Structural evolution of nrDNA ITS in Pinaceae and its phylogenetic implications

Nuclear ribosomal DNA (nrDNA) has been considered as an important tool for inferring phylogenetic relationships at many taxonomic levels. In comparison with its fast concerted evolution in angiosperms, nrDNA is symbolized by slow concerted evolution and substantial ITS region length variation in gymnosperms, particularly in Pinaceae. Here we studied structure characteristics, including subrepeat composition, size, GC content and secondary structure, of nrDNA ITS regions of all Pinaceae genera. The results showed that the ITS regions of all taxa studied contained subrepeat units, ranging from 2 to 9 in number, and these units could be divided into two types, longer subrepeat (LSR) without the motif (5'-GGCCACCCTAGTC) and shorter subrepeat (SSR) with the motif. Phylogenetic analyses indicate that the homology of some SSRs still can be recognized, providing important informations for the evolutionary history of nrDNA ITS and phylogeny of Pinaceae. In particular, the adjacent tandem SSRs are not more closely related to one another than they are to remote SSRs in some genera, which may imply that multiple structure variations such as recombination have occurred in the ITS1 region of these groups. This study also found that GC content in the ITS1 region is relevant to its sequence length and subrepeat number, and could provide some phylogenetic information, especially supporting the close relationships among Picea, Pinus, and Cathaya. Moreover, several characteristics of the secondary structure of Pinaceae ITS1 were found as follows: (1) the structure is dominated by several extended hairpins; (2) the configuration complexity is positively correlated with subrepeat number; (3) paired subrepeats often partially overlap at the conserved motif (5'-GGCCACCCTAGTC), and form a long stem, while other subrepeats fold onto itself, leaving part of the conserved motif exposed in hairpin loops.     

Comparative mapping in the Pinaceae

A comparative genetic map was constructed between two important genera of the family Pinaceae. Ten homologous linkage groups in loblolly pine (Pinus taeda L.) and Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) were identified using orthologous expressed sequence tag polymorphism (ESTP) and restriction fragment length polymorphism (RFLP) markers. The comparative mapping revealed extensive synteny and colinearity between genomes of the Pinaceae, consistent with the hypothesis of conservative chromosomal evolution in this important plant family. This study reports the first comparative map in forest trees at the family taxonomic level and establishes a framework for comparative genomics in Pinaceae.     

Early evolution of the bilateria

The phylogeny of the Bilateria and especially the early steps in the evolution of the bilaterian bauplan are still a controversial topic. In this context the relationships of the platyhelminths and the nematodes play a crucial role. Previous molecular studies of the relationships of these groups, which were based on 18S ribosomal DNA sequences, yielded conflicting results. In the present study a new framework is developed for the phylogenetic analysis of bilaterian relationships, using concatenated amino acid sequences of several nuclear genes. In this analysis, the rhabditophoran platyhelminths are probably the sister group of all other analyzed Bilateria, the Eubilateria, which are characterized by a one-way intestine with an anus. The Eubilateria are split into the nematode lineage and the coelomates. The phylogenetic results of the present study indicate that genetic features found in the model organisms Caenorhabditis and Drosophila might be found in all Eubilateria. Estimations of the divergence times show that the major bilaterian phyla did not originate in an explosive radiation during the Cambrian but rather that the Bilateria have a several hundred million years long Precambrian history.     

Early tetrapod evolution

Tetrapods include the only fully terrestrial vertebrates, but they also include many amphibious, aquatic and flying groups. They occupy the highest levels of the food chain on land and in aquatic environments. Tetrapod evolution has generated great interest, but the earliest phases of their history are poorly understood. Recent studies have questioned long-accepted hypotheses about the origin of the pentadactyl limb, the phylogeny of tetrapods and the environment in which the first tetrapods lived.     

Phenylpropanoids in mycorrhizas of the Pinaceae

Tissue-specific accumulation of phenylpropanoids was studied in mycorrhizas of the conifers, silver fir (Abies alba Mill.), Norway spruce [Picea abies (L.) Karst.], white pine (Pinus strobus L.), Scots pine (Pinus silvestris L.), and Douglas fir [Pseudotsuga menziesii (Mirbel) Franco], using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble flavanols (catechin and epicatechin), proanthocyanidins (mainly dimeric catechins and/or epicatechins), stilbene glucosides (astringin and isorhapontin), one dihydroflavonol glucoside (taxifolin 3′-O-glucopyranoside), and a hydroxycinnamate derivative (unknown ferulate conjugate). In addition, a cell wall-bound hydroxycinnamate (ferulate) and a hydroxybenzaldehyde (vanillin) were analysed. Colonisation of the root by the fungal symbiont correlated with the distribution pattern of the above phenylpropanoids in mycorrhizas suggesting that these compounds play an essential role in restricting fungal growth. The levels of flavanols and cell wall-bound ferulate within the cortex were high in the apical part and decreased to the proximal side of the mycorrhizas. In both Douglas fir and silver fir, which allowed separation of inner and outer parts of the cortical tissues, a characteristic transversal distribution of these compounds was found: high levels in the inner non-colonised part of the cortex and low levels in the outer part where the Hartig net is formed. Restriction of fungal growth to the outer cortex may also be achieved by characteristic cell wall thickening of the inner cortex which exhibited flavanolic wall infusions in Douglas fir mycorrhizas. Long and short roots of conifers from natural stands showed similar distribution patterns of phenylpropanoids and cell wall thickening compared to the respective mycorrhizas. These results are discussed with respect to co-evolutionary adaptation of both symbiotic partners regarding root structure (anatomy) and root chemistry. Received: 25 May 1998 / Accepted: 6 November 1998     

Early hominid physical evolution

Hindrances against bipedalism evolution are localized in obstetrical constraints, maternal mortality rates, infant birth trauma and unsafe pregnancy. Analysis of infant survival probability shows that a shift to bipedalism could occur as a necessary consequence of the process of body fur reduction, in a balance between such hindrances and safe infant transportation. Fur reduction is proposed to correlate with cooling mechanism in intra-species physical fights. The triggering of a feed-back mechanism connecting reduction of body fur to canine reduction would be responsible for a passage from threat displays to actual physical fights. The proposed scenario for such changes is the transition from uni-male to multi-male social structures among Hominoidea. The implications of the approach adopted are discussed.     

Vesicular endophytes in roots of the Pinaceae

Vesicles and hyphae typical of vesicular-arbuscular mycorrhizae (VAM) were common in seedlings of Pseudotsuga menziesii, Abies lasiocarpa and Tsuga mertensiana growing in openings where herbaceous hosts of these fungi were common. Seedlings of A. lasiocarpa, Tsuga heterophylla, and T. mertensiana growing under closed forest canopies also had vesicles but at a much lower incidence than seedlings in the openings. The Pinaceae are generally assumed to be ectomycorrhizal, but Glomus-type colonizations occurred on the same seedlings as the ectomycorrhizae. The ecological significance of abundant VAM-type endophytes in otherwise ectomycorrhizal hosts deserves comprehensive study.     

Identification of COS markers in the Pinaceae

Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Early evolution and the origin of eukaryotes

Our understanding of evolutionary relationships in the eukaryotic world has been revolutionized by molecular systematics. Phylogenies based upon comparisons of rRNAs define five major eukaryotic assemblages plus a series of paraphyletic protist lineages. Comparison of conserved genes that were duplicated prior to the divergence of eubacteria, archaebacteria, and eukaryotes, positions the root of the universal tree within the eubacterial line of descent. In this review a novel model is presented which uses the rRNA and protein based phylogenies to describe the evolutionary origins of eukaryotes.     

Early events in the evolution of spider silk genes

Silk spinning is essential to spider ecology and has had a key role in the expansive diversification of spiders. Silk is composed primarily of proteins called spidroins, which are encoded by a multi-gene family. Spidroins have been studied extensively in the derived clade, Orbiculariae (orb-weavers), from the suborder Araneomorphae ('true spiders'). Orbicularians produce a suite of different silks, and underlying this repertoire is a history of duplication and spidroin gene divergence. A second class of silk proteins, Egg Case Proteins (ECPs), is known only from the orbicularian species, Lactrodectus hesperus (Western black widow). In L. hesperus, ECPs bond with tubuliform spidroins to form egg case silk fibers. Because most of the phylogenetic diversity of spiders has not been sampled for their silk genes, there is limited understanding of spidroin gene family history and the prevalence of ECPs. Silk genes have not been reported from the suborder Mesothelae (segmented spiders), which diverged from all other spiders >380 million years ago, and sampling from Mygalomorphae (tarantulas, trapdoor spiders) and basal araneomorph lineages is sparse. In comparison to orbicularians, mesotheles and mygalomorphs have a simpler silk biology and thus are hypothesized to have less diversity of silk genes. Here, we present cDNAs synthesized from the silk glands of six mygalomorph species, a mesothele, and a non-orbicularian araneomorph, and uncover a surprisingly rich silk gene diversity. In particular, we find ECP homologs in the mesothele, suggesting that ECPs were present in the common ancestor of extant spiders, and originally were not specialized to complex with tubuliform spidroins. Furthermore, gene-tree/species-tree reconciliation analysis reveals that numerous spidroin gene duplications occurred after the split between Mesothelae and Opisthothelae (Mygalomorphae plus Araneomorphae). We use the spidroin gene tree to reconstruct the evolution of amino acid compositions of spidroins that perform different ecological functions.     

Early evolution of the MFT-like gene family in plants

Angiosperm genes sharing a conserved phosphatidylethanolamine-binding (PEPB) domain have been shown to be involved in the control of shoot meristem identity and flowering time. The family is divided into three subfamilies, FT-like, TFL1-like and MFT-like. This study is focused on the evolution of the MFT-like clade, suggested to be ancestral to the two other clades. We report that the bryophyte Physcomitrella patens and the lycopod Selaginella moellendorfii contain four and two MFT-like genes respectively. Neither species have any FT or TFL1-like genes. Furthermore, we have identified a new subclade of MFT-like genes in Angiosperms. Quantitative expression analysis of MFT-like genes in Physcomitrella patens reveals that the expression patterns are circadian and reaches maximum in gametangia and sporophytes. Our data suggest that the occurrence FT and TFL1-like genes, is associated with the evolution of seed plants. Expression data for Physcomitrella MFT-like genes implicates an involvement in the development of reproductive tissues in the moss.     

Early evolution of MHC polymorphism

There is unwarranted satisfaction with the view that MHC polymorphism evolved because there was a selective advantage in having a variety of MHC proteins to bind a variety of peptide subsets for presentation to T cells. While this may, in part, explain its maintainance, polymorphism may have evolved initially to reject foreign virus "grafts". The possession of similar membranes promotes aggregation between "like" cells, but it also promotes aggregation between the cells and viruses which retain membrane components of their previous host. The selection pressure afforded by hostile virus "grafts" would favour cells which developed polymorphic membrane components (since "like" will not aggregate with "not-like"). This polymorphism would have evolved before the appearance of multicellular organisms. Thus, the evolution of modern immune systems would have been imposed upon pre-existing polymorphic systems. A path this evolution may have taken involves the development of mechanisms for intracellular distinction between self and not-self.     

Promotion of flowering in the Pinaceae by gibberellins

Significant female flowering of 6- to 11-year-old seedlings and grafted ramets of sexually mature scions of lodgepole pine (Pinus contorta Dougl.) was promoted by both topical and spray applications of a gibberellin (GA) A_4/7 mixture (1.6 to c. 5 mg per plant in total) during that period (June to September) when sexual differentiation of lateral primordia would be expected to take place. Girdling was used in most experiments to enhance the GA_4/7 effect, as was the auxin, naphthaleneacetic acid (NAA). Average frequency of flowering branches on treated plants over all experiments ranged from 27 to 59% (control ranged from 0 to 36%) and average number of female strobili was increased from 2- to 6-fold by growth regulator treatment, relative to controls. Within an experiment, clonal or family frequency of flowering for treated plants ranged from 11 to 67% (controls were 0 to 28%), and number of female strobili was increased from 2- to 14-fold by growth regulator treatment, relative to controls. Movement of the flowering stimulus from the point of application was apparent in several experiments, the response in adjacent branches being correlated positively with increasing dosage of GA_4/7. Significant male flowering occurred only in one experiment, girdling and GA_4/7 treatment being promotive factors. The use of spray applications of GA_4/7+ NAA is warranted to induce early and enhanced flowering in lodgepole pine seedlings and vegetative propagules for genetic improvement programs.     

Promotion of flowering in the Pinaceae by gibberellins

Significant male and female flowering (cone bud production) by girdled branches of 6-year-old Douglas fir (Pseudotsuga menziesii (Mirb. Franco) seedlings was promoted by applications (mid-April to June) of 1.6 or 3.2 mg per branch (in total) of certain non-polar gibberellins (GA's). Girdling alone was ineffective. When tested alone, a mixture of GA_4/7 was most effective. GA₉ less so, while GA₅ and the more polar GA₃ were essentially ineffective. For female cone buds GA_4/7+ GA₉ were synergistically effective, but for male cone buds GA_4/7 alone was best. The auxin naphthaleneacetic acid (NAA) was not tested alone, but at low dosage (0.175 mg/branch in total) NAA enhanced the flowering efficacy of GA's for both sexes; at a high dosage (0.875 mg/branch in total) male cone bud production was further enhanced, but only at the expense of females. For female flowering the best treatment (90% frequency of flowering 6.8 cone buds/branch), was GA_4/7+ GA₉+ low NAA; for male flowering, it was GA_4/7+ high NAA (30% frequency and 4.2 cone buds/branch. Frequency of flowering for controls was 18% and 0%, average number of cone buds/branch was 0.9 and 0, for females and males, respectively. The successful treatments did not affect promordia initiation, rather they caused the differentiation of previously initiated, but undetermined, lateral primordia into cone and latent buds at the expense of vegetative bud differentiation. The lack of success reported by earlier workers in promoting flowering in Pinaceae species by GA's appears to be the unfortunate result of selecting GA₃ for initial testing. The practical implications of this early and enhanced flowering by non-polar GA's seedlings of a commercially important conifer are discussed in relation to accelerating the processes of tree improvement.     

Promotion of Flowering in the Pinaceae by Gibberellins

Flowering was significantly promoted in 4-year-old grafts of mature coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) clones by exogenous gibberellins (GAs) A₄ and A₇ (as a mixture) applied alone and in combination with A₅ and A₉. Biweekly applications of 400 μg GA_4/7 per branch between late March and late June gave a 5-fold increase in ovulate and 3-fold increase in staminate strobilus production over untreated controls. ⁶N-benzyladenine and 2,3,5-triiodobenzoic acid applied in combination with GAs had no consistent effect on strobilus production. Non-destructive branch girdling, ineffective by itself as a cultural treatment, enhanced the GA benefit to flowering. Exogenous application of GA_4/7 is effective and appears to be a practical method for promotion of early and enhanced flowering in grafted Douglas-fir seed orchards.