Genetic differentiation and adaptive evolution at reproductive loci in incipient Drosophila species.

Accessory gland proteins (Acps) are part of the seminal fluid of male Drosophila flies. Some Acps have exceptionally high evolutionary rates and evolve under positive selection. Proper interactions between Acps and female reproductive molecules are essential for fertilization. These observations lead to suggestions that fast evolving Acps could be involved in speciation by promoting reproductive incompatibilities between emerging species. To test this hypothesis, we used population genetics data for three sibling species: D. mayaguana, D. parisiena and D. straubae. The latter two species are morphologically very similar and show only incipient reproductive isolation. This system allowed us to examine Acp evolution at different time frames with respect to speciation and reproductive isolation. Comparing data of 14 Acp loci with data obtained for other genomic regions, we found that some Acps show extraordinarily high levels of divergence between D. mayaguana and its two sister species D. parisiena and D. straubae. This divergence was likely driven by adaptive evolution at several loci. No fixed nucleotide differences were found between D. parisiena and D. straubae, however. Nevertheless, some Acp loci did show significant differentiation between these species associated with signs of positive selection; these loci may be involved in this early phase of the speciation process.

When North and South don't mix: genetic connectivity of a recently endangered oceanic cycad, Cycas micronesica, in Guam using EST-microsatellites.

Subject to environmental changes and recurrent isolation in the last ca. 250 Ma, cycads are often described as relicts of a previously common lineage, with populations characterized by low genetic variation and restricted gene flow. We found that on the island of Guam, the endemic Cycas micronesica has most of the genetic variation of 14 EST-microsatellites distributed within each of 18 genetic populations, from 24 original sampling sites. There were high levels of genetic variation in terms of total number of alleles and private alleles, and moderate levels of inbreeding. Restricted but ongoing gene flow among populations within Guam reveals a genetic mosaic, probably more typical of cycads than previously assumed. Contiguous cycad populations in the north of Guam had higher self-recruitment rates compared to fragmented populations in the south, with no substantial connection between them except for one population. Guam's genetic mosaic may be explained by the influence of forest continuity, seed size, edaphic differences, and human transport of cycads. Also important are the extent of synchrony among flushes of reproductive female seed-bearing sporophylls and restricted pollen movement by an obligate mutualist and generalist insects. An NADH EST-locus under positive selection may reflect pressure from edaphic differences across Guam. This and three other loci are ideal candidates for ecological genomic studies. Given this species' vulnerability due to the recent introduction of the cycad aulacaspis scale, we also identify priority populations for ex situ conservation, and provide a genetic baseline for understanding the effects of invasive species on cycads in the Western Pacific, and islands in general.

Is the microbial tree of life verificationist?

The field of microbial phylogenetics has questioned the feasibility of using a tree-like structure to the describe microbial evolution. This debate centres on two main points. First, because microorganisms are able to transfer genes from one to another in zero generations (horizontal gene transfer, or HGT), the use of molecular characters to perform phylogenetic analyses will yield an erroneous topology and HGT clearly makes the evolution of microorganisms non tree-like. Second, the use of concatenated gene sequences in a total evidence approach to phylogenetic systematics is a verificationist endeavour, the aim of which is to bolster support. However, the goal of the total evidence approach to phylogenetic research is based in the idea of increasing explanatory power over background knowledge through test and corroboration, rather than to bolster support for nodes in a tree. In this context, the testing of phylogenetic data is a falsificationist endeavour that includes the possibility of not rejecting the null hypothesis that there is no tree-like structure in molecular phylogenetic data. We discuss several tests that aim to test rigorously the hypothesis that a tree of life exists for microorganisms. We also discuss the philosophical ramifications of background knowledge and corroboration in microbial studies that need to be considered when suggesting that HGT confounds the tree of life. © The Willi Hennig Society 2009.

DNA sequences from a fossil termite in Oligo-Miocene amber and their phylogenetic implications.

DNA was extracted from the fossil termite Mastotermes electrodominicus preserved in Oligo-Miocene amber (25 million to 30 million years old). Fragments of mitochondrial [16S ribosomal DNA (rDNA)] and nuclear (18S rDNA) genes were amplified by polymerase chain reaction. Phylogenetic analysis of fossil and extant 18S rDNA confirmed morphological cladistic analyses of living dictyopterans (termites, cockroaches, and mantids). The fossil termite shares several sequence attributes with Mastotermes darwiniensis. Addition of this fossil to living-species phylogeny is required to substantiate Mastotermes monophyly and affects molecular phylogenetic hypotheses of termites in this, the oldest DNA yet characterized.

Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata.

DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover 'cryptic species'. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be 'character based', whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.

Very old DNA.

The verification of DNA sequences obtained from very old tissue sources as indeed ancient is a major point of discussion in the ancient DNA field. Proper controls and the use of the phylogenetic approach are the general methods employed for verification of the ancient DNA. Most studies have reported the recovery of extremely small amounts of nucleic acids which are sheared into rather small fragments. In addition, problems such as 'PCR jumping' can produce spurious sequence information. These observations suggest that random amplification techniques and the development of primers for highly informative but short target regions are essential for the further development of the ancient DNA field.

How the fruit fly changed (some of) its spots.

Recent results show that the establishment of wing melanization patterns in Drosophila depends on the veins. While several genes have been shown to play a role in melanization, changes in a single gene - the one encoding tyrosine hydroxylase - are sufficient to generate novel pigmentation patterns.

Genes for tight adherence of Actinobacillus actinomycetemcomitans: from plaque to plague to pond scum.

The Gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans forms an extremely tenacious biofilm on solid surfaces such as glass, plastic and hydroxyapatite. This characteristic is likely to be important for colonization of the oral cavity and initiation of a potentially devastating form of periodontal disease. Genetic analysis has revealed a cluster of tad genes responsible for tight adherence to surfaces. Evidence indicates that the tad genes are part of a locus encoding a novel secretion system for the assembly and release of long, bundled Flp pili. Remarkably similar tad loci appear in the genomes of a wide variety of Gram-negative and Gram-positive bacteria, including many significant pathogens, and in Archaea. We propose that the tad loci are important for microbial colonization in a variety of environmental niches.

The molecular through ecological genetics of abnormal abdomen. II. Ribosomal DNA polymorphism is associated with the abnormal abdomen syndrome in Drosophila mercatorum.

Restriction endonuclease cleavage analyses of cloned and genomic DNA samples indicate that the structure of the DNA encoding the large cytoplasmic RNAs (rDNAs) is altered in Drosophila mercatorum lines which exhibit an abnormal abdomen (aa) phenotype. In a majority of the rDNA repeat units from aa flies, the 28S coding sequence is interrupted by a large [5-6 kilobase pairs (kbp)] insert. A subclone containing this inserted DNA (ins 3) hybridizes primarily to rDNA-containing sequences in in situ and genomic blot hybridization experiments. Additionally, genomic nitrocellulose blot hybridization analyses show that ins- containing rDNA repeat units are clustered in a spontaneously arising aa mutant. This rDNA alteration in D. mercatorum flies with the aa phenotype more closely resembles the bobbed (bb) defect of D. hydei than the bb defect of D. melanogaster, which involves alterations in rDNA copy number. By analogy with the other Drosophila systems, we propose that the altered D. mercatorum rDNA repeat units are defective in rRNA production at a critical stage. The lowered levels of rRNA ultimately would limit the concentration of ribosomes needed to produce large quantities of a protein (in these cases, juvenile hormone esterase) needed for normal development.

The molecular through ecological genetics of abnormal abdomen. III. Tissue-specific differential replication of ribosomal genes modulates the abnormal abdomen phenotype in Drosophila mercatorum.

The abnormal abdomen (aa) syndrome in Drosophila mercatorum is controlled by two major X-linked genetic elements. We have previously shown that the major X-linked element of aa is associated with the presence of large inserts in the 28S gene of the ribosomal RNA (rDNA) genes. We show that, in polytene tissue of wild-type D. mercatorum, the uninterrupted rDNA repeats are overreplicated relative to interrupted repeats. Uninterrupted rDNA repeats are also overreplicated in polytene tissue of hybrid larval offspring from wild-type and aa parents. This overreplication of uninterrupted repeats is not observed in diploid tissues of wild-type hybrids (of wild-type and aa parents) and homozygous aa larvae or in polytene tissue of aa larvae. Furthermore, molecular analysis of an aa line that has reverted to the wild type indicates that the reversion phenomenon is associated with the ability to overreplicate uninterrupted rDNA repeats in polytene tissues. The patterns of differential replication of rDNA genes in wild-type hybrids and aa larvae of D. mercatorum offer a possible mechanism for the tissue-specific control of the aa phenotype and suggest that the molecular basis for the second X-linked genetic element of aa is involved in the control of differential replication in polytene tissues.

The molecular through ecological genetics of abnormal abdomen. IV. Components of genetic variation in a natural population of Drosophila mercatorum.

Natural populations of Drosophila mercatorum are polymorphic for a phenotypic syndrome known as abnormal abdomen (aa). This syndrome is characterized by a slow-down in egg-to-adult developmental time, retention of juvenile abdominal cuticle in the adult, increased early female fecundity, and decreased adult longevity. Previous studies revealed that the expression of this syndrome in females is controlled by two closely linked X chromosomal elements: the occurrence of an R1 insert in a third or more of the X-linked 28S ribosomal genes (rDNA), and the failure of replicative selection favoring uninserted 28S genes in larval polytene tissues. The expression of this syndrome in males in a laboratory stock was associated with the deletion of the rDNA normally found on the Y chromosome. In this paper we quantify the levels of genetic variation for these three components in a natural population of Drosophila mercatorum found near Kamuela, Hawaii. Extensive variation is found in the natural population for both of the X-linked components. Moreover, there is a significant association between variation in the proportion of R1 inserted 28S genes with allelic variation at the underreplication (ur) locus such that both of the necessary components for aa expression in females tend to cosegregate in the natural population. Accordingly, these two closely linked X chromosomal elements are behaving as a supergene in the natural population. Because of this association, we do not believe the R1 insert to be actively transposing to an appreciable extent. The Y chromosomes extracted from nature are also polymorphic, with 16% of the Ys lacking the Y-specific rDNA marker. The absence of this marker is significantly associated with the expression of aa in males. Hence, all three of the major genetic determinants of the abnormal abdomen syndrome are polymorphic in this natural population.

Temporal and spatial heterogeneity of mtDNA polymorphisms in natural populations of Drosophila mercatorum.

Restriction endonuclease analysis of mtDNA was used to examine the genetic relatedness of several geographically separated isolines of the Drosophila mercatorum subgroup. In addition, we examined the temporal and spatial distribution of two mtDNA restriction site polymorphisms produced by the enzymes BstEII and BstNI at a single locality--Kamuela, Hawaii. Due to small sample sizes of some collections and the undesirable dependance of the estimation of polymorphism frequency on its variance, an arcsin square root transformation of the frequency data was used. We also use an Fst estimator of our transformed frequencies to demonstrate considerable spatial and temporal differentiation within the Kamuela population. In contrast, isozyme data from the same population reveals no pattern of differentiation. The temporal and geographic heterogeneity and population subdivision detected with mtDNA analysis also is consistent with the known dispersal behavior and ecological constraints of this species. The mtDNA data in conjunction with the isozyme data show that the population structure of the Kamuela D. mercatorum is close to the boundary line separating panmixia from subdivision, a conclusion that could not be made from isozyme data alone.

Polytene chromosomes as indicators of phylogeny in several species groups of Drosophila.

BACKGROUND: Polytene chromosome banding patterns have long been used by Drosophila evolutionists to infer degree of relatedness among taxa. Recently, nucleotide sequences have preempted this traditional method. We place the classical Drosophila evolutionary biology tools of polytene chromosome inversion analysis in a phylogenetic context and assess their utility in comparison to nucleotide sequences. RESULTS: A simultaneous analysis framework was used to examine the congruence of the chromosomal inversion data with more recent DNA sequence data in four Drosophila species groups - the melanogaster, virilis, repleta, and picture wing. Inversions and nucleotides were highly congruent with one another based on incongruence length difference and partitioned Bremer support values. Inversion phylogenies were less resolved because of fewer numbers of characters. Partitioned Bremer supports, corrected for the number of characters in each matrix, were higher for inversion matrices. CONCLUSIONS: Polytene chromosome data are highly congruent with DNA sequence data and, when placed in a simultaneous analysis framework, are shown to be more information rich than nucleotide data.

Homologues of the engrailed gene from five molluscan classes.

We used the polymerase chain reaction (PCR) to amplify, clone, and sequence 10 engrailed homeodomains from 8 species in the five major molluscan classes, including the serially organized chiton (Polyplacophora) lineage. The Drosophila melanogaster gene engrailed (en) is one of several genes involved in embryonic segment polarity determination. Studies of engrailed sequence and expression in molluscs are of interest due to questions regarding the evolution and homology of segmentation in these taxa. Nucleotide and deduced amino acid sequence comparisons reflect evolutionary conservation within helices of the en homeodomain and ancient divergences in the region 3' to the homeodomain.

Collection and storage of invertebrate samples.

The validity of any comparative study is dependent on the reliability of the identification of the samples in the study. Not all researchers are experts in the field of identification of samples, nor do all researchers have quick and ready access to expert systematists who can accomplish the task of identification. The importance of verification of sample identity for comparative studies is vital. We describe several methods by which researchers can obtain and identify samples from the wild, and we suggest methods by which voucher samples can be obtained for future reference to these collected samples. We outline alternatives to collection of samples from the wild, such as purchase from stock centers and biological supply companies. Museum collections can also be extremely helpful in obtaining complete organismal samples for comparative studies.

Computational problems in molecular systematics.

The development of extremely powerful computer programs and the ready availability of microcomputers has revealed several computational problems with data analysis. These problems occur in the handling of systematic data in general and molecular systematic data in particular. This paper examines three areas of controversy in molecular systematics resulting from increased computer power. We start by examining the first step in DNA sequence analysis, the establishment of homology via sequence alignment. Next we examine several problems in phylogenetic analysis that have arisen in the last few years due to use of the PAUP (Swofford, 1991), HENNIG86 (Farris, 1988), and PHYLIP programs. These problems include limitations on the number of taxa examined in a given analysis and the accuracy of the parsimony trees in such analyses. The final subject is an examination of programs used for assessing tree robustness. We concentrate on certain programs (such as MALIGN (Wheeler and Gladstein, 1993), PAUP (Swofford, 1991), HENNIG86 (Farris, 1988), PHYLIP (Felsenstein, 1990), CLADOS (Nixon, 1993), MacClade (Maddison and Maddison, 1993), etc.), but similar comments about other programs could also be made.

Commemoration of the "Molecules and morphology in Systematics" meetings held in paris, france, march 24-march 28, 1997

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The potential of distance-based thresholds and character-based DNA barcoding for defining problematic taxonomic entities by CO1 and ND1.

The mitochondrial CO1 gene (cytochrome c oxidase I) is a widely accepted metazoan barcode region. In insects, the mitochondrial NADH dehydrogenase subunit 1 (ND1) gene region has proved to be another suitable marker especially for the identification of lower level taxonomic entities such as populations and sister species. To evaluate the potential of distance-based thresholds and character-based DNA barcoding for the identification of problematic species-rich taxa, both markers, CO1 and ND1, were used as test parameters in odonates. We sequenced and compared gene fragments of CO1 and ND1 for 271 odonate individuals representing 51 species, 22 genera and eight families. Our data suggests that (i) the combination of the CO1 and ND1 fragment forms a better identifier than a single region alone; and (ii) the character-based approach provides higher resolution than the distance-based method in Odonata especially in closely related taxonomic entities.

The plant proteome folding project: structure and positive selection in plant protein families.

Despite its importance, relatively little is known about the relationship between the structure, function, and evolution of proteins, particularly in land plant species. We have developed a database with predicted protein domains for five plant proteomes (http://pfp.bio.nyu.edu) and used both protein structural fold recognition and de novo Rosetta-based protein structure prediction to predict protein structure for Arabidopsis and rice proteins. Based on sequence similarity, we have identified ~15,000 orthologous/paralogous protein family clusters among these species and used codon-based models to predict positive selection in protein evolution within 175 of these sequence clusters. Our results show that codons that display positive selection appear to be less frequent in helical and strand regions and are overrepresented in amino acid residues that are associated with a change in protein secondary structure. Like in other organisms, disordered protein regions also appear to have more selected sites. Structural information provides new functional insights into specific plant proteins and allows us to map positively selected amino acid sites onto protein structures and view these sites in a structural and functional context.