increases our understanding of the processes driving disease transmission. We report the first study to use high-throughput sequencing (HTS) to elucidate the population genetic structure of the hermaphroditic snail Bulinus truncatus (Gastropoda, Heterobranchia) on a regional (17-150 km) and inter-regional (1000-5400 km) scale. This snail species acts as an intermediate host of Schistosoma haematobium and Schistosoma bovis, which cause human and animal schistosomiasis respectively. METHODS: Bulinus truncatus snails were collected in Senegal, Cameroon, Egypt and France and identified through DNA barcoding. A single-end genotyping-by-sequencing (GBS) library, comprising 87 snail specimens from the respective countries, was built and sequenced on an Illumina HiSeq 2000 platform. Reads were mapped against S. bovis and S. haematobium reference genomes to identify schistosome infections, and single nucleotide polymorphisms (SNPs) were scored using the Stacks pipeline. These SNPs were used to estimate genetic diversity, assess population structure and construct phylogenetic trees of B. truncatus. RESULTS: A total of 10,750 SNPs were scored and used in downstream analyses. The phylogenetic analysis identified five clades, each consisting of snails from a single country but with two distinct clades within Senegal. Genetic diversity was low in all populations, reflecting high selfing rates, but varied between locations due to habitat variability. Significant genetic differentiation and isolation by distance patterns were observed at both spatial scales, indicating that gene flow is not strong enough to counteract the effects of population bottlenecks, high selfing rates and genetic drift. Remarkably, the population genetic differentiation on a regional scale (i.e. within Senegal) was as large as that between populations on an inter-regional scale. The blind GBS technique was able to pick up parasite DNA in snail tissue, demonstrating the potential of HTS techniques to further elucidate the role of snail species in parasite transmission. CONCLUSIONS: HTS techniques offer a valuable toolbox to further investigate the population genetic patterns of intermediate schistosome host snails and the role of snail species in parasite transmission.

Population genomics of introduced Nile tilapia Oreochromis niloticus (Linnaeus, 1758) in the Democratic Republic of the Congo: Repeated introductions since colonial times with multiple sources.

During colonial times, Nile tilapia Oreochromis niloticus (Linnaeus, 1758) was introduced into non-native parts of the Congo Basin (Democratic Republic of the Congo, DRC) for the first time. Currently, it is the most farmed cichlid in the DRC, and is present throughout the Congo Basin. Although Nile tilapia has been reported as an invasive species, documentation of historical introductions into this basin and its consequences are scant. Here, we study the genetic consequences of these introductions by genotyping 213 Nile tilapia from native and introduced regions, focusing on the Congo Basin. Additionally, 48 specimens from 16 other tilapia species were included to test for hybridization. Using RAD sequencing (27,611 single nucleotide polymorphisms), we discovered genetic admixture with other tilapia species in several morphologically identified Nile tilapia from the Congo Basin, reflecting their ability to interbreed and the potential threat they pose to the genetic integrity of native tilapias. Nile tilapia populations from the Upper Congo and those from the Middle-Lower Congo are strongly differentiated. The former show genetic similarity to Nile tilapia from the White Nile, while specimens from the Benue Basin and Lake Kariba are similar to Nile tilapia from the Middle-Lower Congo, suggesting independent introductions using different sources. We conclude that the presence of Nile tilapia in the Congo Basin results from independent introductions, reflecting the dynamic aquaculture history, and that their introduction probably leads to genetic interactions with native tilapias, which could lower their fitness. We therefore urge avoiding further introductions of Nile tilapia in non-native regions and to use native tilapias in future aquaculture efforts.

Application of frozen Thiel-embalmed specimens for radiotherapy delineation guideline development: a method to create accurate MRI-enhanced CT datasets.

PURPOSE: Thiel embalming followed by freezing in the desired position and acquiring CT + MRI scans is expected to be the ideal approach to obtain accurate, enhanced CT data for delineation guideline development. The effect of Thiel embalming and freezing on MRI image quality is not known. This study evaluates the above-described process to obtain enhanced CT datasets, focusing on the integration of MRI data obtained from frozen, Thiel-embalmed specimens. METHODS: Three Thiel-embalmed specimens were frozen in prone crawl position and MRI scanning protocols were evaluated based on contrast detail and structural conformity between 3D renderings from corresponding structures, segmented on corresponding MRI and CT scans. The measurement error of the dataset registration procedure was also assessed. RESULTS: Scanning protocol T1 VIBE FS enabled swift differentiation of soft tissues based on contrast detail, even allowing a fully detailed segmentation of the brachial plexus. Structural conformity between the reconstructed structures on CT and MRI was excellent, with nerves and blood vessels imported into the CT scan never intersecting with the bones. The mean measurement error for the image registration procedure was consistently in the submillimeter range (range 0.77-0.94 mm). CONCLUSION: Based on the excellent MRI image quality and the submillimeter error margin, the procedure of scanning frozen Thiel-embalmed specimens in the treatment position to obtain enhanced CT scans is recommended. The procedure can be used to support the postulation of delineation guidelines, or for training deep learning algorithms, considering automated segmentations.

A masculinizing supergene underlies an exaggerated male reproductive morph in a spider.

In many species, individuals can develop into strikingly different morphs, which are determined by a simple Mendelian locus. How selection shapes loci that control complex phenotypic differences remains poorly understood. In the spider Oedothorax gibbosus, males develop either into a 'hunched' morph with conspicuous head structures or as a fast-developing 'flat' morph with a female-like appearance. We show that the hunched-determining allele contains a unique genomic fragment of approximately 3 megabases that is absent in the flat-determining allele. This fragment comprises dozens of genes that duplicated from genes found at the same as well as different chromosomes. All functional duplicates, including a duplicate of the key sexual differentiation regulatory gene doublesex, show male-specific expression, which illustrates their integrated role as a masculinizing supergene. Our findings demonstrate how extensive indel polymorphisms and duplications of regulatory genes may contribute to the evolution of co-adapted gene clusters, sex-limited reproductive morphs and the enigmatic evolution of exaggerated sexual traits in general.

Biomechanical comparison of Thiel embalmed and fresh frozen nerve tissue.

The aim of this study was to determine the effect of Thiel embalming on the biomechanical properties of nerve tissue, to validate the use of Thiel embalmed bodies as a reliable model system for obtaining biomechanical data to supplement neurodynamic models, for anesthesiological and neurosurgical training and for future preclinical test set-ups involving nerve tissue. Upon the arrival of a body at the anatomy department, a fresh median nerve was harvested, the harvest site was sutured and following the Thiel embalming procedure the Thiel embalmed median nerve of the opposing wrist was harvested. Micro CT was performed to establish the cross-sectional area and biomechanical tensile testing was performed to compare the Young's modulus/elasticity of fresh frozen and Thiel embalmed nerves. Thiel embalming did not cause a significant difference in elasticity when comparing Thiel embalmed and fresh frozen specimens. A correlation was found between the cross-sectional area of Thiel embalmed nerve specimens and their Young's modulus. Thiel embalming does not significantly alter the elasticity of nerve tissue compared to fresh frozen nerve tissue. Similar shapes were observed when comparing the stress/strain curves of both specimen types. This indicates that Thiel embalmed nerve tissue is a viable alternative for using fresh frozen specimens when investigating biomechanical principles/mechanisms. Some specimens showed a reversed trend in Young's modulus that could be related to slight differences in embalming outcome, so caution is advised when Thiel embalmed specimens are used to obtain raw numerical data for direct application in the clinic.

First genomic study on Lake Tanganyika sprat Stolothrissa tanganicae: a lack of population structure calls for integrated management of this important fisheries target species.

BACKGROUND: Clupeid fisheries in Lake Tanganyika (East Africa) provide food for millions of people in one of the world's poorest regions. Due to climate change and overfishing, the clupeid stocks of Lake Tanganyika are declining. We investigate the population structure of the Lake Tanganyika sprat Stolothrissa tanganicae, using for the first time a genomic approach on this species. This is an important step towards knowing if the species should be managed separately or as a single stock. Population structure is important for fisheries management, yet understudied for many African freshwater species. We hypothesize that distinct stocks of S. tanganicae could be present due to the large size of the lake (isolation by distance), limnological variation (adaptive evolution), or past separation of the lake (historical subdivision). On the other hand, high mobility of the species and lack of obvious migration barriers might have resulted in a homogenous population. RESULTS: We performed a population genetic study on wild-caught S. tanganicae through a combination of mitochondrial genotyping (96 individuals) and RAD sequencing (83 individuals). Samples were collected at five locations along a north-south axis of Lake Tanganyika. The mtDNA data had low global FST and, visualised in a haplotype network, did not show phylogeographic structure. RAD sequencing yielded a panel of 3504 SNPs, with low genetic differentiation (FST = 0.0054; 95% CI: 0.0046-0.0066). PCoA, fineRADstructure and global FST suggest a near-panmictic population. Two distinct groups are apparent in these analyses (FST = 0.1338 95% CI: 0.1239,0.1445), which do not correspond to sampling locations. Autocorrelation analysis showed a slight increase in genetic difference with increasing distance. No outlier loci were detected in the RADseq data. CONCLUSION: Our results show at most very weak geographical structuring of the stock and do not provide evidence for genetic adaptation to historical or environmental differences over a north-south axis. Based on these results, we advise to manage the stock as one population, integrating one management strategy over the four riparian countries. These results are a first comprehensive study on the population structure of these important fisheries target species, and can guide fisheries management.

Evolution at two time frames: Polymorphisms from an ancient singular divergence event fuel contemporary parallel evolution.

When environments change, populations may adapt surprisingly fast, repeatedly and even at microgeographic scales. There is increasing evidence that such cases of rapid parallel evolution are fueled by standing genetic variation, but the source of this genetic variation remains poorly understood. In the saltmarsh beetle Pogonus chalceus, short-winged 'tidal' and long-winged 'seasonal' ecotypes have diverged in response to contrasting hydrological regimes and can be repeatedly found along the Atlantic European coast. By analyzing genomic variation across the beetles' distribution, we reveal that alleles selected in the tidal ecotype are spread across the genome and evolved during a singular and, likely, geographically isolated divergence event, within the last 190 Kya. Due to subsequent admixture, the ancient and differentially selected alleles are currently polymorphic in most populations across its range, which could potentially allow for the fast evolution of one ecotype from a small number of random individuals, as low as 5 to 15, from a population of the other ecotype. Our results suggest that cases of fast parallel ecological divergence can be the result of evolution at two different time frames: divergence in the past, followed by repeated selection on the same divergently evolved alleles after admixture. These findings highlight the importance of an ancient and, likely, allopatric divergence event for driving the rate and direction of contemporary fast evolution under gene flow. This mechanism is potentially driven by periods of geographic isolation imposed by large-scale environmental changes such as glacial cycles.

Supplementary feeding increases nestling feather corticosterone early in the breeding season in house sparrows.

Several studies on birds have proposed that a lack of invertebrate prey in urbanized areas could be the main cause for generally lower levels of breeding success compared to rural habitats. Previous work on house sparrows Passer domesticus found that supplemental feeding in urbanized areas increased breeding success but did not contribute to population growth. Here, we hypothesize that supplementary feeding allows house sparrows to achieve higher breeding success but at the cost of lower nestling quality. As abundant food supplies may permit both high- and low-quality nestlings to survive, we also predict that within-brood variation in proxies of nestling quality would be larger for supplemental food broods than for unfed broods. As proxies of nestling quality, we considered feather corticosterone (CORT f), body condition (scaled mass index, SMI), and tarsus-based fluctuating asymmetry (FA). Our hypothesis was only partially supported as we did not find an overall effect of food supplementation on FA or SMI. Rather, food supplementation affected nestling phenotype only early in the breeding season in terms of elevated CORT f levels and a tendency for more variable within-brood CORT f and FA. Early food supplemented nests therefore seemed to include at least some nestlings that faced increased stressors during development, possibly due to harsher environmental (e.g., related to food and temperature) conditions early in the breeding season that would increase sibling competition, especially in larger broods. The fact that CORT f was positively, rather than inversely, related to nestling SMI further suggests that factors influencing CORT f and SMI are likely operating over different periods or, alternatively, that nestlings in good nutritional condition also invest in high-quality feathers.

Persistent inter- and intraspecific gene exchange within a parallel radiation of caterpillar hunter beetles (Calosoma sp.) from the Galápagos.

When environmental gradients are repeated on different islands within an archipelago, similar selection pressures may act within each island, resulting in the repeated occurrence of ecologically similar species on each island. The evolution of ecotypes within such radiations may either result from dispersal, that is each ecotype evolved once and dispersed to different islands where it colonized its habitat, or through repeated and parallel speciation within each island. However, it remains poorly understood how gene flow during the divergence process may shape such patterns. In the Galápagos islands, three phenotypically similar species of the beetle genus Calosoma occur at higher elevations of different islands, while lowlands are occupied by a fourth species. By genotyping all major populations within this radiation for two nuclear and three mitochondrial gene fragments and seven microsatellite markers, we found strong support that the oldest divergence separates the highland species of the oldest island from the remaining species. Despite their morphological distinctness, highland species of the remaining islands were genetically closely related to the lowland population on each island and within the same magnitude as lowland populations sampled at different islands. Repeated evolution of highland ecotypes out of the lowland species appears the most likely scenario and estimates of geneflow rates revealed extensive admixture among ecotypes within islands, as well as between islands. These findings indicate that gene exchange among the different populations and species may have shaped the phylogenetic relationships and the repeated evolution of these ecotypes.

Sex-biased dispersal at different geographical scales in a cooperative breeder from fragmented rainforest.

Dispersal affects both social behavior and population structure and is therefore a key determinant of long-term population persistence. However, dispersal strategies and responses to spatial habitat alteration may differ between sexes. Here we analyzed spatial and temporal variation in ten polymorphic microsatellite DNA loci of male and female Cabanis's greenbuls (Phyllastrephuscabanisi), a cooperative breeder of Afrotropical rainforest, to quantify rates of gene flow and fine-grained genetic structuring within and among fragmented populations. We found genetic evidence for female-biased dispersal at small spatial scales, but not at the landscape level. Local autocorrelation analysis provided evidence of positive genetic structure within 300 m distance ranges, which is consistent with behavioral observations of short-distance natal dispersal. At a landscape scale, individual-based autocorrelation values decreased over time while levels of admixture increased, possibly indicating increased gene flow over the past decade.

Asymmetry of the ULNT1 elbow extension range-of-motion in a healthy population: consequences for clinical practice and research.

OBJECTIVES: To investigate the effect of isolated muscular variance, side and hand dominance on elbow-extension range-of-motion (EE-ROM) of the median nerve upper limb neurodynamic test (ULNT1). This study analyzes these variables potential to influence ULNT1 EE-ROM symmetry and the possible consequences for clinical practice and research. STUDY DESIGN: Controlled laboratory study, cross-sectional. BACKGROUND: No normative data exist to interpret correctly EE-ROM. Clinical interpretation is based on bilateral comparison. This procedure assumes natural EE-ROM symmetry, with lack of scientific evidence. METHODS: Nineteen participants with Langer's axillary arch (LAA), a muscular variant bridging the brachial plexus, were selected from 640 healthy volunteers, together with a matched control group. ULNT1 EE-ROM's were measured using the Vicon(®) optoelectronic system. RESULTS: A full mixed model revealed no significant effects on EE-ROM for LAA and the variable side. Significant differences were found in EE-ROM between dominant and non-dominant sides (standard ULNT1 test position: 2.84° ± 1.60°, p = 0.0004; ULNT1 with differentiating maneuver: 3.05° ± 1.98°, p = 0.003). Approximately 30% of the subjects showed clinically detectable restriction (≥10°) of the dominant side EE-ROM. CONCLUSION: Hand dominance is significantly associated with restriction of EE-ROM, which results in a clinically detectable asymmetry. This compromises the clinical procedure of comparing the patient's EE-ROM to the opposite side. Erroneous conclusions could result in side to side analyses, if the effect is not taken into account in neurodynamic research.

Microsatellite resources for Passeridae species: a predicted microsatellite map of the house sparrow Passer domesticus.

We identified microsatellite sequences of potential utility in the house sparrow (Passer domesticus) and assigned their predicted genome locations. These sequences included newly isolated house sparrow loci, which we fully characterized. Many of the newly isolated loci were polymorphic in two other species of Passeridae: Berthelot's pipit Anthus berthelotii and zebra finch Taeniopygia guttata. In total, we identified 179 microsatellite markers that were either isolated directly from, or are of known utility in, the house sparrow. Sixty-seven of these markers were designed from unique sequences that we isolated from a house sparrow genomic library. These new markers were combined with 36 house sparrow markers isolated by other studies and 76 markers isolated from other passerine species but known to be polymorphic in the house sparrow. We utilized sequence homology to assign chromosomal locations for these loci in the assembled zebra finch genome. One hundred and thirty-four loci were assigned to 25 different autosomes and eight loci to the Z chromosome. Examination of the genotypes of known-sex house sparrows for 37 of the new loci revealed a W-linked locus and an additional Z-linked locus. Locus Pdoµ2, previously reported as autosomal, was found to be Z-linked. These loci enable the creation of powerful and cost-effective house sparrow multiplex primer sets for population and parentage studies. They can be used to create a house sparrow linkage map and will aid the identification of quantitative trait loci in passerine species.

Spatial heterogeneity in genetic relatedness among house sparrows along an urban-rural gradient as revealed by individual-based analysis.

Understanding factors that shape patterns of kinship in sedentary species is important for evolutionary ecologists as well as conservation biologists. Yet, how patterns of relatedness are hierarchically structured in space remains poorly known, even in common species. Here, we use information from 16 polymorphic microsatellite DNA markers to study how small-scale kinship structure varies among house sparrows (Passer domesticus) along an urban-rural gradient. Average levels of relatedness were higher among urban individuals than among individuals from rural areas, suggesting lower rates of dispersal in more built-up habitats. Comparison of observed levels of relatedness with simulated distributions of known kinship values showed that central urban individuals had the highest proportion of closely related conspecifics in their immediate neighbourhood. Spatial auto-correlograms supported this small-scale genetic structure and further indicated stronger effects of genetic drift and/or limited dispersal in urban populations. Results of this study underscore the importance of individual-level analyses as a complementary approach to traditional population-level analyses when studying genetic population structure over small spatial scales.

Developmental stability covaries with genome-wide and single-locus heterozygosity in house sparrows.

Fluctuating asymmetry (FA), a measure of developmental instability, has been hypothesized to increase with genetic stress. Despite numerous studies providing empirical evidence for associations between FA and genome-wide properties such as multi-locus heterozygosity, support for single-locus effects remains scant. Here we test if, and to what extent, FA co-varies with single- and multilocus markers of genetic diversity in house sparrow (Passer domesticus) populations along an urban gradient. In line with theoretical expectations, FA was inversely correlated with genetic diversity estimated at genome level. However, this relationship was largely driven by variation at a single key locus. Contrary to our expectations, relationships between FA and genetic diversity were not stronger in individuals from urban populations that experience higher nutritional stress. We conclude that loss of genetic diversity adversely affects developmental stability in P. domesticus, and more generally, that the molecular basis of developmental stability may involve complex interactions between local and genome-wide effects. Further study on the relative effects of single-locus and genome-wide effects on the developmental stability of populations with different genetic properties is therefore needed.

The impact of muscular variation on the neurodynamic test for the median nerve in a healthy population with Langer's axillary arch.

OBJECTIVE: The neurodynamic test of the median nerve (ULNT1) is frequently used to assess the mechanics and physiology of the brachial plexus and median nerve. The present study looks for a positive ULNT1 in a healthy population with Langer's axillary arch (LAA) and analyzes whether LAA affects the elbow extension range of motion (EE-ROM) of the ULNT1. METHOD: Of 640 volunteers screened, 26 LAA sides were finally included. Additional history taking revealed "minor symptoms" in some subjects. Minor symptoms do not qualify as a disorder because there is no interference with daily activities and no medical advice is sought. This study investigates whether the ULNT1 can (re)produce minor symptoms or abnormal responses in subjects with LAA. The EE-ROM was compared between the subjects' left and right side, and the subtraction angle-which is the effect of placing the cervical spine in contralateral lateral flexion-was compared between LAA sides and controls. RESULTS: Langer's axillary arch sides showed a significant increase in the occurrence of minor symptoms and positive ULNT1, but no influence was observed on the EE-ROM. CONCLUSIONS: These findings suggest that LAA may be capable of transiently provoking the axillary neurovascular bundle. The unaffected EE-ROM may be the consequence of a vascular origin of the minor symptoms or the consequence of an ulnar nerve/medial cord response to the ULNT1.