Ecology

Geospatial dataSampling locations were established, flagged, and recorded in June 2016, using a Trimble Geo7X global navigation satellite system (GNSS) receiver using the Trimble® VRS Now real-time kinematic (RTK) correction. Location accuracies were verified to within ±2 cm. Points were imported into a geodatabase using Esri ArcMap (Advanced license, Version 10.5) and projected using the Universal Transverse Mercator (UTM), Zone 17 North projection, with the 1983 North American datum (NAD83). Field investigators navigated to the flagged locations by visually locating them in the field or by using recreational grade GNSS receivers with the locations stored as waypoints.Plant tissue sampling and preparationMiscanthus x giganteus grows in clumps of bamboo-like canes. A single cane was cut at soil level from each of the five sample collection points in each circular plot, individually labelled, and brought to the lab for processing (Fig. 2). Each stem was measured from the cut at the base to the last leaf node, and the length was recorded. Green, fully expanded leaves were cut from each stem and leaves and stems from each plant were placed in separate paper bags and dried at 60 °C. The dry leaf and stem tissues were ground to pass a 1 mm screen (Wiley Mill Model 4, Thomas Scientific, Swedesboro, New Jersey, USA). Subsamples of the ground material were analyzed for total carbon (C) and nitrogen (N), acid-digested for the analysis of total macro- and micronutrients, and water-extracted for spectroscopic analysis and the characterization of the water extractable organic matter (WEOM) (Fig. 2).Fig. 2Images of field samples, and diagram of plant tissue processing. Center panel – flow chart outlining the procedures for plant tissue processing, the kinds of analyses performed, and the type of data generated. Upper left inset panel – ground level picture of Miscanthus x giganteus circular plots. Upper right inset panel – some plant samples on the day of collection.Full size imageTotal carbon and nitrogenDried and ground leaf and stem material (~4–6 mg) was analyzed for total C and N content by combustion (Vario EL III, Elementar Americas Inc., Mt. Laurel, New Jersey, USA). The instrument was calibrated using an aspartic acid standard (36.08% C ± 0.52% and 10.53% N ± 0.18%). Validation by inclusion of two aspartic acid samples as checks in each autosampler carousel (80 wells) resulted in a net positive bias of 1.44 and 1.68% for C and N, respectively. The mean C and N concentrations and standard deviations for the sample set are presented in Table 1.Table 1 Giant miscanthus composition including leaf (L) and stem (S) dry weight, length, and carbon (C) and nitrogen (N) concentrations (n = 165). Values are reported as means ± standard deviations.Full size tableMacro- and micronutrientsPlant tissue samples were analyzed for a suite of macro- and micronutrients including aluminum (Al), arsenic (As), boron (B), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), nickel (Ni), phosphorus (P), lead (Pb), sulfur (S), selenium (Se), silicon (Si), titanium (Ti), vanadium (V), and zinc (Zn) using Inductively Coupled Plasma with Optical Emission Spectroscopy (ICP-OES). Samples (0.5 g) were digested using 10 mL of trace metal grade nitric acid (HNO3) in a microwave digestion system (Mars 6, CEM, Matthews, North Carolina, USA). During the digestion procedure (CEM Mars 6 Plant Material Method), the oven temperature was increased from room temperature to 200 °C in 15 minutes and held at 200 °C for 10 minutes. The pressure limit of the digestion vessels was set to 800 psi although it was not monitored during individual runs. Sample digestates were transferred quantitatively to centrifuge tubes, diluted to 50 mL with 2% HNO3 (prepared with lab grade deionized water), and centrifuged at 2500 rpm for 10 min (Sorvall ST8 centrifuge, Thermo Fisher Scientific, San Jose, California, USA). The digestates were decanted into clean centrifuge tubes and analyzed using an iCAP 7400 ICP-OES Duo equipped with a Charge Injection Device detector (Thermo Fisher Scientific, San Jose, California, USA). An aliquot of digested sample was aspirated from the centrifuge tube using a CETAC ASX-520 autosampler (Teledyne CETAC Technologies, Omaha, Nebraska, USA) and passed through a concentric tube nebulizer. The resulting aerosol was then swept through the plasma using argon as the carrier gas with a flow rate of 0.5 L/min and a nebulizer gas flow rate of 0.7 L/min. Macro- and micronutrients were quantified by monitoring the emission wavelengths (Em λ) reported in Table 2.Table 2 Macro- and micronutrients measured, and emission wavelengths (Em λ) used to quantify them in the miscanthus leaves (L) and stems (S), the total number and percentage detected (n = 150 for leaves and 162 for stems), the mean detected concentration ± standard deviation, and the mean method detection limit (MDL) ± standard deviation.Full size tableCharacterization of the water extractable organic matter (WEOM)The WEOM of the giant miscanthus leaves and stems was isolated by extracting the plant material with deionized water at room temperature6. The water extractions were performed by mixing ~0.2 g of dry, ground leaves and stems with 100 mL of deionized water in 125 mL pre-washed brown Nalgene bottles. All brown Nalgene bottles used for these extractions were pre-washed by soaking them for 24 hours in a 10% hydrochloric acid solution followed by 24 hours in a 10% sodium hydroxide solution, and a thorough rinse with deionized water. The bottles containing the extraction solution were shaken on an orbital shaker at 180 rpm for 24 hours. The extract was vacuum filtered using 0.45 µm glass fibre filters (GF/F, Whatman) into pre-washed 60 mL brown Nalgene bottles. The filtered water extracts containing the WEOM were stored in the dark in a refrigerator (4 °C) until analysis by UV-Visible and fluorescence spectroscopy. Samples were visually inspected just prior to analysis to ensure no colloids or precipitates had formed during storage. Samples that had become visually cloudy were re-filtered.On the day of analysis, the water extracts were removed from the refrigerator and allowed to warm up to room temperature. Chemical characteristics of the WEOM were assessed through the analysis of optical properties on an Aqualog spectrofluorometer (Horiba Scientific, New Jersey, USA) equipped with a 150 W continuous output Xenon arc lamp. Excitation-emission matrix (EEM) scans were acquired in a 1 cm quartz cuvette with excitation wavelengths (Ex λ) scanned using a double-grating monochrometer from 240 to 621 nm at 3 nm intervals. Emission wavelengths (Em λ) were scanned from 246 to 693 nm at 2 nm intervals and emission spectra were collected using a Charge Coupled Device (CCD) detector. All fluorescence spectra were acquired in sample over reference ratio mode to account for potential fluctuations and wavelength dependency of the excitation lamp output. Samples were corrected for the inner filter effect7 and each sample EEM underwent spectral subtraction with a deionized water blank to remove the effects due to Raman scattering. Rayleigh masking was applied to remove the signal intensities for both the first and second order Rayleigh lines. Instrument bias related to wavelength-dependent efficiencies of the specific instrument’s optical components (gratings, mirrors, etc.) was automatically corrected by the Aqualog software after each spectral acquisition. The fluorescence intensities were normalized to the area under the water Raman peak collected on each day of analysis and are expressed in Raman-normalized intensity units (RU). All sample EEM processing was performed with the Aqualog software (version 4.0.0.86).The optical data obtained from the EEM scans were used to calculate several indices representative of WEOM chemical composition (Table 3) including the absorbance at 254 nm (Abs254), the ratio of the absorbance at 254 to 365 nm (Abs254:365), the ratio of the absorbance at 280 to 465 nm (Abs280:465), the spectral slope ratio (SR), the fluorescence index (FI), the humification index (HIX), the biological index (BIX), and the freshness index (β:α). The SR was calculated as the ratio of two spectral slope regions of the absorbance spectra (275–295 and 350–400 nm)8. The FI was calculated as the ratio of the emission intensities at Em λ 470 and 520 nm, at an Ex λ of 370 nm9. The HIX was calculated by dividing the emission intensity in the 435–480 nm region by the sum of emission intensities in the 300–345 and 435–480 nm regions, at an Ex λ of 255 nm10. The BIX was calculated as the ratio of emission intensities at 380 and 430 nm, at an Ex λ of 310 nm11. The freshness index β:α was calculated as the emission intensity at 380 nm divided by the maximum emission intensity between 420 and 432 nm, at an Ex λ of 310 nm12. To further characterize the giant miscanthus WEOM, the fluorescence intensity at specific excitation-emission pairs was also identified. The fluorescence peaks identified here have previously been reported for surface water samples and water extracts13 and include peak A (Ex λ 260, Em λ 450), peak C (Ex λ 340, Em λ 440), peak M (Ex λ 300, Em λ 390), peak B (Ex λ 275, Em λ 310), and peak T (Ex λ 275, Em λ 340). A brief description of these optical indices is provided in Table 3.Table 3 Description of the optical indices calculated from the excitation-emission matrix (EEM) fluorescence scans and used to analyze the WEOM composition of giant miscanthus leaves and stems.Full size table More

Rasmussen, R. S. & Morrissey, M. T. Application of DNA-based methods to identify fish and seafood substitution on the commercial market. Compr. Rev. Food Sci. Food Saf. 8, 118–154 (2009).CAS 
Article 

Google Scholar 
Chiu, M.-C., Huang, C.-G., Wu, W.-J. & Shiao, S.-F. A new horsehair worm, Chordodes formosanus sp. N. (Nematomorpha, Gordiida) from Hierodula mantids of Taiwan and Japan with redescription of a closely related species, Chordodes japonensis. ZooKeys 160, 1–22 (2011).Article 

Google Scholar 
Robins, J. H. et al. Phylogenetic species identification in Rattus highlights rapid radiation and morphological similarity of new Guinean species. PLoS One 9, e98002. https://doi.org/10.1371/journal.pone.0098002 (2014).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Sutherland, W. J., Roy, D. B. & Amano, T. An agenda for the future of biological recording for ecological monitoring and citizen science. Biol. J. Linn. Soc. 115, 779–784 (2015).Article 

Google Scholar 
Ho, J. K. I., Puniamoorthy, J., Srivathsan, A. & Meier, R. MinION sequencing of seafood in Singapore reveals creatively labelled flatfishes, confused roe, pig DNA in squid balls, and phantom crustaceans. Food Control 112, 107144. https://doi.org/10.1016/j.foodcont.2020.107144 (2020).CAS 
Article 

Google Scholar 
Elson, J. & Lightowlers, R. Mitochondrial DNA clonality in the dock: Can surveillance swing the case?. Trends Genet. 22, 603–607 (2006).CAS 
PubMed 
Article 

Google Scholar 
Bernt, M., Braband, A., Schierwater, B. & Stadler, P. F. Genetic aspects of mitochondrial genome evolution. Mol. Phylogenet. Evol. 69, 328–338 (2013).CAS 
PubMed 
Article 

Google Scholar 
Blaxter, M. L. The promise of a DNA taxonomy. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 669–679 (2004).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Waugh, J. DNA barcoding in animal species: progress, potential and pitfalls. BioEssays 29, 188–197 (2007).CAS 
PubMed 
Article 

Google Scholar 
Grandjean, F. et al. Rapid recovery of nuclear and mitochondrial genes by genome skimming from Northern Hemisphere freshwater crayfish. Zool. Scr. 46, 718–728 (2017).Article 

Google Scholar 
Trevisan, B., Alcantara, D. M. C., Machado, D. J., Marques, F. P. L. & Lahr, D. J. G. Genome skimming is a low-cost and robust strategy to assemble complete mitochondrial genomes from ethanol preserved specimens in biodiversity studies. PeerJ 7, e7543. https://doi.org/10.7717/peerj.7543 (2019).Article 
PubMed 
PubMed Central 

Google Scholar 
Franco-Sierra, N. D. & Díaz-Nieto, J. F. Rapid mitochondrial genome sequencing based on Oxford Nanopore Sequencing and a proxy for vertebrate species identification. Ecol. Evol. 10, 3544–3560 (2020).PubMed 
PubMed Central 
Article 

Google Scholar 
Baeza, J. A. Yes, we can use it: a formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics and barcoding research using the Caribbean spiny lobster Panulirus argus. BMC Genomics 21, 882. https://doi.org/10.1186/s12864-020-07292-5 (2020).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Phillips, A. R., Robertson, A. L., Batzli, J., Harris, M. & Miller, S. Aligning goals, assessments, and activities: An approach to teaching PCR and gel electrophoresis. CBE Life Sci. Educ. 7, 96–106 (2008).PubMed 
PubMed Central 
Article 

Google Scholar 
Dhorne-Pollet, S., Barrey, E. & Pollet, N. A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants. BMC Genomics 21, 785. https://doi.org/10.1186/s12864-020-07183-9 (2020).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: Delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239. https://doi.org/10.1186/s13059-016-1103-0 (2016).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Krehenwinkel, H. et al. Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale. GigaScience 8, giz006. https://doi.org/10.1093/gigascience/giz006 (2019).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Srivathsan, A. et al. ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone. BMC Biol. 19, 217. https://doi.org/10.1186/s12915-021-01141-x (2021).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Prost, S. et al. Education in the genomics era: Generating high-quality genome assemblies in university courses. GigaScience 9, giaa058. https://doi.org/10.1093/gigascience/giaa058 (2020).Article 
PubMed 
PubMed Central 

Google Scholar 
Salazar, A. N. et al. An educational guide for nanopore sequencing in the classroom. PLoS Comput. Biol. 16, e1007314. https://doi.org/10.1371/journal.pcbi.1007314 (2020).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Watsa, M., Erkenswick, G. A., Pomerantz, A. & Prost, S. Portable sequencing as a teaching tool in conservation and biodiversity research. PLoS Biol. 18, e3000667. https://doi.org/10.1371/journal.pbio.3000667 (2020).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Egeter, B. et al. Speeding up the detection of invasive bivalve species using environmental DNA: A Nanopore and Illumina sequencing comparison. Mol. Ecol. Resour. https://doi.org/10.1111/1755-0998.13610 (2022).Article 
PubMed 

Google Scholar 
Oxford Nanopore. Flongle. https://nanoporetech.com/products/flongle. Last accessed 05 May 2022 (2022).Oxford Nanopore. MinION. https://nanoporetech.com/products/minion. Last accessed 05 May 2022 (2022).Baeza, J. A. & García-De León, F. J. Are we there yet? Benchmarking low-coverage nanopore long-read sequencing for the assembling of mitochondrial genomes using the vulnerable silky shark Carcharhinus falciformis. BMC Genomics 23, 320. https://doi.org/10.1186/s12864-022-08482-z (2022).Article 
PubMed 
PubMed Central 

Google Scholar 
Ghiselli, F. et al. Molluscan mitochondrial genomes break the rules. Philos. Trans. R. Soc. B Biol. Sci. 376, 20200159. https://doi.org/10.1098/rstb.2020.0159 (2021).Article 

Google Scholar 
Zhang, Z.-Q. Animal biodiversity: An introduction to higher-level classification and taxonomic richness. Zootaxa 3148, 7–12 (2011).Article 

Google Scholar 
Bouchet, P., Bary, S., Héros, V. & Marani, G. How many species of molluscs are there in the world’s oceans, and who is going to describe them? In Tropical Deep-Sea Benthos 29 (eds Héros, V. et al.) 9–24 (Muséum national d’histoire naturelle, 2016).
Google Scholar 
Reese, D. S. Palaikastro shells and bronze age purple-dye production in the Mediterranean Basin. Annu. Br. Sch. Athens 82, 201–206 (1987).Article 

Google Scholar 
Lardans, V. & Dissous, C. Snail control strategies for reduction of schistosomiasis transmission. Parasitol. Today 14, 413–417 (1998).CAS 
PubMed 
Article 

Google Scholar 
Baker, G. M. (ed.) Molluscs as Crop Pests. (CABI, 2002). https://doi.org/10.1079/9780851993201.0000Mannino, M. A. & Thomas, K. D. Depletion of a resource? The impact of prehistoric human foraging on intertidal mollusc communities and its significance for human settlement, mobility and dispersal. World Archaeol. 33, 452–474 (2002).Article 

Google Scholar 
Carter, R. The history and prehistory of pearling in the Persian Gulf. J. Econ. Soc. Hist. Orient 48, 139–209 (2005).Article 

Google Scholar 
Vilariño, M. L. et al. Assessment of human enteric viruses in cultured and wild bivalve molluscs. Int. Microbiol. Off. J. Span. Soc. Microbiol. 12, 145–151 (2009).
Google Scholar 
Tedde, T. et al. Toxoplasma gondii and other zoonotic protozoans in Mediterranean mussel (Mytilus galloprovincialis) and blue mussel (Mytilus edulis): A food safety concern?. J. Food Prot. 82, 535–542 (2019).CAS 
PubMed 
Article 

Google Scholar 
Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Sayers, E. W. GenBank. Nucleic Acids Res. 44, D67–D72 (2016).CAS 
PubMed 
Article 

Google Scholar 
Grande, C., Templado, J. & Zardoya, R. Evolution of gastropod mitochondrial genome arrangements. BMC Evol. Biol. 8, 61. https://doi.org/10.1186/1471-2148-8-61 (2008).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Formenti, G. et al. Complete vertebrate mitogenomes reveal widespread repeats and gene duplications. Genome Biol. 22, 120. https://doi.org/10.1186/s13059-021-02336-9 (2021).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Buchfink, B., Reuter, K. & Drost, H.-G. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat. Methods 18, 366–368 (2021).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Kolmogorov, M., Yuan, J., Lin, Y. & Pevzner, P. A. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37, 540–546 (2019).CAS 
PubMed 
Article 

Google Scholar 
Meng, G., Li, Y., Yang, C. & Liu, S. MitoZ: A toolkit for animal mitochondrial genome assembly, annotation and visualization. Nucleic Acids Res. 47, e63. https://doi.org/10.1093/nar/gkz173 (2019).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Bernt, M. et al. MITOS: Improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313–319 (2013).PubMed 
Article 

Google Scholar 
Chaisson, M. J. P., Wilson, R. K. & Eichler, E. E. Genetic variation and the de novo assembly of human genomes. Nat. Rev. Genet. 16, 627–640 (2015).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Alexander, J. & Valdés, A. The ring doesn’t mean a thing: Molecular data suggest a new taxonomy for two pacific species of sea hares (Mollusca: Opisthobranchia, Aplysiidae). Pac. Sci. 67, 283–294 (2013).Article 

Google Scholar 
WoRMS Editorial Board. World Register of Marine Species. https://www.marinespecies.org at VLIZ. Accessed 10 Jan 2022 (2022).Barco, A. et al. A molecular phylogenetic framework for the Muricidae, a diverse family of carnivorous gastropods. Mol. Phylogenet. Evol. 56, 1025–1039 (2010).CAS 
PubMed 
Article 

Google Scholar 
Houart, R. Description of eight new species and one new genus of Muricidae (Gastropoda) from the Indo-West Pacific. Novapex 18, 81–103 (2017).
Google Scholar 
Shao, K.-T. & Chung, K.-F. The National Checklist of Taiwan (Catalogue of Life in Taiwan, TaiCoL). GBIF. https://www.gbif.org/dataset/1ec61203-14fa-4fbd-8ee5-a4a80257b45a (2021).Gaitán-Espitia, J. D., González-Wevar, C. A., Poulin, E. & Cardenas, L. Antarctic and sub-Antarctic Nacella limpets reveal novel evolutionary characteristics of mitochondrial genomes in Patellogastropoda. Mol. Phylogenet. Evol. 131, 1–7 (2019).PubMed 
Article 
CAS 

Google Scholar 
Feng, J. et al. Comparative analysis of the complete mitochondrial genomes in two limpets from Lottiidae (Gastropoda: Patellogastropoda): rare irregular gene rearrangement within Gastropoda. Sci. Rep. 10, 19277. https://doi.org/10.1038/s41598-020-76410-w (2020).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Xu, T., Qi, L., Kong, L. & Li, Q. Mitogenomics reveals phylogenetic relationships of Patellogastropoda (Mollusca, Gastropoda) and dynamic gene rearrangements. Zool. Scr. 51, 147–160 (2022).Article 

Google Scholar 
Ranjard, L. et al. Complete mitochondrial genome of the green-lipped mussel, Perna canaliculus (Mollusca: Mytiloidea), from long nanopore sequencing reads. Mitoch. DNA Part B 3, 175–176 (2018).Article 

Google Scholar 
Sun, J. et al. The Scaly-foot Snail genome and implications for the origins of biomineralised armour. Nat. Commun. 11, 1657. https://doi.org/10.1038/s41467-020-15522-3 (2020).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Dixit, B., Vanhoozer, S., Anti, N. A., O’Connor, M. S. & Boominathan, A. Rapid enrichment of mitochondria from mammalian cell cultures using digitonin. MethodsX 8, 101197. https://doi.org/10.1016/j.mex.2020.101197 (2021).Article 
PubMed 

Google Scholar 
Wanner, N., Larsen, P. A., McLain, A. & Faulk, C. The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing. BMC Genomics 22, 726. https://doi.org/10.1186/s12864-021-08046-7 (2021).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Malukiewicz, J. et al. Genomic skimming and nanopore sequencing uncover cryptic hybridization in one of world’s most threatened primates. Sci. Rep. 11, 17279. https://doi.org/10.1038/s41598-021-96404-6 (2021).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Kipp, E. J. et al. Nanopore adaptive sampling for mitogenome sequencing and bloodmeal identification in hematophagous insects. bioRxiv. https://doi.org/10.1101/2021.11.11.468279 (2021).Article 
PubMed 
PubMed Central 

Google Scholar 
Sereika, M. et al. Oxford Nanopore R10.4 long-read sequencing enables near-perfect bacterial genomes from pure cultures and metagenomes without short-read or reference polishing. bioRxiv. https://doi.org/10.1101/2021.10.27.466057 (2021).Article 

Google Scholar 
Oxford Nanopore. Nanopore Community. https://nanoporetech.com/community. Last accessed 05 May 2022 (2022).Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Li, H. Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Vaser, R., Sović, I., Nagarajan, N. & Šikić, M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 27, 737–746 (2017).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Oxford Nanopore. medaka. https://github.com/nanoporetech/medaka. Last accessed 05 May 2022 (2022).Walker, B. J. et al. Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9, e112963. https://doi.org/10.1371/journal.pone.0112963 (2014).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Faust, G. G. & Hall, I. M. SAMBLASTER: Fast duplicate marking and structural variant read extraction. Bioinformatics 30, 2503–2505 (2014).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Pedersen, B. S. & Quinlan, A. R. Mosdepth: Quick coverage calculation for genomes and exomes. Bioinformatics 34, 867–868 (2018).CAS 
PubMed 
Article 

Google Scholar 
Tsai, I. J. Genome skimming exercise (last updated 2022.04.14). https://introtogenomics.readthedocs.io/en/latest/emcgs.html (2022).Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Vaidya, G., Lohman, D. J. & Meier, R. SequenceMatrix: Concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27, 171–180 (2011).PubMed 
Article 

Google Scholar 
Edler, D., Klein, J., Antonelli, A. & Silvestro, D. raxmlGUI 2.0: A graphical interface and toolkit for phylogenetic analyses using RAxML. Methods Ecol. Evol. 12, 373–377 (2021).Article 

Google Scholar 
Rabiee, M., Sayyari, E. & Mirarab, S. Multi-allele species reconstruction using ASTRAL. Mol. Phylogenet. Evol. 130, 286–296 (2019).PubMed 
Article 

Google Scholar 
Rambaut, A. FigTree, version 1.4.4. http://tree.bio.ed.ac.uk/software/figtree/ (2018).Hackl, T. & Ankenbrand, M. J. gggenomes: A Grammar of Graphics for Comparative Genomics. https://github.com/thackl/gggenomes (2022).Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).CAS 
PubMed 
Article 

Google Scholar 
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549 (2018).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar  More

Theodor, J. M., Erfort, J. & Métais, G. The earliest artiodactyls: Diacodexeidae, Dichobunidae, Homacodontidae, Leptochoeridae and Raoellidae. in Evolution of Artiodactyls (eds. Prothero, D.R. & Foss, S. E.). 32–58. (Johns Hopkins University, 2007).Badiola, A. et al. The role of new Iberian finds in understanding European Eocene mammalian paleobiogeography. Geol. Acta. 7(1–2), 243–258 (2009).
Google Scholar 
Boivin, M. et al. New material of Diacodexis (Mammalia, Artiodactyla) from the early Eocene of Southern Europe. Geobios 51(4), 285–306 (2018).Article 

Google Scholar 
Ellenberger, P. Sur les empreintes de pas des gros mammiféres de l’Eocene supérieur de Garrigues-Ste-Eulalie (Gard). Palaeovertebr. Mém. Jubil. R. Lavocat. 13, 37–78 (1980).
Google Scholar 
Santamaría, R. L. G. & Casanovas-Cladellas, M. L. Nuevos yacimientos con icnitas de mamíferos del Oligoceno de los alrededores de Agramunt (Lleida, España). Paleont. Evol. 23, 141–152 (1990).
Google Scholar 
Sarjeant, W. A. S. & Langston, W. Jr. Vertebrate footprints and invertebrate traces from the Chadronian (Late Eocene) of Trans-Pecos. Texas. Mem. Mus. Bull. 36, 1–86 (1994).
Google Scholar 
Costeur, L., Balme, C. & Legal, S. Early Oligocene mammal tracks from southeastern France. Hist. Biol. 16(4), 257–267. https://doi.org/10.1080/10420940902953197 (2009).Article 

Google Scholar 
Wroblewski, A.F.-J. & Gulas-Wroblewski, B. E. Earliest evidence of marine habitat use by mammals. Sci. Rep. 11, 8846. https://doi.org/10.1038/s41598-021-88412-3 (2021).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Fornós, J. J. & Pons-Moya, J. Icnitas de Myotragus balearicus del yacimiento de Ses Piquetes (Santanyi, Mallorca). Bol. Soc. Hist. Nat. Balears 26, 135–144 (1982).
Google Scholar 
Flor, G. Estructuras de deformación por pisadas de cérvidos en la duna cementada de Gorliz (Vizcaya, N de España). Rev. Soc. Geol. Esp. 2(1–2), 23–29 (1989).
Google Scholar 
Fornós, J. J., Bromley, R. G., Clemmensen, L. B. & Rodríguez-Perea, A. Tracks and trackways of Myotragus balearicus Bate (Artiodactyla, Caprinae) in Pleistocene aeolianites from Mallorca (Balearic Islands, Western Mediterranean). Palaeogr. Palaeocl. Palaeoecol. 180, 277–313 (2002).ADS 
Article 

Google Scholar 
Neto de Carvalho, C. Vertebrate tracksites from the Mid-Late Pleistocene eolianites of Portugal: The first record of elephant tracks in Europe. Geol. Q. 53(4), 407–414 (2009).
Google Scholar 
Neto de Carvalho, C., Saltão, S., Ramos, J. C. & Cachão, M. Pegadas de Cervus elaphus nos eolianitos plistocénicos da ilha do Pessegueiro (SW Alentejano, Portugal). Ciênc. Terra 5, 36–40 (2003).
Google Scholar 
Neto de Carvalho, C., Figueiredo, S. & Belo, J. Vertebrate tracks and trackways from the Pleistocene eolianites of SW Portugal. Commun. Geol. 103(1), 101–116 (2016).CAS 

Google Scholar 
Neto de Carvalho, C. et al. Paleoecological implications of large-sized wild boar tracks recorded during the Last Interglacial (MIS 5) at Huelva (SW Spain). Palaios https://doi.org/10.2110/palo.2020.058 (2020).Article 

Google Scholar 
Neto de Carvalho, C. et al. First vertebrate tracks and palaeoenvironment in a MIS-5 context in the Doñana National Park (Huelva, SW Spain). Quat. Sci. Rev. https://doi.org/10.1016/j.quascirev.2020.106508 (2020).Article 

Google Scholar 
Cardoso, J. L. Les grands mammifères du Pléistocène supérieur du Portugal. Essai de synthése. Geobios 29(2), 235–250 (1996).Article 

Google Scholar 
Sala, M. T. N., Pantoja, A., Arsuaga, J. L. & Algaba, M. Presencia de bisonte (Bison priscus Bojanus, 1827) y uro (Bos primigenius Bojanus, 1827) en las cuevas del Búho y de la Zarzamora (Segovia, España). Munibe 61, 43–55 (2010).
Google Scholar 
Figueiredo, S. D. & Sousa, M. F. O registo de bovídeos plistocénicos em Portugal. in Livro de Resumos das IV Jornadas de Arqueologia do Vale do Tejo. Vol. 10. (Centro Português de Geo-História e Pré-História, 2017).Barr, K. & Bell, M. Neolithic and Bronze age ungulate footprint-tracks of the Severn Estuary: Species, age, identification and the interpretation of husbandry practices. Environ. Archaeol. 22(1), 1–15 (2017).Article 

Google Scholar 
Bell, M. Making One’s Way in the World (Oxbow Books, 2020).Book 

Google Scholar 
Díaz-Martínez, I. et al. Multi-aged social behavior based on artiodactyl tracks in an early Miocene palustrine wetland (Ebro Basin, Spain). Sci. Rep. 10, 1099. https://doi.org/10.1038/s41598-020-57438-4 (2020).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Quintana, J. Descripción de un rastro de Myotragus e icnitas de Hypnomys del yacimiento cuaternario de Ses Penyes d’es Perico (Ciutadella de Menorca, Balears). Paleont. Evol. 26–27, 271–279 (1993).
Google Scholar 
Muñiz, F. et al. Following the last Neanderthals: Mammal tracks in Late Pleistocene coastal dunes of Gibraltar (S Iberian Peninsula). Quat. Sci. Rev. 217, 297–309 (2019).ADS 
Article 

Google Scholar 
Altuna, J. Fauna de mamíferos de los yacimientos prehistóricos de Guipúzcoa. Con catálogo de los mamíferos cuaternarios del Cantábrico y del Pirineo occidental. Munibe 24, 1–464 (1972).
Google Scholar 
López González, F., Vila Taboada, M. & Grandal d’Anglade. Sobre los grandes bóvidos pleistocenos (Bovidae, Mammalia) en el NO de la Península Ibérica. Cad. Lab. Xeol. Laxe 24, 57–71 (1999).Sommer, R. S., Kalbe, J., Ekström, J., Benecke, N. & Liljengren, R. Range dynamics of the reindeer in Europe during the last 25,000 years. J. Biogeogr. 41, 298–306. https://doi.org/10.1111/jbi.12193 (2014).Article 

Google Scholar 
Whittle, A., Antoine, S., Gardiner, N., Milles, A. & Webster, A. Two Later Bronze Age occupations and an Iron Age channel on the Gwent foreshore. Bull. Board Celt. Stud. 36, 200–223 (1989).
Google Scholar 
Aldhouse-Green, S. et al. Prehistoric human footprints from the Severn Estuary at Uskmouth and Magor Pill, Gwent, Wales. Archae. Cambr. 141, 4–55 (1992).
Google Scholar 
Allen, J. R. L. Subfossil mammalian tracks (Flandrian) in the Severn Estuary, S.W. Britain: Mechanics of formation, preservation and distribution. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 352(1352), 481–518 (1997).ADS 
PubMed Central 
Article 

Google Scholar 
Bell, M. Prehistoric coastal communities: the Mesolithic in western Britain. in CBA Research Report. Vol. 149. (Council for British Archaeology, 2007).Bell, M. The Bronze Age in the Severn estuary. in Research Report. Vol. 172. (Council for British Archaeology, 2013).Scales, R. Footprint tracks of people and animals. in Prehistoric Coastal Communities: The Mesolithic in Western Britain (ed. Bell, M.). Vol. 149. 139–147. CBA Research Report 149. (Council of British Archaeology, 2007).Roberts, G. Ephemeral, subfossil mammalian, avian and hominid footprints within Flandrian sediment exposures at Formby Point, Sefton Coast, North West England. Ichnos 16, 33–48 (2009).Article 

Google Scholar 
Waddington, C. Low Hauxley, Northumberland: A review of archaeological interventions and site condition. Archael. Res. Serv. 2010/25 (2010).Eadie, G. & Waddington, C. Rescue recording of an eroding inter-tidal peat bed at Lower Hauxley, Northumberland (6109). Archael. Res. Serv. 2013/17 (2013).Burns, A. The prehistoric footprints at Formby. in Sefton Coast Landscape Partnership Scheme (2014).Pandolfi, L., Petronio, C. & Salari, L. Bos primigenius Bojanus, 1827 from the Early Late Pleistocene deposit of Avetrana (southern Italy) and the variation in size of the species in southern Europe: Preliminary report. J. Geol. Res. https://doi.org/10.1155/2011/245408 (2011).Article 

Google Scholar 
Currant, A. P. A review of the Quaternary mammals of Gibraltar. in Neanderthals on the Edge: 150th Anniversary Conference of the Forbes’ Quarry Discovery, Gibraltar (eds. Stringer, C. B., Barton, R. N. E. & Finlayson, J.C.). 201–206. (Oxbow, 2000).Penela, A. J. M. Los grandes mamíferos del yacimiento acheulense de la Solana del Zamborino, Fonelas (Granada, España). Antr. Paleoecol. Hum. 5, 29–187 (1988).
Google Scholar 
Bataille, G. Prehistoric Painting. Lascaux or the Birth of Art (MacMillan, 1980).
Google Scholar 
Zazo, C. et al. Palaeoenvironmental evolution of the Barbate-Trafalgar coast (Cadiz) during the last ~140 ka: Climate, sea-level interactions and tectonics. Geomorphology 100, 212–222 (2008).ADS 
Article 

Google Scholar 
Zazo, C. et al. Landscape evolution and geodynamic controls in the Gulf of Cadiz (Huelva coast, SW Spain) during the Late Quaternary. Geomorphology 68, 269–290. https://doi.org/10.1016/j.geomorph.2004.11.022 (2005).ADS 
Article 

Google Scholar 
García de Domingo, A., González Lastra, J., Hernaiz Huerta, P. P., Zazo Cardeña, C. & Goy Goy, J. L. Mapa Geológico de la Hoja No. 1073 (Vejer de la Frontera). Mapa Geológico de España a Escala 1:50.000. Segunda Serie (MAGNA). http://info.igme.es/cartografiadigital/geologica/Magna50Hoja.aspx?Id=1073&language=es (©Instituto Geológico y Minero de España (IGME), 1990).Demathieu, G., Ginsburg, L., Guérin, C. & Truc, G. Étude paléontologique, ichnologique et paléoécologique du gisêment oligocène de Saignon (bassin d’Apt, Vaucluse). Bull. Mus. Natl. Hist. Nat. 6(2), 153–183 (1984).
Google Scholar 
Bang, P. & Dahlstrøm, P. Animal Tracks and Signs (Oxford University Press, 2001).
Google Scholar 
Wright, E. The History of the European Aurochs (Bos primigenius) from the Middle Pleistocene to Its Extinction: An Archaeological Investigation of Its Evolution, Morphological Variability and Response to Human Exploitation. (PhD. Thesis, University of Sheffield, 2013).Koenigswald, W. V., Sander, P. M. & Walders, M. The Upper Pleistocene tracksite Bottrop-Welheim (Germany). Acta Zool. Cracov. 39(1), 235–244 (1996).
Google Scholar 
Martínez-Navarro, B., Rook, L., Papini, M. & Libsekal, Y. A new species of bull from the Early Pleistocene paleoanthropological site of Buia (Eritrea): Parallelism on the dispersal of the genus Bos and the Acheulian culture. Quat. Intern. 212(2), 169–175. https://doi.org/10.1016/j.quaint.2009.09.003 (2010).Article 

Google Scholar 
Van Vuure, C. Retracing the Aurochs: History, Morphology and Ecology of an Extinct Ox (Coronet Books, 2005).
Google Scholar 
Franks, J. W. Interglacial deposits at Trafalgar Square, London. N. Phytologist 59(2), 145–152 (1960).Article 

Google Scholar 
Estévez, J. & Saña, M. Auerochsenfunde auf der Iberischen Halbinsel. in Archäologie und Biologie des Auerochsen (ed. Weniger, G.-C.) (Neanderthal Museum, 1999).Mona, S. et al. Population dynamic of the extinct European aurochs: Genetic evidence of a north-south differentiation pattern and no evidence of post-glacial expansion. BMC Evol. Biol. 10, 1–13 (2010).Article 
CAS 

Google Scholar 
Rodríguez-Vidal, J. et al. Undrowning a lost world—The Marine isotope stage 3 landscape of Gibraltar. Geomorphology 203, 105–114 (2013).ADS 
Article 

Google Scholar 
Pfeiffer, T. Systematic relationship between the Bovini with special references to the fossil taxa Bos primigenius Bojanus and Bison priscus Bojanus. in Archäologie und Biologie des Auerochsen (ed. Weniger, G.-C.). 59–70. (Neanderthal Museum, 1999).Zazula, G. D. et al. A late Pleistocene steppe bison (Bison priscus) partial carcass from Tsiigehtchic, Northwest Territories, Canada. Quat. Sci. Rev. 28(25–26), 2734–2742 (2009).ADS 
Article 

Google Scholar 
Boeskorov, G. G. et al. The Yukagir Bison: The exterior morphology of a complete frozen mummy of the extinct steppe bison, Bison priscus from the early Holocene of northern Yakutia, Russia. Quat. Intern. 406, 94–110. https://doi.org/10.1016/j.quaint.2015.11.084 (2016).Article 

Google Scholar 
Ekström, J. The Late Quaternary History of the Urus (Bos primigenius Bojanus 1827) in Sweden. PhD. Thesis. (Lund University, 1993).Grange, T. et al. The evolution and population diversity of Bison in Pleistocene and Holocene Eurasia: Sex matters. Diversity 10(3), 65. https://doi.org/10.3390/d10030065 (2018).Article 

Google Scholar 
Castaños, J., Castaños, P. & Murelaga, X. First complete skull of a Late Pleistocene Steppe Bison (Bison priscus) in the Iberian Peninsula. Ameghiniana 53(5), 543–551. https://doi.org/10.5710/amgh.03.06.2016.2995 (2016).Article 

Google Scholar 
Álvarez-Lao, D. J., Kahlke, R.-D., García, N. & Mol, D. The Padul mammoth finds: On the southernmost record of Mammuthus primigenius in Europe and its southern spread during the Late Pleistocene. Palaeogeogr. Palaeocl. Palaeoecol. 278(1–4), 57–70 (2009).ADS 
Article 

Google Scholar 
Loope, D. B. Recognizing and utilizing vertebrate tracks in cross section: Cenozoic hoofprints from Nebraska. Palaios 1, 141–151 (1986).ADS 
Article 

Google Scholar 
Albarella, U., Dobney, K. & Rowley-Conwy, P. Size and shape of the Eurasian wild boar (Sus scrofa), with a view to the reconstruction of its Holocene history. Environ. Archaeol. 14, 103–136 (2009).Article 

Google Scholar 
Davis, S. J. M. The effects of temperature change and domestication on the body size of Late Pleistocene to Holocene mammals of Israel. Palaeobiology 7, 101–114 (1981).Article 

Google Scholar 
Cerilli, E. & Petronio, C. Biometrical variations of Bos primigenius Bojanus 1827 from middle Pleistocene to Holocene. in Proceedings of the International Symposium on ‘Ongulés/Ungulates’, Toulouse. 37–42. (1991).Davis, S. J. M. & Mataloto, R. Animal remains from Chalcolithic of São Pedro (Redondo, Alentejo): Evidence for a crisis in the Mesolithic. Rev. Port. Arqueol. 15, 47–85 (2012).
Google Scholar 
Mariezkurrena, K. & Altuna, J. Biometría y diformismo sexual en el esqueleto de Cervus elaphus würmiense, postwürmiense y actual del Cantábrico. Munibe (Antr.-Arkeol.) 35, 203–246 (1983).
Google Scholar 
Davis, S. J. M. The mammals and birds from the Gruta do Caldeirão, Portugal. Rev. Port. Arqueol. 5, 29–98 (2002).CAS 

Google Scholar 
Barr, K. Prehistoric Avian, Mammalian and H. sapiens Footprint—Tracks from Intertidal Sediments as Evidence of Human Palaeoecology. PhD. Thesis. (University of Reading, 2018).Hall, J. G. A comparative analysis of the habitat of the extinct aurochs and other prehistoric mammals in Britain. Ecography 31, 187–190 (2008).Article 

Google Scholar 
Bicho, N. F., Gibaja, J. F., Stiner, M. & Manne, T. L. Paléolithique supérieur au sud du Portugal: Le site du Vale do Boi. L’antropologie 114, 48–67 (2010).
Google Scholar 
Bicho, N. & Haws, J. The Magdelian in central and southern Portugal: Human ecology at the end of the Pleistocene. Quatern. Int. 272–273, 6–16 (2012).Article 

Google Scholar 
Cortés-Sánchez, M. et al. Palaeoenvironmental and cultural dynamics of the coast of Málaga (Andalusia, Spain) during the Upper Pleistocene and early Holocene. Quatern. Sci. Rev. 27, 2176–2193 (2008).ADS 
Article 

Google Scholar 
Bohórquez, A. M., Ruiz, C. B., Caparrós, M. & Moigne, A. M. Una aproximación a la compreensión de la fauna de macromamiferos de la Cueva de Zafarraya (Alcaucín, Málaga). Menga Rev. Prehist. Andalucía 3, 83–105 (2012).
Google Scholar 
Ripoll, M. P. & Maroto, J. L. fauna mediterránea durante el Pleistoceno superior del Mediterráneo Ibérico. Kobie Serie Anejo 18, 27–38 (2021).
Google Scholar 
Lazo, A. Ranging behaviour of feral cattle (Bos taurus) in Doñana National Park, S.W. Spain. J. Zool. 236(3), 359–369. https://doi.org/10.1111/j.1469-7998.1995.tb02718.x (1995).Article 

Google Scholar 
AliceVision. Meshroom: V2021.1.0. GNU-GPL. https://alicevision.org/ (2020).OpenDroneMap Authors ODM. A Command Line Toolkit to Generate Maps, Point Clouds, 3D Models and DEMs from Drone, Balloon or Kite Images. OpenDroneMap/ODM GitHub Page. https://github.com/OpenDroneMap/WebODM (2020).Cignoni, P., Callieri, M., Corsini, M., Dellepiane, M., Ganovelli, F. & Ranzuglia, G. MeshLab: an open-source mesh processing tool. in Sixth Eurographics Italian Chapter Conference. 129–136. MeshLab V. 2020.12. https://www.meshlab.net/ (2008).CloudCompare. V2.11.0. GNU-GPL. https://www.cloudcompare.org (2020).Zhukov, S., Iones, A. & Kronin, G. An ambient light illumination model. Render. Tech. 98, 45–55 (1998).Article 

Google Scholar 
Vergne, R., Pacanowski, R., Barla, P., Granier, X., & Schlick, C. Radiance scaling for versatile surface enhancement. in Proceedings of the 2010 ACMSIGGRAPH Symposium on Interactive 3D Graphics and Games.143–150. (2010). More

Genome characteristics of ancient obligate symbiontsWe first tested the hypothesis that each of the ant lineages sequenced in our study (Cardiocondyla, Formica, and Plagiolepis) hosts its own ancient strictly vertically transmitted symbiont that have co-speciated with its host, which has been shown previously in the Camponotus- Blochmannia symbiosis [17]. To address this aim, we compared the genomes of symbionts from 13 species of ants, 8 from our study combined with 5 previously published genomes, representing four independently evolved symbioses. This includes symbionts from three Formica, two Plagiolepis, and an additional three Cardiocondyla species that we sequenced, in addition to four previously published genomes from Blochmannia, the obligate symbiont of Camponotus ants, and the one pre-existing Westeberhardia genome from Cardiocondyla obscurior [8, 18,19,20,21].We found the gene order of single copy orthologs in symbionts is highly conserved in ant species belonging to the same genus (Fig. 1). This type of structural stability of genomes is typically found in symbionts that have been strictly vertically transmitted within a matriline [22] and has been documented in the obligate symbionts of whiteflies, psyllids, cockroaches, and aphids [23,24,25,26]. In contrast, genome structure differed substantially between symbionts from different ant genera (Fig. 1, Fig. S1). We also find that the host and symbiont phylogenies are in general concordance in Cardiocondyla (TreeMap: p = 0.00100 CI95% = [0.00000, 0.00424]), and in Formica the topologies suggest co-segregation, although there were too few nodes to confirm this statistically (Fig. S2). Together, this strongly suggests the symbioses in all four ant lineages are independently acquired ancient associations that have co-speciated with their hosts.Fig. 1: Structural stability of ant symbiont genomes.A Ant lineages known to host bacteriocyte-associated symbionts (red font) and lineages not known to (black font), based on [91]. Outgroup (grey font) not examined in this study. B Visualisation of symbiont genomes showing conservation of gene order in the symbionts of ant species that belong to the same genus. Blocks show the locations of single copy orthologs in the symbiont genome, lines connect shared single copy orthologs between genomes. All genomes and annotations were generated in this study except the Blochmannia symbionts and the Westeberhardia strain from C. obscurior [8, 18,19,20,21]. *Evidence of symbionts were detected in embryos of Anoplolepis [91] but it is unclear if they are localised in bacteriocytes in larvae and adults.Full size imageIn addition, our phylogenetic analysis reveals that all four symbiont lineages originate from a single clade, the Sodalis-allied bacteria (Fig. 2). This demonstrates that ant lineages that host bacteriocytes-associated symbionts have convergently acquired related bacteria, which differs from previous findings based on limited taxa and genes [27]. All of the symbionts have evidence of advanced genome reduction, which is characterized by reduced genome size, GC content, and number of coding sequences, similar to other ancient obligate symbionts of insects [4]. The three strains of Westeberhardia we analysed have extremely small (0.45–0.53 Mb) GC depleted genomes (22–26%) that are similar to the figures reported for the strain in Cardiocondyla obscurior [8]; confirming that they have some of the smallest genomes of any known gammaproteobacterial endosymbiont (Fig. 2). By comparison, the symbionts in Formica and Plagiolepis have genomes around twice the size (1.37–1.38 Mb) and GC content (~41%) of Westeberhardia (Fig. 2) raising the possibility that they are in an earlier stage of genome reduction than both Westeberhardia and Blochmannia. The Formica and Plagiolepis symbionts have a similar size, GC range, and number of coding sequences as known obligate symbionts such as Candidatus Doolittlea endobia [28], and several Serratia symbiotica lineages that are co-obligate symbionts in aphids [29].Fig. 2: Phylogenetic origins of the bacteriocyte-associated symbionts of ants.A pruned phylogeny of gammaproteobacterial endosymbionts based on Fig. S8. The phylogeny is based on a dayhoff6 recoded amino acid alignment of 72 genes analysed using phylobayes. Bar plots represent the size (in Mbp) and GC content of symbiont genomes. Bars are colour coded to represent hypothesised relationships between symbionts and hosts. Species names highlighted in red in the phylogeny indicate the four bacteriocyte-associated symbionts of ants. Genomes sequenced and assembled for this paper are referenced as ‘novel symbiont’ lineages. Full phylogenies with node support and branch lengths are available as Fig. S8 and Fig. S9, respectively.Full size imageBacteriocyte-associated endosymbiontsUsing fluorescent in situ hybridisation, we determine whether the Sodalis-allied symbionts we sequenced are localised in bacteriocytes to confirm they are the associations first observed by Lillienstern and Jungen in the early 1900’s [10, 11].Consistent with Lilienstern’s findings [11], we found the Sodalis symbiont in Formica ants is distributed in bacteriocytes surrounding the midgut in adult queens (Fig. 3A). The symbionts are also found in eggs and ovaries of adult queens, indicating they are vertically transmitted from queens to offspring (Fig. 3B–C). Sectioning of F. cinerea larvae shows the bacteriocytes to be arranged in a single layer of cells surrounding the midgut, as well as in clusters of bacteriocytes closely situated to the midgut (Fig. 3D–D’). In adult Plagiolepis queens, the symbiont was not present in bacteriocytes around the midgut, suggesting the symbiont may play a more substantive role in larval development or pupation and then migrates to the ovaries prior to or during metamorphosis. Apart from that, the localisation of the symbiont in Plagiolepis was the same as in Formica – symbionts in larval midgut bacteriocytes, ovaries and eggs (Fig. S3) – supporting Jungen’s cytological findings [10]. Bacteriocytes are also found surrounding the midgut in Camponotus and Cardiocondyla ants [8, 30, 31] indicating the symbionts are localised in a similar manner in all four ant lineages.Fig. 3: Anatomical localisation of symbiont in Formica ants.Fluorescent in situ hybridisation (FISH) generated images showing the localisation of symbionts in Formica ants. A–C Whole mount FISH of Formica fusca: queen gut (A, crop and proventriculus on the right, midgut in the middle, hindgut and Malpighian tubules on the left), ovaries (B) and egg (C). DAPI staining of host tissue in blue, symbiont stained in red. D–D’. FISH on transverse cytological sections of Formica cinerea larva midgut. DAPI staining only, showing host nuclei of bacteriocytes in a single layer surrounding the midgut (D), and a magnified region highlighting symbionts in red localised within bacteriocytes and in a bacteriome (D’). A FISH image of the symbiont-free midgut of a Formica lemani queen is available as Fig. S11.Full size imageConservation of metabolic functions in ant endosymbiontsDespite on-going genome reduction, obligate symbionts of insects typically retain gene networks required for maintaining the symbiosis with their host, such as pathways for synthesising essential nutrients. This has resulted in the symbionts of sap- and blood-feeding insects converging on genomes that have retained the same sets of metabolic pathways – to synthesise essential nutrients missing in their hosts’ diets [32, 33]. Here we compare the metabolic pathways retained in the reduced genomes of the four bacteriocyte-associated symbionts of ants to test the hypothesis that have been acquired to perform similar functions. For this, we assess whether they have consistently retained metabolic pathways to synthesise the same key nutrients. Two major patterns stand out.The first major pattern we find is that the four ant symbionts have all retained the shikimate pathway, which produces chorismate, along with most of the steps necessary to produce tyrosine from this precursor (Tables 1 and S2). Both the symbiont of Formica and Westeberhardia each lack one of the genes required to produce tyrosine. However, in Westeberhardia it is believed the host encodes the missing gene, supplying the enzyme to fulfil the final step of the pathway [8]. Intriguingly, we find that this gene is also present in the Formica ant genomes (Fig. S4). In addition, all symbionts except Westeberhardia can produce phenylalanine which is a precursor that can be converted to tyrosine by their hosts [5, 34, 35]. Tyrosine is important for insect development as it is used to produce L-DOPA, which is a key component of insect cuticles [5]. Tyrosine is also a precursor for melanin synthesis, which is important in protection against pathogens, and plays a fundamental role in neurotransmitters and hormone production [36, 37]. In several species of ants, weevils, and other beetles, symbionts are believed to provision hosts with tyrosine, and it has been shown experimentally in several of these species that removal or inhibition of their symbionts causes cuticle development to suffer [38,39,40,41,42,43,44,45,46,47]. A thicker cuticle has been shown to help symbiont-carrying grain beetles resist desiccation [43], and defend against natural enemies [48]. However, female reproduction is delayed at higher humidity, suggesting a metabolic cost to carrying their Bacteroidetes symbiont. Tyrosine provisioning is also the likely function of Westeberhardia in Cardiocondyla ants, as this is one of the few nutrient pathways retained in this symbiont. Our analysis confirms the shikimate pathway, and the symbiont portions of the tyrosine pathway, have been retained in Westeberhardia from three phylogenetically diverse Cardiocondyla lineages, providing additional support for this hypothesis. In addition to tyrosine, most of the symbionts have retained the capacity to produce vitamin B9 (tetrahydrofolate) and all can perform the single step conversions necessary to produce alanine and glycine. However, our gene enrichment analysis indicates that tyrosine, and the associated chorismate biosynthetic process, are the only enriched vitamin or amino acid pathways that are shared by all of the symbiont genomes (Table S1). This suggests that provisioning of tyrosine by symbionts, or tyrosine precursors, is of general importance across all bacteriocyte-associated symbioses of ants.Table 1 Comparison of the retention and losses of metabolic pathways for key nutrients across ant symbionts.Full size tableThe second major pattern emerging from our comparative analysis is that there are clear differences in the pathways lost or retained across symbionts (Tables 1 and S2). This is most evident when comparing Blochmannia with Westeberhardia, the latter of which has lost the capacity to synthesise most essential nutrients. The symbionts of Formica or Plagiolepis, in contrast, have retained the capacity to synthesise many of the same amino acids and B vitamins as Blochmannia, suggesting they may perform similar functions for their hosts. However, Blochmannia has retained more biosynthetic pathways, particularly those involved in the synthesis of essential amino acids. Previous experimental studies have confirmed that Blochmannia provisions hosts with essential amino acids [1]. The absence of several core essential amino acids in the Formica and Plagiolepis symbionts may reflect differences in the dietary ecology of the different ant genera. The retention of the full complement of essential amino acids biosynthetic pathways in the highly reduced genome of Blochmannia does however indicate it plays a more substantive nutrient-provisioning role for its hosts than the other ant symbionts we investigated.Previous work on the extracellular gut symbionts of several arboreal ant lineages identified nitrogen recycling via the urease operon as a function that may be of key importance for ant symbioses [1, 2, 49, 50]. However, we do not find any evidence that the symbionts of Formica, Plagiolepis, or Cardiocondyla play a role in nitrogen recycling via the urease operon (Table 1). This suggests that nitrogen recycling may play an important role for more strictly herbivorous ants, such as Cephalotes. Our results indicate tyrosine supplementation by symbionts may be universally required for essential physiological process across a broader range of ant lineages.The origins and losses of symbioses in Formica and Cardiocondyla
We investigated the presence of the symbiont in phylogenetically diverse Formica and Cardiocondyla species to identify the evolutionary origins and losses of the symbiosis. Although the symbiont in Plagiolepis was present in P. pygmaea and two unknown Plagiolepis species we investigated, we did not have sufficient phylogenetic sampling to assess the origins of the symbiosis.In Formica, we find the symbiont is restricted to a single clade in the paraphyletic Serviformica group (Fig. 4A). The species in this clade are socially polymorphic, forming both multi-queen and single-queen colonies [51]. Based on a previously dated phylogeny of Formica ants, we estimate the symbiosis originated approximately 12–22 million years ago [52]. In Cardiocondyla, the symbiosis is widespread throughout the genus. The prevalence of the symbiont in Cardiocondyla, in combination with its highly reduced genome, suggests it is a very old association that likely dates back to the origins of the ant genus some 50–75 million years ago [53]. The symbiont was also absent in two clades, the argentea and palearctic groups (Fig. 4B). This may represent true evolutionary losses in these clades. It may be that these losses are linked to a notable change in social structure in these two Cardiocondyla clades, having gone from the ancestral state of maintaining multi-queen colonies to single-queen colonies [54], however it is not clear how this could impact the symbiosis.Fig. 4: Phylogenetic distribution of symbionts in queens of Formica and Cardiocondyla ants.Pie charts represent the proportion of Formica (A) and Cardiocondyla (B) queens sampled that carried the symbiont (red) and those that did not (grey). Numbers represent the number of queens positive for the symbiont over the total number of queens sampled (intracolony infection frequencies in Table S5). See the supplementary material for the statistical testing of differences in prevalence within Serviformica Clade 1. The Formica phylogeny is based on [81] and the Cardiocondyla phylogeny is based on [83], with major clades highlighted. Dashed lines indicate species added to the original source phylogeny based on additional published phylogenies (specified in the Taxonomic Analysis section of the methods). Starred names are provisional names of a recognised morphospecies to be described by B. Seifert.Full size imageEvidence of variation in colony-level dependence on symbiontsObservations from individual studies on F. cinerea and F. lemani [10, 11], as well as Cardiocondyla obscurior [8], reported rare cases of ant queens not harbouring their symbionts in nature. This called into question the degree to which these insects depend on symbionts for nutrients, and whether the symbiosis may be breaking down in certain host lineages. However, given the limited number of species and populations studied, it is unclear how often colonies are maintained with uninfected queens, and whether this varies across species, suggesting species may differ in their dependence on their symbiont. To answer this question, we assessed the presence of the symbionts in 838 samples from 147 colonies of phylogenetically diverse Formica and Cardiocondyla species collected across 8 countries.Our investigation reveals the natural occurrence of uninfected queens is a widespread phenomenon in many Formica and Cardiocondyla species (Fig. 4). We confirmed the absence of symbionts in queens, and that they have not been replaced with another bacterial or fungal symbiont, using multiple approaches including diagnostic PCR, metagenomic and deep-coverage amplicon sequencing (Tables S3,  S4, Figs. S5, S6). Wolbachia was high in relative abundance, especially in Formica ants, but was not sufficiently present across samples to be a feasible replacement. There was also clear evidence of variation across host species. In Formica, queens and workers of F. fusca always carried the symbiont, whereas queens and workers of F. lemani, F. cinerea, and F. selysi showed varying degrees of individuals not carrying the symbionts (Fig. 4A, Table S5). A similar pattern can be seen in Cardiocondyla, where queens of several species, such as C. obscurior, always carry the symbiont, compared to lower incidences in other species (Fig. 4B). Klein et al [8] identified a single C. obscurior colony with uninfected queens in Japan. However, queens of this species nearly always carry the symbiont in nature.The degradation and eventual loss of symbionts from bacteriocytes has been reported in males, and in sterile castes of aphids and ants [55], which do not transmit symbionts to offspring. In reproductive females, bacteriocytes may degrade as a female ages; however, symbionts are typically retained at high bacterial loads in the ovaries, as this is required to maintain the symbionts within the germline [31]. All of the symbiotic ant species we investigated maintain multi-queen colonies, and the vast majority had at least one queen, often more, within a colony that carried the symbiont (Table S5). We hypothesize that species that maintain colonies with uninfected queens may be able to retain sufficient colony-level fitness with only a fraction of queens harbouring the symbiont and receiving its nutritive benefits.Dependence on symbionts in a socioecological contextThe retention of symbionts in queens and workers of some species, but not others, suggests species either differ in their dependence on symbiont-derived nutrients, or that symbionts have lost the capacity to make nutrients in certain host lineages. Our analysis of symbiont genomes did not reveal any structural differences, such as the disruption of metabolic pathways, which could explain differences in symbiont retention between host species (Table S2). This suggests differences in the retention of symbionts may reflect differences in host ecologies.In ants, which occupy a wide range of feeding niches, reliance on symbiont-derived nutrients will largely depend on lineage-specific feeding ecologies. For example, several species of arboreal Camponotus ants have been shown to be predominantly herbivorous [56]. Blochmannia, in turn, has retained the capacity to synthesise key nutrients lacking in their plant-based diets, such as essential amino acids [1]. Blochmannia is also always present in queens and workers [31], which is a testament to the importance of these nutrients for the survival of its primarily herbivorous host [13]. In contrast, Formica and Cardiocondyla species are thought to have a more varied diet [14]. Diet flexibility and altered foraging efforts may therefore reduce their reliance on a limited number of symbiont-derived nutrients allowing colonies of some species to persist with uninfected queens in certain contexts. Silvanid beetles and grain weevils, for example, can survive in the absence of their tyrosine-provisioning symbionts [38, 57, 58] when provided nutritionally balanced diets, in the laboratory [57] or in cereal grain elevators [59, 60]. Similarly, studies on Cardiocondyla and Camponotus ants have shown they can maintain sufficient colony health in the absence of their symbionts, if provided a balanced diet [31, 61]. It would be interesting to know whether species of Formica and Cardiocondyla that always carry the symbiont in nature, such as F. fusca and C. obscurior, have more restricted diets with less access to nutrients such as tyrosine, as this may explain their dependence on their symbiont for nutrients and tendency to harbour them in queens.Although it is unusual for bacteriocyte-associated symbionts to be absent in reproductive females, the fact that it is simultaneously occurring in phylogenetically diverse species from many locations suggests the symbiosis may have persisted in this manner over evolutionary time. Perhaps through diet flexibility colonies can be maintained with uninfected queens in some contexts, however we expect them to be disadvantaged in other ecological scenarios. Fluctuating environmental conditions may therefore eventually purge asymbiotic queens from lineages, allowing the symbiosis to be retained over longer periods of evolutionary time. The multiple-queen colony lifestyle in all symbiotic Formica and Cardiocondyla species we investigated may also provide an additional social buffer that limits the costs to individual queens being asymbiotic. Workers will still nourish larvae and queens without symbionts and colony fitness may be maintained through the reproductive output of nestmate queens that carry the symbiont. There may also be an adaptive explanation for the losses if, for example, metabolic costs to maintain the symbiosis trade off in a context dependent manner [44, 62, 63]. Under this scenario, maintaining a mix of infected and uninfected queens may benefit a colony by allowing for optimal reproduction under a broader range of environmental scenarios.Our data suggest that symbiotic relationships can evolve to solve common problems but also rapidly break down if the symbiosis is no longer required, or potentially when costs are too high [44]. We have identified tyrosine provisioning as a possible unifying function across bacteriocyte-associated symbionts of ants. But we have also shown species can vary in how much they depend on symbionts for nutrients. Our results demonstrate that ants have a unique labile symbiotic system, allowing us to better understand the evolutionary forces that influence the persistence and breakdown of long-term endosymbiotic mutualisms.
Candidatus Liliensternia hugann and Candidatus Jungenella plagiolepisWe propose the names Candidatus Liliensternia hugann for the Sodalis-allied symbiont found in Formica. The genus name is in honour of Margarete Lilienstern who first identified the symbiont [11]. The species name is derived from the combined first names of the first authors parents. Similarly, we propose the name of Candidatus Jungenella plagiolepis for the Plagiolepis-bound symbiont. The genus name is in honour of Hans Jungen who originally discovered the symbiont [10], and the species name is derived from Plagiolepis, the genus in which the symbiont can be found. More

Tolussi, C. E., Hilsdorf, A. W. S., Caneppele, D. & Moreira, R. G. The effects of stocking density in physiological parameters and growth of the endangered teleost species piabanha, Brycon insignis (Steindachner, 1877). Aquaculture 310, 221–228 (2010).
Google Scholar 
Wang, Y. et al. Effects of stocking density on growth, serum parameters, antioxidant status, liver and intestine histology and gene expression of largemouth bass (Micropterus salmoides) farmed in the in-pond raceway system. Aquac. Res. 51, 5228–5240 (2020).CAS 

Google Scholar 
Zahedi, S., Akbarzadeh, A., Mehrzad, J., Noori, A. & Harsij, M. Effect of stocking density on growth performance, plasma biochemistry and muscle gene expression in rainbow trout (Oncorhynchus mykiss). Aquaculture 498, 271–278 (2019).CAS 

Google Scholar 
Yousefi, M., Paktinat, M., Mahmoudi, N., Pérez-Jiménez, A. & Hoseini, S. M. Serum biochemical and non-specific immune responses of rainbow trout (Oncorhynchus mykiss) to dietary nucleotide and chronic stress. Fish Physiol. Biochem. 42, 1417–1425 (2016).CAS 
PubMed 

Google Scholar 
Duan, Y., Dong, X., Zhang, X. & Miao, Z. Effects of dissolved oxygen concentration and stocking density on the growth, energy budget and body composition of juvenile Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel). Aquac. Res. 42, 407–416 (2011).CAS 

Google Scholar 
Castillo-Vargasmachuca, S. et al. Effect of stocking density on growth performance and yield of subadult pacific red snapper cultured in floating sea cages. N. Am. J. Aquac. 74, 413–418 (2012).
Google Scholar 
Upadhyay, A. et al. Stocking density matters in open water cage culture: influence on growth, digestive enzymes, haemato-immuno and stress responses of Puntius sarana (Ham, 1822). Aquaculture 547, 737445 (2021).
Google Scholar 
Kumar, V. et al. Assessment of the effect of sub-lethal acute toxicity of Emamectin benzoate in Labeo rohita using multiple biomarker approach. Toxicol. Rep. 9, 102–110 (2022).CAS 
PubMed 
PubMed Central 

Google Scholar 
Rebl, A. et al. The synergistic interaction of thermal stress coupled with overstocking strongly modulates the transcriptomic activity and immune capacity of rainbow trout (Oncorhynchus mykiss). Sci. Rep. 10, 1–15 (2020).ADS 

Google Scholar 
Braun, N., de Lima, R. L., Baldisserotto, B., Dafre, A. L. & de Oliveira Nuñer, A. P. Growth, biochemical and physiological responses of Salminus brasiliensis with different stocking densities and handling. Aquaculture 301, 22–30 (2010).CAS 

Google Scholar 
Refaey, M. M., Tian, X., Tang, R. & Li, D. Changes in physiological responses, muscular composition and flesh quality of channel catfish Ictalurus punctatus suffering from transport stress. Aquaculture 478, 9–15 (2017).CAS 

Google Scholar 
Liu, G. et al. Influence of stocking density on growth, digestive enzyme activities, immune responses, antioxidant of Oreochromis niloticus fingerlings in biofloc systems. Fish Shellfish Immunol. 81, 416–422 (2018).CAS 
PubMed 

Google Scholar 
Kumar, G. & Engle, C. R. Technological advances that led to growth of shrimp, salmon, and tilapia farming. Rev. Fish. Sci. Aquac. 24, 136–152 (2016).
Google Scholar 
Sundin, L. Hypoxia and blood flow control in fish gills. In Biology of tropical fishes (eds Val, A. L. & Almeida-Val, V. M. F.) 353–362 (Manaus INPA, 1999).
Google Scholar 
Beveridge, M. C. M. Cage Aquaculture Vol. 5 (John Wiley & Sons, 2008).
Google Scholar 
Valenti, W. C., Barros, H. P., Moraes-Valenti, P., Bueno, G. W. & Cavalli, R. O. Aquaculture in Brazil: past, present and future. Aquac. Rep. 19, 100611 (2021).
Google Scholar 
Das, A. K., Meena, D. K. & Sharma, A. P. Cage farming in an Indian Reservoir. World Aquac. 45, 56–59 (2014).
Google Scholar 
Sarkar, U. K. et al. Status, prospects, threats, and the way forward for sustainable management and enhancement of the tropical Indian reservoir fisheries: an overview. Rev. Fish. Sci. Aquac. 26, 155–175 (2018).
Google Scholar 
Singh, A. K. & Lakra, W. S. Culture of Pangasianodon hypophthalmus into India: impacts and present scenario. Pakistan J. Biol. Sci. 15, 19 (2012).CAS 

Google Scholar 
Jena, J. et al. Evaluation of growth performance of Labeo fimbriatus (Bloch), Labeo gonius (Hamilton) and Puntius gonionotus (Bleeker) in polyculture with Labeo rohita (Hamilton) during fingerlings rearing at varied densities. Aquaculture 319, 493–496 (2011).
Google Scholar 
Liu, B., Jia, R., Han, C., Huang, B. & Lei, J.-L. Effects of stocking density on antioxidant status, metabolism and immune response in juvenile turbot (Scophthalmus maximus). Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 190, 1–8 (2016).CAS 

Google Scholar 
Wu, F. et al. Effect of stocking density on growth performance, serum biochemical parameters, and muscle texture properties of genetically improved farm tilapia, Oreochromis niloticus. Aquac. Int. 26, 1247–1259 (2018).CAS 

Google Scholar 
Andrade, T. et al. Evaluation of different stocking densities in a Senegalese sole (Solea senegalensis) farm: implications for growth, humoral immune parameters and oxidative status. Aquaculture 438, 6–11 (2015).CAS 

Google Scholar 
Qi, C. et al. Effect of stocking density on growth, physiological responses, and body composition of juvenile blunt snout bream, Megalobrama amblycephala. J. World Aquac. Soc. 47, 358–368 (2016).CAS 

Google Scholar 
Shao, T. et al. Evaluation of the effects of different stocking densities on growth and stress responses of juvenile hybrid grouper♀ Epinephelus fuscoguttatus×♂ Epinephelus lanceolatus in recirculating aquaculture systems. J. Fish Biol. 95, 1022–1029 (2019).CAS 
PubMed 

Google Scholar 
Adineh, H., Naderi, M., Hamidi, M. K. & Harsij, M. Biofloc technology improves growth, innate immune responses, oxidative status, and resistance to acute stress in common carp (Cyprinus carpio) under high stocking density. Fish Shellfish Immunol. 95, 440–448 (2019).CAS 
PubMed 

Google Scholar 
Fazelan, Z., Vatnikov, Y. A., Kulikov, E. V., Plushikov, V. G. & Yousefi, M. Effects of dietary ginger (Zingiber officinale) administration on growth performance and stress, immunological, and antioxidant responses of common carp (Cyprinus carpio) reared under high stocking density. Aquaculture 518, 734833 (2020).CAS 

Google Scholar 
Hoseini, S. M., Yousefi, M., Hoseinifar, S. H. & Van Doan, H. Effects of dietary arginine supplementation on growth, biochemical, and immunological responses of common carp (Cyprinus carpio L.), stressed by stocking density. Aquaculture 503, 452–459 (2019).CAS 

Google Scholar 
Adineh, H., Naderi, M., Nazer, A., Yousefi, M. & Ahmadifar, E. Interactive effects of stocking density and dietary supplementation with nano selenium and garlic extract on growth, feed utilization, digestive enzymes, stress responses, and antioxidant capacity of grass carp, Ctenopharyngodon idella. J. World Aquac. Soc. 52, 789–804 (2021).CAS 

Google Scholar 
Zhao, H. et al. Transcriptome and physiological analysis reveal alterations in muscle metabolisms and immune responses of grass carp (Ctenopharyngodon idellus) cultured at different stocking densities. Aquaculture 503, 186–197 (2019).CAS 

Google Scholar 
Frisso, R. M., de Matos, F. T., Moro, G. V. & de Mattos, B. O. Stocking density of Amazon fish (Colossoma macropomum) farmed in a continental neotropical reservoir with a net cages system. Aquaculture 529, 735702 (2020).CAS 

Google Scholar 
Tammam, M. S., Wassef, E. A., Toutou, M. M. & El-Sayed, A.-F.M. Combined effects of surface area of periphyton substrates and stocking density on growth performance, health status, and immune response of Nile tilapia (Oreochromis niloticus) produced in cages. J. Appl. Phycol. 32, 3419–3428 (2020).CAS 

Google Scholar 
Zaki, M. A. A. et al. The impact of stocking density and dietary carbon sources on the growth, oxidative status and stress markers of Nile tilapia (Oreochromis niloticus) reared under biofloc conditions. Aquac. Reports 16, 100282 (2020).
Google Scholar 
Rowland, S. J., Mifsud, C., Nixon, M. & Boyd, P. Effects of stocking density on the performance of the Australian freshwater silver perch (Bidyanus bidyanus) in cages. Aquaculture 253, 301–308 (2006).
Google Scholar 
Mohler, J. W., King, M. K. & Farrell, P. R. Growth and survival of first-feeding and fingerling Atlantic sturgeon under culture conditions. N. Am. J. Aquac. 62, 174–183 (2000).
Google Scholar 
Mirghaed, A. T., Hoseini, S. M. & Ghelichpour, M. Effects of dietary 1, 8-cineole supplementation on physiological, immunological and antioxidant responses to crowding stress in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 81, 182–188 (2018).
Google Scholar 
Hoseini, S. M., Mirghaed, A. T., Iri, Y. & Ghelichpour, M. Effects of dietary cineole administration on growth performance, hematological and biochemical parameters of rainbow trout (Oncorhynchus mykiss). Aquaculture 495, 766–772 (2018).CAS 

Google Scholar 
Barton, B. A., Morgan, J. D. & Vijayan, M. M. Physiological and condition-related indicators of environmental stress in fish. In Biological Indicators of Aquatic Ecosystem Stress (ed. Adams, S. M.) 111–148 (American Fisheries Society, 2002).
Google Scholar 
Varela, J. L. et al. Dietary administration of probiotic Pdp11 promotes growth and improves stress tolerance to high stocking density in gilthead seabream Sparus auratus. Aquaculture 309, 265–271 (2010).CAS 

Google Scholar 
Costas, B., Aragão, C., Dias, J., Afonso, A. & Conceição, L. E. C. Interactive effects of a high-quality protein diet and high stocking density on the stress response and some innate immune parameters of Senegalese sole Solea senegalensis. Fish Physiol. Biochem. 39, 1141–1151 (2013).CAS 
PubMed 

Google Scholar 
Long, L. et al. Effects of stocking density on growth, stress, and immune responses of juvenile Chinese sturgeon (Acipenser sinensis) in a recirculating aquaculture system. Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 219, 25–34 (2019).CAS 

Google Scholar 
Sadhu, N., Sharma, S. R. K., Joseph, S., Dube, P. & Philipose, K. K. Chronic stress due to high stocking density in open sea cage farming induces variation in biochemical and immunological functions in Asian seabass (Lates calcarifer, Bloch). Fish Physiol. Biochem. 40, 1105–1113 (2014).CAS 
PubMed 

Google Scholar 
Zahran, E., Risha, E., AbdelHamid, F., Mahgoub, H. A. & Ibrahim, T. Effects of dietary Astragalus polysaccharides (APS) on growth performance, immunological parameters, digestive enzymes, and intestinal morphology of Nile tilapia (Oreochromis niloticus). Fish Shellfish Immunol. 38, 149–157 (2014).CAS 
PubMed 

Google Scholar 
Aruoma, O. I. Free radicals, oxidative stress, and antioxidants in human health and disease. J. Am. Oil Chem. Soc. 75, 199–212 (1998).CAS 
PubMed 
PubMed Central 

Google Scholar 
Haridas, H. et al. Enhanced growth and immuno-physiological response of genetically improved farmed Tilapia in indoor biofloc units at different stocking densities. Aquac. Res. 48, 4346–4355 (2017).CAS 

Google Scholar 
Ruane, N. M., Carballo, E. C. & Komen, J. Increased stocking density influences the acute physiological stress response of common carp Cyprinus carpio (L.). Aquac. Res. 33, 777–784 (2002).
Google Scholar 
Wang, X. et al. Effects of stocking density on growth, nonspecific immune response, and antioxidant status in African catfish (Clarias gariepinus). (2013).Johnson, K. M. & Lema, S. C. Tissue-specific thyroid hormone regulation of gene transcripts encoding iodothyronine deiodinases and thyroid hormone receptors in striped parrotfish (Scarus iseri). Gen. Comp. Endocrinol. 172, 505–517 (2011).CAS 
PubMed 

Google Scholar 
El-Khaldi, A. T. F. Effect of different stress factors on some physiological parameters of Nile tilapia (Oreochromis niloticus). Saudi J. Biol. Sci. 17, 241–246 (2010).CAS 
PubMed 
PubMed Central 

Google Scholar 
Sharma, A., Devi, S., Singh, K. & Prabhakar, P. K. Correlation of body mass index with thyroid-stimulating hormones in thyroid patient. Asian J. Pharm. Clin. Res. 11, 65–68 (2018).
Google Scholar 
Li, D., Liu, Z. & Xie, C. Effect of stocking density on growth and serum concentrations of thyroid hormones and cortisol in Amur sturgeon, Acipenser schrenckii. Fish Physiol. Biochem. 38, 511–520 (2012).CAS 
PubMed 

Google Scholar 
Park, J.-W. et al. The thyroid endocrine disruptor perchlorate affects reproduction, growth, and survival of mosquitofish. Ecotoxicol. Environ. Saf. 63, 343–352 (2006).CAS 
PubMed 

Google Scholar 
Refaey, M. M. et al. High stocking density alters growth performance, blood biochemistry, intestinal histology, and muscle quality of channel catfish Ictalurus punctatus. Aquaculture 492, 73–81 (2018).CAS 

Google Scholar 
Reinecke, M. et al. Growth hormone and insulin-like growth factors in fish: where we are and where to go. Gen. Comp. Endocrinol. 142, 20–24 (2005).CAS 
PubMed 

Google Scholar 
Salas-Leiton, E. et al. Dexamethasone modulates expression of genes involved in the innate immune system, growth and stress and increases susceptibility to bacterial disease in Senegalese sole (Solea senegalensis Kaup, 1858). Fish Shellfish Immunol. 32, 769–778 (2012).CAS 
PubMed 

Google Scholar 
Dyer, A. R. et al. Correlation of plasma IGF-I concentrations and growth rate in aquacultured finfish: a tool for assessing the potential of new diets. Aquaculture 236, 583–592 (2004).CAS 

Google Scholar 
Kajimura, S. et al. Dual mode of cortisol action on GH/IGF-I/IGF binding proteins in the tilapia, Oreochromis mossambicus. J. Endocrinol. 178, 91–99 (2003).CAS 
PubMed 

Google Scholar 
Ren, Y., Wen, H., Li, Y. & Li, J. Stocking density affects the growth performance and metabolism of Amur sturgeon by regulating expression of genes in the GH/IGF axis. J. Oceanol. Limnol. 36, 956–972 (2018).ADS 
CAS 

Google Scholar 
Salas-Leiton, E. et al. Effects of stocking density and feed ration on growth and gene expression in the Senegalese sole (Solea senegalensis): potential effects on the immune response. Fish Shellfish Immunol. 28, 296–302 (2010).CAS 
PubMed 

Google Scholar 
Vijayan, M. M., Aluru, N. & Leatherland, J. F. Stress response and the role of cortisol. Fish Dis. Disord. 2, 182–201 (2010).
Google Scholar 
Hegazi, M. M., Attia, Z. I. & Ashour, O. A. Oxidative stress and antioxidant enzymes in liver and white muscle of Nile tilapia juveniles in chronic ammonia exposure. Aquat. Toxicol. 99, 118–125 (2010).CAS 
PubMed 

Google Scholar 
Kpundeh, M. D., Xu, P., Yang, H., Qiang, J. & He, J. Stocking densities and chronic zero culture water exchange stress’ effects on biological performances, hematological and serum biochemical indices of GIFT tilapia juveniles (Oreochromis niloticus). J. Aquac. Res. Dev. 4, 2 (2013).
Google Scholar 
Tan, C. et al. Effects of stocking density on growth, body composition, digestive enzyme levels and blood biochemical parameters of Anguilla marmorata in a recirculating aquaculture system. Turk. J. Fish. Aquat. Sci. 18, 9–16 (2018).
Google Scholar 
Ni, M. et al. The physiological performance and immune responses of juvenile Amur sturgeon (Acipenser schrenckii) to stocking density and hypoxia stress. Fish Shellfish Immunol. 36, 325–335 (2014).CAS 
PubMed 

Google Scholar 
Abdel-Tawwab, M. Effects of dietary protein levels and rearing density on growth performance and stress response of Nile tilapia, Oreochromis niloticus (L.). Int. Aquat. Res. 4, 1–13 (2012).
Google Scholar 
Chatterjee, N. et al. Effect of stocking density and journey length on the welfare of rohu (Labeo rohita Hamilton) fry. Aquac. Int. 18, 859–868 (2010).
Google Scholar 
Pakhira, C., Nagesh, T. S., Abraham, T. J., Dash, G. & Behera, S. Stress responses in rohu, Labeo rohita transported at different densities. Aquac. Rep. 2, 39–45 (2015).
Google Scholar 
Tahmasebi-Kohyani, A., Keyvanshokooh, S., Nematollahi, A., Mahmoudi, N. & Pasha-Zanoosi, H. Effects of dietary nucleotides supplementation on rainbow trout (Oncorhynchus mykiss) performance and acute stress response. Fish Physiol. Biochem. 38, 431–440 (2012).CAS 
PubMed 

Google Scholar 
Montero, D. et al. Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture 171, 269–278 (1999).CAS 

Google Scholar 
Urbinati, E. C., de Abreu, J. S., da Silva Camargo, A. C. & Parra, M. A. L. Loading and transport stress of juvenile matrinxã (Brycon cephalus, Characidae) at various densities. Aquaculture 229, 389–400 (2004).
Google Scholar 
Evans, D. H. Cell signaling and ion transport across the fish gill epithelium. J. Exp. Zool. 293, 336–347 (2002).CAS 
PubMed 

Google Scholar 
McCormick, S. D. Endocrine control of osmoregulation in teleost fish. Am. Zool. 41, 781–794 (2001).CAS 

Google Scholar 
Postlethwaite, E. & McDonald, D. Mechanisms of Na+ and C-regulation in freshwater-adapted rainbow trout (Oncorhynchus mykiss) during exercise and stress. J. Exp. Biol. 198, 295–304 (1995).CAS 
PubMed 

Google Scholar 
Liu, P., Du, Y., Meng, L., Li, X. & Liu, Y. Metabolic profiling in kidneys of Atlantic salmon infected with Aeromonas salmonicida based on 1H NMR. Fish Shellfish Immunol. 58, 292–301 (2016).CAS 
PubMed 

Google Scholar 
Hosfeld, C. D., Hammer, J., Handeland, S. O., Fivelstad, S. & Stefansson, S. O. Effects of fish density on growth and smoltification in intensive production of Atlantic salmon (Salmo salar L.). Aquaculture 294, 236–241 (2009).
Google Scholar 
Wagner, E. I., Miller, S. A. & Bosakowski, T. Ammonia excretion by rainbow trout over a 24-hour period at two densities during oxygen injection. Progress. Fish-Culturist 57, 199–205 (1995).
Google Scholar 
Dong, J. et al. Effect of stocking density on growth performance, digestive enzyme activities, and nonspecific immune parameters of Palaemonetes sinensis. Fish Shellfish Immunol. 73, 37–41 (2018).CAS 
PubMed 

Google Scholar 
Wang, Y. et al. Effects of stocking density on the growth performance, digestive enzyme activities, antioxidant resistance, and intestinal microflora of blunt snout bream (Megalobrama amblycephala) juveniles. Aquac. Res. 50, 236–246 (2019).CAS 

Google Scholar 
Trenzado, C. E. et al. Effect of dietary lipid content and stocking density on digestive enzymes profile and intestinal histology of rainbow trout (Oncorhynchus mykiss). Aquaculture 497, 10–16 (2018).CAS 

Google Scholar 
Li, X., Liu, Y. & Blancheton, J.-P. Effect of stocking density on performances of juvenile turbot (Scophthalmus maximus) in recirculating aquaculture systems. Chin. J. Oceanol. Limnol. 31, 514–522 (2013).ADS 
CAS 

Google Scholar 
Ezhilmathi, S. et al. Effect of stocking density on growth performance, digestive enzyme activity, body composition and gene expression of Asian seabass reared in recirculating aquaculture system. Aquac. Res. https://doi.org/10.1111/are.15725 (2022).Article 

Google Scholar 
Bolasina, S., Tagawa, M., Yamashita, Y. & Tanaka, M. Effect of stocking density on growth, digestive enzyme activity and cortisol level in larvae and juveniles of Japanese flounder, Paralichthys olivaceus. Aquaculture 259, 432–443 (2006).CAS 

Google Scholar 
Hoseini, S. M., Hoseinifar, S. H. & Van Doan, H. Effect of dietary eucalyptol on stress markers, enzyme activities and immune indicators in serum and haematological characteristics of common carp (Cyprinus carpio) exposed to toxic concentration of ambient copper. Aquac. Res. 49, 3045–3054 (2018).CAS 

Google Scholar 
Ni, M. et al. Effects of stocking density on mortality, growth and physiology of juvenile Amur sturgeon (Acipenser schrenckii). Aquac. Res. 47, 1596–1604 (2016).CAS 

Google Scholar 
Abdel-Tawwab, M., Hagras, A. E., Elbaghdady, H. A. M. & Monier, M. N. Dissolved oxygen level and stocking density effects on growth, feed utilization, physiology, and innate immunity of Nile Tilapia, Oreochromis niloticus. J. Appl. Aquac. 26, 340–355 (2014).
Google Scholar 
Toko, I., Fiogbe, E. D., Koukpode, B. & Kestemont, P. Rearing of African catfish (Clarias gariepinus) and vundu catfish (Heterobranchus longifilis) in traditional fish ponds (whedos): effect of stocking density on growth, production and body composition. Aquaculture 262, 65–72 (2007).
Google Scholar 
Suárez, M. D. et al. Influence of dietary lipids and culture density on rainbow trout (Oncorhynchus mykiss) flesh composition and quality parameter. Aquac. Eng. 63, 16–24 (2014).
Google Scholar 
Santín, A., Grinyó, J., Bilan, M., Ambroso, S. & Puig, P. First report of the carnivorous sponge Lycopodina hypogea (Cladorhizidae) associated with marine debris, and its possible implications on deep-sea connectivity. Mar. Pollut. Bull. 159, 111501 (2020).PubMed 

Google Scholar 
Jørpeland, G., Imsland, A., Stien, L. H., Bleie, H. & Roth, B. Effects of filleting method, stress, storage and season on the quality of farmed Atlantic cod (Gadus morhua L.). Aquac. Res. 46, 1597–1607 (2015).
Google Scholar 
Bulow, F. J. RNA-DNA ratios as indicators of growth in fish: a review. In The Age and growth of fish (eds Summerfelt, R. C. & Hall, G. E.) 45–64 (Iowa State University Press, Ames, Iowa, 1987).
Google Scholar 
Regnault, M. & Luquet, P. Study by evolution of nucleic acid content of prepuberal growth in the shrimp Crangon vulgaris. Mar. Biol. 25, 291–298 (1974).CAS 

Google Scholar 
Tanaka, H. K. M. et al. High resolution imaging in the inhomogeneous crust with cosmic-ray muon radiography: the density structure below the volcanic crater floor of Mt. Asama, Japan. Earth Planet. Sci. Lett. 263, 104–113 (2007).ADS 
CAS 

Google Scholar 
Gwak, W. S. & Tanaka, M. Developmental change in RNA: DNA ratios of fed and starved laboratory-reared Japanese flounder larvae and juveniles, and its application to assessment of nutritional condition for wild fish. J. Fish Biol. 59, 902–915 (2001).CAS 

Google Scholar 
Ali, M., Iqbal, R., Rana, S. A., Athar, M. & Iqbal, F. Effect of feed cycling on specific growth rate, condition factor and RNA/DNA ratio of Labeo rohita. African J. Biotechnol. 5, 1551–1556 (2006).CAS 

Google Scholar 
Zehra, S. & Khan, M. A. Dietary lysine requirement of fingerling Catla catla (Hamilton) based on growth, protein deposition, lysine retention efficiency, RNA/DNA ratio and carcass composition. Fish Physiol. Biochem. 39, 503–512 (2013).CAS 
PubMed 

Google Scholar 
Misra, H. P. & Fridovich, I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175 (1972).CAS 
PubMed 

Google Scholar 
Takahara, S. et al. Hypocatalasemia: a new genetic carrier state. J. Clin. Invest. 39, 610–619 (1960).CAS 
PubMed 
PubMed Central 

Google Scholar 
Rick, W. & Stegbauer, H. P. α-Amylase measurement of reducing groups. In Methods of Enzymatic Analysis (ed. Bergmeyer, H. S.) 885–890 (Elsevier, 1974).
Google Scholar 
Cherry, I. S. & Crandall, L. A. Jr. The specificity of pancreatic lipase: its appearance in the blood after pancreatic injury. Am. J. Physiol. Content 100, 266–273 (1932).CAS 

Google Scholar 
Drapeau, G. R. [38] Protease from Staphyloccus aureus. In Methods in Enzymology (eds Jura, N. & Murphy, J. M.) 469–475 (Elsevier, 1976).
Google Scholar 
AOAC. Official Methods of Analysis of AOAC International. (Association of Official Analytical Chemists Washington, DC, 2005).Bosworth, B. G., Small, B. C. & Mischke, C. Effects of transport water temperature, aerator type, and oxygen level on channel catfish Ictalurus punctatus fillet quality. J. World Aquac. Soc. 35, 412–419 (2004).
Google Scholar 
Ma, L. Q., Qi, C. L., Cao, J. J. & Li, D. P. Comparative study on muscle texture profile and nutritional value of channel catfish (Ictalurus punctatus) reared in ponds and reservoir cages. J. Fish. China 38, 532–537 (2014).
Google Scholar 
APHA. Standard Methods for the Examination of Water and Wastewater. (American Public Health Association, American Water Works Association, Water Environment Federation, 2012). More

Danovaro, et al. Ecological variables for developing a global deep-ocean monitoring and conservation strategy. Nat. Ecol. Evol. 4(2), 181–192. https://doi.org/10.1038/s41559-019-1091-z (2020).Danovaro, R., Snelgrove, P. V. R. & Tyler, P. Challenging the paradigms of deep-sea ecology. Trends Ecol. Evol. 29(8), 465–475. https://doi.org/10.1016/j.tree.2014.06.002 (2014).Article 
PubMed 

Google Scholar 
Collin, S. P. The neuroecology of cartilaginous fishes: sensory strategies for survival. Brain Behav. Evol. 80(2), 80–96. https://doi.org/10.1159/000339870 (2012).Article 
PubMed 

Google Scholar 
Carrier, J. C., Musick, J. A., & Heithaus, M. R. (Eds.). Biology of sharks and their relatives. CRC (2012).Musick, J. A. & Cotton, C. F. Bathymetric limits of chondrichthyans in the deep sea: a re-evaluation. Deep Sea Res. Part II 115, 73–80. https://doi.org/10.1016/j.dsr2.2014.10.010 (2015).Article 

Google Scholar 
Treberg, J. R. & Speers-Roesch, B. Does the physiology of chondrichthyan fishes constrain their distribution in the deep sea?. J. Exp. Biol. 219(5), 615–625. https://doi.org/10.1242/jeb.128108 (2016).Article 
PubMed 

Google Scholar 
Didier, D. A., Kemper, J. M. & Ebert, D. A. Phylogeny, biology and classification of extant holocephalans. In Biology of Sharks and Their Relatives, 2nd edn (Carrier, J. C., Musick, J. A. & Heithaus, M. R., eds), pp. 97–124. New York, NY: CRC Pres. (2012).Weigmann, S. Annotated checklist of the living sharks, batoids and chimaeras (Chondrichthyes) of the world, with a focus on biogeographical diversity. J. Fish Biol. 88(3), 837–1037. https://doi.org/10.1111/jfb.12874 (2016).CAS 
Article 
PubMed 

Google Scholar 
Coates, M. I., Gess, R. W., Finarelli, J. A., Criswell, K. E. & Tietjen, K. A symmoriiform chondrichthyan braincase and the origin of chimaeroid fishes. Nature 541(7636), 208–211. https://doi.org/10.1038/nature20806 (2017).ADS 
CAS 
Article 
PubMed 

Google Scholar 
Lisney, T. J. A review of the sensory biology of chimaeroid fishes (Chondrichthyes; Holocephali). Rev. Fish Biol. Fisheries 20(4), 571–590. https://doi.org/10.1007/s11160-010-9162-x (2010).Article 

Google Scholar 
Finucci, B. et al. Ghosts of the deep–biodiversity, fisheries, and extinction risk of ghost sharks. Fish Fish. 22(2), 391–412. https://doi.org/10.1111/faf.12526 (2021).Article 

Google Scholar 
Newton, K. C., Gill, A. B. & Kajiura, S. M. Electroreception in marine fishes: chondrichthyans. J. Fish Biol. 95(1), 135–154. https://doi.org/10.1111/jfb.14068 (2019).Article 
PubMed 

Google Scholar 
Crampton, W. G. Electroreception, electrogenesis and electric signal evolution. J. Fish Biol. 95(1), 92–134. https://doi.org/10.1111/jfb.13922 (2019).Article 
PubMed 

Google Scholar 
Whitehead, D. L. Ampullary organs and electroreception in freshwater Carcharhinus leucas. J. Physiol.-Paris 96(5–6), 391–395. https://doi.org/10.1016/S0928-4257(03)00017-2 (2002).Article 
PubMed 

Google Scholar 
Raschi, W. G., & Gerry, S. Adaptations in the elasmobranch electroreceptive system. Fish Adaptations. Enfield, NH: Scientific Publishers, 233–258 (2003).Atkinson, C. J. L. & Bottaro, M. Ampullary pore distribution of Galeus melastomus and Etmopterus spinax: possible relations with predatory lifestyle and habitat. J. Mar. Biol. Assoc. UK 86(2), 447–448. https://doi.org/10.1017/S0025315406013336 (2006).Article 

Google Scholar 
Kempster, R. M. & Collin, S. P. Electrosensory pore distribution and feeding in the basking shark Cetorhinus maximus (Lamniformes: Cetorhinidae). Aquat. Biol. 12(1), 33–36. https://doi.org/10.3354/ab00328 (2011).Article 

Google Scholar 
Kempster, R. M., McCarthy, I. D. & Collin, S. P. Phylogenetic and ecological factors influencing the number and distribution of electroreceptors in elasmobranchs. J. Fish Biol. 80(5), 2055–2088. https://doi.org/10.1111/j.1095-8649.2011.03214.x (2012).CAS 
Article 
PubMed 

Google Scholar 
Whitehead, D. L., Gauthier, A. R., Mu, E. W., Bennett, M. B. & Tibbetts, I. R. Morphology of the Ampullae of Lorenzini in juvenile freshwater Carcharhinus leucas. J. Morphol. 276(5), 481–493. https://doi.org/10.1002/jmor.20355 (2015).Article 
PubMed 

Google Scholar 
Gauthier, A. R. G., Whitehead, D. L., Tibbetts, I. R., Cribb, B. W. & Bennett, M. B. Morphological comparison of the Ampullae of Lorenzini of three sympatric benthic rays. J. Fish Biol. 92(2), 504–514. https://doi.org/10.1111/jfb.13531 (2018).CAS 
Article 
PubMed 

Google Scholar 
Fields, R. D., Bullock, T. H. & Lange, G. D. Ampullary sense organs, peripheral, central and behavioral electroreception in Chimeras (Hydrolagus, Holocephali, Chondrichthyes). Brain Behav. Evol. 41(6), 269–289. https://doi.org/10.1159/000113849 (1993).CAS 
Article 
PubMed 

Google Scholar 
Didier, D.A. Phylogenetic systematics of extant chimaeroid fishes (Holocephali, Chimaeroidei). American Museum Novitates; n. 3119 (1995).Serena, F. Field identification guide to the sharks and rays of the Mediterranean and Black Sea (Food and Agriculture Organization, 2005).
Google Scholar 
Holt, R. E., Foggo, A., Neat, F. C. & Howell, K. L. Distribution patterns and sexual segregation in chimaeras: implications for conservation and management. ICES J. Mar. Sci. 70(6), 1198–1205. https://doi.org/10.1093/icesjms/fst058 (2013).Article 

Google Scholar 
Ragonese, S., Vitale, S., Dimech, M., & Mazzola, S. Abundances of demersal sharks and chimaera from 1994–2009 scientific surveys in the central Mediterranean Sea. PloS one, 8(9). https://doi.org/10.1371/journal.pone.0074865 (2013).Vacchi, M., & Orsi, L. R. Alimentazione di Chimaera monstrosa L. sui fondi batiali liguri. Atti della Società Toscana di Scienze Naturali, Memorie serie B, 86, 388–391 (1979).Macpherson, E. Food and feeding of Chimaera monstrosa, Linnaeus, 1758, in the western Mediterranean. ICES J. Mar. Sci. 39(1), 26–29. https://doi.org/10.1093/icesjms/39.1.26 (1980).Article 

Google Scholar 
Mauchline, J. & Gordon, J. D. M. Diets of the sharks and chimaeroids of the Rockall Trough, northeastern Atlantic Ocean. Mar. Biol. 75(2–3), 269–278. https://doi.org/10.1007/BF00406012 (1983).Article 

Google Scholar 
Albo-Puigserver, et al. Feeding ecology and trophic position of three sympatric demersal chondrichthyans in the northwestern Mediterranean. Mar. Ecol. Prog. Ser. 524, 255–268. https://doi.org/10.3354/meps11188( (2015).ADS 
Article 

Google Scholar 
Priede, I. G. Deep-sea fishes: biology, diversity, ecology and fisheries. Cambridge University Press (2017).Ferrando, S. et al. First description of a palatal organ in Chimaera monstrosa (Chondrichthyes, Holocephali). Anat. Rec. 299(1), 118–131. https://doi.org/10.1002/ar.23280 (2016).Article 

Google Scholar 
Garza-Gisholt, E., Hart, N. S., & Collin, S. P. Retinal morphology and visual specializations in three species of chimaeras, the deep-sea R. pacifica and C. lignaria, and the Vertical Migrator C. milii (Holocephali). Brain, behavior and evolution, 92(1–2), 47–62. https://doi.org/10.1159/000490655 (2018).Pethybridge, H., Daley, R. K. & Nichols, P. D. Diet of demersal sharks and chimaeras inferred by fatty acid profiles and stomach content analysis. J. Exp. Mar. Biol. Ecol. 409(1–2), 290–299. https://doi.org/10.1016/j.jembe.2011.09.009 (2011).Article 

Google Scholar 
Rivera-Vicente, A. C., Sewell, J. & Tricas, T. C. Electrosensitive spatial vectors in elasmobranch fishes: implications for source localization. PLoS ONE 6(1), e16008. https://doi.org/10.1371/journal.pone.0016008 (2011).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Kajiura, S. M., Cornett, A. D. & Yopak, K. E. Sensory adaptations to the environment: electroreceptors as a case study. Biol. Sharks Relatives 2, 393–434 (2010).Article 

Google Scholar 
Raschi, W. A morphological analysis of the Ampullae of Lorenzini in selected skates (Pisces, Rajoidei). J. Morphol. 189(3), 225–247. https://doi.org/10.1002/jmor.1051890303 (1986).Article 
PubMed 

Google Scholar 
Jordan, L. K. et al. Linking sensory biology and fisheries bycatch reduction in elasmobranch fishes: a review with new directions for research. Conserv. Physiol. 1(1), cot002. https://doi.org/10.1093/conphys/cot002 (2013).Wueringer, B. E., Peverell, S. C., Seymour, J., Squire Jr, L., Kajiura, S. M., & Collin, S. P. Sensory systems in sawfishes. 1. The ampullae of Lorenzini. Brain, behavior and evolution, 78(2), 139–149. https://doi.org/10.1159/000329515 (2011).Bird C.S. The tropho-spatial ecology of deep-sea sharks and chimaeras from a stable isotope perspective. PhD thesis – University of Southampton, UK (2017).Andres, K. H. & Von Düring, M. Comparative anatomy of vertebrate electroreceptors. Prog Brain Res 74, 113–131. https://doi.org/10.1016/S0079-6123(08)63006-X (1998).Article 

Google Scholar 
Crooks, N. & Waring, C. P. A study into the sexual dimorphisms of the Ampullae of Lorenzini in the lesser-spotted catshark, Scyliorhinus canicula (Linnaeus, 1758). Environ. Biol. Fishes 96(5), 585–590. https://doi.org/10.1016/S0079-6123(08)63006-X (2013).Article 

Google Scholar 
Didier, D. A. Phylogeny and classification of extant Holocephali. Biol. Sharks Relatives 4, 115–138 (2004).Article 

Google Scholar 
Wueringer, B. E. & Tibbetts, I. R. Comparison of the lateral line and ampullary systems of two species of shovelnose ray. Rev. Fish Biol. Fisheries 18(1), 47–64. https://doi.org/10.1007/s11160-007-9063-9 (2008).Article 

Google Scholar 
Theiss, S. M., Collin, S. P. & Hart, N. S. Morphology and distribution of the ampullary electroreceptors in wobbegong sharks: implications for feeding behaviour. Mar. Biol. 158(4), 723–735. https://doi.org/10.1007/s00227-010-1595-1 (2011).Article 

Google Scholar 
Schäfer, B. T. et al. Morphological observations of Ampullae of lorenzini in Squatina guggenheim and S. occulta (Chondrichthyes, Elasmobranchii, Squatinidae). Microscopy Res Tech. 75(9), 1213–1217. https://doi.org/10.1002/jemt.22051 (2012).Brown, B. R. Sensing temperature without ion channels. Nature 421(6922), 495–495. https://doi.org/10.1038/421495a (2003).ADS 
CAS 
Article 
PubMed 

Google Scholar 
Fields, R. D., Fields, K. D. & Fields, M. C. Semiconductor gel in shark sense organs?. Neurosci. Lett. 426(3), 166–170. https://doi.org/10.1016/j.neulet.2007.08.064 (2007).CAS 
Article 
PubMed 
PubMed Central 
MATH 

Google Scholar 
Brown, B. R. Temperature response in electrosensors and thermal voltages in electrolytes. J. Biol. Phys. 36(2), 121–134. https://doi.org/10.1007/s10867-009-9174-8 (2010).Article 
PubMed 

Google Scholar 
Josberger, E. E. et al. Proton conductivity in Ampullae of Lorenzini jelly. Sci. Adv. 2(5), e1600112. https://doi.org/10.1126/sciadv.1600112 (2016).ADS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Froese, R. and Pauly D. https://www.fishbase.de/ (2021).Sims, D. W. The biology, ecology and conservation of elasmobranchs: recent advances and new frontiers. J. Fish Biol. 87(6), 1265–1270. https://doi.org/10.1111/jfb.12861 (2015).CAS 
Article 
PubMed 

Google Scholar 
Heithaus, M. R., Frid, A., Wirsing, A. & Worm, B. Predicting ecological consequences of marine top predator declines. Trends Ecol. Evol. 23, 202–210. https://doi.org/10.1016/j.tree.2008.01.003 (2008).Article 
PubMed 

Google Scholar 
Dymek, J., Muñoz, P., Mayo-Hernández, E., Kuciel, M. & Żuwała, K. Comparative analysis of the olfactory organs in selected species of marine sharks and freshwater batoids. Zool. Anz. 294, 50–61. https://doi.org/10.1016/j.jcz.2021.07.013 (2021).Article 

Google Scholar 
Bellono, N. W., Leitch, D. B. & Julius, D. Molecular tuning of electroreception in sharks and skates. Nature 558(7708), 122. https://doi.org/10.1038/s41586-018-0160-9 (2018).ADS 
CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
Luchetti, E. A., Iglésias, S. P., & Sellos, D. Y. Chimaera opalescens n. sp., a new chimaeroid (Chondrichthyes: Holocephali) from the north‐eastern Atlantic Ocean. J. Fish Biol., 79(2), 399–417. https://doi.org/10.1111/j.1095-8649.2011.03027.x (2011).Marranzino, A. N. & Webb, J. F. Flow sensing in the deep sea: the lateral line system of stomiiform fishes. Zool. J. Linn. Soc. 183(4), 945–965. https://doi.org/10.1093/zoolinnean/zlx090 (2018).Article 

Google Scholar 
Yopak, K. E. & Montgomery, J. C. Brain organization and specialization in deep-sea chondrichthyans. Brain Behav. Evol. 71(4), 287–304. https://doi.org/10.1159/000127048 (2008).Article 
PubMed 

Google Scholar 
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9(7), 671–675. https://doi.org/10.1038/nmeth.2089 (2012).CAS 
Article 
PubMed 
PubMed Central 

Google Scholar 
R Core Team, R. A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/ (2021).Wickham, H. ggplot2: Elegant Graphics for Data Analysis. Springer, New York (2016). More

Carvalho, G. R. & Hauser, L. Molecular genetics and the stock concept in fisheries. In Molecular Genetics in Fisheries (eds Carvalho, G. R. & Pitcher, T. J.) 55–79 (Springer Netherlands, 1995). https://doi.org/10.1007/978-94-011-1218-5_3.Chapter 

Google Scholar 
Avise, J. C. Conservation genetics in the marine realm. J. Hered. 89, 377–382 (1998).Article 

Google Scholar 
Waples, R. S. Separating the wheat from the chaff: Patterns of genetic differentiation in high gene flow species. J. Hered. 89, 438–450 (1998).Article 

Google Scholar 
Pecoraro, C. et al. The population genomics of yellowfin tuna (Thunnus albacares) at global geographic scale challenges current stock delineation. Sci. Rep. 8, 13890 (2018).ADS 
PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Nikolic, N. et al. Connectivity and population structure of albacore tuna across southeast Atlantic and southwest Indian Oceans inferred from multidisciplinary methodology. Sci. Rep. 10, 15657 (2020).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Anderson, G., Lal, M., Hampton, J., Smith, N. & Rico, C. Close kin proximity in yellowfin tuna (Thunnus albacares) as a driver of population genetic structure in the tropical western and central Pacific Ocean. Front. Mar. Sci. 6, 341 (2019).Article 

Google Scholar 
Collette, B. B. & Nauen, C. E. Scombrids of the World: An Annotated and Illustrated Catalogue of Tunas, Mackerels, Bonitos, and Related Species Known to date v.2 (FAO, 1983).
Google Scholar 
Majkowski, J., Arrizabalaga, H. & Carocci, F. C1. Tuna and Tuna-like Species. Review of the state of World Fisheries Resources (FAO, 2005).Mahon, R. Fisheries and research for tunas and tuna-like species in the Western Central Atlantic: implications of the agreement for the implementation of the provisions of the United Nations Convention on the Law of the Sea of the 10 December 1982 relating to the conservation and management of straddling fish stocks and highly migratory fish stocks. (FAO Fisheries Technical Paper, 1996).Doray, M., Stéquert, B. & Taquet, M. Age and growth of blackfin tuna (Thunnus atlanticus ) caught under moored fish aggregating devices, around Martinique Island. Aquat. Living Resour. 17, 13–18 (2004).Article 

Google Scholar 
Arocha, F., Barrios, A. & Marcano, J. Blackfin tuna (Thunnus atlanticus) in the Venezuelan fisheries. Collect. Vol. Sci. Pap ICCAT 68(3), 1253–1260 (2012).
Google Scholar 
Mathieu, H., Pau, C. & Reynal, L. Chapter 2.1.10.7 THON A NAGEOIRES NOIRES. ICCAT ICCAT Manual. International Commission for the Conservation of Atlantic Tuna. 15 (2013).Maghan, W. B. & Rivas, L. R. The blackfin tuna (Thunnus atlanticus) as an underutilized fishery resource in the tropical western Atlantic Ocean. FAO Fish. Rep. 71(2), 163–172 (1971).
Google Scholar 
De Sylva, D. P., Rathjen, W. F. & Higman, J. B. Fisheries development for underutilized Atlantic tunas: Blackfin and little tunny. NOAA Technical Memorandum NMFS-SEFC-191 (1987).Richardson, D. E., Llopiz, J. K., Guigand, C. M. & Cowen, R. K. Larval assemblages of large and medium-sized pelagic species in the Straits of Florida. Prog. Oceanogr. 86, 8–20 (2010).ADS 
Article 

Google Scholar 
Freire, K. M. F., Lessa, R. & Lins-Oliveira, J. E. Fishery and biology of blackfin tuna Thunnus atlanticus off northeastern Brazil. Gulf Caribb. Res. 17, 15–24 (2005).Article 

Google Scholar 
Vieira, K. R., Oliveira, J. E. L. & Barbalho, M. C. Aspects of the dynamic population of blackfin tuna (Thunnus atlanticus-Lesson, 1831) caught in the Northeast Brazil. Collect. Vol. Sci. Pap ICCAT 58(5), 1623–1628 (2005).
Google Scholar 
FJ Mather, I. I. I. Tunas (genus Thunnus) of the western North Atlantic. Part III. Distribution and behavior of Thunnus species. World Sci. Meeting Biol. Tunas Exper. Pap. Vol. 8, 1–23 (1962)Cornic, M. & Rooker, J. R. Influence of oceanographic conditions on the distribution and abundance of blackfin tuna (Thunnus atlanticus) larvae in the Gulf of Mexico. Fish. Res 201, 1–10 (2018).Article 

Google Scholar 
Block, B. A. et al. Electronic tagging and population structure of Atlantic bluefin tuna. Nature 434, 1121–1127 (2005).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Luckhurst, B. E., Trott, T. & Manuel, S. Landings, seasonality, catch per unit effort, and tag-recapture results of yellowfin tuna and blackfin tuna at Bermuda. Am. Fish. Soc. Symp. 25, 225–234 (2001).
Google Scholar 
Singh-Renton, S. & Renton, J. CFRAMP’s large pelagic fish tagging program. Gulf Caribb. Res. Vol 19, (2007).Cermeño, P. et al. Electronic tagging of Atlantic bluefin tuna (Thunnus thynnus, L.) reveals habitat use and behaviors in the Mediterranean Sea. PLoS ONE 10, e0116638 (2015).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Begg, G. A., Friedland, K. D. & Pearce, J. B. Stock identification and its role in stock assessment and fisheries management: An overview. Fish. Res 43, 1–8 (1999).Article 

Google Scholar 
Saxton, B. Historical demography and genetic population structure of theBlackfin tuna (Thunnus atlanticus) from the Northwest Atlantic Ocean and the Gulf of Mexico. Texas A&M University (2009).Antoni, L., Luque, P. L., Naghshpour, K., Reynal, L. & Saillant, E. A. Development and characterization of microsatellite markers for blackfin tuna (Thunnus atlanticus) with the use of Illumina paired-end sequencing. Fish. Bull. 112, 322–325 (2014).Article 

Google Scholar 
Weir, B. S. & Cockerham, C. C. Estimating F-statistics for the analysis of population structure. Evolution 38, 1358 (1984).CAS 
PubMed 

Google Scholar 
Goudet, J. FSTAT (Version 1.2): A computer program to calculate F-statistics. J. Hered 86, 485–486 (1995).Article 

Google Scholar 
Rousset, F. Genepop’007: A complete re-implementation of the genepop software for Windows and Linux. Mol. Ecol. Resour. 8, 103–106 (2008).PubMed 
Article 

Google Scholar 
Guo, S. W. & Thompson, E. A. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48, 361–372 (1992).CAS 
PubMed 
MATH 
Article 

Google Scholar 
Van Oosterhout, C., Huthinson, W. F., Wills, D. P. M. & Shipley, P. Micro-checker: Software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4, 535–538 (2004).Article 
CAS 

Google Scholar 
Excoffier, L., Smouse, P. E. & Quattro, J. M. Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics 131, 479–491 (1992).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Excoffier, L. & Lischer, H. E. L. Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol. Ecol. Resour. 10, 564–567 (2010).PubMed 
Article 

Google Scholar 
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B (Methodol.) 57, 289–300 (1995).MathSciNet 
MATH 

Google Scholar 
Pritchard, J. K., Stephens, M. & Donnelly, P. Inference of population structure using multilocus genotype data. Genetics 155, 945–959 (2000).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Falush, D., Stephens, M. & Pritchard, J. K. Inference of population structure using multilocus genotype data: Linked loci and correlated allele frequencies. Genetics 164, 1567–1587 (2003).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Hubisz, M. J., Falush, D., Stephens, M. & Pritchard, J. K. Inferring weak population structure with the assistance of sample group information. Mol. Ecol. Resour. 9, 1322–1332 (2009).PubMed 
PubMed Central 
Article 

Google Scholar 
Jombart, T., Devillard, S. & Balloux, F. Discriminant analysis of principal components: A new method for the analysis of genetically structured populations. BMC Genet. 11, 94 (2010).PubMed 
PubMed Central 
Article 

Google Scholar 
Dupanloup, I., Schneider, S. & Excoffier, L. A simulated annealing approach to define the genetic structure of populations. Mol. Ecol. 11, 2571–2581 (2002).CAS 
PubMed 
Article 

Google Scholar 
Peakall, R. & Smouse, P. E. GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28, 2537–2539 (2012).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Smouse, P. E. & Peakall, R. Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure. Heredity 82(Pt 5), 561–573 (1999).PubMed 
Article 

Google Scholar 
Rousset, F. Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance. Genetics 145, 1219–1228 (1997).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Bezerra, N. P. A. et al. Reproduction of Blackfin tuna Thunnus atlanticus (Perciformes: Scombridae) in Saint Peter and Saint Paul Archipelago, Equatorial Atlantic, Brazil. Rev. Biol. Trop. 61, 1327–1339 (2013).PubMed 
Article 

Google Scholar 
Fitzpatrick, B. M. Power and sample size for nested analysis of molecular variance. Mol. Ecol. 18, 3961–3966 (2009).PubMed 
Article 

Google Scholar 
Ely, B. et al. Consequences of the historical demography on the global population structure of two highly migratory cosmopolitan marine fishes: The yellowfin tuna (Thunnus albacares) and the skipjack tuna (Katsuwonus pelamis). BMC Evol. Biol. 5, 19 (2005).PubMed 
PubMed Central 
Article 

Google Scholar 
Alvarado Bremer, J. R., Viñas, J., Mejuto, J., Ely, B. & Pla, C. Comparative phylogeography of Atlantic bluefin tuna and swordfish: The combined effects of vicariance, secondary contact, introgression, and population expansion on the regional phylogenies of two highly migratory pelagic fishes. Mol. Phylogenet. Evol. 36, 169–187 (2005).CAS 
PubMed 
Article 

Google Scholar 
Hedgecock, D., Barber, P. & Edmands, S. Genetic approaches to measuring connectivity. Oceanography 20, 70–79 (2007).Article 

Google Scholar 
Pruett, C. L., Saillant, E. & Gold, J. R. Historical population demography of red snapper (Lutjanus campechanus) from the northern Gulf of Mexico based on analysis of sequences of mitochondrial DNA. Mar. Biol. 147, 593–602 (2005).CAS 
Article 

Google Scholar 
Saillant, E., Bradfield, S. C. & Gold, J. R. Genetic variation and spatial autocorrelation among young-of-the-year red snapper (Lutjanus campechanus) in the northern Gulf of Mexico. ICES J. Mar. Sci 67, 1240–1250 (2010).Article 

Google Scholar 
Robledo-Arnuncio, J. J. & Rousset, F. Isolation by distance in a continuous population under stochastic demographic fluctuations. J. Evol. Biol. 23, 53–71 (2010).CAS 
PubMed 
Article 

Google Scholar 
Rocha, L. A., Craig, M. T. & Bowen, B. W. Phylogeography and the conservation of coral reef fishes. Coral Reefs 26, 501–512 (2007).ADS 
Article 

Google Scholar 
Vasconcellos, A. V., Vianna, P., Paiva, P. C., Schama, R. & Solé-Cava, A. Genetic and morphometric differences between yellowtail snapper (Ocyurus chrysurus, Lutjanidae) populations of the tropical West Atlantic. Genet. Mol. Biol. 31, 308–316 (2008).CAS 
Article 

Google Scholar 
Vieira, K. R., Oliveira, J. E. L. & Barbalho, M. C. Reproductive characteristics of blackfin tuna Thunnus atlanticus (Lesson, 1831), in northeast Brazil. Collect. Vol. Sci. Pap ICCAT 58, 1629–1634 (2005).
Google Scholar 
Nielsen, E. E. et al. Genomic signatures of local directional selection in a high gene flow marine organism; the Atlantic cod (Gadus morhua). BMC Evol. Biol. 9, 276 (2009).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Lamichhaney, S. et al. Population-scale sequencing reveals genetic differentiation due to local adaptation in Atlantic herring. Proc. Natl. Acad. Sci. USA 109, 19345–19350 (2012).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Latch, E. K., Dharmarajan, G., Glaubitz, J. C. & Rhodes, O. E. Relative performance of Bayesian clustering software for inferring population substructure and individual assignment at low levels of population differentiation. Conserv. Genet. 7, 295–302 (2006).Article 

Google Scholar 
Brophy, D., Rodríguez-Ezpeleta, N., Fraile, I. & Arrizabalaga, H. Combining genetic markers with stable isotopes in otoliths reveals complexity in the stock structure of Atlantic bluefin tuna (Thunnus thynnus). Sci. Rep. 10, 14675 (2020).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar  More

Spalding, M. D. & Grenfell, A. M. New estimates of global and regional coral reef areas. Coral Reefs 16, 225–230 (1997).Article 

Google Scholar 
Moberg, F. & Folke, C. Ecological goods and services of coral reef ecosystems. Ecol. Econ. 29, 215–233 (1999).Article 

Google Scholar 
Roberts, C. M. et al. Marine Biodiversity Hotspots and Conservation Priorities for Tropical Reefs. Science 295, 1280–1284 (2002).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Johannes, R., Wiebe, W., Crossland, C., Rimmer, D. & Smith, S. Latitudinal limits of coral reef growth. Mar. Ecol. Prog. Ser. 11, 105–111 (1983).ADS 
Article 

Google Scholar 
Kleypas, J. A., Mcmanus, J. W. & Meñez, L. A. B. Environmental Limits to Coral Reef Development: Where Do We Draw the Line? Am. Zool. 39, 146–159 (1999).Article 

Google Scholar 
Yamano, H., Hori, K., Yamauchi, M., Yamagawa, O. & Ohmura, A. Highest-latitude coral reef at Iki Island, Japan. Coral Reefs 20, 9–12 (2001).Article 

Google Scholar 
Guan, Y., Hohn, S. & Merico, A. Suitable Environmental Ranges for Potential Coral Reef Habitats in the Tropical Ocean. PLOS ONE 10, e0128831 (2015).PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Bellwood, D. R. & Hughes, T. P. Regional-Scale Assembly Rules and Biodiversity of Coral Reefs. Science 292, 1532–1535 (2001).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Connolly, S. R., Bellwood, D. R. & Hughes, T. P. Indo-Pacific Biodiversity of Coral Reefs: Deviations from a Mid-Domain Model. Ecology 84, 2178–2190 (2003).Article 

Google Scholar 
Bellwood, D. R., Hughes, T. P., Connolly, S. R. & Tanner, J. Environmental and geometric constraints on Indo‐Pacific coral reef biodiversity. Ecol. Lett. 8, 643–651 (2005).Article 

Google Scholar 
Kiessling, W., Simpson, C., Beck, B., Mewis, H. & Pandolfi, J. M. Equatorial decline of reef corals during the last Pleistocene interglacial. Proc. Natl Acad. Sci. 109, 21378–21383 (2012).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Veron, J. E. N. et al. Delineating the Coral Triangle. Galaxea. J. Coral Reef. Stud. 11, 91–100 (2009).Article 

Google Scholar 
Briggs, J. C. Marine Longitudinal Biodiversity: Causes and Conservation. Divers. Distrib. 13, 544–555 (2007).Article 

Google Scholar 
Renema, W. et al. Hopping Hotspots: Global Shifts in Marine Biodiversity. Science 321, 654–657 (2008).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Kiessling, W. Paleoclimatic significance of Phanerozoic reefs. Geology 29, 751–754 (2001).ADS 
Article 

Google Scholar 
Wallace, C. & Rosen, B. Diverse staghorn corals (Acropora) in high-latitude Eocene assemblages: Implications for the evolution of modern diversity patterns of reef corals. Proc. Biol. Sci. 273, 975–982 (2006).PubMed 
PubMed Central 

Google Scholar 
Perrin, C. & Kiessling, W. Latitudinal trends in Cenozoic reef patterns and their relationship to climate. Carbonate Syst. Oligocene–Miocene Clim. Transit. 17–33 (Wiley-Blackwell, 2010).Kiessling, W. Habitat effects and sampling bias on Phanerozoic reef distribution. Facies 51, 24–32 (2005).Article 

Google Scholar 
Kiessling, W. Reef expansion during the Triassic: Spread of photosymbiosis balancing climatic cooling. Palaeogeogr. Palaeoclimatol. Palaeoecol. 290, 11–19 (2010).Article 

Google Scholar 
Ziegler, A. M., Hulver, M. L., Lotts, A. L. & Schmachtenberg, W. F. Uniformitarianism and palaeoclimates: inferences from the distribution of carbonate rocks. In: Fossils and Climate (ed. Brenchley, P. J.), 3–25 (Wiley, Chichester, 1984).Crame, J. A. & Rosen, B. R. Cenozoic palaeogeography and the rise of modern biodiversity patterns. Geol. Soc. Lond. Spec. Publ. 194, 153–168 (2002).ADS 
Article 

Google Scholar 
Leprieur, F. et al. Plate tectonics drive tropical reef biodiversity dynamics. Nat. Commun. 7, 1–8 (2016).Article 
CAS 

Google Scholar 
Zaffos, A., Finnegan, S. & Peters, S. E. Plate tectonic regulation of global marine animal diversity. Proc. Natl Acad. Sci. U. S. A. 114, 5653–5658 (2017).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Roberts, G. G. & Mannion, P. D. Timing and periodicity of Phanerozoic marine biodiversity and environmental change. Sci. Rep. 9, 6116 (2019).ADS 
PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Valentine, J. W. & Moores, E. M. Global Tectonics and the Fossil Record. J. Geol. 80, 167–184 (1972).ADS 
Article 

Google Scholar 
Pellissier, L., Heine, C., Rosauer, D. F. & Albouy, C. Are global hotspots of endemic richness shaped by plate tectonics? Biol. J. Linn. Soc. 123, 247–261 (2017).Article 

Google Scholar 
Chittaro, P. M. Species-area relationships for coral reef fish assemblages of St. Croix, US Virgin Islands. Mar. Ecol. Prog. Ser. 233, 253–261 (2002).ADS 
Article 

Google Scholar 
Tittensor, D. P., Micheli, F., Nyström, M. & Worm, B. Human impacts on the species–area relationship in reef fish assemblages. Ecol. Lett. 10, 760–772 (2007).PubMed 
Article 

Google Scholar 
Tittensor, D. P. et al. Global patterns and predictors of marine biodiversity across taxa. Nature 466, 1098–1101 (2010).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Huntington, B. E. & Lirman, D. Species-area relationships in coral communities: evaluating mechanisms for a commonly observed pattern. Coral Reefs 31, 929–938 (2012).ADS 
Article 

Google Scholar 
Kiessling, W., Simpson, C. & Foote, M. Reefs as cradles of evolution and sources of biodiversity in the Phanerozoic. Science 327, 196–198 (2010).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Pandolfi, J. M. et al. Global Trajectories of the Long-Term Decline of Coral Reef Ecosystems. Science 301, 955–958 (2003).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Hoegh-Guldberg, O. Coral reef ecosystems and anthropogenic climate change. Reg. Environ. Change 11, 215–227 (2011).Article 

Google Scholar 
Hughes, T. P. et al. Global warming and recurrent mass bleaching of corals. Nature 543, 373–377 (2017).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Kim, S. W. et al. Refugia under threat: Mass bleaching of coral assemblages in high-latitude eastern Australia. Glob. Change Biol. 25, 3918–3931 (2019).ADS 
Article 

Google Scholar 
Pörtner, H.-O. et al. IPCC special report on the ocean and cryosphere in a changing climate. IPCC Intergov. Panel Clim. Change Geneva Switz. 1, 1–755 (2019).Sully, S., Burkepile, D. E., Donovan, M. K., Hodgson, G. & van Woesik, R. A global analysis of coral bleaching over the past two decades. Nat. Commun. 10, 1264 (2019).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Couce, E., Ridgwell, A. & Hendy, E. J. Future habitat suitability for coral reef ecosystems under global warming and ocean acidification. Glob. Change Biol. 19, 3592–3606 (2013).ADS 
Article 

Google Scholar 
Hoegh-Guldberg, O., Poloczanska, E. S., Skirving, W. & Dove, S. Coral Reef Ecosystems under Climate Change and Ocean Acidification. Front. Mar. Sci. 4, 1–20 (2017).O’Neill, B. C. et al. The Scenario Model Intercomparison Project (ScenarioMIP) for CMIP6. Geosci. Model Dev. 9, 3461–3482 (2016).ADS 
Article 

Google Scholar 
Precht, W. F. & Aronson, R. B. Climate flickers and range shifts of reef corals. Front. Ecol. Environ. 2, 307–314 (2004).Article 

Google Scholar 
Greenstein, B. J. & Pandolfi, J. M. Escaping the heat: range shifts of reef coral taxa in coastal Western Australia. Glob. Change Biol. 14, 513–528 (2008).ADS 
Article 

Google Scholar 
Pellissier, L. et al. Quaternary coral reef refugia preserved fish diversity. Science 344, 1016–1019 (2014).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Vilhena, D. A. & Smith, A. B. Spatial Bias in the Marine Fossil Record. PLoS ONE 8, 1–7 (2013).Article 
CAS 

Google Scholar 
Close, R. A., Benson, R. B. J., Saupe, E. E., Clapham, M. E. & Butler, R. J. The spatial structure of Phanerozoic marine animal diversity. Science 368, 420–424 (2020).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Jones, L. A., Dean, C. D., Mannion, P. D., Farnsworth, A. & Allison, P. A. Spatial sampling heterogeneity limits the detectability of deep time latitudinal biodiversity gradients. Proc. R. Soc. B Biol. Sci. 288, 20202762 (2021).Article 

Google Scholar 
Jones, L. A. & Eichenseer, K. Uneven spatial sampling distorts reconstructions of Phanerozoic seawater temperature. Geology (2021) https://doi.org/10.1130/G49132.1.Stolarski, J. et al. The ancient evolutionary origins of Scleractinia revealed by azooxanthellate corals. BMC Evol. Biol. 11, 1–11 (2011).Article 

Google Scholar 
Frankowiak, K. et al. Photosymbiosis and the expansion of shallow-water corals. Sci. Adv. 2, e1601122 (2016).ADS 
PubMed 
PubMed Central 
Article 
CAS 

Google Scholar 
Phillips, S. J., Anderson, R. P., Dudík, M., Schapire, R. E. & Blair, M. E. Opening the black box: an open-source release of Maxent. Ecography 40, 887–893 (2017).Article 

Google Scholar 
Phillips, S. J., Anderson, R. P. & Schapire, R. E. Maximum entropy modeling of species geographic distributions. Ecol. Model. 190, 231–259 (2006).Article 

Google Scholar 
Swets, J. A. Measuring the accuracy of diagnostic systems. Science 240, 1285–1293 (1988).ADS 
MathSciNet 
CAS 
PubMed 
MATH 
Article 

Google Scholar 
Boyce, M. S., Vernier, P. R., Nielsen, S. E. & Schmiegelow, F. K. A. Evaluating resource selection functions. Ecol. Model. 157, 281–300 (2002).Article 

Google Scholar 
Hirzel, A. H., LeLay, G., Helfer, V., Randin, C. & Guisan, A. Evaluating the ability of habitat suitability models to predict species presences. Ecol. Model. 199, 142–152 (2006).Article 

Google Scholar 
Elith, J., Kearney, M. & Phillips, S. The art of modelling range-shifting species. Methods Ecol. Evol. 1, 330–342 (2010).Article 

Google Scholar 
Miller, K. G. et al. The Phanerozoic Record of Global Sea-Level Change. Science 310, 1293–1298 (2005).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Hallam, A., Grose, J. A. & Ruffell, A. H. Palaeoclimatic significance of changes in clay mineralogy across the Jurassic-Cretaceous boundary in England and France. Palaeogeogr. Palaeoclimatol. Palaeoecol. 81, 173–187 (1991).Article 

Google Scholar 
Gröcke, D. R., Price, G. D., Ruffell, A. H., Mutterlose, J. & Baraboshkin, E. Isotopic evidence for Late Jurassic–Early Cretaceous climate change. Palaeogeogr. Palaeoclimatol. Palaeoecol. 202, 97–118 (2003).Article 

Google Scholar 
Royer, D. L., Berner, R. A., Montañez, I. P., Tabor, N. J. & Beerling, D. J. CO2 as a primary driver of Phanerozoic climate. GSA Today 14, 1–10 (2004).
Google Scholar 
Grabowski, J. et al. Magnetic susceptibility and spectral gamma logs in the Tithonian–Berriasian pelagic carbonates in the Tatra Mts (Western Carpathians, Poland): Palaeoenvironmental changes at the Jurassic/Cretaceous boundary. Cretac. Res. 43, 1–17 (2013).Article 

Google Scholar 
Vickers, M. L. et al. The duration and magnitude of Cretaceous cool events: Evidence from the northern high latitudes. GSA Bull. 131, 1979–1994 (2019).CAS 
Article 

Google Scholar 
Hay, W. W. & Floegel, S. New thoughts about the Cretaceous climate and oceans. Earth-Sci. Rev. 115, 262–272 (2012).ADS 
CAS 
Article 

Google Scholar 
Tennant, J. P., Mannion, P. D. & Upchurch, P. Sea level regulated tetrapod diversity dynamics through the Jurassic/Cretaceous interval. Nat. Commun. 7, 12737 (2016).ADS 
CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Schouten, S. et al. Onset of long-term cooling of Greenland near the Eocene-Oligocene boundary as revealed by branched tetraether lipids. Geology 36, 147 (2008).ADS 
Article 

Google Scholar 
Zachos, J. C., Dickens, G. R. & Zeebe, R. E. An early Cenozoic perspective on greenhouse warming and carbon-cycle dynamics. Nature 451, 279–283 (2008).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Crame, J. A. Taxonomic diversity gradients through geological time. Divers. Distrib. 7, 175–189 (2001).
Google Scholar 
Mannion, P. D., Upchurch, P., Benson, R. B. J. & Goswami, A. The latitudinal biodiversity gradient through deep time. Trends Ecol. Evol. 29, 42–50 (2014).PubMed 
Article 

Google Scholar 
Fenton, I. S. et al. The impact of Cenozoic cooling on assemblage diversity in planktonic foraminifera. Philos. Trans. R. Soc. B Biol. Sci. 371, 1–12 (2016).Article 
CAS 

Google Scholar 
Saupe, E. E. et al. Climatic shifts drove major contractions in avian latitudinal distributions throughout the Cenozoic. Proc. Natl Acad. Sci. 116, 12895–12900 (2019).CAS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Hall, R. Cenozoic geological and plate tectonic evolution of SE Asia and the SW Pacific: computer-based reconstructions, model and animations. J. Asian Earth Sci. 20, 353–431 (2002).ADS 
Article 

Google Scholar 
Hall, R. Southeast Asia’s changing palaeogeography. Blumea 54, 148–161 (2009).Article 

Google Scholar 
Gaboriau, T. et al. Ecological constraints coupled with deep-time habitat dynamics predict the latitudinal diversity gradient in reef fishes. Proc. R. Soc. B Biol. Sci. 286, 20191506 (2019).Article 

Google Scholar 
Saupe, E. E. et al. Extinction intensity during Ordovician and Cenozoic glaciations explained by cooling and palaeogeography. Nat. Geosci. 13, 65–70 (2020).ADS 
Article 
CAS 

Google Scholar 
Lunt, D. J. et al. DeepMIP: model intercomparison of early Eocene climatic optimum (EECO) large-scale climate features and comparison with proxy data. Clim 17, 203–227 (2021).ADS 

Google Scholar 
Freeman, L. A., Kleypas, J. A. & Miller, A. J. Coral Reef Habitat Response to Climate Change Scenarios. PLoS ONE 8, 1–14 (2013).
Google Scholar 
Foster, G. L., Royer, D. L. & Lunt, D. J. Future climate forcing potentially without precedent in the last 420 million years. Nat. Commun. 8, 1–8 (2017).ADS 
Article 
CAS 

Google Scholar 
Farnsworth, A. et al. Past East Asian monsoon evolution controlled by paleogeography, not CO2. Sci. Adv. 5, 1–13 (2019).Article 
CAS 

Google Scholar 
Zhang, L. et al. Consensus Forecasting of Species Distributions: The Effects of Niche Model Performance and Niche Properties. PLoS ONE 10, 1–18 (2015).
Google Scholar 
Harrison, S. P. et al. Evaluation of CMIP5 palaeo-simulations to improve climate projections. Nat. Clim. Change 5, 735–743 (2015).ADS 
Article 

Google Scholar 
Seo, C., Thorne, J. H., Hannah, L. & Thuiller, W. Scale effects in species distribution models: implications for conservation planning under climate change. Biol. Lett. 5, 39–43 (2009).PubMed 
Article 

Google Scholar 
Couce, E., Ridgwell, A. & Hendy, E. J. Environmental controls on the global distribution of shallow-water coral reefs. J. Biogeogr. 39, 1508–1523 (2012).Article 

Google Scholar 
Laborel, J. West African reef corals: an hypothesis on their origin. in Proceedings of the Second International Coral Reef Symposium vol. 1 425–443 (Great Barrier Reef Committee Brisbane, 1974).Spalding, M., Spalding, M. D., Ravilious, C. & Green, E. P. World Atlas of Coral Reefs. (University of California Press, 2001).Block, S. et al. Where to Dig for Fossils: Combining Climate-Envelope, Taphonomy and Discovery Models. PLoS ONE 11, 1–16 (2016).Jones, L. A. et al. Coupling of palaeontological and neontological reef coral data improves forecasts of biodiversity responses under global climatic change. R. Soc. Open Sci. 6, 182111 (2019).ADS 
PubMed 
PubMed Central 
Article 

Google Scholar 
Kusumoto, B. et al. Global distribution of coral diversity: Biodiversity knowledge gradients related to spatial resolution. Ecol. Res. 35, 315–326 (2020).Article 

Google Scholar 
Muir, P. R., Wallace, C. C., Done, T. & Aguirre, J. D. Limited scope for latitudinal extension of reef corals. Science 348, 1135–1138 (2015).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Sillero, N. & Barbosa, A. M. Common mistakes in ecological niche models. Int. J. Geogr. Inf. Sci. 35, 213–226 (2021).Article 

Google Scholar 
Valdes, P. J. et al. The BRIDGE HadCM3 family of climate models:HadCM3@Bristol v1.0. Geosci. Model Dev. 10, 3715–3743 (2017).ADS 
CAS 
Article 

Google Scholar 
Sheppard, C. R. C. Predicted recurrences of mass coral mortality in the Indian Ocean. Nature 425, 294–297 (2003).ADS 
CAS 
PubMed 
Article 

Google Scholar 
Saupe, E. E. et al. Macroevolutionary consequences of profound climate change on niche evolution in marine molluscs over the past three million years. Proc. R. Soc. B Biol. Sci. 281, 1–9 (2014).
Google Scholar 
Haywood, A. M. et al. What can Palaeoclimate Modelling do for you? Earth Syst. Environ. 3, 1–18 (2019).Article 

Google Scholar 
Sellwood, B. W. & Valdes, P. J. Mesozoic climates: General circulation models and the rock record. Sediment. Geol. 190, 269–287 (2006).ADS 
Article 

Google Scholar 
Waterson, A. M. et al. Modelling the climatic niche of turtles: a deep-time perspective. Proc. R. Soc. B Biol. Sci. 283, 1–9 (2016).
Google Scholar 
Chiarenza, A. A. et al. Ecological niche modelling does not support climatically-driven dinosaur diversity decline before the Cretaceous/Paleogene mass extinction. Nat. Commun. 10, 1–14 (2019).CAS 
Article 

Google Scholar 
Dunne, E. M., Farnsworth, A., Greene, S. E., Lunt, D. J. & Butler, R. J. Climatic drivers of latitudinal variation in Late Triassic tetrapod diversity. Palaeontology 64, 101–117 (2020).Article 

Google Scholar 
Lyster, S. J., Whittaker, A. C., Allison, P. A., Lunt, D. J. & Farnsworth, A. Predicting sediment discharges and erosion rates in deep time—examples from the late Cretaceous North American continent. Basin Res. 1–27 (2020) https://doi.org/10.1111/bre.12442.Lunt, D. J. et al. Palaeogeographic controls on climate and proxy interpretation. Clim 12, 1181–1198 (2016).ADS 

Google Scholar 
Vasquez, V. L., de Lima, A. A., dos Santos, A. P. & Pinto, M. P. Influence of spatial extent on habitat suitability models for primate species of Atlantic Forest. Ecol. Inform. 61, 101179 (2021).Article 

Google Scholar 
Collins, D. S. et al. Controls on tidal sedimentation and preservation: Insights from numerical tidal modelling in the Late Oligocene–Miocene South China Sea, Southeast Asia. Sedimentology 65, 2468–2505 (2018).Article 

Google Scholar 
Dean, C. D., Collins, D. S., van Cappelle, M., Avdis, A. & Hampson, G. J. Regional-scale paleobathymetry controlled location, but not magnitude, of tidal dynamics in the Late Cretaceous Western Interior Seaway, USA. Geology 47, 1083–1087 (2019).ADS 
CAS 
Article 

Google Scholar 
Markwick, P. J. & Valdes, P. J. Palaeo-digital elevation models for use as boundary conditions in coupled ocean–atmosphere GCM experiments: a Maastrichtian (late Cretaceous) example. Palaeogeogr. Palaeoclimatol. Palaeoecol. 213, 37–63 (2004).Article 

Google Scholar 
Elith, J. et al. Novel methods improve prediction of species’ distributions from occurrence data. Ecography 29, 129–151 (2006).Article 

Google Scholar 
Sillero, N. What does ecological modelling model? A proposed classification of ecological niche models based on their underlying methods. Ecol. Model. 222, 1343–1346 (2011).Article 

Google Scholar 
Guisan, A., Thuiller, W. & Zimmermann, N. E. Habitat suitability and distribution models: with applications in R. (Cambridge University Press, 2017).Kearney, M. R., Wintle, B. A. & Porter, W. P. Correlative and mechanistic models of species distribution provide congruent forecasts under climate change. Conserv. Lett. 3, 203–213 (2010).Article 

Google Scholar 
Owens, H. L. et al. Constraints on interpretation of ecological niche models by limited environmental ranges on calibration areas. Ecol. Model. 263, 10–18 (2013).Article 

Google Scholar 
Franklin, J. Mapping Species Distributions: Spatial Inference and Prediction. (Cambridge University Press, 2010). https://doi.org/10.1017/CBO9780511810602.Liu, C., White, M. & Newell, G. Selecting thresholds for the prediction of species occurrence with presence-only data. J. Biogeogr. 40, 778–789 (2013).Article 

Google Scholar 
Liu, C., Newell, G. & White, M. On the selection of thresholds for predicting species occurrence with presence-only data. Ecol. Evol. 6, 337–348 (2016).PubMed 
Article 

Google Scholar 
Kiessling, W. & Krause, M. C. PARED—An online database of Phanerozoic reefs. https://www.paleo-reefs.pal.uni-erlangen.de/ (2021).Jones, L. A., Mannion, P. D., Farnsworth, A., Bragg, F. & Lunt, D. J. Code from ‘Climatic and tectonic drivers shaped the tropical distribution of coral reefs’. Zenodo (2022) https://doi.org/10.5281/zenodo.6458366. More