Philippa Kaur

Study species and area
The blacktip shark (Carcharhinus limbatus; Fig. 1B) is a cosmopolitan species, encountered globally in tropical and subtropical waters34,35,36. It is a commercially and recreationally important species in the southeast U.S. and Gulf of Mexico shark fisheries, as well as being one of the top four species in the global shark fin trade37,38. The species has moderately slow growth rates and low fecundity; producing pups in alternating years after a 10–12 month gestation period, giving birth to 4–7 pups34,39. This leads to low natural recruitment abilities, rendering their populations susceptible to stock collapses from overexploitation. However, the U.S. C. limbatus population is well managed and sustainably fished. In this study we refer to four life-stage categories: neonate, young of the year, immature, and mature, following the definitions of Castro35. Neonates are animals with an open umbilical scar, these scars close within the first 2–3 weeks of life. Young of the year (YOY) have closed umbilical scars, which are typically still visible. YOY can also be distinguished from older immature animals based on size40. Mature males are distinguished from immature males based on claspers that are elongated and calcified, while for females, 50% size-at-maturity was used to distinguish between immature and mature animals39. Along the Florida Gulf Coast, neonate, YOY, and immature C. limbatus seasonally use inshore nursery areas including Terra Ceia Bay1,41,42. Terra Ceia Bay is a small (~ 5 km × 1.5 km), shallow (~ 4 m maximum depth) bay with a narrow (~ 1 km) opening into Tampa Bay (Fig. 1A). As C. limbatus is a migratory species that prefers temperatures above 21 °C, there is an influx of immature sharks into the bay during April after an absence for most of the winter. Gravid females enter the bay for parturition in May and June1,33,34. Natural mortality of neonates is highest within the first 15 weeks of life, which is reflected in a sharp decrease in their abundance from July into August. The remaining young of the year and immature animals leave the bay and migrate southward during the fall months1,33. These patterns have held with continued sampling in the bay until the present, although some immature animals now stay in the bay until later in the year and return earlier, possibly because waters have warmed in recent years (Gardiner, unpublished data).
Figure 1

(A) Map of sampling locations in Terra Ceia Bay (FL, USA). Indicated are in green the eDNA sampling sites, in blue the gillnet sites in 2018 and 2019, and in red circles the longline sampling sites in 2019. Sampling events are aggregated per year and method (i.e. not all sites were sampled every month, see Supplementary Material 1 for detail). (B) A juvenile Carcharhinus limbatus, caught, tagged and released in Terra Ceia Bay (photo credit: T. R. Wiley). Map was generated with R v 3.4.0 (https://www.R-project.org/) using polygons extracted from Google Earth, (https://earth.google.com/web/).

Full size image

Shark sampling
Shark surveys in Terra Ceia Bay were conducted as part of the GulfSPAN survey (www.fisheries.noaa.gov/southeast/endangered-species-conservation/shark-and-sawfish-surveys-and-tagging), a fishery-independent effort to assess shark diversity and relative abundance along the Florida Gulf Coast in order to inform stock assessments. All protocols for the handling and use of animals were approved by the Institutional Animal Care and Use Committee at the University of South Florida (protocol # W IS00004541) and sampling was conducted in accordance with Florida Fish and Wildlife Conservation Commission Special Activities Licenses SAL-18-1666-SRP and SAL-19-1666-SRP. The surveys are conducted each year from April to October. In order to conserve limited resources for this survey, sampling is not conducted from November to March when shark abundances are known to be very low due to decreased water temperatures during these winter months. The primary sampling method of this project for 2018 was monofilament gillnet. However, as this method primarily captures neonate and YOY sharks, it was considered not to be an appropriate comparison on its own to eDNA, which is shed by all life stages. Therefore, bottom longline gear was also deployed on a monthly basis in 2019 to sample a wider variety of life stages and to provide a better comparison to eDNA when combined with gillnets. For each month of sampling in 2019, catches were summed over 3–4 longline and gillnet stations and used to calculate monthly catch-per-unit-effort (CPUE), defined as the number of individuals caught per station per hour. All sampling coordinates and details of the fishing gear are summarized in Supplementary Material 1 and Supplementary Material 2, respectively.
eDNA collection and processing
Water samples were collected on a monthly basis concurrent with GulfSPAN surveys during June–October 2018 and April–October (except July) 2019. Four to six water samples were collected at each sampling location per monthly survey, immediately after gear is set in order to avoid contamination from captured animals. eDNA samples were collected in mid-water to avoid catching suspended sediment, ensuring that only recently released eDNA was collected43,44. A total of 58 water samples of 2 L each (Supplementary Material 1) were collected with a Kemmerer type water sampler and vacuum filtration was carried out immediately after collection with a Pegasus Alexis peristaltic pump (www.fondriest.com). The cups containing the hydrophilic polyethersulfone (PES) filters (Pall Corporation; 47 mm diameter; 0.45 µm pore size) were placed in a clean, iced cooler (containing only eDNA samples) for transport to the laboratory. The filters containing sample filtrates were stored in sterile 5.0 mL cryogenic screw-cap vials containing silica beads. The silica beads function as a desiccator, drying out the filters, preventing the DNA from degradation26. The filters were then stored at − 20 °C until extraction. DNA extraction from half of each filter was performed using the DNeasy PowerSoil Kit (www.qiagen.com), following the manufacturers’ protocol, with three additional specifications. As the outer circumference of the filter does not contain any DNA (due to placement of the filter cup), it was cut off and discarded. The remainder of the half filter was cut into small pieces prior to being added to the bead tubes for the first step of the extraction process. The vortexing (bead-beating) step of the protocol was performed in a shaker at 58 °C (for 10 min) instead of at room temperature, in order to maximize the DNA yield. Genomic DNA was eluted into 100 μL and frozen at − 20 °C until further processing.
Real-time PCR assay development
Primer design
Primers were designed to target a 149 bp fragment of the mitochondrial (mtDNA) nicotine adenine dinucleotide dehydrogenase subunit 2 (NADH2) gene within the C. limbatus genome, while excluding cross amplification with other elasmobranch species known to co-occur in the study area (Rhizoprionodon terraenovae, R. porosus, Carcharhinus brevipinna, C. acronotus, C. leucas, Sphyrna mokarran, S. lewini, S. zygaena, and S. tiburo). Primers were designed manually using Geneious Prime, version 2019.2.3 (https://www.geneious.com). NADH2 sequences from C. limbatus and the non-target shark species were downloaded from GenBank (Supplementary Material 3) and aligned using the Muscle 3.8.425 plugin in Geneious45. Forward (588 F-limbatus-NADH2: 5′-TGCCCCCAATCTCACCTTAC-3′) and reverse (776 R-limbatus-NADH2: 5′-CCGGAAAGTGGGGGTAATCC-3′) primers were designed to amplify the fragment of interest exclusively in C. limbatus. This was accomplished by maximizing the number of mismatches in the ligation sites of both primers in the nine exclusion species. In order to confirm primer specificity, they were first tested by applying conventional PCR to total genomic DNA (gDNA) extracted from eight C. limbatus individuals and from eight individuals per each of the nine species known to co-occur in the study area. Genomic DNA was extracted from tissue samples using the DNeasy Blood & Tissue kit (www.qiagen.com) following the manufacturer’s protocol. Each amplification reaction was performed in a total volume of 15 µL and consisted of: 7.5 µL of Master Mix ‘Applied 2×’ (Applied Biosystems), 3.5 µL of DNase/RNase-free water (Fisher Scientific), 1 µL of each primer (10 µM) and 2 µL of genomic DNA (10 ng/µL). The PCR profile included an initial denaturing step of 95 °C for 2 min, 35 cycles of 95 °C for 30 s, 63 °C for 30 s and 72 °C for 30 s and a final extensions step of 72 °C for 5 min. The quality of all amplifications was assessed by electrophoresis, running the products through a 1.5% agarose gel stained with Gel Red (Gentaur, Kampenhout, Belgium) and visualized on a UV light platform. Subsequently, the primers were tested on the real-time PCR platform for amplification of tissue-derived gDNA from C. limbatus and the non-target species. PCR products were sequenced in both directions to confirm species identity, using an ABI 3730 genetic analyzer (Applied Biosystems). Sequences were checked using Geneious.
Real-time PCR
Concentrations of C. limbatus NADH2 fragments in our eDNA samples were checked by running six replicate reactions for each sample, using the Chai Open PCR System platform (Chai Biotechnologies, Santa Clara, CA, USA). The real-time PCR recipe was optimized to a final volume of 15 µL, containing 7.5 µL of PowerUp SYBR Green Master Mix (Applied Biosystems), 2.5 µL of DNase/RNase-free water (Fisher Scientific), 1 µL of each primer (5 µM) and 3 µL of eDNA template. The real-time PCR program consisted of 50 °C for 2 min, an initial denaturation step at 95 °C for 2 min, followed by 80 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s. A final melting curve analysis step at 60 °C for 1 min was followed by an increasing ramp temperature of 5 °C per second to reach 95 °C for 5 min. Using 80 amplification cycles in real-time PCR is not standard protocol when working with tissue extractions, but with appropriate negative controls it has been found to be useful for detecting the presence of eDNA that which is usually present in very low concentrations16. The melt curve protocol was used to detect primer dimers, to check specificity by determining that a single amplicon was produced, and to obtain a melting temperature (Tm) value for that sample, which may then be compared with the standard curve of C. limbatus DNA. Each real-time PCR run consisted of 16 reactions (two 8-well PCR strips per run), including two positive (13.1 × 10−1 ng/μL gDNA), and two negative (DNase/RNase-free water) controls. Cq values of the standard concentrations were compared between independent runs to check for consistency and replicability of results.
Primer efficiency, LOD and LOQ
Primer efficiency, limit of detection (LOD), and limit of quantification (LOQ) were assessed following the protocol and curve fitting method described in Klymus et al.46. For the standard curve, tenfold dilutions (in ddH2O) of C. limbatus gDNA derived from tissue extractions were used. The dilution series spanned seven orders of magnitude, ranging from 13.1 × 10−1 to 13.1 × 10−7 ng/μL, measured with a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Each dilution was run ten times and reported Cq (cycle quantification) values were used to determine primer efficiency, LOD, and LOQ46. LOD and LOQ were determined at a concentration of 13.1 × 10−6 ng/μL of gDNA (the conc. at which at least 95% of the replicates amplified with a CV of 54% or less, see “Results” section). Amplification efficiency was determined by plotting Cq values against gDNA dilutions and calculating the linear slope and the coefficient of determination (R2) value.
Contamination control
Strict adherence to contamination control procedures was followed during all field and laboratory stages. In the field, disposable gloves were used and changed between each sample. A section of the boat was allocated as a clean area for water collection and filtration. Work surfaces, water collection and filtration equipment were cleaned prior to and after each use with a 50% bleach solution. To prevent cross-contamination between samples, filtration was performed using disposable, single-use filter funnels, which were subsequently stored individually in two layers of double sealed plastic bags prior to being stored on ice. All eDNA sample processing and analyses were conducted in the Crustacean Genomics and Systematics Lab at Florida International University where there had never been any shark tissue or PCR product present. Extractions, pre-PCR preparations and (post)PCR procedures were physically and temporally separated. All laboratory equipment was cleaned with bleach solution and subsequently exposed to UV sterilization (including ddH2O used for PCR reactions) for 15 min, before and after each use. Pertaining to filter extractions, bags containing the individual filters were cleaned on the outside with bleach prior to entering the lab. All work surfaces were cleaned with 50% bleach solution prior to and after each use, and between processing of individual samples. All equipment used for the extractions was likewise cleaned with bleach between each individual sample. Disposable gloves were used and changed between each sample or in addition, more often when deemed appropriate. Aerosol barrier pipette tips were used for all laboratory procedures. C. limbatus tissue extraction was performed months prior to the onset of the eDNA laboratory work, in a separate dedicated tissue extraction room. Separate laboratory rooms, located in different parts of the building, were used for PCR and real-time PCR experiments. No equipment was shared between the laboratories and commuting between the different laboratories within the same day was prohibited, this included colleagues who were not part of this study. In order to identify potential contamination, negative controls were added at multiple stages. Negative controls were collected in the field by filtering 2 L of tap water, processed as standard samples. In the laboratory, DNA extraction blanks (elution buffer from extraction kit) and PCR blanks were included.
Data analysis
Cq and melting temperature (Tm) values were used to determine the presence/absence of C. limbatus eDNA in a sample, by comparison with those of the standard curves and the positive controls. The Cq value indicates the cycle at which the fluorescence produced by the DNA amplification in a sample surpasses a preset threshold of fluorescence background noise, which is a proxy for the quantity of eDNA present in a sample. When a sample has a Cq value, it means that target DNA is present assuming there is no non-specific amplification. The Cq value represents the PCR cycle at which amplification is first detected and is positively correlated with the template DNA present in the sample. Filters were considered positive when at least two out of the six replicates produced a Cq value, in combination with an associated Tm value close (± 2 °C) to the mean Tm of the standard curve (78.2 °C). For each filter sample, the average Cq of all positive replicates were calculated to obtain a single value per filter.
Because many detections presented Cq values below LOQ and LOD (Cq >42.09, see “Results” section), they were treated as qualitative data (presence or absence of target DNA on the filter)46. A Chi square proportion test was performed to test for a difference in the proportion of C. limbatus positive filters between the high (April–July) and low abundance (August–October) seasons over 2018 and 2019, and the catch data (CPUE) was also compared between the same two seasons in 2018 and 2019 using the Wilcoxon signed-rank test. In order to determine the correlation between the proportion of positive filters and catch data (CPUE) per month, a non-parametric Spearman correlation was performed. All statistical analyses were performed in R v 3.4.0 (https://www.R-project.org/). More

Industrial milk production results in waste products and pollutants including greenhouse gas emissions, product packaging, wastewater and effluents. Sustainability indicators are poorly defined for the industry, making efforts to measure and monitor sustainability challenging. Alexandre André Feil, from Universidade do Vale do Taquari, Brazil, and colleagues conducted a systematic review to identify and assess the characteristics of sustainability indicators used in the dairy sector to date.

The literature search covered a 50-year period of publication, from 1969 to the present. Although 130 publications containing the phrases ‘sustainability indicator’ and ‘dairy industry’ were included in the review, the authors found that only about 5% of these studies directly addressed sustainability indicators. The ‘triple bottom line’ of sustainability — environmental, social and economic factors — were used by Feil and colleagues to assess indicators used in the literature. The environmental indicators that emerged included ozone depletion, water eutrophication, energy consumption, ecotoxicity, abiotic depletion, global warming, water acidification, photochemical oxidation, human toxicity and water consumption. Social indicators included product delivery capacity, quality of raw materials and product, traceability systems, workers’ health and safety, noise pollution and traffic accidents. Among economic indicators were profit margin, participation in milk processing, costs of production, storage and distribution. Feil and colleagues note that the frequency of these indicators and their composite assessment in the literature to date is low, as is literature considering the triple bottom line. But, the theme of sustainability is, they note, incipient in the dairy industry.

Establishing a holistic approach to sustainability in the dairy industry is crucial. In this systematic review and analysis, Feil and colleagues also note the importance of tailoring sustainability indicators to regional issues — a strategy that will be important as emerging economies are set to increase their dairy production in the coming decades. More

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We performed double-digest restriction site associated DNA (ddRAD) sequencing on 122 dromedary DNA samples from 18 countries (Supplementary Data 1) representative of the species distribution range. We included one Bactrian camel to test for potential interspecific hybridization, as this continues to be a widespread practice in Central Asia that might have started as early as pre-Roman times11. Higher numbers of reads mapping to the Bactrian camel were detected in three individuals from Iran and in six from Kazakhstan (see “Methods”), and we decided to remove these samples from downstream analysis due to potential introgression from Bactrian camel (Supplementary Data 2). After stringent filtering for genotype and individual missingness, minor allele frequency and relatedness, the final dataset consisted of 95 dromedaries and 22,721 SNPs present in at least 75% of the individuals.
Moderate genome-wide diversity and low population structure
With 22,721 SNPs, we estimated expected (HE = 0.27 ± 0.17; mean ± SD) and observed (HO = 0.25 ± 0.17) heterozygosities in the global dromedary population (npop = 17; nind = 95). Separating the samples according to their continental origins, both Asian (nind = 49, HE = 0.27 ± 0.17/HO = 0.25 ± 0.17) and African dromedaries (nind = 46, HE = 0.26 ± 0.17/HO = 0.25 ± 0.18) showed similar genomic diversity. The mean HE (t = −2.2641, df = 45,398, P = 0.02) and inbreeding coefficients (t = −2.5159, df = 43,024, P = 0.01) were higher in Asian than African dromedaries, but mean HO (t = −1.2791, df = 45,385, P = 0.2) was not different between continents, according to Welch′s t test. Complete diversity and inbreeding values are given in Supplementary Table 1. In comparison with other domestic species, i.e., sheep (HE = 0.22–0.32)26 or cattle (HE = 0.24–0.30)27, we consider the genome-wide diversity in dromedaries as moderate at the best. Several bottlenecks during the last glacial period (see demographic analysis below, and Fitak et al.24) and during domestication left modern dromedaries with a minimum of only six maternal lineages1 and limited genome-wide diversity. This will have implications on future intensification of breeding and genomic selection in dromedaries from regions with increasing desertification.
In general, the genome-wide differentiation within the global dromedary population was very low. Analysis of Molecular Variance (AMOVA) revealed that most of the variation, ~94.3%, is explained within individuals (Supplementary Table 2). Allelic richness (AR) was similar between countries (AR = 0.25–0.27) with exception of Kenya which was lower (AR = 0.21). The pairwise fixation index between African and Asian individuals was very low (FST = 0.006; P  More

Diverse microbial communities contribute to the removal of various pollutants
Revisiting the history of biological wastewater treatment, with the removal of carbon, nitrogen, and phosphorus from wastewater, the key is to provide different microbial niches to enrich diverse functional microorganisms, such as heterotrophs, nitrifiers, denitrifiers, and polyphosphate accumulating organisms (PAOs). Figure 1 shows the redox potential distribution of typical reactions carried out by different types of functional microorganisms. Reactions for the biological nitrogen cycle usually occur at potentials ranging from 0.34 to 0.97 V, while reactions for anaerobic sulfate reduction and methanogenesis occur at potentials ranging from −0.22 to −0.14 V and −0.43 to −0.25 V, respectively. By suitable management, these functional microorganisms can carry out biological metabolisms sequentially in time or space, which can be applied to achieve successful wastewater purification9,10,11,12.
Fig. 1: Redox potentials distribution of typical reactions, and element cycles of sulfur, carbon, and nitrogen.

The dotted lines in the carbon cycle represent the aerobic reaction. The lower right corner shows an example of how these cycles are correlated with each other. Sulfate reduction/denitrification enables the simultaneous carbon removal and sulfate/nitrogen removal; the sulfammox process enables the simultaneous sulfate removal and nitrogen removal.

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For the removal of organic carbon, anaerobic treatment shows a good example of how functional microorganisms cooperate with each other to achieve the conversion from organic carbon to renewable bioenergy methane (CH4) (Fig. 1). During anaerobic treatment, fermenting bacteria first degrade complex organic substrates such as protein and sugar into monomers which are subsequently utilized by acidogenic bacteria to produce acetate and hydrogen. Finally, methanogens consume acetate and hydrogen/carbon dioxide (CO2) to generate the end product CH4. Success to maintain the microbial population and the growth of these microorganisms is the primary cause of anaerobic system stability. In addition, in aerobic biological wastewater treatment processes, organic carbon is mainly degraded by heterotrophs to produce CO2 and synthesize biomass.
For typical municipal wastewater treatment, cultivating microbial communities through a serial of anaerobic, anoxic, and aerobic (A2O) reactors enable the enrichment of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), denitrifiers, and PAOs, resulting in the efficient removal of organic carbon, nitrogen, and phosphorus from wastewater13. In the anaerobic reactor, volatile fatty acids (VFAs) could be stored by PAOs with energy supplied from intracellular stored polyphosphate. In the anoxic reactor, denitrification will occur with organic carbon in wastewater as the electron donor, and recirculated oxidized nitrogen as the electron acceptor. In the aerobic reactor, phosphorus is uptaken by PAOs, ammonia is nitrified by nitrifiers, and also activities of heterotrophs will occur for organic carbon removal. Through activities of all these diverse microorganisms, wastewater can be efficiently treated.
When treating sulfate-containing wastewater, sulfate-reducing bacteria (SRB) which regulate the sulfidogenic bioprocess will become crucial microbes for sulfate removal (Fig. 1). During sulfate reduction, sulfate is first reduced to sulfite and then to sulfide by SRB. The reduction of sulfite to sulfide can be accomplished via the direct pathway in which sulfite is directly reduced to sulfide by receiving six electrons or another pathway that trithionate and thiosulfate are acted as intermediates14. Carbon sources as electron donors can be involved in the SRB metabolism. For example, some SRB metabolize organic compounds as electron donors through Acetyl CoA or a modified TCA pathway15. Many intermediate products originating from anaerobic fermentation/hydrolysis such as amino acids, sugars, long-chain fatty acids, and VFAs, can also be metabolized by SRB14. In this case, organic carbon can be simultaneously removed efficiently with sulfate.
Furthermore, the synergistic removal of contaminants may be completed by cooperative interactions, and the biological element cycle could be interlinked to each other (Fig. 1). It is well-known that denitrification can remove carbon and nitrogen simultaneously, sulfur-based denitrification can remove sulfur and nitrogen, and denitrifying PAOs can remove organic carbon, nitrogen, and phosphorus together. Rios-Del Toro et al.16 found that anaerobic denitrification and ammonium oxidation could be coupled with the reduction of sulfate in marine sediments (sulfammox). Free sulfide, elemental sulfur, and sphalerite were produced during the ammonium oxidation with the reduction of sulfate16. To achieve the niche development of sulfammox, it is obvious that certain concentrations of sulfate and ammonium should be present in wastewater. However, it remains difficult to connect specific microbes to these functions. Metagenomic analysis needs to be implemented to discover uncultivated functional microbes for further application. On the other hand, for the coupling of sulfate removal and CH4 production, conductive materials were reported to be able to alleviate the inhibition of sulfate on methanogenesis, which can enhance the diverse biogeochemical reactions. Liu et al.17 found that the addition of conductive materials could re-enrich syntrophic partners inactivated by sulfate. This new syntrophic community could efficiently produce CH4 in sulfate-containing environment. In this case, the proper addition of conductive materials in anaerobic systems is the key for achieving the coupling of CH4 production and sulfate removal. All these show that the interconnections between biogeochemical cycles such as carbon, nitrogen and sulfur would be potentially applied for developing novel environmental biotechnologies through the optimization of microbial niches (Fig. 1). Similar concepts could be developed for other biological element cycles.
Deciphering functions of known and unknown microorganisms
Biological treatment processes would be successfully functioned once that targeted microbial communities are enriched through microbial niches optimization. Therefore, the understanding of key microbial players is the fundamental step. With the application of novel molecular and bioinformatics techniques, more and more uncultured microbes and microbial functions have been and will be identified. For instance, the concept that nitrification is carried out by AOB and NOB sequentially has been accepted for more than a century and the known AOB and NOB are phylogenetically not closely related. However, some Nitrospira (NOB) species were found to possess all genes encoding enzymes necessary for ammonia oxidation via nitrite to nitrate, completely revising the picture of the nitrogen cycle18,19. The expression of genes during growth through ammonia oxidation to nitrate suggested that Nitrospira might be the key bacteria responsible for nitrification, and metabolic labor division in nitrification is not strictly required.
The niches of novel functional microbes may be different from ‘conventional’ microbes, thus investigating metabolic kinetics, diversity, and microbial interactions of these new microorganisms are crucial for developing novel wastewater treatment technologies based on the optimization of microbial niches.
Clarification of novel microbial metabolic mechanisms
Wastewater treatment processes can be improved through clarifying biological metabolic mechanisms. For example, interspecies hydrogen and formate transfer have been considered as the common pathways for syntrophic methanogenesis. Recently, it has been reported that some syntrophic bacteria and methanogens could exchange electrons directly by conductive pili or outer membrane cytochromes for syntrophic CH4 production20,21. Since electron carriers are not required during direct interspecies electron transfer (DIET), it was considered as a faster and potentially more energy-conserving pathway for CH4 production22. Therefore, DIET may be a crucial approach to improve the energy conversion from wastewater.
By discovery of this new microbial mechanism, several strategies have been proposed that can be potentially applied to achieve the stimulation of DIET so as to improve methanogenesis. The first one is the microbiology-based regulation. A high abundance of DIET-capable microorganisms often implies the good performance of DIET. Enriching DIET players by optimizing their niches can result in the dominance of DIET pathway in methanogenic systems. For example, the well-known bacteria with the DIET ability, Geobacter, which syntrophically consumes ethanol as the organic substrate for growth, could be enriched in an up-flow anaerobic sludge blanket reactor treating brewery wastewater21. In many cases, high CH4 production efficiency could be attributed to the high abundance of Geobacter. Conducting the pretreatment of ethanol-type fermentation may be a useful approach for cultivating Geobacter species23. Second, promoting the excretion of extracellular compounds and adjusting the syntrophic interaction could be also applied for better DIET performance24. Finally, applying conductive materials as electron conduits in methanogenic systems can provide a good external conductive environment for syntrophic partners with the DIET ability. In this case, electrons released from syntrophic bacteria can be directly transferred to methanogens via conductive materials without contacting closely, enhancing the efficiency of CH4 production25. In the wastewater treatment system, the addition of conductive materials could enhance the conductivity of anaerobic sludge, and stimulate the activity of respiratory chain and the extracellular electron transfer rate of syntrophic partners, thereby promoting the methanogenic efficiency26.
Strategies for emerging compounds removal through microbial niche tuning
Providing suitable niches for specific microbes can also enhance the removal of emerging compounds and alleviate their toxicity. Conventional AOB can remove EDCs due to their enzyme of ammonia monooxygenase, which can degrade certain types of micro-pollutants, and heterotrophs could be also responsible for the degradation of synthetic estrogen10. In addition, the recently discovered complete ammonia-oxidizing bacteria which could oxidize ammonia to nitrate via nitrite were also found to be able to degrade micro-pollutants27. Therefore, by tuning all these functional microorganisms, not only conventional pollutants will be removed efficiently, but also emerging compounds will be well controlled.
On the other hand, suitable microbial niches could be applied to alleviate the biological toxicity induced by emerging compounds. For example, the alternate operation of aerobic and extended anaerobic treatment resulted in the enhanced removal of endocrine activities and better control of biological toxicity4. Different redox situations of wastewater under aerobic and anaerobic conditions might be one of the reasons for promoting endocrine degradation4. In addition, the change of organic loading rate could lead to a niche variety of microbes as well, thus affecting the removal efficiencies4. Recently, it was confirmed that cysteine produced during the sulfate reduction could alleviate the nano-metal particle toxicity5. This shows that the microbial interactions during biological processes could be functioning diverse for achieving different purposes.
Microbial niche-based design of the wastewater treatment system
For wastewater treatment, if only organic carbon is removed, one aerobic reactor is adequate. While for organic carbon and nitrogen removal, anoxic combined with aerobic reactors would be applied. Furthermore, for organic carbon, nitrogen, and phosphorus removal, anaerobic, anoxic, and aerobic reactors would be adopted. With more types of pollutants removal, the numbers of biological reactors would be extended for wastewater treatment system design.
Besides biological reactors, the microbial niche nexus concept should be incorporated during wastewater treatment system design. For the upgradation of conventional WWTPs, novel microbial communities could be explored and utilized for solving new challenges, including emerging compounds removal. All these could be achieved through microbial niche optimization to enrich diverse microorganisms in the present WWTPs besides to build new infrastructure (Fig. 2). To achieve this purpose, it is essential to further explore the unrevealed biological processes or functions. For example, rare species in biological treatment processes should be paid attention to, which may act as the seed and would be dominant with varied environmental conditions28,29. In some cases, species with a low abundance may also contribute a lot to the key function of a microbial system. For instance, Pester et al.30 reported that Desulfosporosinus with only 0.006% of the total relative abundance was an important sulfate reducer in a peatland system.
Fig. 2: Infrastructure and microbial niche concepts should be considered during WWTP design and upgradation.

Development of wastewater treatment plants based on infrastructure-based design or microbial niche-based design.

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Elephant herbivory in KNP presently prevents a widespread woody palm Hyphaene petersiana from reaching reproductive size. Out of 65 individual palms sampled inside the Nwaxitshumbe elephant exclosure, 60 (32 females, 28 males) were mature (92%). The mean maximum height of individuals within the enclosure was 7.0 m (range 1.5–11 m). This palm reaches maturity between 4–5.3 m in height as evidenced by the mean height of the tallest immature stems per individual as 5.3 m and the mean height of the shortest mature stems as 4 m (n = 20). Outside the exclosure the mean height of the 75 surveyed individuals was only 1.6 m (max 3.2 m, only 30%  > 2 m). Not one of these were reproductive, with most being several (2.5+) m short of being reproductive (Figs. 1, 2, 3). Signs of elephant herbivory of the palm outside the exclosure were widespread, as has been found elsewhere in Africa19. We found no seedlings inside or outside the exclosure (Fig. 3). Outside the exclosure this is due to a widespread lack of reproduction. The absence of elephants and their role in dispersal and germination7,8,9 explain the lack of recruitment inside the exclosure, despite the production of many thousands of fruits annually over several decades.
Figure 1

Arrows indicate short H. petersiana palms outside the 2 m tall electric fence compared to tall palms within the exclosure (A). The large fruits of H. petersiana (B).

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Figure 2

Arrows indicate that on Google earth image (Image 2013 CNES/Airbus) of where Fig. 1 was taken, tall palms are clearly visible within the exclosure (grey-green canopies) but are short outside the fence.

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Figure 3

Size-class distribution of H. petersiana inside and outside the elephant exclosure.

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This simple result of mass sterilization by elephants is important for biodiversity conservation for at least three reasons. Firstly, and critically, sterile plants cannot evolve new adaptations, such as to the looming threat of global change, nor can they disperse seeds to move with moving climate zones. Secondly, without seedling recruitment populations will eventually go extinct, although in the case of this highly persistent resprouting palm, this is only likely with sudden or significant environmental change because this species can live for about a century20. Thirdly, because sterile plants do not produce flowers, fruits and large stems this too has biodiversity implications. We observed ad hoc that the outer layer of the fruits of this palm (Fig. 1) is eaten by vervet monkeys (Chlorocebus pygerythrus), porcupines (Hystrix africaeaustralis) and squirrels (Paraxerus cepapi). Elephants also consume Hyphaene fruits7,8,9. We observed the palm flowers to be heavily visited by pompilid wasps, that African palm swifts (Cypsiurus parvus) were only nesting in tall palms inside the exclosure and that woodpeckers used the tall soft stems for nest sites. Sterilization therefore has diverse biodiversity consequences. These negative impacts are based on data from one location and for only one plant species, but these impacts are likely geographically widespread and to occur on other common woody KNP species. As minimum size to maturity in plant species is well known to scale with their maximum height17,18 and therefore broken, but potentially tall trees are likely sterile, as was the case for H. petersiana. For example, the geographically widely-distributed important savanna tree Colophospermum mopane (“mopane”) can reach 10–25 m tall but is most often a short ( 60 km transect) google earth survey of H. petersiana showed an almost total absence of mature individuals outside of elephant exclosures and a survey of a population of 40 individuals of the congener H. coriacea, indicated that 75% of individuals were sterile.
Since there are only a few antelope (approximately 8 ha per animal during the period 2000–2017 according to SanParks records) within the exclosure, grass biomass is much higher inside than outside. The exclosure is actively burned to maintain the grazing for these rare antelope and although many of the palms inside the exclosure had been burned recently, their canopies had escaped damage because they are several metres above the high grass-biomass fueled fire zone. Many fruits on the ground below mature individuals were damaged by the fire. Outside the exclosure elephant herbivory keeps plants short and therefore when fires take place, fire damages fronds and this exacerbates the lack of plants becoming tall enough to become reproductive. The achievement of reproductive size inside the exclosure is due to the absence of elephants rather than an absence of fire.
The impact of elephant herbivory on reducing the size of this palm outside compared to inside the Nwaxitshumbe exclosure was previously noted by Levick and Rogers12. However, they interpreted elephant herbivory as having a positive impact on this palm, because of a greater relative stem density outside the exclosure. Also, they suggested that tall vegetation in the exclosure “would be less permeable to vectors such as wind and water”12. They missed the dramatic and negative impact on reproductive status despite H. petersiana fruits being conspicuously large (up to 10 cm in length) and individual fruit-loads often exceed 100 fruits (Fig. 1). We suggest this was missed because assessing reproductive condition is not a routine conservation assessment of the impacts of herbivory. The debatable positive impacts of elephant herbivory on this palm suggested by Levick and Rogers12 should be weighed up against more definitely negative impacts on the reproductive status of plants and the additional negative impacts this has, for example on frugivores and pollinators. We suggest that managers consider the conservation impacts of elephants, both positive and negative, on the sexual reproduction of resprouting plants. Although fruiting is less obvious for most plant species than for H. petersiana, given its large fruits, it would nevertheless be relatively easy to assess the minimum size a species needs to be, to be sexually reproductive. Species with tall maximum heights may be a priority. Also, if the present high level of elephant herbivory in KNP is reduced, fruiting by well-established resprouts of this palm could occur within two decades, because they are capable of rapid growth20. However, there is no plan14 to directly control the presently steadily increasing population1, although there are plans to reduce access to artificial waterpoints14 Finally, we emphasize the general conservation problem that resprouting plant species such as H. petersiana present15. Although they are able to increase stem density despite chronic elephant herbivory or persist in situ in the absence of elephants, their loss of reproduction or loss of their dispersal mutualists, means that they are nevertheless presently “the living dead”23 in KNP. More

Plant functional variability
In total, 78 species occurred (cover ≥ 5%) at the different sites, creating the set of species over which CSR strategies were assessed (Fig. 2; Table S2). A clear dominance of relatively stress-tolerant strategies was evident across the sites; indeed, most species showed a proportion of S exceeding 50% (Fig. 2, Supplementary Figs. S1, S2).
Figure 2

CSR classification of four sites related to Dianthus pseudocrinitus (a–d) showing the relative importance of the C, S and R axes for sympatric (non-Dianthus) species within the plant community (left side) and the individuals of D. pseudocrinitus (right side) in each site (a Rein; b Misino; c Biu Pass; d Rakhtian). The species are represented in gray scale according to their mean cover (%). The numbering indicated in the circles corresponds to Table S2. The small triangles show the community weighted mean (CWM) strategies at each site for the sympatric species and the individuals of D. pseudocrinitus.

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Dianthus pseudocrinitus was the only Dianthus species that exhibited general functional divergence, ranging from strong ruderalism at the Rein site (R; C:S:R = 12.0:7.2:80.8%), an intermediate strategy at Rakhtian and Misino (S/SR; C:S:R = 2.8:75.9:21.3%; and C:S:R = 7.4:70.5:22.1%, respectively), to strong stress-tolerance at the Biu Pass site (S; C:S:R = 6.8:82.3:10.9%) (Fig. 2). Differences among D. pseudocrinitus populations at different sites were apparent for S-selection (ANOVA on arcsine transformed data, predictor variables were sites and response variables were the percentage CSR-scores; f = 34.386, dfnumerator = 3, dfdenominator = 37, p = 0.000) and R-selection (f = 43.707, dfnumerator = 3, dfdenominator = 37, p = 0.000) but not for C-selection (f = 2.801, dfnumerator = 3, dfdenominator = 37, p = 0.054), with a Tukey’s post-hoc multiple comparison on data for R-selection (i.e. the highest f-value), suggesting that populations at all sites differed from one another, except for those at Misino and Rakhtian.
In terms of interspecific differences, analysis of variance (ANOVA) showed that D. pseudocrinitus differed significantly from the community mean at the Rein site in terms of R-selection (f = 46.982, dfnumerator = 16, dfdenominator = 146, p = 0.000) and S-selection (f = 44.601, dfnumerator = 16, dfdenominator = 146, p = 0.000; arcsine transformed data, with species (i.e. taxa present in the plant community) as the predictor variables and percentage CSR-scores as the response variables). Crucially, that D. pseudocrinitus exhibited extensive intraspecific variability was evident as extreme values of strategy variance (s2) compared to the intraspecific variability of sympatric species at the Rakhtian and Rein sites (Table 1). Note that the CSR strategy variability evident for sympatric species is presented in greater detail in Fig. S3.
Table 1 Variance (s2) in C-, S-, and R-selection values (%) for D. pseudocrinitus and other species at the (a) Rein and (b) Rakhrian sites, with species ordered according to decreasing variance in R-selection (n = 10).
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Dianthus polylepis subsp. polylepis exhibited an extreme stress-tolerant strategy (C:S:R = 0.1:99.1:0.8%) across all sites (Fig. S1). Most sympatric species at sites of D. polylepis subsp. polylepis represented a broadly stress-tolerant strategy (Fig. S1), but interspecific functional variability was evident, including subordinate species (mean cover percentage 5.5–9.0%) with relatively generalist, intermediate strategies (Fig. S1). Intraspecific differences in Dianthus polylepis subsp. polylepis between sites were apparent for C-selection (ANOVA on arcsine transformed data, predictor variables were sites and response variables the percentage CSR-scores; f = 7.599, dfnumerator = 5, dfdenominator = 48, p = 0.000) and S-selection (f = 6.686, dfnumerator = 5, dfdenominator = 48, p = 0.000) and R-selection (f = 8.099, dfnumerator = 5, dfdenominator = 48, p = 0.000), with a Tukey’s post-hoc multiple comparison on data for R-selection (i.e. the highest f-value) suggesting that the population at Bezd was distinct from other sites.
Dianthus polylepis subsp. binaludensis exhibited an extremely stress-tolerant strategy (C:S:R = 0.5:99.5:0.0%) at all sites except Zoshk, where it exhibited an intermediate S/SR strategy (Fig. S2). Intraspecific differences in D. polylepis subsp. binaludensis between sites were apparent for C-selection (ANOVA on arcsine transformed data, predictor variables were sites and response variables the percentage CSR-scores; f = 2.801, dfnumerator = 4, dfdenominator = 46, p = 0.054), S-selection (f = 25.796, dfnumerator = 4, dfdenominator = 46, p = 0.000) and R-selection (f = 18.476, dfnumerator = 4, dfdenominator = 46, p = 0.000), with a Tukey’s post-hoc multiple comparison on data for S-selection (i.e. the highest f-value) suggesting that the population at Zoshk was distinct from other sites. At Zoshk, Dahane Jaji and Dizbad, D. polylepis subsp. binaludensis exhibited significantly lower C-selection (p ≤ 0.05) with respect to the community mean (t tests within site on arcsine-transformed data).
Site and environmental variables
The canonical correspondence analysis (CCA) (Fig. 3) was constrained by a matrix of soil and topographic data and bioclimatic variables. Seven soil variables (clay, silt, sand, EC, P, CEC and organic carbon) and 15 bioclimatic variables were eliminated from the environmental data set owing to high collinearity (VIF  > 10). Soil organic matter, pH, N, K, lime, elevation, and aspect were the edaphic/topographic variables exhibiting the highest levels of significance (p  More

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