Register      Login
Wildlife Research Wildlife Research Society
Ecology, management and conservation in natural and modified habitats
RESEARCH ARTICLE

Comparison of morphological identification and DNA metabarcoding for dietary analysis of faeces from a subtropical lizard

Takaki Kurita https://orcid.org/0000-0002-2857-3834 A * and Mamoru Toda B
+ Author Affiliations
- Author Affiliations

A Natural History Museum and Institute Chiba, 955-2 Aoba-cho, Chuo-ku, Chiba 260-8682, Japan.

B Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.


Handling Editor: Andrea Taylor

Wildlife Research 50(3) 224-236 https://doi.org/10.1071/WR22054
Submitted: 18 March 2022  Accepted: 9 August 2022   Published: 5 September 2022

© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Abstract

Context: The common methods for detecting prey in faeces are traditional morphological examination of digested prey remains and faecal DNA (fDNA) metabarcoding. Although recent studies have shown that fDNA metabarcoding can identify prey that is excreted without morphologically identifiable remains, it is unclear whether fDNA metabarcoding provides a more comprehensive assessment of diet than morphological analyses.

Aims: We aimed to compare prey detection abilities of morphological and fDNA-metabarcoding assays by evaluating similarities and differences of estimated prey communities. We also aimed to provide the first comprehensive prey composition of an endangered ground-dwelling gecko, Goniurosaurus kuroiwae, which will contribute to the conservation management of this species.

Methods: We identified the prey consumed by G. kuroiwae by morphological examination and fDNA metabarcoding using the same series of faecal samples. For these two methods, we compared the overall community composition of prey items and the detection rate for each prey type. We also tested for environmental and ontogenetic variation of the composition of prey items consumed.

Key results: When comparing the two methods there were significant differences in the composition of prey communities that were identified from the same series of faecal samples. The fDNA metabarcoding analyses showed the higher rate of detection of earthworms, amphipods and coleopteran and lepidopteran larvae, which lack hard exoskeleton or have small body sizes. The morphological assay allowed identification of a few prey types, including spiders and earwigs, more frequently than fDNA metabarcoding. Combined use of both methods revealed that G. kuroiwae consumed large-sized invertebrates with a slight ontogenetic shift from small-sized invertebrates to large-sized ones.

Conclusions: Although fDNA metabarcoding showed the higher overall prey detection rate, it did not include all the prey items detected by morphological analysis. The complementary use of both methods can yield better understanding of the prey composition of invertebrate-consuming animals represented by reptiles and amphibians.

Implications: We have shown that the most comprehensive reconstruction of prey composition from faeces will be achieved by combining results from both fDNA metabarcoding and morphological analyses.

Keywords: DNA metabarcoding, endangered species, faeces, gecko, Goniurosaurus kuroiwae, invertebrates, morphology, prey identification, Ryukyu Archipelago.


References

Anderson, MJ (2001). A new method for non-parametric multivariate analysis of variance. Austral Ecology 26, 32–46.
A new method for non-parametric multivariate analysis of variance.Crossref | GoogleScholarGoogle Scholar |

Anderson, MJ (2006). Distance-based tests for homogeneity of multivariate dispersions. Biometrics 62, 245–253.
Distance-based tests for homogeneity of multivariate dispersions.Crossref | GoogleScholarGoogle Scholar |

Ando, H, Mukai, H, Komura, T, Dew, T, Ando, M, and Isagi, Y (2020). Methodological trends and perspectives of animal dietary studies by noninvasive fecal DNA metabarcoding. Environmental DNA 2, 391–406.
Methodological trends and perspectives of animal dietary studies by noninvasive fecal DNA metabarcoding.Crossref | GoogleScholarGoogle Scholar |

Azama, Y (1989). Effects of logging of natural forests in Kunigami area of Okinawa Hontô to animal communities. Journal of Okinawa Association of Biology Education 22, 3–12.

Azuma, S, Sasaki, T, and Itô, Y (1997). Effects of undergrowth removal on the species diversity of insects in natural forests of Okinawa Hontô. Pacific Conservation Biology 3, 156–160.
Effects of undergrowth removal on the species diversity of insects in natural forests of Okinawa Hontô.Crossref | GoogleScholarGoogle Scholar |

Bjorndal, KA, Bolten, AB, Lagueux, CJ, and Jackson, DR (1997). Dietary overlap in three sympatric congeneric freshwater turtles (Pseudemys) in Florida. Chelonian Conservation Biology 2, 430–433.

Boyer, F, Mercier, C, Bonin, A, Le Bras, Y, Taberlet, P, and Coissac, E (2016). obitools: a UNIX-inspired software package for DNA metabarcoding. Molecular Ecology Resources 16, 176–182.
obitools: a UNIX-inspired software package for DNA metabarcoding.Crossref | GoogleScholarGoogle Scholar |

Brown, DS, Jarman, SN, and Symondson, WOC (2012). Pyrosequencing of prey DNA in reptile faeces: analysis of earthworm consumption by slow worms. Molecular Ecology Resources 12, 259–266.
Pyrosequencing of prey DNA in reptile faeces: analysis of earthworm consumption by slow worms.Crossref | GoogleScholarGoogle Scholar |

Callahan, BJ, McMurdie, PJ, Rosen, MJ, Han, AW, Johnson, AJA, and Holmes, SP (2016). DADA2: high-resolution sample inference from Illumina amplicon data. Nature Methods 13, 581–583.
DADA2: high-resolution sample inference from Illumina amplicon data.Crossref | GoogleScholarGoogle Scholar |

Camacho, C, Coulouris, G, Avagyan, V, Ma, N, Papadopoulos, J, Bealer, K, and Madden, TL (2009). BLAST+: architecture and applications. BMC Bioinformatics 10, 421.
BLAST+: architecture and applications.Crossref | GoogleScholarGoogle Scholar |

Chao, A, and Jost, L (2012). Coverage-based rarefaction and extrapolation: standardizing samples by completeness rather than size. Ecology 93, 2533–2547.
Coverage-based rarefaction and extrapolation: standardizing samples by completeness rather than size.Crossref | GoogleScholarGoogle Scholar |

Chen, S, Zhou, Y, Chen, Y, and Gu, J (2018). fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890.
fastp: an ultra-fast all-in-one FASTQ preprocessor.Crossref | GoogleScholarGoogle Scholar |

Corse, E, Tougard, C, Archambaud-Suard, G, Agnèse, J-F, Messu Mandeng, FD, Bilong Bilong, CF, Duneau, D, Zinger, L, Chappaz, R, Xu, CCY, Meglécz, E, and Dubut, V (2019). One-locus-several-primers: a strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies. Ecology and Evolution 9, 4603–4620.
One-locus-several-primers: a strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies.Crossref | GoogleScholarGoogle Scholar |

da Silva, LP, Mata, VA, Lopes, PB, Pereira, P, Jarman, SN, Lopes, RJ, and Beja, P (2019). Advancing the integration of multi-marker metabarcoding data in dietary analysis of trophic generalists. Molecular Ecology Resources 19, 1420–1432.
Advancing the integration of multi-marker metabarcoding data in dietary analysis of trophic generalists.Crossref | GoogleScholarGoogle Scholar |

de Cáceres, M, and Legendre, P (2009). Associations between species and groups of sites: indices and statistical inference. Ecology 90, 3566–3574.
Associations between species and groups of sites: indices and statistical inference.Crossref | GoogleScholarGoogle Scholar |

de Cáceres, M, Legendre, P, and Moretti, M (2010). Improving indicator species analysis by combining groups of sites. Oikos 119, 1674–1684.
Improving indicator species analysis by combining groups of sites.Crossref | GoogleScholarGoogle Scholar |

Deagle, BE, Thomas, AC, McInnes, JC, Clarke, LJ, Vesterinen, EJ, Clare, EL, Kartzinel, TR, and Eveson, JP (2019). Counting with DNA in metabarcoding studies: how should we convert sequence reads to dietary data? Molecular Ecology 28, 391–406.
Counting with DNA in metabarcoding studies: how should we convert sequence reads to dietary data?Crossref | GoogleScholarGoogle Scholar |

Dial, BE (1978). Aspects of the behavioral ecology of two Chihuahuan desert geckos (Reptilia, Lacertilia, Gekkonidae). Journal of Herpetology 12, 209–216.
Aspects of the behavioral ecology of two Chihuahuan desert geckos (Reptilia, Lacertilia, Gekkonidae).Crossref | GoogleScholarGoogle Scholar |

Dunham AE (1983) Realized niche overlap, resource abundance and intensity of interspecific competition in lizard ecology. In ‘Lizards Ecology: Studies of Model Organism’. (Eds. RD Huey, ER Pianka, TW Schoener) pp. 261–280. (Harvard University Press: Cambridge, Cambridgeshire, UK)
| Crossref |

Egeter, B, Bishop, PJ, and Robertson, BC (2015). Detecting frogs as prey in the diets of introduced mammals: a comparison between morphological and DNA-based diet analyses. Molecular Ecology Resources 15, 306–316.
Detecting frogs as prey in the diets of introduced mammals: a comparison between morphological and DNA-based diet analyses.Crossref | GoogleScholarGoogle Scholar |

Elbrecht, V, Braukmann, TWA, Ivanova, NV, Prosser, SWJ, Hajibabaei, M, Wright, M, Zakharov, EV, Hebert, PDN, and Steinke, D (2019). Validation of COI metabarcoding primers for terrestrial arthropods. PeerJ 7, e7745.
Validation of COI metabarcoding primers for terrestrial arthropods.Crossref | GoogleScholarGoogle Scholar |

Enoki, T, Kusumoto, B, Igarashi, S, and Tsuji, K (2014). Stand structure and plant species occurrence in forest edge habitat along different aged roads on Okinawa Island, southwestern Japan. Journal of Forest Research 19, 97–104.
Stand structure and plant species occurrence in forest edge habitat along different aged roads on Okinawa Island, southwestern Japan.Crossref | GoogleScholarGoogle Scholar |

Ficetola, GF, Manenti, R, and Taberlet, P (2019). Environmental DNA and metabarcoding for the study of amphibians and reptiles: species distribution, the microbiome, and much more. Amphibia-Reptilia 40, 129–148.
Environmental DNA and metabarcoding for the study of amphibians and reptiles: species distribution, the microbiome, and much more.Crossref | GoogleScholarGoogle Scholar |

Gill, V, Pinho, CJ, Aguiar, CAS, Jardim, C, Rebelo, R, and Vasconcelos, R (2020). Questioning the proverb ‘more haste, less speed’: classic versus metabarcoding approaches for the diet study of a remote island endemic gecko. PeerJ 8, e8084.
Questioning the proverb ‘more haste, less speed’: classic versus metabarcoding approaches for the diet study of a remote island endemic gecko.Crossref | GoogleScholarGoogle Scholar |

Hamanaka, K, Mori, A, and Moriguchi, H (2014). Literature survey on food habit of snakes in Japan: revisited. Bulletin of Herpetological Society of Japan 2014, 167–181.

Hasegawa, M, Sasaki, T, Sato, H, and Abe, S (2015). Effects of roads on collembolan community structure in subtropical evergreen forests on Okinawa Island, southwestern Japan. Pedobiologia 58, 13–21.
Effects of roads on collembolan community structure in subtropical evergreen forests on Okinawa Island, southwestern Japan.Crossref | GoogleScholarGoogle Scholar |

Hibitts, TJ, Pianka, ER, Huey, RB, and Whtting, MJ (2005). Ecology of the common barking gecko (Ptenopus garrulus) in southern Africa. Journal of Herpetology 39, 509–515.
Ecology of the common barking gecko (Ptenopus garrulus) in southern Africa.Crossref | GoogleScholarGoogle Scholar |

Honda, M, and Ota, H (2017). On the live colouration and partial mitochondrial DNA sequences in the topotypic population of Goniurosaurus kuroiwae orientalis (Squamata: Eublepharidae), with description of a new subspecies from Tokashikijima Island, Ryukyu Archipelago, Japan. Asian Herpetological Research 8, 96–107.
On the live colouration and partial mitochondrial DNA sequences in the topotypic population of Goniurosaurus kuroiwae orientalis (Squamata: Eublepharidae), with description of a new subspecies from Tokashikijima Island, Ryukyu Archipelago, Japan.Crossref | GoogleScholarGoogle Scholar |

Huey, RB, and Pianka, ER (1981). Ecological consequences of foraging mode. Ecology 62, 991–999.
Ecological consequences of foraging mode.Crossref | GoogleScholarGoogle Scholar |

IUCN (2021) The IUCN Red List of Threatened Species. Version 2021-3. Available at https://www.iucnredlist.org. [Accessed on 17 July 2022]

Kamenova, S, Mayer, R, Rubbmark, OR, Coissac, E, Plantegenest, M, and Traugott, M (2018). Comparing three types of dietary samples for prey DNA decay in an insect generalist predator. Molecular Ecology Resources 18, 966–973.
Comparing three types of dietary samples for prey DNA decay in an insect generalist predator.Crossref | GoogleScholarGoogle Scholar |

Kartzinel, TR, and Pringle, RM (2015). Molecular detection of invertebrate prey in vertebrate diets: trophic ecology of Caribbean island lizards. Molecular Ecology Resources 15, 903–914.
Molecular detection of invertebrate prey in vertebrate diets: trophic ecology of Caribbean island lizards.Crossref | GoogleScholarGoogle Scholar |

Kidera N, Ota H (2017) Goniurosaurus kuroiwae. The IUCN Red List of Threatened Species 2017:e.T98152257A96877452 [html]. Available at
| Crossref |

Kurita, T, Honda, M, and Toda, M (2018). Species delimitation and biogeography of the Ryukyu ground geckos, Goniurosaurus kuroiwae ssp. (Squamata: Eublepharidae), by use of mitochondrial and nuclear DNA analyses. Journal of Zoological Systematics and Evolutionary Research 56, 209–222.
Species delimitation and biogeography of the Ryukyu ground geckos, Goniurosaurus kuroiwae ssp. (Squamata: Eublepharidae), by use of mitochondrial and nuclear DNA analyses.Crossref | GoogleScholarGoogle Scholar |

Liang, B, Zhou, RB, Liu, YL, Chen, B, Grismer, LL, and Wang, N (2018). Renewed classification within Goniurosaurus (Squamata: Eublepharidae) uncovers the dual roles of a continental island (Hainan) in species evolution. Molecular Phylogenetics and Evolution 127, 646–654.
Renewed classification within Goniurosaurus (Squamata: Eublepharidae) uncovers the dual roles of a continental island (Hainan) in species evolution.Crossref | GoogleScholarGoogle Scholar |

Martin, M (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12.
Cutadapt removes adapter sequences from high-throughput sequencing reads.Crossref | GoogleScholarGoogle Scholar |

Meglécz, E (2022). COInr and mkCOInr: building and customizing a non-redundant barcoding reference database from BOLD and NCBI using a lightweight pipeline. bioRxiv , .
COInr and mkCOInr: building and customizing a non-redundant barcoding reference database from BOLD and NCBI using a lightweight pipeline.Crossref | GoogleScholarGoogle Scholar |

Mori, A, and Moriguchi, H (1988). Food habits of the snake in Japan: a critical review. Snake 20, 98–113.

Mumma, MA, Adams, JR, Zieminski, C, Fuller, TK, Mahoney, SP, and Waits, LP (2016). A comparison of morphological and molecular diet analyses of predator scats. Journal of Mammalogy 97, 112–120.
A comparison of morphological and molecular diet analyses of predator scats.Crossref | GoogleScholarGoogle Scholar |

Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O’Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoecs E, Wagner H (2020) vegan: community ecology package. R package version 2.5-7. Available at https://CRAN.R-project.org/package=vegan

Ômine, T, and Itô, Y (1998). Abundance and diversity of soil macrofauna of forests in Yanbaru, northern montane part of Okinawa Island, with special reference to removal of undergrowth. Okinawa Daigaku Kiyo 15, 131–159.

Ota, H (1998). Geographic patterns of endemism and speciation in amphibians and reptiles of the Ryukyu Archipelago, Japan, with special reference to their paleogeographical implications. Researches of Population Ecology 40, 189–204.
Geographic patterns of endemism and speciation in amphibians and reptiles of the Ryukyu Archipelago, Japan, with special reference to their paleogeographical implications.Crossref | GoogleScholarGoogle Scholar |

Ota, H (2000). Current status of the threatened amphibians and reptiles of Japan. Population Ecology 42, 5–9.
Current status of the threatened amphibians and reptiles of Japan.Crossref | GoogleScholarGoogle Scholar |

Pérez-Mellado, V, Pérez-Cembranos, A, Garrido, M, Luiselli, L, and Corti, C (2011). Using faecal samples in lizard dietary studies. Amphibia-Reptilia 32, 1–7.
Using faecal samples in lizard dietary studies.Crossref | GoogleScholarGoogle Scholar |

Pertoldi, C, Schmidt, JB, Thomsen, PM, Nielsen, LB, de Jonge, N, Iacolina, L, Muro, F, Nielsen, KT, Pagh, S, Lauridsen, TL, Andersen, LH, Yashiro, E, Lukassen, MB, Nielsen, JL, Elmeros, M, and Bruhn, D (2021). Comparing DNA metabarcoding with faecal analysis for diet determination of the Eurasian otter (Lutra lutra) in Vejlerne, Denmark. Mammal Research 66, 115–122.
Comparing DNA metabarcoding with faecal analysis for diet determination of the Eurasian otter (Lutra lutra) in Vejlerne, Denmark.Crossref | GoogleScholarGoogle Scholar |

Pianka, ER, and Huey, RB (1978). Comparative ecology, resource utilization and niche segregation among gekkonid lizards in the southern Kalahari. Copeia 1978, 691–701.
Comparative ecology, resource utilization and niche segregation among gekkonid lizards in the southern Kalahari.Crossref | GoogleScholarGoogle Scholar |

Pincheira-Donoso, D (2008). Testing the accuracy of fecal-based analyses in studies of trophic ecology in lizards. Copeia 2008, 322–325.
Testing the accuracy of fecal-based analyses in studies of trophic ecology in lizards.Crossref | GoogleScholarGoogle Scholar |

Pinkas, L, Oliphant, MS, and Inverson, ILK (1971). Food habits of albacore, bluefin tuna, and bonito in Californian waters. Fisheries Bulletin 152, 11–105.

R Core Team (2020) ‘R: A language and environment for statistical computing.’ R Foundation for Statistical Computing, Vienna, Austria. Available at https://www.R-project.org/

Rohland, N, and Reich, D (2012). Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Research 22, 939–946.
Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture.Crossref | GoogleScholarGoogle Scholar |

Schneider, CA, Rasband, WS, and Eliceiri, KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671–675.
NIH Image to ImageJ: 25 years of image analysis.Crossref | GoogleScholarGoogle Scholar |

Schober, P, Boer, C, and Schwarte, LA (2018). Correlation coefficients: appropriate use and interpretation. Anesthesia & Analgesia 126, 1763–1768.
Correlation coefficients: appropriate use and interpretation.Crossref | GoogleScholarGoogle Scholar |

Shen, W, Le, S, Li, Y, and Hu, F (2016). SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS ONE 11, e0163962.
SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation.Crossref | GoogleScholarGoogle Scholar |

Soulsbury, CD, Gray, HE, Smith, LM, Braithwaite, V, Cotter, SC, Elwood, RW, Wilkinson, A, and Collins, LM (2020). The welfare and ethics of research involving wild animals: a primer. Methods in Ecology and Evolution 11, 1164–1181.
The welfare and ethics of research involving wild animals: a primer.Crossref | GoogleScholarGoogle Scholar |

Tanaka S (1996) Goniurosaurus kuroiwae. In ‘The Encyclopedia of Animals in Japan 5: Amphibians, Reptiles, Chondrichthyes’. (Eds T Hidaka, S Sengoku, T Hikida, M Matsui, K Nakaya) pp. 67–68, 71. (Heibonsha: Tokyo, Japan) (in Japanese)

UNESCO (2022) Amami-Oshima Island, Tokunoshima Island, Northern part of Okinawa Island, and Iriomote Island. Available at https://whc.unesco.org/en/list/1574. [Accessed 28 August 2022]

Vamos, EE, Elbrecht, V, and Leese, F (2017). Short COI markers for freshwater macroinvertebrate metabarcoding. Metabarcoding and Metagenomics 1, e14625.
Short COI markers for freshwater macroinvertebrate metabarcoding.Crossref | GoogleScholarGoogle Scholar |

Watanabe, I, Akiyama, T, and Sano, S (2011). Distribution of mercury in ecosystems of Yanbaru, northern part of Okinawa Island, Japan and its relationships with other elements. Chikyukagaku (Geochemistry) 45, 29–42.
Distribution of mercury in ecosystems of Yanbaru, northern part of Okinawa Island, Japan and its relationships with other elements.Crossref | GoogleScholarGoogle Scholar |

Werner, YL, Takahashi, H, Yasukawa, Y, and Ota, H (2006). Factors affecting foraging behaviour, as seen in a nocturnal ground lizard, Goniurosaurus kuroiwae kuroiwae. Journal of Natural History 40, 429–459.
Factors affecting foraging behaviour, as seen in a nocturnal ground lizard, Goniurosaurus kuroiwae kuroiwae.Crossref | GoogleScholarGoogle Scholar |