Variations and interrelationships of foliar hydraulic and photosynthetic traits for Larix gmelinii北大核心CSCD

Aims: Variations and potential trade-offs of leaf hydraulic and photosynthetic traits are essential for assessing and predicting the effect of climate change on tree survival, growth and distribution. Our aims were to examine variations and interrelationships of leaf hydraulic and photosynthetic traits in response to changes in site conditions for Dahurian larch (Larix gmelinii)-a dominant tree species in Chinese boreal forests. Methods: This study was conducted at the Maoershan Forest Ecosystem Research Station. A transect of 27 year-old Dahurian larch plantation was established that consisted of five plots extending from the valley to the ridge of a slope. The predawn leaf water potential (ψpre), area- and mass-based leaf hydraulic conductance (Karea and Kmass, respectively), resistance to embolism capacity (P50), leaf mass per area (LMA), net photosynthetic rate (A), and leaf nitrogen content (N) were measured in August 2016. Important findings: The ψpre, Karea, Kmass, P50, A, LMA, and N all varied significantly among the plots (p < 0.05), indicating significant intra-specific variations in these traits in response to the changes in site conditions. The P50 was significantly (p < 0.05) correlated with ψpre, Karea or Kmass, suggesting that a trade-off between hydraulic efficiency and safety exist within the species to some degree. There were significant (p < 0.05) pairwise correlations between A, LMA, and N. Nevertheless, there was no significant (p < 0.05) correlation between the measured photosynthetic traits and hydraulic traits. We concluded that the intra-specific variations and multiple interrelationships of the leaf hydraulic and photosynthetic traits for the larch reflect the plasticity of its leaf traits and strategies of its survival and growth as a result of its acclimation to diverse site conditions.     

苏门白酒草的繁殖特征与入侵性的关系以及某些进化含义

Conyza sumatrensis （Retz.） E. Walker, a member of Asteraceae, is a highly invasive species. However, its reproduction biology remains poorly known. To understand the role of reproductive traits in successful invasion of the species, we studied several traits of its reproductive system： the miniature capitulum and gynomonoecious sexual system, the biology and phenology of capitula and florets, pollen/ovule ratio, the mating system （selfcompatibility）, flower visitors, physical traits and dispersal potential of achenes, germination potential of achenes from manually pollinated capitula, and the association of these traits with invasiveness. Our study showed that the reproductive traits of autonomous seed production, versatile mating system of self- and cross-pollination, and generalized pollination system might contribute to the species＇ successful invasive capability. The invasiveness was further enhanced by the high and rapid production of achenes, as well as the high percentage, rapid germination rate and high dispersal capability of achenes. It was concluded that in annual or winter-annual weeds, autonomous seed production contributed significantly to the invasiveness of the species.     

Addition of Ptyas nigromarginata （Blyth, 1854） （Squamata： Colubridae） to the Snake Fauna of Thailand with Preliminary Remarks on Its Distribution

The first record of Ptyas nigromarginata(Blyth,1854) from Thailand and a new record from China are reported.Literature and internet sources were searched for previous records of this species to establish its geographic and altitudinal distributions in Asia.The distribution in India was adapted to the new state division of the northern part of the country.The results show that the species almost exclusively occurs in hill evergreen and montane forests of the Himalayan foothills and mountain ranges continuous with it,at altitudes of 1000–2300 m above sea level.     

DNA Barcode ITS Effectively Distinguishes the Medicinal Plant Boerhavia diffusa from Its Adulterants

Boerhavia diffusa (B. diffusa), also known as Punarnava, is an indigenous plant in India and an important component in traditional Indian medicine. The accurate identification and collection of this medicinal herb is vital to enhance the drug’s efficacy and biosafety. In this study, a DNA barcoding technique has been applied to identify and distinguish B. diffusa from its closely-related species. The phylogenetic analysis was carried out for the four species of Boerhavia using barcode candidates including nuclear ribosomal DNA regions ITS, ITS1, ITS2 and the chloroplast plastid gene psbA-trnH. Sequence alignment revealed 26% polymorphic sites in ITS, 30% in ITS1, 16% in ITS2 and 6% in psbA-trnH, respectively. Additionally, a phylogenetic tree was constructed for 15 species using ITS sequences which clearly distinguished B. diffusa from the other species. The ITS1 demonstrates a higher transition/transversion ratio, percentage of variation and pairwise distance which differentiate B. diffusa from other species of Boerhavia. Our study revealed that ITS and ITS1 could be used as potential candidate regions for identifying B. diffusa and for authenticating its herbal products.     

Accidental discovery and isolation of Zika virus in Uganda and the relentless epidemiologist behind the investigations

INTRODUCTION Zika virus(ZIKV)has captured the attention of the world because of its potential to infect neural cells and its teratogenic effects on foetuses and the new born.The virus seems to have various modes of transmission and has been the subject of many reviews in the literature(example,Musso and Gubler,2016,Wang et al.,2016).ZIKV was first isolated in1947 but remained almost innocuous     

SPATIAL DISTRIBUTION OF ZOOPLANKTON IN THE MAIN STEM OF THE MIDDLE YANGTZE RIVER北大核心CSCD

To better understand zooplankton distribution and its relationship with the physical-chemical factors in middle Yangtze River, we collected 20 zooplankton samples from segments at Yichang, Jingzhou, Yueyang, Wuhan and Hukou in October, 2016. A total of 23 species that belong to 13 families and 14 genera were identified, among which 16 species belong to Rotifera, 4 to Copepoda and 3 to Cladocera. Among the five segments, the highest number of zooplankton species was detected at Hukou (9 species), while the lowest was at Yueyang (5 species). The average density at Wuhan (10.94±5.81) ind./L was higher than that at Hukou and the other segments. Rotifers (3.41±0.21) ind./L were dominant in the zooplanktonic community, and Keratella valga, Synchacta atylata and Keratella cochlearis were the dominant species. The average density of copepods (mainly nauplius) was (0.75±0.07) ind./L. Cladocera had the lowest average density. Similarly, the zooplankton biomass at Wuhan was also higher than that at Hukou and the other three segments. Comparing with studies at other segments of Yangtze River, we detected lower zooplankton diversity in our investigation. Spearman correlations indicated that the biomass and diversity of zooplankton were significantly and positively correlated (P<0.05) to chlorophyll a. © 2019, Institute of Hydrobiology, Chinese Academy of Sciences. All rights reserved.     

Carbon storage and potentials of the broad-leaved forest in alpine region of the Qinghai-Xizang Plateau,China北大核心CSCD

Aims Our objective was to explore the vegetation carbon storages and their variations in the broad-leaved forests in the alpine region of the Qinghai-Xizang Plateau that includes Qinghai Province and Xizang Autonomous Region. Methods Based on forest resource inventory data and field sampling, this paper studied the carbon storage, its sequestration rate, and the potentials in the broad-leaved forests in the alpine region of the Qinghai-Xizang Plateau. Important findings The vegetation carbon storage in the broad-leaved forest accounted for 310.70 Tg in 2011, with the highest value in the broad-leaved mixed forest and the lowest in Populus forest among the six broad-leaved forests that include Quercus, Betula, Populus, other hard broad-leaved species, other soft broad-leaved species, and the broadleaved mixed forest. The carbon density of the broad-leaved forest was 89.04 Mg•hm2, with the highest value in other hard broad-leaved species forest and the lowest in other soft broad-leaved species forest. The carbon storage and carbon density in different layers of the forests followed a sequence of overstory layer > understory layer > litter layer > grass layer > dead wood layer, which all increased with forest age. In addition, the carbon storage of broad-leaved forest increased from 304.26 Tg in 2001 to 310.70 Tg in 2011. The mean annual carbon sequestration and its rate were 0.64 Tg•a1 and 0.19 Mg•hm2•a1, respectively. The maximum and minimum of the carbon sequestration rate were respectively found in other soft broad-leaved species forest and other hard broad-leaved species forest, with the highest value in the mature forest and the lowest in the young forest. Moreover, the carbon sequestration potential in the tree layer of broad-leaved forest reached 19.09 Mg•hm2 in 2011, with the highest value found in Quercus forest and the lowest in Betula forest. The carbon storage increased gradually during three inventory periods, indicating that the broad-leaved forest was well protected to maintain a healthy growth by the forest protection project of Qinghai Province and Xizang Autonomous Region.     

Chloroplast DNA Underwent Independent Selection from Nuclear Genes during Soybean Domestication and Improvement

正The chloroplast is one of the most important organs in plants because of its essential role in photosynthesis.Studies have shown that the chloroplast was once a free-living cyanobacteria and was integrated into the host species through endosymbiosis(Goksoyr.1967).after which a large number of its genes had been donated to the host nuclear genome(Heins and     

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.     

Potential Risk of Hybridization in Ex Situ Collections of Two Endangered Species of Sinojackia Hu （Styracaceae）

Spontaneous hybridization in ex situ facilities can undermine the genetic integrity of ex situ collections and potentially contaminate open-pollinated seeds or seedlings destined for the reintroduction of endangered plant species into the wild. In the present study, the potential risk of hybridization between two endangered Chinese endemic species, namely Sinojackia xylocarpa Hu and S. rehderiana Hu, which are naturally allopatric species but were conserved ex situ in Wuhan Botanical Garden （WBG）, Wuhan, China, were investigated over three consecutive years from 2003 to 2005. The entire overlapping flowering period of the two species was 14-20 d and the two species shared the same pollinator insects during the entire flowering season in WBG. The floral isolation between the two species was not an issue in the ex sltu collection at WBG. The results suggest an opportunity for pollen transfer between species and a potential risk of genetic Introgression and loss of genetic identity of open-pollinated seeds produced in the ex sltu Collection of these two endangered species. An artificial reciprocal cross between S xylocarpa and S. rehderlana confirmed that the two congener species could readily set seeds, indicating no post-pollination barriers to hybridization and the importance of spatial isolation as a barrier to inter-specific crossing. Therefore, to manage these crossable species with overlapping flowering times and shared pollination vectors in ex situ facilities, spatial isolation should be carefully considered to minimize the possibility of spontaneous hybridization.     

Impacts of variability of habitat factors on species composition of ichthyoplankton and distribution of fish spawning ground in the Changjiang River estuary and its adjacent waters

During June, August and October 2006, there were three multi-disciplinary surveys carried out in the Changjiang River estuary and its adjacent waters (122°00′–125°00′E, 27°50′–34°00′N) by R/V Beidou to study the species composition and abundance of ichthyoplankton (including fish eggs, larvae and juveniles), the spatial distribution of fish spawning ground and their relationship with habitat factors. There were 29, 29 and 25 grid stations sampled in the three cruises, respectively. The ichthyoplankton samples were collected by horizontally towing with a macro-plankton net (mouth diameter 80 cm, length 270 cm, mesh size 0.50 mm) at the sea surface, and the towing speed was 3.0 n mile/h at each sampling station. The towing lasted for 10 min. After hauling for each station, habitat factors including temperature and salinity were measured by Sea Bird-25 CTD. Samples were preserved in 5% formaldehyde solution immediately after sampling for analysis in laboratory. Since the trawl speed could not be accurately evaluated due to the effects of ocean currents and wind-induced wave, the amount of ichthyoplankton was evaluated by actual number of the sampling haul in each station. Ichthyoplankton collected were divided into three categories: dominant species, important species and main species by the index of relative importance (IRI). There are 71 species (including 1200 fish eggs and 2575 fish larvae and juveniles) collected during 3 cruises and 59 species have been correctly identified to species level, which belongs to 50 genera, 37 families and 9 orders; while 5 species can only be identified to genera level, 1 species only identified to family level and 6 species identified to order level. These 59 species identified to species level and 5 species identified to genera level are divided into three ecological patterns, i.e., brackish water species, neritic water species and coastal water species. Warm water species have 34 species in those 59 species identified to species level, accounting for 57.63%, warm temperature species have 25 species, accounting for 42.37%. According to the analysis of IRI, the dominant species are Engraulis japonicus (in June and August, that is important species in October), Scomber japonicus (in August), and Johnius grypotus (in October) during the survey; important species are Cynoglossus joyneri (in June and August), Trichiurus lepturus (in June, August and October), Gonorhynchus abbreviatus (in August), Stolephorus commersonii (in October), Saurida undosquamis (in October) and Saurida elongate (in October), and main species have 12 species in June, 9 species in August and 10 species in October, respectively. The amount of fish eggs and larvae of the dominant species, important species and main species (28 species) are 97.50% and 97.13% of the total amount of fish eggs and larvae, respectively, which are the important composition of fish eggs and larvae in the Changjiang River estuary and its adjacent waters.
In June and August of 2006, if compared with that in corresponding months in 1986, there are great changes in the habitat factors especially for temperature and salinity in the investigating areas: high salinity water from off-shelf is much closer to the coastal areas which results in the dramatic increase of sea surface salinity during all three surveys. Sea surface temperature, on the other hand, decreases distinctively in June, but significantly increases in August. The run-off of the Changjiang River greatly reduced due to the long-term drought in summer 2006, which is responsible for the great changes of habitat factors in the Changjiang River estuary and its adjacent waters. The habitat of the Changjiang River estuary is greatly changed, which consequently has significant influences on the spawning, breeding and the spatial distribution of spawning ground of neritic water species, such as Sardinella zunasi, Thryssa kammalensis, Thryssa mystax, Setipinna taty and S. commersonii ect, and coastal water species, such as Ilisha elongate and Konosirus punctatus ect.     

Environmental friendly nitrogen fertilization

With the huge intensification of agriculture and the increasing awareness to human health and natural resources sustainability, there was a shift towards the development of environmental friendly N application approaches that support sustainable use of land and sustain food production. The effectiveness of such approaches depends on their ability to synchronize plant nitrogen demand with its supply and the ability to apply favored compositions and dosages of N-species. They are also influenced by farming scale and its sophistication, and include the following key concepts: (i) Improved application modes such as split or localized ("depot") application; (ii) use of bio-amendments like nitrification and urease inhibitors and combinations of (i) and (ii); (iii) use of controlled and slow release fertilizers; (iv) Fertigation-fertilization via irrigation systems including fully automated and controlled systems; and (v) precision fertilization in large scale farming systems. The paper describes the approaches and their action mechanisms and examines their agronomic and environmental significance. The relevance of the approaches for different farming scales, levels of agronomic intensification and agro-technical sophistication is examined as well.     

Impacts of variability of habitat factors on species composition of ichthyoplankton and distribution of fish spawning ground in the Changjiang River estuary and its adjacent waters

During June, August and October 2006, there were three multi-disciplinary surveys carried out in the Changjiang River estuary and its adjacent waters (122°00′–125°00′E, 27°50′–34°00′N) by R/V Beidou to study the species composition and abundance of ichthyoplankton (including fish eggs, larvae and juveniles), the spatial distribution of fish spawning ground and their relationship with habitat factors. There were 29, 29 and 25 grid stations sampled in the three cruises, respectively. The ichthyoplankton samples were collected by horizontally towing with a macro-plankton net (mouth diameter 80 cm, length 270 cm, mesh size 0.50 mm) at the sea surface, and the towing speed was 3.0 n mile/h at each sampling station. The towing lasted for 10 min. After hauling for each station, habitat factors including temperature and salinity were measured by Sea Bird-25 CTD. Samples were preserved in 5% formaldehyde solution immediately after sampling for analysis in laboratory. Since the trawl speed could not be accurately evaluated due to the effects of ocean currents and wind-induced wave, the amount of ichthyoplankton was evaluated by actual number of the sampling haul in each station. Ichthyoplankton collected were divided into three categories: dominant species, important species and main species by the index of relative importance (IRI). There are 71 species (including 1200 fish eggs and 2575 fish larvae and juveniles) collected during 3 cruises and 59 species have been correctly identified to species level, which belongs to 50 genera, 37 families and 9 orders; while 5 species can only be identified to genera level, 1 species only identified to family level and 6 species identified to order level. These 59 species identified to species level and 5 species identified to genera level are divided into three ecological patterns, i.e., brackish water species, neritic water species and coastal water species. Warm water species have 34 species in those 59 species identified to species level, accounting for 57.63%, warm temperature species have 25 species, accounting for 42.37%. According to the analysis of IRI, the dominant species are Engraulis japonicus (in June and August, that is important species in October), Scomber japonicus (in August), and Johnius grypotus (in October) during the survey; important species are Cynoglossus joyneri (in June and August), Trichiurus lepturus (in June, August and October), Gonorhynchus abbreviatus (in August), Stolephorus commersonii (in October), Saurida undosquamis (in October) and Saurida elongate (in October), and main species have 12 species in June, 9 species in August and 10 species in October, respectively. The amount of fish eggs and larvae of the dominant species, important species and main species (28 species) are 97.50% and 97.13% of the total amount of fish eggs and larvae, respectively, which are the important composition of fish eggs and larvae in the Changjiang River estuary and its adjacent waters.
In June and August of 2006, if compared with that in corresponding months in 1986, there are great changes in the habitat factors especially for temperature and salinity in the investigating areas: high salinity water from off-shelf is much closer to the coastal areas which results in the dramatic increase of sea surface salinity during all three surveys. Sea surface temperature, on the other hand, decreases distinctively in June, but significantly increases in August. The run-off of the Changjiang River greatly reduced due to the long-term drought in summer 2006, which is responsible for the great changes of habitat factors in the Changjiang River estuary and its adjacent waters. The habitat of the Changjiang River estuary is greatly changed, which consequently has significant influences on the spawning, breeding and the spatial distribution of spawning ground of neritic water species, such as Sardinella zunasi, Thryssa kammalensis, Thryssa mystax, Setipinna taty and S. commersonii ect, and coastal water species, such as Ilisha elongate and Konosirus punctatus ect.     

A study on the diversity of the moth community in the North Dagang Wetland Nature Reserve, Tianjin, China

North Dagang Wetland Nature Reserve is situated in the southeast of Tianjin, China. It is the biggest wetland nature reserve in Tianjin. An investigation into the diversity of the moth community was carried out from June 12 to October 14, 2001, and then from April 12 to October 30, 2002. The investigation was conducted in order to identify the moth species in wetlands, study their dynamic laws, select indicator insects to indicate wetland quality, and assist in the study and protection of wetlands. A total of 132 moth species were collected, which belonged to 105 genera in 17 families. The data about insects which was collected during the survey was listed and constructed in terms of species-abundance, indices of diversity (H′), evenness (J′), specific richness (S) and individual number (N) using the Microsoft Excel Program. The status of the wetlands was also analyzed using the moth community species-abundance curve, indices of diversity, the connection between evenness and richness indices, etc. The results show that moth species in the wetlands were not rich, and the moth community was unstable. In other words, the living environment of the moths and the environmental quality of the wetlands were unstable due to the serious degeneration of the wetland environment and human activities.     

Monitoring of algae in Dutch rivers: does it meet its goals?

Observations on phytoplankton in the lower reaches of the rivers Rhine and Meuse were carried out in the framework of the national monitoring programme of the main water systems in The Netherlands. Seasonal changes in density and species composition were analysed to detect the major variables and to discuss whether this monitoring meets its goals. Phytoplankton reached peak densities of 140 and 65 μg L^-1 Chl a in the Rhine and Meuse, respectively. Overall density was correlated with seasonal variation in water discharge rather than with nutrient concentrations, that are high in both rivers. The position of sampling sites in relation to the downstream development of the plankton was very important, especially in the regulated Meuse. Despite hydrographic and chemical differences between the two rivers, many species, predominantly diatoms and green algae, were shared. The occurrence of the diatom Skeletonema subsalsum in the Rhine and the green algae Neodesmus danubialis, Micractinium pusillum and Pseudotetrastrum punctatum in the Meuse (1992, but not 1996) was interpreted as a feature related, respectively, to the high salinity of the Rhine and specific riverine conditions of the Meuse. In general the potamoplankton was characterised as an opportunistic assemblage exploiting the high nutrient contents and disturbed hydrography of both rivers. Nevertheless, the phytoplankton contributed to the characterisation of the present river communities, ones that differ rather strongly in historic records. Although year to year variation in phytoplankton density is mainly related to variation in water discharge, phytoplankton biomass did increase as a result of eutrophication over a period of decades. The widespread nature of many of today's potamoplankton species in the two rivers render these organisms less useful for indicating short-term changes in water quality. However the importance of phytoplankton in trophic relationships merits its inclusion in the monitoring of riverine ecosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.     

Pea leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) uses vibrational duets for efficient sexual communication

The pea leafminer (Liriomyza huidobrensis) is a notorious pest of vegetables and ornamental plants worldwide. Despite a large number of studies on its biology and ecology, the courtship behavior and sexual communication of this species remain unclear. Here, we studied vibrational communication in the sexual interaction of the pea leafminer. On host plant leaves, females and males behaviorally displayed the bobbing-quivering alternation, which finally led to copulation. Moreover, records of laser vibrometry revealed threesignal duets underlying the behavioral alternation. Sexually mature males spontaneously emitted calls (MCs) to initiate the duets. The females rapidly responded to MCs by emitting replies (FRs) that are longer in duration. The FRs further triggered male replies (MRs) in their search for potential partners. Leafminer-produced vibrational signals convey efficient information to partners and generate pair formation on stretched substrates, such as plant leaves and nylon mesh, but cannot elicit responses on dense substrates, such as glass and plastic. Vibrational playbacks of both MCs and FRs can elicit replies in females and males, respectively. This study completely characterizes substrate-borne vibrational duets in a dipteran insect. The discovery of vibrational sex signals in the pea leafminer provides new insights for the development of novel approaches to control the pest and its relative species.     

Genetic Analysis of Multiple Paternity in an Endangered Ovoviviparous Lizard Shinisaurus crocodilurus

The crocodile lizard(Shinisaurus crocodilurus) is an ovoviviparous lizard belonging to a monotypic family that originated during the end of the quaternary ice age. A rare species in the wild, the crocodile lizard was listed in CITES Appendix II. Knowledge of the reproductive biology and mating system of this species is important for designing conservation strategies and improving genetic variation. To investigate the paternity of the crocodile lizards and to interpret their reproductive behaviour, we collected saliva from females, potential fathers and offspring in a seminatural enclosure experiment and analyzed the paternity of the crocodile lizard using 12 microsatellite genetic loci. The overall observed incidence of multiple paternity was 42.9%(6 of 14 clutches) and Fis was 0.089 ± 0.056. These results indicate that the primary mating mode of the crocodile lizard is that males are polygynous while with females are polyandrous, and there is multiple paternity among offspring of the same mother.