Corresponding author: Thomas Wesener (
Academic editor: Pavel Stoev
Three populations of the pill millipede genus
Some of the most ornamented members of the pill millipedes (order
The position of
Even more problematic than the position of
Finding and collecting
Adding to these morphological peculiarities, sequencing of
In 1984, an isolated population of
For a comprehensive analysis and evaluation of the British
To investigate the morphological and molecular diversity of the British
As near outgroups, two female specimens of undetermined Italian
All animals were collected by hand. Two slightly different preservation methods were utilized: (1) Immediately after capture, specimens were put into 1.8 ml screw top vials with 98% ethanol; British
Before the disintegration of the specimens for the removal of tissue and SEM preparations, colored multi-layer photographs of the enrolled specimens were taken under a Leica Z6 Imaging-System. A total of 11 specimens from the Isle of Wight population, and 8 from the Welsh population were photographed. For optimal depth of field, the 10-15 single photographs taken from each specimen were put together into one multi-layer photograph using the software Auto-Montage.
The tiny size of the specimens made DNA extraction difficult. In at least one case, the animal was filled with a long and massive nematomorph >4x the length of the host specimen. Due to the possible presence of parasites and the fact that no sequences of the genus (or family) were present on NCBI GenBank for comparison, no whole body extraction was utilized, but every specimen was carefully dissected. Intersegmental muscle tissue was chosen as the extraction target. Due to the small size of the specimens, the specimen was pulled apart along the tergite margins and divided into several parts, where the muscle tissue binding the tergites could be removed with fine forceps. Dissected muscle tissue was washed in a dish of ethanol to remove attached particles of the intestine, which are usually filled with cephaline gregarines.
The muscle tissue was processed with a DNAeasy Blood & Tissue kit from Qiagen following the manufacturer’s extraction protocol, except that two times 50 µl elution buffer were used to heighten the DNA yield of the extraction. DNA was extracted from a total of 44 specimens: 13 specimens of
To gain insight into the genetic diversity of the British
Purified PCR products from 34 specimens were outsourced for double-strand sequencing to a contract sequencing facility (Macrogen, Seoul, Korea) on an ABI3730 XL automatic DNA sequencer, using the same primer sets as for PCR. Sequences of a total of 27 specimens (13x Isle of Wight, 12x Wales, 2x Italy) could be obtained, while the sequences for the French
Sequencing reads were assembled with Bioedit 7.1.3. (
The analysis involved 32 nucleotide sequences (COI from 32 specimens), with a total of 660 positions in the final dataset. The number of base substitutions per site from between sequences were determined using MEGA (v. 5.2,
The tree with the highest log likelihood (-2177.4216) is shown below (Analysis section). The percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrap). The tree is drawn to scale, with branch lengths reflecting the number of substitutions per site.
To evaluate the intra- and interspecific variation of closely related
A total of 15 (11, 4 of which separately given for male and female) morphological characters commonly employed in the taxonomy of
Addtional individual information in Table
Additional individual information in Table
Additional individual information in Table
Additional individual information in Table
Additional individual information in Table
Additional individual information in Table
Despite differences in the morphology and coloration (see below), no variation in the mitochondrial haplotypes were found within
All
The close relationship of the UK and French
Most of the studied somatic characters show considerable variation (see Table
The only unambiguous morphological characters that neither show great variation within a species, nor are constant between closely related species, are the characters 8–12 (Table
The telopods of the studied
The UK
Success of DNA extraction and PCR was generally low, indicating the necessity of special treatments to ensure success. Some specimens rolled-up so tight that ethanol was not able to penetrate the specimen, and muscle tissue was already partly decayed. We thus recommend to conserve specimens individually and open them shortly after conservation (i.e., a few minutes after death at best) so that ethanole can enter the organism. Furthermore, it is reasonable to not extract sequences from whole specimens but only from muscular and other tissues free of contamination from gut content or parasites. PCR success was improved (i.e., obtaining positive bands at all) with another deviation from standard protocols, namely using 5µl of DNA instead of the usual 1-2 µl. We were not able to study the reason for this necessity and can thus only speculate. Possibilities are insufficient primer matching, decay, or other, inherent peculiarities.
The differing results of sequencing attempts at the ZFMK and BGI despite of presumably standardized methods highlight the persistent difficulties of obtaining glomerid DNA sequences. Here, we deem a further investigation of best practice methods for
For studies of the intraspecific variation of
Between
Telopods need to be studied and illustrated with great care. Slight variations of the angle of view can provide very different observations (compare Figs
Since trachysphaeran surface structures are tiny, complex, and manifold, their evaluation as well as drawing conclusions is obfuscated. Many characters are too fine-scaled or complex to be evaluated in a discrete fashion and it is hard to define categories due to variability in quasi-countable characters. The possible inner variation of extended structures (e.g., endotergum) confounds decisions of species identification based on small details of the structure. However, even small details vary in their peculiarities: large sclerotized protuberances (LSPs; Fig.
Problems did not only arise from the structural diversity itself but also from the method of documentation. Too few pictures were taken and not all angles were available due to fixation. Comparing all specimens, not all structures were shot from the same angle. A better chance to find true species-specific characters and evaluate them properly would be given by zoomable 3D images, as can be obtained by micro-CT studies. Additionally, physical influences may have distorted the evaluation. LSPs and similar extended structures (e.g., setae) especially may have been evaluated inconsistently due to prior abrasion (during struggle in life, dissection, mounting and sputtering). Grooves may have been evaluated incorrectly if clogged by dirt. Here, testing whether ultra-sonic cleaning is feasible would be beneficial (we did not try due to the risk of loosing of one specimen). Given these complications and our relatively small sample size, further corroboration of our results is desirable. Meanwhile, our conclusions have to be considered as preliminary, but nevertheless relevant.
Both, barcoding and the morphological study identify the British
We thank Desmond Kime for his advice and the laborious collecting efforts of French
Body part and tergite nomenclature. SEM micrographs of a female of
lateral view, showing slightly unrolled specimen
same specimen, view of the opening between anal shield and thoracic shield, exposing head and collum (reduced first tergite) usually held inside the sphere when rolled up
Maximum Likelihood Tree of 32 COI sequences of
Habitus. Preserved
TW15:
TW18:
TW20:
TW26:
TW31:
TW32:
TW1:
TW1:
TW29:
TW29:
TW29:
Telopods. SEM micrographs of
anterior view (MBiID
anterior view (MBiID
posterior (anal) view (MBiID
anterior view (MBiID
Telopods. SEM micrographs of
anterior view (MBiID
anterior view (MBiID
posterior (anal) view (MBiID
posterior (anal) view (MBiID
Telopods. SEM micrographs of
Taxonomically informative character examples.
TW30:
GL07:
Localities and method application. Locality ID [LocID] as given in Checklist Materials, Extracted specimens [# Extracted], PCR and sequencing success [# PCR success, # Sequencing success] and number of
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1 | Isle of Wight, GB | 13 | 13 | 13 | 5 |
2 | South Wales, GB | 15 | 15 | 12 | 8 |
3 | Génis, FR | 10 | 4 (2) | - (2) | - |
4 | Grotte de l'Estellas, FR | 1 | - | - | 2 |
5 | Leitza, ESP | 1 | - | - | 1 |
6 | Sare, FR | 1 | - | - | 2 |
7 | Oropa, IT | 2 | 2 | 2 | - |
8 | Velika-Kapela, CRO | 1 | - (1) | - (1) | - |
Characters and states. Character numbers [C #], states are exemplified with SEM images partly in figures, partly with links to MorphBank, then given as MBiIDs.
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Color of freshly preserved specimen. | (0) brownish (Fig. |
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Collum (reduced tergite 1), number of toothed ridges (Fig. |
(0) 4/5 ( |
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Thoracic shield (enlarged tergite 2) anterior margin, number of rows of sclerotized nodules (Fig. |
Real number |
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Thoracic shield, number of rows of large sclerotized protuberances (Fig. |
Real number |
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Endotergum (underside of posterior margin of tergites), structure. | (0) simple margin with one row of sclerotized nodules, and single row of short setae (Fig. |
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Endotergum, number of rows of setae. | (0) 1 (Fig. |
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Endotergum, number of rows of sclerotized nodules. | (0) 1 (Fig. |
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Male tergite 10, posterior margin, number of rows of large bacilli. | (0) 1 ( |
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Female tergite 10, posterior margin, number of rows of large bacilli (Fig. |
(0) 1 ( |
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Male anal shield, shape. | (0) well-rounded ( |
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Female anal shield, shape (Fig. |
0) well-rounded ( |
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Male anal shield, setae at posterior margin. | (0) isolated, 1-2 rows, ( |
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Female anal shield, setae at posterior margin (Fig. |
(0) isolated, 1-2 rows, ( |
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Male anal shield, large circular grooves. | (0) absent or hard to see ( |
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Female anal shield, large circular grooves (Fig. |
(0) absent or hard to see ( |
Character matrix.
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0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |
1 | 0 | 0 | 1 | 0 | 0 | 0 | n/a | 0 | n/a | 1 | n/a | 0 | n/a | 1 | |
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0 |
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0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | |
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0 | 0 |
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0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 |
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0 |
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0 | 0 | 0 | 1 | 1 | 0 | 0 |
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Additional sample information for
Material specimen # | Extraction voucher # | MBspecimenID | GenBank accession # |
a | TW01 |
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b | TW02 | – |
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c | TW03 | – |
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d | TW04 | – |
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e | TW05 | – |
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f | TW11 | – |
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g | TW12 |
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h | TW13 |
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i | TW14 |
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j | TW15 | – |
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k | TW16 | – |
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l | TW17 |
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m | TW18 | – |
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n | TW19 | – |
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o | TW20 | – |
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p | TW21 | – | |
q | TW22 |
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r | TW23 | – |
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s | TW24 |
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t | TW25 |
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u | TW26 |
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v | TW27 | – | |
w | TW28 | – |
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x | TW29 |
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y | TW30 |
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z | TW31 | – |
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aa | TW32 | – |
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ab | TW33 | – | – |
ac | TW47 | – | – |
ad | TW48 | – | – |
ae | TW49 | – | – |
af | TW50 | – | – |
ag | TW51 | – |
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ah | TW52 | – |
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ai | TW53 | – | – |
aj | TW54 | – | – |
ak | TW55 | – | – |
al | TW57 | – | – |
Additional sample information for
Material specimen # | Extraction voucher # | MBspecimenID | GenBank accession # |
a | GEU157 | – | |
b | GEU157 | – |
Additional sample information for
Material specimen # | Extraction voucher # | MBspecimenID | GenBank accession # |
a | GL017 | – |
Additional sample information for
Material specimen # | Extraction voucher # | MBspecimenID | GenBank accession # |
a | GEU158 | – | |
b | GEU158 | – |
Additional sample information for
Material specimen # | Extraction voucher # | MBspecimenID | GenBank accession # |
a | BGI-MYR-16 | – |
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Additional sample information for
Material specimen # | Extraction voucher # | MBspecimenID | GenBank accession # |
a | TW06 | – | – |
b | TW07 | – |
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