Phylogeography and genetic diversity of the Scapholeberis kingii species complex (Cladocera: Daphniidae) in China.

There is increasing interest in the diversity and phylogeography of aquatic invertebrate zooplankton in the Eastern Palearctic, yet this topic remains largely unexplored in China. Here, we investigated the lineage diversity and phylogeography of an important cladoceran taxon, the Scapholeberis kingii (Cladocera: Daphniidae) species complex, members of which live in the surface layers of freshwater ecosystems. We identified only the S. smirnovi morphospecies from this species complex in 29 of 491 Chinese water bodies examined. Its phylogenetic position was verified using both a mitochondrial (mitochondrial cytochrome c oxidase subunit I; COI) and a nuclear marker (the nuclear large subunit ribosomal RNA gene; 28S). Pronounced geographical separation among three S. smirnovi mitochondrial lineages was observed in China: only a single lineage (Lineage A) was present in the Eastern Plain, whereas Lineages B and C were restricted to the Inner Mongolia-Xinjiang Plateau and the Qinghai-Tibetan Plateau respectively. This deep mtDNA divergence and the substantial genetic differentiation among S. smirnovi populations from different regions is likely a result of the rapid uplift of the Qinghai-Tibetan Plateau and associated ecological changes. This study contributes to an understanding of the genetic diversity of the S. kingii complex, a key component of neustonic zooplankton.

Ancestral genetic variation in phenotypic plasticity underlies rapid evolutionary changes in resurrected populations of waterfleas.

The role of phenotypic plasticity in adaptive evolution has been debated for decades. This is because the strength of natural selection is dependent on the direction and magnitude of phenotypic responses to environmental signals. Therefore, the connection between plasticity and adaptation will depend on the patterns of plasticity harbored by ancestral populations before a change in the environment. Yet few studies have directly assessed ancestral variation in plasticity and tracked phenotypic changes over time. Here we resurrected historic propagules of Daphnia spanning multiple species and lakes in Wisconsin following the invasion and proliferation of a novel predator (spiny waterflea, Bythotrephes longimanus). This approach revealed extensive genetic variation in predator-induced plasticity in ancestral populations of Daphnia It is unlikely that the standing patterns of plasticity shielded Daphnia from selection to permit long-term coexistence with a novel predator. Instead, this variation in plasticity provided the raw materials for Bythotrephes-mediated selection to drive rapid shifts in Daphnia behavior and life history. Surprisingly, there was little evidence for the evolution of trait plasticity as genetic variation in plasticity was maintained in the face of a novel predator. Such results provide insight into the link between plasticity and adaptation and highlight the importance of quantifying genetic variation in plasticity when evaluating the drivers of evolutionary change in the wild.

Genome Assembly of a Relict Arabian Species of Daphnia O. F. Müller (Crustacea: Cladocera) Adapted to the Desert Life.

The water flea Daphnia O.F. Müller 1776 (Crustacea: Cladocera) is an important model of recent evolutionary biology. Here, we report a complete genome of Daphnia (Ctenodaphnia) arabica (Crustacea: Cladocera), recently described species endemic to deserts of the United Arab Emirates. In this study, genome analysis of D. arabica was carried out to investigate its genomic differences, complexity as well as its historical origins within the subgenus Daphnia (Ctenodaphnia). Hybrid genome assembly of D. arabica resulted in ~116 Mb of the assembled genome, with an N50 of ~1.13 Mb (BUSCO score of 99.2%). From the assembled genome, in total protein coding, 5374 tRNA and 643 rRNA genes were annotated. We found that the D. arabica complete genome differed from those of other Daphnia species deposited in the NCBI database but was close to that of D. cf. similoides. However, its divergence time estimate sets D. arabica in the Mesozoic, and our demographic analysis showed a great reduction in its genetic diversity compared to other Daphnia species. Interestingly, the population expansion in its diversity occurred during the megadrought climate around 100 Ka ago, reflecting the adaptive feature of the species to arid and drought-affected environments. Moreover, the PFAM comparative analysis highlights the presence of the important domain SOSS complex subunit C in D. arabica, which is missing in all other studied species of Daphnia. This complex consists of a few subunits (A, B, C) working together to maintain the genome stability (i.e., promoting the reparation of DNA under stress). We propose that this domain could play a role in maintaining the fitness and survival of this species in the desert environment. The present study will pave the way for future research to identify the genes that were gained or lost in this species and identify which of these were key factors to its adaptation to the harsh desert environment.

Ceriodaphnia (Cladocera: Daphniidae) in China: Lineage diversity, phylogeography and possible interspecific hybridization.

The distribution and species/lineage diversity of freshwater invertebrate zooplankton remains understudied in China. Here, we explored the species/lineage diversity and phylogeography of Ceriodaphnia species across China. The taxonomy of this genus is under-explored. Seven morphospecies of Ceriodaphnia (C. cornuta, C. laticaudata, C. megops, C. pulchella, C. quadrangula, C. rotunda and C. spinata) were identified across 45 of 422 water bodies examined. Rather little morphological variation was observed within any single morphospecies regardless of country of origin. Nevertheless, we recognized that some or all of these morphospecies might represent species complexes. To investigate this, phylogenetic relationships within and among these morphospecies were investigated based on mitochondrial (partial cytochrome c oxidase subunit I gene) and nuclear (partial 28S rRNA gene) markers. The mitochondrial marker placed these populations in nine lineages corresponding to the morphospecies: C. laticaudata and C. pulchella were each represented by two lineages, suggesting that both are species complexes. The remaining five morphospecies were each represented by a single mtDNA lineage. Three of the nine mitochondrial lineages (belonging to C. pulchella, C. rotunda and C. megops) are newly reported and exhibited a restricted distribution within China. The nuclear-DNA phylogeny also recognized seven Ceriodaphnia taxa within China. We detected occasional mito-nuclear discordances in Ceriodaphnia taxa across China, suggesting interspecific introgression and hybridization. Our study contributes to an understanding of the species/lineage diversity of Ceriodaphnia, a genus with understudied taxonomy.

Diversity of the Bosmina (Cladocera: Bosminidae) in China, revealed by analysis of two genetic markers (mtDNA 16S and a nuclear ITS).

BACKGROUND: China is an important biogeographical zone in which the genetic legacies of the Tertiary and Quaternary periods are abundant, and the contemporary geography environment plays an important role in species distribution. Therefore, many biogeographical studies have focused on the organisms of the region, especially zooplankton, which is essential in the formation of biogeographical principles. Moreover, the generality of endemism also reinforces the need for detailed regional studies of zooplankton. Bosmina, a group of cosmopolitan zooplankton, is difficult to identify by morphology, and no genetic data are available to date to assess this species complex in China. In this study, 48 waterbodies were sampled covering a large geographical and ecological range in China, the goal of this research is to explore the species distribution of Bosmina across China and to reveal the genetic information of this species complex, based on two genetic markers (a mtDNA 16S and a nuclear ITS). The diversity of taxa in the Bosmina across China was investigated using molecular tools for the first time. RESULTS: Two main species were detected in 35 waterbodies: an endemic east Asia B. fatalis, and the B. longirostris that has a Holarctic distribution. B. fatalis had lower genetic polymorphism and population differentiation than B. longirostris. B. fatalis was preponderant in central and eastern China, whereas B. longirostris was dominated in western China. The third lineage (B. hagmanni) was only detected in a reservoir (CJR) of eastern China (Guangdong province). Bosmina had limited distribution on the Tibetan plateau. CONCLUSIONS: This study revealed that the biogeography of Bosmina appear to be affected by historical events (Pleistocene glaciations) and contemporary environment (such as altitude, eutrophication and isolated habitat).

New lineages and old species: Lineage diversity and regional distribution of Moina (Crustacea: Cladocera) in China.

The distribution and genetic diversity of freshwater zooplankton is understudied in the Eastern Palearctic. Here, we explored the lineage diversity and regional distribution of the genus Moina in China. Members of this genus are often keystone components of freshwater ecosystems and have been frequently subjected to toxicological and physiological studies. Four species of Moina were identified, based on morphology, in 50 of 113 Chinese water bodies examined, and their phylogenetic position was analyzed using both a mitochondrial (mitochondrial cytochrome c oxidase subunit I; COI) and a nuclear marker (the nuclear internal transcribed spacer; ITS-1). Both molecular markers identified four clades corresponding broadly to the morphological species. Mitochondrial DNA analysis showed the presence of four species complexes with eleven lineages across China, five of which were new. However, some lineages (and even individual haplotypes) were widespread in Eurasia, suggesting an ability to disperse over long distances. In contrast, a few lineages exhibited restricted distributions. The nuclear phylogeny also recognized four species of Moina within China and seven very distinct clades. Interestingly, one specimen possessing Moina cf. micrura mtDNA had ITS-1 alleles of the M. cf. brachiata clade. This discordance between mtDNA and nuclear ITS-1 phylogenies is indicative of interspecific introgression and hybridization. Additionally, our COI phylogeny showed apparent paraphyly in two Moina species groups, suggesting introgression of their mitochondrial genomes. Our data shows the regional distribution/diversity of the Moina species complex in a Eurasian context.

Acute toxicity of bisphenol A and its structural analogues and transcriptional modulation of the ecdysone-mediated pathway in the brackish water flea Diaphanosoma celebensis.

Bisphenol A (BPA) is a representative endocrine disrupting chemical (EDC) that has estrogenic effects in aquatic animals. In recent years, due to the continuing usage of BPA, its analogues have been developed as alternative substances to replace its use. The molting process is a pivotal point in the development and reproduction of crustaceans. However, studies of the effects of EDCs on molting in crustaceans at the molecular level are scarce. In the present study, we examined the acute toxicity of BPA and its analogues bisphenol F (BPF) and S (BPS) to the brackish water flea Diaphanosoma celebensis. We further identified four ecdysteroid pathway - related genes (cyp314a1, EcRA, EcRB, and USP) in D. celebensis, and investigated the transcriptional modulation of these genes during molting and after exposure to BPA and its analogues for 48 h. Sequencing and phylogenetic analyses revealed that these four genes are highly conserved among arthropods and may be involved in development and reproduction in the adult stage. The mRNA expression patterns of cyp314a1, EcRA and USP were matched with the molting cycle, suggesting that these genes play a role in the molting process in the adult stage in cladocerans. Following relative real-time polymerase chain reaction (RT-PCR) analyses, BPA and its analogues were found to modulate the expression of each of these four genes differently, indicating that these compounds can disrupt the normal endocrine system function of D. celebensis. This study improves our understanding of the molecular mode of action of BPA and its analogues in D. celebensis.

Species and hybrids in the genus Diaphanosoma Fischer, 1850 (Crustacea: Branchiopoda: Cladocera).

Cladocerans are well-studied planktonic crustaceans, especially those of the genus Daphnia in which interesting evolutionary questions have been addressed on speciation processes. The aim of the present study is to demonstrate that other genera of cladocerans show similar levels of cryptic diversity, intraspecific gene flow, and thus become useful model systems for comparison. In order to do so, we chose the genus Diaphanosoma, widespread in tropical and temperate areas. We started with a survey of species diversity in the genus Diaphanosoma in Asia using a morphological approach, then obtained sequences from a mitochondrial and a nuclear marker from multiple individuals of different species, performed tests on DNA taxonomy and molecular phylogenies, and assessed the role of hybridization in explaining the cases of mitonuclear discordance. The results are that cryptic diversity occurs in Diaphanosoma, and mitonuclear discordance was found in about 6% of the sequenced animals. Past hybridization is supported as the most likely explanation for the discordance: no evidence was found of first generation hybrids with heterozygous sequences. Our analysis on patterns of genetic diversity in Diaphanosoma supports similarities and differences with what is known in Daphnia.

De novo transcriptome assembly of brackish water flea Diaphanosoma celebensis based on short-term cadmium and benzo[a]pyrene exposure experiments.

To develop a brackish water flea as a promising model for marine monitoring, Diaphanosoma celebensis were exposed to two pollutants, cadmium (Cd) and benzo[a]pyrene (BaP), which have different chemical characteristics and distinct modes of metabolic action on aquatic animals. Twenty-four hours after exposure to Cd (2 mg/L) or BaP (25 µg/L), whole body transcriptomes were analyzed. In total, 99.6 Mbp were assembled from nine libraries, resulting in 98,458 transcripts with an N50 of 1883 bp and an average contig length of 968 bp. Functional gene annotations were performed using Gene Ontology, Eukaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Cd significantly modulated endocrine and digestive enzyme system. Following BaP treatment, DNA repair and circadian rhythm related metabolisms were significantly modulated. Both the chemicals induced stress response and detoxification metabolism. This brackish water flea genomic information will be useful to monitor estuaries and coastal regions, as water fleas have been confirmed as promising sentinel models in freshwater ecosystems.

Comparative Transcriptome Analysis for Understanding Predator-Induced Polyphenism in the Water Flea Daphnia pulex.

The crustacean Daphnia pulex is one of the best model organisms for studying inducible defense mechanisms due to their inducible morphology in response to the predator Chaoborus larvae. In this study, multiple developmental stages of D. pulex were exposed to C. flavicans larvae and transcriptome profiles of samples from late embryo to fifth instar were sequenced by the RNA-seq technique to investigate the genetic background underlying inducible defenses. In comparison, differentially expressed genes between defensive and normal morphs were identified, including 908 genes in late embryo, 1383 genes in the first-third (1⁻3) instar, and 1042 genes in fourth-fifth (4⁻5) instar. Gene ontology enrichment analysis showed that structural constituents of the cuticle and structural molecule activity genes were prominent up-regulated genes in late embryos. Down-regulated genes in late embryos and 1⁻3 instar comprised metabolic process, hydrolase activity, and peptidase activity gene classes. Pathway analysis indicated that small molecule neurotransmitter pathways were potentially involved in the development of inducible defenses. The characterization of genes and pathways in multiple developmental stages can improve our understanding of inducible defense responses of D. pulex to predation at the molecular level.

Rapid evolution mitigates the ecological consequences of an invasive species (Bythotrephes longimanus) in lakes in Wisconsin.

Invasive species have extensive negative consequences for biodiversity and ecosystem health. Novel species also drive contemporary evolution in many native populations, which could mitigate or amplify their impacts on ecosystems. The predatory zooplankton Bythotrephes longimanus invaded lakes in Wisconsin, USA, in 2009. This invasion caused precipitous declines in zooplankton prey (Daphnia pulicaria), with cascading impacts on ecosystem services (water clarity). Here, we tested the link between Bythotrephes invasion, evolution in Daphnia and post-invasion ecological dynamics using 15 years of long-term data in conjunction with comparative experiments. Invasion by Bythotrephes is associated with rapid increases in the body size of Daphnia Laboratory experiments revealed that such shifts have a genetic component; third-generation laboratory-reared Daphnia from 'invaded' lakes are significantly larger and exhibit greater reproductive effort than individuals from 'uninvaded' lakes. This trajectory of evolution should accelerate Daphnia population growth and enhance population persistence. We tested this prediction by comparing analyses of long-term data with laboratory-based simulations, and show that rapid evolution in Daphnia is associated with increased population growth in invaded lakes.

NMDA receptor activation upstream of methyl farnesoate signaling for short day-induced male offspring production in the water flea, Daphnia pulex.

BACKGROUND: The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex. RESULTS: To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N-methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids. CONCLUSIONS: We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex. This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.

Molecular cloning of doublesex genes of four cladocera (water flea) species.

BACKGROUND: The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species. RESULTS: We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5' upstream regulatory elements are preserved in D. magna and D. pulex. CONCLUSIONS: The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.

Plastic response to a proxy cue of predation risk when direct cues are unreliable.

Responses to proximate cues that directly affect fitness or cues directly released by selective agents are well-documented forms of phenotypic plasticity. For example, to reduce predation risk, prey change phenotype in response to light level (e.g., moon phase) when light affects predation risk from visual predators, and to chemical cues (kairomones) released by predators. Less well understood is the potential for organisms to perceive predation risk through "proxy cues": proximate cues that correlate with, but do not directly affect predation risk. Previous field studies indicate that body and spine length of an invasive cladoceran in Lake Michigan, Bythotrephes longimanus (the spiny water flea), increase during the growing season, coincident with a decrease in clutch size. Although the cause of seasonal trait changes is not known, changes are associated with warmer water temperature and increased predation risk from gape-limited fish (i.e., fish whose ability to consume Bythotrephes is limited by mouth size). Using a laboratory experiment, we found no effect of fish (Perca flavescens) kairomones on Bythotrephes morphology or life history. In contrast, higher water temperature led to longer absolute spine and body length, increased investment in morphological defense of offspring (measured as the ratio of spine-to-body length), and decreased clutch size and age at reproduction. These plastic responses are unlikely to be adaptive to temperature per se, but rather our findings indicate that temperature serves as a proxy cue of fish predation risk. Temperature correlates with risk of gape-limited fish predation due to growth of fish from larval stages incapable of consuming Bythotrephes early in the season, to larger sizes by midseason increasingly capable of consuming Bythotrephes, but limited by gape size to consuming smaller individuals. We argue that for Bythotrephes, temperature is a more reliable cue of predation risk than fish kairomones, because fish kairomones are present throughout the season due to continual presence of non-gape-limited adult fish, to which plastic response would have little effect. Organisms may, therefore, not only respond to changes in an environmental factor because the factor directly affects risk, but also when the environmental factor serves as a proxy signaling change in predation risk.

Molecular approach to identify sibling species of the Ceriodaphnia cornuta complex (Cladocera: Daphniidae) from Australia with notes on the continental endemism of this group.

Taxonomy of the genus Ceriodaphnia Dana, 1853 (Cladocera: Daphniidae) has been uncertain for a long time, the species richness was often underestimated due to (1) a morphological similarity among the species and (2) their great morphological inter- and intra- populational variability. Support for this conclusion comes from the first analysis of three molecular markers for Australian representatives of this genus, two mitochondrial (COI and 16s) and one nuclear (28s) genes. Sequence analysis indicates the existence of three sibling Australian species belonging to the complex. Further work is required to establish species boundaries and investigate potential morphological diagnoses. Comparison of COI sequences with all other published sequences from the genus Ceriodaphnia revealed no common clades among continents pointing to the regional endemism within this group, which could suggest its Mesozoic differentiation.

Micromoina arboricola n. gen., n. spec. (Crustacea: Cladocera), a new moinid living in a forest tree-hole in Minas Gerais, Brazil.

With a maximum size of ca 0.5 mm, Micromoina arboricola is among the smallest moinids known to date. It was discovered in a flooded treehole in a forest in the Medio Rio Doce Valley, Minas Gerais, Brazil, where it mainly feeds on particulate organic matter derived from the vinhatico tree . However, it is easily cultured in the lab on a diet of green algae plus yeast and pelleted fish food. Structurally, it is a miniature version of a moinid, distinguished by characters on the antennules (both sexes) and the postabdomen. The latter is peculiar in shape, in lacking a basal spine, and in having only three lateral plumose setae. A comparative investigation of the barcoding fragment of the COI gene in a number of moinids confirms the family Moinidae as composed of several genera, as well as the status of the new taxon.

De Novo Transcriptomic and Life-History Responses of Moina Micrura Under Stress Environment Conditions.

Moina micrura represents a promising model species for ecological and ecotoxicological investigations in tropical freshwater ecosystems. Illumina NovaSeq™ 6000 sequencing was employed in this study to analyze M. micrura across three distinct developmental stages: juvenile, adult, and male. Current study successfully annotated 51,547 unigenes (73.11%) derived from seven (7) different databases. A total of 554 genes were found to be significantly upregulated, while 452 genes showed significant downregulation between juvenile and male. Moreover, 1001 genes were upregulated, whereas 830 genes exhibited downregulation between the adult and male. Analysis of differentially expressed genes revealed upregulation of chitin, cuticle, myosin (MYO), mitogen-activated protein kinases (MAPK), fibrillin (FBN), cytochrome (CYP), glutathione s-transferase (GST), vitellogenin (VTG), acetylcholinesterase (AChE), and transforming growth factor beta (TGFB) under unfavorable environmental conditions (male), as compared to favorable environmental conditions (juveniles and adults). These alterations in gene expression significantly impact the phenological and life-history traits of M. micrura. Furthermore, the upregulation of hemoglobin (HMB), doublesex (DSX), juvenile hormone analogs (JHA), heat shock protein (HSP), and methyltransferase (METT) genes in males initiates the sex-switching effects observed in M. micrura. These findings hold substantial value for researchers interested in determining M. micrura sequences for future investigations of gene expression and comparative reproductive genome analysis within the Moina genus and cladoceran families.

Chromosome-scale genome assembly of the freshwater cladoceran crustacean Chydorus sphaericus: A resource for discovery of genes responsive to ecological challenges.

The genus Chydorus Leach 1816 (family Chydoridae) is a diverse and ecologically important taxon within freshwater ecosystems. Despite having been widely used in ecological, evolutionary and eco-toxicological studies, no high-quality genomic resource is available for any member of the genus. Here, we present a high-quality chromosome-level assembly of the C. sphaericus genome by combining 7.40 Gb (∼ 50 × coverage) PacBio reads, 19.28 Gb (∼ 135 × coverage) Illumina paired-end reads, and 34.04 Gb Hi-C reads. Our genome assembly is approximately 151 Mb, with contig and scaffold N50 lengths of 1.09 Mb and 13.70 Mb, respectively. The assembly captured 94.9% of the complete eukaryotic BUSCO. Repetitive elements accounted for 17.6% of the genome, and 13,549 protein-coding genes were predicted (based on transcriptome sequencing data, ab-initio or homology-based prediction), of which 96.4% have been functionally annotated in the NCBI-NR database. We identified 303 gene families specific to C. sphaericus, mainly families enriched in functions related to immune response, visual senses and detoxification. Interestingly, we also found 53 significantly expanded gene families in C. sphaericus, mostly with functions related to detoxification. This high-quality assembly genome will act as a reference genome for C. sphaericus and benefit studies on functional and comparative genomics of Chydorus and other crustaceans.

Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans.

Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete-scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete-scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and division pattern. We show for the first time that branchiopod neuroblasts divide in the same pattern as insect and malacostracan neuroblasts. Furthermore, in contrast to D. melanogaster, neuroblasts are not selected from proneural clusters in the branchiopod. Snail rather than ASH is the first gene to be expressed in the nascent neuroblasts suggesting that ASH is not required for the selection of neuroblasts as in D. melanogaster. The prolonged expression of ASH in D. magna furthermore suggests that it is involved in the maintenance of the neuroblasts in the neuroepithelium. Based on these and additional data from various representatives of arthropods we conclude that the selection of neural precursors from proneural clusters as well as the segregation of neural precursors represents the ancestral state of neurogenesis in arthropods. We discuss that the derived characters of malacostracans and branchiopods - the absence of neuroblast segregation and proneural clusters - might be used to support or reject the possible groupings of paraphyletic crustaceans.

Genome-wide identification of 194 G protein-coupled receptor (GPCR) genes from the water flea Daphnia magna.

In crustaceans, G protein-coupled receptors (GPCRs) are the largest transmembrane receptor family and function by mediating various environmental stimuli in cells. Understanding GPCR signaling is crucial to better understanding of crustacean endocrinology. GPCRs evolved from early eukaryotes, and genome-wide identification of GPCRs in a particular taxon can provide insight into evolutionary tendencies and adaptive strategies of GPCR response to environmental stimuli. Here, we identified 194 full-length GPCR genes in the water flea Daphnia magna that can be divided into five distinct classes (A, B, C, F, and other). A strong orthologous relationship for amine, neuropeptide, and opsin receptors was found in the phylogenetic comparison of D. magna GPCRs to those of humans and two well-known insects (Drosophila melanogaster and Solenopsis invicta). Our results based on phylogenetic relationships suggest that most GPCRs subfamilies have undergone sporadic evolutionary processes for adaptation to environmental pressures. Despite the dynamics of GPCR evolution, some GPCRs are highly conserved between species. This study provides a better understanding of the evolution of GPCRs and expands our knowledge of the potential physiological mechanisms in D. magna in response to various environmental stimuli.