Preliminary results on the molecular study of fish-eating by ‘trawling Myotis ’ bat species in Europe

Preliminary results on the molecular study of fish-eating by ‘trawling Myotis ’ bat species in Europe

Robert S. Sommer

*, Michael Hofreiter

, Frauke Krüger

, Björn M. Siemers

, Johanna L. A. Paijmans

, Chenhong Li

& Matthias F. Geiger

1 Institute of Biosciences, University of Rostock, Universitätsplatz 2, 18055 Rostock, Germany — ² Institute of Biochemistry and Biology, Karl-Liebknecht-Str. 24 – 25, 14476 Potsdam, Germany — ³ Hamann and Schulte Environmental Planning GbR, Koloniestraße 16, 45897 Gel senkirchen, Germany — ⁴ Max Planck Institute of Ornithology, 82319 Seewiesen, Germany — ⁵ College of Fisheries and Life Science, Shang hai Ocean University, Shanghai, China — ⁶ Zoological Research Museum Alexander Koenig, Adenauerallee 160, 53113 Bonn, Ger- many — ⁷ deceased on 23^th of May 2012 — *Corresponding author: robert.sommer@uni-rostock.de

Submitted March 19, 2018.

Accepted December 5, 2018.

Published online at www.senckenberg.de/vertebrate-zoology on February 15, 2019.

Published in print on February 27, 2019.

Editor in charge: Clara Stefen

Abstract

Piscivory in vespertilionid bat species in the genus MyotisDQGWUDZOLQJDVW\SLFDOHOHPHQWRIEHKDYLRXULQWKHKXQWLQJÀLJKWDERYHWKHZDWHU VXUIDFHDUHZLGHO\DFNQRZOHGJHG+RZHYHUDPRQJWKH(XURSHDQEDWIDXQDRQO\ORQJ¿QJHUHGEDWVMyotis capaccinii) are known to con- VXPH¿VK,Q'DXEHQWRQ¶VEDWMyotis daubentoniiDVZHOODVWKHSRQGEDWMyotis dasycneme), both also members of the guild of European

‘trawling Myotis¶VSHFLHVSLVFLYRU\LVSRVVLEOHEXWKDVQRW\HWEHHQVXI¿FLHQWO\UHFRUGHGE\PRUSKRORJLFDODQDO\VHVRISUH\LQIDHFDOSHO- OHWV3UHYLRXVH[SHULPHQWDOVWXGLHVKDYHVKRZQWKDW¿VKUHPDLQVDUHGLI¿FXOWWRGHWHFWLQIDHFDOSHOOHWVRIM. daubentonii; thus, piscivory PD\QRWDOZD\VEHUHOLDEO\GHWHFWHGE\PRUSKRORJLFDOLGHQWL¿FDWLRQRISUH\UHPDLQV:HDQDO\VHGWKH'1$RIIDHFDOVDPSOHVRIDOOWKUHH trawling MyotisVSHFLHVIURPGLIIHUHQWUHJLRQVRI(XURSHE\3&5DPSOL¿FDWLRQDQG6DQJHUVHTXHQFLQJRIWKHPROHFXODUPDUNHUV651$

DQGWKH'1$EDUFRGHUHJLRQRIWKHF\WRFKURPHR[LGDVHVXEXQLW,&2,IRUGHWHFWLRQRISRVVLEOH¿VK'1$DQGWD[RQRPLFLGHQWL¿FDWLRQ RIVSHFLHV,QRQRIWKHVDPSOHVZHZHUHDEOHWRDPSOLI\¿VK'1$EDVHGRQ&2,LQIDHFHVRIM. capaccinii and M. daubentonii from

%XOJDULDDQGLQXVLQJWKH651$ORFXVIURPM. dasycneme faeces from Germany. Based on technical problems of our molecular DSSURDFKRXUGDWDGRQRWUHYHDODGH¿QLWLYHDQGUHOLDEOHPROHFXODUUHFRUGRIGLHWGHULYHG¿VK'1$LQWKHIDHFDOSHOOHWVRIWKHLQYHVWLJDWHG trawling Myotis species nor proof for piscivory in M. dasycneme and M. daubentonii2XUUHVXOWVPD\EHDVWLPXODWLRQDQGGUDIWIRURWKHU UHVHDUFKHUVWRWHVWWKHGHWHFWLRQRIGLHWGHULYHG¿VK'1$LQWUDZOLQJMyotis with improved molecular genetics approaches.

Key words

Feeding ecology, hunting behaviour, Myotis, piscivory, trawling bat species.

Introduction

Three species of the guild of ‘trawling Myotis¶ VSHFLHV DUH NQRZQ IURP (XURSH WKH ORQJ¿QJHUHG EDW Myo- tis capaccinii 'DXEHQWRQ¶V EDW Myotis daubentonii) DQG WKH SRQG EDW Myotis dasycneme). Through several experiments it was documented that all three mentioned (XURSHDQVSHFLHVXVHXQLTXHIRUDJLQJWHFKQLTXHVGXULQJ KXQWLQJÀLJKWDERYHWKHZDWHUVXUIDFHWKH\XVHWKHLUIHHW and tail membrane to catch insects, such as arthropods, ZKLFKÀRDWRQRUÀ\DERYHWKHZDWHUK^ALKO & S^CHNITZ-

LERS^IEMERS et alDS^IEMERS & S^CHNITZLER,

$GGLWLRQDOO\DOOWKUHHKDYHUHODWLYHO\ODUJHIHHWLQ proportion to their body dimensions. They share this mor- phological feature with other trawling bats, among others with N. leporinus. Recent studies on the feeding ecology of M. daubentonii and M. dasycneme in Europe, which were based on morphological and molecular analyses of the bat guano, show that both species feed on insects and, WRDPXFKOHVVHUGHJUHHRQVSLGHUVEXWQRVLJQVRI¿VK remains could be detected in the guano of the two spe- FLHVCIECHANOWSKI & Z^APARTK^RÜGER et al

N^ISSEN et al,QFRQWUDVWWRWKHVHWZRVSH- FLHV WKH ORQJ¿QJHUHG EDWM. capaccinii, distributed in Mediterranean regions of Europe is known to, in addition WR LQVHFWLYRU\ DOVR H[KLELW SLVFLYRU\ A^IHARTZA et al., ; L^EVIN et al B^ISCARDI et al5HFHQW work has also provided insight into how M. capaccinii discern prey and adjust their movements during trawling ZKHQDWWDFNLQJ¿VKA^IZPURUA et al

The similar hunting behaviour of trawling Myotis spe- FLHVDQGWKHLUWLJKWFRQQHFWLRQWRDTXDWLFHFRV\VWHPVOHG to the assumption that piscivory could be possible also DPRQJ'DXEHQWRQ¶VEDWDQGWKHSRQGEDWLQ&HQWUDODQG QRUWKHUQ(XURSH$VVKRZQE\H[SHULPHQWVS^IEMERS et alE'DXEHQWRQ¶VEDWLVDEOHWRFDWFKVPDOO¿VKHV XQGHUODERUDWRU\FRQGLWLRQVZKHUHLWJUDVSHG¿VKHVZLWK LWVIHHW7KH¿VKZDVWDNHQGLUHFWO\IURPWKHFODZLQWRWKH PRXWKDQGZDVHDWHQS^IEMERS et alE:HNQRZ from the experiments of S^IEMERS et alDWKDWDIWHU a specimen of M. daubentonii had consumed a lot of in- VHFWVDQGDSSUR[LPDWHO\VPDOO¿VKGXULQJDIRXUZHHN period, only insect remains were found in the guano, but RQO\WZR¿VKVFDOHVDQGRQHERQHUHPDLQHG%HFDXVHWKH UHPDLQV RI WKH VPDOO ¿VK DUH VR GLI¿FXOW WR LGHQWLI\ LQ faeces, probably because of demineralisation in the stom- DFKWKHODWH%M|UQ6LHPHUVFRQFOXGHGWKDWSLVFLYRU\ZLOO EHYHU\GLI¿FXOWWRDVVHVVTXDQWLWDWLYHO\LQWKH¿HOGIRU the European trawling MyotisS^IEMERS et alD,Q contrast to that, B^ROSSET & D^ELMAREFODLPHGWR KDYHIRXQG¿VKUHPDLQVLQWKHJXDQRRIM. daubentonii in France several decades ago.

$QDOWHUQDWLYHWRPRUSKRORJLFDODQDO\VHVRIIDHFHVIRU GLHWDU\VWXGLHVXVHV'1$UHPDLQVIURPSUH\LWHPVLQWKH IDHFHVHJP^OMPANON et alK^RÜGER et al This method allows the detection of dietary elements that DUHGLI¿FXOWWRLGHQWLI\E\PRUSKRORJLFDOIHDWXUHV+HUH we used a molecular genetic approach to guano samples of M. dasycneme, M. capaccinii and M. daubentonii in RUGHUWRGHWHFW¿VK'1$DQGXOWLPDWHO\WHVWRXUK\SRWK- esis that M. daubentonii and M. dasycneme also prey on

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Material and Methods

Fieldwork and sample selection

'XULQJ 0D\$XJXVW LQ DQG M. capaccinii, M. daubentonii and M. dasycneme were caught at differ- ent localities using mist nets placed over the water sur- IDFHVXSSOHPHQWDU\WDEOH6%DWLQGLYLGXDOVZHUHNHSW separately in soft cotton bags for a maximum of one hour and released after taking measurements and collecting IDHFDOVDPSOHVFIK^RÜGER et al7KHVHVDPSOHV contain “individual samples” in M. capaccinii ± pellets of one bat individual) and “mixed samples” from M. daubentonii ±SHOOHWVRIWZRLQGLYLGXDOVIURP Bulgaria and “mixed samples” from M. dasycneme

SHOOHWVRIWZRLQGLYLGXDOVIURPQRUWKHUQ*HUPDQ\6XS- SOHPHQWDU\ ,QIRUPDWLRQ 7DE 6 )URP QRUWKHUQ *HU- many we used also samples from nursing colonies of M. dasycneme JHQHUDOO\ WHQ IDHFDO SHOOHWV SHU VDPSOH IURP WZR GLIIHUHQW ORFDOLWLHV LQ DQG DQ XQNQRZQ QXPEHU RI FRQWULEXWLQJ DQLPDOV 6DPSOHV ZHUH HLWKHU VWRUHGGULHGDQGIUR]HQRULQHWKDQRODWURRPWHP- SHUDWXUHIXUWKHULQIRUPDWLRQVVXSSOHPHQWDU\LQIRUPD- WLRQ7DE6

Laboratory procedures

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±DQG±VXSSOHPHQWDU\LQIRUPDWLRQ7DE6 in dedicated cleanroom facilities at the University of York, where no previous work was performed on either EDWVRU¿VKIRUPHUODERUDWRU\RI0+RIUHLWHU8QLYHUVLW\

RI<RUN$IWHUSURFHVVLQJHDFKVDPSOHVHULHVZHFOHDQHG WKHEHQFKDQGDOOVXUURXQGLQJODERUDWRU\HTXLSPHQWDQG irradiated the whole cleanroom with UV light for at least KRXUV WR DYRLG SRVVLEOH FURVVFRQWDPLQDWLRQ EHWZHHQ WKH VXEVHTXHQW VDPSOH VHULHV :H H[WUDFWHG '1$ IURP SHOOHW VDPSOHV XVLQJ WKH 4,$DPS '1$ 6WRRO 0LQL .LW 4LDJHQ 8. DFFRUGLQJ WR WKH PDQXIDFWXUHU¶V LQVWUXF- WLRQV 4,$DPS '1$ VWRRO KDQGERRN ^th HGLWLRQ$SULO ,QRUGHUWRGHWHFWSRVVLEOHFRQWDPLQDWLRQGXULQJ '1$LVRODWLRQZHLQFOXGHGDEODQNVDPSOH,' :HFKRVHWRDQDO\VHWKHIDHFDOVDPSOHVIRUWKHSUHV- ence of two different molecular markers: the relatively FRQVHUYHG651$DQGWKH'1$EDUFRGHUHJLRQRIF\- WRFKURPHR[LGDVHVXEXQLW,&2,ZKLFKXVXDOO\DOORZV IRU KLJKHU WD[RQRPLF UHVROXWLRQ V^ENCES et al 3ULPHUV ZHUH GHVLJQHG EDVHG RQ DYDLODEOH '1$ VH- TXHQFHVRIWKH6DQG&2,UHJLRQVRIGLIIHUHQWFRPPRQ (XURSHDQ¿VKVSHFLHVRutilus rutilus, Scardinius eryth- URSKWKDOPXV$EUDPLVEUDPD3HUFDÀXYLDWLOLV&\SULQXV FDUSLR$OEXUQXVDOEXUQXV7LQFDWLQFD&DUDVVLXVFDUDV- sius, Esox lucius, Gasterosteus aculeatus, Thymallus thy- mallus, and Salmo trutta). For primer design we chose WKHFRPPRQ¿VKIRXQGLQ(XURSHEXWWKH\DOVRFRYHUHG a wide taxonomic range, from salmonids to cyprinids and percids. From our experience, primers designed on a wide taxonomic range of species should amplify other species not used for primer design. Even though, we still PD\PLVVVRPH¿VK'1$WDUJHWLIWKH\DUHQRWLQWKHVSH- cies we used for primer design and they have mutations LQWKHSULPLQJVLWHV6FUHHQLQJIRUVXLWDEOHPLQLEDUFRGH UHJLRQV FDESV ZLWK FRQVHUYHG ÀDQNLQJ SULPHU binding sites was performed manually from the resulting PXOWLSOHVHTXHQFHDOLJQPHQW:KHUHQHFHVVDU\GHJHQHU- DWHVLWHVZHUHLQFOXGHGLQWKHSULPHUVHTXHQFHWRLQFUHDVH DPSOL¿FDWLRQXQLYHUVDOLW\

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Validation of the designed mini-barcodes

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PCR products of all reactions showing a clear band of H[SHFWHGSURGXFWVL]HRQDQDJDURVHJHOVXSSOHPHQWDU\

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Results and Discussion

Sequencing results

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¿VKVSHFLHVEXWZDVDVVLJQHGWRDQRQWDUJHWJURXSLH mammals: BoVKLWV HomR2YLVDQGLQWKUHH FDVHVEDW'1$UHPDLQVZKLFKZHUHDVVLJQHGWRWKH Myo- tis species, M. emarginatus and M. capaccinii 7DE Except for the latter, these species are very common con- taminants, often present at low concentrations in reagents DQGSODVWLFZDUHFIL^EONARD et alRU±LQWKHFDVH RIKXPDQ'1$±FDQEHLQWURGXFHGGXULQJH[SHULPHQWV GHVSLWHDOOSUHFDXWLRQVWDNHQ'XHWRRXUKLJKO\VHQVLWLYH 3&5VHWXSZLWKF\FOHVDOVRVPDOOHVWWUDFHDPRXQWV RI'1$FDQVHUYHDVWHPSODWHVDQGOHDGWRDPSOLFRQVWKDW FDQEHVXFFHVVIXOO\VHTXHQFHG$SSURDFKHVWRDYRLGWKLV W\SH RI FRQWDPLQDWLRQ KDYH EHHQ GHYHORSHG C^HAMPLOT et alEXWGXHWRWKHSLORWQDWXUHRIRXUVWXG\±ZH ZDQWHGWRLQYHVWLJDWHLI¿VK'1$QRUPDOO\QRFRPPRQ FRQWDPLQDQWZDVSUHVHQWDWDOOLQWKHVDPSOHV±ZHGLG not employ these approaches.

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Fish species identification by DNA bar- coding and biogeographical context

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ID species origin number of positive PCR products and used primers

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ȝO ȝO ȝO ȝO

M. capaccinii Bulgaria Ƒ Ƒ Ƒ Ƒ

2 M. capaccinii Bulgaria Ƒ Ƒ Ƒ Ƒ

M. capaccinii Bulgaria Ƒ Ƒ Ƒ Ƒ

M. capaccinii Bulgaria Ŷ Ŷ Ƒ Ƒ M. capaccinii

M. capaccinii Bulgaria Ŷ Ŷ Ŷ Ŷ × M. capaccinii Squalius sp.

M. capaccinii Bulgaria Ŷ Ŷ Ƒ Ƒ H. sapiens

M. capaccinii Bulgaria Ŷ Ŷ Ƒ Ŷ M. emarginatus

8 M. capaccinii Bulgaria Ŷ Ŷ Ƒ Ŷ × H. sapiens Gobio sp.

M. daubentonii Bulgaria Ŷ Ŷ Ƒ Ŷ × H. sapiens &RWWXVsp.

M. daubentonii Bulgaria Ƒ Ŷ Ƒ Ƒ H. sapiens,

B. taurus

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ŷ Ŷ Ƒ Ƒ H. sapiens

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ŷ Ƒ Ƒ H. sapiens

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ŷ Ƒ Ƒ H. sapiens

M. dasycneme Germany Ŷ Ŷ Ƒ Ƒ H. sapiens

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ŷ Ƒ Ƒ H. sapiens

M. dasycneme Germany Ƒ Ŷ Ƒ Ƒ —

22 M. dasycneme Germany Ƒ Ŷ Ƒ Ƒ —

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ŷ Ŷ Ƒ Ƒ ×

Phoxinus phoxinus, H. sapiens

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

28 M. dasycneme Germany Ŷ Ŷ Ŷ Ŷ H. sapiens,

B. taurus —

M. dasycneme Germany Ƒ Ŷ Ŷ Ŷ B. taurus —

M. dasycneme Germany Ŷ Ŷ Ŷ Ŷ B. taurus —

M. dasycneme Germany Ŷ Ŷ Ŷ Ŷ B. taurus —

M. dasycneme Germany Ŷ Ŷ Ŷ Ŷ B. taurus B. taurus

M. dasycneme Germany Ƒ Ƒ Ŷ Ŷ H. sapiens —

M. dasycneme Germany Ŷ Ƒ Ŷ Ŷ B. taurus B. taurus

^blank Ƒ Ƒ Ƒ Ƒ — —

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ — —

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ — —

M. dasycneme Germany Ƒ Ŷ Ƒ Ŷ × 2YLVDULHV Leuciscus sp.

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ — —

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ — —

M. dasycneme Germany Ƒ Ŷ Ƒ Ƒ × Barbatula

barbatula —

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

M. dasycneme Germany Ƒ Ƒ Ƒ Ƒ

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7KHVHFRQG¿VK'1$SRVLWLYHPDWFKZDVUHFRYHUHG IURP D VLQJOH EDW ,' DM. capaccinii from northern Bulgaria, s. Tab. 2) and could be unambiguously assigned to the gudgeon species complex GobioVSSZLWK VHTXHQFH LGHQWLW\ 7KH '1$ EDUFRGH EDVHG PROHFXODU WD[RQRPLFXQLW%,1%2/'$$&FRQWDLQVRYHU specimens of different Gobio species from throughout

&HQWUDO(XURSHZLWKXSWRSGLVWDQFHXQFRUUHFW- ed genetic distances) between them, and the taxonomic UHVROXWLRQRIWKHVPDOO&2,IUDJPHQWREWDLQHGGRHVQRW allow an assignment to species level with high probabil- LW\$ PDQXDO FRPSDULVRQ WR '1$ EDUFRGHV EHORQJLQJ to the geographical closely occurring *RELRNRYDWVFKHYL

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LWHP$VWKHUHDUHVHYHUDORWKHUGobio lineages occurring in neighbouring Romania and other Balkan countries, the TXHVWLRQ ZKLFK VSHFLHV RI JXGJHRQ ZDV SUREDEO\ FRQ- sumed cannot be answered.

7KHWKLUGLQFLGHQFHRISRVLWLYH¿VK'1$IDHFDOSHO- lets occurred in a mixed sample from two M. dauben- tonii%XOJDULDV7DE DQGLQGLFDWHVWKHSUHVHQFHRI EXOOKHDG&RWWXVVSUHPDLQVLQWKHIDHFHV$SDUWLFXODU

&RWWXV VSHFLHV FDQQRW EH LGHQWL¿HG EDVHG RQ WKH VKRUW

&2,IUDJPHQWZKLFKLVDOVRGXHWRDJHQHUDOSUREOHPLQ GHOLQHDWLQJ VFXOSLQ VSHFLHV ZLWK '1$ EDUFRGHV ZKHUH it has been shown that several species share the same KDSORW\SHV G^EIGER et al KNEBELSBERGER et al.,

2IWKHWKUHHSRVLWLYHO\WHVWHG*HUPDQVDPSOHVVWHP- ming from mixed pellets of nursing colonies of M. da- sycneme RQH ,' FRXOG EH DVVLJQHG WR D PLQQRZ SUREDEO\ WKH FRPPRQ PLQQRZ Phoxinus phoxinus) E\ FRPSDULQJ WKH REWDLQHG VKRUW 6 IUDJPHQW ZLWK GDWD IURP 1&%, *HQ%DQN VHTXHQFH LGHQWLW\ YLD D %/$671 VHDUFK7KH VHFRQG IDHFDO VDPSOH ,' EHORQJLQJ WR WKH :LVPDU0JJHQEXUJ QXUVLQJ FRORQ\

WKDWZDVIRXQGWRFRQWDLQ'1$UHPDLQVRI¿VKLQGLFDWHV WKH SRWHQWLDO LQJHVWLRQ RI GDFH Leuciscus sp.), as de- PRQVWUDWHG E\ WKH 6 VHTXHQFH IUDJPHQW VLPLODULW\ RI WR'1$VHTXHQFHVLQ*HQ%DQN7KHH[DFWVSHFLHV L. idus or L. leuciscus) cannot be determined unambigu- ously, also because both species have exchanged mito- FKRQGULDOVHTXHQFHVRYHUODUJHDUHDVRIWKHLUGLVWULEXWLRQ C^OSTEDOAT et al)LQDOO\VDPSOH,'IURPWKH VDPHQXUVLQJFRORQ\DVWKHIRUPHUM. dasycneme) was SRVLWLYHO\WHVWHGIRUWKHSUHVHQFHRI6'1$IUDJPHQWV DVVLJQHGWRDQHPDFKHLOLGORDFK%/$671LGHQWLW\

to Barbatula barbatulaIURP6ZHGHQ

Interpretation of the sequencing results:

arguments for piscivory?

:LWK VL[ RXW RI JXDQR VDPSOHV RI WUDZOLQJMyotis VSHFLHVUHYHDOLQJXQDPELJXRXVO\'1$UHPDLQVRI¿VKHV ZHKDYHSURYLGHGHYLGHQFHIRU¿VK'1$LQWKHLUVFDWEXW RQO\LQDOLPLWHGQXPEHURIVDPSOHV2XUWHFKQL- cal approach with an extremely sensitive PCR appears to be highly vulnerable for accumulating errors during the PCR and thus the likelihood of amplifying residual IUDJPHQWV RI HQYLURQPHQWDO '1$ LV YHU\ KLJK 7KLV LV LQGLFDWHGE\WKHPDMRULW\RIWKHREWDLQHG'1$VHTXHQFHV QRW PDWFKLQJ ¿VK VSHFLHV HYHQ LI ZH XVHG ¿VKVSHFLI- LFSULPHUV7DE,WZRXOGEHSRVVLEOHWRFLUFXPYHQW these issues using several approaches. For example, to DYRLGDPSOL¿FDWLRQRIWKHPRVWFRPPRQFRQWDPLQDQWLQ RXU GDWD VHW QRW XQH[SHFWHGO\ KXPDQ '1$ EORFNLQJ SULPHUVB^OESSENKOL et alFRXOGEHXVHG$OWHU- natively, hybridization capture could be used for target enrichment as it is both highly sensitive and compara- WLYHO\SURPLVFXRXVZLWKUHJDUGWRSRWHQWLDOWDUJHWVK^O-

ZAREWA et al$QGWKLUGLWZRXOGEHSRVVLEOHWR DYRLGFRQWDPLQDWLRQIURPUHDJHQWVFIC^HAMPLOT et al., EXW ZH FDXWLRQWKDWWKH PDMRULW\RI FRQWDPLQDQW VHTXHQFHV EDW DQG KXPDQ OLNHO\ GHULYHG IURP WKH EDW H[FUHPHQWVHLWKHUGLUHFWO\EDWRUGXHWRKDQGOLQJKX- mans), and would thus not be prevented by decontamina- tion of reagents. Moreover, all these approaches are more time- and money-intensive than our simple, high-sensi- tive PCR and thus not ideal for pilot-study work. Finally, ZHVKRXOGSRLQWRXWWKDWDEVHQFHRIDPSOL¿FDWLRQSURG- XFWVGRHVQRWQHFHVVDULO\PHDQDEVHQFHRI¿VK'1$LQD faeces sample. Thus, we are not able to offer an exhaus- WLYH PROHFXODU UHFRUG RI ¿VK'1$ LQ WKH IDHFDO SHOOHWV of the investigated trawling MyotisVSHFLHV2QWKHRWKHU KDQG WKH ¿VK VSHFLHV UHYHDOHG E\ WKH '1$ VHTXHQFHV

¿WWRWKHW\SLFDOHQYLURQPHQWRIWUDZOLQJMyotis species DWWKHQHWWLQJVLWHV,QWHUHVWLQJO\DOOUHFRYHUHG¿VKVSH- cies are typically associated with river systems and not ZLWKVWDQGLQJZDWHUVXFKDVSRQGV7KHEXOOKHDGV&. go- bio (XUDVLDQ PLQQRZV P. phoxinus) and nemacheilid ORDFKHVSUREDEO\B. barbatula as no other nemacheilid loach occurs in Germany) are associated with gravel to VWRQHERWWRPDQGIDVWÀRZLQJFROGZDWHUZLWKHOHYDWHG OHYHOVRIR[\JHQ,QFRQWUDVWWRWKDWLGHRUGDFHL. idus or L. leuciscus DQG FKXE Squalius sp.) prefer usually the lower reaches of larger streams or rivers with lower ZDWHU FXUUHQW7KXV ZH YLHZ RXU UHVXOWV DV D ¿UVW VWHS towards a more comprehensive investigation of trawling bats dietary spectrum.

Possible contamination with environmental DNA and transfer path of fish DNA

$V ¿VKHV ¿VK ODUYDH DQG DOVR ¿VK HJJV DUH SDUW RI WKH FRPSOH[IRRGZHERIDTXDWLFRUJDQLVPVVHYHUDOWUDQVIHU SDWKVRI¿VK'1$LQWRWKHIDHFHVRIEDWVPXVWEHFRQ- sidered.

%HVLGHVWKHSRVVLELOLW\WKDWVPDOO¿VKRUODUYDHGHDG or alive) could have been picked up from the water sur- face during trawling, several other possibilities must be considered, much as in forensics, where secondary trace '1$WUDQVIHULVDQXQGLVSXWHGLVVXHHJS^ZUTA et al., 2QHSRVVLELOLW\IRUFRQWDPLQDWLRQRIWKHEDWIDH- FDOSHOOHWVZLWK¿VK'1$ZRXOGEH¿VKODUYDHGHDGRU DOLYH HDWHQ FRPSOHWH RU SDUWLDOO\ E\ SLVFLYRURXV LQ- sects, which themselves became later on prey for the WUDZOLQJEDWVDQGVHUYHGDVYHFWRUVIRU¿VK'1$:DWHU ERDWPHQ &RUL[LGDH DUH UHJXODUO\ IRXQG LQ WKH GLHW RI M. dasycneme and M. daubentonii ZLWK D IUHTXHQF\ XS WRLQWKHIDHFDOSHOOHWVK^RÜGER et al

$V &RUL[LGDH RFFDVLRQDOO\ IHHG RQ ¿VK DQG ¿VK ODUYDH M^CC^ORMICK & P^OLISLWFDQQRWEHUXOHGRXWWKDW VSHFLPHQVRIWKLVSUH\VHUYHDVYHFWRUIRU¿VK'1$DQG LWVFRQVXPSWLRQOHGWRDVHFRQGDU\WUDQVIHURI¿VKWLVVXH WRWKHVWRPDFKDQGJXWFRQWHQWRIWUDZOLQJEDWV$GGLWLRQ- DOO\ FRQWDPLQDWLRQ ZLWK HQYLURQPHQWDO '1$ H'1$

PD\EHWKHUHDVRQIRUWKH¿VK'1$LQWKHDQDO\VHGSHO- OHWV,WLVNQRZQIURPUHFHQWVWXGLHVWKDWWKHDQDO\VLVRI water content using environmental metabarcoding is a SRZHUIXOWRROIRUDTXDWLFVSHFLHVGHWHFWLRQV^ALENTINI et al )UHH ¿VK '1$ RU FHOOV LQ WKH ZDWHU FROXPQ could adhere to the prey and pose a possibility for sec- RQGDU\FRQWDPLQDWLRQDOWKRXJKLWLVTXHVWLRQDEOHLIVXFK ORZDPRXQWVRIIUHH'1$ZRXOGVWLOOEHGHWHFWDEOHDIWHU passage of the digestive system.

Conclusions

2XUGDWDGRQRWSURYLGHDGH¿QLWLYHPROHFXODUUHFRUGRI GLHWGHULYHG¿VK'1$LQWKHIDHFDOSHOOHWVRIWKHLQYHV- tigated trawling MyotisVSHFLHVQRUGH¿QLWLYHSURRIIRU piscivory in M. dasycneme and M. daubentonii. Rather, ZHFRQVLGHUWKHDPSOL¿HGVHTXHQFHVRI¿VK'1$IURP bat guano as genuine pilot data. However, as we also VXFFHVVIXOO\ DPSOL¿HG ¿VK '1$ IURP IDHFDO SHOOHWV RI M. capacciniiZKLFKLVNQRZQWRIHHGRQ¿VKZHVSHFX- ODWHWKDWWKH¿VK'1$ZHDPSOL¿HGZLWKWKHVDPHPR- lecular approach from the guano of M. dasycneme and M. daubentonii, are not unlikely to be an indication for piscivory in those species. Further studies with a more UH¿QHG PROHFXODU JHQHWLF DSSURDFK OLNH IDHFDO '1$

PHWDEDUFRGLQJ FIR^AZGOURET et al P^OMPANON et alRUK\EULGL]DWLRQFDSWXUHDSSURDFKHVVKRXOG XOWLPDWHO\EHDEOHWRDQVZHUWKHTXHVWLRQRISLVFLYRU\LQ M. dasycneme and M. daubentonii.

Acknowledgements

:H DUH JUDWHIXO WR -R[HUUD$LKDUW]D DQG WZR DQRQ\PRXV UHYLHZ- ers for helpful comments and suggestions to an earlier version of WKHPDQXVFULSW:HWKDQN*'UU66RPPHU,+DUPV)*OR]D 5DXVFK$ 6HHEHQV 0*|WWVFKH +9DOTXL DV ZHOO DV 3 DQG /

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Supplementary Information Supplementary information Tab. S1:3HOOHWVDPSOLQJVDPSOLQJVLWHVDQGPHWKRGV,QYHVWLJDWHGIHFDOVDPSOHVRIMyotis dasycneme, Myotis capaccinii and Myotis daubentonii IURPGLIIHUHQWUHJLRQVRI(XURSH/HJHQGVWRUHGDWURRPWHPSHUDWXUHDEEUHYLDWLRQVIRUVDPSOHFROOHFWRU%6%6LHPHUV,+,+DUPV).).UJHU5656RPPHUDQG6666RPPHU6DPSOHQR LVQRWOLVWHGEHFDXVHWKLV,'SRVLWLRQZDVXVHGIRUDEODQN'1$LVRODWLRQ IDspeciessexsamplingno. of bat individ.no. pelletspellet storing Date of samplingcountrylocalitysample collector M. capacciniifemnettingGULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 2M. capacciniimalenettingGULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. capacciniimalenettingGULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. capacciniimalenettingGULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. capacciniimalenetting8GULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. capacciniimalenettingGULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. capacciniimalenettingGULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 8M. capacciniimalenettingGULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. daubentoniimalenetting2GULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. daubentoniimalenetting2GULHG±ƒ&Bulgaria5XVVHQVNL/RP%6 M. dasycnememalenetting22DOFRKROGermanyRastorf,+).56 M. dasycnemefemnetting22DOFRKROGermany:DKOVWRUI,+).56 M. dasycnemefemnetting22DOFRKROGermany:DKOVWRUI,+).56 M. dasycnememalenetting22DOFRKROGermany:DKOVWRUI,+).56 M. dasycnemefemnetting22DOFRKROGermany:DKOVWRUI,+).56 M. dasycnemefemnetting22DOFRKROGermany:DKOVWRUI,+).56 M. dasycnememalenetting22DOFRKROGermanyRastorf,+).56 M. dasycnememalenetting22DOFRKROGermany3RVWVHH3UHH],+).56 M. dasycnemefemnetting22DOFRKROGermanyRastorf,+).56 M. dasycnememalenetting22DOFRKROGermany:DKOVWRUI,+).56 M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). 22M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). 28M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH). M. dasycneme—nursing colony—SODVWLFEDJGermany:HVWHQGH).

IDspeciessexsamplingno. of bat individ.no. pelletspellet storing Date of samplingcountrylocalitysample collector M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666 M. dasycneme—nursing colony—SODVWLFEDJGermany:LVPDU0JJHQEXUJ5666

Tab. S1 continued.

Supplementary information Tab. S2:5HFRYHUHG'1$VHTXHQFHVWKDWFRXOGEHXQDPELJXRXVO\DVVLJQHGWR¿VKYLD%/$67VHDUFKLQFOXGLQJWKHSRVLWLYHVFRQWUROVSRV IDsample origin PDUNHUDQGUHFRYHUHG'1$VHTXHQFHDIWHUPDQXDOWULPPLQJZLWKOHQJWKLQESV¶¶ pos&. carpio&2,SRVLWLYH TGCCTGAGCCGGGATAGTAGGAACCGCCTTAAGCCTCCTCATTCGGGCCGAACTTAGCCAACCCGGGTCGCTTCTAGGTGATGACCAAATTTATAACGTTATCGTCACNG VLQJOHUYUHDG posG. gobio&2,SRVLWLYH TCCTCTTCGAGCTGAGTTGAGCCACCTGGCTCACTTCTAGGTGATGACCAAATTTATAATGTAATCGTTACTGCCCACGCCTTCGTAATAATT VLQJOHUYUHDG posO. mykiss&2,SRVLWLYH TGCCTGAGCCGGGATAGTAGGCACCGCCCTGAGTCTACTGATTCGGGCGGAACTAAGCCAGCCGGGCGCTNCTNTGGGGGATGACCAAATCTATAACGTGATCGTCACAGC VLQJOHUYUHDG posR. rutilus&2,SRVLWLYH TCCTTATTCGGGCCGAACTAAGCCACCCGGGTCACTTTTAGGCGATGACCAAATTTATAATGTCATCGTTACCGCCCACGCCTTCGTAATAATT VLQJOHUYUHDG M. capaccinii

&2,Squalius sp.): GGGGACTGCCCTAAGCCTCCTTATTCGGGCCGAACTAAGCCAACCTGGGTCACTTTTAGGCGATGACCAAATTTATAATGTCNTTGTCNCCGCCCACGCCTTCGTAATAA TTTTCTTTATAGTAATGCCAATTCT DVVHPEOHG 8M. capaccinii&2,Gobio sp.): GGGGACTGCTTTAAGCCTCCTCATTCGAGCTGAGTTGAGCCAACCTGGCTCACTTCTAGGTGATGACCNANTTTATAATGTAATCGTTACTGCCCACGCCTTCGTAATGATT DVVHPEOHG M. daubentonii&2,&RWWXV sp.): AGGCACAGCTTTAAGCCTCCTAATTCGAGCAGAACTAAGCCAACCCGGCGCCCTTTTGGGGGACGACCAGATCTATAATGTAATTGTTACAGCCCATGCTTTCGTAATAATT DVVHPEOHG M. dasycneme6Phoxinus phoxinus): GAGCTTAAGGTACAAACTTAATCACGTTAAACGACTTCTAAAGAGCAAGAACTTAGTGGCGATAAGACTTTACC DVVHPEOHG M. dasycneme6Leuciscus sp.): GAGCTTAAGGTACAAAGTTCAACCACGTTAAACGACTCCACAGAAAGCAAGAACTTAGTGGCAAATGAAACTTTACC DVVHPEOHG M. dasycneme6Barbatula barbatula): GAGCTTAAGGTACAGGCCCAACCGCGTTAAACAACTTATTAAATAAGTCTTAAACATAGCGGAATGTGGGACCTTACC DVVHPEOHG