Analysis of six chloroplast genomes provides insight into the evolution of Chrysosplenium (Saxifragaceae).

BACKGROUND: Chrysosplenium L. (Saxifragaceae) is a genus of plants widely distributed in Northern Hemisphere and usually found in moist, shaded valleys and mountain slopes. This genus is ideal for studying plant adaptation to low light conditions. Although some progress has been made in the systematics and biogeography of Chrysosplenium, its chloroplast genome evolution remains to be investigated. RESULTS: To fill this gap, we sequenced the chloroplast genomes of six Chrysosplenium species and analyzed their genome structure, GC content, and nucleotide diversity. Moreover, we performed a phylogenetic analysis and calculated non-synonymous (Ka) /synonymous (Ks) substitution ratios using the combined protein-coding genes of 29 species within Saxifragales and two additional species as outgroups, as well as a pair-wise estimation for each gene within Chrysosplenium. Compared with the outgroups in Saxifragaceae, the six Chrysosplenium chloroplast genomes had lower GC contents; they also had conserved boundary regions and gene contents, as only the rpl32 gene was lost in four of the Chrysosplenium chloroplast genomes. Phylogenetic analyses suggested that the Chrysosplenium separated to two major clades (the opposite group and the alternate group). The selection pressure estimation (Ka/Ks ratios) of genes in the Chrysosplenium species showed that matK and ycf2 were subjected to positive selection. CONCLUSION: This study provides genetic resources for exploring the phylogeny of Chrysosplenium and sheds light on plant adaptation to low light conditions. The lower average GC content and the lacking gene of rpl32 indicated selective pressure in their unique habitats. Different from results previously reported, our selective pressure estimation suggested that the genes related to photosynthesis (such as ycf2) were under positive selection at sites in the coding region.

Extreme diversification of floral volatiles within and among species of Lithophragma (Saxifragaceae).

A major challenge in evolutionary biology is to understand how complex traits of multiple functions have diversified and codiversified across interacting lineages and geographic ranges. We evaluate intra- and interspecific variation in floral scent, which is a complex trait of documented importance for mutualistic and antagonistic interactions between plants, pollinators, and herbivores. We performed a large-scale, phylogenetically structured study of an entire plant genus (Lithophragma, Saxifragaceae), of which several species are coevolving with specialized pollinating floral parasites of the moth genus Greya (Prodoxidae). We sampled 94 Lithophragma populations distributed across all 12 recognized Lithophragma species and subspecies, and four populations of related saxifragaceous species. Our results reveal an unusually high diversity of floral volatiles among populations, species, and clades within the genus. Moreover, we found unexpectedly major changes at each of these levels in the biosynthetic pathways used by local populations in their floral scents. Finally, we detected significant, but variable, genus- and species-level patterns of ecological convergence in the floral scent signal, including an impact of the presence and absence of two pollinating Greya moth species. We propose that one potential key to understanding floral scent variation in this hypervariable genus is its geographically diverse interactions with the obligate specialized Greya moths and, in some species and sites, more generalized copollinators.

Chloroplast genome analyses and genomic resource development for epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae), using genome skimming data.

BACKGROUND: Epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae) have an epilithic habitat (rocky slopes) and a parapatric distribution in East Asia, which makes them an ideal model for a more comprehensive understanding of the demographic and divergence history and the influence of climate changes in East Asia. However, the genetic background and resources for these two genera are scarce. RESULTS: The complete chloroplast (cp) genomes of two Oresitrophe rupifraga and one Mukdenia rossii individuals were reconstructed and comparative analyses were conducted to examine the evolutionary pattern of chloroplast genomes in Saxifragaceae. The cp genomes ranged from 156,738 bp to 156,960 bp in length and had a typical quadripartite structure with a conserved genome arrangement. Comparative analysis revealed the intron of rpl2 has been lost in Heuchera parviflora, Tiarella polyphylla, M. rossii and O. rupifraga but presents in the reference genome of Penthorum chinense. Seven cp hotspot regions (trnH-psbA, trnR-atpA, atpI-rps2, rps2-rpoC2, petN-psbM, rps4-trnT and rpl33-rps18) were identified between Oresitrophe and Mukdenia, while four hotspots (trnQ-psbK, trnR-atpA, trnS-psbZ and rpl33-rps18) were identified within Oresitrophe. In addition, 24 polymorphic cpSSR loci were found between Oresitrophe and Mukdenia. Most importantly, we successfully developed 126 intergeneric polymorphic gSSR markers between Oresitrophe and Mukdenia, as well as 452 intrageneric ones within Oresitrophe. Twelve randomly selected intergeneric gSSRs have shown that these two genera exhibit a significant genetic structure. CONCLUSIONS: In this study, we conducted genome skimming for Oresitrophe rupifraga and Mukdenia rossii. Using these data, we were able to not only assemble their complete chloroplast genomes, but also develop abundant genetic resources (cp hotspots, cpSSRs, polymorphic gSSRs). The genomic patterns and genetic resources presented here will contribute to further studies on population genetics, phylogeny and conservation biology in Saxifragaceae.

Pseudo-parallel patterns of disjunctions in an Arctic-alpine plant lineage.

Disjunct distributions have intrigued biologists for centuries. Investigating these biogeographic patterns provides insight into speciation and biodiversity at multiple spatial and phylogenetic scales. Some disjunctions have been intensively studied, yet others have been largely overlooked and remain poorly understood. Among the lesser-known disjunction patterns is that between the mountain ranges of western North America. Flora and fauna endemic to the mountains of this region provide important systems for investigating causes and results of disjunctions, given the relatively recent geological formation of this area and the intense climatic fluctuations that have occurred since its formation. In Micranthes (Saxifragaceae), which has high rates of montane endemism, two species, M. bryophora and M. tolmiei, show this biogeographical pattern. By reconstructing a time-calibrated phylogeny based on 518 low-copy nuclear markers and including multiple populations of each species from the Coast Ranges, Cascades, Sierra Nevada, and Rocky Mountains, this study provides a biogeographical and temporal framework for the evolution of Micranthes in western North America. Strongly supported east-west differentiated clades are recovered for M. bryophora and M. tolmiei in both maximum likelihood and coalescent-based species tree reconstructions. Biogeographic analysis suggests different patterns of dispersal for both taxa and the dating analyses recovered contrasting ages for each clade. Due to both the different geographic patterns and the timing of the initial diversification of each taxon corresponding to different geologic and climatic events, the disjunction patterns shown for these taxa are suggested to be an example of biogeographical pseudocongruence.

Floral organ MADS-box genes in Cercidiphyllum japonicum (Cercidiphyllaceae): Implications for systematic evolution and bracts definition.

The dioecious relic Cercidiphyllum japonicum is one of two species of the sole genus Cercidiphyllum, with a tight inflorescence lacking an apparent perianth structure. In addition, its systematic place has been much debated and, so far researches have mainly focused on its morphology and chloroplast genes. In our investigation, we identified 10 floral organ identity genes, including four A-class, three B-class, two C-class and one D-class. Phylogenetic analyses showed that all ten genes are grouped with Saxifragales plants, which confirmed the phylogenetic place of C. japonicum. Expression patterns of those genes were examined by quantitative reverse transcriptase PCR, with some variations that did not completely coincide with the ABCDE model, suggesting some subfunctionalization. As well, our research supported the idea that thebract actually is perianth according to our morphological and molecular analyses in Cercidiphyllum japonicum.

Evolutionary radiations in the species-rich mountain genus Saxifraga L.

BACKGROUND: A large number of taxa have undergone evolutionary radiations in mountainous areas, rendering alpine systems particularly suitable to study the extrinsic and intrinsic factors that have shaped diversification patterns in plants. The species-rich genus Saxifraga L. is widely distributed throughout the Northern Hemisphere, with high species numbers in the regions adjacent to the Qinghai-Tibet Plateau (QTP) in particular the Hengduan Mountains and the Himalayas. Using a dataset of 297 taxa (representing at least 60% of extant Saxifraga species), we explored the variation of infrageneric diversification rates. In addition, we used state-dependent speciation and extinction models to test the effects of geographic distribution in the Hengduan Mountains and the entire QTP region as well as of two morphological traits (cushion habit and specialized lime-secreting glands, so-called hydathodes) on the diversification of this genus. RESULTS: We detected two to three rate shifts across the Saxifraga phylogeny and two of these shifts led to radiations within two large subclades of Saxifraga, sect. Ciliatae Haworth subsect. Hirculoideae Engl. & Irmsch. and sect. Porphyrion Tausch subsect. Kabschia Engl. GEOSSE analyses showed that presence in the Hengduan Mountains had a positive effect on diversification across Saxifraga. Influence of these mountains was strongest in Saxifraga sect. Ciliatae subsect. Hirculoideae given its pronounced distribution there, and thus the radiation in this group can be classified at least partially as geographic. In contrast, the evolution of the cushion life form and lime-secreting hydathodes had positive effects on diversification only in selected Saxifraga sections, including sect. Porphyrion subsect. Kabschia. We therefore argue that radiation in this group was likely adaptive. CONCLUSIONS: Our study underlines the complexity of processes and factors underpinning plant radiations: Even in closely related lineages occupying the same life zone, shifts in diversification are not necessarily governed by similar factors. In conclusion, alpine plant radiations result from a complex interaction among geographical settings and/or climatic modifications providing key opportunities for diversification as well as the evolution of key innovations.

[Newly records of medicinal species in Jiangxi].

Seven species were reported as new records of Jiangxi province, which collected from Wugongshan region, including Huperzia kunmingensis(Lycopodiaceae), Hydrangea mangshanensis(Saxifragaceae), Itea glutinosa(Saxifragaceae), Stellaria monosperma var. japonica(Lycopodiaceae), Youngia pratti(Compositae), and Calanthe henryi(Orchidaceae), Collabium formosanum(Orchidaceae). Among these species, H. kunmingensis, H. mangshanensis, I. glutinosa, Y. pratti and C. henryi are endemic in China. C. henryi is stenotopic distribution in China and regarded as vulnerable endangered plant by IUCN. The report is of great significance to the plant diversity and floristic composition in Jiangxi. All the specimens examined are preserved in Jishou University (JIU) and Sun yat-sen University (SYS).

Genetic Diversity and Spatial Genetic Structure of the Grassland Perennial Saxifraga granulata along Two River Systems.

Due to changes in land use, the natural habitats of an increasing number of plant species have become more and more fragmented. In landscapes that consist of patches of suitable habitat, the frequency and extent of long-distance seed dispersal can be expected to be an important factor determining local genetic diversity and regional population structure of the remaining populations. In plant species that are restricted to riparian habitats, rivers can be expected to have a strong impact on the dynamics and spatial genetic structure of populations as they may enable long-distance seed dispersal and thus maintain gene flow between fragmented populations. In this study, we used polymorphic microsatellite markers to investigate the genetic diversity and the spatial genetic structure of 28 populations of Saxifraga granulata along two rivers in central Belgium. We hypothesized that rivers might be essential for gene flow among increasingly isolated populations of this species. Genetic diversity was high (HS = 0.68), which to a certain extent can be explained by the octoploid nature of S. granulata in the study area. Populations along the Dijle and Demer rivers were also highly differentiated (G"ST = 0.269 and 0.164 and DEST = 0.190 and 0.124, respectively) and showed significant isolation-by-distance, indicating moderate levels of gene flow primarily between populations that are geographically close to each other. Along the river Demer population genetic diversity was higher upstream than downstream, suggesting that seed dispersal via the water was not the primary mode of dispersal. Overall, these results indicate that despite increasing fragmentation populations along both rivers were highly genetically diverse. The high ploidy level and longevity of S. granulata have most likely buffered negative effects of fragmentation on genetic diversity and the spatial genetic structure of populations in riparian grasslands.

Cryptic introgression into the kidney saxifrage (Saxifraga hirsuta) from its more abundant sympatric congener Saxifraga spathularis, and the potential risk of genetic assimilation.

BACKGROUND AND AIMS: Although hybridization can play a positive role in plant evolution, it has been shown that excessive unidirectional hybridization can result in replacement of a species' gene pool, and even the extinction of rare species via genetic assimilation. This study examines levels of introgression between the common Saxifraga spathularis and its rarer congener S. hirsuta, which have been observed to hybridize in the wild where they occur sympatrically. METHODS: Seven species-specific single nucleotide polymorphisms (SNPs) were analysed in 1025 plants representing both species and their hybrid, S. × polita, from 29 sites across their ranges in Ireland. In addition, species distribution modelling was carried out to determine whether the relative abundance of the two parental species is likely to change under future climate scenarios. KEY RESULTS: Saxifraga spathularis individuals tended to be genetically pure, exhibiting little or no introgression from S. hirsuta, but significant levels of introgression of S. spathularis alleles into S. hirsuta were observed, indicating that populations exhibiting S. hirsuta morphology are more like a hybrid swarm, consisting of backcrosses and F2s. Populations of the hybrid, S. × polita, were generally comprised of F1s or F2s, with some evidence of backcrossing. Species distribution modelling under projected future climate scenarios indicated an increase in suitable habitats for both parental species. CONCLUSIONS: Levels of introgression observed in this study in both S. spathularis and S. hirsuta would appear to be correlated with the relative abundance of the species. Significant introgression of S. spathularis alleles was detected in the majority of the S. hirsuta populations analysed and, consequently, ongoing introgression would appear to represent a threat to the genetic integrity of S. hirsuta, particularly in areas where the species exists sympatrically with its congener and where it is greatly outnumbered.

Phylogeny, divergence times, and historical biogeography of the angiosperm family Saxifragaceae.

Saxifragaceae (Saxifragales) contain approximately 640 species and 33 genera, about half of which are monotypic. Due to factors such as morphological stasis, convergent morphological evolution, and disjunct distributions, relationships within Saxifragaceae have historically been troublesome. The family occurs primarily in mountainous regions of the Northern Hemisphere, with the highest generic and species diversity in western North America, but disjunct taxa are known from southern South America. Here, we integrate broad gene (56 loci) and taxon (223 species) sampling strategies, both the most comprehensive to date within Saxifragaceae, with fossil calibrations and geographical distribution data to address relationships, divergence times, and historical biogeography among major lineages of Saxifragaceae. Two previously recognized main clades, the heucheroids (eight groups+Saniculiphyllum) and saxifragoids (Saxifraga s.s.), were re-affirmed by our phylogenetic analyses. Relationships among the eight heucheroid groups, as well as the phylogenetic position of Saniculiphyllum within the heucheroids, were resolved with mostly high support. Divergence time estimates indicate that Saxifragaceae began to diversify ca. 38.37 million years ago (Mya; 95% HPD=30.99-46.11Mya) in the Mid-Late Eocene, and that the two major lineages, the heucheroids and saxifragoids, began to diversify approximately 30.04Mya (95% HPD=23.87-37.15Mya) and 30.85 Mya (95% HPD=23.47-39.33Mya), respectively. We reconstructed ancestral geographic areas using statistical dispersal-vicariance (S-DIVA). These analyses indicate several radiations within Saxifragaceae: one in eastern Asia and multiple radiations in western North America. Our results also demonstrate that large amounts of sequence data coupled with broad taxon sampling can help resolve clade relationships that have thus far seemed intractable.

On the evolutionary and biogeographic history of Saxifraga sect. Trachyphyllum (Gaud.) Koch (Saxifragaceae Juss.).

Arctic-alpine plants in the genus Saxifraga L. (Saxifragaceae Juss.) provide an excellent system for investigating the process of diversification in northern regions. Yet, sect. Trachyphyllum (Gaud.) Koch, which is comprised of about 8 to 26 species, has still not been explored by molecular systematists even though taxonomists concur that the section needs to be thoroughly re-examined. Our goals were to use chloroplast trnL-F and nuclear ITS DNA sequence data to circumscribe the section phylogenetically, test models of geographically-based population divergence, and assess the utility of morphological characters in estimating evolutionary relationships. To do so, we sequenced both genetic markers for 19 taxa within the section. The phylogenetic inferences of sect. Trachyphyllum using maximum likelihood and Bayesian analyses showed that the section is polyphyletic, with S. aspera L. and S bryoides L. falling outside the main clade. In addition, the analyses supported several taxonomic re-classifications to prior names. We used two approaches to test biogeographic hypotheses: i) a coalescent approach in Mesquite to test the fit of our reconstructed gene trees to geographically-based models of population divergence and ii) a maximum likelihood inference in Lagrange. These tests uncovered strong support for an origin of the clade in the Southern Rocky Mountains of North America followed by dispersal and divergence episodes across refugia. Finally we adopted a stochastic character mapping approach in SIMMAP to investigate the utility of morphological characters in estimating evolutionary relationships among taxa. We found that few morphological characters were phylogenetically informative and many were misleading. Our molecular analyses provide a foundation for the diversity and evolutionary relationships within sect. Trachyphyllum and hypotheses for better understanding the patterns and processes of divergence in this section, other saxifrages, and plants inhabiting the North Pacific Rim.

Phylogeny and systematics of Micranthes (Saxifragaceae): an appraisal in European territories.

Recent phylogenetic studies have shown that Saxifraga, as currently understood, must be divided into two genera: Saxifraga L. sensu stricto and Micranthes Haw. To better understand the evolutionary history of these two genera, we performed phylogenetic analyses inferred from the nuclear ribosomal sequences from the internal transcribed spacer and the sequences of the plastid DNA (rbcL). Our molecular data confirmed the monophyly of the genus Micranthes and the consistency of the existing systematic treatments based on morphological criteria. Moreover, Micranthes species native from the Iberian Peninsula (i.e. M. clusii, M. lepismigena and M. stellaris) should be included into Micranthes sect. Arabisa.

Tales of the unexpected: phylogeography of the arctic-alpine model plant Saxifraga oppositifolia (Saxifragaceae) revisited.

Arctic-alpine biota occupy enormous areas in the Arctic and the northern hemisphere mountain ranges and have undergone major range shifts during their comparatively short history. The origins of individual arctic-alpine species remain largely unknown. In the case of the Purple saxifrage, Saxifraga oppositifolia, an important model for arctic-alpine plants, phylogeographic studies have remained inconclusive about early stages of the species' spatiotemporal diversification but have provided evidence for long-range colonization out of a presumed Beringian origin to cover today's circumpolar range. We re-evaluated the species' large-scale range dynamics based on a geographically extended sampling including crucial areas such as Central Asia and the (south-)eastern European mountain ranges and employing up-to-date phylogeographic analyses of a plastid sequence data set and a more restricted AFLP data set. In accordance with previous studies, we detected two major plastid DNA lineages also reflected in AFLP divergence, suggesting a long and independent vicariant history. Although we were unable to determine the species' area of origin, our results point to Europe (probably the Alps) and Central Asia, respectively, as the likely ancestral areas of the two main lineages. AFLP data suggested that contact areas between the two clades in the Carpathians, Northern Siberia and western Greenland were secondary. In marked contrast to high levels of diversity revealed in previous studies, populations from the major arctic refugium Beringia did not exhibit any plastid sequence polymorphism. Our study shows that adequate sampling of the southern, refugial populations is crucial for understanding the range dynamics of arctic-alpine species.

Isolation and characterization of microsatellite markers from Tiarella polyphylla (Saxifragaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Tiarella polyphylla in Korea and Japan to study the genetic diversity and genetic structure of the species. METHODS AND RESULTS: We successfully isolated a total of nine polymorphic simple sequence repeat loci. These nine loci contained from two to eight alleles per locus. The observed and expected heterozygosities ranged from 0.000 to 0.250 and 0.000 to 0.974, respectively. CONCLUSIONS: The markers developed in this study may be used to estimate genetic diversity, genetic structure, and historical and recent gene flow in T. polyphylla, which requires conservation and management.

Phylogenetic placement of the enigmatic and critically endangered genus Saniculiphyllum (Saxifragaceae) inferred from combined analysis of plastid and nuclear DNA sequences.

Saniculiphyllum, a monotypic genus distributed in Southwest China, was thought to be extinct before our recent rediscovery. The taxonomic position of this genus has been enigmatic ever since its publication. It was originally treated as the only member of a distinct tribe Saniculiphylleae in the family Saxifragaceae. Some proposed a new family, Saniculophyllaceae, to accommodate this genus, although its affinities are clearly with members of Saxifragaceae. Here we analyzed six DNA regions, the nuclear ribosomal ITS and 26S rDNA and the plastid rbcL, matK, trnL-trnF, psbA-trnH genes, spacers, and intron to explore the phylogenetic position of Saniculiphyllum within Saxifragaceae. The combined nuclear and chloroplast dataset includes 63 ingroup species, representing all genera but Hieronymusia in the family. Results from likelihood, parsimony and Bayesian phylogenetic methods corroborate earlier results. Two clades of Saxifragaceae, the Heucheroid and Saxifragoid clades, were recovered. The topologies obtained from different analyses confirm the placement of Saniculiphyllum in Saxifragaceae, but our analyses reveal that Saniculiphyllum is embedded within the large Heucheroid clade. However, the closest relatives of Saniculiphyllum within the Heucheroid clade remain unclear. Combined with morphological data, our results suggest that Saniculiphyllum should best be regarded as a highly distinctive lineage within the Heucheroid clade of Saxifragaceae. Morphological novelties and conservation status of Saniculiphyllum are also presented.

[ISSR analysis of genetic diversity in Rodgersia aesculifolia germplasm resources].

OBJECTIVE: To study the genetic diversity of germplasm resources for Rodgersia aesculifolia. METHODS: 24 species of germplasm resources for Rodgersia aesculifolia were analyzed by ISSR molecular markers. To make up the systematic diagram of genetic relationship by NTSYSpc-2.11 software, clustered by UPGMA method and establish the dendrogram. RESULTS: A total of 109 ISSR bands was obtained by 8 primers, among which 96 were polymorphic bands. The average percentage of polymorphic bands was 88.1%. The genetic distance (GD) of the 24 Rodgersia aesculifolia accessions ranged from 0.115 to 0.877, and average 0.421. Two groups and five inferior groups were clustered by the digital data of polymorphic bands. By cluster analysis, the geographical distribution is obvious. CONCLUSION: The diversity level of the different germplasm resources for Rodgersia aesculifolia higher and the relationship of Rodgersia aesculifolia correlates with the geographical location in some way.

Unveiling cryptic species diversity of flowering plants: successful biological species identification of Asian Mitella using nuclear ribosomal DNA sequences.

BACKGROUND: Although DNA sequence analysis is becoming a powerful tool for identifying species, it is not easy to assess whether the observed genetic disparity corresponds to reproductive isolation. Here, we compared the efficiency of biological species identification between nuclear ribosomal and chloroplast DNA sequences, focusing on an Asian endemic perennial lineage of Mitella (Asimitellaria; Saxifragaceae). We performed artificial cross experiments for 43 pairs of ten taxonomic species, and examined their F1 hybrid pollen fertility in vitro as a quantitative measure of postzygotic reproductive isolation. RESULTS: A nonlinear, multiple regression analysis indicated that the nuclear ribosomal DNA distances are sufficient to explain the observed pattern of F1 hybrid pollen fertility, and supplementation with chloroplast DNA distance data does not improve the explanatory power. Overall, with the exception of a recently diverged species complex with more than three biological species, nuclear ribosomal DNA sequences successfully circumscribed ten distinct biological species, of which two have not been described (and an additional one has not been regarded as a distinct taxonomic species) to date. CONCLUSION: We propose that nuclear ribosomal DNA sequences contribute to reliable identification of reproductively isolated and cryptic species of Mitella. More comparable studies for other plant groups are needed to generalize our findings to flowering plants.

Parallel floral adaptations to pollination by fungus gnats within the genus Mitella (Saxifragaceae).

The widespread pattern of parallel flower evolution as an adaptation for particular pollinator agents, known as "pollination syndromes", has long drawn attention from evolutionary biologists. Here, we report parallel evolution of saucer-shaped flowers and an associated unusual pollination system within the lineage Heucherina, a group of saxifragaceous genera. Field observations reveal that 18 of 28 plant species studied are pollinated almost exclusively by fungus gnats (Mycetophilidae). Among the 18 species with a fungus-gnat pollination system, 13 have characteristic saucer-shaped flowers and are pollinated mainly by several unspecialized mycetophilid genera with short mouthparts. We performed phylogenetic analyses using nucleotide sequences of external and internal transcribed spacers of nuclear ribosomal DNA and reconstructed ancestral floral morphologies with an establishment of the model of floral character evolution under a maximum-likelihood framework. Our analysis indicates that there is significant directionality in the evolutionary shifts of floral forms in the Heucherina. The inferred phylogeny further supports four origins of saucer-shaped flowers, which is shared among 14 species that are traditionally classified into the genus Mitella. In addition, our analysis indicates the extensive polyphyly of genus Mitella, as also suggested previously. The results suggest that the flower-visiting fungus gnats have caused convergent selection for the saucer-shaped flower repeatedly evolved within Heucherina.

Natural hybridization in Saxifraga callosa Sm.

Saxifraga callosa Sm. is an evergreen perennial species distributed from Eastern Spain, through the Western Alps and the Apennines, to southern Italy. The existence of high morphological variation within different subspecies indicates that phenotypic characters are useful but not sufficient taxonomic tools. Indeed, available morphological data already suggested that S. callosa subentity lantoscana may be an outcross between S. callosa and S. cochlearis. In this work, by analyzing ITS (Internal Transcribed Sequences), AFLP (Amplified Fragment Length Polymorphisms), and cpDNA (chloroplast DNA) markers, a comprehensive study of the genomic relationships among S. callosa and related species has been carried out. The sequence of the ITS region of S. callosa subentity lantoscana gave no conclusive results on the taxonomy status of S. callosa subentity lantoscana. On the other hand, the use of the "NewHybrids" software to analyze an AFLP data-set (208 polymorphic amplified fragments) supported a significant posterior probability that S. callosa subentity lantoscana individuals are natural hybrids between S. callosa and S. cochlearis. The level of introgression of genes from alien genomes was confirmed by a simpler and quick methodology that analyze length variation in cpDNA sequences.

Chloroplast DNA phylogeography of the arctic-montane species Saxifraga hirculus (Saxifragaceae).

The genetic structure of populations of an arctic-montane herb, Saxifraga hirculus (Saxifragaceae), was analysed by means of chloroplast restriction fragment-length polymorphism. Sampled populations were distributed across Europe and North America (Alaska and Colorado). There was no evidence for geographically structured genetically divergent lineages, and although no haplotypes were shared between North America and Europe, the haplotypes from different continents were intermixed on a minimum spanning tree. European populations were much more highly differentiated and had much lower levels of haplotype diversity than their Alaskan counterparts. Centres of haplotype diversity were concentrated in those Alaskan populations located outside the limits of the last (Wisconsin) glaciation, suggesting that they may have acted as refugia during the Pleistocene. It was not possible to identify putative migration routes or corresponding refugia in the European genepool. One British population, from the Pentland Hills, was genetically very distant from all the others, for reasons that are as yet unknown.