Genetic diversity and differentiation of allopolyploid Dactylorhiza (Orchidaceae) with particular focus on the Dactylorhiza majalis ssp. traunsteineri/lapponica complex

Genetic differentiation of Dactylorhiza majalis ssp. traunsteineri from the Alps, Scandinavia, and Britain was studied and compared with other allotetraploid members of the systematically challenging genus Dactylorhiza . One-hundred and eleven populations from altogether 18 taxa were analysed for eight polymorphic plastid markers and two size-variable fragments from the nuclear internal transcribed spacer (ITS) region. In total, 60 plastid haplotypes and six ITS alleles were found among the 737 individuals analysed. No clear differentiation between populations of ssp. traunsteineri from the three regions was revealed. However, ssp. traunsteineri was genetically differentiated from Dactylorhiza baumanniana , Dactylorhiza elata , and D. majalis ssp. sphagnicola , although the majority of allotetraploid taxa remained inseparable. Judging from the degree of concerted evolution in ITS, D. majalis ssp. alpestris may be regarded as a relatively old allotetraploid, whereas ssp. baltica and ssp. purpurella may be considerably younger. Based on plastid data, the Alp region had the highest genetic diversity followed by Scandinavia and Britain. The geographic distribution of haplotypes provided support for possible refugial areas around the Alps and for several independent immigration routes into Scandinavia after the last ice age.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 52–67.     

Relationships in Patagonian species of Berberis (Berberidaceae) based on the characterization of rDNA internal transcribed spacer sequences

Sequence analysis of the internal transcribed spacer (ITS) of the 18S(ITS1)-5.8S-26S(ITS2) rDNA region was performed in order to analyse the phylogenetic relationships between 13 Patagonian species of the genus Berberis (Berberidaceae). The divergence values between the pairwise sequence in the studied Patagonian species were in the range 2.9–22.9%. The lengths of the ITS1 and ITS2 sequences were in the range 227–231 bp and 220–224 bp, respectively, and the 5.8S sequence was 159 bp throughout all species . B. microphylla sensu Landrum does not appear to be monophyletic based on current sampling. Indeed, we suggest that B. microphylla should be distinguished from B. buxifolia , B. parodii , and B. heterophylla . ITS sequences, together with data obtained from morphological, biochemical, amplified fragment length polymorphism, and cytological characterizations, support the existence of diploid and polyploid hybrid speciation in the genus.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 321–328.     

Molecular and morphological evidence for hybridization between endemic Canary Island Convolvulus

Morphological data and molecular data from the chloroplast trn H- psb A region and nuclear ribosomal ITS region are used to test the hypothesis that the problematic Canary Island endemic Convolvulus floridus var. virgatus is a hybrid between the endemic species C. floridus and C. scoparius . Analysis of mean leaf length and width of 58 individuals indicates that accessions referable to C. floridus var. virgatus are intermediate between the parental taxa in leaf dimensions. Direct sequencing of the ITS region of C. scoparius and C. floridus revealed two species-specific ribotypes distinguished by 10 base differences. Examination of ITS chromatograms for putative hybrids revealed polymorphisms at those sites that are diagnostic between species in all except one putative hybrid. Morphological intermediacy and ITS additivity therefore support the hybrid status of C. floridus var. virgatus . An analysis of intraindividual ITS variation confirmed the co-occurrence of both parental ribotypes in putative hybrids and demonstrated that some hybrid individuals contained chimaeric ITS types. It is proposed that chimaeric ITS types are the result of recombination following backcrossing. Sequencing of the trn H- psb A region revealed four haplotypes. Three were, for the most part, confined to C. floridus and putative hybrids although one C. scoparius accession was also found to share this haplotype. The remaining haploype was only found in C. scoparius and putative hybrid accessions. Patterns of haplotype distribution between parental and hybrid accessions suggest multiple, bidirectional hybridization events between C. floridus and C. scoparius . The nomenclature of the hybrid is discussed and the necessary new name C.  ×  despreauxii is proposed.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 187–204.     

A revision and phylogenetic analysis of Stapeliopsis (Apocynaceae)

A survey of morphological characters is carried out for Stapeliopsis . The information obtained from this is combined with molecular data from the plastid trn L-F DNA region and ITS1 of the nuclear encoded 18S−26S rRNA cistron, to obtain a hypothesis of the evolutionary relationships among the species. It is shown that Stapeliopsis is monophyletic in a combined molecular and morphological analysis. Stapeliopsis is sister to a clade containing Huernia , Orbea and Tromotriche . The species of Stapeliopsis group into two clades. One contains S. khamiesbergensis , S. neronis and S. urniflora , and this is highly supported. The remaining species fall into an unsupported clade in which S. exasperata is sister to the others. The genera Hermanschwartzia Plowes and Neopectinaria Plowes are rejected. It is shown that a synapomorphy for Stapeliopsis is the laterally flattened inner corona-lobes, which touch the anthers only at their bases. Eight species of Stapeliopsis are recognized, with no subgeneric divisions.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 125–155.     

Intraspecific divergences of Rhodiola alsia (Crassulaceae) based on plastid DNA and internal transcribed spacer fragments

In the present study, we used two maternally inherited plastid DNA intergenic spacers, rpl20‐rps12 and trnS‐trnG, and the biparentally inherited nuclear ribosomal internal transcribed spacer (ITS) region to explore genetic variation and phylogeographical history of Rhodiola alsia, a herb endemic to the Qinghai‐Tibetan Plateau (QTP). Based on range‐wide sampling (18 populations and 227 individuals), we detected 45 plastid DNA haplotypes and 19 ITS sequence types. Only three plastid DNA haplotypes were widespread; most haplotypes were restricted to single sites or to neighbouring populations. Analysis of molecular variance revealed that most of the genetic variance was found within populations (51.24%) but that populations were also distinct (F_ST = 0.48759). We found three areas with relatively high plastid DNA diversity and these could further be recognized as potentially isolated divergence centres based on the ITS sequence type distribution. These represent three potentially isolated glacial refugia for R. alsia: one of them has long been recognized as an important refugium on the south‐eastern edge of the QTP, whereas the others are new and located in the north and south of the Tanggula Mountains on the plateau platform. Divergence time estimates based on ITS suggest that the main lineages of R. alsia diverged from each other 0.35–0.87 Mya, indicating that climatic oscillations during the Pleistocene may have been an important driver of intraspecific divergence in R. alsia. Rhodiola alsia probably experienced a phylogeographical history of retreat to isolated glacial refugia during Quaternary glaciations that led to different degrees of allopatric intraspecific divergence. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 204–215.     

Comparison of three organelle markers for phylogeographic inference in Batrachospermum helminthosum (Batrachospermales, Rhodophyta) from North America

Phylogeographic signal provided by the newly developed 23S plastid rRNA marker (universal plastid amplicon, UPA) and the cytochrome oxidase subunit I gene marker (COI) in the freshwater red alga Batrachospermum helminthosum, throughout its range in North America, was investigated. These markers were compared in individuals from a previous study using the cytochrome oxidase 2–3 spacer region ( cox 2–3), which has yielded the most useful data to date with 13 haplotypes among geographic locations. Five haplotypes were resolved for the UPA, differing by only one to two base pairs (bp), and we conclude that this marker may be more appropriate for studying interspecific variation. In contrast, the COI gene revealed 16 haplotypes, differing from one to 44 base pairs or up to 6.6% sequence variation. The intraspecific variation of COI in this taxon is much greater than that reported thus far for marine red algae (generally <5 bp). The intraspecific variation within B. helminthosum is in accord with levels shown in Batrachospermum macrosporum (48 bp within distant locations in Brazil). The COI gene is comparable to the cox 2–3 spacer for phylogeographic studies as the haplotype networks were similar and showed the same geographic patterns. To our knowledge, this is the first comparison of these three regions for phylogeographic research in the red algae.     

Molecular variation and speciation in Antitrichia curtipendula s.l. (Leucodontaceae, Bryophyta)

Molecular variation in Antitrichia curtipendula (Hedw.) Brid. s.l. was studied based on the nuclear internal transcribed spacer (ITS) and the chloroplast markers trn L- trn F and rpl 16, and analysed by neighbour joining (for ITS; recombination present), maximum parsimony (chloroplast markers) and TCS (haplotype network). Old World plus E North American populations belong to a different lineage than those of W North America. These are molecularly well differentiated and are treated as A. curtipendula and A. gigantea (Sull. & Lesq.) Kindb. Two distinct groups of Old World haplotypes are separated by one 'missing' haplotype and are interpreted as cryptic species. Tropical African populations share one ITS deletion and form a lineage within one of the cryptic species. Molecular variation within A. gigantea , within each of the two cryptic Old World plus E North American (except tropical African) haplotype groups, and among tropical African populations are of similar magnitude, suggesting that analogous mechanisms and similar time spans explain the found variation. Events related to Pleistocene climatic oscillations are suggested as having caused this differentiation within each group, whereas the African lineage was probably split off before this. Identical tRNA-Gly sequences were found in 33 specimens; new primers were designed for rpl 16 and ITS 1+2.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 341–354.     

Taxonomic status of two South American sympatric squat lobsters, Munida gregaria and Munida subrugosa (Crustacea: Decapoda: Galatheidae), challenged by DNA sequence information

We investigated the taxonomic status of two sympatric morphospecies of squat lobsters from southern South America (Beagle Channel, Strait of Magellan, and Burdwood Bank), Munida gregaria and Munida subrugosa , by DNA sequence analysis of three mitochondrial (mt)DNA gene fragments [416 bp of 16S rDNA(165), 566 bp of cytochrome c oxidase subunit I(COI) and 418 bp of NADH dehydrogenase subunit 1 (ND1)]; and the nuclear rDNA internal transcribed spacer (ITS) 1 (883–952 bp). We obtained a total of 79 sequences from 32 individuals. The 16S sequences of all M. gregaria and M. subrugosa were invariant and identical, whereas COI and ND1 showed 12 and 15 variable sites, respectively. These polymorphisms were shared between morphospecies. Interspecific Tamura–Nei distances for COI and ND1 sequences were 0.0024 and 0.0032, respectively, and were not significantly different from intraspecific distances (Kruskal–Wallis tests: P  = 0.58 and P  = 0.69, for COI and ND1, respectively). Similar to the results obtained from the mtDNA sequences, no relationship was found between the ITS1 maximum parsimony tree topology and the morphologic classification of specimens in M. gregaria and M. subrugosa . We conclude that M. gregaria and M. subrugosa from southern South America may either represent a case of a dimorphic species, or a case of incomplete lineage sorting. The fact that these two morphospecies did not show fixed differences over a total of 1947 bp analysed reinforces the hypothesis of a single dimorphic species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 421–434.     

Potential refugium on the Qinghai–Tibet Plateau revealed by the chloroplast DNA phylogeography of the alpine species Metagentiana striata (Gentianaceae)

Metagentiana striata is an alpine annual herbaceous plant endemic to the east of the Qinghai–Tibet (Q–T) Plateau and adjacent areas. The phylogeography of M. striata was studied by sequencing the chloroplast DNA (cpDNA) trn S– trn G intergenic spacer. Ten haplotypes were identified from an investigation of 232 individuals of M. striata from 14 populations covering the entire geographical range of this species. The level of differentiation amongst populations was very high ( G _ST = 0.746; N _ST = 0.774) and a significant phylogeographical structure was observed ( P  < 0.05). An analysis of molecular variance found a high variation amongst populations (76%), with F _ST = 0.762 (highly significant, P  < 0.001), indicating that little gene flow occurred amongst the different regions; this was explained by the isolation of populations by high mountains along the Q–T Plateau and adjacent areas ( N _m = 0.156). Only one ancestral haplotype (A) was common and widespread throughout the distributional range of M. striata. The populations of the Hengduan Mountains region of the south-eastern Q–T Plateau showed high diversity and uniqueness of haplotypes. It is suggested that this region was the potential refugium of M. striata during the Quaternary glaciation, and that interglacial and postglacial range expansion occurred from this refugium. This scenario was in good agreement with the results of nested clade analysis, which inferred that the current spatial distribution of cpDNA haplotypes and populations resulted from range expansion, together with past allopatric fragmentation events.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 125–140.     

Geographical diversification of the genus Cicer (Leguminosae: Papilionoideae) inferred from molecular phylogenetic analyses of chloroplast and nuclear DNA sequences

Cicer L. (Leguminosae: Papilionoideae) consists of 42 species of herbaceous or semi-shrubby annuals and perennials distributed throughout the temperate zones of the Northern Hemisphere. The origin and geographical relationships of the genus are poorly understood. We studied the geographical diversification and phylogenetic relationships of Cicer using DNA sequence data sampled from two plastid regions, trnK / matK and trnS - trnG , and two nuclear regions, the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions of nuclear ribosomal DNA, from 30 species. The results from the phylogenetic analyses of combined nuclear and chloroplast sequence data revealed four well-supported geographical groups: a Middle Eastern group, a West-Central Asian group, an Aegean–Mediterranean group, and an African group. Age estimates for Cicer based on methods that do not assume a molecular clock (for example, penalized likelihood) demonstrate that the genus has a Mediterranean origin with considerable diversification in the Miocene/Pliocene epochs. Geological events, such as mountain orogenesis and environmental changes, are major factors for the dispersal of Cicer species. The early divergence of African species and their geographically distinct region in the genus suggest a broader distribution pattern of the genus in the past than at present.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 154 , 175–186.     

Systematics of the tribe Podalyrieae (Fabaceae) based on DNA, morphological and chemical data

Phylogenetic relationships among nine genera and 28 species of the southern African tribe Podalyrieae were estimated from sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA as well as morphological and chemical data. Morphological and ITS sequence data produced cladograms with similar topologies, both supporting the monophyly of Podalyrieae (excluding Hypocalyptus ). The combined data sets indicate that subtribe Xiphothecinae are monophyletic, but embedded within Podalyriinae. The high degree of congruence between previous taxonomic hypotheses and those based on DNA data provides further evidence for the utility of ITS sequences in studying phylogeny.  © 2002 The Linnean Society of London , Botanical Journal of the Linnean Society , 2002, 139 , 159–170.     

A new Pyrenean hybrid Cirsium (Asteraceae) as revealed by morphological and molecular analyses

A new nothosubspecies Cirsium  ×  vivantii is described after a molecular and morphological characterization of eight individuals collected in two separate valleys of the French central Pyrenees. Both hypervariable Amplified Fragment Length Polymorphism (AFLP) markers and nuclear rDNA (ITS) and plastid ( trn L-F, TRNT-L) DNA sequences were analysed. The profiles of these hybrid samples were compared to those of 43 individuals belonging to their presumed parental taxa C. carniolicum ssp. rufescens and C. palustre . A total of 133 AFLP bands were scored from three primer-pair combinations. All 130 AFLP bands that amplified in the hybrid samples were present in either C. carniolicum ssp. rufescens , C. palustre or both taxa, supporting the participation of these plant genomes in the resulting hybrids. Several Additive Polymorphic Sites (APS) detected in the ITS sequences of the hybrid samples also confirmed their derived origins from ribotypes of the two parental taxa. The lack of exclusive AFLP markers and the nonconcerted evolution of rDNA polymorphisms towards either of the parental ribotypes indicated their recent origin. Plastid trn T-L sequences were used to identify C. palustre as the maternal parent of six of these hybrid individuals; either parent could have acted as the plastid genome donor for the other two individuals. The morphological study revealed that all hybrid individuals were morphologically intermediate between their parents showing largely lobed and less spiny basal leaves as in C. carniolicum ssp. rufescens and decurrent leaf bases and pinkish corollas as in C. palustre .  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 421–434.     

Distinct geographical structure across species units evidenced by chloroplast DNA haplotypes and nuclear ribosomal ITS genotypes of Corylopsis (Hamamelidaceae) in the Japanese islands

The geographical distribution of chloroplast DNA (cpDNA) haplotypes and nuclear ribosomal internal transcribed spacer (nrITS) genotypes of Japanese Corylopsis (Hamamelidaceae), which consists of four species, was investigated. Two hundred and five individuals belonging to four species from 30 populations, covering the entire geographical range, were studied. Based on approximately 1108 bp of the three non-coding regions of cpDNA, nine haplotypes were detected, and each was distinguished from adjacent haplotypes by one substitution. Based on approximately 507-bp nrITS sequences, 47 genotypes were detected, for which three clades were identified in the phylogenetic analysis. There was inconsistency between the cpDNA haplotypes, nrITS genotypes, and classification of Corylopsis taxa, possibly because of incomplete lineage sorting or introgressive hybridization. The distribution of the haplotypes was highly structured geographically, and N _ST (0.893) was significantly greater than G _ST (0.819), implying that the current distribution of Corylopsis species was structured phylogeographically during Quaternary climatic oscillations. The haplotype composition and results of analysis of molecular variance showed that the populations in Hokuriku were highly divergent, suggesting that they are long-term persistent populations arising from refugia during the Quaternary climatic oscillations. Refugial populations in Chugoku and Shikoku may have lost genetic diversity because of a bottleneck resulting from a small population size, followed by post-glacial range expansion. Pre-existing refugia may have been so small that the subsequent range expansion replaced the pre-existing genetic structure.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 501–518.     

Genetic variability in the complete mitochondrial control region of the Eurasian otter (Lutra lutra) in the Iberian Peninsula

Sequencing of the complete mitochondrial DNA control region from 31 samples of the Eurasian otter, Lutra lutra , enabled us to establish the length and structure of this fragment, as well as to describe, for the first time, the RS3 repetitive region located at the 3' end. In addition, genetic variability of the 5' end was examined in 63 individuals, 57 of which were wild otters from the Iberian Peninsula and six captive reared otters. This analysis resulted in extremely low variability. All the samples from the Iberian Peninsula share a single haplotype, Lut 1, the most common haplotype in Europe. Captive otters showed two haplotypes: Lut 3, which has been described in wild otters from eastern Germany, and Lut 6, an haplotype not described to date. Higher variability was observed in the repetitive RS3 region. The tandem repeat was composed of an array of ten repeat units of 22 bp with differences in the repetitive motifs that differed in the arrays of different specimens. In total, 20 different haplotypes from 31 individuals were found. However, the geographical distribution of these haplotypes did not generate a phylogeographical signal. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 86 , 397–403.     

Inclusion of the Vietnamese endemic genus Grushvitzkya in Brassaiopsis (Araliaceae): evidence from nuclear ribosomal ITS and chloroplast ndhF sequences

The phylogenetic position of the recently described monotypic Vietnamese genus Grushvitzkya is evaluated using nuclear ribosomal ITS and chloroplast ndh F sequence data . Grushvitzkya stellata has often been considered as intermediate between Brassaiopsis and Trevesia , but differs in having a 5-locular ovary rather than a 2- or 6–16-locular ovary, respectively. The molecular data indicate that Grushvitzkya is deeply nested within Brassaiopsis and has a close relationship with B. phanrangensis from central and southern Indochina, and B. stellata from the regions bordering China and Vietnam. The Kimura 2-parameter distances of the ITS sequences between G. stellata and B. phanrangensis and B. stellata are 0.403 and 0.945%, respectively. Furthermore, G. stellata and B. phanrangensis have an identical ndh F sequence profile. Grushvitzkya stellata differs from B. stellata by a single nucleotide in their ndh F sequences. The available evidence is compatible with the hypothesis that Grushvitzkya diverged recently from B. phanrangensis and B. stellata . Although these three closely related species all possess stellate trichomes, G. stellata and B. phanrangensis have highly similar trichome micromorphology, with almost no difference in size, shape and branching pattern, supporting a very close relationship between the two taxa. The higher number of locules in the ovary of Grushvitzkya appears to be a secondary increase despite earlier interpretations that higher numbers are evolutionarily primitive. Grushvitzkya is herein merged with Brassaiopsis ; because the combination B. stellata is occupied, a new name, Brassaiopsis grushvitzkyi , is proposed.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 455–463.     

Island-like radiation of Saussurea (Asteraceae: Cardueae) triggered by uplifts of the Qinghai–Tibetan Plateau

Increasing evidence suggests that geological or climatic events in the past triggered the radiative diversification of both animals and plants on islands as well as continents. The Qinghai–Tibetan Plateau (QTP) has been extensively uplifted since the Miocene, but there is little information on possible links between these events and biological diversification in this and adjacent regions. Partly to explore such links, we have examined the diversification of Saussurea (Asteraceae: Cardueae), a species-rich genus that is mostly endemic to QTP, but also occurs in arid highlands elsewhere in the Northern Hemisphere. The phylogenetic analyses were conducted on the basis of the nuclear (internal transcribed spacer, ITS) and plastid ( trnL-F and psbA-trnH ) sequences from 55 species, representing 19 sections from all six subgenera of Saussurea , and species from 15 genera of the Cardueae. The results suggest that the currently circumscribed genus Saussurea ( s.l. ) is a polyphyletic group and that five sections should be excluded from the genus. Samples from the other 14 sections (representing five subgenera) clustered as a monophyletic group (here designated the Saussurea s.s. lineage, SSSL) with high statistical support. However, none of the analyses (nuclear, plastid or combined) resolved SSSL's infrageneric phylogeny, and the parallel clades of the lineage indicate that island-like adaptive radiation occurred. Furthermore, this radiation appears to have occurred 14–7 Mya, during the period of the major uplift events of QTP. Thus, our results support the hypothesis that geological events may play important roles in driving biological diversification through continental radiation.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 893–903.     

Mosaic variation in allozyme and plastid DNA markers in the European ranges of Silene vulgaris and its partially sympatric relative S. uniflora (Caryophyllaceae)

The perennial herbs Silene vulgaris and S. uniflora are closely related, partially sympatric and interfertile, yet morphologically distinct. We used nuclear (allozyme) and plastid (polymerase chain reaction–restriction fragment length polymorphism) DNA markers to investigate whether these species have a shared history of postglacial colonization and hybridization in Europe, as inferred from large‐scale patterns of geographic variation. The majority of plastid haplotypes and allozyme alleles were widespread and patchily distributed within both species and there was no geographic structure in the distributions of shared allozymes or haplotypes. The mosaic variation is consistent with a scenario in which repeated episodes of interspecific hybridization pre‐dated the largely allopatric range expansion of the two species during the postglacial period. Our overall results are not consistent with a scenario of extensive hybridization and introgression during the postglacial range expansion of the species or within their current areas of sympatry, but we found some evidence for local, postglacial evolution and hybridization in the Baltic region. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166 , 127–148.     

A new species of Achetaria (Plantaginaceae) from south-eastern Brazil

A new species, Achetaria latifolia V.C.Souza, is described and illustrated. It is characterized by its broadly ovate leaves, indument of stems and leaves of noncapitate hairs, and pubescent capsule. Achetaria latifolia is known only in the coastal dune-area of Cabo Frio region, Rio de Janeiro State, Brazil.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 73–75.