Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin

Background and Aims

In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed.

Methods

Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization.

Key Results

This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome).

Conclusions

It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.     

Divergence and Polymorphism Under the Nearly Neutral Theory of Molecular Evolution

The nearly neutral theory attributes most nucleotide substitution and polymorphism to genetic drift acting on weakly selected mutants, and assumes that the selection coefficients for these mutants are drawn from a continuous distribution. This means that parameter estimation can require numerical integration, and this can be computationally costly and inaccurate. Furthermore, the leading parameter dependencies of important quantities can be unclear, making results difficult to understand. For some commonly used distributions of mutant effects, we show how these problems can be avoided by writing equations in terms of special functions. Series expansion then allows for their rapid calculation and, also, illuminates leading parameter dependencies. For example, we show that if mutants are gamma distributed, the neutrality index is largely independent of the effective population size. However, we also show that such results are not robust to misspecification of the functional form of distribution. Some implications of these findings are then discussed.     

Allopolyploid origin of the Balkan endemic Ranunculus wettsteinii (Ranunculaceae) inferred from nuclear and plastid DNA sequences

The Balkan Peninsula, characterized by high rates of endemism, is recognised as one of the most diverse and species-rich areas of Europe. However, little is known about the origin of Balkan endemics. The present study addresses the phylogenetic position of the Balkan endemic Ranunculus wettsteinii, as well as its taxonomic status and relationship with the widespread R. parnassiifolius, based on nuclear DNA (internal transcribed spacer, ITS) and plastid regions (rpl32-trnL, rps16-trnQ, trnK-matK and ycf6-psbM). Maximum parsimony and Bayesian inference analyses revealed a well-supported clade formed by accessions of R. wettsteinii. Furthermore, our phylogenetic and network analyses supported previous hypotheses of a likely allopolyploid origin for R. wettsteinii between R. montenegrinus and R. parnassiifolius, with the latter as the maternal parent.     

Breeding systems and crossing experiments in Anemone patens and in the Anemome pulsatilla group (Ranunculaceae)

Breeding system studies of Anemone patens, A. pulsatilla ssp. pulsatilla, A. pulsatilla ssp. gotlandica , and A. pulsatilla ssp. grandis in cultivation showed that all taxa are self-compatible. Fruit and seed set were usually low after auto-deposition of pollen but increased in artificially self-pollinated flowers. Cross-pollination usually resulted in increased fruit and seed set compared with self-pollination, but the level varied noticeably among crossing combinations and for different material due probably to the great variation in the frequency of fruit abortion. Plants of A. pulsatilla ssp. pulsatilla derived from one site showed especially low fruit set. Resource limitation is probably an important component determining reproductive success. No crossing barriers between taxa could be established. Significant differences between the three subspecies of A. pulsatilla were found for several morphological characters.     

Multiple origins of Southern Hemisphere Anemone (Ranunculaceae) based on plastid and nuclear sequence data

 Using two molecular data sets, the plastid atpB-rbcL intergenic spacer region and the nuclear ribosomal internal transcribed spacer regions (ITS), the taxonomic affinities of two newly available Anemone species from the Southern Hemisphere were tested. From previous work based on morphology and geographic distribution, it was assumed that A. tenuicaulis from New Zealand was most closely related to the Tasmanian A. crassifolia, whereas the affinity of A. antucensis from Chile and Argentina was regarded as uncertain. Analyses of molecular sequence data from these and 18 other species of Anemone s.lat. (with Clematis as outgroup) result in trees largely congruent with past analyses based on morphology and plastid restriction site data. They strongly support A. richardsonii and A. canadensis (with boreal distributions in the Northern Hemisphere) as paraphyletic to a well supported Southern Hemisphere clade consisting of A. antucensis and A. tenuicaulis. This group of four species is part of an otherwise predominantly Northern Hemisphere assemblage (subgenus Anemonidium s.lat., chromosome base number x=7), including A. narcissiflora, A. obtusiloba, A. keiskeana and A. (=Hepatica) americana. All other austral species included in the present sampling, A. crassifolia (Tasmania), A. knowltonia (=Knowltonia capensis), and A. caffra (both South African), form a separate clade, sister to A. (=Pulsatilla) occidentalis and other Northern Hemisphere anemones (subgenus Anemone s.lat., x=8). Possible phytogeographical links of the Southern Hemisphere species are discussed. Received April 23, 2001 Accepted October 4, 2001     

The reproductive biology of Anemone canadensis (Ranunculaceae): breeding system and facilitation of sexual selection

Our data show that Anemone canadensis L. is xenogamous and has adaptations that should increase the opportunity for sexual selection and minimize interference between female and male sexual function. The flowers of plants within insect exclosures and hand self-pollinated flowers set very few fruits compared to those that were open-pollinated and hand crosspollinated. Examination of styles with uv-fluorescence microscopy showed that self pollen tubes grew into the transmission tissue but rarely entered the ovary, i.e., there is a gametophytic self-incompatibility system. A number of traits should enhance sexual selection. First, pollen grains arrived on the stigmas beginning on day 1, germinated, but the pollen tubes grew very slowly until day 4 or 5 when the transmission tissue matured. This allowed large numbers of pollen grains to reach the stigmas, presumably from a number of pollen parents. Second, basipetal maturation of the transmission tissue, which underlies the elongate stigmatic surfaces, allows pollen tubes originating near the tip of the style to initiate rapid growth prior to those lower in the style. Thus, rapidly growing pollen tubes from the top of the style were able to catch up with those lower in the style. In essence, all the pollen tubes in a style have an equal opportunity to reach the ovule. Large stigmatic pollen loads and high fruit set suggest there is minimal interference between male and female sexual function. This may be a consequence of the spatial separation of the pistils and stamens, which increases during the life of a flower. Synchrony of function among the pistils of a flower in conjunction with large and/or multipatemal pollen loads may reduce the variance in competition among developing embryos and result in fruits of equivalent size.     

Nectar production in the pollen flower of Anemone nemorosa in comparison with other Ranunculaceae and Magnolia (Magnoliaceae)

The observation that the flowers of Anemone nemorosa offer nectar to pollinating bee-flies (Bombylius major) prompted this investigation into the site of nectar secretion and nectary tissue. To allow comparison on a broader basis, other nectar-secreting pollen flowers of the Ranunculaceae and Magnolia (Magnoliaceae) were included in the analysis. The contradictory information available on the function of the mouthparts of bee-flies during nectar and pollen feeding motivated us to investigate the proboscis structure in detail by SEM. Our investigations in Anemone nemorosa proved, for the first time, nectar secretion in the genus Anemone s.s. (i.e. other than the Pulsatilla group) and in addition, within the family, a new type of a carpellary nectary. The latter is an epithelial nectary involving the whole epidermis of the ovarian part of the carpel. The nectary of Anemone nemorosa resembles that of Magnolia (e.g. M. stellata), which we re-investigated. In both Anemone nemorosa and Magnolia stellata, nectar production is limited mainly to the female phase of the proterogynous flower. In this way, the attractiveness of the flower is also assured in the non-pollen presenting phase. Especially in Magnolia, with its numerous carpels arranged on a cone-like receptacle, the economic disadvantage of a choricarpous- compared to a coenocarpous-gynoecium is compensated for by nectar secretion by each carpel. When licking up the nectar droplets from the carpel surfaces, contact of the insect's body with each stigma may be achieved.     

Evolution of Functional Genes in Cetaceans Driven by Natural Selection on a Phylogenetic and Population Level

Cetaceans represent an evolutionary lineage marked by drastic morphological and physiological changes during their adaptation to an exclusively marine existence. In addition, several cetacean species exhibit geographical ranges that encompass different marine environments, with genetic breaks being sometimes consistent with environmental breaks. As such, genes that underwent adaptation during the land-sea transition can also be potential candidates for adaptation to different oceanic environments. In this study, we analysed 3 milk protein genes (β-casein, κ-casein, and α-lactalbumin) and 2 immunity related genes (MHC DQβ1 and γ-fibrinogen) for selection based on available phylogenetic datasets of both mammals and cetaceans, and used the results from this analysis to assess adaptation to different environments on a population level in the European common dolphin (Delphinus delphis). We found that evidence for positive selection could be detected in all genes in the phylogenetic analyses, with β-casein showing a further increase in selective pressure in the cetacean lineage. At the population level, both the immune system locus DQβ1 and β-casein genes showed patterns of variation consistent with divergent selection, and in each case the same populations showed differentiation. One of these populations was also differentiated at neutral markers, while the other was not. We discuss possible inference, and the potential for the further development of these ideas using genomic technologies.     

Natural Selection During Functional Divergence to LMP7 and Proteasome Subunit X (PSMB5) Following Gene Duplication

The LMP7 and PSMB5 genes were created through an ancient gene duplication event of their ancestral locus. These proteins contain an active site of proteolysis, and LMP7 replaces PSMB5 as a component of the 20S proteasome after stimulation of cells by interferon-. Replacement of PSMB5 by LMP7 changes the profile of the products of 20S proteasome processing, predisposing digested peptides for transport to and display by the immune system. The purpose of this study is to investigate evolutionary forces influencing functional divergence between LMP7 and PSMB5 following duplication. Levels of synonymous and nonsynonymous substitution rates are estimated to infer differences in levels of natural selection. Estimates of substitution rates indicate that natural selection elevated rates of nonsynonymous substitution in LMP7 following gene duplication, whereas PSMB5 experienced an increase in substitution rate that was not likely due to diversifying natural selection following duplication. Following initial divergence, nearly neutral mutations have dominated gene evolution in both lineages. The LMP7 gene locus provides a rare example of a protein with specialized function arising from duplication and divergence of a housekeeping protein by way of natural selection.Reviewing Editor: Dr. Rasmus Nielsen     

Climatic Factors Drive Population Divergence and Demography: Insights Based on the Phylogeography of a Riparian Plant Species Endemic to the Hengduan Mountains and Adjacent Regions

Quaternary climatic factors have played a significant role in population divergence and demography. Here we investigated the phylogeography of Osteomeles schwerinae, a dominant riparian plant species of the hot/warm-dry river valleys of the Hengduan Mountains (HDM), Qinling Mountains (QLM) and Yunnan-Guizhou Plateau (YGP). Three chloroplast DNA (cpDNA) regions (trnD-trnT, psbD-trnT, petL-psbE), one single copy nuclear gene (glyceraldehyde 3-phosphate dehydrogenase; G3pdh), and climatic data during the Last Interglacial (LIG; c. 120–140 ka), Last Glacial Maximum (LGM; c. 21 ka), and Current (c. 1950–2000) periods were used in this study. Six cpDNA haplotypes and 15 nuclear DNA (nDNA) haplotypes were identified in the 40 populations of O. schwerinae. Spatial Analysis of Molecular Variance, median-joining networks, and Bayesian phylogenetic trees based on the cpDNA and nDNA datasets, all suggested population divergence between the QLM and HDM-YGP regions. Our climatic analysis identified significant heterogeneity of the climatic factors in the QLM and HDM-YGP regions during the aforementioned three periods. The divergence times based on cpDNA and nDNA haplotypes were estimated to be 466.4–159.4 ka and 315.8–160.3 ka, respectively, which coincide with the time of the weakening of the Asian monsoons in these regions. In addition, unimodal pairwise mismatch distribution curves, expansion times, and Ecological Niche Modeling suggested a history of population expansion (rather than contraction) during the last glaciation. Interestingly, the expansion times were found being well consistent with the intensification of the Asian monsoons during this period. We inferred that the divergence between the two main lineages is probably caused by disruption of more continuous distribution because of weakening of monsoons/less precipitation, whilst subsequent intensification of the Asian monsoons during the last glaciation facilitated the expansion of O. schwerinae populations.     

Genetic structure and evolution of Alpine polyploid complexes: Ranunculus kuepferi (Ranunculaceae) as a case study

The alpine white-flowered buttercup, Ranunculus kuepferi Greuter & Burdet, is a polyploid complex with diploids endemic to the southwestern Alps and polyploids – which have been previously described as apomictic – widespread throughout European mountains. Due to the polymorphic status of both its ploidy level and its reproductive mode, R. kuepferi represents a key species for understanding the evolution of polyploid lineages in alpine habitats. To disentangle the phylogeography of this polyploid taxon, we used cpDNA sequences and AFLP (amplified fragment length polymorphism) markers in 33 populations of R. kuepferi representative of its ploidy level and distribution area. Polyploid individuals were shown to be the result of at least two polyploidization events that may have taken place in the southwestern Alps. From this region, one single main migration of tetraploids colonized the entire Alpine range, the Apennines and Corsica. Genetic recombination among tetraploids was also observed, revealing the facultative nature of the apomictic reproductive mode in R. kuepferi polyploids. Our study shows the contrasting role played by diploid lineages mostly restricted to persistent refugia and by tetraploids, whose dispersal abilities have permitted their range extension all over the previously glaciated Alpine area and throughout neighbouring mountain massifs.     

国产毛莨科银莲花族十七种植物的细胞学研究

研究了国产毛茛科银莲花族Trib.Anemoneae17种植物的染色体数目和核型。10种银莲花属AnemoneL.植物中,1种（西南银莲花A.davidii)为x=8的四倍体（2n=4x=32）,5种（匍枝银莲花A.stolonifera、草玉酶A.rivularis、卵叶银莲花A.begoniifolia、水棉花A.hupehensis f.allba、大火草A.tomentosa）为x=8的二倍体（2n=2x=16）,4种（鹅掌草A.flaccida、湿地银莲花A.rupestris、蓝匙叶银莲花A.trullifolia var.colestina、拟条叶银莲花A.trulifolia var.holophylla、展毛银莲花A.rupestris）为x=7的二倍体（2n=2x=14)。罂粟莲花Anemoclema glaucifolium为x=8的二倍体。6种铁线莲属ClematisL.植物（滇川铁线莲C.hkockiana、长花钱线莲C.rehderiana、金毛铁线莲A.chrysocoma）均为x=8的二倍体。银莲花花属中x=7的种类核型彼此十分相似,均由6对大型具中部着丝点的染色体和1对具端部着丝点的染色体组成;x=8的二倍体种类的核型与罂粟莲花属和铁线莲属植物的核型十分相似,均由5对大型具中部着丝点和3对具端部或近端部着丝点的染色体组成。     

The First Comprehensive Phylogeny of Coptis (Ranunculaceae) and Its Implications for Character Evolution and Classification

Coptis (Ranunculaceae) contains 15 species and is one of the pharmaceutically most important plant genera in eastern Asia. Understanding of the evolution of morphological characters and phylogenetic relationships within the genus is very limited. Here, we present the first comprehensive phylogenetic analysis of the genus based on two plastid and one nuclear markers. The phylogeny was reconstructed using Bayesian inference, as well as maximum parsimony and maximum likelihood methods. The Swofford-Olsen-Waddell-Hillis and Bayesian tests were used to assess the strength of the conflicts between traditional taxonomic units and those suggested by the phylogenetic inferences. Evolution of morphological characters was inferred using Bayesian method to identify synapomorphies for the infrageneric lineages. Our data recognize two strongly supported clades within Coptis. The first clade contains subgenus Coptis and section Japonocoptis of subgenus Metacoptis, supported by morphological characters, such as traits of the central leaflet base, petal color, and petal shape. The second clade consists of section Japonocoptis of subgenus Metacoptis. Coptis morii is not united with C. quinquefolia, in contrast with the view that C. morii is a synonym of C. quinquefolia. Two varieties of C. chinensis do not cluster together. Coptis groenlandica and C. lutescens are reduced to C. trifolia and C. japonica, respectively. Central leaflet base, sepal shape, and petal blade carry a strong phylogenetic signal in Coptis, while leaf type, sepal and petal color, and petal shape exhibit relatively higher levels of evolutionary flexibility.     

Palatability of Selected Alpine Plant Litters for the Decomposer Lumbricus rubellus (Lumbricidae)

On alpine pastureland the decline in large-bodied earthworm numbers and biomass after abandonment of management might be the result of a shift from highly palatable grass litter to poorly digestible leaf litter of dwarf shrubs. To test this hypothesis, we analysed nitrogen, phosphorous and total phenolic contents of fresh and aged litter of eight commonly occuring alpine plant species and compared consumption rates of these food sources in a controlled feeding experiment with Lumbricus rubellus (Lumbricidae). Furthermore, we analysed the microbial community structure of aged litter materials to check for a relationship between the microbial characteristics of the different plant litter types and the food choice of earthworms. Plant litters differed significantly in their chemical composition, earthworms, however, showed no preference for any litter species, but generally rejected fresh litter material. Microbial community structures of the litter types were significantly different, but we could find no evidence for selective feeding of L. rubellus. We conclude that L. rubellus is a widespread, adaptable ubiquist, which is able to feed on a variety of food sources differing in quality and palatability, as long as they have been exposed to wheathering.     

Population genetics and conservation priorities for the critically endangered island endemic Delphinium pentagynum subsp. formenteranum (Ranunculaceae)

Isozyme electrophoresis was used to evaluate levels of genetic diversity and population genetic structure of the critically endangered (CR) perennial larkspur, Delphinium pentagynum subsp. formenteranum (Ranunculaceae), endemic to the island of Formentera (Balearic Islands, Spain). There is only one known population for this taxon, containing only 480 individuals. Moderate values of diversity were detected (P = 40.7%, A = 1.6 and H _e = 0.180), within the range observed in other surveyed larkspurs, but higher than most island endemics. Moderate levels of inbreeding were detected, probably as a consequence of the population's genetic structuring (biparental inbreeding). Threats to this taxon are mainly anthropogenic (fires, grazing, pathway works, and building pressures), although given that only one population exists, stochastic risks cannot be ignored. Conservation of D. pentagynum subsp. formenteranum requires in situ strategies, such as implementing a monitoring program and establishing a botanical reserve, and ex situ strategies, such as collection and long-term storage of seeds.     

Epigenetic Differentiation Persists after Male Gametogenesis in Natural Populations of the Perennial Herb Helleborus foetidus (Ranunculaceae)

Despite the importance of assessing the stability of epigenetic variation in non-model organisms living in real-world scenarios, no studies have been conducted on the transgenerational persistence of epigenetic structure in wild plant populations. This gap in knowledge is hindering progress in the interpretation of natural epigenetic variation. By applying the methylation-sensitive amplified fragment length polymorphism (MSAP) technique to paired plant-pollen (i.e., sporophyte-male gametophyte) DNA samples, and then comparing methylation patterns and epigenetic population differentiation in sporophytes and their descendant gametophytes, we investigated transgenerational constancy of epigenetic structure in three populations of the perennial herb Helleborus foetidus (Ranunculaceae). Single-locus and multilocus analyses revealed extensive epigenetic differentiation between sporophyte populations. Locus-by-locus comparisons of methylation status in individual sporophytes and descendant gametophytes showed that ∼75% of epigenetic markers persisted unchanged through gametogenesis. In spite of some epigenetic reorganization taking place during gametogenesis, multilocus epigenetic differentiation between sporophyte populations was preserved in the subsequent gametophyte stage. In addition to illustrating the efficacy of applying the MSAP technique to paired plant-pollen DNA samples to investigate epigenetic gametic inheritance in wild plants, this paper suggests that epigenetic differentiation between adult plant populations of H. foetidus is likely to persist across generations.     

Patterns of Population Differentiation and Natural Selection on the Celiac Disease Background Risk Network

Celiac disease is a common small intestinal inflammatory condition induced by wheat gluten and related proteins from rye and barley. Left untreated, the clinical presentation of CD can include failure to thrive, malnutrition, and distension in juveniles. The disease can additionally lead to vitamin deficiencies, anemia, and osteoporosis. Therefore, CD potentially negatively affected fitness in past populations utilizing wheat, barley, and rye. Previous analyses of CD risk variants have uncovered evidence for positive selection on some of these loci. These studies also suggest the possibility that risk for common autoimmune conditions such as CD may be the result of positive selection on immune related loci in the genome to fight infection. Under this evolutionary scenario, disease phenotypes may be a trade-off from positive selection on immunity. If this hypothesis is generally true, we can expect to find a signal of natural selection when we survey across the network of loci known to influence CD risk. This study examines the non-HLA autosomal network of gene loci associated with CD risk in Europe. We reject the null hypothesis of neutrality on this network of CD risk loci. Additionally, we can localize evidence of selection in time and space by adding information from the genome of the Tyrolean Iceman. While we can show significant differentiation between continental regions across the CD network, the pattern of evidence is not consistent with primarily recent (Holocene) selection across this network in Europe. Further localization of ancient selection on this network may illuminate the ecological pressures acting on the immune system during this critically interesting phase of our evolution.