Tina Wöhrmann

Development of microsatellite markers in Fosterella rusbyi (Bromeliaceae) using 454 pyrosequencing

PREMISE OF THE STUDY Polymorphic microsatellite markers were developed for Fosterella rusbyi (Bromeliaceae) to evaluate the population genetic structure and genetic diversity of natural populations of F. rusbyi and other Fosterella species in Bolivia. METHODS AND RESULTS 454 pyrosequencing technology was used to generate 73027 sequence reads from F. rusbyi DNA, which together contained 2796 perfect simple sequence repeats (SSRs). Primer pairs were designed for 30 loci, of which 15 were used to genotype 30 F. rusbyi plants from two geographical areas in Bolivia. All markers were polymorphic, with two to nine alleles in the overall sample. Cross-species amplification was tested in 10 additional Fosterella species. Seven loci showed consistent amplification in six or more species. CONCLUSIONS The 15 SSR markers developed for F. rusbyi are promising candidates for population genetic analyses within F. rusbyi and other species of Fosterella.

Reproduction biology and chloroplast inheritance in Bromeliaceae: a case study in Fosterella (Pitcairnioideae)

We applied a series of intra- and interspecific in situ cross-pollination experiments under greenhouse conditions to evaluate the breeding systems in four Fosterella species (Pitcairnioideae s.str.; Bromeliaceae). Viable hybrids were produced between each pair of the investigated species, suggesting that reproduction barriers may be low also under natural conditions. Seed germination rates proved to be high in each crossing treatment, indicating a high viability of the artificial hybrids. Large numbers of seeds were produced after both closed and open pollination treatments, suggesting that autogamy may be a major reproductive strategy in the genus. Our results support the concept that self-compatibility is an appropriate way to avoid natural hybridization in Bromeliaceae and could assist in maintaining species integrity in the presence of pollen flow. Paternity was verified in all crosses by genotyping parents and offspring with a set of polymorphic nuclear microsatellite markers. To study the mode of chloroplast inheritance, we developed a novel set of 24 chloroplast microsatellite markers using 454 pyrosequencing technology, and applied four of these markers for genotyping parents and offspring from all crosses. Our results clearly demonstrated a maternal inheritance of plastids.

Genetic variability in wild populations of Prunus divaricata Ledeb. in northern Iran evaluated by EST-SSR and genomic SSR marker analysis

A population genetic analysis based on eight genomic SSR markers and three EST-SSR (expressed sequence tags) markers developed in peach (Prunus persica (L.) Batsch) and Japanese plum (Prunus salicina Lindl.) was carried out in 12 wild populations of cherry plum (Prunus divaricata Ledeb.) sampled along the Iranian coast of the Caspian Sea. A total of 184 alleles (3–31 per locus) were detected with a mean value of 16.7 alleles per locus. None of the loci or populations showed deviation from Hardy–Weinberg equilibrium, and all markers proved to be unlinked. The mean values for the observed and the expected heterozygosity were 0.66 and 0.73, respectively. There was very little genetic differentiation among populations, as was indicated by low overall values of Wright’s FST (0.03) and Nei’s GST (0.08). An analysis of molecular variance (AMOVA) showed that 96.8% of the total variance was attributable to differences between individuals within populations. Genetic and geographic distances were nevertheless positively correlated, as evidenced by a Mantel test. The high level of genetic diversity and the apparent lack of genetic structure in wild P. divaricata may be attributed to frequent long distance gene flow through frugivorous birds and possibly humans, as has been documented for other Prunus species.

Development of 15 nuclear microsatellite markers in the genus Dyckia (Pitcairnioideae; Bromeliaceae) using 454 pyrosequencing

The genus Dyckia (Bromeliaceae) comprises 147 species that are distributed in Brazil and adjacent countries. Many species are rare and narrow endemics. We used 454 pyrosequencing to isolate 1,587 microsatellite loci in Dyckia marnier-lapostollei. Of 50 loci that were selected for primer design, 15 markers proved to be polymorphic in five populations from three heterologous species, Dyckia dissitiflora, Dyckia pernambucana and Dyckia limae. Numbers of alleles per locus varied from 3 to 30, and expected and observed heterozygosities ranged from 0.21 to 0.53 and from 0.16 to 0.44, respectively, in the overall sample. The 15 new microsatellite markers are promising tools for studying population genetics in Dyckia species.

A set of plastid microsatellite loci for the genus Dyckia (Bromeliaceae) derived from 454 pyrosequencing

UNLABELLED PREMISE OF THE STUDY Phylogeographical analyses of Dyckia (Bromeliaceae) suffer from low levels of sequence variation. Plastid microsatellite markers were developed to achieve a better-resolved genus-wide plastid genealogy of Dyckia. • METHODS AND RESULTS Approximately 84% of the D. marnier-lapostollei plastome was sequenced using 454 technology. Flanking primer pairs were designed for 34 out of 36 chloroplast simple sequence repeats (cpSSRs) detected, and 12 loci were further characterized by genotyping Dyckia samples at the level of populations and species. Three, five, and six cpSSRs were polymorphic among four individuals of D. limae, 12 individuals of D. dissitiflora, and 12 of D. pernambucana, respectively, with two to three alleles per locus and species. All loci were polymorphic among 19 different Dyckia species, with three to 10 alleles per locus. Ten primer pairs cross-amplified with bromeliad genera from five subfamilies. • CONCLUSIONS The set of 12 cpSSR markers provides a toolbox to analyze phylogeographical patterns of Dyckia species.

Population genetic structure of the rock outcrop species Encholirium spectabile (Bromeliaceae): The role of pollination vs. seed dispersal and evolutionary implications

PREMISE OF THE STUDY Inselbergs are terrestrial, island-like rock outcrop environments that present a highly adapted flora. The epilithic bromeliad Encholirium spectabile is a dominant species on inselbergs in the Caatinga of northeastern Brazil. We conducted a population genetic analysis to test whether the substantial phenotypic diversity of E. spectabile could be explained by limited gene flow among populations and to assess the relative impact of pollen vs. seed dispersal on the genetic structure of the species. METHODS Nuclear and chloroplast microsatellite markers were used to genotype E. spectabile individuals from 20 rock outcrop locations, representing four geographic regions: northern Espinhaço Range, Borborema Plateau, southwestern Caatinga and southeastern Caatinga. F-statistics, structure, and other tools were applied to evaluate the genetic makeup of populations. KEY RESULTS Considerable levels of genetic diversity were revealed. Genetic structuring among populations was stronger on the plastid as compared with the nuclear level, indicating higher gene flow via bat pollination as compared with seed dispersal by wind. structure and AMOVA analyses of the nuclear data suggested a high genetic differentiation between two groups, one containing all populations from the southeastern Caatinga and the other one comprising all remaining samples. CONCLUSIONS The strong genetic differentiation between southeastern Caatinga and the remaining regions may indicate the occurrence of a cryptic species in E. spectabile. The unique genetic composition of each inselberg population suggests in situ conservation as the most appropriate protection measure for this plant lineage.

Mechanisms of Speciation in Southeast Asian Ant-Plants of the Genus Macaranga (Euphorbiaceae)

The palaeotropic genus Macaranga (Euphorbiaceae) is an excellent model system to analyze co-evolutionary processes associated with myrmecophytism, a mutualistic interaction between plants and ants. Ant-plants like Macaranga provide nesting space and feed their partners, whereas the ants protect the plants from herbivores and competitors such as lianas. We used genome-based evidence to investigate speciation mechanisms in Macaranga ant-plants, and their co-evolution with ants from the genus Crematogaster. Our previous work had shown that myrmecophytic Macaranga species show little genetic differentiation, suggesting an adaptive radiation. We hypothesized that the obligatory symbiosis with ants may reduce gene flow among plant populations, eventually enhancing allopatric speciation. To test this hypothesis, we verified the monophyly of the investigated plant lineages by phylogenetic analyses, reconstructed parsimony networks based on chloroplast DNA (cpDNA) variation, and assessed population genetic parameters using nuclear microsatellites and cpDNA haplotypes. Our data provided evidence for vicariant events as well as for hybridization and cpDNA introgression among closely related Macaranga species. The extent of population differentiation within myrmecophytic versus non-myrmecophytic species proved to be in a similar range, indicating that our working hypothesis of enhanced allopatric speciation in myrmecophytes cannot be sustained by the present evidence. Nevertheless, the mutualistic interactions of Macaranga and associated ants may be a key innovation that opened an adaptive zone putatively exploited by the divergence of Macaranga.

Isolation and characterization of 11 new microsatellite markers for Macaranga (Euphorbiaceae)

We provide primer sequences for 11 new polymorphic microsatellite markers developed in the tropical ant–plant genus Macaranga (Euphorbiaceae), after enrichment cloning of Macaranga tanarius and Macaranga hypoleuca. Allele numbers per locus ranged from two to 16 among 20 accessions of M. tanarius, and from three to 10 among 22 accessions of M. hypoleuca. Observed and expected heterozygosities ranged from 0.150 to 0.900 and from 0.375 to 0.894 in M. tanarius, and from 0.545 to 1.000 and from 0.434 to 0.870 in M. hypoleuca, respectively. Six of the 11 primer pairs successfully cross‐amplified polymorphic polymerase chain reaction products in Macaranga winkleri.

Microsatellites from Fosterella christophii (Bromeliaceae) by de Novo Transcriptome Sequencing on the Pacific Biosciences RS Platform

Premise of the study: Microsatellite markers were developed in Fosterella christophii (Bromeliaceae) to investigate the genetic diversity and population structure within the F. micrantha group, comprising F. christophii, F. micrantha, and F. villosula. Methods and Results: Full-length cDNAs were isolated from F. christophii and sequenced on a Pacific Biosciences RS platform. A total of 1590 high-quality consensus isoforms were assembled into 971 unigenes containing 421 perfect microsatellites. Thirty primer sets were designed, of which 13 revealed a high level of polymorphism in three populations of F. christophii, with four to nine alleles per locus. Each of these 13 loci cross-amplified in the closely related species F. micrantha and F. villosula, with one to six and one to 11 alleles per locus, respectively. Conclusions: The new markers are promising tools to study the population genetics of F. christophii and to discover species boundaries within the F. micrantha group.

Development of 15 nuclear microsatellite markers in Deuterocohnia (Pitcairnioideae; Bromeliaceae) using 454 pyrosequencing

Premise of the Study Microsatellite markers were developed in Deuterocohnia longipetala (Bromeliaceae) to investigate species and subspecies boundaries within the genus and the genetic diversity of natural populations. Methods and Results We used 454 pyrosequencing to isolate 835 microsatellite loci in D. longipetala. Of 64 loci selected for primer design, 15 were polymorphic among 23 individuals of D. longipetala and 76 individuals of the heterologous subspecies D. meziana subsp. meziana and D. meziana subsp. carmineo‐viridiflora. Twelve and 13 of these loci were also polymorphic in one population each of D. brevispicata and D. seramisiana, respectively. Numbers of alleles per locus varied from two to 14 in D. longipetala, two to 12 in D. meziana, one to nine in D. brevispicata, and one to 10 in D. seramisiana. STRUCTURE analyses clearly separated the taxa from each other. Conclusions The 15 new microsatellite markers are promising tools for studying population genetics in Deuterocohnia species.