Steps Towards a Phylogeny of the Pill Millipedes: Non-Monophyly of the Family Protoglomeridae, with an Integrative Redescript
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A peer-reviewed open-access journal ZooKeys 510: 49–64 (2015) Integrative redescription of Eupeyerimhoffia archimedis 49 doi: 10.3897/zookeys.510.8675 RESEARCH ARTICLE http://zookeys.pensoft.net Launched to accelerate biodiversity research Steps towards a phylogeny of the pill millipedes: non- monophyly of the family Protoglomeridae, with an integrative redescription of Eupeyerimhoffia archimedis (Diplopoda, Glomerida) Jan Philip Oeyen1, Thomas Wesener1 1 Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute for Animal Biodiversity, Center for Taxonomy and Evolutionary Research (Section Myriapoda), Adenauerallee 160, D-53113 Bonn, Germany Corresponding author: Jan Philip Oeyen ([email protected]) Academic editor: Ivan H. Tuf | Received 30 September 2014 | Accepted 7 May 2015 | Published 30 June 2015 http://zoobank.org/175DD451-08A4-477B-8A06-9AB5F09F65BA 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. doi: 10.3897/zookeys.510.8675 Abstract 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 ad- dition 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%) betweenEupeyerimhoffia and the other genera in the order, as well as its unusual morphological characters, including unique mor- phological adaptations to roll into a ball, are probably an indication of the old age of the group. Keywords COI, Glomerida, integrative, taxonomy, redescription, Sicily Copyright 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. 50 Jan Philip Oeyen & Thomas Wesener / ZooKeys 510: 49–64 (2015) Introduction The pill millipedes of the order Glomerida comprise about 290 species in 34 genera (Mauriès 2005, Golovatch et al. 2010, Wesener 2010, 2012) and exhibit a Holarctic distribution, with species found in North America, Europe and North Africa, and Asia with the exception of India south of the Himalayas (Shelley and Golovatch 2011). The Glomerida are currently divided into three families (Mauriès 1971, 2005), the two spe- cies-poor families Glomeridellidae and Protoglomeridae, and the family Glomeridae, which contains the majority of species and genera (~240 species in 27 genera) (Mauriès 2005, Wesener 2012). 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 (Mauriès 2005). Here we redescribe the little-known species Eupeyerimhoffia archimedis (Strasser, 1965), and describe the male telopods for the first time. Additionally, we illustrate sev- eral 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. Material and methods 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. 1C) and further samples were collected at a new locality (East of Palazzolo Acreide, Sicily). Ex- act coordinates are provided in Table 1. All samples were conserved in 98% EtOH for further analyses and deposited in the collection of the Zoological Research Museum Alexander Koenig (ZFMK, Bonn, Germany). Morphological analysis 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 (Hi- tachi LTD, Japan) and digital images were captured using DISS5 (point electronic GmbH, Germany). Integrative redescription of Eupeyerimhoffia archimedis 51 Figure 1. Eupeyerimhoffia archimedis (Strasser, 1965) female in situ and habitat. A E. archimedis female rolled up, in situ B E. archimedis female in situ C Habitat of E. archimedis, close to the type locality Ferla. Molecular analysis 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 1) were also downloaded. Specimens from which DNA was extracted were stored as vouchers at the ZFMK. Accession numbers, locality data and voucher information for all samples included in the study are displayed in Table 1. 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 (Saiki et al. 1988) using the Nancy (Simon 52 Jan Philip Oeyen & Thomas Wesener / ZooKeys 510: 49–64 (2015) Table 1. Sample information with voucher numbers (ZFMK = Zoological Research Museum Alexander Koenig, Bonn, Germany. ZSM = Bavarian State Collection of Zoology. NHMC = Natural History Mu- seum of Crete), GenBank accession numbers (Acc.#) and locality information. Samples where sequences were downloaded from GenBank are marked with an asterisk. Specimen Species Acc. # Locality Voucher *Glomeris marginata ZFMK Germany, Nordrhein-Westfalen, Bonn, FJ409909 (Villers, 1789) MYR0009 Venusberg, coll. T. Wesener, IX.2007 Slovenia, Lower Sava, Brežice, Prilipe, dry creek *Glomeridella minima ZFMK JQ074181 valley, 45.8773°N, 15.6246°E, 150 m, coll. H. (Latzel, 1884) MYR0003 Reip, 17.x.2009. *Geoglomeris subterranea BC ZSM JQ350441 Switzerland, Aargau Verhoeff, 1908 MYR 00370 Italy, Piemonte, Biella, NW Sanctuary of Oropa, ZFMK *Trachysphaera sp. JQ074180 Fagus forest with stones, 45.62947°N, 7.98168°E, MYR0006 1200 m, coll. T. Wesener, 14.iv.2011 U.S.A., California, Mendocino County, between *Glomeroides primus ZFMK Fort Bragg and Whiskey Springs, 39.3976°N, JQ074182 (Silvestri, 1929) MYR0004 123.6946°W, 35 m, coll E. Garcia, C. Richart & A. Schönhofer, 29.iii.2011. Italy, Trentino-Südtirol, Prov. Trient, Madonna Onychoglomeris tyrolensis ZFMK di Campiglio, Beech forest, 46.2209528°N, KP205571 (Latzel, 1884) MYR1276 010.8296250°E, 1553 m, coll. T. Wesener, 04.x.2012. Spain, Galicia, Ribadeo, Trabada, deep and moist Protoglomeris vasconica ZFMK KP205572 creek valley with deciduous forest, 43.4295°N, (Brölemann, 1897) MYR0934 7.2290°E, coll. H. Reip, 29.vii.2012. Glomerellina laurae ZFMK Europe, Greece, Rhodos, Kapi - Profitis Ilias, coll. KP205573 Silvestri, 1908 MYR2260 NHMC, 01.i.2000. Italy Sicily, Province Syracuse, South of Ferla, Eupeyerimhoffia archimedis ZFMK Southern slope, deciduous forest, 37.1151333°N, KP205574 (Strasser, 1965) 1 MYR1876 014.9403667°E, coll. J.P. Oeyen & P. Erkeling, 10.vii.2013. Italy, Sicily, Province Syracuse, East of Palazzolo Eupeyerimhoffia archimedis ZFMK Acreide, Ravine, deciduous forest, 37.0997667°N, KP205575 (Strasser, 1965) 2 MYR1965 015.0232000°E, coll. J.P. Oeyen & P. Erkeling, 13.vii.2013. et al. 1994) and LCO (Folmer et al. 1994) primer pair following previously published protocols (Wesener et al. 2010). Both strands were sequenced by Macrogen (Macrogen Europe Laboratory, Amsterdam, The Netherlands), following the Sanger sequencing method (Sanger et al. 1977). Sequencing reads were assembled and aligned by hand with Bioedit 7.1.3 (Hall 1999) and confirmed with BLAST searches (Altschul et al. 1997). Sequences were uploaded to GenBank (Accession numbers: KP205571 to KP205557). Mean pairwise distances between terminals (transformed into percentages)