DOI: 10.5061/dryad.2z34tmpj7
title: Data from: Refining trophic dynamics through multi-factor Bayesian mixing models: A case study of subterranean beetles

description: Food web dynamics are vital in shaping the functional ecology of
 ecosystems. However, trophic ecology is still in its infancy in
 groundwater ecosystems due to the cryptic nature of these environments. To
 unravel trophic interactions between subterranean biota, we applied an
 interdisciplinary Bayesian mixing model design (multi-factor BMM) based on
 the integration of faunal C and N bulk tissue stable isotope data (δ13C
 and δ15N) with radiocarbon data (Δ14C), and prior information from
 metagenomic analyses. We further compared outcomes from multi-factor BMM
 with a conventional isotope double proxy mixing model (SIA BMM), triple
 proxy (δ13C, δ15N and Δ14C, multi-proxy BMM) and double proxy combined
 with DNA prior information (SIA+DNA BMM) designs. Three species of
 subterranean beetles (Paroster macrosturtensis, Paroster mesosturtensis
 and Paroster microsturtensis) and their main prey items Chiltoniidae
 amphipods (AM1: Scutachiltonia axfordi and AM2: Yilgarniella sturtensis),
 cyclopoids and harpacticoids from a calcrete in Western Australia were
 targeted. Diet estimations from stable isotope only models indicated
 homogeneous patterns with modest preferences for amphipods as prey items.
 Multi-proxy BMM suggested increased - and species-specific - predatory
 pressures on amphipods coupled with high rates of scavenging/predation on
 sister species. SIA+DNA BMM showed marked preferences for amphipods AM1
 and AM2 and reduced interspecific scavenging/predation on Paroster
 species. Multi-factorial BMM revealed the most precise estimations (lower
 overall SD and very marginal beetles’ interspecific interactions),
 indicating consistent preferences for amphipods AM1 in all the beetles’
 diets. Incorporation of genetic priors allowed crucial refining of the
 feeding preferences, while integration of more expensive radiocarbon data
 as a third proxy (when combined with genetic data) produced more precise
 outcomes but close dietary reconstruction to that from SIA+DNA BMM.
 Further multidisciplinary modelling from other groundwater environments
 will help elucidate the potential behind these designs and bring light to
 the feeding ecology of one the most vital ecosystems worldwide.
description: Supporting information Sacco et al., 2020 Ecology and
 Evolution Extraction of DNA: a
 total of 15 individuals for each of the three <i>Paroster</i>
 species (<i>P. macrosturtensis </i>(B), <i>P.
 mesosturtensis </i>(M)<i>, </i><i>P.
 microsturtensis </i>(S)) were sorted into triplicate stygobitic
 pools (i.e. 5 individuals per pool) for DNA extraction. Prior to DNA
 extraction, stygobitic animals (5 individuals per pool;
 <i>n</i>=18) were placed in a petri dish containing ultrapure
 water and UV sterilised for 10 minutes to eliminate any potential eDNA
 that may be contained on the exoskeleton. Immediately post-UV treatment,
 the animals were placed into tissue lysis tubes with 180 µL ATL and 20 µL
 Proteinase K and homogenised using Minilys® tissue homogeniser
 (ThermoFisher Scientific, Australia) at high speed for 30 seconds. Lysis
 tubes, inclusive of one laboratory control, were incubated at 56°C using
 an agitating heat block (Eppendorf ThermoStat™ C, VWR, Australia) for 5
 hours. Following incubation, DNA extraction was carried out using DNeasy
 Blood and Tissue Kit (Qiagen; Venlo, Netherlands eluted off the silica
 column in 50 µL AE buffer. PCR: The
 quality and quantity of DNA extracted from each stygobitic pool was
 measured using quantitative PCR (qPCR), insect 16S and COI genes. PCR
 reactions to assess the quality and quantity of the DNA target of interest
 were carried out <i>via</i> qPCR (Applied Biosystems [ABI],
 USA) in 25 µL reaction volumes consisting of 2 mM
 MgCl<sub>2</sub> (Fisher Biotec, Australia), 1 x 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 (MZArtF and MZArtR Zeale, Butlin, & Jones 2011;
 Ins16S_1shortF and Ins16S_1shortR Clarke, Soubrier, & Cooper
 2014), 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 minutes, followed by 40 cycles of 95°C
 for 30 seconds, 52°C for 30 seconds, 72°C for 30 seconds, and a final
 extension at 72°C for 10 minutes. 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 6-8bp multiplex identifier tag (MID-tag) with the 16S and COI
 primer set. Independent MID-tag qPCR for each stygobitic pool were carried
 out in 25 µL reactions containing 1 x PCR Gold Buffer, 2.5 mM
 MgCl<sub>2</sub>, 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 (160-600bp) 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 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 v10.2.5.
 MID-tag and primer sequences were trimmed from the sequence reads allowing
 for no mismatch in length or base composition. Each stygobitic pool for
 the two genes amplified were searched using BLASTn (Altschul, Gish,
 & Lipman 1990), against the NCBI GenBank nucleotide database to
 enable taxonomic identification. This was automated in the internet-based
 bioinformatics workflow environment through Pawsey Supercomputing (REF).
 The BLAST results obtained were imported into MEtaGenome Analyzer v4
 (MEGAN; Huson, Auch, & Schuster 2007), where they were
 taxonomically assigned using the LCA-assignment algorithm (min. bit score
 = 65.0, top percentage = 5%, min. support = 5).
 Additional references Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of molecular biology, 215(3), 403-410. Clarke, L. J., Soubrier, J., Weyrich, L. S., & Cooper, A. (2014). Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias. Molecular ecology resources, 14(6), 1160-1170. Huson, D. H., Auch, A. F., Qi, J., & Schuster, S. C. (2007). MEGAN analysis of metagenomic data. Genome research, 17(3), 377-386. Zeale, M. R., Butlin, R. K., Barker, G. L., Lees, D. C., & Jones, G. (2011). Taxon‐specific PCR for DNA barcoding arthropod prey in bat faeces. Molecular ecology resources, 11(2), 236-244.
description: Note S1 B, M and S are sister species
 characterised by high genetic diversity (Guzik, Cooper, & Austin
 2009), with analyses suggesting they diversified in groundwater from a
 stygobiont ancestor (Langille, Tierney, & Cooper, 2019). The
 species live permanently within the groundwater, where they respire
 directly through their cuticle (Jones et al, 2019). They usually show
 predatory and opportunistic (i.e. scavenger, see Hyde, 2018) feeding
 behaviours (Saccò et al., 2020) and are characterised by different
 corporal features. B has the largest body (3.6-4.1 mm) with thin antennae
 and prominent elytral shoulders (Fig. 1a). M is 2.0-2.3 mm, with a
 relatively small head and pronotum, and small posterior legs (Fig. 1b),
 the specimens of this species showing marked sexual dimorphism. S is
 1.7-1.8 mm with boat-shaped bodies and mesofemurs (strongly sinuated) and
 metafemurs (heavily scalloped) with distinctive characters (Watts
 & Humphreys 2009) (Fig. 1c). Amphipods AM1 and
 AM2 belong to the family Chiltoniidae that, together with
 <i>Scutachiltonia bradfordae </i>(King et al., 2012) (AM3),
 inhabit the Sturt Meadows calcrete. They have distinct stygobiontic
 traits: elongated antennae and uropods, and lack of both pigmentation and
 eyes. Molecular phylogenetic analyses (Bradford, Adams, & Cooper
 2010) indicate that these amphipods are not sister clades, but belong to
 three different lineages genetically more similar to other Yilgarn and
 non-Yilgarn amphipods than to each other. AM1 is the
 largest species and its distinctive traits are elongated coxae and setose
 pereiopods (Fig. 1d). It is a unique morphotype in calcrete aquifers and
 is endemic to the Sturt Meadows calcrete (King et al., 2012). AM2 has the
 least stygobiotic-like morphology with medium size body features (Fig.
 1e). AM3 is the rarest species in the calcrete (King et al. 2012), and it
 was not included in the isotopic analysis due to insufficient
 material. The orders Cyclopoida (C) and Harpacticoida
 (H) belong to the two taxa of copepods that most successfully invaded
 inland groundwater worldwide (Galassi, 2001) and display peculiar traits
 adaptive to the interstitial life. Cyclopoids differ from other copepods
 by having first antennae shorter than the length of the head and thorax,
 and uniramous second antennae (Lowry, 1999) (Fig. 1f). Harpacticoids
 display a very short pair of first antennae, biramous second pair of
 antennae and a wide abdomen, with a worm-like body (Barnes, 1987) (Fig.
 1g). Additional references Barnes, R. D. (1987). Invertebrate zoology (No. Ed. 5). WB Saunders company. Bradford, T., Adams, M., Humphreys, W. F., Austin, A. D., & Cooper, S. J. B. (2010). DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia’s arid zone. Molecular Ecology Resources, 10(1), 41-50. Galassi, D. M. (2001). Groundwater copepods: diversity patterns over ecological and evolutionary scales. Hydrobiologia, 453(1), 227-253. Guzik, M. T., Cooper, S. J. B., Humphreys, W. F., & Austin, A. D. (2009). Fine‐scale comparative phylogeography of a sympatric sister species triplet of subterranean diving beetles from a single calcrete aquifer in Western Australia. Molecular Ecology, 18(17), 3683-3698. Langille, B. L., Tierney, S. M., Austin, A. D., Humphreys, W. F., & Cooper, S. J. (2019). How blind are they? Phototactic responses in stygobiont diving beetles (Coleoptera: Dytiscidae) from calcrete aquifers of Western Australia. Austral Entomology, 58(2), 425-431. Lowry, J. K. (1999). Crustacea, the Higher Taxa: Description, Identification, and Information Retrieval. Version 2 October 1999.