James J. Smith

Allopatric genetic origins for sympatric host-plant shifts and race formation in Rhagoletis.

Tephritid fruit flies belonging to the Rhagoletis pomonella sibling species complex are controversial because they have been proposed to diverge in sympatry (in the absence of geographic isolation) by shifting and adapting to new host plants. Here, we report evidence suggesting a surprising source of genetic variation contributing to sympatric host shifts for these flies. From DNA sequence data for three nuclear loci and mtDNA, we infer that an ancestral, hawthorn-infesting R. pomonella population became geographically subdivided into Mexican and North American isolates ≈1.57 million years ago. Episodes of gene flow from Mexico subsequently infused the North American population with inversion polymorphism affecting key diapause traits, forming adaptive clines. Sometime later (perhaps ±1 million years), diapause variation in the latitudinal clines appears to have aided North American flies in adapting to a variety of plants with differing fruiting times, helping to spawn several new taxa. Thus, important raw genetic material facilitating the adaptive radiation of R. pomonella originated in a different time and place than the proximate ecological host shifts triggering sympatric divergence.

Conditions for sympatric speciation: A diploid model incorporating habitat fidelity and non-habitat assortative mating

SummaryThree types of genes have been proposed to promote sympatric speciation: habitat preference genes, assortative mating genes and habitat-based fitness genes. Previous computer models have analysed these genes separately or in pairs. In this paper we describe a multilocus model in which genes of all three types are considered simultaneously. Our computer simulations show that speciation occurs in complete sympatry under a broad range of conditions. The process includes an initial diversification phase during which a slight amount of divergence occurs, a quasi-equilibrium phase of stasis during which little or no detectable divergence occurs and a completion phase during which divergence is dramatic and gene flow between diverging habitat morphs is rapidly eliminated. Habitat preference genes and habitat-specific fitness genes become associated when assortative mating occurs due to habitat preference, but interbreeding between individuals adapted to different habitats occurs unless habitat preference is almost error free. However, ‘nonhabitat assortative mating’, when coupled with habitat preference can eliminate this interbreeding. Even when several loci contribute to the probability of expression of non-habitat assortative mating and the contributions of individual loci are small, gene flow between diverging portions of the population can terminate within less than 1000 generations.

Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures

Abstract Dilute salt solutions eluted peroxidase and hydroxyproline-rich glycoproteins (HRGPs) very rapidly (60 % within 10s) from the surface of intact tomato cells grown in suspension culture. Further purification of the HRGPs based on (a) their solubility in 10% trichloroacetic acid and (b) chromatography on carboxymethyl cellulose, gave two components (P1 and P2) rich in serine, tyrosine, lysine and arabinosylated hydroxyproline. The sum of the hydroxyproline arabinoside profiles of P1 and P2 approximated that of the wall. P1, unlike P2, was histidine-rich and also contained proline. Significantly, isodityrosine (IDT) was absent from P1 and P2 but present in cell wall hydrolysates where, the Hyp:IDT molar ratio was ca 15: 1. In cells 4 days after subculture, 3 H-proline pulse-chase data indicated turnover of P1 and P2 presumably resulting from covalent attachment to the wall as neither P1 nor P2 appeared in the growth medium. At day four the cell mean generation time (MGT) was 4.6 days, the cell hydr oxyproline content was 0.7 % (w/w), the half lives of P1 and P2 were both ca 12 hr, and the combined CaCl 2 elutable P1 and P2 precursor pools contained ca 400 μg Hyp/g cells (dry weight). Calculated from the MGT and Hyp content, the cell demand was 44.μg Hyp/g cells (dry weight)/hr. The precursor pool size was therefore sufficient for 9 hours growth. However the pool turnover calculated from half life and pool size was 5.6 %/hr or 22.4μg Hyp/g cells (dry weight)/hr. Thus the supply of P1 and P2 precursors met > 50 % of the cell wall demand. Corroborative experiments showed that after depletion of the P1 and P2 pools by salt elution, washed cells resuspended in growth medium repleted the precursor pools at a rate corresponding to a synthesis of 43μg Hyp/g cells (dry weight)/hr, or 98 % of the demand. These data allow us to make the following suggestions: P1 and P2 represent monomeric extensin precursor subunits. Salt elution of P1 and P2 indicates their ionic binding by pectic carboxyl groups. The rapidity of elution indicates a high diffusivity of these extended rodlike macromolecules through the cell wall. This may imply a preferred orientation for P1 and P2 perpendicular rather than parallel to the plane of the wall. The lack of IDT in P1 and P2 implies that IDT forms in muro , possibly via peroxidase. We speculate that some of these IDT residues may crosslink an extensin precursor ‘tweft’ around a cellulose microfibrillar ‘twarp’. Such formation of heteromultimeric extensin interpenetrated by microfibrils would create a mechanically coupled extensin-cellulose network.

Sequential Sympatric Speciation Across Trophic Levels

A major cause for biodiversity may be biodiversity itself. As new species form, they may create new niches for others to exploit, potentially catalyzing a chain reaction of speciation events across trophic levels. We tested for such sequential radiation in the Rhagoletis pomonella (Diptera: Tephritidae) complex, a model for sympatric speciation via host plant shifting. We report that the parasitic wasp Diachasma alloeum (Hymenoptera: Braconidae) has formed new incipient species as a result of specializing on diversifying fly hosts, including the recently derived apple-infesting race of R. pomonella. Furthermore, we show that traits that differentially adapt R. pomonella flies to their host plants have also quickly evolved and serve as ecological barriers to reproduction, isolating the wasps. Speciation therefore cascades as the effects of new niche construction move across trophic levels.

The Evolution and Function of Carotenoid Hydroxylases in Arabidopsis

To gain insight into the evolution of xanthophyll synthesis in Arabidopsis thaliana, we analyzed two pairs of duplicated carotenoid hydroxylase enzymes in Arabidopsis thaliana: the cytochrome P450 enzymes CYP97A3 and CYP97C1, and non-heme di-iron enzymes, BCH1 and BCH2. Hydroxylated carotenes did not accumulate in a quadruple mutant for these four genes, demonstrating that they encode the full complement of carotenoid hydroxylases in A. thaliana. We were thus able to infer definitively the activity of each enzyme in vivo based on the phenotypes of selected double and triple mutant genotypes. The CYP97 and BCH gene pairs are primarily responsible for hydroxylation of alpha- and beta-carotenes, respectively, but exhibit some overlapping activities, most notably in hydroxylation of the beta-ring of alpha-carotene. Surprisingly, triple mutants containing only CYP97C1 or CYP97A3 activity produced 74 and 6% of the wild-type lutein level, indicating that CYP97C1 can efficiently hydroxylate both the beta- and epsilon-rings of alpha-carotene and that CYP97A3 also has low activity toward the epsilon-ring of alpha-carotene. The modes of functional divergence for the gene pairs appear distinct, with the CYP97 duplicates being strongly co-expressed but encoding enzymes with different in vivo substrates, while the BCH duplicates encode isozymes that show significant expression divergence in reproductive organs. By integrating the evolutionary history and substrate specificities of each extant enzyme with the phenotypic responses of various mutant genotypes to high light stress, we propose two likely scenarios for the evolution of alpha-xanthophyll biosynthesis in plants from ancestral organisms.

Tomato extensin precursors P1 and P2 are highly periodic structures

Abstract Intact cultured tomato cells eluted with dilute salt solutions yielded two soluble precursors (P1 and P2) to the bound extensin network. HF-deglycosylation of P1 and P2 (with methanol as scavenger) followed by tryptic degradation and HPLC on Hamilton PRP-1, gave unique tryptic peptide maps. The P1 map consisted predominantly of the deca- and hexadecapeptides, H5 and H20: (P1-H5) Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, and (H20) Ser-Hyp-Hyp-Hyp-Hyp-Val-Lys-Pro-Tyr-His-Pro-Thr-Hyp-Val-Tyr-Lys; the P2 map consisted almost entirely of the di- and octapeptides, H3 and H4: (P2-H3) Tyr-Lys; (H4) Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys, and small amounts of a closely related decapeptide containing intramolecularly-linked isodityrosine: (H11) Ser-Hyp-Hyp-Hyp-Hyp-Val- 1 2 IDT-Lys- 1 2 IDT-Lys. Both P1 and P2 are therefore highly periodic structures: P1 consists to a considerable extent of repeated H5 and H20 peptide blocks, while P2 may consist entirely of a single repeating decapeptide Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys-Tyr-Lys with occasional isoleucine for valine substitutions, and varying only in the extent of intramolecular IDT formation, which could stiffen the molecular rod. In P1, single tyrosine residues occur within the hexapeptide Val-Lys-Pro-Tyr-His-Pro region ofH20. This nonglycosylated region is sterically unhindered. Assuming a polyproline II conformation, all the basic residues of H20 lie in the same plane (available for pectic binding), while the hexapeptide tyrosine residue lies out of that plane and is therefore a prime candidate for the postulated inter molecular isodityrosine crosslink. Our sequences also reveal fundamental tetrapeptide and tripeptide periodicities of tomato extensin precursors: the contiguous decamers and hexadecamers consist of hydroxyproline tetrapeptides separated by a relatively few tripeptide sequences, (notably Tyr-Lys-Ser, Val-Tyr-Lys, Thr-Hyp-Val, Val-Lys-Pro, and Tyr-His-Pro). Furthermore, the presence of the pentapeptide Ser-Hyp-Hyp-Hyp-Hyp in all the repeat peptides shows that extensin, like many synthetic block copolymers, consists of relatively rigid domains (glycosylated Ser-Hyp-Hyp-Hyp-Hyp) separated by intervening (non-glycosylated) flexible spacers. Thus extensin is well-designed for its suggested role in mechanically coupling the cellulosic load-bearing polymers of the primary cell wall through formation of a network of defined porosity.

The genetics and ecology of sympatric speciation: a case study

Mating occurs on the larval host plant in allRhagoletis species (Diptera: Tephritidae). We show how this attribute, when coupled with certain differences in other biological traits, strongly influences the mode of speciation. In species of thesuavis species group, host shifts have never occurred during speciation, and larvae feed in the husks of any walnut species(Juglans spp.), which are highly toxic. Taxa are allopatric or parapatric and exhibit deep phylogenetic nodes suggesting relatively ancient speciation events. Traits responsible for species and mate recognition, particularly in parapatric species, are morphologically distinct and strongly sexually dimorphic. All aspects of their biology, genetics and distribution are consistent with a slow rate of allopatric speciation followed by morphological divergence in secondary contact. In contrast, speciation in thepomonella species group has always involved a shift to a new, usually unrelated, non-toxic host, and all taxa within these groups are sympatric, monophagous and morphologically indistinguishable from one another. Phylogenetic nodes are very shallow, indicating recent sympatric speciation. Sympatric divergence is promoted by genetic variation which allows a portion of the original species to shift to a new habitat or host. Evidence suggests that changes in a few key loci responsible for host selection and fitness on a new host may initiate host shifts. By exploiting different habitats, competition for resources between diverging populations is reduced or avoided. We provide evidence that in phytophagous and parasitic insects sufficient intrinsic barriers to gene flow can evolve between sister populations as they adapt to different habitats or hosts to allow each population to establish independent evolutionary lineages in sympatry.

Genetic Analysis of a Pathogenic Erwinia sp. Isolated from Pear in Japan.

ABSTRACT Four Erwinia strains, originally isolated in Japan from pear trees with bacterial shoot blight symptoms, were analyzed to determine their genetic relationship with Erwinia amylovora and E. pyrifoliae. When genomes were characterized with amplified fragment length polymorphism markers and by comparative groEL sequence analysis, the Japanese Erwinia sp. and South Korean E. pyrifoliae strains were placed in the same group, which was phylogenetically distinct from a group of 15 strains of E. amylovora. Sequencing of the 29,593-bp plasmid pEJ30 from Erwinia strain Ejp556 revealed that this plasmid was nearly identical to plasmid pEP36 from E. pyrifoliae and was closely related to the nontransferable ubiquitous plasmid pEA29 from E. amylovora. Twenty-one presumptive genes and their order in pEP36 were highly conserved in pEJ30; however, transposon Tn5394, which was present in pEP36, was not found in pEJ30. Short-sequence DNA repeats were conserved between pEJ30 and pEP36, and were different from short-sequence repeats in pEA29. Despite base-pair mismatches, primer pairs used in pEA29 polymerase chain reaction assays for E. amylovora amplified plasmid DNA from the Japanese Erwinia Ejp556 and Ejp562. Like E. pyrifoliae and a few strains of E. amylovora, Japanese Erwinia Ejp617 contained plasmids related to E. pyrifoliae ColE1-related plasmid pEP2.6. Based on these genetic analyses, we conclude that the Erwinia pathogen of pear in Japan is closely related to E. pyrifoliae and that both of these pathogens are demonstrably distinct from E. amylovora.

Ribosomal DNA polymorphism in Colletotrichum lindemuthianum

Genetic divergence among 57 isolates of Colletotrichum lindemuthianum collected in north, central and south America and The Netherlands was assessed on the basis of PCR-RFLP analysis and sequencing of the nuclear rDNA region of the two internal transcribed spacers (ITS 1 and ITS 2) and the 5.8S rRNA gene. A reproducible 0.58 kb fragment was amplified in all 57 isolates. Races of C. lindemuthianum formed two groups based on the restriction of the PCR-amplified ITS regions. Group I consisted mainly of Middle American races (65%), whereas 85% of Andean races belonged in group II. Genetic distances calculated from the sequence polymorphism in the rDNA region ranged from 0.2 to 1.8% among 14 isolates of C. lindemuthianum. The neighbour-joining and parsimony analyses of the sequence data showed no association of any particular ITS genotype with host gene pool, virulence or geographic origin of races. These data suggest that virulence can arise in different geographic areas at different times, independent of genetic background. Molecular polymorphism among isolates of races 7, 17, 31 and 73 collected in different countries was demonstrated by RFLP analysis of the ITS regions. A similar lack of agreement was observed in the sequence data between isolates of race 73 from Mexico and the United States and the Jukes—Cantor genetic distance between the isolates was large (0.9%). These findings support a level of molecular variability within C. lindemuthianum greater than the variability previously characterized by virulence analysis and suggest independent evolution of specific virulence patterns.

Sequential divergence and the multiplicative origin of community diversity

Significance Understanding how new life forms originate is a central question in biology. Population divergence is usually studied with respect to how single lineages diverge into daughter taxa. However, populations may not always differentiate in isolation; divergence of one taxon could create new niche opportunities in higher trophic levels, leading to the sequential origin of many new taxa. Here, we show that this may be occurring for three species of parasitoid wasps attacking Rhagoletis fruit flies. As flies shift and adapt to new host plants, wasps follow suit and diverge in kind, resulting in a multiplicative increase of diversity as the effects of ecologically based divergent selection cascade through the ecosystem. Biodiversity therefore may potentially beget increasing levels of biodiversity. Phenotypic and genetic variation in one species can influence the composition of interacting organisms within communities and across ecosystems. As a result, the divergence of one species may not be an isolated process, as the origin of one taxon could create new niche opportunities for other species to exploit, leading to the genesis of many new taxa in a process termed “sequential divergence.” Here, we test for such a multiplicative effect of sequential divergence in a community of host-specific parasitoid wasps, Diachasma alloeum, Utetes canaliculatus, and Diachasmimorpha mellea (Hymenoptera: Braconidae), that attack Rhagoletis pomonella fruit flies (Diptera: Tephritidae). Flies in the R. pomonella species complex radiated by sympatrically shifting and ecologically adapting to new host plants, the most recent example being the apple-infesting host race of R. pomonella formed via a host plant shift from hawthorn-infesting flies within the last 160 y. Using population genetics, field-based behavioral observations, host fruit odor discrimination assays, and analyses of life history timing, we show that the same host-related ecological selection pressures that differentially adapt and reproductively isolate Rhagoletis to their respective host plants (host-associated differences in the timing of adult eclosion, host fruit odor preference and avoidance behaviors, and mating site fidelity) cascade through the ecosystem and induce host-associated genetic divergence for each of the three members of the parasitoid community. Thus, divergent selection at lower trophic levels can potentially multiplicatively and rapidly amplify biodiversity at higher levels on an ecological time scale, which may sequentially contribute to the rich diversity of life.