Preliminary Study of Temperature Effects on Size and Shape in the Modern Spinicaudatan Eulimnadia texana (Crustacea: Branchiopoda).

Studies of temperature effects on morphology in Spinicaudata have focused on length, with no data on shape. To fill this gap, size and shape variability in response to temperature fluctuations was investigated by rearing the modern spinicaudatan Eulimnadia texana. Two days after hydration, juvenile individuals were separated into four different temperature treatments: 20°C, 23°C, 26°C, and 29°C. Hermaphrodite size and shape were analysed by looking at linear combinations of size variables and using Fourier shape analysis; methods that are also used to describe fossil size and shape for better comparison. Size differences were considerable, with reduced growth at low and high temperatures and accelerated growth at the optimum temperature of 26°C, revealing that the reaction of size to increasing temperature is non-linear. The height of the dorsal margin, which is associated with space for egg production in Eulimnadia texana, accounts for a high amount of size variability in this species and, presumably, in most of the Limnadiidae. Hermaphrodite shapes reared under temperatures of 20°C and 29°C are statistically distinct, while intermediate temperatures yield intermediate shapes. The rate of shape change along temperature is comparatively low between 23°C and 26°C and accelerated at lower and higher temperatures. With increasing temperature, the highest point of the dorsal margin is shifted towards the anterior of the carapace, while it assumes a median position at 20°C. Our result that temperature has strong effects on carapace size and shape implies considerable ecophenotypic variability in Spinicaudata.

Testing Weissman's Lineage Selection Model for the Maintenance of Sex: The Evolutionary Dynamics of Clam Shrimp Reproduction over Geologic Time.

One of the most perplexing questions within evolutionary biology is: "why are there so many methods of reproduction?" Contemporary theories assume that sexual reproduction should allow long term survival as dispersal and recombination of genetic material provides a population of organisms with the ability to adapt to environmental change. One of the most frustrating aspects of studying the evolution of reproductive systems is that we have not yet been able to utilize information locked within the fossil record to assess breeding system evolution in deep time. While the fossil record provides us with information on an organism's living environment, as well as some aspects of its ecology, the preservation of biological interactions (reproduction, feeding, symbiosis, communication) is exceedingly rare. Using both information from extant taxa uncovered by a plethora of biological and ecological studies and the rich representation of the Spinicaudata (Branchiopoda: Crustacea) throughout the fossil record (from the Devonian to today), we address two hypotheses of reproductive evolutionary theory: (1) that unisexual species should be short lived and less speciose than their outcrossing counterparts and (2) that androdioecy (mixtures of males and hermaphrodites) is an unstable, transitionary system that should not persist over long periods of time. We find no evidence of all-unisexual spinicaudatan taxa (clam shrimp) in the fossil record, but do find evidence of both androdioecious and dioecious clam shrimp. We find that clades with many androdioecious species are less speciose but persist longer than their mostly dioecious counterparts. These data suggest that all-unisexual lineages likely do not persist long whereas mixtures of unisexual and sexual breeding can persist for evolutionarily long periods but tend to produce fewer species than mostly sexual breeding.

Population Density Effects on Carapace Growth in Clam Shrimp: Implications for Palaeontological Studies.

Fossil morphological data are time-averaged and generally reflect an overlap of different sources of carapace variability. To examine whether a proposed relationship between size and population density in fossil spinicaudatans is biologically meaningful, we set up rearing experiments involving two extant species: Eulimnadia texana and Eocyzicus argillaquus. Three and five days after hydration, clam shrimp were transferred into cups of various population densities that ranged between 1 and 15 inds/400 ml. Size and shape were measured 14 and 16 days after hydration, respectively. Every second day, we recorded length and sex of E. texana, which matured faster in lower-density cups. According to our growth model, population density and maximal carapace length follow a logarithmic relationship. At maturity, hermaphrodites yielded similar lengths across all population densities (~4.7 mm at 24°C), independent of age. Hence, clam shrimp can put off reproductive maturity as a response to decreased growth under higher density conditions. Growth rate generally decreases at maturity, but that effect is more pronounced in clam shrimp of high population densities, while low-density adults keep growing. For both species, multivariate analyses reveal that carapace size of low-density individuals is significantly larger than carapace size of higher-density individuals, while size values of intermediate densities cannot be distinguished. Shape distinction is strong in hermaphrodites of E. texana: 39.8% of the density-dependent shape variation is associated with relative umbo height, which is generally higher in individuals of smaller population densities. The H/L ratio, which is often used as a simple shape indicator, does not contribute to the main variation in shape, but it forms one of several ratios significant for 18.3% of the shape variability. In turn, the H/L ratio drives 30% of the shape variation in E. argillaquus. In addition, higher densities triggered shifts in ontogenetic growth trajectories in one third of the individuals, which led to aberrant morphologies. The present rearing experiment shows that some of the morphological variability on fossil bedding planes can be explained by population density. Also, it implies a considerable amount of ecophenotypic variability in Spinicaudata that affects our understanding of fossil taxonomy and palaeoecology.

A New Standard for Crustacean Genomes: The Highly Contiguous, Annotated Genome Assembly of the Clam Shrimp Eulimnadia texana Reveals HOX Gene Order and Identifies the Sex Chromosome.

Vernal pool clam shrimp (Eulimnadia texana) are a promising model system due to their ease of lab culture, short generation time, modest sized genome, a somewhat rare stable androdioecious sex determination system, and a requirement to reproduce via desiccated diapaused eggs. We generated a highly contiguous genome assembly using 46× of PacBio long read data and 216× of Illumina short reads, and annotated using Illumina RNAseq obtained from adult males or hermaphrodites. Of the 120 Mb genome 85% is contained in the largest eight contigs, the smallest of which is 4.6 Mb. The assembly contains 98% of transcripts predicted via RNAseq. This assembly is qualitatively different from scaffolded Illumina assemblies: It is produced from long reads that contain sequence data along their entire length, and is thus gap free. The contiguity of the assembly allows us to order the HOX genes within the genome, identifying two loci that contain HOX gene orthologs, and which approximately maintain the order observed in other arthropods. We identified a partial duplication of the Antennapedia complex adjacent to the few genes homologous to the Bithorax locus. Because the sex chromosome of an androdioecious species is of special interest, we used existing allozyme and microsatellite markers to identify the E. texana sex chromosome, and find that it comprises nearly half of the genome of this species. Linkage patterns indicate that recombination is extremely rare and perhaps absent in hermaphrodites, and as a result the location of the sex determining locus will be difficult to refine using recombination mapping.

The role of androdioecy and gynodioecy in mediating evolutionary transitions between dioecy and hermaphroditism in the animalia.

Dioecy (gonochorism) is dominant within the Animalia, although a recent review suggests hermaphroditism is also common. Evolutionary transitions from dioecy to hermaphroditism (or vice versa) have occurred frequently in animals, but few studies suggest the advantage of such transitions. In particular, few studies assess how hermaphroditism evolves from dioecy or whether androdioecy or gynodioecy should be an "intermediate" stage, as noted in plants. Herein, these transitions are assessed by documenting the numbers of androdioecious and gynodioecious animals and inferring their ancestral reproductive mode. Both systems are rare, but androdioecy was an order of magnitude more common than gynodioecy. Transitions from dioecious ancestors were commonly to androdioecy rather than gynodioecy. Hermaphrodites evolving from sexually dimorphic dioecious ancestors appear to be constrained to those with female-biased sex allocation; such hermaphrodites replace females to coexist with males. Hermaphrodites evolving from sexually monomorphic dioecious ancestors were not similarly constrained. Species transitioning from hermaphroditic ancestors were more commonly androdioecious than gynodioecious, contrasting with similar transitions in plants. In animals, such transitions were associated with size specialization between the sexes, whereas in plants these transitions were to avoid inbreeding depression. Further research should frame these reproductive transitions in a theoretical context, similar to botanical studies.

Ancient androdioecy in the freshwater crustacean Eulimnadia.

Among the variety of reproductive mechanisms exhibited by living systems, one permutation--androdioecy (mixtures of males and hermaphrodites)--is distinguished by its rarity. Models of mating system evolution predict that androdioecy should be a brief stage between hermaphroditism and dioecy (separate males and females), or vice versa. Herein we report evidence of widespread and ancient androdioecy in crustaceans in the genus Eulimnadia, based on observations of over 33,000 shrimp from 36 locations from every continent except Antarctica. Using phylogenetic, biogeographical and palaeontological evidence, we infer that androdioecy in Eulimnadia has persisted for 24-180 million years and has been maintained through multiple speciation events. These results suggest that androdioecy is a highly successful aspect of the life history of these freshwater crustaceans, and has persisted for orders of magnitude longer than predicted by current models of this rare breeding system.

When males and hermaphrodites coexist: a review of androdioecy in animals.

Androdioecy (populations consisting of males and hermaphrodites) is a rare mating system in plants and animals: up to 50 plants and only 36 animals have been described as being androdioecious, with most of the latter being crustaceans. To date, a thorough comparative analysis of androdioecy in animals has not been undertaken. Herein we present such an analysis. Androdioecy has only been extensively surveyed in 2 animal taxa: the nematode Caenorhabditis and the clam shrimp Eulimnadia. The other major taxon having androdioecious species is the Cirripedia (barnacles), but there are only limited studies on androdioecy in this group. In animals, androdioecy is found either in species that have morphologically and ecologically distinct sexes (that is, hermaphrodites and small, "complemental" males) that are derived from hermaphroditic ancestors (that is, the barnacles) or in species that have similarly-sized males and hermaphrodites that have been derived from dioecious ancestors (the remaining androdioecious species). We suggest that the barnacles have evolved a sexual specialization in the form of these complemental males that can more efficiently use the constrained habitats that these barnacles often experience. For the remaining species, we suggest that androdioecy has evolved as a response to reproductive assurance in species that experience episodic low densities. Additionally, we hypothesize that the development of mechanisms allowing reproductive assurance in species with a number of sexually differentiated traits is most likely to result in androdioecy rather than gynodioecy (mixtures of females and hermaphrodites), and that these species may be developmentally constrained to stay androdioecious rather than being capable of evolving into populations solely consisting of efficient, self-compatible hermaphrodites. We conclude by suggesting several areas in need of further study to understand more completely the evolution and distribution of this interesting mating system in animals.

INBREEDING DEPRESSION IN A SELF-COMPATIBLE, ANDRODIOECIOUS CRUSTACEAN, EULIMNADIA TEXANA.

The observation that offspring produced by the mating of close relatives are often less fit than those produced by matings between unrelated individuals (i.e., inbreeding depression) has commonly been explained in terms of the increased probability of expressing deleterious recessive alleles among inbred offspring (the partial dominance model). This model predicts that inbreeding depression should be limited in regularly inbreeding populations because the deleterious alleles that cause inbreeding depression (i.e., the genetic load) should be purged by regularly exposing these alleles to natural selection. We indirectly test the partial dominance model using four highly inbred populations of an androdioecious crustacean, the clam shrimp Eulimnadia texana. These shrimp are comprised of males and hermaphrodites, the latter capable of either self-fertilizing or mating with a male (i.e., outcrossing between hermaphrodites is impossible). Hermaphrodites are further subdivided into monogenics (produced via self-fertilization) and amphigenics (produced via self-fertilization or outcrossing). Electrophoretic evidence suggests significant differences in heterozygosity among populations, but that selfing rates were not statistically different (average s = 0.67). Additional electrophoretic analyses reveal that three previously described sex-linked loci (Fum, Idh-1, and Idh-2) are all tightly linked to each other, with crossing over on the order of 1% per generation. Although selfing rates are clearly high, we present evidence that early inbreeding depression (hatching rates, juvenile survival, and age at sexual maturity) exists in all four populations. For all of these factors, inbreeding depression was inferred by the positive correlation of multilocus heterozygosity and fitness. Cumulative inbreeding depression (8) is between 0.41 and 0.47 across all populations, which appears to be too low to limit the effects of purging via identity disequilibrium. Instead, we suggest that the maintenance of inbreeding depression in these populations is due to the observed linkage group, which we suggest contains a large number of genes including many related to fitness. Segregation of such a large linkage group would explain our observations of the predominance of amphigenic hermaphrodites in our field samples and of survival differences between monogenics and amphigenics within selfed clutches. We propose that a modified form of the overdominance model for inbreeding depression operating at the level of linkage groups maintains the observed levels of inbreeding depression in these populations even in the face of high rates of selfing.

Phenotypic plasticity of life-history traits in clonal and sexual fish (Poeciliopsis) at high and low densities.

Models of resource allocation strategies predict an array of life-history responses of individuals living in resource-stressed versus non-stressed environments. I tested a number of these predictions using three fish strains (a sexual and two clonal strains) in high and low density treatments. To examine the plasticity of life-history traits in females raised in these two environments, I measured survival, growth, egg production, egg size, and proportion mature at 10 weeks of age. Survival was not affected by density treatment. However, both growth and overall egg production were lower in females from the high density treatments, and reproductive maturity was significantly delayed at the high density for all strains. Egg production per unit size was not affected by density in any strain, signifying that differences in the numbers of eggs produced was merely a reflection of the differences in size of fish in the two density treatments. Egg size was also unaffected by density in all strains. These results are related to models of resource allocation in stressful environments. There was a consistent pattern of increased reproductive investment in the sexual strain relative to the two clonal strains. The sexual strain matured earlier, produced more eggs per unit body weight, and had larger eggs than either clone at both densities. These results are interpreted by considering the predicted adaptive responses of these three strains to the long-term environmental differences in their natural habitats.

Competition in phenotypically variable and uniform populations of the tadpole shrimp Triops longicaudatus (Notostraca: Triopsidae).

Life-history parameters (growth, reproduction, and survival) were measured from one genetically heterogeneous and three homogeneous populations of tadpole shrimp (Triops longicaudatus) raised under three competitive levels: 5, 10, and 16 shrimp per 38-1 aquarium. Comparisons were made between the homogeneous populations (three monomorphic selfing lines) and a heterogeneous population (a mixture of the three lines) to test for increased productivity in genetically variable populations, a prediction of the tangled bank hypothesis for a short-term advantage to sexual reproduction. In most comparisons, the mixture of inbred lines performed better than expected from their individual performances in pure cultures but did not outperform the best performing line in any comparison. Decomposition of the mixed culture's performance into performances of the component inbred lines showed that not all lines experienced release from competition in the mixed culture. No genotype X environment interaction was found for any of the three fitness correlates, indicating that all three lines performed similarly under the three competitive conditions. These results provide little support for the tangled bank hypothesis and suggest that the levels of heterogeneity necessary for detectable resource partitioning in these shrimp may involve factors not considered in this study.

GENOTYPIC AND ENVIRONMENTAL COMPONENTS OF VARIATION IN GROWTH AND REPRODUCTION OF FISH HEMICLONES (POECILIOPSIS: POECILIIDAE).

The frozen-niche-variation model was proposed to account for the coexistence of genetically related clones in naturally occurring unisexual populations. This model is based on two assumptions: 1) ecologically different clones have multiple independent origins from sexual ancestors; and 2) the population of sexual ancestors contains genetic variability for ecologically relevant traits. To test these assumptions, we produced 14 new "hemiclones" (nonrecombining haploid genotypes) of fish (Poeciliopsis: Poeciliidae). Our ability to synthesize many new hemiclones demonstrates the feasibility of multiple independent origins of nonrecombining genotypes. A substantial proportion (10-50%) of the phenotypic variation among hemiclones in size at birth, juvenile growth rate, and fecundity had a genetic basis. Thus, we conclude that multiple origins can give rise to an assemblage of genetically distinct hemiclones, each with a unique combination of life-history traits. Additionally, a comparative analysis of two natural hemiclones revealed that the synthetic strains represent a broad field of variation from which natural hemiclones can be selected.