Origin of the Qinghai-Tibetan Plateau endemic Milula (Liliaceae): further insights from karyological comparisons with Allium

Milula, a monotypic genus endemic to the Qinghai-Tibetan Plateau, was found to be nested deeply within Allium by the molecular phylogeny despite the aberrant morphology. It remains unknown what had contributed to the rapid evolution of morphology and origin of this exceptional species. In contrast to a previous report of its karyotypes with 2n = 16 = 8M+8SM (2SAT), similar to most species of Allium, a rather different karyotype, 2n = 20 = 4M +10SM+6T (2SAT), was found in examined 31 individuals from 6 populations of M. spicata distributed in the central Tibet. Karyotypes of 7 Allium species occurring in the Qinghai-Tibetan Plateau were further reported. The basic number x = 8 was confirmed for all of them and their karyotypes consist mainly of metacentric and submetacentric chromosomes with rare subterminal and terminal chromosomes. The karyotype of M. spicata is distinctly different from that of most Allium species occurring in the plateau through a complete comparison of all available species in this region and adjacent areas. However, the same chromosome number and similar karyotypic structure were found in A. fasciculatum of Sect. Bromatorrhiza, indicating a possible close relationship between them. But this similarity is contradictory to the preliminary molecular phylogenetic analysis that Milula was closely related to A. cyathophorum of Sect. Bromatorrhiza with x=8, but the other species with x=10 and 11 in this section were clearly placed in the other clade. We therefore suggested that the paralleling evolution from x=8 to x=9, 10 and 11 with increasing asymmetry of karyotype possibly due to the chromosomal Robertsonian translocation might occur separately in the two recognized phylogenetic lineages of Allium. In addition to aneuploidy and following change of the chromosomal structures, the habitat isolation due to the recent uplift of the Qinghai-Tibetan Plateau and the Quaternary climatic oscillation, plays a greater role in origin of Milula and other endemic species (genera) with aberrant morphology from their progenitors.

Radiation and diversification within the Ligularia-Cremanthodium-Parasenecio complex (Asteraceae) triggered by uplift of the Qinghai-Tibetan Plateau

The Ligularia-Cremanthodium-Parasenecio (L-C-P) complex of the Tussilagininae (Asteraceae: Senecioneae) contains more than 200 species that are endemic to the Qinghai-Tibetan Plateau in eastern Asia. These species are morphologically distinct; however, their relationships appear complex. A phylogenetic analysis of members of the complex and selected taxa, of the tribe Senecioneae was conducted using chloroplast (ndhF and trnL-F) and nuclear (ITS) sequences. Phylogenetic trees were constructed from individual and combined datasets of the three different sequences. All analyses suggested that Doronicum, a genus that has been included in the Tussilagininae, should be excluded from this subtribe and placed at the base of the tribe Senecioneae. In addition, the Tussilagininae should be broadly circumscribed to include the Tephroseridinae. Within the expanded Tussilagininae containing all 13 genera occurring in eastern Asia, Tussilago and NSPetasites diverged early as a separate lineage, while the remaining I I genera comprise an expanded L-C-P complex clade. We suggest that the L-C-P clade, which is largely unresolved, most likely originated as a consequence of an explosive radiation. The few monophyletic subclades identified in the L-C-P clade with robust support further suggest that some genera of Tussilagininae from eastern Asia require generic re-circumscriptions given the occurrence of subclades containing species of the same genus in different parts of the phylogentic tree due to homoplasy of important morphological characters used to delimit them. Molecular-clock analyses suggest that the explosive radiation of the L-C-P complex occurred mostly within the last 20 million years, which falls well within the period of recent major uplifts of the Qinghai-Tibetan Plateau between the early Miocene to the Pleistocene. It is proposed that significant increases in geological and ecological diversity that accompanied such uplifting, most likely promoted rapid and continuous allopatric speciation in small and isolated populations, and allowed fixation or acquisition of similar morphological characters within unrelated lineages. This phenomenon, possibly combined with interspecific diploid hybridization because of secondary sympatry during relatively stable stages between different uplifts, could be a major cause of high species diversity in the Qinghai-Tibetan Plateau and adjacent areas of eastern Asia. (c) 2005 Elsevier Inc. All rights reserved.

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

Background and Aims The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences.Methods Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae.Key Results The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya.Conclusions The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.

Molecular phylogeny, recent radiation and evolution of gross morphology of the rhubarb genus Rheum (Polygonaceae) inferred from chloroplast DNA trnL-F sequences

Background and Aims Rheum, a highly diversified genus with about 60 species, is mainly confined to the mountainous and desert regions of the Qinghai-Tibetan plateau and adjacent areas. This genus represents a good example of the extensive diversification of the temperate genera in the Qinghai-Tibetan plateau, in which the forces to drive diversification remain unknown. To date, the infrageneric classification of Rheum has been mainly based on morphological characters. However, it may have been subject to convergent evolution under habitat pressure, and the systematic position of some sections are unclear, especially Sect. Globulosa, which has globular inflorescences, and Sect. Nobilia, which has semi-translucent bracts. Recent palynological research has found substantial contradictions between exine patterns and the current classification of Rheum. Two specific objectives of this research were (1) to evaluate possible relationships of some ambiguous sections with a unique morphology, and (2) to examine possible occurrence of the radiative speciation with low genetic divergence across the total genus and the correlation between the extensive diversification time of Rheum and past geographical events, especially the recent large-scale uplifts of the Qinghai-Tibetan Plateau.Methods The chloroplast DNA trnL-F region of 29 individuals representing 26 species of Rheum, belonging to seven out of eight sections, was sequenced and compared. The phylogenetic relationships were further constructed based on the sequences obtained.Key Results Despite the highly diversified morphology, the genetic variation in this DNA fragment is relatively low. The molecular phylogeny is highly inconsistent with gross morphology, pollen exine patterns and traditional classifications, except for identifying all samples of Sect. Palmata, three species of Sect. Spiciformia and a few species of Sect. Rheum as corresponding monophyletic groups. The monotypic Sect. Globulosa showed a tentative position within the clade comprising five species of Sect. Rheum. All of the analyses revealed the paraphyly of R. nobile and R. alexandrae, the only two species of Sect. Nobilia circumscribed by the possession of large bracts. The crude calibration of lineages based on trnL-F sequence differentiation implied an extensive diversification of Rheum within approx. 7 million years.Conclusions Based on these results, it is suggested that the rich geological and ecological diversity caused by the recent large-scale uplifts of the Qinghai-Tibetan Plateau since the late Tertiary, coupled with the oscillating climate of the Quaternary stage, might have promoted rapid speciation in small and isolated populations, as well as allowing the fixation of unique or rare morphological characters in Rheum. Such a rapid radiation, combined with introgressive hybridization and reticulate evolution, may have caused the transfer of cpDNA haplotypes between morphologically dissimilar species, and might account for the inconsistency between morphological classification and molecular phylogeny reported here.

A new species of Saussurea (Asteraceae) from Tibet and its systematic position based on ITS sequence analysis

A new species of Saussurea, S. erecta S. W Liu, J. T Pan A J. Q. Liu sp. nov., is described from Tibet. It resembles S. kingii but may be distinguished by having distinct stems and glabrous achenes. Saussurea kingii was placed in sect. Pseudoeriocoryne of subgen. Eriocoryne; this section was circumscribed by acaulescence and an inflorescence with congested capitula surrounded by a rosette of leaves. The discovery of S. erecta with distinct stems, cauline leaves and corymbose capitula blurred the delimitation of sect. Pseudoeriocoryne and suggested that the section may be polyphyletic. Both the close relationship and the significant difference between S. erecta and S. kingii were confirmed by analyses of nrDNA ITS sequences. The resulting phylogenies based on ITS data further suggest that Saussurea sect. Pseudoeriocoryne, as traditionally defined, does not constitute a monophyletic group. The rapid radiation and speciation of Saussurea in the Qinghai-Tibetan Plateau, as inferred from ITS phylogeny, are discussed. (c) 2005 The Linnean Society of London.

Molecular phylogeny and biogeography of the Qinghai-Tibet Plateau endemic Nannoglottis (Asteraceae)

All taxa endemic to the Qinghai-Tibet Plateau are hypothesized to have originated in situ or from immediately adjacent areas because of the relatively recent formation of the plateau since the Pliocene, followed by the large-scaled biota extinction and recession caused by the Quaternary ice sheet. However, identification of specific progenitors remains difficult for some endemics, especially some endemic genera. Nannoglottis, with about eight species endemic to this region, is one such genus. Past taxonomic treatments have suggested its relationships with four different tribes of Asteraceae. We intend to identify the closest relatives of Nannoglottis by evaluating the level of monophyly, tribal delimitation, and systematic position of the genus by using molecular data from ndhF gene, trnL-F, and ITS region sequences. We find that all sampled species of Nannoglottis are a well-defined monophyly. This supports all recent taxonomic treatments of Nannoglottis, in which all sampled species were placed in one broadly re-circumscribed genus. Nannoglottis is most closely related to the Astereae, but stands as an isolated genus as the first diverging lineage of the tribe, without close relatives. A tentative relationship was suggested for Nannoglottis and the next lineage of the tribe was based on the ITS topology, the "basal group," which consists of seven genera from the Southern Hemisphere. Such a relationship is supported by some commonly shared plesiomorphic morphological characters. Despite the very early divergence of Nannoglottis in the Astereae, the tribe must be regarded to have its origin in Southern Hemisphere rather than in Asia, because based on all morphological, molecular, biogeographical, and fossil data, the Asteraceae and its major lineages (tribes) are supposed to have originated in the former area. Long-distance dispersal using Southeast Asia as a steppingstone from Southern Hemisphere to the Qinghai-Tibet Plateau is the most likely explanation for this unusual biogeographic link of Nannoglottis. The 23-32-million-year divergence time between Nannoglottis and the other Astereae estimated by DNA sequences predated the formation of the plateau. This estimation is further favored by the fossil record of the Asteraceae and the possible time of origin of the Astereae. Nannoglottis seems to have reached the Qinghai-Tibet area in the Oligocene-Eocene and then re-diversified with the uplift of the plateau. The molecular infragenetic phylogeny of the genus identifies two distinct clades, which reject the earlier infrageneric classification based on the arrangement of the involucral bracts and the length of the ligules, but agree well with the habits and ecological preferences of its current species. The "alpine shrub" vs. "coniferous forest" divergence within Nannoglottis was estimated at about 3.4 million years ago when the plateau began its first large-scale uplifting and the coniferous vegetation began to appear. Most of the current species at the "coniferous forest" clade of the genus are estimated to have originated from 1.02 to 1.94 million years ago, when the second and third uprisings of the plateau occurred, the climate oscillated and the habitats were strongly changed. The assumed evolution, speciation diversity, and radiation of Nannoglottis based on molecular phylogeny and divergence times agree well with the known geological and paleobotanical histories of the Qinghai-Tibet Plateau. (C) 2002 Elsevier Science (USA). All rights reserved.

Karyomorphology of Sinadoxa (Adoxaceae) and its systematic significance

Karyomorphology of Sinadoxa corydalifolia and the karyotype of the diploid Adoxa moschatellina are investigated for the first time. Karyomorphological data indicate that Sinadoxa is directly akin to other two genera of the Adoxaceae, rather than related to Araliales; and three genera of the Adoxaceae should be a natural group. The karyomorphological combinations of the Adoxaceae delimit it from all taxa assumed to be related to it. The karyotype characteristics of all taxa of the Adoxaceae are compared and summarized. The mechanisms of speciation in the Adoxaceae are discussed.

中国南方含硒建造中硒稳定同位素与化学形态的地质意义

氧化还原敏感元素硒的地球化学循环非常复杂, 它的循环主要受氧化还原势、酸碱度、温度、压力等条件的控制。长期以来，关于硒的主要研究集中在环境生物地球化学领域。对于硒的地质地球化学行为的研究，学者们认为硒不可能独立形成矿床,它主要以伴生元素的形式赋存于一些热液矿床（如，拉尔玛硒-金热液矿床等）。 二十世纪末, 我国学者开始关注硒的矿床地球化学行为, 发现了目前唯一的渔塘坝沉积型的独立硒矿床。另外，下寒武统底部黑色岩系（如，遵义牛蹄塘组）处于地球发展演化的关键时期且富集了众多的有用金属元素, 因此它引起了全世界广大地质学家的普遍关注和重视。这些黑色岩系的共同特点是含有大量的有机质和丰富的金属元素(PGE，Cu，Ni，Mo，Au，U，V，Mn，Fe，Co，Bi，Cr，Se等)。但遵义牛蹄塘组中Ni-Mo多金属层的成因一直争论不休。 本文以拉尔玛硒-金热液矿床、渔塘坝独立硒矿床、遵义黄家湾含Ni-Mo-Se多金属层剖面为研究对象，主要通过硒稳定同位素的测试和不同化学形态的分析结合其他的地球化学参数（如，C-S-Fe体系，氧化还原敏感元素），示踪富硒地层的沉积环境，研究不同地质体系中硒稳定同位素的组成，进而完善硒同位素的理论体系，探讨硒富集过程中的形态迁移途径，并为矿床的形成提供更完善的地球化学证据。通过一系列的研究工作，我们取得了以下几点认识： (1) 通过不同的消解方法准确测定了低硒,高硒样品的总硒含量,相对标准偏差小于10%，而且不同的消解方法测得同样的结果, 满足了地质样品中微量元素的测试要求。由于不同消解方式存在自身的优缺点,我们建议根据不同的样品类型,不同的研究目的选取合适的消解方法。 (2) 通过巯基棉吸附装置达到了纯化富集样品硒的目的, 硒的回收率一般大于90%, 满足同位素测试的需要。应用自制的氢化物发生器与Nu-MC-ICP-MS联用,实现了在线气体进样测试硒稳定同位素的目的。 采用标准样品匹配测试方法校正仪器测试过程中的质量分馏。 硒稳定同位素的测试精度为2δ=0.30‰, 标准NIST SRM 3149采用与样品同样的处理方法,没有发现前处理过程的同位素分馏。 (3) 传统地球化学参数对沉积环境的指示意义。通过氧化还原敏感微量元素及其与TOC的关系和C-S-Fe体系的分析，对渔塘坝独立硒矿床的多元素富集及沉积环境, 遵义黑色岩系沉积环境进行了详细的解释。TS-TFe的关系表明样品中有过量的硫存在，可能为有机硫或为其他亲硫元素提供有利的沉淀条件。C-S-Fe体系及微量元素指数V/(V + Ni) 说明渔塘坝矿区的硅质岩和页岩的沉积环境为缺氧到静海环境， Ni/Co、V/Cr几乎不能作为该区缺氧环境的指示参数, 后者可能由于Cr的外源输入（碎屑、热液等）所致。 C-S-Fe体系及微量元素指数V/(V + Ni)体系指示遵义黑色岩系的沉积环境为微含氧到缺氧的条件, 但对于Ni-Mo矿而言, V/(V + Ni)体系指示其为含氧条件沉积，这可能与Ni-Mo多金属层的成因有关。但铁的硫化度可以指示Ni-Mo多金属层极端的缺氧还原环境。 (4) 硒的形态对古氧化还原条件和矿物质来源的示踪意义。黑色岩系的硒形态分布不同于海洋沉积物，体现了成岩作用对硒形态分布的改变。黑色岩系中硒的主要形态为有机结合态和硫化物/硒化物结合态。硫化物/硒化物结合态比例与铁的硫化程度（DOS）之间明显的相关关系说明在海洋环境中硒主要通过氧化还原反应富集在富有机质的沉积物与沉积岩中。这种相关关系与岩石类型没有关系，这使得将硫化物、硒化物结合的Se（-II）比例作为一个氧化还原条件示踪剂更加可行。DOS与硫化物/硒化物结合态硒的关系，及Se（IV）与Se（-II）的关系均说明遵义牛蹄塘组的K-斑脱岩形成于碱性的氧化环境，Ni-Mo多金属层沉积于微酸性的极端还原环境，而渔塘坝矿床形成于微碱性的还原缺氧环境。生物的同化作用与异化作用之间本身存在互补关系，但这种互补关系却存在不同的转化趋势。不同的转化趋势可能主要受氧化还原反应和酸碱度的控制，其他的地质作用也可能起着重要作用（如，硅酸盐、Fe（II）的含量，硫化物、有机质含量，风化程度等）。同时我们发现相同的氧化还原条件下可能存在不同的富集途径。Ni-Mo多金属层中极少的有机结合态硒暗示海洋同生沉积主要被生物异化还原控制，而沉积物从海洋富集硒的途径主要为直接由Se（VI）和Se（IV）到Se（-II）的还原途径，从高价态到元素态的还原途径可能偶有发生但强度很小。 (5) 硒稳定同位素对矿床成因及物质来源的示踪意义。热液或表生环境中,硒的再次活化迁移对硒同位素的大范围分馏是很重要的。较少的硫化物结合态硒和较大范围的硒同位素组成说明渔塘坝矿床形成时经历了多次氧化还原过程。干酪根硒同位素组成与全岩相似, 而且样品富集轻同位素暗示该矿床硒来的富集主要通过海洋硒的生物吸收同化异化还原与多次无机氧化还原实现。对于拉尔玛金硒矿床而言，未蚀变岩体的硫化物结合态硒富集轻同位素，而后期低温流体的蚀变作用导致蚀变岩体中硫化物结合态硒富集重同位素。干酪根的硒同位素组成暗示热液输入的硒为该矿床硒的主要来源，硒从热液直接进入干酪根的机制是一致的。随着硫含量的增加岩石和矿体中的硒逐渐富集重同位素, 说明海水对矿床富集的贡献是很有限的。遵义黑色岩系中硒同位素组成与Se（-II）之间的关系, 单质硒的缺乏，硒的富集与同位素的关系说明，热液为主要的硒来源，而早寒武世海水中的硒主要通过Se（VI）和Se（IV）到Se（-II）的直接生物还原实现。三个区域的S/Se比值综合说明硫与硒的共同沉降过程中不存在硒的同位素分馏。不同化学形态的分布与不同形态中硒稳定同位素的结合可能会更好的解释硒的全球地球化学循环。 (6) 根据目前硒的同位素分馏体系及所测得的数据,我们初步建立了不同时代、不同成因黑色岩系中硒稳定同位素的分馏模式。

湖北恩施表生高硒环境中有机结合态硒的形态研究

硒是人体和动物必需的微量元素，摄入过高或过低均可导致不同的健康效应。已有的研究表明，硒的生物有效性不仅与地质环境中的总硒有关，更取决于硒的形态分布。不同形态的硒具有显著差异的地球化学特性，影响着环境中硒的迁移、循环、生物有效性和毒性。渔塘坝是中国唯一发生过人群硒中毒爆发性流行的地区，该地区风化富硒碳质岩和高硒土壤中有机结合态硒可达到总硒的60%以上，而有机结合态硒的进一步研究有助于深入了解硒的分布和生物可利用性，阐明富硒碳质岩风化及其成土过程中硒的生物地球化学次生富集过程与循环机制。本论文利用改进的7步连续化学浸提技术和优化的有机结合态硒提取方案，使用氢化物发生－原子荧光光谱法，研究了恩施表生高硒环境中有机结合态硒的形态分布，得出了以下几点结论： 1. 在硒七步连续提取方案的基础上，对影响硒提取的因素如液固比、提取剂浓度等进行了优化、并对不同结合态（相态）硒提取中的若干问题进行了讨论。研究结果表明，硒的连续化学提取过程中，液固比为20:1到50:1时能够满足不同结合态硒提取的要求；在水浴加热条件下，NaOH浓度0.1M～0.5M时能有效提取有机结合态硒；1M Na2SO3溶液是元素硒的较好提取剂；而对于硫化物/硒化物结合态硒，合适配比的NaOH＋H2O2混合液有利于H2Se的完全吸收，500～600mL/min的载气气流能保证其被CrCl2+HCl混合液还原所产生H2Se气体的完全吸收。 2. 利用优化的连续化学浸提方案提取风化富硒碳质岩石和高硒土壤样品的有机结合态硒，进而分离出富里酸硒和胡敏酸硒，并用高压密闭消化－氢化物原子荧光光谱法测定了硒的含量。该方案的回收率为84.4～104.2%，平均回收率为96.1%；有机结合态硒与胡敏酸富里酸硒加和值之间有良好的一致性，线性相关系数为0.999（n＝14），斜率为1.0026。而后使用自制的氢化物反应发生和吸收装置进一步分离了与富里酸（胡敏酸）吸附的Se(IV)和强结合的硒，其中分离富里酸硒的回收率为86.3%～107.6%，平均值为97.1%；分离胡敏酸硒的回收率为85.5%～105.9%，平均值为97.9%。表明本论文所采用的实验方案是切实可行的。 3. 风化富硒碳质岩石样品中胡敏酸硒比例较高，所占比例为36.3%～75.8%，平均值为58.1%，但胡敏酸硒并不是占有绝对的优势，且各个样品之间存在较大差异。高硒土壤样品中富里酸硒占有机结合态比例在25.4%～85.0%，除个别样品外，多数样品的富里酸硒所占比例均在70%～80%，表明富里酸硒是渔塘坝土壤有机结合态硒的一种主要存在形式，在适当的条件下（如环境的pH-Eh变化），能够被转化从而被植物吸收利用。并且这种岩石中胡敏酸硒含量高、土壤中富里酸硒含量高的分布特征指示了硒在风化过程中可能的迁移和转化规律。 4. 富里酸硒和胡敏酸硒的形态分析表明，不同岩石样品中各有机结合态硒的形态分布差异较大，分布规律不明显，这可能与岩石样品的风化程度有关；而对于土壤样品，不论是在富里酸还是胡敏酸中，吸附Se(IV)的含量都大于强结合硒，尤其是富里酸中的吸附Se(IV)，平均值为83.4%，最高达95.4%。这部分吸附的Se(IV)相对于强结合的富里酸硒更容易被释放，而这也进一步佐证了富里酸硒易于受环境变化（pH-Eh）和倍半氧化物等的影响而转变为生物可利用态硒。 5. 无论是胡敏酸强结合硒（HA-Se）还是富里酸强结合硒（FA-Se）均在有机结合态硒中以一定的比例存在，对这部分硒的研究还仅局限于定量，而其究竟是以何种形态、以何种方式与腐殖质结合尚不清楚。因此，有必要对这部分硒进行进一步研究。

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with a matrix of carbon nanotubes for the analysis of low-mass compounds in environmental samples

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for environmental analysis has been mainly focused on qualitative analysis of high-mass molecules, such as toxins, humic acid, and microorganisms. Herein,we describe a novel MALDI-TOF-MS method with a matrix of oxidized carbon nanotubes for analysis of low-mass compounds in environmental samples. A number of chemicals in the environment were qualitatively analyzed by the present method, and it was found that most of them, especially the highly polar chemicals, were measurable with high sensitivity. With the intrinsic ability to measure high-mass chemicals, this method can compensate for the current shortage of methods for environmental analysis for the measurement of highly polar or high-mass chemicals. For sample analysis, arsenic speciation in Chinese traditional medicines was qualified and diphenylolpropane in water samples was quantified. With the relatively high tolerance of the method to interfering molecules, a simple pretreatment or even no pretreatment could be employed before MS detection. Furthermore, this method can be employed in a high-throughput format.

Analiza specjacyjna arsenu w wodzie techniką wysokosprawnej chromatografii cieczowej połączonej ze spektrometrią mas z jonizacją w indukowanej plazmie

Wydział Chemii: Pracownia Analizy Spektroskopowej Pierwiastków

An investigation into the tropospheric chemistry of acidic aerosols and ammonia in the laboratory

This thesis describes a broad range of experiments based on an aerosol flow-tube system to probe the interactions between atmospherically relevant aerosols with trace gases. This apparatus was used to obtain simultaneous optical and size distribution measurements using FTIR and SMPS measurements respectively as a function of relative humidity and aerosol chemical composition. Heterogeneous reactions between various ratios of ammonia gas and acidic aerosols were studied in aerosol form as opposed to bulk solutions. The apparatus is unique, in that it employed two aerosol generation methods to follow the size evolution of the aerosol while allowing detailed spectroscopic investigation of its chemical content. A novel chemiluminescence apparatus was also used to measure [NH4+]. SO2.H2O is an important species as it represents the first intermediate in the overall atmospheric oxidation process of sulfur dioxide to sulfuric acid. This complex was produced within gaseous, aqueous and aerosol SO2 systems. The addition of ammonia, gave mainly hydrogen sulfite tautomers and disulfite ions. These species were prevalent at high humidities enhancing the aqueous nature of sulfur (IV) species. Their weak acidity is evident due to the low [NH4+] produced. An increasing recognition that dicarboxylic acids may contribute significantly to the total acid burden in polluted urban environments is evident in the literature. It was observed that speciation within the oxalic, malonic and succinic systems shifted towards the most ionised form as the relative humidity was increased due to complete protonisation. The addition of ammonia produced ammonium dicarboxylate ions. Less reaction for ammonia with the malonic and succinic species were observed in comparison to the oxalic acid system. This observation coincides with the decrease in acidity of these organic species. The interaction between dicarboxylic acids and ‘sulfurous’/sulfuric acid has not been previously investigated. Therefore the results presented here are original to the field of tropospheric chemistry. SHO3-; S2O52-; HSO4-; SO42- and H1,3,5C2,3,4O4-;C2,3,4O4 2- were the main components found in the complex inorganic-organic systems investigated here. The introduction of ammonia produced ammonium dicarboxylate as well as ammonium disulfite/sulfate ions and increasing the acid concentrations increased the total amount of [NH4+].

Osmotic stress tolerance mechanisms in Lactococcus lactis

The response of Lactococcus lactis subsp. cremoris NCDO 712 to low water activity (aw) was investigated, both in relation to growth following moderate reductions in the aw and in terms of survival following substantial reduction of the aw with NaCI. Lc.lactis NCDO 712 was capable of growth in the presence of ≤ 4% w/v NaCI and concentrations in excess of 4% w/v were lethal to the cells. The presence of magnesium ions significantly increased the resistance of NCDO 712 to challenge with NaCI and also to challenge with high temperature or low pH. Survival of Lc.lactis NCDO 712 exposed to high NaCI concentrations was growth phase dependent and cells were most sensitive in the early exponential phase of growth. Pre-exposure to 3% w/v NaCI induced limited protection against subsequent challenge with higher NaCI concentrations. The induction was inhibited by chloramphenicol and even when induced, the response did not protect against NaCI concentrations> 10% w/v. When growing at low aw, potassium was accumulated by Lc. lactis NCDO 712 growing at low aw, if the aw was reduced by glucose or fructose, but not by NaCI. Reducing the potassium concentration of chemically defined medium from 20 to 0.5 mM) produced a substantial reduction in the growth rate, if the aw was reduced with NaCI, but not with glucose or fructose. The reduction of the growth rate correlated strongly with a reduction in the cytoplasmic potassium concentration and in cell volume. Addition of the compatible solute glycine betaine, partially reversed the inhibition of growth rate and partially restored the cell volume. The potassium transport system was characterised in cells grown in medium at both high and low aw. It appeared that a single system was present, which was induced approximately two-fold by growth at low aw. Potassium transport was assayed in vitro using cells depleted of potassium; the assay was competitively inhibited by Na+ and by the other monovalent cations NH4+, Li+, and Cs+. There was a strong correlation between the ability of strains of Lc. lactis subsp. lactis and subsp. cremoris to grow at low aw and their ability to accumulate the compatible solute glycine betaine. The Lc. lactis subsp. cremoris strains incapable of growth at NaCI concentrations> 2% w/v did not accumulate glycine betaine when growing at low aw, whereas strains capable of growth at NaCI concentrations up to 4% w/v did. A mutant, extremely sensitive to low aw was isolated from the parent strain Lc. lactis subsp. cremoris MG 1363, a plasmid free derivative of NCDO 712. The parent strain tolerated up to 4% w/v NaCI and actively accumulated glycine betaine when challenged at low aw. The mutant had lost the ability to accumulate glycine betaine and was incapable of growth at NaCI concentrations >2% w/v or the equivalent concentration of glucose. As no other compatible solute seemed capable of substitution for glycine betaine, the data suggest that the traditional; phenotypic speciation of strains on the basis of tolerance to 4% w/v NaCI can be explained as possession or lack of a glycine betaine transport system.

Trophic role of small cetaceans and seals in Irish waters

Understanding the role of marine mammals in specific ecosystems and their interactions with fisheries involves, inter alia, an understanding of their diet and dietary requirements. In this thesis, the foraging ecology of seven marine mammal species that regularly occur in Irish waters was investigated by reconstructing diet using hard parts from digestive tracts and scats. Of the species examined, two (striped and Atlantic white-sided dolphin) can be considered offshore species or species inhabiting neritic waters, while five others usually inhabit more coastal areas (white-beaked dolphin, harbour porpoise, harbour seal and grey seal); the last species studied was the bottlenose dolphin whose population structure is more complex, with coastal and offshore populations. A total of 13,028 prey items from at least 81 different species (62 fish species, 14 cephalopods, four crustaceans, and a tunicate) were identified. 28% of the fish species were identified using bones other than otoliths, highlighting the importance of using all identifiable structures to reconstruct diet. Individually, each species of marine mammal presented a high diversity of prey taxa, but the locally abundant Trisopterus spp. were found to be the most important prey item for all species, indicating that Trisopterus spp. is probably a key species in understanding the role of these predators in Irish waters. In the coastal marine mammals, other Gadiformes species (haddock, pollack, saithe, whiting) also contributed substantially to the diet; in contrast, in pelagic or less coastal marine mammals, prey was largely comprised of planktivorous fish, such as Atlantic mackerel, horse mackerel, blue whiting, and mesopelagic prey. Striped dolphins and Atlantic white-sided dolphins are offshore small cetaceans foraging in neritic waters. Differences between the diet of striped dolphins collected in drift nets targeting tuna and stranded on Irish coasts showed a complex foraging behaviour; the diet information shows that although this dolphin forages mainly in oceanic waters it may occasionally forage on the continental shelf, feeding on available prey. The Atlantic white-sided dolphin diet showed that this species prefers to feed over the continental edge, where planktivorous fish are abundant. Some resource partitioning was found in bottlenose dolphins in Irish waters consistent with previous genetic and stable isotope analysis studies. Bottlenose dolphins in Irish waters appears to be generalist feeders consuming more than 30 prey species, however most of the diet comprised a few locally abundant species, especially gadoid fish including haddock/pollack/saithe group and Trisopterus spp., but the contribution of Atlantic hake, conger eels and the pelagic planktivorous horse mackerel were also important. Stomach content information suggests that three different feeding behaviours might occur in bottlenose dolphin populations in Irish waters; firstly a coastal behaviour, with animals feeding on prey that mainly inhabit areas close to the coast; secondly an offshore behaviour where dolphins feed on offshore species such as squid or mesopelagic fish; and a third more complex behaviour that involves movements over the continental shelf and close to the shelf edge. The other three coastal marine mammal species (harbour porpoise, harbour seal and grey seal) were found to be feeding on similar prey and competition for food resources among these sympatric species might occur. Both species of seals were found to have a high overlap (more than 80%) in their diet composition, but while grey seals feed on large fish (>110mm), harbour seals feed mostly on smaller fish (<110mm), suggesting some spatial segregation in foraging. Harbour porpoises and grey seals are potentially competing for the same food resource but some differences in prey species were found and some habitat partitioning might occur. Direct interaction (by catch) between dolphins and fisheries was detected in all species. Most of the prey found in the stomach contents from both stranded and by catch dolphins were smaller sizes than those targeted by commercial fisheries. In fact, the total annual food consumption of the species studied was found to be very small (225,160 tonnes) in comparison to fishery landings for the same area (~2 million tonnes). However, marine mammal species might be indirectly interacting with fisheries, removing forage fish. Incorporating the dietary information obtained from the four coastal species, an ECOPATH food web model was established for the Irish Sea, based on data from 2004. Five trophic levels were found, with bottlenose dolphins and grey and harbour seals occurring at the highest trophic level. A comparison with a previous model based on 1973 data suggests that while the overall Irish Sea ecosystem appears to be “maturing”, some indices indicate that the 2004 fishery was less efficient and was targeting fish at higher trophic levels than in 1973, which is reflected in the mean trophic level of the catch. Depletion or substantial decrease of some of the Irish Sea fish stocks has resulted in a significant decline in landings in this area. The integration of diet information in mass-balance models to construct ecosystem food-webs will help to understand the trophic role of these apex predators within the ecosystem.

No evidence of host specialization in a parasitic pea-crab exploiting two echinoid hosts

The pinnotherid crab Dissodactylus primitivus lives parasitically on 2 burrowingechinoid species, Meoma ventricosa and Plagiobrissus grandis. The fecundity of female crabsvaries between hosts, and is higher when parasitizing P. grandis than M. ventricosa. Moreover, thehosts present great variations in morphology (size and density of spines). These characteristicssuggest the potential to differentiate crabs according to host species. We investigated the genetic(microsatellites) and morphometric (outline analysis) differentiation of this parasitic crab between2 host species at 1 Jamaican site (Western Lagoon, Discovery Bay), and compared it with geographicdifferentiation among 4 sites along the north coast of Jamaica. Greater genetic differencesbetween parasites of the 2 sympatric hosts than between parasites of a single host at different geographiclocations would indicate host differentiation. Genetic analyses (microsatellites) did notdetect spatial differentiation (probably due to local hydrography) or differentiation according tohost species. This lack of host differentiation could be explained by mobility of adult crabsbetween hosts. However, there was weak but significant morphological differentiation betweenfemale crabs from the 2 hosts. This morphological difference may reflect constraints due to hostmorphology.