Philippa Kaur

arising from: P. B. Ngor et al.; Scientific Reports https://doi.org/10.1038/s41598-018-27340-1 (2018).
As one of the richest sources of fisheries-related data in the lower Mekong basin, the Tonle Sap dai fishery has received considerable attention in the literature in recent years as concerns grow over the impacts of hydropower dams on fisheries, which are important for livelihoods and food security1,2,3.
Ngor et al.4 reported a decline since 2000 in the catch of larger species which tend to occupy higher trophic levels; compensatory increases in the catch of smaller species; and declines in the mean body weight (and length) of common species in the Tonle Sap dai fishery, as evidence of the effects of indiscriminate fishing or “fishing-down” of the multi-species fish assemblage in the lower Mekong basin. We provide evidence below that suggest that these apparent recent changes are more likely to reflect changing hydrological conditions than fishing-down effects, possibly caused by climate change and recently also by hydropower development.
The dai fishery has been reliably monitored since 1997–98. Without explanation, Ngor et al. excluded the first three seasons (1997–98 to 1999–2000) of monitoring data which include one of the driest fishing seasons on record (1998–99). The authors thereby created a time series beginning with the three wettest seasons (largest floods) since monitoring began (2000–1 to 2002–3) that were followed by 12 seasons of variable, but decreasing flows caused by hydropower dam construction, low rainfalls possibly resulting from climate change, and abstractions for agriculture5,6 (Fig. 1).
Figure 1

Source: Mekong River Commission Secretariat.

The flood index (FI) or flood pulse14 in the Tonle Sap Great Lake System (1997/08–2014/15). The FI is a measure of the flood extent and duration, calculated as the sum of the flooded area days above the mean flooded area from April to March of the following year2. Whilst highly variable, a downward decline (p-value = 0.06) in the FI is observed between 2000/01 (Year 2001) and 2014/15 (Year 2015) shown by solid circles. Adding the most recent data for 2016–2018 (not shown here), confirmed that a downward linear trend in the FI since the 2000/01 season is statistically significant (p-value  45 cm) excluding those with zero catch in any year. These 28 species formed approximately 16% of the total catch during the study period. We also found negative regression coefficients for all 28 species, supporting the findings of Ngor et al. However, the combined annual catch of these 28 species did not decline significantly through time (R2 = 0.22; p-value = 0.07).
We did however find that the combined annual catch of these 28 larger species varied significantly with the annual flood index (FI)—a measure of flood extent and duration (R2 = 0.46; p-value  45 cm) species and the flood index (R2 = 0.46; p-value  More

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Continuous and discrete quantity information are important in guiding animal behaviour in virtually all aspects of life. The capacity to evaluate continuous (uncountable) quantities, such as length, area, weight, or duration, is widespread and can be found in organisms with relatively simple nervous systems, such as annelids, crustaceans, or gastropds1,2,3. This quantitative information takes part in decision-making processes in different contexts. For example, animals may gauge body sizes of rivals or prospective mates, assess distances from home, or estimate the extent of a food patch4,5,6.
Several vertebrates, from teleost fishes to primates, can also process discrete (countable) information. For example, many species are capable of accurately estimating the number of elements in a set and comparing the numerosity of different sets7,8. Studies conducted in nature or in the laboratory have shown that numerical abilities serve important adaptive functions. For example, in guppies, New Zealand robins, and macaques, quantity discrimination is used to select the patch containing the larger number of food items9,10,11. Conversely, some predators, namely lions and striped field mice, use this ability to select the smallest prey groups because they are more vulnerable to predation12,13. Various group-living mammals, including chimpanzees, lions, and hyenas, gauge the relative number of opponents before deciding whether to attack or withdraw14,15,16. Gregarious fish use the same ability to select the social group that provides the best protection from predators17,18,19. Some species, including eastern mosquitofish, brown-headed cowbirds, and American coots, use their quantitative abilities to increase reproductive success20,21,22.
Cognitive psychologists have shown that in these cases it is not necessary to assume the existence of a true numerical estimation system because an animal can use continuous cues, such as the amount of movement, the cumulative surface occupied by items, or the convex hull of the set as a proxy for number23,24. Inferring the existence of a numerical system requires a series of careful laboratory control experiments in which the animal is subjected to numerical tasks, while the access to non-numerical information is simultaneously prevented8,25. This process is not always straightforward and studies often fail to reach a firm conclusion even after numerous experiments are performed. In fact, convincing evidence of the presence of a numerical system exists only for a small fraction of the species investigated (e.g., guppy26, chicken27, and rhesus monkeys10).
It is not known whether numerical abilities have similar selective advantages in other phyla and whether numerical systems are widespread outside the vertebrate group. To date, this issue has been investigated only in a handful of species, and there is convincing evidence of a true numerical system for only one of them, the honeybee28,29,30. Honeybees, Apis mellifera, can be trained to discriminate different numbers of dots to obtain a food reward31,32. They are able to accomplish this task even when main continuous cues are controlled, thus it is suggested that they possess a numerical system analogous to that of vertebrates. Honeybees can also use ordinal information and learn the correct position in a sequence of artificial flowers when distance cues are made irrelevant33. Similar evidences have been recently provided for another social bee, the bumblebee, Bombus terrestris34,35. The function of cardinal and ordinal numerical abilities in social bees is unclear, but it has been suggested that they mainly serve to recognise flowers from the number of petals and to learn the location of food around their hives, respectively.
Circumstantial evidence suggests the ability to estimate the quantity of conspecifics in three other arthropod species. The juvenile spiders of Portia africana have been reported to take into account the number of competitors present when choosing between two patches of food36. Males of the coleopteran Tenebrio molitor are able to discriminate different numbers of females based on the odours they emit37. Ants (Formica xerophila) perceiving themselves as part of a large group are more aggressive towards another species than ants perceiving themselves as isolated individuals38. Controls are difficult to perform in these types of experiments, and it is unknown whether these three species are actually estimating the number of individuals or they are using other types of information as a proxy of number.
Recently, a mollusc, the cuttlefish, was observed to prefer the larger quantity of shrimps up to 4 versus 5 items39. Although authors manipulate some continuous cues (i.e., density and total activity of preys), it is unclear whether cuttlefish are really counting prey or are using other cues, such as the cumulative area occupied by shrimps or the convex hull of the groups.
Theba pisana is a small terrestrial snail inhabiting the dunes of the Mediterranean coasts. Similar to most snails, it is active mainly at night. This species has a considerable thermal tolerance, with an upper lethal limit that lies, depending on exposure time, between 46 °C and 50 °C40. However, during sunny days, the sandy ground can reach temperatures that largely exceed this lethal limit (up to 75 °C). To avoid these adverse conditions at sunrise, dune snails climb the stem of tall vegetation, where the temperature rarely exceeds 30 °C, and remain inactive until night. If placed on the ground during the day, these snails rapidly regain an elevated position by orienting towards nearby stems and climbing on them (Fig. 1a; Supplementary Video S1). At our site of capture, snails were collected mainly from vertical, unbranched stems of live or dead inedible plants and herbs (e.g., Puccinellia palustris, P. distans, and Juncus maritimus).
Figure 1

(a) Example of a dune snail T. pisana climbing on the stem of tall vegetation. (b) The circular arena used for investigating quantity discrimination ability in laboratory.

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Zanforlin showed that it is possible to simulate this behaviour in the laboratory41. After placing dune snails on a brightly lit arena, they rapidly orient towards a black cardboard shape on a white background and climb on it. With this setup, it was possible to study shape preference by placing two shapes at 60° angle from the centre and releasing the snail from the centre of the arena. He found that, confronted with similar geometric figures (e.g., two rectangles), snails oriented consistently towards the stimulus with the largest area. When area was kept constant, no particular preference for shape was observed, although there was a tendency to prefer the figure with a longer perimeter or with wider axes.
In all the experiments of the former study, snails were required to choose between two single shapes. In nature, however, stems are frequently arranged in clusters. All things being equal, there are several potential advantages in heading towards a large cluster of stems. In a cluster, there is greater probability of finding the stem with the most suitable features, such as a correct diameter or an optimal orientation to shelter from wind and sun40. In addition, not all the stems are accessible due to the presence of intricate or thorny vegetation at the base. Heading towards a group of stems increases the chances that at least one stem can be reached and climbed. Furthermore, most predators (mainly passerine birds, wall lizards, and rats) are small and catch only one or few preys at a time, and hence, sheltering in clusters could determine a dilution effect on predation risk42,43.
Based on the above considerations, we made the prediction that natural selection in T. pisana should favour the ability to discriminate between a single stem and a cluster and discriminate among clusters, based on the quantity of stems. The aim of the first experiment was to test this hypothesis. In the laboratory, we simulated stems used by dune snails as refuges by using black vertical bars on a white background (Fig. 1b; Supplementary Video S2). As we found that dune snails discriminate rather accurately between quantities of stems, in a series of subsequent experiments, we investigated the mechanism involved. Specifically, we tried to figure out if snails were using a true numerical system or if they used continuous quantitative information that co-varied with numerosity, such as the cumulative area occupied by items, their density or the convex hull they spanned.
Experiment 1: discrimination of the quantity of refuges
A previous study on dune snails investigated the choice between single objects that differed in shape and size41. However, based on their ecology, we predict that snails searching for protection from the heat also should focus on number and should move towards the largest available group of stems. In Experiment 1a, we studied whether dune snails prefer a group of refuges to a single one (Fig. 2a), and in Experiment 1b, we measured their accuracy to discriminate among groups of refuges differing in numerosity (Fig. 2b). To obtain reference data about snails’ general discriminatory abilities, in Experiment 1c, we measured the accuracy of dune snails to discriminate two equally shaped objects that differ in surface area (Fig. 3c).
Figure 2

(a–c) Stimuli used in Experiment 1a, 1b and 1c, respectively.

Full size image

Figure 3

(a) Percentage of snails choosing the stimulus with larger quantity of bars in Experiment 1a and 1b. Snails showed a significant preference for larger quantity up to 4 versus 5 bars. There was a significant difference amongst the numerical ratios (P  More

Data
Data were collected in 2015 through a survey deployed as part of the Feed the Future Buena Milpa project (http://www.cimmyt.org/project-profile/buena-milpa/). The project’s goal was to reduce food insecurity and malnutrition by fostering sustainable, resilient, and innovative maize-based farming systems in the WHG.
We conducted the survey, with support from local researchers and agronomy students of the University of San Carlos (USAC), in summer of 2015 in 989 maize-growing farm households in 64 communities of 16 municipalities of the WHG. The number of surveys at the departmental level was: Totonicapán (226), Quiche (350), Quetzaltenango (187) and Huehuetenango (226) (See Supplementary Table 1 and Supplementary Fig. 1 for details). Criteria to select communities were: location within the targeted municipalities in the Buena Milpa project, the active work of project partners within the communities, and their distribution within four selected watersheds that included farming systems at different altitudes, ranging from 1400 to 3200 masl. For household selection, enumerators walked radial transects to survey household members, choosing only households that explicitly agreed to participate and had agricultural land on at least part of which maize was grown. The survey was a closed questionnaire with 139 questions in 18 sections including 5 project themes: milpa-maize germplasm improvement, natural resource conservation in farming system, farming system diversification, agricultural innovation systems and social inclusion.
Milpa diversity and extent
We used survey findings regarding the previous year’s crop production (2014) for all 989 surveyed households to understand the importance and diversity of the milpa system. The 989 households reported crop production on a total of 1,541 plots, 1,324 of which included maize. The other 217 plots grew potato (85 plots), coffee (50), vegetables (31), bean (19), fruit trees (12), faba bean (7), forestry trees (6), pea (2), oats, wheat, etc. Given the study’s focus on smallholder farmers, we discarded other 119 plots that had unrealistically large values for plot size or maize production levels for the region (i.e. more than ten times the average plot size or calculated maize yield,  > 2 ha and  > 11 ton ha−1 year−1).
For the resulting 1205 plots we constructed a tree depicting maize-system diversity in the WHG, with each node indicating the main cropping associations. The tree was nested, so the first node displays all maize plots, with successive splits for monocrop vs intercropped maize and with crop names presented according to how often they are grown (overall: maize  > common beans  > potatoes  > squash  > faba beans  > fruit trees  > vegetables). So, in plots where maize is grown with potatoes and common beans, the association is termed maize-bean-potatoes.
Milpa and food security
We assessed maize yield differences for monocropping and intercropping to detect a yield penalty or advantage for maize (see below), including survey information only for households from which we had available yield data for all crops. In the survey, total production for each crop was recorded regardless of the number of plots on which it was grown and therefore, for 398 households (40.2% of the sample) that had more than one plot under maize or any of the milpa crops, it was impossible to calculate the yield and had to be discarded from the analysis due to a lack of available plot-level information. The results were further screened for complete crop information and unrealistic values on crop production levels (i.e. ten times higher than the average yield for the different crops—See Supplemental Table 2), resulting in a usable sample of 368 plots.
For statistical analysis, only those crop combinations with a sample size equal to or greater than 9 plots were selected, resulting in 357 plots with, in descending order, the following numbers of 300 plots sown to each cropping combination: maize monocrop (163), maize-bean (109), maize-bean-squash (30), maize-bean/faba (12), maize-potato (13), maize-squash (11), maize-bean-potato (10), maize-faba (9). Other crop combinations with very low sample sizes were maize-squash-faba (4), maize-potato-faba (3), maize-bean-squash-faba (2), maize-bean-potato-faba (1) and maize-bean-potato-squash (1), making it difficult to include them in further statistical analyses.
Maize yields for plots under monocropped maize (163) were first compared to maize yields from intercropped plots (194). To choose the most appropriate statistical test, we checked if the outcome variable, maize yield, met the assumptions required for a parametric test. Although we had independent samples, large sample sizes, and homogeneous variances (F = 1.1809, p-value = 0.267), maize yield proved to be non-normally distributed after Shapiro-Wilks normality test was significant (W = 0.911, p-value  1]{ }}}{{text{N}}}{ } times {overline{text{s}}},{[18]}$$

where (N) is the number of nutrients considered (14), (s_{i}) is the fraction of potentially nourished male adults by a given nutrient, and (overline{s}) is the average of potentially nourished persons for all nutrients. To calculate s we multiplied the yield of each crop reported in the survey (kg ha−1) by the nutrient composition of each crop, using the content per 100 g of the 14 different nutrients for each crop, as per the INCAP Food Composition Table for Central America32 and using the INCAP RDA for an adult male33 (See Supplementary Tables 3 and 4). All values were calculated per hectare and year. PNA levels across cropping associations were also compared. First, we checked if PNA data met the assumptions required for a parametric test. PNA variances proved to be non-homogeneous (F = 14.5, p-value =   More

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Study area
The field work was carried out at the Sturt Meadows calcrete aquifer (28°41′ S 120°58′ E) located on Sturt Meadows pastoral station, Western Australia, ~ 42 km from the settlement of Leonora (833 km northeast of Perth, see Fig. 1a). The study area is a calcrete aquifer lying in the Raeside paleodrainages in the Yilgarn region of Western Australia (Fig. 1a). The vegetation of the area is Acacia woodlands, primarily Acacia aneura (F.Muell. ex Benth.), and is subjected to combined grazing pressure from domestic stock, feral animals and macropods. The aquifer is accessible through a bore grid comprising 115 bore holes of between 5 and 11 m depth (Fig. 1b).
Figure 1

Map of the Sturt Meadows calcrete. (a) Location within the Yilgarn craton region and detailed paleodrainages/cacretes in the area and (b) the grid map showing the location of the boreholes sampled for stygofauna together with probe samples, water samples (in light blue) and the combination of both. Map was produced in ArcGIS Desktop 10.679 and edited in Adobe Illustrator 25.080.

Full size image

Three sampling campaigns were carried out, two of them (LR1: 26/07/2017 and LR2: 07/11/2017) corresponding to low rainfall periods24 and one during the wet season (high rainfall, HR; two consecutive days of sampling collection: 17-18/03/2018) (Supplementary Fig. 1). The well-studied stygofaunal community of the area is composed of 11 main stygofaunal taxa belonging to five Classes: Oligochaeta (family Tubificidae (Vejdovský 1884)), subcohort Hydrachnidia, Maxillopoda (two species of harpacticoids: Novanitocrella cf. aboriginesi (Karanovic, 2004), Schizopera cf. austindownsi (Karanovic, 2004) and four species of cyclopoids: Halicyclops kieferi (Karanovic, 2004), Halicyclops cf. ambiguous (Kiefer, 1967), Schizopera slenderfurca (Karanovic & Cooper, 2012) and Fierscyclops fiersi (De Laurentiis et al., 2001)), Malacostraca: Amphipoda (species Scutachiltonia axfordi (King, 2012), Yilgarniella sturtensis (King, 2012) and Stygochiltonia bradfordae (King, 2012)) and Insecta: Coleoptera: Dytiscidae (species Paroster macrosturtensis (Watts & Humphreys 2006), Paroster mesosturtensis (Watts & Humphreys 2006) and Paroster microsturtensis (Watts & Humphreys 2006) and respective larvae).
Field work procedures and sample preparation
Given the sensitivity of the hydrological dynamics in shallow calretes23,25, extensive water extraction along the bores was avoided, and preliminary tests on the bores with the highest water depth were carried out to quantify potential risk of dewatering the calcrete. During the field campaigns LR2 and HR, 20 water samples in total (two samples for stable isotope analysis on DOC (Dissolved Organic Carbon) and DIC (Dissolved Inorganic Carbon), three samples for radiocarbon analysis on DOC, one sample for radiocarbon analysis on DIC, and two samples for stable isotope and radiocarbon analyses on POC (Particulate Organic Carbon)) were collected from bores D13 and W4 (Fig. 1b), which are representative of the two main geological conformations of the area—calcrete (W4) and clay (D13) (Supplementary Fig. 2)—and host stable hydrological and biotic conditions7. Water samples were collected using a submersible centrifugal pump (GEOSub 12 V Purging Pump) after wells were purged of three well-volumes and stabilisation of in-field parameters was observed, according to the methodology in Bryan et al.26.
Samples for 14CDIC analysis were filtered through 0.45 μm filters and collected in 1 L high density poly-ethylene (HDPE) bottles. δ13CDIC samples were filtered through 0.2 μm filters, collected in 12 mL glass vials (Exetainers) and refrigerated after sampling. δ 13CDOC samples were filtered through 0.2 μm filters, collected in 60 mL HDPE bottles and frozen after sampling.14CDOC samples were filtered through 0.2 μm filters, collected in 3 L HDPE bottles and frozen after sampling.
In order to investigate 14C and δ 13C content of POC, two extra liters were collected from the same bores (D13, W4) and kept frozen (− 20 °C) until further analyses. 14CPOC δ13CPOC samples were then filtered through pre-combusted GF/F filters (12 h at 450 °C), washed with 1.2 N HCl to remove any inorganic carbon, and subsequently dried at 60 °C for 24 h. All samples were closed with sealing tape after collection to limit atmospheric exchange and kept in darkness.
Temperature, pH, ORP, salinity, DO and depth were measured in situ using a portable Hydrolab Quanta Multi‐Probe Meter across 30 bores during LR1, LR2 and HR23 (presented in Supplementary Table 8). Adult and larval stygofaunal specimens were collected from the same bores by hauling a weighted plankton net (mesh 100 μm27) five times through the water column (Fig. 1b). All biological samples were kept frozen (− 20 °C) in darkness until laboratory processing. Individual organisms were counted and identified (and consequently separated) to the lowest taxonomic level via optical microscopy and reference to specific taxonomic keys. Plant material, sediment samples and fauna were each separated during the sorting in the laboratory and each taxon pooled according to sampling campaign (LR1, LR2 or HR) and subsequently washed with Milli-Q water to remove surface impurities from their bodies. Sediment samples were soaked in acid (0.1 N HCl) to remove inorganic carbon, and together with the other samples were then oven dried at 60 °C overnight and ground until obtaining a homogeneous fine powder and stored at − 20 °C until further analyses.
Previous investigations on the ecological niche trends at Sturt Meadows indicated that all stygofauna characterize similar niche occupations under low rainfall regimes (LR1 and LR2)23. Stygofaunal specimens from the two low rainfall sampling events were combined to form sample LR to address the competing requirements between isotopic detection limits, analytical replicates and cost, while maintaining the main taxonomic and biological classifications7.
Bulk isotope and 14C analyses
Water δ13CDIC and δ13CPOC isotopic ratios were analysed by Isotope Ratio Mass Spectrometer—WABC at The University of Western Australia using a GasBench II coupled with a Delta XL Mass Spectrometer (Thermo-Fisher Scientific)—and the results, with a precision of ± 0.10‰, were reported as ‰ deviation from the NBS19 and NSB18 international carbonate standard28.
δ13CDOC isotopic ratios of waters were analysed via Liquid Chromatography Isotope Ratio Mass Spectrometer (LC-IRMS) at the La Trobe Institute for Molecular Sciences (LIMS, La Trobe University, Melbourne, Australia) comprising an Accela 600 pump connected to a Delta V Plus Isotope Ratio Mass Spectrometer via a Thermo Scientific LC Isolink (Thermo Scientific).
C and N bulk SIA on homogenised samples of sediment, roots, stygofauna and copepods (cyclopoids and harpacticoids) were performed at the Australian Nuclear Science and Technology Organisation (ANSTO, Sydney, Australia). Samples were loaded into tin capsules and analysed with a continuous flow isotope ratio mass spectrometer (CF-IRMS), model Delta V Plus (Thermo Scientific Corporation, U.S.A.), interfaced with an elemental analyser (Thermo Fisher Flash 2000 HT EA, Thermo Electron Corporation, USA) following the procedure published by Mazumder et al.29.
For radiocarbon analyses, samples (sediment, roots, copepods, ants, stygofauna) were treated with 1 M HCl for 2 h to remove all possible carbonate contamination. These pre-treated samples together with 14CPOC, 14CDOC and 14CDIC samples were subjected to CO2 extraction and graphitization following the methodology published by Hua et al.30. 14C content of samples was determined by means of the Accelerator Mass Spectrometry (AMS) at ANSTO.
Carbon CSIA
Carbon CSIA followed the procedure described in Saccò et al.7. Samples of roots and stygofaunal specimens were hydrolysed under vacuum with 0.5 to 1 mL of amino acid-free 6 M HCl (Sigma-Aldrich) at 110 °C for 24 h. The protein hydrolysates were dried overnight in a rotary vacuum concentrator and stored in a freezer. Prior to analysis, the samples were dissolved in Milli-Q water and 10 μL of 1-mmol solution of 2-aminoisobutyric acid (Sigma-Aldrich) as internal standard. The sample stock had a concentration of approximately 8 to 10 mg/mL, which was further diluted as needed. Single amino acid carbon isotope analysis was carried out at the La Trobe Institute for Molecular Sciences (LIMS, La Trobe University, Melbourne, Australia) using an Accela 600 pump connected to a Delta V Plus Isotope Ratio Mass Spectrometer via a Thermo Scientific LC Isolink (Thermo Scientific).
The amino acids were separated using a mixed mode (reverse phase/ion exchange) Primesep A column (2.1 × 250 mm, 100 °C, 5 μm, SIELC Technologies) following the chromatographic method described in Mora et al.31 after Smith et al.32. Mobile phases are those described in Mora et al.33. Percentage of Phases B and C in the conditioning run, as well as flow rate of the analytical run and timing of onset of 100% Phase C were adjusted as needed. Samples were injected onto the column in the 15 μL—partial loop or no waste—injection mode, and measured in duplicate or triplicate.
To elucidate carbon flows through the stygofaunal community we focused on the essential amino acids Valine (Val), Phenylalanine (Phe) and Arginine (Arg), as these compounds must be integrated through diet and cannot be synthetised internally by the fauna14,34. In addition, to distinguish between terrestrial and aquatic carbon sources, the ratio between Val and Phe signals (δ13CVal-Phe), a widely employed index in archaeology and freshwater biology35, was calculated for roots, water mites, aquatic worms, amphipods and beetles (larvae and adults).
Microbial taxonomic and functional gene analyses
Consumer amphipods (Scutachiltonia axfordi (AM1), Yilgarniella sturtensis (AM2), S. bradfordae (AM3)), cyclopoids and harpacticoids, together with predator stygobiotic beetles (Paroster macrosturtensis (B), P. mesosturtensis (M) and P. microsturtensis (S)) (see Saccò el at 7. for further details on the trophic characterisation of the stygofaunal community at Sturt Meadows), were used for gut microbiome bacterial 16S metabarcoding and microbial functional analysis. A total of 16 AM1, 16 AM2, 16 AM3, 20 cyclopoids and 20 harpaticoids and 20 of each one of the three Paroster species (B, M and S), were sorted into duplicates of stygobiotic pools of 3–5 individuals from both LR and HR events for DNA extraction. Prior to DNA extraction stygobitic animals (3–5 individuals per pool; n = 40) were placed in a petri dish containing ultrapure water and UV sterilized in a UV oven for 10 min to eliminate any bacterial species that may be contained on the exoskeleton as this study targeted the gut microbiome. Immediately post-UV treatment, the animals were placed into tissue lysis tubes with 180 μL tissue lysis buffer (ATL) and 20 μL Proteinase K and homogenised using Minilys tissue homogeniser (ThermoFisher Scientific, Australia) at high speed for 30 s. Lysis tubes, inclusive of two laboratory controls, were incubated at 56 °C using an agitating heat block (Eppendorf ThermoStat C, VWR, Australia) for 5 h.
Following the incubation, the analytical procedure was adapted from Saccò et al.22 and DNA extraction was carried out using DNeasy Blood and Tissue Kit (Qiagen; Venlo, Netherlands) and eluted off the silica column in 30–50 μL AE buffer. The quality and quantity of DNA extracted from each stygobitic pool was measured using quantitative PCR (qPCR), targeting the bacterial 16S gene. PCR reactions were used to assess the quality and quantity of the DNA target of interest via qPCR (Applied Biosystems [ABI], USA) in 25 μL reaction volumes consisting of 2 mM MgCl2 (Fisher Biotec, Australia), 1 × PCR Gold Buffer (Fisher Biotec, Australia), 0.4 μM dNTPs (Astral Scientific, Australia), 0.1 mg bovine serum albumin (Fisher Biotec, Australia), 0.4 μM of each primer (Bact16S_515F and Bact16S_806R36,37), and 0.2 μL of AmpliTaq Gold (AmpliTaq Gold, ABI, USA), and 2 μL of template DNA (Neat, 1/10, 1/100 dilutions). The cycling conditions were: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min.
DNA extracts that successfully yielded DNA of sufficient quality, free of inhibition, as determined by the initial qPCR screen (detailed above), were assigned a unique 6–8 bp multiplex identifier tag (MID-tag) with the bacterial 16S primer set. Independent MID-tag qPCR for each stygobiotic pool were carried out in 25 μL reactions containing 1 × PCR Gold Buffer, 2.5 mM MgCl2, 0.4 mg/mL BSA, 0.25 mM of each dNTP, 0.4 μM of each primer, 0.2 μL AmpliTaq Gold and 2–4 μL of DNA as determined by the initial qPCR screen. The cycling conditions for qPCR using the MID-tag primer sets were as described above. MID-tag PCR amplicons were generated in duplicate and the library was pooled in equimolar ratio post-PCR for DNA sequencing. The final library was size selected (160–600 bp) using Pippin Prep (Sage Sciences, USA) to remove any MID-tag primer-dimer products that may have formed during amplification. The final library concentration was determined using a QuBitTM 4 Fluorometer (Thermofischer, Australia) and sequenced using a 300 cycle V2 kit on an Illumina MiSeq platform (Illumina, USA).
MID-tag bacterial 16S sequence reads obtained from the MiSeq were sorted (filtered) back to the stygobitic pool based on the MID-tags assigned to each DNA extract using Geneious v10.2.538. MID-tag and primer sequences were trimmed from the sequence reads allowing for no mismatch in length or base composition.
Filtered reads were then input into a containerised workflow comprising USEARCH39 and BLASTN40, which was run on a high-throughput HPC cluster at Pawsey supercomputing centre. The fastx-uniques, unoise3 (with minimum abundance of 8) and otutab commands of USEARCH were applied to generate unique sequences, ZOTUs (zero-radius Operational Taxonomic Units) and abundance table, respectively. The ZOTUs were compared against the nucleotide database using the following parameters in BLASTN: perc_identity ≥ 94, evalue ≤ 1e−3, best_hit_score_edge 0.05, best_hit_overhang 0.25, qcov_hsp_perc 100, max_target_seqs = 5. An in-house Python script was used to assign the ZOTUs to their lowest common ancestor (LCA)41. The threshold for dropping a taxonomic assignment to LCA was set to perc_identity ≥ 96 and the difference between the % of identity of the two hits when their query coverage is equal was set to 1. Results on the microbial taxonomic diversity detected in ground water samples from a previous study on carbon inputs in the aquifer22 were incorporated in this work to allow qualitative comparison with the stygofaunal gut diversity.
To investigate functional activity involved in carbon cycling, the 16S metabarcoding data were fed to the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) software package to generate predicted metagenome profiles42. These profiles were clustered into Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthologs (KOs)43 and MetaCyc pathway abundances44 focusing on the relative abundances of four carbon metabolisms: carbon fixation in prokaryotes, carbohydrates, lipids and amino acid metabolisms. Relative abundance of pathways linked with methane, nitrogen and sulfur metabolisms were also investigated.
Statistical analyses
The Phyloseq package in R45,46 was used to plot the ZOTU abundance for the styfofaunal specimens at the order level under LR and HR periods. The Statistical Analysis of Metagenomic Profiles (STAMP) bioinformatics software package was used to carry out Principal Components Analysis (PCA) to assess the differences between functional genomic fingerprints based on the KEGG orthologs function prediction between copepods (C and H) and amphipods (AM1, AM2 and AM3), and determine statistically significant results from the PICRUSt2 output47. Clustering patterns according to rainfall periods (LR and HR) and major consumers taxonomic groups (cyclopoids, harpacticoids and amphipods) were assessed through Permutational multivariate analysis of variance (PERMANOVA, R-package46 ‘vegan’) and pairwise post hoc pairwise multilevel comparisons48.
For comparison of potential shifts in abundances of microbial metabolic pathways within groundwater samples, copepods and amphipods across rainfall periods, analysis of variance (ANOVA) was performed on the abundance data (two replicates per each group) on the predicted pathways depicting carbon fixation, carbohydrate, lipid, amino acid, methane, nitrogen and sulfur metabolisms. ANOVAs coupled with Tukey’s HSD pairwise comparisons (R-package46 ‘stats’) were employed to inspect significant differences between bulk SIA (δ13C and δ15N) and essential amino acid (δ13CPhe, δ13CArg, δ13CVal and δ13CVal-Phe) data from the stygofaunal taxa within the different rainfall conditions (LR and HR). PERMANOVAs (R-package46 ‘vegan’) were also performed to investigate the potential clustering trends within the stygofaunal taxa across rainfall periods from the combination of radiocarbon (Δ14C) and carbon SIA (δ13C) isotopic fingerprints.
SIMM (Stable Isotope Mixing Models, R-package46 ‘simmr’) were used to estimate dietary proportions of copepods and amphipods within a Bayesian framework. Due to the lack of species-specific trophic discrimination factors for stygofauna, we employed the widely accepted values of 3.4 ± 2‰ for nitrogen and 0.5 ± 1‰ for carbon49. Markov chain Monte Carlo (MCMC) algorithms were used for simulating posterior distributions in SIMM, and MCMC convergence was evaluated using the Gelman-Rubin diagnostic by using 1.1 as a threshold value for analysis validation. More