Patterns of Intraspecific DNA Variation in the Daphnia Nuclear Genome

Understanding nucleotide variation in natural populations has been a subject of great interest for decades. However, many taxonomic groups, especially those with atypical life history attributes remain unstudied, and Drosophila is the only arthropod genus for which DNA polymorphism data are presently abundant. As a result of the recent release of the complete genome sequence and a wide variety of new genomic resources, the Daphnia system is quickly becoming a promising new avenue for expanding our knowledge of nucleotide variation in natural populations. Here, we examine nucleotide variation in six protein-coding loci for Daphnia pulex and its congeners with particular emphasis on D. pulicaria, the closest extant relative of D. pulex. Levels of synonymous intraspecific variation, π_s, averaged 0.0136 for species in the Daphnia genus, and are slightly lower than most prior estimates in invertebrates. Tests of neutrality indicated that segregating variation conforms to neutral model expectations for the loci that we examined in most species, while K_a/K_s ratios revealed strong purifying selection. Using a full maximum-likelihood coalescent-based method, the ratio of the recombination rate to the mutation rate (c/u), averaged 0.5255 for species of the Daphnia genus. Lastly, a divergence population-genetics approach was used to investigate gene flow and divergence between D. pulex and D. pulicaria.     

Transcontinental Phylogeography of the Daphnia pulex Species Complex

Daphnia pulex is quickly becoming an attractive model species in the field of ecological genomics due to the recent release of its complete genome sequence, a wide variety of new genomic resources, and a rich history of ecological data. Sequences of the mitochondrial NADH dehydrogenase subunit 5 and cytochrome c oxidase subunit 1 genes were used to assess the global phylogeography of this species, and to further elucidate its phylogenetic relationship to other members of the Daphnia pulex species complex. Using both newly acquired and previously published data, we analyzed 398 individuals from collections spanning five continents. Eleven strongly supported lineages were found within the D. pulex complex, and one lineage in particular, panarctic D. pulex, has very little phylogeographical structure and a near worldwide distribution. Mismatch distribution, haplotype network, and population genetic analyses are compatible with a North American origin for this lineage and subsequent spatial expansion in the Late Pleistocene. In addition, our analyses suggest that dispersal between North and South America of this and other species in the D. pulex complex has occurred multiple times, and is predominantly from north to south. Our results provide additional support for the evolutionary relationships of the eleven main mitochondrial lineages of the D. pulex complex. We found that the well-studied panarctic D. pulex is present on every continent except Australia and Antarctica. Despite being geographically very widespread, there is a lack of strong regionalism in the mitochondrial genomes of panarctic D. pulex – a pattern that differs from that of most studied cladocerans. Moreover, our analyses suggest recent expansion of the panarctic D. pulex lineage, with some continents sharing haplotypes. The hypothesis that hybrid asexuality has contributed to the recent and unusual geographic success of the panarctic D. pulex lineage warrants further study.     

Evolutionary history of alpine and subalpine Daphnia in western North America

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Mitochondrial DNA diversity in an apomictic Daphnia complex from the Canadian High Arctic

Cyclic parthenogenesis is the ancestral mode of reproduction in the cladoceran crustacean, Daphnia pulex, but some populations have made the transition to obligate parthenogenesis and this is the only mode of reproduction known to occur in arctic populations. Melanism and polyploidy are also common in arctic populations of this species. Prior allozyme studies of arctic D. pulex revealed substantial levels of clonal diversity on a regional scale. Clonal groupings based on cluster analysis of allozyme genotypes do not conform to groupings based on the presence/absence of melanin or on ploidy level. In order to further elucidate genetic relationships among arctic D. pulex clones, mitochondrial DNA (mtDNA) variation was examined in 31 populations from two Canadian high-arctic sites. The data were also compared to a previous study of mtDNA variation in populations from a Canadian low-arctic site. Cladistic analysis of restriction site variation of the entire mitochondrial genome and nucleotide sequence variation of the mitochondrial control region was used to construct genetic relationships among mitochondrial genotypes. Three distinct mitochondrial lineages were detected. One lineage was associated with diploid, nonmelanic clones and is the same as the lineage that is found in temperate populations of D. pulex. The other two lineages (A & B) were associated with polyploid, melanic clones. Sequence divergence between the A and B lineages was 2.4%. Sequence divergence between D. pulex and either of these two lineages exceeded 3%. It is suggested that the melanic, polyploid clones are hybrids between males of D. pulex (and/or a closely related congener, D. pulicaria) and females of either of two ancestral melanic species that have mitochondrial lineages A and B. Geographic patterns of mitochondrial diversity in ‘melanic’ lineage B support the hypothesis of an high-arctic refuge for the ancestral species during the last glacial period.     

Morphological evolution and genetic differentiation in Daphnia species complexes

Despite many ecological and evolutionary studies, the history of several species complexes within the freshwater crustacean genus Daphnia (Branchiopoda, Anomopoda) is poorly understood. In particular, the Daphnia longispina group, comprising several large-lake species, is characterized by pronounced phenotypic plasticity, many hybridizing species and backcrossing. We studied clonal assemblages from lakes and ponds comprising daphnids from several species complexes. In order to reveal patterns of reticulate evolution and introgression among species, we analysed three data sets and compared nuclear, mtDNA and morphological divergence using animals from 158 newly established clonal cultures. By examining 15 nuclear and 11 mitochondrial (12S/16S rDNA) genetic characters (allozymes/restriction enzymes), and 48 morphological traits, we found high clonal diversity and discontinuities in genotypic and morphological space which allowed us to group clones by cytonuclear differentiation into seven units (outgroup D. pulex). In contrast to six groups emerging from nuclear divergence (related to three traditional species, D. cucullata, D. galeata, D. hyalina and three pairwise intermediate hybrids), a seventh group of clones was clearly resolved by morphological divergence: distinct mtDNA haplotypes within one nuclear defined cluster, ‘D. hyalina’, resembled traditional D. hyalina and D. rosea phenotypes, respectively. In other nuclear defined clusters, association between mtDNA haplotype and morphology was low, despite hybridization being bidirectional (reciprocal crosses). Morphological divergence was greatest between young sister species which are separated on the lake/pond level, suggesting a significant role for divergent selection during speciation along with habitat shifts. Phylogenetic analyses were restricted to four cytonuclear groups of clones related to species. mtDNA and nuclear phylogenies were consistent in low genetic divergence and monophyly of D. hyalina and D. rosea. Incongruent patterns of phylogenies and different levels of genetic differentiation between traditional species suggest reticulate evolutionary processes.     

Population genetic structure of three pond-inhabiting Daphnia species on a regional scale (Flanders, Belgium)

1. Only a few studies have compared patterns of genetic variation among populations of different Daphnia species on a regional scale. The present study addresses this gap and examines the relationship between diversity as revealed by allozyme variation and habitat size for populations of Daphnia pulex, D. obtusa and D. curvirostris in Flanders (Belgium). In addition, we examined whether patterns of isolation‐by‐distance could be observed in each of these three Daphnia species. 2. The relationship between genetic diversity and habitat size varied among Daphnia species that occur in the same region. In D. pulex and D. obtusa populations, a positive relationship between local genetic diversity and habitat size was found, whereas the relationship was negative in D. curvirostris populations. 3. Regional genetic diversity was lower than expected from patterns of local genetic diversity in D. pulex and D. obtusa populations in Flanders. This suggests that the subdivision of local Daphnia populations in a region did not obviously increase genetic diversity. 4. Genetic differentiation among populations of these three species in Flanders was moderate and comparable with values observed in other Daphnia species. Patterns of isolation‐by‐distance could be observed, but the scatter was high (D. pulex) or the slope was very low (D. obtusa).     

Evolutionary Genomics of a Subdivided Species

The ways in which genetic variation is distributed within and among populations is a key determinant of the evolutionary features of a species. However, most comprehensive studies of these features have been restricted to studies of subdivision in settings known to have been driven by local adaptation, leaving our understanding of the natural dispersion of allelic variation less than ideal. Here, we present a geographic population-genomic analysis of 10 populations of the freshwater microcrustacean Daphnia pulex, an emerging model system in evolutionary genomics. These populations exhibit a pattern of moderate isolation-by-distance, with an average migration rate of 0.6 individuals per generation, and average effective population sizes of ∼650,000 individuals. Most populations contain numerous private alleles, and genomic scans highlight the presence of islands of excessively high population subdivision for more common alleles. A large fraction of such islands of population divergence likely reflect historical neutral changes, including rare stochastic migration and hybridization events. The data do point to local adaptive divergence, although the precise nature of the relevant variation is diffuse and cannot be associated with particular loci, despite the very large sample sizes involved in this study. In contrast, an analysis of between-species divergence highlights positive selection operating on a large set of genes with functions nearly nonoverlapping with those involved in local adaptation, in particular ribosome structure, mitochondrial bioenergetics, light reception and response, detoxification, and gene regulation. These results set the stage for using D. pulex as a model for understanding the relationship between molecular and cellular evolution in the context of natural environments.     

Proteomic Profile of Daphnia pulex using Data‐Independent Acquisition Mass Spectrometry and Ion Mobility Separation

Daphnia pulex is a keystone species for aquatic habitats and an ecological/evolution model organism. Although significant progress has been made on characterizing its genome, the D. pulex proteome remains largely uncharacterized partially due to abnormally high protein degradation during homogenization and emphasis on genomic analysis. In this study, various sample preparation and mass spectrometry acquisition methods are performed for the purpose of improving D. pulex proteome exploration. Benefits for employing both in‐gel and in‐solution methods of trypsin digestion are observed. Furthermore, acquisition methods employing ion mobility separation greatly increase peptide identification and more than doubled the proteome coverage. Bioinformatic analysis suggests that mitochondrial and hydrolytic activities are enriched in D. pulex compared to closely related invertebrates or Homo sapiens. Also, novel D. pulex proteins possessing putative genome modifying functional domains are identified. Data are available via ProteomeXchange with identifier PXD008455.     

Predator-induced defences in <Emphasis Type="Italic">Daphnia pulex</Emphasis>: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

Background  

The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.     

In Silico Prediction and In Vivo Validation of Daphnia pulex Micrornas

Daphnia pulex, the crustacean with the first sequenced genome, is an important organism that has been widely used in ecological and toxicological research. MicroRNAs (miRNAs) are 21–25 nucleotide small non-coding RNAs that are involved in a myriad of physiological processes. In this research, we predicted 75 D. pulex miRNAs by sequence homology and secondary structure identification from the full genome sequence. Fourteen predicted miRNAs were selected for quantitative real time polymerase chain reaction (RT-PCR) validation. Out of these, eight (mir-8, mir-9, mir-12, mir-92, mir-100, mir-133, mir-153 and mir-283) were successfully amplified and validated. Next, expression levels were quantified at three different life stages (days 4, 8 and 12 of age) using U6 spliceosomal RNA as a reference gene. The expression of mir-8, mir-9, mir-12, mir-92 and mir-100 significantly differed across time suggesting these microRNAs might play a critical role during D. pulex development. This is the first study to identify and validate miRNAs in D. pulex, which is an important first step in further studies that evaluate their roles in development and response to environmental and ecological stimuli.     

Copy Number of the Transposon,Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.     

Molecular genetic identification and phylogeny of Daphnia species (Crustacea, Cladocera) from water bodies of the Lake Chany basin

The data on the molecular genetic identification of Daphnia species from the water bodies of the Lake Chany basin are presented. Phylogenetic relationships between these species have been established. The fragments of the mitochondrial DNA genes were used as genetic markers. According to the data obtained, the water bodies examined were inhabited by five Daphnia species, including Daphnia (Daphnia) galeata Sars, D. (D.) longispina O. F. Müller, D. (D.) curvirostris Eylmann, D. (D.) pulex Leydig, and D. (Ctenodaphnia) magna Straus. In addition, longispina a-like individuals that form a separate phylogenetic lineage was identified.     

The possible role of genome activity changes in the sex determination of Daphnia pulex

Summary Sex-dependent differences in the state of the nuclear chromatin of somatic cells were found in Daphnia pulex. It is suggested that the genome of Daphnia pulex has two developmental programmes based on identical chromosome sets. The female programme consistently functions under a wide range of ecological conditions, whereas the male programme is turned on by specific ecological stimuli. The genes controlling the activation and function of the male programme may be phenotypically latent for many parthenogenetic generations.     

Frequency and Pattern of Heteroplasmy in the Complete Human Mitochondrial Genome

Determining the levels of human mitochondrial heteroplasmy is of utmost importance in several fields. In spite of this, there are currently few published works that have focused on this issue. In order to increase the knowledge of mitochondrial DNA (mtDNA) heteroplasmy, the main goal of this work is to investigate the frequency and the mutational spectrum of heteroplasmy in the human mtDNA genome. To address this, a set of nine primer pairs designed to avoid co-amplification of nuclear DNA (nDNA) sequences of mitochondrial origin (NUMTs) was used to amplify the mitochondrial genome in 101 individuals. The analysed individuals represent a collection with a balanced representation of genders and mtDNA haplogroup distribution, similar to that of a Western European population. The results show that the frequency of heteroplasmic individuals exceeds 61%. The frequency of point heteroplasmy is 28.7%, with a widespread distribution across the entire mtDNA. In addition, an excess of transitions in heteroplasmy were detected, suggesting that genetic drift and/or selection may be acting to reduce its frequency at population level. In fact, heteroplasmy at highly stable positions might have a greater impact on the viability of mitochondria, suggesting that purifying selection must be operating to prevent their fixation within individuals. This study analyses the frequency of heteroplasmy in a healthy population, carrying out an evolutionary analysis of the detected changes and providing a new perspective with important consequences in medical, evolutionary and forensic fields.     

Engines of change: Transposable element mutation rates are high and variable within Daphnia magna

Transposable elements (TEs) represent a major portion of most eukaryotic genomes, yet little is known about their mutation rates or how their activity is shaped by other evolutionary forces. Here, we compare short- and long-term patterns of genome-wide mutation accumulation (MA) of TEs among 9 genotypes from three populations of Daphnia magna from across a latitudinal gradient. While the overall proportion of the genome comprised of TEs is highly similar among genotypes from Finland, Germany, and Israel, populations are distinguishable based on patterns of insertion site polymorphism. Our direct rate estimates indicate TE movement is highly variable (net rates ranging from -11.98 to 12.79 x 10⁻⁵ per copy per generation among genotypes), differing both among populations and TE families. Although gains outnumber losses when selection is minimized, both types of events appear to be highly deleterious based on their low frequency in control lines where propagation is not limited to random, single-progeny descent. With rate estimates 4 orders of magnitude higher than base substitutions, TEs clearly represent a highly mutagenic force in the genome. Quantifying patterns of intra- and interspecific variation in TE mobility with and without selection provides insight into a powerful mechanism generating genetic variation in the genome.     

Predictors of invasion success by Daphnia species: influence of food, temperature and species identity

Non-indigenous North American Daphnia ‘pulex’ has recently invaded lakes and reservoirs in South Island, New Zealand, that formerly contained only native Daphnia carinata. New Zealand is characterised by a wide range of freshwater ecosystems and low species diversity of planktonic crustaceans, particularly Cladocera. The potential success of a species to invade and establish in a new community is likely to be predicted more accurately when reproductive response norms and fitness of key resident species, as well as the non-indigenous species, have been established under a range of relevant environmental conditions. Based on the results of experiments to test aspects of reproduction and fitness of the invader, D. ‘pulex’, and D. carinata when grown together at a range of relevant temperatures (8–23 °C) and related photoperiods, I predict the species-specific potential of D. ‘pulex’ to be dispersed and colonise New Zealand lentic habitats, and the potential of D. carinata to persist with the invader in these habitats. Larger population densities of D. ‘pulex’ compared to D. carinata at higher temperatures and food level, and larger densities of D. carinata at low temperatures, imply a potential for both species to coexist in New Zealand lakes, facilitated by seasonal succession; increased water temperature and nutrient input associated with climate and land use changes appear likely to promote the wider establishment of D. ‘pulex’, with both negative and positive implications for the conservation and management of New Zealand’s freshwater ecosystems.     

Competitive abilities and distribution ranges of four asexual Daphnia pulex lineages and Daphnia mitsukuri in Eurasian continental islands

Daphnia pulex and D. mitsukuri are morphologically similar and distributed in small lowland Japanese lakes. Daphnia pulex in Japan reproduces by obligate parthenogenesis and is composed of four different lineages (JPN1–JPN4) with North American origins. Although this species is found throughout Japan, JPN1 has the largest distribution range, followed by JPN2. Daphnia mitsukuri is a putatively endemic species that is now rarely found in Japan. Since Daphnia share the same algal food, the difference in distribution ranges among the four asexual D. pulex lineages and D. mitsukuri may be caused by exploitative competition for algal food. To examine this possibility, we measured the threshold food concentration (TFC) at zero-net growth rate and the food concentration required for 50% of the individuals to survive (FC50). In addition, we examined the effects of temperature on the somatic growth rate (SGR) and the mortality rates of four asexual D. pulex lineages and D. mitsukuri. The experiments showed that TFC was lowest in D. pulex JPN1 and highest in D. mitsukuri, supporting the idea that the largest distribution range of JPN1 is due to its superiority in inter-lineage competition, and that the distribution area of D. mitsukuri is reduced through exploitative competition with D. pulex. However, although JPN2 was found in relatively large areas, it had higher TFC than JPN3 and JPN4, which appear in very limited areas. These results clearly showed that the difference in distribution ranges among D. pulex lineages are not necessarily determined by their competitive superiority alone.     

Reproductive allocation in Daphnia exposed to toxic cyanobacteria

We investigated experimentally how resources were allocated to reproduction in Daphnia pulex and Daphnia longispina when varying levels of toxic Microcystis were added to higher quality food. We used multiple regression models to estimate mean offspring size and clutch size in relation to maternal size and clutch number, and analysed effects of treatments on residuals from the models. We also measured variation in per offspring investment. At a high cyanobacterial level, D.pulex was virtually unable to reproduce. At a lower level, D.pulex produced small clutches. However, the regression model residuals indicated that the presence of cyanobacteria increased the portion of available resources allocated to reproduction. The observed allocation may be a means to maximize reproduction under diminished longevity. Effects on mean offspring size were marginal in D.pulex but variation in per offspring investment sometimes decreased in cyanobacterial exposures. Daphnia longispina was affected by a higher cyanobacterial level only, where offspring sized was reduced. Deviations from the regression model indicated that effects on maternal size alone do not explain this effect. Clutch size residuals and per offspring investment were unaffected by treatments in D.longispina. The observed responses differ from theoretical models on reproductive allocation under food imitation.