Evolutionary Comparison of the Complete Chloroplast Genomes in Convallaria Species and Phylogenetic Study of Asparagaceae.

The genus Convallaria (Asparagaceae) comprises three herbaceous perennial species that are widely distributed in the understory of temperate deciduous forests in the Northern Hemisphere. Although Convallaria species have high medicinal and horticultural values, studies related to the phylogenetic analysis of this genus are few. In the present study, we assembled and reported five complete chloroplast (cp) sequences of three Convallaria species (two of C. keiskei Miq., two of C. majalis L., and one of C. montana Raf.) using Illumina paired-end sequencing data. The cp genomes were highly similar in overall size (161,365-162,972 bp), and all consisted of a pair of inverted repeats (IR) regions (29,140-29,486 bp) separated by a large single-copy (LSC) (85,183-85,521 bp) and a small single-copy (SSC) region (17,877-18,502 bp). Each cp genome contained the same 113 unique genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Gene content, gene order, AT content and IR/SC boundary structure were nearly identical among all of the Convallaria cp genomes. However, their lengths varied due to contraction/expansion at the IR/LSC borders. Simple sequence repeat (SSR) analyses indicated that the richest SSRs are A/T mononucleotides. Three highly variable regions (petA-psbJ, psbI-trnS and ccsA-ndhD) were identified as valuable molecular markers. Phylogenetic analysis of the family Asparagaceae using 48 cp genome sequences supported the monophyly of Convallaria, which formed a sister clade to the genus Rohdea. Our study provides a robust phylogeny of the Asparagaceae family. The complete cp genome sequences will contribute to further studies in the molecular identification, genetic diversity, and phylogeny of Convallaria.

Intracellular DNA transfer events restricted to the genus Convallaria within the Asparagaceae family: Possible mechanisms and potential as genetic markers for biographical studies.

Intracellular gene transfer among plant genomes is a common phenomenon. Due to their high conservation and high plastid membrane integrity, chloroplast (cp) genomes incorporate foreign genetic material very rarely. Convallaria is a small monocotyledonous genus consisting of C. keiskei, C. majalis and C. montana. Here, we characterized, analyzed and identified 3.3 and 3.7 kb of mitochondrial DNA sequences in the plastome (MCP) of C. majalis and C. montana, respectively. We identified 6 bp and 23 bp direct repeats and mitochondrial pseudogenes, with rps3, rps19 and rpl10 identified in the MCP region. Additionally, we developed novel plastid molecular genetic markers to differentiate Convallaria spp. based on 21 populations. BEAST and biogeographical analyses suggested that Convallaria separated into Eurasian and North American lineages during the middle Pliocene and originated in East Asia. Vicariance in the genus was followed by dispersal into Europe and southeastern North America. These analyses indicate that the MCP event was restricted to the genus Convallaria of Asparagaceae, in contrast to similar events that occurred in its common ancestors with other families of land plants. However, further mitochondrial and population studies are necessary to understand the integration of the MCP region and gene flow in the genus Convallaria.

Development of 19 novel microsatellite markers of lily-of-the-valley (Convallaria, Asparagaceae) from transcriptome sequencing.

The lily-of-the-valley Convallaria (Asparagaceae) consists of three herbaceous perennial species. The plants are commonly found in northern hemisphere, and are best-known for their ornamental and pharmaceutical value. In order to assess the genetic structure, diversity and demographic history of Convallaria species, 19 novel microsatellite markers were developed based on transcriptome data of C. keiskei. Polymorphism and cross-amplification of the markers were tested in three populations of C. keiskei and one population each of C. majalis and C. montana. The transferability rate in two species was both 89.5%. The average number of alleles detected per locus was 7.7, 3.3 and 2.7 in C. keiskei, C. majalis and C. montana, respectively, and the polymorphism information content correspondingly varied from 0.067 to 0.730, from 0.071 to 0.637 and from 0.195 to 0.680 at the population level. The observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.833, respectively. Seven of the 19 loci showed significant deviation from Hardy-Weinberg equilibrium. The availability of these markers will provide a useful molecular tool for further population genetics, phylogeographic and breeding studies of Convallaria species.

Evidence of mitochondrial DNA in the chloroplast genome of Convallaria keiskei and its subsequent evolution in the Asparagales.

DNA transfer between internal organelles such as the nucleus, mitochondrion, and plastid is a well-known phenomenon in plant evolution, and DNA transfer from the plastid and mitochondrion to the nucleus, from the plastid to the mitochondrion, and from the nucleus to the mitochondrion has been well-documented in angiosperms. However, evidence of the transfer of mitochondrial DNA (mtDNA) to the plastid has only been found in three dicotyledons and one monocotyledon. In the present study, we characterised and analysed two chloroplast (cp) genome sequences of Convallaria keiskei and Liriope spicata, and found that C. keiskei has the largest cp genome (162,109 bp) in the Asparagaceae. Interestingly, C. keiskei had a ~3.3-kb segment of mtDNA in its cp genome and showed similarity with the mt gene rpl10 as a pseudogene. Further analyses revealed that mtDNA transfer only occurred in C. keiskei in the Nolinoideae, which diverged very recently (7.68 million years ago (mya); 95% highest posterior density (HPD): 14.55-2.97 mya). These findings indicate that the C. keiskei cp genome is unique amongst monocotyledon land plants, but further work is necessary to understand the direction and mechanism involved in the uptake of mtDNA by the plastid genome of C. keiskei.

The impact of extensive clonal growth on fine-scale mating patterns: a full paternity analysis of a lily-of-the-valley population (Convallaria majalis).

BACKGROUND AND AIMS: The combination of clonality and a mating system promoting outcrossing is considered advantageous because outcrossing avoids the fitness costs of selfing within clones (geitonogamy) while clonality assures local persistence and increases floral display. The spatial spread of genetically identical plants (ramets) may, however, also decrease paternal diversity (the number of sires fertilizing a given dam) and fertility, particularly towards the centre of large clumped clones. This study aimed to quantify the impact of extensive clonal growth on fine-scale paternity patterns in a population of the allogamous Convallaria majalis. METHODS: A full analysis of paternity was performed by genotyping all flowering individuals and all viable seeds produced during a single season using AFLP. Mating patterns were examined and the spatial position of ramets was related to the extent of multiple paternity, fruiting success and seed production. KEY RESULTS: The overall outcrossing rate was high (91 %) and pollen flow into the population was considerable (27 %). Despite extensive clonal growth, multiple paternity was relatively common (the fraction of siblings sharing the same father was 0·53 within ramets). The diversity of offspring collected from reproductive ramets surrounded by genetically identical inflorescences was as high as among offspring collected from ramets surrounded by distinct genets. There was no significant relationship between the similarity of the pollen load received by two ramets and the distance between them. Neither the distance of ramets with respect to distinct genets nor the distance to the genet centre significantly affected fruiting success or seed production. CONCLUSIONS: Random mating and considerable pollen inflow most probably implied that pollen dispersal distances were sufficiently high to mitigate local mate scarcity despite extensive clonal spread. The data provide no evidence for the intrusion of clonal growth on fine-scale plant mating patterns.

Extremely low genotypic diversity and sexual reproduction in isolated populations of the self-incompatible lily-of-the-valley (Convallaria majalis) and the role of the local forest environment.

BACKGROUND AND AIMS: Clonal growth is a common phenomenon in plants and allows them to persist when sexual life-cycle completion is impeded. Very low levels of recruitment from seed will ultimately result in low levels of genotypic diversity. The situation can be expected to be exacerbated in spatially isolated populations of obligated allogamous species, as low genotypic diversities will result in low availability of compatible genotypes and low reproductive success. Populations of the self-incompatible forest herb lily-of-the-valley (Convallaria majalis) were studied with the aim of inferring the relative importance of sexual and asexual recruitment. Then the aim was to establish a relationship between genotypic diversity, sexual reproduction and the local forest environment. METHODS: Highly polymorphic microsatellite markers were used to investigate clonal diversities and population genetic structure of 20 populations of C. majalis in central Belgium. KEY RESULTS: Most of the populations studied consisted of a single genotype and linkage disequilibrium within populations was high, manifesting clonal growth as the main mode of reproduction. A population consisting of multiple genotypes mainly occurred in locations with a thin litter layer and high soil phosphorus levels, suggesting environment-mediated sporadic recruitment from seed. Highly significant genetic differentiation indicated that populations are reproductively isolated. In agreement with the self-incompatibility of C. majalis, monoclonal populations showed very low or even absent fruit set. CONCLUSIONS: Lack of sexual recruitment in spatially isolated C. majalis populations has resulted in almost monoclonal populations with reduced or absent sexual reproduction, potentially constraining their long-term persistence. The local forest environment may play an important role in mediating sexual recruitment in clonal forest plant species.

Reproductive demography of ramets and genets in a rhizomatous clonal plant Convallaria keiskei.

Clonal growth occurring below the ground makes it difficult to identify individuals and demonstrate the demographic features of a focal plant species. In this study, genotypically identified ramets of a rhizomatous clonal herb, Convallaria keiskei Miq., were monitored for their growth, survival, and reproduction from 2003 to 2006. After the monitoring period, their subterranean organs were excavated to explore the underground connections of established ramets and the direction of clonal growth. We then combined data on the fate of the monitored ramets with the information of rhizome connections, clarifying reproductive demography at both the ramet and genet levels. Although each ramet initiated both sexual reproduction (via flowering) and clonal growth, clonal growth tended to precede sexual reproduction. In a surveyed genet, 51.0% of ramets produced flowers and 29.6% generated clonal offspring during the study period. Consequently, we clarified the reproductive demography of C. keiskei: clonal growth tended to precede flowering in a ramet, and a genet can keep reproducing every season at the genet level, despite a ramet not having inflorescence every year.

Phylogenetic analyses and chromosome evolution in Convallarieae (Ruscaceae sensu lato), with some taxonomic treatments.

The tribe Convallarieae, comprising 10 genera and 95 species, has recently been transferred from its own family to Ruscaceae sensu lato. In this study, sequence data from trnK and rbcL were analyzed for 19 species in 8 genera, and chromosome morphology was analyzed for 17 species in 7 genera. The parsimony analysis of trnK and rbcL sequences showed that Convallarieae are monophyletic. Although early branches did not receive strong bootstrap support, Convallaria diverged at the first branch, followed by Speirantha. The rest of the tribe was split into three, well-supported clades: one with Reineckea, the second with Campylandra and Rohdea, and the third with Tupistra, Tricalistra, and Aspidistra. Two monotypic genera, Rohdea and Tricalistra, were embedded in a clade of Campylandra and of Tupistra, respectively. Three karyotypes were distinguished in the tribe on the basis of the basic number and morphology of metaphase chromosomes: Convallaria type (with x=19 and unimodal chromosome length), Tupistra type (with x=19 and trimodal chromosome length), and Aspidistra-elatior type (with x=18 and trimodal chromosome length). The character-state distribution in the molecular tree showed that the Convallaria type is plesiomorphic, from which was derived the Tupistra type and subsequently the Aspidistra-elatior type. Taxonomic treatments to transfer Campylandra to Rohdea and Tricalistra to Tupistra are also given.