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New genetic data reveals a new species of Zospeum in Bosnia (Gastropoda, Ellobioidea, Carychiinae)
expand article infoThomas Inäbnit, Adrienne Jochum§|, Raijko Slapnik#, Eike Neubert§|
‡ University of Potsdam, Potsdam, Germany
§ Natural History Museum of the Burgergemeinde Bern, Bern, Switzerland
| University of Bern, Bern, Switzerland
¶ Senckenberg Research Institute and Natural History Museum, Frankfurt, Germany
# Unaffiliated, Kamnik, Slovenia
Open Access

Abstract

Recent integrative investigations of the terrestrial ellobiid genus, Zospeum, have revealed significant findings concerning its Alpine-Dinaric evolution and taxonomy. Due to the expected discrepancy between the useful, but limited, 1970s’ classification system based on shell data and the results of recent genetic analyses in the latest investigation, a revision of the entire radiation was undertaken, and a new classification system was devised by the present authors in an earlier paper. Concurrent to this work, molecular sequences from two Austrian caves were published independently of our revision by another research group. By incorporating these genetic data within our phylogenetic framework here, we show that the Austrian individuals are genetically most similar to Zospeum amoenum and consequently, classify them within that species. We additionally reveal two new genetic lineages from the largely under-sampled southern extension of Zospeum’s known distributional range. The first lineage, deriving from the region of Dubrovnik, Croatia, is a potential candidate for genetically clarifying Zospeum troglobalcanicum. The second lineage derives from the municipality of Tomislavgrad, Bosnia-Herzegovina and is herein, described a new species: Zospeum simplex Inäbnit, Jochum & Neubert, sp. nov.

Keywords

Dinarides, microsnails, molecular phylogenetics, shell variability, subterranean ecology, troglobitic microsnails

Introduction

The carychiid genus, Zospeum, consists of tiny (0.9–2.6 mm), troglobitic snail species that are distributed in two disjunct areas: a western zone, comprising the western Pyrenees and the Cantabrian mountains of Spain and France (Jochum et al. 2015a, 2019) and an eastern zone, encompassing the southeastern Alps and Dinarides of northeastern Italy, southern Austria, Slovenia, Croatia, Bosnia-Herzegovina and Montenegro (see Inäbnit et al. 2019). This work addresses the species rich, eastern radiation of Zospeum.

Until recently, the eastern radiation of Zospeum was largely classified using a scheme devised by Bole (1974), based solely on shell morphology. More recent studies (Weigand et al. 2011; Weigand et al. 2013; Jochum et al. 2015b), however, found Bole’s (1974) scheme, though effective for its time, now incongruent with genetic data, leading to a thorough revision by Inäbnit et al. (2019). They subdivided the eastern Zospeum radiation into 25 species that could be divided genetically into five species groups: the Z. spelaeum group (northeastern Italy, Slovenia, north-western Croatia; five species), the Z. alpestre group (Slovenian Alps and adjacent regions in Italy and Austria; four species), the Z. obesum group (southwestern Slovenia and adjacent Croatia; two species), the Z. pretneri group (Croatia, more or less close to the Adriatic coast; four species), and the Z. frauenfeldii group (southern Slovenia, northwestern Croatia, northwestern Bosnia-Herzegovina; five species); five species could not be assigned to any of the five groups due to lack of molecular data.

One of the issues raised in Inäbnit et al. (2019) is that Zospeums eastern distribution has been unevenly sampled throughout its history. Most studies covered almost only Slovenian (e.g., Frauenfeld 1854, 1856; Freyer 1855; Bole 1974; Weigand et al. 2013), Italian (Pezzoli 1992 and papers cited therein) and northwestern Croatian populations (Slapnik and Ozimec 2004; Inäbnit et al. 2019). The consequence of this sampling disparity is that we have very limited records from southern Croatia, Bosnia-Herzegovina and Montenegro (see Inäbnit et al. 2019: fig. 1a), none of which include genetic data. In fact, the only species described from the southern half of the Zospeum’s distribution range is Zospeum troglobalcanicum Absolon 1916. Shells that obviously belong to different species exist in museum collections (see Inäbnit et al. 2019: fig. 10W-Z; Gittenberger 1975), but genetic data from these southernmost populations is still lacking for a contemporary, integrative taxonomic assessment. In the current study, we add new sequences from 12 specimens, collected in southern Croatia and Bosnia-Herzegovina to the existing genetic dataset.

Approximately the same time as the revision by Inäbnit et al. (2019) was published, Kruckenhauser et al. (2019) published the results of a small barcoding study of specimens from Austria (for locations see Fig. 1). Due to this unfortunate overlap, their results could not be incorporated into the classification system proposed by Inäbnit et al. (2019). We have however, included these results in our work here.

Figure 1. 

Map showing the distribution of the Zospeum pretneri group and the Zospeum alpestre group (except Z. isselianum). Austrian specimens from Kruckenhauser et al. (2019) are labelled as “Z. cf. amoenum”.

Materials and methods

Material is housed in the following collections:

AJC Adrienne Jochum Collection, Kelkheim, Germany;

MCSMNH Malacological Collection of the Slovenian Museum of Natural History (former CSR SASA, MZBI & SMNH) Ljubljana, Slovenia;

NHMW Naturhistorisches Museum Wien, Wien, Austria;

NMBE Naturhistorisches Museum der Burgergemeinde Bern, Bern, Switzerland;

RSC Rajko Slapnik Collection, Kamnik, Slovenia;

SMF Senckenberg Forschungsinstitut und Naturmuseum, Frankfurt am Main, Germany.

In order to preserve the shell from dissolution during the extraction, our DNA extraction protocol was based on a method initially described in Schizas et al. (1997) and partially modified after Böttger-Schnack and Machida (2011). DNA extraction was conducted on 12 ethanol-preserved individuals (NMBE 568052-568063). Each specimen was inserted into a 0.2-ml PCR-tube and dried at room temperature. Eight μl ddH2O and 2 μl 5× PCR-buffer (Promega 5× Colorless GoTaq Reaction Buffer) were added and the mixture was heated at 94 °C for 2 min. whereby 1.3 μl proteinase K solution (from the DNEasy Blood and tissue kit, Qiagen) were then added and the solution was homogenised and then incubated in a PCR-thermocycler at 55 °C for 15 min., afterwards at 70 °C for 10 min. The incubation was repeated once. Ten μl of Gene Releaser (Bioventures Inc.) were then added and the mixture was inserted into a thermocycler with the following protocol: 65 °C for 30 s, 8 °C for 30 s, 65 °C for 1.5 min., 97 °C for 3 min., 8 °C for 1 min., 65 °C for 3 min., 97 °C for 1 min., 65 °C for 1 min., 80 °C for 5 min. The mixture, including the intact shell, was centrifuged for 1 min. using a table centrifuge and the clear phase with the DNA was transferred to another 0.2 mL PCR-tube, where 15 μl of AE-Buffer (DNeasy Kit, Qiagen) was added. The shell was cleaned from the remains of the Gene Releaser chemicals by rinsing with 80% EtOH.

We used five markers, two mitochondrial (COI (658 bp), 16S (483 bp)) and three nuclear markers (H3 (330 bp), ITS2 (809 bp), 28S (590 bp)) with a total length of 2870 bp (for primers, see Table 1).

Table 1.

Primers used in this study.

Marker Primer Name Primer sequence Reference
COI LCO1490 (F) 5‘-GGTCAACAAATCATAAAGATATTGG-3‘ Folmer et al. (1994)
COI HCO2198 (R) 5‘-TAAACTTCAGGGTGACCAAAAAATCA-3‘ Folmer et al. (1994)
16S 16S F 5‘-CGGCCGCCTGTTTATCAAAAACAT-3‘ Palumbi et al. (1991)
16S 16S R 5‘-GGAGCTCCGGTTTGAACTCAGATC-3‘ Palumbi et al. (1991)
28S LSU-2 (F) 5‘-GGGTTGTTTGGGAATGCAGC-3‘ Wade and Mordan (2000)
28S LSU-4 (R) 5‘-GTTAGACTCCTTGGTCCGTC-3‘ Wade and Mordan (2000)
ITS2 ITS2ModA (F) 5’-GCTTGCGGAGAATTAATGTGAA-3’ Bouaziz-Yahiatene et al. (2017)
ITS2 ITS2ModB (R) 5’-GGTACCTTGTTCGCTATCGGA-3’ Bouaziz-Yahiatene et al. (2017)
H3 H3-F 5‘-ATGGCTCGTACCAAGCAGAC(ACG)GC-3‘ Colgan et al. (1998)
H3 H3-R 5‘-ATATCCTT(AGGGCAT(AG)AT(AG)GTG-3‘ Colgan et al. (1998)

The PCR-solution included the following admixture: 2 μl template, 12.5 μl GoTaq (Promega) polymerase, 8.5 μl of nuclease-free water, and 1 μl of both forward and reverse primer (10 μmol) respectively. In cases where the PCR signal was judged too weak, the reaction was repeated using 3 μl template DNA, 3 μl of the previous PCR product, and 5.5 μl of nuclease-free water. The amount of GoTaq and primers remained the same. The amplification was conducted using the following cycling protocols: For COI, the admixture was first heated up to 95 °C for 1 min, followed by 30 cycles of 30 s (of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 1 min), and a final extension at 72 °C for 3 min. For 16S, the protocol started with 2:30 min at 90 °C, followed by 10 cycles of 30 s at 92 °C, 30 s at 44 °C, and 40 s at 72 °C, followed again by 30 s at 92 °C, 40 s at 48 °C, and 40 s at 48 °C. The protocol for 28S started with 1 min at 96 °C, then went into 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 1 min at 72 °C, finishing with 10 min at 72 °C. The ITS2 protocol started with 1 min at 96 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 44 °C, and 1 min at 72 °C, ending with 10 min at 72 °C. For H3, the admixture was first heated up to 95 °C for 3 min, followed by 40 cycles of 45 s at 94 °C, 45 s at 50 °C, and 2 min at 72 °C, finishing with 10 min at 72 °C. The protocols for COI and H3 could be used for both markers. The PCR products were sequenced at the LGC Genomics GmbH (Berlin, Germany) using their standard protocol.

Sequences received from LGC were imported into the Geneious 5.4.7 software (Kearse et al. 2012). The forward and reverse sequences for each gene and individual were combined and edited. In addition to the sequences that were generated during this study, we used the sequences previously used and generated in Inäbnit et al. (2019), as well as those generated by Kruckenhauser et al. (2019). The name of some of the Spanish specimens were updated based on the results of Jochum et al. (2019). A total list of samples can be found in Table 2. For each marker, sequences were aligned in Geneious using the MAFFT multiple sequence alignment plugin version 1.3.6 (based on MAFFT v7.308; Katoh et al. 2002; Katoh and Standley 2013), allowing the program to choose the most appropriate algorithm. The sequence length of each alignment was standardised to the length mentioned above.

Table 2.

Specimens used in this study. Italicised accession numbers indicate sequences taken from BOLD, not italicised numbers are from GenBank.

Species Source Collection number Locality Coordinates COI 16S H3 28S ITS2
Carychium tridentatum (Risso, 1826) Inäbnit et al 2019 NMBE 549936 Taunus, Eppstein, Germany 50.1601, 8.3846 MH383001 MH382969 MH383018 MH382989 MH383038
Z. vasconicum Prieto, De Winter, Weigand, Gómez & Jochum, 2015 Weigand et al. 2013 AJC 1875a Cueva del Cráneo, Dima, Bizkaia, Spain 43.1287, -2.7348 BARCA206-12 KC206116 KC206249
Weigand et al. 2013 AJC 1874b Cueva Silibranka II, Manaria, Bizkaia, Spain 43.287, -2.945 BARCA204-12 KC206117 KC206248
Weigand et al. 2013 AJC 1847c Cueva de Ermita de Sandaili, Valle de Araotz, Bizkaia, Spain 42.9994, -2.4381 KM281092 KC206119 KC206247
Z. cf. schaufussi Weigand et al. 2013 AJC 1878a Cueva de Las Paúles, Monte Santiago, Castilla y León, Spain 43.1282, -2.7362 BARCA194-12 KC206113 KC206252
Weigand et al. 2013 AJC 1844b Cueva de los Cuervos, Barranco de Aranaga, Bizkaia, Spain 43.2829, -3.2588 BARCA192-12 KC206120 KC206246
Z. praetermissum Jochum, Prieto & De Winter 2019 Weigand et al. 2013; Romero et al. 2017 AJC 1842a Cueva del Bosque, Inguanzo, Asturias, Spain 43.3123, -4.8724 KM281091 KC206121 KC206245 KM281051
Z. zaldivarae Prieto, De Winter, Weigand, Gómez & Jochum, 2015 Weigand et al. 2013 AJC 1876c Cueva de Las Paúles, Monte Santiago, Castilla y León, Spain 43.1282, -2.7362 BARCA209-12 KC206114 KC206251
Weigand et al. 2013 AJC 1876b Cueva de Las Paúles, Monte Santiago, Castilla y León, Spain 43.1282, -2.7362 BARCA208-12 KC206115 KC206250
Z. costatum Freyer, 1855
Weigand et al. 2013 NMBE 553383 Jama 2 pri Jabljah, Loka pri Mengšu, Slovenia 46.1426, 14.5533 HQ171599 KC206158 KC206208
Weigand et al. 2013 NMBE 553383 Jama 2 pri Jabljah, Loka pri Mengšu, Slovenia 46.1426, 14.5533 HQ171601 KC206159 KC206207
Z. spelaeum (Rossmaessler, 1838) Weigand et al. 2013 NMBE 553316 Grotte Bac, Trieste Municipality, Trieste Prov., Italy 45.6361, 13.8717 BARCA182-12 KC206110 KC206255
Weigand et al. 2013 AJC 1898a Grotte Bac, Trieste Municipality, Trieste Prov., Italy 45.6361, 13.8717 BARCA184-12 KC206108 KC206257
Weigand et al. 2013 NMBE 553316 Grotte Bac, Trieste Municipality, Trieste Prov., Italy 45.6361, 13.8717 KC206109 KC206256
Weigand et al. 2013 NMBE 553314 Grotte d‘Ercole, near Gabrovizza, Trieste Prov., Italy 45.731, 13.7261 BARCA181-12 KC206111 KC206254
Weigand et al. 2013 NMBE 553311 Velika Pasica, Gornji Ig, Slovenia 45.9189, 14.4934 BARCA179-12 KC206135 KC206231
Inäbnit et al. 2019 NMBE 554396 Hotiške Ponikve, Hotičina, Slovenia 45.5735, 14.0309 MH382992 MH382954 MH383022 MH382974 MH383024
Z. isselianum Pollonera, 1887 Weigand et al. 2013 NMBE 553389 Turjeva jama, Robič, Kobarid, Slovenia 46.2435, 13.5046 HQ171594 KC206097 KC206268
Z. amoenum (Frauenfeld, 1856) Inäbnit et al. 2019 RS 2037 Ihanščica, Ihan, Ljubljana, Slovenia 46.1216, 14.6476 MH383003 MH382971 MH383020
Weigand et al. 2013 NMBE 553378 Konečka zijalka, Šmihel nad Mozirjem, Mozirje, Slovenia 46.4024, 14.9393 BARCA123-10 KC206179 KC206187
Weigand et al. 2013 NMBE 553378 Konečka zijalka, Šmihel nad Mozirjem, Mozirje, Slovenia 46.4024, 14.9393 BARCA124-10 KC206178 KC206188
Jochum et al. 2015 MCSMNH 40600a Potočka zijalka, Olševa, Slovenia 46.4493, 14.6693 BARCA211-13
Z. amoenum (Frauenfeld, 1856) Jochum et al. 2015 MCSMNH 40600a-2 Potočka zijalka, Olševa, Slovenia 46.4493, 14.6693 BARCA212-13
Z. cf. amoenum Kruckenhauser et al. 2019 NHMW109000/AL/01821/8139 Steiner Lehmhöhle, Austria 46.42228, 14.53462 AMOL570-19
Kruckenhauser et al. 2019 NHMW109000/AL/01821/8140 Steiner Lehmhöhle, Austria 46.42228, 14.53462 AMOL571-19
Kruckenhauser et al. 2019 NHMW109000/AL/01822/8141 Hafnerhöhle, Austria 46.51200, 14.21623 AMOL572-19
Kruckenhauser et al. 2019 NHMW109000/AL/01822/8142 Hafnerhöhle, Austria 46.51200, 14.21623 AMOL573-19
Z. alpestre (Freyer, 1855) Weigand et al. 2013 NMBE 553391 Jama pod Mokrico, Kamniška Bistrica, Slovenia 46.3093, 14.5832 HQ171593 KC206099 KC206266
Inäbnit et al. 2019 MCSMNH 40651a Jelenska zijalka, Raduha, Slovenia 46.3656, 14.7567 MH383002 MH382970 MH383019 MH382990 MH383039
Z. kupitzense A. Stummer, 1984 Weigand et al. 2013; Romero et al. 2017 NMBE 553393 Ložekarjeva zijalka, Solčava, Slovenia 46.4268, 14.624 BARCA125-10 KC206150 KC206216 KM281049
Z. exiguum Kusčer, 1932 Inäbnit et al. 2019 NMBE 548774 Jama Borušnjak 3, Lupoglav, Ćićarija, Istra 45.3702, 14.1841 MH382994 MH382959 MH383009 MH382979 MH383030
Weigand et al. 2013 NMBE 553384 Križna jama, Lož, Cerknica, Slovenia 45.7452, 14.4673 HQ171582 KC206162 KC206204
Weigand et al. 2013 NMBE 553384 Križna jama, Lož, Cerknica, Slovenia 45.7452, 14.4673 HQ171585 KC206163 KC206203
Z. obesum (Frauenfeld, 1854) Weigand et al. 2013 NMBE 553409 Krška jama, Krška vas, Slovenia 45.8899, 14.7711 BARCA177-12 KC206136 KC206230
Weigand et al. 2013 NMBE 553409 Krška jama, Krška vas, Slovenia 45.8899, 14.7711 BARCA175-12 KC206137 KC206229
Z. pretneri Bole, 1960 Weigand et al. 2013 AJC 1370 Donja Cerovačka špilja, Kesići, Gračac, Croatia 44.2701, 15.8855 HQ171595 KC206151 KC206215
Z. tholussum Weigand, 2013 Weigand 2013 SMF 341633 Lukina jama – Trojama, Krasno, Croatia 44.7621, 15.0296 BARCA120-10
Z. manitaense Inäbnit, Jochum & Neubert 2019 Inäbnit et al. 2019 NMBE 548800 Manita peć, Starigrad, Croatia 44.311, 15.4792 MH382963 MH383012 MH382983
Inäbnit et al. 2019 NMBE 548811 Manita peć, Starigrad, Croatia 44.311, 15.4792 MH383000 MH382968 MH383017 MH382988 MH383037
Z. aff. troglobalcanicum Absolon 1917 This work NMBE 568052 Špilja Jezero, Cavtat, Konavle, Croatia 42.5858, 18.2569 MW786768 MW796484 MW784525 MW784537
This work NMBE 568053 Špilja Jezero, Cavtat, Konavle, Croatia 42.5858, 18.2569 MW786767 MW796485 MW784524 MW784536
Z. simplex sp. nov. Inäbnit, Jochum & Neubert This work NMBE 568054 Špilja Dahna, Omerovići, Bosnia and Herzegovina 43.6572, 17.2078 MW796475
This work NMBE 568055 Jama u kamenolomu, Cebara, Bosnia and Herzegovina 43.6517, 17.2133 MW786764 MW784509 MW796481 MW784526 MW784530
This work NMBE 568056 Jama u kamenolomu, Cebara, Bosnia and Herzegovina 43.6517, 17.2133 MW786765 MW784510 MW796478 MW784521 MW784532
This work NMBE 568057 Jama u kamenolomu, Cebara, Bosnia and Herzegovina 43.6517, 17.2133 MW786766 MW784511 MW796476 MW784520 MW784531
This work NMBE 568058 Jama u kamenolomu, Cebara, Bosnia and Herzegovina 43.6517, 17.2133 MW786763 MW784512 MW796477 MW784529
Z. simplex sp. nov. Inäbnit, Jochum & Neubert This work NMBE 568059 Vranjača, Grabovica, Bosnia and Herzegovina 43.6625, 17.1039 MW786762 MW784513 MW796486 MW784522
This work NMBE 568060 Jama Dobravljevac, Gornji Brišnik, Bosnia and Herzegovina 43.6347, 17.2328 MW786761 MW784515 MW796482 MW784527 MW784535
This work NMBE 568061 Jama Dobravljevac, Gornji Brišnik, Bosnia and Herzegovina 43.6347, 17.2328 MW786760 MW784516 MW796479 MW784523 MW784533
This work NMBE 568062 Jama Dobravljevac, Gornji Brišnik, Bosnia and Herzegovina 43.6347, 17.2328 MW786759 MW784514 MW796483 MW784534
This work NMBE 568063 Jama Dobravljevac, Gornji Brišnik, Bosnia and Herzegovina 43.6347, 17.2328 MW786758 MW784517 MW796480 MW784519 MW784528
Z. subobesum Bole, 1974 Weigand et al. 2013 NMBE 553326 Tounjčica, Tounj, Ogulin, Croatia 45.2439, 15.3253 HQ171602 KC206152 KC206214
Weigand et al. 2013 NMBE 553326 Tounjčica, Tounj, Ogulin, Croatia 45.2439, 15.3253 HQ171604 KC206153 KC206213
Weigand et al. 2013 NMBE 553328 Jopićeva špilja, Brebovnica, Krnjak, Karlovac, Croatia 45.2951, 15.5939 BARCA172-12 KC206125 KC206241
Z. frauenfeldii (Freyer, 1855) Weigand et al. 2013 NMBE 553388 Podpeška jama, Podpeč, Dobrepolje, Slovenia 45.8393, 14.6863 HQ171587 KC206160 KC206206
Weigand et al. 2013 NMBE 553388 Podpeška jama, Podpeč, Dobrepolje, Slovenia 45.8393, 14.6863 HQ171589 KC206161 KC206205
Inäbnit et al. 2019 NMBE 548771 Hrustovača špilja, Hrustovo, Sanski Most, Bosnia and Herzegovina 44.6607, 16.7285 MH383006 MH382976 MH383027
Z. bucculentum Inäbnit, Jochum & Neubert 2019 Inäbnit et al. 2019 NMBE 548801 Jama na Škrilama, Netretić, Croatia 45.5277, 15.3476 MH382997 MH382964 MH383013 MH382984 MH383033
Inäbnit et al. 2019 NMBE 548772 Pivnica špilja, Žakanje, Croatia 45.6108, 15.3617 MH382957 MH383007 MH382977 MH383028
Inäbnit et al. 2019 NMBE 548806 Vrelić špilja, Donje Dubrave, Ogulin, Croatia 45.3114, 15.352 MH382966 MH383015 MH382986 MH383035
Z. pagodulum Inäbnit, Jochum & Neubert 2019 Inäbnit et al. 2019 NMBE 548805 Kučka jama, Lovran, Učka, Istra, Croatia 45.2985, 14.2135 MH382998 MH382965 MH383014 MH382985 MH383034
Inäbnit et al. 2019 NMBE 548807 Grnjača špilja, Lovran, Učka, Istra, Croatia 45.2835, 14.2381 MH382999 MH382967 MH383016 MH382987 MH383036
Z. robustum Inäbnit, Jochum & Neubert 2019 Inäbnit et al. 2019 NMBE 554397 Tonkovića špilja, Ogulin, Croatia 45.3359, 15.2541 MH382953 MH383004 MH382973 MH383023
Inäbnit et al. 2019 NMBE 548773 Budina špilja, Studenci, Croatia 44.7121, 15.3639 MH382993 MH382958 MH383008 MH382978 MH383029
Inäbnit et al. 2019 NMBE 548777 Markov ponor, Lipovo polje, Croatia 44.7606, 15.1797 MH382995 MH382961 MH383010 MH382981 MH383032
Inäbnit et al. 2019 NMBE 548787 Markov ponor, Lipovo polje, Croatia 44.7606, 15.1797 MH382996 MH382962 MH383011 MH382982
Inäbnit et al. 2019 NMBE 548776 Vrlovka, Kamanje, Croatia 45.6319, 15.3934 MH382960 MH382980 MH383031
Inäbnit et al. 2019 RS 2210a Vrlovka, Kamanje, Croatia 45.6319, 15.3934 MH382972 MH383021 MH382991 MH383040
Inäbnit et al. 2019 NMBE 554399 Židovske kuće, Cerovica, Žumberak, Croatia 45.8, 15.48 MH382955 MH382975 MH383025
Inäbnit et al. 2019 NMBE 554400 Pušina jama, Jezemice, Žumberak, Croatia 45.7369, 15.3606 MH382956 MH383005 MH383026

Topologies were estimated using two different phylogenetic methods: Maximum Likelihood (ML) and Bayesian Inference (BI). The five markers were set as partitions in both of these methods, using a distinct model for the third codon in protein-coding genes (COI, H3). The maximum likelihood (ML) topology was estimated using the RAxML 7.2.8 (Stamatakis 2014) plugin of Geneious with the GTR gamma nucleotide model and 1000 bootstrap replicates. An additional ML tree was calculated for the Z. pretneri group (with Z. robustum NMBE 548777 as an outgroup) without H3 and 28S.

The Bayesian tree was reconstructed with MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001) using the substitution models suggested by PartitionFinder (Lanfear et al. 2016, Lanfear et al. 2012, Guindon et al. 2010), a Markov Chain Monte Carlo (MCMC) chain length of 10000000 generations, a subsampling frequency of every 4000 generations, the first 100000 generations were discarded as burn-in, four heated chains and a chain temperature parameter of 0.2. Calculations were performed on the UBELIx (http://www.id.unibe.ch/hpc), the HPC cluster at the University of Bern.

The single gene alignments of COI, 16S, and ITS2 were imported into MEGA X 10.1.7 (Kumar et al. 2018) and the various sequences grouped into species. The average evolutionary divergence between sequence pairs within species (subsequently referred to as within-species divergence) was estimated where possible (only for species with more than one sequence present) using the Maximum Composite Likelihood model (Tamura et al. 2004) on standard settings. The Maximum Composite Likelihood model was also used to estimate the average evolutionary divergence between sequence pairs between species (subsequently referred to as between-species divergence). The focus of the analyses lay on the Z. pretneri group (as defined by Inäbnit et al. 2019; all markers) and the Z. alpestre group (only COI, with the Austrian specimens from Kruckenhauser et al. 2019) classified as separate species or included in Z. amoenum.

Additionally, an Automatic Barcode Gap Discovery (ABGD; Puillandre et al. 2011; https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html) analysis was performed on the COI alignments of the Z. pretneri group and of the Z. alpestre group using the default settings (Pmin = 0.001, Pmax = 0.1, Steps = 10, X = 1.5, Nb bins = 20, distance = Jukes-Cantor).

A map (Fig. 1) was constructed using the Natural Earth dataset in QGIS 3.16.3. Most locality data was taken from Inäbnit et al. (2019), and the coordinates for the Austrian sites were taken from Kruckenhauser et al. (2019). Locality data of the specimens sequenced in this study were provided by the various collectors.

Results

Phylogenetic trees

Both the ML and the BI trees (see Fig. 2 for the latter) are more or less identical. The specimens sequenced in this study clustered with Z. pretneri, Z. tholussum, and Z. manitaense. In both trees they form a badly supported monophyletic group that splits again into two groups in accordance with their geographical distribution (see Fig. 1) and could be separated at the species level: the two specimens from the region of Dubrovnik, Croatia (Špilja Jezero; referred to as Z. aff. troglobalcanicum), and the remaining specimens from Bosnia-Herzegovina (Jama u kamenolomu, Vranjača, Jama Dobravljevac; described as Z. simplex sp. nov. herein). The latter group is not supported in either tree but recovered in both. An additional specimen (NMBE 568054, Špilja Dahna), from which we were only able to amplify H3, didn’t cluster with any species within the Z. pretneri group. The two groups were also recovered, though here with high node support, in the additional ML tree (Suplementary tree 1) calculated for the Z. pretneri group. The Austrian specimens from Kruckenhauser et al. (2019) form a strongly supported monophyletic group within Z. amoenum.

Figure 2. 

Bayesian tree of the genus Zospeum. Node support values of both the Bayesian Inference (front) and the Maximum Likelihood analysis (back) are given. Branches are coloured to denote the informal species groups within the eastern radiation of Zospeum following Inäbnit et al. (2019). Coloured sample names indicate specimens not included in the tree in Inäbnit et al. (2019): blue: Austrian specimens from Kruckenhauser et al. (2019); dark green: Zospeum troglobalcanicum; light green: Zospeum simplex sp. nov.

Divergences

For most markers, intraspecific divergences among the species in the Z. pretneri group are clearly smaller than the interspecific divergences (Table 3). This indicates that these species comprise separate lineages, especially the specimens classified as Z. aff. troglobalcanicum and those collected in Bosnia (henceforth referred to as Z. simplex sp. nov.), which were not included in previous genetic studies (see Inäbnit et al. 2019).

Table 3.

The number of base substitutions per site from averaging over all sequence pairs within (within-species divergences) and between (between-species divergences) species within the Z. pretneri group. Results shown for each marker separately. Between-species distances are listed below the black, empty boxes, the Standard errors above.

COI
Species No. of sequences Within-species divergences Between-species divergences
Divergence Standard Error Z. tholussum Z. pretneri Z. manitaense Z. simplex sp. nov. Z. aff. troglobalcanicum
Z. tholussum 1 0.0126 0.0152 0.0148 0.0142
Z. pretneri 1 0.0602 0.0123 0.0148 0.0123
Z. manitaense 1 0.0849 0.0618 0.0161 0.0167
Z. simplex sp. nov. 9 0.0034 0.0018 0.0765 0.0779 0.0882 0.0133
Z. aff. troglobalcanicum 2 0.0288 0.0078 0.0777 0.0628 0.0974 0.0724
16S
Species No. of sequences Within-species divergences Between-species divergences
Divergence Standard Error Z. pretneri Z. manitaense Z. simplex sp. nov.
Z. pretneri 1 0.0079 0.0097
Z. manitaense 2 0.0045 0.0031 0.0302 0.0078
Z. simplex sp. nov. 9 0.005 0.0022 0.0389 0.0301
ITS2
Species No. of sequences Within-species divergences Between-species divergences
Divergence Standard Error Z. simplex sp. nov. Z. manitaense Z. aff. troglobalcanicum
Z. simplex sp. nov. 8 0.012 0.003 0.0055 0.0056
Z. manitaense 1 0.0226 0.0074
Z. aff. troglobalcanicum 2 0.0072 0.0035 0.0219 0.0278

Zospeum amoenum shows a high intraspecific divergence when compared to other members of the Z. alpestre group (see Table 4), though other species (such as Z. aff. troglobalcanicum, see Table 3) show similarly high intraspecific divergence. When the Austrian populations from Kruckenhauser et al. (2019) are aligned within Z. amoenum, the interspecific divergence within the Z. alpestre group ranges between 0.0564–0.067. The between-group divergence amongst Z. amoenum sensu Inäbnit et al. (2019) and the specimens from Kruckenhauser et al. (2019) was smaller (0.0348±0.0071) than that amidst the other species within the Z. alpestre group, but still higher than the within-group divergence in both Z. amoenum and the Austrian specimens.

Table 4.

The number of base substitutions per site from averaging over all sequence pairs within (within-species divergences) and between (between-species divergences) species within the Z. alpestre group for the marker COI. Shown are results, where the four Austrian specimens were considered a separate species and where the Austrian specimens were considered conspecific with Z. amoenum. Between-species distances are listed below the black, empty boxes, the Standard errors above.

Austrian populations treated as a separate species
Species No. of sequences Within-species divergences Between-species divergences
Divergence Standard Error Z. amoenum Austrian pops. Z. alpestre Z. isselianum Z. kupitzense
Z. amoenum 5 0.0203 0.0048 0.0071 0.0105 0.0104 0.0118
Austrian pops. 4 0.0062 0.0026 0.0348 0.0117 0.0107 0.0126
Z. alpestre 2 0.0098 0.0039 0.0564 0.0629 0.0133 0.013
Z. isselianum 1 0.0554 0.0524 0.0693 0.0131
Z. kupitzense 1 0.067 0.0704 0.075 0.0718
Austrian populations included in Z. amoenum
Species No. of sequences Within-species divergences Between-species divergences
Divergence Standard Error Z. amoenum Z. alpestre Z. isselianum Z. kupitzense
Z. amoenum 9 0.02599 0.0055 0.0109 0.0099 0.0112
Z. alpestre 2 0.0098 0.004 0.0593 0.013 0.0129
Z. isselianum 1 0.0541 0.0693 0.0127
Z. kupitzense 1 0.0685 0.075 0.0718

Automatic Barcode Gap Discovery (ABGD)

The ABGD run on the Z. pretneri-group COI alignment yielded two different possible subdivision schemes: one where the alignment was subdivided into five groups (five groups scheme; prior maximal distance P = 7.74e-03; barcode gap distance: 0.043) and a second where the alignment was subdivided into seven groups (seven groups scheme; prior maximal distance P = 4.64e-03; barcode gap distance: 0.003). Both subdivision schemes considered the previously published sequences of Z. pretneri, Z. tholussum, and Z. manitaense as separate groups. The five-group scheme separated the individuals sequenced in this study into a Croatian group (Špilja Jezero) and a Bosnian group (Jama Dobravljevac, Jama u kamenolomu, Vranjača), while the seven-group scheme separated those individuals into two Croatian groups (one for each of the two specimens from Špilja Jezero) and two Bosnian groups (1: specimens from Jama u kamenolomu; 2: specimens from Jama Dobravljevac and Vranjača).

The ABGD run on the Z. alpestre-group COI alignment yielded one subdivision scheme with seven groups (prior maximal distance P = 4.64e-03; barcode gap distance: 0.016): Z. isselianum, Z. alpestre, Z. kupitzense, Z. amoenum from Ihanščica, Z. amoenum from Konečka zijalka, Z. amoenum from Potočka zijalka and Zospeum sp. from Austria.

Taxonomic implications

Zospeum simplex Inäbnit, Jochum & Neubert, sp. nov.

Figures 1, 3

Type specimens

Holotype: NMBE 568060, Jama Dobravljevac, 25.08.2019, leg. R. Slapnik & J. Valentinčič; Paratypes: NMBE 568061–568063; SMF 349425, 4 shells; RSC 3760, 6 shells; Jama Dobravljevac, 25.08.2019, leg. R. Slapnik & J. Valentinčič.

Specimens examined

NMBE 568054, Špilja Dahna, 03.09.2009, leg. A. Schoenhoffer; NMBE 568055–568058, Jama u kamenolomu, 24.08.2019, leg. R. Slapnik & J. Valentinčič; NMBE 568059, Vranjača, 24.08.2019, leg. R. Slapnik & J. Valentinčič.

Diagnosis

Shell usually ca. 1.3 mm in height, transparent, conical, peristome thickened, roundish, with a differentiated parietal shield, lamellae not present.

Measurements (n = 9): Shell height: 1.26–1.42 mm (mean: 1.378 ± 0.047 mm); shell width: 0.93–1.04 mm (mean: 0.976 ± 0.035 mm); aperture height: 0.54–0.67 mm (mean: 0.6 ± 0.037); aperture width: 0.54–0.65 mm (mean: 0.601 ± 0.033 mm); number of whorls: 5–5.5.

Description

Shell conical, translucent when fresh; suture deep; aperture somewhat roundish to reniform; parietal shield clearly differentiated from the rest of the lip, straight and thin; no lamellae present.

Differing from Z. pretneri and Z. tholussum by its broader shell and the differentiated parietal shield; differs from Z. manitaense by the absence of a visible parietalis in the aperture; barely differs from Z. aff. troglobalcanicum morphologically, on average with reduced shell broadness and a slightly deeper suture (see Remarks).

Distribution

Known from four caves (Jama Dobravljevac, Špilja Dahna, Jama u kamenolomu, Vranjača) in the municipality of Tomislavgrad in Bosnia-Herzegovina.

Etymology

Named simplex (= simple, unsophisticated) due to the lack of any form of shell sculpture or lamellae.

Remarks

Difficult to separate from Z. troglobalcanicum without genetic data (which is not uncommon in Zospeum; see Inäbnit et al. 2019). Both species have a nondescript shell without prominent shell sculpture or lamellae within the aperture. Absolon’s (1916) description of Z. troglobalcanicum consisted out of a photograph depicting multiple specimens haphazardly clustered together in various positions and a legend that established the name and type locality. The lack of a written characterisation of the species in the original description and the fact that the specimens in the photograph weren’t depicted in any standardised position makes a characterisation of the species fairly challenging (putative syntype specimen, collected by K. Absolon from the type locality, was only located very recently by AJ in Vienna (NHMW Mol.Coll.Edlauer 32.749) and couldn’t be studied yet). From the photograph in Absolon (1916), the species can be characterised as similar to Z. manitaense in shell shape, without any visible lamella in the aperture and with a comparatively large parietal shield. The larger parietal shield might serve as a distinguishing character between Z. simplex and Z. troglobalcanicum, though the illustration of a topotypic specimen in Bole (1974; fig. 3h) might indicate that this character is variable within the population. The two specimens we preliminarily assigned to Z. troglobalcanicum (Fig. 3, NMBE 568052; Inäbnit et al. 2019: fig. 7u) only have a small parietal shield. As of now, the shell height:shell width ratio seems to be the most effective way of separating the two specimens from Z. simplex (Z. simplex: generally higher than 1.3 (one exception); Z. aff. troglobalcanicum: below 1.3), but that might just be due to the low sample sizes. Investigation of the inner aspects of the shells will be presented in a later work.

Figure 3. 

Specimens sequenced in this study. Zospeum troglobalcanicum: NMBE 568052 & 568053 (both from Špilja Jezero); Zospeum simplex sp. nov.: NMBE 568054 (Špilja Dahna), NMBE 568055–568057 (Jama u kamenolomu), NMBE 568059 (Vranjača), NMBE 568060 (Holotype, Jama Dobravljevac), NMBE 568061–568063 (Paratypes, Jama Dobravljevac)

Discussion

The phylogenetic tree reconstructions (Fig. 2) agree mostly with those figured in Inäbnit et al. (2019). The main difference is that the node support values within the Z. pretneri group and in that of Z. amoenum are now fairly low and the topology is different. This can be explained by the high number of new specimens that sometimes are only represented by one marker (especially in Z. amoenum). It should also be noted that our current trees resolve Z. robustum, for which we didn’t have any new specimens, with a significant node support as a monophyletic group (node support was not significant in Inäbnit et al. 2019, but the classification as an independent species could be justified via species delimitation methods). Since its position was not resolved with significant node support in either tree, the specimen from Tonkovića špilja is not included in Z. robustum in this tree, as was the case in Inäbnit et al. (2019). Due to lack of additional material, the classification within Z. robustum remains unchanged in this work.

The 12 Zospeum individuals from Bosnia-Herzegovina and Croatia, are the first to be molecularly assessed from the greatly understudied, southern extension of Zospeum’s distribution. Within the phylogenetic trees (Fig. 2, Suppl. material 1), these specimens form a monophyletic group with a deep split between the two specimens from Croatia and nine of the ten specimens from Bosnia-Herzegovina (the remaining specimen from Špilja Dahna is only represented by a sequence of the conservative histone H3 gene, which doesn’t usually resolve to species level).While recovered in all phylogenetic trees calculated for this work, this arrangement only has high node support values in the Suppl. material 1, which was calculated without the conservative H3 and 28S nuclear markers. This result might indicate that conservative markers may have a destabilising effect on species level phylogeny within this group. Both ABGD schemes support the separation of the Croatian and Bosnia-Herzegovina individuals from each other at species level, though the seven-group scheme further subdivided the specimens from both geographical regions. We prefer to use the five-group scheme for the following reasons here: a) The barcode gap of the seven-group scheme is much lower (0.003) than the barcode gap (0.032) that was detected in the Carychiidae alignment in Weigand et al. (2011), while the barcode gap in the five-group scheme was slightly higher (0.043) than in Weigand et al. (2011); b) both individuals from Croatia (considered separate groups in the seven-group scheme) derive from the same cave and are unambiguously recovered as monophyletic and closely related in all trees, making their status as separate taxa unlikely. The divergence analysis further corroborates the results of the ABGD five-group scheme whereby the between-group divergence between the Croatian and the Bosnian groups (see Table 3) was within the general range of interspecific divergence within the Z. pretneri group. We thus, propose separating the individuals sequenced in this study into two species:

  • A species encompassing all ten specimens from Bosnia-Herzegovina. This species is described as Z. simplex sp. nov. above. Since we do not have enough molecular and morphological data for the individual from Špilja Dahna, we cannot confidently place it within Z. simplex right now. However, due to its close geo graphical proximity (less than 1 km) to one of the caves with genetically identified specimens (Jama u kamenolomu), we expect it could well be assignable to Z. simplex as no external morphological inconsistencies separate it from other Z. simplex specimens in our study.
  • A species comprising two specimens from Špilja Jezero in the region of Dubrovnik. This locality is fairly close (around 22 km) to the type locality (Benetina pećina) of Z. troglobalcanicum Absolon, 1916. The sequenced specimens do not show any major external morphological differences from the specimen identified as Z. troglobalcanicum (as figured in Bole 1974: fig. 3h) and from those imaged in Inäbnit et al. 2019: fig. 7u), though the adult specimen clearly has a smaller parietal shield than the specimens figured in Absolon (1916). We propose tentatively classifying those specimens within Z. troglobalcanicum until genetic material from the type locality can clarify its status and the morphological investigation of the singular syntype (NHMW Mol.Coll.Edlauer 32.749) of this species can be taxonomically and nomenclaturally clarified in a separate work.

Even if it is not as large as the between-group divergence of other species pairs within the Z. alpestre group, our divergence analysis revealed that the between-group divergence between Z. amoenum and the two Austrian populations is greater than the within-group divergence of either lineage. Our analysis also found that the within-group divergence in Z. amoenum is only slightly increased if the Austrian populations are included within this species. These results agree with the tree reconstruction published in Kruckenhauser et al. (2019), which resolved the Austrian population as the sister group of Z. amoenum. Our trees, as mentioned above, lack the resolution to separate the Austrian populations from Z. amoenum and can thus, not confirm this conclusion. The ABGD scheme for the Z. alpestre group recovers the Austrian population as a separate group from Z. amoenum and splits the latter species into three groups. The barcode gap in this scheme is, however, much lower (0.016) than the one proposed for Carychiidae in Weigand et al. (2011), which was used for species classification within the Z. alpestre group before (e.g., in Weigand et al. 2013). We are thus, reluctant to draw conclusions regarding Z. amoenum and the Austrian specimens from the ABGD scheme. It may indicate some large intraspecific genetic variability within Z. amoenum (with the possibility of the presence of several species) that might coincide with the large morphological variation found in this species (Inäbnit et al. 2019), which would need to be addressed in a separate study with better sampling.

Zospeum amoenum described in Inäbnit et al. (2019) bears either a small parietalis that does not expand within the shell or it is lacking completely. Kruckenhauser et al. (2019) did not figure a specimen in which the configuration of the parietalis within the last whorl could be seen, but Gittenberger (1982) figured one specimen from the Hafnerhöhle (one of the two caves sampled by Kruckenhauser et al. 2019), where the parietalis was exposed. The parietalis of this specimen is slightly broadened three quarters of a whorl into the shell and seems to decrease expansion again further into the shell. Though the syntype of Z. amoenum (see Inäbnit et al. 2019: fig. 6L) shows a similar configuration of the parietalis, it is not congruent with the description of this structure in Z. amoenum assessed in Inäbnit et al. (2019).

Our study suggests that a final species assignment for the two Austrian populations is not possible until further supporting information becomes available. Until then, we classify these two Austrian populations as Z. amoenum, avoiding the now outdated classification of these populations with Z. isselianum (as was done in Kruckenhauser et al. 2019).

Acknowledgements

We thank Estée Bochud for her help in data retrieval. We also thank Jana Valentinčič for her assistance in the field and Axel Schoenhoffer for providing us with samples for analysis.

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