Hawaiian Drosophila Research Articles

Phylogenetic analysis of DNA length mutations in a repetitive region of the Hawaiiandrosophila yolk protein geneYp2

Nucleotide sequence analysis has demonstrated that interspecific size variation in the YP2 yolk protein among Hawaiian Drosophila is due to in-frame insertions and deletions in two repetitive segments of the coding region of the Yp2 gene. Sequence comparisons of the complex repetitive region close to the 5' end of this gene across 34 endemic Hawaiian taxa revealed five length morphs, spanning a length difference of 21 nucleotides (nt). A phylogenetic character reconstruction of the length mutations on an independently derived molecular phylogeny showed clade-specific length variants arising from six ancient events: two identical insertions of 6 nt, and four deletions, one of 6 nt, one of 12 nt, and two identical but independent deletions of 15 nt. These mutations can be attributed to replication slippage with nontandem trinucleotide repeats playing a major role in the slipped-strand mispairing. Geographic analysis suggests that the 15 nt deletion which distinguishes the planitibia subgroup from the cyrtoloma subgroup occurred on Oahu about 3 million years ago. The homoplasies observed caution against relying too heavily on nucleotide insertions/deletions for phylogenetic inference. In contrast to the extensive repeat polymorphisms within other Drosophila and the human species, the more complex 5' Yp2 repetitive region analyzed here appears to lack polymorphism among Hawaiian Drosophila, perhaps due to founder effects, low population sizes, and hitchhiking effects of selection on the immediately adjacent 5' region.

Environmental influences on male courtship and implications for female choice in a lekking HawaiianDrosophila

This study examined how variation in a critical environmental variable, protein content in the adult diet, influenced male mating signals, mating success and male ‘quality’ in a lekking Hawaiian Drosophila, D. grimshawi . Specifically, the effects of low- and high-protein-containing diets on male physical condition, courtship behaviour, copulation success and adult offspring production were investigated. Potential differences in attractiveness to females were investigated by recording times to mating. Males fed high-protein diets were in better physical condition, courted more vigorously, and mated more often and sooner than males fed low-protein diets. Matings involving males fed a high-protein diet resulted in more eclosing offspring than matings involving low-protein-fed males. Because females control when mating occurs in this species, these results indicate that females may have preferred males fed high-protein diets. The results are discussed in light of recent questions regarding female choice and the possible benefits that females of lekking species might gain by discriminating between males.

SEQUENTIAL RADIATIONS AND PATTERNS OF SPECIATION IN THE HAWAIIAN CRICKET GENUS LAUPALA INFERRED FROM DNA SEQUENCES

The tremendous diversity of endemic Hawaiian crickets is thought to have originated primarily through intraisland radiations, in contrast to an interisland mode of diversification in the native Hawaiian Drosophila. The Hawaiian cricket genus Laupala (family Gryllidae) is one of several native genera of flightless crickets found in rain-forest habitat across the Hawaiian archipelago. I examined the phylogenetic relationships among mitochondrial DNA (mtDNA) sequences sampled from 17 species of Laupala, including the 12S ribosomal RNA (rRNA), transfer RNA (RNA)val and 16S rRNA regions. The distribution of mtDNA variants suggests that species within Laupala are endemic to single islands. The phylogenetic estimate produced from both maximum likelihood and maximum parsimony supports the hypothesis that speciation in Laupala occurred mainly within islands. The inferred biogeographical history suggests that diversification in Laupala began on Kauai, the oldest rain-forested Hawaiian island. Subsequently, colonization to younger islands in the archipelago resulted in a radiation of considerable phylogenetic diversity. Phylogenetic patterns in mtDNA are not congruent with prior systematic or taxonomic hypotheses. Hypotheses that may explain the conflict between the phylogenetic patterns of mtDNA variation and the species taxonomy are discussed.

809 Electrophysiological analysis of cerebellar Purkinje neuron in mice lacking type I inositol 1,4,5-trisphosphate receptor (II)

There are several mechanisms for the determination of sex. Sexual behaviour is part of the sex-determination cascade, and in Drosophila melanogaster male courtship is controlled in part by the fruitless gene. As part of a study of sexual behaviour in Hawaiian Drosophila, we have cloned the neural sex-determination gene fru from the Hawaiian picture-wing species Drosophila heteroneura. The fru gene has at least seven exons covering a region of 18 kb and encodes three transcripts, fruA, fruB and fruC. Each transcript encodes a single ORF of 841, 678 and 691 aa, respectively. The FRUA and FRUB proteins have a BTB protein–protein-binding domain and two zinc finger-like domains and are well conserved with the D. melanogaster proteins. The FRUC protein has a BTB domain but no zinc finger-like domains. The fru gene is expressed in 1–7 day old adult males as a 5.1 kb transcript. This transcript is not seen in adult females, so the fru gene has a different pattern of sex-differential expression in the Hawaiian Drosophila compared with D. melanogaster.

Pattern of ecological shifts in the diversification of Hawaiian Drosophila inferred from a molecular phylogeny.

The endemic Hawaiian drosophilids, a unique group that are remarkable for their diversity and rapid proliferation, provide a model for analysis of the process of insular speciation. Founder events and accompanying random drift, together with shifts in sexual selection, appear to explain the dramatic divergence in male morphology and mating behaviour among these flies, but these forces do not account for their spectacular ecological diversification into a wide array of breeding niches. Although recognized as contributing to the success of this group, the precise role of adaptive shifts has not been well defined. To delineate the pattern of ecological diversification in the evolution of Hawaiian Drosophila, we generated a molecular phylogeny, using nucleotide sequences from the yolk protein gene Yp1, of 42 endemic Hawaiian and 5 continental species. By mapping ecological characters onto this phylogeny, we demonstrate that monophagy is the primitive condition, and that decaying leaves were the initial substrate for oviposition and larval development. Shifts to decaying stems, bark and tree fluxes followed in more derived species. By plotting female reproductive strategies, as reflected in ovarian developmental type, on the molecular tree, we also demonstrate a phylogenetic trend toward increasing fecundity. We find some statistical support for correlations between ecological shifts and shifts in female reproductive strategies. Because of the short branches at the base of the phylogram, which lead to ecologically diverse lineages, we conclude that much of the adaptive radiation into alternate breeding substrates occurred rapidly, early in the group's evolution in Hawaii. Furthermore, we conclude that this ecological divergence and the correlated changes in ovarian patterns that adapt species to their ecological habitats were contributing factors in the major phyletic branching within the Hawaiian drosophilid fauna.

Molecular phylogeny and divergence times of drosophilid species.

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.

Organ and cell allometry in Hawaiian Drosophila: how to make a big fly.

The importance of body size in predicting many aspects of an animal's biology has become well established in recent years. However, little is known about how body size evolves at the cellular level. Some published data suggest that it is cell number and not cell size that accompanies changes in organ and body size across taxa. We examined organ and cell allometry in the wing, eye and basitarsus of adult Hawaiian Drosophila, ranging in body length from 0.2 mm to 0.8 mm. Linear measurements of all three structures exhibit a positive allometry with body length. Exponents of the allometric equation were 0.96, 0.55 and 1.50 for wing, eye and basitarsus, respectively. Surface markers were used to quantify cell size of each organ. The allometric exponents for cell size as a function of organ size were 0.53, 0.68 and 0.33 for wing, eye and basitarsus, respectively. In contrast to reports in the literature on other systems, our results for Hawaiian Drosophila indicate that cell size may contribute between one third and two thirds to evolutionary changes in organ and body size.

Tests of hypotheses for lek formation in a Hawaiian Drosophila

Abstract. Several hypotheses for the formation of leks were tested in laboratory experiments using a Hawaiian fruit fly, Drosophila grimshawi. Males formed leks spontaneously when placed into large enclosures, making it possible to test hypotheses for lek formation that are difficult to test in the field. The signalling, lek choice, hotspot, and search-cost hypotheses were addressed. Results indicated that several selection pressures may be responsible for the evolution of leks in D. grimshawi. The 'hotspot' hypothesis was strongly supported, but reduced search cost and 'hotshot' mechanisms may also apply. No evidence was found to support the lek-choice or signalling hypotheses. Results from mating experiments indicated that D. grimshawi females probably discriminate among potential mates once at a lek, but female choice was concluded to be too weak to be a significant force for male aggregation.

Evolution of regulatory genes and patterns: relationships to evolutionary rates and to metabolic functions.

In an effort to understand the forces shaping evolution of regulatory genes and patterns, we have compared data on interspecific differences in enzyme expression patterns among the rapidly evolving Hawaiian picture-winged Drosophila to similar data on the more conservative virilis species group. Divergence of regulatory patterns is significantly more common in the former group, but cause and effect are difficult to discern. Random fixation of regulatory variants in small populations and/or during speciation may be somewhat more likely than divergence driven by selection. Within the picture-winged group, we also have compared enzymes that fulfill different metabolic roles. There are highly significant differences between individual enzymes, but no obvious correlations to functional categories.

Phylogenetic Pattern and Developmental Process in Drosophila

Recent molecular and morphological results on the phylogenetics of Drosophila and other Drosophilidae are reviewed. There is some consensus on the redefinition of Drosophila to make it a monophyletic group, except for the controversial placement of the Hawaiian Drosophila and the subgenus Sophophora. The possibility of Hawaiian Drosophila as a group that is morpho? logically atavistic (e.g., with many character reversals) is discussed. Drosophila development is reviewed, with focus on understanding the developmental genetic and molecular bases of char? acter convergence and reversal in drosophilid phylogenetics and its applications to homology decisions. The clustering of convergent characters on cladograms is discussed as a consequence of developmental constraints. The convergent evolution of broadened head shape in male dro- sophilids is presented as an example. Molecular and developmental analysis of homoplastic characters pivotal to phylogenetic schemes, such as eye setae in Drosophila sensu stricto and the Hawaiian species, can be of fundamental importance for homology assessment. (Drosophila; homeotic genes; homology assessment; systematics; molecular evolution.) The fruit fly Drosophila is well known for its importance in experimental and mech? anistic biology. In this article, we stress the impact that the study of Drosophila has had in revolutionizing many aspects of modern biology and how this bears on phyloge? netic reconstruction. Our central theme is

CHEMICAL COMMUNICATION IN HAWAIIAN DROSOPHILA.

We are interested in elucidating the extent to which lekking Hawaiian Drosophila species have diverged from their continental counterparts, which engage in sexual behavior at communal food sources, with regard to the chemical communication systems that the flies employ. Accordingly, we have analyzed flies from three closely related Hawaiian Drosophila species in the adiastola subgroup. These species are of interest because the males engage in a unique behavior: while courting, they raise their abdomens over their heads and emit anal droplets. Analysis of the flies' behavior, the hydrocarbons in males' anal droplets, and males' cuticular hydrocarbons suggest that females' responses to males may be mediated by cuticular pheromones and/or pheromones in males' extruded droplets that enable the females to distinguish conspecific from heterospeciflc males. Conversely, perception of cuticular hydrocarbons from conspecific females enables D. adiastola males to distinguish females from a closely related species from conspecific females. On the basis of these observations, we suggest that the adiastola subgroup species are unique among drosophilids in that they utilize an anal droplet-mediated pheromone communication system, some or all components of which are species specific. However, the lekking Hawaiian Drosophila species are similar to D. melanogaster and related continental species in that the Hawaiian flies employ a cuticular pheromone communication system, some components of which are sex and species-specific.

Ultrastructural diversity in the egg chorion of Hawaiian Drosophila and Scaptomyza: ecological and phylogenetic considerations

Formation of the egg shell (chorion) in Drosophila and Scaptomyza (Diptera : Drosophilidae) is a complex developmental process involving coordinated synthesis and secretion of multiple proteins by the monolayer of follicle cells surrounding the egg. Using scanning electron microscopy, the ultrastructure of the chorion in 37 endemic Hawaiian drosophilids, representing the genera Drosophila and Scaptomyza, were analyzed and compared with 7 representative species of continental Drosophila. The detailed structure of the chorion was described for 8 chorionic regions: the respiratory filaments, follicle imprints, operculum, micropyle, dorsal ridge, ventral rim, posterior pole, and the chorion cross-section. The morphology of each region is similar among related species, but strikingly different among groups. The main functions of the chorion are to protect the developing embryo from the vicissitudes of the environment and to provide channels for gas exchange during embryogenesis. Adaptation to the diverse ovipositional substrates used by Drosophila in general, and the Hawaiian species in particular, has resulted in extraordinary diversity in the various chorionic structures. The respiratory filaments differ in number and have evolved to different lengths and degrees of porosity. Furthermore, other regions also involved in respiratory exchange (the operculum, follicle imprints, the pole region, and the dorsal ridge) have diverged in parallel to the ecological divergence. The thickness and complexity of the outer endochorion are dramatically different in various groups, providing varying degrees of mechanical strength to the eggshell, which promotes embryonic survival in the diverse microenvironments. These varied chorionic structures have been found to provide useful morphological characters for phylogenetic analyses of the drosophilids.

Sexual selection in a lekking Hawaiian Drosophila: the roles of male competition and female choice in male mating success

The importance of sexual selection in Drosophila grimshawi, a lek-forming Hawaiian Drosophila, was analysed in the laboratory by studying details of male and female mating behaviour at spontaneouslyforming leks in large and in small observation chambers. Sexual selection was found to be intense, as is often hypothesized for Hawaiian Drosophila. However, little evidence was found for a significant role of active female choice in male mating success although females can reject attempted copulations. Male body size was significantly related to mating success, but mostly because large males spent more time sexually active at lek sites, which resulted in increased mating success, and not because large males physically dominated smaller ones. Variation in larval environmental quality significantly affected male size, and thus, environmental factors may have a large, but indirect, influence on male mating sucess.

Sexual dimorphism in remating in Hawaiian Drosophila species

Males and females have been predicted to show similar patterns of return to receptivity after mating, as a consequence of the genetic correlation between these traits in the two sexes. This hypothesis was tested by comparing mating and remating behaviour among several species of Hawaiian Drosophila. The species have diverged in their probability of first-time matings as well as in other aspects of their reproductive behaviour. Drosophila differens was monomorphic in remating behaviour. The two phylogenetically younger species, D. silvestris and D. heteroneura, both showed sexual dimorphism in remating behaviour, with males being equally likely to mate on 2 successive days and females being much less likely to mate on the first day after a copulation. Each species is sexually dimorphic in secondary sexual morphology, but the species differ in secondary sexual characters. With so few taxa, it is difficult to infer whether monomorphism or dimorphism is the ancestral condition. However, the existence of other dimorphisms that differ between species demonstrates that sufficient time has passed since population divergence to allow dimorphic behaviour to evolve independently in each species. Consequently, although we cannot rule out the hypothesis that males and females showed similar remating behaviour when each species was first formed, even in these very young species, sufficient time has passed for dimorphisms to evolve and for the effects of genetic correlations between the sexes to be rendered insignificant in maintaining monomorphisms.

The mtDNA genealogy of closely related Drosophila silvestris.

Genetic, morphological, and behavioral analyses have been used to examine the evolutionary dynamics and phylogeny of the rare Hawaiian Drosophila species, D. silvestris. Critical to understanding the evolution of this species is the examination of the distribution of populations of D. silvestris on the Big Island of Hawaii. Behavioral analysis using mating asymmetries and the Kaneshiro hypothesis as an indicator of ancestral behavioral state has suggested that flies from the northern part of the island are ancestral to those on the southern part of the island. Consequently, a sequential pattern of colonization going from north to south is predicted for these flies on the east side of the Island of Hawaii. We have examined this prediction using mitochondrial DNA (mtDNA) restriction site analysis with four-base cutters and DNA sequencing. The resulting mtDNA phylogeny based on 23 phylogenetically informative restriction sites and two phylogenetically informative DNA sequence characters agrees in part with the phylogeny predicted from the behavioral data.

Complementary DNA-DNA hybridization in Drosophila.

We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.

The molecular evolution of the alcohol dehydrogenase locus and the phylogeny of Hawaiian Drosophila.

DNA sequences in the alcohol dehydrogenase genes of flies representing the major groups of Hawaiian Drosophila are used to clarify the relationships of these groups, among themselves and with mainland Drosophila. The topology of the tree derived from these sequences agrees with karyotypic and morphological data but disagrees, in part, with the results of an earlier study that used immunological comparisons between variants of a larval hemolymph protein. A time scale, derived from a comparison of closely related Hawaiian Drosophila species, provides divergence-time estimates that are substantially more recent than those obtained from the immunological studies, although they are still within the bounds set by fossil and biogeographical evidence. The two major lineages of Hawaiian Drosophila, the scaptomyzoids and the drosophiloids, are shown to be widely separated from one another. The scaptomyzoids appear to have diverged early in the history of the subgenus Drosophila, greater than 25 Mya. While hundreds of scaptomyzoid species are found in the Hawaiian archipelago, many fewer are found elsewhere around the world, suggesting that they could have originated outside Hawaii. The drosophiloid lineage is strictly endemic to Hawaii and originated little more than 10 Mya, according to the alcohol dehydrogenase molecular clock. Thus, Drosophila apparently inhabited the Hawaiian archipelago (greater than or equal to 5 Myr before the emergence of the oldest existing high island, Kauai, 5 Mya.

ASYMMETRICAL MATING PATTERNS BETWEEN GEOGRAPHIC STRAINS OF DROSOPHILA MERCATORUM: A TEST OF THE KANESHIRO HYPOTHESIS.

Kaneshiro (1976) and Ohta (1978) observed asymmetrical behavioral isolation between species pairs of Hawaiian Drosophila whose phylogenetic relationships had previously been inferred from cytology, morphology, and biogeography. The mating patterns were onesided in that males from putative ancestral populations confined with ancestral and derived females exhibited either random mating or heterogamic mating, while derived males in the same situation mated homogamically. These observations were seminal to the development of Kaneshiro's hypothesis that such ancestral/ descendent asymmetries are the norm and thus may provide a basis for determining the direction of evolution among a group of closely related species or populations of the same species. The original hypothesis suggested that some elements of male courtship behavior are lost during founder events leading to the establishment of a new population. Derived females recognize and accept the courtship repertoire of ancestral males, which is complete, but ancestral females discriminate against derived males due to their deficient courtship sequence. Ohta (1978) proposed that founder events might also involve selection for decreased female discrimination, and this idea was subsequently incorporated into the hypothesis (Kaneshiro 1980, 1983; Kaneshiro and Giddings, 1987). A basic assumption was that mating propensities were equal in ancestral and derived populations. Thus, the hypothesis explained the results of male choice experiments in terms of female mate choice decisions, and relied heavily on the role of female discrimination. If this model describes a typical sequence of events, then asymmetrical mating patterns should demonstrate unambiguous directions of evolution, and would be of significant value in phylogenetic analysis. Summary discussions concerning this possibility appeared recently (Kaneshiro and Giddings, 1987; Ehrman and Wasserman, 1987; DeSalle and Templeton, 1987). Giddings and Templeton (1983) presented evidence supporting the model, but included two new constraints which they deemed essential for its applica-

Rates of DNA change and phylogeny from the DNA sequences of the alcohol dehydrogenase gene for five closely related species of Hawaiian Drosophila.

The sequence of 1.6 kb of DNA surrounding the alcohol dehydrogenase (Adh) gene from five species of the Planitibia subgroup of the Hawaiian picture-winged Drosophila, with estimated divergence times of 0.4-5.1 Myr, has been determined. The gene trees which were found by using the sequence divergence from different regions of the sequences are generally in accord with the phylogeny proposed for these species when chromosomal inversions and island of origin are used. One of the species (D. picticornis) appears to be more distant from the other species in this group than they are from a member of the Grimshawi group (D. affinidisjuncta) which is chromosomally more distant. Two of the species (D. differens and D. plantibia) show heterogeneity in the nucleotide changes in the Adh coding region, heterogeneity which is interpreted to be due to a gene conversion or recombination after hybridization between the two species. The minimal rate of nucleotide substitution of synonymous nucleotides and of nontranscribed nucleotides downstream from the coding region is estimated as 1.5 x 10(-8) and 1.1 x 10(-8) substitutions/nucleotide/year, respectively. This rate is two to three times the maximal rate estimated for mammalian synonymous substitutions.

Evolution of the autosomal chorion cluster inDrosophila. IV. The HawaiianDrosophila: Rapid protein evolution and constancy in the rate of DNA divergence

Autosomal chorion genes s18, s15, and s19 are shown to diverge at extremely rapid rates in closely related taxa of Hawaiian Drosophila. Their nucleotide divergence rates are at least as fast as those of intergenic regions that are known to evolve more extensively between distantly related species. Their amino acid divergence rates are the fastest known to date. There are two nucleotide replacement substitutions for every synonymous one. The molecular basis for observed length and substitution mutations is analyzed. Length mutations are strongly associated with direct repeats in general, and with tandem repeats in particular, whereas the rate for an average transition is twice that for an average transversion. The DNA sequence of the cluster was used to construct a phylogenetic tree for five taxa of the Hawaiian picture-winged species group of Drosophila. Assignment of observed base substitutions occurring in various branches of the tree reveals an excess of would-be homoplasies in a centrally localized 1.8-kb segment containing the s15 gene. This observation may be a reflection of ancestral excess polymorphisms in the segment. The chorion cluster appears to evolve at a constant rate regardless of whether the central 1.8-kb segment is included or not in the analysis. Assuming that the time of divergence of Drosophila grimshawi and the planitibia subgroup coincides with the emergence of the island of Kauai, the overall rate of base substitution in the cluster is estimated to be 0.8% million years, whereas synonymous sites are substituted at a rate of 1.2% million years.