Hawaiian Drosophila as an Evolutionary Model Clade: Days of Future Past.

The Hawaiian Drosophila have been a model system for evolutionary, ecological, and ethological studies since the inception of the Hawaiian Drosophila Project in the 1960s. Here we review the past and present research on this incredible lineage and provide a prospectus for future directions on genomics and microbial interactions. While the number of publications on this group has waxed and waned over the years, we assert that recent systematic, biogeographic, and ecological studies have reinvigorated Hawaiian Drosophila as an evolutionary model system. The characteristics that distinguish good model clades from good model organisms (e.g., Drosophila melanogaster) are somewhat different so we first define what constitutes a good evolutionary model. We argue that the Hawaiian Drosophila possess many desired aspects of a good evolutionary model, describe how this group of geographically isolated flies have been used in the past, and propose some exciting avenues for future evolutionary research on this diverse, dynamic clade of Drosophila.

Genetic variability among native and introduced strains of the parasitic nematode Deladenus siricidicola.

Sirex noctilio is an invasive Eurasian woodwasp that can kill pine (Pinus spp.) trees and has been introduced to areas of the Southern Hemisphere where plantations of introduced pines are grown. The main method of control of this invasive pest has been introduction and augmentation of a parasitic nematode, Deladenus siricidicola. The strain of D. siricidicola used for biological control of S. noctilio in the Southern Hemisphere originated in Sopron, Hungary. The genotype of D. siricidicola used for biological control sterilizes females of the strain of S. noctilio present in Australia. However, different strains of S. noctilio have been introduced to different geographic areas that have been invaded and different combinations of D. siricidicola and S. noctilio genotypes vary in whether these nematodes sterilize female S. noctilio. Moreover, even in the event of sterilization, partial sterilization can occur, where not all woodwasp eggs are compromised. Sirex noctilio has now invaded North America accidentally, putatively accompanying D. siricidicola, but these host/parasite pairings do not result in female sterilization. More information is needed about the genetic diversity of D. siricidicola both where it is native and introduced. In addition, the host range of these nematodes is necessary to understand to evaluate their potential use in areas where pine communities are native. We collected and evaluated Deladenus parasitizing S. noctilio, S. juvencus, and associated insects in Hungary, Denmark, Spain, and Italy, as well as in the United States. Phylogenetic analyses were unable to fully provide fine resolution, although some community structure was evident. Many D. siricidicola samples from Hungary had identical COI and ITS sequences to the strain of D. siricidicola accidentally introduced to North America putatively when S. noctilio invaded. The same or similar strains of D. siricidicola parasitize two different Sirex species that utilize pines as well as a Sirex parasitoid and a pine-boring beetle, demonstrating some limited variability in host specificity of this species. These results highlight the genetic diversity of Deladenus siricidicola in its native range in Europe.

Diversification in Hawaiian long-legged flies (Diptera: Dolichopodidae: Campsicnemus): biogeographic isolation and ecological adaptation.

Flies in the genus Campsicnemus have diversified into the second-largest adaptive radiation of Diptera in the Hawaiian Islands, with 179 Hawaiian endemic species currently described. Here we present the first phylogenetic analysis of Campsicnemus, with a focus on the Hawaiian fauna. We analyzed a combination of two nuclear (CAD, EF1α) and five mitochondrial (COI, COII, 12S, 16S, ND2) loci using Bayesian and maximum likelihood approaches to generate a phylogenetic hypothesis for the genus Campsicnemus. Our sampling included a total of 84 species (6 species from Europe, 1 from North America, 7 species from French Polynesia and 70 species from the Hawaiian Islands). The phylogenies were used to estimate divergence times, reconstruct biogeographic history, and infer ancestral ecological associations within this large genus. We found strong support for a South Pacific+Hawaiian clade, as well as for a monophyletic Hawaiian lineage. Divergence time estimates suggest that Hawaiian Islands were colonized approximately 4.6 million years ago, suggesting that most of the diversity within Campsicnemus evolved since the current high islands began forming ∼5 million years ago. We also observe a novel ecotype within the Pacific Campsicnemus; a widespread obligate water-skating form that has arisen multiple times across the Pacific Islands. Together, these analyses suggest that a combination of ecological, biogeographic and temporal factors have led to the impressive diversity of long-legged flies in Hawaii and elsewhere in the Pacific.

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.

Diversification and dispersal of the Hawaiian Drosophilidae: the evolution of Scaptomyza.

The genus Scaptomyza is emerging as a model lineage in which to study biogeography and ecological adaptation. To place future research on these species into an evolutionary framework we present the most comprehensive phylogeny of Scaptomyza to date, based on 5042 bp of DNA sequence data and representatives from 13 of 21 subgenera. We infer strong support for the monophyly of almost all subgenera with exceptions corroborating hypotheses of conflict inferred from previous taxonomic studies. We find evidence that the lineage originated in the Hawaiian Islands and subsequently dispersed to the mainland and other remote oceanic islands. We also identify that many of the unique ecological niches exploited by this lineage (e.g., herbivory, spider predation) arose singly and independently.

A New Species of Anticheta (Diptera: Sciomyzidae) from Mexico.

Anticheta patzcuaroensis Pote, new species (Diptera: Sciomyzidae), from Lake Pátzcuaro, Michoacán, Mexico, is described and illustrated. The most recent key to the genus Anticheta Haliday in the Nearctic region is edited to include the new species. Information is given about the Sciomyzidae holdings in the Cornell University Insect Collection.

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Host-plants shape insect diversity: phylogeny, origin, and species diversity of native Hawaiian leafhoppers (Cicadellidae: Nesophrosyne).

Herbivorous insects and the plants on which they specialize, represent the most abundant terrestrial life on earth, yet their inter-specific interactions in promoting species diversification remains unclear. This study utilizes the discreet geologic attributes of Hawai'i and one of the most diverse endemic herbivore radiations, the leafhoppers (Hemiptera: Cicadellidae: Nesophrosyne), as a model system to understand the role of host-plant use in insect diversification. A comprehensive phylogeny is reconstructed to examine the origins, species diversification, and host-plant use of the native Hawaiian leafhoppers. Results support a monophyletic Nesophrosyne, originating from the Western Pacific basin, with a sister-group relationship to the genus Orosius. Nesophrosyne is characterized by high levels of endemicity according to individual islands, volcanoes, and geologic features. Clades demonstrate extensive morphologically cryptic diversity among allopatric species, utilizing widespread host-plant lineages. Nesophrosyne species are host-plant specific, demonstrating four dominant patterns of specialization that shape species diversification: (1) diversification through host switching; (2) specialization on widespread hosts with allopatric speciation; (3) repeated, independent shifts to the same hosts; and, (4) absence or low abundance on some host. Finally, evidence suggests competing herbivore radiations limit ecological opportunity for diversifying insect herbivores. Results provide evolutionary insights into the mechanisms that drive and shape this biodiversity.

Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny of the Drosophila repleta species group.

We present a revised molecular phylogeny of the Drosophila repleta group including 62 repleta group taxa and nine outgroup species based on four mitochondrial and six nuclear DNA sequence fragments. With ca. 100 species endemic to the New World, the repleta species group represents one of the major species radiations in the genus Drosophila. Most repleta group species are associated with cacti in arid or semiarid regions. Contrary to previous results, maximum likelihood and Bayesian phylogenies of the 10-gene dataset strongly support the monophyly of the repleta group. Several previously described subdivisions in the group were also recovered, despite poorly resolved relationships between these clades. Divergence time estimates suggested that the repleta group split from its sister group about 21millionyears ago (Mya), although diversification of the crown group began ca. 16Mya. Character mapping of patterns of host plant use showed that flat leaf Opuntia use is common throughout the phylogeny and that shifts in host use from Opuntia to the more chemically complex columnar cacti occurred several times independently during the history of this group. Although some species retained the use of Opuntia after acquiring the use of columnar cacti, there were multiple, phylogenetically independent instances of columnar cactus specialization with loss of Opuntia as a host. Concordant with our proposed timing of host use shifts, these dates are consistent with the suggested times when the Opuntioideae originated in South America. We discuss the generally accepted South American origin of the repleta group.

Standardized terminology and visual atlas of the external morphology and terminalia for the genus Scaptomyza (Diptera: Drosophilidae).

The genus Scaptomyza is one of the two Drosophilidae genera with Hawaiian endemic species. This genus is an excellent model for biogeographic studies since it is distributed throughout the majority of continents, including continental islands, the Hawaiian Islands, and many other remote oceanic islands. This genus currently comprises 273 described species, 148 of which are endemic to the Hawaiian Islands. However, most descriptions were published before efforts to standardizing the morphological terminology across the Diptera were made in the 1980's. Since research groups developed their own set of terminologies independently, without considering homologies, multiple terms have been used to refer to the same characters. This is especially true for the male terminalia, which have remarkable modifications within the family Drosophilidae. We reviewed the Scaptomyza literature, in addition to other studies across the Drosophilidae and Diptera, compiled the English synonyms, and provided a visual atlas of each body part, indicating how to recognize the morphological characters. The goal of the present study is to facilitate species identification and propose preferred terms to be adopted for future Scaptomyza descriptions.

Whole mitochondrial genome phylogeny of Drosophilidae.

A total of 241 mitochondrial genomes were assembled and annotated from the SRA database to reconstruct a mtDNA genome phylogeny for the genus Drosophila, the family Drosophilidae, and close relatives. The resulting mtDNA genome phylogeny is largely congruent with previous higher-level analyses of Drosophila species with the exception of the relationships between the melanogaster, montium, anannassae, saltans and obscura groups. Although relationships within these species groups are congruent between nuclear and mtDNA studies, the mtDNA genome phylogeny of the groups is different when compared to earlier studies. Monophyly of known species groups within the genus Drosophila are highly supported and, as in previous work, the genera Lordiphosa, Hirtodrosophila, Zaprionus and Scaptomya are all imbedded within the genus Drosophila. Incongruence and partitioned support analyses indicate that DNA sequences are better at resolving the phylogeny than their translated protein sequences. Such analyses also indicate that genes on the minus strand of the circular molecule (Lrrna, Srrna, ND4, ND4L and ND5) provide most of the support for the overall phylogenetic hypothesis.

A standardized nomenclature and atlas of the female terminalia of Drosophila melanogaster.

The model organism Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.

Phylogenetic relationships in the spoon tarsus subgroup of Hawaiian Drosophila: conflict and concordance between gene trees.

The Hawaiian Drosophilidae contains approximately 1000 species, placed in species groups and subgroups based largely on secondary sexual modifications to wings, forelegs and mouthparts. Members of the spoon tarsus subgroup possess a cup-shaped structure on the foretarsi of males. Eight of the twelve species in this subgroup are found only on the Big Island of Hawaii, suggesting that they have diverged within the past 600,000 years. This rapid diversification has made determining the relationships within this group difficult to infer. We use 13 genes, including nine rapidly evolving nuclear loci, to estimate relationships within the spoon tarsus species, as well as to test the monophyly of this subgroup. A variety of analytical approaches are used, including individual and concatenated analyses, Bayesian estimation of species trees and Bayesian untangling of concordance knots. We find widespread agreement between phylogenetic estimates derived from different methods, although some incongruence is present. Notably, our analyses suggest that the spoon tarsus subgroup, as currently defined, is not monophyletic.

Phylogenetic and ecological relationships of the Hawaiian Drosophila inferred by mitochondrial DNA analysis.

The Hawaiian Drosophilidae are comprised of an estimated 1000 species, all arising from a single common ancestor in the last 25 million years. This group, because of its species diversity, marked sexual dimorphism and complex mating behavior, host plant specificity, and the well-known chronology of the Hawaiian Archipelago, is an excellent model system for evolutionary studies. Here we present a phylogeny of this group based on ~2.6 kb of mitochondrial DNA sequence. Our taxon sampling is the most extensive to date, with nearly 200 species representing all species groups and most subgroups from the larger clades. Our results suggest that the picture wing and modified mouthpart species, long believed to be derived within this radiation, may actually occupy a basal position in the phylogeny. The haleakale species group, in contrast, is strongly supported as sister to the AMC clade. We use the phylogenetic results to examine the evolution of two important ecological characters, the host family and type of substrate used for oviposition and larval development. Although both host and substrate transitions are common in the group, oviposition substrate is more conserved among species groups than host plant family. While the ancestral host plant family is equivocally reconstructed, our results suggest that the ancestor of this group may have used rotting bark as a primary oviposition substrate.

Sperm Cyst "Looping": A Developmental Novelty Enabling Extreme Male Ornament Evolution.

Postcopulatory sexual selection is credited as a principal force behind the rapid evolution of reproductive characters, often generating a pattern of correlated evolution between interacting, sex-specific traits. Because the female reproductive tract is the selective environment for sperm, one taxonomically widespread example of this pattern is the co-diversification of sperm length and female sperm-storage organ dimension. In Drosophila, having testes that are longer than the sperm they manufacture was believed to be a universal physiological constraint. Further, the energetic and time costs of developing long testes have been credited with underlying the steep evolutionary allometry of sperm length and constraining sperm length evolution in Drosophila. Here, we report on the discovery of a novel spermatogenic mechanism-sperm cyst looping-that enables males to produce relatively long sperm in short testis. This phenomenon (restricted to members of the saltans and willistoni species groups) begins early during spermatogenesis and is potentially attributable to heterochronic evolution, resulting in growth asynchrony between spermatid tails and the surrounding spermatid and somatic cyst cell membranes. By removing the allometric constraint on sperm length, this evolutionary innovation appears to have enabled males to evolve extremely long sperm for their body mass while evading delays in reproductive maturation time. On the other hand, sperm cyst looping was found to exact a cost by requiring greater total energetic investment in testes and a pronounced reduction in male lifespan. We speculate on the ecological selection pressures underlying the evolutionary origin and maintenance of this unique adaptation.

The genus Drosophila as a model for testing tree- and character-based methods of species identification using DNA barcoding.

DNA barcoding has recently been proposed as a promising tool for the (1) rapid assignment of unknown samples to described species by non-expert workers and (2) a potential method of new species discovery based on degree of DNA sequence divergence. Two broad methods have been used, one based on degree of DNA sequence variation, within and between species and another requiring the recovery of species as discrete clades (monophyly) on a phylogenetic tree. An alternative method relies on the identification of a set of specific diagnostic nucleotides for a given species (characters). The genus Drosophila has long served as a model system in genetics, development, ecology and evolutionary biology. As a result of this work, species boundaries within this genus are quite well delimited, with most taxa being defined by morphological characters and also conforming to a biological species concept (e.g., partial or complete reproductive isolation has used to erect and define species). In addition, some of the species in this group have also been subjected to phylogenetic analysis, yielding cases where taxa both conform and conflict with a phylogenetic species concept. Here, we analyzed 1058 COI sequences belonging to 68 species belonging to Drosophila and its allied genus Zaprionus and with more than a single representative to assess the performance of the three DNA barcoding methods. 26% of the species could not be defined using distance methods, i.e. had a barcoding gap of ≤ 0, and 23% were not monophyletic. We focused then on four groups of closely-related species whose taxonomy is well-established on non-molecular basis (e.g., morphology, geography, reproductive isolation) and to which most of the problematic species belonged. We showed that characters performed better than other approaches in the case of paraphyletic species, but all methods failed in the case of polyphyletic species. For these polyphyletic species, other sources of evidence (e.g., morphology, geography, reproductive isolation) are more relevant than COI sequences, highlighting the limitation of DNA barcoding and the needs for integrative taxonomy approaches. In conclusion, DNA barcoding of Drosophila shows no reason to alter the 250 years old tradition of character-based taxonomy, and many reasons to shy away from the alternatives.

Evolution of Reproductive Behavior.

Behaviors associated with reproduction are major contributors to the evolutionary success of organisms and are subject to many evolutionary forces, including natural and sexual selection, and sexual conflict. Successful reproduction involves a range of behaviors, from finding an appropriate mate, courting, and copulation, to the successful production and (in oviparous animals) deposition of eggs following mating. As a consequence, behaviors and genes associated with reproduction are often under strong selection and evolve rapidly. Courtship rituals in flies follow a multimodal pattern, mediated through visual, chemical, tactile, and auditory signals. Premating behaviors allow males and females to assess the species identity, reproductive state, and condition of their partners. Conflicts between the "interests" of individual males, and/or between the reproductive strategies of males and females, often drive the evolution of reproductive behaviors. For example, seminal proteins transmitted by males often show evidence of rapid evolution, mediated by positive selection. Postmating behaviors, including the selection of oviposition sites, are highly variable and Drosophila species span the spectrum from generalists to obligate specialists. Chemical recognition features prominently in adaptation to host plants for feeding and oviposition. Selection acting on variation in pre-, peri-, and postmating behaviors can lead to reproductive isolation and incipient speciation. Response to selection at the genetic level can include the expansion of gene families, such as those for detecting pheromonal cues for mating, or changes in the expression of genes leading to visual cues such as wing spots that are assessed during mating. Here, we consider the evolution of reproductive behavior in Drosophila at two distinct, yet complementary, scales. Some studies take a microevolutionary approach, identifying genes and networks involved in reproduction, and then dissecting the genetics underlying complex behaviors in D. melanogaster Other studies take a macroevolutionary approach, comparing reproductive behaviors across the genus Drosophila and how these might correlate with environmental cues. A full synthesis of this field will require unification across these levels.

Evidence for both sequential mutations and recombination in the evolution of kdr alleles in Aedes aegypti.

BACKGROUND: Aedes aegypti is a globally distributed vector of human diseases including dengue, yellow fever, chikungunya, and Zika. Pyrethroid insecticides are the primary means of controlling adult A. aegypti populations to suppress arbovirus outbreaks, but resistance to pyrethroid insecticides has become a global problem. Mutations in the voltage-sensitive sodium channel (Vssc) gene are a major mechanism of pyrethroid resistance in A. aegypti. Vssc resistance alleles in A. aegypti commonly have more than one mutation. However, our understanding of the evolutionary dynamics of how alleles with multiple mutations arose is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We examined the geographic distribution and association between the common Vssc mutations (V410L, S989P, V1016G/I and F1534C) in A. aegypti by analyzing the relevant Vssc fragments in 25 collections, mainly from Asia and the Americas. Our results showed all 11 Asian populations had two types of resistance alleles: 1534C and 989P+1016G. The 1534C allele was more common with frequencies ranging from 0.31 to 0.88, while the 989P+1016G frequency ranged from 0.13 to 0.50. Four distinct alleles (410L, 1534C, 410L+1534C and 410L+1016I+1534C) were detected in populations from the Americas. The most common was 410L+1016I+1534C with frequencies ranging from 0.50 to 1.00, followed by 1534C with frequencies ranging from 0.13 to 0.50. Our phylogenetic analysis of Vssc supported multiple independent origins of the F1534C mutation. Our results indicated the 410L+1534C allele may have arisen by addition of the V410L mutation to the 1534C allele, or by a crossover event. The 410L+1016I+1534C allele was the result of one or two mutational steps from a 1534C background. CONCLUSIONS/SIGNIFICANCE: Our data corroborated previous geographic distributions of resistance mutations and provided evidence for both recombination and sequential accumulation of mutations contributing to the molecular evolution of resistance alleles in A. aegypti.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

Spread of an introduced vector-borne banana virus in Hawaii.

Emerging diseases are increasing in incidence; therefore, understanding how pathogens are introduced into new regions and cause epidemics is of importance for the development of strategies that may hinder their spread. We used molecular data to study how a vector-borne banana virus, Banana bunchy top virus (BBTV), spread in Hawaii after it was first detected in 1989. Our analyses suggest that BBTV was introduced once into Hawaii, on the island of Oahu. All other islands were infected with isolates originating from Oahu, suggesting that movement of contaminated plant material was the main driving factor responsible for interisland spread of BBTV. The rate of mutation inferred by the phylogenetic analysis (1.4 x 10(-4) bp/year) was similar to that obtained in an experimental evolution study under greenhouse conditions (3.9 x 10(-4) bp/year). We used these values to estimate the number of infections occurring under field conditions per year. Our results suggest that strict and enforced regulations limiting the movement of banana plant material among Hawaiian islands could have reduced interisland spread of this pathogen.