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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 ., Competing Interests: Conflict of Interest StatementThe authors have no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., Competing Interests: Conflict of interest statementThe authors have no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Briševac et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The evolutionary trajectories and genetic architectures underlying ecological divergence with gene flow are poorly understood. Sympatric timing types of the intertidal insect Clunio marinus (Diptera) from Roscoff (France) differ in lunar reproductive timing. One type reproduces at full moon, the other at new moon, controlled by a circalunar clock of yet unknown molecular nature. Lunar reproductive timing is a magic trait for a sympatric speciation process, as it is both ecologically relevant and entails assortative mating. Here, we show that the difference in reproductive timing is controlled by at least four quantitative trait loci (QTL) on three different chromosomes. They are partly associated with complex inversions, but differentiation of the inversion haplotypes cannot explain the different phenotypes. The most differentiated locus in the entire genome, with QTL support, is the period locus, implying that this gene could not only be involved in circadian timing but also in lunar timing. Our data indicate that magic traits can be based on an oligogenic architecture and can be maintained by selection on several unlinked loci., Competing Interests: DB, CP, TK No competing interests declared, (© 2023, Briševac et al.)

Adaptive ecotype formation can be the first step to speciation, but the genetic underpinnings of this process are poorly understood. Marine midges of the genus Clunio (Diptera) have recolonized Northern European shore areas after the last glaciation. In response to local tide conditions they have formed different ecotypes with respect to timing of adult emergence, oviposition behavior and larval habitat. Genomic analysis confirms the recent establishment of these ecotypes, reflected in massive haplotype sharing between ecotypes, irrespective of whether there is ongoing gene flow or geographic isolation. QTL mapping and genome screens reveal patterns of polygenic adaptation from standing genetic variation. Ecotype-associated loci prominently include circadian clock genes, as well as genes affecting sensory perception and nervous system development, hinting to a central role of these processes in ecotype formation. Our data show that adaptive ecotype formation can occur rapidly, with ongoing gene flow and largely based on a re-assortment of existing alleles., Competing Interests: TK, NF, CP No competing interests declared, (© 2023, Kaiser et al.)

Circalunar clocks, which allow organisms to time reproduction to lunar phase, have been experimentally proven but are still not understood at the molecular level. Currently, a new generation of researchers with new tools is setting out to fill this gap. Our essay provides an overview of classic experiments on circalunar clocks. From the unpublished work of the late D. Neumann we also present a novel phase response curve for a circalunar clock. These experiments highlight avenues for molecular work and call for rigor in setting up and analyzing the logistically complex experiments on circalunar clocks. Re-evaluating classic experiments, we propose that (1) circalunar clocks in different organisms will have divergent mechanisms and physiological bases, (2) they may have properties very different from the well-studied circadian clocks and (3) they may have close mechanistic and molecular relations to seasonal rhythms and diapause., (© 2021 The Authors. BioEssays published by Wiley Periodicals LLC.)

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.

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., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

An animal's fitness strongly depends on successful feeding, avoidance of predators and reproduction. All of these behaviours commonly involve chemosensation. As a consequence, when species' ecological niches and life histories differ, their chemosensory abilities need to be adapted accordingly. The intertidal insect Clunio marinus (Diptera: Chironomidae) has tuned its olfactory system to two highly divergent niches. The long-lived larvae forage in a marine environment. During the few hours of terrestrial adult life, males have to find the female pupae floating on the water surface, free the cryptic females from their pupal skin, copulate and carry the females to the oviposition sites. In order to explore the possibility for divergent olfactory adaptations within the same species, we investigated the chemosensory system of C. marinus larvae, adult males and adult females at the morphological and molecular level. The larvae have a well-developed olfactory system, but olfactory gene expression only partially overlaps with that of adults, likely reflecting their marine vs. terrestrial lifestyles. The olfactory system of the short-lived adults is simple, displaying no glomeruli in the antennal lobes. There is strong sexual dimorphism, the female olfactory system being particularly reduced in terms of number of antennal annuli and sensilla, olfactory brain centre size and gene expression. We found hints for a pheromone detection system in males, including large trichoid sensilla and expression of specific olfactory receptors and odorant binding proteins. Taken together, this makes C. marinus an excellent model to study within-species evolution and adaptation of chemosensory systems.

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing., Competing Interests: The authors declare no competing financial interests.

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., Competing Interests: The authors have declared that no competing interests exist.

Circadian rhythms pre-adapt the physiology of most organisms to predictable daily changes in the environment. Some marine organisms also show endogenous circalunar rhythms. The genetic basis of the circalunar clock and its interaction with the circadian clock is unknown. Both clocks can be studied in the marine midge Clunio marinus (Chironomidae, Diptera), as different populations have different local adaptations in their lunar and diurnal rhythms of adult emergence, which can be analyzed by crossing experiments. We investigated the genetic basis of population variation in clock properties by constructing the first genetic linkage map for this species, and performing quantitative trait locus (QTL) analysis on variation in both lunar and diurnal timing. The genome has a genetic length of 167-193 centimorgans based on a linkage map using 344 markers, and a physical size of 95-140 megabases estimated by flow cytometry. Mapping the sex determining locus shows that females are the heterogametic sex, unlike most other Chironomidae. We identified two QTL each for lunar emergence time and diurnal emergence time. The distribution of QTL confirms a previously hypothesized genetic basis to a correlation of lunar and diurnal emergence times in natural populations. Mapping of clock genes and light receptors identified ciliary opsin 2 (cOps2) as a candidate to be involved in both lunar and diurnal timing; cryptochrome 1 (cry1) as a candidate gene for lunar timing; and two timeless (tim2, tim3) genes as candidate genes for diurnal timing. This QTL analysis of lunar rhythmicity, the first in any species, provides a unique entree into the molecular analysis of the lunar clock.

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.

The marine midge Clunio marinus (Chironomidae, Diptera) is characterized by a one-dimensional distribution along the European Atlantic coast, where its lunar and circadian emergence rhythms are genetically adapted to the local tidal regimes, resulting in a series of 'temporal races'. Clunio marinus is restricted to rocky coasts and thus the temporal races occur in different rocky patches. We studied 10 populations of Clunio marinus from five different regions, spanning the major rocky mainland coasts from Spain to Norway, using amplified fragment length polymorphisms (AFLP), microsatellites and mitochondrial cytochrome oxidase I (COI) sequences. Star-like patterns of COI haplotypes within regions indicate postglacial colonization. A high degree of shared polymorphisms in AFLP markers suggests colonization from a single source, implying postglacial evolution of timing adaptations in relation to the local tidal regime. In contrast, no COI haplotypes are shared among regions. We hypothesize that different levels of differentiation of nuclear vs. mitochondrial markers in the source region were carried forward during postglacial expansion. Despite the recent origin of populations, all markers reveal distinct genetic differentiation between rocky coasts on a scale of 650-1150 km. Differentiation between rocky coasts is not correlated to timing adaptations, suggesting that geographic isolation is prevalent between rocky coasts and that this facilitated the evolution of local timing adaptations. At the same time there is little genetic differentiation within rocky coasts on a scale of 2-6 km; leaving open the possibility that within rocky coasts with large variation in tidal regimes, temporal adaptations evolved in the face of gene flow.

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., (© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.)