Targeting optimal introns for phylogenetic analyses in non-model taxa: experimental results in Asian pitvipers

Nuclear introns are increasingly used as phylogenetic markers. Here, we present a multidisciplinary approach towards optimal locus selection and amplification using Asian pitvipers as an example of a non‐model taxon, and raise the profile of length variant heterozygotes (LVHs) in intron loci. Taxon‐specific primers were identified using a bioinformatic approach, and also designed from existing exon primed, intron crossing (EPIC) primer amplifications. Eleven further universal EPIC primer pairs were assayed using a range of PCR optimization strategies. Taxon‐specific primers yielded the most consistent amplifications, but assaying a large number of universal EPIC primers yielded another appropriate locus for phylogenetic purposes. Modified Taq DNA polymerases such as JumpStart?Taq either significantly improved the specificity and yield of EPIC PCR amplifications (of low copy number nuclear targets), or resulted in amplifications that were not significantly worse than those derived from a generic Taq DNA polymerase. Finally, LVHs were detected in all loci that were sequenced suggesting that they are relatively common in introns. This study provides an efficient and cost effective template for the successful identification of intron markers for molecular systematics which is universally applicable to other non‐model taxon groups. © The Willi Hennig Society 2005.     

Glowing in the dark: discriminating patterns of bioluminescence from different taxa during the Arctic polar night

Research since 2009 has shown that despite almost total darkness during the Arctic polar night, there is much more biological activity than previously assumed, both at the sea surface, water column and sea floor. Here, we describe in situ monitoring of the bioluminescent fraction of the zooplankton community (dinoflagellates, copepods, krill and ctenophores) as a function of time and space. In order to examine the relative contribution of each selected taxon and any diurnal patterns in the relative signals, a time series platform capable of detecting in situ bioluminescent flashes was established in Kongsfjord, Svalbard, during the polar night in January 2013. Combined with laboratory-controlled measurements of animals collected next to the time series platform, we present both taxon-specific and community characteristics of the bioluminescence signal from a location at 79°N and from the middle of the polar night. Based on this 51-h time series, we conclude that the bioluminescent fraction of the zooplankton does not maintain a diurnal signal. Rather, the frequency of bioluminescence flashes from the entire bioluminescent community remained steady throughout the sampling period. Furthermore, we conclude that bioluminescence flash kinetic characteristics have a strong potential for in situ taxa recognition of zooplankton.     

Development of a procedure for discriminating among Escherichia coli isolates from animal and human sources

Counts of Escherichia coli cells in water indicate the potential presence of pathogenic microbes of intestinal origin but give no indication of the sources of the microbial pollution. The objective of this research was to evaluate methods for differentiating E. coli isolates of livestock, wildlife, or human origin that might be used to predict the sources of fecal pollution of water. A collection of 319 E. coli isolates from the feces of cattle, poultry, swine, deer, goose, and moose, as well as from human sewage, and clinical samples was used to evaluate three methods. One method was the multiple-antibiotic-resistance (MAR) profile using 14 antibiotics. Discriminant analysis revealed that 46% of the livestock isolates, 95% of the wildlife isolates, and 55% of the human isolates were assigned to the correct source groups by the MAR method. Amplified fragment length polymorphism (AFLP) analysis, the second test, was applied to 105 of the E. coli isolates. The AFLP results showed that 94% of the livestock isolates, 97% of the wildlife isolates, and 97% of the human isolates were correctly classified. The third method was analysis of the sequences of the 16S rRNA genes of the E. coli isolates. Discriminant analysis of 105 E. coli isolates indicated that 78% of the livestock isolates, 74% of the wildlife isolates, and 80% of the human isolates could be correctly classified into their host groups by this method. The results indicate that AFLP analysis was the most effective of the three methods that were evaluated.     

Estimating species richness from quadrat sampling data: a general approach

We consider the problem of estimating the number of species (denoted by S) of a biological community located in a region divided into n quadrats. To address this question, different hierarchical parametric approaches have been recently developed. Despite a detailed modeling of the underlying biological processes, they all have some limitations. Indeed, some assume that n is theoretically infinite; as a result, n and the sampling fraction are not a part of such models. Others require some prior information on S to be efficiently implemented. Our approach is more general in that it applies without limitation on the size of n, and it can be used in the presence, as well as in the absence, of prior information on S. Moreover, it can be viewed as an extension of the approach of Dorazio and Royle (2005, Journal of the American Statistical Association 100, 389-398) in that n is a part of the model and a prior distribution is placed on S. Despite serious computational difficulties, we have perfected an efficient Markov chain Monte Carlo algorithm, which allows us to obtain the Bayesian estimate of S. We illustrate our approach by estimating the number of species of a bird community located in a forest.     

Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.     

PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data

Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?     

SMOOTH: a statistical method for successful removal of genotyping errors from high-density genetic linkage data

High-density genetic linkage maps can be used for purposes such as fine-scale targeted gene cloning and anchoring of physical maps. However, their construction is significantly complicated by even relatively small amounts of scoring errors. Currently available software is not able to solve the ordering ambiguities in marker clusters, which inhibits the application of high-density maps. A statistical method named SMOOTH was developed to remove genotyping errors from genetic linkage data during the mapping process. The program SMOOTH calculates the difference between the observed and predicted values of data points based on data points of neighbouring loci in a given marker order. Highly improbable data points are removed by the program in an iterative process with a mapping algorithm that recalculates the map after cleaning. SMOOTH has been tested with simulated data and experimental mapping data from potato. The simulations prove that this method is able to detect a high amount of scoring errors and demonstrates that the program enables mapping software to successfully construct a very accurate high-density map. In potato the application of the program resulted in a reliable placement of nearly 1,000 markers in one linkage group.     

Quasispecies in retrotransposons: a role for sequence variability in Tnt1 evolution

Retroviral replication is a very error-prone process. Replication of retroviruses gives rise to populations of closely related but different genomes referred to as ‘quasispecies’. This huge swarm of different sequences constitutes a reservoir of potentially useful genomes in case of an environmental change, endowing retroviruses with extreme adaptability. Retrotransposons are mobile genetic elements closely related to retroviruses, and retrotransposition is as error prone as retroviral replication. The Tnt1 retrotransposon is present in hundreds of copies in the genome of tobacco that show a high level of sequence heterogeneity. When Tnt1 is expressed, its RNA is not a single sequence but a population of sequences displaying a quasispecies-like structure. This population structure gives to Tnt1, as in the case of retroviruses, a high sequence plasticity and an adaptive capacity. We propose this adaptivity as the major reason for Tnt1 maintenance in Nicotiana genomes and we discuss in this paper the importance of sequence variability for Tnt1 evolution. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enzyme variation and taxonomy: the estimation of sampling errors in measurements of interspecific genetic similarity

The uses of biochemical genetics as an aid to taxonomy are discussed briefly. A survey is made of published data to establish expected levels of genetic similarity or identity between conspecific populations, between congeneric species and between species of different genera. Existing measures of genetic similarity, identity or distance are discussed and some of the difficulties involved in their use in taxonomy are outlined. A new measure of genetic similarity is proposed specifically designed for use in making interspecific comparisons for taxonomic purposes. This measure offers considerable advantages in ease of calculation and also greatly simplifies the estimation of sampling errors. An empirical comparison is made, using published data, of the measures and confidence limits obtained by the proposed memod against published measures of genetic variation between species.     

Simple sequence repeat (SSR) analysis for assessment of genetic variability in apricot germplasm

Thirty SSR primer combinations, developed from peach SSR-enriched genomic libraries and BAC libraries of peach [ Prunus persica (L.) Batsch.], were tested for cross amplification with 74 apricot ( Prunus armeniaca L.) germplasm accessions. Twelve primer pairs amplified 14 polymorphic SSR loci useful for discriminating most apricot cultivars, as well as for investigating patterns of variation in apricot germplasm. Levels of polymorphism were higher than the levels described using other codominant marker systems (i.e., isozymes, RFLP markers). Overall, 107 alleles were identified, and all but 11 accessions were unambiguously discriminated. Genetic differentiation of native germplasm into traditional ecogeographical groups was low, with a high level of genetic identity (> 0.75) between the groups. However, neighbor joining cluster analysis of marker distances between cultivars reflected the complex history of apricot domestication, producing groupings not evidently based on the geographical origin of the cultivars. Distant positioning of Chinese cultivars on UPGMA and neighbor joining dendrograms supports the authors' consideration of Chinese apricots as subspecies, Prunus armeniaca var. ansu Maxim., rather than a separate species.     

Sustaining genetic variation in a small population: evidence from the Mauritius kestrel

We obtained measures of genetic diversity in 10 kestrel species at a suite of 12 microsatellite loci. We estimated the relative effective size (Ne) of the species using a Markov chain Monte Carlo (MCMC) approach, which jointly estimated the locus specific mutation rates as nuisance parameters. There was surprisingly high genetic diversity found in museum specimens of the Mauritius kestrel. Being an endemic species on a small island, it is known to have a long history of small population size. Conversely, kestrels with a continental distribution had Ne estimates that were only one order of magnitude larger and similar to each other, despite having current population sizes that were between one and three orders of magnitude larger than the Mauritius kestrel. We show how many of the theoretical results describing the effective size of a subdivided population can be captured in terms of three rates which describe the branching pattern of the gene genealogy, and that they are useful in estimating the time to migration-drift and mutation-drift equilibrium. We use this approach to argue that population subdivision has helped retain genetic diversity in the Mauritius kestrel, and that the continental species' genetic diversity has yet to reach equilibrium after the range changes following the last ice age. We draw parallels with Hewitt's observation that genetic variation seems to survive species' range compression and is rather vulnerable to range expansion.     

Molecular basis for skeletal variation: insights from developmental genetic studies in mice

Skeletal variations are common in humans, and potentially are caused by genetic as well as environmental factors. We here review molecular principles in skeletal development to develop a knowledge base of possible alterations that could explain variations in skeletal element number, shape or size. Environmental agents that induce variations, such as teratogens, likely interact with the molecular pathways that regulate skeletal development.     

Estimating genetic benefits of polyandry from experimental studies: a meta‐analysis

The consequences of polyandry for female fitness are controversial. Sexual conflict studies and a meta‐analysis of mating rates in insects suggest that there is a longevity cost when females mate repeatedly. Even so, compensatory material benefits can elevate egg production and fertility, partly because polyandry ensures an adequate sperm supply. Polyandry can therefore confer direct benefits. The main controversy surrounds genetic benefits. The argument is analogous to that surrounding the evolution of conventional female mate choice, except that with polyandry it is post‐copulatory mechanisms that might bias paternity towards males with higher breeding values for fitness. Recent meta‐analyses of extra‐pair copulations in birds have cast doubt on whether detectable genetic benefits exist. By contrast, another meta‐analysis showed that polyandry elevates egg hatching success (possibly due to a fertilization bias towards sperm with paternal genes that elevate embryo survival) in insects. A detailed summary of whether polyandry elevates other components of offspring performance is lacking. Here we present a comprehensive meta‐analysis of 232 effect sizes from 46 experimental studies. These experiments were specifically designed to try to quantify the potential genetic benefits of polyandry by controlling fully for the number of matings by females assigned to monandry and polyandry treatments. The bias‐corrected 95% confidence intervals for egg hatching success (d = ?0.01 to 0.61), clutch production (d = 0.07 to 0.45) and fertility (d = 0.04 to 0.40) all suggest that polyandry has a beneficial effect (although P values from parametric tests were marginally non‐significant at P = 0.075, 0.052 and 0.058, respectively). Polyandry was not significantly beneficial for any single offspring performance trait (e.g. growth rate, survival, adult size), but the test power was low due to small sample sizes (suggesting that many more studies are still needed). We then calculated a composite effect size that provides an index of general offspring performance. Depending on the model assumptions, the mean effect of polyandry was either significantly positive or marginally non‐significant. A possible role for publication bias is discussed. The magnitude of the reported potential genetic benefits (d = 0.07 to 0.19) are larger than those from two recent meta‐analyses comparing offspring sired by social and extra‐pair mates in birds (d = 0.02 to 0.04). This difference raises the intriguing possibility that cryptic, post‐copulatory female choice might be more likely to generate ‘good gene’ or ‘compatible gene’ benefits than female choice of mates based on the expression of secondary sexual traits.     

Shotgun haplotyping: a novel method for surveying allelic sequence variation

Haplotypic sequences contain significantly more information than genotypes of genetic markers and are critical for studying disease association and genome evolution. Current methods for obtaining haplotypic sequences require the physical separation of alleles before sequencing, are time consuming and are not scaleable for large surveys of genetic variation. We have developed a novel method for acquiring haplotypic sequences from long PCR products using simple, high-throughput techniques. This method applies modified shotgun sequencing protocols to sequence both alleles concurrently, with read-pair information allowing the two alleles to be separated during sequence assembly. Although the haplotypic sequences can be assembled manually from the resultant data using pre-existing sequence assembly software, we have devised a novel heuristic algorithm to automate assembly and remove human error. We validated the approach on two long PCR products amplified from the human genome and confirmed the accuracy of our sequences against full-length clones of the same alleles. This method presents a simple high-throughput means to obtain full haplotypic sequences potentially up to 20 kb in length and is suitable for surveying genetic variation even in poorly-characterized genomes as it requires no prior information on sequence variation.     

Estimating the uncertainty in annual net ecosystem carbon exchange: spatial variation in turbulent fluxes and sampling errors in eddy-covariance measurements

Above forest canopies, eddy covariance (EC) measurements of mass (CO₂, H₂O vapor) and energy exchange, assumed to represent ecosystem fluxes, are commonly made at one point in the roughness sublayer (RSL). A spatial variability experiment, in which EC measurements were made from six towers within the RSL in a uniform pine plantation, quantified large and dynamic spatial variation in fluxes. The spatial coefficient of variation (CV) of the scalar fluxes decreased with increasing integration time, stabilizing at a minimum that was independent of further lengthening the averaging period (hereafter a ‘stable minimum’). For all three fluxes, the stable minimum (CV=9–11%) was reached at averaging times (τ_p) of 6–7 h during daytime, but higher stable minima (CV=46–158%) were reached at longer τ_p (>12 h) during nighttime. To the extent that decreasing CV of EC fluxes reflects reduction in micrometeorological sampling errors, half of the observed variability at τ_p=30 min is attributed to sampling errors. The remaining half (indicated by the stable minimum CV) is attributed to underlying variability in ecosystem structural properties, as determined by leaf area index, and perhaps associated ecosystem activity attributes. We further assessed the spatial variability estimates in the context of uncertainty in annual net ecosystem exchange (NEE). First, we adjusted annual NEE values obtained at our long‐term observation tower to account for the difference between this tower and the mean of all towers from this experiment; this increased NEE by up to 55 g C m^?2 yr^?1. Second, we combined uncertainty from gap filling and instrument error with uncertainty because of spatial variability, producing an estimate of variability in annual NEE ranging from 79 to 127 g C m^?2 yr^?1. This analysis demonstrated that even in such a uniform pine plantation, in some years spatial variability can contribute ～50% of the uncertainty in annual NEE estimates.     

Somaclonal variation — a novel source of variability from cell cultures for plant improvement

Summary It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.     

Stress-induced variation in evolution: from behavioural plasticity to genetic assimilation

Extreme environments are closely associated with phenotypic evolution, yet the mechanisms behind this relationship are poorly understood. Several themes and approaches in recent studies significantly further our understanding of the importance that stress-induced variation plays in evolution. First, stressful environments modify (and often reduce) the integration of neuroendocrinological, morphological and behavioural regulatory systems. Second, such reduced integration and subsequent accommodation of stress-induced variation by developmental systems enables organismal 'memory' of a stressful event as well as phenotypic and genetic assimilation of the response to a stressor. Third, in complex functional systems, a stress-induced increase in phenotypic and genetic variance is often directional, channelled by existing ontogenetic pathways. This accounts for similarity among individuals in stress-induced changes and thus significantly facilitates the rate of adaptive evolution. Fourth, accumulation of phenotypically neutral genetic variation might be a common property of locally adapted and complex organismal systems, and extreme environments facilitate the phenotypic expression of this variance. Finally, stress-induced effects and stress-resistance strategies often persist for several generations through maternal, ecological and cultural inheritance. These transgenerational effects, along with both the complexity of developmental systems and stressor recurrence, might facilitate genetic assimilation of stress-induced effects. Accumulation of phenotypically neutral genetic variance by developmental systems and phenotypic accommodation of stress-induced effects, together with the inheritance of stress-induced modifications, ensure the evolutionary persistence of stress-response strategies and provide a link between individual adaptability and evolutionary adaptation.