Drastic reduction of plastome size in the mycoheterotrophic Thismia tentaculata relative to that of its autotrophic relative Tacca chantrieri.

PREMISE OF THE STUDY: Heterotrophic angiosperms tend to have reduced plastome sizes relative to those of their autotrophic relatives because genes that code for proteins involved in photosynthesis are lost. However, some plastid-encoded proteins may have vital nonphotosynthetic functions, and the plastome therefore may be retained after the loss of photosynthesis. METHODS: We sequenced the plastome of the mycoheterotrophic species Thismia tentaculata and a representative of its sister genus, Tacca chantrieri, using next-generation technology, and we compared sequences and structures of genes and genomes of these species. KEY RESULTS: The plastome of Tacca chantrieri is similar to those of other autotrophic taxa of Dioscoreaceae, except in a few local rearrangements and one gene loss. The plastome of Thismia tentaculata is ca. 16 kbp long with a quadripartite structure and is among the smallest known plastomes. Synteny is minimal between the plastomes of Tacca chantrieri and Thismia tentaculata. The latter includes only 12 candidate genes, with all except accD involved in protein synthesis. Of the 12 genes, trnE, trnfM, and accD are frequently among the few that remain in depauperate plastomes. CONCLUSIONS: The plastome of Thismia tentaculata, like those of most other heterotrophic plants, includes a small number of genes previously suggested to be essential to plastome survival.

Phylogeny and evolution of bracts and bracteoles in Tacca (Dioscoreaceae).

Most species in the genus Tacca (Dioscoreaceae) feature green to black purple, conspicuous inflorescence involucral bracts with variable shapes, motile filiform appendages (bracteoles), and diverse types of inflorescence morphology. To infer the evolution of these inflorescence traits, we reconstructed the molecular phylogeny of the genus, using DNA sequences from one nuclear, one mitochondrial, and three plastid loci (Internal Transcribed Spacer (ITS), atpA, rbcL, trnL-F, and trnH-psbA). Involucres and bracteoles characters were mapped onto the phylogeny to analyze the sequence of inflorescence trait evolution. In all analyses, species with showy involucres and bracteoles formed the most derived clade, while ancestral Tacca had small and plain involucres and short bracteoles, namely less conspicuous inflorescence structures. Two of the species with the most elaborate inflorescence morphologies (T. chantrieri in southeast China and T. integrifolia in Tibet), are predominantly self-pollinated, indicating that these conspicuous floral displays have other functions rather than pollinator attraction. We hypothesize that the motile bracteoles and involucres may facilitate selfing; display photosynthesis in the dim understory, and protect flowers from herbivory.

The fewer and the better: prioritization of populations for conservation under limited resources, a genetic study with Borderea pyrenaica (Dioscoreaceae) in the Pyrenean National Park.

Taxa considered under low International Union for the Conservation of Nature categories of extinction risk often represent cases of concern to conservation biology. Their high relative abundance precludes management of the entire range due to limited economical resources. Therefore, they require a cost-effective management plan. Borderea pyrenaica (Dioscoreaceae), an endemic plant of the Central Pyrenees and pre-Pyrenees, reaches the French side of the Central Pyrenees on its narrow northernmost boundary at Gavarnie (Parc National des Pyrenées, PNP, France), where it is protected as Vulnerable and considered a priority species. We have used nuclear microsatellite population genetic data to design a management strategy for the 11 populations of B. pyrenaica present in this area and to identify Relevant Genetic Units for its Conservation. The 18 SSR loci analysed identified 56 alleles, 24 of which fulfilled the rarity criterion for this set of populations. Genetic structuring of populations and representativity values derived from regression analyses of probabilities of loss of rare alleles together support differentiation of the B. pyrenaica populations into different management units. Estimates derived from G(ST) values indicate that five populations would adequately represent the 99.9% of the variation relative to most common alleles whereas calculations based on representativity values indicated that these five populations should equate the proportion 2:2:1 from the three different phylogeographical subdivisions of Gavarnie (Western, Eastern-1 and Eastern-2 ranges). This scheme would allow the preservation of 98.21% of the total B. pyrenaica alleles present in Gavarnie, according to the post glacial history of its populations. This conservation genetic approach could be applied to other low-extinction risk categories of extremely rare and subalpine plants in need of regulatory plans in European National Parks and Natural Reserves.

Genetic diversity and geographic differentiation in Tacca chantrieri (Taccaceae): an autonomous selfing plant with showy floral display.

BACKGROUND AND AIMS: Despite considerable investment in elaborate floral displays, Tacca chantrieri populations are predominantly selfing. It is hypothesized that this species might possess considerable spatial or temporal variation in outcrossing rates among populations. To test this hypothesis, genetic variability and genetic differentiation within and among T. chantrieri populations were investigated to find out if they are in agreement with expectations based on a predominantly inbred mating system. METHODS: Genetic diversity was quantified using inter-simple sequence repeats (ISSR) in 303 individuals from 13 populations taken from known locations of T. chantrieri in China, and from one population in Thailand. KEY RESULTS: Of the 113 primers screened, 24 produced highly reproducible ISSR bands. Using these primers, 160 discernible DNA fragments were generated, of which 145 (90.62 %) were polymorphic. This indicated considerable genetic variation at the species level. However, there were relatively low levels of polymorphism at population levels, with percentages of polymorphic bands (PPB) ranging from 8.75 % to 55 %. A high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: G(ST) = 0.5835; AMOVA analysis: F(ST) = 0.6989). Furthermore, based on levels of genetic differentiation, the 14 populations clustered into two distinct groups separated by the Tanaka Line. CONCLUSIONS: High levels of differentiation among populations and low levels of diversity within populations at large spatial scales are consistent with earlier small-scale studies of mating patterns detected by allozymes which showed that T. chantrieri populations are predominantly selfing. However, it appears that T. chantrieri has a mixed-mating system in which self-fertilization predominates, but there is occasional outcrossing. Significant genetic differences between the two distinct regions might be attributed to vicariance along the Tanaka Line. Finally, possible mechanisms of geographic patterns based on genetic differentiation of T. chantrieri are discussed.

A Bayesian approach for discriminating among alternative inheritance hypotheses in plant polyploids: the allotetraploid origin of genus Borderea (Dioscoreaceae).

Polyploidy is a common phenomenon occurring in a vast number of land plants. Investigations of patterns of inheritance and the origins of plants (i.e., autopolyploidy vs. allopolyploidy) usually involve cytogenetic and molecular studies of chromosome pairing, chromosome mapping, and marker segregation analysis through experimental crosses and progeny tests. Such studies are missing for most wild species, for which artificial crosses are difficult, not feasible, or unaffordable. We report here a Bayesian method to discriminate between alternative inheritance patterns in the two extant, tetraploid species of the monocot genus Borderea (Dioscoreaceae), which does not involve progeny array tests. Our approach is based on the screening of a large number of SSR genotypes, which were obtained from successful amplifications of 17 microsatellite regions in individuals of both B. chouardii and B. pyrenaica. We tested for tetrasomic vs. disomic modes of inheritance, using the Bayes factor test. Assignment of genotypes under both alternatives could be unequivocally done for 14 and 11 of the 17 studied microsatellite regions in B. chouardii and B. pyrenaica, respectively, totaling 9502 analyzed genotypes. The comparison of posterior probabilities for the two competing hypotheses across the surveyed loci clearly favored a disomic inheritance pattern. Linkage tests indicated that none of the studied SSR loci were in linkage disequilibrium, thus representing independent samples of the Borderea genome. These results, along with previous allozyme data, support the allotetraploid origin of this paleoendemic genus and reveal the lowest reported chromosome base number for the family of the yams.

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants.

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.

On the verge of extinction: genetics of the critically endangered Iberian plant species, Borderea chouardii (Dioscoreaceae) and implications for conservation management.

Borderea chouardii is a relictual and dioecious, strictly sexually reproducing, long-living geophyte of the Dioscoreaceae family. Previous biological and demographic studies have indicated the existence of a uniformly distributed panmictic population of this taxon at the southernmost Spanish pre-Pyrenean mountain ranges where it occurs in rather inaccessible crevices of a single limestone cliff. However, individuals of B. chouardii are spatially subdivided into two subpopulations located, respectively, on the upper and lower parts of the cliff, and vertically separated 150 m. Because of its extreme rarity, B. chouardii was the first Iberian taxon to have a specific conservation plan and has been included in several red lists under the category of critically endangered (CR). However, no previous attempts have been conducted to analyse the fine scale evolutionary mechanisms involved in its present microspatial distribution. Genetic diversity and population structure have been investigated through the analysis of neutral hypervariable markers such as simple sequence repeats (SSRs) and randomly amplified polymorphic DNAs (RAPDs) to unravel the impact of life history traits in the differentiation of the two subpopulations. Both types of molecular markers were unequivocal in distinguishing two genetically distinct groups of individuals corresponding to their spatial separation. However, SSRs detected a higher level of subpopulation differentiation (F(ST) = 0.35, R(ST) = 0.32) than RAPDs (F(ST) = 0.21). SSR data indicated significant deviation from random dispersal of genes and genotypes between the two groups, suggesting that mating occurs mainly among individuals within subpopulations, thus, favouring the divergence between the two groups. This microevolutionary differentiation scenario might have been caused by a coupled effect of past genetic drift and reproductive isolation, as a result of strong glacial age bottlenecks and inefficient dispersal system of pollen and seeds, respectively. The identification of such genetic structure in this narrow endemic prompts a modification of the management strategies of its single extant population.

Characterization of seven (CTT)(n) microsatellite loci in the Pyrenean endemic Borderea pyrenaica (Dioscoreaceae): remarks on ploidy level and hybrid origin assessed through allozymes and microsatellite analyses.

We present the identification and characterization of microsatellite loci in the Pyrenean endemic Borderea pyrenaica Miégeville (Dioscoreaceae). Seven microsatellite loci were isolated from a (CTT)(n)-enriched partial genomic library. Electropherograms patterns suggest that B. pyrenaica is a tetraploid species, as is its congener B. chouardii. One microsatellite locus was monomorphic, whereas the remaining ones presented from 2 to 10 alleles when analyzed in a sample of 60 individuals. Microsatellites have revealed higher levels of genetic variability than those in previous studies based on allozymes. Levels of genetic diversity are discussed in terms of tetrasomic (autotetraploidy) or duplicated disomic (allotetraploidy) modes of allele segregation. According to the first hypothesis, mean levels of genetic variability (H(min)-H(max)) range between 0.36 and 0.41, whereas, according to the second hypothesis, the 7 primer pairs amplified 11 chromosomal loci, and mean levels of observed and expected heterozygosities were 0.217 and 0.229, respectively, and did not differ significantly from HW expectations. These results suggest a hybrid allopolyploid origin for the Borderea taxa.