A NEW SPECIES OF THE FAMILY BATHYNELLIDAE ( CRUSTACEA , SYNCARIDA ) FROM SPAIN

A. I. Camacho, B. A. Dorda & I. Rey. 2013. Integrated DNA and morphological taxonomy to describe a new species of the Family Bathynellidae (Crustacea, Syncarida) from Spain. Graellsia, 69(2): 179-200. A new species of Bathynellidae Grobben, 1905 is described from Spain. Vejdovskybathy-nella vasconica sp. nov. displays an exclusive feature within the genus: eightsegmented antenna. Besides, the new species has a unique combination of morphological characters, including medial seta on exopod of antenna, antennule length similar to antenna, three-segmented mandibular palp without sexual dimorphism, four segments on endopod of thoracopod I to VII, three spines on the sympod of uropod, two claws on the endopod of uropod, first spine longer than the rest on the furcal rami, exopod smaller than endopod on female thoracopod VIII, a long frontal projection and medium size outer protuberance on penial region of male thoracopod VIII, and a medium size frontal crest with a small “spur” on basipod of male thoracopod VIII. Partial sequences from mitochondrial gene cytochrome oxidase I (COI) and 18S ribosomal RNA (rRNA) gene have been obtained from specimens of the type locality of the new species. The analyses of molecular data demonstrate the presence of two highly divergent genetic units within the Bathynellidae, corresponding to two morphologically well differenciated genera. urn:lsid:zoobank.org:pub:B30D62AD-CE8F-482B-BF4A-8979CD98A31D


Introduction
In traditional taxonomy a unique combination of morphological features is used to define a given taxon.Nevertheless, this approach does not seem to differentiate some species within certain groups such as the Bathyllenacea, an order of groundwater crustaceans where in some cases morphology is not fully satisfactory to distinguish between closely related species.Therefore, in order to conduct proper taxonomic and evolutionary studies in Bathynellacea it is higly recommended to obtain DNA sequences from different species to establish comparisons, combining a molecular and a morphological approach.This especially holds true for the Bathynellidae, where differences between species are particularly subtle and very difficult to find.
In this paper we describe morphologically a new species of the genus Vejdovskybathynella (Bathynellidae) from the Iberian Peninsula.We obtained sequences of mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) and of nuclear DNA, 18S ribosomal RNA (rRNA) genes, from several specimens of the new species and of representatives of other taxa, some identified down to species level and others not, from the families Bathynellidae and Parabathynellidae.We have compared the divergent clades that can help to assign specimens to genera and to elucidate the phylogenetic relationships between taxa at both higher levels (family and genus) and at the species level.

Material and methods
TAXONOMIC SAMPLING/SPECIMEN COLLECTION Specimens studied were found in 4 samples from Goikoetxe cave, Vizcaya (northern Spain).All samples were collected with a 0.1 mm mesh hand net (plankton type) from water in small gours and pools in the epikarstic zone.The specimens used in the morphological study were fixed in 4% buffered formalin and stored in etanol (70%).Specimens used for the molecular study were directly frozen at -20ºC, in 400 ml of distilled water.
The morphological and molecular descriptions are based on the type series.
MORPHOLOGICAL STUDY  A total of 23 specimens were used in the morphological study: 16 females and seven males collected in 29 April 2011.They constitute the type series of the new species described herein.
A complete dissection of all anatomical parts of several specimens was done and, together with whole specimens, kept as permanent slides (special metal slides, glycerine gelatine stained with Methylene Blue as mounting medium).Anatomical observations were performed using an oil immersion lens (100X) with a Zeiss interference microscope provided with a drawing tube.The material is deposited in the Museo Nacional de Ciencias Naturales, Madrid (MNCN).
We follow the terminology proposed by Serban (1972 and following papers) to name Md and male Th VIII.Serban worked in depth on the family Bathynellidae (Serban 1989a(Serban , b, 1992;;Serban & Leclerc, 1984;Serban et al., 1971), revised and described many genera, and we think that his terminology is the most accurate and intuitive.

SPECIMEN COLLECTION FOR DNA EXTRACTION
Twenty eight specimens were used for DNA extraction, but succesful DNA extraction was only posible from six, and are part of the type series.
This type material, the DNA extracted from the six specimens, is deposited in the Tissues and DNA Collection of the Museo Nacional de Ciencias Naturales, Madrid (MNCN).Voucher numbers are shown in Table 1.
In order to examine the phylogenetic relationships between bathynellids, we used partial DNA sequences of the mtDNA gene COI (508 bp) and the nuclear 18S rRNA (997 bp).The small subunit (SSU) 18S rRNA gene is one of the most frequently used genes in phylogenetic studies and an important marker for random target PCR in environmental biodiversity screening (Chenuil, 2006) and in general, rRNA gene sequences are easy to access due to their highly conserved flanking regions allowing the use of universal primers (Hillis & Dixon, 1991).COI sequences were obtained from two individuals, and 18S rRNA sequences were obtained from six specimens.

DNA EXTRACTION, AMPLIFICATION, AND SEQUENCING
Extraction was carried out with Chelex following Walsh et al. (1991).Fresh specimens were cut in two in distilled water, and were placed in the wall of a 1.5 ml microcentrifuge tube with a sterile needle.Each tube contained 100 ml Chelex 100 (Bio-Rad; 5% in distilled water) and 400 ml of distilled water.The specimens were incubated overnight at 56ºC, followed by 10 minutes at 100ºC and centrifuged at 16,000g for 10 minutes.
A 510 base pair (bp) region of the COI gene was amplified with the primers C1-J-1718 (5'-GGAG-GATTTGGAAATTGATTAGTTCC-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAA AATCA-3') (Folmer et al., 1994;Simon et al., 1994).A 997 base pair (bp) fragment of the 18S rRNA region was amplified in two fragments, using the primers 1F (5'-TACCTGGTTGATCCT-GCCAG TAG-3') and 3R (5'-AGGCTCCCTCTC-CGGAATCGAAC-3') and 3F (5'-GTTCGATTCC GGAGAGGGA-3') (Giribet et al., 1996) and bi (5'-GAGTCTCGTTCGTTATCGGA-3') (Whiting et al., 2002).Three ml of the DNA solution were used as a template.Other components of the 25 ml PCR reaction were: 1x of the corresponding buffer (75 mM Tris HCl, pH 9.0; 50 mM KCl and 20 mM (NH 4 ) 2 SO 4 ), 2 mM MgCl 2 ), 10 mM dNTPs mix, 0.1 mM of both primers, 0.02% BSA, and 0.125 units AmpliTaq Gold® DNA Polymerase (Applied Biosystems).Six ml of PCR products were electrophoresed through a 1.5% agarose gel and visualized with Ethidium Bromide under ultraviolet light.PCR products were purified by treatment with ExoSAP-IT (USB Amersham, Buckinghamshire, UK) in a 5:1 amplicon: enzyme ratio and incubated at 37 ºC for 45 min, followed by 15 min at 80 ºC to inactivate the enzyme.The purified PCR product was then used to sequence in both directions using the BigDye Terminator v3.1 sequencing kit (Applied Biosystems Inc., Foster City, USA) in a 10 ÌL volume, containing 15-20 ng of purified product and 3 pmol of primer.To verify that the sequences obtained came from a bathynellacean, they were compared with sequences from GenBank using Blast (Altschul et al., 1997).The alignment of all bathynellacean COI and 18S gene sequences generated in our lab was performed and edited manually using MEGA 4.0 (Tamura et al., 2007).Fine adjustments were made by eye, as the COI does not present any gaps.All sequences were submitted to GenBank (see    CSIC (Spain) and GenBank accession numbers of specimens.* specimens used in phylogenetic reconstruction (Fig. 4A).Tabla 1.-Localidades de las muestras estudiadas de Bathynellacea, número de la colección de Tejidos y ADN del MNCN y número de acceso a Genbank de cada uno de los ejemplares estudiados.*ejemplares usados en el analisis filogenético (Fig. 4A).PHYLOGENETIC AND DNA SEQUENCE ANALYSIS Phylogenetic reconstruction based on COI and 18S rRNA sequence data involved Bayesian Inference (BI), maximum likelihood (ML) and maximum parsimony (MP) approaches, using separate data sets.To examine relationships between species of Bathynellidae, we analysed the mtDNA COI sequences obtained by us from several species of Vejdovskybathynella Serban et Leclerc, 1984(V. edelweiss Camacho, 2007and V. caroloi Camacho, 2007), Paradoxiclamousella Camacho, Dorda et Rey, 2013(P. fideli Camacho, Dorda et Rey, 2013 and P. cf fideli) and other undetermined genera from Spain.The Parabathynellid Iberobathynella imuniensis from Spain was chosen as out-group (GenBank accession number HQ659850).To examine relationships between families and genera, we used 18S rRNA sequences obtained by us.Genera of Parabathynellidae included Iberobathynella Schminke, 1973, Paraiberobathynella Camacho et Serban, 1998and Hexabathynella Schminke, 1972, all from Spain.In the case of the Bathynellidae, we used the genera Vejdovskybathynella, Paradoxiclamousella, plus other not yet determined material from Spain (voucher and GenBank accession numbers shown in Table 1).The Anaspidid Syncarida Anaspides tasmaniae Thomson, 1893 was chosen as out-group (GenBank accession number DQ310660).Pairwise comparisons of observed proportional sequence divergence (p-distance) (Tables 2 and 3) and corrected sequence divergence (Kimura-2parameter model), were determined using PAUP*4.0b10(Swofford, 2002).To test for possible saturation of nucleotide substitutions (only with COI sequences), we plotted p-distance (y) versus corrected estimates of proportional sequence divergence (x) for first, second and third codon positions, as well as for transitions and transversions separately (not shown).We initially explored the dataset using distance analyses (neighbour joining, NJ) with PAUP*4.0b10.Phylogenetic analyses were conducted using Maximum Likelihood (ML; Felsenstein, 1981Felsenstein, , 1985) ) and Bayesian inference (BI) (Huelsenbeck & Ronquist, 2001;Ronquist & Huelsenbeck, 2003).All characters were equally weighted.Modeltest 3.7 (Posada & Crandall, 1998) were used to identify the model of sequence evolution that best fit the data, based on Akaike information criteria (AIC), for use in the phylogenetic (ML) and distance analyses (NJ).The general timereversible model of evolution (GTR) with gamma parameter and a proportion of invariable positions (GTR+G+I) was selected as the best fit and was used for ML (Yang, 1994;Gu et al., 1995;Swofford et al., 1996) and BI analyses.ML analyses with empirical base frequencies were performed using Garli (Zwickl, 2006;Zwickl & Balhoff, 2006).We used nonparametric bootstrapping (500 pseudoreplicates) to assess the stability of internal branches in the resulting topologies (Felsenstein, 1985;Felsenstein & Kishino, 1993).BI analysis was performed with MrBayes 3. 1.2 (Ronquist & Huelsenbeck, 2003) assuming six discrete gamma categories.Bayesian analyses were initiated with random starting trees and run for 1,000,000 generations sampled every 100 generations.The convergence occurred during the first million generations, the likelihood values converged to relative stability after approximately 100,000 generations; subsequently we conservatively discarded all samples obtained during the first hundred thousand (10%) generations as burnin.Robustness of the observed clades was assessed with Bayesian posterior probabilities.

Systematic account
According to Serban (2000), it was assumed that the largest part of France and the Iberian Peninsula is populated by representatives of the subfamily Gallobathynellinae Serban et al., 1971.This subfamily consists of four tribes (the differences are shown in Table 4), two subtribes, 11 genera, and 25 species including the new species described herein (Camacho, 2007;Camacho et al., 2013).The majority of the species occur in France, two are known from Italy, one from Switzerland, one from Germany, and eight, including the new species, from Spain.
The new species belongs to the Tribe Vejdovskybathynellini Serban, 1989 (see Table 4) based on the number of main teeth on the Mandible (5), the three-segmented mandibular palp, the four segmented endopod of the thoracopods I to VII, an antennule similar in size to antenna, basipod of the male thoracopod VIII vertical and with frontal crest with frontal spur and penial region with a lobe with outer protuberance, a developed frontal projection and one outer lobe of similar length.This Tribe has a single genus, Vejdovskybathynella Serban et Leclerc, 1984, to which the new species belongs.Table 5 summarizes the main features of the different genera from subtribes Gallobathynellina and Meridiobathynellina, as well as the genus Vejdovskybathynella, clearly identifying the differences between them.Tribe Vejdovskybathynellini Serban, 1989 DIAGNOSIS (translated from Serban 1989a, 1989b and modified) A. I and A. II ordinary.Md.: mandibular palp with three segments with the setae longer in the males than in the females or not.Th.I-VII: endopod with four segments in all the pairs.Male Th.VIII: penial region with one lobe that shows an elongated frontal projection and an outer lobe, vertical basipod with a very prominent frontal crest (Fr.crt.) and with a distal region curved on the external side, forming a spur (''eperon'') (S. fr.crt.); endopod a single segment, elongated and shorter than the exopod.Female Th.VIII ordinary, coxal seta elongated or absent (see Table 4).
Genus Vejdovskybathynella Serban et Leclerc, 1984 DIAGNOSIS (modified from Serban andLeclerc 1984 andCamacho, 2007) A. I: with seven segments.Md.: mandibular palp with or without sexual dimorphism.Male Th.VIII: penial region with frontal projection and a large outer lobe; the basipod has a vertical position and a prominent frontal crest (Fr.crt.).Female Th.VIII: with an exopod simplified.Uropod: sympod with three or four spines and endopod with two or three spines.Furcal rami: first or second spine the longest (see Table 5).TYPE SPECIES.Vejdovskybathynella balazuci Serban et Leclerc, 1984.

DESCRIPTION
Body.Total length of males 0.65-0.79mm, of females 0.56-0.85mm.Body elongated, segments slightly widening towards posterior end; each approximately nine times as long as wide.Head twice as long as wide.Pleotelson with one small smooth dorsal seta at each side.All drawings are of the holotype (male) except for Th VIII female, and one figure of Md that belongs to the allotype.
Antennule (Fig. 1A).As long as antenna, sevensegmented; length of first three segments slightly longer than the other four segments; fourth and sixth segmentssimilar and smaller than seventh; fifth smallest; inner flagellum rectangular; setation as in Fig. 1A; segment six and seven with two terminal aesthetascs, all similar in size.
Paragnaths (Fig. 1D).Elongated and with a projection on the distal part; with fine setules around distal part.
Mandible (Figs. 1E, F).Palp with three segments, terminal segment with two equal and barbed claws.Masticatory part: pars molaris with five simple main teeth, processus incisivus accessorius with one tooth and incisor process (pars incisiva) with two teeth.
Maxillule (Fig. 1G).Proximal endite with four setae; distal endite with six teeth, four of which provided with denticles othertwo simple, seta-like; three plumose setae, of similar size, on the outer margin of endite.
Male thoracopod VIII (Fig. 3C, D).Outer lobe (O.lb.), similar to the frontal projection and not exceeding the basipod, both with transverse circular section; expanded outer protuberance (O.prt.); vertical rectangular basipod (Bsp.)without seta; frontal crest of basipod not very prominent and with a very small spur; exopod with six setae; endopod one-half the size of the exopod, with two small terminal setae of similar length.
Female thoracopod VIII (Fig. 3E).Coxa without setae; very large epipod reaching, beyond distal end of exopod; endopod small and slightly more slender than exopod, single segment with two apical smooth setae; exopod with two apical unequal smooth setae of different lengths.
Uropod (Fig. 3G).Sympod 1.6 times longer than wide and 1.6 times longer than endopod, with three equal spines; endopod slightly longer than exopod, with two strong similar claws, terminally with two barbed setae (one of them very long) and with two plumose setae located dorsolaterally; exopod with two barbed terminal setae, of which external very long and two small barbed medial setae.
Furcal rami (Fig. 3H).Almost square, bearing five spines; dorsal spine a little shorter than second      and third spine but thinner, a little longer than fourth spine, and 1.7 times shorter than the first, which is the longest, but not very long, attaining 2.5 times length of furca.
ETYMOLOGY.The species name, vasconica, is derived from the Basque Country, northeastern Spain, where the Goikoetxe cave is located and where the new species has been found.It is the first species of Bathynellacea described from the Basque Country.
In the female Th.VIII the exopod is larger than the endopod, except in V. edelweiss and V. vasconica sp.nov., where they are similar in size or shorter.Only the new species lacks setae on the coxopod.
The male Th.VIII of V. edelweiss and V. vasconica sp.nov. is more similar to V. balazuci and V. espattyensis than to the Spanish species, though they have a less-developed frontal projection and a more rectangular basipod than the French species.The male Th.VIII of V. pascalis and V. caroloi have a more square aspect.
The pleopod of the new Spanish species has four setae on the second segment like two of the French species, whereas the third French species has six setae and the other Spanish species have five.
V. vasconica sp.nov. is the only species of the genus with three spines on the sympod of the uropod.The new species and V. pascalis have two claws on the endopod of the uropod like the French species; the rest of Spanish species display three claws.
V. edelweiss and V. pascalis have dorsal spines on the furcal rami that are longer than the first spine; in the case of the new species they are smaller and in the rest of the species they are all similar; in V. vasconica sp.nov. the first spine of the furcal rami is longest, whereas the second spine is longest in the rest of species of the genus and is very long in V. balazuci, V. espattyensis, and V. caroloi (four times longer than the first spine).

NUCLEOTIDE ANALYSIS
The alignment of all Bathynellacean COI gene sequences (15 specimens in total) resulted in a consensus length of 508 bp, of which 58% were variable.The models of sequence evolution selected for the mtDNA were GTR + I + G.No stop codons or gaps were observed in any of the translated amino acid sequences suggesting that the genuine mtDNA COI gene was sequenced.The base frequencies were as follows: A= 0.29, C= 0.13, G= 0.14 and T= 0.44.
The alignment of all Bathynellacean 18S rRNA gene sequences (16 specimens) comprised 45% variable sites; base frequencies were similar in all cases, about 25% (24.9% A, 24.2% C and T and 26.7% G).

GENETIC DIVERGENCES
The genetic divergence of COI is relevant at the species level, with significant results found within the different morphospecies (Camacho et al., 2011(Camacho et al., , 2012)).The uncorrected sequence divergence esti- Within the same genus, the new species showed a genetic distance of 14.2 % to 16.7 with respect to other species of Vejdovskybathynella, the maximum being with V. caroloi and the minimum with V. edelweiss (Table 2).The divergence of the new species with different populations of Paradoxiclamousella fideli ranged from 15.7% to 16.7%, while the divergence with the only known COI sequence of an Australian species of the family Bathynellidae showed the highest genetic distance (from 18.7% to 19.3%).The lowest genetic distance found between species was with the assigned specimen from Imunia cave (12.8%).In the case of the specimens from Río Chico cave and Erizo rivers in the Ojo Guareña cave, genetic distances ranged from 13% to 13.6%.Finally, the genetic distance between the two specimens studied of V. vasconica sp.nov.was only 0.6%.
The uncorrected sequence divergence estimates of 18S rRNA between all the specimens studied and the outgroup taxa are summarized in Table 3.The genetic divergence between the outgroup used, Anaspides tasmaniae (Thomson, 1893) and both families of the Bathynellacea ranges from 11.44% to 14.98%.The average uncorrected sequence divergence estimates between families and genera, and with the outgroup taxa are summarized in Table 9. Anaspides shows more genetic divergence with the Bathynellidae than with the Parabathynellidae.Genetic divergence between genera, in both families, are normally between 6% and 8%, with the exception of the distance shown between Iberobathynella and Paraiberobathynella, which is only about 2%, a value more typical of intrageneric genetic divergence.The lowest distance between both families occurred between I. parasturiensis and V. edelweiss (7.60%), and the highest between Pi. cf.maghrebensis and P.cf.fideli (12.06%).
In both families, the lowest distance between two species in the same genus occurred in the Paraiberobathynella (0.41%), and the highest between Iberobathynella burgalensis (from the North of Spain) and I. celiana (from southern Spain)  (2.17%), followed by 2.15% between Vejdovskybathynella edelweiss and V. caroloi.

PHYLOGENETIC ANALYSES
The results of the phylogenetic analyses (ML, Bayesian) are summarized in Fig. 4A and B. The COI mtDNA sequence data analysis produced a tree (A) in which all samples of Bathynellidae are clearly separated from Iberobathynella imuniensis (Parabathynellidae) and Bathynellidae sp1 from Australia.There are two groups supported in ML and Bayesian analyses (> 100% Bayesian posterior probability and 93% and 87% bootstrap values).The phylogenetic reconstruction revealed that the major clades were clearly grouped in at least two different genera: Paradoxiclamousella and Vejdovskybathynella.The first robust group (93% bootstrap) is formed by P. fideli from CO69 cave, the population of CO209 cave and an unassigned Bathynellidae from La Nava cave (probably belonging to Paradoxiclamousella genus).The other clade (87% bootstrap) is formed by the specimens of Vejdovskybathynella edelweiss (Ojo Guareña and Huesos caves), the new species (Vejdovskybathynella vasconica) and two subgroups formed by unassigned Bathynellidae from Imunia and Redonda caves and Erizo river and Río Chico caves respectively (probably belonging to Vejdovskybathynella). The species from La Gándara cave, Vejdovskybathynella caroloi, appears as the only phylogenetic incongruence in the topologies observed in the gene tree for COI.The type locality of the new species is geographically farther from Paradoxiclamousella populations than Vejdovskybathynella edelweiss and V. caroloi populations (see Figure 5).V. vasconica sp.nov.shows enough genetic distance with respect to V. edelweiss (about 15%) and V. caroloi (15.75% to 16.73%)as to be separated into a different clade (see Table 2).
The 18S rRNA sequence data analysis produced a tree (B) in which there are two groups supported in ML and Bayesian analyses (> 100% Bayesian posterior probability and 99% and 100% bootstrap values).All samples of Bathynellidae are clearly separated from the Parabathynellidae.The phylogenetic reconstruction of the Parabathynellidae family revealed that the major clades clearly grouped, at least, two different genera: Hexabathynella and Iberobathynella.Paraiberobathynella appears nested within the group of Iberobathynella species, and although the bootstrap value is not very high (61%), this indicates a small phylogenetic incongruence.The phylogenetic reconstruction of the Bathynellidae revealed that the first robust group (100% bootstrap) is formed by P. fideli from CO69 cave and the population of CO209 cave.The other group is formed by Vejdovskybathynella (including the new species described herein) and various unassigned specimens from several populations, but the support is somewhat weak (27%).

General discussion and conclusion
According to Serban (2000), none of the Iberian species belong to the genus Bathynella Vejdovsky, 1882, which ranges mainly to the northeast of France and never beyond the Pyrenees (Camacho, 2007).Current knowledge suggests that other representatives of the subfamily Gallobathynellinae Serban, Coineau et Delamare Deboutteville, 1971, populate most of France and the entire Iberian Peninsula.Here we describe a new species from northern Spain belonging to Vejdosvkybathynella.This is the fourth species of this genus found in Spain, all of them being limited to the north of the Iberian Peninsula (Fig. 5).
The new species lives in groundwater of epikartic gours and pools in a Basque cave and shows very subtle morphological differences with other Vejdosvkybathynella species.
In this study we have complemented the morphological analysis with a molecular analysis to support the validity of the new species described.We present all the morphological characters needed to establish a new taxon, as required by classical taxonomy, but we also have added new characters (mtDNA and 18S rRNA sequences) obtained with molecular techniques.
The genetic divergences observed in partial COI mtDNA sequences of the new species, 14.17 to 16.73%, should be enough to discriminate between congeneric species.At the moment, we still lack clear taxon definitions, and the demarcation of species using genetic divergences based on a single mitochondrial sequence is still imprecise (Meier et al., 2008).In a previous paper, we found values of divergence between 14 and 17% for different morphospecies of the genus Vejdovskybathynella (Camacho et al., 2011(Camacho et al., , 2012)), and values ranging between 15.8 and 23.6% between morphospecies of Iberobathynella Schminke, 1973 (Parabathynellidae) (Camacho et al., 2012).Nevertheless, since genetic divergence thresholds can vary among organisms, currently it is not clear what level of divergence designates a significant difference within and among lineages.Within crustaceans, Lefébure et al. (2006) investigated the relationship between morphospecies and genetically diverse species and identified 16% genetic divergence to be a consistent threshold for distinct species.Costa et al. (2007) suggested that a 17% divergence assessed different species between decapoda crustaceans.Abrams et al. (2012)    ger, ranging from 15.8% to 24.6%, .For the purpose of this study, the genetic divergences found and the robustness of the main clades assure us that our new species, V. vasconica sp.nov., is certainly valid and belongs to the genus Vejdovskybathynella.The results also show that the specimens from Erizo river and Río Chico cave probably belong to Vejdovskybathynella, and that the species of these two caves, is closer to V. vasconica sp.nov.than to V. edelweiss.In addition, the phylogenetic position established for some unassigned taxa provides a first indication of their relationship both to each other and to other species currently morphologically well identified and supported by their geographical distribution (see map of the Fig. 5).
Besides the COI sequences, partial sequences of 18S rRNA (about 1000 bp) have been obtained in this study.This is the first time this is done in Spanish Bathynellacea, so we do not have previous data to establish comparisons.Abrams et al. (2012) obtained partial sequences (about 700 bp) of several species belonging to three Australian genera of Parabathynellidae, and one more sequence of one unassigned specimen of Australian Bathynellidae.They found on average 18S rRNA sequence divergence among genera between 3.1% and 8.8%, and an average divergence between all Australian parabathynellid species of 4%.In our study, the genetic divergences observed in partial 18S rRNA sequences between the new species and other Spanish Bathynellidae ranged from 1.63 to 6.12% (see Table 3).We have not included the Genbank sequences of Australian species in our data-set since the combined number of base pairs was very small to offer good results.In the phylogenetic tree obtained using only our data several small incongruences within two major clades that correspond to both families were found.For example, Iberobathynella burgalensis, I. parasturiensis and I. imuniensis appear in the same group, but Paraiberobathynella appears nested within them.In addition, the group corresponding to Vejdovskybathynella is not very well supported, despite the geographical proximity of species (see Fig. 5).
It would be necessary to obtain longer sequences of these genes, as well as to consider more genes in order to expand the molecular studies.This should be done in parallel to further morphological analyses to outline the populations composed by specimens of mixed species, of different or the same genera.The use of both classical taxono-mic tools and molecular techniques will generate more precise knowledge on the taxonomy of a group of groundwater invertebrates that is proving to be much more diverse than previously considered.

Table 1 .
-Localities of the studied samples of Bathynellacea, voucher numbers of the Tissues and DNA Collection of the Museo Nacional de Ciencias Naturales,

Table 2 .
-COI genetic distances among and within species groups of the Spanish and Australian Bathynellidae.Tabla 2.-Distancia genética encontrada en el gen COI entre y dentro de grupos de especies de la familia Bathynellidae de España y Australia.

Table 3 .
-18S genetic distances among and within species groups of the Spanish Bathynellacea.Tabla 3.-Distancia génetica en el gen 18S entre y dentro de grupos de especies españolas de la familia Bathynellidae.

Table 8 .
-Summary of genetic divergence (%) for COI between different level of taxa of populations of Bathynellidae from Northern of Spain.Outgroup used: Iberobathynella imuniensis of the Parabathynellidae family.Tabla 8.-Resumen de la divergencia genética encontrada en el gen COI entre diferentes poblaciones de la familia Bathynellidae del Norte de España, pertenecientes a varios géneros y especies.Grupo externo usado: Iberobathynella imuniensis de la familia Parabathynellidae.