Molecular phylogenetic analysis of Violaceae (Malpighiales) based on plastid and nuclear DNA sequences

A phylogenetic analysis of Violaceae is presented using sequences from rbcL, atpB, matK and 18S rDNA from 39 species and 19 genera. The combined analysis of four molecular markers resulted in only one most parsimonious tree, and 33 of all 38 nodes within Violaceae are supported by a bootstrap proportion of more than 50%. Fusispermum is in a basal-most position and Rinorea, Decorsella, Rinoreocarpus and the other Violaceae are successively diverged. The monogeneric subfamily Fusispermoideae is supported, and it shares a number of plesiomorphies with Passifloraceae (a convolute petal aestivation, actinomorphic flowers and connate filaments). The other monogeneric subfamily Leonioideae is sunken within the subfamily Violoideae and is sister to Gloeospermum, sharing some seed morphological characteristics. The present molecular phylogenetic analysis suggests that the convolute, apotact and quincuncial petal aestivation is successively derived within the family. The evolutionary trends of the other morphological characteristics, such as a filament connation, the number of carpels and floral symmetry, are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phylogenetic relationships in Myrceugenia (Myrtaceae) based on plastid and nuclear DNA sequences

Myrceugenia is a genus endemic to South America with a disjunct distribution: 12 species occurring mainly in central Chile and approximately 25 in southeastern Brazil. Relationships are reconstructed within Myrceugenia from four plastid markers (partial trnK-matK, rpl32-trnL, trnQ-5'rps16 and rpl16) and two ribosomal nuclear regions (ETS and ITS) using maximum parsimony and Bayesian analyses. Relationships inferred previously from morphological data are not completely consistent with those from molecular data. All molecular analyses support the hypothesis that Myrceugenia is monophyletic, except for M. fernadeziana that falls outside the genus. Chilean species and Brazilian species form two separate lineages. Chilean species form three early diverging clades, whereas Brazilian species are a strongly supported monophyletic group in a terminal position. Least average evolutionary divergence, low resolution, short branches, and high species diversity found in the Brazilian clade suggest rapid radiation. Geographical distributions and phylogenetic reconstructions suggest that extant Myrceugenia species arose in northern Chile followed by colonization southward and finally to the Juan Fernández Islands and southeastern Brazil.     

A phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and plastid DNA sequences

Background and Aims

Laeliinae are a neotropical orchid subtribe with approx. 1500 species in 50 genera. In this study, an attempt is made to assess generic alliances based on molecular phylogenetic analysis of DNA sequence data.

Methods

Six DNA datasets were gathered: plastid trnL intron, trnL-F spacer, matK gene and trnK introns upstream and dowstream from matK and nuclear ITS rDNA. Data were analysed with maximum parsimony (MP) and Bayesian analysis with mixed models (BA).

Key Results

Although relationships between Laeliinae and outgroups are well supported, within the subtribe sequence variation is low considering the broad taxonomic range covered. Localized incongruence between the ITS and plastid trees was found. A combined tree followed the ITS trees more closely, but the levels of support obtained with MP were low. The Bayesian analysis recovered more well-supported nodes. The trees from combined MP and BA allowed eight generic alliances to be recognized within Laeliinae, all of which show trends in morphological characters but lack unambiguous synapomorphies.

Conclusions

By using combined plastid and nuclear DNA data in conjunction with mixed-models Bayesian inference, it is possible to delimit smaller groups within Laeliinae and discuss general patterns of pollination and hybridization compatibility. Furthermore, these small groups can now be used for further detailed studies to explain morphological evolution and diversification patterns within the subtribe.Key words: Laeliinae, Orchidaceae, ITS, trnL intron, trnL-F spacer, matK     

A re-evaluation of basal phylogenetic relationships within trogons (Aves: Trogonidae) based on nuclear DNA sequences

The avian clade Trogonidae (trogons) consists of approximately 40 species distributed pantropically in the Neotropical, Afrotropical and Indomalayan zoogeographical regions. In this study, we evaluate the basal phylogenetic relationships within the trogons based on DNA sequences from three nuclear introns [myoglobin intron 2, β -fibrinogen intron 7 and glyceraldehydes-3-phosphodehydrogenase (G3PDH) intron 11]. In addition, previously published cytochrome b and 12S sequences were re-analysed and combined with the nuclear data set. The analysis of the three nuclear genes combined suggests a sister group relationship between the Afrotropical ( Apaloderma ) and Indomalayan ( Harpactes ) clades, whereas the Neotropical taxa ( Trogon , Pharomachrus , and Priotelus ) form an unresolved polytomy basal to these two groups. In addition, two of the three individual gene trees also support a sister group relationship between the Afrotropical and Indomalayan trogons. This is at odds with previously published studies based on mitochondrial sequence data and DNA–DNA hybridization. The third nuclear intron (G3PDH), however, suggests that the Afrotropical trogons are basal relative the other trogons. This was also suggested by the mitochondrial data set, as well as the analysis of the combined nuclear and mitochondrial data. Both of these conflicting hypotheses are supported by high posterior probabilities. An insertion in β -fibrinogen further supports a basal position of the Afrotropical clade. Analyses of the myoglobin intron with additional outgroups place the root differently and strongly support monophyly of each of the zoogeographical regions (including the Neotropics), and these three clades form a basal trichotomy. This suggests that that rooting is a serious problem in resolving basal phylogenetic relationships among the trogons.     

Species boundaries and phylogenetic relationships in the critically endangered Asian box turtle genus Cuora

Turtles are currently the most endangered major clade of vertebrates on earth, and Asian box turtles (Cuora) are in catastrophic decline. Effective management of this diverse turtle clade has been hampered by human-mediated, and perhaps natural hybridization, resulting in discordance between mitochondrial and nuclear markers and confusion regarding species boundaries and phylogenetic relationships among hypothesized species of Cuora. Here, we present analyses of mitochondrial and nuclear DNA data for all 12 currently hypothesized species to resolve both species boundaries and phylogenetic relationships. Our 15-gene, 40-individual nuclear data set was frequently in conflict with our mitochondrial data set; based on its general concordance with published morphological analyses and the strength of 15 independent estimates of evolutionary history, we interpret the nuclear data as representing the most reliable estimate of species boundaries and phylogeny of Cuora. Our results strongly reiterate the necessity of using multiple nuclear markers for phylogeny and species delimitation in these animals, including any form of DNA "barcoding", and point to Cuora as an important case study where reliance on mitochondrial DNA can lead to incorrect species identification.     

Molecular phylogenetic analysis of Passifloraceae sensu lato (Malpighiales) based on plastid and nuclear DNA sequences

A phylogenetic analysis of Passifloraceae sensu lato was performed using rbcL, atpB, matK, and 18S rDNA sequences from 25 genera and 42 species. Parsimony analyses of combined data sets resulted in a single most parsimonious tree, which was very similar to the 50% majority consensus tree from the Bayesian analysis. All nodes except three were supported by more than 50% bootstrap. The monophyly of Passifloraceae s.l. as well as the former families, Malesherbiaceae, Passifloraceae sensu stricto, and Turneraceae were strongly supported. Passifloraceae s.s. and the Turneraceae are sisters, and form a strongly supported clade. Within Passifloraceae s.s., the tribes Passifloreae and Paropsieae are both monophyletic. The intergeneric relationships within Passifloraceae s.s. and Turneraceae are roughly correlated with previous classification systems. The morphological character of an androgynophore/gynophore is better used for characterizing genera grouping within Passifloraceae s.s. Other morphological characters such as the corona and aril are discussed.     

Species boundaries,phylogenetic relationships,and ecological differentiation inAnthriscus (Apiaceae)

Species boundaries and phylogenetic relationships of 17 taxa ofAnthriscus (Apiaceae), with special emphasis on the critical sect.Cacosciadium, were explored using morphological data with principal component analysis, phenetics, and phylogenetics. The analyses did not provide satisfactory resolution of taxa from sect.Cacosciadium and only four species were retained. The total number of species was reduced to nine. Sect.Cacosciadium is distinguished by only two synapomorphies while sects.Anthriscus andCaroides are better supported. Present geographic and ecological variation suggests that the radiation ofAnthriscus occurred through divergence of peripheral isolated populations adapting to different habitats: high montane meadows and screes, shady climax forests, and seasonally dry habitats at lower altitudes. The adaptive significance of particular morphological traits is discussed.     

Species boundaries within Saprolegnia (Saprolegniales, Oomycota) based on morphological and DNA sequence data

Saprolegnia is a common and widespread genus of Oomycetes, however species identifications are difficult and uncertain. To test whether keys based on morphological characters could identify species as determined by molecular characters we determined partial DNA sequences for the 28S rRNA gene and the complete internal transcribed spacer (ITS) region for 55 isolates belonging to Saprolegnia and one isolate of Protoachlya hypogyna that exhibited saprolegnoid zoospore discharge in water culture. Phylogenetic analyses of the combined sequence data yielded 10 robustly supported clades that probably represent separate species. Morphological analyses of all isolates revealed that each DNA-based clade could be delimited from others by autapomorphic or unique combinations of morphological character states but not without employing several features previously not used at the species level. Taxonomic implications of these results are discussed and recommendations for less equivocal characterization of new Saprolegnia species are made.     

Mitochondrial DNA sequences of six sturgeon species and phylogenetic relationships within Acipenseridae

Studies on mitochondrial DNA variability amongst six sturgeon species reared in Italian aquaculture plants are reported. Restriction analysis by Rsal of mitochondrial cytochrome b and D-loop fragments amplified by PCR permitted identification of interspecific variations that would be suitable as markers for species diagnosis. Data obtained by partial sequencing of the cytochrome b gene, analyzed through maximum parsimony, neighbour joining, and UPGMA distance methods, revealed species clusters that support previous morpho-meristic and geographical data for sturgeon classification into sub-genera. According to our cluster analysis, the genera Acipenser and Huso are monophyletic. Moreover, these and previous cytogenetic data suggest that three 120-chromosome species (H. huso, H. dauricus and A. ruthenus) are at the base of sturgeon differentiation, which occurred through two different events of polyploidization.     

Molecular phylogenetic relationships of the deep-sea fish genus Coryphaenoides (Gadiformes: Macrouridae) based on mitochondrial DNA

In order to characterize the phylogenetic relationship and deep-sea adaptation process of the deep-sea fish genus Coryphaenoides, the nucleotide sequences of the mitochondrial (mt) 12 S rRNA and COI gene sequences for seven Coryphaenoides species were analyzed. Our molecular phylogenetic tree shows a new arrangement of seven Coryphaenoides species, which form two distinct groups, abyssal and nonabyssal species, and differs from the results of previous taxonomic studies. Using the mutation rate of mitochondrial genes, the divergence time between abyssal and nonabyssal Coryphaenoides was found to be 3.2-7.6 million years ago. Our study suggests that hydraulic pressure plays an important role in the speciation process in the marine environment.     

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1-->3)-beta-d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked beta-d-galactopyranose residues connected by (1-->6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1-->3) and (1-->6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals (R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.     

A phylogenetic hypothesis for the Aizoaceae (Caryophyllales) based on four plastid DNA regions

The Aizoaceae is the largest family of leaf succulent plants, and most of its species are endemic to southern Africa. To evaluate subfamilial, generic, and tribal relationships, we produced two plastid DNA data sets for 91 species of Aizoaceae and four outgroups: rps16 intron and the trnL-F gene region (both the trnL intron and the trnL-F intergenic spacer). In addition, we generated two further plastid data sets for 56 taxa restricted to members of the Ruschioideae using the atpB-rbcL and the psbA-trnH intergenic spacers. In the combined tree of the rps16 intron and trnL-F gene region, three of the currently recognized subfamilies (Sesuvioideae, Mesembryanthemoideae, and Ruschioideae) are each strongly supported monophyletic groups. The subfamily Tetragonioideae is polyphyletic, with Tribulocarpus as sister to the Sesuvioideae and Tetragonia embedded in the Aizooideae. Our study showed that the group consisting of the Sesuvioideae, Aizooideae, and Tetragonioideae does not form a monophyletic entity. Therefore, it cannot be recognized as a separate family in order to accommodate the frequently used concept of the Mesembryanthemaceae or "Mesembryanthema," in which the subfamilies Mesembryanthemoideae and Ruschioideae are included. We also found that several genera within the Mesembryanthemoideae (Mesembryanthemum, Phyllobolus) are not monophyletic. Within the Ruschioideae, our study retrieved four major clades. However, even in the combined analysis of all four plastid gene regions, relationships within the largest of these four clades remain unresolved. The few nucleotide substitutions that exist among taxa of this clade point to a rapid and recent diversification within the arid winter rainfall area of southern Africa. We propose a revised classification for the Aizoaceae.     

Species relationships within Diplotaxis (Brassicaceae) and the phylogenetic origin of D. muralis

Nineteen from the ca. 30 Diplotaxis species including all known haploid chromosome numbers have been analysed for isoelectric focusing patterns of Rubisco, allozymes and RAPDs. D. erucoides (n=7) was clearly separated from all other species as were D. harra and D. crassifolia (n=13 each). Taxa with n=8 had different IEF patterns, but allozyme data grouped D. siettiana, D. ibicensis and D. brevisiliqua together. Species with n=9 were characterised by different IEF patterns, and their position was neither resolved in the allozyme nor in the RAPD tree. Only the D. catholica accessions were strongly clustered together. D. viminea and D. siifolia (both n=10) were kept separate, whereas the n=11 taxa D. tenuifolia, D. cretacea and D. simplex grouped together. Data confirm D. viminea as maternal parent of the allotetraploid D. muralis, and D. tenuifolia as the likely parent.     

A phylogenetic analysis of Diurideae (Orchidaceae) based on plastid DNA sequence data

DNA sequence data from plastid matK and trnL-F regions were used in phylogenetic analyses of Diurideae, which indicate that Diurideae are not monophyletic as currently delimited. However, if Chloraeinae and Pterostylidinae are excluded from Diurideae, the remaining subtribes form a well-supported, monophyletic group that is sister to a "spiranthid" clade. Chloraea, Gavilea, and Megastylis pro parte (Chloraeinae) are all placed among the spiranthid orchids and form a grade with Pterostylis leading to a monophyletic Cranichideae. Codonorchis, previously included among Chloraeinae, is sister to Orchideae. Within the more narrowly delimited Diurideae two major lineages are apparent. One includes Diuridinae, Cryptostylidinae, Thelymitrinae, and an expanded Drakaeinae; the other includes Caladeniinae s.s., Prasophyllinae, and Acianthinae. The achlorophyllous subtribe Rhizanthellinae is a member of Diurideae, but its placement is otherwise uncertain. The sequence-based trees indicate that some morphological characters used in previous classifications, such as subterranean storage organs, anther position, growth habit, fungal symbionts, and pollination syndromes have more complex evolutionary histories than previously hypothesized. Treatments based upon these characters have produced conflicting classifications, and molecular data offer a tool for reevaluating these phylogenetic hypotheses.     

Phylogenetic relationships among middle American pocket gophers (genus Orthogeomys) based on mitochondrial DNA sequences.

Relationships among members representing each of the three subgenera of the Middle American rodent genus Orthogeomys (Rodentia: Geomyidae) were studied by comparing DNA sequence data from two regions of the mitochondrial genome. Results from 527 bp from the 16 S rDNA region and a 402-bp fragment of the cytochrome b gene indicate that the three subgenera are well differentiated genetically, with the subgenus Orthogeomys being distantly related to Macrogeomys and Heterogeomys, and Macrogeomys appearing as the most derived. Within the subgenus Macrogeomys, O. heterodus and O. cherriei form a distinct clade, as do O. dariensis and O. cavator. As with previous protein-electrophoretic studies, the placement of O. underwoodi could not be determined definitively within the subgenus Macrogeomys. We interpret our inability to determine phylogenetic relationships among these three clades as evidence for a rapid phyletic radiation within this subgenus. Sequence divergence estimates indicate that the Macrogeomys radiation took place following the time of completion of the Panamanian land bridge (1.9-2.9 mya). Additionally, the near identity of sequences of a newly described species, O. thaeleri, with those of O. dariensis (percentage sequence divergence = 0.3%) suggests that the two may be conspecific.     

Molecular phylogenetics of Meliaceae (Sapindales) based on nuclear and plastid DNA sequences

Phylogenetic analyses of Meliaceae, including representatives of all four currently recognized subfamilies and all but two tribes (32 genera and 35 species, respectively), were carried out using DNA sequence data from three regions: plastid genes rbcL, matK (partial), and nuclear 26S rDNA (partial). Individual and combined phylogenetic analyses were performed for the rbcL, matK, and 26S rDNA data sets. Although the percentage of informative characters is highest in the segment of matK sequenced, rbcL provides the greatest number of informative characters of the three regions, resulting in the best resolved trees. Results of parsimony analyses support the recognition of only two subfamilies (Melioideae and Swietenioideae), which are sister groups. Melieae are the only tribe recognized previously that are strongly supported as monophyletic. The members of the two small monogeneric subfamilies, Quivisianthe and Capuronianthus, fall within Melioideae and Swietenioideae, respectively, supporting their taxonomic inclusion in these groups. Furthermore, the data indicate a close relationship between Aglaieae and Guareeae and a possible monophyletic origin of Cedreleae of Swietenioideae. For Trichilieae (Melioideae) and Swietenieae (Swietenioideae) lack of monophyly is indicated.     

Species relationships in the genus Vasconcellea (Caricaceae) based on molecular and morphological evidence

Validity of the taxa currently recognized in the genus Vasconcellea was analyzed by investigating morphological and molecular data from 105 specimens of this genus and six specimens of the related genus Carica. Taxon identification of these specimens was compared with clustering in two phenetic dendrograms generated with 36 morphological characters and 254 amplified fragment length polymorphic (AFLP) markers. Moreover, cytoplasmic haplotypes were assessed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of one mitochondrial and two chloroplast DNA regions. Results show that the morphological data set, containing mainly vegetative characteristics, merely reveals external resemblance between specimens, which is not directly associated with genetic relationships and taxon validity. Phenotypic plasticity and intercompatibility between several species are likely to confuse morphological delimitation of the taxa. Based on the results of our study, several specimens that could not be identified with the currently used identification key (1) could be attributed to a known taxon, which should be extended to include a higher range of morphological variability or (2) could be hypothesized to be of hybrid origin. Because of the high intraspecific variation within V. microcarpa and V. × heilbornii, revision of these taxa is recommended.     

A new species of Anisopappus (Asteraceae, Inuleae) and its phylogenetic relationships within the genus

A new species of the genus Anisopappus (Asteraceae, Inuleae) from Angola, A. fruticosus , is described. The principal differences with respect to A. chinensis are discussed, and phylogenetic relationships with the other species of the genus are investigated by means of a cladistic analysis using morphological characters.