A field test of a model for the stability of androdioecy in the freshwater shrimp, Eulimnadia texana.

The evolution of hermaphroditism from dioecy is a poorly studied transition. Androdioecy (the coexistence of males and hermaphrodites) has been suggested as an intermediate step in this evolutionary transition or could be a stable reproductive mode. Freshwater crustaceans in the genus Eulimnadia have reproduced via androdioecy for 24+ million years and thus are excellent organisms to test models of the stability of androdioecy. Two related models that allow for the stable maintenance of males and hermaphrodites rely on the counterbalancing of three life history parameters. We tested these models in the field over three field seasons and compared the results to previous laboratory estimates of these three parameters. Male and hermaphroditic ratios within years were not well predicted using either the simpler original model or a version of this model updated to account for differences between hermaphroditic types ('monogenic' and 'amphigenic' hermaphrodites). Using parameter estimates of the previous year to predict the next year's sex ratios revealed a much better fit to the original relative to the updated version of the model. Therefore, counter to expectations, accounting for differences between the two hermaphroditic types did not improve the fit of these models. At the moment, we lack strong evidence that the long-term maintenance of androdioecy in these crustaceans is the result of a balancing of life history parameters; other factors, such as metapopulation dynamics or evolutionary constraints, may better explain the 24+ million year maintenance of androdioecy in clam shrimp.

Sex chromosome evolution in the clam shrimp, Eulimnadia texana.

Chromosomes that determine sex are predicted to evolve differently than autosomes: a lack of recombination on one of the two sex chromosomes is predicted to allow an accumulation of deleterious alleles that eventually leads to reduced functionality and potential physical degradation of the nonrecombining chromosome. Because these changes should occur at an elevated evolutionary rate, it is difficult to find appropriate species in which to test these evolutionary predictions. The unique genetic sex-determining mechanism of the crustacean Eulimnadia texana prevents major chromosome degeneration because of expression of both 'proto-sex' (i.e. early stage of development) chromosomes in homozygous form (ZZ and WW). Herein, we exploit this unique genetic system to examine the predicted accumulation of deleterious alleles by comparing both homogametic sexual types to their heterogametic counterpart. We report differences in crossing over in a sex-linked region in the ZW hermaphrodites (approximately 3%) relative to the ZZ males (approximately 21%), indicative of cross-over suppression in the ZW hermaphrodites. Additionally, we report that both ZZ and WW genotypes have reduced fitness relative to ZW hermaphrodites, which is consistent with the prediction of harboured recessive mutations embedded on both the Z and the W chromosomes. These results suggest that the proto-sex chromosomes in E. texana accumulate recessive deleterious alleles. We hypothesize that recessive deleterious alleles of large effect cannot accumulate because of expression in both ZZ and WW individuals, keeping both chromosomes from losing significant function.

Evolutionary transitions among dioecy, androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata).

Examinations of breeding system transitions have primarily concentrated on the transition from hermaphroditism to dioecy, likely because of the preponderance of this transition within flowering plants. Fewer studies have considered the reverse transition: dioecy to hermaphroditism. A fruitful approach to studying this latter transition can be sought by studying clades in which transitions between dioecy and hermaphroditism have occurred multiple times. Freshwater crustaceans in the family Limnadiidae comprise dioecious, hermaphroditic and androdioecious (males + hermaphrodites) species, and thus this family represents an excellent model system for the assessment of the evolutionary transitions between these related breeding systems. Herein we report a phylogenetic assessment of breeding system transitions within the family using a total evidence comparative approach. We find that dioecy is the ancestral breeding system for the Limnadiidae and that a minimum of two independent transitions from dioecy to hermaphroditism occurred within this family, leading to (1) a Holarctic, all-hermaphrodite species, Limnadia lenticularis and (2) mixtures of hermaphrodites and males in the genus Eulimnadia. Both hermaphroditic derivatives are essentially females with only a small amount of energy allocated to male function. Within Eulimnadia, we find several all-hermaphrodite populations/species that have been independently derived at least twice from androdioecious progenitors within this genus. We discuss two adaptive (based on the notion of 'reproductive assurance') and one nonadaptive explanations for the derivation of all-hermaphroditism from androdioecy. We propose that L. lenticularis likely represents an all-hermaphrodite species that was derived from an androdioecious ancestor, much like the all-hermaphrodite populations derived from androdioecy currently observed within the Eulimnadia. Finally, we note that the proposed hypotheses for the dioecy to hermaphroditism transition are unable to explain the derivation of a fully functional, outcrossing hermaphroditic species from a dioecious progenitor.

Levels of inbreeding depression over seven generations of selfing in the androdioecious clam shrimp, Eulimnadia texana.

Androdioecy (mixtures of males and hermaphrodites) is a rare mating system in both plants and animals. Theory suggests that high levels of inbreeding depression can maintain males in androdioecious populations if hermaphrodites commonly self-fertilize. However, if inbreeding depression (delta) can be 'purged' from selfing populations, maintaining males is more difficult. In the androdioecious clam shrimp, Eulimnadia texana, delta is estimated to be as high as 0.7. Previous work suggests that this high level is maintained in the face of high levels of inbreeding due to an associative overdominance of fitness-related loci with the sex-determining locus. Such associative overdominance would make purging of inbreeding depression difficult to impossible. The current experiment was designed to determine if delta can be purged in these shrimp by tracking fitness across seven generations in selfing and outcrossing treatments. Evidence of purging was found in one of four populations, but the remaining populations demonstrated a consistent pattern of delta across generations. Although the experimental design allowed ample opportunity for purging, the majority of populations were unable to purge their genetic load. Therefore, delta in this species is likely due to associative overdominance caused by deleterious recessive alleles linked to the sex determining locus.

Relative fitness of two hermaphroditic mating types in the androdioecious clam shrimp, Eulimnadia texana.

Androdioecy (populations of males and hermaphrodites) is a rare reproductive form, being described from only a handful of plants and animals. One of these is the shrimp Eulimnadia texana, which has populations comprised of three mating types: two hermaphroditic types (monogenics and amphigenics) and males. In a recent study, the amphigenic hermaphrodites were found to be in greater abundance than that predicted from a model of this mating system. Herein, we compare the relative fitness of offspring from amphigenic and monogenic siblings, attempting to understand the greater relative abundance of the former. Populations started with offspring from selfed monogenic hermaphrodites had a net reproductive rate (R) 87% that of offspring from their amphigenic siblings. Additionally, within populations of amphigenic offspring (which included males, monogenics and amphigenics), amphigenics survived longer than monogenics. These differences help to explain the increased relative abundance of amphigenics in natural populations, but amphigenics continue to be more abundant than expected.

Maintenance of androdioecy in the freshwater shrimp, Eulimnadia texana: estimates of inbreeding depression in two populations.

Androdioecy is an uncommon form of reproduction in which males coexist with hermaphrodites. Androdioecy is thought to be difficult to evolve in species that regularly inbreed. The freshwater shrimp Eulimnadia texana has recently been described as both androdioecious and highly selfing and is thus anomalous. Inbreeding depression is one factor that may maintain males in these populations. Here we examine the extent of "late" inbreeding depression (after sexual maturity) in these clam shrimp using two tests: (1) comparing the fitness of shrimp varying in their levels of individual heterozygosity from two natural populations that differ in overall genetic diversity; and (2) specifically outcrossing and selfing shrimp from these same populations and comparing fitness of the resulting offspring. The effects of inbreeding differed within each population. In the more genetically diverse population, fecundity, size, and mortality were significantly reduced in inbred shrimp. In the less genetically diverse population, none of the fitness measures was significantly lowered in selfed shrimp. Combining estimates of early inbreeding depression from a previous study with current estimates of late inbreeding depression suggests that inbreeding depression is substantial (delta = 0.68) in the more diverse population and somewhat lower (delta = 0.50) in the less diverse population. However, given that males have higher mortality rates than hermaphrodites, neither estimate of inbreeding depression is large enough to account for the maintenance of males in either population by inbreeding depression alone. Thus, the stability of androdioecy in this system is likely only if hermaphrodites are unable to self-fertilize many of their own eggs when not mated to a male or if male mating success is generally high (or at least high when males are rare). Patterns of fitness responses in the two populations were consistent with the hypothesis that inbreeding depression is caused by partially recessive deleterious alleles, although a formal test of this hypothesis still needs to be conducted.

Lack of IL-2 cytokine expression despite Il-2 messenger RNA transcription in tumor-infiltrating lymphocytes in primary human breast carcinoma: selective expression of early activation markers.

Tumor-infiltrating lymphocytes (TIL) are found in most human infiltrating ductal breast carcinomas. In studies of other tumors, TIL were capable of activation by IL-2, both in vitro and in vivo, to produce selective tumor cytolysis. Specific TIL-mediated tumor cytolysis in human breast tumors has recently been reported. The large numbers of TIL within human breast cancers imply that an immune response is occurring, since many of these cells express HLA class II as a late activation marker. However, the degree of early activation of the native TIL in breast tumors has not been fully investigated. Early activation markers CD69, CD43, and CD38 together with the IL-2R (CD25) and IL-2 cytokine were examined using mAbs and tissue section immunohistology. In situ hybridization was used to detect IL-2 mRNA (IL-2 mRNA) in parallel with immunohistochemical localization of IL-2. The results revealed the expression of CD69, CD43, and CD38, but markedly low CD25 (IL-2R) and IL-2 protein expression by the TIL. This strongly indicates that the TIL are an activated population of T cells that shows a deficiency in IL-2 protein and IL-2R expression despite adequate levels of IL-2 mRNA. The mechanism for apparent inhibition of IL-2 production and IL-2R expression in the presence of IL-2 mRNA is currently unclear; however, this may explain the relative anergic state of native TIL.

Evolutionary models of quantitative disease risk factors.

Numerous mutations are now known that have significant effects on various phenotypes; many of these mutations are of interest because they influence quantitative risk factors for major diseases. Such diversity raises the question of how much genetic heterogeneity we should expect to find in the effects of alleles, that is, the size of the effects, the number of severe alleles, and their frequency in the population. Can evolutionary models suggest a general pattern? In this article we examine what is currently known about several basic aspects of the problem. These include the distribution of quantitative effects of new mutations on a phenotype, the distribution of allelic effects that would be found in a natural population, and the relationship between these effects and Darwinian fitness. We discuss these issues in light of various models that have been proposed and the existing relevant data. Then we consider how these points relate to the distribution of genetic effects on an important human trait, the cholesterol ratio, an important risk factor for coronary heart disease. The complexities of quantitative traits and inadequacies in the available data prevent definitive models that can directly connect the mutational effects, allelic effects, and fitness distributions from being developed, and we consider how sample limitations and the nonequilibrium of human populations caused by our demographic history make rigorous solutions difficult. However, based on what is currently known, we argue that for human quantitative chronic disease risk factors the nearly neutral models of allelic evolution at single loci probably apply reasonably well. In general, and although much is still speculative, the data available for such risk factors are consistent with these expectations and may enable us to predict many aspects of etiologic heterogeneity for human disease.

The effects of recurrent clonal formation on clonal invasion patterns and sexual persistence: a Monte Carlo simulation of the frozen niche-variation model.

The interaction of sexual and asexual organisms in a heterogeneous environment was explored using a Monte Carlo simulation. The model was designed to address sexual persistence and the pattern of clonal invasion in a species that periodically produces clonal mutants. The parameters of the model were the mutation rate of outcrossed sexuals to obligate asexuality, the number of progeny per parent, the within-genotype niche width, and the carrying capacity for nine separate resources. The inclusion of recurrent clonal invasion due to meiosis-disrupting mutations drove the sexual species extinct in temporally stable environments, at a rate dependent on the mutation frequency, sexual niche breadth, and the relative magnitude of the number of progeny per parent and the carrying capacity. In simulations with uniform resource distributions, clonal invasion was distinctly nonrandom. The pattern of clonal invasion was "centripetal'': mutant clones that captured or "froze" the rarely recombined (or marginal) sexual phenotypes were more successful initially than clones freezing frequently recombined (or central) sexual phenotypes. The long-term persistence of the sexuals was confined to simulations that included temporal resource fluctuations. In such instances, sexuals and asexuals coexisted in a mutation/extinction equilibrium, where asexuals were continually produced by mutation and lost by short-term random extinctions. Increased within-genotype niche width reduced the probability of clonal extinction and thus restricted the likelihood of sexual/clonal coexistence.

The genetic mechanism of sex determination in the conchostracan shrimp Eulimnadia texana.

We report the results of a laboratory pedigree analysis describing the unique sex-determining mechanism of the conchostracan shrimp, Eulimnadia texana. Natural populations of E. texana are mixtures of self-compatible hermaphrodites and males and represent one of the few known cases of androdioecy in animals. Hermaphrodites are of two types: amphigenic (producing both male and hermaphroditic offspring) and monogenic (producing only hermaphroditic offspring). We propose a simple genetic model to explain this polymorphism and show by genetic analysis that males, amphigenics, and monogenics can be interpreted as three alternative phenotypes of a one-locus system of sex determination. We discuss the implications of this novel system of sex determination for understanding the evolution of reproductive systems.