Biochemical Systematics and Ecology

Monophyly and intratribal relationships of the tribe Trypetini were tested using mitochondrial 16S ribosomal RNA gene from 16 species of Trypetini, six tephritid outgroups, and two non-tephritid outgroups. The number of aligned sites was 1279 bp, but 1165 bp were used for analysis after excluding sites with missing data or gaps. Among these 1165 sites, 447 sites were variable and 305 were informative for parsimony analysis. Phylogenetic information was extracted from this data set using neighbor-joining and maximum parsimony methods and compared to a phylogenetic hypothesis proposed from the morphological literature. My molecular data suggest: (1) monophyly of the tribe Trypetini; (2) monophyly of the Chetostoma group; (3) monophyly of the Trypeta group; and (4) non-monophyletic nature of the previous concept of Hemilea. I suggest that more sensitive genetic markers with less A+T bias are needed to bring about finer resolution within the tribe Trypetini, and additional tephritid lineages should be sampled to identify the sister-group relationship of the tribe Trypetini.

The expression patterns of 14 enzymatic systems in skeletal muscle, liver and heart tissues of three species of Hypostomus from the Iguaçu River basin (Brazil) were investigated. Although the patterns were similar for the three species, different tissues showed differential expressions, and the data showed that differential tissue expressions of isoperoxidases may be due to preferential combination or association of polypeptide subunits. The detected patterns for SOD isozymes showed that the quaternary structures of these enzymes were in disagreement with the subunit number reported for the majority of other vertebrate groups. Tissue-specific restriction on heterotetramer formation also were described in LDH and MDHP isozymes. Thus, these tissue-specific gene expression character in the species of Hypostomus have the greatest potential to be recognized and applied in systematic studies among species of Hypostomus.

Polymorphism at 11 enzyme loci was used to compare the four evergreen oak species of the Mediterranean Basin and to establish their taxonomical relationships. As a comparison, two evergreen oak species from Tibet, located in the primary distribution area of evergreen oaks, were analysed at the same loci. Cladistic analyses led to the separation of a cluster of four species, namely the three Mediterranean Q. ilex, Q. coccifera and Q. alnifolia and the Tibetan Q. aquifolioides. The other Tibetan species, Q. semicarpifolia, and Q. suber, from the Western Mediterranean Basin, were very distinct genetically from the four other species. These results were not consistent with previous taxonomical treatment, based on morphology alone, which classified Q. ilex and Q. coccifera in "ilex group" (=subgenus Sclerophyllodrys O. Schwartz), and the other four species in "cerris group" (subgenus Cerris, according to Schwartz). Allozyme variation has thus provided useful information to clarify the taxonomy of evergreen oaks.

The anamorph determination of Cordyceps sinensis remains problematic due to the lack of clear links between the sexual and conidial forms of the fungus. In this study, we applied molecular approaches to analyze the genetic variation of Cordyceps sinensis and its allies to identify the anamorph-teleomorph connection. The sequences of the internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal RNA gene of Cordyceps sinensis (teleomorph) collected from Qingzang plateau (altitude over 4000m), Tibet and several related asexual conidial forms were determined. The sequence comparison showed that Cordyceps sinensis was most closely related to Hirsutella sinensis, and was clearly divergent from Paecilomyces sinensis, Stachybotrys sp. or Tolypocladium sp.; distance values, estimated according to Kimura two-parameter models between Cordyceps sinensis and Hirsutella sinensis, were extremely low (<0.02), whereas distance values between Cordyceps sinensis and Paecilomyces sinensis, Stachybotrys sp. and Tolypocladium sp. were 0.34, 0.21 and 0.25, respectively. Taken together, Hirsutella sinensis and Cordyceps sinensis are the different stages of the life cycle stages of the same organism. Hirsutella sinensis is therefore the anamorph of Cordyceps sinensis, rather than Paecilomyces sinensis or other species. The possible reasons as to why different taxa can be obtained when culturing Cordyceps sinensis are also discussed.

9'Z-(3S,5R,6R,3'S,5'R,6'S)-Neoxanthin reisolated from spinach (Spinacea oleracea) and characterized by HPLC, VIS, MS, and 2D (1)H NMR, has been submitted to photoinduced stereomutation in the presence of iodine or diphenyl diselenide at conditions not involving isomerization of the allenic bond. The six individual geometrical isomers, all-E,9Z,9'Z,13Z,13'Z,15Z and three minor di-Z-isomers, presumably 9,9'-di-Z,9',13-di-Z and 9',13'-di-Z, present in the equilibrium mixture have been characterized by HPLC, VIS data, 1H NMR and reversibility tests. Judged by the quantitative composition of the equilibrium mixture the naturally occurring 9'Z-isomer is thermodynamically less stable than the all-E-isomer. The availability of these isomers facilitates future search in natural sources. 9'Z-(6R90% of total neoxanthin in spinach and broccoli (Brassica oleracea var. italica), consistent with previous findings of its abundance in chloroplasts. all-E90% of total violaxanthin in the same sources. It is postulated that a neoxanthin Delta9'-isomerase is present and involved in the biosynthesis of abscisic acid in higher plants. Allenic S-isomers are of interest as postulated biosynthetic precursors of R-allenes. All-E-(6S)- and 9'Z-(6S)-neoxanthin were available as semi-synthetic model compounds. The allenic (6S)-diastereomers could not be detected in spinach or broccoli.

DNA sequence and morphological data were analyzed for specimens of twenty-five species of acanthomorph fishes and two specimens representing the outgroups Aulopiformes and Myctophiformes. A 572 base-pair (bp) segment of the 12S ribosomal mitochondrial gene, 1112 bp from three regions of the 28S ribosomal nuclear gene, and 38 morphological transformation series were analyzed under the criterion of maximum parsimony. The total evidence analysis resulted in a set of four most parsimonious trees. Relationships common to all trees are largely congruent with the hypothesis articulated by Johnson and Patterson (1993. Bull. Mar. Sci. 52, 554-626).

The composition of the essential oils of Thymus pulegioides L. plants growing wild in Vilnius district (Lithuania) with "ester", lemon and carvacrol (phenol) odours were collected in one location and investigated by GC, GC/MS and IR-spectroscopy. The essential oils with lemon and carvacrol odour belong to corresponding known citral-geraniol and carvacrol chemotypes. The essential oil of plants with "ester" odour containing 50-70% of alpha-terpenyl acetate belong to alpha-terpenyl acetate chemotype, which was not noticed earlier in Thymus pulegioides L. species.

The presence and identity of very long chain polyunsaturated fatty acids from three freshwater crustacean species, Bathynella natans, B. baicalensis and Baicalobathynella magna from Lake Baikal and caves of central Europe were determined by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS with APCI). LC-MS with APCI enabled the identification of more than 50 very long chain polyunsaturated fatty acids. These acids were described in the crustaceans for the first time, predominantly 26:5n6, 28:7n6, 30:7n3 and 40:7n6. A hypothesis for the biosynthesis of these acids is proposed.

Using enzyme electrophoresis and nematocyst analysis, the sympatrically occurring "light" and "dark" color morphs of the sea anemone Anthopleura orientalis from the Sea of Japan were shown to be two valid species. The "light" morph was identified as A. orientalis (Averintsev, 1967 Issledovaniya fauny morei: Vyp. 5 (13). Nanka, Leningrad, pp. 62-77), while the "dark" morph was designated as Anthopleura sp. The analysis of 21 isozyme loci revealed high value of Nei's genetic distance (D=1.284) between the two species, which are indistinguishable in their external morphology. The mean values of observed and expected heterozygosities for A. orientalis and Anthopleura sp. are high (H(o)=0.252+/-0.061, H(e)=0.250+/-0.061 and H(o)=0.327+/-0.052, H(e)=0.351+/-0.054, respectively). The species differ significantly in the size of spirocysts and nematocysts, among which the atrichs from acrorhagi and the basitrichs from actinopharynx contribute most to the observed difference. Strong qualitative difference is revealed between distributions of nematocysts in mesenteric filaments of the two sea anemone species studied. The possible conspecificity of Anthopleura sp. with Anthopleura artemisia (Dana, 1848) is discussed and the conclusion made that these are two separate species.

Allozyme analyses were performed to determine patterns of genetic variation and phylogenetic relationships within the genus Notropis in southern Mexico. Products of 28 gene loci were resolved in 24 geographic samples belonging to four putative species. These species represent the southern limit of the Cyprinidae in North America. Five loci were found monomorphic and 11 were diagnostic among species when the outgroup, Phoxinus phoxinus, was not taken into account. Four groups were identified, but these do not correspond perfectly to the four nominal species. Notropis sallaei was identified by allozyme analyses being the most basal among southern Mexican Notropis and the most genetically divergent. Notropis imeldae, was also diagnosed on the basis of allozymic variation, however a population sample representing the Balsas drainage was genetically divergent and is considered as an undescribed new species (N. n. sp.). No genetic differences were found between the samples of N. boucardi and N. moralesi. Therefore, we recommend that N. moralesi should be considered as a junior synonym of N. boucardi. A broad geographic sampling strategy was employed across all the distribution range of N. boucardi representing rivers from three different drainages, which cover both Mexican slopes. The slight divergence found among the headwater populations of N. boucardi permitted us to predict a model of paleohydrographic relationships of these three drainages. Two alternative hypotheses are postulated to explain the current distribution pattern of populations of N. boucardi in southern Mexico.

The cyanogenic polymorphism in Trifolium repens is caused by the variation in two genes, the interaction of which produces four distinct cyanotypes. Along the Atlantic coasts of Bretagne, T. repens is sometimes found in populations mixed with the related species Trifolium occidentale, although the latter species usually occurs only in a narrow fringe along the coast, whereas T. repens is a more inland species. No plants of T. occidentale have ever been reported to have linamarase activity. Indeed, of 763 T. occidentale plants studied, none contained linamarase activity. However, the variation in the proportion of cyanotypes in T. repens was enormous, even between sites less than 2km apart. Our results confirm the presumption that T. repens and T. occidentale are indeed separate species. Both the fact that T. occidentale plants never contain linamarase activity, and the difference in proportion of plants with cyanoglucosides in mixed stands show that gene flow between the species must be rare. These dissimilar distributions strongly indicate that cyanotypic frequencies in adjacent and mixed populations of the very closely related species T. occidentale and T. repens are regulated by different mechanisms

Plains zebras (Equus quagga antiquorum) occur in few large, but many small, isolated populations in KwaZulu-Natal. Problems identified in small populations include reduced striping patterns on hind quarters, smaller size, elevated mortality rates and high number of still-births. Inbreeding may be implicated. Population viability analysis (PVA) was conducted with a computer model (VORTEX), and DNA and allozyme analyses were conducted to test the findings of the model. Using standard methods, DNA (PCR-RAPD) and allozyme diversity was assessed in blood samples from 72 plains zebra from four KwaZulu-Natal Nature Conservation Services (KZN-NCS) protected areas: Umfolozi Game Reserve (UGR), Albert Falls (AFNR), Vernon Crookes (VCNR) and Harold Johnson (HJNR) Nature Reserves. Populations of the latter three, small-sized (9-110 individuals) populations were seeded from the same source population (UGR: current population of 2000) during the past 25 years. Information from PCR-RAPD and allozyme analyses were compared with each other as well as to that predicted by population genetic modelling (using VORTEX). Allozyme heterozygosities were consistently high in all populations (12.1-12.9%), with no observable losses associated with reduced population size. On the other hand, percentage loss of polymorphism (20-39%) calculated from the PCR-RAPD study appeared to be positively correlated with the loss of heterozygosity predicted by population viability analysis (PVA), and negatively correlated with population size. On the basis of the above results, a policy of translocation was advocated for small, intensely managed populations of zebras, whereby a harem should be translocated every five years for a population size of nine (HJNR), while for a population size of 110 (VCNR) translocations should take place every 15 years if heterozygosity is to be maintained at more than 90% within each population over 100 years.

The two common southern African mice species (Mastomys coucha and M. natalensis) are morphologically almost identical, making field identification impossible at present. Specimens from two localities were collected and tissue and blood samples taken. The habitat type of each locality was studied, and a distribution map compiled. A definite correlation between biome-type and species range was found to be present. Three isozyme markers were identified: glucose phosphate isomerase in liver, and two general (non-specific) protein coding loci in muscle. In addition, we also identified species characteristic haemoglobin components in both species. This is the first study to report genetic variation within, and differentiation between these species. Our results are of medical importance because Mastomys coucha carries bubonic plague and M. natalensis carries Lassa Fever.

The leaf essential oil of Wollemia nobilis (Wollemi Pine) has been investigated and compared with other members of the family Araucariaceae. All araucaroids examined yielded steam volatile oils in low yields. The oil from Wollemia nobilis was composed mainly of (+)-16-kaurene (60%), together with alpha-pinene (9%) and germacrene-D (8%). Oils from Agathis species endemic to Australia were high in monoterpenes, in contrast to those isolated from extra-Australian species. The major constituents of A. atropurpurea oil were phyllocladene (13%) and 16-kaurene (19%), followed by alpha-pinene (8%) and delta-cadinene (9%). A. microstachya yielded oil in which alpha-pinene (18%) was the major component; the only other components in excess of 5% were myrcene (7%), bicyclogermacrene (6%) and delta-cadinene (6%). A. robusta oil contained spathulenol (37%) and rimuene (6%). Approximately 40% of the oil was unidentified sesquiterpenes. A. australis oil contained 16-kaurene (37%), sclarene (5%) and an unidentified oxygenated diterpene K (12%) as major components; the only other compound in excess of 5% was germacrene-D (9%). 5,15-Rosadiene (60%), and 16-kaurene (7%) were the major constituents of A. macrophylla oil. A. moorei oil was rich in sesquiterpenes, but the only compounds in excess of 5% were allo-aromadendrene (6%), germacrene-D, delta-cadinene (10%), an unidentified sesquiterpene (12%) and 16-kaurene (6%). In A. ovata oil the most significant compounds were caryophyllene oxide (15%) and phyllocladene (39%). Araucaria angustifolia contained germacrene-D (9%) and the diterpenes hibaene (30%) and phyllocladene (20%) as major components of its essential oil. Oils of A. bidwillii, A. columnaris and A. cunninghamii were all low in mono- and sesquiterpenes and high in diterpenes. In the first, hibaene (76%) was the major constituent; the second contained hibaene (9%), sclarene (6%), luxuriadiene (13-epi-dolabradiene)(23%) and two unidentified diterpene hydrocarbons (B) (33%) and (E) (10%). In the last, 16-kaurene (53%) was the most significant component followed by hibaene (29%). A. heterophylla was unusual in that over half the oil was made up of the monoterpenoid alpha-pinene (52%), with phyllocladene (32%) being the only other compound of significance. alpha-Pinene (18%) was a significant component of A. hunsteinii oil; sclarene (11%) and germacrene-D (5%) were the only other compounds present in concentrations of more than 5%. A. luxurians oil was composed of 5,15-rosadiene (20%) and luxuriadiene (13-epi-dolabradiene) (66%), previously unreported from natural sources. The major components of A. montana were phyllocladene (61%) and 16-kaurene (23%). Sclarene (20%), luxuriadiene (19%) and the unidentified diterpene hydrocarbons (B) (25%) and (E) (10%) were the most important constituents of A. muelleri oil. A. scopulorum contained large amounts of 16-alpha-phyllocladanol (41%) as well as luxuridiene (10%) and delta-cadinene and alpha-copaene, both at 6%.