Daphnia hybridization along ecological gradients in pelagic environments: the potential for the presence of hybrid zones in plankton

The relative homogeneity of pelagic environments has been regarded as the reason for the absence of hybrid zones for hybridizing planktonic Daphnia (Crustacea: Cladocera); occasional dominance of interspecific hybrids over parental species was explained by their temporal superiority in fluctuating environments. However, water bodies with spatially varying environmental conditions might facilitate the formation of hybrid zones in plankton. We studied the distribution of species and hybrids of the Daphnia longispina complex in 11 canyon-shaped reservoirs, localities characterized by horizontal environmental gradients (particularly of food supply and size-selective predation); we also analysed patterns of carapace size and fecundity among coexisting taxa. Spatial distribution of taxa agreed with their ecological characteristics; those showing different affinities along longitudinal reservoir profiles differed in size according to the presumed fish predation gradient. Only hybrids of Daphnia galeata with Daphnia cucullata and D. longispina (=hyalina) were recorded. The latter two species preferred opposite ends of gradients, such spatial segregation probably explaining the absence of their hybrids. Distributional patterns were relatively stable in two consecutive summers, apart from a substantial decline of D. galeata X cucullata in the second year. The observed pattern of a hybrid-dominated zone in intermediate conditions suggests that local Daphnia hybrid zones may indeed form within reservoirs.     

Interspecific hybridization in Daphnia: distinction and origin of hybrid matrilines

Three coexisting Daphnia species belonging to the D. longispina group (D. galeata, D. hyalina, and D. cucullata) form species-hybrid complexes by producing interspecific hybrids in several lakes in Germany and The Netherlands. To evaluate the genetic consequences of interspecific hybridization, I studied the patterns of mitochondrial DNA (mtDNA) sequence variation. The directionality of interspecific hybridization and divergence of hybrids from parental species was tested, using the DNA sequences of a segment of mtDNA. Via the polymerase chain reaction, it was possible to investigate single animals and even single resting eggs. A species-specific marker was established, using restriction patterns of amplified cytochrome b segments. mtDNA genotypes of hybrids revealed unidirectional mitochondrial gene flow for two hybrids, which were investigated by using multiple clones. No evidence for introgression of mtDNA was found. On the basis of a phylogenetic analysis, the species exhibit considerable distinctness, whereas differences between clones within species and between hybrids and maternal species tend to be very low. These results indicate a recent origin of hybrids and suggest that the radiation of the D. longispina group occurred > 5 Mya.      

Spatial distribution of the Daphnia longispina species complex and other planktonic crustaceans in the heterogeneous environment of canyon-shaped reservoirs

Canyon-shaped reservoirs are often characterised by longitudinalgradients of environmental factors (including trophic level,phytoplankton and zooplankton biomass and abundance of planktivorousfish) affecting the taxonomic composition of the pelagic community.We tested the hypothesis that the spatial distribution of differentspecies and interspecific hybrids of the Daphnia longispinaspecies complex is non-random under such conditions. Duringthe summer stratification, we sampled crustacean zooplanktonfrom 11 reservoirs, covering both longitudinal (upstream, middle,dam) and vertical (epi-, meta- and hypolimnion) environmentalgradients. Allozyme electrophoresis was used to discriminateamong different Daphnia taxa. All three frequently hybridizingEuropean species of the complex (galeata, cucullata, longispina= hyalina) and hybrids with Daphnia galeata were commonly recorded.Smaller-bodied Daphnia cucullata and its hybrids, when present,preferred mostly the nutrient- and food-rich upstream regions;D. longispina and its hybrids were more commonly found in thedownstream part, and often dominated in the meta- or hypolimnion.Redundancy analyses confirmed significant differences in theDaphnia taxon composition, as well as in spatial distributionof other crustacean species, along both gradients. For the firsttime, we demonstrate regular patterns in the horizontal distributionof Daphnia species and hybrids within a water body, thus acceptingour hypothesis. Such spatial distributional patterns may stronglyimpact local hybridization processes.     

Hybridization in the Daphnia galeata complex: are hybrids locally produced?

Within the species complex of Daphnia galeata,D. cucullata and D. hyalina variouscombinations of hybrids and parental taxa occur inlakes throughout Europe. Since daphnids are cyclicparthenogens and mostly reproduce asexually, hybridpopulations can be maintained by asexual reproductionand without recurrent hybridization events. Therefore,it is possible that hybridization events have beenrare, with range expansion occurring by dispersal ofhybrids.Allozyme data from seven European populations wereused to compare genetic variation within and betweenhybrid and parental taxa. An UPGMA cluster analysis ofgenetic distances showed that D. cucullata × galeatahybrids from different lakes grouped indifferent clusters according to the lake from whichthey were isolated, suggesting multiple hybridizationevents. Clonal diversity within hybrid taxa wascomparable to parental taxa. Furthermore, evidence wasfound for introgression of the Pgi-S allele fromD. cucullata to D. galeata in three lakes.These results indicate that multiple hybridizationevents within this species complex are likely, andthat hybrid taxa can reproduce sexually.     

Analysis of reticulate relationships within the Daphnia longispina species complex. Allozyme phenotype and morphology

A two-step method is proposed to get reliable associations between morphology and genotype in clonal assemblages in which more than two predominantly parthenogenetic species are thought to coexist with hybrids. In dataset 1, the genetic relationships among clones of the Daphnia longispina hybrid complex from seven prealpine lakes in southern Germany were studied based on the variation at 21 enzyme loci. The spatial arrangement in the multidimensional scaling plot revealed a reticulate pattern among three presumably parental species, D. cucullata, D. galeata and D. hyalina, and three hybrid groups, D. cucullata/galeata, D. cucullata/hyalina and D. galeata/hyalina. The Got1 locus was believed to be a discriminating factor between species and hybrids (cf. Wolf and Mort, 1986). However, this locus is more variable, and 57% of the clones would have been misidentified using it. Moreover, the morphological variation within genetically defined groups is also higher than previously assumed. In dataset 2, the revision of morphological and genetic markers greatly improved the association between morphology and genotype in newly collected animals. The spatial arrangement of clones in multidimensional scaling plots and morphological asymmetries to parents suggest both, different degrees of introgression and bidirectional hybridization. Most unexpected genotypes were found in the cxh hybrid group, suggesting that F1-hybrids are fertile. The results showed (1) that the clonal diversity was very high (2) that detailed analyses of multiple morphological and allozyme markers are necessary to resolve taxonomic relationships within clonal assemblages consisting of multiple species, hybrids and differently introgressed backcrosses, and (3) that the three original species seem to have sufficient within-species recombination and a low enough rate of backcrossing to allow taxonomic identification. It must remain undecided if the present situation is locally restricted, if it is stable or represents a transient situation which could lead to either a consolidation of the three species by gradual elimination of the hybrids, to a taxonomic breakdown, or to hybrid speciation.     

Genetic assessment of species boundaries in the North American Daphnia longispina complex (Crustacea: Daphniidae)

Species boundaries in the North American Daphnia longispina group have proved difficult to establish on the basis of morphology alone. This confusion may be due to hydridization, phenotypic plasticity or the existence of sibling species. We therefore used genetic analysis to delineate species boundaries by examining 27 North American populations belonging to the longispina complex for variation at 15–26 allozyme loci. The populations consisted of Daphnia thorata from two western sites and two eastern sites, Daphnia galeata mendotae from its type location and seven sites across its range, and Daphnia rosea from eight temperate and seven arctic sites. Two populations from the Eurasian longispina complex were also included for reference. Populations assigned to D. galeata mendotae formed a genetically cohesive group, whereas a genetic dichotomy was found between temperate and arctic D. rosea , suggesting that this taxon includes two species. Genetic analysis also confirmed the distinctness of western D. thorata from other members of the longispina group. Unexpectedly, eastern populations resembling D. thorata were genetically more similar to temperate D. rosea than to any helmeted species ( D. galeata, Daphnia hyalina or D. thorata ). Our results suggest that the helmet character is a poor indicator of phylogenetic relationships, as the genetic ability to produce this feature has been lost or acquired several times in the evolution of the longispina group.     

Molecular genetic diagnostics of some Daphnia species (Crustacea,Cladocera) from the Volga river

Population genetic structure of the widespread Daphnia species from Kuibyshev and Saratov reservoirs was examined by use of RAPD-PCR technique with four mitochondrial DNA (16S rRNA) specific primers. One of the examined Daphnia clones from the Volga region appeared to be conspecific to the North American Daphnia galeata, while another clone was most likely a hybrid between D. galeata and D. cucullata.     

Predator-induced life-history changes and the coexistence of five taxa in a Daphnia species complex

Interspecific hybridization is common in water fleas of the Daphnia galeata species complex (e.g. D. galeata , D. cucullata , D. hyalina and their interspecific hybrids). We studied the effect of fish on the life histories of five taxa of this species complex originating from the Plußsee, northern Germany. Using four clones per taxon, we found that fish kairomones reduce size at birth and size at maturity significantly. For size at maturity larger taxa showed a significantly stronger reaction to fish kairomones than the smaller taxa. With respect to the intrinsic rate of increase, r , we compared two predation regimes (positive size selective and not selective). We found that under the fish predation regime most clones had a higher r when cultured with fish kairomones, leading to a higher r , and a stronger reaction for the smaller taxa. We conclude that fish predation might be an important factor influencing the co-occurrence of Daphnia parental taxa with their hybrids.     

Identifying hybridizing taxa within the <Emphasis Type="Italic">Daphnia longispina</Emphasis> species complex: a comparison of genetic methods and phenotypic approaches

Daphnia galeata Sars, D. longispina O. F. Müller and D. cucullata Sars (Crustacea: Cladocera) are closely related species which often produce interspecific hybrids in natural populations. Several marker systems are available for taxon determination in this hybridizing complex, but their performance and reliability has not been systematically assessed. We compared results from identifications by three molecular methods. More than 1,200 individuals from 10 localities in the Czech Republic were identified as parental species or hybrids by allozyme electrophoresis and the analysis of the restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP); over 440 of them were additionally analyzed and identified by 12 microsatellite loci. Identification by microsatellite markers corresponded well with allozyme analyses. However, consistent discrepancies between ITS-RFLP and other markers were observed in two out of 10 studied localities. Although some marker discrepancies may have been caused by occasional recent introgression, consistent deviations between ITS-RFLP and other markers suggest a long-term maintenance of introgressed alleles. These results warn against its use as a sole identification method in field studies. Additionally, we quantitatively evaluated the discriminatory power of geometric morphometric (elliptic Fourier) analysis of body shapes based on photos of over 1,300 individuals pre-classified by allozyme markers. Furthermore, a randomly selected subset of 240 individuals was independently determined from photos by several experts. Despite a tendency for morphological divergence among parental Daphnia species, some taxa (especially D. galeata, D. longispina, and their hybrids) substantially overlapped in their body shapes. This was reflected in different determination success for particular species and hybrids in discriminant analysis based on shape data as well as from photographs.     

Respiratory electron transport system (ETS) activity as an estimator of the thermal tolerance of two Daphnia hybrids

The influence of temperature on the activity of the respiratoryelectron transport system (ETS) was measured in one clone ofDaphnia hyalina x galeata and one of Daphnia cucullata x galeata,isolated from Lake Bled (Slovenia). The ETS activity of ovigerousfemales acclimated to 7, 20 and 25°C, was measured at 5,10, 15, 20, 25 and 30°C. Population growth experiments showedthat D. cucullata x galeata grew better at high rather thanlow temperatures. Daphnia hyalina x galeata, however, grew moresuccessfully at low temperatures than did D. cucullata x galeata.The highest Q₁₀ of ETS activity of D. cucullata x galeata atthe lowest temperature range of 5–15°C indicated theabsence of enzymes that could function sufficiently well atlow temperatures. The ETS activity of the warm-acclimated hybridD. hyalina x galeata reached a maximum at an incubation temperatureof 20°C, while D. cucullata x galeata had maximal ETS activityat 25°C. Thus D. cucullata x galeata has a more efficientenzyme system than D. hyalina x galeata at the higher temperature.The higher Arrhenius activation energy (E_a) for D. cucullatax galeata than for D. hyalina x galeata indicates that enzymesfrom D. cucullata x galeata are more temperature sensitive thanthose from D. hyalina x galeata. In conclusion, the ETS of D.cucullata x galeata is adapted to a higher temperature and tonarrower temperature fluctuations than that of D. hyalina xgaleata.     

Fine-scale genetic analysis of Daphnia host populations infected by two virulent parasites - strong fluctuations in clonal structure at small temporal and spatial scales

Numerous theoretical studies suggest that parasites impose a strong selection pressure on their host, driving genetic changes within host populations. Yet evidence of this process in the wild is scarce. In the present study we surveyed, using high resolution microsatellite markers, the genetic structure of cyclically parthenogenetic Daphnia hosts within two different Daphnia communities belonging to the Daphnia longispina hybrid complex. One community, consisting of a single host species, was infected with the protozoan parasite Caullerya mesnili. The second community consisted of two parental Daphnia spp. and their hybrids, and was infected with the yeast parasite Metschnikowia. Significant differences in the clonal composition between random and infected sub-samples of Daphnia were detected on several occasions within both communities, indicating that host genotypes differ in resistance to both parasites. In addition, one parental species in the multi-taxon community was consistently under-infected, compared with the other taxa. Overall, our field data confirm that infection patterns are strongly affected by host genetic composition in various Daphnia-microparasite systems. Thus, parasite-driven selection operates in natural Daphnia populations and microparasites influence the clonal structure of host populations.     

Allochronic differentiation among Daphnia species, hybrids and backcrosses: the importance of sexual reproduction for population dynamics and genetic architecture

Seasonal dynamics of the abundance, sexual reproduction and genetic architecture in a Daphnia hyalina-galeata hybrid complex were studied in the large and deep Lake Constance. We found evidence for the occurrence of first and second order hybridization. Our study revealed strong differences between the parental species not only regarding their seasonal dynamics, genetic architecture and diversity, but also their sexual reproductive behaviour. The overwintering D. hyalina showed low genetic diversity, no genetic differentiation during the season, and reproduced sexually in autumn, whereas D. galeata reached higher levels of genetic diversity, reproduced sexually in early summer, and exhibited changes in genetic structure during the season, but was only present from spring to autumn. However, in both species sexual reproduction was a rare event, and daphnids, including hybrids, reproduced predominantly asexually. This allows long-term persistence of hybrids as well without continuing hybridization events. Within all variables studied, F1 and F2 hybrids showed an intermediate pattern, whereas proposed backcross hybrids were more similar to their respective parentals. These differences in phenotype as well as significant differences in pairwise Fst values between parentals suggest that gene flow seems to be relatively low in the Lake Constance hybrid system. We found evidence for unidirectional introgression by backcrossing from D. galeata to D. hyalina and found a decrease in at least one of the proposed introgressed alleles in the hyalina-backcross while the season progressed. Our findings suggest allochronic differentiation within this hybrid population and different microevolutionary trajectories of the parental species, which will be discussed in the light of the ongoing reoligotrophication process of Lake Constance.     

Reticulate evolution of the Daphnia pulex complex as revealed by nuclear markers

The study of species complexes is of particular interest to understand how evolutionary young species maintain genomic integrity. The Daphnia pulex complex has been intensively studied as it includes species that dominate freshwater environments in the Northern hemisphere and as it is the sole North American complex that shows transitions to obligate parthenogenesis. Past studies using mitochondrial markers have revealed the presence of 10 distinct lineages in the complex. This study is the first to examine genetic relationships among seven species of the complex at nuclear markers (nine microsatellite loci and one protein-coding gene). Clones belonging to the seven species of the Daphnia pulex complex were characterized at the mitochondrial NADH dehydrogenase (ND5) gene and at the Lactate dehydrogenase (LDH) locus. K-means, principal coordinate analyses and phylogenetic network analyses on the microsatellite data all separated European D. pulicaria, D. tenebrosa, North American D. pulex, D. pulicaria and their hybrids into distinct clusters. The hybrid cluster was composed of diploid and polyploid hybrids with D. pulex mitochondria and some clones with D. pulicaria mitochondria. By contrast, the phylogeny of the D. pulex complex using Rab4 was not well resolved but still showed clusters consisting mostly of D. pulex alleles and others of D. pulicaria alleles. Incomplete lineage sorting and hybridization may obscure genetic relationships at this locus. This study shows that hybridization and introgression have played an important role in the evolution of this complex.     

p-loci: a computer program for choosing the most efficient set of loci for parentage assignment

Determining how many and which codominant marker loci are required for accurate parentage assignment is not straightforward because levels of marker polymorphism, linkage, allelic distributions among potential parents and other factors produce differences in the discriminatory power of individual markers and sets of markers. p-loci software identifies the most efficient set of codominant markers for assigning parentage at a user-defined level of success, using either simulated or actual offspring genotypes of known parentage. Simulations can incorporate linkage among markers, mating design and frequencies of null alleles and/or genotyping errors. p-loci is available for windows systems at http://marineresearch.oregonstate.edu/genetics/ploci.htm.     

Life-history variation in a hybrid species complex ofDaphnia

Summary Life-history variation was examined among members of theDaphnia longispina group, which consists ofD. galeata, D. hyalina, D. cucullata, and hybrids. Factorial experiments were conducted at two temperatures (14° and 20° C) and two food concentrations (0.2 and 1.0 mg Cl⁻¹). Differences in life-history features (size at maturity, age at first reproduction, size of first clutch, offspring size in first clutch) under the different environmental conditions were assessed among eightDaphnia clones, which represented members of this species complex. Significant differences between parentals and hybrids for most life-history features were observed under various treatments; generally, hybrid clones showed intermediate life-history traits when compared with parentals. When comparisons were made among clones within a given species (i.e.D. galeata, D. galeata xcucullata, D. cucullata), clonal differences were also noted for certain life-history traits. The data are discussed with reference to the formation and maintenance of hybrid species complexes in nature. This paper is dedicated to the memory of Hans Georg Wolf, who died suddenly in May 1990 at the age of 39 years.     

New microsatellite primers for Daphnia galeata mendotae

In order to study genetic changes in populations of Daphnia galeata mendotae, I characterized seven polymorphic microsatellite loci. Primers to amplify these loci were tested on individual eggs from the resting egg bank of Onondaga Lake, NY. Levels of polymorphism and cross‐amplification in D. g. galeata indicate that they will be useful markers for ecological genetic studies on both adults and diapausing eggs of these species.     

Clonal diversity,clonal persistence and rapid taxon replacement in natural populations of species and hybrids of the Daphnia longispina complex

Hybridization is common among cyclical parthenogens, especially in zooplankton species assemblages of the genus Daphnia. To explore hybridization dynamics and the extent of clonal diversity in the Daphnia longispina complex, we analysed population structure in eight permanent lakes. Based on 15 microsatellite loci, three major taxonomic units emerged: two species, D. galeata and D. longispina and their F1 hybrids, supported by factorial correspondence analysis and two Bayesian methods. At the same time, the detection of backcross classes differed between methods. Mean clonal diversity was lowest in the F1 hybrids, as expected from the high rate of asexual reproduction. Within taxa, replicated genotypes were of clonal origin, but clonal lineages persisted in subsequent years in only one of three resampled lakes. In another lake, the taxon composition changed from being dominated by hybrids to complete dominance by one parental taxon. Such a year‐to‐year taxon replacement has not been reported for the D. longispina complex before. Our data on this hybrid complex illustrate that high‐resolution genotyping is essential for the understanding of ecological and evolutionary outcomes of hybridization in partially clonal taxa.     

Development of twelve novel polymorphic microsatellite DNA markers for the Boeseman’s rainbowfish (Melanotaenia boesemani) and tests for their cross-utility in 21 rainbowfish species from West Papua (Indonesia)

We developed 12 microsatellite markers for the endangered Boeseman’s rainbowfish (Melanotaenia boesemani). Twenty-eight individuals from the type locality at Ayamaru Lake were examined, and all loci were polymorphic with a number of alleles per locus varying from 3 to 18. Average observed and expected heterozygosities were 0.681 and 0.678, respectively. Cross-species amplification was successfully obtained for 21 Melanotaenia species, with a number of alleles per locus ranging between 1 and 20. Average observed and expected heterozygosities varied between 0.105 and 0.708 and 0.118–0.755, respectively. Only 21 inbreeding coefficient (Fis) values presented a significant homozygote excess among the 264 locus-by-locus calculated values. Tests for genotyping errors revealed that four of these 21 significant Fis values could be explained by the presence of null alleles. These new microsatellite markers appear highly reliable for further conservation purposes or population genetic studies of the many rainbowfish endangered species.