Biosystematics and conservation: a case study with two enigmatic and uncommon species of Crassula from New Zealand.

BACKGROUND AND AIMS: Crassula hunua and C. ruamahanga have been taxonomically controversial. Here their distinctiveness is assessed so that their taxonomic and conservation status can be clarified. METHODS: Populations of these two species were analysed using morphological, chromosomal and DNA sequence data. KEY RESULTS: It proved impossible to differentiate between these two species using 12 key morphological characters. Populations were found to be chromosomally variable with 11 different chromosome numbers ranging from 2n = 42 to 2n = 100. Meiotic behaviour and levels of pollen stainability were both variable. Phylogenetic analyses showed that differences exist in both nuclear and plastid DNA sequences between individual plants, sometimes from the same population. CONCLUSIONS: The results suggest that these plants are a species complex that has evolved through interspecific hybridization and polyploidy. Their high levels of chromosomal and DNA sequence variation present a problem for their conservation.

Nuclear DNA variation, chromosome numbers and polyploidy in the endemic and indigenous grass flora of New Zealand.

BACKGROUND AND AIMS: Little information is available on DNA C-values for the New Zealand flora. Nearly 85 % of the named species of the native vascular flora are endemic, including 157 species of Poaceae, the second most species-rich plant family in New Zealand. Few C-values have been published for New Zealand native grasses, and chromosome numbers have previously been reported for fewer than half of the species. The aim of this research was to determine C-values and chromosome numbers for most of the endemic and indigenous Poaceae from New Zealand. SCOPE: To analyse DNA C-values from 155 species and chromosome numbers from 55 species of the endemic and indigenous grass flora of New Zealand. KEY RESULTS: The new C-values increase significantly the number of such measurements for Poaceae worldwide. New chromosome numbers were determined from 55 species. Variation in C-value and percentage polyploidy were analysed in relation to plant distribution. No clear relationship could be demonstrated between these variables. CONCLUSIONS: A wide range of C-values was found in the New Zealand endemic and indigenous grasses. This variation can be related to the phylogenetic position of the genera, plants in the BOP (Bambusoideae, Oryzoideae, Pooideae) clade in general having higher C-values than those in the PACC (Panicoideae, Arundinoideae, Chloridoideae + Centothecoideae) clade. Within genera, polyploids typically have smaller genome sizes (C-value divided by ploidy level) than diploids and there is commonly a progressive decrease with increasing ploidy level. The high frequency of polyploidy in the New Zealand grasses was confirmed by our additional counts, with only approximately 10 % being diploid. No clear relationship between C-value, polyploidy and rarity was evident.

When does intraspecific C-value variation become taxonomically significant?

AIMS: To examine what possible role intraspecific DNA C-value variation may play in plant taxonomy. SCOPE: Although many of the original examples of intraspecific C-value variation have been shown to be the result of experimental variation, new examples using the appropriate standards and controls continue to be published. The evidence that intraspecific C-value variation alters phenotypes can be equivocal, and detailed studies are needed to clarify any possible relationship. However, populations within species have been shown to have varying DNA amounts that can be correlated with eco-geographic variables, suggesting that the variation is adaptive and that these may be examples of incipient speciation. CONCLUSIONS: Where intraspecific C-value variation appears most significant for taxonomy is as an indicator of taxonomic heterogeneity, pointing to the need for a re-evaluation of the delimitation of the species in question. There is also the need to test whether intraspecific C-value variants produce fertile F(1) hybrids or not, as this would be a good indication of whether they belong in the same biological species.

Two recessive gene inheritance for triallate resistance in Avena fatua L.

Extensive use of the preemergence herbicide triallate over the last three decades has selected for resistant (R) Avena fatua L. populations in several areas of the United States and Canada. R plants are also cross-resistant to the unrelated pyrazolium herbicide difenzoquat. We made reciprocal crosses between inbred R and susceptible (S) lines to determine the genetic basis of triallate resistance. Seeds from parental lines and F(2) populations were treated with soil applications of 0.275, 0.55, or 1.1 kg/ha triallate in the greenhouse and plant heights recorded after 37 days. Surviving F(2) plants were selfed and the resulting F(3) families were screened with 1.1 kg/ha triallate. In the F(2) populations, assortment of S and R phenotypes fit a 15:1 segregation ratio, suggesting that resistance was controlled by the two independently segregating recessive genes TRR1 and TRR2. None of the 912 F(3) progeny from 51 R F(2) individuals was susceptible to triallate treatment, further supporting a two-gene mode of inheritance. There was a possible maternal effect on susceptibility at the highest triallate rate tested.

Are ecological and evolutionary theories scientific?

Scientists observe nature, search for generalizations, and provide explanations for why the world is as it is. Generalizations are of two kinds. The first are descriptive and inductive, such as Boyle's Law. They are derived from observations and therefore refer to observables (in this case, pressure and volume). The second are often imaginative and form the axioms of a deductive theory, such as Newton's Laws of Motion. They often refer to unobservables (e.g. inertia and gravitation). Biology has many inductive generalizations (e.g. Bergmann's Rule and 'all cells arise from preexisting cells') but few, if any, recognized universal laws and virtually no deductive theory. Many biologists and philosophers of biology have agreed that predictive theory is inappropriate in biology, which is said to be more complex than physics, and that one can have nonpredictive explanations, such as the neo-Darwinian Theory of Evolution by Natural Selection. Other philosophers dismiss nonpredictive, explanatory theories, including evolutionary 'theory', as metaphysics. Most biologists do not think of themselves as philosophers or give much thought to the philosophical basis of their research. Nevertheless, their philosophy shows in the way they do research. The plethora of ad hoc (i.e. not universal) hypotheses indicates that biologists are reluctant inductivists in that the search for generalization does not have a high priority. Biologists test their hypotheses by verification. Theoretical physicists, in contrast, are deductive unifiers and test their explanatory hypotheses by falsification. I argue that theoretical biology (concerned with unobservables, such as fitness and natural selection) is not scientific because it lacks universal laws and predictive theory. In order to make this argument, I review the differences between verificationism and falsificationism, induction and deduction, and descriptive and explanatory laws. I show how these differ with a specific example of a successful and still useful (even if now superseded as explanatory) deductive theory, Newton's Theory of Motion. I also review some of the philosophical views expressed on these topics because philosophers seem to be even more divided than biologists, which is not at all helpful. The fact that biology does not have predictive theories does not constitute irrefutable evidence that it cannot have them. The only way to falsify this philosophical hypothesis, however, is to produce a predictive theory with universal biological laws. I have proposed such a theory, but it has been presented piecemeal. At the end of this paper, I bring the pieces together into a deductive theory on the evolution of life history traits (e.g. clutch size, mating relationships, sexual size dimorphism).

Low outcrossing rates and shift in pollinators in New Zealand pohutukawa (Metrosideros excelsa; Myrtaceae).

New Zealand pohutukawa (Metrosideros excelsa), a member of the Myrtaceae, is a large, mass-flowering tree endemic to northern New Zealand coastlines. Mainland populations have been reduced to fragmented stands, and the original suite of bird pollinators has been largely replaced by introduced species. The native pollinator fauna on several offshore islands is largely intact and includes three species of the New Zealand honeyeaters (Meliphagidae) and native, solitary bees. We estimated multilocus outcrossing rates for three mainland and two island populations and found that they were among the lowest in the Myrtaceae (t(m) = 0.22-0.53). The shift in pollinators had no measurable effect on the mating system. Mass-flowering facilitates geitonogamous selfing, and inbreeding depression in seedling height was detectable at 6 mo of growth. F(s) [Wright's (1965) Fixation Index] was consistently higher than F(m) in all populations, indicating that selection may eliminate selfed offspring from populations prior to achieving reproductive maturity. Results suggest that increased selfing in mainland populations due to pollinator changes is not responsible for current patterns of poor regeneration of this species.

Major changes in chromatin condensation suggest the presence of an apoptotic pathway in plant cells.

A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was confirmed by analyses of (i) DNA accessibility to DNase I, (ii) histone disassociation induced by HCl, (iii) saturation of binding sites by the PI fluorochrome (iv), and (v) visual inspection by fluorescence and confocal microscopy. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed us to identify different stages in an apoptosis-like pathway in plants. The initial step of chromatin condensation which occurred prior to endonucleolytic DNA degradation was detected by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry. The initial chromatin condensation appears to be a reversible step in the early stage of apoptosis. The loss of reversibility of chromatin condensation observed subsequently may be a critical point in the cascade of apoptotic events, leading to further irreversible changes during apoptosis in plants.

An improved method for preparing the chromosomes of pines and other gymnosperms.

A simple technic is described to produce well spread gymnosperm chromosomes. Root tip meristems are digested with a pectinase:cellulase mixture to produce a cell suspension which then is squashed to yield flat, well spread chromosome complements that can be stained or used for in situ hybridization.

Parental genome separation and asynchronous centromere division in interspecific F1 hybrids in Lathyrus.

Chromosomes were studied in root-tip metaphase cells of several F1 interspecific Lathyrus hybrids including: L. hirsutus x L. cassius (H x C), L. cassius x L. hirsutus (C x H), L. cassius x L. odoratus (C x O), and their parents, all with 2n = 2x = 14. Two types of morphologically distinct centromeres were identified in the hybrids on the basis of the degree of contraction of the primary constriction. At least 12 well-defined centromeres were seen in all cells of L. hirsutus, L. cassius and L. odoratus, and about 80% of cells had 14. The hybrids were more variable than the species. H x O contained between six and 14 well-defined centromeres, while cells of H x C, C x H and C x O all had seven well-defined and seven weakly defined centromeres. These were used as markers to plot their spatial disposition in two dimensions on metaphase spreads. In H x C, C x H and C x O the two types of centromeres showed a significant tendency to occupy two spatially distinct and concentrically arranged domains on the metaphase plate (P < 0.005). Owing to shortage of material subsequent work was restricted to H x C and C x H. Six or seven chromosomes of one parental genome were selectively labelled by in situ hybridization using biotinylated total genomic DNA from either parent as a probe. Moreover, there was a very strong correlation between centromere type and genomic origin (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Solid organic residues produced by irradiation of hydrocarbon-containing H2O and H2O/NH3 ices: infrared spectroscopy and astronomical implications.

Methane clathrate (CH4 nH2O)--expected in cometary nuclei, in outer Solar System satellites, and perhaps in interstellar grains--as well as ices prepared from other combinations of CH4, C2H6, or C2H2 with H2O (and sometimes with NH3) were irradiated at 77 degrees K by plasma discharge. CH4 clathrate and other H2O/hydrocarbon ices color and darken noticeably after a dose approximately 10(8) to approximately 10(9) erg cm-2 over a period of 1-10 hr. Upon evaporation of the now yellowish to tan irradiated ices, a colored solid film adheres to the walls of the reaction vessel at room temperature. Transmission measurements of these organic films were made from 2.5 to 50 micrometers wavelength. The residue left after CH4 nH2O irradiation exhibits IR bands which we tabulate and identify with alkane, aldehyde, alcohol, and perhaps alkene and substituted aromatic functional groups. Aldehydes are especially well indicated, and may be related to recent claims of polyoxymethylene (H2CO)n in the coma of Comet Halley. Spectra presented here are compared with previous studies of UV or proton-irradiated, nonenclathrated hydrocarbon-containing ices may be useful for interpreting infrared features found in the spectra of comets and interstellar grains.

Coloration and darkening of methane clathrate and other ices by charged particle irradiation: applications to the outer solar system.

Methane clathrate is expected to be an important carbon-containing ice in the outer solar system. We investigate the effect of electron irradiation by coronal discharge on several simple hydrocarbons enclathrated in or mixed with H2O or H2O+NH3 in simulation of the effects of the solar wind, planetary magnetospheric particles, and cosmic rays on surfaces containing these ices in the outer solar system and interstellar space. H2O+CH4 clathrate, H2O+C2H6, H2O+CH4+NH3, H2O+C2H6+NH3, and H2O+C2H2 are all initially white ices, and all produce yellowish to brownish organic products upon charged particles irradiation. Significant coloration occurs with doses of 10(9) erg cm-2, corresponding to short interplanetary irradiation times. Uranian magnetospheric electrons penetrate to approximately 1 mm depth and deposit this dose in 8, 30, 65, 200, and 500 years into the surfaces of Miranda, Ariel, Umbriel, Titania, and Oberon, respectively. Further irradiation of the laboratory ice surface results in a progressive darkening and a more subdued color. For a conversion efficiency to solids G approximately equal to 1 molecule keV-1, the upper limit for the time for total destruction of CH4 and other simple hydrocarbons in the upper 1 mm is 5 x 10(4) years (Miranda) to 3 x 10(6) years (Oberon). Remote detection of CH4 is possible only when its replenishment rate exceeds the destruction rate at the depth probed by spectroscopy. Reflection spectroscopy or irradiated H2O+CH4 frost is compared with the spectra of several outer solar system objects and to other relevant organic and inorganic materials. Ultraviolet-visible and infrared transmission spectroscopy of the postirradiation residues is presented. Persistence of color and of CH4 ice bands on Triton and Pluto suggests ongoing surface activity and/or atmospheric haze. Over 4 x 10(9) year time scales, > or = 10 m of satellite and cometary surface material is processed by cosmic rays to a radiation-hardened ice-tholin mixture devoid of CH4. Preaccretional chemistry, exogenous materials, and endogenous organic chemistry all contribute to the spectral properties of icy satellites which accreted simple CH(O) molecules. Radiation darkening traces the deposition of mobilized or impact-exposed carbon-bearing volatiles on these satellites. More exhaustive experiments are necessary to work out the detailed relationships between initial composition, exposure age, and color/albedo.

Quantitative analysis of diploid translocation heterozygotes: test of models and equations.

Equations have been derived for two different models of chromosome pairing and chiasmata distribution. The first model represents the normal condition and assumes complete synapsis of homologous bivalents and the arms of interchange quadrivalents. This is followed by a nonrandom distribution of chiasmata among bivalents and multivalents such that each bivalent or bivalent-equivalent always has at least one chiasma. Univalents occur only as part of a III, I configuration at diakinesis or metaphase I. The second model assumes that a hologenomic mutation is present in which all chromosomes of a genome are equally affected. Two different assumptions can be made for such a mutation, and both give the same results: (1) homologous or homoeologous chromosome arms may be randomly paired or unpaired, but synapsis always leads to a crossover; (2) homologous or homoeologous arms always pair, but chiasmata are randomly distributed among the arms. The meiotic configurations at diakinesis or metaphase I are the same for both assumptions. Meiotic configurations of normal diploid interchange heterozygotes show good agreement with numbers predicted by the equations for nonrandom chiasmata distribution among configurations. Inter-specific hybrids with supernumerary chromosomes produced meiotic configurations frequencies in agreement with predictions of equations for random chiasmata distribution, but a hybrid without supernumeraries fitted the nonrandom expectations.

Colchicine induced quadrivalent formation in Helianthus: evidence of ancient polyploidy.

The premeiotic treatment of microsporocytes of Helianthus annuus and H. laciniatus (x= 17) results in the formation of multivalent chromosome configurations at meiosis in these 'diploid' species. This is interpreted to mean that colchicine has disrupted the normal attachment of genomes to the nuclear membrane and allowed synapsis of the ancestral genomes, indicating the polyploid origin of these 'diploid' species. This technique should be of importance in generating intergenomal recombination in classical allopolyploid agronomic species because it could produce new genotypic combinations normally impossible to obtain.

Studies on heteromorphic self-incompatibility systems: Physiological aspects of the incompatibility system of Primula obconica.

In Primula obconica, a species with a heteromorphic self-incompatibility system, the distinction between compatible and incompatible pollen tubes takes place on the stigma surface in thrum flowers, self tubes growing randomly over the papillar cells. No differences were seen between self and cross tube behaviour on the pin stigma surface, but self tubes were inhibited within the stigmatic tissue with differences in tube length evident after 24 h. The stigma surface bears a proteinaceous pellicle and binds the lectin Concanavalin A. Removal of the stigma removes the incompatibility barrier in mature gynoecia. Bud pollination shows that pollen tubes cannot grow in a normal manner on immature stigmas; the random growth of tubes over the stigma surface resembles that of mature thrum selfs. Fewer compatible tubes reach the style base of young gynoecia and smaller numbers of seeds are set than in mature flowers. Pin and thrum pollen grains germinate and grow in aqueous media, thrum tubes growing longer than pin. The presence of H3BO4 and CaCl2 in the growth medium promotes tube elongation and lengths equivalent to compatible styles can be obtained. The pollen grains have proteinaceous materials in their walls which diffuse out on moistening. Prolonged washing in aqueous media removes these materials but the incompatibility reaction remains unchanged. Thus the incompatibility reaction is between pollen tubes and stigmatic tissue and differs from the homomorphic, sporophytic system where pollen wall proteins elicit the incompatibility response.

Studies on heteromorphic self-incompatibility systems: the cytochemistry and ultrastructure of the tapetum of Primula obconica.

This paper describes the cytochemistry and ultrastructure of the developing tapetum in Primula obconica, a plant with a heteromorphic, sporophytic self-incompatibility system. The tapetum is of the secretory type and cytochemical tests have shown that when it breaks down proteinaceous (esterase) and lipidic components are deposited on the developing pollen grains. Acid phosphatase, a marker of gametophytic enzyme activity, is confined to the cytoplasm and intine of the developing pollen. Ultrastructural studies show that prior to its dissolution the tapetum undergoes a number of changes. In the early stages of development the tapetum is rich in ribosomes and rough endoplasmic reticulum, but following the breakdown of the tapetal cell wall the main components of the cytoplasm are densely staining spherical bodies surrounded by ribosomes and orbicular bodies, which appear to be confined to the cell periphery. As the cells break down, rod-like fibrils can be seen amongst the degenerate organelles and within the bacular cavities of the pollen. On dehiscence the pollen has a lipidic coating in addition to the fibrillar material in the pollen wall and the remnants of the tapetum can be seen adhering to the fibrous layer of the anther wall. Thus the mature, binucleate pollen of P. obconica is demonstrated to carry wall materials of sporophytic origin.

Chromosome number and flavonoid synthesis in Briza L. (Gramineae).

Following a recent reidentification of the apigenin C-glycosides in diploid Briza media, the luteolin C-glycosides were reexamined and three acylated derivatives of a luteolin C-glycoside were found. In an attempt to identify the chromosome or group of chromosomes responsible for the change in flavonoid synthesis from 4'-hydroxy- in diploid plants to 3',4'-dihydroxyflavone C-glycosides in autotetraploid plants, leaf flavonoids of artificially produced aneuploids of B. media were examined. Among these plants three different leaf flavonoid profiles were recognized: diploid, tetraploid, and a "modified" tetraploid pattern. All the aneuploids with the normal or "modified" tetraploid pattern were trisomic for one of the small acrocentric chromosomes. Induced polyploids of other Briza species were usually found to have similar flavonoid patterns in the two chromosome races. Flavonoid sulfates were found in three South American species but are absent from all the European species.