Apochromic populations of Dactylorhiza incarnata s.l. (Orchidaceae): diversity and systematic significance as revealed by allozyme markers and morphology

Apochromic forms of the Eurasian Dactylorhiza incarnata s.l. were studied in northern Europe to reveal their genetic (allozyme) and morphological diversity and to assess their systematic significance. The study included eight localities with sympatric populations of plants with anthocyanin‐pigmented and apochromic flowers. Parallel samples of the two morphs were taken from each locality. Genetic variation was only found at the allozyme loci pgd, pgi and ugpp. Statistically significant differences in allele frequencies between the two colour morphs were found in two localities and demonstrate that the occurrence of apochromic individuals in D. incarnata s.l. is not always because of spontaneous mutation. At least in some localities the apochromic plants form distinct breeding groups (but local populations of different colour morphs may also be composed of several more or less distinct breeding groups). Based on molecular and morphometric data, it is proposed that the apochromic study populations from calcareous fens should be referred to D. incarnata var. ochroleuca, whereas the apochromic study populations from non‐calcareous fens are better treated as aberrant local populations of var. incarnata s.l. Possible evolutionary patterns and processes are discussed and guidelines for identification of var. ochroleuca from morphological features are given. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 396–407.     

Amplified fragment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae)

The utility of the PCR-based AFLP technique (polymerase chain reaction; amplified fragment length polymorphisms) was explored in elucidating details of polyploid evolution in the Eurasian orchid genus Dactylorhiza. We emphasized Swedish taxa but also included some material from the British Isles and elsewhere in Europe. Three different sets of primers, amplifying different subsets of restriction fragments, independently revealed similar patterns for relationships among the Dactylorhiza samples investigated. The AFLP data support the general picture of polyploid evolution in Dactylorhiza, i.e., that allotetraploid derivatives have arisen repeatedly as a result of hybridization beween the two parental groups D. incarnata s.l. (sensu lato; diploid marsh orchids) and the D. maculata group (spotted orchids). Within the incarnata s.l. group, morphologically defined varieties were interdigitated. The D. maculata group consisted of two distinct subgroups, one containing autotetraploid D. maculata subsp. maculata and the other containing diploid D. maculata subsp. fuchsii. Allotetraploids showed a high degree of additivity for the putative parental genomes, and relationships among them were partly correlated to morphologically based entities, but also to geographic distribution. Thus, allotetraploid taxa from the British Isles clustered together, rather than with morphologically similar plants from other areas.     

Bryophyte flora of Uganda. 3. Lophoziaceae

Abstract

We studied infraspecific morphological variation within European Dicranum majus Sm. A principal components analysis based on six leaf characters scored in 82 specimens revealed two distinct plant types. Plants with bistratose submarginal upper leaf lamina cells, numerous spine-like dorsal lamina projections, a costa that is dorsally rough far down and has two layers of guide cells in its basal part, and falcate or strongly falcate, long leaves are mainly of a relatively southern origin. Specimens with a unistratose, smooth upper leaf lamina, a costa that is dorsally rough only above and has one layer of guide cells, and with slightly curved to straight, short leaves were only collected in northern Europe. Even if most authors do not formally recognize variation within D. majus, our results suggest that the two kinds of plants should be recognized at least at the variety level. In view of the confusing nomenclature in Dicranum it is beyond the scope of this paper to find a name for the northern plant. Type material of D. majus var. orthophyllum A. Braun ex Milde, a name that was frequently used for northern North American plants, belongs to the southern phenotype.     

Allozyme variation and genetic integrity of Dactylorhiza incarnata (Orchidaceae)

Eleven Danish populations of the diploid Dactylorhiza incarnata s.lat. have been examined for allozyme variation in DIA, PGD, PGI, PGM, SKD, TP1, and UGPP. The results reveal a deficiency of heterozygotes in several populations. Possible reasons for this deficiency are considered. The results also indicate that no genetic barrier seems to prevent gene flow between D. incarnata var. incarnata , var. dunensis , and var. ochrantha — and some indication exists that a considerable amount of gene exchange takes place between sympatric populations of var. incarnata and var. ochrantha . On the other hand, there is no sign of recent introgression between D. incarnata s.lat. and any of the sympatric tetraploid species ( D. maculata, D. majalis, D. purpurella s.lat.). It is suggested that knowledge on the various degrees of genetic integrity of morphologically recognizable entities should be elaborated and subsequently utilized for a biosystematic approach to Dactylorhiza .     

Plastid DNA haplotype variation in Dactylorhiza incarnata (Orchidaceae): evidence for multiple independent colonization events into Scandinavia

The early marsh orchid, Dactylorhiza incarnata (L.) Soó s. l., grows in medium-rich to rich fens and marshes over much of Europe and parts of Asia. The species is highly polymorphic and different forms may grow together at the same site. In the present study, I tested the hypothesis that these forms represent different migrant populations that have colonized Scandinavia independently of each other, possibly from different source areas. Accessions from Scandinavia and elsewhere were screened for variation at three size-variable plastid marker loci, one polyA repeat, one polyA-polyTA-polyT repeat and one 9 bp indel. Ten haplotypes were defined on basis on the combined variation pattern. The common occurrence of several haplotypes in southern Scandinavia and adjacent areas to the south and the east of the Baltic Sea suggests that D. incarnata has been dispersed on repeated occasions across the Baltic. Also, there was some correlation between haplotype composition and morphological form on the island of Gotland, in agreement with the independent colonization hypothesis. Material from northernmost Sweden, Finland and northwest Russia was fixed for a single widespread haplotype, indicating that populations in this area are located farther away from the Pleistocene refugia. Dactylorhiza incarnata ssp. lobelii from southwest Norway was characterized by a haplotype that was not encountered elsewhere in Scandinavia. Given its proximity to British populations dominated by the same haplotype, it is suggested that D. incarnata ssp. lobelii was established independently of the other Scandinavian populations, from coastal refugia located in western Europe.     

Transferrin C subtypes and ethnic heterogeneity in Sweden

Transferrin (TF) C subtypes were studied in Swedish Lapps (Saami) and in Swedes from northern, central and southern Sweden, and the allele frequencies were compared with those in other European populations. The Swedish Lapps were found to have the lowest frequency of the TF*C3 allele (1-2%) so far observed in Europe. Most European populations have TF*C3 allele frequencies between 5 and 7%. Finns differ by having high TF*C3 frequencies (13-14%). The relatively high TF*C3 frequencies found in northeastern Sweden (13%) and in central Sweden (9%) are most likely due to eastern influence. Unlike other genetic markers of eastern influence (e.g. TF*DCHI), which are of Asiatic Mongoloid origin, TF*C3 appears to originate from Finno-Ugric populations.     

Allozyme variation and racial differentiation in Swedish Carex lepidocarpa s.l. (Cyperaceae)

Two morphological races have previously been recognized within the sedge, Carex lepidocarpa , in Sweden. These largely allopatric races are accorded specific status, as C. lepidocarpa s.s. and C. jemtlandica , in Scandinavian floras. A study of allozyme variation in populations from 80 Swedish sites supports the morphological evidence for racial differentiation within C. lepidocarpa. The two races differ from each other in terms of allele frequencies at polymorphic loci and also show different levels of within-population genetic diversity. Material that is morphologically referable to C. lepidocarpa s.s. is characterized by relatively high levels of allozyme variation, both within and between populations. Carex lepidocarpa s.s. is widespread in southern Sweden. In contrast, material that is morphologically assignable to C. jemtlandica shows low levels of within-population genetic diversity, and there is little differentiation between the geographically separated isolates of C. jemtlandica in northern Sweden and on the Baltic island of Gotland. The high degree of morphological similarity and moderate levels of genetic differentiation between the two races within C. lepidocarpa indicate that it is more appropriate to recognize the races as subspecies than as species. The low levels of genetic variation in C. jemtlandica , both within and between populations, suggest that C. jemtlandica may have arisen from C. lepidocarpa (or a near ancestor of C. lepidocarpa) as a result of population fragmentation and isolation in glacial refugia, or during the process of post-glacial colonization of Scandinavia. Lack of allozyme evidence for extensive hybridization between the two races of C. lepidocarpa , despite their ability to hybridize freely where their ranges overlap at present, supports the suggestion that the two races have had separate post-glacial histories.     

Structure of allozyme variation in Nordic Silene nutans (Caryophyllaceae): population size, geographical position and immigration history

We investigated allozyme variation in 34 populations of the perennial herb Silene nutans from Sweden and northern Finland, areas that were ice-covered during the last (Weichselian) glaciation. The present geographical structure of genetic variation in S. nutans in Sweden and northern Finland appears to have been mainly shaped by ancient historical processes. Patterns of variation in allele frequencies suggest two major postglacial immigration routes into Sweden, with populations entering the area from both the south and the east and forming a contact zone with admixed populations in central Sweden. While estimates of within-population genetic diversity and allelic richness are significantly correlated with present population size and geographical position (latitude), population size is not correlated with latitude. Low genetic diversity in the northern populations is more likely to have resulted from ancient stochastic events during the process of immigration than from recent population fragmentation. F _IS values are high and increase with latitude. Evidence of recent bottlenecks was detected in several southern Swedish populations: these can be interpreted in terms of population fragmentation as a result of anthropogenic disturbance. Soil pH is uncorrelated with population size and position.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 81 , 357–371.     

Molecular investigations of cytochrome c oxidase subunit I (COI) and the internal transcribed spacer (ITS) in the poultry red mite, Dermanyssus gallinae, in northern Europe and implications for its transmission between laying poultry farms

Samples of Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) from more than 49 Norwegian and Swedish laying poultry farms, and additional samples collected from Scottish, Finnish, Danish and Dutch layer farms, were compared genetically. Analysis of partial mitochondrial gene cytochrome c oxidase subunit I (COI) sequences of mites from Norway and Sweden revealed 32 haplotypes. Only single haplotypes were found on most farms, which suggests that infections are recycled within farms and that transmission routes are few. Both Norwegian and Swedish isolates were found in the two major haplogroups, but no haplotypes were shared between Norway and Sweden, indicating little or no recent exchange of mites between these countries. There appears to be no link between haplotypes and geographical location as identical haplotypes were found in both the northern and southern Swedish locations, and haplotypes were scattered in locations between these extremes. The current data suggest that wild birds in Sweden are not a reservoir for D. gallinae infection of layer farms as their mites were genetically distinct from D. gallinae of farm layer birds. Transmission of the poultry red mite in Scandinavia is thus likely to depend on synantropic factors such as the exchange of contaminated material or infested birds between farms or facilities.     

Tyrosinase and tyrosinase related protein 1 alleles specify domestic cat coat color phenotypes of the albino and brown loci

The genes encoding enzymes of the tyrosinase family are strong candidates for coat color variation in mammals. To investigate their influence in domestic cat coat color, we determined the complete nucleotide coding sequence of the domestic cat genes tyrosinase (TYR)--a plausible candidate gene for the albino (C) locus, and tyrosinase related protein 1 (TYRP1)--a candidate gene for the brown (B) locus. Sequence variants between individuals exhibiting variation in pigmentation were submitted to association studies. In TYR, two nonsynonymous substitutions encoding TYR-G301R and TYR-G227W were associated with the siamese and burmese phenotypes of the albino locus, respectively. TYRP1 was mapped on chromosome D4 within 5 cM of a highly polymorphic microsatellite, previously found to be fixed in a cat breed selected for the chocolate (b) allele of the B locus, which reinforced TYRP1 as a candidate gene for the B locus in the domestic cat. Two DNA polymorphisms, one leading to a TYRP1-A3G substitution in the signal peptide and another to an in-frame insertion TYRP1-421ins17/18 caused by a donor splice site mutation in intron 6, were associated with the chocolate (b) allele. A premature UAG stop codon at position 100 of TYRP1 was associated with a second allele of the B locus, cinnamon (b(l)). The results provide very strong evidence that the specific nucleotide variants of feline TYR (chromosome D1) are causative of the siamese (c(s)) and burmese (c(b)) alleles of the albino locus, as well as nucleotide variants of TYRP1 (chromosome D4) as specifying the chocolate (b) and cinnamon (b(l)) alleles of the B locus.     

Geographic structure of genetic variation in the widespread woodland grass Milium effusum L. A comparison between two regions with contrasting history and geomorphology.

Allozyme variation in the forest grass Milium effusum L. was studied in 21-23 populations within each of two equally sized densely sampled areas in northern and southern Sweden. In addition, 25 populations from other parts of Eurasia were studied for comparison. The structure of variation was analysed with both diversity statistics and measures based on allelic richness at a standardised sample size. The species was found to be highly variable, but no clear geographic patterns in the distribution of alleles or in overall genetic differentiation were found, either within the two regions or within the whole sample. Thus, no inferences about the direction of postglacial migration could be made. Obviously, migration and gene flow must have taken place in a manner capable of randomising the distribution of alleles. However, there were clear differences in levels and structuring of the variation between the two regions. Levels of variation, both in terms of genetic diversity and allelic richness, were lower in northern Sweden as compared with southern Sweden. In contrast, different measures of geographic structure all showed higher levels of population differentiation in the northern region. This is interpreted as due to different geomorphological conditions in the two regions, creating a relatively continuous habitat and gene flow in the southern region as compared with the northern region where the species, although common, is confined to narrow and mutually isolated corridors in the landscape.     

Environmental response of Galium palustre s. lat. in a coastal area in Central Sweden

Galium palustre s. lat. is a polymorphic taxon. The differences in morphology are correlated with the level of ploidy, but there is also a certain amount of phenotypic plasticity. The plasticity probably contributes to this taxon's successful colonization of a wide variety of habitats. The amount of plasticity was studied for plants from different kinds of habitats in Sweden, both for diploids (G. palustre s. str.) and octoploids (G. elongatum ), which were cloned. This cloned material was then transplanted to three different localities in Sweden: a sunny exposed sea-shore, a ditch, and a shady alder swamp. The general pattern of morphology was similar for all material, independent of its origin and level of ploidy, with smaller plants (smaller in several vegetative characters) and a more abundant flowering on the exposed seashore than in the ditch, and the largest plants with a fairly sparse flowering in the alder swamp. The octoploid plants originating from the wooded wetland are the least plastic ones but are very variable within the localities. G. palustre var. balneum shows a similar variation pattern as the others, but there are also genetically fixed differences in some characters between it and G. palustre var. palustre . The changes in morphology of the plants in the different localities occur fairly rapidly (in one month), although the differences are more spectacular one year later.     

Systematics and conservation genetics of Dactylorhiza majalis ssp. elatior (Orchidaceae) on Gotland

A tall allotetraploid member of the Dactylorhiza incarnata/maculata complex with unspotted leaves and large pinkish flowers from the island of Gotland in the Baltic was examined for molecular variation patterns at five nuclear microsatellite loci, nuclear ITS and in plastid haplotypes. The allotetraploid was well separated from allopatric allotetraploids of similar appearance, including the western European D. majalis ssp. integrata (syn. D. praetermissa) and forms of D. majalis ssp. lapponica from mainland Sweden. It also differed from other allotetraploids distributed in the Baltic Sea region, including D. majalis ssp. baltica and D. majalis ssp. lapponica. It is here recognized as D. majalis ssp. elatior (Fr.) Hedrén & H. A. Pedersen. Dactylorhiza osiliensis Pikner, described from Saaremaa (Estonia) is regarded as a synonym. The distribution covers Gotland, Saaremaa and possibly Hiiumaa. Dactylorhiza majalis ssp. elatior may have one or several recent origins within its present distribution area, and it contains no other molecular markers than those found in the parental D. incarnata var. incarnata and D. maculata ssp. fuchsii in the same area. It appears to have weak barriers towards secondary hybridization with its parental lineages. The situation is reminiscent to that of other young allotetraploids in the D. majalis s.l. complex, suggesting that introgression may be an underestimated process explaining the accumulation of genetic diversity in evolving allopolyploid plants.     

南岭南北坡灰蝶的区系组成与生态分布

南岭是我国重要的动植物发源地和保存地,研究南岭蝴蝶的区系和生态分布对保护蝴蝶的多样性具有重要意义。通过对南岭中段南北坡的灰蝶进行多年的调查,结果表明:南岭保护区共记录到灰蝶101种,82.178%的种分布在东洋区。按中国动物区系划分,南岭灰蝶主要分布在华南、华中、西南三个区。从灰蝶在南岭南北坡的分布来看,2012-2014年在南北坡调查点共发现灰蝶865头,分别属于31个种,其中南坡共发现470头(隶属于27个种)、北坡228头(隶属于13个种)。这说明南坡的灰蝶种数多,是北坡种数的2倍;南坡的种群密度也比北坡高,是北坡的2.06倍。从同一坡向不同海拔来看,南坡1000-1100 m与500-600 m两个海拔的灰蝶种类数相同,但前者的种群密度为后者的1.55倍;北坡灰蝶种数与种群密度1000-1100 m均高于500-600 m;山顶灰蝶种类少。     

Subspecific relationships and genetic structure in the spotted owl

Hierarchical genetic structure was examined in the three geographically-defined subspecies of spotted owl (Strix occidentalis) to define relationships among subspecies and quantify variation within and among regional and local populations. Sequences (522 bp) from domains I and II of the mitochondrial control region were analyzed for 213 individuals from 30 local breeding areas. Results confirmed significant differences between northern spotted owls and the other traditional geographically defined subspecies but did not provide support for subspecific level differences between California and Mexican spotted owls. Divergence times among subspecies estimated with a 936 bp portion of the cytochrome b gene dated Northern and California/Mexican spotted owl divergence time to 115,000–125,000 years ago, whereas California/Mexican spotted owl divergence was estimated at 15,000 years ago. Nested clade analyses indicated an association between California spotted owl and Mexican spotted owl haplotypes, implying historical contact between the two groups. Results also identified a number of individuals geographically classified as northern spotted owls (S. o. caurina) that contained haplotypes identified as California spotted owls (S. o. caurina). Among all northern spotted owls sampled (n=131), 12.9% contained California spotted owl haplotypes. In the Klamath region, which is the contact zone between the two subspecies, 20.3% (n=59) of owls were classified as California spotted owls. The Klamath region is a zone of hybridization and speciation for many other taxa as well. Analyses of population structure indicated gene flow among regions within geographically defined subspecies although there was significant differentiation among northern and southern regions of Mexican spotted owls. Among all areas examined, genetic diversity was not significantly reduced except in California spotted owls where the southern region consists of one haplotype. Our results indicate a stable contact zone between northern and California spotted owls, maintaining distinct subspecific haplotypes within their traditional ranges. This supports recovery efforts based on the traditional subspecies designation for the northern spotted owl. Further, although little variation was found between California and Mexican spotted owls, we suggest they should be managed separately because of current isolation between groups.     

On the distinction of Dactylorhiza baltica and D. pardalina (Orchidaceae) and the systematic affinities of geographically intermediate populations

The eastern European Dactylorhiza baltica (Klinge) N. I. Orlova and the western European D. pardalina (Pugsl.) Aver. (= D. praetermissa var. junialis (Verm.) Sengh) are usually considered to have non-overlapping geographic distributions, for which reason it has rarely been realized that they are morphologically similar. They have not previously been thoroughly compared by molecular methods, and no existing flora or revision has convincingly demonstrated that they can be distinguished by morphological characters. In reality, they might be 'political' rather than natural taxa. Prompted by the recent discovery of geographically intermediate populations (in eastern Denmark), originally identified as D. baltica , we have addressed this problem by analysis of morphometric data as well as molecular data from allozyme markers, plastid haplotypes, nuclear ITS alleles and nuclear microsatellites. Dactylorhiza baltica and D. pardalina turned out to be clearly distinguished genetically, and although they are morphologically similar, a few characters were identified that distinguish with 81–85% certainty between the two taxa. Molecular and morphometric data place the geographically intermediate populations in D. pardalina . Both taxa were confirmed to be allotetraploids combining diploid genomes from the D. incarnata s.l. and D. maculata s.l. lineages, and they should therefore be recognized as infraspecic taxa under D. majalis s.l. Thus, D. baltica should be called D. majalis subsp. baltica ; D. pardalina is identical with D. praetermissa var. junialis , but the nomenclatural consequences for D. praetermissa , if treated as subspecies under D. majalis , are still unresolved.     

Placental enzyme polymorphisms in Canadian populations.

Phenotype distributions and allele frequencies of adenylate kinase and esterase D were determined for four Canadian populations. In two population samples from south-western Ontario, allele frequencies at both loci were similar to those of European populations. In two northern, indigenous populations, the allele AK2 was not detected. There was variation at the EsD locus with EsD2 having a frequency of 0.176 in an Indian population, and 0.156 in an Eskimo population.     

Polymorphic populations of Dactylorhiza incarnata s.l. (Orchidaceae) on the Baltic island of Gotland: morphology, habitat preference and genetic differentiation

Background and Aims

Organisms may be polymorphic within natural populations, but often the significance and genetic background to such polymorphism is not known. To understand the colour polymorphism expressed in the diploid marsh-orchids Dactylorhiza incarnata, morphological, habitat and genetic differentiation was studied in mixed populations on the island of Gotland, supplemented with genetic marker data from adjacent areas.

Methods

A total of 398 accessions was investigated for plastid haplotype and three nuclear microsatellites. Morphometric data and vegetation data were obtained from a subset of 104 plants.

Key Results

No clear pattern of habitat differentiation was found among the colour morphs. Within sites, the yellow-flowered morph (ochroleuca) was slightly larger than the others in some flower characters, whereas the purple-flowered morph with spotted leaves (cruenta) was on average smaller. However, populations of the same colour morph differed considerably between sites, and there was also considerable overlap between morphs. Morphs were often genetically differentiated but imperfectly separated within sites. Most populations were characterized by significant levels of inbreeding. The ochroleuca morph constitutes a coherent, highly homozygous sublineage, although introgression from purple-flowered morphs occurs at some sites. The cruenta morph was genetically variable, although Gotland populations formed a coherent group. Purple-flowered plants with unspotted leaves (incarnata in the strict sense) were even more variable and spanned the entire genetic diversity seen in the other morphs.

Conclusions

Colour polymorphism in D. incarnata is maintained by inbreeding, but possibly also by other ecological factors. The yellow-flowered morph may best be recognized as a variety of D. incarnata, var. ochroleuca, and the lack of anthocyanins is probably due to a particular recessive allele in homozygous form. Presence of spotted leaves is an uncertain taxonomic character, and genetic differentiation within D. incarnata would be better described by other morphological characters such as leaf shape and stature and size and shape of lip and spur.Key words: Dactylorhiza incarnata, cruenta, ecology, genetic differentiation, Gotland, microsatellites, ochroleuca, plastid DNA, polymorphism     

Enzyme variation and inheritance in Glechoma hederacea (Lamiaceae), a diploidized tetraploid

The chromosome number of the polyploid species Glechoma hederacea was found to be 2n = 36 in a sample of 93 ramets derived from 27 sites in N and C Europe. Variation in 10 enzymes was surveyed in material from S Sweden and S Czech Republic. The genetic control of variation was investigated using segregating progeny from crosses and self-fertilized heterozygous plants. The genetic analysis comprised 30 of 32 putative alleles detected in the geographical survey. Five loci (Aat-2, Tpi-1, Tpi-2, Pgd-2 and Mnr) behaved as isoloci with one copy of a locus being monomorphic for a common allele, the other di-allelic for a common allele and a variant allele. In four isoloci (Pgd-1, Pgi-2, Mdh-2 and Adh), both copies of the duplicated locus were polymorphic, with one allele common to both copies and with another allele unique for each copy except for Pgd-1 where both copies were tri-allelic. Three loci, Pgm-3, Skd-1 and Skd-2 were regarded as being non-duplicated. Segregation ratios for all enzyme loci were in close agreement with expectations based on disomic inheritance. Our data suggest that the tetraploid G. hederaca is a diploidized autotetraploid.