Isa Schön

Asexual reproduction in nonmarine ostracods

Asexual reproduction has evolved repeatedly in nonmarine ostracods and takes a variety of forms from ancient asexuals to species in which sexual and asexual lineages coexist. Clonal diversity is highly variable. There is evidence that some of this diversity is maintained by ecological differentiation. Hybridization between asexual females and males, of the same or related species, contributes to clonal diversity. Molecular data suggest that some clonal lineages are surprisingly old (more than 5 Myr). In the ancient asexual Darwinula stevensoni, from a lineage that has apparently been without sex for more than 100 Myr, a remarkable lack of sequence variation in ITS1 may be explained by occasional automixis, gene conversion or somatic recombination, or by efficient DNA repair. Overall, the ostracods provide an excellent system in which to study the evolution of reproductive modes.

Cryptic Species in Putative Ancient Asexual Darwinulids (Crustacea, Ostracoda)

Background Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied. Methodology/Principal Findings We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies. Conclusions/Significance Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.

The life-cycle of the asexual ostracod Darwinula stevensoni (Brady & Robertson, 1870) (Crustacea, ostracoda) in a temporate pond

The life-cycle of the ancient asexual ostracod Darwinula stevensoni was studied during 1 year in a eutrophic pond in Belgium. The reproductive period of this species started in March and was effectively completed by September of the same year. All changes in population structure took place during the spring and summer months and a rapid turnover of the instars was observed. The life-cycle of Darwinula stevensoni appears to take one year or less in Belgium and this is considerably shorter than the 4 years which had been reported previously from subarctic populations. The difference to the present study is most likely temperature-related. Maximal densities of D. stevensoni were observed in June and July and attained 105 ind. m−2. During winter, densities were lower with a mean of 104 ind. m−2. Consequently, the calculated population size of each month was high throughout the year. Together with the low mutation rate, such a large population size could effectively counteract the stochastic loss of mutation-free genotypes as predicted by Mullers ratchet. D. stevensoni is a brooder; the maximum number of embryos and juvenile instars (up to third stage) found within a single female was 11.

Cryptic species of the Eucypris virens species complex (Ostracoda, Crustacea) from Europe have invaded Western Australia.

Eucypris virens, an ostracod with mixed reproduction and Holarctic distribution, forms a species complex with more than 35 cryptic species in Europe. Here, we analysed COI and LSU DNA sequence data from Western Australian E. virens to distinguish between the possibilities that vicariant processes have led to the formation of Australian E. virens species or that these ostracods have been introduced into Western Australia. Phylogenetic reconstructions, genetic networks and estimates of genetic distances all show clearly that Western Australian and European E. virens are very closely related. Some haplotypes are identical, others are only separated by one or two mutational steps. Among the Western Australian representatives of E. virens, three phylogenetic clades can be distinguished. We identified three European cryptic species as ancestors for two of the Western Australian clades and one close relative to the third Western Australian clade. We therefore conclude that E.virens has been introduced into Western Australia, most likely from western Europe, and did not diverge in Australia. In Europe, E. virens shows a typical pattern of geographic parthenogenesis while we found only asexual populations in Western Australia.

Darwinulid Ostracods: Ancient Asexual Scandals or Scandalous Gossip?

Whereas studies of putative ancient asexuals could help solve the paradox of sex, most research on such groups still focuses on consolidating their status. The evidence for the darwinulid ostracods is as yet inconclusive. Recent males have been found in a single species, but their functionality is uncertain and their morphology highlights the erroneous assignment of male status to a single individual of Darwinula stevensoni, presently the best candidate for an ancient asexual darwinulid. Previous records of putative fossil males for the past 200 million years have been rejected. Genetic signatures of ancient asexuality are equally inconclusive: there is no Meselson effect in the darwinulids, but neither the presence nor the absence of the Meselson effect does provide conclusive evidence for or against sex. However, it would seem that a combination of a general purpose genotype with powerful homogenising genetic mechanisms (gene conversion, DNA repair) could counter the deleterious effects of the absence of sex in at least a number of darwinulid species.

The Elusive Clone – In Search of Its True Nature and Identity

Sex in eukaryotes involves a combination of meiosis and syngamy; absence of these processes leads to asexual reproduction of which there are different kinds (Box 9.1). Lineages comprising subsequent generations of such asexually reproducing individuals are nearly invariably referred to as clones. In addition, they are mostly identified by genetic techniques using different molecular markers and with differing powers of resolution. Here, we demonstrate that various clonal concepts are being used without clear discrimination, which can significantly impede both the repeatability of, and comparisons between, studies. An example of the resulting dilemma is that, according to one concept, the more than 350 species of obligate asexual bdelloid rotifers all belong to one clone, whereas according to another concept, each of these species comprises several clones.

On the Strandesia obtusata/elliptica lineage (Ostracoda, Cyprididae) in the alluvial valley of the upper Paraná River (Brazil), with the description of three new species

We redescribe Strandesia tolimensis Roessler, 1990, originally described from Colombia, and describe three new species, Strandesia lansactohai n. sp., S. velhoi n. sp. and S. nupelia n. sp. from lakes, channels and rivers in the alluvial valley of the upper Parana River. Mainly valve morphology and shape and size of the attachment of the caudal ramus are used to characterize these species, as other soft parts are uninformative. All populations studied here are parthenogenetic, so male morphologies are not available. Sequences of the mitochondrial COI-gene are used to construct a phylogeny, as well as a genetic network of specimens, and in both analyses the four species are well-supported. In addition, we also delimit the four species with genetic distances using Birky’s K/θ method and conclude that it supports the identity of the species under consideration.

High cryptic diversity and persistent lineage segregation in endemic Romecytheridea (Crustacea, Ostracoda) from the ancient Lake Tanganyika (East Africa)

Ostracods form a substantial part of the endemic fauna of ancient lakes. Here, we have investigated the phylogenetic and phylogeographic patterns and genetic diversities of species of the endemic genus Romecytheridea from the Southern and Central part of Lake Tanganyika. We found that ostracod populations from four different localities are genetically highly differentiated from each other when analyzing the mitochondrial 16S region, while they are almost identical with genetic markers from the nuclear genome (D1-D2 from the large ribosomal subunit (LSU) and ITS). The criteria of the K/θ method for the evolutionary species concepts are fulfilled when analyzing 16S DNA sequence data, indicating that these populations are in fact different (cryptic) species with allopatric distribution. We discuss various hypotheses on how this high diversity could have originated. The complete lineage segregation can partly be explained by geographic isolation during periods of low lake level stands. But, other factors must have contributed to genetic isolation and speciation, as the two closest populations (Chimba and Katoto) from shallow parts of the Southern basin of Tanganyika are also geographically fully segregated.

Clonal diversity in the ancient asexual ostracod **Darwinula stevensoni** assessed by RAPD-PCR

As Darwinulidae (Ostracoda) are considered to be ancient asexuals with a wide geographical and ecological distribution, they are expected to have accumulated mutations during a long timeframe. However, previous studies on genetic variability suggested a low genetic divergence within the darwinulid species Darwinula stevensoni.Here, overall genotopic diversity of D. stevensoni is estimated with the Random Amplified Polymorphic DNA (RAPD) technique. Using six primers revealing 47 consistently scorable polymorphic loci, substantial clonal diversity within this species is detected. Five of the seven surveyed populations are multiclonal. Moreover, the seven populations have a different clonal composition with almost all of the observed clonal genotypes being restricted to single populations, indicating the absence of a single widespread ‘clone’.The observed clonal diversity seems to refute the existence of a widespread general purpose genotype for D. stevensoni. However, in light of previously detected uniformity at functional loci, we reconsider the definition of a GPG. We suggest that it need not imply a genome-wide fixed genotype, but rather consists of a set of ecologically relevant genes.

Lessons to Learn from Ancient Asexuals

This chapter reviews current hypotheses on the prevalence of sex in the eukaryotic world and presents four examples of putative ancient asexuals. We evaluate theoretical and practical concepts on how to demonstrate long-term asexuality. These are either derived from classical biological research (e.g. absence of males in fossil or recent populations) or from molecular biology (e.g. the Meselson effect, presence of functional transposable elements). Testing for the presence of conserved meiotic core proteins is a novel and especially promising way to verify ancient asexuality. We further re-evaluate statistical methods that utilize existing DNA sequence information on how to test for meiotic recombination. Molecular mechanisms counteracting the accumulation of deleterious mutations might be the most important avenue for ancient asexuals to persist in long, evolutionary time frames. Such mechanisms are reassessed and linked to molecular data from putative ancient asexuals. In particular, the purging of deleterious mutations by ameiotic recombination appears to be more effective than previously anticipated.