Data from: Metabarcoding reveals the dietary patterns of bats within a unique European habitat, the pasture‐dominated landscapes of Ireland
Abstract
Considered keystone predators, insectivorous bats play essential roles in maintaining the functioning of ecosystems. Investigating how bat species' diets vary across landscapes is crucial for understanding bat ecology and their role in ecosystem health. Here, we characterised the predator–prey interactions of two common bat species with different foraging strategies, the Brown long-eared bat (Plecotus auritus) and Soprano pipistrelle (Pipistrellus pygmaeus), across the unique pastureland-dominated landscape of Ireland. Over 3 years (2021–2023), faecal samples (n = 4627 in total) were collected annually at three time points (gestation, lactation, post-lactation) from 12 maternity roosts and analysed using metabarcoding and next-generation sequencing. Both bat species showed broad diet diversity, with 392 and 350 arthropod species identified for the Brown long-eared bat and Soprano pipistrelle, respectively, primarily Lepidoptera and Diptera. The Brown long-eared bat exhibited a generalist diet, demonstrating dietary flexibility. Lepidoptera interactions were more frequent overall (62%) compared to Diptera (31%), but interactions with Diptera species increased markedly at one specific roost, suggesting that this species can opportunistically feed on available prey species. In contrast, the Soprano pipistrelle exhibited a more specialised diet, with 83% consisting of Diptera species. Both spatial and temporal factors significantly influenced dietary richness and composition in both species. Surrounding land cover, in particular, played an important role in shaping diet composition. Our findings suggest that the Brown long-eared bat exhibits a broad foraging strategy, acting as a generalist with a preference for Lepidoptera, while the Soprano pipistrelle shows a consistent reliance on Diptera. Our study provides new insights into bat diet variation in pastureland landscapes, contributing to the understanding of their ecological role.
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1 Metabarcoding reveals the effect of land cover and temporal variations on the diet of Brown long-eared and Soprano pipistrelle bats within a unique European habitat, the pasture dominated landscapes of Ireland. Hurpy1., G., Aughney2. T., Skujina1,3. I., Roche4. N. and Teeling1. E. C*. 1 School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Ireland. 2 Bat Eco Services Limited, Virginia, Co. Cavan, Ireland. 3 Genome Stability Laboratory, School of Biological and Chemical Sciences, University of Galway, University Road, Galway, Ireland. 4 Bat Conservation Ireland Carmichael House 4-7, North Brunswick Street, Dublin 7, Ireland . *Corresponding author: [email protected] Table of Contents Page 2 Supporting Table S1 Page 3 Supporting Table S2 Page 4 Supporting Figure S1 Page 15 Supporting Figure S2 Page 16 Supporting Figure S3 Page 19 Supporting Table S3 Page 20 Supporting Figure S4
2 Supporting Table S1: Number of faecal samples collected at each bat maternity roost over the three years of sampling (2021-2023). 1st sampling period : Gestation ; 2nd sampling period : Lactation ; 3rd sampling period : Post-lactation. Numbers of faecal samples included in the analyses, after that bat species was confirmed, are indicated between parentheses. The sub-total lines show the total of number of faecal samples collected per bat species. The total line presents the combined total for the two bat species. The numbers indicated in the “roost column” correspond to the numbers assigned to each roost in Fig. 1. 2021 sampling collections 2022 sampling collections 2023 sampling collections TOTAL Bat species Roost 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd Soprano pipistrelle Pipistrellus pygmaeus Glendalough (1) 16(16) 135(20) 77(20) 25(20) 42(20) 91(20) 23(20) 45(20) 36(20) 490(176) Kilafin (2) / / / 7(7) 13(13) 11(10) 5(1) 20(19) 23(20) 79(70) Birr (3) 30(0) 41(12) 36(15) 11(7) 26(13) 15(10) 45(20) 23(20) 12(12) 239(109) Dromore wood (4) / / / 54(20) 72(20) 29(20) 30(20) 40(20) 28(20) 253(120) Sub-total 46(16) 176(32) 113(35) 97(54) 153(66) 146(61) 103(61) 128(79) 99(72) 1061(475) Brown long-eared bat Plecotus auritus Ennisnag (5) 31(17) 10(2) 6(5) 43(16) 36(20) 20(6) 17(14) 33(12) 27(15) 223(107) Glengarriff (6) 39(18) 47(16) 52(12) 19(18) 11(11) 22(20) 50(20) 38(20) 63(18) 341(153) Inagh (7) 197 (20) 103(20) 97(20) 11(11) 119(20) 55(20) 56(20) 46(20) 48(20) 732(171) Kinvarra (8) 78(20) 108(20) 168(20) 68(20) 171(20) 76(20) 79(20) 33(20) 71(20) 852(180) Letterfrack (9) 11(10) 19(18) 182(19) 20(20) 52(20) 64(20) 54(20) 97(20) 45(20) 544(167) Carra James (10) 43(20) 46(20) 30(20) 26(20) 30(20) 30(20) 33(20) 27(20) 23(20) 288(180) Milltown (11) 35(19) 26(0) 48(17) 26(20) 30(20) 30(20) 30(20) 34(20) 38(20) 297(156) Killmore (12) 11(11) 96(16) 17(17) 21(17) 54(20) 22(14) 12(12) 24(16) 32(20) 289(143) Sub-total 445 (135) 455 (112) 600 (130) 234 (142) 503 (151) 319 (140) 331 (146) 332 (148) 347 (153) 3566 (1257) TOTAL 491 (151) 631 (145) 713 (165) 331 (196) 656 (217) 465 (201) 434 (207) 460 (228) 446 (225) 4627 (1732)
3 Supporting Table S2: NucleoSpin Plant kit extraction protocol (adapted from Zarzoso-Lacoste et al., 2018). NucleoSpin Plant kit extraction protocol (adapted from Zarzoso-Lacoste et al., 2018) 1. Sample preparation Add carefully 0.01 gr of pooled faeces into 2mL tube 2. Cell lysing Buffer PL2 and PL3 Add 400 μL Buffer PL2 and 10 μL RNase to each tube strips containing faeces sample and close using new cap strips. Mix by vigorous shaking for 15–30 s. Spin for 30 s at 11 000 rpm to collect any sample from the Cap Strips. Incubate samples at 60°C overnight, on dry bath. Carefully add 100 μL Buffer PL3 to each sample and close the tubes, mix thoroughly, and incubate for 15 min on ice to precipitate sodium dodecyl sulfate completely. 3. Filtrate lysate Place NucleoSpin filter (violet ring) into new collection tube 2mL Load all lysate and centrifuge for 2min at 11 000 g. Collect filtered lysate into a new 1.5 Eppendorf tube and discard filter 4. Adjust DNA binding condition Predispose 450 μL Buffer PC in 1.5mL Eppendorf Add 400 μL clear lysate and mix thoroughly by pipetting five times 5. Bind DNA Label green column and place it on new column Transfer 700 μL of lysate to column and centrifuge for 2 min at 14 000 rpm and discard flow through 6. Wash and dry silica membrane Add 400 μL PW1 Buffer and centrifuge for 1 min at 14 000 rpm Add 700 μL PW2 Buffer and centrifuge for 1 min at 14 000 rpm Add 700 μL PW2 Buffer and centrifuge for 1 min at 14 000 rpm Centrifuge at 14 000 rpm for 10 min 7. Elute DNA Place column in labelled 1.5 Eppendorf Dispose PE Buffer 75 μL into column (pre-heated at 70°C) and incubate at 70°C for 2 min Centrifuge 2 min at 14 000 rpm Dispose PE Buffer 75 μL into column (pre-heated at 70°C) and incubate at 70°C for 2 min Centrifuge 2 min at 14 000 rpm Dispose in - 20°C freezer of use for PCR immediately
4 Supporting Figure S1. Sample completeness curves for each maternity roost by year and for each sampling period. Curves were created with iNEXT and ggiNEXT packages, with q = 0. Rarefaction curve (solid line) represents the expected sample completeness as the sample size increases. Extrapolation curve (dashed line) predicts the potential sample completeness beyond the observed sample size. A - H : Brown long-eared bat roosts; I - L : Soprano pipistrelle roosts..
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16 Supplementary Figure S3 Web plots showing interactions between the Brown long-eared bat (S.3A) and Soprano Pipistrelle (S.3B) and prey of Lepidoptera (a) and Diptera (b) families across maternity roosts. Percentages show the percentage of each order in diets. The web plot was created using the ‘bipartiteD3’ function from the bipartiteD3 package.
17 Supplementary Figure S.3A Carra James Ennisnag Glengarriff Inagh Killmore Kinvarra Letterfrack Miltown ROOST LEPIDOPTERA FAMIL ab Carra James Ennisnag Glengarriff Inagh Killmore Kinvarra Letterfrack Miltown ROOST Coleophoridae0.12 Blastobasidae0.22 Arhyresthiidae 0.22 Crambidae1.3 Erebidae . Drepanidae0.1 Depressariidae 0.0 Geometridae . 3 Gelechiidae0.0 Hepialidae . Lasiocampidae 0. 3 Nepticulidae 0.02 Noctuidae . 0 Nolidae0. 3 ecophoridae0.2 0.3 Notodontidae Tortricidae 0. ponomeutidae 0.02 Pyralidae 0.12 Praydidae 0.0 Plutellidae 0.02 Tipulidae .0 Tachinidae 1. 0 Tabanidae0. Syrphidae 0. 3 Scathophagidae . 3 Sciaridae Stratiomyidae 0.0 Simuliidae 0.0 0.0 Muscidae13.3 Sarcophagidae 0. 3 Rhagionidae 0. Psychodidae Polleniidae 0. 1 1.33 Pediciidae 0. Pallopteridae 0. Mycetophilidae 0.1 Limoniidae 2.1 Lauxaniidae 0. 0 Heleomy idae0.20 Empididae 1. Fanniidae0.20 Chironomidae 0. 3 Dolichopodidae0.0 Chaoboridae 0. Ceratopogonidae0.20 Calliphoridae1.0 Bibionidae0.2 Anthomyiidae2. Anisopodidae 1. 0 DIPTERA FAMIL Brown long-eared bat Brown long-eared bat
18 Supplementary Figure S.3B ROOST LEPIDOPTERA FAMIL ab ROOSTDIPTERA FAMIL Soprano pipistrelle Soprano pipistrelle Birr Dromore wood Glendalough Kilafin psolophidae 0. ponomeutidae 1. Tortricidae 12.02 Tineidae 0.2 Pyralidae 0. ecophoridae 3. 2 Noctuidae 13. Hepialidae 1 .13 Gracillariidae . Geometridae .0 Gelechiidae 2.0 Ericraniidae 0.2 Erebidae 0.2 Elaschistidae0.2 Drepanidae 0.2 Depressariidae 0.2 Crambidae 0. Colephoridae 1. Blastobasidae 2 .1 Argyresthiidae 3.23 Adelidae 0.2 Birr Dromore wood Glendalough Kilafin Trichoceridae 0.11 Tipulidae .13 Psychodidae23.2 Sepsidae 0.3 Scathophagidae 3.0 Sciaridae 0.3 Tephritidae 0.0 Syrphidae 0.11 Stratiomyidae 0.11 Simuliidae 0. Rhagionidae0.11 Muscidae 2. Limoniidae 1 . 1 Lonchopteridae 0.0 Chironomidae 22. Chaoboridae .0 Culicidae 1. 1 Ceratopogonidae 2. 2 Anisopodidae .0 Agromy idae 0.0 Anthomyiidae 0. Bibionidae 0.21 Calliphoridae 0.0 Empididae 2. Drosophilidae 0.11 Dolichopodidae 0.0 Hybotidae Heleomy idae 0.2 0.32 Keroplatidae 0.0 Lauxaniidae 0.1 Psilidae 0.1 Polleniidae 0.0 Pediciidae 0.0 Pallopteridae0.0 Mycetophilidae 0.
19 Supporting Table S3: Details of outliers discarded for the nMDS analysis. SampleID Bat species Roost Year Sampling period nMDS1 nMDS2 D1586_21 Soprano pipistrelle Glendalough 2023 Gestation -2879.1 1153.415 D1631_23 Brown long-eared bat Killmore 2023 Post-lactation -10.7066 -1.50344 D3_2 Brown long-eared bat Killmore 2021 Gestation -2.88731 -2.34257 D1426_13 Brown long-eared bat Killmore 2022 Post-lactation -0.34752 -1.21338 D1426_12 Brown long-eared bat Killmore 2022 Post-lactation -0.31472 -1.21219 D1118_14 Brown long-eared bat Glengarriff 2022 Gestation -0.28079 -1.22061 D1155_5 Soprano pipistrelle Kilafin 2022 Lactation -0.02778 -1.21098 D589_14 Brown long-eared bat Killmore 2021 Post-lactation -0.27187 -1.2112 D589_16 Brown long-eared bat Killmore 2021 Post-lactation -0.26608 -1.2135 D589_6 Brown long-eared bat Killmore 2021 Post-lactation -0.26603 -1.2135 D51_31 Brown long-eared bat Miltown 2021 Gestation -0.26425 -0.21315 D779_14 Brown long-eared bat Letterfrack 2021 Post-lactation 3350.001 991.1094 D1625_19 Brown long-eared bat Letterfrack 2023 Post-lactation -0.02771 -1.5773
20 Supporting Information Figure S4 Non-metric multidimensional scaling plot showing the dissimilarity in diet composition across bat species (Top) and Brown long-eared with all samples included (Bottom).