Molecular evidence for naturalness of genera in the tribe Antirrhineae (Scrophulariaceae) and three independent evolutionary lineages from the New World and the Old

The tribe Antirrhineae consists of 29 genera distributed in the New World and the Old. Phylogenetic analyses of ITS and ndhF sequences served to recognize six main lineages: Anarrhinum group (Anarrhinum, Kickxia); Linaria group (Linaria); Maurandya group (Cymbalaria, Asarina, Maurandella, Rhodochiton, Lophospermum); Schweinfurthia group (Pseudorontium, Schweinfurthia); Antirrhinum group (Antirrhinum, Pseudomisopates, Misopates, Acanthorrhinum, Howeliella, Neogarrhinum, Sairocarpus, Mohavea, Galvezia); Chaenorrhinum group (Chaenorrhinum, Albraunia, Holzneria). Parsimony (cladistics), distance-based (Neighbor-Joining), and Bayesian inference reveal that: (1) the tribe is a natural group; (2) genera such as Linaria, Schweinfurthia, Kickxia, and Antirrhinum also form natural groups; (3) three Antirrhineae lineages containing genera from the New and Old World are the result of three intercontinental disjunctions displaying similar levels of ITS-sequence divergence and differentiation times (Oligocene-Miocene); (4) evolution of flower shapes is not congruent with primitiveness of personate flowers; (5) both polyploidy and dysploidy appear to be responsible for most variation in chromosome number in the six main lineages. Nuclear and chloroplast evidence also supports the split of American and Mediterranean species of Antirrhinum into different genera, a result that should be contemplated in the interest of a more natural (monophyletic) taxonomy. Nucleotide additivity causes poor resolution in the ITS analysis of 22 species of Mediterranean Antirrhinum and lead us to interpret extensive hybridization in the Iberian Peninsula.This research was supported by the Spanish Dirección General de Investigación Científica y Técnica (DGICYT) through the project Flora Iberica (PS91-0070-C03-01). We thank Jorge Martínez and Emilio Cano for his assistance in the lab, Rafael Rubio and Omar Fiz for helping on the Bayesian inferences, the curators/collection managers of BCF, E, JACA, MA, SALA, SALAF, SEV, TARI, VAL for loans and access to specimens, and two anonymous reviewers for improving the quality of this publication.     

A new section of Linaria (Scrophulariaceae: Antirrhineae)

An investigation of species of the Antirrhineae (Scrophulariaceae), primarily from the point of view of seed morphology, has indicated that the inclusion of Linaria chalepensis and L. armeniaca in section Versicolores is unsatisfactory. Several unusual features of both the seeds and other parts of the plants provide a basis for recognizing a distinct section to include these two species. An overlooked infrageneric taxon of Linaria has been reinstated as section Macrocentrum (C. Koch) D. Sutton. All taxa included in this section are described and illustrated. The significance of the unusual seed morphology is discussed in relation to the affinities of the genus.     

Phylogeny of Bonatea (Orchidaceae: Habenariinae) based on molecular and morphological data

The genus Bonatea is widely distributed throughout southern and eastern Africa. Considerable debate surrounds the generic status of Bonatea, but there have been neither previous studies of evolutionary relationships among Bonatea species, nor any tests of the monophyly of the genus in relation to its close relative Habenaria. We investigated phylogenetic relationships between Bonatea and selected Habenaria species using morphology, as well as sequences of the chloroplast gene matK and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. A fully resolved cladogram was obtained using morphological data, but neither the ITS, matK, nor combined data sets yielded well-resolved and well-supported phylogenetic structure for Bonatea. There is poor congruence between ITS and matK data for interspecific relationships in Bonatea, whilst the morphological results are largely congruent with the ITS analysis. Relative to Habenaria, there is little sequence variation between Bonatea species, which could indicate a recent and rapid radiation of Bonatea. Although the sampled Bonatea species form a distinct clade, more extensive sampling of Habenaria would be required to establish unambiguously whether or not Bonatea is monophyletic.     

Phylogeny of the Calliergonaceae (Bryophyta) based on molecular and morphological data

Phylogeny of the Calliergonaceae is reconstructed based on nuclear and chloroplast sequence data, and morphology. Depending on treatment of insertions and deletions, the total number of informative characters was 194 or 163. The study yielded good support for two major clades, one with Hamatocaulis, Scorpidium, and possibly Hygrohypnum ochraceum (Wils.) Loeske, and one with Calliergon, Loeskypnum, Straminergon, and Warnstorfia. Inclusion of H. ochraceum in the ingroup increased tree length, markedly decreased consistency and retention indices, and decreased support for the structure of the Hamatocaulis-Scorpidium clade, and its position within the family is challenged. When H. ochraceum was excluded, good support is found for the genera Hamatocaulis and Scorpidium. Within the other major subclade the monophyly of a clade with Loeskypnum, Straminergon, and Warnstorfia, of a portion of Calliergon, and of Warnstorfia fluitans plus W. pseudostraminea are well supported.     

Phylogeny and biosystematics ofPseudomonotes (Dipterocarpaceae) based on molecular and morphological data

The placement of a recently discovered South American monotypic genus,Pseudomonotes tropenbosii, in subfam.Monotoideae (Dipterocarpaceae) extends the geographical range of the subfamily from Africa to the Neotropics. Although morphological and anatomical evidence suggest similarities betweenPseudomonotes andMonotes, the close alliance of these two genera was questionable due to their disjunct distribution and a lack of phylogenetic analysis. In the present study, we reconstructed the phylogeny ofPseudomonotes and other putatively related taxa usingrbcL sequence data. The analysis ofrbcL sequences of 20 taxa belonging to 15 genera and eight families recovered a single most parsimonious tree. The genusSarcolaena (Sarcolaenaceae) formed a clade sister to the monophyleticDipterocarpaceae clade.Monotes andPseudomonotes formed a strongly supported group, sister to the monophyletic clade withPakaraimaea and the remaining Asiatic dipterocarp species studied. The study strongly supports the placement ofPseudomonotes within subfam.Monotoideae of theDipterocarpaceae.     

Phylogeny of Schisandraceae based on morphological data: evidence from modern plants and the fossil record

Schisandraceae are traditionally subdivided in two genera, Schisandra and Kadsura, based on differences in the organisation of the floral receptacle, the carpels, and the presence or absence of a ``pseudostigma'. Recently, phylogenetic analyses utilizing ITS sequence data and morphological data resulted in incongruent tree topologies, with the morphological trees suggesting monophyly of the two genera, whereas ITS trees did not resolve Schisandra and Kadsura as monophyletic clades. In the present paper we study seed morphology and leaf epidermal features of 22 species of Schisandraceae in order to provide additional data for a morphological data matrix. Seed morphological characters are highly homoplastic and do not yield further evidence for monophyly of the two genera. Instead, a number of characters appear to support sister group relationships between taxa within the genera, such as, for instance, for K. coccinea and K. scandens, both of which have large seeds along with a multi-layered mesotesta. Considering leaf epidermal characteristics, species of Kadsura were found to be consistently amphistomatic, whereas species of Schisandra are always hypostomatic. Phylogenetic analysis using the extended data matrix resulted in weakly supported Kadsura and Schisandra clades with five and four synapomorphies indicating monophyly of Kadsura and Schisandra, respectively. Fossils ascribed to Schisandraceae date back to the Late Cretaceous. These are tri-and hexacolpate pollen types displaying a combination of features found in modern Schisandraceae and partly also in Illiciaceae. Leaf remains from this period are poorly preserved and difficult to ascribe to Schisandraceae because of the lack of synapomorphies for the family. In the Early Cainozoic, leaf and seed remains from North America and Europe unambiguously belong to the family. Seeds from the Eocene of North America show some similarities to the modern Schisandra glabra from North America, while fossils from Europe show more similarities to modern Asian species.     

Phylogeny of Castanea (Fagaceae) based on chloroplast trnT-L-F sequence data

Species in the genus Castanea are widely distributed in the deciduous forests of the Northern Hemisphere from Asia to Europe and North America. They show floristic similarity but differences in chestnut blight resistance especially among eastern Asian and eastern North American species. Phylogenetic analyses were conducted in this study using sequences of three chloroplast noncoding trnT-L-F regions. The trnT-L region was found to be the most variable and informative region. The highest proportion of parsimony informative sites, more and larger indels, and higher pairwise distances between taxa were obtained at trnT-L than at the other two regions. The high A+T values (74.5%) in the Castanea trnT-L region may explain the high proportion of transversions found in this region where as comparatively lower A+T values were found in the trnL intron (68.35%) and trnL-F spacer (70.07%) with relatively balanced numbers of transitions and transversions. The genus Castanea is supported as a monophyletic clade, while the section Eucastanon is paraphyletic. C. crenata is the most basal clade and sister to the remainder of the genus. The three Chinese species of Castanea are supported as a single monophyletic clade, whose sister group contains the North American and European species. There is consistent but weak support for a sister–group relationship between the North American species and European species.     

A search for the phylogenetic position of the seven-son flower (Heptacodium, Dipsacales): Combining molecular and morphological evidence

A first report on the problematic phylogenetic position ofHeptacodium (2 spp.; China) using molecular data from chloroplast DNA is presented. Amplification of ORF2280 homolog region was executed in a number of representative taxa in order to determine ifHeptacodium shows similar structural rearrangements as other Dipsacales. DNA sequences ofndhF were generated to clarify the phylogenetic position ofHeptacodium among Caprifoliaceae (s.l.). Six outgroup taxa and fifteen representatives of Dipsacales were sampled and more than 2100 basepairs ofndhF sequence were used in a cladistic analysis. Parsimony analysis produced two shortest trees and showedHeptacodium as sister to all members of Caprifoliaceae (s.str.), although weakly supported. Additionally, trees were constructed withndhF data supplemented with availablerbcL sequences and a morphological data set. Results of all analyses support an unresolved basal position forHeptacodium among Caprifoliaceae (s.l.), which in part explains the difficulty experienced previously in classifying the genus.     

A phylogenetic analysis ofDuguetia (Annonaceae) based on morphological data

In this paper we present a phylogeny based on a study of the general morphology of 61 of the ca. 80 species ofDuguetia. Analyses were performed with PAUP. The resulting phylogeny is compared with the classification ofDuguetia as suggested byFries (1934, 1937, 1939, 1959). The results are partly corresponding withFries's classification. SectionAlcmene includingD. rionegrensis, and sects.Geanthemum, Synsepalantha, andCalothrix withoutD. uniflora, are upheld by this analysis. It is concluded that sect.Xylopipetalum should be united with sect.Duguetia. Before other taxonomic decisions at the section level can be made, more solid data must be available. Most dissimilarities found are considered to be not strong enough to propose alterations.Studies inAnnonaceae XXIII. For 22nd part seeZuilen & Maas (1994).     

Phylogeny of Carpinus and subfamily Coryloideae (Betulaceae) based on chloroplast and nuclear ribosomal sequence data

Phylogenetic studies were conducted for Carpinus and the subfamily Coryloideae (Betulaceae) using sequences of the chloroplast matK gene, the trnL-trnF region (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer) and the psbA-trnH intergenic spacer, and the nuclear ribosomal ITS regions. The combined analyses of the three chloroplast regions suggest that Coryloideae is monophyletic; Ostryopsis is sister to the Carpinus - Ostrya clade; Corylus is monophyletic and sister to the Ostrya - Carpinus - Ostryopsis clade; Ostrya is paraphyletic; and within Carpinus, species of sect. Carpinus from eastern Asia form a monophyletic group, whereas the positions of C. betulus from Europe and C. caroliniana from eastern North America are unresolved within the Carpinus clade. The cpDNA tree generated in this study is largely congruent with the previously published ITS results, but the ITS tree places Carpinus sect. Distegocarpus as sister to the Ostrya - Carpinus sect. Carpinus clade. Future work is needed to examine the relationships within the Ostrya - Carpinus clade, evaluate the generic status of Ostrya, and test the phylogenetic position of Ostryopsis.     

Phylogeny of the genus Hesperodiaptomus (Copepoda) based on nucleotide sequence data of the nuclear ribosomal gene

The genus Hesperodiaptomus Light, 1938, one of the most diverse groups of freshwater copepods that occur in North and Central America, plays a major role in the food webs of the alpine freshwater communities. Phylogenetic relationships of these taxa remain poorly understood due to difficulties in obtaining reliable morphological characters for phylogenetic analyses. To understand the phylogenetic relationships within this group, we reconstructed a partial phylogeny of the genus Hesperodiaptomus based on nuclear ribosomal gene sequences. Phylogenetic analyses based upon the taxa examined supported the monophyly the genus and revealed two clades. The eiseni clade comprised species that are morphologically similar to Hesperodiaptomus eiseni (Lilljeborg, 1889), and the shoshone clade included species morphologically similar to Hesperodiaptomus shoshone (S.A. Forbes, 1882). The two groups can be distinguished by a modification of the right basis, the arrangement of spinules on the distal pad of the second exopod, and the degree of presence the inner lamellar expansion of the right coxa. Handling editor: P. Spaak     

Phylogeny ofCyclamen L. (Primulaceae): Evidence from morphology and sequence data from the internal transcribed spacers of nuclear ribosomal DNA

A molecular phylogenetic study of the genusCyclamen L. (Primulaceae) has been undertaken, based on sequence data from the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. Sequence data from 15 species ofCyclamen and seven outgroup taxa selected from Primulaceae and Myrsinaceae were analyzed phylogenetically. A second analysis based on a combined morphological and molecular dataset was performed to evaluate earlier hypotheses of character evolution in the genus. The results indicate that four monophyletic subgroups may be recognized in the genus, viz.Cyclamen, Psilanthum Schwarz,Eucosme Schwarz andGyrophoebe Schwarz. Each of the four subgenera is diagnosed by distinct basic chromosome number, as well as by morphological and molecular characteristics.     

Phylogenetic signal common to three data sets: Combining data which initially appear heterogeneous

This study makes use of three sources of data, morphology and two chloroplast DNA sequences,ndhF andrbcL, to resolve relationships in Gesneriaceae. Cladograms from each of the three data sets separately are not topologically congruent. Statistical indices suggest that each data set is congruent with thendhF data althoughrbcL and morphology are themselves incongruent. Consensus methods provide no resolution of taxonomic relationships when trees from the different data sets are combined. Combining data sets generally results in cladograms that are more fully resolved than each of the data sets analyzed separately and support for the clades increases based on higher decay index and bootstrap values. These results indicate that there is a phylogenetic signal common to each of the data sets, however, the noise (errors due to homoplasy, mis-scoring, etc.) unique to each data source masks this signal. In combining the data, the evidence for the common evolutionary history in each data set overcomes the noise and is apparent in the resulting trees.     

Sorghum laxiflorum and S. macrospermum, the Australian native species most closely related to the cultivated S. bicolor based on ITS1 and ndhF sequence analysis of 25 Sorghum species

Australian species make up seventeen of the worlds twenty-five recognised species of Sorghum, with the genus separated into five sections: Eu-sorghum, Chaetosorghum, Heterosorghum, Para-sorghum and Stiposorghum. Whereas the genetic relationships within section Eu-sorghum are well known, little is known about the genetic relationships and crossabilities outside the primary genepool. We made a detailed investigation of phylogenetic relationships within Sorghum to identify wild species most closely related to cultivated sorghum (with outgroups Zea mays and Saccharum officinarum). The ribosomal ITS1 gene of ten species and the chloroplast ndhF gene from nineteen species were sequenced. Independent and combined analyses of the ITS1 and ndhF data sets were undertaken. The Eu-sorghum species were resolved into a strongly supported lineage by all three analyses, and included the Australian natives S. laxiflorum and S. macrospermum in the ITS1 and combined analyses. All remaining sorghum species were resolved into a second well-supported lineage in the combined analyses, although some internal relationships within this second lineage remain unresolved. We identified S. laxiflorum and S. macrospermum as the Australian species most closely related to cultivated sorghum. Our data support a reduction in the number of subgeneric sections from five to three: Eu-sorghum (unchanged); a combined Chaetosorghum/Heterosorghum to reflect the very close relationship between these two species; and a combined Para-sorghum/Stiposorghum section, thereby removing the unclear taxonomic and genetic boundaries between these species.     

Floral ontogeny inMazus pumilus (Scrophulariaceae)

InMazus pumilus, all the floral appendages are initiated in acropetal sequence in the second cell layer (except stamens) of the floral primordium by periclinal divisions. The actinomorphic calyx tube is formed due to zonal growth. The zygomorphy in corolla is evident from the inception of petal primordia which arise sequentially as independent units in order of one anterior, a pair of anterio-lateral followed by a pair of posterio-lateral. Later these primordia exhibit differential growth because of which zygomorphy becomes more pronounced. The upper corolla tube is formed by interprimordial growth and lower corolla tube by zonal growth. Stamens are initiated in the third layer of the floral apex. Unlike sepals and petals, in the development of stamens (4) underlying cells of corpus also contribute. Posterior stamen is absent. The stamens become epipetalous because of interprimordial and zonal growth in the common region below the bases of petals as well as stamens. The two carpel primordia arise as crescent shaped structures which become continuous due to interprimordial growth. The ovary is formed by a ring of zonal meristem. The style develops later between stigma and ovary because of intercalary growth. The residual apex grows vertically along with the ovary and forms the septum of the ovary. All the floral appendages exhibit similar pattern of histogenesis and early growth suggesting thereby the appendicular nature of these appendages.     

The taxonomic relationships within the genus Excoecaria L. (Euphorbiaceae) based on leaf morphology and rDNA sequence data

The tropical Indo-Pacific genus Excoecaria L. (Euphorbiaceae) has several closely related species in Australia whose taxonomic relationships are unclear. The most widely reported species in Australia is the mangrove species Excoecaria agallocha L. (type species), whose taxonomic and geographic limits are difficult to define from its closely related species or sub-species. Two additional taxa have also been described but not clearly differentiated from the type species: Excoecaria dallachyana Baillon and Excoecaria ovalis Endl. This project aimed to determine the taxonomic relationships of the Australian Excoecaria species using both leaf morphological data and DNA sequence data from the internal transcribed spacer (ITS) region of ribosomal genes. The nucleotide differences in the examined ITS1 region show that E. agallocha from eastern Australia and E. ovalis from Western Australia respectively, are genetically uniform within species but differ from each other consistently, thus supporting species status. The leaf morphological data also support this view: single factor analysis of variance consistently separated E. ovalis from E. agallocha on the basis of leaf width, leaf length and length of petiole. In contrast, E. ovalis from the Gulf of Carpentaria differs only slightly from E. ovalis in Western Australia, but no evidence was found to suggest any leaf morphological differentiation within this species. The analysis also suggests that E. dallachyana is not closely related to either mangrove species E. agallocha or E. ovalis, despite superficial morphological similarities.     

Marbleseeds are gromwells – Systematics and evolution of Lithospermum and allies (Boraginaceae tribe Lithospermeae) based on molecular and morphological data

Phylogenetic relationships are complex within the Lithospermeae, a large subgroup of the Boraginaceae s.str. The relationships of New World Lasiarrhenum, Macromeria, Nomosa, Onosmodium, Perittostoma, and Psilolaemus to subcosmopolitan and much larger Lithospermum have not been critically investigated in the recent past. No molecular data on the phylogeny of these genera and Lithospermum have so far been published. We investigated the relationships within Lithospermeae using three loci (nuclear ITS plus 5.8S rRNA, chloroplast trnL-F-spacer, and trnS-G-spacer) and micromorphological character traits (pollen, nutlets). Lithospermum s.l. constitutes the sistergroup of Asian Ulugbekia and is monophyletic only when its American segregates “Macromeria”, monotypic Nomosa, and Onosmodium are included. Both the African and the South American species groups of Lithospermum are monophyletic, but North American representatives are not resolved in a single clade. Morphological characters that have been considered as important for generic delimitation in the past (such as large, yellow corollas without faucal scales, particular pollen types, coarsely veined leaves, shrubby habit) have evolved in at least two only distantly related lineages within Lithospermum s.l. The reduction of American “Macromeria”, Nomosa, and Onosmodium as well as Asian Ulugbekia under Lithospermum is proposed to render the latter monophyletic. This redefined Lithospermum s.l. appears to have undergone a type of recent “island radiation” in the Americas, reflected in a morphological diversity far exceeding that found in the Old World.     

Evolution of novel morphological and reproductive traits in a clade containing Antirrhinum majus (Scrophulariaceae)

Phylogenetic analysis of DNA sequences of the chloroplast genes rcbL and ndhf revealed a highly supported clade composed of the families Plantaginaceae, Callitrichaceae, and Hippuridaceae in close association with the model organism Antirrhinum majus and other members of family Scrophulariaceae. Plantago has miniature actinomorphic wind-pollinated flowers that have evolved from zygomorphic animal-pollinated precursors. The aquatic Hippuridaceae have reduced windpollinated flowers with one reproductive organ per whorl, and three, rather than four, whorls. In monoecious aquatic Callitrichaceae, further reduction has occurred such that there is only one whorl per flower containing a single stamen or carpel. Optimization of character states showed that these families descended from an ancestor similar to Antirrhinum majus. Recent studies of plant developmental genetics have focused on distantly related species. Differences in the molecular mechanisms controlling floral development between model organisms are difficult to interpret due to phylogenetic distance. In order to understand evolutionary changes in floral morphology in terms of their underlying genetic processes, closely related species exhibiting morphological Variation should be examined. Studies of genes that regulate morphogenesis in the clade described here could aid in the elucidation of a general model tot such fundamental issues as how changes in floral symmetry, organ number, and whorl number are achieved, as well as providing insight on the evolution of dicliny and associated changes in pollination syndrome.