TrnL–trnF Intergenic Spacer and trnL Intron Define Major Clades Within Luzula and Juncus (Juncaceae): Importance of Structural Mutations

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL–trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion o within a pseudogene) within the chloroplast trnL–trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The Southern Hemisphere clade was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5 acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL–trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.     

A Preliminary Molecular Phylogeny of the Rhynchosporeae (Cyperaceae)

The tribe Rhynchosporeae comprises the genera Rhynchospora and Pleurostachys and has never been studied using molecular techniques. The objective of this study was to use an analysis of trnL-F sequences to evaluate the hypothesized taxonomic divisions within the Rhynchosporeae including the monophyly of the genera and the soundness of the subgenera and sections. A total of 44 ingroup species were studied, 41 of Rhynchospora representing 22 of Kükenthal’s 28 sections, and three of Pleurostachys. Five outgroup species from other genera were also included. The cladistic analysis of 50 trnL intron and trnL-F intergenic spacer sequences resulted in 16 trees and a strict consensus tree. The Rhynchosporeae form two well-supported primary clades with several well-supported smaller clades, many of which agree with previously hypothesized sections. Pleurostachys is embedded within the second primary clade.     

The architecture of the chloroplast psbA-trnH non-coding region in angiosperms

The psbA-trnH intergenic region is among the most variable regions in the angiosperm chloroplast genome. It is a popular tool for plant population genetics and species level phylogenetics and has been proposed as suitable for DNA barcoding studies. This region contains two parts differing in their evolutionary conservation: 1) the psbA 3′UTR (untranslated region) and 2) the psbA-trnH intergenic non-transcribed spacer. We compared the sequence and RNA secondary structure of the psbA 3′ UTR across angiosperms and found consensus motifs corresponding to the stem portions of the RNA stem-loop structures and a consensus TTAGTGTATA box. The psbA-trnH spacer exhibited patterns that can be explained by the independent evolution of large inversions in the psbA 3′UTR and mutational hot spots in the remaining portion of the psbA-trnH spacer. We conclude that a comparison of chloroplast UTRs across angiosperms offer clues to the identity of putative regulatory elements and information about selective constraints imposed on the chloroplast non-coding regions.     

Cytoplasmic diversity,phylogenetic relationships and molecular evolution of Tunisian Citrus species as inferred from mutational events and pseudogene of chloroplast trnL-trnF spacer

The genus Citrus L. is among the most important fruit trees in the world. In this report, cytoplasmic polymorphism of twenty seven Tunisian Citrus cultivars was explored using the chloroplast trnL-trnF intergenic spacer. Chloroplast sequences showed variation in length and nucleotide content. Haplotype and nucleotide diversity showed low variations. Molecular phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups distinguishing between mandarin and pummelo groups. The trnL-trnF intergenic spacer showed one copy of pseudogene of the original trnF gene in 27 Citrus species at position 275 bp with a size varying from 49 to 63 bp. The anticodon domain was identified as the most conserved element, but one transversion (T−>C) was found in the D-domain. Meanwhile, one transversion (T−>A) and one transition (T−>G) were found in the T-domain. Neutrality tests (Tajima, Fu & Li and Fu) which revealed positive and non-significant values and Pi and θ_W assume a neutral model of evolution and advocated a constant population size. The study demonstrates the resolving power of trnL-trnF sequence data to prove both pummelo and mandarin gene pool’s contribution in the development of Tunisian secondary species and inferring their genetic and phylogenetic relationships.     

Chloroplast DNA-relationship in palaeoaustralLopidium concinnum (Hypopterygiaceae, Musci). An example of stenoevolution in mosses Studies in austral temperate rain forest bryophytes 2

Evolutionary relationship between disjunct populations of the palaeoaustral moss taxonLopidium concinnum (Hypopterygiaceae) from New Zealand and southern South America were studied using non-coding chloroplast DNA sequences. No or only slight changes could be observed within the sequences oftrnT_UGU —trnL_UAA 5exon intergenic spacer,trnL_UAA intron andtrnL_UAA 3exon —trnF_GAA intergenic spacer. This indicates nearly no genetic divergence between extant New Zealand and Chilean populations, i.e. no significant differing pathways of evolution within the 80–60 million years of disrupted areas with interrupted gene flow. Molecular data support the idea of an old Gondwanan relict species of stenoevolutionary character. Ecological data on short-range dispersal strengthen this assessment.     

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.     

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.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morphology always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermediate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence comparison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) intergenic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we examined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species. __________ Translated from Acta Botanica Yunnanica, 2007, 29 (1): 91–97 [译自:云南植物研究]     

Major differences between the rrnA operons of two strains of Agrobacterium vitis

The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554. Compared to the earlier obtained rrnA sequence of A. vitis strain S4, several important differences were noted: the sequences diverged at the 5′-flanking region, within the 16S–23S intergenic region, and within the 23S rRNA sequence. The B8 stem-loop structure at the 5′-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4. These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons. Received: 16 February 1996 / Accepted: 26 April 1996     

Schottarum (Schismatoglottideae: Araceae) substantiated based on combined nuclear and plastid DNA sequences

Recent studies on Schismatoglottideae have resulted in the recognition of four new monophyletic genera, the resurrection of two additional genera following clarification of their monophyly, and the publication of many taxonomically novel species. However, generic boundaries among some parts of Schismatoglottideae remain unclear owing to several reasons: (1) more taxa are being revealed through our on-going fieldwork, now expanded to previously unsampled localities on Borneo; (2) established occurrence of a high level of homoplasies among the morphological characteristics hitherto used to delimitate genera; and (3) gene regions used in previous studies contradicted some of current taxonomic placements. Among the unsolved groups from previous studies a clade comprising Schismatoglottis sarikeensis and S. josefii needs further investigation. Therefore, phylogenetic analyses were carried out to investigate the position of these two species using the nuclear region, internal transcribed spacer and combined plastid regions: trnL intron and trnL-F intergenic spacer, coding matK+partial 3′ trnK, intergenic spacer trnH-psbA. A total of 23 accessions representing 16 taxa of Schismatoglottideae and Philonotieae were included in the study. Phylogenetic analyses of a total 4,658 bp combined dataset using parsimony, maximum likelihood, and Bayesian methods revealed that S. sarikeensis and S. josefii do not belong to Schismatoglottis, and therefore are transferred to Schottarum (≡Hottarum sarikeense ≡ Schismatoglottis sarikeense). Flowering mechanism, pollination strategy, and fruitset of S. sarikeense are also presented in the paper.     

Postglacial population expansion of Dacrydium pectinatum (Podocarpaceae) in Hainan, southem China, based on cpDNA trnL-F nocoding sequence data

This study determined the sequences of chloroplast DNA (cpDNA) trnL-F non-coding regions of individuals of a tropical coniferous species, Dacrydium pectinatum, collected from 12 natural populations located in Hainan Province, southern China. Sequence length varied from 868 bp to 876 bp, indicating length polymorphism. Base composition in the sequences was high in A+T content between 64.17% and 64.95%, and no recombination event occurred (Rm = 0). Thirty haplotypes were identified based on statistical parsimony algorithm by running the TCS program. Populations of D. pectinatum in Hainan were lacking genetic differentiation. Such a deduction was supported by the observed F _ST values (0.00), AMOVA (24.17% of molecular variance attributed to difference among populations, P>0.05), high values of Nm (ranging from 1.92 to 2.50) and the branching structure in neighbor-joining (NJ) tree constructed from haplotypes. A ‘star-like’ pattern was exhibited in the TCS network of trnL-F haplotypes, and majority of the haplotypes coalesced near the tips in NJ tree. Gene genealogies of cpDNA haplotypes proposed a recent population expansion of D. pectinatum in Hainan, which was further supported by the results from Tajima’s D test and mismatch distribution analysis. Our data, in conjunction with geological and palynological evidences, showed that in the Holocene, due to global warming, refugee populations of D. pectinatum in Hainan might experience a range expansion. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2005, 44(5): 70–74 [译自: 中山大学学报 (自然科学版), 2005, 44(5): 70–74]     

Duplication and Diversification of the Apolipoprotein CI (APOCI) Genomic Segment in Association with Retroelements

We have previously shown that several multicopy gene families within the major histocompatibility complex (MHC) arose from a process of segmental duplication. It has also been observed that retroelements play a role in generating diversity within these duplicated segments. The objective of this study was to compare the genomic organization of a gene duplication within another multicopy gene family outside the MHC. Using new continuous genomic sequence encompassing the APOE-CII gene cluster, we show that APOCI and its pseudogene, APOCI′, are contained within large duplicated segments which include sequences from the hepatic control region (HCR). Flanking Alu sequences are observed at both ends of the duplicated unit, suggesting a possible role in the integration of these segments. As observed previously within the MHC, the major differences between the segments are the insertion of sequences (approximately 200–1000 bp in length), consisting predominantly of Alu sequences. Ancestral retroelements also contribute to the generation of sequence diversity between the segments, especially within the 3′ poly(A) tract of Alu sequences. The exonic and regulatory sequences of the APOCI and HCR loci show limited sequence diversity, with exon 3 being an exception. Finally, the typing of pre- and postduplication Alus from both segments indicates an estimated time of duplication of approximately 37 million years ago (mya), some time prior to the separation of Old and New World monkeys. Received: 17 July 1999 / Accepted: 6 November 1999     

Pollination by fungus gnats (Diptera: Mycetophilidae) and self-recognition sites in Tolmiea menziesii (Saxifragaceae)

Coleeae (Bignoniaceae) are a tribe almost entirely restricted to Madagascar. Coleeae have previously been placed in neotropical Crescentieae due to species with indehiscent fruits, a character otherwise unusual in Bignoniaceae. A phylogeny based on three chloroplast regions (ndhF, trnT-L spacer, trnL-F spacer) identifies a monophyletic Coleeae that is endemic to Madagascar and surrounding islands of the Indian Ocean (Seychelles, Comores and Mascarenes). African Kigelia is not a member of Coleeae, rather it is more closely related to a subset of African and Southeast Asian species of Tecomeae. The molecular phylogeny indicates that indehiscent fruit have arisen repeatedly in Bignoniaceae: in Coleeae, Kigelia and Crescentieae. The characteristic fleshy fruits of species of Coleeae likely arose autochthonously in Madagascar. Within Coleeae Colea and Ophiocolea are sisters, Phyllarthron is sister to Colea + Ophiocolea, and Rhodocolea is sister to the rest of the tribe.     

Comparative Analysis of Evolution in a Rodent Histone H2a Pseudogene

Sequences were obtained from five species of rodents that are orthologous to an H2a histone pseudogene from Mus musculus. The pseudogene is part of the cluster of replication-dependent histone genes found on Mus musculus chromosome 13. Comparative analysis of these five sequences together with the previously published sequence from M. musculus shows that this gene has likely been a pseudogene throughout the evolution of the genus Mus, while the gene from Rattus norvegicus is likely functional. Three large (>20 bp) deletions were found among the Mus pseudogenes, a feature that is very unusual compared to surveys of processed pseudogenes. In addition, there are two single-base deletions and one 4-bp insertion among the Mus pseudogenes. The species distributions of one of the large deletions and the 4-bp insertion require either independent insertions of an identical sequence, independent deletions with identical boundaries, or a deletion followed by precise reintegration of the original sequence. The evidence favors the hypothesis of multiple deletions with identical boundaries. The ``coding' regions of the Mus pseudogenes show a much reduced level of among-species variability in the 3′ half of the pseudogene, compared both to the 5′ half and to flanking sequences. This supports a hypothesis that the 3′ end of the pseudogene is the target of frequent gene conversion by functional H2a genes. Received: 1 April 1997 / Accepted: 12 June 1997     

Evolution of Growth Hormone in Primates: The GH Gene Clusters of the New World Monkeys Marmoset (Callithrix jacchus) and White-Fronted Capuchin (Cebus albifrons)

The GH gene cluster in marmoset, Callithrix jacchus, comprises eight GH-like genes and pseudogenes and appears to have arisen as a consequence of gene duplications occurring independently of those leading to the human GH gene cluster. We report here the complete sequence of the marmoset GH gene locus, including the intergenic regions and 5′ and 3′ flanking sequence, and a study of the multiple GH-like genes of an additional New World monkey (NWM), the white-fronted capuchin, Cebus albifrons. The marmoset sequence includes 945 nucleotides (nt) of 5′ flanking sequence and 1596 nt of 3′ flanking sequence that are “unique”; between these are eight repeat units, including the eight GH genes/pseudogenes. The breakpoints between these repeats are very similar, indicating a regular pattern of gene duplication. These breakpoints do not correspond to those found in the much less regular human GH gene cluster. This and phylogenetic analysis of the repeat units within the marmoset gene cluster strongly support the independent origin of these gene clusters, and the idea that the episode of rapid evolution that occurred during GH evolution in primates preceded the gene duplications. The marmoset GH gene cluster also differs from that of human in having fewer and more evenly distributed Alu sequences (a single pair in each repeat unit) and a “P-element” upstream of every gene/pseudogene. In human there is no P-element upstream of the gene encoding pituitary GH, and these elements have been implicated in placental expression of the other genes of the cluster. The GH gene clusters in marmoset and capuchin appear to have arisen as the consequence of a single-gene duplication event, but in capuchin there was then a remarkable expansion of the GH locus, giving at least 40 GH-like genes and pseudogenes. Thus even among NWMs the GH gene cluster is very variable. [Reviewing Editor: Nicolas Galtier]     

Phylogeny of the Madagascan endemic family Didiereaceae

A molecular phylogeny of the Didiereaceae was produced through parsimony analysis of chloroplastrpl16 intron andtrnL-trnF andtrnT-trnL intergenic spacer sequences of all eleven species of the Didiereaceae and several outgroup taxa from the Portulacaceae. Results indicated that: 1) the Didiereaceae were embedded within the Portulacaceae, withCalyptrotheca as the sister group of the family; 2) present generic limits were supported; 3)Alluaudiopsis was the most basal lineage; 4) at least two separate episodes of polyploidization within the genusAlluaudia had occurred, and 5) unusually low amounts of variation were present in rapidly evolving noncoding plastid sequences.     

The correlation between development of atypical bisexual flowers and phylogeny in the Aroideae (Araceae)

 In the intermediate zone of the inflorescence of genera of Aroideae one can find flowers with male and female characteristics. Until now, two types of developmental sequences of atypical bisexual flowers (ABFs) have been recognized: the Philodendron type and the Cercestis type. In the Philodendron type, bisexual flowers generally consist of functional carpels and staminodes inserted on the same whorl. In the Cercestis type, the gynoecium and stamens are inserted on two different whorls. These different ontogenetic patterns represent two different pathways in the evolution of unisexual flowers in this subfamily. A molecular phylogenetic analysis of 33 genera of Araceae, based on the chloroplast trnL intron and trnL–F intergenic spacer sequences was carried out. We use this phylogenetic analysis and those published by French et al. (1995) and Mayo et al. (1997) to examine the distribution of the two types of ABFs in selected genera. Our results suggest that the two developmental patterns of ABFs in Aroideae sensu Mayo et al. (1997) do not correspond to two separate evolutionary lineages but rather are more or less consistent within clades. Although this new molecular phylogeny does not include all aroid genera, it corroborates in general, at the subfamily level, the molecular analysis of French et al. (1995) based on chloroplast DNA restriction site data and the analysis of Mayo et al. (1997) based on morphological and anatomical data. Received March 15, 2001 Accepted October 11, 2001