Abstract The Iberian Peninsula represents a hot spot of cyphophthalmid (mite harvestman) disparity, with four of the eight genera currently recognized in the family Sironidae represented in the region - a generic diversity and morphological disparity not found in any other region of the World so far. From these, two genera ( Iberosiro and Odontosiro) are monotypic, and are restricted to the western side of the peninsula. Parasiro is restricted to the north-east region, from the Catalonian Coastal Ranges and both sides of the Eastern Pyrenees, in areas where the annual rainfall surpasses 1000 mm, and mostly restricted to areas with Paleozoic and Variscan rocks, with other species of the genus extending to Corsica, Sardinia, and the Italian Peninsula. A second species of the genus Paramiopsalis, Paramiopsalis eduardoi sp.

From Fragas do Eume, is described here along with a re-diagnosis of the genus. Paramiopsalis species, together with Odontosiro, inhabit the north-west corner of the Iberian Peninsula, an area with some of the highest recorded annual rainfall, and with Paleozoic rocks from the Iberian Massif or Variscan granitoid rocks. A phylogenetic analysis of the members of the family Sironidae using four molecular markers, despite not including all of the Iberian genera, clearly shows the non-monophyly of the Iberian Cyphophthalmi, indicating that the Iberian Peninsula is home to multiple ancient lineages of mite harvestmen. The two Paramiopsalis species form a sister clade to the Balkan genus Cyphophthalmus, whereas Parasiro constitutes the first lineage of the sironids represented. INTRODUCTION Cyphophthalmi are present in virtually all continental land masses or on islands of continental origin.

Within these land masses, peaks of species diversity have been described or observed in such places as New Zealand, Sumatra, Borneo, or the Balkans. These radiations often account for single lineages, although in some cases up to three lineages of independent origin have been observed. Such is the case for New Zealand, which has perhaps the best-studied cyphophthalmid fauna, from a taxonomic and evolutionary point of view (; ). Only two lineages occur in the much larger land mass of Australia (), whereas places such as Sumatra and Borneo may host a single or at most two lineages each, despite the large number of species that inhabit those large islands ().

In this article we focus on a land mass that has been isolated from other terranes during a long period of history: the Iberian Peninsula (). This territory is home to just a few species of Cyphophthalmi, which nonetheless belong to four genera, exhibiting greater morphological disparity than any other land mass of equivalent size. Furthermore, a new species of Cyphophthalmi from Galicia is described and illustrated.

Gluing can be a highly efficient mechanism of prey capture, as it should require less complex sensory–muscular feedback. Whereas it is well known in insects, this mechanism is much less studied in arachnids, except spiders. Soil-dwelling harvestmen (Opiliones, Nemastomatidae) bear drumstick-like glandular.

Satellite view centered on the Iberian Peninsula showing the known localities for the Iberian species of Cyphophthalmi. Type localities are identified with the bold font. An asterisk denotes the localities of the specimens used in the molecular study. Localities identified as ‘ Paramiopsalis sp.’ are based on specimens listed by as Paramiopsalis ramulosus, but have not been examined by the authors.

The type locality for Parasiro coiffaiti is indicated with a question mark because the locality listed for the type material is not from the Girona Province (see ). Satellite view centered on the Iberian Peninsula showing the known localities for the Iberian species of Cyphophthalmi. Type localities are identified with the bold font. An asterisk denotes the localities of the specimens used in the molecular study. Localities identified as ‘ Paramiopsalis sp.’ are based on specimens listed by as Paramiopsalis ramulosus, but have not been examined by the authors. The type locality for Parasiro coiffaiti is indicated with a question mark because the locality listed for the type material is not from the Girona Province (see ).

H ISTORY OF CYPHOPHTHALMID RESEARCH IN THE I BERIAN P ENINSULA A recent catalogue of the Iberian Opiliones () lists four species of Cyphophthalmi for this biogeographical area, namely Parasiro coiffaiti, Odontosiro lusitanicus, Paramiopsalis ramulosus, and Iberosiro distylos. Detailed accounts for these species have been provided elsewhere (; ). Older accounts also report the presence of Cyphophthalmus duricorius Joseph, 1868 (as Siro duricorius) as a result of an error perpetuated in the literature (; ), which was corrected later on by: 146–147; see also; ). Our knowledge on the Iberian Cyphophthalmi is mostly derived from the work of two of the most prominent European opilionologists: the French Christian Juberthie and the Catalan Maria Rambla. The first three known species from the Iberian Peninsula were all described by C. Juberthie in three different genera, two of which were monotypic (). The fourth species also belongs to a monotypic genus ().

In this article we revise the Iberian cyphophthalmid fauna, adding a second species for the former monotypic genus Paramiopsalis, and provide a phylogenetic analysis of selected species of the family Sironidae, using four molecular markers in order to test for monophyly of the Iberian lineages. MATERIAL AND METHODS M ORPHO- ANATOMY The male holotype and a female paratype of the new species, and the male lectotype of Parasiro coiffaiti, were photographed in dorsal, ventral, and lateral positions using a JVC KY-F70B digital camera mounted on a Leica MZ 12.5 stereomicroscope. A series of images was taken at different focal planes and assembled with the dedicated software package Auto-Montage Pro v5.00.0271 (Syncroscopy).

A male paratype was examined using an FEI Quanta 200 Scanning Electron Microscope (SEM), after sputter coating with gold/palladium. Observation and description of Opiliones genitalia are classically conducted using light microscopy. In this paper, we used a novel method of spermatopositor observation using confocal laser scanning microscopy (CLSM; ).

This study presents the first use of CLSM to describe the structure of a new species. The method relies on the natural fluorescence property of the arthropod cuticle to obtain a stack of 2D sections that can be combined into a 3D object. Quarkxpress 9 Free Download Trial Version. Regarding the extremely small size and fragility of the structure, observation with light microscopy and preparation for electron microscopy was impossible.

In addition, to be relatively fast and easy (with the whole process taking approximately 1 h), this technique is also non-destructive, which is a great advantage when working with collection specimens. After dissection, the spermatopositor was mounted in glycerin between a glass slide and a cover slip, as for observation with light microscopy. Details of experimental protocols can be found in and. The structure was imaged on a Zeiss LSM 510 meta confocal microscope. Three-dimensional visualization was carried out using Maximum Intensity Projection in the Zeiss LSM Image Browser software. T AXON SELECTION In this study, we use 14 species represented by 16 specimens in the molecular data set, including all the available representatives of the Iberian Peninsula, as well as other selected Cyphophthalmi (see ). All previous analyses of cyphophthalmid phylogeny show that Metasiro, once classified within Sironidae, belongs to the family Neogoveidae, and therefore it is used to root the trees.

Furthermore, many of our analyses indicate that the genera Parasiro and Suzukielus do not form a clade with the other sironids, or that they are early offshoots of the sironid lineage (; ), thereby justifying our choice of outgroups. Family Species 18S rRNA 28S rRNA 16S rRNA COI Neogoveidae DNA101532 Metasiro americanus DQ825542 DQ825595 DQ825616 DQ825645 Sironidae DNA100487 Cyphophthalmus duricorius AY639461 DQ513120 AY639526 AY639556 Sironidae DNA100499 Cyphophthalmus ere AY639462 AY639499 AY639527 AY639557 Sironidae DNA100494 Cyphophthalmus martensi AY639470 AY639505 AY639535 AY639562 Sironidae DNA100493 Cyphophthalmus minutus AY639473 DQ825591 AY639537 AY639565 Sironidae DNA101878 Paramiopsalis eduardoi sp. EU638284 EU638287 EU638281 EU638288 Sironidae DNA102080 Paramiopsalis eduardoi sp.

Family Species 18S rRNA 28S rRNA 16S rRNA COI Neogoveidae DNA101532 Metasiro americanus DQ825542 DQ825595 DQ825616 DQ825645 Sironidae DNA100487 Cyphophthalmus duricorius AY639461 DQ513120 AY639526 AY639556 Sironidae DNA100499 Cyphophthalmus ere AY639462 AY639499 AY639527 AY639557 Sironidae DNA100494 Cyphophthalmus martensi AY639470 AY639505 AY639535 AY639562 Sironidae DNA100493 Cyphophthalmus minutus AY639473 DQ825591 AY639537 AY639565 Sironidae DNA101878 Paramiopsalis eduardoi sp. Gowin Deluxe 2011 Serial Killers. EU638284 EU638287 EU638281 EU638288 Sironidae DNA102080 Paramiopsalis eduardoi sp. In addition to the phylogenetic analyses, we used three additional specimens of Paramiopsalis ramulosus collected in May 2008 for calculating within- and among-species molecular distances.

M OLECULAR DATA Molecular markers included two nuclear ribosomal genes (complete 18S rRNA, 18S hereafter, and a 1-kb fragment of 28S rRNA, 28S hereafter), one mitochondrial protein-encoding gene, cytochrome c oxidase subunit I, and mitochondrial 16S rRNA (16S hereafter). These markers have proven to be informative in many studies on Opiliones systematics (e.g. The DNEasy® tissue kit was used for tissue lysis and DNA purification, following the manufacturer's protocol (Qiagen). Total DNA was extracted by incubating the entire animal or appendage in lysis buffer overnight, as described in. The intact cuticle of the animal was then removed after the lysis step and kept in ethanol. Purified genomic DNA was used as a template for PCR amplification.

The complete 18S ( c. 1.8 kb) was amplified in three overlapping fragments of c. 900 bp each, using primers pairs 1F-5R, 3F-18Sbi, and 18Sa2.0-9R (; ). 1000 bp of the 28S rRNA was amplified using the primer set 28SD1F/28SrD1a-28Sb (;; ), or alternatively with the more internal forward primer 28Sa (). COI was amplified using the primer pair LCO1490/HCOoutout (; ), or alternatively with the more internal reverse primer HCO2198 ().

16S was amplified with the primer pair 16Sar-16Sb (; ). Polymerase chain reactions (PCR; 50 µL) included 2 µL of template DNA, 1 µM of each primer, 200 µM of dinucleotide-triphosphates (Invitrogen), 1× PCR buffer, containing 1.5 mM MgCl 2 (Applied Biosystems), and 1.25 units of AmpliTaq DNA polymerase (Applied Biosystems). The PCR reactions were carried out using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems), and involved an initial denaturation step (5 min at 95 °C), followed by 35 cycles including denaturation at 95 °C for 30 s, annealing (ranging from 44 to 49 °C) for 30 s, and an extension at 72 °C for 1 min, with a final extension step at 72 °C for 10 min.

The double-stranded PCR products were verified by agarose gel electrophoresis (1% agarose), and were then purified with a Perfectprep PCR Cleanup 96 system (Eppendorf). The purified PCR products were sequenced directly with the same primer pairs used for amplification. Each sequence reaction contained a total volume of 10 µL, including 2 µL of PCR product, 1 µM of one of the PCR primer pairs, 2 µL ABI BigDye 5x sequencing buffer, and 2 µL ABI Big Dye Terminator v3.0 (Applied Biosystems).

The sequencing reactions involved an initial denaturation step for 3 min at 95 °C, and 25 cycles (95 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min). The BigDye-labelled PCR products were then cleaned using Performa DTR Plates (Edge Biosystems). The sequence reaction products were then analysed using an ABI Prism 3730xl Genetic Analyzer. Chromatograms were edited, and overlapping sequence fragments for each individual were assembled using the software SEQUENCHER 4.7 (Gene Codes Corporation 1991–2007), after BLAST searches (), as implemented by the NCBI website (), were conducted to check for putative contamination.

The software package MACGDE: Genetic Data Environment for MacOSX () was used to determine fragments based on internal primers and secondary structure features (; ). 18S, 28S, and 16S rRNAs were divided into 2, 9, and 5 fragments, respectively, according to internal primers and secondary structure features. The COI was divided in six fragments. All new sequences have been deposited in GenBank under accession codes EU638281–EU638289 (see ). P HYLOGENETIC ANALYSES Phylogenetic analyses were conducted under Direct Optimization (; ) with POY version 4.0 RC build 2615 (). Tree searches were conducted under parsimony with 20 random-addition sequences, followed by subtree pruning and regrafting (SPR) and tree bissection and reconnection (TBR), and one round of ratcheting ().

A detailed description of the commands used to implement this search strategy with the new version of POY can be found in. In order to assess nodal stability (; ), different parameter sets were used for a range of indel-to-tranversion and transversion-to-transition ratios.

We used ‘111’, ‘121’, ‘211’, and ‘3221’ transformation matrices. It has been argued that the parameter set ‘111’ (often called ‘equal weighting’ or ‘unweighted parsimony’) maximizes explanatory power (), but it requires all indel events to be counted independently. Parameter set ‘3221’ was implemented using the commands transform(tcm:(2,1), gap_opening:3). Nodal support was estimated by jackknifing (; ) with 100 replicates using the command transform (auto_sequence_partition:true), which increases the number of fragments - and thus the number of characters - used for jackknifing. The data for all genes were analysed simultaneously. Molecular evolutionary analyses for estimating p-distances and the standard error associated for COI sequence data were conducted using MEGA v3.1 ().

For these analyses we used three additional specimens of Paramiopsalis ramulosus from northern Portugal. RESULTS P ARASIRO COIFFAITI (). Spermatopositor () small (180-µm long), with 3/3 dorsal long bifurcating microtrichia with enlarged bases, and shorter ventral microtrichia without enlarged bases.

Pars apicalis with hooked mobile digits. Ventral part with four pairs of shorter microtrichia; ventral plate clearly surpasses the pars distalis, only visible from the dorsal side. Ovipositor not studied. Etymology: The species is named after our colleague Eduardo Mateos from the Departament de Biologia Animal, Universitat de Barcelona, who collected the specimens of the new species. Phylogentics: Our molecular phylogenetic analysis resulted in a well-resolved and stable phylogeny. We show the single most parsimonious tree of 2914 steps obtained under equal weighting (), and represent the nodal stability using sensitivity plots. Except for Suzukielus sauteri(Roewer, 1916), which appears as sister to the genus Siro under alternative parameter sets, all nodes appear highly stable.

The two specimens of Paramiopsalis eduardoi sp. Form a clade (100% jackknife proportion), and group with Paramiopsalis ramulosus (100% bootstrap).

The genus Paramiopsalis appears as sister to the genus Cyphophthalmus with strong support (93% jackknife proportion), and under all of the parameter sets examined. Phylogeny of selected members of the family Sironidae, based on the combined analysis of 18S, 28S, cytochrome c oxidase subunit I (COI), and 16S under direct optimization and equal weighting.

The support values on branches indicate the jackknife frequencies. Each weighting scheme is assigned a code corresponding to the ratio of indel/transversion, transversion/transition, and transition values. Tree lengths for the different parameter sets are as follows: 111, 2914; 121, 4585; 211, 3228; 3221, 6168. Black squares indicate monophyly; grey squares indicate that either the group is paraphyletic or the internal relationships are different.