一种新型抗性质粒的构建及其在核黄素生产菌中的运用

枯草芽孢杆菌Bacillus subtilis RF1是课题组前期通过基因工程改造得到的一株核黄素高产菌,为了进一步提高核黄素的产量,需要对该菌株进行进一步的基因工程改造.本研究首先将编码的链丝菌素乙酰基转移酶基因sat克隆到p MA5质粒上,构建具有诺瓦丝菌素Norseothricin(NTC)抗性的重组质粒p MA5-sat,实验证实该重组质粒能够用于B.subtilis RF1的抗性筛选.随后将核黄素合成相关的关键酶葡萄糖-6-磷酸脱氢酶编码基因zwf克隆到重组质粒p MA5-sat上,获得重组质粒p MA5-sat-zwf,并成功构建重组菌B.subtilis RF1/p MA5-sat-zwf.结果显示,重组菌胞内葡萄糖-6-磷酸脱氢酶活力比原始菌提高了近50倍,说明葡萄糖-6-磷酸脱氢酶在重组菌中成功过量表达;根据发酵特性分析,重组菌B.subtilis RF1/p MA5-sat-zwf最终核黄素产量达到12.01 g/L,比原始菌B.subtilis RF1提高了30.3%.综上,本研究构建的新型抗性质粒能够成功运用于核黄素生产菌枯草芽孢杆菌的基因工程改造.     

parDE基因对重组质粒pLM2在Enterobacter gergoviae 57-7中稳定性的影响

将 parDE基因克隆到载有组成型表达nifA基因的质粒 pST10 2 1上 ,得到重组质粒pLM2 .将 pLM2 ,pST10 2 1分别转到玉米联合固氮菌Enteorbactergergoviae 5 7 7(E5 7 7) ,获得转化子E5 7 7(pLM2 ) ,E5 7 7(pST10 2 1) ,二者在c(NH+ 4 ) =5 0mmolL-1的Kp培养基中表达的固氮活性一致 .将pLM2 ,pST10 2 1分别转到E .coliDH5α(pMGFP2 .1) ,培养物用 4 88nm光激发后在 5 10nm均有相同的的荧光特征发射峰 .上述实验表明 ,重组质粒pLM2仍保留了原质粒pST10 2 1中nifA的功能 .但质粒 pLM2和 pST10 2 1在E5 7 7中的稳定性试验表明 ,E5 7 7(pST10 2 1)在无抗生素的LB中继代培养 70代后 ,质粒几乎全部丢失 (仅存 1% ) ;而E5 7 7(pLM2 )继代培养 10 0代后 ,质粒全部存在 (10 0 % ) .图 4表 1参 12     

营养和环境条件对光滑球拟酵母葡萄糖代谢速度的影响

增加培养基中Mg2 浓度,使磷酸果糖激酶和3-磷酸甘油醛脱氢酶活性提高22.2%和23.4%,葡萄糖消耗速率从1.94g L-1h-1提高到2.43g L-1h-1,提高了25%.较低kLa(200h-1)导致胞内ATP处于较低的水平,变构激活糖酵解关键酶活性,与高kLa(450h-1)比较,葡萄糖消耗速率(2.31g L-1h-1)提高了35%,低kLa虽能加快葡萄糖消耗,但不利于丙酮酸产量的提高.烟酸(NA)是细胞合成NAD 的前体,培养基中缺乏NA导致细胞生长微弱,葡萄糖消耗缓慢;NA质量浓度从4mg/L增加到8mg/L时,葡萄糖消耗速度(2.01g L-1h-1)和丙酮酸产量(46.4g/L)分别提高了48.4%和29%,高NA浓度有利于高葡萄糖消耗速度的提高,但降低了丙酮酸对葡萄糖的产率.一定浓度的(0~10mg/L)的外源电子受体乙醛提高了光滑球拟酵母中醇脱氢酶活性(提高8.6%),加速NAD 再生,降低NADH/NAD 比率,从而促进细胞生长和提高葡萄糖消耗速度.以上结果表明,营养和环境条件通过改变细胞胞内辅因子水平,影响糖酵解关键酶活性而改变葡萄糖代谢速度.图3表4参18     

嗜热脂肪芽孢杆菌过氧化氢酶基因perA在大肠杆菌中的高效表达

利用PCR技术得到嗜热脂肪芽孢杆菌 (Bacillusstearothermophilus)过氧化氢酶基因 perA ,将该基因与表达载体 pKK2 2 3 3连接构建重组质粒pK perA ,转化大肠杆菌过氧化氢酶HPⅠ和HPⅡ双缺突变株UM 2 ,得到重组大肠杆菌UM 2 1.酶活测定结果表明 ,表达产物具有正常的生物学活性 .SDS PAGE电泳结果显示出明显的特异性表达条带 ,单体Mr =86× 10 3 ,与嗜热脂肪芽孢杆菌所产酶相同 .实验表明 ,重组质粒在宿主UM 2中有较好的稳定性 ,在无选择压力条件下传代 6 0次基本保持稳定 ,传代 10 0次重组质粒保留 80 %以上 .摇瓶实验确定重组菌的最佳表达条件为 :IPTG浓度 ,0 .75mmol/L ;诱导时间 3h ;培养基起始 pH 6 .5 ;诱导温度 37℃ ;装液量 5 0mL/ 2 5 0mL .在优化条件下 ,重组菌产生的过氧化氢酶占菌体总蛋白的 8% ,酶活力可达 35U/mL ,是原始菌株BacillusstearothermophilusIAM110 0 1的 11.7倍 .图 2表 1参 10     

产琥珀酸重组大肠杆菌的构建和发酵性能

琥珀酸是一种重要的化工产品,微生物发酵法生产琥珀酸具有广阔的应用前景.野生大肠杆菌厌氧发酵产物中琥珀酸含量低,且有大量副产物.为构建高产琥珀酸的重组大肠杆菌,阻断代谢旁路,利用Xer/dif重组酶系统对Escherichia coli CICIM B0013进行代谢途径的调控研究.结果显示,敲除了包括乙酸激酶和磷酸乙酰转移酶基因(ackA-pta)、乳酸脱氢酶基因(ldhA)、丙酮酸甲酸裂解酶基因(pflB)、乙醇脱氢酶基因(adhE)等琥珀酸合成竞争途径中关键酶基因的重组大肠杆菌菌株E.coli CICIM B0013-1040,其发酵液中琥珀酸成为主要产物得到积累;进一步对其PTS系统进行修饰并适当增强PEP羧化酶基因(ppc)表达剂量,成功获得一株具备较强琥珀酸生产能力的菌株E.coli CICIM B0013-1050(pTH-ppc),该菌株厌氧转化葡萄糖36 h,琥珀酸产量达到36.2 g/L,生产强度为1.01 g L-1 h-1,葡萄糖-琥珀酸转化率为64.3%,发酵液经高效液相色谱(HPLC)检测,无乳酸、乙酸、甲酸、乙醇生成.     

赣江流域水体中可溶态镉的时空分布特征及水质评价

为了解赣江流域水体中镉的分布特征,在赣江流域的重点河段采集44个水样(丰水期和枯水期各22个),采用氢化物发生-原子荧光光谱法测定水体中可溶态镉浓度。结果表明,赣江水体ρ(可溶态镉)〔(0.112±0.111)μg·L-1〕较低,丰水期水体ρ(可溶态镉)〔(0.095±0.116)μg·L-1〕显著低于枯水期〔(0.128±0.160)μg·L-1〕(P0.05)。干流水体ρ(可溶态镉)〔(0.116±0.038)μg·L-1〕略低于支流〔(0.121±0.102)μg·L-1〕,且从上游到下游呈逐步下降趋势。从水体可溶态镉浓度的空间分布来看,赣州城区至峡江县城之间应该是赣江流域水体镉污染的重点监控区域。     

解淀粉芽孢杆菌DC-4豆豉溶栓酶成熟肽编码序列的克隆及表达

利用PCR方法从解淀粉芽孢杆菌DC 4总DNA中扩增出豆豉溶栓酶 (DFE)成熟肽编码区片段 .测序结果表明 :DFE成熟肽编码区长 82 5bp,编码 2 75个氨基酸残基 ,分子量为 2 7.7× 10 3 ,推导的N’ -端氨基酸序列与豆豉溶栓酶N’ -端氨基酸测序结果完全一致 ,说明克隆到的基因确实是豆豉溶栓酶基因 .同源性分析表明 ,DFE成熟肽编码区的核苷酸和氨基酸序列与日本纳豆激酶的同源性分别为 80 .0 %和 86 .5 % ,这提示豆豉溶栓酶可能是一种新型的溶栓酶 .将表达质粒pET Nde转化E .coliBL2 1(DE3)中 ,IPTG可诱导表达大量的DFE融合蛋白 ,占菌体可溶性蛋白的 4 0 % ,主要以包涵体的形式存在 .图 3表 1参 19     

嗜热脂肪土芽孢杆菌木聚糖酶基因的合成及其在大肠杆菌中的表达

来自嗜热脂肪土芽孢杆菌的木聚糖内切酶XT6在工业上有着重要的应用,已经成功应用于工业规模的生产试验.本文作者在合成XT6基因全序列的同时对其密码子进行了优化,且构建重组质粒在大肠杆菌中高表达.通过优化表达条件,功能正常的XT6基因在大肠杆菌中成功过量表达,蛋白表达量占细胞中总蛋白的65%.重组表达的木聚糖内切酶XT6特性和天然酶相似,以桦木木聚糖为底物测定细胞提取物中木聚糖酶活性,最大活性高达3 030 U/mL.本文首次报道了来自嗜热脂肪土芽孢杆菌中木聚糖酶基因全序列的合成和在大肠杆菌中成功过量表达.图6表1参19     

纳豆激酶原核表达载体的构建及其活性鉴定

利用引物搭桥的方法,经PCR扩增,获得了纳豆激酶成熟肽基因(NK),从而构建了大肠杆菌表达质粒NK/pTWIN1,NK/PET32a及NK/PML-c2x,经分别转化宿主菌ER2566,BL21和ER2566,获得了转纳豆激酶基因重组菌.结果表明,NK/pTWIN1-ER2566和NK/PET32a-BL21的纳豆激酶蛋白表达量较高.本研究选用NK/pTWIN1-ER2566作为表达菌株,对其进行发酵表达.SDS-PAGE显示,目的蛋白约占菌体总蛋白的36%,该蛋白是以包涵体形式存在的.包涵体经收集、蛋白变性及复性,并经过SP Sepharose分离纯化,得到了纯化的纳豆激酶蛋白,琼脂糖-纤维蛋白平板法测出1mg纳豆激酶干粉的溶栓活性相当于600u尿激酶.本文从基因工程角度研究纳豆激酶基因的克隆、表达及纯化,为利用基因工程菌生产纳豆激酶奠定了基础.图5表1参11     

顺丁烯二酸异构酶工程菌发酵条件优化

为提高重组大肠杆菌中顺丁烯二酸异构酶表达量,通过正交试验设计,对工程菌的生长条件和目的蛋白可溶表达条件进行优化.采用250 mL三角瓶中装有50 mL(Amp 100 mg L-1)的培养基,分别研究培养基中葡萄糖、蛋白胨、酵母浸粉的浓度,培养基pH值以及摇床转速、装液量、接种量等对蛋白可溶表达量的影响.确定顺丁烯二酸异构酶工程菌最优化培养基为:蛋白胨20 g L-1、酵母浸粉2.5 g L-1、K2HPO4·3H2O 3.0 g L-1、KH2PO4 1.5 g L-1、NaCl 6 g L-1、MgSO43 g L-1,培养基pH调至6.5.确定顺丁烯二酸异构酶工程菌可溶性表达最优条件为:37℃下培养至D600 nm值为1.0时,添加终浓度为0.05 mmol L-1的IPTG进行诱导,诱导温度37℃,摇床转速220 r min-1,装液量20%,接种量5%,诱导时长为6 h.利用BioFlo 415发酵罐以最优化的培养基和发酵条件对该工程菌进行了3批发酵实验,与摇瓶实验相比,顺丁烯二酸异构酶的表达量提高了近1.5倍,单位发酵液的酶活力由46 U mL-1发酵液提高到78 U mL-1发酵液.以上数据为顺丁烯二酸异构酶重组工程菌的中试发酵奠定了基础.图7表2参17     

Rapid,recurring, structured survey versus bioblitz for generating biodiversity data and analysis with a multispecies abundance model

A bioblitz inexpensively and quickly generates biodiversity data, but bioblitzes are often conducted with haphazard, unreplicated sampling. Results tend to be taxonomically, geographically, or temporally biased, lack metadata, and consist of lists of observed taxa that do not enable further analyses or correction for imperfect detection. A rapid, recurring, structured survey (RRSS) uses a structured sampling design and temporal and spatial replication to survey randomly selected sites on a conservation property. We participated in a loosely structured bioblitz and a subsequent RRSS at Big Canoe Creek Nature Preserve in Springville (St. Clair County), Alabama (USA) to compare observed richness derived from the 2 survey approaches. The RRSS data structure enabled us to fit models that accounted for imperfect detection to estimate abundances, occupancy probabilities, and habitat associations. The loosely structured bioblitz data could not be used in such models. We present a new integrated multispecies abundance model that we applied to avian RRSS data. Our model extension enables estimation for the community, employs data augmentation to estimate the number of undetected species, and incorporates covariates. The RRSS generated a more comprehensive and less biased list of observed taxonomic richness than the loosely structured bioblitz (e.g., 73 vs. 45 bird species and 104 vs. 63 insect families from the RRSS vs. loosely structured bioblitz, respectively). Models fit to the RRSS data identified seasonal patterns in avian community composition and allowed for estimation of habitat–occupancy relationships for insect taxa. The RRSS protocol has potential for broad transferability as a standardized, quick, and inexpensive way to inventory biodiversity and estimate ecological parameters while providing an outreach opportunity.     

Compliance with and ecosystem function of biodiversity offsets in North American and European freshwaters

Land-use change via human development is a major driver of biodiversity loss. To reduce these impacts, billions of dollars are spent on biodiversity offsets. However, studies evaluating offset project effectiveness that examine components such as the overall compliance and function of projects remain rare. We reviewed 577 offsetting projects in freshwater ecosystems that included the metrics project size, type of aquatic system (e.g., wetland and creek), offsetting measure (e.g., enhancement, restoration, and creation), and an assessment of the projects’ compliance and functional success. Project information was obtained from scientific and government databases and gray literature. Despite considerable investment in offsetting projects, crucial problems persisted. Although compliance and function were related to each other, a high level of compliance did not guarantee a high degree of function. However, large projects relative to area had better function than small projects. Function improved when projects targeted productivity or specific ecosystem features and when multiple complementary management targets were in place. Restorative measures were more likely to achieve targets than creating entirely new ecosystems. Altogether the relationships we found highlight specific ecological processes that may help improve offsetting outcomes.     

A palaeo-hydrogeological model for arsenic contamination in southern and south-east Asia

An argument is presented in which areas of natural arsenic contamination of modern groundwaters throughout Asia have a common origin. Arsenic originally accumulated in oceanic ferro-manganoan sediments of the eastern Palaeo-Tethys. This was further concentrated through oceanic crustal extinction in what later became the south-east Chinese accreted mineralised terrain. Proto-Himalayan uplift of this area created the palaeo-drainage systems of the Ganges – Brahmaputra, Irrawaddy, Mekong, and Red Rivers, with consequent headwater erosion of arsenic-rich sediments. Their downstream deposition as immature and easily redistributed Neogene sandstones, silts, and iron-rich clays has created secondary and tertiary reservoirs of adsorbed and authigenic arsenic, from which the current arsenic-rich groundwaters have evolved. Considering river basins within the above palaeo-hydrogeological framework provides a basis for assessing the risk of arsenic in groundwater basins of south and south-eastern Asia.     

Cost‐effective conservation of an endangered frog under uncertainty

How should managers choose among conservation options when resources are scarce and there is uncertainty regarding the effectiveness of actions? Well‐developed tools exist for prioritizing areas for one‐time and binary actions (e.g., protect vs. not protect), but methods for prioritizing incremental or ongoing actions (such as habitat creation and maintenance) remain uncommon. We devised an approach that combines metapopulation viability and cost‐effectiveness analyses to select among alternative conservation actions while accounting for uncertainty. In our study, cost‐effectiveness is the ratio between the benefit of an action and its economic cost, where benefit is the change in metapopulation viability. We applied the approach to the case of the endangered growling grass frog (Litoria raniformis), which is threatened by urban development. We extended a Bayesian model to predict metapopulation viability under 9 urbanization and management scenarios and incorporated the full probability distribution of possible outcomes for each scenario into the cost‐effectiveness analysis. This allowed us to discern between cost‐effective alternatives that were robust to uncertainty and those with a relatively high risk of failure. We found a relatively high risk of extinction following urbanization if the only action was reservation of core habitat; habitat creation actions performed better than enhancement actions; and cost‐effectiveness ranking changed depending on the consideration of uncertainty. Our results suggest that creation and maintenance of wetlands dedicated to L. raniformis is the only cost‐effective action likely to result in a sufficiently low risk of extinction. To our knowledge we are the first study to use Bayesian metapopulation viability analysis to explicitly incorporate parametric and demographic uncertainty into a cost‐effective evaluation of conservation actions. The approach offers guidance to decision makers aiming to achieve cost‐effective conservation under uncertainty.     

Parasitic wasps orient to green leaf volatiles

Summary Undamaged plants emit low levels of green leaf volatiles (GLVs), while caterpillar-damaged and artificially damaged plants emit relatively higher levels of certain GLVs. Female braconid parasitoids,Microplitis croceipes, oriented to both damaged plants and to individual GLVs in no-choice tests in a wind tunnel, but seldom oriented to undamaged plants. Female ichneumonid parasitoids,Netelia heroica, also oriented to individual GLVs in a wind tunnel. Males of both wasp species failed to orient to the GLVs. These data show that leaf-feeding caterpillars can cause the release of GLVs, and that parasitic wasps can respond to these odors by flying upwind (chemoanemotactic response), which brings the wasps to their caterpillar hosts. This supports the hypothesis that plants communicate with members of the third trophic level,i.e., plants under herbivore attack emit chemical signals that guide natural enemies of herbivores to sites of plant damage. In this interaction, the GLVs serve as tritrophic plant-to-parasitoid synomones. That parasitoids from two different wasp families oriented to GLVs suggests that the response may be widespread among the Hymenoptera.Mention of a commercial or proprietary product does not constitute an endorsement by the U.S. Department of Agriculture     

Potential for Electropositive Metal to Reduce the Interactions of Atlantic Sturgeon with Fishing Gear

Atlantic sturgeon (Acipenser oxyrhynchus) populations have been declared either endangered or threatened under the U.S. Endangered Species Act. Effective measures to repel sturgeon from fishing gear would be beneficial to both fish and fishers because they could reduce both fishery‐associated mortality and the need for seasonal and area closures of specific fisheries. Some chondrostean fishes (e.g., sturgeons and paddlefishes) can detect weak electric field gradients (possibly as low as 5 Μv/cm) due to arrays of electroreceptors (ampullae of Lorenzini) on their snout and gill covers. Weak electric fields, such as those produced by electropositive metals (typically mixtures of the lanthanide elements), could therefore potentially be used as a deterrent. To test this idea, we recorded the behavioral responses of juvenile Atlantic sturgeon (31–43 cm fork length) to electropositive metal (primarily a mixture of the lanthanide elements neodymium and praseodymium) both in the presence and absence of food stimuli. Trials were conducted in an approximately 2.5 m diameter × 0.3 m deep tank, and fish behaviors were recorded with an overhead digital video camera. Video records were subsequently digitized (x, y coordinate system), the distance between the fish and the electropositive metal calculated, and data summarized by compiling frequency distributions with 5‐cm bins. Juvenile sturgeon showed clear avoidance of electropositive metal but only when food was present. On the basis of our results, we conclude that the electropositive metals, or other sources of weak electric fields, may eventually be used to reduce the interactions of Atlantic sturgeon with fishing gear, but further investigation is needed. El Potencial del Metal Electropositivo para Reducir las Interacciones del Esturión Atlántico con Instrumentos de Pesca Bouyoucos, Bushnell & Brill 13–003     

The role of time and species identities in spatial patterns of species richness and conservation

Many conservation actions are justified on the basis of managing biodiversity. Biodiversity, in terms of species richness, is largely the product of rare species. This is problematic because the intensity of sampling needed to characterize communities and patterns of rarity or to justify the use of surrogates has biased sampling in favor of space over time. However, environmental fluctuations interacting with community dynamics lead to temporal variations in where and when species occur, potentially affecting conservation planning by generating uncertainty about results of species distribution modeling (including range determinations), selection of surrogates for biodiversity, and the proportion of biodiversity composed of rare species. To have confidence in the evidence base for conservation actions, one must consider whether temporal replication is necessary to produce broad inferences. Using approximately 20 years of macrofaunal data from tidal flats in 2 harbors, we explored variation in the identity of rare, common, restricted range, and widespread species over time and space. Over time, rare taxa were more likely to increase in abundance or occurrence than to remain rare or disappear and to exhibit temporal patterns in their occurrence. Space–time congruency in ranges (i.e., spatially widespread taxa were also temporally widespread) was observed only where samples were collected across an environmental gradient. Fifteen percent of the taxa in both harbors changed over time from having spatially restricted ranges to having widespread ranges. Our findings suggest that rare species can provide stability against environmental change, because the majority of species were not random transients, but that selection of biodiversity surrogates requires temporal validation. Rarity needs to be considered both spatially and temporally, as species that occur randomly over time are likely to play a different role in ecosystem functioning than those exhibiting temporal structure (e.g., seasonality). Moreover, temporal structure offers the opportunity to place management and conservation activities within windows of maximum opportunity.     

Main nutrient compounds in food bodies of Mexican <Emphasis Type="Italic">Acacia</Emphasis> ant-plants

Summary. Myrmecophytic plants use obligate ant mutualists as a constitutive indirect defence mechanism. These plants often produce cellular food bodies (FBs) to nourish their resident ants. Lipids, proteins, and even highly specialised compounds such as glycogen have been reported from FBs, but detailed chemical analyses of FB composition have so far been presented only for Southeast Asian Macaranga and Central American Piper myrmecophytes. Here we report the chemical composition of FBs of five myrmecophytic Acacia (Fabaceae) species from Mexico using HPLC (carbohydrates and proteins) and GC-MS (lipids). Feeding experiments revealed no hints on any use of external food sources by the inhabiting Pseudomyrmex ants. These ants obviously rely completely on FBs and extrafloral nectar provided by their hosts. The total content of nutrients in Acacia FBs was 15-25% of FB dry mass, being much lower than in Macaranga or Piper FBs. Proteins were dominating (8-14 % dm) in Acacia FBs and thus were present in higher amounts than in Macaranga FBs, yet in lower amounts than in Piper. Lipids contributed 1-9 % of dry mass, showing a lower proportion than in FBs of Macaranga or Piper. Carbohydrates made up 3-11 % dm, reaching in most Acacia species the same range as observed in Macaranga and in Piper FBs. Water content was 18-24 % of FB fresh mass, and structural tissue obviously made up a much higher proportion in Acacia FBs than in Macaranga or Piper FBs. Both characters might represent an adaptation to producing FBs unprotected at the leaf tips under dry conditions. Acacia FBs contain all amino acids and all fatty acids that are considered essential for insects, and their contents of lipids and proteins are higher than in the leaves from which they are ontogenetically derived. This indicates a putatively adaptive enrichment of nutritionally valuable compounds in structures functioning as ant-food.     

Examining the uncertain origin and management role of martens on Prince of Wales Island,Alaska

Conservation biologists are generally united in efforts to curtail the spread of non‐native species globally. However, the colonization history of a species is not always certain, and whether a species is considered non‐native or native depends on the conservation benchmark. Such ambiguities have led to inconsistent management. Within the Tongass National Forest of Alaska, the status of American marten (Martes americana) on the largest, most biologically diverse and deforested island, Prince of Wales (POW), is unclear. Ten martens were released to POW in the early 1930s, and it was generally believed to be the founding event, although this has been questioned. The uncertainty surrounding when and how martens colonized POW complicates management, especially because martens were selected as a design species for the Tongass. To explore the history of martens of POW we reviewed other plausible routes of colonization; genetically and isotopically analyzed putative marten fossils deposited in the late Pleistocene and early Holocene to verify marten occupancy of POW; and used contemporary genetic data from martens on POW and the mainland in coalescent simulations to identify the probable source of the present‐day marten population on POW. We found evidence for multiple routes of colonization by forest‐associated mammals beginning in the Holocene, which were likely used by American martens to naturally colonize POW. Although we cannot rule out human‐assisted movement of martens by Alaskan Natives or fur trappers, we suggest that martens be managed for persistence on POW. More generally, our findings illustrate the difficulty of labeling species as non‐native or native, even when genetic and paleo‐ecological data are available, and support the notion that community resilience or species invasiveness should be prioritized when making management decisions rather than more subjective and less certain conservation benchmarks.