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.     

Amplified Fragment Length Polymorphism (AFLP) Reveals Species-Specific Markers in the Daphnia Galeata–HyalinaSpecies Complex

Daphnia often occur in species complexes that consist of two or more co-occurring species and their hybrids. Hybrid individuals are often capable of sexual reproduction and so backcrossing with introgression occurs. To better understand hybridization and backcrossing frequency, we sought to develop PCR-based, species-specific markers in the Daphnia galeata–hyalina species complex using amplified fragment length polymorphism (AFLP). This technique produces large numbers of reproducible markers for assessing diversity across the nuclear genome and provides several advantages over mtDNA and microsatellite approaches. We examined 28 clones of D. galeata, D. hyalina, and their hybrids isolated from Lake Constance on the Swiss-German border. Using a single AFLP primer combination we found five potential species-specific markers, defined as bands that occurred in >80% of one parental species and <20% of the other. Two bands appeared to be co-dominant and were present (homozygous) in D. galeata, absent in D. hyalina, and heterozygous in the hybrid. We conclude AFLP could provide enough PCR-based, species-specific markers to identify species, hybrids, and backcrosses from even small amounts of tissue (i.e. resting eggs).     

Isolation and development of microsatellite markers for the Japanese dormouse, Glirulus japonicus

Eight microsatellite markers were developed for the Japanese dormouse (Glirulus japonicus), a natural monument and near‐threatened species in Japan. The markers amplify in individuals from all of the mitochondrial lineages detected in a previous study. Numerous polymorphisms were detected in specimens from a local population in central Honshu (11–21 alleles per locus; n = 31) and from the entire distribution range of the species (19–41 alleles per locus; n = 152). These microsatellites will be useful in conservation genetic studies of G. japonicus.     

Eleven polymorphic microsatellite markers for Oedaleus decorus (Orthoptera, Acrididae), an endangered grasshopper in Central Europe

We isolated and characterized 11 microsatellite loci in the grasshopper Oedaleus decorus (Orthoptera: Acrididae), an endangered species in Central Europe. Polymorphism was studied from two populations, one out of two populations known from Switzerland (n = 20 individuals) and one site from south of France (n = 20). The number of alleles and the expected heterozygosity ranged from five to 12 and from 0.559 to 0.898, respectively, in the Swiss population, and from 14 to 23 and from 0.895 to 0.974, respectively, in the French population. These microsatellite markers are suitable for further conservation genetic studies of O. decorus.     

Microsatellite DNA markers for the Chinese wood frog (Rana chensinensis) and tests for their cross-utility in 15 ranid frog species

We developed 22 microsatellite markers for the Chinese wood frog (Rana chensinensis) to study the impact of landscape features on its population structure. Thirty‐four individuals from one breeding site were examined and 14 loci were polymorphic. The number of alleles, expected heterozygosity and observed heterozygosity varied from two to 14, from 0.0833 to 0.9118, and from 0.1376 to 0.8667, respectively. Cross‐species amplification was tested for 15 ranid frog species. The Plateau brown frog, Rana kukunoris (n = 23), was successfully amplified at 18 loci, and 15 were polymorphic with number of alleles varying from two to 18. Ten other species were also amplified at a limited number of loci.     

The role of hybridization in the origin and spread of asexuality in Daphnia

The molecular mechanisms leading to asexuality remain little understood despite their substantial bearing on why sexual reproduction is dominant in nature. Here, we examine the role of hybridization in the origin and spread of obligate asexuality in Daphnia pulex, arguably the best‐documented case of contagious asexuality. Obligately parthenogenetic (OP) clones of D. pulex have traditionally been separated into ‘hybrid’ (Ldh SF) and ‘nonhybrid’ (Ldh SS) forms because the lactase dehydrogenase (Ldh) locus distinguishes the cyclically parthenogenetic (CP) lake dwelling Daphnia pulicaria (Ldh FF) from its ephemeral pond dwelling sister species D. pulex (Ldh SS). The results of our population genetic analyses based on microsatellite loci suggest that both Ldh SS and SF OP individuals can originate from the crossing of CP female F₁ (D. pulex × D. pulicaria) and backcross with males from OP lineages carrying genes that suppress meiosis specifically in female offspring. In previous studies, a suite of diagnostic markers was found to be associated with OP in Ldh SS D. pulex lineages. Our association mapping supports a similar genetic mechanism for the spread of obligate parthenogenesis in Ldh SF OP individuals. Interestingly, our study shows that CP D. pulicaria carry many of the diagnostic microsatellite alleles associated with obligate parthenogenesis. We argue that the assemblage of mutations that suppress meiosis and underlie obligate parthenogenesis in D. pulex originated due to a unique historical hybridization and introgression event between D. pulex and D. pulicaria.     

Polymorphic microsatellite markers for the critically endangered Balearic shearwater, Puffinus mauretanicus

Ten novel polymorphic microsatellite loci were isolated and characterized from the Balearic shearwater (Puffinus mauretanicus), a critically endangered seabird. The developed loci revealed a relatively low number of alleles per locus, as well as low levels of polymorphism (H_O = 0.377 ± 0.241). One of the loci appeared to be W‐linked. All polymorphic loci were successfully amplified in its closely related species, the Yelkouan shearwater (Puffinus yelkouan). These microsatellite markers would be useful for assessing population structure in the Balearic shearwater and the possible hybridization process between both shearwaters species.     

Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Invasibility of a reservoir to exotic Daphnia lumholtzi: experimental assessment of diet selection and life history responses to cyanobacteria

SUMMARY 1. In reservoirs of the south-central United States, the exotic cladoceran Daphnia lumholtzi is common during warm midsummer conditions, when cyanobacteria are abundant and native Daphnia are rare. In the current study, we employed surveys, field experiments, and a life table experiment to investigate the role of food quality in explaining the distribution and phenology of D. lumholtzi , relative to two native species ( Daphnia parvula and Daphnia mendotae ).
2. During May–September 2000 in eutrophic McDaniel Lake, Missouri U.S.A., cyanobacteria (primarily Oscillatoria ) first appeared at 6-m depth and then became abundant throughout the epilimnion.
3. During the May field experiment, D. lumholtzi , D. parvula and D. mendotae all consumed a similar diet of algae, showing positive selection for small greens (chlorophytes and cryptophytes <20 μm). During the July experiment, when the epilimnion exceeded 25 °C and cyanobacteria were common in the lake, D. lumholtzi consumed significantly more total algae and more cyanobacteria than the two native species. Although the Daphnia selected against cyanobacteria, all three species consumed about 25% of this food in their diet.
4. A life table experiment compared the responses of D. lumholtzi and D. parvula with variation in density of high-quality food ( Ankistrodesmus ) and concentration of a toxic strain of cyanobacteria ( Anabaena flos-aquae ). Both Daphnia species showed reduced survivorship, fertility and intrinsic rates of increase in response to elevated concentrations of cyanobacteria, particularly at the higher food level.
5. The results suggest that D. lumholtzi shows similar inhibition from cyanobacteria as does the native Daphnia . However, their continued high in situ feeding rates imply that D. lumholtzi is less affected by midsummer conditions in warm-water reservoirs than are native Daphnia .     

Isolation and characterization of microsatellite loci in the great bustard,Otis tarda

With the aim to study population genetics of the endangered great bustard, Otis tarda, dinucleotide microsatellite loci were isolated using an adapted hybrid‐capture enrichment protocol. This work reports the characterization of a set of six polymorphic microsatellite markers within the great bustard (n = 52). Results from cross‐species amplifications in several other members of the family Otididae demonstrate that five primer pairs also successfully amplified homologous loci outside the species Otis tarda.     

Discrimination of hybrid classes using cross-species amplification of microsatellite loci: methodological challenges and solutions in Daphnia

Microsatellite markers are important tools in population, conservation and forensic studies and are frequently used for species delineation, the detection of hybridization and introgression. Therefore, marker sets that amplify variable DNA regions in two species are required; however, cross-species amplification is often difficult, as genotyping errors such as null alleles may occur. To estimate the level of potential misidentifications based on genotyping errors, we compared the occurrence of parental alleles in laboratory and natural Daphnia hybrids (Daphnia longispina group). We tested a set of 12 microsatellite loci with regard to their suitability for unambiguous species and hybrid class identification using F(1) hybrids bred in the laboratory. Further, a large set of 44 natural populations of D. cucullata, D. galeata and D. longispina (1715 individuals) as well as their interspecific hybrids were genotyped to validate the discriminatory power of different marker combinations. Species delineation using microsatellite multilocus genotypes produced reliable results for all three studied species using assignment tests. Daphnia galeata × cucullata hybrid detection was limited due to three loci exhibiting D. cucullata-specific null alleles, which most likely are caused by differences in primer-binding sites of parental species. Overall, discriminatory power in hybrid detection was improved when a subset of markers was identified that amplifies equally well in both species.     

The outcome of the invasion of Florida lakes by Daphnia lumholtzi

1. We examined 7–12 years of monthly to quarterly historical data from 15 lakes in FL, U.S.A. to determine the extent and outcome of invasion by the alien cladoceran Daphnia lumholtzi Sars. 2. The alien species was found in 10 of the 15 lakes, including Florida’s three largest lakes: Okeechobee, George and Apopka. All the surveyed lakes had resident populations of the smaller native species Daphnia ambigua Scourfield. 3. In most of the lakes, D. ambigua occurred seven to ninefold more often in plankton samples than D. lumholtzi, and at 10‐ to 100‐fold higher maximal densities. One exception was a small lake in central Florida (Lake Jesup), where D. lumholtzi attained high densities on several occasions in the 10 years of sampling. 4. In Lake Okeechobee, where data were of sufficient quality and quantity to perform statistical analyses, the results of canonical correlation analysis indicated that high densities of D. lumholtzi were correlated with lower concentrations of suspended solids, high algal biomass and higher temperature, whereas the opposite conditions were correlated with high densities of D. ambigua. 5. Based on the majority of data, D. lumholtzi has successfully invaded many lakes in Florida, yet it has not become a substantive component of the zooplankton. Additional research is needed to determine whether resources, fish predation or some other factor is responsible for this outcome of invasion.     

Testing the ecological relevance of Daphnia species designations

1. Molecular approaches have increasingly revealed hidden genetic structure within ecologically important species, leading to the creation of sibling species whose ecological relevance is often unclear. A prime example is Daphnia galeata mendotae, which was split into D. dentifera and D. mendotae based on differences at two allozyme loci. 2. In a set of lake populations in Michigan USA, we test the geographical and temporal consistency of the genetic structure underlying this species split. We also test the morphological relevance of this molecular variation and its ecological significance in lakes. In essence, we ask: does recognition of these new species provide valuable information for plankton ecologists? 3. We found that D. dentifera and D. mendotae represent morphologically and ecologically distinct forms that are distributed among lakes in non‐random fashion, which were remarkably stable over 6 years. Key differences between the species concern their body and head shape, vertical habitat use within lakes and distribution among lakes of different size. We hypothesise that these differences represent specialisation to habitats that differ in risk of invertebrate predation. 4. Reproductive barriers alone are insufficient to explain the pattern of genetic structure; in some lakes complete introgression is apparent. However, parent species and hybrids exhibit a stable co‐existence in many lakes, which suggests that ecological specialisation reinforces divergence within this taxon.     

Microsatellite markers for European Daphnia

We present 32 polymorphic microsatellite markers for species of the European Daphnia longispina group: D. galeata, D. hyalina, D. rosea, D. cucullata and D. curvirostris. Microsatellite markers were either isolated from genomic libraries or optimized based on previously published sequence information of sister taxa. Cross‐species tests revealed that all but one of the polymorphic markers are applicable to more than one species, which allows intra‐ and interspecific genetic studies on, i.e. population structure, hybridization events and introgression.     

Sperm number assessed by flow cytometry in species of Daphnia (Crustacea,Cladocera)

Daphnia magna and Daphnia pulex are two important model species in ecotoxicology. In daphniids, studies of the effects of contaminants have mostly focused on female life history traits, yet it would also be important to examine male reproductive traits, particularly in relation to endocrine disruptors. In this study, we developed a protocol that uses flow cytometry to measure sperm number in individual males of different species of Daphnia. We tested our protocol on 114 males from several clones of three common species of Daphnia. Sperm count varied widely among individuals and reached high numbers (up to 1.45 × 10⁵). Positive relationships between male length and sperm number were observed in D. pulex and Daphnia pulicaria, but not in D. magna. Important inter‐clonal differences in sperm production were observed in all species, with some clones producing very little sperm. Duplicated sperm samples showed on average only 6% difference in sperm counts. Sperm counts were stable at least over a 2‐hr period and up to 5 hr for most samples. This sperm isolation protocol and flow cytometric enumeration approach will be of major interest to ecotoxicologists.     

Characterization of nine microsatellite loci in the sea star Astropecten aranciacus and cross-species amplification for related taxa

So far, only few microsatellite markers have been developed and extensively tested for echinoderms. To study the population genetic structure of the sea star Astropecten aranciacus, we developed primers for nine polymorphic microsatellite loci and tested them on two populations from Faro in Portugal (N = 25) and from La Herradura in Spain (N = 20). Within populations, allele numbers varied from four to 20, while expected and observed heterozygosities ranged from 0.593 to 0.936 and from 0.222 to 0.900, respectively. Additional cross‐species amplifications were polymorphic at some loci, indicating their potential usefulness for related taxa.     

Differing effects of suspended sediments on the performance of native and exotic Daphnia

1. Although large cladocerans are usually uncommon in large rivers, Daphnia lumholtzi (an exotic species in North America) is widespread and occasionally abundant. We collected zooplankton on the Illinois River (Illinois, U.S.A.) in 1995 and 1996 and found that the peak density of D. lumholtzi (25 L⁻¹) typically exceeded that of all native species combined. Maximum density occurred during warm periods (up to 27 °C) when concentrations of inorganic suspended sediments were high (>50 mg L⁻¹).
2. Using a life table experiment, we examined the effects of variation in suspended sediment (0 and 80 mg L⁻¹) and food (10⁴ and 10⁵ Ankistrodesmus cells mL⁻¹) on fitness of D. lumholtzi and the native Daphnia parvula. Daphnia lumholtzi had greater survivorship than D. parvula in most treatments and higher life-time fertility than D. parvula in all treatments. Both species achieved their fastest intrinsic rates of growth in treatments with high food, but their responses to suspended solids differed. The growth rate of D. lumholtzi in high food was slightly increased by higher turbidity, whereas that of D. parvula was depressed.
3. Results suggest that the ability of D. lumholtzi to tolerate suspended solids is an important factor contributing to its success in invading North American rivers.     

Development of microsatellite markers for Diaphanosoma dubium (Crustacea,Cladocera) and application to seasonal population dynamics

Diaphanosoma, the “tropical Daphnia”, is common and ubiquitous in South China. Like other ctenopods, Diaphanosoma has a reproductive mode similar to Daphnia’s, but its resting eggs are rarely observed and lack an ephippium. With limited dispersal and reduced buffer effect from resting egg banks, Diaphanosoma is expected to have a population genetic structure different from that of temperate Daphnia. To facilitate genetic comparison, we developed microsatellite markers using next-generation sequencing for the most common species in tropical and subtropical East Asia, Diaphanosoma dubium. Thirty-one polymorphic microsatellite markers were obtained, and 29 of them were efficient for the congeneric species D. excisum, D. orghidani, D. mongolianum and D. chankensis. The markers allowed intra- and interspecific genetic analysis, including population structure, hybridization and introgression. We used 11 selected microsatellite markers to analyze spatial and temporal heterogeneity of genetic diversity in four (sub)tropical D. dubium populations from two large reservoirs and two temporary ponds. In contrast to temperate Cladocera, higher genetic diversity in summer rather than in spring suggested weak contribution from resting eggs in spring. Clustering of DAPC and STRUCTURE analyses indicated a clear-cut genetic structure in the four populations. Variation partitioning revealed that water storage and depth were key factors in genetic differentiation. Within large reservoirs, we detected backward (reversing time) gene flow from resting egg banks. We conclude that resting eggs have an effective contribution to the genetic diversity in large water bodies during growing seasons and that large water bodies can host higher genetic diversity in summer due to environmental heterogeneity and high carrying capacity. Spatial and temporal heterogeneity of genetic diversity detected by our microsatellite markers showed the newly developed markers can be applied for further study of populations of D. dubium and other species of Diaphanosoma at a contemporary scale.

     

Variation of Mini- and Microsatellite DNA Repeats in Parthenogenetic Lizard Darevskia armeniaca as Revealed by DNA Fingerprinting Analysis

Population and family samples of two morphological forms (mutant and normal with respect to dorsal color) of parthenogenetic lizard species Darevskia armeniaca were examined by means of DNA fingerprinting using M13 mini- and (GACA) _n and (TCC) _n microsatellite DNA markers. The morphological forms examined were characterized by clonally inherited, species-specific patterns of the DNA markers, which were different from the species-specific DNA fingerprints of the other parthenogenetic species of the genus Darevskia (D. dahli, D. unisexualis, and D. rostombekovi). The mean index of similarity (S) obtained for a sample of 36 individuals from three isolated populations using three types of DNA markers was 0.966. This was similar to those observed in D. dahli (0.962) (P > 0.05), but higher than that in D. unisexualis (0.950) (P < 0.05) and D. rostombekovi(0.875) (P < 0.01). Inheritance of M13 minisatellite and (TCC) _n microsatellite DNA markers in the F1 offspring of parthenogenetic lizards was examined. It was shown that variability and clonal diversity of the fingerprint phenotypes observed in the populations and families of D. armeniaca could be at least partly explained by RFLP mutations in microsatellite repeats.