Grace A. Wyngaard

Observing copepods through a genomic lens

BackgroundCopepods outnumber every other multicellular animal group. They are critical components of the worlds freshwater and marine ecosystems, sensitive indicators of local and global climate change, key ecosystem service providers, parasites and predators of economically important aquatic animals and potential vectors of waterborne disease. Copepods sustain the world fisheries that nourish and support human populations. Although genomic tools have transformed many areas of biological and biomedical research, their power to elucidate aspects of the biology, behavior and ecology of copepods has only recently begun to be exploited.DiscussionThe extraordinary biological and ecological diversity of the subclass Copepoda provides both unique advantages for addressing key problems in aquatic systems and formidable challenges for developing a focused genomics strategy. This article provides an overview of genomic studies of copepods and discusses strategies for using genomics tools to address key questions at levels extending from individuals to ecosystems. Genomics can, for instance, help to decipher patterns of genome evolution such as those that occur during transitions from free living to symbiotic and parasitic lifestyles and can assist in the identification of genetic mechanisms and accompanying physiological changes associated with adaptation to new or physiologically challenging environments. The adaptive significance of the diversity in genome size and unique mechanisms of genome reorganization during development could similarly be explored. Genome-wide and EST studies of parasitic copepods of salmon and large EST studies of selected free-living copepods have demonstrated the potential utility of modern genomics approaches for the study of copepods and have generated resources such as EST libraries, shotgun genome sequences, BAC libraries, genome maps and inbred lines that will be invaluable in assisting further efforts to provide genomics tools for copepods.SummaryGenomics research on copepods is needed to extend our exploration and characterization of their fundamental biological traits, so that we can better understand how copepods function and interact in diverse environments. Availability of large scale genomics resources will also open doors to a wide range of systems biology type studies that view the organism as the fundamental system in which to address key questions in ecology and evolution.

Patterns of genome size in the copepoda

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28–1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32–24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.

The relationship between genome size, development rate, and body size in copepods

AbstractFreshwater cyclopoid copepods exhibit at least a fivefold range in somatic genome size and a mechanism, chromatin diminution, which could account for much of this interspecific variation. These attributes suggest that copepods are well suited to studies of genome size evolution. We tested the nucleotypic hypothesis of genome size evolution, which poses that variation in genome size is adaptive due to the ‘bulk’ effects of both coding and noncoding DNA on cell size and division rates, and their correlates. We found a significant inverse correlation between genome size and developmental (growth) rate in five freshwater cyclopoid species at three temperatures. That is, species with smaller genomes developed faster. Species with smaller genomes had significantly smaller bodies at 22 °C, but not at cooler and warmer temperatures. Species with smaller genomes developed faster at all three temperatures, but had smaller bodies only at 22 °C. We propose a model of life history evolution that adds genome size and cell cycle dynamics to the suite of characters on which selection may act to mold life histories and to influence the distribution of traits among different habitats.

Phylogeny of the freshwater copepod Mesocyclops (Crustacea: Cyclopidae) based on combined molecular and morphological data, with notes on biogeography.

We combined molecular and morphological characters in a copepod taxon for which obtaining a sufficiently high number of characters that evolve at different rates is a challenge. Few molecular markers are known to resolve evolutionary relationships in the copepods, and thus there is potential for morphology to contribute substantially to phylogenetic reconstruction. We used a morphology based tree of the entire Mesocyclops genus to guide our taxon sampling of 10 species for molecular and combined analyses. Morphology including polymorphic characters, 18S rDNA, and ITS2 sequences were analyzed using parsimony, ML, and Bayesian methods. Strong similarities among topologies were observed regardless of the character type or algorithm, with higher levels of support obtained in combined data analyses. In combined analyses Old World species formed a monophyletic group and New World species formed a paraphyletic group in this freshwater, predominantly (sub)tropical genus. Mesocyclops darwini was the single taxon whose relationships showed conflict among the previous reconstructions using only morphological characters and the tree inferred from the combined data set. Support for these alternative positions of M. darwini were compared using constraint tests, with the result supporting monophyly of Old World taxa.

Geographical variation in dormancy in a copepod: evidence from population crosses

Populations of the freshwater copepod Mesocyclops edax inhabiting Michigan lakes arc dormant during winter, whereas populations inhabiting Florida lakes develop and reproduce continuously throughout the year. A Michigan and a Florida population were exposed to dormancy inducing conditions (low temperature and short photoperiod) in the laboratory and observed for indications of dormancy. All Michigan individuals and a small percentage of the Florida individuals entered dormancy as indicated by prolonged duration of the fourth copepodid instar and cessation of feeding. I suggest that in these population these observations represent diapause, rather than quiescence. The two populations were crossbred to examine the nature of inheritance of dormancy. The F1 hybrids exhibited an incidence of diapause approximately intermediate between the Florida and Michigan parental stocks. The backcrosses of F1 individuals to the Michigan and Florida stocks, respectively, exhibited a high and an intermediate incidence of diapause. Survival of the F2 crosses was very low. The present study presents evidence of genetic differentiation between the Michigan and Florida populations of M. edax with respect to ability to diapause.

Evidence for Endoreduplication: Germ Cell DNA Levels Prior to Chromatin Diminution in Mesocyclops edax

We studied the functional significance of marked differences in the DNA content of somatic cells and germ line nuclei by static Feulgen–DNA cytophotometry for several species of microcrustaceans that exhibit chromatin diminution during very early stages of embryogenesis. Mature females and males showed many gonadal nuclei with elevated amounts of DNA that persist until dispersal of this “extra” DNA throughout the cytoplasm as fragments and coalescing droplets of chromatin during anaphase of the diminution division.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development

BackgroundChromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution — some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 – 75 Gb, 12–74 Gb of which are lost from pre-somatic cell lineages at germline – soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown.ResultsHere, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome.ConclusionsOur results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Genetic architecture of the cryptic species complex of Acanthocyclops vernalis (Crustacea: Copepoda) II. Crossbreeding experiments, cytogenetics and a model of chromosomal evolution

Abstract Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within‐line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n= 8 and one with 2n= 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex

Variability in genetic architecture of the cryptic species complex of Acanthocyclops vernalis (Copepoda) I. Evidence from karyotypes, genome size, and ribosomal DNA sequences

Abstract Populations of the North American cryptic species complex of Acanthocylops vernalis (Fischer 1853) (Copepoda) possess unusually variable karyotypes and levels of reproductive isolation, but are difficult to discern morphologically. We established nine isofemale lines derived from four local and geographically isolated pond populations from Wisconsin and three isofemale lines from a lake in Ohio to explore the variability and relationships of chromosome numbers, genome sizes, and similarity of ribosomal DNA sequences. Five karyotypes (2n = 6, 7, 8, 9 and 10) were observed, although genome sizes were remarkably consistent. A 593 bp region of the 18S ribosomal gene was identical in four Wisconsin and three Ohio lines, but differed among five Wisconsin lines. This variability in 18S rDNA sequences among lines, which is uncharacteristic for well defined cyclopoid species, was used to construct a network of relationships among the isofemale lines. Mapping chromosome number onto this network revealed a cluster of lineages with variable chromosome numbers and a cluster with consistent chromosome number, although the overall pattern was more complex than this. Although 4–5 bivalents were observed in oocytes of the progeny from one isofemale line from Ohio, progeny of the latter had smaller genomes than any of the Wisconsin lines. All Wisconsin and Ohio lines lacked chromatin diminution, a trait previously attributed to some populations of A. vernalis by Standiford (1989, Genetika 79: 207–214) and Akifiev (1974, Priroda (USSR) 9: 49–54). Occurrence of chromosomal aberrations among disjunct populations may account for the unusual genetic variability of this species complex. Such differentiation also may be accelerated in fragmented habitats where genetic isolation seems to be recent and recurrent.

TIMING OF CHROMATIN DIMINUTION IN THE FREE- LIVING, FRESH-WATER CYCLOPIDAE (COPEPODA)

ABSTRACT Chromatin diminution, the fragmentation and elimination of chromosome regions during early embryogenesis, occurs in some species of free-living, fresh-water copepods. We studied the variation in timing of this developmental trait by examining durations of cell stages in several genera of Cyclopidae. Chromatin diminution varied in timing within and among genera, and ranged from the 8-64-cell stage. The timing of chromatin diminution, as measured by the number of hours post fertilization, differs considerably within and among genera. An unusually long cell-stage duration relative to that of other cell stages is indicative of the stage during which chromatin diminution occurs or the preceding stage. Thus, the relative length of cell-stage durations within a species is a reliable assay for the presence or absence of chromatin diminution.