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Molecular ecotoxicology of plants.

Plant molecular exotoxicology investigates ecological implications of genetic and molecular responses to toxins, herbicides, pollutants and natural stress factors. Plant fitness is analysed by examining the relationships between plant genotype and ecological phenotype, enabling regulatory networks formed by second messenger molecules and transcriptional as well as post-transcriptional events to be elucidated. This general approach is illustrated here by specific case studies: detoxification by glucosyl transfer or binding to cell wall macromolecules; roles of the multifunctional formaldehyde dehydrogenase; and abiotic induction of plant immunity through reactive oxygen species. As a practical application of molecular ecotoxicology, the interaction of commercialized transgenic crop plants with potential environmental selection factors is discussed.

Aldehyde Oxidoreductases↗

Malarial vectors in an irrigated rice cultivation area in southern Sri Lanka.

Entomological surveys were carried out from March 1998 to December 1999 to study the prevalence, distribution and abundance of malarial vectors in relation to selected environmental factors and potential mosquito breeding sites in irrigation channels in 15 villages in the Lunugamvehera Irrigation and Settlement Project, a malaria-endemic area of southern Sri Lanka. Mosquito collections were made at monthly intervals using four sampling methods. Thirteen anopheline species were collected. Following monsoonal rains, anopheline breeding took place primarily in rainwater accumulations. During the inter-monsoonal period, pools formed in the irrigation system, semi-permanent pools formed as a result of rainfall and permanent ground pools were the major breeding sites of anophelines. Very little anopheline breeding took place within the irrigation channels. Amongst the seven anopheline species collected from human dwellings, Anopheles subpictus was the most prevalent, followed by A. culicifacies; together these two species comprised more than 99% of the indoor resting population. The number of days of rain was an important macro-epidemiological factor influencing the density of malarial vectors. There was no consistent trend between the amount of water released or the number of days of water release from the reservoir and the outdoor or indoor resting densities of anophelines.

Agriculture↗

Oxidation of pharmaceuticals during water treatment with chlorine dioxide.

The potential of chlorine dioxide (ClO2) for the oxidation of pharmaceuticals during water treatment was assessed by determining second-order rate constants for the reaction with selected environmentally relevant pharmaceuticals. Out of 9 pharmaceuticals only the 4 following compounds showed an appreciable reactivity with ClO2 (in brackets apparent second-order rate constants at pH 7 and T = 20 degrees C): the sulfonamide antibiotic sulfamethoxazole (6.7 x 10(3) M(-1) s(-1)), the macrolide antibiotic roxithromycin (2.2 x 10(2) M(-1) s(-1)), the estrogen 17alpha-ethinylestradiol (approximately 2 x 10(5) M(-1) s(-1)), and the antiphlogistic diclofenac (1.05 x 10(4) M(-1) s(-1)). Experiments performed using natural water showed that ClO2 also reacted fast with other sulfonamides and macrolides, the natural hormones estrone and 17beta-estradiol as well as 3 pyrazolone derivatives (phenazone, propylphenazone, and dimethylaminophenazone). However, many compounds in the study were ClO2 refractive. Experiments with lake water and groundwater that were partly performed at microgram/L to nanogram/L levels proved that the rate constants determined in pure water could be applied to predict the oxidation of pharmaceuticals in natural waters. Compared to ozone, ClO2 reacted more slowly and with fewer compounds. However, it reacted faster with the investigated compounds than chlorine. Overall, the results indicate that ClO2 will only be effective to oxidize certain compound classes such as the investigated classes of sulfonamide and macrolide antibiotics, and estrogens.

Anti-Bacterial Agents↗

Toxicity of the 13 priority pollutant metals to Vibrio fisheri in the Microtox chronic toxicity test.

The Microtox Acute Toxicity Test has been successfully used to measure the toxicity of metals and other pollutants at high concentrations (ppm) in selected environmental samples. However, metals and other toxicants are often found in much lower concentrations (ppb) in many municipal wastewaters and receiving waters. In order to assess the toxicity of these pollutants in these samples, a more sensitive toxicity assay is needed. The Microtox chronic toxicity test has been developed to measure the sublethal effect of toxicants over multiple generations of the test species, Vibrio fisheri. In this study, the toxicity of the 13 priority pollutant metals [i.e. As, Se, Cd, Cr (III and VI), Cu, Pb, Sb, Ag, Tl, Zn, Be, Hg and Ni] to V. fisheri was evaluated using the Microtox chronic toxicity test. In this test, the inhibitory concentration (IC), lowest observable effect concentration (LOEC), and no observable effect concentration (NOEC) were obtained after 22-h of incubation at 27+/-1 degrees C, by comparing the light output of the control to that of the test sample. Among the 13 priority pollutant metals, beryllium (Be) was found to be the most toxic in the test (LOEC=0.742-1.49 microg/l) while thallium (Tl) was the least toxic (LOEC=3840-15300 microg/l). The LOECs for copper (as Cu) and lead (Pb) in reagent (ASTM Type I) water were 6.78-13.6 microg/l and 626-1251 microg/l, respectively. The toxicity of copper sulfate (as Cu) in reagent water was shown and significantly reduced with the addition of natural organic matter (fulvic acid) or EDTA to the sample. The LOEC values for the 13 priority pollutant metals in this test were comparable to or lower than those reported for commonly used aquatic toxicity tests, such as the Ceriodaphnia dubia assay.

Benzopyrans↗

Evolution of a combinatorial transcriptional circuit: a case study in yeasts.

Developing new regulation of existing genes is likely a key mechanism by which organismal complexity arises in evolution. To examine plasticity of gene regulation over evolutionary timescales, we have determined the transcriptional circuit regulating mating type in the human fungal pathogen Candida albicans, and compared it to that of Saccharomyces cerevisiae. Since the two yeasts last shared an ancestor 100-800 million years ago, several major differences in circuitry have arisen. For example, a positive regulator of mating type was retained in C. albicans but lost in S. cerevisiae; this circuit branch was replaced by the modification of an existing negative regulator, thereby conserving the circuit output. We also characterize a tier of mating type transcriptional regulation that is present only in C. albicans, and likely results from the vastly different environmental selections imposed on the two yeasts--in this case, the pressure on C. albicans to survive in a mammalian host.

Candida albicans↗

Ratite reproduction.

The role of the veterinarian in ostrich production in the future will be to develop least-cost management programs. This article focuses on the ostrich as the only current commercially viable ratite species. Managing a successful ranch involves disease prevention, genetic selection, environmental management, and nutrition.

Animals↗

Race, socioeconomic status, and obesity in 9- to 10-year-old girls: the NHLBI Growth and Health Study.

The purpose of this investigation was to determine whether measures of socioeconomic status (SES) are inversely associated with obesity in 9- to 10-year-old black and white girls and their parents. Subjects were participants in the Growth and Health Study (NGHS) of the National Heart, Lung, and Blood Institute. Extensive SES, anthropometric, and dietary data were collected at baseline on 2379 NGHS participants. The prevalence of obesity was examined in the NGHS girls and parents in relation to SES and selected environmental factors. Less obesity was observed at higher levels of household income and parental education in white girls but not in black girls. Among the mothers of the NGHS participants who were seen, lower prevalence of obesity was observed with higher levels of income and education for white mothers, but no consistent patterns were seen in black mothers. Univariate logistic models indicated that the prevalence of obesity was significantly and inversely associated with parental income and education and number of parents in the household in white girls whereas caloric intake and TV viewing were significantly and positively associated with obesity. Among black girls, only TV viewing was significantly and positively associated with the prevalence of obesity. Multivariate logistic regression models revealed that lower parental educational attainment, one-parent household, and increased caloric intake were significantly associated with the prevalence of obesity in white girls; for black girls, only increased hours of TV viewing were significant in these models. It is concluded that socioeconomic status, as measured by education and income, was related to the prevalence of obesity in girls, with racial variation in these associations. A lower prevalence of obesity was seen at higher levels of socioeconomic status in white girls, whereas no clear relationship was detected in black girls. These findings raise new questions regarding the correlates of obesity in black girls.

Black People↗

Molecular phylogeny and evolutionary biology of Acrodipsas (Lepidoptera: Lycaenidae).

Most butterflies in the family Lycaenidae associate with ants but fewer than 3% are myrmecophagous. Larvae of the Australian endemic butterfly genus Acrodipsas parasitise the nests of ants from two subfamilies and, thus, constitute an interesting and uncommon myrmecophagous radiation within the Lepidoptera. Phylogenetic relationships among Acrodipsas species were inferred from fragments of mitochondrial cytochrome oxidase subunits I and II totalling 1155 bp using maximum parsimony and a neighbour joining method. Monophyly of the genus was confirmed, as was the sister genus status of Lucia. Acrodipsas myrmecophila was established as the plesiotypic Acrodipsas species, which together with Acrodipsas brisbanensis parasitises the ancestral dolichoderine host-ants. A speciation event associated with a radical host-ant shift to Myrmicinae occurred in an ancestor of Acrodipsas cuprea but subsequent speciation events may have been driven by climatic fluctuations during the Pleistocene. Modifications to leg morphology in several species were found to be a synapomorphic state, which arose subsequent to the host-shift to Myrmicinae. Minimal genetic variation detected in allopatric species exhibiting divergent morphology suggests that phenotypic variability has been driven by strong environmental selective pressures. As a result, morphological differences between closely related allopatric species have evolved faster than genetic differences, most notably between Acrodipsas arcana and Acrodipsas illidgei. In contrast, sympatric lineages of A. brisbanensis across similar habitats show considerable genetic differentiation, yet have remained phenotypically indistinguishable. Successful amplification of short overlapping fragments of DNA from museum specimens confirms their utility for phylogenetic analysis when the availability of fresh tissue is limited.

Animals↗

Review: Analysis of the evolutionary convergence for high performance swimming in lamnid sharks and tunas.

Elasmobranchs and bony fishes have evolved independently for more than 400 million years. However, two Recent groups, the lamnid sharks (Family Lamnidae) and tunas (Family Scombridae), display remarkable similarities in features related to swimming performance. Traits separating these two groups from other fishes include a higher degree of body streamlining, a shift in the position of the aerobic, red, locomotor muscle that powers sustained swimming to a more anterior location in the body and nearer to the vertebral column, the capacity to conserve metabolic heat (i.e. regional endothermy), an increased gill surface area with a decreased blood-water barrier thickness, a higher maximum blood oxygen carrying capacity, and greater muscle aerobic and anaerobic enzyme activities at in vivo temperatures. The suite of morphological, physiological, and biochemical specializations that define "high-performance fishes" have been extensively characterized in the tunas. This review examines the convergent features of lamnid sharks and tunas in order to gain insight into the extent that comparable environmental selection pressures have led to the independent origin of similar suites of functional characteristics in these two distinctly different taxa. We propose that, despite differences between teleost and elasmobranch fishes, lamnid sharks and tunas have evolved morphological and physiological specializations that enhance their swimming performance relative to other sharks and most other high performance pelagic fishes.

Adaptation, Biological↗

Use of excess solid-phase capacity in immunoassays: advantages for semicontinuous, near-real-time measurements and for analysis of matrix effects.

A flow-based immunoassay system using solid-phase particles with high binding capacity was used for semicontinuous, near-real-time, measurement of 17beta-estradiol (E2). The high binding capacity of the solid phase was exploited to enable (i) a quantitative determination of E2 concentration, based on rate of accumulation of fluorescently labeled anti-E2 antibody on the solid phase, and (ii) the use of a single solid phase for more than a dozen competitive binding measurements. The high binding capacity of the solid phase also permitted the immobilization of a second capture antigen. Biotin was immobilized as a second antigen and used to evaluate a biotin anti-biotin system as a control for matrix effects in the E2 immunoassay. In phosphate-buffered saline, E2 could be quantified (in the range of 10-1000 pM) by using either the summation or ratio of the signals from the labeled anti-E2 and anti-biotin antibody in the presence of biotin at a constant concentration. The same referencing system was applied to estimate the matrix effects in selected environmental samples. Matrix effects that inhibited the binding of the anti-E2 antibody to the solid phase led to false positive responses, but these matrix effects could be identified and partially corrected using the response from the anti-biotin antibody.

Antibodies, Monoclonal↗

Micellar catalyzed degradation of fenitrothion, an organophosphorus pesticide, in solution and soils.

We report on a study of the decomposition of fenitrothion (an organophosphorus pesticide that is a persistent contaminant in soils and groundwater) as catalyzed by cetyltrimethylammonium (CTA+) micelles. The CTA micelles were associated with two types of counterions: (1) inert counterions (e.g. CTABr) and (2) reactive counterions (e.g. CTAOH). The reactive counterion surfactants used were hydroxide anion (HO-) as a normal nucleophile and hydroperoxide anion (HOO-) and the anion of pyruvaldehyde oxime (MINA-) as two alpha-nucleophiles. The reactivity order followed: CTABr < CTAOH < CTAMINA << CTAOOH. Treatment of the rate data using the Pseudo-Phase Ion Exchange (PPIE) model of micellar catalysis showed the ratio k2M/k2w to be less than unity for all the surfactants employed. Rather than arising from a "true catalysis", we attributed the observed rate enhancements to a "concentration effect", where both pesticide and nucleophile were incorporated into the small micellar phase volume. Furthermore, the CTAOOH/CTAOH pair gave an alpha-effect of 57, showing that the alpha-effect can play an important role in micellar systems. We further investigated the effectiveness of reactive counterion surfactants in decontaminating selected environmental solids that were spiked with 27 ppb fenitrothion. The solids were as follows: the clay mineral montmorillonite and SO-1 and S0-2 soils (obtained from the Canadian Certified Reference Materials Project). The reactive counterion surfactant solutions significantly enhanced the rate of fenitrothion degradation in the spiked solids over that obtained when the spiked solid was placed in contact with either 0.02 M KOH or water. The rate enhancements followed the order CTAOOH >> CTAMINA approximately CTAOH > KOH >> water. We conclude that reactive counterion surfactants, especially with alpha-nucleophiles, hold great potential in terms of remediating soils contaminated by toxic organophosphorus esters.

Catalysis↗

Assessing the ecological condition of a coastal plain watershed using a probabilistic survey design.

Using a probabilistic survey design, we assessed the ecological condition of the Florida (U.S.A.) portion of the Escambia River watershed using selected environmental and benthic macroinvertebrate data. Macroinvertebrates were sampled at 28 sites during July-August 1996, and 3414 individuals were identified. Taxonomic data were distilled into the 8 m of the Stream Condition Index (SCI), developed specifically for Florida waterways, and sites were classified on an ordinal scale as very good, good, poor, and very poor. The weighted cumulative distribution function of the SCI showed that 8% (+/- 7) of the stream and river miles in this landscape were in very good condition, while 25% (+/- 7), 51% (+/- 16), and 16% (+/- 15) were in good, poor, and very poor condition, respectively. The only environmental parameter significantly correlated with SCI was dissolved oxygen (DO), and two sites classified as very poor had oxygen levels around or below 2 mg L(-1). However, other sites exhibited similarly low SCI values without the attendant low DO, implying that factors determining site condition were complex and multivariate. The results of this survey corroborate Federal and state assessments demonstrating that many locations within this watershed exhibit significant degradation.

Ecosystem↗

Evidence for multilocus genetic control of preferential fertilisation in maize.

Genetic segregation was studied in more than 1900 seedlings of an F2 between the maize (Zea mays L.) inbred lines T232 and CM37. Significant segregation distortion was observed at 11 of 17 segregating allozyme loci and at a single morphological marker locus distributed on 7 of the 10 chromosomes in the genome. Deviations from genotypic class expectations were small for most loci, and averaged 7.7 per cent. Percent transmission of the allele contributed by T232 varied from 47.7 per cent to 53.3 per cent. The allele donated by T232 was significantly under-represented for loci on chromosomes 1 and 8, whereas the allele contributed by CM37 was deficient for nine of the ten segregating loci on chromosomes 2, 3, and 6. In all cases, the parental origin of the deficient allele was consistent for markers on a chromosome. Evidence is presented that suggests the aberrant ratios arose from linkage of the markers with genetic factors affecting prezygotic transmission, and that a minimum of 5 such factors were operative, one on each of chromosomes 1, 2, 3, 6, and 8. In contrast to the multi-locus and multi-chromosomal distorted segregation observed in the F2, all loci in backcross progenies fit Mendelian expectations. It is suggested that this discrepancy reflects variable environmental selection pressures on genes that influence aspects of gamete competition.

Alleles↗

Genetic polymorphisms in subterranean mammals (Spalax ehrenbergi superspecies) in the near east revisited: patterns and theory.

Allozyme diversity in the superspecies Spalax ehrenbergi has been revisited by studying 36 gene loci in 241 subterranean mole rats from 22 populations and nine chromosomal species, four from Turkey (2n = 52E (east), 52W (west), 56 and 58), four from Israel (2n = 52, 54, 58 and 60), and one from Egypt (2n = 60). The following results were indicated. (1) Genetic patterns: 11 of the 36 loci analysed (30.5 per cent) were monomorphic across the range, fifteen (41.7 per cent) were weakly polymorphic and the remaining 10 loci (27.8 per cent) were strongly polymorphic. (2) Heterozygosity: the average H was 0.051, range 0.00-0.098. In Israel, H increased with aridity and climatic unpredictability towards the northern Negev Desert, and was remarkably high in small steppic semi-isolates and desert isolates. (3) Species discrimination: some of the S. ehrenbergi species can be discriminated qualitatively. (4) Genetic distances (D): between species these values averaged 0.077, range 0.001-0.269, with the highest D between the ancestor Turkish and descendant Israeli and Egyptian species. The phylogenetic tree supports the Turkish origin of the Israeli Spalax ehrenbergi species, and the recent speciation of the Egyptian Spalax. (5) Genetic diversity is mostly (58 per cent) within populations. (6) Allozyme correlates: allozyme diversity was significantly correlated with the external physical (both climatic and edaphic) and biotic (parasite infection and plant cover) environment. (7) Spatial autocorrelation of allozyme frequencies suggests that migration is not influential. (8) Gametic phase disequilibria were significant in four out of five species tested, and were associated with climatic and edaphic factors. These results support the environmental selection hypothesis of genetic diversity including the niche-width variation hypothesis in space and time. Natural selection appears to play a major role in genetic differentiation of proteins in adaptive radiation and speciation.

Animals↗

Utilization of umbilical cords to assess in utero exposure to persistent pesticides and polychlorinated biphenyls.

In support of a study to relate developmental and cognitive effects with prenatal exposure to selected environmental toxicants, we developed and applied an analytical method to determine the concentration of two persistent pesticides, hexachlorobenzene (HCB) and p,p'-dichlorodiphenyldichloroethylene (DDE), and 32 specific polychlorinated biphenyl (PCB) congeners in 316 umbilical cords taken in 1986-1987 from women of the Faroe Islands. The analytical method consisted of homogenization of the cords, partitioning, microsilica gel column chromatography for clean-up, and dual-column capillary gas chromatography (DB-5 and DB-1701) with electron capture detection. Several quality control parameters were followed to monitor the performance of the method. Important criteria used before reporting unknown data were the recovery of in vitro-spiked analytes from a bovine umbilical cord (BUC) and the percentage lipid obtained for a Certified Reference Material (CRM)-350 of mackerel oil (MO). Recoveries of analytes that had been spiked at two concentration ranges (0.26-0.95 ng/g whole weight; 0.35-2.42 ng/g whole weight) into bovine cords ranged from 38.5% to 158% and from 50.4% to 145%, respectively, with a median recovery of 77.7%. Measurement of the percentage lipid for CRM-350 ranged from 73.8% to 107% with a median lipid value of 96.0%. The most prevalent analytes detected (%) in unknown umbilical cords were HCB (100), DDE (100), Ballschmiter/Zell PCBs 153 (100), 138 (98), 180 (98), 170 (93), 118 (88), 187 (86), and 146 (83), with corresponding median concentrations (ng/g whole weight) of 0.17, 1.19, 0.38, 0.30, 0.17, 0.11, 0.12, 0.09, and 0.07, respectively. Total PCB--sum of all measurable PCB congeners--had a median concentration of 1.37 ng/g whole weight. The analytes, which were very low in lipid content were also quantified on a lipid-adjusted basis, which provided an analytical challenge in these umbilical cord samples. The gravimetrically measured lipids in the human specimens ranged from 0.01% to 1.43% (median of 0.18%). In the pooled BUCs, our lipid measurements varied from 0.05% to 0.33% with a median value of 0.13%. The utility of using the umbilical cord as a matrix to assess in utero exposure to persistent environmental pollutants, compared with the use of umbilical cord blood or mother's blood, is worthy of debate.

Adult↗

Salmonella spp. are affected by different levels of water activity in closed microcosms.

Controlling water activity (a(w)) can significantly impact the growth of Salmonella in poultry litter and manure - a phenomenon that was studied quantitatively using two common serotypes of Salmonella. The quantitative effect of changes in levels of a(w) on Salmonella populations was determined using inoculated, frosted glass rectangles placed in closed chambers (microcosms). Glass rectangles with known concentrations of Salmonella enteritidis and S. brandenburg were placed in microcosms maintained at an a(w) level of 0.893 for 24 h at room temperature (RT) and then transferred to other microcosms maintained at the same temperature but with higher a(w) levels (0.932 and 0.987). Salmonella populations on the slides were quantified at 4, 18, 24, and 48 h. Slightly elevated levels of a(w) (<0.1, i.e., 10% equilibrium relative humidity) for 24 h resulted in a 100-fold increase in counts of Salmonella. The data also suggested that in vitro adaptation to dry environments may occur when the organisms are exposed to alternating levels of relatively high and low (0.987 and 0.893) levels of a(w). Any increased tolerance of Salmonella to reduced levels of a(w) could be the result of physico-chemical changes in the organism due to selective environmental pressure, formation of a protective biofilm, and/or entry into a dormant state. Results from this study are compatible with those from previously reported on-farm surveys, reinforcing the contention that maintaining a(w) below 0.85 in and around litter/manure surfaces in poultry or livestock bedding areas may be a critical factor in safe production of food.

Animals↗

The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy is characterized by an increase in ventricular size and impairment of ventricular function. Most cases are believed to be sporadic, and familial dilated cardiomyopathy is usually considered to be a rare and distinct disorder. We studied the proportion of cases of idiopathic dilated cardiomyopathy that were familial in a large sequential series of patients whose first-degree relatives were investigated regardless of whether these relatives had cardiac symptoms. METHODS: We studied relatives of 59 index patients with idiopathic dilated cardiomyopathy of obtaining a family history and performing a physical examination, electrocardiography, and two-dimensional, M-mode, and Doppler echocardiography. A total of 315 relatives were examined. RESULTS: Eighteen relatives from 12 families were shown to have dilated cardiomyopathy. Thus, 12 of the 59 index patients (20.3 percent) had familial disease. There was no difference in age, sex, severity of disease, exposure to selected environmental factors, or electrocardiographic or echocardiographic features between the index patients with familial disease and those with nonfamilial disease. A noteworthy finding was that 22 of 240 healthy relatives (9.2 percent) with normal ejection fractions had increased left ventricular diameters during systole or diastole (or both), as compared with 2 of 112 healthy control subjects (1.8 percent) who were studied separately. CONCLUSIONS: Dilated cardiomyopathy was found to be familial in at least one in five of the patients in this study, a considerably higher percentage than in previous reports. This finding has important implications for family screening and provides direction for further investigation into the causes and natural history of dilated cardiomyopathy.

Aged↗

Phylogeny, evolution, and biogeography of Asiatic Salamanders (Hynobiidae).

We sequenced 15 complete mitochondrial genomes and performed comprehensive molecular phylogenetic analyses to study the origin and phylogeny of the Hynobiidae, an ancient lineage of living salamanders. Our phylogenetic analyses show that the Hynobiidae is a clade with well resolved relationships, and our results contrast with a morphology-based phylogenetic hypothesis. These salamanders have low vagility and are limited in their distribution primarily by deserts, mountains, and oceans. Our analysis suggests that the relationships among living hynobiids have been shaped primarily by geography. We show that four-toed species assigned to Batrachuperus do not form a monophyletic group, and those that occur in Afghanistan and Iran are transferred to the resurrected Paradactylodon. Convergent morphological characters in different hynobiid lineages are likely produced by similar environmental selective pressures. Clock-independent molecular dating suggests that hynobiids originated in the Middle Cretaceous [ approximately 110 million years ago (Mya)]. We propose an "out of North China" hypothesis for hynobiid origins and hypothesize an ancestral stream-adapted form. Given the particular distributional patterns and our molecular dating estimates, we hypothesize that: (i) the interior desertification from Mongolia to Western Asia began approximately 50 Mya; (ii) the Tibetan plateau (at least on the eastern fringe) experienced rapid uplift approximately 40 Mya and reached an altitude of at least 2,500 m; and (iii) the Ailao-Red River shear zone underwent the most intense orogenic movement approximately 24 Mya.

Animals↗