Hopping or Jumping on the Cliffs: The Unusual Phylogeographical and Demographic Structure of an Extremely Narrow Endemic Mediterranean Plant.

Several past and recent climatic and geological events have greatly influenced the current distribution of coastal species around the Mediterranean Basin. As a consequence, the reconstruction of the distributional history of these species is challenging. In this study, we used both chloroplast and nuclear SNPs to assess the levels of genetic differentiation, contemporary/historical levels of gene flow, and demographic history for the three only known (one mainland and two insular) populations of Eokochia saxicola, a rare Mediterranean coastal rocky halophyte. Plastid genome analysis revealed very low intraspecific haplotype variation and partial admixture among Capri and Palinuro populations with at least two independent colonization events for the Strombolicchio islet. Nuclear SNPs variation consistently identified three distinct genetic clusters corresponding to our sampling localities. Furthermore, strong genetic isolation was confirmed by both historical and contemporary levels of migration among the three populations. The DIYABC analysis identified two introductions temporally separated from Palinuro to Capri (ca.25 Mya) and subsequently to Strombolicchio (ca.09 Mya) as the most likely hypothesis for the current distribution of E. saxicola. Regardless of their small population sizes, all study sites supported high-genetic diversity maintained by outcrossing and random mating between individuals owing largely to wind pollination, an exclusive trait among Mediterranean narrow endemics. In conclusion, the patterns observed confirm that some Mediterranean endemics are not necessarily "evolutionary dead-ends" but rather represent species that have extensive demographic stability and a strong evolutionary legacy.

DNA barcoding of native Caucasus herbal plants: potentials and limitations in complex groups and implications for phylogeographic patterns.

DNA barcoding has rapidly become a useful complementary tool in floristic investigations particularly for identifying specimens that lack diagnostic characters. Here, we assess the capability of three DNA barcode markers (chloroplast rpoB, accD and nuclear ITS) for correct species assignment in a floristic survey on the Caucasus. We focused on two herbal groups with potential for ornamental applications, namely orchids and asterids. On these two plant groups, we tested whether our selection of barcode markers allows identification of the "barcoding gap" in sequence identity and to distinguish between monophyletic species when employing distance-based methods. All markers successfully amplified most specimens, but we found that the rate of species-level resolution amongst selected markers largely varied in the two plant groups. Overall, for both lineages, plastid markers had a species-level assignment success rate lower than the nuclear ITS marker. The latter confirmed, in orchids, both the existence of a barcoding gap and that all accessions of the same species clustered together in monophyletic groups. Further, it also allowed the detection of a phylogeographic signal.The ITS marker resulted in its being the best performing barcode for asterids; however, none of the three tested markers showed high discriminatory ability. Even if ITS were revealed as the most promising plant barcode marker, we argue that the ability of this barcode for species assignment is strongly dependent on the evolutionary history of the investigated plant lineage.

Cultivated Tomato (Solanum lycopersicum L.) Suffered a Severe Cytoplasmic Bottleneck during Domestication: Implications from Chloroplast Genomes.

In various crops, genetic bottlenecks occurring through domestication can limit crop resilience to biotic and abiotic stresses. In the present study, we investigated nucleotide diversity in tomato chloroplast genome through sequencing seven plastomes of cultivated accessions from the Campania region (Southern Italy) and two wild species among the closest (Solanum pimpinellifolium) and most distantly related (S. neorickii) species to cultivated tomatoes. Comparative analyses among the chloroplast genomes sequenced in this work and those available in GenBank allowed evaluating the variability of plastomes and defining phylogenetic relationships. A dramatic reduction in genetic diversity was detected in cultivated tomatoes, nonetheless, a few de novo mutations, which still differentiated the cultivated tomatoes from the closest wild relative S. pimpinellifolium, were detected and are potentially utilizable as diagnostic markers. Phylogenetic analyses confirmed that S. pimpinellifolium is the closest ancestor of all cultivated tomatoes. Local accessions all clustered together and were strictly related with other cultivated tomatoes (S. lycopersicum group). Noteworthy, S. lycopersicum var. cerasiforme resulted in a mixture of both cultivated and wild tomato genotypes since one of the two analyzed accessions clustered with cultivated tomato, whereas the other with S. pimpinellifolium. Overall, our results revealed a very reduced cytoplasmic variability in cultivated tomatoes and suggest the occurrence of a cytoplasmic bottleneck during their domestication.

Phenotypic expression of floral traits in hybrid zones provides insights into their genetic architecture.

Information on the genetic architecture of phenotypic traits is helpful for constructing and testing models of the ecoevolutionary dynamics of natural populations. For plant groups with long life cycles there is a lack of line cross experiments that can unravel the genetic architecture of loci underlying quantitative traits. To fill this gap, we propose the use of variation for phenotypic traits expressed in natural hybrid zones as an alternative approach. We used data from orchid hybrid zones and compared expected and observed patterns of phenotypic trait expression in different early-generation hybrid classes identified by molecular genetic markers. We found evidence of additivity, dominance, and epistatic interactions for different phenotypic traits. We discuss the potential of this approach along with its limitations and suggest that it may represent a realistic way to gain an initial insight into the heritability and genomic architecture of traits in organismal groups with complex life history, such as orchids and many others.

The influence of a relict distribution on genetic structure and variation in the Mediterranean tree, Platanus orientalis.

The distribution of plant species around the Mediterranean basin is a product of the influence of both geographical barriers and of climatic changes experienced during the Tertiary, with the transition from a warm to cool periods. Several species, once largely distributed across the Northern Hemisphere, retracted to refugial areas in southern Europe where they are described as Tertiary relicts. Platanus orientalis is a typical representative of Tertiary flora in southwest Eurasia; its distribution spreads from the Caucasus to the Mediterranean, with its western border in Southern Italy and Sicily. We analysed genetic diversity and differentiation in the centre and western part of its distribution range using nuclear microsatellites and compared genetic parameters between core and western populations. We found an overall decrease in genetic diversity estimates (heterozygosity, private allelic richness) from central towards western populations, with those from Southern Italy and Sicily showing the lowest values. The low level of genetic diversity probably results from historic range fragmentation experienced by P. orientalis in its westernmost distribution as confirmed by high level genetic isolation of these populations. Ornamental hybrids were genetically distinguished from P. orientalis as contained private alleles, indicating that gene flow with natural populations is rare. Population assignment and neighbour-joining (NJ) analysis of populations identified four groups belonging to two main phyletic groups (the Southern Italian-Sicilian-Balkan and Cretan-Bulgarian-Turkish lineages) that seem to have different biogeographic origin and also excluded an artificial origin for southern Italian and Sicilian populations as previously suggested. These results show that quantifying the genetic variation of a Tertiary relict in a geographical context, and the potential effect of hybridization with introduced ornamental hybrids, can provide useful insights on factors influencing population genetic structure. Such information is crucial to predict how such taxa will respond to increasing anthropogenic influence on the environment.

The complete plastid genomes of Ophrys iricolor and O. sphegodes (Orchidaceae) and comparative analyses with other orchids.

Sexually deceptive orchids of the genus Ophrys may rapidly evolve by adaptation to pollinators. However, understanding of the genetic basis of potential changes and patterns of relationships is hampered by a lack of genomic information. We report the complete plastid genome sequences of Ophrys iricolor and O. sphegodes, representing the two most species-rich lineages of the genus Ophrys. Both plastomes are circular DNA molecules (146754 bp for O. sphegodes and 150177 bp for O. iricolor) with the typical quadripartite structure of plastid genomes and within the average size of photosynthetic orchids. 213 Simple Sequence Repeats (SSRs) (31.5% polymorphic between O. iricolor and O. sphegodes) were identified, with homopolymers and dipolymers as the most common repeat types. SSRs were mainly located in intergenic regions but SSRs located in coding regions were also found, mainly in ycf1 and rpoC2 genes. The Ophrys plastome is predicted to encode 107 distinct genes, 17 of which are completely duplicated in the Inverted Repeat regions. 83 and 87 putative RNA editing sites were detected in 25 plastid genes of the two Ophrys species, all occurring in the first or second codon position. Comparing the rate of nonsynonymous (dN) and synonymous (dS) substitutions, 24 genes (including rbcL and ycf1) display signature consistent with positive selection. When compared with other members of the orchid family, the Ophrys plastome has a complete set of 11 functional ndh plastid genes, with the exception of O. sphegodes that has a truncated ndhF gene. Comparative analysis showed a large co-linearity with other related Orchidinae. However, in contrast to O. iricolor and other Orchidinae, O. sphegodes has a shift of the junction between the Inverted Repeat and Small Single Copy regions associated with the loss of the partial duplicated gene ycf1 and the truncation of the ndhF gene. Data on relative genomic coverage and validation by PCR indicate the presence, with a different ratio, of the two plastome types (i.e. with and without ndhF deletion) in both Ophrys species, with a predominance of the deleted type in O. sphegodes. A search for this deleted plastid region in O. sphegodes nuclear genome shows that the deleted region is inserted in a retrotransposon nuclear sequence. The present study provides useful genomic tools for studying conservation and patterns of relationships of this rapidly radiating orchid genus.

Strong but permeable barriers to gene exchange between sister species of Epidendrum.

PREMISE OF THE STUDY: The investigation of reproductive barriers between sister species can provide insights into how new lineages arise, and how species integrity is maintained in the face of interspecific gene flow. Different pre- and postzygotic barriers can limit interspecific gene exchange in sympatric populations, and different sources of evidence are often required to investigate the role of multiple reproductive isolation (RI) mechanisms. METHODS: We tested the hypothesis of hybridization and potential introgression between Epidendrum secundum and Epidendrum xanthinum, two Neotropical food-deceptive orchid species, using nuclear and plastid microsatellites, experimental crosses, pollen tube growth observations, and genome size estimates. KEY RESULTS: A large number of hybrids between E. secundum and E. xanthinum were detected, suggesting weak premating barriers. The low fertility of hybrid plants and the absence of haplotype sharing between parental species indicated strong postmating barriers, reducing interspecific gene exchange and the development of advanced generation hybrids. Despite the strength of reproductive barriers, fertile seeds were produced in some backcrossing experiments, and the existence of interspecific gene exchange could not be excluded. CONCLUSIONS: Strong but permeable barriers were found between E. secundum and E. xanthinum. Indeed, haplotype sharing was not detected between parental species, suggesting that introgression is limited by a combination of genic incompatibilities, including negative cytonuclear interactions. Most taxonomic uncertainties in this group were potentially influenced by incomplete RI barriers between species, which mainly occurred sympatrically.

Transitions between self-compatibility and self-incompatibility and the evolution of reproductive isolation in the large and diverse tropical genus Dendrobium (Orchidaceae).

BACKGROUND AND AIMS: The evolution of interspecific reproductive barriers is crucial to understanding species evolution. This study examines the contribution of transitions between self-compatibility (SC) and self-incompatibility (SI) and genetic divergence in the evolution of reproductive barriers in Dendrobium, one of the largest orchid genera. Specifically, it investigates the evolution of pre- and postzygotic isolation and the effects of transitions between compatibility states on interspecific reproductive isolation within the genus. METHODS: The role of SC and SI changes in reproductive compatibility among species was examined using fruit set and seed viability data available in the literature from 86 species and ∼2500 hand pollinations. The evolution of SC and SI in Dendrobium species was investigated within a phylogenetic framework using internal transcribed spacer sequences available in GenBank. KEY RESULTS: Based on data from crossing experiments, estimations of genetic distance and the results of a literature survey, it was found that changes in SC and SI significantly influenced the compatibility between species in interspecific crosses. The number of fruits produced was significantly higher in crosses in which self-incompatible species acted as pollen donor for self-compatible species, following the SI × SC rule. Maximum likelihood and Bayesian tests did not reject transitions from SI to SC and from SC to SI across the Dendrobium phylogeny. In addition, postzygotic isolation (embryo mortality) was found to evolve gradually with genetic divergence, in agreement with previous results observed for other plant species, including orchids. CONCLUSIONS: Transitions between SC and SI and the gradual accumulation of genetic incompatibilities affecting postzygotic isolation are important mechanisms preventing gene flow among Dendrobium species, and may constitute important evolutionary processes contributing to the high levels of species diversity in this tropical orchid group.

Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.

In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants.

Multiple shifts to different pollinators fuelled rapid diversification in sexually deceptive Ophrys orchids.

Episodes of rapid speciation provide unique insights into evolutionary processes underlying species radiations and patterns of biodiversity. Here we investigated the radiation of sexually deceptive bee orchids (Ophrys). Based on a time-calibrated phylogeny and by means of ancestral character reconstruction and divergence time estimation, we estimated the tempo and mode of this radiation within a state-dependent evolutionary framework. It appears that, in the Pleistocene, the evolution of Ophrys was marked by episodes of rapid diversification coinciding with shifts to different pollinator types: from wasps to Eucera bees to Andrena and other bees. An abrupt increase in net diversification rate was detected in three clades. Among these, two phylogenetically distant lineages switched from Eucera to Andrena and other bees in a parallel fashion and at about the same time in their evolutionary history. Lack of early radiation associated with the evolution of the key innovation of sexual deception suggests that Ophrys diversification was mainly driven by subsequent ecological opportunities provided by the exploitation of novel pollinator groups, encompassing many bee species slightly differing in their sex pheromone communication systems, and by spatiotemporal fluctuations in the pollinator mosaic.

An ancient satellite DNA has maintained repetitive units of the original structure in most species of the living fossil plant genus Zamia.

ZpS1 satellite DNA is specific to the genus Zamia and presents repetitive units organized as long arrays and also as very short arrays dispersed in the genome. We have characterized the structure of the ZpS1 repeats in 12 species representative of the whole geographic distribution of the genus. In most species, the clone most common sequences (cMCS) were so similar that a general most common sequence (GMCS) of the ZpS1 repetitive unit in the genus could be obtained. The few partial variations from the GMCS found in cMCS of some species correspond to variable positions present in most other species, as indicated by the clone consensus sequences (cCS). Two species have an additional species-specific variety of ZpS1 satellite. The dispersed repeats were found to contain more mutations than repeats from long arrays. Our results indicate that all or most species of Zamia inherited the ZpS1 satellite from a common ancestor in Miocene and have maintained repetitive units of the original structure till present. The features of ZpS1 satellite in the genus Zamia are poorly compatible with the model of concerted evolution, but they are perfectly consistent with a new model of satellite evolution based on experimental evidences indicating that a specific amplification-substitution repair mechanism maintains the homogeneity and stability of the repeats structure in each satellite DNA originally present in a species as long as the species exists.

Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots.

In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.

Multiple and different genomic rearrangements of the rbcL gene are present in the parasitic orchid Neottia nidus-avis.

In parasitic plants that have lost most, if not all, of their photosynthetic genes, the genome of their plastids has also undergone a dramatic reduction. For example, photosynthetic genes, such as rbcL, frequently become pseudogenes, in which large portions of the gene have been found to be deleted. Orchids are flowering plants with several parasitic lineages. This is consistent with the observation that parasitic orchids can invade pre-existing mutualistic associations between ectomycorrhizal trees and fungi to obtain fixed carbon and nutrients. In addition, some parasitic species are devoid of chlorophyll, and consequently, have lost their photosynthetic capacity. Here, the organization of the plastid genome of the parasitic orchid Neottia nidus-avis (L.) Rich. was investigated using sequencing and hybridization experiments. In particular, genomic rearrangements in the rbcL region of this parasitic orchid were analyzed. At least three distinct rbcL sequences were found to be present as pseudogenes and were likely located in the plastid genome. Based on these results, it is hypothesized that N. nidus-avis contains different plastomes, each with a different pseudogene, and these can exist within the same individual plant.

Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations.

PREMISE OF THE STUDY: We investigated whether four widespread, photosynthetic Mediterranean meadow orchids (Ophrys fuciflora, Anacamptis laxiflora, Orchis purpurea, and Serapias vomeracea) had either nutritional dependency on mycobionts or mycorrhizal fungal specificity. Nonphotosynthetic orchids generally engage in highly specific interactions with fungal symbionts that provide them with organic carbon. By contrast, fully photosynthetic orchids in sunny, meadow habitats have been considered to lack mycorrhizal specificity. METHODS: We performed both culture-dependent and culture-independent ITS sequence analysis to identify fungi from orchid roots. By analyzing stable isotope ((13)C and (15)N) natural abundances, we also determined the degree of autotrophy and mycoheterotrophy in the four orchid species. KEY RESULTS: Phylogenetic and multivariate comparisons indicated that Or. purpurea and Oph. fuciflora featured lower fungal diversity and more specific mycobiont spectra than A. laxiflora and S. vomeracea. All orchid species were significantly enriched in (15)N compared with neighboring non-orchid plants. Orchis purpurea had the most pronounced N gain from fungi and differed from the other orchids in also obtaining C from fungi. CONCLUSIONS: These results indicated that even in sunny Mediterranean meadows, orchids may be mycoheterotrophic, with correlated mycorrhizal fungal specificity.

Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase.

Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants.

Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium.

BACKGROUND AND AIMS: In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids. METHODS: Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL-accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network. KEY RESULTS: Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine. CONCLUSIONS: Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.

Hypervariable plastid locus variation and intron evolution in the Anacamptis palustris lineage.

Data on the organization of a hypervariable chloroplast locus in the Anacamptis palustris (Orchidaceae) lineage are provided and used to infer the pattern of molecular evolution in this group. A large survey of sequence variation in A. palustris and allied taxa reveals several repeat types differing in number and organization that occur in the same plastid region. The resulting repeat type network suggests that at least seven different minisatellite loci evolved near each other in the tRNALEU intron and indicates the presence of at least three main phyletic lines in the A. palustris lineage.

An unusual satellite DNA from Zamia paucijuga (Cycadales) characterised by two different organisations of the repetitive unit in the plant genome.

We have cloned and characterised a highly repetitive DNA family that represents about 5% of the nuclear genome of Zamia paucijuga, a member of Cycadales, an ancient and rare group of seed plants. The unit repeat, cloned from EcoRV digested genomic DNA, has a length of about 323 bp, an high GC content (65-70%) and a subtelomeric localisation. The DNA of Z. paucijuga was digested with various restriction enzymes and analysed by Southern blot using the cloned unit repeat as a probe. These experiments indicated that the repeat is present in the plant genome in an unusual organisation, as tandem arrays typical of satellite DNA and as dispersed elements. The characterisation of these unusual "dispersed satellite DNA" elements required a complex series of experiments using combined Southern blot analyses, PCR, cloning and sequencing. Our results indicate that most of these dispersed elements are formed by few units of the GC-rich satellite DNA repeats arranged in tandem and flanked at both sites by one copy of a 0.6 kb AT-rich direct repeat. This unusual satellite DNA organisation of GC-rich repeats is present in many species of genus Zamia, and, thus, likely represent an ancient rearrangement of the satellite DNA repeats that spread in the genome as dispersed elements.

Molecular evolution of a plastid tandem repeat locus in an orchid lineage.

The molecular evolution of a chloroplast minisatellite locus in the Anacamptis palustris (Orchidaceae) lineage and haplotype variation in two Italian A. palustris populations were investigated. A phylogenetic analyses of the chloroplast tRNA(LEU) intron, where the minisatellite locus is located, revealed that a deletion in the ancestor of the A. palustris lineage led to the formation of two noncontiguous, complementary sequence motifs. We propose a model to explain the initial formation of the minisatellite repeat motif, starting with the two noncontiguous, complementary sequence motifs. A survey of minisatellite variation in four species of the A. palustris lineage revealed several haplotypes that differed not only in repeat number, but also in repeat organization. A haplotype network suggests that three different minisatellite loci evolved independently at the same position in the tRNA(LEU) intron. A secondary structure model revealed that the A. palustris minisatellite repeat forms a stem region of the tRNA(LEU) intron, which allows its notable expansion without negatively affecting splicing. Minisatellite variation was high in the two examined A. palustris populations where 20 haplotypes were detected, whereas no length variation was detected in a neighboring poly (A) microsatellite locus. We estimated a chloroplast minisatellite mutation rate of 3.2 x 10(-3) mutations per generation. Southern blot analyses did not find evidence for chloroplast heteroplasmy. Based on the analysis of the largest known, extant A. palustris population, a stepwise mutation model (SMM) was inferred.