The generic placement of a morphologically enigmatic species in Asteraceae: evidence from ITS sequences

 Bidens cordylocarpa is a high polyploid species restricted in distribution to stream sides in the mountains of Jalisco, Mexico. The morphologically enigmatic species was originally described as a member of the genus Coreopsis, but later transferred to Bidens, largely because the involucral bracts appear most similar to Bidens. Characters of the cypselae, often useful in generic placement, are of no value for this species because the fruits have features not detected in either Bidens or Coreopsis. Sequences from the internal transcribed spacer region of nuclear ribosomal DNA (ITS) were used to assess the relationships of Bidens cordylocarpa. The molecular phylogeny places B. cordylocarpa in a strongly supported clade of Mexican and South American Bidens, and provides more definitive evidence of relationships than morphology, chromosome number, or secondary chemistry. Molecular, morphological, and chromosomal data suggest that B. cordylocarpa is an ancient polyploid, perhaps the remnant of a polyploid complex. Received August 28, 2000 Accepted February 11, 2001     

Phylogenetic relationships of the genus Secale based on the characterisation of rDNA ITS sequences

 Sequence analysis of the internal transcribed spacer of the 18S-5.8S-26S rDNA (ITS-1) region was performed in order to analyse the phylogenetic relationships of eleven taxa of cultivated and wild rye species. The ITS regions were amplified using designed primers. At least ten positive clones of each taxonomic unit were sequenced and compared. Two different ITS sequences were found in three taxa: Secale sylvestre Host, Secale strictum ssp. kuprijanovii Grossh. and Secale strictum ssp. africanum Stapf. Secale sylvestre Host was the species that showed the greatest number of comparative differences in the sequences, and was the most distant of all the taxonomic units analysed. A certain degree of variation was found among all four subspecies of S. strictum analysed. S. strictum Presl ssp. strictum was most closely related to S. strictum ssp. africanum Stapf and S. strictum ssp. kuprijanovii Grossh to S. strictum ssp. anatolicum (Boiss.) Hammer. S. vavilovii showed similarities with this group of subspecies and with the S. cereale group. No differences were found between the weed forms of S. cereale and cultivated rye. Received March 8, 2002; accepted May 31, 2002 Published online: November 20, 2002 Address of the authors: Alfredo De Bustos, Nicolás Jouve (e-mail: nicolas.jouve@uah.es), Department of Cell Biology and Genetics, University of Alcalá, E-28871 Alcalá de Henares (Madrid), Spain.     

Molecular phylogeny supports the morphologically based taxonomic transfer of the “medicagoid”Trigonella species to the genus Medicago L.

 The taxonomic transfer of the 23 Trigonella species previously known as medicagoids to the genus Medicago L. is reanalyzed on the basis of a molecular phylogenetic approach. The internal and external transcribed spacers of 53 Medicago species s. str. and 10 of the 23 medicagoid species were sequenced. Both maximum parsimony or maximum likelihood criteria joined medicagoid species more confidently with Medicago rather than with Trigonella. The basal position of the medicagoid species within the genus Medicago, together with morphologically atypical Medicago species (such as M. radiata and M. platycarpa), raised the question of the expediency to define a new genus and is discussed, relatively to the support of each clade. Using criteria of monophyly and support, it was concluded that the medicagoids are better joined in Medicago rather than placed in a new genus. Received February 6, 2001 Accepted July 17, 2001     

Molecular phylogenetics of Hippophae L. (Elaeagnaceae) based on the internal transcribed spacer (ITS) sequences of nrDNA

 The genus Hippophae comprises 7 species and 8 subspecies according to the latest classification, and has shown enormous ecological, nutrient and medicinal values. Here we analyzed the phylogenetic relationships among 15 taxa of the genus by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). ITS sequences in Hippophae varied in length from 651 bp to 666 bp. The aligned sequences were 690 bp in length and 269 (39.0%) were variable sites with 150 being parsimony-informative. The amount of polymorphism observed within a taxon was extremely low in most taxa except for two putative hybrid species. The aligned sequences were analyzed by maximum parsimony (MP) and neighbor-joining (NJ) methods. In the strict consensus trees of parsimony analysis, the monophyly of Hippophae was supported by 100% bootstrap value. H. tibetana was at the basal position of the genus, and the remaining taxa formed two clades with high bootstrap support. The first clade included subspecies of H.␣rhamnoides and the other one consisted of remaining species. Parsimony analysis also suggested that the species H. tibetana, H. neurocarpa and H.␣salicifolia were all distinct. Although the sequence divergence among subspecies of H. rhamnoides was also remarkably high, the molecular data supported the monophyly of H. rhamnoides when H. rhamnoides subsp. gyantsensis Rousi was excxluded. The NJ trees showed essentially the same topology. The taxonomical arrangement that divided the genus into two sections was not supported based on the ITS sequences. However, the hybrid origin of H. goniocarpa and H. litangensis proposed previously was supported by the present ITS data. Received January 7, 2002; accepted May 10, 2002 Published online: November 22, 2002 Addresses of the authors: Kun Sun, Xuelin Chen, Ruijun Ma, Qin Wang, Institute of Botany, Northwest Normal University, Lanzhou 730070, China. Changbao Li, Song Ge (e-mail: gesong@ns.ibcas.ac.cn or song_ge@hotmail.com), Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.     

A phylogenetic analysis of Primulaceae s.l. based on internal transcribed spacer (ITS) DNA sequence data

 Phylogenetic relationships in Primulaceae were investigated by analysis of nuclear rDNA ITS sequences. Thirty-four species of Primulaceae, two of Myrsinaceae and four outgroup taxa were analyzed. In accordance to the results of recently published papers on the phylogeny of Primulaceae we found the family to be paraphyletic and resolved the positions of some genera. Our results show (a) the rather basal position of Centunculus within Lysimachieae, the genus thus being rather distantly related to Anagallis, (b) the close relationship between Lysimachia sect. Lerouxia, Anagallis, Asterolinon, and Pelletiera, (c) the well-supported monophyly of a group consisting of the four genera Hottonia, Omphalogramma, Bryocarpum, and Soldanella, and (d) the affinity of Stimpsonia to the Myrsinaceae-Lysimachieae-Ardisiandra clade. The ITS sequence data do not provide sufficient information to resolve basal relationships within the Primulaceae s.l. There is evidence against the monophyly of the large genera Primula, Androsace, and Lysimachia. In contrast to the phylogenetic reconstructions based on plastid gene sequences, Cyclamen does not appear as a member of the Myrsinaceae-Lysimachieae clade, but its position remains unclear. Revised July 10, 2002; accepted November 21, 2002 Published online: March 20, 2003     

Chromosome and breeding system evolution of the genus Mercurialis (Euphorbiaceae): implications of ITS molecular phylogeny

 The internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were amplified and sequenced from 19 samples representing all species of the genus Mercurialis and two outgroup species, Ricinus communis and Acalypha hispida. The length of ITS1 in the ingroups ranged from 223 to 246 bp and ITS2 from 210 to 218 bp. Sequence divergence between pairs of species ranged from 1.15% to 25.88% among the ingroup species in the combined data of ITS1 and ITS2. Heuristic phylogenetic analyses using Fitch parsimony on the combined data of ITS1 and ITS2 with gaps treated as missing generated 45 equally parsimonious trees. The strict consensus tree was principally concordant with morphological classification. Within the genus, the ITS sequences recognised two main infrageneric clades: the M. perennis complex including three Eurasian stoloniferous species (M.␣leiocarpa, M. ovata and M. perennis) and the western Mediterranean group including eight both annual and perennial species. Of the western Mediterranean clade, the annual and perennial species grouped respectively into two different groups, and the annual life form is revealed as a synapomorphic character derived from perennial, whereas in the Eurasian clade ITS phylogeny suggested M. leiocarpa as basal clade sister to M.␣perennis and M. ovata. ITS phylogeny failed to resolve the relationships among the different cytotypes of M. ovata and M. perennis. ITS phylogeny also suggested rapid karyotypic evolution for the genus. The karyotypic divergence among the perennial species of western Mediterranean region did not corroborate the nucleotide sequence divergence among the species. Optimisation of chromosome numbers onto the ITS phylogeny suggested x=8 to be the ancestral basic chromosome number of the genus. ITS phylogeny confirmed that the androdioecy of M. ambigua is derived from dioecy. The nucleotide heterozygosity and additivity in ITS sequences clearly confirm the interspecific hybridisation in the genus Mercurialis. Received December 22, 2001; accepted May 21, 2002?Published online: November 14, 2002 Address of the authors: Martin Kr?henbühl, Yong-Ming Yuan (correspondence) and Philippe Küpfer, Institut de Botanique, Laboratoire de botanique évolutive, Université de Neuchatel, Emile-Argand 11, CH-2007 Neuchatel, Suisse. (e-mail: yong-ming.yuan@unine.ch)     

Use of genome-specific repetitive DNA sequences to monitor chromatin introgression from Festuca mairei into Lolium perenne

Repetitive DNA sequences contribute considerably to an understanding of the genomes of higher plants. Repetitive DNA sequences tend to be genome-specific due to the rate of amplification and extent of divergence. Two genome-specific probes from the genomic DNA library of Festuca arundinacea var. genuina Schreb.were selected and characterized. TF521 was found to be P genome-specific since it was able to hybridize with Festuca pratensis Huds. (PP) and Festuca arundinacea var. genuina (PPG₁G₁G₂G₂), but not, or only weakly, with tetraploid Festuca species. TF521 hybridized only with the diploid Festuca and not with the Lolium species (LL). TF436 was specific to tetraploid species of Festuca, such as F. arundinacea var. glauces-cens Boiss. (G₁G₁G₂G₂) and Festuca mairei St. Yves (M₁M₁M₂M₂). By means of Southern hybridization, TF436 was used to detect chromatin introgression of F. mairei in the progenies of the hybrid F. mairei×Lolium perenne L. Potential addition and translocation lines were identified in the BC₁F₁ derivatives of F. mairei×L. perenne. In situ hybridization was used to confirm the genetic identity of these lines. Sequence analyses indicated that TF436 and TF521 were two novel DNA sequences as no homologous sequences were found in Genebank. Received: 22 June 2000 / Accepted: 3 November 2000     

Allozyme variation in Carex sect. Digitatae – Evidence of introgression, genetic distinctiveness and evolution of taxa

 A subset of populations of the European taxa of Carex sect. Digitatae, including Carex digitata, C. pallens, C. ornithopoda, C. pediformis ssp. rhizodes and C. humilis have been investigated for allozyme variation. The (presumably) distantly related C. pendula was used as a reference taxon. Carex digitata, C. pallens and C. ornithopoda on the one hand, and C. humilis and C. pediformis on the other, shared the majority of the alleles. Cluster analyses based on genetic distances grouped populations of C. digitata and C. ornithopoda in a mixed subcluster whereas all populations of C. pallens formed a subcluster distinct from the digitata/ornithopoda cluster. This in spite of the fact that C. ornithopoda is morphologically clearly distinct from C. digitata whereas C. pallens is barely distinguishable from the latter. Carex pediformis and C. humilis appeared not more genetically similar to C. digitata than to the reference taxon. It is concluded that 1) C. digitata, C. pallens and C. ornithopoda are closely related and most probably forms a monophyletic group, 2) C. pallens is a genetically distinct species, 3) C. pediformis and C. humilis are not closely related to the rest of C. sect. Digitatae, 4) C. pediformis and C. humilis are relatively closely related, 5) introgression do occur between the investigated species but not to such an extent that it can explain major geographic patterns of variation in C. digitata. Received December 21, 2001; accepted November 14, 2002 Published online: March 24, 2003     

The phylogeny of Patrinieae sensu Graebner (Valerianaceae) revisited: additional evidence from ndhF sequence data

 DNA sequences of both 5′ and 3′ regions of the plastid ndhF gene were generated in order to study the position of Patrinia and Nardostachys, to check the potential paraphyletic nature of Patrinieae, and to evaluate the possible link between the tribe and Linnaeaceae. Parsimony analysis showed very strong support for Patrinia as sister to all members of Valerianaceae (including Nardostachys) and indicated the paraphyletic nature of the tribe Patrinieae. Additionally, trees were constructed from available rbcL data separately and supplemented with ndhF sequences. Topologies of these combined cladograms are in agreement with the ndhF phylogeny, suggesting that the traditionally circumscribed Patrinieae can no longer be recognized but must be considered as part of a basal grade in Valerianaceae. Parsimony analysis based on a morphological data set supported a monophyletic Patrinieae; combination with the molecular data showed a paraphyletic Patrinieae. Furthermore, the possible link between Patrinieae and Linnaeaceae is evaluated. Received July 12, 2001 Accepted February 25, 2002     

Sectional relationships in the genus Rhododendron (Ericaceae): evidence from matK and trnK intron sequences

 Phylogenetic relationships among all eight subgenera and 12 sections in Rhododendron as well as its related genera were inferred from matK and trnK intron sequences. The results of this study provided the following insights: (1) Rhododendron is paraphyletic because Menziesia is nested within Rhododendron. (2) Subgenus Therorhodion forms a basal lineage of tribe Rhodoreae. (3) Subgenera Hymenanthes and Tsutsusi are monophyletic. (4) Subgenera Azaleastrum and Pentanthera are polyphyletic. (5) Subgenus Rhododendron is monophyletic, if section Rhododendron subsection Ledum is excluded. Received January 8, 1999, in definite form December 22, 2000 Accepted April 12, 2001     

The phylogeny of Lens (Leguminosae): new insight from ITS sequence analysis

 Lens includes L. culinaris subsp. culinaris (the cultivated lentil) and several wild species distributed from the Mediterranean region to western Asia. We compared sequence variation in the ITS region among species of Lens in an effort to end persisting uncertainty regarding the phylogeny of the genus. The parsimony analysis revealed a single minimum-length tree with a topology congruent with patterns derived by previous studies of nuclear and chloroplast DNA RFLPs. The basal and highly divergent status of the L. nigricans clade is depicted, and the progenitor-derivative relationship between L. culinaris subsp. orientalis and L. culinaris subsp. culinaris is reaffirmed. Resolution in the tree was improved by combining the ITS data set with a pre-existing set of chloroplast DNA restriction site data obtained from the same group of samples. Received May 8, 2000 Accepted October 26, 2001     

Molecular approach to the phylogeny and systematics of Cytisus (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-trnF intergenic spacer)

 Phylogenetic relationships of Cytisus and allied genera (Argyrocytisus, Calicotome, Chamaecytisus, Cytisophyllum, and Spartocytisus) were assessed by analysis of sequences of the nrDNA internal transcribed spacer (ITS) and the cpDNA trnL-trnF intergenic spacer. Genera of the Genista-group (Chamaespartium, Echinospartum, Genista, Pterospartum, Spartium, Teline and Ulex) were included to check the position of Cytisus species transferred to Teline. The tree obtained by combining both sets of data indicates that the Genista and Cytisus groups form two separate clades. Cytisus heterochrous and C. tribracteolatus are more closely related to the Cytisus-group, thus their transfer to Teline is not supported by molecular data. Cytisus fontanesii (syn. Chronanthos biflorus) groups with Cytisophyllum sessilifolium and Cytisus heterochrous within the Cytisus-group. Similarly, Argyrocytisus battandieri falls within the Cytisus-group as a well differentiated taxon. All these taxa seem to have early diverged from the Cytisus-group. Their taxonomic rank should be reconsidered to better reflect their phylogenetic separation from Cytisus. On the contrary, Chamaecytisus proliferus and Spartocytisus supranubius enter in the main core of Cytisus, and they should better be included in sections of Cytisus (sect. Tubocytisus and Oreosparton, respectively). Sect. Spartopsis is not monophyletic and the position of several species, currently included in this section, deserves reevaluation: C. arboreus aggregate is closely related to C. villosus (sect. Cytisus) and to Calicotome; C. striatus is closely related to Cytisus sect. Alburnoides; and the position of C. commutatus (incl. C. ingramii) remains unclear. The relationships and positioning of several minor taxa (C. transiens, C. megalanthus, and C. maurus) are also discussed. Received November 22, 2001; accepted March 16, 2002 Published online: October 14, 2002 Addresses of the authors: Paloma Cubas (e-mail: cubas@farm.ucm.es) and Cristina Pardo (e-mail: cpardo@farm.ucm.es), Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense, E-28040 Madrid, Spain. Hikmat Tahiri Faculté des Sciences, Université Mohammed V, BP 1014 Rabat, Morocco (e-mail: tahiri@ fsr.ac.ma).     

A reassessment of tribal affinities of Cratystylis and Haegiela (Asteraceae) based on three chloroplast DNA sequences

 The tribal affinities of Cratystylis and Haegiela were assessed using three chloroplast DNA sequences, the trnL/F spacer, the trnL intron and the matK coding region. The outgroup was represented by two species of the subfamily Barnadesioideae, whereas one to seven genera (45 species including Cratystylis and Haegiela) of the tribes of the Asteroideae [Anthemideae (6 genera), Astereae (7), Calenduleae (2), Gnaphalieae (7), Heliantheae s.l. (5), Inuleae s.str. (3), Plucheeae (3), Senecioneae (4)] and Cichorioideae, [Arctotideae (1), Cardueae (2), Lactuceae (2), Liabeae (1), Mutisieae (1) and Vernonieae (1)] comprise the ingroup. Phylogenetic analysis indicates that Cratystylis has strong support as a member of the tribe Plucheeae, whereas Haegiela is a member of Gnaphalieae. At some point in their taxonomic history, both genera have been placed in these tribes and there are good morphological and chemical characters that justify these placements. The monotypic Haegiela was once included in Epaltes (Plucheeae) and this study has confirmed the need for the separation of the two genera. The genus Cratystylis appears to be monophyletic. Received August 26, 2002; accepted September 19, 2002 Published online: February 7, 2003     

The complex history of the genus Ilex L. (Aquifoliaceae): evidence from the comparison of plastid and nuclear DNA sequences and from fossil data

 A plastid phylogeny of the genus Ilex based on three different loci (the atpB-rbcL spacer, trnL-trnF and rbcL) is compared with its nuclear phylogeny based on two different loci (the ribosomal ITS and the 5S RNA spacer). These two sets of molecular data are then compared to geographical and temporal data from the fossil record. The plastid phylogeny is strongly correlated with the geographic distribution of extant species. However, the nuclear phylogeny is strongly incongruent with the plastid phylogeny, suggesting frequent interlineage hybridizations. Moreover, the comparison of the ribosomal ITS tree and the 5S RNA spacer tree indicates also possible lineage sorting. Particularly interesting is the finding of two different Ilex lineages in the plastid American clade showing different biogeographic patterns in South America. One of them has a simple North American/South American biogeographical relationship. The other has complex biogeographical relationships, some species showing direct Asian/South American biogeographical relationships. During its history, the genus Ilex probably experienced frequent lineage sorting and interlineage hybridization with subsequent nuclear or cytoplasmic introgression, making the study of its history very complex. Received September 24, 2001; accepted August 19, 2002 Published online: November 28, 2002 Addresses of the authors: Jean-Fran?ois Manen (e-mail: manen@cjb.ville-ge.ch), Yamama Naciri-Graven, Conservatoire et Jardin Botaniques, Impératrice 1, CH-1292 Chambésy/Genève, Switzerland. Michael C. Boulter, Palaeobiology Research Unit, University of East London, Romford Road, London E15 4LZ, UK.     

Karyological evolution and molecular phylogeny in Macaronesian dendroid spurges (Euphorbia subsect. Pachycladae)

 Euphorbia subsect. Pachycladae is a taxon of primarily Macaronesian distribution, defined by morphological and biogeographical criteria. On the basis of morphological data, it is a heterogeneous group within which at least three complexes of species can be distinguished. To ascertain whether it is a natural group and discover its phylogenetic relations, we performed a cladistic analysis of the sequences of ribosomal nuclear DNA and a karyological study. The results of the two studies are concordant and show that the sub-section is polyphyletic and includes three different groups. The first monophyletic group is made up of the Macaronesian endemics E. atropurpurea complex and E. lamarckii complex, which form a polytomy with E. dendroides as the basal species. The lauroid species E. longifolia and E. stygiana represent the second monophyletic group, which derive from Mediterranean forms of E. sect. Helioscopia Dumort. Both species are paleopolyploid (2n=44) with highly symmetrical karyotypes. Finally, E. balsamifera, with a Canarian, African and Arabian distribution, remains isolated in a basal position. Its karyotype, with 2n=20 chromosomes, differs from the Macaronesian model and displays analogies with African cactiform spurges. On the basis of the results, some hypotheses are formulated about speciation processes in the three groups. Received March 3, 2001 Accepted October 28, 2001     

Circumscription and phylogeny of Saxifraga sect. Ciliatae: Evidence from nrDNA ITS sequences

The largest section of the genus Saxifraga (Saxifragaceae), sect. Ciliatae, consists of 175 morphologically diverse species. This section is mainly distributed in the Qinghai-Tibetan Plateau and adjacent regions of southwest China and more than 80% of the total number of species are endemic to this region. It remains unknown whether this section is monophyletic and up to now no study has been conducted on the infra-sectional phylogeny. In this study, ITS sequences of the nuclear ribosomal DNA were firstly determined for 33 species mainly from this section and related sections. We further downloaded the corresponding sequences of the same DNA region for the other 22 species of Saxifraga and Mitella from GenBank. All sequences were together used to construct the phy-logenetic trees. The main implications of the phylogenetic analyses include: (1) sect. Ciliatae, as traditionally defined, constitutes as a monophyletic clade and its sister group is a well supported clade that includes species from 8 sections such as sect. Porphyrion, sect. Saxifraga and sect. Mesogyne; (2) three morphological subsections, i.e., subsect. Gemmiparae, subsect. Hirculoideae and subsect. Rosulares were tentatively recovered despite the relatively low statistic bootstrap support for the last one; however, subsect. Flagellares and subsect. Hemi-sphaericae were not recognized as separate entities, and nested within subsect. Gemmiparae; (3) subsect. Hircu-loideae and subsect. Rosulares clustered together as sister subclades while subsect. Gemmiparae diverged early. In addition, our results suggest that the paired variation of ITS sequences in sect. Ciliatae is relatively low between the sampled species in spite of their diverse morphology. It is suggested that such a scenario may mirror rapid speciation in this section that probably trigged by the uplifts of the Qinghai-Tibetan Plateau and the extensive selection pressure under the alpine environments.     

Phylogenetic analysis of a polyphyletic African genus of Caesalpinioideae (Leguminosae): Monopetalanthus Harms

 To assess the new taxonomy of Monopetalanthus proposed by Wieringa, a phylogenetic study was performed using the chloroplast trnL intron and psbA-trnH spacer, and the nuclear ITS region of the 18-26S rDNA. The phylogeny clearly indicates the polyphyletic nature of Monopetalanthus. The molecular data support the transfer of M. longiracemosus to Tetraberlinia and the newly defined Aphanocalyx, which now includes all the previous Monopetalanthus species having leaflets with a marginal vein. Our analyses do not support the monophyly of the newly described genus Bikinia, which includes four new species and six species transferred from Monopetalanthus. Bikinia occurs either as paraphyletic with Tetraberlinia (chloroplast DNA data) or as a monophyletic group that also includes the new monotypic genus Icuria sister to Tetraberlinia (ITS data). Nonetheless, the molecular phylogeny generally supports the taxonomy of Wieringa in subdividing the genus Monopetalanthus into distinct groups, none of which retains the name Monopetalanthus. Received May 28, 2001; accepted July 6, 2002 Published online: November 20, 2002 Address of the authors: G. Y. Fannie Gervais, Anne Bruneau (e-mail: bruneaua@irbv.umontreal. ca), Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke est, Montréal, Québec, Canada, H1X2B2.