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Continental lake-dwelling zooplanktonic organisms have long been considered cosmopolitan species with little geographic variation in spite of the isolation of their habitats. Evidence of morphological cohesiveness and high dispersal capabilities support this interpretation. However, this view has been challenged recently as many such species have been shown either to comprise cryptic species complexes or to exhibit marked population genetic differentiation and strong phylogeographic structuring at a regional scale. Here we investigate the molecular phylogeny of the cosmopolitan passively dispersing rotifer Brachionus plicatilis (Rotifera: Monogononta) species complex using nucleotide sequence variation from both nuclear (ribosomal internal transcribed spacer 1, ITS1) and mitochondrial (cytochrome c oxidase subunit I, COI) genes. Analysis of rotifer resting eggs from 27 salt lakes in the Iberian Peninsula plus lakes from four continents revealed nine genetically divergent lineages. The high level of sequence divergence, absence of hybridization, and extensive sympatry observed support the specific status of these lineages. Sequence divergence estimates indicate that the B. plicatilis complex began diversifying many millions of years ago, yet has showed relatively high levels of morphological stasis. We discuss these results in relation to the ecology and genetics of aquatic invertebrates possessing dispersive resting propagules and address the apparent contradiction between zooplanktonic population structure and their morphological stasis.

We describe the application of a simple, low-cost, and effective method of DNA extraction (hot sodium hydroxide and Tris, HotSHOT) to the diapausing propagules of continental aquatic invertebrates for its use in PCR amplification. We illustrate the use of the technique in cladocerans, rotifers, anostracans, notostracans, and copepod diapausing eggs. We compare the performance of the HotSHOT technique to the currently most widely used method for DNA extraction of zooplankton eggs and individuals, the chelating resin (or Chelex) technique. The HotSHOT technique overcomes several of the problems posed by Chelex and permits easy optimization for its use with 96-well plates for high-throughput DNA extraction and subsequent genetic characterization. We foresee a wide use of this technique in the future from DNA barcoding of diapausing stages to the genetic characterization of the diapausing egg banks of continental aquatic invertebrates.

We investigated the phylogeography of the salt water rotifer Brachionus plicatilis, a cyclical parthenogen with passive dispersal mechanisms, using resting eggs recovered from saline lake sediments. Individual resting eggs were obtained from a large selection of lakes which were representative of five endorheic basins and the chain of coastal ponds in the Iberian Peninsula. The novel use of resting eggs allows the integration of seasonal and annual variations as well as the impact of stochastic effects such as drift and local extinction. A 653 bp fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was sequenced from 98 eggs. Our results revealed a deep phylogeographical structure in this species, with a division into two main lineages with distinct geographical distributions, which probably diverged at the beginning of the Pleistocene period. Most of the mitochondrial DNA haplotypes were restricted to single lakes. Nested clade analysis supported Early Pleistocene fragmentation of populations, low gene flow and some long-distance colonization. These conclusions contrast strongly with previous ideas on rotifer biogeography and this pattern is consistent with a recolonization of the Iberian Peninsula from two glacial refugia. The results provide new insights into the processes responsible for the genetic diversification of passive dispersers, a life-history trait typical of zooplanktonic biotas.

Understanding patterns and processes in biological diversity is a critical task given current and rapid environmental change. Such knowledge is even more essential when the taxa under consideration are important ecological and evolutionary models. One of these cases is the monogonont rotifer cryptic species complex Brachionus plicatilis, which is by far the most extensively studied group of rotifers, is widely used in aquaculture, and is known to host a large amount of unresolved diversity. Here we collate a dataset of previously available and newly generated sequences of COI and ITS1 for 1273 isolates of the B. plicatilis complex and apply three approaches in DNA taxonomy (i.e. ABGD, PTP, and GMYC) to identify and provide support for the existence of 15 species within the complex. We used these results to explore phylogenetic signal in morphometric and ecological traits, and to understand correlation among the traits using phylogenetic comparative models. Our results support niche conservatism for some traits (e.g. body length) and phylogenetic plasticity for others (e.g. genome size).

Cyclically parthenogenetic rotifers are a valuable model for investigating the relationship between reproductive mode and population structure, although advances in this field have been hindered by low allozyme variability in these organisms. A high genotypic diversity is predicted after population establishment, which would be eroded by clonal selection during the parthenogenetic phase. The resting egg bank, produced sexually, is presumed to store high levels of genetic diversity, with subsequent effects on planktonic population structure. Here, we provide the first application of microsatellite markers to a rotifer planktonic population and its associated resting egg bank. Seven polymorphic microsatellite loci were screened in populations of the rotifer Brachionus plicatilis in a temporary pond to analyse: (i) the genetic structure of the resting egg bank; (ii) the changes in the genetic structure of rotifer populations during the parthenogenetic phase; and (iii) the population structure after its initiation from resting eggs. Microsatellites proved to be a useful tool for clone identification, revealing a surprisingly high clonal diversity in rotifer populations. The last sample in the parthenogenetic phase showed evidence of clonal selection, as indicated by a low observed clonal diversity and the appearance of linkage disequilibria. The resting egg bank, analysed comprehensively for the first time in any zooplankter, is in Hardy-Weinberg and linkage equilibrium, and contains a high genotypic diversity. Unexpectedly, the resting egg bank differed from the planktonic population in its allelic composition, suggesting that resting egg hatching is biased.

Brachionus plicatilis (Müller, 1786) is a monogonont rotifer that has long been considered as an ecological generalist with a cosmopolitan distribution in inland and coastal marine habitats (Walker, 1981). Among rotifers, B. plicatilis is probably one of the best-studied taxa. It has been widely used as a model for physiological and ecological studies [for examples see (