Species Of Hawaiian Drosophila Research Articles

The Transmission Patterns of the Endosymbiont Wolbachia within the Hawaiian Drosophilidae Adaptive Radiation.

The evolution of endosymbionts and their hosts can lead to highly dynamic interactions with varying fitness effects for both the endosymbiont and host species. Wolbachia, a ubiquitous endosymbiont of arthropods and nematodes, can have both beneficial and detrimental effects on host fitness. We documented the occurrence and patterns of transmission of Wolbachia within the Hawaiian Drosophilidae and examined the potential contributions of Wolbachia to the rapid diversification of their hosts. Screens for Wolbachia infections across a minimum of 140 species of Hawaiian Drosophila and Scaptomyza revealed species-level infections of 20.0%, and across all 399 samples, a general infection rate of 10.3%. Among the 44 Wolbachia strains we identified using a modified Wolbachia multi-locus strain typing scheme, 30 (68.18%) belonged to supergroup B, five (11.36%) belonged to supergroup A, and nine (20.45%) had alleles with conflicting supergroup assignments. Co-phylogenetic reconciliation analysis indicated that Wolbachia strain diversity within their endemic Hawaiian Drosophilidae hosts can be explained by vertical (e.g., co-speciation) and horizontal (e.g., host switch) modes of transmission. Results from stochastic character trait mapping suggest that horizontal transmission is associated with the preferred oviposition substrate of the host, but not the host's plant family or island of occurrence. For Hawaiian Drosophilid species of conservation concern, with 13 species listed as endangered and 1 listed as threatened, knowledge of Wolbachia strain types, infection status, and potential for superinfection could assist with conservation breeding programs designed to bolster population sizes, especially when wild populations are supplemented with laboratory-reared, translocated individuals. Future research aimed at improving the understanding of the mechanisms of Wolbachia transmission in nature, their impact on the host, and their role in host species formation may shed light on the influence of Wolbachia as an evolutionary driver, especially in Hawaiian ecosystems.

Ultrastructural variation and adaptive evolution of the ovipositor in the endemic Hawaiian Drosophilidae.

Ecological diversification of the endemic Hawaiian Drosophilidae has been accompanied by striking divergence in egg morphology, and ovarian structure and function. To determine how these flies successfully oviposit in a variety of breeding substrates, we used Scanning Electron Microscopy to examine the ultrastructure of the ovipositor of a sample of 65 Drosophila species and five Scaptomyza species of this hyperdiverse monophyletic group. The Drosophila species analyzed included representatives of the fungus-breeding haleakalae group, the leaf-breeding antopocerus and modified tarsus groups, the modified mouthparts species group, the nudidrosophila, and the picture wing clade; the latter sample of 41 species from four species groups included stem- and bark-breeders, as well as tree sap flux-breeders. Ovipositor length was found to vary more than 12-fold among Hawaiian drosophilids, with the longest ovipositors observed in the bark-breeding species and the shortest among the Scaptomyza and fungus-breeders. More noteworthy is the striking variation in overall shape and proportions of the ovipositor, in the shape of the apical region, and in the pattern of sensory structures or ovisensilla. Ultrastructural observations of the pair of long subapical sensilla on the ventral side identify these, for the first time, as taste bristles. Ovipositor form correlates strongly with the oviposition substrate used by the species, being of a distinctive shape and size in each case. We infer that the observed morphological divergence in the ovipositor is adaptive and the product of natural selection for successful reproduction in alternate microhabitats. The array of ovipositor forms that have evolved among the Hawaiian Drosophila species represent a series of ecomorphs that along with other divergent traits of the female reproductive system, have contributed to the successful adaptive radiation of this remarkable fauna.

A Test for Gene Flow among Sympatric and Allopatric Hawaiian Picture-Winged Drosophila

The Hawaiian Drosophila are one of the most species-rich endemic groups in Hawaii and a spectacular example of adaptive radiation. Drosophila silvestris and D. heteroneura are two closely related picture-winged Drosophila species that occur sympatrically on Hawaii Island and are known to hybridize in nature, yet exhibit highly divergent behavioral and morphological traits driven largely through sexual selection. Their closest-related allopatric species, D. planitibia from Maui, exhibits hybrid male sterility and reduced behavioral reproductive isolation when crossed experimentally with D. silvestris or D. heteroneura. A modified four-taxon test for gene flow was applied to recently obtained genomes of the three Hawaiian Drosophila species. The analysis indicates recent gene flow in sympatry, but also, although less extensive, between allopatric species. This study underscores the prevalence of gene flow, even in taxonomic groups considered classic examples of allopatric speciation on islands. The potential confounding effects of gene flow in phylogenetic and population genetics inference are discussed, as well as the implications for conservation.

Hawaiian Drosophila genomes: size variation and evolutionary expansions.

This paper reports genome sizes of one Hawaiian Scaptomyza and 16 endemic Hawaiian Drosophila species that include five members of the antopocerus species group, one member of the modified mouthpart group, and ten members of the picture wing clade. Genome size expansions have occurred independently multiple times among Hawaiian Drosophila lineages, and have resulted in an over 2.3-fold range of genome sizes among species, with the largest observed in Drosophila cyrtoloma (1C = 0.41 pg). We find evidence that these repeated genome size expansions were likely driven by the addition of significant amounts of heterochromatin and satellite DNA. For example, our data reveal that the addition of seven heterochromatic chromosome arms to the ancestral haploid karyotype, and a remarkable proportion of ~70 % satellite DNA, account for the greatly expanded size of the D. cyrtoloma genome. Moreover, the genomes of 13/17 Hawaiian picture wing species are composed of substantial proportions (22-70 %) of detectable satellites (all but one of which are AT-rich). Our results suggest that in this tightly knit group of recently evolved species, genomes have expanded, in large part, via evolutionary amplifications of satellite DNA sequences in centric and pericentric domains (especially of the X and dot chromosomes), which have resulted in longer acrocentric chromosomes or metacentrics with an added heterochromatic chromosome arm. We discuss possible evolutionary mechanisms that may have shaped these patterns, including rapid fixation of novel expanded genomes during founder-effect speciation.

Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics.

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Estimating Divergence Dates and Substitution Rates in the Drosophila Phylogeny

An absolute timescale for evolution is essential if we are to associate evolutionary phenomena, such as adaptation or speciation, with potential causes, such as geological activity or climatic change. Timescales in most phylogenetic studies use geologically dated fossils or phylogeographic events as calibration points, but more recently, it has also become possible to use experimentally derived estimates of the mutation rate as a proxy for substitution rates. The large radiation of drosophilid taxa endemic to the Hawaiian islands has provided multiple calibration points for the Drosophila phylogeny, thanks to the "conveyor belt" process by which this archipelago forms and is colonized by species. However, published date estimates for key nodes in the Drosophila phylogeny vary widely, and many are based on simplistic models of colonization and coalescence or on estimates of island age that are not current. In this study, we use new sequence data from seven species of Hawaiian Drosophila to examine a range of explicit coalescent models and estimate substitution rates. We use these rates, along with a published experimentally determined mutation rate, to date key events in drosophilid evolution. Surprisingly, our estimate for the date for the most recent common ancestor of the genus Drosophila based on mutation rate (25–40 Ma) is closer to being compatible with independent fossil-derived dates (20–50 Ma) than are most of the Hawaiian-calibration models and also has smaller uncertainty. We find that Hawaiian-calibrated dates are extremely sensitive to model choice and give rise to point estimates that range between 26 and 192 Ma, depending on the details of the model. Potential problems with the Hawaiian calibration may arise from systematic variation in the molecular clock due to the long generation time of Hawaiian Drosophila compared with other Drosophila and/or uncertainty in linking island formation dates with colonization dates. As either source of error will bias estimates of divergence time, we suggest mutation rate estimates be used until better models are available.

Evolution of Cuticular Hydrocarbons of Hawaiian Drosophilidae

Hawaiian Drosophila offer an excellent model for adaptive evolution. More than 500 species are reported in Hawaiian islands, and there is considerable diversity in behavior and morphology. Such diversity is mainly driven by sexual selection. In this study qualitative and quantitative chemical compositions of cuticular hydrocarbons (CHCs) in 138 flies belonging to 27 Hawaiian Drosophila species, picture-winged and non picture-winged, were analyzed regarding sexual dimorphism, differences in saturation, branching position, and lengths of CHCs. We found significant variation in the CHC patterns. In several subgroups, new species show decreases in unsaturated hydrocarbons, and gradual increases in branched compounds, monomethylalkanes and dimethylalkanes, not commonly found in Drosophila. Moreover, branching positions gradually shifted towards internal carbons, and chain lengths increased in the new species. The long-term evolution of CHCs in the light of the recent evolutionary migration and adaptation history of Hawaiian Drosophila species along the developing archipelago was discussed.

Population structure and genetic diversity in two species of Hawaiian picture-winged Drosophila

Over the last several decades many picture-winged Drosophila have become less common in both geographical distribution and local population size (pers. obs., Foote pers. comm., Montgomerey pers. comm.). Here we report on a study of two Hawaiian Drosophila species, D. engyochracea, and D. hawaiiensis, to determine the impact that changes in population sizes over the past thirty years have had on the genetic diversity of these species. D. engyochracea is known from only two locations on the Island of Hawai‘i (Kipuka Ki and Kipuka Pua‘ulu), while D. hawaiiensis is currently more wide spread across Hawai‘i Island. We collected 65 D. hawaiiensis and 66 D. engyochracea from two forest patches (kipuka) isolated by a 400 year old volcanic ash deposit. DNA sequence data for 515 bases of the mitochondrial gene COII was analyzed for both species to estimate relative total genetic diversity as well as inter-kipuka gene flow. The more wide spread species, D. hawaiiensis, has more genetic diversity (23 vs. 11 unique haplotypes) than the rarer species, D. engyochracea. The distribution of haplotypes in the kipuka is consistent with more gene flow in D. engyochracea than in D. hawaiiensis. Phylogenetic analysis indicates a small number of individuals morphologically identified as one species but have DNA sequence diagnostic for the other species. These results are consistent with these individuals being descendant from hybrids between species.

A Database of Wing Diversity in the Hawaiian Drosophila

BackgroundWithin genus Drosophila, the endemic Hawaiian species offer some of the most dramatic examples of morphological and behavioral evolution. The advent of the Drosophila grimshawi genome sequence permits genes of interest to be readily cloned from any of the hundreds of species of Hawaiian Drosophila, offering a powerful comparative approach to defining molecular mechanisms of species evolution. A key step in this process is to survey the Hawaiian flies for characters whose variation can be associated with specific candidate genes. The wings provide an attractive target for such studies: Wings are essentially two dimensional, and genes controlling wing shape, vein specification, pigment production, and pigment pattern evolution have all been identified in Drosophila.Methodology/Principal FindingsWe present a photographic database of over 180 mounted, adult wings from 73 species of Hawaiian Drosophila. The image collection, available at FlyBase.org, includes 53 of the 112 known species of “picture wing” Drosophila, and several species from each of the other major Hawaiian groups, including the modified mouthparts, modified tarsus, antopocerus, and haleakalae (fungus feeder) groups. Direct image comparisons show that major wing shape changes can occur even between closely related species, and that pigment pattern elements can vary independently of each other. Among the 30 species closest to grimshawi, diverse visual effects are achieved by altering a basic pattern of seven wing spots. Finally, we document major pattern variations within species, which appear to result from reduced diffusion of pigment precursors through the wing blade.Conclusions/SignificanceThe database highlights the striking variation in size, shape, venation, and pigmentation in Hawaiian Drosophila, despite their generally low levels of DNA sequence divergence. In several independent lineages, highly complex patterns are derived from simple ones. These lineages offer a promising model system to study the evolution of complexity.

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

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

SEM search for sound production and sound perception organs in a variety of Drosophila species.

The discovery of new song types in Hawaiian Drosophila species has raised a question of alternative sound production and sound perception mechanisms in Drosophila. For example D. disjuncta males, which produce song with a carrier frequency of 5,000-6,000 Hz, do not vibrate their wings but only tremble them in a very small amplitude while singing. In our SEM search we found at the wing base of the flies of this species a special structure which could play a part in song production. Our search for auditory sense organs in Drosophila species revealed that the aristae on fly antennae are structurally not as simple as has been assumed. In Hawaiian D. planitibia subgroup species, the fly aristae are more regular than in other Drosophila species and they are also covered with small hairs. We did not find any sign of tympanic hearing organs in any of the studied species, but we discovered on fly legs and wings sensilla which could play a part in sound/vibration perception. Our SEM survey provides a good starting point in the search for alternative pathways for sound production and perception via transmission electron microscopy combined with behavioral studies and electrophysiological recordings in Hawaiian Drosophila species.

SEM search for sound production and sound perception organs in a variety ofDrosophila species

The discovery of new song types in Hawaiian Drosophila species has raised a question of alternative sound production and sound perception mechanisms in Drosophila. For example D. disjuncta males, which produce song with a carrier frequency of 5,000-6,000 Hz, do not vibrate their wings but only tremble them in a very small amplitude while singing. In our SEM search we found at the wing base of the flies of this species a special structure which could play a part in song production. Our search for auditory sense organs in Drosophila species revealed that the aristae on fly antennae are structurally not as simple as has been assumed. In Hawaiian D. planitibia subgroup species, the fly aristae are more regular than in other Drosophila species and they are also covered with small hairs. We did not find any sign of tympanic hearing organs in any of the studied species, but we discovered on fly legs and wings sensilla which could play a part in sound/vibration perception. Our SEM survey provides a good starting point in the search for alternative pathways for sound production and perception via transmission electron microscopy combined with behavioral studies and electrophysiological recordings in Hawaiian Drosophila species.

Multiple sources of character information and the phylogeny of Hawaiian drosophilids.

Relationships among representatives of the five major Hawaiian Drosophila species groups were examined using data from eight different gene regions. A simultaneous analysis of these data resulted in a single most-parsimonious tree that (1) places the adiastola picture-winged subgroup as sister taxon to the other picture-winged subgroups, (2) unites the modified-tarsus species group with flies from the Antopocerus species group, and (3) places the white-tip scutellum species group as the most basal taxon. Because of the different gene sources used in this study, numerous process partitions can be erected within this data set. We examined the incongruence among these various partitions and the ramifications of these data for the taxonomic consensus, prior agreement, and simultaneous analysis approaches to phylogenetic reconstruction. Separate analyses and taxonomic consensus appear to be inadequate methods for dealing with the partitions in this study. Although detection of incongruence is possible and helps elucidate particular areas of disagreement among data sets, separation of partitions on the basis of incongruence is problematic for many reasons. First, analyzing all genes separately and then either presenting them all as possible hypotheses or taking their consensus provides virtually no information concerning the relationships among these flies. Second, despite some evidence of incongruence, there are no clear delineations among the various gene partitions that separate only heterogeneous data. Third, to the extent that problematic genes can be identified, these genes have nearly the same information content, within a combined analysis framework, as the remaining nonproblematic genes. Our data suggest that significant incongruence among data partitions may be isolated to specific relationships and the "false" signal creating this incongruence is most likely to be overcome by a simultaneous analysis. We present a new method, partitioned Bremer support, for examining the contribution of a particular data partition to the topological support of the simultaneous analysis tree.

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.

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.

Insemination patterns in HawaiianDrosophila species (Diptera: Drosophilidae) correlate with ovarian development

In 35 Hawaiian Drosophilaspecies collected from natural populations, all reproductively mature females were inseminated. Onset of receptivity to insemination broadly correlated with the vitellogenic stage of ovarian development, but species and species groups varied in the exact stage of vitellogenesis at which earliest insemination took place. A striking exception was observed in two picture-winged species of the adiastolasubgroup, which were precociously inseminated in previtellogenesis, close to the time of adult eclosion. For a given species, the range of ovarian stages from earliest receptivity to insemination of all females, termed the “insemination period,” also varied among species. This variability may be due to physiological variation between females in hormonal levels, low population densities, and lek behavior of males, or it may reflect variation in degree of female discrimination toward male courtship attempts. Consideration of the quantities of sperm observed stored in the female sperm storage organs, relative to the numbers of eggs produced, indicates obligatory multiple insemination for some species, notably the fungus breeders. In other species groups, there is no apparent necessity for remating, although it may occur.

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.

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.

STATISTICAL TESTS OF PHENOGRAMS BASED ON GENETIC DISTANCES.

Methods used for estimating the confidence levels of UPGMA-type phenograms based on molecular-genetic data are examined. The methods based on internodal variances or on bootstrapping over characters are compared by simulating a three-species phylogeny with a trifurcation. The bootstrap method seems slightly better in this comparison. Weighting of OTU's when constructing the phenogram is also examined. A method that weights each OTU according to the estimated independent evolutionary information, a modified WPGMA, appears slightly better than UPGMA in estimating the branching points and branch lengths. The methods are applied to the data on restriction sites in eight Hawaiian Drosophila species. The significance of the clusters among them differ from those reported earlier.