Corresponding author: Thomas Wesener (
Academic editor: Ivan H. Tuf
As part of the German Barcode of Life (GBOL)
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
The German Barcode of Life –
Here, we show the preliminary results of one of the largest barcoding datasets compiled for centipedes of the order
The taxonomic situation of the type species of
Molecularly, little was done in
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,
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 |
|
SMNG VNR016755-1 |
|
Saxony, Hirschfelde, Neißetal. |
ZFMK-TIS-1449 |
|
ZFMK MYR3840 |
|
Saxony, Leipzig, Leipziger Auwald, Revierort „Die Nonne“ |
ZFMK-TIS-1520 |
|
ZFMK MYR3871 |
|
Saxony, Jähstadt, Annaberger Ratswald. |
ZFMK-TIS-1560 |
|
ZFMK MYR3875 |
|
Bavaria, Donaustauf. |
ZFMK-TIS-1647 |
|
ZFMK MYR3720 |
|
Saxony-Anhalt, Ilsenburg, Ilsetal. |
ZFMK-TIS-1656 |
|
ZFMK MYR3725 |
|
Saxony-Anhalt, Ilsenburg, Ilsensteinhang. |
ZFMK-TIS-2519834 |
|
ZFMK MYR3813 |
|
Saxony-Anhalt, Ilsenburg, Ilsensteinhang. |
ZFMK-TIS-1413 |
|
ZFMK MYR3653 |
|
Saxony-Anhalt, Nordharz, Heudeber. |
ZFMK-TIS-1518 |
|
ZFMK MYR3673 |
|
Saxony-Anhalt, Halberstadt, Athenstedt. |
ZFMK-TIS-1650 |
|
ZFMK MYR3723 |
|
Saxony-Anhalt, Ilsenburg, Dreisageblocksberg. |
ZFMK-TIS-19414 |
|
ZFMK MYR2107 |
|
North Rhine-Westphalia, Windeck, Stromberg. |
ZFMK-TIS-1468 |
|
ZFMK MYR3850 |
|
Saxony, Zwickau, Brückeberg. |
ZFMK-TIS-1525 |
|
ZFMK MYR3676 |
|
Saxony-Anhalt, Schönhausen (Elbe). |
ZFMK-TIS-1603 |
|
ZFMK MYR3705 |
|
Saxony-Anhalt, Gerbstedt, Friedeburg. |
ZFMK-TIS-6359 |
|
ZFMK MYR3536 |
|
Saxony, Gröditz, Weißenberg. |
ZFMK-TIS-15516 |
|
ZFMK MYR1004 |
|
North Rhine-Westphalia, Bonn, Oberkassel Steinbruch. |
ZFMK-TIS-15764 |
|
ZFMK MYR1060 |
|
North Rhine-Westphalia, Wuppertal, NSG ‚Im Hölken‘ |
ZFMK-TIS-15774 |
|
ZFMK MYR1070 |
|
North Rhine-Westphalia, Siebengebirge, Löwenburg. |
ZFMK-TIS-15821 |
|
ZFMK MYR1117 |
|
North Rhine-Westphalia, Wuppertal, Dolinengelände Krutscheid. |
ZFMK-TIS-19577 |
|
ZFMK MYR1526 |
|
North Rhine-Westphalia, Bonn, Kottenforst. |
ZFMK-TIS-19591 |
|
ZFMK MYR1543 |
|
North Rhine-Westphalia, Heimbach, Meuchelberg. |
ZFMK-TIS-19602 |
|
ZFMK MYR1554 |
|
North Rhine-Westphalia, Königswinter, Südhang Wolkenburg. |
ZFMK-DNA-112780112 |
|
ZSM-JSP100815-007 |
|
North Rhine-Westphalia, Bielefeld, Brackweder Wald. |
ZFMK-DNA-112780116 |
|
ZSM-JSP120413-004 |
|
Baden-Württemberg, Bad Urach, St. Johann Fohlenhof. |
ZFMK-DNA-112780042 |
|
ZSM-JSP120413-002 |
|
Baden-Württemberg, Bad Urach, St. Johann Fohlenhof. |
ZFMK-TIS-19495 |
|
ZFMK MYR1630 |
|
North Rhine-Westphalia, Bonn, Oberkassel Steinbruch. |
ZFMK-TIS-1450 |
|
ZFMK-TIS-1450 |
|
Saxony, Leipzig, Leipziger Auwald, Revierort „Die Nonne“. |
ZFMK-TIS-15771 |
|
ZFMK MYR1067 |
|
North Rhine-Westphalia, Wuppertal, NSG ‚Im Hölken‘. |
ZFMK-TIS-15861 |
|
ZFMK MYR1157 |
|
North Rhine-Westphalia, Wuppertal, Dolinengelände Krutscheid. |
ZFMK-TIS-19430 |
|
ZFMK MYR2030 |
|
Rheinland-Pfalz, Altenkirchen, Seelbach bei Hamm. |
ZFMK-DNA-112780045 |
|
ZSM-JSP120412-003 |
|
Baden-Württemberg, Esslingen, St. Bernhard. |
ZFMK-DNA-112780062 |
|
ZSM-JSP100514-021 |
|
Bavaria, Dachau, palace garden. |
ZFMK-DNA-112780066 |
|
ZSM-JSP120411-001 |
|
Baden-Württemberg, Esslingen, St. Bernhard. |
ZFMK-DNA-112780069 |
|
ZSM-JSP120408-007 |
|
Baden-Württemberg, Hegnach, Hardtwald. |
ZFMK-DNA-112780093 |
|
ZSM-JSP120408-002 |
|
Baden-Württemberg, Stuttgart, SW Max-Eyth-See. |
GBOL11002 |
|
ZSM-JSP141102-010 |
|
Bavaria, Regensburg |
GBOL10999 |
|
ZSM-JSP141102-004 |
|
Bavaria, Regensburg |
ZFMK-TIS-19423 |
|
ZFMK MYR2119 |
|
North Rhine-Westphalia, Euskirchen, Bad Münstereifel, Gilsdorf. |
ZFMK-TIS-2538216 |
|
ZFMK MYR3467 |
|
Saxony, Dresden, Gruna |
ZFMK-TIS-1645 |
|
ZFMK MYR3878 |
|
Saxony, Zwickau, Brueckeberg |
GBOL12266 |
|
SMNG-VNR016704-1 |
|
North Rhine-Westphalia. Bochum, Tippelsberg |
GBOL12450 |
|
ZSM-JSP150117-056 |
|
Baden-Württemberg, Breisgau, Badenweiler |
GBOL12421 |
|
SMNG-MYR016705-1 |
|
North Rhine-Westphalia. Unna, Selm |
GBOL11224 |
|
ZSM-JSP141113-005 |
|
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
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 (
Maximum likelihood tree, 1000 bootstrap replicates. L1–L3 =
All 13 specimens of
Within
The distance analysis shows a first cluster of intraspecific distances ranging from 0–2.8%, with a
Frequency distribution of pairwise intraspecific (blue) and interspecific (red) distances. Blue circle = intraspecific distances of
Clear intraspecific distances in German
The three German
Map of
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
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
Specimen determination: As done by
Our analysis shows the importance of COI barcode data in the detection of taxonomic problems inside the centipede order
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
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
Measurement
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.