Characterization of microsatellite markers in sycamore (Acer pseudoplatanus L.)

Sycamore (Acer pseudoplatanus L.) is a tetraploid European hardwood tree species. The reproduction system of the insect‐pollinated trees and patterns of genetic variation are largely unknown. We isolated and characterized eight polymorphic microsatellite markers for Acer pseudoplatanus L. The high degree of polymorphism observed at these markers makes them useful to observe genetic variation patterns at various spatial scales and to analyse gene flow and the mating system. Primers developed for the amplification of microsatellites in A. pseudoplatanus were tested for 21 different species of genus Acer. Amplification products of the expected size were obtained in most cases.     

Isolation and characterization of microsatellite markers from Zale galbanata (Lepidoptera: Noctuidae) and amplification in other members of the genus

The noctuid moth Zale galbanata is described as feeding on maples (Acer sp.) in general, but its primary food plant is box elder (Acer negundo). We isolated five microsatellite loci to investigate the association of food plant use and the genetic similarity of populations in these moths. These markers are polymorphic to various degrees (5–13 alleles), and are presented here. Our initial tests show that some of these loci also work on other Zale species.     

Development of microsatellite markers in Acer capillipes

Acer capillipes is an insect‐pollinated tree species that grows in temperate regions of Japan. We isolated seven polymorphic microsatellite loci from this species using a dual‐suppression polymerase chain reaction (PCR) technique. The number of alleles per locus ranged from two to 10, and the expected heterozygosity ranged from 0.042 to 0.828. Cross‐species amplification from 14 other Acer species was successful for the majority of the isolated loci, suggesting that these loci may be useful for the characterization of other maple species.     

Membrane phospholipid composition during maturation of seeds of Acer platanoides and Acer pseudoplatanus in relation to desiccation tolerance

Membrane phospholipid composition was investigated in seeds of two species from the genus Acer: Norway maple (Acer platanoides L.) — tolerant to desiccation, and sycamore (Acer pseudoplatanus L.) — intolerant to desiccation, during their maturation, from 1 August to 25 September 1995, at weekly intervals. Seeds of Norway maple acquire tolerance to desiccation at the end of August ie. about 125 days after flowering (DAF). Phospholipid composition during development revealed marked differences between studied seeds. Seeds of Norway maple after acquiring tolerance to desiccation contained much more phosphatidylcholine (PC) and phosphatidylethanolamine (PE), compared to sycamore. The ratio of PC/PE in mature Norway maple seeds was evidently higher than those in sycamore. The level of unsaturated fatty acids in the phospholipid fraction substantially increased in Norway maple seeds during development and the saturation of PC and PE was less than in sycamore. The results suggest that phospholipid composition may be involved in desiccation tolerance of Norway maple seeds.     

Characterization of microsatellite loci in White‐chinned Petrel (Procellaria aequinoctialis) and cross‐amplification in six other procellariiform species

Six polymorphic dinucleotide microsatellite loci were isolated and characterized from the White‐chinned Petrel Procellaria aequinoctialis, using a degenerate primer and PCR‐based technique to construct and screen an enriched genomic library. Preliminary data on three populations show heterozygosity levels ranging from 0.22 to 0.67 and allele numbers from three to nine. Preliminary data also suggest genetic distance between these three populations (F_ST 0.088). Cross‐species amplification of these six microsatellite loci and one further locus were tested in six other procellariiform species of the genus Procellaria, Macronectes, Thalassarche and Diomedea.     

Isolation and characterization of microsatellite loci in Acer opalus (Aceraceae), a sexually-polymorphic tree, through an enriched genomic library

An enrichment protocol was used to isolate and characterize microsatellite loci in Acer opalus, a Mediterranean tree species. Highly polymorphic microsatellite loci were required for paternity analyses in a population of this species. Eight microsatellite loci were amplified and a total of 87 alleles were detected in a sample of 142 individuals from one population, allowing the identification of each individual with a unique multilocus genotype. The paternity exclusion probabilities varied from 0.261 for locus Aop820 to 0.806 for locus Aop450, and the parent-pair exclusion probabilities varied from 0.433 for Aop820 to 0.940 for Aop450. The cumulative probabilities of exclusion for paternity and parentage of the eight loci were both higher than 0.999, supporting the usefulness of these microsatellite loci for future paternity and parentage analyses in A. opalus. Cross-species transferability was also assayed, supporting their potential use in other eight Acer species.     

Nine polymorphic microsatellite loci in the Neotropical electric eel Eigenmannia (Teleostei: Gymnotiformes)

Ten microsatellite loci were isolated from a species of the Neotropical electric eel, Eigenmannia, a genus of freshwater fish characterized by small populations and low vagility. Nine microsatellites were polymorphic, the number of alleles ranging from seven to 27, and values of observed heterozygosity ranging from 0.327 to 0.741. These loci were developed for population genetic studies of Eigenmannia sp. 2, however, cross‐amplification carried out with other species of this genus as well as other Gymnotiformes genera indicate that these molecular markers are also potentially useful for population‐level studies in closely related species.     

Polymorphic microsatellite loci for species of Australian freshwater cod, <Emphasis Type="Italic">Maccullochella</Emphasis>

The Australian freshwater cod genus, Maccullochella is represented by three species: Murray cod, M. peelii peelii, eastern freshwater cod, M. ikei, and trout cod, M.macquariensis. Seven novel microsatellite loci from M. ikei and six previously published loci from M. peelii peelii were tested on wild populations of Murray, eastern and trout cod. Levels of polymorphism varied between species with 13 loci polymorphic in Murray cod, 9 in trout cod and 7 in eastern cod. Observed heterozygosities ranged from 0.053 to 0.842. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of Maccullochella.     

Microsatellite Variation in Two Populations of Free-Ranging Yellow Baboons (Papio hamadryas cynocephalus)

We investigated genetic variation at six microsatellite (simple sequence repeat) loci in yellow baboons (Papio hamadryas cynocephalus) at two localities: the Tana River Primate Reserve in eastern Kenya and Mikumi National Park, central Tanzania. The six loci (D1S158, D2S144, D4S243, D5S1466, D16S508, and D17S804) were all originally cloned from and characterized in the human genome. These microsatellites are polymorphic in both baboon populations, with the average heterozygosity across loci equal to 0.731 in the Tana River sample and 0.787 in the Mikumi sample. The genetic differentiation between the two populations is substantial. Kolmogornov–Smirnov tests indicate that five of the six loci are significantly different in allele frequencies in the two populations. The mean F _ST across loci is 0.069, and Shriver's measure of genetic distance, which was developed for microsatellite loci (Shriver et al., 1995), is 0.255. This genetic distance is larger than corresponding distances among human populations residing in different continents. We conclude that (a) the arrays of alleles present at these six microsatellite loci in two geographically separated populations of yellow baboons are quite similar, but (b) the two populations exhibit significant differences in allele frequencies. This study illustrates the potential value of human microsatellite loci for analyses of population genetic structure in baboons and suggests that this approach will be useful in studies of other Old World monkeys.     

Changes in soluble sugars in relation to desiccation tolerance and effects of dehydration on freezing characteristics of Acer platanoides and Acer pseudoplatanus seeds

The role of soluble sugars in desiccation tolerance was investigated in seeds of two species from the genus Acer: Norway maple (Acer platanoides L.) — tolerant and sycamore (Acer pseudoplatanus L.) — intolerant to dehydration. During two years of observations it was found that seeds of Norway maple acquire desiccation tolerance at the end of August i.e. about 125 days after flowering (DAF). During seed development, the transition from intolerant to tolerant state in Norway maple seeds was accompanied by the accumulation in seed tissues of raffinose, stachyose and sucrose. The sucrose/raffinose ratio in Norway maple seeds was lower than in sycamore. In mature Norway maple seeds sucrose and raffinose contents were higher than in sycamore. It was concluded, that soluble sugars such as sucrose, raffinose and stachyose may play an important role in desiccation tolerance and/or intolerance of Norway maple and sycamore seeds. Differential thermal analysis (DTA) was used to study the relationship between desiccation sensitivity and the state of water in seed tissues. The level of non-freezable water was the same in both analysed seed species, but the temperature of water crystallization during desiccation was lower in sycamore seeds.     

Association of Protective Proteins with Dehydration and Desiccation of Orthodox and Recalcitrant Category Seeds of Three Acer Genus Species

Mature and dried seeds from three species of the Acer genus, which differed in desiccation tolerance, were analyzed. The three species investigated were as follows: Acer platanoides L. (Norway maple, orthodox, A1 and A2 seedlots); Acer pseudoplatanus L. (sycamore, recalcitrant, B1 and B2 seedlots); and Acer saccharinum (silver maple, recalcitrant, C1 and C2 seedlots). We compared the appearance of dehydrins and small heat shock proteins in seedlots originating from cropping years that differed in weather conditions, which were monitored in detail during seed development. The experiments showed that three main dehydrins with approximate molecular weights of 46, 35, and 23?kDa were characteristic of all examined Acer species seeds. The three proteins were present in the A1 and A2 seedlots of the orthodox category Norway maple seeds and were noted either individually or together in the B1, B2, C1, and C2 seedlots of recalcitrant category seeds. It was found that one major small heat shock protein existed with a molecular mass of 22?kDa and was detectable at high concentrations in all seeds of the studied Acer species; after the seeds were dried, the content of this protein significantly increased. The potential modulation of dehydrin expression by environmental factors such as developmental heat sum and rainfall is discussed in the present work. The influence of water removal, which is caused by seed drying, in seeds of the same genus and belonging to the orthodox and recalcitrant categories is also explored.     

Development and characterization of the first microsatellite markers in the mangrove species Pelliciera rhizophorae Triana & Planchon

Pelliciera rhizophorae is a unique Neotropical mangrove species belonging to Pelliciera genus. We isolated eight microsatellite loci from this species. All loci were polymorphic and showed three to nine alleles per locus in Colombian Pacific and Caribbean populations. Polymorphic information content ranged from 0.46 to 0.69. Two loci (PeRh‐14 and PeRh‐19) showed null alleles on the Caribbean coast, which suggest genetic differentiation between Pacific and Caribbean populations of P. rhizophorae. Development of these microsatellite loci constitutes a new molecular tool to carry out studies in the genome of the species and to evaluate its population dynamics.     

Microsatellite markers in a complex of Horismenus sp. (Hymenoptera: Eulophidae), parasitoids of bruchid beetles

Parasitoids of the genus Horismenus (Hymenoptera: Eulophidae) are the main natural enemies of bruchid beetles that feed on several species of Phaseolus beans. Samples of Horismenus depressus, H. missouriensis and H. butcheri were collected from seeds of three Phaseolus species throughout Mexico to examine the impact of plant variability on the genetic structure of parasitoid populations. For this purpose, six microsatellite loci were isolated and characterized. These loci are of great interest in understanding the taxonomy of the genus Horismenus, the most important Eulophid genus in the Neotropics.     

Development and characterization of novel tetra-, tri- and di-nucleotide microsatellite markers in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

The cacao plant, Theobroma cacao L., produces white seeds (beans) that form the major ingredient of processed chocolate. A great deal of research effort has been expended to the development of new genetically modified cacao plants with improved productivity and resistance and beans of good industrial quality. The availability of suitable genetic markers is an important aspect of the efficient selection and breeding of this perennial species. We describe the development of 123 microsatellite loci of cacao. An optimized protocol was used to construct and screen a microsatellite-enriched genomic library from which we isolated 64 di-nucleotide, 45 tri-nucleotide and 14 tetra-nucleotide microsatellite loci. The primers were tested on samples from five different T. cacao accessions, one accession from T. grandiflorum and one accession from Herranea sp. Among the 123 loci, 54 were polymorphic, 61 were monomorphic and eight did not present an amplification product. These new markers will be useful in future studies by increasing the accuracy of genotypic assessments in diverse cocoa tree populations as well as in other species of the Theobroma genus.     

Isolation and characterization of polymorphic microsatellite markers from fireweed,Senecio madagascariensis Poir. (Asteraceae)

Ten polymorphic microsatellite loci were isolated and characterized from invasive fireweed (Senecio madagascariensis) populations in the Hawaiian archipelago. These loci provided markers with polymorphism of six to 24 alleles per locus within 96 individuals collected from two populations from the island of Maui. The expected and observed heterozygosities ranged from 0.31 to 0.91 and from 0.056 to 1. These markers should be useful to study the importance of genetic diversity in invasion success of this species.     

Isolation and characterization of microsatellites in the woody shrub,Calothamnus quadrifidus (Myrtaceae)

Microsatellite loci were developed for genetic analysis of the bird pollinated woody shrub Calothamnus quadrifidus. A genomic library was constructed and screened with dinucleotide and trinucleotide repeat sequences. Ten dinucleotide microsatellite markers were developed, and polymorphism in a population of C. quadrifidus was investigated for six of these markers, which showed an average of 12.7 alleles per locus. Mendelian inheritance of the loci was confirmed through analysis of open pollinated progeny arrays of 10 plants. These loci will be used to study gene flow patterns between isolated populations of this species in southwest Western Australia.     

Size-dependency in hydraulic and photosynthetic properties of three <Emphasis Type="Italic">Acer</Emphasis> species having different maximum sizes

Recent studies suggest that physiological traits can be affected by tree size due to stronger hydraulic limitation in taller trees. As trees vary greatly in size, both within and among species, the adaptive responses to hydraulic limitation may be different among species with different maximum sizes. To investigate this, we explored size-dependency in photosynthetic and hydraulic parameters of three Acer species (Acer mono Maxim., Acer amoenum Carr and Acer japonicum Thunb.) using trees of various sizes under well-lit conditions. Leaf stomatal conductance of the Acer species decreased with tree size, implying that water supply to leaves decreases as trees grow. In contrast, content of nitrogen increased with tree size, which may compensate for the decrease in stomatal conductance to maintain the photosynthetic rate. Although the increase in nitrogen and leaf mass per area were larger in species with larger statures, the size-dependency in stomatal conductance was not different among species, and photosynthetic rate and hydraulic conductance were maintained in the three Acer species. Therefore, we suggest that hydraulic limitation on gas exchange does not necessarily depend on the maximum height of the species and that maintenance of photosynthesis and hydraulic properties is a fundamental physiological process during tree growth.     

Isolation and characterization of highly polymorphic microsatellite markers in Hypochaeris radicata (Asteraceae)

We developed five highly polymorphic dinucleotide microsatellite loci for the grassland species Hypochaeris radicata (Asteraceae). Polymorphism of these markers was examined in six populations in the Netherlands. All loci were polymorphic in all populations. The number of alleles per locus varied between 18 and 43. Expected heterozygosity was between 0.86 and 0.91. Cross‐species amplification was tested in six Hypochaeris species and was successful for three different loci in four species. These microsatellites are a useful tool in population genetic, dispersal and metapopulation studies or in testing levels of inbreeding.     

Microsatellite variation and differentiation among local populations of Castanopsis species in Japan

Microsatellite variations in Castanopsis species in Japan were examined to clarify the genetic relationships among 25 local populations according to the difference in the number of layers of adaxial epidermis in the leaves. Six microsatellite loci were assayed for 629 seedlings from the populations, and these seedlings were classified into five types according to the state of the leaf epidermis. Remarkable differences in the allele frequency of the six microsatellite loci were observed among these local populations. The coefficients of genetic differentiation, R_ST, of each locus ranged from 0.209 to 0.388. An unweighted pair-group method (UPGMA) phenogram constructed on the population pairwise R_ST over the loci revealed three clusters (A–C), and six sub-clusters. These clusters reflected the differences in the occurrence frequency of seedlings in each epidermis type within a population. Our findings suggest that clusters A and C are the local populations dominated by Castanopsis sieboldii and Castanopsis cuspidata, respectively, while local populations of cluster B are composed of the two Castanopsis species and/or include many individuals derived by hybridization. The six sub-clusters were found to reflect the geographic relationship among the populations, suggesting a different process for geographic population dynamics during the postglacial period.     

Low genetic diversity and recovery implications of the vulnerable Bankoualé Palm Livistona carinensis (Arecaceae), from North-eastern Africa and the Southern Arabian Peninsula

The Bankoualé Palm, Livistona carinensis is the only known species of Livistona occurring in Africa and is currently classified as vulnerable (IUCN 2004). This extreme outlier species of the genus is restricted to Yemen, Somalia and Djibouti, where all populations are in rapid decline. In Djibouti the palm is confined to three valley systems within the upland plateau of the Goda Massif. This study used microsatellite markers to investigate the genetic diversity and relationships within the species. At the species level L. carinensis contained very low genetic diversity. Most variation was due to the variation between the samples from Yemen and Somalia compared with those in Djibouti. The Djibouti populations were almost monomorphic across the nine loci tested. Interestingly, and despite the small sample sizes, the individuals from botanic gardens collections of the Yemen and Somalia populations were more genetically diverse than the Djibouti populations. This study indicates that the populations in Yemen and Somalia are highly significant for the conservation of the species genetic diversity. Given the lack of genetic diversity both within and among L. carinensis populations in Djibouti, plants could be cultivated for in-situ population enhancement from any seed that is available from within Djibouti with no significant genetic impacts of provenance mismatch. Clearly the populations from Djibouti, Somalia and Yemen are different genetic provenances raising some issues for the conservation and recovery of L. carinensis.