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Fig. 8. Ophiotaenia tkachi n in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 8. Ophiotaenia tkachi n. sp. from Nerodia fasciata confluens, host US 952 (MHNG-PLAT-0130134) (A, B, E, F) and N. erythrogaster, host US 1118 (G, H), and O. perspicua La Rue (1911) (C, D) from Nerodia rhombifer rhombifer (US 951), all samples from Oklahoma, USA. A, B – cross sections at the testicular and ovarian level, respectively; C, D – eggs; E–H – unripe eggs (without fully formed oncospheres), drawn in distilled water.

Fig. 10 in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 10. Maximum likelihood phylograms of cox1 data (Alignment 3; 1608 nt) for selected members of the Proteocephalidae. Bootstrap support from maximum likelihood (ML) and Bayesian inference (BI) nodal support are indicated as ML/BI; values <0.90 (BI) and <70 (ML) are not shown. The scale bar indicates the expected number of substitutions per site. The newly generated sequences are highlighted in bold. Abbreviations of the U.S. states where the samples were collected: AR – Arkansas; MS – Mississippi; OK – Oklahoma. Abbreviations of biogeographical regions: NEA – Nearctic; NEO – Neotropical.

Fig. 7. Ophiotaenia tkachi n in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 7. Ophiotaenia tkachi n. sp. from Nerodia fasciata confluens, host USA 21, Louisiana, USA, holotype (MHNG-PLAT-0063340). A, B – pregravid proglottids, ventral view; C, D – vaginal canal of unusual structure, ventral view (C) and en face view (D).

Fig. 5. Ophiotaenia laruei n in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 5. Ophiotaenia laruei n. sp. (A, B) from Nerodia rhombifer rhombifer, Illinois, USA (USNM 1696448) and Ophiotaenia sp. (C) from Thamnophis sirtalis, Michigan, USA (USNM 1351911). A – mature proglottid of holotype, dorsal view; B, C – terminal genitalia, dorsal view.

Fig. 4 in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 4. Ophiotaenia perspicua La Rue, 1911 from Nerodia rhombifer rhombifer, host US 951, Oklahoma, USA (MHNG-PLAT-0130131). A – mature proglottid, dorsal view; B – pregravid proglottid, ventral view; C, D – cross sections of at the testicular and ovarian level, respectively; E – terminal genitalia (cirrus sac and terminal part of the vagina), dorsal view.

Fig. 2 in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 2. Scolices of Ophiotaenia perspicua La Rue, 1911 (A–C) and O. laruei n. sp. (D–F) from Nerodia rhombifer, Illinois, USA (USNM 1351794); note larger size of scolices and almost triangular suckers in O. laruei n. sp. (D, E – holotype of O. laruei; USNM 1696448).

Fig. 1 in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 1. Ophiotaenia perspicua La Rue, 1911 (USNM 1351794) (A) and O. laruei n. sp. (B) from Nerodia rhombifer, Illinois, USA (USNM 1696448), Ophiotaenia sp. (C) from Thamnophis sirtalis, Michigan, USA (USNM 1351911), and O. perspicua (D) from N. rhombifer and N. fasciata, Illinois, Alabama, Texas and Louisiana, USA. A–C – unmounted specimens from vials; D – mounted specimens; note erroneous labelling of the holotype of O. perspicua (USNM 1348631; dotted) as Proteocephalus variabilis Brooks, 1978.

Fig. 6 in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 6. Ophiotaenia spp. from watersnakes (Colubridae) in North America. A–C – early pregravid proglottid, gravid proglottid and terminal genitalia of Ophiotaenia currani n. sp. from Nerodia fasciata confluens (USA 22), Mississippi, USA (MHNG-PLAT-0063341), ventral view; D, E – terminal genitalia of O. tkachi n. sp. from N. fasciata confluens (US 952), Oklahoma, USA, and holotype of O. variabilis (Brooks, 1978) species inquirenda from N. rhombifer, Louisiana, USA (USNM 1369891), dorsal view.

Fig. 3 in Discovering high species diversity of Ophiotaenia tapeworms (Cestoda: Proteocephalidae) of watersnakes (Colubridae) in North America

Fig. 3. Scolices of Ophiotaenia spp. from watersnakes (Colubridae) in North America. A, B – holotype and voucher of O. perspicua La Rue, 1911, Illinois, USA (USNM 1348631); C, D – O. perspicua from host US 951, Oklahoma, USA (MHNG-PLAT-0130131); E – O. perspicua from host USA 13, Tennessee (MHNG-PLAT-0035370); F, G – anterior parts and scolices of O. perspicua from the same host individual as holotype, Illinois, USA (USNM 1351794); H – holotype of Ophiotaenia laruei n. sp., Illinois, USA (USNM 1351794); note large, almost triangular suckers; all specimens from Nerodia rhombifer rhombifer; I–K – Ophiotaenia currani n. sp. from host USA 22, Mississippi, USA (MHNG-PLAT-0063341); L – holotype of Ophiotaenia tkachi n. sp. from host USA 21, Louisiana, USA; all specimens from Nerodia fasciata confluens; M, N – holotype (?) and voucher of Ophiotaenia variabilis (Brooks, 1978) species inquirenda from N. rhombifer, Louisiana, USA (USNM 1369891); O, P – paratypes of O. variabilis species inquirenda from Nerodia cyclopion, Louisiana, USA (USNM 1369892).

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Fig. 2 in Specificity of Stenurus (Metastrongyloidea: Pseudaliidae) infections in odontocetes stranded along the north-west Spanish coast

Fig. 2. (A–B) Stenurus minor: posterior end of a male (A) with an undivided bursa and broad spicules and of a female (B) with a finger-shaped cuticular process on the anterior lip of the vulva (➨) and first stage larvae (L1) in the uterus (⇨). (C–D) Stenurus globicephalae: posterior end of a male (C) and a female (D) with multiple cuticular swellings (➨) and L1 in the uterus (⇨). (E–F) Stenurus ovatus: posterior end of a male (E) with a divided bursa showing three rays and a female (F) showing a conical terminal process (➨) and a cuticular swelling (⇨).

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Fig. 1 in Specificity of Stenurus (Metastrongyloidea: Pseudaliidae) infections in odontocetes stranded along the north-west Spanish coast

Fig. 1. Nematodes found in the pterygoid sinuses during the necropsy of a short-finned pilot whale (Globicephala macrorhynchus).

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Fig. 3 in Specificity of Stenurus (Metastrongyloidea: Pseudaliidae) infections in odontocetes stranded along the north-west Spanish coast

Fig. 3. Bipartite multigraph relating the three Stenurus species with their hosts. The width of the links (gray arrows) represents the mean parasitic load on each host.

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Fig. 3 in Opening a can of lungworms: Molecular characterization of Dictyocaulus (Nematoda: Dictyocaulidae) infecting North American bison (Bison bison)

Fig. 3. Maximum likelihood analysis of cytochrome oxidase c subunit 1 (cox1) sequence data of Dictyocaulus spp. Analysis was run with TN93 + G as best nucleotide substitution model and 1,000 bootstraps. Angiostrongylus vasorum = outgroup.

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Fig. 1 in Opening a can of lungworms: Molecular characterization of Dictyocaulus (Nematoda: Dictyocaulidae) infecting North American bison (Bison bison)

Fig. 1. Sites of bison fecal collections; WHH: White Horse Hill National Game Preserve; RMA: Rocky Mountain Arsenal National Wildlife Refuge; NBR: National Bison Range; NSM: Neal Smith National Wildlife Refuge; WMW: Wichita Mountains Wildlife Refuge.

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Fig. 2 in Opening a can of lungworms: Molecular characterization of Dictyocaulus (Nematoda: Dictyocaulidae) infecting North American bison (Bison bison)

Fig. 2. Maximum likelihood analysis of internal transcribed spacer 2 (ITS2) sequence data of Dictyocaulus spp. Analysis was run with T92 as best nucleotide substitution model and 1,000 bootstraps. Angiostrongylus vasorum = outgroup.

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The North Pacific Eukaryotic Gene Catalog: metatranscriptome assemblies with taxonomy, function and abundance annotations

This data continues with the development of the unprocessed NPEGC Trinity de novo metatranscriptome assemblies, uploaded to this Zenodo repository for raw assemblies: <a href="../records/7332796">The North Pacific Eukaryotic Gene Catalog: Raw assemblies from Gradients 1, 2 and 3</a> A full description of this data is published in Scientific Data, available here: <a href="https://www.nature.com/articles/s41597-024-04005-5" target="_blank" rel="noopener">The North Pacific Eukaryotic Gene Catalog of metatranscriptome assemblies and annotations</a>. Please cite this publication if your research uses this data: Groussman, R. D., Coesel, S. N., Durham, B. P., Schatz, M. J., & Armbrust, E. V. (2024). The North Pacific Eukaryotic Gene Catalog of metatranscriptome assemblies and annotations. Scientific Data, 11(1), 1161. Excerpts of key processing steps are sampled below with links to the detailed code on the main github code repository: <a href="https://github.com/armbrustlab/NPac_euk_gene_catalog">https://github.com/armbrustlab/NPac_euk_gene_catalog</a> Processing and annotation of protein-level NPEGC metatranscripts is done in 6 primary steps: 1. Six-frame translation into protein sequences 2. Frame-selection of protein-coding translation frames 3. Clustering of protein sequences at 99% sequence identity 4. Taxonomic annotation against MarFERReT v1.1 + MARMICRODB v1.0 multi-kingdom marine reference protein sequence library with DIAMOND 5. Functional annotation against Pfam 35.0 protein family HMM profiles using HMMER3 6. Functional annotation against KOfam HMM profiles (KEGG release 104.0) using KofamScan v1.3.0 <code># Define local NPEGC base directory here:</code> <code>NPEGC_DIR="/mnt/nfs/projects/armbrust-metat"</code> <code># Raw assemblies are located in the /assemblies/raw/ directory</code> <code># for each of the metatranscriptome projects</code> <code>PROJECT_LIST="D1PA G1PA G2PA G3PA G3PA_diel"</code> <code># raw Trinity assemblies:</code> <code>RAW_ASSEMBLY_DIR="${NPEGC_DIR}/${PROJECT}/assemblies/raw"</code> Translation We began processing the raw metatranscriptome assemblies by six-frame translation from nucleotide transcripts into three forward and three reverse reading frame translations, using the transeq function in the EMBOSS package. We add a cruise and sample prefix to the sequence IDs to ensure unique identification downstream (ex, `>TRINITY_DN2064353_c0_g1_i1_1`  to `>G1PA_S09C1_3um_TRINITY_DN2064353_c0_g1_i1_1` for the S09C1_3um sample in the G1PA assemblies). See <a href="https://github.com/armbrustlab/NPac_euk_gene_catalog/blob/main/scripts/aa_data/NPEGC.6tr_frame_selection_clustering.sh">NPEGC.6tr_frame_selection_clustering.sh</a> for full code description. Example of six-frame translation using transeq <code>transeq -auto -sformat pearson -frame 6 -sequence 6tr/${PREFIX}.Trinity.fasta -outseq 6tr/${PREFIX}.Trinity.6tr.fasta</code> Frame selection We use a custom frame-selection python script <a href="https://github.com/armbrustlab/marferret/blob/main/scripts/python/keep_longest_frame.py">keep_longest_frame.py</a> to determine the longest coding length in each open reading frame and retain this sequence (or multiple sequences if there is a tie) for downstream analyses.  See <a href="https://github.com/armbrustlab/NPac_euk_gene_catalog/blob/main/scripts/aa_data/NPEGC.6tr_frame_selection_clustering.sh">NPEGC.6tr_frame_selection_clustering.sh</a> for full code description. Clustering by sequence identity To reduce sequence redundancy and near-identical sequences, we cluster protein sequences at the 99% sequence identity level and retain the sequence cluster representative in a reduced-size FASTA output file. See <a href="https://github.com/armbrustlab/NPac_euk_gene_catalog/blob/main/scripts/aa_data/NPEGC.6tr_frame_selection_clustering.sh">NPEGC.6tr_frame_selection_clustering.sh</a> for full code description of linclust/mmseqs clustering. Sample of linclust clustering script: core mmseqs function <code>function NPEGC_linclust {</code> <code># make an index of the fasta file:</code> <code>$MMSEQS_DIR/mmseqs createdb $FASTA_PATH/$FASTA_FILE NPac.$STUDY.bf100.db</code> <code># cluster sequences at $MIN_SEQ_ID</code> <code>$MMSEQS_DIR/mmseqs linclust NPac.${STUDY}.bf100.db NPac.${STUDY}.clusters.db NPac_tmp --min-seq-id ${MIN_SEQ_ID}</code> <code># retieve cluster representatives:</code> <code>$MMSEQS_DIR/mmseqs result2repseq NPac.${STUDY}.bf100.db NPac.${STUDY}.clusters.db NPac.${STUDY}.clusters.rep</code> <code># generate flat FASTA output with cluster reps</code> <code>$MMSEQS_DIR/mmseqs result2flat NPac.${STUDY}.bf100.db NPac.${STUDY}.bf100.db NPac.${STUDY}.clusters.rep NPac.${STUDY}.bf100.id99.fasta --use-fasta-header</code> <code>}</code> Corresponding files uploaded to this repository: Gzip-compressed FASTA files after translation, frame-selection, and clustering at 99% sequence identity (.bf100.id99.aa.fasta.gz)     NPac.G1PA.bf100.id99.aa.fasta.gz     NPac.G2PA.bf100.id99.aa.fasta.gz     NPac.G3PA.bf100.id99.aa.fasta.gz     NPac.G3PA_diel.bf100.id99.aa.fasta.gz     NPac.D1PA.bf100.id99.aa.fasta.gz MarFERReT + MARMICRODB taxonomic annotation with DIAMOND Taxonomy was inferred for the NPEGC metatranscripts with the DIAMOND fast read alignment software against the <a href="../records/10586950">MarFERReT v1.1 + MARMICRODB v1.0 multi-kingdom marine reference protein sequence library (v1.1)</a>, a combined database of the <a href="https://doi.org/10.1038/s41597-023-02842-4">MarFERReT v1.1 marine microbial eukaryote sequence library</a> and <a href="https://doi.org/10.5281/zenodo.3520509">MARMICRODB v1.0 </a>prokaryote-focused marine genome database. See <a href="https://github.com/armbrustlab/NPac_euk_gene_catalog/blob/main/scripts/aa_data/NPEGC.diamond_taxonomy.log.sh">NPEGC.diamond_taxonomy.log.sh</a> for full description of DIAMOND annotation. Excerpt of core DIAMOND function: <code>function NPEGC_diamond {</code> <code># FASTA filename for $STUDY</code> <code>FASTER_FASTA="NPac.${STUDY}.bf100.id99.aa.fasta"</code> <code># Output filename for LCA results in lca.tab file:</code> <code>LCA_TAB="NPac.${STUDY}.MarFERReT_v1.1_MMDB.lca.tab"</code> <code>echo "Beginning ${STUDY}"</code> <code>singularity exec --no-home --bind ${DATA_DIR} \</code> <code>        "${CONTAINER_DIR}/diamond.sif" diamond blastp \</code> <code>        -c 4 --threads $N_THREADS \</code> <code>        --db $MFT_MMDB_DMND_DB -e $EVALUE --top 10 -f 102 \</code> <code>        --memory-limit 110 \</code> <code>        --query ${FASTER_FASTA} -o ${LCA_TAB} >> "${STUDY}.MarFERReT_v1.1_MMDB.log" 2>&1</code> <code>}</code> Corresponding files uploaded to this repository: Gzip-compressed diamond lowest common ancestor predictions with NCBI Taxonomy against a combined MarFERReT + MARMICRODB taxonomic library (*.Pfam35.domtblout.tab.gz)     NPac.G1PA.MarFERReT_v1.1_MMDB.lca.tab.gz     NPac.G2PA.MarFERReT_v1.1_MMDB.lca.tab.gz     NPac.G3PA.MarFERReT_v1.1_MMDB.lca.tab.gz     NPac.G3PA_diel.MarFERReT_v1.1_MMDB.lca.tab.gz     NPac.D1PA.MarFERReT_v1.1_MMDB.lca.tab.gz Pfam 35.0 functional annotation using HMMER3 Clustered protein sequences were annotated against the Pfam 35.0 collection of 19,179 protein family Hidden Markov Models (HMMs) using <a href="http://hmmer.org/">HMMER 3.3 </a> with the <a href="https://academic.oup.com/nar/article/49/D1/D412/5943818">Pfam 35.0 protein family database</a>. Pfam annotation code is documented here: <a href="https://github.com/armbrustlab/NPac_euk_gene_catalog/blob/main/scripts/aa_data/NPEGC.hmmer_function.sh">NPEGC.hmmer_function.sh</a> Excerpt of core hmmsearch function: <code>function NPEGC_hmmer {</code> <code># Define input FASTA</code> <code>INPUT_FASTA="NPac.${STUDY}.bf100.id99.aa.fasta"</code> <code># hmmsearch call:</code> <code>hmmsearch --cut_tc --cpu $NCORES --domtblout $ANNOTATION_DIR/${STUDY}.Pfam35.domtblout.tab $HMM_PROFILE ${INPUT_FASTA}</code> <code># compress output file:</code> <code>gzip $ANNOTATION_DIR/${STUDY}.Pfam35.domtblout.tab</code> <code>}</code> Corresponding files uploaded to this repository: Gzip-compressed hmmsearch domain table files for Pfam35 queries (*.Pfam35.domtblout.tab.gz)     G1PA.Pfam35.domtblout.tab.gz     G2PA.Pfam35.domtblout.tab.gz     G3PA.Pfam35.domtblout.tab.gz     G3PA_diel.Pfam35.domtblout.tab.gz     D1PA.Pfam35.domtblout.tab.gz KEGG functional annotation using KofamScan v1.3.0 Clustered protein sequences were annotated against the KEGG collection (release 104.0) of 20,819 protein family Hidden Markov Models (HMMs) using <a href="https://github.com/takaram/kofam_scan" target="_blank" rel="noopener">KofamScan </a>and KofamKOALA. Kofam annotation code is documented here: <a href="https://github.com/armbrustlab/NPac_euk_gene_catalog/blob/main/scripts/aa_data/NPEGC.kofamscan_function.sh">NPEGC.kofamscan_function.sh</a> Excerpt of core NPEGC_kofam function: <code># Core function to perform KofamScan annotation</code> <code>function NPEGC_kofam {</code> <code>    # Define input FASTA</code> <code>    local INPUT_FASTA="NPac.${STUDY}.bf100.id99.aa.fasta"</code> <code>    # KofamScan call</code> <code>    ${KOFAM_DIR}/kofam_scan-1.3.0/exec_annotation -f detail-tsv -E ${EVALUE} -o ${ANNOTATION_DIR}/NPac.${STUDY}.bf100.id99.aa.tsv ${FASTA_DIR}/${INPUT_FASTA}</code> <code>    # Keep best hit (data is already sorted by KofamScan)</code> <code>    sort -uk1,1 ${ANNOTATION_DIR}/NPac.${STUDY}.bf100.id99.aa.tsv > ${ANNOTATION_DIR}/NPac.${STUDY}.bf100.id99.aa.best.kofam.tsv</code> <code>    # Compress output file</code> <code>    gzip ${ANNOTATION_DIR}/NPac.${STUDY}.bf100.id99.aa.tsv</code> <code>    # Compress best.kofam output file</code> <code>    gzip ${ANNOTATION_DIR}/NPac.${STUDY}.bf100.id99.aa.best.kofam.tsv</code> <code>}</code> <code># filter hits with a score > 30 in R</code> Corresponding files uploaded to this repository: Gzip-compressed KofamScan domain table files for Kofam queries (*.best.Kofam.incT30.csv.gz):     NPac.G1PA.bf100.id99.aa.best.Kofam.incT30.csv.gz     NPac.G2PA.bf100.id99.aa.best.Kofam.incT30.csv.gz     NPac.G3PA.UW.bf100.id99.aa.best.Kofam.incT30.csv.gz     NPac.G3PA.diel.bf100.id99.aa.best.kofam.incT30.csv.gz     NPac.D1PA.diel.bf100.id99.aa.best.kofam.incT30.csv.gz The full kofamscan tables with score >30 are deposited here: <a title="The North Pacific Eukaryotic Gene Catalog: KOfam protein function annotations" href="../records/13743267" target="_blank" rel="noopener">https://zenodo.org/records/13743267</a>

Fig. 10. A–E in The oldest known Miocene snake fauna from Central Europe: Merkur-North locality, Czech Republic

Fig. 10. A–E. Elapidae gen. et sp. indet. from the Early Miocene (MN 3a) of Merkur−North; trunk vertebra (SGDB 7408/MI−89) in lateral (A), dorsal (B), ventral (C), cranial (D), and caudal (E) views. Scale bar 2 mm.

Fig. 11. A–E in The oldest known Miocene snake fauna from Central Europe: Merkur-North locality, Czech Republic

Fig. 11. A–E. Vipera sp. from the Early Miocene (MN 3a) of Merkur−North; precaudal vertebra (SGDB Ah−610) in lateral (A), dorsal (B), ventral (C), cranial (D), and caudal (E) views. Scale bar 2 mm.

Fig. 8 in The oldest known Miocene snake fauna from Central Europe: Merkur-North locality, Czech Republic

Fig. 8. Natrix merkuriensis sp. nov. from the Early Miocene (MN 3a) of Merkur−North. A. Left ectopterygoid (SGDB Ah−313, paratype). B. Right quadrate (SGDB Ah−312). C. Right maxillary (SGDB Ah−314). D1, D2. Right compound bone (SGDB Ah−315, holotype). In dorsal (A), postero−lateral (B), ventral (C), labial (D1), and lingual (D2) views. Scale bar 2 mm.

Fig. 7 in The oldest known Miocene snake fauna from Central Europe: Merkur-North locality, Czech Republic

Fig. 7. Natrix sansaniensis (Lartet, 1851) from the Early Miocene (MN 3a) of Merkur−North. A1–A3. Left compound bone (SGDB Ah−291). B. Right compound bone (SGDB Ah−620). C1–C5. Precaudal vertebra (SGDB Ah−296). In labial (A1, B), lingual (A2), dorsal (A3, C4), lateral (C1), cranial (C2), ventral (C3), and caudal (C5) views. Scale bar 2 mm.