Jeffrey P. Hill

CHROMOSOMAL REARRANGEMENTS AND THE GENETICS OF REPRODUCTIVE BARRIERS IN MIMULUS (MONKEY FLOWERS)

Chromosomal rearrangements may directly cause hybrid sterility and can facilitate speciation by preserving local adaptation in the face of gene flow. We used comparative linkage mapping with shared gene‐based markers to identify potential chromosomal rearrangements between the sister monkeyflowers Mimulus lewisii and Mimulus cardinalis, which are textbook examples of ecological speciation. We then remapped quantitative trait loci (QTLs) for floral traits and flowering time (premating isolation) and hybrid sterility (postzygotic isolation). We identified three major regions of recombination suppression in the M. lewisii × M. cardinalis hybrid map compared to a relatively collinear Mimulus parishii × M. lewisii map, consistent with a reciprocal translocation and two inversions specific to M. cardinalis. These inferences were supported by targeted intraspecific mapping, which also implied a M. lewisii‐specific reciprocal translocation causing chromosomal pseudo‐linkage in both hybrid mapping populations. Floral QTLs mapped in this study, along with previously mapped adaptive QTLs, were clustered in putatively rearranged regions. All QTLs for male sterility, including two underdominant loci, mapped to regions of recombination suppression. We argue that chromosomal rearrangements may have played an important role in generating and consolidating barriers to gene flow as natural selection drove the dramatic ecological and morphological divergence of these species.

The monosaccharide transporter gene family in land plants is ancient and shows differential subfamily expression and expansion across lineages

BackgroundIn plants, tandem, segmental and whole-genome duplications are prevalent, resulting in large numbers of duplicate loci. Recent studies suggest that duplicate genes diverge predominantly through the partitioning of expression and that breadth of gene expression is related to the rate of gene duplication and protein sequence evolution.Here, we utilize expressed sequence tag (EST) data to study gene duplication and expression patterns in the monosaccharide transporter (MST) gene family across the land plants. In Arabidopsis, there are 53 MST genes that form seven distinct subfamilies. We created profile hidden Markov models of each subfamily and searched EST databases representing diverse land plant lineages to address the following questions: 1) Are homologs of each Arabidopsis subfamily present in the earliest land plants? 2) Do expression patterns among subfamilies and individual genes within subfamilies differ across lineages? 3) Has gene duplication within each lineage resulted in lineage-specific expansion patterns? We also looked for correlations between relative EST database representation in Arabidopsis and similarity to orthologs in early lineages.ResultsHomologs of all seven MST subfamilies were present in land plants at least 400 million years ago. Subfamily expression levels vary across lineages with greater relative expression of the STP, ERD6-like, INT and PLT subfamilies in the vascular plants. In the large EST databases of the moss, gymnosperm, monocot and eudicot lineages, EST contig construction reveals that MST subfamilies have experienced lineage-specific expansions. Large subfamily expansions appear to be due to multiple gene duplications arising from single ancestral genes. In Arabidopsis, one or a few genes within most subfamilies have much higher EST database representation than others. Most highly represented (broadly expressed) genes in Arabidopsis have best match orthologs in early divergent lineages.ConclusionThe seven subfamilies of the Arabidopsis MST gene family are ancient in land plants and show differential subfamily expression and lineage-specific subfamily expansions. Patterns of gene expression in Arabidopsis and correlation of highly represented genes with best match homologs in early lineages suggests that broadly expressed genes are often highly conserved, and that most genes have more limited expression.

Heteroblastic Root Development in Ceratopteris richardii (Parkeriaceae)

Heteroblastic variation among leaves arising from the shoot system is a well‐known phenomenon, but relatively little effort has been directed at assessing whether comparable variation occurs among shoot‐borne roots in homorhizic plants. A morphological and anatomical study of shoot‐borne root formation in the homosporous fern Ceratopteris richardii revealed two developmentally distinct root populations. Roots in the first population were produced once each shoot plastochron, with a single organ originating just beneath each leaf. Among the first six of these roots produced by young shoots, the root plastochron and individual growth rate, the final organ size (diameter and length), the anatomy, and the onset of lateral root initiation all varied as a function of shoot age. The positional differences observed among these organs constituted the first portion of a root heteroblastic series that continued into the adult shoot. The second root population arose from leaf bases beginning near the eighth node, with roots becoming more numerous on leaves at successive nodes. This secondary form of shoot‐borne rooting behavior constituted another aspect of heteroblastic variation, but it is attributed to differences in developmental potential among leaves along the shoot and not to root heteroblasty in a strict sense. The results underscore basic differences in the morphological relationship between root and shoot systems in homorhizic ferns compared with seed plants and show that C. richardii is a useful model for investigating aspects of phase change related to rooting behavior that are not represented in flowering plants.

MERISTEM DEVELOPMENT AT THE SPOROPHYLL PINNA APEX IN CERATOPTERIS RICHARDII

Sporophylls of the homosporous fern Ceratopteris richardii have naked pinna meristems that allow surface growth at the pinna apex to be observed nondestructively over time. Sequential scanning electron microscopy was combined with conventional anatomical approaches to determine the patterns of development in the abaxial epidermis. There were consistent themes in the spatial arrangement of clonal cell patches in the epidermal layer, but the exact anatomical pattern was variable within and between pinnae. Two sporangial cell lineages that run down the abaxial flanks of each pinna always appeared to originate from subapical positions just behind the pinna tip. During pinna growth, the principal direction of tip elongation varied occasionally, independent of the existing cell‐tiling patterns near the pinna apex. These local changes in growth direction resulted in protodermal cells just behind the apex changing their fates to correspond with the new growth vector, independent of their recent cell lineage histories. In particular, the position of sporangial initials shifted to follow the prevailing direction of growth. A model for fern pinna development is proposed to fit these observations, one that emphasizes the existence of developmental parameters that control organogenesis above the cellular level.

Developmental anatomy of the fifth shoot-borne root in young sporophytes of Ceratopteris richardii

Young sporophytes of the homosporous fern Ceratopteris richardii produce a single shoot-borne root below each leaf. The developmental anatomy of the fifth sporophyte root is described using scanning electron microscopy and histological techniques. Three merophyte orthostichies in the body of the root originate from three proximal division faces of a tetrahedral root apical cell. Eight or nine divisions occur in a relatively regular sequence within each merophyte and produce a characteristic radial anatomical pattern in the root. The exact number of early divisions within a merophyte depends on the merophyte’s position within the root as a whole. Predictable inter-merophyte differences arise because a 2-fold (diarch) anatomical symmetry that is characteristic of mature roots is superimposed on a 3-fold radial symmetry that originates behind the apical cell. Before early formative divisions within a merophyte are completed, additional proliferative divisions begin to increase the number of cells within previously established tissue zones. The cellular parameters of early fifth root development in C. richardii are relatively invariant, and are reminiscent of patterns previously described for the heterosporous fern Azolla. Young sporophytes of C. richardii provide a useful model to further investigate the genetic regulation of root development in a non-seed plant, where the anatomy of meristem organization differs from that seen in flowering plant species.

The genetic architecture of traits associated with the evolution of self‐pollination in Mimulus

Quantitative trait locus (QTL) mapping is a first step toward understanding the genetic basis of adaptive evolution and may also reveal reproductive incompatibilities unique to hybrids. In plants, the shift from outcrossing to self-pollination is common, providing the opportunity for comparisons of QTL architecture among parallel evolutionary transitions. We used QTL mapping in hybrids between the bee-pollinated monkeyflower Mimulus lewisii and the closely related selfer Mimulus parishii to determine the genetic basis of divergence in floral traits and flowering time associated with mating-system evolution, and to characterize hybrid anther sterility. We found a moderately polygenic and highly directional basis for floral size evolution, suggesting adaptation from standing variation or in pursuit of a moving optimum, whereas only a few major loci accounted for substantial flowering-time divergence. Cytonuclear incompatibilities caused hybrid anther sterility, confounding estimation of reproductive organ QTLs. The genetic architecture of floral traits associated with selfing in M. parishii was primarily polygenic, as in other QTL studies of this transition, but in contrast to the previously characterized oligogenic basis of a pollinator shift in close relatives. Hybrid anther sterility appeared parallel at the molecular level to previously characterized incompatibilities, but also raised new questions about cytonuclear co-evolution in plants.

Carbon-use efficiency in green sinks is increased when a blend of apoplastic fructose and glucose is available for uptake

Understanding how green sink strength is regulated in planta poses a difficult problem because non-structural carbohydrate (NSC) levels can have integrated, simultaneous feedback effects on photosynthesis, sugar uptake, and respiration that depend on specific NSC moieties. Photosynthetic gametophytes of the fern Ceratopteris richardii provide a simple land plant model to assess how different NSCs imported from the apoplast of intact plants affect green sink strength. Sink strength was quantified as the amount of exogenous sugar that plants grown in low light depleted from their liquid media, and the relative contributions of carbon assimilation by photosynthesis and sugar uptake was estimated from stable isotope analysis of plant dry mass. Gametophytes absorbed fructose and glucose with equal affinity when cultured on either hexose alone, or in the presence of an equimolar blend of both sugars. Plants also depleted sucrose from the surrounding media, although a portion of this disaccharide that was hydrolysed into fructose and glucose by putative cell wall invertase activity remained in the media. The δ13C in plant dry masses harvested from sugar treatments were all close to –18‰, indicating that 25–39% of total plant carbon was from C3 photosynthesis (δ13C=–29‰) and 61–75% was from uptake of exogenous sugars (δ13C=–11‰). Carbon-use efficiency (i.e. carbon accumulated/carbon depleted) was significantly improved when plants had a blend of exogenous sugars available compared with plants grown in a single hexose alone. Plants avoided complete down-regulation of photosynthesis even though a large excess of exogenous carbon fluxed through their cells.

Opportunistic heterotrophy in gametophytes of the homosporous fern Ceratopteris richardii

Fern gametophytes are extremely shade-tolerant, potentially existing for long periods under conditions of ex- treme light limitation. Many previous studies have demonstrated an increase in gametophyte growth and incidence of spon- taneous transition to sporophyte morphology (apogamy) under culture on media containing exogenous sugar. However, these studies did not verify sugar uptake or quantify relative growth on media containing different sugar types. Here, we examine the extent of heterotrophy and underlying mechanisms of sugar transport in photosynthetic gametophytes of the fern Ceratopteris richardii Brongn. Exogenous sugar uptake, growth, and sugar transport were evaluated with assays of exogenous glucose depletion, experimental culture of gametophytes under different sugar and light conditions, and bioin- formatic approaches. The glucose from the growth media was significantly depleted by gametophytes growing under all conditions, especially those in the dark compared with those exposed to higher light. Gametophyte area increased similarly when cultured on equimolar concentrations of either glucose or the disaccharide sucrose, likely due to preferential uptake of one of the monomers of sucrose. Although at least one gene with similarity to sucrose transporters is expressed in ger- minating spores, our results suggest a reliance on monosaccharide transport for exogenous sugar uptake. Glucose assimila- tion in both light and dark conditions constitutes nutritional opportunism and may enhance gametophyte survival in very low light.

Plasticity of female reproductive resource allocation depends on the presence or absence of prior environmental sex determination in Ceratopteris richardii

Abstract Resource allocation plasticity enables individuals to alter patterns of nutrient use between reproductive and vegetative output to better fit their current environment. In sexually labile plant species, abiotic environmental factors can influence expression of dimorphic gender, resulting in environmental sex determination (ESD), which potentially reduces the need for plasticity of resource allocation by preemptively matching an individual’s future nutrient demands to resource availability in its location. Ceratopteris richardii gametophytes exhibit gender‐dependent differences in relative carbon and nitrogen content, and ESD in certain nutrient environments. This study examined whether prior ESD in C. richardii gametophyte populations reduced subsequent plasticity of reproductive allocation compared to instances where no ESD occurred, by quantifying phenotypic responses to reduced P, N, or CO 2 availabilities. All three nutrient‐limited environments resulted in decreased size of egg‐bearing (meristic) gametophytes compared to nonlimited environments, but gametophytes failed to respond to N and CO 2 limitation at the time of sex determination, resulting in no ESD. N limitation resulted in a predictable allometric re‐allocation of resources based on small gametophyte size, whereas CO 2 limitation caused a change in reproductive output consistent with true plasticity. Withholding exogenous P caused ESD and had no effect on relative reproductive output of resultant meristic gametophytes because the size decrease was minor. Under P limitation, ESD matched the resource demands of gender phenotypes to their environment before the onset of developmental dimorphism, reducing the need for large allocation adjustments after sex determination.