New microsatellite markers for Xerophyta dasylirioides (Velloziaceae), an endemic species on Malagasy inselbergs.

PREMISE: Microsatellite markers were developed for Xerophyta dasylirioides (Velloziaceae), a species endemic to the Malagasy inselbergs, to explore the impact of its island-like distribution on genetic diversity and gene flow. METHODS AND RESULTS: A total of 7110 perfect microsatellite loci were recovered by shotgun sequencing on an Illumina MiSeq platform. Primer pairs were designed for 40 arbitrarily selected loci. Fifteen primer pairs that generated distinct PCR products were used to genotype 80 individuals of X. dasylirioides from three inselberg populations. All markers were polymorphic, revealing two to 17 alleles in the overall sampling. Levels of observed and expected heterozygosity per locus ranged from zero to 1.000 and from zero to 0.850, respectively. Success rates of cross-amplification in 10 additional species of Xerophyta (X. croatii, X. decaryi, X. isaloensis, X. labatii, X. lewisiae, X. pinifolia, X. retinervis, X. setosa, X. spekei, X. tulearensis) ranged from zero to 70%. CONCLUSIONS: Fifteen newly developed microsatellite markers provide a toolkit for assessing population genetic parameters of X. dasylirioides in its unique island-like habitats.

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.

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.

No evidence for effects of infection with the amphibian chytrid fungus on populations of yellow-bellied toads.

The parasitic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause the lethal disease chytridiomycosis in amphibians and therefore may play a role in population declines. The yellow-bellied toad Bombina variegata suffered strong declines throughout western and northwestern parts of its range and is therefore listed as highly endangered for Germany and the federal state of Hesse. Whether chytridiomycosis may play a role in the observed local declines of this strictly protected anuran species has never been tested. We investigated 19 Hessian yellow-bellied toad populations for Bd infection rates, conducted capture-mark-recapture studies in 4 of them over 2 to 3 yr, examined survival histories of recaptured infected individuals, and tested whether multi-locus heterozygosity of individuals as well as expected heterozygosity and different environmental variables of populations affect probabilities of Bd infection. Our results show high prevalence of Bd infection in Hessian yellow-bellied toad populations, but although significant decreases in 2 populations could be observed, no causative link to Bd as the reason for this can be established. Mass mortalities or obvious signs of disease in individuals were not observed. Conversely, we show that growth of Bd-infected populations is possible under favorable habitat conditions and that most infected individuals could be recaptured with improved body indices. Neither genetic diversity nor environmental variables appeared to affect Bd infection probabilities. Hence, genetically diverse amphibian specimens and populations may not automatically be less susceptible for Bd infection.

Ecological range shift in the polyploid members of the South American genus Fosterella (Bromeliaceae).

Background and Aims: The distribution of polyploidy along a relatively steep Andean elevation and climatic gradient is studied using the genus Fosterella L.B. Sm. (Bromeliaceae) as a model system. Ecological differentiation of cytotypes and the link of polyploidy with historical biogeographic processes such as dispersal events and range shift are assessed. Methods: 4',6-Diamidino-2-phenylindole (DAPI) staining of nuclei and flow cytometry were used to estimate the ploidy levels of 159 plants from 22 species sampled throughout the distribution range of the genus. Ecological differentiation among ploidy levels was tested by comparing the sets of climatic variables. Ancestral chromosome number reconstruction was carried out on the basis of a previously generated phylogeographic framework. Key Results: This study represents the first assessment of intrageneric, intraspecific and partially intrapopulational cytotype diversity in a genus of the Bromeliaceae family. In Fosterella , the occurrence of polyploidy was limited to the phylogenetically isolated penduliflora and rusbyi groups. Cytotypes were found to be ecologically differentiated, showing that polyploids preferentially occupy colder habitats with high annual temperature variability (seasonality). The combined effects of biogeographic history and adaptive processes are presumed to have shaped the current cytotype distribution in the genus. Conclusions: The results provide indirect evidence for both adaptive ecological and non-adaptive historical processes that jointly influenced the cytotype distribution in the predominantly Andean genus Fosterella (Bromeliaceae). The results also exemplify the role of polyploidy as an important driver of speciation in a topographically highly structured and thus climatically diverse landscape.

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 18 polymorphic microsatellite markers for Vinca minor (Apocynaceae) via 454 pyrosequencing.

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed in Vinca minor (Apocynaceae) to evaluate the level of clonality, population structure, and genetic diversity of the species within its native and introduced range. METHODS AND RESULTS: A total of 1371 microsatellites were found in 43,565 reads from 454 pyrosequencing of genomic V. minor DNA. Additional microsatellite loci were mined from publicly available cDNA sequences. After several rounds of screening, 18 primer pairs flanking di-, tri-, or tetranucleotide repeats were identified that revealed high levels of genetic diversity in two native Italian populations, with two to 11 alleles per locus. Clonal growth predominated in two populations from the introduced range in Germany. Five loci successfully cross-amplified in three additional Vinca species. CONCLUSIONS: The novel polymorphic microsatellite markers are promising tools for studying clonality and population genetics of V. minor and for assessing the historical origin of Central European populations.

DNA fingerprinting in botany: past, present, future.

Almost three decades ago Alec Jeffreys published his seminal Nature papers on the use of minisatellite probes for DNA fingerprinting of humans (Jeffreys and colleagues Nature 1985, 314:67-73 and Nature 1985, 316:76-79). The new technology was soon adopted for many other organisms including plants, and when Hilde Nybom, Kurt Weising and Alec Jeffreys first met at the very First International Conference on DNA Fingerprinting in Berne, Switzerland, in 1990, everybody was enthusiastic about the novel method that allowed us for the first time to discriminate between humans, animals, plants and fungi on the individual level using DNA markers. A newsletter coined "Fingerprint News" was launched, T-shirts were sold, and the proceedings of the Berne conference filled a first book on "DNA fingerprinting: approaches and applications". Four more conferences were about to follow, one on each continent, and Alec Jeffreys of course was invited to all of them. Since these early days, methodologies have undergone a rapid evolution and diversification. A multitude of techniques have been developed, optimized, and eventually abandoned when novel and more efficient and/or more reliable methods appeared. Despite some overlap between the lifetimes of the different technologies, three phases can be defined that coincide with major technological advances. Whereas the first phase of DNA fingerprinting ("the past") was dominated by restriction fragment analysis in conjunction with Southern blot hybridization, the advent of the PCR in the late 1980s gave way to the development of PCR-based single- or multi-locus profiling techniques in the second phase. Given that many routine applications of plant DNA fingerprinting still rely on PCR-based markers, we here refer to these methods as "DNA fingerprinting in the present", and include numerous examples in the present review. The beginning of the third phase actually dates back to 2005, when several novel, highly parallel DNA sequencing strategies were developed that increased the throughput over current Sanger sequencing technology 1000-fold and more. High-speed DNA sequencing was soon also exploited for DNA fingerprinting in plants, either in terms of facilitated marker development, or directly in the sense of "genotyping-by-sequencing". Whereas these novel approaches are applied at an ever increasing rate also in non-model species, they are still far from routine, and we therefore treat them here as "DNA fingerprinting in the future".

Habitat fragmentation and recent bottlenecks influence genetic diversity and differentiation of the Central European halophyte Suaeda maritima (Chenopodiaceae).

PREMISE OF THE STUDY: Central European salt habitats are mainly restricted to the maritime coast but scattered occurrences can also be found inland. In inland habitats, human activities have caused losses and reductions in the size of natural salt sites but have also created new anthropogenic habitats around potash mining dumps colonized by halophytic species in the last 30 yr. We aimed to investigate the effects of bottlenecks, isolation, and ongoing habitat fragmentation on the genetic variation of a species commonly growing in these special habitats. METHODS: We used 10 microsatellite markers to compare genetic diversity and differentiation of 31 populations of Suaeda maritima (Chenopodiaceae) from Central European coasts and inland habitats. Two approaches were applied to analyze the tetraploid data based on allele frequencies directly derived from microsatellite data and from transformed binary data. KEY RESULTS: In comparison to the coastal populations from the North Sea and the English Channel, significantly reduced genetic variation and increased between-population differentiation was revealed for populations from the German inland and the Baltic Sea coast. Genetic structure analyses clearly separated coastal and inland populations. CONCLUSIONS: Our results indicate that gene flow is restricted among populations from inland salt sites and the Baltic Sea coast, presumably due to their isolation, small sizes, genetic bottlenecks and/or founder events. Patterns of allele distribution indicate some occasional genetic exchange among habitat types in the past. Anthropogenic salt sites may facilitate gene flow among inland salt habitats preventing endangered inland halophyte populations from genetic erosion.

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

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.

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.

In silico mining for simple sequence repeat loci in a pineapple expressed sequence tag database and cross-species amplification of EST-SSR markers across Bromeliaceae.

A collection of 5,659 expressed sequence tags (ESTs) from pineapple [Ananas comosus (L.) Merr.] was screened for simple sequence repeats (EST-SSRs) with motif lengths between 1 and 6 bp. Lower thresholds of 15, 7 and 5 repeat units were used to define microsatellites of the mono-, di-, and tri- to hexanucleotide repeat type, respectively. Based on these criteria, 696 SSRs were identified among 3,389 EST unigenes, together representing 2,840 kb. This corresponds to an average density of one SSR every 4.1 kb of non-redundant EST sequences. Dinucleotide repeats were most abundant (38.4% of all SSRs) followed by trinucleotide repeats (38.1%). Flanking primer pairs were designed for 537 EST-SSR loci, and 49 of these were screened for their functionality in 12 accessions of A. comosus, 14 accessions of 5 additional Ananas species and 1 species of Pseudananas. Distinct PCR products of the expected size range were obtained with 36 primer pairs. Eighteen loci analyzed in more detail were all polymorphic in pineapple, and primer pairs flanking these loci also generated PCR products from a wide range of genera and species from six subfamilies of the Bromeliaceae. The potential to reveal polymorphism in a heterologous target species was demonstrated in Deuterocohnia brevifolia (subfamily Pitcairnioideae).

Phylogenetic analysis of Fosterella L.B. Sm. (Pitcairnioideae, Bromeliaceae) based on four chloroplast DNA regions.

The about 31 species of Fosterella L.B. Sm. (Bromeliaceae) are terrestrial herbs with a centre of diversity in the central South American Andes. To resolve infra- and intergeneric relationships among Fosterella and their putative allies, we conducted a phylogenetic analysis based on sequence data from four chloroplast DNA regions (matK gene, rps16 intron, atpB-rbcL and psbB-psbH intergenic spacers). Sequences were generated for 96 accessions corresponding to 60 species from 18 genera. Among these, 57 accessions represented 22 of the 31 recognized Fosterella species and one undescribed morphospecies. Maximum parsimony and Bayesian inference methods yielded well-resolved phylogenies. The monophyly of Fosterella was strongly supported, as was its sister relationship with a clade comprising Deuterocohnia, Dyckia and Encholirium. Six distinct evolutionary lineages were distinguished within Fosterella. Character mapping indicated that parallel evolution of identical character states is common in the genus. Relationships between species and lineages are discussed in the context of morphological, ecological and biogeographical data as well as the results of a previous amplified fragment length polymorphism (AFLP) study.

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.

Microsatellite markers for the tetraploid halophyte Suaeda maritima (L.) Dumort. (Chenopodiaceae) and cross-species amplification in related taxa.

We developed 12 polymorphic microsatellite markers for the tetraploid halophyte Suaeda maritima (Chenopodiaceae). Population genetic parameters were estimated for three populations from different habitats (coastal and inland), using the program Tetrasat. Between two and 15 alleles per locus were observed. Mean expected heterozygosities (H(E) ) and Shannon-Wiener Diversity Indices (H') per locus and population ranged from zero to 0.852, and from zero to 2.990, respectively. The two inland populations were less diverse than the coastal one at most of the loci. All markers cross-amplified in the closely related Suaeda salsa, and all but one were transferable to Suaeda spicata and Suaeda salinaria.

Microsatellite markers for Spergularia media (L.) C. Presl. (Caryophyllaceae) and their cross-species transferability.

We developed 11 polymorphic microsatellite markers for Spergularia media (Caryophyllaceae), a perennial halophyte of coastal salt meadows and continental areas of western Eurasia. The number of alleles per locus observed in a single population of 20 individuals from the German North Sea coast ranged from 3 to 20. Observed and expected heterozygosities ranged from 0.200 to 0.850 and from 0.278 to 0.936 respectively. Observed heterozygosities were lower than expected heterozygosities at all loci, presumably as a consequence of inbreeding. All markers cross-amplified in the closely related S. salina. Of these, nine were polymorphic in the heterologous species. Only two primer pairs generated PCR products in S. diandra.

Single nucleotide sequence analysis: a cost- and time-effective protocol for the analysis of microsatellite- and indel-rich chloroplast DNA regions.

We present a simple method to screen for DNA sequence variation in microsatellite- and indel-rich regions of the chloroplast genome. The single nucleotide sequence (SNS) analysis provides a trade-off between the time- and cost-effective, but less informative and homoplasy-sensitive electrophoretic detection of microsatellite and indel size variation on the one hand, and more costly, but also more accurate methods like DNA sequencing on the other. The principle of the SNS method is to sequence one instead of all four nucleotides of a target region amplified by polymerase chain reaction. By careful selection of the respective nucleotide, almost the same amount of information can be retrieved from these partial sequences as could be from complete sequences; however, only a third to a fourth of the money and time resources are needed.

AFLP analysis of genetic relationships in the genus Fosterella L.B. Smith (Pitcairnioideae, Bromeliaceae).

The neotropical genus Fosterella L.B. Smith (Pitcairnioideae, Bromeliaceae) comprises about 30 species, with a centre of diversity in semiarid to humid habitats of the Andean slopes and valleys of Bolivia. Morphologic differentiation of species is difficult because of a paucity of diagnostic characters, and little is known about the infrageneric phylogeny. Here, we present the results of an amplified fragment length polymorphism (AFLP) analysis of 77 Fosterella specimens, covering 18 recognized species and 9 as-yet undescribed morphospecies. Eight primer combinations produced 310 bands, which were scored as presence/absence characters. Neighbour-joining tree reconstruction revealed 12 clusters (A-L) with various levels of support. Well-supported species groups were also recovered by a principal coordinates analysis. With few exceptions, morphologically defined species boundaries were confirmed by the molecular data. Phylogenetic relationships between species groups remained ambiguous, however, because of short internal branch lengths. The AFLP data were complemented by a survey of the leaf anatomy of 19 Fosterella species. Species concepts and assemblages are discussed in the context of molecular, morphologic, anatomic, ecologic, and biogeographic data. The data suggest that accidental long-distance dispersal and founder events have been important for Fosterella speciation.

A chloroplast genealogy of myrmecophytic Macaranga species (Euphorbiaceae) in Southeast Asia reveals hybridization, vicariance and long-distance dispersals.

Macaranga (Euphorbiaceae) includes about 280 species with a palaeotropic distribution. The genus not only comprises some of the most prominent pioneer tree species in Southeast Asian lowland dipterocarp forests, it also exhibits a substantial radiation of ant-plants (myrmecophytes). Obligate ant-plant mutualisms are formed by about 30 Macaranga species and 13 ant species of the genera Crematogaster or Camponotus. To improve our understanding of the co-evolution of the ants and their host plants, we aim at reconstructing comparative organellar phylogeographies of both partners across their distributional range. Preliminary evidence indicated that chloroplast DNA introgression among closely related Macaranga species might occur. We therefore constructed a comprehensive chloroplast genealogy based on DNA sequence data from the noncoding ccmp2, ccmp6, and atpB-rbcL regions for 144 individuals from 41 Macaranga species, covering all major evolutionary lineages within the three sections that contain myrmecophytes. A total of 88 chloroplast haplotypes were identified, and grouped into a statistical parsimony network that clearly distinguished sections and well-defined subsectional groups. Within these groups, the arrangement of haplotypes followed geographical rather than taxonomical criteria. Thus, up to six chloroplast haplotypes were found within single species, and up to seven species shared a single haplotype. The spatial distribution of the chloroplast types revealed several dispersals between the Malay Peninsula and Borneo, and a deep split between Sabah and the remainder of Borneo. Our large-scale chloroplast genealogy highlights the complex history of migration, hybridization, and speciation in the myrmecophytes of the genus Macaranga. It will serve as a guideline for adequate sampling and data interpretation in phylogeographic studies of individual Macaranga species and species groups.

Development and characterization of chloroplast microsatellite markers in Macaranga (Euphorbiaceae).

As part of our study on the phylogeography of the ant-plant genus Macaranga, we have screened for polymorphic regions in the chloroplast genome. Initially, ten universal PCR primer pairs targeted at chloroplast microsatellite loci were applied to a small set of specimens, covering various taxonomic levels from intrafamilial to intraspecific. Eight primer pairs produced PCR fragments that behaved as single and discrete bands on agarose gels. The five most promising candidate pairs were further analysed with an extended set of DNA templates, and PCR products were separated on sequencing gels. The number of size variants per locus varied from two to eight, combining into 17 haplotypes among 29 Macaranga accessions from 10 species. Comparative sequencing demonstrated that microsatellites were responsible for the observed size variation at three of five loci, whereas variation at the other loci was caused by larger insertions and (or) deletions (indels). In addition to poly(A) and poly(T) repeats, which are typically found in chloroplast DNA, we also identified a variable (CT)n repeat, with n = 4 to n = 8. Sequencing revealed three examples of size homoplasy, one of which was caused by a single base substitution that raised the actual number of haplotypes to 18. Relationships between haplotypes were assessed by phenetic analyses of size variants and by constructing a parsimony network based on sequence variation. For both types of analysis, the distribution of haplotypes correlated with geographically circumscribed regions rather than with taxonomic boundaries.