Molecular Clock Research Articles

Overlapping Central Clock Network Circuitry Regulates Circadian Feeding and Activity Rhythms in Drosophila.

The circadian system coordinates multiple behavioral outputs to ensure proper temporal organization. Timing information underlying circadian regulation of behavior depends on a molecular circadian clock that operates within clock neurons in the brain. In Drosophila and other organisms, clock neurons can be divided into several molecularly and functionally discrete subpopulations that form an interconnected central clock network. It is unknown how circadian signals are coherently generated by the clock network and transmitted across output circuits that connect clock cells to downstream neurons that regulate behavior. Here, we have exhaustively investigated the contribution of clock neuron subsets to the control of two prominent behavioral outputs in Drosophila: locomotor activity and feeding. We have used cell-specific manipulations to eliminate molecular clock function or induce electrical silencing either broadly throughout the clock network or in specific subpopulations. We find that clock cell manipulations produce similar changes in locomotor activity and feeding, suggesting that overlapping central clock circuitry regulates these distinct behavioral outputs. Interestingly, the magnitude and nature of the effects depend on the clock subset targeted. Lateral clock neuron manipulations profoundly degrade the rhythmicity of feeding and activity. In contrast, dorsal clock neuron manipulations only subtly affect rhythmicity but produce pronounced changes in the distribution of activity and feeding across the day. These experiments expand our knowledge of clock regulation of activity rhythms and offer the first extensive characterization of central clock control of feeding rhythms. Despite similar effects of central clock cell disruptions on activity and feeding, we find that manipulations that prevent functional signaling in an identified output circuit preferentially degrade locomotor activity rhythms, leaving feeding rhythms relatively intact. This demonstrates that activity and feeding are indeed dissociable behaviors, and furthermore suggests that differential circadian control of these behaviors diverges in output circuits downstream of the clock network.

Modulation of Neuronal Excitability and Plasticity by BHLHE41 Conveys Lithium Non-Responsiveness.

Many bipolar disorder (BD) patients are non-responsive to lithium. The mechanisms underlying lithium (non-)responsiveness are largely unknown. By using gene-set enrichment analysis methods, we found that core clock gene-sets are significantly associated with lithium response. Among the top hits was BHLHE41, a modulator of the molecular clock and homeostatic sleep. Since BHLHE41 and its paralog BHLHE40 are functionally redundant, we assessed chronic lithium response in double-knockout mutant mice (DKO). We demonstrated that DKOs are non-responsive to lithium's effect in various behavioral tasks. Cellular assays and patch clamp recordings revealed lowered excitability and reduced lithium-response in prefrontal cortical layer 2/3 DKO neurons and on hippocampal long-term potentiation. Single-cell RNA sequencing identified that lithium deregulated mitochondrial respiration, cation channel and postsynapse associated gene-sets specifically in upper layer excitatory neurons. Our findings show that lithium acts in a highly cell-specific way on neuronal metabolism and excitability and modulates synaptic plasticity depending on BHLHE40/41.

Functional and regulatory diversification of Period genes responsible for circadian rhythm in vertebrates.

The Period genes (Per) play essential roles in modulating the molecular circadian clock timing in a broad range of species, which regulates the physiological and cellular rhythms through the transcription-translation feedback loop. While the Period gene paralogs are widely observed among vertebrates, the evolutionary history and the functional diversification of Per genes across vertebrates are not well known. In this study, we comprehensively investigated the evolution of Per genes at the copy number and sequence levels, including de novo binding motif discovery by comparative genomics. We also determined the lineage-specific transcriptome landscape across tissues and developmental stages and phenotypic effects in public RNA-seq data sets of model species. We observed multiple lineage-specific gain and loss events Per genes, though no simple association was observed between ecological factors and Per gene numbers in each species. Among salmonid fish species, the per3 gene has been lost in the majority, whereas those retaining the per3 gene exhibit not a signature of relaxed selective constraint but rather a signature of intensified selection. We also determined the signature of adaptive diversification of the CRY-binding region in Per1 and Per3, which modulates the circadian rhythm. We also discovered putative regulatory sequences, which are lineage-specific, suggesting that these cis-regulatory elements may have evolved rapidly and divergently across different lineages. Collectively, our findings revealed the evolution of Per genes and their fine-tuned contribution to the plastic and precise regulation of circadian rhythms in various vertebrate taxa.

Whole genome phylogenomics helps to resolve the phylogenetic position of the Zygothrica genus group (Diptera, Drosophilidae) and the causes of previous incongruences

Incomplete Lineage Sorting (ILS) and introgression are among the two main factors causing incongruence between gene and species trees. Advances in phylogenomic studies have allowed us to overcome most of these issues, providing reliable phylogenetic hypotheses while revealing the underlying evolutionary scenario. Across the last century, many incongruent phylogenetic reconstructions were recovered for Drosophilidae, employing a limited sampling of genetic markers or species. In these studies, the monophyly and the phylogenetic positioning of the Zygothrica genus group stood out as one of the most controversial questions. Thus, here, we addressed these issues using a phylogenomic approach, while accessing the influence of ILS and introgressions on the diversification of these species and addressing the spatio-temporal scenario associated with their evolution. For this task, the genomes of nine specimens from six Neotropical species belonging to the Zygothrica genus group were sequenced and evaluated in a phylogenetic framework encompassing other 39 species of Drosophilidae. Nucleotide and amino acid sequences recovered for a set of 2,534 single-copy genes by BUSCO were employed to reconstruct maximum likelihood (ML) concatenated and multi-species coalescent (MSC) trees. Likelihood mapping, quartet sampling, and reticulation tests were employed to infer the level and causes of incongruence. Lastly, a penalized-likelihood molecular clock strategy with fossil calibrations was performed to infer divergence times. Taken together, our results recovered the subdivision of Drosophila into six different lineages, one of which clusters species of the Zygothrica genus group (except for H. duncani). The divergence of this lineage was dated to Oligocene ∼ 31 Mya and seems to have occurred in the same timeframe as other key diversification within Drosophila. According to the concatenated and MSC strategies, this lineage is sister to the clade joining Drosophila (Siphlodora) with the Hawaiian Drosophila and Scaptomyza. Likelihood mapping, quartet sampling, reticulation reconstructions as well as introgression tests revealed that this lineage was the target of several hybridization events involving the ancestors of different Drosophila lineages. Thus, our results generally show introgression as a major source of previous incongruence. Nevertheless, the similar diversification times recovered for several of the Neotropical Drosophila lineages also support the scenario of multiple and simultaneous diversifications taking place at the base of Drosophilidae phylogeny, at least in the Neotropics.

Amoebozoan testate amoebae illuminate the diversity of heterotrophs and the complexity of ecosystems throughout geological time

Heterotrophic protists are vital in Earth's ecosystems, influencing carbon and nutrient cycles and occupying key positions in food webs as microbial predators. Fossils and molecular data suggest the emergence of predatory microeukaryotes and the transition to a eukaryote-rich marine environment by 800 million years ago (Ma). Neoproterozoic vase-shaped microfossils (VSMs) linked to Arcellinida testate amoebae represent the oldest evidence of heterotrophic microeukaryotes. This study explores the phylogenetic relationship and divergence times of modern Arcellinida and related taxa using a relaxed molecular clock approach. We estimate the origin of nodes leading to extant members of the Arcellinida Order to have happened during the latest Mesoproterozoic and Neoproterozoic (1054 to 661 Ma), while the divergence of extant infraorders postdates the Silurian. Our results demonstrate that at least one major heterotrophic eukaryote lineage originated during the Neoproterozoic. A putative radiation of eukaryotic groups (e.g., Arcellinida) during the early-Neoproterozoic sustained by favorable ecological and environmental conditions may have contributed to eukaryotic life endurance during the Cryogenian severe ice ages. Moreover, we infer that Arcellinida most likely already inhabited terrestrial habitats during the Neoproterozoic, coexisting with terrestrial Fungi and green algae, before land plant radiation. The most recent extant Arcellinida groups diverged during the Silurian Period, alongside other taxa within Fungi and flowering plants. These findings shed light on heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems, using testate amoebae as a proxy.

The role of N6-methyladenosine RNA methylation in the crosstalk of circadian clock and neuroinflammation in rodent suprachiasmatic nuclei.

N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark that regulates the fate of RNA molecules. Recent studies have revealed a bidirectional interaction between m6A modification and the circadian clock. However, the precise temporal dynamics of m6A global enrichment in the central circadian pacemaker have not been fully elucidated. Our study investigates the relationship between FTO demethylase and molecular clocks in primary cells of the suprachiasmatic nucleus (SCN). In addition, we examined the effects of lipopolysaccharide (LPS) on Fto expression and the role of FTO in LPS-induced reactive oxygen species (ROS) production in primary SCN cell culture. We observed circadian rhythmicity in the global m6A levels, which mirrored the rhythmic expression of the Fto demethylase. Silencing FTO using siRNA reduced the mesor of Per2 rhythmicity in SCN primary cells and extended the period of the PER2 rhythm in SCN primary cell cultures from PER2::LUC mice. When examining the immune response, we discovered that exposure to LPS upregulated global m6A levels while downregulating Fto expression in SCN primary cell cultures. Interestingly, we found a loss of circadian rhythmicity in Fto expression following LPS treatment, indicating that the decrease of FTO levels may contribute to m6A upregulation without directly regulating its circadian rhythm. To explore potential protective mechanisms against neurotoxic inflammation, we examined ROS production following LPS treatment in SCN primary cell cultures pretreated with FTO siRNA. We observed a time-dependent pattern of ROS induction, with significant peak at 32 h but not at 20 h after synchronization. Silencing the FTO demethylase abolished ROS induction following LPS exposure, supporting the hypothesis that FTO downregulation serves as a protective mechanism during LPS-induced neuroinflammation in SCN primary cell cultures.

Molecular circadian clock disruption in the leukocytes of individuals with type 2 diabetes and overweight, and its relationship with leukocyte–endothelial interactions

Aims/hypothesisAlterations in circadian rhythms increase the likelihood of developing type 2 diabetes and CVD. Circadian rhythms are controlled by several core clock genes, which are expressed in nearly every cell, including immune cells. Immune cells are key players in the pathophysiology of type 2 diabetes, and participate in the atherosclerotic process that underlies cardiovascular risk in these patients. The role of the core clock in the leukocytes of people with type 2 diabetes and the inflammatory process associated with it are unknown. We aimed to evaluate whether the molecular clock system is impaired in the leukocytes of type 2 diabetes patients and to explore the mechanism by which this alteration leads to an increased cardiovascular risk in this population.MethodsThis is an observational cross-sectional study performed in 25 participants with type 2 diabetes and 28 healthy control participants. Clinical and biochemical parameters were obtained. Peripheral blood leukocytes were isolated using magnetic bead technology. RNA and protein lysates were obtained to assess clock-related gene transcript and protein levels using real-time PCR and western blot, respectively. Luminex XMAP technology was used to assess levels of inflammatory markers. Leukocyte–endothelial interaction assays were performed by perfusing participants’ leukocytes or THP-1 cells (with/without CLK8) over a HUVEC monolayer in a parallel flow chamber using a dynamic adhesion system.ResultsParticipants with type 2 diabetes showed increased BMAL1 and NR1D1 mRNA levels and decreased protein levels of circadian locomotor output cycles kaput (CLOCK), cryptochrome 1 (CRY1), phosphorylated basic helix-loop-helix ARNT like 1 (p-BMAL1) and period circadian protein homologue 2 (PER2). Correlation studies revealed that these alterations in clock proteins were negatively associated with glucose, HbA1c, insulin and HOMA-IR levels and leukocyte cell counts. The leukocyte rolling velocity was reduced and rolling flux and adhesion were enhanced in individuals with type 2 diabetes compared with healthy participants. Interestingly, inhibition of CLOCK/BMAL1 activity in leukocytes using the CLOCK inhibitor CLK8 mimicked the effects of type 2 diabetes on leukocyte–endothelial interactions.Conclusions/interpretationOur study demonstrates alterations in the molecular clock system in leukocytes of individuals with type 2 diabetes, manifested in increased mRNA levels and decreased protein levels of the core clock machinery. These alterations correlated with the impaired metabolic and proinflammatory profile of the participants with type 2 diabetes. Our findings support a causal role for decreased CLOCK/BMAL1 activity in the increased level of leukocyte–endothelial interactions. Overall, our data suggest that alterations in core clock proteins accelerate the inflammatory process, which may ultimately precipitate the onset of CVD in patients with type 2 diabetes.Graphical

Importance of Circadian Rhythms in the Ocular Surface.

Circadian rhythms are a ubiquitous feature throughout the organism. Accumulating evidence suggests that the dysfunction of circadian rhythms due to genetic mutations or environmental factors contributes to the genesis and progress of multiple diseases. The physiological homeostasis of the ocular surface, like any other tissue or organ, is also orchestrated by circadian rhythms. In this review, we summarize the molecular clocks and the expression of clock-controlled genes in the mammalian ocular surface. Based on the circadian expression of these genes, we conclude the diurnal oscillations of cellular biological activities in the mammalian ocular surface. Moreover, we evaluate the factors entraining circadian oscillators in the ocular surface. Finally, we further discuss the latest development of the close correlation between circadian rhythms and ocular health. Briefly, this review aimed to synthesize the previous studies to aid in understanding the importance of circadian rhythms in the ocular surface and the possible opportunities for circadian rhythm-based interventional strategies to restore the homeostasis of the ocular surface.

Morphometrics and Phylogenomics of Coca (Erythroxylum spp.) Illuminate Its Reticulate Evolution, With Implications for Taxonomy.

South American coca (Erythroxylum coca and E. novogranatense) has been a keystone crop for many Andean and Amazonian communities for at least 8,000 years. However, over the last half-century, global demand for its alkaloid cocaine has driven intensive agriculture of this plant and placed it in the center of armed conflict and deforestation. To monitor the changing landscape of coca plantations, the United Nations Office on Drugs and Crime collects annual data on their areas of cultivation. However, attempts to delineate areas in which different varieties are grown have failed due to limitations around identification. In the absence of flowers, identification relies on leaf morphology, yet the extent to which this is reflected in taxonomy is uncertain. Here, we analyze the consistency of the current naming system of coca and its four closest wild relatives (the "coca clade"), using morphometrics, phylogenomics, molecular clocks, and population genomics. We include name-bearing type specimens of coca's closest wild relatives E. gracilipes and E. cataractarum. Morphometrics of 342 digitized herbarium specimens show that leaf shape and size fail to reliably discriminate between species and varieties. However, the statistical analyses illuminate that rounder and more obovate leaves of certain varieties could be associated with the subtle domestication syndrome of coca. Our phylogenomic data indicate extensive gene flow involving E. gracilipes which, combined with morphometrics, supports E. gracilipes being retained as a single species. Establishing a robust evolutionary-taxonomic framework for the coca clade will facilitate the development of cost-effective genotyping methods to support reliable identification.

Searching for new fundamental interactions via isotopic shifts in molecular lattice clocks

Precision measurements with ultracold atoms and molecules are primed to probe beyond-the-standard model physics. Isotopologues of homonuclear molecules are a natural testbed for new Yukawa-type mass-dependent forces at nanometer scales, complementing existing mesoscopic-body and neutron scattering experiments. Here, we propose using isotopic shift measurements in molecular lattice clocks to constrain these new interactions from new massive scalar particles in the keV/c2 range: The new interaction would impart an extra isotopic shift to molecular levels on top of one predicted by the standard model. For the strontium dimer, a Hz-level agreement between experiment and theory could constrain the coupling of the new particles to hadrons by up to an order of magnitude over the most stringent existing experiments. Published by the American Physical Society 2024

Human primary cells can tell body time: Dedicated to Steven A. Brown.

The field of chronobiology has advanced significantly since ancient observations of natural rhythms. The intricate molecular architecture of circadian clocks, their hierarchical organization within the mammalian body, and their pivotal roles in organ physiology highlight the complexity and significance of these internal timekeeping mechanisms. In humans, circadian phenotypes exhibit considerable variability among individuals and throughout the individual's lifespan. A fundamental challenge in mechanistic studies of human chronobiology arises from the difficulty of conducting serial sampling from most organs. The concept of studying circadian clocks in vitro relies on the groundbreaking discovery by Ueli Schibler and colleagues that nearly every cell in the body harbours autonomous molecular oscillators. The advent of circadian bioluminescent reporters has provided a new perspective for this approach, enabling high-resolution continuous measurements of cell-autonomous clocks in cultured cells, following in vitro synchronization pulse. The work by Steven A. Brown has provided compelling evidence that clock characteristics assessed in primary mouse and human skin fibroblasts cultured in vitro represent a reliable estimation of internal clock properties in vivo. The in vitro approach for studying molecular human clocks in cultured explants and primary cells, pioneered by Steve Brown, represents an invaluable tool for assessing inter-individual differences in circadian characteristics alongside comprehensive genetic, biochemical and functional analyses. In a broader context, this reliable and minimally invasive approach offers a unique perspective for unravelling the functional inputs and outputs of oscillators operative in nearly any human tissue in physiological contexts and across various pathologies.

Lateral redox variability in ca. 1.9 Ga marine environments indicated by organic carbon and nitrogen isotope compositions.

The stepwise oxygenation of Earth's surficial environment is thought to have shaped the evolutionary history of life. Microfossil records and molecular clocks suggest eukaryotes appeared during the Paleoproterozoic, perhaps shortly after the Great Oxidation Episode at ca. 2.43 Ga. The mildly oxygenated atmosphere and surface oceans likely contributed to the early evolution of eukaryotes. However, the principal trigger for the eukaryote appearance and a potential factor for their delayed expansion (i.e., intermediate ocean redox conditions until the Neoproterozoic) remain poorly understood, largely owing to a lack of constraints on marine and terrestrial nutrient cycling. Here, we analyzed redox-sensitive element contents and organic carbon and nitrogen isotope compositions of relatively low metamorphic-grade (greenschist facies) black shales preserved in the Flin Flon Belt of central Canada to examine open-marine redox conditions and biological activity around the ca. 1.9 Ga Flin Flon oceanic island arc. The black shale samples were collected from the Reed Lake area in the eastern part of the Flin Flon Belt, and the depositional site was likely distal from the Archean cratons. The black shales have low Al/Ti ratios and are slightly depleted in light rare-earth elements relative to the post-Archean average shale, which is consistent with a limited contribution from felsic igneous rocks in Archean upper continental crust. Redox conditions have likely varied between suboxic and euxinic at the depositional site of the studied section, as suggested by variable U/Al and Mo/Al ratios. Organic carbon and nitrogen isotope compositions of the black shales are approximately -23‰ and +13.7‰, respectively, and these values are systematically higher than those of broadly coeval continental margin deposits (approximately -30‰ for δ13Corg and +5‰ for δ15Nbulk). These elevated values are indicative of high productivity that led to enhanced denitrification (i.e., a high denitrification rate relative to nitrogen influx at the depositional site). Similar geochemical patterns have also been observed in the modern Peruvian oxygen minimum zone where dissolved nitrogen compounds are actively lost from the reservoir via denitrification and anammox, but the large nitrate reservoir of the deep ocean prevents exhaustion of the surface nitrate pool. Nitrogen must have been widely bioavailable in the ca. 1.9 Ga oceans, and its supply to upwelling zones must have supported habitable environments for eukaryotes, even in the middle of oceans around island arcs.

Molecular Dating of the Teleost Whole Genome Duplication (3R) Is Compatible With the Expectations of Delayed Rediploidization.

Vertebrate evolution has been punctuated by three whole genome duplication events that have been implicated causally in phenotypic evolution, from the origin of phenotypic novelties to explosive diversification. Arguably, the most dramatic of these is the 3R whole genome duplication event associated with the origin of teleost fishes which comprise more than half of all living vertebrate species. However, tests of a causal relationship between whole genome duplication and teleost diversification have proven difficult due to the challenge of establishing the timing of these phenomena. Here we show, based on molecular clock dating of concatenated gene alignments, that the 3R whole genome duplication event occurred in the early-middle Permian (286.18 to 267.20 million years ago; Ma), 52.02 to 12.84 million years (Myr) before the divergence of crown-teleosts in the latest Permian-earliest Late Triassic (254.36 to 234.16 Ma) and long before the major pulses of teleost diversification in Ostariophysi and Percomorpha (56.37 to 100.17 Myr and at least 139.24 to 183.29 Myr later, respectively). The extent of this temporal gap between putative cause and effect precludes 3R as a deterministic driver of teleost diversification. However, these age constraints remain compatible with the expectations of a prolonged rediploidization process following whole genome duplication which, through the effects of chromosome rearrangement and gene loss, remains a viable mechanism to explain the evolution of teleost novelties and diversification.

Pharmacological Modulation of the Cytosolic Oscillator Affects Glioblastoma Cell Biology

The circadian system is a conserved time-keeping machinery that regulates a wide range of processes such as sleep/wake, feeding/fasting, and activity/rest cycles to coordinate behavior and physiology. Circadian disruption can be a contributing factor in the development of metabolic diseases, inflammatory disorders, and higher risk of cancer. Glioblastoma (GBM) is a highly aggressive grade 4 brain tumor that is resistant to conventional therapies and has a poor prognosis after diagnosis, with a median survival of only 12–15 months. GBM cells kept in culture were shown to contain a functional circadian oscillator. In seeking more efficient therapies with lower side effects, we evaluated the pharmacological modulation of the circadian clock by targeting the cytosolic kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1 ε/δ (CK1ε/δ) with specific inhibitors (CHIR99021 and PF670462, respectively), the cryptochrome protein stabilizer (KL001), or circadian disruption after Per2 knockdown expression in GBM-derived cells. CHIR99021-treated cells had a significant effect on cell viability, clock protein expression, migration, and cell cycle distribution. Moreover, cultures exhibited higher levels of reactive oxygen species and alterations in lipid droplet content after GSK-3 inhibition compared to control cells. The combined treatment of CHIR99021 with temozolomide was found to improve the effect on cell viability compared to temozolomide therapy alone. Per2 disruption affected both GBM migration and cell cycle progression. Overall, our results suggest that pharmacological modulation or molecular clock disruption severely affects GBM cell biology.

Oxygenation of Earth's atmosphere induced metabolic and ecologic transformations recorded in the Lomagundi-Jatuli carbon isotopic excursion.

The oxygenation of Earth's atmosphere represents the quintessential transformation of a planetary surface by microbial processes. In turn, atmospheric oxygenation transformed metabolic evolution; molecular clock models indicate the diversification and ecological expansion of respiratory metabolisms in the several hundred million years following atmospheric oxygenation. Across this same interval, the geological record preserves 13C enrichment in some carbonate rocks, called the Lomagundi-Jatuli excursion (LJE). By combining data from geologic and genomic records, a self-consistent metabolic evolution model emerges for the LJE. First, fermentation and methanogenesis were major processes remineralizing organic carbon before atmospheric oxygenation. Once an ozone layer formed, shallow water and exposed environments were shielded from UVB/C radiation, allowing the expansion of cyanobacterial primary productivity. High primary productivity and methanogenesis led to preferential removal of 12C into organic carbon and CH4. Extreme and variable 13C enrichments in carbonates were caused by 13C-depleted CH4 loss to the atmosphere. Through time, aerobic respiration diversified and became ecologically widespread, as did other new metabolisms. Respiration displaced fermentation and methanogenesis as the dominant organic matter remineralization processes. As CH4 loss slowed, dissolved inorganic carbon in shallow environments was no longer highly 13C enriched. Thus, the loss of extreme 13C enrichments in carbonates marks the establishment of a new microbial mat ecosystem structure, one dominated by respiratory processes distributed along steep redox gradients. These gradients allowed the exchange of metabolic by-products among metabolically diverse organisms, providing novel metabolic opportunities. Thus, the microbially induced oxygenation of Earth's atmosphere led to the transformation of microbial ecosystems, an archetypal example of planetary microbiology.IMPORTANCEThe oxygenation of Earth's atmosphere represents the most extensive known chemical transformation of a planetary surface by microbial processes. In turn, atmospheric oxygenation transformed metabolic evolution by providing oxidants independent of the sites of photosynthesis. Thus, the evolutionary changes during this interval and their effects on planetary-scale biogeochemical cycles are fundamental to our understanding of the interdependencies among genomes, organisms, ecosystems, elemental cycles, and Earth's surface chemistry through time.

The chloroplast genomes of two medicinal species (Veronica anagallis-aquatica L. and Veronica undulata Wall.) and its comparative analysis with related Veronica species

Veronica anagallis-aquatica L. and Veronica undulata Wall. are widely used ethnomedicinal plants in China. The two species have different clinical efficacies, while their extremely similar morphology and unclear interspecific relationship make it difficult to accurately identify them, leading to increased instances of mixed usage. This article reports on the complete chloroplast genomes sequence of these two species and their related Veronica species to conduct a comparative genomics analysis and phylogenetic construction. The results showed that the chloroplast (cp) genomes of Veronica exhibited typical circular quadripartite structures, with total lengths of 149,386 to 152,319 base pairs (bp), and GC content of 37.9 to 38.1%, and the number of genes was between 129–134. The total number of simple sequence repeats (SSRs) in V. anagallis-aquatica and V. undulata is 37 and 36, while V. arvensis had the highest total number of 56, predominantly characterized by A/T single bases. The vast majority of long repeat sequence types are forward repeats and palindromic repeats. Selective Ka/Ks values showed that three genes were under positive selection. Sequence differences often occur in the non-coding regions of the large single-copy region (LSC) and small single-copy region (SSC), with the lowest sequence variation in the inverted repeat regions (IR). Seven highly variable regions (trnT-GGU-psbD, rps8-rpl16, trnQ-UUG, trnN-GUU-ndhF, petL, ycf3, and ycf1) were detected, which may be potential molecular markers for identifying V. anagallis-aquatica and V. undulata. The phylogenetic tree indicates that there is a close genetic relationship between the genera Veronica and Neopicrorhiza, and V. anagallis-aquatica and V. undulata are sister groups. The molecular clock analysis results indicate that the divergence time of Veronica may occur at ∼ 9.09 Ma, and the divergence time of these two species occurs at ∼ 0.48 Ma. It is speculated that climate change may be the cause of Veronica species diversity and promote the radiation of the genus. The chloroplast genome data of nine Veronica specie provides important insights into the characteristics and evolution of the chloroplast genome of this genus, as well as the phylogenetic relationships of the genus Veronica.

Effects of chronic light cycle disruption during adolescence on circadian clock, neuronal activity rhythms, and behavior in mice.

The advent of artificial lighting, particularly during the evening and night, has significantly altered the predictable daily light and dark cycles in recent times. Altered light environments disrupt the biological clock and negatively impact mood and cognition. Although adolescents commonly experience chronic changes in light/dark cycles, our understanding of how the adolescents' brain adapts to altered light environments remains limited. Here, we investigated the impact of chronic light cycle disruption (LCD) during adolescence, exposing adolescent mice to 19 h of light and 5 h of darkness for 5 days and 12 L:12D for 2 days per week (LCD group) for 4 weeks. We showed that LCD exposure did not affect circadian locomotor activity but impaired memory and increased avoidance response in adolescent mice. Clock gene expression and neuronal activity rhythms analysis revealed that LCD disrupted local molecular clock and neuronal activity in the dentate gyrus (DG) and in the medial amygdala (MeA) but not in the circadian pacemaker (SCN). In addition, we characterized the photoresponsiveness of the MeA and showed that somatostatin neurons are affected by acute and chronic aberrant light exposure during adolescence. Our research provides new evidence highlighting the potential consequences of altered light environments during pubertal development on neuronal physiology and behaviors.

Contribution of neurons that express fruitless and Clock transcription factors to behavioral rhythms and courtship.

Animals need to integrate information across neuronal networks that direct reproductive behaviors and circadian rhythms. In Drosophila, the master regulatory transcription factors that direct courtship behaviors and circadian rhythms are co-expressed in a small set of neurons. In this study we investigate the role of these neurons in both males and females. We find sex-differences in the number of these fruitless and Clock -expressing neurons ( fru ∩ Clk neurons) that is regulated by male-specific Fru. We assign the fru ∩ Clk neurons to the electron microscopy connectome that provides high resolution structural information. We also discover sex-differences in the number of fru -expressing neurons that are post-synaptic targets of Clk -expressing neurons, with more post-synaptic targets in males. When fru ∩ Clk neurons are activated or silenced, males have a shorter period length. Activation of fru ∩ Clk neurons also changes the rate a courtship behavior is performed. We find that activation and silencing fru ∩ Clk neurons impacts the molecular clock in the sLNv master pacemaker neurons, in a cell-nonautonomous manner. These results reveal how neurons that subserve the two processes, reproduction and circadian rhythms, can impact behavioral outcomes in a sex-specific manner.

Diversity, morphology, and phylogeny of freshwater mussels of the genus Nodularia (Bivalvia: Unionidae) from China, with descriptions of four new species

AbstractFreshwater bivalves (Bivalvia, Unionida) are one of the most threatened groups of animals in the world. Defining species boundaries and understanding the phylogeny and genetic diversity of these species is key to guiding their conservation and management. However, the presence of significant phenotypic plasticity and convergence within this group complicates species delimitation. This includes the freshwater mussel genus Nodularia, endemic to East Asia, for which a comprehensive understanding of species diversity and phylogenetic relationships remains elusive due to inadequate sampling in previous studies, particularly in China, a widely recognized biodiversity hotspot for freshwater mussels. Here, we conduct comprehensive taxonomic and phylogenetic analyses of Nodularia species based on extensive sampling across 23 provinces in China and multiple data sources, including shell morphology, soft body anatomy, six‐gene (COI + ND1 + 16S + 18S + 28S + histone H3) and mitogenome datasets. The integrative systematics approach used here reveals 10 distinct species in this genus, four of which are new to science, i.e. Nodularia hanensis sp. nov., Nodularia huana sp. nov., Nodularia fusiformans sp. nov., Nodularia dualobtusus sp. nov. and two of which are new records for China, i.e. Nodularia dorri (Wattebled [Journal de Conchyliologie, 34, 1886, 54]) and Nodularia micheloti (Morlet [Journal de Conchyliologie, 34, 1886, 75]). We also propose that the nominal species Nodularia jourdyi (Morlet [Journal de Conchyliologie, 34, 1886, 75]) syn. nov. is a new synonym for Nodularia douglasiae (Griffith & Pidgeon, 1833) based on molecular data. BI, ML, and BEAST analyses based on the six‐gene dataset and mitochondrial phylogenomics consistently support the following phylogenetic relationships: (N. dorri + (N. hanensis sp. nov. + N. micheloti)) + (N. breviconcha + (N. huana sp. nov. + (N. fusiformans sp. nov. + ((N. nuxpersicae + N. nipponensis) + (N. dualobtusus sp. nov. + N. douglasiae))))). The molecular clock with fossil calibration indicates that Nodularia originated in the Late Cretaceous period (ca. 73.78 Mya). It then diverged into two independent clades during the Middle Paleogene (ca. 45.01 Mya), followed by a rapid burst of extant speciation during the Neogene (mean age 28.28 to 4.79 Mya). Nodularia breviconcha is the earliest differentiated taxon among the 10 Nodularia taxa, appearing during the Paleogene‐Neogene transition (28.28 Mya; 95% HPD = 14.35–48.44 Mya). Taken together, we provide a robust systematic framework for Nodularia species, addressing phylogenetic relationships, taxonomy, and evolutionary history of this group.

A somatic genetic clock for clonal species

Age and longevity are key parameters for demography and life-history evolution of organisms. In clonal species, a widespread life history among animals, plants, macroalgae and fungi, the sexually produced offspring (genet) grows indeterminately by producing iterative modules, or ramets, and so obscure their age. Here we present a novel molecular clock based on the accumulation of fixed somatic genetic variation that segregates among ramets. Using a stochastic model, we demonstrate that the accumulation of fixed somatic genetic variation will approach linearity after a lag phase, and is determined by the mitotic mutation rate, without direct dependence on asexual generation time. The lag phase decreased with lower stem cell population size, number of founder cells for the formation of new modules, and the ratio of symmetric versus asymmetric cell divisions. We calibrated the somatic genetic clock on cultivated eelgrass Zostera marina genets (4 and 17 years respectively). In a global data set of 20 eelgrass populations, genet ages were up to 1,403 years. The somatic genetic clock is applicable to any multicellular clonal species where the number of founder cells is small, opening novel research avenues to study longevity and, hence, demography and population dynamics of clonal species.