Resequencing studies of nonmodel organisms using closely related reference genomes: optimal experimental designs and bioinformatics approaches for population genomics

Decreasing costs of next‐generation sequencing (NGS) experiments have made a wide range of genomic questions open for study with nonmodel organisms. However, experimental designs and analysis of NGS data from less well‐known species are challenging because of the lack of genomic resources. In this work, we investigate the performance of alternative experimental designs and bioinformatics approaches in estimating variability and neutrality tests based on the site‐frequency‐spectrum (SFS) from individual resequencing data. We pay particular attention to challenges faced in the study of nonmodel organisms, in particular the absence of a species‐specific reference genome, although phylogenetically close genomes are assumed to be available. We compare the performance of three alternative bioinformatics approaches – genotype calling, genotype–haplotype calling and direct estimation without calling genotypes. We find that relying on genotype calls provides biased estimates of population genetic statistics at low to moderate read depth (2–8×). Genotype–haplotype calling returns more accurate estimates irrespective of the divergence to the reference genome, but requires moderate depth (8–20×). Direct estimation without calling genotypes returns the most accurate estimates of variability and of most SFS tests investigated, including at low read depth (2–4×). Studies without species‐specific reference genome should thus aim for low read depth and avoid genotype calling whenever individual genotypes are not essential. Otherwise, aiming for moderate to high depth at the expense of number of individuals, and using genotype–haplotype calling, is recommended.     

Differential sensitivity to mutations in a single peptide by two TCRs having identical beta-chains and closely related alpha-chains

The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR alpha- and ss-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vss8.2 chains, but differ in all three CDR of their related Valpha2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2alpha, and an antagonist residue at the C terminus, which interacts with the CDR3ss, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR alpha- and ss-chains.     

DRG represents a family of two closely related GTP-binding proteins

In a previous publication we identified a novel human GTP-binding protein that was related to DRG, a developmentally regulated GTP-binding protein from the central nervous system of mouse. Here we demonstrate that both the human and the mouse genome possess two closely related drg genes, termed drg1 and drg2. The two genes share 62% sequence identity at the nucleotide and 58% identity at the protein level. The corresponding proteins appear to constitute a separate family within the superfamily of the GTP-binding proteins. The DRG1 and the DRG2 mRNA are widely expressed in human and mouse tissues and show a very similar distribution pattern. The human drg1 gene is located on chromosome 22q12, the human drg2 gene on chromosome 17p12. Distantly related species including Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae also possess two drg genes. In contrast, the genomes of archaebacteria (Halobium, Methanococcus, Thermoplasma) harbor only one drg gene, while eubacteria do not seem to contain any. The high conservation of the polypeptide sequences between distantly related organisms indicates an important role for DRG1 and DRG2 in a fundamental pathway.     

Mycoplasma pneumoniae and Mycoplasma genitalium: a comparison of two closely related bacterial species

The rapid progress in sequencing large quantities of DNA will provide an increasing number of complete genome sequences of closely related bacterial species as well as of pairs of isolates from the same species with different features, such as a pathogenic and an apathogenic representative. This opens the way to apply subtractive comparative analysis as a tool to select from the large pool of all bacterial genes a relatively small set of genes that can be correlated with the expression of a certain phenotype. These selected genes can then be the target for further functional analyses.     

Differential targeting of closely related ECM glycoproteins: the pherophorin family from Volvox.

The alga Volvox carteri represents one of the simplest multicellular organisms. Its extracellular matrix (ECM) is modified under developmental control, e.g. under the influence of the sex-inducing pheromone that triggers development of males and females at a concentration below 10(-16) M. A novel ECM glycoprotein (pherophorin-S) synthesized in response to this pheromone was identified and characterized. Although being a typical member of the pherophorins, which are identified by a C-terminal domain with sequence homology to the sex-inducing pheromone, pherophorin-S exhibits a completely novel set of properties. In contrast to the other members of the family, which are found as part of the insoluble ECM structures of the cellular zone, pherophorin-S is targeted to the cell-free interior of the spherical organism and remains in a soluble state. A main structural difference is the presence of a polyhydroxyproline spacer in pherophorin-S that is linked to a saccharide containing a phosphodiester bridge between two arabinose residues. Sequence comparisons indicate that the self-assembling proteins that create the main parts of the complex Volvox ECM have evolved from a common ancestral gene.     

Seed size: phylogeny and adaptation in two closely related Proteaceae species-pairs

Summary We studied seed size, seed nutrient status and seedling growth of two closely related fynbos Proteaceae species-pairs growing on juxtaposed soils of different nutrient and moisture status. Seeds had a greater mass and higher phosphorus and nitrogen contents for species occurring on limestone (higher nutrient and moisture contents) than those on the colluvial sands (lower nutreints and moisture). This trend was found within, but not across genera, stressing the importance of phylogeny in interpreting adaptations. It would be difficult to test for the effects of either nutrients or moisture separately, since the same advantage of enhanced seedling size, and hence survival in a stressed environment, applies to both factors. The increased root: shoot ratios (using lengths) of the Leucadendron species relative to the Protea species are interpreted as an attempt to overcome a phylogenetic constraint that results in smaller seed size in the former genus.     

Assembly and comparison of two closely related Brassica napus genomes

As an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the de novo assembly of the B. napus cultivar Tapidor and comparison with an improved assembly of the Brassica napus cultivar Darmor‐bzh. Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor‐bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome.     

Demographic mechanisms in the coexistence of two closely related perennials in a fluctuating environment

The demographic variability and life history differentiation of two closely related shrubs (Atriplex canescens and A. acanthocarpa) were investigated in the Chihuahuan Desert, and the results were interpreted in the context of theories of coexistence in fluctuating environments. Demographic information was recorded during three annual intervals and analyzed employing matrix projection models. A. canescens had lower λ (finite rate of population increase), higher longevity and generation time and slower convergence to a stable population structure than A. acanthocarpa. In favorable years for recruitment (those when, for both species, λ > 1), the λ of A. acanthocarpa was higher than that of A. canescens; in unfavorable years (when λ < 1), the reverse was true. Regardless of conditions (year), A. acanthocarpa had a type 2 survivorship curve (constant rate of mortality with age), while A. canescens had a type 3 survivorship curve (declining mortality with age). Elasticity analyses highlighted the larger influence that fecundity and growth would have in modifying the λ of A. acanthocarpa in comparison to that of A. canescens. In contrast, survival would have a larger influence on the λ of A. canescens. Atriplex acanthocarpa behaved as an opportunistic species that benefitted from sporadic favorable conditions and declined rapidly when conditions deteriorated. In contrast, A. canescens behaved as a tolerant species able to withstand years when conditions were poor, but which could not gain any advantage over A. acanthocarpa when conditions improved. By each having a relative advantage over the other on opposite ends of the contrasting climatic conditions experienced in the Chihuahuan Desert, they are able to coexist. Their contrasting life histories agreed with the theoretical predictions for the operation of the two mechanisms of species coexistence in fluctuating environments: the storage effect and the relative non-linearity of competition. Based on these results, we conclude by speculating on the nature of succession in arid communities.     

Strain-specific differences in the genetic control of two closely related mycobacteria

The host response to mycobacterial infection depends on host and pathogen genetic factors. Recent studies in human populations suggest a strain specific genetic control of tuberculosis. To test for mycobacterial-strain specific genetic control of susceptibility to infection under highly controlled experimental conditions, we performed a comparative genetic analysis using the A/J- and C57BL/6J-derived recombinant congenic (RC) mouse panel infected with the Russia and Pasteur strains of Mycobacterium bovis Bacille Calmette Guérin (BCG). Bacillary counts in the lung and spleen at weeks 1 and 6 post infection were used as a measure of susceptibility. By performing genome-wide linkage analyses of loci that impact on tissue-specific bacillary burden, we were able to show the importance of correcting for strain background effects in the RC panel. When linkage analysis was adjusted on strain background, we detected a single locus on chromosome 11 that impacted on pulmonary counts of BCG Russia but not Pasteur. The same locus also controlled the splenic counts of BCG Russia but not Pasteur. By contrast, a locus on chromosome 1 which was indistinguishable from Nramp1 impacted on splenic bacillary counts of both BCG Russia and Pasteur. Additionally, dependent upon BCG strain, tissue and time post infection, we detected 9 distinct loci associated with bacillary counts. Hence, the ensemble of genetic loci impacting on BCG infection revealed a highly dynamic picture of genetic control that reflected both the course of infection and the infecting strain. This high degree of adaptation of host genetics to strain-specific pathogenesis is expected to provide a suitable framework for the selection of specific host-mycobacteria combinations during co-evolution of mycobacteria with humans.     

Behavioural isolation between two closely related Hawaiian Drosophila species: the role of courtship

Behavioural isolation plays a critical role in several recent models of speciation. A detailed understanding of the process of speciation requires analysis of taxonomic groups that have not completed reproductive isolation. We studied D. silvestris and D. heteroneura because they are still in the process of divergence: behavioural isolation between them is incomplete, and neither postzygotic nor ecological isolation has been detected. Behavioural isolation is due to the failure of courtships between male D. silvestris and female D. heteroneura: there is no postzygotic isolation from either parental species. The F1 hybrids are as successful in courtship with parental individuals as same-species pairs, which suggests that the hybrids resemble male D. heteroneura or female D. silvestris in some behaviour patterns that are crucial to mating success. We searched for this crucial resemblance by examining courtship between F1 hybrids and the parental adults. We found that successful F1 males are somewhat more similar than unsuccessful F1 males to D. heteroneura males, but nevertheless they were intermediate between males of the two species. We also found that in both species the presence of female wings is necessary for courtship to proceed to copulation. These results reinforce an earlier report that behavioural isolation between these species is largely attributable to the decision as to whether to court at all, rather than to the details of courtship. Copyright 2000 The Association for the Study of Animal Behaviour.     

A chromosomal investigation of two closely related species of Spodoptera

This paper reports the first cytogenetic study of the two closely related species of noctuid moths, Spodoptera latifascia and S. descoinsi. Chromosomes were prepared using a spreading technique on warm slides. Both gonads and larval brains, which are innovating in the Lepidoptera, were used. Out of 100 specimens observed, 35 showed mitotic metaphases, which allowed chromosomes to be counted. For both species and F1 hybrids, the diploid chromosome number was 2n = 62. The chromosomes of the two species appeared dot-shaped, more rarely rod-shaped, showing little variation in size or morphology. The preparations from larval brains also suggested the existence of two levels of ploidy (haploidy and diploidy) in some nuclei. This result will need further investigation. For the first time in the Lepidoptera, in situ hybridization with Drosophila rDNA as a probe was carried out on S. latifascia and S. descoinsi. It revealed the presence of nucleolar organizing regions located at the distal part of one chromosome pair. Though no sophisticated characterization of S. latifascia and S. descoinsi was possible, it seemed that there was no major chromosomal difference. No karyotypic element could be identified as being involved in reproductive isolation between the two species.     

Interactions between two closely related phytal harpacticoid copepods, asymmetric positive and negative effects

Competition for food is generally thought to exert a strong evolutionary pressure, driving trophic niche separation, either by specialization and/or by widening the choice of potential food resources. Harpacticoid copepods are common inhabitants of phytal assemblages, where several closely related species of the so-called phytal dwelling families often co-occur. However, direct competition among phytal harpacticoids has been thought to be unlikely, due to the abundant and continuously available food supplies. We conducted a series of field and laboratory studies to assess the role of competition in the abundance distribution of two closely related harpacticoid species, Mesochra rapiens and M. aestuari. We found that the abundance of both species co-varied on several seaweed species in the northern Baltic Sea, during a 3-month period. Stable isotope ratios in the green alga Cladophora glomerata field samples indicated different resource utilization of the two species, both in fresh and deteriorated C. glomerata, and in drifting algae. We tested in the laboratory if resource utilization was different between the species in sympatry and allopatry. We used enriched stable carbon isotope ratios (¹³C/¹²C) of the diatom Phaeodactylum tricornutum to trace the uptake in both species. Results from these experiments showed a much higher assimilation by M. aestuari in sympatry with M. rapiens, while the latter species showed a higher assimilation in allopatry. Our results show that while there is no apparent competition for resources between these two species in the field, there seems to be an asymmetric reaction when in sympatry and provided one single resource in the laboratory. We suggest that M. rapiens may facilitate assimilation by M. aestuari and discuss the mechanisms by which this may take place.     

Different pharmacological profile of two closely related endocannabinoid ester analogs

The pharmacological and neuroprotective properties of two ester analogs of the endocannabinoids, arachidonoylethyleneglycol (AA-EG) and alpha,alpha,-dimethyl arachidonoylethyleneglycol (DMA-EG), were investigated. We examined the interaction of both compounds with cannabinoid receptors (CB1 and CB2) and their efficacy in functional assays. In competition binding assays, AA-EG and DMA-EG had low potency to displace the CB1/CB2 agonist [3H]CP-55,940 in membrane preparations expressing rodent or human receptors. Binding data correlate with low efficacy of both compounds as regards to inhibition of adenylyl cyclase activity. It was also shown that DMA-EG resists hydrolysis by rat brain membranes while AA-EG undergo complete splitting under these conditions. In the cannabinoid tetrad, AA-EG induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating cannabimimetic activity. By contrast, DMA-EG was completely inactive in the same models. DMA-EG and AA-EG potently protected rat cortical neurons in culture against oxygen deprivation at nanomolar concentrations. In glutamate-induced damage, the compounds were less active protecting neurons at micromolar concentrations. The data obtained indicate that the ester endocannabinoid template can be used for the development of new compounds with potent biological activity lacking some of the undesirable behavioral side effects.     

Adjustability of the circadian clock in the cockroaches: a comparative study of two closely related species, Blattella germanica and Blattella bisignata.

A single 2h light pulse (250 lux) was given at various times to phase shift the locomotor circadian rhythm of two species of closely related cockroaches, Blattella bisignata and Blatella germanica. The phase-response curve (PRC) of both species showed a similar pattern. Phase delays and advances were induced by light pulse during the early and late subjective night, respectively, while no clear phase shifting was elicited during the subjective day. However, the magnitude of the phase delay (1.89h +/- 0.66h) and advance (0.69h +/- 0.36h) of B. bisignata was significantly larger than that of B. germanica (0.78h +/- 0.38h and 0.35h +/- 0.18h, respectively). This result indicates the superior adjustability of the circadian clock in B. bisignata. The period-response curve (PdRC) was also constructed for both species. Although both species did not show great flexibility in circadian period changes, the phase shifts were significantly correlated with the period changes in the advance zone of B. bisignata (r = 0.72, P < .1). This allowed the circadian clock of B. bisignata to display better entrainability since the phase advance adjustment was significantly more difficult than that of phase delay. The results indicate the overall adjustability of the circadian clock of B. germanica is inferior to that of B. bisignata. The significance of this finding is discussed from an ecological perspective.     

Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies

The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.     

Intra- and interspecific host discrimination in two closely related egg parasitoids

Intraspecific host discrimination is frequently found in solitary parasitoids, but interspecific host discrimination, where female parasitoids recognize hosts already parasitized by females of other species, is rare. This particular behaviour appears to be adaptive only under specific circumstances. In this paper, we quantified intraspecific host discrimination in Anaphes n. sp. (Hymenoptera: Mymaridae), an endoparasitoid of the eggs of Listronotus oregonensis (LeConte) (Coleoptera: Curculionidae) and interspecific host discrimination toward eggs parasitized by Anaphes sordidatus (Girault), a sympatric species competing for the same resource in similar habitats. To examine host discrimination, choice experiments were used where the females had to choose between different categories of eggs (unparasitized, parasitized by Anaphes n. sp. or A. sordidatus). Superparasitism and multiparasitism were avoided in experiments where the female had a choice between unparasitized hosts and hosts parasitized by the same female, by a conspecific or by a female A. sordidatus. When all hosts available were parasitized, conspecific superparasitism occurred more often than self-superparasitism or multiparasitism. These results indicated that females Anaphes n. sp. were capable of self-, conspecific and interspecific discrimination. Self-discrimination followed recognition of an external marking while interspecific discrimination occurred mostly after insertion of the ovipositor. Interspecific discrimination could result from the recent speciation of these species and could be associated with a genotypic discrimination. This behavior appears to be adaptive because of the competition for common hosts between the two parasitoid species.