EphA4 is necessary for spatially selective peripheral somatosensory topography.

Somatosensation is the primary sensory modality employed by rodents in navigating their environments, and mystacial vibrissae on the snout are the primary conveyors of this information to the murine brain. The layout of vibrissae is spatially stereotyped and topographic connections faithfully maintain this layout throughout the neuraxis. Several factors have been shown to influence general vibrissal innervation by trigeminal neurons. Here, the role of a cell surface receptor, EphA4, in directing position-dependent vibrissal innervation is examined. EphA4 is expressed in the ventral region of the presumptive whisker pad and EphA4(-/-) mice lack the ventroposterior-most vibrissae. Analyses reveal that ventral trigeminal axons are abnormal, failing to innervate emerging vibrissae, and resulting in the absence of a select group of vibrissae in EphA4(-/-) mice. EphA4's selective effect on a subset of whiskers implicates cell-based signaling in the establishment of position-dependent connectivity and topography in the peripheral somatosensory system.

Independent parcellation of the embryonic visual cortex and thalamus revealed by combinatorial Eph/ephrin gene expression.

The visual cortex in primates is parcellated into cytoarchitectonically, physiologically, and connectionally distinct areas: the striate cortex (V1) and the extrastriate cortex, consisting of V2 and numerous higher association areas [1]. The innervation of distinct visual cortical areas by the thalamus is especially segregated in primates, such that the lateral geniculate (LG) nucleus specifically innervates striate cortex, whereas pulvinar projections are confined to extrastriate cortex [2--8]. The molecular bases for the parcellation of the visual cortex and thalamus, as well as the establishment of reciprocal connections between distinct compartments within these two structures, are largely unknown. Here, we show that prospective visual cortical areas and corresponding thalamic nuclei in the embryonic rhesus monkey (Macaca mulatta) can be defined by combinatorial expression of genes encoding Eph receptor tyrosine kinases and their ligands, the ephrins, prior to obvious cytoarchitectonic differentiation within the cortical plate and before the establishment of reciprocal connections between the cortical plate and thalamus. These results indicate that molecular patterns of presumptive visual compartments in both the cortex and thalamus can form independently of one another and suggest a role for EphA family members in both compartment formation and axon guidance within the visual thalamocortical system.

Analysis of character correlations among wood decay mechanisms, mating systems, and substrate ranges in homobasidiomycetes.

Homobasidiomycetes include the majority of wood-decaying fungi. Two basic forms of wood decay are known in homobasidiomycetes: white rot, in which lignin and cellulose are degraded, and brown rot, in which lignin is not appreciably degraded. An apparent correlation has been noted between production of a brown rot, decay of conifer substrates, and possession of a bipolar mating system (which has a single mating-type locus, in contrast to tetrapolar systems, which have two mating-type loci). The goals of this study were to infer the historical pattern of transformations in decay mode, mating type, and substrate range characters, and to determine if a causal relationship exists among them. Using nuclear and mitochondrial rDNA sequences, we performed a phylogenetic analysis of 130 species of homobasidiomycetes and performed ancestral state reconstructions by using parsimony on a range of trees, with various loss:gain cost ratios. We evaluated pairwise character correlations by using the concentrated changes test (CCT) of Maddison and the maximum likelihood (ML) method of Pagel. White rot, tetrapolar mating systems, and the ability to decay conifers and hardwoods appear to be plesiomorphic in homobasidiomycetes, whereas brown rot, bipolar mating systems, and exclusive decay of conifers appear to have evolved repeatedly. The only significant correlation among characters was that between brown rot (as the independent character) and exclusive decay of conifer substrates (P < 0.03). This correlation was supported by the CCT on a range of plausible trees, although not with every reconstruction of ancestral states, and by the ML test. Our findings suggest that the evolution of brown rot has promoted repeated shifts to specialization for confier substrates.

Phylogeny of Malpighiaceae: evidence from chloroplast ndhF and trnl-F nucleotide sequences.

The Malpighiaceae are a family of ∼1250 species of predominantly New World tropical flowering plants. Infrafamilial classification has long been based on fruit characters. Phylogenetic analyses of chloroplast DNA nucleotide sequences were analyzed to help resolve the phylogeny of Malpighiaceae. A total of 79 species, representing 58 of the 65 currently recognized genera, were studied. The 3' region of the gene ndhF was sequenced for 77 species and the noncoding intergenic spacer region trnL-F was sequenced for 65 species; both sequences were obtained for the outgroup, Humiria (Humiriaceae). Phylogenetic relationships inferred from these data sets are largely congruent with one another and with results from combined analyses. The family is divided into two major clades, recognized here as the subfamilies Byrsonimoideae (New World only) and Malpighioideae (New World and Old World). Niedenzu's tribes are all polyphyletic, suggesting extensive convergence on similar fruit types; only de Jussieu's tribe Gaudichaudieae and Anderson's tribes Acmanthereae and Galphimieae are monophyletic. Fleshy fruits evolved three times in the family and bristly fruits at least three times. Among the wing-fruited vines, which constitute more than half the diversity in the family, genera with dorsal-winged samaras are fairly well resolved, while the resolution of taxa with lateral-winged samaras is poor. The trees suggest a shift from radially symmetrical pollen arrangement to globally symmetrical pollen at the base of one of the clades within the Malpighioideae. The Old World taxa fall into at least six and as many as nine clades.

Evolutionary instability of ectomycorrhizal symbioses in basidiomycetes.

Mycorrhizae, the symbiotic associations of plant roots and fungal hyphae, are classic examples of mutualisms. In these ecologically important associations, the fungi derive photosynthetic sugars from their plant hosts, which in turn benefit from fungus-mediated uptake of mineral nutrients. Early views on the evolution of symbioses suggested that all long-term, intimate associations tend to evolve toward mutualism. Following this principle, it has been suggested that mycorrhizal symbioses are the stable derivatives of ancestral antagonistic interactions involving plant parasitic fungi. Alternatively, mutualisms have been interpreted as inherently unstable reciprocal parasitisms, which can be disrupted by conflicts of interest among the partners. To determine the number of origins of mycorrhizae, and to assess their evolutionary stability, it is necessary to understand the phylogenetic relationships of the taxa involved. Here we present a broad phylogenetic analysis of mycorrhizal and free-living homobasidiomycetes (mushroom-forming fungi). Our results indicate that mycorrhizal symbionts with diverse plant hosts have evolved repeatedly from saprotrophic precursors, but also that there have been multiple reversals to a free-living condition. These findings suggest that mycorrhizae are unstable, evolutionarily dynamic associations.

Seed plant phylogeny: Demise of the anthophyte hypothesis?

Recent molecular phylogenetic studies indicate, surprisingly, that Gnetales are related to conifers, or even derived from them, and that no other extant seed plants are closely related to angiosperms. Are these results believable? Is this a clash between molecules and morphology?

The root of angiosperm phylogeny inferred from duplicate phytochrome genes.

An analysis of duplicate phytochrome genes (PHYA and PHYC) is used to root the angiosperms, thereby avoiding the inclusion of highly diverged outgroup sequences. The results unambiguously place the root near Amborella (one species, New Caledonia) and resolve water lilies (Nymphaeales, approximately 70 species, cosmopolitan), followed by Austrobaileya (one species, Australia), as early branches. These findings bear directly on the interpretation of morphological evolution and diversification within angiosperms.

Molecular gradients and compartments in the embryonic primate cerebral cortex.

The mature cerebral cortex is divided into morphologically distinct, functionally dedicated and stereotypically connected cortical areas. How might such functional domains arise during development? To investigate possible intrinsic programs within the embryonic cerebral cortex we examined patterns of gene expression early in corticogenesis. We performed these studies using the developing macaque monkey because of the size, complexity, areal make-up and the extended nature of its cortical development. Here, we present results for two types of molecules. (i) Transcription factors -- gene products that bind DNA and activate transcription, directing cellular fates through cascades of gene expression. We find that the transcription factors TBr-1, Lhx-2, Emx-1 and a novel POU domain-containing gene are differentially expressed within the forming primate forebrain, and are present in gradients across the neocortex. (ii) The EphA receptor tyrosine kinases -- gene products that mediate cellular recognition in many embryonic systems. Individual members of this family are expressed during primate corticogenesis in pronounced gradients and/or well-defined compartments with distinct boundaries. Together, these results suggest that at least two modes of grouping cells within the neocortex exist: the graded patterning of cells across its full anteroposterior extent and the parcellation of cells into defined domains. Moreover, emergence of molecular differences between regions of the cortical plate, prior to the arrival of afferent and formation of efferent connections, suggests that the initial cellular parcellation in the telencephalon is cell-autonomously regulated. This initial independence from peripheral influences supports the existence of an intrinsic protomap that may function both to differentially attract and respond to specific afferents, thus predicting the functional map of the mature cortex.

Molecular evidence for the early specification of presumptive functional domains in the embryonic primate cerebral cortex.

To identify molecules that may play a role in the initiation of cerebral cortical area formation, we examined the expression of the Eph receptors and their ligands, the ephrins, during primate corticogenesis. We selected the macaque monkey neocortex because of its clear areal subdivisions, large surface area, protracted development (gestation = 165 d), and similarity to the human brain. In situ hybridizations, performed at early [embryonic day 65 (E65)], middle (E80), and late (E95) stages of cortical development, revealed that EphA system family members are expressed in distinct gradients and laminar and areal domains in the embryonic neocortex. Indeed, several regionally restricted molecular patterns are already apparent within the cortical plate at E65, before the formation of thalamocortical connections, suggesting that the initial expression of some EphA system members is regulated by programs intrinsic to cortical cells. For example, EphA3, EphA6, and EphA7 are all selectively expressed within the presumptive visual cortex. However, although EphA6 and EphA7 are present throughout this region, EphA3 is only expressed in the prospective extrastriate cortex, suggesting that cortical cells harbor functional biases that may influence the formation of appropriate synaptic connections. Although several patterns of early gene expression are stable (e.g., EphA3, EphA4, and EphA6), others change as development proceeds (e.g., EphA5, EphA7, ephrin-A2, ephrin-A3, and ephrin-A5), perhaps responding to extrinsic cues. Thus, at E95, after connections between the cortical plate and thalamus have formed, receptor subtypes EphA3, EphA5, EphA6, and EphA7 and the ligand ephrin-A5 are expressed in posterior regions, whereas EphA4 and ephrin-A2 and ephrin-A3 are either uniformly distributed or anteriorly biased. Taken together, our results demonstrate molecular distinctions among cells of the embryonic primate neocortex, revealing hitherto unrecognized compartmentalization early in corticogenesis.

Species names in phylogenetic nomenclature.

Linnaean binomial nomenclature is logically incompatible with the phylogenetic nomenclature of de Queiroz and Gauthier (1992, Annu. Rev. Ecol. Syst. 23:449-480): The former is based on the concept of genus, thus making this rank mandatory, while the latter is based on phylogenetic definitions and requires the abandonment of mandatory ranks. Thus, if species are to receive names under phylogenetic nomenclature, a different method must be devised to name them. Here, 13 methods for naming species in the context of phylogenetic nomenclature are contrasted with each other and with Linnaean binomials. A fundamental dichotomy among the proposed methods distinguishes those that retain the entire binomial of a preexisting species name from those that retain only the specific epithet. Other relevant issues include the stability, uniqueness, and ease of pronunciation of species names; their capacity to convey phylogenetic information; and the distinguishability of species names that are governed by a code of phylogenetic nomenclature both from clade names and from species names governed by the current codes. No method is ideal. Each has advantages and drawbacks, and preference for one option over another will be influenced by one's evaluation of the relative importance of the pros and cons for each. Moreover, sometimes the same feature is viewed as an advantage by some and a drawback by others. Nevertheless, all of the proposed methods for naming species in the context of phylogenetic nomenclature provide names that are more stable than Linnaean binomials.

Stomatal plugs of Drimys winteri (Winteraceae) protect leaves from mist but not drought.

Two outstanding features of the flowering plant family Winteraceae are the occlusion of their stomatal pores by cutin plugs and the absence of water-conducting xylem vessels. An adaptive relationship between these two unusual features has been suggested whereby stomatal plugs restrict gas exchange to compensate for the presumed poor conductivity of their vesselless wood. This hypothesized connection fueled evolutionary arguments that the vesselless condition is ancestral in angiosperms. Here we show that in Drimys winteri, a tree common to wet forests, these stomatal occlusions pose only a small fixed resistance to water loss. In addition, they modify the humidity response of guard cells such that under high evaporative demand, leaves with plugs lose water at a faster rate than leaves from which the plugs have been experimentally removed. Instead of being adaptations for drought, we present evidence that these cuticular structures function to maintain photosynthetic activity under conditions of excess water on the leaf surface. Stomatal plugs decrease leaf wettability by preventing the formation of a continuous water film that would impede diffusion of CO2 into the leaf. Misting of leaves had no effect on photosynthetic rate of leaves with plugs, but resulted in a marked decrease ( approximately 40%) in leaves from which the plugs had been removed. These findings do not support a functional association between stomatal plugs and hydraulic competence and provide a new perspective on debates surrounding the evolution of vessels in angiosperms.

Duplicate genes and the root of angiosperms, with an example using phytochrome sequences.

The root of the angiosperm tree has not yet been established. Major morphological and molecular differences between angiosperms and other seed plants have introduced ambiguities and possibly spurious results. Because it is unlikely that extant species more closely related to angiosperms will be discovered, and because relevant fossils will almost certainly not yield molecular data, the use of duplicate genes for rooting purposes may provide the best hope of a solution. Simultaneous analysis of the genes resulting from a gene duplication event along the branch subtending angiosperms would yield an unrooted network, wherein two congruent gene trees should be connected by a single branch. In these circumstances the best rooted species tree is the one that corresponds to the two gene trees when the network is rooted along the connecting branch. In general, this approach can be viewed as choosing among rooted species trees by minimizing hypothesized events such as gene duplication, gene loss, lineage sorting, and lateral transfer. Of those gene families that are potentially relevant to the angiosperm problem, phytochrome genes warrant special attention. Phylogenetic analysis of a sample of complete phytochrome (PHY) sequences implies that an initial duplication event preceded (or occurred early within) the radiation of seed plants and that each of the two resulting copies duplicated again. In one of these cases, leading to the PHYA and PHYC lineages, duplication appears to have occurred before the diversification of angiosperms. Duplicate gene trees are congruent in these broad analyses, but the sample of sequences is too limited to provide much insight into the rooting question. Preliminary analyses of partial PHYA and PHYC sequences from several presumably basal angiosperm lineages are promising, but more data are needed to critically evaluate the power of these genes to resolve the angiosperm radiation.

Leaf size, sapling allometry, and Corner's rules: phylogeny and correlated evolution in maples (Acer).

We studied the evolution of leaf size, sapling canopy allometry, and related traits in 17 Acer species growing in the understory of temperate deciduous forests, using parsimony methods, randomization tests, and independent contrasts calculated on a phylogeny inferred from nuclear ribosomal internal transcribed spacer (ITS) sequences. Bivariate correlations and multivariate analyses indicated two independent suites of coevolving traits, and the results were robust over a range of alternative phylogenies. The first suite consisted of strong positive correlations among twig thickness, leaf size, inflorescence length, and branch spacing (Corner's rules). Seed size and mature height were also weakly correlated with these traits. The second suite reflected aspects of sapling crown allometry, including crown size, stem diameter, and total leaf area, which appear to be related to shade tolerance. There was a weak negative correlation between sapling crown size and mature height, but no correlation with leaf or seed size. Most correlations were similar in magnitude for ahistorical and independent contrasts analyses, and discrepancies between these two measures were greater in traits with lower levels of convergent evolution. The evolutionary correlations among twig, leaf, seed, inflorescence, and canopy dimensions emphasize the need for integrated theories of evolution and function of these disparate traits.

Step matrices and the interpretation of homoplasy.

Assumptions about the costs of character change, coded in the form of a step matrix, determine most-parsimonious inferences of character evolution on phylogenies. We present a graphical approach to exploring the relationship between cost assumptions and evolutionary inferences from character data. The number of gains and losses of a binary trait on a phylogeny can be plotted over a range of cost assumptions, to reveal the inflection point at which there is a switch from more gains to more losses and the point at which all changes are inferred to be in one direction or the other. Phylogenetic structure in the data, the tree shape, and the relative frequency of states among the taxa influence the shape of such graphs and complicate the interpretation of possible permutation-based tests for directionality of change. The costs at which the most-parsimonious state of each internal node switches from one state to another can also be quantified by iterative ancestral-state reconstruction over a range of costs. This procedure helps identify the most robust inferences of change in each direction, which should be of use in designing comparative studies.

Evolution of alcohol dehydrogenase genes in peonies (Paeonia): phylogenetic relationships of putative nonhybrid species.

Alcohol dehydrogenase genes were amplified by PCR, cloned, and sequenced from 11 putative nonhybrid species of the angiosperm genus Paeonia. Sequences of five exons and six intron regions of the Adh gene were used to reconstruct the phylogeny of these species. Two paralogous genes, Adh1A, and Adh2, were found; an additional gene, Adh1B, is also present in section Moutan. Phylogenetic analyses of exon sequences of the Adh genes of Paeonia and a variety of other angiosperms imply that duplication of Adh1 and Adh2 occurred prior to the divergence of Paeonia species and was followed by a duplication resulting in Adh1A and Adh1B. Concerted evolution appears to be absent between these paralogous loci. Phylogenetic analysis of only the Paeonia Adh exon sequences, positioning the root of the tree between the paralogous genes Adh1 and Adh2, suggests that the first evolutionary split within the genus occurred between the shrubby section Moutan and the other two herbaceous sections Oneapia and Paeonia. Restriction of Adh1B genes to section Moutan may have resulted from deletion of Adh1B from the common ancestor of sections Oneapia and Paeonia. A relative-rate test was designed to compare rates of molecular change among lineages based on the divergence of paralogous genes, and the results indicate a slower rate of evolution within the shrubby section Moutan than in section Oneapia. This may be responsible for the relatively long branch length of section Oneapia and the short branch length between section Moutan and the other two sections found on the Adh, ITS (nrDNA), and matK (cpDNA) phylogenies of the genus. Adh1 and Adh2 intron sequences cannot be aligned, and we therefore carried out separate analyses of Adh1A and Adh2 genes using exon and intron sequences together. The Templeton test suggested that there is not significant incongruence among Adh1A, ITS, and matK data sets, but that these three data sets conflict significantly with Adh2 sequence data. A combined analysis of Adh1A, ITS, and matK sequences produced a tree that is better resolved than that of any individual gene, and congruent with morphology and the results of artificial hybridization. It is therefore considered to be the current best estimate of the species phylogeny. Paraphyly of section Paeonia in the Adh2 gene tree may be caused by longer coalescence times and random sorting of ancestral alleles.

Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences.

Homobasidiomycete fungi display many complex fruiting body morphologies, including mushrooms and puffballs, but their anatomical simplicity has confounded efforts to understand the evolution of these forms. We performed a comprehensive phylogenetic analysis of homobasidiomycetes, using sequences from nuclear and mitochondrial ribosomal DNA, with an emphasis on understanding evolutionary relationships of gilled mushrooms and puffballs. Parsimony-based optimization of character states on our phylogenetic trees suggested that strikingly similar gilled mushrooms evolved at least six times, from morphologically diverse precursors. Approximately 87% of gilled mushrooms are in a single lineage, which we call the "euagarics." Recently discovered 90 million-year-old fossil mushrooms are probably euagarics, suggesting that (i) the origin of this clade must have occurred no later than the mid-Cretaceous and (ii) the gilled mushroom morphology has been maintained in certain lineages for tens of millions of years. Puffballs and other forms with enclosed spore-bearing structures (Gasteromycetes) evolved at least four times. Derivation of Gasteromycetes from forms with exposed spore-bearing structures (Hymenomycetes) is correlated with repeated loss of forcible spore discharge (ballistospory). Diverse fruiting body forms and spore dispersal mechanisms have evolved among Gasteromycetes. Nevertheless, it appears that Hymenomycetes have never been secondarily derived from Gasteromycetes, which suggests that the loss of ballistospory has constrained evolution in these lineages.

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae).

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1-5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.

Distinct regulatory elements control muscle-specific, fiber-type-selective, and axially graded expression of a myosin light-chain gene in transgenic mice.

The fast alkali myosin light chain 1f/3f (MLC1f/3f) gene is developmentally regulated, muscle specific, and preferentially expressed in fast-twitch fibers. A transgene containing an MLC1f promoter plus a downstream enhancer replicates this pattern of expression in transgenic mice. Unexpectedly, this transgene is also expressed in a striking (approximately 100-fold) rostrocaudal gradient in axial muscles (reviewed by J. R. Sanes, M. J. Donoghue, M. C. Wallace, and J. P. Merlie, Cold Spring Harbor Symp. Quant. Biol. 57:451-460, 1992). Here, we analyzed the expression of mutated transgenes to map sites necessary for muscle-specific, fiber-type-selective, and axially graded expression. We show that two E boxes (myogenic factor binding sites), a homeodomain (hox) protein binding site, and an MEF2 site, which are clustered in an approximately 170-bp core enhancer, are all necessary for maximal transgene activity in muscle but not for fiber-type- or position-dependent expression. A distinct region within the core enhancer promotes selective expression of the transgene in fast-twitch muscles. Sequences that flank the core enhancer are also necessary for high-level activity in transgenic mice but have little influence on activity in transfected cells, suggesting the presence of regions resembling matrix attachment sites. Truncations of the MLC1f promoter affected position-dependent expression of the transgene, revealing distinct regions that repress transgene activity in neck muscles and promote differential expression among intercostal muscles. Thus, the whole-body gradient of expression displayed by the complete transgene may reflect the integrated activities of discrete elements that regulate expression in subsets of muscles. Finally, we show that transgene activity is not significantly affected by deletion or overexpression of the myoD gene, suggesting that intermuscular differences in myogenic factor levels do not affect patterns of transgene expression. Together, our results provide evidence for at least nine distinct sites that exert major effects on the levels and patterns of MLC1f expression in adult muscles.