Literature DB >> 32793779

Dataset supporting the use of nematodes as bioindicators of polluted sediments.

Janina Schenk¹, Sebastian Höss^1,2, Marvin Brinke³, Nils Kleinbölting⁴, Henrike Brüchner-Hüttemann¹, Walter Traunspurger¹.

Abstract

We provide the dataset supporting the research article "Nematodes as bioindicators of polluted sediments using metabarcoding and microscopic taxonomy" [1]. Nematodes are frequently used as bioindicators and the NemaSPEAR[%] is an validated index that is originally based on morphological data. The index was compared to molecular sequence data for the 28S rDNA, 18S rDNA and COI gene for 7 locations. This dataset includes chemical analyses of the sediments for 33 different substances. The sequence data for OTU-based analyses for the 28S rDNA, 18S rDNA and COI gene is given, together with the read distribution during bioinformatics processing. We furthermore include alternative ASV data, based on a cluster-independent approach. The morphological data is presented, including the biomass for each species, as well as an overview about whether the species is represented in the NCBI database. Furthermore, rarefaction analysis is given for the morphological data, and furthermore NMDS plots for the species and genus level based on morphological and molecular data. The correlation between the mean PEC-Q and the NemaSPEAR[%] values is given in order to compare the efficiency of the index, based on morphological and molecular data.

Entities: Chemical

Keywords: Bioindicator; Nemaspear[%]; Nematodes; Polluted sediments

Year: 2020 PMID： 32793779 PMCID： PMC7412863 DOI： 10.1016/j.dib.2020.106087

Source DB: PubMed Journal: Data Brief ISSN： 2352-3409

Specifications table

Value of the data

This dataset supports a pilot study evaluating suitability of molecular-based data for bioindication purposes with nematodes. This dataset provides benefit for molecular-based studies with biomonitoring aims, especially in the field of meiofauna, which is often difficult to identify morphologically. This data can be used to further verify this approach, e.g. with the use of bulk samples instead of isolated nematode communities. This dataset will give further insight into nematode-based bioindication using the NemaSPEAR[%] index.

Data description

This dataset contains the chemical analyses for the seven locations for 33 substances traceable in the sediment samples (Table 1). The number of analysed nematodes for each sample and replicate is given, together with the information if samples were combined due to low specimen numbers (Table 2). For the molecular approach, using Operational Taxonomic Units (OTU) clustering, the list of the final OTU inventory is given for three genetic markers (Tables 4, 5, 6), together with the read distribution during the bioinformatic pipeline using mothur (Table 7). Amplicon Sequence Variants (ASVs) for an alternative cluster-independent approach are given for the three genetic markers in Table 3. For the COI gene, OTUs and ASVs phylogenetic tress show the relationship between the sequence variants, as the majority of OTUs and ASVs in the analyses was classified as “unknown” (Fig. 4 and 5). The morphological approach includes a list of all species found by microscopic analysis, including the biomass, based on measured specimens from this study, for each species (separated for female and male) and the information whether a reference sequence can be found at National Center for Biotechnology Information (NCBI) (Table 8). A rarefaction analysis based the morphological data provides insights into the (extrapolated) number of species in order to compensate that different numbers of individuals were analysed for the locations, due to samples with rare individual numbers (Fig. 1). For each of the Nematode Species at Risk (NemaSPEAR) a NCBI inspection was performed to validate if reference sequences are available for the genus and the species level (Table 9). For molecular-based and morphology-based data, NMDS plots were calculated comparing the nematodes communities for each replicate at the species and genus level (Fig. 2). The NemaSPEAR[%] values for morphological and molecular data were plotted against mean probable-effect-concentration quotients (mean PEC-Q), in order to give insights about the coherences between the indices and the toxic potential of the sediment (Fig. 3).

Table 1

Substance	Unit	PEC	RM1	LU1	CUM1	HI1	VE1	ÖR1	FB1
As	mg/kg dw	50	14	64	25	34	1	<0.5	<2.5
Pb	mg/kg dw	118	48	333	124	107	<2.5	<2.5	1.6
Cd	mg/kg dw	7.8	0.5	45	3.1	4.9	<0.05	<0.05	<0.1
Cr	mg/kg dw	68	77	1174	124	97	2	4	2.8
Cu	mg/kg dw	60	48	571	97	101	1	3	<0.5
Ni	mg/kg dw	32	52	308	53	60	1	3	1.5
Hg	mg/kg dw	1.2	0.46	2.75	3.43	1.67	<0.025	<0.025	0.05
Zn	mg/kg dw	800	169	3507	772	950	<5	<5	6
Naphthalene	mg/kg dw	6.6	0.2	6.6	0.088	0.22	<0.005	<0.005	<0.005
Acenaphthylene	mg/kg dw	5.2	0.035	0.55	0.013	0.031	<0.005	<0.005	<0.005
Acenaphthene	mg/kg dw	3.3	0.025	2	0.02	0.047	<0.005	<0.005	<0.005
Fluorene	mg/kg dw	0.26	0.053	2.6	0.025	0.066	<0.005	<0.005	<0.005
Phenanthrene	mg/kg dw	0.89	0.41	4.4	0.18	0.4	0.012	0.01	0.015
Anthracene	mg/kg dw	0.17	0.13	1.7	0.048	0.12	<0.005	<0.005	<0.005
Fluoranthene	mg/kg dw	1.2	0.81	3.9	0.31	0.75	0.026	0.018	0.022
Pyrene	mg/kg dw	0.94	0.61	4.5	0.26	0.61	0.015	0.01	0.012
Benzo(a)anthracene	mg/kg dw	0.6	0.3	0.71	0.14	0.32	0.01	<0.005	0.011
Chrysene	mg/kg dw	0.83	0.27	0.44	0.18	0.35	<0.005	<0.005	0.01
Benz(b)fluoranthene	mg/kg dw	0.66	0.23	0.47	0.18	0.32	<0.005	<0.005	<0.005
Benzo(k)fluoranthene	mg/kg dw	0.32	0.083	0.17	0.076	0.13	<0.005	<0.005	0.005
Benzo(a)pyrene	mg/kg dw	0.6	0.21	0.25	0.11	0.26	<0.005	<0.005	0.011
Dibenz(a,h)anthracene	mg/kg dw	0.12	0.022	0.044	0.02	0.044	<0.005	<0.005	<0.005
Indeno(1,2,3-cd)pyrene	mg/kg dw	0.48	0.13	0.07	0.1	0.2	<0.005	<0.005	<0.005
Benzo(ghi)perylene	mg/kg dw	0.45	0.17	0.23	0.13	0.25	<0.005	<0.005	<0.005
PCB 28	µg/kg dw	2	0.13	26	1.2	2.9	<0.025	<0.025	<0.005
PCB 52	µg/kg dw	4.6	0.24	71	1.4	2.7	<0.025	<0.025	<0.005
PCB 101	µg/kg dw	6.7	0.41	88	3.1	4.4	<0.025	<0.025	<0.005
PCB 118	µg/kg dw	6.9	0.24	110	1.8	2.7	<0.025	<0.025	<0.005
PCB 138	µg/kg dw	7.5	0.51	100	9.7	11	<0.025	<0.025	<0.005
PCB 153	µg/kg dw	9.7	0.58	78	10	13	<0.025	<0.025	<0.005
PCB 180	µg/kg dw	5.5	0.17	28	7.9	11	<0.025	<0.025	<0.005
p,p'-DDD	µg/kg dw	3.2	0.9	210	11	44	<0.025	<0.025	<0.005
p,p'-DDE	µg/kg dw	6.8	0.91	87	5.1	13	<0.025	<0.025	<0.005

Abbreviation for locations: Rischmühle (RM), Luppe (LU), Cumlosen (CUM), Hitzacker (HI), Veerse (VE), Örtze (ÖR) and Furlbach (FB).

Table 2

Overview about the number of nematodes for each replicate for the seven locations and the morphological and molecular approach. A black box indicates that these replicates were combined into a new replicate due to low individual number. For the location VE more than five replicates were available for morphological analysis, no further samples were available for molecular analysis, though.

Replicate	FB	VE	ÖR	RM	CUM	HI	LU
Morph.
I	87	26	95	98	99	102	99
II	100	98	101	98	100	102	100
III	98	9	101	104	110	100	92
IV	100	71	102	99	96	99	100
V	100	10	100	99	98	101	100
VI		99
VII		100
VIII		73
IX		33
SUM	485	516	499	498	503	504	491
Mol.
I	500	80	200	200	200	200	50
II		26	200	200	200	200	50
III		15	100	200	200	200	50
IV		9		100	200	200	40
V						200	60
SUM	500	130	500	700	800	1000	250

Table 4

List of OTUs determined for the 28S rDNA gene fragment, using the primer pairs 1274/706 for the seven locations (CUM, HI, LU, ÖR, RM, VE and FB). Clustering was performed at 99%.

OTU 1274/706	CUM	HI	LU	ÖR	RM	VE	FB
Acrobeloides sexlineatus	5.39	2.47	0.00	0.16	0.06	0.00	0.00
Anaplectus granulosus	0.37	0.62	0.00	0.00	0.01	0.00	10.22
Aphanolaimus aquaticus	0.00	0.00	0.00	0.00	0.00	0.00	0.04
Aphelenchus spec	0.14	0.17	0.00	0.00	0.01	0.00	0.00
Aporcelaimellus obtusicaudatus	32.23	42.80	0.07	0.82	2.29	0.00	0.00
Basiria sp. SAN-2005	0.04	0.00	0.00	0.00	0.00	0.00	0.00
Boleodorus sp. 2 TJP-2012	0.03	0.00	0.00	0.00	0.00	0.00	0.00
Bursilla sp. PS1179	0.15	1.05	0.00	0.01	0.05	0.00	0.00
Cephaloboides cf. armata SB363	0.00	0.00	0.00	0.00	0.31	0.00	0.00
Cephalobus cubaensis	1.41	0.80	0.00	0.03	0.01	0.00	0.00
Chromadorita leuckarti	0.00	0.20	0.45	0.16	30.98	0.08	6.92
Coslenchus costatus	12.63	1.03	0.00	0.54	0.09	0.00	0.00
Diplogastrellus sp. NK-2010	0.00	0.87	5.50	0.00	3.68	0.00	0.00
Dorylaimus stagnalis	0.00	0.06	0.00	0.16	0.50	0.00	0.00
Epitobrilus medius	0.02	0.02	0.00	0.01	0.00	0.00	0.00
Ethmolaimus pratensis	0.22	0.20	0.00	1.17	0.00	0.27	7.11
Eucephalobus cf oxyuroides	37.53	26.20	0.00	1.13	0.99	0.00	0.00
Eucephalobus striatus	0.45	1.66	0.02	0.01	0.04	0.00	0.00
Eudorylaimus sp. 2 KH-2018	0.26	0.00	0.00	0.00	0.00	0.00	0.00
Eumonhystera filiformis	0.00	0.16	0.80	0.00	0.26	0.25	0.00
Ironus longicaudatus	0.00	0.00	0.00	0.00	0.00	0.00	0.12
Ironus tenuicaudatus	0.00	0.00	0.00	0.06	0.00	0.00	0.00
Meloidogyne arenaria	0.03	0.00	0.00	0.00	0.00	0.00	0.00
Mesocriconema curvatum	0.02	0.83	0.44	0.00	0.06	0.00	0.00
Mesodorylaimus bastiani	0.13	0.62	0.00	0.00	0.00	0.00	0.00
Monhystera paludicola	0.00	0.00	0.00	0.08	0.36	0.05	0.04
Monhystera spec	0.00	0.00	0.00	0.04	0.00	0.00	0.00
Mononchoides sp. 1 VS-2014	0.05	0.00	0.00	0.00	0.00	0.00	0.00
Mononchus aquaticus	1.33	0.58	0.00	0.00	0.02	0.00	0.00
Mononchus truncatus	0.02	0.10	0.00	0.05	0.55	0.48	0.00
Neotobrilus longus	0.63	0.02	0.00	0.52	0.01	1.71	29.59
Panagrolaimus detritophagus	0.00	0.05	0.00	0.00	0.00	0.00	0.00
Panagrolaimus sp. PS1159	0.00	0.02	0.00	0.00	0.00	0.00	0.00
Panagrolaimus	0.03	0.00	0.00	0.00	0.00	0.00	0.00
Paractinolaimus sp.	0.07	0.01	0.00	0.01	0.00	0.00	0.00
Paratylenchus microdorus	0.00	0.03	0.00	0.00	0.00	0.00	0.00
Pellioditis sp. VS-2014	0.00	0.04	0.00	0.00	0.00	0.00	0.00
Plectus aquatilis	0.02	0.00	0.00	0.00	0.00	0.00	2.75
Plectus exinocaudatus	0.09	0.05	0.22	0.00	0.05	0.08	0.16
Plectus opisthocirculus	0.00	0.05	0.00	0.00	0.00	0.00	0.08
Plectus velox	0.01	0.10	0.00	0.00	0.01	0.00	0.00
Pratylenchoides ritteri	0.02	0.07	0.00	0.00	0.00	0.00	0.00
Pratylenchoides sp. 1 MP-2013	1.60	15.80	0.00	0.04	0.40	0.00	0.00
Pratylenchus convallariae	0.06	0.00	0.00	0.00	0.00	0.00	0.00
Prionchulus punctatus	0.01	0.04	0.00	0.00	0.00	0.00	0.00
Prismatolaimus dolichurus	0.34	0.27	0.00	0.01	0.01	0.00	0.08
Prismatolaimus intermedius	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Prodesmodora circulata	0.00	0.00	0.00	0.00	0.00	1.11	0.00
Protolitonotus magnus	0.00	0.00	0.00	0.01	0.00	0.00	0.00
Rhabditidoides sp. NK-2010	0.00	0.00	0.00	0.00	0.02	0.00	0.00
Rhabditis sp. DF5059	0.04	0.02	0.01	0.00	0.00	0.00	0.00
Rhabditoines regina	0.00	0.00	0.00	0.00	0.00	0.00	0.08
Rotylenchus robustus	0.81	0.09	0.00	0.02	0.00	0.00	0.00
Semitobrilus pellucidus	0.19	0.03	2.28	89.37	0.98	88.46	0.00
Theristus agilis	0.00	0.05	0.00	4.77	1.07	6.86	38.35
Tobrilus cf. helveticus KR-2013	0.04	0.00	0.00	0.00	0.00	0.00	0.00
Tobrilus gracilis	1.59	1.89	90.21	0.72	56.58	0.51	0.00
Tobrilus sp. BI-LSU-318	0.00	0.00	0.00	0.00	0.00	0.04	0.00
Trichodorus arasbaranensis	0.17	0.00	0.00	0.00	0.00	0.11	0.00
Tripyla glomerans	0.00	0.14	0.00	0.04	0.57	0.00	0.00
Tripyla setifera	0.00	0.07	0.00	0.03	0.00	0.00	4.48
unknown_1	0.03	0.00	0.00	0.00	0.00	0.00	0.00
unknown_10	0.07	0.00	0.00	0.00	0.00	0.00	0.00
unknown_11	0.02	0.00	0.00	0.00	0.00	0.00	0.00
unknown_12	0.03	0.00	0.00	0.00	0.00	0.00	0.00
unknown_13	0.14	0.00	0.00	0.00	0.00	0.00	0.00
unknown_2	0.00	0.04	0.00	0.00	0.00	0.00	0.00
unknown_3	0.02	0.00	0.00	0.00	0.00	0.00	0.00
unknown_4	0.08	0.10	0.00	0.00	0.00	0.00	0.00
unknown_5	0.07	0.00	0.00	0.00	0.00	0.00	0.00
unknown_6	0.05	0.00	0.00	0.00	0.00	0.00	0.00
unknown_7	0.00	0.04	0.00	0.00	0.00	0.00	0.00
unknown_8	0.02	0.00	0.00	0.00	0.00	0.00	0.00
unknown_9	0.05	0.00	0.00	0.00	0.00	0.00	0.00
Zeldia punctata	1.19	0.52	0.00	0.03	0.02	0.00	0.00

Table 5

List of OTUs determined for the 18S rDNA gene fragment, using the primer pairs 3NDf/C_1132f for the six locations (CUM, HI, LU, ÖR, RM and VE). Clustering was performed at 99%.

OTU_3NDf/C_1132f	CUM	HI	LU	ÖR	RM	VE
Cephalobus sp./Acrobeloides nanus	46.64	24.14	0.00	5.19	1.85	0.00
Amplimerlinius macrurus	0.50	0.46	0.00	0.00	0.00	0.00
Anaplectus sp. PDL-2005	0.05	0.23	0.00	0.00	0.00	0.00
Aphelenchoides bicaudatus	0.00	0.00	0.00	0.00	0.15	0.00
Aphelenchoides sp. Asp6753	0.00	0.07	0.00	0.00	0.00	0.00
Aphelenchoides sp. BE1	0.00	0.00	0.00	0.00	0.00	0.00
Aphelenchus sp. JH-2004	0.72	0.48	0.00	0.00	0.00	0.00
Aporcelaimellus sp. AMS-2013	0.29	0.00	0.00	0.00	0.00	0.00
Basiria duplexa	10.72	0.49	0.00	0.41	0.06	0.00
Boleodorus thylactus	0.07	0.00	0.00	0.00	0.00	0.00
Cephaloboides cf. armata SB363	0.00	0.00	0.00	0.00	11.43	0.00
Cephalobus persegnis	0.00	2.72	0.00	0.00	0.08	0.00
Chromadorina bioculata	0.00	0.00	0.00	0.00	0.71	0.00
Coslenchus turkeyensis	0.05	0.31	0.00	0.00	0.00	0.00
Daptonema hirsutum	0.00	0.00	0.00	0.00	1.13	0.00
Daptonema sp. 1255	0.00	0.12	0.00	0.00	7.85	0.00
Diphterophora communis	7.09	0.00	0.00	0.42	0.00	0.00
Diplogaster rivalis	0.14	5.35	26.11	0.01	13.91	0.00
Ditylenchus sp. 10 JH-2014	1.20	2.22	0.00	0.10	0.07	0.00
Ditylenchus sp. 6 JH-2014	0.00	0.14	0.00	0.00	0.00	0.00
Ditylenchus sp. 8 JH-2014	0.00	0.11	0.00	0.00	0.00	0.00
Ethmolaimus pratensis	0.00	0.00	0.00	0.13	0.00	0.00
Eumonhystera sp. 1 JH-2014	0.00	0.00	0.00	8.53	0.00	7.60
Filenchus vulgaris	0.50	0.00	0.00	0.00	0.00	0.00
Geocenamus quadrifer	0.00	6.70	0.00	0.00	0.27	0.00
Hemileius microclava	0.00	0.00	0.00	0.00	0.03	0.00
Heterocephalobus elongatus	1.03	0.86	0.08	0.02	0.00	0.00
Ironus sp.	0.00	0.00	0.00	0.00	0.02	0.00
Merlinius brevidens	0.97	1.15	0.00	0.00	0.03	0.00
Merlinius joctus	0.00	0.44	0.00	0.00	0.00	0.00
Mesorhabditis sp. FL-Type-10	1.07	4.24	0.00	0.00	0.88	0.00
Monhystera cf. paludicola JH-2014	0.00	0.00	0.00	0.50	0.16	0.00
Monhystera sp. 1 JH-2014	0.00	0.00	0.00	0.00	1.84	0.00
Mononchus aquaticus_100c	0.00	0.00	0.01	12.55	0.01	17.73
Neotobrilus longus	0.00	0.00	0.00	2.20	0.00	2.33
Neotobrilus longus	2.27	1.00	8.88	3.06	1.48	3.70
Oscheius tipulae	0.63	4.43	0.00	0.00	1.27	0.00
Panagrolaimus cf. rigidus AF40	0.00	0.91	0.00	0.00	0.00	0.00
Panagrolaimus sp. AS01	0.00	0.50	0.00	0.00	0.00	0.00
Paratylenchus dianthus	0.00	0.15	0.00	0.00	0.00	0.00
Phasmarhabditis sp. SA2	0.63	0.00	0.00	0.00	0.00	0.00
Phasmarhabditis sp. SA4	1.12	0.62	0.00	0.00	0.25	0.00
Plectus opisthocirculus	0.00	0.07	0.00	0.00	0.00	0.00
Plectus sp. 3 JS-2016	0.00	0.06	0.00	0.00	0.03	0.00
Prismatolaimus cf. dolichurus JH-2004	0.41	0.00	0.00	0.02	0.00	0.00
Prismatolaimus dolichurus	0.14	0.73	0.00	0.00	0.01	0.00
Pristionchus lheritieri	0.98	0.00	0.00	0.13	0.00	0.00
Punctodora ratzeburgensis	0.00	0.00	0.00	0.00	5.28	0.00
Rhabditis sp. DF5059	12.40	23.22	1.62	1.05	2.36	0.00
Rhabditis sp. RA5	8.14	14.50	0.00	0.00	2.13	0.00
Rhynchoscolex simplex	0.00	0.00	0.00	0.31	0.00	0.00
Semitobrilus pellucidus	0.00	0.00	0.00	0.00	0.00	1.67
Semitobrilus pellucidus /Tobrilidae sp. 1 JH-2014	0.14	0.00	0.00	63.54	0.00	62.82
Tobrilus cf. zakopanensis 1 JH-2014	0.00	0.00	0.16	0.00	0.03	0.00
Tobrilus gracilis/Tobrilus sp. 1 JH-2014	0.34	2.38	62.80	1.49	45.39	0.00
Trichodorus similis	1.09	0.00	0.00	0.00	0.00	0.00
Tridentulus sp. PDL-2005	0.00	0.01	0.34	0.00	0.00	4.15
Tripyla glomerans	0.00	0.49	0.00	0.06	0.80	0.00
Tripyla sp. JH-2004	0.00	0.17	0.00	0.00	0.00	0.00
unknown_13	0.00	0.00	0.00	0.00	0.51	0.00
unknown_16	0.00	0.17	0.00	0.00	0.00	0.00
unknown_17	0.00	0.09	0.00	0.00	0.00	0.00
unknown_19	0.31	0.01	0.00	0.00	0.00	0.00
unknown_21	0.00	0.00	0.00	0.27	0.00	0.00
unknown_22	0.12	0.00	0.00	0.00	0.00	0.00
unknown_4	0.00	0.00	0.00	0.00	0.00	0.00
unknown_6	0.00	0.27	0.00	0.00	0.00	0.00
unknown_9	0.21	0.00	0.00	0.00	0.00	0.00

Table 6

List of OTUs determined for the COI gene, using the primer pairs mlCOIint/dgHCO for the six locations (CUM, HI, LU, ÖR, RM, and VE). Clustering was performed at 97%.

COI_OTU	CUM	HI	LU	ÖR	RM	VE
Acanthocyclops vernalis	0.00	0.00	0.00	0.01	0.01	0.00
Amphichaeta raptisae	0.02	0.00	0.00	0.49	0.00	0.00
Arthropoda environmental sample	0.00	0.00	0.00	0.06	0.00	0.00
Asellus aquaticus	0.06	2.29	0.00	0.00	0.00	0.00
Blastomussa loyae	0.00	0.00	0.00	0.00	0.00	6.77
Bos taurus	0.64	11.20	0.12	0.08	0.18	0.59
Candona candida	0.00	0.00	0.00	0.00	0.01	0.00
Chironomus melanescens	0.00	0.00	0.00	0.20	0.00	0.00
Chironomus pallidivittatus	0.00	0.00	0.11	0.01	0.01	0.00
Chydoridae sp. SHDT150804	0.00	0.00	0.00	0.01	0.00	0.00
Coccinella trifasciata perplexa	0.00	5.80	0.00	0.00	0.56	0.00
Corynoneura kadalinka	0.00	0.00	0.00	0.00	0.35	0.00
Cricotopus sylvestris	0.05	0.00	0.00	0.02	0.01	0.00
Endochironomus albipennis	0.00	0.00	0.00	0.00	0.01	0.00
Eucyclops cf. serrulatus ZISP 11SNM-547	0.00	0.00	0.00	0.00	0.03	0.00
Eucyclops cf. serrulatus ZISP 11SNM-549	0.00	0.00	0.00	0.07	0.02	0.00
Fejervarya granosa	0.00	1.30	0.00	0.00	0.20	0.00
Homo sapiens	0.15	0.00	0.00	0.00	0.00	0.00
Limnodrilus claparedianus	0.00	0.00	0.00	0.81	0.58	0.00
Limnodrilus hoffmeisteri complex lineage IX	0.00	0.00	0.00	0.18	0.00	0.00
Limnodrilus hoffmeisteri complex lineage X	0.00	0.00	0.00	0.00	0.20	0.00
Naididae sp. JCOCH052–10	0.00	0.00	0.00	0.87	0.00	0.00
Nais communis	0.00	0.00	0.00	0.02	0.00	0.00
Nais sp. N12 RV-2017	0.00	0.01	0.00	0.00	0.04	0.00
Ophidonais serpentina	0.00	0.00	0.00	0.00	0.04	0.00
Parasitidae sp. BOLD:AAF9233	0.00	0.00	0.00	0.03	0.00	0.00
Penicillium sclerotiorum	0.01	3.33	0.00	0.02	0.02	0.00
Polypedilum albinodus	0.00	0.00	0.00	0.01	0.00	0.00
Polypedilum pullum	0.22	1.44	0.85	0.06	1.24	20.46
Potamothrix heuscheri	0.00	0.00	0.00	0.01	0.01	0.00
Potamothrix moldaviensis	0.00	0.00	0.00	0.13	0.00	0.00
Prodiamesa olivacea	0.01	0.00	0.00	0.15	0.00	0.00
Propappus volki	0.00	0.00	0.00	0.00	0.05	0.00
Pseudo-nitzschia subfraudulenta	0.00	0.00	0.00	0.00	0.00	0.40
Tanytarsus pallidicornis	0.00	0.00	0.00	0.04	0.00	0.00
Tubifex tubifex	0.00	0.00	3.39	1.63	0.37	0.00
Tubificinae sp. 1 RV-2016	0.00	0.00	0.00	0.14	0.00	0.00
Tubificinae sp. T2_336	0.00	0.00	0.00	0.01	0.00	0.00
uncultured fungus	0.15	0.00	0.00	0.01	0.00	0.00
unknown_10	0.00	0.00	0.00	28.46	0.00	0.00
unknown_102	0.00	4.91	0.00	0.00	0.09	0.00
unknown_104	0.00	0.00	1.06	0.00	0.00	0.00
unknown_105	0.00	0.00	0.00	0.02	0.01	0.00
unknown_107	0.62	0.00	0.00	0.02	0.00	0.00
unknown_109	0.00	0.00	0.00	0.32	0.00	0.00
unknown_11	0.00	0.00	0.00	0.00	1.37	0.00
unknown_110	0.00	0.00	0.00	0.00	0.29	0.12
unknown_112	0.00	0.00	0.00	1.18	0.00	0.00
unknown_113	0.00	0.00	0.00	0.00	0.14	0.00
unknown_114	0.00	0.00	0.03	0.00	0.00	0.00
unknown_115	0.11	0.00	0.00	0.00	0.00	0.00
unknown_116	0.58	0.59	0.00	0.00	0.10	0.00
unknown_117	0.00	0.00	0.00	0.00	0.01	1.73
unknown_118	0.00	0.19	0.00	0.00	0.00	0.00
unknown_12	1.18	0.00	0.00	0.05	0.00	0.00
unknown_120	0.00	0.00	0.00	0.00	0.02	0.00
unknown_121	0.00	0.00	0.00	0.00	10.68	0.00
unknown_122	0.01	0.00	0.00	0.02	0.00	0.03
unknown_123	0.00	0.00	4.55	0.00	0.00	0.00
unknown_124	0.05	0.00	0.00	0.00	0.00	0.00
unknown_125	0.00	0.03	0.00	0.00	0.00	0.00
unknown_127	0.01	0.00	0.00	0.03	0.00	0.00
unknown_128	0.00	0.00	0.00	0.02	0.00	0.00
unknown_129	0.01	0.00	0.01	0.00	0.00	0.00
unknown_13	2.76	0.00	0.00	0.06	0.00	0.00
unknown_131	0.00	0.00	0.00	0.00	0.19	1.11
unknown_132	0.00	0.01	0.08	0.00	0.35	0.00
unknown_133	0.00	0.00	0.24	0.00	0.11	0.00
unknown_134	14.74	0.00	0.91	0.74	0.00	0.00
unknown_15	0.02	0.00	0.00	52.83	0.08	67.54
unknown_16	0.00	0.00	0.00	0.00	0.03	0.03
unknown_17	0.00	0.00	0.00	0.01	0.00	0.00
unknown_18	0.00	0.62	0.00	0.00	0.02	0.00
unknown_2	0.00	0.00	0.00	0.00	0.17	0.00
unknown_21	0.00	0.07	0.00	0.00	0.00	0.00
unknown_22	0.00	0.00	0.00	0.00	0.20	0.00
unknown_23	0.00	0.00	0.00	0.04	0.01	0.09
unknown_24	0.13	0.01	0.00	0.00	0.01	0.00
unknown_25	0.00	0.00	0.00	0.02	0.03	0.00
unknown_26	0.00	0.00	0.03	0.00	0.00	0.00
unknown_27	2.43	0.00	0.00	0.04	0.00	0.00
unknown_28	36.22	0.31	0.00	0.31	0.04	0.00
unknown_3	10.11	0.00	0.00	0.22	0.00	0.00
unknown_30	1.25	0.96	0.00	0.06	0.01	0.00
unknown_31	0.00	0.00	0.00	0.15	0.00	0.00
unknown_32	0.13	0.00	0.00	0.00	0.00	0.00
unknown_34	0.00	0.11	0.00	0.00	0.02	0.00
unknown_35	0.00	0.00	0.00	0.00	0.01	1.08
unknown_36	0.00	0.00	0.02	0.00	0.00	0.00
unknown_37	1.04	0.00	0.00	0.08	0.00	0.00
unknown_38	0.00	0.39	0.00	0.00	0.01	0.00
unknown_39	0.00	0.00	0.10	0.00	0.00	0.00
unknown_4	10.11	0.00	0.00	0.24	0.00	0.00
unknown_41	0.00	0.39	0.00	0.00	0.01	0.00
unknown_43	0.00	1.10	0.00	0.00	0.02	0.00
unknown_44	0.43	0.00	0.00	0.00	0.00	0.00
unknown_45	0.30	1.64	0.00	0.00	0.00	0.00
unknown_46	0.00	0.00	0.02	0.00	0.00	0.00
unknown_47	0.28	0.00	0.00	0.00	0.00	0.00
unknown_48	0.15	0.00	20.89	0.00	0.00	0.00
unknown_49	0.00	0.00	0.00	0.24	0.00	0.00
unknown_50	0.02	3.97	4.67	0.00	5.68	0.00
unknown_51	0.00	0.00	0.00	0.08	0.00	0.00
unknown_52	0.00	0.00	0.00	0.00	0.02	0.00
unknown_53	0.00	0.43	0.00	0.00	0.02	0.00
unknown_55	0.00	0.00	16.83	0.00	0.01	0.03
unknown_56	0.00	0.00	0.00	0.00	0.01	0.00
unknown_57	0.00	0.05	0.00	0.00	0.00	0.00
unknown_59	2.63	5.54	0.00	0.11	0.72	0.00
unknown_6	3.85	3.25	0.00	0.13	0.00	0.00
unknown_60	0.00	0.00	0.00	0.00	0.48	0.00
unknown_61	0.00	1.18	0.00	0.00	0.03	0.00
unknown_62	0.00	11.55	0.00	0.00	0.26	0.00
unknown_63	0.00	0.00	0.10	0.00	0.00	0.00
unknown_64	0.00	5.57	0.00	0.00	0.09	0.00
unknown_66	0.00	0.93	0.00	0.00	0.03	0.00
unknown_68	0.32	0.00	0.00	0.00	0.00	0.00
unknown_70	0.00	1.29	0.00	0.00	0.02	0.00
unknown_71	0.82	0.01	0.00	0.00	0.00	0.00
unknown_72	0.00	0.01	16.30	0.00	0.01	0.00
unknown_75	4.02	0.00	0.00	0.13	0.00	0.00
unknown_76	0.00	0.11	0.00	0.00	0.00	0.00
unknown_77	0.07	0.00	0.00	0.00	0.00	0.00
unknown_79	0.24	0.00	0.00	0.01	0.00	0.00
unknown_8	0.00	0.00	0.00	0.00	0.01	0.00
unknown_81	0.00	0.00	0.00	0.02	0.00	0.00
unknown_83	0.00	0.00	0.00	0.07	0.00	0.00
unknown_84	0.00	0.00	0.00	8.28	0.00	0.00
unknown_85	0.02	0.37	0.00	0.00	0.00	0.00
unknown_86	0.12	0.00	21.31	0.75	25.03	0.00
unknown_87	0.00	0.00	0.00	0.00	0.00	0.00
unknown_89	0.12	0.00	0.00	0.00	21.52	0.00
unknown_9	0.00	0.02	8.29	0.00	0.64	0.00
unknown_90	0.00	0.00	0.00	0.00	0.74	0.00
unknown_91	0.00	0.17	0.00	0.00	1.21	0.00
unknown_92	0.00	0.00	0.00	0.00	0.13	0.00
unknown_94	0.00	0.00	0.07	0.00	0.00	0.00
unknown_95	0.08	0.06	0.00	0.00	0.00	0.00
unknown_96	0.56	0.00	0.00	0.01	0.00	0.00
unknown_97	0.00	0.06	0.00	0.00	0.10	0.00
unknown_98	0.03	25.79	0.01	0.01	25.00	0.00
unknown_99	3.16	2.96	0.00	0.12	0.29	0.00

Table 7

Distribution of reads within the bioinformatic pipeline using mothur. Given is the number of reads at each current step of the analysis for each sample sequenced for the three genetic markers (1274/706 = 28S rDNA, mlCOIint/dgHCOCOI, 3NDf/C_1132f = 18S rDNA).

1274/706	total	Cum1-N	Cum4-N	Cum8-N	Cum9-N	Hi1-N	Hi2-N	Hi3-N	Hi4-N	Hi5-N
Reads	939,932	33,297	40,542	41,783	39,667	20,260	20,468	32,322	43,424	40,865
After Merging	664,456	23,157	27,574	29,279	25,755	11,774	13,932	23,267	30,755	27,636
After filtering of long or short reads/homopolymers	664,456	23,157	27,574	29,279	25,755	11,774	13,932	23,267	30,755	27,636
After filtering of not fitting reads	598,184	21,414	26,353	27,435	17,702	11,076	10,800	17,782	24,452	25,978
After uchime	505,353	15,849	20,226	20,712	13,709	8860	6768	12,015	19,517	21,399
After removal of rares	461,690	13,595	18,177	18,249	11,596	8073	5491	10,223	17,474	19,660

1274/706	LU1-N	LU2-N	OE1-N	OE10-N	OE2-N	RM10-N	RM2-N	RM3-N	RM4-N	VE1-N
Reads	83,965	60,945	67,117	60,600	83,511	67,836	53,470	39,092	40,649	70,119
After Merging	60,262	43,294	46,395	44,289	63,039	47,965	37,983	28,172	28,203	51,725
After filtering of long or short reads/homopolymers	60,262	43,294	46,395	44,289	63,039	47,965	37,983	28,172	28,203	51,725
After filtering of not fitting reads	59,015	42,881	45,941	43,803	62,745	44,758	28,141	13,683	23,067	51,158
After uchime	52,309	37,938	42,381	37,418	57,892	39,016	25,006	9457	17,141	47,740
After removal of rares	48,053	35,766	40,195	33,875	54,537	36,025	23,152	7870	15,095	44,584

mlCOIintF/dgHCO	total	Cum1-N	Cum4-N	Cum8-N	Cum9-N	Hi1-N	Hi2-N	Hi3-N	Hi4-N	Hi5-N
Reads	219,693	4732	6793	4174	19,136	2675	13,087	11,210	4270	3889
After Merging	156,481	2721	2879	1374	13,759	78	5548	7199	2033	1397
After filtering of long or short reads/homopolymers	156,481	2721	2879	1374	13,759	78	5548	7199	2033	1397
After filtering of not fitting reads	156,481	2721	2879	1374	13,759	78	5548	7199	2033	1397
After uchime	155,930	2714	2879	1369	13,749	78	5542	7192	2033	1397
After removal of rares	155,473	2691	2829	1350	13,706	73	5522	7178	2012	1369
mlCOIintF/dgHCO	LU1-N	LU2-N	OE1-N	OE10-N	OE2-N	RM10-N	RM2-N	RM3-N	RM4-N	VE1-N
Reads	41,914	6133	16,755	9593	8252	11,629	30,348	8156	11,088	5859
After Merging	38,149	4024	13,830	7100	5804	9172	25,265	4899	7993	3257
After filtering of long or short reads/homopolymers	38,149	4024	13,830	7100	5804	9172	25,265	4899	7993	3257
After filtering of not fitting reads	38,149	4024	13,830	7100	5804	9172	25,265	4899	7993	3257
After uchime	37,690	4010	13,827	7094	5804	9158	25,262	4899	7976	3257
After removal of rares	37,657	4007	13,801	7074	5766	9131	25,233	4881	7958	3235

3NDf/C_1132f	total	Cum1_N	Cum4_N	Cum8_N	Cum9_N	Hi1_N	Hi2_N	Hi3_N	Hi4_N	Hi5_N
Reads	526,519	22,607	25,268	24,394	33,179	22,067	22,673	23,612	23,519	24,289
After Merging	112,014	5666	4285	6265	4356	5879	6479	7263	6142	3330
After filtering of long or short reads/homopolymers	112,014	5666	4285	6265	4356	5879	6479	7263	6142	3330
After filtering of not fitting reads	110,098	5587	4214	6196	4224	5799	6372	7187	6066	3269
After uchime	105,997	5312	3999	5818	3969	5653	5957	6737	5889	3058
After removal of rares	56,835	2906	1913	3233	1955	3177	3185	3960	3236	1403

3NDf/C_1132f	LU1_N	LU2_N	OE10_N	OE1_N	OE2_N	RM10_N	RM2_N	RM3_N	RM4_N	VE1_N
Reads	39,345	29,816	31,399	25,150	36,830	31,745	29,051	25,816	31,383	24,376
After Merging	9663	8764	6809	3420	6902	8243	2453	3408	8666	4021
After filtering of long or short reads/homopolymers	9663	8764	6809	3420	6902	8243	2453	3408	8666	4021
After filtering of not fitting reads	9525	8680	6669	3351	6731	8142	2383	3266	8554	3883
After uchime	9397	8575	6463	3285	6595	7984	2216	3084	8182	3824
After removal of rares	5297	5419	2903	1564	3226	4446	888	1250	4898	1976

Table 3

Results of the cluster-independent bioinformatic approach. Amplicon Sequence Variants (ASVs) are given for the three genetic markers (COI = mlCOIint/dgHCO, 28S rDNA = 1274/706, and 18S rDNA= 3NDf/C_1132f). Numbers are given for all samples and replicates combined, as this is only an alternate approach, which was not regarded further.

ASV (mlCOIint/dgHCO)	overall	ASV (1274/706)	overall	ASV (3NDf/C_1132f)	overall
unknown_4	19,900	Nematoda environmental sample	67,051	Diplogaster rivalis	2717
unknown_5	13,129	Aporcelaimellus obtusicaudatus	25,065	Homalozoon vermiculare	553
unknown_6	11,719	Tobrilus gracilis;Tobrilus sp. BI-LSU-312	23,119	Oscheius tipulae	399
unknown_7	10,593	Eucephalobus oxyuroides	19,260	Mesorhabditis belari	290
unknown_9	8837	Tobrilus gracilis	15,565	invertebrate environmental sample	274
unknown_12	6880	Tobrilus pellucidus	11,906	Semitobrilus pellucidus	75
unknown_2	5113	unknown_1	7706	56c	24
unknown_18	3899	Merlinius sp. MerlSp1	5177	unknown_9	24
unknown_19	3809	unknown_2	4139	Punctodora ratzeburgensis	21
unknown_22	3355	Rhabditis sp. DF5059	2857	unknown_11	21
unknown_21	2992	Rhynchoscolex simplex	2798	Bacillidium sp.	19
unknown_23	2929	unknown_3	2733	unknown_12	19
unknown_20	2851	Acrobeloides tricornis	2548	unknown_3	16
unknown_24	2802	unknown_4	1931	Microsporidium sp. BPAR7	16
unknown_25	2693	Mononchus truncatus	1052	Aphelenchoides bicaudatus	14
Polypedilum pullum	2657	unknown_5	969	unknown_14	13
unknown_26	2636	uncultured Paractinolaimus sp.	895	Daptonema sp. 1255	12
unknown_11	2325	Amplimerlinius paraglobigerus	813	uncultured fungus	11
unknown_27	2022	unknown_6	735	Mesorhabditis sp. WB-2009	10
unknown_28	1787	Ethmolaimus pratensis	648	Tobrilus cf. zakopanensis 1 JH-2014	9
unknown_29	1729	unknown_7	615	unknown_19	9
unknown_32	1654	Mononchus aquaticus	610	Frontonia leucas	7
Coccinella trifasciata perplexa	1648	Cephalobus cubaensis	596	Oscheius sp. AW-2011	6
unknown_33	1585	Mesodorylaimus bastiani	578	unknown_13	6
unknown_35	1447	Zeldia punctata	512	unknown_26	6
unknown_37	1315	Eumonhystera filiformis	418	Chromadorina bioculata	6
Limnodrilus hoffmeisteri	1145	unknown_8	412	unknown_27	5
unknown_38	1131	unknown_9	405	unknown_29	3
unknown_39	1017	unknown_10	398	Rhabditis sp. DF5059	3
unknown_40	1004	Eucephalobus striatus	337	Basiria duplexa	3
unknown_42	941	Monhystera paludicola;Monhystera stagnalis	267	uncultured eukaryote	2
Tubifex tubifex	927	unknown_11	262	Acrobeloides_nanus-7–3NDf	2
Limnodrilus claparedianus	867	Homalozoon vermiculare	259	Ironus sp. 199_89c	2
unknown_45	821	Tripyla glomerans	249	Aporcelaimellus sp. SSU_3	2
unknown_46	801	unknown_12	224
unknown_47	769	Anaplectus granulosus	220
unknown_48	687	Pratylenchus convallariae	204
unknown_49	681	unknown_13	197
unknown_50	666	unknown_14	184
unknown_51	619	Monhystera paludicola/Monhystera sp.	178
Nais communis	594	Prodesmodora circulata	166
unknown_53	585	unknown_15	166
Phasmarhabditis papillosa	582	uncultured Tobrilus	164
unknown_54	496	Oscheius tipulae	163
unknown_55	486	Mesorhabditis sp. 'Bursilla' PS1179	155
unknown_56	483	unknown_16	148
unknown_43	482	unknown_17	142
unknown_57	464	unknown_18	121
Fejervarya granosa	418	unknown_19	118
unknown_60	410	unknown_20	117
unknown_61	379	unknown_21	117
unknown_62	376	unknown_22	116
unknown_63	360	unknown_23	109
unknown_65	325	unknown_24	107
unknown_66	312	unknown_25	104
unknown_67	300	unknown_26	104
unknown_70	265	Aphelenchus sp. OH-2016	102
unknown_69	261	unknown_27	100
Bos taurus	247	unknown_28	100
Limnodrilus hoffmeisteri complex lineage VIII	227	unknown_29	89
Meloidogyne naasi	223	unknown_30	82
unknown_72	223	Cephaloboides cf. armata SB363	81
unknown_73	220	Dorylaimus stagnalis	80
unknown_76	209	unknown_31	79
unknown_71	204	unknown_32	79
Blastomussa loyae	203	unknown_33	75
Naididae sp. JCOCH052–10	201	unknown_34	75
unknown_78	188	unknown_35	71
unknown_79	186	unknown_36	71
unknown_80	186	Boleodorus sp. 2 TJP-2012	64
unknown_81	185	unknown_37	60
unknown_86	156	unknown_38	54
unknown_88	152	Rhabditis brassicae	53
Gammarus pulex	145	unknown_39	52
unknown_89	140	unknown_40	51
unknown_90	139	unknown_41	50
unknown_92	128	unknown_42	50
unknown_93	128	unknown_43	49
unknown_84	127	unknown_44	49
unknown_94	127	unknown_45	46
unknown_95	123	unknown_46	43
unknown_97	115	unknown_47	43
unknown_98	106	unknown_48	42
unknown_99	106	unknown_49	39
unknown_100	105	Plectus exinocaudatus	38
Amphichaeta raptisae	104	unknown_50	38
Rhabditida sp. 3028ed	103	unknown_51	37
unknown_101	102	Trichodorus similis	36
unknown_103	101	unknown_52	36
unknown_104	101	unknown_53	34
Tubificinae sp. 1 RV-2016	100	unknown_54	34
unknown_105	100	unknown_55	33
Enochrus ater	88	unknown_56	28
unknown_106	88	Coslenchus costatus	27
unknown_107	87	Plectus velox	26
unknown_109	86	unknown_57	25
Rhabditida sp. 3003ed	84	Chiloplectus andrassyi	22
unknown_111	84	Plectus opisthocirculus	21
unknown_112	84	unknown_58	20
unknown_113	81	unknown_59	19
unknown_114	81	Trichodorus arasbaranensis	18
unknown_115	80	unknown_60	18
unknown_116	80	unknown_61	18
unknown_117	79	unknown_62	18
unknown_119	76	unknown_63	17
unknown_120	74	unknown_64	17
unknown_122	73	unknown_65	17
unknown_124	66	Trischistoma sp. 2 ZQZ-2010a	16
unknown_126	65	unknown_66	16
unknown_127	65	unknown_67	16
unknown_128	65	unknown_68	16
Corynoneura carriana	64	unknown_69	16
Limnodrilus hoffmeisteri complex lineage X	64	Plectus aquatilis/Plectus acuminatus	15
unknown_129	64	unknown_70	15
unknown_130	61	unknown_71	15
unknown_118	59	unknown_72	15
unknown_132	58	unknown_73	15
unknown_133	58	Pellioditis sp. VS-2014	14
unknown_135	56	unknown_74	14
unknown_137	55	unknown_75	14
unknown_138	53	unknown_76	14
unknown_136	52	unknown_77	14
unknown_15	51	unknown_78	13
unknown_140	48	unknown_79	13
unknown_141	48	Acrobeloides sp. ES-2017	12
unknown_142	48	Meloidogyne graminicola	12
Limnodrilus hoffmeisteri complex lineage IX	47	unknown_80	12
unknown_143	47	unknown_81	12
unknown_144	46	unknown_82	12
unknown_139	45	unknown_83	12
unknown_145	45	unknown_84	12
Chironomus melanescens	42	unknown_85	11
unknown_125	39	unknown_86	11
unknown_149	39	unknown_87	11
unknown_150	38	unknown_88	11
unknown_151	38	unknown_89	11
unknown_152	38	Prismatolaimus dolichurus	10
unknown_148	37	unknown_90	10
Cricotopus sylvestris	36	unknown_91	10
unknown_17	36	unknown_92	10
unknown_153	35	unknown_93	10
unknown_154	34	unknown_94	10
unknown_155	34	unknown_95	10
unknown_156	34	unknown_96	10
unknown_158	33	unknown_97	10
unknown_159	32	unknown_98	10
unknown_160	31	unknown_99	10
unknown_36	31	unknown_100	9
unknown_161	30	unknown_101	9
unknown_162	29	unknown_102	9
unknown_163	29	unknown_103	9
unknown_164	29	unknown_104	9
unknown_165	28	unknown_105	8
unknown_166	28	unknown_106	8
unknown_167	28	unknown_107	8
unknown_168	28	unknown_108	8
Prodiamesa olivacea	27	unknown_109	8
unknown_169	27	Frontonia sp. 4 WS-2014	7
unknown_171	27	unknown_110	7
Propappus volki	25	unknown_111	7
unknown_172	25	unknown_112	7
unknown_34	25	unknown_113	7
unknown_173	24	unknown_114	7
unknown_175	24	unknown_115	6
unknown_176	24	unknown_116	6
unknown_178	24	unknown_117	6
unknown_180	23	unknown_118	6
unknown_58	23	unknown_119	6
unknown_184	22	unknown_120	6
unknown_31	22	unknown_121	6
unknown_189	21	unknown_122	6
unknown_170	20	unknown_123	6
unknown_190	20	unknown_124	6
unknown_191	20	unknown_125	6
unknown_192	20	unknown_126	6
unknown_193	20	unknown_127	6
unknown_194	20	unknown_128	6
unknown_195	20	Arabidopsis thaliana	5
unknown_197	19	uncultured Asellus sp.	5
Potamothrix moldaviensis	18	unknown_129	5
unknown_198	18	unknown_130	5
unknown_199	18	unknown_131	5
unknown_200	18	unknown_132	5
unknown_201	18	unknown_133	5
Tanytarsus pallidicornis	17	unknown_134	5
unknown_202	17	unknown_135	5
unknown_203	17	unknown_136	4
unknown_204	17	unknown_137	4
unknown_205	17	unknown_138	4
uncultured fungus	16	unknown_139	4
unknown_206	16	unknown_140	4
unknown_207	16	unknown_141	4
unknown_209	16	unknown_142	4
unknown_210	16	Daubaylia potomaca	3
Arthropoda environmental sample	15	Tobrilus medius	3
Ophidonais serpentina	15	unknown_143	3
unknown_212	15	unknown_144	3
unknown_213	15	unknown_145	3
unknown_214	15	unknown_146	3
unknown_215	15	unknown_147	3
Asellus aquaticus	14	unknown_148	3
Penicillium sclerotiorum	14	Bos indicus x Bos taurus	2
unknown_216	14	Mesocriconema xenoplax	2
unknown_217	14	unknown_149	2
unknown_218	14	unknown_150	2
unknown_219	14	unknown_151	2
unknown_220	13	unknown_152	2
unknown_223	13	unknown_153	2
unknown_224	13	unknown_154	2
unknown_225	13	unknown_155	2
unknown_226	13	unknown_156	2
Homo sapiens	12	unknown_157	2
unknown_227	12	unknown_158	2
unknown_229	12	unknown_159	2
unknown_230	12	unknown_160	2
Nais sp. N12 RV-2017	11	unknown_161	2
unknown_231	11	unknown_162	2
unknown_232	11	unknown_163	2
unknown_234	11
unknown_235	11
unknown_238	11
unknown_182	10
unknown_239	10
unknown_240	10
Parasitidae sp. BOLD:AAF9233	9
unknown_157	9
unknown_221	9
unknown_241	9
unknown_242	9
unknown_244	9
unknown_245	9
unknown_247	9
unknown_44	9
Eucyclops cf. serrulatus ZISP 11SNM-549	8
unknown_249	8
unknown_250	8
Chironomus pallidivittatus	7
Micospina auribohnorum	7
unknown_181	7
unknown_208	7
unknown_251	7
unknown_252	7
unknown_254	7
unknown_255	7
unknown_258	7
Acanthocyclops vernalis	6
Anomala ruficapilla	6
Paragordius tricuspidatus	6
unknown_260	6
unknown_261	6
unknown_262	6
unknown_263	6
unknown_264	6
unknown_266	6
Potamothrix heuscheri	5
unknown_196	5
unknown_271	5
unknown_272	5
unknown_273	5
unknown_274	5
unknown_275	5
unknown_276	5
unknown_277	5
unknown_278	5
unknown_279	5
unknown_280	5
unknown_3	5
Eucyclops cf. serrulatus ZISP 11SNM-547	4
unknown_281	4
unknown_282	4
unknown_284	4
unknown_285	4
unknown_286	4
unknown_287	4
unknown_291	4
unknown_292	4
unknown_293	4
unknown_294	4
unknown_295	4
unknown_68	4
Chydoridae sp. SHDT150804	3
Habrotrocha constricta	3
unknown_296	3
unknown_297	3
unknown_298	3
unknown_299	3
unknown_300	3
unknown_301	3
unknown_302	3
unknown_303	3
unknown_304	3
unknown_305	3
unknown_306	3
unknown_307	3
unknown_308	3
unknown_309	3
unknown_310	3
unknown_311	3
unknown_312	3
unknown_314	3
unknown_1	2
unknown_315	2
unknown_316	2
unknown_318	2
unknown_319	2
unknown_320	2
unknown_321	2
unknown_322	2

Fig. 4

Maximum likelihood tree based on the OTU inventory for the COI gene. The numbers at the branches represent the support values for the nodes.

Fig. 5

Maximum likelihood tree based on the ASV inventory for the COI gene. The numbers at the branches represent the support values for the nodes.

Table 8

Inspection of NCBI reference database for the Nematode SPEcies At Risk (NemaSPEAR[%]) as defined by Höss et al. [3] at the species and at the genus level. An” x” indicates whether the species or genera is found at NCBI for the 18S rDNA or 28 rDNA gene, while an “n” indicates that no reference sequence was found. The number of deposited sequences is given, the numbers in parentheses indicate reference sequences that were generated in our lab but are not yet published.

NemaSPEAR	18S	n	28S	n	NemaSPEAR _genus	18S	n	28S	n
Achromadora ruricola	x	1	n		Achromadora	x	17	n
Achromadora terricola	x	1	n		Aglenchus	x	30	x	6
Aglenchus agricola	x	25	x	2	Alaimus	x	18	x	3
Alaimus meyli	n		n		Amphidelus	x	1	n
Alaimus parvus	x	2	n		Aphanolaimus	x	3	x	5
Alaimus primitivus	n		n		Aphelenchus	x	>50	x	>50
Amphidelus cf elegans	n		n		Aporcelaimellus	x	>50	x	38
Aphanolaimus aquaticus	x	3	x	3	Bastiania	x	4	n
Aporcelaimellus obtusicaudatus	x	24	x	17	Cephalenchus	x	>50	x	>50
Coslenchus costatus	x	6	x	3	Coslenchus	x	30	x	20
Cuticularia oxycerca	n		n		Crassolabium	n		x	3
Cylindrolaimus communis	x	1	n		Criconema	n	51	x	15
Epidorylaimus agilis	n		n		Cylindrolaimus	x	6	x	12
Epitobrilus medius	x	1	x	17	Diplogasteritus	x	6	x	12
Epitobrilus steineri	n		n		Epidorylaimus	x	9	x	3
Ethmolaimus pratensis	x	3	x	7(+7)	Ethmolaimus	x	3	x	7
Eucephalobus oxyuroides	x	12	x	5	Eudorylaimus	x	>50	x	18
Eudorylaimus acuticauda	n		n		Fictor	x	13	x	25
Eudorylaimus carteri	x	5	x	4	Hemicycliophora	x	30	x	>50
Eumonhystera andrassy	n		n		Hirschmanniella	x	28	x	32
Eumonhystera barbata	n		n		Hofmaenneria	n		n
Eumonhystera longicaudatula	x	1	n		Ironus	x	15	x	7
Eumonhystera simplex	x	1	n		Monhystrella	x	2	n
Eumonhystera vulgaris	x	1	n		Paramphidelus	x	4	n
Helicotylenchus pseudorobustus	x	61	x	38	Paraphanolaimus	x	4	n
Hemicycliophora typica	x	4	x	2	Paraplectonema	x	1	x	2
Hirschmaniella gracilis	n		n		Plectus	x	>50	x	>50
Hofmaenneria brachystoma	n		n		Pratylenchus	x	>50	x	>50
Ironus ignavus	n		n		Prismatolaimus	x	>50	x	8
Ironus longicaudatus	x	2	x	2	Prodesmodora	x	7	x	3
Ironus tenuicaudatus	n		x	3	Prodorylaimus	x	6	x	4
Mesodoryaimus conurus	n		n		Punctodora	x	5	n
Mesodoryaimus subtiliformis	n		n		Rhabditis	x	>50	x	>50
Monhystera lemani	n		n		Rhabdolaimus	x	>50	n
Monhystrella paramacrura	n		n		Semitobrilus	x	4	x	1
Mononchus truncatus	x	5	x	3	Theristus	x	33 (+>50)	x	3 (+>50)
Neotobrilus longus	x	3	x	16	Thornia	x	1	n
Panagrolaimus cf thienemanni	n		n		Trischistoma	x	19	x	8
Paramphidelus dolichurus	n		n
Paraphanolaimus anisitsi	n		n
Paraplectonema pedunculatum	x	1	n
Plectus aquatilis	x	7	x	5
Plectus cirratus	x	1	x	2
Plectus opisthocirculus	x	1	x	1
Plectus rhizophilus	x	2	x
Prismatolaimus dolichurus	x	3	x	7
Prismatolaimus intermedius	x	3	n
Prismatolaimus tenuicaudatus	n		n
Prodesmodora circulata	x	4	x	3
Punctodora dudichi	n		n
Punctodora ratzeburgensis	x	5	n
Rhabditis gracilicauda	n		n
Rhabdolaimus terrestris	x	1	n
Semitobrilus pellucidus	x	2	x	16
Theristus agilis	x	3	(x)	(>150)
Theristus vesentiniae	n		n
Thornia propinqua	n		n
Trischistoma monhystera	n		n

Fig. 1

Rarefaction analyses based on morphological data for the 7 locations (CUM, HI, ÖR, VE, FB and RM). Given is the species diversity that was observed, as well as extrapolated estimates for up to 1000 individuals inspected (dashed lines).

Table 9

Species inventory for the seven locations (VE, ÖR, RM, HI, LU, CUM, FB). Given are the species identified by morphological inspection, together with the biomass for a male (m biomass) and female (fm biomass) based on measured specimen in the dataset. It is indicated if a NCBI sequence for the 28S rDNA or 18S rDNA gene is found, an “n” showing that this species is missing.

Species	NCBI	fm biomass	m biomass	VE	ÖR	RM	HI	LU	CUM	FB
Achromadora cf. terricola	18S	0.93		0.00	0.60	0.00	0.00	0.00	0.59	1.24
Achromadora longicauda	n	0.05		0.19	0.00	0.00	0.00	0.00	0.00	0.00
Achromadora ruricola	18S	0.19	0.14	4.27	0.00	0.00	0.00	1.02	0.00	0.00
Achromadora sp.	18S	0.13		0.00	0.00	0.00	0.99	0.20	0.00	0.00
Achromadora micoletzky	n	0.17	0.08	11.06	0.00	0.60	4.96	0.41	14.31	0.00
Aglenchus sp.	18S,28S	0.11	0.10	0.00	0.00	0.40	0.00	0.00	0.00	0.00
Alaimus parvus	18S	0.13		0.00	0.00	0.00	0.00	0.00	0.40	0.00
Alaimus primitivus	n	0.34	0.12	0.59	0.00	0.00	0.40	0.00	0.79	0.00
Amphidelus dudichi	n	1.34		0.19	0.00	0.00	0.00	0.00	0.00	0.00
Amphidelus elegans	n	0.45		0.19	0.00	0.00	0.00	0.00	0.40	0.00
Anaplectus grandepapillatus	18S	2.34	1.72	0.00	0.00	0.00	0.00	0.00	0.00	18.56
Anaplectus granulosus	28S	0.92	0.69	0.00	0.00	0.00	2.38	0.00	1.00	0.00
Aphanolaimus aquaticus	18S,28S	0.82	0.43	0.00	0.00	0.80	0.00	0.00	0.00	0.00
Aphelenchoides bicaudatus	18S	0.07	0.06	0.19	0.00	0.00	0.20	0.00	0.00	0.00
Aphelenchoides parietinus	28S	0.23	0.20	0.00	0.00	0.00	0.99	0.00	0.79	0.00
Aphelenchus cf. avenae	18S,28S	0.21	0.25	0.00	0.00	0.00	0.20	0.00	0.40	0.21
Aporcelaimellus obtusicaudatus	18S,28S	9.99		0.97	0.00	0.00	3.37	0.00	2.98	0.00
Bastiania gracilis	18S	0.14	0.11	0.19	0.00	0.00	0.00	0.00	0.00	0.00
Bitylenchus dubius	18S	0.29	0.14	0.00	0.00	0.20	9.91	0.00	3.18	0.00
Bursilla monhystera	n	0.63	0.13	0.00	0.00	0.20	0.40	0.00	1.20	0.00
Cephalenchus hexalineatus	18S,28S	0.12	0.10	0.00	0.00	0.00	0.20	0.00	0.00	0.00
Cephalobus persegnis	18S,28S	0.43	0.23	0.19	0.00	0.60	20.22	0.61	11.52	0.00
Chromadorina bioculata	18S,28S	0.27	0.22	0.00	0.00	2.01	0.20	0.61	0.00	0.00
Chromadorita leuckarti	18S,28S	1.48	0.80	2.14	0.40	0.00	0.20	2.03	0.00	10.10
Chromadorina viridis	28S	0.79	0.54	0.00	0.00	1.81	0.00	0.00	0.00	0.00
Chrysonema holsaticum	n	1.90	0.55	0.00	0.00	0.00	0.00	0.00	0.20	0.00
Clarkus papillatus	18S,28S	1.46	1.95	0.00	0.00	0.00	0.40	0.00	0.00	0.00
Coslenchus costatus	18S,28S	0.11	0.07	0.00	0.00	0.00	2.18	0.61	1.59	0.00
Crassolabium ettersbergensis	n	0.30		0.00	0.00	0.00	0.00	0.00	0.59	0.00
Crocodoylaimus flavomaculatus	n	1.27	1.23	0.00	0.00	0.00	0.00	0.00	0.40	0.00
Cryptonchus tristis	18S	1.57	1.71	0.38	0.00	0.00	0.00	0.00	0.00	0.00
Cylindrolaimus melancholicus	n	1.36	0.99	0.00	0.00	0.00	0.00	0.00	0.00	0.82
Daptonema dubium	n	5.06	2.20	0.00	0.00	21.90	3.37	0.00	0.00	0.00
Diplogaster rivalis	18S,28S	1.92	0.65	0.00	0.00	0.00	0.40	1.02	0.00	0.00
Ditylenchus sp.	18S,28S	0.92	0.46	0.00	0.00	0.00	0.99	0.00	0.00	0.00
Dorylaimidae sp.	18S,28S	3.41	2.50	0.00	0.00	0.20	0.00	0.20	0.00	0.00
Dorylaimoides limnophilus	18S,28S	1.22		0.00	0.00	0.20	0.20	0.00	0.00	0.00
Dorylaimus stagnalis	18S,28S	47.64	42.07	0.00	0.00	0.00	0.40	0.61	0.40	0.00
Epidorylaimus agilis	n	2.35		0.38	0.00	0.20	0.00	0.00	0.00	0.21
Epitobrilus stefanskii	18S	3.27	2.51	0.19	0.40	0.20	0.00	1.63	0.00	0.00
Ethmolaimus pratensis	18S,28S	0.68	0.44	0.19	9.02	0.00	0.00	0.00	0.00	1.03
Eucephalobus oxyuroides	18S,28S	0.26	0.14	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Eudorylaimus acuticauda	n	3.94	2.69	0.00	0.00	0.00	0.99	0.00	0.79	0.00
Eudorylaimus carteri	18S,28S	4.65	3.27	0.00	0.00	0.00	0.00	0.00	0.00	0.21
Eudorylaimus centrocercus	28S	3.71	1.72	0.00	0.00	0.00	0.00	0.00	0.40	0.00
Eumonhystera barbata	n	0.12		0.19	0.00	0.20	0.00	0.00	0.00	0.21
Eumonhystera dispar	n	0.43		1.16	0.20	0.20	0.40	1.22	0.00	0.82
Eumonhystera filiformis	18S,28S	0.27	0.22	5.43	6.01	6.22	6.74	26.88	0.40	0.00
Eumonhystera longicaudatula	18S	0.22		1.94	17.43	3.41	0.99	0.00	0.00	1.86
Eumonhystera pseudobulbosa	n	0.04		1.75	0.80	1.41	0.59	2.03	0.40	0.21
Eumonhystera simplex	18S	0.03		2.71	0.00	1.00	1.19	0.00	3.38	0.00
Eumonhystera vulgaris	18S	0.19		6.41	19.24	0.00	0.59	1.42	0.00	2.27
Eumonhystera sp.	18S	0.14		18.62	0.00	0.00	0.00	0.00	0.00	0.00
Euteratocephalus palustris	18S	0.24		0.00	0.00	0.00	0.20	0.00	0.20	0.00
Eutobrilus grandepapillatus	n	10.80	5.89	0.00	0.00	0.00	0.20	0.00	0.00	0.00
Fictor fictor	n	1.58	0.59	0.19	0.00	0.00	0.20	0.00	0.00	0.00
Filenchus sp.	18S,28S	0.09		0.00	0.00	0.00	0.00	0.00	0.00	0.21
Filenchus vulgaris	18S,28S	0.10	0.09	5.43	0.00	4.62	8.33	0.61	16.69	0.00
Helicotylenchus pseudorobustus	18S,28S	0.35		0.38	0.00	0.00	0.20	0.00	1.00	0.00
Hemicycliophora typica	18S,28S	0.52		4.27	0.00	0.00	0.40	0.41	0.20	0.00
Heterocephalobus elongatus	18S	0.41	0.32	0.00	0.00	0.00	0.59	0.00	0.20	0.00
Hirschmaniella gracilis	n	1.36	1.02	0.19	0.00	0.00	0.00	0.00	0.00	0.00
Hofmaenneria niddensis	n	0.58	0.41	0.00	0.00	0.00	0.00	0.00	0.00	1.65
Ironus longicaudatus	18S,28S	1.00		0.19	0.00	0.00	0.00	0.00	0.00	0.00
Laimaphelenchus penardi	18S	0.16	0.13	0.19	0.00	0.00	0.00	0.00	0.00	0.00
Malenchus bryophilus	18S,28S	0.06	0.03	0.00	0.00	0.00	0.40	0.41	0.20	0.00
Mermithidae				0.19	0.00	0.40	0.00	0.00	0.00	0.41
Mesodorylaimus bastiani	18S,28S	1.45	1.04	0.19	0.00	0.00	0.00	0.00	1.79	0.00
Mesodorylaimus paetzoldi	n	2.81		0.00	0.00	0.00	0.00	0.20	0.00	0.00
Mesodorylaimus sp.	18S,28S	1.94		0.00	0.20	0.00	0.00	0.20	0.00	0.00
Monhystera paludicola	18S,28S	0.86	0.56	5.04	2.00	1.81	0.00	2.44	0.00	0.41
Monhystera stagnalis	18S,28S	1.24	0.68	0.00	0.00	0.80	0.00	0.20	0.00	0.21
Monhystera sp.	18S,28S	0.43		0.00	0.00	3.01	0.00	0.00	0.00	0.00
Monhystrella macrura	n	0.16	0.08	0.00	0.00	0.20	0.00	0.00	0.00	0.00
Monhystrella paramacrura	n	0.04	0.06	0.00	0.00	5.82	0.20	0.20	0.79	0.00
Mononchus aquaticus	18S,28S	4.21	4.33	0.19	0.40	0.00	0.20	0.20	0.00	0.00
Mononchus truncatus	18S,28S	4.94	3.21	0.00	0.00	0.20	0.40	0.00	0.00	0.00
Mylonchulus brachyuris	18S	1.38	1.06	0.19	0.00	0.00	1.19	0.00	0.00	0.00
Mylonchulus sigmaturus	18S	1.86	1.07	0.00	0.00	0.00	1.78	0.00	0.20	0.00
Neotobrilus diversipapillatus	n	5.33	1.54	0.00	0.00	0.00	0.20	0.00	0.00	0.00
Panagrolaimus rigidus	18S,28S	1.07	0.54	0.59	0.00	0.00	2.57	0.41	1.59	0.00
Paractinolaimus macrolaimus	18S,28S	10.82	7.87	0.00	0.00	0.00	0.00	0.41	0.00	0.00
Paraplectonema pedunculatum	18S	0.68		0.00	0.00	0.00	0.20	0.00	0.00	0.00
Pellioditis pellioides	n	7.97	3.11	0.38	0.00	0.00	0.00	0.00	0.00	0.00
Plectus aquatilis	18S,28S	1.30		0.19	0.00	0.00	0.00	0.20	0.20	0.00
Plectus opisthocirculus	18S,28S	0.13		0.59	0.20	0.80	0.99	2.24	0.00	0.00
Plectus parvus	18S,28S	0.17		0.59	0.20	0.00	0.00	0.20	1.00	0.00
Plectus rhizophilus	18S	0.76		0.00	0.00	0.00	0.00	0.81	0.00	0.00
Plectus tenuis	18S	0.64		0.00	0.00	0.00	0.00	0.61	0.00	0.00
Pratylenchus sp.	18S,28S	0.09		0.00	0.00	0.00	0.40	0.00	0.00	0.00
Prismatolaimus dolichurus	18S,28S	0.47	0.33	0.00	0.00	0.00	0.20	0.00	0.40	0.00
Prismatolaimus intermedius	18S	0.10	0.14	1.55	0.20	0.80	6.15	0.00	9.34	0.21
Prismatolaimus sp.	18S	0.37		0.00	0.00	0.00	0.59	0.00	0.00	0.00
Prodesmodora arctica	n	0.07		0.59	0.00	0.00	0.00	0.00	0.00	0.00
Prodesmodora circulata	18S,28S	0.33		0.38	0.00	0.00	0.20	0.00	0.20	0.00
Prodorylaimus fliiarium	n	2.31	2.12	0.38	0.00	0.00	0.00	0.00	0.00	0.00
Prodorylaimus sp.	18S,28S	3.52		0.00	0.20	0.00	0.00	1.02	0.00	0.00
Punctodora ratzeburgensis	18S	0.53	0.34	0.00	0.00	19.29	0.20	0.00	0.00	0.00
Rhabditidae	18S,28S	1.00		0.97	0.00	0.00	0.00	0.00	1.39	0.00
Rhabditidae sp.	18S	0.42		0.00	0.00	0.00	1.98	0.61	0.20	0.00
Rhabditis gracilicauda	n	0.51	0.66	0.19	0.00	0.00	0.20	0.00	0.20	0.00
Rhabdolaimus aquaticus	18S	0.08		0.00	0.00	0.20	0.00	0.00	0.00	0.00
Rhabdolaimus terrestris	18S	0.11		2.33	0.00	0.00	0.00	0.00	0.00	0.00
Semitobrilus pellucidus	18S,28S	4.86	5.08	10.47	41.89	0.20	0.00	0.00	0.00	9.07
Species 1		0.51	0.42	0.19	0.00	0.00	0.00	0.00	0.00	0.00
Species 2 (Cephalobidae)		0.46	0.37	0.00	0.00	0.00	0.00	0.00	0.59	0.00
Teratocephalus tenuis	n	0.10	0.05	0.00	0.00	0.00	0.20	0.00	0.20	0.00
Teratocephalus terrestris	18S	0.08		0.19	0.00	0.00	0.20	0.00	1.20	0.00
Theristus agilis	18S	1.19	0.68	0.19	0.00	0.00	0.20	0.00	0.00	37.31
Theristus vesentiniae	n	0.21	0.15	0.00	0.00	0.00	0.00	0.00	0.79	3.30
Tobrilus gracilis	18S,28S	6.32	4.19	0.38	0.20	19.09	0.00	47.04	0.00	0.00
Trichodorus sparsus	18S,28S	0.94	0.60	1.36	0.00	0.00	0.00	0.00	0.00	0.00
Tripyla glomerans	18S,28S	17.79	12.80	0.00	0.00	0.60	0.20	0.61	0.00	0.00
Tripyla setifera	28S	1.61	1.43	0.38	0.20	0.00	0.00	0.00	0.00	9.48
Trischistoma monohystera	n	1.19	0.74	0.97	0.00	0.00	0.20	0.00	0.40	0.00
Tylenchidae	18S,28S	0.43	0.33	0.00	0.00	0.00	0.00	0.00	0.59	0.00
Tylencholaimellus affinis	18S	1.80	1.33	0.19	0.00	0.00	0.00	0.00	0.00	0.00
Tylencholaimus teres	18S	0.48	0.44	0.19	0.00	0.00	0.00	0.00	2.58	0.00
Tylenchus davanei	n	0.69	0.54	0.00	0.20	0.40	6.35	0.41	9.93	0.00
Tylenchus sp. 1	18S,28S	0.69	0.51	0.19	0.00	0.00	0.00	0.00	0.20	0.00
Tylenchus sp. 2		0.26		0.38	0.00	0.00	0.40	0.00	0.00	0.00
Tylenchus sp. 3		0.19		0.00	0.00	0.00	0.00	0.00	1.39	0.00
Wilsonema otophorum	18S,28S	0.06	0.03	0.00	0.00	0.00	0.00	0.00	0.20	0.00

Fig. 2

-nMDS plots based on not-transformed relative abundances of species (a, b, c) and genera (d, e, f) found in sediments sampled from the seven locations based on taxonomic data for the 28S marker (a, d), the 18S marker (b, e) and the morphological analysis (c, f); Bray-Curtis similarity was applied; each location is encoded by a different color and symbol.

Fig. 3

NemaSPEAR[%] (a, c, e) and NemaSPEAR[%]genus (b, d, f) calculated based on morphological (morph; a, b) and molecular (28S-rDNA: c, d, 18S-rDNA: e, f) taxonomic nematode species and genus data of river sediment sampled at 7 sites (6 sites for 18S-rDNA) plotted against the mean PEC-Q values calculated for the respective sediments; a: r2 = 0.71; p = 0.017; b: r2 = 0.66; p = 0.027; c: r2 = 0.65; p = 0.029; d: r2 = 0.78; p = 0.008; e: r2 = 0.60; p = 0.070; f: r2 = 0.88; p = 0.006.

Chemical analysis of sediment samples. Given is the measured value for each of the 33 substances for each of the seven locations investigated; PEC = consensus-based probable effect concentrations as defined by De Deckere et al. [2] as “Consensus 2; dw = dry weight; values with Abbreviation for locations: Rischmühle (RM), Luppe (LU), Cumlosen (CUM), Hitzacker (HI), Veerse (VE), Örtze (ÖR) and Furlbach (FB). Overview about the number of nematodes for each replicate for the seven locations and the morphological and molecular approach. A black box indicates that these replicates were combined into a new replicate due to low individual number. For the location VE more than five replicates were available for morphological analysis, no further samples were available for molecular analysis, though. List of OTUs determined for the 28S rDNA gene fragment, using the primer pairs 1274/706 for the seven locations (CUM, HI, LU, ÖR, RM, VE and FB). Clustering was performed at 99%. List of OTUs determined for the 18S rDNA gene fragment, using the primer pairs 3NDf/C_1132f for the six locations (CUM, HI, LU, ÖR, RM and VE). Clustering was performed at 99%. List of OTUs determined for the COI gene, using the primer pairs mlCOIint/dgHCO for the six locations (CUM, HI, LU, ÖR, RM, and VE). Clustering was performed at 97%. Distribution of reads within the bioinformatic pipeline using mothur. Given is the number of reads at each current step of the analysis for each sample sequenced for the three genetic markers (1274/706 = 28S rDNA, mlCOIint/dgHCOCOI, 3NDf/C_1132f = 18S rDNA). Results of the cluster-independent bioinformatic approach. Amplicon Sequence Variants (ASVs) are given for the three genetic markers (COI = mlCOIint/dgHCO, 28S rDNA = 1274/706, and 18S rDNA= 3NDf/C_1132f). Numbers are given for all samples and replicates combined, as this is only an alternate approach, which was not regarded further. Maximum likelihood tree based on the OTU inventory for the COI gene. The numbers at the branches represent the support values for the nodes. Maximum likelihood tree based on the ASV inventory for the COI gene. The numbers at the branches represent the support values for the nodes. Inspection of NCBI reference database for the Nematode SPEcies At Risk (NemaSPEAR[%]) as defined by Höss et al. [3] at the species and at the genus level. An” x” indicates whether the species or genera is found at NCBI for the 18S rDNA or 28 rDNA gene, while an “n” indicates that no reference sequence was found. The number of deposited sequences is given, the numbers in parentheses indicate reference sequences that were generated in our lab but are not yet published. Rarefaction analyses based on morphological data for the 7 locations (CUM, HI, ÖR, VE, FB and RM). Given is the species diversity that was observed, as well as extrapolated estimates for up to 1000 individuals inspected (dashed lines). Species inventory for the seven locations (VE, ÖR, RM, HI, LU, CUM, FB). Given are the species identified by morphological inspection, together with the biomass for a male (m biomass) and female (fm biomass) based on measured specimen in the dataset. It is indicated if a NCBI sequence for the 28S rDNA or 18S rDNA gene is found, an “n” showing that this species is missing. -nMDS plots based on not-transformed relative abundances of species (a, b, c) and genera (d, e, f) found in sediments sampled from the seven locations based on taxonomic data for the 28S marker (a, d), the 18S marker (b, e) and the morphological analysis (c, f); Bray-Curtis similarity was applied; each location is encoded by a different color and symbol. NemaSPEAR[%] (a, c, e) and NemaSPEAR[%]genus (b, d, f) calculated based on morphological (morph; a, b) and molecular (28S-rDNA: c, d, 18S-rDNA: e, f) taxonomic nematode species and genus data of river sediment sampled at 7 sites (6 sites for 18S-rDNA) plotted against the mean PEC-Q values calculated for the respective sediments; a: r2 = 0.71; p = 0.017; b: r2 = 0.66; p = 0.027; c: r2 = 0.65; p = 0.029; d: r2 = 0.78; p = 0.008; e: r2 = 0.60; p = 0.070; f: r2 = 0.88; p = 0.006.

Experimental design, materials, and methods

Seven locations were sampled, with five replicates for morphological and molecular analysis each. Each replicate was comprised from five subsamples. The detailed process is described in ref [1]. Sediment samples were processed as described by Higgins and Thiel [4]. From each of the five replicates 100 nematodes were isolated for morphological analyses, if possible. For the location VE more replicates were analysed, due to low nematode densities (Table 2). For molecular analyses 200 nematodes per replicate were isolated, as this gave more DNA yield in previous studies [5]. For several replicates with low nematodes densities, two or more replicates were combined as shown in Table 2. At each of the seven locations, for analysis of chemical contamination 5–10 subsamples of sediment were collected with a stainless-steel grab sampler and then pooled in a stainless-steel tub to avoid the effects of local variability. After the removal of large debris, the sediment material was homogenized, and 1 kg was transferred to glass containers. For each pooled sample, chemical analyses were conducted for 33 substances and according to the following methods: arsenic (As; ISO 17,294–2-E29:2005–02), 7 metals (Cd, Cu, Pb, Cr, Ni, Zn; ISO 17,294–2-E29:2005–02 and Hg; ISO 16,772:2005–06), 16 polycyclic aromatic hydrocarbons (PAHs according to the US EPA; ISO 18,287), 7 polychlorinated biphenols (PCB 28, 52, 101, 118, 138, 153, 180; EN 15,308), p,p'-DDD and p,p'-DDE (both ISO 10,382); Table 1).

Morphological data

Nematodes were assigned to the lowest taxonomic level as described in ref [1]. All specimens were measured and for each species the biomass contribution was calculated following Andrassyi [6], differentiating between male, female and juvenile stages 1 and 4, as also described in ref [1]. A rarefaction curve was calculated with R, using the package “iNEXT” [7] and the functions “iNEXT” and “ggiNEXT”. It was furthermore checked, whether a NCBI sequence was available for the morphological identified species, by using the species as search term and verifying if a 28S rDNA, 18S rDNA or COI sequence was found (Table 8).

Molecular data

Molecular data was analysed with mothur [8], following the standard protocol with alterations for the BLAST identification [9]. For the 18S rDNA and 28S rDNA gene fragment a cluster threshold of 99% was chosen, while for the COI gene 97% were chosen, due to the accelerated mutation rate. The distribution of reads in the process of bioinformatics was summed up after every filtering step, including merging of paired-end reads, filtering of long/short and homopolymer reads, and filtering of reads not fitting the alignment. The final OTU inventory for the three markers is listed (Table 4, 5, 6), based on BLAST with a cut-off of 95% identity. The results of an alternative approach, using no clustering, is given in Table 3. Data was generated with DADA2 [10], using default parameters with slight adaptions (length cutoff (280,250), maxEE = c (2,2), truncQ = 2). For the 18S gene fragment no length cut-off was chosen, as the overlap was very low due to the long amplicon length (around 540 bp). The BLAST comparison was run with an identity cut-off of 97%. For the COI phylogenetic trees were built in MEGA7 [11] for the ASV and OUT inventory, using maximum likelihood with 500 bootstrap replicates and the Jukes-Cantor model.

Statistical data

NMDS plots were generated with PRIMER_v6 [12], using all replicates for the molecular and morphological data for the seven locations. The data was not transformed and Bray-Curtis similarity was applied. NMDS plot was generated with default parameters in PRIMER. Correlations between the NemaSPEAR[%] values for the morphological approach, the 28S rDNA gene fragment and the 18S rDNA gene fragment for the species and genus level against the logarithmized mean PEC-Q values were plotted using SigmaPlot11 (Fig. 3).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.

Subject	Pollution
Specific subject area	An nematode-based index (NemaSPEAR[%]), which is based on morphological data, is validated with molecular data, amplifying fragments of the 28S rDNA, 28S rDNA and COI gene.
Type of data	Table
	Graph
	Figure
How data were acquired	Data was acquired using a Zeiss microscope and Illumina MiSeq sequencing (V3 chemistry; 2 × 300 bp; 2.5 M read pairs).
	Software used for further processing: PRIMER_v6, R Studio, mothur, cutadapt, SigmaPlot 11
Data format	NGS data is deposited under PRJNA513975 and PRJNA608650.
	Raw morphological data is provided here.
	Analyzed
	Filtered
Parameters for data collection	Sediment samples were collected at seven locations across Germany, whose pollution status was analysed before.
Description of data collection	Sediment samples were taken in 2017 and 2018 using a corer (2.6 cm diameter), using five subsamples per replicate.
Data source location	Institution: Bielefeld University
	City: Bielefeld
	Country: Germany
	Sample collection:
	Furlbach: N51°53.724 E008°42.931
	Veerse: N53°08.483 E009°30.060
	Oertze: N53°00.944 E010°04.974
	Saale-Rischmühle: N51°21.038 E012°00.213
	Elbe-Hitzacker: N53°09.643 E011°02.787
	Elbe-Cumlosen: N53°02.432 E011°38.592
	Luppe: N51°23.116 E012°00.526
Data accessibility	With the article and under PRJNA513975 and PRJNA608650
	https://www.ncbi.nlm.nih.gov/bioproject/PRJNA513975
	https://www.ncbi.nlm.nih.gov/bioproject/PRJNA608650
Related research article	Janina Schenk, Sebastian Höss, Marvin Brinke, Nils Kleinbölting, Henrike Brüchner-Hüttemann, Walter Traunspurger
	Title: Nematodes as bioindicators of polluted sediments using metabarcoding and microscopic taxonomy
	Journal: Environment International
	DOI: https://doi.org/10.1016/j.envint.2020.105922

6 in total

1. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.

Authors: Koichiro Tamura; Glen Stecher; Daniel Peterson; Alan Filipski; Sudhir Kumar
Journal: Mol Biol Evol Date: 2013-10-16 Impact factor: 16.240

2. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.

Authors: Patrick D Schloss; Sarah L Westcott; Thomas Ryabin; Justine R Hall; Martin Hartmann; Emily B Hollister; Ryan A Lesniewski; Brian B Oakley; Donovan H Parks; Courtney J Robinson; Jason W Sahl; Blaz Stres; Gerhard G Thallinger; David J Van Horn; Carolyn F Weber
Journal: Appl Environ Microbiol Date: 2009-10-02 Impact factor: 4.792