Dr. Africa Gómez, Universitat de València

Africa Gómez (1998)

Hydrobiologia, in press

Allozyme electrophoresis is a well established technique for revealing genetic variation that has given useful results in a wide range of organisms. However, in rotifers it has been applied scarcely and only in a few species. In this paper the methods of acetate allozyme electrophoresis are introduced, including laboratory set up equipment and staining recipes that have successfully applied to brachionid rotifers. In addition, the literature published on allozyme electrophoresis in rotifers is reviewed and main results and prospects of the technique are discussed. I conclude that, despite the availability of DNA techniques, allozyme electrophoresis is promising and can yield important results in rotifer population genetics, ecology and systematics.

Manuel Serra, África Gómez, & María José Carmona (1998)

Hydrobiologia, in press

In this paper we review previous studies on sympatric Brachionus populations in Torreblanca Marsh as a model of evolutionary and ecological relationships between closely related species. The marsh is a wetland in the Mediterranean coast of Spain with high spatial and temporal heterogeneity. Allozyme and morphometric analysis showed that Brachionus group plicatilis (formerly, Brachionus plicatilis and currently split into B. plicatilis and B. rotundiformis) was composed by three groups of genotypes with no evidence of gene flow between them (B. plicatilis, B. rotundiformis SM and B. rotundiformis SS). Correlation between seasonal and spatial distributions, on one hand, and temperature and salinity, on the other hand, were consistent with the results of experimental studies of population dynamics. Accordingly, B. plicatilis is an eurihaline, low temperature group, B. rotundiformis SM is better adapted to high temperature and low salinity, and B. rotundiformis SS is adapted at high temperature and high salinity conditions. The groups had different mictic response to density, salinity and temperature, which can be explained to some extent as an adaptive escape response, given their different ecological preferences. These differences imply a partial ecological barrier for male-female encounter between groups. Mating experiments showed that most copulations occurred within group. B. plicatilis had a mating recognition system different to either B. rotundiformis SM and SS, whereas the two B. rotundiformis groups had partially differentiated mating preferences. Cross-mating experiments performed in the laboratory failed to produce any hybrids. We conclude that three sympatric sibling species inhabit Torreblanca Marsh. The remarkable covariation between genetic differences among clonal groups, and their ecological preferences, mixis response and mating behavior is hypothesized to play a role in stable sympatry, and give insight into the evolution of genetic divergence and speciation in rotifers.

Africa Gómez, Gary Cecchine & Terry W. Snell (1997)

Aquatic Toxicology, 37, 271-282.

The effect of pentachlorophenol on the predator-prey relationship of two rotifers (Asplanchna girodi and Brachionus calyciflorus) was studied using Asplanchna predatory behavior as an endpoint. The experimental design included three treatments: prey and predator exposure, only prey exposure, and only predator exposure. This enabled us to distinguish toxic effects on the predator from those on the prey and to detect interactions. The main toxic effects observed were on the predator and involved the number of predator-prey encounters. Encounters decreased in a dose-dependent manner, with a no observed effect threshold at 0.11 mg PCP/L. Decrease in encounter rate reduced the number of captures, ingestions, and time to first ingestion, parameters that are directly related to the predator's fitness. The importance of these results in assessing potential damage to functional relationships in aquatic ecosystems is discussed.

Africa Gómez, María José Carmona and Manuel Serra (1997)

Oecologia, 111, 350-356.

We investigated how adaptation to salinity and temperature acts as reproductive barriers in three sympatric species from the Brachionus plicatilis species complex. These species co-occur in a salt marsh in Spain, and a previous electrophoretic study of variation revealed no hybrids between them. A factorial experiment was designed to test for differences in population growth rates and patterns of bisexual reproduction. The design combined representative strains from each species in different salinity and temperature conditions, representing the range over which these rotifers are found in their natural environment. We found differences in the growth response of the three species to both factors and in the pattern of bisexual reproduction. These differences help to explain succession patterns occurring in the field. We conclude that these ecological factors together with mate recognition systems account for the absence of gene flow in these sympatric species.

Manuel Serra, Agustí Galiana and Africa Gómez (1997)

Hydrobiologia, 358, 63-70.

Monogonont rotifers are cyclical parthenogens living in limnic habitats with considerable seasonal variation and often with island-like features. The occurrence of bisexual reproduction in these organisms makes it feasible to define 'fields for gene recombination', i.e., biological species. In this paper, we analyze the structure of diversity in monogonont rotifers using several data sets: taxonomic and intraspecific diversity as reported in identification keys, morphological variation reported in ecological studies, and allozyme and mating behavior patterns. We also study the role of dispersal, mate recognition systems and cyclical parthenogenesis in processes driving speciation. Our analysis shows that a great deal of organic diversity is present in low taxonomic levels, and suggests that sibling species are frequent. Monogonont rotifers seem to have conditions for an active speciation, which may be particularly promoted by seasonal specialization and timing of bisexual reproduction.

Africa Gómez and Terry W. Snell (1996)

Journal of Evolutionary Biology, 9, 953-964.

An attempt is made to identify sibling species within the Brachionus plicatilis complex (Rotifera). Allozyme and morphological data for nine strains from all over the world are provided. Although the analysis of morphological data classifies individuals from nine strains in two groups, cluster analysis using genetic distance data obtained from allozyme data reveal at least three groups of strains. A male choice design is described for the first time in rotifers and used to test for male mate preferences using sympatric male strains which belong to three distinct species. The results suggest that the B. plicatilis complex is actually composed of at least three sibling species.

Africa Gómez & Manuel Serra (1996)

Functional Ecology, 10, 681-687.

1. Some proximate mechanisms that increase the probability of successful copulation are examined in Brachionus plicatilis (Rotifera), a cyclical parthenogen. Using behavioural tests, we analysed first whether male choice exists concerning female age. Second, we examined male direct discrimination of sexual (mictic) and parthenogenetic (amictic) females on the basis of male behaviour after encountering both kinds of females.

2. Results showed that the probability of male mating initiation decreases with the age of female, and that males copulate almost exclusively with females less than 24 h old. Preference for sexual females was also found in the probability of mating initiation, but no preference for copulating sexual females was found.

3. As only young, sexual females are fertilisable, these mechanisms would enhance the likelihood of a male copulating with a sexually receptive female, and, thus, male fitness.

4. A theoretical model shows that the surprisingly low degree of male preference for sexual females can be adaptively explained on the basis of relative frequency of young females and mictic females in the population when the sexual phase occurs.

África Gómez, Miguel Temprano & Manuel Serra (1995)

Journal of Evolutionary Biology, 8, 601-622.

Populations of the rotifer Brachionus plicatilis inhabiting three temporary ponds in Torreblanca Marsh (Castellón, Spain) were regularly screened for allozyme variation, sexual reproduction levels and population densities during an annual cycle. Relevant ecological parameters in the ponds were also recorded. The electrophoretic survey of the three ponds (Poza Sur, Poza Norte and Canal Central) revealed a high level of overall genetic polymorphism in four marker loci, but only 13 multilocus genotypes were found. According to their four-locus genotypes, we classified clones into three clonal groups (SS, SM, L) characterized by unique arrays of alleles in the four marker loci, and significant differences in body shape and size. Clonal group succession took place in Poza Sur; SM clones occurring in spring, SS clones from spring to fall, and L clones from fall to spring. Despite the partial overlapping of sexual periods, the absence of heterozygotes indicates that gene flow is strongly restricted between clonal groups. In the transition periods, the population was far from Hardy-Weinberg equilibrium due to heterozygote deficiencies in all four markers. In clonal group L, the only polymorphic locus, Pgi, was in Hardy-Weinberg equilibrium. In clonal group SM, both polymorphic loci were in Hardy-Weinberg equilibrium in Poza Norte, but not in Poza Sur. Factor analysis on the limnological parameters recorded during field sampling indicates that genetically different clonal groups are also ecologically specialized. These findings suggest that the taxonomical species B. plicatilis is in fact a species complex. The complete genetic discontinuity model of rotifer population succession is supported by these data. Our results are similar to those found in cladocerans inhabiting temporary habitats.

María José Carmona, Africa Gómez and Manuel Serra (1995)

Hydrobiologia, 313/314, 365-371.

Populations of the rotifer Brachionus plicatilis were monitored in three small ponds in a marsh on the Mediterranean coast. Samples were taken approximately every three weeks from July 1992 to November 1993. Salinity, temperature, conductivity, pH and oxygen concentration were measured in the field. Population density was determined from preserved quantitative samples. Individuals were classified as mictic females, amictic females, non-ovigerous females, and males, differentiating between two morphotypes ("S" and "L"). From these counts, a level of mixis was calculated. We also determined the proportion of mictic females in natural populations by culturing females isolated from fresh samples. From these data, mictic patterns over time and correlation between levels of mixis and environmental and population parameters were analyzed. From a previous study "S" and "L" morphotypes were known to correspond to genetically different clonal groups. Our data showed that reproduction was predominantly parthenogenetic in these clonal groups, but mictic females were found in most samples, the proportion of mictic females ranging from 0 to 29%. The clonal groups showed different patterns of mixis. L clonal group presented a continuous sexual reproductive pattern. In contrast, S clones showed a rather punctuated mictic pattern. A positive correlation between levels of sexual reproduction and population density was found for S and L groups. However, they differed in their density threshold for mictic reproduction. The adaptive meaning of these patterns and their implications in maintaining genetic diversity within and between populations are discussed.

Africa Gómez & Manuel Serra (1995)Hydrobiologia, 313/314, 111-119.

In this study, we present results on cross-mating experiments using Brachionus plicatilis strains collected in three ponds of a coastal marsh (Torreblanca Marsh, Castellón, Spain). These strains were known to differ widely both in morphology and allozyme patterns from a previous study, where they were grouped into three genetically different clonal groups. Although some of the strains co-occurred in the same pond and sexual periods overlapped, no trace of gene flow was found among them. Our first objective was to determine whether behavioral reproductive isolation was responsible for the absence of interbreeding. A second objective was to explore the relationship between sexual isolation and genetic divergence. We performed two experiments. In Experiment 1, we tested five strains from different clonal groups; in Experiment 2, we added a strain from a congeneric species, and strains from different ponds. We recorded male mating behavior in all possible male-female strain pairings. Our data show that males of a strain tend to mate with females of the same strain or genetically similar strains, regardless of the pond they come from. We also found a high positive correlation between isolation distance and genetic distance. These results support the view that mating behavior acts as an important isolating mechanism giving cohesion to the clonal groups, and structuring populations of this rotifer, and that Brachionus plicatilis is a taxon composed of more than one well defined biological species.

Africa Gómez & Gary R. Carvalho

(submitted to Molecular Ecology)

Cyclically parthenogenetic rotifers offer a valuable opportunity to investigate the relationship between reproductive mode and population structure, though advances in this field have been hindered by the low variability in allozyme loci in these organisms. A high genotypic diversity in genetic equilibrium is predicted after sexual reproduction, 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 in equilibrium. 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 (1) the genetic structure of the resting egg bank, (2) the changes in the genetic structure of rotifer populations during the parthenogenetic phase, (3) 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 rotifer population became more structured during the parthenogenetic phase. The last sample in the parthenogenetic phase shows evidence of clonal selection as indicated by a low observed clonal diversity and the appearance of linkage disequilibrium. The resting egg bank, analysed comprehensively for the first time in any zooplankter, is in genetic 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.