Viola suavis, a new species in the Nordic flora, with analyses of the relation to other species in the subsection Viola (Violaceae)

Evidence from cytology, enzyme markers and morphometry suggest that a species of Viola (Violaceae) collected in southeastern Norway should be referred to V. suavis , although differing in some phenotypic traits. V. suavis is not previously known from the Nordic countries, but must now be regarded as an established member of the Norwegian flora. It was probably introduced more than a century ago, providing an interesting analogy to the situation of V. alba in Öland (Sweden). Thus, it seems like human activity have played a role for expanding the distribution areas of several of the species within Viola subsection Viola (V. alba, V. hirta, V. odorata and V. suavis ). Electrophoretic evidence showed that the basal chromosome number of the subsection is x=5. V. suavis (2n=40) is allo-octoploid, and closely related tetraploid taxa were easily recognized. However, it is difficult to trace its origin to lower-ploid ancestors, and it seems likely that the complex may have contained extinct taxa with other allelic combinations that better explain the origin of V. suavis .     

Hybridization between Viola canina and V. persicifolia in Norway

Hybridization in Norway between Viola canina and V persicifolia was investigated by analysis of root tip mitosis, pollen stainability and morphological variation. The chromosome number was proved to be 2n = 40 (tetraploid) for V. canina ssp. montana , 2n = 20 (diploid) for V. persicifolia and 2n = 30 (triploid) for the hybrid, V. canina × persicifolia . No deviating chromosome numbers were found. The tetraploids and the diploids had a high frequency, and the triploids a low frequency, of apparently good pollen grains. Canonical variate analysis were used to investigate the pattern of morphological differentiation in the material. The specimens clearly separate into three groups in accordance with the three ploidy levels. No introgression was demonstrated.     

On the origin of the sweet-smelling Parma violet cultivars (Violaceae): wide intraspecific hybridization, sterility, and sexual reproduction

Parma violets are reputed for their double, fragrant flowers and have been cultivated for centuries in Europe. However, due to a rather atypical morphology their taxonomic affinity has not been clarified. Authors have proposed an origin from three possible species, Viola alba, V. odorata, or V. suavis, or a hybrid origin. Using both ITS sequence variation and allozyme variation in 14 putative loci, we showed that the Parma violet cultivars have their origin within Viola alba and that they are best included in the Mediterranean subsp. dehnhardtii. There is no trace of interspecific hybridization. However, the cultivars appear to have a single origin in a wide hybrid within V. alba, involving parental plants from the eastern and western Mediterranean region; historical literature sources seem to indicate Turkey and Italy, respectively. The Parma violet cultivars possess high levels of allozyme heterozygosity and to some extent also within-individual ITS sequence variation. Losses of heterozygosity and within-individual ITS sequence variation in some of the cultivars indicate subsequent rare events of sexual reproduction, presumably through cleistogamous seed set. We unambiguously identify the closest wild relative of this group of cultivars, allowing growers to develop new selection procedures, and show a peculiar molecular process associated with human selection.     

Interspecific hybridization between Vigna radiata (L.) Wilczek and V. glabrescens

Summary Interspecific hybrids of the mungbean, Vigna radiata (L.) Wilczek (2n=22) and V. glabrescens (2n=44) were generated with the aid of embryo culture. V. glabrescens x V. radiata hybrids were recovered via germination of the immature embryos. Reciprocal hybrids were obtained via shoot formation from embryonic callus. The authenticity of the hybrids was determined by morphological characteristics, chromosome number, and isozyme patterns. The hybrids were highly sterile upon selfing, but backcrossing to the diploid parent yielded viable seeds. Some of the plants resembled the diploid parent morphologically while others resembled neither parent. The backcross plants were sufficiently fertile to give a large number of mature, selfed seeds. Plants obtained differed morphologically and in their isozyme patterns from either parent, indicating introgression. These progeny populations will be used as bridging materials to transfer pest resistance from the wild tetraploid to the cultivated mungbean.     

Intraspecific variation in Viola suavis in Europe: parallel evolution of white-flowered morphotypes

Background and Aims

Viola species are commonly grown for their ornamental flowers, but their evolutionary history and taxonomy are often complicated and have been poorly explored so far. This is a study of the polymorphic, typically blue-flowered species Viola suavis, concentrating on the white-flowered populations of uncertain taxonomic assignment that occur in Spain and central and south-eastern Europe. The aim was to resolve their origin and taxonomic status and to study the intraspecific structure and (post)glacial history of this species.

Methods

Viola suavis and five close relatives were sampled from multiple locations and subjected to molecular (AFLP, sequencing of nrDNA ITS) and morphometric analyses. Data on ploidy level and pollen fertility were also obtained, to address an assumed hybrid origin of the white-flowered populations.

Key Results

In V. suavis a strong intraspecific genetic split into two groups was observed, indicating that there has been a long-term isolation and survival in distinct glacial refugia. The white-flowered populations could be placed within the variation range of this species, and it is clear that they evolved independently in two distant areas. Their parallel evolution is supported by both morphological and genetic differentiation. The strongly reduced genetic variation and absence of unique AFLP fragments suggest their derived status and origin from the typical, blue-flowered populations.

Conclusions

These results suggest that intraspecific variation in V. suavis has been largely shaped by population isolations during the last glaciation and subsequent recolonizations, although cultivation and vegetative spread by humans have affected the present picture as well.Key words: AFLP, central Europe, flow cytometry, ITS sequences, multivariate morphometrics, parallel evolution, Spain, Violaceae     

New distributional and molecular information call into question the systematic position of the West Asian Viola sintenisii (Violaceae)

The west Asian Viola sintenisii W.Becker, described from temperate woodlands of northern Iran and south-western Turkmenistan on the Caspian coast, was long considered a vicariant geographical subspecies of the mainly south European V. alba Besser. The new findings of V. sintenisii in four geographically separated stations in north-eastern Azerbaijan significantly expands its range of distribution. These occurrences are within the range of V. alba ssp. alba . Here the two taxa frequently co-occur but remain distinct and V. sintenisii is therefore justified at species level. It is likely that the two species are also sympatric in the Hyrcanian region, a range previously attributed uniquely to the latter. Viola sintenisii seems to be fairly common in the eastern parts of the Great Caucasus, but there are reasons to suggest that it has there been misinterpreted as V. suavis . The chromosome number of V. sintenisii , 2 n  = 20, is reported here for the first time.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 91–98.     

Genetic structure among and within peripheral and central populations of three endangered floodplain violets

Understanding the partitioning of genetic variance in peripheral and central populations may shed more light on the effects of genetic drift and gene flow on population genetic structure and, thereby, improve attempts to conserve genetic diversity. We analysed genetic structure of peripheral and central populations of three insect-pollinated violets (Viola elatior, Viola pumila, Viola stagnina) to evaluate to what extent these patterns can be explained by gene flow and genetic drift. Amplified fragment length polymorphism was used to analyse 930 individuals of 50 populations. Consistent with theoretical predictions, peripheral populations were smaller and more isolated, differentiation was stronger, and genetic diversity and gene flow lower in peripheral populations of V. pumila and V. stagnina. In V. elatior, probably historic fragmentation effects linked to its specific habitat type were superimposed on the plant geographic (peripheral-central) patterns, resulting in lower relative importance of gene flow in central populations. Genetic variation between regions (3-6%), among (30-37%) and within populations (60-64%) was significant. Peripheral populations lacked markers that were rare and localized in central populations. Loss of widespread markers in peripheral V. stagnina populations indicated genetic erosion. Autocorrelation within populations was statistically significant up to a distance of 10-20 m. Higher average genetic similarity in peripheral populations than in central ones indicated higher local gene flow, probably owing to management practices. Peripheral populations contributed significantly to genetic variation and contained unique markers, which made them valuable for the conservation of genetic diversity.     

Asymmetrical introgression between two Morus species (M. alba, M. rubra) that differ in abundance

Asymmetrical introgression is an expected genetic consequence of hybridization when parental taxa differ in abundance; however, evidence for such effects in small populations is scarce. To test this prediction, we estimated the magnitude and direction of hybridization between red mulberry (Morus rubra L.), an endangered species in Canada, and the introduced and more abundant white mulberry (Morus alba L.) using nuclear (randomly amplified polymorphic DNA) and cytoplasmic (chloroplast DNA sequence) markers. Parentage of 184 trees (n = 42 using cpDNA) from four sympatric populations was estimated using a hybrid index and related to six morphological characters and population frequencies of the parental classes. Overall, the frequency of nuclear hybrids was 53.7% (n = 99) and ranged from 43% to 67% among populations. The parental and hybrid taxa differed with respect to all of the morphological traits. Sixty-seven percent of all hybrids contained more nuclear markers from M. alba than M. rubra (hybrid index x = 0.46); among populations, the degree of M. alba bias was correlated with the frequency of M. alba. In addition, the majority of hybrids (68%) contained the chloroplast genome of white mulberry. These results suggest that introgression is bidirectional but asymmetrical and is related, in part, to the relative frequency of parental taxa.     

Morphological versus genetic diversity of Viola reichenbachiana and V. riviniana (sect. Viola,Violaceae) from soils differing in heavy metal content

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non‐metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore‐bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non‐polluted sites, high levels of intra‐ and inter‐population variability, and the occurrence of inter‐specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter‐specific hybrids from non‐polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal‐polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.     

Offspring performance in three cleistogamous Viola species

Seed germination and seedling/juvenile fitness in the cleistogamous perennials Viola hirta, V. mirabilis, and V. riviniana were investigated during three growing seasons, to compare the performance of chasmogamously (CH) and cleistogamously (CL) derived progeny. For V. hirta and V. mirabilis the effects of sibling competition were examined, for V. riviniana the effects of interspecific (grass) competition. Seed abortion and seed weight were also taken into account as fitness measures.In none of the species, seed abortion rate differed between CH and CL capsules. In V. mirabilis and V. riviniana, CL seeds had a lower germination rate than CH seeds. In V. hirta the two seed types did not differ in germinability. Mortality did not differ between the two seedling types in any of the species. In V. hirta and V. riviniana, CL progeny had shorter mean length of largest leaf than CH progeny. In V. mirabilis plant size did not differ between progeny types. Sibling competition had little effect on offspring performance, but grass competition increased mortality and reduced plant size of V. riviniana progeny. The two progeny types did not differ in their response to sibling and grass competition.The differences in performance between progeny types could be attributable to inbreeding depression in the CL phase, but the slightly lower fitness of CL offspring is probably balanced by their lower production costs. It is suggested that a dimorphic reproductive system is maintained in perennial Viola species to maximize total seed output in the face of environmental variation.     

Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Bt cry1Ac and gfp transgenes

Concerns exist that transgenic crop x weed hybrid populations will be more vigorous and competitive with crops compared with the parental weed species. Hydroponic, glasshouse, and field experiments were performed to evaluate the effects of introgression of Bacillus thuringiensis (Bt) cry1Ac and green fluorescent protein (GFP) transgenes on hybrid productivity and competitiveness in four experimental Brassica rapa x transgenic Brassica napus hybrid generations (F1, BC1F1, BC2F1 and BC2F2). The average vegetative growth and nitrogen (N) use efficiency of transgenic hybrid generations grown under high N hydroponic conditions were lower than that of the weed parent (Brassica rapa, AA, 2n = 20), but similar to the transgenic crop parent, oilseed rape (Brassica napus, AACC, 2n = 38). No generational differences were detected under low N conditions. In two noncompetitive glasshouse experiments, both transgenic and nontransgenic BC2F2 hybrids had on average less vegetative growth and seed production than B. rapa. In two high intraspecific competition field experiments with varied herbivore pressure, BC2F2 hybrids produced less vegetative dry weight than B. rapa. The competitive ability of transgenic and nontransgenic BC2F2 hybrids against a neighbouring crop species were quantified in competition experiments that assayed wheat (Triticum aestivum) yield reductions under agronomic field conditions. The hybrids were the least competitive with wheat compared with parental Brassica competitors, although differences between transgenic and nontransgenic hybrids varied with location. Hybridization, with or without transgene introgression, resulted in less productive and competitive populations.     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.     

Morphological, cytogenetic, and molecular evidence for introgressive hybridization in birch.

Extensive morphological variation of tetraploid birch (Betula pubescens) in Iceland is believed to be due to gene flow from diploid dwarf birch (B. nana) by means of introgressive hybridization. A combined morphological and cytogenetic approach was used to investigate this phenomenon in two geographically separated populations of natural birch woodland in Iceland. The results not only confirmed introgressive hybridization in birch, but also revealed bidirectional gene flow between the two species via triploid interspecific hybrids. The populations showed continuous morphological variation connecting the species, but karyotypically they consisted of only three types of plants: diploids, triploids, and tetraploids. No aneuploids were found. Some of the tetraploid plants had B. pubescens morphology as expected, but most of them had intermediate characters. Most of the diploid plants were B. nana, but some were intermediates and a few had B. pubescens morphology. The triploid plants were either intermediates or they resembled one of the two species. Similar introgressive variation was observed among the diploid and triploid progeny of open-pollinated B. nana in a garden. Birch samples including field plants and artificial hybrids were further examined using a molecular method based on genomic Southern hybridization. The experiments verified introgression at the DNA level.     

Species introduction promotes hybridization and introgression in Coregonus: is there sign of selection against hybrids?

Species introductions are considered one of the major drivers of biodiversity loss via ecological interactions and genetic admixture with local fauna. We examined two well-recognized fish species, native whitefish (Coregonus lavaretus) and introduced vendace (Coregonus albula), as well as their morphological hybrids in a single lake to test for selection against hybrids and backcrosses in the wild. A representative random subsample of 693 individuals (27.8%) was taken from the total catch of coregonids. This subsample was examined with the aim to select c. 50 individuals of pure whitefish (n = 52), pure vendace (n = 55) and putative hybrid (n = 19) for genetic analyses. The subsequent microsatellites and mitochondrial (mt) DNA analyses provided compelling evidence of hybridization and introgression. Of the 126 fish examined, four were found to be F(1) , 14 backcrosses to whitefish and seven backcrosses to vendace. The estimates of historical gene flow suggested higher rates from introduced vendace into native whitefish than vice versa, whereas estimates of contemporary gene flow were equal. Mitochondrial introgression was skewed, with 18 backcrosses having vendace mtDNA and only three with whitefish mtDNA. Hybrids and backcrosses had intermediate morphology and niche utilization compared with parental species. No evidence of selection against hybrids or backcrosses was apparent, as both hybrid and backcross growth rates and fecundities were high. Hybrids (F(1) ) were only detected in 2 year-classes, suggesting temporal variability in mating between vendace and whitefish. However, our data show that hybrids reached sexual maturity and reproduced actively, with backcrosses recorded from six consecutive year-classes, whereas no F(2) individuals were found. The results indicate widespread introgression, as 10.8% of coregonids were estimated to be backcrosses.     

Hybridization of Senecio squalidus and S. viscosus and Introgression of Genes from Diploid into Tetraploid Senecio Species

Natural triploid hybrids (Senecio x londinensis Lousley) betweenS. squalidus L. (2n = 20) and 5. viscosus L. (2n = 40) are fairlyfrequently found in Britain. Under glasshouse conditions bothnatural and artificial hybrids displayed very low levels ofseed fertility and gave rise to morphologically diverse F₂ plantsat about the triploid or pentaploid chromosome levels. By theF₄ generation, progeny of a F₂ pentaploid plant had somaticchromosome numbers near to the tetraploid level and considerablyincreased pollen and seed fertilities. Such fertile tetraploidsegregants of S. x londinensis permit the introgression of S.squalidus genes into S. viscosus, and may indicate the courseof introgression into other tetraploid species of Senecio. Senecio, hybridization, introgression     

基于数字化标本和种群形态变异特征试论块茎堇菜的分类地位

块茎堇菜是青藏高原及其邻近地区的一种特有两型闭花植物,目前其分类地位存在诸多争议。据此,本文对块茎堇菜及其近缘类群鳞茎堇菜的主要数字化标本以及同域分布的几个自然种群的叶片形态变异特征进行了分析。结果显示,所有参试数字化标本被分为两个明显不同的集群,一个集群符合块茎堇菜的叶片形态,另一集群则符合鳞茎堇菜;同样,同域分布的6个自然种群也分为两个集群。此外,叶片形态变异分析还显示叶片先端和叶基部形态参数ΔW、AL、BL和LWR在块茎堇菜/鳞茎堇菜同一类群不同种群间或种群内变异幅度较小且较为稳定,然而它们在不同类群间差异却极为显著（P<0.01）。因此,块茎堇菜和鳞茎堇菜理应作为两个不同的类群处理,这也表明叶片形态特征完全可以作为鳞茎堇菜和块茎堇菜的重要分类学依据。     

Introgressive hybridization between a native and an introduced species:Viola lutea subsp.Sudetica versusV. Tricolor

A hybrid swarm betweenViola lutea subsp.sudetica (2n=50, native species) andViola tricolor (2n=26, introduced species) originated in the 1970’s in the Krkono?e Mts. Analyses of chromosome numbers, reproductive biology, morphology, and ecology gave the following results: (1) Compared to the plants found in the 1970’s, the number of colour morphs have decreased and the types now prevailing in the field are morphologically closer toV. lutea subsp.sudetica, forming a continuum. (2) In the field, hybrids having approximately the same chromosome number as the primary hybrids are most common. Some plants of the hybrid swarm have certain characters unknown to their parents. (3) In the field, autogamous types similar toV. tricolor were found. (4) A limited number of plants from the field and culture have higher somatic chromosome numbers thanV. lutea subsp.sudetica; there was a tendency towards increasing chromosome numbers in their progeny (up to 2n=62). These plants have some new morphological characters (a small proportion of hexacolpate pollen) not found in the parents (nor in the other hybrids with prevailing tetracolpate and pentacolpate pollen grains) and higher pollen fertility in comparison to the other hybrids. These plants also have the highest germination rate. (5) There is a tendency for chromosome numbers to decline in the progeny of open pollinated hybrid plants in the lowland experimental graden. (6) The phenology of the plants in the mountain grasslands and the lowland garden is different; the parents behave in a totally contrasting manner. (7) In the field at least some hybrids extend outside the geographical and ecological ranges of the parental species, invading new communities. (8) Seeds ofV. tricolor do not show any dormancy and germinate in the year of production; most of theV. lutea subsp.sudetica seeds germinate during the spring of the following year. Hybrids with intermediate chromosome numbers had both types of germination strategy. The germination rate of intermediates with high chromosome numbers was even higher than that ofV. tricolor.     

长萼堇菜多倍体复合体种群的分布格局研究

采用常规压片方法观察了长萼堇菜Viola inconspicua Blume多倍体复合体18个种群的染色体数目,发现其中4个种群的染色体为2n=4x=24,14个种群的染色体为2n=8x=48.讨论了长萼堇菜四倍体和八倍体种群的分布,并推测其可能的起源和造成目前分布格局的原因.     

Introgressive hybridization in fishes: the biochemical evidence

Biochemical methods can detect variation at individual genetic loci, making possible the direct assessment of natural hybridization and introgression between fish populations. Protein electro-phoresis has been used to confirm and extend knowledge of many situations where species hybrids have been detected by morphological analyses. New cases of natural hybridization, including some at the subspecies level, have also been identified. Biochemical studies have provided the first conclusive evidence of natural post F₁ hybrids and of introgression between fish taxa. The strongest cases for introgression have used a combined analysis of nuclear protein genes and taxaspecific maternally inherited mitochondrial DNA variation. Information on the significance of introgression as a source of gene flow between taxa, particularly below the species level where sympatric subspecies and sibling species are involved, should expand in the future as the numbers and types of nuclear and mitochondrial DNA loci which can be assayed for variation increase. The full importance of introgressive hybridization in speciation may then be understood.