1 M usemn © £ ComparatiYe US ISSN 0006-9698 Cambridge, Mass. 9 February 2015 Number 543 CYPHOPHTHALMUS SOLENTIENS1S SP. NOV. (CYPHOPHTHALMI, SIRONIDAE), A NEW ENDOGEAN MITE HARVESTMAN SPECIES FROM CROATIA, WITH AN APPLICATION OF CONFOCAL LASER MICROSCOPY TO ILLUSTRATE GENITALIA IN OPILIONES Taras B. Dreszer,1 Tonci Rada,2 and Gonzalo Giribet1 Abstract. The genus Cyphophthalmus is one of the most diverse genera of Cyphophthalmi and has been used as a model to study diversification in the Balkan region. However, the taxonomy of the group is deficient and type material is not available for study. Here we describe a new species, Cyphophthalmus solentiensis sp. nov., from the coastal region of Croatia using state-of-the-art techniques for illustrating species of Cyphophthalmi. The species, phylogenetically close to C. gjorgjevici on the basis of a molecular data analysis of four markers, is illustrated by means of stereomicroscopy and scanning electron microscopy, and the genitalia are imaged using confocal laser microscopy and three-dimensional reconstruction techniques, allowing unparalleled visualization of Opiliones genitalia. We hope that this description stimulates research in this diverse but still obscure genus of Cyphophthalmi. Key words: Opiliones; Arachnida; genitalia; phylogeny; Balkans; Mediterranean region; confocal laser microscopy; molecular data INTRODUCTION Among the most diverse genera of Cy- phophthalmi is the sironid Cyphophthalmus Joseph, 1868, with 32 species currently recog- nized (Karaman, 2009), distributed from 1 Museum of Comparative Zoology, Department of Organismic and Evolutionary Biology, Harvard Univer- sity, 26 Oxford Street, Cambridge, Massachusetts 02138, U.S.A.; e-mail: ggiribet@g.harvard.edu. 2 Speleological Society “Spiljar”, Varazdinska 53, 21000 Split, Croatia. Austria to Turkey along the Mediterranean region. The group has undergone an inter- esting biogeographical history due to its ancient age and because it diversified explosively in the Balkan region, giving origin to at least three phylogenetic lineages (Boyer et al, 2005; Murienne et al, 2010) whose evolution could be related to the paleogeographic history of the Adria micro- plate (Murienne et al., 2010). Among these three clades, the gjorgjevici lineage is one of the poorest-known lineages, and includes C. The President and Fellows of Harvard College 2015. BREVIORA No. 543 7 gjorgjevici (Hadzi, 1933) and undescribed species Irom Macedonia (Karaman, 2009; Murienne et al., 2010). However, despite the large diversity of this clade and its impor- tance for understanding the biogeography of the Balkan region, the group has a large number of missing types for the older species (see Giribet, 2000), and has substan- dard descriptions of recent species (Kara- man, 2008, 2009), with types kept in a private collection not broadly available to other researchers. Fortunately, advances in molecular phylogenetics and species delim- itation techniques allow assessment of spe- cies molecularly, which has helped to identify a new species of Cyphophthalmus recently collected in endogean habitats in Croatia. The species, closely related to other endogean species, C. gjorgjevici , is here described and fully illustrated by means of light stereomicroscopy, scanning electron microscopy (SEM), and confocal laser microscopy to highlight informative charac- ters of interest in describing new diversity of Cyphophthalmus . MATERIALS AND METHODS Stereomicroscope imaging The male holotype and one female para- type were cleansed with ultrasounds for 5 min and imaged using a JVC KY-F70B digital camera mounted on a Leica MZ 12.5 stereoscope with the 0.5-4.0X objective. A series of 10-20 images was taken at different focal lengths, and then assembled using the software package Auto-Montage Pro Ver- sion 5.00.0271. Each specimen was photo- graphed in dorsal, ventral, and lateral positions. SEM imaging Two males and one female paratype were cleansed as explained above and mounted on SEM stubs with biadhesive carbon tape. These samples were sputter coated with an EMS 300T D dual-headed sputter coater at the Harvard Center for Nanoscale Systems. A 5-nm platinum/palladium layer was ap- plied. Samples were imaged with a Carl Zeiss Ultra Plus FESEM using the SE2 detector. Images were then edited using Adobe Photo- shop CS5. Autofluorescence imaging Following Murienne and Giribet (2009), we took advantage of the autofluorescence of the arthropod cuticle (Klaus et al, 2003, 2014) to image the spermatopositor and ovipositor organs of the new species. Three spermatopositors and one ovipositor were dissected out and placed in lactic acid for 1- 24 hours. Subsequently, the organs were mounted in glycerin on microscope slides. The specimens were imaged using the Zeiss Elyra microscope at the Harvard Center for Biological Imaging, set to the Plan Apo- chromat 20X/0.8 Ph2 objective. The images recorded the autofluorescence of the samples by laser excitation. A filter prevented laser light from reaching the detector but allowed fluorescence. A laser wavelength of 561 nm was used, and autofluorescence of all wave- lengths above that were recorded. Gain was adjusted for maximum clarity. The sperma- topositor was imaged using a gain of 565, and the ovipositor was imaged using a gain of 614. Images were recorded as stacks in the z- axis. This was done by imaging the same sample 50-150 times at different focal planes. Carl Zeiss Zen software (Black Edition v. 2010) was then used to create a three- dimensional automontage of the images. Molecular methods To test the phylogenetic position of the new species, we conducted a standard 2015 A NEW CYPHOPHTHALMUS SPECIES 3 Table 1 . Taxa and Markers Used in This Study with MCZ and GenBank Accession Numbers. GenBank Accession Numbers in Bold Indicate New Sequences for this Study MCZ # 18S rRNA 28S rRNA COI 16S rRNA Metasiro americanus IZ-1 33799 DQ825542 DQ825595 JF786394 DQ825616 Cyphophthalmus duricorius IZ- 135009 KJ857509 KJ857512 KJ857527 KJ857515 Cyphophthalmus ere IZ-1 3501 8 AY639462 DQ825593 AY639557 AY639527 Cyphophthalmus gjorgjevici IZ-1 350 17 AY639464 DQ825587 AY639559 AY639529 Cyphoph thalmus gordani IZ- 1 35014 AY639467 DQ825592 - AY639532 Cyphophthalmus hlavaci IZ-1 35052 - - - KJ857544 Cyphophthalmus markoi IZ- 1 350 1 6 AY639469 AY639504 AY639561 AY639534 Cyphophthalmus martensi IZ- 1 350 1 3 AY639471 DQ825589 AY639563 AY639536 Cyphophthalmus minutus IZ-1 35012 AY639473 DQ825591 AY639565 AY639537 Cyphoph thalmus ognjenovici IZ-1 35027 AY639475 DQ825594 AY639567 - Cyphophthalmus rumijae IZ- 1 350 1 1 AY639477 DQ825588 AY639569 AY639539 Cyphophthalmus teyrovskyi IZ-1 35025 AY639482 DQ5131 18 AY639571 AY639544 Cyphophthalmus trebinjanum IZ-1 35026 AY639483 DQ5131 19 AY639572 - Cyphophthalmus zetae IZ-1 35022 AY639485 AY639515 AY639574 AY639546 Cyphophthalmus solentiensis sp. nov. IZ-129787 KJ857518 KJ857522 KJ857528 KJ857532 Cyphophthalmus solentiensis sp. nov. IZ-1 35079 KJ857519 KJ857523 KJ857529 KJ857533 Iberosiro sp. IZ- 1 35072 KJ857520 KJ857524 KJ857530 KJ857534 Paramiopsalis eduardoi IZ-135034 EU638284 EU638287 EU638288 EU638281 Paramiopsalis ramulosus IZ-1 35006 AY639489 DQ513121 DQ825641 AY639550 Paramiopsalis sp. IZ-1 35070 JF934957 JF934991 JF786390 JF935024 Parasiro coiffaiti IZ-132372 AY918872 DQ513122 DQ825642 AY918877 Parasiro minor IZ-1 32374 JF934958 JF934992 JF786391 JF935025 Siro a car o ides IZ-1 34454 AY639490 DQ513128 DQ825643 AY639551 Siro carpaticus IZ-1 3507 1 KJ857536 KJ857539 KJ857542 KJ857545 Siro clousi IZ-1 30003 KJ857537 KJ857540 KJ857543 - Siro exilis IZ- 1 3455 1 AY639491 DQ825585 AY639579 - Siro kamiakensis IZ- 1 32388 KJ857538 KJ857541 - - Siro rubens IZ- 1 3239 1 AY428818 DQ825584 DQ5131 1 1 - Siro shasta IZ-1 30004 KJ857521 KJ857525-6 KJ857531 KJ857535 Siro valleorum IZ-1 35008 AY639492 DQ513123 AY639580 AY639552 Suzukielus sauteri IZ-1 32256 DQ5131 38 DQ5131 16 DQ513108 DQ518086 Suzukielus sauteri IZ-1 32263 DQ825541 DQ825583 DQ825640 DQ825615 phylogenetic analysis using four polymerase chain reaction (PCR)-amplified markers, the nuclear ribosomal genes 18S ribosomal RNA and 28S rRNA, and two mitochondri- al genes, the ribosomal 16S rRNA and the protein-encoding cytochrome c oxidase sub- unit I. DNA extraction, PCR amplification, and sequencing follow previous work on Cyphophthalmi (e.g., Giribet and Shear, 2010; Giribet et al., 2012). Single-step (i.e., direct optimization) and two-step (alignment + tree inference) phylogenetic analyses were conducted on a sironid data set (Table 1), rooted with the neogoveid Metasiro amer- icanus , mostly following published recent analyses of centipede and arachnid data sets of similar characteristics (Giribet and Edge- combe, 2013; Giribet et al , 2014). For the direct optimization analyses (Wheeler, 1996) we used POY v.5.1.1 (Wheeler et al , 2014), exploring six parameter sets (Table 2). All input files were unaligned and sequences were treated as a single unpartitioned frag- ment. Tree searches were conducted using the timed search function in POY, i.e., multiple cycles of (a) building Wagner trees. 4 BREVIORA No. 543 Table 2. Weighted Steps for the Analysis of the Six Parameter Sets (First Column) for the Direct Optimization Analyses for the Four Markers and the Combined Analyses (MOL), with wILD Values. Italicized Numbers Indicate Values for Parameter Set that Minimize Incongruence Among Data Partitions. 18S 28S 16S COI MOL WILD 111 85 748 1654 2641 5192 0.01233 121 111 1133 2650 4020 8019 0.01309 211 87 893 1878 2696 5625 0.01262 221 115 1406 3055 4101 8807 0.01476 3211 113 1202 2761 4072 8253 0.01272 3221 172 1576 3464 5383 10728 0.01240 (b) subtree pruning and regrafting, (c) tree bisection and reconnection, (d) ratcheting (Nixon, 1999), and (e) tree-fusing (Goloboff, 1999, 2002) [command: search (max_time: 00:01:00, min_time:00:00:10, hits:20, memor- y:gb:2)j. For the individual partitions, timed searches of 1 hour were run on four processors under six parameter sets, as in Giribet et al. (2012) (see Table 2). For the combined analysis of the four markers we started with the same search strategy, giving a preliminary tree as input, and the resulting trees were given as input for a second round of analyses (sensitivity analy- sis tree fusing, SATF), as described by Giribet (2007), and continued until the tree lengths stabilized (Giribet et al., 2012). The optimal parameter set was estimated using the modified WILD metrics (Wheeler, 1995; Sharma et al, 2011) as a proxy for the parameter set that minimizes overall incon- gruence among data partitions (Table 2). Nodal support for the optimal parameter set was estimated via jackknifing (100 replicates), with a probability of deletion of e~> (Farris et al., 1996) using auto_sequence_partition, as discussed in earlier work (Giribet et al, 2012). Maximum likelihood (ML) analyses were conducted on static multiple sequence alignments inferred in MUSCLE v. 3.6 (Edgar, 2004) through the EMBL-EBI server (http://www.ebi.ac.uk/Tools/msa/muscle/). The MUSCLE alignments were conducted for each gene independently and hypervariable regions in the data set were subsequently trimmed with Gblocks v. 0.91b (Castresana, 2000; Talavera and Castresana, 2007) to cull positions of ambiguous homology. Data sets were concatenated with SequenceMatrix (Vaidya et al, 2011). Maximum likelihood analyses were con- ducted using RAxML ver. 7.2.7 (Stamatakis et al, 2008b) in the CIPRES server (Miller et al, 2010). For the searches, a unique general time reversible (GTR) model of sequence evolution with corrections for a discrete gamma distribution (GTR + T) was specified for each independent data partition, and 100 independent searches were conducted. Nodal support was estimated via the rapid boot- strap algorithm (1,000 replicates) using the GTR-CAT model (Stamatakis et al, 2008a). Bootstrap resampling frequencies were there- after mapped onto the optimal tree from the independent searches. RESULTS The SATF analyses with POY stabilized after two to five rounds, depending on the parameter set. The parameter set that min- imized wILD was 111, where all nucleotide and indel transformations are equally weighted. The resulting tree was nearly identical to those found under the other explored parameter sets. This tree (Fig. 1) shows monophyly of Cyphophthalmus and a 2015 A NEW CYPHOPHTHALMUS SPECIES 5 Metasiro americanus 100 Parasiro coiffaiti Parasiro minor 100 . — Suzukielus sauteri IZ- 132263 74 EL' Suzukielus sauteri IZ- 132256 58 , Siro acaroides 51 Siro shasta 85 C Siro exilis 97 Siro kamiakensis Siro clousi Siro valleorum Siro carpaticus 78 Siro rubens 84 91 100 Iberosiro sp. Paramiopsaiis ramulosus 1 00 Paramiopsaiis eduardoi 97 98 100 c. 98 Paramiopsaiis sp. Cyphophthalmus gjorgjevici Cyphophthalmus solentiensis sp. nov. IZ-1 29787 Cyphophthalmus solentiensis sp. nov. IZ-1 35079 Cyphophthalmus ere Cyphophthalmus duricorius Cyphophthalmus rumijae — Cyphophthalmus martensi Cyphophthalmus zetae Cyphophthalmus hlavaci 94 54 CT 91 Cyphophthalmus markoi Cyphophthalmus gordani Cyphophthalmus teyrovskyi — Cyphophthalmus trebinjanum 84 1 Cyphophthalmus minutus Cyphophthalmus ognjenovici 90.0 Figure 1. Direct optimization tree under parameter set 111 for the combined analysis of all four markers (5.192 steps; wILD = 0.01233). Numbers on branwches represent jackknife support values. basal split between the clade containing C. gjorgjevici and C. solentiensis sp. nov. and the other Cyphophthalmus species. This result is also obtained with the static alignment analyzed under ML (Fig. 2). Both trees find Cyphophthalmus as the sister group to the clade including Iberosiro and Paramiopsaiis (an Iberian clade), and mostly differ in some of the unsupported internal relationships of Cyphophthalmus. Interestingly, C. solentien- sis sp. nov. does not group with C. hlavaci , the closest species geographically. Although sequence data for C. hlavaci are restricted to the 16S rRNA, individual analysis of this gene continues to place these two species in clearly separate clades. TAXONOMY Family Sironidae Simon, 1879 Genus Cyphophthalmus Joseph, 1868 Cyphophthalmus solentiensis Dreszer, Rada & Giribet sp. nov. Figures 3-7 Type specimens Hob type. Male (Museum of Comparative Zoology [MCZ] IZ-1 35079; ex DNA107119) from cave Bratska jama (43.34475, 16.3410), 6 B REV I ORA No. 543 Metasiro americanus 100 Parasiro coiffaiti Parasiro minor 58 54 78 100 r— Suzukielus sauteri IZ- 132256 Suzukielus sauteri IZ- 132263 Siro clousi 4T 100 93 • Siro valieorum 67 Siro rubens 94 100 Siro car pat ic us Siro kamiakensis Siro exilis 84 ■ Siro acaroides ■ Siro shasta 91 99 ■ Iberosiro sp. 100 Paramiopsalis ramulosus ioo i — Paramiopsalis sp. 100 Paramiopsalis eduardoi Cyphophthalmus gjorgjevici 1 oo i— Cyphophthalmus solentiensis sp. nov. IZ- 135079 Cyphophthalmus solentiensis sp. nov. IZ- 129787 Cyphophthalmus duricorius Cyphophthalmus rumijae Cyphophthalmus ere Cyphophthalmus zetae ■ Cyphophthalmus markoi Cyphophthalmus hlavaci 0.05 Cyphophthalmus martens i Cyphophthalmus gordani Cyphophthalmus teyrovskyi Cyphophthalmus trebinjanum Cyphophthalmus ognjenovici 90 1 Cyphophthalmus minutus Figure 2. Maximum likelihood phylogenetic hypothesis of the concatenated trimmed aligned data (-In L = 22,1 18.686904). Numbers on nodes indicate bootstrap support values. Gornje Selo, Solta Island, Middle Dalmatia, Croatia, Leg. Tonci Rada, 20.iii.2012 (Figs. 3A-C). Par a types. Two males, one female mount- ed for SEM (MCZ IZ-1 35079); one female imaged (Figs. 3D-F), two males dissected for genitalia, eight males, five females in 96% EtOFI (MCZ IZ-1 35079); same collecting data as holotype. Nineteen specimens from Podaspilje village, near Omis, Middle Dal- matia, Croatia (MCZ IZ-1 29787), Leg. Tonci Rada, 24. iv. 201 3. One male and one female mounted for SEM (MCZ IZ-1 29787); one male dissected for genitalia (MCZ IZ- 129787). Etymology. The species is named after the island of Solta, its type locality, on the basis of its Latin name, Solent, Solentia, Solen- tium. Diagnosis. Cyphophthalmus with a longi- tudinal carina of the male anal plate low and without ornamentation, and without the heavily granulated “rostrum” of C. gjorgje- vici, its closest species phylogenetically. Spermatopositor with three microtrichiae ventrales positioned in the edges of a V, the central one being more basal, a character not yet described in any other Cyphophthalmus. Description of Male. Total length of male holotype (in mm): 2.09; largest width at 2015 A NEW CYPHOPHTHALMUS SPECIES 7 Figure 3. Cyphophthalmus solentiensis sp. nov. IZ- 135079. A-C, male holotype in A. dorsal, B, lateral, and C, ventral views. D-F, female paratype in D, dorsal, E, lateral, and F, ventral views. Scale bars = 1 mm. second opisthosomal segment: 1.13; length/ width ratio 1.85; width across tip of ozo- phores: 0.91; prosomal width: 1.11. Body brown-orange and legs slightly lighter (in ethanol). Cuticle with light tuberculate- microgranulate surface (Figs. 4-6) ( sensu Murphree, 1988). Ozophore conical of type 2 ( sensu Juberthie, 1970), completely ornamented (Fig. 5F); with a subterminal ozopore. Eyes absent. Ventral prosomal complex (Figs. 4A, B, 5A, B) with coxae I and II free, coxae III and IV fused; gonostome semicircular (130 pm wide X 90 pm long), with two triangular projections on its posterior angles (Fig. 5A); sternum absent. Proximal end of coxae I to IV all meeting along the midline. Endites of coxae of legs II and III and of legs III and IV running along their suture; coxal pores present in endites between coxae III and IV, with two projections of the coxae IV endite near the coxal pore (Fig. 5A); endites of coxae IV running adjacent to midline suture for a length approximate to that of 8 B REV I ORA No. 543 Figure 4. Cyphophthalmus solentiensis sp. nov. IZ-1 35079. A, paratype male in ventral position; B. paratype female in ventral position. Scale bars = 0.5 mm. 2015 A NEW CYPHOPHTHALMUS SPECIES 9 Figure 5. Cyphophthalmus solentiensis sp. nov. paratypes IZ- 1 35079. A, male and B, female thoracic complex, scale bars = 80 pm. C, male and D, female anal region, scale bars = 200 pm. E, male spiracle, scale bar = 30 pm. F, male ozophore, scale bar = 50 pm. G, male chelicer and H, male palp, scale bars = 200 pm. 10 B REV 1 ORA No. 543 Figure 6. A-H, J, Cyphophthalmus solentiensis sp. nov. paratype male IZ- 1 35079 and I, female IZ- 135079. A, leg I of male; B, leg II of male; C, leg III of male; D, leg IV of male; E, metatarsus and tarsus I; F, metatarsus and tarsus II; G, metatarsus and tarsus III; H, metatarsus and tarsus IV of male; I, metatarsus and tarsus I of female; J, detail of claw I. (A D, scale bars = 300 pm. E-I, scale bars = 100 pm. J, scale bar = 50 pm.) 2015 A NEW CYPHOPHTHALMUS SPECIES 11 Figure 7. Cyphophthalmus solentiensis sp. nov. A, B, spermatopositor of paratype male IZ- 1 35079. A, dorsal view; B, ventral view (v indicates microtrichiae ventrales). C-E, ovipositor of paratype female IZ- 135079. C, detail of ovipositor tip, dorsal view; D, detail of ovipositor tip, ventral view; E, whole ovipositor. (A-D, scale bars = 100 pm. E, scale bar = 200 pm). 12 B REV 1 ORA No. 543 Table 3. Leg Measurements (Length/Width, mm) of Male Paratype Mounted for SEM. Leg Trochanter Femur Patella Tibia Metatarsus Tarsus Total Length I 0.27/0.15 0.67/0.15 0.33/0.14 0.46/0.14 0.27/0.12 0.61/0.15 2.61 II 0.20/0.13 0.58/0.15 0.28/0.15 0.37/0.14 0.23/0.11 0.54/0.13 2.20 III 0.16/0.14 0.45/0.13 0.25/0.15 0.33/0.14 0.21/0.10 0.49/0.12 1.89 IV 0.23/0.13 0.60/0.15 0.31/0.16 0.38/0.16 0.23/0.11 0.56/0.18 2.31 gonostome. Opisthosomal mid-dorsal longi- tudinal sulcus conspicuous (Figs. 3A, B). Spiracles circular (Fig. 5E). Ventral opistho- somal area without exocrine glands; opisthosomal exocrine glands with a pair of openings on tergite VIII (Fig. 5C). Opisthosomal tergite IX and sternites 8 and 9 fused into a corona analis (Fig. 5C). Anal plate oval, with a smooth mid- longitudinal ridge with setae (Fig. 5C). Proximal cheliceral segment 505 pm long, ornamented, with a tuberculate surface for most of its length, but smooth near the distal tip, without a dorsal crest or a ventral process (Fig. 5G). Second cheliceral segment 893 pm long; mobile digit 301 pm. Widest part of the second cheliceral segment near articulation with mobile digit; cheliceral distal segments with uniform dentition (Fig. 5G). Pedipalp (Fig. 5H) 2.709 mm long; trochanter without ventral apophysis. Pedipalp measurements of male paratype (in pm): trochanter 229; femur 448; patella 296; tibia 389; tarsus 374. Legs slender (Figs. 6A-D; measurements in Table 3); leg formula I, II, IV, III. Except for tarsi I-IV and metatarsi I— II, all articles ornamented (Figs. 6A-D). All legs with setae, the highest concentration occurring along the ventral side of the tarsus of all four walking legs (Figs. 6E-I). Tarsus I without a distinct solea (Figs. 6A, E). Tarsus of leg IV entire (Figs. 6D, FI), with a lamelliform adenostyle positioned toward the first third of the dorsal side on tarsus IV. All claws smooth, without dentition or lateral pegs. Spermatopositor (/? = 3; Fig. 7A; see suppl. videos in http://mczbase.mcz.harvard.edu/ guid/MCZ:IZ: 135079 and http://mczbase. mcz. harvard. edu/guid/MCZ:IZ: 129787). Dis- tal margin of terminal lobe semicircular, ca. 80 pm in diameter, terminally with small denticles and two pairs of microtrichiae3 terminales, evenly spaced, not touching at the base, the two median ones longer than the lateral ones. Lateral movable fingers very broad at the base (ca. 20 pm) and clearly hooked, not surpassing the terminal lobe. Dorsal microtrichiae in two symmetrical groups, a lateral one with three short micro- trichiae (the longest 106 pm) coming from a common lateral lobe, and a dorsal one with two long microtrichiae (ca. 130 pm), with enlarged bases, touching at the base, up to ca. 20 pm wide X 50 pm high. With three short microtrichiae ventrales, ca. 50 pm long, the central one in a more basal position than the lateral ones (Fig. 7B). Description of Female. Total length 2.22 mm long, 1.14 mm maximum wide, at second opisthosomal segment. Ventral pro- somal complex (Fig. 5B) with coxal lobes II narrower (at their narrowest part) than long (sensu Karaman, 2009). Anal plate without conspicuous modifications (Fig. 5D). Tarsus IV narrow and elongate (Fig. 61), without glandular pores or other modifications. 3 We follow here Schwendinger & Giribet (2005) in using microtrichiae instead of setae for the spermatopositor organ. Thus, instead of setae terminales we use microtrichiae terminales, and so on. 2015 A NEW CYPHOPHTHALMUS SPECIES 13 Ovipositor (Figs. 7C-E; see suppl. videos in http://mczbase.mcz.harvard.edu/guid/MCZ: IZ: 135079) composed of 15 annular seg- ments plus apical lobes, measured at 1.1 mm long extended. Each annulus with eight simple setae, equidistant, the three lateral ones longer than the ventral ones, and with the ventral setae being shorter than the dorsal ones. Setae of annulus 15 much longer than the others (150 vs. 55 pm long between the lateral setae of annuli 15 and 14, respectively). Apical lobes 270 pm long, with saccate receptacles occupying ca. 100 pm. Each apical lobe bears one multibranched sensorial pro- cess with a very wide base, opening from a lateral depression, and two long terminal simple setae, ca. 100 pm long, and eight to nine shorter simple setae. Distribution. Known only from two local- ities in Croatia. Notes. Cyphophthalmus solentiensis sp. nov. is phylogenetically closely related to C. gjorgjevici (Hadzi, 1933), although they differ in the dorsal side of the prosoma, and overlaps geographically with C. hlavaci Karaman, 2009. Unfortunately the latter species is poorly illustrated. However, there seems to be major differences in several characters, including the longitudinal carina of the male anal plate, which is narrow and pronounced in C. hlavaci (although it is not illustrated in the original description), but low and without ornamentation in C. solen- tiensis sp. nov. The new species seems to be larger and has much more slender append- ages than C. hlavaci (Karaman, 2009: figure 33). DISCUSSION Molecular phylogenetic analysis of a sironid data set clearly places C. solentiensis sp. nov. with C. gjorgjevici, a species de- scribed from Skopje, more than 500 km away, and not with its geographically close species C. hlavaci. The phylogenetic results place these two species in separate clades with high support. Although we think that a diagnostic character of our species is the V- shaped disposition of the three microtrichiae ventrales from the spermatopositor organ, most authors did not illustrate the sperma- topositor in Cyphophthalmus, and some have only illustrated spermatopositors in dorsal view (Juberthie, 1970; Karaman, 2008, 2009). Gruber (1969) illustrated the ventral side of the spermatopositor of several species he described as subspecies of C. duricorius, and in these species the microtrichiae ventrales are lined up in a row, or the central one is more distal than the lateral ones, contrary to C. solentiensis sp. nov., but no other proper illustrations are available for the ventral side of any other Cyphophthalmus species. Un- fortunately, no information on the genitalia of C. gjorgjevici is available. Here we provided state-of-the-art images and accessory videos for a new species of Cyphophthalmus with the aim to improve the deficient taxonomy of this group of biogeographical importance. We hope that the new imaging techniques for Opilones genitalia can be rapidly applied to many other species to generate data sets on par with the technologies available to many researchers. ACKNOWLEDGMENTS Erin McIntyre assisted with the molecular work, which was funded by the MCZ. The Harvard Center for Biological Imaging provided access to the Zeiss Elyra laser confocal microscope. The Division of Sci- ence funded the usage fees through its undergraduate imaging account. Douglas Richardson provided training and subse- quent assistance with the Elyra microscope. The Harvard Center for Nanoscale Systems provided access to the Zeiss FESEM Ultra 14 BREVIORA No. 543 Plus scanning electron microscope, and the usage fees were funded by a Grant for Undergraduate Research through the MCZ. 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