Investigating biogeographic boundaries of the Sunda shelf: A phylogenetic analysis of two island populations of Macaca fascicularis.

OBJECTIVES: Cyclical submergence and re-emergence of the Sunda Shelf throughout the Pleistocene served as a dynamic biogeographic landscape, across which long-tailed macaques (Macaca fascicularis) have migrated and evolved. Here, we tested the integrity of the previously reported continental-insular haplotype divide reported among Y and mitochondrial DNA lineages across multiple studies. MATERIALS AND METHODS: The continental-insular haplotype divide was tested by heavily sampling wild macaques from two important biogeographic regions within Sundaland: (1) Singapore, the southernmost tip of continental Asia and (2) Bali, Indonesia, the southeastern edge of the Indonesian archipelago, immediately west of Wallace's line. Y DNA was haplotyped for samples from Bali, deep within the Indonesian archipelago. Mitochondrial D-loop from both islands was analyzed against existing data using Maximum Likelihood and Bayesian approaches. RESULTS: We uncovered both "continental" and "insular" Y DNA haplotypes in Bali. Between Singapore and Bali we found 52 unique mitochondrial haplotypes, none of which had been previously described. Phylogenetic analyses confirmed a major haplogroup division within Singapore and identified five new Singapore subclades and two primary subclades in Bali. DISCUSSION: While we confirmed the continental-insular divide among mtDNA haplotypes, maintenance of both Y DNA haplotypes on Bali, deep within the Indonesian archipelago calls into question the mechanism by which Y DNA diversity has been maintained. It also suggests the continental-insular designation is less appropriate for Y DNA, leading us to propose geographically neutral Y haplotype designations.

Reproductive isolation in Drosophila: how close are we to untangling the genetics of speciation?

Our understanding of the genetic basis of reproductive isolation in Drosophila has progressed rapidly over the past decade. Details of the genetic structure of hybrid sterility have been revealed and a general consensus has been reached concerning the genetic bases of Haldane's rule. Genetic analyses now reach beyond hybrid sterility and inviability, allowing us to make important comparisons across different traits involved in reproductive isolation. Expansion of genetic studies to include rescue of hybrid incompatibilities has opened the door for more detailed molecular and developmental analyses of reproductive isolation than has ever before been possible.

All in the family: evolutionary and functional relationships among death receptors.

Over the last decade, significant progress has been made towards identifying the signaling pathways within mammalian cells that lead to apoptosis mediated by death receptors. The simultaneous expression of more than one death receptor in many, if not all, cell types suggests that functional innovation has driven the divergence of these receptors and their cognate ligands. To better understand the physiological divergence of the death receptors, a phylogenetic analysis of vertebrate death receptors was conducted based upon amino-acid sequences encoding the death domain regions of currently known and newly identified members of the family. Evidence is presented to indicate an ancient radiation of death receptors that predates the emergence of vertebrates, as well as ongoing divergence of additional receptors both within several receptor lineages as well as modern taxonomic lineages. We speculate that divergence among death receptors has led to their functional specialization. For instance, some receptors appear to be primarily involved in mediating the immune response, while others play critical roles during development and tissue differentiation. The following represents an evolutionary approach towards an understanding of the complex relationship among death receptors and their proposed physiological functions in vertebrate species.

The molecular through ecological genetics of abnormal abdomen in Drosophila mercatorum. VI. The non-neutrality of the Y chromosome rDNA polymorphism.

An association between quantitative variation of rDNA on the Y chromosome and male expression of the juvenilized, adult cuticle of the abnormal abdomen syndrome has been found for Drosophila mercatorum. Many pleiotropic effects of this syndrome have been described previously for females, but little was known about possible pleiotropic effects in males. The effects on males open up new avenues for the action of natural selection operating on the system. In females, the syndrome causes an increase in egg-to-adult development time, precocious sexual maturation, increased fecundity and decreased longevity. In addition to the cuticle phenotype, in males abnormal abdomen causes delayed sexual maturation, increased longevity, and decreased mating success, yet no change in egg-to-adult development time. Thus the syndrome has opposing fitness effects in the two sexes, which may help explain the genetic polymorphism observed in this system. Although investigated intensively, associations between naturally occurring Y-linked polymorphism and fitness phenotypes have not been found in Drosophila melanogaster.

Reexamination of alcohol dehydrogenase structural mutants in Drosophila using protein blotting.

Using protein blotting and an immuno-overlay procedure, we have reexamined the cross-reacting material produced by ADH null-activity mutants generated with ethyl methanesulfonate (EMS). Of the 13 mutants, 11 have an immunodetectable polypeptide of wild-type size. The native and urea denatured isoelectric points (pI) establish that 7 of 13 of the mutations have no effect on protein charge. The electrophoretic mobilities of each variant on increasing percent acrylamide gels (Ferguson analysis), reveal that 9 of the 11 immunodetectable mutants have retained the ability to form dimers under native conditions. None of the inactive mutant proteins has the ability to form the "adduct-bound" isozyme. We have found no correlation between protein pI and in vivo stability. The observed frequencies of specific charge class alterations do not dispute the propensity of G:A transitions previously found for EMS mutagenesis.

Partial correction of structural defects in alcohol dehydrogenase through interallelic complementation in Drosophila melanogaster.

Alcohol dehydrogenases (ADH) from the F1 progeny of all pairwise crosses between 12 null-activity mutants and crosses between these mutants and four active variants, ADHn5 ADHF, ADHD and ADHS, were analyzed for the presence of active or inactive heterodimers. Gels were stained for ADH enzyme activity, and protein blots of duplicate gels were probed with ADH-specific antibody to detect cross-reacting material. Crosses between the three major electrophoretic variants. ADHF, ADHS and ADHD, all produced active heterodimers. Four mutant proteins (ADHn2, ADHn4, ADHn10 and ADHn13) did not form heterodimers with any other ADH subunit tested. Of the 28 crosses involving the remaining null activity mutants, 22 produce heterodimers. Twelve of these exhibit partial restoration of enzyme activity. In five cases of active heterodimers from null-activity crosses, Adhn11 supplied one of the subunits. In two crosses involving the active variant ADHD, the null activity mutant subunits (ADHn8 and ADHn3) destabilized the heterodimer sufficiently to cause inactivation of the ADHD subunit. In the cross between AdhF and Adhn3, the activity of the ADHF subunit was also greatly reduced in association with the ADHn3 subunit. Two crosses (Adhn1 X Adhn11 and Adhn5 X Adhn12) result in partial restoration of one of the homodimeric proteins (ADHn1 and ADHn12, respectively), as well as forming active heterodimers.

The genetics of reproductive isolation in the Drosophila simulans clade: X vs. autosomal effects and male vs. female effects.

A strong effect of homozygous autosomal regions on reproductive isolation was found for crosses between the species in the Drosophila simulans clade. Second chromosome regions were introgressed from D. mauritiana and D. sechellia into D. simulans and tested for their homozygous effects on hybrid male and hybrid female sterility and inviability. Most introgressions are fertile as heterozygotes, yet produce sterile male offspring when made homozygous. The density of homozygous autosomal factors contributing to hybrid male sterility is comparable to the density of X chromosome factors for this level of resolution. Female sterility was also revealed, yet the disparity between male and female levels of sterility was great, with male sterility being up to 23 times greater than female sterility. Complete hybrid inviability was also associated with some regions of the second chromosome, yet there were no strong sex differences. In conclusion, we find no evidence to support a strong X chromosome bias in the evolution of hybrid sterility or inviability but do find a very strong sex bias in the evolution of hybrid sterility. In light of these findings, we reevaluate the current models proposed to explain the genetic pattern of reproductive isolation.

The molecular through ecological genetics of abnormal abdomen in Drosophila mercatorum. V. Female phenotypic expression on natural genetic backgrounds and in natural environments.

The abnormal abdomen (aa) syndrome in Drosophila mercatorum depends on the presence of R1 inserts in a third or more of the X-linked 28S rDNA genes and the absence of selective underreplication of inserted repeats in polytene tissues that is controlled by an X-linked locus (ur) half a map unit from the rDNA complex. This syndrome affects both life history and morphology in the laboratory. Because abnormal morphologies are rarely encountered in nature, the purpose of this study is to see if the female life history traits are still affected under more natural genetic backgrounds and environmental conditions. Two outbred stocks were extracted from the natural population living near Kamuela, Hawaii: KaaX that has only X chromosomes with uraa alleles, and K+X that has only ur+ alleles. These two stocks have nonoverlapping distributions of insert proportions, indicating strong disequilibrium between the ur locus and the rDNA complex. The KaaX stock had almost no morphological penetrance of uraa, indicating that genetic background is important. KaaX expressed longer female egg-to-adult developmental times, increased early adult female fecundity, and decreased female adult longevity compared with K+X. By bagging natural rots of the cactus Opuntia megacantha near Kamuela, Hawaii, it was shown that egg-to-adult developmental time is slowed down by 0.92 days in females bearing uraa alleles in nature, with no detectable slowdown in uraa males. The bagged rot data also indicate that females bearing uraa alleles have a strong fecundity advantage in nature under some ecological conditions but not others.

The effects of interspecific Y chromosome replacements on hybrid sterility within the Drosophila simulans clade.

We attempted to introgress Y chromosomes between three sibling species of Drosophila: D. simulans, D. sechellia and D. mauritiana. Four D. sechellia Y chromosomes were introgressed into D. simulans without loss of fertility whereas the four reciprocal introgressions (D. simulans Y introgressed into D sechellia) all result in sterility. Both reciprocal Y introgressions of D. simulans and D. mauritiana (four of each) also result in sterility. Compared with D. simulans males, the males with the D. sechellia Y chromosome in D. simulans background had lower productivity but only after multiple matings with virgin females. These males also were inferior compared with pure species males in sperm displacement and/or remating ability. The two different Y genotype males, however, were comparable in viability, longevity and mating success in female choice tests. We also use our results to estimate the effective number of autosomal loci interacting with X-linked genes to produce hybrid male sterility.

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.

Incipient speciation by sexual isolation in Drosophila melanogaster: extensive genetic divergence without reinforcement.

The collection of Drosophila melanogaster from Zimbabwe and nearby regions (the Z-type) yield females who would not mate with the cosmopolitan D. melanogaster males (the M-type). To dissect the genetic basis of this sexual isolation, we constructed 16 whole-chromosome substitution lines between two standard Z- and M-lines. The results were as follows: (1) All substitution lines appear normal in viability and fertility in both sexes, indicating no strong postmating isolation. (2) The genes for the behaviors are mapped to all three major chromosomes with the same ranking and comparable magnitude of effects for both sexes: III > II >> X > or = 0 (III, II and X designate the effects of the three chromosomes). The results suggest less evolution on the X than on autosomes at loci of sexual behavior. (3) The genes for "Z-maleness" are many and somewhat redundant. Whole-chromosome effects for Z-maleness appear nearly additive and show little dominance. (4) In contrast, "Z-femaleness" has less redundancy as partial genotypes never exhibit full phenotypic effects. Epistatic interactions and incomplete dominance can sometimes be detected. (5) The extensive genetic divergence underlying sexual isolation has evolved in the absence of detectable reduction in hybrid fitnesses. Sexual selection has apparently been a driving force of multiple facets of speciation at the nascent stage without reinforcement.

Recent radiation of endemic Caribbean Drosophila of the dunni subgroup inferred from multilocus DNA sequence variation.

Studies of island endemism provide a unique opportunity to elucidate fundamental mechanisms of speciation. Here we examine intra- and interspecific DNA sequence variation at four unlinked genetic loci among populations of the Drosophila dunni subgroup to provide a detailed genealogical portrait of the process of speciation among these island endemic species. Our data indicate two major rounds of diversification that have shaped the D. dunni subgroup. The first occurred 1.6-2.6 million years ago and separated three major lineages, one in Puerto Rico and the Virgin Islands, a second in the northern Lesser Antilles and Barbados, and a third in St. Vincent and Grenada. A second round of diversification occurred in the last 96,000 years in the northern Lesser Antilles and Barbados. The four distinct species that resulted from this recent round of diversification maintain relatively high amounts of genetic variation, similar to that of a closely related mainland species, and share extensive ancestral polymorphism. These data suggest a minimal role for population bottlenecks linked to founder events in the history of the D. dunni subgroup. Further, the recent divergence of these island populations highlights the extremely rapid development of reproductive isolation and distinct patterns of abdominal pigmentation that has occurred in these species.

Evolution of abdominal pigmentation differences across species in the Drosophila dunni subgroup.

The Drosophila dunni subgroup displays a nearly perfect latitudinal cline in abdominal pigmentation that likely resulted from selective forces acting in the habitat of each species during speciation. Here we characterize the nature of this clinal variation by developing a quantitative measure to assess variation in abdominal pigmentation within and between the D. dunni subgroup species. Using discriminant analysis, we confirm the existence of a cline and find that our quantitative measure of pigmentation distinguishes each of the species with singular efficacy. We then combine our quantitative phenotypic analysis of pigmentation with the phylogeny of the D. dunni subgroup species and map the species relationships into the three-dimensional morphological space defined by our pigmentation measures. In this manner, we can visualize how the species have traversed the morphological pigmentation space during the course of speciation. Our analysis reveals that natural selection has caused overall intensity of pigmentation among the northernmost species of the cline to converge. Along with this convergence in phenotype has been a relaxation in expression of sexual dimorphism in these species, indicating a possible shift in the relative intensity of natural and sexual selection. Our analysis indicates an accelerated rate of change in pigmentation for the darkest species in addition to this species evolving a novel abdominal pigmentation trait.

Genetic and developmental analysis of abdominal pigmentation differences across species in the Drosophila dunni subgroup.

Abdominal pigmentation pattern varies dramatically among the species of the Drosophila dunni subgroup across the islands of the Caribbean. Previously, we developed a quantitative measure of abdominal pigmentation to assess phenotypic variation within and between species of this group. In this paper, we use this quantitative measure in an interspecific genetic analysis to decipher the underlying genetic basis of pigmentation differences between one of the lightest and the darkest species in the group. Our analysis shows that pigmentation expression in different areas of the abdomen is under separate genetic control. For these different abdominal regions, we detected a wide range of genetic effects, including X-linked, autosomal additive, near single-gene dominant, and sex-specific effects. Combining these genetic results with our earlier phenotypic and phylogenetic analyses, we present a simple conceptual model to explain how change in the control of expression of pigmentation has evolved throughout the D. dunni subgroup.

Island hopping in Drosophila: patterns and processes.

Radiation of Drosophila along the Hawaiian archipelago has resulted in an astounding array of diversity. The speciation in this group corresponds well to the geological history of the region and colonization events appear to have been a major contributing factor. Although much less impressive in terms of diversity, Drosophila have also radiated throughout the Caribbean islands. In contrast to the pattern exhibited in Hawaii, major changes that distinguish the species in the Caribbean are not always coupled to colonization events. The patterns of speciation for these two island groups are compared and contrasted in light of founder effect speciation models.

Mobile elements and the genesis of microsatellites in dipterans.

Factors that influence the genesis and genomic distribution of microsatellite DNA are poorly understood. We have identified a novel class of Dipteran mobile elements, mini-me elements, which help elucidate both of these issues. These retroposons contain two internal proto-microsatellite regions that commonly expand into lengthy microsatellite repeats. These elements are highly abundant, accounting for approximately 1.2% of the Drosophila melanogaster genome, giving them the potential to be a prolific source of microsatellite DNA variation. They also give us the opportunity to observe the outcomes of multiple microsatellite genesis events (initiating from the same proto-microsatellite) at separate mini-me loci. Based on these observations, we determined that the genesis of microsatellites within mini-me elements occurs through two separate mutational processes: the expansion of preexisting tandem repeats and the conversion of sequence with high cryptic simplicity into tandemly repetitive DNA. These modes of microsatellite genesis can be generalized beyond the case of mini-me elements and help to explain the genesis of microsatellites in any sequence region that is not constrained by selection.

Drosophila melanogaster and D. simulans rescue strains produce fit offspring, despite divergent centromere-specific histone alleles.

The interaction between rapidly evolving centromere sequences and conserved kinetochore machinery appears to be mediated by centromere-binding proteins. A recent theory proposes that the independent evolution of centromere-binding proteins in isolated populations may be a universal cause of speciation among eukaryotes. In Drosophila the centromere-specific histone, Cid (centromere identifier), shows extensive sequence divergence between D. melanogaster and the D. simulans clade, indicating that centromere machinery incompatibilities may indeed be involved in reproductive isolation and speciation. However, it is presently unclear whether the adaptive evolution of Cid was a cause of the divergence between these species, or merely a product of postspeciation adaptation in the separate lineages. Furthermore, the extent to which divergent centromere identifier proteins provide a barrier to reproduction remains unknown. Interestingly, a small number of rescue lines from both D. melanogaster and D. simulans can restore hybrid fitness. Through comparisons of cid sequence between nonrescue and rescue strains, we show that cid is not involved in restoring hybrid viability or female fertility. Further, we demonstrate that divergent cid alleles are not sufficient to cause inviability or female sterility in hybrid crosses. Our data do not dispute the rapid divergence of cid or the coevolution of centromeric components in Drosophila; however, they do suggest that cid underwent adaptive evolution after D. melanogaster and D. simulans diverged and, consequently, is not a speciation gene.

A test of reciprocal X-Y interactions as a cause of hybrid sterility in Drosophila.

Elucidation of the nature of the gene interactions that underly the sterility of interspecific hybrids is important in evolutionary biology. The interactions between the heterospecific X and Y (or Z and W) chromosomes are often used as an explanation for two reasons. First, the fertility of the hybrids of the heterogametic sex is much more often affected than that of the homogametic sex (Haldane's rule) and X-Y interactions are specific to the heterogametic sex. Second, sex chromosomes, especially the X chromosome, are often considered to be of special importance in determining the fertility of hybrids. X-Y interactions have been addressed in studies of males with a heterospecific Y chromosome in a mixed genetic background. A more stringent test of the X-Y interaction model requires each X chromosome sterility factor to be tested separately for its interaction with the Y chromosome in a homogeneous background of the pure species. Here we report such a test of the X-Y interaction model and conclude that X-Y interactions should not be assumed to be the only or even the most common cause of hybrid sterility.

Natural selection and ribosomal DNA in Drosophila.

Natural populations of Drosophila mercatorum are variable for the number of X-linked 28S ribosomal genes bearing a 5-kilobase insert. A separate polymorphic X-linked gene controls whether 28S repeats bearing the insert are preferentially underreplicated during the formation of polytene tissue. Female flies having at least a third of their 28S genes bearing the insert and lacking the ability to preferentially underreplicate inserted repeats display the abnormal abdomen syndrome. The syndrome is characterized by retention of juvenile abdominal cuticle into the adult, a slowdown in larval developmental time, and an increase in early female fecundity. The life history traits are expressed in nature and provide a basis for strong natural selection. The abnormal abdomen syndrome should be favored whenever the adult age structure is skewed towards young individuals, and field studies confirm this prediction. The closely related species, Drosophila hydei, also bears these inserts and appears to be subject to similar selection. However, D. mercatorum responds to this selection primarily through the allelic variation that controls preferential underreplication, whereas D. hydei responds primarily through adjustment of the proportion of inserted 28S genes. This is interpreted to mean that the evolution of a multigene family arises from the interaction of population-level and DNA-level processes.

Characterization of defects in adult germline development and oogenesis of sterile and rescued female hybrids in crosses between Drosophila simulans and Drosophila melanogaster.

Crosses between Drosophila melanogaster and D. simulans normally result in progeny that are either inviable or sterile. Recent discovery of strains that rescue these inviability and sterility phenotypes has made it possible to study the developmental basis of reproductive isolation between these two species in greater detail. By producing both rescued and unrescued hybrids and examining the protein product staining patterns of genes known to be involved in early germline development and gametogenesis, we have found that in crosses between D. simulans and D. melanogaster, hybrid female sterility results from the improper control of primordial germline proliferation, germline stem cell maintenance, and cystoblast formation and differentiation during early oogenesis. Rescued hybrid females are fertile, yet they generally have lower amounts of adult germline from the outset and show a premature degeneration of adult germline cells with age. In addition, older rescued hybrid females also exhibit mutant egg phenotypes associated with defects in dorso-ventral patterning which may result from the improper partitioning of cytoplasmic factors during early oogenesis that could stem from the early defect. Although a variety of germline and oogenic defects are described for the hybrid females, all of them can potentially result from the same underlying primary defect. Hybrid males from these same crosses, on the other hand, have no detectable germline in adult reproductive tissues, even when hybrid sterility rescue strains are used, indicating that male sterility and female sterility stem from distinctly different developmental defects.