Nesticus baeticus sp. n., a new troglobitic spider species from south-west Europe (Araneae, Nesticidae)

Abstract A new troglobitic species, Nesticus baeticus sp. n. (♂♀), inhabiting the karst landscapes of the high part of the Cazorla, Segura and Las Villas Natural Park (NE Jaén, Spain) where it has been found in 8 caves is diagnosed and described, its distribution and habitat are also analyzed.The new species belongs to the Iberian species group that includes Nesticus luquei, Nesticus lusitanicus and Nesticus murgis. Evolutionary relationships of the Iberian Nesticus species are discussed on the basis of morphological and molecular data (cox1 and rrnL).


Introduction
Th e genus Nesticus Th orell, 1869 is distributed worldwide except for south-eastern Asia and Australia and comprises 125 species and 8 subspecies (Platnick 2011). In Europe Nesticus is represented by 23 species, of which fi ve are known from Iberia. Unlike N. cellulanus (Clerck, 1757), a species with a holarctic distribution, the four others are endemic to Iberia, being cavernicolous species with more or less evident troglomorphic features.
Th e fi rst species described from Iberia was N. obcaecatus Simon, 1907, found only from a single locality: Cueva del Molino de Aso (Huesca), on the southern slopes of the Central Pyrenees (Simon 1907). Th e species description was originally based on a single female specimen. Th e male was described several decades later (Ribera 1979), from a series of both sexes collected from the same cave. Th e second Iberian endemic is N. lusitanicus Fage, 1931, a native species of the karst landscape in central Portugal. Th is species was described on the basis of females (Fage 1931), its male was found half of a century later (Ribera 1988). Th e third species, which was found in several caves in Asturias and Cantabria (north-western Iberian Peninsula) (Ribera and Guerao, 1995), is N. luquei Ribera & Guerao, 1995. Finally, N. murgis Ribera & De Mas, 2003 was described from a cave in the province of Almeria (Ribera and De Mas 2003). Overall, ranges of these species are rather small, in two species (N. obcaecatus and N. murgis) being restricted to a single cave.
Th is work describes a new cavernicolous species whose distribution includes several karst landscapes of the diff erent mountain formations that make up the Sistema Bético, the ridge in southern Iberian Peninsula.
Female vulva was removed and treated with 30% KOH. After observation and drawing, the vulva was washed in distilled water and stored in 70% ethanol. Left male palps were illustrated in all cases. We follow Coyle and McGarity (1992) for describing the paracymbium, and Huber (1993) and Agnarsson et al. (2007) for other parts of male and female copulatory organs. Holotype and paratypes have been deposited in the Arachnida Collection of the CRBA (Centre de Recursos de Biodiversitat Animal) at the University of Barcelona. Catalogue numbers are given in brackets.
A Nikon Coolpix 4500 digital camera attached to a stereomicroscope was used to capture images. Following Coleman (2003) and with the aid of a Trust Scroll Tablet TB-4200 the samples were drawn with repeated reference to the specimen under the microscope. Th e specimens used for SEM studies were dehydrated with alcohol gradi-ent dehydration and ultrasonically cleaned. Th ey were then critical-point dried and were mounted and covered with gold and examined using a HITACHI S-2300 Scanning Electron Microscope (SEM) (SCT, Universitat de Barcelona, Spain).

Phylogeny
Taxonomic sampling. Taxa analyzed in the present study are listed in Appendix 1. All the Iberian species are included except N. murgis due to impossibility to obtain fresh material for DNA analysis. N. eremita Simon, 1879 from Croatia andN. ionescui Dumitrescu, 1979 from Romania are also included to test the monophyly of Iberian species. Sequence from Nesticus sp. from China (Arnedo et al. 2004) was also included in the analysis as a more distantly related Nesticus species that was used to root the tree.
Sample Storage and DNA Extraction.Specimens were preserved in 95% or absolute ethanol and stored at 4°C. Total genomic DNA was extracted from legs of a single specimen using the QIamp® DNA Mini Kit (QIAGEN) following the manufacturer's protocols. Th e approximate concentration and purity of the DNA obtained were verifi ed using 1% agarose/TBE gel electrophoresis.
A Perkin-ElmerCetus Moldel 480 thermocycler was used to perform 35 iterations of the following cycle: 30s at 95°C, 45s at 45°C, and 1 min at 72°C, beginning with an additional step of 3 min at 95°C, and ending with another step of 10 min at 72°C. PCR results were visualized by means of a 1% agarose/TBE gel. Amplifi ed products were purifi ed using Microcon PCR columns following the manufacturer's specifi cations. Purifi ed products were directly cycle-sequenced from both strands using ABI BigDye (Applied Biosystems) chemistry, precipitated in DyeEx Spin kit (Qiagen, Chatsworth, CA) columns, and run out on ABI Prism 377 (Applied Biosystems) automated sequencers. Sequencing reactions were performed in our lab with the forward and reverse PCR primers. Resulting product were run and analyzed at the Serveis Científi co-Tècnics of the Universitat de Barcelona.
Alignment. Raw sequences were compared against chromatograms and complementary contigs built and edited using the Geneious Pro 3.6.2 software (http://www. genious.com). Sequences were manipulated and preliminary manual alignments constructed using BioEdit V.7.0.5.3 (Hall 1999). Alignment of the cox1 gene fragments was trivial due to the absence of length polymorphism. However, there were some length diff erences among the rrnL fragments, suggesting the occurrence of insertion/ deletion events during the evolution of these sequences. Automatic alignment algorithms have been considered as superior to manual protocols due to their objectivity and repeatability (Giribet et al. 2002). Automatic alignments for the rrnL data set were constructed with the program MAFFT v 6.240 (Katoh and Toh 2007) Th e alignment was constructed using the manual strategy option set Q-INS-I, the most accurate multiple sequence alignment, whit default options. All analyses were performed by coding gaps as absence/presence character following Simmons and Ochoterena's simple coding method (Simmons and Ochoterena 2000), as implemented in the software GAPCODER (Young and Healy 2002). Th is method allows the inclusion of gap information in phylogenetic inference, minimizing the eff ect of increasing the weight of overlapping multiple non-homologous gaps that results from scoring gaps as 5 th state (Pons and Vogler 2006).
Phylogenetic analyses. Parsimony analyses of the combined data matrices were conducted with the program Winclada v.1.00.08 (Nixon 2002) using the following heuristic tree search strategy: 1000 iterations of 10 Wagner trees constructed with random addition taxa and subsequent TBR branch swapping, holding a total maximum of 10000 trees. Th is program facilitated combination of the diff erent gene fragments in a single data set for simultaneous analyses and also provided additional statistics for those trees (CI and RI values). Clade support was assessed via Bootstrap (Felsenstein 1985) as implemented in Winclada, based on 1000 bootstrap replicates with 20 iterations and 10 starting trees per replica. Uncorrected genetic distances between taxa of cox 1 gene from terminal taxa were assessed with the program MEGA v.3.0 (Kumar et al. 2004). Etymology. Th e Latin name 'baeticus' means 'from Baetica' (the south of Spain) and refers to the 'Sistema Bético', the ridge containing the karst landscapes from where the new species was collected.

Nesticus baeticus
Diagnosis. Males clearly diff er from those of other Nesticus species in the shape of paracymbium (8)(9)(10) and in the TTA structure (11)(12). In females, the development of the median septum of the vulva (Figs14-16), the shape of the spermathecae and adjoining structures are also diagnostic (Fig. 17). Th e degree of ocular reduction of the AM eyes (Fig. 2) is also characteristic compared to other Iberian species.
Comments. On the basis of morphology, N. baeticus sp. n. lies within the group including N. murgis (known from Almería) and N. luquei (an endemic to northwestern Spain). Th e shape and arrangement of the median apophysis (Figs 5, 11-12), the embolus (Figs 3-7, 11-12) and the paracymbial processes of male palp (Figs 8-10), plus the location and structure of the spermathecae and vulval glands of the female (Fig 17) are similar in all three species. N. baeticus sp. n. diff ers more signifi cantly from N. lusitanicus, both in the morphology of the copulatory organs of both sexes.

Distribution.
Nesticus baeticus sp. n. inhabits the karst landscapes of the high part of the Cazorla, Segura and Las Villas Natural Park (NE Jaén, Spain) where it has been found in 8 caves. Most of the material studied comes from the area surrounding the Tranco's Reservoir, in Hornos, Jaén. Th e area is calcareous, lush and quite humid, with numerous, medium-sized caves, both horizontal and vertical. Th e specimens were generally located within the fi rst few meters of the dark zone, their presence reaching towards the cave interiors, which were sampled more intensively.

Discussion
Specimens and sequences, with corresponding Genbank accession numbers, analyzed in the present study, are listed in Appendix 1. Alignments of two mitochondrial genes and gap scores as presence/absence were merged resulting in a combined data matrix of 950 characters (cox1=472, rrnL=456 and 22 gap characters). Uncorrected cox1 genetic divergences among terminal taxa are summarized in Appendix 2. Parsimony analyses of the combined data matrix yielded a single most-parsimonious tree of 705 steps (CI = 73 and RI = 62) (Fig. 1). Th e results show that the Iberian species do not constitute a monophyletic group. N. luquei, N. lusitanicus and N. baeticus sp. n. form a clade with high bootstrap support (99%), while N. cellulanus is nested within a clade that also includes N. ionescui, from Romania, and N. eremita, from Croatia. N. obcaecatus is the sister group of the remaining species of the ingroup. Th is topology, along the high genetic divergences observed between N. obcaecatus, N. cellulanus and the remaining Iberian species suggest the existence of tree independent colonization to the Iberian Peninsula. Preliminary results of a more extensive phylogenetic analysis, including almost all the Mediterranean species of Nesticidae (our work in progress), support this hypothesis.
Th e morphology of both the male and female copulatory organs of this Iberian group of species (N. luquei, N. lusitanicus, N. murgis and N. baeticus sp. n.) shows important diff erences as compared to N. cellulanus, the type species of the genus Nesticus, as well as to the Carpathonesticus species. Th us, the absence of paracymbial ramifi cation, the shape and size of the median apophysis, plus clear diff erences in size and arrangement of the TTA processes constitute the major diff erences in the males. Th e number of spermathecae is the most noticeable character in the females.
With regards to N. obcaecatus, signifi cant diff erences in the shape and structure of the paracymbium, the median apophysis and the TTA (see Ribera 1988), as well as in the shape of the epigynum and the arrangement of the spermathecae (see Fage 1931), indicate that this species is most distantly-related to all the Iberian endemics known to date. On the basis of the aforementioned characters N. obcaecatus seems to be more closely-related to N. idriacus Roewer, 1931 known from the eastern area of the Alps and to N. morisii Brignoli, 1975 known from Italy. Besides, the conformation of the copulatory organs of these three species are very similar to Typhlonesticus absoloni (Kratochvil, 1933) known from Montenegro. Th us, all three species are likely to belong with Typhlonesticus as well. Yet, in order to prove or reject this assumption, a molecular phylogeny of all the Mediterranean Nesticidae is to be performed. Our future research is focused on resolving the phylogeny of the Mediterranean Nesticidae (work in progress) and will include most of the Mediterranean species to test the monophyletic status of current genera.
N. obcaecatus shows highly troglomorphic characters, such as complete depigmentation, reduction of the eye size and their number (only six eyes), and is known from a single cave. Th ese data alongside its phylogenetic uniqueness (basal position and a deep genetic distance from other Iberian congeners) suggest that this species may be considered a relict representative of an old colonization to Iberia, and should be a candidate for protection.
Climatic relict hypothesis assume that adaptation and speciation to caves are mainly driven by climatic factors. Th e Pleistocene glacial cycles has been identifi ed as de main driver of the evolution of cave-dwelling fauna in the Paleartic (Barr 1968;Vandel 1958Vandel , 1964. Th e uncorrected genetic distances between N. baeticus, N. lusitanicus and N. luquei range between 14.3 and 15.5%. Assuming an average substitution rate for arthropod mitochondrial genes between 2% (DeSalle et al. 1987) to 2.3% (Brower 1994) we can conclude that the origin of these species preceded the Pleistocene glacial cycles and, hence that other climatic or environmental factors may have been responsible for the evolution of these taxa.