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Allozyme variation and genetic integrity of Dactylorhiza incamata (Orchidaceae)

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Abstract

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.

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... Ряд систематиков считает необходимым рассматривать D. ochroleuca в качестве самостоятельного вида, так как по их мнению таксон с бледно-желтыми цветками отличается от формы D. incarnata с чисто белыми цветками [1,2,5,6]. Согласно другим исследователям [3,4,[10][11][12], D. ochroleuca -это только альбиносная форма D. incarnata. Данная проблема существует потому, что, несмотря на четкие морфологические различия между D. ochroleuca и D. incarnata, молекулярно-генетических маркеров для разделения представителей данных таксонов (на примере изученных образцов из Скандинавии и Западной Европы) выявлено не было. ...
... Полиморфизм оценивали по изменчивости частот аллелей восьми генных локусов: фосфоглюкоизомеразы (PGI, EC 5.3.1.9), NADH-дегидрогеназы (NADHD, EC 1.6.99.5), шикиматдегидрогеназы (SKDH, EC 1. В исследовании использован материал: D. incarnata -из восьми популяций с Урала (201 особь), пяти популяций из Сибири (130) и семи популяций из Беларуси (193); D. ochroleuca -из двух популяций с Урала (47 особей), трех популяций из Беларуси (53), одной популяции из Сибири (11). В анализ были включены также выборки нетипичных особей из окрестностей озер Нарочь и Сервечь (Беларусь, рис. ...
... Тем самым был найден генный маркер, по которому близкие таксоны D. ochroleuca и D. incarnata различаются. К сожалению, локус GDH не использовался зарубежными коллегами [3,4,10,11] при изучении популяций D. ochroleuca и D. incarnata из Скандинавии и Западной Европы. Поскольку морфологические различия между особями данных таксонов очевидны, можно ожидать существование и других таксон-специфичных маркеров. ...
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25.07.2016 г. С использованием аллозимного анализа изучены полиморфизм и генетическая структура популя-ций D. incarnata и D. ochroleuca в местах их совместного произрастания в России и Беларуси. Пока-зано, что особи D. ochroleuca из популяций с Урала и Сибири, удаленных от основной части ареала, не отличаются от особей из основной части ареала (Беларусь) по аллельному составу восьми генных локу-сов. Выявлено, что D. ochroleuca и D. incarnata дифференцированы по разным аллелям локуса GDH. Тем самым был установлен генный маркер, по которому эти близкие таксоны различаются. Кроме локуса GDH особи D. ochroleuca и D. incarnata в зоне их совместного произрастания различаются по аллель-ной структуре PGI и NADHD. Несмотря на то что у D. incarnata с Урала и из Сибири оба локуса были полиморфными, а из Беларуси-один из них (PGI), все особи D. ochroleuca, произрастающие сов-местно с полиморфными особями D. incarnata, оказались гомозиготными по одинаковым аллелям. Сделан вывод о генетической обособленности и существовании механизма изоляции D. ochroleuca от D. incarnata даже при их совместном произрастании. Обнаружено, что локус GDH у D. incarnata полиморфен только в популяциях, которые произрастают совместно с D. ochroleuca, за исключени-ем единичных примеров. Было сделано заключение, что изменчивость локуса GDH у D. incarnata связана с гибридизацией с D. ochroleuca. Ключевые слова: Dactylorhiza incarnata, D. ochroleuca, аллозимный анализ, межвидовые интрогрессив-но гибридные комплексы, генетический полиморфизм.
... Ряд систематиков считает необходимым рассматривать D. ochroleuca в качестве самостоятельного вида, так как по их мнению таксон с бледно-желтыми цветками отличается от формы D. incarnata с чисто белыми цветками [1,2,5,6]. Согласно другим исследователям [3,4,[10][11][12], D. ochroleuca -это только альбиносная форма D. incarnata. Данная проблема существует потому, что, несмотря на четкие морфологические различия между D. ochroleuca и D. incarnata, молекулярно-генетических маркеров для разделения представителей данных таксонов (на примере изученных образцов из Скандинавии и Западной Европы) выявлено не было. ...
... Полиморфизм оценивали по изменчивости частот аллелей восьми генных локусов: фосфоглюкоизомеразы (PGI, EC 5.3.1.9), NADH-дегидрогеназы (NADHD, EC 1.6.99.5), шикиматдегидрогеназы (SKDH, EC 1. В исследовании использован материал: D. incarnata -из восьми популяций с Урала (201 особь), пяти популяций из Сибири (130) и семи популяций из Беларуси (193); D. ochroleuca -из двух популяций с Урала (47 особей), трех популяций из Беларуси (53), одной популяции из Сибири (11). В анализ были включены также выборки нетипичных особей из окрестностей озер Нарочь и Сервечь (Беларусь, рис. ...
... Тем самым был найден генный маркер, по которому близкие таксоны D. ochroleuca и D. incarnata различаются. К сожалению, локус GDH не использовался зарубежными коллегами [3,4,10,11] при изучении популяций D. ochroleuca и D. incarnata из Скандинавии и Западной Европы. Поскольку морфологические различия между особями данных таксонов очевидны, можно ожидать существование и других таксон-специфичных маркеров. ...
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We carried out an allozyme analysis to investigate polymorphism and genetic structure of the populations of D. incarnata and D. ochroleuca in regions of their joint growth in Russia and Belarus. We found that D. ochroleuca individuals in the populations of the Urals and Siberia, which are distant fragments from the main range of the species, do not differ significantly from individuals within the main part of the area (Belarus) on the basis of the allelic composition of eight gene loci. We revealed that D. ochroleuca and D. incarnata are differentiated by different alleles of the GDH locus. Thus, we established a genetic marker suitable to distinguish these closely related taxa. In addition to the GDH locus, D. ochroleuca and D. incarnata in the places of their joint growth, differ in the allelic structure of the PGI and NADHD loci. D. incarnata from the Urals and Siberia were polymorphic for both loci, and individuals from Belarus were polymorphic for one locus (PGI). In contrast, all D. ochroleuca individuals growing in sympatric populations with polymorphic D. incarnata were homozygous for the same alleles. Thus, comparison of the genetic structure of D. ochroleuca and D. incarnata points to the existence of a genetic isolation and a functioning isolation mechanism even under conditions of their joint growth. We found that the GDH locus in D. incarnata is polymorphic only in populations which grow together with D. ochroleuca, with exception a few examples. Thus, we conclude that variability of the GDH locus in D. incarnata is associated with hybridization with D. ochroleuca.
... ochroleuca Jagiello and Kuusk -with leaves without spots and with yellow flowers. Additionally, subsequent studies using allozyme markers [15,16] and the AFLP method [9] demonstrated that the aforementioned varieties of D. incarnata show very low genetic variation, high levels of inbreeding, and in terms of the analyzed loci they are almost identical. Furthermore, Hedrén and Nordström argued that these varieties represent distinct inbred lines and gene flow across them is rare [17]. ...
... A relatively low level of genetic diversity was observed in the northern part of the early marsh orchid range [14,15,41]. These results indicated that the level of allozyme variation for the populations of the early marsh orchid is the lowest among the studied species within the Dactylorhiza genus. ...
Article
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The genetic structure of Dactylorhiza incarnata var. incarnata populations is shaped not only by historical events such as recolonization after ice sheet retreat or limited seed and pollen dispersal, but also the bottleneck effect. During the last decade, D. incarnata var. incarnata has also experienced a strong decline in population numbers and sizes, due to habitat loss and fragmentation. In the present research genetic diversity was examined in eight populations located in northern Poland, using six nuclear microsatellites loci. At the species level our results showed a moderate mean level of genetic diversity (A = 4.67; Ae = 2.73; Rs = 4.48; Ho = 0.438; FIS = 0.224), which varied among the studied populations (A: 2.17–3.67; Ae: 1.55–2.69; Rs: 1.31–1.61; Ho: 0.292–0.631; FIS: −0.283–0.340). A considerable overabundance of homozygotes was detected in four populations (FIS within the range of 0.067–0.340), and in the remaining populations an excess of heterozygotes was observed. The average apparent out-crossing rate was also calculated (ta = 0.980), and primarily indicated a tendency to out-cross within the species. Moderate genetic differentiation was found among the studied populations (FST = 0.149; RST = 0.174; p < 0.05). The differentiation of the populations corresponded to relatively low gene flow value (Nm = 0.426) among populations, which amounted to only one migrant every second generation.
... [44,[61][62] and based on species presence-only observations. The list of locations in which the Dactylorhiza populations were found was prepared based on available literature data [14,26,29,30,35,[63][64][65][66][67][68] and information gathered during the field works [69]. Only these localities which could be precisely placed on the map (Fig 1) were used in the analysis and the duplicate presence records were removed. ...
... ochroleuca). Allozyme data clearly indicated that Turkish populations of D. incarnata s.l. were far more genetically variable than populations from northern Europe, because the species appears to have lost genetic variation during its recolonization from southern refugia after the Weichselian glaciation [18,[22][23]64,83]. Pillon et al. [28] revealed a higher level of genetic diversity in the Caucasus and the Mediterranean Basin than in western Europe using plastid and nuclear DNA sequence data. ...
Article
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The intensively discussed taxonomic complexity of the Dactylorhiza genus is probably correlated with its migration history during glaciations and interglacial periods. Previous studies on past processes affecting the current distribution of Dactylorhiza species as well as the history of the polyploid complex formation were based only on molecular data. In the present study the ecological niche modeling (ENM) technique was applied in order to describe the distribution of potential refugia for the selected Dactylorhiza representatives during the Last Glacial Maximum. Additionally, future changes in their potential habitat coverage were measured with regard to three various climatic change scenarios. The maximum entropy method was used to create models of suitable niche distribution. A database of Dactylorhiza localities was prepared on the grounds of information collected from literature and data gathered during field works. Our research indicated that the habitats of majority of the studied taxa will decrease by 2080, except for D. incarnata var. incarnata, for which suitable habitats will increase almost two-fold in the global scale. Moreover, the potential habitats of some taxa are located outside their currently known geographical ranges, e.g. the Aleutian Islands, the western slopes of the Rocky Mountains, Newfoundland, southern Greenland and Iceland. ENM analysis did not confirm that the Balkans, central Europe or central Russia served as the most important refugia for individual representatives of the Dactylorhiza incarnata/maculata complex. Our study rather indicated that the Black Sea coast, southern Apennines and Corsica were the main areas characterized by habitats suitable for most of the taxa.
... This pattern was interpreted as ongoing local differentiation that could possibly result in sympatric speciation (Vallius et al., 2004). Thirdly, studies of allozyme variation in D. incarnata (Pedersen, 1998; Hedrén, 2001a) have shown that populations of D. incarnata are characterized by high levels of inbreeding (Hedrén, 2001a). It is possible therefore that morphs of D. incarnata represent inbred lines, and gene flow across varieties is rare for this reason. ...
... ochroleuca is sometimes narrowly circumscribed such that only the most morphologically typical plants are included (Delforge, 2001). Atypical plants are then regarded as occasional forms of purpleflowered incarnata, forma ochrantha (Pedersen, 1998; Delforge, 2001; Haggar, 2005b), implying that these forms have no closer relationship with each other than with purpleflowered forms. Similarly, Kreutz (1993) published a report on the orchid flora of Gotland where he claimed that genuine ochroleuca is rare on Gotland and that most yellowflowered populations should instead be regarded as ochrantha. ...
Article
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. 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. 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. 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.
... Как известно, равновесие может нарушаться из-за избирательного характера скрещиваний и других важнейших эволюционных факторов, таких как отбор и поток генов [25,32,33]. Преобладание гомозигот в субпопуляциях D. incarnata в Европе связывают с инбридингом [5,7]. В случае изученных локальных популяций в Забайкальском крае и Бурятии представить наличие инбридинга довольно сложно. ...
Article
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28.03.2016 г. Представлены результаты изучения полиморфизма и генетической структуры популяций Dacty-lorhiza salina и D. incarnata, произрастающих в Забайкальском крае и Бурятии, по данным аллозим-ного анализа восьми генных локусов (PGI, NADHD, SKDH, GDH, PGM, DIA, ADH, IDH). Установле-на специфичность аллельной структуры локусов SKDH, PGM и IDH для D. salina и D. incarnata, по которым виды достоверно отличаются друг от друга. Показано, что в Забайкалье сформировались межвидовые интрогрессивно гибридные комплексы, имеющие разную генетическую структуру. В местах массового произрастания D. incarnata встречаются единичные растения D. salina, межвидо-вые гибриды первого и последующих поколений. В местах массового произрастания D. salina обна-ружены только гибриды, не являющиеся гибридами первого поколения. Они были гетерозиготны-ми не по трем локусам с дифференцирующими аллелями обоих родителей-SKDH, PGM и IDH, а лишь по одному из них. Степень генетической дифференциации между пятью популяциями D. sa-lina в среднем составляла 7.5%, а для D. incarnata-7.1%, что в соответствии со шкалой оценки Райта относится к средним значениям. Среднее значение F ST для всех изученных популяций двух близких видов рода Dactylorhiza составило 0.478, что свидетельствует об очень высокой степени генетической дифференциации между D. salina и D. incarnata, произрастающих в Забайкалье. Наиболее сильно виды различаются по аллельной структуре локусов SKDH, PGM и IDH (F ST равнялся 0.705, 0.976 и 0.762, соответственно). Дисперсионный анализ данных (AMOVA) показал, что между популяциями D. salina и D. incarnata, в зоне перекрывания их ареалов в Забайкальском крае и Бурятии, имеются существенные различия, и в варьировании частот аллелей восьми локусов (71%, d.f. = 9), и в измен-чивости генотипов (61%, d.f. = 9). Несмотря на то что между D. salina и D. incarnata существует явный поток генов в результате межвидовой гибридизации, генетическая дифференциация популяций этих близких видов сохраняется на высоком уровне. Ключевые слова: Dactylorhiza salina, D. incarnata, аллозимный анализ, межвидовые интрогрессивно гибридные комплексы, генетический полиморфизм.
... Как известно, равновесие может нарушаться из-за избирательного характера скрещиваний и других важнейших эволюционных факторов, таких как отбор и поток генов [25,32,33]. Преобладание гомозигот в субпопуляциях D. incarnata в Европе связывают с инбридингом [5,7]. В случае изученных локальных популяций в Забайкальском крае и Бурятии представить наличие инбридинга довольно сложно. ...
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The results of studying the polymorphism and genetic structure of populations of D. salina and D. incarnata growing in Zabaykalsky krai and Buryatia are represented according to the data of allozyme analysis of eight genetic loci (PGI, NADHD, SKDH, GDH, PGM, DIA, ADH, and IDH). The specificity of the allelic structure of loci SKDH, PGM, and IDH is established, for which D. salina and D. incarnata reliably differ from each other. It is shown that interspecies introgressive hybrid complexes with different genetic structures were formed in Transbaikalia. Places of mass growth of D. incarnata were observed to have single plants of D. salina, the interspecies hybrids of the first and subsequent generations. Places of mass growth of D. salina were observed to contain only the hybrids that are not hybrids of the first generation. They were heterozygous not for three loci with differentiating alleles of both parents, SKDH, PGM, and IDH, but for only one of them. The degree of genetic differentiation among five populations of D. salina was on average 7.5% and that of D. incarnata was 7.1%, which in accordance with Wright’s estimation relates to mean values. The average value of FST for all studied populations of the two related species of the genus Dactylorhiza was 0.478, indicating a very high degree of genetic differentiation between D. salina and D. incarnata growing in Transbaikalia. The greatest differences between the species are for the allelic structure of loci SKDH, PGM, and IDH (FST was equal to 0.705, 0.976, and 0.762, respectively). Analysis of molecular variance (AMOVA) showed that populations of D. salina and D. incarnata in the zone where their ranges in Zabaykalsky krai and Buryatya overlap have essential differences both for the variation of alleles frequencies of eight loci (71%, d.f. = 9) and for the variability of genotypes (61%, d.f. = 9). Despite the fact that D. salina and D. incarnata explicitly share a gene flow as a result of interspecies hybridization, the genetic differentiation of populations of these related species remains at a high level.
... Lower average F IS value for D. fuchsii than for D. incarnata (0.19 vs. 0.61) is likely connected with higher proportion of self-pollination in flowers of D. incarnata. Such values accurately correspond to the values that have been detected in those species by Hedrén (2001a: 20), who observed F IS = 0.11 for D. fuchsii and 0.73 for D. incarnata, and correspond well to the data about deficiency of heterozygotes in those species that was observed by Pedersen (1998a). Allopolyploids always display higher values of F IS , confirming non-recombinant inheritance of parental genomes. ...
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Morphological and allozyme analyses of Siberian dactylorchids have revealed the presence of a new, previously undescribed allopolyploid that is described here as species new to science. The genetic constitution as revealed by allozyme analysis of this species confirms that its parents are the diploid Siberian D. fuchsii and D. incarnata, and it is 2n=80, representing the first such count from the Asiatic Russia. In most literature surveys of Siberia, the new species has been identified as D. baltica, which is different in the allele composition of the pgi locus. Morphologically, both allotetraploids are similar; D. sibirica is distinguishable due to the absence of spots on the leaves and a narrower lip with narrower lateral lobes. A brief summary of Dactylorhiza allopolyploids from Siberia (Russia) is presented.
... Neiland and Wilcock (2000) found inter-specific pollination to be frequent in mixed populations of orchids in Scotland. Pedersen (1998) also argued that no effective pre-pollination barrier had been demonstrated between D. incarnata var. incarnata and the white-flowered var. ...
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Dactylorhiza orchids are known for their high variation in morphology and distinct varieties have been named in D. incarnata s.l. However, it is not known how these varieties interact in mixed populations and why they remain stable. We conducted three field experiments in West-Estonian populations of D. incarnata to examine if the two most common varieties co-occurring these are separated from each other either by pre-pollination or post-pollination reproductive barriers. We found that pollinia were far more frequently transferred between the purple-flowered var. incarnata and the pale-flowered var. ochroleuca than between plants of the same variety. Furthermore, in hand-pollination and germination experiments concerning the same two varieties, we found that pollen source (self-pollination, within- or between-varieties pollination) did not affect seed production or probability of fungus infection of the germinating seeds. These two varieties of D. incarnata thus had no pre-pollination or early functioning post-pollination reproductive barriers. Post-pollination barriers may, however, act later in seedling or adult stage.
... Dactylorhiza incarnata s.l. is variable and includes distinct color morphs, which may be sympatric, and also geographical races, which are often treated as subspecies or separate species. However, allozyme analyses (Hedrén, 1996c;Pedersen, 1998a), AFLP analysis (Hedrén et al., 2001), or plastid DNA variation (Hedrén, 2003;Pillon et al., in press) reveal that D. incarnata has astonishingly little genetic variation and that morphologically defined taxa are poorly correlated to genetic differentiation patterns. In contrast, D. maculata s.l. is variable both in morphology and at molecular marker loci, and it includes diploid as well as autotetraploid cytotypes. ...
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Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.
Article
Principal components analysis (PCA) was applied to summarize variation in 23 morphological characters scored from nine late-flowering dune populations of Dactylorhiza incarnata (Orchidaceae) in Wales, The Netherlands, and Denmark. The Dutch and Danish groups of populations were vaguely separated in a PCA conducted on populations, and a very broad overlap between them was found in a PCA conducted on individual specimens. On the other hand, a clear morphological distinction between the Welsh and continental groups of populations appeared from both PCAs. The probability of each character to distinguish correctly between the main groups of populations was estimated. Three characters were found to distinguish reliably (success ≥ 90 %) between the Welsh and continental groups of populations, while no character was found to distinguish reliably between the Dutch and Danish groups of populations. It is concluded that the Welsh and Dutch/Danish populations represent two distinct taxa distributed in the British Isles and in continental Northwestern Europe, respectively. The British taxon is recognized as D. incarnata subsp. coccinea and the continental one as D. incarnata subsp. lobelii (stat. nov.). Brief taxonomic accounts are provided.
Article
Morphometric and allozyme data were collected from 11 populations of Dactylorhiza majalis s.l. in the Ardennes (Belgium, France) and North Jutland (Denmark). All study populations were growing in obviously acid habitats. Genetic distances were calculated and illustrated by UPGMA dendrograms, while principal components analyses (PCAs) were used to summarize morphological variation patterns. Based on the PCAs, the probability of each character to distinguish correctly between the main groups of populations was estimated. The study populations from the Ardennes and from the Danish island of Læsø seem to agree with populations from Sweden previously studied by Hedrén—and to belong to D. majalis subsp. sphagnicola (comb. nov.). It should be noted that allozyme data suggest a polytopic origin of this taxon. The study populations from the Danish region of Thy differ morphologically from the others and exhibit much less genetic variation. They are described as a new subspecies, D. majalis subsp. calciñigiens. The evolution and biogeography of the two taxa are discussed, and brief taxonomic accounts are provided.
Article
In order to prepare for a biosystematic treatment of Dactylorhiza, a search is made for applicable taxonomic concepts (species, subspecies, variety)–aiming at a hierarchical system in which a unique kind of biological unity applies to each level in the hierarchy. The degree of reproductive isolation between D. purpurella s.str. and each of D. maculata s.str., D. majalis s.str., and D. purpurella ssp. majaliformis (delimitation and nomenclature according to Løjtnant 1993) is assessed from morphometric data. These three cases represent three putative levels in a phylogenetic hierarchy. The results are compared to previously published data on corresponding groups of taxa (mainly from northern Europe), and a positive correlation is found between the putative level of phylogenetic relationship and the degree of reproductive isolation within the genus. Furthermore, taxa defined on each level are found to be morphologically recognizable. The three empirically defined categories are matched with general species concepts from the literature, subsequently leading to recommendation of the following taxonomic concepts for Dactylorhiza (slightly shortened): (1) As “species” are designated taxa complying with the biological species concept in a modern, botani-cally focused sense; (2) as “subspecies” are designated taxa complying with the ecological, but not with the biological species concept; (3) as “varieties” are designated taxa complying with the phenetic, but neither with the biological nor the ecological species concept. In a postscript the taxonomic consequences for the taxa specifically examined in the case studies are briefly sketched, and reference is made to a forthcoming revision of the “purpurella complex”.
Article
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.
Article
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.
Article
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.
Article
Full-text available
Although the potential for gene flow between species with large differences in chromosome numbers has long been recognized, only few studies have thoroughly investigated in situ hybridization across taxa with different ploidy levels. We combined morphological, cytological, and genetic marker data with pollination experiments to investigate the degree, direction, and spatial pattern of hybridization between the diploid Dactylorhiza incarnata and its tetraploid derivative, D. praetermissa. To identify hybrids, 169 individuals were genotyped using AFLPs and morphologically characterized. Individuals were clustered on the basis of their AFLP profile using the program Structure. To reduce the dimensionality of the plant-trait matrix, PCA was applied. The origin of suspected hybrid individuals was verified using flow cytometry. An AMOVA and spatial autocorrelation analysis were used to indirectly infer the extent of gene flow. Only five individuals were regarded as putative hybrids on the basis of the AFLP data; all had been assigned to the D. praetermissa morphotype. Only one had deviating DNA content and was presumably a triploid. High Φ(ST) values between different subpopulations and significant spatial genetic structure were observed, suggesting localized gene flow. Using combined data to study hybridization between D. incarnata and D. praetermissa, very few unequivocal hybrids were observed. We propose several non-mutually exclusive explanations. Localized pollen flow, in combination with different microhabitat preferences, is probably one of the reasons for the low frequency of hybrids. Also, the triploid first-generation hybrids may experience difficulties in successful establishment, as a result of genic incompatibilities.
Book
Still widely used as gene markers, isozymes detected by zymogram techniques have proven valuable in a range of other biological applications over the last few years. Along with these new applications, many new techniques have also emerged. Yet more than eight years since the Handbook of Detection of Enzymes on Electrophoretic Gels was first published, it remains the only book completely devoted to zymogram methods. The time has come to bring its contents up to date. New in the Second Edition: • An overview of new applications of enzyme electrophoresis and zymogram techniques • Zymogram techniques for approximately 100 enzymes not included in the first edition, bringing the total to more than 900 methods for detecting more than 400 different enzymes • Information on subunit structure included within the enzyme sheets to facilitate interpretation of isozyme patterns detected on zymograms • An appendix containing information on the buffer systems most commonly used for enzyme electrophoresis in starch, cellulose acetate, and polyacrylamide gels. The second edition of this bestselling handbook makes significant additions to the set of reliable gene markers suitable for electrophoretic analysis. It also strengthens the value of enzyme electrophoresis as a powerful tool proven successful in solving a variety of problems encountered across many biological areas, including the post-genome biology.
Article
During meiosis in naturally occurring triploid hybrids between the diploid Orchis fuchsii Druce (2n = 4O) and the two tetraploids, O. purpurella Steph. and O. praetermissa Druce (2n = 8O), there is a regular formation of 20 bivalents and 20 bivalents. Since the two tetraploid species themselves show typical ‘diploid’ behaviour in synapsis and fertility, they are considered to be allopolyploids, and the hybrid pairing to be allosyndetic. The implication is therefore that both tetraploids are amphidiploids of which O. fuchsii has been one progenitor. It is suggested that varieties of the polytypic diploid O. latifolia L. sec. Pugsl. may have been the other progenitors. A feature of interest in the microsporogenesis of both parents and hybrids is the close synchronization of nuclear events in the pollen massulae, which behave as physiological units throughout meiosis and pollen-mitosis. In the triploids, although numerous dysploid nuclei are produced, none dies prematurely, probably because of mutual compensation within what is, in effect, a common cytoplasmic matrix.
Chapter
The ability to observe allelic variation at isozyme loci has revolutionized research in the fields of biochemical genetics, population genetics, and evolution. This variation, called allozymic polymorphism, has been used in plants to examine genetic processes at every stage of the life cycle and to ascertain genetic diversity in all major crops as well as many other species. Yet the potential for using allozymes as genetic markers was not immediately predicted when isozyme variability was initially described (Hunter and Marker, 1957; Markert and Moller, 1959). In fact, the extent and prevalence of allozyme polymorphism was a rather disconcerting surprise to evolutionary biologists. Classical evolutionary theory had predicted that the most “efficient” form of an enzyme should, over time, become predominant in isolated populations, with an occasional rare allele produced through mutation. The discovery in many populations of relatively high levels of polymorphism at isozyme loci has forced a major reconsideration of evolutionary theory (Kimura and Crow, 1964; Koehn et al., 1983; Kimura, 1983).
Chapter
Gel electrophoresis of proteins has become a standard and powerful research tool for application in a multitude of biological disciplines. One form of protein electrophoresis, isozyme analysis, has become particularly prominent in systematic and evolutionary biology as well as agronomy (Tanksley and Orton, 1983). Isozymes, or multiple molecular forms of enzymes, are enzymes that share a common substrate but differ in electrophoretic mobility (Markert and Moller, 1959). They are revealed when tissue extracts are subjected to electrophoresis in various types of gels and subsequently submersed in solutions containing enzyme-specific stains. Genetic analysis may indicate that some of the variant electromorphs are encoded by alternate alleles at a single locus, in which case the allelic products are termed allozymes (Prakash et al., 1969). Data retrieved from electrophoretic gels consist of the number and relative mobilities of various enzyme products, which with appropriate genetic analyses become transformed into single or multilocus genotypes for each individual analyzed. Reasons are many for the popularity of electrophoretic data (Avise, 1975; Gottlieb, 1977; Crawford, 1983), but foremost among these is that isozymes provide a series of readily scored, single-gene markers.
Article
(1) Nearly all bees collected pollen on some trips and nectar only on others; most of the exceptions were observed for a few trips only. (2) Only a few of the local species were visited extensively for pollen. Bees foraging on these showed a greater constancy per trip than bees foraging on other species. The most popular pollen often occurred as a minor constituent of loads that were predominantly of other pollens. (3) Bombus lucorum foragers visited fewer species and had purer loads than B. agrorum foragers. Pollen-gatherers from two B. lucorum colonies made different use of the surrounding flora, suggesting that the colony somehow influences an individual bee's choice of forage. (4) B. lucorum foragers showed day-to-day constancy and about 70% collected their original pollen type on the tenth day after the first observation. Inconstant bees sometimes collected the same combination of pollens on successive trips.
Article
A field method for conducting insect choice experiments shows that bumblebees in Rocky Mountain subalpine meadows are flower-constant to a limited extent when visiting superficially similar composite flower heads.
Article
Bumblebees of any one species in Maine forage for pollen and/or nectar from a large variety of morphologically diverse flowers, but individuals have limited foraging repertoires at any one time. Unspecialized individuals were sometimes unsuccessful in extracting nectar and/or pollen from highly rewarding flowers. In any one area with a variety of concurrently blooming plants, the bumblebees had apparent species preferences. Superimposed on the species preferences were individual preferences. Individuals had primary foraging specialties (their majors) and secondary specialities (their minors). Minors were often bridges to new majors. Queens in Maine necessarily have several successive majors during their lifetime since the blooming time of the plants they utilize are brief relative to their life-span as foragers. However, the blooming time of most plants available to Bombus fervidus workers are long relative to their lifetime. Switching was rarely observed in these bees, even in some individuals observed daily for up to 1 mo at the same foraging area containing other plant species in bloom that were highly attractive to other individuals of the same bumblebee species. On a per flower basis, those flowers producing the most food rewards generally had the largest number of bees majoring from them, and the food rewards available were roughly comparable between different kinds of flowers, regardless of their differences in rates of nectar production. Specializing was usually preceded by sampling a variety of rewarding as well as nonrewarding flowers. When the flowers from which bees were majoring in an area were experimentally removed many of the bees sampled flowers of other concurrently blooming plants, but they generally did not switch to flowers from which the food rewards were being depleted by specialists, unless these were experimentally fortified with syrup. Upon finding superior food rewards in enriched blossoms, they switched immediately. Flower-specificity is related to site-specificity. Many bees shared the same foraging area, but different individual bees at the same site utilized the flowers of different plant species. When the foraging area contained landmarks, the bees visited clumps of flowers in a sequence (foraging path) that was generally repeated several times on the same foraging trip when the foraging site was small. The foraging behavior of bumblebees is discussed from a comparative standpoint with other bees and in relation to food distribution and availability in the environment.
Article
1. The flower constancy of honeybees on successive days has been studied by removing and identifying pollen collected by marked pollen-gatherers. Removing pollen from the bees decreased their tendency to collect it later; chilling the bees before marking did not influence their foraging behaviour; neither treatment affected their constancy to the kind of pollen collected. No sequence of nectar or pollen collection with age of bee, was found. 2. In each experiment most of the bees collected only a few of the pollens available to them. Bees collecting the most common pollens tended to be the most constant. In general, the proportion of bees collecting their original pollen decreased as the number of foraging days increased, and only about half were doing so after 1 week; the rate of decrease differed in different experiments. No bee regularly collected different pollens at different times of the day. When the pollen they were accustomed to collect was unavailable for a day, most foraged for nectar only or remained at home. Most bees that changed to another pollen probably did so when the pollen they had previously collected was scarce or unattractive for longer periods. 3. About 6% of the loads each contained more than one species of pollen. Bees that collected mixed loads were more inclined to do so later; probably they were dissatisfied with the crops they were working and were sampling others. 4. When a colony was moved to another site with a similar flora, the bees tended to visit the same species as before, but when one species predominated, bees that had not visited it before tended to do so.
Article
Data are presented to show that there are three serum fl-globulin types in laboratory mice controlled by a pair of alleles. Each allele appears to give rise to three electrophoretically distinct zones in starch gel. Within the inbred strain AI AGS there was variation between mice in the intensity of staining of the three zones. Reciprocal mating data gave no evidence of an effect of fl-globulin type on segregation ratios as has been reported for cattle.
Article
Multiple paternity occurred frequently in a sample of Raphanus sativus involving all of the assayed parents and at least 85% of the fruits overall. Minimum paternal donor number ranged from 1-4 (mean 2.27). Likely sources of multiple paternity within radish fruits are pollen carry-over and multiple visits by pollen vectors. -from Author
Article
Evidence from all ozyme markers suggests that the NW European Dactylorhiza purpurella (Orchidaceae) is an allotetraploid which originated from taxa closely related to the present-day D. incarnata s. I. and D. fuchsii/D. maculata. However, Dactylorhiza purpurella deviates more strongly from the allotetraploid condition than other taxa previously investigated in Dactylorhiza (i.e., D. majalis, D. traunsteineri, D. sphagnicola, and D. lapponica), in that the characteristic incarnata alleles occur at lower frequencies than expected at two loci. It is suggested that D. purpurella arose from parents slightly different from those giving rise to the other allotetraploids, or that the tetraploid genome in D. purpurella has been modified by rare recombination events between homoeologous chromosomes, replacing segments of the incarnata chromosomes with the fuchsii/maculata genome.
Article
Swedish material of Dactylorhiza incarnara s. l. shows little variation at commonly investigated allozyme loci. However, interpretable variation was found at one esterase locus. All plants investigated of D. incarnata var. cruenta from southern Sweden (with spotted leaves) were homozygous for allele a at this locus, whereas all plants investigated of D. incarnata var. ochroleuca were fixed for allele b. In D. incarnata var. incarnata, both alleles were found, although the b allele dominated. In northern Swedish material of D. incarnata s. l., only allele b was found, regardless whether the material had spotted leaves (sometimes referred to as var. cruenta), or had unspotted leaves. These results indicate that there is restricted gene flow between var. cruenta and the other varieties in southern Sweden, although they often grow in mixed populations. The northern Swedish material with spotted leaves appears not to be related to the southern var. cruenta.
Article
Taxa endemic to North-western Europe are rare, but the orchid genusDactylorhiza contains several species restricted to this area. Evidence from morphological and cytological studies have indicated that some species may have arisen recently and may be of hybrid origin. In the present report, I use allozymes to characterize the genomes in various species ofDactylorhiza and evaluate the possibilities for rapid evolutionary change in the genus. Allotetraploid species have evolved repeatedly from two principal diploid ancestral lineages. These lineages include extant diploid and autotetraploid species, from which allotetraploid derivatives may still arise. It is suggested that allotetraploidization dominates over introgression as speciation mechanism in the genus. The more common and widespread allotetraploid species could be characterized by their allozyme characters over considerable distances, indicating that each of them may have a unique origin and that they have spread from their ancestral populations to the present distribution areas. However, it is also possible that some allotetraploid species contain local populations that have been independently derived from the ancestral lineages.
Article
Ten species of orchid plants belonging to the generaOrchis (7),Dactylorhiza (2), andGymnadenia (1) were analyzed by enzyme electrophoresis. Each species can be identified by a combination of enzyme bands different from those of all other species examined. The electrophoretic data were used for the construction of phenetic and phylogenetic trees with the help of computer programs. The trees were almost identical regardless which method was used. Our results differ considerably from a classification based on morphological evidence. The electrophoretic data indicate that the genusOrchis is not a monophyletic group.
Article
Relative mobilities (Rm's) of peroxidase and acid phosphatase isozymes were examined in leaves of flax (Linum usitatissimum L.). The leaves were sampled from four equidistantly spaced positions from main stem base to apex in various genotypes. Rm's for the two slowest-migrating isozymes of each enzyme system changed in a simple, coherent fashion in leaves from stem bases toward apices. The Rm trends up the stem seen in two highly branched flax types were somewhat different from those in two sparsely branched types. The coherent Rm trends in the four types, suggesting a smooth continuum and a potentially large number of slightly different forms of these isozymes, are discussed in relation to other data for such Rm trends. In the study reported here, both enzyme systems behaved similarly. This fact and the simple Mendelian genetical system with no codominance controlling Rm's in flax peroxidases and acid phosphatases suggest posttranscriptional or posttranslational modifications as plausible mechanisms underlying the numerous, presumably small molecular changes generating the small, consistent changes in Rm's.
Ubersicht iiber die Bastarde schweizerischer Orchideen mit Beriicksichtigung der moglichen Kombinationen
  • H R Reinhard
Reinhard, H. R. 1967. Ubersicht iiber die Bastarde schweizerischer Orchideen mit Beriicksichtigung der moglichen Kombinationen. -Ber. Schweiz. Bot. Ges. 77: 103-127 & 2 enclosures.
Interspecific relationship of ten European orchid species as revealed by enzyme electrophoresis. -PI Starch gel electrophoresis of ferns: A compilation of grinding buffers, gel and electrode buffers, and staining schedules. -Amer
  • M Schlegel
  • G Steinbriick
  • K Hahn
  • B Rottger
Schlegel, M., Steinbriick, G., Hahn, K. & Rottger, B. 1989. Interspecific relationship of ten European orchid species as revealed by enzyme electrophoresis. -PI. Syst. Evol. 163: Soltis, D. E., Haufler, C. H., Darrow, D. C. & Gastony, G. J. 1983. Starch gel electrophoresis of ferns: A compilation of grinding buffers, gel and electrode buffers, and staining schedules. -Amer. Fern J. 73: 9-27.
Zusammenstellung aller bisher bekannten Bastarde der in Deutschland verbreiteten Orchideen
  • E Peitz
Peitz, E. 1972. Zusammenstellung aller bisher bekannten Bastarde der in Deutschland verbreiteten Orchideen. -In:
A hybrid swarm between the diploid Dactylorhiza fuchsii (Druce) So6 and the tetra-ploid D. purpurella (T. & T. A. Steph.) Sob in Durham
  • R M Lord
  • A J Richards
Lord, R. M. & Richards, A. J. 1977. A hybrid swarm between the diploid Dactylorhiza fuchsii (Druce) So6 and the tetra-ploid D. purpurella (T. & T. A. Steph.) Sob in Durham. -Watsonia 1 1 : 205-2 10. 81 9-828. 201: 31-55. 127-134.
Genetic differentiation, polyploidization and hybridization in northern European Dactylorhiza (Orchidaceae): evidence from allozyme markers
  • M Hedrtn
Zusammenstellung aller bisher bekannten Bastarde der in Deutschland verbreiteten Orchideen
  • E Peitz
  • K Senghas
  • H Sundermann
Guide des orchid 6es d'Europe, d'Afrique du Nord et du Proche-Orient
  • P Delforge