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Phylogenetic reconstruction is fundamental to study evolutionary biology and historical biogeography. However, there was not a molecular phylogeny of gymnosperms represented by extensive sampling at the genus level, and most published phylogenies of this group were constructed based on cytoplasmic DNA markers and/or the multi-copy nuclear ribosomal DNA. In this study, we use LFY and NLY, two single-copy nuclear genes that originated from an ancient gene duplication in the ancestor of seed plants, to reconstruct the phylogeny and estimate divergence times of gymnosperms based on a complete sampling of extant genera. The results indicate that the combined LFY and NLY coding sequences can resolve interfamilial relationships of gymnosperms and intergeneric relationships of most families. Moreover, the addition of intron sequences can improve the resolution in Podocarpaceae but not in cycads, although divergence times of the cycad genera are similar to or longer than those of the Podocarpaceae genera. Our study strongly supports cycads as the basal-most lineage of gymnosperms rather than sister to Ginkgoaceae, and a sister relationship between Podocarpaceae and Araucariaceae and between Cephalotaxaceae-Taxaceae and Cupressaceae. In addition, intergeneric relationships of some families that were controversial, and the relationships between Taxaceae and Cephalotaxaceae and between conifers and Gnetales are discussed based on the nuclear gene evidence. The molecular dating analysis suggests that drastic extinctions occurred in the early evolution of gymnosperms, and extant coniferous genera in the Northern Hemisphere are older than those in the Southern Hemisphere on average. This study provides an evolutionary framework for future studies on gymnosperms.
Citation: Lu Y, Ran J-H, Guo D-M, Yang Z-Y, Wang X-Q (2014) Phylogeny and Divergence Times of Gymnosperms Inferred from Single-Copy Nuclear Genes. PLoS ONE 9(9): e107679. https://doi.org/10.1371/journal.pone.0107679
Editor: Sven Buerki, Royal Botanic Gardens, Kew, United Kingdom
Received: March 7, 2014; Accepted: August 19, 2014; Published: September 15, 2014
Copyright: © 2014 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All gene sequences are deposited in NCBI and the GenBank accession numbers are shown in Table S1. All LFY and NLY gene sequences determined in this study are deposited in NCBI under GenBank accession numbers KF377856-KF377901, KF377904-KF377918 and KF377921-KF377963, and the trees and alignments are deposited in TreeBase (number S16207).
Funding: This work was supported by the National Natural Science Foundation of China (Grant Nos. 31170197, 31330008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
A solid organismal phylogeny is fundamental to study evolutionary biology and historical biogeography. In recent years, the angiosperm phylogeny group (APG) III system has provided an evolutionary framework for studying angiosperms . However, phylogenetic relationships of the main lineages of gymnosperms, either classified into four subclasses (Cycadidae, Ginkgoidae, Gnetidae and Pinidae) by Christenhusz et al.  or into the widely accepted five clades (cycads, ginkgos, cupressophytes, Pinaceae and gnetophytes), are still in hot debate. Gymnosperms, which have been resolved as the sister group of angiosperms by increasing evidence from morphological, molecular phylogenetic and evolutionary developmental studies –, bear important information of seed-plant evolution, and represent an important link in the evolution of many gene families and biological pathways. Therefore, a better understanding of evolutionary relationships within gymnosperms can also help us to interpret the evolution of seed plants, and even molecular evolution in land plants.
Gymnosperms have a rich fossil record that is very useful for phylogenetic reconstruction, but this group suffered a dramatic extinction in the Cenozoic  and currently comprises 12 families, 83 genera, and only a little more than 1,000 species , which makes it difficult to resolve some interfamilial and intergeneric relationships (see review by Wang and Ran ). The early molecular phylogenetic studies of gymnosperms only sampled a small part of the recognized genera , , –, and in particular most published molecular phylogenies were constructed based on uniparentally inherited cytoplasmic DNA markers and/or the multi-copy nuclear ribosomal DNA , , –. Despite that 53 genera representing all extant main lineages of gymnosperms were studied in Ran et al. , the main focus of the study was the fast evolution of the mitochondrial gene rps3 in Conifer II (cupressophytes) and the underlying mechanisms. Some other studies of gymnosperms mainly focused on individual families or clades, such as conifers , , Cupressaceae –, Pinaceae  and cycads , . Although great progress has been made on understanding the phylogeny of gymnosperms in recent years, more interesting phylogenetic hypotheses have been proposed and hotly debated (see review by Wang and Ran ), like the phylogenetic position of Gnetales and the relationship between cycads and ginkgos , , , . Till now, there is not a molecular phylogeny of gymnosperms that is reconstructed based on a complete sampling of all extant genera, although this ancient and widespread plant group has huge ecological and economic value. Also, it would be interesting to know whether the phylogenetic relationships of gymnosperms inferred from cytoplasmic DNA are supported by evidence from the nuclear genome, given the complex inheritance patterns of organellar genes in this group . Moreover, phylogenetic relationships within some lineages, such as Pinaceae , , Podocarpaceae  and Zamiaceae , , , need to be further resolved.
Due to the fast development of genome sequencing technologies, phylogenomic analyses have been increasingly used in reconstructing the tree of life, and the efficiency of using multiple single- or low-copy nuclear genes for phylogenetic analysis has been widely recognized . However, this is still difficult for gymnosperms with large and complex nuclear genomes characterized by long introns and numerous gene-like fragments . For example, based on ESTs, Lee et al.  analyzed millions of amino acid sites from 150 species across land plants, and placed Gnetales as sister to the rest of the gymnosperms, but their dataset suffered greatly from missing data and poor alignment (our unpublished analysis). Nevertheless, Yang et al.  successfully used two sister nuclear genes LEAFY (LFY) and NEEDLY (NLY), which originated from an ancient gene duplication in the common ancestor of seed plants and encode transcription factors regulating the development of reproductive structures in gymnosperms , , to reconstruct the phylogeny of Cupressaceae comprising all its 32 genera. They also confirmed that both LFY and NLY exist as single copy in gymnosperms, even in the polyploid species, and are excellent markers for studying the phylogeny and evolution of gymnosperms .
In this study, on the basis of Yang et al. , we use LFY and NLY gene sequences to reconstruct the phylogeny of gymnosperms based on a complete sampling of extant genera, in effort to provide an evolutionary framework for future studies on this important group. In addition, some controversial interfamilial and intergeneric relationships are resolved and discussed. Moreover, benefiting from the rich fossil record, we estimate the divergence times of different lineages, which would further help us understand the diversification history of gymnosperms.
Materials and Methods
Ninety species representing all recognized genera of extant gymnosperms were sampled. Most genera were represented by one species each, since the coding sequences of LFY and NLY used to reconstruct the phylogeny of gymnosperms are very conserved among congeneric species. If using introns of the two genes, the sequences are unalignable between the main clades of gymnosperms , and most congeneric species do not form monophyletic groups, respectively, due to the wide interspecific sharing of alleles as reported in Pinus . Therefore, the addition of more congeneric species can not significantly improve the resolution of intergeneric relationships of gymnosperms when using single-copy nuclear genes like LFY and NLY. Nevertheless, we sampled two species of Pinus to represent its two subgenera with an ancient divergence, and more species from the Juniperus-Cupressus- Callitropsis-Xanthocyparis-Hesperocyparis clade, in which the generic division is controversial . The origins of materials, including the data downloaded from NCBI, are shown in Table S1.
DNA and RNA extraction, PCR and RT-PCR amplification, cloning and sequencing
Total DNA was extracted from silica-gel dried leaves using either the modified CTAB method  or the DNAsecure Plant Kit (Tiangen, Beijing, China). Young leaves and reproductive organs of Ephedra equisetina were collected for total RNA extraction, which followed the modified Trizol method (Tiangen). The first-strand cDNA was produced using the 5′ RACE system (Invitrogen) and the 3′ RACE kit (Tiangen). Polymerase chain reaction (PCR) was conducted in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA) or an Eppendorf Mastercycler (Eppendorf Scintific, Westbury, NY, USA), in a volume of 25 µl containing 50–200 ng of DNA or cDNA template, 6.25 pmol of each primer, 0.2 mM of each dNTP, 2 mM MgCl2, and 0.75 U of ExTaq DNA polymerase (Takara Biotechnology, CO., Ltd. Dalian, China). PCR cycles were as follows: one cycle of 4 min at 94°C, four cycles of 1 min at 94°C, 30 s at 55–58°C, and 1.5–6.0 min at 72°C, followed by 32 cycles of 30 s at 94°C, 30 s at 53–55°C and 1.5–6.0 min at 72°C, with a final extension step for 10 min at 72°C.
After separation by 1.5% agarose gel electrophoresis, the PCR products were purified using the TIANgel Midi Purification Kit (Tiangen) and identified by direct sequencing with the PCR primers. Then, the correct PCR products were cloned with the pGEM-T Easy Vector System II (Promega, Madison, USA). Ten clones with the correct insertion, confirmed by EcoR I digestion, were picked for each species and screened for variation by sequencing with T7 primer. All distinct clones were further sequenced using SP6 and internal primers. Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit., and the sequencing products were separated on a 96-capillary 3730XL DNA analyzer (Applied Biosystems). All newly sequenced LFY and NLY genes, totaling 104 sequences, are deposited in NCBI under GenBank accession numbers KF377856-KF377901, KF377904-KF377918 and KF377921-KF377963 (Table S1). The primers used for amplifying and sequencing the LFY and NLY genes are shown in Table S2.
DNA sequence analysis
Sequence alignments were generated with CLUSTAL X  and manually refined. The variable sites and variability of conspecific clones were calculated using MEGA5  and BioEdit v7.2.0 , respectively. Introns of the two nuclear genes could not be reliably aligned among distantly related gymnospermous families, and thus were excluded when constructing the entire phylogeny of gymnosperms. However, some intron regions are relatively conserved and alignable within cycads and Podocarpaceae, respectively, and thus were included in the alignments to infer the intergeneric relationships of these groups. The aligned sequences were further trimmed using the Gblocks server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html).
We used the software DAMBE  to test substitution saturation for the two datasets LFY and NLY, and the results showed that none of them was substitutionally saturated. To determine whether the two gene datasets can be combined, we checked variation of clones in each species, and found that many species did not show clone polymorphism of LFY and NLY and no more than two distinct clones occurred in the same individual. In particular, the conspecific clones showed a high sequence similarity of over 95%. Then, we tried to conduct separate phylogenetic analyses for LFY and NLY that included all distinct clones, and the results showed that conspecific clones grouped together except two LFY clones from the tetraploid species Fitzroya cupressoides that were discussed in Yang et al. . Therefore, we randomly selected one clone from each species for the further analyses. The incongruence length difference test (ILD) , implemented in PAUP* 4.0b10 , CONCATERPILLAR (a hierarchical likelihood ratio test) , and CADM (a test of congruence among distance matrices)  were performed to assess congruence between different datasets. According to the three tests, no significant incongruence existed between LFY and NLY (Table 1), so we combined the two genes for phylogenetic analysis.
Initially, we used the LFY + NLY coding sequences (CDS) and the 1st+2nd codon positions, respectively, to reconstruct the phylogeny of all sampled gymnosperms. The fern Angiopteris lygodiifolia was used as outgroup for two reasons. First, as mentioned in the introduction, the two nuclear genes of gymnosperms originated from a duplication event in the common ancestor of seed plants, and the NLY gene was lost in angiosperms. Thus, the LFY gene of ferns may represent an ancestral state of the two genes. Second, the LFY gene sequence cannot be reliably aligned between gymnosperms and angiosperms, although a sister relationship between the two groups is supported by most recent studies (see review by Wang and Ran ). The results showed that the phylogenetic trees generated from different methods all supported cycads as a monophyletic group and the basal-most clade of gymnosperms (Fig. S1). To avoid long-branch attraction (LBA) artifacts, the phylogeny of gymnosperms was further reconstructed using cycads as functional outgroups. In addition, to better resolve the intergeneric relationships within cycads and Podocarpaceae that were controversial, we conducted separate phylogenetic analyses for the two lineages with combined LFY + NLY sequences, and compared gene trees generated from CDS and CDS+intron, respectively. The sister groups were chosen as outgroups, including Ginkgo biloba for cycads , as well as Araucaria heterophylla and Agathis robusa for Podocarpaceae . When introns were included, the sequences could not be aligned among different gymnospermous families, and therefore the generated trees were not rooted or rooted with a functional outgroup, such as Cycas for cycads. The details of all datasets used for phylogenetic analyses are shown in Table 2. The trees and alignments are deposited in TreeBase (number S16207).
Phylogenetic relationships were reconstructed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI), respectively. The MP analyses were implemented in PAUP* 4.0b10 , using heuristic searches with 1000 random addition sequence replicates, starting trees obtained via stepwise addition, tree-bisection-reconnection (TBR) branch swapping, MulTrees and Collapse options in effect, and a maximum of 2000 trees saved for each replicate. Robustness of the nodes (50% majority-rule consensus) was tested by the bootstrap analysis  using 1000 replicates with the same settings as above. The evolutionary models for the ML and BI analyses were optimized in jModeltest 2.0  and MrModeltest 2.3 , using Akaike Information Criterion (AIC), respectively. The best models for analyses are shown in Table 2. The ML analyses were carried out in PHYML version 2.4.4  with a BIONJ tree as a starting point, and support values for the nodes were calculated based on 100 bootstrap replicates. The Bayesian inference was performed with MrBayes 3.1.2 . One cold and three heated Markov chain Monte Carlo chains were run for 10,000,000 generations with random initial trees, and every 1000 generations were sampled. The first 20% of the samples were discarded as burn-in and a 50% majority-rule consensus tree was generated based on the trees sampled after generation 2,000,000.
The Shimodaira-Hasegawa test (SH test)  and the Kishino-Hasegawa test (KH test) , implemented in PAUP* 4.0b10, were used to test alternative phylogenetic hypotheses for the deep lineages with controversial phylogenetic positions. The different positions of three taxa, including Ginkgoaceae (sister to cycads or conifers + Gnetales), Gnetales (sister to conifers, Conifer II, Pinaceae, or other gymnosperms), and Sciadopityaceae (sister to Cupressaceae + Taxaceae + Cephalotaxaceae or Araucariaceae + Podocarpaceae), were compared. Alternative tree topologies were generated in PhyML 2.4.4 , and the tree files were run in PAUP to calculate the p-value for each topology.
Divergence time estimation
Based on the LFY + NLY coding sequences, the divergence times of gymnosperms were estimated using the Markov chain Monte Carlo (MCMC) method, which was implemented in BEAST v1.7.5 , under an uncorrelated lognormal-relaxed clock model of rate variation among lineages. The topology was constrained to reflect the ML tree, and a GTR+I+G substitution model was used. Mean substitution rates were allowed to vary. Sauquet et al.  suggested that more age constraints could lead to improved time estimates, but risky age constraints might strongly influence estimated ages. Hence, we incorporated 11 fossil constraints that were widely recognized and used in previous molecular dating of gymnosperms or seed plants , , , and nearly each main lineage of gymnosperms was calibrated by at least one fossil record (For details, see Table S3).
For the most recent common ancestor (MRCA) of gymnosperms (A), a minimum age of 306.2 Ma was set based on Cordaixylon iowensis, the oldest cordaitean coniferophyte found in the Laddsdale Coals (Cherokee Group, Desmoinesian Series; 307.2±1.0 Ma) near What Cheer of Iowa, and a maximum age of 366.8 Ma was set based on the well-documented first appearance of seeds (in the form of preovules) in the Upper Fammenian (Upper Devonian) VCo Spore Biozone –. In cycads, the stem age of Lepidozamia (B) was constrained to a minimum age of 33.9 Ma based on the fossil of Lepidozamia leaves from the Eocene of Australia, which possesses cuticular characters that are unique to Lepidozamia . In the family Pinaceae, the stem age of Picea (C) was constrained by Picea burtonii from the Apple Bay locality, Vancouver Island, British Columbia, dated to the Valanginian Stage of the Early Cretaceous (≥133 Ma). This seed cone fossil shares multiple morphological and anatomical characteristics with extant Picea, especially in the distribution and branching pattern of resin canals in the ovule scale . For Gnetales, its crown node (D) was calibrated based on Eoantha zherikhinii (≥125 Ma), which is a reproductive organ with whorls of scales and is considered closely related to Gnetum and Welwitschia , . In Conifer II, we set a minimum age of 172 Ma for the Araucariaceae-Podocarpaceae split (E) based on the first appearance of Araucarites phillipsii-Brachyphyllum mammilare from the Aalenian (172–176 Ma) , and 28 Ma for the Podocarpus-Retrophyllum split (F) based on Retrophyllum australe from the West Dale Flora of southwestern Australia (dated to 28–48 Ma) . The two calibrations were also used in Leslie et al. . Araucarites phillipsii, with seed cones similar to those in Araucariaceae, was considered as the first unambiguous evidence for the stem or crown of the plant family, and Brachyphyllum mammilare was found to have pollen cones that produced relatively large, non-saccate pollen comparable to modern Araucaria and foliages that contained oval sclereids similar to those in extant Araucaria cunninghamii. In addition, Retrophyllum australe had distinctive heterofacially flattened foliage similar to Nageia and Afrocarpus. For the split of Taxaceae-Cupressaceae (G), a minimum age of 197 Ma was set based on Palaeotaxus rediviva from the Skromberga Colliery in Scania, Sweden (dated to 197–201 Ma) , which showed an axillary short shoot that terminated in a single ovule and bore helically arranged sterile scales on seed cones identical to extant Austrotaxus and Taxus. The remaining fossil constraints were used to set a minimum age for four nodes in Cupressaceae s.l., as in Yang et al. , including the MRCAs of Sequoia-Metasequoia-Sequoiadendron (H, 140 Ma, Sequoia in early Cretaceous) , , Glyptostrobus-Taxodium (I, 99 Ma, Glyptostrobus in Cretaceous) , , Diselma-Fitzroya-Widdringtonia (J, 95 Ma, Widdringtonia in Cretaceous) , and Juniperus-Cupressus-Hesperocyparis (K, 33.9 Ma, Juniperus in the Eocene/Oligocene boundary) .
Since the age estimates by BEAST are usually older than those by PL (penalized likelihood) and the ages estimated with lognormal priors are slightly younger than those estimated with either uniform or exponential priors, Sauquet et al.  suggested that using lognormal priors can decrease the uncertainty in age estimates. Therefore, in this study, all fossil constraints were given lognormal prior distributions in the BEAST estimate. For the root constraint, we used a stdev of 0.5, a prior mean of 3.6, and an offset of 290.7 Ma. For a better comparability of our results with previous divergence time estimates of gymnosperms, the other constraints were set following Leslie et al.  and Yang et al. . The minimum age was set by the age of fossil, with a 95% confidence interval of the probability distribution extending 20 or 40 million years earlier than this minimum age, since the test by Leslie et al.  found that the fossil calibrations associated with the two prior age distributions led to very similar divergence time estimates. We ran four independent MCMC runs of 100 million generations, sampling every 2,500 generations. Tracer v1.5 was used to check convergence of the chains to the stationary distribution, ensuring the Effective Sample Size (ESS) >200. The first 20% of the generations were discarded as burn-in and trees were summarized with TreeAnnotator. The final tree and divergence times were visualized using FigTree v1.4.0.
In this study, we cloned and sequenced the LFY and NLY genes from 41 genera of 7 families (Table S1). These new data combined with the sequences downloaded from GenBank (mostly reported in Yang et al. ) completely represented all extant genera of gymnosperms. In Parasitaxus usta, the only parasitic conifer, we only got a pseudogene of NLY, in which several indels in the second exon led to an ORF shift. It is interesting that, by RT-PCR, we obtained cDNA sequences of both LFY and NLY genes from Ephedra equisetina and the LFY gene had two clone types that differed by a 9-bp deletion.
Both LFY and NLY sequences amplified from genomic DNA comprised three exons and two introns, and almost covered the full length of the two genes. The exon length was conserved, totally about 1000 bp, but the intron length varied greatly among different groups. A long repeat occurred in the first intron of the NLY gene of four Taxaceae genera (Pseudotaxus, Austrotaxus, Amentotaxus and Torreya), making it difficult to sequence the full length of the gene. Also, the first NLY intron of Cathaya and Pseudotsuga, two genera of the pine family, was difficult to sequence due to long length or complex structures. The detailed information of the sequence alignments for phylogenetic analyses, including sequence lengths and numbers of variable and parsimony-informative sites, is shown in Table 2.
Since the MP analysis is more easily affected by long branch attraction (LBA) than the ML and BI analyses –, we did not show the MP trees in this study. As mentioned earlier, when Angiopteris lygodiifolia was used as outgroup, all phylogenetic trees generated supported cycads as a monophyletic and basal-most group of gymnosperms, followed by Ginkgo (see the ML and BI trees in Fig. S1). When cycads were used as functional outgroups, the ML and BI trees generated from combined LFY and NLY CDS were topologically identical to each other, except for some branches with low statistical support. In the ML tree (Fig. 1), Ginkgoaceae was sister to the remaining gymnosperms excluding cycads, and Pinaceae was sister to a clade that was further divided into two sister subclades, i.e., Gnetales and conifer II (Cupressophytes). The conifer II was split into two lineages. One consisted of Sciadopityaceae, Podocarpaceae and Araucariaceae, and Sciadopityaceae was weakly supported to be sister to Podocarpaceae-Araucariaceae. Within the other lineage, Cephalotaxaceae was embedded in Taxaceae, and the two families formed a monophyletic group sister to Cupressaceae. In addition, this nuclear gene tree provided a relatively good resolution for intergeneric relationships in some families such as Pinaceae and Cupressaceae.
Numbers associated with branches are bootstrap percentages higher than 50%. The cycads were used as functional outgroups.
Although the trees generated with different rooting or from different codon positions (all CDS vs. 1st+2nd codons) were similar in topology, they differed in the positions of Gnetales and Sciadopityaceae (Fig. 2). For instance, in the phylogenetic tree generated from the first and second codon positions and rooted with cycads (Fig. 2D), Gnetales was weakly supported to be sister to Pinaceae, and Sciadopityaceae sister to a well-supported clade containing Cupressaceae and Taxaceae-Cephalotaxaceae. Moreover, many intra-familial relationships were poorly resolved (tree not shown), perhaps due to the declined phylogenetic signals caused by the removal of the third codon positions.
A and C, All three codon positions were used; B and D, 1st and 2nd codon positions were used. A and B, Angiopteris lygodiifolia was used as outgroup; C and D, The cycads were used as functional outgroups. Numbers associated with branches are bootstrap percentages of ML higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively.
Since the phylogenetic positions of some genera of cycads and Podocarpaceae were controversial in previous studies , , –, –, here we reconstructed internal relationships of the two groups, respectively. When only the LFY and NLY CDS was used, the phylogenetic signals were insufficient to resolve some intergeneric relationships (Figs. 3, 4), therefore we added the conserved intron regions of the two genes into analysis. For cycads, the addition of introns neither changed the tree topology nor greatly improved the resolution (Fig. 3), and the generated trees suggested a basal position of Dioon in Zamiaceae, a sister relationship between Zamia and Microcycas, and a close relationship among Encephalartos, Lepidozamia and Macrozamia. However, the resolution of internal relationships of Podocarpaceae was improved by adding intron sequences, with high support values for most nodes (Fig. 4). A large clade was strongly supported and well resolved, containing Microcachrys, Saxegothaea, Pherosphaera, Acmopyle, Dacrycarpus, Dacrydium, Falcatifolium, Afrocarpus, Podocarpus, Nageia and Retrophyllum. Within the clade, there existed two monophyletic sister groups. One was the ‘dacrydioid’ group comprising Dacrycarpus, Dacrydium and Falcatifolium, and the other was the ‘podocarpoid’ group including the Retrophyllum-Nageia subclade and the Afrocarpus-Podocarpus subclade. In addition, a close relationship among Manoao, Lagarostrobos and Parasitaxus was revealed (Fig. 4).
A, CDS; B, CDS+Intron. Numbers associated with branches are bootstrap percentages of ML and MP higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. Ginkgo biloba was used as outgroup in Fig. 3A.
A, CDS; B, CDS+Intron. Numbers associated with branches are bootstrap percentages of ML and MP higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. Araucaria heterophylla and Agathis robusa were used as outgroups in Fig. 4A.
The results of the SH and KH tests are shown in Table S4. The trees placing Ginkgoaceae with conifers + Gnetales were better than the trees placing the family sister to cycads, but the trees placing Sciadopityaceae with Podocarpaceae + Araucariaceae were not significantly different from the trees placing Sciadopityaceae sister to Cupressaceae + Taxaceae + Cephalotaxaceae. The sister relationship between Gnetales and the other gymnosperms was rejected by both SH and KH tests for the CDS dataset, and by the KH test for the dataset of the 1st+2nd codon positions. In addition, the topology placing Gnetales sister to conifers was rejected by the KH test for the CDS dataset. There was not significant difference in ln score between the other two topologies (Gnetales sister to Conifer II or Pinaceae).
Divergence time estimation
The divergence time estimation based on combined LFY and NLY CDS suggested a Triassic-Jurassic origin of the crown group for most families (Fig. 5). The mean ages and 95% HPDs are shown in Table S5. The most recent common ancestor (MRCA) of cycads was dated to the Middle Jurassic (158.1 Ma), and that of Pinaceae to the Lower Triassic (198.4 Ma). The divergence time between Cupressaceae and Taxaceae s.l. was close to that between Podocarpaceae and Araucariaceae, i.e., in the Late Triassic to the Early Jurassic. Most gymnosperm genera originated in the Cretaceous to the Cenozoic (Fig. 5).
A time scale is shown at the bottom. A–K indicate fossil calibration points. 1–10, A, D, E and G indicate some nodes of interest. Median ages of nodes are shown, with horizontal bars indicating the 95% highest posterior density intervals.
Evolution and phylogenetic utility of the LFY and NLY genes in gymnosperms
Our study indicates that both LFY and NLY genes occur in all extant genera of gymnosperms except that NLY has not been found in Gnetum (Table S1). Frohlich and Parker  found that the LFY-NLY gene pair originated from a duplication event in the common ancestor of seed plants, and then both paralogous genes were remained in gymnosperms while NLY was lost in angiosperms. Using this information of gene evolution as strong evidence, they also proposed the mostly male theory of flower origin given the important role of LFY in flower development, although this theory is not supported by the study of Vazquez-Lobo et al. . The study of Frohlich and Parker  only sampled a few species from gymnosperms, and supposed the existence of NLY in Gnetum. Frohlich  further mentioned the occurrence of both LFY and NLY in Ephedra (his unpublished observations), a close relative of Gnetum. Our present study has covered all extant gymnospermous genera, and the results suggest that each studied species harbors both LFY and NLY genes, except that NLY is still not found in Gnetum. Therefore, our study further supports that the LFY-NLY gene pair originated from an ancient gene duplication, at least before the divergence of gymnosperms. In addition, we have successfully obtained the LFY and NLY genes of Ephedra by RT-PCR and RACE. Moreover, the selection test suggests that both LFY and NLY genes have experienced strong purifying selection in gymnosperms (our unpublished data), implying their conserved functions.
Currently, functions of LFY and NLY in gymnosperms are still not very clear , –, but it is clear that both of them exist as single-copy genes suitable for phylogenetic reconstruction in gymnosperms based on the present study and Yang et al. . No more than two distinct clones were found in the same individual. Although the NLY sequence obtained from the parasitic Parasitaxus usta represents a pseudogene, its exon region shares a high similarity (over 90%) with that of other Podocarpaceae species, and thus still could be used in phylogenetic analysis. Actually, the LFY gene has been successfully utilized in phylogenetic and biogeographic studies of several gymnosperm groups, including Gnetum , Thuja , and Pseudotsuga . In particular, the intergeneric relationships of Cupressaceae s.l. have been well resolved by the LFY and NLY genes .
Interfamilial relationships of gymnosperms
Our study provides the first molecular phylogeny of gymnosperms covering all extant families and genera, and the phylogeny is based on two single-copy nuclear genes LFY and NLY (Fig. 1). This nuclear gene phylogeny is topologically largely consistent with most previous phylogenies of gymnosperms constructed based on cytoplasmic and/or nuclear ribosomal DNA , , . That is, the cycads diverged first, followed by Ginkgoaceae, and then conifers plus Gnetales. Within conifers, Podocarpaceae is sister to Araucariaceae, and Cephalotaxaceae-Taxaceae sister to Cupressaceae (Fig. 2). However, we did not found a sister relationship between cycads and Ginkgoaceae as suggested by chloroplast phylogenomic analyses ,  as well as genome-scale nuclear and plastid data .
The present study seems to supports the monophyly of Taxaceae s.l. that includes Cephalotaxus (Figs. 1, S1), which is consistent with the study of Leslie et al.  based on rbcL, matK, 18S and PHYP. To confirm whether the topology is really constant, we further conducted phylogenetic analyses for the Cephalotaxaceae-Taxaceae lineage using two species (Taiwania cryptomerioides and Cunninghamia lanceolata) of its sister group Cupressaceae as outgroups. The results indicate that Cephalotaxus is strongly supported to be sister to Taxaceae based on either LFY or LFY + NLY CDS, but is nested within Taxaceae with a weak support based on NLY (Fig. 6). The inconsistent positions of Cephalotaxaceae in different analyses could be caused by LBA artifacts or insufficient resolution of the markers. Actually, the evolutionary relationship of Cephalotaxaceae and Taxaceae has been controversial for a long time. All molecular studies based on chloroplast and/or nuclear ribosomal DNA suggested a sister relationship between the two families –, but many morphological studies supported the merge of them (see review by Ghimire and Heo ). A more broadly defined Taxaceae including Cephalotaxaceae has been suggested by Quinn et al.  based on rbcL and matK sequence analyses, and by Ghimire and Heo  based on a cladistic analysis of morphological characters. Also, in the new gymnosperm classification scheme of Christenhusz et al. , Cephalotaxaceae was merged into Taxaceae, and this taxonomic treatment has been adopted by Lang et al.  in the revision of Cephalotaxus. As discussed above, more studies are still needed to resolve the relationship between Cephalotaxaceae and Taxaceae.
A, combined LFY and NLY; B, LFY; C, NLY. Numbers associated with branches are bootstrap percentages of ML and MP higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. Taiwania cryptomerioides and Cunninghamia lanceolata were used as outgroups.
The systematic position of Gnetales has been debated for several decades, which involves six main hypotheses (see reviews by Braukmann et al.  and Wang and Ran ), i.e., anthophyte, gnetales-other seed plants, gnetales-other gymnosperms, Gnetifer, Gnecup and Gnepine , , , , , –. The last three hypotheses all support a close relationship between Gnetales and conifers. In particular, the Gnepine hypothesis (Gnetales sister to Pinaceae) is supported by more and more molecular phylogenetic studies after eliminating bias in data analyses (see review by Wang and Ran ), despite the fact that the Gnecup hypothesis (Gnetales sister to conifer II or cupressophytes) is still supported by a couple of recent phylogenomic studies using all chloroplast genes , . According to the present study, the Gnetales has a close relationship with conifers, although it has not been resolved whether the Gnecup or Gnepine hypothesis is correct. In the trees generated from combined LFY and NLY CDS, Gnetales is strongly supported as sister to conifer II (Fig. 2A and 2C), which is corroborated by the SH and KH tests (Table S4). However, when excluding the third codon positions and using cycads as functional outgroups, the most popular Gnepine hypothesis is recovered with low support (Fig. 2D, Table S4). A similar phenomenon is also observed in Sciadopityaceae. When all CDS sequences are used, this family is moderately supported as sister to the Podocarpaceae-Araucariaceae clade (Fig. 2A and 2C), but when excluding the third codon positions it is revealed as sister to the Taxaceae-Cephalotaxaceae-Cupressaceae clade (Fig. 2B and 2D) as found in most previous studies , –, . The topological conflicts on phylogenetic positions of Gnetales and Sciadopityaceae may be attributed to LBA artifacts that could occur when the fast-evolving third codon positions are included in analyses. Zhong et al.  also found that the LBA artifacts and parallel changes could mislead the phylogenetic placement of Gnetales when using chloroplast genome data, and the removal of fast-evolving genes can effectively alleviate the LBA artifacts, thereby recovering a sister relationship between Gnetales and Pinaceae.
Intergeneric relationships within gymnospermous families
The combined LFY and NLY CDS phylogeny provides a good resolution for intergeneric relationships within four families including Cupressaceae, Pinaceae, Taxaceae and Araucariaceae (Figs. 1, S1). The LFY + NLY phylogeny of Cupressaceae has been discussed in detail by Yang et al. . For Pinaceae, all of the eleven genera form two strongly supported clades. One clade comprises Cedrus, Pseudolarix, and two pairs of sister genera, i.e., Nothotsuga-Tsuga and Keteleeria-Abies, while the other clade includes the sister genera Pseudotsuga and Larix, and the three closely related genera Pinus, Cathaya and Picea (Figs. 1, S1). The revealed intergeneric relationships are largely congruent with the finding of Wang et al. , and are generally consistent with the results of morphological and anatomical analyses (see review by Farjón ). However, Wang et al.  did not completely resolve the systematic position of Cedrus. According to the present study, Cedrus is sister to the Nothotsuga-Tsuga-Pseudolarix-Keteleeria-Abies clade (Figs. 1, S1), which is consistent with most recent molecular phylogenetic studies , . Moreover, like Wang et al. , our study supports the monotypic genus Cathaya as sister to Picea (Figs. 1, S1), rather than to Pinus as suggested by Lin et al. . In Taxaceae, Torreya is sister to Amentotaxus, and Austrotaxus is closely related to the sister genera Pseudotaxus and Taxus (Figs. 1, 6, S1), corroborating previous studies , –. For Araucariaceae, the previous rbcL gene analysis suggested a basal position of Wollemia in the family . However, the present study supports Wollemia as sister to Agathis (Figs. 1, S1), consistent with more recent studies , , , .
The concatenated LFY and NLY CDS can not resolve some intergeneric relationships of cycads and Podocarpaceae very well (Figs. 1, S1). It is interesting that the addition of intron sequences can improve the resolution in Podocarpaceae but not in cycads (Figs. 3, 4), although divergence times of the cycad genera are similar to or longer than those of the Podocarpaceae genera (Fig. 5) , . Consistent with most previous studies , , , , the phylogeny of cycads inferred from either CDS or CDS+Intron sequences of LFY and NLY supports the genus Dioon from tropical America as the basal-most lineage in Zamiaceae (Fig. 3), rather than sister to the Bowenia-Ceratozamia-Stangeria-Microcycas-Zamia clade in the PHYP tree constructed by Nagalingum et al. . Actually, despite low support values in some clades, the present LFY + NLY gene tree is topologically very similar to the recently reconstructed phylogeny of cycads based on five single-copy nuclear genes . For instance, the two genera Zamia and Microcycas, also from tropical America, have a sister relationship and form a clade sister to Stangeria, while the African Encephalartos and the Australian Lepidozamia form a clade sister to Macrozamia from Australia (Fig. 3). Moreover, our study also does not support the establishment of the family Boweniaceae or Stangeriaceae that was based on morphological analyses , , since the two genera Bowenia and Stangeria are nested within Zamiaceae and do not form a monophyletic clade (Fig. 3), as found in most previous molecular phylogenetic analyses , , , .
Compared to the CDS dataset, the CDS+Intron dataset provides a much better resolution for intergeneric relationships of Podocarpaceae (Fig. 4), a large family comprising 19 genera with a wide distribution in the tropics, especially in the Southern Hemisphere , . Our study strongly supports a large clade comprising 11 genera, of which the Australian Microcachrys and the South American Saxegothaea diverged first, followed by the two Australian genera Pherosphaera and Acmopyle, and then the three genera Dacrycarpus, Dacrydium and Falcatifolium (all distributed in Asia and Australia) forming the ‘dacrydioid’ group sister to the ‘podocarpoid’ group that include Retrophyllum, Nageia, Afrocarpus and Podocarpus. In addition, we found a close relationship among the three Australian genera Manoao, Lagarostrobos and Parasitaxus and a sister relationship between Prumnopitys and Sundacarpus (Fig. 4B). This phylogeny of Podocarpaceae constructed from nuclear genes is topologically highly consistent with those inferred from plastid DNA fragments  and from a combined analysis of nrITS1, NLY intron 2 and rbcL sequences as well as anatomical and morphological data . However, our nuclear gene phylogeny strongly supports two pairs of sister genera Retrophyllum-Nageia and Afrocarpus-Podocarpus (Fig. 4B). The genus Phyllocladus is nested within Podocarpaceae (Fig. 4), and thus the family status of Phyllocladaceae is not supported.
Divergence times of gymnosperms
The divergence time estimation is very helpful to interpret the temporal evolution of organisms. Previous studies have provided divergence time estimates for different gymnospermous groups, such as Pinaceae , cycads , Podocarpaceae , Cupressaceae , , and conifers . However, only Crisp and Cook  estimated divergence times of gymnosperms as a whole using molecular clock, and in their study many extant genera were not sampled.
Our present study provides divergence time estimates for gymnosperms based on a sampling of all extant families and genera (Fig. 5). The estimated crown ages of some groups such as Pinaceae, cycads and Podocarpaceae are approaching to those reported in previous studies , , . However, the estimated crown age of Cupressaceae and divergence times of most genera of this family are a little younger than those reported in Yang et al.  and Mao et al. . This could be attributed to the discrepancy of different dating methods and delineation of different fossil calibrations.
Based on the molecular dating analysis, all of the extant five lineages of gymnosperms (cycads, ginkgos, cupressophytes, Pinaceae and gnetophytes) originated at least before 300 Ma (in the Carboniferous), but the crown ages of all families except Ginkgoaceae and Sciadopityaceae are younger than 200 Ma (Fig. 5), indicating that drastic extinctions occurred in the early evolution of gymnosperms, which might be caused by the two extreme cooling events in the Carboniferous and Triassic . After 200 Ma, the divergence speed of genera is moderate when extinction is not considered (Fig. 7), although recent studies showed that the pulse of extinction and speciation in the Cenozoic, even in the late Tertiary, shaped today's species diversity of gymnosperms , . Leslie et al.  found that lineages of conifers that diversified mainly in the Southern Hemisphere show a significantly older distribution of divergence ages than their counterparts in the Northern Hemisphere. However, interestingly, we found that extant coniferous genera in the Northern Hemisphere are older than those in the Southern Hemisphere on average (Fig. 8A). In fact, if excluding the several genera that originated before 150 Ma, the distribution of divergence ages of the remaining genera is very similar between the two hemispheres (Fig. 8B). Of great interest is to investigate why more ancient genera survive in the Northern Hemisphere than in the Southern Hemisphere. Moreover, to get a more accurate estimation of the divergence times and a solid reconstruction of the evolutionary dynamics of gymnosperms, more nuclear genes or genome sequences should be used in future studies, and more reliable fossils are needed to be found.
The divergence times was based on the median ages of the nodes from the BEAST analysis (see Fig. 5).
A, boxplot comparison of all genera; B, dot plot comparison of each genus. The calculation of divergence times was based on the median ages of the nodes from the BEAST analysis as shown in Fig. 5.
The ML and BI trees of gymnosperms constructed from combined LFY and NLY sequences. Numbers associated with branches are bootstrap percentages of ML higher than 50% and Bayesian posterior probabilities greater than 0.90, respectively. A, ML tree from the CDS sequences with Angiopteris lygodiifolia as outgroup; B, BI tree from the CDS sequences with Angiopteris lygodiifolia as outgroup; C, ML tree from the 1st+2nd codon positions with Angiopteris lygodiifolia as outgroup; D, BI tree from the 1st+2nd codon positions with Angiopteris lygodiifolia as outgroup; E, BI tree from the CDS sequences with cycads as functional outgroups; F, ML tree from the 1st+2nd codon positions with cycads as functional outgroups; G, BI tree from the 1st+2nd codon positions with cycads as functional outgroups.
Sources of materials.
Primers used for the PCR amplification and sequencing.
The eleven calibration points used in divergence time estimation for gymnosperms. All constraints were given lognormal prior distributions, where the minimum age was set by the age of the fossil constraint and 95% confidence interval of the probability distribution extending 20 or 40 million years earlier than the minimum age.
Results of the Shimodaira-Hasegawa (SH) test and the Kishino-Hasegawa (KH) test.
Ages of selected clades.
We are indebted to Profs. Chad Husby (Montgomery Botanical Center, USA), Frank Van Caekenberghe (National Botanic Garden of Belgium), Peter Brownless and Peter Hollingsworth (Royal Botanic Garden Edinburgh), Christopher Quinn (Royal Botanic Gardens of Australia), Shou-Zhou Zhang (Shenzhen FairyLake Botanical Garden, China), and Zhiduan Chen (Institute of Botany, Chinese Academy of Sciences) for their kind help in sampling some genera of gymnosperms. We also thank Prof. Robert P. Adams (Baylor University, USA) and the Royal Botanic Garden, Kew (UK) for providing some DNA samples, Ms. Wan-Qing Jin for her assistance in DNA sequencing, and the two anonymous reviewers for their insightful comments and suggestions on the manuscript.
Conceived and designed the experiments: XQW. Performed the experiments: YL. Analyzed the data: JHR YL XQW DMG. Contributed reagents/materials/analysis tools: XQW YL ZYY JHR. Wrote the paper: XQW YL JHR.
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- 88. Won H, Renner SS (2006) Dating dispersal and radiation in the gymnosperm Gnetum (Gnetales) - Clock calibration when outgroup relationships are uncertain. Syst Biol 55: 610–622.
- 89. Peng D, Wang X-Q (2008) Reticulate evolution in Thuja inferred from multiple gene sequences: implications for the study of biogeographical disjunction between eastern Asia and North America. Mol Phylogenet Evol 47: 1190–1202.
- 90. Wei X-X, Yang Z-Y, Li Y, Wang X-Q (2010) Molecular phylogeny and biogeography of Pseudotsuga (Pinaceae): insights into the floristic relationship between Taiwan and its adjacent areas. Mol Phylogenet Evol 55: 776–785.
- 91. Cheng Y, Nicolson RG, Tripp K, Chaw SM (2000) Phylogeny of Taxaceae and Cephalotaxaceae genera inferred from chloroplast matK gene and nuclear rDNA ITS region. Mol Phylogenet Evol 14: 353–365.
- 92. Wang X-Q, Shu Y-Q (2000) Chloroplast matK gene phylogeny of Taxaceae and Cephalotaxaceae, with additional reference to the systematic position of Nageia. Acta Phytotax Sin 38: 201–210.
- 93. Hao DC, Xiao PG, Huang BL, Ge GB, Yang L (2008) Interspecific relationships and origins of Taxaceae and Cephalotaxaceae revealed by partitioned Bayesian analyses of chloroplast and nuclear DNA sequences. Plant Syst Evol 276: 89–104.
- 94. Ghimire B, Heo K (2014) Cladistic analysis of Taxaceae s.l.. Plant Syst Evol 300: 217–223.
Quinn CJ, Price RA, Gadek PA (2002) Familial concepts and relationships in the conifers based on rbcL and matK sequence comparisons. Kew Bull 57 513–531.
- 96. Lang XD, Su JR, Lu SG, Zhang ZJ (2013) A taxonomic revision of the genus Cephalotaxus (Taxaceae). Phytotaxa 84: 1–24.
- 97. Donoghue MJ, Doyle JA (2000) Seed plant phylogeny: Demise of the anthophyte hypothesis? Curr Biol 10: R106–109.
- 98. Doyle JA, Donoghue MJ (1986) Seed plant phylogeny and the origin of angiosperms - an experimental cladistic approach. Bot Rev 52: 321–431.
- 99. Schmidt M, Schneider-Poetsch HA (2002) The evolution of gymnosperms redrawn by phytochrome genes: the Gnetatae appear at the base of the gymnosperms. J Mol Evol 54: 715–724.
- 100. Burleigh JG, Mathews S (2007) Assessing among-locus variation in the inference of seed plant phylogeny. Int J Plant Sci 168: 111–124.
- 101. Zhong B, Deusch O, Goremykin VV, Penny D, Biggs PJ, et al. (2011) Systematic error in seed plant phylogenomics. Genome Biol Evol 3: 1340–1348.
Farjón A (1990) Pinaceae: Drawings and Descriptions of the Genera Abies, Cedrus, Pseudolarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix and Picea. Königstein, Germany: Koeltz Scientific Books.
- 103. Setoguchi H, Osawa TA, Pintaud JC, Jaffre T, Veillon JM (1998) Phylogenetic relationships within Araucariaceae based on rbcL gene sequences. Am J Bot 85: 1507–1516.
- 104. Liu N, Zhu Y, Wei ZX, Chen J, Wang QB, et al. (2009) Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes. Chinese Sci Bull 54: 2648–2655.
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Eckenwalder JE (2009) Conifers of the World: the Complete Reference. London: Timber Press.
Farjón A (2010) A Handbook of the World Conifers. Vol. 1, 2. Leiden: Brill Press.
- 110. Biffin E, Brodribb TJ, Hill RS, Thomas P, Lowe AJ (2012) Leaf evolution in Southern Hemisphere conifers tracks the angiosperm ecological radiation. Proc Roy Soc B-Biol Sci 279: 341–348.
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domain: literature


text:
Free forum : A friendly group for lovers of Psp and siggy piggies who love tags. Lots of challenges, games and friendship. Everyone is welcome.
Free forum : PSP community, signature tags, challenges, having a blast
PSP and Tagmaking community that offers friendship, Challenges, Tutorials, and Signature Tags.
Free forum : A place to share our creations and make friends and laugh and most important have a good time.
We are a group of friends who love to make and share tags. We have daily theme threads, PSP Challenges and more. Please join us!
Free forum : The Sav is Back! Welcome to Savage Island where you can kick back and relax. This is a place for both taggers and non-taggers to escape from reality. Welcome!
We are a group of women who enjoy tags, psp and friendship. We have daily theme threads, some games, a wonderful chat thread, psp challenges and more! Come check us out! Members MUST be active upon jo
Free forum : PSP, TAGS, SIGS, TUTORIALS. Free forum : Wicked Cute Tutorials
Forum gratis : Free forum : Just a place to have all my tags and backgrounds
Acesta este forumul OFICIAL al serverului SunShine
Siggies, Snags, Tags, Gift Baskets and More
Free forum : A kick butt forum with awesome designers & members! We offer WWO's, Tags, Siggies, Tag Auctions, Member Points, Snags, Goodies for Facebook & Blogs, Chat, Friendship & tons more.
The Gates are open to the GFX world.
Siggy Tags Offers, And Lots More.
Free forum : Paint Shop Pro Hobbies
Free forum : We are a friendship and psp group We offer tags wwo lots of ways to win tubes and tags for pspers and non pspers
Free forum. Free forum : The Everybody Forum. Free forum,
Official Group for MtA Licensing Company on MSN--**Open to Pspers, Non-PSPers and those with or without licenses. Siggy Piggys Welcome!**. MtA is a new
Free forum : A relaxing forum for all scrappers, taggers, tubers and those who love to collect tags and join in the fun with a heap of freebies, competitions, challenges & more
Personal business web pages (develop and designed), digital signatures, digital name tags, personal/business layouts, myspace layouts, myspace codes, glitz enhancements, natural enhancements, photography enhancements
Giving Inf. CE,Clan Tags,Room Saves,And More To Non-Hackers
PSP Group offering tags, snags, WWO, games, tutorials, graphics & much, much more!! We are an open group so please feel free to join at any time.
This forum will let you role play animals. There are, pack, clans, heards, and more.
Favorite artist tubes created in tags, siggys sets, banners and more.
For people who have an addiction to making tags and collecting them just the same.
Free forum : Looking for a year round game of secret Santa using sig tags???
We're just a few girls looking for a place to make tags!
Free forum : :::Welcome·To·Gorjuss·Girls·Group:::Our Group Have PSP, Tags, Tuts Scraps and Pressies. And With Dedicated of Beautiful Artwork of : Suzanne Woolcott And Artwork of: AngelBear's Bear
Here is a PSP location for taggers & Snaggers. only offers. all the time. Want to request? Join us :0 Want to offer? Join Us :) No Pressure site

domain: hobby


text:
When I was only as tall as your knees,
Sundown was a well-established routine:
bath and pajamas and prayers and sleep.
At first I’d wail, my little fists flailing into
the depths of your arms; warm, coaxing, as
you’d whisper tales of elves and fairies,
drifting me off into deep, sweet slumber.
These days your knees bend and ache;
your eyes and ears aren’t what they once were, and
you forget a little more with each passing day.
At first it was where you put your keys, then
what year, then how to speak, and finally, me.
There’s not much to slow your memory from
fading into a world that only you know.
Now it’s my turn to anticipate the Sundown –
you grow confused as the light fades, and I
prepare the bath and pajamas and prayers and sleep.
At first you protest, pacing and anxious, scared
until we reach the bed, comforting and familiar.
This is what our nights have become, yet
I still cherish our Sundown.

domain: artistic


Goods and Services (the trademark is registered for the following goods and services)
Class 1 : Filtering materials; oils for industry
Class 4 : Fuels; solid fuels; coke; oils; gas; charcoal; candles; lighter fluids.
Class 7 : Filtration apparatus; filters.
Class 9 : Protective clothing; protective masks; vacuum cleaners.
Class 11 : Air conditioning apparatus and systems; filters for air conditioning; filters for drinking water; air filters; water purifiers; dehumidifiers; air ionisers; cooker hood filters; barbecues; disposable barbecues.
Class 19 : Non-metallic building materials; aggregates; sand; clays for building; ready-mixed concrete; concrete; bricks; blocks.
Class 36 : Export/import finance and insurance advisory services; customs clearance and documentation services.
Class 37 : Refractory repair services; installation of filtration systems; mining; hire services for industrial equipment; extraction of landfill gas; repair, maintenance and installation services all relating to fuel usage; emergency repair services relating to apparatus which utilise fuel; mineral extraction.
Class 39 : Distribution services; transportation services; bulk transportation services; transportation services utilising inland waterways; freight services; import/export cargo services; export packing services; fuel delivery and top up services; oil delivery and top up services; distribution of landfill gas for power/heating; supply of electricity.
Class 40 : Processing of bulk mineral products; mineral separation, processing, handling and blending services; fine materials recovery; effluent treatment; power generation and utilisation facilities; custom assembly of plants; carbon reactivation services; filter recycling services; conversion of landfill gas into electricity.
Class 42 : Environmental monitoring services; pollution monitoring services; analysis of landfill gas; analytical services; analysis of surface, ground and waste water, technical plant design services; land reclamation services; health and safety monitoring; health and safety assessment services relating to hazardous materials and the working environment; site surveys; contaminated land assessments; effluent analysis; occupational health and hygiene consultancy services; site evaluation services; bespoke filter design services; filtration consultancy and advisory services; technical advisory services relating to processing of bulk mineral products, mineral separation, processing, handling and blending services, fine materials recovery, effluent treatment, power generation and utilisation facilities, custom assembly of plants, carbon reactivation services and filter recycling services.... More


text:
NE Philly restaurants (Grant and Academy Area)
My husband is currently working evenings at the NE Philly Airport. We to meet for dinner every once in awhile, and we usually go for cheesesteaks (yum). I'd like to try something different. Any suggestions?
Miller's & 3 Monkey's is good. Santucci's Pizza is at Woodhaven & Knights Road. Or if you want bbq, you could try Sweet Lucy's. If you want Japanese, there's Fuji San on the Blvd between Red Lion & Haldemann - there's an I-Hop and Party City next to it. And I think there's a Paddywhack's at Comly & the Blvd.

domain: food


Please visit our new athletics website, www.bethanybison.com
BETHANY BEATS THOMAS MORE TO ADVANCE TO PAC VOLLEYBALL FINAL
Nov. 4 - BEAVER FALLS, Pa. – The Bethany College volleyball team advanced to the PAC Tournament Championship match for the first time in school history by outlasting Thomas More for a 3-2 victory (25-18, 29-27, 19-25, 18-25, 15-11) Friday night in the conference semifinals, which were held at Geneva.
The Bison (28-5), who tied the single-season school record for victories, will take on top-seeded Geneva (26-8) in the title match Saturday at 4 p.m. Both teams will be looking for their first PAC championship, as the Golden Tornadoes are in their first year in the conference after transitioning from the NAIA.
In the opening game, the Bethany hitters controlled the action, collecting 17 kills. Senior Carrie Talkington (Moundsville, W.Va./John Marshall) pounded down seven to lead the way, including three straight to give the Green and White a 16-14 lead they never relinquished. The kills also ignited a 12-4 run to close the game, as the Bison went in front 1-0.
The Saints looked like they would draw even in the second, as they took a 23-18 lead. After a pair of BC points, Thomas More scored again and was serving for the match at 24-20. Bethany stormed back with five consecutive points to go in front 25-24. The teams began exchanging points, which led to a 27-27 tie. Following a TMC error, the Bison finally claimed the match on an ace by senior Tiffany Hoffman (Cambridge, Ohio/John Glenn) and BC was one game away from moving on to the title match.
That win would not come easily. Thomas More recovered from a 0-2 deficit in Tuesday’s quarterfinal to beat #6 Saint Vincent and looked to rewrite that script. In the third game, four straight points by the Saints gave them a 14-8 lead and Bethany could not get closer than four the rest of the way before TMC closed out a 25-19 victory. In the fourth game, Thomas More used a 7-1 spurt to take a 21-12 advantage and held off a Bison rally for a 25-18 win to force the fifth and deciding game.
The final game went back-and-forth throughout. The Bison scored the first four points, which was answered by three in a row by Thomas More. Bethany tallied three straight, but the Saints ripped off seven of the next eight to take their largest lead of the game at 10-8. The Bison made the final run of the night, scoring seven of the final 10 points to pull out the win. The final score came on a kill by junior Taylor Cassidy (Barboursville, W.Va./Cabell Midland), sending BC into the championship match and giving them 28 wins to tie the total first posted by the 1987 team that went 28-22.
Four Bison compiled double-digit kills in the victory, led by Talkington with 19 and she also posted five blocks. Junior Lauren Goodwill (Canton, Ohio/Jackson) delivered 14 kills and Cassidy and freshman Breana Harris (Severn, Md./Old Mill) both recorded 11. Junior setter Jessica Zavatchen (Uniontown, Pa./Laurel Highlands) triggered the offense with another big night, passing out 54 assists and also finished with 14 digs and three blocks.
Bethany’s backrow efforts were sparked by Tiffany Hoffman with 30 digs. In addition, senior Jessica Cole (Connellsville, Pa.) chipped in 21 digs and senior Megan Hoffman (Cambridge, Ohio/John Glenn) amassed 16.
Saturday’s Bethany-Geneva contest will be the rubber match between the teams this year. The Bison won the first meeting between the teams on Oct. 1 by a 3-1 score behind 16 kills from Talkington and 41 assists from Zavatchen, which was the only loss suffered by the Golden Tornadoes in the conference this year. The GTs earned their revenge with a 3-1 triumph in Beaver Falls on Oct. 20.


text:
Mike Bauer live with Band, Zebulon Krol, and Obey the Wolves
Thursday, June 28th, 2018
2906 Sunset Blvd, Los Angeles, CA 90026
Mike Bauer and his band return to the stage for the first time in 2018, supporting headliner Zebulon Krol and Obey the Wolves. Don't miss this event at the storied Silverlake Lounge.


text:
Jordyn Woods makes her acting debut in a sneak peek for this week’s episode of Grown-ish.
Premiered by Entertainment Tonight on Monday, the clip shows Woods playing a freshman student named Dee, who lives in Hawkins Hall and gathers with the rest of the residents in the aftermath of the scare.
In the scene, Dee engages in an open conversation about mental health awareness, led by Aaron, R.A. of the dorm. "I understand that you guys think that what I'm trying to do is whack, but I'm really just trying to create an environment so we can have an open dialogue about what's really going on in our lives,” Aaron says in the scene.
“So please, bear with me. Are any of you experiencing a sense of isolation or de-personalization?"
When the group of students responds with looks of confusion, Woods’ character chimes in, "Um, I'm sorry, I don't even know what that means.”
When Kid Cudi's 2010 song, "Pursuit of Happiness," is mentioned, things start to click together for Dee, who notes, "Yeah, OK... in that case, I've been hella de-personalized before.”
Grown-ish airs Wednesdays at 8 p.m. on Freeform.

domain: entertainment


A massive march and rally are planned for this Saturday, Aug. 13 in Harlem. The Millions March will be held along the Malcolm X Boulevard corridor, starting at 110th Street. Under the banner of “Pan-Africanism or Perish,” the day’s focus will be on three leading issues: ending the bombing of Libya, ending the illegal economic sanctions in Zimbabwe and ending Mayor Michael Bloomberg’s destruction of education, housing and healthcare.
Nation of Islam leader Minister Louis Farrakhan will be the keynote speaker.
In an interview with the Amsterdam News, the minister encouraged tri-state area residents to attend in great number to hear words of wisdom, encouragement and practical solution building.
Farrakhan laid out the key issues that will be dealt with on Saturday:
“Sister Viola Plummer and the December 12th Movement, along with many of our nationalist brothers and sisters, see the effort of the Western nations in the bombing of Libya and their step toward the recolonization of the Continent of Africa. There is a relationship between what Western nations are doing to Libya and Africa and what is being done to the Black, Brown and white poor in America. We must address this in America.”
March convenors will hold a press conference on Thursday, Aug. 11 at noon at the National Black Theatre, 2031 Fifth Ave. at the corner of 125th Street. Speakers will include Plummer, Min. Abdul Hafeez Muhammad of the Nation of Islam, Shaka Cousins of the All African Peoples Revolutionary Party, radio personality Bob Law and others.
Farrakhan and other speakers will lay out the case for Libya’s Col. Muammar al-Gaddafi and Zimbabwe’s Robert Mugabe. They will touch on Somalia and the famine in the Horn of Africa and the snatching up of Africa’s essential resources by outside nations.
And the current burning of cities in Britain by disaffected youth is something folks on this side of the pond should pay close attention to, said the minister.
“The budgetary problems of England, America, France, Greece, Italy, Spain, Portugal, Germany and Canada…are forcing these governments to use austere measures to try and balance their budgets. These austere measures usually are made on the backs of the poor, and so the killing of a young Black man in London was a trigger that ignited the passion of the dissatisfied people of the United Kingdom.
“With what was signed into law by our president, with trillions of dollars cut, with no taxation of the rich to help, these cuts will be on the back of poor and middle class whites. Blacks and Browns of America are fast becoming the new poor. Unfortunately, what we see in England, Greece and Israel will be going on in America.”
You think so, Minister?
“I know so.”
Does the United States government know this?
“Yes, they do. This is why they are firing teachers and hiring more police and dressing them like soldiers in Iraq and Afghanistan, giving them automatic weapons with armor piercing bullets-because they know the poor will rise because they can’t take it.”
Farrakhan qualified his predictions, saying that, “If you study the Bible and the Qur’an, all of these things are written. It is prophecy. May God help us. Unfortunately, we have reached a time of trouble like there never was since there was a nation and a time.”
Back to Saturday: a united front of far-reaching grassroots organizations has come together. The Millions March in Harlem Coalition comprises many leading national and international organizations that have fought for African peoples’ human right to self-determination for decades.
“Not only I, but every speaker that will come and each performer that will perform will be feeding the public that which inspires us to do better and rise higher in our pursuit of freedom, justice and equality,” Farrakhan assured the Amsterdam News.
As for the young people who might be thinking about wiling their time away doing other things on Saturday, the Chicago-based minister insisted, “Our young people are in turmoil because we as the elders are enamored by the wealth of the modern pharaoh. Unfortunately, we have neglected to plan effectively for our future in the time of pharoah. God allowed the elders to die in the wilderness because of their cowardice and love of pharaoh, and he took their children and it was they who inhabited the promised land. We as elders should be directed by them.”
It won’t be a talk shop but a rallying of the spirit and sense of purpose. not unlike that felt in the aftermath of Farrakhan’s 1995 Million Man March. “Our people will be energized to want to unite with each other, respect each other, love each other more. With our unity, there will be nothing that we will not be able to accomplish.”
Harlem will be the spot on Saturday.
“In the Bible, it says, ‘My people are destroyed for the lack of knowledge,’” said Farrakhan. “So on Saturday-God willing-much knowledge will be imparted. For those who attend-and it is free of charge-to sacrifice a few hours to become more knowledgeable may help us to discharge our duties to self and family in a better way.
“God willing, I will be able to visit with Brother Gil Noble [the ‘Like It Is’ host is currently in critical condition after suffering a major stroke]. He is a great soldier and a great helper. To lose him would be a great loss.”
For more information, contact Millions March in Harlem at (718) 398-1766 or www.millionsmarchharlem.com.


When Madonna recently taped an appearance on Ellen she had a surprise in store for her son Rocco.
As part of the show she volunteered her 12-year-old son to take a seat in the dunk tank, which would help raise $10,000 for breast cancer research, something Ellen DeGeneres has been doing all month long.
When Ellen asked Rocco, who is a dancer on his mother’s MDNA tour, if his mother was too strict, he replied: “Um, yeah, she’s a good mother.” Madonna of course kept pressing her son, questioning what that really meant.
In the end, Rocco explained that she is strict but he doesn’t mind: “She’s a good mother. Yes. That’s all I have to say. She’s very strict, but in a good way.”
When asked how he feels about his famous mom flashing her lady parts to her fans though, the young boy was very clear: “I actually don’t see that.”Madonna confirmed, explaining he’s underneath the stage getting ready when she does all of that.
Watch Rocco get dunked below. The full episode with the Material Girl airs Monday, October 29.
– Cicely C. Mitchell, Mix 96.5, Houston
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DUATHLON: Sapperton’s Sheard is British champion
A King’s School student from Sapperton has become British Duathlon champion.
Seventeen-year-old Seamus Sheard emerged as under-20 age group champion following his winning performance in the British Duathlon Championships, held at Windsor.
The Windsor Duathlon standard distance race (10km run/40km cycle/5km run) was the first UK domestic championships of 2016, featuring more than 750 of Britain’s finest age group duathletes.
A member of Sleaford Triathlon Club, Seamus completed the event in 2hr 17min 15sec, a time that also saw him finish 103th overall, despite poor conditions with a boggy transition and mud causing issues for many competitors.
This win followed up his inaugural Team GB international performance last October when he competed at the 2015 ITU Duathlon World Championship in Adelaide where, although out of the medals, he finished a creditable 17th in his group and 117th overall.
His next goal is to qualify for this year’s ITU World Triathlon Series Grand final in Cozumel, Mexico.
Speaking after the event, the King’s School sixth former wished to thank his club coaches and local sponsors who have provided both support and financial assistance over the past year, enabling him to reach the point he is now at, and commented: “I hope this is the start of another exciting season and can’t wait to see what else I can achieve.”


text:
The HMS team has been back in Canada about a week now.
The past few days have been filled with debrief, reflection and worship.
Last night while we had our final barbeque, and while we worshiped
I had two thoughts.
Arriving and Breaking
First off, I have realized that
I need a good breaking.
To be exposed
made raw and sensitive
to even the most tender touch of God.
To His leading.
To the breaking of hearts.
Where my heart has become calloused
and does not know anymore how to recognize
the soft touch of hand in need that reaches out
and the finger of God that points me where to love,
that is where I need a good breaking.
I began to ponder, while sitting in circle of students
that just spent the past year on an incredible journey,
what is arriving?
Something true for almost all people
in this life,
is that they want to arrive somewhere.
To reach a goal,
a final place.
Somewhere that we can work towards reaching,
something that we can plan on accomplishing.
But, I think, if you want to arrive,
you need to stop heading somewhere else
outside of the present moment.
We so easily wander into thoughts of reaching a place different
than where we are today.
But to be here,
to be present and to realize its impact on everything that is to come.
That is what I think it means to arrive.
To allow yourself to catch up with yourself,
or to pull yourself back from getting too far ahead.
now. Really be here.
See the the textures of the colors on the wall.
See the way the light hits the corners of the room
casting shadows and highlights.
Hear the sound of worship,
hear it truly and deeply.
Here the rise and falls and the intonations of voices
being lifted up
Arrive here, where you are now.
Was the beginning of a semester,
a journey leading to this place?
But only because the present
builds on the present to reach another day.
Each day of your semester,
is an arrival.
Your destination is to be where you are now in Christ.


text:
Thursday 17 September 2020
It all started with an education program
Monday 14 September 2020
A season under high surveillance
Monday 07 September 2020
It all started with an Ocean Initiative
Friday 04 September 2020
It all started with a demonstration
Tuesday 01 September 2020
Back to School with Ocean Campus
Wednesday 26 August 2020
Everything started with one lobbying action
Le Havre, France: Progress made in Dollemard’s old landfill sites
For several years now Dollemard’s former landfill site has been sparking off outrage among Le Havr...
Less than a month left to say no to microplastics in our products in Europe
Surfrider Europe calls for a broad mobilisation of civil society and progressive stakeholders to res...
Chemical pollution in the Gulf of Fos, France: Surfrider Foundation Europe takes to court
Following a ferric chloride solution leak from a company called Kem One at Lavéra in Martigues, Fra...
Chemical pollution, forgotten from bathing water quality follow-up
Coral, fish, plankton, crustaceans: this is how you picture the ocean floor, a deep and mysterious w...
Surfrider’s Ocean Friendly Restaurants program launches in Europe
Staying true to one’s sustainable habits can be challenging outside the household, and restaurant ...
Plastic pollution: beware of plastic fake outs
More than 400 million tons of plastic are produced worldwide every year, a figure making heads spin....

domain: news


Posted on September 12, 2018
Once upon a time, a long, long time ago, there weren't 50-jillion different television channels. There weren't streaming services of any kind, there weren't even humble ways to record a TV show, and if you go back far enough, there wasn't even color on TV!
Cue the gasps! But it's all true.
On this date in 1959, the first regularly scheduled program in color premiered. It was the cowboy family show Bonanza.
The show was set in Nevada, near beautiful Lake Tahoe (which is located on the border of California and Nevada). I wonder how many people decided to visit Lake Tahoe because of those beautiful full-color shots being displayed in their living rooms?
Guess what? When Bonanza first appeared, it got okay ratings. There were basically only three television stations then, and Bonanza usually ended up in the middle slot for its audience share. But it was expensive to make, and the NBC folks considered cancelling it.
But they didn't - because RCA owned NBC, and RCA was trying to sell color TV sets. And having a well-crafted show airing in color REALLY helped convince people to buy them! (I mean, why buy a color set if all the shows were shot in B&W?)
So NBC kept Bonanza in the lineup, and it started to get more and more popular. It turned out to be the second longest western show ever, and one of the top-10 longest running live-action TV shows, AND one of the top-50 greatest shows of all time!! Bonanza ran for 14 seasons!
Also on this date:
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GM looking to bring carbon fiber to mainstream cars
On Friday, GM signed a partnership agreement with Teijin Limited, a Japanese company that specializes in carbon fiber reinforced thermoplastic technology.
Teijin has developed an automotive molding process that cuts the cycle times of its carbon fiber components to under a minute. Other carbon fiber companies use thermosetting resin processes that take more than five times longer to set, according to Teijin. These longer molding times are a primary reason why carbon fiber has been so expensive and limited to high-end performance cars.
Teijin won both a Frost & Sullivan 2011 Global Automotive Carbon Composites Technology Innovation Award and a Best Product Innovation Award at the ICIS Innovation Awards 2011 for its technology.
GM believes that with Teijin's foundation in carbon fiber, the companies will be able to effectively mass produce and apply carbon fiber, making it less expensive, and practical for applications beyond expensive cars. GM doesn't just envision using carbon fiber on the likes of Cadillacs and Corvettes, but also on mainstream, everyday cars, trucks and crossovers.
"Our relationship with Teijin provides the opportunity to revolutionize the way carbon fiber is used in the automotive industry, said GM Vice Chairman Steve Girsky. "This technology holds the potential to be an industry game changer and demonstrates GM's long-standing commitment to innovation."
Next year, Teijin will break ground on the Teijin Composites Application Center in the northern part of the United States so that it can collaborate more closely with GM.
Carbon fiber is both 10 times stronger than regular-grade steel and a quarter of the weight. Exotic automakers like McLaren and Lamborghini have capitalized on this combination of properties to create cars that have the structural integrity and light weight necessary to get as much out of their big, powerful engines as possible.
The Lamborghini Sesto Elemento, a concept-turned-limited-production-model, is a showcase for the performance potential of carbon fiber and other composites. The car uses carbon fiber-reinforced plastics throughout major components like the chassis, suspension and body, along with a stripped-down build (the interior was basically just the raw carbon tub with seats) to cut weight down to 2,202 lbs. (999 kg). Resulting performance, as provided by Lamborghini, is a 2.5-second dash to 62 mph (100 km/h), making the 570-hp (419 kW) car every bit as quick as the Bugatti Veyron.
GM, of course, isn't interested in using carbon fiber to build bullet-quick supercars, but to take advantage of the other benefit of low weight: cleaner, more efficient driving. Using carbon fiber will help GM to greatly reduce the weight of its vehicles, which in turn will increase the fuel economy of its fleet, helping the automaker to meet government-imposed restrictions. More efficiency is also a big selling point for consumers.
The companies haven't detailed any specific applications for their automotive carbon fiber work but stated that such applications will be announced closer to market readiness.
We look forward to seeing how this partnership develops. A push to bring carbon fiber to the mass market is certainly an intriguing turn.


text: This fast-moving sequel to Piggie Pie! (1995) follows demanding, bunny-eating Gritch the witch as she (following a crash-landing on her broom) shops for new transport. After consulting a wittily rendered address book (among the entries is one ""Bea Witch, Samantha Lane, Darrin, Conn.""), she ends up at a wacky car dealership. More frightening than Gritch is the foxy salesman's plaid jacket as he goes through a ""Who's on First?.""--style routine with brooms labeled ""Witch 1"" and ""Witch 2."" Finally the warty, purple-clad shopper settles on ""something hip. Something hot. Something happening!"" She peels out in a fast car and absconds with the fox, and his future doesn't look very secure. The book, like its predecessor, is silly, fun, and revved-up for reading aloud.

domain: library


Want to help the people affected by the fires?
The Creek, Rye, and Skirball fires have damaged or destroyed dozens of structures and spurred hundreds of thousands evacuations across Los Angeles County. In Ventura County, the Thomas Fire has been even more devastating, claiming at least 150 structures and forcing 100,000 people to evacuate.
The smoke in the air, the wind howling through the trees, and the alerts ringing in our phones have put us all a little on edge. It is easy to feel helpless when natural disasters rip through our community, but there are ways to do some good.
Open up your wallet
If you have the means, this is one of the easiest ways to help. Even a small donation can make a difference.
The Red Cross, which is operating multiple evacuation centers in LA County, is accepting donations through its website or by text. To make a contribution, text REDCROSS to 90999.
The Salvation Army, now providing food and supplies to evacuees in Ventura County, is also requesting donations, which can be made online or by phone (1 800 SAL ARMY).
Donate your time and skills
Looking to get involved right away? Head up to Ventura County, where the United Way, Red Cross, Sheriff’s Department are teaming up to place individual volunteers in places where their efforts will have the most impact.
The Red Cross accepts volunteer applications on its website year-round, and it’s a great way to get involved in your community during times of need.
Many of the evacuation centers and animal shelters housing people and pets displaced by the fires are accepting donations of food, water, and other supplies. A list of evacuation centers and animal shelters for the Creek Fire can be found here (and here for the smaller Skirball Fire).
Make sure to call ahead and check what might be needed.
Foster an animal
Support the LA fire department
Want to support the brave women and men that fight the fires and protect our communities? Donate directly to the Los Angeles Fire Department Foundation.


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This is a real classic Vacchetta made in Tuscany, with an exquisite vintage effect.
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This watch strap is fully hand made and hand stitched with linen thread. Leather will collect great patina. Edges polished with beeswax and special compound using traditional technique.
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