Comparative analysis of a translocated copy of the trnK intron in carnivorous family Nepenthaceae

During phylogenetic analysis of the Nepenthaceae cpDNA trnK intron, it became apparent that a second non-functional copy of the locus was present in most of the investigated taxa. The translocation event was older than the radiation of all recent Nepenthaceae, and the translocated pseudogenized copy was conserved in nearly all members of the plant family. Using single chloroplast PCR and inverse PCR, we could exclude a plastom location for the second copy. Although translocation into the nucleus is possible, mitochondrial localization seems more likely based on these data. In total, the translocated sequence contained at least 3525 base pairs (bp) that were homologous to the Spinacia oleracea chloroplast genome. Comparative phylogenetic analysis of the non-functional copy revealed a high amount of homoplasies compared to topologies from the cpDNA trnK intron phylogenetic reconstruction. Therefore, this copy proved to be insufficient for phylogenetic reconstruction of the family. Since two different paralogs of the non-functional copy were found in one species, it is feasible that different paralogs were conserved in different groups and that paralogous sequences were included in the data matrix. These data demonstrate that phylogenetic analyses of pseudogenized copies of phylogenetically relevant loci should be performed with great caution. In addition, pseudogenized copies can exist in nearly every member of a plant family, and can be PCR-amplified at levels comparable to the specific copy. In this case, the inclusion of such copies can easily remain unnoticed, thus leading to faulty hypotheses.     

Deep genomic analysis of Coelastrella saipanensis (Scenedesmaceae,Chlorophyta): comparative chloroplast genomics of Scenedesmaceae

Many species belonging to the coccoid green algae genus Coelastrella are considered potential candidates for the large-scale production of natural pigments and biofuels. However, little is known about the structural, functional and molecular aspects of the chloroplast genomes (cpDNAs) of this genus. In the present study, the complete sequence of the cpDNA of strain FACHB-2138, which was further identified as Coelastrella saipanensis Hanagata based on morphological and molecular analyses, was elucidated. The 196 140 bp cpDNA sequence that was assembled as a circular map was found to possess the typical quadripartite structure. The two identical copies of 11 897 bp inverted repeat (IR) sequences were separated from one another by single copy regions. The large single copy region (LSC) was 104 949 bp, whereas the small single copy region (SSC) was 67 397 bp. The cpDNA encoded a total of 96 unique genes, which included 67 protein-coding genes, three rRNA genes and 26 tRNA genes. A total of 19 group I introns were annotated in this genome. Comparative analyses with three species from the family Scenedesmaceae showed C. saipanensis had a slightly expanded genome, higher GC content and less skewed distribution of its genes between the two DNA strands than that of the other three species. The cpDNA data deduced from the present study helps to expand our present understanding of plant systematics and phylogenetic reconstruction, and identify the possible biotechnological applications of the species belonging to the studied taxa.     

Phylogeny and diversification of Chinese Araliaceae based on nuclear and plastid DNA sequence data

Chinese Araliaceae consist of 20 genera and ca. 175 species. To assess the evolutionary relationships of Araliaceae and their biogeographic diversification in China, the phylogeny of Chinese Araliaceae was constructed by sampling 96 accessions representing 20 genera and 50 species of Chinese Araliaceae and 45 closely related taxa using sequences of the nuclear ribosomal internal transcribed spacer (ITS) region and six plastid regions (the ndhF gene, the trnL-trnF region, the rps16intron, the atpB-rbcL intergenic spacer, the rpl16 intron, and the psbA-trnH intergenic spacer). Phylogenetic analyses of the combined plastid and ITS data supported the results of the previously studies that the Chinese members of Araliaceae were scattered within the Asian Palmate group and the Aralia-Panax group withOsmoxylon at the base of core Araliaceae. The generic status of Pentapanax and Tupidanthus is not supported. Our analysis clearly places them in Aralia and AsianSchefflera, respectively. In a broader phylogenetic framework of Araliaceae, based on the fossil-calibrated Bayesian dating, Chinese Araliaceae was inferred to have originated in Asia and underwent a rapid radiation in its evolutionary history. Its diversification is hypothesized to have been driven largely by the orogenies in Asia during the Cenozoic. In China, the distribution pattern of the phylogenetic diversity of Araliaceae corresponds with its taxonomic diversity across the entire region.     

Extensive intraspecific chloroplast DNA (cpDNA) variation in the alpine Draba aizoides L. (Brassicaceae): haplotype relationships and population structure

Chloroplast DNA (cpDNA) sequence variation is currently the most widely used tool for the inference of phylogenetic relationships among plants at all taxonomic levels. Generally, noncoding regions tend to evolve faster than coding sequences and have recently been applied to the study of phylogenetic relationships among closely related taxa. An implicit assumption of many of these studies is that intraspecific cpDNA variation is either absent or low and therefore will not interfere with the reconstruction of interspecific relationships. A survey of cpDNA sequence variation in the common alpine plant species Draba aizoides L. was undertaken to assess levels of intraspecific cpDNA sequence variation. These levels were compared to levels of interspecific sequence divergence between D. aizoides and related alpine Draba species. Intraspecific cpDNA sequence divergence was extensive in D. aizoides, and intraspecific differences were often larger than interspecific differences. cpDNA haplotype relationships were explored using a maximum parsimony approach and minimum-spanning networks. Results from both methods were largely congruent but comparisons provided interesting insights into the presumed evolutionary history of cpDNA haplotypes. A combined effect of cpDNA introgression and complex lineage sorting was inferred to explain the pattern of cpDNA variation found in D. aizoides. Our results suggest that intraspecific cpDNA variation can be extensive and that intraspecific variation needs to be taken into account when inferring phylogenetic relationships among closely related taxa.     

Polyphyletic origin in <Emphasis Type="Italic">Pimpinella</Emphasis> (Apiaceae): evidence in Western Europe

The genus Pimpinella L. comprises about 150 species, being one of the largest genera within the family Apiaceae (subfamily Apioideae). Previous molecular phylogenetic studies have shown that Pimpinella is a taxonomically complex group. In this study, evolutionary relationships among representatives from Western Europe have been inferred from phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer (ITS 1 and ITS 2) and plastid sequences (trnL intron and the trnL-F spacer), with a representative sampling included (168 accessions in the ITS analysis, representing 158 species; and 42 accessions in the cpDNA analysis representing 35 taxa of Pimpinella and closely related species). All analyses resolved that Pimpinella is a non-monophyletic group, and Pimpinella’s taxa that grow in Western Europe are part of phylogenetically independent groups that correspond to three different tribes of the subfamily Apioideae: Pimpinelleae (core group), Pyramidoptereae and Smyrnieae.     

Widespread genealogical nonmonophyly in species of Pinus subgenus Strobus

Phylogenetic relationships among Pinus species from subgenus Strobus remain unresolved despite combined efforts based on nrITS and cpDNA. To provide greater resolution among these taxa, a 900-bp intron from a late embryogenesis abundant (LEA)-like gene (IFG8612)was sequenced from 39 pine species, with two or more alleles representing 33 species. Nineteen of 33 species exhibited allelic nonmonphyly in the strict consensus tree, and 10 deviated significantly from allelic monophyly based on topology incongruence tests. Intraspecific nucleotide diversity ranged from 0.0 to 0.0211, and analysis of variance shows that nucleotide diversity was strongly associated (P < 0.0001)with the degree of species monophyly. Although species nonmonophyly complicates phylogenetic interpretations, this nuclear locus offers greater topological support than previously observed for cpDNA or nrITS. Lacking evidence for hybridization, recombination, or imperfect taxonomy, we feel that incomplete lineage sorting remains the best explanation for the polymorphisms shared among species. Depending on the species, coalescent expectations indicate that reciprocal monophyly will be more likely than paraphyly in 1.71 to 24.0 x 10(6) years, and that complete genome-wide coalescence in these species may require up to 76.3 x 10(6) years. The absence of allelic coalescence is a severe constraint in the application of phylogenetic methods in Pinus, and taxa sharing similar life history traits with Pinus are likely to show species nonmonophyly using nuclear markers.     

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

Chickpea (Cicerarietinum, Leguminosae), an important grain legume, is widely used for food and fodder throughout the world. We sequenced the complete plastid genome of chickpea, which is 125,319bp in size, and contains only one copy of the inverted repeat (IR). The genome encodes 108 genes, including 4 rRNAs, 29 tRNAs, and 75 proteins. The genes rps16, infA, and ycf4 are absent in the chickpea plastid genome, and ndhB has an internal stop codon in the 5'exon, similar to other legumes. Two genes have lost their introns, one in the 3'exon of the transpliced gene rps12, and the one between exons 1 and 2 of clpP; this represents the first documented case of the loss of introns from both of these genes in the same plastid genome. An extensive phylogenetic survey of these intron losses was performed on 302 taxa across legumes and the related family Polygalaceae. The clpP intron has been lost exclusively in taxa from the temperate "IR-lacking clade" (IRLC), whereas the rps12 intron has been lost in most members of the IRLC (with the exception of Wisteria, Callerya, Afgekia, and certain species of Millettia, which represent the earliest diverging lineages of this clade), and in the tribe Desmodieae, which is closely related to the tribes Phaseoleae and Psoraleeae. Data provided here suggest that the loss of the rps12 intron occurred after the loss of the IR. The two new genomic changes identified in the present study provide additional support of the monophyly of the IR-loss clade, and resolution of the pattern of the earliest-branching lineages in this clade. The availability of the complete chickpea plastid genome sequence also provides valuable information on intergenic spacer regions among legumes and endogenous regulatory sequences for plastid genetic engineering.     

Development of PCR primer sets for intron 1 of the low-copy gene LEAFY in Davalliaceae

? Premise of the study: Primers were designed for amplifying intron 1 of the single-copy nuclear LEAFY gene for species of Davalliaceae. ? Methods and Results: New primer sets were designed and successfully amplified for intron 1 of the LEAFY gene in 13 species representing the five genera of Davalliaceae. The orthology of these sequences was further confirmed by phylogenetic analyses. Site variation in LEAFY intron 1 sequences across genera of the Davalliaceae and among accessions of the Humata repens complex were 18% and 8%, respectively. Such variation was greater than that for the cpDNA atpB-rbcL intergenic spacer region across the same taxa and accessions. ? Conclusions: Using our newly designed primers, intron 1 of the LEAFY gene could be amplified for all species tested. In addition, this single-copy, biparentally inherited, and quickly evolving region showed considerable potential for addressing infraspecific-level questions.     

Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium (Liliaceae; Liliales) and the infrageneric classification of Fritillaria

We present phylogenetic analyses of 37 taxa of Fritillaria (Liliaceae), 15 species of Lilium, and several outgroup taxa from Liliaceae s.s. to investigate the generic delimitation of Fritillaria in relation to Lilium as well as infrageneric relationships within Fritillaria. We used DNA sequences from the maturase-coding plastid matK gene and the trnK intron, the intron of the ribosomal protein-coding rpl16 plastid gene, and the nuclear ribosomal internal transcribed spacers (ITS). Phylogenetic analysis using maximum parsimony defined Fritillaria and Lilium (the latter including Nomocharis) as sister taxa. Fritillaria sections Fritillaria and Liliorhiza are supported in part, and some of the most enigmatic species usually included in Fritillaria (sections Petilium and Theresia and the monotypic genus Korolkowia) are closely related. The results support the new classification of Fritillaria proposed by Rix. We postulate independent origins of the underground bulbils found in Fritillaria davidii and the remainder of subgenus Liliorhiza.     

Inferring phylogeny at low taxonomic levels: utility of rapidly evolving cpDNA and nuclear ITS loci

Plant molecular systematic studies of closely related taxa have relied heavily on sequence data from nuclear ITS and cpDNA. Positive attributes of using ITS sequence data include the rapid rate of evolution compared to most plastid loci and availability of universal primers for amplification and sequencing. On the other hand, ITS sequence data may not adequately track organismal phylogeny if concerted evolution and high rDNA array copy number do not permit identification of orthologous copies. Shaw et al. (American Journal of Botany 92: 142-166) recently identified nine plastid regions that appear to provide more potentially informative characters than many other plastid loci. In the present study, sequences of these loci and ITS were obtained for six taxonomic groups in which phylogenetic relationships have been difficult to establish using other data. The relative utility of these regions was compared by assessing the number of parsimony informative characters, character congruence, resolution of inferred trees, clade support, and accuracy. No single locus emerged as the best in all lineages for any of these measures of utility. Results further indicated that in preliminary studies, sampling strategy should include at least four exemplar taxa. The importance of sampling data from independent distributions is also discussed.     

When is enough,enough in phylogenetics? A case in point from Hordeum (Poaceae)

Direct optimization was used to reconstruct the phylogeny of the 26 diploid taxa included in the genus Hordeum. The total data set was composed of 16 nucleotide sequence regions from the nuclear as well as the plastid genome. The nine nuclear regions were from single‐copy, protein coding genes located on six of the seven chromosome pairs in the diploid H. vulgare genome. The seven plastid regions comprise protein coding genes as well as intergenic regions. Studies of character congruence between data partitions showed no correlation between chromosomal location and congruence among the nuclear sequences and a level of congruence among the plastid sequences comparable with the level among the nuclear sequences. Combined analysis of all data resolved the phylogeny completely with most clades being robust and well supported. However, due to incongruence among data partitions some relationships are still and likely to remain ambiguously inferred. Rather than adding still more genes to the phylogenetic analyses, patterns of incongruence may be better explored by adding data from multiple specimens per taxon. For some species relationships the plastid data appear positively misleading, emphasizing the need for caution if plastid data are the only or dominant type of data used for phylogenetic reconstruction and subsequent re‐classification.
© The Willi Hennig Society 2011.     

Giants and dwarfs: molecular phylogenies reveal multiple origins of annual spurges within Euphorbia subg. Esula

Euphorbia (Euphorbiaceae) comprises over 2150 species and is thus the second-largest genus of flowering plants. In Europe, it is represented by more than 100 species with highest diversity in the Mediterranean area; the majority of taxa belong to subgenus Esula Pers., including about 500 taxa. The few available phylogenetic studies yielded contrasting results regarding the monophyly of subg. Esula, and the phylogenetic relationships among its constituents remain poorly understood. We have sampled DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid trnT-trnF region from about 100, predominantly European taxa of subg. Esula in order to infer its phylogenetic history. The plastid data support monophyly of subg. Esula whereas the ITS phylogeny, which is generally less resolved, is indecisive in this respect. Although some major clades have partly incongruent positions in the ITS and plastid phylogenies, the taxonomic content of the major terminal clades is congruent in both trees. As traditional sectional delimitations are largely not corroborated, an improved classification is proposed. Character state reconstruction illustrates that the annual life form developed independently several times in different clades of subgenus Esula from perennial ancestors, and that several morphological traits used in previous classifications of Euphorbia developed in parallel in different lineages.     

Genome size and plastid trnK-matK markers give new insights into the evolutionary history of the genus Lavandula L.

The genus Lavandula L. consists of 39 species distributed from the North Atlantic Islands, across the Mediterranean Basin to India. We analysed 36 taxa of the genus Lavandula representing two of the three subgenera and six of the eight sections according to the most recent classification (Upson & Andrews 2004). We achieved a phylogenetic reconstruction from partial sequences from plastid trnK and matK genes; the genome size was estimated by flow cytometer measurements. The primary aim was to track phylogenetic patterns through the maternal inherited marker at the sectional level and identify possible genome duplications. The cpDNA tree shows the phylogenetic relationships between subgenus, sections and also elucidates for the first time the relationships between the endemic species of Macaronesia, Morocco and Arabia. The ancestral split between the two subgenera could be explained by dispersal followed by an early vicariance event. The C-value shows genome up-sizing within several phylogenetic clades and geographical areas. An ancestral genome-up sizing is characterized at the node of section Dentatae and Lavandula. The cpDNA tree suggests that the taxa L. angustifolia subsp. pyrenaica (DC.) Guinea and L. stoechas subsp. luiseiri are best treated as a distinct species.     

Molecular evolution of insertions and deletion in the chloroplast genome of silene

Insertions, deletions, and inversions in the chloroplast genome of higher plants have been shown to be extremely useful for resolving phylogenetic relationships both between closely related taxa and among more basal lineages. Introns and intergenic spacers from the chloroplast genome are now increasingly used for phylogenetic and population genetic studies of populations from a single species, and it is therefore interesting to know whether indels can provide useful data and hence increase the power of intraspecific studies. Here, we show that indels in three cpDNA intergenic spacers and one cpDNA intron for two species of Silene evolve at slightly higher rates than base pair substitutions. Repeat indels appear to have the highest rate of evolution and are thus more prone to homoplasy. We show that coded indel data have high information content for phylogenetic analysis, and indels thus provide useful information to infer phylogenetic relationships at the intraspecific level.     

Reticulate phylogenetics and phytogeographical structure of Heliosperma (Sileneae, Caryophyllaceae) inferred from chloroplast and nuclear DNA sequences

The Balkan Peninsula is known to be one of the most diverse and species-rich parts of Europe, but its biota has gained much less attention in phylogenetic and evolutionary studies compared to other southern European mountain systems. We used nuclear ribosomal internal transcribed spacer (ITS) sequences and intron sequences of the chloroplast gene rps16 to examine phylogenetic and biogeographical patterns within the genus Heliosperma (Sileneae, Caryophyllaceae). The ITS and rps16 intron sequences both support monophyly of Heliosperma, but the data are not conclusive with regard to its exact origin. Three strongly supported clades are found in both data sets, corresponding to Heliosperma alpestre, Heliosperma macranthum and the Heliosperma pusillum clade, including all other taxa. The interrelationships among these three differ between the nuclear and the plastid data sets. Hierarchical relationships within the H. pusillum clade are poorly resolved by the ITS data, but the rps16 intron sequences form two well-supported clades which are geographically, rather than taxonomically, correlated. A similar geographical structure is found in the ITS data, when analyzed with the NeighbourNet method. The apparent rate of change within Heliosperma is slightly higher for rps16 as compared to ITS. In contrast, in the Sileneae outgroup, ITS substitution rates are more than twice as high as those for rps16, a situation more in agreement with what has been found in other rate comparisons of noncoding cpDNA and ITS. Unlike most other Sileneae ITS sequences, the H. pusillum group sequences display extensive polymorphism. A possible explanation to these patterns is extensive hybridization and gene flow within Heliosperma, which together with concerted evolution may have eradicated the ancient divergence suggested by the rps16 data. The morphological differentiation into high elevation, mainly widely distributed taxa, and low elevation narrow endemics is not correlated with the molecular data, and is possibly a result of ecological differentiation.     

Phylogenetic Use of Noncoding Regions in the GenusGentianaL.: ChloroplasttrnL (UAA) Intron versus Nuclear Ribosomal Internal Transcribed Spacer Sequences

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA)trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genusGentianaL. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in thetrnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least inGentiana,ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNAtrnL intron seems to be preferable at the intergeneric level.     

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.