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Many organisms inhabiting the interface between land and sea have evolved biological clocks corresponding to the period of the semilunar (14.77 days) or the lunar (29.53 days) cycle. Since tidal amplitude is modulated across the lunar cycle, these circasemilunar or circalunar clocks not only allow organisms to adapt to the lunar cycle, but also to specific tidal situations. Biological clocks are synchronized to external cycles via environmental cues called zeitgebers. Here, we explore how light at night sets the circalunar and circasemilunar clocks of Clunio marinus, a marine insect that relies on these clocks to control timing of emergence. We first characterized how moonlight intensity is modulated by the tides by measuring light intensity in the natural habitat of C. marinus. In laboratory experiments, we then explored how different moonlight treatments set the phase of the clocks of two C. marinus strains, one with a lunar rhythm and one with a semilunar rhythm. Light intensity alone does not affect the phase of the lunar rhythm. Presenting moonlight during different 2-h or 4-h windows during the night shows that (1) the required duration of moonlight is strain-specific, (2) there are strain-specific moonlight sensitivity windows and (3) timing of moonlight can shift the phase of the lunar rhythm to stay synchronized with the lowest low tides. Experiments simulating natural moonlight patterns confirm that the phase is set by the timing of moonlight. Simulating natural moonlight at field-observed intensities leads to the best synchronization. Taken together, we show that there is a complex and strain-specific integration of intensity, duration and timing of light at night to precisely entrain the lunar and semilunar rhythms. The observed fine-tuning of the rhythms under natural moonlight regimes lays the foundation for a better chronobiological and genetic dissection of the circa(semi)lunar clock in C. marinus. [ABSTRACT FROM AUTHOR]

Introduction DNA barcoding is a powerful and cost-effective tool for species identification (Adamowicz et al. 2019; Candek and Kuntner 2015; Hebert et al. 2003). The formally accepted DNA barcode for [...], DNA barcodes are widely used for species identification and biogeographic studies. Here, we compare the use of full mitochondrial genomes versus DNA barcodes and other mitochondrial DNA fragments for biogeographic and ecological analyses. Our dataset comprised 120 mitochondrial genomes from the genus Clunio (Diptera: Chironomidae), comprising five populations from two closely related species (Clunio marinus and Clunio balticus) and three ecotypes. We extracted cytochrome oxidase c subunit I (COI) barcodes and partitioned the mitochondrial genomes into non-overlapping windows of 750 or 1500 bp. Haplotype networks and diversity indices were compared for these windows and full mitochondrial genomes (15.4 kb). Full mitochondrial genomes indicate complete geographic isolation between populations, but do not allow for conclusions on the separation of ecotypes or species. COI barcodes have comparatively few polymorphisms, ideal for species identification, but do not resolve geographic isolation. Many of the similarly sized 750 bp windows have higher nucleotide and haplotype diversity than COI barcodes, but still do not resolve biogeography. Only when increasing the window size to 1500 bp, two windows resolve biogeography reasonably well. Our results suggest that the design and use of DNA barcodes in biogeographic studies must be carefully evaluated for each investigated species. Keywords: DNA barcoding, mitochondrial genome, cytochrome oxidase I, haplotype network, diversity, ecotype. Les codes a barres de l'ADN sont largement employes pour identifier des especes et mener des etudes biogeographiques. Dans ce travail, les auteurs comparent l'utilisation de genomes mitochondriaux complets contre les codes a barres et autres sequences d'ADN mitochondrial pour des analyses ecologiques et biogeographiques. Le jeu de donnees comprend 120 genomes mitochondriaux chez le genre Clunio (Diptera: Chironomidae), incluant cinq populations de deux especes proches (Clunio marinus et Clunio balticus) ainsi que trois ecotypes. Les auteurs ont extrait les sequences de la sous-unite I de la cytochrome c oxydase et divise les genomes mitochondriaux en fenetres non-chevauchantes de 750 ou 1500 pb. Les reseaux d'haplotypes et les indices de diversite ont ete compares pour ces fenetres ainsi que pour les genomes complets (15,4 kb). Les genomes complets revelent un isolement geographique complet entre les populations, mais ne permettent pas de conclure a la separation des ecotypes ou des especes. Les codes a barres COI, en comparaison, presentent peu de polymorphismes, une situation ideale pour l'identification des especes, mais ne permettent pas de deceler l'isolement geographique. Plusieurs des fenetres de taille semblable (750 pb) affichaient une diversite nucleotidique et haplotypique superieures a celles du code a barre COI, mais ne permettaient toujours pas de resoudre la biogeographie. C'est seulement en augmentant la taille des fenetres a 1500 pb que deux des fenetres ont pu resoudre la biogeographie de maniere raisonnable. Ces resultats suggerent que la conception et l'utilisation des codes a barres dans le contexte d'etudes biogeographiques doivent etre evalues attentivement pour chaque espece etudiee. [Traduit par la Redaction] Mots-cles: codage a barres de l'ADN, genome mitochondrial, cytochrome oxydase I, reseau d'haplotypes, diversite, ecotype.

Semilunar rhythms are found in numerous marine organisms, but the molecular mechanism and functional principles of endogenous circasemilunar clocks remain elusive. Here, we explore the connection between the free-running circasemilunar clock and the circadian clock in the marine midge Clunio marinus with three different chronobiological assays. First, we found that the free-running circasemilunar period of the adult emergence rhythm in C. marinus changes linearly with diel T-cycle length, supporting a day-counting mechanism. Second, under LD 6:6, periods of circasemilunar and circadian emergence were comparable to those under LD 12:12, indicating that the circasemilunar counter in C. marinus relies on endogenous circadian oscillations rather than external T-cycles. Finally, when desynchronizing the circadian clock with constant light, the free-running circasemilunar emergence rhythm disappeared as well, suggesting that it requires a synchronized circadian clock. These results oppose the long-held view that C. marinus' free-running circasemilunar clock operates independently of the circadian clock. In a broader evolutionary context, our results strengthen the idea that the circasemilunar clocks of dipterous insects are based on different functional principles compared to the circasemilunar or circalunar clocks of marine annelids and algae. These divergent clock principles may indicate multiple evolutionary origins of circasemilunar and circalunar clocks. [ABSTRACT FROM AUTHOR]

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

Genetic variants underlying traits that become either non-adaptive or selectively neutral are expected to have altered evolutionary trajectories. Uncovering genetic signatures associated with phenotypic loss presents the opportunity to discover the molecular basis for the phenotype in populations where it persists. Here we study circalunar clocks in populations of the marine midge Clunio marinus. The circalunar clock synchronizes development to the lunar phase, and it is set by moonlight and tidal cycles of mechanical agitation. Two out of ten studied populations have lost their sensitivity to mechanical agitation while preserving sensitivity to moonlight. Intriguingly, the F1 offspring of the two insensitive populations regained the sensitivity to mechanical entrainment, implying a genetically independent loss of the phenotype. By combining quantitative trait locus mapping and genome-wide screens, we explored the genetics of this phenotypic loss. QTL analysis suggested an oligogenic origin with one prevalent additive locus in one of the strains. In addition, it confirmed a distinct genetic architecture in the two insensitive populations. Genomic screens further uncovered several candidate genes underlying QTL regions. The strongest signal under the most prominent QTL contains a duplicated STAT1 gene, which has a well-established role in development, and CG022363, an ortholog of the Drosophila melanogaster CG32100 gene, which plays a role in gravitaxis. Our results support the notion that adaptive phenotypes have a complex genetic basis with mutations occurring at several loci. By dissecting the most prevalent signals, we started to reveal the molecular machinery responsible for the entrainment of the circalunar clock. Author summary: Biological clocks allow organisms to synchronize their physiology and behaviour with their environment. A key part of this mechanism are reliable environmental cues that play a role in setting the clock. Here we study the circalunar clock of Clunio marinus, a marine insect that inhabits the intertidal zone. The circalunar clock is set by moonlight as well as mechanical agitation caused by the tides. Interestingly, in two out of ten studied Clunio populations, mechanical agitation was not sufficient to set the circalunar clock. We found that this loss of sensitivity has a genetic basis. Furthermore, we use the insensitive populations as natural mutants. Finding the mutations that lead to the loss of sensitivity allows us to uncover the molecular mechanisms underlying the setting of the yet unexplored molecular clockwork. Additionally, by uncovering the genetic basis for the loss of sensitivity in two independent populations, we get insights into the processes underlying the evolutionary adaptation of biological clocks. [ABSTRACT FROM AUTHOR]

Abstract Background The intertidal zone of seacoasts, being affected by the superimposed tidal, diurnal and lunar cycles, is temporally the most complex environment on earth. Many marine organisms exhibit lunar rhythms in reproductive behaviour and some show experimental evidence of endogenous control by a circalunar clock, the molecular and genetic basis of which is unexplored. We examined the genetic control of lunar and diurnal rhythmicity in the marine midge Clunio marinus (Chironomidae, Diptera), a species for which the correct timing of adult emergence is critical in natural populations. Results We crossed two strains of Clunio marinus that differ in the timing of the diurnal and lunar rhythms of emergence. The phenotype distribution of the segregating backcross progeny indicates polygenic control of the lunar emergence rhythm. Diurnal timing of emergence is also under genetic control, and is influenced by two unlinked genes with major effects. Furthermore, the lunar and diurnal timing of emergence is correlated in the backcross generation. We show that both the lunar emergence time and its correlation to the diurnal emergence time are adaptive for the species in its natural environment. Conclusions The correlation implies that the unlinked genes affecting lunar timing and the two unlinked genes affecting diurnal timing could be the same, providing an unexpectedly close interaction of the two clocks. Alternatively, the genes could be genetically linked in a two-by-two fashion, suggesting that evolution has shaped the genetic architecture to stabilize adaptive combinations of lunar and diurnal emergence times by tightening linkage. Our results, the first on genetic control of lunar rhythms, offer a new perspective to explore their molecular clockwork.

Genetic divergence of populations in the presence of gene flow is a central theme in speciation research. Theory predicts that divergence can happen with full range overlap – in sympatry – driven by ecological factors, but there are few empirical examples of how ecologically divergent selection can overcome gene flow and lead to reproductive isolation. In the marine midge Clunio marinus (Diptera: Chironomidae) reproduction is ecologically restricted to the time of the lowest tides, which is ensured through accurate control of development and adult emergence by circalunar and circadian clocks. As tidal regimes differ along the coastline, locally adapted timing strains of C. marinus are found in different sites across Europe. At the same time, ecologically suitable low tides occur at both full and new moon and twice a day, providing C. marinus with four nonoverlapping temporal niches at every geographic location. Along the coast of Brittany, which is characterized by a steep gradient in timing of the tides, we found an unusually large number of differentially adapted timing strains, and the first known instances of sympatric C. marinus strains occupying divergent temporal niches. Analysis of mitochondrial genotypes suggests that these timing strains originated from a single recent colonization event. Nuclear genotypes show strong gene flow, sympatric timing strains being the least differentiated. Even when sympatric strains exist in nonoverlapping temporal niches, timing adaptations do not result in genome‐wide genetic divergence, suggesting timing adaptations are maintained by permanent ecological selection. This constitutes a model case for incipient ecological divergence with gene flow. [ABSTRACT FROM AUTHOR]

Ten polymorphic microsatellite loci were cloned and characterized for the marine midge Clunio marinus (Chironomidae, Diptera). The number of alleles ranged from three to 31, the observed heterozygosity ranged from 0.06 to 0.83. During preliminary tests for polymorphisms, we identified subtly differing alleles due to the heteroduplexes they formed on nondenaturing gels. This allowed for the use of unlabelled primers in standard lower resolution PAGE and thus saved time and money in primer testing. [ABSTRACT FROM AUTHOR]