Survival in nunatak and peripheral glacial refugia of three alpine plant species is partly predicted by altitudinal segregation.

Mountain biota survived the Quaternary cold stages most probably in peripheral refugia and/or ice-free peaks within ice-sheets (nunataks). While survival in peripheral refugia has been broadly demonstrated, evidence for nunatak refugia is still scarce. We generated RADseq data from three mountain plant species occurring at different elevations in the southeastern European Alps to investigate the role of different glacial refugia during the Last Glacial Maximum (LGM). We tested the following hypotheses. (i) The deep Piave Valley forms the deepest genetic split in the species distributed across it, delimiting two peripheral refugia. (ii) The montane to alpine species Campanula morettiana and Primula tyrolensis survived the LGM in peripheral refugia, while high-alpine to subnival Saxifraga facchinii likely survived in several nunatak refugia. (iii) The lower elevation species suffered a strong population decline during the LGM. By contrast, the higher elevation species shows long-term stability of population sizes due to survival on permanently ice-free peaks and small population sizes at present. We found peripheral refugia on both sides of the Piave Valley, which acted as a major genetic barrier. Demographic modelling confirmed nunatak survival not only for S. facchinii but also for montane to alpine C. morettiana. Altitudinal segregation influenced the species' demographic fluctuations, with the lower elevation species showing a significant population increase at the end of the LGM, and the higher elevation species either showing decrease towards the present or stable population sizes with a short bottleneck. Our results highlight the role of nunatak survival and species ecology in the demographic history of mountain species.

Comparative Analysis of Chloroplast Genomes within Saxifraga (Saxifragaceae) Takes Insights into Their Genomic Evolution and Adaption to the High-Elevation Environment.

Saxifraga species are widely distributed in alpine and arctic regions in the Northern hemisphere. Highly morphological diversity within this genus brings great difficulties for species identification, and their typical highland living properties make it interesting how they adapt to the extreme environment. Here, we newly generated the chloroplast (cp) genomes of two Saxifraga species and compared them with another five Saxifraga cp genomes to understand the characteristics of cp genomes and their potential roles in highland adaptation. The genome size, structure, gene content, GC content, and codon usage pattern were found to be highly similar. Cp genomes ranged from 146,549 bp to 151,066 bp in length, most of which comprised 130 predicted genes. Yet, due to the expansion of IR regions, the second copy of rps19 in Saxifraga stolonifera was uniquely kept. Through sequence divergence analysis, we identified seven hypervariable regions and detected some signatures of regularity associated with genetic distance. We also identified 52 to 89 SSRs and some long repeats among seven Saxifraga species. Both ML and BI phylogenetic analyses confirmed that seven Saxifraga species formed a monophyletic clade in the Saxifragaceae family, and their intragenus relationship was also well supported. Additionally, the ndhI and ycf1 genes were considered under positive selection in species inhabiting relatively high altitudes. Given the conditions of intense light and low CO2 concentration in the highland, the products of these two genes might participate in the adaptation to the extreme environment.

Comparative analysis of spatial genetic structures in sympatric populations of two riparian plants, Saxifraga acerifolia and Saxifraga fortunei.

PREMISE: The genetic structure between plant populations is facilitated by the spatial population arrangement and limited dispersal of seed and pollen. Saxifraga acerifolia, a local endemic species in Japan, is a habitat specialist that is confined to waterfalls in riparian environments. Its sister species, Saxifraga fortunei, is a generalist that is widely distributed along riverbanks. Here, we examined sympatric populations of the two Saxifraga species to test whether the differences in habitat preference and colonization process influenced regional and local genetic structures. METHODS: To reveal genetic structures, we examined chloroplast microsatellite variations and genome-wide nucleotide polymorphisms obtained by genotyping by sequencing. We also estimated the gene flow among and within populations and performed landscape genetic analyses to evaluate seed and pollen movement and the extent of genetic isolation related to geographic distance and/or habitat differences. RESULTS: We found strong genetic structure in the specialist S. acerifolia, even on a small spatial scale (<1 km part); each population on a different waterfall in one river system had a completely different predominant haplotype. By contrast, the generalist S. fortunei showed no clear genetic differentiation. CONCLUSIONS: Our findings suggest that the level of genetic isolation was increased in S. acerifolia by the spatially fragmented habitat and limited seed and pollen dispersal over waterfalls. Habitat differentiation between the sister taxa could have contributed to the different patterns of gene flow and then shaped the contrasting genetic structures.

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.

Degradation of key photosynthetic genes in the critically endangered semi-aquatic flowering plant Saniculiphyllum guangxiense (Saxifragaceae).

BACKGROUND: Plastid gene loss and pseudogenization has been widely documented in parasitic and mycoheterotrophic plants, which have relaxed selective constraints on photosynthetic function. More enigmatic are sporadic reports of pseudogenization and loss of important photosynthesis genes in lineages thought to be fully photosynthetic. Here we report the complete plastid genome of Saniculiphyllum guangxiense, a critically endangered and phylogenetically isolated plant lineage, along with genomic evidence of reduced chloroplast function. We also report 22 additional plastid genomes representing the diversity of its containing clade Saxifragales, characterizing gene content and placing variation in a broader phylogenetic context. RESULTS: We find that the plastid genome of Saniculiphyllum has experienced pseudogenization of five genes of the ndh complex (ndhA, ndhB, ndhD, ndhF, and ndhK), previously reported in flowering plants with an aquatic habit, as well as the surprising pseudogenization of two genes more central to photosynthesis (ccsA and cemA), contrasting with strong phylogenetic conservatism of plastid gene content in all other sampled Saxifragales. These genes participate in photooxidative protection, cytochrome synthesis, and carbon uptake. Nuclear paralogs exist for all seven plastid pseudogenes, yet these are also unlikely to be functional. CONCLUSIONS: Saniculiphyllum appears to represent the greatest degree of plastid gene loss observed to date in any fully photosynthetic lineage, perhaps related to its extreme habitat specialization, yet plastid genome length, structure, and substitution rate are within the variation previously reported for photosynthetic plants. These results highlight the increasingly appreciated dynamism of plastid genomes, otherwise highly conserved across a billion years of green plant evolution, in plants with highly specialized life history traits.

Effects of genetic variation and environmental factors on bergenin in Rodgersia sambucifolia Hemsl.

ETHNOPHARMACOLOGICAL RELEVANCE: Bergenin is a well-known active compound that exhibits antioxidant, antiarrhythmic, hepatoprotective, and anti-inflammatory activities. However, the resource reserve of Rodgersia sambucifolia, one of the main raw materials for extracting bergenin, have sharply declined, and the bergenin content in different germplasms differs vastly, resulting in a serious shortage of the market supply of bergenin. AIM OF THE STUDY: To investigate the influence of genetic diversity and environmental factors on bergenin content in Rodgersia sambucifolia. MATERIALS AND METHODS: Fifty Rodgersia sambucifolia samples with a growth period of 2-3 years were collected from different areas across China and the bergenin content was determined via HPLC. Meanwhile the total genomic DNA was extracted and ISSR was performed. The bergenin content as measured using HPLC and the environmental data gathered from the meteorological stations and field work were combined and analyzed using correlation tests in XLSTAT 2018 to detect the key factors affecting bergenin content. The genetic UPGMA tree constructed based on genetic distances of the 50 samples and the chemical dendrogram constructed according to the distance between the bergenin content were compared to determine the correlation between genetic and chemical differentiation. RESULTS: Among the 50 individuals, bergenin content varied from 2.83 to 12.54%, with the highest content being 4.43-fold that of the lowest content. The survey of the 50 individuals produced a total of 193 amplified bands, 187 of which were polymorphic (96.89%). In the study, bergenin content was positively correlated with annual mean temperature (AMT) (r = 0.583, P < 0.0001) and 1-12 month monthly mean temperature (MMT) (P < 0.0001). A comparison of the genetic dendrogram with the AHC dendrogram found no corresponding relationship between them. Mantel correlation analyses also showed that there was no significant correlation between them (r = 0.144). CONCLUSIONS: There were large differences in bergenin content among different germplasms that were not correlated with the high genetic variation in Rodgersia sambucifolia but were significantly correlated with environmental factors, such as temperature. This study lays the foundation for subsequent superior germplasm selection and artificial breeding of Rodgersia sambucifolia to improve the bergenin content and meet market demands.

Bridging population genetics and the metacommunity perspective to unravel the biogeographic processes shaping genetic differentiation of Myriophyllum alterniflorum DC.

The degree to which dispersal limitation interacts with environmental filtering has intrigued metacommunity ecologists and molecular biogeographers since the beginning of both research disciplines. Since genetic methods are superior to coarse proxies of dispersal, understanding how environmental and geographic factors influence population genetic structure is becoming a fundamental issue for population genetics and also one of the most challenging avenues for metacommunity ecology. In this study of the aquatic macrophyte Myriophyllum alterniflorum DC., we explored the spatial genetic variation of eleven populations from the Iberian Plateau by means of microsatellite loci, and examined if the results obtained through genetic methods match modern perspectives of metacommunity theory. To do this, we applied a combination of robust statistical routines including network analysis, causal modelling and multiple matrix regression with randomization. Our findings revealed that macrophyte populations clustered into genetic groups that mirrored their geographic distributions. Importantly, we found a significant correlation between genetic variation and geographic distance at the regional scale. By using effective (genetic) dispersal estimates, our results are broadly in line with recent findings from metacommunity theory and re-emphasize the need to go beyond the historically predominant paradigm of understanding environmental heterogeneity as the main force driving macrophyte diversity patterns.

Divergent gene expression levels between diploid and autotetraploid Tolmiea relative to the total transcriptome, the cell, and biomass.

PREMISE OF THE STUDY: Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea, we present an integrated approach for cross-ploidy comparisons that account for differences in transcriptome size and cell density and make multiple comparisons of transcript abundance. METHODS: We use RNA spike-in standards in concert with cell size and density to identify and correct for differences in transcriptome size and compare levels of gene expression across multiple scales: per transcriptome, per cell, and per biomass. KEY RESULTS: In total, ~17% of all loci were identified as differentially expressed (DEGs) between the diploid and autopolyploid species. The per-transcriptome normalization, the method researchers typically use, captured the fewest DEGs (58% of total DEGs) and failed to detect any DEGs not found by the alternative normalizations. When transcript abundance was normalized per biomass and per cell, ~66% and ~82% of the total DEGs were recovered, respectively. The discrepancy between per-transcriptome and per-cell recovery of DEGs occurs because per-transcriptome normalizations are concentration-based and therefore blind to differences in transcriptome size. CONCLUSIONS: While each normalization enables valid comparisons at biologically relevant scales, a holistic comparison of multiple normalizations provides additional explanatory power not available from any single approach. Notably, autotetraploid loci tend to conserve diploid-like transcript abundance per biomass through increased gene expression per cell, and these loci are enriched for photosynthesis-related functions.

Contrasting evolutionary origins of two mountain endemics: Saxifraga wahlenbergii (Western Carpathians) and S. styriaca (Eastern Alps).

BACKGROUND: The Carpathians and the Alps are the largest mountain ranges of the European Alpine System and important centres of endemism. Among the distinctive endemic species of this area is Saxifraga wahlenbergii, a Western Carpathians member of the speciose genus Saxifraga. It was frequently considered a taxonomically isolated Tertiary palaeopolyploid and palaeoendemic, for which the closest relatives could not yet be traced. A recently described narrow endemic of the Eastern Alps, S. styriaca, was hypothesized to be closely related to S. wahlenbergii based on shared presence of peculiar glandular hairs. To elucidate the origin and phylogenetic relationships of both species we studied nuclear and plastid DNA markers based on multiple accessions and analysed the data in a wide taxonomic context. We applied Sanger sequencing, followed by targeted next-generation sequencing (NGS) for a refined analysis of nrITS variants to detect signatures of ancient hybridization. The ITS data were used to estimate divergence times of different lineages using a relaxed molecular clock. RESULTS: We demonstrate divergent evolutionary histories for the two mountain endemics. For S. wahlenbergii we revealed a complicated hybrid origin. Its maternal parent belongs to a Western Eurasian lineage of high mountain taxa grouped in subsect. Androsaceae and is most likely the widespread S. androsacea. The putative second parent was most likely S. adscendens, which belongs to the distantly related subsect. Tridactylites. While Sanger sequencing of nrITS only showed S. adscendens-related variants in S. wahlenbergii, our NGS screening revealed presence of sequences from both lineages with clear predominance of the paternal over the maternal lineage. CONCLUSIONS: Saxifraga styriaca was unambiguously assigned to subsect. Androsaceae and is not the sister taxon of S. wahlenbergii. Accordingly, the similarity of the glandular hairs observed in both taxa rests on parallelism and both species do not constitute an example of a close evolutionary link between the floras of the Western Carpathians and Eastern Alps. With the origin of its paternal, S. adscendens-like ITS DNA estimated to ca. 4.7 Ma, S. wahlenbergii is not a relict of the mid-Tertiary climate optimum. Its hybrid origin is much younger and most likely took place in the Pleistocene.

[Development and application of polymorphic microsatellite markers in Saxifraga genus].

In order to develop genomic-SSR markers for species of Saxifraga genus, a mixed plant genomic DNA sample was sequenced based on high-throughput Illumina MiSeq platform. According to genomic sequencing data, SSR loci were identified with MISA software, and then primers were designed with Primer 3 software. A total of 120 pairs of primers were randomly synthesized and amplified in genomic DNA of a few plant samples. Those primers who have yielded polymorphic bands and were considered easy to amplify were identified. After that, transferability of these primers was evaluated, and phylogenetic relationship of 25 species of Saxifraga genus was analyzed with UPGMA (unweighted pair group method analysis). In our results, 587 256 sequences containing SSRs were identified from a total of 1 881 979 combined read pairs obtained in genomic sequencing. Primers were designated to amplify SSRs containing two to six nucleotide repeat units, screened in a small portion of species. Finally, 17 pairs of primers which have produced abundant of polymorphic bands with little problem were amplified in 25 species of Saxifraga genus. A total of 2 687 polymorphic bands were obtained, the average polymorphic rate was 158 bands per pairs of primers. The transferability rate was ranging from 88.0% to 100% across 25 species of Saxifraga. In phylogenetic analysis, the clustering of 25 species based on 17 pairs of SSR primers was different from morphological classification. Our analysis has provided molecular data for genetic relationship of Saxifraga genus, and the transferable and polymorphic SSRs have provided information for genetic diversity research.

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.

Historical introgression among the species of Rodgersia (Saxifragaceae) in mountainous forests of southwest China.

In the present study, we used genetic data and ecological niche modelling to explore possible historical introgressions among the species of Rodgersia (Saxifragaceae) in central-southwest China. Markedly differentiated chloroplast haplotypes were found in R. aesculifolia, R. sambucifolia and the Lijiang (LJ) population of R. pinnata, respectively, and differentiated chloroplast haplotypes within each of them showed the closest relationships with haplotypes from different species. ITS cloning did not reveal any shared ribotype between R. aesculifolia and the remaining species. Historical introgression between R. aesculifolia and R. sambucifolia (or R. pinnata) seems to be the most plausible explanation according to the geographical pattern and derivative status of putative introgressed chloroplast haplotypes, and also from morphological evidence. Introgressions were also found among R. sambucifolia, R. pinnata, and R. henricii from Yunnan. Frequent gene exchanges may have promoted the diversity of leaf shapes in this genus. Ecological niche modelling indicated that past secondary contact following range shifts during Pleistocene cold periods may have provided opportunities for ancient introgression between R. aesculifolia and adjacent species.

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.

The influence of habitat on the evolution of plants: a case study across Saxifragales.

BACKGROUND AND AIMS: Organismal evolution tends to be closely associated with ecological conditions. However, the extent to which this association constrains adaptation or diversification into new habitats remains unclear. We studied habitat evolution in the hyper-diverse angiosperm clade Saxifragales. METHODS: We used species-level phylogenies for approx. 950 species to analyse the evolution of habitat shifts as well as their influence on plant diversification. We combined habitat characterization based on floristic assignments and state-of-the art phylogenetic comparative methods to estimate within- and across-habitat diversification patterns. KEY RESULTS: Our analyses showed that Saxifragales diversified into multiple habitats from a forest-inhabiting ancestor and that this diversification is governed by relatively rare habitat shifts. Lineages are likely to stay within inferred ancestral ecological conditions. Adaptation to some habitat types (e.g. aquatic, desert) may be canalizing events that lineages do not escape. Although associations between increased diversification rates and shifts in habitat preferences are occasionally observed, extreme macroevolutionary rates are closely associated with specific habitats. Lineages occurring in shrubland, and especially tundra and rock cliffs, exhibit comparatively high diversification, whereas forest, grassland, desert and aquatic habitats are associated with low diversification. CONCLUSIONS: The likelihood of occupation of new habitats appears to be asymmetric. Shifts to aquatic and desert habitats may be canalizing events. Other habitats, such as tundra, might act as evolutionary sources, while forests provide the only habitat seemingly colonized easily by lineages originating elsewhere. However, habitat shifts are very rare, and any major environmental alteration is expected to have dramatic evolutionary consequences.

Niche divergence between diploid and autotetraploid Tolmiea.

PREMISE OF STUDY: Polyploidy is common in eukaryotes and is of major evolutionary importance over both short and long time-scales. Compared to allopolyploids, autopolyploids remain understudied; they are often morphologically cryptic and frequently remain taxonomically unrecognized, although there is increasing recognition of the high frequency of autopolyploidy in angiosperms. While autopolyploidy can serve as an instant speciation mechanism, little is known about the ecological consequences of this process. We describe the ecological divergence of a diploid-autotetraploid species pair in Tolmiea. METHODS: We investigated whether abiotic niche divergence has shaped the current allopatric distribution of diploid T. diplomenziesii and its autotetraploid derivative, T. menziesii, in the Pacific Northwest of North America. We employed field measures of light availability, as well as niche modeling and a principal component analysis of environmental space. Within a common garden, we also investigated physiological responses to changes in soil moisture. KEY RESULTS: Diploid and autotetraploid Tolmiea inhabit significantly different climatic niche spaces. The climatic niche divergence between these two species is best explained by a shift in precipitation availability, and we found evidence of differing physiological response to water availability between these species. CONCLUSIONS: We found that spatial segregation of T. diplomenziesii and T. menziesii was accompanied by adaptation to changes in climatic regime. Tolmiea menziesii is not a nascent autotetraploid, having persisted long enough to be established throughout the Pacific Northwest, and therefore both polyploidization and subsequent evolution have contributed to the observed differences between T. menziesii and T. diplomenziesii.

Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales.

Cynomoriaceae, one of the last unplaced families of flowering plants, comprise one or two species or subspecies of root parasites that occur from the Mediterranean to the Gobi Desert. Using Illumina sequencing, we assembled the mitochondrial and plastid genomes as well as some nuclear genes of a Cynomorium specimen from Italy. Selected genes were also obtained by Sanger sequencing from individuals collected in China and Iran, resulting in matrices of 33 mitochondrial, 6 nuclear, and 14 plastid genes and rDNAs enlarged to include a representative angiosperm taxon sampling based on data available in GenBank. We also compiled a new geographic map to discern possible discontinuities in the parasites' occurrence. Cynomorium has large genomes of 13.70-13.61 (Italy) to 13.95-13.76 pg (China). Its mitochondrial genome consists of up to 49 circular subgenomes and has an overall gene content similar to that of photosynthetic angiosperms, while its plastome retains only 27 of the normally 116 genes. Nuclear, plastid and mitochondrial phylogenies place Cynomoriaceae in Saxifragales, and we found evidence for several horizontal gene transfers from different hosts, as well as intracellular gene transfers.

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.

Divergent selection along climatic gradients in a rare central European endemic species, Saxifraga sponhemica.

BACKGROUND AND AIMS: The effects of habitat fragmentation on quantitative genetic variation in plant populations are still poorly known. Saxifraga sponhemica is a rare endemic of Central Europe with a disjunct distribution, and a stable and specialized habitat of treeless screes and cliffs. This study therefore used S. sponhemica as a model species to compare quantitative and molecular variation in order to explore (1) the relative importance of drift and selection in shaping the distribution of quantitative genetic variation along climatic gradients; (2) the relationship between plant fitness, quantitative genetic variation, molecular genetic variation and population size; and (3) the relationship between the differentiation of a trait among populations and its evolvability. METHODS: Genetic variation within and among 22 populations from the whole distribution area of S. sponhemica was studied using RAPD (random amplified polymorphic DNA) markers, and climatic variables were obtained for each site. Seeds were collected from each population and germinated, and seedlings were transplanted into a common garden for determination of variation in plant traits. KEY RESULTS: In contrast to previous results from rare plant species, strong evidence was found for divergent selection. Most population trait means of S. sponhemica were significantly related to climate gradients, indicating adaptation. Quantitative genetic differentiation increased with geographical distance, even when neutral molecular divergence was controlled for, and QST exceeded FST for some traits. The evolvability of traits was negatively correlated with the degree of differentiation among populations (QST), i.e. traits under strong selection showed little genetic variation within populations. The evolutionary potential of a population was not related to its size, the performance of the population or its neutral genetic diversity. However, performance in the common garden was lower for plants from populations with reduced molecular genetic variation, suggesting inbreeding depression due to genetic erosion. CONCLUSIONS: The findings suggest that studies of molecular and quantitative genetic variation may provide complementary insights important for the conservation of rare species. The strong differentiation of quantitative traits among populations shows that selection can be an important force for structuring variation in evolutionarily important traits even for rare endemic species restricted to very specific habitats.

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.