The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera, and species groups in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred a species tree with highly supported groups for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via cooption of parts of an ancestral appendage gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster most closely with antennae, relative to multiple other tissues. Furthermore, three genes essential for eyespot development, Distal-less (Dll), spalt (sal), and Antennapedia (Antp), share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots, antennae, legs, and also wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely reused an ancient GRN for their development, a network also previously implicated in the development of antennae, legs, and wings. Significance Where do butterfly eyespots come from? One of the long-standing questions in the field of evolution concerns addressing where novel complex traits come from. Here we show that butterfly eyespots, a novel complex trait, likely originated from the redeployment of a preexisting gene-regulatory network regulating antennae, legs, and wings, to novel locations on the wing.

Two ratiometric near-infrared fluorescent probes have been developed to selectively detect mitochondrial pH changes based on highly efficient through-bond energy transfer (TBET) from cyanine donors to near-infrared hemicyanine acceptors. The probes consist of identical cyanine donors connected to different hemicyanine acceptors with a spirolactam ring structure linked via a biphenyl linkage. At neutral or basic pH, the probes display only fluorescence of the cyanine donors when they are excited at 520 nm. However, acidic pH conditions trigger spirolactam ring opening, leading to increased π-conjugation of the hemicyanine acceptors, resulting in new near-infrared fluorescence peaks at 740 nm and 780 nm for probes A and B, respectively. This results in ratiometric fluorescence responses of the probes to pH changes indicated by decreases of the donor fluorescence and increases of the acceptor fluorescence under donor excitation at 520 nm due to a highly efficient TBET from the donors to the acceptors. The probes only show cyanine donor fluorescence in alkaline-pH mitochondria. However, the probes show moderate fluorescence decreases of the cyanine donor and considerable fluorescence increases of hemicyanine acceptors during the mitophagy process induced by nutrient starvation or under drug treatment. The probes display rapid, selective, and sensitive responses to pH changes over metal ions, good membrane penetration, good photostability, large pseudo-Stokes shifts, low cytotoxicity, mitochondria-targeting, and mitophagy-tracking capabilities.

A near-infrared reactive cyanine platform (probe A) was prepared by condensation of 9-chloro-1,2,3,4-tetrahydro-10-methyl-acridinium iodide with Fisher's aldehyde. A near-infrared fluorescent probe (probe B) was prepared by modifying a reactive chlorine atom of probe A with tert-butyl(2-aminoethyl)carbamate through a substitution reaction. The deprotection of the Boc group of probe B was achieved under an acidic condition, affording an amine-functionalized cyanine dye (probe C). A near-infrared ratiometric fluorescent probe (probe D) for mitochondrial pH detection was synthesized by conjugating a FRET coumarin donor to a FRET cyanine acceptor (probe C) through an amide bond connection. Probe A shows low fluorescence of 2% due to an electron-withdrawing chlorine atom, while probes B–D display high fluorescence quantum yields of 60%, 32%, and 35% in aqueous solutions containing 10% ethanol, respectively. Probes B–D show strong fluorescence with push–pull molecular structures in neutral and basic pH conditions. However, protonation of the probe's second amine at the 9-position under acidic condition disrupts the push–pull feature of the probes, resulting in fluorescence quenching of the new cyanine fluorophores. The probes can selectively stain mitochondria, while probe D was employed to detect pH changes in HeLa cells and Drosophila melanogaster first-instar larvae.