Origin of the Woody Macaronesian Sempervivoideae and the Phylogenetic Position of the East African Species of Aeonium

Evolutionary relationships among the genera of Macaronesian Sempervivoideae, Aeonium, Aichryson, Greenovia, and Monanthes, were studied using sequence variation of the chloroplast DNA trnL (UAA) - trnF (GAA) spacer and the nuclear ribosomal Internal Transcribed Spacer 2 (ITS2). Phylogenetic analysis indicates that the Moroccan Sedum sect. Monanthoidea is the sister taxon of the Macaronesian Sempervivoideae. In combination with the terminal position in the Macaronesian Sempervivoideae of the East African A. leucoblepharum, which has formerly been assumed to be the sister taxon of the other species of Macaronesian Sempervivoideae (i.e. Aichryson and Monanthes), a recent remigration to Africa is suggested. Statistical support for the terminal position of A. leucoblepharum using only spacer sequences is without homoplasy but not high since only single mutations in both the chloroplast and nuclear sequence characterize the clade containing A. leucoblepharum. A. leucoblepharum and the Canarian species with a similar growth-form share 50% of the RAPDs. Within a clade comprising woody species with yellow flowers and a herbaceous rosette, the highest genetic divergence, as determined with RAPDs, is found between A. simsii and the woody Macaronesian and African species. The extremely close genetic ties among the woody and branched (sub)shrubs indicate that, when compared to the other species of the genus, the woody, African Aeonium species are not the sister group of the Macaronesian Sempervivoideae and substantiate the view that an ancestor of A. leucoblepharum recently migrated from the Canary Islands to East Africa and Arabia through long distance dispersal, rather than being a relict of an African Aeonium flora from the Tertiary.     

Phylogenetics and evolution of the Macaronesian members of the genus Aichryson (Crassulaceae) inferred from nuclear and chloroplast sequence data

Aichryson is a genus of annual or perennial herbs, comprising approximately 13 species. The genus is nearly endemic to Macaronesia, with the center of diversity on the Canary Islands. Previous studies indicate that Aichryson is monophyletic and sister to a clade comprising two other genera, Monanthes and Aeonium. However, phylogenetic relationships within the genus have yet to be investigated. Phylogenetic relationships were estimated for Aichryson using DNA sequence data from both the nuclear and chloroplast genome. Parsimony analyses were conducted using separate nuclear and chloroplast data sets, as well as a combined data set. These combined analyses provide a well-resolved topology that is used to investigate patterns of evolution within the genus. For example, although typically herbaceous in habit, two members display a woody growth form. Our analyses indicate that woodiness is a derived feature in Aichryson that has arisen once. Furthermore, the data presented suggest that the Aichryson pachycaulon group, a group of five subspecies, are in fact not monophyletic and that a reexamination of classification is necessary. The present study indicates that the biogeographical patterns exhibited by Aichryson appear to be rare, occurring in only one other known genus.The authors would like to thank Susana Fontinha of the Madeiran Botanical Garden for plant material of Madeiran Aichryson; Drs. D. Crawford and J. Archibald, as well as two anonymous reviewers for helpful comments; Eastern Illinois Undergraduate Honors Research Fund, and University of Kansas Plant Biology Research Award. Additional financial support for this work was provided by the Department of Ecology and Evolutionary Biology and the Museum of Natural History and Biodiversity Research Center, University of Kansas and a Kansas NSF EPSCoR First Award (NSF 32171) to MEM.     

Phylogenetic relationships in the Crassulaceae inferred from chloroplast DNA restriction-site variation

A restriction-site analysis of chloroplast DNA from 44 species, representing 19 genera and all six subfamilies of the Crassulaceae was conducted using 12 restriction endonucleases. A total of 969 variable sites was detected, 608 of which were phylogenetically informative and used in parsimony analysis. Estimated values of nucleotide sequence divergence were used to construct a distance tree by the neighbor-joining method. Maximum sequence divergence in the family was ~7%. Different tree inference methods yielded only moderately different topologies. The amount of support for the monophyletic groups obtained in the Wagner parsimony analysis was evaluated by bootstrap and decay analysis. There is very strong support for a basal division of the family, which separates the monophyletic subfamily Crassuloideae from all other taxa. Four of the six traditionally recognized subfamilies are indicated to be polyphyletic. These include the Cotyledonoideae, Sempervivoideae, Sedoideae, and Echeverioideae. The Kalanchoideae and the genera Cotyledon and Adromischus exhibit low levels of cpDNA sequence divergence relative to one another, suggesting a relatively recent radiation. The genera Sedum and Rosularia are indicated to be polyphyletic. Sedum comprises sister taxa of most of the other genera of the family.     

The importance of phylogeny and ecology in microgeographical variation in the morphology of four Canarian species of Aeonium (Crassulaceae)

The relative importance of natural selection in the diversification of organisms can be assessed indirectly using matrix correspondence. The present study determines the environmental and genetic correlates of microgeographical variation in the growth form, leaf form and flower morphology in populations of four Aeonium species from section Leuconium using partial regression methods. The phylogeny of the four species and the other 12 species in the section was deduced from amplified fragment length polymorphism (AFLP). Pubescence of floral organs and flower size correlate with the phylogeny while traits related to growth form, leaf form, flower construction and inflorescence size correlate with ecological factors. The variation in the latter four traits may therefore reflect selection by current ecological conditions while variation in pubescence and flower size may reflect historical events like neutral mutations, founder events and drift. Additionally, the morphological analyses revealed a large amount of variation in all traits within populations. This suggests a possible influence of microhabitat on the variation in morphology of Aeonium in the Canary Islands.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 76 , 521–533.     

Molecular phylogeny, recent radiation and evolution of gross morphology of the rhubarb genus Rheum (Polygonaceae) inferred from chloroplast DNA trnL-F sequences

BACKGROUND AND AIMS: Rheum, a highly diversified genus with about 60 species, is mainly confined to the mountainous and desert regions of the Qinghai-Tibetan plateau and adjacent areas. This genus represents a good example of the extensive diversification of the temperate genera in the Qinghai-Tibetan plateau, in which the forces to drive diversification remain unknown. To date, the infrageneric classification of Rheum has been mainly based on morphological characters. However, it may have been subject to convergent evolution under habitat pressure, and the systematic position of some sections are unclear, especially Sect. Globulosa, which has globular inflorescences, and Sect. Nobilia, which has semi-translucent bracts. Recent palynological research has found substantial contradictions between exine patterns and the current classification of Rheum. Two specific objectives of this research were (1) to evaluate possible relationships of some ambiguous sections with a unique morphology, and (2) to examine possible occurrence of the radiative speciation with low genetic divergence across the total genus and the correlation between the extensive diversification time of Rheum and past geographical events, especially the recent large-scale uplifts of the Qinghai-Tibetan Plateau. METHODS: The chloroplast DNA trnL-F region of 29 individuals representing 26 species of Rheum, belonging to seven out of eight sections, was sequenced and compared. The phylogenetic relationships were further constructed based on the sequences obtained. KEY RESULTS: Despite the highly diversified morphology, the genetic variation in this DNA fragment is relatively low. The molecular phylogeny is highly inconsistent with gross morphology, pollen exine patterns and traditional classifications, except for identifying all samples of Sect. Palmata, three species of Sect. Spiciformia and a few species of Sect. Rheum as corresponding monophyletic groups. The monotypic Sect. Globulosa showed a tentative position within the clade comprising five species of Sect. Rheum. All of the analyses revealed the paraphyly of R. nobile and R. alexandrae, the only two species of Sect. Nobilia circumscribed by the possession of large bracts. The crude calibration of lineages based on trnL-F sequence differentiation implied an extensive diversification of Rheum within approx. 7 million years. CONCLUSIONS: Based on these results, it is suggested that the rich geological and ecological diversity caused by the recent large-scale uplifts of the Qinghai-Tibetan Plateau since the late Tertiary, coupled with the oscillating climate of the Quaternary stage, might have promoted rapid speciation in small and isolated populations, as well as allowing the fixation of unique or rare morphological characters in Rheum. Such a rapid radiation, combined with introgressive hybridization and reticulate evolution, may have caused the transfer of cpDNA haplotypes between morphologically dissimilar species, and might account for the inconsistency between morphological classification and molecular phylogeny reported here.     

Phylogenetics and diversification of Cotyledon (Crassulaceae) inferred from nuclear and chloroplast DNA sequence data

Crassulaceae includes approximately 35 genera and 1500 species of leaf and stem succulent flowering plants. The family is nearly cosmopolitan in distribution, but is particularly diverse in southern Africa, where five genera comprising approximately 325 species are found. One of these genera, Cotyledon, includes 10 species that are largely confined to South Africa, where they are commonly found on rocky hillsides, coastal flats, and cliff faces. Species of Cotyledon are characterized by five-parted, pendulous, sympetalous flowers, but the genus is highly diverse in growth form, flower color and size, and leaf morphology. One particularly variable species, C. orbiculata, has been divided into five varieties based on leaf morphology and biogeography; however, the monophyly of this species as well as the relationships among the varieties have not previously been investigated. Parsimony analyses of a combined data set of DNA sequences from chloroplast and nuclear genome provided the first estimate of phylogeny for Cotyledon, and resulted in two minimum-length trees and a fully resolved phylogeny for the genus. Results indicate that C. orbiculata is not monophyletic and suggest the need for additional studies and a revised classification within the genus.     

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.     

Glacial-induced altitudinal migrations in Armeria (Plumbaginaceae) inferred from patterns of chloroplast DNA haplotype sharing

In contrast to northern European areas where large-scale migrations occurred to recolonize territories after glacial periods, species in southern regions survived and diverged without large geographical displacements. As a result of the importance of orography in much of the southern areas, such displacements must have involved populations ascending or descending mountains. The present study provides support for glacial-induced altitudinal migrations from chloroplast phylogeographic patterns in Armeria (Plumbaginaceae) in southeast Spain. One hundred and five sequences of the trnL-F spacer were obtained from seven species. Fifteen different haplotypes were recognized, their genealogy was inferred, and associations with geography were explored using nested clade analysis. Seven instances were detected in which the same haplotype is shared by two or three species within a particular massif. In all the cases, at least one of the species involved displayed different haplotypes in other areas; in most, the haplotype shared is predominant either in one of the species involved or in the massif. These patterns of haplotype sharing strongly suggest horizontal transfer between species. In one of the massifs (Sierra Nevada) the three species involved in haplotype sharing (A. splendens, A. filicaulis ssp. nevadensis, A. villosa ssp. bernisii) occur at markedly different altitudinal belts. It is argued that altitudinal migrations within the contraction-expansion model provide the best explanation for the current pattern, and that at least in one case it resulted in the formation of a new hybrid taxon, A. filicaulis ssp. nevadensis.     

Evolution ofSedum seriesRupestria (Crassulaceae): Evidence from chloroplast DNA and biosystematic data

Evolutionary relationships within the compariumSedum seriesRupestria (Crassulaceae) were investigated by phylogenetic analyses of restriction site variation of the chloroplast genome and morphological variation. DNAs were digested with 21 restriction enzymes and hybridized withS. album cpDNA probes covering the entire genome. A total of 141 ingroup variable restriction sites was identified. Morphological variation was observed in a total of 66 characters. Both datasets were analysed using parsimony methods and compared with evidence from cytology, artificial hybridization and biogeography to infer evolutionary relationships. The results 1) indicate a relatively high level of nucleotide sequence divergence withinS. ser.Rupestria; 2) indicate three main lines of plastome differentiation, which are in agreement with morphology, basic chromosome numbers, and centres of diversity; 3) do not provide a compelling solution for basal relationships; 4) support an earlier hypothesis on reticulation and provide evidence for a third case of allopolyploidy in the series; 5) suggest that interspecific gene flow inS. ser.Rupestria is funnelled through the presumably allopolyploidS. rupestre subsp.erectum.     

Paternal inheritance of chloroplast DNA in interspecific hybrids in the genus Larrea (Zygophyllaceae)

The mode of chloroplast DNA (cpDNA) inheritance was investigated in the genus Larrea (Zygophyllaceae) by polymerase chain reaction (PCR) amplification of cpDNA fragments using three pairs of chloroplast universal primers. A total of 20 F(1)s from interspecific crosses among five different taxa in the section Bifolium was examined. Twelve F(1)s were from six crosses between L. cuneifolia (4x) and L. divaricata (2x) (Peru or Argentina) or L. tridentata (2x or 4x). Eight F(1)s were from two sets of reciprocal crosses between L. divaricata (2x) (Argentina) and L. tridentata (2x). Length polymorphism was observed in all three regions of cpDNA that separated L. cuneifolia parents from L. divaricata and L. tridentata parents and in one of the three cpDNA regions that differentiated L. divaricata (Argentina) parents from L. tridentata (2x) parents. In each case, it was the paternal cpDNA marker that appeared in the F(1) individuals. This was further confirmed by restriction fragment length polymorphism (RFLP) analysis of the amplified cpDNA fragments. Larrea may be the fifth genus reported in angiosperms with a paternal bias in cpDNA transmission. Possible mechanisms that may result in paternal cpDNA inheritance were briefly reviewed. Based on the observed uniparental paternal inheritance of cpDNA, restriction analysis of the three cpDNA regions and previous cytogenetic studies, L. divaricata was probably the maternal progenitor of L. cuneifolia.     

Historical biogeography and phylogenetic relationships of the genus Chamaecyparis (Cupressaceae) inferred from chloroplast DNA polymorphism

The genus Chamaecyparis comprises five species and one variety native to Taiwan, Japan, Canada, and USA, which demonstrates a classical eastern Asian, western North American, and eastern North American disjunct distributional pattern. The phylogenetic relationships of the species of Chamaecyparis were inferred by comparing 1130 bp of the combined data set of chloroplast trnV intron and petG-trnP intergenic spacer. The phylogenetic tree shows that Chamaecyparis nootkatensis (Cupressus nootkatensis or Xanthocyparis nootkatensis) is clearly diverged from other Chamaecyparis species. For Chamaecyparis species, C. thyoides is sister to C. formosensis and C. pisifera and these together form a monophyletic group. C. lawsoniana is sister to C. obtusa and C. taiwanensis; and these form another monophyletic group. Homogeneity in evolutionary rates was found among species in these two monophyletic groups. Results indicate the divergent evolution of C. taiwanensis and C. formosensis and molecular evidence in this investigation supports C. taiwanensis as a variety of C. obtusa. Utility of cpDNA intergenic spacer petG-trnP in Chamaecyparis is also discussed. Several biogeographical implications were inferred: (1) at least two divergence events have produced the eastern Asian, and both western and eastern North American disjunct distribution in Chamaecyparis; (2) intercontinental sister species pairs are found in Chamaecyparis; (3) cpDNA divergence between two intercontinental sister pairs of C. thyoides and C. pisifera, and C. lawsoniana and C. obtusa is 2.8% and 1.1%, which suggest an estimated divergence time of 14 and 5.5 million years ago during middle and late Miocene, respectively; (4) cpDNA divergence of two Asian Chamaecyparis groups between C. obtusa and C. taiwanensis, and between C. pisifera and C. formosensis is 0.25% and 0.57%, which suggest an estimated divergence time of 1.3 and 2.9 million years ago during Pleistocene and late Pliocene, respectively; these estimated divergence times suggest a relatively recent migration of Chamaecyparis to Taiwan from the Japanese Archipelago; (5) that climatic deterioration caused the disappearance of Chamaecyparis in continental Asia is probable.     

Phylogenetic patterns in the genus Manihot (Euphorbiaceae) inferred from analyses of nuclear and chloroplast DNA regions

From a phylogenetic perspective, the genus Manihot can be considered as an orphan group of plants, and the scientific knowledge acquired has been mainly related to cassava, one of the most important crops in poor tropical countries. The goal of the majority of evolutionary studies in the genus has been to decipher the domestication process and identify the closest relatives of cassava. Few investigations have focused on wild Manihot species, and the phylogeny of the genus is still unclear. In this study the DNA sequence variation from two chloroplast regions, the nuclear DNA gene G3pdh and two nuclear sequences derived from the 3'-end of two cassava ESTs, were used in order to infer the phylogenetic relationships among a subset of wild Manihot species, including two species from Cnidoscolus as out-groups. Maximum parsimony and Bayesian analyses were conducted for each data set and for a combined matrix due to the low variation of each region when analyzed independently. A penalized likelihood analysis of the chloroplast region trnL-trnF, calibrated with various age estimates for genera in the Euphorbiaceae extracted from the literature was used to determine the ages of origin and diversification of the genus. The two Mesoamerican species sampled form a well-defined clade. The South American species can be grouped into clades of varying size, but the relationships amongst them cannot be established with the data available. The age of the crown node of Manihot was estimated at 6.6 million years ago. Manihot esculenta varieties do not form a monophyletic group that is consistent with the possibility of multiple introgressions of genes from other wild species. The low levels of variation observed in the DNA regions sampled suggest a recent and explosive diversification of the genus, which is confirmed by our age estimates.     

Phylogenetic relationships in Monanthes (Crassulaceae) based on morphological,chloroplast and nuclear DNA variation

Phylogenetic relationships of all 10 recognized taxa of the genus Monanthes which is endemic to the Canary Islands and Salvage Islands, were investigated using the four data sets: morphology, sequences of the chloroplast DNA trnL (UAA) — trnF (GAA) intergenic spacer, ITS2 sequences of the nuclear ribosomal region and Random Amplified Polymorphic DNAs (RAPDs). In contrast to the molecular data, the morphological data were internally inconsistent which probably resulted from parallel or convergent evolution of morphological characters. The molecular data sets indicated that the genus is not monophyletic due to inclusion of the annual M. icterica which is the putative sister taxon of Aichryson, that M. polyphylla is the sister taxon of the perennial species of the genus, that M. muralis from Hierro is of allotetraploid origin and that M. lowei, M. minima, M. brachycaulos, M. laxiflora, M. anagensis and M. muralis from La Palma are closely related. Combined ITS2 sequences, trnL — trnF sequences and morphological data indicated that the relationships among three types of perennial growth forms, i.e. tiny rosettes, small, branched shrubs and diffuse branches shrublets, are highly dependent on the outgroup used. After deletion of the most distant outgroup and a taxon of alleged hybrid origin (M. muralis) relationships among the growth forms of Monanthes still could not be consistently resolved.     

Intraspecific differentiation of Allium wallichii (Amaryllidaceae) inferred from chloroplast DNA and internal transcribed spacer fragments

In the present study, we used two chloroplast DNA (cpDNA) fragments (trnL-F and rps16) and the nuclear ribosomal internal transcribed spacer (ITS) sequence data to examine intraspecific differentiation and phylogeographical history of Allium wallichii. Based on wide scale sampling (28 populations and 174 individuals) across the entire distribution range of this species, 33 cpDNA haplotypes and 25 ITS ribotypes were detected in our investigation. These cpDNA haplotypes were divided into three major lineages, which was further supported by the ITS phylogenetic results. High haplotype/ribotype diversity and population differentiation, together with most of the haplotypes/ribotypes being exclusive to single populations, implied restricted gene flow among populations and significant geographical isolation. Nearly all populations with high haplotype/ribotype diversity were found in the Hengduan Mountain Region (HMR), whereas the populations of the Himalayas and Nanling Mountains showed a lower level, suggesting the HMR might serve as a potential divergence center for A. wallichii. The main lineages of A. wallichii diverged from each other between Mid–Late Pliocene and Early Quaternary based on two sets of molecular markers, indicating that the Quaternary climatic fluctuation could not have contributed greatly to the divergence of the main lineages of A. wallichii. Instead, the intricate topography and heterogeneous habitats resulting from the drastic uplift of the Qinghai–Tibet Plateau from the Late Pliocene could be responsible for the intraspecific differentiation of A. wallichii. The present study further highlights the importance of geographic isolation and habitat heterogeneity in shaping and maintaining high species diversity within the HMR.     

A phylogeny of the European gentians inferred from chloroplast trnL (UAA) intron sequences

Phylogenetic relationships between the European species of the genus Gentiana L. (Gentianaceae) were inferred from chloroplast trnL , (UAA) intron sequence data. The phylogeny obtained is largely in accordance with the classification of species into sections Gentiana, Megalanthe and Calathianae. Few synapomorphies support the branching of the main lineages and thus could suggest a rapid radiation following the colonization of Europe. Within section Gentiana , our results are highly congruent with the previous distinction of G. montserratii Vivant from G. lutea L. Section Megalanthe is divided into two well separated lineages, both of which comprise calcicole and calcifuge species. The 'star phylogeny' obtained in section Calathianae suggests that most of the taxa speciated almost simultaneously. Relative-rate tests between two lineages suggested that section Chondropliyllae displays higher mutation rates than the rest of the genus Gentiana and that cpDNA can violate assumptions of rate constancy at lower taxonomic level.     

Phylogeny of the basal angiosperm genus Pseuduvaria (Annonaceae) inferred from five chloroplast DNA regions, with interpretation of morphological character evolution

Phylogenetic relationships within the magnoliid basal angiosperm genus Pseuduvaria (Annonaceae) are investigated using chloroplast DNA sequences from five regions: psbA-trnH spacer, trnL-F, matK, rbcL, and atpB-rbcL spacer. Over 4000 nucleotides from 51 species (of the total 53) were sequenced. The five cpDNA datasets were analyzed separately and in combination using maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. The phylogenetic trees constructed using all three phylogenetic methods, based on the combined data, strongly support the monophyly of Pseuduvaria following the inclusion of Craibella phuyensis. The trees generated using MP were less well resolved, but relationships are similar to those obtained using the other methods. ML and Bayesian analyses recovered trees with short branch lengths, showing five main clades. This study highlights the evolutionary changes in seven selected morphological characters (floral sex, stamen and carpel numbers, inner petal color, presence of inner petal glands, flowering peduncle length, and monocarp size). Although floral unisexuality is ancestral within the genus, several evolutionary lineages reveal reversal to bisexuality. Other phylogenetic transitions include the evolution of sapromyophily, and fruit-bat frugivory and seed dispersal, thus allowing a wide range of adaptations for species survival.