Widely targeted metabolomics and flavoromics reveal the effect of Wickerhamomyces anomalus fermentation on the volatile and nonvolatile metabolites of black garlic juice.

Black garlic has a variety of biological activities, but many consumers cannot accept it because of the garlic odor and the bitter taste. In this study, fermentation with yeast Wickerhamomyces anomalus was adopted to improve the flavor of black garlic juice. Although fermentation reduced antioxidant activities, the garlicky odor and bitter taste were weakened. Metabolomic analysis revealed 141 metabolites were significantly differentially regulated. The upregulated metabolites were mainly related to nucleotides, organic acids and their derivatives, while the downregulated metabolites were mainly related to amino acids, lipids and their derivatives. Flavoromics analysis revealed that 137 metabolites were significantly differentially regulated, particularly garlicky and pungent volatiles were significantly downregulated. Correlation analysis indicated that esters are most closely related to nonvolatile metabolites, and lipids degradation was significantly correlated with volatiles. The results indicated that W. anomalus fermentation is an effective strategy to improve the flavor of black garlic juice.

Effect of peach gum polysaccharide on the rheological and 3D printing properties of gelatin-based functional gummy candy.

Excellent 3D printing materials must exhibit good extrudability and supportability, but these two characteristics are often contradictory. In this study, peach gum polysaccharide (PGP) was added to gelatin to prepare a 3D-printed functional gummy candy encapsulating curcumin. Rheology tests indicated that adding PGP could effectively improve the apparent viscosity and thermal stability and consequently improve the 3D printability and supportability of the products. When PGP addition was 6 %, the printing accuracy was higher than 90 %. Texture and microstructure analysis further revealed that PGP addition promoting a dense gel structure formed and the water holding capacity and supportability of gel materials were enhanced. Furthermore, the in vitro gastrointestinal digestion tests showed that after 6 h of simulated gastrointestinal fluid digestion, the retention rate of curcumin was nearly 80 %. The above results indicated that the composite gel of PGP and gelatin is a good 3D printing base material for nutrient delivery.

Effects of grafting methods and raw materials on the physicochemical properties and biological activities of phenolic acids grafted oat ß-glucan.

Phenolic acids are commonly used as food biological preservatives. Grafting phenolic acids onto polysaccharides could effectively enhance their biological activities and environmental stability to varying degrees. However, grafting methods and raw materials could affect the physical properties and biological activities of the phenolic acid-grafted polysaccharides. In this study, caffeic acid (CA) and gallic acid (GA) were grafted onto oat ß-glucan (OG) and hydrolyzed oat ß-glucan (OGH) through N,N'-carbonyldiimidazole-mediated (CDI) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride coupling N-hydroxysuccinimide (EDC/NHS) methods. Graft modification decreased the crystallinity and thermal stability of the conjugates, but retained good bioactivities for the conjugates. The antioxidant and bacteriostatic activities of the conjugates prepared by the EDC method were better than those of the CDI method, and the OGH-conjugates showed better biological activities than OG-conjugates. EDC-GAOGH showed best DPPH (89.78%) and ABTS (92.32%) scavenging activities. The inhibitory effect of EDC-GAOGH on Escherichia coli was significantly better than that of EDC-CAOGH, but for Staphylococcus aureus, the results are opposite, which indicating that different phenolic acid grafting products have different inhibitory effects on pathogenic microbes. In general, grafting phenolic acids onto OGH using EDC method is an effective strategy for preparing food biological preservative.

Formation, Evolution, and Antioxidant Activity of Melanoidins in Black Garlic under Different Storage Conditions.

Melanoidins (MLDs) are formed through the reaction of carbonyl compounds and amino compounds in the Maillard reaction (MR) during the heating or storage of food. In this study, the formation, chemical composition, and structural characteristics of black garlic (BG) MLDs stored at different temperatures (4 °C, 20 °C, and 35 °C) over a period of 6 months were investigated. The initial products of the MR formed more often at 4 °C and 20 °C, while higher temperatures (35 °C) promoted the reaction in the middle and late stages of the MR. The higher temperature promoted an increase in molecular weight and MLD content, which can be attributed to the increase in protein and phenolic content. Elemental analysis confirmed an increase in nitrogen (N) content and the continuous incorporation of nitrogen-rich substances into the skeleton. Amino acids, particularly aspartic acid and threonine, were the primary N-containing compounds involved in MLD formation. Additionally, the infrared analysis revealed that the changes in MLDs during storage were characterized by amide I and amide II groups. The MR enhanced the yields of heterocyclic compounds (from 56.60% to 78.89%), especially that of O-heterocyclic compounds, at the higher temperature according to Py-GC-MS analysis. Furthermore, the higher temperature enhanced the molecular weight, maximum height, and roughness of MLDs compared to the control. The antioxidant ability of MLDs was positively correlated with storage temperatures. In summary, temperature had an impact on the formation, evolution, and antioxidant activity of MLDs.

Identification of potential hub genes linked to immune and metabolic alterations in postoperative systemic inflammatory dysregulation.

Background: Postoperative systemic inflammatory dysregulation (PSID) is characterised by strongly interlinked immune and metabolic abnormalities. However, the hub genes responsible for the interconnections between these two systemic alterations remain to be identified. Methods: We analysed differentially expressed genes (DEGs) of individual peripheral blood nucleated cells in patients with PSID (n = 21, CRP > 250 mg/L) and control patients (n = 25, CRP < 75 mg/L) following major abdominal surgery, along with their biological functions. Correlation analyses were conducted to explore the interconnections of immune-related DEGs (irDEGs) and metabolism-related DEGs (mrDEGs). Two methods were used to screen hub genes for irDEGs and mrDEGs: we screened for hub genes among DEGs via 12 algorithms using CytoHubba in Cytoscape, and also screened for hub immune-related and metabolic-related genes using weighted gene co-expression network analysis. The hub genes selected were involved in the interaction between changes in immunity and metabolism in PSID. Finally, we validated our results in mice with PSID to confirm the findings. Results: We identified 512 upregulated and 254 downregulated DEGs in patients with PSID compared with controls. Gene enrichment analysis revealed that DEGs were significantly associated with immune- and metabolism-related biological processes and pathways. Correlation analyses revealed a close association between irDEGs and mrDEGs. Fourteen unique hub genes were identified via 12 screening algorithms using CytoHubba in Cytoscape and via weighted gene co-expression network analysis. Among these, CD28, CD40LG, MAPK14, and S100A12 were identified as hub genes among both immune- and metabolism-related genes; these genes play a critical role in the interaction between alterations in immunity and metabolism in PSID. The experimental results also showed that the expression of these genes was significantly altered in PSID mice. Conclusion: This study identified hub genes associated with immune and metabolic alterations in patients with PSID and hub genes that link these alterations. These findings provide novel insights into the mechanisms underlying immune and metabolic interactions and new targets for clinical treatment can be proposed on this basis.

Effect of honeysuckle leaf extract on the physicochemical properties of carboxymethyl konjac glucomannan/konjac glucomannan/gelatin composite edible film.

Honeysuckle leaves are rich in bioactive ingredients, but often considered as agro-wastes. In this study, honeysuckle leaf extract (HLE) was added to the carboxymethyl konjac glucomannan/konjac glucomannan/gelatin composite edible film (CMKH). Compared to films without HLE addition (CMK), the water vapor barrier properties of CMKH slightly decreased, but the transmittance of the CMKH films in UV region (200-400 nm) as low as zero. The elongation at break of CMKH film was 1.39 âˆ¼ 1.5 fold higher than those of CMK films. The DPPH and ABTS scavenging activity of CMKH-Ⅱ was 85.75% and 90.93%, respectively, which is similar to the equivalent content of Vc. The inhibition rate of CMKH-Ⅰ and CMKH-Ⅱ against Escherichia coli and Listeria monocytogenes were close to 90%, and the inhibition rate against Staphylococcus aureus were up to 96%. The results emphasized that the composite film containing 25% (v/v) HLE has potential application value in food preservation.

Directional Activated Exciton Highway via Fractal Electric Field Modulation for Ultrasensitive Carbon Nanotube-Based Sensors.

The electrical vapor sensor based on carbon nanotubes (CNTs) has attracted wide attention due to its excellent conductivity, stable interfacial structure, and low dimensional quantum effects. However, the conductivity and contact interface activity were still limited by the random distribution of coated CNTs, which led to limited performance. We developed a new strategy to unify the CNT directions with image fractal designing of the electrode system. In such a system, directional aligned CNTs were gained under a well-modulated electric field, leading to microscale CNT exciton highways and molecule-scale host-guest site activation. The carrier mobility of the aligned CNT device is 20-fold higher than that of the random network CNT device. With excellent electrical properties, such modulated CNT devices based on fractal electrodes behave as an ultrasensitive vapor sensor for methylphenethylamine, a mimic of illicit drug methamphetamine. The detection limit reached as low as 0.998 ppq, 6 orders of magnitude sensitive than the reported 5 ppb record based on interdigital electrodes with random distributed CNTs. Since the device is easily fabricated in wafer-level and compatible with the CMOS process, such a fractal design strategy for aligned CNT preparation will be widely applied in a variety of wafer-level electrical functional devices.

Simultaneously enhanced stability and biological activities of chlorogenic acid by covalent grafting with soluble oat ß-glucan.

Chlorogenic acid (CA) has a wide range of biological activities but the chemical structure is extremely unstable. In this study, CA was grafted onto a soluble oat ß-glucan (OßGH) to improve the stability. Although the crystallinity and thermal stability of CA-OßGH conjugates reduced, the storage stability of CA significantly improved. The DPPH and ABTS scavenging ability of CA-OßGH IV (graft ratio 285.3 mg CA/g) were higher than 90 %, which is closed to activities of equivalent concentration of Vc (93.42 %) and CA (90.81 %). The antibacterial abilities of CA-OßGH conjugates are improved compared to the equivalent content of CA and potassium sorbate. Particularly, the inhibition rate of CA-OßGH for gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes) are significantly higher than that of gram-negative bacteria (Escherichia coli). The results demonstrated that covalent grafted CA with soluble polysaccharide is an effective strategy to enhance its stability and biological activities.

Preparation of garlic stem cellulose nanocrystal/leaf extract/chitosan film for black garlic preservation by electrostatic spraying.

In this study, a novel active chitosan (CH) packaging film that incorporates garlic leaf extract (GL) and stem cellulose nanocrystals (CNC) was prepared. The addition of CNC to the CH film increased its tensile strength, hydrophilicity, thermal stability, and water/oxygen barrier and decreased its water contact angle and weight-loss rate, while the addition of GL greatly enhanced its antioxidant and antibacterial activities. SEM and AFM analyses showed that the CNC agglomerates and deposits in the lower layer and the surface roughness of the film was the highest at 1.2 % concentration. The optimal composition of the film was determined to be 0.8 % CNC and 4 % GL by the fuzzy mathematics evaluation method. Then, black garlic was preserved with the optimized coating by electrostatic spraying and was found to slow water loss and migration, while its excellent antioxidant activities decreased the degree of browning during 90 d of storage.

Characterization of whey protein isolate-(-)-epigallocatechin-3-gallate conjugates prepared by non-enzymatic and enzymatic methods and their application in stabilizing ß-carotene emulsion.

(-)-Epigallocatechin-3-gallate (EGCG) was conjugated to whey protein isolate (WPI) using alkaline treatment, free radical grafting and tyrosinase-catalyzed oxidation. The structure and properties of the obtained conjugates and their application as antioxidant emulsifiers in stabilizing ß-carotene emulsion were systematically characterized. Fourier transform infrared spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis verified the covalent linking between WPI and EGCG. The highest grafting efficiency was obtained for enzyme conjugate, followed for alkaline conjugate. Conjugation of EGCG decreased the α-helix content and fluorescence intensity of protein, and these changes depend on both EGCG conjugation and the cross-linking methods. Due to their improved emulsifying properties and antioxidant activity, WPI-EGCG conjugates formed smaller emulsion droplets and protect the encapsulated ß-carotene more effectively, and the protective property is positively correlated with EGCG content in conjugates.

Use of Transcriptomics to Reveal the Joint Immunotoxicity Mechanism Initiated by Difenoconazole and Chlorothalonil in the Human Jurkat T-Cell Line.

It is very important to evaluate the immunotoxicity and molecular mechanisms of pesticides. In this study, difenoconazole and chlorothalonil were evaluated for immunotoxicity by using the human Jurkat T-cell line, and the EC50 were 24.66 and 1.17 mg/L, respectively. The joint exposure of difenoconazole and chlorothalonil showed a synergistic effect at low concentrations (lower than 10.58 mg/L) but an antagonistic effect at high concentrations (higher than 10.58 mg/L). With joint exposure at a concentration of EC10, the proportion of late apoptotic cells was 2.26- and 2.91-fold higher than that with exposure to difenoconazole or chlorothalonil alone, respectively. A transcriptomics analysis indicated that the DEGs for single exposure are associated with immunodeficiency disease. Single exposure to chlorothalonil was mainly involved in cation transportation, extracellular matrix organization, and leukocyte cell adhesion. Single exposure to difenoconazole was mainly involved in nervous system development, muscle contraction, and immune system processes. However, when the joint exposure dose was EC10, the DEGs were mainly involved in the formation of cell structures, but the DEGs were mainly involved in cellular processes and metabolism when the joint exposure dose was EC25. The results indicated that the immunotoxicological mechanisms underlying joint exposure to difenoconazole and chlorothalonil are different under low and high doses.

Comparative Transcriptome Analysis to Investigate the Immunotoxicity Mechanism Triggered by Dimethomorph on Human Jurkat T Cell Lines.

Dimethomorph (DMM) is a broad-spectrum fungicide used globally in agricultural production, but little is known regarding the immunotoxicity of DMM in humans. In this study, the immunotoxicity of DMM on human Jurkat T cells was evaluated in vitro. The results indicated that the half-effective concentration (EC50) of DMM for Jurkat cells was 126.01 mg/L (0.32 mM). To further elucidate the underlying mechanism, transcriptomics based on RNA sequencing for exposure doses of EC25 (M21) and EC10 (L4) was performed. The results indicated that compared to untreated samples (Ctr), 121 genes (81 upregulated, 40 downregulated) and 30 genes (17 upregulated, 13 downregulated) were significantly differentially regulated in the L4 and M21 samples, respectively. A gene ontology analysis indicated that the significantly differentially expressed genes (DEGs) were mostly enriched in the negative regulation of cell activities, and a KEGG pathway analysis indicated that the DEGs were mainly enriched in the immune regulation and signal transduction pathways. A quantitative real-time PCR for the selected genes showed that compared to the high-dose exposure (M21), the effect of the low-dose DMM exposure (L4) on gene expression was more significant. The results indicated that DMM has potential immunotoxicity for humans, and this toxicity cannot be ignored even at low concentrations.

Single nucleotide polymorphism-based analysis of the genetic structure of the Min pig conserved population.

OBJECTIVE: The study aims to uncover the genetic diversity and unique genetic structure of the Min pig conserved population, divide the nucleus conservation population, and construct the molecular pedigree. METHODS: We used KPS Porcine Breeding Chip v1 50K for SNP detection of 94 samples (31♂, 63♀) in the Min pig conserved population from Lanxi breeding Farm. RESULTS: The polymorphic marker ratio (PN), the observed heterozygosity (Ho), and the expected heterozygosity (He) were 0.663, 0.335, and 0.330, respectively. The pedigree-based inbreeding coefficients (FPED) was significantly different from those estimated from runs of homozygosity (FROH) and single nucleotide polymorphism (FSNP) based on genome. The Pearson correlation coefficient between FROH and FSNP was significant (p<0.05). The effective population content (Ne) showed a continuously decreasing trend. The rate of decline was the slowest from 200 to 50 generations ago (r = 0.95), then accelerated slightly from 50 to 5 generations ago (1.40

The combined effect of protein hydrolysis and Lactobacillus plantarum fermentation on antioxidant activity and metabolomic profiles of quinoa beverage.

Lactic acid bacteria fermentation is a commonly applied technique to produce nutritional, functional, and organoleptic enhanced foods. In the present study, protein hydrolysis and Lactobacillus plantarum fermentation were coupled to develop quinoa beverages. Protein hydrolysis effectively promoted the growth and fermentation of L. plantarum. Fermentation alone did not significantly improve antioxidant activity, but the combined use of protein hydrolysis and L. plantarum fermentation significantly improved the antioxidant activity of the quinoa beverage. Nontargeted metabolomics based on UHPLC-Q Exactive HF-X/MS and multivariate statistical analysis were performed to reveal the metabolite profile alterations of the quinoa beverage by different processing methods. A total of 756 metabolites were identified and annotated, which could be categorized into 12 different classes. The significant differentially abundant metabolites were mainly involved in primary metabolite metabolism and secondary metabolite biosynthesis. Many of these metabolites were proven to be vitally important to the function and taste formation of the quinoa beverage. Most importantly, the coupled use of protein hydrolysis and L. plantarum fermentation significantly increased some functional ingredients compared with protein hydrolysis and L. plantarum fermentation alone. The above results indicate that protein hydrolysis coupled with L. plantarum fermentation is an effective strategy to develop functional quinoa beverages.

The Efficiency of Lemon Essential Oil-Based Nanoemulsions on the Inhibition of Phomopsis sp. and Reduction of Postharvest Decay of Kiwifruit.

Essential oils (EOs) have excellent antibacterial activity and are generally recognized as safe (GRAS) for use in food preservatives. However, the application of EOs is limited because of their strong volatility and easily oxidized. Encapsulation of EOs into nanoemulsions could effectively prevent oxidative deterioration. In this study, lemon essential oil-based nanoemulsion (LEO/NE) was prepared by high-pressure homogenization. FT-IR and encapsulation efficiency analysis indicated that LEO was effectively encapsulated in the nanoemulsion. The results of zeta potential changes after 35 d storage indicated that LEO/NE exhibits good stability at room temperature. The effect of LEO/NE on the main soft rot pathogens of kiwifruit Phomopsis sp. was investigated, and the results showed that LEO/NE significantly inhibited spore germination and mycelia growth of Phomopsis sp. by promoting ROS accumulation, intracellular antioxidant enzyme activities, and cell apoptosis. The preservation experiment was carried out by inoculating Phomopsis sp. spores into fresh kiwifruit, and the LEO/NE effectively inhibited soft rot development in kiwifruit in a LEO dose dependent manner. LEO/NE with 1% LEO loading amount has a good effect on preventing postharvest decay of kiwifruit caused by Phomopsis sp.

Encapsulation of Zanthoxylum bungeanum essential oil to enhance flavor stability and inhibit lipid oxidation of Chinese-style sausage.

BACKGROUND: Zanthoxylum bungeanum essential oil (ZBEO) is a popular seasoning, commonly used in the food industry. It contains many easily degraded and highly volatile bioactive substances. Control of the stability of the bioactive substances in ZBEO is therefore very important in the food industry. RESULTS: In this study, microencapsulation was applied to improve ZBEO stability. The key parameters for microcapsule preparation were optimized by the Box-Behnken design method, and the optimum conditions were as follows: ratio of core to wall, 1:8; ratio of hydroxypropyl-α-cyclodextrin (HPCD) to soy protein isolate (SPI), 4; total solids content, 12%; and homogenization speed, 12 000 rpm. Antioxidant experiments have indicated that tea polyphenols (TPPs) effectively inhibited hydroxy-α-sanshool degradation in ZBEO microcapsules. Application of ZBEO microcapsules in Chinese-style sausage effectively inhibited lipid oxidation in sausages and protected hydroxy-α-sanshool and typical volatiles from volatilization and degradation during sausage storage. CONCLUSION: The results suggested that ZBEO microencapsulation is an effective strategy for improving the stability of its bioactive components and flavor ingredients during food processing. © 2022 Society of Chemical Industry.

Effect of cooking methods on the edible, nutritive qualities and volatile flavor compounds of rabbit meat.

BACKGROUND: Rabbit meat is a good edible meat source with high nutritional values. Cooking has a significant impact on the edible properties, nutritional qualities and flavor characteristics of meat. Studying the effect of cooking methods on rabbit meat qualities could encourage more understanding and acceptance of rabbit meat by consumers, and could also provide some reference for rabbit meat processing. Therefore, the effects of boiling, sous-vide cooking, steaming, microwaving, roasting, frying and pressure cooking on the edible, nutritive and volatile qualities of rabbit meat were investigated. RESULTS: The sous-vide cooked rabbit meat sample showed higher moisture content, water-holding capacity and lower cooking losses than other samples, but the results of roasted rabbit meat sample were the opposite, and scanning electron microscopy observations also verified the results. There was no significant difference in 2-thiobarbituric acid reactive substance (TBARS) value in the cooked samples except for roasting. Microwaving, roasting and frying exhibited stronger antioxidant activity than the other cooked samples after in vitro digestion. A total of 38 volatiles were identified in the cooked meat samples, and the samples were well divided into four groups by principal component analysis, and 13 volatiles were considered discriminatory variables for the cooked rabbit meat. CONCLUSION: The physicochemical characteristics of cooked meat differed significantly between the processing methods. Roasted meat showed lower TBARS value and stronger antioxidant activity after simulated digestion compared to the other meats. However, pressure cooked meat detected the most volatile components while roasting the least. © 2022 Society of Chemical Industry.

Preparation and characterization of feruloylated oat ß-glucan with antioxidant activity and colon-targeted delivery.

Ferulic acid (FA) is an effective chemopreventive and therapeutic agent for colorectal cancer. However, FA cannot stably reach the colon through human digestive system, and it can be grafted into oligosaccharides to improve its digestion stability. Therefore, in this study, different degrees of substitution of feruloylated oat ß-glucan (FA-OßG) were prepared by grafting FA onto water soluble oat ß-glucan. FA grafting changed the crystallinity and surface morphology of OßG, and the thermal stability of the FA-OßG improved. As the DS increased, the antioxidant activity of FA-OßG increased, and FA-OßG III with DS of 0.184 showed the same antioxidant activities compared to the equal amount of free FA. The FA-OßG showed higher stability under gastrointestinal and colonic conditions than free FA. Furthermore, the FA-OßG conjugates exhibited good in vitro anticancer activity against human colorectal cancer cells, while FA-OßG III showed better anticancer activity than an equal amount of free FA.

Whey protein isolate-gum Acacia Maillard conjugates as emulsifiers for nutraceutical emulsions: Impact of glycation methods on physicochemical stability and in vitro bioaccessibility of ß-carotene emulsions.

The physicochemical stability and in vitro bioaccessibility of ß-carotene emulsions stabilized by the whey protein isolate (WPI)-gum Acacia (GA) conjugates prepared by dry-heating (Con WPI (D)) and ultrasound (Con WPI (U)) Maillard reaction were investigated in this study. The conjugate-stabilized emulsions have a larger mean size than the WPI-stabilized emulsion. The stability of emulsions under freeze-thaw treatment, high ionic strength conditions, thermal treatment and pH conditions near the isoelectric point of WPI were improved by glycation. The chemical stability of ß-carotene in emulsions was also enhanced by WPI-GA conjugates. The WPI-GA conjugate-stabilized emulsions have better stability for droplet aggregation during in vitro simulated gastrointestinal digestion, which increased ß-carotene bioaccessibility. However, the Con WPI (U)-stabilized emulsion shows poor thermal stability and lower ß-carotene bioaccessibility than the Con WPI (D)-stabilized emulsion, which may be attributed to ultrasound-induced structural changes during the process of glycation.

Dispersal and mating patterns determine the fate of naturally dispersed populations: evidence from Bombina orientalis.

BACKGROUND: In contrast to the explosive increase of a population following biological invasion, natural dispersal, i.e., when a population disperses from its original range into a new range, is a passive process that is affected by resources, the environment, and other factors. Natural dispersal is also negatively impacted by genetic drift and the founder effect. Although the fates of naturally dispersed populations are unknown, they can adapt evolutionarily over time to the new environment. Can naturally dispersed populations evolve beneficial adaptive strategies to offset these negative effects to maintain their population in a stable state? RESULTS: The current study addressed this question by focusing on the toad Bombina orientalis, the population of which underwent natural dispersal following the Last Glacial Maximum in Northeast Asia. Population genetic approaches were used to determine the genetic structure, dispersal pattern, and mating system of the population of B. orientalis in northeast China (Northern population). The results showed that this northern population of B. orientalis is a typical naturally dispersed population, in which the stable genetic structure and high level of genetic diversity of the population have been maintained through the long-distance biased dispersal behavior of males and the pattern of promiscuity within the population. CONCLUSIONS: Our findings suggest that naturally dispersed populations can evolve effective adaptive strategies to maintain a stable population. Different species may have different strategies. The relevance of these maintenance mechanisms for naturally dispersed populations provide a new perspective for further understanding the processes of speciation and evolution.