A new endogean, anophthalmous species of Parazuphium Jeannel from Northern Morocco (Coleoptera, Carabidae), with new molecular data for the tribe Zuphiini

Abstract A new species of the genus Parazuphium (Coleoptera, Carabidae, Zuphiini), Parazuphium aguilerai sp. n., is described from the Tingitan peninsula in North Morocco. The only known specimen was found under a large deeply buried boulder, and belongs to an anophthalmous, depigmented and flattened species. This is the second species of blind Parazuphium known so far, the other being Parazuphium feloi Machado 1998 from a lava tube in the Canary Islands. Molecular data of the only known Parazuphium aguilerai sp. n. specimen are provided, and a reconstructed phylogeny based on these molecular data confirms its inclusion inside Zuphiini within Harpalinae. Identification keys to the Mediterranean and Macaronesian species of Parazuphium are provided.

The species of the genus seem to be associated with deep soil or the soil crevices near rivers or temporary flooded areas (Baehr 1985, Machado 1992, and generally show a flattened habitus, some degree of depigmentation and microphthalmy. Some species are known from caves, one of them being the only previously known blind species of the genus (P. feloi Machado, from the Canary islands) (Machado 1998).
During an entomological expedition to North Morocco we found the single specimen of a new species of Parazuphium, anophthalmous and with strong modifications apparently related to its endogean habitat. Despite an attempt to collect additional material the following year no other specimen was found, possibly due to the endogean habits of this species. We describe the species here, and provide some molecular data to characterize it and to postulate its phylogenetic position among the Zuphiini for which genetic data are available (Ribera et al. 2006).

Material and methods
The unique specimen was killed and stored in absolute ethanol in the field, and total DNA was extracted using the QIAGEN Dneasy tissue kit (Qiagen, Hilden, Germany), without destroying the external cuticle. The extracted specimen was mounted in DMHF (Dimethyl Hydantoin-Formaldehyde) on a transparent acetate label. For the morphological study and photographs we used a Zeiss Stemi 2000C Trinocular Zoom Stereomicroscope with Spot Insight Firewire digital camera and software.

Molecular methods
Total genomic DNA for the single specimen of Parazuphium aguilerai sp. n. was extracted using QIAGEN Dneasy tissue kit (Qiagen, Hilden, Germany). To characterize the new species we amplified fragments of six genes, four mitochondrial and two nuclear: 3' end of cytochrome c oxidase subunit (cox1); a single fragment including the 3' end of the large ribosomal unit (rrnL), the whole tRNA-Leu gene (trnL) and the 5' end of the NADH dehydrogenase 1 (nad1); 5' end of the small ribosomal unit, 18S rRNA (SSU); and an internal fragment of the large ribosomal unit, 28S rRNA (LSU). Primers used are given in Table 1. Additionally, we extracted DNA from one specimen of Parazuphium cf. baeticum (K. and J. Daniel 1898), Zuphium olens Rossi 1790, Ildobates neboti Español 1966 and several other outgroups among Carabidae (Table 2), which were amplified for the same molecular gene fragments. PCR reactions were made using PuReTaq Ready-To-Go PCR beads (GE Healthcare, UK) and standard conditions [39 cycles using 48-50°C as annealing temperature]. New sequences have been deposited in GenBank (NCBI) with Acc. Nos JF778779-JF778845. Each individual gene matrix was aligned in MAFFT with the Q-ins-i option and default parameters. The four genes fragments were concatenated to get a final dataset of 20 taxa and 3376 bp that was employed in phylogenetic analyses. Table 2 shows taxa information, source and accession number for each DNA sequence.

Phylogenetic methods
Bayesian phylogenetic analyses (BA) were performed with MrBayes v.3.1. Ronquist 2001, Ronquist andHuelsenbeck 2003), partitioning by gene with a GTR+G model applied to each partition. Two independent runs of 20,000,000 generations were conducted, each with three hot and one cold chain, whereby trees were sampled every 100 generations. Sampled trees were analysed with Tracer v.1.5 (Rambaut and Drummond 2007) and their half compact consensus tree was calculated with a burning value of 10% with node posterior probabilities used as support values, checking for an appropriate degree of convergence between chains with the effective sample size in Tracer v.1.5. MrBayes was run on-line at the freely available computational service of Bioportal (www.bioportal.uio.no Type locality. Souk-Khemis-des-Anjra, Tetuan, Morocco (Fig. 4).

Especie
Legs. Pro-and meso-femora dilated proximally, forming an obtuse interior angle (Figs 2i, k). Metafemora with a strong acute tooth on the interior margin (Fig. 2m). Front tibia with antennal cleaner (toilette organ), as reported in other species of the genus (Fig. 2i). Meta-tibia long and straight, with an internal spine at apex. Meso and meta tibiae with a circle of seta round the apex. Pro-tarsomeres 1-4 dilated (Fig. 2j). First meso-and meta-tarsomeres as long as 2° to 4° combined (Figs 2l, n). Fourth tarsomere cordiform. Trochanters without tooth or any other special structure.
Aedeagus. Median lobe as in Fig. 3, short and robust with a ventral constriction between the basal and the distal part as described for the genus. Basal margin arcuate, bisinuate, with the apex rounded. Internal sac with two small sclerites. Parameres asymmetric, as in other species of the genus.
Etymology. The specific epithet is a Latinized eponym, genitive case, based on the name of our late friend Pedro Aguilera, who collected the specimen with us during his last trip to Morocco.
Recognition and comparisons. Parazuphium aguilerai sp. n. can be clearly distinguished from any other species of the genus through the combinations of the following characters: lack of eyes, reduced size (2.7 mm), length and proportions of 2°, 3° and 4° antennomeres (0.1, 0.13 and 0.14mm respectively) and the presence of a tooth on metafemora. Parazuphium feloi from the Canary Islands is also anophthalmous, but it is larger than P. aguilerai sp. n. and without a tooth on the hind femora (Machado 1998). Parazuphium ramirezi J. and E. Vives from south Spain shows the same tooth on the metafemora, but is also larger, and with reduced eyes (Vives and Vives 1976). There are also some differences in the shape of the head and pronotum: in P. aguilerai sp. n. the head is more parallel-sided, the anterior angles of the pronotum are less rounded, and the anterior margin not straight.

identification key
Key to adults of the West Mediterranean and Macaronesian Parazuphium species, modified from Antoine (1962) and Hürka (1982) Third antennal segment not twice as long as 2nd and distinctly shorter than 4th, legs short and robust, metaibiae curved, strongly so in male. North Africa,

Phylogenetic analysis of molecular data
The cox1 gene fragment was aligned with no gaps, and its correct translation to amino acids confirmed. Alignment of the three ribosomal markers resulted in several gaps, which were included in the analyses as obtained from MAFFT without further modifications. Bayesian analysis reached a convergence value of 0.0005 after 20 million generations. The initial 10% saved trees were removed as a burning value and the half consensus tree was built with the "sumt" option in MrBayes v.3.1. Figure 5 represents the obtained phylogeny, were most of nodes showed very high Bayesian posterior probabilities, which are interpreted as Bayesian support.
We recovered a monophyletic Zuphiini, with the two studied species of Parazuphium as sisters, and sister to Zuphium (Fig. 5). Zuphiini was sister to Drypta, in a monophyletic Dryptinae (sensu Serrano 2003).

Relationships of Parazuphium
The genus Parazuphium is currently included in subtribe Zuphiina (tribe Zuphiini), together with Ildobates, Zuphium and Polistichus among the Palaearctic fauna (Baehr 2003). Although the scarcity of data does not allow a comprehensive study, our molecular results support this taxonomic position, with both studied Parazuphium species clustered together as a sister group of Zuphium (Fig. 5). Zuphium and Parazuphium species are recovered as related to Ildobates neboti, which was found as belonging to the Zuphiini by Ribera et al. (2006). Our data confirm the close relationship of Zuphium and Parazuphium, while a more detailed phylogeny would be needed to establish the position of Ildobates within Zuphiini.
The subgenus Neozuphium was described by Hürka (1982) based on the relative length of the 2nd to 4th antennomeres and the shape of the legs, more robust and with curved tibia and enlarged femora in the males in Neozuphium. Parazuphium aguilerai sp. n. has the third antennomere only slightly longer than the 2nd and slightly shorter than the 4th (Fig. 2e), so it would agree with Neozuphium (species of Parazuphium s.str. have the 3rd antennomere double than the 2nd, and similar to the 4th, Hürka 1982, Mateu 1988). However, the shape and size of the legs do not agree with the diagnostic characters of Neozuphium, as the males have straight metatibia (Fig. 2m) and they are in general slender and long in comparison to P. (Neozuphium) damascenum (Figs 2i-n). These are, in any case, characters with dubious phylogenetic information, so instead of redefining the subgenera, or describing additional taxa, we opt to follow Serrano (2003),treating the subgenus Neozuphium Hürka as consubgeneric with Parazuphium Jeannel, and the former name as a junior synonym of the latter.

Endogean way of life in Zuphiini
Parazuphium aguilerai sp. n. differs from all other known species of the genus in its clear adaptations to an endogean way of life. Other species are regularly found in soil crevices, specially among the cracks of the dried substratum of areas which are regularly inundated (Baehr 1985, Machado 1992, Lencina and Serrano 1995. These species have some modifications suggesting an adaptation to this cryptic way of life (small size, flattened body, some degree of depigmentation, microphthalmy, Jeannel 1942), but not to the extent of P. aguilerai sp. n., which was found in company of other typical endogean insects (Leptanilla sp., Amblyopone sp., Torneuma sp.) below a deeply buried large stone in a hillside. The only other anophthalmous species of the genus (P. feloi) was found in a cave, and it is larger and with longer appendages (Machado 1998), as is typical of cave fauna inhabiting larger open spaces. Parazuphium aguilerai sp. n. shares with P. feloi, P. chevrolati and P. vaucheri the presence of a spine in the metatibia (Machado 1998), although at the moment it is not possible to assert the phylogenetic value of this character.