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Systematics |
2Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK; 3Department of Botany, Natural History Museum, Cromwell Road, London, SW7 5BD, UK
Received for publication November 6, 2003. Accepted for publication April 22, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: combined analysis • Dactylorhiza • microsatellites • Orchidaceae • systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 1989
, 1990a
, b
, 1991
, 1992
) based on previous classifications (Vermeulen, 1947
; Senghas, 1968
), contained a system in which the genus is divided into four sections and seven subsections. Many specialists (e.g., Delforge, 1995
) broadly accept the species placement of Averyanov's system but use informal "groups" instead of formal "sections" and "subsections."
Subsequent investigations of allozyme markers (e.g., Hedrén, 2002
) confirmed that some species of Dactylorhiza are diploid (e.g., D. fuchsu and D. incarnata), at least one other is apparently autopolyploid (D. maculata sensu stricto [s.s.]), and a third group of species has an allotetraploid origin (D. majalis and others). Recent molecular studies, based on internal transcribed spacer (ITS) sequencing (Bateman et al., 1997
, 2003
) and amplified fragment length polymorphism (AFLP) analysis (Hedrén et al., 2001
; Hedrén, 2002
), have shown that the genus is sister to Gymnadenia sensu lato (s.l.) (Bateman et al., 1997
) and consists of five groups (Bateman and Denholm, 2003
; Bateman et al., 2003
): "Dactylorhiza incarnata group," including D. euxina and D. umbrosa (diploids); "Dactylorhiza maculata group," including D. fuchsii, D. saccifera, D. foliosa (diploids), and D. maculata (autotetraploid); "Dactylorhiza majalis group," including the allotetraploid species D. traunsteineri, D. baltica, D. russowii, D. praetermissa, D. purpurella, and associated infraspecific taxa; "Dactylorhiza sambucina group," including D. romana and D. flavescens (all diploid); and putatively "primitive" diploids, such as Dactylorhiza aristata, D. viridis (= Coeloglossum viride), and D. iberica.
The number of species (12–75) recognized varies significantly among authors (Pedersen, 1998
). Their underlying taxonomic concepts differed, and many species are poorly defined. Some authors, for example, accepted morphologically different allotetraploid forms as different species (Averyanov, 1988, 1989, 1990a, b, 1991, 1992; Tyteca and Gathoye, 1999,
2000
), but others (Bateman and Denholm, 1983
; Hedrén et al., 2001
) insist that most of them are subspecies (or even varieties) of D. majalis s.l. Another example is D. "cruenta,"; which we treated only as a form of D. incarnata in this study. This situation can be explained by a high frequency of hybridization and polyploidization events (Vermeulen, 1947
; Heslop-Harrison, 1968
) and significant polymorphism of most morphological characters widely used in diagnoses.
Smoljaninova (1976)
listed nine species of Dactylorhiza from European Russia, whereas Averyanov (1988
, 1989
, 1990a
, b
, 1991
, 1992
) recognized 14 species plus a further eight from the Russian part of the Caucasus. Averyanov united these species in "species aggregata," six in European Russia and five in the Caucasus. According to Cherepanov (1995)
, there are 13 species of Dactylorhiza from European Russia and six from the Russian Caucasus. Later, Averyanov (2000)
accepted only seven species from European Russia.
The main taxonomic problems of Russian dactylorchids are similar to those previously elucidated for western Europe: (1) relations within and between members of the Dactylorhiza maculata complex, (2) taxonomic status of allotetraploids (notably D. traunsteineri s.l. and D. baltica) and their origin, and (3) taxonomic status of different forms of the D. incarnata aggregate. Here we use the separate names for members of the D. maculata complex, and where we state D. maculata, we are referring to the narrowly defined autotetraploid rather thatn the whole complex (i.e., including D. fuchsii and other diploids).
Unfortunately, there are fewer taxonomic studies of dactylorchids in Russia than in western Europe. Most investigators have used the so-called "classical" method, based on analysis of herbarium specimens, which over the last 30 years has been challenged by the adoption of morphometric approaches and more recently by the advent of multivariate statistical methods. Fortunately, molecular methods are also fully quantitative and therefore can be used in conjunction with morphometry. For Russian material, morphometric studies are rare, and molecular analyses have never been performed. The goal of our work was to establish a morphometric and molecular framework for an extensive investigation of European Russian Dactylorhiza, simultaneously utilizing both morphological and molecular data in the "demographic" approach advocated by Bateman (2001; i.e., limits of taxa are established by looking at variation within and between populations)
.
Choice of molecular marker
Plastid DNA is widely used in molecular phylogenetic and phylogeographic studies. These regions evolve relatively slowly and hence can help to determine patterns of geographical distribution, although some polymorphic regions have proved useful for distinguishing among closely related species and even populations (Soliva and Widmer, 1999
; Fay and Cowan, 2001
). Plastid DNA also offers the ability to determine the maternal parent of an allopolyploid because orchids have uniparental (maternal) inheritance of plastids (Corriveau and Coleman, 1988
).
The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is another valuable tool for understanding infrageneric taxonomy in orchid systematics (Bateman et al., 1997
, 2003
), but polyploidy, hybridization, and gene conversion (Franzke and Mummenhoff, 1999
; Chase et al., 2003
) in Dactylorhiza can lead to undesirable complexity (Bateman et al., 2003
). On the other hand, ITS offers the possibility of elucidating hybrid origins and maternity when combined with plastid sequences (e.g., Bateman and Hollingsworth, 2004
). In this study, we use a combined approach based on all three data types: morphometric, plastid haplotypes (from four regions), and ITS alleles (assessed on the basis of two length variable regions).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), but without an RNA treatment. Polymerase chain reactor (PCR) was performed with a set of primers (Y. Pillon, M. F. Fay, and M. W. Chase, unpublished data) designed to amplify four polymorphic plastid loci (Table 2) in three plastid DNA regions: the trnS-trnG spacer, the trnL intron, and the trnL-trnF spacer.
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, but with the addition of dimethyl sulfoxide (DMSO) in the PCR reactions to reduce problems in primer annealing caused by the typical secondary structure (stems and loops) of ribosomal DNA. Sequence editing and assembly were done with Sequence Navigator and AutoAssembler (Applied Biosystems [ABI] Warrington, Cheshire, UK). Alignment was performed by eye because the level of sequence divergence was low and insertions/deletions (indels) were rare. As a result, we observed that Dactylorhiza maintains several ITS alleles (Bateman et al., 2003
) that differed from one another by both base substitutions and characteristic indels; these indels alone are good markers for these alleles (Fig. 1). Two pairs of primers were designed to amplify length-polymorphic regions and permit us to detect the presence of the different ITS alleles (Table 3). Thus, the characterization of particular ITS alleles was first accomplished via direct sequencing of PCR products containing these amplified regions and cloning if more than one allele was present, but then in the later stages by amplification of the two length-variable fragments described above, which permitted us to determine which alleles were present in each plant accession.
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). This network was visualized using Graphviz version 1.10 (Gansner and North, 2000
).
Morphometric material
Many previous investigators have used a set of morphological characters in alliance with multivariate statistical analysis for characterization of Dactylorhiza accessions (Bateman and Denholm, 1983
, 1985
, 1989
, 2003
; Reinhard, 1990
; Dufrêne et al., 1991
; Pedersen, 1998
; Tyteca and Gathoye, 1999
, 2000
; Foley, 2000
). Principal components analysis (PCA) was used in a majority of these studies and also several more recent papers in which morphological and DNA data were combined at the population level, as was recently done for pit vipers (Puorto et al., 2001
). Principal components analysis is a well-known technique that uses eigenvalue matrix calculations to visualize multidimensional data and compute loadings for each character. Another useful approach is principal coordinates analysis (PCoA), which uses a distance matrix instead of the data matrix for PCA. We chose 14 morphological characters (Table 4), most of which were measured in nature on the same plants subsequently used for DNA extractions (in a few cases we measured neighboring plants occurring in the same population). We analyzed these data using both PCA and multidimensional scaling (MDS, the expanded variant of PCoA). All statistical calculations used the R program, version 1.71 for Windows and Linux (Venables et al., 2002
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There is a clear association between species and haplotype, with only a few exceptions. Most northern Russian D. maculata specimens share their haplotype with D. fuchsii and do not have haplotype B (or N or X), which is typical for D. maculata from central Russia and western Europe. All samples collected as "D. cruenta" have haplotype E, which is typical of D. incarnata. Haplotype H (initially found in Georgian material) was also found in several D. incarnata samples from central Russia. Dactylorhiza saccifera (= D. amblyopoda) and some of the D. flavescens samples from western Caucasus have haplotypes (G and R3, respectively) typical of these species. Dactylorhiza viridis (= Coeloglossum viride) samples have haplotypes V3 and V4.
Our putative tetraploid specimens (D. traunsteineri, D. russowii, and D. baltica) had the A haplotype most commonly found in D. fuchsii. Some northern plants with the A and Q haplotypes (collected as either "D. maculata" or "D. fuchsii") could also in fact be allotetraploids (see Discussion).
ITS
The data from nuclear ITS fragments are much more diverse, as expected for a DNA region with biparental inheritance (Table 5). In addition, tetraploids can possess up to four alleles, and we observed the conversion of the ITS allele to the parental type in many plants (some fragments were much lower in intensity than would otherwise have been expected in hybrids). We again immediately noted that the distribution of ITS alleles and previously used species concepts (Table 1) were highly correlated.
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Morphology
Both PCA and MDS of the morphological data revealed similar structure that corroborates haplotype distribution (Fig. 3); the samples form four groups. Most samples belong to group I in the upper left (one member of this group falls into group II), which contains D. maculata, D. fuchsii, and D. traunsteineri samples with haplotypes A, Q, and RU1, mostly from the Russian North. Others are group II in the lower left (one member falls into group IV), which contains D. incarnata with haplotype E; group III with two subgroups in upper right and center of the ordination, which contains D. maculata with haplotypes B and X; and group IV in lower right, which contains D. fuchsii, D. baltica, and putative allotetraploids with haplotype A, most from central Russia.
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With the aim of understanding morphological differences between these groups, we analyzed morphometric information from samples with the A haplotype by discriminant analysis and regression trees. Discriminant analysis of the morphological characters supported both group I and group IV with 100% probability (
2= 36, P << 0.01). The most important discriminant characters were (a) leaf width and (b) plant height. Regression trees (Breiman et al., 1984
) can describe the character values that predict classification, and in this case leaf width ("less than" vs. "more than 22.5 mm") clearly divides the existing samples.
Geographic patterns
Plastid data
We found a significant geographical pattern of haplotype distribution from south to north (Kruskal-Wallis 2 = 21.4529, P << 0.05). The distribution of haplotypes in Russia also has similarities to patterns observed in the western European flora. For example, the dactylorchid flora of central Russia has some links with Sweden (haplotype X), but rarer haplotypes (N, Q) are distributed only in central Europe (Y. Pillon, M. F. Fay, and M. W. Chase, unpublished data). The Caucasian haplotype RU3 is similar to Turkish D. euxina haplotypes K and I, and RU4 is similar to the Mediterranean R3 haplotype.
The ITS data
When the ITS data were used for PCA analysis alone, the structure of the graph was consistent only with distribution by latitudinal zones (i.e., with the sites categorized according to latitude in 5° intervals: from 50° to 55°, 55° to 60°, and so on). We used linear regression analysis to estimate the relationship between latitudinal zone and heterogeneity within populations (the measure of heterogeneity was the standard deviation of fragment length types) as a dependent variable, and a significant relationship was found (F = 24.46 on 64 df, P << 0.05; see also Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) is probably an identification error because both populations of supposed D. romana sampled have the morphology, ITS alleles, and haplotype typical of D. flavescens. Thus, we conclude that D. romana does not exist in the Russian Federation, and its distribution in eastern Europe is restricted to the Crimea. In the light of the position of haplotype RU5 in the network, one can hypothesize that this specimen belongs to a group with characteristics of both D. incarnata and D. euxina. Hedrén (2001)
showed that some plants intermediate in morphology between D. incarnata and D. euxina from Turkey should be referred to a separate tetraploid species, D. armeniaca. The diagnosis of D. armeniaca is congruent with morphometric data from our specimen (voucher from Tuapse, Krasnodar region), so we have probably here the first record of this species in Russia.
Dactylorhiza maculata and D. fuchsii from northern Russia are difficult to distinguish (L. Averyanov, Botanical Institute, Saint-Petersburg, personal communication), so the possession of A, Q, and RU1 haplotypes (all from the D. fuchsii group; Fig. 2) for all of these plants requires an explanation. Plants initially identified as D. traunsteineri (an allotetraploid) from these sites have similar morphologies and genotypes (some Scandinavian D. traunsteineri specimens also have the A haplotype: Y. Pillon, M. F. Fay, and M. W. Chase, unpublished data). Most of these plants grow in Sphagnum bogs, the typical habitat of D. maculata. On the other hand, the morphology of these specimens differs from that of typical D. maculata (Fig. 3). The discriminant and regression analyses show that morphologically our specimens with the A haplotype are clearly divided into northern plants with narrow leaves (group I; Figs. 3, 5) and southern plants with broad leaves (group IV). The ITS alleles from the first group are those of both D. fuchsii and D. maculata. All of these northern plants are tetraploids according to several cytological observations (Averyanov, 1990a
). This leads us to speculate that most northern Russian specimens with A, Q, and RU1 haplotypes are allotetraploids formed by D. maculata and D. fuchsii, probably with the participation of unreduced gametes from the D. fuchsii parent (Ramsey and Schemske, 1998
).
The C haplotype, found in many of the western European and Turkish allotetraploids (Y. Pillon, M. F. Fay, and M. W. Chase, unpublished data), is absent from Russia. Many of these plants also have the D. incarnata ITS allele. Our D. russowii (= D. traunsteineri s.l.) specimen from South Karelia has the D. incarnata allele but combined with the haplotype A. Other putative allotetraploids (including several specimens referred to D. baltica) with D. incarnata alleles also have the A haplotype and are morphologically similar to typical D. fuchsii (group IV; Fig. 3). However, this tentative conclusion could reflect the limited range of morphometric characters used.
Two group III samples are distant from the remainder on the PCA plot (Fig. 3). The basis for such a position is their wide lip, but this character is often found in typical D. maculata (Bateman and Denholm, 1989
). The ITS alleles of all specimens in group III are similar (Table 5).
Dactylorhiza "cruenta" is now often accepted as a form of D. incarnata (e.g., Bateman and Denholm, 1985
; Hedrén et al., 2001
), a view supported by our data. This "taxon" only has the E haplotype and an ITS allele consistent with D. incarnata; it also belongs to the same group (group II; Fig. 3) as D. incarnata. This group is homogeneous with two exceptions: first (the point in the right top of the group) is the "northern tetraploid" with extremely small flowers, but all other plants measured (but not sampled for DNA) from this population have flowers typical of group I. The second exception (the point on the bottom of the graph) is a mammoth plant of D. incarnata with unusually large flowers, but again all other plants from this population have typical D. incarnata morphology. Both of these outliers fall in their expected groups in the analysis containing all three data matrices (Fig. 5).
We did not find heterogeneity in plastid haplotypes within populations, even though for more than a half of them several samples were examined. The intrapopulation diversity of ITS alleles is considerable and shows a clear south–north geographic cline (Fig. 6), with the latter being significantly more heterogeneous.
The correspondence of the morphological PCA graph to haplotypes and a priori delimited species clearly shows that the morphological data correlate with plastid haplotypes better than with standard taxonomic circumscriptions that have been used for species description. This also indicates that characters used in descriptions and keys for this species require further revision. The agreement between different categories of data is greater in simultaneous analysis of morphology and haplotypes than for morphology and ITS data, which supports the use of plastid data as a good species marker in this group of plants in spite of its strictly maternal inheritance. Most workers would not expect plastid DNA patterns and morphological variation to be better correlated than a biparentally inherited region like ITS, but the congruence of plastid haplotypes and morphology in this case could be due to the fact that the location of plants during the last glacial maximum (their refugium) is more important to their morphological characteristics than their present location and with which other plants they are interacting at present.
This study is the first of which we are aware in plants to sample DNA and measure morphological characters for the same individuals. Because of the high level of congruence between the two, relatively robust statements can be made about which characters are reliable for distinguishing taxa and which are not helpful for these purposes. In addition, a strong case is made for collecting morphological data from plants destined to be used in molecular studies and for combining these categories of data to produce clearer ideas about how taxa should be circumscribed and identified in the field.
The geographical patterns revealed by this study of plastid DNA are straightforward because most of European Russia has a typical postglacial flora (e.g., Tikhomirov et al., 1987– 1988
), which means that immigration was likely from refugia in western Europe and/or the Caucasus and Crimea. The clear correlation between latitude and heterogeneity of the ITS data demonstrates that either ITS gene conversion in northern regions of European Russia is much slower or (more probably) the time since hybridization occurred in northern Dactylorhiza populations is less than in southern populations.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Averyanov L. V. 1989 A review of genus Dactylorhiza Neck. ex Nevski (Orchidaceae), 2. Novosti sistematiki vysshikh rastenij 26: 47-56 [In Russian.]
Averyanov L. V. 1990a A review of genus Dactylorhiza Neck. ex Nevski (Orchidaceae), 3. Novosti sistematiki vysshikh rastenij 27: 32-62 [In Russian.]
Averyanov L. V. 1990b A review of the genus Dactylorhiza. In J. Arditti (ed.), Orchid biology: reviews and perspectives, 159–206. Timber Press, Portland, Oregon, USA
Averyanov L. V. 1991 A review of genus Dactylorhiza Neck. ex Nevski (Orchidaceae), 4. Novosti sistematiki vysshikh rastenij 28: 33-42 [In Russian.]
Averyanov L. V. 1992 A review of genus Dactylorhiza Neck. ex Nevski (Orchidaceae), 5. Novosti sistematiki vysshikh rastenij 29: 14-25 [In Russian.]
Averyanov L. V. 2000 Orchids (Orchidaceae) of Middle Russia. Turchaninovia 3: 30-53 [In Russian.]
Bateman R. M. 2001 Evolution and classification of European orchids: insights from molecular and morphological characters. Journal Europäischer Orchideen 33: 33-119
Bateman R. M. I. Denholm 1983 A reappraisal of the British and Irish dactylorchids, 1. The tetraploid marsh-orchids. Watsonia 14: 347-376
Bateman R. M. I. Denholm 1985 A reappraisal of the British and Irish dactylorchids, 2. The diploid marsh-orchids. Watsonia 15: 321-355
Bateman R. M. I. Denholm 1989 A reappraisal of the British and Irish dactylorchids, 3. The spotted orchids. Watsonia 17: 319-349
Bateman R. M. I. Denholm 2003 The heath spotted-orchid (Dactylorhiza maculata (L.) Soó) in the British Isles: a cautionary case-study in delimiting infraspecific taxa and inferring their evolutionary relationships. Journal Europäischer Orchideen 35: 3-36
Bateman R. M. P. M. Hollingsworth In press Morphological and molecular delimitation of Anacamptis (Orchidaceae: Orchidineae), with particular reference to A. x albuferensis (A. fragrans x A. robusta), a new hybrid from Mallorca, Spain. Taxon 53: 43-54
Bateman R. M. P. M. Hollingsworth J. Preston Y.-B. Luo A. M. Pridgeon M. W. Chase 2003 Molecular phylogenetics and evolution of Orchidinae and selected Habenariinae (Orchidaceae). Botanical Journal of Linnean Society 142: 1-40[CrossRef]
Bateman R. M. A. M. Pridgeon M. W. Chase 1997 Phylogenetics of subtribe Orchidineae (Orchidoideae, Orchidaceae) based on nuclear ITS sequences. 2. Infrageneric relationships and reclassification to achieve monophyly of Orchis sensu stricto. Lindleyana 12: 113-141
Breiman L. J. H. Friedman R. A. Olshen C. J. Stone 1984 Classification and regression trees. Chapman and Hall, New York, New York, USA
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Cherepanov S. K. 1995 Vascular plants of Russia and adjacent territories. Mir i Semya, St.-Petersburg, Russia. [In Russian.]
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Fay M. F. R. S. Cowan 2001 Plastid microsatellites in Cypripedium calceolus (Orchidaceae): genetic fingerprints from herbarium specimens. Lindleyana 16: 151-156
Foley M. J. Y. 2000 Dactylorhiza incarnata (L.) Soó subsp. ochroleuca (Wüstnei ex Boll) P. F. Hunt and Summerh. (Orchidaceae): a comparison of British and European plants. Watsonia 23: 299-303
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Heslop-Harrison J. 1968 Genetic system and ecological habit as factors in dactylorchid variation. Jahresberichte des Naturwissenschaftlichen Vereins in Wuppertal 21: –22 20-27
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Puorto G. M. G. Salomao R. D. G. Theakston R. S. Thorpe D. A. Warrell W. Wuester 2001 Combining mitochondrial sequences and morphological data to infer species boundaries: phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae). Journal of Evolutionary Biology 14: 527-538[CrossRef][ISI]
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Tyteca D. J.-L. Gathoye 2000 Morphometric analysis of the Dactylorhiza majalis group in France and western Europe with description of Dactylorhiza parvimajalis Tyteca et Gathoye, spec. nov. Journal Europäischer Orchideen 32: 471-511
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Zernov A. S. 2002 Diagnostic book of north-western Trans-Caucasus plants. KMK Scientific Press, Moscow. [In Russian.]
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