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.

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.

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.

Daphnia japonica sp. nov. (Crustacea: Cladocera) an eastern Palearctic montane species with mitochondrial discordance.

The Daphnia longispina complex (Crustacea: Cladocera) contains several keystone freshwater species such as D. longispina O.F. Müller (D. rosea Sars is a junior synonym), D. galeata Sars, D. cucullata Sars, and D. dentifera Forbes. The complex is common throughout the Holarctic, but there are several geographic regions where local forms have been assigned to European species names based on a superficial morphological resemblance. Here we examine the species status of a form that was previously assigned to D. rosea from a montane bog pond on Honshu, Japan. We used two nuclear non-coding loci (nDNA), mitochondrial sequences (the ND2 protein-coding region) and morphology for evidence. The mitochondrial gene evidence supported the existence of a divergent lineage that is more closely related to D. galeata than to D. dentifera. However, morphology and the nuclear DNA data indicated a lineage that is most closely related to D. dentifera. As our evidence supported the existence of a cohesive divergent lineage, we described a new species, Daphnia japonica sp. nov. Recognition of local and subalpine diversity in this group is critical as ongoing anthropogenic disturbance has been associated with introductions, local extirpations, and hybridization.

Stochastic variation in gut bacterial community affects reproductive rates in the water flea Daphnia magna.

It is well-documented that perturbation of the gut bacterial community can influence the reproductive rates of the host. Less is known about how natural ecological processes can change the bacterial composition in the gut and how such changes influence the reproductive rate of the host. Here, we provide novel experimental insights into such processes using the clonally reproducing water flea, Daphnia magna. A total of 20 replicate cultures were reared for 5 weeks (Phase 1) to allow for divergence of bacterial communities through stochastic processes (i.e. drift, founder effects, and/or colonization). Duplicate cultures created from each of these were reared for 21 days (Phase 2) while recording reproductive rates. There was a significant repeatability in reproductive rates between these duplicates, suggesting that divergence of the bacterial communities during Phase 1 translated into reproductive rate effects during Phase 2. This was further supported by significant differences in the relative abundance of gut bacteria (investigated by amplicon sequencing of a part of the 16S rRNA gene) between cultures with high and low reproductive rate in Phase 2. These results are consistent with the hypothesis that stochastic processes can cause natural variation in the bacterial composition in the gut, which in turn affect host reproductive rates.

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.

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.

Exploring mitogenome evolution in Branchiopoda (Crustacea) lineages reveals gene order rearrangements in Cladocera.

The class Branchiopoda, whose origin dates back to Cambrian, includes ~ 1200 species which mainly occupy freshwater habitats. The phylogeny and systematics of the class have been debated for long time, until recent phylogenomic analyses allowed to better clarify the relationships among major clades. Based on these data, the clade Anostraca (fairy and brine shrimps) is sister to all other branchiopods, and the Notostraca (tadpole shrimps) results as sister group to Diplostraca, which includes Laevicaudata + Spinicaudata (clam shrimps) and Cladoceromorpha (water fleas + Cyclestherida). In the present analysis, thanks to an increased taxon sampling, a complex picture emerges. Most of the analyzed mitogenomes show the Pancrustacea gene order while in several other taxa they are found rearranged. These rearrangements, though, occur unevenly among taxa, most of them being found in Cladocera, and their taxonomic distribution does not agree with the phylogeny. Our data also seems to suggest the possibility of potentially homoplastic, alternative gene order within Daphniidae.

Complete mitochondrial genome of Ovalona pulchella (Branchiopoda, Cladocera) as the first representative in the family Chydoridae: Gene rearrangements and phylogenetic analysis of Cladocera.

Chydoridae are phytophilic-benthic microcrustaceans that make up a significant proportion of species diversity and play an important role in the littoral zone of freshwater ecosystems worldwide. Here, we provide the complete mitochondrial genome of Ovalona pulchella (King, 1853), determined by next-generation sequencing. The entire mitochondrial genome is 15,362 bp in length; this is the first sequenced mitochondrial genome in the family Chydoridae. The base composition and codon usage were typical of Cladocera species. The mitochondrial gene arrangement (37 genes) was not consistent with that of other Branchiopoda. Both maximum likelihood and Bayesian analyses supported each suborder and family of Branchiopoda as monophyletic groups. The relationships among the families were as follows: [(Leptestheriidae + Limnadiidae) + (Sididae + (Bosminidae + (Chydoridae + Daphniidae)))] + Triopsidae. The newly sequenced O. pulchella was most closely related to the family Daphniidae. The complete mitochondrial genome of O. pulchella also provides valuable molecular information for further analysis of the phylogeny of the Chydoridae and the taxonomic status of the Branchiopoda.

New insight into the taxonomic resolution of the genus Bythotrephes Leydig (Crustacea: Cladocera) based on molecular data from Central Europe.

The taxonomic status of the genus Bythotrephes Leydig (Crustacea: Cladocera) has been debated since the second half of the XIX century. The most widespread view of recent decades has been that Bythotrephes is a monotypic genus, which was support by preliminary molecular data. However, the recent detailed morphological revision of this genus clearly distinguishes at least seven species. Therefore, we performed a multi-lake survey in Central Europe to give new insight into the taxonomic status of Bythotrephes by combining genetic analysis with traditional morphology-based taxonomy. Based on the morphology we identified two species in Central Europe, B. brevimanus and B. lilljeborgi, as well as hybrid forms. For the genetic analysis, we used newly obtained 113 sequences of mtDNA COI gene of the 535-bp length Bythotrephes from Central Europe and sequences downloaded from GenBank. There were no significant differences between all analyzed sequences, which supports the hypothesis that Bythotrephes is a monotypic genus, with only one highly polymorphic species. On the other hand, the results of our work could point out that the COI gene is insufficient to evaluate the taxonomic status of Bythotrephes. Nonetheless, we have identified 29 new haplotypes of mtDNA COI, and one which was the same as the haplotype found in North America and Finland. Furthermore, this haplotype was the source variant from which most other haplotypes were derived.

Genome-wide identification of 216 G protein-coupled receptor (GPCR) genes from the marine water flea Diaphanosoma celebensis.

G protein-coupled receptors (GPCRs) are considered to have originated from early evolution of eukaryotic species, therefore, the genome-wide identification of GPCR genes can provide insight into the adaptive strategy and evolutionary tendency in an animal taxon. Here, we identified a total 216 full-length GPCR genes in the marine water flea Diaphanosoma celebensis genome, which were classified into five distinct classes (A, B, C, F, and other). Phylogenetic comparison of GPCRs in D. celebensis to those in humans (Homo sapiens), fruitfly (Drosophila melanogaster), and freshwater water flea (Daphnia magna) reveals a high level of orthological relationship of amine, neuropeptide, and opsin receptor repertoire, while purinergic and chemokine receptors were highly differentiated in humans. Our findings suggest sporadic evolutionary processes within the GPCR gene families identified in D. celebensis. In this study, these results may provide a better understanding on the evolution of GPCRs, and expand our knowledge of the cladoceran GPCR gene repertories which in part, mediate cell physiological mechanisms in response to various environmental stimuli.

Phylogenetic analysis of eight species of Anomopoda based on transcriptomic and mitochondrial DNA sequences.

Anomopoda is the widespread planktonic microcrustacean, which plays a crucial role in aquatic ecosystem. There are few studies about the evolutionary relationships among various Anomopoda basing on molecular data. In the present study, phylogenetic analysis of eight Anomopoda was carried out. Firstly, the culture system was developed to breed cladocerans. By using this system, eight species (Daphnia magna, D. pulex, D. sinensis, Ceriodaphnia reticulata, Moina micrura, Scapholeberis kingi, Simocephalus vetulus and Eurycercus lamellatus) were purified and cultured stably in the laboratory. Then, transcriptomic sequences and partial mitochondrial DNA sequences were both used to reconstruct the phylogenetic tree among 8 species. Transcriptomic sequences were sequenced on Illumina Hiseq 2500 platform. After assembly and annotation, transcriptomic sequences were spliced together and aligned for phylogenetic analysis. Basing on the orthologous genes derived from transcriptomic sequences, the phylogenetic analysis showed that 4 genera of Daphniidae were clustered into one group, and among the 4 genera, Ceriodaphnia was closer to Daphnia than Simocephalus, while Scapholeberis was farthest from other species. In addition, Eurycercidae was closer to Daphniidae than Moinidae. The phylogenetic trees based on both 12S rRNA and 16S rRNA sequences were similar with that based on transcriptomic sequences. Meanwhile, the phylogenetic tree based on 16S rRNA sequences was more suitable than that based on 12S rRNA sequences. These results suggested that the phylogenetic analysis basing on the transcriptomic sequences was available in cladocerans, which will help us to effectively understand the phylogenetic relationships among various cladocerans.

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation.

Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

The genome of the marine water flea Diaphanosoma celebensis: Identification of phase I, II, and III detoxification genes and potential applications in marine molecular ecotoxicology.

To assemble the genome of the marine water flea Diaphanosoma celebensis, a sentinel model for marine environmental monitoring, we constructed a high-quality genome using PromethION and HiSeq 2500 platforms. The total length of the assembled genome was 100.08 Mb, with N50 = 2.56 Mb (benchmarking universal single-copy orthologs, 96.9%) and consisted of 179 scaffolds. A total of 15,427 genes were annotated, and orthologous gene clusters in D. celebensis were analyzed and compared with those of the cladocerans Daphnia magna and Daphnia pulex. In addition, phase I, II, and III detoxification gene families of cytochrome P450s, glutathione S-transferases, and ATP-binding cassette were fully identified and revealed lineage-specific gene loss and/or expansion, suggesting that the evolution of detoxification gene families likely modulates fitness and susceptibility in response to environmental stressors. The study improves our understanding of the detoxification-related gene system and should contribute to future studies of molecular ecotoxicology in cladoceran species and their responses to emerging pollutants.

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.

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).

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.

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.