Evolution Of Vascular Plants Research Articles

Phytochelatin Synthases of the Model Legume Lotus japonicus. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants

The biosynthesis of phytochelatins and homophytochelatins has been studied in nodulated plants of the model legume Lotus (Lotus japonicus). In the first 6 to 24 h of treatment with cadmium (Cd), roots started to synthesize elevated amounts of both polypeptides, with a concomitant increase of glutathione and a decrease of homoglutathione, indicating the presence of active phytochelatin synthase (PCS) genes. Screening of transformation-competent artificial chromosome libraries allowed identification of a cluster of three genes, LjPCS1, LjPCS2, and LjPCS3, which were mapped at 69.0 cM on chromosome 1. The genes differ in exon-intron composition and responsiveness to Cd. Gene structures and phylogenetic analysis of the three protein products, LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N, are consistent with two sequential gene duplication events during evolution of vascular plants. Two sites for alternative splicing in the primary transcripts were identified. One of them, involving intron 2 of the LjPCS2 gene, was confirmed by the finding of the two predicted mRNAs, encoding LjPCS2-7R in roots and LjPCS2-7N in nodules. The amino acid sequences of LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share 90% identity, but have only 43% to 59% identity with respect to the typical PCS1 enzymes of Lotus and other plants. The unusual LjPCS2-7N and LjPCS3-7N proteins conferred Cd tolerance when expressed in yeast (Saccharomyces cerevisiae) cells, whereas the alternatively spliced form, LjPCS2-7R, differing only in a five-amino acid motif (GRKWK) did not. These results unveil complex regulatory mechanisms of PCS expression in legume tissues in response to heavy metals and probably to other developmental and environmental factors.

Analysis of a xyloglucan endotransglycosylase/hydrolase (XTH) from the lycopodiophyte Selaginella kraussiana suggests that XTH sequence characteristics and function are highly conserved during the evolution of vascular plants

A tissue print followed by a xyloglucan endotransglycosylase assay revealed that XET activity is present at sites of cell elongation in both roots and shoots of the lycopodiophyte Selaginella kraussiana. This paper provides the first report and analysis of a xyloglucan endotransglycosylase/hydrolase (XTH) cDNA sequence, isolated from a club moss. In silico analysis of the deduced amino acid sequence revealed a strong conservation of the XET-domain described in higher plants. The catalytic site (DEIDLEFLG) varies in only one amino acid compared with the consensus sequence and was shown to be functional after recombinant expression of Sk-XTH1 in Pichia pastoris. Sk-XTH1 displays xyloglucan endotransglycosylase activity over a broad pH (4.5-7.5) and temperature range (4-30 degrees C), but it shows no hydrolase activity. The catalytic site is followed by a consensus sequence for N-linked glycosylation. Four terminal cysteines were shown to stabilize a putative XET-C terminal extension region, which includes conserved amino acids, involved in the recognition and binding of the substrates. The N-linked sugar interactions as well as the disulphide bridges were shown to be necessary to perform XET activity. The presence of a highly conserved XTH sequence and function in a microphyllophyte suggests that XTHs were present before the divergence of lycopodiophytes and euphyllophytes. It also points to a possible key role for XTHs in the production of a cell wall that allowed the further evolution of land plants.

Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii

BackgroundThe lycophyte Selaginella moellendorffii is a member of one of the oldest lineages of vascular plants on Earth. Fossil records show that the lycophyte clade arose 400 million years ago, 150–200 million years earlier than angiosperms, a group of plants that includes the well-studied flowering plant Arabidopsis thaliana. S. moellendorffii has a genome size of approximately 100 Mbp, as small or smaller than that of A. thaliana. S. moellendorffii has the potential to provide significant comparative information to better understand the evolution of vascular plants.ResultsWe sequenced 2181 Expressed Sequence Tags (ESTs) from a S. moellendorffii cDNA library. One thousand three hundred and one non-redundant sequences were assembled, containing 291 contigs and 1010 singletons. Approximately 75% of the ESTs matched proteins in the non-redundant protein database. Among 1301 clusters, 343 were categorized according to Gene Ontology (GO) hierarchy and were compared to the GO mapping of A. thaliana tentative consensus sequences. We compared S. moellendorffii ESTs to the A. thaliana and Physcomitrella patens EST databases, using the tBLASTX algorithm. Approximately 60% of the ESTs exhibited similarity with both A. thaliana and P. patens ESTs; whereas, 13% and 1% of the ESTs had exclusive similarity with A. thaliana and P. patens ESTs, respectively. A substantial proportion of the ESTs (26%) had no match with A. thaliana or P. patens ESTs.ConclusionWe discovered 1301 putative unigenes in S. moellendorffii. These results give an initial insight into its transcriptome that will aid in the study of the S. moellendorffii genome in the near future.

Lichen-Like Symbiosis 600 Million Years Ago

The fossil record of fungi and lichens is scarce. Here we report the discovery of lichen-like fossils, involving filamentous hyphae closely associated with coccoidal cyanobacteria or algae, preserved in marine phosphorite of the Doushantuo Formation (between 551 and 635 million years old) at Weng'an, South China. These fossils indicate that fungi developed symbiotic partnerships with photoautotrophs before the evolution of vascular plants.

COMPUTER MODELS OF EARLY LAND PLANT EVOLUTION

▪ Abstract Computer models are used to mimic the early evolution of ancient vascular plants (tracheophytes). These models have three components: (a) an N-dimensional domain of all mathematically conceivable ancient morphologies (a morphospace); (b) a numerical assessment of the ability (fitness) of each morphology to intercept light, maintain mechanical stability, conserve water, and produce and disperse spores; and (c) an algorithm that searches the morphospace for successively more fit variants (an adaptive walk). Beginning with the most ancient known plant form, evolution is simulated by locating neighboring morphologies that progressively perform one or more tasks more efficiently. The resulting simulated adaptive walks indicate that early tracheophyte evolution involved optimizing the performance of many tasks simultaneously rather than maximizing the performance of one or only a few tasks individually, and that the requirement for optimization accelerated the tempo of morphological evolution in the Silurian and Devonian.

Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants.

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.

Assessing the redundancy of MADS-box genes during carpel and ovule development.

Carpels are essential for sexual plant reproduction because they house the ovules and subsequently develop into fruits that protect, nourish and ultimately disperse the seeds. The AGAMOUS (AG) gene is necessary for plant sexual reproduction because stamens and carpels are absent from ag mutant flowers. However, the fact that sepals are converted into carpelloid organs in certain mutant backgrounds even in the absence of AG activity indicates that an AG-independent carpel-development pathway exists. AG is a member of a monophyletic clade of MADS-box genes that includes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), indicating that these four genes might share partly redundant activities. Here we show that the SHP genes are responsible for AG-independent carpel development. We also show that the STK gene is required for normal development of the funiculus, an umbilical-cord-like structure that connects the developing seed to the fruit, and for dispersal of the seeds when the fruit matures. We further show that all four members of the AG clade are required for specifying the identity of ovules, the landmark invention during the course of vascular plant evolution that enabled seed plants to become the most successful group of land plants.

Monolignol radical–radical coupling networks in western red cedar and Arabidopsis and their evolutionary implications

The discovery of a nine-member multigene dirigent family involved in control of monolignol radical–radical coupling in the ancient gymnosperm, western red cedar, suggested that a complex multidimensional network had evolved to regulate such processes in vascular plants. Accordingly, in this study, the corresponding promoter regions for each dirigent multigene member were obtained by genome-walking, with Arabidopsis being subsequently transformed to express each promoter fused to the β-glucuronidase (GUS) reporter gene. It was found that each component gene of the proposed network is apparently differentially expressed in individual tissues, organs and cells at all stages of plant growth and development. The data so obtained thus further support the hypothesis that a sophisticated monolignol radical–radical coupling network exists in plants which has been highly conserved throughout vascular plant evolution.

Evolutionary Aspects of MADS‐box Genes in the Eusporangiate Fern Ophioglossum

Abstract: The MIKC‐type MADS‐box genes of seed plants encode transcription factors which are typically expressed in a tissue‐ or organ‐specific way. In contrast, the MIKC genes of the fern Ceratopteris isolated so far, show fairly ubiquitous expression in both reproductive and non‐reproductive organs. In order to determine whether this is a feature which is unique to a lineage of highly derived, leptosporangiate ferns, we initiated a characterization of the family of MIKC‐type MADS‐box genes of the eusporangiate “fern”Ophioglossum. Within the large clade of ferns and their allies, Ophioglossum is only very distantly related to Ceratopteris. cDNAs were isolated which represent at least four different Ophioglossum MIKC‐type genes, termed OPM1, OPM3 ‐ OPM5. Hybridization of genomic DNA gel blots, however, revealed that appreciably more MADS‐box genes are present in the Ophioglossum genome. As for the MIKC‐type genes from Ceratopteris, phylogeny reconstructions did not reveal orthology to seed plant genes for any of these. For OPM1 and OPM5, however, a relatively close relationship to some Ceratopteris genes was moderately supported. Semi‐quantitative RT‐PCR revealed that OPM1, OPM3 and OPM5 are expressed in both the vegetative (trophophore) and the reproductive (sporophore) part of the Ophioglossum leaf. In contrast, expression of OPM4 is restricted to the sporophore, suggesting a specific role in generative development for that gene. These findings suggest that relatively ubiquitous expression is a typical, but not absolute, feature of MIKC‐type MADS‐box genes from ferns and their allies. Taken together, the data outlined here corroborate the view that the phylogeny of MIKC‐type MADS‐box genes took different pathways in ferns and their allies on the one hand, and seed plants on the other hand, involving numerous independent gene duplications in both lineages, and distinct differences in the ubiquity of the expression patterns and, by inference, function. The implications of these findings for a better understanding of the evolution of vascular plants are discussed.

Evolution of endemic vascular plants of Ulleungdo and Dokdo in Korea - floristic and cytotaxonomic characteristics of vascular flora of Dokdo -

A total of 48 vascular plant taxa belonging to 27 families, 46 genera, 41 species, and 7 varieties are recognized in Island Dokdo. Five species of Plantago asiatica, Schizophragma hydrangeoides, Rubus phoenicolasius, Viola kusanoana, and Ampelopsis brevipedunculata are regarded as already extinct and previously reported 21 species cannot be reconfirmed in the island. Most Plants are dry and salt tolerant species and no taxon confined to this island is found. In terms of species composition, Dokdo is most closely related to Ulleungdo. Only limited number of plant taxa can inhabit by the lack of diverse environments and dry and salty conditions, however, more recently, frequent visit of people and transplantation of alien species cause to introduction of weeds to the island. The results of examining chromosomes of 16 taxa indicate that no alteration of chromosome numbers in the island has been occurred. The triploid level of Cyrtomium falcatum is the first report for the species

Evolution of MADS-box gene induction by FLO/LFY genes.

Some MADS-box genes function as floral homeotic genes. The Arabidopsis LFY gene is a positive regulator of floral homeotic genes, and homologs of the FLO/LFY gene family in other angiosperms and gymnosperms are likely to have a similar function. To investigate the origin of the floral homeotic gene regulatory cascade involving the FLO/LFY gene, FLO/LFY homologs were cloned from a leptosporangiate fern (Ceratopteris richardii), two eusporangiate ferns (Angiopteris lygodiifolia and Botrychium multifidum var. robustum), three fern allies (Psilotum nudum, Equisetum arvense, and Isoetes asiatica), and a moss (Physcomitrella patens). The FLO/LFY gene phylogenetic tree indicates that both duplication and loss of FLO/LFY homologs occurred during the course of vascular plant evolution. The expression patterns of the Ceratopteris LFY genes (CrLFY1 and 2) were assessed. CrLFY1 expression was prominent in tissues including shoot tips and circinate reproductive leaves, but very weak in other tissues examined. Expression of CrLFY2 was also prominent in tissues, including shoot tips and circinate reproductive leaves. These patterns of expression are dissimilar to that of any Ceratopteris MADS-box gene previously reported, suggesting that the induction of MADS-box genes by FLO/LFY is not established at the stage of ferns.

The role of land plants, phosphorus weathering and fire in the rise and regulation of atmospheric oxygen

AbstractThe evolution of vascular plants and their spread across the land surface, beginning ∼420 Ma, progressively increased the rate of weathering of phosphorus from rocks. This phosphorus supply promoted terrestrial and marine productivity and the burial of organic carbon, which has been the major source of O2 over geological timescales. Hence, it is predicted that the rise of plants led to an increase in the O2 content of the atmosphere from ∼12 vol %, 570–400 Ma to its present level of ∼21 vol % by ∼340 Ma. Previous modelling studies suggest that O2 then rose to ∼35 vol % ∼300 Ma. Such high concentrations are difficult to reconcile with the known persistence of forests, because rising O2 increases the frequency and intensity of vegetation fires, tending to decrease biomass and cause ecological shifts toward faster regenerating ecosystems. Rising O2 also directly inhibits C3 photosynthetic carbon assimilation and increases the production of toxic reactive oxygen species in cells. These effects suppress plant‐induced phosphorus weathering and hence organic carbon burial, providing a sensitive negative feedback on O2. A revised model predicts that this mechanism could have regulated atmospheric O2 within the range 15–25 vol % for the last 350 million years.

Paleobotany: some aspects of non‐flowering and flowering plant evolution

SummaryDilcher, D. L.: Paleobotany: some aspects of non‐flowering and flowering plant evolution. ‐Taxon 50: 697–711. 2001. ‐ ISSN 0040‐0262.When an examination of some aspects of the evolution of vascular land plants is made across broad sweeps of Paleozoic, Mesozoic and Tertiary time, three basic patterns occur in unrelated groups in vascular land plant evolution: independent evolution of similar morphologies, nearly contemporaneous evolution of similar structures and strategies, and repeated evolution of similar features or strategies at different times. Examples are given in non‐flowering plants and also in the evolution of angiosperms. A database is presented to illustrate the presence of angiosperms in the Jurassic/Lower Cretaceous fossil record and the diversity and abundance of organs recovered from these early angiosperms. These data are presented as working documents with the anticipation that they may grow into a major database for interested scholars world‐wide.

Oxidation of cinnamyl alcohols and aldehydes by a basic peroxidase from lignifying Zinnia elegans hypocotyls

The xylem of 26-day old Zinnia elegans hypocotyls synthesizes lignins derived from coniferyl alcohol and sinapyl alcohol with a G/S ratio of 43/57 in the aryl-glycerol-β-aryl ether core, as revealed by thioacidolysis. Thioacidolysis of Z. elegans lignins also reveals the presence of coniferyl aldehyde end groups linked by β- 0-4 bonds. Both coniferyl and sinapyl alcohols, as well as coniferyl and sinapyl aldehyde, are substrates of a xylem cell wall-located strongly basic peroxidase, which is capable of oxidizing them in the absence and in the presence of hydrogen peroxide. This peroxidase shows a particular affinity for cinnamyl aldehydes with k M values in the μM range, and some specificity for syringyl-type phenols. The affinity of this strongly basic peroxidase for cinnamyl alcohols and aldehydes is similar to that shown by the preceding enzymes in the lignin biosynthetic pathway (microsomal 5-hydroxylases and cinnamyl alcohol dehydrogenase), which also use cinnamyl alcohols and aldehydes as substrates, indicating that the one-way highway of construction of the lignin macromolecule has no metabolic “potholes” in which the lignin building blocks might accumulate. This fact suggests a high degree of metabolic plasticity for this basic peroxidase, which has been widely conserved during the evolution of vascular plants, making it one of the driving forces in the evolution of plant lignin heterogeneity.

Genetic engineering and lignin biosynthetic regulation in forest tree species

Genetic engineering of forest tree species is regarded as a strategy to reduce worldwide pressure on natural forests, to conserve genetic resources and ameliorate stress on global climate, and to meet growing demand for forest wood and timber products. Genetic engineering approaches toward the control or management of fungal pathogens, arthropod herbivores, bacterial and viral diseases, the use of pest resistance genes, and weed competitors are being studied. Although the production of transgenic trees is relatively recent and only a few species have been successfully genetically engineered in forest tree species, very useful and valuable information is available on the application of transgenic trees. Genes involved in important agricultural traits such as herbicide resistance, insect resistance, and wood quality have been isolated and have been used to genetically engineer trees. New technologies of plant molecular biology and genomics now make it possible high-efficient genetic improvement of forest trees. Genetic engineering promises to expand greatly the potential for genetic manipulation as new genes of commercial interest are discovered and utilized. Lignification is a process essential to the nature and evolution of vascular plants that is still poorly understood, even though it has been studied for more than a century. Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Rines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. It is also unusual as a plant polymer in that there are no plant enzymes for its degradation. These results have significant implications regarding the tradiational definition of lignin, and highlight the need for a better understanding of the lignin precursor biosynthetic pathway. In this review, we describe the progress made recently in genetic engineering of forest tree species.

Brassinolide plays in the hypoxia band

The evolution of vascular plants took place in an oxygen-enriched environment, thus, not surprisingly, oxygen is involved in hundreds of metabolic reactions, including the biosynthesis and metabolism of plant growth regulators (e.g. gibberellins, ethylene and brassinolides), as well as in multiple metabolic pathways (i.e. respiration and photorespiration). Although previous publications have described the effect of hypoxia on metabolism, little is known about the capacity of non-aquatic plants to grow in sub-optimal oxygen concentrations or the way hypoxia affects plant development.

Nuclear DNA Amounts in Pteridophytes

DNA amounts (C-value and genome size) are much-used biodiversity characters. A workshop held at Kew in 1997 identified major gaps in our knowledge of plant DNA amounts, recommending targets for new work to fill them. Murray reviewed non-angiosperm plants noting that representation of pteridophyte species (approx. 0.42%) was poor, while locating C-value data for them was very difficult. The workshop confirmed the need to make data for other groups besides angiosperms accessible for reference purposes. This paper pools DNA C-values for 48 pteridophyte species from eight original sources into one reference source, and fulfils a key workshop recommendation for this group. Comparing these data shows that nuclear 1C-values in pteridophytes vary approx. 1000-fold, from 0.055pg in Selaginella species to about 55pg in Ophioglossum petiolatum. Genome size estimates for 25 pteridophytes vary approx. 200-fold from 0.055 to 10.7pg, and the mean genome sizes in diploids and polyploids (5.15 and 4.59pg, respectively) are not significantly different. Wider comparisons show that ranges of genome sizes in the major groups of land plants are very different. Those in bryophytes and pteridophytes are narrow compared with those in gymnosperms and angiosperms. The data indicate that the origin of land plants possibly involved a first major increase in genome size in the evolution of vascular plants, while a second such increase occurred later in gymnosperms. C-values for pteridophytes remain very few, but conversely opportunities for new work on them are many.

Alpha-expansins in the semiaquatic ferns Marsilea quadrifolia and Regnellidium diphyllum: evolutionary aspects and physiological role in rachis elongation.

To investigate the evolutionary history of expansins and their role in cell elongation in early land plants, we isolated two alpha-expansin genes, Mq-EXP1 and Rd-EXP1, respectively, from the semiaquatic ferns Marsilea quacdrifolia L. and Regnellidium diphyllum Lindm. The deduced amino acid sequences of the fern expansins exhibit a high degree of identity to those of seed plants, showing that expansin genes were conserved during the evolution of vascular plants. Gel-blot analysis of M. quadrifolia and R. diphyllum genomic DNA indicated that, in both ferns, alpha-expansins are encoded by multigene families. Expression of alpha-expansin genes probed with Mq-EXP1 was confined to the elongating region of the Marsilea rachis. Cell-wall proteins of M. quadrifolia induced in-vitro extension of acidified cucumber cell walls. In R. diphyllum, expression of Rd-EXP1 increased when elongation of the rachis was enhanced by submergence or ethylene. These results indicate that alpha-expansins act as wall-loosening proteins in ferns, as has been proposed for angiosperms. In addition, Rd-EXP1 may play a role in mediating elongation of the rachis in submerged plants.

The relationships of vascular plants.

Recent phylogenetic research indicates that vascular plants evolved from bryophyte-like ancestors and that this involved extensive modifications to the life cycle. These conclusions are supported by a range of systematic data, including gene sequences, as well as evidence from comparative morphology and the fossil record. Within vascular plants, there is compelling evidence for two major clades, which have been termed lycophytes (clubmosses) and euphyllophytes (seed plants, ferns, horsetails). The implications of recent phylogenetic work are discussed with reference to life cycle evolution and the interpretation of stratigraphic inconsistencies in the early fossil record of land plants. Life cycles are shown to have passed through an isomorphic phase in the early stages of vascular plant evolution. Thus, the gametophyte generation of all living vascular plants is the product of massive morphological reduction. Phylogenetic research corroborates earlier suggestions of a major representational bias in the early fossil record. Mega-fossils document a sequence of appearance of groups that is at odds with that predicted by cladogram topology. It is argued here that the pattern of appearance and diversification of plant megafossils owes more to changing geological conditions than to rapid biological diversification.

Ordovician and Pennsylvanian Berthierine-Bearing Flint Clays

We report here the first occurrence of berthierine-bearing flint clays, one Ordovician and one Pennsylvanian in age. They are characterized by a berthierine-kaolinite-boehmite (bkb) assemblage. The Pennsylvanian flint clay from northeastern Kentucky is more typical in that it occurs in association with coal measures. The Ordovician occurrence from northwestern Illinois is the oldest flint clay of which we are aware. Because it is Ordovician, it formed before the evolution of terrestrial vascular plants. All previous reports of flint clays point to a genetic connection between flint-clay formation and the growth and decay of plants (Bohor and Triplehorn, 1993). Except for the high berthierine content and greenish color, the physical properties of this flint clay are similar to those of other kaolinitic flint clays. Some samples of the Ordovician flint clay are nearly pure berthierine. The Ordovician flint clay is a facies of the Neda Formation (~440 Ma), the uppermost formation of the Ordovician Maquoketa Group, which, in northern Illinois, northeastern Iowa, and southern Wisconsin, is unconformably overlain by Silurian carbonates. This unconformity is exposed in a narrow northwest-southeast corridor in the vicinity of the twin cities of Sterling and Rock Falls, Illinois. This corridor was formed between two glacial till sheets, one deposited by an Illinoian glacier that reached its southern limit to form the north side of the corridor, and the other by another lobe of Illinoian ice that pushed generally to the southwest so the northern edge of this tongue forms the southern margin of the corridor (Figure 1⇓). This work began when Dr. Thomas Berres of Northern Illinois University brought this material to our attention. Native Americans of the Archaic (~5000–2000 BP) and Woodland (~2000 BP) periods used this flint clay from northwestern Illinois as a pipestone, i.e., for carving artifacts including ceremonial pipes (Berres, …