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.

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.

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.

Microplegia vs 4:1 Blood Cardioplegia: Effectiveness and Cost Savings in Complex Cardiac Operations.

BACKGROUND: Microplegia has been studied during isolated coronary artery bypass grafting and valve surgery but not in more complex operations. Objectives of this study were to demonstrate safety and effectiveness of microplegia relative to Buckberg cardioplegia during these operations. METHODS: From January 2012 to January 2017, 242 patients underwent multicomponent operations with simplified microplegia delivered via syringe pump and 10,512 with modified Buckberg cardioplegia. Operations included aortic root, arch, or ascending aorta replacement in 424 (94%) patients, aortic valve surgery in 324 (72%) patients, and concomitant coronary artery bypass grafting in 47 (10%) patients. Outcomes were compared in 226 propensity-matched pairs. RESULTS: There was no difference in median postoperative troponin T between groups after adjusting for aortic clamp time. Microplegia patients received significantly less crystalloid with their cardioplegia (mean 27 ± 8.0 mL/operation vs 735 ± 357 mL/operation; P < .001) and had lower peak intraoperative glucose (196 ± 40 mg/dL vs 248 ± 69 mg/dL; P < .001). Microplegia and Buckberg groups had similar in-hospital mortality (2.7% [n = 6] vs 2.2% [n = 5]; P = .8), stroke (2.2% [n = 5] vs 3.6% [n = 8]; P = .4), renal failure (8% [n = 18] vs 5.8% [n = 13]; P = .4), prolonged ventilation (23% [n = 51] vs 24% [n = 54]; P = .7), median postoperative length of stay (both 8.1 days; P > .9), and median red cell units administered to patients requiring transfusion (4 units vs 3 units; P = .14). The mean cost of cardioplegia per case with microplegia was 1/26th that of Buckberg cardioplegia. CONCLUSIONS: Our simplified microplegia technique offers several advantages over Buckberg cardioplegia without compromising myocardial protection or safety in complex, multicomponent operations with extended aortic clamp times.

Phylogeny of the Genus Drosophila.

Understanding phylogenetic relationships among taxa is key to designing and implementing comparative analyses. The genus Drosophila, which contains over 1600 species, is one of the most important model systems in the biological sciences. For over a century, one species in this group, Drosophila melanogaster, has been key to studies of animal development and genetics, genome organization and evolution, and human disease. As whole-genome sequencing becomes more cost-effective, there is increasing interest in other members of this morphologically, ecologically, and behaviorally diverse genus. Phylogenetic relationships within Drosophila are complicated, and the goal of this paper is to provide a review of the recent taxonomic changes and phylogenetic relationships in this genus to aid in further comparative studies.

Studies in Hawaiian Diptera III: New Distributional Records for Canacidae and a New Endemic Species of Procanace.

The distributions of Hawaiian Canacidae, comprising nearly 800 individual collection events, are reviewed and a total of four new island records are reported. These include Canaceoides angulatus from Kahoolawae and Procanace bifurcata from Molokai and Maui, and Procanace constricta from Oahu. A new species from Kauai, Procanace hardyi O'Grady and Pak, is described. This species is closely related to P. constricta from Oahu, Maui, Molokai and Hawaii and shares a similar constriction of the abdomen between tergites four and five but differs in the configuration of the seventh abdominal tergite. Detailed distribution maps for all species are included.

Using near-infrared spectroscopy to monitor lower extremities in patients on venoarterial extracorporeal membrane oxygenation.

Patients on peripheral extracorporeal membrane oxygenation (ECMO) are at risk for lower extremity ischemia. Effective monitoring is needed to identify complications quickly and allow timely correction. Near-infrared spectroscopy has been used extensively in cerebral monitoring during cardiac surgery. We present its use in monitoring lower extremity perfusion in patients on ECMO. Five patients on ECMO had near-infrared spectroscopy monitors placed on the calf of both lower extremities. Continuous real-time tissue oxygen saturation data (stO2) was displayed and recorded. Two patients had lower extremity complications in the leg with the arterial cannula. The patients with complications had lower stO2 in the cannulated leg at the time of ECMO insertion, larger differences in stO2 between the legs at the time of insertion, lower nadir stO2s, and larger peak differences in stO2 between the legs than patients without limb complications. The use of near-infrared spectroscopy for continuous monitoring of tissue oxygenation in the lower extremities in patients on ECMO may allow early identification of patients with lower extremity complications.

Phylogenetics of the antopocerus-modified tarsus clade of Hawaiian Drosophila: diversification across the Hawaiian Islands.

The Hawaiian Drosophilidae radiation is an ecologically and morphologically diverse clade of almost 700 described species. A phylogenetic approach is key to understanding the evolutionary forces that have given rise to this diverse lineage. Here we infer the phylogeny for the antopocerus, modified tarsus and ciliated tarsus (AMC) clade, a lineage comprising 16% (91 of 687 species) of the described Hawaiian Drosophilidae. To improve on previous analyses we constructed the largest dataset to date for the AMC, including a matrix of 15 genes for 68 species. Results strongly support most of the morphologically defined species groups as monophyletic. We explore the correlation of increased diversity in biogeography, sexual selection and ecology on the present day diversity seen in this lineage using a combination of dating methods, rearing records, and distributional data. Molecular dating analyses indicate that AMC lineage started diversifying about 4.4 million years ago, culminating in the present day AMC diversity. We do not find evidence that ecological speciation or sexual selection played a part in generating this diversity, but given the limited number of described larval substrates and secondary sexual characters analyzed we can not rule these factors out entirely. An increased rate of diversification in the AMC is found to overlap with the emergence of multiple islands in the current chain of high islands, specifically Oahu and Kauai.

Studies in Hawaiian Diptera II: New Distributional Records for Endemic Scatella (Ephydridae).

Here we summarize the known distributional data for the Hawaiian Scatella (Ephydridae). We report on four new island records; Scatellaamnica and Scatellastagnalis from Kauai, Scatellaoahuense from Lanai, and Scatellaterryi from Maui. A list of material present, comprising over 3100 individual specimen records in the collections of the Bernice Pauahi Bishop Museum, University of Hawaii at Manoa, and Essig Musuem of Entomology at UC Berkeley is included, along with details distributional maps for the Hawaiian endemic species.

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.

Studies in hawaiian Diptera I: new distributional records for endemic asteia (asteiidae).

New island records are reported for five species of Asteia endemic to the Hawaiian Islands (Asteiahawaiiensis, Asteiamauiensis, Asteiamolokaiensis, Asteiapalikuensis, Asteiasabroskyi). These new records expand our understanding of distributions in Asteia, change the percentage of single island endemics from 78% to 33%, and have significance in how we view the process of diversification acting in this lineage. We also present a list of the known rearing records for two species in this group. Asteiamontgomeryi has been recorded from Erythrina and Asteiasabroskyi has been reared from Pisonia, Urera, Charpentiera and Hibiscadelphus.

Microbial interactions and the ecology and evolution of Hawaiian Drosophilidae.

Adaptive radiations are characterized by an increased rate of speciation and expanded range of habitats and ecological niches exploited by those species. The Hawaiian Drosophilidae is a classic adaptive radiation; a single ancestral species colonized Hawaii approximately 25 million years ago and gave rise to two monophyletic lineages, the Hawaiian Drosophila and the genus Scaptomyza. The Hawaiian Drosophila are largely saprophagous and rely on approximately 40 endemic plant families and their associated microbes to complete development. Scaptomyza are even more diverse in host breadth. While many species of Scaptomyza utilize decomposing plant substrates, some species have evolved to become herbivores, parasites on spider egg masses, and exploit microbes on living plant tissue. Understanding the origin of the ecological diversity encompassed by these nearly 700 described species has been a challenge. The central role of microbes in drosophilid ecology suggests bacterial and fungal associates may have played a role in the diversification of the Hawaiian Drosophilidae. Here we synthesize recent ecological and microbial community data from the Hawaiian Drosophilidae to examine the forces that may have led to this adaptive radiation. We propose that the evolutionary success of the Hawaiian Drosophilidae is due to a combination of factors, including adaptation to novel ecological niches facilitated by microbes.

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.

Diversity and phylogenetic relationships of Wolbachia in Drosophila and other native Hawaiian insects.

Wolbachia is a genus of parasitic alphaproteobacteria found in arthropods and nematodes, and represents on of the most common, widespread endosymbionts known. Wolbachia affects a variety of reproductive functions in its host (e.g., male killing, cytoplasmic incompatibility, parthenogenesis), which have the potential to dramatically impact host evolution and species formation. Here, we present the first broad-scale study to screen natural populations of native Hawaiian insects for Wolbachia, focusing on the endemic Diptera. Results indicate that Wolbachia infects native Hawaiian taxa, with alleles spanning phylogenetic supergroups, A and B. The overall frequency of Wolbachia incidene in Hawaiian insects was 14%. The incidence of infection in native Hawaiian Diptera was 11% for individuals and 12% for all species screened. Wolbachia was not detected in two large, widespread Hawaiian dipteran families-Dolichopodidae (44 spp screened) and Limoniidae (12 spp screened). Incidence of infection within endemic Hawaiian lineages that carry Wolbachia was 18% in Drosophilidae species, 25% in Caliphoridae species, > 90% in Nesophrosyne species, 20% in Drosophila dasycnemia and 100% in Nesophrosyne craterigena. Twenty unique alleles were recovered in this study, of which 18 are newly recorded. Screening of endemic populations of D. dasycnemia across Hawaii Island revealed 4 unique alleles. Phylogenetic relationships and allele diversity provide evidence for horizontal transfer of Wolbachia among Hawaiian arthropod lineages.

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.

Fungal diversity associated with Hawaiian Drosophila host plants.

Hawaiian Drosophila depend primarily, sometimes exclusively, on specific host plants for oviposition and larval development, and most specialize further on a particular decomposing part of that plant. Differences in fungal community between host plants and substrate types may establish the basis for host specificity in Hawaiian Drosophila. Fungi mediate decomposition, releasing plant micronutrients and volatiles that can indicate high quality substrates and serve as cues to stimulate oviposition. This study addresses major gaps in our knowledge by providing the first culture-free, DNA-based survey of fungal diversity associated with four ecologically important tree genera in the Hawaiian Islands. Three genera, Cheirodendron, Clermontia, and Pisonia, are important host plants for Drosophila. The fourth, Acacia, is not an important drosophilid host but is a dominant forest tree. We sampled fresh and rotting leaves from all four taxa, plus rotting stems from Clermontia and Pisonia. Based on sequences from the D1/D2 domain of the 26S rDNA gene, we identified by BLAST search representatives from 113 genera in 13 fungal classes. A total of 160 operational taxonomic units, defined on the basis of ≥97% genetic similarity, were identified in these samples, but sampling curves show this is an underestimate of the total fungal diversity present on these substrates. Shannon diversity indices ranged from 2.0 to 3.5 among the Hawaiian samples, a slight reduction compared to continental surveys. We detected very little sharing of fungal taxa among the substrates, and tests of community composition confirmed that the structure of the fungal community differed significantly among the substrates and host plants. Based on these results, we hypothesize that fungal community structure plays a central role in the establishment of host preference in the Hawaiian Drosophila radiation.

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.

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.

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.