Research Article |
Corresponding author: Jan Philip Oeyen ( janphilipoyen@gmail.com ) Academic editor: Ivan H. Tuf
© 2015 Jan Philip Oeyen, Thomas Wesener.
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:
Oeyen JP, Wesener T (2015) Steps towards a phylogeny of the pill millipedes: non-monophyly of the family Protoglomeridae, with an integrative redescription of Eupeyerimhoffia archimedis (Diplopoda, Glomerida). In: Tuf IH, Tajovský K (Eds) Proceedings of the 16th International Congress of Myriapodology, Olomouc, Czech Republic. ZooKeys 510: 49-64. https://doi.org/10.3897/zookeys.510.8675
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Eupeyerimhoffia archimedis (Strasser, 1965) is redescribed based on several specimens collected at a number of sites close to the type locality on Sicily, Italy. Scanning electron microscopy is used to illustrate several unusual morphological characters for a member of the Glomerida for the first time. A fragment of the mitochondrial COI gene (668bp) is sequenced for the first time in Eupeyerimhoffia to provide a species-specific barcode and to gain first insights into the genetic distances between the genera in the widespread family Protoglomeridae. The novel sequences are compared to representatives of all other genera of the family: Protoglomeris vasconica (Brölemann, 1897) from northern Spain, the dwarfed Glomerellina laurae Silvestri, 1908 from Italy and Glomeroides primus (Silvestri, 1929) from western North America. The addition of COI sequences from the two other families of the Glomerida renders the family Protoglomeridae paraphyletic with Glomeroides primus being more closely related to Glomeridella minima (Latzel, 1884) than to the other genera in the family. The large genetic distances (13.2–16.8%) between Eupeyerimhoffia and the other genera in the order, as well as its unusual morphological characters, including unique morphological adaptations to roll into a ball, are probably an indication of the old age of the group.
COI, Glomerida, integrative, taxonomy, redescription, Sicily
The pill millipedes of the order Glomerida comprise about 290 species in 34 genera (
While the two genera of the Glomeridellidae are Mediterranean, the four genera and 20 species of the Protoglomeridae show a disjunct distribution, partly European, in Spain, the eastern Mediterranean, Algeria and Sicily, and partly in the New World from Guatemala to California (
Here we redescribe the little-known species Eupeyerimhoffia archimedis (Strasser, 1965), and describe the male telopods for the first time. Additionally, we illustrate several unusual (and potentially apomorphic) morphological characters of a member of the family Protoglomeridae for the first time using scanning electron microscopy. To complete our integrative approach, we also analyze the genetic distances between the four genera of the family using the common barcoding fragment, COI.
Samples of Eupeyerimhoffia archimedis were collected by hand in July 2013. A single male and several females were collected close to the type locality (Ferla; Fig.
Sample information with voucher numbers (ZFMK = Zoological Research Museum Alexander Koenig, Bonn, Germany. ZSM = Bavarian State Collection of Zoology. NHMC = Natural History Museum of Crete), GenBank accession numbers (Acc.#) and locality information. Samples where sequences were downloaded from GenBank are marked with an asterisk.
Species | Specimen Voucher | Acc. # | Locality |
---|---|---|---|
*Glomeris marginata (Villers, 1789) | ZFMK MYR0009 | FJ409909 | Germany, Nordrhein-Westfalen, Bonn, Venusberg, coll. T. Wesener, IX.2007 |
*Glomeridella minima (Latzel, 1884) | ZFMK MYR0003 | JQ074181 | Slovenia, Lower Sava, Brežice, Prilipe, dry creek valley, 45.8773°N, 15.6246°E, 150 m, coll. H. Reip, 17.x.2009. |
*Geoglomeris subterranea Verhoeff, 1908 | BC ZSM MYR 00370 | JQ350441 | Switzerland, Aargau |
*Trachysphaera sp. | ZFMK MYR0006 | JQ074180 | Italy, Piemonte, Biella, NW Sanctuary of Oropa, Fagus forest with stones, 45.62947°N, 7.98168°E, 1200 m, coll. T. Wesener, 14.iv.2011 |
*Glomeroides primus (Silvestri, 1929) | ZFMK MYR0004 | JQ074182 | U.S.A., California, Mendocino County, between Fort Bragg and Whiskey Springs, 39.3976°N, 123.6946°W, 35 m, coll E. Garcia, C. Richart & A. Schönhofer, 29.iii.2011. |
Onychoglomeris tyrolensis (Latzel, 1884) | ZFMK MYR1276 | KP205571 | Italy, Trentino-Südtirol, Prov. Trient, Madonna di Campiglio, Beech forest, 46.2209528°N, 010.8296250°E, 1553 m, coll. T. Wesener, 04.x.2012. |
Protoglomeris vasconica (Brölemann, 1897) | ZFMK MYR0934 | KP205572 | Spain, Galicia, Ribadeo, Trabada, deep and moist creek valley with deciduous forest, 43.4295°N, 7.2290°E, coll. H. Reip, 29.vii.2012. |
Glomerellina laurae Silvestri, 1908 | ZFMK MYR2260 | KP205573 | Europe, Greece, Rhodos, Kapi - Profitis Ilias, coll. NHMC, 01.i.2000. |
Eupeyerimhoffia archimedis (Strasser, 1965) 1 | ZFMK MYR1876 | KP205574 | Italy Sicily, Province Syracuse, South of Ferla, Southern slope, deciduous forest, 37.1151333°N, 014.9403667°E, coll. J.P. Oeyen & P. Erkeling, 10.vii.2013. |
Eupeyerimhoffia archimedis (Strasser, 1965) 2 | ZFMK MYR1965 | KP205575 | Italy, Sicily, Province Syracuse, East of Palazzolo Acreide, Ravine, deciduous forest, 37.0997667°N, 015.0232000°E, coll. J.P. Oeyen & P. Erkeling, 13.vii.2013. |
A female and the single male from the type locality were dissected under an Olympus SZX12 stereomicroscope with Dumont 5 Inox B forceps. Samples were dehydrated in 100% EtOH for 12 hrs, mounted on aluminum stubs, dried for 12 hrs at 45 °C and sputter coated with 50 nm of pure gold in a Hummer VI sputtering system (Anatech LTD, USA). Samples were observed with a Hitachi S-2460N SEM (Hitachi LTD, Japan) and digital images were captured using DISS5 (point electronic GmbH, Germany).
Muscle tissue was removed from specimens of Onychoglomeris tyrolensis (Latzel, 1884), Protoglomeris vasconica (Brölemann, 1897), Glomerellina laurae Silvestri, 1908, and Eupeyerimhoffia archimedis (Strasser, 1965). Sequences of Glomeroides primus (Silvestri, 1929) were downloaded from GenBank. Additionally, sequences from GenBank of Glomeridella minima (Latzel, 1884), a member of the basal family Glomeridellidae, as well as of Glomeris marginata (Villers, 1789), Geoglomeris subterranea Verhoeff, 1908 and Trachysphaera sp. from the family Glomeridae (Table
Total genomic DNA was extracted using the Qiagen DNAeasy Blood&Tissue kit following the standard protocol. A fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified via PCR (
Mean pairwise distances between terminals (transformed into percentages) were determined using MEGA5.2 (
While the genetic marker used does not allow a study of the phylogeny of the group, first insights into the separation of the genera are provided.
# | Species | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
---|---|---|---|---|---|---|---|---|---|---|
1 | Glomeris marginata | |||||||||
2 | Glomeridella minima | 16.0 | ||||||||
3 | Geoglomeris subterranea | 17.4 | 15.6 | |||||||
4 | Trachysphaera sp. | 15.0 | 13.2 | 15.6 | ||||||
5 | Glomeroides primus | 16.3 | 14.2 | 16.4 | 15.0 | |||||
6 | Onychoglomeris tyrolensis | 14.3 | 13.2 | 16.8 | 13.2 | 15.3 | ||||
7 | Protoglomeris vasconica | 14.8 | 12.0 | 15.3 | 13.5 | 15.0 | 13.2 | |||
8 | Glomerellina laurae | 18.8 | 16.0 | 18.3 | 15.3 | 16.7 | 16.3 | 15.8 | ||
9 | Eupeyerimhoffia archimedis 1 | 16.1 | 15.2 | 15.0 | 13.1 | 16.5 | 15.0 | 13.2 | 16.8 | |
10 | Eupeyerimhoffia archimedis 2 | 16.2 | 15.3 | 15.0 | 13.2 | 16.7 | 15.2 | 13.4 | 16.8 | 0.2 |
The uncorrected pairwise distances between genera included in the present study are relatively high. The genetic distances are not lower between species within the same family than between species of different families. The distances range from 18.8% between Glomerellina laurae (Protoglomeridae) and Glomeris marginata (Glomeridae) to 12.0% between Protoglomeris vasconica (Protoglomeridae) and Glomeridella minima (Glomeridellidae). The two Eupeyerimhoffia archimedis samples show a 0.2% sequence divergence, but also show both the highest (16.8%: G. laurae) and lowest distance (13.2 and 13.4%: P. vasconica) to other species within the family.
The maximum likelihood tree receives little to no support, most nodes remain unresolved and all taxa are separated by long branches (Fig.
Diagnosis. Simple telopods with four podomeres distal to syncoxite, forming pincers. Telopoditomeres 1–3 lacking trichosteles. Telopoditomere 2 with a non-membranous immovable finger located almost parallel to telopoditomere 3. Here we follow the typological system of
Eupeyerimhoffia Brölemann, 1913: 166–174 (first description);
Trinacriomeris Strasser, 1965: 10–14. syn.
Tergite 11 fused to anal shield. Telopod simple with four podomeres. Process of telopoditomere 2 of telopod short and stout. Male leg-pair 17 with four podomeres. Coxa of male leg-pair 18 not fused to syncoxite. Lateral palpi of gnathochilarium as large as inner palpi. One of the largest Glomerida, 18–22 mm long. Mandible with large condylus. Members might be mistaken in habitus, size and color with the species of the genus Onychoglomeris Verhoeff, 1909, whose species differ greatly in the telopods and many other characteristics.
Eupeyerimhoffia algerina Brölemann, 1913 from Algeria
Eupeyerimhoffia archimedis (Strasser, 1965) from southern Sicily.
Trinacriomeris archimedis
Trinacriomeris archimedis
Trinacriomeris archimedis
Eupeyerimhoffia archimedis
Trinacriomeris archimedis
1 F, MHNG, lectotype (designated herewith), labeled paratype, in 70% ethanol, Italie (Sicile): Siracuse: Avola pr. Siracuse. 1 F, MHNG 3460, dried and mounted, Italie (Sicile): Siracuse: Avola pr. Siracuse; 1 F, ZFMK MYR01879, 1 M, ZFMK MYR01875, Italy, Sicily at type locality, south of Ferla, 37.1151333°N, 014.9403667°E, coll. J.P. Oeyen & P. Erkeling, 10.vii.2013; 1 F, ZFMK MYR 1965, Italy, Sicily, Province Syracuse, East of Palazzolo Acreide, Ravine, deciduous forest, 37.0997667°N, 015.0232000°E, coll. J.P. Oeyen & P. Erkeling, 13.vii.2013.
A second female type specimen from Ferla, Sicily was, according to the first description, stored at the University of Catania, Institute of Zoology, Italy.
Can easily be distinguished from the other Sicilian Glomerida species by size and color. It is the largest and only light brown species on the island. It can be distinguished from its congener E. algerina in having: (1) Single continuous anterior stria on collum, posterior stria divided in lateral parts; (2) thoracic shield with single continuous stria reaching the lateral lobes on both sides.
General coloration (living specimen) light brown, almost copper. Collum, head, antennae, posterior margin and lateral speckled fields of tergites lighter, almost golden cream color (Fig.
Head sparsely covered with minute setae, >10 supralabral setae (Fig.
Eupeyerimhoffia archimedis (Strasser, 1965) male, SEM. A Head, frontal view B Head, ventral view C Head, detail of lateral area D Antenna, posterior viewE Antenna, antennomere 6 and 7 F Antenna, apical edge of antennomere 3 G Mandible, mesal view H Gnathochilarium, ventral view I Endochilarium, dorsal view J Gnathochilarium, lateral and inner palpi, dorsal view K Endochilarium, detail of median area, dorsal view. Abbreviations: 1-7 = Antennal segments number 1-7; 4iT = 4-combed inner tooth; AC = Apical cone; AF = Antennal fossa; Ca = Cardine; Co = Condylus; cP = Central pads; CT = Central tooth; eT = External tooth; fS = fringed seam; Gu = Gula; iA = Intermediate area; iL = Incisura lateralis; iP = Inner palpi; L = Labrum; LL = Lamella linguales; LP = Lateral palpi; M = Mentum; MP = Molar plate; O = Ocellaria; O1-O4 = Ocelli 1-4; pL = Pectinate lamellae; pmL = paramedian lobe; TO = Organ of Tömösváry; SC = Sensory clusters; St = Stipites. Arrows mark sensilla basiconica. Scale bar: 400 μm (A, B); 200 μm (C); 150 μm (D); 25 μm (E); 10 μm (F); 100 μm (G); 250 μm (H, I); 40 μm (J); 50 μm (K).
Labrum wide, with 19 marginal setae (Fig.
Epipharynx with pronounced central tooth and two lateral membranous lobes, covered densely in cuticular scales (Fig.
Ocellaria black, 3+1 convex lenses (Fig.
Antennae with four apical cones (Fig.
Organ of Tömösváry recessed, elongate, curved ventrally (Fig.
Gnathochilarium ventrally with 8 large setae on lamella linguales, 12 large setae on each stipites (Fig.
Endochilarium with large anterior membranous paramedian lobes (pmL), densely covered with cuticular scales (Fig.
Mandible with single large outer tooth and four-combed inner tooth (Fig.
Collum with one continuous anterior and two posterior lateral striae (Fig.
Eupeyerimhoffia archimedis (Strasser, 1965) male, SEM. A Thoracic shield, dorso-lateral view B Thoracic shield, meso-lateral C Thoracic shield, schism detail, ventro-lateral view D Tergite, detail of peg, antero-lateral view E Tergite, detail of depression, lateral view F Tergite, ventral view G Tergite, dorso-lateral view H Tergite, ozopore, dorsal view I Collum, dorsal view J Tergite 11 and anal shield, detail of furrow, ventro-lateral view K Tergite 11 and anal shield, left side, anterior view L Tergite 11 and anal shield, right side, posterior view. Abbreviations: AS = Anal shield; Dp = Depression; F = Furrow; Oz = Ozopore; LDp = Lateral depression; P = Peg; Sch = Schisma; stri = striae; T11 = Tergite 11. Arrows point anteriorly. Scale bar: 400 μm (A, H, I, K, L); 300 μm (B, F); 100 μm (C, D, J); 50 μm (E); 500 μm (G).
Thoracic shield with very small schism (Fig.
Tergites 3–10 covered with minute recessed setae, with single complete transverse anterior stria and short lateral striae anteriorly circumventing a depression (Fig.
Ozopore simple, neither with special sutures nor other structures (Fig.
Tergite 11 and anal shield completely fused but both dorsally and ventrally distinguishable by a pronounced furrow (Fig.
Pleurites evenly covered with small setae, bulge at anterior edge widest medially narrowing towards proximal edge. Pleurite 1.2 times wider than long.
Stigmatic plates reaching around coxa on both anterior and posterior sides. 1.5 times wider than long, almost pentagonal in shape. Plate with regular margin, lacking any projections. Spiracle inconspicuous, protected by small knob.
Midbody legs sparsely covered with minute setae (Fig.
Eupeyerimhoffia archimedis (Strasser, 1965) male and female, SEM. A Leg-pair 1, male, right side, posterior view B Leg-pair 2, female, right side, posterior view C Leg-pair 2, male, left side, posterior view D Leg-pair 2, female, right vulva, ventral view E Leg-pair 2, male gonopore, posterior view F Leg-pair 9, male, right side, posterior view G Leg-pair 17, left side, anterior view H Leg-pair 18, right side, posterior view I Telopod with syncoxite, anterior view J Telopod, inner horn of syncoxite K Telopod, posterior view. Abbreviations: Cx = Coxa; Pre = Prefemur; Fem = Femur; Post = Postfemur; Tib = Tibia; Tar = Tarsus; Vu = Vulva; Go = Gonopore; Mp = Median plate; Lp = Lateral plate; Op = Operculum; 1-4 = Podomere 1-4; cL = Central lobe; h = Inner horn; Scx = Syncoxite; Tr = Trichostele. Scale bar: 400 μm (A, B); 250 μm (C); 100 μm (D, H); 25 μm (E); 200 μm (F, I); 150 μm (G, K); 50 μm (J).
Male sexual characters.
Male tergite 11 and anal shield do not show any special structures (Fig.
Male first leg-pair sparsely covered with minute setae (Fig.
Male second leg-pair similar to midbody legs, but with a bulbous medial coxal protrusion carrying two spines (Fig.
Male gonopore clam-shaped and mesally protruding from posterior side of coxa 2 (Fig.
Male leg 17 reduced with 4 podomeres (Fig.
Male leg 18 reduced, but to a lesser extent than leg-pair 17 (Fig.
Telopod (male leg 19) stout, syncoxite likewise (Fig.
Female sexual characters.
Female second leg-pair similar to midbody legs, but coxa with two spines on separate medial protrusions which are fused basally (Fig.
Female vulva large, attached to posterior side of coxa via membranes (Fig.
Not enough samples present to describe morphological variation. The populations from Ferla and Palazzolo Acreide have two different haplotypes, differing at one base pair position.
As described by
All of the samples were collected during the day in deciduous forests. Specimens were mainly found in the leaf litter or under small stones. Curiously, some were also found in close proximity to ant nests (Hymenoptera) and under moss growing directly on an exposed rocky surface.
The described position of the vulva operculum might be an artifact, as the structural integrity of membranous structures was not preserved by critical point drying. This should be considered for future studies of glomerid vulvae.
The sampling within the present study did not allow for a description of the morphological variation within the species. However, the 0.2% sequence divergence between the two reported localities shows that there is variability within the species, with at least two haplotypes present on the island.
Eupeyerimhoffia archimedis shows several interesting characters. The mandible with a large condylus and flat molar plate lacking a groove (Fig.
The volvation strategy of Eupeyerimhoffia is another striking and possibly unique feature of the genus inside the order Glomerida. Similar pegs on the tergites have been reported for members of the genera Epiromeris (
As stated above, the COI fragment is not well suited to study the group’s phylogeny. Therefore it is not surprising that the COI tree lacks resolution and receives little statistical support. Nonetheless, together with the distance analysis, it is sufficient to observe that the members of the family Protoglomeridae are not each other’s closest relatives (e.g. Glomeroides primus grouping with Glomeridella minima from the separate suborder Glomeridelloidea) and possibly that the family does not constitute a monophyletic unit. Similar results have also been reported by
We thank K. Ulmen (ZFMK: SEM) and C. Etzbauer (ZFMK: Molecular lab) for help in the lab, H. Reip (Jena) for the specimens of Protoglomeris vasconica, S. Simaiakis (NHMC) for the specimens of Glomerellina laurae, and A. Schönhofer for the specimens of Glomeroides primus. We also thank P. Schwendinger (MHNG) for his advice and for hosting one of us (TW) during a stay at Geneva in the summer of 2012. We thank the Alexander-Koenig-Gesellschaft (AKG) for funding the trip to re-collect Eupeyerimhoffia archimedis in Sicily and P. Erkeling for his help in the field. Sergei Golovatch and a second reviewer provided useful comments that enhanced the quality of the here presented article. Lastly, we thank P.B. Frandsen (Rutgers University, USA) for proofreading and giving constructive comments on the manuscript.