Long-Read Sequencing of Benthophilinae Mitochondrial Genomes Reveals the Origins of Round Goby Mitogenome Re-Arrangements

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Long-read sequencing of benthophilinae mitochondrial genomes reveals the origins of round goby mitogenome re-arrangements

Author(s): Gutnik, Silvia; Walser, Jean-Claude; Adrian-Kalchhauser, Irene

Publication Date: 2019-01-08

Permanent Link: https://doi.org/10.3929/ethz-b-000317088

Originally published in: Mitochondrial DNA Part B: Resources 4(1), http://doi.org/10.1080/23802359.2018.1547133

Rights / License: Creative Commons Attribution 4.0 International

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ISSN: (Print) 2380-2359 (Online) Journal homepage: https://www.tandfonline.com/loi/tmdn20

Long-read sequencing of benthophilinae mitochondrial genomes reveals the origins of round goby mitogenome re-arrangements

Silvia Gutnik, Jean-Claude Walser & Irene Adrian-Kalchhauser

To cite this article: Silvia Gutnik, Jean-Claude Walser & Irene Adrian-Kalchhauser (2019) Long-read sequencing of benthophilinae mitochondrial genomes reveals the origins of round goby mitogenome re-arrangements, Mitochondrial DNA Part B, 4:1, 408-409, DOI: 10.1080/23802359.2018.1547133 To link to this article: https://doi.org/10.1080/23802359.2018.1547133

Published online: 08 Jan 2019.

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tmdn20 MITOCHONDRIAL DNA PART B: RESOURCES 2019, VOL. 4, NO. 1, 408–409 https://doi.org/10.1080/23802359.2018.1547133

ARTICLE Long-read sequencing of benthophilinae mitochondrial genomes reveals the origins of round goby mitogenome re-arrangements

Silvia Gutnika, Jean-Claude Walserb and Irene Adrian-Kalchhauserc aBiozentrum, Department Growth & Development, University of Basel, Basel, Switzerland; bGenetic Diversity Centre Zurich, ETH Zurich, Zurich, Switzerland;cProgram Man-Society-Environment, Department of Environmental Sciences, University of Basel, Basel, Switzerland

ABSTRACT ARTICLE HISTORY Genetic innovation may be linked to evolutionary success, and indeed, the invasive round goby mito- Received 7 September 2018 chondrial genome sequence carries two novel features not previously described in Benthophilinae. Accepted 2 November 2018 First, the round goby mitochondrial genome carries a rearrangement of the tRNA cluster Ile-Glu-Met. KEYWORDS Second, the round goby mitochondrial genome features a 1250 bp non-coding sequence insertion Neogobius melanostomus; downstream of the D-loop region. In this publication, we test where in the goby phylogeny the novel mitogenome; D-loop; tRNA arrangement first arose and whether the sequence insertion is associated with invasive popula- transfer RNA; phylogeny tions only or a genuine feature of the species. We sequence native and invasive populations in Europe and North America, and show that all round gobies carry the sequence insertion. By sequencing the tRNA cluster in selected Gobiidae, we show that the tRNA arrangement arose at the root of the Benthophilinae species radiation.

Introduction In this publication, we trace the phylogenetic origin of these two novel features. We test (a) whether the novel tRNA Recently, two novel mitochondrial genome features were arrangement first arose in the round goby, or originated ear- reported in the round goby (Adrian-Kalchhauser et al. 2017). lier in the goby phylogeny, and (b) whether the sequence The round goby is an invasive, small, benthic Ponto-Caspian insertion is a universal feature present in all round gobies fish species. It is native to the Black and Caspian Sea and their tributaries, and belongs to one of three tribes within or an anomaly associated with certain invasive populations the family of Benthophilinae (Neilson and Stepien 2009a). only. To locate the origin of the tRNA re-arrangement, we Many species of this family have colonized rivers and coasts compared existing and newly sequenced tRNA cluster outside the native range in Europe and North America arrangements in 10 gobiid species (5 members of the (Kornis et al. 2012; Roche et al. 2013). However, the round Benthophilinae, and 5 members of other gobiid families). To goby Neogobius melanostomus is the most successful invader determine whether the non-coding insert is a genuine fea- among them (Hirsch et al. 2015). ture of the species Neogobius melanostomus or an anomaly Genetic innovation may be linked to evolutionary success, associated with the Swiss invasive population, we analyzed a and indeed, the round goby mitochondrial genome sequence 7.5 kb region of the mitochondrial genome using Oxford carries two novel features. First, the round goby mitochon- Nanopore long read technology in individuals from the drial genome carries a rearrangement of the tRNA cluster native region and from three globally distributed between the ND1 and ND2 genes. In gobies generally, the invaded sites. cluster contains tRNA Isoleucine (forward orientation), tRNA Glutamine (reverse orientation) and tRNA Methionine (forward orientation), without any spacer sequence. In the round Materials and methods goby, the position of tRNA Isoleucine and tRNA Glutamine Origin of the round goby Gln-Ile-Met tRNA are swapped to yield the sequence Gln (rev) Ile (fw) Met (fw), rearrangment and tRNA genes are separated by up to 42 nucleotides of noncoding spacer sequence. Second, the round goby mito- Representative goby species were chosen for analysis based chondrial genome features a 1250 bp non-coding sequence on previous phylogenetic analyses (Neilson and Stepien insertion downstream of the D-loop region. The insert bears 2009a) to cover major branches of the Benthophilinae and only minimal similarities to D-loop repeats or any known sister groups. Sequences of Ponticola kessleri, Odontobutis sequence, and is flanked on both sides by potentially func- obscura and Gillichthys mirabilis were available at NCBI tional genes for tRNA Phenylalanine. (Sequence accession numbers: NC_025638.1, KT438552.1 and

CONTACT Irene Adrian-Kalchhauser [email protected] University of Basel, Vesalgasse 1, 4051 Basel, Switzerland. ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MITOCHONDRIAL DNA PART B: RESOURCES 409

NC_012906.1, respectively). Sequences of Neogobius fluviatilis, acknowledgements). Samples were shipped as tissue samples Babka gymnotrachelus, Proterorhinus semilunaris, Zosterisessor in ethanol. ophiocephalus and Gobius niger were generated de novo in DNA isolations were done as described above. Then, a 7.5 this study. kb fragment of the mitochondrial genome spanning Tissue samples were obtained from collaborators (see Cytochrome b, the control region, and the region coding for acknowledgements) and shipped in 100% EtOH. DNA was 12S and 16S ribosomal RNA (Figure 2) was amplified using V isolated with standard phenol-chloroform extraction and pre- the LongAmp R Taq2xMastermix (New England Biolabs) with cipitation in the following way: tissues were lysed in ATL lysis forward primer SG082 (CCACCAACCCCAACAATAAG) and buffer from the DNeasy Blood & Tissue kit from QIAGEN reverse primer SG083 (AAGCATAGTCAAGGGGAGGAG) accord- according to the manufacturer’s instruction. For each 25 mg ing to the manufacturer’s instructions. PCR cycling conditions of tissue, 180 mL lysis buffer and 20 mL Proteinase K were were 94 C for 30 sec, followed by 35 cycles of amplification used. After lysis, residual debris was removed by centrifuga- with 94 C for 30 sec, 62.5 C for 1 min, 65 C for 7 min, fol- tion at 4 C for 10 min at 13,000 g in an Eppendorf lowed by an elongation step at 65 C for 10 min and a final Centrifuge 5423R. Then, proteins were removed by two 4 C step. Amplicon size was controlled by agarose gel rounds of phenol-chloroform-isoamylalcohol extraction electrophoresis. (25:24:1; Roth) and one round of chloroform extraction. DNA Then, samples were prepared for long-range sequencing was precipitated by adding 1/10 volume of sodium acetate with MinION technology (Oxford Nanopore). In a first step, (3 M, pH 5.2), and 2.5 volumes of 100% ethanol, incubating PCR reactions were cleaned using a 1:1 ratio of Agencourt m at 80 C for several hours, and centrifuging at 13,000 g for AMPure XP beads (Beckman Coulter) and eluted with 50 L 30 min in an Eppendorf Centrifuge 5423R. DNA pellets were of ddH2O. Then, samples were dA-tailed using the NEBNext washed by centrifugation in the presence of 1 ml of 70% dA-Tailing Module (New England Biolabs) according to the ‘ ’ EtOH for 10 min and then dried and dissolved in 100 mL protocol 1D PCR barcoding (96) genomic DNA (SQK-LSK108) using 45 mL DNA, 7 mL buffer, 3 mL Ultra II End-prep enzyme, ddH2O. DNA concentrations were measured using a TM 5 mL ddH2O, and incubated for 5 min at 20 C and 5 min at NanoDrop 2000 spectrophotometer. A 1850 bp fragment containing the genes for tRNAs Gln, Ile 65 C in a PCR machine. Residual nucleotides were removed and Met (Figure 1) was amplified using primers by cleaning with Agencourt AMPure XP beads (Beckman m fragment1_fw_mtGenome (CCCGATTCCGATATGACCAAC) and Coulter) as above. Half of the beads were eluted with 10 L of the respective barcode adaptor from the PCR 96 barcoding SG015 (CCACAGGTAAAATGGCTGAG) for Babka gymnotrachelus Kit (Oxford Nanopore) (instead of water; to reduce sample and Proterorhinus semilunaris,andprimersSG006 (ATGAGTGCGA volume and save enzyme in the next step), the other half GCCTCCTACC) and SG015 (see above) for Neogobius fluviatilis, was kept as backup. 10 mL of Blunt/TA Ligase Master Mix Proterorhinus semilunaris, Zosterisessor ophiocephalus and Gobius (New England Biolabs) was added and samples were incu- niger. The PCR mix was as follows: 14.4 mLddHO, 2 mL10 2 bated for 30 min at RT. Reactions were cleaned using a 0.4 Buffer þ MgCl ,1.6mL 2.5 mM dNTPs, 1 mLDNA(1–100 ng), 0.2 2 ratio of Agencourt AMPure XP beads (Beckman Coulter) and mLFastStartTaq(Roche)and0.4mL10mMprimereach.PCR eluted with 25 mL of ddH O. cycling conditions were denaturation at 94 C for 4 min, fol- 2 Barcoding-PCR was performed using the lowed by 35 cycles of amplification with 94 C for 30 sec, 50 C VR LongAmp Taq2xMastermix and the corresponding barcoding for 30 sec and 72 C for 2 min, followed by an elongation step at PCR primer. PCR cycling conditions were 95 C for 3 min, fol- 72 C for 10 min, and a final 4 C step. Amplicon size was con- lowed by 22–29 cycles of amplification with 95 C for 15 sec, trolled by agarose gel electrophoresis. 62.5 C for 15 sec, 65 C for 7 min, followed by an elongation PCR products were then cloned for sequencing. About 1 step at 65 C for 10 min and a final 4 C step. Most often, 22 mL of fresh PCR product was directly used in TA-cloning reac- VR cycles yielded sufficient amounts of product. If the band was tions (TOPO TA Cloning kit for sequencing, Thermo Fisher faint, cycle numbers were increased stepwise. m VR Scientific) using 0.5 L TOPO vector and incubation times of For sequencing, 10 mL of each product were combined. A more than 30 min at RT. The TA cloning reactions were trans- 0.4 ratio of Agencourt AMPure XP beads (Beckman Coulter) a formed by heat shock into competent DH5 bacteria pre- was used for clean-up. 45 mL of the combined sample were pared in-house. Candidate colonies were mini-prepped using then used in a dA-tailing reaction as done previously, with 5 the ZR Plasmid Miniprep Classic kit (Zymo Research) and mL internal control DNA provided in the kit added as spike. ’ sequenced at Microsynth with the company s M13F and The reaction was cleaned up using DNA-low binding tubes M13R primers. Sequences were analysed and inspected using and a 1:1 ratio of Agencourt AMPure XP beads (Beckman ApE (A plasmid Editor, freely available at http://jorgensen. Coulter). The sample was then mixed on a thermomixer at 22 biology.utah.edu/wayned/ape/). C for 5 min and eluted using 35 mL of ddH2O. 30 mL (846 ng) of DNA were used in the adaptor ligation m Origin of the large round goby non-coding reaction. 20 L adaptor mix (Ligation sequencing kit 1D, m mitochondrial genome insertion Oxford Nanopore) and 50 L Blunt/TA Master Mix were added and incubated for 30 min at RT. A 0.4 ratio of Round goby samples were obtained from the native region Agencourt AMPure XP beads (Beckman Coulter) was used for and from the invasive range in North America and from clean-up and the library was eluted with 20 mL of ABB buffer the European invasive range from collaborators (see (Ligation sequencing kit 1D, Oxford Nanopore). Loading of 410 S. GUTNIK ET AL.

Ponticola kessleri

Babka gymnotrachelus* *** Proterorhinus semilunaris* ND1 GlnIle Met ND2 10-31nt 22-34nt 22-47nt

* see supplementary information Benthophilinae for spacer sequences

Neogobius melanostomus Neogobius fluviatilis* Zosterisessor ophiocephalus*

Gobius niger*

Odontobutis obscura

ND1 Ile Gln Met ND2

Gillichtys mirabilis

Pomatoschistus minutus tree modified from Neilson & Stepien 2009 * generated de novo in this study

Benthophilinae (ND1, tRNA Gln, tRNA Ile, tRNA Met, ND2) Non-Benthophilinae (ND1, tRNA Ile, tRNA Gln, tRNA Met, ND2)

Neogobius melanostomus ------GCACTAGCCCTAGTTATTTGACATCTGTCCCTCCCCTTAGC Odontobutis obscura CCTGATCTGAAAAAATTTCCTTCCCATCACCCTCACACTCATAATCTGACACCTAGCCCT Neogobius fluviatilis ------ACACTAGCCCTAGTTATTTGACATCTATCCCTCCCCCTGGC Zosterisessor ophiocephalus TCTAATCTGAAAGAGTTTCCTTCCCCTCACCCTCGCCCTGGTGATCTGGCACTTGTCTCT Proterorhinus semilunaris ------ACGATTGCCATAGTTATTTGaCACTTATCACTCCCCCTGGC Gobius niger CTTAATCTGGAAAAACTTTCTTCCTCTCACCCTRGCCATGGTCATTTGACACTTGTCCCT Babka gymnotrachelus ------ACAATTACCCTAGTTATTTGACACCTATCCCTCCCACTCGC Gillichtys mirabilis TCTTATCTGGAAGAACTTTCTCCCCCTCACTCTCTCCCTGGTCATCTGACACTTTTCACT Ponticola kessleri ------ACAATTGCTATGGTTATTTGACACCTATCTCTCCCCCTTGC Pomatoschistus minutus CCTTCTCTGGAAAAGCTTCTTGCCCCTCACGCTGGCCCTGGTTGTCTGACACCTCTCGCT * * * * *********** * ** ***** * ** * **** ** * ** * ** **** ** * * * * ** *** * * **

Neogobius melanostomus CCTCGCTGGACTTCCCCCCCTACTCTAATCCAAA-----ACAAGTCATAATATATGTCCT Odontobutis obscura CCCGATTGCATCCGCAGGGCTCCCCCCGCATCACAACTA-GGG-CAGTGCCCGAA-ACA- Neogobius fluviatilis CTTCGCCGGACTTCCCCCCCAACTTTAACGCGAGTCCAAAAAGCCCTACATTATACTCCT Zosterisessor ophiocephalus CCCGTTGGCTTTCGCCGGTCTCCCCCCGCAGCTATAAG--GGAGCTGTGCCTGAAT-CCG Proterorhinus semilunaris CTTTGCCGGGCTTCCCCCAATCCTGACATAA------ACTTATCTTCC Gobius niger CCCTYTRGCCTTTRRCGGTCTTCCYCCCCARCTTTAACT-GGAGCTGTGCCTGAATA--- Babka gymnotrachelus CTTTGCCGGACTACCCCCATCACACTAA------CTCAAAACAATTGCC Gillichtys mirabilis CCCCCTTGCACTTGCTGGTCTACCCCCCCAACTTTAACACGGAGCCGTGCCTGAATA--- Ponticola kessleri CTTTGCCGGGCTACCACCTCAATGTTAA------AT--AAGGCACTATT Pomatoschistus minutus TCCCCTAGCCTTTGCCGGCCTCCCCCCTCAAATGTAAG--GGAGCTGTGCCTGAA-A--- * * ** ** ** ** ** * * ** * ** ** ** ** ** ** *

Neogobius melanostomus TAGAAAGAAGGGACTTGAACCCTACCTAAAGAGATCAAAACTCTTAGTGCTTCCACTACA Odontobutis obscura AAGGATCACTTTGATAGAGTGAATTATGGGGGTTAAAATCCCCCCTTCTCCTTAGAAAAA Neogobius fluviatilis TAGAAAGAAGGGAATTGAACCCTACCTAAAGAGATCAAAACTCTTAGTGCTTCCACTACA Zosterisessor ophiocephalus AAGGGCCACTTTGATAGAGTGAAACATGGGGGTTAGAGTCCCCCCAACTCCTTAGAAAGA Proterorhinus semilunaris TAGAAAGAAGGGGTTCGAACCCTACCTCAAGAGATCAAAACTCTTAGTGCTTCCACTACA Gobius niger AAGGGCCACTTTGATAGAGTGAACCATGGGGGTTAAACTCCCCCCAGCTCCTTAGAAAGA Babka gymnotrachelus TAGAAAGAAGGGATTTGAACCCTACCTTAAGAGATCAAAACTCTTGGTGCTTCCACTACA Gillichtys mirabilis AAGGGCCACTTTGATAGAGTGACTAATGGGGGTTAAAATCCCCCCGCCTCCTTAGAAAGA Ponticola kessleri TAGAAAGAAGGGATTTGAACCCAACCTTAAGAGATCAAAACTCTTGGTGCTTCCACTACA Pomatoschistus minutus AAGGGCCACTTTGATAGAGTGACTTATGAGGGTTAAAGCCCCTCCAACTCCTTAGAAAGA ************ * ****** **** ***************** ************** **** **************** *** ****** * *** ** *********** *

Neogobius melanostomus CCACTTCCTATTATTTGTACCCTTAAAGGCCCATATCGAGCCAAGGAGTTGTGCCTGAAC Odontobutis obscura AGGGACTCGAACCCTACCTGAAGAGCTCAAAACTCTTAGTGCTTCCACTACACCACTTCC Neogobius fluviatilis CCACTTCCTATACCT-ATACCCTTAGGACCTCACATCAGACCAAGGAGTTGTGCCTGAAC Zosterisessor ophiocephalus AGGGACTCGAACCCTACCTTAAGAGATCAAAACTCTTGGTGCTTCCACTACACCACTTCC Proterorhinus semilunaris CCACTTCCTACATCCTAA----GTTT------ACCTAGCTCTGGGGCTGTGCCTGAAC Gobius niger AGGGRTTCGAACCCTACCTTAAGAGATCAAAACTCTTGGTGCTTCCRCTACACCACTTCC Babka gymnotrachelus CCACTTCCTAGTGTAAATAT--TTTA------GGACTATCCTGGAGCTGTGCCTGAAC Gillichtys mirabilis AGGGACTTGAACCCTACCTGAAGAGATCAAAACTCTTAGTGCTTCCACTACACCACTTCC Ponticola kessleri CCACTTACTAGCATAATAAATTTTTA------GATTACTTCTGGAGCTGTGCCTGAAC Pomatoschistus minutus AGGGACTCGAACCCTACCTGAAGAGATCAAAACTCTTAGTGCTTCCACTACACCACTTCC ****** *** * ** * *********** **** * *********** ***** *********** ******** *************

Neogobius melanostomus CAAAGGGCCACTTTGATAGGGTGAATCATGGGGGTTAGAGTCCCCCCAACTCCTGCACGA Odontobutis obscura TAGTAAAGTCAGCTAAACAAGCTTTTGGGCCCATACCCCAAATATGTTGGTTAAATTCCC Neogobius fluviatilis CAAAGGGCCACTTTGATAGAGTGAATTATAGGGGTTAAAGCCCCCTCAACTCCTATACAC Zosterisessor ophiocephalus TAGTAGAATCAGCTAAATAAGCTTTTGGGCCCATACCCCAAACATGTTGGTTAAAGTCCT Proterorhinus semilunaris TAAAGGGCCACTT TGATAGAGTGAACTATGGGGGTTAAAGTCCCCTCATCCCCTAGAACA Gobius niger TAGTAAGGTCAGCTAAATAAGCTTTTGGGCCCATACCCCAAACATGTCGGTTAAAGTCCT Babka gymnotrachelus CAAAGGGCCACTTTGATAGAGTGA ATTATGAGGGTTAGAGTCCCCCCAACTCCTATAAAA Gillichtys mirabilis TAGTAAAGTCAGCTAAACAAGCTTTTGGGCCCATACCCCAAACATGTTGGTTAAACCCCC Ponticola kessleri CAAAGGGCCACTTTGATAGAGTGAAC TATAGGGGTTAAAGCCCCCTCAACTCCTGTGCAT Pomatoschistus minutus TAGTAAGGTCAGCTAAAAAAGCTTTTGGGCCCATACCCCAAACATGTTGGTTAAACCCCT ****************** ***** ** ****** ** **** ** * *** ***** ********* ************************ **** ******* **

Neogobius melanostomus AAACATAACAAATCAGTAGGACTAAACACCACACCTTGCTTAGTAAGGTCAGCTAA-AAT Odontobutis obscura TCCTTTACTTATGAGCCCCTACATCTACATTATACTACTATTTTCCCTAGGCCTAGGCAC Neogobius fluviatilis AAACTGACA------CGAAATATTTTACACTTTACTGAGTAAGGTCAGCTAA-AAT Zosterisessor ophiocephalus TCTTCCACTAATGAGCCCCTACATTTTACCCCTCTTTTTCTCGGCTTTAGGCCTAGGGAC Proterorhinus semilunaris TCTATTAT----TATAG------GTCGCCCATAGTAAGGTCAGCTAACGTT Gobius niger TCCTTTACTAATGAACCCCTATATTGTACCACTTTTTTTYTCAGCACTGGGMYTGGGYAC Babka gymnotrachelus TGCCAAATCATCTTTGA------CCTCCCGACAGTAAGGTCAGCTAATGTC Gillichtys mirabilis TCCTTTACTAATGCACCCCTACACACTTACCCTCCTTTTCTTTGGCCTTGTTCTAGGCAC Ponticola kessleri A------AGTATA------ATTCCCTATAGTAAGGTCAGCTAATGTT Pomatoschistus minutus TCCTTTGCTAATGAACCCGTACATTTTAGCCCTCCTACTGTTTGGCCTGCTGCTGGGCAC *************** ** * ** *** *** ** * * * * * * * ** **

Neogobius melanostomus AAGCTTTTGGGCCCATGCCCCAAGCATGCCGGTTAAAATCCAGCCTTTACTAGTGAACCC Neogobius fluviatilis AAGCTTTTGGGCCCATACCCCAAACATGCCGGTTAAATTCCCGCCTTTACTAATGAGCCC Proterorhinus semilunaris AAGCTTTTGGGCCCATA CCCCAAACATGCAGGTTAAACTCCCGCCTTTACTAATGAACCC Babka gymnotrachelus AAGCTTTTGGGCCCATACC CCAAATATGCCGGTTAAATTCCCGCCTTTACTAATGAACCC Ponticola kessleri AAGCTTTTGGGCCCATACCCCAAACATGCCGGTTAAATTCCCGCCTTTACTAATGAGCCC **************** ****** **** ******* *** ********** *** ***

Neogobius melanostomus CTACGTTATATTCCTATTTTTGTCCGCCCTAG------Neogobius fluviatilis CTACATTATATTTTTATTTTTATCCGCCCTGG------Proterorhinus semilunaris TTACGTTATTTCATTATTTTTATTTACTCTAG------Babka gymnotrachelus TTATATTATTTCATTATTTTTATGTACACTAG------Ponticola kessleri TTATATTATTTCATTATTTTTGTTTACACTAG------** **** * ******* * * ** * Figure 1. Top: Origin of the re-arranged tRNA cluster Gln, Ile, Met. Most Gobiidae carry the arrangement Ile, Gln, Met without spacers. Benthophilinae however carry the arrangement Gln, Ile, Met, and feature variable length spacers between the genes. Bottom: Alignment of Benthophilinae tRNA sequences. Spacer sequences vary between species, but display more similarities among Neogobius species (Neogobius melanostomus, Neogobius fluviatilis) and Ponticola species (Proterorhinus semilunaris, Babka gymontrachelus, Ponticola kessleri), respectively. MITOCHONDRIAL DNA PART B: RESOURCES 411

(A) North America Europe N

Ba S

Rh St Bl

St Rh Bl St. Lawrence River at Montreal River Rhine at Basel Black Sea at Odessa (native region) Ba Baltic Sea at Szczecin

(B)

Cyt B D-loop Insert 12S 16S

SNP SNP density (bandwidth: 500bp) tRNA non-coding insert reported in Adrian-Kalchhauser et al, 2016 Figure 2. Global occurrence of the novel sequence insertion. (A) Map of sampling sites in North America (St. Lawrence river) and Europe (Rhine river, Baltic Sea, and native region Black Sea). (B) Density of single nucleotide polymorphisms observed in the novel round goby non-coding insert, the adjacent D-loop, and adjacent expressed coding and non-coding sequences.

the MinION device was done as described by the 1D PCR obscura, Gillichtys mirabilits and Pomatoschistus minutus) and barcoding (96) genomic DNA protocol (Oxford Nanopore). found that all analysed Benthophilinae species share the re- Sequencing (flowcell FLO-MIN106) was performed at the arranged organisation of the tRNA cluster. They feature the Genetic Diversity Centre Zurich. ONT Albacore Sequencing arrangement Gln (rev), Ile (fw), Met (fw) (Figure 1), and dis- Pipeline Software (version 2.1.7) was use to basecall, demulti- play non-conserved spacers of 10–47 nucleotides between plex and aligned the fast5 files. NanoPack (De Coster et al. the tRNA genes (Figure 1). All non-Benthophilinae species on 2018) was used to create statistical summaries. We used sam- the other hand feature the arrangement Ile (fw), Gln (rev), tools (V1.8, https://github.com/samtools/samtools) to com- Met (fw) without spacers (Figure 1). bine sam files of the same barcode, convert the sam files to This change in gene arrangement yields further evidence bam files, and sort the bam files. The bam files were for an evolutionary radiation at the root of the Benthophilinae inspected and consensus sequences extracted with IGV (Neilson and Stepien 2009a). The spacer sequences display (Robinson et al. 2011; Thorvaldsdottir et al. 2013). substantial divergence between species (Figure 1). Since spacer sequences are more similar among the two Neogobius species and among the three Proterorhinus, Babka and Results and discussion Ponticola species,respectively, they support the previously We compared the Ile-Glu-Met tRNA cluster in five observed division of Benthophilinae into a Neogobius and a Benthophilinae (Ponticola kessleri, Babka gymnotrachelus, Ponticola lineage (Neilson and Stepien 2009a;Medvedevetal. Proterorhinus semilunaris, Neogobius melanostomus and 2013). The spacer sequences appear to evolve quite freely and Neogobius fluviatilis) and in five non-Benthophilinae goby may be useful to (1) clarify currently debated relationships species (Zosterisessor ophicephalus, Gobius niger, Odontobutis among Ponticola species (Neilson and Stepien 2009a; 412 S. GUTNIK ET AL.

Medvedev et al. 2013) and to (2) confirm the identity of puta- Funding tive cryptic tubenose goby species (Neilson and Irene Adrian-Kalchhauser is a Marie Heim Vogtlin€ research fellow of the Stepien 2009b). Swiss National Research Foundation. The Genetic Diversity Centre Zurich Using long read technology, we investigated the origin of partially sponsered the sequencing. a recently detected 1250 bp sequence insertion in the round goby mitochondrial genome (Adrian-Kalchhauser et al. 2017), and found that individuals from across the native and inva- Author contributions sive range carry the insert (Figure 2a). Accordingly, the insert IAK and SG designed research, IAK and SG organized samples, SG per- did not arise recently during the invasion, and was not main- formed lab experiments, JCW, SG and IAK analysed data and wrote tained by drift, but rather constitutes a genuine feature of the manuscript. the Neogobius melanostomus mitochondrial genome. This incites questions on its potential functional significance and Permissions potential reasons for retention in an otherwise economized mitochondrial genome. Expression analyses and replication Fish used in this work were caught in accordance with permission 2-3-6- assays may reveal whether the sequence plays a role in tran- 4-1 from the Cantonal Office for Environment and Energy, Basel Stadt. scription or replication of the round goby mitochondrial genome. References To understand whether the inserted sequence experiences mutation restraints, we quantified the number of single Adrian-Kalchhauser I, Svensson O, Kutschera VE, Alm Rosenblad M, Pippel M, Winkler S, Schloissnig S, Blomberg A, Burkhardt-Holm P. nucleotide polymorphisms (SNPs) detected between the four 2017. The mitochondrial genome sequences of the round goby and analysed mitochondrial genomes. We find fewer SNPs within the sand goby reveal patterns of recent evolution in gobiid fish. BMC expressed sequences (Cytochrome B, 12S and 16S) than in Genomics. 18:177. the D-loop and the sequence insertion (Figure 2b). De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. Accordingly, a function of the insert that requires sequence NanoPack: visualizing and processing long-read sequencing data . – conservation is unlikely. We find that SNPs accumulate in the Bioinformatics. 34:2666 2669. Hirsch PE, N’Guyen A, Adrian-Kalchhauser I, Burkhardt-Holm P. 2015. D-loop following the TAS sites (Adrian-Kalchhauser et al. What do we really know about the impacts of one of the 100 worst 2017), indicating the presence of a hypervariable region in invaders in Europe? A reality check. Ambio. 45(3):267–279. the round goby control region similar to human Kornis MS, Mercado-Silva N, Vander Zanden MJ. 2012. Twenty years of Hypervariable Segments I and II (Stoneking 2000). invasion: a review of round goby Neogobius melanostomus biology, – Hypervariable sites are potentially useful markers for stud- spread and ecological implications. J Fish Biol. 80:235 285. Medvedev DA, Sorokin PA, Vasil’ev VP, Chernova NV, Vasil’eva ED. 2013. ies focusing on short time evolutionary frames. Mutation Reconstruction of phylogenetic relations of Ponto-Caspian gobies rates in human hypervariable segments are four to six times (Gobiidae, Perciformes) based on mitochondrial genome variation and higher than at average positions (Meyer et al. 1999). The some problems of their taxonomy. J Ichthyol. 53:702–712. round goby hypervariable regions may therefore be a useful Meyer S, Weiss G, von Haeseler A. 1999. Pattern of nucleotide substitu- source of haplotypes in future phylogenetic studies. tion and rate heterogeneity in the hypervariable regions I and II of human mtDNA. Genetics. 152:1103–1110. Previously, the region has been challenging to sequence due Neilson ME, Stepien CA. 2009a. Escape from the Ponto-Caspian: evolution to its highly repetitive nature. Our long-read approach cir- and biogeography of an endemic goby species flock (Benthophilinae: cumvents this issue and highlights the power of single mol- Gobiidae: Teleostei). Mol Phylogenet Evol. 52:84–102. ecule long-read sequencing for short-term mitochondrial Neilson ME, Stepien CA. 2009b. Evolution and phylogeography of the phylogenies and for biogeographical analyses. tubenose goby genus Proterorhinus (Gobiidae: Teleostei): evidence for new cryptic species. Biol J Linnean Soc. 96:664–684. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz Acknowledgements G, Mesirov JP. 2011. Integrative genomics viewer. Nat Biotechnol. 29: 24–26. We are grateful to Yuriy Kvach, Mariella Rasotto, Alexander Cerwenka, Roche KF, Janac M, Jurajda P. 2013. A review of Gobiid expansion along Przemysław Czerniejewski, and Pierre Magnan for providing tissue sam- the Danube-Rhine corridor - geopolitical change as a driver for inva- ples and to Silvia Kobel for technical support. sion. Knowl Manag Aquat Ecosyst. 411:01. Stoneking M. 2000. Hypervariable sites in the mtDNA control region are mutational hotspots. Am J Human Genet. 67:1029–1032. Disclosure statement Thorvaldsdottir H, Robinson JT, Mesirov JP. 2013. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and The authors have no conflict of interest to declare. exploration . Brief Bioinform. 14:178–192.