Composition of meiobenthonic Platyhelminthes from brackish environments of the Galician and Cantabrian coasts of Spain with the description of a new species of Djeziraia (Polycystididae, Kalyptorhynchia)

From 1997 to 1999, the fauna of free‐living Platyhelminthes of the rias ecosystem was studied along the Galician and Cantabrian coast in northern Spain. In total, 72 platyhelminth species are listed in this study. Forty‐two species represent new records for the Iberian Peninsula, three of which represent new genera records. A new species belonging to the genus Djeziraia (Polycystididae, Kalyptorhynchia), Djeziraia longistyla sp. nov., is described in this paper. In this broad‐scale study, a large data set (27 localities) of the estuaries of northern Spain allowed an analysis of the turbellarian species assemblages and the relation of species distributions to salinity, conductivity, oxygen, temperature, and sediment characteristics. Species assemblages (species diversity) of each habitat of the brackish water ecotone are shown. The present study contributes to knowledge on the ability of adaptation of free‐living Platyhelminthes to regimes of brackish water ecotones.


Introduction
Research on the taxonomy and ecology of meiobenthonic organisms is well established for estuaries of northern and western Europe (Gourbault 1981;Warwick and Gee 1984;Ax 1991;Warwick et al. 1991;Mettam et al. 1994;Smol et al. 1994;Soetaert et al. 1994;Attrill and Thomas 1996;Blome 1996;Blome and Faubel 1996). In contrast, the meiobenthonic fauna of estuaries of the Iberian Peninsula is relatively unknown (Soetaert et al. 1995). Most studies in this region have been carried out in the delta of the Ebro River and deal with ecological aspects (Muñ oz 1990;Ibañ ez et al. 1995Ibañ ez et al. , 1996Ibañ ez et al. , 1997Guillen and Palanques 1997;Mikhailova 2003), whereas studies on the Galician and Cantabrian coasts of Spain have been focused on the distribution and composition of meiofaunal and macrofaunal communities (Palacio et al. 1992;Curras et al. 1993;García-Á lvarez et al. 1993;Lastra et al. 1993;Sanchez-Mata et al. 1993;Garmendia et al. 1996;Arroyo et al. 2004;Borja et al. 2004;Garcia-Arberas and Rallo 2004). In Europe, taxonomic investigations on free-living Platyhelminthes have been mostly conducted in aquatic environments of Fennoscandia and estuarine areas of the North Sea (Nasonov 1926;Luther 1943Luther , 1960Luther , 1962Luther , 1963Karling 1963Karling , 1974Den Hartog 1977;Armonies 1987;Ax 1956a;1995;Faubel and Warwick 2005). Comparable investigations are relatively scarce in Spain. Until now, only a few species were known and described from the Iberian Mediterranean coast (Gieysztor 1931;Steinbö ck 1954).
Hence, it was the aim of the present investigation to contribute to the knowledge of species composition and distribution of estuarine brackish water turbellarians along the Galician and Cantabrian coast. A brief account is given of the relationships between species distribution and community structure, and intrinsic abiotic factors during the study are shown. A new species, Djeziraia longistyla sp. nov. is described.

Material and methods
The northern region of the Iberian Peninsula, specifically the Galician and Cantabrian coast (Figure 1), is characterized by more than 26 rias of different sizes and depths. Rias are estuaries with important variations in salinity in the superficial waters due to evaporation or to the contribution of freshwater from rivers. These areas are usually very fertile, which, apart from the stratification, results in high oxygen consumption and therefore oxygen depletion, especially in the depressions on the estuary bottom (e.g. Ria de Vigo, Galicia) where anaerobic sediments have been deposited. In addition, the rias are subjected twice a day to tides, one of which is generally of greater amplitude. During high tide, the meiobenthonic organisms descend 5-10 mm into the sediment (Margalef 1989).
During the years 1997-1999, samples were taken (Project Fauna Iberica: SEUI-DGES PB95-0235) in the rias of the coastal region of northern Spain. Sixty-one sample sites were selected within the Comunidad de Galicia (March 1998 andMarch 1999), Comunidad de Asturias (October 1998), Comunidad de Cantabria (September 1997), and Comunidad de el País Vasco (July 1998). In addition to the sampling in the rias, the investigations were extended to the most important rivers contributing a great amount of freshwater to the system (e.g. Arnoia, Ribadil, Miñ o, Carvallo, Miñ or, Oubia, Ferreras, Uncín, Ason, Ebro, Mieras, and Lea) (Table I; Figure 1).
The samples were obtained from sandy or muddy sediments in the eulittoral zone along the coastline and from the mouths of rivers during low tide using a small shovel to take surface sediments of about 320 cm 3 from an area of about 2068 cm and a depth of 1-2 cm (samples 4, 5, 7-10, 13, 14, 18, 22-27; Table I; Figure 1). Three replicates were taken for each sediment sample. In general, below a sediment depth of 2 cm, the chemocline showed a strong contrast from aerobic to anaerobic sediment layers in the eulittoral and shallow sublittoral areas. Therefore, free-living Platyhelminthes were found only in the first 2 cm of sediment. Samples from stagnant waters (limnetic, brackish or marine), such as rock-pools covered with aquatic vegetation or detritus, were extracted with a plankton-net (mesh size 125 mm) (samples 12, 15, 16, 19-21; Table I). The Karaman-Chappuis method (Chappuis 1942) was applied to obtain samples from the gravel of river banks (samples 1-3, 6, 11, 17; Table I). The water obtained with this method was filtered (approximately 30 litres) with a sieve of mesh size 125 mm. The individual number of each turbellarian species was recorded for all samples taken.
The samples were deposited in storage jars and transported to the laboratory. Individuals were separated under a dissecting microscope. Sexually mature specimens were identified alive and in squash preparations, i.e. flattened under the increasing pressure of the cover slip as the preparation dries. For anatomical study of the hard-structures such as stylets, whole mounts (polyvinyl-lactophenol) were prepared. For histological observation, specimens were fixed in Bouin's fixative. Sagittally prepared serial sections (4 mm thick) of sexually mature specimens were stained with Azan. Most of the individuals were also photographed (Video Graphic Printer UP-86 OCE) and filmed (Canovision EX-HI 8) Figure 1. Location of the sample sites, with turbellarians (black points) and without turbellarians (white circles). The locality numbers correspond to those given in Tables I-III. under a microscope. The type material has been deposited in the Museo Nacional de Ciencias Naturales (MNCN), Madrid.
For characterization of the habitats, the abiotic factors of conductivity, temperature, oxygen concentration, and salinity (refractometric determination) were measured mainly in the water column (conductivity, Tu: CRISON CDTM-523; oxygen, Tu: YSI 33-SCT). Salinity regimes and classification were based on the Venice System (Karling 1974): limnetic (,0.5%), oligohaline (0.5-5%), mesohaline (5-18%), polyhaline (18-30%), euhaline (30-40%). Table II shows the distribution of the principal species found in localities along the Galician and Cantabrian coastal regions. Microturbellarians were found in 27 of the 61 sampling sites. Therefore, only these sites are considered in the faunistic approach presented. The total number of Turbellaria amounted to 72 species. From the total species number, 48 could be identified to species level and 13 could not be clearly identified (sexually immature juveniles) and are designated as ''cf''. Eleven taxa could be taxonomically determined to   (Karling, 1935) 24 Brackish (meso) the genus level. One species found in the polyhaline area of Ria de Arosa is new to science (Djeziraia longistyla sp. nov., description below). All species found are new records for the Galician and Cantabrian coast and 43 species represent new records for the Iberian Peninsula. The two sampling visits indicate high species richness for the area, with most species occurring in the freshwater/oligohaline and polyhaline/marine zones. There are no species common to all Spanish districts. Promesostoma marmoratum is the species with the broadest distribution, always found in habitats with salinities between 0.5-5%. Another species with a wide distribution found in oligohaline habitats is Mecynostomum auritum. Forty-seven species were found only in one of the sample localities (Table II) and 18 species live exclusively in limnetic areas (up to 0.5%).

Meiobenthonic composition
In the study area, one species, Rhynchoscolex simplex, appears to be ubiquitous without any definite relationship to a specific substrate. However, Rhynchoscolex simplex is a characteristic representative of freshwater sands, although the species is also abundant in stony areas of running waters (ubiquist- Table III). The species settles in the sandy interspaces between the stones and pebbles.
Some species were captured in only one type of habitat, and are therefore characteristic for the limnetic, brackish, or marine areas. Between them the most abundant species for the marine area was Archilina papillosa, in the limnetic environments Stenostomum leucops and Microdalyellia tennesseensis, and in the brackish environments Macrostomum rostratum.
On the other hand, the brackish habitats are characteristically overlapping zones for the marine and limnetic species. Therefore, 17 species captured in this study belong to these olig, oligohaline; meso, mesohaline; poly, polyhaline. a Locality numbers correspond to those given in Table I. Meiobenthonic Platyhelminthes from brackish environments overlapping areas. Within this group we find three species originally described for marine and brackish environments: Archilopsis unipunctata, Monocelis lineata, and Promesostoma marmoratum.
Another group is formed by species that can be found in different types of substratum, such as Bothrioplana semperi, Microdalyellia fusca, Phaenocora unipunctata in limnetic habitats and, for example, Proxenetes flabellifer or Ptychopera westbladi (see Table III) within brackish water habitats.
Alien species includes species that were found only once or in very small numbers, such as Nematoplana cf. nigrocapitula, a marine species, captured in a limnetic habitat (brook in America Beach, Ria de Bayona), and Paramecynostmum diversicolor and Pelophila pachymorpha, marine species but captured in brackish habitats.
The casual species represent a group of species with low individual number (sometimes only one individual). Their presence in the studied habitats is casual and their association with the locality cannot be asserted. Within this group we find species such as Archimonotresis limophila, Brunetia camarguensis, and Castrella truncata (Table III).
Etymology. The species name refers to the long stylet of the male copulatory organ.
Description. Sexually mature individuals 0.9-1.2 mm long ( Figure 2A). Body conical, rounded posteriorly. Colourless or light grey due to subepidermal pigment layer; whitish at posterior end due to well-developed caudal glands (in incident light). Well-developed proboscis glands and brain between the proboscis and the pharynx (Figure 2A). Two dark eyes clearly separated, posterior to the proboscis and surrounded by the brain. The ciliary cover forms narrow furrows that run parallel to the longitudinal axis, only visible in slightly squashed individuals (Figure 2A, posterior end of the body).
The general organization of the epidermis and the basal membrane corresponds to the organization described for Djeziraia pardii Schockaert, 1971(Schockaert 1971. The proboscis is very small (0.045 mm long in living animals) and at the anterior end of the body. The pharynx (0.112 mm diameter) is directed forward, and lies in the anterior half of the body.
Male reproductive system with two elongated testes dorso-laterally at the posterior end of the pharynx. The male copulatory organ is composed of an unpaired vesicula seminalis, a vesicula granulorum or prostatic vesicle, and a long slender stylet ( Figure 2B, C). The spherical vesicula seminalis is surrounded by a thin epithelium and weakly developed diagonal muscle layers while the prostatic vesicle is smaller and surrounded with a strong muscular layer consisting of spiral fibres. Both vesicles are clearly separated by a sphincterlike constriction. Extra-vesicular prostatic glands enter the prostatic vesicle proximally at the constriction. The stylet is a straight delicate tube, 250-300 mm long, slightly curved and with a terminal opening ( Figure 2B, C).
In the female reproductive system, the bilateral vitellaria represent two large tubes running from the posterior end of the pharynx to the posterior end of the body (Figure 2A). The ovaries lie at the middle of the body, postero-laterally to the male copulatory organ. Ovaries connected with the single receptaculum seminis via short oviducts ( Figure 2B). The receptaculum seminis passes communicates with the atrium genitale communis via the female duct (type II, after Artois and Schockaert 2005). The ovoid bursa is dorso-caudally located, and provided with a well-developed, muscular stalk or female duct type I and a distal common oviduct ( Figure 2B). The female duct type I connects the bursa with the atrium genitale while the common oviduct connects the bursa with the receptaculum seminis. It is not discernible whether the distal part of the common oviduct and the proximal part of the female duct type I join the distal bursa together or separately ( Figure 2B). In one animal the bursa contains a ''ball'' of compact sperm. The uterus lies ventrally and its opening is just ventral to the opening of the female duct type II. Eggs were not observed in the studied animals.
Female and male atrial organs (male copulatory organ, female ducts I and II, and uterus) open independently into the latero-frontal section of the small atrium genitale communis ( Figure 2B). A long narrow genital duct leads from the distal part of the genital Figure 2. Djeziraia longistyla sp. nov. (A) General organization in dorsal view (from the living animal); (B) atrial organs; (C) stylet (photograph from the living animal). b, bursa; cg, caudal glands; cod, common oviduct; fd-I, female duct type I; fd-II, female duct type II; ga, common genital atrium; gd, genital duct; gp, gonopore; ov, ovary; ovd, oviduct; p, proboscis; pg, prostatic glands; ph, pharynx; pv, prostatic vesicle; rs, receptaculum seminis; st, stylet; te, testis; u, uterus; vi, vitellaria; vs, vesicula seminalis; vd, vitelloduct. atrium to the common genital pore. This pore is situated at the beginning of the last third of the body.
The most conspicuous difference between Djeziraia longistyla sp. nov., D. pardii, and D. euxinica is the length of the stylet. The two last mentioned species have short stylets: 85 mm in D. pardii and 105-120 mm in D. euxinica. In D. longistyla sp. nov. the stylet is 250-300 mm long. The presence of a long, slender stylet (.150 mm) is shared with D. incana. Djeziraia incana has a stylet of 162-198 mm (up to 295 mm in one specimen) in length. Based on the length of the stylet, D. longistyla sp. nov. and D. incana are the most similar species within the genus Djeziraia. The differences between D. longistyla sp. nov. and D. incana concern the shape of the prostatic vesicle and the vesicula seminalis. Djeziraia longistyla sp. nov. shows a prostatic vesicle with a coat of strong spiral muscle layers, as in D. pardii, whereas D. incana has a thin-walled prostatic vesicle. As previously mentioned, both prostatic and seminalis vesicles are clearly separated in D. longistyla sp. nov. through a sphincter-like constriction. This prominent division between the vesicles is present in D. pardii, but not in D. incana.
On the other hand, Djeziraia longistyla sp. nov. and D. incana share a common oviduct that is surrounded by a thick circular muscle layer, as is the case in D. pardii (''diverticulum'' after Schockaert 1971, or ''insemination duct'' after Artois andSchockaert 2001). However, the peculiar horns at the connection of the common oviduct to the recepaculum seminis (Schockaert 1971;Artois and Schockaert 2001) are absent in both D. longistyla sp. nov. and D. incana.

Environmental remarks
Relationships between environmental factors and turbellarian presence in the sample sites during the study are presented in Figures 3-5. The number of specimens is plotted against temperature, conductivity, and oxygen.
High levels of conductivity did generally indicate high levels of salinity. The sites with lower salinity values are areas that correspond to the brackish water ecotone. The substrates consisted of muddy, stony, or sandy sediments. In these areas Turbellaria abundance shows the highest values (cf. Figure 3). The maximum value at site 12 was essentially caused by the high abundance of Macrostomum rostratum (.30 individuals).
The temperature recordings (Figure 4) reflect the actual values at the time of sampling, which varied between 11.6 and 27.8uC at different times of day.
Dissolved oxygen ranged between 1 and 10 mg l 21 (Figure 4). The upper sediment layers (0-2 cm sediment depth) were well oxygenated and contrasted well against the lower greyish chemocline in which the oxygen values rapidly declined to very low values. Low values of oxygen correspond with sites from which turbellarian species were generally absent or which were very poor in species richness (Figure 4). That holds for the limnetic and marine areas, but in the brackish water ecotone there were some sites (10, 12, 25, and 27) with low oxygen values and high abundance values. This may be a result of a favourable combination of abiotic and biological factors.
In Figure 5, the percentage of species in each of the different types of substrate is shown. Habitats consisting of sand or mud each accounted for 29% of the species. The dominant Figure 3. Number of turbellarians found in relation to the conductivity and to the salinity (conductivity data of the brackish localities were not considered, because they were punctual measurements). substrate covering the bottoms of the sites sampled was mud, sometimes blended with sand. In relation to the occurrence of sandy-gravel sites (5% of the total of sample sites), the abundance of species appears to be relatively higher than in muddy habitats (Figure 4).
With respect to species richness (Figure 4), a clear gradient occurs from habitats arranged according to sand, stony, sandy stony, sandy muddy, and muddy. The lowest numbers of species were found in sands and in stony habitats. However, it is generally the case that muddy sediments are most densely settled. These results suggest that the presence of ''Turbellaria'', at the species level only, is closely related to characteristic types of substrates.

Discussion
The sampling sites were located on the coast and riverbanks of the rias along the Galician and Cantabrian coasts where the habitats range from limnetic (salinity between 0 and 0.5%) to euhaline areas (e.g. Ria de Ajo, Ria de Rada, salinity of up to 30%; Figure 3). The habitats considered as brackish (with variable salinity, between 0.5 and 30%) are characterized by short-term salinity variations (shock-biotopes after Den Hartog 1964). These variations are normal occurrences in the rias. Therefore, only species with a high degree of adaptation to salinity variations would be expected to occur (Meixner 1943;Reuter 1961;Ax and Ax 1970).
For the distribution of the turbellarians and colonization of brackish habitats, two environmental factors appear to be fundamental: the substratum and the salinity. Most of the species were found in sandy, sandy-gravel, or sandy-stony substratum ( Figure 5). Consequently, the interstitium as a tidal shelter and colonization medium appears to have been verified (Karaman 1955;Ax 1956a;Riemann 1968;Karling 1974). On the other hand, muddy bottom is also an adequate habitat for Platyhelminthes, but only in the first few centimetres of sediment and with the simultaneous presence of green algae or periphyton ( Figure 5; Table I).
In relation to the salinity and the studied turbellarian fauna, some species were exclusive to limnetic habitats, a fact that coincides with previous records (e.g. Figure 5. Percentage of turbellarians in relation to the substrate type. M, muddy; S, sandy; SG, sandy-gravel; SM, sandy-muddy; SST, sandy-stony; ST, stony.