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£ 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.

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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

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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.

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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

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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),

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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

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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

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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.

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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.

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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.)

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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).

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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.

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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

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Plus scanning electron microscope, and the usage fees were funded by a Grant for Undergraduate Research through the MCZ. David Lange provided training on this microscope. A Harvard College Research Program grant provided a stipend for the imaging work done for this paper. Three anonymous reviewers provided comments that helped to improve this paper.

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