云南酸性土壤中度嗜酸链霉菌的多样性

[目的]了解酸性土壤环境里中度嗜酸链霉菌的多样性,调查其物种资源.[方法]用分散和差速离心法及选择性分离培养基从14份云南酸性土壤样品中分离到367株具有链霉菌培养特征的放线菌,并进行了颜色分群.从各颜色类群中选取代表菌株共97株,通过显微形态观察和pH梯度生长实验确定其中的中度嗜酸链霉菌.进一步从中筛选出16株中度嗜酸链霉菌代表菌株,进行16SrRNA基因序列的相似性和系统发育分析,并结合基因组DNA-DNA相关性数据.[结果]分离菌株归为12同的颜色类群,其中80%属于中度嗜酸链霉菌,其代表菌株在系统发育树上形成了8个距离较远且与已知种不同的进化分枝,可能代表链霉菌属内至少8个不同的新基因种.[结论]用以上方法筛选出的中度嗜酸链霉菌可归为8个不同于已知种的进化群,说明云南酸性土壤含有丰富多样的中度嗜酸链霉菌新物种.     

武陵山放线菌多样性

[目的]为了探究武陵山放线菌多样性,以便从新放线菌菌株中发现新的潜在药物先导化合物.[方法]从武陵山采集280份土样,采用纯培养的方法,用4种培养基分离到1134株放线菌.选择其中30株代表菌进行了初步分类鉴定;以3株细菌和7株农作物致病真菌作为指示菌,检测其抑菌活性;利用特异性引物扩增的方法,检测是否具有聚酮合酶(PKS Ⅰ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和多烯类化合物合成酶(CYP)基因.[结果]分离到的武陵山放线菌中,链霉菌占70%以上,还有小单孢菌等8个科13个属,其中有5个菌株是潜在的新种.选取的30株实验菌对细菌、真菌有不同程度的抗菌活性;其中含有4类化合物合成基因的菌株占23%～60%.[结论]武陵山原始森林土壤中,放线菌多样性很丰富,且存在很多未开发的稀有类群.有抑菌活性的菌株,可用于进一步的药物开发利用.     

药用植物内生放线菌的分离和生物学特性

[目的]探索药用植物内生环境可培养放线菌的分离、培养方法,总结药用植物内生放线菌的生物学特性,探讨其物种多样性,挖掘新的微生物资源.[方法]采用10种分离培养基对37个新鲜的药用植物样品进行内生放线菌的分离;通过比较,选择适合植物内生放线菌生长的培养条件;根据菌落形态和细胞特征观察结果,选择其中174株菌测定16S rRNA基因序列,分析药用植物内生放线菌的多样性;应用Biolog GENⅢ微孔培养、API 50CH以及API ZYM试剂条测试27株代表菌株的生理生化特性.[结果]分离得到940株植物内生菌,分属于47个属,30个科,其中放线菌600余株,分属于34个属,共发现潜在的新分类单元有7个;本研究中药用植物内生放线菌的培养条件是:PYG培养基、pH7.2、28℃- 32℃;菌株间的生物学特性的差异与菌株系统进化关系呈正相关关系;不同环境植物的内生菌菌株的生物学特性差异较大,相同环境的不同植物内生菌的生物学特性差异较小.[结论]药用植物内生放线菌物种丰富多样;药用植物内生放线菌在唯一碳源利用、发酵碳源产酸及酶学活性等生理生化特性方面没有表现出和宿主植物的直接相关性,而是呈现出和宿主植物的地理分布有一定的相关性.     

青藏高原阿里、那曲和海西地区土壤可培养放线菌的多样性

【目的】为了研究青藏高原北部地区土壤可培养放线菌的多样性,并比较不同选择性分离培养基对高原土壤放线菌的分离效果。【方法】使用9种分离培养基,并尝试添加藤黄微球菌发酵液,对采集自阿里、那曲和海西地区的14份土壤样品中的放线菌进行选择性分离。通过16S r RNA基因序列分析对分离菌株进行初步分类鉴定,并在不同分类水平上统计所分离得到的放线菌多样性。【结果】分离得到去重复后的放线菌255株,分布于放线菌门的8个目,14个科,23个属,包含94个可能的物种。其中至少25个物种可能为新种,分布于13个属。链霉菌属的菌株108株,可能的物种28个,是最主要的优势菌属。分离培养基中添加藤黄微球菌发酵液明显增加了放线菌分离菌株的数量和多样性,稀释的葡萄糖酵母麦芽汁培养基适合分离链霉菌,淀粉甘油脯氨酸培养基、丙酸钠酪蛋白培养基等则适合分离稀有放线菌。【结论】青藏高原北部土壤放线菌多样性非常丰富,并且存在较多的新颖放线菌类群;添加藤黄微球菌发酵液是提高放线菌分离效率的有效手段。     

泥河湾大长梁遗址和马圈沟Ⅰ文化层放线菌的分离及系统发育分析

为分析泥河湾盆地大长梁遗址和马圈沟Ⅰ文化层中可培养放线菌的物种多样性,本实验通过平板稀释涂布法对该区域土样进行放线菌的物种分离,得到197株放线菌纯培养物。采用链霉菌属特异引物对放线菌纯培养物进行初步鉴定,确定143株放线菌归属于链霉菌属。对未被鉴定的54株放线菌进行16S rDNA全序列测定并分析其系统发育关系。最终,将197株放线菌归属于放线菌纲下的5个亚目,6个科,10个属。其中,马圈沟Ⅰ文化层的分离菌株12-9与Nocardioides albus KCTC 9186T之间的16S rDNA序列相似性是93.73%,可能是一个潜在新属;大长梁遗址的分离菌株18-64与Haloglycomyces albus YIM 92370T的16S rDNA序列相似性是93.64%,可能是一个潜在新属。     

云南干热环境可培养高温放线菌多样性及产纤维素酶活性评价

[目的]探索云南东川干热河谷、元谋土林以及昆明周边高温堆肥、热泉等环境可培养高温放线菌的多样性及其产纤维素酶的潜力.[方法]利用稀释涂布平板法从采集于上述环境的样品中分离得到菌株500余株,通过形态去重复后对300余株进行16SrRNA基因测序分析,并对获得的菌株利用刚果红染色的方法进行纤维素酶活性初步筛选.[结果]分离到的菌株共分布于放线菌纲下9个亚目15个科33个属,其中候选新属2个、候选新种3个.451株菌的纤维素酶筛选结果显示57％具有纤维素酶活性,其中链霉菌、小单孢菌、野野村氏菌在纤维素酶活性菌株中占较大比例.[结论]云南干热环境下蕴含着丰富的放线菌资源,纤维素酶初步筛选显示出了良好的降解活性,为下一步的深入研究提供良好的菌源.     

放射污染区古菌分离及多样性分析

【目的】研究放射污染区古菌多样性。【方法】放射污染区采集土样,采用甘油-精氨酸培养基(GJ)、甘油-天冬氨酸培养基(C1)、海藻糖-肌酸培养基(B7)、甘露醇-丙氨酸培养基(Z5)、干酪素-甘露醇培养基(CMKA)、壳聚糖-天冬酰胺培养基(F6)、甘露醇-酸水解酪蛋白培养基(GW1)、CM培养基、HP培养基和KC培养基10种分离培养基,采用梯度稀释法对古菌进行分离,将分离获得的菌株经形态特征,16S rRNA基因片段扩增及限制性内切酶酶切,选取酶切图谱中存在差异性的条带进行测序,最终通过序列比对,聚类分析,获得不同种类的古菌资源。【结果】从该土样中共获得了256株古菌,最终筛选出71株不同类型的古菌,这71株古菌均属于广古菌门,盐杆菌纲,盐杆菌目,盐杆菌科,分布于盐陆生菌属(Haloterrigena)、纳白菌属(Natrialba)、盐球菌属(Halococcus)、盐红菌属(Halorubrum)、盐长寿菌属(Halovivax)、纳线菌属(Natrinema)、盐碱球菌属(Natronococcus)、盐二型菌属(Halobiforma)、盐惰菌属(Halopiger)、盐池栖菌属(Halostagnicola)、富盐菌属(Haloferax)11个属,26个种,其中31株菌的16S rRNA基因序列与已有效发表菌株的序列相似性小于98%,Haloterrigena为该土样的优势菌属。对于分离效果较好的F6培养基采用了梯度营养成分的稀释,最终获得了19株古菌,这些菌株相互之间存在一定的差异性。【结论】本次分离获得了大量的古菌,表明放射污染区存在着较为丰富的古菌资源,其中蕴藏着多种新的物种类型,具有较大的研究价值。     

西藏地区土壤放线菌种群多样性及拮抗活性研究

从西藏不同海拔、不同气候条件的5个地区采集的10份土样中,使用选择分离培养基、ISP 2（Yeast extract_malt extract agar）和高氏一号培养基,通过分散差速离心法（Dispersion and differential centrifugation,DDC）分离得到放线菌156株。根据形态和培养特征将其归入32个类群,并从中选出65个代表菌株进行全细胞壁氨基酸组分分析,结果表明：９株菌为非链霉菌胞壁类型,其余为链霉菌胞壁类型。16S rDNA扩增片段长度多态性（ARDRA）分析得到不同带型,对其序列分析结果表明：分离菌株分属链霉菌属（Streptomyces）和5个稀有放线菌属。同时测试了代表菌株抗耐药细菌和真菌的活性,其中38.5%的菌株具有拮抗活性。     

聚酮类化合物生物合成途径基因阳性菌株生物多样性研究

从中国云南省采集土样,采用GPY培养基、淀粉_酪素琼脂培养基和甘油天门冬酰胺琼脂培养基分离得到了876株细菌放线菌菌株。经聚酮类化合物基因筛选得到75株Ⅰ型和Ⅱ型聚酮类化合物生物合成基因双阳性的菌株,经抗菌活性、形态、生理等结果分析比较,选取其中的10株进行了16S rDNA序列分析。在物种多样性方面,分离到的菌分布至少有7个科,8个属。其中有链霉菌科的链霉菌属,分枝杆菌科的分枝杆菌属,链孢囊菌科的链孢囊菌属和野野村菌属,诺卡氏菌科的诺卡氏菌属,微球菌亚目的一新属、产碱杆菌科的无色杆菌属和β_亚纲中与紫色杆菌属紧邻的一革兰氏阴性菌新属。综合其表型、基因型特征,初步确定其中8株为新物种。研究表明利用新的思路和程序从自然环境分离、鉴定未知菌是微生物资源开发利用的关键。     

青海盐碱环境中具抗肿瘤活性放线菌的筛选和多样性研究

从我国青海省采集盐碱土样或泥样,用添加1.0~3.0mol/L NaCl的GPY琼脂培养基和ISP2琼脂培养基分离到145株典型放线菌菌株。采用6种肿瘤细胞株对分离菌株的发酵产物进行体外筛选,得到26株抗肿瘤活性阳性菌株(17.9%),19株为拟诺卡氏属(Nocardiopsis)菌株,7株为链霉菌属(Streptomyces)菌株。在抗肿瘤活性、形态特征、生理生化特性和全细胞水解物氨基酸组分分析等实验结果的基础上,选取差异较大的8株抗肿瘤活性阳性菌株进行16S rRNA基因序列测定和系统发育多样性分析。结果表明,2株属于链霉菌属(Streptomyces)的1个已知物种和1个潜在新种;6株属于拟诺卡氏菌属(Nocardiopsis),可能代表该属的4个新种。研究表明青海盐碱环境中存在产生抗肿瘤活性物质的重要放线菌资源,也提示其中蕴藏着较丰富的微生物多样性。     

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN ORCHIS MASCULA AND ORCHIS PAUCIFLORA

Studies of the strength and nature of reproductive isolation (RI) between species can greatly contribute to our understanding of speciation. Although the role of RI in speciation is well recognized, there is a dearth of information on the contributions of different barriers between related plant species. Here, we estimated multiple components of RI between two Mediterranean orchid sister species (Orchis mascula and Orchis pauciflora), disentangling the strength and absolute contributions of seven different isolating mechanisms. Our survey includes one prepollination, two postpollination prezygotic (pollen–stigma incompatibility, conspecific pollen precedence), two intrinsic postzygotic (embryo mortality and hybrid sterility) and two extrinsic postzygotic (hybrid habitat differentiation and hybrid pollination) isolating mechanisms. We found strong RI between the investigated species, although none of the barriers were able to completely impede gene flow. Five isolating mechanisms contributed positively to the maintenance of species boundaries. Contrary to most surveys of isolating mechanisms, our data speak against a clear predominance of prepollination or of prezygotic barriers but confirm the emerging pattern of multiple barriers contributing to the maintenance of species integrity. These findings suggest an allopatric condition during early phases of species divergence. We discuss our data in the wider context of previous studies carried out in this orchid group by using a comparative approach.     

Drivers of population genetic differentiation in the wild: isolation by dispersal limitation,isolation by adaptation and isolation by colonization

Empirical population genetic studies have been dominated by a neutralist view, according to which gene flow and drift are the main forces driving population genetic structure in nature. The neutralist view in essence describes a process of isolation by dispersal limitation (IBDL) that generally leads to a pattern of isolation by distance (IBD). Recently, however, conceptual frameworks have been put forward that view local genetic adaptation as an important driver of population genetic structure. Isolation by adaptation (IBA) and monopolization (M) posit that gene flow among natural populations is reduced as a consequence of local genetic adaptation. IBA stresses that effective gene flow is reduced among habitats that show dissimilar ecological characteristics, leading to a pattern of isolation by environment. In monopolization, local genetic adaptation of initial colonizing genotypes results in a reduction in gene flow that fosters the persistence of founder effects. Here, we relate these different processes driving landscape genetic structure to patterns of IBD and isolation by environment (IBE). We propose a method to detect whether IBDL, IBA and M shape genetic differentiation in natural landscapes by studying patterns of variation at neutral and non‐neutral markers as well as at ecologically relevant traits. Finally, we reinterpret a representative number of studies from the recent literature by associating patterns to processes and identify patterns associated with local genetic adaptation to be as common as IBDL in structuring regional genetic variation of populations in the wild. Our results point to the importance of quantifying environmental gradients and incorporating ecology in the analysis of population genetics.     

Isolation of single cell suspensions from the rat mammary gland: Separation,characterization, and primary culture of various cell populations

Summary Isolation and characterization of a single cell suspension from the rat mammary gland was achieved by combining selective enzymatic digestion and the mechanical agitation of a Stomacher laboratory blender with immunohistological identification of cell-specific markers. Utilizing this procedure we were able to isolate single cell suspensions of high yield (10 to 15×10⁶ cells/rat) and viability (>98%) with a concurrent decrease in isolation time and the amount of proteolytic enzymes required. Five distinct cell fractions were isolated from the mammary gland cell suspension after banding on discontinuous Percoll gradients. These populations were characterized both before and after primary cell culture by a combination of histological, immunohistological, and autoradiographic techniques. Fractions two and three were found to be enriched for mammary epithelial cells, as identified by their high binding of antikeratin antibodies. These populations also exhibited a minimal degree of binding to actin, myosin, and fibronectin antibodies. Fraction three also exhibited a high labeling index as measured by autoradiography following in vivo administration of [methyl-³H]thymidine. The remaining fractions were found to contain higher percentages of myoepithelial cells or other mammary cell types. Inasmuch as there is a direct correlation between mammary gland cell types and susceptibility to mammary gland carcinomas, further studies of these cell populations may provide new insights into the mechanisms underlying mammary gland carcinogenesis. This work was supported by grant R809580 from the U. S. Environmental Protection Agency, Office of Research Grants and Centers, Washington, D. C.     

Evolution of reproductive isolation in stickleback fish

To understand how new species form and what causes their collapse, we examined how reproductive isolation evolves during the speciation process, considering species pairs with little to extensive divergence, including a recently collapsed pair. We estimated many reproductive barriers in each of five sets of stickleback fish species pairs using our own data and decades of previous work. We found that the types of barriers important early in the speciation process differ from those important late. Two premating barriers—habitat and sexual isolation—evolve early in divergence and remain two of the strongest barriers throughout speciation. Premating isolation evolves before postmating isolation, and extrinsic isolation is far stronger than intrinsic. Completing speciation, however, may require postmating intrinsic incompatibilities. Reverse speciation in one species pair was characterized by significant loss of sexual isolation. We present estimates of barrier strengths before and after collapse of a species pair; such detail regarding the loss of isolation has never before been documented. Additionally, despite significant asymmetries in individual barriers, which can limit speciation, total isolation was essentially symmetric between species. Our study provides important insight into the order of barrier evolution and the relative importance of isolating barriers during speciation and tests fundamental predictions of ecological speciation.     

Sexual isolation evolves faster than hybrid inviability in a diverse and sexually dimorphic genus of fish (Percidae: Etheostoma)

Abstract .Theory predicts that sexual (or behavioral) isolation will be the first form of reproductive isolation to evolve in lineages characterized by sexual selection. Here I directly compare the rate of evolution of sexual isolation with that of hybrid inviability in a diverse and sexually dimorphic genus of freshwater fish. The magnitude of both sexual isolation and hybrid inviability were quantified for multiple pairs of allopatric species. Rates of evolution were inferred by comparing genetic distances of these species pairs with the magnitude of each form of reproductive isolation: the slope of the regression of genetic distance on the magnitude of reproductive isolation represents the rate of evolution. Of the two forms of isolation, the magnitude of sexual isolation exhibited the steeper slope of regression, indicating that sexual isolation will tend to evolve to completion earlier than hybrid inviability, strictly as a by-product of evolution in geographically isolated populations. Additional evidence from the literature is used to qualitatively compare rates of evolution of sexual isolation with that of other forms of reproductive isolation. Preliminary comparisons support the prediction that sexual isolation will evolve more rapidly than other forms. Because Etheostoma is characterized by striking sexual dimorphism, these results are consistent with the hypothesis that sexual selection for exaggerated mate-recognition characters causes the relatively rapid evolution of sexual isolation.     

Impact of ancestral populations on postzygotic isolation in allopatric speciation

Postzygotic isolation evolves due to an accumulation of substitutions (potentially deleterious alleles in hybrids) in populations that have become geographically isolated. These potentially deleterious alleles might also be maintained in ancestral populations before geographic isolation. We used an individual-based model to examine the effect of the genetic state of an ancestral population on the evolution of postzygotic isolation after geographic isolation of a population. The results showed that the number of loci at which degenerative alleles are fixed in an ancestral population at equilibrium significantly affects the evolutionary rates of postzygotic isolation between descendant allopatric populations. Our results suggest that: (1) a severe decrease in population size (e.g., less than ten individuals) is not necessary for the rapid evolution of postzygotic isolation (e.g., <10,000 generation); (2) rapid speciation can occur when there is a large difference in the equilibrium number of accumulated degenerative alleles between ancestral and descendant populations; and (3) in an ancestral population maintained at a small effective population size for a long period of time, postzygotic isolation rarely evolves if back mutations that restore the function of degenerative alleles are limited.     

Reproductive isolation among allopatric Drosophila montana populations

An outstanding goal in speciation research is to trace the mode and tempo of the evolution of barriers to gene flow. Such research benefits from studying incipient speciation, in which speciation between populations has not yet occurred, but where multiple potential mechanisms of reproductive isolation (RI: i.e., premating, postmating‐prezygotic (PMPZ), and postzygotic barriers) may act. We used such a system to investigate these barriers among allopatric populations of Drosophila montana. In all heteropopulation crosses we found premating (sexual) isolation, which was either symmetric or asymmetric depending on the population pair compared. Postmating isolation was particularly strong in crosses involving males from one of the study populations, and while sperm were successfully transferred, stored, and motile, we experimentally demonstrated that the majority of eggs produced were unfertilized. Thus, we identified the nature of a PMPZ incompatibility. There was no evidence of intrinsic postzygotic effects. Measures of absolute and relative strengths of pre‐ and postmating barriers showed that populations differed in the mode and magnitude of RI barriers. Our results indicate that incipient RI among populations can be driven by different contributions of both premating and PMPZ barriers occurring between different population pairs and without the evolution of postzygotic barriers.     

Evolution of postmating isolation: comparison of three models based on possible genetic mechanisms

In this study, we simulated the process of the evolution of postmating isolation using three models in which postmating isolation is caused by (1) genetic divergence through collaborative coevolution, (2) genetic divergence through antagonistic coevolution resulting from sexual conflict, and (3) genetic divergence through combinational incompatibility. The collaborative coevolution model and the combinational incompatibility model showed a similar decreasing pattern of hybrid compatibility over generations depending on population size and mutation rates. The antagonistic coevolution model showed that reproductive isolation can evolve rapidly depending on the intensity of selection. In the combinational incompatibility model, the increasing number of loci that interact and result in incompatibility would have both promoting and inhibiting effects on the formation of hybrid incompatibility in the earlier stage of isolation. Mutation rates for genes causing incompatibility significantly affect the number of generations required for postmating isolation, which indicates that models assuming high mutation rates (e.g., μ = 10⁻⁴) might predict much faster evolution for reproductive isolation than those observed in real populations. Received: January 29, 2001 / Accepted: July 4, 2001     

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN NORTH AMERICAN PHEROMONE STRAINS OF THE EUROPEAN CORN BORER

Of 12 potential reproductive isolating barriers between closely related Z‐ and E‐pheromone strains of the European corn borer moth (Ostrinia nubilalis), seven significantly reduced gene flow but none were complete, suggesting that speciation in this lineage is a gradual process in which multiple barriers of intermediate strength accumulate. Estimation of the cumulative effect of all barriers resulted in nearly complete isolation (>99%), but geographic variation in seasonal isolation allowed as much as ～10% gene flow. With the strongest barriers arising from mate‐selection behavior or ecologically relevant traits, sexual and natural selection are the most likely evolutionary processes driving population divergence. A recent multilocus genealogical study corroborates the roles of selection and gene flow ( Dopman et al. 2005 ), because introgression is supported at all loci besides Tpi, a sex‐linked gene. Tpi reveals strains as exclusive groups, possesses signatures of selection, and is tightly linked to a QTL that contributes to seasonal isolation. With more than 98% of total cumulative isolation consisting of prezygotic barriers, Z and E strains of ECB join a growing list of taxa in which species boundaries are primarily maintained by the prevention of hybridization, possibly because premating barriers evolve during early stages of population divergence.