Research Article |
Corresponding author: Thomas Wesener ( twesener@uni-bonn.de ) Academic editor: Ivan H. Tuf
© 2015 Thomas Wesener, Karin Voigtländer, Peter Decker, Jan Philip Oeyen, Jörg Spelda, Norman Lindner.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wesener T, Voigtländer K, Decker P, Oeyen JF, Spelda J, Lindner N (2015) First results of the German Barcode of Life (GBOL) – Myriapoda project: Cryptic lineages in German Stenotaenia linearis (Koch, 1835) (Chilopoda, Geophilomorpha). In: Tuf IH, Tajovský K (Eds) Proceedings of the 16th International Congress of Myriapodology, Olomouc, Czech Republic. ZooKeys 510: 15-29. https://doi.org/10.3897/zookeys.510.8852
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As part of the German Barcode of Life (GBOL) Myriapoda program, which aims to sequence the COI barcoding fragment for 2000 specimens of Germany’s 200 myriapod species in the near future, 44 sequences of the centipede order Geophilomorpha are analyzed. The analyses are limited to the genera Geophilus Leach, 1814 and Stenotaenia Koch, 1847 and include a total of six species. A special focus is Stenotaenia, of which 19 specimens from southern, western and eastern Germany could be successfully sequenced. The Stenotaenia data shows the presence of three to four vastly different (13.7–16.7% p-distance) lineages of the genus in Germany. At least two of the three lineages show a wide distribution across Germany, only the lineage including topotypes of S. linearis shows a more restricted distribution in southern Germany. In a maximum likelihood phylogenetic analysis the Italian species S. ‘sorrentina’ (Attems, 1903) groups with the different German S. linearis clades. The strongly different Stenotaenia linearis lineages within Germany, independent of geography, are a strong hint for the presence of additional, cryptic Stenotaenia species in Germany.
Barcode, biodiversity, COI, cryptic diversity
The German Barcode of Life – Myriapoda project aims to sequence part of the mitochondrial cytochrome c oxidase subunit I gene known as the barcode fragment for all approximately 200 Myriapoda species in Germany (
Here, we show the preliminary results of one of the largest barcoding datasets compiled for centipedes of the order Geophilomorpha, with a special focus on the recently revised Stenotaenia Koch, 1847 (
The taxonomic situation of the type species of Stenotaenia, S. linearis, is slightly confused, as the original Koch type specimens from Regensburg, Germany are apparently lost (
Molecularly, little was done in Stenotaenia. One specimen of S. linearis was used for the Fauna Bavarica project (
Specimens were determined and collected by the authors of the study by hand, and either directly or after a few days transferred to vials containing 95% undenatured ethanol. The vials contain an individual GBOL number with which the specimens can be connected to the accompanying data. After conservation the specimens were either sent to the GBOL facility at the Museum Koenig, Bonn, Germany (ZFMK) or to the corresponding laboratory at the Bavarian State collection of Zoology, Munich, Germany (ZSM). Upon arrival, all specimens were photographed (images will be uploaded to BOLD, http://www.boldsystems.org/), and a tissue sample was removed for DNA extraction. All specimens will later be stored as vouchers in 95% undenatured ethanol, either at the ZFMK, the SMNG (Senckenberg Museum für Naturkunde, Görlitz) or the ZSM (see Table
GBOL numbers, Genbank codes, locality data. GBOL number refers to DNA extraction and BOLD registration. SMNG = Senckenberg Museum für Naturkunde, Görlitz, Germany; ZFMK = Zoological Research Museum A. Koenig, Bonn, Germany; ZSM = Zoologische Staatssammlung München, Germany.
GBOL | GenBank | Voucher | Species | Locality |
---|---|---|---|---|
ZFMK-TIS-1318 | KM999124 | SMNG VNR016755-1 | Geophilus alpinus | Saxony, Hirschfelde, Neißetal. |
ZFMK-TIS-1449 | KM999119 | ZFMK MYR3840 | Geophilus alpinus | Saxony, Leipzig, Leipziger Auwald, Revierort „Die Nonne“ |
ZFMK-TIS-1520 | KM999120 | ZFMK MYR3871 | Geophilus alpinus | Saxony, Jähstadt, Annaberger Ratswald. |
ZFMK-TIS-1560 | KM999118 | ZFMK MYR3875 | Geophilus alpinus | Bavaria, Donaustauf. |
ZFMK-TIS-1647 | KM491674 | ZFMK MYR3720 | Geophilus alpinus | Saxony-Anhalt, Ilsenburg, Ilsetal. |
ZFMK-TIS-1656 | KM491579 | ZFMK MYR3725 | Geophilus carpophagus | Saxony-Anhalt, Ilsenburg, Ilsensteinhang. |
ZFMK-TIS-2519834 | KM491622 | ZFMK MYR3813 | Geophilus carpophagus | Saxony-Anhalt, Ilsenburg, Ilsensteinhang. |
ZFMK-TIS-1413 | KM491587 | ZFMK MYR3653 | Geophilus electricus | Saxony-Anhalt, Nordharz, Heudeber. |
ZFMK-TIS-1518 | KM491687 | ZFMK MYR3673 | Geophilus electricus | Saxony-Anhalt, Halberstadt, Athenstedt. |
ZFMK-TIS-1650 | KM491673 | ZFMK MYR3723 | Geophilus electricus | Saxony-Anhalt, Ilsenburg, Dreisageblocksberg. |
ZFMK-TIS-19414 | KM491636 | ZFMK MYR2107 | Geophilus electricus | North Rhine-Westphalia, Windeck, Stromberg. |
ZFMK-TIS-1468 | KM999123 | ZFMK MYR3850 | Geophilus flavus | Saxony, Zwickau, Brückeberg. |
ZFMK-TIS-1525 | KM491642 | ZFMK MYR3676 | Geophilus flavus | Saxony-Anhalt, Schönhausen (Elbe). |
ZFMK-TIS-1603 | KM491670 | ZFMK MYR3705 | Geophilus flavus | Saxony-Anhalt, Gerbstedt, Friedeburg. |
ZFMK-TIS-6359 | KM491617 | ZFMK MYR3536 | Geophilus flavus | Saxony, Gröditz, Weißenberg. |
ZFMK-TIS-15516 | KM491627 | ZFMK MYR1004 | Geophilus flavus | North Rhine-Westphalia, Bonn, Oberkassel Steinbruch. |
ZFMK-TIS-15764 | KM491602 | ZFMK MYR1060 | Geophilus flavus | North Rhine-Westphalia, Wuppertal, NSG ‚Im Hölken‘ |
ZFMK-TIS-15774 | KM491626 | ZFMK MYR1070 | Geophilus flavus | North Rhine-Westphalia, Siebengebirge, Löwenburg. |
ZFMK-TIS-15821 | KM491693 | ZFMK MYR1117 | Geophilus flavus | North Rhine-Westphalia, Wuppertal, Dolinengelände Krutscheid. |
ZFMK-TIS-19577 | KM491685 | ZFMK MYR1526 | Geophilus flavus | North Rhine-Westphalia, Bonn, Kottenforst. |
ZFMK-TIS-19591 | KM491632 | ZFMK MYR1543 | Geophilus flavus | North Rhine-Westphalia, Heimbach, Meuchelberg. |
ZFMK-TIS-19602 | KM491649 | ZFMK MYR1554 | Geophilus flavus | North Rhine-Westphalia, Königswinter, Südhang Wolkenburg. |
ZFMK-DNA-112780112 | KM491570 | ZSM-JSP100815-007 | Geophilus flavus | North Rhine-Westphalia, Bielefeld, Brackweder Wald. |
ZFMK-DNA-112780116 | KM999125 | ZSM-JSP120413-004 | Geophilus flavus | Baden-Württemberg, Bad Urach, St. Johann Fohlenhof. |
ZFMK-DNA-112780042 | KM999126 | ZSM-JSP120413-002 | Geophilus ribauti | Baden-Württemberg, Bad Urach, St. Johann Fohlenhof. |
ZFMK-TIS-19495 | KM999122 | ZFMK MYR1630 | Stenotaenia linearis | North Rhine-Westphalia, Bonn, Oberkassel Steinbruch. |
ZFMK-TIS-1450 | KM999121 | ZFMK-TIS-1450 | Stenotaenia linearis | Saxony, Leipzig, Leipziger Auwald, Revierort „Die Nonne“. |
ZFMK-TIS-15771 | KM491663 | ZFMK MYR1067 | Stenotaenia linearis | North Rhine-Westphalia, Wuppertal, NSG ‚Im Hölken‘. |
ZFMK-TIS-15861 | KM491574 | ZFMK MYR1157 | Stenotaenia linearis | North Rhine-Westphalia, Wuppertal, Dolinengelände Krutscheid. |
ZFMK-TIS-19430 | KM491573 | ZFMK MYR2030 | Stenotaenia linearis | Rheinland-Pfalz, Altenkirchen, Seelbach bei Hamm. |
ZFMK-DNA-112780045 | KM491689 | ZSM-JSP120412-003 | Stenotaenia linearis | Baden-Württemberg, Esslingen, St. Bernhard. |
ZFMK-DNA-112780062 | KM491558 | ZSM-JSP100514-021 | Stenotaenia linearis | Bavaria, Dachau, palace garden. |
ZFMK-DNA-112780066 | KM491631 | ZSM-JSP120411-001 | Stenotaenia linearis | Baden-Württemberg, Esslingen, St. Bernhard. |
ZFMK-DNA-112780069 | KM491658 | ZSM-JSP120408-007 | Stenotaenia linearis | Baden-Württemberg, Hegnach, Hardtwald. |
ZFMK-DNA-112780093 | KM491637 | ZSM-JSP120408-002 | Stenotaenia linearis | Baden-Württemberg, Stuttgart, SW Max-Eyth-See. |
GBOL11002 | KP698104 | ZSM-JSP141102-010 | Stenotaenia linearis | Bavaria, Regensburg |
GBOL10999 | KP698105 | ZSM-JSP141102-004 | Stenotaenia linearis | Bavaria, Regensburg |
ZFMK-TIS-19423 | KR559681 | ZFMK MYR2119 | Stenotaenia linearis | North Rhine-Westphalia, Euskirchen, Bad Münstereifel, Gilsdorf. |
ZFMK-TIS-2538216 | KR559680 | ZFMK MYR3467 | Stenotaenia linearis | Saxony, Dresden, Gruna |
ZFMK-TIS-1645 | KR559679 | ZFMK MYR3878 | Stenotaenia linearis | Saxony, Zwickau, Brueckeberg |
GBOL12266 | KR736251 | SMNG-VNR016704-1 | Stenotaenia linearis | North Rhine-Westphalia. Bochum, Tippelsberg |
GBOL12450 | KR736248 | ZSM-JSP150117-056 | Stenotaenia linearis | Baden-Württemberg, Breisgau, Badenweiler |
GBOL12421 | KR736250 | SMNG-MYR016705-1 | Stenotaenia linearis | North Rhine-Westphalia. Unna, Selm |
GBOL11224 | KR736249 | ZSM-JSP141113-005 | Stenotaenia linearis | Baden-Württemberg, Ulm, Kiesental |
At the ZFMK, DNA was extracted from the tissue samples using the BioSprint96 magnetic bead extractor by Qiagen (Germany). After the extraction, samples were outsourced for PCR and sequencing (BGI China). For PCR and sequencing, HCO/LCO primer pairs (
However, for more than five S. linearis and more than 10 Geophilus specimens no sequences could be obtained. Sequences were obtained for 19 Stenotaenia and 25 Geophilus specimens. Sequence identities were confirmed with BLAST searches (
Sequences were aligned by hand in Bioedit (
The evolutionary history was inferred by using the maximum likelihood method based on the selected Tamura-Nei model (
The number of pairwise base differences per site were calculated in MEGA6 (
Geophilus is not clearly separated from Stenotaenia in our analysis (Fig.
All 13 specimens of G. flavus show little genetic distance (0–2.4%) to one another. Within the group containing the remaining Geophilus species, G. ribauti Brölemann, 1908, a species formerly treated as a member of the genus Brachygeophilus Brölemann, 1908, is in a basal position to a weakly supported clade (64% statistical support) including G. electricus (Linné, 1758), G. carpophagus Leach, 1814, and G. alpinus Meinert, 1870. In this clade, G. electricus (100% statistical support) is opposed to the sister-taxa G. carpophagus and G. alpinus (83% statistical support). Inside G. electricus, the one specimen from western Germany is opposed to the three from Saxony-Anhalt (Table
Within Stenotaenia, a basal trichotomy separates the specimens into (1) S. linearis L1, (2) S. ‘sorrentina’, and (3) the weakly supported (56% bootstrap support) S. linearis L2 (including the topotypes) together with S. linearis L3 (Fig.
The distance analysis shows a first cluster of intraspecific distances ranging from 0–2.8%, with a G. electricus outlier at 4.9% (Fig.
Clear intraspecific distances in German Geophilomorpha range from 0–5% (Fig.
The three German Stenotaenia lineages are only weakly geographically separated (Fig.
Map of S. linearis samples studied during GBOL (large dots), as well as other S. linearis records from Edaphobase, the ZSM and ZFMK collection (small dots, status 10.2014). Yellow = S. linearis L1; Blue = S. linearis L2; Green = S. linearis L3. (A) S. linearis in the field, photo: J. Spelda, specimen from Stuttgart-Hofen, Zuckerberg.
Stenotaenia L2 represents topotypic material from Regensburg, a specimen from the Kiesental near Ulm, as well as a single specimen from Dachau in southern Germany. All three localities are more than 100 km apart but only the specimen from Dachau differs by 1.4%. S. L2 differs significantly (13.7–16.7%) from other German Stenotaenia. This clade might be characterised as of southeastern German origin along the Danube river system.
Both clades of lineage 3, one from western and eastern Germany (L3a), the other from SW Germany (L3b) show identical haplotypes, but differ from one another by 9.4% (Fig.
Whether or not the apparent sympatric distribution of the three different lineages of Stenotaenia in Germany (Fig.
Such a large project faces a set of predictable technical problems, which can potentially cause wrong results.
Specimen collections: According to the main aim of the project (get approx. 10 specimens from at least five localities for each species to capture the estimated German-wide COI variation), the different collectors preferred localities where they could find many myriapod specimens easily – a potential collection bias. The amount of successfully sequenced S. linearis specimens as well as G. alpinus specimens and their different positions and deep splits within the maximum likelihood tree (Fig.
Specimen determination: As done by
Our analysis shows the importance of COI barcode data in the detection of taxonomic problems inside the centipede order Geophilomorpha. However, it also illustrates that barcode data alone does not clarify taxonomic problems. Only a thorough morphological study of the Stenotaenia species, including the types, plus the addition of nuclear markers, may be able to solve the complex picture of this genus.
As a result of the voucher-based barcoding effort, all analyzed specimens, and even their DNA extracts, are available for loan and should be incorporated into any future study of Stenotaenia.
Collection permits for natural protection areas were thankfully provided by the following people: H. Mölleken (Ressort Umweltschutz, Stadt Wuppertal), W. Wasch (Personal- und Organisationsamt, Bundesstadt Bonn), M. Ehling (Struktur- und Genehmigungsdirektion Nord, Rhineland-Palatine), F. Makiolczyk (Amt für Natur- und Landschaftsschutz, Rhein-Sieg-Kreis). T. Klug (ZFMK) provided assistance in collecting and determining the specimens. B. Rulik, J. Thormann, L. von der Mark form the GBOL-Team in Bonn and photographed, extracted and sequenced the ZFMK specimens, their invaluable help is greatly appreciated. M. Geiger assisted with the upload of the sequence data to GenBank. Special thanks go to M. Balke, F. Glaw, A. Hausmann, O. Hawlitschek, R. Melzer, J. Moriniere, I. Stöger, S. Schmidt for discussion, F. Ceseña, S. Friedrich, T. Lehmann, T. Meier, V. Svara, S. Swoboda and U. Biener-Miller (all ZSM) for sorting and preparing the ZSM samples. H. E. Wesener thankfully corrected the English of the manuscript. Two anonymous reviewers provided numerous comments that improved the quality of the here presented work.
This is a publication of the German Barcode of Life (GBOL) project of the Humboldt Ring, financed by the German Federal Ministry for Education and Research.
Table. Estimates of Evolutionary Divergence between Sequences
Data type: Measurement
Explanation note: The number of base differences per site from between sequences are shown. The analysis involved 45 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 658 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.